IN-VITRO ANTIMALARIAL ACTIVITY BY SYBR GREEN-I BASED ...

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Jatakiya et al. World Journal of Pharmacy and Pharmaceutical Sciences

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





Table-2


% 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



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






Table-3


% 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




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






Table-4


% 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




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


J-20 was dissolved in methanol/1-butanol/ionic liquid (triethyl methyl ammonium methyl


water bath was done for 30 minutes in case of methanol, 40 minutes in case of 1-butanol and


J-20

11 12

13

14

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






Table-5


% 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


J-21 R= -OCH3 J-22 R= -CH3

J-23 R= -SO2NH2 J-24 R= -Cl

15


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): 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






Table-6


% 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





): 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)

16


2670


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






Table-7


% yield (%w/w) 88.4% 66% 99.45%

Rf value 0.55 0.55 0.55

Melting point 286-288 286-288 286-288





): 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)

17


2671



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






Table-8


% yield (%w/w) 88% 65.6% 99.7%

Rf value 0.51 0.51 0.51

Melting point 276-278 276-278 276-278





): 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)


Series J-3: (2-amino-5-bromobenzoyl chloride series)5-7

18


2672


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)

19 20

21


2673


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


J-30 was dissolved in Methanol/1-butanol/ionic liquid (triethyl methyl ammonium methyl




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

22

23


2674







Table-9


% yield (%w/w) 88% 66% 99.68%

Rf value 0.45 0.45 0.45

Melting point 244-246 244-246 244-246





): 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





24


2675



Table-10


% yield (%w/w) 86.8% 65% 99.58%

Rf value 0.41 0.41 0.41

Melting point 214-216 214-216 214-216





): 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






Table-11


% 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


25


2676





): 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)


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






Table-12


% 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




26


2677



): 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)


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


J-40 was dissolved in Methanol/1-butanol/ionic liquid (triethyl methyl ammonium methyl




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

27 28


2678


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






Table-13


% yield (%w/w) 86.8% 64% 99.58%

Rf value 0.28 0.28 0.28

Melting point 156-158 156-158 156-158




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


29

30


2679






Table-14


% yield (%w/w) 85.8% 63% 99.56%

Rf value 0.25 0.25 0.25

Melting point 134-136 134-136 134-136





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






Table-15


% 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


31


2680





): 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)


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





Table-16


% yield (%w/w) 87.2% 64% 99.58%

Rf value 0.30 0.30 0.30

Melting point 168-170 168-170 168-170




32


2681


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


2682


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.


2683


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


2684


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


2685


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


2686


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




2687


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


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


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


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


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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