{"paper_id":"1ca7d0f7-cb0d-438a-b10b-2eb3dea27aab","body_text":"R E S E A R C H A R T I C L E Open Access\nBiotransformation of oral contraceptive\nethynodiol diacetate with microbial and plant\ncell cultures\nSalman Zafar 1,2, Sammer Yousuf 1, Hammad A Kayani 1, Saifullah Saifullah 1*, Saifullah Khan 1, Abdullah M Al-Majid 3\nand M Iqbal Choudhary 1,3*\nAbstract\nBackground: Biotransformation by using microbial and plant cell cultures has been applied effectively for the\nproduction of fine chemicals on large scale. Inspired by the wealth of literature available on the biotransformation\nof steroids, we decided to investigate the biotransformation of ethynodiol diacetate ( 1) by using plant and\nmicrobial cultures.\nResults: The biotransformation of ethynodiol diacetate ( 1) with Cunninghamella elegans and plant cell suspension\ncultures of Ocimum basilicum and Azadirachta indica is being reported here for the first time. Biotransformation of 1\nwith Cunninghamella elegans yielded three new hydroxylated compounds, characterized as\n17α-ethynylestr-4-en-3β,17β-diacetoxy-6α-ol ( 2), 17 α-ethynylestr-4-en-3β,17β-diacetoxy-6β-ol ( 3), and\n17α-ethynylestr-4-en-3β,17β-diacetoxy-10β-ol ( 4) and a known metabolite, 17 α-ethynyl-17β-acetoxyestr-4-en-3-one\n(5). The biotransformation of 1 with Ocimum basilicum included hydrolysis of the ester group, oxidation of alcohol\ninto ketone, and rearrangement of the hydroxyl group. Thus four major known metabolites were characterized as\n17α-ethynyl-17β-acetoxyestr-4-en-3-one ( 5), 17 α-ethynyl-17β-hydroxyestr-4-en-3-one ( 6), 17α-ethynyl-3\nβ-hydroxy-17β-acetoxyestr-4-ene ( 7) and 17 α-ethynyl-5α,17β-dihydroxyestr-3-ene ( 8). Biotransformation of 1 with\nAzadirachta indica culture yielded compounds 5 and 6. Spectroscopic data of compound 8 is being reported for\nthe first time. Structure of compound 6 was unambiguously deduced through single-crystal x-ray diffraction studies.\nConclusion: Biotransformation of an oral contraceptive, ethynodiol diacetate ( 1), by using microbial and plant cell\ncultures provides an efficient route to the synthesis of a library of new steroids with potential contraceptive\nproperties. These methods can be employed in the production of such compounds with high stereoselectivity.\nKeywords: Ethynodiol diacetate, Microbial transformation, Biotransformation, Ocimum basilicum , Azadirachta indica ,\nCunninghamella elegans , Cell suspension culture, Norethisterone, 17 α-Ethynylestr-4-en-3β,17β-diacetoxy-6α-ol,\n17α-Ethynylestr-4-en-3β,17β-diacetoxy-6β-ol, 17 α-Ethynylestr-4-en-3β,17β-diacetoxy-10β-ol\nBackground\nDevelopment of efficient, environmental friendly and\ncost effective routes to synthesize fine chemicals is the\nneed of the day. Biotransformation has been an exciting\narea of research for decades. Enzymes from various\nsources, i.e., microorganisms, animal and plant cells, have\nbeen employed for carrying out reactions at chemically\ninaccessible positions of organic compounds. Microbial\ntransformation of steroids has been extensively investi-\ngated [1] and various hydroxylated derivatives have been\nproduced on large scale [2]. Cell suspension cultures of\nplants have also been efficiently employed for the bio-\ntransformation of organic compounds, e.g., steroids [3],\nterpenes [4,5], alkaloids [6] and flavonoids [7]. The reac-\ntions carried out by microbial and plant cultures include\nhydroxylation, oxidation and reduction of alcohols,\nketones and C = C bond [8].\n* Correspondence: saif_sahir@yahoo.com; iqbal.choudhary@iccs.edu\n1H. E. J. Research Institute of Chemistry, International Center for Chemical\nand Biological Sciences, University of Karachi, Karachi 75270, Pakistan\n3Department of Chemistry, College of Science, King Saud University, PO Box\n2455, Riyadh 11451, Saudi Arabia\nFull list of author information is available at the end of the article\n© 2012 Zafar et al.; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative\nCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and\nreproduction in any medium, provided the original work is properly cited.\nZafar et al. Chemistry Central Journal 2012, 6:109\nhttp://journal.chemistrycentral.com/content/6/1/109\n\nOcimum basilicum L. (Lamiaceae) (sweet basil) is\nfound mostly in Asia. It is used as a flavorant in food,\nperfumery, cosmetics and medicines [9]. There are some\nreports of biotransformation of chemical compounds\nwith O. basilicum culture [10]. Azadirachta indica A.\nJuss. (Meliaceae) (Neem) is native to tropical and semi-\ntropical Asia. Cell suspension culture of Azadirachta\nindica has also been previously recruited for the struc-\ntural transformation of dydrogesterone [11].\nEthynodiol diacetate (1) is a semi synthetic steroidal drug,\nused as an oral contraceptive . It inhibits the ovulation\nprocess, and serves as a potent progestin. It provides ad-\nequate control of menstrual cyclicity in combination with\nan estrogen, and thus has a co mplete contraceptive prop-\ne r t y ,e v e ni nl o wd o s e s[ 1 2 ] .T h eb i o t r a n s f o r m a t i o no f1 has\nbeen previously studied in vivo in rhesus monkey [13] and\nbaboon [14], and in vitro by rat and human liver cells [15].\nBiotransformation of 1 with microbial and plant cell cul-\ntures has not been reported earlier. During the current\nstudy, we investigated the metabolism of compound 1 with\na fungal and two plant cell cultures, which resulted in a\nnumber of new 2–4 and known 5–8 metabolites.\nResults\nMicrobial transformation of ethynodiol diacetate with\nC. elegans\nThe 1H- and 13C-NMR chemical shifts of compounds\n2–5 are presented in Tables 1 and 2, respectively. Other\ndata is presented below:\n17α-Ethynylestr-4-en-3β,17β-diacetoxy-6α-ol (2).\nColorless amorphous solid (5 mg, 0.5%). 1H-NMR\n(CD3OD, 300 MHz): Table 1, 13C-NMR (CD3OD, 75\nMHz): Table 2. EI-MS m/z (rel. int., %): 400 (15, M+),\nTable 1 1H-NMR data of compounds 1-8 at 300 (compounds 2,3,4,5,7), 400 (compound 8) and 500 (compound 6) MHz;\nδ in ppm, J and W 1/2 in Hz\nCOMPOUNDS\nCarbon 1 2 3 4 5 6 7 8\n1 1.41, 2.05 1.12, 2.07, 1.36, 1.70 1.38, 1.94 1.52, 2.24 1.54, 2.25 1.74, 1.96 1.75, 1.92\n2 2.01, 2.27 dt, J=13.4, 2.8 1.33, 1.94 1.46, 2.01 1.55, 1.83 2.21, 2.38 2.28, 2.37 1.95, 2.23 1.91, 1.98\n3 5.20 br s,\nW1/2=19.6\n4.05, m\n(W1/2=17.4 Hz)\n5.20, m\n(W1/2=22.8 Hz)\n4.01, m\n(W1/2=15.6 Hz)\n- - 4.14 m,\nW1/2=16.8\n5.85d J =9.6\n4 5.32 5.55, br s\n(W1/2=9.7 Hz)\n5.55, br s,\n(W1/2=17.2 Hz)\n5.40, br s\n(W1/2=9.37 Hz)\n5.81 s 5.81 s 5.37 5.50 d J=9.6\n5 - ---- - - -\n6 0.95, 1.68 4.12, br s\n(W1/2=9.6 Hz)\n4.16, br s,\n(W1/2=17.1 Hz)\n2.05, 2.41 2.27, 2.49 2.29, 2.45 dt,\nJ=14.5, 3.28\n1.67, 1.82 1.77, 1.82\n7 1.17, 1.80 2.05, 2.65 1.13, 1.87 0.90, 1.75 1.12, 1.83 1.06, 1.82 0.94, 1.73 1.15, 1.78\n8 1.25 1.94 1.82 1.72 1.36 1.35 1.25 0.82\n9 0.71 0.60 0.65 0.80 0.85 0.86 0.67 1.08\n10 1.77 1.80 2.20 - 2.06 2.07 td, J=10.5, 4.7 1.75 1.47\n11 1.25, 1.84 1.23, 1.85 1.59, 1.67 1.22, 1.91 1.23, 1.88 1.13, 2.02 0.78, 1.51\n12 1.67, 1.82 1.65, 1.83 1.66, 1.84 1.66, 1.82 1.70, 1.87 1.63, 1.75 1.27, 2.04 1.54, 1.68\n1 3 - ---- - - -\n14 1.51 1.50 1.52 1.48 1.54 1.51 1.50 1.42\n15 1.31, 1.72 1.37, 1.67 1.27, 1.29 1.37, 1.72 1.33, 1.75 1.27, 1.54 1.32, 1.77 1.28, 1.67\n16 1.98, 2.72 2.15, 2.65 2.04, 2.65 2.05, 2.65 1.97, 2.73 ddd,\nJ=15, 9.6, 5.7\n1.98, 2.27 1.99, 2.70 ddd\nJ=15, 6.0, 3.6\n1.94, 2.27\n1 7 - ---- - -\n18 0.87, s 0.94, s 0.95, s 0.93, s 0.91, s 0.89, s 0.87, s 0.84, s\n2 0 - ---- - - -\n21 2.55, s 2.94, s 2.97, s 2.95, s 2.57, s 2.55, s 2.55, s 2.55, s\n2 2 - ---- -\n23 2.02 2.01, s 2.00, s 1.99, s 2.02 s 2.01 s\n24\n25 2.02 2.01, s 2.00, s 1.99, s\nNote: Assignments based on COSY, HMBC and HMQC spectra. Assignments shown without multiplicity means multiplet.\nZafar et al. Chemistry Central Journal 2012, 6:109 Page 2 of 8\nhttp://journal.chemistrycentral.com/content/6/1/109\n\n340 (95), 298 (37), 280 (35), 231 (50), 119 (39), 110\n(72), 91 (100), 79 (75), 55 (65). HREI-MS m/z (mol.\nformula, calcd value): 400.2065 (C 24H32O5, 400.2038).\n17α-Ethynylestr-4-en-3β,17β-diacetoxy-6β-ol (3).\nColorless amorphous solid (10 mg, 1.0%). 1H-NMR\n(CD3OD, 300 MHz): Table 1, 13C-NMR (CD3OD, 75\nMHz): Table 2. EI-MS m/z (rel. int., %): 400 (15, M+),\n340 (95), 298 (37), 280 (35), 231 (50), 119 (39), 110\n(72), 91 (100), 79 (75), 55 (65). HREI-MS m/z (mol.\nformula, calcd value): 400.2065 (C 24H32O5, 400.2038).\n17α-Ethynylestr-4-en-3β,17β-diacetoxy-10β-ol (4) .\nColorless amorphous solid (5 mg, 0.5%). 1H-NMR\n(CD3OD, 300 MHz): Table 1, 13C-NMR (CD3OD, 75\nMHz): Table 2. EI-MS m/z (rel. int., %): 400 (15, M+),\n340 (95), 298 (37), 280 (35), 231 (50), 119 (39), 110\n(72), 91 (100), 79 (75), 55 (65). HREI-MS m/z (mol.\nformula, calcd value): 400.2065 (C\n24H32O5, 400.2038).\n17α-Ethynyl-17β-acetoxyestr-4-en-3-one (5).\nColorless crystalline solid (14 mg, 1.4%). M. P. 161–\n163°C\n(lit. 161 –162°C [ 16]). 1H-NMR (CDCl3, 300 MHz):\nTable 1, 13C-NMR (CDCl3, 75 MHz): Table 2. EI-MS:\nm/z (rel. int., %) 340 (90, M+,C 22H28O3), 298 (37),\n231 (47), 119 (39), 110 (69), 91 (100), 79 (75),\n55 (65).\nResults of biotransformation of ethynodiol diacetate with\nO. basilicum\nThe 1H- and 13C-NMR chemical shifts of compounds\n6–8 are presented in Tables 1 and 2, respectively. Other\ndata is presented below:\n17α-Ethynyl-17β-hydroxyestr-4-en-3-one (6) .\nColorless crystalline solid (20 mg, 3.3%). M. P. 201–\n203°C (lit. 203 –204°C [ 17]). 1H-NMR (CDCl3, 500\nMHz): Table 1, 13C-NMR (CDCl3, 150 MHz): Table 2.\nEI-MS: m/z (rel. int., %) 298 (81, M+,C 20H26O2), 231\n(71), 160 (40), 135 (44), 110 (85), 91 (100), 79 (77), 55\n(60). Crystal data :C 20H26O2, Mr = 298.41,\nOrthorhombic, space group P2 12121, a = 6.5463(5) Å ,\nb = 12.1646(10) Å , c = 20.7743(17)Å, α, β, γ =9 0o,\nV = 1654.3(2) Å3 , Z =4 , ρcalc = 1.198 mg/m3, F\n(000) = 648, μ (Mo K α) = 0.71073 Å, max/min\ntransmission 0.9881/ 0.9669, crystal size 0.45 x 0.17 x\n0.16, 1.94° < θ< 25.5°, 9821 reflections were collected,\nof which 3,601 reflections were judged observed\n(Rint = 0.0274). The R values were: R1 = 0.0367,\nwR2 = 0.0860 for I > 2 σ(I), and R1 = 0. 0.0424,\nwR2 = 0.890 for all data; max/min residual electron\ndensity: -0.148 eA ˚/-0.148 eA ˚−3. The structure was\nsolved by the direct methods, expanded by using Fourier\ntransformation techniques [18] and refined by a full-\nmatrix least-square calculation on F\n2 with the aid of\nSHELXL97 program [19]. Crystallographic data for\ncompound 6 has been deposited in the Cambridge\nCrystallographic Data Center. The crystallographic\ninformation can directly be obtained free of charge from\nCCDC data center (CCDC 837461 reference code).\n17α-Ethynyl-3β-hydroxy-17β-acetoxyestr-4-ene\n(7).\nColorless amorphous solid (3.5 mg, 0.58%). 1H-NMR\n(CDCl3, 300 MHz): Table 1, 13C-NMR (CDCl3,\n75 MHz): Table 2. EI-MS: m/z (rel. int., %) 342 (100,\nM+,C 22H30O3), 255 (5), 185 (4), 145 (15), 105 (30), 91\n(43), 81 (51), 55 (49).\n17α-Ethynyl-5α,17β-dihydroxyestr-3-ene(8). Colorless\namorphous solid (2.7 mg, 0.45%). 1H-NMR (CDCl3,4 0 0\nMHz): T able1, 13C-NMR (CDCl3,1 0 0M H z ) :T a b l e2.\nEI-MS: m/z (rel. int., %) 300 (22, M+,C 20H28O2), 282\n(37), 199 (63), 149 (73), 91 (100), 81 (89), 55 (86).\nResults of biotransformation of ethynodiol diacetate with\nA. indica\nBiotransformation of 1 with A. indica afforded two\nknown metabolites 5 and 6, which have been discussed\nearlier.\nTable 2 13C-NMR data of compounds 1 –8, MHz; δ in ppm\nCompounds\nC 12345678\n1 27.7 27.0 24.1 34.7 26.6 26.6 25.6 19.2\n2 34.9 32.5 28.2 29.1 36.5 36.5 34.9 20.8\n3 70.3 68.0 71.2 68.1 199.8 199.9 67.4 132.0\n4 119.9 129.0 124.0 128.1 124.7 124.6 124.3 132.4\n5 144.8 143.0 145.0 143.0 166.3 166.5 142.8 69.7\n6 31.3 73.5 73.0 32.2 35.4 35.5 32.9 39.7\n7 25.7 38.3 39.0 33.0 30.7 30.6 31.4 26.6\n8 41.2 32.2 35.5 37.0 40.7 41.0 41.2 41.2\n9 49.4 51.4 50.5 55.0 48.9 49.1 49.7 40.9\n10 41.6 48.9 38.3 70.8 42.5 42.5 41.8 49.4\n11 25.2 26.5 26.2 20.8 26.2 26.2 25.7 27.8\n12 32.9 34.2 34.1 34.0 32.8 32.4 32.1 32.7\n13 47.7 49.0 48.8 48.8 47.5 46.9 47.6 47.0\n14 47.6 48.9 49.0 49.5 47.6 49.2 47.7 45.9\n15 23.4 24.2 30.6 24.2 23.4 22.9 23.4 22.9\n16 37.3 38.2 38.1 38.4 37.2 38.8 37.3 38.9\n17 84.5 85.9 86.0 86.0 84.3 79.7 84.5 79.9\n18 13.4 14.0 13.8 14.0 13.4 12.7 13.4 12.7\n20 83.3 83.0 84.0 84.0 83.2 87.2 83.4 87.6\n21 74.8 76.5 76.5 76.8 75.0 74.2 74.5 73.9\n22 169.6 171.5 171.0 171.2 169.5 169.6\n23 21.4 21.2 21.2 21.5 21.4 21.5\n24 170.9 171.5 172.0 171.2\n25 21.4 21.2 21.2 21.5\nZafar et al. Chemistry Central Journal 2012, 6:109 Page 3 of 8\nhttp://journal.chemistrycentral.com/content/6/1/109\n\nDiscussion\nIn the current study, biotransformation of ethynodiol\ndiacetate ( 1)C 24H32O4, with C. elegans is being carried\nout for the first time, affording three new 2–4 and a\nknown 5 metabolite. Biotransformation of 1 was also\ninvestigated with cell cultures of O. basilicum yielding\nfour known metabolites 5–8. Substrate 1 was also sub-\njected to biotransformation with A. indica and two\nknown metabolites 5 and 6 were obtained.\nThe molecular formula for metabolite 2 (C24H32O5)\nwas obtained from the HREI-MS [ M+ m/z 400.2065\n(calcd 400.2038)], which was 16 a.m.u. higher than the\nsubstrate 1. The compound was found to be UV in-\nactive. The IR spectrum showed the presence of an ester\ncarbonyl (1742 cm\n-1), and an -OH (3433 cm -1) func-\ntional groups.\nThe 16 a.m.u. increment in the M+ of the metabolite\n2, as compared to substrate 1, could be attributed to the\naddition of an oxygen atom. The 1H-NMR of 2 (Table 1)\nshowed a methyl singlet at δ 0.94, and a six-proton sing-\nlet (2 x CH 3)a t δ 2.01. This suggested that both ester\ngroups remain intact. Therefore the change was assumed\nto be the hydroxylation of substrate 1. A downfield pro-\nton signal at δ 4.12 (br. s., W1/2 = 9.6 Hz) with its corre-\nsponding carbon at δ 73.5 appeared in the HSQC\nspectrum. Another downfield proton signal at δ 4.05 (m,\nW1/2 = 17.4 Hz) showed HMBC correlation with the\nester carbonyl carbon ( δ 171.5). This proton was there-\nfore assigned to H-3. The H-3 showed COSY interaction\nwith the olefinic proton ( δ 5.55, br. s., W1/2 = 9.7 Hz),\nwhich was assigned to H-4. H-4 in turn, showed a weak\nallylic coupling with the hydroxyl-bearing methine\nproton ( δ 4.12) in COSY spectrum. This suggested that\nthe hydroxylation had occurred at C-6 of the steroidal\nskeleton. This was further confirmed by the HMBC cor-\nrelations of H-6 ( δ 4.12) with C-4 ( δ 129.0), and C-10\n(δ 48.9). H-8 ( δ 1.94) showed NOESY interactions with\nH-6 ( δ 4.12) indicating that the C-6 proton was\nβ-oriented, thus the geminal hydroxyl group was\nα-oriented. The structure of 2 was thus deduced as\n17α-ethynylestr-4-en-3β,17β-diacetoxy-6α-ol.\nMetabolite 3 had the same molecular composition\n(C\n24H32O5)a st h a to f 2, as deduced from the HREI-MS\n[M+ m/z 400.2065 (calcd 400.2038)]. The compound was\nfound to be UV inactive. The IR spectrum showed\nabsorptions for the ester carbonyl (1740 cm -1), and hy-\ndroxyl (3433 cm -1) groups.\nThe 1H- and 13C-NMR spectra of metabolite 3 were\nvery similar to 2. A downfield hydroxyl-bearing methine\nproton signal at δ 4.16 (br. s., W1/2 = 17.1 Hz) with its\ncorresponding carbon at δ 73.0 appeared in the spectra\nof 3.T h eC - 4o l e f i n i cp r o t o n(δ 5.55, br. s., W1/2 = 17.2 Hz)\nshowed a weak allylic coupling with the hydroxyl-bearing\nmethine proton ( δ 4.16). This suggested that the hydro-\nxylation had occurred at C-6 of the steroidal skeleton. The\nNOESY spectrum did not show any correlation between\nH-6 ( δ 4.16), and H-8 ( δ 1.82, axial). Therefore it was\nassigned an equatorial orientation (α-orientation). The rest\nof the proton and carbon values were distinctly similar to\nmetabolite 2. Metabolite 3 was characterized as a new\ncompound (17 α-ethynylestr-4-en-3β,17β-diacetoxy-6β-ol)\nFigure 1.\nThe molecular composition C\n24H32O5 for metabolite 4\nwas obtained from the HREI-MS [ M+ m/z 400.2065\nH\nO\nH\nO\nHHO\nO\nOH\nO\nH\nO\nHHO\nO\nH\nO\nH\nO\nHHO\nO\nOH\nH\nO\nH\nO\nHHO\nOH\nO\nH\nO\nH\nO\nHH\nO\n1\n23 4 5\n1\n20\n3 5 7\n9\n11 13\n14 16\n18\n21\n2223\n24 25\n12 Days\nshaking incubator\nFigure 1 Biotransformation of ethynodiol diacetate (1) with Cunninghamella elegans.\nZafar et al. Chemistry Central Journal 2012, 6:109 Page 4 of 8\nhttp://journal.chemistrycentral.com/content/6/1/109\n\n(calcd 400.2038)], 16 mass units higher than substrate 1.\nThe compound was found to be UV inactive, suggesting\nlack of any conjugated system. The IR spectrum showed\nthe presence of ester carbonyl (1740 cm\n-1), and -OH\n(3433 cm -1) groups.\nThe molecular formula and the IR spectrum of 4\nsuggested the hydroxylation of substrate 1, but the 1H-\nNMR spectrum (Table 1) of 4 did not show any\ndownfield hydroxyl-bearing methylene proton signal. A\ndownfield multiplet at δ 4.01 ( W\n1/2 = 15.6 Hz), and a\nbroad singlet at δ 5.40 ( W1/2 = 9.4 Hz) were vicinally\ncoupled in the COSY 45 o spectrum. These were\nassigned to H-3 ( δ 4.01), and H-4 ( δ 5.40), with corre-\nsponding carbons at δ 68.1 and 128.1, respectively. A\ndownfield quaternary carbon signal at δ 70.8 was\nHMBC correlated with H-4 ( δ 5.40). The only position\nthus available for hydroxylation was C-10. The hy-\ndroxyl group at C-10 was assigned axial orientation in\ncorrespondence with those of previously reported\ncompounds, 3-ethyl-6 β,17β-dihydroxy-18,19-dinor-17α\n-pregn-4-en-20-yn-3-on [20], 13-ethyl-6 β,10β,17β-trihy-\ndroxy-18,19-dinor-17α -pregn-4-en-20-yn-3-on [21],\nand 10 β-hydroxy-19-nor-testosterone [22]. The rest of\nthe spectrum closely resembled with the substrate 1,\nas well as metabolites 2 and 3. The\n13C-NMR\nspectrum of 4 had one CH less than the substrate,\nand an additional downfield quaternary carbon ( δ 70.8)\nwhich further supported the proposed structure,\n17α-ethynylestr-4-en-3β,17β-diacetoxy-10β-ol for me-\ntabolite 4.\nThe M+ of compound 5 (m/z 340, C 22H28O3), 43 amu\nless than compound 1, suggested the loss of an acetyl\nmoiety, either from C-3 or C-17. The compound showed\nflorescence under the UV light indicative of the conver-\nsion of the ester into an unsaturated ketone, through hy-\ndrolysis followed by oxidation. This also confirmed that\nthe ester at C-3 had been hydrolyzed, while C-17 ester\nremains intact. This was confirmed with the help of\n1H-\nand 13C-NMR spectra. The broad singlet at δ 5.20 (H-3)\nwas absent in the 1H-NMR spectrum of 5. 13C-NMR\nshowed the presence of a new ketonic carbonyl signal at\nδ 199.8 (C-3) and the absence of the ester carbonyl at δ\n170.9 (C-24). The compound was thus characterized as\nnorethisterone acetate. It is a potent oral progestational\nagent. Compound 5 has been reported earlier as an\nin vitro metabolite of ethynodiol diacetate by rat and\nhuman liver cells [15].\nThe EI-MS of 6 (C20H26O2) showed the M+ at m/z\n298. The 85 amu decrease in molecular weight suggested\nthe hydrolysis of both the ester groups. The UV flores-\ncence indicated the oxidation of the hydroxyl group,\nformed through hydrolysis of C-3 ester followed by oxi-\ndation into the corresponding α, β-unsaturated ketone.\nThe M\n+ of 6 was 42 amu less than 5, suggesting the\nhydrolysis of C-17 ester group. The rest of the spectrum\nwas in close correspondence with metabolite 5. The\ncompound was characterized as norethisterone. It is a\nprogestin used as oral contraceptive pills. Single-crystal\nX-ray diffraction analysis was carried out to establish the\nstructure of compound 6 (Figures 2 and 3). The ORTEP\nH\nO\nH\nO\nHHO\nO\nH\nO\nH\nO\nHH\nO H\nHO\nH\nO\nHH\nOH\nO\nH\nOH\nHH\nHH\nOH\nHH\nOH\n1\n5 67 8\n1\n20\n3 5 7\n9\n11 13\n14 16\n18\n21\n22\n23\n24 25\nA\nB\nC D\n10-20 Days\nshaking incubator\nFigure 2 Biotransformation of ethynodiol diacetate (1) with cell suspension cultures of Ocimum basilicum (compounds 5–8, in 20 days)\nand Azadirachta indica (compounds 5 and 6, in 10 days).\nZafar et al. Chemistry Central Journal 2012, 6:109 Page 5 of 8\nhttp://journal.chemistrycentral.com/content/6/1/109\n\ndiagram of 6 (Figure 3) showed four trans fused rings A,\nB, C, and D with chair, half chair , chair, and envelop\nconformations, respectively. The C-17 -OH and acetyl-\nene groups existed in pseudo-equatorial and pseudo-\naxial orientations, respectively. All the bond angles and\nlengths were within the normal range. The figure was\nplotted with the aid of ORTEPII program [23]. Earlier\nin vitro incubation of 1 with rat and human liver cells by\nFreudenthal et. al . has led to the formation of 6 [15].\nThe EI-MS of 7 (C\n22H28O2) showed the M+ at m/z\n342, 2 amu higher than 5, attributed to the hydrolysis\nof the C-3 ester into an -OH which did not oxidized\ninto a ketone, as in 5.T h e 1H-NMR also showed a\nbroad singlet at δ 4.14 ( W1/2 = 16.8 Hz, H-3), while\nother broad singlet at δ 5.20 (proton geminal to ester\ngroup in 1) was absent. The spectrum also showed a\nsinglet for methyl group at δ 2.01 (H-23), further indi-\ncating that the ester at C-17 remained intact. The rest\nof the spectrum was distinctly similar to substrate 1.\nCompound 7 was unambiguously identified as 17 α-\nethynyl-3β-hydroxy-17β-acetoxyestr-4-ene. Metabolite 7\nhas earlier been obtained from the in vitro biotrans-\nformation of ethynodiol diacetate ( 1) with rat and\nhuman liver cells [15].\nThe M+ of compound 8 (C20H28O2) appeared at m/z\n300 (EI-MS). The 1H-NMR spectrum of 8 showed two\nolefinic proton doublets at δ 5.85 ( J3,4 = 9.6 Hz) and\n5.84 ( J4,3 = 9.6 Hz). The olefinic protons belonged to\nadjacent carbon atoms as inferred from the COSY\nspectrum and assigned to H-3 and H-4, respectively.\nThe spectrum was also devoid of any hydroxyl-bearing\nmethine proton signal. The\n13C-NMR spectrum of 8\nshowed no ketonic carbonyl signal, but two tertiary\nhydroxyl carbon signals, appeared at δ 69.7 and 79.9.\nThe signal at δ 79.9 was assigned to C-17, in compari-\nson with metabolite 6. Carbon resonating at δ 69.7\nwas HMBC correlated with the proton at δ 5.85 (H-4)\nand thus assigned to C-5. The metabolite 8 was thus\nidentified as 17 α-ethynyl-5α,17β-dihydroxyestr-3-ene.\nCompound 8 was earlier obtained from the photosen-\nsitized oxidation of 19-nor-17 α-pregn-4-en-20-yn-17-ol\n[24]. The spectroscopic data of this compound was\nnot reported previously.\nExperimental\nGeneral\nEthynodiol diacetate ( 1) was purchased from Sigma-\nAldrich. Thin layer chromatography was carried out on\nprecoated plates (Silica gel, Merck, PF 254). Column chro-\nmatography (CC) was performed by using silica gel (E.\nMerck, Germany).\n1H- and 13C-NMR spectra were\nrecorded in CDCl 3 and CD 3OD on Bruker Avance-NMR\nspectrometers. The chemical shifts ( δ values) are pre-\nsented in ppm and the coupling constants ( J values) are\nin Hertz. JEOL (Japan) JMS-600H mass spectrometer\nwas used for recording EI-MS in m/z (rel. %). Single-\ncrystal X-ray diffraction data was collected on Bruker\nSmart APEX II, CCD 4-K area detector diffractometer\n[25]. Data reduction was performed by using SAINT\nprogram. The structure was solved by direct methods\n[26], and refined by full-matrix least squares on F2 by\nusing the SHELXTL-PC package [27]. The figures were\nplotted with the aid of ORTEP program [20].\nMicrobial and callus cultures\nCulture of Cunninghamella elegans was purchased from\nNRRL (1392), grown on Saboraud dextrose agar (SDA).\nThe culture medium for C. elegans was prepared by dis-\nsolving glucose (40 g), yeast extract (20 g), peptone (20 g),\nNaCl (20 g), KH 2PO4 (20 g) and glycerol (40 mL) in dis-\ntilled water (4.0 L).\nPlant material of Ocimum basilicum and Ocimum\nsanctum were obtained from the greenhouse facility of\nthe H. E. J. Research Institute of Chemistry, University\nFigure 3 Computer-generated ORTEP diagram of metabolite 6. Hydrogens are omitted for clarity.\nZafar et al. Chemistry Central Journal 2012, 6:109 Page 6 of 8\nhttp://journal.chemistrycentral.com/content/6/1/109\n\nof Karachi. Callus culture of the plant was derived from\nyoung leaves which were cultivated in 300 mL jars, con-\ntaining 25 mL of Murashige and Skoog (MS) media [28],\neach supplemented with 2% sucrose, 0.5 mg/L 2,4-\ndiphenoxy acetic acid (2,4-D), 2.5 mg/L naphthalene\nacetic acid anhydride (NAA), 0.01 g/L ascorbic acid and\nsolidified by 0.6% agar at 25 ± 1°C in the dark.\nThe callus culture of the Azadirachta indica , also\nobtained from the greenhouse facility of the H. E. J. Re-\nsearch Institute of Chemistry, was established from\nyoung leaves, cultivated in 300 mL jars having 25 mL of\nMurashige and Skoog media [28], enriched with sucrose\n(30 g/L), 3-indole butyric acid (4 mg/L), 6-benzyl amino-\npurine (1 mg/L), and agar (6 g/L) at 25 ± 1°C under\ncomplete darkness.\nFermentation of ethynodiol diacetate (1) with C. elegans\nand purification of metabolites\n4.0 L of culture medium for C. elegans was prepared as\ndescribed earlier and distributed evenly among 40\nErlenmyer flasks (100 mL each). The flasks were plugged\nwith cotton swab and sterilized in an autoclave at 121°C\nfor 15 minutes. Spores of the fungus were transferred\ninto 10 flasks under sterilized conditions in a laminar\nflow cabinet to prepare the seed flasks. These innocu-\nlated flasks were kept on a rotary shaker for two days and\nthen the seed flasks were used to inoculate the remaining\n30 flasks with spores of C. elegans which were again kept\non shaker for incubation. After enough growth, the sub-\nstrate ( 1, 1.0 g) dissolved in acetone (20 mL), was trans-\nferred equally to all the flasks under sterilized conditions.\nThe flasks were again kept on shaker for fermentation\nand time course study was conducted by harvesting the\ncontent of one flask and checking the extent of trans-\nformation on TLC. The fermentation was continued for\n12 days. The culture medium was then filtered to separ-\nate mycelium from broth, and filtrate was extracted with\ndichloromethane (DCM) (4 L × 3). The organic phase\nwas collected, dried (Na\n2SO4), and concentrated in vacuo\nto obtain a brown gum (1.6 g). This gum was fractionated\non silica gel with petroleum ether and ethyl acetate as\nmobile phase. Main fractions were subjected to silica gel\ncolumn chromatography by using gradient eluent sys-\ntems of pet. ether/ ethyl acetate to obtain metabolites 2,\n3 and 4 at 30% and metabolite 5 at 40% ethyl acetate in\npet. ether.\nFermentation of ethynodiol diacetate (1) with O. basilicum\nand purification of metabolites\nCell suspension cultures were derived from static cultured\ncalli in Erlenmeyer flasks (1 L), containing 400 mL of\nthe culture medium. The flasks were placed on a shaker\n(100 rpm) with a 16 hours photoperiod at 25 ± 1°C\nfor 15 days of pre-culturing. A solution of compound 1\n(600 mg) in acetone (100 mg/mL) was added to each flask\nthrough a 0.2 μM membrane filter (millipore) and the\nflasks were again placed on shaker for 20 days. Negative\n(containing only plant cell suspension culture) and posi-\ntive (compound 1 in the medium) controls were also pre-\npared. Time course study was carried out on a daily basis\nand the extent of bioconversion was analyzed by TLC.\nThe fermentation media was filtered and filtrate was\nextracted thrice with DCM, dried over anhydrous\nNa\n2SO4, and evaporated in vacuo. The extract (2.0 g) was\nsubjected to fractionation with 10% gradient of pet. ether/\nacetone (P.E./Ac.), followed by further column chroma-\ntography to obtain metabolites 5 (8:2 P.E./Ac.), 6, 7 (7:3\nP.E./Ac.) and 8 (6:4 P.E./Ac.) in appreciable quantities.\nFermentation of ethynodiol diacetate (1) with A. indica and\npurification of metabolites\nCell suspension culture was derived from static calli, cul-\ntured in Erlenmeyer flasks (1 L), each containing 400 mL\nof the Murashige and Skoog media, supplemented with\ningredients as mentioned above, except BA and agar. After\n20 days of pre-culturing on a shaker (100 rpm) and 16\nhours of photoperiod at 25 ± 1°C, a solution of substrate\n(100 mg in 1 mL of acetone) was added to each flask\nthrough a 0 .2 μM membrane filter and the flasks were\nplaced on a shaker for 10 days. The time course study was\nperformed and the course of biotransformation was moni-\ntored by TLC. Positive and negative controls were also run\nalong with the main experiment in order to differentiate\nthe transformed products from metabolites. After 10 days\nof incubation, the cells and the media were separated by fil-\ntration. The filtrate (~2 L) was extracted with CH\n2Cl2 (3 ×\n2 L) at r. t. The combined extract were dried over anhyd-\nrous Na2SO4, and concentrated in vacuo, which afforded a\nbrown residue (1.1 g). The transformed metabolites were\nisolated from this gummy crude by using repeated column\nchromatography (silica gel) with petroleum ether/EtOAc\ngradient, affording compounds 5 (6:4 P.E./EtOAc) and 6\n(1:1 P.E./EtOAc).\nConclusion\nIn conclusion, the biotransformation of oral contraceptive\nethynodiol diacetate (1)w i t hC. elegans, O. basilicum and\nA. indica was investigated for the first time which pro-\nvided an efficient route to several metabolites. Biotrans-\nformation of 1 with C. elegans led to the formation of\nthree new and one known metabolites, while biotrans-\nformation with cell suspension cultures of O. basilicum\nand A. indica afforded four known metabolites. Metabol-\nite 5 was obtained in all three experiments. Single-crystal\nX-ray structure of metabolite 6 and spectroscopic data of\nmetabolite 8 are being reported here for the first time.\nMetabolites 5, 6 and 7 were reported previously as\nZafar et al. Chemistry Central Journal 2012, 6:109 Page 7 of 8\nhttp://journal.chemistrycentral.com/content/6/1/109\n\nin vitro metabolites of ethynodiol diacetate ( 1)f r o mr a t\nand human liver cells.\nCompeting interests\nTwo of the authors, S. Zafar and H. A. Kayani, acknowledge the Higher\nEducation Commission, Pakistan, for providing financial support through the\nHEC indigenous Ph. D. scholarship program.\nAuthors’ contributions\nSZ Carried out the microbial transformation by using Cunninghamella\nelegans, purified all the metabolites and solved the spectroscopic data. SY\nconducted the single-crystal X-ray crystallographic studies. HAK carried out\nthe biotransformation by using cell cultures of Ocimum basilicum . Saifullah\ncarried out the biotransformation by using cell suspension cultures of\nAzadirachta indica . SK helped in the biotransformation experiments with\nplant cell cultures. AAM helped in the preparation of the manuscript. MIC\nconceived the original study, supervised the research, helped in solving the\nspectroscopic data, and finalized the manuscript. All authors read and\napproved the final manuscript.\nAcknowledgments\nWe acknowledge the support of the Higher Education Commission, Pakistan,\nfor providing necessary funding for the research work.\nAuthor details\n1H. E. J. Research Institute of Chemistry, International Center for Chemical\nand Biological Sciences, University of Karachi, Karachi 75270, Pakistan.\n2Department of Chemistry, Abdul Wali Khan University, Mardan 23200,\nPakistan. 3Department of Chemistry, College of Science, King Saud University,\nPO Box 2455, Riyadh 11451, Saudi Arabia.\nReceived: 25 July 2012 Accepted: 20 September 2012\nPublished: 29 September 2012\nReferences\n1. Mahato SB, Garai S: Advances in microbial steroid biotransformation.\nSteroids 1997, 62:332–345.\n2. Abourashed EA, Clark AM, Hufford CD: Microbial models of mammalian\nmetabolism of xenobiotics: An updated review. Curr Med Chem 1999,\n6:359–374.\n3. Hamada H, Konishi H, Williams HJ, Scott AI: Biotransformation of\ntestosterone isomers by a green cell suspension culture of Marchantia\npolymorpha. Phytochemistry 1991, 30:2269–2270.\n4. Drawert F, Berger RG, Godelmann R: Regioselective biotransformation of\nvalencene in cell suspension cultures of Citrus sp. Plant Cell Rep 1984,\n3:37–40.\n5. Nasib A, Musharraf SG, Hussain S, Khan S, Anjum S, Ali S, Atta-ur-Rahman,\nChoudhary MI: Biotransformation of ( −)-ambrox by cell suspension\ncultures of Actinidia deliciosa . J Nat Prod 2006, 69:957–959.\n6. Dorisse P, Gleye J, Loiseau P, Puig P, Edy A, Henry M: Papaverine\nbiotransformation in plant cell suspension cultures. J Nat Prod 1988,\n51:532–536.\n7. Frydman A, Weisshaus O, Huhman DV, Sumner LW, Bar-Peled M, Lewinsohn\nE, Fluhr R, Gressel J, Eyal Y: Metabolic engineering of plant cells for\nbiotransformation of hesperedin into neohesperidin, a substrate for\nproduction of the low-calorie sweetener and flavor enhancer NHDC.\nJ Agric Food Chem 2005, 53:9708–9712.\n8. Sakamakia H, Itoh KI, Taniaib T, Kitanakac S, Takagid Y, Chaie W, Horiuchie\nCA: Biotransformation of valencene by cultured cells of Gynostemma\npentaphyllum. J Mol Cat B Enzym 2005, 32:103–106.\n9. Sahoo Y, Remien YN, Yao RS: In vitro clonal propagation of an aromatic\nmedicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot\nproliferation. In vitro Cell Devel Biol Plant Largo 1997, 33:293–296.\n10. Itoh K, Nakamura K, Utsukihara T, Sakamaki H, Horiuchi CA: Stereoselective\noxidation of racemic 1-arylethanols by basil cultured cells of Ocimum\nbasilicum cv. Purpurascens . Biotechnol Lett 2008, 30:951–954.\n11. Azizuddin S, Khan S, Choudhary MI, Atta-ur-Rahman: Biotransformation of\ndydrogesterone by cell suspension cultures of Azadirachta indica. Turk J\nChem 2008, 32:141–146.\n12. Pincus G, Garcia CR, Paniagua M, Shepard J: Ethynodiol diacetate as a new,\nhighly potent oral inhibitor of ovulation. Science 1962, 138:439–440.\n13. Lewis CJ, Vose CW, Spalton PN, Ford GC, Haskins NJ, Palmer RF: Metabolism\nof ethynodiol diacetate in the rhesus monkey before and after\nadministration of rifampicin. Xenobiotica 1980, 10:705–713.\n14. Ishihara M, Osawa Y, Kirdani RY, Sandberg AA: Metabolic fate of ethynodiol\ndiacetate in the baboon. Steroids 1975, 25:829–847.\n15. Freudenthal RI, Cook CE, Forth J, Rosenfeld R, Wall ME: The metabolism of\nethynodiol diacetate by rat and human liver. J Pharmacol Exp Ther 1977,\n177:468–473.\n16. Iriarte J, Djerassi C, Ringold HJ: Steroids. CVII. Δ5(6)–19-nor steroids, a new\nclass of potent anabolic agents. J Am Chem Soc 1959, 81:436–438.\n17. Djerassi C, Miramontes L, Rosenkranz G, Sondheimer F: Steroids. LIV.\nSynthesis of 19-Nov-17 α-ethynyltestosterone and 19-nor-17 α-\nmethyltestosterone. J Am Chem Soc 1954, 76:4092–4094.\n18. Beurskens PT, Admiraal G, Beurskens G, Bosman WP, Gelder R, Israel R, Smits\nJMM: The DIRDIF-94 program system, technicall report of the crystallography\nlaboratory. Netherlands: University of Nijmegen; 1994.\n19. Sheldrick GM: A program for refinement of crystal structures. SHELXL 97 .\nGermany: University of Göttingen; 1997.\n20. Choudhary MI, Sarfaraz MA, Nawaz A, Fatmi MQ, Atta-ur-Rahman: The\nmicrobial hydroxylation of levonorgestrel. Nat Prod Lett 2006,\n20(20):1074–1081.\n21. Shang-hui H, Xu-fang T, Han guang-dian: Novel microbial hydroxylation of\n13-ethyl-6β,10β,17β-trihydroxy-18,19-dinor-17α -pregn-4-en-20-yn-3-on.\nSteroids 1998, 63:88–92.\n22. Huszcza E, Dmochowska-Gladysz J: Transfomation of testosterone and\nrelated steroids in Absidia glauca culture. J Basic Microbiol 2003,\n43(2):113–120.\n23. Johnson CK: ORTEP II. Report ORNL-5138 . Tennessee, USA: Oak Ridge\nNational Laboratory; 1976.\n24. Sedee A, Van Henegouwen GB: Photosensitized decomposition of\ncontraceptive steroids: a possible explanation for the observed (Photo)\nallergy of the oral contraceptive pill. Arch Pharm (Weinheim) 1985,\n318:111–119.\n25. Siemens: SMART and SAINT . WI, USA: Madison: Siemens Analytical X-Ray\nInstruments Inc.; 1996.\n26. Altomare A, Cascarano G, Giacovazzo C, Guagliardi A: Completion and\nrefinement of crystal structures with SIR92. J Appl Cryst 1993, 26:343–350.\n27. Sheldrick GM: SHELXTL-PC (Version 5.1) . WI, USA: Madison: Siemens Analytical\nInstruments Inc; 1997.\n28. Murashige T, Skoog F: A revised medium for rapid growth and bioassays\nwith tobacco tissue cultures. Physiol Plantarum 1962, 15:473–497.\ndoi:10.1186/1752-153X-6-109\nCite this article as: Zafar et al. : Biotransformation of oral contraceptive\nethynodiol diacetate with microbial and plant cell cultures. Chemistry\nCentral Journal 2012 6:109.\nOpen access provides opportunities to our \ncolleagues in other parts of the globe, by allowing \nanyone to view the content free of charge.\nPublish with ChemistryCentral and every\nscientist can read your work free of charge\nW. Jeffery Hurst, The Hershey Company.\navailable free of charge to the entire scientific community\npeer reviewed and published immediately upon acceptance\ncited in PubMed and archived on PubMed Central\nyours     you keep the copyright\nSubmit your manuscript here:\nhttp://www.chemistrycentral.com/manuscript/\nZafar et al. Chemistry Central Journal 2012, 6:109 Page 8 of 8\nhttp://journal.chemistrycentral.com/content/6/1/109","source_license":"CC-BY-4.0","license_restricted":false}