Lipase-catalyzed preparation of corticosteroid 17α-esters endowed with antiandrogenic activity

Article abstract of DOI:10.1016/j.tetlet.2008.05.086

Several 17α-monoesters of cortexolone and its Δ⁹-derivative are endowed with antiandrogenic activity. Their synthesis can be accomplished by means of a lipase-catalyzed chemoselective alcoholysis of the corresponding 17α,21-diesters.

Full text of DOI:10.1016/j.tetlet.2008.05.086

Tetrahedron Letters 49 (2008) 4610–4612
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Tetrahedron Letters
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Lipase-catalyzed preparation of corticosteroid 17
with antiandrogenic activity
a-esters endowed
Patrizia Ferraboschi , Maria De Mieri , Laura Ragonesi b
a,*
a
a
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano, Via Saldini, 50-20133 Milano, Italy
COSMO Pharmaceutical SpA, Via Colombo, 1-20020 Lainate, Milano, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Several 17
Their synthesis can be accomplished by means of a lipase-catalyzed chemoselective alcoholysis of the
corresponding 17 ,21-diesters.
a
-monoesters of cortexolone and its D9-derivative are endowed with antiandrogenic activity.
Received 2 April 2008
Revised 15 May 2008
Accepted 19 May 2008
Available online 23 May 2008
a
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Lipase
Corticosteroid
Chemoselective alcoholysis
Antiandrogen
1
. Introduction
Parent compounds 1 and 2 are devoid of antiandrogenic
activity, the acylation of 17
(Fig. 1).
The acylation of tertiary 17-hydroxy group can be achieved,
according to a published method, by acidic hydrolysis of 17,21-
a-hydroxy group being then essential
Recently, the antiandrogenic activity of a series of 17
cortexolone (also named Reichstein’s substance S) 1 and
texolone 2, some of them known in the past only as anti-inflamma-
a-esters of
9
D
-cor-
3
tory or progestational compounds, has been reported. Particularly,
orthoester 5, in turn prepared by reaction of the 17a,21-diol with
among the screened esters 3a–d and 4, cortexolone 17
a
-propio-
a commercially available orthoester in the presence of p-toluen-
4
nate 3b showed a potent antiandrogenic topical activity, in the
sulfonic acid. 21-Monoester 6, derived from the well-known acyl
1
9
5
absence of the systemic one, whereas 17
a
-butanoate of
D
-deriv-
transfer from 17
a
position to 21 is the main by-product, present
2
ative 4 displayed an almost exclusive systemic activity.
up to 3%. 17?21 Migration is, also in vitro and in vivo, one of
OH
O
OH
O
OH
OH
O
O
OH
OH
OCOR
2 2 3
OCO(CH ) CH
O
O
O
O
1
2
3
4
a. R = CH3 b. R = CH CH
2
3
c. R = (CH ) CH d. R = (CH ) CH
3
2
2
3
2 3
Figure 1.
*
Corresponding author. Tel.: +39 02 50316052; fax: +39 02 50316040.
E-mail address: patrizia.ferraboschi@unimi.it (P. Ferraboschi).
0
040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.05.086

P. Ferraboschi et al. / Tetrahedron Letters 49 (2008) 4610–4612
4611
OCOR
O
esters 3 and 4. Use of lipases for selective transformations of ste-
roids is well documented and examples of acylation of hydroxy
groups as well as of ester hydrolysis, for this class of compounds,
O
O
R
O
OR'
OH
8,9
are reported.
2
. Results and discussion
The difficulty to selectively esterify the hindered 17a-tertiary
O
O
alcohol, in the presence of the primary 21 one, suggested us to pre-
5
6
10
pare a series of 17
and to try to selectively remove the acyl group from the primary
1-alcohol, using a lipase. In fact traditional basic hydrolyses are
a,21-diesters by means of a reported method,
Figure 2.
2
3
b
not selective since, as reported by Gardi and Ercoli, mixtures of
1
1–14
15
the main metabolic transformations of 17-monoesters, leading
products are obtained. The desired diesters 7a–d
and 8 were
1
6
to 21-monoesters devoid of antiandrogenic activity. In order to
obtained by reaction of 1 or 2 with the suitable anhydride, in the
presence of the corresponding acid and PTSA (about 70% yields).
obtain, following the reported method,3 17
,4
a-monoesters 3 suit-
10
ably pure for biological tests and pharmacological applications
>99%), a carefully and laborious purification procedure is usually
(
necessary (Fig. 2).
Table 2
The known properties of lipases6,7 to maintain the activity in
CCL-catalyzed alcoholysis of 7a, 7c and 7d in toluene
organic solvents, to selectively transform polyfunctional com-
pounds and to require mild reaction conditions, prompted us to
study a chemoenzymatic approach to the preparation of above
Substrate
n-Butanol
Conversiona (%)
n-Octanol
Conversiona (%)
Time (h)
Time (h)
7
7
7
a
c
d
24
74
74
98
98
81
24
24
48
90
98
97
Table 1
a
1
Lipase-catalyzed alcoholysis of 7b to 3b
Evaluated from H NMR spectra on the basis of the integration of H-21 signals of
3
2
a, 3c, 3d and 7a, 7c, 7d, respectively. Signals due to H-21 of cortexolone
1-monesters were not detectable.
Lipase
Solvent
Alcohol
Time (h)
Conversiona (%)
PPL
PFL
Toluene
n-Octanol
Methanol
n-Octanol
n-Octanol
n-Butanol
n-Butanol
n-Butanol
Methanol
Ethanol
120
24
76
76
96
96
96
80
80
24
24
13
0
91
77
91
86
18
100
86
100
100
Chloroform
Acetonitrile
Toluene
Acetonitrile
Tetrahydrofuran
Chloroform
Toluene
Toluene
Toluene
Toluene
CALB
CALB
CCL
CCL
CCL
CCL
CCL
CCL
CCL
Table 3
CCL-catalyzed alcoholysis of 8 in toluene
Alcohol
Time (h)
Conversiona (%)
Methanol
Ethanol
n-Propanol
n-Butanol
53
53
53
53
79
28
100
100
n-Butanol
n-Octanol
a
1
a
1
Evaluated from H NMR spectra on the basis of the integration of H-21 signals of
Evaluated from H NMR spectra on the basis of the integration of H-21 signals of
9
3
b and 7b. Signals due to H-21 of 21-monopropionate were not detectable.
4 and 8.
D
-Cortexolone 21-butyrate was not present.
OCOR
O
OH
O
OCOR
OCOR
CCL
R'OH
toluene
O
O
7
3
a. R = CH3 b. R = CH CH3
2
c. R = (CH ) CH d. R = (CH ) CH
3
2
2
3
2 3
OCOCH
O
2 2 3
CH CH
OH
O
2 2 3
OCOCH CH CH
2 2 3
OCOCH CH CH
CCL
R'OH
toluene
O
O
8
4
Scheme 1.

4
612
P. Ferraboschi et al. / Tetrahedron Letters 49 (2008) 4610–4612
Then, starting from 17
a
,21-dipropionate 7b,12 precursor of the
Acknowledgements
monoester endowed with the highest topical antiandrogenic activ-
1
ity, we screened several commercially available lipases under
This work was partially financially supported by Università
degli Studi di Milano. We thank Professor Fiamma Ronchetti for
helpful discussions.
alcoholysis conditions in organic solvents; these experimental con-
ditions were jugded more suitable, than the aqueous ones, to be
used for lipophilic substrates as steroids. Lipases from Pseudomo-
nas fluorescens (PFL) and from porcine pancreas (PPL) afforded neg-
ative results: PFL was able to transform only a little amount (10%)
of starting material 7b while no formation of monoester 3b was
observed, using PPL. Lipase from Candida antarctica B (CAL B)
showed to selectively remove the 21-acyl group, 77 and 91% con-
References and notes
1.
2.
3.
4.
Celasco, G.; Moro, L.; Bozzella, R.; Ferraboschi, P.; Bartorelli, L.; Quattrocchi, C.;
Nicoletti, F. Arzneim.-Forsch. 2004, 54, 881–886.
Celasco, G.; Moro, L.; Bozzella, R.; Ferraboschi, P.; Bartorelli, L.; Di Marco, R.;
Quattrocchi, C.; Nicoletti, F. Arzneim.-Forsch. 2005, 55, 581–587.
(a) Gardi, R.; Vitali, R.; Ercoli, A. Gazz. Chim. Ital. 1963, 93, 434–450; (b) Ercoli,
A.; Gardi, R. U.S. Patent 3,152,154, 1964.
version to 17
trile, respectively (see Table 1). Best results were observed when
,21-diester 7b was treated with lipase from Candida cylandra-
a-monoester 3b being achieved in toluene or acetoni-
Gardi, R.; Vitali, R.; Ercoli, A. Gazz. Chim. Ital. 1963, 93, 413–430.
1
7
a
5. Anderson, B. D.; Conradi, R. A.; Lambert, W. J. J. Pharm. Sci. 1984, 73, 604–611.
6. Carrea, G.; Riva, S. Angew. Chem., Int. Ed. 2000, 30, 2226–2254.
cea (CCL), with conversions depending on the chosen alcohol and
the solvent: in fact, using butanol or octanol as the acyl acceptor
and toluene as the solvent, a complete conversion was observed
after 24 h, and pure 17
90% yields (Scheme 1). The CCL-catalyzed alcoholysis is less effi-
7.
8.
9.
Gotor-Fernández, V.; Brieva, R.; Gotor, V. J. Mol. Catal. B: Enzym. 2006, 40, 111–
20.
Riva, S. In Applied Biocatalysis; Blanch, H. W., Clark, D. J., Eds.; M. Dekker: New
York, 1991; Vol. 1, pp 179–220.
Ferrero, M.; Gotor, V. In Stereoselective Biocatalysis; Patel, R. N., Ed.; M. Dekker:
New York, 2000; pp 579–631.
1
1
7
18
a-propionate 3b was recovered in
>
cient if methanol or ethanol are used as acyl acceptors or if more
polar solvents are chosen. The results are summarized in Table 1.
1
0. Turner, R. B. J. Am. Chem. Soc. 1953, 75, 3489–3492.
11. Cortexolone, 17
,21-diacetate 7a: 1H NMR (500 MHz, CDCl
5.71 (br s, 1H, H-4), 4.86 (d, 1H, H-21, J 16.5 Hz), 4.61 (d, 1H, H-21, J 16.5 Hz),
a
3
): selected data d
,21-diacetate 7a,11 dibutyrate 7c and divalerate 7d
13
14
Also 17a
2
1
.14 (s, 3H, CH
8CH ).
3
CO), 2.08 (s, 3H, CH ), 0.73 (s, 3H,
,21-dipropionate 7b: 1H NMR (500 MHz, CDCl
): selected data d
3
3
CO), 1.17 (s, 3H, 19CH
3
were submitted to CCL-catalyzed alcoholysis in order to prepare
the other biologically active monoesters. Selective hydrolysis of
1-ester was confirmed, 17a
obtained as unique products (Scheme 1); no relevant differences in
the alcoholysis procedure were observed: only, the use of butanol
showed a slower conversion rate of dibutyrate 7c and divalerate
d. The results are collected in Table 2.
Finally, we prepared
derivative endowed with a high systemic antiandrogenic activity.
Also in this case the CCL-catalyzed transformation of diester 8 was
performed in toluene with different alcohols, comparing the con-
version rate at the same time. Best results, like for dipropionate
3
12. Cortexolone, 17
5.77 (br s, 1H, H-4), 4.93 (d, 1H, H-21, J 16.6 Hz), 4.65 (d, 1H, H-21, J 16.6 Hz),
.22 (s, 3H, 19CH ), 1.16–1.22 (t + t, 6H, CH ), 0.79 (s, 3H, 18CH ).
3. Cortexolone, 17 ,21-dibutyrate 7c: H NMR (500 MHz, CDCl ): selected data d
5.77 (br s, 1H, H-4), 4.92 (d, 1H, H-21, J 16.6 Hz), 4.64 (d, 1H, H-21, J 16.6 Hz),
.21 (s, 3H, 19CH ), 0.97–1.04 (t + t, 6H, CH ), 0.78 (s, 3H, 18CH ).
4. Cortexolone, 17 ,21-divalerate 7d: H NMR (500 MHz, CDCl ): selected data d
.77 (br s, 1H, H-4), 4.92 (d, 1H, H-21, J 16.5 Hz), 4.63 (d, 1H, H-21, J 16.5 Hz),
a
-monoesters 3a,19 3c and 3d being
20
21
2
1
3
3
3
1
1
a
3
1
3
3
3
1
1
a
3
7
5
9
D
-cortexolone 17
a
-butyrate 4, that is, the
1.22 (s, 3H, 19CH ), 0.92-0.97 (t + t, 6H, CH ), 0.79 (s, 3H, 18CH ).
3
3
3
2
9
,21-dibutyrate 8: 1H NMR (500 MHz, CDCl
15.
D
-Cortexolone, 17
a
3
): selected data d
5
1
1
.78 (br s, 1H, H-4), 5.59 (m, 1H, H-11), 4.94 (d, 1H, H-21, J 16.6 Hz), 4.66 (d,
H, H-21, J 16.6 Hz), 1.36 (s, 3H, 19CH ), 0.96–1.03 (t + t, 6H, CH ), 0.74 (s, 3H,
8CH ).
is commercially available (Aldrich); its D9-derivative was
3
3
3
16. Cortexolone
1
prepared according a reported procedure starting from cortisol: Chamberlin,
E. M.; Tristram, E. W.; Utne, T.; Chemerda, J. M. J. Org. Chem. 1960, 25, 295.
7. Typical procedure of CCL-catalyzed alcoholysis: To a solution of cortexolone,
7
b, were observed using butanol or octanol as acyl acceptors, a
complete conversion of diester 8 to monoester 4 being achieved
22
1
after 53 h (Table 3, Scheme 1).
17
a,21-dipropionate 7b (0.5 g, 1.09 mmol) in toluene (35 mL), n-butanol (0.4 g,
5.45 mmol) and CCL (23 g, 3.86 U/mg, FLUKA) were added. The mixture was
kept at 30 °C under stirring, monitoring the reaction progress by TLC
3
. Conclusion
(PhCH /AcOEt) until starting material disappearance (24 h). The enzyme was
3
removed by filtration on a Celite pad. After evaporation of solvent at reduced
pressure pure cortexolone, 17a-propionate 3b was recovered (0.420 g, 96%).
Crystallization from isopropylether afforded 3b with the required purity for
The CCL-catalyzed alcoholysis in organic solvents, selectively
removing the 21-acyl group, allowed us to prepare 17
a-mono-
biological test (>99% by HPLC).
3
a ): selected data d 5.78
-propionate 3b: 1H NMR (500 MHz, CDCl
esters 3a–d and 4 in good yields and with the required purity
>99%), avoiding the 17?21 acyl migration.
Additionally, it is important to underline that CCL can be recy-
cled at least four times, without sensible decrease of enzymatic
activity: in fact the recovered enzyme from alcoholysis of 7b to
b was reused, under the same reaction conditions (toluene as
the solvent, butanol as the acyl acceptor and 24 h as the reaction
time), and, after the fourth cycle, we observed only a lowered con-
version from 100% to 94%.
Moreover, this chemoenzymatic approach represents a simple
preparative method that could be used, after the suitable develop-
18. Cortexolone, 17
br s, 1H, H-4), 4.32 (dd, 1H, H-21, J 18.3 and 4.9 Hz), 4.25 (dd, 1H, H-21, J 18.3
and 4.9 Hz), 1.22 (s, 3H, CH -19), 1.17 (t, 3H, CH , J 7.6 Hz), 0.72 (s, 3H, CH -18).
Mp 133 °C (t-butylmethylether).
19. Cortexolone, 17
-acetate 3a: 1H NMR (500 MHz, CDCl
s, 1H, H-4), 4.20–4.35 (d + d, 2H, H-21), 2.10 (s, 3H, CH
9), 0.70 (s, 3H, CH -18). Mp 195 °C (acetone/diethylether).
0. Cortexolone, 17 -butyrate 3c: H NMR (500 MHz, CDCl
(br s, 1H, H-4), 4.32 (dd, 1H, H-21, J 18.0 and 4.5 Hz), 4.26 (dd, 1H, H-21, J 18.0
and 4.5 Hz), 1.22 (s, 3H, CH -19), 0.99 (t, 3H, CH , J 7.5 Hz), 0.71 (s, 3H, CH -18).
Mp 135 °C (isopropylether).
(
(
3
3
3
a
3
): selected data d 5.76 (br
CO), 1.21 (s, 3H, CH
3
3
-
1
3
3
1
2
2
2
a
3
): selected data d 5.78
3
3
3
-valerate 3d: 1H NMR (500 MHz, CDCl
1. Cortexolone, 17
(br s, 1H, H-4), 4.31 (dd, 1H, H-21, J 18.1 and 4.5 Hz), 4.26 (dd, 1H, H-21, J 18.1
and 4.5 Hz), 1.21 (s, 3H, CH -19), 0.94 (t, 3H, CH , J 7.2 Hz), 0.72 (s, 3H, CH -18).
Mp 114 °C (isopropylether).
a
3
): selected data d 5.77
3
3
3
ment, for a future scaling up (i.e., in a continuous packed-bed
9
-butyrate 4: 1H NMR (500 MHz, CDCl
(br s, 1H, H-4), 5.57 (m, 1H, H-11), 4.29 (dd, 1H, H-21, J 18.0 and 4.5 Hz), 4.24
(dd, 1H, H-21, J 18.0 and 4.5 Hz), 1.37 (s, 3H, CH -19), 0.98 (t, 3H, CH , J 7.5 Hz),
.66 (s, 3H, CH -18). Mp 136 °C (acetone/hexane).
2.
D
-Cortexolone, 17
a
3
): selected data d 5.77
9
reactor) of the synthesis of cortexolone and
D
-cortexolone 17
a
-
monoesters, recently recognized as antiandrogenic compounds,
in good yields and purity.
3
3
0
3