Synthesis and antiproliferative evaluation of some novel B-nor-D-homosteroids

Steroids 98 (2015) 138–142

Contents lists available at ScienceDirect

Steroids journal homepage: www.elsevier.com/locate/steroids

Synthesis and antiproliferative evaluation of some novel B-nor-D-homosteroids Jianguo Cui, Qifu Lin, Chunfang Gan, Junyan Zhan, Wei Su, Dandan Zhao, Binbin Qi, Yanmin Huang ⇑ College of Chemistry and Materials Science, Guangxi Teachers Education University, Nanning 530001, China

a r t i c l e

i n f o

Article history: Received 31 October 2014 Received in revised form 17 February 2015 Accepted 12 March 2015 Available online 23 March 2015 Keywords: Dehydroepiandrosterone 3b-Hydroxy-5-androsten-17-one B-nor-D-homosteroids Antiproliferative activity

a b s t r a c t Using 3b-hydroxy-5-androsten-17-one as a starting material, a series of novel nitrogen-containing B-norD-homosteroids were designed and synthesized by the oximation, Beckman rearrangement, ozonation, cyclization and condensation reaction. The structures of all new compounds were determined by analysis of their NMR, MS and IR spectra. The antiproliferative activity of compounds was evaluated against HT-29 (colonic carcinoma), HeLa (human cervical carcinoma) and Bel 7404 (human liver carcinoma) cells. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Dehydroepiandrosterone (DHEA) is endogenous steroidal prohormone with important physiological activities, such as anti-aging and assimilation of protein. It is also a very important intermediate in the synthesis of steroid drugs. Previously, besides applying to hormonal drugs, several modified steroid compounds, such as D-homo-aza steroids that contain the NHCO group inside D-ring, were used in the synthesis of steroids ester carrying alkylator [1–4]. The result of the investigation of D-homo-aza-androsterone alkylator indicated that the presence of characteristic group, –NH–CO–, in the homo-aza steroid molecule is very important in reducing the acute toxicity and enhancing the anti-tumor activity for the steroid compounds [5–7]. B-norsteroids are also unordinary steroidal compounds possessing a novel skeleton of [6-5-6-5] fused rings. These compounds were discovered and constantly synthesized since 1998 [8–13]. The results of bioactivity assay had shown that these kinds of compounds displayed an excellent antiproliferative activity and good inhibitory activity against Mycobacterium tuberculosis. In our previous studies, we synthesized some novel A-homo [14–15], B-homo [16], C-homo [17], D-homosteroidal lactams [18] and B-norsteroids [19–20], and investigated their cytotoxic activity against different types of cancer cells. The results showed that some A-homo, B-homo steroidal lactams and B-norsteroids with a cholesteric side chain and D-homosteroidal lactams ⇑ Corresponding author. Tel.: +86 771 3908065; fax: +86 771 3908308. E-mail address: [email protected] (Y. Huang). http://dx.doi.org/10.1016/j.steroids.2015.03.012 0039-128X/Ó 2015 Elsevier Inc. All rights reserved.

possessing the 3-, 6-hydroxyl or hydroximino group displayed distinct antiproliferative activity against some cancer cells through inducing cancer cell apoptosis by activation of the intrinsic pathway [21]. Basing on the knowledge of literature and our previous work, in order to investigate the bioactivity of new steroidal derivatives as a new anticancer drug, some novel steroidal compounds possessing a B-nor structure at B ring and a D-homo lactam at D ring were designed and synthesized by combining the structure of B-norsteroid with D-homosteroid, and their antiproliferative activities were assayed against HT-29 (colonic carcinoma), HeLa (human cervical carcinoma) and Bel 7404 (human liver carcinoma) cells. 2. Results and discussion 2.1. Chemistry Scheme 1 outlines the synthetic procedures of compounds (5– 13). First, the structure of D-homo lactam at D ring was constructed. Compound 1 was transformed into the corresponding (17E)-hydroximino-5-androsten-3-ol acetate (2) by the condensation reaction with hydroxylamine hydrochloride. Beckman rearrangement of compound 2 in SOCl2/THF gave 3b-acetoxy-17a-aza-D-homo5-androsten-17-one (3) in 83% yield. The structure of 3 was confirmed by analysis of their NMR, MS and IR spectra [18]. Next, B-nor skeleton at B ring was built. The ozonolysis of compound 3 was performed in CH2Cl2 at 78 °C. After bubbling O2 to

139

J. Cui et al. / Steroids 98 (2015) 138–142 N OH

O

b

a AcO

AcO

2

1 H N

H N

O

O

d

c AcO

AcO

3

O

4

AcO

CHO

5 H N

OH

CHO

H N

O

e

O

f AcO

OH

6

HO

N H

NHCSNH 2 H N

AcO

g-l

H N

O

OH

R H

O

OH

7

N H

NHCSNH 2

8. R = NNHCSNHCH3-(E) 9. R = NNHCSNHCH2CH3-(E) 10. R = NNHCSNHPh-(E) 11. R = NOH-(E) 12. R = NOCH3-(E) 13. R = NOCH2Ph-(E)

Scheme 1. Reagents and conditions: a: NH2OHHCl/95%EtOH/NaOAc3H2O; b: SOCl2/THF; c: O3/CH2Cl2, Me2S; d: Al2O3/C6H6; e: H2NNHCSNH2/EtOH/HOAc; f: 13% K2CO3/ CH3OH; g: H2NNHCSNHCH3/EtOH/HOAc; h: H2NNHCSNHCH2CH3/EtOH/HOAc; i: H2NNHCSNHPh/EtOH/HOAc; j: NH2OHHCl/95%EtOH/ NaOAc3H2O; k: NH2OCH3HCl/ 95%EtOH/NaOAc3H2O; l: NH2OCH2PhHCl/95%EtOH/NaOAc3H2O.

expel the excess O3 and adding Me2S to decompose the produced ozonide, compound 4 was obtained. The structure of 4 was confirmed by analysis of the 13C NMR chemical shifts at C-5 and C-6. Resonances showing of C-5 at 215.6 ppm and C-6 at 201.5 ppm demonstrated that 5,6-double bond in 3 had been broken to yield 5- and 6-carbonyl groups. The chemical shift of C6-H at 9.62 ppm in 1 H NMR confirmed further the formation of 6-formyl group. B-nor skeleton of compound 5 bearing the 5b-OH and 6b-formyl was generated by the intramolecular aldol condensation of 4 with neutral alumina. The structure of 5 was established by analysis of the carbon NMR chemical shifts at C-5. The chemical shift of C-5 at 83.4 ppm for 5, and the vanishing of the chemical shift of C-5 at 215.6 ppm for 4, showed that 5-carbonyl of 4 had been converted to 5-hydroxyl of 5. The configurations of C-5 and C-6 in compound 5 had been described in Refs. [22–24]. Last, compound 6 and compounds 8–13 were obtained by the reaction of 5 with different nitrogen-containing agents. The structures of all compounds were confirmed by analysis of their NMR, MS and IR spectra. For example, in the 13C NMR spectra, the disappearing of the signal at 202.8 ppm (C-6) and appearing of the signals at 150.9 ppm (C-6) and 177.8 ppm (C@S) in 6 indicated that 6-formyl group in 5 had been converted to 6-thiosemicarbazone group. In 1H NMR spectrum of 6, the broad singlets at 7.54, 7.98 and 11.06 ppm showed further the presence of @NNHCSNH2. The HREI mass spectra exhibited molecular ion peak at m/z 451.2393 (M+H)+ for C22H35N4O4S. Furthermore, the deacetylation of 6 with alcoholic K2CO3 gave compound 7 in very good yield. The structure of 7 was confirmed by analysis of their NMR, MS and IR spectra. All of the synthesized compounds (6–13) were in the E-configuration, which was confirmed using 1H-NMR spectroscopy. The signal of the N-NHCS group in compounds 6–10 is in the range of 10–11 ppm, in comparison to Z-isomer, which possesses a characteristic NH signal in the range of 14–15 ppm [25–26], and the

downfield chemical shift of C6-H at 7.41, 7.35 and 7.44 ppm due to the influence of OH and OR in compounds 11–13 indicated the presence of E-configuration. 2.2. Biological results and discussion To evaluate the antiproliferative activity of the compounds, we determined their IC50 values against Bel-7404 (human liver carcinoma), HeLa (human cervical carcinoma) and HT-29 (colonic carcinoma) cancer cells using a MTT assay. MTT is a compound that can be taken up by viable cells and reduced by a mitochondrial dehydrogenase forming a formazan product in living cells. The absorbance of the formazan product at 492 nm is in linear proportion to cell numbers. The results were summarized as IC50 values in lmol/L in Table 1. As shown in Table 1, compound 5 with a structure of B-nor and D-homo lactam didn’t increase its cytotoxicity against HeLa cancer

Table 1 In vitro antiproliferative activities (IC50 in lmol/L) of some B-nor-D-homosteroids. Comp.

Bel-7404

HeLa

HT-29

3 4 5 6 7 8 9 10 11 12 13 Cisplatin

>100 >100 15.9 >100 >100 >100 >100 >100 >100 >100 83.0 23.2

37.6 95.5 >100 >100 71.1 64.7 >100 >100 >100 >100 >100 10.1

ND 34.5 >100 >100 >100 15.1 ND >100 >100 >100 16.6 ND

ND: not determined.

140

J. Cui et al. / Steroids 98 (2015) 138–142

cells comparing with its precursor 3 possessing a structure of D-homo lactam. However, the cytotoxicity of 5 obtained a remarkable enhancement against Bel-7404 cell. Here, changing B-ring into B-nor structure in steroidal nucleus did not increase the cytotoxic activities of these nitrogen-containing D-homosteroids. Compounds 6–13 were almost inactive against Bel-7404 and HeLa cells. Nonetheless, compound 8 with a 6-(40 -methyl)thiosemicarbazone and 13 with 6-O-benzyloxime group showed a selective antiproliferative activity against HT-29 cells with IC50 values of 15.1 and 16.6 lM, respectively. In conclusion, we have developed an efficient method for the preparation of B-nor-D-homesteroids by the oximation, Beckman rearrangement, ozonation, cyclization and condensation reaction using dehydroepiandrosterone as the starting material. Some novel nitrogen-containing B-nor-D-homo-steroids had been designed and synthesized, and their structures were characterized by analysis of NMR, MS and IR spectra. The cytotoxicity of the synthesized compounds against Bel-7404 (human liver carcinoma), HeLa (human cervical carcinoma) and HT-29 (colonic carcinoma) cancer cells were investigated. The results showed that compounds 8 and 13 displayed a selective antiproliferative activity to the HT-29 tumor cell lines. Our findings provide new evidences showing the relationship between the chemical structure and biological function. The information obtained from the studies is valuable for the design of novel steroidal chemotherapeutic drugs. 3. Experimental 3.1. Chemistry 3.1.1. Reagent and instrument The sterols were purchased from the Sinopharm Chemical Reagent Co., Ltd, Shanghai, China. All chemicals and solvents were analytical grade. Melting points were determined on an X4 apparatus (Beijing Tech Instrument Co. Ltd., Beijing, China) and were uncorrected. Infrared spectra were measured with a Nicolet FT360 Spectrophotometer (Thermo Scientific, America). The 1H and 13 C NMR spectra were recorded in CDCl3 on a Bruker AV-300 spectrometer at working frequencies 300 and 75 MHz, respectively. Chemical shifts are expressed in parts per million (d) values and coupling constants (J) in Hertz. HREIMS were measured on a Agilent 6210 TOFMS instrument. The cell proliferation assay was undertaken by a MTT method using 96-well plates on MLLTISKAN MK3 analysis spectrometer (Thermo Scientific, Shanghai, China). 3.1.2. 3b-Acetoxy-17-aza-D-homo-5-androsten-17-one (3)[18] The solution of thionyl chloride (8.0 mL) was added slowly to a solution of compound 2 (905 mg, 2.62 mmol) in dry THF (20 mL) under argon. The solution was stirred under anhydrous condition for 2 h at 0 °C. After completion of the reaction as indicated by TLC (Vpetroleum ether:Vethyl acetate = 1:2), some water was added to the solution. The solution was neutralized with ammonia and the product was extracted with CH2Cl2 (20  3 mL). The combined extract was washed with water, 5% NaHCO3, and saturated brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure to give a crude product which was chromatographed on silica gel (elution:ethyl acetate/petroleum ether = 1/1) to give faint yellow solid 748 mg. Yield: 83%, mp 165–167 °C. IR (KBr) m/cm1: 3179, 2954, 2823, 1740, 1675, 1622, 1434, 1389, 1238, 1033, 964, 903, 780, 604; 1H NMR (300 MHz, CDCl3) d: 1.01 (3H, s, 19CH3), 1.18 (3H, s, 18-CH3), 2.04 (3H, s, CH3CO–), 2.45 (1H, ddd, J = 9.0, 7.5, 1.8, C16-bH), 4.56–4.66 (1H, m, C3-bH), 5.39 (1H, d, J = 5.1, C6-H), 6.23 (1H, br s, –NH); 13C NMR (75 MHz, CDCl3) d: 171.8 (C-17), 170.5 (C-10 ), 139.5 (C-5), 121.8 (C-6), 73.6 (C-3), 54.2 (C-9), 49.4 (C-13), 47.9 (C-14), 39.6 (C-7), 37.8 (C-10), 36.8

(C-1), 36.7 (C-4), 32.2 (C-12), 31.2 (C-16), 30.8 (C-2), 27.6 (C-8), 22.0 (C-18), 21.4 (C-20 ), 20.9 (C-15), 20.0 (C-11), 19.2 (C-19). 3.1.3. 3b-Acetoxy-5,6,17-trioxo-17a-aza-D-homo-5,6-secoandrostane (4) A flow of 2 L/min of O3 in O2 through a sintered glass dispersion tube was bubbled into a solution of 3 (464 mg, 1.34 mmol) in a mixture of CH2Cl2 (50 mL) and MeOH (3 mL) at 78 °C for 30 min until the solution turned pale blue (the progress of the reaction was monitored by TLC). The mixture was purged with O2 for 1.5 h, and then Me2S (3 mL) was added. The mixture was allowed to warm to room temperature and was stirred overnight. After removing solvent and dimethylsulfide under reduced pressure, appropriate water was added to the mixture and extracted by CH2Cl2 (3  15 mL), and the organic layer was washed with water and saturated brine. After drying over anhydrous sodium sulfate, solvent was removed under reduced pressure, and the resulting crude product was purified by chromatography on silica gel using petroleum ether (60–90 °C)/EtOAc (1:3) as eluent to give 346 mg (68%) of 4 as a colorless oil; IR (KBr) m/cm1: 3428, 2942, 2864, 1728, 1634, 1438, 1373, 1246, 1164, 1061, 952; 1H NMR (300 MHz, CDCl3) d: 0.99 (s, 3H, 19-CH3), 1.16 (s, 3H, 18-CH3), 2.01 (s, 3H, CH3CO–), 2.59 (dd, 1H, J = 14.7, 4.8, C4-H), 3.01 (dd, 1H, J = 14.7, 4.5, C4-H), 5.32–5.39 (m, 1H, C3-aH), 6.54 (br s, 1H, –NH), 9.62 (s, 1H, C6-H); 13C NMR (75 MHz, CDCl3) d: 215.6 (C5), 201.5 (C-6), 171.5 (C-17), 170.1 (C-10 ), 73.2 (C-3), 54.1 (C-14), 52.4 (C-10), 44.9 (C-13), 43.2 (C-7), 42.3 (C-9), 41.6 (C-4), 39.8 (C-12), 35.2 (C-16), 34.3 (C-8), 30.5 (C-1), 25.0 (C-18), 22.9 (C-2), 21.6 (C-15), 21.2 (C-11), 21.1 (C-20 ), 17.4 (C-19); HREIMS: m/z 378.2257 [M+H]+ (calcd for C21H32NO5, 378.2281). 3.1.4. 3b-Acetoxy-5b-hydroxy-6b-formyl-17a-aza-B-nor-D-homoandrost-17-one (5) Neutral Al2O3 (3.46 g, unactivated) was added to the solution of 4 (346 mg, 0.92 mmol) in benzene (30 mL). The solution was stirred at room temperature for 12 h, and the suspension was poured over a silica gel column and eluted with CH2Cl2 until TLC showed no product remained on Al2O3. After evaporation of the solvent, the residue was purified by flash column chromatography (CH2Cl2/MeOH = 30:1) to afford 242 mg of 5 as pale yellow solid, Yield: 70%. mp 112–114 °C; IR (KBr) m/cm1: 3403, 2954, 2864, 1712, 1630, 1458, 1401, 1258, 1058, 666; 1H NMR (300 MHz, CDCl3) d: 0.90 (s, 3H, 19-CH3), 1.25 (s, 3H, 18-CH3), 2.08 (s, 3H, CH3CO–), 2.33–2.41 (m, 3H, C16-H and C7-H), 5.10–5.19 (m, 1H, C3-aH), 6.53 (br s, 1H, –NH), 9.74 (d, 1H, J = 2.4, C6-H); 13C NMR (75 MHz, CDCl3) d: 202.8 (C-6), 171.9 (C-17), 169.6 (C-10 ), 83.4 (C-5), 70.2 (C-3), 62.7 (C-7), 55.5 (C-13), 49.5 (C-14), 47.8 (C-8), 45.7 (C-9), 42.5 (C-10), 40.1 (C-4), 38.9 (C-12), 30.4 (C-16), 26.6 (C-1), 24.4 (C-2), 22.7 (C-18), 21.6 (C-15), 21.4 (C-11), 21.4 (C-20 ), 18.3 (C-19); HREIMS: m/z 378.2277 [M+H]+ (calcd for C21H32NO5, 378.2281). 3.1.5. General procedure for the synthesis of compounds (6, 8–10) The compound 5 (1.04 mmol) was dissolved in 30 mL of CH3CH2OH. After the mixture was heated to 60 °C, the solution was adjusted to pH  3–5 by adding a few drops of glacial acetic acid, and thiosemicarbazide or 4-substituted thiosemicarbazide (1.24 mmol) was added. The mixture was stirred for 1 h at 60 °C until no starting material (the progress of the reaction was monitored by TLC). Then the reaction was terminated by adding a little water and the majority of solvent was evaporated under reduced pressure. After appropriate water was added to the residue, the solution was extracted by CH2Cl2 (3  15 mL) and the organic layer was washed with water, saturated NaHCO3 solution and saturated brine. After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the crude product was

J. Cui et al. / Steroids 98 (2015) 138–142

purified by chromatography on silica gel using CH2Cl2/MeOH (30:1) as eluent to give the corresponding target products. 3.1.5.1. 3b-Acetoxy-5b-hydroxy-6b-thiosemicarbazone-17a-aza-Bnor-D-homo-androst-17-one (6). White solid, Yield: 63%, mp 233– 234 °C; IR(KBr) v/cm1: 3424, 2925, 2852, 1724, 1626, 1524, 1450, 1373, 1250, 1172, 1017; 1H NMR (300 MHz, DMSO) d: 0.82 (s, 3H, 19-CH3), 1.08 (s, 3H, 18-CH3), 1.97 (s, 3H, CH3CO–), 2.10– 2.17 (m, 2H, C16-H) 2.52–2.50 (m, 1H, C7-H and C8-H), 4.27 (br s, 1H, –OH), 4.92–4.81 (m, 1H, C3-aH), 7.31 (d, 1H, J = 7.2, C6-H), 7.36 (br s, 1H, –CONH–), 7.54 (br s, 1H, –NH2), 7.98 (br s, 1H, –NH2), 11.06 (s, 1H, –NH–); 13C NMR (75 MHz, DMSO) d: 177.8 (C@S), 170.4 (C-17), 170.3 (C-10 ), 150.9 (C-6), 82.2 (C-5), 69.3 (C-3), 55.1 (C-13), 54.9 (C-14), 51.2 (C-7), 47.4 (C-9), 44.5 (C-8), 42.9 (C-10), 39.3 (C-12), 31.0 (C-4), 30.8 (C-16), 25.0 (C-1), 22.6 (C-18), 22.0 (C-2), 21.7 (C-15), 21.7 (C-11), 21.6 (C-20 ), 18.9 (C-19); HREIMS: m/z 451.2393 [M+H]+ (calcd for C22H35N4O4S, 451.2379). 3.1.5.2. 3b-Acetoxy-5b-hydroxy-6b-(40 -methyl)thiosemicarbazone17a-aza-B-nor-D-homo-androst-17-one (8). White solid, Yield: 65%, mp 195–197 °C; IR (KBr) m/cm1: 3387, 2925, 2852, 1708, 1622, 1548, 1450, 1389, 1266, 1185, 1127, 1066, 1021, 952; 1H NMR (300 MHz, CDCl3) d: 0.92 (s, 3H, 19-CH3), 1.18 (s, 3H, 18CH3), 2.04 (s, 3H, CH3CO–), 2.37–2.22 (m, 4H, C4-H, C16-H), 2.59 (br s, 1H, C7-H), 3.17 (d, 3H, J = 4.8, –NCH3), 5.12–5.01 (m, 1H, C3-aH), 7.13 (br s, 1H, –NHCO), 7.22 (d, 1H, J = 7.5, C6-H), 7.40 (q, 1H, J = 4.8, –NHCH3), 10.14 (d, 1H, J = 6.9, @NNHCSA); 13C NMR (75 MHz, CDCl3) d: 178.1 (C@S), 172.1 (C-17), 170.0 (C-10 ), 148.6 (C-6), 82.8 (C-5), 69.9 (C-3), 55.5 (C-14), 55.1 (C-13), 51.7 (C-7), 47.9 (C-9), 44.9 (C-8), 42.9 (C-10), 40.7 (C-12), 40.0 (C-4), 31.0 (C-16), 30.4(–NHCH3), 29.7 (C-1), 29.5 (C-18), 24.7 (C-2), 22.6 (C-15), 21.8 (C-11), 21.4 (C-20 ), 18.3 (C-19); HREIMS: m/z 465.2559 [M+H]+ (calcd for C23H37N4O4S, 465.2536). 3.1.5.3. 3b-Acetoxy-5b-hydroxy-6b-(40 -ethyl)thiosemicarbazone-17aaza-B-nor-D-homo-androst-17-one (9). White solid, Yield: 67%, mp 243–245 °C; IR (KBr) m/cm1: 3367, 2929, 2852, 1724, 1642, 1532, 1454, 1377, 1315, 1242, 1074, 1021, 948, 805, 727; 1H NMR (300 MHz, CDCl3) d: 0.90 (s, 3H, 19-CH3), 1.18 (s, 3H, 18-CH3), 1.24 (t, 3H, J = 7.5, –NCH2–), 2.04 (s, 3H, CH3CO–), 2.35–2.23 (m, 3H, C4-H and C16-H), 2.62 (br s, 1H, C7-H), 3.71–3.62 (m, 2H, –NCH2–), 5.11–5.01 (m, 1H, C3-aH), 7.20 (br s, 1H, –NHCO), 7.25 (d, 1H, J = 6.9, C6-H), 7.31 (t, 1H, J = 5.4, –NHCH2–), 10.18 (br s, 1H, @NNHCSA); 13C NMR (75 MHz, CDCl3) d: 177.0 (C@S), 172.1 (C-17), 170.0 (C-10 ), 148.6 (C-6), 82.7 (C-5), 69.9 (C-3), 55.5 (C-14), 54.8 (C-13), 51.4 (C-7), 47.9 (C-9), 44.9 (C-8), 42.7 (C-10), 40.8 (–NCH2–), 39.9 (C-12), 39.2 (C-4), 30.4 (C-16), 29.3 (C-1), 24.7 (C-2), 22.6 (C-18), 21.8 (C-15), 21.4 (C-11), 21.4 (C-20 ), 18.3 (C-19), 14.5 (–NCH2CH3); HREIMS: m/z 479.2691 [M+H]+ (calcd for C24H39N4O4S, 479.2692). 3.1.5.4. 3b-Acetoxy-5b-hydroxy-6b-(40 -phenyl)thiosemicarbazone17a-aza-B-nor-D-homo-androst-17-one (10). White solid, Yield: 64%, mp 199–200 °C; IR (KBr) m/cm1: 3309, 2929, 2860, 1736, 1646, 1589, 1536, 1450, 1377, 1258, 1193, 1058, 1025, 948, 756, 694; 1H NMR (300 MHz, CDCl3) d: 0.92 (s, 3H, 19-CH3), 1.18 (s, 3H, 18-CH3), 2.04 (s, 3H, CH3CO–), 2.40–2.30 (m, 3H, C4-H and C16-H), 2.70 (br s, 1H, C7-H), 5.16–5.03 (m, 1H, C3-aH), 7.20 (t, 1H, J = 6.9, p-Ph-H), 7.32 (d, 1H, J = 4.2, C6-H), 7.41–7.35 (m, 2H, m-Ph-H), 7.62 (d, 2H, J = 7.5, o-Ph-H), 9.13 (s, 1H, –NH-Ph), 10.74 (s, 1H, @NNHCS–); 13C NMR (75 MHz, CDCl3) d:175.6 (C@S), 172.2 (C-17), 170.0 (C-10 ), 149.8 (C-6), [138.0, 128.6, 128.6, 125.8, 124.2, 124.2] (Ph-C), 82.8 (C-5), 69.9 (C-3), 55.5 (C-14), 55.0 (C-13), 51.2 (C-7), 47.9 (C-9), 45.0 (C-8), 42.7 (C-10), 41.0 (C-12),

141

39.9 (C-4), 30.5 (C-16), 29.7 (C-1), 28.9 (C-18), 24.7 (C-2), 22.6 (C-15), 21.8 (C-11), 21.5 (C-20 ), 18.4 (C-19); HREIMS: m/z 527.2692 [M+H]+ (calcd for C28H39N4O4S, 527.2692). 3.1.6. 3b,5b-Dihydroxy-6b-thiosemicarbazone-17a-aza-B-nor-Dhomo-androst-17-one (7) After compound 6 (123 mg, 0.26 mmol) was dissolved in 25 mL of CH3OH, 4 mL of 13% K2CO3 solution was added. The reaction mixture was stirred at room temperature for 1 h until no starting material was observed (controlled by TLC). The majority of solvent was evaporated under reduced pressure. Appropriate water was added to the mixture and extracted by CH2Cl2 (3  15 mL), and the organic layer was washed with water and saturated brine. After drying over anhydrous sodium sulfate, solvent was removed under reduced pressure, and the resulting crude product was purified by chromatography on silica gel using CH2Cl2/MeOH (30:1) as eluent to give 75 mg (63%) of 7 as white solid. mp 221–222 °C; IR(KBr) v/cm1: 3420, 2942, 2848, 1626, 1540, 1454, 1385, 1262, 1005; 1H NMR (300 MHz, DMSO) d: 0.79 (s, 3H, 19-CH3), 1.07 (s, 3H, 18-CH3), 2.19–2.08 (m, 3H, C4-H and C16-H), 2.55–2.47 (m, 2H, C7-H and C8-H), 3.93–3.82 (m, 1H, C3-aH), 4.15 (br s, 1H, –OH), 4.88 (s, 1H, –OH), 7.33 (br s, 1H, –CONH–), 7.35 (d, 1H, J = 7.2, C6-H), 7.54 (s, 1H, –NH2), 7.96 (s, 1H, –NH2), 11.01 (s, 1H, –NH–); 13C NMR (75 MHz, DMSO) d: 177.8 (C@S), 170.4 (C-17), 151.0 (C-6), 83.1 (C-5), 65.5 (C-3), 55.2 (C-13), 54.5 (C-14), 50.4 (C-7), 47.7 (C-9), 44.9 (C-8), 43.4 (C-10), 42.8(C-4), 39.3 (C-12), 31.0 (C-16), 29.1 (C-1), 28.2 (C-2), 22.7 (C-18), 22.1 (C-15), 21.5 (C-11), 19.3 (C-19); HREIMS: m/z 409.2273 [M+H]+ (calcd for C20H33N4O3S, 409.2273). 3.1.7. General procedure for the synthesis of compounds (11–13) The compound 5 (0.22 mmol) was dissolved in 20 mL of 95% CH3CH2OH. After the mixture was heated to 60 °C, CH3COONa3H2O (0.24 mmol) and NH2OHHCl or alkoxyammonium chloride (0.24 mmol) were added. The mixture was stirred for 1 h at 60 °C. Then the reaction was terminated by adding a little water and the majority of solvent was evaporated under reduced pressure. Appropriate water was added into the reaction mixture, and the product was extracted with CH2Cl2. The combined extracts were washed with water and saturated brine, dried with anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using CH2Cl2/MeOH (40:1) as eluent to afford the target products 11–13. 3.1.7.1. 3b-Acetoxy-5b-hydroxy-6b-hydroximino-17a-aza-B-nor-Dhomoandrost-17-one (11). White solid, Yield: 73%, mp 241– 243 °C; IR (KBr) m/cm1: 3424, 2925, 2856, 1732, 1630, 1516, 1446, 1385, 1242, 1185, 1029, 935; 1H NMR (300 MHz, CDCl3) d: 0.94 (s, 3H, 19-CH3), 1.18 (s, 3H, 18-CH3), 2.06 (s, 3H, CH3CO), 2.44–2.28 (m, 3H, C7-H and C16-H), 5.16–5.06 (m, 1H, C3-aH), 6.80 (br s, 1H, –NHCO), 7.41 (d, 1H, J = 8.4, C6-H), 9.63 (br s, 1H, @NOH); 13C NMR (75 MHz, CDCl3) d: 172.9 (C-17), 169.9 (C-10 ), 153.1 (C-6), 81.7 (C-5), 70.1 (C-3), 55.5 (C-13), 53.3 (C-14), 51.4 (C-7), 48.1 (C-9), 44.9 (C-8), 42.9 (C-10), 41.4 (C-12), 39.9 (C-4), 30.3 (C-16), 29.7 (C-1), 28.6 (C-2), 24.7 (C-18), 22.5 (C-15), 21.6 (C-11), 21.4 (C-20 ), 18.2 (C-19); HREIMS: m/z 393.2390 [M+H]+ (calcd for C21H33N2O5, 393.2390). 3.1.7.2. 3b-Acetoxy-5b-hydroxy-6b-O-methyloxime-17a-aza-B-norD-homoandrost-17-one (12). White solid, Yield: 72%, mp 113–115 °C; IR (KBr) m/cm1: 3436, 2938, 2852, 1708, 1650, 1454, 1385, 1279, 1258, 1046, 731; 1H NMR (300 MHz, CDCl3) d: 0.93 (s, 3H, 19-CH3), 1.19 (s, 3H, 18-CH3), 2.05 (s, 3H, CH3CO–), 2.46–2.27 (m, 4H, C4-H and C16-H), 2.63 (br s, 1H, C7-H), 3.83 (s, 3H, –OCH3), 5.11 (br s, 1H, C3-aH), 6.70 (br s, 1H, –NHCO), 7.35

142

J. Cui et al. / Steroids 98 (2015) 138–142

(d, 1H, J = 8.1, C6-H); 13C NMR (75 MHz, CDCl3) d: 172.2 (C-17), 169.7 (C-10 ), 152.6 (C-6), 81.8 (C-5), 70.1 (C-3), 61.4 (–OCH3), 55.5 (C-13), 52.7 (C-14), 50.9 (C-7), 48.2 (C-9), 45.0 (C-8), 42.9 (C-10), 41.5 (C-12), 40.0 (C-4), 30.5 (C-16), 28.0 (C-1), 24.6 (C-2), 22.7 (C-18), 21.6 (C-15), 21.4 (C-11), 20.9 (C-20 ), 18.4 (C-19); HREIMS: m/z 407.2545 [M+H]+ (calcd for C22H35N2O5, 407.2546).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.steroids.2015.03. 012. References

3.1.7.3. 3b-Acetoxy-5b-hydroxy-6b-O-benzyloxime-17a-aza-B-nor-Dhomoandrost-17-one (13). White solid, Yield: 61%, mp 180–181 °C; IR (KBr) m/cm1: 3489, 3346, 2929, 2860, 1699, 1642, 1454, 1373, 1274, 1176, 1062, 1017, 927, 727; 1H NMR (300 MHz, CDCl3) d: 0.91 (s, 3H, 19-CH3), 1.13 (s, 3H, 18-CH3), 2.06 (s, 3H, CH3CO–), 2.30–2.20 (m, 3H, C4-H and C16-H), 2.37 (br s, 1H, C7-H), 5.06 (s, 2H, –OCH2Ph), 5.07–5.12 (m, 1H, C3-aH), 6.50 (br s, 1H, –NHCO), 7.36–7.27 (m, 5H, Ph-H), 7.44 (d, 1H, J = 8.1, C6-H); 13C NMR (75 MHz, CDCl3) d: 172.1 (C-17), 169.7 (C-10 ), 153.3 (C-6), [138.0, 128.3, 128.3, 128.2, 128.2, 127.8] (Ph-C), 81.7 (C-5), 75.5 (–OCH2), 70.1(C-3), 55.4 (C-13), 52.7 (C-14), 50.9 (C-7), 48.1 (C-9), 45.0 (C-8), 42.7 (C-10), 41.5 (C-12), 40.1 (C-4), 30.5 (C-16), 28.1 (C-1), 24.6 (C-2), 22.6 (C-18), 21.6 (C-15), 21.4 (C-20 ), 20.7 (C-11), 18.3 (C-19); HREIMS: m/z 483.2859 [M+H]+ (calcd for C28H39N2O5, 483.2859).

3.2. Antiproliferative activity 3.2.1. Materials Stock solutions of the compounds were prepared in sterile dimethyl sulfoxide (DMSO) (Sigma) at a concentration of 10 mg/ mL and afterward diluted with complete nutrient medium (RPMI-1640) supplemented with 10% heat inactivated fetal bovine serum and 0.1 g/L penicillin G + 0.1 g/L streptomycin sulfate.

3.2.2. Cell culture Bel 7404, HeLa and HT-29 cancer cells were grown in the medium (RPMI-1640) supplemented with 10% heat inactivated fetal bovine serum and 0.1 g/L penicillin G + 0.1 g/L streptomycin sulfate in a humidified atmosphere of 5% CO2 at 37 °C.

3.2.3. Assay for cell viability The cell proliferation assay was undertaken by a MTT method using 96-well plates. Using cisplatin as a positive control, the antiproliferative activity of the compounds was determined. Briefly, cells (3  104 cells per well) were seeded in 96-wells plates. One day after seeding, cells in the wells were respectively treated with target compounds at various concentrations. An equal amount of DMSO was added to the cells used as negative controls. All were treated in triplicate. After reincubated for 72 h, 20 lL of the tetrazolium dye (MTT) (5 mg/mL) solution were added to each well, and the cells were incubated for an additional 4 h. After the supernatant was discarded, 200 lL of DMSO were added to dissolve the purple formazan crystals formed. The absorbance values (A) at 492 nm were determined using a MLLTISKAN MK3 analysis spectrometer (Thermo Scientific Co.). The IC50 values were calculated as the concentration of drug yielding 50% cell survival.

Acknowledgments The authors acknowledge the financial support of the National Natural Science Foundation of China, China (No. 21462009) and the Natural Science Foundation of Guangxi Province, China (No. 2014GXNSFAA118052).

[1] Dimitrios TPT, George DG, Catherine K, Athanasios P, Panaviotis K, Charalambos C. Lactandrate: a D-homo-aza-androsterone alkylator in the treatment of breast cancer. Breast Cancer Res Treat 2006;97:17–31. [2] Anna IK, Manolis AF, Evagelia SA, Athanasios P, George NP, Sotiris SN. Rational design, synthesis, and in vivo evaluation of the antileukemic activity of six new alkylating steroidal esters. Bioorg Med Chem 2008;16:5207–15. [3] Dimitrios TPT. Hybrid aza-steroid alkylators in the treatment of colon cancer. Cancer Lett 2006;243:202–10. [4] Karapidaki I, Bakopoulou A, Papageorgiou A, Iakovidou Z, Mioglou E, Nikolaropoulos S, et al. Genotoxic, cytostatic, antineoplastic and apoptotic effects of newly synthesized antitumour steroidal esters. Mutat Res 2009;675:51–9. [5] Dhingraa N, Bhardwaj TR, Mehta N, Mukhopadhyay T, Kumar A, Kumar M. Synthesis, antiproliferative, acute toxicity and assessment of antiandrogenic activities of some newly synthesized steroidal lactams. Eur J Med Chem 2010;45:2229–36. [6] Woo LWL, Fischer DS, Sharland CM, Trusselle M, Foster PA, Chander SK, et al. Anticancer steroid sulfatase inhibitors: synthesis of a potent fluorinated second-generation agent, in vitro and in vivo activities, molecular modeling, and protein crystallography. Mol Cancer Ther 2008;7:2435–44. [7] Aggarwal S, Thareja S, Bhardwaj TR, Haupenthal J, Hartmann RW, Kumar M. Synthesis and biological evaluation of novel unsaturated carboxysteroids as human 5a-reductase inhibitors: a legitimate approach. Eur J Med Chem 2012;54:728–39. [8] Lin WH, Fang JM, Cheng YS. Diterpenoids and steroids from Taiwania cryptomerioides. Phytochemistry 1998;48:1391–7. [9] Miyamoto T, Kodama K, Aramaki Higuchi YR, Van Soest RWM. Orostanal, a novel abeo-sterol inducing apoptosis in leukemia cell from a marine sponge, Stelletta hiwasaensis. Tetrahedron Lett 2001;42:6349–51. [10] Liu B, Zhou W. The first stereoselective synthesis of orostanal, a novel abeosterol inducing apoptosis in leukemia cells. Tetrahedron Lett 2002;43:4187–9. [11] Liu B, Zhou W. The first stereoselective synthesis of orostanal isolated from a marine sponge Stelletta hiwasaensis. Tetrahedron 2003;59:3379–84. [12] Wei X, Rodríguez AD, Wang Y, Franzblau SG. Novel ring B abeo-sterols as growth inhibitors of Mycobacterium tuberculosis isolated from a Caribbean Sea sponge, Svenzea zeai. Tetrahedron Lett 2007;48:8851–4. [13] Wei X, Rodríguez AD, Wang Y, Franzblau SG. Synthesis and in vitro biological evaluation of ring B abeo-sterols as novel inhibitors of Mycobacterium tuberculosis. Bioorg Med Chem Lett 2008;18:5448–50. [14] Huang YM, Chen SJ, Cui JG, Gan CF, Liu ZP, Wei YL, et al. Synthesis and cytotoxicity of A-homo-lactam derivatives of cholic acid and 7-deoxycholic acid. Steroids 2011;76:690–4. [15] Huang YM, Cui JG, Chen SJ, Gan CF, Zhou AM. Synthesis and antiproliferative activity of some steroidal lactams. Steroids 2011;76:1346–50. [16] Huang YM, Cui JG, Chen SJ, Lin QF, Song HC, Gan CF, et al. Synthesis and evaluation of some new aza-B-homocholestane derivatives as anticancer agents. Mar Drugs 2014;12:1715–31. [17] Huang YM, Cui JG, Chen SJ, Gan CF, Yao QC, Lin QF. Synthesis and antiproliferative activity of C-homolactam derivatives of 7-deoxycholic acid. Bioorg Med Chem Lett 2013;23:2265–7. [18] Huang YM, Cui JG, Zhong ZG, Gan CF, Zhang WY, Song HC. Synthesis and cytotoxicity of 17a-aza-D-homoandroster-17-one derivatives. Bioorg Med Chem Lett 2011;21:3641–3. [19] Gan CF, Fan LH, Cui JG, Huang YM, Jiao YX, Wei WX. Synthesis and in vitro antiproliferative evaluation of some ring B abeo-sterols. Steroids 2012;77:1061–8. [20] Gan CF, Fan LH, Huang YM, Liu ZP, Cui JG. Synthesis of novel ring B abeo-sterol derivatives and their antiproliferative activities. Med Chem 2013;9:846–54. [21] Huang YM, Cui JG, Zeng QX, Zeng C, Chen Q, Zhou AM. 6-Hydroximino-4-azaA-homo-cholest-3-one and related analogue as a potent inducer of apoptosis in cancer cells. Steroids 2012;77:829–34. [22] Liu B, Zhou WS. The first stereoselective synthesis of orostanal isolated from a marine sponge Stelletta hiwasaensis. Tetrahedron 2003;59:3379–84. [23] Wentworth Jr P, Nieva J, Takeuchi C, Galve R, Wentworth AD, Dilley RB, et al. Evidence for Ozone Formation in Human Atherosclerotic Arteries. Science 2003;302:1053. [24] Natalie KC, Johanna CS, Terry DB, Wentworth Jr P. Adduction of cholesterol 5,6-secosterol aldehyde to membrane-bound myelin basic protein exposes an immunodominant epitope. Biochemistry 2011;50:2092–100. [25] Maciej S, Anna MW, Josef J, Matus P, Katarina K, Marcela V, et al. Investigation of the biological properties of (hetero)aromatic thiosemicarbazones. Molecules 2012;17:13483–502. [26] Débora CR, Angel ARD, Jeferson GDS, Nayane FS, Camila FV, Isolda CM, et al. Structural studies and investigation on the activity of imidazole-derived thiosemicarbazones and hydrazones against crop-related fungi. Molecules 2013;18:12645–62.