Oxidation of lynestrenol by the fungus Cunninghamella elegans

Article abstract of DOI:10.1080/14786410701614507

Transformation of lynestrenol (19-nor-17α-pregn-4-en-20-yn-17β- ol) (1) was carried out by incubation with Cunninghamella elegans to obtain 19-nor-17α-pregn-4-en-20-yn-3-one-10β,17β-diol (2), 19-nor-17α-pregn-4-en-20-yn-3-one-6β,17β-diol (3), and 19-nor-17α-pregn-4-en-20-yn-3β,6β,17β-triol (4). Metabolite 4 was identified as a new compound. These metabolites were structurally characterised on the basis of spectroscopic techniques.

Full text of DOI:10.1080/14786410701614507

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Oxidation of lynestrenol by the fungus
Cunninghamella elegans
M. Iqbal Choudhary a , M. Atif a & Atta-Ur-Rahman a
a International Center for Chemical Sciences , H.E.J. Research
Institute of Chemistry , University of Karachi, Karachi–75270,
Pakistan
Published online: 09 Dec 2009.
To cite this article: M. Iqbal Choudhary , M. Atif & Atta-Ur-Rahman (2010) Oxidation of lynestrenol
by the fungus Cunninghamella elegans , Natural Product Research: Formerly Natural Product
Letters, 24:1, 1-6, DOI: 10.1080/14786410701614507
To link to this article: http://dx.doi.org/10.1080/14786410701614507
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Natural Product Research
Vol. 24, No. 1, 10 January 2010, 1–6
Oxidation of lynestrenol by the fungus Cunninghamella elegans
*
M. Iqbal Choudhary , M. Atif and Atta-Ur-Rahman
International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of
Karachi, Karachi – 75270, Pakistan
(Received 27 September 2004; final version received 24 March 2005)
Transformation of lynestrenol (19-nor-17ꢀ-pregn-4-en-20-yn-17ꢁ-ol) (1) was
carried out by incubation with Cunninghamella elegans to obtain 19-nor-17ꢀ-
pregn-4-en-20-yn-3-one-10ꢁ,17ꢁ-diol (2), 19-nor-17ꢀ-pregn-4-en-20-yn-3-one-
6ꢁ,17ꢁ-diol
(3),
and
19-nor-17ꢀ-pregn-4-en-20-yn-3ꢁ,6ꢁ,17ꢁ-triol
(4).
Metabolite 4 was identified as a new compound. These metabolites were
structurally characterised on the basis of spectroscopic techniques.
Keywords: microbial transformation; oxidation; Cunninghamella elegans;
lynestrenol (19-nor-17ꢀ-pregn-4-en-20-yn-17ꢁ-ol); 19-nor-17ꢀ-pregn-4-en-20-yn-
3-one-10ꢁ,17ꢁ-diol; 19-nor-17ꢀ-pregn-4-en-20-yn-3-one-6ꢁ,17ꢁ-diol; 19-nor-17ꢀ-
pregn-4-en-20-yn-3ꢁ,6ꢁ,17ꢁ-triol
1. Introduction
Microbial transformation frequently produces chemically inaccessible products with high
stereo-selectivity (Charney & Herzog, 1967). Lynestrenol (1), used as an oral contraceptive
(Van den Driessche, Le Bars, & Chamban, 1980), shares various activities with the natural
hormone progesterone. It has a strong progestational influence on the endometrium, as
well as suppressing ovulation and menstruation (Castro-Vazquez, Macome, De Carli, &
Rosner, 1971). In a continuation of our work on bioactive-steroids and progestagen-type
compounds (Atta-ur-Rahman, Choudhary, Asif, Farooq, & Yaquoob, 1998a; Atta-ur-
Rahman, Choudhary, Asif, Farooq, & Yaqoob, 2000; Atta-ur-Rahman et al., 1998b;
Choudhary, Azizuddin, & Atta-ur-Rahman, 2002; Choudhary, Musharraf, Ali, Atif, &
Atta-ur-Rahman, 2004; Choudhary, Musharraf, Shaheen, & Atta-ur-Rahman, 2002;
Choudhary, Shah, Musharraf, Shaheen, & Atta-ur-Rahman, 2003a; Choudhary et al.,
2003b) we now report that the transformation of lynestrenol (1) by Cunninghamella elegans
resulted in the formation of compounds 2, 3, and 4. Compounds 2 and 3 were obtained
earlier by the microbial transformation of norethiesterone by Rhizopus nigricans (Zakelj-
Mavric, Belic, & Gottlieb, 1986). Compound 4 was found to be a new metabolite.
*Corresponding author. Email: hej@cyber.net.pk
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410701614507
http://www.informaworld.com

2
M.I. Choudhary et al.
18 OH
21
20
16
12
17
15
13
14
11
9
H
H
1
4
10
2
3
8
H
H
7
6
5
1
12 Days
OH
OH
HO
OH
H
H
H
H
OH
H
H
H
H
H
H
H
O
O
OH
OH
4
2
3
Scheme 1. Fungal hydroxylation of lynestrenol (1) by C. elegans.
2. Results and discussion
Three oxidised metabolites were detected by the analysis of the fermentation broth of
lynestrenol (1), C20H28O, with C. elegans. The metabolites were isolated as described in
the experimental section and characterised through detailed spectroscopic analysis as
19-nor-17ꢀ-pregn-4-en-20-yn-3-one-10ꢁ,17ꢁ-diol (2), 19-nor-17ꢀ-pregn-4-en-20-yn-3-one-
6ꢁ,17ꢁ-diol (3), and 19-nor-17ꢀ-pregn-4-en-20-yn-3ꢁ,6ꢁ,17ꢁ-triol (4) (Scheme 1).
The high-resolution electron impact mass spectrum (HREI MS) of metabolite 2
showed a molecular ion (Mþ) at m/z 314.1863, 30 a.m.u. greater than the substrate 1 and
corresponding to the molecular formula C20H26O3 (Calcd 314.1881). The IR spectrum
showed strong absorptions at 3399 and 1729 cmꢀ1, indicating the presence of hydroxyl and
ketonic carbonyl groups, respectively. The UV spectrum exhibited an absorption at
232 nm, indicating the presence of a conjugated ketonic function. The 1H-NMR spectrum
showed the downfield shift of the H-4 olefinic signal from ꢂ 5.31 to 5.76, as compared to
substrate 1, thus indicating an oxidation at the vicinal C-3. The 13C-NMR broadband
decoupled spectrum (Table 1) showed a carbonyl signal at ꢂ 199.4 for a C-3 ketonic group.
An additional quaternary carbon at ꢂ 68.2 indicated the presence of a hydroxyl group. The
location of the hydroxyl group at C-10 was deduced on the basis of the heteronuclear
multiple bond connectivity (HMBC) interaction of H-4 (ꢂ 5.76) with C-10 (ꢂ 68.2). The ꢁ
orientation of the OH-group at C-10 was deduced from the downfield chemical shifts of
C-1 and C-9, and by the upfield shifts of C-6, C-8, and C-11 (ꢀ4.2, ꢀ6.1 and ꢀ2.9 ppm,
respectively) as compared to the substrate 1, as well as a chemical shift comparison
with the reported data of a closely related compound, 13-ethyl-10ꢁ,17ꢁ-dihydroxy-18,

Natural Product Research
3
Table 1. 13C-NMR data of compounds 1–4 (ppm, 100 MHz).
Carbon
1
2
3
4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
28.8, t
22.1, t
25.5, t
120.0, d
140.3, s
35.5, t
32.6, t
42.0, d
49.9, d
41.6, d
26.0, t
31.6, t
47.0, s
49.4, d
22.9, t
38.8, t
79.9, s
12.7, q
–
33.7, t
32.1, t
201.0, s
124.8, d
168.6, s
31.3, t
29.7, t
35.9, d
52.1, d
68.2, s
23.1, t
31.9, t
46.8, s
49.5, d
20.1, t
38.9, t
79.1, s
12.6, q
–
26.2, t
36.3, t
200.2, s
125.4, d
164.8, s
71.9, d
37.7, t
34.1, d
49.0, d
38.1, d
26.0, t
32.4, t
47.1, s
49.1, d
22.8, t
38.8, t
77.2, s
12.7, q
–
25.6, t
32.6, t
67.1, d
128.1, d
162.2, s
72.6, d
37.6, t
34.6, d
49.4, d
37.2, d
27.2, t
29.1, t
47.1, s
49.6, d
22.8, t
38.9, t
80.1, s
12.8, q
–
87.6, s
73.8, d
88.0, s
74.2, d
88.0, s
74.1, d
87.4, s
73.9, d
Note: Carbon multiplicities were determined by DEPT experiments; s ¼ quaternary, d ¼ methine,
t ¼ methylene, q ¼ methyl.
19-dinor-17ꢀ-pregn-4-en-20-yn-3-one (Hu, Tian, Sun, & Han, 1996). Compound 2
was therefore identified as 19-nor-17ꢀ-pregn-4-en-20-yn-3-one-10ꢁ,17ꢁ-diol (Zakelj-
Mavric et al., 1986).
The HREI MS of compound 3 showed an Mþ at m/z 314.1893, which corresponds to
the molecular formula C20H26O3 (Calcd 314.1881), 30 a.m.u. greater than the parent
compound 1. The IR, UV, and 1H-NMR spectra were very similar to compound 2, but the
1H-NMR spectrum showed an additional methine proton signal at ꢂ 4.37 (brs), due to a
hydroxyl-bearing methine proton. The H-4 also exhibited a downfield shift to ꢂ 5.88 as
compared to substrate 2, which indicated the presence of a hydroxyl group at C-6.
The 13C-NMR spectra (Table 1) of compound 3 (broadband decoupled and DEPT)
showed a new methine carbon signal at ꢂ 71.9 (C-6). The proposed structure 3 was
3
supported by JCH heteronuclear couplings of H-4 (ꢂ 5.88) with C-6, and of H-6 (ꢂ 4.37)
with C-4 (ꢂ 125.4), as well as a chemical shift comparison with the reported data of a
closely related compound, 13-ethyl-6ꢁ,17ꢁ-dihydroxy-18,19-dinor-17ꢀ-pregn-4-en-20-yn-
3-one (Hu, Tian, & Han, 1998). The stereochemistry of the newly introduced hydroxyl
group at C-6 was deduced to be ꢁ on the basis of a nuclear overhauser enhancement
spectroscopy (NOESY) correlation between geminal H-6 (ꢂ 4.37) and H-9 (ꢂ 1.98), which is
always ꢀ-oriented in this class of steroids. Reduction of 3 with NaBH4-MeOH yielded
a diasteromeric mixture of C-3 reduced products. One of them was identified as compound
4 by co-TLC method. The metabolite 3 was thus characterised as 19-nor-17ꢀ-pregn-4-en-
20-yn-3-one-6ꢁ,17ꢁ-diol (Zakelj-Mavric et al., 1986).
The HREI MS of compound 4 showed an Mþ at m/z 316.2005, which was 32 a.m.u.
higher than that of compound 1 and corresponded to the formula C20H28O3

4
M.I. Choudhary et al.
(Calcd 316.2038). The IR spectrum did not show any carbonyl absorption. The UV
spectrum exhibited end absorption at 202 nm, indicating the lack of a conjugated
1
chromophore. The H-NMR of compound 4 was found to be different from that of
metabolite 3, especially in two aspects. Firstly, the appearance of an additional methine
proton signal at ꢂ 4.18, and secondly, the upfield shift of the olefinic proton from ꢂ 5.88 to
5.61 (H-4). The 13C-NMR spectra (Table 1) showed the disappearance of the C-3 ketonic
resonance and the appearance of an additional OH-bearing methine carbon atom at ꢂ 67.1.
The reduction of the C-3 ketonic carbonyl was further inferred by the correlated
spectroscopy (COSY 45ꢁ) interaction of the methine H-3 (ꢂ 4.18) with the olefinic H-4
(ꢂ 5.61). The stereochemistry of the newly introduced hydroxyl group at C-3 was deduced
to be ꢁ on the basis of a large W1/2 value (20 Hz) of geminal H-signal, which has dihedral
angles of 180ꢁ and 60ꢁ with the C-2 methylene protons. The above spectral data led to the
structure of compound 4 as 19-nor-17ꢀ-pregn-4-en-20-yn-3ꢁ,6ꢁ,17ꢁ-triol.
In this study, three metabolites were obtained when compound 1 was treated with
C. elegans. Metabolite 2 was obtained in 13.4 mg, while metabolites 3 and 4 were obtained
in 11.2 and 9.7 mg quantities, respectively. A large amount of the starting material
remained unchanged. C. elegans effected a mainly hydroxylated and regioselective keto
formation of compound 1. The presence of 17ꢀ-ethynyl group in substrate 1 has a
profound influence on the site of oxidation. C-10, C-6, and C-15 were found to be
the favourable sites of oxidation with Cunninghamella species in 19-nor series of steroids
(Hu et al., 1998).
3. Experimental
3.1. General experimental procedures
The EI MS were measured on a Varian MAT 311A mass spectrometer. The HREI MS
1
spectra were recorded on a Jeol JMS-600H mass spectrometer. The H-NMR spectra
(ꢂ ppm, J in Hz) were obtained in deuterated solvent (CDCl3) at 400 MHz on a Bruker
Avance-400 NMR spectrometer, while 13C-NMR spectra were recorded in CDCl3 on the
same instrument at 100 MHz. The FT-IR were recorded on a Shimadzu FTIR-8900
spectrophotometer. UV absorptions were measured on an Avance UV spectrophotometer.
Optical rotation measurements were made on a Jasco DIP-360 digital polarimeter. Melting
points were recorded on a Buchi 535 apparatus. The metabolites were purified by column
chromatography on Si gel. The purity of the samples were checked on precoated TLC plates
(Si gel 60, PF254, 20 ꢂ 20, 0.25 mm, E. Merck). Compound 1 was isolated from Orgametril, a
product of P.T. Organon, Indonesia. The authenticity of the drug was established through
spectral comparison with its reported data (Van Dijck, Lankwerden, & Vermer, 1977).
3.2. Preparation of the medium
The medium for C. elegans (TSY-0865) was prepared by mixing the following ingredients
in distilled water (4 L): glucose (40 g), glycerol (40 mL), peptone (20 g), yeast extract (20 g),
KH2PO4 (20 g), and NaCl (20 g); the pH was maintained at 5.6.
3.3. Culture and fermentation procedure
The following procedure was used for the fermentation of the fungus: two-day old spores
of the fungus were aseptically transferred into broth medium flasks containing 100 mL of

Natural Product Research
5
freshly prepared and autoclaved medium. The seed flasks thus obtained were incubated on
a shaker at 30ꢁC for two days. Two-day old broth cultures from a 100 mL-seed flask were
inoculated aseptically into 40 media flasks (250 mL) containing 100 mL of medium, and
fermentation was continued for a further 48 h. Compound 1 (800 mg) was diluted with
acetone (20 mL) and the resulting solution was evenly distributed among 40 conical flasks
in shake cultures, and the fermentation was continued on a shaker at room temperature
(30ꢁC) for 12 days. The mycelium was filtered, washed with ethyl acetate and the broth
thus obtained was extracted with ethyl acetate. The extracts were dried over anhydrous
sodium sulfate and concentrated in vacuo to afford a gum that was adsorbed on equal
quantities of Si gel, and eluted with solvent gradients of petroleum ether and EtOAc of
increasing polarity.
3.3.1. 19-Nor-17ꢀ-pregn-4-en-20-yn-3-one-10ꢁ,17ꢁ-diol (2)
Colourless amorphous solid; Yield 13.4 mg; m.p. 207–210ꢁC; [ꢀ]25D: þ211ꢁ (CHCl3,
c 0.18); UV (CHCl3) ꢃmax: 232 nm; IR (CHCl3) ꢄmax: 3399 (OH), 1729 (CO, ketonic), 1663
1
(C¼C) cmꢀ1; H-NMR (CDCl3, 400 MHz): ꢂ 5.76 (1H, s, H-4), 2.61 (1H, m, Ha-2), 2.32
(1H, m, Hb-2), 2.55 (1H, brs, H-21), 2.19 (1H, m, Ha-1), 1.96 (1H, m, Hb-1), 1.11 (1H, m,
H-11), 0.90 (3H, s, Me-18); 13C-NMR (CDCl3, 100 MHz) ꢂ: see Table 1; EI-MS: m/z (rel.
int.,%) 314 [Mþ] (66), 296 (21), 228 (100), 213 (38), 173 (15); HREI-MS: m/z 314.1863
(C20H26O3, Calcd 314.1881).
3.3.2. 19-Nor-17ꢀ-pregn-4-en-20-yn-3-one-6ꢁ,17ꢁ-diol (3)
Colourless crystalline solid; Yield 11.2 mg; m.p. 196–198ꢁC; [ꢀ]25D: ꢀ52ꢁ (CHCl3, c 0.2);
UV (CHCl3) ꢃmax: 235 nm; IR (CHCl3) ꢄmax: 3396 (OH), 1725 (CO, ketonic), 1660
1
(C¼C) cmꢀ1; H-NMR (CDCl3, 400 MHz): ꢂ 5.88 (1H, s, H-4), 4.37 (1H, brs, H-6), 2.37
(1H, m, Ha-2), 2.25 (1H, m, Hb-2), 2.55 (1H, brs, H-21), 2.01 (1H, m, Ha-7), 1.32 (1H, m,
Hb-7), 1.98 (1H, m, H-9), 0.91 (3H, s, Me-18); 13C-NMR (CDCl3, 100 MHz) ꢂ: see Table 1;
EI-MS: m/z (rel. int.,%) 314 [Mþ] (89), 296 (42), 228 (67), 213 (64), 158 (56), 55 (100);
HREI-MS: m/z 314.1893 (C20H26O3, Calcd 314.1881).
3.3.3. 19-Nor-17ꢀ-pregn-4-en-20-yn-3ꢁ,6ꢁ,17ꢁ-triol (4)
Colourless crystalline solid; Yield 9.7 mg; m.p. 151–157ꢁC; [ꢀ]25D: ꢀ65ꢁ (CHCl3, c 0.7); UV
(CHCl3) ꢃmax: 202 nm; IR (CHCl3) ꢄmax: 3300 (OH), 1667 (C¼C) cmꢀ1; 1H-NMR (CDCl3,
400 MHz): ꢂ 5.61 (1H, s, H-4), 4.23 (1H, brs, H-6), 4.18 (1H, m, W1/2 ꢃ 20 Hz, H-3), 2.05
(1H, m, Ha-2), 1.31 (1H, m, Hb-2), 2.53 (1H, brs, H-21), 1.99 (1H, m, Ha-7), 1.27 (1H, m,
Hb-7), 1.91 (1H, m, H-9), 0.82 (3H, s, Me-18); 13C-NMR (CDCl3, 100 MHz) ꢂ: see Table 1;
EI-MS: m/z (rel. int.,%) 316 [Mþ] (54), 298 (75), 280 (63), 215 (100), 197 (84), 122 (67), 55
(45); HREI-MS: m/z 316.2005 (C20H28O3, Calcd 316.2038).
References
Atta-ur-Rahman, Choudhary, M.I., Asif, F., Farooq, A., & Yaqoob, M. (1998a). Microbial
transformations of testosterone. Natural Product Letters, 12, 255–261.
Atta-ur-Rahman, Choudhary, M.I., Asif, F., Farooq, A., & Yaqoob, M. (2000). Fungal
transformations of steroids by Cephalosporium aphidicola and Trichothecium roseum.
Natural Product Letters, 14, 217–224.

6
M.I. Choudhary et al.
Atta-ur-Rahman, Choudhary, M.I., Asif, F., Farooq, A., Yaqoob, M., & Dar, A. (1998b).
Microbial transformation of sarsasapogenin by Fusarium lini. Phytochemistry, 49, 2341–2342.
Castro-Vazquez, A., Macome, C.J., De Carli, N.D., & Rosner, M.J. (1971). On the mechanism of
action of oral contraceptives. I. Effects of lynestrenol on ovum implantation and oviducal
morphology in the rat. Fertity and Sterility, 22, 741–744.
Charney, W., & Herzog, H.L. (1967). Microbial Transformations of Steroids (p. 5). New York:
Academic Press.
Choudhary, M.I., Azizuddin, & Atta-ur-Rahman. (2002). Microbial transformation of danazol.
Natural Product Letters, 16, 101–106.
Choudhary, M.I., Musharraf, S.G., Ali, R.A., Atif, M., & Atta-ur-Rahman. (2004). Microbial
transformation of antifertility agents, norethisterone and 17ꢀ-ethynylestradiol. Zeitschrift fur
Naturforschung B, 59, 319–323.
Choudhary, M.I., Musharraf, S.G., Shaheen, F.,
¨
& Atta-ur-Rahman. (2002). Microbial
transformation of (+)-androsta-1,4-diene-3,17-dione by Cephalosporium aphidicola. Natural
Product Letters, 16, 377–382.
Choudhary, M.I., Shah, S.A.A., Musharraf, S.G., Shaheen, F., & Atta-ur-Rahman. (2003a).
Microbial transformation of dehydroepiandrosterone. Natural Product Letters, 17, 215–220.
Choudhary, M.I., Sultan, S., Yaqoob, M., Musharraf, S.G., Yasin, A., & Shaheen, F. (2003).
Microbial transformation of cortisol and prolyl endopeptidase inhibitory activity of its
transformed products. Natural Product Research, 17, 389–395.
Hu, S.-H., Tian, X.-F., & Han, G.-D. (1998). Novel microbial hydroxylation of 13-ethyl-17ꢁ-
hydroxy-18,19-dinor-17ꢀ-pregn-4-en-20-yn-3-one. Steroids, 63, 88–92.
Hu, S.-H., Tian, X.-F., Sun, Y.-H., & Han, G.-D. (1996). Microbial hydroxylation of 13-ethyl-17ꢁ-
hydroxy-18,19-dinor-17ꢀ-pregn-4-en-20-yn-3-one. Steroids, 61, 407–410.
´ ´ ´
Van den Driessche, J., Le Bars, S., & Chamban, Y. (1980). Comptes Rendus des Seances de la Societe
de Biologie et de ses Filiales, 174, 347.
Van Dijck, L.A., Lankwerden, B.J., & Vermer, J.G.C.M. (1977). Recueil des Travaux Chimiques des
Pays-Bas, 96, 200.
Zakelj-Mavric, M., Belic, I., & Gottlieb, H.E. (1986). The bioconversion of 17ꢀ-ethynyl steroids with
11ꢀ-hydroxylase of Rhizopus nigricans. FEMS Microbiology Letters, 33, 117–120.