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Contents lists available at ScienceDirect

Steroids

journal homepage: www.elsevier .com/locate /s teroids

Synthesis and anti-tumor evaluation of B-ring substituted steroidalpyrazoline derivatives

0039-128X/$ - see front matter � 2013 Published by Elsevier Inc.http://dx.doi.org/10.1016/j.steroids.2013.09.006

⇑ Corresponding author. Tel.: +91 9411003465.E-mail addresses: shamsuzzaman9@gmail.com, shams_chem@rediffmail.com

(Shamsuzzaman).

Please cite this article in press as: Shamsuzzaman et al. Synthesis and anti-tumor evaluation of B-ring substituted steroidal pyrazoline derivatives. S(2013), http://dx.doi.org/10.1016/j.steroids.2013.09.006

Shamsuzzaman a,⇑, Hena Khanam a, Ashraf Mashrai a, Asif Sherwani b, Mohammad Owais b,Nazish Siddiqui c

a Department of Chemistry, Aligarh Muslim University, Aligarh 202002, Indiab Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, Indiac Department of Illmul Advia Ajmal Khan Tibbiya College, Aligarh Muslim University, Aligarh 202002, India

a r t i c l e i n f o

303132333435363738

Article history:Received 13 February 2013Received in revised form 31 August 2013Accepted 7 September 2013Available online xxxx

Keywords:Cholest-5-en-7-onePyrazolinesAnticancerMTTSEM

a b s t r a c t

The synthesis and anti-tumor activity screening of new steroidal derivatives (4–18) containing pharma-cologically attractive pyrazoline moieties are performed. During in vitro anticancer evaluation, the newlysynthesized compounds displayed moderate to good cytotoxicity on cervical and leukemia cancer celllines. In addition these compounds were found to be nontoxic to normal cell (PBMCs) (IC50 > 50 lM).The structure–activity relationship is also discussed. The most effective anticancer compound 9 wasfound to be active with IC50 value of 10.6 lM. It demonstrated significant antiproliferative influence onJurkat cell lines. The morphological changes and growth characteristics of HeLa cells treated with com-pound 4 were analyzed by means of SEM.

� 2013 Published by Elsevier Inc.

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1. Introduction

Cancer is becoming the biggest health hazard for the world.Despite recent advances in early diagnosis, prevention and ther-apy, cancer still affects millions of people worldwide and is oneof the leading causes of death. In 2008, 7.6 million people diedfrom cancer according to the World Health Organization (WHO)and without immediate action, the global number of deaths fromcancer will increase by nearly 80% by 2030 [1]. Although cancerchemotherapy has established a new era of molecularly targetedtherapeutics, the efficacy of the existing drugs for the treatmentof various cancers is rather limited [2], and there is a need to devel-op new therapeutic agents to overcome the limitations with thecurrent therapy. That’s why there is an intense effort in cancerresearch to design new, potent, selective and less toxic anticanceragents that are capable of rapid destruction of tumor vasculatureleading to tumor necrosis and anti-tumor efficacy [3,4]. In vitrostudies, using a variety of human cancer cell lines, have been em-ployed to evaluate the effectiveness of new medicinal compoundsagainst these cancers.

Steroids are an important class of natural products which havehigh ability to penetrate cells and bind to nucleus and membrane

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receptors. They include great variations in structure and play avery important role in life [5,6]. Structurally diverse cytotoxicand cytostatic steroids are very relevant as lead compounds andmolecular probes for anticancer drug discovery and cancermolecular mechanisms elucidation. The chemistry of steroids hasmotivated extensive investigation through decades and a compre-hensive review on the syntheses of novel bioactive steroids hasbeen recently published [7]. The investigation of modified steroidderivatives condensed with various heterocyclic rings has drawngreat attention [8]. A variety of steroids with unusual and interest-ing structures have been synthesized and evaluated for their anti-tumor [9,10], antimicrobial [11] and anti-parasitic activities [12].Hybrid anti-cancer agents, which combine two active compoundsin one, such as steroidal alkylators, contain steroidal moiety as bio-logical vectors for anti-tumor agents in order to diminish toxicityand to enhance specificity, were recently demonstrated [13]. Suchtypes of agents attain duplicate effects on cancer cells. Thesemerged molecules may act on multiple therapeutic targets and of-fer the possibility of circumventing drug resistance. In addition, thehybrids may also minimize unwanted side effects and allow forsynergic action [14].

Pyrazolines present an interesting group of compounds, whichhas been known to possess wide spread pharmacological proper-ties [15]. Recently, different authors worldwide have reported anti-tumor, antiproliferative or anticancer potential of pyrazolinederivatives [16–18]. These derivatives are also well known for their

teroids

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Compound 4

Compound 7

Compound 9

Compound 18

Fig. 1. Dose-dependent effects of steroidal pyrazolines (4,7,9,18) on cell viability ofHeLa, Jurkat and PBMC cell lines. Data shown are mean ± standard error of at leastthree independent experiments.

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pronounced anti-inflammatory, analgesic [19], antidepressant [20]and antimicrobial activities [21]. In addition, antidiabetic activitiesof many compounds containing pyrazoline rings have been re-viewed [22]. The above application of steroids as well as pyrazolinederivatives gave us immense confidence to prepare some new ste-roidal pyrazoline derivatives by combining heterocyclic moiety ofpotent cytotoxic activity with steroid skeleton. In continuation ofour programme towards synthesis of new steroidal derivatives[23], we wish to report herein the facile synthesis of new B-ringsubstituted steroidal pyrazoline derivatives 4–18 starting from ste-roidal a, b-unsaturated ketones 1–3. The evaluation of their anti-tumor activity was carried out in vitro against human cancer celllines (HeLa and Jurkat) and normal cells (PBMCs) using MTT assay.HeLa cells treated with compounds 4, 9 and 18 were observed un-der Fluorescence Microscope (Fig. 2).The surface morphology ofthe treated (with compound 4) and untreated fixed HeLa cells werealso studied (Fig 3).

2. Experimental

2.1. Chemistry

2.1.1. GeneralAll glass apparatus were oven-dried prior to use. Chemicals and

solvents used in this study were of ACS grade and used directlywithout additional steps of purification. Melting points were deter-mined on a Kofler apparatus and are uncorrected. The IR spectrawere recorded on KBr pellets with Interspec 2020 FT-IR Spectrom-eter spectro Lab and values are given in cm�1. 1H and 13C NMRspectra were run in CDCl3/DMSO-d6 on a Bruker Avance II 400NMR Spectrometer (operating at 400 MHz for 1H and at 100 MHzfor 13C NMR) with tetramethylsilane (TMS) as internal standardand values are given in parts per million (ppm) (d). Splitting pat-terns are described as singlet (s), doublet (d), triplet (t) and multi-plet (m). Mass spectra were recorded on a JEOL D-300 massspectrometer. Elemental analyses of all the new compounds wererecorded on Perkin Elmer 2400 CHN Elemental Analyzer. Fluores-cence images were observed under Zeiss Imager M2, Gottingen(Germany) Fluorescence microscope. Scanning electron micro-graph (SEM) was obtained using JSM 6510LV scanning electronmicroscope (JEOL, Tokyo, Japan) at an accelerating voltage of 10and 20 kV. HPLC analysis was performed by LC-100 HPLC instru-ment. Thin layer chromatography (TLC) plates were coated withsilica gel G and exposed to iodine vapors to check the homogeneityas well as the progress of reaction. Sodium sulfate (anhydrous) wasused as a drying agent.

2.1.2. General procedure for the synthesis of steroidal pyrazolinederivatives 4–18

To a solution of cholest-5-en-7-one 1–3 (1.0 mmol) in dichloro-methane/methanol (1:4) (10 ml), hydrazine hydrate (1.5 mmol)was added followed by acetyl chloride (compounds 7–9)/formicacid (compounds 10–12)/benzoic acid (compounds 13–15)/mer-captoacetic acid (compounds 16–18) (3.5 mmol). The reaction mix-ture was refluxed for 3–11 h. The progress as well as completion ofreaction was monitored by TLC. After completion of the reaction,the reaction mixture was cooled to room temperature. The precip-itate thus obtained was filtered, washed with water, air dried andmonitored through TLC for the purity. Thin layer chromatographyrevealed just a single spot which proved the presence of a singleproduct. For further purification, the product was recrystallizedfrom methanol to give product as solid powder. The compounds4–6 were prepared by the same method without addition of anyacid.

The purity of compounds was analyzed by HPLC (Supplemen-tary Fig. SH). Twenty microliter of each sample were injected into

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the HPLC analysis system by manual injection after they were dis-solved in chloroform. The mobile phase for separation was a mix-ture of methanol and acetonitrile (60:40, vol/vol), and lasted for10 min; the solvent mixture was maintained at a flow rate of

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Fig. 2. Micrographs showing effect of steroidal pyrazoline derivatives on HeLa Cells. Micrographs captured after 48 h of incubation in bright field under FluorescenceMicroscope. A = Untreated control, B = Treated with comp 18, C = Treated with comp 9, D = treated with comp 4.

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1.000 mL/min. UV detection was performed simultaneously at230 nm wavelength.

2.1.2.1. 3b-Acetoxy-5a-cholestano-[5,7-c d]-pyrazoline (4).Mp: 203–205 �C; Anal. Calc. for C29H48N2O2: C, 76.27, H, 10.59, N, 6.13.Found: C, 76.45, H, 10.46, N, 6.01; IR (KBr) m cm�1: 3417 (N–H),1740 (C@O), 1244 (C–O), 1641 (C@N); 1H NMR (400 MHz, CDCl3):d 2.17 (1H, s, NH, exchangeable with D2O), 4.70 (1H, m, C3-aH, W1/

2 = 18 Hz, axial), 2.02 (3H, s, OCOCH3), 1.00 (3H, s, C10-CH3), 0.71(3H, s, C13-CH3), 0.92 & 0.85 (other methyl protons); 13C NMR(100 MHz, CDCl3): d 171.0, 153.1, 72.0, 53.2, 49.5, 48.1, 42.4,38.5, 38.1, 37.4, 37.3, 36.2, 35.2, 35.1, 34.5, 27.3, 26.7, 26.6, 26.4,22.8, 22.3, 21.8, 21.6, 20.4, 20.1, 19.6, 17.4, 16.7, 11.2; MS (ESI):m/z 456.37 [M+] (Calcd for C29H48N2O2, 456.71).

2.1.2.2. 3b-Chloro-5a-cholestano-[5,7-c d]-pyrazoline (5).Mp: 194–196 �C; Anal. Calc. for C27H45ClN2: C, 74.87, H, 10.47, N, 6.47.

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Found: C, 74.67, H, 10.65, N, 6.31; IR (KBr) m cm�1: 3423 (N–H),1640 (C@N), 720 (C–Cl); 1H NMR (400 MHz, CDCl3): d 2.0 (1H, s,NH, exchangeable with D2O), 3.51 (1H, m, C3-aH, W1/2 = 16 Hz, ax-ial), 1.10 (3H, s, C10-CH3), 0.81 (3H, s, C13-CH3), 0.92 & 0.86 (othermethyl protons); 13C NMR (100 MHz, CDCl3): d 152.0, 55.2, 54.8,51.7, 50.0, 45.1, 44.3, 43.5, 40.0, 39.3, 38.7, 37.3, 37.1, 36.2, 35.7,33.2, 28.2, 28.0, 27.4, 23.8, 23.3, 22.8, 22.6, 21.0, 19.0, 17.0, 12.2;MS (ESI): m/z 432.33/434.32 [M+] (Calcd for C27H45ClN2, 433.12/435.12).

2.1.2.3.5a-Cholestano-[5,7-c d]-pyrazoline (6).Mp: 208–210 �C; Anal.Calcd for C27H46N2: C, 81.34, H, 11.63, N, 7.03. Found: C, 81.52, H,11.47, N, 7.19; IR (KBr) m cm�1: 3410 (N–H), 1635 (C@N); 1HNMR (400 MHz, DMSO-d6): d 2.11 (1H, s, NH, exchangeable withD2O), 1.12 (3H, s, C10-CH3), 0.71 (3H, s, C13-CH3), 0.91 & 0.85(other methyl protons); 13C NMR (100 MHz, DMSO-d6): d 156.0,55.7, 54.6, 50.3, 49.7, 43.0, 39.7, 39.5, 39.4, 39.3, 38.1, 36.2, 35.1,

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Fig. 3. SEM micrographs of surface ultrastructural characteristics of untreated (a) and treated (b) HeLa cells with compound 4.

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33.3, 28.6, 28.0, 27.6, 27.4, 23.7, 22.8, 22.6, 22.2, 21.1, 20.6, 19.0,17.0, 12.2; MS (ESI): m/z 398.37 [M+] (Calcd for C27H46N2, 398.67).

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2.1.2.4. 20-Acetyl-3b-acetoxy-5a-cholestano-[5,7-c d]-pyrazoline (7).Mp: 225–227 �C; Anal. Calc. for C31H50N2O3: C, 74.65, H, 10.10, N,5.62. Found: C, 74.47, H, 10.26, N, 5.47; IR (KBr) m cm�1: 1662 (CH3-

C@O), 1733 (OCOCH3), 1623 (C@N), 1262 (C–O); 1H NMR (CDCl3,400 MHz): d 2.01(3H, s, CH3CO), 2.02 (3H, s, OCOCH3), 4.67 (1H,m, C3-aH, W1/2 = 17 Hz, axial), 1.13 (3H, s, C10-CH3), 0.71 (3H, s,C13-CH3), 0.92 & 0.85 (other methyl protons). 13C NMR (CDCl3,100 MHz): d 171.0, 165.1, 154.0, 73.0, 53.5, 49.5, 48.3, 42.1, 38.6,38.4, 37.4, 37.3, 37.2, 35.3, 35.1, 34.7, 27.5, 26.9, 26.5, 26.4, 23.1,22.8, 22.5, 21.8, 21.5, 20.4, 20.1, 19.8, 17.6, 16.7, 11.2; MS (ESI):m/z 498.38 [M+] (Calc. for C31H50N2O3, 498.75).

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2.1.2.5. 20-Acetyl-3b-chloro-5a-cholestano-[5,7-c d]-pyrazoline (8).Mp: 215–217 �C; Anal. Calc. for C29H47ClN2O: C, 73.31, H, 9.97, N,5.90. Found: C, 73.48, H, 9.79, N, 6.05; IR (KBr) m cm�1: 1670 (CH3-

C@O), 1630 (C@N); 1H NMR (CDCl3, 400 MHz): d 2.05 (3H, s, CH3-

CO), 3.49 (1H, m, C3-aH, W1/2 = 16 Hz, axial), 1.12 (3H, s, C10-CH3), 0.74 (3H, s, C13-CH3), 0.91 & 0.86 (other methyl protons).13C NMR (CDCl3, 100 MHz): d 165, 154.0, 54.8, 55.2, 51.7, 50.0,45.3, 44.3, 43.5, 40.0, 39.5, 38.9, 37.1, 36.3, 36.2, 35.7, 33.2, 28.6,28.0, 27.8, 23.8, 23.3, 22.8, 22.6, 21.5, 21.0, 19.0, 17.0, 12.3; MS(ESI): m/z 474.34/476.33 [M+] (Calc. for C29H47ClN2O, 475.15/477.15).

2.1.2.6. 20-acetyl-5a-cholestano-[5,7-c d]-pyrazoline (9).Mp: 222–224 �C; Anal. Calc. for C29H48N2O: C, 79.03, H, 10.98, N, 6.36.

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Table 1Antiproliferative activity (IC50 ± SD values) of steroids (1–18) in human tumor celllines and normal cell lines. Doxorubicin (Dox) and 5-Fluorouracil (5-Fu) are drugs ofreference.

IC50 (lM)

Compounds HeLa Jurkat PBMC

1 49.7 ± 3.5 45.1 ± 0.7 602 42.1 ± 0.5 44.5 ± 2.6 55.73 44.2 ± 3.2 46.3 ± 3.0 58.94 15.3 ± 0.3 24.4 ± 2.8 50.15 31.1 ± 1.2 34.5 ± 1.4 50.46 31.6 ± 3.6 42.8 ± 0.1 54.17 39.7 ± 3.2 12.8 ± 0.3 52.18 25.1 ± 0.6 23.9 ± 1.1 50.59 22.5 ± 2.1 10.6 ± 0.1 54.7

10 31.2 ± 2.2 25.2 ± 4.1 53.211 30.3 ± 2.1 32.6 ± 2.6 51.712 40.4 ± 3.4 31.9 ± 0.9 50.113 26.4 ± 0.5 14.8 ± 0.9 53.614 26.9 ± 2.6 23.5 ± 1.7 54.315 33.5 ± 1.7 35.1 ± 1.7 53.116 30.4 ± 2.6 19.4 ± 0.7 51.317 21.4 ± 1.3 34.7 ± 1.9 55.018 20.3 ± 0.5 14.8 ± 0.3 52.4Dox 4.1 ± 0.1 4.3 ± 0.4 –5-Fu 8.1 ± 0.3 9.0 ± 0.6 –

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Found: C, 79.19, H, 10.83, N, 6.54; IR (KBr) m cm�1: 1680 (C@O),1631 (C@N); 1H NMR (CDCl3, 400 MHz): d 2.02 (3H, s, CH3CO),1.10 (3H, s, C10-CH3), 0.81 (3H, s, C13-CH3), 0.92 & 0.85 (othermethyl protons). 13C NMR (CDCl3, 100 MHz): d 165.0, 153.0, 55.7,54.6, 50.3, 49.7, 43.0, 39.7, 39.5, 39.4, 39.3, 38.1, 36.2, 35.1, 33.3,28.6, 28.0, 27.6, 27.4, 23.8, 23.7, 22.8, 22.6, 22.2, 21.1, 20.6, 19.0,16.0, 12.2; MS (ESI): m/z 440.38 [M+]. (Calc. for C29H48N2O, 440.71).

2.1.2.7. 20-Formyl-3b-acetoxy-5a-cholestano-[5,7-c d]-pyrazoline(10). Mp: 260–262 �C; Anal. Calcd for C30H48N2O3: C, 74.34, H,9.98, N, 5.78. Found: C, 74.54, H, 9.80, N, 5.95; IR (KBr) m cm�1:1691(HC@O), 1738 (OCOCH3), 1636 (C@N), 1239 (C–O); 1H NMR(CDCl3, 400 MHz): d 6.4 (1H, s, CHO), 4.71 (1H, m, C3-aH, W1/

2 = 17 Hz, axial), 2.04 (3H, s, OCOCH3), 1.12 (3H, s, C10-CH3), 0.71(s, 3H, C13-CH3), 0.92 & 0.85 (other methyl protons). 13C NMR(CDCl3, 100 MHz): d 170.0, 159.0, 151.1, 72.0, 53.5, 49.5, 48.3,42.1, 38.6, 38.4, 37.4, 37.3, 37.2, 35.2, 35.1, 34.7, 27.5, 26.9, 26.5,26.4, 22.8, 22.5, 21.8, 21.5, 20.4, 20.1, 19.8, 17.9, 16.7, 11.2; MS(ESI): m/z 484.37 [M+] (Calc. for C30H48N2O3, 484.72).

2.1.2.8. 20-Formyl-3b-chloro-5a-cholestano-[5,7-c d]-pyrazoline (11).Mp: 198–200 �C; Anal. Calc. for C28H45ClN2O: C, 72.93, H, 9.84, N,6.08. Found: C, 72.75, H, 9.99, N, 5.93; IR (KBr) m cm�1: 1689(HC@O), 1630 (C@N), 725 (C–Cl); 1H NMR (CDCl3, 400 MHz): d6.5 (1H, s, CHO), 2.62 (1H, m, C3-aH, W1/2 = 15 Hz, axial), 1.01(3H, s, C10-CH3), 0.71 (s, 3H, C13-CH3), 0.91 & 0.85 (other methylprotons). 13C NMR (CDCl3, 100 MHz): d 161.1, 151.0, 54.8, 55.2,51.7, 49.1, 45.3, 44.3, 43.5, 40.0, 39.5, 38.9, 37.1, 36.3, 36.2, 35.7,33.2, 28.6, 28.0, 27.8, 23.8, 23.3, 22.8, 22.6, 21.0, 19.0, 17.0, 12.3;MS (ESI): m/z 460.32/462.32 [M+] (Calc. for C28H45ClN2O, 461.13/463.13).

2.1.2.9. 20-Formyl-5a-cholestano-[5,7-c d]-pyrazoline (12).Mp: 218–220 �C; Anal. Calc. for C28H46N2O: C, 78.82, H, 10.87, N, 6.57.Found: C, 78.64, H, 11.00, N, 6.41; IR (KBr) m cm�1: 1690 (HC@O),1631 (C@N); 1H NMR (CDCl3, 400 MHz): d 6.4 (1H, s, CHO), 1.12(3H, s, C10-CH3), 0.71 (3H, s, C13-CH3), 0.91 & 0.85 (other methylprotons). 13C NMR (CDCl3, 100 MHz): d 160.0, 156.1, 55.7, 54.6,50.3, 49.7, 43.0, 39.7, 39.5, 39.4, 39.3, 38.1, 36.2, 35.1, 33.3, 28.6,28.0, 27.6, 27.4, 23.7, 22.8, 22.6, 22.2, 21.1, 20.6, 19.0, 17.8, 12.2;MS (ESI): m/z 426.36 [M+] (Calc. for C28H46N2O, 426.68).

2.1.2.10. 20-Benzoyl-3b-acetoxy-5a-cholestano-[5,7-c d]-pyrazoline(13).Mp: 225–227 �C; Anal. Calc. for C36H52N2O3: C, 77.10, H, 9.35,N, 5.00. Found: C, 77.28, H, 9.18, N, 5.18; IR (KBr) m cm�1: 1690 (C6-

H5C@O), 1736 (OCOCH3), 1633 (C@N), 1244 (C–O), 3104 (C–H, aro-matic); 1H NMR (CDCl3, 400 MHz): d 4.69 (1H, m, C3-aH, W1/

2 = 18 Hz, axial), 7.4–7.9 (5H, m, aromatic), 2.05 (3H, s, OCOCH3),1.14 (3H, s, C10-CH3), 0.81 (3H, s, C13-CH3), 0.92 & 0.85 (othermethyl protons). 13C NMR (CDCl3, 100 MHz): d 170.0, 160.0,155.1, 120.0, 118.5, 112.3, 112.0, 110.2, 100.1, 72.0, 53.5, 49.5,48.3, 42.1, 38.6, 38.4, 37.4, 37.3, 37.2, 35.3, 35.1, 34.7, 27.5, 26.9,26.5, 26.4, 23.1, 22.8, 22.5, 21.8, 20.4, 20.1, 19.8, 17.6, 16.7, 11.2;MS (ESI): m/z 560.40 [M+] (Calc. for C36H52N2O3, 560.82).

2.1.2.11. 20-Benzoyl-3b-chloro-5a-cholestano-[5,7-c d]-pyrazoline(14).Mp: 230–232 �C; Anal. Calc. for C34H49ClN2O: C, 76.01, H, 9.19,N, 5.21. Found: C, 76.18, H, 9.03, N, 5.37; IR (KBr) m cm�1: 1686 (C6-

H5C@O), 1635 (C@N), 3120 (C–H, aromatic), 720 (C–Cl); 1H NMR(CDCl3, 400 MHz): d 3.43 (1H, m, C3-aH, W1/2 = 17 Hz, axial), 7.5–7.9 (5H, m, aromatic), 1.13 (3H, s, C10-CH3), 0.71 (3H, s, C13-CH3), 0.91 & 0.84 (other methyl protons). 13C NMR (CDCl3,100 MHz): d 160.0, 152.0, 130.0, 129.1, 125.9, 125.6, 123.4, 120.0,55.2, 54.8, 51.7, 50.0, 45.3, 44.3, 43.5, 40.0, 39.5, 38.9, 37.1, 36.3,36.2, 35.7, 33.2, 28.6, 28.0, 27.8, 23.8, 22.8, 22.6, 21.5, 21.0, 19.0,

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17.0, 12.3; MS (ESI): m/z 536.35/538.35 [M+] (Calc. for C34H49ClN2-

O, 537.22/539.22).

2.1.2.12. 20-Benzoyl-5a-cholestano-[5,7-c d]-pyrazoline (15).Mp:214–216 �C; Anal. Calc. for C34H50N2O: C, 81.22, H, 10.02, N, 5.57.Found: C, 81.06, H, 10.18, N, 5.42; IR (KBr) m cm�1: 1670 (C6H5-

C@O), 1637 (C@N), 3056 (C–H, aromatic); 1H NMR (CDCl3,400 MHz): 7.5–7.9 (5H, m, aromatic), 1.12 (3H, s, C10-CH3), 0.72(3H, s, C13-CH3), 0.92 & 0.84 (other methyl protons). 13C NMR(CDCl3, 100 MHz): d 166.0, 155.0, 123.0, 119.3, 116.5, 112.5,109.2, 100.0, 55.7, 54.6, 50.3, 49.7, 43.0, 39.7, 39.5, 39.4, 39.3,38.1, 36.2, 35.1, 33.3, 28.6, 28.0, 27.6, 27.4, 23.7, 22.8, 22.6, 22.2,21.1, 20.6, 19.0, 18.0, 12.2; MS (ESI): m/z 502.39 [M+] (Calc. forC34H50N2O, 502.78).

2.1.2.13. 20-Mercaptoacetyl-3b-acetoxy-5a-cholestano-[5,7-c d]-pyr-azoline (16).Mp: 190–192 �C; Anal. Calc. for C31H50N2O3S: C, 70.14,H, 9.49, N, 5.28. Found: C, 70.34, H, 9.31, N, 5.44; IR (KBr) m cm�1:2540 (SH), 1739 (OCOCH3), 1692 (CH2CON), 1640 (C@N), 1243 (C–O); 1H NMR (CDCl3, 400 MHz): d 6.30 (2H, s, CH2), 4.71 (1H, m, C3-aH, W1/2 = 17 Hz, axial), 2.04 (3H, s, OCOCH3), 1.51 (1H, s, SH), 1.14(3H, s, C10-CH3), 0.71 (3H, s, C13-CH3), 0.92 & 0.85 (other methylprotons). 13C NMR (CDCl3, 100 MHz): d 170.4, 162.2, 152.4, 72.5,54.6, 50.5, 49.3, 43.0, 39.7, 39.5, 38.7, 38.5, 38.4, 38.2, 37.3, 36.2,36.1, 35.7, 28.5, 28.0, 27.5, 27.4, 25.3, 23.9, 22.8, 22.5, 21.4, 20.9,18.9, 17.7, 12.2; MS (ESI): m/z 530.35 [M+]. (Calc. for C31H50N2O3S,530.82).

2.1.2.14. 20-Mercaptoacetyl-3b-chloro-5a-cholestano-[5,7-c d]-pyraz-oline (17).Mp: 148–150 �C; Anal. Calc. for C29H47ClN2OS: C, 68.67,H, 9.34, N, 5.52. Found: C, 68.84, H, 9.18, N, 5.69; IR (KBr) mcm�1: 2530 (SH),1686 (CH2CON), 1633 (C@N), 727 (C–Cl); 1HNMR (CDCl3, 400 MHz): d 6.32 (2H, s, CH2), 3.87 (1H, m, C3-aH,W1/2 = 16 Hz, axial), 1.62 (1H, s, SH), 1.12 (3H, s, C10-CH3), 0.71(3H, s, C13-CH3), 0.91 & 0.85 (other methyl protons). 13C NMR(CDCl3, 100 MHz): d 160.2, 153.1, 54.4, 50.7, 49.0, 47.2, 43.5,39.5, 39.4, 38.3, 38.2, 38.1, 38.0, 36.7, 36.5, 35.7, 28.1, 28.0, 27.4,27.2, 25.5, 23.7, 22.8, 22.6, 21.3, 20.2, 19.0, 17.6, 12.1; MS(ESI):m/z 506.31/508.31 [M+]. (Calc. for C29H47ClN2OS, 507.22/509.22).

2.1.2.15. 20-Mercaptoacetyl-5a-cholestano-[5,7-c d]-pyrazoline (18).Mp: 160–162 �C; Anal. Calc. for C29H48N2OS: C, 73.67, H, 10.23,

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eflu

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1 OAc - 10 OAc H

2 Cl - 11 Cl H

3 H - 12 H H

4 OAc - 13 OAc C6H5

5 Cl - 14 Cl C6H5

6 H - 15 H C6H5

7 OAc - 16 OAc SHCH2

8 Cl - 17 Cl SHCH2

9 H - 18 H SHCH2

Scheme 1. Synthesis of steroidal pyrazoline derivatives (4–18).

Table 2Effect of solvent in the synthesis of steroidal N-substituted pyrazolines 10–12.

Products Solvent

CH3OH CH2Cl2 (CH3)2CHOH CH2Cl2/CH3OH

Timea (h) Yieldb (%) Timea (h) Yieldb (%) Timea (h) Yieldb (%) Timea (h) Yieldb (%)

10 12 60 15 62 17 65 8 7511 15 68 14 70 18 70 11 8512 9 65 8 62 12 67 6 82

a Reaction progress monitored by TLC.b All yields refer to recrystallized products.

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N, 5.93. Found: C, 73.85, H, 10.07, N, 6.07; IR (KBr) m cm�1: 2532(SH), 1690 (CH2CON), 1631 (C@N); 1H NMR (CDCl3, 400 MHz): d6.27 (2H, s, CH2), 1.40 (1H, s, SH), 1.13 (3H, s,C10-CH3), 0.71 (3H,s, C13-CH3), 0.91 & 0.85 (other methyl protons). 13C NMR (CDCl3,

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100 MHz): d 160.2, 152.0, 54.3, 50.2, 49.0, 47.5, 43.3, 39.6, 39.5,38.4, 38.2, 38.1, 38.0, 36.4, 36.3, 35.3, 28.2, 28.1, 27.5, 27.1, 25.3,23.7, 22.4, 22.3, 21.1, 20.2, 19.0, 17.5, 12.6; MS (ESI): m/z 472.35[M+] (Calc. for C29H48N2OS, 472.78).

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2.2. Cytotoxicity assay

The cytotoxic potential of steroidal pyrazolines as well as start-ing compounds, against two cancer cell lines, viz. HeLa (cervical),Jurkat (leukemia) (obtained from NCCS Pune, Maharashtra) andnormal cells was assessed by determining the number of viablecells surviving after their incubation with drug for stipulated timeperiod using MTT (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method [24].The tumor cell lines (HeLa and Jurkat) and normal cells (PBMC)were maintained in RPMI 1640 culture medium supplementedwith 10% heat-inactivated fetal calf serum. The cells were platedat a density of 5 � 104 cells per well in a 96-well plate, and cul-tured for 24 h at 37 �C. Stock solutions of the synthesized steroidswere prepared in 1:1 mixture of DMSO and THF [25]. The cellswere subsequently exposed to drugs. The plates were incubatedfor 48 h, and cell proliferation was measured by adding 20 lL ofMTT dye (5 mg/mL in phosphate-buffered saline) per well. Theplates were incubated for a further 4 h at 37 �C in a humidifiedchamber containing 5% CO2. Formazan crystals formed due toreduction of dye by viable cells in each well were dissolved in150 lL dimethyl sulfoxide, and absorbance was read at 570 nm.The absorption values were expressed as the cell viability (%),according to the control group as 100%. Assays were performedin triplicate on three independent experiments. The concentrationrequired for 50% inhibition of cell viability (IC50) was calculatedusing the software ‘‘Prism 3.0’’.

2.2.1. Blood peripheral mononuclear cell isolationFresh blood (20–15 mL) was kindly provided by Blood bank

Jawahar Lal Nehru Medical College, AMU Aligarh. The blood samplewas diluted with the same volume of PBS. After that, the dilutedblood sample was carefully layered on Ficoll-Histopaque. The mix-ture was centrifuged under at 400g for 30 min at 20–22 �C. Theundisturbed lymphocyte layer was carefully transferred out. Thelymphocyte was washed and pelleted down with three volumesof PBS for twice and resuspended RPMI-1640 media with antibioticand antimycotic solution 10%, v/v fetal calf serum (FCS). Cell count-ing was performed to determine the PBMC cell number with equalvolume of trypan blue [26].

2.2.2. Fluorescence Microscopy/Scanning Electron MicroscopyThe HeLa cell line was maintained in RPMI 1640 culture med-

ium supplemented with 10% heat-inactivated fetal calf serum.The cells were plated at a density of 104 cells on glass cover slips,and cultured for 24 h at 37 �C. These were subsequently exposedwith compounds for 48 h. Cells were fixed by 2% paraformaldehydefor 2 h followed by washing with HBSS. The HeLa cells treated withcompounds 4, 9 and 18 were observed under Fluorescence Micro-scope (Fig. 2). The surface morphology of the treated (with com-pound 4) and untreated fixed HeLa cells are shown in Fig. 3.

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3. Results and discussion

3.1. Chemistry

The generation of molecules/molecular assemblies possessingwell defined biological functions remains an extremely challengingtask. In the view of significance of different nitrogen containingsteroidal heterocycles, a series of some new N-substituted steroi-dal pyrazoline derivatives have been rationally designed and syn-thesized and evaluated as potential anticancer agents. Thestarting materials 3b-acetoxycholest-5-en-7-one 1, 3b-chlorocho-lest-5-en-7-one 2 and cholest-5-en-7-one 3 were synthesized byknown literature method [27]. These were allowed to react with

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hydrazine hydrate and acetyl chloride/formic acid/benzoic acid/mercaptoacetic acid in dichloromethane/methanol (1:4) under re-flux conditions for the synthesis of new types of steroids attachedto nitrogen heterocycles 4–18 (Scheme 1). To determine theoptimal reaction conditions, nature of solvents were investigated(Table 2). Initially, the reaction was carried out in methanol (onlycompounds 10–12 were tested as model reaction) but, unfortu-nately, the expected products were not observed in good yield.Similar results were found with CH2Cl2. The use of isopropanol in-creased the chemical yield but, reduced the reaction rate. When weused dichloromethane/methanol (1:4) mixture as solvent, the rateas well as yield of products was improved. We applied this solventsystem for other compounds also. Interestingly, better chemicalyields and fast reaction rate were examined. Spectral data for allthe compounds (4–18) can be found in Supplementary information(Figs. S1–S30).

The structures of the target compounds, 4–18 were readilyidentified by their correct elemental analyses and compatible IR,1H NMR, 13C NMR and MS spectral data. All the analytical and spec-tral data of compounds 4–18 were in accordance with the pro-posed structure. The diagnostic signals at 3410–3423 cm�1 and1635–1641 appeared in the IR spectra of compounds 4–6 wereattributed to N–H and C@N stretching vibrations respectively.The 1H NMR spectra of compounds displayed singlet, at d = 2.0–2.17 ppm, due to N–H proton. The IR spectra of compounds 10–12 showed characteristic absorption peaks at 1689–1691 (HC@O)and 1630–1636 cm�1 (C@N). The 1H NMR spectra of compoundsrevealed the presence of singlet at d 6.4–6.5 which is characteristicfor the formyl proton. The signal at d 159–161 ppm in 13C NMRspectra further supported the presence of formyl group while peakat 150.9–156.7 was due to C@N function. Other compounds werecharacterized in the same way.

A conceivable mechanism for the synthesis of steroidal pyrazo-lines 4–18 is represented in Scheme 2 [28]. a, b-unsaturated ke-tones react with hydrazine hydrate to furnish the intermediate‘c’, which has been trapped by t-butyl carbazate (SupplementaryFig. S). This intermediate reacts with acid/acetyl chloride to endowthe corresponding products 4–18.

We confirmed the general pattern of reaction by reacting 3-methyl-2-cyclohexenone with NH2NH2�H2O,NH2NH2�H2O/CH3-

COCl,NH2NH2�H2O/HCOOH,NH2NH2�H2O/C6H5COOH and NH2NH2-

�H2O/SHCH2COOH (Supplementary Fig. Si–Svi).The obtainedproducts (compounds a–e) were also screened against cancer celllines (HeLa, Jurkat) and normal cell lines (PBMC) (SupplementaryTable S).

3.1.1. StereochemistryThe stereochemical assignation of C5–N bond has been estab-

lished on the basis of mechanism as well as on NMR spectral anal-ysis of the compounds. During the course of reaction, thenucleophilic attack of N of the reagent at C-5 does occur preferablyfrom less hindered (a) side because of the steric encumbrance im-posed by axial (b) methyl group at C-10, resulting axial (a) orienta-tion of C5–N bond and trans to C10-axial methyl group (A/B ringjunction trans) [23a]. In addition the ring fusion stereochemistryin angularly methylated six-membered ring compounds can bedetermined by NMR spectroscopy. The half band width (W1/2) val-ues of C3-axial proton in the 1H NMR spectra of the synthesizedcompounds clearly suggest that A/B ring junction is trans [29].13C NMR values of C-19 are strongly dependent on the ring fusionstereochemistry. The cis and trans steroids differ most significantlyat C-19 and this signal will surely characterize the nature of thering junction [30]. In the compounds 4–18, C-19 chemical shiftvalues were observed in the range of 16–18 ppm, which is consis-tent with the values obtained for trans steroids [31].

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+

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HN NH

H

X

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H

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OH

HN N H

-H2O

H2N

X

H

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HN N

Path a Path b

-H2O

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X

H

H

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N NC

O

RCH3/

RCOOH/ CH3COCl

Scheme 2. A provisional mechanism for the synthesis of steroidal pyrazoline derivatives (4–18).

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3.2. Anticancer activity and structure–activity relationship

To gain insight on how modifications on ring-B can affect cyto-toxicity, 15 steroidal pyrazoline derivatives with a variety of func-tionalities attached to N, namely, hydrogen, formyl, acetyl, benzoyland mercaptoacetyl, were synthesized and assayed in vitro forcytotoxicity in human cancer and normal cell lines, using theMTT assay. The cancer cells encompassed HeLa (cervical cancercells) and Jurkat (leukemia) while PBMCs were used as normalcells. A period of 48 h of drug exposure was chosen to test cytotox-icity. Doxorubicin (Dox) and 5-Fluorouracil (5-Fu) were used ascytotoxic drugs of reference. The cytotoxicity (IC50) of the synthe-sized steroidal pyrazoline derivatives along with starting steroid,against cancer lines (HeLa and Jurkat) as well as normal cells aredetailed in Table 1, whereas the curves of dose-dependent effects

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of most active compounds (4,7,9 and 18) are displayed in Fig. 1and remaining are given in Supplementary data. A number of cor-relations can be made from the data given in the Table 1. It is evi-dent from the IC50 values, that all the compounds showedmoderate to good activity while compounds 4, 7, 9, 13, 16 and18 elicited a marked inhibitory activity (IC50 < 19 lM) against boththe cell lines. All the compounds were found to be nontoxic to nor-mal cells (IC50 > 50 lM). Its noteworthy point that compound 4was found to be specific against HeLa while compounds 7, 9, 13,16 and 18 showed selectivity towards Jurkat cells. Substituent at3b-position as well as groups attached to N of the pyrazoline ringplayed a crucial role in determining activity. It is manifested fromthe data that acetoxy group at 3b-position impart greater activity(compound 4; HeLa, 10, 7 and 13; Jurkat) as compared to H andCl (compound 9 is unexpectedly more potent than 7). But presence

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of bulky substituent attached to pyrazoline ring nullified this effectand D5 derivatives were found to be more active (compounds 9and 18). Nature of the substituent over the pyrazoline ring alsoinfluenced the relative toxicity. This could be attributed to theirdifferences in either polarity which changes their lipophilicity orthe conformation which alters the target protein binding proper-ties present within the cell or on the cell membrane. HeLa cellstreated with compounds 4, 9 and 18 were observed under Fluores-cence Microscope (Fig. 2). These images clearly designated reduc-tion in cancer cell count, thus showing cytotoxicity of abovementioned steroids. Fig. 3 shows SEM micrograph of untreatedHeLa cells (a) and HeLa cells treated (b) with compound 4. The con-torted-looking state of the treated cells showed distinct morpho-logical changes corresponding to typical apoptosis, includingcellular blebbing and the formation of apoptotic bodies. [32]. Itcould be concluded that newly synthesized steroidal pyrazolineswere able to produce distinctive morphological features of celldeath that might correspond to apoptosis.

552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607608609610

4. Conclusion

In summary, we have demonstrated an efficient, expedient andconvenient approach for the synthesis of steroidal pyrazolinederivatives. Moreover, fast reaction rate, simple experimentationand better yields of the products are the advantages of this reac-tion. The investigated compounds 4, 7, 9, 13, 16 and 18 exertedinteresting antiproliferative behavior by showing low values ofinhibition count (IC50). In particular, compounds 9 (IC50 = 10.6 lM)and 7 (IC50 = 12.8 lM) showed a better cytotoxic profile among allthe tested compounds and were found to be potent inducer of celldeath in cultured Jurkat cell lines. Furthermore, all the compoundswere found to be non toxic to normal cell lines (PBMC).

Acknowledgements

We sincerely thank, the chairman, Department of Chemistry,Aligarh Muslim University, Aligarh for providing necessary re-search facilities. HK acknowledges UGC, New Delhi India for pro-viding BSR fellowship (R. No. Acad/D-742/MR). Authors alsothank the SAIF, Punjab University Chandigarh for providing spec-tral data.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.steroids.2013.09.006.

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