Synthesis, characterization, and cytotoxicity of N-2

Synthesis, characterization, and cytotoxicity of N-2′-hydroxyethylsubstituted azastigmastanes Mahboob Alam, Shahab A. A. Nami, Sumbul Rehman, Dong-Ung Lee & Soonheum Park Medicinal Chemistry Research ISSN 1054-2523 Med Chem Res DOI 10.1007/s00044-013-0795-x

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Author's personal copy MEDICINAL CHEMISTRY RESEARCH

Med Chem Res DOI 10.1007/s00044-013-0795-x

ORIGINAL RESEARCH

Synthesis, characterization, and cytotoxicity of N-20 -hydroxyethyl-substituted azastigmastanes Mahboob Alam • Shahab A. A. Nami • Sumbul Rehman • Dong-Ung Lee • Soonheum Park

Received: 30 March 2013 / Accepted: 12 September 2013 Ó Springer Science+Business Media New York 2013

Abstract The in vitro cytotoxicity of N-20 -hydroxyethylsubstituted azastigmastanes and its precursor steroidal ketones against cancer cell lines: MCF-7, HepG2, and HCT 116; has been carried out using MTT assay. The N-20 hydroxyethyl-substituted azastigmastanes were synthesized by bespoke version of Schmidt reaction using common Lewis acids like BF3–OEt2, SnCl4 and H2SO4 as catalysts in substantial yields. Sulphuric acid was found to be the most suitable catalyst in terms of reaction yield and time, while SnCl4 was found to be the weakest in case of 3b-acetoxy-N-20 -hydroxyethyl-6-aza-B-homo-5a-stigmastan-7-one and 3b-chloro-N-20 -hydroxyethyl-6-aza-B-homo5a-stigmastan-7-one and almost inactive in case of N-20 -hydroxyethyl-6-aza-B-homo-5a-stigmastan-7-one. The products were obtained in semi-solid state and characterized by spectroscopic techniques and microanalytical data. Moreover, on the basis of IC50, compound 5 was found to inhibit the cancer cells most effectively in conformity with our previous findings.

M. Alam D.-U. Lee (&) Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea e-mail: [email protected] S. A. A. Nami (&) Department of Kulliyat, Faculty of Unani Medicine, Aligarh Muslim University, Aligarh 202002, India e-mail: [email protected]; [email protected] S. Rehman Department of Ilmul Advia, Faculty of Unani Medicine, Aligarh Muslim University, Aligarh 202002, India S. Park Department of Chemistry, Dongguk University, Gyeongju 780-714, Republic of Korea

Keywords Cytotoxicity Steroidal ketone Stigmastane Lewis acids Schmidt reaction

Introduction The quest for the development of anticancer agents with minimal side effects and maximum efficacy has been an area of intense research for the past few decades with the discovery of cisplatin (Rosenberg et al., 1965). In this arena, steroids and their derivatives owe special significance because of their broad biological relevance (Norman et al., 2004). Plants derived steroids such as stigmasterol has a profound effect on the immune function and a supplemented diet with plant sterols have found to lower the serum IL-6 concentrations appreciably (de Jonga et al., 2003; Bouic, 2001). Recently, polyfunctionalized stigmastane derivatives have shown to possess in vitro antiviral activity against several pathogen viruses (Wachsman et al., 2000, 2002). Particularly, (22S,23S)-3b-bromo-5a,22,23-trihydroxy-stigmastan-6-one prevents Herpes simplex virus type 1 multiplication and viral spreading in a human conjunctival cell line, with no cytotoxicity (Michelini et al., 2008). Although substantial amount of work has been reported on the anticancer activity of stigmastane derivatives including their oxygenated side chain analogs (Misharin et al., 2008) but to the best of our search no such work has been done on the hydroxyethyl-substituted azastigmastane. Hence, in continuation of our earlier work on the cytotoxicity of azacholestanes (Nami et al., 2013) we herein report the cytotoxicity, synthesis, and characterization of novel hydroxyethyl-substituted azastigmastane starting from easily accessible steroidal ketones. Interestingly, based on IC50 values, it is inferred that the azastigmastane derivatives have better cytotoxicity than their corresponding starting

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Author's personal copy Med Chem Res

steroidal ketones. However, the cytotoxicity of 3b-chloro-N20 -hydroxyethyl-6-aza-B-homo-5a-stigmastan-7-one (5) is relatively high and comparable to the standard drug doxorubicin. Here, we have employed a modified version of Schmidt reaction that has been successfully employed by Aube et al. (Gracias et al., 1995) where hydroxyalkylazide is used in combination with BF3–OEt2, SnCl4 or H2SO4 as catalysts. In the present study, we report the cytotoxicity, synthesis, and spectral characterization of N-hydroxyalkyl azastigmastane from easily accessible 3b-acetoxy-5a-stigmastan-6-one (1) (Ahmad et al., 1978), 3b-chloro-5a-stigmastan-6-one (2) (Shoppee, 1948), and 5a-stigmastan-6-one (3) (Ahmad et al., 1978). On reaction with hydroxyalkylazide in the presence of different Lewis acids (BF3–OEt2, SnCl4) and protonic acid (H2SO4) it gives 3b-acetoxy-N-20 -hydroxyalkyl-6-aza-B-homo-5a-stigmastan-7-one (4) and its analogs 5 and 6, respectively.

Experimental Apparatus and reagents Melting points were determined on a Kofler apparatus and are uncorrected. IR spectra were recorded as neat with Pye Unicam SP-3-100-spectrophotometer and its values are given in cm-1. Elemental analyses (C, H, and N) were carried out with a Carlo Erba EA-1108 analyzer. 1H-NMR spectra were measured in CDCl3 on a Bruker AC 300 (300 MHz) with TMS as internal standard and its values are given in ppm (d) (s, singlet; br, broad and mc, multiplet centered at). 13C NMR spectra were recorded on a Bruker Avance II 400 spectrophotometer, with chemical shifts reported in parts per million relative to the residual deuterated solvent. Mass spectra were measured on VG micromass model ZAB-IF apparatus at 70 eV ionization voltage. Thin-layer chromatography (TLC) was performed on glass plates precoated with silica gel G and exposed to iodine vapors to monitor the reactions and to certify the purity of the reaction products. Silica gel (mesh size 60–120, BDH) was used for (*25 g for each gram of material) purification using gravity column chromatography. All the reactions were performed under the anhydrous conditions. Petroleum ether refers to a fraction of b.p. 60–80 °C. Sodium sulfate (anhydrous) was used as a drying agent for organic extracts after reaction workup. All the solvents were distilled prior to use. In vitro cytotoxicity The in vitro cytotoxicity of starting steroidal stigmastane (1–3) and their corresponding substituted azastigmastanes, 3b-acetoxy-N-20 -hydroxyethyl-6-aza-B-homo-5a-stigmastan-

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7-one (4), 3b-chloro-N-20 -hydroxyethyl-6-aza-B-homo-5astigmastan-7-one (5), and N-20 -hydroxyethyl-6-aza-B-homo5a-stigmastan-7-one (6) was performed by employing the human breast carcinoma cell line: MCF-7; human liver hepatocellular carcinoma cell line: HepG2; and human colon carcinoma cell line: HCT 116; using a standard 3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction assay (Cory et al., 1991). Cells in exponential growth were seeded into 96-well plates in a serum-free environment at concentration of 5 9 105 cells/200 ll/well and allowed to grow in specific medium containing 5 % FCS. After 24 h, cells were treated with various concentrations of test compound at a concentration range of 0–25 lM. Control (ethanol only) and positive control (doxorubicin) cells were cultured using the identical conditions. After 96 h of incubation, the medium was removed and replaced with fresh medium. MTT reagent (5 mg/ml in PBS) was added to each well at a volume of 1:10 and incubated for 2–3 h at 37 °C. After treatment, 100 ml of DMSO was added to each well after carefully aspirating the supernatants. Absorbance was measured at 620 nm in a multi-well plate reader. Triplicate wells were prepared for each individual concentration. Dose–response curves were plotted as percentages of the cell absorbances. Drug sensitivity was expressed in terms of the concentration of drug required for a 50 % reduction of cell viability (IC50). The IC50 value was defined as the concentration of test sample resulting in a 50 % reduction of absorbance as compared with untreated controls that received a serial dilution of the solvent in which the test samples were dissolved, and was determined by linear regression analysis. Care must be taken while handling H2SO4, BF3–OEt2, or SnCl4 as these are irritant and corrosive to skin. However, we have used smaller amounts and no such effect was observed. General procedure of synthesis of N-substituted azastigmastane (Hashmi, 2001; Smith et al., 2000; Wrobleski and Aube, 2001; Badiang and Aube, 1996; Gracias et al., 1997; Milligan et al., 1995; Aube and Milligan, 1991; Mossman and Aube, 1996; Desai et al., 2000; Hewlett et al., 2004; Schildknegt et al., 1998) Steroidal ketone 1 (0.45 mmol) in CH2Cl2 (6 ml) was treated with 2-azidoethanol (3 mmol) and cooled to 0 °C. H2SO4 (*0.25 ml), BF3–OEt2, or SnCl4 (*0.5 ml), respectively, was then added drop wise over a period of 5–6 min and the subsequent evolution of gas was recorded. The reaction was kept at 0 °C for 30 min and allowed to attain room temperature and then stirred for 6 h. The resulting solution was concentrated, and a


saturated solution of NaHCO3 was added to the residual oil. The reaction mixture was then stirred for another 1 h at room temperature. After concentration, 75 ml of CH2Cl2 was added and then the organic layer was separated, dried, and concentrated to afford oil 4, which was chromatographed over silica gel column. Elution with petroleum ether–acetone (7:3) afforded product 4 in a semi-solid state. However, several attempts were made to crystallize them in common organic solvents and their mixtures, but failed. 3b-Acetoxy-N-20 -hydroxyethyl-6-aza-B-homo-5astigmastan-7-one (4) [Found: C 74.39; H 10.96; N 2.55. C33H57NO4 calcd: C 74.53; H 10.80; N 2.63]. IR (KBr) mmax: 3,465 (OH), 1,720 (ester), 1,660 (amide) cm-1. 1H NMR (CDCl3) dH: 4.3 (m, 1H, C3–aH, W1/2 = 16 Hz), 3.5 (t, 2H, J = 5.4 Hz, CH2– N), 3.0 (t, 2H, J = 5.9 Hz, CH2–OH), 2.9 (br, 1H, C5–aH), 2.2 (d, 2H, J = 6.8 Hz, C7a–H), 2.1 (s, 3H, CH3COO), 2.0 (s, 1H, OH), 1.2 (CH3-19), 0.67 (CH3-18), 0.87 (d, 3H, CH3-26, J = 7.4 Hz), 0.83 (d, 3H, CH3-27, J = 6.8 Hz), 0.90 (t, 3H, CH3-29, J = 7.8 Hz) and 0.96 (d, 3H, CH3-21, J = 6.5 Hz). 13C NMR (CDCl3) dC: 173.1 (OCOCH3), 171.2 (N–C=O), 71.0 (C-3), 58.1 (C-14), 57.2 (C-17), 56.5 (C0 -1), 51.1 (C-9), 46.2 (C-24), 48.1 (C0 -2), 43.2 (C-13), 39.9 (C-5), 39.8 (C-12), 38.4 (C-8), 37.5 (C-1), 35.9 (C20), 35.8 (C-22), 36.2 (C-10), 37.1 (C-7a), 32.1 (C-4), 27.6 (C-16), 29.1 (C-25), 28.2 (C-2), 24.8 (C-23), 24.3 (C-15), 23.5 (C-28), 22.9 (C-11), 22.4 (C-27), 22.5 (C-21), 22.1 (OCOCH3), 19.8 (C-26), 19.8 (C-19), 11.9 (C-29) and 11.8 (C-18), EI-MS(m/z): 531 [M?], 390 [M–C10H21]?. Under similar reaction conditions steroidal ketones 2 and 3 gave compound 5 and 6, respectively, as semi-solid after elution with petroleum ether–acetone (8:2) in the silica column, respectively. 3b-Chloro-N-20 -hydroxyethyl-6-aza-B-homo-5astigmastan-7-one (5) [Found: C 73.01; H 10.85; N 2.66. C31H54NO2Cl calcd: C 73.26; H 10.71; N 2.75]. IR (KBr) mmax: 3,480 (OH), 1,688 (amide) cm-1. 1H NMR (CDCl3) dH: 4.4 (m, 1H, C3–aH, W1/2 = 18 Hz), 3.8 (t, 2H, J = 5.5 Hz, CH2–N), 3.7 (m, 2H, CH2–OH), 2.8 (m, 1H, C5–aH), 2.6 (d, 2H, J = 6.5 Hz, C7a–H), 2.2 (s, 1H, OH), 1.1 (CH3-19), 0.66 (CH3-18), 0.86 (d, 3H, CH3-26, J = 7.1 Hz), 0.85 (d, 3H, CH3-27, J = 6.6 Hz), 0.89 (t, 3H, CH3-29, J = 7.4 Hz) and 0.93 (d, 3H, CH3-21, J = 6.3 Hz). 13C NMR (CDCl3) dC: 169.5 (N–C=O), 60.2 (C-3), 54.1 (C0 -1), 49.8 (C-9), 47.6 (C0 -2) and other 13C NMR signals are in close proximity with compound 4. EI-MS(m/z): 507/509 [M?], 366/368 [M-C10H21]?.

N-20 -Hydroxyethyl-6-aza-B-homo-5a-stigmastan-7one (6) [Found: C 81.08; H 12.26; N 2.95. C31H55NO calcd: C 81.33; H 12.11; N 3.06]. IR (KBr) mmax: 3,496 (OH), 1,692 (amide) cm-1. 1H NMR (CDCl3) dH: 3.9 (t, 2H, J = 5.2 Hz, CH2–N), 3.4 (t, 2H, J = 5.1 Hz, CH2–OH), 2.9 (m, 1H, C5–aH), 2.7 (d, 2H, J = 6.6 Hz, C7a–H), 2.0 (s, 1H, OH), 1.3 (CH3-19), 0.69 (CH3-18), 0.87 (d, 3H, CH3-26, J = 7.2 Hz), 0.89 (d, 3H, CH3-27, J = 6.8 Hz), 0.92 (t, 3H, CH3-29, J = 7.6 Hz) and 0.95 (d, 3H, CH3-21, J = 6.4 Hz). 13C NMR (CDCl3) dC: 167.3 (N–C=O), 55.1 (C0 -1), 50.3 (C-9), 48.2 (C0 -2), 25.5 (C-3), and other 13C NMR signals are in close accordance with compound 4. EIMS(m/z): 473 [M?], 332 [M-C10H21]?.

Results and discussion The in vitro cytotoxicity of N-substituted azastigmastane and their precursors, steroidal ketones (1–3) against three cancer cell lines has been the subject of this paper. The substituted azastigmastanes (4, 5, and 6) were obtained in substantial yields by employing a modified Schmidt reaction starting from steroidal ketones. The easily accessible steroidal ketones (1, 2, and 3) were synthesized by the literature method (Ahmad et al., 1978; Shoppee, 1948). The experiments were carried out between ketones (1, 2, and 3) and 2-azidoethanol in dichloromethane. Thus, when 2-azidoethanol was added to a solution of steroidal ketone (1) in BF3–OEt2, SnCl4 or H2SO4, respectively, vigorous gas evolution was observed immediately. Then a saturated aqueous NaHCO3 solution was added in the reaction mixture followed by standard workup resulting in the formation of desired N-substituted azastigmastane (4) (Scheme 1). The yields of purified compounds (4, 5, and 6), starting from steroidal ketones (1, 2, and 3) are given in Table 1. Of the several Lewis acids and protonic acid, H2SO4 was found to be the most convenient in terms of availability, effectiveness, and its relative ease of workup. The structure of compound 4 was ascertained by IR, 1H NMR, 13C NMR and MS spectra, and microanalysis. Compound 4 gave diagnostic IR bands at 3,465 (OH), 1,720 (ester group) and 1,241 (CH3COO), 1,660 cm-1 (amide band) supporting the insertion of nitrogen atom into steroidal nucleus. The 1H-NMR spectrum is in conformity with the structure and exhibits diagnostic peak at d 4.3 (W1/2 = 16 Hz) ascribed to C3–aH as a multiplet, suggesting that junction A/B is trans. A peak observed at d 2.9 may be due to C5–aH, while a triplet observed at d 3.5 (J = 5.4 Hz) may be ascribed to CH2–N. Similarly a singlet at d 3.0 (J = 5.9 Hz) for two protons corresponding to

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Author's personal copy Med Chem Res Scheme 1 N-substituted azastigmastane

C10H21 C10H21

N3CH2CH2OH X

A,

_ 0 0C X

H

H

N

H

O

H

O

HO X AcO Cl H

(1) (2) (3)

Table 1 Yields of the reaction between protonic acid and lewis acids with 2-azidoethanol starting from ketone 1, 2 or 3 Compounds H2SO4 (% yield) BF3–OEt2 (% yield) SnCl4 (% yield) 4

82

61

14

5

77

58

10

6

73

54

Nil

CH2–OH, while the singlet at d 2.1 may be due to three hydrogen of acetate. Interestingly one singlet was observed at d 2.0 which gets vanished when exchanged with deuterium (the hydroxy proton resonance observed at d 2.0 was disappeared on the addition of D2O). However, the angular methyl protons (CH3-19), (CH3-18) were observed as singlet and side chain methyl protons (CH3-21), (CH3)2-26,27 and (CH3-29) as doublets at d 1.2, 0.67, 0.96, 0.87, 0.83, and 0.90 ppm, respectively. These spectral studies are in accordance with the formation of 4. The possibility of formation of 7-azastigmastane (7) can be ruled out on the basis of the 1H NMR spectrum. The position of C5–aH may be successfully employed to differentiate peak of 6-aza (4, 5, and 6) and 7-azastigmastane (7) (Scheme 2) as evident from a large number of reports. If C5–aH was observed at B3.0 ppm then there is a formation of 6-azastigmastane (4, 5 and 6) while any value greater than d 3.0 results in the formation of 7-azastigmastane (7). The C5–aH protons in 6-azastigmastane generally resonate at Bd 3.0 while those peaks in 7-azastigmastane commonly appear at Cd 3.0. We have observed C5–aH at d 2.9 ppm, which clearly indicates the regioselectivity expansion of B-ring of stigmastane and leads to the formation of 6-azastigmastane (4, 5 and 6). Generally, the regioselectivity found in the Schmidt reaction is rooted in the migratory aptitudes of the groups

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A BF3-OEt2 SnCl4 H2SO4

X AcO Cl H

(4) (5) (6)

attached to the carbonyl moiety. It is commonly observed that for acyclic or cyclic ketones, the more electrons donating substituent attached to the carbonyl will preferentially migrate. Moreover, in the 13C NMR spectrum, the peak appearing in the range of d 167–171 ppm corresponds to –N–C=O of the lactam implying the insertion of nitrogen atom of the azidoethanol reagent into steroid B-ring, while other characteristics of 13C NMR signals appeared at d 173.1 (CH3COO), 56.5 (C0 -1), 48.1 (C0 -2), and 37.1 ppm (C-7a), respectively. Hence, it has been characterized as 3b-acetoxy-N-20 -hydroxyalkyl-6-aza-B-homo-5a-stigmastan-7-one (4). Products 5 and 6 were also characterized based on similar frequencies of the respective functional groups. Moreover, the mass spectrum of compound (4) further establishes its formation and gave ion peaks for respective fragments. Molecular ion peak was observed at m/z 531 [M?] and other notable peaks include m/z 390 (M–C10H21). The compounds 1–6 were screened against three human cancer cell lines MCF-7, HepG2, and HCT116. The IC50 values for these compounds were compared to doxorubicin, a well-known anticancer drug. An enhancement in terms of cytotoxicity has been observed that the compounds 4–6 inhibit various cancer cell lines in a dosedependent manner. However, the IC50 of 3b-chloro-N-20 hydroxyethyl-6-aza-B-homo-5a-stigmastan-7-one (5) is found to be comparable with doxorubicin. Notably, all the compounds are found to be most effective against the human breast carcinoma cell line: MCF-7 as evident from Table 2. The antitumor efficacy of compound 5 may be attributed to its binding to cellular Fe pools. This inactivates ribonucleotide reductase, the enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides. A strong positive correlation was established between RR

Author's personal copy Med Chem Res Scheme 2 Migration pathway of a, b carbon

C10H21

X H

N H

O

C10H21 (a)

HO (4, 5 and 6) C10H21

X

a H

b

O

N

N2

(b)

X N H

H O

OH

(7)

Table 2 Cytotoxicity of compounds 4–6 against the cell lines Compounds

Cytotoxicity of cell lines (lM) MCF-7

HepG2

HCT116

1

3.54 ± 0.02

4.87 ± 0.02

5.02 ± 0.02

2

3.01 ± 0.02

3.56 ± 0.02

3.15 ± 0.02

3

3.34 ± 0.02

5.22 ± 0.02

4.51 ± 0.02

4

3.02 ± 0.02

4.52 ± 0.02

4.24 ± 0.02

5

2.55 ± 0.02

3.02 ± 0.02

2.72 ± 0.02

6

2.98 ± 0.02

4.68 ± 0.02

3.96 ± 0.02

Doxorubicin

2.44 ± 0.02

2.68 ± 0.02

2.56 ± 0.02

The highest concentration tested was 25 lM for compounds 1–6, and all the values are an average of three observations

activity and the rate of replication of tumor cells. The inhibition of RR prevents the production of de oxyribonucleotides. As a consequence these compounds interfere with DNA synthesis, thus decreasing the rate of replication of tumor cells and inhibiting tumor growth. The antitumor activity seems to be due to an inhibition of

DNA synthesis in cancer cells produced by modification in reductive conversion of ribonucleotides to deoxyribonucleotides (Babu et al., 2006).

Conclusion The present work explores the in vitro cytotoxicity of N-20 hydroxyethyl-substituted azastigmastanes against human breast carcinoma cell line: MCF-7, human liver hepatocellular carcinoma cell line: HepG2, and human colon carcinoma cell line: HCT 116, using MTT assay against doxorubicin as standard drug. The substituted azastigmastanes were synthesized by employing bespoke version of Schmidt reaction and are obtained as semi-solids. Spectroscopic techniques such as FT-IR, 1HNMR and mass spectra, and microanalytical data were used to characterize the final products. On the basis of IC50, 3b-chloro-N-20 hydroxyethyl-6-aza-B-homo-5a-stigmastan-7-one was found to inhibit the three different types of cancer cells most effectively.

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