Synthetic Studies of Some Biologically Important Molecules
Transcript of Synthetic Studies of Some Biologically Important Molecules
Synthetic Studies of Some Biologically Important M olecules
A Thesis presented in partial fulfillm ent o f the degree of Doctor o f Philosophy from the University of London
b y
Peter George Robins
UCL Chemistry Department, 20 Gordon Street,
London.
October 1994
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A b s t r a c t
A b s t r a c t
Potass ium channels play a crucial part in ce llu la r hom eostasis and
disruptions in their function are implicated in a variety of disease states
including diabetes and cardiac arrhythmias. Consequently , a wide variety
of potassium channel modulators are in therapeutic use but few of them
appear to act as antagonists. However, apamin, from bee venom has been
shown to be an extremely potent blocker of the small conductance,
calcium-activated potassium channel (SK) and the main body of this thesis
is concerned with ex tend ing previous w ork on the s truc tu re -ac tiv i ty
rela tionships of this compound. Two salient, pos itively-charged nitrogen
atoms on ad jacent arginine residues in the pep tide , arranged at an
appropriate spacing seem to be the seat of activity and the assumption that
this is the pharmacophore provides the basis of the work. Thus a series of
bis-quaternary ammonium salts were synthesised by the substitu tion of
various amines onto one of a number of rigid, aromatic frameworks which
were chosen because they arranged the cationic centres approximately 11
 apart, the posited crucial distance, as well as providing a hydrophobic
b r id g e . 2 ,6- and 2 ,7 -d im e th y l a n th ra c e n e , 3 ,6- and 1,6 -d im e th y l -
ph en an th ren e and t r a n s - 2 , 4 ' - and t r a n s 4 , 4 ' - d im e th y I s t i lb e n e w ere
synthesised according to established procedures. All compounds, including
p -x y len e and 2 ,6 -d im ethy l n a p h th a le n e , w ere b i s -b ro m in a te d in the
benzylic position with N-bromosuccinimide or bromine. The first three
fram ew orks in the list above were then substi tued with p iperid ine ,
pyrrolidine and morpholine and quaternised with iodom ethane to give 9
compounds of medium to negligible activity in blocking the SK current in
ra t s y m p a th e t ic n e u ro n e s . S u b s t i tu t io n o f a m ix tu re of 2 ,5 -
dim ethylpyrrolid ines followed by iodom éthylation produced a series of
compounds with good activity whilst the synthesis of a series of compounds
co n ta in in g cf j -2 ,6 -d im e th y lp cou ld not be com ple ted . 1,4-
diazabicyclo[2 .2 .2]octane (DABCO) and N-substitu ted derivatives, together
with quinuclidine were also used as substituents, but did not improve on
previous results. An attempt to make tricationic derivatives of benzene
could not be completed in the time available. The results of the assay and
A b s t r a c t
in fe ren c e s are p resen ted toge the r with spec tra l da ta for a ll new
c o m p o u n d s .
The second part of the thesis deals with a series of attempts to
generate the lithium dianion of pyruvic acid and effect its addition to
various electrophiles. No evidence was found for the presence of the ion so
a ttem pts were made to stabilise it by the use o f esters and hindered
th ioes te rs o f the acid which included ter t-buty l and 1, 1-d ie thy lpropy l
th iopyruvate and methyl and ethyl pyruvate. A num ber o f 0 - tr ia lky ls i ly l
enol ethers w a 5 also synthesised and their reaction^ with electrophiles in
the presence of Lewis acids were investigated. No conditions were found
under which reaction was observed to occur so the Diels-Alder and inverse-
dem and D ie ls -A lde r reac tions o f the silyl enol e thers were brief ly
i n v e s t ig a t e d .
A c k n o w l e d g e m e n t
A c k n o w l e d g e m e n t
I would like to thank my supervisor, P. J. Garratt, for the support he
has provided over the years of my attendance at UCL and for his help
during an often trying time.
I would also like to thank the various members of my group who
have provided the usual supply of chemicals, amity and conflict that have
made this course so varied. These include Stefan, Rob, Ashley, Sundram and
Najeeb. Many other people, too numerous to mention, were also on hand to
provide respite, but especially Aneela, Dan, Bruce, Duncan, Tara and Dave.
Thanks to Stefan for various nmr spectra, Karen for the nOe spectrum, and
Keith Wibley for drawing the M E? and energy-minimised diagrams.
F u n d in g for this course was p rov ided by the S c ience and
Engineering Research Council and I would like to extend my thanks to
them, and those responsible for securing my grant.
To Graham: thank you for supporting me over the years
The errors of a wise man make your rule
Rather than the perfections of a fool.
- William Blake
This thesis is dedicated to my father.
C o n t e n t s
Contents
A b s t r a c t .............................................................................................................................................. 2
A c k n o w l e d g e m e n t ..........................................................................................................................4
Dedication.............................................................................................................................................. 5
Table of Contents............................................................................................................................... 6
G l o s s a r y ................................................................................................................................................9
1. In t ro d u c t io n ............................................................................................................................... 11
1.1. The Basis of the Cellular Potassium Current............................................12
1.1.1. The Action Potential............................................................................. 12
1.1.2. Discovery of the Role of K+................................................................14
1.1.3. Classification of Potassium Channels........................................... 15
1.2. Potassium Channel diversity............................................................................. 17
1.3. Potassium Channel Structure............................................................................ 20
1.3.1. The Selectivity Filter............................................................................20
1.3.2. Use of Channel Antagonists..............................................................21
1.3.3 Detailed Channel Structure D educed From SAR- and 24
Point Mutation-Studies
1.4. Electrophysiological Characterisation........................................................... 28
1.5. Specific Potassium Channel Modulators......................................................31
1.5.1. Animal Toxins..........................................................................................31
1.5.2. Apamin-A specific Blocker of SK ...................................................32
1.5.3. Characterisation of Apamin Binding Sites ..................................33
1.6 The Structure of Apamin............................................................. 33
1.6.1. Primary Sequence.................................................................................. 33
1.6.2. Structure/Activity Relationships.................................................... 34
1.6.3. Configuration of Apamin................................................................... 38
1.6.4. Molecules Containing Bis-Quaternary N itrogen ..................... 42
Atoms as Mimics of Apamin
1.7. Therapeutic Applications of Potassium Channel A ntagonists 44
1.7.1. Mechanisms of Interaction With the C hannel..................... 44
1.7.2. Clinical Applications.............................................................................46
1.8. References.................................................................................................................. 48
2. Results and Discussion............................................................................................................ 52
2.1 Routes to Dimethylated Aromatic Hydrocarbons.................................... 53
C o n t e n t s
2.1.1. D im ethylphenanthrenes.......................................................................54
2.1.2. Dimethylanthracenes.............................................................................57
2.2. Benzylic Functionalisation of Aromatic H ydrocarbons ..................... 63
2.3. Synthesis of D im ethyl-phenanthrenes and -an th racenes.................. 67
2.3.1. Phenanthrenes.................................................................................. 67
2.3.2. Anthracenes.............................................................................................. 71
2.4. Other Hydrocarbons................................................................................................ 75
2.5. Formation of Amines..............................................................................................76
2.5.1. Derivatives of Piperidine, Pyf/olidine and M orpholine..76
2.5.2. Quaternisation of Amines................................................................... 80
2.5.3. Derivatives of 2,5-Dimethylpyrrolidine..................................... 82
2.5.4. Derivatives of DABCO..........................................................................84
2.5.5. Derivatives of Quinuclidine.............................................................. 88
2.5.6. Attempted Synthesis of Derivatives of c is -2 ,6 - ....................... 89
Dimethylpiperidine
2.5.7. Other Compounds...................................................................................90
2.6. References................................................................................................................... 92
3. Pharmacological Evaluation...................................................................................................95
Glossary.................................................................................................................................96
3.1. Introduction................................................................................................................ 97
3.2. Preparation of Rat Sympathetic Ganglia...................................................100
3.3. Derivatives of Piperidine, Pyrrolidine and M orpholine .................103
3.4. Derivatives of 2,5-Dimethylpyrrolidine...................................................... 104
3.5. A Comparison of Electrostatic Potentials................................................... 107
3.6. 2 ,6-B is[(cis-2 ,6-dim ethyl)piperidom ethyl]- ............................................... 114
Dimethiodide
3.7. Derivatives of DABCO and Quinuclidine.....................................................115
3.8. The Effect of N-N Distance.............................................................................. 119
3.9. Conclusion..................................................................................................................122
3.10. References............................................................................................................... 122
4. Experimental..............................................................................................................................124
4.1. Apparatus and Reagents......................................................................................125
4.2. Experimental.............................................................................................................126
4.3. References................................................................................................................. 181
5. N u c le o p h i le Addition of Pyruvic Acid Synthons.................................................182
5.1.Introduction................................................................................................................. 183
C o n t e n t s
5.1.1. Carboxylic Acid Dianions...............................................................183
5.1.2. Pyruvic Acid......................................................................................... 187
5.1.3. Aims of the Project...........................................................................190
5.2. Results and Discussion.......................................................................................193
5.3. Experimental...........................................................................................................210
5.3.1. Apparatus and Reagents.................................................................. 210
5.3.2. Experiments........................................................................................... 211
5.4. References................................................................................................................218
Appendix........................................................................................................................................... 221
Corrigenda and Addendum....................................................................................................... 225
G l o s s a r y
Glossary
KC - potassium channel;
ICF - intracellular fluid;
ECF - extracellular fluid;
ATP - adenosine triphosphate;
gx ’ conductance of x in a given medium;
AP - action potential;
C a j 2 + - intracellular Ca^+;
Nai'*’ - intracellular Na"*";
A T Pi - intracellular ATP;
I k - current associated with a given potassium channel K;
K m - muscarine-sensitive potassium channel;
K r / K i - inward rectifying potassium channel;
K v - outward (delayed) rectifying potassium channel;
K a - potassium channel carrying the transient A-current;
K a T P / ^ g - adenosine triphosphate-sensitive potassium channel;
BK - large conductance, Ca^"*"-activated potassium channel;
SK - small conductance, Ca^'*’-activated potassium channel;
TEA - tetraethylammonium ion;
QA - quaternary ammonium salt;
K d (v ) - voltage-dependent affinity of a putative blocker for a given
channel recep tor;
5 - (i) parts per million of applied field, relative to tetramethylsilane;
(ii) electrical distance;
z - ionic charge;
SAR - s tructure/activity relationship;
Sn - membrane-spanning segment of a potassium channel;
H5 - extracellular connecting region between S5 and S6 of most potassium
channels;
Shaker, RCK - two closely related families of genes from d r o s o p h i l a
melanogaster which encode a series of A- and delayed
rectifier-type channels;
L D 50 - median lethal dose; the dose required to effec t 50% mortality in vivo'.
G l o s s a r y
A23187 - a Ca^"^ ionophore used to stimulate response of Ca^'^-dependent
p ro c e s s e s ;
nOe - nuclear Overhauser effect;
HPLC - high-pressure liquid chromatography;
DABCO - l,4-diazabicyclo[2.2.2]octane;
HIV - human immunodeficiency virus;
FAB - fast atom bombardment mass spectrometry;
Cq - quaternary carbon atom;
Ct - tertiary carbon atom;
Cs - secondary carbon atom;
Gly, G - glycine;
Tyr, Y - tyrosine;
Asp, D - aspartic acid;
Thr, T - threonine;
Met, M - methionine;
Val, V - valine;
His, H - histidine;
Cys, C - cysteine;
Asn, N - asparagine;
Pro, P - proline;
Glu, E - glutamic acid;
Leu, L - leucine;
Arg, R - arginine;
S - serine;
F - phenylalanine;
K - lysine;
I - isoleucine;
A - alanine;
W - tryptophan;
Har - homoarginine;
Orn - ornithine;
T h r-11 - eleventh amino acid residue from the C-terminus.
10
Chapter 1
In trod u ction
I n t r o d u c t i o n
I n t r od u c t i on
1.1 T he Basis O f T he C e l lu la r P o ta s s iu m C u r r e n t
1.1.1 T he A c t io n P o te n t i a l
All living cells exhibit a resting membrane potential, but a characteristic
of excitable cells (that is, muscle cells and neurons ) is that they are able to
a l te r th a t po ten t ia l by va r ia t ions in the io n -p e rm e a b i l i ty o f the
m e m b r a n e ^ . In a normal cell, this potential difference at rest is 50-100 mV
with the interior of the cell negative and is due to the uneven distribution
of ions between the intracellular fluid (ICF) and the extracellu lar fluid
(ECF). This difference is maintained by a slow but continuous process of
active transport whereby enzymes powered by the metabolism of adenosine
triphosphate (ATP-ases) pump Na+ ions from the cell and K+ ions into the
cell resulting in concentrations of K+ in the ECF and ICF of 4 and 150 mM
respectively. The diffusion rate for this process is small, typically 1 p m o 1
m ’ ^ s ’ ^, but very much more rapid ion flow occurs when the stimulus
applied to an excitable cell (the release of acetylcholine at a synapse or
motor end-plate is the usual means by which this occurs) is sufficiently
strong to generate the so-called action potential (AP) (figure 1 .1 ) . The
stim ulus reduces the negative resting m em brane po ten t ia l to a less
negative value, a process called depolarisation. When a critical voltage, the
threshold potential, is reached activation of the voltage dependent Na+
channels occurs, causing a sudden, large increase in the Na+ conductance (gN a) of the cell membrane, and a fast Na+ influx. During this phase of the
AP, the negative state inside the cell is not only reversed but the membrane
potential even reaches positive values (overshoot). Prior to this, however, gNa begins to decrease (this “inactivation” occurs after approximately 0.1
ms) accompanied by a slow rise in K"*" conductance (g ^ ) . This permits K+
efflux and leads to the re-estab lishm ent o f the negative potentia l orrepolarisation. For a few milliseconds before g ^ returns to its resting value,
the m em brane po ten tia l may be even m ore n ega tive than at rest
(hyperpo larisa tion) . Besides m uscle contraction and the p ropagation of
nerve impulses, the AP is associated with a number of phenomena such as
12
I n t r o d u c t i o n
— - 90 mV
1. Resting state ^ + 20 mV
A
2. Depolarization
jovershoo t
3. Repolarization
Threshold 4 ' / \/ \
Hyperpolarization
A. Depolarization and repolarization
Restingmembranepotential
Time
Figure 1.1: The Action Potential
13
I n t r o d u c t i o n
an increase in the intracellular concentration of ATP or excretion from
endocrine cells, and these form the basis of any clinical exploitation.
1.1.2 Discovery Of The Role Of K+
The nature of the action potential has been known for a number of years.
The rôle of Na+ was established first since it was known that the external
c o n c e n tra t io n of the ca tion was m uch g rea te r than the in te rn a l
concen tra tion , rendering a ltera tions in experim ental cond it ions a more
fac ile p rocess . A series o f experim ents on a num ber of c e llu la r
preparations such as frog muscle^ and squid giant axon^ revealed that if
this concentration fell below a certain level, or was replaced by a Na**‘- f r e e
medium, then progressive depression of the amplitude of the AP, leading
ultimately to the inexcitability of the cell was observed. This was easily
m easured using voltage-c lam p techniques. It was apprec ia ted , as the
importance of Na"*" in the AP was established, that if the early phase of the
AP was due to the influx of N a + , then some degree of rectification was
necessary in order to explain the falling phase of the AP and also to restore
the potential of the cell membrane to a resting value. This was rather more
d iff icu lt to a ttr ibute experim entally to K"*" since the flow of ions was
outward and the later part of the current during constant depolarisation
was found to be broadly the same, regardless of the composition of the
external medium^.
H od g k in and H ux ley m oun ted an e x te n s iv e s tudy o f the
electrophysiology of the delayed rectifier channel in the giant axon of the
squid, Lol igo fo rbes i , first establishing the relationship between current
and voltage, then dissecting the contributions due to Na"^ and Under
vo ltage-c lam p cond it ions , when the res t ing m em brane po ten t ia l was
subjected to a sudden displacement, then held constant for 10-50 ms, the
transient inward current due to the influx of Na"^ was observed to pass over
into a long-lasting outward current, the magnitude of which depended on
the s treng th of the depo la r isa tion and the tem pera tu re . Though the
ou tw ard curren ts reached in a vo ltage-c lam p experim en t were often
considerably greater than those observed in the AP, this was thought to be
because the duration of the AP was not sufficient to allow the outward
current to reach its maximum value, and it seemed that this current
14
I n t r o d u c t i o n
associated with prolonged depolarisation was the same current responsible
for the falling phase of the AP. There was strong evidence that the latter
(and therefore the former) was caused by K+ leaving the axon^, and,
together with the results of tracer experiments^, the rôle of K" in the AP
was thus established.
1 .1 .3 C la s s i f ic a t io n O f P o ta s s iu m C h a n n e l s
The rem arkable feature o f all ion channels is the ir se lec tiv ity to a
particular ion and in the case of potassium channels, the heteromorphism
that can lead to the presence of several different types within the same
c e l l ^ . There are at least 30 distinguishable types o f potassium channel
( K C ) ^ , differing in the means through which they are regulated or “gated”
and this forms the basis for an ad hoc classification. Though designation of
a particular channel depends on the criteria used for electrophysiological
c h a r a c t e r i s a t i o n ^ , three basic types of gating have been identif ied ,
a c c o r d in g to w h ic h the f o l l o w in g c h a n n e l ty p e s m ay be
d isc r im in a te d ^ ® ’ 1, 12.
(i) V o l ta g e - s e n s i t iv e c h a n n e ls , a c t iv a te d by c h a n g e s in
membrane potential throughout the AP;
(ii) Ion-activated channels which are gated by changes in the
i n t r a c e l lu la r ion c o n c e n t r a t io n s . C a i^ + is capable of
activating and Nai+ of blocking some classes of K"*" channels;
( ii i) K+ c h a n n e ls g a ted by n e û ro t r a n s m i t te r s such as
a c e ty lc h o l in e .
The best characterised of these channels are given in table 1 . 1
along with conductance measurements and those agents which are known
to block individual channels. Conductance can also be used as a distinct
basis for classification but varies according to tissue type and the method
by which it is obtained. For these purposes, conductances are given in
picoSiemens (pS) as determined by the patch-clamp technique (see section
1 .4 ) where a channel with a conductance of 10 pS will carry 1 pA of
current when the potential is 100 mV away from equilibrium^^.
It is now well established that potassium channels are also present in
most non-excitab le cells. For instance, the rhythm ic e lec tr ica l activ ity
evoked by glucose is closely involved in the regulation of insulin secretion
15
T y p e Single C h a n n e l C o n d u c t a n c e Blocking Agents
V o l t a g e - S e n s i t i v e C h a n n e l s
Oulward Rectifier (Ik v )Inward Rectifier (Ir / Ik i ) Transient Outward A-Current
5-60 ps TEA, aminopyridines, Cs^, Ba^+, Zn^+, stryehninc, quinine5-30 ps TEA, Cs+ (potent), Rb+, Na+, Li+, Sr^+, formaldehyde20 ps Aminopyridines (potent), TEA (weak), dendrotoxins, quinidine
L i g a n d - A c t i v a t e d C h a n n e l s
On
High Conductance Ca^^-Activated(BK) Intermediate Conductanee Ca^+ Activated (IK) Low Conductance Ca^^-Aetivatcd (SK) ATP-Sensitive ChanneI(KATP/Kc)5-HT Inactivated Channel (Is)Na+-Activated K+ Channel (K^a)
100-250 ps TEA (potent), charybdotoxin, quinine, Ba^ +18-50 ps Quinine, eharybdotoxin, Cs+, dicarboeyanine dyes10-14 ps Apamin, neuromuscular blockers20-60 ps Tolbutamide, glibenclamide, TEA (weak), lignocaine, tetroeaine 55 ps Cs+ (partial), Ba^+, TEA (weak), 4-aminopyridine (weak)220 ps TEA, 4-aminopyridine, tetrodotoxin (reduces Na^ entry)
oa .
R e c e p t o r - C o u p l e d C h a n n e l s
Atrial (AChm) Channel 25-50 ps Cs+, Ba^+, 4-aminopyridine
Tabic 1.1; Classification Of Potassium Channels (adapted from ref. I I )
I n t r o d u c t i o n
and depends on variations in the K"*" permeability of the p-cells in the
p a n c r e a s ( s e e section 1 .7 .2 ) . The rates of K"*" flux in non-excitable cells
are clearly independent of the action potential and may be modulated by
hormonal effects (such as those induced by insulin or epinephrine) or by
sympathetic stimulation, but the specific physiological function of some of
the channels is still not clear. However, there are no broad differences
between the electronic properties of channels found in non-excitable cells
and their excitable counterparts. Recombinant DNA techniques have been
important in establishing the molecular basis of KC diversity, but also the
structural homology seen in an enormously diverse range of species^
M ost work has involved the so-called “Shaker” gene o f the fru it-f ly
Drosophi la melanogaster which, upon cloning and expression in X e n o p u s
oocytes yields a family of voltage-gated KCs with distinct biophysical and
pharm acological properties but sharing a core of transm em brane domains
flanked by variable N- and C-terminal r e g i o n s ^ W i t h the addition of the
genes Shal, Shab and Shaw, which, together with Shaker comprise an
extended gene family encoding A- and delayed rectif ier-type channels^ ^
(see next section), four subfamilies have been defined. The fact that shared
amino acid identity is greater between proteins encoded by the individual
d rosoph ila genes and those encoded by m am m alian hom ologues than
between the drosophila genes of the four subfamilies suggests that these
arose be fore the d ivergence o f vertebra te and in v er teb ra te species.
Further, the discovery of genes homologous to Shaker in plant tissue lends
weight to the idea that voltage-gated channels at least, ubiquitous in
eukaryotic cells, have evolved as structural variations of channels encoded
by a gene which dates from the Precambrian era, before the divergence of
P la n ta e and A n im a l ia .
1.2 P o tass iu m Channel D ive r s i ty
KCs thus play a crucial rôle in de te rm in ing the res t ing m em brane
potential, time course amplitude and polarity of electrical changes in most
types of cells. Their exceptional diversity makes it instructive to detail,
beyond the information given in Table 1 .1 , the range of channel types and
the importance of the current they carry.
17
I n t r o d u c t i o n
(i) M-current,
The M -current is a small, sub-threshold, voltage-dependent, outward
K + current. It determines the general level of excitability since it becomes
activated in the region between the resting and threshold potentials and
can therefore limit repetitive activity and firing frequency. It has been
i d e n t i f i e d ^ ^ in a num ber of different ganglia and neurons^® and is
inhibited by m uscarinic ACh-receptor agonists, including muscarine itse lf
(hence M -cu rren t) .
(ii) Inward (Anomalous) Rectifier, Ir / I k i
Unusually, opening of this channel permits an inward K+ current^ ^.
The m echanism of action of the channel is thought to be a voltage- dependen t steric b lockade by M gj^ + ; h y p e rp o la r isa t io n at po ten t ia ls
negative to E r causes Kg"*" to enter the cell, leading to displacement of the
ion and an increase in gR . At depolarisation, M g^+ blocks the channel
again. Assigning a function to the channel is difficult but it could play a
rôle in countering local elevations in intercellular K"*" concentra tion .
(iii) Delayed (Outward) Rectifier, I ^ y
As discussed, the delayed rectifier channel was the first kind of
potassium channel to be characterised. It carries the curren t which is
mainly responsible for the repolarisation phase of the AP. It is activated
there fo re by d epo la r is ing voltages and occurs , on average , severa l
milliseconds after depolarisation, hence the name. They are also ubiquitous
in non-exc itab le cells^® but the function of the current in these cases is
u n c l e a r .
(iv) Transient K"** or A-current, I ^
Along with Ij^ and I r , the A-current contributes significantly to the
resting m em brane potential in vertebrate and invertebrate neurons. It is
active in the sub-th resho ld region of the m em brane po ten tia l , being
activated by depolarisation after a period of hyperpolarisation. It therefore
plays a role in de term ining the firing frequency and is observed to
prolong the inter-spike interval during bursts in various neurons^^.
(v) ATP-sensitive K+ Channels, Kyi^Tp/^G
18
I n t r o d u c t i o n
These channels are sensitive to the concentration of ATPj and occur
in the heart, pancreatic (3-cells and skeletal muscle; they are associated
with specific physio log ica l functions in each case. The ac tivation of ca rd iac has been suggested as a possible source o f therapeutic
in tervention for ischaemic hypoxia; the subsequent loss of K+ would
coun terac t the increased excitability of the heart and shorten the time
during which it is susceptible to arrhythmias of ischaemic origin.Harrison and Ashcroft identified a channel, K q , at pancreatic p - c e l l s
which appeared to be m odulated by glucose concen tra tion^^ . It was later
suggested that glucose, whose m etabolism increases in trace l lu la r ATP concentration, leads to closure of K ^ t p » which was therefore identical to
Kg -
(vi) Stretch-Activated K"*" Channels
Ion channels sensitive to the tension of the cell m em brane are
known in a number of tissues but only a few are -selective. The channels
show sim ilar properties to the intermediate conductance C a ^ " ^ -a c t iv a te d
channels and it is generally assumed that they play a crucial rôle in
volume regulation and cell death^'^’^^.
(vii) Ca2+-activated K+ Channels
Given their rôle in the generation of the AP, it is not surprising that
these channels are widespread in excitable cells. Two types may be clearly
d istinguished , v/z., voltage-dependent channels of large conductance (BK),
with conductances of the order of 100-250 pS, and channels o f small
conductance (10-14 pS) with little or no voltage-dependence (SK). Channels
with intermediate conductances are also widespread, but are less defined in
their function. BK is the best characterised of the three since its large
conductance yields a high signal/noise ratio during record ing and these
channels have been iden tif ied by the patch-clam p techn ique in nearly
every cell type^^ . The SK channel is much less extensive and its small
conductance means that it is often overlooked unless suppression of BK
with tetraethylammonium ion (TEA, a general KC blocker discussed in the
ne x t s e c t io n ) is c a r r ie d o u t ^ ^ . It is re sp o n s ib le fo r a slow afte rhyperpo larisa tion curren t (AHP) so is often ca lled K y ^ y p ; AHP is
19
I n t r o d u c t i o n
known to trigger repetitive firing in a num ber of cells such as rat
sym pathe tic n e u ro n s and cultured cells from rat skeletal muscle^^. This
current is selectively blocked by apamin.
1.3. P o t a s s iu m Channe l S t r u c tu r e
1.3.1 The Se lect iv ity F i l ter
Potassium channels, like all ion channels, are water-filled pores but are
a lso enzym es w hich ca ta ly se the norm ally im p o ss ib le t ra n sp o rt of
potassium ions across the lipid bilayer. Potassium movem ent through the
channel is rapid (conductances correspond to a rate of 10^ - 1 0 s"^) and the
activation energy is low so is, physically speaking, s im ilar to aqueous
d i f f u s io n ^ ^ . However, any model of channel architecture must account for
the observed limiting of K+ diffusion at high concentrations and the fact
that flux can be competitively inhibited by other cations, much as enzymes
are inh ib ited by substrate analogues. After in tensive study, however, a
model of gross channel architecture has emerged.
The most in tr igu ing aspect of potassium channels is their high
selectivity in allowing only K'*' to pass, particularly in view of the smaller
radius of the sodium cation (95 pm against 133 pm for K"^). This “selectivity
filter” occurs at the narrowest part of the channel and its mode of action is
electrostatic in basis^.
Ions in aqueous solution interact strongly with the water around
them; a positively charged ion attracts the negative end of the water dipole,
i . e. the oxygen atom, and though the water m olecules are in therm al
ag ita tion , they m ain ta in their hydrogen-bonded s truc tu re as m uch as
possible and point the oxygen, on average, towards the ion. This interaction
lowers the energy of the ion significantly; alkali metal cations prefer this
environm ent to à vacuum by approximately 70-130 kcal mol" ^ , an energy
comparable to that of a covalent bond. Measurement of flux rates indicate
that K+ experiences energy changes of, at most, a few kcal mol" and the
channel must therefore compensate for the lost energy of hydration almost
completely by electrostatic and chemical interactions. The work required to
move the ion to the interior of the channel is thus the difference between
two energy terms and these depend inversely on the size of the ion.
20
I n t r o d u c t i o n
E isenm ann and Horn^^'^ explained the equilibrium binding selectivity of
cation-selective glass electrodes as a model for the channel binding site by
calculating the interaction of a cation of radius r with an anionic site of
radius rA and deduced that the energy of interaction is proportional to
( r + r A ) '^ - For a large anionic site (a low field-strength site) with rA >» r, the
importance of ion radius is minimised and the barrier to entry dominated
by hydration energies. Since this is less for larger ions, this provides the
necessary selectivity of K+ over Na+.
S im ilarly Bezanilla and Armstrong^ suggested a bracelet of oxygen
atoms ringing the narrowest part of the channel, which would substitute
for the water molecules and select for the ion with the closest fit. This is
s im ilar to E isenm ann’s low field-strength model since it m inimises the
effect o f ion radius by delocalising negative charge.
The high selectivity of potassium channels is not however consistent
with the high transport rates that are observed. A crude calculation based
on the bulk resistivity of a salt solution bathing the channel^ reveals that,
if a pore 6 A in diameter and 50 A long were filled with 120 mM KCl, its
calculated resistance would be 18 GO (corresponding to a conductance of 55
pS) or 20 GG (g = 50 pS) if access resistance due to diffusion up to the mouth
of the pore is taken into account. Since the resis tance of some real
channels is as low as 4 G Q , these channels must only have a short, narrow
region with large antechambers at one or both ends that m inimise the
contribu tion of diffusion. This allows one to construct a p icture of a
“typica l” potassium channel (figure 1 . 2 ).
1.3.2 Use O f C h a n n e l A n ta g o n is t s
C ruc ia l to the unders tand ing o f channel a rch i tec tu re has been the
availability of blocking agents. These provide a ready means of disturbing
channel function in ways which can be controlled and, more importantly,
quantified. A problem has traditionally hampered elec trophysio logica l and
b iophysica l charac te r isa tion of potassium channels how ever, vi z . , those
m odu la to rs ava ilab le were typ ica lly unselec tive and often lacking in
potency. Of those that have been used, by far the most useful have been the
a m i n o p y r i d i n e s , 4 - a m i n o p y r i d in e an d 3 ,4 - d i am i n o p y r i d i n e , and
quaternary ammonium ions (QA), derivatives of TEA (figure 1 .3 ) .
21
I n t r o d u c t i o n
The binding affinity of many potassium channel blockers is voltage-
mmrnm
m m ## # # #
F igure 1.2: Diagram of general potassium channel structure inferred
from conductance measurements and showing tunnel (T),
mouths (M) and antechambers (A).
N.
NH21
Et —
Et
N — Et
Et
3
Figure 1.3; Classical potassium channel blockers: 4-aminopyridine 1,
3, 4-diaminopyridine 2 and tetraethylammonium ion (TEA) 3.
d e p e n d en t^ ^ . For instance, TEA itself blocks the squid axon delayed rectifier
channel when perfused inside the cell and it is found that its affinity is
higher if the inner electrical potential is made more positive^^. Woodhull
devised a model which quantified this voltage dependence and related it to
I n t r o d u c t i o n
the position of the site of the blocking a c t i o n ^ I n t u i t i v e l y , we may
imagine that a more positive potential inside pushes the ion outward (i.e.
into the channel). The blocking ion will partition according to a Boltzmann
distribution so that the voltage-dependent affinity, K j(V ), is given by:
Kd(V) = Kd(0)e(zôFV/RT) eq. 1.1
where Kd(0) is the affinity with no applied voltage, z is the valence of the
ion, F, R and T have the standard meaning (RT/F = 25 mV at 25 °C) and 5 is the
fraction of the field traversed by the blocker in moving to the blocking
s i te 3 5 ,3 6 this last quantity, sometimes called the electrical distance, we
have a way of assessing the relative penetration of various blockers into
the channel and thus a means to infer the size and shape of its entrance.
A set of experiments by Miller^ supports the notion that the blocking
site is within the membrane field and relates the electrical distance, S, to
the physica l distance. W orking with the delayed rec tif ie r channel from
rabbit sarcoplasm ic reticulum, he used a series o f bis-quaternary amines
(two trim ethylam ino groups linked by an alkyl chain of varying length)
and noted that for short chain lengths, the ions behaved as simple divalent
c o m p o u n d s ( z 6 = 1.3) with twice the effective valence of trimethyl
amm onium ion (TMA, for which z5 = 0.65). As the length was increased,
however, the effective valence gradually decreased to that of TMA; whilst it
may have been expected that the aggregate effective valence would be
given by ô = 6 % + 6 2 , it seems that one end was entering as far into the
channel as possible whilst the other, repelled by the positive charge of the
first, stayed as far out as possible. A chain length of 4-5 carbon atoms was
found to be just enough to get the second end out of the field (Ô2 = 0, z5 = 6 1 ).
Thus 65% of the field falls within a distance of 6Â. Given that the
m em brane is some 50 Â thick, this implies the fo llow ing s tructura l
f e a t u r e s :
(i) an antechamber so large that little potential drops across it and
whose resistance is negligible compared to the narrowest part of the
c h a n n e l ;
(ii) a mouth which the QA enters and blocks;
(iii) a tunnel which only the smallest ions can enter.
This accords well with the model predicted on the basis o f conductance
23
I n t r o d u c t i o n
m e a s u r e m e n t s .
Besides voltage, there is one other, perhaps more important factor,
which affects the affinity of quaternary am m onium derivatives for the
c h a n n e l , v i z . , the length of the hydrocarbon side chains that are
subs ti tu ted for the ethyl groups of From TEA to
trie thylnonylam m onium ion (TEnonA) affinity increases 600 cal mol"^ for
each added carbon atom, suggesting that the leng thened side-chain is
binding to a hydrophobic moiety adjacent to the channel m outh^^. Though
it now seems probable that the walls of the channel are lined by a - h e l i c e s
that have hydrophilic or charged sidegroups, it is possible that the long
arm o f T E nonA ex tends beyond the h y d ro p h i l ic l in in g in to the
hydrophobic material behind, thus contributing to the b inding energy^
This e ffec t can also be produced by adding a benzene ring whilst
hyd roph il ic groups, like hydroxy l, reduce the b ind ing a ff in ity . This
feature, a QA site that binds a wide range of differently sized ions with the
same 5 and that prefers hydrophobic groupings, is seen in a wide range of
c h a n n e l s .
1.3.3 Deta i l ed Channel Structure Deduced From SAR And Point
M u t a t i o n S t u d i e s
Studies of the action of TEA on voltage-gated channels have revealed that
the in ternal and external binding sites for TEA are distinct; whilst, as
discussed, in ternal binding is dependent on m em brane potentia l, external
binding is insensitive to such changes. Mutational analysis o f the channels
encoded by the Shaker gene from drosophila has given im portant clues
about the nature of the TEA binding site and thus about the channel pore. It
is important to see this in terms of the generally accepted model of KC
s tru c tu re , how ever . The express ion of genes f rom severa l species ,
including mouse, rat, human and d r o s o p h i l a itself has produced a series of
po tass ium channel-form ing proteins with very sim ilar prim ary sequences.
A lthough there is little crystallographic information available on intrinsic
m em brane proteins it is generally believed that hydrophobic stretches of
n in e te en or m ore am ino acids in any sequence , d e te rm in ed from
h y d ro p a th y p lo ts , are m em brane-spanning'^^*'^^. This has consequences
24
I n t r o d u c t i o n
for channel structure since these regions are seen in all known sequences.
Voltage-gated KCs belong to a superfamily of voltage-gated and secondary
m e s s e n g e r -g a te d c h a n n e l s ^ ^ including the voltage-dependent Na+ and
C a 2 + channels and C a- + -activated KCs which, because of sequence
sim ilarities, are thought to contain either one or four copies of an
underly ing structural motif"^^. This consists of six m em brane-spanning
segments (S1-S6), five of which are hydrophobic, the remaining segment
(S4) being positively charged and amphipathic (Figure 1 .4 ) .
ex trace llu lar
T h e p r o p o s e d m e m b r a n e - s p a n n i ng or i en ta t i on o f one protein subuni t o f a vo l tage- gated KC: four units probably ase mbl e to make a funct ional c h a n n e l . C - t e r m i n i o f t e n c o n t a i n a p h o s p h o r y l a t i o n s i t e ( P) and the s e q u e n c e b e t we e n s e g me n t s SI and 52 is f r eq ue nt ly N - g l y c o s y l a t e d , ind icated in this diagram by smal l branches .
in tracellu lar
Figure 1.4: Diagram of a voltage-gated potassium channel deduced
from hydropathy plots and site-directed mutagenesis studies (from ref. 45)
There is also an extended hydrophobic loop (H5) connecting segments S5
and S6 which is tucked into the lipid bilayer from the extracellular side.
The Shaker-like KCs are proposed to consist of four subunits arranged into
an outer cylinder of sixteen a -helices (formed from segments S1-S3 and
S5), surrounding an inner cylinder of eight a -helices (S6 and 54)^^. The
results of site-directed mutagenesis studies have provided insight into
which of these domains are involved in gating, selectivity and inactivation.
For instance, the positively charged 54 is most likely to be the voltage
25
I n t r o d u c t i o n
sensor of voltage-gated KC’s, moving towards the extracellular surface by a
helical screw mechanism (figure 1 .5 ) during the transition o f the channel
from a closed to an activated state^^.
0. ' ' © '
At the resting membrane potential, all positively-charged residues are paired with fixed negative charges on other transmembrane segments of the channel and the transmembrane segment is held in that position by the negative internalmembrane potential. Depolarisation reduces the force holding the positive charges in their inward position. The S4helix is then proposed to undergo a screw-like motion through a rotation of approximately 60® and an outward displacement of approximately 5Â. This movement leaves an unpaired negative charge on the inward surface of themembrane and reveals an unpaired positive charge on the outward surfaceto give net charge transfer of +1.
F ig u re 1.5: The helical screw mechanism of the putative voltage
sensor of voltage-gated potassium channels (from ref. 47).
S45, the intracellular loop connecting S4 and S5, is also thought to form an
am phipathic helix that takes part in the regulation o f the gating of the
channel; its positive charges point towards the centre of the pore, forming
an e lec tros ta t ic barrier to the passage of cations whilst m ovem ent away
from the pore would cause opening. This latter m ovem ent implies a
rotation of the helix which would move its positive charges away from the
pore and b r ing in a negative charge which could take part in the
formation o f a negatively charged ring at the intracellular to the pore.
The H5 loop is strongly implicated in pore formation"^^. It is highly
conserved am ong known KC proteins and invariably consists o f nineteen
amino acids with a Gly-Tyr-Gly-Asp sequence motif towards the C-terminus
and a Thr-Met-Thr-Thr-Val motif in the middle (figure 1 .6 ) .
26
I n t r o d u c t i o n
X
ou tsideKj
w)
T h e d i ag r a m s h o w s , in s ing l e letter code, the S 5 - S 6 l inker region o f v o l t a g e - g a t e d Sh ak er channe l s wi th the c o n s e r v e d H5 r e g i on i nser ted into the m e mb r a n e from the o u t s i d e . T h e p r e s u m e d pore f ormi ng res idue , t yros i ne ( Y) can be seen whi l s t those res idues with d i a g o n a l h a t c h i n g s e e m to be respons i bl e for b i ndi ng T E A from the intracel lular s ide.
Figure 1.6: The amino acid sequence of the H5 loop
The pore is assumed to be formed from four (3-hairpin structures of
H5, one from each of the subunits. The narrowest part of the pore, the part
that separates intracellular and extracellular space, may be formed by four
tyrosine residues (again, one from each subunit) since tyrosine occurs in
the H5 region of every known KC subunit sequence. Variations in the H5
region are m ajor determ inants of KC pharm aco logy , a ltering the
sensitivity of a given channel toward either internal or external blockers.
Experiments have also indicated that the H5 region is the location of
the binding site for both internally- and externally- applied TEA. The
series of channels encoded by the Shaker-related RCK family of genes
showed distinct affinities for external TEA and characterisation of the
proteins revealed substantial differences at residue 19 of the otherwise
h ig h ly - c o n s e r v e d H5 r e g i o n ^ B . S ite -d irec ted m utagenes is to give
positively-charged residues at position 1 also caused a significant, but
much less substantial alteration to the sensitivity to external TEA. This
suggests that a negatively-charged residue 1 attracts TEA, directing it to
residue 19 which then takes over with a van-der-Waals type interaction.
The most useful information regarding the internal blocking site
has come from chimeric KCs in which the H5 region of one is replaced by
a n o t h e r ^ ^ . The fact that the S5-S6 linker region carries properties such as
27
I n t r o d u c t i o n
TEA sensitivity and single channel conductance over from its parent also
supports the idea that H5 is the pore-forming region. Substitution of Thr-11
specifically alters block of Shaker channels by internal TEA as well as the
se lec tiv ity properties of the pore and this residue has therefore been
suggested as participating in the form ation o f the selectiv ity f il te r of
Shaker K C ’s as well as providing a binding site for internal TEA.
U nfo r tu n a te ly , s i te -d irec ted m utagenes is s tud ies have not been
carried out on SK, the small conductance, C a^"^-ac tiva ted KC b locked
specifically by apamin. The problem arises out of this specificity, however,
since no channels have yet been cloned which have been found to be
blocked by the peptide. The value of mutational analysis may be seen from
such work carried out on voltage-gated channels encoded by Shaker and
RCK using dendrotoxin and charybdotoxin where, despite the difficulties
involved in elucidating the structure of the peptides themselves because of
their size, much is now known about the receptor site (the majority of
which seems to be located on the H5 region for both toxins, with some
degree of overlap of the two)^®.
The paradigm of KC topology illustrated here by the Shaker A-
channels has however been found to hold for almost all the KC proteins
expressed so far, though the m olecular features of many do, admittedly,
remain to be elucidated. Recently, however. Ho et. al. expressed an ATP-
regulated KC from rat kidney whose structure represents a major departure
from this model^^. Analysis of the protein’s primary structure allowed the
prediction of a structural model (figure 1 .7 ) which con ta ined only two
m e m b ra n e -sp a n n in g segm en ts bu t w hich c o n se rv e s an am ino ac id
segment analogous to H5 of voltage-gated KCs, providing further evidence
that this region forms the ion-permeation pathway.
1 .4 . E l e c t r o p h y s i o l o g i c a l C h a r a c t e r i s a t i o n
S AR studies o f KC modulators have been important in establishing the
common features and, presumably, ancestry of potassium channels but this
lack o f specif ic ity has f rus tra ted attem pts to ch a ra c te r ise ind iv idual
channels . One o f the most im portant advances has been the increasing
soph is t ica tion of e lec trophysio log ica l techniques. These even allow the
properties of a single channel to be investigated in the absence
28
I n t r o d u c t i o n
] P O 4 l o o p
C O O H
Figure 1.7: Diagram of an ATP-regulated potassium channel from rat
kidney (from ref. 15).
of noise from other ion channels which, under standard voltage-clamp
conditions, may obscure in terpreta tion of the resu lts . In the past,
macroscopic currents due to potassium ion flux have been characterised,
but the problem now arises of dissecting them and ascertaining the
contributions due to the various classes of single channels. The observed
variability of single channel properties with respect to conductance, ion
29
I n t r o d u c t i o n
se lec tiv ity , open probability and k inetics , as well as dependence on
a g o n is t s , a n ta g o n is ts , s e c o n d a ry m e s s e n g e rs , c y to p la s m ic c a lc iu m
concentration and even mechanical stress, has increased enormously as a
result o f research into this problem, but has created an incongruity: far
more ion channel types have been identified than there are known ion
currents. Thus any macroscopic current must consist of contributions from
several populations of channels.
Two methods, more than any others, have been instrum ental in
these advances. In one of a num ber of patch-clamp techniques developed
by N eher and S a k m a n n ^ a micropipette is attached to a patch of cell
membrane then disturbances to potassium channel function are caused and
the ion flux (current) is m onito red with tim e v ia changes in the
com position o f the saline solution contained within the p ipette (figure
1 .8 ) . Single channels can be detected electrically because they have a very
high turnover rate ( 10^ ions s" giving rise to a current of approximately
20 pA). This is now the most common method for measurem ent of ion
c h a n n e l c u rre n t .
bath solution
ATPpipette solution
no transmitter)
c -A MP
PROTEI Si KINASE
cytoplasm
receptor
transmitter receptor complex
channel
Diagram illustrating the suitability of patch-clamp current recording to investigate channel regulation by transmitters acting via secondary messengers. By applying the transmitter to the cell membrane outside the pipette opening, the modulation of membrane channels of the patch in the pipette tip can be examined. In this example, The hypothetical pathway of KC regulation by serotonin in A p l y s i a sensory neurones is illustrated.
Figure 1.8: P a tc h -c la m p re c o rd in g
30
I n t r o d u c t i o n
A lternative ly , ion-conducting molecules can be studied in model
p h o s p h o l i p i d m e m b r a n e s ^ ^ T h e s e are typ ica lly art if ic ia l p lanar
bilayers formed across a 150 pM diameter hole in a Teflon f ilm ^^’^^. Using
this method, the voltage and the peptide or protein concentration can be
adjusted to allow the measurement of single ion channels. The large size
and complexity of channel proteins, which has made elucidation of detailed
channel structure so difficult, makes this technique particularly attractive.
A “minimalist” approach to the design of protein analogues may be posited:
first, the structural basis for a given function is predicted then a simplified
pep tide or pro te in sequence is designed which con ta ins the putative
functional features whilst d iscard ing , or rep lac ing with sequences of
m inim al com plex ity , those considered to be sup e rf lu o u s^ ^ . This latter
technique has been particularly useful in establishing a great deal of the
secondary structure of channel proteins.
1.5 Spec i f i c Po tass ium C h anne l M o d u la to r s
1.5.1 An im al Toxins
Ultimately, the lack of specificity of the QA ions and the ubiquity of the
binding site meant that the results of studies which used them had to be
in terpreted cautiously. M oreover, both QA ions and 4-am inopyridine are
known to block receptors for a wide variety of neurotransmitters including
muscarinic-, Ü2 -, a l - , a 2-, 5 H T i a - and 5HT2 -recep to rs^^ . Other compounds,
such as quinine, local anaesthetics and some ions, like Cs2+ and Zn^+ also
effectively block all, or at least some, types o f potassium channel, but they
also have additional, often more specific, effects on other classes of ion
c h a n n e l .
A number of animal toxins have been discovered to act as potent,
specific antagonists of KCs however. For instance, dendrotoxin, comprising
about 2.5% of the total venom protein o f the Eastern green mamba,
D e n d ro a p s is a u g u s tic e p s , is a peptide of 7077 Da which blocks transient
outward KCs^^. Similarly, charybdotoxin, a minor constituent of the venom
of the Mideastern scorpion, L e iu ru s q u in q u e s tr ia tu s var. hebraeus with a
mass of 4353 Da is selective for large conductance, Ca^"^-activated KCs^^.
31
I n t r o d u c t i o n
1.5.2 A p am in , A Specific B locke r O f SK
A pam in, in com parison with o ther p ro te inous venom cons ti tuen ts , is
relatively small with a mass of only 2039 Da. It is a specific blocker for a
class of small conductance, Ca^ + -activa ted KCs and has an extensively
studied pharmacology. It is isolated from the venom of the Honey bee. A p is
m e lli fe r a , which, besides apamin, contains several toxic components, such
as h istam ine and phospholipase A, and o ther peptide toxins, such as
m elittin and m ast-cell degranulating peptide, all o f which have been
p u r i f ie d to h o m o g e n e i ty by H a b e rm a n n and c o -w o rk e r s ^ ® . Its
exceptionally small size (see section 1 .6 . 1 ) means that it is the only peptide
known to cross the blood-brain barrier.
Although the neurotoxicity of apamin is high (LD50 = 4 mg/kg upon
in travenous adm in is tra t ion in m ouse)^^ , the first indication of its site of
action came from studies on non-neuronal cell cultures of the guinea-pig.
A pam in was found to be se lec tive in in h ib i t in g the A T P -induced
hyperpolarisation of in testinal smooth muscle ce lls^^ as well as K" loss
from hepatocytes^^ . The K+ loss observed with the Ca^+ ionophore A23187
was also inhibited by apamin, leading Banks and co-workers to suggest that
it was acting on a Ca^ + -d e p e n d e n t K C ^^ . The fact that apamin did not
however inhibit a Ca^''"-dependent potassium current in erythrocytes gave
early evidence of the m ultifarious nature of this fam ily o f channels,
iK(Ca). This finding was followed by the observation that charybdotoxin
s p e c if ic a l ly b locked a Ca^ -a c t iv a te d KC c h a ra c te r i s e d by large64conductance (100-250ps) as previously described . However, the target for
apamin in cultured rat skeletal muscle was postulated as a C a ^ '* '-ac t iv a ted ,27QA-insensitive channel of much smaller conductance (10-14ps) . Further,
this channel showed only a weak voltage dependence and much greater
sensitivity to C a ^ ^ concentration at negative membrane potentials than the
charybdotox in-sensitive channel. That the d ifferen t Ca^ " ' '-dependent K"*"
currents are due to different channels is now well-established and it is
accepted that apamin is highly selective for ju s t one of them: the Ca^'*'-
ac tiva ted , Q A -res is tan t , nearly v o l ta g e - in d e p en d e n t ch an n e l o f sm all
conductance. These characteristics make it ideally suited to generate the
long-lasting after-hyperpolarisation that is seen in the many cells which5 8are known to contain this channel
32
I n t r o d u c t i o n
1.5.3 C harac ter i sa t ion Of A p am in B in d in g Sites
A pam in may be radiolabelled with iodine at its h istid ine residue^
w hils t re ta in ing its b io logica l activity . This has a llow ed rad iograph ic
localisation and quantification of binding sites for ^ ^ I ] - m o n o i o d o a p a m i n
in the central nervous system of a number of species with an affinity that
accords well with its neurotoxicity. The binding to peripheral organs was
observed to be much less, though there were exceptions. Cook and Haylett
were the first to observe the binding of ^ ^ I] -m o n o io d o ap a m in to in tac t
ce lls unde r p h ys io log ica l cond it ions us ing an assay o f g u in ea -p ig
h e p a to c y te s ^ G . The dissociation constant, K j , for the iodinated derivative
(350 pM) compared well with that for native apamin (K j = 376 pM for the
same assay). It is known that a-adrenoreceptor agonists cause a loss of
from hepa tocy tes and the same workers dem onstra ted that increas ing
co n cen tra t io n s o f apam in p roduced a p rog ress ive dep ress io n o f the
dose/response curve to (-)-phenylephrine, measured as a function of K"*"
loss from a suspension of the cells. These two results strongly suggested a
binding o f labelled apamin that was directly related to the presence of
a p a m in -s e n s i t iv e , Ca^ +-activated KCs and that this was consistent with
simple competition at a single class of binding site. The binding of
m onoiodoapamin to rat hepatocytes and human erythrocytes was observed
to be much less and could not be reliably quantified, suggesting the lack of
apam in-sensitive Ca^+-activated KCs in these cell types.
1.6. The Structure O f Ap amin
1.6.1 P r im a r y S e q u e n c e
Given its potency, that the structure of apamin has been the source of a
great deal of research is not surprising. It was established by Habermann^ ^
to be a peptide containing eighteen residues with two disulphide bridges
whilst Callewaert and co-workers^^ deduced its amino acid sequence as that
shown in figure 1 .9 .
33
I n t r o d u c t i o n
I ICys-Asn-Çys-Lys-AIa-Pro-GIu-Thr-Ala-Leu-Cys-AIa-Arg-Arg-<Zys-Gln-Gln-His-NH 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8
4*F igu re 1.9: A p a m i n
The position of the disulphide bridge was unequivocally established
by the same w o r k e r s ^ T r y p s i c hydrolysis of native apamin gave only one
peptide and arginine. Since N-term inal analysis revea led only alanine,
then cleavage must have occurred between positions 4 and 5, 13 and 14, and
14 and 15, i.e. the bridges could not have been between residues 1 and 3 and
residues 11 and 15. Of the two remaining choices, the bridge was found to be
as shown in figure 1 .9 . A complete cycle of Edman degradation removed
cyste ine-1 and thus destroyed one bridge. Trypsic hydrolysis of the product
gave arginine and a single peptide, a result which could only have arisen
if a disulphide bridge was linking cysteine-3 and cyste ine-15.
1 .6 .2 S t r u c t u r e / A c t i v i t y R e l a t i o n s h i p S t u d i e s
A number of teams have investigated the SAR of apamin using a number of
m odified peptides and synthetic analogues. V incen t and co-w orkers^ ®
undertook a series of specific chemical modifications o f the peptide and
found that most did not have a great effect on peptide activity, determined
by the median lethal dose (LD5 0 ) of subcutaneous in jec tion in mouse.
Transformation of the e-amino group of lysine-4 5 into hom oarginine
34
I n t r o d u c t i o n
NHH (CH2)4NH2 NH2
(i) y + CH3 0 — < ( +^ y HSO4- ^ ^ 2
H (C H 2>4N H ^ y NH2+ CH3OH
< Vly s in e
5H Œ 2S-
( ii) XHjN y
o o H CH2S
MeCNH ' y + CH3COOH
c y s te in e
7O
O
H (C H 2)4N H 2 o o
/ y • - J - . x
ly s in e 5
H (CH2)4NHCCH3 + CH3COOH
< V9 n
( i i i ) H (CH2)2C 02H h y ELN=C=N(CH2)3NMe2 H (CH2)2CNHCH2C02Et
. A . A ,Vglutamic acid
1 0
(iv)
H2N COOEt
O
+ (CH3CH20C)20
h is t id in e
NH-, O
H (CH2)3NH ( +
1 3
OEt
+ MeCH20C02H
OH
H (CH2)3NH (
y . /arg in in e
1 4
OH
1 5
Figu r e 1.10
(H ar) using m ethy lisourea hydrogen su lpha te (f igu re 1 .1 0 ( 1 ) ) had no
discernible effect, whilst variously acétylation of lysine-4 and of the a -
am in o g ro u p o f c y s te in e -1 7 w ith a c e t ic a n h y d r id e ( f ig u re
1 .1 0 ( i i ) ) p r o d u c e d a reduction in activity o f only 2.5 tim es. Similarly,
modification of the carbonyl of glutamine-7 10 by formation of an amide
bond with glycine ethyl ester (figure 1 .1 0 (1 1 !) ) produced only a 50%
decrease in activity whilst carbéthoxylation of h is t id ine-18 12 with diethyl
pyrocarbonate (figure 1 . 10(1 v))produced no detectable loss o f activity at
35
I n t r o d u c t i o n
all. These effects were synergistic, however; acétylation of cysteine-1 and
lysine-4 together with carbéthoxylation of histidine-18 produced a peptide
devoid of neurotoxicity. Reduction of the disulphide bridges with NaBH^.
fo llow ed by a lkylation of the resulting thiol with iodoacetam ide also
e lim inated toxicity , confirming an earlier result by Habermann^®. Most
interesting, however, was that modification of arginine-13 and arginine-14
with cyc lohexaned ione (figure l . l O ( v ) ) or trypsic cleavage, resulting in
the removal of arginine-14 and the formation of a two-chain peptide (see
Callewaert, above) also destroyed the toxicity, giving the first indication of
the singular importance of this part of the sequence.
These observations were expanded by G ran ier et a lP Using a
method that they had established in the solid-phase synthesis of apamin, a
num ber of synthetic analogues, differing in the arginine region by one,
two or three residues from apamin itself, were synthesised. Replacement of
either o f the arginine residues in positions 13 and 14 with lysine resulted
in values for the LD50 (in the same assay as the previous workers) very
close to that of apamin. Replacement of both, however, led to a peptide with
an activity that was very faint but which could be enhanced by reaction
with 0 -methylisourea sulphate, to give homoarginine residues in positions
4, 13 and 14 and a relative activity 0.15 times that of apamin, indicating the
favourable effect of the guanidinium structure on activity. The com par ison
with [Har-4]-apamin, prepared by Vincent and which shows no loss of
activity, suggests that positive charge in positions 13 and 14 is not of
unique importance and that the length of the arm bearing the functional
group is crucial. The synthesis of [O rnith ine-13, O rn i th in e -14]-apamin
(figure 1 . 1 1 ) and its complete lack of neurotoxicity would seem to support
th is^ ^ .
H CH2-CH2-CH2-NH2
''''COOH
16F ig u re 1.11: O r n i t h i n e
G a n e l l in and co -w o rk e rs^ synthesised a series of com pounds
containing a bis-guanidinium grouping and a number of small peptides with
ad jacent a rg in ine or arg in ine-lysine residues. O f the la t te r group of
36
I n t r o d u c t i o n
compounds, all were found to be almost completely lacking in activity,
su g g e s t in g that this d ip ep tid e s tru c tu re does not convey enough
information for effective recognition by the receptor site. The possibility
of steric hindrance from derivatising groups was discounted by the lack of
activ ity o f the conform ationa lly f lex ib le 1, 10-b isguan idodecane (figure
1 . 1 2 ), synthesised because the decane chain was considered as equivalent
to the eleven bonds separating the guanidine groups in the peptide.
NH
NH
F igu r e 1.12
It is possible that the inactivity of the compounds was due to the inability
of the groups to achieve an appropriate spatial separation, but it seems
more likely that the guanidinium groups are a necessary, but not sufficient
contribution to the toxicity of the peptide.
L abbé-Ju lié et also found it difficult to correlate the observed
high affinity of apamin (K j = 10 pM) solely with the position of two
guanidinium groups in positions 13 and 14 and undertook a study to
determine which other of apam in’s structural features were responsible, at
least in part, for its action. For this, they were helped by two very sensitive
assays which were able to determine activities below the threshold of
detectability of previous studies, v iz ., inhibition of I ] - m o n o i o d o a p a m i n
b ind ing to rat brain synaptic m em branes and in trace reb ro ven tr icu la r
injection in mice.
Firstly, it was known by this time from proton NMR studies that Leu-
10, the only hydrophobic residue in apamin, was in proxim ity to the
arg in ine s ide -cha ins^^ , but replacement of this residue by alanine led only
to slight loss of potency, implying no close interaction with the receptor.
However, a more profound effect was noted by the sequential shortening of
the C-terminus. Loss of His-18 led to a peptide with 16 % of the toxic effect
and 1 % of the binding activity of apamin. Since these values were lower
than those obtained for a peptide in which His-18 had been chem ically
m o d i f i e d ^ ® ’^ ^ , it seem ed that there was a po ten t ia l ly s ign if ican t
37
I n t r o d u c t i o n
involvement of the histidine residue.
Loss of GIn-17 and GIn-16 led to a greatly decreased activity but since
both gave similar values (0.35 and 0.43% toxicity respectively and 0.01%
binding efficiency), G in-17 was implicated as an important element in the
interaction of apamin with its receptor, probably through the amide side
chain. D espite the drastic loss o f activ ity how ever, bo th behaved as
com plete apamin agonists, with a dose-response curve paralle l to that
ob ta ined fo r na tive apamin in the b ind ing assay. G ln-17 could not
therefore be an essential residue for the specific activity o f apamin.
The m odification o f Arg-13 and Arg-14 using cyc lohexaned ione
produced a peptide with a previously undetectable activity (see above) but
measurable in the new assays. As was the case for the glycine-deficient
p e p tid e s , th is ana logue was ab le to sp e c i f ic a l ly in h ib i t ^ I ] -
m ono iodoapam in binding and exh ib ited the charac te r is t ic sym ptom s of
apamin poisoning in mice (0.002 and 0.04% relative activity compared to
apamin). Though the rôle o f the positive charges o f the arginine side-
chains was well established, being considered as part o f a well-defined
conformation which fitted a specific pocket on the receptor with negative
charges at the bottom, this was the first time that other forces of attraction
around Arg-13 and Arg-14 were shown to be involved in the specific
activity of apamin. It appears that the side-chains o f the arginine residues
are involved in hydrophobic bonding to the receptor, com plem enting the
ionic a ttrac tion .
1 .6 .3 C o n f i g u r a t i o n
N aturally enough, in the light of these observations, the secondary and
tertiary structure of apamin was of great interest. Early studies on the
c ircular dichroism spectrum of apamin by M iroshnikov et a l J ^ indicated
that a large part of the molecule existed as an a -he lix , and also showed that
the c o n f i g u r a t i o n was not greatly influenced by m odification of the side-
c h a in s . T h e s e w o rk e rs d e m o n s t ra te d th a t r e l a t iv e c o n s ta n c y o f
c o n f i g u r a t i o n observed over a wide pH range and in the presence of
organic solvents and 6M guanidinium HCl, indicating a stability thought to
be due to the established presence of the d isulphide links. Subsequent
studies concentrated on locating the a -he lix . Bystrov et a l J ^ undertook an
38
I n t r o d u c t i o n
extensive NMR study of the peptide in D%0 solution and, stressing the
helical nature of fhe c o n f ig u r a t io n ,p r o p o s e d a tentative structure based on
the m easu red shifts, sp in-spin couplings and to rs ional angles, (figure
1 .1 3 ) .
Thi
Alq
NHAla
F ig u re 1.13; Proposed Structure of Apamin (from ref. 75)
H igh-resolution NMR studies revealed that alm ost all o f the amide NH
protons o f the molecule resolved into individual signals. By observing the
exchange rates with D 2 O, it was found that the individual amide groups
possessed m arkedly d ifferen t ha lf- lives , a fac t a t t r ibu ted to hydrogen
bonding and/or screening of these groups from the solvent molecules. This
latter explanation was later refuted by Englander and K allenbach^^ who
suggested that apam in’s small size would almost certainly perm it solvent
accessibility and therefore slow amide exchange was most likely due solely
to hydrogen bonding and not to screening o f the am ide group by the
peptide architecture. The rigidity of the molecule was therefore due to a
combination of disulphide linkage and strong hydrogen bonding.
H id e r and R agnarsson^® predicted that it was not possible to
construct the disulphide linkages if the entire 4-17 peptide segment was in
39
I n t r o d u c t i o n
an a -h e l ic a l configuration. However, limiting it to segments 8-17 or 4-12
pe rm itted c ross l ink ing . The fo rm er was p refe rred , b e in g s trong ly in
accord with the NMR study o f Bystrov et and allowed for the
formation of two p-turns in the N-terminal octapeptide which would orient
the half-cysteines into the correct position for linkage.
Subsequently , Bystrov et a l j ^ refined their prediction to include an
a -h e l ix in the region o f peptides 6-13 and suggested the most plausible
system of in tram olecular hydrogen bonding, to include the a - h e l ix and
three p-turns in the C-terminal residue, based on the m easurements of the
exchange rates of the NH groups of Ala-5, Cys-15 and Gln-16.(figure 1 ,14)
-H* • 0
H -N
H -N
H -N
F ig u re 1.14: Diagram of in tram olecular H-bonds in apamin
This allowed construction of a spatial structure which also took account of
the coupling constants of the side-chains and their p red ic ted geometry,
(figure 1 .1 5 )
This did not fu lly reso lve the crucial ques tion o f the precise
conformation of the guanidinium groups of the two arginine residues. In
40
I n t r o d u c t i o n
CH3NH-
:n h
F i g u r e 1 .15
an extensive 2D NMR study, Pease and Wemmer^^ posited a structure that
was genera lly consistent with previous models (figure 1 .1 6 ) but in which
the s ide -cha ins were shown to have far fewer constra in ts and exhibit
grea ter variability in their movements than did the backbone that held
t h e m .
TeA9
L i e
E7
P6R13
A12
AS
N 2R14'
0 1 7
K40 1 6
C 3
H 1 8
F ig u re 1.16: Ribbon Diagram of Apamin (from ref. 81).
41
I n t r o d u c t i o n
In particular, the lack of nOe signals involving the side-chain proton of
the a rg in in e res idues made it im poss ib le to d e te rm in e the exac t
conform ation of the side-chains and suggested that both were mobile in
solution. It would seem that, providing the residues are positioned in a well-
defined way, then the inherent flexibility of the side-chains allows the
correc t pos ition ing of the guanidinium groups in the recep to r pocket,
corroborating the SAR studies of Granier et al
1 .6 .4 M o le c u le s C o n ta in in g B i s - q u a t e r n a r y N i t r o g e n A to m s As
M im ics O f A p a m in
In 1985, Cook and Haylett^^ correlated the ability of several compounds to
inhibit ^^I]-m onoiodoapam in binding with their ability to inhibit Ca^ + -
m ediated K+ efflux from guinea-pig hepatocytes. Tetraethylam m onium ion
and q u in in e were e ffec tive only in h igh c o n c e n t ra t io n w h ils t 9-
am inoacrid ine , quinacrine and chloroquine were slightly m ore effective.
By far the most active compounds, besides apam in i tse lf , were the
n e u ro m u sc u la r b lock ing agents tubocura rine 1 8 , pancuron ium 21 and
a tracu r ium 2 0 , the structures of which are given in fig 1.17 along with
the inhibition constants against [^^^I]-monoiodo-apamin b ind ing for these
and re la ted compounds. As previously d iscussed, the neu ro tox ic ity of
apamin is crucially dependent on the two adjacent, posit ive ly charged
amino acid residues in positions 13 and 14 and it was therefore postulated
that the ability of these compounds to behave in such a way may depend on
the distance between the two charged nitrogen atoms, present in them all
and which were thought to mimic the active peptide m oiety. Certainly,
there was a high correlation between their affinity for apam in binding
sites and their ability to inhibit K+ efflux from hepatocytes and, with the
compelling evidence that these binding sites corresponded to KCs, that the
compounds were acting as competitive antagonists at the apamin binding
site in the SK channel seemed an obvious conclusion. The subsequent
discovery that dequalinium inhibited [^^^I]-m ono iodoapam in b ind ing even
m ore e ffec tiv e ly supported this and led to the sy n th es is o f 3,6-
b is (p ip e r id o m eth y l)p h en an th ren e d im eth iod ide 23 . This was selected
42
I n t r o d u c t i o n
O M e“l
OH
MeO OH Me
2+ Me Me
N-(CH2) io -N ^ y - N H z
1 8
Tubocurarine Chloride; K , = 9.2±0.4
“ O
1 9
Dequalinium Iodide; K j = 1.1 ±0.1
CH2CH2C0(CH2)50CCH2CH2>^N. ^
Me Me
OMeOMe OMe
2Ct'2 0
Atracurium Chloride; K j = 4.3±0.2 MeCOO
2+
MeMe
Me
H
2+
2Br'
Pancuronium Bromide (R=Me), Vecuronium Bromide (R=H) 2 1 2 2
Ki = 3.2+0.2 Kj = 3.6±0.5
Me Me
2+
2r2 3
3,6-Bis(piperidomethyl)phenanthrene Dimethiodide; K , = 9.9±1.3
F igure 1.17: Potent, selective blockers of SK (all values in | iM )
43
I n t r o d u c t i o n
because its rigid framework was proposed to space the two quaternary
n itrogen atom s at an appropriate distance with the m ethy lene bridges
affo rd ing a s light degree of freedom that could enhance any supposed
in te rac t io n with the receptor, whilst the p ip e r id in e ring was chosen
because , with the exception of dequalin ium , it is a s truc tura l feature,
w hether substitu ted or unsubstitu ted , of all the neu rom uscu la r b lockers
illustrated. Though it was 3-fold less effective at inh ib iting radiolabelled
apam in b ind ing than pancuronium , it appeared that, d esp ite the large
difference in the nature of the backbone, the interaction was of the same
nature, and provided a starting point for SAR studies of this class of
c o m p o u n d .
1.7. Therapeut ic Appli ca t ions of Potassium C h a n n e l A n ta g o n i s t s .
1.7.1 M ech an ism s O f Interac t io n With The Channel
V oltage-gated KCs exist in several conform ational sta tes which may be
regarded as closed resting, closed activated, open and inactivated. At normal
resting potential, most KCs are closed. Upon a shift o f m embrane potential
to more positive values, a voltage-dependent conform ational change takes
place, but the subsequent opening and closing of the activated channel is
voltage independent. These transitions may be sum m arised in the state
diagram in figure 1 .18 . Two modes of interaction are well understood®^: N-
type and C-type inactivation, referring to which terminus o f the protein is
C : closed resting state; II € * , C **, activated closedIt \ states; O , open state; I,
^ P * _^ P** ^ P ^ , inactivated state. C-type^ u ^ icN and N-type ( I n )
inatpena'n. | | ^ inac t iva t io n may occurV Tl / y independently or s imul
taneously ( I c n )-
F ig u re 1.18: Transition state diagram of a voltage-gated potassium
c h a n n e l
44
I n t r o d u c t i o n
involved. The N-terminal sequence is thought to behave like a ball that
blocks the pore by swinging in and out of a cytoplasm ic receptor site
be tw een segm ents S4 and S5, w hilst C -type in ac t iv a t io n is due to
c o n fo rm at io n a l changes in the reg ion tow ards the C -term inus which
involves a constriction of the channel’s extracellu lar mouth. Deletions or
a lte ra tions in the carboxy-term inal cytoplasm ic dom ain do not markedly
affect this process, so it is unlikely to involve the terminus itself in a ball
and chain mechanism like that suggested for N -type inactivation. Both
term ini are believed to be in tracellu lar which has im portant consequences
for any attempted pharmacological modification since the large majority of
the protein is located on the inner side of the cell, with the exception of a
few res idues betw een a lte rna te pairs of m em brane -spann ing segments.
Therefore, any putative modulators which cannot penetrate the membrane
can only bind to a small area of the protein. The fact that a large part of
this area is the H5 segment which, as a lready d iscussed , varies little
between different channel proteins is one of the reasons why the variety
o f KC blockers is much less than the number of channels themselves, and
also accounts for the ubiquity of the QA binding site.
H - drugs
îpriiîîiin i/;/iLAJiiiimivi
m f \ Y J
k m _A_ j
blockade of open closed
ion channels
ch an g eof
channel gating
Possible modes of interaction of drugs with voltage- or Ca^+- activated KCs: drugs may directly block open or closed channels (left). Alternatively, a change in channel gating can occur, resulting in an increase or decrease of channel open time or the probability of channel opening; this may result from drug binding to one or more allosteric sites at subunits of the channel protein or from alterations in the lipid environment of the channel (right).
F i g u r e 1 .19
45
I n t r o d u c t i o n
We may posit four ways in which drugs may interact with KCs^^:
(i) by binding directly to the open channel, thereby blocking the
current, but also preventing closure of the channel;
(ii) by binding to the closed channel, preventing opening;
(iii) by binding to an allosteric site on the protein and thereby
changing the channel’s gating properties;
(iv) by altering the lipid environment of the channel.
These are summarised in figure 1 .19 .
1 .7 .2 C l in ic a l A p p l i c a t i o n s
Several marketed drugs appear to function as selective KC b l o c k e r s ^ F o r
instance, as a result of the SAR of TEA derivatives, clofilium 24 (figure
1 . 2 0 ) was found to be an effective treatment for disrhythmias of the heart.
E t
C l— < ^ ( C H 2 ) 4 N ( C H 2 ) 6 C H 3
2 4
F i g u r e 1 .20
It was the first specifically designed class III antiarrhythmic and works by
b lock ing card iac delayed rec tif ie r channels , thus leng then ing the AP
duration of cardiac cells in a selective manner.
Am ong the most im portant of the known KC blockers are the
antid iabetic sulphonylureas g lyburide 25 , g l ip iz id e 26, and tolbutamide 2 7
(figure 1 . 2 1 )
\ / '-”"N(CH2)2— \ S—N -C —N\ = / H \ _ / ^ H H
OCH3 2 5
glyburide
46
I n t r o d u c t i o n
/ ~ \ y / — \ V 9
2 6
g lip iz id e
— \ ° °H3C—^ a— S -N -C — N— (CH 2)30 H 3^ ^ H H
2 7to lbutam ide
F ig u r e 1.21
It is known that increases in intracellular glucose in pancreatic p - c e l l s
leads to a corresponding increase in cytoplasmic ATP concentration. This
causes the closure of Ka t p » resulting in membrane depolarisation and the
opening of vo ltage-dependen t Ca^+ channels. The resultant increase in
C a j 2 + c o n c en tra t io n causes ex o c y to t ic se c re t io n o f in su l in . The
su lphony lu reas apparen tly function by b lock ing the pancrea tic ATP-
dependent KC.
The historical lack of potent antagonists has meant, however, that
the field of phanvitfcological modulation of KCs compared, say, to that for
sodium channels, is still in relative infancy. Current industrial research
centres around the su lphonylureas as an tid iabetics , and derivatives of
sotalol, a mixed class I l/class III antiarrhythm ic whose class III effect
seems to reside in the D-isomer. It seems likely, however, that as the
architec ture o f potassium channels becomes clearer, drug design should
become more sophisticated and produce a range of modulators that reflects
the diversity and pharmacological importance of the channels.
47
I n t r o d u c t i o n
1.8 . R e f e r e n c e s
(1 ) Despopoulos, A.; Silbemagl, F. Color A tlas O f Physiology; 2nd éd.;
Springer Verlag: NY, 1986.
( 2 ) Overton, E. Pfleugers Arch. ges. Physiol. 1902 , 92 , 346.
( 3 ) Hodgkin, A. L.; Katz, B. J. Physiol 1 9 4 9 ,7 9 5 , 37.
( 4 ) Hodkin, A. L.; Huxley, A. F. J. Physiol 1 9 5 2 ,116, 449.
( 5 ) Hodgkin, A. L.; Huxley, A. F. J. Physiol 1952, 116, 473.
( 6 ) Hodgkin, A. L. Biol. Rev. 1 9 5 1 ,2 6 , 339.
( 7 ) Latorre, R.; Miller, C. J. J. Membr. Biol. 1983, 76, 197.
( 8 ) Yellen, G. Ann. Rev. Biophys. Biophys. Chem. 1987 , 76, 227.
(9 ) Steinberg, M. L; Robertson, D. W. J. Med. Chem. 1 9 9 0 ,3 3 , 1529.
(1 0 ) Hille, B. lonic Channels O f Excitable Membranes', Sinauer:
Sunderland, MA, 1984.
(1 1 ) Cook, N. S. TIPS 1988, 9 ,2 1 .
(12 ) Robertson, D. W.; Steinberg, M. I. J. Med. Chem. 1990, 55, 1529.
(1 3 ) Ruff, R. L. Muscle Nerv. 1 9 8 6 ,9 , 767.
(1 4 ) Petersen, O. H.; Findlay, I. Physiol. Rev. 1987, 67, 1054.
(1 5 ) Ho, K.; Nichols, C. G.; Jonathan Lederer, W.; Lytton, J.; Vassilev, P. M.;
Kanazirska, M. V.; Hebert, S. C. Nature 1993, 562 , 31.
(1 6 ) Papazian, D. M.; Schwarz, T. L.; Tempel, B. L.; Jan, Y. N.; Jan, Y. L.
Science 1987, 257, 749.
(1 7 ) Butler, A.; Wei, A.; Baker, K.; Salkoff, L. Science 1989, 245 , 943.
(1 8 ) Jan, L. Y.; Jan, Y. N. Cell 1992, 69, 715.
(1 9 ) Brown, D. A. Trends Neurosci. 1 9 8 8 ,7 7 , 294.
(2 0 ) Nowak, L. M.; MacDonald, R. L. Neurosci. Lett. 1983, 35, 85.
(2 1 ) Sakmann, B. Nature 1 9 8 3 ,5 0 5 , 250.
(2 2 ) Halliwell, J. V. Proc. Natl. Acad. Sci. USA 1986, 55, 493.
(2 3 ) Ashcroft, F. M.; Harrison, D. E.; Ashcroft, S. J. H. Nature 1984, 572, 446.
(2 4 ) Hoffmann, E. K. J. Membr. Biol. 1 9 8 4 ,7 5 , 211.
(2 5 ) Sarkadi, B.; Cheung, R.; Mack, E.; Grinstein, S.; Gelfand, E. W.;
Rothstein, A. Am. J. Physiol 1985, 248, C480.
(2 6 ) Kolb, H. A. Rev. Physiol. Biochem. Pharmacol. 1990 , 7 75, 52.
(2 7 ) Blatz, L. A.; Magleby, K. L. Nature 1986, 525 , 718.
(2 8 ) Kawai, T.; Watanabe, M. Br. J. Pharmacol. 1986, 57, 225.
48
I n t r o d u c t i o n
(29 ) Eisenmann, G.; Horn, R. J. Membr. Biol. 1983, 7(5, 197.
(3 0 ) Eisenmann, G. Biophys. J. 1962, 2, 259.
(3 1 ) Bezanilla, F.; Armstrong, C. M. J. Gen. Physiol. 1 9 7 2 , 6 0 , 588.
(32 ) Hille, B. Prog. Biophys. M ol Biol. 1 9 7 0 ,2 7 , 1.
(3 3 ) French, R. J.; Shoukimas, J. J. Biophys. J. 1981, 34 , 271.
(3 4 ) Armstrong, C. M.; Binstock, L. J. Gen. Physiol. 1965, 48, 859.
(3 5 ) Woodhull, A. M. J. Gen. Physiol. 1 9 7 3 ,6 7 , 687.
(3 6 ) Coronado, R.; Rosenberg, R. G.; Miller, C. J. Gen. Physiol. 1980, 76, 425.
(3 7 ) Miller, C. J. Gen. Physiol. 1982, 79, 869.
(3 8 ) French, R. J.; Shoukimas, J. J. J. Gen. Physiol. 1985, 85, 669.
(3 9 ) Armstrong, C. M. J. Gen. Physiol 1971, 58, 413.
(4 0 ) Swenson Jr., R. P. J. Gen. Physiol. 1981, 77, 255.
(4 1 ) Armstrong, C. M. Potassium Channels: Basic Function And
Therapeutic Aspects', Alan R. Liss Inc.: NY, 1990.
(4 2 ) Eisenberg, D. Ann. Rev. Biochem. 1984, 53 , 595.
(4 3 ) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 757, 105.
(44 ) Jan, L. Y.; Jan, Y. N. Nature 1990, 345, 672.
(45 ) Pongs, O. TIPS 199 2 ,7 5 , 359.
(46 ) Âkerfeldt, K. S.; Lear, J. D.; Wasserman, Z. R.; Chung, L. A.; DeGrado, W.
F. Acc. Chem. Res. 1993, 26, 191.
(4 7 ) Catterall, W. A. Ann. Rev. Biochem. 1986, 55, 953.
(4 8 ) Stuhmer, W. EMBO 1 9 8 9 ,5 , 3235.
(4 9 ) Hartmann, H. Nature 1 9 9 1 ,2 5 7 , 942.
(5 0 ) MacKinnon, R.; Miller, C. Science 1989, 245 , 1382.
(5 1 ) Sakmann, B.; Neher, E. Ann. Rev. Physiol. 1 9 8 4 ,4 6 , 455.
(5 2 ) Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J.
Pfleugers Arch. 1981 , 3 9 1 , 85.
(5 3 ) Miller, C. Ion Channel Reconstitution; Plenum: NY, 1986.
(5 4 ) Latorre, R. Ionie Channels In Cells And M odel Systems; Plenum: NY,
1986
(5 5 ) Mueller, P.; Rudin, D. O.; Tien, H. T.; Wescott, W. C. Nature 1962, 194,
979.
(5 6 ) Montai, M.; Mueller, P. Proc. Natl. Acad. Sci. USA 1 9 7 2 ,6 9 , 3561.
(5 7 ) Drukarch, B.; Kits, K. S.; Leysen, J. E.; Stoof, J. C.; Schepens, E. Br. J.
Pharmacol. 1 9 8 9 ,9 5 , 113.
(5 8 ) Dreyer, F. Rev. Physiol. Biochem. Pharmacol 1990, 775, 94.
49
I n t r o d u c t i o n
(5 9 ) Moczydlowski, E.; Lucchese, K.; Ravindram, A. J. Membr. Biol. 1988,
705, 95.
(6 0
(61
(62
(63
(64
(65
(66(67
(68
(69
(70
(71
(72
(73
(74
(75
(76
(77
(78
(79
Habermann, E. Science 1972 , 7 77, 314.
Habermann, E. Naunym Schm iedeberg 's Arch. Pharm acol. 1 9 7 7 , 3 0 0 ,
189.
Banks, B.; Brown, C.; Burgess, G. M.; Claret, M.; Cocks, T. M.; Jenkinson,
D. H. Nature 1979, 282, 415.
Burgess, G. M.; Clavet, M.; Jenkinson, D. H. J. Physiol. 1 9 8 1 ,5 7 7, 67.
Miller, C.; Moczydlowski, E.; Latorre, R.; Phillips, M. Nature 1985, 313,
316.
Hugues, M.; Duval, D.; Kitabgi, P.; Lazdunski, M.; Vincent, J. P. J. Biol.
Chem. 1982, 257, 2762.
Cook, N. S.; Haylett, D. G. J. Physiol. 1985, 358, 373.
Habermann, E.; Reiz, K. G. Biochem. Z. 1965, 343 , 192.
Shipolini, R.; Bradbury, A. P.; Callewaert, G. L.; Vemon, C. A. Chem.
Commun. 1967, 679.
Callewaert, G. L.; Shipolini, R.; Vemon, C. A. FEBS Lett. 1 9 6 8 ,7 , 111.
Vincent, J. P.; Schweitz, H.; Lazdunski, M. Biochem istry 1975, 14,
2521.
Granier, C.; Pedroso Muller, E.; Van Rietschoten, J. Eur. J. Biochem.
1978, 82, 293.
Cosand, W. C.; Merrifield, R. B. Proc. Natl. Acad. Sci. USA 1977, 74,
2771.
Demonchaux, P.; Ganellin, C. R.; Dunn, P. M.; Haylett, D. G.; Jenkinsef^
D. H. Eur. J. Med. Chem. 1991, 26, 915.
Labbé-Jullié, C.; Granier, C.; Albericio, P.; Defendini, M.-L.; Ceard, B.;
Rochat, H.; Van Rietschoten, J. Eur. J. Biochem. 1991, 196, 639.
Bystrov, V. P.; Okhanov, V. V.; Miroshnikov, A. P.; Ovchinnikov, Y. A.
FEBS.Lett. 1980 ,779 , 113.
Habermann, E.; Pisher, K. A dvances In C ytopharm acology, Raven
Press: NY, 1979.
Miroshnikov, A. L; Elyakova, E. G.; Kudelin, A. B.; Senyavina, A. B.
Bioor g. Khim. 1978, 4, 1022.
Bystrov, V. P.; Arseniev, A. S.; Gavrilov, Y. D. J. Magn. Res. 1978, 30,
151.
Englander, J. W.; Kallenbach, N. R. Q. Rev. Biophys. 1984, 16, 521.
50
I n t r o d u c t i o n
(8 0 ) Hider,
(8 1 ) Pease,
(8 2 ) Hoshi,
51
Chapter 2
Results and Discussion
Re s u l t s a n d D i s c u s s i o n
2. Results And Discussion
T he m o d e s t s u c c e s s o f 3 , 6 - b i s ( p i p e r i d o m e t h y l ) p h e n a n t h r e n e
dimethiodide as an apamin antagonist led us to wonder how the structure of
the compound could be altered to improve its binding affinity. The two most
obvious ways in which modifications could be achieved were by changing:
(i) the substitu ted amine; (ii) the spacing fram ew ork . The envisaged
synthesis (scheme 2 . 1 ) of analogues of 23 made the former a simple task
since this merely required the use of different amines in the S n 2 reaction
in the penultimate stage of the synthesis whilst the la tte r required the
availability of a number of dimethylated aromatic hydrocarbons.
PN-Br
bMe ■Me BrH jC-f-V
2 8 2 9
RlNHzC-\ CHjI +
-j|— CH^NR; - R 2N(CH3)H2C t - J —CH2(CH3)NR2
^ 21'
3 0 3 1
S c h e m e 2.1
These are, generally speaking , quite rare and w here com m erc ia lly
ava ilab le are often p roh ib itive ly expensive , so it was necessa ry to
synthesise the majority of those that were needed. The synthetic routes to
such compounds are broadly the same as those used to obtain the parent
hydrocarbons, but with the additional problem of reg iospecific ity of the
m ethyl groups.
2.1 R o u te s To D im e th y la te d A r o m a t i c H y d r o c a r b o n s
The means by which these are obtained fall usually into one of two
categories: cyclodehydration of ortho-substi tu ted deriva tives (3 2 , scheme
2 .2 . 1 ) or photolytically-induced cyclisation of suitably substitu ted acyclic
precursors (35 , scheme 2 .2 .2 ) .
53
4 - R
Re su l t s a n d D i s c u s s i o n
Rt—
X = COOH, CHO 3 2
R
R
S c h e m e 2 .2 .1
hv/I]
S c h e m e 2 .2 .2
2 .1 .1 D i m e t h y l p h e n a n t h r e n e s
Early investiga tions into the synthesis of phenanthrenes were prom pted
by the need to identify, by comparison with authentic samples, certain
hydrocarbons obtained by the dehydrogenation of bile acid; these were
predicted, on the basis of of absorption spectra and X-ray crystallographic
m e a s u re m e n ts , to be d e r iv a t iv e s o f p h e n a n th r e n e ^ . B a rd h am and
S e n g u p t a ^ d e ve loped a syn thes is based on e th y lc y c lo h e x a n o n e -2 -
carboxylate 37 (scheme 2 .3 ) .
C0 2 Et
O
EtOzC
(i) K (s)/C6Hô
^ (ii)37
Br
(i) 10% KOH/A
(ii) HVH2O
(iii) H 2O38
Na/Et 2O
= / H2O
P20s/A/6mm Hg = \ Se, 300-320'C
41
Scheme 2.3
42
The potassium enolate of this was reacted with p-phenylethyl bromide, then
hydrolysed to the cyclohexanone 3 $ . Reduction with sodium followed by
cyclisation with phosphorus pentoxide and dehydrogenation with selenium
gave phenan threne 42 in unstated yield. Using appropria te ly substituted
54
R e su l t s a n d D i s c u s s i o n
m a te r i a l s , they w ere a lso ab le to s y n th e s i s e 1,4- and 1,7-
d im e thy lphenan th rene . By using a s im ila r but in d ep en d en tly derived
route , H aw orth and co-w orkers^ extended this to include all the 9,10-
unsubstituted analogues with the exception of the 3,5- and 4,5-derivatives,
but observed a troublesome shift of the methyl group between positions 4
and 1 during the dehydrogenation of some of the compounds.
3 ,6 - D im e th y lp h e n a n th r e n e 4 4 was synthesised by Sengupta and
C h a t t e r j e e ^ via the ca ta ly t ic d e h y d ro g e n a tio n o f 7 -m e th y l - l ,2 ,3 ,4-
t e t r a h y d r o n a p h th a l e n e - 2 - s p i r o ( 3 ’- m e th y lc y c lo p e n ta n e ) 4 3 (scheme 2 .4 ) .
Me.Me
43
Pt/C(cat.)
300-310 'C 36%
S c h e m e 2 .4
2,6- and 2,7-Dimethylanthracene were also formed as side products but in
yields too low to make separation viable. Later, Sengupta and co-workers^
synthesised the same phenanthrene by an alternative route (scheme 2 .5 ) .
Me Me0
Xylene.0
OMe Me
46 4745
Me
10% Pd/C
300-3 20“C
Me44
(i) KOH
(ii) sodalime
Me
S c h e m e 2 .5
4 ,5 -d im e th y lp h e n a n th re n e 54 is of interest since twisting of the sp^
framework can be achieved by alkyl substitution at these positions and 4,5-
d isubstitu ted phenanthrenes are the smallest polycyclic m olecules with an
enforced helical structure; if the barrier to racém isation is sufficiently
high, the compounds may be resolved. The strain on the framework is one
55
Re s ul t s a n d D i s c us s io n
of the reasons why this phenanthrene was the last of its family to be
s y n t h e s i s e d ^ ; Newman and W hitehouse ’s syn thes is s ta r ted with the
o z o n o ly s is o f py rene 49 then p roceeded as show n to g ive the
phenan th rene 54 in reasonable yield (Scheme 2 .6 ) .
L iA lW u / = ( > =
EtOH,
H+(cat.) 76% C2H5O
P, HI
165°CH3C Œ 3
CgHg, EhO ^90% HOH2C CH2OH 90%
5 2
S c h e m e 2.6
4,5-Dimethylphenanthrene was also prepared in low yield by Frim and co-
w o r k e r s ^ ’ using a method which is now the most com m only used to
prepare phenanthrenes , i .e ., formation of a stilbene by the Wittig reaction
of an aldehyde and the ylide derived from the phosphonium salt of a benzyl
halide, followed by photocyclisation (scheme 2 .7 ) .
PPh r Br
(i)n-BuLi, THF
( T o i 2h. Me
10%
C-C(3Hi2» I2» h v /==■
20-50%
56 Me
Scheme 2.7
Me Me
Couture and co-w orkers^ used a directly analogous method to prepare 3,6-
and 1,8-dim ethylphenanthrene, the latter in relatively m odest yield (45%)
which they attributed to the presence of the two m ethyl groups in the
ortho pos it ions in the parent s tilbene. This m ethod has also been
e x te n s iv e ly used to p repare 3 ,6 -d im ethy l p h e n a n th re n e in y ie ld s o f
between 46 and 819&10.11 and its use is now preferred since it is quite
general and cyclisation is successful with stilbenes bearing halo, methoxy,
trifluoromethyl, phenyl and carboxy substituents (though not nitro, acetyl
56
R e s u l t s a n d D i s c u s s i o n
or d i m e t h y l a m i n o ) ^ 2 The general procedure for the photoirradiation step
was e s ta b l ish e d by M allo ry who found that the most sa tisfac tory
conditions were using 0.01 moles of the stilbene dissolved in 1 litre of
cyclohexane under an atmosphere of air and with iodine as oxidant.
2 . 1 . 2 D i m e t h y l a n t h r a c e n e s
D im e th y la n th ra c e n e s w ere h is to r ic a l ly sy n th e s ise d by F r ie d e l -C ra f ts
reactions of toluene with aluminium chloride and various alkyl or benzyl
halides but most of these syntheses resulted in isom eric m i x t u r e s ^ T h e
first workers to devise a specific synthesis o f dimethylanthracenes by
the use of a Friedel-Crafts approach were Morgan and Coulson^^. Their
route to 2,6- and 2,7-dim ethylanthracene (scheme 2 .8 ) was established in
order to obtain reference samples of the two h y d r o c a r b o n s after studies
on the constituents of low-boiling tar and also to settle the confusion
surrounding the structures of products of earlier syntheses.
Me57(a) R=3-Me 57(b) R=4-Me
Me
AICI3 .CS2
82%Rir
58Me 'Me
59(a) R = 4-Me 59(b) R = 5-Me
6-12 hrs r4 -
60(a) R=6-Me 60(b) R=7-Me
Scheme 2.8
Thus, 2 ,4 ,4 '- and 2 ,5 ,4 '- tr im ethylbenzophenone 59 were obtained by
the reaction of p-to luoyl ch lo ride and the app rop ria te xylene. These
benzophenones were reported to cyclise with loss of w ater by prolonged
dry reflux to give the respective d imethylanthracene. The smoothness of
the reac tion was la ter con tes ted by Pepper and co -w orkers^ ^ , who
demonstrated that a large part of the crystalline precipitate observed upon
cooling of the reaction m ixture was in fact d im ethy lan th rone , though
reduc tion o f this com pound was easily effected . In fact, substitu ted
57
R e s u l t s a n d D i s c u s s i o n
an th racenes have often been synthesised by the red u c t io n o f the
co rre sp o n d in g an th rone or an th raqu inone but aga in , ea rly syn theses
were p lagued by ambiguity. In an early m ethod, for instance. Seer^ 4 obtained anthraquinones by the Friedel-C rafts condensa tion o f m -toluoyl
chloride 61 (scheme 2 .9 ) but he was not able to unequivocally assign the
structure o f the products until Morgan and Coulson characterised the two
anthraquinones formed by oxidation o f their anthracenes.
H3C COCl
61
AICI3. 130-140X
62
Scheme 2.9
M eth o d s fo r the red u c t io n o f the a p p ro p r ia te a n th ro n e or
anthraquinone include the classical Clemmensen m ethod for the reduction
of aromatic k e t o n e s ( s c h e m e 2 . 1 0 ).
63
Z n, H 2 O, HCl
A, 48hrs
Scheme 2.10
H . H
H H
This system is weak enough to give only the 9,10-dihydroanthracene 64 but
a modification by M artin^^, using an alkaline two-phase system, proceeds
to completion and is useful for compounds not appreciably soluble in the
acidic mixture above or molten at the reflux temperature (scheme 2 . 1 1 ).
65
Zn, N aO H , P hM e
A, 12hrs, 93%
Scheme 2.11
66
58
R e s u l t s a n d D i s c u s s io n
A m ong o th e r rea g e n ts tha t have been u sed is a lu m in iu m
tr is (cyc lohexy l) oxide which G aylord and S tepân^^ used to specifically
reduce a num ber of m ethylated anthraquinones, including both 2 ,6- and
2 ,7 -d im e th y lan th raq u in o n e (schem e 2 ,1 2 ) .
Me MeAl, HgCl 2, CeHiiOH
CCI 4(cat), A, 2h, 69%
60(b)
Scheme 2.12
A stra ightforw ard and apparently general m ethod was established
by C risw ell and Klandermann^®, the later part o f which simply required
re f lu x o f the in te rm e d ia te an th ro n e w ith so d iu m b o ro h y d r id e in
isopropanol (scheme 2 ,1 3 ) .
H H
H+, H2O, A
^ 6 CH3OH. NaBH4 Me
A, 88%
Me MePrOH, NaBH4
A, 24-36hrs
Scheme 2.13
a GH
HO H
60(b)
Klemm, Kohlik and Desai^^ obtained disubstituted anthraquinones by
the D ie ls -A lder reaction o f 4 -m ethy lbenzoqu inone 71 and isoprene 7 2 ,
followed by base-assisted dehydrogenation of the adduct (scheme 2 .1 4 ) .
(i) Eton, 75*C Me
-
72
(ii) 5% KOH, EtOH O 2, 46 hrs
Scheme 2.14
“rMe
59
R e s u l t s a n d D i s c u s s io n
This gave an isomeric mixture of 2,6- and 2,7-dim ethylanthraquinone from
which the isom ers were separa ted by f rac tiona l c ry s ta l l is a t io n from
ethanol. Reduction with zinc dust in ethanoic acid gave the respective
anthracenes, but the overall yield for the synthesis was very low.
1,5- and 1 ,8-D im ethylan thracenes 76 and have been formed by
the cyclodehydration of the appropriate o-benzoylbenzoic acid by Cristol
and Caspar^^ (scheme 2.15).
Me OH 73
C.H 2SO4, 95'C 3-4 hrs, 73%
O Me
Zn, C.NH3 50'C, 16 hrs
72%Me
Zn, C.NH3 50‘C, 2hrs
65% Me
Me 76 78
Scheme 2.15
The acid catalysed cyclisation of either of the benzoic acid derivatives led to
a good yield (the same, regardless of which starting material) of a
60
R e s u l t s a n d D i s c u s s io n
Me O Me OMe O
82 O Me81 O Me
Me OH Me CO
Me OMe OH
87 O83 O Me7 9 0 M e
Me O MeMe
O
Figure 2.1: The Hayashi Rearrangement
m ixture o f the two anthraquinones due to a H ayashi rearrangem ent^
(figure 2 .1 )
NH2 NH,NBS, DMF
91-95%
BrCHO
8 8 8 9
(i) NaNO 2 . H+
(ii) C H 2 =N 0 H
69%9 0
89%
HO H
Br Mb Mb
N aB H 4 (i) CuCNCF3 COOH
94%
(i) CuCN NMS, A, 4 hrs
9 2 (ii) NH 3 , H2 O 9 3 69%
CH O
— ' o c o “. . i ' c ô a “(i) NaOH. EtOH, A 0-20“C THF 69%(ii)H 2Û,H+ ^ 6 3
82%6 0 ( b )
S c h e m e 2 .1 6
61
R e su l t s a n d D i s c u s s io n
The two com pounds were separated by f rac tional c rys ta ll isa t ion from
ethanol prior to reduction with zinc dust in concentrated aqueous ammonia
s o lu t io n .
M ost recently , Lai and Peck^^ devised a syn thesis of 2 ,7 -d im ethy l
an th racene w hich is po ten t ia l ly genera l (schem e 2 . 1 6 ) , Again, the
a n th ra ce n e is ob ta ined from the an th rone , i t s e l f ob ta in ed by the
cyclodehydration of the carboxylic acid 94 . This is conceptually similar to
Cristal and Caspar’s method (see scheme 2 .1 5 , above) but represents an
improvement since that of the earlier workers relied on the availability of
a selectively substituted phthalic anhydride. The new method involves the
reaction of the Grignard reagent derived from p-chlorotoluene and 3-
bromotoluAlckhyJe 90 (easily obtained as shown) followed by dehydration and
conversion to, first, the nitrile 93 then the acid 9 4 . Yields in the synthesis
are good to excellent and though long, it could prove to have considerable
p o t e n t i a l .
A num ber of compounds are known to give d im ethylan thracenes
when pyrolysed under a variety of conditions. For instance, Errede and
C a s s i d y r e p o r t e d that 2 ,6-d im ethylan thracene 6 0 ( b ) was formed in
good y ield from the low -pressu re , fas t-f low py ro ly s is o f p - to ly l-p -
xy ly lm ethane 95 (scheme 2 .1 7 ) .
Me
Me95
Me 970°C,0.03s
lmmHg,55%
S c h e m e 2 .17
60(b)
Trahanovsky and Suker^^ observed that dimers ofo-quinodim ethane 9 6
gave su b s ti tu ted an th racenes under s im ila r cond it ions , bu t with an
interesting alteration of regiochem istry (scheme 2 .1 8 ) .
920“C
60(b)
S c h e m e 2 .1 8
62
R e s u l t s a n d D is c u s s io n
The conversion was found to be highly specific and indicated that the
dimer was not reverting to the monomeric state. S im ilarly , l ,2 ,4 ,5 -d i(3 -
m e th y lb e n z o )c y c lo h e p ta n e 97 was found to give 1, 8-dimethylanthracene
7 8 upon pyrolysis, but with a retention of re la tive s tereochem istry
(scheme 2 .1 9 ) .
2 7
Me Me
97
730’C
78
S c h e m e 2 .19
2.2 B e n z y l ic F u n c t i o n a l i s a t i o n O f A r o m a t i c H y d r o c a r b o n s
O ne of the easiest ways to functionalise benzylic carbon atoms is by
b ro m in a t io n . This has usua lly been ach ieved by rea c t io n o f the
hydrocarbon in one of two ways and there are many examples of both
th roughou t the literature . The first is the reaction with brom ine in
te t r a c h lo ro m e th a n e so lu t ion under h igh in te n s i ty i l lu m in a t io n . This
method has been used to prepare a number of derivatives such as 9,10-
b i s ( b r o m o m e t h y l ) p h e n a n t h r e n e ^ ^ 9 9 and 9 ,1 0 - b i s ( b r o m o m e th y l ) -
anthracene 1 0 1 ^ ^ ’^® (scheme 2 . 2 0 ).
Br 2, CCI4
hv(200W sunlamp) 77%
Br 2 > CCI 4 , A benzoyl peroxide
99
9h, 500W tungsten lamp 85% 101
Scheme 2.20
63
R e s u l t s a n d D i s c u s s io n
Other compounds have been prepared in this way and it is a useful method
w here m u lt ib ro m i na tion is r e q u i re d or fo r c o m p o u n d s such as
t e t r a m e t h y l f l u o r a n t h e n e ^ 1 1 0 2 , where failure is observed through other
methods (scheme 2 . 2 1 ).
Complex mixtureNBS, e c u initiator ^
Me Me
MeMe
Br], CCI4 500W lamp
56%
103
Br
Scheme 2.21
The form ation of 9 ,10 -b is (b rom om ethy l)an th racene has also been
effected without the aid of ultraviolet i llum ination by H a u p t m a n n ^ and
Berner et a l^^ , who found that gentle heating of the reaction mixture was
merely required to give the p roduct (in unspecif ied yield) but this is
apparently unusual. In any case, the reaction proceeds via a free-radical
chain mechanism and the second major class of reactions differs in the
source and the means by which the radicals are genera ted , though the
mechanism of reaction is the same. N-Bromosuccinimide is now known to
generate a low, steady-state concentration of bromine which is available to
react as described. In the absence of an external energy source, a radical
initiator such as benzoyl peroxide is used and the whole carried out again
in a non-in teracting solvent such as tetrachlorom ethane. These conditions
have been used to prepare b is(b rom om ethy l)phenan threnes by a num ber
of workers.
___ NBS, CCI4 A, 4hrs
\ / \ — / AIBN, benzoyl peroxideMe 570/0 Br 104
Scheme 2.22
64
R e s u l t s a n d D i s c u s s io n
For instance, Staab and co-workers were the first group to synthesise 3,6-
b i s ( b r o m o m e t h y l ) p h e n a n t h r e n e ^ '^ ’^^ 1 0 4 (scheme 2 .2 2 ) and in doing so,
established a method that was used, with small m odifications, by several
s u b s e q u e n t g roups in the sy n th es is of, am ong o th e r c om pounds ,
c y c l o p h a n e s ^ ^ , h e l ic e n e s ^ ^ , ch ira l p h e n a n th re n e d e r iv a t iv e s ^ ^ and
c o r o n e n e ^ ^ . Staab and co-workers carried out the reaction using a 1:1
mixture of benzoyl peroxide and aza-bis(isobutyronitrile), while the latter
in it ia to r alone was used to prepare 2, 7 -b is (b rom om ethy l)phenan th rene
1 0 6 from its bis-iodinated derivative by Jenny and co-workers'^® (schem e
2 .2 3 ) .
1 0 5
N B S , e c u , A
l e h r s , 5 5 % , A IB N
I Br1 0 6
= ' Br
Scheme 2.23
HOOC OOŒI
107 108NaBH4 , diglyme, BFgiEl 2 0 ,50°C,
3hrs, 17%
LiAlH 4 ,THF, Soxhlet, 24hrs
HD CH
109
MeaSiBr, CH C l 3 , 50“C, 8 hrs, 58%
110(b)
Scheme 2.24
Staab and co-workers'* I»'* also synthesised 2 ,7 -b is (b rom om ethy l)-
a n th r a c e n e 1 1 0 ( b ) bu t by a ra the r d i ffe ren t m ethod , p roceed ing
a l te rn a t iv e ly v i a the methyl ester of the carboxylic acid 1 0 8 or the
65
R e s u l t s a n d D i s c u s s io n
an lhroned icarboxylic acid 107 through the b is(hydroxym ethy l) compound
109 (scheme 2 .2 4 ) .
O th e r ro u te s to b is (b ro m o m e th y l ) d e r iv a t iv e s o f a ro m a tic
h y d r o c a r b o n s in c lu d e tha t o f G o ld e n ^ ^ who o b ta in e d 9 ,1 0 -b is -
( b ro m o m e th y l) a n th ra c e n e 101 by the reaction of 9 ,10-bis(chlorom ethyl)-
a n th ra c e n e 1 1 1 with sodium bromide in refluxing acetone to give the
product in good yield (scheme 2 .2 5 ) .
NaBr, Ac ] 0
A, 82%
S c h e m e 2 .25
i n 10 1
The bis(hydroxymethyl) derivative has also been used to produce a variety
o f dibrom inated aromatic compounds (scheme 2 .2 6 ) .
CH2OH CH2OH CH2Br CH2BrC 3 H5 N, PBrj
THF, 2hrs, rt
32% HBr, AcOH
A, 20min, 94%
1 1 3
1 1 4 ^HO
i i )
HOOK1 1 5
HOH2 Ç ÇH2 OH
( iv )
1 1 7
C 5 H 5 N, PBrg, CgHg
50°C, 2hrs, 85%
Br Br1 1 6
BrH2C CH2Br
C5 H5 N. PBrj.CgHg
60°C, 2hrs, 87%
References: (i) 44, 45, 46 (ii) 47; (iii) 48; (iv) 4 9
S c h e m e 2 . 2 6
1 1 8
66
R e s u l t s a n d D i s c u s s io n
2.3 S y n t h e s i s O f Dim e t h y l - p h e n a n t h r e n e s and - a n t h r a c e n e s
Our initia l targets were 3 ,6-dim ethylphenanthrene, the spacing group in
our lead m olecule , and 2,6- and 2 ,7 -d im ethylan thracene . As discussed,
e s tab lished syntheses ex isted for all three com pounds and these were
followed in the first instance.
2 . 3 . 1 P h e n a n t h r e n e s
M allo ry 's m ethod^^ for the preparation of 3 ,6-dim ethylphenanthrene from
the photolytically induced cyclisation of r r a n j - 4 ,4 ' - d im e th y l s t i l b e n e was
used initially. Plentiful supplies of the stilbene were available but, in any
case, it is easily prepared from p-xylene and p-tolualdehyde in good yield.
The yields for the cyclisation, typically carried out on 1.0 g of starting
m aterial in 1 litre of cyclohexane, were satisfactory at around 35% after
recrysta llisa tion from ethanol, but much less than the reported yield of
8 1 % ^ . A modification to the earlier procedure was recently reported by Liu
and Katz^®, developed as part of a general route to [5]-helicenes^ ^ . They
found that cyclisation of molecules contain ing benzylic e ther functions
was spoilt by elimination of the ethers to give alkenes because of the HI
genera ted . Addition of p ropylene oxide to consum e the nascent acid
p r e v e n te d e l im in a t io n w ith the o n ly r e q u i r e m e n t b e in g th a t
stoichiometric amounts of iodine needed to be used since air, in the absence
of the sequestered iodine, was an ineffective oxidant. Interestingly, yields
were found to be improved even for m olecules w ithout benzylic ether
functions and, using this method, a crude yield of 95% was claimed for the
cyclisation of t rans -4 , 4 '-dimethylstilbene. No significant increase in yield
was observed, however, using this methodology when attempts were made
by us to repeat it, possibly due to the presence of small amounts of dissolved
oxygen , and since M allo ry 's m ethod had the advan tage of re la tive
insensitivity to changes in reaction conditions, its use was preferred.
The H nm r spectrum of 3 ,6 -d im ethy lphenan th rene (figure 2 .2 )
illustrates many of the features characteristic of the spectra of polycyclic
arom atic com pounds and their m ethylated derivatives. All the arom atic
protons are well resolved, even at 60 MHz, and a number of broadenings
and splittings are observed.
67
R e s u l t s a n d D is c u s s io n
H9. HIO
HI. H8H4, H5
CDCI3
H2, H7
Q
Figure 2.2
The spectra of such compounds are a mixture o f ortho, meta and para
coupling and long range coupling transmitted through the ring. Alkylated
derivatives also present the possibility of benzylic coupling which, because
o f the much larger differences in the chemical shifts o f the protons
involved, tends to be better documented and more obvious than coupling
betw een ring pro tons. How ever, the fou r-bond coup ling observed in
protons 2 and 7 of 3 ,6-dimethylphenanthrene is probably due to coupling
with protons 4 and 5 respectively which, though themselves singlets, are
observed to be broadened. This is presumably due to a combination of
unreso lved meta, para, benzylic and possibly long-range coupling. Any
multi-bond coupling in protons 9 and 10 is not observed, even at 400 MHz,
and the signal appears as a singlet.
The mass spectrum of this compound is also typical of compounds of
this type and polycyclic aromatics in general. The m olecular ion peak at
m/z 206 forms the baseline and strong peaks are observed at m/z 191 and
176 corresponding to loss of the methyl groups. Peaks at m/z 165 and 150
due to loss of ethylene from each of these ions are also evident as well as
minor peaks resulting from cleavage and further loss of e thene from these
io n s .
The e lec tron ic spectra of po lynuclear a rom atic com pounds are
usually complicated and for this reason they are useful as fingerprints for
the identification of unknown compounds of this type, such as are often
68
R e su l t s a n d D i s c u s s io n
observed in the degradation of natural products. Furtherm ore, substituents,
regardless of their nature or position, have only a small effect on the
lineshape and position of the absorption maxima rela tive to the parent
hydrocarbon. This is i llustrated in figure 2 .3 w here three com pounds,
d i f fe r in g in the pos it ion and e lec tronega tiv ity o f th e ir subs ti tuen ts
produce spectra that are unm istakably phenan threno id in charac te r and
with only minor differences in the wavelength of m aximum absorption.
+ 1 . 00A
0 . 20 0 ( A / [I I V . )
+ 0 . 0 0 A
2 0 0 . 0 1 0 0 . 0 ( M M / D I V . >
N + O
V . V V A'%V',
V y y . y 10 0 . 0 ( NM/ DI V . )+ 2 .
+ ü .
600*!!0
NM ■5 0 0 . 0
Me
>Me
HM
F i g u r e 2 .3
69
R e su l t s a n d D i s c u s s io n
The nex t step in our syn thes is invo lved fu n c t io n a l is a t io n o f the
phenanthrene. Of those methods with the hydrocarbon as substrate , that
using N -brom osucc in im ide seem ed the m ost a t trac tive because of the
apparent ease of reaction, good yields and the fact that, through its use, a
precedent existed for the formation of at least one of the target compounds.
A m odif ica tion of S taab 's m e t h o d ^ w a s em ployed in which catalytic
benzoyl peroxide was used as the sole radical initiator and this gave post
crystallisation yields typically around 75%, something of an improvem ent
on published yields of 5 6 ^ ^ ^ and 57%^"^’^^ . 3 ,6 - B i s ( b r o m o m e th y l ) -
phenanthrene was obtained as a white powder and gave spectra that were
entirely consistent with its structure. The H nmr spectrum has the same
profile as that of the parent phenanthrene except that there is a downfield
shift of 2 ppm for the alkyl protons and 0.5 ppm for the ortho ring protons
at positions 2 and 7 and 4 and 5. The mass spectrum gives the correct
pattern of isotope peaks corresponding to the molecular ion at m/z 364 and
for [M - Br]+ at m/z 283 and 285 with the predicted fragmentation pattern at
m/z 204 and below. The electronic spectrum also has the lineshape typical
o f phenanthrenes and the wavelength of maximum absorp tion appears at
258.5 nm compared to 253.5 nm in the unbrominated compound.
1,6 -D im e th y lp h e n a n th re n e 1 2 3 may be unequivocally synthesised
by the photocyclisation of r r a n 5 -2 ,4 ’-d im ethy ls t i lbene 1 2 2 using M allo ry’s
m e th o d ^ ^ .
Me Me Me
1 1 9 A, 2 hrs 1 2 0
PPh 3+B f
12 1
(i) Li, MeOH
( i i ) Me
Me h v , l 2, C6H .2
12-24 hrs
1 2 2 1 2 3
= benzoyl peroxide
S c h e m e 2.27
70
R e s u l t s a n d D is c u s s io n
The latter compound was prepared as shown in scheme 2 .2 7 using the same
m eth o d o lo g y as that used to prepare the 4 ,4 ’-isom er: p -xy lene was
b ro m in a te d using N -b ro m o su cc in im id e (50% ) or b ro m in e under the
i llum ination of a sodium lamp (11%), then the triphenylphosphonium salt
form ed. Form ation of the stilbene proved to be a d isappoin ting ly low-
yielding reaction for reasons that are not clear but are presumably steric
in origin. Yields were insensitive to changes in reaction conditions such as
d ifferent bases (either Li/MeOH or Na/MeOH) or protracted reaction time
and reflux. Precipitation of the product was also difficult since this rarely
occurred spontaneously to any great extent, so the crude reaction mixtures
w ere co o le d to -78 °C. This p rov ided a m ix tu re o f s ti lbene and
t r ip h e n y lp h o s p h in e ox ide w hich was e as i ly s e p a ra te d by c o lu m n
chrom atography to give the product as white crystals in a yield of only
16%. The final stage o f the synthesis gave, a fte r chrom atography in
hexane, a 36% yield of the phenanthrene as shiny colourless plates; this
yield was not incidentally improved by the use of Liu and K atz’s procedure
in v o lv in g s to ic h io m e tr ic am ounts I2 and p ropylene oxide. The mass
spectrum is almost exactly as that for 3,6-isomer and the UV spectrum has
already been given in this chapter (see figure 2.3)
2 .3 .2 A n t h r a c e n e s
O f the many routes, established and putative, that exist to 2,6- and 2,7-
d im e th y la n th ra c e n e , M organ and C oulson 's m ethod^^ seemed the most
prom ising since, despite the relative crudeness of the reaction conditions,
the route was very short and the starting m aterials cheap and readily
available . M oreover, in contrast to other, earlier m ethods, this could be
used to synthesise both isomers in a manner that was both regiospecific
and po ten t ia l ly h igh-y ie ld ing . The in itia l step (see schem e 2 .8 ) , the
F r ie d e l -C ra f ts acy la tion o f xy lene w ith p - to luoy l c h lo r id e p roceeded
sm oothly to give 2,4,4 '-trim ethylbenzophenone as a pale yellow oil and
2 ,5 ,4 '- tr im ethy lbenzophenone as a low-melting yellow solid in yields of
64% and 77% respectively. The cyclisations proved to be much less facile
than the original paper suggests, however. The authors were alert to the
dange rs in h eren t in a techn ique as fo rc ing as dry ref lux o f the
benzophenones in air (when we carried out the experiment, a Woods metal
71
R e su l t s a n d D i s c u s s io n
bath was used and temperatures as high as 360 °C were observed) and their
suggestion that the product be removed at in tervals from the cooled
reaction mixture proved both advantageous to the overall yield and simple
since the highly crystalline product was easily removed by filtration and
subsequent washing with E t2 0 to remove the more soluble benzophenone.
Extensive decomposition was observed after 12-15 hours' reflux and the
reaction was generally discontinued after three or four cycles of heating,
cooling, filtration and evaporation, but even at this stage, the yield was
much low er than reported. The brown solid obtained was purif ied by
vacuum sublimation giving bright yellow plates that mass spectral analysis
revealed to be a mixture of the anthracene 60 (m/z 206) and a compound
with a molecular mass of 223, presumably the anthrone 6 3 . The H nmr
spectrum showed 60 and 63 to be present in the ratio of approximately 3:7
respectively in a yield that, over many experiments, never exceeded 17%
(based on converted benzophenone). No mention was made of this in
Morgan and Coulson's original paper but it was subsequently repeated by
P e p p e r , H o w e ll and R ob inson^ who s u b je c te d 2 ,4 ,4 ' - t r im e th y l -
benzophenone to the reaction conditions and obtained the two compounds
in unspec if ied proportion . The anthracene was separab le by repeated
fractional crystallisation from ethanol, but the low overall yield made this
unattractive and wasteful. As discussed in section 2 .1 .2 , there are many
methods for the reduction of aromatic ketones, the most attractive of which
was that of Criswell and Klandermann^®, which simply required reflux of
the in term edia te anthrone with sodium borohydride in isopropanol. This
was there fo re carried out on the sublim ed, but u n separa ted reaction
mixtures of both compounds and gave, in each case, a quantitative crude
yield o f the anthracene. The two were purified by recrysta llisa tion from
ethano l and subsequen t colum n chrom atography using hexane as the
eluant g iv ing white, h ighly crysta lline products w ith the in tense blue
fluorescence characteristic o f anthracenes. The H nmr spectrum of each
compound is relatively simple because of the high degree o f symmetry of
the m olecules and again, all the signals are well resolved. Thus, the
spec trum of 2 ,6 -d im e th y lan th racen e 6 0 ( a ) appears as a pair of singlets
and a doublet o f doublets, the higher field pair of which, corresponding to
protons 3 and 7, are observed to be further split into doublets due to
coupling with the methyl groups. Like 3 ,6 -d im ethylphenanthrene 4 4 , the
72
R esu lts a n d D isc u s s io n
Other pair o f ortho protons in pos it ions 1 and 5, do not appear as a doublet ,
but are ob se r ve d to be broadened. Simi larly, the s ignal due to protons 1 and
8 o f 2 , 7 - d i m e t h y l a n t h r a c e n e 6 0 ( b ) is a broad s i n g l e t w i t h the ortho
protons 3 and 6 appearing as a doublet o f doublet s at 57 . 2 7 . Protons 9 and 10
h a v e b e e n a s s i g n e d p r e v i o u s l y in the l i t erature u s i n g n O e e x p e r i m e n t s .
T h e m a s s sp e c tr a o f both c o m p o u n d s are very s i m i l a r w i t h a s trong
m o le c u l a r ion peak and peaks at 191 ( [M - C H 3 ] + ), 178 and 89, whi l s t the
e l e c t r o n i c s p e c t r a e x h i b i t m a x i m u m a b s o r p t i o n at 2 5 5 . 5 nm for both
c o m p o u n d s .
B r o m i n a t i o n o f the two anthracenes was e f f e c t e d o n c e aga in by the
N - b r o m o s u c c i n i m i d e / b e n z o y l p e r o x i d e s y s t e m d e v i s e d by Staab ^^ and was
sa t i s f ac to ry , th ou gh the react ion was much l e s s s m o o th than that o b se r v e d
for the p h e n a n t h r e n e . A s d i s c u s s e d , 2 , 7 - b i s ( b r o m o m e t h y l ) a n t h r a c e n e has
b e e n m a d e b e f o r e by Staab^ ( s e e s c h e m e 2 . 2 4 , s e c t io n 2 . 2 ) but the
s y n t h e s i s w a s not ap p l i ca b le in this ca se s i n ce the b ro m in at ed c o m p o u n d
w a s o b t a i n e d from the d ie s t er and d i c a r b o x y l i c ac i d o f the anth rac ene ,
rather than the h yd ro carb on i t se l f . This was unf ortunate in v i e w o f the
g o o d y i e l d but S t a a b ’s a l t e r n a t iv e m e t h o d w a s s a t i s f a c t o r y und er the
c i r c u m s t a n c e s . Th e two c o m p o u n d s were thus obta i ned , af ter cry s t a l l i s a t i on
f rom a var iety o f so l ve nts , as h i g h- m el t i n g y e l l o w n e e d le s in y i e l d s o f abou!-
$0 % . O n c e aga in , l i tt le ch a n g e is obser ved in the H nmr spectra o f the
c o m p o u n d s c o m p a r e d to the ir p recurso rs , apart f r o m a d o w n f i e l d sh i f t
more n ot ic ab l e in those protons ortho to the subs t ituted m et hy l group. The
m a s s s p e c t r a g i v e s a t i s f a c t o r y i s o t o p e a b u n d a n c e p e a k s for the t w o
b r o m i n e a t o m s a n d are s i m i l a r to t h a t o b s e r v e d f o r 3 , 6 -
b i s ( b r o m o m e t h y l ) p h e n a n t h r e n e . F i n a l l y , the U V s p e c t r a g a v e m a x i m u m
absorpt ion for the 2 , 6 - i s o m e r as 265 .5 nm and 2 62 .5 nm for the 2 , 7 - i s o m e r
w i t h the c h a r a c t e r i s t i c l i n e s h a p e .
T h e sy m m e tr ic a l 1,5- and 1, 8 - d im et hy la n t hr ac en es w e r e a l s o se le c te d
as ta rget s for the in it ial s ta ge o f the s y n t h e s e s s i n c e th e y p r o v id e d an
appropr ia te sp a c i n g for the two methy l groups (e s t im ated to be 10.0 Â and
7 .3 Â r e s p e c t i v e l y co m p a r e d to 9 .5 Â for 3 , 6 - d i m e t h y l p h e n a n t h r e n e ) and
w e r e c o n v e n i e n t l y prepared in Cri s tol and C a s p a r ’s d i v e r g e n t syn th es i s^ “
d e s c r i b e d in s c h e m e 2 .1 5 . T h e H a y a s h i r e ar r a ng e m e nt in the c y c l i s a t i o n
73
R e su l ts a n d D is c u s s io n
m eant tha t both an th raqu inones could be p rep a re d from a s ingle
precursor, 6 -m ethy l-2 -(2 -m ethy lbenzoyl)benzo ic acid and this i tse lf was
prepared in an apparently straightforward three-step procedure based on
work with benzoylbenzoic acid esters by Newman and co-workers^
( s c h e m e 2.28).
C.H2SO4
1 2 4
Me
E^O, A, 4 hrs
O1 2 6
•5°C
Me o Me ^ M g B rV . C6H6. X
E^O, A
12 7 7 3 0 Me
S c h e m e 2.28
When attempts were made by us to repeat the synthesis, the initial Diels-
A lder reaction o f 2 -m ethylfuran 1 2 4 with maleic anhydride gave the
adduc t 6 -m e th y l-2 ,4 -d io x o -3 ,1 0 -d io x a t r ic y c lo [5 ,2 , l ,0 ^ ’^ ]dec-7-ene 126 as a
low-melting, pale pink powder and was routinely accomplished in yields of
70% or grea ter, but the subsequent dehydra tion step p roved to be
c o n s id e ra b ly m ore p ro b le m a t ic . The au tho rs e v id e n t ly e x p e r ie n c e d
difficulty also and report that among the usual reagents found unsuitable
for this step were hydrogen ch loride gas, phospho rus pen tox ide in
benzene, sodium sulphate, acetyl chloride and acetic anhydride whilst the
only reagent found suitable was 90% sulphuric acid. We found that the
temperature of the reaction and thus the degree of agitation of the mixture
was critical and established a slightly modified procedure in which rather
than a ttem pt isolation of the product by fil tra tion o f the neutra lised
reaction mixture (this often did not occur, even despite strong basification)
the product was extracted into chloroform and evaporated. This gave the
product as a dark oil or an unstable brown solid which could be crystallised
from propan-2-ol to give the anhydride as large, white needles, but yields
were never impressive, rarely exceeding 10% from the adduct.
74
R e su l t s a n d D i s c u s s io n
Formation of the acid 73 was accomplished by the addition of the
Grignard reagent derived from o-bromotoluene to an ethereal solution of
the phthalic anhydride giving the benzoic acid as a pale yellow powder in
yields of the order of those reported. Cristol and C aspar’s method for the
cyc lodehydration o f this compound involved heating it in concentra ted
sulphuric acid for at 95 °C for four hours, somewhat stringent conditions
that m erely resulted in the form ation of an in trac tab le oil when we
rep ea ted the experim en t, desp ite the a u th o rs ’ c la im s tha t the two
anthraquinones were stable to the reaction conditions. With the failure of
this reaction, however, it was decided to discontinue attempts to make the
anthracenes since the modest yield at all but the first stage of the synthesis
and the d if f icu l ty in rep l ica t in g e x p e r im e n ta l p ro c e d u re m ade it
uneconomical in view of the limited time available.
2.4 O th e r H y d r o c a r b o n s
r e a c " .
t i m e /
h o u r s
y i e l d /
%
^ J h - h /
Hz
" Jr - h /
Hz
5c H2 ^ m a x /
n m
12 8
2 68 8.2 - 4.51 322.0
1 2 9 V 2 13 8.2 -
4 .51 -H l
4.61-H14 313.5
1 3 01 36 8.3 1.7 4.64 227.0
' O X . .1 3 1
2 51 - - 4.48 218.0
y ^ ^
B r - ^ ^ 13 23 41
9.8
(H 7) -
4 .79-HI"
5 .0 2 -H l ' 256.5
Values fo r are given fo r protons ortho to the methylene group and four-bond coupling o f theseprotons to the ring protons where applicable. For prOton assignments, see experimental section.
Table 2.1
75
R e su l t s a n d D is c u s s io n
O f the remaining four dimethylated hydrocarbons which were used,
two had already been synthesised “en route” to the two phenanthrenes,v/z.
t r a n s - 4 ,4 ' - and rraAZ5-2,4’-d im ethylstiIbene and the rem ain ing pair, 2,6-
d im ethylnaphthalene and p-xylene were com m ercially available. All were
brom inated, with varying degrees of success, using S taab ’s system^'^ and
the yields and reaction time, together with selected spectral data are given
in table 2.1 above.
2,5 Formation O f Amines
2.5 .1 D e r iv a t iv es O f P ip e r id in e , P y r r o l id in e and M o r p h o l in e
As ind ica ted , the next stage in the synthesis o f the b is-quaternary
an a lo g u es of 3 ,6 -b is (p ipe r idom ethy l)phenan th rene d im e th io d id e involved
the reaction of the b is(bromom ethyl) hydrocarbons with various amines.
F o llow ing the earlier synthesis of the p iper ido -substi tu ted phenanthrene
we attempted in the first instance to make a series based on substitution
onto the same skeleton of a number of cyclic amines chosen because of the
h igh crysta llin ity that their semirigidity would hopefully confer on the
product and also because of their structural similarity to portions of the
neurom uscular blockers described in Chapter 1. The conditions employed
in itia lly involved stirring the dibromide with sto ich iom etric amounts of
amine in tetrahydrofuran, but after reaction under these conditions for a
day, the reaction was observed to have turned a dark-brown colour and
work-up produced an oil in the complicated H nmr spectrum of which,
none of the desired amine could be detected. Though it is undesirable to use
an excess of amine in reactions of this kind because, of the risk of competing
side-reactions, an attempt was made using the dibromide and a large excess
of base to determine its effect on the reaction, but once again, only an
in tractable brown oil was produced; this, and the earlier result were the
same regardless of the amine used. However, upon a change of solvent to
ethanol and a reduction in reaction time, work-up again produced a dark-
brown oil. Reverse phase HPLC revealed how ever that this consisted
primarily of a single compound. Moreover, the H nmr spectrum indicated
that this was the bis-aminated phenanthrene. Since the oil would not yield
76
R e su l t s a n d D i s c u s s io n
to c ry s ta ll isa t ion or tr i tu ra tion with a num ber o f so lvents includ ing
ethanol, petroleum spirit and chloroform and would not run on silica gel,
the problem remained of isolating the compound in crystalline form. This
was eventually solved by establishment of the procedure detailed in the
ex p e r im en ta l section: a fte r reac tion , the e thano l was rem oved then
chloroform or ether added to the crude reaction mixture. This was extracted
with 10% HCl(aq) to remove the basic product and any salt that may have
form ed and these extracts made alkaline with aqueous amm onia. The
precip ita te thus formed was exhaustively extracted into ether, dried and
evaporated to give a clear, usually slightly coloured oil and this was then
seeded or triturated to give the product as an off-white or yellow powder.
This procedure produced most of the compounds u ltim ately synthesised
with occasional modifications that are detailed as they were found to be
n e c e s s a r y .
u c ;1 3 3 1 3 4 1 3 5
F i g u r e 2.4
The f irs t three compounds to be thus form ed were 3 ,6 -b is (pyrro lido-
m e th y l ) - , 3 , 6 - b i s ( p ip e r id o m e th y l ) - and 3 ,6 - b i s ( m o r p h o l i n o m e t h y l ) -
p h e n a n th re n e (133, 134, 135, figure 2 .4 ) in yields which were initially
found to be modest but improved with repetition. The three compounds also
gave spectral data completely in accord with the given structures and show
no signs of either monosubstitution or the presence of basic side products.
Thus, the H nmr spectra of the three are midway in character between the
d ib rom ide and the parent phenan threne and 4 -bond coup ling to the
b&Y pro(;b'AS is observed in the ortho protons of positions 2 and 7 in all.
The mass spectra gave molecular ion peaks and the expected fragmentation
pattern corresponding to the loss of the two substituents. Two points are
worthy of note. Firstly, the molecular ion of compound 135 appears at m/z
77
R e s u l t s a n d D is c u s s io n
377 corresponding to [M + H]+. This feature was found to be common to all
the m o rpho lino -subs ti tu ted com pounds subsequen tly syn thes ised and is
rem arkab le not only because of the unpred ic tab le occurrence o f this
phenom enon with the other analogues, but also because a com parison of
the pK a values o f the con juga te acids of the am ines revea ls the
m orpho lin ium ion to be considerably m ore acidic than that fo r the
remaining pair of compounds (Table 2 .2 ) .
pKa
m o r p h o l in i u m ion 8.330
p i p e r i d in i u m ion 11.123
p y r r o l i d i n i u m ion 11.270
From “T he C R C H andbook O f C hem istry A n d P hysics”, 5 9 th ed ., 1 9 7 8
T a b l e 2 .2
Secondly, besides the peak one would expect from the sequential loss of the
substituents at m/z 204, there is a peak of almost equal intensity two units
h ig h e r , c o r re s p o n d in g to [phenanthrene]"*". This is perhaps due to a
parallel decomposition pathway in which a hydrogen atom is transferred
from the protonated amine onto the adjacent carbon atom, prior to the loss
of the amine itself. The electronic spectra of the three compounds again
support the structure and each exhibit essentially the same wavelength of
maximum absorption (255.0 nm for compounds 134 and 1 3 5 and 254.0 nm
for compound 1 3 3 ) .
The piperido-, pyrrolido- and m orpholino-methyl derivatives o f the
two anthracenes were the next targets and the synthesis of most of these
was accomplished with little trouble in yields that were good to very good
u s in g the s tanda rd m eth o d o lo g y . D uring the p re p a ra t io n o f 2 ,7-
b is (p i p e r i do m e th y l) a n th r a c e n e 1 4 4 however, fine, w hite needles were
observed to precipitate from the reaction mixture. These were acid-soluble,
so no fu ther work-up was perform ed on the crysta ls w hich spectra l
analysis revealed to be the desired diamine. Otherwise, the compounds were
obtained as off-white powders with spectral data in accord with the given
78
R e s u l t s a n d D i s c u s s io n
s t r u c tu r e s . In p a r t ic u la r , the H nmr sp ec tra o f a ll bu t 2 ,6-
b i s ( p y r r o l id o m e th y l ) a n t h r a c e n e 1 4 0 exhibit benzylic coupling. All six
compounds gave mass spectra where the molecular ion appears at [M -f- H]+
bu t the f rag m en ta tio n pa tte rns are o therw ise as ex p ec ted and the
e le c tro n ic sp e c tra all e xh ib i t the h igh ly c h a ra c te r is t ic an th ra ce n o id
lineshape (figure 2 .5 ) .
5 0 A
)
A0M M
1 0 0 . 0 ( N M D I U . > 0 0L* 0 0 . 0
F igu re 2.5: Electronic Spectrum Of Compound 154 , Show ing A nthracene
L i n e s h a p e
The wavelengths of maximum absorption are given in table 2 . 3
along with other, selected spectral data.
79
Results and D iscussion
^max nm ^Jh-h/H z "Jh-h/H z 5cH2
OXCClO 258.0 8.7 1.4 3.64
138 (139)(260.0) (8.7) - (4.79)
CTCCLO 258.0 8.6 - 3.84
140 (141)(258.0) (9.2) (1.3) 4.77
oCJXCCLnC? 258.0 8.7 1.2 3.67
142 (143)(260.5) (8.7) - (4.89)
a ^ x x r o 261.5 9.1 1.3 3.71
144 (145)(260.0) (8.8) - (4.93)
a x O T o 262.0 8.5 1.2 3.87
146 (147)(260.5) (8.8) - (4.93)
oOf'XxXr'O 256.0 8.7 1.5 3.71
148 (149)(259.0) (8.3) - (4.89)
r f h 2 5 4 .0 8.1 1.5 3.88
(255.0) (8.1) - (4.35)
\ _ / ^ 134 (23)
2 5 5 .0 7.9 - 3.77
(255.0) (8.0) - (4.85)
0 0 ^ 135 (137)
255.5 9.1 1.4 3.76
(255.5) (8.1) - (4.46)
Figures in brackets refer to di-N-methylated analogue, iso la ted as diiodide.
Table 2.3
2.5.2 Q uatern i sa t ion O f A m ines
I o d o m é t h y l a t i o n o f th e se c o m p o u n d s w a s n e c e s s a r y in or de r to rend er the
n i t r o g e n s q u a t er na r y l i k e t h o s e o f the n e u r o m u s c u l a r b l o c k e r s w e w e r e
a i m i n g to m i m i c and a l so to c o n f e r s o lu b i l i t y in the a s s a y by w h i c h they
w e r e b e i n g tes ted . C lea r l y , wi th a react ion as e s t a b l i s h e d as q ua te r n i sa t io n
80
Results and Discussion
of a nitrogen atom, many methods and reagents exist but the method of
S ten lake et al using iodom ethane or m ethyl m ethane-su lphona te in
ace ton itr ile was selected because of the superf ic ia l s im ilarity of the
com pounds that were used in this reference ( them selves neurom uscular
blocking agents) and the similarities of scale; we intended to carry out
reactions on 10-30 mg of material and the paper detailed precautions that
were necessary as a result. Thus, our compounds were stirred in 1-2 ml of
acetonitrile for a period of time that varied between 24 and 48 hours with a
la rge ex cess o f io d o m e th a n e . The q u a te rn is e d co m p o u n d u sua lly
precipitated from this mixture, often after only 10 minutes, but in any case,
the product was isolated by pouring into dry ether which gave the products
as flocculent white or pale-yellow precipitates which were then isolated by
filtration in yields that were as high as 91% but more often around 50%.
These h igh -m elting salts were com plete ly unde tec tab le using standard
e lec tron im pact mass spectrom etry techniques but y ie lded spectra that
exh ib i ted certa in charac te r is t ic features under fast atom bom bardm ent
conditions. Thus, the molecular ion was observed as [M + I]+ where M refers
to the respective dication (under this nom enclature, the indicated charge
refers to the overall charge of the ion and not to a charge in excess of that
already on M) , then a small or non-existent peak due to M+ and peaks due
to loss of a methyl group, loss of the amine and loss of the two together. The
unsubstituted hydrocarbon framework was also seen as strong peak at m/z
204 and many of the compounds exhibited a prominent peak in this region,
corresponding to No conditions were found under which the diiodide
could be observed or the molecular ion made more prominent, but the
sp e c tra w ere n o n e th e less su f f ic ie n t ly c h a ra c te r i s t ic . The e le c tro n ic
spectra are, for the most part, virtually ind istinguishable from those of
their unquatern ised precursors, retaining the lineshapes and exhibiting a
shift of at most 2 nm. The nmr spectra, on the other hand, illustrate the
effect that increasing the degree of substitution on the nitrogen atom (and
thereby restric ting inversion processes) has on the ring protons of the
amine; the rigid tetrahedral structure means that the equatorial and axial
protons of the heterocyclic ring are no longer equivalent and the signals
due to the |3-protons are now resolved into distinct multiplets for all except
the m eth iodide salts of 2 ,7 -b is (py rro lidom ethy l)an th racene 1 4 7 and 2,6-
b i s ( m o r p h o l i n o m e t h y l ) a n t h r a c e n e 1 4 3 . For both, there is evidence of
81
Results and Discussion
d iffe ren tia t io n which none the less rem ains incom ple te , so the values
quoted for these two compounds in the experim ental section are for a
single multiplet due to all eight protons. Which of the signals was due to
which proton was solved by an nOe experiment: irradiation of the protons
of the methyl groups of compound 141 at 62.98 produced an enhancem ent
of the multiplet at 53.68 which, it is therefore assumed, belongs to the four
pyrrolidinyLprotons syn to the methyl group.
Otherwise, the nmr spectra of the compounds are similar to those of
their unquaternised precursors, though interestingly, most of them fail to
exhibit secondary splitting of the signal due to the ortho protons of the
aromatic ring. One can understand this in terms of the altered electronic
environment in the vicinity of the charged nitrogen atom, but the problem
is compounded by the fact that the extent of the splitting is often so small
that it is simply not fully resolved
2 .5 .3 D e r iv a t iv e s O f 2 , 5 - D i m e t h y l p y r r o l i d i n e
Thus synthesised, this first batch of compounds was submitted for testing
but, as d iscussed in the fo llowing chapter, the results represented no
improvement on any of our lead compounds. Given the structural similarity
of the three amines, substitution of an amine with different steric, rather
than electronic properties seemed the most likely way of influencing these
values w hilst s tay ing w ith in our o rig ina l b r ie f . The serend ip itous
d iscovery of 2 ,5-dim ethylpyrrolid ine and its subsequent substitution onto
the various hydrocarbons led us to just such a compound. Our sample, a
commercially available mixture, consisted of 64% of the cis-isomer and 36%
of the trans-isomer as determined by nmr analysis and comparison with a
pure sample of the cis compound. In the first instance, this amine was
reacted with 2 ,7 -b is(b rom om ethy l)an th racene but this was in itia lly not
without problems. Isolating the compound from the dark oil that was
routinely produced was found to be difficult and the product was eventually
obtained by precipita tion with the careful addition of water and the
removal and washing (with cold ethanol) of the product. Luckily, there
appears to have been a preference for the dibromide to react with the cis-
isomer of the amine since the H nmr spectrum of the product reveals the
product of substitution by this isomer to be six times more abundant than
82
Results and D iscussion
for the trans-isomer. Thus, the syn and anti protons of the pyrrolidine ring
appear as a pair of complex multiplets, the a-p ro tons as a broadened quartet
and the methyl groups as a dominating doublet with a smaller set of peaks
to the side of these corresponding to the less abundant isomer.
The quaternisation of this diamine illustrated a problem that was to
occur with a number of later compounds when it was found that after
pouring of the reaction mixture into ether in the usual way, a precipitate
was obtained but which, upon filtration, collapsed to an oil or a hard, glassy
solid. The problem seemed to be solved by allowing them to dry in a vacuum
so that if, after pouring into ether, the precipita tes were settled, either
through gravity or centrifugation, and the solvent decanted, they could be
obtained by evaporation of the remaining solvent in vacuo. O bv iously ,
because the solvent was largely ether, this had to be carried out with great
care, but the procedure was succesful for all those com pounds which
ex h ib i te d this problem . 2 ,7-Bis [ (2 ,5 -d im e th y l )p y r ro l id o m e th y l ] a n th racene
d im e th iod ide 1 5 3 was thus obtained as a yellow powder whose spectra
exhibited all the characteristics of its previous homologues.
Upon pharmacological testing, this compound was found to be some
twenty times more active than the lead phenanthrene derivative so we set
about the synthesis of a number of derivatives using this amine. The full
series of analogues made in the light of this observation is given in table
2 .4 . These were obtained generally without incident but a num ber of
amines could only be obtained as oils. It was found, after b r ie f
experimentation that they could be quaternised directly with methyl iodide
to give, in all cases, the salt as an off-white or pale-brown solid though the
problem of oiling-out discussed above was also observed for some products.
Prior to this however, when attempts to make the bis-substituted benzene,
1,4 -b is [ (2 ,5 -d im e th y l )p y r ro l id o m e th y l ]b e n z e n e 1 5 8 produced only a dark-
b row n oil, an a l te rn a t iv e p ro ce d u re was d e v ise d in w hich the
dibromoxylene was added to a stoichiometric solution of the pyrrolidine in
sodium methoxide and methanol. No improvement was noted, however and
the com pound, which gave satisfactory data was reacted as described.
S im i la r ly , t ra n s -4 ,4 '- b i s [ ( 2 ,5 -d im e th y l ) p y r r o l id o m e th y l ] s t i lb e n e 1 6 0 was
obtained as a brown oil by the dropwise addition of a solution of the lithium
salt of the pyrrolidine to the d ibrom ide but in satisfactory yield. All
compounds provided consistent spectral data and the pharm acological data
83
Results and Discussion
are discussed in the next chapter.
Yield/% ÔCH2 ^max
Me
150 (151)
4 8
(50)
8.1, 9.1
(9.8, 7.6)
4.05, 4.11
(4.80, 5.15)
2 5 8 . 5
( 2 5 6 . 5 )
Me Metnoccrü152 (153)
92( 12)
8 . 2
(8.6)
1.1 3 . 9 5
( 4 . 88 )
2 6 2 . 5
( 2 6 2 . 0 )
“trpco!Me
154 (155)
5 0
( 92)
8. 7
(8. 8)
1.6
( 1 . 3)
3 . 91
( 4 . 76)
2 5 8 .
( 2 6 0 . 0 )
Me Me
Me
156 (157)
17
(64)
8. 8
(8.2)
0 . 8 3 . 8 7
( 4 . 72 )
2 3 6 . 0
( 23 0 . 5 )
Me
158 (159)
59
(48)
3 . 7 6
( 4 . 58)
2 2 4 . 0
( 22 2 . 0 )
160 (161)
5 0
(38)
8 . 0
(7. 8)
3 . 7 8
( 4 . 53 )
3 1 4 . 0
( 31 6 . 5 )
Me
162 (163)
41
( 39)
7.5
(7.6) -
3.95, 4.03
(4.84, 5.04)
3 0 7 . 5
( 30 7 . 5 )
Figures in brackets refer to di-N-methylated analogue, isolated as diiodide.
T a b le 2.4
2.5.4 D eriva t ives O f D A BC O
It was of interest to us to alter the electronic properties of the compounds
in order to assess the effect on their binding properties and an obvious way
84
Results and Discussion
in which to do this was to in troduce more charge in the term inal
substituents; this seemed sensible in view of the high basicity of the
g u a n id in iu m groups of arginine (pKa for arginine is estimated to be about
12, rendering it h ighly charged at physio log ica l p H ^ ^ ) . F u r th e rm o re ,
dequalin ium , one of our lead compounds, not only contains a pair of
aromatic ring systems, but also has a pair of terminal amine groups which,
though not strictly basic, are likely to greatly alter the electronic profile of
the molecule. The amine we selected was l ,4 -d iaza -[2 .2 .2 ]-b icyc looc tane
(DABCO, figure 2.6) for several reasons.
F igure 2.6: DABCO
F irs t ly , the n i trogen a tom , exposed becau se o f the “ fo ld e d -b a c k ”
substituents , is rendered much more reactive than the equivalen t open-
chain or secondary amine and the consequent expected facility of the
substitu tion reaction was borne out in practice. A lso, the restric tions
placed on the molecule as a result of the rigid structure mean that the lone
pairs on the nitrogen atoms extend in opposite d irections to one another
and the electronic environment at one is unusually insensitive to reaction
at the other; this is further enhanced by the n itrogen atoms being
constrained to adopt a near-tetrahedral structure, m inim ising the effect of
substitution on overall molecular structure. The use of a tertiary amine also
obviates the need for a subsequent quaternisation reaction, an im portant
consideration in view of the small scale at which these syntheses were
carried out. There is also a precedent for the use o f the dibasic DABCO
molecule in the formation of pharmacologically active agents.
O+ ATA + / ---- \ V n R2
V N — ( CH 2 ) n — N— 'R2N-
1 6 5
F i g u r e 2 .7
85
Results and Discussion
Diederich and co-workers^^ prepared a series of tetracationic bis-DABCO
derivatives 1 6 5 of the form shown in figure 2.7 as carriers for nucleotide
5 ’-tr iphosphates - potential chain-term inating inh ib itors o f HIV reverse
transcriptase but hampered by poor cellular uptake. The DABCO derivatives
formed 1:1 complexes with the nucleotides and were found to exhibit the
correct l ipophilic ity to partition into the organic phase during transport
e x p e r i m e n t s
The conditions under which our syntheses were carried out were the
same as those for previous quatern isa tion reactions. Thus, when the
respective dibromide was stirred with an excess of DABCO (this was possible
becouse there was no risk of side-reactions, given the quaternary nature of
the product) then almost instantly, a precipitate was observed to form. The
reactions were nonetheless treated as previous substitu tions and heated
under gentle reflux for four hours in order to e lim inate the risk of
monosubstitution. It was not found necessary to pour the compounds into
ether since precipitates were observed in all cases after the given reaction
time and these were removed by filtration (with no attendant problems)
and washed with ether to give the products as white or off-white powders
in good to excellent yields (table 2 .5 ) .
Yield/% ^Jh-h/ H z "Jh-h/ H z 5cH2 ^max/ nm
S X X t S
1 6 6
92 - - 4 . 5 9 2 1 7 . 5
nS ^ ^ C O ^ nS "
1 6 7
69 8.4 - 4 . 7 8 2 3 1 . 0
1 6 8
96 8.8 1.3 4 . 7 5 2 6 2 . 0
1 6 9
4 1 8.9 - 4 . 8 6 25 9 . 5
1 7 0
58 - - 4 . 5 4 3 1 6 . 5
All compounds isolated as dibromide
T a b l e 2.5
86
Results and Discussion
The 1 H nmr spectra of the com pounds are re la tive ly sim ple since
quaternisation of the nitrogen atom does not give rise to diastereotopic
protons at the ot-carbon atom. Thus, the ring protons of the DABCO moiety
appear as a large pair of triplets, broadened due to their proximity to the
quaternised and the uncharged nitrogen atoms. Benzylic coupling is only
obse rved fo r the 2 ,6 -d im e thy lan th racene d e r iv a tiv e 1 6 8 but the nmr
spectra are otherwise as expected for the individual compounds. The mass
spectra are much the same in character as those for the methiodide salts of
the amines so that peaks are seen for [M + Br]'*’ in the appropriate ratio, a
negligible or absent molecular ion peak and peaks corresponding to the
sequential loss o f the two DABCO units. [DABCO]+ itself appears as a strong
peak at m/z 112 in all spectra and once again, [M^ + ] is observed for a
number of the compounds. Wavelengths of maximum absorption are given
in table 2 .5 .
The synthesis of the compounds 171 and 1 7 2 in figure 2 .8 gave a
pair of compounds which resem bled the nucleotide carriers of Diederich
and c o -w o rk e rs ^ ^ (figure 2 .7 ) . Carrying four charges, these m olecules
were therefore superficially similar t o the DABCO derivatives which, it is
a s s u m e d , will be protonated in vivo , but the substitu tion alters the
lipophilicity and steric profile around these extra charges.
Me +
17 2
F i g u r e 2.8
These com pounds were read i ly p repared by s t i r r in g l ,4 - b i s - ( l ,4 -
d iaza [2 .2 .2 ]b icyclooctano-m ethy l)benzene dibrom ide with iodom ethane and
4 - (b ro m o m e th y l ) to lu e n e (a-b rom o-p-xy lene) respectively for 24 hours in
acetonitrile. The starting material was observed to disappear into solution a
short while after the addition of the halide, followed by the precipitation of
87
Results and Discussion
the product. Isolation was as before, by pouring into ether and filtration'
giving the two compounds as white powders in very good yields. The high
degree o f symm etry of the m olecules and the ir m oie ties resu lts in
com paritively simple nmr spectra, but neither provides a particularly neat
mass spectrum, even under FAB conditions. Identification is made possible,
however, by the presence of fragmentation products
2.5.5 D er ivat ives O f Q u in u c l id in e
The extent to which the basic nitrogen atom within these compounds was
responsible for changes in binding efficiency could be assessed by the
synthesis of a set of homologues in which this atom was not present. The
appropria te base, l -a z a - [2 .2 .2]-b icyclooctane , or qu inuc l id ine , was thus
substituted for DABCO in the previous reaction and generated a new family
of compounds, illustrated with yields and appropriate spectral data in table
2 .6 . The synthesis of the compounds was less straightforward than before
because of problems during filtration and yields were generally lower, but
the spectral details were obviously very similar to those of the DABCO
d e r iv a t iv e s .
Yield/% ^Jh-h/H z ^Jh-h/H z SCH2 ^max
1 7 3
100 - - 4.48 217.0
0 ^1 0 0 :0 17 4
25 8.4 1.1 4.62 230.0
0-XCCX;gi17 5
7 8.9 1.1 4.71 262.5
17 6
10 8.8 - 4.89 267.5
1 7 7 +
64 8.8 1.3 4 .?4 255.0
T a b le 2.6
88
Results and Discussion
2.5.6 A t te m p te d S y n th e s i s O f D e r iv a t iv e s O f c / s - 2 ,6 - D im e th y I p ip e r id in e
2 ,6 -D im ethylp iperid ine may be purchased as its configura tiona lly
pure c i5-form and its substitution onto the hydrocarbon frameworks would
allow us to assess the effect that expansion of the heterocyclic ring would
have on the re la tive potency of the 2 ,5 -d im e th y lp y rro l id o -su b s t i tu ted
compounds. The extra steric strain associated with the a -b ran ch in g on the
larger ring meant that when the reactions were carried out, a h igher
boiling solvent was used, in this case, propan-2-ol. Previously, a number of
u nsuccess fu l a ttempts had been made to subs ti tu te qu inald ine and 4-
aminoquinaldine (the latter, when substituted at positions 1 and 10 of a
decyl chain, giving dequalinium, one of the lead compounds) onto the bis-
bromomethyl compounds in a number of solvents of decreasing volatility,
starting at ethanol and moving onto butanone (methyl ethyl ketone) and 2 ,
6-dimethylheptan-4-one (diisobutylketone) with a boiling point of 169 °C.
At this tem pera tu re , ex tensive decom position o f the d ib rom ides was
observed to occur, and with no attendant reaction, it was clear that
offsetting steric strain by raising the temperature of the reaction was of
only limited use. However, despite the lack of reaction in this case, propan-
2-ol was established as a good compromise since, on the basis of the limited
extent to which decolourisation occurred after reflux, decom position was
kept to a minimum. Upon reaction of s to ich iom etr ic amounts of the
p iperid ine with 2, 7 -b is(b rom om ethy l)an th racene , how ever, a p rec ip ita te
was observed but which analysis revealed to be the salt of the starting
amine. The formation of side products such as this had only been observed
to o ccu r once be fo re w hen, du r ing an a ttem p t to rem ake 2,7-
b is (m o rp h o l in o m e th y l )a n th ra c e n e 1 4 8 , the only product obtained was the
m orpholine salt (presumably the hydrobrom ide). Several fu rther attempts
at substitu ting 2 ,6-dimethy Ipiperidine produced the same result, but the
reaction was found to be successful in producing the derivative 2 ,6-bis[(cis-
2 ,6 - d im e th y l ) p i p e r i d o m e th y l ] n a p h t h a l e n e 1 7 8 when the solvent was
changed to butanone.
89
Results and Discussion
Me Me
F ig u r e 2.9
Me Me
W e w e r e u n a b le to r e p r o d u c e th i s r e s u l t w i th 1 ,4 -
b is (b ro m o m e th y l )b e n z e n e , f ir s t in b u tan o n e , then , fo r c o m p a r iso n ,
ethanol, so it was decided to alter the reaction conditions in an attempt to
synthesise other members of this series. Thus, a solution of the dibromide
in ethanol was stirred with 0.1 M silver nitrate solution and the piperidine
added upon formation of a precipitate of silver bromide. Once again, only
the salt o f the amine was formed, appearing as a highly crystalline residue
in the evaporated mixture, by a mechanism that is unclear. Lack of time,
how ever, did not allow us to investigate further and the naphthalene
derivative remained the only compound of this type that we were able to
synthesise. It was quaternised as before with iodomethane in acetonitrile
and ap a r t from the problem of o iling-ou t, p reven ted as p rev ious ly
described, proceeded smoothly to give the dimethylated compound 179 . The
two com pounds once again illustrate the effect that quaternisation of an
endocyclic n itrogen atom has on the H nmr signals o f the protons
adjacent to it since 1 7 9 displays separate signals co rresponding to the
equatorial and axial protons at positions 3 and 4 of the piperidine ring.
Irradiation of the N-methyl group at 52.85 produces an enhancem ent of the
signal at 51.68 which therefore belongs to the axial proton at this carbon
a tom .
2 .5 .7 O t h e r C o m p o u n d s
The effec t o f increased charge over the whole molecule had previously
been addressed via the synthesis of the DABCO and N-substituted DABCO
derivatives described earlier. By combining this with multiple substitution,
both o f the central hydrocarbon and of the substituents them selves we
conceived the idea of a dendritic “net” that would hopefully provide a
balance o f hydrophilicity and lipophilicity with a degree of flexibility that
would m aximise interaction with the receptor. The concept of this type of
90
Results and Discussion
molecule, referred to as arboranes or dendrimers if polynuclear^ '^, is not
new and synthetic routes to the molecules proceed, not surprisingly, by a
series o f sequential substitutions of a central template. The first targets
(which at this stage, remained unbranched) are shown in figure 2 . 1 0 .
+
180 R = methyl181 R = p-xylyl
6 B r '
R
F ig u r e 2.10
The proven facility of the reaction of DABCO with benzylic halogens meant
that the both the molecules shown could potentially be synthesised in two
steps from 1, 3, 5-tris(bromomethyl)benzene ( a , a ' , a " - t r i b r o m o m e s i t y l e n e ) .
The synthesis of this compound seemed simple given the ease with which
p-xylene had been dibrominated, but our first attempt, in which mesitylene
was reacted with three equivalents of N -brom osuccin im ide in refluxing
te trach lo rom ethane produced a c lear oil which c rys ta ll ised but which
contained several similar products. These were not separable in this state
by rec ry s ta l l i sa t io n or th in - la y e r c h rom a tog raphy us ing any so lven t
system that we were able to find. The alternative method, using liquid
bromine under h igh-in tensity illum ination was prom ising because it had
been used to m ultiply brom inate a num ber of m olecules in precedent
reactions in good yields as d iscussed earl ie r in this chapter. Using
mesitylene, however, the same mixture of products as before was observed,
regardless of the conditions that were employed in the reaction. This is odd
because s to ich io m e tr ic am ounts o f b rom ine would be abso rbed to
colourlessness during the reaction, but whether it was added over five
minutes or two hours a messy H nmr spectrum was still produced with
m any p eak s e x tra n e o u s to the two th a t w ere e x p e c te d . The
tris(b rom om ethy l) com pound has been made before^^ and the method is
tacit acknow ledgem ent o f the failure of the two concerted techniques
91
Results and Discussion
discussed above (scheme 2 .2 9 ) . It begins with the tricarboxylic acid 1 8 2
which is esterified then reduced with lithium alum inium hydride to the
triol 1 8 4 Reaction with phosphorus tribromide gives the tribromide 185 in
45% overall yield
COOH C02MeMe0 H/H2S04(cat) UAIH4/THF
HOOC COOH1 8 2
Me02C^ ^ C02Me 1 8 3
OH
HO. OH
1 8 4
Br
Hr. Br
18 5
PBr 3/Et 2O
S c h e m e 2 .29
It seemed that the best way of controlling this unpredictability but
still using the attractive NBS reaction was by a process of sequential
bromination where, it was reasoned, the addition of only one equivalent of
bromine atoms each time would would limit the number o f possible side-
products and make purification simpler. This was in fact mostly borne out
in practice so that reaction of mesitylene with one equivalent of NBS
produced , a f te r d is t i l la t ion , 5 -b rom om ethy l-m -xy lene 1 8 6 as colourless
l iqu id in good y ield . F u r th e r reac tion gave the b is (b ro m o m e th y l)
deriva tive 187 as white crystals, but much less smoothly and only after
chrom atography in a chloroform /hexane eluant. The final step, however
p roved as frus tra t ing ly im precise as be fore and the syn thes is had,
unfortunately to be abandoned.
2 .6 R e f e r e n c e s
(1 ) Bernal, J. D.; Crowfoot, D. J. Ind. Chem. Soc. 1933, 10, 729.
( 2 ) Bardham, J. C.; Sengupta, S. C. J. Chem. Soc. 1932, 2520.
(3 ) Haworth, R. D.; Mavin, C. R.; Sheldrick, G. J. Chem. Soc. 1934 (I), 454.
(4 ) Sengupta, S. C.; Chatterjee, D. N. J. Ind. Chem. Soc. 1953, iO, 27.
(5 ) Sengupta, S. C.; Sachchidananda, A. B.; Mitra, A. J. Ind. Chem. Soc.
92
Results and Discussion
1960, 37, 597.
6) Newman, M. S.; Whitehouse, H. S. JACS 1949, 71, 3664.
7) Frim, R.; Mannschreck, A.; Rabinovitz, M. Angew. Chem. Int. Ed. Engl.
1 9 9 0 ,2 9 ,9 1 9 .
8) Frim, R.; Goldblum, A.; Rabinovitz, M. J. Chem. Soc. Perkin Trans. 2
1992 , 267.
9) Buquet, A.; Couture, A.; L ab ilehe-C om bier , A. J. Org .C h»m .
1 9 7 9 ,4 4 , 2300.
10) Davy, J. R.; Jessup, P. J.; Reiss, J. A. J. Chem. Ed. 1975, 52, 747.
11) Newman, M. S.; Lilje, K. C. J. Org, Chem. 1 9 7 9 ,4 4 , 4944.(1 2 ) Blackburn, E. V.; Timmons, C. J. Mod. React. Org. Syn. 1970, 188.
13) Wood, C. S.; Mallory. F. B. J. Org. Chem. 1964, 29, 3373.
14) Seer, C. Monatsh. 1 9 1 1 ,5 2 , 143.
15) Morgan, G. T.; Coulson, E. A. J. Chem. Soc. 1929, 2203.
16) Pepper, J. M.; Howell, M.; Robinson, B. P. Can. J. Chem 1 9 6 4 ,4 2 , 1242.
17) Bradlow, H. L.; WanderVerf, C. A. JACS 1 947 ,59 , 1254.
18) Martin, E. L. 5A C S 1 936 ,58 , 1438.
19) Gaylord, N. G.; Stepan, V. Coll. Czech. Chem. Comm. 1974, 59, 1700.
20 ) Criswell, T. R.; Klandermann, B. H. J. Org. Chem. 1974, 59, 770.
21 ) Klemm, I. H.; Kohlik, A. J.; Desai, K. B. J. Org. Chem. 1963, 28,
625.
22 ) Cristol, S. J.; Caspar, M. L. J. Org. Chem. 1968, 55, 2020.
23) Newman, M. S.; Ihrman, K. G. JACS 1958, 80, 3652.
24 ) Lai, Y.-H.; Peck, T.-G. Aust. J. Chem. 1 9 9 2 ,4 5 , 2067.
25 ) Errede, L. A.; Cassidy, J. P. JACS 1960, 82, 3653.
26) Trahanovsky, W. S.; Surber, B. W. JACS 1985, 107, 4995.
27 ) Tsuge, A.; Nago, H.; Mataka, S.; Tashiro, M. J. Chem.Soc. Perkin Trans.
1 1992, 1179.
28) Cheng, S. K. T.; Wong, H. N. C. Synth. Commun. 1990, 20, 3053.
29) Millar, I. T.; Wilson, K. V. J. Chem. Soc. 1964, 2121.
30) De Briijn, P. Compt. Rend. 1950, 257, 295.
31 ) Li, Y. Ph. D. Thesis, Chinese University of Hong Kong, 1993.
32) Hauptmann, S. Chem. Ber. 1960, 95, 2604.
33) Berner, E.; Gramstad, T.; Vister, T. Acta Chem. Scand. 1953, 7, 1255.
34) Staab, H. A.; Meissner, Ü. E.; Meissner, B. Chem. Ber. 1976, 109, 3875.
35) Meissner, U.; Meissner, B.; Staab, H. A. Angew. Chem. Int. Ed. Engl.
93
Results and Discussion
1973, 12, 916.
(3 6 ) Leach, D. N.; Reiss, J. A. Aust. J. Chem. 1 9 7 9 ,5 2 , 361,
(3 7 ) Moradpour, A.; Kagan, H.; Baes, M.; Morren, G.; Martin, R. H.
Terahedron 1 9 7 5 , 5 7 , 2139.
(3 8 ) Laarhoven, W. H.; Peters, W. H. M.; Tinnemans, A. H. A. Terahedron
1978, 34, 769.
(3 9 ) Craig, J. T.; Halton, B.; Lo, S.-F. Aust. J. Chem. 1 9 7 5 ,2 5 , 913.
(4 0 ) Baumgartner, P.; Paioni, R.; Jenny, W. Helv. Chim. Acta 1 9 7 1 , 5 4 , 266.
(4 1 ) Staab, H. A.; Sauer, M. Liebigs Ann. Chem. 1984, 742.
(4 2 ) Sauer, M.; Staab, H. A. Liebigs Ann. Chem. 1984, 615.
(4 3 ) Golden, J. H. J. Chem. Soc. 1961, 3W *
(4 4 ) Akiyama, S.; Misumi, S.; Nakagawa, M. Bull. Chem. Soc. Jpn. 1960, 55,
1293.
(4 5 ) Akiyama, S.; Misumi, S.; Nakagawa, M. Bull. Chem. Soc. Jpn. 1962, 55,
1826.
(4 6 ) Akiyama, S.; Nakagawa, M. Bull. Chem. Soc. Jpn. 1971, 44 , 3158.
(4 7 ) Du Vernet, R, B.; Wennerstrdm, O.; Lawson, J.; Otsubo, T.; Bockelheide,
V. JACS 1978, 700, 2457.
(4 8 ) Badger, G. M.; Campbell, J. E.; Cook, J. W.; Raphael, R. A.; Scott, A. I. J.
Chem. Soc. 1950, 2326.
(49 ) Bergmann, E. D.; Ikan, R. J. Organomet. Chem. 1958, 25 , 907.
(5 0 ) Liu, L.; Yang, B.; Katz, T. J.; Poindexter, M. K. J. Org-
1991, 56, 3769.
(5 1 ) Liu, L.; Katz, T. J. le t. Lett. 19 9 1 ,5 2 , 6831.
(52 ) Newman, M. S.; Me Cleary, C. D. JACS 1941 ,65 , 1537.
(5 3 ) Newman, M. S.; Lord, B. T. JACS 1944, 66, 733.
(5 4 ) Stenlake, J. B.; Waigh, R. D.; Dewar, G. H.; Hughes, R.; Chappie, D. J.;
Coker, G. C. Eur. J. Med. Chem.-Chimica Therapeutica 1981, 16, 515.
(5 5 ) Creighton, T. E. Proteins: Structures and M olecular Properties',
Freeman: NY, 1984.
(5 6 ) Li, T.; Krasne, S. J.; Persson, B.; Kaback, H. R.; Diederich, F. J.
Organomet. Chem. 1993, 58, 380.
(5 7 ) Cochrane, W. P.; Pauson, P. L.; Stevens, T S. J. Chem. Soc. (C) 1968,
630.
94
Chapter 3
Pharmacological Evaluation
Chapter 3
Pharmacological Evaluation
P h a r m a c o l o g i c a l E v a l u a t i o n
G l o s s a r y
Besides the terms given in the glossary on page 9 of this thesis, the
following abbreviations are also used in this chapter:
K j . the inhibition constant, a measure of the ability of a drug to inhibit the
response at a receptor. For practical purposes, Kj and the IC50 , an
empirical measurement of the dose of drug required to effect 50% of
the observed maximal response, are the same.
Hepes - (N -[2 -hyd roxye thy l]p ipe raz ine -N '-[2 -e thanesu lphon ic acid]), used
as a buffer in tissue culture media;
HESS - Hank's Balanced Salt Solution;
U - a measure of enzyme activity: one unit of collagenase hydrolyses 1 pM
of furylacryloyl Leu-Gly-Pro-Ala (FALGPA) per minute at 25 °C at pH 7.5
in the presence of Ca^+ ions;
MEP - molecular electrostatic potential;
V(r) - electrostatic potential at point r.
96
P h arm aco log ica l E valuation
Pharmacological Evaluation
3.1 I n t r o d u c t i o n
JL he sm all-conductance, ca lc ium -activa ted po tass ium channel, SK, has
been found to be widely distributed in excitable cells. Its low conductance
and h igh channel dens ity in m ost m em brane p a tch es , r e su l t in g in
complicated kinetics, has meant that neither the sequence of ion selectivity
nor single channel kinetics are known in detail. However, the current for
which it is responsible, a late after-hyperpolarisation, is readily detectable,
since suppress ion of the large-conductance, ca lc ium -ac t iva ted po tass ium
channel, BK (which is usually present alongside SK), is readily achieved by
the use o f TEA in m illimolar quantities, at which concentra tion SK is
u n a f f e c t e d ^ . The channel has been thus identified in bullfrog sympathetic
g a n g l i o n ^ , cultured cells from rat skeletal muscle^ and m em brane patches
of p r im ary ra t m uscle cu l tu re s^ . The discovery o f apam in and its
apparen tly unique suppression of the AHP current has led to further
d iscoveries . For instance, nanom olar concentrations o f the pep tide can
block this current in both the spinal motorneurones^ and neurones of the
m otor cortex^ of the cat and inhibit the neurotensin induced relaxation of
gu inea-p ig co lon^ .
The presence of apamin binding sites, p resum ed to be apam in-
sensitive potassium channels, in smooth muscle seems to be dependent on
the degree of innervation of the tissue. For instance^, rat m yotubes, known
to contain these channels, when co-cultured with neurones from the rat
spinal cord were found to exhibit action potentials no longer followed by
the AHP. Similarly , an apamin-sensitive AHP was observed in rat leg
muscles two days after transection of the sciatic n« rve^ . It seems that
apamin-sensitive KCs are fully expressed in denervated m ammalian muscle
cells and completely absent in innervated ones, but which factor controls
the expression of this type of channel is not known.
Until the small conductance Ca^'*'-activated KC can be expressed and
th e r e f o r e s e q u e n c e d th ro u g h r e c o m b in a n t D N A t e c h n i q u e s , a ll
inform ation about the channel has of necessity been estab lished through
97
P h arm aco log ica l Evaluation
kinetic measurements or competitive binding studies. Clearly, apamin has
been crucial in this regard and, perhaps more importantly, has provided a
starting point for SAR studies. The first compounds to be studied on this
basis are detailed in Table 3.1 and their structures given in figure 3 .1 . The
firs t colum n of the table lists the d issoc ia tion constan t, Ki of the
recep to r /[^ ^ ^ I] -m ono iodoapam in complex in the presence of the inhibitor,
and the second, the IC50 (that is, the concentration required to effect 50%
inhibition) for angiotensin-evoked K+ loss from guinea-pig hepatocytes
K i (pM ) IC 50 (pM )
Q u in in e 510+78 150
Q u in id in e 340+46 240
Q u in a c r in e 77+11 73
C h lo r o q u in e 140+14 200
P r i m a q u i n e 890+99 970
9 - A m in o a c r id in e 70±10 120
S t r y c h n i n e 180+18 190
A t r a c u r iu m 4.5+0.2 3.0
T u b o c u r a r i n e 7.5±0.7 3.0
P a n c u r o n i u m 6 .8+0 .9 3.5
G a l la m in e 14±2 12
D é c a m é th o n iu m 620±80 450
H e x a m é th o n iu m 760+90 2000
D ib u c a in e 810±100 470
(TEA)+ 5800+1300 7900
B a2+ 14000 _
A p a m in 0.376+0.083x10-3 1.0x10-3
Adapted from ref. 9
T a b le 3.1
Evidently , the greatest degree of block was observed with the three
neu rom uscu lar blockers, atracurium , tubocurarine and pancuronium .
98
P h arm aco log ica l Evaluation
= CHHQ
MeO
quinine(quinidine = dextro isomer)
Me
OMe
MeO
Me
chloroquine
quinacrine
NHz
prim aquine
Os try ch n in e
Q — ( C H 2 ) 2 N E t 3
q(CH2)2NEt3
9-am inoacr id ine
Me3N-(CH2)n"NMe3 ^
NEti
O(CH2)2NEt3
gailamine
hexaméthonium (n = 6)décaméthonium (n = 10) d ib u ca in e
COOMeMe
Me 'N +MeMe
MeCOO
pancuronium
Me OMeMeOH
N +
MeMeO OHtubocurarine
O O
. (CH2)2C0(CH2)50C(CH2)2\'N
atracuriumMeO OMe
F ig u r e 3.1
As p re v io u s ly d i scussed, this abil i ty was though t to c o r re l a t e wi th the
a r r a n g e m e n t o f the two p o s i t i v e ly c h a r g e d n i t r o g e n a t o m s , s p a c e d
99
P h arm aco log ica l Evaluation
approximately 11Â apart in each molecule and was presumed distinct from
their neurom uscular blocking effects. This suggestion was corroborated by
la te r w o rk ^ . D e q u a l in iu m and 3 ,6 - b i s ( p ip e r id o m e t h y l ) p h e n a n th r e n e
d im e th io d id e 2 3 , ne ither of which have an appreciab le neurom uscular
b locking capability, were tested in the same assay described above and
found, particu larly in the form er case, to have a sign ificant b locking
effect (Table 3 .2 ) .
Ki/pM ICso/liM
V e c u r o n iu m 3.6+0.5 4.9+0.3
D e q u a l in iu m 1.1+0.1 1.9+0.1
2 3 9.9+1.3 8.7+1.1
from re fs 9, 10
T a b le 3.2
3.2 P r e p a r a t i o n o f R a t S y m p a th e t i c G a n g l ia
T he choice of compounds such as 3 ,6 -b is (p iper idom ethy l)phenan th rene
d im e th iod ide 23 as suitable for development thus seem ed reasonable and
the consequent syntheses of analogues of this compound are detailed in the
previous chapter. This chapter deals with the results of the assay by which
they were tested for their ability to block SK. Pharm acological data was
collected by Mr P. M. Dunn of the pharmacology department, UCL. This
assay is based on the fact that the AP in rat sym pathetic neurones is
fo llow ed by an apamin-sensitive, SK -m ediated AH P (/. e. the membrane
potential becomes more negative than in the resting ce ll)^^ . This is studied
in single neurones either by using an in tracellu lar m icroelectrode or by
whole-cell patch clamping. Thus seventeen day old Sprague Dawley rats
were k illed by inhalation of a r ising concen tra tion o f nitrous oxide.
S u p e r io r c e rv ica l g an g lia w ere rem o v ed and p la c e d in ic e -c o ld
unsupplemented L-15 medium. The pooled ganglia from 2 to 4 animals were
desheathed and 3 or 4 deep cuts were made in each ganglion using fine
iridectomy scissors. The ganglia were transferred to 4ml Hanks Ca^'*'- and
M g ^ ^ - f re e saline buffered with 10 mM Hepes (pH 7.4) (HBSS), containing
6mg/ml BSA and 372 U/ml collagenase and incubated for 35 minutes at 37
100
P harm aco log ica l E valuation
°C. The ganglia were then incubated for a further 15 minutes in 4 ml HBSS
containing Im g/m l trypsin. The enzyme solution was next rem oved and
enzyme activity stopped by adding 1ml growth medium to the ganglia. The
ganglia were then dissociated by gently passing them 5-10 times through a
fire polished Pasteur pipette. Undissociated pieces of tissue were allowed to
settle out and the supernatan t (conta in ing the ce ll suspension) was
removed, made up to 5 ml and centrifuged at 800 g for 5 minutes. The pellet
was resuspended in 1-2 ml growth medium and d ispensed into prepared
culture dishes containing 1 ml growth medium.
35 mm plastic culture dishes were treated with laminin (10 pg /m l in
HBSS) for 90 minutes then rinsed with HBSS. A glass ring (diameter 13 mm,
height 2 mm) was placed in the centre of each disk (to retain the neurones
in the center of the dish) and 1 ml growth medium was added.
Cells were grown in L-15 medium supplemented with 10% foetal calf
serum, 0.2 mM glutamine, 0.6% (w/v) D-glucose, 0.19% (w/v) N aH C O ],
penicill in (100 U/ml), streptomycin (100 pg /m l) and nerve growth factor
(0.05 pg/ml). Cells were maintained at 37 °C in a humidified atmosphere of
95% O 2 , 5% C O 2 for up to 10 days b e fo re be ing taken for
e le c tro p h y s io log ica l record ing .
Electrical recording was carried out by placing the dishes on the
stage o f an inverted microscope and perfused at a rate of 7 ml/minute with
Krebs solution containing (mM): NaCl, 118; KCl, 4.8; CaCl2 , 4.5; NaHCO], 25;
K H 2P O 4 , 2.28; MgS0 4 , 1.19; glucose, 11; equilibrated with 95% O2, 5% CO2.
Drugs were were applied by perfusing the bath at the required final
c o n c e n t r a t io n . I n t r a c e l lu l a r r e c o r d in g s w e re m a d e w ith g la s s
microelectrodes drawn from 1mm diameter glass tubing, filled with IM KCl
(res is tance 80-120 M Q ) and connected to a bridge balance amplifier to
permit sim ultaneous current in jection and potential recording.
Action potentials were evoked by in jection of 30 ms pulses of
depolarising current at a frequency of 0.2 Hz. The signals were digitised at
1 KHz and averages of 3 or 4 action potentials were obtained before, during
and a f te r drug app lication . The time cou rse o f the d ru g -se n s i t iv e
component of the AHP was determined by subtracting the record obtained
in the presence of the drug from the average of the pre- and post-drug
control records. The reduction in the amplitude of the AHP at the time of
m axim um d iffe rence was exp ressed as a pe rcen tage o f the con tro l
101
P h a rm aco log ica l Evaluation
am plitude at that time point. W here appropriate , this was repeated at
several concentrations and the Hill equation was fitted to the data using an
ite rative least-squares curve-fitt ing routine, providing an estimate of the
IC 50 value.
Obviously, because this assay is different from that originally used to
test the neuromuscular blockers as well as our lead compound, the results
obtained through testing of the new analogues are not strictly comparable,
but the new assay has the great advantage that, unlike the guinea-pig
hepatocyte assay which consists of a suspension o f cells in a physiological
solution, this uses single cells which are thus freely accessible to the drug,
c rea ting no d iffus ion delays. M oreover, several com pounds , including
apamin itself, have been retested on the new assay, providing a basis for
comparison. These values are given in Table 3 .3 .
Ki/|iM IC sq/^M
D e q u a l in iu m 1.1±0.05^^
T u b o c u r a r i n e 11.6±3.3l2 20
A p a m in 3x10-313 3x10-3
T a b le 3.3
A num ber of the compounds were also tested on a separate assay as the
result o f a random screening programme. This consists of a section of
rabbit in tes tine , the jejunum , which contracts spontaneously and whose
a c t iv i ty is in h ib i te d by A T P, b ra d y k in in , n e u ro te n s in and a -
adrenoreceptor agonists. Since these are known to act by opening K(Ca)
channels, the degree of inhibition of contraction by any putative drug is a
measure of its ability to block these channels. Of the three compounds
tested, only one, / r a « j - 4 , 4 ' - b i s [ ( 2 , 5 - d i m e t h y l ) p y r r o l i d o m e t h y 1]s t i l b e n e
dimethiodide, showed reasonable activity, but not of the order to make more
extensive investigation worthwhile. Apamin exhibits an IC50 of 0.92± 0.19
nm in this assay and the results for the three compounds are given in the
appropria te sections below.
102
Pharmacological Evaluation
3 .3 . D e r iv a t iv e s o f P i p e r i d in e , P y r r o l i d i n e a n d M o r p h o l i n e
T T he first group of compounds that were submitted for testing are detailed
in Table 3.4 along with values obtained in the bioassay. The upper figure
refers to the % inhibition of K+ loss at 10 pM whilst the lower is the IC5 0 ,
es tim ated by in terpo la tion of the above figures. The la tter value is
o rd inarily obtained by testing over a range o f concentra tions but the
estimated values reveal these to be too low to make more than one run of
the technically demanding assay viable.
VTOCCr-/
O -C H .
1 4 5
32.1+4.3
20.25pM*
1 3 9
20.4±8.8
37 .OpM*
2 3
8.0+4.0
60.OpM *
C '^ H i
1 4 7
32.9+14.9
20.0pM*
1 4 1
25.1+2.2
29.OpM*
1 3 6
23.8+8.6
30.5pM *
1 4 9
22.0+2.4
33.0pM*
1 4 3
26.7+11.2
27.0pM *
1 3 7
25.7+11.3
28.0pM *
' / C 5 0 value, est im a ted by interpolation
Table 3.4: % Inhibtion at 10 pM of AHP in rat sympathetic neurones for
derivatives of piperidine, pyrrolidine and m orpholine
Figure 3 .2 gives a dose-response curve of the two compounds and 136
against dequalin ium and i llustrates the generally poor activ ity of the
compounds compared to the latter. The most significant detail of table 3 .4
h o w e v e r , is the im p o ten c y o f 3 ,6 - b i s ( p ip e r id o m e th y l )p h e n a n th r e n e
d imethiodide, 23 . This was our lead compound and showed an activity in the
e a rl ie r assay based on gu inea-p ig hepa tocy tes w hich com pared with
dequalinium (see figure 1 .1 7 , Chapter 1). Its drastic loss of activity in this
assay, particularly in light of later results with s im ilar compounds, is
103
Ph arm aco log ica l Evaluation
difficult to ascribe, but in the context of the rather poor activities of all the
compounds listed, is not in itself significant. There are no clear trends in
the table (the estimated values of the IC50 would render these of only
questionable value anyway), but it is interesting to note the slight decrease
in the value for the IC50 in going from the anthracenes (average N-N
distance 12.0 Â) to the phenanthrene (N-N distance 9.5 Â) which accords
with the prem ise o f our project. The figures are o therwise som ew hat
d i s a p p o i n t i n g .
3 .4 . D e r i v a t i v e s o f 2 , 5 - D i m e t h y l p y r r o I i d i n e
T he next group of com pounds to be ana lysed were the (2,5-
d im ethyl)pyrro lidom ethyl analogues of the previously form ed compounds.
As discussed in Chapter 2 , the discovery of this amine was a fortunate
coincidence, but was a nonetheless obvious way of a ltering the steric
p roperties of the com pounds around the n itrogen atom s, the pu tative
pharmacophore. The first of these to be tested, the 2,7-dimethylanthracene
derivative 153 showed a dramatic increase in activity, approaching that of
d equa lin ium (F igure 3 .3 ) . Incidentally , this diagram gives an IC 50 for
dequalinium of 0.75 mM, which was obtained on a different batch of cells
and has been found to be within the experimental error of this assay. This
im provem ent was also observed in the 2 ,6 -d im ethy lan th racene analogue
15 5 . The values obtained in the assay for these two compounds and for the
other derivatives made in the light of these observations are given in Table
3 .5 . The comparable activity of the dimethylanthracene analogues is again
seen and the exponential increase in IC50 going from anthracene through
naphthalene to benzene is again in accord with our initial hypothesis that
activity is in someway dependent on transmolecular distance. The reason
for the marked overall increase in this series of analogues is less obvious
however, but is apparently steric, originating in the a -m e th y l groups of
the p y r ro l id in e r ing ; 2 ,7 -b is (p y r ro l id o m e th y l)a n th ra c e n e d im e th io d id e ,
which merely lacks these methyl groups, is some eighteen times less active.
104
P h arm aco log ica l Evaluation
N-N dist/Â % in h ib " (lOpM) IC5o/|iMMe Me Me Me
C Q O O r j Ô11.9 - 1.1
Me fvie Me
Me 155
12.2 42.8±7.8t 3.2*
Me Me Me 10.0 34.4±3.6t 5.8*
Me Me 159
7.9 32.0 33.0*
Me Me
Me Me 1 6 1
14.2 [45.0] -
Me Me
M e ^ ^ A ^ ^ ^ y U e
^ 163
11.0 51.0 9.2*
^ M e ^ ^ __
Me 151
10.8 40.0 13.5*
Me Me
Me M e 179a
10.0 54.9 8.0*
*estim ated va lu e
t at 3,uM
[ ] va lu e obtained on rabbit jejenu m
Table 3.5: % Inhibition of AHP in rat sympathetic neurones for
derivatives of 2 ,5-dim ethyl pyrrolid ine and 2 ,6-dime thy Ipiperidine^
105
P h a rm a co lo g ica l Evaluation
.o■EJC
X
Me Me100
902 1 *
80
70
60
50Me Me
40I 143 (26pM )
136 (30jiM )
30
20
10
0 L_ 0.01 0.1 101
[ B l o c k e r ] ( u M )
F ig u re 3.2: Dose-response curves of compounds 136 and 143 against
d e q u a l i n i u m
jCC
X
100 MeMe
+ N—(CH2)io —X90153 ( M u M )
2 1 *80
70
60
50
40 Me Me
30Me Me
20
0.01 0.1 1 10
[ B l o c k e r ] ( u M )
F ig u re 3.3: Dose-response curve of compound 153 compared to
d e q u a l i n i u m
106
P h a r m a c o l o g i c a l E v a l u a t i o n
A ssum ing that dequalin ium contorts i t s e l f into a conform ation
w here the term inal qu inald in ium groups are sp a t ia l ly a rranged at a
distance comparable to that in our compounds and that there is negligible
e lec tros ta t ic transmission along the decyl chain, then we may make a
d irec t com parison between the end groups o f the two molecules. The
similarity of the two moieties rests on the substitution around the nitrogen
a tom excep t that one o f the a - m e t h y l g r o u p s in the b is (2 ,5 -
d im ethyO pyrro lido derivative is extended to form part of the ring in
quinald ine (Figure 3 .4 ) .
M e
— N. +
M e
M e
F igure 3.4
O f course , the com parison is not complete s ince the trigonal p lanar
n itrogen atoms of dequalinium mean that the free m ethyl group is co-
p lanar with the quinaldine ring, but the apparent spatial overlap of the
two, together with the fact that the salient guanidinium groups in apamin
also con ta in tetrahedral nitrogen atoms m akes this a likely point of
i n t e r a c t i o n .
3.5 A Com parison of the E lec tros ta t ic P o te n t ia l s
Steric accessibility is always the most crucial aspect of b inding, but most
drugs exert their function via in te rm olecu la r in te ra c t io n s which are
e lec trosta tic in origin. Therefore, for compounds of approx im ate ly equal
steric volume, the molecular electrostatic potential (MEP) can be used to
assess the likelihood of binding to a receptor with affimity for a ligand
whose MEP profile is known^^. It is calculated as the interaction between a
107
P h arm aco log ica l Evaluation
positive unit charge and the molecule at different points around the latter,
using equation 3 .1 :
V(r) = (Z Z a /IR a - rl) - (J p ( r ’)dr7lr’ - rl) eq. 3.1
where V(r) is the electrostatic potential at point r, ( r’) the electron density
function at r ’ and Z& the charge of the atom A found at Ra- By varying the
distance between the unit charge and the molecule, a 3D map is constructed
which serves as an electrostatic “fingerprint” of a compound. The MEPs at
longer d is tances (>3 Â) have been suggested to reflect a “distance
pharm acophore” , essential for aligning the ligand prior to contact, whilst
the M EP at the van der W aal’s contact distance (1.75 Â) constitutes a
“con tac t p h a rm aco p h o re” , requ ired for op tim al b i n d i n g ^ F i g u r e s 3 . 5
gives the 3D MEP diagrams for our most active compounds, viz., 2,6- 155 and
2 ,7 - b is [ (2 ,5 -d im e th y l)p y r ro l id o m e th y l]an th rac e n e d im e th io d id e 153 whilst
figure 3 .6 gives an equivalent diagram of dequalinium . These diagrams,
which were generated on a Silicon Graphics display using the SYBYL
package from TRIPOS associates are based on certain assumptions. Firstly,
the two anthracenes are shown with the methyl groups of the pyrrolidyl
ring cis with respect to each other since this was revealed to be by far the
most abundant isom er after substitution of the d im ethylpyrro lid ine onto
the anthracene nucleus, as previously discussed. Secondly, the two cyclic
methyl groups are themselves anti to the methyl group attached to the
nitrogen atom since the approach of the methyl group from the syn side
would be h ighly d isfavoured . The activity of the two com pounds is
therefore assumed to reside in this configuration, despite the fact that its
purity is not complete (figure 3 .7 ) .
Me/,,
F i g u r e 3.7
The illustated conformation of the two molecules are global energy minima
based on an iterative energy minimisation programme on the COSMIC
108
P h a r m a c o l o g i c a l E v a l u a t i o n
F ig u r e 3.5: MEP Diagrams for 2,7- (top) and 2,6-bis[(2,5-
d im e th y O p y r r o l i d o m e t h y l ] a n th r a c e n e D im e th io d id e .
109
Pharm acological Evaluation
Figure 3.6: MEP Diagram of Dequalinium
110
P h a r m a c o l o g i c a l E v a l u a t i o n
Structure drawing p a c k a g e ^ ^ ^ ^ and these are given in figures 3 .8 and 3 .9 .
As may be expected, these conformations place the ring substituents on
opposite sides with respect to the aromatic ring plane, and the diagrams
give views of the molecules through and perpendicular to the ring plane.
Thirdly, given the large number of degrees of freedom of the decyl
chain of dequalinium, generation of an energy minimised conformation is
not m eaningful. Therefore, an arbitrary conform ation which places the
pair of endocyclic, quaternary nitrogen atoms some 13.5 Â apart has been
used as the basis of the MEP diagram. It is also assumed that dequalinium
remains unprotonated at physiological pH.
Inspection of the diagrams reveals im portant d ifferences between
the essentially indistinguishable hourglass shaped MEP diagrams of the two
anthracenes and the much less uniform diagram of dequalin ium . The
isopotentia l shells of the diagrams are co lour coded according to the
following scheme: red, 20.0; orange, 15.0; yellow 10.0; green, 5.0 kcal mol"^,
c o r r e s p o n d in g to p e rp e n d ic u la r d is ta n c e s from the m o le c u le of
approximately 1, 2, 4 and 7 Â. The transections of the isopotential contour
maps of the two anthracenes are roughly 3 Â above the aromatic ring
plane whilst that of dequalinium is the same distance above the axis of the
decyl chain, which, in this diagram, is linear. The diagrams reveal the
d isrup tion to the e lec trosta tic p rofile that has occurred around the
terminal moieties as a result of the aromatic ring and the para amino
group. Given that these views represent distance pharm acophores for the
compounds, it seems reasonable to conclude, on a purely qualitatitive basis,
that their initial contact with the receptor will be of a d ifferent type
because of the extensive delocalisation in the quinaldine ring system of
dequalinium . W hether the nature of the binding is u ltim ately different
once the ligands have docked is not of course possible to predict but since
MEPs are a representation of what the receptor actually "sees", the
poss ib i li ty exists that this is the case. This hypo thes is could not
u n fo r tu n a te ly be c o n f i rm e d by the te s t in g o f 2 ,6 - and 2 ,7-
b is (q u in a ld in o m e th y l)an th ra c e n e s ince , as d isc u sse d in the p rev ious
chapter, these compounds could not be synthesised. Their synthesis would
have allowed an assessment of the degree to which areas of the molecules
111
P h a r m a c o l o g i c a l E v a l u a t i o n
F ig u r e 3.8: Energy-Minimised Conform at ion of 2 ,7-Bis[(2,5-
d im ethy Opy rro l idom ethyl ] an th ra cene D im e th io d id e 1 5 3
112
P h a r m a c o l o g i c a l E v a l u a t i o n
Figure 3.9: En er gy -M in im ised Con fo rm at ion of 2 ,6-B is[ (2 ,5
d im e th y l ) p y r ro l id o m e th y l ] a n th r a cen e D im e t h i o d id e 1 5 5
113
P h arm aco log ica l Evaluation
remote to the putative pharmacophores were contributing to secondary
binding, but in the absence of this information, it is assumed that the major
determinant o f binding ability o f the 2,5-dim ethyl pyrrolido derivatives is
steric, originating in the area around the charged centres. If, as the weight
of evidence suggests, it is ch iefly the interaction o f two nitrogen atoms
with a receptor site in the base o f well-defined pocket which determines
the degree o f block of apamin and analogous compounds, then it is easy to
understand that the substitution pattern around these atoms w ou ld be
c r u c ia l .
3 .6 . 2 , 6 - B i s [ ( c i 5 - 2 , 6 - d i m e t h y l ) p i p e r i d o n i e t h y 1]
n a p h t h a l e n e D i m e t h i o d i d e
A n obvious modification to the compounds given in Table 3 .5 would be
expansion o f the heterocyclic ring v i a the use o f 2,6-dimethylpiperidine.
T his co m p o u n d has the ad vantage that it m ay be p u rch ased
configurationally pure, thus preventing the formation o f isom ers upon
quaternisation o f the nitrogen atom, a process d iscussed in Chapter 2 .
H ow ever , 2 ,6 - b i s [ ( c i j - 2 ,6 - d im e t h y l )p ip e r id o m e th y l ] -n a p h th a le n e 1 7 9 was
the only member o f this series synthesised due to a persistent problem
whereby the salt o f the amine was formed exclusively in the reaction. The
comparable activity o f this compound to its pyrrolidomethyl analogue 1 5 7
(see table 3 . 5 ) suggests that our assumption that the activity o f the latter
compound rests in the most abundant isomer and not in one o f the minor
isomers, is correct. However, the increased steric volume o f the substituted
piperidine, together with the fact that the a -m e th y l groups are more
proximate to the nitrogen atom mean that the approach o f the cationic
centres to the receptor is occluded and this presumably explains the small
drop in the value o f the IC5 0 . Though one ob v iou sly cannot draw firm
conclusions from a single compound, this result is enough to suggest that
the change in the values o f the IC5 0 of the other compounds in this series,
had they yielded to synthesis, would not have been dramatic.
114
P harmaco log ica l Evaluat ion
3.7 Derivatives of DABCO and Q uin uc l id ine
O ubsVitotion of DABCO onto the hydrocarbons gave a series of compounds
with markedly different electronic and steric profiles from those previous.
Most significantly, there are now two further basic nitrogen atoms in the
molecules, which we considered significant given the high basicity of the
guanidiniorn moiety of arginine (pKa is estimated to be about 12) and the
high charge it consequently carries at physiological pH. As discussed in
Chap ter 1, there is presumably an effect due to this contributing to the
action of apamin at its binding site since there is no pharmacological effect
observed when the two arginine residues o f the peptide are substituted
with ornithine (in which the guanidine group is replaced by the much less
basic amino group).
% inhibition IC50 (pM)
at lOpM
gross' 0 532Br' 166
y
grcoug?2 B f 167
11.4
[ 12]*
47
* Value obtained on rabbit jejenum
T ab le 3.6: % Inhibition of AHP in rat sympathetic neurones
by derivatives of DABCO
Assuming, therefore, that the second set of nitrogen atoms of the DABCO
moieties are also protonated in vivo then the use of this amine provides a
way of introducing this increased charge, despite the fact that it is not
delocalised as in arginine. Five compounds were synthesised in this series,
but the results with the first two compounds tested indicated that there was
little value in further application of the d ifficult assay (table 3 .6 ) ; the
small reduction in the IC50 between the benzene and the naphthalene
derivatives suggested that the values for the anthracenes would not be
significantly higher, and given their low values anyway, it was apparent
115
Pharmaco log ica l Evaluat ion
that a modified approach was needed. Therefore, two sets of compounds
were syn thes ised in which the res idual bas ic ity o f the b is-D A B C O
compounds was removed by: (i) replacement of the second nitrogen atom
with carbon to give derivatives of quinuclidine, lacking the excess charge
of the previous analogues; (ii) substitution at the nitrogen atom to give a
set o f com pounds which re ta ined this charge but su rrounded by a
lipophilic environm ent. The first series, form ed by the substitu tion of
quinuclidine instead of DABCO onto the bis-bromomethyl compounds, gave
a set o f compounds that are in all respects but one the same as the bis-
DABCO derivatives above. This assumes that, in the former series, the second
nitrogen atom makes a discrete contribution to the electronic profile of the
m olecule since there is unlikely to be significant transm ission along the
aliphatic bridges which connect it to the rest o f the molecule (and as
previously discussed, the disposition of the nitrogen atom places its lone
pair away from the m olecule , and quatern isa tion does little to affect
m o lecu la r s truc ture). Use o f qu inuclid ine therefo re p rov ides a d irec t
measure of the contribution of this atom to the binding efficiency of the
bis-substituted DABCO derivatives. The second set of analogues are a pair of
compounds formed by substitution of a hydrocarbon onto the bis-DABCO
deriva tive of p-xylene to give an extended te traca tion ic species with
several degrees of freedom and alternating areas of hydrophilic ity and
lipophilic ity . The high overall charge of the m olecule would seem to
m it iga te ag a in s t op tim al b ind ing e ff ic iency s ince the re is a lso a
hydrophobic contribution to the binding of apamin. It has further been
suggested that the inability of certain peptide analogues to mimic the
blocking action of the dicationic neurom uscular blocking agents initially
cited by Cook and Haylett was because they were too polar in comparison to
the latter which have a large, hydrophobic spacer between the two ionic
c e n t r e s ^ . It was hoped, however, that a ba lance betw een the two
properties would be achieved in our compounds, as had been done in the
structurally sim ilar nucleotide carriers of Diederich^^. The values obtained
for the IC50 in the rat ganglion assay for both types of compound are given
in table 3 .7 . The importance of the steric contribution to to binding is
further underlined by the near-complete inactivity of the bis-DABCO and
N-substituted bis-DABCO derivatives given in tables 3 .6 and 3 .7 . Since the
second nitrogen atom of the DABCO moiety will be protonated also at
116
Pha rm aco log ica l Evaluat ion
physiological pH (pKa for DABCO = 8.8 and 3.0), all these compounds are
te tracation ic and therefore have electrostatic profiles which have certain
elements in common with dequalinium.
% inhib ition
at lOpM
IC50 (pM )
2 B f 1731 1 .0 ± 8 4 7
2 B f 1742 1 .9 ± 2 3 3
2 B f 1 7 55 3 .0 ± 6 8 .8
- -
” 3*= 2 B f 1 7 11 5 ± 1 1 4 0 .5
2 B f , 2 r
1 7 2
2 3 ± 1 0
[ 6 *]
3 0 .5
Values for % inhibition are given as the average of 3 - 5 experim ents
* % inhibition o f ATP responses in rabbit jejunum at lOpM
Table 3.7
Unlike the previous compounds however, they all lack branching at the 2-
positions o f the rings and are considerably more ste r ica lly congested
around the putative pharm acophore. Given that the 3D geom etry of a
structure is only a necessary, but not sufficient, cause of activity (since
e lectronic and hydrophobic forces are, as d iscussed, also required for
117
Phar maco log ica l Evaluat ion
response), this would seem to suggest that the nature of the binding of
dequa lin ium and our pa ir o f active m olecu les is, at least part ia l ly ,
electrostatically distinct. W hether these results further suggest that these
bindings are qualitatively different or indeed, occurs at distinct areas of
the receptor, is too early to say, however.
Though becom ing a l i t tle rem oved from our in it ia l rem it, the
com pounds 180 and 181 (figure 3 .1 0 ) would have provided an interesting
extension of the concept that lay behind the the synthesis o f the previous
compounds. Again, the overall charge of the molecule is high, certainly
rendering the molecule too polar to cross the b lood-b ra in ba rr ier for
instance, but it was reasoned that the presence o f the cationic centres over
a relatively wide area coupled with an enhanced flexibility would provide
an opportunity for any two of them to position them selves over the
receptor site in the optimum position for block.
Me
_ Me
3 B r , 31 *
Me 180
M e
6Br*
181
Me"
Figure 3.10
Unfortunately, synthesis of the compounds was not achieved in the time
available, but compounds of the type shown in figure 3 .1 0 are the subject
of continued investigation by other workers.
118
Pharmacological Evaluation
3.8 T he E f fe c t o f N i t r o g e n - N i t r o g e n D is ta n c e
Our original hypothesis, based on the observation by Cook and Haylett, was
that, in the absence of m odula ting fac tors , then the t ran sm o lecu la r
distance between the two nitrogen atoms in our compounds was of singular
im portance. Leaving aside the e lectronic and steric con tr ibu tions which
are inferred as equally important, it is helpful to assess the extent to which
the h y p o th es is holds. V alues for these in te rc a t io n ic d is tan ces are
essentia lly unaffected by the nature o f the subs ti tuen t and though the
early m easurem ents which suggested the su itab il i ty o f our com pounds
were from Dreiding models, the values quoted in table 3 .8 were obtained on
the CO SM IC structure drawing package. It is assum ed that d iffe ren t
substituents on the hydrocarbon fram ework will have only a neglig ible
effect on the transm olecular distance and so the m easurem ents , which
were taken as the average of the ten lowest energy conformations of the
b is[(2 ,5 -d im ethy l)py rro lidom ethy l] analogues, are quo ted as genera l for
each hydrocarbon spacer.
Average N-N distance/Â
3,6-dimethylphenanthrene 9.5
1,6-dimethyIphenanthrene 10.8
fraAij-4,4'-dimethylstilbene 142
rranj-2,4’-dimethylstiIbene 11.0
2,6-dimethylnaphthalene 10.0
2,7-dimethylanthracene 11.9
p-xylene 7.9
2,6-dimethylanthracene 12.2
Table 3.8
U nfortuna te ly , the limited set o f data means that not all c lasses o f
compound can be assessed for this correlation but the results for two sets of
com pounds are given below. The first, the de riva tives of pyrro lid ine ,
provide an excellent corroboration of the hypothesis, despite the limited
activity of the compounds (figure 3 .1 1 )
119
Pha rm aco log ica l Evaluat ion
B is (p y rro l id o m eth y l) d e r iv a t iv e s
y = - 21.341 + 4.8384X - 0 .22283x^2 R^2 = 1 .0005.0
■ Column 24.9 -
oU
4 . 8 -
4 . 7 -
4 . 6 -
4.510 1 1 12 1 39
N-N distance
F i g u r e 3 .11
These da ta p red ic t tha t op tim um b in d in g o f b i s (p y r ro l id o m e th y l )
substituted compounds will occur when the distance betw een the nitrogen
atoms is approxim ate ly 10.8 Â. S im ilarly , a p lo t o f the ca lcu la ted
transmolecular distances versus -log IC50 for our three b is(p iperidom ethy l)
substitu ted compounds (figure 3 .1 2 ) provides a figure of the same order,
approximately 11 Â. These diagrams provide reasonable evidence of the
veracity of the hypothesis in some cases, but the poor activity of the six
compounds above underlines the fact that it cannot be used to predict
absolute or even relative activity; obviously, two cationic nitrogen atoms
are not in themselves a sufficient constituent of the pharmacophore.
120
oU
y =
Phar maco log ica l Evaluat ion
B is (p ip e r id o m eth y l) d e r iv a t iv e s 41.725 + 8.5448% - 0.39049x^2 R ^2 = 1.000
5.0 ■ Colum n 2
4.9
4.8
4.7
4.6
4.5
4.4
4.3
4.29 1 0 1 1 1 2 13
y =
N -N distance
F igu r e 3.12
B is (2 ,5 -D im eth y lp y rro l id o m e th y l) D e r iv a t iv e s
71.330 + 23.094X - 2.3196x''2 + 7.7457e-2x^3 R' 2 = 0.7206
■ Column 2
5
48 97 10 1 1 1 2 1 3
olo
00o
N -N distance
F i g u r e 3 .1 3
121
P h a r m a c o l o g i c a l E v a l u a t i o n
In the case of our most active set of compounds, the derivatives of 2,5-
dimethylpyrrolidine, the contribution of these atoms to the activity and its
correlation with their relative disposition becomes even more tenuous and
a s im ple second order polynom ial function cannot be constructed that
gives an optimum value for the transmolecular distance (figure 3.13 g i v e s
the third order function which exhibits a m inim um at an N-N distance
corresponding to 10.6 Â). The efficacy of these compounds in blocking SK
is c learly m itiga ted by factors o ther than the d is tance betw een the
nitrogen atoms which is in accord with the generally accepted view of the
nature of the binding of apamin to its receptor^
3.9 . C o n c l u s i o n
L is quite clear from this limited data set that a pair of cationic nitrogen
atoms is a useful starting point for construction of compounds with SK
agonistic properties. However, the only lim ited success with a set of
compounds synthesised using this as the sole criterion implies that the
steric contribution to the binding of apamin mimics is greater than this
simple theory would suggest. The fact that dequalin ium , apparently the
most potent non-peptide blocker of the small conductance, C a^"^ -ac tiva ted
KC so far discovered^ ^ , and 2 ,7 - b i s [ ( 2 ,5 - d im e th y l ) p y r r o l id o m e th y l ) ]
an th racene d im eth iod ide 1 5 3 , which has an activity approaching that of
dequalinium, share a degree of steric crowding around the supposed active
site and that, in the absence of these substituents, the activity of the latter
is decimated, gives an indication of a new site of synthetic intervention.
3 .1 0 R e f e r e n c e s
(1 ) Romey, G.; Lazdunski, M. Biochem. Biophys. Res. Commun. 1 9 8 4 ,7 7 5 ,
669.
(2 ) Pennefather, P.; Lancaster, B.; Adams, P. R.; Nicoll, R. A. Proc. Natl.
Acad. Sci. USA 1984, 52, 3040.
(3 ) Blatz, A. L.; Magleby, K. L. Nature 1 9 8 6 ,5 2 5 , 718.
122
P h a r m a c o l o g i c a l E v a l u a t i o n
(4 ) Zhang, L.; Krnjevic, K. Neurosci. Lett. 1987 , 74, 58.
(5 ) Szente, M. B.; Baranyi, A.; Woody, C. D. Brain Res. 1 9 8 8 ,4 6 7 , 64.
( 6 ) Hugues, M.; Duval, D.; Schmid, H.; Kitabgi, P.; Lazdunski, M.; Vincent,
J. P. Life Sci. 1982, 31, 437.
(7 ) Schmid-Antomarchi, H.; Renaud, J.-F.; Romey, G.; Hugues, M.; Schmid,
A.; Lazdunski, M. Proc. Natl. Acad. Sci. USA 1982, 82, 2188.
( 8 ) Castle, N. Ph. D. Thesis, UCL, 1987.
(9 ) Cook, N. S.; Haylett, D. 0 . Br. J. Pharmacol. 1983, 546P.
(1 0 ) Cook, N. S.; Haylett, D. G. J. Physiol. 1 9 8 5 ,5 5 5 , 373.
(1 1 ) Dunn, P. M. Eur. J. Pharmacol. 1994, 252 , 189.
(1 2 ) Kawai, T.; Watanabe, M. Br. J. Pharmacol. 1986 87, 225 .
(1 3 ) Dunn, P. M., Personal Communication.
(1 4 ) Hogberg, T. H.; Norinder, U. In A Textbook o f Drug Design and
D eve lo p m en t', P. Krogsgaard-Larsen and H. Bundgaard, Ed.; Harwood:
Chur, 1991.
(1 5 ) Vintner, J. G.; Davis, A.; Saunders, M. R. J. Comp. Aid. Mol. Design
1987, 7, 31.
(1 6 ) Morley, S. D.; Abraham, R. J.; Haworth, I. S.; Jackson, D. E.; Saunders,
M. R.; Vintner, J. G. J. Comp. Aid. Mol. Design 1991, 5, 475.
(1 7 ) Demonchaux, P.; Ganellin, C. R.; Dunn, P. M.; Haylett, D. G.; Jenkinson,
D. H. Eur. J. Med. Chem. 1991, 26, 915.
(1 8 ) Li, T.; Krasne, S. J.; Persson, B.; Kaback, H. R.; Diederich, F. J. Org.
Chem. 1 9 9 3 ,5 5 , 380.
123
Chapter 4
Exper im en ta l
E x p e r i m e n t a l
4. Experimental
4.1 A p p a r a t u s a n d R e a g e n t s
Chemical reagents were purchased from Aldrich Chemical Co., Lancaster,
Fisons and BDH. Solvents were purified by standard m ethodologies. All
experim ents using water sensitive reagents were carried out under an
a tm osphere of dry nitrogen.
M icroanalysis samples were prepared by d ry ing in vacuo at room
temperature over silica gel. The analyses were carried out by Jill Maxwell
or Alan Stones in the Microanalytical Section of the Chemistry Department,
U niversity College London, with a Perk in -E lm er 2400 CHN Elem ental
A n a ly s e r .
Melting points were determined on a Reichert melting point apparatus.
Both melting and boiling points are uncorrected.
U ltrav io le t (UV) spectra were recorded on a Shim adzu UV 160A
u ltrav io let spectrometer using quartz cells of 1 cm pathlength and in
either absolute ethanol or deionised water as indicated.
Proton nuclear magnetic resonance H nmr) spectra were recorded on
a Varian VXR-400 (400 MHz) or VXR-200 (200 MHz) spectrometer. Chemical
shifts (6 ) and coupling constants (J) are reported in ppm and Hz,
respectively. The spectra were recorded in deuterochloroform (CDCI3 ) or
d im e th y lsu lp h o x id e -d 6 (d^-DMSO) solution. Residual protic solvent ie. CHCI3
( 5 h = 7.26 ppm) or CD3 S O C D 2 H (§ h = 2.52 ppm) was used as internal
reference. The following abbreviations are used in signal assignments: s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad).
Carbon nuclear magnetic resonance (^ ^C -nm r) spectra were recorded at
100 MHz on a Varian VXR-400 spectrometer. Signals are reported as 5 values,
using the resonances of CDCI3 (5 c 77.0 ppm, t) or (CD3 )2 SO (5 c 39.7 ppm,
hep tup le t) as reference. W here ind ica ted , the signal m u ltip lic i ty was
determined by an APT pulse sequence.
Mass spectra were recorded on a VG7070H mass spectrom eter with
Finnigan Incos II data system at University College London, or on a VG ZAB-
2F (EIMS) or VG12-250 (Cl) mass spectrometer at the London School of
Pharm acy . A ccurate Mass de te rm ina tions were made on a V acuum
125
E x p e r i m e n t a l
Generators VG ZAB SE mass spectrometer at the London School of Pharmacy.
For analytical thin layer chromatography (TLC) Merck Kieselgel 60 F 2 5 4
plates were used. Compounds were visualised by ultra-violet light or by
heat development using a p-anisaldehyde-based (350 ml 95 % EtOH, 12 ml
conc. H 2 S O 4 , 8 ml p -an isa ldehyde , 6 ml g lacial acetic acid) s ta in ing
preparation . Column chrom atography was perform ed using M erck flash
silica gel 60 (200-400 mesh) or Sorbsil C60-A (40-60 pm) flash silica.
Apart from the numbering used for assignm ent o f nmr signals the
nam ing and num bering of com pounds th ro u g h o u t the e x p e r im e n ta l
section adheres to Chemical Abstract nomenclature.
4.2 E x p e r i m e n t a l
1. P r e p a r a t i o n of 3 , 6 - D i m e t h y l p h e n a n t h r e n e 44
CH
7’r a / i 5'-4 ,4 '-dimethylstilbene (1.00 g, 4.80 mmol) and iodine (0.10 g,
0.39 mmol) were dissolved in warm cyclohexane (1 litre) then this was
allowed to cool and the turbid solution filtered. The mixture was irradiated
under UV light for 24-48 hours, washed with N a 2 S 2 0 3(aq) and the solvent
removed in vacuo giving a pale brown solid which was chrom atographed
(hexane 67-70 °C) to give the phenanthrene as white flakes (0.20 g, 0.97
mmol, 20%), m. p. 143-145 °C (lit. 146 ° C \ 142 °C^, 142-143 °C^).
126
E x p e r i m e n t a l
2. P r e p a r a t i o n o f 2 , 4 , 4 ' - t r i m e t h y l b e n z o p h e n o n e 59 (b )
CHCH
This was prepared according to the literature method"^ giving, after
distillation, a very pale-yellow oil in 64% yield, b.p. 138-140 °C/0.07 mm Hg
(lit. 169 °C/4 mm Hg).
3. P r e p a r a t i o n O f 2 , 7 - d im e th y l a n t h r a c e n e 60(b)
Dry 2 ,4 ,4 '-trimethylbenzophenone (10.00 g, 44.59 mmol) was heated
to gentle reflux using a W o o d ’s M etal bath and m aintained at this
temperature for 4-6 hours. The precipitate which formed upon cooling of
the ketone was filtered off and washed lightly with E t2 0 to give a brown
solid, then the filtrate was evaporated and returned to reflux. This was
repeated four or five times. The combined precipitates were then vacuum
sublimed at approximately 100 °C to give yellow plates and needles that
consisted of a mixture of the anthracene and the anthrone (1.57 g). This
was reduced directly by adding the unseparated mixture (0.50 g) to sodium
borohydride (0.50 g, 13.21 mmol) in ‘PrOH, then refluxing for 24-48 hours.
The reaction mixture was poured into a slurry of ice (200 ml) made acidic
by the addition of a few drops of conc. HCl, then the yellow precipitate (0.46
g) was filtered off, dried and column chromatographed (hexane) to give the
highly crystalline anthracene (0.089 g, 0.43 mmol, 1% ) m.p. 230-235 °C (lit.
241 °C" , 240-243 °C^, 238-239 °C^).
127
E x p e r i m e n t a l
4. P r e p a r a t i o n o f 2 ,5 ,4 - T r i m e t h y I b e n z o p h e n o n e 5 9 (a )
CH CH
This was prepared according to the literature method^ producing the
title benzophenone as a yellow oil in 11% yield. This crysta llised on
standing and was used without further purification m.p. 4 9 -51°C (lit. 54 °C).
5. P r e p a r a t i o n o f 2, 6 - D i m e t h y l a n t h r a c e n e 6 0 (a )
This was prepared from 2 ,5 ,4 '-trim ethylbenzophenone (18.00 g, 87.3
mmol) using the same method as described in experiment 3, giving a crude
yield of 4.10 g. This an thracene/anthrone m ixture (0.23 g) was then
suspended in *PrOH (25 ml) with sodium borohydride (0.23 g, 6.21 mmol) and
heated to reflux for 24 hours. The product was collected as before, and
recrystallised from hexane (67-70 °C), giving the product as pale-yellow
flakes (0.1085 g, 0.53 mmol, 1%), m. p. 228-230 °C (lit. 230-235 250 °C"^).
6 . P r e p a r a t i o n o f 3 , 6 - B i s ( b r o m o m e t h y l ) p h e n a n t h r e n e 104
Br Br
3,6-Dimethylphenanthrene (1.20 g, 5.81 mmol) was heated to reflux
128
E x p e r i m e n t a l
in e c u (15 ml) with N-bromosuccinimide (2.03 g, 11.40 mmol) and benzoyl
peroxide (36 mg) for 6 hours. Filtration, followed by removal of the solvent
in vacuo produced a pale-yellow powder which was recrysta llised from
ethanol to give the dibromide as an off-white powder (1.73 g, 4.75 mmol,
82%), m. p. 160-162 °C (lit. 173-175 °C^).
7. P r e p a r a t i o n of 2 , 7 - B i s ( b r o m o m e t h y l ) a n t h r a c e n e 110 (b)
2,7-D im ethy lan th racene (0.30 g, 1.45 mmol), N -b rom osucc in im ide
(0.50 g, 2.80 mmol) and benzoyl peroxide (10 mg) were heated to reflux in
C C I 4 for 3 hours. The solvent was removed in vacuo and the yellow solid
obtained was recrystallised from EtOH, giving the dibromide as a yellow
powder (0.1778 g, 5.24 mmol, 34%), m. pt. 190-198 °C (lit. 202-204 °C^).
8 . P r e p a r a t i o n of 2 , 6 < B i s ( b r o m o m e t h y l ) a n t h r a c e n e 1 1 0 ( a )
2 ,6 -D im ethylan thracene (0.10 g, 0.48 mmol), N -b rom osucc in im ide
(0.17 g, 0.95 mmol) and benzoyl peroxide (5 mg) were heated to reflux in
C C I 4 (20 ml) for two hours. The solution was filtered, evaporated in vacuo
and recrystallised from 'PrOH, giving the dibromide as a yellow powder
(49.7 mg, 0.14 mmol, 28%), m.p. 186-188 °C.
nmr: 4.70 (s, 2H, H D ; 7.49 (d, 2H, ^ j ^ . h = 7.7Hz, H3,7);
7.97 (s, 2H, Hl,5); 7.98 (d, 2H, ^ J ^ .h = 8.0Hz, H4,8);
8.37 (s, 2H, H9,10)
^^C nmr 35.7; 125.6; 126.8; 127.5; 127.9; 130.4; 131.1; 134.8
129
E x p e r i m e n t a l
Mass Spectrum (m/z): 366 [(C i6H i 2* 'B r 2)+, 11%]; 364
[ (C i6H i 2*‘B r79Br)+, 22%]; 362 [ (C if iH i2’ ®Br2>+,
11%]; 285 [(C i6H i 2^^Br)+, 100%]; 283
[(C if iH i2™Br)+, 99%]; 204 [ ( C ie H n ) * , 27%]; 203
[ (C l6H i i )+ , 33%]; 202 [(C ieH io )* , 43%]
UV (EtOH): "max = 265.5nm; 8 = 67760
Accurate Mass: Expected for C i ^ H i i B r ] , 362.9302
Found 362.9305
9. P r e p a r a t i o n of 3 , 6 - B i s ( p i p e r i d o m e t h y l ) p h e n a n t h r e n e 134
3,6-Bis(brom om ethyl)phenanthrene (0.10 g, 0.27 mmol) was heated
to reflux in ethanol (5 ml) then piperidine (0.05 g, 0.06 ml, 0.60 mmol),
made up to 1 ml in ethanol, was added dropwise. After reflux for four hours,
the mixture was diluted with Et20 (20 ml) then washed with 10% H C l(aq)
(2x20 ml). The aqueous layer was removed and the product precipitated by
the careful addition of NH](aq). The product was extracted with Et2 0 (3x2 0
ml), d r ied (M gS 0 4 ) then evaporated to give an orange oil which was
triturated with E t2 0 to give a pale orange powder. After washing with cold
petroleum spirit (b.p. 80-100 °C), the title amine was obtained as a white
powder (26.4 mg, 0.07 mmol, 26%), m.p. 108-110 °C.
200MHz iR nmr (CDCI3): 1.45 (m, 4H, H51; 1.63 (m, 8H, H4'); 2.47 (m, 8H,
H3');3.74 (s, 4H, H r ) ; 7.59 (dd, 2H, = 8.1Hz,
4Jh -h = 1.5Hz, H2,7); 7.68 (s, 2H, H9,10); 7.82 (d, 2H,
^JH-H = 8.1 Hz, H l ,8); 8.60 (d, 2H, ^Jh-H = 0.64Hz,
130
E x p e r i m e n t a l
nmr (CDCI3):
H4,5)
24.4; 25.9; 54.6; 64.3; 123.2; 126.3; 128.0; 128.3; 130.0;
131.3; 136.4
Mass Spectrum (m/z): 372 [M'*', 12%]; 288 [(M - Cs H iqN)'^, 27%]; 206
[(C i 6 H i 4)+, 100%); 204 [(C i6H i2)+ , 75%]
UV (EtOH): Xmax = 255.0nm; 8 = 103480
Accurate Mass: Expected for C26H 32N 2, 372.2565
Found, 372.2561
10. Preparat ion of 3,6- Bis( morph ol in ome thy I) p h e n a n t h r e n e 135
M orpholine (0.05 g, 0.05 ml, 0.60 mmol) was treated with 3,6-
b is (b rom om ethy l)phenan th rene (0.10 g, 0 ..27 m m ol) as d esc r ibed in
experim ent 9 , giving a clear oil This was triturated with E t2 0 , giving the
title amine as white crystals (20.0 mg, 0.05 mmol, 20%) m. p. 105-109 °C.
200M Hz iH nmr (CDCI3); 2.53 (m, 8H, H41; 3.7Zf-(m, 8H, H31; 3.77 (s, 4H,
H D ; 7.61 (dd, 2H, ^ J ^ .h = S.2Hz, ^Jh-H = 14Hz,
H2,7); 7.70 (s, 2H, H9,10); 7.84 (d, 2H, = 8.2Hz,
H l ,8); 8.60 (s, 2H, H4,5)
nmr (CDCI3 ): 53.8; 64.0; 67.0; 123.1; 126.4; 127.9; 128.6; 130.0;
131.4; 136.1
Mass Spectrum (m/z): 377 [(M H)+, 100%]; 292 [(M - C4H gN0 )+, 15%];
131
E x p e r i m e n t a l
UV (EtOH):
Accurate Mass
206 [(Ci 6 H i 4)+, 10%]; 204 [(C i6H i2)+ , 4%]
^max - 255.Onm; £ = 81410
Expected for C24H 29N 2O 2, 377.2229
Found, 377.2224
11. P r e p a r a t i o n of 3 , 6 - B i s ( p y r r o l i d o m e t h y l ) p h e n a n t h r e n e 133
Pyrrolid ine (0.04 g, 0.05 ml, 0.60 mmol) was treated with 3,6-
b is (b ro m o m eth y l)p h en an th ren e (0 .10 g, 0.27 m m ol) as d esc r ib ed in
experim ent 9. After work-up and trituration with E t2 0 , the title amine was
obtained as pale brown crystals (23.70 mg, 0.07 mmol, 25%) m. p. 95-98 °C.
200 MHz iH nmr (CDCI3): 1.81 (m, 8H, H41; 2.59 (m, 8H, H3'); 3.88 (s, 4H,
m y , 7.60 (dd, 2H, 3Jh_h = 8.1Hz, ^ J ^ .h = 1.5Hz,
H2,7); 7.67 (s, 2H, H9,10); 7.83 (d, 2H, =
8.0Hz,Hl,8); 8.63 (s, 2H,H4,5)
13c nmr (CDCI3): 23.4; 54.2; 61.1; 123.1; 126.4; 127.8; 128.6; 130.1;
131.4; 136.7
Mass Spectrum (m/z): 34.1k[ M+, 10%]; 2^6- [(M - 9 N)+, 27%]; 206
[(C i6H i4)+ , 100%]; 204[(Ci6H 12)+, 74%]
UV (EtOH): ^max = 254.0nm; e = 109110
Accurate Mass: Expected for C24H 29N 2, 345.2331
Found, 345.2330
132
E x p e r i m e n t a l
12 . P r e p a r a t i o n o f 6 - M e t h y I - 2 , 4 - d i o x o - 3 , 1 0 - d i o x a t r i c y c I o
[5 .2 .1 .0 i*5]dec-7-ene 126
Me
This was prepared according to the literature method^®, giving off-
white crystals in 77% yield, m.p. 58-59 °C (lit. 59-63 °C).
13. P r e p a r a t i o n o f 3 - M e t h y l p h t h a l i c a n h y d r i d e 127
Me
C o m p o u n d 126 (50.00 g, 0.28 mol) was crushed to a fine powder and
added portionwise to vigorously stirred sulphuric acid (500 ml) held at -2
°C. The temperature of the reaction was raised to 10°C, then the mixture was
poured onto ice (1.5 kg). The yellow precipitate which formed was collected
and washed with ice water, then dissolved in chloroform (100 ml). The
aqueous layer was discarded and the organic layer dried (M gS0 4 ) and
evapora ted in vacuo to give a sticky brown solid which was recrystallised
twice from ^PrOH to give large white needles (6.05 g, 0.04 mol, 14%), m.p.
115-118 °C (lit. 10 117-119 °C).
133
E x p e r i m e n t a l
14. P r e p a r a t i o n of 2 - ( 2 - M e t h y l b e n z o y l ) - 6 - m e t h y l b e n z o i c ac id 73
Me OH
This was prepared according to the literature method^®, except 3-
methylphthalic anhydride (5.00 g, 0.031 mol) was added to a solution of o-
tolyl magnesium bromide (c. 1 M in Et2 0 ). This produced an orange powder
which was dissolved in Et20 (50 ml) and washed with 10% HCl(aq) (50 ml).
The organic layer was then dried (M gS0 4 ) and the solvent removed in
v a c u o to give a pale yellow powder (3.68 g, 48%), m. p. 113-115 °C (lit.^®
116-118 °C)
15. Preparation of 3,6 - B i s ( p i p e r i d o m e t h y l ) p h e n a n t h r e n e dimethiodide 23
N++ N-
MeMe 6 '
3 ,6 -B is(p ip e r id o m eth y l)p h en an th ren e (15.0 mg, 0 .04 mmol) was
stirred under N2(g) with iodomethane (0.12 g, 0.05 ml, 0.87 mmol) in freshly
distilled, dry acetonitrile (1 ml) for 24 hours. After pouring into Et2 0 (400
ml), the pale yellow precipitate was collected by filtration and dried (20.0
mg, 0.03 mmol, 76%), m.p. >230°C.
nmr (CDCI3): 1.68 (m, 4H, H51; 1.92 (m, 8H, H41; 2.95 (s. 6H,
H61; 3.43 (m, 8H, H3'); 4.85 (s, 2H, H T); 7.34 (d, 2H,
3Jh-H = 8.0Hz, H2,7); 7.54 (s, 2H, H9,10), 7.68 (d, 2H,
3 Jh -h = 8.1Hz, H l ,8); 8.55 (s, 2H, H4,5)
134
E x p e r i m e n t a l
13c nmr (CDCI3): 19.0; 20.8; 42.8; 60.3; 63.6; 125.6; 128.2; 128.4; 129.6;
129.9; 131.1; 132.4
Mass spectrum (m/z): 529 [(M + I)+, 6%]; 303 [(M - C5H 10N - CH])""", 16%];
204 [(C i6H i 2)+, 100%]; 201 [M2+, 35%]
UV (EtOH):
Analysis (%):
^max = 255.0nm; e = 82150
Calculated for C28H 38N 2I2. C: 51.23; H: 5.83; N: 4.27
Found, C: 50.65; H: 6.14; N: 3.99
16. P r e p a r a t i o n o f 3 , 6 - B i s ( p y r r o l i d o m e t h y l ) p h e n a n t h r e n e
d i m e t h i o d i d e 136
+ .N'
Me
3,6 -B is(py rro l idom ethy l)phenan th rene (20.0 mg, 0 .06 m m ol) was stirred under N 2 (g ) with iodomethane (0.16 g, 0.07 ml, 1.16 mmol) in
acetonitrile (1 ml) for 48 hours. The white precipitate which formed in this
time was removed by filtration, washed with Et2 0 and dried in vacuo, giving
the title salt as a white powder (18.2 mg, 0.06 mmol, 50%) m. p. >230 °C.
nmr (d^-DMSO):
13c nmr (d^-DMSO):
1.68 (m, 8H, m y , 2.52 (s, 6H, H5'); 2.99 (m, 4H,
H3aO; 3.23 (m, 4H, H3sO; 4.35 (s, 4H, HU); 7.38 (d,
2H, 3 J h .h = 8.1Hz, H2,7); 7.55 (s, 2H, H9,10); 7.69
(d, 2H, 3 J h .h = 8.1Hz, H l ,8); 8.63 (s, 2H, H4,5)
20.9; 47.6; 63.0; 65.8; 127.6; 127.7; 127.9; 129.3; 129.5;
130.7; 132.5
135
E x p e r i m e n t a l
Mass Spectrum (m/z): 501 [(M + I)+, 12%]; 359 ((M - CH3)+, 2%]; 289 [(M
C4H iiN )+ , 11%]; 274 [(M - C4H 11N - CH3)+, 3%];
204 [(Ci6H|2)+.15%]; 187 [M2+, 7%]
UV (H2O):
Analysis, %:
^max = 255.0nm; £ = 64620
Calculated for C26H 34N 2I2. C: 49.70; H:5.45; N: 4.46
Found, C: 49.33; H: 5.74; N: 4.19
17. P r e p a r a t i o n of 3, 6 - B i s ( m o r p h o l i n o m e t h y l ) p h e n a n t h r e n e
d i m e t h i o d i d e 137
+ N-
5 Me Me
3,6-B is(m orpholinom ethyl)phenanthrene (15.0 mg, 0.04 mmol) was
quaternised as described in experiment 15 using iodomethane (0.16 g, 0.07
ml, 1.16 mmol). The reaction mixture was then poured into dry Et2 0 (100 ml)
and the resultant precipitate was removed by filtration to give the title salt
as a white powder (20.0 mg, 0.03 mmol, 76%) m. p. >230 °C.
I r nmr (dg-DMSO): 2.00 (m, 8H, H41; 2.67 (s, 6H, H5'); 3.51 (m, 8H, H31;
4.46 (s, 4H); 7.37(d, 2H, = 8.1Hz, H2,7); 7.56
(s,2H, H9,10); 7.69 (d, 2H, = 8.1Hz, H l ,8); 8.57
(m, 2H, H4,5)
13c nmr (d6-D M S0): 45.3; 58.9; 60.0; 125.8; 128.0; 128.5; 129.3; 129.4;
131.3; 132.6
Mass Spectrum (m/z): 533 [(M + I)+, 12%]; 405 [(M - H)"*", 2%]; 391
[(M - CH3)+, 11%]; 305 [(M - C 4 H n N 0 ) + , 10%];
288[(M - C4H 11NO - CH3)+, 5%]; 204 [(C i6H i 2)+,
136
E x p e r i m e n t a l
UV (H2O):
Analysis (%):
48%]; 202 [M^+, 7%]
Imax = 255.5nm; E = 68940
Cklculatedfor C26H 34N2O 2I2, C: 47.29; H: 5.19; N:4.24
Found: C: 46.57; H: 5.29; N: 3.88
18. P r e p a r a t i o n o f 2 , 6 -B is ( p ipe rid om e t h y l ) a n t h r a c e n e 138
2,6-Bis(bromom ethyl)anthracene (0.10 g, 0.27 mmol) was heated to
reflux in ethanol (5 ml) then piperidine (0.05 g, 0.06 ml, 0.60 mmol) in
ethanol (1 ml) was added dropwise. Reflux was continued for four hours
then the mixture was worked up as described in experiment 9 to give a pale
yellow powder (54.1 mg, 0.15 mmol, 54%), m.p. 184-188 °C.
1h nmr (CDCI3):
nmr (CDCI3):
1.43 (m, 4H, H5'); 1.59 (m, 4H, H4'); 2.42 (m, 4H,
H31; 3.64 (s,4H, H I ') ; 7.46 (dd, 2H, ^j ^ . h = 8.7Hz,
4Jh -h = 1.4Hz, H3,7); 7.83 (s, 2H, Hl,5); 7.92 (d, 2H,
3Jh.h = 8.7Hz, H4,8); 8.32 (s,2H, H9,10)
24.4; 25.9; 54.6; 64.1; 125.6; 127.3; 127.4; 127.9; 131.2;
131.4; 135.3
Mass Spectrum (m/z): 373 [(M + H)+, 100%]; 290 [(M - C5HioN)+, 8%]; 206
[ (C i 6H i 4)+, 5%]
UV (EtOH): ^max “ 258.Onm, £ — 110640
Accurate Mass: Expected for C26H 33N 2, 373.2644
Found, 373.2640
137
E x p e r i m e n t a l
19. P r e p a r a t i o n o f 2 , 6 - B i s ( m o r p h o l i n o m e t h y l ) a n t h r a c e n e 142
2,6-Bis(bromom ethyl)anthracene (0.10 g, 0.27 mmol) was treated as
experim ent 17 with morpholine (0.05 g, 0.05 ml, 0.60 mmol). Work up as
before gave a yellow oil which was triturated with Et2 0 to give pale yellow
crystals (55.7 mg, 0.15 mmol, 55%), m.p. 204-206 °C.
nmr (CDCI3): 2.51 (br s, 8H, H4'); 3.67 (s,4H, H I ') ; 3.73 (m,8H,
H3'); 7.48 (dd, 2H, 3j h _h = 8.7Hz, = 12Hz,
H3,7); 7.84 (s,2H, Hl,5); 7.94 (d, 2H, 3J h .h = 8.7Hz,
H4,8); 8.33 (s,2H, H9,10)
13c nmr (CDCI3): 53.7; 63.7; 63.8; 125.6; 125.7; 127.1; 127.6; 128.2;
131.3; 131.4
Mass Spectrum (m/z): 377 [(M + H)+, 100%]; 292 [(M - C4H 8NO + 2H)+,
13%]; 206 [(Ci 6H i 4)+, 7%]
UV (EtOH): Xmax - 258.0; £ = 25000
Accurate Mass: Expected for C24H 28N 2O 2» 376.2150
Found 376.2155
20 . P r e p a r a t i o n o f 2 , 6 - B i s ( p y r r o l i d o m e t h y l ) a n t h r a c e n e 140
138
E x p e r i m e n t a l
2,6-Bis(bromom ethyl)anthracene (0.10 g, 0.27 mmol) was treated as
described in experiment 17 with pyrrolidine (0.04 g, 0.05 ml, 0.60 mmol).
Work up as before gave the title amine as an off-white powder (47.8 mg,
0.14 mmol, 51%), m.p. >230 °C (dec.).
nmr (CDCI3); 1.83 (br s, 8H. H4'); 2.64 (br s, 8H, H31; 3.84 (s,4H,
H I ') ; 7.50 (d, 2H, 3 J h .h = 8.6Hz. H3,7); 7.89 (s, 2H,
H I,5); 7.94 (d, 2H, = 8.6Hz, H4,8); 8.34 (s, 2H,
H9,10)
nmr (CDCI3): 23.4; 54.2; 60.8; 125.8; 127.1; 127.3; 128.3; 131.2;
131.5; 135.2
Mass Spectrum (m/z): 345 [(M + H)+, 100%]; 276 [(M - C4H 8N + 2H)+, 5%];
206 [(Ci6Hi4)+. 6%]
UV (EtOH): Xmax - 258.0nm; £ = 141840
Accurate Mass: Expected for C24H 29N 2, 345.2331
Found 345.2334
21. P r e p a r a t i o n of 2,6- B is ( p ip e r id o m e th y 1)a n t h r a c e n e d im e th io d id e 139
Me
2,6-B is(p iperidom ethyl)an thracene (40.0 mg, 0.11 mol) was stirred
under N 2(g) with iodomethane (0.32 g, 0.14 ml, 2.32 mmol) in acetonitrile (1
ml) for four days. The mixture was then poured into dry E t2 0 and the
resultant precipitate was filtered and dried, giving the title salt as a white
powder (33.4mg, 0.05 mmol, 47%), m.p. >230 °C.
139
E x p e r i m e n t a l
nmr (dg-DMSO):
nmr (d^-DMSO):
1.53 (br m, 2H, H5a'); 1.63 (hr m, 2H, H5s'); 1.92
(br m, 8H, H4'); 3.02 (s, 6H, H6 '); 3.39 (m. 8H, H3');
4.79 (s,4H, H r ) ; 7.65 (d, 2H, = 8.8Hz, H3,7);
8.27 (d. 2H, 3Jh _h = 8.7Hz, H4,8); 8.34 (s, 2H, Hl,5);
8.78 (s, 2H, H9,10)
19.4; 20.9; 43.1; 60.0; 63.4; 125.7; 127.3; 128.9; 129.3;
131.2; 131.3; 134.1
Mass Spectrum (m/z): 529 [(M + I)+, 10%]; 401 [(M - H)+, 2%]; 387 [(M
C H 3)+, 3%]; 303 [(M - C e H n N ) ^ 22%]; 288 [(M -
C6H 13N - CH3)+, 6%]; 204 [ (C iô H n )^ , 100%]
UV (H2O):
Analysis (%):
Xmax = 260.0nm; 6 = 230790
Calculated for C28H 38N 2I2, C: 51.23; H: 5.83; N: 4.27
Found, C: 49.86; H: 5.97; N: 4.15
2 2 . P r e p a r a t i o n o f 2 , 6 - B l s ( m o r p h o l i n o m e t h y l ) a n t h r a c e n e
d i m e t h i o d i d e 143
Me
2 ,6 -B is(m orpho linom ethy l)an th racene (40.0 mg, 0.11 m m ol) was
treated with iodomethane (0.32 g, 0.14 ml, 2.32 mmol) as experiment 20. This
produced the title salt as a pale yellow powder (34.0 mg, 0.05 mmol, 48%),
m.p. >230 °C.
nmr (d^-DMSO): 2.49 (m, 8H, H41; 3.16 (s, 6H, H51; 4.01 (m, 8H,
H31; 4.89 (s, 4H, HU); 7.67 (d, 2H, 3Jh_h = 8.7Hz,
140
E x p e r i m e n t a l
nmr (d^-DMSO):
H3,7); 8.28 (d, 2H. 3Jh_h = 8.8Hz, H4,8); 8.37 (s, 2H,
H I ,5); 8.78 (s, 2H, H9,10)
45.2; 58.8; 59.9; 67.9; 125.2; 127.4; 129.0; 129.3; 131.2;
131.3; 134.3
Mass Spectrum (m/z): 533 [(M + I)+, 17%]; 405 [(M)+; 1%]; 391 [(M -
C H 3)+; 12%); 305 [(M - C5H n N O ) + , 37%]; 288 [(M
C 5H 11NO - CH3>+ 5%]; 204 [(C i6H i 2)+, 95%]; 202 [
M2+, 11%]
UV (H2O):
Analysis (%):
^max — 260.5nm; 6 = 240500
Calculated for C26H34N2O2I2. C: 47.29; H: 5.19; N:4.24
Found: C: 46.38; H: 5.33; N: 3.92
23 P r e p a r a t i o n of 2, 6- B i s ( p y r r o l i d o m e t h y l ) a n t h r a c e n e d im e th io d id e 141
Me
2 ,6 -B is (p y r ro l id o m e th y l )a n th ra c e n e (40 .0 mg, 0 .12 m m ol) was
treated with iodomethane (0.32 g, 0.14 ml, 2.32 mmol) as described in
experiment 20. This produced the title salt as a pale yellow powder (23.8 mg,
0.04 mmol, 32%), m.p. >230 °C.
nmr (dg-DMSO): 2.18 (m, 8H, H40; 2.98 (s, 6H, H5'); 3.47 (m, 4H,
H 3 a l; 3.68 (m, 4H, H3s'); 4.77 (s, 4H, HU); 7.68 (dd,
2H, 3j h _h = 9.2Hz; = 13Hz, H3,7); 8.27 (d, 2H,
3Jh -h = 8.7Hz, H4,8); 8.37 (s, 2H, Hl,5); 8.77 (s, 2H,
H9,10)
141
E x p e r i m e n t a l
nmr (d^-DMSO): 20.9 (C4?); 47.5; 63.0; 65.4; 127.0; 127.3; 128.8; 129.1;
131.26; 131.33; 133.4
Mass Spectrum (m/z): 501 [(M + I)+, 28%]; 359 [(M - CH3>+, 12%]; 289 [(M
- C5H i i N)+, 34%]; 274 [(M - C 5H nN -C H 3)+ 26%];
204 [(Ci 6H i 2)+, 100%]; 202 [M2+, 10%]
UV (H2O):
Analysis, %:
^max - 258.0nm; e = 192510
Calculated for C26H 34N 2I2. C: 49.70; H:5.45; N: 4.46
Found, C: 48.92; H: 5.55; N: 4.39
24 . P r e p a r a t i o n o f 2 , 7 - B i s ( m o r p h o l i n o m e t h y l ) a n t h r a c e n e 148
2 ,7-B is(b rom om ethy l)an th racene (0.10 g, 0.27 m m ol) was reacted
with morpholine (0.05 g, 0.05 ml, 0.60 mmol) using the method described for
2 ,6 -b is (b ro m o m e th y l)a n th ra ce n e in ex p e r im en t 18 . This gave an orange
gum which was triturated with E t2 0 to give the title amine as a yellow
powder (34.8 mg, 0.09 mmol, 34%), m.p. 177-180 °C.
nmr (CDCI3):
13c nmr (CDCI3):
2.54 (m, 8H, H4'); 3.71 (s, 4H, H I ') ; 3.76 (m, 8H,
H3'); 7.50 (dd, 2H, = 8.7Hz, ^j ^ . h = 1.5Hz,
H3,6); 7.87 (s, 2H, Hl,8); 7.96 (d, 2H, 3Jh _h = 8.7Hz,
H4,5); 8.34 (s, IH, H9); 8.38 (s, IH, HIO)
53.7; 63.7; 66.9; 125.6; 125.8; 127.0; 127.7; 128.3;
131.1; 131.6; 135.0;
Mass Spectrum (m/z) 377 [(M + H)+, 100%; 292 [(M - C 4H gN 0)+ , 6%]; 206
[(Ci 6Hi4)+, 2%]
142
E x p e r i m e n t a l
UV (EtOH): Xmax = 256.Onm; £ = 154730
Accurate Mass: Expected for C24H 29N 2O 2, 377.2229
Found, 377.2233
25 . P r e p a r a t i o n o f 2 , 7 - B i s ( p y r r o l i d o m e t h y l ) a n t h r a c e n e 146
2 ,7-B is(b rom om ethy l)an th racene (0.10 g, 0.27 m mol) was trea ted
with pyrrolidine (0.04 g, 0.05 ml, 0.60 mmol) in ethanol (15 ml) using the
m ethod described for 2 ,6-b is(brom om ethyl)an thracene in experim en t 19 .
Work up as before produced a yellow oil which was triturated with E t2 0 to
give the title amine as a pale yellow powder (58.9 mg, 0.17 mmol, 63%), m.p.
182-184 °C.
iH nmr (CDCI3): 1.83 (m, 8H, H4'); 2.61 (m, 8H, H3'); 3.87 (s, 4H,
HU); 7.56 (dd, 2H, % _ H = 8.6Hz, 4Jh_h = 1.2Hz,
H3,6); 7.94 (dd, 2H, = 8.5Hz, ^ J ^ .h = 1.2Hz,
H4,5); 8.35 (s, 3H, HI, H8, H9); 8.38 (s, IH, HIO)
13c nmr (CDCI3): 23.5; 54.2; 61.1; 126.6; 127.2; 128.8; 128.8; 130.6;
130.9; 131.5; 138.1
Mass Spectrum (m/z): 345 [(M + H)+, 20%]; 284 [(M - C4HgN)+, 13%]; 205
[(C i6H i3 )+ , 7%]
UV (EtOH): ^max - 262.0nm; £ = 81470
Accurate Mass: Expected for C24H29N 2, 345.2343
Found, 345.2331
143
E x p e r i m e n t a l
26 . P r e p a r a t i o n o f 2 , 7 - B i s ( p l p e r i d o m e t h y l ) a n t h r a c e n e 14 tf.
2 ,7 -B is(b rom om ethy l)an th racene (0.10 g, 0 .27 m m ol) was reacted
with piperidine (0.05 g, 0.06 ml, 0.60 mmol) using the method described for
2 ,6 -b is (b ro m o m e th y l)a n th ra ce n e in expe rim en t 1 7 . Upon cooling of the
reac tio n m ix tu re , f ine w hite needles o f an ac id so lu b le m ate r ia l
p rec ip ita ted and these were filtered off, washed with cold e thanol and
dried, giving the title amine, (54.2 mg). Partial evaporation of the filtrate
provided a further crop of material (6.9 mg, 0.16 mmol, 61% total yield) m.p.
166-168 °C.
nmr (CDCI3): 1.46 (m, 4H, H5'); 162 (m, 8H, H4'); 3.71 (s, 4H,
HU); 2.45 (m, 8H, H 307.56 (dd, 2H, ^ j ^ . h = 9.1Hz,
% - H = 1.3Hz, H3,6); 7.93 (d, 2H, ^ j ^ . h = 8.6Hz,
H4,5); 8.33 (s, 3H, HI, H8, H9); 8.37 (s, IH, HIO)
nmr (CDCI3): 24.4; 26.0; 54.6; 64.2; 121.5; 126.5; 126.7; 127.3; 128.6;
130.5; 131.5; 137.9
Mass Spectrum (m/z) 345 [(M + H)+, 19%]; 284 [(M - C4HgN)+, 14%]; 205
[(Ci 6H i 3)+. 8%]
UV (EtOH): ^max - 261.5nm; £ = 135850
Accurate Mass: Expected for C26H 33N2 , 373.2641
Found 373.2644
27. P r e p a r a t i o n of 2 ,7 - B i s ( m o r p h o l i n o m e t h y l ) a n t h r a c e n e d im e th io d id e 149
144
E x p e r i m e n t a l
Me
21'
2 ,7 -B is(m orpho linom ethy l)an th racene (35.2 mg, 0 .09 m m ol) was
stirred with iodomethane (0.12 g, 0.06 ml, 0.88 mmol) in acetonitrile (2 ml)
for 24 hours. After pouring the reaction m ixture into dry E t2 0 , the
resultant precipitate was removed by filtration and dried, to give the title
salt as a white powder (36.2 mg, 0.05 mmol, 59%) m.p. >230 ®C.
nmr (d^-DMSO): 2.49 (m, 8H, H4'); 3.15 (s, 6H, H5'); 4.01 (m, 8H,
H30; 4.89 (s, 4H, HK); 7.68 (d, 2H, ^ J ^ .h = 8.3Hz,
H3,6); 8.26 (d, 2H, = 8.1Hz, H4,5); 8.39 (s, 2H,
H l ,8); 8.76 (s, IH, H9); 8.79 (s, IH, HIO)
nmr (dg-DMSO): 43.6; 58.8; 59.9; 67.6; 124.9; 128.90; 128.94; 129.0;
130.6; 130.9; 131.8; 134.4
Mass Spectrum (m/z): 407 [(M + H)+, 2%]; 391 [M - CH3)+, 22%]; 305 [(M -
C 5 H iiN 0 )+ , 10%]; 288 [(M - C5H 11NO - CH3)+, 4%];
204 [(C i6H i 2)- . 23%]
UV (H2O):
Analysis (%):
%max = 259.0nm; £ = 123190
Calculated for C26H34N 2O 2I2» C: 47.29; H: 5.19; N:4.24
Found; C: 46.67; H: 5.29; N: 4.05
145
E x p e r i m e n t a l
28. P r e p a r a t i o n of 2 ,7 -B i s ( p y r r o l i d o m e t h y 1)a n t h r a c e n e d im e t h i o d i d e 147
, Me Me
21'
2,7-Bis(pyrrolidomethyl)anthracene (40.0 mg, 0.12 mmol) was stirred
under N 2(g) in acetonitrile (1 ml) with iodomethane (0.32 g, 0.14 ml, 2.32
mmol) for 48 hours. The mixture was then poured into Et2 0 (100 ml) and the
resultant precipitate filtered and dried, giving the title salt as a white
powder, (11.9mg, 0.02 mmol, 16%) m.p. >230°C.
H nmr (dg-DMSO): 2.19 (m, 8H, H4'); 3.00 (s, 6H, H5'); 3.50 (m, 4H,
H3a'); 3.69 (m, 4H, H 3 s l ; 4.90 (s, 4H, HI ); 7.78 (dd,
2H, 3Jh_h = 8.8Hz, 4Jh_h = 1.3Hz, H3,6); 8.35 (d, 2H,
3 Jh -h = 8.8Hz, H4,5); 8.68 (s, 3H,H3,8,9); 8.91 (s,
IH, HIO)
nmr (d^-DMSO): 20.8; 47.3; 63.0; 65.3; 122.8; 127.7; 129.30; 129.33;
129.7; 130.0; 132.1; 132.3
Mass Spectrum (m/z): 359 [(M - CH3)+, 1%]; 289 [(M - C4H 8N - % ) + , 2%];
206 [(C i6H i 4)+, 6%]; 85 [(C4H 8N + CH3)+, 100%]; 70
[(C4H 8N)+, 25%]
UV (H2O):
Analysis, %:
^max = 260.5nm; £ = 134680
Calculated for C26H 34N 2I2. C: 49.70; H:5.45; N: 4.46
Found, C: 48.34; H: 5.54; N: 4.17
146
E x p e r i m e n t a l
29. P r e p a r a t i o n of 2 , 7 - B i s ( p i p e r i d o m e t h y l ) a n t h r a c e n e d i m e t h i o d i d e 145
Me
21"
2,7-Bis(piperidomethyl)anthracene (40.0 mg, 0.11 mmol) was stirred
with iodomethane (0.16 g, 0.07 ml, 1.16 mmol) in acetonitrile (1 ml) for 48
hours then poured into dry Et2 0 (100 ml). The resultant precipitate was
then filtered and dried, giving the title salt as an off-white powder (64.0
mg, 0.10 mmol, 91%) m. p. >230°C.
nmr (dg-DMSO): 1.75 (m, 4H, H50; 1.94 (m, 8H, H4'); 3.05; (s, 6H,
H60; 3.48 (m, 8H, H3'); 4.93 (s, 4H, HI ); 7.76 (d,
2H, = 8.8Hz, H3,6); 8.34 (d, 2H, ^ J ^ .h = 8.9Hz,
H4,5); 8.67 (s, 3H, Hl,8,9); 8.92 (s, IH, HIO)
nmr (d^-DMSO): 19.4; 20.8; 28.4; 46.2; 60.0; 122.8; 127.6; 128.0; 129.6;
129.76; 129.80; 132.3; 132.9
Mass Spectrum (m/z): 387 [(M - CH3)+, 10%]; 303 [(M - C5H 10N - CH3)+,
5%]; 206 [(C :6H i 2)+, 45%]; 201 [M2+, 5%] 99
[(C5H 10N + CH3)+, 98%]
UV (H2O):
Analysis (%):
A«max “ 260.Onm; 6 — 121400
Calculated for C28H 38N 2I2» C: 51.23; H: 5.83; N: 4.27
Found, C: 50.87; H: 6.01; N: 4.12
147
E x p e r i m e n t a l
30. P r e p a r a t i o n o f 2 , 7 - B i s [ ( 2 , 5 - d i m e t h y l ) p y r r o l i d o m e t h y l ] a n t h r a c e n e 152
Me
Me Me'
2 ,7-B is(brom om ethyl)an thracene (0.10 g, 0.27 mmol) was heated to
reflux in EtOH (10 ml) then 2,5-dimethylpyrrolidine (0.06 g, 0.07 ml, 0.60
mmol) was added. Reflux was continued for one hour then the solvent
vo lum e was halved and w ater carefu lly added to the m ixture until
precipitation just began to occur. After cooling and standing overnight, a
pale yellow precipitate formed which was removed by filtration. This solid
was then dissolved in 10%HCl(aq) and precipitated by the careful addition
of aqueous amm onia solution. Extraction of this precipita te with E t2 0
fo llowed by drying (M gS0 4 ) and vacuum removal of the solvent gave the
title amine as a yellow powder (62.4 mg, 0.16 mmol, 58%), m.p. 142-147 °C.
200 MHz 1 h nmr (CDCI3): 1.05 (d, l OHz, H5c'); 1.08 (d, 6 H, ^ j ^ . h =
1.0 Hz, H5tO; 1.42 (m, 4H, H4'); 1.82 (m, 4H, H41;
2.70 (m, 4H, H3 ); 3.95 (s, 4H, H r ) ; 7..53 (dd, 2H,
3 Jh -H = 8.2Hz, 4 jh _ h = l .lH z, H3,6); 7.90 (d, 2H,
3JH_H = 8.6Hz, H4,5); 8.34 (s, 3H, Hl,8,9); 8.36 (s,
IH, HIO)
13c nmr (CDCI3): 20.8; 31.4; 56.5; 60.5; 126.2; 126.46; 126.50; 127.8;
128.1; 128.2; 130.5; 130.4
Mass Spectrum (m/z): 401 [(M + H)+, 22%]; 304 [(M - C6H 12N + 2H)+,
16%]; 205 [(C i6H i 3)+. 12%]
UV (EtOH) ^max - 262.5nm; £ = 81260
Accurate Mass: C 28H 36N 2 requires 400.2879
Found 400.2873
148
E x p e r i m e n t a l
31. P r e p a r a t i o n o f 2 , 7 - B l s [ ( 2 , 5 - d i m e t h y l ) p y r r o l i d o m e t h y l ] a n t h r a c e n e
d im e th iodide 153
Me5 MeMe
Me Me
21*
2 ,7 -8 is [ (2 ,5 -d im e th y l)p y rro l id o m e th y l] an th racene (35 .0 mg, 0 .09
mmol) was stirred with iodomethane (0.25 g, 0.11 ml, 1.77 mmol) in
acetonitrile (1 ml) for 24 hours. After pouring into E t2 0 (400 ml), the
resultant precipitate was allowed to settle, then the ether was decanted and
the m oist solid carefully dried in vacuo to give the title salt as yellow
powder (7.4mg, 0.01 mmol, 12%), m.p. >230 °C.
nmr (d^-DMSO):
13c nmr (d6-D M S0):
1.43 (d, 12H, 3Jh . h = 6.5Hz, H5'); 168 (m, 4H, H4');
2.08 (m, 4H, H4'); 2.79 (s, 6H, H6'); 3.63 (m, 4H,
H31; 4.88 (s, 4H, H I ') ; 7.68 (d, 2H, ^j ^ . h = 8.6Hz,
H3,6); 8.34 (d, 2H, = 8.7Hz, H4,5); 8.62 (s, 3H,
Hl,8,9); 8.92 (s, IH, HIO)
13.4; 26.5; 36.3; 60.7; 67.6; 125.5; 128.1; 129.3; 129.6;
130.0; 130.7; 132.3; 132.5
Mass Spectrum (m/z): 557 [(M + I)+, 4%]; 401 [(M - CH3)+, 5%]; 317 [(M
C 6H 12N - CH3)+, 15%; 215 [M2+, 5%] ; 204
[(Ci6Hi2)+. 100%]
UV (H2O):
Analysis (%):
Im ax = 262.0nm; e = 135320
Calculated for C28H 42N 2I2. C: 52.64; H; 6.19; N:4.09
Found C: 51.02; H: 6.31; N: 3.87
149
E x p e r i m e n t a l
32. P r e p a r a t i o n of 2 , 6 - B i s [ ( 2 , 5 - d i m e t h y l ) p y r r o l i d o n i e t h y l ] a n t h r a c e n e 154
5 'Me
Me
Me
Me
2,6-Bis(bromom ethyi)anthracene (0.10 g, 0.27 mmol) was treated as
described in experiment 30 . Reflux was maintained for two hours then the
reaction was allowed to cool overnight, producing a precipita te of pale
yellow plates. These were filtered off, washed with cold ethanol and dried,
giving the title amine (53.8 mg, 0.14 mmol, 50%), m. p. 142-146 °C.
nmr (CDCI3): 1.07 (d, 3Jh _h = 2.3Hz, HSc'); 110 (d, 6H, HSt'); 1.43
(hr s, 4H, H40; 1.87 (m, 4H, H4'); 2.70 (m, 4H, H3');
3.91 (s, 4H, H r ) ; 7.45 (dd, 2H, 3Jh _h = 8.7Hz, 4Jh . h =
1.6Hz, H3,7); 7.85 (s, 2H, Hl,5); 7.91 (d, 2H, =
8.5Hz, H4,8); 8.34 (s, 2H, H9,10)
nmr (CDCI3): 20.4; 31.0; 55.8; 60.4; 125.7; 127.5; 127.75; 127.82;
131.2; 131.4; 135.0
Mass Spectrum (m/z); 401 [(M + H)+, 100%]; 304 [(M - C6H 12N + 2H)+,
7%]; 206 [(Ci 6 H i 4)+. 4%]
UV (EtOH): Xmax = 258.0nm; E = 175300
Accurate Mass: C 28H 36N 2 requires 400.2879
Found 400.2876
150
E x p e r i m e n t a l
33. P r e p a r a t i o n of 2 , 6 - B i s [ ( 2 , 5 - d i m e t h y l ) p y r r o l l d o m e t h y l ] a n t h r a c e n e
d im eth io d id e 155
5 MeMe
Me
N +
Me MeMe21*
2 ,6 -B is [ (2 ,5 -d im e th y l )p y r ro l id o m e th y l ]a n th ra c e n e (0 .03 g, 0 .075
mmol) was treated as described in experiment 31 . The reaction mixture was
then poured into dry Et2 0 and the resultant precipitate filtered, giving the
title salt as a white powder (47.3 mg, 0.07 mmol, 92%), m. p. >230 °C.
nmr (d^-DMSO):
nmr (d^-DMSO):
1.43 (d, 12H, = 6.5Hz, H5'); 1.68 (m, 4H, H4');
2.07 (m, 4H, H4'); 2.78 (s, 6H, H6'); 3.65 (m, 4H,
m y , 4.76 (s, 4H, H I ') ; 7.58 (dd, 2H, = 8.8Hz,
4Jh -h = 1.3Hz, H3,7); 8.26 (d, 2H, = 8.9Hz,
H4,8); 8.31 (s, 2H, Hl,5); 8.80 (s, 2H, H9,10)
13.5; 26.4; 36.3; 60.8; 67.4; 125.7; 127.27; 127.29;
129.1; 131.1; 131.2; 133.7
Mass Spectrum (m/z): 557 [(M + I)+, 28%]; 429 [(M - H)+, 5%]; 401 [(M
CH3)+, 3%]; 317 [(M - C 6H i2N )+ , 35%]; 302 [(M -
C6H 12N - CH3)+, 8%], 215 [M2+, 15%]
204 [(C i 6 H i 2)+, 100%]
UV (H2O):
Analysis (%):
A-max ~ 260.Onm, £ — 239340
Calculated for C30H 42N 2I2, C: 52.64; H: 6.19; N:4.09
Found, C: 51.76; H: 6.37; N: 3.87
151
E x p e r i m e n t a l
34 . P r e p a r a t i o n of 2 , 6 - B l s ( b r o m o m e t h y l ) n a p h t h a l e n e 130
2,6-Dimethylnaphthalene (0.30 g, 1.92 mmol) was heated to reflux in
C C I4 (20 ml) with benzoyl peroxide (15.7 mg) and N-bromosuccinimide (0.68
g, 3.81 mmol) for one hour. Filtration of the reaction mixture followed by
evaporation gave a light-sensitive, yellow solid which was recrysta llised
from ethanol to give the title dibromide as white crystals (0.22 g, 0.61 mmol,
32%), m.p. 101-103 °C.
nmr (CDCI3): 4.64 (s, 4H, H I'); 7.50 (dd, 2H, = 8.3Hz,
H = 1.7Hz, H3,7); 7.79 (d, 2H, ^ J^ .h = 8.5Hz, H4,8);
7.80 (s, 2H, Hl,5)
13c nmr (CDCI3): 33.8 ( C r ); 127.4 (Ct); 127.7 (Q ); 128.8 (Q);
132.8 (Cq); 135.9 (Cq)
Mass Spectrum (m/z): 316 [(C i2H i o * ‘B r2)+, 19%]; 314
[ (C i2H i o ’ ^B r8 'B r)+ , 40%]; 312 [ (C i2H io ' '^ B r 2)=
20%]; 235 [(C12H io**Br)+, 99%]; 233
[(Ci2Hio'79Br)+, 99%]; 154 [ (C ,2H io )+ . 100%]
UV (EtOH): ^max = 227.Onm; £ = 79490
Accurate Mass: Expected for C i 2H io B r2 311 .9158
Found 311.9149
152
E x p e r i m e n t a l
35. P r e p a r a t i o n of 2 , 6 - B i s [ ( 2 , 5 - d i m e t h y l ) p y r r o l i d o n i e t h y l ] n a p h t h a l e n e 156
5 M eM e
M eM e
2,6-B is(brom om ethyl)naphthalene (0.10 g, 0.32 mmol) was refluxed
in ethanol (10 ml) with 2, 5-dimethylpyrrolidine (0.06 g, 0.08 ml, 0.65 mmol)
for two hours. After work-up as described in experiment 9 , a sticky yellow
oil was obtained which resisted trituration with a number of solvents and
so was isolated by prec ip ita tion from ethanol solution by the careful
addition of water. This gave a turbid solution which crystallised overnight
to give white flakes of the title amine which were filtered off and dried
(18.6 mg, 0.05 mmol, 17%), m.p. 50-52 °C.
nmr (CDCI3): 1.07 (d, 12H, 3Jh _h = 5.4Hz, H5'); 1.38 (m, 4M, H41;
1.77 (m, 4H, H4'); 2.59 (m, 4H, H3'); 3.87 (s, 4H,
H r ) ; 7.43 (dd, 2H, = 8.8Hz, ^ J ^ .h = 0.8Hz,
H3,7); 7.68 (s, 2H, Hl,5); 7.72 (d, 2H, 3J h _h = 8.3Hz,
H4,8)
nmr (CDCI3): 20.7 ( C4-' , m), 30.0 ( C 5 ' , m); 31.2
(C5‘); 59.8 ( C D ; 127.3 (Q); 127.5 (Q); 128.0 (Q);
132.4 (Cq); 135.2 (Cq)
Mass Spectrum (m/z): 351 [(M + H)+, 100%]; 252 [(M - C6H i 2N)+, 5%]; 154
[ (C i2H io)+ , 3%]
UV (EtOH): ^max = 236.0nm; £ = 65150
Accurate Mass: Expected for C24H 34N 2, 350.2721
Found 350. 2716
153
E x p e r i m e n t a l
36 . P r e p a r a t i o n of 2 ,6 - B i s [ ( 2 ,5 - d i m e t h y l ) p y r r o l id o m e th y l ] n a p h t h a i e n e
d im e th io d id e 157
MeM e
M eM e M e
2 ,6 -B is [ (2 ,5 -d im e th y l)p y rro l id o m e th y l ]n a p h th a le n e (12 .0 mg, 0.03
mmol) was stirred in acetonitrile (1 ml) with iodomethane (60.0 mg, 0.03 ml,
0.44 mmol). A white precipitate formed which gave a f locculent material
after pouring into dry E t2 0 (200 ml). This was filtered off and dried, giving
the title salt as a white powder (14.0 mg, 0.02 mmol, 64%), m. p. >230 °C.
nmr (dg-DMSO): 1.42 (d, 12H, 3Jh _h = 6.3Hz, H5'); 1.66 (m, 4H, H41;
2.04 (m, 4H, H41; 2.75 (s, 6H, H6'); 3.54 (m, 4H,
H3'); 4.72 (s, 4H, HK); 7.64 (d, 2H, ^j ^ . h = 8.3Hz,
H3,7); 8.14 (d, 2H, = 8.0Hz, H4,8); 8.15 (s, 2H,
H l ,5 )
nmr (d^-DMSO): 13.4 ; 26.4 ; 36.3 ; 60.4 ; 67.1 ; 126.9; 129.2; 129.9;
132.68; 132.74
Mass Spectrum (m/z): 267 [(M - C6H 12N - CH3)+, 49%]; 252 [(M - C g H ^ N
2CH3)+, 1%]; 190 [M2+, 24%]; 98 [(C6Hi2N)+, 65%]
UV (H2O):
Analysis (%):
^max = 230.5nm; £ = 192220
Calculated for C26H40N 2I2, C: 49.22; H;6.36; N:4.42
Found, C: 48.87; H: 6.15; N; 4.11
37. P r e p a r a t i o n of T r a w s - 4 , 4 ' - b i s ( b r o m o m e t h y l ) s t i l b e n e 1 2 8
154
E x p e r i m e n t a l
7 raAi5-4 ,4 '-dimethylstilbene (0.50 g, 1.37 mmol) was heated to reflux
in CCI4 (25 ml) with benzoyl peroxide (25 mg) and N-bromosuccinimide
(0.47 g, 2.65 mmol) for 2 hours. Filtration of the reaction mixture followed
by evaporation in vacuo and recrystallisation from EtOH produced the title
dibromide as a pale yellow powder (0.60 g, 0.93 mmol, 68%), m.p. 155-158 °C.
nmr (CDCI3): 4.51 (s, 4H, HI); 7.09 (s, 2H, H6); 7.38 (d, 4H, ^ j ^ . h
= 8.2Hz, H3 or 4); 7.48 (d, 4H, = 8.3Hz, H3 or 4)
13c nmr (CDCI3):
Mass Spectrum (m/z):
33.4 (Cl); 126.9 (Ct); 128.6 (Ct); 129.5 (Ct);
137.2 (Cq); 137.3 (Cq)
368 [(C i6H i48lB r2)+ , 2%]; 366
[ (C i6 H i4 ’ 5Br8lBr)+ , 4%] ; 364 [ ( C i e H u ’ ^Braj-^,
2%]; 287 [(C i6H i48 'B r)+ , 65%]; 285
[(C i6Hi479Br)+, 59%]; 206 [(C :6H i4)+ , 100%]
UV (EtOH): ^max = 322.Onm; £ = 36820
Accurate Mass: Expected for 364.9462
Found, 364.9469
38. Preparation of T r a / i s - 2 , 4 ' - b i s ( b r o m o m e t h y l ) s t i l b e n e 1 2 9
155
E x p e r i m e n t a l
7’r a « 5-2 ,4 '-dimethylstilbene (0.50 g, 2.40 mmol) was refluxed in CCI4
(20 ml) with benzoyl peroxide (20.0 mg) and N-bromosuccinimide (0.85 g,
4.78 mmol) for two hours. Reduction of the crude, filtered mixture gave a
yellow oil which was flash chrom atographed (CH2 C I 2 ) to give a sticky
yellow powder. This was triturated with E t2 0 , giving the title dibromide as a
white powder (0.11 g, 0.31 mmol, 13%), m. p. 90-95 °C.
1 h nmr (CDCI3); 4.51 (s, 2H, HI); 4.61 (s, 2H, H14); 7.06 (d, IH, ^ . h
= 16.2 Hz, H6); 7.24-7.53 (m, 7H); 7.62 (d, IH, ^ j ^ . h
= 7.5 Hz); 7.69 (m, IH)
13c nmr (CDCI3): 32.0; 33.4; 125.7; 126.4; 127.2; 128.1; 128.8; 129.3;
129.5; 130.4; 134.9; 136.7; 137.4; 137.5
Mass Spectrum (m/z): 368 [(C i6H i48lBr2)+ , 7%]; 366
( ( C i 6H i 4’ ’ B r8 'B r )+ , 17%] ; 364 [(C i6H i 4’ ^ B r2)+,
7%]; 287 [(C i6H i 4^ 'B r)+ , 100%]; 285
[(C i6H i479Br)+ , 100%]; 206 [(C i6H l4)+ , 100%]; 205
[ ( C i e H n r , 100%]
UV (EtOH): -max = 313.5 nm; £ = 31250
Accurate Mass: Expected for i 4^ ^B r2, 364.9462
Found, 364.9460
39. Preparat ion of 1,4-Bis [ ( 2 ,5 - d i m e t h y l ) p y r r o l i d o m e t h y l ]b e n z e n e 158
5'MeMe
MeMe
156
E x p e r i m e n t a l
2,5-Dimethylpyrrolidine (0.50 ml, 0.41 g, 4.08 mmol) was dissolved in
a so lu t ion of sodium (0.09 g) in m ethano l (15 ml) then 1,4-
bis(bromomethyl)benzene (0.34 g, 2.03 mmol) was added slowly and with
vigorous stirring. The mixture was then ref luxed for two hours and
standard work-up gave the amine as a brown oil (0.27 g, 2.41 mmol, 59%).
nmr (CDCI3): 1.02 (d, 12H, = 6.0Hz, H5tO; 1.09 (d, 12H,
H = 6.1Hz, H 5c l; 1.40 (m, 4H, H4'); 1.79 (m, 4H,
H4'); 2.59 (br q, 4H, ^ J ^ . h = 5.5Hz, H 31 3.76 (s, 4H,
H I ') ; 7.24 (s, 4H, H2,3,5,6)
13c nmr (CDCI3): 20.3 (C5’); 31.1 (C4’); 54.5 (C3’); 59.3 ( C l ’;
127.4 (C l ,4); 148.4 (C2,3,5,6)
Mass Spectrum (m/z): 301 [(M + H)+, 32%]; 202 [(M - C6H i 2N)-i-,
7%]; 104 [(CgH8)+, 6%]; 98 [(C6Hi2N)+, 10%]
UV (EtOH): ^max = 224.0 nm; e = 28520
Accurate Mass: Expected for C 10H 16N 2, 164.1313
Found: 164.1311
40. Preparat ion of l , 4 -B i s - [ ( 2 ,5 - d im e t h y l ) p y r r o l id o m e t h y l ] b e n z e n e
dimethiodide 159
5 'M e M eM e
M eM e
M e2 1 "
1,4 -B is[(2 ,5 -d im ethy l)py rro lidom ethy l]benzene (0.17 g, 0.55 mmol)
was dissolved in acetonitrile (1 ml) and stirred with iodomethane (0.50 g,
0.22 ml, 3.52 mmol) for 24 hours. During this time, a white precipitate formed and this was removed by filtration, washed with E t2 0 and dried,
157
E x p e r i m e n t a l
giving the title salt as a creamy-white powder (0.15 g, 0.26 mmol, 48%), m.p.
>260 °C (dec.).
nmr (d^-DMSO):
13c nmr (d^-DMSO):
1.36 (d, 6H, 3J h _h = 6.5Hz, H5"); 166 (m, 4H, H4');
2.08 (m, 4H, H4'); 2.71 (s, 6H, H60; 3.56 (m, 4H,
my, 4.58 (s, 4H, H r ) ; 7.59 (s, 4H, H2,3,5,6)
13.4 (C 50; 26.4 (C 40; 36.5 (C6'); 59.7 (C3'); 67.2
(C r ) ; 129.7 (C l,4); 133.2 (C2,3,5,6 )
Mass Spectrum (m/z): 315 [(M - CH3)+, 3%]; 202 [(M - C6H 12N - 2CHs)+,
49%]; 187 [(C i3H i9N )+ , 12%]; 104 [(CgH8)+, 64%];
98 [(C6Hi2N)+, 80%]
UV (H2O):
Analysis (%):
^max “ 222.Onm; 6 — 42540
Calculated for C22H 38N 2I2, C: 45.22; H: 6.55; N:4.80
Found, C: 44.14; H: 6.43; N: 4.33
41. Preparation of rra / i5- 4 ,4 "-bis[(2 ,5 - d im e t h y l ) p y r r o l i d o m e t h y l ]
s t i lb e n e 160
3 M e
M eM e
M e
2,5-Dimethylpyrrolidine (0.07 g, 0.07 ml, 0.57 mmol) was added to
stirred, dry THF (10 ml) at 0 °C under N 2(g) then n-BuLi (0.36 ml of a 1.6 M
solution in hexanes, 0.57 mmol) was added dropwise. After 10 minutes, the
solution was removed with a syringe and added dropwise to a stirred
suspension of f r o n j -4 ,4 '-b is(brom om ethyl)s ti lbene (0.10 g, 0.27 mmol) in
dry THF (5 ml). The mixture was stirred at 0 °C for 30 minutes then standard
158
E x p e r i m e n t a l
work-up gave the title amine as a brown oil (54.3 mg, 0.29 mmol, 50%).
nmr (CDCI3): 1.11 (d, 6H, 3Jh_h = 5.6Hz, H3); 1.39 (m, 4H, HI);
1.78 (m, 4H, HI); 2.60 (m, 4H, H2); 3.78 (s, 4H,
H5); 7.06 (s, 2H, HIO); 7.27 (d, 4H, ^ J ^ .h = 8.0Hz, H7
or 8); 7.43 (d, 4H, 3Jh_h = 8.2Hz, H7 or 8)
nmr (CDCI3): 20.3 (Cl or C3); 31.21 (Cl or C3); 54.4; 59.5; 126.0;
128.0; 129.7; 130.2; 136.0
Mass Spectrum (m/z): 403 [(M + H)+, 11%]; 304 [(M - C6H12N)+, 23%]; 206
[(Ci6Hi4)+, 100%]; 98 [(C6H i 2N)+, 23%]
UV (EtOH):
Accurate Mass:
^max = 314.0nm; e = 57820
Expected for C28H 39N 2, 403. 3113
Found, 403.3108
42. P r e p a r a t i o n of 7 r a w s - 4 ,4 ' - b i s [ ( 2 ,5 - d im e th y l ) p y r r o l id o m e th y l ] s t i l b e n e
d im e th io d id e 161
5 Me
M eM e
2 1 '
M e M e
r r a n 5-4 ,4 '-b is [ (2 ,5 -d im ethy l)py rro lidom ethy l] s ti lbene (54.3 mg.
0.13 mmol) was added to acetonitrile (3 ml) and stirred with iodomethane(0.20 ml, 0.46 g, 3.21 mmol) for 24 hours. After pouring into E t iO (100 ml),
the mixture was centrifuged (x3000, 5minutes) then the E t2 0 decanted. The
moist solid was carefully dried in vacuo giving the title salt as yellow
powder (21.5 mg, 0.05 mmol, 38%), m. p. >230 °C.
159
E x p e r i m e n t a l
nmr (d^-DMSO): 1.37 (d, I2H, 3Jh _h = 6.4Hz, H3); 1.65 (m, 4H, HI);
2.08 (m, 4H, HI); 2.69 (s, 6H, H5); 3.49 (m, 4H,
H2); 4.53 (s, 4H, H6); 7.42 (s, 2H, H l l ) ; 7.45 (d, 4H,
3Jh -H = 7.8Hz, H8 or 9); 7.73 (d, 4H, = 7.7Hz,
H8 or 9)
nmr (d^-DMSO): 13.4; 26.4; 36.2; 60.3; 67.0; 127.1; 127.2; 129.2; 133.1
138.6
Mass Spectrum (m/z); 402 [(M - 2CH3)+, 2%]; 304 [(M - C6H 12N - 2CH3)+,
2%]; 206 [(C i6H i4)+ , 91%]; 201 [M2+, 1%]
UV (H2O):
Analysis (%):
^max = 316.5 nm; e = 31490
Calculated for C30H44N 2I2, C: 52.49, H: 6.46; N: 4.08
Found: C: 51.03; H: 6.23; N: 3.96
43. Preparation of 7>a/î5:-2 ,4 ' -bis[(2 ,5 - d i m e t h y l )pyrro l idom eth yI]
s t i lbe ne 162
Me
3 Me
MeMe
14
7 r a n j - 2 ,4 ’-bis(bromomethyl)stilbene (0.08 g, 0.22 mol) was refluxed
in EtOH (10ml) with 2,5-dimethylpyrrolidine (0.04 g, 0.05 ml 0.44 mmol) for
two hours. Standard work-up gave the title amine as a pale yellow oil
(35.7mg, 0.09 mmol, 41%).
^H nmr (CDCI3): 0.89 (d, 12H, 3JH-H = 5.4Hz, H3); 1.14 (m, 4H, HI);
1.80 (m, 4H, HI); 2.68 (m, 4H, H2); 3.95 (s, 2H, H5);
4.03 (s, 2H, H18); 7.06 (d, IH, ^j ^ . h = 16.0Hz); 7.35
160
E x p e r i m e n t a l
(m, 7H); 7.54 (d, IH, 3JH-H = 7.5Hz); 7.56 (m, IH)
Mass Spectrum (m/z): 403 [(M + H)+, 8%]; 305 [(M + H - CôH nN)"^, 35%];
207 [(C i6H i 5)+, 57%]; 91 [(C?H7)+, 100%]
UV (EtOH):
Accurate Mass:
Xmax = 307.5; e = 28500
Expected for C28H 39N 2, 403. 3113
Found, 403.3112
44. Preparation of r r a n 5- 2 ,4 ' -bis[(2 ,5 -d im e th y I ) p y r r o l i d o m e th y l ] s t i lb e n e
dimethiodide 163
2 i M e
20
M eM e
1421'
7 raA îj-2 ,4 '-b is [(2 ,5 -d im ethy l)py rro lidom ethy l]s t i lbene (13.0 mg, 0.03
mmol) was dissolved in acetonitrile (1 ml) and stirred with iodomethane
(0.34 g, 0.15 ml, 2.41 mmol) for 24 hours. The mixture was then poured into
E t 2 0 (100 ml) and centrifuged (x3500, 4 minutes). Decanting of the solvent
followed by careful drying in vacuo of the moist solid produced the title
salt as a pale brown powder (8.7 mg, 0.01 mmol, 39%), m. p. >230 °C.
^H nmr (d^-DMSO): 1.17 (d, 6H, 3Jh_h = 6.4Hz); 1.28 (d, 6H, 3Jh_h =
6.5Hz); 1.58 (m, 4H, HI); 1.99 (m, 4H, HI); 2.60 (s,
3H, H4 or 19); 2.61 (s, 3H, H4 or 19); 3.52 (m, 4H,
H2,20); 4.84 (s, 2H, H5); 5.04 (s, 2H, H18); 7.43 (d,
IH, 3Jh_h = 16.6Hz); 7.63 (m, 7H); 7.86 (d, IH,
H = 7.6Hz); 7.89 (m, IH)
Mass Spectrum (m/z): 560 [(M + H + I)+, 6%]; 320 [(M - C6H 12N - CH3 +
161
E x p e r i m e n t a l
UV (EtOH):
Analysis (%):
H)+, 75%]; 207 [(C i6H i5)+ , 68%]; 98 [ (C ^H iiN )^ ,
100%]
^max = 307.5 nm; £ = 19220
Calculated for C30H44N 2I2» C: 52.49, H: 6.46; N: 4.08
Found: C: 51.67; H: 6.66; N: 3.96
45. Preparat ion of l , l ’- (P ‘ X y ly l )b i s ( l -aza -4 -azon iab ic yc lo [2 .2 .2 ]oc tan e )
dibromide 166
2Br'
l,4-Bis(bromomethyl)benzene (100.0 mg, 0.38 mmol) was dissolved in
acetonitrile (10 ml) then DABCO (90.0 mg, 0.80 mmol) was added and the
reaction stirred for five minutes. The precipitate which formed was filtered
and washed with E t2 0 giving the title salt as a white powder (131.3 mg, 0.035
mmol, 92%), m.p. >320 °C.
nmr (d^-DMSO):
nmr (d^-DMSO):
3.20 (br t, 12H, ^j ^ . h = 7.3Hz, H4'); 3.50 (br t, 12H,
3Jh -h = 7.3Hz, H31; 4.59 (s, 4H, HU); 7.67 (s, 4H,
H2,3,5,6)
46.8 (C4-); 54.7 (C3'); 70.1 (CU); 131.2 (C l,4); 136.4
(C2,3 5,6)
Mass Spectrum (m/z): 409 [(M + 8lBr)+, 41%]; 407 [(M + 79sr)+ , 42%]; 328
[M+, 0.1%]; 327 [(M - H)+, 5%]; 216 [(M -
C6H i2N 2)+ , 27%]; 215 [(M - C6H 12N 2 - H)+, 31%];
164 [M2+, 8%] 112 [(C6H i 2N 2)+, 100%]; 104
[(CgH8)+, 7%]
162
E x p e r i m e n t a l
UV (H2O):
Analysis (%):
^max = 217.5 nm; £ = 8170
Calculated for € 20X32^ 4 6 :2, C: 49.19, H: 6.61; N: 11.47
Found: C: 49.03; H: 6.68; N: 11.38
46. Preparat ion of l , l ’* ( 2 ,6 -d i m e t h y l n a p h t h a l e n y l ) b i s ( l -a z a - 4 -
azoniabicyc lo [2 .2 .2 ]octane) dibromide 167
2 B r '
2,6-Bis(bromom ethyl)naphthalene (100.0 mg, 0.32 mmol) was heated
to reflux for four hours with DABCO (70.0 mg, 0.64 mmol) in acetonitrile (10
ml). Filtration of the precipitate which formed followed by washing with
E t 2 0 gave the title salt as a white powder (119.0 mg, 0.54 mmol, 69%), m.p.
>320 °C.
nmr (d^-DMSO):
13c nmr (d6-D M S0):
Mass Spectrum (m/z):
UV (H2O):
Analysis (%):
3.05 (br t, 12H, = 6.9Hz, H4'); 3.42 (br t, 12H,
3Jh -h = 6.8Hz, H3'); 4.78 (s, 4H, HU), 7.71 (d, 2H,
3Jh-H = 8.4Hz, H3,7); 8.13 (d, 2H, ^ J ^ .h = 8.4Hz,
H4,8); 8.20 (s, 2H, Hl,5)
44.6 (C40; 51.6 (C3'); 6 6 .2 ( C U ) ; 126.4; 129.0; 130.5;
132.8; 133.3
459 [(M + 8 lBr)+, 3%]; 457 [(M + 79Br)+, 3%]; 266
[(M - C6H i 2N 2)+, 4%]; 265 [(M - C6H 12N 2 - CH3)+,
1%]; 189 [M2+, 0.15%]; 154 [(C i2 H io)+ , 100%]; 112
[(C6H i 2N 2)+]
Xmax = 231.0 nm; £ = 55570
Cklculatedfor C24H32N 4Br2, C: 53.74, H: 6.01; N: 10.45
163
E x p e r i m e n t a l
Found: C: 53.54; H: 6.12; N: 10.32
47. P r e p a r a t i o n o f 1 , 1 -^ ' 'û ws-4 ,4 ' -d im ethyI s t i l b e n y 1) b is ( l - a z a - 4 -
azon i a b i c y c lo [2 .2 .2]oc tan e) d ib r o m id e 170
2Br'
j -4 ,4 '-b is (b ro m o m eth y I)s t i lb en e (100.0 mg, 0.27 mmol) was
treated with DABCO (60.0 mg, 0.55 mmol) as described in experiment 4 6 ,
giving the title salt as a pale yellow powder (74.4 mg, 0.16 mmol, 58%), m. p.
>320 °C.
nmr (d^-DM SO):
nmr (d^-DMSG):
UV (HiO):
Analysis (%):
3.03 (br t, 12H, ^ J ^ . h = 7.1Hz, H40; 3.33 (br t, 12H
3Jh -h = 7.6Hz, H31; 4.54 (s, 4H, HU); 7.45 (s, 2H,
H7); 7.54 (d, 4H, = 8.1Hz, H2 or 3); 7.77 (d, 4H,
= 8.1Hz, H2 or 3)
44.7 (C41; 51.6 (C31; 66.1 (CU); 126.6; 127.0;
129.1; 133.6; 138.5
Mass Spectrum (m/z): 318 [(M - C ôH nN )'' ' , 46%]; 221 [M^+, 13%]; 206
[ (C i 6 H i 4)+, 100%]; 112 [ (C eH n N z )^ , 72%]
X-max — 316.5nm; £ — 20180
Calculated for C2gH3gN4Br2, C: 56.96, H: 6.49; N: 9.49
Found: C: 55.09; H: 6.61; N: 7.96
164
E x p e r i m e n t a l
48. P re p a r a t i o n o f l , l "“( 2 , 6 - d i n i e t h y l a n t h r a c e n y l ) b i s ( l - a z a - 4 -
a z o n i a b i c y c l o [ 2 .2 . 2 ] o c t a n e ) d ib r o m id e 168
2Br"
2,6-Bis(bromom ethyl)anthracene (100.0 mg, 0.27 mmol) was treated
with DABCO (60.0 mg, 0.55 mmol) as described in experiment 46 giving the
title salt as a white powder (124.0 mg, 0.26 mmol, 96%), m.p. >320 °C (dec.).
nmr (d^-DMSO):
nmr (d^-DMSO):
3.04 (br t, 12H, = 7.2Hz, H4'); 3.43 (br t, 12H,
3Jh -h = 7.2Hz, H30; 4.75 (s, 4H, H I ') ; 7.63 (dd, 2H,
3Jh -H = 8.8Hz, 4Jh . h = 13Hz, H3,7); 8.29 (d, 2H,
H = 8.7Hz, H4,8); 8.33 (s, 2H, H I,5), 8.77 (s, 2H,
H9,10)
44.7 (C41; 51.7 (C3'); 66.5 (CT); 125.2; 127.2; 129.0;
129.3; 131.2; 131.3; 134.2
Mass Spectrum (m/z): 509 [(M + 8lBr)+, 30%]; 507 [(M + 79b f )+, 30%]; 427
[(M - H)+, 7%]; 316 [(M - C6Hi2N)+, 60%]; 214
[(M2+, 25%]; 204 [(Ci6Hi2)+, 100%]; 112
[(C6Hi2N2)+, 41%]
UV (HiO):
Analysis (%):
^max = 262.0 nm; e = 57400
Calculated for C2gH36N 4B r2, C:57.14; H:6.17; N: 9.52
Found, C: 56.59; H: 5.88; N: 8.73
49. P r e p a r a t i o n of 1 ,1 - (p -x y ly l ) b i s ( 4 - m e t h y l - 1,4-d iazon iab ic y d o
[2 .2 .2]octane) diiodide d ib ro m id e 171
165
E x p e r i m e n t a l
Me
2 B f 21-
C o m p o u n d 166 (50.0 mg, 0.13 mmol) was added to acetonitrile (5 ml)
with iodomethane (0.40 ml, 0.91 g, 6.43 mmol) and stirred for 24 hours at
room temperature. Filtration of the precipitate which formed followed by
washing with E t2 0 gave the title salt as a pale yellow powder (67.8 mg, 0.13
mmol, 100%) m.p. >320 °C.
1H nmr (d^-DMSO): 3.27 (s, 6H, H5'); 3.94 (m, 24H, H3',4 '); 4.95 (s, 4H,
H D ; 7.73 (s, 4H, H2,3,5,6)
13c nmr (de-DMSO): 50.2 ; 51.8 ; 52.5 ; 65.5 ; 128.8; 133.9
Mass Spectrum (m/z): 358 [M+, 0.2%]; 231 [(M - C6H 12N 2 - CHg)'"', 94%];
142 [(C6H 12N2 + CH3)+, 71%]; 112 [(C6Hi2N2)+,
31%]; 104 [(CgH8)+, 78%]
UV (H2O):
Analysis (%):
X.max “ 223.0 nm; £ — 37400
Qlculatedfor C22H3gN4Br2l2, C: 34.22; H: 4.96; N: 7.26
Found: C: 33.80; H: 4.79; N: 6.86
50 . P r e p a r a t i o n o f l , l - ( p - x y l y l ) b : s ( 4 - p - x y l y l - l , 4 -
d i a z o n i a b i c y c lo [ 2 .2 .2 ] o c t a n e ) t e t r a b r o m i d e 172
4Br"
166
E x p e r i m e n t a l
C o m p o u n d 166 (50.0 mg, 0.13 mmol) was stirred at room temperature
with 4-bromomethyltoluene (50.0 mg, 0.27 mmol) in acetonitrile (5 ml) for 24 hours. Filtration of the precipitate followed by washing with E t2 0 gave
the title salt as a white powder (61.4 mg, 0.08 mmol, 63%), m.p. >280 °C (dec.).
nmr (d^-DMSO): 3.10 (s, 6H); 3.40 (m, 24H); 4.62 (s, 4H); 4.93 (s, 4H);
7.65 (m, 12H)
Mass Spectrum (m/z): 409 [(M - ZCgHg + S 'B r)+ . 30%]; 407 [(M - ZCgHg +
7^Br)+. 31%]; 217 [(M - IC gH g - C6H 12N 2 + H)+
68%]; 112 [(C6Hi2N2)+, 100%]; 105 [(CgH9)+, 45%];
104 [(C8Hg)+, 5%]
UV (H2O):
Analysis (%):
Xmax ~ 218.0 nmj £ — 28220
Calculated for C36H 5oN4Br4, C: 50.37: H: 5.87;N: 6.53
Found: C: 47.12; H: 6.61; N: 6.14
51. Preparation of l , l ' - ( 2 , 7 - d i m e t h y l a n t h r a c e n y l ) b i s ( l - a z a - 4 -
azoniabicyclo [2 .2 .2 ]octane) dibromide 169
2Br
2,7-Bis(bromomethyl)anthracene (0.10 g, 0.27 mmol) was stirred for
two hours with DABCO (60.0 mg, 0.05 mmol) in acetonitrile (10 ml). The
reaction mixture was poured into Et2 0 (200 ml) producing a flocculent
precipitate. A fter settling, the solvent was decanted and the moist solid
carefully dried in vacuo, giving the title salt as a yellow powder (65.0 mg,
0.11 mmol, 41%), m.p. >320 °C.
nmr (d^-DMSO): 3.02 (m, 12H, H4'); 3.41 (m, 12H, H3'); 4.86 (s, 4H,
HI'); 7.72 (d, 2H, 3jH_H = 8.9Hz, H3,6); 8.34 (d, 2H,
167
E x p e r i m e n t a l
nmr (d^-DMSO):
3Jh -H = 8.7Hz, H4,5); 8.62 (s, 3H, H I ,8,9); 8.90 (s, IH,
HID)
44.8; 51.7; 66.3; 122.8; 127.6; 129.7; 129.8; 130.0;
132.4; 133.0; 137.2
Mass Spectrum (m/z): 509 [(M + 8lBr)+, 2%]; 507 [(M + '79Br)+, 2%]; 428
[M+, 1%]; 316 [(M - C 6H i 2N2)+, 4%]; 204
[(C i 6 H i 2)+, 9%]; 112 [(C6Hi2N2)+, 64%]
UV (H2O):
Analysis (%):
%max = 259.5 nm; e = 98400
Calculated for C2sH 36N 4Br2, C:57.14; H:6.17; N: 9.52
Found, C: 55.59; H: 5.93; N: 8.62
52. P r e p a r a t i o n of 2 ,6 - B ls - [ ( c i s - 2 ,6 - d im e th y l )p ip e r id o m e th y 1]
n a p h t h a l e n e 178
6'Me
Me
Me
Me
2,6-Bis(bromomethyl)naphthalene (100.0 mg, 0.32 mmol) and c is -2 ,6 -
dimethylpiperidine (70.0 mg, 0.08 ml, 0.64 mmol) were refluxed in butanone
(10 ml) for two hours. This produced a precipitate which was filtered off
and recrysta llised from isopropanol giving the amine as white needles
(26.2 mg, 0.07 mmol, 22%), m.p. 64-67 °C
^H nmr (CDCI3): 1.14 (d, 12H, = 5.9Hz, H6'); 1.33 (m, 4H, H5');
1.60 (m, 8H, H4'); 2.54 (m, 4H, H31; 3.95 (s, 4H,
H D ; 7.45 (dd, 2H, ^ J ^ . h = 8.4Hz, = 1.2Hz,
H3,7); 7.72 (d, 2H, ^j ^ . h = 8.4Hz, H4,8); 7.80 (s, 2H,
H l ,5 )
168
E x p e r i m e n t a l
13c nmr (CDCI3): 22.1; 24.2; 34.5; 53.6; 57.3; 126.2; 126.7; 127.2; 127.6;
132.3
Mass Spectrum (m/z): 379 [(M + H)+, 100%]; 266 [(M - C 7H i4N )+ , 78%];
154 [(C i 2 H io )+, 69%]; 112 [(CvH m N )^ 36%]
UV (EtOH): ^max = 234nm; e = 46750
Accurate Mass: Expected for C26H 38N 2 378.3035
Found 378.3035
53. P repara t ion of 2 ,6 -Bi^^is -2 ,6 -d imeth y l )p ip e r id o m e th y 1]
n a p h t h a l e n e dimethiodide 179
6' M eM e
M e
M eM e
21" M e
Compound 179 (10.0 mg, 0.03 mmol) was dissolved in dry acetonitrile
(1 ml) with iodomethane (0.16 g, 0.07 ml, 1.16 mmol) and stirred for 24
hours. After pouring into E t2 0 (200 ml), the compound was isolated by
decanting the solvent and placing the near-dry solid under low vacuum,
giving the title salt as an off-white powder (14.2 mg, 0.02 mmol, 80%), m.p.
>230 °C.
nmr (d^-DM SO): 1.10 (m, 2H, H5'e); 144 (m, 2H, H5'a); 1.54 (d, 12H,
^Jh -H = 6.2Hz, H60; 1.62 (m, 4H, H4"g); 168 (m, 4H,
H4'a); 2.85 (s, 6H, H7'); 3.19 (m, 4H, H3'); 4.78 (s,
4H, HU); 7.58 (d, 2H, 3Jh _h = 8.5Hz, H3,7); 8.10 (s,
2H, Hl,5); 8.10 (d, 2H, = 8.4Hz, H4,8)
Mass spectrum (m/z): 535[(M 4- I)+, 2%]; 393 [(M - CH3)+, 1%]; 281 [(M -
C H 3 - C 7H i 4N)+, 29%]; 204 [M^+, 11%]; 154
169
UV (EtOH):
Analysis (%):
E x p e r i m e n t a l
[ (C i 2 H io)+, 100%]
^max = 231.0 nm; e = 192320
Calculated for C28H 44N 2I2» C: 50.76; H: 6.69; N: 4.23
Found, C: 48.82; H: 6.41; N: 3.87
54. Preparat ion of l , l ' - ( p - x y ly l ) b i s ( l - a z o n ia b ic y c lo [ 2 .2 .2 ] o c t a n e )
dibromide 173
2 B r ‘
l,4-Bis(brom omethyl)benzene (80.0 mg, 0.30 mmol) was stirred with
quinuclid ine (70.0 mg, 0.65 mmol) in acetonitrile (10 ml) for 24 hours. The
precipita te which formed was filtered off, washed with E t2 Û and dried,
giving the title salt as a white powder (146.9 mg, 0.30 mmol, 100%),
m.p. >230 °C.
nmr (d^-DMSO):
nmr (d^-DMSO):
Mass Spectrum (m/z):
UV (H2O):
Analysis (%):
1.85 (m, 12H, H4'); 2.04 (br t, 2H, ^j ^ . h = 3.0Hz,
H5'); 3.45 (t, 12H, ^ J ^ .h = 7.6Hz, H3'); 4.48 (s,
4 H ,H D ; 7.61 (s, 4H, H2,3,5,6)
19.5; 23.3; 53.7; 65.5; 129.5; 133.4
407 [(M + 8lBr)+, 48%]; 405 [(M + 79Br)+, 51%]; 325
[(M - H)+, 1%]; 215 [(M - C7H i 3N)+, 18%]; 104
[(CgH8)+, 27%]
A-max “ 217.0 nm; £ = 16980
Calculated for C22H 34N 2B r2, C: 54.33; H: 7.05; N: 5.76
Found, C: 54.17; H: 7.09; N: 5.71
170
E x p e r i m e n t a l
55. P r e p a r a t i o n o f 1 , 1 - ( 2 , 6 - d i m e t h y l n a p h t h a l e n y l ) b : s ( l
a z o n ia b i c y c l o [ 2 .2 . 2 ] o c t a n e ) d ib r o m i d e 174
2 B r ’
2,6-Bis-(bromomethyl)naphthalene (50.0 mg, 0.16 mmol) was reacted
with qu inuc l id ine (40.0 mg, 0.32 mmol) as described in experiment 5 3 ,
giving the title salt as white powder (21.5 mg, 0.04 mmol, 25%), m.p. >230 °C.
nmr (dg-DMSO):
nmr (d^-DMSO):
1.85, (br s, 12H, H4'); 2.04 (m, 2H, H5'); 3.47 (br t,
12H, 3Jh_h = 7.8Hz, H3'); 4.62 (s, 4H, HT); 7.69 (dd,
2H, = 8.4Hz, 4Jh_h = l . lH z, H3,7); 8.11 (d, 2H,
3Jh . h = 8.3Hz, H4,8); 8.17 (s, 2H, Hl,5)
19.5 (CSy, 23.4 (C4-); 53.8 (C31; 66.1 (C l '); 126.8;
128.9; 130.4; 132.7; 133.0
Mass Spectrum (m/z): 457 [(M + 8lBr)+, 78%]; 485 [(M + 79Br)+, 75%]; 375
[(M + H)+, 4%]; 265 [(M - C v H ib N )^ , 100%];
188 [M2+, 19%]; 155 [(C i2H u ) + , 11%]; 112
[(C7H 13N + H)+, 40%]; 111 [(C7H i 3N)+, 36%]
UV (H2O):
Analysis (%):
^max = 230.0 nm; £ = 88350
Calculated for C26H 36N 2B r2, C: 58.22; H: 6.77; N: 5.22
Found, C: 58.87; H: 6.91; N: 5.03
171
E x p e r i m e n t a!
56. P r e p a r a t io n of l , l ' - ( 2 , 7 - d i m e t h y l a n t h r a c e n y I ) b i s ( l -
a z o n i a b i c y c l o [ 2 .2 . 2 ] o c t a n e ) d ib r o m i d e 176
2 B r
2,7-B is(brom om ethyl)an thracene (20.0 mg, 0.05 mmol) was stirred
with quinuclidine (13.3 mg, 0.12 mmol) in acetonitrile (5 ml) for 24 hours.
The mixture was then poured into E t2 0 (200 ml) producing a flocculent
yellow precipitate which was allowed to settle then the solvent decanted
with a pipette. The moist solid was carefully dried in vacuo giving the title
salt as a yellow powder (3.2 mg, 0.01 mmol, 10%), m.p. >230 °C (dec.).
nmr (d^-DMSO): 1.86 (m, 12H, H4'); 2.04 (m, 2H, H5'); 3.48 (br t, 12H,
3Jh-H = 7.6Hz, H3'); 4.89 (s, 4H, H I ') ; 7.70 (d, 2H,
3Jh-H = 8.8Hz, H3,6); 8.32 (brs,2H, H4,5); 8.62 (s, 3H,
H1,8,H9); 8.90 (s, IH, HIO)
Mass Spectrum (m/z): 507 ((M + 5%]; 505 [(M + '^9Br)+, 5%]; 213
[(M - C7H i 3N)+, 12%]; 206 [ (C ie H u ) ^ , 100%]; 204
[ ( C |6H i 2)+. 89%]
U V ( H 2O ): ^max = 267.5 nm; e = 70710
57. Preparation of l , l ’- ( 2 , 6 - d i m e t h y l a n t h r a c e n y l ) b i s ( l -
azoniabicyc lo[2 .2 .2 ]octane) dibromide 175
2 B r ’
172
E x p e r i m e n t a l
2,6-B is(brom om ethyl)an thracene (70.0 mg, 0.19 mmol) was stirred
with quinuclidine (40.0 mg, 0.40 mmol) in acetonitrile (10 ml) then worked-
up as described in experiment 55 to give the title salt as a yellow powder
(8.0 mg, 0.01 mmol, 7%), m.p. >230°C.
nmr (d^-DM SO): 1.86 (m, 12H, H41; 2.05 (m, 2H, H51; 3.51 (m, 12H,
H3 ); 4.72 (s, 4H, H I ') ; 7.71 (dd, = 8.9Hz,
= l .lH z, H3,7); 8.26 (d, 3J h . h = 8.9Hz, H4,8); 8.38 (s,
IH, Hl,5); 8.75 (s, IH, H9,10)
Mass Spectrum (m/z): 507 [(M + 8>Br)+, 11%]; 505 [(M + 13%]; 315
[(M - C7H i 3N)+, 27%]; 213 [M2+, 7%]; 204
[ (C i6H i 2)+. 29%]
UV (H2O);
Analysis (%):
^max - 262.5 nm; e = 15470
Calculated for C3oH3gN2B r2, C: 61.44; H: 6.53; N: 4.78
Found, C: 59.89; H: 6.23; N: 4.52
58. Preparation of l ,6 -b l s [ ( 2 ,5 - d im e th y l ) p y r r o l id o m e t h y I ]
p h e n a n t h r e n e 150
Me
S' Me 2 7 Me
Me
1,6 -Bis(bromomethyl)phenanthrene (50.0 mg, 0.14 mmol) was heated
to reflux for three hours in EtOH (2 ml) with 2,5-dimethylpyrrolidine (0.035
ml, 0 .028 g, 0.29 mmol). After cooling overn ight, the solution was
evaporated and E t2 0 (20 ml) added. This was extracted with 10% HCl(aq) (20
173
E x p e r i m e n t a l
ml) then the aqueous layer was made basic by the addition of NH3(aq) and
extracted with E t2 0 (3x20 ml). Drying (M gS0 4 ) and evaporation of the
organic extracts gave the title amine as a yellow oil (27.0 mg, 0.07 mmol,
489%).
1h nmr (CDCI3): 0.90 (d, 12H, 3 J h .h = 5.5Hz, H5'); 1.17 (m, 4H, H41;
1.85 (m, 4H, H40; 2.74 (m, 4H, H3'); 4.05 (m, 2H,
H r ' ) ; 4.11 (s, 2H, H D ; 7.56 (d, IH, 3Jh _h = 7.6Hz,
H9 or HIO); 7.57 (d, IH, = 8.1Hz, H2); 7.68 (d,
IH, 3Jh _h = 6.8Hz, H9 or HIO); 7.74 (d, IH, ^Jh . r =
9.1Hz, H7); 7.82 (d, IH, ^j ^ . h = 8.1Hz, HI); 8.28 (br
d, IH, 3Jh . h = 8.8Hz, H6); 8.59 (s, IH, H4); 8.62 (d,
IH, ^Jh -H = 8.6Hz, H5)
Mass Spectrum (m/z): 400 [M+, 3%]; 302 [(M - C6Hi2N)+, 91%]; 206
[(C i6H i4)+ , 100%]; 205 [(C i6Hi3)+, 100%]; 204
[ (C i 6 H i 2)+. 75%]
UV (EtOH): Imax = 258.5 nm; e = 13100
Accurate Mass: Expected for C28H 36N 2, 400.2879
Found, 400.2882
59. Preparation of l , 6 - B i s [ ( 2 ,5 - d i m e t h y l ) p y r r o I id o m e t h y l ] p h e n a n t h r e n e
dimeth iodide 151
MeMe
5' Me 2 7 Me
+ N-
Me2 F
Me
174
E x p e r i m e n t a l
1,6 -B is [ (2 ,5 -d im e thy l)py rro l idom ethy l]phenan th rene (12.2 mg, 0.03
mmol) was stirred with iodomethane (0.12 g, 0.05 ml, 0.87 mmol) in
acetonitrile (1 ml) for 72 hours. After pouring into ether (200 ml), the
precipitate was centrifuged (x2000, 5 minutes) then the solvent decanted.
The moist solid was dried in a low vacuum, giving the title salt as an off-
white powder (10.3 mg, 0.02 mmol, 50%), m.p. >320 °C
nmr (d^-DMSO): 1.16 (d, 6H, 3 Jh .h = 5.9Hz); 1.43 (d, 6H, 3Jh_h = 5.6
Hz); 1.67 (m, 4H, H4'); 2.08 (m, 4H, H4'); 2.76 (s, 3H,
H6 ' or 6 "), 2.79 (s, 3H, H6 or 6 "); 3.67 (m, 2H,
H31, 3.95 (m, 2H, H3"); 4.80 (s, 2H, H I ') ; 5.15 (s,2H, H I" ) ; 7.71 (dd, IH, 3 % .^ = 7.8Hz, 4 % . ^ = l.OHz,
H9); 7.79 (d, IH, = 7.6Hz, H7); 7.91 (dd, IH,
^Jr-H = 7.6Hz, '^Jh-H = 0.8Hz, H6); 8.11 (d, IH, ^Jh-H
= 9.0Hz, HI); 8.20 (d, IH, 3Jh_h = 7.8Hz, HIO); 8.53
(dd, IH, 3Jh_h = 9.8Hz, ^ j ^ . h = 0.7Hz, H2); 9.04 (m,
IH, H4); 9.08 (d, IH, = 7.8Hz, H5)
Mass Spectrum (m/z): 415 [(M - CH])"*", 46%]; 318 [(M - CH3 - CgHioN)^,
52%]; 205 [(C i6H i 3)+, 45%]; 215 [M2+, 24%]; 204
[ (C i6H i 2)+, 60%]
UV (H2O):
Analysis (%):
X-max = 256.5 nm; 8 = 44900
Calculated for C30H 42N 2I2» C: 52.64; H: 6.19; N: 4.09
Found, C: 51.79;H: 6.30;N: 3.87
60. Preparation of 1 , 1 - ( 3 , 6 - d i m e t h y l p h e n a n t h r e n y l ) b i s ( l , 4 -
diazoniab icyclo [2 .2 .2 ]octane) dibromide 1?^
2Br*
175
E x p e r i m e n t a l
3,6-Bis(bromomethyl)phenanthrene (50.0 mg, 0.14 mmol) was stirred
in acetonitrile (5 ml). Addition of quinuclidine (0.16 g, 0.14 mol) caused the
dibromide to dissolve. After 10 minutes, a fine white precipitate was formed
and stirring was continued for further 24 hours. The reaction mixture was
poured into Et2 0 (100 ml) then centrifuged (x2000, 5 minutes), decanted and
evaporated to dryness in a low vacuum, producing the title salt as creamy-
white powder (52.8 mg, 0.09 mmol, 64%), m.p. >320 °C.
nmr (d^-DMSO): 1.86 (m, 12H, H4'); 2.06 (m, 2H, H5'); 3.57 (br t,
12H, 3 J h .h = 7.6Hz, H3'); 4.74 (s, 4H, H I ') ; 7.80 (d,
2H, 3 J h .h = 8.1Hz, H2, 7); 8.02 (s, 2H, H9, 10); 8.13
(d, 2H, 3Jh_h = 8.2Hz, HI, 8); 9.28 (s, 2H, H4, 5)
nmr (d^-DMSO): 19.5; 23.4; 54.0; 66.5; 126.5; 127.8; 128.7; 129.1; 129.5;
131.1; 132.4
Mass Spectrum (m/z): 507 [(M 4- 8 lBr)+, 95%]; 505 [M -k 79Br)+, 94%]; 425
[(M - H)+, 4%]; 315 [(M - C yH isN )^ , 3%]; 213 [M2+.
14%]; 204 [(C i6H i 2)+, 100%]; 112 [(CyHisN )^, 33%]
UV (HiO): Xrnax = 255.0 nm; e = 77080
Analysis (%): Calculated for C3oH3gN2Br2, C: 61.44; H: 6.53; N: 4.78
Found, C: 60.61; H: 6.60; N: 4.71
61. Preparation of T r ip h e n y l - p - x y l y l p h o s p h o n i u m bromide 121
PPh 3+ Br
This was prepared according to the literature method, giving the
title salt as a fine white powder in 70% yield, m.p. 265-270 °C (lit.^^ 276-277
°C).
176
E x p e r i m e n t a l
62. P r e p a r a t i o n O f - 2 , 4 ' - d i m e t h y l s t i l b e n e 1 2 2
Me14 Me
Sodium (1.38g, O.Oômol) was dissolved in methanol (150 ml) and then
triphenyl-p-xylyl phosphonium bromide (13.43 g, 30.0 mmol) was added
with stirring followed by o-tolualdehyde (3.60 g, 3.45 ml, 30.0 mmol).
Stirring was continued for three hours, then the crude product was isolated
by crystallisation at -78 °C (4.24 g). This was dissolved in hot cyclohexane
and filtered, then column chromatographed (CHClgiC^H 12, 0:1 rising to 1:10)
giving the title alkene as white crystals (1.39 g, 4.80 mmol, 16%), m.p. 44-47
°C.
nmr (CDCI3): 2.37 (s, 3H, HI); 2.43 (s, 3H, H14); 6.98 (d, IH,
= 16Hz, H6); 7.18 (d, IH, = 7.9Hz); 7.20 (m,
3H); 7.29 (d, IH, 3 J h .h = 8.0Hz); 7.43 (d, IH, =
16.0Hz, H7); 7.51 (d, IH, ^j ^ . h = 7.3Hz)
nmr (CDCI3): l9.9(tj);2J-3(Cs);l7b.2;125.5; 126.2; 126.4; 127.3; 129.4;
129.9; 130.3 (all Q ) ;
134.9; 135.7; 136.5; 137.5 (all Cq)
Mass Spectrum (m/z): 208 [M+, 100%]; 193 [(M - CH3)+, 52%]; 178 [(M -
2C H 3)+ , 52%]; 165 [(Ci3H9)+, 10%]; 115 [(CgH?)^,
32%]; 91 [(C7H7)+, 15%]
UV (EtOH): ^max - 302.0 nm, £ = 24370
Accurate Mass: Expected for C 16H 16, 208.1252
Found 208.1252
177
E x p e r i m e n t a l
63. P re p a r a t i o n of 1 , 6 - d i m e t h y l p h e n a n t h r e n e 123
Me
Me
7'ra/i5-2,4"-dimethylstilbene (0.13 g, 0.62 mmol) was d issolved in
cyclohexane (600 ml) with iodine (20.0 mg, 0.08 mmol) and irradiated for 6 hours. The reaction mixture was then washed with N a 2 S 2 0 g ( a q ) (200 ml),
d r ied (M g S 0 4 ) and evaporated in vacuo to give an orange oil. This was
column chromatographed (hexane, 67-70 °C) to give white crystals of the
phenanthrene (46.5 mg, 0.22 mmol, 36%), m.p. 85-87 °C (lit. 82.5-83.5 °C^,
87-88
1h nmr (CDCI3):
nmr (CDCI3);
2.65 (s, 3H, H I" ) , 2.77 (s, 3H, HT); 7.46 (m, 2H, H7
and H9 orlO); 7.55 (d, IH, ^Jr -H = 8 3Hz, H9 or 10);
7.79 (d, IH, 3Jh _h = 7.8Hz, H2); 7.82 (d, IH, 3J h _h =
7.9Hz, H8); 7.91 (d, IH, % . H = 9.0Hz, H3); 8.52 (s,
IH, H5); 8.60 (d, IH, = 8.4Hz, H4)
20.0; 22.2; 120.8; 121.8; 122.6; 125.8; 126.5; 127.6;
128.1; 128.3; 129.6; 130.0; 130.7; 130.9; 134.7; 136.2
Mass Spectrum (m/z): 206 [M+, 100%]; 191 [(M - % ) + , 65%]; 178 [(M
C 2H4)+, 14%]; 165 [(M - CH3 - C2H2)+, 10%]
UV (EtOH): A.max = 256.5 nm; 8 = 39640
Accurate Mass: Expected for C 16H 14, 206.1096
Found 206.1091
64. P r e p a r a t i o n o f l , 6 - b i s ( b r o m o m e t h y l ) p h e n a n t h r e n e 132
178
E x p e r i m e n t a l
Br
Br
1,6 -Dimethylphenanthrene (0.18 g, 0.87 mmol) was refluxed in CCI4
(10 ml) with N-bromosuccinimide (0.30 g, 1.70 mmol) and benzoyl peroxide
(10.3 mg) for three hours. After cooling and filtration, the solution was
evaporated to give a yellow solid which was recrystallised from EtOH to give
the dibromide as a cream coloured powder (130.8 mg, 0.36 mmol, 41%), m.p.
104-110 °C.
nmr (CDCI3): 4.79 (s, 2H, m ' y , 5.02 (s, 2H, H I ') ; 7.64 (d, IH,
H = 8.4Hz, H9 or HIO); 7.67 (m, 2H, H7 and H9 or
HIO); 7.90 (d, IH, ^ J ^ .h = 9.8Hz, H2); 7.93 (d, IH,
3Jh.H = 8.1Hz, H8); 8.12 (d, IH, ^ J ^ .h = 9.1Hz, H3);
8.70 (s, IH, H5); 8.73 (d, IH, ^ j ^ . h = 8.1Hz, H4)
13c nmr (CDCI3): 31.9; 34.2; 122.8; 123.4; 124.1; 126.4; 127.3; 127.8;
128.7; 129.4; 130.1; 130.5; 130.8; 131.6; 134.0; 136.2
Mass spectrum (m/z): 366 [ (Q 6 H i 2 8l B r 2r , 7%]; 364 [ ( C i e H n ’ ^B rS lB r) '
15%]; 362 [(C i6H i 2’ ’ B r2)+, 7%]; 285
[ (C i6H i 2* 'B r)+ , 100%]; 283 [(C i6H i 2"'’ Br)+,
100%]; 204 [(C i6H i 2)+, 35%]
UV (EtOH); A.max - 256.5nm; 6 = 36040
Accurate Mass: Expected for C i 6H i 2B r2, 362.9302
Found 362.9299
65 . P r e p a r a t i o n o f l , 3 - D i m e t h y l - 5 - ( b r o m o m e t h y l ) b e n z e n e 18 6
179
E x p e r i m e n t a l
M e Me
Mesitylene (1.90 g, 2.20 ml, 0.015 mol) was heated to reflux for four
hours with N-bromosuccinimide (5.34 g, 0.03 mol) and benzoyl peroxide (15
mg) in CCI4 (50 ml). The newly-formed succinimide was then removed by
f itra tion and the filtrate evaporated in vacuo. The crude product was
vacuum distilled (Kugelrohr) to give the title bromide as a colourless oil
(0.48 g, 2.30 mmol, 15%).
66 . P r e p a r a t i o n o f l - M e t h y I - 3 , 5 - b i s ( b r o m o m e t h y l ) b e n z e n e 187
Me
This was prepared acording to experiment 66 from 1,3-dim ethyl-5-
(brom om ethyl)benzene (1.00 g, 5.02 mmol), N -brom osucc in im ide (1.80 g,
10.10 mmol) and benzoyl peroxide (10 mg). The crude product, a sem i
crysta lline yellow oil, was column chromatographed ( 10:1 hexane:CH 2C l 2)
to give a fraction with Rf 0.49 which was evaporated to give white crystals
of the title dibromide (0.12 g, 0.45 mmol, 9%), m.p. 38-42 ®C (lit.^^ 41.5-42.5
®C).
180
E x p e r i m e n t a l
5.3 R e f e r e n c e s
( 1 ) Laarhoven, W. H.; Peters, W. H. M.; Tinnemans, A. H. A. Tetrahedron
1978, 34, 769.
( 2 ) Haworth, R. D.; Mavin, C. R.; Sheldrick, G. J. Chem. Sac. 1934 (I), 454.
( 3 ) Newman, M. S.; Lilje, K. C. J. Org. Chem. 1 9 7 9 ,4 4 , 4944.
( 4 ) Morgan, 0 . T.; Coulson, E. A. J. Chem. Sac. 1929, 2203.
( 5 ) Pepper, J. M.; Howell, M.; Robinson, B. P. Can. J. Chem. 1 9 6 4 ,4 2 , 1242.
( 6 ) Lai, Y.-H.; Peck, T.-G. Aust. J. Chem. 19 9 1 ,4 5 , 2067.
( 7 ) Klemm, I. H.; Kohlik, A. J.; Desai, K. B. J. Org. Chem. 1 9 6 3 ,2 5 , 625.
( 8 ) Du Vernet, R. B.; Wennerstrom, O.; Lawson, J.; Otsubo, T.; Bockelheide,
V. JACS. 1978, 100, 2457.
( 9 ) Staab, H. A.; Sauer, M. Liebigs Ann. Chem. 1984, 742.
(1 0 ) Cristol, S. J.; Caspar, M. L. J. Organomet. Chem. 1968, 33, 2020.
(1 1 ) Davy, J. R.; Jessup, P. J.; Reiss, J. A. J. Chem. Ed. 1975, 52, 747.
(1 2 ) Jacobs, W. A. J. Org. Chem. 1951, 16, 1593.
(13) Vogtle, P.; Zuber, M.; Lichtenthaler, R. G.; Chem. Ber. 1973, 106, 717.
181
Chapter 5
Nucleophilic Addition o f Pyruvic A cid Synthons
N u c le o p h i l i c A d d i t io n o f P y ru v ic A c id S y n th o n s
5. Nucleophilic Addition O f Pyruvic Acid Synthons
5.1 I n t r o d u c t i o n
The chemistry of carbonyl compounds with activated hydrogen atoms is an
e n o rm o u s ly d ive rse and syn th e t ic a l ly im p o r ta n t a sp ec t o f o rgan ic
chemistry. The phenomenon of a-hydrogen acidity in such compounds is
due to the keto-enol tau tom erism they undergo and the subsequen t
resonance stabilisation of the resulting depro tonated anion 1 8 9 (figure
5 .1 ) .
O
R ' Rl H H
18 8
OR,
H
O'
18 9
R Ri
H
F i g u r e 5.1
Historical attempts at generating the a-an ions of carboxylic acids as shown
were largely unsuccessful since decomposition usually occurs under the
cond it ions needed for their form ation. However, recent advances have
made this a facile process for many acids and this introduction provides a
brief overview of the history of such chemistry, together with a review of
the enolate chemistry of synthons of pyruvic acid, a compound which has
so far eluded such advances.
5.1.1 C a rb o x y l ic A cid D ia n io n s
The modern chemistry of carboxylic acid dianions can be traced to an
experim ent in 1938 by Morton, Fulwell and Palmer^ which inferred the
form ation of the a -a n io n of phenylaceta te and hexanoate anions using
p heny lsod ium as base. Thus, reactions with carbon d iox ide produced
p h e n y lm a lo n ic - 1 9 1 and butylm alonic-acids in yields of 60 and 17%
respective ly (scheme 5 .1 ) .
183
N u c le o p h i l ic A d d i t i o n o f P y r u v ic A c i d S y n th o n s
19 0 19 1
(i) CO2
(ii)
H COOH
19 2
S c h e m e 5.1
The pheny lace ta te dianion 1 9 1 was subsequently form ed, in 1956, by
H auser and Chambers^ in a system that consisted of sodium or potassium
amide in liquid ammonia (scheme 5 .2 ) .
H H
2KNH2, NH3(1)
19 0 19 1
S c h e m e 5.2
H rOOHC6H5CH2C 1
Later still in 1958, DePree and Closson^ formed the dianion of acetic
acid 195 using sodium amide at approximately 200 °C (scheme 5 .3 ) .
O
M e ^ O - N a ^ +200 °C
1 9 4
O' Na+
H2C ^ O' Na+
19 5
S c h e m e 5.3
Under these rather forcing conditions, sodium amide acts as both base and
solvent, and the workers claim to have isolated the salt as a dry-air stable
solid which reacted with standard reagents such as benzyl chloride or
carbon dioxide.
H ow ever, the inherent d isadvantages o f such system s, v iz . , the
ex p e r im en ta l d iff icu lty or the in stab il i ty o f the d ian ions under the
p reva i l ing conditions m eant that the syn the tic op p o r tu n it ie s of such
potentially useful chemistry was not explored for a num ber o f years. In
1967 how ever , Creger^ published a paper detailing his use of lithium
diisopropylam ide (LDA) in tetrahydrofuran (THF)Zhexane solution to form
184
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c i d S y n th o n s
the a -a n io n s of sec-butanoic acid 197 and 2-methylbutanoic acid and their
subsequent alkylkation in high yield (scheme 5 .4 ) .
Y RM e /' COOH -------------------^ M e'/^C O O ' Na+ — M e”/ COOHMe Me (ii) H Me
1 9 6 1 9 7 41-89% 1 9 8
S c h e m e 5.4
Though his method was limited in scope (yields of alkylated products from
other straight chain or a -b ranched carboxylic acids were found to be low
in the region of 30-60%), his demonstration of the use of LDA changed the
course o f such reactions and soon, a whole chem istry was constructed
around the use of similar strong, organic-solvent soluble bases. The success
of such bases (typically hindered lithium amides which are easily produced
by the reaction of bu ty ll i th ium with the appropria te am ine in THF
solution) can be considered as arising from three factors:
(i) they are soluble in aprotic solvents, with which no complexation
takes place, and this aids the observed, almost quantitative, metallation of
the a - c e n t r e ^ ;
(ii) the steric hindrance of the groups on the amide reduces the
nucleophilicity of the base, as well as the amine subsequently formed, and
reduces com petitive s ide-reactions^;
( i i i) the low tem pera tu res needed fo r the r e a c t io n p reven t
d eco m p o s i t io n (and, as later d iscussed , reduce the r isk of C la isen
c o n d e n s a t io n s ) .
A significant improvement to Creger’s system was made by Pfeffer
and Silbert who, noting that formation of the lithiated dianion of straight-
chain carboxylic acids produced a cloudy, he terogeneous solution, added
the highly polar aprotic solvent hexamethylphosphoram ide (HMPA) in the
belief that this would facilitate dispersion of the dianion. This produced a
clear solution and immediately raised yields to 90% or grea ter^’ The rôle
that the co-solvent plays can be inferred from the observation that best
results are obtained when it and the dianion are in a 1:1 molar ratio; it is
know n that o rg an o l i th iu m com pounds form h igh m o le c u la r w eight
a g g r e g a te s ^ and that for lithium dianions in THF, polymers may be formed
185
N u c le o p h i l i c A d d i t io n o f P y ru v ic A c id S yn th o ns
where n = 65 - 250 (accounting for their colloidal state)^. HMPA would thus
seem responsible for disrupting the aggregated species and forming what
has been d escr ibed as “ an assoc ia tion be tw een one m olecu le of
hexam ethylphosphoram ide and the counter-ion in a solvent-separated ion-
pa ir^® ” . It is interesting to note however, the deleterious effect that HMPA
has on reactions of dianions of a-branched acids. These are soluble in THF
and addition of the co-solvent produces a sharp decrease in yield as the
length of the side-chain is increased, accompanied by an increase in the
formation of the alkene derived from the alkyl halide. HMPA is somehow
thought to amplify the steric effect of branching but the method of its
agency is unclear.
Concurrent with the use of lithium amides was a second method
which, though not reaching the popularity of the first, has nonetheless
been found to be useful in a number of cases. It was devised by Normant
and Angelo who used as their base aromatic radical anions formed by
d is s o lv in g sod ium in n ap h th a len e or p h e n a n th r e n e ^ ^ . Yields of a -
a lkylated products were quite low for many aliphatic acids but satisfactory
for aromatic acids, such as phenylacetic acid (scheme 5 .5 ) .
^ " Y ^ C O O H (ii) ‘PrBr k j
1 9 0 1 9 9
S c h e m e 5.5
Later, Angelo showed that a similar system could be extended to
include reactions with carbonyl compounds to produce (3-hydroxy acids
(scheme 5 .6 ) .
O (i) L i , lQ lJ , THF, 50-60 °C----------------------/---\ D H
M e - ^ O H (ii) 0 = 0 \ ---0
2 0 0 38% 2 0 1 COOH
S c h e m e 5 .6
186
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c i d S y n th o n s
He found tha t the l i th iu m /n a p h th a le n e sy s tem was p re fe ra b le to
l i th iu m /p h e n a n th re n e or sod ium /naph tha lene sy s tem used b e fo re and
allowed him a greater degree of success with purely aliphatic acids.
5 .1 .2 P y r u v ic A c id
P y ruv ic ac id or 2 -oxo -p ropano ic acid is a c o m m on m o lecu le in
p h y s io lo g ica l processes where, in its enol form , ( f ig u re 5 . 2 ) it is
considered to be a key intermediate in enzymatic reactions catalysed by
py ru v a te k inase , phosphoeno lpyruva te c a rb o x y lase , p y ru v a te phospha te
dikinase, malic enzyme and oxalacetate decarboxylase^^.
0,0H
MeO
2 0 2
OH.OH
H jCO
2 0 3
F i g u r e 5.2
Though the enol form has been generated in solution and is reasonably
stable ( t i /2 = 7 minutes at 20 °C in D2 O s o l u t i o n ^ t h e acid has proved
uny ie ld ing to direct a lkylation using both c lass ica l and more modern
methods. The reasons for this are unclear. Tapia et aZ."^suggest that this may
be due to the insolubility of its dianion in organic solvents, but given the
rem arkab le efficacy of HMPA in solvating a - l i t h i a t e d s t r a ig h t - c h a in
a c id s^ , this seems puzzling. As a consequence of these apparent limitations,
a num ber of synthetic equivalents of pyruvic acid have been prepared.
These vary in their degree of complexity, but all can be regarded as
m olecules of pyruvic acid with their chemistry m odified so that, after
reaction and suitable deprotection, three carbon hom ologation is observed.
Most recently, Tapia et al.^^ showed that conversion of the a - k e t o
function of the acid to its d imethylhydrazone derivative 2 0 4 fac ili ta tes
formation of the dianion 2 0 5 (scheme 5 .7 ) .
187
N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S yn th o n s
O
MeOH M6 2NNH2
O
2 0 2
E^O
N “ U+OH 2MeLi, THE, HMPA, 0 °C
O' LtMe
2 0 4 2 0 5
S c h e m e 5.7
This dianion is a strong nucleophile , reacting with a variety of alkyl
halides as well as aldehydes and ketones in reasonable yield. An added
advantage is that deprotection proceeds spontaneously in the acidic w ork
up (scheme 5 .8 ) .
Me2NN ~ L i +
o2 0 5
OH+
40%
OH
2 0 6
Q d
2 0 7
S c h e m e 5.8
Similar work has been carried out by W illiams and Benbow ^^, who
used tert-butyl esters of pyruvate oxime ethers 2 0 8 to effect C-alkylation
(scheme 5 .9 ) .
M eO . ^O . MeO^ , 0 .N
MeO^Bu
02 0 8
(i) 3LDA, THF, -78 °C
(ii) RBr
5 1 -7 7 %
RN
0 ‘Bu
O2 0 9
S c h e m e 5.9
Three interesting points emerge from this work:
(i) three equivalents of base were needed (use of two equivalents
yielded only unreacted starting material);
(ii) use of the parent oxime gave complete decomposition under a
variety of conditions;
188
N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S y n th o n s
(iii) excess alkylating agent was needed and only benzylic or allylic
h a lid es reac ted su ff ic ien tly rap id ly to c om pe te w ith d eco m p o s i t io n
p a th w a y s .
The problem in (i) was overcome by the addition of two equivalents
of lithium bromide prior to reaction, followed by 1.5 equivalents of base.
This led to good yields, so the workers assumed that the oxime ethers must
have coordinated to two equivalents of lithium base in unreactive clusters
prior to deprotonation.
Classically, oxalacetic acid has been used as a pyruvate equivalent.
C o r n f o r t h , F i r th and G o t t s c h a lk ^ ^ a t te m p te d sy n th e s is o f N-
a c e ty ln e u ra m in ic acid (N A N A , 212.) by the aldol condensation of N-
acetylhexosamine and pyruvic acid, but observed no reaction. By replacing
pyruvic acid with oxaloacetic acid 211 however, in the expectation that the
more reactive methylene group would help drive the reaction, it was found
to be successful, though it proceeded in very low yield. The superfluous
carboxyl group was lost in the pH-regulated medium they used (scheme
5 .9 ) .
CHOH-
HO-H-H-
■NHAc ■H OH OH
O
OH "O2CO2'
CH2OH
210 211
(i) p H 9 - * i l , 2 3 °C, 48hr
(ii) pH 6.8, 0“C
-CO2
S c h e m e 5.9
HH*^ H
HO H H
ÇO2'= 0
HOH NHAc HOH OH
CH2OH
212
A more elaborate approach was developed by Schmidt and B e tz^^’^^.
The first step in their synthesis of the related system 3-deoxy-D-manno-2-
octulosonic acid (KDO) was the reaction of their pyruvate synthon, 2-
benzyloxy-3-(phenylth io )acry lic acid N -m ethylam ide 2 1 3 with 2 ,3 :4 ,5 -d i-0 -
isop ropy lidene-D -arab inose (schem e 5 .1 0 ) .
189
N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S yn th o n s
HNMe
2 1 3
LDA, THF, HMPA, -80 °C
CHO
Ov MeMe O
O Me
M e. P"«
Q, MeMe O
O Me2 1 4
S c h e m e 5 .10
They chose such functionalisa tion fo llow ing investiga tion of a - a l k o x y
s u b s t i tu te d a c ry la te s and fou n d tha t a p - t h i o s u b s t i t u e n t and
m onosubs t i tu ted amide group supported p -carbon lithiation, as well as
in c re a s in g n u c leoph il ic r ea c t iv i ty and d ia s te re o s e le c t iv i ty . Q uench ing
r e v e a le d tha t , in the p re s e n c e o f tw o e q u iv a le n ts o f l i th iu m
diisopropylamide, formation of the dianion was practically quantitative. A
further advantage was the ease of deprotection: heating in high-boiling
petrol followed by treatment with Raney nickel produced good yields at
each stage.
5.1.3 A im s O f T he P ro je c t
3 -D eo x y -D -m an n o -2 -o c tu lo so n ic acid (KDO, see p rev ious section) is
currently the subject of much research interest since it is found in the
lipopolysaccharides (LPS) of all gram -negative bacteria which have been
s t u d i e d ^ ^ . The KDO residues are situated at the reducing ends of the
polysaccharide domains, linking them by ketoside bonds to the fatty-acid
subs ti tu ted 2-am ino-2-deoxy-D -g lucosy l d isaccharides know n as lipid A.
Figure 5 .3 is a block diagram indicating the location of KDO in the LPS
from Salmonella. The incorporation of KDO appears to be an vital step in
LPS biosynthesis and indeed in the growth o f gram negative bacteria,
accounting for the enormous in terest in the b iochem istry and synthetic
carbohydrate chemistry of KDO and derivatives. KDO itself is thought to be
form ed in vivo from D -arabinose-5-phosphate and phosphoenol pyruvate
p r e c u r s o r s .
190
N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S y n th o n s
£iN^£IN p:
NH-P~
(121NH- (U)
P~
- N H
0 i i d e chain Outer core Di h e p lose (KDO)j region reg ion
L i p i d A
■(Hi-■(12)-
- d i ) -i l SJ-
3 - OH' t e t r o d e c a n o ic dodecanoie te t r a d ec o n o c he»adecano<
k acid re i idv ts
B lock diagram representing the con stituents o f the gram -negative bacterial lipopolysaccharide. The inside o f the cell is at the right and the suroundings o f the cell at the left o f the drawing. The diheptose-K DO region is som etim es referred to as the "inner core". P' represents phosphate groups (from ref. 19).
F i g u r e 5.3
S yn the tic approaches to KDO have the re fo re ty p ic a l ly invo lved the
biom im etic addition of some pyruvate equ ivalen t to p ro tec ted arabinose
d e r iv a t iv e s .
COOHOHHO
OHA c
HOOH
N A N A 2 1 2 -
COOHOHHO
OH
HOOH
KDN 2 15
F i g u r e 5 .4
C rich ^ ® has postulated a route to the related sytems N-acetylneuraminic
acid (NANA, 2 1 2 see scheme 5 .9 and figure 5 .4 ) and 3-deoxy-D -m anno-2-
191
N u c le o p h i l i c A d d i t io n o f P y ru v ic A c id S y n th o n s
nonulosonic acid (KDN, 2 1 5 ) (figure 5 .4) which includes a similar addition
of some pyruvate equivalent as the initial step (scheme 5 .1 1 ) .
Me
Me O
[H]
COOH
f" CH2CCOOR" /
Me
CHO
MeMe Me
2 1 6 2 17 2 18
S c h e m e 5.11
Our brief was therefore to investigate conditions under which pyruvic acid
or synthons thereof could be used to facilitate this initial reaction.
192
N u c le o p h i l i c A d d i t i o n o f P y ru v ic A c i d S yn th o n s
5.2 R e s u l ts A nd D isc u ss io n
Given the apparent absence in the l i te ra tu re re la t ing to the d irec t
alkylation of pyruvic acid, this seemed to be a good starting point for
investigations into the pyruvic acid system. Though the failure of standard
m ethodolog ies is im plic it in certain papers^ in itia l attem pts centred
around a mixed THF/hexane/HM PA solvent and lithium diisopropylamide as
base (scheme 5 .1 2 ) .
OMe OH
OJ L OH (i) LDA. THF, HMPA
» N2(g), -78 °CO (ii) Mel L O
2 0 2 2 1 9
S c h e m e 5.12
Several attempts were made, using iodomethane as the alkylating agent, but
all resulted in a complex mixture of products. The H nmr spectra of the
c rude p ro d u c ts were not p a r t ic u la r ly h e lp fu l in " id e n t i fy in g the
com ponents ; com plete decom position of the subs tra te had apparen tly
occurred since there were no signals relating either to pyruvic acid [5 2 .5 2
(s, 3H); 58.31 (s, IH)] or the supposed product, 2-oxo-butanoic acid [51 .04
(t, 2H); 52.84 (q, 3H) and a broad downfield singlet]. Inspection of the
aqueous layer by saturation with sodium chloride, fo llowed by extraction
with E t2 0 revealed only the presence of HMPA. A sim ilar experiment,
conduc ted w ithou t the use o f the co -so lven t p ro d u ced a s im ila r ly
unremarkable result, though peaks due to pyruvic acid are present among
the num erous peaks presum ably due to decom position in the H nm r
spectrum of the product.
We checked that this failure was not due to procedural error by
repeating the reaction using standard procedure on a known substra te^.
Thus, alkylation using 1-bromobutane, of octanoic acid 2 2 0 at 0 °C gave 2-
butyloctanoic acid 221 in 42% yield (scheme 5 .1 3 ) .
193
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c i d S y n th o n s
(i) LDA. THF ,HMPA, N2(g),0 °C
(ii) CH3(CH2)3Br
2 2 0 2 2 1
S c h e m e 5 .13
HMe
All analy tica l details support the s tructure and though the y ield was
rela tively low, it clearly demonstrated the correctness o f the method. A
repeat run was therefore carried out on pyruvic acid under precisely the
same conditions, but the result was the same as for previous reactions of
this type. The mass spectrum is dominated at high m/z by HMPA but at
low er values, strong alkyl signals are present, perhaps ind ica ting the
presence of octane, form ed in a s ide-reaction by the attack of excess
b u ty ll i th ium on brom obutane.
A tten tion was then turned to rela ted pyruvic acid system s. The
m odulating efect that estérification with bulky alkyl groups has on the
reactivity of acids in the alkylation reaction (as well as their propensity to
undergo self-condensation) is well noted 15,22 this, together with the
fac t that Ley and co-w orkers have pub lished w ork on the term ina l
acylation of P-ketothioesters such as 2 2 2 23 ,24,25 (scheme 5 .1 4 ) led us to
believe that similar a-ke to th ioeste r systems may prove susceptible to anion
f o r m a t i o n .
(i) NaH, DME, 0 °CO O (ii) nBuLi. DME .0 °C H O H O O
(ii)
2 2 2 (iii) If 2 2 383%
S c h e m e 5 .14
L e y ’s route was unfortunately not applicable to com pounds of this type
since his procedure involved the attack on diketene o f sodium thio-tert-
butoxide 2 2 5 as shown in scheme 5 .15 .
194
N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c id S y n th o n s
MeNaH, THF, 0 “C
M e ' / ^ S HMe
2 2 4
Me
M e ^ '/^ S 'N a '"Me
2 2 5
H2C
O
O O
63% M e " " ^ ^ ^ ^ ^ S ‘Bu
2 2 3
S c h e m e 5 .15
Therefore, a classical estérification route was chosen, i. e., the addition of
the acid chloride to the thiol, the standard method for preparing thio-
c a rb o x y l ic e s t e r s ^ P y r u v o y l chloride 2 2 8 is a reasonably stable liquid
which resists synthesis from the acid using standard reagents such as
phosphorus trichloride, phosgene, thionyl chloride or oxalyl chloride
The acid chloride has been synthesised from trimethylsilyl pyruvate^ but
the m ost successful synthesis in the literature is due to Ottenheijm and
DeM an who suggested oc,a-dichloromethyl methyl ether 2 2 6 as a suitable
chlorine source (scheme 5 .1 6 ) .
^OH A,30minM e' V + CI2CHOCH3 --------------
O2 0 2
HCl2 2 6
Me
O
O
2 2 8
OO. ,OMe
Me ) <q CI h
2 2 7
OCl + ¥
H OMe
2 2 9
S c h e m e 5 .16
The c h lo r in a te d e ther is o f ten avo ided on accoun t o f its acu te
carcinogenicity, but, it was claimed, was able to give yields of up to 51% in
this reaction. We found that some modification was neeeded to reach these
yields, for instance, p ro longing the reaction time and conducting the
experim ent under a nitrogen atmosphere. Also, the sole H nmr signal,
supposedly at 52.59 in CDCI3 was found actually to be 52.48, but the synthesis
195
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c id S yn th o n s
was nonetheless successful and a typical run appears in the experimental
section of this chapter.
Despite the noted lability of the acid chloride, the reaction with 1,1-
d im e th y le th a n e th io l 2 3 0 proved to be much less facile than anticipated.
Fractional distillation of the thiol was found to be necessary because it
consisted of several compounds, exhibiting several spots on a TLC plate
(this is a noted problem with tertiary thiols and in the absence of base,
yields for the reaction were tiny. Triethylamine was eventually found to be
the most successful base and the novel thioester 2 3 1 was obtained as a
yellow oil in reasonable yield (scheme 5 .1 7 ) .
EfaN, EtzO, A
S c h e m e 5.17
A series of a ttempted alkylations were then carried out on the
thioester under a variety of conditions. LDA was the first base chosen with
the entire reaction carried out at -78 °C. Once again, only the decomposition
products were evident in the crude reaction mixture, the only signals in
the nmr spectrum, apart from those due to HMPA, appearing in the alkyl
region. This is perhaps not surprising, since a study on the synthetic
u til ity o f a series of th ioes te r enolate anions inc lud ing ter t-bu ty l
thioacetate carried out by Edwin Wilson and Hess^®, revealed no reaction of
the m agnesium or lith ium enolates with iodom ethane or m ethyl vinyl
ketone at 0 °C, though the Claisen condensation product was observed in
both cases (scheme 5 .1 8 ) .
9 ( i ) ‘PrMgBr, EfeO, THF, -20— 0 °C/100min O O MeMe
Me (ii) îT, H2OMe
Me2 3 2 2 2 2
S c h e m e 5 .1 8
196
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c id S y n th o n s
No explanation for this preference for self-condensation at the expense of
alkylation was provided so we sought an alternative base.
The proven efficacy of lithium isopropylcyclohexylamide (LICAD) in
generating the a -an ions of a wide variety of esters was demonstrated by
Rathke and Lindert ^, so it was tried next using standard conditions on the
same thioester. This method produced a small quantity of yellow solid which
is almost certainly not the desired product, since the carbonyl stretch in
the infra-red spectrum is negligibly weak and in the wrong region besides.
Its id en t i ty rem ains unc lear. The mass sp ec tru m has a com p lex
f ragm en ta tion pattern (a pers is ten t problem with these system s, with
neither El or FAB methods giving satisfactory results, even with otherwise
fully characterised compounds), so is not useful in helping to identify the
product of this reaction.
A repeat run using the same base produced a further quantity of
crystalline material though with a markedly different melting point (>200
°C against 126-130 °C, the decomposition point of the material yielded in the
previous experiment). The H nmr spectrum is not helpful in identifying
this material but reveals that the solid is contaminated with the thioester
w h ich w h e th e r re g e n e ra te d or u n re a c te d , im p l ie s th a t c o m p le te
decomposition of the substrate has at least been prevented. The infrared
spectrum confirms this presence with a strong signal at 1718 cm" ^
However, neither of the two samples yielded to recrystallisation and
remain unidentified. Use of the base was nonetheless continued for the
reasons given above and the reaction of the thioester with an aldehyde was
investigated. This was chemistry more relevant to the aim of the project
since the first step of the Crich synthesis involved, as d iscussed, the
addition of some pyruvate dianion equivalent to a sugar derivative The
aldehyde chosen in this case was benzaldehyde in order to add some detail
to the previously empty lowfield part of the spectrum (scheme 5 .1 9 ) .
O
Me
(i) LICAD, THF, HMPA
Me ^2(g). -78 °C
O Me Me (ii) PhCHO
2 3 1
S c h e m e 5 .1 9
H OH ?
O M e Me
2 3 3
197
N u c le o p h i l i c A d d i t io n o f P y ru v ic A c id S y n th o n s
The result of this experiment, which, like all those previously, was
carried out at -78 °C, was a mixture of unidentified products, none of which
appeared to be the desired aldol. The 200 MHz H nmr spectrum includes a
profusion of weak signals between 50 and 55 whose origin is unclear whilst
the absence of the normally strong tert-butyl peak around 51.4 is striking
and perhaps indicates that some kind of hydrolysis may have occurred.
D e u te ra t io n and c a rb o n y la t io n are s ta n d a rd te c h n iq u e s fo r
determining the existence and degree of formation of the a - a n i o n s ^ and
water may be used to the same end to reform the substrate in situations
where extensive decomposition is observed to occur. The fact that simple
hydrolysis of the reaction mixture in the experiment above produced only
negligible amounts of starting material meant that we embarked upon the
s y n th e s is o f a new, m ore h indered th io e s e te r . 1 ,1 -D ie th y lp ro p y l
th iopyruvate ( tert-heptyl thiopyruvate) was chosen for the same reasons
as before and its synthesis followed a similar route with the exception of
the fact that the appropriate thiol 2 3 6 was com m ercially unavailab le and
had to be synthesised from the alcohol 2 3 5 . The alcohol i tse lf was
synthesised from 234 by addition to ethyl magnesium bromide in good yield
(>90%) whilst the thiol was prepared in approxim ately 50% yield in a
s tandard substitution reaction using a method after Barton and Crich^
(scheme 5 .2 0 ) .
Me
O (i) Et MgBr, Et^O, A (i) HiS, CH iCh
(ii) P f, H iO (ii)93% 51%
2 3 4
OM eC (0)C 0C k Et,N ^N 2 (g ),E t2 0 , A, 1 h ^
52% Me
2 3 7
S c h e m e 5 .20
198
N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S yn th o n s
The novel thioester 237 was obtained as a sweet-smelling oil in yields of up
to 55% and gave consistent spectral data. It is pertinent at this point to talk
of the nature of the carbonyl stretch in these compounds . In any a , p -
unsa tu ra ted system, two conform ations are possib le and two peaks are
therefore seen in the in fra -red corresponding to the two conform ers
where the unsaturated systems are disposed s-cis or s-trans to one another.
The lower of the two is always assumed to be due to the s-trans form since
delocalisation is expected to be greater. Spectroscopic and crystallographic
data of a -d ik e to systems however, indicates that the s-trans form of the
com pounds is the stable conformer^ Hence, we should only expect one
band in the infra-red. There is also little difference observed between the
IR and R am an ac tive f r e q u e n c ie s ^ ^ , in d ic a t in g l i t t l e m e c h a n ic a l
interaction, (presumably as a result of a fairly weak central C-C bond and a
low degree of conjugation). In pyruvic acid, the two carbonyl groups
absorb at 1745 c rn 'l and appear as one peak and all the systems synthesised
in this project exhibit single absorptions in the carbonyl region of the
i n f r a r e d .
R eac tion o f the th ioes te r with b rom obu tane us ing L ICA D in
T H F /H M P A at -78 ®C p roduced a very e n co u rag in g resu lt . A fte r
chromatography of the mixture, a clear oil was obtained in an apparently
good yield of 47%. The 400 MHz ^H nmr spectrum of the compound seems to
support fo rm ation of the product, 1 ,1-diethylpropyl 2 -oxo-th iohep tanoa te
2 3 8 (scheme 5 .2 1 ) and the IR spectrum shows a strong carbonyl stretch
(similar to the thioester at 1718 cm '^ ) .
OOÏ (i) LICAD, THF, HMPA
Me N2(g). -78 °C Me" ^ ^ ^ Me
Me (") CH3(CH2)3Br ^ ^Me
2 3 7 2 3 8
S c h e m e 5.21
The C nmr spectrum shows only a slight sign o f the two low-field
carbonyl peaks one would expect but the spectrum of the parent thioester
shows these to be very weak. The mass spectrum gives c lear alkyl
f ragm en ta tion from butyl dow nw ards, as well as as the te r t-hep ty l
199
N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S yn th o n s
fragm ent, a fragm ent due to ester cleavage [(C2 H 5 ) 3 0 8 ■*■] and a weak
molecular ion peak at m/z 258. It was found that formation of the presumed
product was arrested if the temperature of the reaction was allowed to rise
to 0 °C prior to the addition of the euectrophile and we had therefore
established, we hoped, a working procedure for the reaction. However, we
were not able to capitalise on this success when the reaction of the
th ioester with benzaldehyde under the same conditions as before produced,
after chrom atography, an oil whose H nm r spectrum was com plete ly
devoid of aromatic signals and whose IR spectrum showed no aromatic
stretches and only a weak carbonyl stretch at 1715 cm-1.
The loss of all signals in the nmr spectrum apart from those in the
alkyl region in this reaction seemed to indicate that alkane formation was
becom ing a prevalent side reaction. It was decided therefore to use 1-
bromodecane as the quenching agent for two reasons:
(i) the formation of any alkane would hopefully lead to material of
h igh enough m o lecu la r w eigh t to g ive a c lea r and c h a ra c te r is t ic
fragm entation pattern in the mass spectrum;
(ii) the higher molecular weight would also give more material by
w e i g h t .
In the subsequent reaction, carried out entirely at -78 ®C, none of
the desired product was apparently formed. The sample displays a triplet at
52.30 in the nmr spectrum, perhaps due to a pair of methylene protons a
to the diketo system (the singlet in the parent thioester occurs at 52.33 and
the a -p ro to n s of bromobutane as a triplet at 53 .40) but once again, no
signals due to the carbonyl groups or the carbon atoms to which they are
attached are present in the infra-red or nmr spectrum.
It was unclear exactly what had been occurring in the preceding
reactions and it was difficult to make sense of the conflicting data. It
seemed fairly reasonable however, to assume that, had the reactions been
working at all, they were doing so in yields that were undetectable and this
led us to change the base system under investigation. The system of Tapia
et al. uses methyllithium as base and the entire reaction is carried out at
0 There is tacit acknowledgm ent of the failure o f this system to
genera te the pyruvate dianion within the paper and the use of the
d im ethy lhyd razone presum ably arose from this observa tion . It becam e
clear however, when we tried to use the system on our thioester that the
200
N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c id S yn th o n s
gain in stability of the dianion caused by functionalisation of the a - k e t o
group and which allowed such relatively mild conditions to be employed,
was not m atched by estérification; tert-hepty l th iopyruva te was reacted
under these conditions and quenched with a tw o-fold excess of various
e le c tro p h i le s inc lud ing b rom odecane , b en z a ld e h y d e and b ro m o b u tan e ,
p ro d u c in g in each case only the r e g e n e ra te d th io e s te r and the
electrophile . Quenching with water produced, after work-up, only an oil
with an IR spectrum identical to the thioester. M odification of the a - k e t o
function, by far the most common m ethod of genera ting pyruvic acid
synthons, would thus seem to be a more effective remedy than the remote
functionalisa tion we had been employing up to this point, though the
single success with tert-heptyl th iopyruvate suggested that perhaps the
two strategies could be made to work in concert.
Enolates have for some time been trapped as their trialkylsilyl enol
ethers, in the expectation that the ether function would be readily cleaved
by a num ber of organometallic reagents to generate a h ighly reactive
enola te , and are easily prepared by the reaction o f the ketone with
trialkylsilyl chlorides in the presence of any of a num ber of bases^^ . A
large, stereospecific chemistry now exists for reaction of these compounds
with electrophiles and there seemed no obvious reason why pyruvic acid or
its derivatives would not be amenable to at least formation of the silyl enol
e th e r , e s p e c ia l ly in view of the s ta b i l i ty o f the d e p ro to n a te d
d im ethy lhydrazone of Tapia et a l^^ . The b is-tr im ethy ls i ly l derivative of
pyruvic acid has been synthesised and was the source of some interesting
kinetic data which deduced that ketonisation of the c leaved ether was
s u f f i c i e n t ly s lo w ^ ^ (see section 5 . 1 .2 ) to suggest that reaction of the
nascent enolate was a real possibility. For our first attempt at synthesis of
the t r im e thy ls i ly l derivative of te r t-hep ty l th iopyruva te , we retu rned to
our original system and attempted generation of the lithium enolate at -78
°C using lithium diisopropylamide in THF, fo llowed by quenching with
ch lo ro tr im e thy ls i lane (TM S-Cl). The fact that this produced only the
th ioeste r was further evidence that the lithium enolate, were it being
generated at all was insufficiently stable under the reaction conditions to
react. A m ore conventional system was then em ployed. Follow ing the
method of House et. al. triethylamine (EtgN), then TMS-Cl were added to a
201
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c id S yn th o ns
solution of the thioester in dimethylformamide. After two hours’ stirring,
extraction with hexane gave a low yield of the silylated enol 2 3 9 (scheme
5 .2 2 ) , easily detectable in the nmr spectrum of the unpurified reaction
mixture because of the pair of doublets around 55.0 due to the newly-formed
pair of vinylic protons (see Appendix, p. 222).
O
Me
O
2 3 7
S ^ ^ E t EtgN, M c 3 SiCl, DM F
E t E t N i(g ) , 2 hours
50%
0SiMc3
o E t E t
2 3 9
S c h e m e 5.22
This material proved to be surprisingly resistant to hydrolysis, unaffected
by D2O after three hours at room temperature or by stirring in water at pH4
for two hours, and this allowed us to employ an aqueous work-up which had
the twin advantage of removing any residual solvent and dissolving the
amine salt which was always formed. Thus, by extracting the mixture with
hexane, then washing with H2O and drying briefly over MgSO^, yields of up
to 54% were possible. The pure material was however unstable over days in
air, so was stored at -20 °C in hexane solution and used in this form. For the
reaction of this compound with electrophiles, we intended to follow the
methods of House et. and Rasmussen^^ which involve metathesis of
the silyl enol ether with methyllithium followed by reaction in situ with an
alkyl or benzyl halide. The reaction was m odified to account for the
p robab le decom position of the anion under the ref lux ing cond it ions
employed by House, and was carried out at -40 °C. A quenching reaction was
the first to be tried (scheme 5 . 2 3 ) which regenerated the th ioester as
expected, but in less than quantitative yield due to decomposition.
OLiE t MeLi, DME
-40°C, H (
40 mins
2 4 02 3 9
HiO, A
30 m ins
34%
H 3C
O Et Et
2 3 7
S c h e m e 5 .23
202
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c id S y n th o n s
This became the overriding pathway when repeated with benzaldehyde and
no product could be detected in the crude reaction m ixture, despite the
disappearance of all nmr signals relating to the silyl enol ether. The
aqueous layer was examined also in the unlikely event that the target aldol
was w a te r - so lu b le , but the p ro d u c t appeared to c o n s is t only o f
benzaldehyde and regenerated thioester.
M u k a iy a m a and c o - w o r k e r s ^ ^ d isc o v e re d tha t t i tan iu m
te t r a c h lo r id e (T iC U ) could be used to promote the reaction of ketone
trim ethylsily l enol ethers with ketones or aldehydes w ithout the problem
of self-condensation that limits the usefulness o f o ther aldol syntheses.
They proposed that the reaction proceeded via the form ation of the
titanium enol ether 2 4 2 (scheme 5 .2 4 ) which added to the electrophile to
give the chelate 2 4 3 .
OSiMea
2 4 1
OTiCl]
R i ' ^ C R j Rs
2 4 2
+ MegSiCl
OI I
R4 CR5
Cb
RiR2 R3
2 4 3
R4
R5
HiO OH
Ri
2 4 4
+ TiCyOH
Sc h e m e 5 .24
Hydrolysis yielded the desired product. Other Lewis acids were later found
capable of promoting the reaction^ \ but we decided to use M ukaiyam a’s
original system and test its suitability for our th ioester derivative. O-
" f r im e th y l s i l y l - t e r t - h e p ty l th io p y r u v a te 2 3 9 was thus reac ted with
benzaldehyde in the presence of TiCU at -78 °C to give a yellow oil that nmr
analysis revealed to consist once again of unreacted benzaldehyde and
regenerated thioester, the latter recovered by chrom atography in almost
q uan ti ta t ive y ield. No change was obse rved upon e lev a tio n o f the
tem pera tu re of reaction though increas ing am ounts o f d ecom pos it ion
became apparent above 40 °C. We checked for any procedural errors by
203
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c id S yn th o n s
s y n th e s i s in g 1- t r im e th y Is i iy I c y c lo h e x e n e f ro m c y c lo h e x a n o n e and
reacting it with benzaldehyde to give the aldol shown in scheme 5 .2 5 , a
reaction carried out in our source paper'^®.
0SiMc3 ÇHO
2 4 5
TiCU, CH2CI2
N 2(g), -78 °C, I hr 33%
S c h e m e 5.25
9 h o H
2 4 6
We obtained the product as yellow crystals (the ratio of the threo isomer to
the erythro isom er was not investigated) though in considerably lower
yield than claimed in the reference. This did however suggest that the
thioester derivative was not suitable for reaction under these conditons.
Further investiga tion of this reac tion was carr ied out on the
trim ethy ls i ly l enol derivatives of methyl and ethyl pyruvate . The two
esters are commercially available and their use at this stage was expedient
given the tim e-consum ing synthesis of the th ioester (the use of which
could be continued at a later stage). The two silyl enol ethers (248 and 2 4 9
for the derivatives of m ethyl and ethyl pyruvate respec tive ly ) were
syn thes ised w ithou t inc iden t as schem e 5 . 2 4 by the reaction of the
respective ester with TMS-Cl and EtgN in DMF at room temperature and
obtained as clear oils in reasonable yield. The reaction of 0-tr im ethylsily l
enol(ethyl pyruvate) 2 4 9 with benzaldehyde at a number of temperatures
using M ukaiyam a's system did not produce markedly different results from
those described above and once again, the tendency to decomposition was
considerably increased above 40 °C.
It was at this point that we discovered a pair of papers by Sugimura
and co-workers which detailed the use of the trimethylsilyl enol ethers of
both these pyruvic acid acid esters as well as tert-butyl pyruvate, in a Lewis
acid-m ediated aldol reaction. Sugimura^^'"^^ was also aware of the potential
for the synthesis of the y - h y d r o x y - a - k e t o function which occurs in KDO
and used his reaction to synthesise a number of 1,4-lactone derivatives of
KDO. His conditions were essentially the same as those of Mukaiyama that
we had adopted though interestingly, of all the Lewis acids used in the
204
N u c le o p h i l i c A d d i t i o n o f P y ru v ic A c i d S yn th o n s
r e a c t io n , T iC l4 was found to be the least effective in promoting the
reaction, B F 3 :E t2 0 the best (scheme 5 .26).
OMe OSiMej Lewis acid, CH 2C I2 9
2 4 7 2 4 9 250 Yield: 10% (TiCU)86% (BI^:0Et2)
S c h e m e 5.26
All reactions were carried out on acetals to prevent the further substitution
which was found to be a problem with unprotected keto functions (scheme
5 .2 7 ) .
O SiM e,
BF,:E^O, CH,Cb
O N 2 (g), -78— 8 °C2 5 1 2 4 9
OH O
252 - 10%
C02Et
253 4 3
S c h e m e 5.27
S u g im u ra ’s m ethod for m aking the silyl enol ethers was also slightly
different; 4-(dimethylam ino)pyridine (DMAP) was found to be an efficient
cata lyst for the formation of the ether in benzene solution and was a
modification of an earlier procedure developed by Sekine^^ who used the
conditions to form bis-( tr im ethy ls ily l)eno l ethyl pyruvate in 80% yield
(com pared to 51%, obtained by Peliska and O'Leary in the absence of a
c a ta ly s t ^ ^ ) . However, we observed no significant increase in the yield of
the ethers com pared to our earlier attempts, also in the absence of a
catalyst, and yields were stable at around 40%. This figure was unaffected if
Sug im ura ’s careful anhydrous and anaerobic work-up was replaced by a
cycle o f rapid washing and drying. For our first attempts at reaction of the
ethers under the new conditions, an aldehyde, benzaldehyde, was used in
place o f the ketals used in the paper since at this stage, m ultip le
205
N u c le o p h i l i c A d d i t io n o f P y ru v ic A c id S yn th o n s
substitutions were not of primary concern to us but the only products of
the reaction , using boron tri fluoride diethyl etherate as promoter, were
u n r e a c te d a ld e h y d e and r e g e n e ra te d e s te r . W hen re p e a te d w ith
benzaldehyde dimethyl acetal 2 5 4 however, the presence of a small amount
o f the c in n am ald éh y d e d e r iv a tiv e 2 6 3 was the first indication of a
successfu l reac tion since the product was p resum ably form ed by the
e l im in a t io n o f m e th a n o l f ro m the a lk o x y k e to e s te r d u r in g
chrom atography of the crude reaction mixture (scheme 5 .2 8 ) .
9^® OSiMes OMe O
° N2(g), -78 °C °2 5 4 2 4 9
H O
H+
2 6 3
S c h e m e 5 .28
Though the rem aining m aterial was the expected m ixture of ester and
aldehyde (regenerated from the acetal under the same conditions that had
p ro d u ce d e lim in a tio n o f the p roduct) this resu l t was encou rag ing .
However, elevation of the temperature or an increased reaction time failed
to p rov ide fu rthe r ev idence of product fo rm ation , desp ite S ug im ura
claiming a yield of 71% for this reaction. In common with him however, we
observed that total consumption of the acid accompanied all attempts at its
reaction and assumed therefore that though cleavage o f the e ther was
occurring as predicted, decomposition pathways were com peting with the
reaction pathway and the reaction was therefore extrem ely sensitive to
a lterations in conditions.
W ith one exception (propanal dimethyl acetal), all of Sug im ura’s
electrophiles were aromatic, so we briefly investigated the reaction of some
aliphatic aldehydes with the silyl enol ethers to assess whether this would
affect the course of the reaction. The use of acetaldehyde at -78 °C on O-
trim ethylsily l-enol(ethylpyruvate) gave only a small amount of brown oil
(scheme 5 .2 9 ) .
206
N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S yn th ons
0 0S iMe3 HO H O1 + 1 O (i) BI^iEtzO, CH2CI2, -78— 0 °C II ^
M e " ^ H 'E t " 'E tO (ii) Pf, H2O ^
2 64- 2 4 9 2 6 5
S c h e m e 5 .29
This exhibited a broad peak in the infrared at 3431 cm"^ but no evidence of
a successful reaction was otherwise obtained. Similarly, the use of hexanal
produced only a complex mixture of products which included unreacted
aldehyde, despite it being used in stoichiometric quantities.
In o n e f in a l a t t e m p t , 0 - t r i m e t h y l s i l y l e n o l ( t e r t - h e p t y l
thiopyruvate) was reacted with benzaldehyde but merely gave a yellow oil
tha t c o n s is ted m ain ly of the regenera ted th io es te r and w hich was
recovered in 36% yield after chromatography in chloroform. Any further
attempts at nucleophilic addition of these compounds was abandoned at this
p o in t .
T hough our attempts at u til is ing these esters and th ioesters o f
pyruvic acid as synthons for the nucleophilic addition of the acid itse lf
were abandoned, we decided to make one final investiga tion into the
chemistry of the silyl enol ethers. Inspection of the compounds reveals a
double bond substi tu ted by two e lec tronega tive g roups, c rea ting , we
imagined, a potentia lly strong dienophile and so the reactiv ity of O-
t r i m e t h y l s i ly l - e n o l ( e th y lp y r u v a te ) 2 4 9 in the D iels-A lder reaction with
cyclopentadiene was investigated (scheme 5 .3 0 )
OMejSi Œ 2CI2, A
ÇH2
2 66 2 4 9
S c h e m e 5.30
OEt
In an tic ipa tion o f a fac ile reaction, we in it ia l ly ref luxed the
subs tra te with freshly cracked cyc lopen tad iene only b r ie f ly but this
procedure merely produced an intractable brown oil. By carrying out the
207
N u c le o p h i l i c A d d i t io n o f P y ru v ic A c i d S yn th o n s
reaction at a lower temperature (-78 °C, rising to 0 °C) no reaction was
observed at all, so the apparent instability of the silyl enol ether under the
reoction conditions was not easily offset by making the conditions more
m ild .
1 ,3-D iphenylisobenzofuran is an excellent diene in the D iels-Alder
r e a c t io n '^ ^ since there is a strong driving force from the aromatisation of
the cyclohexadiene ring, and it therefore seemed a useful alternative to
cyclopen tad iene (scheme 5 .3 1 ) .
2 5 6
OEtaSiO
OEt
CH-
2 5 7
C H iC k , A Ph
OEtPh
2 5 8
S c h e m e 5.31
Upon reflux of 0-tr ie thylsily l-enol(ethyl pyruvate) 2 5 7 in CH2 C I 2 for 1-2
hours, the fluorescent colour of the diene was observed to discharge and
work-up provided a yellow solid that was recrystallised from iPrOH to give
white crystals with a melting point some twenty degrees higher than the
diene. The H nmr spectrum of this compound gave no indication of the
presence of the triethylsilyl function, however. The low melting points of
the two isomers of l ,3 -d ihydro - l ,3 -d ipheny lisobenzofu ran (86-87 ®C and
99.5-100.5 °C for the cis and trans compounds respectively"^^) rule out the
possibility that reduction of the furan had occurred and the absence of a
peak at m/z 272 in the mass spectrum confirm s this (Mr for 1,3-
diphenylisobenzofuran is 270.31). The mass spectrum does display a peak at
m/z 363 and peaks corresponding to the loss of one and two phenyl groups
from this ion at 286 and 209 respectively, but it is not clear to what species
these figures refer since no pattern of f ragm enta tion of the desired
product seems to account for them.
As a final attempt we decided to use hexachlorocyclopentadiene in an
inverse dem and D iels-A lder reaction. Such reactions typ ica lly require
208
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c i d S y n th o n s
som ew hat forcing conditions such as reaction in a Carius tube at high
temperature, but are well established as a way of forcing the reaction of
e le c t ro n - r ic h a lkenes and in the m anufac tu re o f h igh ly c h lo r in a te d
p e s t i c id e s ^ ^ . The efficacy of the reaction was once again established with a
trial run of a method drawn from the literature*^^ so that 1-brom opropene
2 6 0 , after sealing and heating in a Carius tube at 170 °C for 19 hours with
h e x a c h lo r o c y c lo p e n ta d ie n e 2 5 9 , gave the adduct 2 6 1 shown in scheme
5 .3 1 in reasonable yield.
Cl Cl
+ Carius Tube, 170°C , 19hrs Cl^^ < ‘ *Br
H s C ^ H 25%
2 6 0
S c h e m e 5.31
Cl Cl
2 6 1
H o w ev er , the reac tion o f s to ich io m e tr ic am oun ts o f O - t r ie th y ls i ly l
enol(ethyl pyruvate) with the diene in a sealed tube at 150°C for four days
produced only an oily black material with no trace of the silyl enol ether
visible in its nmr spectrum. A repeat run at a higher temperature and for a
shorter time (180 °C for 24 hours) did however provide a solid material.
Breakage of the tube was accompanied by the release of an acidic-smelling
gas and the product was obtained as dry, shiny black flakes. The mass
spectrum of this material, however showed the only volatile constituent to
be h e x a c h lo ro c y c lo p e n ta d ie n e , the rem a in d e r p re su m a b ly be ing h igh
m olecu la r w eight tars form ed from the decom position o f the c learly
unstable silyl enol ether. With this failure, no further reactions o f the
compounds were investigated
209
N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S y n th o n s
5.3 E x p e r i m e n t a l
5.3.1 A p p a r a tu s and R e a g e n t s
All details for this section are as for chapter 4 except:
(i) In fra-red (IR) spectra were recorded on a P erk in -E lm er PE-983
sp e c tro m e te r using a liqu id film and d ich lo rom e thane so lvent. M ain
absorption bands are reported with wavenum ber n in cm" and intensity
(s, strong; m, medium; w, weak; br, broad);
(ii) Purif ica tion of te trahydrofuran for use in a lky la tion experim ents
was by reflux o v e r sodium wire with benzophenone as indicator.
The general procedure for the alkylation reactions described in the
p re v io u s s e c t io n was as fo llo w s : d i is o p ro p y la m in e or iso p ro p y l-
cyclohexylamine was dissolved in dry THF (to approximately 0.5 M) under
nitrogen gas in a two-necked flask connected to a nitrogen bubbler. The
tem pera ture was lowered to -78 °C and a sto ichiom etric amount of n-
butyllithium was added dropwise via the use of a syringe and septum, then
the temperature raised to 0 °C for fifteen minutes. After lowering to -78 °C,
the carbonyl compound was added and allowed to react for fifteen to thirty
minutes at this temperature or at 0 °C. The electrophile (approximately IM
in hexamethylphosphoramide) was next added and allowed to react at -78 °C
or 0 °C for a period of time between thirty minutes and twelve hours.
S tandard work-up refers to quenching with aqueous am m onium chloride,
fo llow ed by ex traction with d iethyl ether. The o rganic ex tracts were
washed with water and saturated sodium chloride solution, then dried and
evaporated in vacuo. All unsuccessful experiments have been indicated in
the previous section by placing the product in square brackets.
210
N u c le o p h i l i c A d d i t io n o f P y ru v ic A c i d S yn th o n s
5 .3 .2 E x p e r i m e n t s
1. P r e p a r a t io n of 1 ,1 -D im e th y le th y l t h io p y r u v a te 231
OMe Me
l* l-D im e th y le th an e th io l (13.5 ml, 10.83 g, 0.12 mol) and dry
triethylamine (3.70 ml, 2.66 g, 0.03 mol) were added to dry ether (15 ml)
under an atmosphere of nitrogen then heated to reflux. Pyruvoyl chloride
(3.70 ml, 0.03 mol), made up to 10ml in dry ether was then added dropwise,
producing a vigorous reaction and a white precipitate. A further portion of
dry e ther (30 ml) was added to facilitate stirring, then reflux was
c o n t in u e d fo r 30 m in u te s . The p r e c ip i t a t e d t r i e th y l a m m o n iu m
hydrochloride was dissolved in the minimum amount of sodium chloride
solution then the organic layer was dried (MgS0 4 ), evaporated in vacuo and
distilled through a Vigreux column, giving the thioester as a yellow oil
(1.32 g, 8.23 mmol, 33%), b.p. 50-70 °C/17 mm Hg.
nmr (CDCI3): 1.51 (s, 9H); 2.38 (s, 3H)
13c nmr (CDCI3); 22.6; 28.4; 62.4; 189.8; 193.3
Mass Spectrum:
IR ( c m ' l ) :
160 [M+, 1%]; 57 [(CMe3)+, 64%]
1014.2 (s); 1093.7 (m); 1164.1 (m); 1210.0 (m);
1363.0 (m); 1448.7 (w); 1659.8 (s); 1717.9 (s); 1766.9
(m); 3994.2 (br)
A n a ly s is : Calculated for C7H 12O 2S, C:52.46; H: 7.56
Found, C: 52.16, H: 7.89
211
N u c le o p h i l i c A d d i t io n o f P y ru v ic A c i d S yn th o n s
2. P r e p a r a t io n of 3 -E t h y l - p e n t a n - 3 -ol 235
MeMeOH
Me
This was prepared according to the l i te ra tu re m ethod^ then
distilled, giving the title alcohol as a clear oil (23,72 g, 0.20 mol, 93%), b.p.
140-144 °C (lit. 140-142 °C).
3. P r e p a r a t i o n of 3 - E t h y l - p e n t a n - 3 - t h i o l 236
Me MeSH
Me
This was prepared according to the l i te ra tu re m ethod^ ^ , then
distilled, giving the title thiol as a yellow oil which was used immediately
without further purification (11.75 g, 0.09 mol, 51%).
4. P r e p a r a t i o n of 1 , 1 - D ie t h y lp r o p y l t h i o p y r u v a t e 237
Me Me
Me
3-Ethyl-pentan-3-thiol (4.00 g, 0.03 mol) and triethylamine (3.10 g,
4.27 ml, 0.03 mol) were added to dry ether (15 ml), followed by pyruvoyl
chloride (3.30 g, 0.03 mol) in dry ether (5 ml). The mixture was refluxed for
1 hour then worked-up by the addition of aqueous ammonium chloride (15
ml). The organic layer was washed with water (15 ml) then dried (MgS0 4 )
and evaporated in vacuo to give a dark oil which was distilled (Kugelrohr),
giving the title thioester as a yellow oil (3.27 g, 16.16 mmol, 52%), b.p. 110-
115 °C/30 mm Hg.
212
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c i d S yn th o n s
I r nmr (CDCI3):
nmr (CDCI3):
Mass Spectrum (m/z):
IR (cm‘ ^):
A n a ly s is :
0.85 (t, 9H, ^ Jh -H = 7.4Hz); 1.82 (q, 6H, ^JR-H =
7.7Rz); 2.33 (s, 3R)
8.2; 23.7; 27.2; 61.3; 190.9; 194.6
202 [M+, 5%]; 99 [(CEt3)+, 67%]; 29 [ (C iR s)^ , 100%]
1011.1 (m); 1130.5 (m); 1350.8 (m); 1378.3 (m);
1454.8 (m); 1655.6 (vs); 1717 .9 (vs); 1756.3 (s);
2874.0 (s); 2935.2 (s)
Calculated for C 10R I 8O 2S, C: 59.37; R: 8.97
Found, C: 59.26; R: 8.91.
5. P r e p a r a t i o n o f 1 ,1 - D i e t h y l p r o p y I 2 - o x o - t h i o l h e p t a n o a t e 238
Me Me
Me
Isopropylcyclohexylamine (0.06 g, 0.08 ml, 0.50 mmol) was added to
dry TRF (1.5 ml) and cooled to -78 °C then n-butyllithium (0.2 ml of a 2.5 M
solution in hexane, 0.50 mmol) was added. The temperature was raised to 0
°C for 15 m inutes then low ered again to -78 °C. 1 ,1 -D ie thy lpropy l
thiopyruvate (0.10 g, 0.49 mmol) in dry TRF (0.5 ml) was added at this
tem perature and stirred for 30 minutes. RM PA (0.59 ml) was then added,
followed 30 minutes later by bromobutane (0.14 g, 0.10 ml, 0.10 mmol) and
after warming to 0 °C and standard work-up, a yellow oil was obtained. This
was co lum n chrom atographed (E t2 0 :petroleum spirit, b.p. 40-60 °C, 0:1
rising to 1:0) to give a clear oil which spectral data indicated to be the title
thioester (0.06 g, 0.23 mmol, 47%).
213
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c i d S y n th o n s
nmr (CDCI3): 0.87 (t, 9H, ^Jh -H = 5.4Hz); 0.89 (t, 3H, ^JR-H =
7.5Hz); 1.43 (m, 12H), 2.30 (t, 2H, ^ J r -R = 7.3Rz)
nmr (CDCI3): 1.0; 8.1; 13.8; 22.4; 26.2; 28.1; 31.5; 53.1; 191.5;
197.2
Mass Spectrum (m/z): 258 [M+, 1%]; 131 [(Et3CS)+, 28%]; 9 9 [ (E t3 0 + , 5%];
57 [(C4R9)+, 100%]; 43 [(C3R 7)+, 76%]; 29 [(C2R5)+,
64%]
IR ( c m ' l ) : 1014.2 (m); 1090.7 (m); 1259.0 (w); 1378.3 (m);
1451.7 (w); 1598.6 (m); 1653.7 (w); 1717.9 (s);
2867.9 (w); 2922.9 (m); 2959 7 (s)
A n a ly s is : Calculated for C 14R 24O 2S, C: 65.58; R: 9.43
Found, C: 65.12; R: 9.12
6. Preparat ion o f 2 -b u ty lo c ta n o ic ac id 221
.COOKMe
Me
This was prepared according to the literature method^, followed by column
c h ro m a to g ra p h y (p e n ta n e :E t2 0 , 1:0 rising to 1:1), giving the title acid as a
clear oil (1.50 g, 7.50 mmol, 42%).
7. Preparation of P yruvoy l C h lo r ide 228
Me
214
N u c le o p h i l i c A d d i t io n o f P y r u v ic A c i d S y n th o n s
This was prepared according to the literature m ethod^ ^ , giving the acid
chloride as a pale green oil (2.36 g, 0.02 mol, 24%), b.p. 24-37 °C/100-105 mm
Hg (lit. 53 °C/126 mm Hg).
8. P r e p a r a t i o n of O - T r i m e t h y l s i l y l -
e n o l ( l , l - d i e t h y I p r o p y l t h i o p y r u v a t e ) 2 3 9
Me
To a solution of ^heptyl thiopyruvate (0.40 g, 2.00 mmol) in dry DMF under
n itrogen was added trie thylam ine (0.63 g, 0 .36 ml, 6 .20 mmol) and
ch lo ro trim ethy ls i lane (0.67 g, 0.79 ml, 6.20 mmol), p roducing a white
precipitate. The mixture was stirred for two hours then dry hexane (10 ml)
was added and stirred and the hexane (upper) layer syringed out. The
m ate r ia l was s tored under n itrogen in hexane so lu t io n at reduced
temperature (0.30g, l.SOmmol, 54%).
^H nmr (CDCI3):
13c nmr (CDCI3):
0.24 (s, 9H); 0.84 (t, 9H, = 7.2Hz); 1.78 (q, 6H,
^JH-H = 7.4Hz); 4.50 (d, IH, 3j h _h = 1.6Hz); 5.32 (d, IH, 3Jh _h = 1.5Hz)
5.8; 7.9; 21.2; 58.8; 101.4; 140.3; 159.7
IR (cm’ l ) : 1075.4 (s); 1252.9 (s); 1399.7 (s); 1497.6 (s);
1611.7 (m); 1682.3 (vs); 1724.9 (vs); 1941.3 (w);
2763.8 (m); 3369.7 (w)
215
N u c le o p h i l ic A d d i t io n o f P y ru v ic A c i d S y n th o n s
9 . P r e p a r a t i o n o f 0 - T r i n i e t h y s i I y l - e n o l ( m e t h y l p y r u v a t e ) 2 4 8
Me
This was prepared according to the literature method'^^^, giving the ether as
a colourless oil (0.87 g, 4.99 mmol, 28%), b.p. 50-55 °C/10 mm Hg (lit. 75-76.5
°C/41 mm Hg).
10. P r e p a r a t i o n of 0 - T r i m e t h y s i l y l - e n o l ( e t h y I p y r u v a t e ) 249
Me
This was prepared according to the literature method'^ giving the ether as
a colourless oil (2.57 g, 13.65 mmol, 43%), b.p. 62-67 °C/15 mm Hg (lit. 69-70
°C/18mm Hg).
11. P r e p a r a t i o n of 0 - T r i e t h y s i l y I - e n o l ( e t h y l p y r u v a t e ) 257
Me
Ethyl pyruvate (5.0 g, 4.72 ml, 0.043 mol) was added to benzene with 4-
dim ethylam inopyrid ine (10 mg) and ch lorotriethylsilane (6.78 g, 7.55 ml,
0.045 mol) and heated to reflux. Triethylamine (4.53 g, 6.27 ml, 0.045 mol)
was then added, causing the immediate formation of a white precipitate.
Heat was maintained for three hours then the mixture was washed with
w ater (5 ml), dried (M g S 0 4 ) and distilled, giving the title ether as a
colourless oil (1.95 g, 0.01 mol, 17%) b.p. 150-155 °C/22 mm Hg.
216
N u c le o p h i l i c A d d i t io n o f P y ru v ic A c id S yn th ons
1 h nmr (CDCI3): 0.67 (q, 6H, ^ J r - H = 8.0Hz); 0.95 (t, 9H, ^ J r - H =
7.9Hz); 1.26 (t, 3R, ^Jh -R = 7.1Hz); 4.17 (q, 2H, 3JH-
H = 7.1Hz); 4.81 (d, IH, ^J j j . r = l.lHz); 5.44 (d, IH,
^JH-H = l .lH z)
13c nmr (CDCI3): 4.7; 6.5; 14.1; 61.0; 103.3; 147.5; 164.4
Mass Spectrum (m/z): 230 [M+, 1%] ; 115 [(SiEt3)+, 76%]; 29 [(C2H 5)+, 100%]
IR (cm-1): 861.2 (m); 1001.9 (m); 1237.6 (m); 1320.2 (s); 1620.0
(s); 1684.3 (s); 1727.1 (s); 3449.2 (br)
A n a ly s is : Calculated for C n H 2 2 0 3 S i , C: 57.34; H: 9.63
Found: C: 56.99; H: 9.52
12. P r e p a r a t io n o f ( 2 -oxo )c ycIoh e xy l pheny l m eth a n o l 246
This was prepared according to the literature m e t h o d ^ g i v i n g a yellow
solid which was recrystallised from EtOAc to give the title aldol as yellow
crystals (0.80 g, 3.92 mmol, 33%), m.p. 103-105 °C (lit. 103.5-104.5 °C for
erythro, 75 °C for threo isom er^^).
13. P r e p a r a t i o n o f 5 - B r o m o - l , 2 , 3 , 4 , 7 , 7 - h e x a c h I o r o - 6 -
m e t h y l n o r b o r n - 2 - e n e 2 6 1
Cl p
217
N u c le o p h i l ic A d d i t io n o f P y r u v ic A c i d S yn th o n s
This was prepared according to the literature method^ ^ from a mixture of
cis- and trans- 1-bromopropene (2.50 g, 1.77 ml, 17.69 mmol), giving the
title compound as a waxy white solid (1.39 g, 4.46 mmol, 25%), m.p. 187-188
°C (lit. 193-195 °C for 5-endo, 6-endo isomer^ ^).
14. At tem pted Preparation of the A d d u c t o f O -T rim ethy l s i ly l -
e n o l ( e t h y l p y r u v a t e ) a n d H e x a c h l o r o c y c l o p e n t a d i e n e
0 -T r im e th y ls i ly l -en o l(e th y l py ruvate ) (1 .78 g, 9 .45 m m ol) and
hexachlorocyclopentadiene (2.58 g, 9.45 mmol) were sealed in a Carius tube
and heated to 180 °C for 24 hours. Breakage of the tube was accompanied by
the release of an acidic-smelling gas. The tube was then rinsed in CH 2 C I 2
and evaporated to give a shiny black powder which consisted of unreacted
hexach lorocyc lopen tad iene and decom position p roducts .
5.4 R e f e r e n c e s
( 1 ) Morton, A. A.; Ful^well Jnr., F.; Palmer, L. JACS 1 9 3 8 ,6 0 , 1426.
(2 ) Hauser, C. R.; Chambers, W. J. JACS 1956, 75, 4942.
(3 ) De Free, D. O.; Closson, R. D. JACS 1958, SO, 2311.
(4 ) Creger, P. L. JACS. 1967, 89, 2500.
(5 ) Pfeffer, P. E.; Silbert, L. S. 36. 1971, 5290 , .
( 6 ) Petragnani, N.; Yonashiro, M. Synthesis. 19 8 2 , 521.
(7 ) Pfeffer, P. E.; Silbert, L. S. J. Org. Chem. 1970, 55, 262.
(8 ) Pfeffer, P. E.; Silbert, L. S.;Chirinko Jr., J. M. J. Org. Chem. 1 9 7 2 ,5 7 ,
451.
(9 ) Malian, J. M.; Bebb, R. L. Chem. Rev. 1969, 69, 693.
(1 0 ) Chan, L. L.; Smid, J. JACS 1968, 90, 4654.
(1 1 ) Normant, H.; Angelo, B. Bull. Chim. Soc. Fr. 1962, 810.
(1 2 ) Angelo, B. Bull. Chim. Soc. Fr. 1970, 1848.
(1 3 ) Peliska, J. A.; O'Leary, M. H. JACS 1 9 9 1 ,1 1 3 , 1841.
(1 4 ) Tapia, I.; Alcazar, V.; Moran, J. R.; Caballero, C.; Grande, M. Chem. Lett.
1990 , 697.
(1 5 ) Williams, D. R.; Benbow, J. W. Tet. Lett. 1 9 9 0 ,5 7 , 5881.
(1 6 ) Comforth, J. W.; Firth, M. E.; Gottschalk, A. Biochem. J. 1 9 5 7 ,6 5 , 57.
218
N u c le o p h i l i c A d d i t io n o f P y ru v ic A c i d S y n th o n s
(1 7 ) Schmidt, R. R.; Betz, R. Angew. Chem. Int. Ed. Engl. 1 9 8 4 ,2 5 , 430.
(1 8 ) Esswein, A. A.; Betz, R.; Schmidt, R. R, Helv. Chim. Acfa.1989, 72, 213.
(1 9 ) Unger, F. M. Adv. Carbohydr. Chem. Biochem. 1 9 8 1 ,5 5 , 323.
(2 0 ) Crich, D. SERC Earmarked Application. 1990.
(2 1 ) "Aldrich Library O f NMR Spectra", Edition II, Vol I . Aldrich
Chemical Co., 1983.
(2 2 ) Hauser, C. R.; Puterbaugh, W. H. JACS 1953, 75, 1068.
(2 3 ) Booth, P. M.; Fox, C. M. J.; Ley, S. V. J. Chem. Soc. Perkin Trans. I. 1987,
121 .
(2 4 ) Clarke, T.; Ley, S. V. J. Chem. Soc. Perkin Trans. I. 1987, 131.
(2 5 ) Ley, S. V.; Woodward, P. R. Tet. Lett. 1987, 28, 345.
(2 6 ) Voss, J. Synthesis o f Thioesters and Thiolactones. Vol. 6 of
Com prehensive O rganic Synthesis. 8 vols. Oxford: Pergamon, 1991.
(2 7 ) Ottenheijm, H. C. J.; De Man, J. M. M. Synthesis. 1975, 163.
(2 8 ) Hausler, J.; Schmidt, U. Chem. Ber. 1974, 107, 145.
(2 9 ) Spencer, M. D.; Ph. D., UCL, 1990.
(3 0 ) Edwin Wilson Jr., G.; Hess, A. J. Org. Chem. 1 9 8 0 ,4 5 , 2766.
(3 1 ) Rathke, M. W.; Lindert, A. JACS 1971, 95, 3318.
(3 2 ) Barton, D. H. R.; Crich, D. J. Chem. Soc. Perkin Trans. I. 1986, 1603.
(3 3 ) Bellamy, L. J. The Infrared Spectra O f Complex M olecules. Vol. 1 of 2
vols. NY: Chapman & Hall, 1975. .
(3 4 ) Rasmussen, R. J.; Tunnicliff, D. D.; Robert Brittain, R. JACS. 1949, 71,
1068.
(3 5 ) Stork, 0 .; Hudrlik, P. F. JACS 1 9 6 8 ,9 0 , 4462.
(3 6 ) House, H. 0 .; Czuba, L. J.; Gall, M.; Olmstead, H. D. J. Org. Chem. 1969 ,54 ,
2324.
(3 7 ) House, H. O.; Gall, M.; Olmstead, H. D. J. Org. Chem. 1 9 7 1 ,5 6 , 2361.
(3 8 ) Rasmussen, J. K. Synthesis. 1977 , 92.
(3 9 ) Mukaiyama, T.; Narasaka, K.; Banno, K. Chem. Lett. 1973, 1011.
(4 0 ) Mukaiyama, T.; Narasaka, K.; Banno, K. JACS 1974, 96, 7503.
(4 1 ) Mukaiyama, T.; Murakami, M. Synthesis. 1987 , 1043.
(4 2 ) Sugimura, H. Noguchi Kenkyusho Jiho. 1989 , 52 , 33.
(4 3 ) Sugimura, H.; Shigekawa, Y.; Uematsu, M. Syn. Lett. 1991, 153.
(4 4 ) Sekine, M.; Futatsugi, T.; Yamada, K.; Hata, T. J. Chem. Soc. Perkin
Trans. I. 1982, 2509.
219
N u c le o p h i l ic A d d i t io n o f P y r u v ic A c i d S y n th o n s
(4 5 ) Greenhouse, R.; Borden, W. T.; Hirotsu, K.; Clardy, J. JACS 1977, 99,
1664.
(4 6 ) Smith, J. G.; McCall, R. B. J. Org. Chem. 1 9 8 0 ,4 5 , 3982.
(4 7 ) Boger, D. L.; Patel, M. Prog. Heterocycl. Chem. 1989, 7, 30.
(4 8 ) Alexander, R.; Davies, D. I. J. Chem. Soc. Perkin Trans. 1. 1973, 83.
220
A p p e n d i x
K>K)to
5
:k"3atv.
1. nmr Spectrum of O-Trimctliylsilyl cnol( I , I -diethylpropyl thiopyruvate) 23 9
K)toOJ
T— 1— I— I— I— I— I— I— r —r
r
T
2. h l mm specl tum ol O-'Irimelhylsi lyl cnoKclhyl pyiiiv:ile) 2 4 V
K>K)
L- V iU l -J L _
r
f
j
T I I j r 1—I—:—r "1 j : i I i i ! i I i J i i ~
? I r'pM
3. 'I I nmr spectrum of 0-Trielhylsilyl enol(elhyl pyruvate) 2 5 7
C or r i gen da
Throughout this thesis:
(i) “pyrrolido” and “pyrrolidyl” should be read as pyrrolidino and
p y r ro l id in y l , resp e c t iv e ly ;
(ii) “piperido” should be read as piperidino;
(iii) “chloroform ” should be read as trichloromethane;
(iv) “ 1, 4-diaza[2.2.2]bicyclooctane” and “ l-aza[2 .2 .2]b icyclooctane”
should be read as 1, 4-diazabicyclo[2.2,2]octane and 1-
azab icyc lo [2 .2 .2 ]oc tane , respec tive ly ;
(v) “/A C 5 ” should be read as J. Am. Chem. Soc.
A d d e n d u m
The express ions “NHg (aq .)” or “aqueous am m onia” refer to an
aqueous ammonia solution at a concentration of approximately 2M.
225