Fungal Mediated Biotransformation of Melengestrol Acetate, and T-Cell Proliferation Inhibitory Activity of Biotransformed Compounds

Bioorganic Chemistry 104 (2020) 104313

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Fungal mediated biotransformation of melengestrol acetate, and T-cell proliferation inhibitory activity of biotransformed compounds

Saira Javed a, Atia-tul-Wahab b,*, Almas Jabeen b, Shynar Zhumagaliyeva c, Zharylkasyn A. Abilov c, Atta-ur-Rahman a, M. Iqbal Choudhary a,b,c,d,* a H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan b Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan c Al-Farabi Kazakh National University, Department of Chemistry and Chemical Technology, Almaty, Kazakhstan d Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Komplek Campus C, Surabaya 60115, Indonesia

ARTICLE INFO ABSTRACT

Keywords: Glomerella fusaroide, and Rhizopus stolonifer were effectively able to transform the steroidal hormone melen Melengestrol acetate gestrol acetate (MGA) (1) into four (4) new metabolites, 17α-acetoxy-11α-hydroxy-6-methyl-16-methyl Biotransformation enepregna-4,6-diene-3,20-dione (2), 17α-acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-1,4,6-triene-3,20- T-cell Proliferation dione (3), 17α-acetoxy-6,7α-epoxy-6β-methyl-16-methylenepregna-4,6-diene-3,20-dione (4), and 17α-acetoxy- Anti-inflammatory 11β,15β-dihydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (5). All these compounds were struc turally characterized by different spectroscopic techniques. The objective of the current study was to assess the anti-inflammatory potential of melengestrol acetate (1), and its metabolites 2–5. The metabolites and the sub strate were assessed for their inhibitory effects on proliferation of T-cells in vitro. The substrate (IC50 = 2.77 ± 0.08 µM) and its metabolites 2 (IC50 = 2.78 ± 0.07 µM), 4 (IC50 = 2.74 ± 0.1 µM), and 5 (IC50 = < 2 µM) exhibited potent T- cell proliferation inhibitory activities, while compound 3 (IC50 = 29.9 ± 0.09 µM) showed a moderate activity in comparison to the standard prednisolone (IC50 = 9.73 ± 0.08 µM). All the metabolites were found to be non-toxic against 3T3 normal cell line. This study thus identifies some potent compounds active against T-cell proliferation. Their anti-inflammatory potential, therefore, deserves to be further investigated.

1. Introduction effectiveness of the process. Biocatalytic reactions involve different types of reactions, such as hydroxylation, reduction, Michael addition, Among various natural and synthetic classes of chemical compounds, oxidation, esterification, epimerization, reverse aldol reaction, and steroid- based drugs are the most extensively used for various health epoxidation [9–15]. Microbial transformations of steroids on a large disorders due to their contraceptive, anti-cancer, anti-androgenic, anti- scale in the synthesis of drugs and other bioactive analogues have been HIV, anti-tumor, anabolic, anti-bacterial, anti-inflammatory, and pro reported earlier [3,16]. Fungal-mediated biotransformation is exten gestational properties [1,2]. The synthesis of analogues of steroids sively used for the conversion of steroids, and other organic compounds. through conventional chemical methods is often difficult [3–7]. How Structurally modified compounds, obtained through biocatalytic re ever, biocatalysts are capable of carrying out regio-, and stereoselective actions, are regio-, and stereo-selective with altered therapeutic index reactions efficiently. [2,3,7,9–12,17–19]. These modified compounds may have higher po Microbial transformation is an effective tool to generate different tency, reduced adverse effects, and longer half-lives in blood, as oxygenated analogues of steroids and other natural products [4,7,8]. compared to their parent substances [2,3]. The enzyme cytochrome Biocatalytic reactions are more favorable than chemical synthesis P450 monooxygenases, found specificallyin fungi, is responsible for the because of their eco-friendly nature, mild reaction conditions, and cost- selective hydroxylation of various complex steroids [6,12,18,20].

* Corresponding authors at: Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan (Atia-tul-Wahab); H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan (M. Iqbal Choudhary). E-mail addresses: [email protected] (Atia-tul-Wahab), [email protected] (M.I. Choudhary). https://doi.org/10.1016/j.bioorg.2020.104313 Received 16 July 2020; Received in revised form 17 September 2020; Accepted 20 September 2020 Available online 24 September 2020 0045-2068/© 2020 Elsevier Inc. All rights reserved. S. Javed et al. Bioorganic Chemistry 104 (2020) 104313

Melengestrol acetate (1) (C25H32O4) is an orally active synthetic to obtain brown gummy crude. For fractionation of obtained crude, progestagenic steroid, capable of suppressing estrus (heat) in heifers column chromatography was employed. Different chromatographic [21]. MGA (1) is a synthetic analogue of medroxyprogesterone acetate techniques were used to purify resulting metabolites from the crude (MPA), which also exhibits glucocorticoid activity. Glucocorticoids are extract. generally used against many inflammatory disorders [22,23]. We have previously reported many structural modificationsof steroids to produce 2.4. Fermentation of melengestrol acetate (1) with Glomerella fusaroide analogues with diverse activities. Microbial transformation of MGA (1) was previously reported with Cunninghamella blakesleeana, and a new Melengestrol acetate (1) (1 g) was dissolved in 25 mL of acetone, and oxygenated analogue was obtained. dispersed in 25 Erlenmyer flasks (1 L), each contained 400 mL of In continuation of our research on microbial transformations, MGA autoclaved media cultured with Glomerella fusaroide. All flasks were ◦ (1) was incubated with Glomerella fusaroide, and Rhizopus stolonifer, incubated for 8 days on rotary shaker at 27 C. After complete fermen which yielded four new metabolites 2–5. Immunosuppressant activity of tation, reaction was quenched by adding ethyl acetate. Each flask was the resulting metabolites was evaluated through the inhibitory effects on then filtered, and extracted by ethyl acetate 3 times. The collected sol the proliferation of T-cells in human blood in vitro. vent layer was evaporated on rotavapor to obtain brown gummy crude material. The crude material was then loaded on a silica gel column for 2. Materials and methods fractionation using solvent gradients of pet. ether, and acetone. Two main fractions were obtained at pet. ether: acetone (70:30), and (67:33). 2.1. Materials. These two fractions were further subjected to recycling HPLC. Metabo lites 2 (Rt = 42 min), and 3 (Rt = 36 min) were purified at M-80, Melengestrol acetate (1) was obtained from Haihang Industry Co., methanol–water (70:30). Ltd. Beijing, China. Precoated TLC plates (silica gel, 0.25 mm) were purchased from Merck, Germany. Ceric sulfate was used as a TLC 2.4.1. 17α-Acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-4,6- staining reagent. Fractions were obtained by column chromatography, diene-3,20-dione (2) utilizing silica gel (70–230 mesh, Merck). Purified metabolites were ◦ [ ]25 = + = White solid: M. p.: 222–225 C, α D 7.5 (c 0.01, CHCl3), UV obtained through recycling preparative HPLC [JAI LC-908 W equipped, 1 (MeOH) λmax nm: 225, and 288 nm. IR (CHCl3) Vmax cm : 3433 (OH), with YMC M-80 column (4–5 μm, 20–250 mm i.d.)]. UV light of 254 nm – 1 13 2944 (CH), and 1739 (C–O), H NMR (CD3OD; 400 MHz) Table-1; C was used. JEOL (Japan) JMS-600H mass spectrometer was used to + NMR (CD3OD; 100 MHz) Table-2; EI-MS: m/z 412.3 [M] , 353 (61), 309 + measure the electron impact mass spectra (EI-MS), and high-resolution (42); HREI-MS m/z 412.2250 [M ] (mol. formula, calcd value: mass spectra (HREI-MS) in m/z (rel. %). Bruker Avance NMR spec C25H32O5, 412.2251). trometers (Bruker, Switzerland) were used to record 1H NMR spectra at 13 400, and 500 MHz, and C NMR spectra at 100, and 125 MHz in 2.4.2. 17α-Acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-1,4,6- CD3OD, respectively. Melting point of each compound was measured on triene-3,20-dione (3) Buchi-560 (Switzerland). For measuring the optical rotation in meth ◦ [ ]25 = + = White solid: M. p.: 238–240 C, α D 5.7 (c 0.008, CHCl3), UV anol, JASCO P-2000 polarimeter (Japan) was used. UV Spectra (in nm) 1 (MeOH) λmax nm: 213, 228, 261, 301 nm. IR (CHCl3) Vmax cm : 3400 were recorded on Hitachi U-3200 spectrophotometer (Japan). FT-IR- – – 1 (OH), 2931 (CH), and 1739 (C–O), and 1650 (C–C), H NMR (CD3OD; 8900, and Bruker VECTOR 22 spectrophotometers (Bruker, France) 13 1 500 MHz) Table-1; C NMR (CD3OD; 125 MHz) Table-2; EI-MS: m/z were used to record IR spectra (cm ). + + 410.3 [M] , 368 (26), 351 (100). HREI-MS m/z 410.2093 [M ] (mol. formula, calcd value: C H O , 410.2075). 2.2. Culture medium. 25 30 5

The growth medium for Glomerella fusaroide was prepared by adding 2.5. Fermentation of melengestrol acetate (1) with Rhizopus stolonifer glucose (100 g), glycerol (100 mL), peptone (50 g), yeast extract (50 g), 1.0 g of MGA (1) dissolved in 25 mL acetone was distributed among KH2PO4 (50 g), and NaCl (50 g) into 10.0 L of distilled H2O. Similarly, the medium for Rhizopus stolonifer (TSY- 0471) was formulated by 27 conical flasks (1 L, each containing 400 mL media with complete growth of Rhizopus stolonifer). All 27 flasks were placed on shaker for mixing following ingredients in 20.0 L of distilled H2O: glucose (200 g), fermentation reaction for 14 days. After 14 days, ethyl acetate was used peptone (100 g), KH2PO4 (100 g), and yeast extract (60 g). for quenching the reaction. Organic layer was then filtered to separate 2.3. General fermentation and extraction conditions biomass, and extracted thrice with ethyl acetate. Organic phase was then concentrated using rotary evaporator to obtain gummy brown crude. The above mentioned ingredients were used to prepare media for Two main fractions were obtained through column chromatography by fungal culture. Prepared media was transferred to 250 mL flasks (each using solvent gradients of pet. ether: acetone. Reversed phase HPLC ◦ – = containing 100 mL media), and autoclaved at 121 C for 20 min. (M 80, 70:30 methanol water) was used to purify metabolites 4 (Rt = Seed flaskswere prepared by using fungal strains of ATCC, and TSY 38 min), and 5 (Rt 26 min). ◦ under sterilized conditions, and then incubated at 25 ± 2 C for 4–5 days α α β on a rotary shaker. Similarly, the remaining flasks were also prepared 2.5.1. 17 -Acetoxy-6,7 -epoxy-6 -methyl-16-methylenepregna-4,6-diene- using the fresh seed flask. After obtaining mature growth of fungal 3,20-dione (4) – ◦ [α]25 = + = culture, particular amount of substrate, dissolved in acetone, was White solid: M. p.: 138 140 C, D 24 (c 0.005, CHCl3), UV 1 dispersed equally, and were again kept on shaker for 12–14 days (MeOH) λmax nm: 229, 291, and 380 nm. IR (CHCl3) Vmax cm : 1240 – – – 1 depending upon the results. Transformations were regularly analyzed by (C O), 1740 (C–O), and 1585 (C–C). H NMR (CD3OD; 500 MHz) 13 + Table-1; C NMR (CD3OD; 125 MHz) Table-2; EI-MS: m/z 412.3 [M] time course study using TLC after different time intervals. Two different + control experiments were also carried out to analyze the fungal me (7), 370 (34), 353 (100). HREI-MS m/z 412.2250 [M ] (mol. formula, tabolites (negative control), and substrate degradation (positive con calcd value: C25H32O5, 412.2249). trol). When the fermentation process was completed, the reaction was quenched by using ethyl acetate, followed by filtration in order to 2.5.2. 17α-Acetoxy-11β,15β-dihydroxy-6-methyl-16-methylenepregna- remove mycelia. The broth was extracted using the same solvent, ethyl 4,6-diene-3,20-dione (5) ◦ [ ]25 = = acetate. The obtained solvent layer was then evaporated on rotavapour White solid: M. p.: 203–205 C, α D -14.2 (c 0.007, CHCl3), UV

2 S. Javed et al. Bioorganic Chemistry 104 (2020) 104313

1 (MeOH) λmax nm: 236, 243, and 248 nm. IR (CHCl3) Vmax cm : 3407 incubated in the presence of 5% CO2 for 24 h at 37 ℃. Exponentially – 1 13 4 (OH), 1721 (C–O), H NMR (CD3OD; 500 MHz) Table-1; C NMR growing harvested cells were counted, using haemocytometer 5 × 10 + (CD3OD; 125 MHz) Table-2; FAB-MS: m/z 429.3 [M] (7), 370 (34), 353 cells/mL concentration was maintained. Freshly prepared media along (100). HRFAB-MS m/z 429.2295 (mol. formula, calcd value): C25H32O6, with compounds of different concentrations were added in each 96-well 429.2277. plate. After incubation of 48 h, 200 μL MTT (0.5 mg/mL) dye was added, and plates were again incubated for 4 h. 100 μL of DMSO was also added to these well plates. Absorbance at 540 nm was measured by microplate 2.6. Biological activity evaluation reader to record the reduction of MTT to formazan in cells. % Inhibition was calculated by using the following formula. 2.6.1. T-lymphocyte proliferation inhibition assay

[ ( ) ] O.D. of test compound O.D. of negative control % Inhibition = 100 × 100 O.D. of positive control O.D. of negative control

T-Lymphocytes were isolated by Ficoll hypaque density gradient method. Briefly,the blood was aseptically collected from healthy human volunteers. 10 mL of blood was mixed with equal volume of incomplete RPMI-1640 (Sigma Aldrich, St. Louis, USA). The mixture containing 3. Results and discussion blood, and RPMI was gently layered on 5 mL of lymphocyte separation medium (LSM) (MP Biomedicals, lllkirch, France), and tubes were then Incubation of melengestrol acetate (1), using two different fungi ◦ centrifuged at 400 g for 20 min at 25 C. The buffy layer collected was Glomerella fusaroide, and Rhizopus stolonifer, yielded four new metabo ◦ mixed with incomplete RPMI-1640, and centrifuged at 4 C for 10 mins lites (Fig. 1). at 300 g. The pellet containing PBMCs was resuspended in 1 mL of RPMI Biotransformation of progestogenic steroid melengestrol acetate (1) containing 5% FBS (Gibco, California, USA). Cell proliferation was using Glomerella fusaroide, and Rhizopus stolonifer is reported here for the conducted by Alamar blue assay. The isolated PMBCs were plated at a firsttime. Fermentation of substrate 1 with Glomerella fusaroide afforded 6 concentration of 2 × 10 cells/mL in a round bottom 96-well tissue metabolites 2, and 3, while with Rhizopus stolonifer resulted in metab culture plates (Iwaki, Chiba, Japan). Peripheral blood T-cells were olites 4, and 5. + activated with 7.5 μg/mL phytohemagglutinin-L (PHA-L; Sigma, St. The HREI-MS of compound 2 exhibited the M at m/z 412.2250 Louis, USA). Three different concentrations of compounds (2.5, 25, and (C25H32O5, calcd 412.2251), which was 16 a.m.u. more than the sub 250 µM) in triplicates were added. The plates were then incubated for strate 1 (m/z 396.1) indicating the addition of oxygen group in the ◦ 48 h at 37 C in 5% CO2. A one-tenth volume of Alamar blue dye was transformed product. The IR spectrum showed absorption bands at then added, and the incubation continued for 4 hrs. Absorbance was 3433, and 1739 cm 1 due to the presence of hydroxyl and ketone read in spectrophotometer at wavelengths of 570, and 600 nm [24]. functionalities, respectively. The 1H NMR (Table 1) spectrum showed an additional methine signal at δ 4.00, suggesting the hydroxylation of the 2.6.2. Cytotoxicity assay substrate 1. A new downfield signal appeared at δ 68.3 in 13C NMR All compounds were subjected to MTT (3-(4,5-dimethyl thiazol-2yl)- spectrum (Table 2). The hydroxyl group was placed at C-11, based on 2,5-diphenyl tetrazolium bromide) assay for cytotoxicity evaluation on the HMBC correlations of H-11 (δ 4.00) with C-10 (δ 39.3), C-12 (δ 43.2), 3 T3 cell in 96-well plates. The culture cells were prepared by adding and C-9 (δ 56.9) (Fig. 2). The COSY spectrum confirmedthe presence of DMEM, supplemented with streptomycin (100 μg/mL), 5% of fetal hydroxyl group at C-11 by showing corelations of H-9 (δ 1.38) with bovine serum, and penicillin (100 IU/mL). These cultured cells were geminal H-11 (δ 4.00). The hydroxylation at C-11 was further deduced

Fig. 1. Biotransformation of melengestrol acetate (1) by Glomerella fusaroide, and Rhizopus stolonifer.

3 S. Javed et al. Bioorganic Chemistry 104 (2020) 104313

Table 1 1H NMR Chemical Shift Assignments of Compounds 1–5 (δ in ppm, J in Hz).

Carbon 1 2 3 4 5

1 2.07, ov; 1.74, ov 2.71, ov; 2.06, ov 7.85,d (J1/2 = 10.5) 1.59, m; 1.70, m 1.70, m; 1.99, ov 2 2.33, ov; 2.63, ov 2.61, ov; 2.35, ov 6.14 dd (J1/2 = 1.5, J1/3 = 10.0) 2.38, m; 2.64, m 1.54, ov; 2.00, m 3 – – – – – 4 5.84, s 5.85, s 6.19, s 6.21, s 6.21, s 5 – – – – – 6 – – – – – 7 6.06, s 6.03, s 5.91, s 3.22, s 6.07, ov 8 2.35, ov 2.38, ov 2.50, m 2.09, ov 2.41, ov 9 1.30, ov 1.38, t (J1/2 = 13.0) 1.59, m 1.21, ov 1.25, ov 10 – – – – – 11 1.73, ov; 1.49, m 4.00, dt (J1/2 = 6.5, J1/3 = 13.5) 4.09, m 2.08, ov; 2.01, ov 3.43, br .s 12 2.02, m; 1.61,m 2.02, ov; 1.88,ov 1.98, ov; 3.32, ov 2.03, ov; 1.50, m 2.64, m; 2.38, ov 13 – – – – – 14 1.96, m 1.98, ov 2.05, ov 2.25, m 2.16, br.s 15 2.64, ov; 2.26, m 2.25, ov; 2.68, ov 2.28, m; 2.63, m 2.29, m; 2.78, dd(J1/2 = 5.0, J2/4 = 15.5) 4.72, br.s 16 – – – – – 17 – – – – – 18 0.76, s 0.76, s 0.76, s 0.72, s 0.76, s 19 1.13, s 1.24, s 1.28, s 1.22, s 1.26, s 20 – – – – – 21 2.11, s 2.12, s 2.12, s 2.14, s 2.12, s 22 – – – – – 23 2.03, s 2.04, s 2.01, s 2.05, s 2.01, s 24 1.80, s 1.86, s 1.94, s 1.43, s 1.44, s 25 5.49, s; 5.44, s 5.50, s; 5.48, s 5.52, s; 5.48, s 5.40, s; 5.50, s 5.83, s; 5.77, s

in the 1H NMR spectrum (Table 1). Furthermore, in 13C NMR (Table 2) broad-band decoupled spectrum, two new olefiniccarbons appeared at δ Table 2 160.5, and 124.5, along with a new methine signal at δ 67.1. The po 13 – δ C NMR Chemical Shift Assignments of Compounds 1 5 ( in ppm). sition of the hydroxyl group at C-11 (δ 67.1) was inferred from the Carbon 1 2 3 4 5 HMBC correlations of H-11 (δ 4.09) with C-12 (δ 42.6), and C-9 (δ 58.0), 1 35.0 37.0 160.5 37.1 37.2 while the placement of the double bond between C-1 and C-2 was 2 34.4 34.6 124.5 34.3 32.2 deduced on the basis of HMBC correlations of H-1 with ketonic C-3 (δ 3 202.8 203.0 189.0 201.4 201.4 189.0), C-5 (δ 133.0), C-19 (δ 20.9), and C-10 (δ 43.2). H-2 showed 4 121.5 121.9 122.1 127.9 127.8 HMBC correlations with C-4 (δ 122.1), and C-10 (δ 43.2) which sug 5 132.5 132.5 133.0 168.1 132.8 α β δ 6 167.2 167.1 166.1 58.0 168.4 gested the , -unsaturation. COSY correlations of H-9 ( 58.0) with H-11 7 140.0 139.4 135.9 68.1 125.0 (δ 4.09) indicated the hydroxylation at C-11 (δ 67.1), while COSY cor 8 37.9 36.2 36.8 36.2 32.7 relation of H-1 (δ 7.85) with H-2 (δ 6.14) indicated the presence of a 9 51.7 56.9 58.0 51.6 51.8 double bond between C-1 (δ 160.5), and C-2 (δ 124.5) (Fig. 2). H-11 (δ 10 37.5 39.3 43.2 38.1 38.3 4.09) showed NOESY correlations with β-oriented H3-19 (δ 1.28), and 11 21.3 68.3 67.1 21.9 68.2 δ α 12 31.9 43.2 42.6 31.6 34.4 H3-18 ( 0.76). Therefore, OH at C-11 was deduced to be -oriented 13 48.0 48.0 48.0 48.8 48.8 (Fig. 3). The new compound was thus deduced to have structure 3. + 14 47.2 46.8 46.6 46.3 50.6 The HREI-MS of metabolite 4 showed the M at m/z 412.2250 15 33.0 33.0 32.7 33.1 71.5 (C25H32O5, calcd 412.2249). The 16 a.m.u. increase in the mass as 16 146.3 146.1 146.0 146.1 152.1 17 95.3 94.8 94.6 95.1 94.9 compared to substrate 1 (m/z 396.1) indicated the addition of an oxygen 18 14.7 15.5 15.5 14.6 14.8 in the transformed product. The IR spectrum showed an absorption band 1 19 16.6 17.4 20.9 17.2 17.2 at 1240 cm , indicating the presence of C–O bonds due to the epoxi 20 204.8 204.4 204.3 204.8 204.2 dation in the substrate 1. The 1H NMR spectrum (Table 1) showed a 21 27.9 27.9 27.9 27.9 27.9 signal at δ 3.22, indicating the addition of an oxymethine. The 13C NMR 22 172.0 171.9 171.8 172.0 172.0 δ 23 21.0 21.0 21.1 20.7 21.0 spectrum (Table 2) showed two new signals at 58.0, and 68.1. HMBC 24 19.9 19.9 19.1 21.0 20.8 correlations indicated the formation of an epoxide between C-6 (δ 58.0), 25 120.3 120.7 120.7 120.4 124.6 and C-7 (δ 68.1). H-7 (δ 3.22) exhibited HMBC correlations with C-8 (δ 36.2), C-9 (δ 51.6), and C-14 (δ 46.3) while H3-24 (δ 1.43) showed HMBC correlations with C-5 (δ 168), C-6 (δ 58.0), and C-7 (δ 68.1). COSY on the basis of NOESY correlations of β-oriented H -19 (δ 1.24), and H - 3 3 correlations of H-7 (δ 3.22), and H-8 (δ 2.09) further indicated the po 18 (δ 0.76) with H-11 (δ 4.00), suggesting the OH group as α (Fig. 3). The sition of epoxide group (Fig. 2). Orientation of epoxide was determined compound was thus identified as a new compound. + by NOESY correlations of β-oriented H -19 (δ 1.22) with H -24 (δ 1.43), The HREI-MS of 3 exhibited the M at m/z 410.2093, supporting the 3 3 and β-oriented H-8 with H-7 (δ 3.22) suggesting an α orientation of molecular formula C H O (calcd 410.2075). EI-MS indicated the 25 30 5 epoxide at C-6 (δ 58.0), and C-7 (δ 68.1) (Fig. 3). addition of 14 a.m.u. to the mass of substrate 1 (m/z 396.1), suggesting + + The HRFAB -MS of compound 5 showed the [M + H] peak at m/z the addition of an oxygen atom with possible unsaturation. In the IR 429.2295 (C H O , calcd. 429.2277), that is 32 a.m.u. more than spectrum, the absorption band at 3400 cm 1 indicated the presence of a 25 32 6 1 – compound 1 (m/z 396.1). IR Spectrum showed absorption band for a hydroxyl, while the band at 1650 cm indicated (C–C) unsaturation. hydroxyl group at 3407 cm 1. The 1H NMR spectrum showed two new An additional signal at δ 4.09 was observed in the 1H NMR spectrum methine signals at δ 4.72, and 3.43, while the 13C NMR spectrum showed (Table 1), while two downfield signals appeared for olefinic methine new methine signals at δ 68.2, and 71.5 indicating the double hydrox protons at δ 7.85 (J = 10.5 Hz), and 6.14 (J = 1.5, J = 10.0 Hz) 1/2 1/2 1/3 ylation of substrate 1. H-11 (δ 3.43) showed HMBC correlations with C-9

4 S. Javed et al. Bioorganic Chemistry 104 (2020) 104313

Fig. 2. Key HMBC ( ), COSY ( ) correlations of metabolites 2–5.

Fig. 3. NOESY correlations ( ) of metabolites 2–5.

(δ 51.8), C-8 (δ 32.7), and C-13 (δ 48.8) indicating hydroxylation at C-11 H-11 (δ 3.43), and H-14 (δ 2.16) with H-15 (δ 4.72) suggested the OH (δ 68.2). Similarly, H-15 (δ 4.72) showed HMBC correlations with C-13 groups at C-11, and C-15 as β-oriented (Fig. 3). (δ 48.8), C-16 (δ 152.1), C-17 (δ 94.9), and C-25 (δ 124.6) indicating the second hydroxylation at C-15 (δ 71.5) (Fig. 2). The COSY correlations between H-14 and H-15 indicated the presence of an OH group at C-15, 3.1. T-cell proliferation inhibitory activity while COSY correlations between H-11 and H-12 was indicative of the presence of OH group at C-11. NOESY correlations of H-9 (δ 1.25) with Melengestrol acetate (1) is a corticosteroid hormone with reported glucocorticoid activity. Glucocorticoids function to reduce

5 S. Javed et al. Bioorganic Chemistry 104 (2020) 104313

Table 3 interests or personal relationships that could have appeared to influence T-cell proliferation inhibitory effect of compounds 1–5. the work reported in this paper.

Compounds T-cell proliferation IC50 (µM) Cytotoxicity Acknowledgements 1 2.77 ± 0.08 Inactive 2 2.78 ± 0.07 Inactive 3 29.9 ± 0.09 Inactive Authors acknowledge the enabling role of the Higher Education 4 2.74 ± 0.1 Inactive Commission, Pakistan, through research project # 7993 entitled, “Syn < 5 2 Inactive thesis of New Analogues of Anti-inflammatory Agents via Microbial Biotechnological Methods” and also financial support of the Searle Company Ltd. Pakistan, through research funding. inflammation.Substrate 1, and its metabolites 2–5 were evaluated for T- Appendix A. Supplementary material cell proliferation inhibitory activity by using Alamar blue dye. Blood of healthy human was used to isolate PBMCs (peripheral blood mono Supplementary data to this article can be found online at https://doi. nuclear cells). Compound 1, and its metabolites 2, 4, and 5 showed at org/10.1016/j.bioorg.2020.104313. least three-fold potent inhibitory activity with IC50 values of 2.77, 2.78, 2.74, and < 2 µM, as compared to the standard drug prednisolone (IC50 References 9.73 ± 0.08 µM) (Table 3), respectively. Metabolite 3 showed a mod [1] M.S. Ahmad, S. Yousuf, A. Jabeen, M.I. Choudhary, Biotransformation of anabolic erate activity with IC50 value of 29.9 µM. compound methasterone with Macrophomina phaseolina, Cunninghamella blakesleeana, and Fusarium lini, and TNF-α inhibitory effect of transformed 3.2. Cytotoxicity assay products, Steroids 128 (2017) 75–84, https://doi.org/10.1016/j. steroids.2017.04.001. – [2] M.S. Ahmad, S. Zafar, M. Bibi, S. Bano, M.I. Choudhary, Biotransformation of Melengestrol acetate (1) and its metabolites 2 5 were evaluated for androgenic steroid mesterolone with Cunninghamella blakesleeana and cytotoxicity against 3T3 (mouse fibroblast)cell line using MTT assay. All Macrophomina phaseolina, Steroids 82 (2014) 53–59, https://doi.org/10.1016/j. tested compounds were found to be noncytotoxic to 3T3 cell line, as steroids.2014.01.001. ´ compared with the standard cytotoxic drug, cyclohexamide. [3] L.F. Bianchini, M.F. Arruda, S.R. Vieira, P. Campelo, A.M. Gregio, E.A. Rosa, Microbial biotransformation to obtain new antifungals, Front Microbiol. 6 (2015) 1433, https://doi.org/10.3389/fmicb.2015.01433. 4. Conclusions [4] A. Al-Aboudi, B.M. Kana’an, M.A. Zarga, S. Bano, K. Javed, M.I. Choudhary, Fungal biotransformation of diuretic and antihypertensive drug spironolactone with Gibberella fujikuroi, Curvularia lunata, Fusarium lini, and Aspergillus alliaceus, In conclusion, biotransformation of MGA (1) with Glomerella fusar Steroids 128 (2017) 15–22, https://doi.org/10.1016/j.steroids.2017.10.003. oide and Rhizopus stolonifer yielded four new metabolites 2–5. During [5] S. Bano, S. Yousuf, A. Jabeen, M.A. Mesaik, M.I. Choudhary, New anti- these transformations, epoxidation at C-6/C-7, dehydrogenation at C-1/ inflammatory metabolites by microbial transformation of medrysone, PloS One 110153951 (4) (2016), https://doi.org/10.1371/journal.pone.0153951. C-2, and hydroxylations at C-11, C-15 were observed. C-11 was the site [6] E. Baydoun, S. Bano, A. Jabeen, S. Yousuf, A. Mesaik, C. Smith, M.I. Choudhary, for α- OH in metabolites 2, and 3, whereas β- OH occurred at C-11, and Fungal transformation and T-cell proliferation inhibitory activity of melengestrol C-15 in metabolite 5. All the compounds were found to have potent acetate and its metabolite, Steroids 86 (2014) 56–61, https://doi.org/10.1016/j. steroids.2014.04.012. activity against the proliferation of T-cells in vitro. All tested compounds [7] E. Baydoun, M. Bibi, M.A. Iqbal, A.T. Wahab, D. Farran, C. Smith, M.I. Choudhary, were found to be noncytotoxic to 3T3-cell line (mouse fibroblast).Based Microbial transformation of anti-cancer steroid exemestane and cytotoxicity of its on their potent anti-inflammatory activity, compounds 1–5, along with metabolites against cancer cell lines, Chem. Central J. 757 (1) (2013), https://doi. org/10.1186/1752-153X-7-57. standard prednisolone will be further studied in silico to understand the [8] E. Baydoun, S. Iqbal, C. Smith, M.I. Choudhary, Biotransformation of drospirenone, structure–activity relationships. In addition, biotransformation of MGA a contraceptive drug, with Cunninghamella elegans, Steroids 126 (2017) 30–34, (1) will be performed with other fungal cell cultures in order to syn https://doi.org/10.1016/j.steroids.2017.07.010. [9] M.I. Choudhary, M.Y. Mohammad, S.G. Musharraf, M. Parvez, A. Al-Aboudi, New thesize diverse libraries of new analogues of substrate 1. Metabolites oxandrolone derivatives by biotransformation using Rhizopus stolonifer, Steroids 74 will be studied in in vivo models as well. (13–14) (2009) 1040–1044, https://doi.org/10.1016/j.steroids.2009.08.003. [10] E. Baydoun, A.T. Wahab, N. Shoaib, M.S. Ahmad, R. Abdel-Massih, C. Smith, M. 5. Compliance with Ethics Requirements I. Choudhary, Microbial transformation of contraceptive drug etonogestrel into new metabolites with Cunninghamella blakesleeana and Cunninghamella echinulata, Steroids 115 (2016) 56–61, https://doi.org/10.1016/j.steroids.2016.08.003. The studies on cells from human blood was carried out after an [11] G.W. Duncan, S.C. Lyster, J.W. Hendrix, J.J. Clark, H.D. Webster, Biologic effects of – approval from Independent Ethics Committee, International Center for melengestrol acetate, Fertil. Steril. 15 (4) (1964) 419 432, https://doi.org/ 10.1016/S0015-0282(16)35287-6. Chemical and Biological Sciences, University of Karachi No.: ICCBS/IEC- [12] J.M. Gao, J.W. Shen, J.Y. Wang, Z. Yang, A.L. Zhang, Microbial transformation of 008-BC-2015/Protocol/1.0 (24). The procedure followed was in accor 3β-acetoxypregna-5,16-diene-20-one by Penicillium citrinum, Steroids 76 (1–2) dance with the Helsinki Declaration of 1975, as revised in 2008. (2011) 43–47, https://doi.org/10.1016/j.steroids.2010.08.006. [13] D.J. Patterson, G.H. Kiracofe, J.S. Stevenson, L.R. Corah, Control of the bovine Informed consent was obtained from human blood donor for being estrous cycle with melengestrol acetate (MGA): A Review, J. Anim. Sci. 67 (8) included in the study. (1989) 1895–1906, https://doi.org/10.2527/jas1989.6781895x. [14] B. Schiffer, A. Daxenberger, K. Meyer, H.H. Meyer, The fate of trenbolone acetate and melengestrol acetate after application as growth promoters in cattle: Author contributions environmental studies, Environ. Health Perspect. 109 (11) (2001) 1145, https:// doi.org/10.1289/ehp.011091145. MIC, AUR, and ZAA contributed to the concept and design of study, [15] S.A.A. Shah, S. Sultan, N.B. Hassan, F.K.B. Muhammad, M.A.B.M. Faridz, F.B. M. Hussain, H.S. Adnan, Biotransformation of 17α-ethynyl substituted steroidal analysis, and interpretation of data, and manuscript writing. ATW drugs with microbial and plant cell cultures: a review, Steroids 78 (14) (2013) contributed in analysis and interpretation of data, study supervision and 1312–1324, https://doi.org/10.1016/j.steroids.2013.10.001. manuscript writing. SJ performed biotransformation experiments, and [16] E. Baydoun, M. Karam, M.S.A. Khan, M.S. Ahmad, C. Smith, R. Abdel-Massih, M. interpretation of data. AJ and SZ biological assays. All authors read and I. Choudhary, Microbial transformation of nandrolone with Cunninghamella echinulata and Cunninghamella blakesleeana and evaluation of leishmaniacidal approved the final manuscript. activity of transformed products, Steroids 88 (2014) 95–100, https://doi.org/ 10.1016/j.steroids.2014.06.020. Declaration of Competing Interest [17] H.N. Bhatti, R.A. Khera, Biological transformations of steroidal compounds: a review, Steroids 77 (12) (2012) 1267–1290, https://doi.org/10.1016/j. steroids.2012.07.018. The authors declare that they have no known competing financial

6 S. Javed et al. Bioorganic Chemistry 104 (2020) 104313

[18] T. Dong, G.W. Wu, X.N. Wang, J.M. Gao, J.G. Chen, S.S. Lee, Microbiological contraceptive drug, desogestrel, and anti-MDR-Staphylococcus aureus activity of its transformation of diosgenin by resting cells of filamentous fungus, Cunninghamella metabolites, Bioorg. Chem. 77 (2018) 152–158, https://doi.org/10.1016/j. echinulata CGMCC 3.2716, J. Mol. Catal. B Enzym. 67 (3–4) (2010) 251–256, bioorg.2017.12.027. https://doi.org/10.1016/j.molcatb.2010.09.001. [22] C. Smith, M.S.A. Khan, M.S. Ahmad, D. Farran, M.I. Choudhary, E. Baydoun, [19] N.T. Khan, S. Zafar, S. Noreen, A.M. Al Majid, Z.A. Al Othman, S.I. Al-Resayes, M. Microbial transformation of oxandrolone with Macrophomina phaseolina, and I. Choudhary, Biotransformation of dianabol with the filamentous fungi and Cunninghamella blakesleeana, Steroids 102 (2015) 39–45, https://doi.org/10.1016/ β-glucuronidase inhibitory activity of resulting metabolites, Steroids 85 (2014) j.steroids.2015.06.008. 65–72, https://doi.org/10.1016/j.steroids.2014.04.004. [23] S. Zafar, M. Bibi, S. Yousuf, M.I. Choudhary, New metabolites from fungal [20] M. Siddiqui, M.S. Ahmad, S. Yousuf, N. Fatima, N.N. Shaikh, M.I. Choudhary, biotransformation of an oral contraceptive agent: Methyloestrenolone, Steroids 78 Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella (4) (2013) 418–425, https://doi.org/10.1016/j.steroids.2013.01.002. blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity [24] S.A. Ahmed, R.M. Gogal Jr.,, J.E. Walsh, A new rapid and simple non-radioactive evaluation of its metabolites, PloS One 12 (2) (2017), e0171476, https://doi.org/ assay to monitor and determine the proliferation of lymphocytes: an alternative to 10.1371/journal.pone.0171476. [3H] thymidine incorporation assay, J. Immunol. Methods 170 (2) (1994) [21] M. Siddiqui, I. Ibrahim, A. Hussain, E.H. Ajandouz, A. Hijazi, E. Baydoun, M. 211–224, https://doi.org/10.1016/0022-1759(94)90396-4. I. Choudhary, Cunninghamella blakesleeana-mediated biotransformation of a