Effective high-pressure cleavage of sterically hindered steroid esters

Article abstract of DOI:10.1002/hlca.200490135

A simple and effective method to deprotect of sterically hindered steroid esters is described. Deprotection was carried out in MeOH in the presence of a catalytic amount of Et3N under high-pressure conditions. Enzymatic, anionite, and high-pressure methods are compared.

Full text of DOI:10.1002/hlca.200490135

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Effective High-Pressure Cleavage of Sterically Hindered Steroid Esters
by Wojciech Kroszczyn¬skia), Ewa Olszewskaa), Piotr Sa¯an¬skib), and Janusz Jurczak*b)c)
a) Agricultural and Teachers University, ul. 3 Maja 54, 08-110 Siedlce
b) Institute of Organic Chemistry, Polish Academy of Sciences, ul. Kasprzaka 44/52, 01-224 Warszawa
c) Department of Chemistry, Warsaw University, ul. Pasteura 1, 02-093 Warszawa, Poland
(phone: ‡48-22-8230944; fax: ‡48-22-8230944; e-mail: Jurczak@icho.edu.pl)
A simple and effective method to deprotect of sterically hindered steroid esters is described. Deprotection
was carried out in MeOH in the presence of a catalytic amount of Et3N under high-pressure conditions.
Enzymatic, anionite, and high-pressure methods are compared.
Introduction. The problem of mild ester hydrolysis is of great importance in the
chemistry of natural compounds. Corticosteroids, which are important anti-inflamma-
tory drugs, seem to be interesting examples. Some esters are formed as superfluous by-
products; in some cases, the ester group is prepared as a protection of a hydroxy group
and then it should be easily removed. Starting materials are very expensive; therefore, a
great need still exists for a versatile and simple process whereby esters may be
hydrolyzed without heating or exposure to extreme pH values. Drastic conditions, such
as the use of KOtBu, are excluded for this family of compounds because of the presence
of a relatively unstable dihydroxyacetone moiety [1 3]. The usual procedure, which
applies a MeOH solution of Na2CO3 or K2CO3 at low temperature [4], fails if the esters
are sterically hindered.
Therefore, many hindered esters of corticosteroids cannot be hydrolyzed without
providing protection for the dihydroxyacetone moiety. Such an operation, followed by
deprotection (e.g., derivatives of vinyl ethers or dihydropyran are obtained and cleft in
the presence of acids, following alkaline hydrolysis) [4], must result in a significant loss
of yield. For this reason, searching for an efficient way of deprotection under neutral
conditions is still justified. Various methods have been tested, such as protection of the
dihydroxyacetone moiety [5] and the use of an anionite and microbiological
approaches [6].
We started from plant enzymes that had been effective in certain simple ester
hydrolyses [7][8]. It seemed probable that these enzymes could exhibit some
selectivity. Then we used the anionite that had been recommended as a very efficient
reagent [9]. Unfortunately, both of these methods failed in the case of hindered steroid
esters. Yamamoto et al. [10] have reported a very effective hydrolysis of b-hydroxy,
keto, and amino esters with aqueous MeCN under high-pressure conditions. In our
case, this method was not efficient enough. Good results obtained earlier in our
laboratory [11] encouraged us to apply a transesterification approach to achieve
hydrolysis of sterically hindered steroid esters.

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Results and Discussion. Steroid esters are sensitive to both acidic and alkaline
conditions. Therefore, the hydrolysis of such systems should be performed in neutral
medium. The esters 1 10 of prednisolone (ˆ(11b)-11,17,21-trihydroxypregna-1,4-
diene-3,20-dione) and budesonide (ˆ(11b,16a)-16,17-[butylidenebis(oxy)]-11,21-di-
hydroxypregna-1,4-diene-3,20-dione) were selected as model compounds (see Fig.).
Unfortunately, because of steric hindrance, which makes the attack at the reaction
center difficult, as well as instability of the systems having protective groups, the yields
of hydrolysis of such systems by means of conventional methods are low. Attempts with
aqueous-alcoholic KOH solution, methanolic ammonia, hydrazine hydrate in ethylene
glycol plus KOH, KOH in a phase-transfer system in the presence of [18]crown-6 as a
catalyst, KOtBu in DMSO, HCl in CHCl3, AcOH in quinoline, and other reagent
systems exhibited slow hydrolysis to prednisolone and simultaneous formation of
decomposition products, whereas the yields of the desired products were low.
Fig. 1. Steroid esters used for investigation of hydrolysis. 1, prednisolone 11b-acetate; 2, prednisolone 11b,17-
diacetate; 3, prednisolone 11b,21-diacetate; 4, prednisolone 11b,17,21-triacetate; 5, prednisolone 21-pivalate
(ˆ2,2-dimethylpropanoate); 6, prednisolone 21-tetrahydrophtalate (a 3 :2 diastereoisomer mixture of hydro-
gen (1R,6S)- and (1S,6R)-cyclohex-3-ene-1,2-dicarboxylate; 7, (11b,16a)-21-(acetyloxy)-16,17-[(1R)-butylide-
nebis(oxy)]-11-hydroxypregna-1,4-diene-3,20-dione; 8, (11b,16a)-21-(acetyloxy)-16,17-[(1S)-butylidenebis-
(oxy)]-11-hydroxypregna-1,4-diene-3,20-dione; 9, 21-(acetyloxy)-16,17-{[(1R)-furan-2-ylmethyl]bis(oxy)}-11-
hydroxypregna-1,4-diene-3,20-dione; 10, 21-(acetyloxy)-16,17-{[(1S)-furan-2-ylmethyl]bis(oxy)}-11-hydroxy-
pregna-1,4-diene-3,20-dione.
For the study of enzymatic hydrolysis, we employed commercially available
enzymatic preparations (Lipase Sigma, Maxatase), as well as those obtained from
bacteria (Pseudomonas aeruginosa and Aeromonas hydrophila), moulds, and milled
and degreased seeds of oat, sunflower, and wheat. The hydrolysis was conducted at
various pH values in the range of pH 6 8. In general, the yields of enzymatic hydrolysis

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Helvetica Chimica Acta Vol. 87 (2004)
of steroid esters were low. The hydrolysis of acetyl groups occurred at the 17a- and 21-
positions of the steroid system but not at the 11b-position. The esters having the bulky
groups (pivalate, tetrahydrophthalate) at the 21-position did not react at all.
The hydrolysis with an anionite (Dowex, Type I) in two solvents (MeOH and
MeCN) gave low yields, irrespective of the solvent type. This protocol enabled also the
hydrolysis of derivatives having a large steric hindrance, but the yield was merely 10%.
The literature and our own experience suggest that high pressure accelerates the
hydrolysis of the systems having large steric hindrance. Therefore, we used a
transesterification (solvolysis) reaction (Eqn. 1) to obtain unesterified steroid systems.
Transesterification of several standard compounds was rapid and gave high yields (see
Table 1).
R'COOR ‡ MeOH ! R'COOMe ‡ ROH
R ˆ steroid system
(1)
Table 1. Yields of Transesterification of Simple Carboxylic Acid Esters in 2% Et3N/MeOH, at Room
Temperature and 11 kbar Pressure for 2 h
Ester
Yield [%]
Ester
Yield [%]
Isopropyl acetate
tert-Butyl acetate
Isopropyl benzoate
tert-Butyl benzoate
100
100
95
Prednisolone 21-acetate
Prednisolone 21-isobutyrate
Prednisolone 21-pivalate (5)
100
96
78
89
Optimization of the high-pressure method was carried out by reacting 1 and 6 as
steroid standards for various periods of time in the presence of Et3N as a catalyst (see
Table 2). The hydrolysis of prednisolone tetrahydrophthalate 6 and 11b-acetate 1 was
slower than that of the standards in Table 1, and the yield of the product depended upon
the reaction time. Unfortunately, prolongation of the reaction time favored formation
of by-products, i.e., of products of decomposition of the substrate and of the primary
product.
Table 2. Yield of Prednisolone and Secondary Products from Prednisolone 11b-Acetate (1) and Prednisolone 21-
Tetrahydrophthalate 6 in 2% Et3N/MeOH at Room Temperature and 11 kbar
22 h
64 h
168 h
prednisolone secondary products prednisolone secondary products prednisolone secondary products
6
1
31
23
49
6
9
81
76
16
19
28a)
45
traces
< 5a)
a) Yield of the reaction in MeOH without Et3N.
A comparison of yields of the hydrolysis of the steroid esters 1 10 by various
methods is summarized in Table 3. For compounds 1 and 6, the yield of prednisolone is

Helvetica Chimica Acta Vol. 87 (2004)
1491
Table 3. Comparison of Yields [%]a) of Various Methods of Deprotection of 1 10
Enzymatic method
Anionite method
High-pressure method
Conditions of high-
pressure methodb)
1
0
5
23
76
25
33
20
75
31
81
76
40
54
84
26 h, A
168 h, A
22 h, A
22 h, A
22 h, A
22 h, A
22 h, A
168 h, A
27 h, B
6 h, B
2
3
4
5
6
0
0
0
< 5
< 5
< 5
30
traces
traces
28
7
8
9
30
25
0
2
traces
0
0
6 h, B
6 h, B
10
0
a) For the hydrolysis products, see text. b) A, 2% Et3N/MeOH, 11 kbar; B, 1% Et3N/MeCN and 10% MeOH/
MeCN, 6 kbar.
given. For compounds 2 4, the main product was prednisolone 11b-acetate. The
epimer ratio of recovered 6 remained close to 3 :2 after application of all methods.
The presented data show that the high-pressure reaction is the best method for
hydrolysis of steroid esters having large steric hindrance. This method allows hydrolysis
of compounds that otherwise do not undergo hydrolysis at all (enzymatically) or
hydrolyze only with difficulty (using anionites).
Experimental Part
General. Prednisolone (Schering) and MeCN (Merck, Lichrosolv grade) were commercially available. The
steroid derivatives were kindly provided by the Pharmaceutical Research Institute in Warsaw. HPLC: Product
samples were transferred to calibrated vessels and diluted with MeOH to a concentration of 0.1 0.5 mg/ml
prior to quant. analysis; Shimadzu LC-6-AV system, Rheodyne 7125 sample injector with a 20ml sample loop,
and CR-6 data processor; detection at 242 nm, 250 Â 4 mm column packed with LichrosorbRP 18 (10 mm);
mobile phase, 45% MeOH/H2O for determination of prednisolone, 40% EtOH/H2O for determination of
budesonide, 60% H2O/MeOH for determination of products from 9 and 10; flow rate 1 ml/min.
Enzymatic Hydrolysis. Hydrolysis of the steroid ester (5 mg) was carried out in phosphate buffer soln.
(1 ml) at pH 6, in the presence of oat seeds or sunflower seeds (10 mg) finely ground and degreased by Et2O
extraction at r.t. After 48 h, the mixture was filtered, the solid washed with MeOH, and the filtrate transferred to
a 25-ml volumetric flask.
Anionite-Catalyzed Hydrolysis. A sample of steroid ester (5 mg) in 90% MeOH/H2O (5 ml) was stirred
under reflux with Dowex 1 Â 8, 100/200 (OH form). After 8 h, the soln. was diluted with MeOH, filtered, and
transferred to a volumetric flask.
High-Pressure Transesterification. The high-pressure apparatus [12] was charged with the Teflon ampoule
containing a soln. of 10 mg of the steroid ester in an appropriate solvent. After closing the vessel with a mobile
piston, the apparatus was placed between the pistons of a hydraulic press and the pressure was raised to 11 kbar.
The mixture was kept under this pressure at r.t. for several hours and then decompressed. After decompression,
the product was diluted with MeOH, filtered, and transferred to a volumetric flask.
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Received March 2, 2004