Biotransformation of danazol by Fusarium solani and Gibberella fujikuorii, and prolyl endopeptidase inhibition studies of transformed products

Article abstract of DOI:10.3906/kim-0912-400

Biotransformation of danazol (17β-hydroxy-17α-pregna-2,4-dien- 20-yno-[2,3-d] isoxazole) (1) on fermentation with Fusarium solani yielded 17β-hydroxy-2-(hydroxymethyl)-17α-pregn-4-en-20-yn-3-one (2) and 17β-hydroxy-2-(hydroxymethyl)-17α-pregna-1,4-dien-20-yn-3-one (3), while the fermentation of 1 with Gibberella fujikuorii yielded compound 2 only. The structures of these compounds were deduced on the basis of modern spectroscopic techniques. Prolyl endopeptidase inhibition activities of danazol (1) and its transformed products 2 and 3 are also studied. TUeBITAK.

Full text of DOI:10.3906/kim-0912-400

Turk J Chem
34 (2010) , 945 – 951.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-0912-400
Biotransformation of danazol by Fusarium solani and
Gibberella fujikuorii, and prolyl endopeptidase
inhibition studies of transformed products
AZIZUDDIN1,∗, Muhammad Iqbal CHOUDHARY2
1Department of Chemistry, Federal Urdu University for Arts, Science & Technology,
Gulshan-e-Iqbal Campus, Karachi, PAKISTAN
e-mail: azizpobox1@yahoo.com
2H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, PAKISTAN
Received 02.01.2010
Biotransformation of danazol (17β -hydroxy-17α-pregna-2,4-dien-20-yno-[2,3-d] isoxazole) (1) on fer-
mentation with Fusarium solani yielded 17β -hydroxy-2-(hydroxymethyl)-17α-pregn-4-en-20-yn-3-one (2)
and 17β -hydroxy-2-(hydroxymethyl)-17α-pregna-1,4-dien-20-yn-3-one (3), while the fermentation of 1 with
Gibberella fujikuorii yielded compound 2 only. The structures of these compounds were deduced on the ba-
sis of modern spectroscopic techniques. Prolyl endopeptidase inhibition activities of danazol (1) and its
transformed products 2 and 3 are also studied.
Key Words: Biotransformation, danazol, Fusarium solani, Gibberella fujikuorii, prolyl endopeptidase
(PEP) inhibition assay
Introduction
Biotransformations of steroids exhibit a vast biochemical potential for the synthesis of new secondary metabo-
lites. Various kinds of steroid modifications, such as hydroxylation, epoxidation, dehydrogenation, double bond
formation, oxidation,1,2 reduction, glycosylation, isomerization, hydrolysis, and acetylation3,4 are now routinely
performed at the industrial level using a wide variety of microorganisms. Through these microbial reactions,
many novel intermediates for the synthesis of new steroidal pharmaceuticals have become available.
∗Corresponding author
945

Biotransformation of danazol by Fusarium solani and Gibberella fujikuorii..., AZIZUDDIN, M. I. CHOUDHARY
Danazol (1) is a heterocyclic compound with an isoxazole ring fused to ring-A of a steroidal nucleus. It
is an orally effective, pituitary gonadotropin inhibitor, and is effectively used in the treatment of endometriosis,
benign fibrocystic mastitis, and precocious puberty.5
Prolyl endopeptidase (PEP) or post-proline cleaving enzyme is now called “proly oligopeptidase” (EC.
3.4.21.26). It catalyzes the cleavage of the peptide bonds at the carbonyl side of proline residue.6,7 This enzyme
is widely distributed in various organs, particularly in the human brain. PEP activity has been reported to be
significantly higher in Alzheimer’s patients than in normal persons. In addition, the abnormal PEP levels may
be related to neuropathological disorders, such as major depression, mania, schizophrenia, and senile dementia
of Alzheimer’s type. PEP also plays a key role in the regulation of blood pressure by participating in the
rennin-angiotensin system through metabolism of bradykinin and angitensins II and I. Thus specific inhibitors
of PEP are expected to have anti-amnesic and other effects. Many PEP inhibitors have been synthesized as
candidates for the treatment of neuropathological disorders.
In continuation of our studies on biotransformation of bioactive compounds,8−10 danazol (1) was incu-
bated with Fusarium solani and Gibberella fujikuorii. Fermentation of danazol (1) with Fusarium solani yielded
compounds 2 and 3. Fermentation of 1 with Gibberella fujikuorii yielded only 2 (Table 1). They were reported
for the first time from these fungal strains. The structures of compounds 2 and 3 were established with the
help of spectroscopic studies and reported data.8 In the present study, we were also interested in examining the
prolyl endopeptidase inhibition activities of danazol (1) and its transformed products 2 and 3.
Table 1. % Yields of metabolites of danazol (1) by fermentation with Fusarium solani and Gibberella fujikuorii
after 12 days.
Microorganisms
Fusarium solani
% Yield of 2 % Yield of 3
10.8
12.6
9.6
-
Gibberella fujikuorii
Experimental
General Methods: The 1 H-NMR spectrum was recorded in CDCl3 on a Bruker AM-400 NMR spectrometer
with TMS as an internal standard using the UNIX operating system at 400 MHz. The 13 C-NMR spectra were
recorded in CDCl3 at 100 MHz on a Bruker AM-400 NMR spectrometer. The HREIMS was recorded on Jeol
JMS 600 and HX 110 mass spectrometers with the data system DA 5000. The IR spectra were recorded on a
Jasco A-302 spectrophotometer. The UV spectra were recorded on a Hitachi U-3200 spectrophotometer. The
optical rotations were measured on a JASCO DIP-360 digital polarimeter. The melting point was determined on
a Buchi 510 apparatus. Column chromatography (CC) was carried on a silica gel column (70-230 mesh). Purity
of the samples was checked by TLC on pre-coated silica gel GF-254 preparative plates (20 × 20 cm, 0.25 mm
thick, Merck), detected under UV light (254 and 366 nm), while ceric sulfate was used as the spraying reagent.
r
Danazol (1) was purified from tablets of Danocrineꢀ (200 mg each, Searle Pharmaceutical Company). Prolyl
endopeptidase (Flavobacterium meningosepticum origin) was purchased from Seikagaku Corporation (Tokyo,
Japan), N -benzyloxycarbonyl-Gly-Pro-pNA was procured from Bachem Fine Chemicals Co., and bacitracin was
purchased from Sigma Co., Ltd. Dr. Hideaki Shimizu (Yakult Central Institute for Microbiological Research,
946

Biotransformation of danazol by Fusarium solani and Gibberella fujikuorii..., AZIZUDDIN, M. I. CHOUDHARY
Tokyo, Japan) kindly gifted a specific inhibitor of PEP, N -benzyloxycarbonyl-pro-prolinal.
Preparation of Fermentation Media: Two liters of medium for F. solani (ATCC 12823) was prepared
by mixing glucose (20 g), peptone (10 g), KH2 PO4 (10 g), yeast extract (10 g), glycerol (20 mL), and NaCl
(10 g). Medium of G. fujikuorii (ATCC 10704) was prepared by mixing glucose (160 g), NH4 NO3 (1 g),
KH2 PO4 (10 g), MgSO4 .7H2 O (2 g), and Giberella trace element solution (4 mL) into distilled water (2 L).
The fermentation medium thus obtained was distributed equally among 20 flasks of 250 mL capacity (100 mL
in each) and autoclaved.
Cultivation of the Microbes: Two-day-old spores of the both microbes were transferred into the broth
medium flasks (250 mL) of their respective media containing freshly prepared and autoclaved media (100 mL).
The seed flasks of the fungi were incubated on a shake table at 30 ◦ C for 2 days.
Inoculation of the Cultures: Broth cultures (100 mL) from 2-day-old seed flasks of the fungi were
equally distributed to 18 media flasks (250 mL) containing the respective media (100 mL). The incubation was
continued for a further 2 days for fungi.
Fermentation of Danazol (1): Danazol (1) (200 mg) was dissolved in DMSO (10 mL) and the
resulting solution was evenly distributed among 18 conical flasks containing shake cultures of microbes, and the
fermentation was continued for 12 days.
Extraction and Isolation: Each mycelium was filtered, washed with EtOAc (500 mL), and the broth
thus obtained was extracted with EtOAc (6 L). The ethyl extract was dried over anhydrous sodium sulfate
and concentrated in vacuo to afford a brown gum (approximately, 1 g for each fungi), which was adsorbed on
flash silica gel (3 g), subjected to column chromatography. Elution with EtOAc:pet. ether (5:5) afforded 2 and
EtOAc:pet. ether (6:4) afforded 3.
Prolyl Endopeptidase Inhibition Assay: PEP inhibition activity was assayed by a modified method
of Yoshimoto et al.;11 100 mM Tris (hydroxymethyl)-aminomethane HCl buffer containing 1 mM EDTA (pH
7.0, 247 μL), PEP (0.02 units/well) 15 μL, and a stock solution of the test compound in MeOH (8 μL, diluted
to the desired range of concentrations) were mixed in 96-well microplate and preincubated for 10 min at 30
◦ C. The reaction was initiated by adding 30 μL of 0.2 mM of N -benzyloxycarbonyl-Gly-Pro-pNA (in 40%
1,4-dioxane) as the substrate. The amount of released p-nitroaniline was measured spectrophotometrically, as
increase in absorption at 410 nm with a 96-well microplate reader (Molecular Devices, SpectraMax 340, USA).
The percentage inhibition was calculated by the following equation:
% Inhibition = 100 – [(O.D. of test compound / O.D. of control) × 100]
The potency of enzyme inhibitory activity was represented by the IC50 values, which were defined as the
concentration of the test compound that resulted in 50% inhibition of the enzyme with respect to the MeOH
control. Z -pro-prolinal was used as a positive control.
Results
17β-Hydroxy-2-(hydroxymethyl)-17α-pregn-4-en-20-yn-3-one (2): Colorless crystalline solid, mp 164-
165 ◦ C, [α]D25 –20◦ (c 0.1, CHCl3). UV (MeOH) λmax (log ε): 242 nm (4.16). IR (CHCI3)νmax : 3412 (OH),
3308 (OH), 1654 (C=O), 1620 cm−1 (C=C). FDMS m/z 342 [M+ , 100%]. EIMS m/z (rel. int. %): 342
947

Biotransformation of danazol by Fusarium solani and Gibberella fujikuorii..., AZIZUDDIN, M. I. CHOUDHARY
[M+ ] (100), 327 (26), 275 (47), 189 (17), 121 (89), 91 (65). HREIMS m/z342.2204 (M+ , calcd 342.2195
for C22 H30 O3). 1 H- (CDCl3 , 400 MHz) and 13 C-NMR (CDCl3 , 100 MHz) data listed in Tables 2 and 3,
respectively.
Table 2. 1 H-NMR (400 MHz, CDCl3)a) chemical shifts of danazol (1) and its metabolites 2 and 3. δ in ppm and J
in Hz.
C. NO.
1
2
3
1
2.50 (d, J = 15.7)
1.52 (m)
6.98 (s)
2.72 (d, J = 15.7)
1.90 (m)
2
3
-
2.56 (m)
-
-
6.17 (d, J = 1.9)
-
-
5.71 (d, J = 1.7)
-
-
6.07 (d, J = 1.2)
-
4
5
6
2.38 (m); 2.43 (m)
1.04 (m); 1.78 (m)
1.50 (m)
2.37 (m); 2.41 (m)
1.05 (m); 1.81 (m)
1.50 (m)
2.37 (m); 2.41 (m)
1.06 (m); 1.84 (m)
1.50 (m)
7
8
9
1.18 (m)
1.15 (m)
1.15 (m)
10
11
12
13
14
15
16
-
-
-
1.62 (m); 1.47 (m)
1.76 (m); 1.67 (m)
-
1.62 (m); 1.47 (m)
1.74 (m); 1.67 (m)
-
1.62 (m); 1.47 (m)
1.72 (m); 1.67 (m)
-
1.53 (m)
1.53 (m)
1.53 (m)
1.74 (m); 1.36 (m)
2.30 (ddd, J = 13.9, 9.6, 5.5)
1.74 (m); 1.36 (m)
2.30 (ddd, J = 13.9, 9.6, 5.5)
1.74 (m); 1.36 (m)
2.28 (ddd, J = 13.9, 9.6, 5.5)
2.00 (ddd, J = 13.9, 11.9, 4.0) 1.98 (ddd, J = 13.9, 11.9, 4.0) 1.98 (ddd, J = 13.9, 11.9, 4.0)
17
18
19
20
21
22
-
-
-
0.92 (s)
0.90 (s)
1.02 (s)
-
0.88 (s)
1.23 (s)
-
1.24 (s)
-
2.57 (s)
8.00 (s)
2.55 (s)
2.53 (s)
3.70 (dd, J = 14.6, 11.3)
3.72 (dd, J = 18.0, 11.3)
4.34 (d, J = 13.2)
4.36 (d, J = 13.2)
aAssignments based on COSY and HMQC.
17β-Hydroxy-2-(hydroxymethyl)-17α-pregna-1,4-dien-20-yn-3-one (3): Colorless solid, mp
214-215 ◦ C; [α]D25 -54◦ (c1.0, CHCl3). UV (MeOH) λmax (log ε): 248 nm (4.22). IR (CHCl3)νmax : 3425
(OH), 3314 (OH), 1667 (C=O), 1622 (C=C), 1608 cm−1 (C=C). EIMS m/z (rel. int. %), 340 [M+ ] (26),
239 (7), 173 (10), 134 (100), 121 (40), 91 (29), 55 (12). HREIMS m/z 340.2102 (M+ , calcd 340.2038 for
C
22 H28 O3). 1 H- (CDCl3 , 400 MHz) and 13 C-NMR (CDCl3 , 100 MHz) data listed in Tables 2 and 3, respec-
tively.
948

Biotransformation of danazol by Fusarium solani and Gibberella fujikuorii..., AZIZUDDIN, M. I. CHOUDHARY
Table 3. 13 C-NMR (100 MHz, CDCl3)a)b) chemical shifts of danazol (1) and its metabolites 2 and 3.
C. NO.
1
1
2
3
32.9 (CH2) 38.9 (CH2)
151.5 (CH)
135.8 (C)
187.0 (C)
123.7 (CH)
169.9 (C)
32.6 (CH2)
32.4 (CH2)
36.2 CH)
52.2 (CH)
43.6 (C)
2
107.5 (C)
164.9 (C)
43.7 (CH)
202.6 (C)
3
4
108.1 (CH) 123.6 (CH)
155.1 (C) 171.8 (C)
5
6
32.0 (CH2) 32.4 (CH2)
30.4 (CH2) 31.4 (CH2)
7
8
36.7 (CH)
53.7 (CH)
40.8 (C)
36.2 (CH)
53.9 (CH)
39.3 (C)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
21.0 (CH2) 20.7 (CH2)
32.9 (CH2) 32.6 (CH2)
22.7 (CH2)
33.2 (CH2)
47.0 (C)
46.4 (C)
46.7 (C)
49.5 (CH)
49.9 (CH)
49.6 (CH)
23.2 (CH2)
38.9 (CH2)
79.6 (C)
22.9 (CH2) 23.1 (CH2)
38.4 (CH2) 38.9 (CH2)
78.9 (C)
79.6 (C)
12.3 (CH3) 12.7 (CH3)
18.4 (CH3) 17.7 (CH3)
12.8 (CH3)
18.8 (CH3)
74.3 (C)
73.2 (C)
74.2 (C)
87.2 (CH)
87.3 (CH)
87.1 (CH)
148.3 (CH) 63.8 (CH2)
62.5 (CH2)
aMultiplicities were determined by DEPT experiments.
bAssignment based on HMQC and HMBC.
Discussion
Biotransformation of danazol (1) using Fusarium solani and Gibberella fujikuorii yielded compounds 2 and 3
(Scheme). The structures of these compounds were identified on the basis of modern spectroscopic methods and
comparison with reported data. They were found to be 17β-hydroxy-2-(hydroxymethyl)-17α-pregn-4-en-20-
yn-3-one (2)8 and 17β-hydroxy-2-(hydroxymethyl)-17α-pregna-1,4-dien-20-yn-3-one (3).8 They were reported
for the first time from these fungal strains.
Transformed products 2 and 3 of danazol (1) were screened for their enzyme inhibitory activity against
prolyl endopeptidase (PEP). These compounds 1-3 displayed inhibitory potential against the PEP enzyme.
Danazol (1) showed the most potent inhibition against the PEP amongst all the compounds, while its metabolic
derivatives 2 and 3 were also found to be significant inhibitors but showing lesser potency than danazol (1).
The activities of compounds 1-3 are shown in Table 4.
949

Biotransformation of danazol by Fusarium solani and Gibberella fujikuorii..., AZIZUDDIN, M. I. CHOUDHARY
OH
CH
C
H
H
HOH2C
O
H
H
OH
CH
C
2
OH
CH
H
12 days
C
2'
H
H
N
HOH2C
O
O
H
H
Danazol (1)
3
Scheme. Biotransformation of danazol (1) by fermentation with F. solani and G. fujikuorii after 12 days.
Table 4. Prolyl endopeptidase (PEP) inhibition activities by compounds 1-3.
Compounds
IC50 (μM) S. E. M.
57.4 0.002
1
2
379.3 0.00081
827.7 0.04
3
Z-pro-prolinal *
880 0.001
IC50 = Concentration of a test compound
S. E. M. = Standard error of the mean of 5 assays
* = Standard inhibitor used for the inhibition of prolyl endopeptidase
Acknowledgements
Azizuddin acknowledges Ms. Amsha Yaseen for conducting prolyl endopeptidase inhibition activities.
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