Microbial transformation of topical corticosteroid: Prednicarbate

Article abstract of DOI:10.14233/ajchem.2014.16548

In the present research, the steroidal antiinflammatory topically used drug prednicarbate (1) was subjected to microbial biotransformation by Cunninghamella elegans. Prednicarbate (1) was transformed into various metabolites. One new and two known metabolites were purified named as prednisolone 17-ethylcarbonate (2) Prednisolone (3) and 4-(4-hydroxybenzyl)-2-(3-methylbut-2-enyl)phenyl methyl carbonate (4). The compound (4) was separated as a new compound and was not reported in literature. Its structure did not show resemblance with prednicarbate so possibly derived from fungal mass. Their structures were elucidated by using modern spectroscopic techniques e.g. ¹³C NMR, ¹H NMR, HMQC, HMQC, COSY, NOESY and mass spectrometry e.g. EI-MS.

Full text of DOI:10.14233/ajchem.2014.16548

Asian Journal of Chemistry; Vol. 26, No. 18 (2014), 6043-6046
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.16548
Microbial Transformation of Topical Corticosteroid: Prednicarbate
1
,*
1
1
1
2
SAEED AHMAD , MUHAMMAD FAHAD MUKHTAR , FARHAN HAMEED KHALIQ , SAJID IRSHAD and JAVED IQBAL
1
2
Faculty of Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
Faculty of Pharmacy, University of Lahore, Lahore 54000, Pakistan
*
Corresponding author: Tel: +92 62 9255243; E-mail: rsahmed_iub@yahoo.com
Received: 19 October 2013; Accepted: 29 November 2013; Published online: 1 September 2014;
AJC-15850
In the present research, the steroidal antiinflammatory topically used drug prednicarbate (1) was subjected to microbial biotransformation
by Cunninghamella elegans. Prednicarbate (1) was transformed into various metabolites. One new and two known metabolites were
purified named as prednisolone 17-ethylcarbonate (2) Prednisolone (3) and 4-(4-hydroxybenzyl)-2-(3-methylbut-2-enyl)phenyl methyl
carbonate (4). The compound (4) was separated as a new compound and was not reported in literature. Its structure did not show resemblance
with prednicarbate so possibly derived from fungal mass. Their structures were elucidated by using modern spectroscopic techniques e.g.
13
1
C NMR, H NMR, HMQC, HMQC, COSY, NOESY and mass spectrometry e.g. EI-MS.
Keywords: Biotransformation, Cunninghamella elegans, Prednicarbate, Topical.
INTRODUCTION
EXPERIMENTAL
Now a day's microbial biotransformation is flourished in
order to find the new modified drug molecules. In microbes,
bacteria and fungi are used to modify existing drug molecules
Prednicarbate (1) was obtained from Stiefel Laboratories
Pakistan (Pvt) Ltd. Thin layer chromatography was carried
out on precoated plates (Silica gel, Merck, PF254). Column
chromatography (CC) was performed by using silica gel (E.
1
with better the rapeutic profile, efficacy and least resistant .
1
13
The factors that govern the microbiological hydroxylation of
steroids involve a combination of effects based on the site,
stereochemistry and nature of the existing functional groups
Merck, Germany). H- and C NMR spectra were recorded in
CDCl on Bruker Avance-NMR spectrometers. The chemical
3
shifts (δ values) are presented in ppm and the coupling
constants (J values) are in Hertz. JEOL (Japan) JMS-600H
mass spectrometer was used for recording EI-MS in m/z.
Microbial culture: Culture of Cunninghamella elegans
was purchased from FCBP (764), grown on Saboraud dextrose
agar (SDA) England. The culture medium for C. elegans was
prepared by dissolving glucose (40 g), yeast extract (20 g),
2
in the substrate . The importance of microbial biotechnology
in the production of steroid drugs and hormones was realized
for the first time in 1952 when the Upjohn Company patented
the process of 11α-hydroxylation of progesterone by Rhizopus
3
species .A variety of steroids are widely used as antiinflamma-
tory, diuretic, anabolic, contractive, antiandrogenic, proges-
4
teronal and anticancer agents as well as in other applications .
2 4
peptone (20 g), NaCl (20 g), KH PO (20 g) and glycerol
Using microorganisms in the biotransformation procedures
has been investigated for decades with the advantages in
(40 mL) in distilled water (4 L).
Fermentation of prednicarbate with Cunninghamella
elegans and purification of metabolites: In experiment forty
250 mL conical flasks for the distribution of 4000 mL media
such that each flask containing 100 mL media. 1 g of predni-
carbate was dissolved in 20 mL acetone and was equally
divided among all the flasks such that (25 mg/0.5 mL/flask),
which were then placed on shaker at 25-28 °C for 14 days.
After completion of fermentation, the fungal mass was filtered
and the filtrate was extracted with dichloromethane. The extract
5,6
production of the molecules with regio- and stereospecificity .
One of the major biotechnological aspects in this area is the
application of a wide range of the microorganisms including
bacteria, fungi and micro algae in converting steroid substances
into the pharmacologically active compounds or other useful
7-9
intermediates . Prednicarbate is quite a new topically applied
corticosteroid drug. Its potency is like hydrocortisone. No
microbial biotransformation has been referenced in the litera-
ture until now. It prescribed for the treatment of inflammatory
2 4
(organic phase) was dehydrated over anhydrous Na SO ,
10
skin diseases, such as atopic dermatitis . It is a non-haloge-
nated corticosteroid, double ester derivative of prednisolone.
evaporated under high vacuum in rotary evaporator to get
brown gummy crude extract which was put to analysis through

6
044 Ahmad et al.
Asian J. Chem.
different chromatographic techniques. Slurry of this crude
extract has been prepared in a petri dish. A wide width column
has been prepared and packed with silica gel and solvent
petroleum ether. Slurry was loaded on to the column while
maintaining its surface and put a cotton onto the slurry for
assuring its surface balance during the experiment. Now this
slurry was eluted with gradient ethyl acetate/petroleum ether
solvent system. Flow rate of the column was quite fast in this
experiment. Fractions obtained were dried in vacuum hood
and subjected to thin layer chromatography and similar frac-
tions were compared and compile. Five fractions were obtained
from the main column after compilation. Fraction 3 was run
on normal phase recycling HPLC.
C
20
H
22
O
4
which was established by EI-MS. Molecular weight
of the compound was 310 and melting point was 390 °C.
Prednisolone 17-ethylcarbonate (2) had an elemental
+
, deduced through HREI-MS (M = m/z
composition C24
H
32
O
7
432.2143, calcd 432.2148) and molecular weight is 432.5067.
The compound showed fluorescence under UV light. This
suggested that the compound has unsaturated carbonyl system.
The UV analysis showed absorbance at (238 nm) for a conju-
13
1
gated ketone. The C and H NMR spectra signaled the presence
1
of the double bond between C-1/C-2 and C-4/C-5. The H and
1
3
13
C NMR data for metabolite (2) is given in Table-1. The C
NMR spectrum (100 MHz, CDCl ) (including BB, dept 90
3
and dept135) of (2) signaled the resonances for three methyl,
seven methylene, seven methine and seven quaternary carbons.
Mass spectra indicated that the mass of this compound is
RESULTS AND DISCUSSION
4
32.2143 Da which is 56 mass unit less than the substrate
488.241) which showed the removal of CH CH CO (56 mass
units). So metabolite (2) was thus identified as prednisolone
Fermentation of prednicarbate (1) with Cunninghamella
elegans for 14 days yielded three known compounds (2,3)
which were identified as prednisolone 17-ethylcarbonate (2),
prednisolone (3) and 4-(4-hydroxybenzyl)-2-(3-methylbut-2-
enyl)phenyl methyl carbonate (4).
(
2
2
11
1
7-ethylcarbonate .
TABLE-1
1
13
H- (400 MHz, CDCl ) AND C NMR (100 MHz, CDCl )
Products resulting from the biotransformation of predni-
carbate are presented in Fig. 1.
3
3
CHEMICAL SHIFT ASSIGNMENTS OF 2
Carbon
No.
δC
Multiplicity δH (J = Hz)
O
C
1
2
3
4
5
6
7
8
9
156.0
127.9
186.5
122.5
169.8
31.9
34.0
31.2
55.3
39.8
70.1
39.8
47.9
51.4
23.8
34.8
89.6
17.0
21.1
204.4
70.2
155.0
64.6
14.2
CH
CH
C
7.23, d (10)
O
O
6.25, dd (1.6,10)
O
O
HO
-
O
H
CH
C
5.99, s
-
H
H
1
CH2
2.3, dd; 2.56, td (3.2,13.2)(4.8,13.6)
O
CH2
1.09, m; 2.10, m
CH
CH
C
2.15, m
1.06, m
Cunninghamellaelegans
Hydrolysis
Hydrolysis
10
11
12
-
CH
CH2
C
4.48, br s
O
1.65, m; 2.05, m
O
O
O
13
14
15
16
-
O
HO
O
O
HO
O
CH
1.62, m
4
H O
H O
H
CH
CH
2
1.48, m; 1.81, m
HO
H
2
1.45, m; 2.75, m
HO
H
H
1
7
8
C
-
H
H
O
1
CH3
CH3
C
0.98, s
O
3
2
19
20
21
1.44, s
*
Fig .1. Drug Prednicarbate (1) is transformed into three metabolites
prednisolone 17-ethylcarbonate (2) and prednisolone (3) by means
of Cunninghamella elegans and one new metabolite not reported
in literataure derived from Cunninghamella elegans as 4-(4-
hydroxybenzyl)-2-(3-methylbut-2-enyl)phenyl methyl carbonate
-
CH2
C
4.82, d; 5.02, d (17.6,17,6)
22
23
24
-
CH2
CH3
4.21, q (7.2)
1.32, t (7.2)
Prednisolone 17-ethylcarbonate (2) was purified on normal
21 28 5
Prednisolone (3) had an elemental composition C H O ,
+
phase HPLC as a colour less solid. R
acetone = 7:3), m.f.: C24 , m.w.: 432.506680 g/mol, HREI-
MS:m/z (m.f., calcd value): 432.2143 (C24 ,432.2148).
Prednisolone (3) was purified on normal phase HPLC as
a white colour crystalline solid. R = 0.25 (petroleum. ether/
acetone = 6:4), m.p.: 235 °C, m.f.: C21 , m.w.: 360.444020
g/mol, HREI-MS:m/z (m.f., calcd value): 360.1923 (C21
60.1937).
-(4-Hydroxybenzyl)-2-(3-methylbut-2-enyl)phenyl
f
= 0.40 (petroleum ether/
deduced through HREI-MS (M = m/z 360.1923, calculated
360.1937) molecular weight is 360.444. The compound
showed fluorescence under UV light. This suggested that the
compound has unsaturated carbonyl system. The UV analysis
showed absorbance at (238 nm) for a conjugated ketone. The
32 7
H O
32 7
H O
f
13
1
H
28
O
5
C and H NMR spectra signaled the presence of the double
1
bond between C-1/C-2 and C-4/C-5. The H and C NMR
13
28 5
H O ,
1
data for metabolite 3 is given in Table-2. The C NMR
3
3
4
spectrum (100 MHz, CDCl ) (including BB, dept 90 and
3
methyl carbonate (4) was isolated as colourless amorphous powder
was soluble in methanol by column chromatography technique.
The molecular formula of the compound was found to be
dept135) of data showed the resonances for two methyl, six
methylene, seven methine and six quaternary carbons. Mass
spectra displayed that the mass of this compound is 360.1923

Vol. 26, No. 18 (2014)
Microbial Transformation of Topical Corticosteroid: Prednicarbate 6045
Da which is 128 mass unit less than the substrate (488.241)
which showed the removal of CH
TABLE-3
H- (400 MHz, CDCl ) CHEMICAL SHIFT
ASSIGNMENTS OF METABOLITE PRODUCED (4)
1
3
2 2
CH CO (56 mass units) and
removal of CH CH OCO (72 mass units). This metabolite was
2
2
12
H NMR
.605 (d)
J = 9
δC
Multiplicity δH (J = Hz)
thus characterized as prednisolone (3) .
7
130.0
CH
CH
CH
158.7 (Q), 130.0 (CH), 39.6
(CH ), 128 (Q)
2
TABLE-2
6.846 (d)
J = 9
116.4
129.7
128 (Q), 158 (Q), 116.4
1
13
H (400 MHz, CDCl ) AND C NMR (100 MHz, CDCl )
3
3
CHEMICAL SHIFT ASSIGNMENTS OF COMPOUND 3
6
.54 (dd)
115.02 (Q), 132.4 (CH),
128.4 (Q), 39.6 (CH2)
Carbon
No.
J = 8.4
J = 2.0
6.47 (d)
J = 8
δC
Multiplicity
δH (J = Hz)
1
2
3
4
5
6
7
8
9
155.8
128.0
186.4
122.6
169.6
31.9
33.9
31.3
55.2
43.9
70.2
39.8
48.1
51.2
23.9
34.2
88.6
17.5
21.1
212.0
67.5
CH
CH
C
7.22, d (10)
6.25, dd (2,10)
115.0
132.4
CH
CH
155.02 (Q), 125.3 (Q), 128.4
(Q)
-
6.00, s
6.41 (d)
J = 2
129.7 (CH), 155.02 (Q), 39.6
CH
C
-
2.32, m; 2.56, td (4.4, 13.2)
5.05 (t)
3.75(s)
3.40 (s)
123.6
53.6
39.6
28.7
25.9
CH
MEO
CH2
CH2
CH3
28.7 (CH ), 132.9 (Q)
2
CH2
CH2
CH
CH
C
172.1 (Q)
1.16, m; 2.08, m
2.12, m
1.08, m
128.4 (Q)
3
.06 (d)
.65 (s)
128.3 (Q), 123.6 (CH)
123.6 (CH), 132.9 (Q), 17.7
1
10
11
12
13
14
15
16
17
18
19
20
21
-
4.48, br, t
1.46, m; 2.02, dd (3.6,14.4)
(
CH3)
CH
1.56 (s)
17.7
CH3
25.9 (CH ), 123.6 (CH), 132.9
3
CH2
(Q)
C
-
1.66, m
CH
CH2
CH2
C
as green chemistry while chemical synthesis involved hazardous
chemicals lead to the environmental pollution. Cunninghamella
elegans had an ability to hydrolyze the compounds due to the
1.48, m; 1.85, m
1.54, m; 2.70, m
-
0.96, s
1.43, s
CH3
CH3
C
13
presence of hydrolase enzymes in its enzyme system . The
reaction involved in the synthesis of metabolites was ester
hydrolysis shown in Fig. 1. It is expected that the new meta-
bolites formed may have more antiinflammatory activity, better
therapeutic profile, safety, efficacy and least resistance and
can be employed in the treatment of asthma as that of the parent
compound.
-
4.27, dd; 4.62, dd (4.8,19.6)
CH2
4-(4-Hydroxybenzyl)-2-(3-methylbut-2-enyl)phenyl methyl
carbonate (4) was isolated as colourless amorphous powder.
The molecular formula of the compound was found to be
It is expected that the new metabolites formed may have
more antiinflammatory activity related to skin, better thera-
peutic profile, safety, efficacy and least resistance and can be
employed in the treatment of inflammatory skin diseases i.e.
dermatitis, eczema, etc. as that of the parent compound. In the
present study we could produce these compounds outside the
body which showed the definite potential of the emerging route
of metabolism of drug as well as new drug discovery from the
existing one.
20 22 4
C H O which was established by EI-MS. Molecular weight
of the compound was 310 and melting point was 390 °C.
1
13
H and C NMR data for metabolite 4 is given in Table-3.
1
The H NMR shows the peak at 7.605 (d) with J-9 and another
peak at 6.846 (d) with J = 9 of benzene ring so it is again a
para substituted ring. It also showed doublet of doublet at
6
.54 (d,d) with J = 8.4, 2 and 6.47 (d) J = 8 so it's ortho in
position and 6.41 (d) with J = 2 so it's meta substituted and
thus benzene ring is tri substituted. It also showed a triplet
methine at 5.05 which attribute double bond present in
molecule. It also shows two methylene group at 3.40 (s) and
Conclusion
3
.06 (d) and two methyl at 1.65 and 1.56 which are singlet so
Microbial biotransformation of prednicarbate by Cunning-
hamella elegans resulted first time from this fungus into two
known and one new metabolite which illustrates the enzymatic
potential of the fungus. The new metabolite was not reported
in literature and its structure did not showed structural
resemblance with substrate so it was assumed to be a fungal
metabolite.
attached to quaternary center. By using the HMBC correlation
and COSY correlation structure was established and important
correlation was given in Table-3 to established structure. The
1
3
C-NMR shows the signals at 130.0 (CH), 116.4 (CH), 129.7
CH), 115 (CH), 132.4 (CH), of benzene ring and 123.6 (CH)
(
of double bond. A signal at 53.6 shows Methoxy and two
methylene signals at 39.6 and 28.7. It also shows two signals
of methyl present at 25.9 and 17.7.
The two known compounds included prednisolone and
prednisolone 17-ethylcarbonate and one new metabolite as
4-(4-hydroxybenzyl)-2-(3-methylbut-2-enyl) phenyl methyl
carbonate. It is expected that new derivative may have more
anti-inflammatory activity than the parent compound. Their
structures of these compounds were elucidated by using
This was potential route of synthesis of these compounds.
Chemical reactions required variety of chemicals and condi-
tions to accomplish the reactions while in biotransformation a
number of reactions can be accomplished in one container
and reactions under mild conditions was required even at room
temperature. As there was no hazardous substances used for
the production of these metabolites so it could be denominated
1
3
1
modern spectroscopic techniques e.g., C NMR, H NMR,
HMQC, HMQC, COSY, NOESY and mass spectrometry e.g.
EI-MS.

6
046 Ahmad et al.
ACKNOWLEDGEMENTS
This project was financially supported by The Islamia
Asian J. Chem.
4
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6
.
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ledged to the technical support of H.E.J., Research Institute
of Chemistry, University of Karachi, Karachi, Pakistan
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