Fungal biotransformation of diuretic and antihypertensive drug spironolactone with Gibberella fujikuroi, Curvularia lunata, Fusarium lini, and Aspergillus alliaceus

Article abstract of DOI:10.1016/j.steroids.2017.10.003

Derivatives of spironolactone (1), a diuretic and antihypertensive drug, were synthesized by using fungal cells for the first time. Ten different fungi were screened for their ability to biotransform 1, four of which were able to produce metabolites 2–8. Gibberella fujikuroi produced canrenone (2), 1-dehydrocanrenone (3), Curvularia lunuta provided compound 2, and 7α-thio-spironolactone (4), Fusarium lini yielded compounds 2, 3, 1β-hydroxycanrenone (5), 1α-hydroxycanrenone (6), 1-dehydro-15α-hydroxycanrenone (7), and 15α-hydroxycanrenone (8), while Aspergillus alliaceus was able to produce all the seven metabolites. Metabolites 5, 6, and 7 were identified as new compounds. Their structures were elucidated by using different spectroscopic techniques. Substrate 1 and its metabolites 2, 3, and 5–8 were also evaluated for α-glucosidase inhibitory activity in vitro. Substrate 1 was found to be strongly active with IC₅₀ = 335 ± 4.3 μM as compared to the standard drug acarbose IC₅₀ = 840 ± 1.73 μM, whereas all of resulting metabolites were found to be inactive.

Full text of DOI:10.1016/j.steroids.2017.10.003

Accepted Manuscript
Fungal biotransformation of diuretic and antihypertensive drug spironolactone
with Gibberella fujikuroi, Curvularia lunata, Fusarium lini, and Aspergillus
alliaceus
Amal Al-Aboudi, Belal Muneeb Kana'an, Musa Abu Zarga, Saira Bano, Atia-
tul-Wahab, Kulsoom Javed, M Iqbal Choudhary
PII:
S0039-128X(17)30183-6
https://doi.org/10.1016/j.steroids.2017.10.003
STE 8167
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 May 2017
4 October 2017
9 October 2017
Please cite this article as: Al-Aboudi, A., Kana'an, B.M., Zarga, M.A., Bano, S., Atia-tul-Wahab, Javed, K.,
Choudhary, M.I., Fungal biotransformation of diuretic and antihypertensive drug spironolactone with Gibberella
fujikuroi, Curvularia lunata, Fusarium lini, and Aspergillus alliaceus, Steroids (2017), doi: https://doi.org/10.1016/
j.steroids.2017.10.003
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Fungal biotransformation of diuretic and antihypertensive drug
spironolactone with Gibberella fujikuroi, Curvularia lunata,
Fusarium lini, and Aspergillus alliaceus
Amal Al-Aboudia*, Belal Muneeb Kana'ana, Musa Abu Zargaa, Saira Banob,
Atia-tul-Wahabc, Kulsoom Javedb, and M Iqbal Choudharyb,c,d
*
aChemistry Department, Faculty of Science, The University of Jordan, Amman-11942, Jordan
bH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
cDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for
Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
dDepartment of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah-21412,
Saudi Arabia
Corresponding authers: alaboudi@ju.edu.jo; iqbal.choudhary@iccs.edu
ABSTRACT
Fungal cells were used for the first time to synthesize derivatives of spironolactone (1), a diuretic
and antihypertensive drug. Ten different fungi were screened for their ability to biotransform 1,
four of which were able to produce metabolites 2-8. Gibberella fujikuroi produced canrenone
(2), 1-dehydrocanrenone (3), Curvularia lunuta provided compound 2, and 7α-thio-
spironolactone (4), Fusarium lini yielded compounds 2, 3, 1β-hydroxycanrenone (5), 1α-
hydroxycanrenone (6), 1-dehydro-15α-hydroxycanrenone (7), and 15α-hydroxycanrenone (8),
while Aspergillus alliaceus was able to produce all the seven metabolites. Metabolites 5, 6, and 7
were identified as new compounds. Their structures were elucidated by using different
spectroscopic techniques. Substrate 1 and its metabolites 2, 3, and 5-8 were also evaluated for α-
glucosidase inhibitory activity in vitro. Substrate 1 was found to be strongly active with IC50 =
335 4.3 µM as compared to the standard drug acarbose IC50 = 840 1.73 µM, whereas all of
resulting metabolites were found to be inactive.

Keywords: Spironolactone (Antihypertensive drug); Aspergillus alliaceus; Curvularia lunuta;
Fusarium lini; Gibberella fujikuroi; α-Glucosidase inhibitory activity
1.
Introduction
Spironolactone (1), a synthetic steroid, is a competitive antagonist of aldosterone. It increases the
secretion of water and sodium, while decreasing the excretion of potassium by competing for the
aldosterone sensitive Na+/K+ channel in the distal tubule of the nephrons. For decades,
spironolactone (1) has been considered as an antagonist at the aldosterone receptors of the
epithelial cells of the kidney, and is clinically used in the treatment of hyper aldosteronism, and
occasionally as a potassium-sparing diuretic agent [1]. Spironolactone (1) is rapidly metabolized
into a large number of derivatives by the liver [2]. The major metabolism of compound 1 results
in the formation of canrenone (2), and 7α-methylthiospironolactone (4) [3], both of which are
belived to mediate the therapeutic action of compound 1.
Inspired by the extensive literature on the biotransformation of steroids, we decided to study the
ability of the enzymatic systems of whole cell fungi to synthesize derivatives of spironolactone
(1). Chemists have found widespread applications of biotransformation in the structural
modifications of steroids with high regio- and stereo-selectivity [4]. Examples of hydroxylation,
oxidation, reduction, isomerization, Michael addition, and reverse aldol reactions are well as
documented in the literature [5].
Enzymes or whole cells biotransformation represent the most efficient catalytic systems known
to carry out conventional chemical reactions [6, 7]. They play a vital role in the preparation of
new oxygenated derivatives with different biological activities [8].
Previously we have reported several steroids having strong inhibitory activity against the enzyme
α-glucosidase [9-12]. The discovery of the new α-glucosidase inhibitors provides an approach in
the management of diabetes and hyperglycemia [13]. In this study, microbial transformation was
used to synthesize derivatives of the diuretic and antihypertensive drug, spironolactone (1).
These derivatives were then evaluated for their α-glucosidase inhibitory activity in vitro.
Initially, we screened spironolactone (1) against various bioactivities but it was found to be
active against only α-glucosidiase enzyme. Therefore, we decided to evaluate transformed
products of 1 against this enzyme.

2.
Material and Methods
General
2.1
Spironolactone (1) (C24H32O4S) was obtained from Jordan Pharmaceutical Manufacturer.
Routine thin-layer chromatography was performed on Precoated TLC plates (silica gel, 20×20,
0.25 mm thick PF, Merck, Germany), and ceric sulfate solution was used as staining reagent.
Column chromatography was performed on silica gel (70–230 mesh, Merck) for fractionation.
Recycling preparative HPLC separation was performed on a JAI LC-908W instrument, equipped
with YMC L-80 (4–5 µm, 20−50 mm i.d.) using MeOH-H2O as the mobile phase, with UV
detection at 254 nm. NMR spectra were recorded on Bruker Avance NMR spectrometers
(Bruker, Spectrospin, Switzerland) at 400, 500 or 600 MHz in CDCl3 or CD3OD. HR-ESI mass
spectra were recorded using a Bruker Daltonics Apex IV, 7.0T Ultra Shield Plus (Bruker
Daltonics, Germany), and a JEOL JMS-600H (JEOL, Japan) mass spectrometer was used to
record the electron impact mass spectra (EI-MS). Specific rotations were measured withMCP
200 polarometer (Germany). UV Spectra were measured in methanol on a Hitachi U-3200
spectrophotometer (Japan). Infrared (IR) spectra were measured using Shimudzu FT IR-8900 or
Thermo-Nicolt Nexus 870FT-IR spectrophotometers (Japan). Melting points were measured
without correction, using an Electrothermal (IA9300) digital melting point apparatus (Japan).
2.2
Microorganism
Fungi cultures were purchased from three international culture collection centers: American
Type Culture Collection (ATCC), Northern Regional Research Laboratories (NRRL) and
Tryptone Soya Yeast Extract (National Institute of Health Sciences, Tokyo-Japan) (TSY). The
fungi used were Aspergillus alliaceus (ATCC 10060), Aspergillus flavus (ATCC 11489),
Aspegillus niger (ATCC 1015), Beauveriabassiana (ATCC 7159), Cunninghamella blakesleeana
(ATCC 8688A), Cunninghamella elegans (ATCC 36114), Curvularia lunata (ATCC 12017),
Fusarium lini (NRRL 2204), Gibberella fujikuroi (ATCC 10704), and Rhizopus stolonifer (TSY
0471).

2.3 Preparation of media
Fungi were grown on Sabouraud dextrose agar (SDA), and preserved at 4 °C. The constituents of
a suitable medium for each fungus (Table-1) were dissolved in 1.0 liter distilled water, and the
pH was then adjusted by 0.1 M NaOH, and 0.1 M HCl solutions.
2.4 General screening protocol
Preparation of second generation culture: The media (100 mL) were transferred into conical
flasks (250 mL), and autoclaved at 124 °C for 15 min. Three-day old slant fungal spores were
added to the media under aseptic conditions, and incubated on a shaker (128 rpm) at 28 °C until
a reasonable biomass was obtained. The mycelia thus formed were distributed into conical flasks
containing 100 mL autoclaved media, and incubated on a shaker at 28 oC for a few days until a
good biomass was obtained.
Addition of the substrate: The substrate was dissolved in 12 mL acetone (25 mg/mL), and
added to flasks containing second stage culture, so that each flask contained about 25 mg of the
substrate. For every screening study a positive control containing the substrate in the media
without the fungus, and a negative control containing the fungus in the media without the
substrate were prepared. The flasks were incubated in a shaker (128 rpm) at 28 °C.
Monitoring the biotransformation: A 10 mL sample of media was taken from the flask and
filtered, and extracted with ethyl acetate. It was then evaporated, and reconstituted with 0.5 mL
of chloroform, and tested by TLC. The TLC results were compared with the positive and
negative controls, which were treated in the same way. The progress of the biotransformation
was monitored every two days for a maximum of three weeks. Each of the ten fungi (Table-1)
was screened for its ability to biotransform the three substrates under study. The results of the
screening tests were re-produced on a larger scale to purify, and identify the biotransformation
products.
2.5
Biotransformation of spironolactone (1)
Each of the ten fungi (Table-1) was screened for their ability to biotransform spironolactone (1)
according to the above screening procedure. The results of the screening tests showed that four
cultures, which include Gibberella fujikuroi, Curvularialunata, Fusarium lini, and Aspergillus

alliaceus, were suitable for biotransformation of compound 1, and these tests were re-produced
on a larger scale to separate, and identify the biotransformation products.
2.5.1 Biotransformation of spironolactone using Gibberella fujikuroi
Compound 1 (1,000 mg/ 20 mL) was distributed in 40 flasks, containing 3-day-old second
generation culture of G. fujikuroi, and kept for 10 days. The mycelia were filtered and extracted
with ethyl acetate. The organic layer was dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The brown gummy material (1.1 g) obtained was subjected to silica gel column
chromatography with gradient polarity of hexane: ethyl acetate mixtures of increasing polarity.
Two main fractions were obtained and detected on thin layer chromatography (TLC). Fraction 1
(30 mg) yielded compound 1 (10 mg) by using preparative silica gel thin layer chromatography
(precoated-TLC) (EtOAc: hexane 60:40 v/v) as a mobile phase. Fraction 2 (300 mg) yielded
compounds 2 and 3 by using EtOAc: hexane (60:40, v/v) as a mobile phase. Compounds 2 (Rt:
49 min, 2 mg), and 3 (Rt: 41 min, 2 mg) were further purified by repeated RPHPLC (L-80,
MeOH: H2O = 70:30, 4 mL/min) (Figure 1).
2.5.2 Biotransformation of spironolactone (1) using Curvularia lunata
Compound 1 (750 mg/ 25 mL acetone) was distributed in 30 flasks containing 3 days old second
generation culture of C. lunata, and kept for fermentation for 14 days. A crude brown gummy
material (0.9 g) was obtained upon following the same steps mentioned above. The crude extract
was then chromatographed over the silica gel column by elution with gradient hexanes-ethyl
acetate solvent system which yielded three main fractions. Fraction 1 (300 mg) was purified to
get spironolactone (1) (20 mg) using EtOAc: hexane 60:40 v/v) as a mobile phase, while fraction
2 (100 mg) was purified to yield metabolite 2 (1.5 mg) using EtOAc: hexane (60:40, v/v) as the
mobile phase. Fraction 3 (100 mg) was stored in a freezer at -4 oC until it was finally purified by
repeated RP-HPLC L-80, MeOH: H2O = 70:30, 4 mL/min) with, 4 mL/ min) to obtained
metabolite 4 (Rt: 19 min, 4 mg) (Figure 2).
2.5.3 Biotransformation of spironolactone using Fusarium lini
Spironolactone (1) (1,500 mg/ 30 mL acetone) and solution was distributed in 60 flasks
containing three days old second generation culture of F. lini, and kept for fermentation for 14
days. The brown gummy material (1.1 g) obtained was subjected to silica gel column

chromatography with gradient polarity of hexane: ethyl acetate mixtures of increasing polarity.
Four main fractions were obtained on the basis of TLC analysis. Each fraction was subjected to
preprative-TLC. Fraction 1 (100 mg) was purified to obtain compound 1 (12 mg) using EtOAc:
hexane (60:40, v/v) as a mobile phase, while fraction 2 was purified to obtain pure metabolites 2
(1 mg), and 3 (1 mg) using EtOAc: hexane 60:40 v/v as mobile phase. Fraction 3 (100 mg)
yielded a mixture using MeOH: CHCl3 (6: 94 with ammonia vapor, v/v), which was then
subjected to repeated RP-HPLC (L-80, MeOH-H2O = 70:30, 4 mL/min), to obtain metabolites 5
(Rt: 28 min, 3 mg), 6 (Rt: 23 min, 5 mg), and 7 (Rt: 21 min, 12 mg). Fraction 4 (35 mg) yielded
metabolite 8, using CHCl3:CH3OH (92:8 v/v with ammonia vapor) as a mobile phase, and further
purified by RP-HPLC (L-80, MeOH-H2O = 70:30, 4 mL/min), afforded metabolite 8 (Rt: 25 min,
5mg) (Figure 3).
2.5.4 Biotransformation of spironolactone (1) using Aspergillus alliaceus
Compound 1 (1,250 mg/ 25 mL of acetone) was distributed in 50 flasks containing 3-day-old
second generation culture of A. alliaceus, and kept for fermentation for 10 days. The brown
gummy material (1.5 g) was obtained after treating the mycelia, and media. Five main fractions
were obtained on the basis of TLC analysis. Fraction 1 (50 mg), eluted from column at 30%
ethyl acetate in hexane, consisted mainly of compound 1 (8 mg), while fraction 2 (100 mg),
eluted from column at 35-40% ethyl acetate in hexane, consisted of metabolites 2 (1 mg) and 3
(1.5 mg). Fraction 3 (15 mg), eluted from column at 45% ethyl acetate in hexane, consisted of
metabolite 4 (0.8 mg). Fraction 4 (75 mg), eluted from column at 60-65% ethyl acetate in
hexane, was subjected to RP-HPLC using the solvent system MeOH: H2O (70:30, v/v) on L-80
column, to obtain metabolites 5 (1 mg), 6 (1.3 mg), and 7 (1.4 mg). Fraction 5 (60 mg), eluted
from column at 70% ethyl acetate in hexane, was also subjected to RP-HPLC, using the solvent
system MeOH: H2O (70:30, v/v) on L-80 column, to afford metabolite 8 (1.8 mg) (Figure 4).
2.6
Spectral data of the isolated compounds
1
The H- and 13C-NMR data of the isolated compounds are summerised in Tables-2 and -3,
respectively. 2D-NMR experiments (HSQC, COSY, HMBC, and NOESY) were used to
determine the structures of all metabolites. Other spectroscopic results are summerised as
follows:

Spironolactone (1)
M.p. = 198–202 °C (lit. [14] 198-197 °C) [ꢀ]ꢂꢁꢃ = –33.5°, UV (MeOH) λmax nm: 240. IR (KBr)
1
13
νmax cm-1:1768 (C=O), 1690 (C=C). H-NMR (CDCl3, 500 MHz): Table-2. C-NMR (CDCl3,
125 MHz): Table-4. EI-MS (rel. int. %): m/z 416.2 [M+], C24H32O4S. HR-ESI-MS: m/z
439.19123 [M+Na+], (C24H32O4SNa, calcd. 439.19135).
Canrenone (2)
o
o
[ ]ꢂꢃ
M.p. = 148-150 C (John, et al [15]148-150 °C). ꢀ : -25.96 (CHCl3), UV (MeOH) λmax nm:
ꢁ
13
283, IR (KBr) νmax cm-1: 1765 (C=O), 1654 (C=C).1H-NMR (CDCl3, 400 MHz): Table-2. C-
NMR (CDCl3, 100 MHz): Table-3. EI-MS (rel. int. %): m/z 340.3 [M+], C22H28O3. HR-ESI-
MS: m/z 363.19254 [M+Na]+, (C22H28O3Na, calcd. 363.19307).
1-Dehydrocanrenone (3)
o
o.
[ ]ꢂꢃ
M.p. = 135-139 C (John, et al [15] 135-139 °C). ꢀ : -27.11 (CHCl3), UV (MeOH) λmax
ꢁ
nm: 298 and 222. IR (KBr) νmax cm-1: 1769 (C=O), 1650 (C=C). 1H-NMR (CDCl3, 400 MHz):
13
Table-2. C-NMR (CDCl3, 100 MHz): Table-3. EI-MS (rel. int. %): m/z338 [M+], C22H26O3
.HR-ESI-MS: m/z 361.17708 [M+Na]+, (C22H26NaO3, calcd. 361.17742).
7α-Thio-spironolactone (4)
o
o
[ ]ꢂꢃ
M.p. = 188-191 C (Karim et al [16] 188-190 °C). ꢀ : -26.96 (CHCl3). UV (MeOH) λmax
ꢁ
nm: 242. IR (KBr) νmax cm-1: 3416 (S-H), 1768 ((C=O), 1663 (C=C).1H-NMR (CDCl3, 600
MHz): Table-2. 13C-NMR (CDCl3, 150 MHz): Table-3. EI-MS (rel. int. %): m/z374.0 [M+],
C22H30O3S. HR-ESI-MS: m/z 373.18420 [M-H]-, (C22H29O3S, calcd. 373.18429).
1β-Hydroxycanronene (5)
o
Amorphous, ꢀ : -30.47 (CHCl3). UV (MeOH) λmax nm: 283. IR (KBr) νmax cm-1: 3385
[ ]ꢂꢃ
ꢁ
(O-H), 1738 (C=O), 1631(C=C). 1H-NMR (CDOD3, 500 MHz): Table-2. 13C-NMR (CDCl3, 125
MHz): Table-3. EI-MS (rel. int. %): m/z 356 [M+]. HR-ESI-MS: m/z 379.18768 [M+ Na]+,
(C22H28O4Na, calcd. 379.18798).
1α-Hydroxycanrenone (6)

o
Amorphous, ꢀ : -27.52 (CHCl3). UV (MeOH) λmax nm: 283. IR (KBr) νmax cm-1: 3407
[ ]ꢂꢃ
ꢁ
13
(O-H), 1750 (C=O), 1630 (C=C).1H-NMR (CDCl3, 400 MHz): Table-2. C-NMR (CDCl3, 100
MHz): Table-3. EI-MS (rel. int. %): m/z 356 [M+]. HR-ESI-MS: m/z 379.18762 [M+ Na]+,
(C22H28O4Na, calcd. 379.18798).1-Dehydro-15α-hydroxycanrenone (7)
o
Amorphous, ꢀ : -26.24 (CHCl3). UV (MeOH) λmax nm: 298 and 222. IR (KBr) νmax cm-1:
[ ]ꢂꢃ
ꢁ
1
3413 (C=O), 1766 (C=O),1652 (C=C). H-NMR (CDOD3, 500 MHz): Table-2. 13C-NMR
(CDCl3, 125 MHz): Table-3. EI-MS (rel. int. %): m/z 354 [M+], C22H26O4. HR-ESI-MS: m/z
377.17238 [M+ Na]+, (C22H26O4Na, calcd. 379.17233).
15α-Hydroxycanrenone (8)
o
o
M.p. = 234-238 C. ꢀ : -24.57 (CHCl3). UV (MeOH) λmax nm: 283.IR (KBr) νmax cm-1:
[ ]ꢂꢃ
ꢁ
13
3396 (O-H), (1738C=O), 1646 (C=C).1H-NMR (CDCl3, 500 MHz): Table-2. C-NMR (CDCl3,
125 MHz): Table-3. EI-MS (rel. int. %): m/z 356.1 [M+] , C22H28O4. HR-ESI-MS: m/z
379.18762 [M+Na]+, (C22H28O4Na, calcd. 379.18798).
2.7 Assay for α-Glucosidase Inhibitory Activity
α-Glucosidase (E.C.3.2.1.20) enzyme inhibition assay was performed according to the slightly
modified method of Matsui [17]. The activity was evaluated by using 0.1 M phosphate buffer
(pH 6.8) at 37 °C. The enzyme (0.2 U/mL) in phosphate buffer saline was incubated with various
concentrations of test compounds at 37 °C for 15 min. The substratep-nitrophenyl-α-d-
glucopyranoside (0.7 mM) was added, and change in absorbance at 400 nm was monitored upto
30 min by multiplate reader. Test compound was replaced with DMSO (7% final) as control.
Acarbose was used as the standard inhibitor. The IC50 value was calculated by using EZ- FIT5
software and the percent inhibition was calculated by the following formula:
% Inhibition = 100 - (OD test well / OD control) × 100.3. Results and Discussion
3.1 Biotransformation of spironolactone (1)
Ten different microorganisms (Table-1) were studied for their ability to transform spironolactone
(1). The screening results showed that four fungi A. alliaceus, G. fujikuroi, C. lunata, and F. lini,
were able to biotransform 1. Large scale fermentation experiments were carried out in order to
isolate metabolites 2-8, produced as a result of whole cell biocatalysis of the enzymatic system of
Gibberella fujikuroi, Curvularia lunata, Fusarium lini, and Aspergillus alliaceus.

Biotransformation of compound 1 with these four fungi resulted in the synthesis of three new (5-
7), and four known metabolites 2-4, and 8.
Compound 1 was fully characterized by HR-ESI-MS, 1D- and 2D-NMR experimentsused as
reference. The results of NMR assignments were in full agreement with the reported data [12].
HR-ESI-MS of 5 showed the [M+Na]+ at m/z 379.18768 (C22H28O4Na, calcd. 379.18798),
indicating the presence of an additional oxygen, and removal of sulfur as compared to compound
1. The IR spectrum showed absorption at 3385 cm-1, indicating the presence of a hydroxyl group.
The spectrum also showed the presence of carbonyls at 1738 cm-1, and olefine C=C at 1630 cm-1.
1
The H- and13C-NMR spectra of compound 5 showed, upon comparison with the spectra of 2, the
disappearance of a methylene signal and the appearance of a methine proton signal at δ 4.06 (dd,
J1ax,2ax= 12.5, J1ax,2eq= 5.0 Hz) connected to a carbon resonating at δ 73.2, which indicated the
presence of a hydroxyl group. The downfield shift of the methylene carbon at δ 43.9 (C-2)
suggested that the hydroxyl group might be at C-1. Presence of OH group at C-1 was deduced from
2D-NMR. The COSY spectrum showed cross peaks between δ 4.06 (H-1) and 2.66, and 2.60 (H2-
2) (Figure 5). HMBC showed correlations of C-1 (δ 73.2) with 19-CH3 (δ 1.14) and H2-2 (δ 2.66,
2.60). In addition, in HMBC H2-2 (δ 2.66, 2.60) also showed correlations with C-3 at δ 196.9
(Figure 5). The β (equatorial) stereochemistry of the newly introduced hydroxyl at C-1 in 5 was
inferred from the multiplicity of H-1 at δ 4.06, which appeared as a dd with one larger coupling
constant, J1ax,2ax=12.5 Hz due to the axial–axial, and a small coupling constant (J1ax,2eq= 5.0 Hz)
due to the axial-equatorial coupling of protons. Finally, the stereochemistry of OH-1 was
established from NOESY spectrum, which showed a correlation between Hax-1(δ 4.06) and Hax-9
(δ1.40) (Figure 6). The structure of metabolite 5 was thus deduced as 1β-hydroxycanrenone.
HR-ESI-MS of compound 6 showed the [M+Na]+ at m/z 379.18762 (C22H28O4Na, calcd.
379.18798) indicating the presence of an additional oxygen, and removal of sulfur compared to
compound 1. The IR spectrum showed absorption at 3407, 1750, and 1630 cm-1 due to the
hydroxyl, carbonyl and olefinic functionalities, respectively. The 13C-NMR (CDCl3) spectrum of
compound 6 showed 22 signals, similar to those of compound 5. The 1H-NMR spectrum showed
an OH-bearing methine proton at δ 4.18 (1H, bs), connected to the carbon at δ 71.7. 2D-NMR
experiments indicated the presence of the hydroxyl group at C-1. The COSY spectrum showed
correlation of H-1 (δ 4.18) with H2-2 (δ 2.58 and δ 2.88) (Figure 5). The HMBC spectrum

showed correlations of H-1 (δ 4.18) with C-19 (δ 17.8) and C-2 (δ 43.0). more H-2 (δ 2.58 and δ
2.88) also showed correlations with C-3 (δ 196.9) (Figure 5). The α-stereochemistry of OH-1
was inferred from the NOESY correlations between Heq-1 (δ 4.18), and CH3-19 (δ 1.13). This
correlation was not seen in 5 due to different stereochemistry of OH-1 (Figure 6). Based on the
above data, the structure of metabolite 6 was deduced as 1α-hydroxycanrenone.
Metabolite 7 showed the [M+Na]+ at m/z 377.17238 (C22H26O4Na, calcd 377.17233) in the
HRESI-MS, indicating an additional oxygen and increased degree of unsaturation compared to
compound 1. IR spectrum showed absorptions at 3396, 1738, and 1646 cm-1 for hydroxyl,
1
carbonyl, and olefinic functionalities, respectively. The H-NMR spectrum showed a new
resonance for an OH-bearing methine proton at δ 4.27 (1H, td, J15ax 16ax/14ax= 10.0, J15ax,16eq =3.0
,
Hz) assigned to H-15. H-15 showed COSY correlation with H2-16 (δ1.83 and 2.83) and H-14 (δ
1.40) (Figure 5), while in HMBC spectrum, the new hydroxylated methine carbon at δ 71.1
showed correlations with H-14 (δ 1.40) and H2-16 (δ 1.83 and 2.83) (Figure 5). The α
(equatorial) orientation of the newly introduced hydroxyl at C-15 was inferred from the
multiplicity of H-15 signal at δ 4.40 as a triplet of doublet (td) with one large coupling constant
due to coupling of the two di-axial coupling (J15ax,16ax
= 10.0 Hz) and a small coupling constant
=3.0 Hz). In addition to the multiplicity of
due to axial–equatorial coupling of protons (J15ax,16eq
Hax-16 (δ 2.83) which appeared as a dd with two large coupling constants due to coupling of the
two geminal protons (J16ax, 16eq=15.0 Hz), and due to coupling of the two di-axial protons (J16ax,
15ax = 10.0 Hz). NOESY spectra showed correlations between Hax-15 (δ 4.40) and H3-18 (δ 1.06)
and Hax–8 (δ 2.52) indicated the α stereochemistry of H-15 (Figure 6). Based on the above
mentioned spectral data, the structure of metabolite 7 was deduced as 1-dehydro-15α-
hydroxycanrenone.
Compounds 2-4, and 8 were characterized as known compounds based on detailed spectral
analyses. Canrenone (2) was reported earlier by Chen et al. [18]. It is worth mentioning that
canrenone (2) is the key intermediate in the synthesis of spironolactone, a widely used
aldosterone antagonist diuretic in the clinic [19]; in addition, canrenone (2) is one of the main
known metabolites of spironolactone in rat serum after oral administration of the drug [20]. 1-
Dehydrocanrenone (3), a known synthetic steroid [14], is used to synthesize 2-oxasteroids, which
are used to enhance the antiandrogenic activity [21]. Although 1-dehydrocanrenone (3) is known

as a synthetic steroid, but it is not known among the reported mammalian metabolites of
spironolactone. Thus, this is the first report of conversion of 1 to 1-dehydrocanrenone using
whole cell microbial transformation. The spectral data of this compound are reported for the first
time, and compared with the values of similar compounds [22]. The 1H- and 13C-NMR chemical
shifts of different hydrogen and carbon atoms of 4 are in full agreement with literature data [18].
7α-Thio-spironolactone (4) is known as an intermediate steroid in synthesis of spironolactone
(1). Moreover, spironolactone (1) and its metabolite 7α-thio-spironolactone (4) were also
determined in pediatric plasma samples by HPLC method [23]. 15α-Hydroxycanrenone (8) was
first reported previously as a biotransformation product of canrenone (2) using Aspergillus
ochraceus, and by efficient hydroxylation of functionalized steroids by Colletotrichum lini ST-1
without determination of the exact stereochemistry of the OH-15 group [24, 25].
3.2
α-Glucosidase Inhibitory Activity
Spironolactone (1) and its metabolites 2, 3, and 5-8 were tested for α-glucosidase inhibitory
activity in vitro. Results indicated that only 1 possess a strong activity with IC50 = 335 4.3 µM,
as compared to the standard drug acabose (IC50 =840 1.73 µM), while its metabolites were
found inactive in this assay.
4.0
Conclusion
In conclusion, we report here an efficient method for the transformation of a diuretic and
antihypertensive drug spironolactone (1) by using various fungal cultures. Biotransformation of
compound 1 with these four fungi resulted in the production of three new 5-7, and four known
metabolites 2-4, and 8. Compounds were also evaluated for α-glucosidase inhibitory activity in
vitro, as a result compound 1 was identified as a strong inhibitor with IC50 = 335 4.3µM, as
compared to the standard drug acabose IC50 = 840 1.73 µM, while its metabolites were found
to be inactive in this assay. This strategy can be used effectively for the synthesis of libraries of
new derivatives of spironolactone (1).
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Figure 1: Biotransformation of spironolactone by Gibberella fujikuroi.
Figure 2: Biotransformation of spironolactone by Curvularia lunata.

Figure 3: Biotransformation of spironolactone by Fusarium lini.

Figure 4: Biotransformation of spironolactone by Aspergillus alliaceus.
O
O
O
O
OH
OH
H
H
H
H
H
H
O
O
6
5
O
O
H
H
7
H
OH
O
Figure 5: Key COSY and HMBC correlations in metabolites 5-7.

Figure 6: Key NOESY correlations in metabolites 5-7.

Table-1: Constituents of the media for each fungus.
Constituents of the media/ L solution
Fungi
Glucose (20.0 g), peptone (5.0 g), yeast
extract (3.0 g), KH2PO4 (5.0 g), pH= 5.6.
Rhizopus stolonifer (TSY 0471)
Fusarium
lini
(NRRL
68751),
Cunninghamella blakesleeana (ATCC Glucose (10.0 g), glycerol (10.0 mL),
8688A), Buvaria bassiana (ATCC 7159), peptone (5.0 g), yeast extract (5.0 g),
Curvularia lunata (ATCC 12017), KH2PO4 (5.0 g), NaCl (5.0 g), pH= 7.6
Cunninghamella elegans (ATCC 36114)
Aspergillus alliaceus (ATCC 10060), Glucose (10.0 g), glycerol (10.0 mL),
1015), peptone (5.0 g), yeast extract (5.0 g),
KH2PO4 (5.0 g), NaCl (5.0 g), pH= 7.4.
Aspergillus
Aspergillus flavus (ATCC 11489)
niger
(ATCC
Glucose (30.0 g), glycerol (10.0 mL),
peptone (5.0 g), Yeast extract (5.0 g),
KH2PO4 (5.0 g), NaCl (5.0 g), pH= 6.4.
Gibberella fujikuroi (ATCC 10704)

Table 2:1H-NMR chemical shift data of spironolactone (1) and its metabolites 2–8.
Carbon
1a
2b
3c
4d
5e
4.06, dd, J=12.5,5.0
2.60, m; 2.66, m
-
6f
7f
8e
1
2
1.67, m; 2.03, m
1.71, m; 2.00, m
7.03, d, J=10.0
1.73, m; 2.03, m
4.18, bs
7.07, d, J=10.0
1.71, m; 2.00, m
2.33, m; 2.37, m
2.42, m; 2.54, m
6.24, d, J=10.0
2.36, m; 2.43, m
2.58, m; 2.88, m
6.27, dd, J=10.0, 1.5
2.42, m; 2.54, m
3
-
-
-
-
-
-
-
4
5.65, s
5.66, bs
-
5.99, bs
-
5.62, s
5.72, bs
5.77, bs
6.00, bs
5.70, bs
5
-
2.35, m; 2.80, m
3.95,
-
-
-
6.19, dd, J=10.0, 3.0
6.09, dd, J=10.0, 1.5
2.28, m
-
6.23, dd, J=10.0, 2.5
6.45, dd, J=10.0, 2.0
2.52, m
-
6.13, dd, J=10.0. 3.0
6.55, dd, J=10.0, 1.5
2.47, m
6
6.12, dd, J=10.0, 2.4 6.25, d, J=10.0
2.42, m; 2.75, m
2.91, m
6.14, d, J =10
6.07, d, J =10
2.32, m
7
6.06, dd, J=10.0,1.6
2.29, m
5.97, d, J=10.0
2.35, m
8
1.98, m
1.92, m
9
0.98, m
1.17, m
1.36, m
1.25, m
1.40, m
1.74, m
1.43, m
1.22, m
10
11
12
13
14
15
-
-
-
-
-
-
-
-
1.66, m; 1.87,
1.43, m; 1.61, m
1.28, m; 1.53, m
-
1.45, m; 1.65, m
1.36, m;1.62, m
-
1.43, m; 1.61, m
1.28, m; 1.54, m
-
1.64, m; 2.12, m
1.60, m; 1.66, m
-
1.47, m; 1.77, m
1.43, m; 1.63, m
-
1.66, m; 1.87, m
1.45, m; 1.63
-
1.43, m; 1.67, m
1.46, m; 1.57, m
-
1.35, m; 1.65,
-
1.37, m
1.34, m
1.42, m
1.65, m
1.35, m
1.40, m
1.40, m
1.38, m
1.57, m; 1.88,
1.36, m; 1.53, m
1.56, m; 1.84, m
1.36, m; 1.65, m
1.57, m; 1.88, m
1.57, m; 1.88, m
4.40, dd, J= 10.0, 3.0
4.33, m
1.88, m; 2.31,
m
1.78dd, J=15.0. 3.5
2.84dd, J=15.0. 9.5
16
1.78, m; 2.19, m
1.88, m; 2.32, m
1.70, m; 2.24, m
1.85, m; 2.27, m
1.86, m; 2.31, m
1.83, m; 2.88, dd, J=15.0. 10.0
17
18
-
-
-
-
0.96, s
1.20
-
-
-
-
1.02, s
0.94, s
1.02, s
1.05, s
1.02, s
1.02
1.06, s
19
1.18, s
1.11, s
1.20, s
1.14, s
1.13
1.22, s
1.12, s
1.90, m; 2.37,
20
1.89, m; 2.33, m
1.89, m; 2.35, m
1.94, 2.29
2.42, 2.60
-
1.90, m; 2.37, m
1.90, m; 2.37, m
2.07, m; 2.46, m
2.09, m; 2.49, m
2.50, m; 2.57, m
-
2.45, m; 2.55,
21
2.45, m; 2.45, m
2.48, m; 2.54, m
2.47, m; 2.58, m
2.48, m; 2.54, m
2.50, m; 2.57, m
22
-
-
-
-
-
-
-
-
-
-
-
-
23- SCO
-
-
-
24-
SCOCH
2.28, s
-
-
-
-
-
-
a500 MHz, CDCl3, b400 MHz, CDCl3, c300 MHz, CDCl3
d600 MHz, CDCl3, e500 MHz, MeOD, f500 MHz, CDCl3

Table-3: 13C-NMR chemical shift data of spironolactone (1) and its metabolites 2–8.
Carbon
1
1b
2a
3a
4c
5b
6a
7c
8c
35.5
33.8
198.5
126.9
165.6
39.8
45.0
38.8
49.2
38.4
20.4
31.0
45.4
45.9
22.2
35.0
95.4
14.5
17.7
31.1
29.1
176.4
194.3
31.3
34.0
33.9
199.3
124.0
163.0
128.3
139.3
37.8
50.4
36.0
20.1
31.1
46.
152.5
128.3
186.2
124.2
161.8
128.2
136.7
38.3
47.2
41.0
21.3
31.1
46.1
48.1
22.4
35.4
95.4
14.5
20.7
31.6
29.2
176.4
-
35.7
34.0
198.5
127.0
166.8
39.7
45.1
40.2
46.9
38.6
20.3
31.0
45.2
44.6
23.0
35.0
95.5
14.7
17.8
30.9
29.2
176.9
-
73.2
43.9
196.9
124.3
162.0
128.3
139.7
38.3
51.2
42.0
22.7
31.9
45.8
47.0
23.2
35.5
95.4
14.4
10.9
31.1
29.2
176.6
-
71.7
43.0
196.4
123.4
159.2
128.8
139.9
37.7
43.6
41.0
19.6
31.5
46.6
47.0
22.5
35.5
95.4
14.4
17.8
31.2
29.3
176.6
-
152.4
127.8
186.2
123.9
161.8
128.3
137.3
37.7
48.0
41.0
21.2
31.6
46.8
55.0
71.1
47.5
92.8
16.1
20.7
31.2
29.1
176.1
-
34.0
33.9
199.4
124.0
162.7
128.0
139.9
37.3
50.3
36.0
20.0
31.7
47.3
54.8
71.3
47.6
92.9
16.0
16.4
31.2
29.1
176.2
-
2
3
4
5
6
7
8
9
10
11
12
13
14
47.0
22.5
35.5
95.3
14.4
16.3
31.6
29.2
176.5
-
15
16
17
18
19
20
21
22
23- SCO
24- SCOCH3
-
-
-
-
-
-
-
a100 MHz, CDCl3
b125 MHz, CDCl3
c150 MHz, CDCl3

22
Table-4: α-Glucosidase Inhibitory Activity of Spironolactone (1) and its Metabolites.
Compounds
% Inhibition
IC50+SEM [µM]
82.8
27.8
36.1
-
335 4.3
1
-
2
-
3
-
4
10.1
11.8
-31.8
-0.6
59.1
-
5
-
6
-
7
-
8
Standard (Acarbose)
840 1.73

23
Graphical Abstract

24
Highlights
• Biotransformation of spironolactone (1) yielded 3 new and 4 known metabolites.
• α-Glucosidase inhibitory activity of substrate 1 and metabolites were evaluated.
• Compound 1 was found to be potent α-glucosidase inhibitor (IC50 = 335 4.3 µM).
• The standard drug (acarbose) has shown IC50 =840 1.73 µM with α-glucosidase.