Nhu Thi Quynh Doan and Søren Brøgger...

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Current Pharmaceutical Design, 2015, 21, 5501-5517 5501

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Thapsigargin, Origin, Chemistry, Structure-Activity Relationships and Prodrug Development

Nhu Thi Quynh Doan and Søren Brøgger Christensen*

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenha-

gen, Copenhagen, Denmark

Abstract: Thapsigargin was originally isolated from the roots of the Mediterranean umbelliferous plant Thapsia garganica in order to characterize the skin irritant principle. Characteristic chemical properties and semi-syntheses are reviewed. The biological activity was related to the subnanomolar affinity for the sarco/endoplasmic reticulum calcium ATPase. Prolonged inhibition of the pump afforded collapse of the calcium homeostasis and eventually apoptosis. Structure-activity relationships enabled design of an equipotent analogue containing a linker. Conjuga-tion of the analogue containing the linker with peptides, which only are substrates for either prostate specific anti-gen (PSA) or prostate specific membrane antigen (PSMA) enabled design of prodrugs targeting a number of cancer diseases including prostate cancer (G115) and hepatocellular carcinoma (G202). Prodrug G202 has under the name of mipsagargin in phase II clinical trials shown promising properties against hepatocellular carcinoma.

Keywords: Thapsigargin, mipsagargin, drug development, clinical trials, structure activity relationships, prostate specific antigen, prostate specific membrane antigen, prodrug, anti-angiogenesis.

1. INTRODUCTION

Already Theophrastos (372 – 287 B.C.) described the skin irri-tant properties of the resin of Mediterranean plant Thapsia gargani-ca L. (Apiaceae, previous Umbelliferae). Later scholars like Dios-corides (approximately 50 A.D.) and Plinius (24 – 79 A.D.) also mentioned the use of preparations from the plant for treatment of e.g. pulmonary diseases, catarrh and as a counterirritant for relief of rheumatic pain [1, 2]. Radix thapsiae and resina thapsiae have been included in several pharmacopeias latest in the French pharmacope-ia from 1937. In spite of this extended traditional use of the plant the first publication describing the constitution was not published before 1978 [3]. The relative configuration of the major skin irritat-ing principle of thapsigargin (1, Tg, Fig. 1) was published in 1980 [4] and 1982 [5] and the absolute configuration in 1985 [6]. A number of cellular assays revealed that the compound was a very potent secretagogue for histamine release from peritoneal mast cells [7, 8] and activated a number of cells belonging to the humane in-flammatory response [9]. In addition to being a skin irritant Tg (1) also showed to be a cocarcinogen facilitating skin cancer develop-ment in mice [10]. Intensive interest for Tg (1), however, first ap-peared when the potent biological activities were related to the ability of inhibiting the Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) [11]. Today Tg (1) has become a positive stand-ard in all experiments for calcium homeostasis in cells [12]. Pro-longed inhibition of the SERCA pump affords a persistent high concentration of calcium ions in the cytosol, which after 12 – 24 hours induces apoptosis [13]. The ubiquitous presence of SERCA in all living cells and the subnanomolar affinity for the SERCA pump therefore makes Tg (1) a potent universal cell toxin. Howev-er, the overexpression of proteolytic enzyme in neovascular tissue in tumors and the presence of prostate specific antigen only in the prostate gland or in prostate cancer tumors have enabled design of

*Address correspondence to this author at the Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences University of Copenhagen, Universitetsparken 2, DK-2100 Copehagen Ø, Denmark; Tel/Fax: +45-3533-6253, +45-3533-6041; E-mail: [email protected]

prodrugs selectively activated in tumors. Mipsagargin a prodrug designed only to be cleaved in neovascular tumors has in phase II clinical trials showed an encouraging effect against sorafenib re-sistant patients.

2. CHEMISTRY

2.1. The Structures and Diversity of Polyoxygenated Guaiano-lides

Thapsigargin (1) was isolated together with the related thapsigargicin (2, Tc, Fig. 1). Besides Thapsigargin (1) several hexaoxygenated guaianolides only differing from Tg (1) by the acyl groups at O-2 and O-8 have been found in the genus Thapsia (2-13, Fig. 1) [14, 15]. Two additional thapsigargins have been found in Laser trilobum Borkh. (14, 15, Fig. 2) [16]. As well the hexa- as the pentaoxygenated guaianolides are only present in either Thapsia species or in L. trilobum (16-18, Fig. 3). Comparison of the 1H and 13C NMR spectra of Tg (1) and the related Tc (2) revealed that both compounds possessed an acetoxy, a butanoyloxy and an an-geoyloxy group present on the hydrogen poor guaianolide skeleton. In addition the presence of an octanoyloxy residue was present in Tg (1) whereas a hexanoyloxy group was present in Tc (2). Com-parison with the spectra of trilobolide (16, Fig. 3) [17], which just had been published at this time, revealed the absence of the two protons at C-2, whereas an additional acyloxy group was present.

A combination of these observations led to the suggestion of the constitution of Tg (1) [4]. The poor number of protons at the guaia-nolide skeleton prevented establishment of the relative configura-tion. Fortunately, treatment of Tg (1) with thionyl chloride afforded a crystalline epoxide 19 (Scheme 1) the structure of which was solved through an X-ray crystallographic analysis [18]. The X-ray analysis did neither enable establishment of the absolute configura-tion nor of the relative configuration at C-7 or C-11.

Resistance towards periodic acid of the debutanoyl thapsigargin 20 (Scheme 2), however, revealed that the three hydroxyl groups had to be trans disposed.

Søren B. Christensen

5502 Current Pharmaceutical Design, 2015, Vol. 21, No. 38 Doan and Christensen

Fig. (1). Structures of naturally occurring hexaoxygenated guaianolides

found in Thapsia [thapsigargins] (1-13) with traditional numbering.

Fig. (2). Structures of the naturally occurring hexaoxygenated guaianolides

found in L. trilobum [thapsigargins] (14-15).

Fig. (3). Structures of naturally occurring pentaoxygenated guaianolides

[trilobolides] (16-18) found T. garganica or L.trilobum.

The 1 -disposed H-1 is typically found in guaianolides isolated from species belonging to Apiaceae (umbelliferous plants) but a few examples have been found in plants belonging to Asteraceae (composites) [19].

2.2. Chemistry of Polyoxygenated Guaianolides

From a chemical point of view the polyoxygentaed guaianolides possess three functional groups: ester groups, tertiary alcohols and a double bond between C-4 and C-5. In spite of this, methods have been developed for selective modifications of the structures.

2.2.1. Epoxide Formation

Treatment of as well pentaoxygenated as hexaoxygenated guai-anolides converts the 7,11-dihydroxy diol into an epoxide with conversion of the stereochemistry at C-11 (Scheme 1). Epoxide formation from a diol is a very seldom reaction and probably locked optimal conformation of the 7- and 11-hydroxy groups enables the reaction [17, 18].

Scheme 1. Conversion of thapsigargin (1) into epoxide (19).

2.2.2. Selective Hydrolysis of the Ester Group at O-8

Treatment of as well penta- as hexaoxygenated guaianolides with triethylamine in a protolytic solvent like methanol affords a selective hydrolysis of the O-8 ester group (Scheme 2) [20]. The hydrolysis of the labile O-8 ester group might be facilitated by the juxtaposed OH-11 since the hydrolysis of the ester group in the epoxide 19 demands harsher reaction conditions. The use of a stronger base like sodium carbonate results in opening of the 6,12-lactone. As a consequence, a mixture of the 6,12- (20) and 8,12- (21) guaianolides are obtained after workup under acidic conditions (Scheme 2) [18].

Scheme 2. Formation of 6,12- (20) and 8,12- (21) guaianolides.

2.2.3. Selective Substitutions of the Ester Group at O-2, O-10 and O-8

Masking of the OH-8 and OH-11 by reaction with 2,2-dime-thoxypropane yields an isopropylidende derivative (22, Scheme 3) in which the polycyclic nature prevents relactonization. Treatment of 22 with strong base affords 23 and traces of 24 (Scheme 3) [20, 21].

1

5 6 7

8910

3

4

O12

15

O

O

O14

O

O

O

OH

13

O

OH11

H

2 R2

R1

Thapsigargin (1): R1 = Oct, R2 = But

Thapsigargicin (2): R1 = Hex, R2 = But

Thapsitranstagin (3): R1 = iVal, R2 = 2-MeBut

Thapsivillosin A (4): R1 = Ang, R2 = Sen

Thapsivillosin B (5): R1 = Ang, R2 = 2-MeBut

Thapsivillosin C (6): R1 = Oct, R2 = 2-Mebut

Thapsivillosin D (7): R1 = 6-MeOct, R2 = Sen

Thapsivillosin E (8): R1 = 6-MeOct, R2 = 2-Mebut

Thapsivillosin G (9): R1 = 6-MeHep, R2 = 2-Mebut

Thapsivillosin H (10): R1 or R2 = Ang or Sen

Thapsivillosin I (11): R1 = Ang, R2 = But

Thapsivillosin J (12): R1 = iVal, R2 = But

Thapsivillosin K (13): R1 = Sen, R2 = 2-MeBut

O

O

O

O

O

Ang But

2-MeBut 6-MeHep

6-MeOct

iVal

Non

O

SenOct

O

O O

O

Hex

1

5 6 7

8910

3

4

O12

15

O

O

O14

O

O

OH

13

O

OH11

H

2

R1

OR2

2-Hydroxy-10-desacetoxytrilobolide (14): R1 = R2 = H

2-Acetoxytrilobolide (15): R1 = R2 = Ac

Trilobolide (16): R = 2-MeButNortrilobolide (17): R = ButThapsivillosin F (18): R = Sen

1

5 6 7

8910

3

4

O12

15

O

O

14O

O

OR

OH

13

O

OH11

H

2

O

O

O

O O

O

O

OH

O

O

H

OH

O

1O

O

O

O O

O

O

O

O

H O

19

O

SOCl2

O

O

O

O O

O

O

OH

O

O

H

OH

O

O

O

O

O O

O

OH

OH

O

O

H

OH

1 20

Et3N

MeOH

HO

O

O

O O

OH

O

O

CH3

HOHO

21

O

Na2CO3 (aq)MeOH

HO

O

O

O O

O

OH

OH

O

H

OH

-O

19

H20

O

Thapsigargin, Origin, Chemistry, Structure-Activity Relationships Current Pharmaceutical Design, 2015, Vol. 21, No. 38 5503

Scheme 3. Masking OH-8 and OH-11.

As expected the , -unsaturated angeloyl group at C-3 is more stable towards basic hydrolysis than the other acyl groups in the molecule. Secondary alcohols are known to react faster with acyl groups than tertiary alcohols. Consequently acylation of OH-2 pro-ceeds faster than acylation of OH-10 enabling selective acylation to give 25, which subsequently can be acylated at O-10 to give 26 (Scheme 4). Removal of the isopropylidene group with acid in an aqueous medium affords 27 in which O-8 selectively can be acylat-ed affording 28 (Scheme 4). In total, the procedure enables selec-tive replacement of all the acyl groups in the starting material ex-cept for the angeloyl group [20, 21].

Surprisingly attempts to convert the debutanoyl nortrilobolide 29 into the corresponding isopropylidene derivative 30 only afford-ed the target compound in trace amounts whereas the major product was the 3-methylether 31 (Scheme 5). The most likely explanation for the different behavior of the hexa- and the pentaoxygenated guaianolides is that the voluminous octanoyloxy group in the 2-position prevents an attack from the methanol on the intermediary formed carbocation 32, whereas the absence of the 2-subsituent in 29 favors the competing reaction.

Scheme 4. Selective substitutions of acyl groups at O-2, O-10 and O-8.

Scheme 5. Formation of isopropylidene derivatives 30 and 31.

2.2.4. Selective Substitution of the Angloyl Group at O-3

Whereas the double bond between C-2 and C-3 in the angeloyl moiety protects the ester bond from saponification the same group also enable selective hydrolysis of this group. Oxidation of the group with reagents like osmium tetraoxide or permanganate fol-lowed by treatment with periodate enables formation of the pyruvic ester 33, which can be solvolyzed under mild conditions like pyri-dine in methanol to give the 3-hydroxy compound 34 (Scheme 6) [20].

Scheme 6. Selective angelate cleavage at O-3.

Again a significant difference between the penta- and the hex-aoxygenated guaianolide has been observed in the selectivity of oxidation of the angeloyl double bond. In the case of the pentaoxy-genated guaianolide, nortrilobolide (17), the C-4-C-5 double bond is sensitive to oxidation, e.g. ozonolysis, affording a cleavage of the five-membered ring to form 35 and 36 (Scheme 7). Again it is as-sumed that the sterical effect of the 2-octanoyl substituent prevents the attack of the C-4-C-5 double bond in the hexaoxygenated guai-anolides.

Scheme 7. Ozonolysis of nortrilobolide (17) to form 35 and 36.

20

O

O

O

O O

O

O

OH

O

O

H

O

O

O

O

HO OH

O

OH

O

H

O

22

23

O

HO

HO OH

O

OH

O

H

O

24

O

O

O

O O

O

OH

OH

O

O

H

OH

MeO OMe

H

Acetone

KOH

MeOH

O

O

HO

O

OH

OH

OH

O

O

29

MeO OMeH

Acetone

O

HO

O

OH

OH

OH

O32

O

HO

O

O

OH

O

O

MeO

31

Major

30

Minor

O

HO

O

O

OH

O

O

O

O

O

O

O

O OO

O

OH

O

O

H

OH

O

1

O

O

O

O OO

O

OH

O

O

H

OH

O

HO HO

O

O

O

O OO

O

OH

O

O

H

OH

O

33

O

O

HO

O OO

O

OH

O

O

H

OH

O

34

MnO4- or OsO4

IO4-

Pyridine

MeOH

O

O

HO

O

O

OH

OH

O

O

17

O

HO

O

O

OH

OH

O35

O

OHO

O

HO

O

O

OH

OH

O

OHO

O

36

O O

O

1) O3 then PPh3

2) Pyridine, MeOH

O

O

O

O OH

O

OH

O

H

O

25

O

R

O

O

O

O O

O

OH

O

H

O

O

R

O

O

O

O O

OH

OH

O

H

OH

O

R

2627

O

O

O

O O

O

OH

O

H

OH

O

R

28

R"

O

O

O

O

HO OH

O

OH

O

H

O

23

(RCO)2O, DMAP

(R'CO)2O,DMAP

O

R'

O

R'

H

MeOH

(R''CO)2O,DMAP

O

R'

2 108


Selective removal of the angeloyl group in the pentaoxygentaed guaianolides, however, might be obtained taking advantage of the possible nucleophilic removal of the angeloyloxy group by reacting nortrilobolide (17) with an acidic aqueous medium (Scheme 8). A similar reaction has been observed in the hexaoxygenated guaiano-lide [14]. As expected a mixture of the two 3-alcohols 37 is ob-tained.

Scheme 8. Selective angelate cleavage of nortrilobolide (17) under acidic

aqueous conditions to yield 37.

2.2.5. Selective Reduction of the Lactone Group

Treatment of Tg (1) with sodium borohydride affords a selec-tive reduction of the lactone carbonyl group to form the hemiacetal 38, although only in modest yield (Scheme 9) [20, 22]. Sodium bis(2-methoxyethoxy)ethoxyaluminium hydride (Red-Al) was found to be superior to sodium borohydride [22].

Scheme 9. Selective reduction of lactone carbonyl at C-12.

2.2.6. Chemistry of the 12-Semiacetal (38)

Attempts to O-12-alkylate 38 with ethyl orthoformate only to some extend afforded the acetal 39, instead the two orthoesteres 40 and 41 were formed as the major products (Scheme 10) [22].

Scheme 10. Alkylation at O-12.

Another unexpected property of the 12-hemiacetal is the reac-tion of the diacetate 42, obtained from 38, with nitriles to form oxazoles 43 (Scheme 11) [23].

Scheme 11. Formation of oxazoles 43 from hemiacetal 38.

2.2.7. Synthesis of Thapsigargin (1)

At the present the annual demand of paclitaxel is approximately 1 ton per year. A successful outcome of the current clinical trials of mipsagargin is expected to create an annual demand of Tg (1) in the same order of magnitude. At the present Tg (1) is only available from the roots or fruits of the wild population of T. garganica. A total synthesis of Tg (1) in 42 steps affording an overall yield of 0.6% has been developed starting form (S)-carvone. (Scheme 12) [24, 25].

Scheme 12. Total synthesis of thapsigargin (1) in 42 steps from (S)-carvone.

In spite of the impressive academic achievement this synthesis, however, is not commercial feasible. An alternative procedure could be semi-synthesis from other available natural products. A few other studies towards synthesis of closely related compounds have been published [26, 27]. Trilobolide (16) might be a possibil-ity as an alternative starting material since this compound is easily available and can be isolated from L. trilobum, which can be grown e.g. in The Czech Republic.

A synthetic route has been developed in order to obtain Tg (1) in four steps starting from nortrilobolide (17) (Scheme 13).

Treatment of nortrilobolide (17) with chromium trioxide and aqueous hydrogen fluoride in an one pot reaction affords the ketone 44 in excellent yield. Oxidation of 44 with manganese(III) acetate in the presence of excess amount of octanoic acid affords stereose-lective the -2-octanoyloxygentaed derivative 45. Reduction of 45 with zinc borohydride gives a mixture of the two epimeric alcohols 46S and 46R, 46S being the major product. Finally, acylation of 46S with the mixed anhydride generated from angelic acid and 2,4,6-trichlorobenzoyl chloride yields thapsigargin (1).

3. THE SARCO/ENDOPLASMIC RETICULUM CALCIUM ATPASE AS A DRUG TARGET

In the resting state any cells maintain a low Ca2+ concentration (100 nM) in the cytosol and a high Ca2+ concentration (0.5 mM) in the sarco/endoplasmic reticulum. In the case of the endo/sarco-plasmic reticulum the Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) maintain this gradient by transporting Ca2+ ions from the cytosol into the organelle. For each ATP consumed by the pump two Ca2+ ions are transported across the membrane

O

O

HO

O

O

OH

OH

O

O

O

HO

O

O

OH

OH

O

HO

17 37

O OH+ (aq)

MeCN

O

O

O

O O

O

O

OH

O

H

OH

O

OH38

O

O

O

O O

O

O

OH

O

H

OH

O

O

39Minor

O

O

O

O O

O

O

OH

O

H O

O

O

O

O

O

O

O O

O

O

OH

O

H O

O

O

O

40Major

41Major

EtO OEt, H

OEt

O

O

O

O O

O

O

OH

O

H

OH

O

OH38

O

O

O

O O

O

O

OH

O

H O

O42

O

OO

O

O

O

O O

O

O

OH

O

H O

43 N

O

OH

R

Ac2O

DMAP

ScCl4

RCN

O

O

O

O O

O

O

OH

O

OH

H

O

O

1

42 steps

O

H

(S)-carvone

O

O

O

O O

O

O

OH

O

O

H

OH

O

1

O

O

O

O O

O

O

OH

O

H

OH

O

OH38

NaBH4

or

Red-Al

12

Red-Al

O

Na

O

Al

H

HOO


Scheme 13. Semi-synthesis of Tg (1) starting from nortrilobolide

(17).

and 2-3 H+ ions released into the cytosol [28]. Blockage of the pump results in a collapse of the Ca2+-gradient. As a consequence the cytosolic Ca2+ concentration increases to 500 nM for about 3 hours where after it again decreases to low nM concentration. A continued blockage of the pump affords a new increase after about 19 hours, but now the concentration reaches 1500 nM. The later burst induces a cascade reaction, which eventually results in apop-tosis [13]. From a chemotherapeutic point of view the cytotoxicity is interesting since cells are killed in a proliferation independent way [13]. Most presently used chemotherapeutics like paclitaxel, vincristine and doxorubicin only kill cells during the proliferation and consequently slowly developing cancer diseases like prostate cancer are not affected [13]. The drawback by targeting SERCA, however, is that this pump is essential for survival of almost all kind of cells meaning that Tg (1) is a general cell toxin. This is supported by the low lethal dosis for mice (0.8 mg/kg) [29]. The use of Tg (1) per se as a drug thus is excluded. However, a prodrug targeting the compound towards cancer tissue has been developed. A prodrug is a drug that by itself is inactive, but is cleaved near the pharmacological target to release the active agent. The moiety used to inactivate the agent is named the promoiety. An optimum bond for conjugating the active drug with the promoiety is a bond that only is cleaved in the target tissue (Fig. 4).

Fig. (4). The prodrug principle. An agent (gray oval) is coupled to an inacti-

vating group (the promoiety, circle) via a linker (wavy line) and a bond

sensitive to cleavage only in the target tissue (bold binding). At the pharma-

cological target the sensitive bond is cleaved and the active agent released.

3.1. Structure-Activity Relationships

A requirement for use of the prodrug approach is an intimate knowledge of structure-activity relationships (SAR). Only such knowledge enables location of the linker to give an agent, which still possesses activity. Furthermore SAR analysis might indicate how structural changes of the molecule might make the compound more drugable. Below are presented Tables 1-10 illustrating the effects of changes of the structure of Tg (1). The data given in the table are mainly taken from the references: [21, 30-35].

3.1.1. Changes at the 8-Position

When interpreting the relative activities presented in Table 1 (analogues 20, 47-72) it is important to remember that the IC50 value of Tg (1) is subnanomolar [23, 35] meaning that even an ana-logue 100 times less active than Tg (1) is a potent compound. The IC50 value is the concentration, in which the SERCA pump is inhib-ited 50%. Considering this fact it can be concluded that even dra-matic changes of the side chain at O-8 only to a minor extent reduc-es activity. Even hydrolysis to give the 8-hydroxy analogue 20 still affords a potent compound. From the point of view of designing a prodrug this finding is encouraging since it means that a broad spectrum of linkers possessing a terminal amino group may be used as linkers.

The key then may be a peptide bond. Noticeable is the analogue terminating with a tert-butyloxycarbonyl protecting group (Boc) 62. The significant lower activity of this compound might indicate that the presence of terminal hydrophilic groups could be of importance. Important for defining the pharmacophore of Tg (1) is the finding that inversion of the stereochemistry at C-8 dramatically reduces the potency as seen for analogue 72 (Table 2).

3.1.2. Changes at the 3-Position

In contrast to changes at O-8 dramatic changes can be seen by changes at O-3 (analogues 34, 73-82, Table 3).

No major reduction of activity is observed by replacing the angeloyl with a benzoyl 76 or a flexible octanoyl group 74 (Table 3). However, introduction of a 4-methylbenzoyl 77 causes a severe drop in activity, whereas a 3-methylbenzoyl 78 is tolerated.

A similar trend is seen by replacement with a 2-phenylbenzoate 79 and a 4-phenylbenzoate 80. Adding even a little flexibility into the side chain as is seen in biphenylacetoyl 81 again regains activi-ty.

As was the case for C-8, inversion of the stereochemistry at C-3 provokes a dramatic decrease in activity as seen in analogue 82 (Table 4).

3.1.3 Changes at the 2-Position

The binding site is very tolerant toward substitution in the 2-position since no dramatic shifts in activity occurs by replacements in this position (analogues 83-86, Table 5 and 6). This conclusion is further confirmed by inspecting the naturally occurring hexao-xygentaed guaianloides (Fig. 1), all of which have very similar activities.

3.1.4. Changes in the 10-Position

In contrast to the 2-position, the 10-position is located in an area in the binding cavity, which severely reacts towards introduc-tion of voluminous groups by reducing the affinity of the ligand (analogues 87-92, Table 7).

3.1.5 Changes at the 7- and 11-Positions

Important to notice is that replacement of the hydroxy groups with an acyl group and thereby preventing the group form being hydrogen donors only to a limiting extent effect the IC50 value (ana-logues 19, 22, 93-101, Table 8 and 9).

Acylation of O-7 even with small acyl group only affords a minor decrease in activity but flexible large substituents decrease activity. The epoxide 19 also still possesses some affinity for the

O

O

HO

O

O

OH

OH

O

O

O

HO

O

O

OH

OH

O

O

17 44

O O

O

HO

O

O

OH

OH

O

O

O

O

HO

O

O

OH

OH

O

HO

OO

O45

O

HO

O

O

OH

OH

O

HO

OO

O

O

HO

O

O

OH

OH

O

O

OO

O

1

O

CrO3, HF (aq)

MeCN

O

O

Mn(OAc3)

Octanoic acid

Zn(BH4)2

2,4,6-trichlorobenzoyl chlorideEt3N, Angelic acid

46S 42R


Table 1. Changes at O-8.

Thapsigargin analogues

Relative IC50*

20 R =

5.4/50 [30] [21]

47 R =

327 [21]

48 R =

81 [21]

49 R =

4.4 [21]

50 R =

1.7 [21]

51 R =

1.9 [21]

52 R =

1.5 [21]

53 R =

1.8 [21]

54 R =

1.3 [21]

55 R =

1.7 [21]

56 R =

35 [31]

57 R =

99 [31]

O

O

O

O

OH

O

O

O

OH

H

ROO

8

H

OHN

O

NH2

O

NH

O

NH2

O

NH

O

NH2

O

NH2

O

NH

O

O

O

NH2

O

NH

O

O

O

HN O

O

ONH2

O

NH

ONH2

O

NH2


(Table 1) Contd....

58 R =

90 [31]

59 R =

17 [31]

60 R =

3 [31]

61 R =

2.6 [31]

62 R =

240 [31]

63 R =

287 [31]

64 R =

3.4 [31]

65 R =

1.2 [31]

66 R =

0.8 [31]

67 R =

30 [35]

68 R =

8.5 [35]

69 R =

11.5 [35]

70 R =

44 [35]

71 R =

70 [35]

*The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

O

NH2

O

NH2

O

NH2

O

NH2

O

N

H

O

O

OHN

ONH2

O

N

H

O

NH2

O

N

H

O

NH2

O

N

H

O

NH2

OH

O

OH

NH

O

O

O

O

HN O

O

O

O

HN

ONH

O

O

O

OO

ONH

O

O

O

OO

ONH

OHN O

O


Table 2. Inversion at C-8.

Thapsigargin analogue Relative IC50*

72

3124 [32]




Relative IC50*

34 R =

565 [21]

73 R =

66.5

[32]

74 R =

11.2 [32]

75 R =

1.53 [32]

76 R =

4.0 [21]

77 R =

220 [21]

78 R =

45 [21]

79 R =

15 [21]

80 R =

350 [21]

81 R =

90 [21]


O

O

O

O

OH

O

O

O

O

OH

H O

8

O

O

O

O

O

OH

O

R

O

O

OH

H OO

3

H

O

O

O O

O

O

OH

O

O

H

OH

O

3

O

O

O

O

O

O

O

O


Table 4. Inversion at C-3.


82

438 [32]




83

8 [21]




84

40 [35]

85

0.1/17.5 [33] [35]

86

0.3 [33]

* The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

O

O

O

O

OH

O

O

O

OH

H OO

3

O

O

O

O

O

OH

O

O

OH

H OO

OO

O

O

O

O

OH

O

OH

H OO O

O

O

O

O

O

OH

O

H

OH

OO

H

O

O

O

O

OH

O

H

OH

OO

H




Relative

IC50*

87 R =

42/135 [32] [21]

88 R =

12.5 [21]

89 R =

80 [21]

90

R =

78 [21]

91 R =

350 [21]

92 R =

135 [21]


Table 8. Changes at O-7 and O-11.


Relative

IC50*

93 R1 =

R2 =

2.8 [32]

94 R1 =

R2 =

55 [23]

95 R1 =

R2 =

65 [23]

96 R1 =

R2 =

42.5 [23]

O

O

O

O

OH

O

O

O

OH

H O

R

O 10

H

O

O

O

O

O

O

O

O

O

O

O

O

O

O

H O

R1

R2

7

11

OO

O

H

O

H

O

H

O

H


(Table 8) Contd....

97 R1 =

R2 =

275 [23]

98 R1 =

R2 =

2.5 [32]

99 R1 =

R2 =

55 [21]

100 R1 =

R2 =

15 [32]

*The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value o thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

Table 9. Changes at O-7 and O-11.


19

100 [Private communi-

cation]

22

95 [21]

101

350 [21]


Table 10. Changes at the 12- and 11-positions.


102

1.06 [34]

O

H

H

O

H

O

O O

O

O

O

O

O

O

O

O

H OOO

O

O

O

O

OH

O

O

O

H

O

O O

O

OH

O

O

O

O

O

O

O

H

O

O

O

O

O

O

OH

O

O

OH

H OO O


(Table 10) Contd....


38R

1.63 [22]

38S

1.63 [22]

39

1.17 [22]

103

1.92 [22]

42

47 [23]

104

5.39 [22]

O

O

O

O

OH

OH

O

O

OH

H OO O

O

O

O

O

OH

O

O

OH

H OO

OH

O

O

O

O

O

OH

O

O

OH

H OO

O

O

O

O

O

O

OH

O

O

O

OH

H OO O

O

O

O

O

O

OH

O

O

O

O

H OO O

O

O

O

O

O

O

OH

O

O

H O

OH

O O




40

2.51 [22]

41

40.4 [22]

105

16.4 [22]

106

582 [22]

107

6312 [22]

108

8.5 [23]

O

O

O

O

OH

O

O

H O

O

O

HO

O O

O

O

O

O

OH

O

O

H O

O

O

HO

O O

O

O

O

O

OH

O

O

H O

O

O

H

O O

O

OH

O

O

OH

O

O

H O

O

O

H

O

O

OH

O

O

OH

HO H O

O

O

H

O

O

O

O

O

OH

O

O

H O

N

O

H

O O




109

100 [23]

110

395 [23]


binding site. However no effect was observed in a whole cell assays indicating that inhibition of the pump was not efficient [8].

3.1.6. Changes at the 12- and 11-Positions

The trend observed for position 10 is observed again in the 12 and 11 positions. Introduction of large non-flexible groups causes reduction in affinity whereas smaller groups are tolerated (ana-logues 38-42, 102-110, Table 10).

3.2. The Pharmacophore of Thapsigargin

The first X-ray structure of SERCA bound to Tg (1) was pub-lished by Toyoshina in 2002 [36]. Important for interpreting the Structure-Activity Relationships is proof of the same binding site for all analogues. X-ray structures have revealed that a number of analogues with high affinity all bind to SERCA at the same binding site [23, 35, 37]. The structures verify that the most important inter-actions between the pump and the analogues are hydrophobic inter-actions. Four hydrophobic interactions have been suggested: 1) the angeloyl group at O-3 and N768, V769 and V263, 2) the acyl group at O-8 and residues P827, L828 and I829, 3) the acetyl group at O-10 and residues Y837 and F834, and 4) the methyl group at C-15 and Q259. The octanoyl at O-2 extends into the lipophilic mem-brane with only limited interaction with the ATPase [35]. A hydro-phobic interaction between the lactone ring and F256 has also been suggested (Fig. 5). In addition, to verifying the same binding site calculations indicate that beside hydrophobic interactions some water molecules also are of significance for correct localization of the ligand in the binding site [23], in particular hydrogen bridge from the carbonyl of the acyl group at O-8 to the backbone amide of L828 or the side chain of E255. In addition a hydrogen bond between OH-7 and the backbone of I829 may stabilize the binding [23]. The importance of hydrophobic interactions from the angeloyl and the acetyl group to the pump explains the drastic drop in affini-ty if either C-3 or C-8 is inverted. After inversion the two acyl group would be prevented from forming hydrophobic interactions with the backbone.

As revealed from Fig. 6 a long flexible substituent at O-8 al-lows affinity for the pump since it is able to adjust between the fifth, third and fourth transmembrane segments. The Boc-group is placed in a cavity between the first, second, third and fourth helices towards the surface of the pump [37]. This binding site explains the poor activity of Ile-N-6-aminohexanoic acid since the nonflexible amid group would be located between the transmembrane residues.

Fig. (5). Hydrophobic interactions between the backbone of the binding site

of SERCA (helices) and angeloyl at O-3 (N768, V769, V263), butanoyl at

O-8 (P827, L828, I829), acetyl at O-10 (Y837) and the lactone ring (residue

F256) of Tg (1) (ball and sticks). The interacting residues are marked in

gray. PDB 4J2T.

Fig. (6). The location of the N-Boc-12-aimnododecanoyl residue of com-

pound 62 in the binding site of SERCA. PDB 2BY4.

O

O

O

O

OH

O

O

H O

N

O

H

O O

O

O

O

O

OH

O

O

H O

N

O

H

O O

O


Whereas a large diversity of substituents at O-2 and O-8 are accepted only a limited number of nonflexible groups are allowed at O-3. Very pronounced is the significant loss of activity by mov-ing the methyl group from the meta to the para position of benzoic acid [21]. A little more diversity is allowed in the cavity near the O-10 [21].

Based on the structure activity-relationships the pharmacophore model shown in Fig. 7 has been developed for Tg (1).

Fig. (7). Pharmacophore model of thapsigargin (1). The ovals indicate resi-

dues that interact with the backbone of SERCA via hydrophobic interactions

and Bu-C=O and O-7 are assumed to be involved in hydrogen bridges from

Tg (1) to to the backbone of SERCA.

4. PRODRUG DESIGN

The potency of analogues with a long but flexible acyl residue at O-8 and the possibility for formation of a peptide bond if a -amino group was introduced was used as a starting point for pro-drug development. Targeting of the analogue towards cancer tissue was obtained taking advantage of proteolytic enzymes expressed to a larger extent in tumors.

4.1. Prostate Specific Antigen

Prostate specific antigen (PSA) is a proteolytic enzyme secreted into the semen of by the epithelial cells of the prostate, with the physiologic function to regulate the viscosity of the ejaculate [38, 39]. A healthy man typically has a serum concentration of less than 4 ng/mL. An steady increase beyond this level might indicate the presence of malign prostate cells [38]. PSA entrance into the blood causes inactivation either by complexation to or proteolysis by ma-jor serum protease inhibitors like 1-antichymotrypsin or 2-macroglobulin [40-42].

4.1.1. Substrate Specificity of PSA

From the point of view of creating prodrugs the substrate speci-ficity of PSA calls attention. PSA cleaves on the C-side of most Tyr residues and certain Leu residues, but not on the C-site of Phe and Trp site [43, 44]. In addition during screening of a number of sub-strates PSA was shown for cleave on the C-site of Gln [45].

4.1.2. PSA Specific Substrates

A few attempts to develop prodrug based on the substrate speci-ficity of PSA have been made [46]. In this case advantage of the ability of PSA to cleave on the C-site of Gln was taken to construct a number of peptides terminating with Gln. Screening of a number of peptides terminating in Gln afforded His-Ser-Ser-Lys-Leu-Gln (HSSKLQ) as a possible choice for a promoiety group. The peptide was conjugated to 7-amino-4-metylcoumarin and the product was cleaved fast in the prostate by PSA but was stable in serum [47].

Conjugation to the peptide, morpholino-Pro-Ser-Ser-Lys-Leu-Gln afforded a prodrug 111 (Fig. 8), which was found not to be a substrate for PSA. However, insertion of an additional Leu residue afforded a prodrug 112 with the wanted cleavage profile to produce 113 after PSA cleavage [47]. The morpholino group was added in order to increase solubility.

Fig. (8). Prodrug developed for cleavage by PSA. Prodrug only cleaved by

PSA and thereby selective toxic in the prostate and in prostate cancer tissue.

The obtained prodrug 112 was tested in mice. No toxicity was observed but inoculated prostate cancer tumors were found to shrink by treatment with the drug. In comparison, tumors on not treated control mice grew exponentially [47]. The compound was under the name of G115 taken into the pipeline of GenSpera in order to develop it into a drug.

4.2. Prostate Specific Membrane Antigen

Like PSA, prostate specific membrane antigen (PSMA) is a proteolytic enzyme but in contrast to PSA PSMA is not secreted into the lumen but is bound to the membrane of the epithelial cells.

Fig. (9). Prodrug developed for cleavage by PSMA. The arrows indicate the

cleavage sites.

O

O

O

O O

O

O

O

O

OH

H

O

O

HOH

HO

H

H

10

83

7

15

2

11

12

O

O

O

O HO

O

O

OH

OH

O

O

HN

O

R

111: R =

HN

NH

HN

NH

HN

N

O

OH2N

O

O

NH2

O

HOO

N

NH

O

O

NH

HN

NH

HN

NH

HN

N

O

OH2N

O

O

NH2

O

HOO

N

NH

O

OO

PSA clevage

112: R =

HO

NH2

O

113: R =

HO

O

O

O

O HO

O

O

OH

OH

O

O

HN

O

R

114 R =

OHN

NH

HN

NH2

O

O

O OH

O OH

O

NH

O OH

OO OH

O

OH

116 R =

O NH2

O

OH

115 R =

OHN

NH2

O

O

O OH

OH

PSMA cleavage


4.2.1. Substrate Specificity of PSMA

PSMA is a carboxypeptidase, which cleaves poly- -glutamoyl peptide bonds in glutamated folates and thereby enables uptake of folate [46, 48]. PSMA is expressed in the prostate but in spite of the name PSMA is overexpressed in neovasculature in a number of tumors including hepatocellular carcinoma, mesothelioma, ovarian cancer, renal cancer, bladder cancer, renal cancer and breast cancer [48].

4.2.2. PSMA Specific Substrates

The substrate specificity of PSMA also have inspired to design prodrugs [46]. The thapsigargin prodrug 114 is shown in Figure 8. Incubation of the prodrug 114 with PSMA afforded a fast cleavage of the three terminal Glu residues and a slower cleavage of the fourth Glu to give 12-ADTAsp 116. Incubation of 12-ADTAsp 116 and 12-ADTAspGlu 115 showed that 12-ADTAsp 116 was almost half as potent a cytotoxin towards TSU and LNCaP cells as Tg (1), whereas 12-ADTAspGlu 115 was 10 times less potent towards LNCaP cells and to a 100 times less potent towards TSU cells [48]. An X-ray study of the complex of 12-ADTAsp 116 bound to SER-CA revealed that 12-ADTAsp 116 was bound very similar as Boc-12ADT 62. The compound was under the name of G202 taken into the pipeline of GenSpera in order to develop it into a drug.

4.3. Development of G202 into a Drug

4.3.1. In Vivo Studies

The in vitro results encouraged to perform in vivo studies. Mice inoculated with PSMA producing human prostate cancer cells (LNCaP and MDA-PCa2b cell lines) were treated with G202 (56 mg/kg) for three consecutive days. Over a 30 days period an aver-age regression of LNCaP on 50% was observed, in contrast the tumors grew exponentially in mice incubated with vehicle and a 100% enlargement of tumors was observed if the mice were treated with docetaxel [48]. In a number of other xenografts with human cancer cells lines, significant regressions were observed. Histologi-cal analyses showed tumor necrosis in the treated animals. The observed necrosis indicated that the cells were not killed by inhibi-tion of the SERCA pump since this would be expected to afford apoptosis. A more likely explanation is the cleavage of the prodrug in the neovascular tissue causing destruction of the capillaries and thereby depriving the tumor for oxygen and nutrients.

After administration of G202, the half-life of G202 in BALB mice was determined to 4.9 hours. Only trace amounts of 12-ADTAsp 116 and 12-ADTAspGlu 115 were found. In contrast, significant levels of the active agent were found in tumors. In mon-keys the half-life was determined to be 9.7 hours and in humans 15.1 hours. Only reversible damages were observed at a dose of 10 mg/kg in monkeys [48].

4.3.2. Clinical Trials

The preclinical data convinced FDA to allow clinical trials per-formed on G202, which now has been given the name Mipsagargin. A phase II clinical trial was performed on 25 patients suffering from hepatocellular carcinoma who had failed the first line treat-ment with the kinase inhibitor sorafenib [49]. Patients were admin-istered episodic dosing of mipsagargin on the first three days of each treatment cycle of 28 days. Study participants experienced a median time to progression of 4.2 months, nearly twice the time demonstrated in prior studies with placebo or ineffective agents. Thirty-five percent of patients received 5 or more cycles of treat-ment with an average time on study of 7.1 months. Additionally, mipsagargin demonstrated decreased blood flow in liver tumors as measured by DCE-MRI [50]. In addition clinical trials against glio-blastomas are presently being performed.

CONCLUSION

Thapsigargin (1) was originally isolated of curiosity in order to understand the skin irritating properties of the resin of T. garganica [51]. Observed tumor promoting properties and general cytotoxicity of the agent made it unlikely that the compound might be a drug candidate [10]. However, after in depth studies to understand the mechanism of action [11] and use of prodrug design, a compound has successfully been developed, which currently is under phase II clinical trials showing promising results. Ingenol mebutate market-ed by Leo Pharma is another example of a drug developed from a cocarcinogen [52].

LIST OF ABBREVIATIONS

12ADT = 8-(12-amindodecanoy)-8-debutanoyl)-thapsigargin

PSA = Prostate specific antigen

PSMA = prostate specific membrane antigen

SERCA = sarco/endoplamsic reticulum calcium ATPase

Tc = thapsigargicin

Tg = thapsigargin

CONFLICT OF INTEREST

SBC is a member of the Scientific Board of GenSpera. NTQD has no conflict of interest. The project has been supported by the Danish Cancer Society, GenSpera and the Danish Strategic Re-search Council.

ACKNOWLEDGEMENTS

NTQD and SBC have contributed equally to the manuscript.

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Received: June 16, 2015 Accepted: August 17, 2015

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