Title of Invention

A NOVEL PROCESS FOR THE PREPARATION OF MYCOPHENOLATE MOFETIL (MMF) BY ENZIMATIC TRANSESTERIFICATION

Abstract The present invention relates to a process for the preparation of mycophenolate mofetil (MMF) in which an ester of mycophenolic acid (MPA) with a low-molecular- weight aliphatic alcohol is transesterif ied with N- (2- hydroxyethyl) morpholine in the presence of Candida antarctica lipase, and in which the MPA esterification reaction is carried out in the presence of Candida antarctica lipase using the corresponding alcohol as the solvent.
Full Text TITLE
"A method for the preparation of mycophenolate mofetil
by enzymatic transesterification"
DESCRIPTION
Mycophenolate mofetil (MMF; Registry Number 128794-94-
5) , the ester of mycophenolic acid (MPA; Registry
Number 24280-93-1) with N-(2-hydroxyethyl)morpholine
(Registry Number 622-40-2), is an immunosuppressant
currently used in the treatment of patients who have
undergone a kidney transplant.

after oral administration, MMF is hydrolyzed with MPA,
which is the real immunosuppressant agent, because it
is a powerful inhibitor of inosine monophosphate
dehydrogenase (B.J.Bornes, A.E.Eakin, R.A.Izydore and
I.H.Hall Biochemical Pharmacology, 62, (2001), p. 91-
100) .
MMF was described for the first time in US patent
4,753,935 (1987, to which the patent EP 028713B1
corresponds): in that patent, the preparation of MMF is
described by conventional methods of esterification of
MPA with N-(2-hydroxyethyl)morpholine. Those methods
provide for condensation between the MPA and the N-(2-
hydroxyethyl)morpholine by means of the acyl chloride
of MPA or by using a condensing agent, such as, for

example, dicyclohexylcarbodiimide. Those synthesis
methods have, however, the disadvantage of leading,
apart from to the desired product, to a series of
secondary MPA polycondensation products owing to the
simultaneous presence on the molecule of a phenolic
hydroxyl and of a lactone function in addition to the
carboxylic group.
In order to overcome that problem, alternative methods
of preparing MMF have recently been developed. Some
methods, described in the patent applications WO 00-
34503(2000) and WO 03-042393 (2003), report on the
preparation of MMF using a biocatalytic method, by
reaction between MPA and N-(2-hydroxyethyl)morpholine
by enzymatic catalysis. For it is known that many
hydrolytic enzymes, such as, for example, lipases,
esterases or proteases, which are nowadays readily
commercially available, used in organic solvents and
with rigorous control of the experimental conditions
(the water content, pH, temperature, presence of
surfactants and water scavengers) are capable not only
of performing the hydrolysis of non-natural substrates
but also of catalyzing the synthesis of esters
(Klibanov A.M. CHEMTECH, 1986, 16, 354; Schopineau J.,
McCafferty F. D., Therisod M., Klibanov A.M.
Biotechnol. and Bioeng. 1988, 31, 208; Therisod M.,
Klibanov A.M. Journal of Chemical American Society,
1987, 109, 3977) .
Nor are those enzymatic methods entirely satisfactory,
however, because the presence of water, which is
produced during the esterification reaction or
contained in the reaction medium, has a considerable
influence on the level of progress of the reaction.

while the use of a surfactant in the reaction medium
may complicate the subsequent procedures of purifying
the MMF from the crude reaction product. In addition,
the type of organic solvent used may have a strong
influence on the reaction kinetics and the catalytic
efficiency of the enzyme, leading in some cases to long
reaction times in order to obtain a level of progress
of the reaction acceptable for the application thereof
in key industrial preparations.
We therefore decided to establish whether it was not
possible to find suitable experimental conditions which
would enable us to carry out the preparation of MMF,
through the use of enzymes, by operating in the absence
of surfactants and without rigorous control of the pH
conditions and of the presence of water in the reaction
medium. Because the literature describes numerous
examples of the application in organic chemistry of
enzymatic transesterification reactions catalyzed by
lipases, we decided to establish whether this approach
could be applied successfully also to the preparation
of MMF (E.Santaniello, P.Ferraboschi and P.Grisenti.
Enzyme Microb. Technol. 1993, vol 15, p.367-382). In
order to do that, it would have been necessary to
identify the most suitable hydrolytic enzyme for
carrying out both the MPA esterification reaction with
a simple alcohol, and the subsequent
transesterification with N-(2-hydroxyethyl)morpholine.
By studying various reaction conditions for the
enzymatic esterification of MPA with various aliphatic
alcohols and by using as the enzymatic catalyst various
lipases (triacyl glycerol lipases, EC 3.1.1.3) we
surprisingly established that the esterification,

catalyzed by Candida antarctica lipase (CAL B, Novozym
435) , of MPA with low-molecular-weight aliphatic
alcohols, such as, for example, ethanol or methanol,
leads quantitatively to the corresponding ethyl or
methyl esters in from 30 to 40 hours. This method,
which uses the same aliphatic alcohol (i.e. methanol or
ethanol) as the only reaction solvent, is not affected
significantly by the formation of water which is
generated in the reaction environment and does not
require the use of a surfactant.
In addition, CAL B under the experimental conditions we
used, but employing as the reaction solvent
isopropanol, 2,2,2-trifluoroethanol, 2,2,2-
trichloroethanol, n-propanol and n-butanol, enabled us
to obtain, analogously to the use of methanol and
ethanol, the corresponding MPA esters.
The subsequent transesterification reaction of these
MPA esters with N-(2-hydroxyethyl)morpholine in
tetrahydrofuran (THF), still catalyzed by CAL B,
proved capable of leading quantitatively to MMF without
providing for the use of a surfactant and in an
anhydrous environment (Figure 1).


The specificity between MPA and CAL B was all the more
surprising if it is borne in mind that, under the same
experimental conditions, other lipases, such as
Pseudomonas cepacia lipase (PCL) or Candida rugosa
lipase (CRL), did not show themselves capable of
leading to the corresponding methyl or ethyl esters or
of catalyzing transesterification reactions with N-(2-
hydroxyethyl)morpholine.
Those lipases, which are known in the literature for
being capable of catalyzing esterification and
transesterification reactions on various non-natural
substrates, are apparently not capable, under the
experimental conditions we used, of accepting MPA in
their active site.
The MPA esterification reaction is carried out in a
typical manner using CAL B as the enzyme and the
appropriate alcohol as the solvent, preferably a C1-C4
alkyl alcohol or a halogenated derivative thereof. The
preferred amount of enzyme used is from 20 to 60 mg per
mmole of MPA, preferably 53 mg. The concentration of

MPA is from 0.05 to 0.2 molar, preferably 0.1 molar.
The reaction is carried out under agitation at a
temperature of from 15 to 45°C, preferably 30°C for a
period of time of from 30 to 40 hours. The reaction is
completed by removing the enzyme by filtration and
concentrating the filtrate under vacuum.
The transesterification reaction of the MPA esters with
N-(2-hydroxyethyl)morpholine is carried out in a
typical manner using CAL B as the enzyme and an aprotic
polar organic solvent, preferably having a log P of
less than 0.5, and even more preferably THF or 1,4-
dioxan.
The preferred amount of enzyme used is from 50 to 150
mg per mmole of substrate, preferably 107 mg. The
concentration of the substrate is from 0.1 to 0.3
molar, preferably 0.25. The molar ratio between the MPA
ester and the N- (2-hydroxyethyl)morphcline is from 0.2
to 0.4, preferably 0.3. The reaction is carried out
under agitation at a temperature of from 15 to 45°C,
preferably at from 25 to 30°C for a period of time of
from 24 to 36 hours and is then completed by removing
the enzyme by filtration and concentrating the filtrate
under vacuum. Alternatively, the MPA esterification
reaction and the subsequent transesterification
reaction with N-(2-hydroxyethyl)morpholine can be
carried out by re-using the same enzyme without any
apparent loss of enzyme activity compared with the use
of a "fresh" enzyme.
In addition, CAL B under the experimental conditions
described above can be used in more use cycles, both in
esterification and in transesterification, without any
appreciable loss of catalytic activity.

Experimental part
Example 1
Preparation of mycophenolic acid ethyl ester (2)
1.01g (3.15 mmoles) of MPA are dissolved under
agitation at a temperature of 30 °C in 37.5 ml of
absolute ethanol, and then 17 0 mg of CAL B are added.
The reaction mixture is maintained under vigorous
agitation for 40 hours at the temperature of 30 °C and
then the reaction is completed: the enzyme is removed
by filtration and the filtered solution is concentrated
under vacuum to give an oily residue. The oily residue
is taken up with dichloromethane (20 ml) and the
organic solution obtained is washed in sequence with a
saturated sodium bicarbonate solution (15 ml) and then
with water. The organic phase is then dried over sodium
sulphate, filtered and concentrated under vacuum to
give 1.042 g (2.99 mmoles; 95% yield) of mycophenolic
acid ethyl ester (2) which is used directly in the next
synthesis step without any further purification.
Elemental analysis calculated for C19H24O6: C=65.50;
H-6.94; O-27.55. Found: 065.42; H=6.90; O=27.44.
Mass spectrum (analytical fragments): 349 (m+1), 348
(molecular ion), 303 (m-45)
1H-NMR (500 MHz)CDCl3: 1.16 (t, 3H, CH3-CH2-) , 1.76 (s,
3H, CH3-C=), 2.15 (s, 3H, CH3-Ar), 2.26 (m, 2H, CH2-C=),
2.35 (m, 2H, CH2CO), 3.35 (d, 2H, CH2Ar) , 3.75 (s, 3H,
OCH3), 4.04 (q, 2H, CH3-CH2O), 5.16 (s, 2H, ArCH2O) ,
5.20 (t, 1H, CH=).
The following MPA esters were also prepared in an
analogous manner: methyl ester, 2,2,2-trifluoroethyl
ester, 2,2,2-trichloroethyl ester, propyl ester, i-
propyl ester, n-butyl ester.

Methyl ester (1)
Elemental analysis calculated for C18H22O6: C=64.66;
H=6.63; 0=28.71. Found: C=64.59; H=6.54; O=28.65
Mass spectrum (analytical fragments): 335 (m+1), 334
(molecular ion), 303 (m-31)
n-propyl ester (3)
Elemental analysis calculated for C2oH26O6: C=66.28;
H=7.23; O=26.49. Found: C=66.18; H=7.15; 0=26.37
Mass spectrum (analytical fragments): 363 (m+1), 362
(molecular ion), 319 (m-43)
Isopropyl ester (4)
Elemental analysis calculated for C20H26O6: C=66.28;
H=7.23; 0=26.49. Found: C=66.20; H=7.17; 0=26.40.
Mass spectrum (analytical fragments): 363 (m+1),
362(molecular ion), 319 (m-43)
2,2,2-trifluoroethyl ester (5)
Elemental analysis calculated for C19H21F3O6: C=56.72;
H=5.26; F=14.17; O=23.86. Found: C=56.63; H=5.18;
F=14.11; 0=23.80.
Mass spectrum (analytical fragments): 403 (m+1), 402
(molecular ion), 303 (m-99)
2,2,2-trichloroethyl ester (6)
Elemental analysis calculated for C19H21Cl3O6: C=50.52;
H=4.69; Cl= 23.54; O=21.25. Found: 050.43; H=4.65; Cl=
23.45; 0=21.14.
Mass spectrum (analytical fragments): 458 (m+6), 456
(m+4), 454 (m+2), 452 (molecular ion), 303 (m-149)
n-butyl ester (7)
Elemental analysis calculated for C21H28O6: C=67.00;
H=7.50; O=25.50. Found: C=66.92; H=7.42; 0=25.40.
Mass spectrum (analytical fragments): 377 (m+1), 376
(molecular ion), 303 (m-73)

Example 2
Preparation of MMF by transesteriflcatlon of
mycophenolic acid ethyl ester
195 mg (0.56 mmole) of mycophenolic acid ethyl ester
are dissolved, under agitation at a temperature of from
25 to 30°C, in anhydrous THF {2 ml). 60 mg of CAL B and
0.23 ml (249 mg, 1.9 mmoles) of N-(2-
hydroxyethyl)morpholine are then added. The reaction
mixture is maintained under agitation at the
temperature of from 25 to 30oC for 35 hours. After this
period, the reaction is completed: the enzyme is
removed by filtration and the filtered solution is
concentrated under vacuum to give an oily residue. The
oily residue is taken up with dichloromethane (20 ml)
and the organic solution obtained is washed in sequence
with a saturated sodium bicarbonate solution (15 ml)
and then with water. The organic phase is then dried
over sodium sulphate, filtered and concentrated under
vacuum to give 206 mg (0.45 mmole; 82% yield) of MMF.
For analytical purposes, this product was purified by
chromatography over silica gel (1/100 = p/p): by
elution with dichloromethane/methanol = 96/4 v/v, 143
mg of purified MMF are recovered.
1H-NMR (500 MHz)CDCl3: 1.80 (s, 3H, CH3-C=), 2.18(s, 3H,
CH3-Ar) , 2.20-2.45 (m, 4H, CH2-N and CH2-C=) , 2.48 (m,
4H, 2 CH2-N), 2.60 (m, 2H, CH2CO), 3.40 (d, 2H, CH2Ar) ,
3.78 (m, 4H, CH20) , 3.80 (s, 3H, OCH3) , 4.20 (t, 2H,
CH20), 5.15-5.30 (m, 3H, CH2O and CH=).
MMF was prepared in an analogous manner by
transesterification also starting from the following
MPA esters: methyl ester, 2,2,2- trifluoroethyl ester,
2,2,2-trichloroethyl ester, n-propyl ester, i-propyl

ester, n-butyl ester.

WE CLAIM:
1. A process for the preparation of mycophenolate mofetil (MMF), wherein an ester of
mycophenolic acid (MPA) with a low-molecular-weight aliphatic alcohol is
transesterified with N-(2-hydroxyethyl)morpholine in the presence of Candida antartica
lipase.
2. A process as claimed in claim 1, wherein the low-molecular-weight aliphatic alcohol is a
C1-C4 alkyl alcohol or a halogenated derivative thereof.
3. A process as claimed in claim 2, wherein the C1-C4 alkyl alcohol or its halogenated
derivative is selected from methanol, ethanol, isopropanol, 2,2,2-trifluoroethanol, 2,2,2,-
trichloroethanol, n-propanol or n-butanol.
4. A process as claimed in any one of the preceding claims, wherein the
transesterification reaction is carried out in an aprotic polar organic solvent.
5. A process as claimed in claim 4, wherein the aprotic polar organic solvent has a log P
of less than 0.5.
6. A process as claimed in claim 4, wherein the aprotic polar organic solvent is selected
from THF and dioxan, preferably THF.
7. A process as claimed in any one of the preceding claims, wherein the
transesterification reaction is carried out without any surfactant.
8. A process as claimed in any one of the preceding claims, wherein the amount of lipase
is from 50 to 150 mg per mmole of substrate, preferably 107 mg.
9. A process as claimed in any one of the preceding claims, wherein the concentration of
substrate is from 0.1 to 0.3 molar, preferably 0.25.
10. A process as claimed in any one of the preceding claims, wherein the molar ratio
between the MPA ester and the N-(2-hydroxyethyl)morpholine is from 0.2 to 0.4,
preferably 0.3.
11.A process as claimed in any one of the preceding claims, wherein the
transesterification reaction is carried out under agitation at a temperature of from 15 to
45°C, preferably at from 25 to 30°C.

12. A process as claimed in any one of the preceding claims, wherein the MPA
esterification reaction is carried out in the presence of Candida antartica lipase using
the corresponding alcohol as the solvent.
13. A process as claimed in claim 12, wherein the amount of lipase is from 20 to 60 mg per
mmole of MPA, preferably 53 mg.
14. A process as claimed in any one of claims 12 and 13, wherein the concentration of
MPA is from 0.05 to 0.2 molar, preferably 0.1 molar.
15. A process as claimed in any one of claims 12 to 14, wherein the esterification reaction
is carried out under agitation at a temperature of from 15 to 45°C, preferably 30°C.
16. A process as claimed in any one of claims 12 to 15, wherein the esterification reaction
is carried out without any surfactant.


The present invention relates to a process for the preparation of mycophenolate mofetil (MMF) in which an ester of
mycophenolic acid (MPA) with a low-molecular- weight aliphatic alcohol is transesterif ied with N- (2- hydroxyethyl) morpholine
in the presence of Candida antarctica lipase, and in which the MPA esterification reaction is carried out in the presence of Candida
antarctica lipase using the corresponding alcohol as the solvent.

Documents:

00731-kolnp-2007-assignment.pdf

00731-kolnp-2007-correspondence-1.1.pdf

00731-kolnp-2007-form-3-1.1.pdf

00731-kolnp-2007-p.a.pdf

00731-kolnp-2007-priority document.pdf

0731-kolnp-2007-abstract.pdf

0731-kolnp-2007-claims.pdf

0731-kolnp-2007-correspondence others.pdf

0731-kolnp-2007-description (complete).pdf

0731-kolnp-2007-form1.pdf

0731-kolnp-2007-form3.pdf

0731-kolnp-2007-form5.pdf

0731-kolnp-2007-international publication.pdf

0731-kolnp-2007-international search authority report.pdf

731-KOLNP-2007-AMANDED CLAIMS.pdf

731-KOLNP-2007-ASSIGNMENT.pdf

731-KOLNP-2007-CORRESPONDENCE.pdf

731-KOLNP-2007-DESCRIPTION (COMPLETE) 1.1.pdf

731-KOLNP-2007-EXAMINATION REPORT.pdf

731-KOLNP-2007-FORM 1-1.1.pdf

731-KOLNP-2007-FORM 18.1.pdf

731-kolnp-2007-form 18.pdf

731-KOLNP-2007-FORM 2.pdf

731-KOLNP-2007-FORM 3-1.1.pdf

731-KOLNP-2007-FORM 3.pdf

731-KOLNP-2007-FORM 5.pdf

731-KOLNP-2007-FORM-27.pdf

731-KOLNP-2007-GRANTED-ABSTRACT.pdf

731-KOLNP-2007-GRANTED-CLAIMS.pdf

731-KOLNP-2007-GRANTED-DESCRIPTION (COMPLETE).pdf

731-KOLNP-2007-GRANTED-FORM 1.pdf

731-KOLNP-2007-GRANTED-FORM 2.pdf

731-KOLNP-2007-GRANTED-SPECIFICATION.pdf

731-KOLNP-2007-OTHERS.pdf

731-KOLNP-2007-OTHERS1.1.pdf

731-KOLNP-2007-PA.pdf

731-KOLNP-2007-PETITION UNDER RULE 137.pdf

731-KOLNP-2007-REPLY TO EXAMINATION REPORT.pdf

731-KOLNP-2007-REPLY TO EXAMINATION REPORT1.1.pdf


Patent Number 250369
Indian Patent Application Number 731/KOLNP/2007
PG Journal Number 52/2011
Publication Date 30-Dec-2011
Grant Date 29-Dec-2011
Date of Filing 27-Feb-2007
Name of Patentee POLI INDUSTRIA CHIMICA SPA
Applicant Address VIA GIUSEPPE MARCORA 11, I-20121 MILANO
Inventors:
# Inventor's Name Inventor's Address
1 GRISENTI, PARIDE VIALE DE CERMENATE 58, I-20141 MILANO
2 PRESTILEO, PAOLO VIA CENISIO 57. I-20154, MILANO
PCT International Classification Number C12P 17/16
PCT International Application Number PCT/EP2005/053629
PCT International Filing date 2005-07-26
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 MI2004A001703 2004-09-03 Italy