Macrophomina Phaseolina Cell Suspension Cultures†

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Seven new metabolites of drostanolone heptanoate by using Beauveria bassiana, and Cite this: RSC Adv., 2020, 10,451 Macrophomina phaseolina cell suspension cultures†

Zahid Hussain,a Atia-tul-Wahab, *b Nusrat Hussain,ac Shabbir Hussain,ad Atta-ur- Rahmana and M. Iqbal Choudhary *abe

The present study reports the biotransformation of an anabolic-androgenic steroid (AAS) drostanolone heptanoate (1) by using two microbial cultures, Beauveria bassiana, and Macrophomina phaseolina. Fermentation of 1 with B. bassiana yielded ﬁve new transformed products 2–6, while with M. phaseolina Received 29th July 2019 it aﬀorded two new 7–8, and two known 9–10 metabolites. The main sites of hydroxylation in the Accepted 6th December 2019 steroidal skeleton of 1 were at C-5, C-7, C-11, C-14, C-15, and C-20, hydrolysis of the ester moiety at C- DOI: 10.1039/c9ra05878h 17, and reduction of the carbonyl group at C-3. The structures of the transformed products were Creative Commons Attribution-NonCommercial 3.0 Unported Licence. rsc.li/rsc-advances determined by using mass, NMR, and other spectroscopic techniques.

1 Introduction Drostanolone heptanoate ( )(C27H44O3) is a dihydrotestosterone (DHT)-derived anabolic androgenic steroid (AAS). Athletes, and body Steroids constitute an important class of biologically active builders use it to increase muscles strength, and body mass without 6 compounds due to their anabolic, anti-inammatory, anti- gaining fat. However, due to its adverse eﬀects on liver, serum lipid, microbial, anti-leishmanial, and anti-cancer properties. There- and reproductive system, the use of drostanolone heptanoate (1), fore, there have been sustained eﬀorts for the synthesis of new, and other anabolic androgenic steroids have now been discontinued This article is licensed under a 7 and derivatizations of existing steroidal drugs with desired by the International Olympic Committee (IOC). In contrary, many pharmacodynamic proles. Currently a bio-catalytic approach research groups have studied the treatment of estrogen-dependent 8–11 has largely been employed for the structural modications of breast carcinoma using 1 as an aromatase inhibitor.

Open Access Article. Published on 02 January 2020. Downloaded 9/25/2021 7:07:21 PM. steroids, particularly due to the ability of microorganisms to Several bio-transformed products of drostanolone from the catalyze functionalization at almost every site of the inert urine of drostanolone-dosed rabbits have been reported in steroidal skeleton.1–3 Moreover, microorganisms can grow, and literature, including hydroxylations at C-15, C-16, and C-17, 12 replicate quickly, and produce a variety of enzymes under reduction at C-3, and oxidation at C-17. Similarly, several ambient conditions. Biotransformations have been successfully metabolites of drostanolone on incubation with cryopreserved 13 employed where chemical methods were found to be diﬃcult human hepatocytes are also reported. and expensive. In addition, the products obtained are highly In continuation of our structural transformation studies on 14–20 regio-, enantio-, and stereo-selective, and the procedures are steroidal drugs, biotransformation of drostanolone hepta- eco-friendly, and economical.4,5 noate (1) was carried out. Based on small scale screening results, compound 1 was subjected to biotransformation by using two microbial cell cultures, Beauveria bassiana, and Macrophomina phaseolina, for the rst time, yielding seven new, aH. E. J. Research Institute of Chemistry, International Center for Chemical and and two known compounds. The purpose of this study was to Biological Sciences, University of Karachi, Karachi-75270, Pakistan. E-mail: iqbal. synthesize structurally diverse analogues of drostanolone hep- [email protected]; Fax: +922134824924-5; Tel: +922134824924-5 tanoate (1) for their potential use in the biomedical research, by b Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center employing mild, and low-cost biotransformation procedures. for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan cDepartment of Chemistry, University of Baltistan Skardu, 16100, Pakistan dDepartment of Chemistry, Karakoram International University, Gilgit-15100, Gilgit- Baltistan, Pakistan Experimental eDepartment of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah- General 21412, Saudi Arabia † Electronic supplementary information (ESI) available. See DOI: All the chemicals used for microbial transformation were 10.1039/c9ra05878h purchased from the Sigma Aldrich (Germany), and Oxoid

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Limited (UK), whereas drostanolone heptanoate (1) was constant ow rate of 4 mL min 1 was maintained throughout purchased from the Shanghai Bettersyn Biotech Co., Ltd., the analysis to isolate pure metabolites 2–10. Shanghai (China). Silica gel pre-coated thin layer chromatog- 2a-Methyl-7a,11a,17b-trihydroxy-5a-androstan-3-one (2). – a 25 ¼ raphy (TLC) plates (E. Merck, Germany), and silica gel column White solid; melting point: 230 233 C; [ ]D 46.2 (c 0.017, 1 chromatography (Germany) were used for analysis, and frac- MeOH); IR (CHCl3) lmax (cm ): 3475, 3330, 3326 (3 O–H),

tionations (initial purication), while preparative recycling 1687 (C]O); retention time (min): 17 (MeOH : H2O; 75 : 25); reverse phase HPLC-LC-908, tted with JAIGEL-ODS-L-80 HRFAB-MS m/z 337.2356 ([M + H] +; calcd. 336.2301); FAB-MS m/ + 1 13 columns (Japan), was used for the nal purication of trans- z 337 [M + H] ; H-NMR (CD3OD, 300 MHz): Table 1; C-NMR

formed products. The FAB-, and HRFAB-MS (JEOL HX110) (CD3OD, 100 MHz): Table 2. (Japan), and ESI-, and HRESI-MS (Bruker, Maxis II) (Germany) 2a-Methyl-7a,15a,17b-trihydroxy-5a-androstan-3-one (3). – a 25 ¼ techniques were used to deduce the molecular formulae of all Colourless solid; melting point: 236 238 C; [ ]D +25 (c 1 13 l 1 – compounds. The H-, and the C-NMR spectra were recorded 0.028, MeOH); IR (CHCl3) max (cm ): 3733, 3375, 3278 (3 O on Bruker Avance spectrometers (300–600 MHz) (Switzerland). H), 1687 (C]O); retention time (min): 19 (MeOH : H2O; 75 : 25); The melting points were recorded using Stuart SMP-10 instru- HRFAB-MS m/z 337.2372 ([M + H]+; calcd. 336.2301); FAB-MS m/ + 1 13 ment (Switzerland). The JASCO P-200 polarimeter was used to z 337 [M + H] ; H-NMR (CD3OD, 300 MHz): Table 1; C-NMR

measure the optical rotations, while the IR data were recorded (CD3OD, 150 MHz): Table 2.

using KBr disks in CHCl3 on the Bruker Vector 22 FT-IR spec- 2a-Hydroxymethyl-11a,17b-dihydroxy-5a-androstan-3-one (4). – a 25 ¼ trometer (Japan). Molibdophosphoric acid (MPA) was used as White solid; melting point: 145 147 C; [ ]D +43.8 (c l 1 – a staining reagent to detect the transformed products on TLCs. 0.021, MeOH); IR (CHCl3) max (cm ): 3737, 3410, 3369 (3 O ] H), 1703 (C O); retention time (min): 18 (MeOH : H2O; 70 : 30); Microbial cultures, and media preparation HRFAB-MS m/z 337.2366 ([M + H]+; calcd. 336.2301); FAB-MS m/ z 337 [M + H]+; 1H-NMR (CD3OD, 300 MHz): Table 1; 13C-NMR Beauveria bassiana (ATCC-7159) was purchased from the

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. (CD3OD, 100 MHz): Table 2. American Type Culture Collection, while Macrophomina pha- 2a-Methyl-7b,11a,17b-trihydroxy-5a-androstan-3-one (5). seolina (KUCC-730) was obtained from the Karachi University 25 Colourless solid; melting point: 202–205 C; [a]D ¼ +78.7 (c Culture Collection. Both cultures were grown on Sabouraud 0.024, MeOH); IR (CHCl ) l (cm 1): 3743, 3429, 3375 (3 O– dextrose agar slants, and maintained at 4 C. 3 max H), 1699 (C]O); retention time (min): 26 (MeOH : H O; 70 : 30); The culture media for the growth of fungi was prepared by 2 HRFAB-MS m/z 337.2365 ([M + H]+; calcd. 336.2301); FAB-MS m/ adding 50 g glucose, 25 g peptone, 25 g yeast extract, 25 g + 1 13 z 337 [M + H] ; H-NMR (CD3OD, 300 MHz): Table 1; C-NMR KH2PO4, 25 g NaCl, and 50 mL glycerol, in distilled water. The (CD3OD, 100 MHz): Table 2. quantity, and recipe for both cultures were the same. 2a-Methyl-11b,15a,17b-trihydroxy-5a-androstan-3-one (6).

This article is licensed under a 25 Colourless solid; melting point: 244–246 C; [a]D ¼ +27.4 (c General fermentation, extraction, and purication protocol 1 0.028, MeOH); IR (CHCl3) lmax (cm ): 3733, 3421, 3305 (3 O– ] The media for the growth of microorganisms was prepared by H), 1689 (C O); retention time (min): 24 (MeOH : H2O; 70 : 30);  +

Open Access Article. Published on 02 January 2020. Downloaded 9/25/2021 7:07:21 PM. adding the speci c amounts of media ingredients in distilled HRFAB-MS m/z 337.2372 ([M + H] ; calcd. 336.2301); FAB-MS m/ + 1 13 water. Media was then transferred to 100 mL Erlenmeyer asks, z 337 [M + H] ; H-NMR (CD3OD, 300 MHz): Table 1; C-NMR cotton plugged, and autoclaved at 121 C, followed by inocula- (CD3OD, 100 MHz): Table 2. tion of spores of fungi from mycelia on SDA slants under ster- 2a-Methyl-3b,5a,17b-trihydroxy-5a-androstane (7). White 25 ilized conditions. Flasks were placed on a rotary shaker at 26 C solid; melting point: 248–251 C; [a]D ¼113 (c 0.013, MeOH); 1 for the maximum growth of fungi. Aer suitable growth, IR (CH3Cl) lmax (cm ): 3373 (broad O–H); retention time (min): + substrate 1 was dissolved in acetone, and equally distributed in 21 (MeOH : H2O; 75 : 25); HRESI-MS m/z 340.2851 ([M + NH4] ; + each fungus-containing asks, and again placed on a rotary calcd. 322.2508); ESI-MS m/z 340.3 ([M + NH4] ; 304 [M H2- + 1 13 shaker at 26 C with 125 rpm for the fermentation process. A O] ); H-NMR (CD3OD, 300 MHz): Table 1; C-NMR (CD3OD, positive control (only substrate in media), and a negative 150 MHz): Table 2. control (microbial culture in media) were prepared to identify 2a-Methyl-3b,14a,17b-trihydroxy-5a-androstane (8). White 25 the degradation of substrate in media, and fungal metabolites, solid; melting point: 272–275 C; [a]D ¼ +21.8 (c 0.02, MeOH); 1 respectively. The fermentation reaction was stopped by adding IR (CHCl3) lmax (cm ): 3377 (O–H); retention time (min): 27 + dichloromethane to each ask, followed by ltration of fungal (MeOH : H2O; 75 : 25); HRESI-MS m/z 323.2586 ([M + H] ; calcd. + 1 biomass. The ltrate was then extracted with dichloromethane, 322.2508); ESI-MS m/z 323.3 [M + H] ; H-NMR (CD3OD, 400 13 dried by adding anhydrous sodium sulfate, ltered, and evap- MHz): Table 1; C-NMR (CD3OD, 100 MHz): Table 2. orated under high vacuum to aﬀord a solid crude material. This 2a-Methyl-17b-hydroxy-5a-androstan-3-one (9). White solid; – a 25 ¼ crude material was then fractionated through silica gel column melting point: 150 152 C; [ ]D +31.9 (c 0.007, MeOH); IR 1 chromatography with gradient hexanes, and acetone elution. (CHCl3) lmax (cm ): 3425 (O–H), 1706 (C]O); HRESI-MS m/z Aer TLC analysis, the fractions of similar polarities were 305.2480 ([M + H]+; calcd. 304.2402); ESI-MS m/z 305.2 [M + H]+; + 1 13 combined together into ten main fractions. These combined 287.2 ([M + H] –H2O); H-NMR (CD3OD, 300 MHz): Table 1; C-

fractions were then subjected to preparative recycling RP-HPLC, NMR (CD3OD, 100 MHz): Table 2. using an isocratic methanol and water solvent systems. A

452 | RSC Adv.,2020,10,451–460 This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 02 January 2020. Downloaded on 9/25/2021 7:07:21 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Paper hsjunli h oa oit fCeity2020 Chemistry of Society Royal The © is journal This 1 Table 1 H-NMR chemical shift assignments of compounds 1–10 (dH, multiplicity [J ¼ Hz])

Positions 12345678910

1 2.08 dd (J ¼ 12.6, 2.44 dd (J ¼ 13.8, 2.10 m; 1.00 m 2.91 dd (J ¼ 13.8, 2.54 br. t (J ¼ 2.44 br. t (J ¼ 1.61 m; 1.04 1.35 m; 1.00 2.08 dd (J ¼ 1.34 m; 1.05 5.7); 1.05 m 5.8); 1.98 m 6.0); 1.32 m 13.8); 1.88 dd 13.8); 1.16 m m br. t (J ¼ 12.8) 13.2, 6.0); m (J ¼ 13.8, 3.0) 1.04 m 2 2.55 m 2.58 m 2.54 m 2.63 m 2.58 m 2.56 m 1.42 overlap 1.41 m 2.55 m 1.51 m 3 ——————3.67 m 3.70 m — 3.69 m 4 2.42 br. t (J ¼ 2.93 d (J ¼ 6.0); 2.46 m; 2.08 m 2.44 br. t (J ¼ 2.82 dd (J ¼ 2.87 dd (J ¼ 13.8, 1.50 overlap; 1.51 m; 1.39 2.87 br. t (J ¼ 1.82 m; 1.54 13.8); 2.00 dd (J 1.19 m 14.1); 2.00 3.8, 4.8); 1.25 5.7); 1.20 m 1.45 m m 13.5); 1.99 m m ¼ 14.1, 3.3) overlap m 5 1.54 m 1.60 m 1.57 m 1.57 m 1.49 m 1.55 m — 1.33 m 0.91 m 0.75 m 6 1.75 m; 1.31 1.56 m; 1.42 m 1.63 m; 1.44 m 1.40 m; 1.37 m 1.57 overlap; 1.37 m; 1.33 m 1.21 m; 1.18 1.41 m; 1.25 1.33 m; 1.30 1.61 m; 1.28 overlap 1.43 m m m m m 7 1.58 m; 1.32 3.24 m 3.30 m 1.55 m; 1.26 m 3.77 m 2.06 m; 1.13 m 1.63 m; 1.53 1.53 m; 1.36 1.74 m; 0.88 1.21 m; 1.16 overlap m m m m 8 1.50 m 1.66 m 1.60 m 1.52 m 1.52 m 1.67 m 1.43 m 1.71 m 1.46 overlap 1.42 m 9 0.77 m 0.88 br. t (J ¼ 0.82 m 0.86 br. t (J ¼ 1.42 m 0.86 br. t (J ¼ 1.73 m 1.48 m 1.47 overlap 1.66 m 10.2) 10.2) 10.2) 10 —————————— 11 1.40 m; 1.33 3.92 dt (J ¼ 10.5, 1.67 m; 1.66 m 3.93 dt (J ¼ 10.8, 3.88 dt 3.87 m 1.51 overlap; 1.56 m; 1.32 1.56 m; 1.40 1.67 m; 0.89 overlap 5.1) 5.0) (J ¼ 10.8, 5.1) 1.41 overlap m m m 12 1.18 dt (J ¼ 12.9, 2.08 dd (J ¼ 12.0, 1.82 m; 1.10 m 2.11 dd (J ¼ 12.0, 2.06 overlap; 2.07 overlap; 1.57 m; 1.44 1.43 m; 1.25 1.85 m; 1.01 1.42 m; 1.00 3.9); 1.71 m 4.8); 1.10 m 4.8); 1.10 m 1.07 br. t 1.16 m m m m m (J ¼ 11.7) 13 —————————— 14 1.08 m 1.20 m 1.03 m 1.08 m 1.40 m 1.05 m 1.55 m — 0.91 m 0.95 m 15 2.12 m; 1.47 m 1.88 m; 1.67 m 4.06 dt (J ¼ 9.0, 1.73 m; 1.70 m 1.68 m; 1.26 3.90 m 1.37 m; 1.33 1.64 m; 1.51 3.90 m 1.57 m; 1.24 4.8) m m m m 16 1.52 m; 1.47 m 1.95 m; 1.44 m 2.04 m; 1.93 m 2.02 overlap; 2.01 m; 1.56 2.05 overlap; 1.98 m; 1.46 2.14 m; 1.52 1.53 m; 1.26 1.94 m; 1.46 1.48 m overlap 1.84 m m m m m 17 4.58 t (J ¼ 8.7) 3.55 t (J ¼ 8.4) 3.74 t (J ¼ 9.0) 3.59 t (J ¼ 8.7) 3.60 t (J ¼ 8.4) 3.78 t (J ¼ 9.0) 3.61 t (J ¼ 8.4) 4.20 t (J ¼ 7.2) 3.55 t (J ¼ 8.7) 3.54 t (J ¼ 8.4) 18 0.83 s 0.77 s 0.78 s 0.76 s 0.75 s 0.76 s 0.72 s 0.81 s 0.74 s 0.71 s 19 1.12 s 1.24 s 1.14 s 1.22 s 1.20 s 1.23 s 0.94 s 0.83 s 1.11 s 0.82 s 20 0.96 d (J ¼ 6.3) 0.94 d (J ¼ 6.6) 0.97 d (J ¼ 6.3) 3.75 dd (J ¼ 10.8, 0.93 d 0.93 d (J ¼ 6.3) 0.91 d (J ¼ 0.91 d (J ¼ 0.94 d (J ¼ 0.91 d 5.1); 3.47 dd (J ¼ 6.3) 5.4) 6.8) 6.6) (J ¼ 6.9) (J ¼ 11.1, 5.7) S Adv. RSC 21 ————— 22 2.29 t (J ¼ 7.2) ———— 23 1.59 m; 1.32 ———— ,2020, overlap 24 1.38 overlap; ———— 1.33 overlap 10 ———— Advances RSC ,451 25 1.30 overlap; View ArticleOnline 1.29 overlap –

6 | 460 26 1.34 overlap; ———— 1.30 overlap 27 0.90 t (J ¼ 6.9) ———— 453 View Article Online RSC Advances Paper

Table 2 13C-NMR chemical shift assignments of compounds 1–10

Carbons 12345678910

1 49.8 45.6 49.3 45.7 45.6 46.1 35.1 42.3 49.9 42.2 2 42.1 42.2 42.0 49.9 42.8 42.3 31.3 33.1 42.1 40.0 3 215.4 215.5 215.0 214.3 215.5 215.9 71.3 71.4 215.6 71.4 4 45.5 51.2 45.0 46.3 51.4 51.7 38.0 37.8 45.6 38.1 5 36.3 47.4 46.6 50.2 42.8 50.5 77.0 48.8 55.2 56.1 6 32.4 40.2 37.8 30.3 38.0 30.4 29.3 29.2 29.8 21.5 7 26.2 75.0 74.8 24.2 67.4 33.0 27.5 27.0 32.5 29.4 8 49.4 43.3 44.8 36.1 40.2 35.9 33.2 40.2 36.6 36.6 9 55.2 59.1 58.5 60.9 52.9 61.0 49.6 39.7 49.6 33.1 10 37.3 38.8 45.8 39.0 44.1 39.3 42.4 37.6 37.7 37.4 11 29.8 69.6 22.1 69.5 69.7 69.2 24.1 20.6 22.2 32.9 12 38.2 49.4 38.2 49.0 48.5 49.6 33.4 30.0 38.0 37.8 13 43.9 44.9 58.4 44.4 49.6 45.6 44.1 49.0 44.1 44.1 14 51.9 51.1 52.1 51.3 45.9 58.5 44.8 84.8 52.2 52.4 15 28.5 27.2 73.1 32.4 23.4 72.5 26.2 32.7 72.5 24.3 16 23.5 31.1 40.5 30.7 30.6 43.0 30.6 30.1 24.3 30.6 17 84.0 81.8 79.5 81.9 82.0 79.2 82.3 79.6 79.2 82.5 18 12.6 12.6 13.3 12.5 12.3 13.9 10.8 15.7 11.6 11.7 19 14.2 12.9 12.7 12.9 11.9 13.0 14.7 12.3 12.6 12.6 20 14.9 15.0 14.9 62.5 15.0 15.0 19.2 19.0 15.0 19.0 21 175.5 ————————— 22 35.3 ————————— 23 26.2 —————————

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 24 29.8 ————————— 25 32.6 ————————— 26 23.5 ————————— 27 14.3 —————————

2a-Methyl-3b,17b-dihydroxy-5a-androstane (10). White Similarly, downeld signals for oxymethine carbons were 25 13 d solid; melting point: 240–242 C; [a]D ¼ +19.6 (c 0.026, MeOH); observed in the C-NMR spectrum at 81.8, 75.0, and 69.6. In l 1 – 2 IR (CHCl3) max (cm ): 3425 (broad O H); HRESI-MS m/z 324. the HMBC spectrum of compound (Fig. 1), H-12 showed

This article is licensed under a + + 2902 ([M + NH4] ; calcd. 306.2599); ESI-MS m/z 324.3 [M + NH4] ; HMBC interactions with C-9, C-11, C-14, C-17, and C-18, sup- 1 13 H-NMR (CD3OD, 300 MHz): Table 1; C-NMR (CD3OD, 125 porting the positions of both OH groups at C-11, and C-17. MHz): Table 2. Similarly, H-9 showed cross peaks with C-7, C-8, C-10, C-11, and C-14, suggested OH groups at C-7, and C-11. The congu- Open Access Article. Published on 02 January 2020. Downloaded 9/25/2021 7:07:21 PM. Results and discussion ration of H-7, H-11, and H-17 were deduced as b, b, and a (Fig. 2), based on NOESY correlations of H-7 with b-oriented

Biotransformation of drostanolone heptanoate (1) with cell H-8, and H-11 with b-oriented H3-19, and H3-18 (Fig. 2). Thus suspension cultures of B. bassiana, and M. phaseolina produced the structure of new metabolite was determined as 2a-methyl- nine transformed products 2–10. B. bassiana yielded ve new 7a,11a,17b-trihydroxy-5a-androstan-3-one (2). metabolites 2–6 (Scheme 1), whereas M. phaseolina aﬀorded two Compound 3 was obtained as optically active, colorless solid new 7 and 8, and two known 9 and 10 metabolites (Scheme 2). by using recycling RP-HPLC. HRFAB-MS showed the [M + H]+ at

The structures of new compounds were deduced by using m/z 337.2372 (calcd. 336.2301, C20H32O4), suggesting hydrolysis diﬀerent spectroscopic techniques, and by comparing their of ester group, and addition of two oxygen atoms. This was 24 spectral data with substrate 1. The structures of known amu lesser than substrate 1. IR showed absorption bands at 1 metabolites were also determined by comparing their spectro- nmax (cm ) 3733, 3375, 3278, and 1687 for three OH, and scopic data with the reported data. a carbonyl groups. The 1H-, and the 13C-NMR spectra of Metabolite 2 was puried as optically active, colorless solid compounds 2, and 3 were found to be distinctly similar. In the through recycling RP-HPLC. HRFAB-MS showed the [M + H]+ at 1H-NMR spectrum, three downeld methine signals at d 4.06 ¼ ¼ ¼ m/z 337.2356 (calcd. 336.2301, C20H32O4), 24 amu lesser than (dt, J15b,16a/b 9.0 Hz, J15b,14a 4.8 Hz), 3.74 (t, J17a,16a/b 9.0 substrate 1. This suggested hydrolysis of ester group, and Hz), and 3.30 (m) were assigned to the protons geminal to the 1 13 addition of two oxygen atoms. Absorption bands at nmax (cm ) OH groups. Consistent with this observations, the C-NMR 3475, 3330, 3326, and 1687 were observed in the IR spectra for spectrum showed methine carbons resonances at d 79.5, 74.8, three OH, and a carbonyl groups. In the 1H-NMR spectrum, and 73.1. However, the HMBC, COSY, and NOESY spectra of  d ¼ ﬀ 3 three down eld methine signals at 3.92 (dt, J11a,9a/12a 10.5 both compounds were found di erent. Compound displayed Hz, J11a,12e ¼ 5.1 Hz), 3.55 (t, J17a,16a/b ¼ 8.4 Hz), and 3.24 (m) HMBC interactions (Fig. 1) of H-9 with C-5, C-7, C-10, and C-19, were appeared, indicating presence of hydroxyl groups. suggesting one of the OH groups at C-7. Similarly, the other two

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Scheme 1 Biotransformation of drostanolone heptanoate (1) with B. bassiana.

a OH groups were placed at C-15, and C-17, based on the HMBC axially-oriented H3-18, and H-17 with -oriented H-14 (Fig. 2). correlations of H-16 with C-13, C-15, and C-17. The congura- Thus the structure of new compound was deduced as 2a- tion of H-7, H-15, and H-17 were deduced as b, b, and, based on methyl-7a,15a,17b-trihydroxy-5a-androstan-3-one (3). NOESY correlations of H-7 with b-oriented H-8, H-15 with

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Scheme 2 Biotransformation of drostanolone heptanoate (1) with M. phaseolina.

Metabolite 4 was obtained as a white solid using recycling Hz) were appeared in the 1H-NMR spectrum. This was further RP-HPLC. The HRFAB-MS of 4 showed the [M + H]+ at m/z established through the HMBC correlations (Fig. 1). The HMBC

337.2366 (calcd. 336.2301, C20H32O4) suggesting the addition of correlations between H-1 and C-2, C-3, C-5, C-10, and C-20, and two oxygen atoms, along with hydrolytic loss of ester moiety. from H-12 to C-9, C-11, C-13, C-14, and C-17 were observed. The IR spectrum revealed the presence of three OH (3737, 3410, These correlations supported the OH groups at C-11, C-17, and and 3369 cm 1), and a carbonyl (1703 cm 1) groups. Signal for C-20. In the COSY spectrum (Fig. 2), the homonuclear 1H–1H the C-20 methyl protons in the 1H-NMR spectrum was also couplings were observed between H-11, and H-9, and H-12,

found missing. An additional downeld signal at d 62.5 for C-20 while H-2 showed the COSY cross peaks with H-1, and H2-20. 13 ¼ ¼ in the C-NMR spectrum also indicated the oxidation of the The coupling constant of H-11 (dt, J11a,9a/12a 10.8 Hz, J11a,12a –  d methyl group as CH2 OH. New down eld signals for H-11 at 5.0 Hz), and its NOESY correlations with H3-18 ( 0.76 s), d ¼ ¼ d a 3.93 (dt, J11a,9a/12a 10.8 Hz, J11a,12a 5.0 Hz), H-17 at 3.59 (t, implied that OH group at C-11 was equatorial ( ) (Fig. 2). J17a,16a/b ¼ 8.7 Hz), and H2-20 at d 3.75 (dd, J20a,20b ¼ 10.8 Hz, Likewise, H-17 showed NOESY cross peaks with H-14, indi-

J20a,2b ¼5.1 Hz), and d 3.47 (dd, J20b,20a ¼ 11.1 Hz, J20b,2b ¼ 5.7 cating a b-orientation of the OH group at C-17. This new

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Fig. 1 Key HMBC interactions of metabolites 2–8.

metabolite was thus identied as 2a-hydroxymethyl-11a,17b- 3743, 3429, 3375, and 1699, for OHs, and a carbonyl, respec- dihydroxy-5a-androstan-3-one (4). tively. The 1H-, and 13C-NMR spectra of compound 5, were Compound 5 was isolated as colorless solid through recy- found to be distinctly similar to compounds 2, and 3. Three new cling RP-HPLC with a retention time of 26 min. It showed the [M downeld signals for oxymethine protons were observed at + +H] at m/z 337.2365 (calcd. 336.2301, C20H32O4) in the HRFAB- d 3.88 (dt, J11a,9a/12a ¼ 10.8 Hz, J11a,12e ¼5.1 Hz), 3.77 (m), and 1 1 MS. The IR spectrum showed absorption bands at ymax (cm ) 3.60 (t, J17a,16a/b ¼ 8.4 Hz) in the H-NMR spectrum. Similarly,

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Fig. 2 Key COSY, and NOESY interactions in metabolites 2–8.

13C-NMR spectrum of 5 showed downeld signals for oxy- was thus deduced as 2a-methyl-7b,11a,17b-trihydroxy-5a- methine carbons at d 69.7, 67.4, and 82.0. The positions of OH androstan-3-one (5). groups at C-11, and C-17 were deduced through the HMBC Metabolites 6 was obtained as colorless solid by using RP- correlations (Fig. 1) of H-12 (d 2.06, overlap; d 1.07 br. t; HPLC with a retention time of 24 min. The molecular formula

J12a,12e/11a ¼ 11.7 Hz) with C-9 (d 52.9), C-11 (d 69.7), C-13 of compound 6 was consistent with C20H32O4, deduced from the (d 49.6), C-14 (d 45.9), and C-17 (d 82.0) indicated the OH HRFAB-MS at m/z 337.2365 (calcd. 336.2301; [M + H]+). y 1 groups at C-11, and C-17. Similarly, OH group at C-5 was deter- Absorption bands in the IR spectrum at max (cm ) 3733, 3421, mined through the HMBC correlations of H-5 (d 1.52, m) with C-4 3305, and 1689, were due to the presence of three OHs, and (d 51.4), C-7 (d 67.4), and C-10 (d 44.1) (Fig. 1). An OH group at C- a carbonyl, respectively. The 1H-, and 13C-NMR spectra of 11 was deduced as a on the basis of NOESY interactions (Fig. 2) of compounds 5, and 6 were found to be distinctly similar to 1 H-11 (d 3.88 dt, J11a,9a/12a ¼ 10.8 Hz, J11a,12e ¼ 5.1 Hz) with axially- compounds 2, and 3. The H-NMR spectrum showed three new

oriented H3-18 (d 0.75 s), and H3-19 (d 1.20 s). Similarly, the b- downeld signals for methine protons at d 3.78 (t, J17a,16a/b ¼ 9.0 orientations of OH groups at C-7, and C-17 in compound 5 were Hz), 3.87 (m), and 3.90 (m). Signals for deshielded methine deduced through the key NOESY correlations of axially-oriented carbons at d 69.2, 72.5, and 79.2 were appeared in the 13C-NMR H-5 with H-7, and axially-oriented H-14 with H-17 (d 3.60 t, spectrum of 6. This suggested the presence of three OH groups.

J17a,16a/b ¼ 8.4 Hz), respectively. The structure of a new metabolite OH Groups at C-11, C-17 were deduced through the HMBC

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correlations (Fig. 1) of H-12 (d 2.07 overlap; d 1.16 m) with C-9 (d rabbits.12 The spectroscopic data of metabolites 9, and 10 was 61.0), C-11 (d 69.2), C-13 (d 45.6), C-14 (d 58.5), and C-17 (d 79.2), consistent with the literature data. Their structures were iden- whereas, OH group at C-15 was determined through the HMBC tied as 2a-methyl-17b-hydroxy-5a-androstan-3-one (9), and 2a- correlations of H-16 with C-13, C-14, C-15, and C-17. In the methyl-3b,17b-dihydroxy-5a-androstane (10). NOESY spectrum (Fig. 2) of compound 6, cross peaks were observed between axially-oriented H-14, and H-17 suggesting b- orientation of OH group at C-17. Similarly, b-stereochemistry of Conclusion OH groups at C-11, and C-15 were also deduced from the NOESY Microbial biotechnology has wide applications in the synthesis correlations of H-11 with axially-oriented H-9, and H-15 with of libraries of structurally diverse analogues of bioactive natural axially-oriented H-8 and H -18. The structure of a new metab- 3 products, and drugs. The present study aﬀorded seven new, and olite was thus deduced as 2a-methyl-11b,15a,17b-trihydroxy-5a- two known metabolites of drostanolone heptanoate (1) by using androstan-3-one (6). two microbial cultures, Beauveria bassiana, and Macrophomina Compound 7 was puried as a white solid through recycling phaseolina. Incubation of 1 with B. bassiana led to the synthesis RP-HPLC with a retention time of 21 min. The HRESI-MS of of ve new metabolites 2–6, whereas M. phaseolina aﬀorded two metabolite 7 showed the [M + NH ]+ at m/z 340.2851 (calcd. 4 new 7, and 8, and two known transformed products 9, and 10. 322.2508, C H O ). This suggested dihydroxylation, along 20 32 3 Hydroxylation, reduction, and hydrolysis were the main reac- with hydrolysis of side chain ester group. The IR spectrum tions during structural transformation of 1. The transformed showed broad absorbances at y 3373 cm 1 for OH groups. max products will be evaluated for potential biological activities in Absence of the bridge methine signals in the 1H-, and the 13C- the future. In addition, metabolites 2–10 will also be studied at NMR spectra of 7 suggested the hydroxylation at tertiary enzymatic level to understand the mechanism involved during carbon. An OH group at C-5 was deduced through the HMBC biocatalysis. correlations of H-3, H3-19, H2-6, and H-9 with C-5 (Fig. 1). b

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Stereochemistry of OH group at C-3 was assigned as , based on NOESY correlations of axially-oriented H-3 with equatorially- Conﬂicts of interest oriented H3-20 (Fig. 2). An OH group at C-5 (d 77.0) was placed as a, based on the comparison of the reported chemical Authors has no conicts of interest. shis for 5a-OH in steroids.15,20 Thus the structure of a new metabolite was identied as 2a-methyl-3b,5a,17b-trihydroxy- androstane (7). Acknowledgements Metabolite 8 was obtained as a white solid by using RP-HPLC Authors acknowledge the nancial support of the Higher with a retention time of 27 min. The HRESI-MS of 8 showed the Education Commission Pakistan through a project of National This article is licensed under a [M + H]+ at m/z at 323.2586 (calcd. 322.2508), indicating Research Program for Universities (NRPU) (# 7993) entitled, hydrolysis of side chain ester group, along with dihydroxylation. “  y 1 Synthesis of new analogues of anti-in ammatory agents via Broad absorption bands at max 3377 cm in the IR spectrum ” 1 13 microbial biotechnological methods .

Open Access Article. Published on 02 January 2020. Downloaded 9/25/2021 7:07:21 PM. were due to the OH groups. The H-, and C-NMR spectra of metabolite 7 were apparently identical to 8. Absence of the bridge methine signals in the 1H-, and 13C-NMR spectra of 8 References suggested the hydroxylation at tertiary carbon. An OH group at C-14 was deduced through the HMBC correlations of H-17, H-8, 1 K. Kontula, O. Janne, R. Vihko, E. de Jager, J. de Visser and

H2-15, H2-16, and H3-18 with C-14 (Fig. 1). The relative stereo- F. Zeelen, Progesterone-binding proteins, in vitro binding chemistry at C-3 in compound 8 was deduced from the NOESY and biological activity of diﬀerent steroidal ligands, Acta interactions between axially-oriented H-3, and equatorially- Endocrinol., 1975, 78, 574–592.

oriented H3-20 (Fig. 2). Similarly, OH group at C-14 was 2 T. Horiike, M. Ohshiro and M. Kuroyanagi, deduced as a on the basis of reported chemical shi for 14a-OH Biotransformation of the germacrane type sesquiterpene in similar steroidal compound.18 Thus the structure of a new curdione by suspension cultured cells of Lonicera japonica, compound was deduced as 2a-methyl-3b,14a,17b-trihydroxy-5a- Phytochemistry, 1997, 44, 627–632. androstane (8). 3 M. I. Choudhary, S. Sultan, S. Jalil, S. Anjum, A. A. Rahman Metabolites 9, and 10 were obtained as white solids by and H. K. Fun, Atta-ur-Rahman. Microbial transformation repeated column chromatography. HRESI-MS of compound 9 of mesterolone, Chem. Biodiversity, 2005, 2, 392–400. + showed the [M + H] at m/z 305.2480 (calcd. 304.2402; C20H30O2) 4 J. E. Leresche and H. P. Meyer, Chemocatalysis and 47 amu less than the substrate 1, suggesting hydrolysis of side biocatalysis (biotransformation): Some thoughts of chain ester group. Whereas the HRESI-MS of metabolite 10 at m/ a chemist and of a biotechnologist, Org. Process Res. Dev., + z 324.2902 (calcd. 306.2599; [M + NH4] ) suggested a molecular 2006, 10(3), 572–580.

composition of C20H34O2. This indicated the reduction of 5 M. Rabinovich, A. Bolobova and L. Vasil'chenko, Fungal ketonic carbonyl group, along with hydrolysis of ester side chain decomposition of natural aromatic structures and of substrate 1. Metabolites 9, and 10 were previously reported by xenobiotics: a review, Appl. Biochem. Microbiol., 2004, 40(1), Templeton and Kim from the urine of drostanolone dosed 1–17.

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