Steroids 76 (2011) 1288–1296

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Steroids

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Biotransformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one by fourfilamentous fungi

M. Iqbal Choudhary a,b,⇑, Saira Erum a, Muhammad Atif a, Rizwana Malik a, Naik Tameen Khan a,Atta-ur-Rahman a,⇑a H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistanb Department of Chemistry, College of Sciences, King Saud University, Riyadh 11451, Saudi Arabia

a r t i c l e i n f o

Article history:Received 3 February 2011Received in revised form 4 June 2011Accepted 21 June 2011Available online 5 July 2011

Keywords:Biotransformation(20S)-20-Hydroxymethylpregna-1,4-dien-3-oneFilamentous fungiCunninghamella elegans11a-hydroxylationCytotoxicity against HeLa cell lines

0039-128X/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.steroids.2011.06.007

⇑ Corresponding authors at: H.E.J. Research InstitutCenter for Chemical and Biological Sciences, UniversiPakistan. Tel.: +92 21 34824924/34824925; fax: +92

E-mail address: hej@cyber.net.pk (Atta-ur-Rahman

a b s t r a c t

Microbial transformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (1) by four filamentousfungi, Cunninghamella elegans, Macrophomina phaseolina, Rhizopus stolonifer, and Gibberella fujikuroi, affor-ded nine new, and two known metabolites 2–12. The structures of these metabolites were characterizedthrough detailed spectroscopic analysis. These metabolites were obtained as a result of biohydroxylationof 1 at C-6b, -7b, -11a, -14a, -15b, -16b, and -17a positions, except metabolite 2 which contain an O-acetyl group at C-22. These fungal strains demonstrated to be efficient biocatalysts for 11a-hydroxyl-ation. Compound 1, and its metabolites were evaluated for the first time for their cytotoxicity againstthe HeLa cancer cell lines, and some interesting results were obtained.

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1. Introduction

Steroidal compounds are reported to have numerous physiolog-ical activities, depending on the functional groups present in theirskeleton [1]. Structural transformation of steroidal compoundsthrough microorganisms has emerged as an important applicationin steroidal drug industry [1–3]. Microbial conversions of steroidsgenerally involve dehydrogenation, hydroxylation, esterification,halogenation, isomerization, methoxylation, and side-chain modi-fication of steroidal skeleton [1,4–7]. Extensive investigations havebeen carried out to get mechanistic insight about the bioconver-sion process catalyzed by microorganisms. Several studies havealso been focused on the use of microorganisms as an in vitro mod-el of mammalian drug metabolism [8,9].

(20S)-20-Hydroxymethylpregna-1,4-dien-3-one (1) is a neuro-chemical, obtained from the degradation of sterols by microorgan-isms [10]. It is used as an intermediate in steroidal drug synthesis[11], as well as in the synthesis of vitamin D metabolites [12]. Pre-viously few reports on the microbial transformation of 1 with var-ious fungi have been reported [13,14]. In continuation of our

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e of Chemistry, Internationalty of Karachi, Karachi 75270,21 34819018/34819019.).

biotransformational studies on various bioactive steroids [2,3,15–17], substrate 1 was initially screened for its cytotoxicity againstvarious cancer cell lines. As compound 1 showed a strong cytotox-icity against HeLa cell lines, we decided to synthesize libraries ofthe lead compound 1, for SAR studies by employing microbialtransformation.

Cytotoxicity of bioactive compounds can be used againstseveral disorders such as AIDS, cancer, infection, inflammation,etc. Through a bioassay-guided isolation strategy, several classesof compounds had been identified with potent cytotoxicity[18,19].

We are reporting here for the first time the metabolism of 1with Cunninghamella elegans, Macrophomina phaseolina, Rhizopusstolonifer, and Gibberella fujikuroi, which resulted in variousmono-, di-, and tri-hydroxylated, as well as acetylated derivatives.Metabolites obtained by microbial transformation were screenedfor their cytotoxic effects against HeLa cell lines, and some inter-esting results were obtained.

2. Experimental

2.1. General

(20S)-20-Hydroxymethylpregna-1,4-dien-3-one (1) was pur-chased from Acros Chemicals (USA). Column chromatography

M.I. Choudhary et al. / Steroids 76 (2011) 1288–1296 1289

was carried out on silica gel (70–230 mesh, Merck). For TLC pre-coated plates with silica gel (20 � 20, 0.25 mm thick PF254,Merck) were used and stained by spraying with ceric sulfatesolution (10% solution in sulfuric acid). JEOL JMS-600H massspectrometer was used to record the mass spectra (EI- andHREI-MS) in m/z (rel.%). A Buchi-535 apparatus was used fordetermining the melting points. UV spectra (nm) were measuredin methanol on a Hitachi U-3200 spectrophotometer. FT-IR-8900spectrophotometer was used to record IR spectra (cm�1). Opticalrotations were measured in chloroform or methanol with adigital polarimeter JASCO P-2000 (JASCO International Co. Ltd.,Japan) by using 10 cm cell tube. 1H and 13C NMR spectra wererecorded on a Bruker Avance NMR spectrometer at 300–600and 75–150 MHz in CDCl3 or CD3OD, respectively. Standard pulsesequences were used for DEPT, and 2D-NMR experiments.Recycling preparative HPLC separation was performed on a JAILC-908W instrument, equipped with YMC L-80 (4–5 lm,20 � 250 mm i.d.) using MeOH–H2O (2:1) as mobile phase, andUV detection at 254 nm.

2.2. Microorganisms and culture conditions

Fungal cultures of C. elegans (TSY-0865), M. phaseolina (KUCC-730), R. stolonifer (TSY-0471), and G. fujikuroi (ATCC-10704) weregrown on Sabouraud dextrose-agar at 25 �C and stored at 4 �C. Glu-cose (50.0 g), glycerol (50.0 mL), peptone (25.0 g), yeast extract(25.0 g), KH2PO4 (25.0 g), and NaCl (25.0 g) were mixed intodistilled H2O (5.0 L) to prepare the media for C. elegans and M.phaseolina.

The medium for R. stolonifer was prepared by adding glucose(100.0 g), peptone (25.0 g), yeast extract (25.0 g), and KH2PO4

(25.0 g) to distilled H2O (5.0 L), maintaining the pH at 5.6. Glucose(80.0 g), KH2PO4 (5.0 g), MgSO4�2H2O (1.0 g), NH4NO3 (0.5 g), and‘‘Gibberella trace-element’’ solution (2 mL) were added into dis-tilled H2O (5.0 L) to make the medium of G. fujikuroi. The solutionof Gibberella trace-element was prepared by mixing Co(NO3)2�6H2O(0.01 g), FeSO4�7H2O (0.1 g), CuSO4�5H2O (0.1 g), ZnSO4�7H2O(0.161 g), MnSO4�4H2O (0.01 g), and Mo(NH4)3 (0.01 g) in distilledH2O (100 mL).

2.3. General fermentation and extraction protocol

Spores from the 3-day-old slant were used to make the seedflasks, which were then incubated for 48 h on rotary shaker at25 �C. Aliquots (5 mL) from the seed flasks were transferred tothe remaining flasks and incubated on rotary shaker (128 rpm) at25 �C. After 2 days, compound 1 in acetone was evenly distributedamong 40–60 flasks, and fermentation was continued on rotaryshaker (128 rpm) at 25 �C. An incubation of the fungus withoutsample 1, and an incubation of 1 in the medium without funguswere also conducted as parallel control experiments. Time coursestudy was carried out after 2 days, and the degree of transforma-tion was analyzed periodically on TLC. After 10–14 days, the cul-ture medium was filtered, and extracted with dichloromethanein three portions. The extract was dried over anhydrous Na2SO4,evaporated under reduced pressures, and the resulting browngum was analyzed by thin-layer chromatography.

2.4. Fermentation of (20S)-20-hydroxymethylpregna-1,4-dien-3-onewith C. elegans (TSY-0865)

Compound 1 (1.5 g/25 mL acetone) was distributed among 50flasks containing 3-day-old culture of C. elegans and kept for fer-mentation for 12 days. A brown gummy material (3.6 g) obtainedafter filtration, extraction, and evaporation, was subjected to col-

umn chromatography over silica gel for fractionation with increas-ing polarity of ethyl acetate in pet. ether. Three main fractions(HMCE-1–3) were obtained on the basis of TLC analysis. When sub-jected to silica gel column chromatography fraction HMCE-1yielded metabolite 2 (06 mg, EtOAc:pet. ether = 17:83), metabolite3 (07 mg, EtOAc:pet. ether = 20:80), and metabolite 4 (130 mg,EtOAc:pet. ether 25:75), while fraction HMCE-2 yielded metabolite5 (09 mg, EtOAc:pet. ether 40:60), and metabolite 6 (30 mg,EtOAc:pet. ether 40:60). For purification fraction HMCE-3 was sub-jected to repeated RPHPLC with solvent system MeOH:H2O (2:1)on L-80 column to afford metabolites 7 (06 mg, Rt: 24 min, 4 mL/min), and 8 (06 mg, Rt: 32 min, 3 mL/min).

2.4.1. (20S)-11a-Hydroxy-20-acetoxymethylpregna-1,4-dien-3-one(2)

Colorless solid: m.p.: 203–205 �C. ½a�25D : +36� (c = 0.016, CHCl3).

UV (MeOH): kmax nm (log e) 247 (3.8). IR (KBr); mmax: 3410, 1736,1658 cm�1. Rf: 0.43 (pet. ether/EtOAc = 9:1). 1H NMR (CDCl3,600 MHz): Table 1. 13C NMR (CDCl3, 150 MHz): Table 3. EI-MS:m/z 386 [M+] (10), 265 (11), 173 (8), 147 (29), 135 (20), 134 (27),133 (12), 122 (100), 121 (82), 91 (25), 69 (22). HREI-MS: m/z386.2464 (M+, [C24H34O4]+; calcd 386.2457).

2.4.2. (20S)-17a-Hydroxy-20-hydroxymethylpregna-1,4-dien-3-one(3)

Colorless solid: m.p.: 197–198 �C. ½a�25D : +32� (c = 0.27, CHCl3).

UV (MeOH): kmax nm (log e) 248 (3.3). IR (KBr); mmax: 3348, 2939,1656, 1610 cm�1. Rf: 0.44 (pet. ether/EtOAc = 8:2). 1H NMR (CDCl3,600 MHz): Table 1. 13C NMR (CDCl3, 150 MHz): Table 3. EI-MS: m/z344 [M+] (20), 327 (23), 326 (10), 225 (11), 223 (32), 209 (13), 167(66), 161 (24), 122 (100), 95 (32), 91 (37). HREI-MS: m/z 344.2319(M+, [C22H32O3]+; calcd 344.2351).

2.4.3. (20S)-11a-Hydroxy-20-hydroxymethylpregna-1,4-dien-3-one(4)

Colorless crystalline solid: m.p.: 211–213 �C. ½a�25D : +24�

(c = 0.01, CHCl3). UV (MeOH): kmax nm (log e) 248 (3.7). IR (KBr);mmax: 3375, 2939, 1656, 1608 cm�1. Rf: 0.45 (Pet. ether/EtOAc = 7.5:2.5). 1H NMR (CDCl3, 600 MHz): Table 1. 13C NMR(CDCl3, 150 MHz): Table 3. EI-MS: 344 [M+] (15), 327 (14), 267(8), 223 (25), 173 (17), 161 (17), 159 (14), 147 (49), 134 (32),122 (100), 107 (23), 95 (25), 55 (17). HREI-MS: m/z 344.2402(M+, [C22H32O3]+; calcd 344.2351).

2.4.4. (20S)-6b,11a-Dihydroxy-20-hydroxymethylpregna-1,4-dien-3-one (5)

Colorless solid: m.p.: 197–199 �C. ½a�25D : +12� (c = 0.02, CHCl3).

UV (MeOH): kmax nm (log e) 248 (3.9). IR (KBr); mmax: 3371,1657 cm�1. Rf: 0.54 (pet. ether/EtOAc = 6:4). 1H NMR (CDCl3,500 MHz): Table 1. 13C NMR (CDCl3, 125 MHz): Table 3. EI-MS:m/z 360 [M+] (12), 342 (6), 324 (2), 314 (4), 283 (6), 241 (2), 224(6), 222 (8), 202 (2), 173 (10), 167 (24), 150 (17), 147 (40), 145(24), 137 (99), 122 (28), 109 (100), 105 (33), 95 (48), 91 (50), 79(67), 55 (87). HREI-MS: m/z 360.2320 (M+, [C22H32O4]+; calcd360.2301).

2.4.5. (20S)-11a,15b-Dihydroxy-20-hydroxymethylpregna-1,4-dien-3-one (6)

White solid: m.p.: 202–204 �C. ½a�25D : +49� (c = 0.023, CHCl3). UV

(MeOH): kmax nm (log e) 243 (3.6). IR (KBr); mmax: 3444, 1655 cm�1.Rf: 0.49 (pet. ether/EtOAc = 6:4). 1H NMR (CDCl3, 600 MHz): Ta-ble 1. 13C NMR (CDCl3, 150 MHz): Table 3. EI-MS: m/z 360 [M+](6), 342 (8), 309 (3), 265 (6), 283 (3), 239 (8), 225 (4), 189 (8),173 (10), 161 (13), 159 (14), 154 (80), 147 (30), 137 (21), 134(63), 122 (60), 121 (100), 119 (20), 109 (21), 95 (22), 91 (40), 81

Table 11H NMR chemical shift data of compounds 2–7 (d in ppm, J and W1/2 in Hz).

No. 2a 3a 4a 5b 6a 7b

1 7.73 d (10.2) 7.03 d (10.2) 7.74 d (10.3) 7.78 d (10.0) 7.73 d (10.2) 7.75 d (10.0)2 6.15 dd (10.2, 1.8) 6.20 dd (10.2, 1.8) 6.13 dd (10.3, 1.8) 6.13 d (10.2) 6.15 d (10.8) 6.14 dd (10.5, 2.0)3 – – – – – –4 6.08 br. s. 6.06 br. s. 6.07 br. s. 6.15 br. s. 6.09 br. s. 6.08 br. s.5 – – – – – –6 2.44, 2.37 m 1.92, 1.79 m 1.96, 1.06 m 4.52 br. s (W1/2 = 11.1) 2.28, 1.15 m 1.75, 1.13 m7 1.95, 1.06 m 2.43, 2.34 m 2.42, 2.37 m 1.88, 1.30 m 2.52, 2.39 m 2.52, 2.39 m8 1.71 m 1.62 m 1.62 m 2.06 m 2.03 m 1.51 m9 1.07 m 1.05 m 1.09 m 1.36 m 1.13 m 1.14 m10 – – – – – –11 4.04 m (W1/2 = 21) 1.73, 1.64 m 4.05 m (W1/2 = 21.7) 4.11 m (W1/2 = 22.2) 4.06 m (W1/2 = 21.0) 4.04 m (W1/2 = 21.2)12 2.33, 1.16 m 1.66, 1.22 m 2.32, 1.17 m 2.36, 1.18 m 2.31, 1.11 m 2.01, 1.71 m13 – – – – – –14 1.22 m 1.67 m 1.24 m 1.08 m 0.94 m 1.82 m15 1.64, 1.22 m 1.72, 1.21 m 1.57, 1.11 m 1.63, 1.21 m 4.20 m (W1/2 = 9.2) 1.61 m16 1.56, 1.24 m 1.96, 1.92 m 1.86, 1.35 m 1.55, 1.33 m 2.44, 1.41 m 1.92, 1.85 m17 1.12 m – 1.11 m 1.20 m 1.18 m –18 0.76 s 0.80 s 0.77 s 0.80 s 1.05 s 0.80 s19 1.29 s 1.22 s 1.30 s 1.51 s 1.33 s 1.29 s20 1.62 m 1.78 m 1.55 m 1.59 m 1.67 m 1.13 m21 1.01 d (6.6) 1.11 d (6.5) 1.04 d (6.5) 1.05 d (6.4) 1.06 d (6.5) 1.14 d (6.4)22 4.03 m, 3.73 dd (10.2, 7.2) 4.04 m, 3.63 m 3.60 m, 3.36 m 3.62 m, 3.36 d (13.9) 3.60 d (10.8) 3.39 dd (10.8, 6.6) 4.03 m, 3.64 m23 – – – – –24 2.03 s – – – –

a 600 MHz, CDCl3.b 500 MHz, CDCl3.

1290 M.I. Choudhary et al. / Steroids 76 (2011) 1288–1296

(66), 55 (44). HREI-MS: m/z 360.2329 (M+, [C22H32O4]+; calcd360.2301).

2.4.6. (20S)-11a,17a-Dihydroxy-20-hydroxymethylpregna-1,4-dien-3-one (7)

Colorless solid: m.p.: 207–208 �C. ½a�25D : +57� (c = 0.013, CHCl3).

UV (MeOH): kmax nm (log e) 244 (3.3). IR (KBr); mmax: 3329,1654 cm�1. Rf: 0.45 (pet. ether/EtOAc = 6:4). 1H NMR (CDCl3,500 MHz): Table 1. 13C NMR (CDCl3, 125 MHz): Table 3. EI-MS:m/z 360 [M+] (20), 342 (44), 309 (16), 283 (11), 265 (14), 260(14), 239 (33), 202 (14), 189 (18), 154 (66), 147 (41), 135 (36),134 (96), 121 (100), 107 (43), 91 (25), 81 (13). HREI-MS: m/z360.2301 (M+, [C22H32O4]+; calcd 360.2301).

2.4.7. (20S)-14a,15b,17a-Trihydroxy-20-hydroxymethylpregna-1,4-dien-3-one (8)

Crystalline solid: m.p.: 201–203 �C. ½a�25D : +31� (c = 0.035,

CHCl3). UV (MeOH): kmax nm (log e) 244 (3.6). IR (KBr); mmax:3317, 3301, 1703 cm�1. Rf: 0.53 (pet. ether/EtOAc = 1:1). 1H NMR(CDCl3, 500 MHz): Table 1. 13C NMR (CDCl3, 100 MHz): Table 3.FAB (+ve): m/z 377. FAB (�ve): m/z 375. HREI-MS: m/z 376.2157(M+, [C22H32O4]+; calcd 376.2137).

2.5. Fermentation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one(1) with M. phaseolina (KUCC-730)

Compound 1 (0.8 g/25 mL acetone) was distributed among 50flasks containing 3-day-old culture of M. phaseolina. Incubationwas continued for 14 days and then filtrate was extracted withDCM (5 L � 3). A brown thick gum (2.6 g) was obtained by concen-trating the extract in rotary evaporator, and then subjected to col-umn chromatography with increasing polarity of ethyl acetate inpet. ether to obtain two fractions (HMJ-1–2). Fraction HMJ-1 affor-ded a pure metabolite 4 (60 mg, EtOAc:pet. ether 25:75), and asub-fraction HMJ-1A, which was subjected to repeated RPHPLCwith solvent system MeOH:H2O (2:1) on L-80 column to yield apure metabolite 9 (05 mg, Rt: 28 min, 4 mL/min). Fraction HMJ-2

yielded, metabolites 6 (40 mg, EtOAc:pet. ether 40:60), 10(09 mg, EtOAc:pet. ether 40:60), and 11 (07 mg, EtOAc:pet. ether50:50) on elution with silica gel column chromatography.

2.5.1. (20S)-7b-Hydroxy-20-hydroxymethylpregna-1,4-dien-3-one (9)Colorless crystalline solid: m.p.: 216–218 �C. ½a�25

D : +44�(c = 0.014, CHCl3). UV (MeOH): kmax nm (log e) 246 (4.1). IR(KBr); mmax: 3381, 1656, 1608 cm�1. Rf: 0.51 (pet. ether/EtOAc = 7:3). 1H NMR (CDCl3, 500 MHz): Table 2. 13C NMR (CDCl3,125 MHz): Table 3. EI-MS: m/z 344 [M+] (22), 329 (2), 327 (33), 267(12), 225 (11), 223 (32), 209 (13), 187 (11), 173 (26), 161 (25), 147(66), 139 (18), 135 (35), 122 (100), 121 (100), 95 (32), 77 (15), 55(21). HREI-MS: m/z 344.2383 (M+, [C22H32O3]+; calcd 344.2351).

2.5.2. (20S)-15b-Hydroxy-20-hydroxymethylpregna-1,4-dien-3-one(10)

Colorless solid: m.p.: 220–222 �C. ½a�25D : +17� (c = 0.034, MeOH).

UV (MeOH): kmax nm (log e) 248 (3.9). IR (KBr); mmax: 3379, 1656,1612 cm�1. Rf: 0.53 (pet. ether/EtOAc = 7:3). 1H NMR (CDCl3,400 MHz): Table 2. 13C NMR (CDCl3, 75 MHz): Table 3. EI-MS:344 [M+] (2), 326 (15), 205 (10), 187 (16), 173 (30), 161 (11),159 (13), 147 (30), 145 (18), 135 (27), 131 (24), 122 (86), 121(100), 95 (22), 91 (43), 81 (28), 67 (25), 55 (57). HREI-MS: m/z344.2343 (M+, [C22H32O3]+; calcd 344.2351).

2.5.3. (20S)-7b,15b-Dihydroxy-20-hydroxymethylpregna-1,4-dien-3-one (11)

Colorless solid: m.p.: 207–209 �C. ½a�25D : +19� (c = 0.018, MeOH).

UV (MeOH): kmax nm (log e) 246 (3.9). IR (KBr); mmax: 3329,1658 cm�1. Rf: 0.63 (pet. ether/EtOAc = 6.5:3.5). 1H NMR (CDCl3,400 MHz): Table 2. 13C NMR (CDCl3, 75 MHz): Table 3. EI-MS: m/z 360 [M+] (9), 342 (4), 324 (19), 291 (3), 265 (18), 237 (5), 223(7), 211 (18), 185 (11), 173 (16), 171 (24), 161 (17), 145 (22),159 (21), 147 (21), 133 (25), 107 (32), 95 (28), 91 (45), 77 (26),69 (35), 55 (100), 53 (25). HREI-MS: m/z 360.2291 (M+,[C22H32O4]+; calcd 360.2301).

Table 21H NMR chemical shift data of compounds 8–12 (d in ppm, J and W1/2 in Hz).

No. 8b 9b 10c 11c 12a

1 7.03, d (10.3) 7.02 d (10.0) 7.04 d (10.2) 7.03 d (10.0) 7.02 d (10.1)2 6.21, dd (10.5, 2.0) 6.22 dd (10.0, 1.5) 6.20 dd (10.4, 2.0) 6.22 dd (10.4, 1.2) 6.21 d (10.1)3 – – – – –4 6.04 br. s. 6.08 br. s. 6.06 br. s. 6.08 br. s. 6.05 br. s.5 – – – – –6 2.05, m; 1.42, m 2.61, 2.52 m 1.99, 1.15 m 2.62, 2.60 m 1.96, 1.25 m7 2.25, m; 1.50, m 3.45 m (W1/2 = 22.1) 2.52, 1.12 m 3.59 m (W1/2 = 23.4) 2.45, 2.30 m8 1.80, m 1.61 m 2.03 m 2.05 m 1.79 m9 1.67, m 1.01 m 1.18 m 1.08 m 1.07 m10 – – – – –11 1.72, m; 1.70,m 1.79, 1.58 m 1.68, 1.65 m 1.73, 1.71 m 1.69, 1.62 m12 2.41, m; 2.38, m 1.26, 1.22 m 2.31, 2.21 m 1.98, 1.13 m 2.25, 2.21 m13 – – – – –14 – 1.16 m 0.85 m 0.97 m 1.50 m15 4.09, m; (W1/2 = 8.6) 1.72, 1.68 m 4.19 m (W1/2 = 7.2) 4.32 t (5.2) 1.98, 1.69 m16 2.61, m; 1.84, m 2.04, 1.18 m 2.43, 1.39 m 2.35, 1.47 m 3.98 m (W1/2 = 18.8)17 – 1.15 m 1.16 m 1.18 m 1.01 m18 1.22, s. 0.76 s 1.03 s 1.04 s 0.77 s19 0.93, m 1.25 s 1.25 s 1.28 s 1.22 s20 1.99, m 1.51 m 1.68 m 1.68 m 1.51 m21 1.12, d (7.0) 1.04 d (6.6) 1.04 d (6.8) 1.05 d (6.5) 1.04 d (6.0)22 4.08, m, 3.60, m 3.63 m, 3.37 m 3.61 m, 3.38 m 3.62 m, 3.43 m 3.58 d (10.3) 3.33 dd (10.3, 1.8)

a 600 MHz, CDCl3.b 500 MHz, CDCl3.c 400 MHz, CDCl3.

Table 313C NMR chemical shift data of compounds 2–13 (CDCl3).

C 2a 3a 4a 5a 6a 7b 8c 9b 10d 11d 12a

1 158.8 155.6 159.0 160.4 159.0 158.8 155.8 155.4 155.9 155.4 155.72 125.2 127.5 125.1 126.6 125.1 125.2 127.6 127.9 127.5 127.9 127.63 186.8 186.5 186.9 186.8 186.9 186.8 186.5 186.2 186.4 186.1 186.44 124.6 123.9 124.5 124.6 124.5 124.6 123.7 124.9 123.8 125.0 123.75 168.0 169.3 168.3 164.8 168.2 168.0 168.7 164.8 169.2 164.3 169.16 33.4e 37.7 33.5e 74.1 32.7e 33.2e 28.4 42.1 40.7 42.7 39.57 33.2e 32.9e 33.2e 39.8 33.1e 33.5e 26.7 75.9 32.9e 75.7 32.88 35.7 35.8 34.3 28.8 30.5 34.5 39.5 42.8 31.7 38.1 35.19 60.4 51.8 60.5 59.8 60.7 60.4 45.2 48.8 52.7e 49.2 52.410 43.0 43.5 42.9 43.1 44.2 44.0 43.3 43.0 43.7 43.0 43.511 68.3 22.6 68.2 68.2 67.9 68.7 21.6 26.8 22.7 23.2 22.712 51.0 31.9e 51.0 51.1 52.2 43.7 32.6 28.1 32.8e 40.5 33.813 44.0 47.7 44.1 44.1 42.5 47.7 50.0 43.7 42.4 42.9 44.014 52.3 49.5 52.0 52.1 58.9 48.8 84.0 51.4 59.7 59.9 50.115 24.3 23.7 24.3 24.3 69.9 23.6 74.0 23.2 70.3 71.3 40.216 27.7 33.6 27.7 27.7 41.3 38.1 49.6 39.3 41.0 38.4 73.617 54.5 86.8 54.6 54.6 52.2 85.9 86.7 54.7 52.6e 52.3 62.618 13.1 14.7 13.1 13.2 15.7 15.6 18.5 12.2 14.7 14.6 13.519 18.7 18.7 18.7 20.3 18.7 18.7 17.8 18.9 18.7 18.8 18.820 34.3 39.6 38.6 38.5 38.2 39.4 39.1 38.5 38.2 38.5 38.221 17.1 12.7 16.7 16.7 16.8 12.8 13.0 16.9 16.8 16.9 16.622 69.1 68.0 67.6 67.7 67.5 68.0 68.2 67.9 67.7 67.7 67.623 171.4 – – – – – – – – – –24 21.1 – – – – – – – – – –

a 150 MHz.b 125 MHz.c 100 MHz.d 75 MHz.e Exchangeable chemical shifts.

M.I. Choudhary et al. / Steroids 76 (2011) 1288–1296 1291

2.6. Fermentation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one(1) with R. stolonifer (TSY-0471)

Compound 1 (1 g/30 mL acetone) was transferred to 60 flaskscontaining the 3-days-old culture of R. stolonifer. After 12 days ofincubation, media was filtered, and extracted with EtOAc, andevaporated to afford a brown gum (3.3 g). Column chromato-graphic purification using silica gel column afforded pure metabo-lites 4 (20 mg, EtOAc:pet. ether 25:75), and 12 (08 mg, EtOAc:pet.ether 35:65).

2.6.1. (20S)-16b-Hydroxy-20-hydroxymethylpregna-1,4-dien-3-one(12)

Crystalline solid: m.p.: 185–187 �C. ½a�25D : +39� (c = 0.096,

MeOH). UV (MeOH): kmax nm (log e) 249 (3.9). IR (KBr); mmax:3381, 1657, 1610 cm�1. Rf: 0.53 (pet. ether/EtOAc = 6.5:3.5). 1HNMR (CDCl3, 600 MHz): Table 1. 13C NMR (CDCl3, 150 MHz): Ta-ble 2. EI-MS: m/z 344 [M+] (17), 327 (13), 267 (9), 225 (8), 223(27), 209 (10), 173 (19), 171 (10), 161 (18), 159 (15), 147 (51),139 (13), 135 (28), 133 (20), 122 (100), 121 (96), 119 (14), 109

1292 M.I. Choudhary et al. / Steroids 76 (2011) 1288–1296

(18), 107 (23), 91 (28), 79 (13), 55 (14), 41 (13). HREI-MS: m/z344.2384 (M+, [C22H32O3]+; calcd 344.2351).

2.7. Fermentation of (20S)-hydroxymethylpregna-1,4-dien-3-one (1)with G. Fujikuroi (ATCC-10704)

A solution of compound 1 (0.8 g/20 mL acetone) was distributedamong 40 flasks containing 48-h-old culture of G. fujikuroi. Incuba-tion was stopped after 10 days. The filtrate was extracted withCH2Cl2, and column chromatographic separation of the organic ex-tract (1.3 g) afforded two main fractions (HMGF-1–2). FractionHMGF-1 yielded metabolite 4 (16 mg, EtOAc:pet. ether 25:75),while fraction HMGF-2 afforded metabolites 5 (06 mg, EtOAc:pet.ether 40:60), 6 (08 mg, EtOAc:pet. ether 40:60), and 12 (04 mg,EtOAc:pet. ether 35:65) by silica gel column chromatography.

2.8. Fermentation of metabolite 4 with C. elegans (TSY-0865)

Metabolite 4 (60 mg/2.0 mL acetone) was incubated in fourflasks with 3-day-old culture of C. elegans and kept for fermenta-tion for 12 days, under same reaction conditions as for 1. Compar-ative TLC analysis of the crude extract showed that compound 4was metabolized to compounds 2, 5, 6, and 7.

2.9. Cytotoxicity experiment

Cytotoxicity experiments were performed using MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide) colori-metric method. Human cervical carcinoma (HeLa) cells (105 cells/mL) were maintained in Minimum Essential Eagle’s Medium(MEM), supplemented with 5% fetal bovine serum (FBS), 100 IU/mL of penicillin and 100 lg/mL of streptomycin. The cells werethen plated onto 96-well plates (100 lL/well) in 5% CO2 incubatorat 37 �C for overnight incubation and the medium was removedand 200 lL fresh medium was added with different concentrationsof compounds (1–50 lM). After 72 h, MTT (2 lg/mL) was added toeach well and incubated for further 4 h, and 100 lL of DMSO wasadded to each well. The extent of MMT reduction to formazanwithin cells was calculated by measuring the absorbance at570 nm, by an ELISA micro plate reader (Spectra Max Plus, Molec-ular Devices, CA, USA). The cytotoxicity was recorded as concentra-tion causing 50% growth inhibition.

3. Results

Small scale preliminary screening with various fungi showedthat filamentous fungi C. elegans, M. phaseolina, R. stolonifer, andG. fujikuroi have the capacity to transform compound 1 into morepolar hydroxylated derivatives as a result of stereo- and regioselec-tive hydroxylations, thus a large scale fermentation with all ofthese fungi was carried out. Incubation of compound 1 with thesefungi resulted in the production of nine new (2, 3, 5–8, 10–12), andtwo known metabolites 4, and 9.

The EI-MS of 2 showed the M+ at m/z 386 suggested the additionof oxygen and an acetoxy group in the substrate. The M+ peak at m/z 386.2464 was observed in the HREI-MS of 2, which was consis-tent with the formula C24H34O4 (calcd m/z 386.2457). The 1HNMR spectrum showed a new downfield methine multiplet at d4.04 (W1/2 = 21 Hz), as well as the downfield shift of H-1 from d7.01 to 7.73, as compared to the substrate 1, indicated the positionof newly introduced hydroxyl group at C-11. A new methyl signalwas also observed in the 1H NMR spectrum of 2 at d 2.03 (s), and H-22 methylene protons were shifted downfield from d 3.60 and 3.33to d 4.03 and 3.73. The 13C NMR spectrum of 2 showed three majordifferences, as compared to the substrate 1. This included a new

quaternary carbon at d 171.4 (C-23), a downfield methyl signal atd 21.1 (C-24), and a methine signal at d 68.3 (C-11) in compound2. The position of the new hydroxyl group was assigned to be atC-11 with the help of COSY 45� interactions of geminal H-11 (d4.04) with H2-12 (d 1.16, 2.33), and H-9 (d 1.07). The introductionof acetoxy group at C-22 was deduced by long-range heteronuclearcouplings of H-22 (d 4.03, 3.73), and Me-24 (d 2.03) with C-23 (d171.4). The stereochemistry of the C-11 hydroxyl group was as-signed to be a (equatorial) on the basis of NOESY interactions ofgeminal H-11 (d 4.04) with Me-18 (d 0.76), and Me-19 (d 1.29).The structure of the new metabolite 2 was thus deduced as(20S)-11a-hydroxy-20-acetoxymethylpregna-1,4-dien-3-one.

The HREI-MS of metabolite 3 displayed an M+ at m/z 344.2398,consistent with the formula C22H32O3 (calcd 344.2351), which was16 a.m.u. higher than the substrate 1, due to the addition of an oxy-gen atom. The 1H NMR spectrum of 3 was quite similar to the sub-strate 1, except the downfield shifts of C-22 methylene protonsfrom d 3.60 and 3.33 to d 4.04 and 3.63, indicating that a hydroxylgroup was introduced in its vicinity. The 13C NMR spectrum of 3displayed additional quaternary carbon at d 86.8, indicated thathydroxylation occurred at a tertiary carbon. The new hydroxylgroup was placed at C-17 on the basis of HMBC correlations ofH-20 (d 1.78), CH3-18 (d 0.80), and CH3-21 (d 1.11) with C-17 (d86.8). The new metabolite 3 was thus characterized as (20S)-17a-hydroxy-20-hydroxymethylpregna-1,4-dien-3-one.

The M+ ion at m/z 344.2402 in the HREI-MS of metabolite 4 cor-responded to the formula C22H32O3 (calcd 344.2351), indicatingthe addition of an oxygen atom in the substrate 1. The 1H NMRspectrum of compound 4 showed a downfield methine multipletat d 4.05 (W1/2 = 21.7 Hz) for a methine proton geminal to an OHgroup, while H-1 also showed the downfield shift from d 7.01 to7.74, indicating that hydroxylation had occured at C-11. The posi-tion of the newly introduced hydroxyl group was further deducedon the basis of HMBC correlations between H-11 (d 4.05), C-9 (d60.5), C-12 (d 51.0), and C-13 (d 44.1). The stereochemistry was de-duced to be a (equatorial) on the basis of NOESY correlations be-tween H-11 (4.05), and CH3-19 (d 1.30) and CH3-18 (0.77). Basedon the above mentioned spectral data, metabolite 4 was character-ized as a known compound (20S)-11a-hydroxy-20-hydroxymthyl-pregna-1,4-dien-3-one. It was earlier reported from thefermentation of (20S)-hydroxymthylpregna-1,4-dien-3-one withAspergillus occhraceus [14].

The HREI-MS of metabolite 5 showed the M+ peak at m/z360.2320, which corresponded to the formula C22H32O4 (calcd360.2301). The 1H NMR spectrum of 5 showed two additionaldownfield methine signals at d 4.11 (m, W1/2 = 22.2 Hz), and 4.52(br. s, W1/2 = 11.1 Hz), which indicated the introduction of two sec-ondary hydroxyl groups in the metabolite 5. The 13C NMR spec-trum of 5 also showed two downfield methine carbon signals, ascompared to the substrate 1, resonating at d 68.2 (C-11), and74.1 (C-6). The positions of newly introduced hydroxyl groups atC-6, and C-11 were further deduced by HMBC correlations of H-12 (d 2.36, 1.18), and H-9 (d 1.08) with C-11 (d 68.2), as well asHMBC correlations of H-6 with C-4 (d 124.6), C-8 (d 28.8), and C-10 (d 43.1). The a-stereochemistry of the newly introduced hydro-xyl group at C-11 was deduced from the NOESY correlations ofgeminal H-11b (d 4.11) with CH3-18b (d 0.80) and CH3-19b (d1.51), while stereochemistry of C-6 OH was assigned to be b (axial)on the basis of its coupling constant (W1/2 = 11.1 Hz) and NOESYcorrelations between H-6a (d 4.52) with H-9a (d 1.36). The newmetabolite 5 was thus deduced as (20S)-6b,11a-dihydroxy-20-hydroxymethylpregna-1,4-dien-3-one.

The HREI-MS of metabolite 6 exhibited the M+ at m/z 360.2329,consistent with the formula C22H32O4 (calcd 360.2301). The 1HNMR spectrum was substantially different from the substrate 1 intwo different ways; first it showed two additional downfield

Table 4Cytotoxicity activities of the compounds against HeLacell lines.

Compounds IC50 (lM)

1 2.52 ± 0.204 N/Aa

5 N/Aa

6 N/Aa

10 35.02 ± 0.6011 N/Aa

Doxorubicin (standard drug)b 3.1 ± 0.20

a Not active.b Standard used.

M.I. Choudhary et al. / Steroids 76 (2011) 1288–1296 1293

methine signals at d 4.06 (m, W1/2 = 21.0), and 4.20 (m, W1/2 = 9.2).Secondly, the downfield shift of H-1 from d 7.02 to 7.73 was also ob-served which indicated a hydroxylation at C-11. The 13C NMR spec-trum of 6 showed the presence of two additional methine carbonsignals, resonated at d 67.9 (C-11), and 69.9 (C-15). The positionof the newly introduced hydroxyl groups at C-11, and C-15 were de-duced on the basis of HMBC correlations of H-11 (d 4.06) with C-12(d 52.2), and C-9 (d 60.7), and H-15 (d 4.20) with C-16 (d 41.3) and C-17 (d 52.2). The NOESY spectrum of metabolite 6 showed interac-tions of H-11b (d 4.06) with CH3-19b (1.33), and CH3-18b (d 1.05),and H-15a (d 4.20) with H-14a (d 0.94), and supported the stereo-chemical assignment of the hydroxyl groups as C-11aOH and C-15bOH. The new compound 6 was thus characterized as (20S)-11a,15b-dihydroxy-20-hydroxymethylpregna-1,4-dien-3-one.

The HREI-MS of compound 7 showed the M+ at m/z 360.1912,which was consistent with the formula C22H32O4 (calcd360.2301). The 1H NMR spectrum showed an additional downfieldmethine signal at d 4.04 (m, W1/2 = 21.2 Hz), and a downfield shiftof C-22 methylene protons from d 3.33 and 3.60 to d 3.64 and 4.03.In the 1H NMR spectrum, H-1 was also shifted from d 7.02 to 7.75.The 13C NMR spectrum displayed an additional downfield methinesignal at d 68.7, and a quaternary carbon signal at d 85.9, corre-sponding to OH-bearing C-11, and C-17, respectively. The locationsof the newly introduced hydroxyl groups at C-11, and C-17 werefurther ascertained by HMBC correlations of H-9 (d 1.14), and H-12 (d 2.01, 1.71) with C-11 (d 68.7), as well as correlations ofCH3-18 (d 0.80), and H-20 (d 1.13) with C-17 (d 85.9). The stereo-chemistry at C-11 was deduced to be a (equatorial) on the basisof NOESY interactions between its geminal H-11 (d 4.04) withCH3-19 (1.29), and CH3-18 (d 0.80). These spectroscopic studiesled to the identification of new metabolite as (20S)-11a,17a-dihy-droxy-20-hydroxymethylpregna-1,4-dien-3-one (7).

The M+ of metabolite 8 at m/z 376.2254 was observed in theHREI-MS (C22H32O5, calcd 376.2349). The 1H NMR spectrum of 8showed the downfield shifts of C-22 methylene protons from d3.60 and 3.33 to d 4.08 and 3.60 indicating an OH at C-17, and adownfield methine proton signal at d 4.09 (m, W1/2 = 8.6 Hz). The13C NMR spectrum displayed two downfield quaternary carbonsignals at d 86.7 and 84.3, alongwith a downfield methine signalat d 74.0. The location of these OH groups was deduced on the basisof HMBC correlations of H-21 (d 1.13), and C-22 (d 68.2), C-20 (d39.1), and C-17 (d 86.7), also Me-18 (d 0.93) showed interactionwith C-17 (d 86.7), C-13 (d 49.9), and C-14 (d 84.0). As well as inthe HMBC spectrum, H-16 (d 2.61, 1.84) showed correlations withC-17 (d 86.7), C-15 (d 74.0) and C-14 (d 84.0) and suggested thenewly incorporated OH groups at C-14, C-15, and C-17 positions.The stereochemistry of the newly introduced OH group at C-15was deduced to be b, on the basis of its coupling constant (W1/

2 = 8.6 Hz), while the orientation of C-14OH group was assignedto be a on the basis of comparison of the 13C NMR data with thereported C-14a-hydroxysteroids [20]. The structure of the newmetabolite 8 was thus characterized as (20S)-14a,15b,17a-trihy-droxy-20-hydroxymethylpregna-1,4-dien-3-one.

The HREI-MS of compound 9 showed the M+ at m/z 344.2383(C22H32O3, calcd 344.2351). The 1H NMR spectrum of metabolite9 showed a downfield methine proton signal at d 3.45 (m, W1/

2 = 22.1 Hz). The 13C NMR spectrum also exhibited a new downfieldmethine signal at d 75.9. The position of the hydroxyl group at C-7was deduced on the basis of HMBC correlations of H-6 (d 2.61,2.52) with C-7 (d 75.9), C-4 (d 124.9), and C-5 (d 164.8), and corre-lations of H-8 (d 1.61) with C-7 (d 75.9). Vicinal couplings betweenH-6 (d 2.61, 2.52) and H-7 (d 3.47), and H-8 (d 1.61) were also ob-served in the COSY 45� spectrum. The stereochemistry of the newOH was deduced to be b (equatorial) at C-7 on the basis of NOESYcorrelations of geminal H-7 (d 3.45), with H-9 (d 1.01) and H-14 (d1.16). On the basis of the above mentioned spectral data, the

known metabolite 9 was identified as (20S)-7b-hydroxy-20-hydroxymethylpregna-1,4-dien-3-one. This compound was earlierreported from the microbial transformation of ursodeoxycholicacid by Pseudomonas sp. [21].

The molecular formula of 10 was deduced as C22H32O3 (calcd344.2351) on the basis of the M+ in HREI-MS (m/z 344.2343). The1H NMR spectrum showed an additional downfield methine protonsignal at d 4.19 (m, W1/2 = 7.2 Hz). An additional downfieldmethine carbon signal was resonated at d 70.3 (C-15), and a b-downfield shift of C-14 (d 59.7) and C-16 (d 41.0) were observedin the 13C NMR spectrum, which indicated an OH at C-15. In theHMBC spectrum, correlations between H-15 (d 4.19) and C-16 (d41.0), and C-17 (d 52.6) further supported the presence of annew –OH at C-15. The stereochemistry of C-15 OH was deducedto be b on the basis of NOESY correlations between H-15 (d 4.19)and H-14 (d 0.85). The new metabolite 10 was thus characterizedas (20S)-15b-hydroxy-20-hydroxymethylpregna-1,4-dien-3-one.

The HREI-MS of metabolite 11 showed the M+ at m/z 360.2291(C22H32O4) (calcd 360.2301), indicating two additional oxygenatoms, as compared to the substrate 1. The 1H NMR spectrumshowed signals for two methine protons resonated at d 3.59 (m,W1/2 = 23.4 Hz), and 4.32 (t, J = 5.2 Hz). The 13C NMR spectrumshowed two additional OH-bearing methine signals at d 75.7 and71.3, as compared to the substrate 1. The positions of the new OHgroups were deduced on the basis of COSY 45�, and HMBC correla-tions. The stereochemistry of C-7 and C-15 was further investigatedby the coupling constant, and the NOESY correlations between H-7(d 3.59) and H-9 (d 1.08), and H-14 (d 0.97), as well as the correlationof H-15 (d 4.32) with H-14 (d 0.97). On the basis of above mentionedspectral data, the new metabolite 11 was characterized as (20S)-7b,15b-dihydroxy-20-hydroxymethylpregna-1,4-diene-3-one.

The molecular formula of metabolite 12 was deduced from theHREI-MS (M+: m/z 344.2384) as C22H32O3 (calcd 344.2351), with 7�unsaturation. A characteristic methine proton signal, geminal to anOH, was resonated at d 3.98 (m, W1/2 = 18.8) in the 1H NMR spec-trum. The 13C NMR spectrum was quite similar to the substrate1, except a downfield methine signal at d 73.6 (C-16). The HMBCspectrum showed correlations between H-16 (d 3.98), and C-14(d 50.1), C-17 (62.6), and C-15 (d 40.2) and thus further supportedthe position of the new OH at C-16. The stereochemistry of the newOH at C-16 was deduced to be b on the basis of the NOESY crosspeaks between geminal H-16 (d 3.98), and H-17 (d 1.01). Thus,the structure of new metabolite 12 was deduced as (20S)-16b-hy-droxy-20-hydroxymethylpregna-1,4-dien-3-one.

Compounds 1, 4, 5, 6, 10, and 11 were evaluated for their anti-cancer activity against the HeLA cancer cell lines and results arepresented in Table 4.

4. Discussion

Previously biotransformation of (20S)-20-hydroxymethylpreg-na-1,4-dien-3-one (1) with various fungi resulted in hydroxylations

O

H

H H

OHH

OH

HO

O

H

H

OAcHHO

H

O

H

H H

OHHHO

O

H

H H

OHH

O

H

H H

OHOH

O

H

H H

OHHHO

OH

O

H

H H

OHOHHO

O

H

H OH

OH

OH

OH

1

2

34

5

76

8

1

3 5 7

9

11 1317

15

18

19

20

21

Fig. 1. A proposed biotransformation pathway of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (1) by Cunninghamella elegans.

O

H

H

OHHHO

H

O

H

H H

OHH

O

H

H H

OHH

OH

O

H

H H

OHHHO

4 10

6

OH

9

OH

O

H

H H

OHH

11OH

OH

+ +

O

H

H H

OHH

1

1

3 5 7

9

11 1317

15

18

19

20

21

10

Fig. 2. A proposed biotransformation pathway of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (1) by Macrophomina phaseolina.

1294 M.I. Choudhary et al. / Steroids 76 (2011) 1288–1296

M.I. Choudhary et al. / Steroids 76 (2011) 1288–1296 1295

of the substrate at C-7b, C-11a, and C-15b positions [13,14], how-ever to the best of our knowledge, this is the first report concerningthe microbial transformation of 1 with four filamentous fungi C. ele-gans, M. phaseolina, R. stolonifer, and G. fujikuroi.

Incubation of compound 1 with C. elegans resulted in theproduction of metabolites 2–8, which were characterized through

O

H

H H

OHH

1

3 5 7

9

11 1317

15

18

19

20

21

O

H

H

OHHHO

H

O

H

H H

OHH

OH

1

4 12

+

Fig. 3. Resulting metabolites from the fermentation of (20S)-20-hydroxymethyl-pregna-1,4-dien-3-one (1) with Rhizopus stolonifer.

O

H

H

OHHO

H

O

HO

O

H

H H

OHH

OH

HO

5

4

O

1

3

Fig. 4. A proposed biotransformation pathway of (20S)-20-hydr

detailed spectral analysis as (20S)-11a-hydroxy-20-acetoxymethyl-pregna-1,4-dien-3-one (2), (20S)-17a-hydroxy-20-hydroxymethyl-pregna-1,4-dien-3-one (3), (20S)-11a-hydroxy-20-hydroxy-methylpregna-1,4-dien-3-one (4), (20S)-6b,11a-dihydroxy-20-hydroxymethylpregna-1,4-dien-3-one (5), (20S)-11a,15b-dihy-droxy-20-hydroxymethylpregna-1,4-dien-3-one (6), (20S)-11a,17a-dihydroxy-20-hydroxymethylpregna-1,4-dien-3-one (7), and(20S)-14a,15b,17a-trihydroxy-20-hydroxymethylpregna-1,4-dien-3-one (8). Compounds 4 (8.6%), and 6 (2%) were obtained as majormetabolites, while compounds 2 (0.40%), 3 (0.43%), 5 (0.6%), 7(0.38%), and 8 (0.32%) were obtained as minor metabolites. A timecourse analysis of transformation of compound 1 with C. elegans re-vealed that metabolites 3, and 4 were formed after 4 days of incuba-tion. Metabolites 2, and 5–7, were obtained after 6 days, whilemetabolite 8 was obtained after 8 days of incubation. The resultsdemonstrated that C. elegans predominantly catalyzed the hydroxyl-ation at C-6b, C-11a, C-15b, C-14a, and C-17a positions. Interest-ingly, fermentation with C. elegans also resulted in the acetylationof primary alcohol of metabolite 4 and afforded a slightly less polarcompound 2. The evidence for the formation of metabolite 2 was fur-ther deduced by the incubation of compound 4 with C. elegans underthe same incubation conditions, which afforded metabolites 2, and5–7. Metabolite 3 was not detected during fermentation of 4 by C.elegans. This small scale experiment suggested that compound 1was first converted to 4, which was then further transformed tometabolites 2, and 5–7, so the major route of transformation initi-ated through C-11a hydroxylation. Previously, in the literature N-acetylation byC. elegans [22], and N-, and O-acetylations by Cunning-hamella echinulata have been reported [23]. The proposed possiblebiotransformation pathway of compound 1 by C. elegans is shown

H

H

H H

OHH

OH

1

12

6

O

H

H H

OHH

OH

+

H

H H

OHH

5 7

9

11 1317

15

18

19

20

21

oxymethylpregna-1,4-dien-3-one (1) by Gibberlla fujikuroi.

1296 M.I. Choudhary et al. / Steroids 76 (2011) 1288–1296

in Fig. 1. There are some other minor products which could not becharacterized due to their insufficient quantities for structure eluci-dation, and 37% unreacted sample was also recovered.

Fermentation of 1 with M. phaseolina yielded three new 6, 10,and 11, and two known metabolites 4, and 9 which were character-ized as (20S)-11a-hydroxy-20-hydroxymethylpregna-1,4-dien-3-one (4), (20S)-11a,15b-dihydroxy-20-hydroxymethylpregna-1,4-dien-3-one (6), (20S)-7b-hydroxy-20-hydroxymethylpregna-1,4-dien-3-one (9), (20S)-15b-hydroxy-20-hydroxymethylpregna-1,4-dien-3-one (10), and (20S)-7b,15b-dihydroxy-20-hydroxymethyl-pregna-1,4-dien-3-one (11). Incubation of compound 1 with M.phaseolina resulted in a slightly different pattern of metabolismas compared to three other fungi, hydroxylating the substrate atC-7b, C-11a, and C-15b positions. Metabolites 4 (7.5%), and 6(5%) were obtained as major, while metabolites 9 (0.67%), 10(1.125%), and 11 (0.87%) were obtained as minor products.Unreacted sample 1 was recovered (34%). The time course studyindicated that metabolite 4, 9, and 10 were obtained after 6 days,while metabolites 6, and 11 were obtained after 10 days ofincubation. A proposed metabolic pathway of 1 by M. phaseolinais suggested in Fig. 2.

On the other hand R. stolonifer metabolized compound 1 intoone new metabolite (20S)-16b-hydroxy-20-hydroxymethylpreg-na-1,4-dien-3-one (12), along with a known metabolite 4 (Fig. 3).Metabolite 4 (2%) was obtained as a major compounds, whilemetabolite 12 (0.81%) was obtained in a minor quantity, togetherwith some unreacted substrate 1 (46%).

Fermentation of compound 1with G. fujikuroi mainly resulted inhydroxylations at C-6b, C-11a, C-15b, and C-16b positions andyielded metabolites 4 (2%), 5 (0.75%), 6 (1.0%), and 12 (0.54%).Unreacted sample 1 (36%) was also recovered. Time course studiesindicated that metabolite 4, and 12 were detected after 6 days,while metabolites 5, and 6 were produced after 10 days of incuba-tion. A proposed metabolic pathway of 1 by G. fujikuroi is presentedin Fig. 4.

There were some more transformed products in crude mixtureof each fungi, but due to their grossly insufficient quantities forpurification and structure elucidation they could not becharacterized.

During the preliminary screening, substrate 1 was found to ex-hibit a strong cytotoxicity against the HeLa cancer cell line with theIC50 value of 2.52 ± 0.20 lM. This is the first report of the anti-tu-mor activity of compound 1 against HeLa cancer cell line. Metabo-lites 4, 5, 6, 10, and 11 were obtained from the microbialtransformation of 1 in sufficient amounts and were screened fortheir anticancer activities. Among the polar metabolites, onlymetabolite 10 showed a moderate cytotoxic activity with an IC50

value of 35.02 ± 0.60 lM.In conclusion, this is the first report on the biotransformation of

(20S)-20-hydroxymethylpregna-1,4-dien-3-one (1) with C. elegans,M. phaseolina, R. stolonifer, and G. fujikuroi, which provided efficientroutes for the mono-, di-, and tri-hydroxylations of the substrate 1in rings B, C, and D. It is worth reporting here that these fungimainly hydroxylated the substrate 1 at C-6b, C-7b, C-11a, C-14a,C-15b, C-16b, and C-17a positions. Metabolites 4, 5, 6, 10, and 11were characterized as the main metabolites, obtained by hydroxyl-ation of the substrate predominantly at C-11a, and C-15bpositions.

Acknowledgment

This work is funded by a financial support through, ‘‘IndigenousPh.D. Fellowship Program (5000 Scholarships)’’ from the HigherEducation Commission, Islamabad, Pakistan.

References

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