Microbial transformation of nandrolone with Cunninghamella echinulata and Cunninghamella blakesleeana and evaluation of leishmaniacidal activity of transformed products

Article abstract of DOI:10.1016/j.steroids.2014.06.020

Therapeutic potential of nandrolone and its derivatives against leishmaniasis has been studied. A number of derivatives of nandrolone (1) were synthesized through biotransformation. Microbial transformation of nandrolone (1) with Cunninghamella echinulata and Cunninghamella blakesleeana yielded three new metabolites, 10β,12β,17β-trihydroxy-19-nor-4-androsten-3-one (2), 10β,16α,17β-trihydroxy-19-nor-4-androsten-3-one (3), and 6β,10β,17β-trihydroxy-19-nor-4-androsten-3-one (4), along with four known metabolites, 10β,17β-dihydroxy-19-nor-4-androsten-3-one (5), 6β,17β-dihydroxy-19-nor-4-androsten-3-one (6) 10β-hydroxy-19-nor-4-androsten-3,17-dione (7) and 16β,17β- dihydroxy-19-nor-4-androsten-3-one (8). Compounds 1-8 were evaluated for their anti-leishmanial activity. Compounds 1 and 8 showed a significant activity in vitro against Leishmania major. The leishmanicidal potential of compounds 1-8 (IC₅₀ = 32.0 ± 0.5, >100, 77.39 ± 5.52, 70.90 ± 1.16, 54.94 ± 1.01, 80.23 ± 3.39, 61.12 ± 1.39 and 29.55 ± 1.14 μM, respectively) can form the basis for the development of effective therapies against the protozoal tropical disease leishmaniasis.

Full text of DOI:10.1016/j.steroids.2014.06.020

Steroids 88 (2014) 95–100
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Microbial transformation of nandrolone with Cunninghamella echinulata
and Cunninghamella blakesleeana and evaluation of leishmaniacidal
activity of transformed products
a
b
c
c
Elias Baydoun a, , Martin Karam , Atia-tul-Wahab , Mahwish Shafi Ahmed Khan , Malik Shoaib Ahmad ,
⇑
Samreen c, Colin Smith a, Roula Abdel-Massih d, M. Iqbal Choudhary b,c,e,
a Department of Biology, American University of Beirut, Beirut 1107 2020, Lebanon
⇑
b Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
c H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
d Department of Biology, University of Balamand, Koura 100, Lebanon
e Department of Biochemistry, Faculty of Science, King Abdulaziz Universisty. Jeddah 21412, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Therapeutic potential of nandrolone and its derivatives against leishmaniasis has been studied. A number
of derivatives of nandrolone (1) were synthesized through biotransformation. Microbial transformation
of nandrolone (1) with Cunninghamella echinulata and Cunninghamella blakesleeana yielded three new
Received 1 February 2014
Received in revised form 6 June 2014
Accepted 28 June 2014
metabolites, 10b,12b,17b-trihydroxy-19-nor-4-androsten-3-one (2), 10b,16a,17b-trihydroxy-19-nor-4-
Available online 8 July 2014
androsten-3-one (3), and 6b,10b,17b-trihydroxy-19-nor-4-androsten-3-one (4), along with four known
metabolites, 10b,17b-dihydroxy-19-nor-4-androsten-3-one (5), 6b,17b-dihydroxy-19-nor-4-androsten-
3-one (6) 10b-hydroxy-19-nor-4-androsten-3,17-dione (7) and 16b,17b-dihydroxy-19-nor-4-andros-
ten-3-one (8). Compounds 1–8 were evaluated for their anti-leishmanial activity. Compounds 1 and 8
showed a significant activity in vitro against Leishmania major. The leishmanicidal potential of com-
pounds 1–8 (IC50 = 32.0 0.5, >100, 77.39 5.52, 70.90 1.16, 54.94 1.01, 80.23 3.39, 61.12 1.39
Keywords:
Nandrolone
Anabolic compound
Cunninghamella echinulata
Cunninghamella blakesleeana
Leishmania major
and 29.55 1.14
lM, respectively) can form the basis for the development of effective therapies against
Leishmanicidal activity
the protozoal tropical disease leishmaniasis.
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
Anabolic steroids are being extensively used for the treatment
of various hormonal disorders. We have been using biotransforma-
tion techniques to synthesize structurally diverse and pharmaceu-
tically important libraries of steroids. Synthesis of some steroidal
derivatives, such as azasterols and amino quinolones, with good
leishmanicidal activity, have been reported in the literature. Simi-
larly, infliximab (antibody) has also showed an enhanced leish-
manicidal activity when it is used in combination with
corticosteroids [13–15].
Nandrolone (19-nortestosterone) (1) is an anabolic steroid,
known as a performance drug [16–18]. Nandrolone (1) has an
androgenic activity. It is also used for the treatment of anxiety
and memory impairment [19,20]. Nandrolone is commercially
marketed as decanoate (deca-durabolin) and phenylpropionate
ester (durabolin) derivatives. Deca-durabolin is used for the treat-
ment of osteoporosis and breast cancer.
Biocatalysis is a valuable tool for chemists. A number of stereo-,
regio- and chemo-selective transformations have been carried out
through biotransformation, which could not be possible through
conventional chemical synthesis [1–3]. Various drugs have been
subjected to biotransformation in order to obtain novel structural
analogues with presumably enhanced biological activities [4,5].
Among biocatalytic agents, the fungi are most extensively used
for the biotransformation of various classes of organic compounds
of synthetic and natural origin. Stereoslective hydroxylation, epox-
idation and oxidation have been reported through the biotransfor-
mation with fungi [6–12].
⇑
Corresponding authors. Addresses: Department of Biology, American University
of Beirut, Beirut 1107 2020, Lebanon (E. Baydoun), H.E.J. Research Institute of
Chemistry, International Center for Chemical and Biological Sciences, University of
Karachi, Karachi 75270, Pakistan (M.I. Choudhary). Tel.: +92 2134824924; fax: +92
2134819018.
Leishmaniasis is the second most common vector born tropical
disease of protozoal origin. Approximately 350 million people in 88
countries are at the risk [21], with 12 million known cases. The
available drugs for the treatment of leishmaniasis, especially for
E-mail addresses: eliasbay@aub.edu.lb (E. Baydoun), iqbal.choudhary@iccs.edu
(M.I. Choudhary).
http://dx.doi.org/10.1016/j.steroids.2014.06.020
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

96
E. Baydoun et al. / Steroids 88 (2014) 95–100
oral applications, are too few and often ineffective. The need of
development of safe and effective leishmanicidal agents is strongly
felt to decrease disease burden and to overcome the side effects
and emerging resistance. We have been focusing on the search of
new therapeutic agents for leishmaniasis, and synthesizing their
structural analogues to enhance their activities [22].
2.5. Fermentation of nandrolone (1) with C. echinulata
Nandrolone (1) (1 g) was subjected to fermentation with C. ech-
inulata for 10 days on a rotatory shaker (121 rpm, 26 2 °C). The
solid brown extract (1.5 g), obtained after filtration, extraction
and evaporation, was fractionated with silica gel column chroma-
tography. The mobile phase was comprised of 10% gradient of hex-
anes and ethyl acetate.
2. Experimental
Four main fractions (1–4) were obtained from column chroma-
tography. The mobile phase comprises of hexanes: ethyl acetate,
and the polarity of mobile phase increases by increasing the ethyl
acetate concentration. Fraction 1 was collected over the mobile
phase of 25% hexanes and 75% ethyl acetate. Fraction 2 was eluted
with 20% hexanes and 80% ethyl acetate. Similarly, fractions 3 and
4 were obtained through column chromatography by using 15%
hexanes and 85% ethyl acetate. Fraction 1 was subjected to reverse
phase recycling HPLC for purification. The mobile phase was 40%
H2O and 60% MeOH. The compound 5 was eluted at 22 min
(2.5 mg) and compound 6 at 31 min (5 mg). Similarly, fraction 2
was injected to normal phase recycling HPLC for the removal of
impurities. The mobile phase was 94% chloroform and 6% 2-propa-
nol. The peak over retention time 184 min was collected and dried
which yielded 6 mg of compound 4. Fraction 3 yielded metabolite
2 (2 mg) on elution of silica gel column with hexanes and ethyl
acetate (15:85). Fraction 4 yielded metabolite 3 (15 mg, RT = 204 -
min) through purification with reverse phase recycling HPLC using
methanol and water as solvent system with 70:30 ratio.
2.1. General experimental conditions
Nandrolone (1) was purchased from Sigma–Aldrich (cat no.
74640, USA). Sabouraud dextrose agar was procured from Merck
KGaA (cat no. 146392, Germany). Thin layer chromatography
(TLC) was carried out on precoated plates (Merck KGaA, PF254
,
20 Â 20, 0.25 mm) (Germany). Flash silica was used for column
chromatography (CC). Recycling preparative HPLC–LC–908,
equipped with JAIGEL–ODS–L–80 (L = 250 mm, I.D. = T20 mm),
was used for the purification of compounds. Optical rotations were
measured with a JASCO P–2000 polarimeter. UV Spectrum was
recorded in chloroform and methanol with an Evolution 300 UV–
visible spectrophotometer. Infrared (IR) spectrum was recorded
in KBr discs and in chloroform in Vector 22 IR spectrophotometer
(Bruker). 1H and 13C NMR spectra were recorded in CD3OD, CDCl3
and DMSO-d6 on Bruker Avance-NMR (300, 500 and 600 MHz).
Electron impact mass spectra (EI-MS) were recorded on Jeol JMS-
600H mass spectrometer. All the solvents and reagents were of
analytical grades.
2.6. Fermentation of 1 with C. blakesleeana
2.2. Microorganisms
Incubation, filtration, and extraction procedures were similar as
described above. Fermentation of 1 with C. blakesleeana yielded
four metabolites 4, 5, 7 and 8. Four main fractions (1–4) were
acquired after fractionation with silica gel column chromatogra-
phy. Fraction 1 yielded metabolite 4 (5 mg) on purification with
recycling HPLC (chloroform: 2-propanol 94:06 RT = 184 min),
whereas compound 5 was purified from 2 by reverse phase recy-
cling using MeOH and H2O solvent (60:40, RT = 42 min). Similarly,
metabolites 7 and 8 were purified with silica gel column chroma-
tography. The mobile phase comprises of hexanes and ethyl ace-
tate and the polarity of mobile phase vary by increasing 10% of
ethyl acetate in concentration. Compound 7 was eluted at 30% hex-
anes and 70% ethyl acetate over silica gel column chromatography.
The microorganisms were obtained from the American Type
Culture Collection (ATCC). Cunninghamella echinulata (ATCC 9244)
and Cunninghamella blakesleeana (ATCC 8688A) were grown on
Sabouraud dextrose agar slant and maintained at 4 °C.
2.3. Media preparation
Culture medium (1 L each) for C. echinulata (ATCC 9244) and C.
blakesleeana (ATCC 8688A) were prepared by mixing the following
ingredients; glucose (10 g), peptone (5 g), KH2PO4 (5 g), NaCl (5 g)
and glycerol (5 mL).
2.4. General fermentation and extraction conditions
2.6.1. 10b,12b,17b-Trihydroxy-19-nor-4-androsten-3-one (2)
White solid; m. p. 212–214 °C; UV kmax 230 nm (MeOH, log
e
25 = À126.2 (c 0.018, MeOH); IR (KBr); tmax 3419 (O–H
5 L of the fungal growth media was prepared and distributed
equally in 250 mL Erlenmeyer flasks (100 mL each) and autoclaved
at 121 °C. The fungal culture was then distributed to 3 seed flasks
and incubated for two days on shaker (121 rpm) at 26 2 °C. After
suitable growth was observed in the seed flasks, the spores were
then transferred to the remaining flasks and placed on shaker
(121 rpm, 26 2 °C). Compound 1 (1 g), dissolved in 50 mL of
methanol, was distributed among the flasks containing four day
old culture of C. echinulata (ATCC 9244) and C. blakesleeana (ATCC
8688A). The flasks were then placed on shaker for 12 days. Analo-
gous control experiments were conducted which included an incu-
bation of the fungus without substrate 1, and incubation of 1 in the
medium without fungus.
At the end of cultivation phase, the fungal mass was separated
by filtration, and washed with dichloromethane (DCM). The filtrate
was extracted with DCM (4 L Â 3), and after drying with sodium
sulfate (anhydrous), concentrated on rotary evaporator to obtain
a brown thick material. The control flasks were also processed in
the same manner and compared with the test flasks to detect the
presence of transformed products.
1.97); [
a]
D
stretching), 1663 (C=O stretching); HREI-MS m/z 306.1842 (M+,
[C18H26O4]+, calc. 306.1831); EI-MS: m/z 306 [M+] (25), 288 (16),
278 (49), 190 (39); 1H NMR (CD3OD, 300 MHz): Table 2; 13C
NMR (CD3OD, 125 MHz): Table 3.
Table 1
Leishmanicidal activity of compounds 1–8.
Compound
Leishmanicidal activity (IC50 in lM)
1
2
32.0 0.5
>100
3
4
5
6
7
8
77.39 5.52
70.90 1.16
54.94 1.01
80.23 3.39
61.12 1.39
29.55 1.14
Standards
Pentamidine
Amphotericin B
5.09 0.09
0.29 0.05

E. Baydoun et al. / Steroids 88 (2014) 95–100
97
Table 3
2.6.2. 10b,16
a,17b-Trihydroxy-19-nor-4-androsten-3-one (3)
13C NMR Chemical Shift Assignments of (d in ppm) compounds 1–8.
White solid; m. p. 203–205 °C; UV kmax 230 nm (MeOH, log
e
3.4); [a]
25 = 62.5 (c 0.01, MeOH); IR (CHCl3); tmax 3388 (O–H
D
Position 1a
2b
34.6
33.1
3c
32.8
34.5
4b
34.7
34.2
5d
33.8
33.7
6b
27.1
37.1
7d
33.7
33.6
8d
26.5
36.6
stretching), 1664 (C=O stretching); HREI-MS m/z 306.1840 (M+,
[C18H26O4]+, calc. 306.1831); EI-MS: m/z 306 [M+] (40), 288 (30),
278 (89), 264 (21), 213 (66); 1H NMR (CD3OD, 300 MHz): Table 2;
13C NMR (CD3OD, 150 MHz): Table 3.
1
2
3
4
27.2
37.7
202.9 202.2 202.3 202.7 199.0 203.3 200.0 199.8
124.7 124.9 124.8 126.1 124.8 125.6 124.9 124.7
170.8 168.0 168.5 162.4 164.2 168.7 163.6 167.5
5
6
7
8
9
10
11
12
13
14
15
16
17
18
36.5
31.9
41.6
51.0
43.8
27.7
37.2
44.1
51.2
24.1
30.6
82.3
11.6
31.9
34.4
35.5
53.3
70.6
29.9
79.8
49.5
49.8
24.0
30.8
82.5
6.5
33.1
34.5
35.9
54.6
70.9
20.9
38.2
43.5
47.9
36.0
70.8
81.9
12.4
73.7
39.4
30.6
54.5
72.3
20.9
37.7
44.2
51.1
24.2
30.6
82.3
11.5
31.9
31.3
35.4
52.6
70.4
20.1
36.1
43.0
50.1
23.4
30.5
81.6
10.9
72.5
39.1
34.9
50.9
39.5
27.1
37.7
44.4
50.9
24.0
30.6
31.7
31.0
34.8
52.6
70.0
19.7
30.6
47.0
50.5
21.8
35.7
35.3
30.5
39.9
49.4
42.5
25.6
36.2
44.2
47.5
33.7
78.5
89.8
12.2
2.6.3. 6b,10b,17b-Trihydroxy-19-nor-4-androsten-3-one (4)
White solid; m. p. 207–210 °C; UV kmax 230 nm (MeOH, log
e
2.8); [a]
25 = À257.5 (c 0.02, MeOH); IR (CHCl3); tmax 3392 (O–H
D
stretching), 1669 (C=O stretching); HREI-MS m/z 306.1819 (M+,
[C18H26O4]+, calc. 306.1831); EI-MS: m/z 306 [M+] (100), 259 (10),
138 (18.8), 133 (16), 91 (14.4); 1H NMR (CD3OD, 300 MHz): Table 2;
13C NMR (CD3OD, 125 MHz): Table 3.
82.3 220.0
11.6 13.7
2.6.4. 10b,17b-Dihydroxy-19-nor-4-androsten-3-one (5)
a
b
c
= 75 MHz.
White solid; m. p. 223–225 °C, (230–234 °C, Flines et al. 1963);
= 125 MHz.
= 150 MHz.
= 100 MHz.
UV kmax 230 nm (MeOH log e = 3.2 [
a]
25 = –25 (c 0.01, CHCl3),
D
(–28, CHCl3 Flines et al. 1963); IR (CHCl3);
tmax 3406 (O–H stretch-
d
ing), 1656 (C=O stretching); HREI-MS m/z 290.1900 (M+,
[C18H26O4]+, calc. 290.1882); EI-MS: m/z 290 [M+] (60.9), 262
(100.0), 248 (21.2), 148 (69.0), 133 (71.0), 99 (21.7); 1H NMR
(CDCl3, 300 MHz): Table 2; 13C NMR (CDCl3, 125 MHz): Table 3.
2.6.7. 16b,17b-Dihydroxy-19-nor-4-androsten-3-one (8)
White solid; m. p. 144–146 °C, (154–156 °C, Flines et al. 1963);
UV kmax 248 nm (MeOH, log 3.62); [
25 = 47 (c 0.005, CHCl3),
(51, CHCl3, Flines et al. 1963); IR (CHCl3);
e
a]
D
t
max 3395 (O–H stretch-
2.6.5. 6b,17b-Dihydroxy-19-nor-4-androsten-3-one (6)
ing), 1660 (C=O stretching); HREI-MS m/z 290.1871 (M+,
[C18H26O3]+, calc. 290.1882); EI-MS: m/z 290 [M+] 290 (6), 272
(5), 149 (15), 110 (35), 83 (70). 1H NMR (CDCl3, 300 MHz): Table 2;
13C NMR (CDCl3, 100 MHz): Table 3.
White solid; m. p. 213–215 °C, (209–213 °C, Flines et al. 1963);
UV kmax 230 nm (MeOH, log
e 3.34); [a]
25 = –55 (c 0.01, EtOH),
D
(–53, EtOH, Flines et al. 1963.); IR (CHCl3); tmax 3389.2 (O–H
stretching), 1666 (C=O stretching); HREI-MS m/z 290.1897 (M+,
[C18H26O3]+, calc. 290.1882); EI-MS: m/z 290 [M+] (23.5), 261
(15.0), 246 (9.3), 213 (27.7), 138 (52.5); 1H NMR (CDCl3,
300 MHz): Table 2; 13C NMR (CD3OD, 150 MHz): Table 3.
2.7. Assay for leishmanicidal activity
Modified NNN biphasic medium, along with normal physiolog-
ical saline, was used for culturing of Leishmania major (DESTO).
RPMI 1640 medium was used for culturing of Leishmania prom-
astigotes, supplemented with foetal bovine serum (FBS) (10% heat
inactivated). Parasites were centrifuged (at log phase) for 10 min at
2000 rpm and washed thrice with saline at same speed and time.
Fresh culture medium was used for the dilution of parasites to
acquire a final density of 106 cells/mL.
2.6.6. 10b-Hydroxy-19-nor-4-androsten-3,17-dione (7)
White solid; m. p. 201–204 °C, (195–197 °C, Flines et al. 1963);
UV kmax 247 nm (MeOH, log
e 3.38); [a]
25 = 147 (c 0.003, CHCl3),
D
(157, CHCl3, Flines et al. 1963); IR (CHCl3); tmax 3413.9 (O–H
stretching), 1723 (C=O stretching); HREI-MS m/z 288.1725 (M+,
[C18H24O3]+, calc. 288.1725); EI-MS: m/z 288 [M+] (7.0), 272
(10.0), 261 (18.8), 138 (53.0); 1H NMR (CDCl3, 300 MHz): Table 2;
13C NMR (CD3OD, 100 MHz: Table 3.
The sample solution was prepared by 1 mg of test compound in
950 lL of RPMI media and 50 lL of DMSO. It was then mixed on
Table 2
1H NMR (300 MHz) chemical shift assignments (d in ppm, J in Hz) of compounds 1–8.
Position
1
2
3
4
5
6
7
8
1
2
3
1.78 m, 1.47 m
1.87 m, 1.10 m
–
2.19 m, 1.92 m
2.66 m, 2.32 m
–
1.86 m, 1.03 m
2.58 m, 2.27 m
–
2.64 m, 2.27 m 2.12 m, 1.95 m 2.31 m, 1.87 m 2.34 m, 1.99 m 1.54 m, 2.24 m
1.87 m, 2.16 m 2.54 m, 2.34 m 2.37 m, 2.27 m 2.53 m, 2.17 m 1.80 m, 1.20 m
–
–
–
–
–
4
5
5.57 m
–
5.72 s
–
5.72 s
–
5.81 s
–
5.57 s
–
5.85 s
–
5.78 s
–
5.56 s
–
6
7
8
9
10
11
12
13
14
15
16
17
18
2.49 m, 2.33 m
1.87 m, 1.03 m
1.41 m
1.01 m
2.21 m
1.53 m, 2.30 m
2.32 m, 2.25 m
–
2.21 m, 1.52 m
2.60 m, 1.28 m
1.80 m
1.15 m
–
1.73 m, 1.64 m
3.45 dd, J = 10.5, 5.7 1.87 m, 1.12 m
–
2.81 m, 2.32 m
2.19 m, 1.87 m
1.87 m
1.07 m
–
4.38 m
2.0 m, 1.25 m
1.50 m
1.08 m
–
2.29 m, 2.58 m 4.28 t, J = 2.64
1.84 m, 1.01 m 1.96 m, 1.23 m 1.88 m, 1.29 m 1.77 m, 1.05 m
2.65 m, 2.34 m 2.46 m, 2.24 m
1.75 m
1.07 m
–
1.90 m
0.87 m
–
1.94 m
1.14 m
–
1.35 m
0.87 m
–
1.73, 1.67 m
1.73 m, 1.63 m 1.64 m, 1.58 m 1.61 m, 1.34 m 1.74 m, 1.68 m 1.84 m, 1.26 m
1.50 m, 1.20 m 1.88 m, 1.12 m 1.85 m, 1.11 m 1.9 m, 1.12 m
–
2.39 m, 2.24 m
–
–
–
–
–
0.85 td, J = 11.6, 4.0 0.96 m
0.85 m
2.18 m,1,27 m
1.06 m
0.98 m
1.01 m
1.30 m
1.39 m
1.62 m, 1.32 m
1.98 m, 1.47 m
3.49 t, J = 8.8
0.81 s
1.63 m, 1.40 m
1.63 m, 1.36 m 1.60 m, 1.31 m 1.61 m, 1.34 m 1.97 m, 1.58 m 1.81 m, 1.21 m
2.20 m, 1.52 m
3.81 t, J = 8.1
0.84 s
4.02 q, J = 12.48,7.5 2.16 m, 2.07 m 2.05 m, 2.49 m 2.11 m, 1.25 m 2.47 m, 2.08 m 4.04 m
3.30 d, J = 7.44
0.88 s
3.50 t, J = 8.4
0.82 s
3.6 t, J = 8.4
3.58 t, J = 8.64
–
3.5 d, J = 5.7
0.79 s
0.81 s
0.93 s
0.81 s

98
E. Baydoun et al. / Steroids 88 (2014) 95–100
vortex and dispensed in a 96-well plate. In 96-well microtiter
plate, the first row received 180 L of medium, while remaining
wells received 100 L medium. A 20 L of the test compound
was added in wells containing medium and serially diluted. A
100 L of parasite culture was transferred in each well. Two rows
were used for negative and positive controls. Only growth medium
was used for negative controls, while standard leishmanicidal
drugs such as amphotericin or pentamidine were used as positive
controls. This plate was then incubated at 21–22 °C for 72 h. The
culture was examined microscopically on an improved Neubauer
counting chamber. Ezfit 5.03 Perella Scientific software (USA)
was used for calculation of IC50 values of test compounds. All
assays were performed in triplicates [23].
H2-2 (d 2.66 m, 2.32 m) showed HMBC correlations with C-10 (d
70.6). Through these interactions, the position of one of the two
hydroxyl groups was inferred at C-10. Similarly, H2-11 (d 1.64,
1.73), H-17 (d 3.81) and H3-18 (d 0.84) showed HMBC correlations
with C-12 (d 79.8), indicating the second OH at C-12 (Fig. 2). The b
(axial) stereochemistry of OH at C-10 was assigned on the basis of
NOE correlation of H-11 (d 1.64 axial) with OH (d 5.01) at C-10, and
NOESY correlation with H-18 (d 0.84). The methine proton (d 3.45)
showed NOESY correlations with H-9, H-14 and H-17, suggesting
the b hydroxylation at C-12 (equatorial) (Fig. 3). The metabolite 2
was identified as 10b,12b,17b-trihydroxy-19-nor-4-androsten-3-
one.
l
l
l
l
The HREI-MS of metabolite 3 showed the [M]+ at m/z 306.1840
(C18H26O4, calc. 306.1831), indicating dihydroxylation in com-
pound 1. IR Spectrum showed absorbances at 3352 (O–H) and
1664 cmÀ1 (C=O). The 1H NMR spectrum showed an additional
methine proton at d 4.02 (q, Je,e = 12.5 Hz, Je,a = 7.5 Hz). The pres-
ence of new quaternary (d 70.9) and methine (d 70.8) carbons in
the 13C NMR spectrum suggests dihydroxylation in compound 3.
The COSY-DFQF spectrum showed the coupling between the new
methine proton (d 4.02) and H-17 methine proton (d 3.30 d,
Ja,e = 7.44 Hz). The H-2 (d 2.58, 2.27) and H-4 (d 5.72) showed
HMBC correlations with C-10 (d 70.9), which indicated a hydroxyl-
ation at C-10. H-15 (d 2.18) showed the HMBC correlations with
new methine carbon (d 70.8) (Fig. 2). This indicated second hydrox-
ylation at C-16. The OH (d 4.59) in 1H NMR recorded in DMSO-d6
showed the NOE correlation with H-8 (d 1.87), indicating a b
hydroxylation at C-10 (Fig. 3). The b stereochemistry of H-16 was
assigned based on NOESY correlation of methine H-16 (d 4.02) with
H3-18 (d 0.88). The structure of compound 3 was deduced as
3. Results and discussion
The biotransformation of nandrolone (1), C18H26O2, is being
reported for the first time with C. echinulata and C. blakesleeana.
Fermentation with C. echinulata afforded five metabolites 2–6,
out of which three compounds 2–4 were found to be new. Bio-
transformation of 1 with C. blakesleeana generated four compounds
4 and 6–8. Compound 4 is being reported for the first time (Fig. 1).
Metabolite 2 was obtained as a white solid. It was assigned the
molecular formula C18H26O4 based on its HREI-MS at m/z 306.1842
[M]+ (calc. 306.1831), 32 amu higher than 1, which indicated
dihydroxylation. The IR absorbance showed the presence of hydro-
xyl (3419 cmÀ1) and carbonyl functionalities (1663 cmÀ1). A down-
field methine proton at d 3.45 (dd, Ja,a = 10.5 Hz, Ja,e = 5.7 Hz)
appeared in 1H NMR spectrum. The 13C NMR spectrum showed
two new downfield carbon signals at d 70.6 and 79.8, indicating
dihyroxylation. The H-4 (d 5.72 s), H2-6 (d 2.20 m, 1.52 m) and
10b,16a,17b-trihydroxy-19-nor-4-androsten-3-one.
OH
OH
OH
OH
OH
H
OH
OH
H
H
H
3
H
OH
O
H
H
H
H
2
O
C. echinulata
OH
O
C. echinulata
18
OH
4
12
17
15
13
14
C. echinulata,
C. blakesleeana
OH
11
9
16
OH
H
H
1
2
10
8
H
H
C. blakesleeana,
C. echinulata
H
H
OH
H
3
5
7
4
6
C. echinulata
O
H
H
H
1
H
5
C. blakesleeana
O
O
C. blakesleeana
O
OH
OH
6
H
OH
OH
H
H
H
H
H
H
O
O
7
8
Fig. 1. Biotransformation of 1 with Cunninghamella echinulata and Cunninghamella blakesleeana.

E. Baydoun et al. / Steroids 88 (2014) 95–100
99
HO
H
H
H
OH
OH
18
18
12
12
17
17
13
H
13
14
11
9
11
9
16
16
H
H
OH
OH
H
OH
14
15
15
1
1
4
H
10
2
8
7
8
10
5
2
H
H
H
H
H
H
3
5
3
7
4
6
6
O
O
H
H
H
H
H
H
2
3
OH
18
12
17
15
13
11
9
16
H
OH
14
1
4
8
10
5
2
3
H
H
7
6
O
H
OH
H
4
Fig. 2. Key HMBC correlations in compounds 2–4.
H
CH3
CH3
18
18
OH
H
OH
OH
H
OH
OH
H
12
12
14
11
13
H
10
17
11
13
15
17
9
14
9
1
1
8
15
10
8
2
2
5
16
7
3
4
16
H
5
7
H
3
4
H
6
6
H
H
O
H
H
O
2
OH
3
CH3
18
OH
H
OH
12
OH
13
11
17
14
9
1
10
8
2
15
6
5
4
3
7
H
6
H
4
H
O
H
H
Fig. 3. Key NOESY correlations in compounds 2–4.
The HREI-MS of metabolite 4 showed the [M]+ at m/z 306.1819
(C18H26O4, calc. 306.1831), which indicated the presence of two
additional OH. IR Spectrum showed absorbances at 3392 (hydroxyl
group) and 1669 cmÀ1 (ketonic carbonyl group). An additional
broad singlet of methine proton was observed at d 4.39 (W1/
2 = 1.2 Hz) in 1H NMR spectrum. The 13C NMR spectrum showed
two new downfield carbon signals at d 73.8 (C-6) and 72.3 (C-
10), further indicating dihydroxylation in compound 4. The H-4
(d 5.81) showed HMBC correlations with C-6 (d 73.8) and C-10 (d
72.3), suggesting OH substituents on C-6 and C-10 (Fig. 2). The
methine proton at d 4.39 showed HMBC correlations with C-4 (d
126.2), C-5 (d 162.4) and C-7 (d 39.4), indicated an OH at C-6.
The C-1 (d 2.64, 1.26) and C-2 (d 2.18, 1.87) protons showed HMBC
correlations with C-10 (d 72.3), hence second hydroxylation was
inferred at C-10 (Fig. 2). The methine proton at d 4.39 showed
NOESY correlations with H-4 (d 5.81) and H2-7 (d 2.01, 1.26).
Therefore, b-OH was placed at C-6. The NOESY correlation of OH
(d 5.01) (DMSO-d6) with C-6 OH (d 4.49) indicated a b-OH at C-
10 (Fig. 3). Thus the metabolite 4 was identified as 6b,10b,17b-tri-
hydroxy-19-nor-4-androsten-3-one.
spectral data of compounds 5 and 6 was previously reported by
Huszcza et al., Kolet et al., and Flines et al. [24–27], whereas, the
data of compounds 7 and 8 was reported by Caspi et al. and Flines
et al. [28,29]. The molecular composition of compounds 5, 6 and 8
were 16 amu higher than 1 which showed mono-hydroxylation.
Compounds 5, 6 and 8 showed the EI-MS [M]+ m/z at 290, whereas
compound 7 was at m/z 288. Compounds 5–8 were identified as
10b,17b-dihydroxy-19-nor-4-androsten-3-one (5), 6b,17b-dihy-
droxy-19-nor-4-androsten-3-one (6), 10b-hydroxy-19-nor-4-
androsten-3,17-dione (7) and 16b,17b-dihydroxy-19-nor-4-andr-
osten-3-one (8) [24–29]. Compounds 5 and 6 were previously
reported by the biotransformation of 1 with Aspergillus wentii
MRC 200316, whereas compound 7 was synthesized from andro-
stene-3, 17, 19-trione by aromatase cytochrome P-450 mediated
transformation. Metabolite 8 was synthesized from 1 on fermenta-
tion with molds.
3.1. Leishmanicidal activity of nandrolone and its derivatives
We have been focusing on the biotransformation of bioactive
steroids and bioactivity evaluation of resulting metabolites. The
leishmanicidal activity of steroidal analogues 2–8, synthesized
The structures of four known metabolites 5–8 were identified
by comparison of their reported data with the observed data. The

100
E. Baydoun et al. / Steroids 88 (2014) 95–100
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[8] Kolet SP, Niloferjahan S, Haldar S, Gonnade R, Thulasiram HV. Biocatalyst
mediated production of 6b,11a-dihydroxy derivatives of 4-ene-3-one steroids.
Steroids 2013;78:1152–8.
(IC50 = 5.09 0.09
Table 1). Compound 8 (IC50 = 29.55 1.14
cant leishmanicidal activity (the activity value within IC50 range
of 5–34 was considered as significant), whereas compound 5
(IC50 = 54.94 1.01
with IC50 value between 50 and 60 were considered as moderately
active). Compounds (IC50 = 70.90 1.16 M) and
(IC50 = 61.12 1.39 M) exhibited a low leishmanicidal activity
(compounds showing activity within the IC50 range of 60–70 were
considered as low active). Some compounds 3 (IC50 = 77.39 5.52 -
l
M) and amphotericine B (IC50 = 0.29 0.05
lM,
lM) showed a signifi-
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Detoxification of terpinolene by plant pathogenic fungus Botrytis cinerea.
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lM) was only moderately active (compounds
4
l
7
l
lM) and 6 (IC50 = 80.23 3.39 lM) were poorly active (compounds
with IC50 above 70 were considered as poorly active), whereas
compound 2 was inactive (compounds with IC50 above 100 were
inactive). The metabolite 8 was found to be more active against
leishmania, as compared to substrate 1 and other metabolites.
4. Conclusion
A number of hydroxylated derivatives 2–8 of nandrolone (1)
were synthesized through microbial fermentation with C. echinula-
ta and C. blakesleeana. It is an efficient method for the mono- and
dihydroxylation of nandolone. The evaluation of nandrolone and
its derivatives through an in vitro leishmanicidal assay showed a
varying range of IC50 values. Compounds 1 and 8 were found to
be the most active among all compounds with IC50 values of
32.0 0.5 and 29.55 1.14 lM, respectively. We are further trying
to enhance their leishmanicidal activity by chemical and biological
derivatization of 1.
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steroids. Natural Product Research 2008;22:1289–96.
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Acknowledgements
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a diospyrin derivative as antileishmanial agent and potential modulator of
We would like to acknowledge the Lebanese CNRS (Lebanese
Council for Scientific Research) for a research grant.
ornithine
2013;135:407–13.
decarboxylase
of
Leishmania
donovani.
Exp
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