Rabia Farooqa,
Nusrat Hussaina,
Sammer Yousufa,
Atia-tul-Wahab*b,
Malik Shoaib Ahmadb,
Atta-ur-Rahmana and
M. Iqbal Choudhary*abc
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. E-mail: iqbal.choudhary@iccs.edu
bDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. E-mail: atia.tulwahab@iccs.edu
cDepartment of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah-21412, Saudi Arabia
First published on 14th June 2018
The microbial transformation of anabolic androgenic steroid mestanolone (1) with Macrophomina phaseolina and Cunninghamella blakesleeana has afforded seven metabolites. The structures of these metabolites were characterized as 17β-hydroxy-17α-methyl-5α-androsta-1-ene-3,11-dione (2), 14α,17β-dihydroxy-17α-methyl-5α-androstan-3,11-dione (3), 17β-hydroxy-17α-methyl-5α-androstan-1,14-diene-3,11-dione (4), 17β-hydroxy-17α-methyl-5α-androstan-3,11-dione (5), 11β,17β-dihydroxy-17α-methyl-5α-androstan-1-ene-3-one (6), 9α,11β,17β-trihydroxy-17α-methyl-5α-androstan-3-one (7), and 1β,11α,17β-trihydroxy-17α-methyl-5α-androstan-3-one (8). All the metabolites, except 5 and 6, were identified as new compounds. Substrate 1 (IC50 = 27.6 ± 1.1 μM), and its metabolites 2 (IC50 = 19.2 ± 2.9 μM) and 6 (IC50 = 12.8 ± 0.6 μM) exhibited moderate cytotoxicity against the HeLa cancer cell line (human cervical carcinoma). All metabolites were noncytotoxic to 3T3 (mouse fibroblast) and H460 (human lung carcinoma) cell lines. The metabolites were also evaluated for immunomodulatory activity, and all were found to be inactive.
Mestanolone (1) (C20H18O2) is a member of the anabolic-androgenic class of steroids. It is weakly anabolic and strongly androgenic. Mestanolone was first synthesized by the oxidation of 17β-methylandrostan-3β,17β-diol. It is used as a starting material for the synthesis of other anabolic steroids, such as 17-methyl-1-testosterone, and oxandrolone.10,11 Compound 1 was earlier subjected to microbial transformation, and several new analogues were obtained.12
In continuation of our research on biotransformation of bioactive compounds, and drug molecules,13–15 mestanolone (1) was incubated with Macrophomina phaseolina, and Cunninghamella blakesleeana, which yielded metabolites 2–8 (Fig. 1 and 2).
Carbon | 1 | 2 | 3 | |||
---|---|---|---|---|---|---|
δC | δH (J in Hz) | δC | δH (J in Hz) | δC | δH (J in Hz) | |
1 | 38.9 | 1.36 (overlap), 2.21 (m) | 162.3 | 7.57 (d, J1,2 = 10.5) | 38.5 | 1.27 (dd, J = 14.0, J = 5.2), 2.87 (ddd, J = 13.2, J = 6.4, J = 2.0) |
2 | 38.6 | 2.20 (m), 2.50 (td, J2a/2e, = 15.0, J2/1a,e = 7.0) | 127.7 | 5.78 (d, J2,1 = 10.5) | 38.7 | 2.17 (overlap), 2.50 (td, J2a/2e, =14.8, J2/1a,e = 6.8) |
3 | 214.6 | — | 202.2 | — | 214.4 | — |
4 | 45.2 | 2.01 (m), 2.35 (t, J4e/4a,5a = 14.1) | 41.4 | 2.16 (overlap), 2.42 (overlap) | 45.1 | 2.01 (dt, J = 14.8, J = 2.8), 2.36 (overlap) |
5 | 47.6 | 1.50 (overlap) | 45.5 | 1.91 (overlap) | 48.3 | 1.50 (overlap) |
6 | 32.6 | 0.94 (td), 1.51 (overlap) | 28.1 | 1.53 (m), 1.42 (m) | 29.2 | 1.37 (overlap), 1.43 (overlap) |
7 | 32.4 | 1.26 (overlap), 1.73 (m) | 32.6 | 1.24 (overlap), 1.84 (overlap) | 27.5 | 1.56 (m), 1.66 (overlap) |
8 | 38.6 | 1.54 (overlap) | 39.3 | 1.85 (overlap) | 43.4 | 2.16 (overlap) |
9 | 55.0 | 0.74 (td, J9a/8a = J9a/11a = 10.5, J9a/11e = 4.0) | 60.2 | 2.17 (overlap) | 59.1 | 2.32 (overlap) |
10 | 37.5 | — | 35.8 | — | 36.5 | — |
11 | 21.9 | 1.32 (overlap), 1.63 (overlap) | 213.3 | — | 214.6 | — |
12 | 29.7 | 1.29 (overlap), 1.36 (overlap) | 51.5 | 2.47 (overlap), 2.13 (overlap) | 47.6 | 1.88 (overlap), 2.93 (d, J12a/e = 12.0) |
13 | 46.8 | — | 50.9 | — | 55.4 | — |
14 | 51.6 | 1.22 (overlap) | 50.7 | 1.96 (overlap) | 83.4 | — |
15 | 24.0 | 1.28 (overlap), 1.61 (overlap) | 23.6 | 1.37 (m), 1.74 (overlap) | 32.6 | 1.83 (m), 1.70 (overlap) |
16 | 39.5 | 1.84 (m), 1.61 (overlap) | 39.2 | 1.82 (overlap), 1.90 (overlap) | 40.0 | 1.90 (overlap), 2.13 (overlap) |
17 | 82.1 | — | 80.8 | — | 81.5 | — |
18 | 14.4 | 0.85 (s) | 15.4 | 0.77 (s) | 20.8 | 0.85 (s) |
19 | 11.4 | 1.06 (s) | 13.9 | 1.26 (s) | 11.8 | 1.23 (s) |
20 | 26.0 | 1.17 (s) | 26.2 | 1.26 (s) | 29.4 | 1.47 (s) |
Carbon | 4 | 5 | 6 | |||
---|---|---|---|---|---|---|
δC | δH (J in Hz) | δC | δH (J in Hz) | δC | δH (J in Hz) | |
1 | 161.6 | 7.45 (d, J1,2 = 10.0) | 38.6 | 2.17 (m), 2.52 (td, J = 15.0, J = 7.0) | 161.4 | 7.42 (d, J1,2 = 10.4) |
2 | 127.8 | 5.79 (d, J2,1 = 10.5) | 38.1 | 1.20 (overlap), 2.74 (ddd, J = 13.0, J = 6.5, J = 2.0) | 127.5 | 5.83 (d, J2,1 = 10.0) |
3 | 202.0 | — | 213.7 | — | 202.8 | — |
4 | 41.2 | 2.19 (overlap), 2.43 (overlap) | 45.0 | 2.01 (m), 2.36 (overlap) | 41.4 | 2.11 (dd, J4a/4e = 17.6, J4a/5a=3.6), 2.40 (dd, J4e/4a = 17.6, J4e/5α = 14.4) |
5 | 45.1 | 1.91 (m) | 48.1 | 1.52 (m) | 46.4 | 1.92 (overlap) |
6 | 27.9 | 1.53 (overlap), 1.59 (overlap) | 29.4 | 1.33 (overlap), 1.36 (overlap) | 28.3 | 1.54 (overlap), 1.40 (overlap) |
7 | 30.5 | 1.59 (overlap), 2.13 (m) | 33.0 | 1.18 (overlap), 1.84 (overlap) | 32.8 | 1.0 (overlap), 1.87 (overlap) |
8 | 36.9 | 2.60 (m) | 39.3 | 1.83 (overlap) | 33.9 | 1.96 (overlap) |
9 | 60.5 | 2.19 (overlap) | 64.4 | 1.90 (overlap) | 55.3 | 1.02 (overlap) |
10 | 39.4 | — | 36.4 | — | 40.7 | — |
11 | 212.6 | — | 214.3 | — | 68.0 | 4.52 (d, J11,9 α = 2.8) |
12 | 51.8 | 2.09 (d, J12a/e = 12), 2.56 (d, J12e/a = 12.0) | 51.7 | 2.06 (d, J = 11.5), 2.42 (overlap) | 41.3 | 1.48 (overlap), 1.73 (dd, J12 a/e=14.0, J12/11 = 2.4) |
13 | 55.6 | — | 48.1 | — | 46.1 | — |
14 | 150.0 | — | 50.9 | 1.91 (overlap) | 53.5 | 1.23 (m) |
15 | 119.7 | 5.45 (dd, J15/16a = 4.5, J15/16e = 2.0) | 23.7 | 1.36 (overlap), 1.71 (m) | 24.3 | 1.33 (overlap), 1.63 (overlap) |
16 | 46.9 | 2.38 (overlap), 2.52 (m) | 39.2 | 1.32 (overlap), 1.77 (overlap) | 39.1 | 1.62 (overlap), 1.89 (overlap) |
17 | 82.0 | — | 80.8 | — | 82.6 | — |
18 | 19.8 | 0.97 (s) | 15.3 | 0.76 (s) | 17.0 | 1.09 (s) |
19 | 13.5 | 1.31 (s) | 11.3 | 1.23 (s) | 15.5 | 1.28 (s) |
20 | 24.9 | 1.23 (s) | 26.1 | 1.25 (s) | 26.3 | 1.14 (s) |
Carbon | 7 | 8 | ||
---|---|---|---|---|
δC | δH (J in Hz) | δC | δH (J in Hz) | |
1 | 32.8 | 2.01 (overlap), 1.70 (overlap) | 77.0 | 3.77 (dd, J1,2β = 10.8, J1,2α = 6.3) |
2 | 38.7 | 2.48 (overlap), 2.22 (overlap) | 47.4 | 2.52 (overlap) |
3 | 214.4 | — | 211.2 | — |
4 | 45.0 | 2.37 (t, J4α,4β/5 = 14.4), 2.01 (overlap) | 45.5 | 2.37 (t, J4α,4β/5 = 14.4), 1.92 (overlap) |
5 | 41.9 | 1.77 (dd, J5,4β = 12.0, J = 2.4) | 43.5 | 1.50 (overlap) |
6 | 28.2 | 1.70 (overlap) | 29.7 | 1.48 (overlap) |
7 | 27.8 | 1.50 (overlap), 1.30 (overlap) | 32.9 | 1.70 (overlap), 0.98 (overlap) |
8 | 34.9 | 2.48 (overlap) | 37.4 | 1.48 (overlap) |
9 | 79.3 | — | 61.7 | 0.98 (overlap) |
10 | 41.9 | — | 44.5 | — |
11 | 69.5 | 3.87, (d, J11,12α = 2.4) | 68.0 | 3.93 (m) |
12 | 39.2 | 1.87 (m), 1.70 (overlap) | 42.9 | 1.92 (overlap), 1.38 (overlap) |
13 | 46.8 | — | 47.6 | — |
14 | 40.7 | 2.22 (overlap) | 50.8 | 1.38 (overlap) |
15 | 23.5 | 1.70 (overlap), 1.30 (overlap) | 24.6 | 1.68 (overlap), 1.38 (overlap) |
16 | 37.9 | 1.61 (dt, J16α,16β = 13.8, J16α,15α = 2.4), 1.50 (overlap) | 39.2 | 1.92 (overlap), 1.70 (overlap) |
17 | 82.3 | — | 81.8 | — |
18 | 13.6 | 1.25 (s) | 15.1 | 0.85 (s) |
19 | 13.5 | 1.16 (s) | 7.3 | 1.11 (s) |
20 | 26.3 | 0.86 (s) | 25.9 | 1.18 (s) |
% inhibition = [100 − {(mean of O. D. of test compound − mean of O. D. of negative control)/(mean of O. D. of positive control − mean of O. D. of negative control) × 100}] |
Metabolite 2 showed the M+ at m/z 316.2039 in the HREI-MS, in agreement with the formula C20H28O3 (calcd 316.2033) with seven degrees of unsaturation. The presence of hydroxyl, and ketonic, and α,β-unsaturated carbonyl functionalities were inferred from the peaks at νmax (cm−1) 3490, 1701, and 1659, respectively, in the IR spectrum. The appearance of downfield doublets for the olefinic methine protons at δ 7.57 (J1,2 = 10.5 Hz) and 5.78 (J2,1 = 10.5 Hz) was observed in the 1H-NMR spectrum (Table 1). This suggested the presence of a double bond between C-1 and C-2, conjugated with C-3 ketonic carbonyl. Furthermore, the downfield shift of C-12 methylene protons at δ 2.47 (overlapped), and 2.13 (overlapped) suggested oxidation at C-11 position. In the 13C-NMR spectrum (Table 1) two new olefinic carbons appeared at δ 162.3 and 127.7, along with a new quaternary carbon at δ 213.3, which suggested the introduction of CC and a ketonic carbon, respectively. The double bond was placed between C-1 and C-2 on the basis of HMBC correlations of H-1 (δ 7.45) with the ketonic C-3 (δ 202.0), C-5 (δ 45.5), and C-10 (δ 35.8). H-2 (δ 5.79) showed HMBC correlations with the C-3 ketonic carbon (δ 202.0), and C-4 (δ 41.4), which suggested an α,β-unsaturation. This was further supported by UV absorbance at λmax 230 nm. The position of new ketonic group (δ 213.3) was inferred from its HMBC correlations with H-9 (δ 2.17), H2-12 (δ 2.12, 2.47), and H-18 (δ 0.77) suggested a carbonyl group at C-11 (Fig. 3). NOESY correlations showed that the stereochemistry of metabolite 2 was retained as in substrate 1. Thus the structure of the new compound 2 was deduced as 17β-hydroxy-17α-methyl-5α-androsta-1-ene-3,11-dione.
The HREI-MS of metabolite 3 showed the M+ at m/z 334.2140, was consistent with the formula C20H30O4 (calcd 334.2144). A 30 a.m.u. increase in mass as compared to the substrate 1 (m/z 334), suggested the addition of two oxygen atoms with the loss of two hydrogen atoms. The IR absorptions at νmax (cm−1) 3523 and 3490 were due to OH groups. The peak at 1692 cm−1 was due to the presence of a ketonic carbonyl group. In the 1H-NMR spectrum (Table 1), C-12 methylene protons appeared downfield at δ 1.88 (overlapped), and a doublet at δ 2.93 (d, J12a/e = 12.0 Hz), suggesting an oxidation at C-11. C-15 methylene protons appeared at δ 1.83 (multiplet) and 1.70 (overlapped), which suggested a change in the chemical environment at vicinal position. In the 13C-NMR spectrum (Table 1), two additional quaternary carbon signals appeared at δ 214.6 (CO), and 83.4 (C–OH). The HMBC correlations of H-9 (δ 2.32) and H2-12 (δ 1.88, 2.93) with the ketonic carbon (δ 214.6) indicated its presence at the C-11 position (Fig. 3). The position of new OH group was deduced through the HMBC correlations of H2-15 (δ 1.83, 1.70) and H-18 (δ 0.85) with the quaternary carbon (δ 83.4), which indicated hydroxylation at C-14 (Fig. 3). The OH-14 (δ 3.70) displayed NOESY correlation with the α-oriented H-9 (δ 2.32) (acetone-d6), suggesting its α-orientation (Fig. 4). NOESY correlations showed that the stereochemistry of metabolite 3 was retained as in substrate 1. The structure of the new metabolite 3 was thus deduced as 14α,17β-dihydroxy-17α-methyl-5α-androstan-3,11-dione.
Metabolite 4 showed the M+ at m/z 314.1862 in the HREI-MS, supporting the formula C20H26O3 (calcd 314.1876), consistent with eight degrees of unsaturation. In the IR spectrum, the peaks at νmax (cm−1) 3460, 1708, and 1658 indicated the presence of hydroxyl, ketonic, and α,β-unsaturated carbonyl groups, respectively. Appearance of the olefinic doublets at δ 7.45 (J1,2 = 10.0 Hz) and 5.79 (J2,1 = 10.5 Hz) in the 1H-NMR spectrum suggested the presence of a double bond between C-1 and C-2 in ring-A conjugated with the C-3 ketonic carbonyl moiety. The 1H-NMR spectrum (Table 2) showed that C-12 methylene protons appeared as two sharp doublets at δ 2.09 (J12a/e = 12.0 Hz) and 2.56 (J12e/a = 12.0 Hz), which suggested an oxidation at C-11 position. The appearance of a double doublet at δ 5.45 (J15/16a = 4.5 Hz, J15/16e = 2.0 Hz) suggested a double bond between C-14 and C-15 in ring-D. The 13C-NMR spectrum (Table 2) also showed three olefinic signals at δ 161.6, 127.8, and 119.7, and two quaternary carbon signals at δ 150.0 and 212.6, indicating the presence of two double bonds, and a ketonic carbonyl group, respectively. COSY and HMBC techniques were used to deduce the final structure of metabolite 4. The presence of an α,β-unsaturated carbonyl was deduced from UV-visible spectrum (λmax 230 nm). Protons H-1 (δ 7.45) and H-2 (δ 5.79) showed HMBC correlations with the ketonic carbon (δ 202.0) and C-10 (δ 39.4) also indicating the presence of an α,β-unsaturated carbonyl moiety in ring-A. H-9 (δ 2.19) and H2-12 (δ 2.09, 2.56) showed HMBC correlations with the ketonic carbon (δ 212.6), and H-19 (δ 1.31) showed correlation with C-9 (δ 60.5). This indicated position of the carbonyl at C-11. The position of the other CC bond between C-14 and C-15 was deduced on the basis of HMBC correlations of H-15 (δ 5.45) with C-13 (δ 55.6), C-16 (δ 46.9), and C-17 (δ 82.0 (Fig. 3). COSY correlations between C-16 methylene (δ 2.38, 2.52) and olefinic protons (δ 5.45) further supported a double bond between C-14 and C-15 in ring-D. Thus the new compound was identified as 17β-hydroxy-17α-methyl-5α-androsta-1,14-diene-3,11-dione.
Metabolite 7 was obtained as a white solid. The molecular formula C20H32O4 was deduced through the HREI-MS, which displayed the M+ at m/z 336.2299 (calcd 336.2301). The 32 a.m.u. increase in molecular weight could be attributed to the addition of two oxygen atoms as hydroxyl groups. The presence of the hydroxyl groups was also inferred from the IR spectrum at νmax (cm−1) 3377, and 3350. A new methine proton signal at δ 3.87 (d, J11,12α = 2.4 Hz) (1H-NMR), and a methine carbon at δ 69.5, and a quaternary carbon signal at δ 79.3 (13C-NMR) suggested a dihydroxylation in mestanolone (1) (Table 3). COSY and HMBC correlations were used to deduce the positions of the newly introduced OH groups. H2-12 (δ 1.87, and 1.70) showed HMBC correlations with the newly formed methine carbon at δ 69.5 (C-11). COSY cross-peaks with the newly formed methine proton at δ 3.87 further supported the hydroxylation at C-11 (Fig. 3). The second OH was placed at C-9 on the basis of HMBC correlations of H3-19 (δ 1.16), and H-8 (δ 2.48) with C-9 (δ 79.3). H-11 (δ 3.87) also displayed HMBC correlations with C-9, which further supported an OH at C-9. H-11 was found to be α-oriented as it showed its NOESY correlation with H-1 (δ 2.22) (Fig. 4). This suggested that the geminal OH-11 has a β-orientation. H-11 appeared as a doublet (J11,12α = 2.4 Hz) which also supported a β-orientation (axial) of C-11 OH. The OH at C-9 was found to be α-oriented, as deduced from the NOESY correlations of OH-9 (δ 4.36) with H-14 (δ 2.23) (acetone-d6) (Fig. 4). Thus the new metabolite 7 was identified as 9α,11β,17β-trihydroxy-17α-methyl-5α-androstan-3-one.
The physical appearance of metabolite 8 was a white amorphous solid. The HREI-MS showed the [M+] at m/z 336.2299 (calcd 336.2301, C20H32O4). The 32 a.m.u. increase in mass than the substrate 1 suggested the addition of two oxygen atoms as OH. The IR absorptions at 3388, and 3370 cm−1 were due to OH groups. The 1H-NMR spectrum (Table 3) displayed signals for two new downfield methine protons at δ 3.93 (m), and 3.77 (dd, J1,2β = 10.8 Hz, J1,2α = 6.3 Hz, H-1) with their corresponding carbons at δ 68.0 (C-11), and 77.0 (C-1), respectively, in the 13C-NMR spectrum (Table 3). This further suggested dihydroxylation in substrate 1. The OH groups were placed on the basis of HMBC and COSY correlations. The C-12 methylene protons (δ 1.92, and 1.38) displayed COSY correlations with the newly formed methine proton at δ 3.93. These correlations indicated that one of the hydroxyl groups was at C-11. The H-9 (δ 0.98) showed HMBC correlations with C-11, and COSY correlations with H-11, thus placing a hydroxyl at C-11. The H-9 also showed HMBC correlations with the new downfield methine carbon at δ 77.0 (C-1). The CH3-19 (δ 1.11) displayed HMBC correlations with carbon at δ 77.0, suggested that the second OH was located at C-1 (Fig. 3). The newly appeared methine proton at δ 3.77 showed NOESY cross-peaks with H-5 (δ 1.50), and H-9 (δ 0.98). As H-5 and H-9 are α-oriented in substrate 1, therefore H-1 was also assumed to be α-oriented. Thus the geminal OH at C-1 was deduced to be β-oriented. H-11 (δ 3.93) showed NOESY correlations with H-8 (δ 1.48), H-18 (δ 0.85), and H-19 (δ 1.11). As these protons are β-oriented in the substrate mestanolone (1), therefore the resonance of α-OH was inferred at C-11. The structure of the new metabolite 8 was thus deduced as 1β,11α,17β-trihydroxy-17α-methyl-5α-androstan-3-one.
Metabolites 5, and 6 were characterized as known metabolites by comparing their spectroscopic data with the previously reported data in literature. These metabolites were identified as 17β-hydroxy-17α-methyl-5α-androstan-3,11-dione (5), and 11β,17β-dihydroxy-17α-methyl-5α-androsta-1-ene-3-one (6). Metabolite 5 was previously reported by Davitishvili et al. through chemical modification of 3α-hydroxy-5α-androst-9(11)-en-17-one.18 Metabolite 6 was also reported in the literature through chemical modification of androstan-17β-ol-3,11-dione.19
The structure of metabolite 6 was unambiguously deduced through the single-crystal X-ray diffraction techniques. X-ray diffraction studies showed that the molecule consists of four fused rings, rings A (C-1–C-5/C-10), B (C-5–C-10), C (C-8–C-9/C-11–C-14), and D (C-13–C-17). The six membered ring A exists in a pseudo chair conformation, while trans-fused rings B and C exist in chair conformation. trans-fused ring D exists in an envelope conformation. The hydroxyl group at C-11 was found to be β-oriented (Fig. 5). The single-crystal X-ray diffraction data was submitted to the Cambridge Crystallographic Data Collection with CCDC 1532897.
Compounds | HeLa Cell line (Cancer cell line) IC50 ± SD [μM] | H460 Cell line (Cancer cell line) IC50 ± SD [μM] | 3T3 Cell line (Normal cell line) IC50 ± SD [μM] |
---|---|---|---|
1 | 27.6 ± 1.1 | >30 | >30 |
2 | 19.2 ± 2.9 | >30 | >30 |
3 | >30 | >30 | >30 |
5 | >30 | >30 | >30 |
6 | 12.8 ± 0.6 | >30 | >30 |
7 | >30 | >30 | >30 |
8 | >30 | >30 | >30 |
Standard drug, doxorubicin (chemotherapy medicine) | 1.2 ± 0.4 | 0.8 ± 0.03 | — |
Standard, cycloheximide | — | — | 0.8 ± 0.2 |
Footnote |
† Electronic supplementary information (ESI) available: 1H, 13C NMR, HSQC, COSY, NOESY and MS data of all products. CCDC 1532897. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c8ra01309h |
This journal is © The Royal Society of Chemistry 2018 |