hybrid pharmacophore-based drug design, synthesis, and antiproliferative activity of...

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Medicinal Chemistry Research ISSN 1054-2523Volume 24Number 4 Med Chem Res (2015) 24:1534-1545DOI 10.1007/s00044-014-1236-1

Hybrid pharmacophore-based drug design,synthesis, and antiproliferative activityof 1,4-dihydropyridines-linked alkylatinganticancer agents

Rajesh K. Singh, D. N. Prasad &T. R. Bhardwaj

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ORIGINAL RESEARCH

Hybrid pharmacophore-based drug design, synthesis,and antiproliferative activity of 1,4-dihydropyridines-linkedalkylating anticancer agents

Rajesh K. Singh • D. N. Prasad • T. R. Bhardwaj

Received: 21 February 2014 / Accepted: 12 August 2014 / Published online: 26 August 2014

� Springer Science+Business Media New York 2014

Abstract Two series of novel substituted 1,4-dihydro-

pyridine derivatives incorporating nitrogen mustard phar-

macophore hybrids without spacer DHP-M (4a–4d) and

with ethyl spacer DHP-L-M (8a–8g) were designed and

synthesized. They were subjected to in silico ADME pre-

diction study to check their drug-like properties and eval-

uated for their cytotoxicity against: A 549 (lung), COLO

205 (colon), U 87 (glioblastoma), and IMR-32 (neuro-

blastoma) human cancer cell lines in vitro using 3-(4,5-

dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide

(MTT) assay against chlorambucil and docetaxel. Majority

of the test compounds exhibited moderate to significant

cytotoxic activity. The highest activity in all the investi-

gated cancer cells was displayed by DHP-M (4a). This may

be due to the less steric hindrance offered by 4a.

Keywords 1,4-Dihydropyridine � Alkylating agent �Nitrogen mustard � ADME � MTT assay

Introduction

Cancer is a complex disease, characterized by out-of-con-

trol cell growth, invasion, and spread of abnormal cells

which often leads to death. It is responsible for about 13 %

(*7.6 million) of deaths worldwide in 2007. In 2014, there

will be an estimated 1,665,540 new cancer cases diagnosed

and 580,720 deaths are expected due to cancer in the USA.

If recent trends continue in the future, the burden of cancer

deaths will set to be double by the year 2030 (Boyle and

Levin, 2008; Cancer Facts & Figure, 2014). Consequently,

the design of molecules employed as anticancer agents has

been a major endeavor of research and development in

contemporary medicinal chemistry. Although a lot of

research has already been executed in sorting out the

molecular, cellular, and genetic processes that leads to

cancer and plenty of chemotherapeutic agents are available

for the treatment of cancer but none of them avail clinical

importance as bifunctional alkylating anticancer agents.

Nitrogen mustards such as chlorambucil, melphalan, and

mechlorethamine are one of the oldest and generally most

valuable classes of alkylating anticancer agents active

against various leukemia and solid tumors. This class

constitutes highly successful drugs over 70 years and

continues to play a central role in cancer chemotherapy.

They exert their strong cytotoxic activity by interstrand

cross-linking of DNA via aziridine formation. Despite their

strong antitumor activity, the clinical usefulness of nitrogen

mustards has been restricted due to their non site-speci-

ficity, poor physicochemical properties, and high chemical

reactivity causing toxicity to healthy normal cells (Denny,

2008).

Since the last two decades, nitrogen mustard moiety has

been transformed in diverse ways with the aim of finding

more active anticancer agents, devoid of undesirable side

R. K. Singh (&) � D. N. Prasad

Department of Pharmaceutical Chemistry, Shivalik College

of Pharmacy, (Affiliated to Punjab Technical University,

Jalandhar), Distt-Rupnagar, Nangal 140126, Punjab, India

e-mail: [email protected]

T. R. Bhardwaj

University Institute of Pharmaceutical Sciences, Panjab

University, Chandigarh 160014, India

Present Address:

T. R. Bhardwaj

Indo-Soviet Friendship College of Pharmacy, Moga 142001,

Punjab, India

123

Med Chem Res (2015) 24:1534–1545

DOI 10.1007/s00044-014-1236-1

MEDICINALCHEMISTRYRESEARCH

Author's personal copy

effects, and of recognizing the structural and stereochem-

ical features required for the display of specific and

selective anticancer activity. A few of these modifications

are design and synthesis of hypoxia-selective mustards

(Denny, 2010), DNA-directed nitrogen mustards (Gourdie

et al., 1990; Fan et al., 1997; Valu et al., 1990), nitrogen

mustards in gene-directed enzyme prodrug therapy (Li

et al., 2003; Hu et al., 2003), nitrogen mustards in anti-

body-directed enzyme prodrug therapy (Springer et al.,

1994; Turner et al., 2000), and mustards activated by

glutathione transferase (Satyam et al., 1996; Rosario et al.,

2000). In addition, other suitable carriers have also been

explored for the targeted delivery of mustards (Reux et al.,

2008; Fousteris et al., 2007). Recent studies revealed that

targeting nitrogen mustard moiety by linking to various

carriers resulted in higher potency and lower peripheral

cytotoxicity than the corresponding untargeted mustards

(Tala et al., 2014; Marvania et al., 2014, 2011; Mourelatos

et al., 2012; Scutaru et al., 2011; Kapuria et al., 2011).

However, rational drug design is becoming now increas-

ingly significant for the selection of appropriate carrier for

specific target. So the aim of this investigation is to select

suitable carriers for the nitrogen mustard alkylating agent

with an intention to enhance the therapeutic efficacy of

nitrogen mustards.

Literature study revealed that 1,4-dihydropyridines (1,4-

DHPs) have been well recognized as ‘‘privileged structure’’

for their multi-receptor affinity. Some members of this

class were identified as potent P-glycoprotein (P-gp)

inhibitors and multi drug resistant (MDR) antagonists. P-gp

is present on the cell membranes of cancer cells and they

prevent the entry of anticancer agents into the cells

(Malkandi et al., 1994; Kiue et al., 1991; Shah et al.,

2000). In recent years, the anticancer property of various

1,4-DHPs particularly some dibenzoyl derivatives have

attracted a great deal of interest and extensive research

works have been carried out (Abbas et al., 2010; Bazargan

et al., 2008; Shah et al., 2008). Miri and Mehdipour in

2008 and Miri et al. 2011 have synthesized some 1,4-DHP

derivatives containing nitro-imidazole moiety on their C-4

position and evaluated on four different cancer cell lines

exhibiting significant cytotoxic activity (Miri and Mehdi-

pour, 2008; Miri et al., 2011). Al-Said et al. (2011) have

synthesized some 1,2-dihydropyridine derivatives and

screened for their in vitro anticancer activity against human

breast cancer cell line (MCF7) with promising results (Al-

Said et al., 2011). Moreover, some studies reported that

1,4-DHPs and its derivatives potentiate antitumoral and

antimetastatic activity of some common cytotoxic drugs

(Morshed et al., 2005; Ohsumi et al., 1995; Zheng et al.,

2010).

The combination of pharmacological agents may enable

synergistic interaction, where the efficacy of a single agent

is enhanced by the addition of a second compound. Typi-

cally, two pharmacophores are conjugated through a linker

unit, creating a single chemical entity forming ‘‘Hybrid’’

which may present advantages over combinations of the

two constituent drugs. These include enhanced uptake of a

one drug component due to the physicochemical properties

of the other drug component, stronger synergism due to

their proximity or improvement of individual pharmaco-

kinetics, stability or side-effect profiles (Capela et al.,

2011).

Taking into account the aspects described above and our

search for more potent alkylating antitumor agents (Singh

et al., 2012, 2013a, b, 2014), in the present study, we

attempted to improve the therapeutic efficacy of the

alkylating agents by utilizing 1,4-DHP derivatives as a

carrier for the cytotoxic nitrogen mustard (Fig. 1).

Although the preliminary study called for working with

racemic mixtures of some final compounds in DHP-L-M

series provided the option of resolution of optical isomers

if biological data of the reacemic mixtures were encour-

aging. The development of these hybrid molecules with

two cytotoxic pharmacophores in a single molecule may

provide the required reactivity and optimize the intrinsic

cytotoxicity so as remove the problems associated with

currently available nitrogen mustards.

Result and discussion

Chemistry

Synthesis of 1,4-dihydropyridine-mustard agents

without spacer DHP-M (4a–4d)

The target 1,4-dihydropyridine-mustard derivatives without

spacer DHP-M (4a–4d) were synthesized as shown in

Scheme 1. The starting compound bis-(2-hydroxyethyl)

aniline (1) and 4-(bis(2-chloroethyl)amino)benzaldehyde

(2) were synthesized by reported procedure (Wiley and Irick,

1961). Synthesis of the title compounds DHP-M (4a–4d) was

accomplished by Hantzsch multicomponent cycloconden-

sation reaction of 4-(bis(2-chloroethyl)amino)benzaldehyde

(2), substituted 1,3-dicarbonyl compounds (3a–3d), and

ammonium acetate in ethanol by conventional and micro-

wave irradiation techniques to optimize the yield. The

microwave irradiation technique gave the better yields

(Table 1).

In the IR spectra, carbonyl stretching bands, which are

characteristic for the compounds DHP-M (4a–4d) were

observed at about 1688, 1685, 1673, and 1660 cm-1,

respectively. Esters generally show C=O band in the range

of 1750–1735 cm-1. The C=O stretching vibrations are

shifted to lower frequency with wide variation for 4a–4d

Med Chem Res (2015) 24:1534–1545 1535

123


due to ab-unsaturation. This compounds 4a–4d also

exhibited a strong band at 755–760 cm-1 due to C–Cl. The1H NMR spectra of these compounds 4a–4d show the

ethylene bridge yielding two triplets integrating for four

protons each. One triplet appeared at d 3.00–3.75 ppm

which was assigned to –CH2 group adjacent to nitrogen.

The other triplet at d 3.5–4.6 ppm was assigned to the

–CH2-group adjacent to the –Cl group which is more

deshielded due to the electronegativity effect of chlorine.

The signals of –CH3 protons present at position 2 & 6 in

compound 4a–4d appeared as singlet around 2.1–2.3 ppm.

Other characteristic peaks due to the other aromatic and

pyridine protons were observed as expected.

Synthesis of 1,4-dihydropyridine-linker-mustard agents

with ethyl spacer DHP-L-M (8a–8h)

Synthesis of the parent 4-aryl-1,4-dihydropyridine skele-

tons (6a–6h) has been carried out in our lab by condensing

corresponding aldehydes with various b-keto esters by

NH2 ClCH2CH2OH N

OH

OH

N

Cl

Cl

OHC

N

Cl

Cl

OHC N

Cl Cl

NH

CC

(1) (2)

(2) (3a-d)

R

O O

OO

RR

DHP-M (4a-d)

R

4a= -OCH3

4b= -OCH2CH3

4c= -CH3

4d=

a b

c

d

Scheme 1 Sequence of steps

for the synthesis of 1, 4-DHP-

mustard DHP-M (4a–4d).Reagents and conditions

(a) CaCO3, H2O (b) POCl3,

DMF (c) Conventional,

(d) Microwave, ammonium

acetate, ethanol

Table 1 Comparison of % yield and time taken by compounds DHP-

M (4a–4d) from conventional to microwave methods

S. no. Compds. Mp Conventional Microwave

(�C) Yield

(%)

Time

(h)

Yield

(%)

Time

(min)

1. DHP-M (4a) yellow oil 62 20 74 5.0

2 DHP-M (4b) yellow oil 56 24 68 6.0

3. DHP-M (4c) brown oil 47 28 62 8.0

4. DHP-M (4d) brown oil 52 28 65 8.0

1,4-Dihydropyridine linked nitrogenmustard with ethyl spacerDHP-L-M (8a-h)

N

Cl Cl

CH3

NH

COOR1

CH3

R1OOC

H3C

CARRIER

NH

R2

R4R3

R1

P-gp INHIBITING+ANTICANCER ACTIVITY

NH

COR1

CH3H3C

R1OC

N

Cl Cl

R1=CH3, C2H5R2 = H/OCH3

N

Cl

Cl

LINKER

PHARMACOPHORECYTOTOXIC

R1=OCH3, OC2H5, CH3, C6H5

1,4-Dihydropyridine linked nitrogenmustard without spacer DHP-M (4a-d)

R2

Fig. 1 Design of 1,

4-dihydropyridines-mustard

anticancer agents

1536 Med Chem Res (2015) 24:1534–1545

123


Hantzsch method using various conventional and green

chemistry approaches (Kaur et al., 2012). These substituted

4-aryldihydropyridines (6a–6h) were further alkylated with

tris(2-chloroethyl)amine hydrochloride (7) in DMF using

anhydrous potassium carbonate and potassium fluoride to

get compounds DHP-L-M (8a–8h) (Scheme 2). For the

series of DHM-L-M (8a–8d), in IR, the disappearance –OH

band at 3200 cm-1 and the appearance of characteristic

bands at 750 cm-1 which were assigned to C–Cl bond,

suggested the condensation of nitrogen mustard with dif-

ferent 1,4-DHP derivatives. In 1H NMR spectra, appearance

of –N(CH2CH2Cl)2, –OCH2CH2–N–, and –N(CH2CH2Cl)2

protons at d 3.0, 3.1, and 3.5 ppm as three triplets,

respectively, and disappearance of broad singlet of –OH

further confirmed the formation of DHP-L-M 8(a–h). The

rest of the aromatic and pyridine protons were observed as

expected.

In silico ADME studies

One of the main goals in drug discovery is the identifica-

tion of potent molecule together with a reasonable

absorption, distribution, metabolism and excretion

(ADME) profile, lead and/or drug-likeness. Such chemical

entities are likely to be able to enter higher phases of the

drug development process. ADME properties were

Step-I

Step-II

Scheme 2 Sequence of steps for the synthesis of 1, 4-DHP-mustard with ethyl spacer DHP-L-M (8a–8h). Reagents and conditions

(a) Ammonium acetate, ethanol (b) tris(2-chloroethylamine)hydrochloride, K2CO3, KF, DMF, stirring at rt

Med Chem Res (2015) 24:1534–1545 1537

123


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1538 Med Chem Res (2015) 24:1534–1545

123


calculated using Qikprop 2.5 tools of the Schrodinger

software program. The 44 physically descriptors and

pharmaceutically relevant properties of dihydropyridine-

mustard derivatives were analyzed and compared with

mechlorethamine and chlorambucil for predicting drug-like

properties of synthesized compounds (Table 2). We eval-

uated the acceptability of the compounds based on Lipin-

ski’s rule of five and Jorgensen’s rules of three, which is

essential for rational drug design. In accordance with

Lipkinski’s rule of five (Lipinski et al., 1997), compound

should have a molecular weight B500 Daltons, B5

hydrogen bond donors, B10 hydrogen bond acceptors and

logP of B5, and rotatable bond B10. Compounds that

satisfy these rules are considered drug-like. According to

this rule, compounds with the number of violations not

more than 1 show good bioavailability. Poor absorption

and permeation are more likely to occur when any two of

the above-mentioned rules are violated. According to Jor-

gensen’s rule of three (Jorgensen, 2009), there should have

QPlogS [ -5.7, QPPCaco [ 22 nm/s, # Primary Metab-

olites \ 7. On the basis of in silico ADME study, com-

pounds 4a, 4b, 4c, 8a, and 8b are obeying Lipinski’s rule

of five and Jorgensen’s rules of three and all other syn-

thesized compounds have one or two violations of rule of

five and rule of three. The range of human oral absorption

of synthesized compounds is 85–100 %. The entire com-

pounds show good intestinal absorption and hence orally

active, as indicated by their optimum PSA within the

acceptable range (Clark, 1999; Kelder et al., 1999).

In vitro anticancer screening

The newly synthesized compounds were evaluated for

their in vitro cytotoxic activity against human cancer cell

lines: A 549 (lung), COLO 205 (colon), U 87 (primary

glioblastoma cells), and IMR-32 (neuroblastoma cell lines)

using chlorambucil and docetaxel as the reference drugs.

The parameter used here is IC50, which corresponds to the

concentration required for 50 % inhibition of cell viability.

From the results in Table 3, it was found that majority

of the compounds exhibited moderate antitumor activity.

Structurally, final compounds belong to two series: DHP-M

(4a–4d) series and DHP-L-M (8a–8h) series. Careful

examination of the effect of ethyl linker at various m and

p position showed that the presence of ethyl linker slightly

decreases the activity of the compounds. This may be

because of more number of rotatable bonds. Compound 4a

with the carbmethoxy group at 3rd and 5th positions

exhibited the most potent cytotoxicity in the series of DHP-

M (4a–4d) against all cancer cell lines. This may be due to

the less steric hindrance offered by 4a. It is also noted that

presence of carbmethoxy and carbethoxy groups has ben-

eficial effects on the activity of the said compounds 4a and

4b as they are more cytotoxic than acetyl and benzoyl

groups at C-3 and C-5 positions. All the compounds in

series DHP-M (4a–4d) showed good cytotoxic activity

against A549 cancer cell line.

In DHP-L-M (8a–8h) series (Table 3), compounds

having carbmethoxy groups at C-3 and C-5 positions of

pyridine nucleus, i.e., DHP-L-M (8a–8d) have better

cytotoxic activity than compounds having carbethoxy

groups DHP-L-M (8e–8h). This may be owing to less steric

hindrance offered by carbmethoxy group as compared to

carbethoxy group and hence better antiproliferative activ-

ity. Interestingly, 8e bearing carbethoxy group at the 3rd

and 5th position was active against all tested cancer cell

lines. Further analysis revealed that a methoxy group at 3

or 4 position of aromatic ring had a notable increase in

cytotoxicity of DHP-L-M (8c) and (8d). It is interesting to

note that 8c exhibited better selectivity for COLO-205 cell

lines against standard, which have the making of good

drugs for colorectal cancer.

Conclusion

In conclusion, this investigation describes the synthesis and

cytotoxicity studies of two series of 1,4-DHPs-linked

nitrogen mustard agents. The synthesized compounds were

characterized by suitable analytical techniques such as UV,

IR, NMR, Mass, and elemental analyses, and the data

obtained were in full agreement of the proposed structures.

All compounds were submitted for an in silico ADME

prediction study required for drug-likeness. The in vitro

antiproliferative activity of the newly synthesized com-

pounds against human A 549, COLO 205, U 87, and IMR-

32 was investigated. The majority of the synthesized

compounds exhibited moderate to significant antiprolifer-

ative activity. This study revealed than the compounds are

suitable candidate for further exploration. Further modifi-

cation of 1,4-DHPs scaffold by substitution of different

hydrophilic and hydrophobic side groups at various posi-

tions of pyridine ring as well as benzene ring may provide

more potent anticancer agents in future.

Experimental protocol

Chemistry

All chemicals and reagent were obtained from commercial

suppliers and were used without further purification. The

melting points were determined on Veego-programmable

melting point apparatus (microprocessor based) and are

uncorrected. 1H-NMR spectra were obtained using Bruker

Avance-II, 400 MHz spectrometer and are reported in parts

Med Chem Res (2015) 24:1534–1545 1539

123


per million (ppm), downfield from tetramethylsilane as

internal standard. Infrared (IR) spectra were recorded on

Perkin Elmer model 1600 FT-IR RX-I spectrometer as KBr

disks. The ultraviolet spectra were recorded on Shimadzu,

UV-1800 spectrophotometer. The TOF-MS-ES? spectra of

the compounds were recorded on Waters micromass Q-TOF

Mass spectrometer. Elemental analyses for C, H, and N were

performed on Thermo-flash EA-1112 CHNS-O analyzer.

Anhydrous sodium sulfate was used as drying agents. A

computational study of titled compounds was performed for

prediction of ADME properties by QikProp 3.2 tools avail-

able in Schrodinger 9.0 version.

General method for the synthesis of compounds DHP-

M (4a–4d)

Conventional method

The various 1, 3 dicarbonyl compounds (0.02 mol) and

4-{bis(2-chloroethyl)amino}benzaldehyde (2) (0.01 mol)

were taken into a round bottom flask and dissolved in

ethanol (25 mL). To this solution, ammonium acetate

(0.02 mol) was added with stirring and the reaction mixture

was refluxed for 20–28 h. After completion of the reaction

(monitored by TLC), the ethanol was distilled off under

vacuo and the residue was dissolved in ice-water and

extracted with DCM (3 9 30 mL). The organic layer was

separated and dried over anhydrous sodium sulfate, filtered,

and evaporated under reduced pressure. The residue

obtained passed through the column to obtain desired

compounds DHP-M (4a–4d) as dark yellow oil.

Microwave irradiation method

A 1,3 dicarbonyl compounds (0.02 mol), 4-{bis(2-chloro-

ethyl)amino}benzaldehyde (2) (0.01 mol), and ammonium

acetate (0.02 mol) were taken into a flask and dissolved in

a minimum quantity of ethanol (5 mL). A funnel was

placed on the flask and covered with a watch glass. The

reaction mixture was subjected to microwave irradiation at

360 W for 5–8 min with a pulse rate of 60 s in a micro-

wave oven. The solvent was removed and the residue was

cooled and triturated with crushed ice and extracted with

DCM (3 9 10 mL). The organic layer was separated and

dried over anhydrous sodium sulfate, filtered, and evapo-

rated under reduced pressure. The residue obtained passed

through the column to obtain desired compounds DHP-M

(4a–4d) as dark yellow oil.

Dimethyl 4-{4-(bis(2-chloroethyl)amino)pheny}-1,4-dihydro-

2,6-dimethylpyridine-3,5-dicarboxylate DHP-M (4a) Yellow

oil (This compound 4a was prepared from 4-{bis(2-chlo-

roethyl)amino}benzaldehyde (2) (2.46 g, 0.01 mol) and

methyl acetoacetate (2.32 g, 2.14 mL, 0.02 mol)); UV

(MeOH) kmax 345; IR KBr (cm-1): 3362 (N–H), 2972

(C–H), 1688 (C=O), 1228, 1122 (C–N) and 758 (C–Cl);1H-NMR (CDCl3) d (ppm): 2.2 (s, 6H, 2 9 CH3), 3.6 (t,

4H, J = 6.0 Hz, –N(CH2CH2Cl)2), 3.8 (t, 4H, J = 4.0 Hz,

Table 3 The antiproliferative activity data assessed by the MTT reduction assay of synthesized compounds DHP-M (4a–4d) and DHP-L-M

(8a–8h)

Compd. name IC50 value ± S.D.a

A549 COLO 205 U87 IMR32

1. DHP-M (4a) 32.293 ± 2.04 36.54 ± 2.99 28.443 ± 2.13 29.174 ± 1.85

2. DHP-M (4b) 33.276 ± 2.17 41.426 ± 2.44 32.106 ± 2.54 32.57 ± 2.71

3. DHP-M (4c) 34.44 ± 2.23 55.246 ± 2.69 47.334 ± 2.51 39.124 ± 2.41

4. DHP-M (4d) 32.81 ± 2.19 47.663 ± 3.30 34.324 ± 2.90 43.244 ± 2.76

5. DHP-L-M (8a) 37.726 ± 2.03 42.07 ± 2.45 28.504 ± 1.73 35.023 ± 2.61

6. DHP-L-M (8b) 33.873 ± 2.43 36.236 ± 1.79 24.61 ± 2.92 37.07 ± 2.55

7. DHP-L-M (8c) 38.55 ± 1.64 27.72 ± 2.16 23.664 ± 2.88 31.174 ± 2.92

8. DHP-L-M (8d) 32.176 ± 1.91 40.596 ± 2.12 28.19 ± 1.94 33.903 ± 3.07

9. DHP-L-M (8e) 28.88 ± 2.64 38.656 ± 1.76 25.256 ± 2.05 28.376 ± 1.78

10. DHP-L-M (8f) 35.463 ± 1.75 63.194 ± 2.12 35.406 ± 3.30 57.556 ± 1.86

11. DHP-L-M (8g) 72.876 ± 2.53 43.03 ± 3.28 51.19 ± 2.25 38.88 ± 2.63

12. DHP-L-M (8h) 74.383 ± 2.08 40.286 ± 2.53 43.367 ± 2.23 34.576 ± 1.89

13. DOCETAXEL 27.256 ± 1.75 30.706 ± 1.72 20.72 ± 1.19 23.71 ± 1.73

14. CHLORAMBUCIL 21.09 ± 1.72 26.67 ± 1.09 18.82 ± 1.09 20.09 ± 1.03

a All experiments were carried out three times, independently. The data obtained were expressed in terms of mean, standard deviation (SD)

values. Wherever appropriate, the data were also subjected to unpaired two tailed student’s t test. A value of p \ 0.05 was considered as

significant

1540 Med Chem Res (2015) 24:1534–1545

123


–N(CH2CH2Cl)2), 3.6 (s, 6H, 2 9 COOCH3), 4.8 (s, 1H,

–CH of DHP), 5.5 (brs, 1H, exchangeable, NH of DHP), 6.

5 (d, 2H, J = 8.8, ArH meta to pyr ring), 7.0 (d, 2H, J = 4.

8, ArH ortho to pyr ring); 13C-NMR (CDCl3) d (ppm): 16.8

(C–CH3), 40.2 (C–N), 43.2 (C–Cl), 44.8 (ArC-PyC), 53.2

(COOCH3), 128.5, 129.2 136.0, 139.8 (ArC), 105.0

(C–COOCH3), 150.0 (–C–CH3), 165.8 (C=O); MS (ESI):

m/z 442 (M?H)?; Anal. calcd for C21H26N2O4Cl2 : C %

57.15, H % 5.94, N % 6.35; Found: C % 57.62, H % 6.48,

N % 6.72

Diethyl 4-{4-(bis(2-chloroethyl)amino)pheny}-1,4-dihydro-

2,6-dimethylpyridine-3,5-dicarboxylate DHP-M (4b) Yel-

low oil (This compound 4b was prepared from 4-{bis(2-

chloroethyl)amino}benzaldehyde (2) (2.46 g, 0.01 mol)

and ethyl acetoacetate (2.60 g, 2.56 mL, 0.02 mol)); UV

(MeOH) kmax 350; IR (KBr) (cm-1): 3352 (N–H), 2960

(C–H), 1685 (C=O), 1222, 1120 (C–N) and 756 (C–Cl);1H-NMR (CDCl3) d (ppm): 1.2 (t, 6H, 2 9 COOCH2CH3),

2.3 (s, 6H, 2 9 CH3), 3.4-3.5 (m, 8H, –N(CH2CH2Cl)2), 4.

0 (m, 4H, 2 9 COOCH2CH3), 4.9 (s, 1H, –CH of DHP), 5.

5 (brs, 1H, exchangeable, NH of DHP), 7.0 (d, 2H, J = 4.0,

ArH meta to pyr ring), 7.2 (d, 2H, J = 2.5, ArH ortho to

pyr ring); 13C-NMR (CDCl3) d (ppm): 14.8 (CO-

OCH2CH3), 16.5 (C–CH3), 39.2 (C–N), 43.5 (C–Cl), 43.6

(ArC-PyC), 62.5 (COOCH2CH3), 115.3, 114.2, 132.5, 146.

4 (ArC), 102.8 (C–COOCH2CH3), 150.6 (–C–CH3), 168.5

(C=O); MS (ESI): m/z 470 (M?H)?; Anal. calcd for

C23H30Cl2N2O4: C % 58.85, H % 6.44, N % 5.97; Found:

C % 59.15, H % 6.87, N % 5.84

3, 5-Diacetyl-1,4-dihydro-2,6-dimethyl-4-{4-(bis(2-chloro-

ethyl)amino)pheny}pyridine DHP-M (4c) Brown oil

(This compound 4c was prepared from 4-{bis(2-chloro-

ethyl)amino}benzaldehyde (2) (2.46 g, 0.01 mol) and acetyl

acetone (2.0 g, 2.04 mL, 0.02 mol)); UV (MeOH) kmax 338;

IR (KBr) (cm-1): 3345 cm-1 (N–H), 2982 (C–H), 1673 (C=

O), 1228, 1128, 1023 (C–N) and 759 (C–Cl); 1H-NMR

(CDCl3) d (ppm): 2.2 (s, 6H, 2 9 CH3), 2.4 (s, 6H,

2 9 COCH3), 3.35–3.50 (m, 8H, –N(CH2CH2Cl)2), 4.9 (s,

1H, –CH of DHP), 5.7 (brs, 1H, exchangeable, NH of DHP),

7.0 (d, 2H, J = 2.8, ArH meta to pyr ring), 7.1 (d, 2H, J = 2.

0, ArH ortho to pyr ring); 13C-NMR (CDCl3) d (ppm): 16.8

(C-CH3), 28.2 (COCH3), 40.5 (C–N), 43.2 (C–Cl), 38.6

(ArC-PyC), 103.4 (C-COCH3), 114.2, 131.5, 133.4, 146.4

(ArC), 148.6 (–C–CH3), 192.5 (C=O); MS (ESI): m/z 410

(M?H)?; Anal calcd for C21H26Cl2N2O2 : C % 61.62, H %

6.40, N % 6.84; Found: C % 62.05, H % 6.90, N % 6.98

3,5-Dibenzoyl-1,4-dihydro-2,6-dimethyl-4-{4-(bis(2-chloro-

ethyl)amino)pheny}pyridine DHP-M (4d) Brown oil (This

compound 4d was prepared from 4-{bis(2-chloro-

ethyl)amino}benzaldehyde (2) (2.46 g, 0.01 mol) and

benzoyl acetone (3.24 g, 2.97 mL, 0.02 mol)); UV (MeOH)

kmax 365; IR (KBr) (cm-1):3345 cm-1 (N–H), 2961 (C–H),

1660 (C=O), 1224, 1126, 1017 (C–N) and 759 (C–Cl); 1H-

NMR (CDCl3) d (ppm): 2.3 (s, 6H, 2 9 CH3), 3.5–3.7 (m,

8H, –N(CH2CH2Cl)2), 4.9 (s, 1H, –CH of DHP), 6.6–7.9 (m,

14H, ArH), 5.7 (brs, 1H, exchangeable, NH of DHP); 13C-

NMR (CDCl3) d (ppm): 15.8 (C–CH3), 40.5 (C–N), 43.4

(C–Cl), 37.8 (ArC-PyC), 104.4 (C–COPh), 114.2, 129.6,

131.2, 132.5, 133.4, 134.5, 143.5, 146.7 (ArC), 146.6

(–C–CH3), 188.5 (C=O); MS (ESI): m/z 535 (M?H)?; Anal.

calcd for C31H30Cl2N2O2 : C % 69.79, H % 5.67, N % 5.25;

Found: C % 70.80, H % 6.02, N % 5.08

General method for the synthesis of Diethyl 4-{4-(2-(bis-

(2-chloroethyl)amino)ethoxy)phenyl}-1,4-dihydro-2,

6-dimethylpyridine-3, 5-dicarboxylate DHP-L-M (8a–8h)

Tris (2-chloroethylamine) hydrochloride (7) (19.2 g,

0.08 mol), dry powdered potassium carbonate (0.12 mol),

and KF (0.04 mol) were collectively dissolved in DMF

(10 mL) in RBF and stirred for 1 h. A solution of substi-

tuted 1,4-DHPs (6a–6h) (0.02 mol) was dissolved in 5 mL

DMF and added in the above solution drop wise and it was

stirred at room temperature for 2 days. After completion of

the reaction monitored by TLC, the reaction mixture was

poured into ice cold water and extracted with ethyl acetate

(3 9 20 mL). The organic layer was combined, washed

with ice water and dried, evaporated in vacuo, and residue

passes through column to get desired compounds as yellow

gummy mass DHP-L-M (8a–8h).

Dimethyl 4-{4-(2-(bis-(2-chloroethyl)amino)ethoxy)phenyl}-

1,4-dihydro-2, 6-dimethylpyridine-3, 5-dicarboxylate DHP-L-

M (8a) Yellow gummy mass (This compound 8a was

prepared from tris (2-chloroethylamine) hydrochloride (7)

(19.2 g, 0.08 mol), and dimethyl-1,4-dihydro-4(4-hydroxy-

phenyl)-2,6-dimethylpyridine-3,5-dicarboxylate (9.7 g, 0.02

mol)); Yield = 74 %; UV (MeOH) kmax 360; IR KBr

(cm-1): 3350 (N–H), 2984 (C–H), 1681 (C=O), 1488 (Ar

C=C), 1026,1119, 1214 (C–N/C–O) and 750 (C–Cl); 1H-

NMR (CDCl3) d (ppm): 2.3 (s, 6H, 2 9 CH3), 2.7–2.8 (m,

6 H, –CH2N(CH2CH2Cl–)2), 3.25 (t, 4H, J = 8.0 Hz,

–N(CH2CH2Cl)2), 3.5 (s, 6H, 2 9 COOCH3), 3.6 (t, 2H,

J = 6.0 Hz, –OCH2CH2–), 4.9 (s, 1H, –CH of DHP), 5.6

(s, 1H, NH of DHP), 6.6–7.2 (m, 4H, ArH); 13C-NMR

(CDCl3) d (ppm): 16.6 (C–CH3), 42.2 (C–Cl), 53.2 (C–N),

56.7 (–N–C), 65.3 (O–C), 44.8 (ArC-PyC), 52.6

(COOCH3), 114.5, 129.2 136.0, 153.8 (ArC), 105.0


m/z 485 (M) ?; Anal. calcd for C23H30Cl2N2O5: C % 56.

91, H % 6.23, N % 5.77; Found: C % 56.62, H % 6.18,

N % 5.78

Med Chem Res (2015) 24:1534–1545 1541

123


Dimethyl 4-{3-(2-(bis-(2-chloroethyl)amino)ethoxy)phenyl}-


M (8b) Yellow gummy mass (This compound 8b was


(19.2 g, 0.08 mol), and dimethyl-1,4-dihydro-4(3-hydroxy-

phenyl)-2,6-dimethylpyridine-3,5-dicarboxylate (9.7 g, 0.02

mol)); Yield = 67 %; UV (MeOH) kmax 360; IR KBr

(cm-1): 3345 (N–H), 2986 (C–H), 1684 (Ester C=O), 1480

(Ar C=C), 1028, 1121, 1221 (C–N/C–O) and 755 (C–Cl);1H-NMR (CDCl3) d (ppm): 2.3 (s, 6H, 2 9 CH3), 2.7–2.8

(m, 6 H, –CH2N(CH2CH2Cl–)2), 3.24 (t, 4H, J = 7.5 Hz,

–N(CH2CH2Cl)2), 3.6 (s, 6H, 2 9 COOCH3), 3.8 (t, 2H,

J = 6.0 Hz, –OCH2CH2–), 4.93 (s, 1H, –CH of DHP), 5.4

(s, 1H, NH of DHP), 6.7–6.8 (m, 4H, ArH); 13C-NMR

(CDCl3) d (ppm): 16.3 (C–CH3), 41.8 (C–Cl), 53.2 (C–N),

56.7 (–N–C), 64.8 (O–C), 44.8 (ArC-PyC), 52.6

(COOCH3), 112.0, 114.5, 120.2, 130.0, 143.3, 156.8 (ArC),

105.0 (C–COOCH3), 150.0 (–C–CH3), 167.2 (C=O); MS

(ESI): m/z 486 (M?H)?; Anal. calcd for C23H30Cl2N2O5:

C % 56.91, H % 6.23, N % 5.77; Found: C % 55.96, H %

6.17, N % 5.85

Dimethyl 4-{4-(2-(bis-(2-chloroethyl)amino)ethoxy-3-methox-

y)phenyl}-1,4-dihydro-2, 6-dimethylpyridine-3, 5-dicarboxyl-

ate DHP-L-M (8c) Yellow gummy mass (This compound

8c was prepared from tris (2-chloroethylamine) hydro-

chloride (7) (19.2 g, 0.08 mol), and dimethyl 1,4-dihydro-

4-(4-hydroxy-3-methoxyphenyl)-2, 6-dimethylpyridine-3,

5-dicarboxylate (10.3 g, 0.02 mol)); Yield = 69 %; UV

(MeOH) kmax 368; IR KBr (cm-1): 3329 (N–H), 2952

(C–H), 1680 (C=O), 1484 (C=C), 1016, 1123, 1259 (C–N/

C–O) and 759 (C–Cl); 1H-NMR (CDCl3) d (ppm): 2.2 (s,

6H, 2 9 CH3), 2.7–2.8 (m, 6 H, –CH2N(CH2CH2Cl–)2), 3.

2 (t, 4H, J = 8.0 Hz, –N(CH2CH2Cl)2), 3.7 (s, 6H,

2 9 COOCH3), 3.7–3.8 (m, 5H, –OCH2CH2– ? –OCH3),

4.8 (s, 1H, –CH of DHP), 5.4 (s, 1H, NH of DHP), 6.6–6.7

(m, 3H, ArH); 13C-NMR (CDCl3) d (ppm): 16.6 (C–CH3),

42.2 (C–Cl), 53.4 (C–N), 56.5 (OCH3), 56.7 (–N–C), 65.5

(O–C), 44.8 (ArC-PyC), 52.8 (COOCH3), 114.2, 116.0,

122.2, 135.0, 142.4, 150.2 (ArC), 105.0 (C–COOCH3),

149.4 (–C–CH3), 165.8 (C=O); MS (ESI): m/z 516

(M?H)?; Anal. calcd for C24H32Cl2N2O6: C % 55.93,

H % 6.26, N % 5.43; Found: C % 55.72, H % 6.32, N % 5.

80

Dimethyl 4-{3-(2-(bis-(2-chloroethyl)amino)ethoxy-4-methox-

y)phenyl}-1,4-dihydro-2, 6-dimethylpyridine-3, 5-dicarboxyl-

ate DHP-L-M (8d) Yellow gummy mass (This compound

8d was prepared from tris (2-chloroethylamine) hydro-

chloride (7) (19.2 g, 0.08 mol), and dimethyl 1,4-dihydro-



(MeOH) kmax 368: IR KBr (cm-1): 3329 (N–H), 2952

(C–H), 1680 (C=O), 1484 (C=C), 1026, 1259 (C–N/C–O)

and 756 (C–Cl); 1H-NMR (CDCl3) d (ppm): 2.2 (s, 6H,

2 9 CH3), 2.7–2.8 (m, 6 H, –CH2N(CH2CH2Cl–)2), 3.2

(t, 4H, J = 8.0 Hz, –N(CH2CH2Cl)2), 3.7 (s, 6H,

2 9 COOCH3), 3.7–3.8 (m, 5H, –OCH2CH2– ? –OCH3),

4.9 (s, 1H, –CH of DHP), 5.5 (s, 1H, NH of DHP), 6.7–6.8

(m, 3H, ArH); 13C-NMR (CDCl3) d (ppm): 16.4 (C–CH3),

42.6 (C–Cl), 53.4 (C–N), 56.5 (OCH3), 56.5 (–N–C), 65.5

(O–C), 44.8 (ArC-PyC), 52.8 (COOCH3), 114.7, 15.5, 122.

4, 135.0, 146.4, 147.2 (ArC), 105.0 (C–COOCH3), 149.4

(–C–CH3), 167.4 (C=O); MS (ESI): m/z 516 (M?H)?;

Anal. calcd for C24H32Cl2N2O6: C % 55.93, H % 6.26,

N % 5.43; Found: C % 56.72, H % 6.38, N % 5.42

Diethyl 4-{4-(2-(bis-(2-chloroethyl)amino)ethoxy)phenyl}-1,4-

dihydro-2, 6-dimethylpyridine-3, 5-dicarboxylate DHP-L-M

(8e) Yellow gummy mass (This compound 8e was pre-

pared from tris (2-chloroethylamine) hydrochloride (7) (19.

2 g, 0.08 mol), and diethyl-1,4-dihydro-4(4-hydroxy-

phenyl)-2,6-dimethylpyridine-3,5-dicarboxylate (10.27 g,

0.02 mol)); Yield = 73 %; UV (MeOH) kmax 362: IR KBr

(cm-1): 3378 (N–H), 3015, 2960 (C–H), 1692 (C=O),

1646, 1484 (C=C), 1281, 1285 (C–N/C–O) and 751

(C–Cl); 1H-NMR (CDCl3) d (ppm): 1.2 (t, 6H,

2 9 OCH2CH3), 2.3 (m, 6H, 2 9 CH3), 3.0 (t, 4H, –N

(CH2CH2Cl)2), 3.1 (t, 2H, –OCH2CH2–N), 3.5 (t, 4H,

–N(CH2CH2Cl)2), 4.0 (m, 6H, 2xCOOCH2CH3 ? –OCH2–

), 4.8 (s, 1H, –CH of DHP), 6.6–7.0 (m, 4H, ArH) and 8.3

(brs, 1H, –NH of DHP); 13C-NMR (CDCl3) d (ppm): 14.8

(COOCH2CH3), 16.4 (C–CH3), 42.8 (C–Cl), 53.7 (C–N),

56.5 (–N–C), 65.9 (O–C), 43.8 (ArC-PyC), 62.2

(COOCH2CH3), 114.7, 130.6, 134.6, 155.0 (ArC), 103.0


m/z 513 (M)?; Anal. calcd for C25H34Cl2N2O5: C % 58.48,

H % 6.67, N % 5.46; Found: C % 57.94, H % 6.63, N % 5.

51

Diethyl 4-{3-(2-(bis-(2-chloroethyl)amino)ethoxy)phenyl}-


M (8f) Yellow gummy mass (This compound 8f was


(19.2 g, 0.08 mol), and diethyl-1,4-dihydro-4(3-hydroxy-

phenyl)-2,6-dimethylpyridine-3,5-dicarboxylate (10.27 g,

0.02 mol)); Yield = 67 %; UV (MeOH) kmax 362: IR KBr

(cm-1): 3375 (N–H), 2958 (C–H), 1688 (C=O), 1268

(C–N/C–O) and 756 (C–Cl); 1H-NMR (CDCl3) d (ppm): 1.

2 (t, 6H, 2 9 OCH2CH3), 2.3 (m, 6H, 2 9 CH3), 2.9

(t, 4H, –N(CH2CH2Cl)2), 3.2(t, 2H, –OCH2CH2–N), 3.5

(t, 4H, –N(CH2CH2Cl)2), 3.9–4.1 (m, 6H,

2 9 COOCH2CH3 ? –OCH2–), 4.8 (s, 1H, –CH of DHP),

6.5–6.9 (m, 4H, ArH) and 8.6 (brs, 1H, –NH of DHP);13C-NMR (CDCl3) d (ppm): 14.7 (COOCH2CH3), 16.4

(C–CH3), 42.6 (C–Cl), 53.6 (C–N), 56.5 (–N–C), 65.9

1542 Med Chem Res (2015) 24:1534–1545

123


(O–C), 43.8 (ArC-PyC), 62.2 (COOCH2CH3), 112.7, 113.

6, 121.6, 130.7, 142.5, 157.6 (ArC), 103.2.0 (C–COOCH3),

149.4 (–C–CH3), 167.8 (C=O); MS (ESI): m/z 513 (M)?;

Anal. calcd for C25H34Cl2N2O5: C % 58.48, H % 6.67,

N % 5.46; Found: C % 58.52, H % 6.62, N % 5.49

Diethyl 4-{4-(2-(bis-(2-chloroethyl)amino)ethoxy-3-meth-

oxy)phenyl}-1,4-dihydro-2, 6-dimethylpyridine-3, 5-dicar-

boxylate DHP-L-M (8g) Yellow gummy mass (This

compound 8g was prepared from tris (2-chloroethylamine)

hydrochloride (7) (19.2 g, 0.08 mol), and diethyl 1,4-dihydro-



(MeOH) kmax 370: IR KBr (cm-1):3338 (N–H), 2955 (C–H),

1684 (C=O), 1485 (C=C), 1030, 1260 (C–N/C–O) and 755

(C–Cl); 1H-NMR (CDCl3) d (ppm): 1.2 (t, 6H, 2 9

OCH2CH3), 2.3 (m, 6H, 2 9 CH3), 2.8–2.9 (m, 6H, –N(CH2

CH2Cl)2 ? –OCH2CH2–N), 3.5 (t, 4H, –N(CH2CH2Cl)2), 3.6

(s, 3H, –OCH3), 4.1 (m, 6H, 2 9 COOCH2CH3), 4.2 (t, 2H,

–OCH2), 4.9 (s, 1H, –CH of DHP), 5.9 (brs, 1H, –NH of DHP)

and 6.7–6.9 (m, 3H, ArH); 13C-NMR (CDCl3) d (ppm): 14.3

(COOCH2CH3), 16.4 (C–CH3), 42.8 (C–Cl), 53.7 (C–N), 56.5

(–N–C), 56.8 (O–CH3), 65.9 (O–C), 43.8 (ArC-PyC), 62.2

(COOCH2CH3), 113.5, 115.6, 124.6, 134.5, 143.8, 150.6

(ArC), 102.3 (C-COOCH3), 149.4 (–C–CH3), 167.4 (C=O);

MS (ESI): m/z 545 (M?H)?; Anal. calcd for C26H36Cl2N2O6:

C % 57.46, H % 6.68, N % 5.15; Found: C % 57.08, H % 6.

63, N % 5.05

Diethyl 4-{3-(2-(bis-(2-chloroethyl)amino)ethoxy-4-meth-

oxy)phenyl}-1,4-dihydro-2, 6-dimethylpyridine-3, 5-dicar-

boxylate DHP-L-M (8h) Yellow gummy mass (This

compound 8h was prepared from tris (2-chloroethylamine)

hydrochloride (7) (19.2 g, 0.08 mol), and diethyl 1,4-dihy-

dro-4-(3-hydroxy-4-methoxyphenyl)-2, 6-dimethylpyri-

dine-3, 5-dicarboxylate (10.9 g, 0.02 mol)); Yield = 67 %;

UV (MeOH) kmax 370: IR KBr (cm-1):3332 (N–H), 2950

(C–H), 1680 (C=O), 1478 (C=C), 1032, 1259 (C–N/C–O)

and 756 (C–Cl); 1H-NMR (CDCl3) d (ppm): 1.2 (t, 6H,

2 9 OCH2CH3), 2.3 (m, 6H, 2 9 CH3), 2.8–3.0 (m, 6H,

–N(CH2CH2Cl)2 ? –OCH2CH2–N), 3.6 (t, 4H, –N(CH2

CH2Cl)2), 3.7 (s, 3H, –OCH3), 4.1 (m, 6H, 2xCOOCH2CH3),

4.2 (t, 2H, –OCH2), 4.9 (s, 1H, –CH of DHP), 6.7–6.8 (m, 4H,

ArH), 8.5 (brs, 1H, –NH of DHP); 13C-NMR (CDCl3) d(ppm): 14.2 (COOCH2CH3), 16.3 (C–CH3), 42.8 (C–Cl), 53.

3 (C–N), 56.6 (–N–C), 56.4 (O–CH3), 65.9 (O–C), 43.8

(ArC-PyC), 62.2 (COOCH2CH3), 114.2, 115.2, 122.6,

134.5, 145.8, 147.6 (ArC), 102.3 (C–COOCH3), 149.3

(–C–CH3), 167.8 (C=O); MS (ESI): m/z 544 (M)?; Anal.

calcd for C26H36Cl2N2O6: C % 57.46, H % 6.68, N % 5.15;

Found: C % 57.43, H % 6.70, N % 5.13

In silico ADME prediction

ADME properties were calculated using Qikprop 2.5 tools

of Schrodinger software program designed by Professor

William L. Jorgensen. It evaluates the acceptability of

analogs based on Lipinski’s rule of five which is essential

to ensure a drug-like pharmacokinetics profile while using

rational drug design. All the analogs were neutralized

before being used by Qikprop.

In vitro antiproliferative assay

Cell culture

The following established in vitro human cancer cell lines

were applied: A 549 (Human lung cancer cells), COLO 205

(Human colon cancer cells), U 87 (Human primary glio-

blastoma cells), and IMR-32 (Human neuroblastoma cell

lines). All cell lines were obtained from NCCS Pune. All

chemicals and solvents were purchased from Himedia. Cell

lines were maintained in desired media supplemented with

10 % inactivated fetal bovine serum, 100U/mL penicillin

and 100 lL/mL streptomycin incubated at 37 �C, and 5 %

CO2 in humidifier incubator. After attaining 80 %, con-

fluence cells were subcultured by trypsinization with

0.25 % trypsin solution under sterile conditions.

MTT assay

The cytotoxicity of synthesized compounds was deter-

mined by a tetrazolium-based colorimetric assay (MTT

assay) as per the reported procedure (Mosmann, 1983). The

cells of all cell lines were plated out 24 h prior to testing in

96 well plates at a density of 3000 cells/well in 100 lL of

the medium. After overnight incubation, triplicate wells

were treated with varying concentration of compounds

ranging from (1–100 lg/mL) and incubated with standard

chlorambucil and docetaxel for 3 days. The cells were

continuously exposed for a period of 72 h. After 3 days,

medium was replaced with 2 lL of 3-(4,5-dimethylthia-

zole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/

mL) and cells were incubated for 3 h. The relative per-

centage of metabolically active cells compared with

untreated controls was determined on the basis of mito-

chondrial conversion of MTT to formazan crystals dis-

solved in dimethylsulfoxide. Spectrophotometric

absorbance of the sample was determined by micro plate

reader (BIORAD) at 570–630 nm. Concentrations of

sample showing a 50 % reduction in cell viability (i.e.

IC50) were then calculated. An OD value of control cells

(unexposed cells) was taken as 100 % viability (0 %

cytotoxicity).

Med Chem Res (2015) 24:1534–1545 1543

123


Statistical analyses

All experiments were carried out three times, indepen-

dently. The data obtained were expressed in terms of mean,

SD deviation values. Wherever appropriate, the data were

also subjected to unpaired two tailed student’s t test. A

value of p \ 0.05 was considered as significant.

% inhibition ¼ OD of controlð Þ½� OD of treatedð Þ= OD of controlð Þ� � 100:

Acknowledgments The authors are thankful to the College Man-

aging Committee, SCOP, Nangal for providing necessary facilities to

carry out research work. The authors wish to express their gratitude to

Dr. Manoj Kumar, Professor of Pharmaceutical Chemistry, UIPS,

Panjab University, Chandigarh to carry out an ADME study at his lab.

The authors are also thankful to ISFAL, ISF College of Pharmacy,

Moga (Punjab) for carrying out cell line studies and SAIF, Panjab

University, Chandigarh for cooperation in getting the spectral data.

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