Microbial transformation of antifertility agents, norethisterone and 17α-ethynylestradiol

Article abstract of DOI:10.1515/znb-2004-0314

The microbial transformation of oral contraceptive norethisterone (1) by Cephalosporium aphidicola afforded an oxidized metabolite, 17α- ethynylestradiol (2), while the microbial transformation of 2 by Cunninghamella elegans yielded several metabolites, 19-nor-17α-pregna-1,3,5 (1O)-trien-20-yne-3,4,17β-triol (3), 19-nor-17β-pregna-1,3,5 (10)-trien-20-yne-3,7α,17β-triol (4), 19-nor-17α-pregna-1,3,5 (10)-trien-20-yne-3,11α,17β-triol(5), 19-nor-17α-pregna-1,3,5 (10)-trien-20-yne-3,6β,17β-triol (6) and 19-nor-17α-pregna-1,3,5 (10)-trien-20-yne-3,17β-diol-6β-methoxy (7). Metabolite 7 was found to be a new compound. These metabolites were structurally characterized on the basis of spectroscopic techniques.

Full text of DOI:10.1515/znb-2004-0314

Microbial Transformation of Antifertility Agents, Norethisterone and
17α-Ethynylestradiol
Muhammad I. Choudhary, Syed G. Musharraf, Rahat A. Ali, Muhammad Atif, and
Atta-ur-Rahman
H. E. J. Research Institute of Chemistry, International Center for Chemical Sciences, University of
Karachi, Karachi-75270, Pakistan
Reprint requests to M. I. Choudhary. Fax: (92-21) 9243190-9243191.
E-mail: hej@cyber.net.pk
Z. Naturforsch. 59b, 319 – 323 (2004); received July 7, 2003
The microbial transformation of oral contraceptive norethisterone (1) by Cephalosporium aphidi-
cola afforded an oxidized metabolite, 17α-ethynylestradiol (2), while the microbial transforma-
tion of 2 by Cunninghamella elegans yielded several metabolites, 19-nor-17α-pregna-1,3,5 (10)-
trien-20-yne-3,4,17β-triol (3), 19-nor-17α-pregna-1,3,5 (10)-trien-20-yne-3,7α,17β-triol (4), 19-
nor-17α-pregna-1,3,5 (10)-trien-20-yne-3,11α,17β-triol (5), 19-nor-17α-pregna-1,3,5 (10)-trien-20-
yne-3,6β,17β-triol (6) and 19-nor-17α-pregna-1,3,5 (10)-trien-20-yne-3,17β-diol-6β-methoxy (7).
Metabolite 7 was found to be a new compound. These metabolites were structurally characterized on
the basis of spectroscopic techniques.
Key words: Norethisterone, 17α-Ethynylestradiol, Microbial Transformation,
Cephalosporium aphidicola, Cunninghamella elegans
Introduction
Results and Discussion
Screening scale experiments have shown that the
Cephalosporium aphidicola (IMI 68689) was capa-
ble of converting norethisterone (1) C20H26O2 into
product 2. Scale up of this reaction afforded a single
metabolite 2.
Norethisterone (17α-ethynyl-19-nortestestrone) (1)
is a potent progestogen, widely used in oral con-
traceptive pills, as a antifertility agent [1]. It has a
wide variety of uses including the delay in menstru-
ation and the treatment of other menstrual disorders
such as endometriosis. Among the female contracep-
Compound 2 was obtained as a white solid. HREI
tives, norethisterone is considered to be the safest. In MS of 2 displayed the M+ at m/z 296.1614 (C20H24O2
continuation of our microbial transformation studies calcd. 296.1616) which was 2 a.m.u. lesser than the
on bioactive chemical compounds [2 – 11], we stud- substrate 1. The IR spectrum of 2 did not show any
ied the metabolism of norethisterone by Cephalospo- ketonic absorption, but two additional bands at 1603
rium aphidicola. Metabolic studies of 1 have already and 1517 cm−1 (aromatic C=C) were observed. The
been reported by various fungi and bacteria which af- UV spectrum exhibited an absorption at 204 nm, in-
1
forded its hydroxylated metabolites at C-1α, C-1β, C- dicating the lack of conjugated chromophore. The H
6β, C-10β and C-11β positions [12, 13]. In the present NMR spectrum of compound 2 was remarkably differ-
study Cephalosporium aphidicola was used to trans- ent from substrate. It exhibited additional olefin pro-
form compound 1 into a less polar metabolite 2 (17α- tons signals at δ 7.12 (d, J = 8.4 Hz) and 6.60 (dd,
ethynylestradiol). Compound 2, is a well known estro- J = 8.4 Hz, J = 2.7 Hz), while the C-4 proton ap-
gen which is also used as oral contraceptive [14]. In peared as a doublet at δ 6.54 (J = 2.6 Hz), indicating
this paper we also report the microbial transformation the aromatization of ring A. H-4 showed COSY 45◦
of 17α-ethynylestradiol (2) by Cunninghamella ele- interactions with H-2 (δ 6.60), while H-2 also showed
gans, resulting in the formation of known compounds interaction with H-1 (δ 7.12). These assignment were
3 – 6 and a new metabolite 7. Compound 6 was already further supported by the HMBC spectrum of com-
reported as metabolite of 2 by Penicillium chryso- pound 2. The ortho and meta-coupling constant val-
genum [15].
ues for H-1/H-2 and H-2/H-4 signals, further indicated
c
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M. I. Choudhary et al. · Microbial Transformation of Antifertility Agents
the presence of an aromatic ring in the molecule. The stants of H-11 signal indicated an α (equatorial) con-
aromatization of ring A through ∆ 1,2 dehydrogenation figuration of geminal hydroxy group.
The HREI MS of compound 6 showed the M+ at
m/z 312.1729, corresponding to their molecular for-
mula C20H24O3 (calcd. 312.1725). The 1H NMR spec-
trum of compound 6 was very similar to compound 2,
but with an additional methine proton triplet at δ 4.52
(J = 3.6 Hz). The H-4 exhibited a downfield shift at
δ 6.77, which indicated the presence of a hydroxyl
group at C-6. This assignment was further supported
by homoallylic coupling of H-4 with H-6 (δ 4.52) in
in steroids, is a known microbial reaction, achieved
by a number of fungi and bacteria [16]. The struc-
ture of compound 2 was further confirmed by com-
parison with the published data [17], and comparative
TLC with commercially available 17α-ethynylestra-
diol.
In another subsequent experiment, compound 2 was
subjected to screening experiments and it was found
that Cunninghamella elegans (NRRL 1392) was able
to convert compound 2 into several polar metabolites.
Scale-up of this microbial transformation reaction af-
forded five polar metabolites 3 – 7.
2
the COSY 45 ◦ spectrum. H-6 also exhibited J and
3J hetronuclear interactions with C-5 (δ 140.1) and C-
4 (δ 116.0), respectively. The stereochemistry of hy-
droxyl group at C-6 was deduced to be β on the basis
of NOESY correlation between H-6α (δ 4.52) and H-
9α (δ 1.95).
The HREI MS of compound 3 showed the M+
at m/z 312.1719, corresponding to C20H24O3 (calcd.
312.1725), indicated that metabolite 3 contained one
1
Compound 7 exhibited the M+ at m/z 326, which
was 30 a.m.u. greater than the substrate 2 and 14 a.m.u.
greater than the metabolite 6. In addition, a signifi-
cant ion was observed at m/z 294 [M+ - CH3OH].
The HREI MS of compound 7 showed the M+ at
m/z 326.1952, corresponding to molecular formula
C21H26O3 (calcd. 326.1882). The 1H NMR of com-
pound 7 was different from 6, especially in two as-
pects. First, the appearance of an additional three-
proton singlet at δ 3.43, secondly, an upfield shift of
H-6 signal at δ 4.18 (which appeared at δ 4.52 in
6), which indicated the methylation of C-6 hydroxyl
group. The 13C NMR spectra of 7 (Broad-band de-
coupled and DEPT) exhibited resonances for 21 car-
bons including two methyls, five methylenes, eight me-
thines and six quaternary carbons (Table 1). An up-
field oxygen-bearing C-6 methine signal resonated at
δ 76.4 (∆δ + 8.2 ppm vs 6), along with a methyl sig-
nal at δ 56.7, further supporting the proposed struc-
ture 7. Stereochemistry at C-6 was assigned to be α
on the basis of NOESY interaction between H-6α (δ
4.18) and H-9α (δ 2.19). Heteronuclear interaction
of oxygen-bearing methyl protons (δ 3.43) with C-6
(δ 76.4) in the HMBC spectrum, further supported the
biologically methylation at C-6 hydroxyl group which
is chemically less reactive than C-3 phenolic group
(Scheme 1).
additional oxygen atom. In the H-NMR spectrum, the
C-2 proton appeared at δ 6.75, with a simplified split-
ting pattern as an ortho coupled doublet (J = 8.3 Hz)
with H-1 (δ 6.68). This suggested the presence of a
hydroxyl group at C-4. The 13C NMR (broad-band de-
coupled and DEPT) spectra showed a new quaternary
carbon signal at δ 141.2 and disappearance of C-4 me-
thine carbon atom as compared to compound 2. New
3
carbon signal at δ 141.2, (C-4) showed J heteronu-
clear interactions with H-2 (δ 6.75) which further indi-
cated the presence of catechol moeity in compound 3.
The HREI MS of compound 4 showed the M+ at
m/z 312.1774 (C20H24O3 calcd. 312.1725) was found
to be 16 a.m.u. higher than that of compound 2. The
1H-NMR spectrum of 4 was found to be closely related
to compound 2, with an additional signal at δ 4.09 (t,
J = 2.7 Hz), which could be assigned to H-7 on the
basis of its homonuclear coupling with 2H-6 (δ 3.01
and 2.84) in COSY 45◦ spectrum. The configuration of
newly introduced hydroxyl group at C-7 was assigned
to be α on the basis of NOESY interaction between
H-7β (δ 4.09) and H-8β (δ 1.21).
The HREI MS of compound 5 showed the M+ at
m/z 312.1738, corresponding to the molecular formula
C20H24O3 (calcd. 312.1725), and indicated the pres-
1
ence of an additional oxygen atom. The H NMR spec-
trum of 5 was different from compound 2 in two as-
pects. First the C-1 proton exhibited a downfield shift
at δ 7.78 (which appeared at δ 7.12 in 2), and sec-
ond the appearance of a downfield signal at δ 4.18
(1H, ddd, J11ax,9ax = 15.2 Hz, J11ax,12ax = 10.2 Hz,
J11ax,12eq = 4.2 Hz), which indicated the introduction
of a hydroxyl group at C-11. The large coupling con-
Experimental Section
General experimental procedure
IR Spectra were recorded in CHCl3 on a FT IR-8900 spec-
trophotometer. MPs were determined on a Buchi 535 melt-
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M. I. Choudhary et al. · Microbial Transformation of Antifertility Agents
321
Table 1. 13C and 1H NMR spectral data of new metabolite 7.
Position
δC
δH (J in Hz)
1
2
3
4
5
126.5, d
116.5, d
153.5, s
115.2, d
137.3, s
76.4, d
32.7, t
7.17, d (8.5)
6.72, dd (8.4, 2.8)
–
6.78, d (2.7)
–
4.18, dd (2.3, 3.7)
1.87, m, 1.72, m
6
7
8
9
33.8, d
43.7, d
131.8, s
25.9, t
1.92, m
2.19, m
–
1.51, m, 1.22, m
2.14, m, 1.47, m
-
1.8, m
1.74, m, 1.23, m
2.32, m, 2.04, m
–
0.85, s
–
2.58, s
3.43, s
10
11
12
13
14
15
16
17
18
20
21
OCH3
31.1, t
47.3, s
49.0, d
22.6, t
38.9, t
79.7, s
12.7, q
87.4, s
74.0, d
56.7, q
Carbon multiplicities were determined by DEPT experiments; s =
quaternary, d = methine, t = methylene, q = methyl carbons.
ingredients into distilled water (3.0 l); glucose (150 g),
KH2PO4 (3 g), KCl (3 g), MgSO4.7H2O (6 g), glycine
(6 g), and Gibberella trace element solution (6 ml). The
Gibberella trace element solution was prepared by dis-
solving Co(NO3)2.6H2O (0.01 g), FeSO4.7H2O (0.1 g),
CuSO4.5H2O (0.1 g), ZnSO4.7H2O (0.161 g), MnSO4
.4H2O (0.01 g) and NH4 molybdate (0.01 g) in to distilled
water (100 ml). The medium for Cunninghamella elegans
(NRRL 1392) was prepared by mixing the following ingre-
dients into distilled H2O (3.0 l); glucose (30.0 g), peptone
(15.0 g), yeast extract (15.0 g), KH2PO4 (15.0 g), and NaCl
(15.0 g).
Scheme 1. Metabolism of compounds
Cephalosporium aphidicola and Cunninghamella elegans,
respectively.
1 and 2 by
ing point apparatus. Optical rotations were measured on a
Jasco DIP-360 digital polarimeter. UV Spectra were recorded
in CHCl3 on a Hitachi U-3200 spectrophotometer. The 1H-
and 13C NMR spectra were recorded in CDCl3 solutions on
Bruker Avance- 400 NMR at 400 and 100 MHz, respec-
tively. The EI and HREI MS were measured on Jeol JMS-
600H mass spectrometer. TLC were performed with pre-
coated plates (Silica gel 60, PF254, 0.2 mm, Merck). Com-
pounds 1 was isolated from Primolut N, a product of Scher-
ing AG, Berlin, Germany.
General fermentation and extraction protocol
The fermentation media thus obtained was distributed
among 30 flasks of 250 ml capacity (100 ml in each) and
autoclaved. The fermentation was carried out according to
a standard two-stage protocol [18]. Substrates were dis-
solved in DMSO and were evenly divided into 30 flasks
(20 mg/0.5 ml in each flask), containing 24-h-old stage II cul-
tures and fermentation continued for further additional time
on a rotatory shaker (200 rpm) at 29◦C. During the fermen-
tation period, aliquots from flasks were taken daily and ana-
lyzed by TLC in order to determine the degree of transforma-
Organism and media
Microbial cultures were obtained from either Interna- tion of substrate. In all experiments, one control flask without
tional Mycological Institute (IMI), Institute of Fermenta- biomass (for checking substrate stability) and one flask with-
tion (IFO) or Northern Regional Research Laboratories out exogenous substrate (for checking endogenous metabo-
(NRRL). All cultures were maintained on SDA and stored at lite) were used. The culture media and mycelium were sepa-
4 ◦C prior to use. The medium for Cephalosporium aphidi- rated by filtration. The mycelium were washed with CH2Cl2
cola (IMI 68689), was prepared by mixing the following (1 l) and the filtrate was extracted with CH2Cl2 (3 × 1.5 l).
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M. I. Choudhary et al. · Microbial Transformation of Antifertility Agents
The combined organic extract was washed with brine and 400 MHz): δ = 6.68 (1H, d, Jortho = 8.4 Hz, H-1), 6.75 (1H,
dried over anhydrous Na2SO4, evaporated under reduced d, Jortho = 8.3 Hz, H-2), 2.58 (1H, s, H-20), 0.85, (3H, s,
pressure, and analyzed by thin layer chromatography. Con- Me-18). – 13C NMR (CDCl3, 100 MHz): δ = 117.1 (C-1),
trol flasks were also harvested and compared by TLC, to de- 112.4 (C-2), 140.7 (C-3), 141.2 (C-4), 123.5 (C-5), 134.0 (C-
tect the bio-transformed products.
10). – MS (EI, 70 eV): m/z (%) = 312 (53) [M+], 286 (12),
229 (100), 188 (12), 176 (42), 149 (24), 115 (21), 91 (18),
55 (34). – MS (HREI): m/z = 312.1719 (C20H24O3, calcd.
312.1725).
Fermentation of norethisterone (1) with Cephalosporium
aphidicola (IMI 68689)
19-Nor-17α-pregna-1,3,5(10)-trien-20-yne-3,7α,17β-
triol (4) was obtained as a colorless crystalline solid. –
M. p. 197 – 198◦C. – [α]2D5: −2.4◦ (c = 0.19, MeOH). – IR
(CHCl3): νmax = 3441, 2974 and 2816 cm−1. – 1H NMR
(CDCl3, 400 MHz): δ = 7.18 (1H, d, Jortho = 8.5 Hz, H-1),
6.64 (1H, dd, Jortho = 8.5, Jmeta = 2.8 Hz, H-2), 6.55 (1H,
d, Jmeta = 2.6 Hz, H-4), 4.09 (1H, t, J = 2.7 Hz, H-7β),
2.59 (1H, s, H-20), 3.01 (1H, dd, J1 = 15.9 Hz, J2 = 2.4 Hz,
Ha-6), 2.84 (1H, d, J = 16.4 Hz, Hb-6), 0.85 (3H, s, Me-18).
Compound 1 (600 mg), dissolved in 15 ml DMSO,
was evenly distributed among 30 flasks containing stage II
cultures. Fermentation was stopped after 8 days, together
with the control flasks. The organic metabolites were ex-
tracted from the medium and evaporated to afford a brown
gum (3.1 gm). The crude residue was subjected to column
chromatography. Elution with gradient of petroleum ether
and EtOAc yielded compound 2 (164 mg, petroleum ether-
EtOAc 81:19). The spectral data of compound 2 was already
reported [17].
–
13C NMR (CDCl3, 100 MHz): δ = 126.8 (C-1), 117.2
(C-2), 156.2 (C-3), 114.3 (C-4), 137.4 (C-5), 133.9 (C-10),
67.8 (C-7), 39.1 (C-6). – MS (EI, 70 eV): m/z (%) = 312
(71) [M+], 294 (50) [M+ – H2O], 261 (36), 226 (100), 211
(74), 158 (53), 145 (42), 91 (41), 55 (69). – MS (HREI):
m/z = 312.1774 (C20H24O3, calcd. 312.1725).
19-Nor-17α-pregna-1,3,5 (10)-trien-20-yne-3,4,17β-diol
(2) was obtained as a white solid. – M. p. 181 – 182◦C.
– [α]2D5: −131◦ (c = 0.1, MeOH). – IR (CHCl3): νmax
=
3294, 2927, 2866, 1603, 1517 cm−1. – 1H NMR (CDCl3,
400 MHz): δ = 7.12 (1H, d, Jortho = 8.4 Hz, H-1), 6.60
(1H, dd, Jortho = 8.4, Jortho = 2.7 Hz, H-2), 6.54 (1H, d,
Jmeta = 2.6 Hz, H-4), 2.58 (1H, s, H-21), 0.85, (3H, s, Me-
18). – 13C NMR (CDCl3, 100 MHz): δ = 126.3 (C-1), 112.6
(C-2), 154.0 (C-3), 115.2 (C-4), 137.9 (C-5), 131.7 (C-10). –
MS (EI, 70 eV): m/z (%) = 298 [M+] (57), 252 (2), 228 (16),
213 (100), 160 (41), 134 (26), 107 (26), 55 55 (32). – MS
(HREI): m/z = 296.1614 (C20H24O2, 296.1616).
19-Nor-17α-pregna-1,3,5(10)-trien-20-yne-3,11α,17β-
triol (5) was obtained as a white crystalline solid. – M. p.
139 – 140 ◦C. – [α]2D5: −93.3 (c = 0.15, MeOH). – IR
(CHCl3): νmax = 3288, 2925 and 2864 cm−1. – 1H NMR
(CDCl3, 400 MHz): δ = 7.78 (1H, d, Jortho = 8.5 Hz, H-1),
6.62 (1H, dd, Jortho = 8.4 Hz, Jmeta = 2.2 Hz, H-2), 6.57 (1H,
d, Jmeta = 2.1 Hz, H-4), 4.18 (1H, ddd, J11ax,9ax = 15.2 Hz,
J11ax,12ax = 10.2 Hz, J11ax,12eq = 4.2 Hz, H-11β), 2.60 (1H,
brs, H-21), 0.84, (3H, s, Me-18). – 13C NMR (CDCl3, 100
MHz): δ = 127.6 (C-1), 112.6 (C-2), 153.6 (C-3), 114.9 (C-
4), 132.4 (C-5), 49.1 (C-9), 139.4 (C-10), 71.0 (C-11). –
MS (EI, 70 eV): m/z (%) = 312 (12.1) [M+], 294 (38), 260
(22), 224 (19), 211 (61), 157 (100), 141 (82), 91 (77.6), 55
(83.3). − MS (HREI): m/z = 312.1738 (C20H24O3, calcd.
312.1725).
19-Nor-17α-pregna-1,3,5(10)-trien-20-yne-3,6β,17α-
triol (6) was obtained as a white crystalline solid. – M. p.
201 – 202 ◦C. – [α]2D5: −81.4 (c = 0.12, MeOH). – IR
(CHCl3): νmax = 3296, 2933 and 2864 cm−1: – 1H NMR
(CDCl3, 400 MHz): δ = 7.14 (1H, d, Jortho = 8.5 Hz, H-1),
6.67 (1H, dd, Jortho = 8.4, Jmeta = 2.4 Hz, H-2), 6.77 (1H, d,
Jmeta = 2.3 Hz, H-4), 4.52 (1H, t, J = 3.6 Hz, H-6α), 2.89
(1H, brs, H-21), 0.89 (3H, s, Me-18). – 13C NMR (CDCl3,
100 MHz): δ = 127.1 (C-1), 117.4 (C-2), 156.3 (C-3). 116.0
(C-4), 140.1 (C-5), 132.6 (C-10), 68.2 (C-6), 37.6 (C-7). –
MS (EI, 70 eV): m/z (%) = 312 (41) [M+], 250 (9), 226 (14),
200 (31), 157 (25), 145 (70), 91 (56), 53 (100). – MS (HREI):
m/z = 312.1729 (C20H24O3, calcd. 312.1725).
Fermentation of 17α-ethynylestradiol (2) with Cunning-
hamella elegans (NRRL 1392)
Compound 2 was added as a solution in DMSO
(20 mg/0.5 ml in each flask), among 30 flasks containing
stage II cultures. Fermentation was continued for 12 days.
Culture filtrate was extracted with CH2Cl2. The resulting
organic extract was dried to afford a brown gum (2.6 g).
The crude residue was subjected to column chromatography.
Elution with gradient system of petroleum ether and ethyl
acetate afforded metabolites 3 (17.4 mg, petroleum ether-
EtOAc, 76:24), 7 (11.3 mg, petroleum ether-EtOAc, 71:29)
and 4 (8.8 mg, petroleum ether-EtOAc, 61:29), while elution
with petroleum ether (57%)-EtOAc (43%), afforded impure
fraction containing compounds 5 and 6, which were purified
by TLC using EtOAc-petroleum ether (4:6) as mobile phase,
where pure compounds 5 (21.7 mg) and 6 (31.4 mg) were
obtained.
19-Nor-17α-pregna-1,3,5 (10)-trien-20-yne-3,4,17β-triol
(3) was obtained as a colorless crystalline solid. – M.p. 174 –
175 ◦C. – [α]2D5: −2.1◦ (c = 0.1, MeOH). – IR (CHCl3):
19-Nor-17α-pregna-1,3,5(10)-trien-20-yne-3,17α-diol-
νmax = 3286, 2927 and 2869 cm−1. – 1H NMR (CDCl3, 3β-methoxy (7), was obtained as a white crystalline solid.
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M. I. Choudhary et al. · Microbial Transformation of Antifertility Agents
323
– M. p. 178 – 179◦C. – [α]2D5: −1.8 (c = 0.21, MeOH). – Table 1. – MS (EI, 70 eV): m/z (%) = 326 (71.2) [M+], 308
IR (CHCl3): νmax = 3294, 2925 and 2858 cm−1. – UV/vis (4.3) [M+ -H2O], 294 (45) [M+ -CH2OH], 234 (4), 211 (98),
1
(CHCl3): λmax (lgε) = 202 nm (3.4). – H NMR (CDCl3, 157 (100), 115 (29), 81 (39), 55 (12). – MS (HREI): m/z =
400 MHz): see Table 1. – 13C NMR (CDCl3, 100 MHz): see 326.1952 (C21H26O3, calcd. 326.1882).
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