Professional Documents
Culture Documents
Steroids: Haq Nawaz Bhatti, Rasheed Ahmad Khera
Steroids: Haq Nawaz Bhatti, Rasheed Ahmad Khera
Steroids
journal homepage: www.elsevier.com/locate/steroids
Review
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: [email protected], [email protected] (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- tive than microbial transformation in conventional media. A novel
pounds are currently favored when compared to their natural coun- single step microbial transformation process for the production of
terparts due to some therapeutic advantages, such as high potency, testosterone (2) from cholesterol (1) by Lactobacillus bulgaricus [29]
longer half-lives in blood, simple delivery methods and reduced side has also been reported. Moreover, it was observed that addition of
effects [11]. A variety of steroids are widely used as anti-inflamma- cyclodextrin (CD) significantly increased the biotransformations of
tory, immunosuppressive, progestational, diuretic, anabolic and con- cholesterol (1). These biotransformations of cholesterol are shown
traceptive agents [4–9,12,13]. They have also used for the treatment in Scheme 1.
of some forms of breast and prostate cancer [14,15], as replacement It is a well-known fact that a wide range of microorganisms is
agents in the treatment of adrenal insufficiencies [16], in the preven- capable of utilizing sterols such as cholesterol and phytosterols as
tion of coronary heart disease [17], as antifungal agents [18], as active the sole carbon source for the growth and propagation. It is also well
ingredients in anti-obesity agents [19] and in the treatment of de- documented that in the sterol degrading microorganisms, the ring
clared AIDS [20]. Recently, a steroidal glycoside exhibited anti-viral nucleus and the side chain are metabolized by different enzyme sys-
activity on herpes virus type I [21]. Although introduction of new ste- tems simultaneously and independently [30]. Based on these facts,
roids into commerce is now limited, there is scope for improvement Yan et al. [31] reported the microbial transformation of 3-hydroxy-
in the yields of the desired metabolites as well as preparation of novel 5,6-cyclopropanocholestanes-an alter route to 6-methylsteroids.
steroids. At present steroid manufacture has become an important Incubation of 3b-hydroxy-5, 6a-cyclopropano-5a-cholestane (6)
branch of health care industry affected by biotechnology. As far as with Mycobacterium sp. (NRRL B-3805) gave three metabolites, 5,
market value is concerned, steroid manufacturing is second to antibi- 6a-cyclopropano-5a-androsta-3, 17-dione (7), 3b-hydroxy-5,
otic production [22]. 6a-cyclopropano-5a-androsta-17-one (8) and androsta-4-ene-3,
The areas which are now receiving attention in microbial tech- 17-dione (5). Fermentation of 3b-hydroxy-5, 6b-cyclopropano-5b-
nology are: application of newer concepts of genetic engineering of cholestane (9) with Mycobacterium sp. afforded the metabolite 5,
microbes with improved characteristics, solubility improvement 5, 6b-cyclopropano-5b-androsta-3, 17-dione (10) and 3a-hydroxy-
for carrying out biotransformation of substrates that are insoluble 5, 6b-cyclopropano-5b-androsta-17-one (11). Similarly, incubation
in water, immobilization of enzymes or whole cells in a suitable of 3b-hydroxy-5, 6a-cyclopropano-5a-cholest-7-ene (12) with
matrix for economic utilization, development of a continuous pro- Mycobacterium sp. yielded a mixture of side chain cleaved 17-keto
cess for better and economic product recovery; and manipulation steroids as the major products, viz. the cyclopropyl eliminated
of culture media for improvement in product yields by use of product 5, 5, 6a-cyclopropano-5a-androst-7-ene-3, 17-dione (13),
cyclodextrin [9]. 3b-hydroxy-5, 6a-cyclopropano-5a-androst-4-ene-17-one (14), 5,
The previous reviews [2,5–8] covered literature of steroidal bio- 6a-cyclopropano-5a-androst-8(14)-ene-3, 7, 17-trione (15), 5,
transformation up to 1993. This review article attempts to present 6a-cyclopropano-5a-androst-8-ene-3, 7, 17-trione (16) and 6a,
mainly the structures of metabolites of steroids in vitro in a variety 7a-cyclopropanoandrost-4-ene-3, 17-dione (17) as described in
of biological media during the period 1994–2011. Scheme 2.
Ambrus et al. [32] investigated the effect of the different stereo-
2. Microbial transformation of sterols chemistry of C-24 on the C-26 microbial oxidation of sterol side-
chain using genetically modified Mycobacterium sp. BCS 396 to
Phytosterols, together with sapogenins such as diosgenin, hec- transform ergosterol (18). Incubation of ergosterol (18) with
ogenin and solasodine, are established in the pharmaceutical Mycobacterium sp. yielded 26-oxygenated metabolites: 3-oxo-4,
industry as starting materials for the production of steroidal 22-ergostadien-26-oic acid methyl ester (19), 3-oxo-1, 4, 22-ergo-
hormones. Stigmasterol, b-sitosterol and campesterol are the most statrien-26-oic acid methyl ester (20) and 3-oxo-1, 4, 22-ergosta-
commonly used sterols. Stigmasterol and crude sitosterol (a mix- trien-26-oic acid (21). The genetically modified Mycobacterium
ture of b-sitosterol and campesterol) are extracted mainly from sp. BCS 396 strain has also been used to transform sterols with
soybeans. Cholesterol (1), another employed starting material, ob- stigmastane side chain in order to obtain 26-oxidized metabolites.
tained from animal fats and oils, such as lard, beef, tallow, milk fat Microbial transformation of b-sitosterol (22) by Mycobacterium sp.
or fish oil and sitosterol are microbially cleaved to 17-ketosterols. [33] afforded 4-stigmasten-3-one (23), 26-hydroxy-4-stigmasten-
The production of testosterone (2), a male sex hormone or andro- 3-one (24) and 3-oxo-4-stigmasten-26-oic acid (25). Similarly
gen, from cholesterol via a single step microbial transformation incubation of stigmasterol (26) with Mycobacterium sp. BCS 396
process was investigated by Liu et al. [23] and Liu and Lo [24]. Incu- [33] yielded 4,22 stigmastadien-3-one (27), 6b-hydroxy-4, 22
bation of cholesterol (1) with Mycobacterium sp. NRRL B-3805 stigmastadien-3-one (28), 26-hydroxy-4, 22 stigmastadien-3-one
resulted in the isolation of testosterone (2). (29), 3-oxo-4, 22 stigmastadien-26-oic acid methyl ester (30) and
In recent years, much attention has been given to the biocon- 3-oxo-1, 4, 22 stigmastatrien-26-oic acid methyl ester (31). Some
version of steroids in aqueous/organic solvent two-phase systems mutant strains of Mycobacterium sp. are also able to cleave sterol
[25,26]. The main advantage of using such systems is the possibil- side chain giving androstenedione (AD) as the major product.
ity of operating with high concentrations of poor water-soluble Microbial conversion of b-sitosterol (22) to androstenedione (5)
reactants and products in the reactor while avoiding inhibition of was achieved with Mycobacterium sp. VKM Ac-1815D [34,35]. A
the reaction by either reactant or product. The incubation of phytosterol assimilating and AD-producing mutant, B-3805S de-
cholesterol (1) with free cells of Arthrobacter simplex [27] afforded rived from Mycobacterium sp. NRRL B-3805 was also used to trans-
cholest-4-en-3-one (3) in an aqueous organic solvent two-phase form phytosterol (campesterol: stigmasterol: b-sitosterol = 8:1:11,
system. Similarly a novel approach, microbial transformation in molar ratio) to androstenedione (5) [36]. These biotransformations
cloud point system (CPS), has developed to avoid practical difficul- are shown in Scheme 3.
ties, such as substrate solubility and product inhibition. The system Biotransformation of 8a, 9a-epoxytetrahydrolanosterol (32)
could provide microbial cells with aqueous environment and could and lanosta-7, 9 (11)-dien-3b-ol (35) with Mycobacterium sp. NRRL
dissolve substrates and products in surfactant phase. A microbial B-3805 was reported by Wang et al. [37]. Incubation of compound
transformation of cholesterol (1) to androst-1, 4-dien-3-17-dione 32 with Mycobacterium sp. yielded the metabolites, 20(S)-hydroxy-
(ADD) (4) and androst-4-en-3,17-dione (5) has been investigated methyl-12a-hydroxypregn-4-en-3-one (33) and 20(S)-hydroxy-
in a system consisting of non-ionic surfactants Triton X-100 and methyl-4, 4, 14a-trimethypregna-7, 9(11)-dien-3b-ol (34).
Triton X-114 [28]. It was observed that this system was more affec- 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
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
Mycobacterium sp. resulted in the isolation of androsta-4, normally found in cardioactive steroids. Microbial transformation
8(14)-dien-3, 17-dione (36), lanosta-7, 9 (11)-dien-3-one (37), of progesterone (40) by Thamnostylum piriforme yielded 14a-
androsta-8(14)-en-3, 17-dione (38) and methyl 12a-hydrox- hydroxyprogesterone (41) and 9a-hydroxyprogesterone (42). A
ybisnorchola-4, 17(20)-dien-22-oate (39) as described in similar preparation with Mucor griseocyanus afforded 41, 6b, 14a-
Scheme 4. dihydroxyprogesterone (43) and 7a, 14a-dihydroxyprogesterone
(44) [41]. The filamentous fungus, Aspergillus fumigatous [42] effi-
3. Microbial transformation of steroidal hormones ciently hydroxylated 40 producing 11a-hydroxyprogesterone
(45), 15b-hydroxyprogesterone (46), 7b-hydroxyprogesterone
C-3 glycoside derivatives of 14-hydroxy-5b, 14b-pregnane show (47), 7b–15b-dihydroxyprogesterone (48) and 11a–15b-dihydr-
strong interaction with cardiac glycoside receptor of heart muscle oxyprogesterone (49) (Scheme 6). Incubation of progesterone (40)
[38]. Such compounds have been mainly obtained by multistep with Saprolegnia hypogyna yielded 4-androstene-3, 17-dione (50),
chemical transformations of digitoxine, digitoxigenin and related testosterone (2) and testololactone (51) [43]. Transformation of
compounds that contain the 5b, 14b-configuration [38,39] (see 40 with Fusarium culmorum culture [44] yielded a mixture of 15a-
Scheme 5). Conversely, the 14a-hydroxylation of progesterone hydroxyprogesterone (52) and 12b–15a-dihydroxyprogesterone
[40] and other steroids by several microorganisms is a well- (53) [Scheme 7]. However when 40 was fermented with thermo-
documented fact and several fungi have been shown to introduce, philic bacterium, Bacillus stearothermophilus [46], three monohydr-
in reasonable yields, a hydroxyl group at the 14a-position of pro- oxylated metabolites and a new metabolite, were isolated and
gesterone and some other steroids; the 14a-hydroxy configuration identified. These were identified as 20a-hydroxyprogesterone
can then be transformed chemically to the 14b-hydroxy substituent (54), 6b-hydroxyprogesterone (55), 6a-hydroxyprogesterone (56)
1270 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290
COOCH 3
O COOCH 3
19
O
21
O
23
CH2 OH
My cobacterium sp.
HO O
24
22 COOH
O
25
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 afforded 71, 3b, 7b-dihydroxypregn-5-en-20-one (75) and 7-oxo-
Scheme 7. pregnenolone (76) as the major metabolites (Scheme 10). Choudh-
Madyasth and Joseph [46] reported the biotransformations of ary et al. [52] also investigated the metabolism of pregnenolone
17a-hydroxyprogesterone (58) and 16-dehydroxyprogesterone (70) with different fungi. Incubation of 70 with Cunninghamella
(66) with Mucor piriformis. The organism transforms 17a-hydroxy- elegans yielded metabolites, 3b, 7b, 11a-trihydroxypregn-5-en-
progesterone (58) into 17a, 20a-dihydroxypregn-4-en-3-one (59), 20-one (77), 3b, 6b, 11a-trihydroxypregn-4-en-20-one (78) and
7a, 17a-dihydroxypregn-4-en-3, 20-dione (60), 6b, 17a, 20a-tri- 3b, 6a, 11a, 12b, 15b-pentahydroxypregn-4-en-20-one (79), while
hydroxypregn-4-en-3-one (61) and 11a, 17a, 20a-trihydroxy- incubation with Gibberella fujikuroi afforded 77 and 6b, 15b-
pregn-4-en-3-one (62). However, incubation of 58 with F. dihydroxypregn-4-en-3, 20-dione (80). Similarly fermentation of
culmorum [44] yielded 6b, 17a-dihydroxyprogesterone (63) and 70 by Fusarium oxysporum var. cubense [52] yielded 71. These met-
15a, 17a-dihydroxyprogesterone (64) (Scheme 8). Fermentation abolic pathways are shown in Scheme 11.
of 65 by Mucor piriformis yielded 14a-hydroxypregna-4, 16- Microbial transformation of 5b-pregnane-3, 20-dione (81) and
dien-3, 20-dione (66), 7a, 14a-dihydroxypregna-4, 16-dien-3, 3b-hydroxy-5b-pregnane-20-one (85) were investigated by Hu et
20-dione (67), 3b, 7a, 14a-trihydroxypregn-16-en-20-one (68) al. [41]. Bioconversion of (81) by Thamnostylum piriforme yielded
and 3a, 7a, 14a-trihydroxypregn-16-en-20-one (69) as shown 14a-hydroxy-5b-pregnane-3, 20-dione (82), 14a, 15b-dihydroxy-
in Scheme 9. 5b-pregnane-3, 20-dione (83) and 3b, 14a-dihydroxy-5b-preg-
Pregnenolone (70) is a major hormone in human nerve tissues nane-20-one (84). 3b-hydroxy-5b-pregnane-20-one (85) was
and its therapeutic role in repairing defective neurons is well slowly transformed by T. piriforme yielding the metabolites, 3b–
documented [47]. Moreover, it is a precursor of many steroidal 14a-dihydroxy-5b-pregnane-20-one (86) and 3b, 9a, 14a-trihy-
hormones. Microbial transformation of pregnenolone (70) by droxy-5b-pregnane-20-one (87). However, incubation of 86 with
Mucor piriformis [48] resulted in the isolation of two metabolites, Actinomucor elegans resulted in the isolation of 87, 88 and a minor
3b, 7a-dihydroxypregn-5-en-20-one (71) and 3b, 7a, 11a-trihydr- metabolite, 3b, 9a-dihydroxy-5b-pregnane-20-one (88) are shown
oxypregn-5-en-20-one (72). Fermentation of 70 with the fungus in Scheme 12.
Botrytis cinerea [49] yielded two new oxidized metabolites of preg- Wilson et al. [53] used Fusarium oxysporum var. cubense to
nenolone. These were identified as 3b, 11a, 16b-trihydroxypregn- investigate the metabolism of 17a, 21-dihydroxypregn-4-ene-3,
5-en-20-one (73) and 11a, 16b-dihydroxypregn-4-en-3, 20-dione 11, 20-trione (89) and 17a, 21-dihydroxypregna-1, 4-diene-3, 11,
(74). Metabolism of pregnenolone (70) by Bacillus strains [50,51] 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
yielded 17a, 20, 21-trihydroxypregn-4-ene-3, 11-dione (90) and were unable to differentiate the two enantiomers of 95 in the
androst-4-ene-3, 11,17-trione (91), while incubation of 92 with F. course of these hydroxylation reactions. The microbial transforma-
oxysporum var. cubense resulted in the isolation of metabolites, tion of the dl and d-enantiomers of 95 were also reported using
17a, 20, 21-trihydroxypregna-1, 4-diene-3, 11-dione (93) and and- Cunninghamella species [56]. Transformation of 97 by Cunningham-
rost-1, 4-diene-3, 11, 17-trione (94), respectively (Scheme 13). ella blakesleeana yielded 96, 97, 13-ethyl-6b, 17b-dihydroxy-18,
13-Ethyl-17b-hydroxy-11-methylene-18, 19-dinor-17a-pregn- 19-dinor-17a-pregn-4-en-20-yn-3-one (98), 13-ethyl-15a, 17b-
4-en-20-yne (desogestrel) and its 3-oxo derivatives act as powerful dihydroxy-18, 19-dinor-17a-pregn-4-en-20-yn-3-one (99) and
progestogens and are used as oral contraceptives. Such derivatives 13-ethyl-6b, 10b, 17b-trihydroxy-18, 19-dinor-17a-pregn-4-en-
are mainly obtained by multistep chemical transformations of 20-yn-3-one (100). These transformation reactions of 95 are
estrane derivatives [54]. 13-Ethyl-17b-hydroxy-18, 19-dinor-17a- shown in Scheme 14.
pregn-4-en-20-yn-3-one (95) a less potent progestogens, is com- Danazol (17b-hydroxy-17a-pregna-2, 4-dien-20-yno-[2,3-d]
mercially available and inexpensive. The microbial transformation isoxazole (101) is an orally effective, pituitary gonadotropin inhib-
of a racemic mixture of 95 was investigated using the fungi, Rhizo- itor which is effectively used in the treatment of endometriosis, be-
pus nigricans, R. arrhizus, Aspergillus niger, A. ochraceus and Curvu- nign fibrocystic mastitis and precocious puberty [57,58].
laria lanata [55]. Bioconversion of 95 by R. arrhizus gave a major Choudhary et al. [59] used four different fungi and bacteria to
product, (±)-13-ethyl-10b, 17b-dihydroxy-18, 19-dinor-17a- study the transformation of danazol (101). Fermentation of dana-
pregn-4-en-20-yn-3-one (96). Transformation of 95 by R. nigricans, zol (17b-hydroxy-17a-pregna-2, 4-dien-20-yno-[2,3-d] isoxazole
A. niger and C. lunata was slow and inefficient and also afforded the (101) with Fusarium lini, Aspergillus niger and Cephalosporium
metabolite 96. Compound 95 was slowly metabolized by A. ochrac- aphidicola afforded 17b-hydroxy-2-(hydroxymethyl)-17a-pregn-
eus affording a single metabolite, (±)-13-ethyl-7b, 17b-dihydroxy- 4-en-20-yn-3-one (102) and 17b-hydroxy-2-(hydroxymethyl)-
18, 19-dinor-17a-pregn-4-en-20-yn-3-one (97). All these fungi 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
101 with Bacillus cerus yielded the metabolite 102 only as de- and 14a-hydroxyandrost-4-en-3, 17-dione (105). Conversely, T.
scribed in Scheme 15. piriforme produced lower amounts of 14a-hydroxylated deriva-
Testosterone (17b-hydroxyandrost-4-en-3-one) (2) is a male tives and yielded 104, 6b, 17b-dihydroxyandrost-4-en-3-one
sex hormone responsible for the development of secondary male (106), 9a, 17b-dihydroxyandrost-4-en-3-one (107) and 6b-
characteristics. The hydroxylation of steroids is important because hydroxyandrost-4-en-3, 17-dione (108). Microbial transformation
of its physiological role in mammalian organisms and also for prac- of testosterone (2) by Nectria haematococca [59] afforded 1-ene-
tical reasons [2]. Testosterone (2) was actively metabolized by M. testosterone (109) and 11a-hydroxy-1-ene-testosterone (110)
griseocyanus and T. piriforme [41]. M. griseocyanus produced a (Scheme 16). Incubation of 2 with Fusarium culmorum [45] yielded
major metabolite, 14a, 17b-dihydroxyandrost-4-en-3-one (104) 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
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
The fungus Cephalosporium aphidicola [60] hydroxylated testoster- Curvularia lunata, Pleurotus oestreatus [61] yielded only the
one (2) to metabolites 104 and 106. The metabolites 106, 111, and- metabolites 112 and 113. The plant pathogenic fungus Botrytis
rost-4-ene-3, 17-dione (112) and 15a-hydroxyandrost-4-ene-3, cinerea [62] yielded only the metabolite 7b, 17b-dihydroxyand-
17-dione (113) were obtained when compound 2 was metabolized rost-3-one (114). Biotransformation of testosterone (104) by
with the fungus F. oxysporum var. cubense [52], while the fungi 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
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
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- 17-dione (119) was investigated by Angelova et al. [65] in nutrient
rost-4-en-3, 17-dione (108) and 6a-hydroxyandrost-4-en-3, 17- deficient buffer medium by resting Rhodococcus sp. Incubation of
dione (116). These biotransformations are shown in Scheme 17. 112 with Bacillus strain HA–V6-3 [66] yielded the metabolites, 2,
Madyastha [64] studied the biotransformation of androstenedi- 105, 106, 108, 117 6b, 14a-dihydroxyandrost-4-en-3, 17-dione
one (androst-4-ene-3, 17-dione) (112) using the fungus Mucor pir- (120), 11a-hydroxyandrost-4-en-3, 17-dione (121), 4-androsten-
iformis. The organism transformed 112 into testosterone (2), 14a, 3, 6,17-trione (122) and 5a-androst-3, 6, 17-trione (123) (Scheme
17b-dihydroxyandrost-4-en-3-one (104), 14a-hydroxyandrost-4- 18). Bioconversion of 112 by C. aphidicola [60] gave 105 and 108.
en-3, 17-dione (105), 7a-hydroxyandrost-4-en-3, 17-dione (117), Fermentation of 112 with Curvularia lunata [67] yielded 109, 121,
and 7a, 14a, 17b-trihydroxyandrost-4-en-3-one (118). When 112 androsta-1,4-diene-3,17-dione (124), 11a,17b-dihydroxyandrost-
was fermented with T. piriforme [41], it gave testosterone (2), 4-en-3-one (125) and 15a-hydroxyandrosta-1,4-dien-3,17-dione
14a-hydroxyandrost-4-en-3, 17-dione (105), 6b-hydroxyandrost- (126). Microbial transformation of 112 by the fungus Beauveria
4-en-3, 17-dione (108) 7a-hydroxyandrost-4-en-3, 17-dione bassiana [68] has been investigated using pH 6.0 and 7.0 media.
(117) and 7a, 14a, 17b-trihydroxyandrost-4-en-3-one (118). The Two metabolites were obtained with the pH 6.0 medium and iden-
9a-hydroxylated steroids are valuable intermediates in the tified as 125 and 6b, 11a-dihydroxyandrost-4-ene-3, 17-dione
synthesis of highly effective anti-inflammatory pharmaceuticals. (127). On the other hand, four hydroxylated and/or reduced
Microbial hydroxylation of 112 into 9a-hydroxyandrost-4-en-3, 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
HO CH HO CH
C C HO CH
C
OH H
HOH2 C
H
N HOH2 C
O O +
101 O
102 103
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
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, trost-4-en-3-one (106) and 6b, 11a-dihydroxyantrost-4-en-3-one
17b-trihydroxy-5a-androstane (129). These biotransformation (132) as described in Scheme 20.
reactions are shown in Scheme 19. Microbial reactions are unique in their diversity and impor-
Kolek and Świzder [44] investigated the biotransformation of tance. They offer access to otherwise inaccessible sites in the
androst-4-en-3-one (130) with the fungus F. culmorum. The fungus natural as well as synthetic products. Fermentation of (+)- andro-
F. culmorum fermented the compound 130 and yielded 15a- sta-1,4-diene-3, 17-dione (124) with Cephalpsporium aphidicola
hydroxyandrost-4-en-3, 17-dione (113) and 12b, 15a-dihydrox- [69] for 8 days afforded oxidative and reductive metabolites. These
yantrost-4-en-3-one (131). However, fermentation of 130 with C. metabolites were identified as 1-ene- testosterone (109), androst-
aphidicola [60] afforded the metabolites, 6b, 17b-dihydroxyan- 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
O
O O
HO HO
C. aphidicola
109 + 110 + 112 + 125 +
+
O O
124 O
133 134
17-dione (133)ß 11a-hydroxyandrost-4-ene-3, 17-dione (134)ß (139) and 3b, 7a, 17b-trihydroxyandrost-5-ene (140). Bioconver-
11a, 17b-dihydroxyandrost-4-ene-3, 7-dione (125) and 11a, 17b- sion of 135 was also carried out with plant pathogen Rhizopus
dihydroxyandrosta-1, 4-diene-3ione (110) (Scheme 21). stolonifer [70], which afforded 7 metabolites. These metabolites
Dehydroepiandrosterone (3b-hydroxyandrost-5-en-17-one) were identified as 3b, 17b-dihydroxyandrost-5-ene (136), 3b,
(135) is the source of all sex and steroid hormones in the body. 17b-dihydroxyandrost-4-ene (141), 17b-hydroxyandrost-4-ene-3-
Low level of this compound is associated with the risk of pre- one (2), 3b, 11b-dihydroxyandrost-4-ene-17-one (142), 3b,
menopausal breast cancer and hence this compound has clinical 7a-dihydroxyandrost-5-ene-17-one (137), 3b, 7a, 17b-trihydroxy-
importance regarding inhibition of mammary cancer. Incubation androst-5-ene (140) and 11b-hydroxyandrost-4, 6-diene-3, 17-one
of 135 with the fungus M. piriformis [48] resulted in the produc- (143). Microbial transformation of 3b-hydroxyandrost-5-en-17-
tion of 5 metabolites, 3b, 17b-dihydroxyandrost-5-ene (136), 3b, one (135) by Fusarium oxysporum and Colletotrichum musae [53]
7a-dihydroxyandrost-5-ene-17-one (137), 3b-hydroxyandrost-5- yielded 136, 137, 138 and 140 respectively. These biotransforma-
ene-7, 17-dione (138), 3b, 17b-dihydroxyandrost-5-ene-7-one 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
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 fungal transformation of 166 by different fungi. Fermentation of
synthetic method for obtaining access to rare steroids [71]. The 166 by Cephalosporium aphidicola resulted in the formation of
cyclopropane ring of the 3a-5-cyclosteroids is sensitive to a variety androsta-1, 4-dien-3, 11, 17-trione (167), 17b-hydroxyandrost-
of reagents limiting many chemical reactions. The fission of a 4-en-3, 11-dione (168) and 17b-hydroxyandrosta-1, 4-dien-3,
cyclopropane ring in the presence of an adjacent radical has 11-dione (169). Two other fungal species Fusarium lini and
provided a probe for enzyme mechanism [72]. Incubation of 6b-hy- Trichothecium roseum were also tested for biotransformations of
droxy-3a, 5-cycloandrostan-17-one (145) with C. aphidicola [73] adrenosterone (166). Metabolites 167 and 169 were obtained with
for 8 days yielded six metabolites that were identified as Fusarium lini, while metabolite 169 was produced using Trichothe-
14a-hydroxy-3a,5-cycloandrostane-6, 17-dione (145), 3b, 14a- cium roseum. The structures of these metabolites are shown in
dihydroxyandrost-5-en-17-one (146), 6b, 14a-dihydroxy-3a, Scheme 26.
5-cycloandrostan-17-one (147), 3b, 7a-dihydroxyandrost-5-en- Mesterolone ((1a, 5a, 17b)-17-hydroxy-1-methylandrostan-3-
17-one (148), 3b, 7b-dihydroxyandrost-5-en-17-one (149) and one) (170) is a steroidal drug used in the treatment of conditions
3b, 5a, 6b-trihydroxyandrostan-17-one (150) (Scheme 23). Simi- caused by deficient endogenous androgen formation, which can
larly, incubation of 3a, 5-cycloandrostane-6, 17-dione (151) with develop gradually with increasing age. Choudhary et al. [80,81]
C. aphidicola [73] yielded 145 and 2a-hydroxy-3a, 5-cycloandros- investigated the microbial transformations of mesterolone (170)
tane-6, 17-dione (152), while 3a, 5-cycloandrost-6-en-17-one using different fungi. Metabolites (1a, 5a)-1-methylandrostane-3,
(153) afforded 148 and 6b, 7a-dihydroxy-3a, 5-cycloandrostane- 17-dione (171), (1a, 3b, 5a, 17b)-1-methylandrostane-3, 17-diol
17-one (154) with the same fungus [73]. These microbial conver- (172) and (1a, 5a, 15a)-15-hydroxy-1-methylandrostane-3, 17-
sions are shown in Scheme 23. dione (174) were produced by Cephalosporium aphidicola, while
Regiospecificity of the microbial hydroxylation of steroids has metabolites, (5a)-1-methylandrost-1-en-3, 17-dione (173), 174,
been rationalized in terms of the location of existing hydroxyl (1a, 5a, 6a, 17b)-6, 17-dihydroxy-1-methylandrostan-3-one
and carbonyl groups on the carbon skeleton, which may act as (175), (1a, 5a, 15a, 17b)-15, 17-dihydroxy-1-methylandrostan-3-
binding groups. These groups serve to orient the steroid within one (178) and (5a, 15a, 17b)-15, 17-dihydroxy-1-methylandrost-
the hydroxylase and place a CAH bond close to the oxidant co- 1-en-3-one (179) were produced by Fusarium lini. Similarly,
enzyme [74]. The position of the hydroxylation on ring C appeared Rhizopus stolonifer yielded metabolites 171, 175, (1a, 5a, 7a,
to be affected by the ring D substituent. Hanson and Kiran [75] 17b)-7, 17-dihydroxy-1-methylandrostan-3-one (176), (1a, 5a,
investigated this effect by fermenting 17-chloroandrosta-4, 16- 11a, 17b)-11, 17-dihydroxy-1-methylandrostan-3-one (177) and
dien-3-one (155) with the fungus C. aphidicola. Incubation of 179. These transformations are shown in Scheme 27.
compound 155 with C. aphidicola for 10 days gave three metabo- Biotransformation of 3-hydroxyestra-1, 3, 5 (10)-trien-17-one
lites. The metabolites were identified as 17-chloro-6b, 11a- (180) was investigated with the fungus Fusarium oxysporum var.
dihydroxyandrosta-4, 16-dien-3-one (156), 17-chloro-6b, 15b- cubense [53]. 3-Hydroxyestra-1, 3, 5 (10)-trien-17-one (180) was
dihydroxyandrosta-4, 16-dien-3-one (157) and 17-chloro-6b, 11a, biotransformed to 3, 17b-dihydroxyestra-1, 3, 5 (10)-triene (181)
15b-trihydroxyandrosta-4, 16-dien-3-one (158) (Scheme 24). and 3, 15a-dihydroxyestra-1, 3, 5 (10)-triene (182). (Scheme 28).
Microbial transformation of ethisterone (17a-ethynyl-17b- Mestranol (183) is an oral contraceptive and widely used in com-
hydroxyandrost-4-en-3-one) (159) and 17a-ethyl-17b-hydroxy- bination with other estrogens and progestogens in antifertility
androst-4-en-3-one) (162) by the fungi Cephalosporium aphidicola medicines. Incubation of mestranol (183) with Cunninghamella ele-
and Cunninghamella elegans were reported by Choudhary et al. gans [82] yielded two hydroxylated metabolites, 6b-hydroxymestr-
[76]. Fermentation of compound 159 with C. aphidicola yielded anol (184) and 6b, 12b-dihydroxymestranol (185) (Scheme 28).
the metabolite, 17a-ethynyl-17b-hydroxyandrosta-1,4-dien-3- Northisterone (17a-ethynyl-19-nortestosterone) (186) is a po-
one) (160), while with C. elegans afforded 17a-ethynyl-11a,17b- tent progestogen, widely used in oral contraceptive pills, as an
dihydroxyandrost-4-en-3-one (161). On the other hand, compound antifertility agent. It has a wide variety of uses including the delay
162 with fungus C. aphidicola afforded 17a-ethyl-17b-hydroxyan- in menstruation and treatment of other menstrual disorder such as
drosta-1,4-dien-3-one (164). However, when compound 162 was endometriosis. Fungal transformation of nothisterone (186) by
incubated with C. elegans, two new metabolites, 17a-ethyl-11a, Cephalosporium aphidicola [83] yielded an oxidized metabolite,
17b-dihydroxyandrost-4-en-3-one (164) and 17a-ethyl-6a, 17b- 17a-ethynylestradiol (187), while the fungal transformation of
dihydroxy-5a-androstan-3-one (165) as reported in Scheme 25. 187 by Cunninghamella elegans afforded several metabolites, 19-
Adrenosterone (166) is used as estrogen synthetase inhibitor, nor-17a-pregna-1, 3, 5 (10)-trien-20-yne-3, 4, 17b-triol (188),
which has potential clinical importance for managing the estro- 19-nor-17a-pregna-1, 3, 5 (10)-trien-20-yne-3, 7a, 17b-triol
gen-mediated events such as ovulation and the growth of estrogen (189), 19-nor-17a-pregna-1, 3, 5 (10)-trien-20-yne-3, 11a, 17b-
dependent tumors [77,78]. Musharraf et al. [79] reported the triol (190), 19-nor-17a-pregna-1, 3, 5 (10)-trien-20-yne-3, 6b,
Cl Cl
HO
Cl
OH
O O
C. aphidicola OH 157 OH
156 Cl
O HO
155
OH
O
OH 158
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
O
O OH
O
O
O
+
O
C. aphidicola O
167 168
O OH
O
166
O
169
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).
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
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
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
4. Microbial transformations of miscellaneous steroids of epilepsy, ulcers, helminthus, rheumatoid arthritis and hyperlipe-
mia in the ancient Indian system of medicine. It also exhibited
E-Guggulsterone (214), a pregnane-type steroid, has been iso- cytotoxic activity [91] against the human lung cancer cell line
lated from the gums of Ailanthus grandis [89] and Commiphora mu- (NCI-H-226). Bioconversion of E-guggulsterone (pregna-4, 17
kul [90]. The gum resin of C. mukul has been used in the treatment (20)-cis-diene-3, 16-dione) (214) by Aspergillus niger [92] resulted
1284 H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290
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).
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
O O O
O R2 O O
R1
OH
C. cassiicola
O O
241 242
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
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
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
was investigated by Ye et al. [106]. Fermentation of compound 247 [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-
by A. alternata resulted in the isolation of six metabolites, 12b-hy-
pregnadiene-16a-carboxylate, methyl 21-desoxy-21-chloro-11b -hydroxy-
droxy-cinobufagin (248), 12b-hydroxy-desacetylcinobufagin (249), 3,20-dioxo-1,4-pregnadiene-16a-carboxylate, and their 9a-fluoro
3-oxo-12b-hydroxy-cinobufagin (250), 3-oxo-12b-hydroxy-des- derivatives. Steroids 2000;65:210–8.
acetylcinobufagin (251), 12-oxo-cinobufagin (252) and 3-oxo- [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
12a-hydroxyl cinobufagin (253). However, incubation of 247 with prostate cancer. Steroids 2010;75:1024–32.
Mucor spinosus [107] afforded 248, 1b-hydroxy cinobufagin (254) [15] Dıaz-Chico BN, Rodrıguez FG, Gonzalez A, Ramırez R, Bilbao C, Leon AC, Jaime
and 1b, 12b-dihydroxy-cinobufagin (255) as described in Scheme AA, Chirino R, Navarro D. Androgens and androgen receptors in breast cancer.
J Steroid Biochem Mol Biol 2007;105:1–15.
39. [16] Hohnston JO. Aromatase inhibitors. In: Parish EJ, Nes WD, editors.
Bufalin (256) is another major bufadienolide isolated from the Biochemistry and function of sterols. Boca Raton, FL: CRC Press; 1987. p.
traditional Chinese drug Chan’Su [104]. Incubation of bufalin 23–53.
[17] Frye LL, Leonard DA. Lanosterol analog: dual action inhibitors of cholesterol
(256) with Mucor spinosus [108] yielded 12 metabolites, 7b-hydro- biosynthesis. In: Parish EJ, Nes WD, editors. Biochemistry and function of
xyl bufalin (257), 3-epi-7b-hydroxyl bufalin (258), 1b-hydroxyl sterols. Boca Raton, FL: CRC Press; 1987. p. 23–53.
bufalin (259), 15a-hydroxyl bufalin (260), 15b-hydroxyl bufalin [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
(261), telocinobufagin (262), 11b-hydroxyl bufalin (263), 12b-hy- 1998;52:15899–914.
droxyl bufalin (264), 1b, 7b-dihydroxyl bufalin (265), 16a-hydroxyl [19] Suzuki K, Nakata T, Shimizu T, Enomoto K. US Patent, 1998, 5, 712,109.
bufalin (266), 7b, 16a-dihydroxyl bufalin (267) and 1b, 12b- [20] Dombrowski AW, Hazuda DJ, Polishook JD, Felock PJ, Singh SB, Zink DL. 2000,
WO Patent, 0036132.
dihydroxyl bufalin (268). The microbial transformation of bufalin
[21] Arthan D, Svasti J, Kittakoop P, Pittayakhachonwut D, Tanticharoen M,
(269) was also carried out using two strains of filamentous fungi, Thebtaranonth Y. Antiviral isoflavonoid sulfate and steroidal glycosides from
Cunninghamella blakesleana and Mucor spinosus [109]. Cunningham- the fruits of Solanum torvum. Phytochemistry 2002;59:459–63.
ella blakesleana catalyzed the specific hydroxylation of bufalin and [22] Brown WE. The impact of biotechnology to health care industry. Ann Rep
Fermen Proc 1984;7:135.
produced 12a-hydroxyl bufalin (269), 7b, 12a-dihydroxybufalin [23] Liu WH, Kui CW, Wu KL, Lee CY, Hsu WY. Transformation of cholesterol to
(270), 7b-hydroxybufalin (257) and 12b-hydroxybufalin (264). testosterone by Mycobacterium sp.. J Ind Microbiol 1994;13:167–71.
However, fermentation of compound 256 with Mucor spinosus [24] Liu WH, Lo CK. Production of testosterone from cholesterol using a single-
step microbial transformation of Mycobacterium sp.. J Ind Microbiol
afforded 7b, 15a-dihydroxybufalin (271) and 5b, 7b-dihydroxybuf- Biotechnol 1997;19:269–72.
alin (272). These metabolized products are shown in Scheme 40. [25] Phase N, Patil S. Natural oils are better than organic solvents for the
Resibufogenin (271) is a typical bufadienolide isolated from the conversion of soybean sterols to 17-ketosteroids by Mycobacterium fortuitum.
World J Microbiol Biotechnol 1994;10:228–9.
traditional Chinese medicine Chansu, a product of the skin secre- [26] Dias ACP, Cabral JMS, Pinheiro HM. Sterol side-chain cleavage with
tions of local toads such as Bufo bufo gragarizans Cantor or Bufo immobilized Mycobacterium cells in water-immiscible organic solvents.
melanostictus Schneider [110]. It shows a wide range of activities Enzyme Microb Technol 1994;16:708–14.
[27] Liu WH, Horng WC, Tsai MS. Bioconversion of cholesterol to cholest-4-en-3-
such as cardiotonic, antitumor, blood pressure stimulating and one in aqueous/organic solvent two-phase reactors. Enzyme Microb Technol
immunoregulatory [111–114]. Microbial transformation of 1996;18:184–9.
resibufogenin (273) by Mucor subtilissimus [115] resulted in the [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.
isolation of 11 metabolites, 12b-hydroxyresibufogenin (274),
[29] Kumar R, Dahiya JS, Singh D, Nigam P. Biotransformation of cholesterol using
11b-hydroxyresibufogenin (275), 16a-hydroxyresibufogenin Lactobacillus bulgarius in a glucose-controlled bioreactor. Bioresour Technol
(276), 12b-hydroxy-3-epi-resibufogenin (277), 12a-hydroxyresi- 2001;78:209–11.
bufogenin (278), 1b, 11b-dihydroxyresibufogenin (279), 12b, 16a- [30] Kieslich K. Microbial side-chain degradation of sterols. J Basic Microbiol
1985;25:461–74.
dihydroxyresibufogenin (280), 12a, 16a-dihydroxyresibufogenin [31] Yan JL, Lee SS, Wang KC. Microbial transformation of 3-hydroxy-5, 6-
(281), 7a-hydroxyresibufogenin (282), 1b, 12b-dihydroxyresibuf- cyclopropanocholestanes-An alternative route to 6-methylsteroids. Steroids
ogenin (283) and resibufogenin 12-O-b-D-glucoside (284). These 2000;65:863–70.
[32] Ambrus G, Jekkel A, Ilko }y E, Horváth G, Böscskei Z. Novel 26-oxygenated
biotransformations are reported in Scheme 41. 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
References 1995;60:621–5.
[34] Egorova OV, Gulevskaya SA, Puntus IF, Filonov AE, Donova MV. Production of
[1] Mensah-Nyagan AG, Meyer L, Schaeffer V, Kibaly C, Patte-Mensah C. Evidence androstenedione using mutants of Mycobacterium sp.. J Chem Technol
for a key role of steroids in the modulation of pain. Psychoneuroendocrinology Biotechnol 2002;77:141–7.
2009;34:169–77. [35] Malaviya A, Gomes J. Androstenedione production by biotransformation of
[2] Sedlaczek L. Biotransformations of steroids. Crit Rev Biotechnol phytosterols. Biores Technol 2008;99:6725–37.
1988;7:187–236. [36] Liu WH, Pan CP, Lo CK. Transformation of phytosterol to testosterone by a
[3] Charney W, Herzong LH. Microbial transformations of steroids. New Mycobacterium sp. in an airlift fermentor. Food Sci Agric Chem
York: Academic Press; 1976. pp. 5–73. 2001;4:139–42.
[4] Hogg JA. Steroids, the steroid community and Upjohn in perspective: a profile [37] Wang KC, You BJ, Yan JL, Lee SS. Microbial transformation of lanosterol
of innovation. Steroids 1992;257:593–616. derivatives with Mycobacterium sp. (NRRL B-3805). J Nat Prod
[5] Mahato Mukherjee. Steroid transformations by microorganisms. Phytochemistry 1995;58:1222–7.
1984;23:2131–54. [38] Templeton JF, Liang Y, Zeglam TH, LaBella FS. Synthesis of 20-hydroxy-, 20-
[6] Mahato SB, Mukherjee A. Steroid transformations by microorganisms-II. amino- and 20-nitro-14-hydroxy-21-nor-5 beta, 14 beta-pregnane C-3
Phytochemistry 1985;24:1403–21. glycosides and related derivatives: structure–activity relationships of
[7] Mahato SB, Banergee S, Mazumder I. Metabolism of progesterone by a Bacillus pregnanes that bind to the digitalis receptor. J Med Chem 1993;36:42–5.
species. J Chem Res Synop 1989;6:184–5. [39] Templeton JF, Setiloane P, Kumar VPS, Ling Y, Jin J, Boehmer MA, et al.
[8] Ahmad S, Garg SK, Johri BN. Biotransformation of sterols: selective cleavage of Synthesis of 3b,14-dihydroxy-5b, 14b-pregnan-20-one C-3 derivatives:
the side chain. Biotechnol Adv 1992;10:1–67. ozonolysis of digitoxin and digitoxigenin and their derivatives followed by
[9] Mahato SB, Mazumder I. Current trends in microbial steroid biotransformation. zinc-acetic acid reduction to the C-21 methyl ketone. J Chem Soc Perkin Trans
Phytochemistry 1995;34:883–98. 1 1991;4:823–9.
[10] Mahato SB, Garai S. Advances in microbial steroid biotransformation. Steroids [40] Manosroi J, Saowakhon S, Manosroi A. Enhancement of 17 a -
1997;62:332–45. hydroxyprogesterone production from progesterone by biotransformation
[11] Fernandes P, Cruz A, Angelova B, Pinheiro HM, Cabral JMS. Microbial using hydroxypropyl-b-cyclodextrin complexation technique. J Steroid
conversion of steroid compounds: recent developments. Enzyme Microb Biochem Mol Biol 2008;112:201–4.
Technol 2003;32:688–705. [41] Hu SH, Genain G, Azerad R. Microbial transformation of steroids: contribution
[12] Tuba Z, Barden CW, Dancsi A, Francsics-Czinege E, Molnar EC, Csorgei J, Falkay to 14a-hydroxylations. Steroids 1995;60:337–52.
G, Koide SS, Kumar N, Sundaram K, Dukat-Abrok V, Balogh G. Synthesis and [42] Smith KE, Ahmed F, Williams RAD, Kelly SL. Microbial transformations of
biological activity of a new progestogen, 16-methylene-17alpha-hydroxy-18- steroids-VIII. Transformation of progesterone by whole cells and microsomes
methyl-19-norpregn-4-ene-3, 20-dione acetate. Steroids 2000;65:266–74. of Aspergillus fumigatus. J Steroid Biochem Mol Biol 1994;49:93–100.
H.N. Bhatti, R.A. Khera / Steroids 77 (2012) 1267–1290 1289
[43] Khalil AM, Eman MM. Microbiological transformation of progesterone by [75] Hanson JR, Kiran I. The microbiological hydroxylation of 17-chloroandrosta-
some zoosporic fungi. J Basic Microbiol 1996;4:255–9. 4,16-dien-3-one by Cephalosporium aphidicola. J Chem Res 2003; 130–1.
[44] Kolek T, Świzder A. Biotransformation XLV. Transformations of 4-ene-3-oxo [76] Choudhary MI, Sultan S, Khan MTH, Atta-ur-Rehman. Microbial
steroids in Fusarium culmorum culture. J Steroid Biochem Mol Biol transformation of 17a-ethynyl- and 17a-ethylsteroids and tyrosinase
1998;67:63–9. inhibitory activity of transformed products. Steroids 2005;70:798–802.
[45] Al-Awadi S, Afzal M, Oommen S. Studies on Bacillus stearithermophillus. Part I. [77] Yashihama M, Tamura K, Nakakoshi M, Nakamura J, Fujise N, Kawanishe G. 14
Transformation of progesterone to a new metabolite 9,10-seco-4-pregnene- alpha-Hydroxyandrost-4-ene-3,6,17-trione as a mechanical-based irreversible
3,9,20-trione. J Steroid Biochem Mol Biol 2001;78:493–8. inhibitor of estrogen biosynthesis. Chem Pharm Bull 1990;38:2834–7.
[46] Madyasth KM, Joseph T. Transformation of 16-dehydroprogesterone and 17a- [78] Matsumoto T, Nakamura AM, Mori K, Akiyama I, Hirose H, Takahashi Y. Gland
hydroxyprogesterone by Mucor piriforme. Appl Microbiol Biotechnol of the pulmonate snail Lymnaea stagnalis. Gen Comp Endocrinol
1994;41:170–7. 2007;151:195–201.
[47] Baulieu EE, Robel P, Fellous A, Duchossoy Y, Fontaine-Lenoir V, David S. [79] Musharraf SG, Atta-ur-Rehman, Choudhary MI, Sultan S. Microbial
Toward a novel approach of neuroprotection and treatment of Alzbeimerś transformation of (+)-adrenosterone. Nat Prod Lett 2002;16:345–9.
disease. J Mol Neurosci 2004;24:63–5. [80] Choudhary MI, Sultan S, Jalil S, Anjum S, Rahman AA, Fun HK, Atta-ur-
[48] Madyasth KM, Joseph T. Transformation of dehydroepianrosterone Rehman. Microbial transformation of mesterolone. Chem Biodivers
pregnenolone by Mucor piriforme. Appl Microbiol Biotechnol 1995;44: 2005;2:392–400.
339–43. [81] Choudhary MI, Musharraf SG, Siddiqui ZA, Khan NT, Ali RA, Atta-ur-Rehman.
[49] Farooq A, Tahara S. Biotransformation of testosterone and pregnenolone Microbial transformation of mestranol by Cunninghamella elegans. Chem
catalyzed by the fungus Botrytis cinerea. J Nat Prod 2000;63:489–91. Pharm Bull 2005;53:1011–3.
[50] Schaaaf O, Dettner K. Transformation of steroids by Bacillus strains isolated [82] Choudhary MI, Musharraf SG, Ali RA, Atif M, Atta-ur-Rehman. Microbial
from the foregut of water beetles (Coleoptera: Dytiscidae): II. Metabolism of transformation of antifertility agents, norethisterone nad 17 a -
3b-hydroxypregn-5-en-20-one (pregnenolone). J Steroid Biochem Mol Biol ethylnylestradiol. Z Naturforsch 2004; 59B: 319–23.
2000;75:187–99. [83] Choudhary MI, Shah SA, Atta-ur-Rahman, Khan SN, Khan MT. Steroids
[51] Fernandes D, Loi B, Porte C. Biosynthesis and metabolism of steroids in 2010;75:956–66.
molluscs. J Steroid Biochem Mol Biol 2011;127:189–95. [84] Faramarzi MA, Yazdi MT, Shafiee A, Zarrini G. Microbial transformation of
[52] Choudhary MI, Batool I, Shah SAA, Nawaz SA, Atta-ur-Rehman. Microbial hydrocortisone by Acremonium strictum PTCC 5282. Steroids 2002;67:
hydroxylation of pregnenolone derivatives. Chem Pharm Bull 2005;11: 869–72.
1455–9. [85] Hardman JG, Limbird LE, Gilam AG. The pharmacological basis of
[53] Wilson MR, Gallimore WA, Reese PB. Steroid transformations with Fusarium therapeutics. Medicinal Publishing Division, New York; 2001. p. 733.
oxysporum var. cubense and Colletotrichum musae. Steroids 1999;64:834–43. [86] Choudhary MI, Siddiqui ZA, Musharraf SG, Nawaz SA, Atta-ur-Rehman.
[54] Schwarz S, Ring S, Weber G, Teichmüller Palmer H-J, Pfeiffer C, Undentsch B, Microbial transformation of prednisone. Nat Prod Res 2005;19:311–7.
et al. Synthesis of 13-ethyl-11-methylene-18, 19-dinor-17a-pregn-4-en-20- [87] Choudhary MI, Sultan S, Yaqoob M, Musharraf SG, Yasin A, Shaheen F, Atta-
yn-17-ol (desogestrel) and its metabolite 3-oxo desogestrel. Tetrahedron ur-Rehman. Microbial transformation of cortisol and prolyl endopeptidase
1994;50:10709–20. inhibitory activities of its transformed products. Nat Prod Res
[55] Hu SH, Tian XF, Sun YH, Han GD. Microbial hydroxylation of 13-ethyl-17b- 2003;17:389–95.
hydroxy-18, 19-dinor-17a-pregn-4-en-20-yn-17-one. Steroids 1996;61:407–10. [88] Kiran I, Hanson JR, Hunter AC. The microbiological hydroxylation of some
[56] Hu SH, Tian XF, Han GD. Novel microbial hydroxylation of 13-ethyl-17b- methoxysteroids by Cephalosporium aphidicola. J Chem Res 2004;5:362–3.
hydroxy-18, 19-dinor-17a-pregn-4-en-20-yn-3-one. Steroids [89] Hung T, Stuppner H, Ellmerer-Muller EP, Scholz D, Eigner D, Manandhar MP.
1998;63:88–92. Steroids and terpenoids from the gum resin of Ailanthus grandis.
[57] Rosi D, Neuman HC, Christiansen RG, Schane HP, Potts GO. Isolation, Phytochemistry 1995;39:1403–9.
synthesis, and biological activity of five metabolites of danazol. J Med [90] Marg KSK. The Wealth of India, National Institute of Science Communication,
Chem 1977;20:349–52. vol. 2. CSIR, New Delhi; 2001. p. 162.
[58] Ahmed F, Williams RAD, Smith KE. Microbial transformations of steroids-X. [91] Malıkova J, Swaczynova J, Kolar Z, Strnad M. Anticancer and antiproliferative
Cytochromes P-450 11a-hydroxylase and C17–C20 lyase and A 1-ene activity of natural Brassinosteroids. Phytochemistry 2008;69:418–26.
dehydrogenase transform steroids in Nectria haematococca. J Steroid [92] Atta-ur-Rehman, Choudhary MI, Shaheen F, Ashraf M, Jahan S. Microbial
Biochem Mol Biol 1996;58:337–49. transformations of hypolipemic E-guggulsterone. J Nat Prod 1998;61:428–31.
[59] Choudhary MI, Azizuddin, Atta-ur-Rehman. Microbial transformation of [93] Choudhary MI, Shah SAA, Sami A, Ajaz A, Shaheen F, Atta-ur-Rehman. Fungal
danazol. Nat Prod Lett 2002;16:101–6. metabolites of (E)-guggulsterone and their antibacterial and radical
[60] Hanson JR, Nasir H, Pervez A. The hydroxylation of testosterone and some scavenging activities. Chem Biodivers 2005;2:516–24.
derivatives by Cephalosporium aphidicola. Phytochemistry 1996;42:411–5. [94] Jekkel A, Ilko } y E, Horváth G, Pallagi I, Süto } J, Ambrus G. Microbial
[61] Atta-ur-Rehman, Choudhary MI, Asif F, Farooq A, Yaqoob M. Microbial hydroxylation of 13b-ethyl-4-gonene-3, 17-dione. J Mol Catal B
transformations of testosterone. Nat Prod Lett 1998;12:255–61. 1998;5:385–7.
[62] Farooq A, Tahara S. Biotransformation of testosterone. J Nat Prod [95] Weier RM, Hofmann LF. Synthesis and antimineralocorticoid activities of
2000;63:489–91. some 6-substituted 7a-carboalkoxy steroidal spirolactones. J Med Chem
[63] Al-Awadi S, Afzal M, Oommen S. Studies of Bacillus stearothermophilus. Part III. 1977;20:1304–8.
Transformation of testosterone. Appl Microbiol Biotechnol 2003;62:48–52. [96] Preisig CL, Laakso JA, Mocek UM, Wang PT, Baez J, Byng G. Biotransformations
[64] Madyastha KM. Novel microbial transformations of steroids. Adv Exp Med of the cardiovascular drugs mexrenone and canrenone. J Nat Prod
Biol 1995;405:259–70. 2003;66:350–6.
[65] Angelova B, Mutafov S, Avramova T, Dimova I, Boyadjieva L. 9a- [97] Antoun MD, Abramson D, Tyson LR, Chang-jer C, MacLaughlin JL, Peck G, et al.
Hydroxylation of 4-androstene-3,17-dione by resting Rhodococcus sp. cells. Potential antitumor agents. XVII. Physalin B and 25,26-epidihydrophysalin C
Process Biochem 1996;31:179–84. from Witheringia coccoloboides. J Nat Prod 1981;44:579–84.
[66] Schaaaf O, Dettner K. Transformation of steroids by Bacillus strains isolated [98] Soares MBP, Bellintani MC, Ribeiro MI, Tomassini TC, Ribeiro DSR. Inhibition
from the forefut of water beetles (Coleoptera: Dytiscidae): 1. Metabolism of of macrophage activation and lipopolysaccharide-induced death by
androst-4-en-3,17-dione (AD). J Steroid Biochem Mol Biol 1998;67:451–65. seco-steroids purified from Physalis angulata L.. Eur J Pharmacol
[67] Choudhary MI, Sultanl S, Khan MTH, Yasin A, Shaheen F, Atta-ur-Rehman. 2003;459:107–12.
Biotransformation of (+)-androst-4-ene-3, 17-dione. Nat Prod Res [99] Pietro RC, Kashima SD, Sato N, Januario AH, Franca SC. In vitro
2004;18:529–35. antimycobacterial activities of Physalis angulata L.. Phytomedicines
[68] Xiong Z, Wei Q, Chen H, Chen S, Xu W, Qiu Liang GS, et al. Microbial 2000;7:335–8.
transformation of androst-4-ene-3, 17-dione by Beauveria bassiana. Steroids [100] Budhiraja RD, Garg KN, Sudhir S, Arora B. Protective effect of 3-ss-hydroxy-
2006;71:979–83. 2,3-dihydrowithanolide F against CCl4-induced hepatotoxicity. Planta Med
[69] Choudhary MI, Musharraf SG, Shaheen F, Atta-ur-Rehman. Microbial 1986;52:28–32.
transformation of (+)-androsta-1, 4-diene-3, 17-dione by Cephalosporium [101] Glotter E. Withanolides and related ergostane-type steroids. Nat Prod Rep
aphidicola. Nat Prod Lett 2002;16:377–82. 1991;8:415–40.
[70] Choudhary MI, Shah SAA, Musharraf SG, Shaheen F, Atta-ur-Rehman. [102] Choudhary MI, Yousaf S, Samreen, Shah SAA, Ahmad S, Atta-ur-Rehman.
Microbial transformation of dehydroepiandrosterone. Nat Prod Res Biotransformation of physalin H and leishmanicidal activity of its
2003;17:215–20. transformed products. Chem Pharm Bull 2006;54:927–30.
[71] Mahato SB, Mujumdar I. Current trends in microbial steroid biotransformation. [103] Krenn L, Kopp B. Bufadienolides from animal and plant sources.
Phytochemistry 1993;34:883–98. Phytochemistry 1998;48:1–29.
[72] Nonhebel DC. The chemistry of cyclopropylmethyl and related radicals. Chem [104] Steyn PS, Van Heerden FR. Bufadienolides of plant and animal origin. Nat Prod
Soc Rev 1993;22:347–59. Rep 1998;15:397–413.
[73] Bensasson CS, Hanson JR, Huerou YL. The microbiological hydroxylation of [105] Nogawa T, Kamano Y, Yamashita A, Pettit GR. Isolation and structure of five
3a,5-cycloandrostanes by Cephalosporium aphidicola. Phytochemistry new cancer cell growth inhibitory bufadienolides from the Chinese
1999;52:1279–82. traditional drug Ch, an Su. J Nat Prod 2001;64:1148–52.
[74] Holland HL. The mechanism of the microbial hydroxylation of steroids. Chem [106] Ye M, Qu GQ, Guo HZ, Guo DA. Specific 12b-hydroxylation of cinobufagin by
Soc Rev 1982;11:371–95. 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 [112] Xie JT, Dey L, Wu JA, Lowell TK, Yuan CS. Cardiac toxicity of resibufogenin:
patterning in the urinary bladder. Differentiation 2007;75:871–82. electrophysiological evidence. Acta Pharmacol Sin 2001;22:289–97.
[108] Ye M, Qu G, Guo H, Guo D. Novel cytotoxic bufadienolides derived from [113] Kamano Y, Kotaki A, Hashima H, Inoue M, Morita H, Takeya K. Structure–
bufalin by microbial hydroxylation and their structure-activity relationships. toxicity relationship for the toad poison bufadienolides. Bioorg Med Chem
J Steroid Biochem Mol Biol 2004;91:87–98. 1998;6:1103–15.
[109] Ye M, Han J, Tu G, An D, Guo D. Microbial hydroxylation of bufalin by [114] Terness P, Novalon D, Dufter C, Kopp B, Opelz G. The T-cell suppressive effect
Cunninghamella blakesleana and Mucor spinosus. J Nat Prod 2005;68:628. of bufadienolides: structural requirements for the immunoregulatory
[110] Krenn L, Kopp B. Bufadienolides from animal and plant sources. activity. Int Immunopharmacol 2001;1:119–34.
Phytochemistry 1998;48:1–29. [115] Zhang W, Ye M, Qu GQ, Wu WY, Chin YJ, Guo DA. Microbial hydroxylation of
[111] Xie JT, Wang HS, Attele AS, Yuan CS. Effect of resibufogenin from toad venom cinobufagin by Mucor spinosus. J Asian Nat Prod Res 2005;7:225–9.
on isolated purkinje fibres. Am J Chin Med 2000;28:187–96.