US008273739B2

( 1 2) Ulllted States Patent ( 1 0) Patent N 0. : US 8, 273, 739 B2

De Pater ( 4 5 ) Date o f Patent: Sep . 25 , 201 2
( 5 4 ) METHOD FOR THE PURIFICATION OF ( 5 2) US. Cl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 4 /233. 5 ; 5 4 4 /1 5 3
MYCOPHEN OLATE MOFETIL ( 5 8) Field o f Classi?catio n Sear ch . . . . . . . . . . . . . . . . . . . . . . . . N o ne
( 75 ) Inv ento r : Ro b er tu s Mattheu s De Pater , Delf t See ap p hcano n ?le f o r Co m p lete Sear ch hlsto r y '
( N L) ( 5 6 ) Ref er ences Cited
( 73) Assig nee: DSM Sino chem Phar m aceu ticals
N ether lands B. V . , Delf t ( N L) U' S' PATEN T DOCUMEN TS
4 , 75 3, 935 A * 6 /1 988 N elso n et al. . . . . . . . . . . . . . . 5 1 4 /2335
( * ) N o tice: Su b j ect to any disclaim er , the ter m o f this 5 , 24 7, 083 A 9/1 993 Kno x et a1 :
p atent is extended o r adj u sted u nder 35 2004 /01 6 71 30 A1 8/2004 Lee et a1
U30 1 5 4 0) ) b y 297 day s- FOREIGN PATEN T DOCUMEN TS
( 21 ) Ap p l N O _ 1 2/6 6 6 84 6 W0 W0 02/1 0085 5 1 2/2002
. . . ,
OTHER PUBLICATION S
( 22) PCT Filed: J u n. 24 , 2008
Inter natio nal Sear ch Rep o r t f o r PCT/EP2008/05 8021 , m ailed Dec. 2,
( 86 ) PCT N o . : PCT/EP2008/05 8021 2003
371 ( c) ( l) , * cited b y exam iner
( 2) , ( 4 ) Date: Dec. 28, 2009
Pr im ar y Exam iner * J aso n M N o lan
( 87) PCT Pu b - N OJ W02009/000834 Assistant Exam iner * Michael Bar ker
PCT Pu b , Date; Dec, 31 , 2008 ( 74 ) Atto r ney , Ag ent, o r Fir m * N ixo n & V ander hy e RC.
( 6 5 ) Pr io r Pu b licatio n Data ( 5 7) ABSTRACT
Us 201 0/01 9078 5 A1 J u l_ 29 201 0 The p r esent inv entio n p r o v ides a m etho d f o r the p r ep ar atio n
o f m y co p heno late m o f etil Wher ein m y co p heno lic acid o r an
( 30) Fo r eig n Ap p licatio n Pr io r ity Data am ine salt o f m y co p heno lic acid is ester i?ed With 2- m o r
p ho lino ethano l, the r esu lting m ixtu r e is extr acted into Water
J u n. 27, 2007 ( EP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 071 1 1 1 99 at a p H- v alu e b etWeen 1 . 0 and 3. 0, and m y co p heno late
m o f etil is b ack- extr acted in a Water - im m iscib le so lv ent at a
( 5 1 ) Int. Cl. p H- v alu e b etWeen 3. 0 and 5 . 0.
A6 1 K 31 /5 3 77 ( 2006 . 01 )
C07D 4 1 3/1 2 ( 2006 . 01 ) 7 Claim s, N o Dr awing s
US 8, 273, 739 B2
1
METHOD FOR THE PURIFICATION OF
MYCOPHEN OLATE MOFETIL
This ap p licatio n is the Us. natio nal p hase o f Inter natio nal
Ap p licatio n N o . PCT/EP2008/05 8021 ?led 24 J u n. 2008
Which desig nated the Us. and claim s p r io r ity to EP Ap p lica
tio n N o . 071 1 1 1 991 ?led 27 J u n. 2007, the entir e co ntents o f
each o f Which ar e her eb y inco r p o r ated b y r ef er ence.
FIELD OF THE IN V EN TION
The p r esent inv entio n r elates to a m etho d f o r the p u r i?ca
tio n o f m y co p heno late m o f etil.
BACKGROUN D OF THE IN V EN TION
My co p heno lic acid ( MPA, also kno Wn as 6 - ( 4 - hy dr o xy - 6
m etho xy - 7- m ethy l- 3- o xo - 5 - p hthalany l) - 4 - m ethy l- 4 - hex
eno ic acid, 6 - ( 1 , 3- dihy dr o - 4 - hy dr o xy - 6 - m etho xy - 7- m ethy l
3- o xo - 5 - iso b enZ o f u r any l) - 4 - m ethy l- 4 - hexeno ic acid,
Cl7H2OO6 , CAS 24 280- 93- 1 ) is a co m p o u nd With v ar io u s
adv antag eo u s p r o p er ties. N ext to antib io tic activ ity , MPA
also disp lay s antif u ng al, antiv ir al and antitu m o r p r o p er ties
and the co m p o u nd has b een u sed in the tr eatm ent o f p so r iasis
and r ecently as im m u no su p p r essant. The 2- m o r p ho lino ethy l
ester o f MPA, also kno Wn as m y co p heno late m o f etil ( MPM,
C23H31 N O7, CAS 1 28794 - 94 - 5 ) , is a p r o dr u g o f MPA and
has sim ilar adv antag eo u s p r o p er ties. The chem ical str u ctu r e
o f MPM is:
cH3 o H
MPM can b e p r ep ar ed b y ester i?catio n o f MPA With
2- m o r p ho lino ethano l. In Us. Pat. N o . 4 , 75 3, 935 an acid
halide co ndensatio n r o u te has b een descr ib ed. This is a tWo
step p r o cess r eq u ir ing to xic r eag ents f o r f o r m ing the halide o f
MPA and/o r o f 2- m o r p ho lino ethano l. In EP 6 4 9, 4 22 B1 , an
im p r o v ed r o u te Was disclo sed co ncer ning r e?u xing MPA
With 2- m o r p ho lino ethano l in an iner t o r g anic so lv ent cap ab le
o f aZ eo tr o p ic r em o v al o f Water , Witho u t the u se o f additio nal
r eag ents. One o f the m aj o r p r o b lem s asso ciated With the
sy nthesis o f MPM is the f o r m atio n o f u nWanted im p u r ities.
One o f tho se im p u r ities is Im p u r ity B ( C29H4 2N 2O9) Which
has the f o llo Wing chem ical str u ctu r e:
o /\
k/ N )
CH3 0
H300
o /W o O
k/ N \ /\ ( ) /
CH3 0H 0
20
25
30
35
4 0
4 5
5 0
5 5
6 5
2
The o r ig in o f Im p u r ity B is b eliev ed to r eside in the p r o
du ctio n p r o cess leading to MPA, Wher e a hy dr o xy lated
der iv ativ e o f MPA ( MPA- OH, C l7H2 1 07) is f o r m ed Which is
then co nv er ted to Im p u r ity B du r ing ester i?catio n With
2- m o r p ho lino ethano l, ho Wev er also o ther hy p o theses ar e f ea
sib le. The chem ical str u ctu r e o f MPA- OH is:
cH3
o H
H3co
o
0
Ho /
0
CH3 o H
Reg ar dless o f the o r ig in o f Im p u r ity B, sy nthetic and/o r
do Wnstr eam p r o cessing m etho ds to War ds the p r ep ar atio n o f
MPM With accep tab le lev els o f Im p u r ity B ar e hig hly desir
ab le. Acco r ding to the Eu r o p ean Phar m aco p o eia no t m o r e
than 0. 2% Im p u r ity B m ay b e p r esent in MPM. Kno wn
ap p r o aches to War ds lo Wer ing the am o u nt o f Im p u r ity B ar e
Well- kno wn techniq u es su ch as r ecr y stalliz atio n and/ o r chr o
m ato g r ap hic p u r i?catio n as f o r instance su g g ested in Us.
Pat. N o . 5 , 24 7, 083. Ho Wev er , su ch ap p r o aches r esu lt in sig
ni?cant lo sses o f the desir ed p r o du ct MPM and b o th ar e
cu m b er so m e and exp ensiv e, the last issu e b eing p ar ticu lar ly
tr u e f o r chr o m ato g r ap hic p u r i?catio n. Hence, ther e is a need
f o r an ef ?cient and cheap m etho d f o r lo Wer ing the am o u nt o f
Im p u r ity B in MPM.
DETAILED DESCRIPTION OF THE IN V EN TION
Du r ing ester i?catio n o f MPA With 2- m o r p ho lino ethano l
f o llo Wed b y iso latio n o f the r esu lting MPM, in m any cases the
p r o du ct co ntains to o hig h a lev el o f Im p u r ity B. It Was su r
p r ising ly f o u nd that the am o u nt o f Im p u r ity B co u ld b e lo W
er ed sig ni?cantly b y a sim p le and hither to u np r ecedented
extr actio n p r o cedu r e car r ied o u t du r ing do Wnstr eam p r o cess
ing o f the ester i?catio n m ixtu r e.
In the ?r st asp ect o f the inv entio n MPA o r an am ine salt o f
MPA is ester i?ed in a co nv er sio n With 2- m o r p ho lino ethano l.
Pr ef er ab ly said ester i?catio n is car r ied o u t in a so lv ent, p r ef
er ab ly at elev ated tem p er atu r es. The so lv ent u sed f o r ester i
?catio n o f MPA can b e a so lv ent su ch as b enZ ene and su b sti
tu ted b enZ enes like ethy l b enZ ene, m eta- xy lene, o r tho
xy lene, p ar a- xy lene and to lu ene, chlo r o f o r m , m ethy lene
chlo r ide, ether s su ch as dialky l ether s like dib u ty l ether and
diiso p r o p y l ether , keto nes su ch as aceto ne, cy clo hexano ne,
cy clo p entano ne, dip r o p y l keto ne, m ethy liso b u ty l keto ne,
m ethy lp r o p y l keto ne and m ixtu r es o f these so lv ents. Pr e
f er r ed so lv ents ar e xy lene, dib u ty l ether and cy clo hexano ne.
Pr ef er ab ly the ester i?catio n is car r ied o u t u nder aZ eo tr o p ic
sep ar atio n o f Water and u nder u se o f an excess o f 2- m o r p ho li
no ethano l, f o r instance 1 . 00 to 20 m o lar eq u iv alents, p r ef er
ab ly 1 . 01 to 1 0 m o lar eq u iv alents, m o r e p r ef er ab ly 1 . 02 to 5
m o lar eq u iv alents, m o st p r ef er ab ly 1 . 03 to 3 m o lar eq u iv a
lents, still m o st p r ef er ab ly 1 . 04 to 2 m o lar eq u iv alents.
Altho u g h the p r esent inv entio n is m o st su itab ly car r ied o u t
u nder r e?u xing co nditio ns, also ester i?catio n r eactio ns car
r ied o u t at lo Wer tem p er atu r es than the b o iling p o int can b e
f u r ther o p tim iZ ed b y the p r esence o f the chelating ag ent. The
adv antag es o f ester i?catio n at a tem p er atu r e b elo W the b o il
ing p o int ar e that eq u ip m ent f o r co ndensing so lv ent v ap o r s
and r etu r ning these co ndensed v ap o r s ar e no lo ng er r eq u ir ed
and the ener g y inp u t r eq u ir ed to r each and m aintain the b o il
US 8, 273, 739 B2
3
ing p o int, Which no r m ally is su b stantial, can b e cir cu m
v ented. Fu r ther m o r e, f o r m atio n o f u nwanted b y - p r o du cts
g ener ally is lo Wer at lo Wer r eactio n tem p er atu r es.
The m etho d o f the p r esent inv entio n f o r the p r ep ar atio n o f
m y co p heno late m o f etil co m p r ises the step s o f :
( a) ester i?catio n o f m y co p heno lic acid o r an am ine salt o f
m y co p heno lic acid With 2- m o r p ho lino ethano l;
( b ) co ntacting the m ixtu r e o b tained in step ( a) With Water at
a p H- v alu e b etWeen 1 . 0 and 3. 0;
( c) sep ar ating the aq u eo u s p hase f r o m the m ixtu r e o b tained
in step ( b ) ;
( d) co ntacting the aq u eo u s p hase o b tained in step ( c) With
a Water - im m iscib le so lv ent;
( e) sep ar ating the no n- aq u eo u s p hase f r o m the m ixtu r e
o b tained in step ( d) , char acter iz ed in that step ( d) is
car r ied o u t at a p H- v alu e b etWeen 3. 0 and 5 . 0.
In the co ntext o f the p r esent inv entio n the ter m Water
im m iscib le so lv ent r ef er s to a so lv ent Which, When m ixed
With Water , f o r m s a tWo - p hase sy stem and Which disso lv es in
Water to an extent that the r esu lting aq u eo u s p hase co ntains
less than 1 0% b y Weig ht o f the so lv ent, p r ef er ab ly less than
1 % b y Weig ht o f the so lv ent, m o r e p r ef er ab ly less than 0. 5 %
b y Weig ht o f the so lv ent. While o ne can im ag ine cer tain
ino r g anic liq u ids su ch as silico ne ?u ids and halo car b o n liq
u ids Which m eet the de?nitio n and Which ar e inclu ded in the
de?nitio n o f Water - im m iscib le so lv ents , the f ar m o r e co m
m o n and thu s p r ef er r ed Water - im m iscib le so lv ents ar e
o r g anic so lv ents, esp ecially so lv ents co m p r ising hy dr o car
b o ns and/ o r halo hy dr o car b o ns. Rep r esentativ e su itab le
Water - im m iscib le so lv ents inclu de C4 to C l 4 b r anched, cy clic,
and str aig ht chain satu r ated and u nsatu r ated alip hatic hy dr o
car b o ns; C6 to C1 2 alkar y l hy dr o car b o ns; and halo hy dr o car
b o ns co ntaining u p to ab o u t 4 halo g en ato m s, esp ecially
chlo r ine, and f r o m 1 to ab o u t 8 car b o n ato m s. It is also v er y
su itab le to em p lo y m ixtu r es o f these m ater ials o r distillatio n
f r actio ns co m p o sed p r im ar ily o f these m ater ials. Thu s, r ep
r esentativ e Water - im m iscib le so lv ents inclu de su itab le f r e
o ns, car b o n tetr achlo r ide, chlo r o f o r m , m ethy lene chlo r ide,
tr ichlo r o ethy lene, dichlo r o p r o p ane, and sim ilar halo hy dr o
car b o ns, n- p entane, n- hexane, cy clo hexane, 2- m ethy lp en
tane, hex- 1 - ene, b enZ ene, n- hep tane, m ethy lcy clo hexane,
cy clo p entano ne, cy clo hexano ne, b r anched hep tanes and hep
tenes, to lu ene, the no r m al and b r anched o ctanes and o ctenes,
the xy lenes, ethy lb enZ ene, n- no nane and the b r anched
no nanes, the v ar io u s decanes, the do decanes and like hy dr o
car b o ns, C6 - C7, C6 - C8 and C7- C8 nap htha f r actio ns, m ixed
xy lene- ethy lb enZ ene f r actio ns and the like, C4 to C l 4
b r anched, cy clic, and str aig ht chain alco ho ls, ester s and
keto nes. Pr ef er r ed Water - im m iscib le so lv ents ar e the C6 to C8
hy dr o car b o ns inclu ding alip hatics like n- hexane, cy clo hex
ane, n- hep tane and n- o ctane and f r actio ns co m p o sed in su b
stantial p ar t b y these alip hatics and the ar o m atics su ch as
b enZ ene, to lu ene, ethy lb enZ ene, xy lenes and f r actio ns co m
p o sed in su b stantial p ar t b y these ar o m atics.
The p H- v alu e u sed in step ( b ) can b e f u r ther o p tim iZ ed to
co m b ine m inim al lo ss o f p r o du ct r esu lting f r o m deg r adatio n
With m axim al extr actio n y ield. It has b een f o u nd that the
p H- v alu e sho u ld b e b etWeen 1 . 0 and 3. 0, p r ef er ab ly b etWeen
1 . 5 and 2. 7, m o r e p r ef er ab ly b etWeen 1 . 8 and 2. 4 and m o st
p r ef er ab ly b etWeen 2. 0 and 2. 2.
The p H- v alu e u sed in step ( d) can b e f u r ther o p tim iZ ed to
co m b ine m axim al r edu ctio n o f Im p u r ity B With m axim al
extr actio n y ield o f the desir ed MPM. It has b een f o u nd that
the p H- v alu e sho u ld b e b etWeen 3. 0 and 5 . 0, p r ef er ab ly
b etWeen 3. 75 and 4 . 75 , m o r e p r ef er ab ly b etWeen 4 . 0 and 4 . 5
and m o st p r ef er ab ly b etWeen 4 . 2 and 4 . 3. It has b een f o u nd
that at the p H- r ang es m entio ned ab o v e ther e is an u nexp ected
20
25
30
35
4 0
4 5
5 0
5 5
6 0
6 5
4
dif f er ence in extr actio n b ehav io u r b etWeen MPM and Im p u
r ity B Wher e the f o r m er ap p ear s to disso lv e to a v er y lar g e
extent in the o r g anic p hase Wher eas the latter disso lv es to a
lar g e extent in the aq u eo u s p hase.
In US 2004 / 1 6 71 30 it Was su g g ested to add acid to cr u de
m y co p heno late m o f etil to f o r m an acid salt o f m y co p heno late
m o f etil to b e so lu b le in the aq u eo u s so lu tio n. Ho Wev er , US
2004 / 1 6 71 30 do es no t sp ecif y exactly Which p H v alu es ar e
r eq u ir ed and the do cu m ent neither indicates any ef f ect o n the
p r esence, ab sence o r dim inishing o f im p u r ities. The sp eci?c
p H r eg im e descr ib ed ab o v e su r p r ising ly in?u ences the
am o u nt o f Im p u r ity B p r esent in the ?nal p r o du ct.
In a ?r st em b o dim ent o f the p r esent inv entio n MPA is u sed
in the f o r m o f a salt. Su itab le salts ar e am ines and alkali m etal
salts. In case o f alkali m etal salts, also an acid sho u ld b e
p r esent in a m o lar am o u nt that is at least eq u al to that o f the
m o lar am o u nt o f the MPA alkali m etal salt. In case o f am ine
salts, additio n o f acid is no t m andato r y , altho u g h acid can also
b e added in o r der to decr ease co nv er sio n tim es and/o r
incr ease y ields. Exam p les o f su itab le am ine salts o f MPA ar e,
b u t ar e no t lim ited to , salts f r o m am ines su ch as ter t- b u ty
lam ine, cy clo hexy lam ine, dib enZ y lam ine, N , N - diiso p r o p y l
ethy lam ine, N , N - dim ethy lcy clo hexy lam ine, N , N - dim ethy l
iso p r o p y lam ine, N - m ethy l- p ip er idine, m o r p ho line, ter t
o cty lam ine, p ip er idine, iso - p r o p y lam ine, N , N , N ' , N ' - tetr a
m ethy lb u ty lenediam ine, N , N , N ' , N '
tetr am ethy lethy lenediam ine, tr ib u ty lam ine, tr iethy l- am ine
and tr ip r o p y lam ine. Su itab le alkali m etal salts o f MPA ar e
salts f r o m lithiu m and p o tassiu m , p r ef er ab ly f r o m so diu m .
In a seco nd em b o dim ent o f the p r esent inv entio n, ester i?
catio n o f MPA o r an MPA salt can b e p o sitiv ely in?u enced
( i. e. r edu ctio n o f r eactio n tim e, incr ease o f m axim u m co nv er
sio n) b y the additio n o f su b stances that ar e cap ab le o f ab so r b
ing Water . These su b stances can b e p r esent in the m ixtu r e o f
MPA, so lv ent and 2- m o r p ho lino ethano l. Ho Wev er , these su b
stances m ay also b e p r esent in the v ap o r p hase o f said m ix
tu r e; desp ite the f act that the p r esent inv entio n deals With a
m etho d f o r ester i?catio n in no n- b o iling m ixtu r es, a v ap o r
p hase nev er theless is u su ally p r esent ab o v e su ch no n- b o iling
m ixtu r es. Su b stances that ar e cap ab le o f ab so r b ing Water ar e
f o r instance salts o f alkali and ear th alkali m etals and u su ally
these salts ar e car b o nates, halides o r su lf ates. Su itab le
exam p les ar e CaCl2, CaSO4 , K2CO3, K2SO4 , Mg SO4 ,
N a2CO3, N aZ SO4 and the like. Pr ef er r ed o ther su b stances ar e
m o lecu lar siev es, p r ef er ab ly tho se With p o r e siZ es r ang ing
f r o m 0. 1 - 0. 6 nm , m o r e p r ef er ab ly r ang ing f r o m 0. 2- 0. 5 nm ,
m o st p r ef er ab ly r ang ing f r o m 0. 3- 0. 4 nm .
In a seco nd asp ect o f the inv entio n, MPM is o b tained
acco r ding to the m etho d o f the ?r st asp ect. Said MPM co n
tains less than 0. 05 % b y Weig ht o f Im p u r ity B, p r ef er ab ly
b etWeen 0. 001 % and 0. 03% b y Weig ht o f Im p u r ity B, m o r e
p r ef er ab ly b etWeen 0. 002% and 0. 02% b y Weig ht o f Im p u r ity
B, m o st p r ef er ab ly b etWeen 0. 005 % and 0. 01 % b y Weig ht o f
Im p u r ity B.
In a thir d asp ect o f the p r esent inv entio n, MPM o b tainab le
acco r ding to the ?r st asp ect can b e u sed in p har m aceu tical
co m p o sitio ns, f o r instance in antif u ng al, antiv ir al and/o r anti
tu m o r co m p o sitio ns, b u t also in co m p o sitio ns u sef u l in the
tr eatm ent o f p so r iasis and as im m u no su p p r essant. Acco r d
ing ly , said p har m aceu tical co m p o sitio ns hav e the adv antag e
that the am o u nts o f Im p u r ity B p r esent in said co m p o sitio ns
ar e at hither to u np r ecedented lo W lev els.
EXAMPLES
Gener al Metho ds
HPLC analy sis Was p er f o r m ed o n a Water s HPLC\ MS
sy stem ( Alliance HT 2795 sep ar atio n m o du le; Dio de ar r ay
detecto r , m o del 996 ) With the f o llo Wing sp eci?cs:
US 8, 273, 739 B2
5
Co lu m n: Water s Su n?r e C1 8, 1 5 0> < 4 . 6 m m , 3. 5 p m
Co lu m n tem p : 4 00 C.
Flo W r ate: 1 . 0 m l/m in
UV - detectio n 25 1 nm ( and 21 4 nm f o r the deter m inatio n o f 5
xy lene)
Inj ectio n v o lu m e: 5 u l ( u se ?xed lo o p )
Mo b ile p hase A: So r ensen b u f f er /Water ( 30/70)
Mo b ile p hase B: So r ensen b u f f er /ACN ( 30/70)
Gr adient: T = 0 m in. 5 0% B
T = 1 2 m in. 70% B
T = 1 6 m in 1 00% B
T=21 . 4 m in 1 00%B
T=21 . 5 m in 5 0%B
T = 28 m in 5 0% B
The chem icals ar e Water ( Milli- Q p u r i?ed o r HPLC g r ade) ,
aceto nitr ile ( ACN , g r adient g r ade, Mer ck 1 . 00030) , KHZ PO4
( p . a. , Mer ck 1 . 04 873) , N a2HPO4 . 2H2O ( p . a. , Mer ck
1 . 06 5 80) .
Mo b ile Phases:
Pho sp hate so lu tio nA: 3. 026 g o f KHZ PO4 Was disso lv ed in
1 L MilliQ Water .
Pho sp hate so lu tio n B: 3. 95 87 g o f N a2HPO4 . 2H2O Was
disso lv ed in 1 L MilliQ Water .
So r ensen b u f f er ( 0. 022 M, p H 6 . 4 ) : 700 m L p ho sp hate
so lu tio n A Was m ixed With 300 m L p ho sp hate so lu tio n
B.
Ref er ences, Standar ds and Co ntr o ls:
Standar d: PH Eu r r ef er ence f o r p eak identi?catio n CRS
( co ntains m y co p heno late m o f etil With im p u r ities A, B, D, E,
F, G and H) .
Exam p le 1
Co nv er sio n o f MPA- Tr iethy lam ine Salt ( MPA- TEA)
Into MPM
MPA- TEA ( 1 5 . 0 g ; 73. 5 % MPA; 1 . 1 % MPA- OH; 34 . 5
m m o l MPA) and 2- m o r p ho lino - ethano l ( 2. 25 m l; 1 8. 3 m m o l)
Wer e su sp ended in xy lene ( 6 0 m l) . The m ixtu r e Was heated to
1 20- 1 25 0 C. A p o sitiv e nitr o g en Ho w Was ap p lied du r ing the
r eactio n. Af ter 6 ho u r s additio nal 2- m o r p ho lino ethano l ( 1 . 5 5
m l; 1 2. 6 m m o l) Was added. Af ter 22 ho u r s ano ther p o r tio n o f
2- m o r p ho lino ethano l ( 0. 7 m l; 5 . 7 m m o l) Was added ( in to tal
4 . 5 m l 2- m o r p ho lino ethano l; 36 . 6 m m o l) . The r eactio n Was
f o llo Wed b y HPLC:
Reactio n Mo lar excess Co nv er sio n to Ratio Im p u r ity
tim e ( h) 2- m o 1 p ho lino ethano l MPM ( %) B/MPM ( W/W %)
3 0. 5 24 . 5 0. 1 6
6 0. 5 39. 7 0. 23
22 0. 9 74 . 9 0. 6 0
29 1 . 1 81 . 0 0. 5 8
4 6 . 5 1 . 1 87. 1 0. 6 5
5 4 1 . 1 88. 4 0. 6 3
71 1 . 1 90. 3 0. 6 4
Af ter the analy tical sam p le at 71 h, the r eactio n m ixtu r e
Was co o led to 1 21 30 C. and the m ixtu r e thu s o b tained co n
tained 91 . 4 % ( W %) MPM and 0. 5 8% ( W %) Im p u r ity B.
20
25
30
35
4 0
4 5
5 0
6 0
6 5
6
Exam p le 2
Pu r i?catio n o f MPM b y Extr actio n at Dif f er ent p H
V alu es
The r eactio n m ixtu r e o b tained in Exam p le 1 ( 6 0 m l) Was
dilu ted With Water ( 6 0 m l) . Under stir r ing the p H Was adj u sted
to 2 With 6 N H2SO4 at 1 21 30 C. The p hases Wer e sep ar ated.
The aq u eo u s p hase Was extr acted thr ee tim es With EtOAc ( 4 0
m l) at p H 2. 2 at 1 21 30 C. in o r der to r em o v e u nco nv er ted
MPA. The aq u eo u s p hase Was div ided into six p o r tio ns o f 20
m l. To each p o r tio n EtOAc ( 4 0 m l) Was added and the p H o f
the six m ixtu r es Was adj u sted to 3. 5 , 3. 75 , 4 . 0, 4 . 25 , 4 . 5 and
5 . 0, r esp ectiv ely u sing 4 N N aOH at r o o m tem p er atu r e. The
p hases Wer e sep ar ated, g iv ing EtOAc p hases 1 and Water
p hases 1 ; these p hases Wer e analy Z ed b y HPLC.
All EtOAc p hases 1 Wer e Washed With 20 m l Water at the
sam e p H as the f o r e- g o ing extr actio n ( 3. 5 , 3. 75 , 4 . 0, 4 . 25 , 4 . 5
and 5 . 0, r esp ectiv ely ) at r o o m tem p er atu r e. The p hases Wer e
sep ar ated g iv ing EtOAc p hases 2 and Water p hases 2; also
these p hases Wer e analy Z ed b y HPLC.
The r esu lts ar e as f o llo Ws:
Extr actio n at p H 3. 5
MPM Im p u r ity B Yield MPM Im p u r ity B/MPM
Phase ( g /l) ( g /l) ( %) ( %)
EtOAc 1 22. 79 0 5 4 . 0 0
Water 1 4 6 . 31 0. 6 7 4 6 . 0 1 . 4 5
EtOAc 2 1 9. 6 1 0 79. 4 0
Water 2 8. 86 0. 01 4 5 20. 6 0. 1 6
At p H 3. 5 Im p u r ity B is no t extr acted into EtOAc.
Yield MPM o v er ?r st extr actio n is 5 4 %.
Yield lo ss o v er Washing o f the EtOAc- extr act is 21 %.
MPM- y ield o v er extr actio n and Wash- step is 4 3%.
Extr actio n at p H 3. 75
MPM Im p u r ity B Yield MPM Im p u r ity B/MPM
Phase ( g /l) ( g /l) ( %) ( %)
EtOAc 1 35 . 6 9 0 84 . 2 0
Water 1 1 6 . 39 0. 6 7 1 5 . 8 4 . 1
EtOAc 2 34 . 03 0 87. 5 0
Water 2 8. 4 2 0. 021 8 1 2. 5 0. 26
At p H 3. 75 Im p u r ity B is no t extr acted into EtOAc.
Yield MPM o v er ?r st extr actio n is 84 %.
Yield lo ss o v er Washing o f the EtOAc- extr act is 1 3%.
MPM- y ield o v er extr actio n and Wash- step is 74 %.
Extr actio n at p H 4 . 0
MPM Im p u r ity B Yield MPM Im p u r ity B/MPM
Phase ( g /l) ( g /l) ( %) ( %)
EtOAc 1 4 0. 20 0. 021 3 94 . 4 0. 05
Water 1 5 . 87 0. 6 6 5 . 6 1 1 . 24
EtOAc 2 38. 94 0 89. 8 0
Water 2 7. 39 0. 031 3 1 0. 2 0. 4 2
At p H 4 . 0 Im p u r ity B is alm o st no t extr acted into EtOAc.
The am o u nt is b elo W 0. 1 %.
Yield MPM o v er ?r st extr actio n is 94 %.
Yield lo ss o v er Washing o f the EtOAc- extr act is 1 0%.
MPM- y ield o v er extr actio n and Wash- step is 85 %.
US 8, 273, 739 B2
Extr actio n at p H 4 . 25
MPM Im p u r ity B Yield MPM Im p u r ity B/MPM
Ph?s? ( g /l) ( g /l) ( %) ( %)
EtOAc 1 39. 79 0. 035 97. 5 0. 09
Water l 2. 5 3 0. 5 5 2. 5 21 . 7
EtOAc 2 39. 75 0 94 . 0 0
Water 2 4 . 4 0 0. 0835 6 . 0 1 . 9
A?er extr actio n at p H 4 . 25 0. 1 % o f Im p u r ity B is f o u nd in the ?r st EtOAc- extr act.
Yield MPM o v er ?r st extr actio n is 98%.
Yield lo ss o v er Washing o f the EtOAc- extr act is 6 %.
MPM- y ield o v er extr actio n and Wash- step is 92%.
Extr actio n at p H 4 . 5
MPM Im p u r ity B Yield MPM Im p u r ity B/MPM
Phase ( 90 ( g /l) ( %) ( %)
EtOAc 1 4 3. 30 0. 086 98. 3 0. 20
Water 1 1 . 6 7 0. 4 5 1 . 7 26 . 9
EtOAc 2 4 4 . 4 6 0 96 . 0 0
Water 2 3. 22 0. 1 5 8 4 . 0 4 . 9
A?er extr actio n at p H 4 . 5 , 0. 2% o f Im p u r ity B is in the ?r st EtOAc- extr act.
Yield MPM o v er ?r st extr actio n: 98%.
Yield lo ss o v er Washing o f the EtOAc- extr act: 4 %.
Im p u r ity B can b e r edu ced to < 0. l% b y this step .
MPM- y ield o v er extr actio n and Wash- step is 94 %.
Extr actio n at p H 5 . 0
MPM Im p u r ity B Yield MPM Im p u r ity B/MPM
Phase ( 90 ( g /l) ( %) ( %)
EtOAc 1 4 1 . 5 4 0. 225 99. 3 0. 5 4
Water 1 0. 77 0. 1 21 0. 7 1 5 . 7
EtOAc 2 4 3. 1 8 0. 225 99. 4 0. 5 2
Water 2 0. 4 4 0. 04 38 0. 6 9. 9
A?er extr actio n at p H 5 . 0, 0. 5 % o f Im p u r ity B is f o u nd in the ?r st EtOAc- extr act.
Yield MPM o v er ?r st extr actio n is 99%.
Yield lo ss o v er Washing o f the EtOAc- extr act is 1 %.
A?er this Wash- step 0. 5 % Im p u r ity B is f o u nd in the extr act.
MPM- y ield o v er extr actio n and Wash- step is 99%.
Exam p le 3
Iso latio n o f MPM
The r eactio n m ixtu r e o b tained in Exam p le 1 ( 6 0 m l) Was
dilu ted With Water ( 6 0 m l) . Under stir r ing the p H Was adj u sted
to 2 With 6 N H2SO4 ( 4 . 7 m l) at 1 21 30 C. The p hases Wer e
sep ar ated and the aq u eo u s p hase Was extr acted thr ee tim es
With EtOAc ( 3x4 0 m l) at p H 2. 1 at 1 21 3 C. EtOAc ( 1 6 0 m l)
Was added to the Water p hase and the p H Was adj u sted to 4 . 25
With 4 N N aOH ( 8. 3 m l) . The p hases Wer e sep ar ated and Wer e
analy z ed b y HPLC ( Water p hase 1 and EtOAc p hase 1 ) . The
EtOAc p hase Was Washed With 80 m l Water at p H 4 . 5 ( p H
adj u stm ent With 20 u l 6 N H2SO4 ) . The p hases Wer e sep ar ated
g iv ing Water p hase 2 and EtOAc p hase 2. Bo th p hases Wer e
analy z ed b y HPLC. Water ( 80 m l) Was added to EtOAc p hase
2 and the p H Was adj u sted to 8. 1 5 With 4 N N aOH ( ~ 0. 1 m l) .
The p hases Wer e sep ar ated and the EtOAc p hase 3 Was ana
ly z ed b y HPLC as su m m ar iz ed in the Tab le b elo w. Finally
EtOAc p hase 3 Was Washed With Water at p H ~ 6 .
Z etaCar b o n p o Wder R5 5 SP f r o m Cu no ( 0. 75 g ) Was added
and the m ixtu r e Was stir r ed f o r 1 ho u r . The car b o n Was ?lter ed
20
25
30
35
4 0
4 5
5 0
5 5
6 0
6 5
8
o f f and Washed With 5 0 m l EtOAc. The ?ltr ate Was ev ap o r ated
u nder v acu u m at 700 C. ( b ath tem p er atu r e) and the r esidu e
Was disso lv ed in 1 5 m l EtOAc and 75 m l l- p r o p ano l at 5 00 C.
Under stir r ing the m ixtu r e Was g r adu ally co o led to 0- 5 0 C.
( nu cleatio n at 37- 380 C. ) . The cr y stals Wer e ?lter ed o f f ,
Washed With tWo cake- v o lu m es l- p r o p ano l o f 0- 5 0 C. , and
dr ied u nder v acu u m at 4 0- 4 5 0 C. , y ielding 1 1 . 08 g ( y ield
74 . 3%) MPM as White cr y stals co ntaining 99. 9% MPM and
0. 006 % Im p u r ity B.
MPM Im p u r ity B Yield MPM Im p u r ity B/MPM
Phase ( g /l) ( g 1 ) ( %) ( W/W %)
EtOAc 1 6 3. 0 4 8 97. 4 0. 08
Water 1 3. 87 6 80 2. 6 1 7. 6
EtOAc 2 6 3. 7 1 5 98. 4 0. 02
Water 2 2. 02 5 5 1 . 6 2. 7
EtOAc 3 6 6 . 3 21 99. 4 0. 03
The inv entio n claim ed is:
1 . Metho d f o r the p r ep ar atio n o f m y co p heno late m o f etil
co m p r ising the step s o f :
( a) ester i?catio n o f m y co p heno lic acid o r an am ine salt o f
m y co p heno lic acid With 2- m o r p ho lino ethano l;
( b ) co ntacting the m ixtu r e o b tained in step ( a) With Water at
a p H- v alu e b etween 1 . 0 and 3. 0;
( c) sep ar ating the aq u eo u s p hase f r o m the m ixtu r e o b tained
in step ( b ) ;
( d) co ntacting the aq u eo u s p hase o b tained in step ( c) With
a Water - im m iscib le so lv ent;
( e) sep ar ating the no n- aq u eo u s p hase f r o m the m ixtu r e
o b tained in step ( d) , char acter iz ed in that step ( d) is
car r ied o u t at a p H- v alu e b etWeen 3. 0 and 5 . 0.
2. Metho d acco r ding to claim 1 Wher ein step ( a) is car r ied
o u t in a Water - im m iscib le so lv ent.
3. Metho d acco r ding to claim 2 Wher ein step ( a) is car r ied
o u t u nder az eo tr o p ic r em o v al o f Water .
4 . Metho d acco r ding to claim 1 Wher ein step ( d) is car r ied
o u t at a p H- v alu e b etWeen 3. 5 and 4 . 5 .
5 . A co m p o sitio n co m p r ising m y co p heno late m o f etil and
b etWeen 0. 002% ( W/W) and 0. 05 % ( W/W) o f a co m p o u nd o f
f o r m u la ( 1 ) :
( 1 )
CH3 0]
H3CO
o /w o o
N
k/ \ /\ O /
CH3 OH O
US 8, 273, 739 B2
9 1 0
6 . A p har m aceu tical co m p o sitio n co m p r ising m y co p heno - 7. A m edicam ent Which co m p r ises m y co p heno late m o f etil
late m o f etil and b etWeen 0. 002% ( W/W) and 0. 05 % ( W/W) o f and b etWeen 0. 002% ( W/W) and 0. 05 % ( W/W) o f a co m p o u nd
a co m p o u nd o f f o r m u la ( 1 ) : o f f o r m u la ( 1 ) :
( 1 ) ( 1 )
CH3 0
o
H3CO O O H3CO
0/ o O
0N O 1 5 01 M /
\ /\ O / O O
CH o n
CH3 OH O 3