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IN-VITRO ANTIMALARIAL ACTIVITY BY SYBR GREEN-I BASED
FLUORESCENCE ASSAY OF N-SUBSTITUTED PIPERAZINE
DERIVATIVES SYNTHESISED BY HIGHER GREENER SOLVENTS
AND IONIC LIQUIDS ON PLASMODIUM FALCIPARUM
Viraj P. Jatakiya*, Nadim M. R. Chhipa and Prof. Dr. Dhrubo Jyoti Sen
Department of Pharmaceutical Chemistry, Shri Sarvajanik Pharmacy College, Gujarat
Technological University, Arvind Baug, Mehsana-384001, Gujarat, India.
ABSTRACT
N-substituted novel piperazine derivatives in four series were planned
to synthesize by comparison study for the solvents with higher greener
values and lower greener values for that yield percentage and kinetic
studies were performed and evaluated for their capacity to inhibit
the growth of Plasmodium falciparum 3D7 strain in culture. N -ethyl
piperazine has been conjugated with substituted aromatic acid
chlorides in J-1 to J-3 series and in J-4 with higher homologous
unsubstituted acid chloride and then converted in to Schiff’s base
by substituted aromatic amines by conventional method (methanol)
and green solvents (1-butanol and ionic liquid: triethyl methyl
ammonium methyl sulphate). Rate of reaction was tested for all
compounds by kinetic study and all compounds were characterized
by spectral studies. The synthesised compounds have been screened by in-vitro method by
SYBR Green-I assay for Plasmodium falciparum. Final compounds % yield in methanol
was found around 88%, in 1-butanol around 66% and in ionic liquid above 99%. Overall
rate of reaction was found 2nd
order in kinetic study. Rate of reaction k in methanol was
found = 0.0004 µg-1
sec-1 and in 1-butanol it was = 0.0001 µg
-1sec
-1, half life of reaction
was 50 seconds in methanol and 1000 seconds in 1-butanol while overall reaction time in
ionic liquid was only 20 seconds. In antimalarial evaluation series J-3 (5-bromo-2-
aminobenzoyl chloride series) was found to have highest activity in terms of IC50 (µM).
WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS
VVoolluummee 33,, IIssssuuee 22,, 22665533--22669922.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357
Article Received on
19 November 2013,
Revised on 23 December
2013,
Accepted on 27 January
2014
*Correspondence for Author
Viraj P. Jatakiya
Department of Pharmaceutical
Chemistry, Shri Sarvajanik
Pharmacy College, Gujarat
Technological University,
Arvind Baug, Mehsana-
384001, Gujarat, India.
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(Compound J-33: 4-{-[(2-amino-5-bromophenyl)(4-ethylpiperazin-1-yl) methylidene]
amino} benzene-sulfonamide) was found most active having IC50 0.076 µM and SI
>2631.57. % yield and rate of reaction is highest in ionic liquid compared to other two
solvents so reaction is more ecofriendly and more acceptable in this
environmental benign solvent. Among all four series J-3 is more active and among all
compounds J-33 is most active compounds against Plasmodium falciparum 3D7. So, the
activities of compounds constitute a strong rationale for further investigation.
Keywords: Imidazolo-piperazine, Ethyl piperazine, Schiff’s base, Green solvents, Ionic
liquid, Plasmodium falciparum 3D7 strain, SYBR Green-I assay, IC50, CC50, Selectivity
Index.
INTRODUCTION
Wu T. N. et al. has developed novel imidazolo-piperazine hit series of dual acting
antimalarial and tested against wild type and drug resistant parasites. Starting from a hit
series from a GNF (Genomics Institute of the Novartis Research Foundation) compound
library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a
novel imidazolo-piperazine scaffold was optimized. They focused on optimization of cellular
potency against wild-type and drug resistant parasites and improvement of physiochemical
and pharmacokinetic properties. The lead compounds in this series showed good potencies
in-vitro and decent oral exposure levels in-vivo. In a Plasmodium berghei mouse infection
model, one lead compound lowered the parasitemia level by 99.4% after administration of
100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days.
N
NN
O
NH2
NH
O
O
1
The lead compounds were also well-tolerated in the preliminary in-vitro toxicity studies and
represents an interesting lead for drug development. Below compound is first generation
imidazolo-piperazine with more promising activity and better pharmacokinetics.1
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N
NN
O
NH2
NH
FR
NH2
Where R= H or F
2
Chatterjee A. K. et al. did research on the basis of the initial success of optimization of a
novel series of imidazolo-piperazines, a second generation of compounds involving changes
in the core piperazine ring was synthesized to improve antimalarial properties. These
changes were carried out to further improve the potency and metabolic stability of the
compounds by leveraging the outcome of a set of in-vitro metabolic identification studies.
The optimized 8,8-dimethyl imidazolo-piperazine analogues exhibited improved potency, in-
vitro metabolic stability profile and, as a result, enhanced oral exposure in-vivo in mice. The
optimized compounds were found to be more efficacious than the current antimalarials in a
malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies
to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology
studies.2
REVIEW OF PIPERAZINE DERIVATIVES AS AN ANTIMALARIAL:
1. Louis M. et al. has performed Synthesis and in-vitro and in-vivo Antimalarial Activity
of N1-(7-chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine derivatives. Three series of
monoquinolines consisting of a 1,4-bis(3-aminopropyl)piperazine linker and a large variety
of terminal groups were synthesized and examined for antimalarial activity for
resistance species. Among them eleven compounds displayed a higher selectivity index
(ratio CC50/IC50 activity) than chloroquine and one of them cured mice infected by
Plasmodium berghei.3
2. Mendoza A. et al. did synthesis of aryl Piperazine and pyrrolidine derivatives were
synthesised and evaluated for their capacity to inhibit the growth of Plasmodium falciparum
chloroquine-resistant (FCR-3) strain in culture. The combined presence of a hydroxyl
group, a propane chain and a fluoro were shown to be crucial for the antiplasmodial activity.
The most active compound 1-(4-fluoronaphthyl)-3-[4-(4-nitro-2-trifluoromethylphenyl)
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piperazin-1-yl] propan-1-ol was almost 20–40 times more active on P. falciparum (IC50:
0.5 lM) than on tumorogenic and non-tumorogenic cells. In- silico molecular docking
study and molecular electrostatic potential calculation revealed that this compound bound to
the active site of Plasmodium plasmepsin II enzyme.4
3. Sergheraert C. et al. has described Synthesis and evaluation of the activity of a new
family of 1,4-bis(3-aminopropyl) piperazine derivatives against a chloroquine-resistant strain
of Plasmodium falciparum, and as inhibitors of β-hematin formation. The highest
antimalarial activities were obtained for compounds displaying the highest predicted
vacuolar accumulation ratios and the best potencies as inhibitors of β-hematin formation.
The most potent compound displayed an activity 3-fold better than chloroquine for
acomparable selectivity index upon MRC-5 cells. Therefore, in this series, the replacement
of the 7-chloroquinoline group can constitute a strong rationale for further investigation.5
4. Melnyk P. et al. had performed synthesis and evaluation of the activity of new N1
-(7-
chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine derivatives against a chloroquine-
resistant strain of Plasmodium falciparum. This work provided additional structure–
activity and structure–cytotoxicity information in the N1
-(7- chloro-4-quinolyl)-1,4-bis(3-
aminopropyl)piperazine family and proved that a number of substitutions lead to
compounds with high activities and reduced cytotoxicities.6
5. Fattorusso C. et al .did design, synthesis, and antiplasmodial activity of antimalarial
heterodimers based on the 1,4-bis(3-aminopropyl)piperazine linker. Biological evaluation
included determination of activity against chloroquine- sensitive and chloroquine-resistant
Plasmodium falciparum strains. Some of the novel compounds presented high activity in-
vitro against chloroquine-resistant strains, more potent than chloroquine and clotrimazole.7
6. Castro E. A. et al. offered linear regression models on a set of aryl-piperazine
derivatives that are obtained by exploring a pool containing 1497 Dragon molecular
descriptors, in order to establish the best relationships linking the molecular structure
characteristics to their exhibited potencies against chloroquine resistant and chloroquine
sensitive strains of Plasmodium falciparum parasite. The adjustment of the training
molecular set together with the performance achieved during the internal and external
validation processes leads to predictive QSAR models. In addition, they derive alternative
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linear models based on the Coral methodology, which lead to satisfactory results. They
applied the final equations to predict the activity on some unknown compounds having
non-observed activities.8
RATIONALE OF WORK
The treatment of malaria is become very challenging now a day due to drug resistance. As
per the report of WHO and NATIONAL INSTITUTE OF MALARIA RESEARCH-INDIA,
resistance is developing with almost all antimalarial drugs except Artemisinin. So, there is a
need to develop some more effective anti-malarial with new moieties and geometry to
target the resistant species and to save millions of people from death. So here is a plan to
synthesize new anti-malarials with core piperazine ring in accordance with the SAR of
imidazolo-piperazine derivatives with less reaction steps and with high yield. Also there is a
need to develop a new project in accordance to “Green Guidelines” to protect the
environment and human from various hazards. The whole research plan will be carried out in
solvents with higher green values those are 1-butanol and ionic liquid. Methanol is common
solvent for this synthesis process. So against it 1-butanol and ionic liquid has been taken
to develop the process of synthesis. 1-butanol have high boiling point than methanol so has
low environmental issues while ionic liquids are environmentally benign solvents.
PROJECT DESIGN
Structure design
As per the report of WHO and National Institute of Malaria Research, India resistance
covered almost all anti-malarial drugs except Artemisinine and its derivatives. So, there is a
need to develop newer antimalarial agents with new moieties and increased potency.9,10
So,
after reviewing the article of GNF (Genomics of Novartis Research Foundation) we have
focused on those compounds in terms of their activity, synthesis steps, % yield and
pharmacokinetics. GNF made library of thousands of compounds contain imidazolo-
piperazine as core ring and new geometry. In the research of finding newer anti-malarial,
they extracted some compounds with good potency, very good activity and very good
pharmacokinetic profile. The compounds showed very good anti-malarial activity on
resistant strain of the parasites of plasmodium derivatives.1,2 So, we focused on these
compounds containing different geometry and designed the new structure of compounds with
core piperazine ring by the use of the principles of drug discovery those are mainly structural
scissoring and structural pruning. For this, structural activity relationship of reviewed
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compounds was carried out by deep examination of their structures, geometry and activity.
Structural activity relationship of the reviewed compounds is mentioned below. Correlation
of designed compound series with reviewed compound is given below.
Figure-1: Correlation of designed compounds series with reviewed compound (Centre)
In this novel approach we assume that the nitrogen of imidazole ring may act as H-bond
acceptor. So, in series no.2 (J-21 to J-24) –OH substitution in phenyl ring and in series no.3
(J-31 to J-34) –NH2 substitution on phenyl ring has been kept. Both substituted group can
act as H-bond acceptor as well as H-bond donator. Articles of anti-malarials contain core
piperazine ring gave us inspiration that our designed compounds might have good anti-
malarial activity on the resistant genus of plasmodium. The part of aim is also to design the
synthesis scheme of the compounds with minimum reaction steps and high % yield. So,
accordingly scheme is designed as below.
Synthetic scheme of series:5-7
N
NH
CH3
+
ClO
R2
R1
R3 N
N
O
R2
R1
R3
CH3
N-Ethy l
PiperazineSubstituted
Acid chloride
Condensation
Keto-Piperazine intermediate
NH2
R
Substituted Aniline
-HCl
Methanol/
Butanol/
Ionic liquids
H2SO4
H2O
Final compound
N
N
N
R2
R1
R3
R
CH3
+
Acid chloride substitutions:
Series-J-1 R1= -Cl, R
2= -H R
3= -H
Series-J-2 R1= -H, R
2= -OH, R
3= -Br
Series-J-3R1= -H, R
2= -NH2, R
3= -Br
Aniline substitutions:
Where R= -OCH3, -CH3, -SO2NH2, -Cl
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Synthetic scheme of series 4:5-7
+
N-Ethy l
Piperazine
Condensation
Keto-Piperazine intermediate
Substituted Aniline
-HCl
Methanol/
Butanol/
Ionic liquids
H2SO4
H2O
Final compound
+
N
NH
CH3 COCl
Phenacety l chloride
N
N
CH3
O
NH2
R
N
N
CH3
N
R
MECHANISMS OF REACTION STEPS5-7
After preparation of acid chloride the synthetic part takes only two steps to prepare final
compounds. First step involves the reaction of N-ethyl piperazine with different substituted
acid chlorides to prepare keto-piperazine intermediate. The mechanism involves in this step is
simple condensation reaction. The reaction completes with liberation of hydrochloric acid.
Second step involves reaction of keto-piperazine intermediates with different substituted
anilines to yield final products of all four series. It requires a proper solvent for completion.
The mechanism involves in this is Neucleophilic addition followed by dehydration. Nitrogen
of the –NH2 group of the different substituted anilines do attack on the carbonyl carbon of the
keto-piperazine intermediate which is partially positive charged and give carbinolamine
intermediate. Further use of dehydrating agent gives iminium intermediate and neutralization
gives final imine.
Reaction mechanisms of both steps are given below with their respective intermediates.
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N
N
CH3
H
Cl
O
+
-N
N
CH3
H
CCl
O-
+
-HCl
N
N
CH3
O
Mechanism of step-1: Condensation reaction
C
O: :
+ :NH2R C
O:-
NH2R+
:. .
C
OH
NHR
:. .
. .
Carbinolamine
H---A
C
OH2
+
NHR. .
:
C
NHR+
-H2O
A-
C
NR+ H---A
Iminium IonImine
Mechanism of step-2: Neucleophilic addition followed by dehydration
EXPERIMENTAL EXPLORATION
About experiment and parameters to be evaluated
In this section synthetic part, procedure of synthesis in detail, reaction synthesis steps
structure of the final compounds. IUPAC name of final compounds and different evaluation
parameters are discussed.
Evaluation is done for various parameters those are described below:
Solubility Melting point
% yield
Thin layer chromatography
UV/Visible spectrophotometry
Infra-red spectroscopy
Mass spectroscopy
Nuclear Magnetic Resonance (1H-NMR) spectroscopy
Reaction steps, procedure and evaluation of all final compounds are described individually
for each four series. Also comparison of % yield of final compounds which are synthesised in
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methanol, 1-butanol and ionic liquid is also given. Series name are given according to acid
chlorides used.
Chemicals
All chemicals for synthesis work were procured from below listed companies
Sigma Aldrich, U.S.A.
Loba Chemie, Mumbai.
S.D. Fine chem., Mumbai.
Finar chem. Ltd., Ahmedabad.
Instruments
All the melting points were determined in open capillaries on chemiline CL-725 melting
point apparatus and are uncorrected.
Thin layer chromatography was performed on microscopic plates (2×7.5cm) coated with
silica-gel-60 F254 prepared by Merck KGaA, Germany and spots were visualized under UV
light and by exposure to iodine vapour.
UV spectra were recorded in UV-1800 Shimadzu spectrophotometer.
IR spectra of all compounds was recorded on FT-IR 8400S Shimadzu
spectrophotometer using KBr.
Mass spectra was obtained using 2010EV MS Shimadzu instrument.
The 1H-NMR was recorded on Bruker Advance-II NMR 400MHz instruments using
DMSO-d6 as solvent and Tetra methyl silane (TMS) as internal standard, chemical shifts
were expressed as δ values (part per million). Splitting patterns are as follows: s (singlet), d
(doublet), m (multiplet), b (broad), dd (doublet in doublet).
Series J-1: (4-chlorobenzoyl chloride series)5-7
1. Preparation of 4-chlorobenzoyl chloride from 4-chlorobenzoic acid: 0.1 mole 4-
chlorobenzoic acid was reacted with 0.1 mole of thionyl chloride in round-bottom flask.
Little amount of extra thionyl chloride was added to prevent anhydride formation. Reaction
was refluxed for 1 hour at 50 and reaction was monitored by thin layer chromatography.
The compound was isolated as liquid and purified by distillation.
Boiling point: 102-104 , Rf value: 0.62 (Ethyl acetate: hexane- 1:1)
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Cl
ClO
4-chlorobenzoyl chloride
O OH
Cl
4-chlorobenzoic acid
+ SOCl2
Thiony l chloride
Reflux
50 °C, 1 hr.
2. Preparation of keto-piperazine derivative from 4-chlorobenzoyl chloride and N-ethyl
piperazine: 0.1 mole 4-chlorobenzoyl chloride was reacted with 0.1 mole of N-ethyl
piperazine drop wise. Though the reaction is explosive, care should be taken while
performing the reaction and also it was carried out in ice bath. The product was purified by
washing with diethyl ether. The product was isolated as solid and reaction was monitored by
thin layer chromatography.
Melting point: 56 , Rf value: 0.74 (Ethyl acetate), Mass: m/z 252.9 (M)
N
N
CH3
H
+
N-ethy l piperazine
COCl
Cl
4-chlorobenzoyl chloride
Condensation
Ice bath
-HCl N
N
CH3
O
Cl
(4-chlorophenyl)(4-ethylpiperazin-1-yl)methanone
3. Preparation of final product from J-10 and different substituted anilines: 0.1 mole of
J-10 was dissolved in methanol/1-butanol/ionic liquid (triethyl methyl ammonium methyl
sulphate). In that 0.1 mole of different substituted anilines were added separately. Heating on
water bath was done for 30 minutes in case of Methanol, 40 minutes in case of 1-butanol and
reaction was carried out at room temperature in case of ionic liquid. A drop of H2SO4 was
added as dehydrating agent to isolate solid product. Isolated compound was purified by
washing with diethyl ether and ethyl acetate. Used anilines are given below in reaction. Exact
time of reaction for all solvents is mentioned in next chapter of kinetic study. Evaluation
parameters are discussed of all four compounds of this series are mentioned after reaction
with their respective code.
J-10
3 4
5 6
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N
N
CH3
O
Cl
(4-chlorophenyl)(4-ethylpiperazin-1-yl)methanone
+
NH2
R
Substituted anilines
Methanol/1-butanol/ionic liquid
H2SO4
-H2O
N
N
CH3
N
Cl
R
4. Evaluation of compounds belongs to series J-1
Evaluation of J-11:
N
N
CH3
N
O
Cl
CH3
Compound Code: J-11
Molecular formula: C20H24N3OCl
IUPAC name: 1-(4-chlorophenyl)-1-(4-ethylpiperazin-1-yl)-N-(4 methoxyphenyl)
methanimine
Molecular weight: 357.87 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-1
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 87.4% 66% 99.7%
Rf value 0.32 0.32 0.32
Melting point 192-194 192-194 192-194
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 228.50 nm
Mass Spectroscopy report: m/z 359.1(M+1), 360 (M+2)
J-11 R= -OCH3 J-12 R= -CH3
J-13 R= -SO2NH2 J-14 R= -Cl
7
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NMR Spectroscopy report:1H NMR (DMSO): δ 7.475-7.414 (dd, 4H, Ar-H), 7.271-7.249
(m, 2H, Ar-H), 6.894-6.872 (d, 2H, Ar-H), 3.714-3.682 (d, 3H, CH3), 2.920-2.591 (m, 8H,
piperazine C4H8N2), 2.585-2.552 (q, 2H, CH2), 1.165-1.119 (t, 3H, CH3)
Evaluation of J-12:
N
N
CH3
N
Cl
CH3
Compound Code: J-12
Molecular formula: C20H24N3Cl
IUPAC name: 1-(4-chlorophenyl) -1- (4-ethylpiperazin-1-yl) -N- (4-methylphenyl)
methanimine
Molecular weight: 341.87gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-2
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 86.2% 62.6% 99.58%
Rf value 0.27 0.27 0.27
Melting point 176-178 176-178 176-178
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 226 nm
Mass Spectroscopy report: m/z 341 (M), 343 (M+2)
8
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Evaluation of J-13:
N
N
CH3
N
Cl
S
O
O
NH2
Compound Code: J-13
Molecular formula: C19H23N4O2SCl
IUPAC name: 4-{-[(4-chlorophenyl) (4-ethylpiperazin-1-yl) methylidene] amino}
benzenesulfonamide
Molecular weight: 406.92 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-3
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 88.2% 66.2% 99.7%
Rf value 0.36 0.36 0.36
Melting point 226-228 226-228 226-228
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 268 nm
Infra-red Spectroscopy report (KBr ν cm-1
): 3460.06-3303.83 (-NH2), 2935.46 (aromatic
C-H), 1641.31 (noncyclic C=N), 1595.02-1502.44 (-SO2), 1149.50 (aromatic C-Cl Para),
1006.77-979.77 (aromatic C-H bending)
Mass Spectroscopy report: m/z 407 (M)
NMR Spectroscopy report: 1H NMR (DMSO): δ 8.182-8.126 (d, 2H, SO2NH2) 7.586-7.518
(dd 4H, Ar-H), 7.282-7.276 (d, 2H, Ar-H), 7.127-7.076 (m, 2H, Ar-H), 2.870-2.751 and
2.531-2.441 (mm, 8H, piperazine, C4H8N2 and 2H, CH2), 1.189-0.961 (t, 3H, CH3)
9
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Evaluation of J-14
N
N
CH3
N
Cl
Cl
Compound Code: J-14
Molecular formula: C19H21N3Cl2
IUPAC name: N,1-bis(4-chlorophenyl)-1-(4-ethylpiperazin-1-yl)methanimine
Molecular weight: 362.29 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-4
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 87.8% 65.4% 99.68%
Rf value 0.31 0.31 0.31
Melting point 218-220 218-220 218-220
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 254 nm
Mass Spectroscopy report: m/z 262.9 (M), 265 (M+2)
Series J-2: (5-bromo-2-hydroxybenzoyl chloride series)5-7
1. Preparation of 4-bromo-2-(chlorocarbonyl)phenyl acetate from 2-(acetyloxy)-5-
bromobenzoic acid: 0.1 mole of 5-bromo-2-hydroxybenzoic acid was dissolved in glacial
acetic acid and in that 0.1 mole of acetic anhydride was added to acetylate phenolic group.
Product was isolated as solid by adding above mixture in cold water. This will prevent
dimerization in further step of making acid chloride. 0.1 mole 2-(acetyloxy)-5-bromobenzoic
acid was reacted with 0.1 mole of thionyl chloride in round-bottom flask. Little amount of
extra thionyl chloride was added to prevent anhydride formation. Reaction was refluxed for
10
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1.5 hour at 50 and reaction was monitored by thin layer chromatography. The compound
was isolated as liquid and purified by distillation.
Boiling point: 132-134 , Rf value: 0.54 (Ethyl acetate: hexane- 1:1)
Br
O
O OH
O
CH3
2-(acetyloxy)-5-bromobenzoic acid
O OH
Br
OH
5-bromo-2-hydroxybenzoic acid
Glacial acetic acid
Acetic anhydride
-CH3COOH
Br
O
O OH
O
CH3
+ SOCl2Reflux
50 °C, 1.5 hr
Br
O
O Cl
O
CH3
2-(acetyloxy)-5-bromobenzoic acid 4-bromo-2-(chlorocarbonyl)phenyl acetateThiony l chloride
2. Preparation of keto-piperazine derivative from 4-bromo-2-(chlorocarbonyl) phenyl
acetate and N-ethyl piperazine: 0.1 mole 4-bromo-2-(chlorocarbonyl) phenyl acetate was
reacted with 0.1 mole of N-ethyl piperazine drop wise. Though the reaction is explosive, care
should be taken while performing the reaction and also it was carried out in ice bath. The
product was purified by washing with diethyl ether. The product was isolated as solid and
reaction was monitored by thin layer chromatography.
Melting point: 38-40 , Rf value: 0.70 (Ethyl acetate)
N
N
CH3
H
+
COCl
O
Br
O
CH3
N-ethy l piperazine 4-bromo-2-(chlorocarbonyl)phenyl acetate
condensation
ice bath
-HClN
N
CH3
O
O
Br
O CH3
4-bromo-2-[(4-ethylpiperazin-1-yl)carbonyl]phenyl acetate
3. Preparation of final product from J-20 and different substituted anilines: 0.1 mole of
J-20 was dissolved in methanol/1-butanol/ionic liquid (triethyl methyl ammonium methyl
sulphate). In that 0.1 mole of different substituted anilines were added separately. Heating on
water bath was done for 30 minutes in case of methanol, 40 minutes in case of 1-butanol and
reaction was carried out at room temperature in case of ionic liquid. A drop of H2SO4 was
J-20
11 12
13
14
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added as dehydrating agent to isolate solid product. Excess H2SO4 was added to do
simultaneous hydrolysis of acetylated product and aqueous NaOH solution was added till
neutralization. Isolated compound was purified by washing with diethyl ether and ethyl
acetate. Used anilines are given below in reaction. Evaluation parameters are discussed of all
four compounds of this series are mentioned after reaction with their respective code.
N
N
CH3
O
O
Br
O CH3
+
NH2
R
4-bromo-2-[(4-ethylpiperazin-1-yl)carbonyl]phenyl acetate
Substituted aniline
N
N
CH3
N
OH
Br
R
methanol/1-butanol/
ionic liquid
H2SO4 excess
-H2O
-CH3COOH
4. Evaluation of compounds belongs to series J-2
Evaluation of J-21:
N
N
CH3
N
O
OH
Br
CH3
Compound Code: J-21
Molecular formula: C20H24N3O2Br
IUPAC name: 4-bromo-2-{-(4-ethylpiperazin-1-yl)[(4-methoxyphenyl)imino] methyl}
phenol
Molecular weight: 418.32 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-5
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 88.2% 65.6% 99.68%
Rf value 0.48 0.48 0.48
Melting point 256-258 256-258 256-258
*Triethyl methyl ammonium methyl sulphate
J-21 R= -OCH3 J-22 R= -CH3
J-23 R= -SO2NH2 J-24 R= -Cl
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Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 237 nm
Infra-red Spectroscopy report (KBr ν cm-1
): 3581.56-3305.76 (phenolic-O-H), 2264.27-
2081.05 (N-C=N), 1232.43-1220.86 (-C-O-H), 1093.56-1083.92 (aromatic C-Br meta),
1039.65-983.63 (aromatic C-H)
Mass Spectroscopy report: - m/z 418.2 (M), 420 (M+2), 422 (M+4)
Evaluation of J-22:
N
N
CH3
N
CH3
OH
Br
Compound Code: J-22
Molecular formula: C20H24N3OBr
IUPAC name: 4-bromo-2-{-(4-ethylpiperazin-1-yl)[(4-methylphenyl)imino] methyl}phenol
Molecular weight: 402.32 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-6
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 87.2% 64.6% 99.6%
Rf value 0.42 0.42 0.42
Melting point 230-232 230-232 230-232
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 238.50 nm
Infra-red Spectroscopy report (KBr ν cm-1
): 3581.56-3402.20 (phenolic-O-H), 2985.60
(aromatic C-H), 2081.05 (N-C=N), 1236.29-1209.28 (-C-O-H), 1132.14-1093.56 (aromatic
C-Br meta), 1012.56-954.70 (aromatic C-H bending)
Mass Spectroscopy report: m/z 404 (M+2)
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NMR Spectroscopy report:1H NMR (DMSO): δ 7.580 (s, 1H, Ar-H), 7.278-7.264 (dd, 1H,
Ar-H), 7.123-7.083 (m, 4H, Ar-H), 6.848 (d, 1H, Ar-H), 5.042 (s, 1H, OH), 2.927-2.617 (m,
8H, piperazine C4H8N2), 2.589-2.557 (q, 2H, CH2), 2.200 (s, 3H, CH3), 1.201-1.121 (t, 3H,
CH3)
Evaluation of J-23:
N
N
CH3
N
OH
Br
S
O
O
NH2
Compound Code: J-23
Molecular formula: C19H23N4O3SBr
IUPAC name: 4-{-[(5-bromo-2-hydroxyphenyl)(4-ethylpiperazin-1-yl)methylidene] amino}
benzene sulfonamide
Molecular weight: 467.37 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-7
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 88.4% 66% 99.45%
Rf value 0.55 0.55 0.55
Melting point 286-288 286-288 286-288
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 262.50 nm
Infra-red Spectroscopy report (KBr ν cm-1
): 3500.56-3402.20 (phenolic O-H), 3330.84-
3244.05 (-SO2NH2), 3024.18 (aromatic C-H), 2177.48-2084.91 (N-C=N), 1596.95-1556.45 (-
SO2NH2), 1303.79-1232.43 (-C-O-H), 1101.28-1091.63 (aromatic C-Br meta), 1039.56-
906.48 (aromatic C-H bending)
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Mass Spectroscopy report: m/z 468 (M+1)
NMR Spectroscopy report: 1H NMR (DMSO): δ 8.180-8.130 (d, 2H, SO2NH2), 7.580-
7.495 (d, 2H, Ar-H), 7.264 (d, 1H, Ar-H), 7.123-7.083 (m, 3H, Ar-H), 6.848 (d, 1H, Ar-H),
5.042 (s, 1H, OH), 2.741-2.731 and 2.589-2.557 (m, 8H, piperazine C4H8N2 and 2H CH2),
1.201-1.138 (t, 3H, CH3)
Evaluation of J-24:
N
N
CH3
N
Cl
OH
Br
Compound Code: J-24
Molecular formula: C19H21N3O2ClBr
IUPAC name: 4-bromo-2-[{(4-chlorophenyl)imino}(4-ethylpiperazin-1-yl)methyl] phenol
Molecular weight: 422.74 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-8
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 88% 65.6% 99.7%
Rf value 0.51 0.51 0.51
Melting point 276-278 276-278 276-278
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 259 nm
Infra-red Spectroscopy report (KBr ν cm-1
): 3593.14-3404.13 (phenolic O-H), 3014.53-
2775.38 (aromatic C-H), 2266.20-2065.62 (N-C=N), 1249.79 (C-O-H), 1137.92 (aromatic C-
Cl Para), 1081.99-1053.06 (aromatic C-Br meta), 1002.92-914.20 (aromatic C-H bending)
Mass Spectroscopy report: - m/z 422.4 (M), 424 (M+2), 426 (M+4)
Series J-3: (2-amino-5-bromobenzoyl chloride series)5-7
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1. Preparation of 2-(acetylamino)-5-bromobenzoyl chloride from 2-(acetylamino)-5-
bromobenzoic acid: 0.1 mole of 2-amino-5-bromobenzoic acid was dissolved in glacial
acetic acid and in that 0.1 mole of acetic anhydride was added to acetylate phenolic group.
Product was isolated as solid by adding above mixture in cold water. This will prevent
dimerization in further step of making acid chloride.
0.1 mole of 2-(acetylamino)-5-bromobenzoic acid was reacted with 0.1 mole of thionyl
chloride in round-bottom flask. Little amount of extra thionyl chloride was added to prevent
anhydride formation. Reaction was refluxed for 1.5 hour at 50 and reaction was monitored
by thin layer chromatography. The compound was isolated as liquid and purified by
distillation.
Boiling point: 156-158 , Rf value: 0.51 (Ethyl acetate: hexane-1:1)
O OH
NH2
Br
glacial acetic acid
acetic anhydride
-CH3COOH
O OH
NH
Br
O
CH3
2-amino-5-bromobenzoic acid 2-(acetylamino)-5-bromobenzoic acid
O OH
NH
Br
O
CH3
O Cl
NH
Br
O
CH3
2-(acetylamino)-5-bromobenzoic acid 2-(acetylamino)-5-bromobenzoyl chloride
+ SOCl2
Thiony l chloride
reflux
50 °C, 1.5 hr.
2. Preparation of keto-piperazine derivative from 2-(acetylamino)-5-bromobenzoyl
chloride and N-ethyl piperazine: 0.1 mole of2-(acetylamino)-5-bromobenzoyl chloride was
reacted with 0.1 mole of N-ethyl piperazine drop wise. Though the reaction is explosive, care
should be taken while performing the reaction and also it was carried out in ice bath. The
product was purified by washing with diethyl ether. The product was isolated as solid and
reaction was monitored by thin layer chromatography.
Melting point: 34-36 , Rf value: 0.62 (ethyl acetate)
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O Cl
NH
Br
O
CH3
2-(acetylamino)-5-bromobenzoyl chloride
N
N
CH3
H
N-ethy l piperazine
+
condantation
ice bath
-HClN
N
CH3
O
NH
Br
O CH3
N-{4-bromo-2-[(4-ethylpiperazin-1-yl)carbonyl]phenyl}acetamide
3. Preparation of final product from J-30 and different substituted anilines: 0.1 mole of
J-30 was dissolved in Methanol/1-butanol/ionic liquid (triethyl methyl ammonium methyl
sulphate). In that 0.1 mole of different substituted anilines were added separately. Heating on
water bath was done for 30 minutes in case of Methanol, 40 minutes in case of 1-butanol and
reaction was carried out at room temperature in case of ionic liquid. A drop of H2SO4 was
added as dehydrating agent to isolate solid product. Excess H2SO4was added to do
simultaneous hydrolysis of acetylated product and aqueous NaOH solution was added till
neutralization. Isolated compound was purified by washing with diethyl ether and ethyl
acetate. Used anilines are given below in reaction. Evaluation parameters are discussed of all
four compounds of this series are mentioned after reaction with their respective code.
N
N
CH3
O
NH
Br
O CH3
+
R
NH2
Substituted aniline
N
N
CH3
N
NH2
Br
RN-{4-bromo-2-[(4-ethylpiperazin-1-yl)carbonyl]phenyl}acetamide
methanol/1-butanol/
ionic liquid
Excess H2SO4
-H2O
-CH3COOH
4. Evaluation of compounds belongs to series J-3:
Evaluation of J-31:
N
N
CH3
N
O
NH2
Br
CH3
Compound Code: J-31
Molecular formula: C20H25N4OBr
IUPAC name: 4-bromo-2-[{(4-methoxyphenyl)imino}(4-ethylpiperazin-1-yl)methyl]aniline
J-30
J-31 R= -OCH3 J-32 R= -CH3
J-33 R= -SO2NH2 J-34 R= -Cl
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Molecular weight: 417.34 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-9
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 88% 66% 99.68%
Rf value 0.45 0.45 0.45
Melting point 244-246 244-246 244-246
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 290 nm
Infra-red Spectroscopy report (KBr ν cm-1
): 3402.20-3328.91 (aromatic N-H), 2106.12-
2094.55 (N-C=N), 1240 (aromatic C-N-H), 1130.21-1091.63 (aromatic C-Br meta), 1026.06-
983.63 (aromatic C-H bending)
Mass Spectroscopy report:- m/z 418 (M), 422 (M+2)
Evaluation of J-32:
N
N
N
NH2
Br
CH3
CH3
Compound Code: J-32
Molecular formula: C20H25N4Br
IUPAC name: 4-bromo-2-{(4-ethylpiperazin-1-yl)[(4-methylphenyl)imino] methyl}aniline
Molecular weight: 401.34 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
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Comparison of % yield, Rf value and melting point in different solvents
Table-10
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 86.8% 65% 99.58%
Rf value 0.41 0.41 0.41
Melting point 214-216 214-216 214-216
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 283.50 nm
Infra-red Spectroscopy report (KBr ν cm-1
): 3402.20-3201.61 (aromatic N-H), 3024.18-
2719.44 (aromatic C-H), 2179.41-2100.34 (N-C=N), 1251.72-1240.14 (aromatic C-N-H),
1170.71-1056.92 (aromatic C-Br meta), 1006.77-997.13 (aromatic C-H bending)
Mass Spectroscopy report:m/z 401 (M), 403 (M+2)
Evaluation of J-33:
N
N
N
NH2
Br
CH3
S
O
O
NH2
Compound Code: J-33
Molecular formula: C19H24N5O2SBr
IUPAC name: 4-{-[(2-amino-5-bromophenyl)(4-ethylpiperazin-1-yl)methylidene] amino}
benzene sulfonamide
Molecular weight: 466.39 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-11
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 88.4% 66.4% 99.66%
Rf value 0.54 0.54 0.54
Melting point 266-268 266-268 266-268
*Triethyl methyl ammonium methyl sulphate
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Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 257 nm
Infra-red Spectroscopy report (KBr ν cm-1
): 3433.06-3384.55 (aromatic N-H& -NH2),
2092.62 (N-C=N), 1265.22-1257.50 (aromatic C-N-H), 1064.63 (aromatic C-Br meta),
995.20-968.20 (aromatic C-H bending)
Mass Spectroscopy report: m/z 267 (M+1)
NMR Spectroscopy report: 1H NMR (DMSO): δ 9.156 (s, 2H, SO2NH2), 7.942-7.896 (m,
3H, Ar-H), 7.464-7.445 (d, 2H, Ar-H), 7.269-7.254 (d, 1H, Ar-H), 6.874-6.832 (m, 1H, Ar-
H), 4.429 (s, 2H. NH2), 2.919-2.609 (m, 8H, piperazine C4H8N2), 2.582-2.550 (q, 2H, CH2),
1.160-1.124 (t, 3H, CH3)
Evaluation of J-34:
N
N
N
NH2
Br
Cl
CH3
Compound Code: J-34
Molecular formula: C19H24N4ClBr
IUPAC name: 4-bromo-2-[-[(4-chlorophenyl)imino](4-ethylpiperazin-1-yl)methyl]aniline
Molecular weight: 421.76 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-12
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 87.8% 66.4% 99.70%
Rf value 0.48 0.48 0.48
Melting point 254-256 254-256 254-256
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 292 nm
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Infra-red Spectroscopy report (KBr ν cm-1
): 3305.76-3145.68 (aromatic N-H), 2864.09
(aromatic C-H), 2235.34-2092.62 (N-C=N), 1257.50-1242.07 (aromatic C-N-H), 1078.13
(aromatic C-Br meta), 1008.70-914.20 (aromatic C-H bending)
Mass Spectroscopy report: - m/z 420.8 (M), 422 (M+2), 424 (M+4)
Series J-4: (Phenacetyl chloride series)5-7
1. Preparation of keto-piperazine derivative from phenacetyl chloride and N-ethyl
piperazine:
0.1 mole of phenacetyl chloride was reacted with 0.1 mole of N-ethyl piperazine drop wise.
Though the reaction is explosive, care should be taken while performing the reaction and also
it was carried out in ice bath. The product was purified by washing with diethyl ether. The
product was isolated as solid and reaction was monitored by thin layer chromatography.
Melting point: 82-84 , Rf value: 0.74 (Ethyl acetate), Mass- M- 233 m/z
N
N
CH3
H
+
COCl
N-ethy l piperazine phenylacetyl chloride
condansation
ice bath
-HCl
N
N
CH3
O
1-(4-ethylpiperazin-1-yl)-2-phenylethanone
2. Preparation of final product from J-40 and different substituted anilines: 0.1 mole of
J-40 was dissolved in Methanol/1-butanol/ionic liquid (triethyl methyl ammonium methyl
sulphate). In that 0.1 mole of different substituted anilines were added separately. Heating on
water bath was done for 30 minutes in case of Methanol, 40 minutes in case of 1-butanol and
reaction was carried out at room temperature in case of ionic liquid. A drop of H2SO4 was
added as dehydrating agent to isolate solid product. Isolated compound was purified by
washing with diethyl ether and ethyl acetate. Used anilines are given below in reaction.
Evaluation parameters are discussed of all four compounds of this series are mentioned after
reaction with their respective code.
N
N
CH3
O
1-(4-ethylpiperazin-1-yl)-2-phenylethanone
NH2
R
+
Substituted aniline
methanol/1-butanol/
ionic liquid
H2SO4
-H2O
N
N
CH3
N
R
3. Evaluation of compounds belongs to series J-4:
J-40
J-41 R= -OCH3, J-42 R= -CH3
J-43 R= -SO2NH2 J-44 R= -Cl
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Evaluation of J-41:
N
N
N
CH3
OCH3
Compound Code: J-41
Molecular formula: C21H27N3O
IUPAC name: 1-[-1-(4-methoxyphenyl)-3-phenylprop-1-en-2-yl]-4-ethylpiperazine
Molecular weight: 337.45 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-13
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 86.8% 64% 99.58%
Rf value 0.28 0.28 0.28
Melting point 156-158 156-158 156-158
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 224 nm
Mass Spectroscopy report: m/z 337 (M)
Evaluation of J-42:
N
N
N
CH3
CH3
Compound Code: J-42
Molecular formula: C21H27N3
IUPAC name: 1-ethyl-4-[(-1-(4-methylphenyl)-3-phenylprop-1-en-2-yl]piperazine
Molecular weight: 321.44 gm/mol
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Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-14
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 85.8% 63% 99.56%
Rf value 0.25 0.25 0.25
Melting point 134-136 134-136 134-136
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 218 nm
Mass Spectroscopy report: m/z 323 (M+2)
Evaluation of J-43:
N
N
N
S
O
O
NH2
CH3
Compound Code: J-43
Molecular formula: C20H26N4O2S
IUPAC name: 4- {[1-(4-methylpiperazin -1- yl) -2- phenylethylidene] amino}
benzenesulfonamide
Molecular weight: 386.51 gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-15
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 87.2% 64.4% 99.62%
Rf value 0.33 0.33 0.33
Melting point 186-188 186-188 186-188
*Triethyl methyl ammonium methyl sulphate
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Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 256 nm
Infra-red Spectroscopy report (KBr ν cm-1
): 3344.34-3207.40 (-SO2NH2), 3146.97 2941.24
(aromatic C-H), 2866.02-2700.16 (C-H), 1552.59 (-SO2NH2), 1053.60 939.27 (aromatic C-
H)
Mass Spectroscopy report: m/z 387 (M+1)
NMR Spectroscopy report: 1
H NMR (DMSO) : δ 8.175-8.123 (d, 2H, SO2NH2), 7.575-
7.526 (d, 2H, Ar-H), 7.285-7.278 (d, 2H, Ar-H), 7.135-7.071 (m, 5H Ar-H), 2.868-2.753
and 2.537-2.440 (m, 8H, piperazine C4H8N2 and 2H, CH2), 2.3 (s, 1 H, CH2), 1.187-0.963 (t,
3H, CH3)
Evaluation of J-44:
N
N
N
CH3
Cl
Compound Code: J-44
Molecular formula: C20H24N3Cl
IUPAC name: N-(4-chlorophenyl)-1-(4-ethylpiperazin-1-yl)-2-phenylethanimine
Molecular weight: 341.87gm/mol
Solvents used for purification: Diethyl ether, Ethyl acetate
Solvents used for recrystallization: Methanol:water- 80:20
Solubility: Water, DMSO
Comparison of % yield, Rf value and melting point in different solvents
Table-16
Solvents Methanol 1-butanol Ionic liquid*
% yield (%w/w) 87.2% 64% 99.58%
Rf value 0.30 0.30 0.30
Melting point 168-170 168-170 168-170
*Triethyl methyl ammonium methyl sulphate
Mobile phase for TLC: CH2Cl2: Methanol-Ammonium hydroxide- 1:1(v/v)-1 drop
UV Visible Spectroscopy report: λmax: 248 nm
32
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Mass Spectroscopy report: m/z 342.2 (M+1), 343 (M+2)
NMR Spectroscopy report: 1H NMR (DMSO): δ 7.498-7.452 (m, 4H, Cl-Ar-H), 7.284-
7.248 (m, 5H Ar-H), 2.921-2.620 (m, 8H, piperazine C4H8N2 and 2H, CH2), 2.579-2.558 (q,
2H, CH2), 1.150-1.126 (t, 3H, CH3)
ANTI-MALARIAL EVALUATION14-19
Method: P.falciparum drug sensitivity assay using SYBR® Green I V1.0
This procedure describes the SYBR®Green I-based antimalarial drug susceptibility
fluoroassay for laboratory/field use which is optimized for minimal infrastructure and
technical equipment requirements while achieving appropriate sensitivity levels. The assay
measures the DNA content of malaria-infected erythrocytes by fluorochrome staining the
parasite DNA.
Materials and equipment
Materials
Amber 1.5 mL micro tubes, Eppendorf
Clear, cell culture-treated 96-well micro titer
Centrifuge tube
Conical Tubes, 15 mL
Conical Tubes, 50 mL, Flat top, Polypropylene
Cylinder
Gloves: non-powdered
Micropipette tips, sterile
Nalgene MF75 Series 0.2 μM Disposable Sterilization Filter Units: 500mL and 1 L
Nalgene MF75 Series Filter Unit Receivers, 250 mL
Needles
Parafilm® sealing film
Pasteur pipettes
1 mL, 2mL, 5mL, 10mL and 25mL serological pipettes
Sterile plugged and unplugged glass 9 inch Pasteur pipettes
Super frost Disposable Microscope Slides, 75×25mm
Transfer pipettes
Vacuum pump
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Volumetric flasks
Weighing boat
Equipment
Centrifuge with swinging bucket rotor
Class II Bio safety cabinet
Fluorescence reader
Freezers
Liquid handler
Medical grade gas bottles and manometer
Refrigerator
Vacuum trap
Water bath
Water-jacketed incubator set at 36–38°C
Weighing balance
Reagents:
0.5 M EDTA pH 8.0
10% CMS
70% ethanol
Giemsa Modified Azure Blend, EM
Human erythrocytes, compatible with plasma, stored at 2–8 ºC
Medical grade gas: 90% N2, 5% CO2, 5%O2
Methanol
Saponin
SYBR Green I supplied by in-vitro gen as 10000×concentrate in DMSO; stored at –20 ºC.
Triton X-100
Water for cell culture applications
Antimalarial drugs–for example quinine sulphate, desethylamodiaquine, piperaquine,
pyronaridine, mefloquine, lumefantrine, chloroquine diphosphate, dihydroartemisinin, and
primaquine phosphate, atovaquone, artemisinin, artesunic acid.
Sample
Parasitized whole blood from P. falciparum infected subjects P. falciparum culture.
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Procedure
Aseptic procedures
All procedures (except centrifugation) have been performed in a level II biosafety cabinet
with the biosafety cabinet surface which has been wiped down with aseptic solution at
the beginning and the end of every day. The valves on gas cylinders at the end of each day
have been closed. The incubator and the storage surfaces were cleaned at least every 3 months.
Preparation of lysis buffer (1 L)
15.76 gm of Tris-HCl has been dissolved completely in about 700 mL cell culture water
using a magnetic stirrer. pH has been adjusted to 7.5 using concentrated hydrochloric acid. 20
mL 0.5 M EDTA was added to give a final concentration of 10mM (2% w/v). 160 mg
saponin (0.016 % w/v final) has been added. 16.0 mL Triton X-100 (1.6 % v/v final) has been
mixed. Cell culture has been added in water to bring the final volume to 1 Litre. The solution
was mixed thoroughly, avoiding the creation of bubbles. Vacuum filtration has been done
with the solution using 0.2μ pore to remove any particulate matter and store indefinitely at
RT.
SYBR Green I stock solution
10000x SYBR Green I concentrate has been thawed at room temperature in laminar flow
hood in a darkened room. Aliquot 30 μL into amber-colored Eppendorf tubes, label with the
day’s date and store at -20 ºC for up to 6 months.
Lysis buffer containing SYBR Green I (15 mL)
This solution should be made fresh in a darkened room. Thawed one 30 μL aliquot of SYBR
Green I. Added 30 μL SYBR Green I to 15 mL lysis buffer (20x final SYBR Green
concentrations). 15 mL lysis buffer is adequate for one 96 plate. Pipetted to mix, avoiding the
creation of bubbles.
Preparation of malaria cultures and sensitivity assay
Determined % parasitaemia of malarial culture. For fresh field isolates ≤ 0.3%, run the
assay at 2% hematocrit in complete medium without reducing the parasitaemia. If
parasitaemia of culture-adapted samples or fresh field isolates are >0.3, dilute to 0.3% or
0.15% parasitaemia using complete culture medium for 72 or 96hr incubations respectively at
2% hematocrit in complete medium. A 72hr assay is adequate for most drugs; 96hr
incubation can be used for slow acting drugs like antibiotics. Fresh field isolate are not
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washed prior to the assay. Using automated liquid handler or manually, add 100 μL
malaria-infected erythrocytes to each well on a pre-dosed drug plate. Incubate cultures for
72hr or 96hr at 37 ºC in a humidified chamber, under a gas mixture of 90% N2, 5% O2, and
5% CO2 or in a candle jar. After the 72hr or 96hr incubation, added 100 μL lysis buffer
containing 20x SYBR Green I to each well, in a dark room. Incubated the plates at RT in the
dark for 24 hrs. Fluorescence on a fluorescence plate reader has been recorded with
excitation and emission wavelength bands centered at 485 and 530 nm, respectively. Data
was transferred into a graphic programme (EXCEL) and IC50 values were obtained by
Logit regression analysis. Chloroquine diphosphate (SIGMA) was used as the standard
reference drugs.
Cytotoxicity assay
Cytotoxicity of the extracts was carried out using Vero cell line (C1008; Monkey
kidney fibroblast) following the method as mentioned in Sashidhara et al. (2012).18,19 The
cells were incubated with test sample dilutions for 72h and MTT was used as reagent for
detection of cytotoxicity. 50% cytotoxic concentration (CC50) was determined using non-
linear regression analysis. Selectivity Index (SI) was calculated as:
SI = CC50 / IC50
This procedure is designed for use by appropriately-equipped laboratories working on in-
vitro drug susceptibility testing of P. falciparum. Training is required to perform the
procedure successfully.
So, synthesized compounds have been sent to CDRI-Central Drug Research
Institute, Lucknow for evaluation under the expertization of Dr. S. K. Puri and Dr.
Kumkum Shrivastava.
RESULT & DISCUSSION
RESULT OF ANTIMALARIAL ACTIVITY
Mentioned results include antimalarial activity against 3D7 strain of P. falciparum in terms
of IC50, cytotoxic assay on Vero cell line in terms of CC50 and Selectivity Index.
Table-17: Result of antimalarial activity
Sample Code IC50 (µM) CC50 (µM) SI CC50/IC50
J-11 0.5 140.31 280.62
J-12 0.7 145.21 207.44
J-13 0.13 168.56 1296.61
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J-14 0.42 130 309.52
J-21 0.23 36.76 159.82
J-22 0.52 53.76 103.38
J-23 0.096 66 687.5
J-24 0.27 59.3 219.62
J-31 0.15 170 1133.33
J-32 0.35 >200 >571.42
J-33 0.076 >200 >2631.57
J-34 0.11 182.11 1665.54
J-41 >1.0 50.32 <50.32
J-42 >1.0 92.12 <92.12
J-43 0.80 87.65 109.56
J-44 >1.0 47.21 <47.21
Chloroquine 0.005 125 25000
Figure-2 Figure-3
Figure-2: Graph of comparison of IC50 values of compounds
Figure-3: Graph of comparison of CC50 values of compounds
Figure-4: Graph of comparison of selectivity index of compounds
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DISCUSSION OF ANTIMALARIAL ACTIVITY
Synthesis of novel N-substituted piperazine derivatives was carried out in different four
series. All were evaluated for antimalarial activity against Plasmodium falciparum (3D7
strain) by in-vitro testing method named Malaria SYBR Green I-based fluorescence (MSF)
assay. IC50 value of all compounds was calculated by Logit regression analysis. Cytotoxicity
assay was also carried out by using Vero cell line (C1008; Monkey kidney fibroblast) and
50% cytotoxic concentration (CC50) was determined using non-liner regression
analysis.18,19 Selectivity index was also calculated as: SI = CC50/IC50
Intra-series comparison
Series J-1 (4-chlorobenzoyl chloride series): IC50 of compounds were found to be 0.13
for J-13, 0.42 for J-14, 0.5 for J-11 and 0.7 for J-12 so compound J-13 has lower IC50 than
others in the series so possess higher antimalarial activity among four. CC50 of compounds
were found to be 168.56 for J-13, 145.21 for J-12, 140.31 for J-11 and 130 for J-14 so
compound J-13 has higher CC50 value than others in the series so possess low toxicity.
Selectivity index of compounds were calculated from above data and were found to be
1296.61 for J-13, 309.52 for J-14, 280.62 for J-11 and 207.44 for J-12 so compound J-13
has higher selectivity index than others so possess high selectivity towards parasites. So
overall J-13 was found to be best in the series in terms of all parameters for that it was
evaluated because it has higher antimalarial activity low toxicity and higher selectivity
among four compounds of the series.
Series J-2 (5-bromo 2-hydroxy benzoyl chloride series): IC50 of compounds were found
to be 0.096 for J-23, 0.23 for J-21, 0.27 for J-24 and 0.52 for J-22 so compound J-23 has
lower IC50 than others in the series so possess higher antimalarial activity among four.
CC50 of compounds were found to be 66 for J-23, 59.3 for J-24, 53.76 for J-22 and 36.76
for J-21 so compound J-13 has higher CC50 value than others in the series so possess low
toxicity. Selectivity index of compounds were calculated from above data and were found
to 687.5 for J-23, 219.62 for J-24, 152.82 for J-21 and 103.38 for J-22 so compound J-23
has higher selectivity index than others so possess high selectivity towards parasites. So
overall J-23 was found to be best in the series in terms of all parameters for that it was
evaluated because it has higher antimalarial activity low toxicity and higher selectivity
among four compounds of the series.
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Series J-3 (5-bromo 2-amino benzoyl chloride series): IC50 of compounds were found to
be 0.076 for J-33, 0.11 for J-34, 0.15 for J-31 and 0.35 for J-32 so compound J-33 has lower
IC50 than others in the series so possess higher antimalarial activity among four. CC50 of
compounds were found to be >200 for J-32, >200 for J-33, 182.11 for J-34 and 170 for
J-31 so compound J-32 and J-33 has higher CC50 value than others in the series so possess
low toxicity. Selectivity index of compounds were calculated from above data and were
found to >2631.57 for J-33, 1665.54 for J-34, 1133.33 for J-31 and >571.42 for J-32 so
compound J-33 has higher selectivity index than others so possess high selectivity towards
parasites. So overall J-33 was found to be best in the series in terms of all parameters for that
it was evaluated because it has higher antimalarial activity low toxicity and higher selectivity
among four compounds of the series.
Figure-5
Series J-4 (phenacetyl chloride series): IC50 of compounds were found to be 0.8 for J-43,
>1.0 for J-41, >1.0 for J-42 and >1.0 for J-44 so compound J-43 has lower IC50 than others in
the series so possess higher antimalarial activity among four. CC50 of compounds were
found to be 92.12 for J-42, 87.62 for J-43, 50.32 for J-41 and 47.21 for J-44 so compound J-
42 has higher CC50 value than others in the series so possess low toxicity. Selectivity index
of compounds were calculated from above data and were found to 109.56 for J-43, <92.12
for J-42, <50.32 for J-41 and <47.21 for J-44 so compound J-23 has higher selectivity index
than others so possess high selectivity towards parasites.
So overall J-43 was found to be best in the series in terms of all parameters for that it was
evaluated because it has higher antimalarial activity low toxicity and higher selectivity
among four compounds of the series. So compounds which are active in their respective
series have some similarity which can be explained by common structure. (Figure-5)
Compounds which have substituted aniline with sulphonamide group at para position possess
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higher antimalarial activity and higher selectivity with low toxicity compare to methoxy,
methyl and chloro substitution at same position so sulphonamide substitution is found
necessary for better activity and selectivity.
Inter-series comparison
Inter-series comparison of the activity of compounds provides the idea of best active series
among all. As per the results and comparison graph of IC50, CC50 and Selectivity Index of all
synthesised compounds, Compounds of series 3 were found best among all series in terms of
all three parameters. So series of 5-bromo 2-amino benzoyl chloride was found active. Inter-
series comparison of IC50 is depicted in the form of table below:
Comparison of IC50 of first compound of all four series
Table-18
Compound code J-11 J-21 J-31 J-41
IC50 0.5 0.23 0.15 >1.0
Comparison of IC50 of second compound of all four series
Table-19
Compound code J-12 J-22 J-32 J-42
IC50 0.7 0.52 0.35 >1.0
Comparison of IC50 of third compound of all four series
Table-20
Compound code
J-13 J-23 J-33 J-43
IC50 0.13 0.096 0.076 0.8
Comparison of IC50 of forth compound of all four series
Table-21
Compound code J-14 J-24 J-34 J-44
IC50 0.42 0.27 0.11 >1.0
From above inter-series comparison, series 3 was found more active towards P.
falciparum 3D7 strain; series 2 was found 2nd
most active series; series 1 was found 3rd
among active series and series 4 was found least active series. This result can be explained
by comparing structure of compounds. It is due to difference in substitution on the acid
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chloride used to prepare the individual series. In series J-1; p-chlorobenzoyl chloride was
used, in series J-2; 5-bromo-2-hydroxy benzoyl chloride was used, in series J-3; 5-bromo-2-
aminobenzoyl chloride was used and in series J-4; phenacetyl chloride was used. Acid
chloride of series J-2 contain hydroxyl group at 2nd
position and series J-3 contain amino
group at 2nd
position. Both group can act as H-bond donor as well as H-bond acceptor at
binding site that’s why these series are more active than series J-1 and J-4 but among series
J-2 and J-3, 3rd
is more active so it can be predicted that amino substitution at 2nd
position
of acid chloride gives better antimalarial activity with high selectivity (Table-17) compare
to all. From above two intra-series and inter-series comparison of all synthesised
compounds, compound J-33 was found most active antimalarial agent against Plasmodium
falciparum 3D7 strain because of its lowest IC50 value among all that is 0.076.
Figure-6
Toxicity of J-33 was also found less compare to all other compounds of all series because
CC50 value of compound is >200 which was found highest. Selectivity index of J-33 is
>2631.57 and it was found greater than all synthesised compounds.
So overall, compound J-33 was found best in terms of activity and selectivity.
Figure-7
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Structure activity relationship of compound can be carried out as below after
examination of results
Figure-8
Substitution at R with electron donating group like –OCH3, -CH3, -SO2NH2, -Cl is
required for activity. Among four –SO2NH2 gives better antimalarial activity.
Substitution at R2 and R3 with electron donating group is essential for activity.
Compounds without substitution at same position may give poor activity or may
inactive.
Substitution at R1 with such electron donating group which can act as H-bond donor as
well as H-bond acceptor increases the antimalarial activity as well as selectivity. It can be
substituted with hydroxyl or amino group. Among them amino substitution gives better
activity.
Comparison with standard drug
Chloroquine was selected as a standard drug and IC50 of it was found 0.005, CC50 was found
125 and Selectivity Index was found 25000. The same values of synthesised compounds are
lower than chloroquine. So it can be said that synthesised compounds have antimalarial
activity but none of them are as potential as chloroquine.
CONCLUSION
For antimalarial study
After performing all relevant experiments it has been concluded that synthesised compounds
of all four series have antimalarial activity. Among them series J-3 (5-bromo-2-
hydroxybenzoyl chloride series) is found more active in terms of IC50, CC50 and Selectivity
Index. Compound J-33: 4-{- [(2-amino-5-bromophenyl) (4-ethylpiperazin-1-yl)
methylidene] amino}benzenesulfonamide has been found more active, less toxic and more
selective among all synthesised compounds but the same values of synthesised compounds
are lower than chloroquine apart from that activity of compounds constitute a strong
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rationale for further investigation.
ACKNOWLEDGEMENT
All India GPAT scholar Mr. Viraj P. Jatakiya did his M.Pharm. project (2011-2013) on
Synthesis of N-substituted piperazine derivatives by higher greener solvents and it’s
antimalarial activity under the guidance of Prof. Dr. Dhrubo Jyoti Sen and part of his
project is In-vitro antimalarial activity by SYBR Green-I based fluorescence assay of N-
substituted piperazine derivatives synthesised by higher greener solvents and ionic liquids on
plasmodium falciparum. This work has been co-assisted by another all India GPAT scholar
Mr. Nadim M. R. Chhipa who is currently doing M.Pharm. project (2012-2014) on
Synthesis and anti-HIV activity of chicoric acid analogues under the same guide in the
department of Pharmaceutical Chemistry of Shri Sarvajanik Pharmacy College, Mehsana.
Both of these two M.Pharm. projects have been fully funded by RSC Research Grant of
United Kingdom. The Royal Society of Chemistry, Great Britain has awarded £2000 to Prof.
Sen as RSC Fund in 2009.
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