nonsteroidal anti-inflammatory drug-based n -allylidene benzohydrazides and 1-acyl-2-pyrazolines:...

Month 2014 Nonsteroidal Anti-inflamma
tory Drug-based N-AllylideneBenzohydrazides and 1-Acyl-2-pyrazolines: Their Synthesis as Potential
Cytotoxic Agents In Vitro

Kavitha Kankanala,a,b V. Ranga Reddy,c Yumnam Priyadarshini Devi,d Lakshmi Narasu Mangamoori,d D. Rambabu,e

K. Mukkanti,a and Sarbani Palb*

aJNTUH, Kukatpally, Hyderabad-500085, Andhra Pradesh, IndiabDepartment of Chemistry, MNR Degree and PG College, Kukatpally, Hyderabad-500085, Andhra Pradesh, India

cDr. Reddy’s Laboratories Limited, Integrated Product Development, Bachupally, Hyderabad-500055, Andhra Pradesh, IndiadCentre for Biotechnology, IST, JNTUH, Kukatpally, Hyderabad-500085, Andhra Pradesh, India

eInstitute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500046, Andhra Pradesh, India
*E-mail: [email protected] November 21, 2012
DOI 10.1002/jhet.1993Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).

A series of 1-acyl-2-pyrazoline derivatives derived from nonsteroidal anti-inflammatory drugs wasdesigned as potential anticancer agents. Synthesis of these compounds was carried out via the condensationreaction of chalcones and acid hydrazides under heating. The methodology did not require the use of anycostly reagents or catalysts, and the acid hydrazide reactants were readily prepared from mefenamic acidor ibuprofen. A variety of 1-acyl-2-pyrazolines was prepared in good to excellent yields. An N-allylidenebenzohydrazide intermediate was isolated during the reaction optimization study, the structure of whichwas confirmed unambiguously by X-ray single crystal data. A range of N-allylidene benzohydrazides werealso prepared in good yields. Some of the compounds synthesized showed promising cytotoxic activitieswhen tested against HCT-15 human colon cancer cell line in vitro.

J. Heterocyclic Chem., 00, 00 (2014).

INTRODUCTION

The 2-pyrazoline (4,5-dihydro-1H-pyrazole,A) (Figure 1)framework plays an important role in medicinal/heterocyclicchemistry and has attracted particular attention in drug dis-covery. Derivatives of A have shown diverse pharmacologi-cal activities [1–10]. For example, 1-acyl-2-pyrazolinederivatives B, C, and D have shown antibacterial [2], aminooxidase inhibitory [3], and anticancer [7] properties. Severalnonsteroidal anti-inflammatory drugs (NSAIDs), on the otherhand, have shown promising cytotoxic activities [11]. Forexample, well-known anti-inflammatory drugs piroxicamand mefenamic acid have shown selective in vitro cytotoxiceffects on several cancer cell lines [12]. Thus, a combinationof A and an appropriate NSAID in a single molecule isexpected to provide a new template E (Figure 2) for thedesign and identification of novel anticancer agents.Prompted by this idea and because of our longstandinginterest in the chemical modification of NSAIDs [13–19],we became interested in the synthesis and pharmacologicalevaluation of a library of compounds based on E. Herein,we report in vitro cytotoxic activities of novel 1-acyl-2-pyrazoline derivatives based onE incorporating the structural

© 2014 HeteroC

features of known NSAIDs, for example, mefenamic acidand ibuprofen. Because the free carboxylic acid moiety ofthese NSAIDs are known to be partially responsible for theirincreased risk of stomach ulcers and gastrointestinal bleeding[20], hence, new compounds were designed by linking thecarboxylic acid moiety of mefenamic acid/ibuprofen to theNH group of 2-pyrazoline in the form of an amide bond.To the best of our knowledge, the design, synthesis, andin vitro evaluation of this class of compounds as potentialanticancer agents have not been reported earlier.

In spite of their medicinal and other importance,only few methods have been reported for the synthesisof 1-acyl-2-pyrazolines. These include synthesis ofN1-propanoyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole deriv-atives via the reaction of chalcone with hydrazine hydrate andpropionic acid in the same pot [9]. Alternatively, 1-benzoyl-2-pyrazolines were prepared via a linear synthesis involvingthe reaction of chalcone and hydrazine hydrate followed bytreatment with benzoyl chloride in pyridine [10]. The synthe-sis of isoniazide-based 1-acyl-2-pyrazolines has been carriedout via the reaction of chalcone with isonicotinohydrazide inthe presence of acetic acid in EtOH under conventional con-ditions [21]. We adopted a similar strategy to prepare our

orporation

Figure 1. 2-Pyrazoline (A) and its 1-acyl derivatives (B–D) possessing pharmacological activities.

Figure 2. Design of novel nonsteroidal anti-inflammatory drug-based 1-acyl-2-pyrazolines E.

K. Kankanala, V. R. Reddy, Y. P. Devi, L. N. Mangamoori, D. Rambabu, K. Mukkanti, and S. Pal Vol 000

target molecules based onE. Thus, appropriate chalcones (1)were reacted with acid hydrazides (2) prepared frommefenamic acid or ibuprofen in glacial acetic acid under re-flux to give the desired 1-acyl-2-pyrazolines (4) (Scheme 1).

RESULTS AND DISCUSSION

Initially, the synthesis of a representative compound,for example, (2-(2,3-dimethylphenylamino)phenyl)(4,5-dihydro-5-phenyl-3-(4-propoxyphenyl)pyrazo-1-yl)methanone(4a), was examined via the condensation of 2-(2,3-dimethylphenylamino)benzohydrazide (2a) and (E)-3-phenyl-1-(4-propoxyphenyl)prop-2-en-1-one (1a) in glacialacetic acid at room temperature for 25 h (Scheme 2). Afterusual work up and purification, the structure of the

Scheme 1. Synthesis of nonsteroidal anti-inflam

Scheme 2. Preparation of 4a via t

Journal of Heterocyclic Chemi

compound was elucidated by MS, IR, 1H, and 13C NMRspectroscopic measurements as well as single crystal X-raydata. To our surprise, the product isolated in 96% yield wasidentified as an open chain intermediate, that is, (1E)-2-(2,3-dimethylphenylamino)-N0-((Z)-3-phenyl-1-(4-propoxyphenyl)allylidene) benzohydrazide 3a (Scheme 1)instead of the desired pyrazoline 4a. The MS spectrum of3a showed a protonated molecular ion peak at m/z 504,whereas its IR spectrum displayed a strong absorption at1657, 1604, and 1500 cm�1 due to C═O, C═N, and C═Cstretching, respectively. The 1H NMR spectrum of 3adisplayed two singlets at d 9.20 and 9.14 due to two NHgroups that disappeared during D2O exchange experiments.Signals in the range of d 7.41–6.45 were due to the aromaticand olefinic protons, whereas the protons of OCH2 moiety

matory drug-based 1-acyl-2-pyrazolines 4.

he condensation of 1a and 2a.

stry DOI 10.1002/jhet

Month 2014 Nonsteroidal Anti-inflammatory Drug-based N-Allylidene Benzohydrazides and1-Acyl-2-pyrazolines: Their Synthesis as Potential Cytotoxic Agents In Vitro

were observed at d 4.02 ppm as a triplet having J=6.0Hz.The 13C NMR spectrum of 3a showed a signal at168.4 ppm corresponding to carbonyl carbon (C═O) alongwith signals at 22.5 and 20.6 ppm for the methyl groups.Finally, the structure of 3a was confirmed unambiguouslyby single crystal X-ray analysis [22] (Figure 3) that showedE and Z-geometry of the C═C and C═N, respectively.It was evident that the acid hydrazide 2a underwent a

simple Schiff-base formation reaction (via nucleophilicattack on the carbonyl group of 1a followed by eliminationof water molecule) instead of 1,4-addition with the chalcone1a in a regioselective manner to give the open chain product3a. In a separate experiment, a solution of compound 3a inacetic acid was refluxed for 18 h when 3a underwent cycliza-tion via an intramolecular nucleophilic attack by the NHmoiety of hydrazide to the C═C (cf a Michael type reaction)to afford the 5-membered aza heterocycle pyrazoline 4a. The

Figure 3. ORTEP representation of compound 3a. (The

Figure 4. 1H NMR (CDCl3, 4


direct formation of 4a from the reaction of 1a and 2a at roomtemperature was not observed; perhaps, the intramolecularcyclization of the intermediate 3a was not favored at roomtemperature. The poor nucleophilicity of the NH moiety(i.e., the less availability of lone pair due to the adjacentcarbonyl group) of hydrazide and the bulky phenyl group(that may cause some steric crowding) attached to the doublebond could be the reason for this observation. The intramo-lecular cyclization of 3a therefore required additional energythat was supplied by increasing the reaction temperature in aseparate pot. Nevertheless, the structure of 4a wasconfirmed by MS, IR, 1H, and 13C NMR spectra. The MSspectrum of 4a showed a protonated molecular ion peakat m/z 504, whereas the IR spectrum displayed strongabsorptions at 1745 cm�1 due to C═O stretching and at1627 cm�1 due to C═N stretching. The 1H NMR spectraof 4a (Figure 4) showed the two diastereotopic protons

rmal ellipsoids are drawn at 50 % probability level).

00 MHz) spectrum of 4a.



attached to C-4 and one proton at C-5 of the pyrazoline ringthat appeared as three characteristic doublet of doublets(ABX spin system) at d 5.83 (dd, J= 11.7, 4.9Hz, 1H),3.77 (dd, J=17.1, 11.7Hz, 1H), and 3.17 (dd, J=15.6,4.9Hz, 1H) for HX, HA, and HB, respectively. This is incon-sistent with the reported 1H NMR data for HA, HB, and HX

protons of a pyrazoline ring (of a range of compounds) thatappeared as doublets of doublets at 3.10–3.30, 4.00–4.10,and 5.60–5.70 ppm (JAB= 17.07, JAX= 6.30, JBX= 11.05Hz), respectively [23]. A D2O exchangeable singletappeared near d 8.14 indicated the presence of an NH group,whereas signals due to the methyl and propoxy substituentswere appeared at appropriate places (Figure 4). The 1-acyl-2-pyrazoline structure was further supported by the 13CNMR chemical shift values at 171.5 (CO), 167.3 (C-3),and 69.6 (C-5) ppm.We then examined the reaction of various chalcones 1 and

the acid hydrazide 2a at room temperature results of whichare summarized in Table 1. A range of N-allylidenebenzohydrazides 3 were obtained in these cases in good toexcellent yields.On the basis of results presented in Scheme 2, we exam-

ined the reaction of chalcones 1 with hydrazides 2 for lon-ger duration when 1-acyl-2-pyrazolines (4) were obtainedin good yields. Results of these studies are summarizedin Table 2. The hydrazide reactants were readily preparedfrom mefenamic acid or ibuprofen. Thus, a number ofchalcones and hydrazides were employed to afford a vari-ety of 1-acyl-2-pyrazolines (4). In an alternative method,refluxing of an appropriate N-allylidene benzohydrazide(3a–e) in glacial acetic acid for 10–18 h depending on thenature of the reactant employed also provided the corre-sponding 1-acyl-2-pyrazoline (4) in good yield.Having synthesized a range ofN-allylidene benzohydrazides

(3) and 1-acyl-2-pyrazolines (4), the in vitro cytotoxic evalu-ation of some of these compounds was performed againsthuman HCT-15 for colon cancer cells. Cancer, accordingto WHO, is the second leading cause of death worldwide af-ter cardiovascular disease [24], whereas colon (or colorectal)cancer is widespread in theWestern world. Although varioustherapies are available to treat different types of cancerincluding colon cancer, the worldwide deaths, however,due to cancer has been projected to increase over 11 millionin 2030 according to WHO. Thus, identification and devel-opment of new and suitable agents to treat various types ofcancer is of particular interest. Colon cancer being our majorfocus, we have used human colon cancer cell line HCT-15for our in vitro assay [25]. Doxorubicin, a knownanthracycline antibiotic, was used as a reference compound.All the test compounds were examined for their ability toinhibit the cancerous cells (HCT-15) based on a 3,4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT)assay. All these compounds were tested at five different con-centrations, for example, 1, 2, 5, 10, and 25mg/mL, and the


percentage of cell death measured for each compound alongwith IC50 values of active compounds are summarized inTable 3. It is evident from Table 3 that N-allylidenebenzohydrazides, for example, 3b, 3c, and 3d, and 1-acyl-2-pyrazolines, for example, 4c, 4e, and 4f, showed goodactivities especially at higher dose. All these compoundsshowed IC50 values in the range 13–22mg/mL (Table 3)compared with doxorubicin’s IC50 value of 50mg/mL(0.09mM). Thus, the present N-allylidene benzohydrazidesand 1-acyl-2-pyrazoline class of compounds have potentialfor the development of new anticancer agents especially forcolon cancer.

CONCLUSIONS

In conclusion, 1-acyl-2-pyrazoline derivatives derivedfrom NSAIDs were designed as potential anticanceragents. Synthesis of these compounds was carried out viathe condensation reaction of chalcones and acid hydrazidesunder heating. The methodology did not require any costlyreagents or catalysts, and the acid hydrazide reactants werereadily prepared from mefenamic acid or ibuprofen. Avariety of 1-acyl-2-pyrazolines was prepared in good to ex-cellent yields. We were also able to isolate an N-allylidenebenzohydrazide intermediate (during the reaction optimi-zation of a chalcone and acid hydrazide), the structure ofwhich was confirmed unambiguously by X-ray singlecrystal data. This not only helped us to establish the 1,2-nucleophilic addition pathway of this reaction but alsoallowed us to prepare a range ofN-allylidene benzohydrazidesin good yields. A number of compounds synthesized weretested for their cytotoxicity in vitro against HCT-15 humancolon cancer cell line, and some of them showed promisingactivities compared with doxorubicin. Thus, the presentN-allylidene benzohydrazides and 1-acyl-2-pyrazoline classof compounds represent new templates for the identificationand development of novel anticancer agents.

EXPERIMENTAL

ChemistryGeneral methods. Melting points were determined by open

glass capillary method on a Cintex melting point apparatus andare uncorrected. IR spectra were recorded on a Perkin-Elmerspectrometer using KBr pellets. 1H NMR spectra were recordedon a Varian 400MHz spectrometer using CDCl3 or DMSO-d6,with reference to tetramethylsilane as an internal reference. 13CNMR spectra were recorded on a 100MHz spectrometer. Massspectra were recorded on a Jeol JMC D-300 instrument byusing electron ionization at 70 ev. All reactions were monitoredby TLC on precoated silica gel plates. Column chromatographywas performed on 100–200 mesh silica gel (SRL, India) using10–20 times (by weight) of the crude product.

Mefenamic acid and ibuprofen are commercially available.Methyl esters of mefenamic acid and ibuprofen were prepared


Table 1

Preparation of N-allylidene benzohydrazides 3 from 1 and 2a.a

Entry Chalcone (1) Product (3) Time (h) Yieldb (%)

1 25 96

2 15 98

3 15 84

4 12 85

5 15 89

aAll the reactions were performed using chalcone 1 (5mmol) and acid hydrazide 2a (10mmol) in glacial acetic acid (10mL).bIsolated yield.


according to the known methods [26]. Acid hydrazides of theseacids from esters were prepared following the methods availablein literature [27].


Generalmethods for synthesis ofN-allylidene benzohydrazides3. A mixture of chalcone 1 (5mmol) and acid hydrazide 2a(2.5 g, 10mmol) in glacial acetic acid (10mL) was stirred at room


Table 2

Preparation of 1-acyl-2-pyrazolines 4 from chalcones 1 and hydrazides 2.a

Entry Chalcone (1) Hydrazide (2) Product (4) Time Yieldb (%)

1 1a 2a 18 h 90

2 1b 2a 15 h 98

3 1c 2a 4 h 97

4 1d 2a 15 h 96

5 1b 2 h 89

6 1c 35min 88

(Continues)


Journal of Heterocyclic Chemistry DOI 10.1002/jhet

Table 2

(Continued)

Entry Chalcone (1) Hydrazide (2) Product (4) Time Yieldb (%)

7 1d 15min 93

aAll the reactions were performed using chalcone 1 (5mmol) and an hydrazide 2 (10mmol) in glacial acetic acid (10mL) under reflux.bIsolated yield.

Table 3

The in vitro anticancer properties of some of the N-allylidene benzohydrazides (3) and 1-acyl-2-pyrazolines (4) synthesized.

Entry Compounds

% of cell death at various concentrations

1mg/mL 2 mg/mL 5 mg/mL 10 mg/mL 25mg/mL IC50 (mg/mL)

1 3b 16.8 27.2 33.0 45.7 61.8 13.92 3c 25.6 30.3 35.1 39.3 63.6 16.63 3d 15.0 32.2 33.6 43.6 64.0 14.74 4c 24.8 29.1 30.3 38.2 52.9 22.05 4e 18.5 27.7 32.4 35.8 54.0 21.86 4f 29.1 33.7 46.0 46.5 57.0 15.0

All the values are the average of the experiments performed in triplicates. The cell line used is HCT-15 human colon cancer cell line. Doxorubicin[IC50 = 50mg/mL (0.09mM)] was used as a reference compound.


temperature for the time indicated in Table 1 (the progress of thereaction was monitored by TLC). After completion of thereaction, the mixture was filtered off, washed with petroleum ether(2� 10mL), and dried to afford the pure compound.

(1E)-2-(2,3-Dimethylphenylamino)-N0-((Z)-3-phenyl-1-(4-propoxyphenyl)allylidene)benzohydrazide (3a). Off whitesolid; mp 159–160�C; Rf 0.38 (petroleum ether/EtOAc=8.5:1.5),IR (KBr) nmax/cm

�1: 3230, 2959, 1657, 1604, 1500, 1248; 1HNMR (400MHz, CDCl3) d 9.20 (bs, 1H, NH, D2Oexchangeable), 9.14 (bs, 1H, NH, D2O exchangeable), 741–6.91(m, 16H), 6.58 (t, J=7.4Hz, 1H), 6.45 (d, J=16.4Hz, 1H), 4.02(t, J=6.0Hz, 2H), 2.32 (s, 3H), 2.19 (s, 3H), 1.90 (m, 2H), 1.10(t, J=7.5Hz, 3H); 13C NMR (100MHz, CDCl3): d 168.3, 160.2,154.5, 148.0, 139.1, 138.1, 137.8, 136.2, 132.8, 130.2, 129.7,128.8, 128.7, 128.6, 127.1, 125.8, 125.6, 122.2, 120.9, 116.6,115.7, 114.9, 69.7, 22.5, 20.6, 13.8, 10.5; MS (m/z): 504[M+H]+, (100%).

(1E)-2-(2,3-Dimethylphenylamino)-N0-((E)-1,3-diphenylallylidene)benzohydrazide (3b). White solid; mp 166–167 C;Rf 0.45 (petroleum ether/EtOAc=8.5:1.5). IR (KBr) nmax/cm

�1:3286, 3242, 1660; 1H NMR (400MHz, CDCl3): d 9.25 (bs, 1H,NH, D2O exchangeable), 9.03 (bs, 1H, NH, D2O exchangeable),7.66–7.56 (m, 3H), 7.45–7.27 (m, 6H), 7.19–7.14 (m, 3H), 7.13–7.04 (m, 3H), 6.96–6.90 (m, 3H), 6.55 (t, J=7.4Hz, 1H), 6.42(d, J= 16.1Hz, 1H), 2.32 (s, 3H), 2.19 (s, 3H); 13C NMR(100MHz, DMSO+CDCl3): d 174.4, 154.1, 145.5, 140.0, 139.4,137.2, 135.4, 132.2, 130.2, 129.4, 129.1, 128.8, 128.3, 128.1,


127.5, 127.3, 127.2, 126.4, 125.3, 125.1, 119.9, 117.3, 116.1,115.1, 20.1, 13.3; MS (m/z): 446 [M+H]+, (100%).

(1E)-2-(2,3-Dimethylphenylamino)-N0-((E)-3-(4-methoxyphenyl)-1-phenylallylidene)benzohydrazide (3c). Off white solid; mp140–141�C; Rf 0.27 (petroleum ether/EtOAc=8.5:1.5); IR (KBr)nmax/cm

�1: 3283, 3238, 1655, 1505; 1H NMR (400MHz,CDCl3): d 9.25 (bs, 1H, NH, D2O exchangeable), 8.98 (bs, 1H,NH, D2O exchangeable), 7.64–7.55 (m, 3H), 7.36–7.13 (m, 7H),7.06 (t, J=7.4Hz, 1H), 6.96–6.85 (m, 5H), 6.54 (t, J=7.3Hz,1H), 6.37 (d, J=16.1Hz, 1H), 3.81 (s, 3H), 2.32 (s, 3H), 2.19 (s,3H); 13C NMR (100MHz, DMSO-d6): d 167.3, 158.8, 154.3,146.1, 139.4, 137.8, 133.8, 132.0, 131.4, 130.0, 129.4, 129.1,128.6, 128.4, 128.2, 127.9, 125.9, 125.0, 120.1, 118.2, 116.2,114.8, 114.3, 114.2, 55.3, 20.3, 13.5; MS (m/z): 476 [M+H]+,(100%).

(1E)-2-(2,3-Dimethylphenylamino)-N0-((E)-3-(4-chlorophenyl)-1-phenylallylidene)benzohydrazide (3d). Brownish yellowsolid; mp 146–147�C; Rf 0.47 (petroleum ether/EtOAc=8.5:1.5);IR (KBr) nmax/cm

�1: 3254, 2922, 1661; 1H NMR (400MHz,CDCl3): d 9.20 (bs, 1H, NH, D2O exchangeable), 9.03 (bs, 1H,NH, D2O exchangeable), 7.63–7.58 (m, 3H), 7.40–7.28 (m,6H) 7.17–6.89 (m, 7H), 6.54 (t, J = 7.3Hz, 1H), 6.36 (d,J = 16.5 Hz, 1H), 2.31 (s, 3H), 2.18 (s, 3H); 13C NMR(100MHz, CDCl3): d 167.1, 158.5, 154.5, 148.5, 139.0, 138.1,136.4, 134.6, 132.9, 130.6, 130.2, 129.9, 129.4, 129.0, 128.2,125.9, 125.7, 121.0, 116.6, 115.0, 29.7, 20.6; MS (m/z): 480[M+H]+, (100%).



(1E)-2-(2,3-Dimethylphenylamino)-N0-((E)-3-(3-nitrophenyl)-1-phenylallylidene)benzohydrazide (3e). Pale yellow solid;mp 168–170 �C; Rf 0.37 (petroleum ether/EtOAc=8:2); IR (KBr)nmax/cm

�1: 2920, 1650; 1H NMR (400MHz, CDCl3): d 9.32 (bs,1H, NH, D2O exchangeable), 9.10 (bs, 1H, NH, D2Oexchangeable), 8.18 (s, 1H), 8.11 (d, J=8.3Hz, 1H), 7.78–6.87(m, 14H), 6.58 (t, J=7.4Hz, 1H), 6.46 (d, J=16.2Hz, 1H), 2.32(s, 3H), 2.19 (s, 3H); MS (m/z): 491 [M+H]+, (100%).

General procedure for the synthesis of 1-acyl-2-pyrazolines 4Method A. A mixture of chalcone 1 (5mmol) and an

appropriate hydrazide 2 (10mmol) in glacial acetic acid (10mL)was refluxed for the time indicated in Table 2 (the progress ofthe reaction was monitored by TLC). After completion of thereaction, the mixture was cooled and poured into crushed ice(25 g). The separated solid was filtered and dried. The crudeproduct was purified by column chromatography on silica usingpetroleum ether–EtOAc as eluent.

Method B. Appropriate N-allylidene benzohydrazide (3a–e)(10mmol) in glacial acetic acid was refluxed for 10–18 hdepending on the nature of the reactant employed. Theprogress of the reaction was monitored by TLC. Aftercompletion of the reaction, the mixture was poured intocrushed ice (25 g). The separated solid was filtered, dried, andpurified by column chromatography on silica using petroleumether–EtOAc as eluent.

(2-(2,3-Dimethylphenylamino)phenyl)(4,5-dihydro-5-phenyl-3-(4-propoxyphenyl)pyrazo-1-yl)methanone (4a). Off whitesolid; mp 85–88�C; Rf 0.55 (petroleum ether/EtOAc=8.5:1.5); IR(KBr) nmax/cm

�1: 2923, 1745, 1627, 1607, 1582; 1H NMR(400MHz, CDCl3): d 8.14 (bs, 1H, NH, D2O exchangeable), 7.93(d, J=7.8Hz, 1H), 7.64 (d, J=8.8Hz, 2H), 7.33–6.79 (m, 13H),5.83 (dd, J=11.7, 4.9Hz, 1H), 3.95 (t, J=6.3Hz, 2H), 3.77 (dd,J=17.1, 11.7Hz, 1H), 3.17 (dd, J=15.6, 4.9Hz, 1H), 2.28 (s,3H), 2.10 (s, 3H), 1.82 (m, 2H), 1.04 (t, J=7.4Hz, 3H); 13CNMR (100MHz, CDCl3): d 171.5, 167.3, 161.0, 146.3, 142.0,140.0, 131.4, 130.1, 128.9, 128.4, 127.6, 125.7, 125.4, 124.9,123.7, 120.2, 116.6, 115.1, 114.6, 69.6, 60.8, 41.8, 22.5, 20.6,13.9, 10.5; MS (m/z): 504 [M+H]+, (100%).

(2-(2,3-Dimethylphenylamino)phenyl)(4,5-dihydro-3,5-diphenylpyrazo-1-yl)methanone (4b). White solid; mp80–82�C; Rf 0.61 (petroleum ether/EtOAc=8.5:1.5); IR (KBr)nmax/cm

�1: 3338, 2922, 1627, 1579; 1H NMR (400MHz,CDCl3): d 8.16 (bs, 1H, NH, D2O exchangeable), 7.93 (d,J=7.4Hz, 1H), 7.71 (d, J=3.0Hz, 2H), 7.42–7.33 (m, 7H), 7.24–7.20 (m, 2H), 7.11 (d, J=7.8Hz, 1H), 7.02 (t, J= 7.8Hz, 1H),6.89 (d, J=8.3Hz, 2H), 6.82 (t, J=8.3Hz, 1H), 5.86 (dd,J=11.7, 4.9Hz, 1H), 3.80 (dd, J=17.6, 11.7Hz, 1H), 3.20 (dd,J=17.6, 4.9Hz, 1H), 2.29 (s, 3H), 2.11 (s, 3H); MS (m/z): 446[M+H]+, (100%).

(2-(2,3-Dimethylphenylamino)phenyl)(4,5-dihydro-5-(4-methoxyphenyl)-3-phenylpyrazo-1-yl)methanone (4c). Offwhite solid; mp 95–98�C; Rf 0.45 (petroleum ether/EtOAc=8.5:1.5); IR (KBr) nmax/cm

�1: 3559, 3418, 2923, 1628;1H NMR (400MHz, CDCl3): d 8.14 (bs, 1H, NH, D2Oexchangeable), 7.89 (d, J=7.9Hz, 1H), 7.73 (d, J=6.7Hz, 1H),7.41 (d, J=1.8Hz, 2H), 7.40 (d, J=1.8Hz, 1H), 7.27–7.25 (m,3H), 7.23–7.19 (m, 1H), 7.11 (d, J=7.9Hz, 1H), 7.02 (t,J=7.9Hz, 1H), 6.89 (d, J=8.5Hz, 1H), 6.85 (d, J=8.5Hz, 2H),6.80 (t, J=8.0Hz, 2H), 5.81 (dd, J=11.6, 4.9Hz, 1H), 3.78 (dd,J=11.6, 17.7Hz, 1H), 3.77 (s, 3H), 3.20 (dd, J=17.7, 4.9Hz,1H), 2.29 (s, 3H), 2.11 (s, 3H); 13C NMR (100MHz, CDCl3): d


167.6, 159.0, 154.6, 146.4, 139.9, 137.8, 134.1, 132.1, 131.5,131.4, 130.4, 130.1, 128.7, 126.8, 126.7, 125.6, 125.0, 120.3,118.4, 116.6, 115.1, 114.3, 60.4, 55.3, 41.7, 20.6, 13.9; MS (m/z):476 [M+H]+, (100%).

(2-(2,3-Dimethylphenylamino)phenyl)(5-(4-chlorophenyl)-4,5-dihydro-3-phenylpyrazo-1-yl)methanone (4d). Paleyellow solid; mp 108–110�C; Rf 0.61 (petroleum ether/EtOAc=8.5:1.5); IR (KBr) nmax/cm

�1: 2921, 1627, 1585; 1HNMR (400MHz, CDCl3): d 8.13 (bs, 1H, NH, D2Oexchangeable), 7.91 (d, J=7.9Hz, 1H), 7.71 (d, J=7.4Hz, 2H),7.42–7.41 (m, 3H), 7.28–7.21 (m, 5H), 7.12–7.10 (m, 1H), 7.05(t, J = 7.4 Hz, 1H), 6.90 (d, J = 7.8 Hz, 2H), 6.81 (t,J = 7.4Hz, 1H), 5.81 (dd, J = 12.1, 5.1 Hz, 1H), 3.80 (dd,J = 17.9, 11.8 Hz, 1H), 3.17 (dd, J = 17.6, 5.0 Hz, 1H), 2.29(s, 3H), 2.10 (s, 3H); 13C NMR (100MHz, CDCl3): d 167.6,154.5, 146.5, 140.4, 139.9, 137.9, 133.4, 132.0, 131.8,131.1, 130.5, 130.1, 129.2, 128.7, 126.9, 126.8, 125.7,125.1, 120.2, 118.0, 116.6, 115.2, 60.4, 41.5, 20.6, 13.8.MS (m/z): 480 [M+H]+, (100%).

1-(4,5-Dihydro-3,5-diphenylpyrazol-1-yl)-2-(4-isobutyl phenyl)propan-1-one (4e). White solid; mp 151–152�C; Rf 0.71(petroleum ether/EtOAc = 8:2); IR (KBr) nmax/cm

�1: 2930,1660, 1625; 1H NMR (400MHz, CDCl3): d 7.48–7.22 (m,12H), 7.15 (d, J = 8.6 Hz, 2H), 5.43 (dd, J = 11.5, 4.6 Hz,1H), 4.78 (q, 1H), 3.60 (dd, J = 17.5, 11.8 Hz, 1H), 3.15(dd, J = 17.7, 4.8 Hz, 1H), 2.44 (d, J = 7.0 Hz, 2H), 1.80–1.90(m, 1H), 1.58 (d, J = 7.2Hz, 3H), 0.86 (d, J = 7.9Hz, 6H);13C NMR (100MHz, CDCl3): d 176.7, 1 48.6, 140.8, 138.6,135.1, 129.6, 129.4, 129.2, 128.9, 128.7, 128.5, 128.4,128.3, 127.7, 127.2, 127.1, 116.4, 45.1, 41.3, 30.2, 22.4,18.5; MS (m/z): 411 [M+H]+, (100%).

1-(4,5-Dihydro-5-(4-methoxyphenyl)-3-phenylpyrazol-1-yl)-2-(4-isobutyl phenyl)propan-1-one (4f). White solid; mp116–118�C; Rf 0.54 (petroleum ether/EtOAc = 8:2); IR (KBr)nmax/cm

�1: 3282, 1713, 1589, 1508; 1H NMR (400MHz,CDCl3): d 7.72 (m, 2H), 7.44–7.42 (m, 3H), 7.33 (d, J = 8.3Hz,2H), 7.17 (d, J = 8.6Hz, 2H), 7.05 (d, J = 8.3 Hz, 2H), 6.85 (d,J = 8.6Hz, 2H), 5.45 (dd, J = 11.6, 4.6 Hz, 1H), 4.77 (q,J = 7.4Hz, 1H), 3.78 (s, 3H), 3.61 (dd, J = 17.7, 11.9 Hz, 1H),3.12 (dd, J = 17.7, 4.9 Hz, 1H), 2.40 (d, J = 7.0Hz, 2H), 1.81(q, 1H), 1.47 (d, J = 7.3Hz, 3H), 0.86 (d, J = 7.9Hz, 6H); 13CNMR (100MHz, CDCl3): d 172.3, 159.0, 153.4, 139.9, 138.9,134.4, 131.6, 130.1, 129.0, 128.6, 127.6, 126.9, 126.6, 114.3,59.9, 55.2, 45.1, 42.7, 42.0, 30.1, 22.4, 18.8; MS (m/z): 441[M+H]+, (100%).

(4-Chlorophenyl)(5-(4-chlorophenyl)-4,5-dihydro-3-phenylpyrazo-1-yl)methanone (4g). Off white solid; mp 172–174�C;Rf 0.50 (petroleum ether/EtOAc = 8:2); IR (KBr) nmax/cm

�1:2925, 1640, 1580; 1H NMR (400MHz, CDCl3): d 7.9 (d,J= 8.4Hz, 2H), 7.70 (d, J= 7.3Hz, 2H), 7.45–7.41 (m, 5H),7.33–7.28 (m, 4H), 5.76 (dd, J= 11.5, 5.1Hz, 1H), 3.80 (dd,J = 17.6, 11.7 Hz, 1H), 3.19 (dd, J = 17.6, 5.1 Hz, 1H); MS(m/z): 395 [M+H]+, (100%).

Single crystal X-ray data for compound 3a. Single crystalsuitable for X-ray diffraction of 3a was grown from methanol.The crystals were carefully chosen using a stereo zoommicroscope supported by a rotatable polarizing stage. The datawere collected at room temperature on Bruker Kappa APEX- ΙΙCCD DUO diffractometer with graphite monochromatedMo-Ka radiation (0.71073Å). The crystals were glued to a thin



glass fiber using FOMBLIN immersion oil and mounted on adiffractometer. The intensity data were processed using Broker’ssuite of data processing programs (SAINT), and absorptioncorrections were applied using SADABS [28]. The crystalstructure was solved by direct methods using SHELXS-97,and the data was refined by full matrix least-squaresrefinement on F2 with anisotropic displacement parametersfor non-H atoms, using SHELXL-97 [29].

Crystal data of 3a: molecular formula =C33H33N3O2, formulaweight = 503.62, crystal system=monoclinic, space group =P21/c, a= 17.386(3)Å, b= 20.968(5)Å, c= 15.394(3)Å, V= 5586(2) Å3, T= 100(2)K, Z= 8, Dc = 1.198Mgm�3, m(Mo-Ka) = 0.08mm�1, 21063 reflections measured, 4057 independent reflec-tions, 2850 observed reflections [I> 2.0 s (I)], R1_obs = 0.078,goodness of fit = 1.133.

Biological activityChemical and reagents. RPMI-1640, L-glutamine,

streptomycin, and penicillin were obtained from Sigma-Aldrich,USA. Trypsin-versene, fetal bovine serum was procured fromPAA Biotech, Germany. All other fine chemicals/reagents usedin this study were of cell culture grade and obtained fromSigma-Aldrich and/or Merck.

Preparation of test compounds. Test compounds weredissolved in DMSO and were diluted appropriately withculture media before treatment of cells. The finalconcentration of DMSO used in the culture medium was lessthan 0.2%.

Cell lines and culture conditions. The cell line used in thiswork was obtained from the National Centre for Cell Science,Pune, India. The cell line is human colon cancer cell line,HCT15. The cells were grown in RPMI 1640 culturemedium supplemented with 2mM L-glutamine, 10% fetalbovine serum, penicillin (50 IU/mL), and streptomycin(50 mg/mL) at 37�C in a humidified incubator with a 5%CO2 atmosphere and passage thrice weekly to maintain asubconfluent state.

MTT assay for cytotoxicity. The effect of test compoundson cell viability was measured using an MTT assay, which isbased on the reduction of MTT by the mitochondrialdehydrogenase of intact cells to a purple formazan product [25].Doxorubicin, a known anticancer drug, was used as a referencecompound in this assay. Cells (1� 104) were seeded in a 96-well plate. After 24 h, they were treated with differentconcentration (1–25mg/mL) of various test compounds dilutedappropriately with culture media for 48 h. Cells grown in mediacontaining equivalent amount of DMSO served as positivecontrol and cells in medium without any supplementation wereused as negative control. After the treatment, medium-containingcompounds were carefully removed by aspiration. An amount of100mL of 0.4mgmL�1 MTT in phosphate-buffered solution wasadded to each well and incubated in the dark for 4 h. An amountof 100 mL of DMSO was added to each well and kept in anincubator for 4 h for dissolution of the formed formazancrystals. An amount of formazan was determined bymeasuring the absorbance at 540 nm using an enzyme-linkedimmuno sorbent assay plate reader. The data were presented aspercent dead cells, whereas the absorbance from nontreatedcontrol cells was defined as 100% live cells. The percent deadcells was plotted (Y-axis) against concentration (X-axis) ofcompounds, when IC50 values could be interpolated fromthe graph.


Acknowledgments. The authors (S. P. and K. K.) thank Mr. M.N. Raju, the chairman of M.N.R. Educational Trust for hisconstant encouragement and G. R. Krishna and Dr. C. M.Reddy of IISER, Kolkata, India for X-ray single crystal data.

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