Steroids 77 (2012) 1267–1290

Contents lists available at SciVerse ScienceDirect

Steroids
journal homepage: www.elsevier.com/locate/steroids

Review

Biological transformations of steroidal compounds: A review

Haq Nawaz Bhatti ⇑, Rasheed Ahmad Khera
Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad 38040, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Microbial transformation is an important tool for structural modification of organic compounds, espe-
Received 24 May 2012 cially natural products with complex structures like steroids. It can be used to synthesize chemical struc-
Received in revised form 15 July 2012 tures that are difficult to obtain by ordinary methods and as a model of mammalian metabolism due to
Accepted 26 July 2012
similarity between mammalian and microbial enzyme systems. During recent years research has been
Available online 13 August 2012
focused on the structural modifications of bioactive steroids by using various microorganisms, in order
to obtain biologically potent compounds with diverse structures. Steroidal compounds are responsible
Keywords:
for important biological functions in the cells and manifest a variety of activities. This article covers
Biotransformation
Fungi
the microbial transformation of sterols, steroidal hormones and some new types of steroids known as
Hormones bufadienolides. Emphasis has placed on reporting metabolites that may be of general interest and on
the practical aspects of work in the field of microbial transformations. The review covers the literature
from 1994 to 2011.
Ó 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267
2. Microbial transformation of sterols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1268
3. Microbial transformation of steroidal hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1269
4. Microbial transformations of miscellaneous steroids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1283
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288

1. Introduction growth, and proliferation and are precursors of bile acids and hor-
monal steroids. Some steroidal compounds are used as an impor-
Steroids (Greek, stereos = solids) are solid alcohols that are tant source of raw materials for the production of steroids of the
widely distributed in the animal and plant kingdom. The basic androstane, pregnane and estrane series [2].
skeleton consists of 17 carbon atoms arranged in the form of a Microbial transformation is an effective tool for the preparation
perhydrocyclopentanophenanthrene. They include great variations of compounds, which may be otherwise difficult to prepare by con-
in structure and encompass compounds of vital importance to life, ventional synthetic methods. This has been extensively used for
such as cholesterol, the bile acids, sex hormones, vitamin D, corti- the bioconversion of steroids [3]. The research efforts in this field
coid hormones, cardiac aglycones, antibiotics, and insect molting were triggered around 1950, with the announcement of the phar-
hormones. Some of the most potent toxins are steroidal alkaloids. macological effects of cortisol and progesterone and with the iden-
Steroidal compounds are responsible for important biological tification of 11a-hydroxylation activity of a Rhizopus species, a
functions in the cells, e.g. steroids of androstane, pregnane and decisive step in the development of the practical synthesis of ste-
estrane series exhibit various hormonal activities [1]; bile acids roids with useful biological activity [4]. The great versatility of
are essential for lipid digestion and absorption and cardiac agly- microbial systems in the production of commercially valuable ste-
cones are used for heart failure treatment. Sterols are important roids for the pharmaceutical industry is now well recognized [5].
constituents of cell membranes, essential for their stability, cell Several microbial transformations of steroids have been reported
[2,5–10]. Bioconversions for the transformation of steroids have
⇑ Corresponding author. Fax: +92 41 9200764. proliferated and specific microbial transformation steps have been
E-mail addresses: hnbhatti2005@yahoo.com, haq_nawaz@uaf.edu.pk (H.N. Bhatti). incorporated into numerous partial syntheses of new steroidal

0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.07.018
1268 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290
compounds for evaluation as hormone or drug. These modified com-
pounds are currently favored when compared to their natural coun-
terparts due to some therapeutic advantages, such as high potency,
longer half-lives in blood, simple delivery methods and reduced side
effects [11]. A variety of steroids are widely used as anti-inflamma-
tory, immunosuppressive, progestational, diuretic, anabolic and con-
traceptive agents [4–9,12,13]. They have also used for the treatment
of some forms of breast and prostate cancer [14,15], as replacement
agents in the treatment of adrenal insufficiencies [16], in the preven-
tion of coronary heart disease [17], as antifungal agents [18], as active
ingredients in anti-obesity agents [19] and in the treatment of de-
clared AIDS [20]. Recently, a steroidal glycoside exhibited anti-viral
activity on herpes virus type I [21]. Although introduction of new ste-
roids into commerce is now limited, there is scope for improvement
in the yields of the desired metabolites as well as preparation of novel
steroids. At present steroid manufacture has become an important
branch of health care industry affected by biotechnology. As far as
market value is concerned, steroid manufacturing is second to antibi-
otic production [22].
The areas which are now receiving attention in microbial tech-
nology are: application of newer concepts of genetic engineering of
microbes with improved characteristics, solubility improvement
for carrying out biotransformation of substrates that are insoluble
in water, immobilization of enzymes or whole cells in a suitable
matrix for economic utilization, development of a continuous pro-
cess for better and economic product recovery; and manipulation
of culture media for improvement in product yields by use of
cyclodextrin [9].
The previous reviews [2,5–8] covered literature of steroidal bio-
transformation up to 1993. This review article attempts to present
mainly the structures of metabolites of steroids in vitro in a variety
of biological media during the period 1994–2011.
2. Microbial transformation of sterols
Phytosterols, together with sapogenins such as diosgenin, hec-
ogenin and solasodine, are established in the pharmaceutical
industry as starting materials for the production of steroidal
hormones. Stigmasterol, b-sitosterol and campesterol are the most
commonly used sterols. Stigmasterol and crude sitosterol (a mix-
ture of b-sitosterol and campesterol) are extracted mainly from
soybeans. Cholesterol (1), another employed starting material, ob-
tained from animal fats and oils, such as lard, beef, tallow, milk fat
or fish oil and sitosterol are microbially cleaved to 17-ketosterols.
The production of testosterone (2), a male sex hormone or andro-
gen, from cholesterol via a single step microbial transformation
process was investigated by Liu et al. [23] and Liu and Lo [24]. Incu-
bation of cholesterol (1) with Mycobacterium sp. NRRL B-3805
resulted in the isolation of testosterone (2).
In recent years, much attention has been given to the biocon-
version of steroids in aqueous/organic solvent two-phase systems
[25,26]. The main advantage of using such systems is the possibil-
ity of operating with high concentrations of poor water-soluble
reactants and products in the reactor while avoiding inhibition of
the reaction by either reactant or product. The incubation of
cholesterol (1) with free cells of Arthrobacter simplex [27] afforded
cholest-4-en-3-one (3) in an aqueous organic solvent two-phase
system. Similarly a novel approach, microbial transformation in
cloud point system (CPS), has developed to avoid practical difficul-
ties, such as substrate solubility and product inhibition. The system
could provide microbial cells with aqueous environment and could
dissolve substrates and products in surfactant phase. A microbial
transformation of cholesterol (1) to androst-1, 4-dien-3-17-dione
(ADD) (4) and androst-4-en-3,17-dione (5) has been investigated
in a system consisting of non-ionic surfactants Triton X-100 and
Triton X-114 [28]. It was observed that this system was more affec-
tive than microbial transformation in conventional media. A novel
single step microbial transformation process for the production of
testosterone (2) from cholesterol (1) by Lactobacillus bulgaricus [29]
has also been reported. Moreover, it was observed that addition of
cyclodextrin (CD) significantly increased the biotransformations of
cholesterol (1). These biotransformations of cholesterol are shown
in Scheme 1.
It is a well-known fact that a wide range of microorganisms is
capable of utilizing sterols such as cholesterol and phytosterols as
the sole carbon source for the growth and propagation. It is also well
documented that in the sterol degrading microorganisms, the ring
nucleus and the side chain are metabolized by different enzyme sys-
tems simultaneously and independently [30]. Based on these facts,
Yan et al. [31] reported the microbial transformation of 3-hydroxy-
5,6-cyclopropanocholestanes-an alter route to 6-methylsteroids.
Incubation of 3b-hydroxy-5, 6a-cyclopropano-5a-cholestane (6)
with Mycobacterium sp. (NRRL B-3805) gave three metabolites, 5,
6a-cyclopropano-5a-androsta-3, 17-dione (7), 3b-hydroxy-5,
6a-cyclopropano-5a-androsta-17-one (8) and androsta-4-ene-3,
17-dione (5). Fermentation of 3b-hydroxy-5, 6b-cyclopropano-5b-
cholestane (9) with Mycobacterium sp. afforded the metabolite 5,
5, 6b-cyclopropano-5b-androsta-3, 17-dione (10) and 3a-hydroxy-
5, 6b-cyclopropano-5b-androsta-17-one (11). Similarly, incubation
of 3b-hydroxy-5, 6a-cyclopropano-5a-cholest-7-ene (12) with
Mycobacterium sp. yielded a mixture of side chain cleaved 17-keto
steroids as the major products, viz. the cyclopropyl eliminated
product 5, 5, 6a-cyclopropano-5a-androst-7-ene-3, 17-dione (13),
3b-hydroxy-5, 6a-cyclopropano-5a-androst-4-ene-17-one (14), 5,
6a-cyclopropano-5a-androst-8(14)-ene-3, 7, 17-trione (15), 5,
6a-cyclopropano-5a-androst-8-ene-3, 7, 17-trione (16) and 6a,
7a-cyclopropanoandrost-4-ene-3, 17-dione (17) as described in
Scheme 2.
Ambrus et al. [32] investigated the effect of the different stereo-
chemistry of C-24 on the C-26 microbial oxidation of sterol side-
chain using genetically modified Mycobacterium sp. BCS 396 to
transform ergosterol (18). Incubation of ergosterol (18) with
Mycobacterium sp. yielded 26-oxygenated metabolites: 3-oxo-4,
22-ergostadien-26-oic acid methyl ester (19), 3-oxo-1, 4, 22-ergo-
statrien-26-oic acid methyl ester (20) and 3-oxo-1, 4, 22-ergosta-
trien-26-oic acid (21). The genetically modified Mycobacterium
sp. BCS 396 strain has also been used to transform sterols with
stigmastane side chain in order to obtain 26-oxidized metabolites.
Microbial transformation of b-sitosterol (22) by Mycobacterium sp.
[33] afforded 4-stigmasten-3-one (23), 26-hydroxy-4-stigmasten-
3-one (24) and 3-oxo-4-stigmasten-26-oic acid (25). Similarly
incubation of stigmasterol (26) with Mycobacterium sp. BCS 396
[33] yielded 4,22 stigmastadien-3-one (27), 6b-hydroxy-4, 22
stigmastadien-3-one (28), 26-hydroxy-4, 22 stigmastadien-3-one
(29), 3-oxo-4, 22 stigmastadien-26-oic acid methyl ester (30) and
3-oxo-1, 4, 22 stigmastatrien-26-oic acid methyl ester (31). Some
mutant strains of Mycobacterium sp. are also able to cleave sterol
side chain giving androstenedione (AD) as the major product.
Microbial conversion of b-sitosterol (22) to androstenedione (5)
was achieved with Mycobacterium sp. VKM Ac-1815D [34,35]. A
phytosterol assimilating and AD-producing mutant, B-3805S de-
rived from Mycobacterium sp. NRRL B-3805 was also used to trans-
form phytosterol (campesterol: stigmasterol: b-sitosterol = 8:1:11,
molar ratio) to androstenedione (5) [36]. These biotransformations
are shown in Scheme 3.
Biotransformation of 8a, 9a-epoxytetrahydrolanosterol (32)
and lanosta-7, 9 (11)-dien-3b-ol (35) with Mycobacterium sp. NRRL
B-3805 was reported by Wang et al. [37]. Incubation of compound
32 with Mycobacterium sp. yielded the metabolites, 20(S)-hydroxy-
methyl-12a-hydroxypregn-4-en-3-one (33) and 20(S)-hydroxy-
methyl-4, 4, 14a-trimethypregna-7, 9(11)-dien-3b-ol (34).
However, fermentation of lanosta-7, 9(11)-dien-3b-ol (35) with
 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1269

OH

H H
O O
3
2
My cobacter ium sp.
O
O
HO
1

O
O
4 5

Scheme 1. Microbial transformation of cholesterol (1).

O
O

My cobacter ium sp.

5 + +
6 O
HO HO
7 8
O O

Mycobacterium sp.
5 + +
O HO
HO 10 11
9
O O O

+ +
O HO O O
13 14 15
My cobact er ium sp.
5 +
O O
HO
12
+
O O O
16 17

Scheme 2. Microbial transformation of 3-hydroxy-5, 6-cyclopropanocholestanes.

Mycobacterium sp. resulted in the isolation of androsta-4,
8(14)-dien-3, 17-dione (36), lanosta-7, 9 (11)-dien-3-one (37),
androsta-8(14)-en-3, 17-dione (38) and methyl 12a-hydrox-
ybisnorchola-4, 17(20)-dien-22-oate (39) as described in
Scheme 4.
3. Microbial transformation of steroidal hormones
C-3 glycoside derivatives of 14-hydroxy-5b, 14b-pregnane show
strong interaction with cardiac glycoside receptor of heart muscle
[38]. Such compounds have been mainly obtained by multistep
chemical transformations of digitoxine, digitoxigenin and related
compounds that contain the 5b, 14b-configuration [38,39] (see
Scheme 5). Conversely, the 14a-hydroxylation of progesterone
[40] and other steroids by several microorganisms is a well-
documented fact and several fungi have been shown to introduce,
in reasonable yields, a hydroxyl group at the 14a-position of pro-
gesterone and some other steroids; the 14a-hydroxy configuration
can then be transformed chemically to the 14b-hydroxy substituent
normally found in cardioactive steroids. Microbial transformation
of progesterone (40) by Thamnostylum piriforme yielded 14a-
hydroxyprogesterone (41) and 9a-hydroxyprogesterone (42). A
similar preparation with Mucor griseocyanus afforded 41, 6b, 14a-
dihydroxyprogesterone (43) and 7a, 14a-dihydroxyprogesterone
(44) [41]. The filamentous fungus, Aspergillus fumigatous [42] effi-
ciently hydroxylated 40 producing 11a-hydroxyprogesterone
(45), 15b-hydroxyprogesterone (46), 7b-hydroxyprogesterone
(47), 7b–15b-dihydroxyprogesterone (48) and 11a–15b-dihydr-
oxyprogesterone (49) (Scheme 6). Incubation of progesterone (40)
with Saprolegnia hypogyna yielded 4-androstene-3, 17-dione (50),
testosterone (2) and testololactone (51) [43]. Transformation of
40 with Fusarium culmorum culture [44] yielded a mixture of 15a-
hydroxyprogesterone (52) and 12b–15a-dihydroxyprogesterone
(53) [Scheme 7]. However when 40 was fermented with thermo-
philic bacterium, Bacillus stearothermophilus [46], three monohydr-
oxylated metabolites and a new metabolite, were isolated and
identified. These were identified as 20a-hydroxyprogesterone
(54), 6b-hydroxyprogesterone (55), 6a-hydroxyprogesterone (56)
1270 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290
Mycobact er ium sp.
HO
18
My cobacterium sp.
HO
22
Scheme 3. Microbial transformation of ergosterol (18) and b-sitosterol (22) by Mycobacterium sp.
and 9, 10-sco-4-pregnene-3, 9, 20-trione (57) as shown in
Scheme 7.
Madyasth and Joseph [46] reported the biotransformations of
17a-hydroxyprogesterone (58) and 16-dehydroxyprogesterone
(66) with Mucor piriformis. The organism transforms 17a-hydroxy-
progesterone (58) into 17a, 20a-dihydroxypregn-4-en-3-one (59),
7a, 17a-dihydroxypregn-4-en-3, 20-dione (60), 6b, 17a, 20a-tri-
hydroxypregn-4-en-3-one (61) and 11a, 17a, 20a-trihydroxy-
pregn-4-en-3-one (62). However, incubation of 58 with F.
culmorum [44] yielded 6b, 17a-dihydroxyprogesterone (63) and
15a, 17a-dihydroxyprogesterone (64) (Scheme 8). Fermentation
of 65 by Mucor piriformis yielded 14a-hydroxypregna-4, 16-
dien-3, 20-dione (66), 7a, 14a-dihydroxypregna-4, 16-dien-3,
20-dione (67), 3b, 7a, 14a-trihydroxypregn-16-en-20-one (68)
and 3a, 7a, 14a-trihydroxypregn-16-en-20-one (69) as shown
in Scheme 9.
Pregnenolone (70) is a major hormone in human nerve tissues
and its therapeutic role in repairing defective neurons is well
documented [47]. Moreover, it is a precursor of many steroidal
hormones. Microbial transformation of pregnenolone (70) by
Mucor piriformis [48] resulted in the isolation of two metabolites,
3b, 7a-dihydroxypregn-5-en-20-one (71) and 3b, 7a, 11a-trihydr-
oxypregn-5-en-20-one (72). Fermentation of 70 with the fungus
Botrytis cinerea [49] yielded two new oxidized metabolites of preg-
nenolone. These were identified as 3b, 11a, 16b-trihydroxypregn-
5-en-20-one (73) and 11a, 16b-dihydroxypregn-4-en-3, 20-dione
(74). Metabolism of pregnenolone (70) by Bacillus strains [50,51]
COOCH 3
O COOCH 3
19
O COOH
20
O
21
O
23
CH2 OH
O
24
COOH
O
25
afforded 71, 3b, 7b-dihydroxypregn-5-en-20-one (75) and 7-oxo-
pregnenolone (76) as the major metabolites (Scheme 10). Choudh-
ary et al. [52] also investigated the metabolism of pregnenolone
(70) with different fungi. Incubation of 70 with Cunninghamella
elegans yielded metabolites, 3b, 7b, 11a-trihydroxypregn-5-en-
20-one (77), 3b, 6b, 11a-trihydroxypregn-4-en-20-one (78) and
3b, 6a, 11a, 12b, 15b-pentahydroxypregn-4-en-20-one (79), while
incubation with Gibberella fujikuroi afforded 77 and 6b, 15b-
dihydroxypregn-4-en-3, 20-dione (80). Similarly fermentation of
70 by Fusarium oxysporum var. cubense [52] yielded 71. These met-
abolic pathways are shown in Scheme 11.
Microbial transformation of 5b-pregnane-3, 20-dione (81) and
3b-hydroxy-5b-pregnane-20-one (85) were investigated by Hu et
al. [41]. Bioconversion of (81) by Thamnostylum piriforme yielded
14a-hydroxy-5b-pregnane-3, 20-dione (82), 14a, 15b-dihydroxy-
5b-pregnane-3, 20-dione (83) and 3b, 14a-dihydroxy-5b-preg-
nane-20-one (84). 3b-hydroxy-5b-pregnane-20-one (85) was
slowly transformed by T. piriforme yielding the metabolites, 3b–
14a-dihydroxy-5b-pregnane-20-one (86) and 3b, 9a, 14a-trihy-
droxy-5b-pregnane-20-one (87). However, incubation of 86 with
Actinomucor elegans resulted in the isolation of 87, 88 and a minor
metabolite, 3b, 9a-dihydroxy-5b-pregnane-20-one (88) are shown
in Scheme 12.
Wilson et al. [53] used Fusarium oxysporum var. cubense to
investigate the metabolism of 17a, 21-dihydroxypregn-4-ene-3,
11, 20-trione (89) and 17a, 21-dihydroxypregna-1, 4-diene-3, 11,
20-trione (92). Bioconversion of 89 with F. oxysporum var. cubense
 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1271

O
27

O
28 OH
CH2 OH

Mycobacterium sp.

HO O
29
26 COOCH 3

O
30

COOCH 3

O
31

OH CH 2OH

O
33
Mycobacterium sp. CH 2OH
O
HO

32
HO
34

Scheme 4. Microbial transformation of stigmasterol (26) and (32) by Mycobacterium sp.

yielded 17a, 20, 21-trihydroxypregn-4-ene-3, 11-dione (90) and
androst-4-ene-3, 11,17-trione (91), while incubation of 92 with F.
oxysporum var. cubense resulted in the isolation of metabolites,
17a, 20, 21-trihydroxypregna-1, 4-diene-3, 11-dione (93) and and-
rost-1, 4-diene-3, 11, 17-trione (94), respectively (Scheme 13).
13-Ethyl-17b-hydroxy-11-methylene-18, 19-dinor-17a-pregn-
4-en-20-yne (desogestrel) and its 3-oxo derivatives act as powerful
progestogens and are used as oral contraceptives. Such derivatives
are mainly obtained by multistep chemical transformations of
estrane derivatives [54]. 13-Ethyl-17b-hydroxy-18, 19-dinor-17a-
pregn-4-en-20-yn-3-one (95) a less potent progestogens, is com-
mercially available and inexpensive. The microbial transformation
of a racemic mixture of 95 was investigated using the fungi, Rhizo-
pus nigricans, R. arrhizus, Aspergillus niger, A. ochraceus and Curvu-
laria lanata [55]. Bioconversion of 95 by R. arrhizus gave a major
product, (±)-13-ethyl-10b, 17b-dihydroxy-18, 19-dinor-17a-
pregn-4-en-20-yn-3-one (96). Transformation of 95 by R. nigricans,
A. niger and C. lunata was slow and inefficient and also afforded the
metabolite 96. Compound 95 was slowly metabolized by A. ochrac-
eus affording a single metabolite, (±)-13-ethyl-7b, 17b-dihydroxy-
18, 19-dinor-17a-pregn-4-en-20-yn-3-one (97). All these fungi
were unable to differentiate the two enantiomers of 95 in the
course of these hydroxylation reactions. The microbial transforma-
tion of the dl and d-enantiomers of 95 were also reported using
Cunninghamella species [56]. Transformation of 97 by Cunningham-
ella blakesleeana yielded 96, 97, 13-ethyl-6b, 17b-dihydroxy-18,
19-dinor-17a-pregn-4-en-20-yn-3-one (98), 13-ethyl-15a, 17b-
dihydroxy-18, 19-dinor-17a-pregn-4-en-20-yn-3-one (99) and
13-ethyl-6b, 10b, 17b-trihydroxy-18, 19-dinor-17a-pregn-4-en-
20-yn-3-one (100). These transformation reactions of 95 are
shown in Scheme 14.
Danazol (17b-hydroxy-17a-pregna-2, 4-dien-20-yno-[2,3-d]
isoxazole (101) is an orally effective, pituitary gonadotropin inhib-
itor which is effectively used in the treatment of endometriosis, be-
nign fibrocystic mastitis and precocious puberty [57,58].
Choudhary et al. [59] used four different fungi and bacteria to
study the transformation of danazol (101). Fermentation of dana-
zol (17b-hydroxy-17a-pregna-2, 4-dien-20-yno-[2,3-d] isoxazole
(101) with Fusarium lini, Aspergillus niger and Cephalosporium
aphidicola afforded 17b-hydroxy-2-(hydroxymethyl)-17a-pregn-
4-en-20-yn-3-one (102) and 17b-hydroxy-2-(hydroxymethyl)-
17a-pregn-1,4-dien-20-yn-3-one (103), while the fermentation of
1272 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290

O
O

36

O
My cobacter ium sp.
HO 37 O

35

O
38
OH COOMe

O
39

Scheme 5. Microbial transformation of 8a, 9a-epoxytetrahydrolanosterol (32) and lanosta 7, 9 (11)-dien-3b-ol (35).

O
O O

T. pir if or me
+ OH
OH
O O O
40 41 42

O
O O

M. griseocy anus
41 +
+ OH
OH
O O OH
O
40 44
43 OH

O
O O
HO

+ +
O
OH O OH
O O 47
45 46
A.f umigat ous
O
O O
HO
40

+ OH
OH
O
O OH 49
48

Scheme 6. Microbial transformation of progesterone (40) by T. piriforme, M. griseocyanus and A. fumigatous.

101 with Bacillus cerus yielded the metabolite 102 only as de-
scribed in Scheme 15.
Testosterone (17b-hydroxyandrost-4-en-3-one) (2) is a male
sex hormone responsible for the development of secondary male
characteristics. The hydroxylation of steroids is important because
of its physiological role in mammalian organisms and also for prac-
tical reasons [2]. Testosterone (2) was actively metabolized by M.
griseocyanus and T. piriforme [41]. M. griseocyanus produced a
major metabolite, 14a, 17b-dihydroxyandrost-4-en-3-one (104)
and 14a-hydroxyandrost-4-en-3, 17-dione (105). Conversely, T.
piriforme produced lower amounts of 14a-hydroxylated deriva-
tives and yielded 104, 6b, 17b-dihydroxyandrost-4-en-3-one
(106), 9a, 17b-dihydroxyandrost-4-en-3-one (107) and 6b-
hydroxyandrost-4-en-3, 17-dione (108). Microbial transformation
of testosterone (2) by Nectria haematococca [59] afforded 1-ene-
testosterone (109) and 11a-hydroxy-1-ene-testosterone (110)
(Scheme 16). Incubation of 2 with Fusarium culmorum [45] yielded
106 and 15a-hydroxytestosterone (111) as the major metabolites.
 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1273

O OH O O

H
S.hy pogy na +
+ H H
40 O
O O 51
50 2

O OH O

F.Culmorum +
OH OH
40 O
O
52 53

OH O

O O
55
54 OH
B.st earothermophilus
40
O
O
O CH3
CH 3
+ H 3C
O
OH 57
56

Scheme 7. Microbial transformation of progesterone (40) by S. hypogyna, F. culmorum and B. stearothermophilus.

O
OH OH
OH

+
O OH
O O
OH 59 60

M.pir if or mis OH
OH
HO OH
O OH

58 +

O
O
61
OH 62

O O O
OH OH OH

F.culmorum +
OH
O O O
OH
58 63 64

Scheme 8. Microbial transformation of 17a-hydroxyprogesterone (58).

The fungus Cephalosporium aphidicola [60] hydroxylated testoster-
one (2) to metabolites 104 and 106. The metabolites 106, 111, and-
rost-4-ene-3, 17-dione (112) and 15a-hydroxyandrost-4-ene-3,
17-dione (113) were obtained when compound 2 was metabolized
with the fungus F. oxysporum var. cubense [52], while the fungi
Curvularia lunata, Pleurotus oestreatus [61] yielded only the
metabolites 112 and 113. The plant pathogenic fungus Botrytis
cinerea [62] yielded only the metabolite 7b, 17b-dihydroxyand-
rost-3-one (114). Biotransformation of testosterone (104) by
Bacillus stearothermophilus [63] resulted in the formation of 106,
1274 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290

O
O

+ OH
O OH O OH
O 67
66 O
M.pir if or mis O HO

O
65 +
OH
OH HO OH
HO OH 69
68

Scheme 9. Microbial transformation of 16-dehydroxyprogesterone (65).

O
O O
HO

M.pirif or mis +

HO OH
HO HO OH 72
70 71
O
O O
HO
HO OH
OH

B.ciner ea +
O
HO HO 74
70 73

O
O O

+
Bacillus sp. 72 +
HO O
HO HO OH 76
70 75

Scheme 10. Microbial transformations of pregnenolone (70) M. piriformus, B. cinerea and Bacillus sp.

O O
HO HO

O +
HO OH HO
77 78 OH
C.elegans
OH O
HO HO
70
+
OH
HO
79 OH

O O

G.fujikuroi 75 + OH
HO O
70 80 OH

Scheme 11. Microbial transformations of pregnenolone (70) C. elegans and G. fujikuroi.

 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1275

O O

O
+
OH OH OH
O
T.pirif or me H O
H 83
82 O
O
H
81

HO
HO
H 84

O O O

T .pir if or me +
OH OH OH
HO HO HO
H H H
85 86 87

O
O

A .elegans
86 + 87 +
OH
HO
H 85 HO
H 88

Scheme 12. Microbial transformations of 5b-pregnane-3, 20-dione (81) and 3b-hydroxy-5b-pregnane-20-one (85).

HO HO
O OH
O
OH O OH O
O

F.oxy spor um +

89
O O 90 O 91

HO HO
O OH

OH O
O OH O O

F.oxysporum +

O O O
92 94
93

Scheme 13. Microbial transformations of 17a, 21-dihydroxypregn-4-ene-3, 11, 20-trione (89) and 17a, 21-dihydroxypregna-1, 4-diene-3, 11, 20-trione (92).

112, 6a, 17b-dihydroxyandrost-4-en-3-one (115), 6b-hydroxyand-
rost-4-en-3, 17-dione (108) and 6a-hydroxyandrost-4-en-3, 17-
dione (116). These biotransformations are shown in Scheme 17.
Madyastha [64] studied the biotransformation of androstenedi-
one (androst-4-ene-3, 17-dione) (112) using the fungus Mucor pir-
iformis. The organism transformed 112 into testosterone (2), 14a,
17b-dihydroxyandrost-4-en-3-one (104), 14a-hydroxyandrost-4-
en-3, 17-dione (105), 7a-hydroxyandrost-4-en-3, 17-dione (117),
and 7a, 14a, 17b-trihydroxyandrost-4-en-3-one (118). When 112
was fermented with T. piriforme [41], it gave testosterone (2),
14a-hydroxyandrost-4-en-3, 17-dione (105), 6b-hydroxyandrost-
4-en-3, 17-dione (108) 7a-hydroxyandrost-4-en-3, 17-dione
(117) and 7a, 14a, 17b-trihydroxyandrost-4-en-3-one (118). The
9a-hydroxylated steroids are valuable intermediates in the
synthesis of highly effective anti-inflammatory pharmaceuticals.
Microbial hydroxylation of 112 into 9a-hydroxyandrost-4-en-3,
17-dione (119) was investigated by Angelova et al. [65] in nutrient
deficient buffer medium by resting Rhodococcus sp. Incubation of
112 with Bacillus strain HA–V6-3 [66] yielded the metabolites, 2,
105, 106, 108, 117 6b, 14a-dihydroxyandrost-4-en-3, 17-dione
(120), 11a-hydroxyandrost-4-en-3, 17-dione (121), 4-androsten-
3, 6,17-trione (122) and 5a-androst-3, 6, 17-trione (123) (Scheme
18). Bioconversion of 112 by C. aphidicola [60] gave 105 and 108.
Fermentation of 112 with Curvularia lunata [67] yielded 109, 121,
androsta-1,4-diene-3,17-dione (124), 11a,17b-dihydroxyandrost-
4-en-3-one (125) and 15a-hydroxyandrosta-1,4-dien-3,17-dione
(126). Microbial transformation of 112 by the fungus Beauveria
bassiana [68] has been investigated using pH 6.0 and 7.0 media.
Two metabolites were obtained with the pH 6.0 medium and iden-
tified as 125 and 6b, 11a-dihydroxyandrost-4-ene-3, 17-dione
(127). On the other hand, four hydroxylated and/or reduced
metabolites were obtained with the pH 7.0 medium, viz. 2, 125,
1276 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290

HO CH
OH C
C CH
OH
H R.arrhizus

O
O 95
96
HO CH
OH C
C CH H
H A.ochraceus

O OH
O 95 97
HO CH
HO CH C
C
H
H
OH + OH
C CH O 99
H O 98
OH
C.blakesleeana
96 + 97 + HO CH
O 95 C
OH

+
O 100
OH

Scheme 14. Microbial transformations of 13-ethyl-17b-hydroxy-18, 19-dinor-17a-pregn-4-en-20-yn-3-one (95).

HO CH HO CH
C C HO CH
C
OH H
HOH2 C
H
N HOH2 C
O O +
101 O
102 103

Scheme 15. Microbial transformations of danazol (101).

OH O
OH

M.griseocynas
+
OH OH
O O
O 104 105
2

OH OH O
OH

T.piriforme OH +
+
O O O
O 2 106 OH 107 OH 108

OH OH
OH
HO
N.haematococca
+

O O
O 110
2 109

Scheme 16. Microbial transformations of testosterone (2) by M. griseocyanus, T. piriforme and N. haematococca.
 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1277

OH
OH

F.culmorum
106 +
OH
O 111
O 2
OH O O

F.oxy spor um
106 + 111 + +
OH
O 2 O O
112 113

OH OH

B.cinerea

O 2 O OH
114
OH O
OH

B.stear other mophilus

106 + 108 + 112 + +
O O
O
2 115 OH OH 116

Scheme 17. Microbial transformations of testosterone (2) by F. culmorum, F. oxysporum, B. cinerea and B. stearithermophilus.

O O OH

M. pirif ormis
2 + 104 +105 + + OH
O 112 O O
OH OH
117 118
O
O

Rhodococcus sp.
O OH
O
112 119
O
O

+
O OH
O
O
O 122
Bacillus sp. OH 120
2 + 105 + 106 + 108 + 117
O O
O HO

112
+

O O
121 H 123
O

Scheme 18. Microbial transformations of androst-4-ene-3, 17-dione (112) by M. piriformis, T. piriforme, Rhodococcus sp. and Bacillus strain.

6b, 11a, 17b-trihydroxyandrost-4-ene-3-one (130) and 3a, 11a,
17b-trihydroxy-5a-androstane (129). These biotransformation
reactions are shown in Scheme 19.
Kolek and Świzder [44] investigated the biotransformation of
androst-4-en-3-one (130) with the fungus F. culmorum. The fungus
F. culmorum fermented the compound 130 and yielded 15a-
hydroxyandrost-4-en-3, 17-dione (113) and 12b, 15a-dihydrox-
yantrost-4-en-3-one (131). However, fermentation of 130 with C.
aphidicola [60] afforded the metabolites, 6b, 17b-dihydroxyan-
trost-4-en-3-one (106) and 6b, 11a-dihydroxyantrost-4-en-3-one
(132) as described in Scheme 20.
Microbial reactions are unique in their diversity and impor-
tance. They offer access to otherwise inaccessible sites in the
natural as well as synthetic products. Fermentation of (+)- andro-
sta-1,4-diene-3, 17-dione (124) with Cephalpsporium aphidicola
[69] for 8 days afforded oxidative and reductive metabolites. These
metabolites were identified as 1-ene- testosterone (109), androst-
4-ene-3, 17-dione (112), 11a-hydroxyandrosta-1, 4-diene-3,
1278 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290

O O OH
HO
C.lunat a
109 + 121 + + +
O 112 O O
124 O 125

OH
O
126

O
HO

pH 6
O 125 +
O
127
OH
B. bassiana
OH OH
O HO HO
112
pH 7
2 + 125 + +
O HO
H
128 OH 129

Scheme 19. Microbial transformations of androst-4-ene-3, 17-dione (112) by C. lunata and Beauveria bassiana.

O OH

F. culmor um
+
OH OH
O O O
130 113 131

HO

C. aphidicola
106 +

O O
132
130 OH

Scheme 20. Microbial transformations of androst-4-en-3-one (130).

O
O O
HO HO

C. aphidicola
109 + 110 + 112 + 125 +
+
O O
124 O
133 134

Scheme 21. Microbial transformations of (+)-androsta-1, 4-diene-3, 17-dione (124).

17-dione (133)ß 11a-hydroxyandrost-4-ene-3, 17-dione (134)ß
11a, 17b-dihydroxyandrost-4-ene-3, 7-dione (125) and 11a, 17b-
dihydroxyandrosta-1, 4-diene-3ione (110) (Scheme 21).
Dehydroepiandrosterone (3b-hydroxyandrost-5-en-17-one)
(135) is the source of all sex and steroid hormones in the body.
Low level of this compound is associated with the risk of pre-
menopausal breast cancer and hence this compound has clinical
importance regarding inhibition of mammary cancer. Incubation
of 135 with the fungus M. piriformis [48] resulted in the produc-
tion of 5 metabolites, 3b, 17b-dihydroxyandrost-5-ene (136), 3b,
7a-dihydroxyandrost-5-ene-17-one (137), 3b-hydroxyandrost-5-
ene-7, 17-dione (138), 3b, 17b-dihydroxyandrost-5-ene-7-one
(139) and 3b, 7a, 17b-trihydroxyandrost-5-ene (140). Bioconver-
sion of 135 was also carried out with plant pathogen Rhizopus
stolonifer [70], which afforded 7 metabolites. These metabolites
were identified as 3b, 17b-dihydroxyandrost-5-ene (136), 3b,
17b-dihydroxyandrost-4-ene (141), 17b-hydroxyandrost-4-ene-3-
one (2), 3b, 11b-dihydroxyandrost-4-ene-17-one (142), 3b,
7a-dihydroxyandrost-5-ene-17-one (137), 3b, 7a, 17b-trihydroxy-
androst-5-ene (140) and 11b-hydroxyandrost-4, 6-diene-3, 17-one
(143). Microbial transformation of 3b-hydroxyandrost-5-en-17-
one (135) by Fusarium oxysporum and Colletotrichum musae [53]
yielded 136, 137, 138 and 140 respectively. These biotransforma-
tions are shown in Scheme 22.
 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1279

OH O O

O +
+
HO HO OH HO O
136 137 138
M. pir if or mis
HO OH OH
135

+
HO O HO OH
139 140

O
OH O O
HO HO

R. stolonif er
2 +136 +137 + 140 + + +
HO
135 HO HO O
141 142 143

Scheme 22. Microbial transformations of dehydroepiandrosterone (135).

HO OH

O 149
C. aphidicola

HO
OH
OH 150
la
C. co
HO OH ap id i
h id ph
148 ico O C .a O
la

C. aphidicola OH

a 144 OH 145 O
ol
C

c
di
.a

hi
ph

ap
id

.
i

C
co
la

O
O

OH
OH
OH HO
147 146

O O

HO
C. aphidicola
145 +

151 O 152 O
O O

C. aphidicola
148 +
OH
153 154 OH

Scheme 23. Microbial transformations of 6b-hydroxy-3a, 5-cycloandrostan-17-one (144), 3a, 5-cycloandrostane-6, 17-dione (151) and 3a, 5-cycloandrost-6-en-17-one
(153).
1280 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290
Microbial hydroxylation of steroids provides a useful mild
synthetic method for obtaining access to rare steroids [71]. The
cyclopropane ring of the 3a-5-cyclosteroids is sensitive to a variety
of reagents limiting many chemical reactions. The fission of a
cyclopropane ring in the presence of an adjacent radical has
provided a probe for enzyme mechanism [72]. Incubation of 6b-hy-
droxy-3a, 5-cycloandrostan-17-one (145) with C. aphidicola [73]
for 8 days yielded six metabolites that were identified as
14a-hydroxy-3a,5-cycloandrostane-6, 17-dione (145), 3b, 14a-
dihydroxyandrost-5-en-17-one (146), 6b, 14a-dihydroxy-3a,
5-cycloandrostan-17-one (147), 3b, 7a-dihydroxyandrost-5-en-
17-one (148), 3b, 7b-dihydroxyandrost-5-en-17-one (149) and
3b, 5a, 6b-trihydroxyandrostan-17-one (150) (Scheme 23). Simi-
larly, incubation of 3a, 5-cycloandrostane-6, 17-dione (151) with
C. aphidicola [73] yielded 145 and 2a-hydroxy-3a, 5-cycloandros-
tane-6, 17-dione (152), while 3a, 5-cycloandrost-6-en-17-one
(153) afforded 148 and 6b, 7a-dihydroxy-3a, 5-cycloandrostane-
17-one (154) with the same fungus [73]. These microbial conver-
sions are shown in Scheme 23.
Regiospecificity of the microbial hydroxylation of steroids has
been rationalized in terms of the location of existing hydroxyl
and carbonyl groups on the carbon skeleton, which may act as
binding groups. These groups serve to orient the steroid within
the hydroxylase and place a CAH bond close to the oxidant co-
enzyme [74]. The position of the hydroxylation on ring C appeared
to be affected by the ring D substituent. Hanson and Kiran [75]
investigated this effect by fermenting 17-chloroandrosta-4, 16-
dien-3-one (155) with the fungus C. aphidicola. Incubation of
compound 155 with C. aphidicola for 10 days gave three metabo-
lites. The metabolites were identified as 17-chloro-6b, 11a-
dihydroxyandrosta-4, 16-dien-3-one (156), 17-chloro-6b, 15b-
dihydroxyandrosta-4, 16-dien-3-one (157) and 17-chloro-6b, 11a,
15b-trihydroxyandrosta-4, 16-dien-3-one (158) (Scheme 24).
Microbial transformation of ethisterone (17a-ethynyl-17b-
hydroxyandrost-4-en-3-one) (159) and 17a-ethyl-17b-hydroxy-
androst-4-en-3-one) (162) by the fungi Cephalosporium aphidicola
and Cunninghamella elegans were reported by Choudhary et al.
[76]. Fermentation of compound 159 with C. aphidicola yielded
the metabolite, 17a-ethynyl-17b-hydroxyandrosta-1,4-dien-3-
one) (160), while with C. elegans afforded 17a-ethynyl-11a,17b-
dihydroxyandrost-4-en-3-one (161). On the other hand, compound
162 with fungus C. aphidicola afforded 17a-ethyl-17b-hydroxyan-
drosta-1,4-dien-3-one (164). However, when compound 162 was
incubated with C. elegans, two new metabolites, 17a-ethyl-11a,
17b-dihydroxyandrost-4-en-3-one (164) and 17a-ethyl-6a, 17b-
dihydroxy-5a-androstan-3-one (165) as reported in Scheme 25.
Adrenosterone (166) is used as estrogen synthetase inhibitor,
which has potential clinical importance for managing the estro-
gen-mediated events such as ovulation and the growth of estrogen
dependent tumors [77,78]. Musharraf et al. [79] reported the
HO
Cl
O O
C. aphidicola
156
O HO
155
O
Scheme 24. Microbial transformations of 17-chloroandrosta-4, 16-dien-3-one (155).
fungal transformation of 166 by different fungi. Fermentation of
166 by Cephalosporium aphidicola resulted in the formation of
androsta-1, 4-dien-3, 11, 17-trione (167), 17b-hydroxyandrost-
4-en-3, 11-dione (168) and 17b-hydroxyandrosta-1, 4-dien-3,
11-dione (169). Two other fungal species Fusarium lini and
Trichothecium roseum were also tested for biotransformations of
adrenosterone (166). Metabolites 167 and 169 were obtained with
Fusarium lini, while metabolite 169 was produced using Trichothe-
cium roseum. The structures of these metabolites are shown in
Scheme 26.
Mesterolone ((1a, 5a, 17b)-17-hydroxy-1-methylandrostan-3-
one) (170) is a steroidal drug used in the treatment of conditions
caused by deficient endogenous androgen formation, which can
develop gradually with increasing age. Choudhary et al. [80,81]
investigated the microbial transformations of mesterolone (170)
using different fungi. Metabolites (1a, 5a)-1-methylandrostane-3,
17-dione (171), (1a, 3b, 5a, 17b)-1-methylandrostane-3, 17-diol
(172) and (1a, 5a, 15a)-15-hydroxy-1-methylandrostane-3, 17-
dione (174) were produced by Cephalosporium aphidicola, while
metabolites, (5a)-1-methylandrost-1-en-3, 17-dione (173), 174,
(1a, 5a, 6a, 17b)-6, 17-dihydroxy-1-methylandrostan-3-one
(175), (1a, 5a, 15a, 17b)-15, 17-dihydroxy-1-methylandrostan-3-
one (178) and (5a, 15a, 17b)-15, 17-dihydroxy-1-methylandrost-
1-en-3-one (179) were produced by Fusarium lini. Similarly,
Rhizopus stolonifer yielded metabolites 171, 175, (1a, 5a, 7a,
17b)-7, 17-dihydroxy-1-methylandrostan-3-one (176), (1a, 5a,
11a, 17b)-11, 17-dihydroxy-1-methylandrostan-3-one (177) and
179. These transformations are shown in Scheme 27.
Biotransformation of 3-hydroxyestra-1, 3, 5 (10)-trien-17-one
(180) was investigated with the fungus Fusarium oxysporum var.
cubense [53]. 3-Hydroxyestra-1, 3, 5 (10)-trien-17-one (180) was
biotransformed to 3, 17b-dihydroxyestra-1, 3, 5 (10)-triene (181)
and 3, 15a-dihydroxyestra-1, 3, 5 (10)-triene (182). (Scheme 28).
Mestranol (183) is an oral contraceptive and widely used in com-
bination with other estrogens and progestogens in antifertility
medicines. Incubation of mestranol (183) with Cunninghamella ele-
gans [82] yielded two hydroxylated metabolites, 6b-hydroxymestr-
anol (184) and 6b, 12b-dihydroxymestranol (185) (Scheme 28).
Northisterone (17a-ethynyl-19-nortestosterone) (186) is a po-
tent progestogen, widely used in oral contraceptive pills, as an
antifertility agent. It has a wide variety of uses including the delay
in menstruation and treatment of other menstrual disorder such as
endometriosis. Fungal transformation of nothisterone (186) by
Cephalosporium aphidicola [83] yielded an oxidized metabolite,
17a-ethynylestradiol (187), while the fungal transformation of
187 by Cunninghamella elegans afforded several metabolites, 19-
nor-17a-pregna-1, 3, 5 (10)-trien-20-yne-3, 4, 17b-triol (188),
19-nor-17a-pregna-1, 3, 5 (10)-trien-20-yne-3, 7a, 17b-triol
(189), 19-nor-17a-pregna-1, 3, 5 (10)-trien-20-yne-3, 11a, 17b-
triol (190), 19-nor-17a-pregna-1, 3, 5 (10)-trien-20-yne-3, 6b,
Cl Cl
OH
OH 157 OH
Cl
OH
OH 158
 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1281

OH CH OH CH
C C OH CH
C

C. elegans C. aphidicola
O O
O
161 159
160
OH OH
C 2 H5 C 2H 5

C. aphidicola
O O
162 163

OH OH OH
C 2H 5 C 2H 5 C2 H 5
HO

C. elegans
+
O O O
162 164
H 165
OH

Scheme 25. Microbial transformations of 17a-ethynyl-17b-hydroxyandrost-4-en-3-one (159) and 17a-ethyl-17b-hydroxyandrost-4-en-3-one) (162).

O
O OH
O
O
O
+
O
C. aphidicola O
167 168
O OH
O
166

O
169

Scheme 26. Microbial transformations of adrenosterone (166).

OH
OH

O
H
171
172
OH HO
O H
H
179 c) O
a), c)
a)
,b

OH
)

OH
a),b)
b) O
H 173

O b) O
OH 170 ,c)
H
O a)
H 178
a),

a)
b)

OH
OH
HO
O
OH H 174
OH

O
H 177

O OH 175
H O
176 H
OH

Scheme 27. Microbial transformations of mesterolone (170) by (a) R. stolonifer, (b) F. lini, and (c) C. aphodicola.
1282 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290

O OH O

F. oxy spor um +
OH
HO HO HO
181 182
180
OH OH OH OH

C.elegans +
MeO MeO MeO
183 OH 185 OH
184

Scheme 28. Microbial transformations of 3-hydroxyestra-1, 3, 5 (10)-trien-17-one (180) and mestranol (183).

OH 11b, 17b-dihydroxyandrost-4-en-3-one (194), 11b, 17a, 20b, 21-

tetrahydroxypregn-4-en-3-one (195) and 21-acetoxy-17b, 17a,
H 20-trihydroxypregn-4-en-3-one (196) (Scheme 30). Prednisone
(17a, 21-dihydroxypregna-1, 4-diene-3, 11, 20-trione) (197) is
O commonly used in the treatment of asthma, rheumatic disorders,
renal disorders and diseases of inflammatory bowel, skin. gastroin-
C. aphid icola

186 testinal tract [85]. Microbial transformation of prednisone (197) by

Cunninghamella elegans [86] afforded two metabolites, 17a, 21-
dihydroxy-5a-pregn-1-ene-3, 11, 20-trione (198) and 17a, 20S,
OH
21-trihydroxy-5a-pregn-1-ene-3, 11-dione (199), while fermenta-
tion of 197 by Fusarium lini, Rhizopus stolonifer and Curvularia
lunata yielded a metabolite 1, 4-pregnadiene-17a, 20S, 21-triol-
3,11-dione (200) (Scheme 31).
HO
187 Biotransformations of steroids have proliferated and specific
C. elegans

microbial transformation steps have been incorporated into

numerous partial syntheses of new steroids for evaluation as
drugs and hormones. A variety of steroids are widely used as
diuretic, anabolic, contraceptive, anti-androgenic, anti-inflamma-
tory, anti-cancerous agents as well as in other applications. Corti-
OH sol (201) is an important anabolic hormone clinically used in
topical anti-inflammatory and antiallergic medicines. It is also
used in the synthesis of many steroidal hormones. Fermentation
OH
of cortisol (201) with Gibberella fujikuruoi [87] yielded 11b-
HO hydroxyandrost-4-en-3, 17-dione (202), while incubation with
188 Bacillus subtilis and Rhizopus stolonifer afforded 11b, 17a, 20, 21-
OH
+ OH tetrahydroxy- (20S)-pregnt-4-en-3-one (203). The metabolites
HO
11b, 17a, 21-trihydroxy-5a-pregnan-3, 20-dione (204) and 3b,
191 OH
11b, 17a, 21-tetrahydroxy-5a-pregnan-20-one (205) were ob-
+ tained by fermentation of 201 with Bacillus cerus. These microbial
OH
HO OH transformations of compound 201 are shown in Scheme 32.
The microbial hydroxylation of steroids has been rationalized in
189 +
OH
terms of a triangular relationship between two binding sites on the
HO steroid framework and the site of hydroxylation [74]. The dimen-
HO
sions of this triangle vary with the organism but typically the bind-
OMe
192 ing sites might be C-3 and C-17 with hydroxylation taking place at
C-6b and C-11a. The binding groups are typically hydroxyl or
HO
190 carbonyl groups. Kiran et al. [88] studied the effect of replacing a
hydroxyl group by a methoxyl group on the microbial hydroxyl-
Scheme 29. Microbial transformations of nothisterone (186). ation of some steroids by the fungus, Cephalosporium aphidicola.
Incubation of 17b-methoxy-5a-androstan-3-one (206) with C.
aphidicola yielded the metabolites, 17b-methoxy-5a-androstan-
17b-triol (191) and 19-nor-17a-pregna-1, 3, 5 (10)-trien-20-yne-3, 3b-ol (207) and 6b, 11a-dihydroxy-17b-methoxy-5a-androstan-
17b-diol-6b-methoxy (192). These biotransformations are shown 3-one (208). Incubation of 17b-methoxyestra-4-en-3-one (209)
in Scheme 29. with C. aphidicola gave one major metabolite, 6b-hydroxy-
Microorganisms have been widely applied for steroid biotrans- 17b-methoxyestra-4-en-3-one (210). Similarly, fermentation of
formations to prepare derivatives, which are difficult to prepare, by 3b-methoxyandrostan-5-en-17-one (211) gave a mixture of
chemical methods. Faramarzi et al. [84] used Acremonium strictum 7a-hydroxy-3b-methoxyandrostan-5-en-17-one (212) and 7b-hy-
to transform hydrocortisone (193) into its derivatives. The droxy-3b-methoxyandrostan-5-en-17-one (213) as reported in
fermentation of compound 193 by Acremonium strictum afforded Scheme 33.
 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1283

HOH 2C CHOH
OH OH
HO HO
CH2 OH
C=O
HO OH
O O 195
194
A. str ictum
AcOH 2C CHOH
O
193 HO OH

O
196

Scheme 30. Microbial transformations of hydrocortisone (193).

HOH2 C O OH
O HOH2 C HOH2 C
O OH O OH OH
O

C. elegans
+
O O
197 H O
198 H 199
F. lini
C. lunata
R. stolonif er

OH
HOH2 C

O OH

O
200

Scheme 31. Microbial transformations of prednisone (197).

OH O
HOH2 C HOH 2C
O
OH HO OH
HO HO
B.subtilus
R.stolonifer G.fujikuruoi

O O O
203 201 202
B. ceru s

O O
HOH2 C HOH2 C

OH HO OH
HO
+

O HO
H H

205
204

Scheme 32. Microbial transformations of cortisol (201).

4. Microbial transformations of miscellaneous steroids
E-Guggulsterone (214), a pregnane-type steroid, has been iso-
lated from the gums of Ailanthus grandis [89] and Commiphora mu-
kul [90]. The gum resin of C. mukul has been used in the treatment
of epilepsy, ulcers, helminthus, rheumatoid arthritis and hyperlipe-
mia in the ancient Indian system of medicine. It also exhibited
cytotoxic activity [91] against the human lung cancer cell line
(NCI-H-226). Bioconversion of E-guggulsterone (pregna-4, 17
(20)-cis-diene-3, 16-dione) (214) by Aspergillus niger [92] resulted
1284 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290

OMe OMe OMe

HO

C. aphidicola
+
O HO O
H H H
206 207 208 OH
OMe OMe

H H
C. aphidicola

O HO
209 210 OH
O O O

C. aphidicola
+
MeO MeO OH MeO OH
211 212 213

Scheme 33. Microbial transformations of 17b-methoxy-5a-androstan-3-one (206), 17b-methoxyestra-4-en-3-one (209) and 3b-methoxyandrostan-5-en-17-one (211).

7b-hydroxypregna-4, 17 (20)-cis-diene-3, 16-dione) (216), 7b-

hydroxypregn-4-ene-3, 16-dione) (217) and 7b, 15b-dihydroxy-
O pregn-4-ene-3, 16-dione) (218). The biotransformation of 214 with
Cephalosporium aphidicola [92] afforded 11a-hydroxypregna-4, 17
O
(20)-trans-diene-3, 16-dione) (219), 11a-hydroxypregna-4, 17
214 (20)-cis-diene-3, 16-dione) (220), 11a, 15b-dihydroxypregna-4,
17 (20)-trans-diene-3, 16-dione) (221) and 11a, 15b-dihydroxy-
pregna-4, 17 (20)-cis-diene-3, 16-dione) (222) as described in
Scheme 34.
C. aphid icola

Fungal biotransformation of E-guggulsterone (214) was also

A. niger

investigated using the fungi like Rhizopus stolonifer, Fusarium lini,

Cunninghamella elegans or Gibberella fujikuroi [93]. Incubation of
compound 214 with R. stolonifer for 6 days yielded the five hydro-
xyl metabolites, 219, 220, 12a-hydroxypregn-4-ene-3, 16-dione
(223), (6b, 11a, 17Z)-6, 11-dihydroxypregna-4, 17-diene-3, 16-
HO
dione (224) and (6b, 11a, 17E)-6, 11-dihydroxypregna-4, 17-
O O
diene-3, 16-dione (225), and incubation with F. lini afforded
metabolite 220 in good yield. Fermentations of 214 by C. elegans
O O OH for 6 days yielded the metabolites, 219, 220, (6b, 11a)-6,
219 215 11-dihydroxypregn-4-ene-3, 16-dione (226) and (11a, 16b)-6,
11-dihydroxypregn-4-ene-3-one (227). Compound 214 was also
HO
fermented with the fungus G. fujikuori, which resulted in the isola-
O
O tion of the five hydroxylated metabolites 219, 220, (7b, 11a, 12a)-
7, 11, 12-trihydroxypregn-4-ene-3, 16-dione (228), (1b, 11a, 12b,
O OH
17Z)-1, 11, 12-trihydroxypregna-4, 17-diene-3, 16-dione (229)
O and (1b, 11a, 12b, 17E)-1, 11, 12-trihydroxypregna-4, 17-diene-3,
216
220 16-dione (230). Theses biotransformations are shown in Scheme
35.
HO
O Microbial hydroxylation of steroid is industrially important
O
since it involves the synthesis of many intermediates necessary
OH for the synthesis of many steroidal drugs. While every site in a ste-
O OH
O
217 roid molecule is accessible for microbial hydroxylation, the 11a-,
221
11b-, a15- and 16a-hydroxylations are accomplished now in the
HO steroid chemistry mainly by microbial transformations. Jekkel et
O O al. [94] investigated the microbial hydroxylation of 13 b-ethyl-4-
gonene-3, 17-dione (232). Incubation of compound 232 with Fusar-
OH OH
O O OH ium nivale afforded 15a-hydroxy-13 b-ethyl-4-gonene-3, 17-dione
222 218 (232) and 7b, 15a-dihydroxy-13 b-ethyl-4-gonene-3, 17-dione
(233). However, fermentation of 231 by Mortierella pusilla afforded
Scheme 34. Microbial transformations of E-guggulsterone (212) by A. niger and C.
232, 10b-hydroxy-13b-ethyl-4-gonene-3, 17-dione (234) and 6b-
aphidicola.
hydroxy-13b-ethyl-4-gonene-3, 17-dione (235) (Scheme 36).
Mexrenone (236) and canrenone (241) belong to steroid group
in the formation of four new hydroxylated metabolites identified containing a C-17 spirolactone side chain. They are diuretics and
as 7b-hydroxypregna-4, 17 (20)-trans-diene-3, 16-dione) (215), mineralocorticoid receptor antagonists, which have been used for
 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1285

OH OH
HO HO
OH OH
O O

O OH 219
O
230
229
220

)
,d
OH

c)
d) d)

,
a)
HO )
c ) ,d
O ), OH
,b
a)
O O

d)
O OH
a)
228
O
O 214 223
c)
a)
HO
HO
O
OH c) a)

O 224
O
OH
227
HO HO
O O

O O
226 OH OH
225

Scheme 35. Microbial transformations of E-guggulsterone (214) by (a) R. stolonifer, (b) F. lini, (c) C. elegans and (d) G. fujikuori.

O
O O

F. nivale + OH
OH O OH
O O 233
231 232
O
O O

OH
M. pusilla +
O
O O
235 OH
231 234

Scheme 36. Microbial transformations of 13 b-ethyl-4-gonene-3, 17-dione (231).

O O O
O R2 O O
R1

O COOCH 3 O COOCH 3 O COOCH3

236 R3 240
237: R1 =OH, R2 =R3 =H
O 238: R1 =R 3 =H, R 2=OH O
239: R1 =R 2 =H, R 3=OH
O O

OH
C. cassiicola

O O
241 242

Scheme 37. Microbial transformations of Mexrenone (236) and canrenone (241).

1286 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290

H H Biotransformation with whole cell cultures is extensively em-

O O O O
O O
O O
HO O H HO
O O
O O
Cl
R inflammatory, immunostimulating, immunomodolatory and insect
243: R= OH
244: R=H, 6,7
245: R=H
Scheme 38. Microbial transformations of physalin H (243).
the treatment of edematous states such as congestive heart
failure and for treatment of essential hypertension [95]. Preisig et
al. [96] reported the biotransformation results of 236 and 241.
Bioconversion of 236 by Beauveria bassiana yielded one major
monohydroxylated metabolite, 11a-hydroxymexrenone (237),
while fermentation of 236 by Mortierella isabellina afforded the
metabolites 12b-hydroxymexrenone (238) and 6b-hydroxymexre-
none (239) However, incubation of 236 with B. cyclo-oxydans gave
D1,2 mexrenone (240). The fermentation of canrenone (241) with
Corynespora cassiicola resulted in the isolation of 9a-hydroxycanre-
none (242) (Scheme 37).
O O
ployed as an industrial method to synthesize many steroidal drugs,
chiral building blocks and for modifications in natural products
O H
with potent biological activities. Physalin H (243) is a steroidal
lactone, isolated from Physalis angulata, exhibits various biological
activities including antitumor, antifungal, antimycobacterial, anti-
246
repellent [97–101]. Choudhary et al. [102] investigated the metab-
olism of physalin H (243) by Rhizopus stolonifer and Cunninghamella
elegans, targeting the leishmanicidal activity of the transformed
products. Incubation of physalin H (243) with Rhizopus stolonifer
yielded a new dehydrated metabolite, 6,7-dehydrophysalin H
(244). When compound 243 was fermented with Cunninghamella
elegans for 12 days, the metabolites, 6-deoxyphysalin H (245) and
isophysalin B (246) were obtained (Scheme 38).
Microbial catalysis is a useful biotechnology in studies on natu-
ral products, by which a diversity of novel or active compounds can
be obtained. Bufadienolides are steroids with a characteristic a-
pyrone ring at the C-17 position. They are a new type of natural
steroids isolated from the traditional Chinese drug Chan’Su
[103,104] and possess potent antitumor activities [83]. Moreover,
they have cadiotonic, blood pressure stimulating, antiviral and
local anesthetic activities [105]. Microbial transformation of the
cytotoxic steroid cinobufagin (247) by fungus Alternaria alternata

O
O

OH O

O
O OAc
OH
O
O
OH OH
O
250
OH O
A. alternata

HO
O 249
O
251 ta
na
er
A.

l t
.a O
al t

O O A
e
r na

O
O O
ta

OH
O
OAc
OAc OAc
A. alternat a A. alternat a O
O O HO
HO HO 248
H M.
252 247 sp
i no
su
A

s
.a

M. spinosus
lt

M.
er n

sp

248
ata

in o

O O
su

O
s

O
OH OH
O
OAc OH OAc
O

O
O OH HO
OAc
255
253
+ O
HO
254

Scheme 39. Microbial transformations of cinobufagin (247).

 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1287

O O O
O O O

R1 R3

OH OH OH R
HO R2 HO OH HO
H H H
256: R1 =R2 =R 3 =H 258 260: R=α -OH
257: R1 =R3 =H,R2 =OH 261: R= β -OH
259: R1 =OH, R2 =R3 =H O O
265: R1 =R2 =OH, R3 =H
266: R1 =R2 =H,R3 =OH O O
267: R1 =H, R 2 =R 3 =OH
OH
R2
R

OH OH
HO HO
R1 H
262: R1 =OH, R2 =H 264: R=H
263: R1 =H, R 2 =OH 268: R=OH
O O

O O

R1

C. blak esleana M. spinosus

256 OH R2
OH
HO R2 HO OH
H R1
268: R 1=OH, R 2 =H 271: R1 =H,R 2=OH
269: R 1=R 2=OH 272: R1 =OH,R 2=H

Scheme 40. Microbial transformations of bufalin (256).

O O

O O

R3
274: R1 =H,R 2 =H,R 3=OH
R2 275: R1 =H,R 3 =H,R 2=OH
R1 279: R1 = R2 =OH,R 3=H
284: R1 =R 2=H, R 3 = O-glc
O O
OH
HO HO
H 273 H O
Glc= HO
HO
O OH
O
O O
OH R1
R2
OH 276: R 1=R 2 =H
280: R 1=OH,R 2 =H
O O 281: R 1=H,R2 =OH
HO HO
H H
O O
277 O
O O
O
OH OH

OH

O O
O
HO HO HO
H OH H
H
283
278 282

Scheme 41. Microbial transformations of resibufogenin (273).

1288 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290
was investigated by Ye et al. [106]. Fermentation of compound 247
by A. alternata resulted in the isolation of six metabolites, 12b-hy-
droxy-cinobufagin (248), 12b-hydroxy-desacetylcinobufagin (249),
3-oxo-12b-hydroxy-cinobufagin (250), 3-oxo-12b-hydroxy-des-
acetylcinobufagin (251), 12-oxo-cinobufagin (252) and 3-oxo-
12a-hydroxyl cinobufagin (253). However, incubation of 247 with
Mucor spinosus [107] afforded 248, 1b-hydroxy cinobufagin (254)
and 1b, 12b-dihydroxy-cinobufagin (255) as described in Scheme
39.
Bufalin (256) is another major bufadienolide isolated from the
traditional Chinese drug Chan’Su [104]. Incubation of bufalin
(256) with Mucor spinosus [108] yielded 12 metabolites, 7b-hydro-
xyl bufalin (257), 3-epi-7b-hydroxyl bufalin (258), 1b-hydroxyl
bufalin (259), 15a-hydroxyl bufalin (260), 15b-hydroxyl bufalin
(261), telocinobufagin (262), 11b-hydroxyl bufalin (263), 12b-hy-
droxyl bufalin (264), 1b, 7b-dihydroxyl bufalin (265), 16a-hydroxyl
bufalin (266), 7b, 16a-dihydroxyl bufalin (267) and 1b, 12b-
dihydroxyl bufalin (268). The microbial transformation of bufalin
(269) was also carried out using two strains of filamentous fungi,
Cunninghamella blakesleana and Mucor spinosus [109]. Cunningham-
ella blakesleana catalyzed the specific hydroxylation of bufalin and
produced 12a-hydroxyl bufalin (269), 7b, 12a-dihydroxybufalin
(270), 7b-hydroxybufalin (257) and 12b-hydroxybufalin (264).
However, fermentation of compound 256 with Mucor spinosus
afforded 7b, 15a-dihydroxybufalin (271) and 5b, 7b-dihydroxybuf-
alin (272). These metabolized products are shown in Scheme 40.
Resibufogenin (271) is a typical bufadienolide isolated from the
traditional Chinese medicine Chansu, a product of the skin secre-
tions of local toads such as Bufo bufo gragarizans Cantor or Bufo
melanostictus Schneider [110]. It shows a wide range of activities
such as cardiotonic, antitumor, blood pressure stimulating and
immunoregulatory [111–114]. Microbial transformation of
resibufogenin (273) by Mucor subtilissimus [115] resulted in the
isolation of 11 metabolites, 12b-hydroxyresibufogenin (274),
11b-hydroxyresibufogenin (275), 16a-hydroxyresibufogenin
(276), 12b-hydroxy-3-epi-resibufogenin (277), 12a-hydroxyresi-
bufogenin (278), 1b, 11b-dihydroxyresibufogenin (279), 12b, 16a-
dihydroxyresibufogenin (280), 12a, 16a-dihydroxyresibufogenin
(281), 7a-hydroxyresibufogenin (282), 1b, 12b-dihydroxyresibuf-
ogenin (283) and resibufogenin 12-O-b-D-glucoside (284). These
biotransformations are reported in Scheme 41.
References
[1] Mensah-Nyagan AG, Meyer L, Schaeffer V, Kibaly C, Patte-Mensah C. Evidence
for a key role of steroids in the modulation of pain. Psychoneuroendocrinology
2009;34:169–77.
[2] Sedlaczek L. Biotransformations of steroids. Crit Rev Biotechnol
1988;7:187–236.
[3] Charney W, Herzong LH. Microbial transformations of steroids. New
York: Academic Press; 1976. pp. 5–73.
[4] Hogg JA. Steroids, the steroid community and Upjohn in perspective: a profile
of innovation. Steroids 1992;257:593–616.
[5] Mahato Mukherjee. Steroid transformations by microorganisms. Phytochemistry
1984;23:2131–54.
[6] Mahato SB, Mukherjee A. Steroid transformations by microorganisms-II.
Phytochemistry 1985;24:1403–21.
[7] Mahato SB, Banergee S, Mazumder I. Metabolism of progesterone by a Bacillus
species. J Chem Res Synop 1989;6:184–5.
[8] Ahmad S, Garg SK, Johri BN. Biotransformation of sterols: selective cleavage of
the side chain. Biotechnol Adv 1992;10:1–67.
[9] Mahato SB, Mazumder I. Current trends in microbial steroid biotransformation.
Phytochemistry 1995;34:883–98.
[10] Mahato SB, Garai S. Advances in microbial steroid biotransformation. Steroids
1997;62:332–45.
[11] Fernandes P, Cruz A, Angelova B, Pinheiro HM, Cabral JMS. Microbial
conversion of steroid compounds: recent developments. Enzyme Microb
Technol 2003;32:688–705.
[12] Tuba Z, Barden CW, Dancsi A, Francsics-Czinege E, Molnar EC, Csorgei J, Falkay
G, Koide SS, Kumar N, Sundaram K, Dukat-Abrok V, Balogh G. Synthesis and
biological activity of a new progestogen, 16-methylene-17alpha-hydroxy-18-
methyl-19-norpregn-4-ene-3, 20-dione acetate. Steroids 2000;65:266–74.
[13] Ko D, Heiman AS, Chen M, Lee HJ. New steroidal anti-inflammatory
antedrugs: methyl 21-desoxy-21-chloro-11b,17a-dihydroxy-3,20-dioxo-1,4-
pregnadiene-16a-carboxylate, methyl 21-desoxy-21-chloro-11b -hydroxy-
3,20-dioxo-1,4-pregnadiene-16a-carboxylate, and their 9a-fluoro
derivatives. Steroids 2000;65:210–8.
[14] Siemes C, Visser LE, De-Jong FH, Jan-Willem Coebergh W, Uitterlinden AG,
et al. Cytochrome P450 3A gene variation, steroid hormone serum levels and
prostate cancer. Steroids 2010;75:1024–32.
[15] Dıaz-Chico BN, Rodrıguez FG, Gonzalez A, Ramırez R, Bilbao C, Leon AC, Jaime
AA, Chirino R, Navarro D. Androgens and androgen receptors in breast cancer.
J Steroid Biochem Mol Biol 2007;105:1–15.
[16] Hohnston JO. Aromatase inhibitors. In: Parish EJ, Nes WD, editors.
Biochemistry and function of sterols. Boca Raton, FL: CRC Press; 1987. p.
23–53.
[17] Frye LL, Leonard DA. Lanosterol analog: dual action inhibitors of cholesterol
biosynthesis. In: Parish EJ, Nes WD, editors. Biochemistry and function of
sterols. Boca Raton, FL: CRC Press; 1987. p. 23–53.
[18] Chung SK, Ryoo CH, Yang HW, Shim JY, Kang MG, Lee KW, Kang HI. Synthesis
and bioactivities of steroid derivatives as antifungal agents. Tetrahedron
1998;52:15899–914.
[19] Suzuki K, Nakata T, Shimizu T, Enomoto K. US Patent, 1998, 5, 712,109.
[20] Dombrowski AW, Hazuda DJ, Polishook JD, Felock PJ, Singh SB, Zink DL. 2000,
WO Patent, 0036132.
[21] Arthan D, Svasti J, Kittakoop P, Pittayakhachonwut D, Tanticharoen M,
Thebtaranonth Y. Antiviral isoflavonoid sulfate and steroidal glycosides from
the fruits of Solanum torvum. Phytochemistry 2002;59:459–63.
[22] Brown WE. The impact of biotechnology to health care industry. Ann Rep
Fermen Proc 1984;7:135.
[23] Liu WH, Kui CW, Wu KL, Lee CY, Hsu WY. Transformation of cholesterol to
testosterone by Mycobacterium sp.. J Ind Microbiol 1994;13:167–71.
[24] Liu WH, Lo CK. Production of testosterone from cholesterol using a single-
step microbial transformation of Mycobacterium sp.. J Ind Microbiol
Biotechnol 1997;19:269–72.
[25] Phase N, Patil S. Natural oils are better than organic solvents for the
conversion of soybean sterols to 17-ketosteroids by Mycobacterium fortuitum.
World J Microbiol Biotechnol 1994;10:228–9.
[26] Dias ACP, Cabral JMS, Pinheiro HM. Sterol side-chain cleavage with
immobilized Mycobacterium cells in water-immiscible organic solvents.
Enzyme Microb Technol 1994;16:708–14.
[27] Liu WH, Horng WC, Tsai MS. Bioconversion of cholesterol to cholest-4-en-3-
one in aqueous/organic solvent two-phase reactors. Enzyme Microb Technol
1996;18:184–9.
[28] Wang Z, Zhao F, Hao X, Chen D, Li D. Microbial transformation of hydrophobic
compound in cloud point system. J Mol Catal B 2004;27:147–53.
[29] Kumar R, Dahiya JS, Singh D, Nigam P. Biotransformation of cholesterol using
Lactobacillus bulgarius in a glucose-controlled bioreactor. Bioresour Technol
2001;78:209–11.
[30] Kieslich K. Microbial side-chain degradation of sterols. J Basic Microbiol
1985;25:461–74.
[31] Yan JL, Lee SS, Wang KC. Microbial transformation of 3-hydroxy-5, 6-
cyclopropanocholestanes-An alternative route to 6-methylsteroids. Steroids
2000;65:863–70.
[32] Ambrus G, Jekkel A, Ilko }y E, Horváth G, Böscskei Z. Novel 26-oxygenated
products in microbial degradation of ergosterol. Steroids 1995;60:626–9.
[33] Ambrus G, Ilko } y E, Jekkel A, Horváth G, Böscskei Z. Microbial transformation
of b-sitosterol and stigmasterol into 26-oxygenated derivatives. Steroids
1995;60:621–5.
[34] Egorova OV, Gulevskaya SA, Puntus IF, Filonov AE, Donova MV. Production of
androstenedione using mutants of Mycobacterium sp.. J Chem Technol
Biotechnol 2002;77:141–7.
[35] Malaviya A, Gomes J. Androstenedione production by biotransformation of
phytosterols. Biores Technol 2008;99:6725–37.
[36] Liu WH, Pan CP, Lo CK. Transformation of phytosterol to testosterone by a
Mycobacterium sp. in an airlift fermentor. Food Sci Agric Chem
2001;4:139–42.
[37] Wang KC, You BJ, Yan JL, Lee SS. Microbial transformation of lanosterol
derivatives with Mycobacterium sp. (NRRL B-3805). J Nat Prod
1995;58:1222–7.
[38] Templeton JF, Liang Y, Zeglam TH, LaBella FS. Synthesis of 20-hydroxy-, 20-
amino- and 20-nitro-14-hydroxy-21-nor-5 beta, 14 beta-pregnane C-3
glycosides and related derivatives: structure–activity relationships of
pregnanes that bind to the digitalis receptor. J Med Chem 1993;36:42–5.
[39] Templeton JF, Setiloane P, Kumar VPS, Ling Y, Jin J, Boehmer MA, et al.
Synthesis of 3b,14-dihydroxy-5b, 14b-pregnan-20-one C-3 derivatives:
ozonolysis of digitoxin and digitoxigenin and their derivatives followed by
zinc-acetic acid reduction to the C-21 methyl ketone. J Chem Soc Perkin Trans
1 1991;4:823–9.
[40] Manosroi J, Saowakhon S, Manosroi A. Enhancement of 17 a -
hydroxyprogesterone production from progesterone by biotransformation
using hydroxypropyl-b-cyclodextrin complexation technique. J Steroid
Biochem Mol Biol 2008;112:201–4.
[41] Hu SH, Genain G, Azerad R. Microbial transformation of steroids: contribution
to 14a-hydroxylations. Steroids 1995;60:337–52.
[42] Smith KE, Ahmed F, Williams RAD, Kelly SL. Microbial transformations of
steroids-VIII. Transformation of progesterone by whole cells and microsomes
of Aspergillus fumigatus. J Steroid Biochem Mol Biol 1994;49:93–100.
 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290
[43] Khalil AM, Eman MM. Microbiological transformation of progesterone by
some zoosporic fungi. J Basic Microbiol 1996;4:255–9.
[44] Kolek T, Świzder A. Biotransformation XLV. Transformations of 4-ene-3-oxo
steroids in Fusarium culmorum culture. J Steroid Biochem Mol Biol
1998;67:63–9.
[45] Al-Awadi S, Afzal M, Oommen S. Studies on Bacillus stearithermophillus. Part I.
Transformation of progesterone to a new metabolite 9,10-seco-4-pregnene-
3,9,20-trione. J Steroid Biochem Mol Biol 2001;78:493–8.
[46] Madyasth KM, Joseph T. Transformation of 16-dehydroprogesterone and 17a-
hydroxyprogesterone by Mucor piriforme. Appl Microbiol Biotechnol
1994;41:170–7.
[47] Baulieu EE, Robel P, Fellous A, Duchossoy Y, Fontaine-Lenoir V, David S.
Toward a novel approach of neuroprotection and treatment of Alzbeimerś
disease. J Mol Neurosci 2004;24:63–5.
[48] Madyasth KM, Joseph T. Transformation of dehydroepianrosterone
pregnenolone by Mucor piriforme. Appl Microbiol Biotechnol 1995;44:
339–43.
[49] Farooq A, Tahara S. Biotransformation of testosterone and pregnenolone
catalyzed by the fungus Botrytis cinerea. J Nat Prod 2000;63:489–91.
[50] Schaaaf O, Dettner K. Transformation of steroids by Bacillus strains isolated
from the foregut of water beetles (Coleoptera: Dytiscidae): II. Metabolism of
3b-hydroxypregn-5-en-20-one (pregnenolone). J Steroid Biochem Mol Biol
2000;75:187–99.
[51] Fernandes D, Loi B, Porte C. Biosynthesis and metabolism of steroids in
molluscs. J Steroid Biochem Mol Biol 2011;127:189–95.
[52] Choudhary MI, Batool I, Shah SAA, Nawaz SA, Atta-ur-Rehman. Microbial
hydroxylation of pregnenolone derivatives. Chem Pharm Bull 2005;11:
1455–9.
[53] Wilson MR, Gallimore WA, Reese PB. Steroid transformations with Fusarium
oxysporum var. cubense and Colletotrichum musae. Steroids 1999;64:834–43.
[54] Schwarz S, Ring S, Weber G, Teichmüller Palmer H-J, Pfeiffer C, Undentsch B,
et al. Synthesis of 13-ethyl-11-methylene-18, 19-dinor-17a-pregn-4-en-20-
yn-17-ol (desogestrel) and its metabolite 3-oxo desogestrel. Tetrahedron
1994;50:10709–20.
[55] Hu SH, Tian XF, Sun YH, Han GD. Microbial hydroxylation of 13-ethyl-17b-
hydroxy-18, 19-dinor-17a-pregn-4-en-20-yn-17-one. Steroids 1996;61:407–10.
[56] Hu SH, Tian XF, Han GD. Novel microbial hydroxylation of 13-ethyl-17b-
hydroxy-18, 19-dinor-17a-pregn-4-en-20-yn-3-one. Steroids
1998;63:88–92.
[57] Rosi D, Neuman HC, Christiansen RG, Schane HP, Potts GO. Isolation,
synthesis, and biological activity of five metabolites of danazol. J Med
Chem 1977;20:349–52.
[58] Ahmed F, Williams RAD, Smith KE. Microbial transformations of steroids-X.
Cytochromes P-450 11a-hydroxylase and C17–C20 lyase and A 1-ene
dehydrogenase transform steroids in Nectria haematococca. J Steroid
Biochem Mol Biol 1996;58:337–49.
[59] Choudhary MI, Azizuddin, Atta-ur-Rehman. Microbial transformation of
danazol. Nat Prod Lett 2002;16:101–6.
[60] Hanson JR, Nasir H, Pervez A. The hydroxylation of testosterone and some
derivatives by Cephalosporium aphidicola. Phytochemistry 1996;42:411–5.
[61] Atta-ur-Rehman, Choudhary MI, Asif F, Farooq A, Yaqoob M. Microbial
transformations of testosterone. Nat Prod Lett 1998;12:255–61.
[62] Farooq A, Tahara S. Biotransformation of testosterone. J Nat Prod
2000;63:489–91.
[63] Al-Awadi S, Afzal M, Oommen S. Studies of Bacillus stearothermophilus. Part III.
Transformation of testosterone. Appl Microbiol Biotechnol 2003;62:48–52.
[64] Madyastha KM. Novel microbial transformations of steroids. Adv Exp Med
Biol 1995;405:259–70.
[65] Angelova B, Mutafov S, Avramova T, Dimova I, Boyadjieva L. 9a-
Hydroxylation of 4-androstene-3,17-dione by resting Rhodococcus sp. cells.
Process Biochem 1996;31:179–84.
[66] Schaaaf O, Dettner K. Transformation of steroids by Bacillus strains isolated
from the forefut of water beetles (Coleoptera: Dytiscidae): 1. Metabolism of
androst-4-en-3,17-dione (AD). J Steroid Biochem Mol Biol 1998;67:451–65.
[67] Choudhary MI, Sultanl S, Khan MTH, Yasin A, Shaheen F, Atta-ur-Rehman.
Biotransformation of (+)-androst-4-ene-3, 17-dione. Nat Prod Res
2004;18:529–35.
[68] Xiong Z, Wei Q, Chen H, Chen S, Xu W, Qiu Liang GS, et al. Microbial
transformation of androst-4-ene-3, 17-dione by Beauveria bassiana. Steroids
2006;71:979–83.
[69] Choudhary MI, Musharraf SG, Shaheen F, Atta-ur-Rehman. Microbial
transformation of (+)-androsta-1, 4-diene-3, 17-dione by Cephalosporium
aphidicola. Nat Prod Lett 2002;16:377–82.
[70] Choudhary MI, Shah SAA, Musharraf SG, Shaheen F, Atta-ur-Rehman.
Microbial transformation of dehydroepiandrosterone. Nat Prod Res
2003;17:215–20.
[71] Mahato SB, Mujumdar I. Current trends in microbial steroid biotransformation.
Phytochemistry 1993;34:883–98.
[72] Nonhebel DC. The chemistry of cyclopropylmethyl and related radicals. Chem
Soc Rev 1993;22:347–59.
[73] Bensasson CS, Hanson JR, Huerou YL. The microbiological hydroxylation of
3a,5-cycloandrostanes by Cephalosporium aphidicola. Phytochemistry
1999;52:1279–82.
[74] Holland HL. The mechanism of the microbial hydroxylation of steroids. Chem
Soc Rev 1982;11:371–95.
1289
[75] Hanson JR, Kiran I. The microbiological hydroxylation of 17-chloroandrosta-
4,16-dien-3-one by Cephalosporium aphidicola. J Chem Res 2003; 130–1.
[76] Choudhary MI, Sultan S, Khan MTH, Atta-ur-Rehman. Microbial
transformation of 17a-ethynyl- and 17a-ethylsteroids and tyrosinase
inhibitory activity of transformed products. Steroids 2005;70:798–802.
[77] Yashihama M, Tamura K, Nakakoshi M, Nakamura J, Fujise N, Kawanishe G. 14
alpha-Hydroxyandrost-4-ene-3,6,17-trione as a mechanical-based irreversible
inhibitor of estrogen biosynthesis. Chem Pharm Bull 1990;38:2834–7.
[78] Matsumoto T, Nakamura AM, Mori K, Akiyama I, Hirose H, Takahashi Y. Gland
of the pulmonate snail Lymnaea stagnalis. Gen Comp Endocrinol
2007;151:195–201.
[79] Musharraf SG, Atta-ur-Rehman, Choudhary MI, Sultan S. Microbial
transformation of (+)-adrenosterone. Nat Prod Lett 2002;16:345–9.
[80] Choudhary MI, Sultan S, Jalil S, Anjum S, Rahman AA, Fun HK, Atta-ur-
Rehman. Microbial transformation of mesterolone. Chem Biodivers
2005;2:392–400.
[81] Choudhary MI, Musharraf SG, Siddiqui ZA, Khan NT, Ali RA, Atta-ur-Rehman.
Microbial transformation of mestranol by Cunninghamella elegans. Chem
Pharm Bull 2005;53:1011–3.
[82] Choudhary MI, Musharraf SG, Ali RA, Atif M, Atta-ur-Rehman. Microbial
transformation of antifertility agents, norethisterone nad 17 a -
ethylnylestradiol. Z Naturforsch 2004; 59B: 319–23.
[83] Choudhary MI, Shah SA, Atta-ur-Rahman, Khan SN, Khan MT. Steroids
2010;75:956–66.
[84] Faramarzi MA, Yazdi MT, Shafiee A, Zarrini G. Microbial transformation of
hydrocortisone by Acremonium strictum PTCC 5282. Steroids 2002;67:
869–72.
[85] Hardman JG, Limbird LE, Gilam AG. The pharmacological basis of
therapeutics. Medicinal Publishing Division, New York; 2001. p. 733.
[86] Choudhary MI, Siddiqui ZA, Musharraf SG, Nawaz SA, Atta-ur-Rehman.
Microbial transformation of prednisone. Nat Prod Res 2005;19:311–7.
[87] Choudhary MI, Sultan S, Yaqoob M, Musharraf SG, Yasin A, Shaheen F, Atta-
ur-Rehman. Microbial transformation of cortisol and prolyl endopeptidase
inhibitory activities of its transformed products. Nat Prod Res
2003;17:389–95.
[88] Kiran I, Hanson JR, Hunter AC. The microbiological hydroxylation of some
methoxysteroids by Cephalosporium aphidicola. J Chem Res 2004;5:362–3.
[89] Hung T, Stuppner H, Ellmerer-Muller EP, Scholz D, Eigner D, Manandhar MP.
Steroids and terpenoids from the gum resin of Ailanthus grandis.
Phytochemistry 1995;39:1403–9.
[90] Marg KSK. The Wealth of India, National Institute of Science Communication,
vol. 2. CSIR, New Delhi; 2001. p. 162.
[91] Malıkova J, Swaczynova J, Kolar Z, Strnad M. Anticancer and antiproliferative
activity of natural Brassinosteroids. Phytochemistry 2008;69:418–26.
[92] Atta-ur-Rehman, Choudhary MI, Shaheen F, Ashraf M, Jahan S. Microbial
transformations of hypolipemic E-guggulsterone. J Nat Prod 1998;61:428–31.
[93] Choudhary MI, Shah SAA, Sami A, Ajaz A, Shaheen F, Atta-ur-Rehman. Fungal
metabolites of (E)-guggulsterone and their antibacterial and radical
scavenging activities. Chem Biodivers 2005;2:516–24.
[94] Jekkel A, Ilko } y E, Horváth G, Pallagi I, Süto } J, Ambrus G. Microbial
hydroxylation of 13b-ethyl-4-gonene-3, 17-dione. J Mol Catal B
1998;5:385–7.
[95] Weier RM, Hofmann LF. Synthesis and antimineralocorticoid activities of
some 6-substituted 7a-carboalkoxy steroidal spirolactones. J Med Chem
1977;20:1304–8.
[96] Preisig CL, Laakso JA, Mocek UM, Wang PT, Baez J, Byng G. Biotransformations
of the cardiovascular drugs mexrenone and canrenone. J Nat Prod
2003;66:350–6.
[97] Antoun MD, Abramson D, Tyson LR, Chang-jer C, MacLaughlin JL, Peck G, et al.
Potential antitumor agents. XVII. Physalin B and 25,26-epidihydrophysalin C
from Witheringia coccoloboides. J Nat Prod 1981;44:579–84.
[98] Soares MBP, Bellintani MC, Ribeiro MI, Tomassini TC, Ribeiro DSR. Inhibition
of macrophage activation and lipopolysaccharide-induced death by
seco-steroids purified from Physalis angulata L.. Eur J Pharmacol
2003;459:107–12.
[99] Pietro RC, Kashima SD, Sato N, Januario AH, Franca SC. In vitro
antimycobacterial activities of Physalis angulata L.. Phytomedicines
2000;7:335–8.
[100] Budhiraja RD, Garg KN, Sudhir S, Arora B. Protective effect of 3-ss-hydroxy-
2,3-dihydrowithanolide F against CCl4-induced hepatotoxicity. Planta Med
1986;52:28–32.
[101] Glotter E. Withanolides and related ergostane-type steroids. Nat Prod Rep
1991;8:415–40.
[102] Choudhary MI, Yousaf S, Samreen, Shah SAA, Ahmad S, Atta-ur-Rehman.
Biotransformation of physalin H and leishmanicidal activity of its
transformed products. Chem Pharm Bull 2006;54:927–30.
[103] Krenn L, Kopp B. Bufadienolides from animal and plant sources.
Phytochemistry 1998;48:1–29.
[104] Steyn PS, Van Heerden FR. Bufadienolides of plant and animal origin. Nat Prod
Rep 1998;15:397–413.
[105] Nogawa T, Kamano Y, Yamashita A, Pettit GR. Isolation and structure of five
new cancer cell growth inhibitory bufadienolides from the Chinese
traditional drug Ch, an Su. J Nat Prod 2001;64:1148–52.
[106] Ye M, Qu GQ, Guo HZ, Guo DA. Specific 12b-hydroxylation of cinobufagin by
filamentous fungi. Appl Environ Microbiol 2004;70:3521–7.
1290 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290
[107] William A, Ricke A, Wang Y, Gerald R. Smooth muscle differentiation and
patterning in the urinary bladder. Differentiation 2007;75:871–82.
[108] Ye M, Qu G, Guo H, Guo D. Novel cytotoxic bufadienolides derived from
bufalin by microbial hydroxylation and their structure-activity relationships.
J Steroid Biochem Mol Biol 2004;91:87–98.
[109] Ye M, Han J, Tu G, An D, Guo D. Microbial hydroxylation of bufalin by
Cunninghamella blakesleana and Mucor spinosus. J Nat Prod 2005;68:628.
[110] Krenn L, Kopp B. Bufadienolides from animal and plant sources.
Phytochemistry 1998;48:1–29.
[111] Xie JT, Wang HS, Attele AS, Yuan CS. Effect of resibufogenin from toad venom
on isolated purkinje fibres. Am J Chin Med 2000;28:187–96.
[112] Xie JT, Dey L, Wu JA, Lowell TK, Yuan CS. Cardiac toxicity of resibufogenin:
electrophysiological evidence. Acta Pharmacol Sin 2001;22:289–97.
[113] Kamano Y, Kotaki A, Hashima H, Inoue M, Morita H, Takeya K. Structure–
toxicity relationship for the toad poison bufadienolides. Bioorg Med Chem
1998;6:1103–15.
[114] Terness P, Novalon D, Dufter C, Kopp B, Opelz G. The T-cell suppressive effect
of bufadienolides: structural requirements for the immunoregulatory
activity. Int Immunopharmacol 2001;1:119–34.
[115] Zhang W, Ye M, Qu GQ, Wu WY, Chin YJ, Guo DA. Microbial hydroxylation of
cinobufagin by Mucor spinosus. J Asian Nat Prod Res 2005;7:225–9.