U.S. patent application number 13/015825 was filed with the patent office on 2011-05-19 for aminopropanol derivatives.
Invention is credited to Rainer ALBERT, Eric Francotte, Frederic Zecri, Markus Zollinger.
Application Number | 20110118210 13/015825 |
Document ID | / |
Family ID | 34276809 |
Filed Date | 2011-05-19 |
United States Patent
Application |
20110118210 |
Kind Code |
A1 |
ALBERT; Rainer ; et
al. |
May 19, 2011 |
AMINOPROPANOL DERIVATIVES
Abstract
Compounds of formula I: ##STR00001## wherein R.sub.1, R.sub.2, n
and m are as defined in the specification, processes for their
production, their uses and pharmaceutical compositions containing
them.
Inventors: |
ALBERT; Rainer; (Basel,
CH) ; Francotte; Eric; (Nuglar, CH) ; Zecri;
Frederic; (Bartenheim, FR) ; Zollinger; Markus;
(Mohlin, CH) |
Family ID: |
34276809 |
Appl. No.: |
13/015825 |
Filed: |
January 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12391298 |
Feb 24, 2009 |
7902175 |
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13015825 |
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10569696 |
Feb 27, 2006 |
7528120 |
|
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PCT/EP04/09589 |
Aug 27, 2004 |
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12391298 |
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Current U.S.
Class: |
514/80 ; 514/114;
514/92; 548/112; 548/414; 558/166; 558/89 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 3/10 20180101; A61P 1/16 20180101; A61P 9/10 20180101; C07F
9/653 20130101; A61P 17/06 20180101; A61P 31/12 20180101; A61P
37/04 20180101; Y10T 436/163333 20150115; A61P 9/08 20180101; A61P
37/00 20180101; A61P 37/08 20180101; A61P 31/18 20180101; C07D
263/14 20130101; A61P 21/04 20180101; A61P 27/02 20180101; A61P
1/04 20180101; A61P 11/02 20180101; A61P 29/00 20180101; A61P 31/04
20180101; C07F 9/5728 20130101; C07F 9/094 20130101; A61P 17/14
20180101; A61P 25/00 20180101; A61P 17/00 20180101; A61P 13/12
20180101; A61P 19/02 20180101; A61P 37/02 20180101; A61P 37/06
20180101; A61P 9/00 20180101; A61P 5/14 20180101; A61P 7/06
20180101; A61P 35/00 20180101; A61P 35/02 20180101; C07D 209/46
20130101; A61P 31/00 20180101 |
Class at
Publication: |
514/80 ; 548/414;
558/89; 558/166; 514/114; 548/112; 514/92 |
International
Class: |
A61K 31/675 20060101
A61K031/675; C07F 9/06 20060101 C07F009/06; A61K 31/683 20060101
A61K031/683; A61P 29/00 20060101 A61P029/00; A61P 37/06 20060101
A61P037/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 28, 2003 |
GB |
0320196.9 |
Oct 15, 2003 |
GB |
0324206.2 |
Claims
1. (canceled)
2. A compound of formula II: ##STR00035## wherein each of m and n,
independently, is 1, 2 or 3; X is O or a direct bond; R.sub.1 is a
phenylalkyl wherein alkyl is a straight- or branched C.sub.6-20
carbon chain; or a phenylalkyl wherein alkyl is a straight- or
branched (C.sub.1-30)carbon chain wherein said phenylalkyl is
substituted at the phenyl residue by a straight- or branched
(C.sub.6-20)carbon chain optionally substituted by halogen, a
straight- or branched (C.sub.6-20)alkoxy chain optionally
substituted by halogen, a straight- or branched
(C.sub.6-20)alkenyloxy, phenylalkoxy, halophenylalkoxy,
phenylalkoxyalkyl, phenoxyalkoxy or phenoxyalkyl, cycloalkylalkyl
substituted by C.sub.6-20alkyl, heteroarylalkyl substituted by
C.sub.6-20alkyl, heterocyclic C.sub.6-20alkyl or heterocyclic alkyl
substituted by C.sub.2-20alkyl, and wherein the alkyl moiety may
have in the carbon chain, a bond or a heteroatom selected from a
double bond, a triple bond, O, S, sulfinyl, sulfonyl, or NR.sub.5,
wherein R.sub.5 is H, alkyl, aralkyl, acyl or alkoxycarbonyl, and
as a substituent alkoxy, alkenyloxy, alkynyloxy, aralkyloxy, acyl,
alkylamino, alkylthio, acylamino, alkoxycarbonyl,
alkoxycarbonylamino, acyloxy, alkylcarbamoyl, nitro, halogen,
amino, hydroxyl or carboxy, and R.sub.2 is ##STR00036## wherein
each of R.sub.3 and R.sub.4, independently is H or C.sub.1-4alkyl
wherein alkyl is optionally substituted by 1, 2 or 3 halogen atoms;
or a pharmaceutically acceptable salt thereof; and wherein greater
than 70% of the compound by weight is in the form of the R or S
enantiomer.
3. A compound of formula II according to claim 2 in the form of the
substantially pure enantiomer having the following 3-dimensional
configuration. ##STR00037##
4. (canceled)
5. A process for producing a compound as defined in claim 2,
comprising deprotecting a compound of formula III, IIIa or IIIb,
wherein greater than 70% by weight of the compound of formula III,
IIIa or IIIb is in the form of the R or S enantiomer: ##STR00038##
wherein n, m, X, R.sub.1 and R.sub.2 are as defined above, R.sub.6
is an amino protecting group, and R.sub.6' is a simultaneous OH and
amino protecting group; and, where required, converting the
compounds of formula II obtained in free form into the desired salt
form, or vice versa.
6. A method according to claim 7, wherein the HPLC of step (b) is
using a chiral ion-exchange phase based on quinine carbamate or
quinidine carbamate.
7. A method for determining the amount of the R and/or S isomers of
a compound of formula II present in a sample, comprising (a)
reacting the compound of formula II present in the sample with an
aromatic 1,2-dicarbaldehyde to form a compound of formula I
##STR00039## wherein m, n, x, R.sub.1 and R.sub.2 are as defined in
formula II and (b) separating the R and S isomers of the compound
of formula I by HPLC.
8. (canceled)
9. A method for preventing or treating acute or chronic transplant
rejection or T-cell mediated inflammatory or autoimmune diseases in
a subject in need of such treatment, which method comprises
administering to said subject an effective amount of a compound as
defined in claim 2, or a pharmaceutically acceptable salt
thereof.
10. A compound of formula III, IIIa, IIIb: ##STR00040## wherein n,
m, x, R.sub.1 and R.sub.2 are as defined in claim 1 and R.sub.6' is
a simultaneous OH and amino protecting group; or a pharmaceutically
acceptable salt thereof.
11. A compound of formula II according to claim 3, wherein said
compound comprises the S enantiomer of phosphoric acid
mono-[2-amino-2-hydroxymethyl-4-(4-octyl-phenyl)-butyl]ester.
12. A compound of formula II according to claim 2, wherein greater
than 90% of the compound by weight is in the form of the R or S
enantiomer.
13. A compound of formula II according to claim 2, wherein greater
than 95% of the compound by weight is in the form of the R or S
enantiomer.
14. A compound of formula II according to claim 2, wherein greater
than 99% of the compound by weight is in the form of the R or S
enantiomer.
Description
[0001] The present invention relates to organic compounds, a
process for their production and pharmaceutical compositions
containing them.
[0002] More particularly the present invention provides a compound
of formula I:
##STR00002##
wherein each of m and n, independently, is 1, 2 or 3; X is O or a
direct bond;
R.sub.1 is
[0003] a phenylalkyl wherein alkyl is a straight- or branched
(C.sub.6-20)carbon chain; or [0004] a phenylalkyl wherein alkyl is
a straight- or branched (C.sub.1-30)carbon chain wherein said
phenylalkyl is substituted at the phenyl residue by [0005] a
straight- or branched (C.sub.6-20)carbon chain optionally
substituted by halogen, [0006] a straight- or branched
(C.sub.6-20)alkoxy chain optionally substituted by halogen, [0007]
a straight- or branched (C.sub.6-20)alkenyloxy, [0008]
phenylalkoxy, halophenylalkoxy, phenylalkoxyalkyl, phenoxyalkoxy or
phenoxyalkyl, [0009] cycloalkylalkyl substituted by
C.sub.6-20alkyl, [0010] heteroarylalkyl substituted by
C.sub.6-20alkyl, [0011] heterocyclic C.sub.6-20alkyl or [0012]
heterocyclic alkyl substituted by C.sub.2-20alkyl, and wherein the
alkyl moiety may have [0013] in the carbon chain, a bond or a
heteroatom selected from a double bond, a triple bond, O, S,
sulfinyl, sulfonyl, or NR.sub.5, wherein R.sub.5 is H, alkyl,
aralkyl, acyl or alkoxycarbonyl, and [0014] as a substituent
alkoxy, alkenyloxy, alkynyloxy, aralkyloxy, acyl, alkylamino,
alkylthio, acylamino, alkoxycarbonyl, alkoxycarbonylamino, acyloxy,
alkylcarbamoyl, nitro, halogen, amino, hydroxy, or carboxy, and
R.sub.2 is
[0015] ##STR00003## [0016] wherein each of R.sub.3 and R.sub.4,
independently, is H or C.sub.1-4alkyl, wherein alkyl is optionally
substituted by 1, 2 or 3 halogen atoms; in free form or in salt
form.
[0017] Halogen is F, Cl, Br or I. Alkyl or alkoxy may be straight
or branched chain.
[0018] Cycloalkyl is preferably C.sub.3-10cycloalkyl, more
preferably C.sub.3-8cycloalkyl and includes, for example,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or
cycloheptyl.
[0019] Acyl may be a residue R.sub.y--CO-- wherein R.sub.y is
C.sub.1-6alkyl, C.sub.3-6cycloalkyl, phenyl or
phenyl-C.sub.1-4alkyl.
[0020] When in the compounds of formula I the carbon chain as
R.sub.1 is substituted, it is preferably substituted by halogen,
nitro, amino, hydroxy or carboxy. When the carbon chain is
interrupted by an optionally substituted phenylene, the carbon
chain is preferably unsubstituted. When the phenylene moiety is
substituted, it is preferably substituted by halogen, nitro, amino,
methoxy, hydroxy or carboxy.
[0021] In the compounds of the invention, the following
significances are preferred individually or in any sub-combination:
[0022] 1. m and n are each 1 or 2, preferably 1. [0023] 2. X is O.
[0024] 3. R.sub.1 is C.sub.13-20alkyl, optionally substituted by
nitro, halogen, amino, hydroxy or carboxy, and, more preferably
those wherein R.sub.1 is phenylalkyl substituted by
C.sub.6-14-alkyl chain optionally substituted by halogen and the
alkyl moiety is a C.sub.1-6alkyl optionally substituted by hydroxy.
More preferably, R.sub.1 is phenyl-C.sub.1-6alkyl, e.g.
phenyl-C.sub.1-6alkyl, e.g. phenyl-C.sub.2alkyl, substituted on the
phenyl by a straight or branched, preferably straight,
C.sub.6-14alkyl chain. The C.sub.6-14alkyl chain may be in ortho,
meta or para, preferably in para. [0025] 4. each of R.sub.3 and
R.sub.4 is H.
[0026] A particularly preferred compound is phosphoric acid
mono-[2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoindol-2--
yl)-butyl]ester.
[0027] Compounds of formula I may exist in free form or in salt
form, e.g. addition salts with e.g. inorganic acids, such as
hydrochloride, hydrobromide or sulfate, salts with organic acids,
such as acetate, fumarate, maleate, benzoate, citrate, malate,
methanesulfonate or benzenesulfonate salts; when R.sub.3 or R.sub.4
is H, R.sub.2 may also be present in salt form, e.g. an ammonium
salt or salts with metals such as sodium, potassium, calcium, zinc
or magnesium, or a mixture thereof. Compounds of formula I and
their salts in hydrate or solvate forms are also part of the
invention.
[0028] The compounds of formula I have one or more asymmetric
centers in the molecule, and thus various optical isomers may be
obtained. The present invention also encompasses enantiomers,
racemates, diastereoisomers and mixtures thereof. The central
asymmetric carbon atom may have the R or S configuration. Moreover,
when the compounds of formula I include geometric isomers, the
present invention embraces cis-compounds, trans-compounds and
mixtures thereof. Similar considerations apply in relation to
starting materials exhibiting asymmetric carbon atoms or
unsaturated bonds as mentioned above.
[0029] A compound of formula I may be prepared by reacting a
compound of formula II:
##STR00004##
wherein m, n, X, R.sub.1 and R.sub.2 are as defined in formula I;
with an aromatic 1,2-dicarbaldehyde, e.g.
benzene-1,2-dicarbaldehyde, and recovering the resulting compound
of formula I in free or salt form.
[0030] The process may be performed according to methods known in
the art, e.g. as described in the examples.
[0031] A compound of formula II (e.g. a racemic mixture thereof)
may be obtained as described in WO 02/18395 or WO 02/076995.
[0032] The present invention also provides a compound of formula I
or formula II, wherein greater than 70% by weight of the compound
is in the form of the S enantiomer, or greater than 70% by weight
of the compound is in the form of the R enantiomer, e.g. greater
than 90% is in the form of the R or S enantiomer. More preferably
greater than 95% by weight, e.g. greater than 99% by weight of the
compound is in the form of the R or S enantiomer. Thus the
invention may relate to the substantially pure R or S enantiomer
(e.g. the S enantiomer substantially free of the R enantiomer or
vice versa), preferably the S enantiomer, of a compound of formula
I or formula II. Particularly preferred are the substantially pure
(e.g. greater than 99% by weight) R or S enantiomers, especially
the S enantiomers, of phosphoric acid
mono-[2-amino-2-hydroxymethyl-4-(4-octyl-phenyl)-butyl]ester
(FTY720-phosphate) and phosphoric acid
mono-[2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoindol-2--
yl)-butyl]ester.
[0033] Compounds having the following 3-dimensional configuration
are generally preferred:
##STR00005##
[0034] Enantiomers of the compounds of formula I and II cannot be
satisfactorily separated by standard methods. According to the
present invention, separation of the enantiomers is achieved by the
use of novel separation techniques and synthesis strategies.
[0035] A compound of formula I, wherein greater than 70% by weight
of the compound is in the form of the R or S enantiomer, e.g. the
substantially pure R or S enantiomer, may be obtained by:
a) separation of the S enantiomer from the R enantiomer in a
racemic mixture of a compound of formula I, using chromatography on
a chiral stationary phase; or b) reacting a compound of formula II,
wherein greater than 70% by weight of the compound is in the form
of the R or S enantiomer, e.g. the substantially pure R or S
enantiomer of a compound of formula II, with an aromatic
1,2-dicarbaldehyde e.g. benzene-1,2-dicarbaldehyde.
[0036] According to method a), the chromatographic separation is
preferably carried out using a chiral ion-exchange phase based on
quinine carbamate or quinidine carbamate as chiral selector, e.g. a
quinine carbamate phase (8S,9R) available commercially under the
tradename ProntoSIL Chiral AX QN-1:
##STR00006##
[0037] A compound of formula II, wherein greater than 70% by weight
of the compound is in the form of the R or S enantiomer, may be
obtained by deprotecting a compound of formula III, wherein greater
than 70% by weight of the compound of formula III is in the form of
the R or S enantiomer:
##STR00007##
wherein m, n, X, R.sub.1 and R.sub.2 are as defined above and
R.sub.6 is an amino protecting group, and, where required,
converting the compounds of formula I obtained in free form into
the desired salt form, or vice versa.
[0038] Examples of suitable amino protecting groups as R.sub.6 are
e.g. as disclosed in "Protective Groups in Organic Synthesis" T. W.
Greene, J.Wiley & Sons NY, 2.sup.nd ed., chapter 7, 1991, and
references therein, e.g. acyl, e.g. tert.-butoxy-carbonyl,
benzyloxycarbonyl, 9-fluorenyl methoxy carbonyl, trifluoroacetyl,
trimethylsilylethanesulfonyl and the like.
[0039] Alternatively and more preferably a compound of formula II,
wherein greater than 70% by weight of the compound is in the form
of the R or S enantiomer, may be obtained by deprotecting a
compound of formula IIIa or IIIb, wherein greater than 70% by
weight of the compound of formula IIIa or IIIb is in the form of
the R or S enantiomer:
##STR00008##
wherein n, m, X, R.sub.1 and R.sub.2 are as defined above and
R.sub.6' is a simultaneous OH and amino protecting group, e.g. such
that R.sub.6' together with the O and N atoms to which it is
attached, the asymmetric carbon atom and 1 to 3 further carbon
atoms forms a cyclic residue, e.g. a 5 to 7-membered heterocyclic
ring, e.g oxazolidin-2-one (in IIIa R.sub.6' is --C(O)--) or
2-methyl-4,5-dihydro-oxazole (in IIIb R.sub.6' is
--C(CH.sub.3)--).
[0040] The removal of the R.sub.6 or R.sub.6' protecting group in a
compound of formula III, IIIa or IIIb may conveniently be performed
according to methods known in the art, e.g. by hydrolysis, e.g. in
a basic medium, for example using a hydroxide such as barium
hydroxide. It may also be performed by hydrogenolysis, e.g. in the
presence of Pearlman's catalyst, e.g. as disclosed in J. Org.
Chem., 1998, 63, 2375-2377.
[0041] Thus in a further alternative aspect the present invention
provides a compound of formula III, IIIa of IIIb as defined above,
in free or salt form. The compounds of formula III, IIIa or IIIb
have one or more asymmetric centers in the molecule, and thus
various optical isomers may be obtained. The present invention also
encompasses enantiomers, racemates, diastereoisomers and mixtures
thereof.
[0042] The removal of the R.sub.6 or R.sub.6' protecting group in a
compound of formula III, IIIa or IIIb may conveniently be performed
according to methods known in the art, e.g. by hydrolysis, e.g. in
a basic medium, for example using a hydroxide such as barium
hydroxide. It may also be performed by hydrogenolysis, e.g. in the
presence of Pearlman's catalyst, e.g. as disclosed in J. Org.
Chem., 1998, 63, 2375-2377.
[0043] A compound of formula III, IIIa or IIIb comprising greater
than 70% by weight of the R or S enantiomer may be obtained by
separating the S enantiomer from the R enantiomer in a racemic
mixture of a compound of formula III, IIIa or IIIb, using
chromatography (MPLC, HPLC, SFC) or simulated moving bed
(multi-column) chromatography with a polysaccharide-based chiral
stationary phase, preferably an amylose-type phase, e.g. amylose
tris[(S)-.alpha.-methylbenzyl carbamate coated on silica gel
substrate, as available under the tradename CHIRALPAK AS and shown
below, or in an immobilized form as prepared according to the
processes described in WO 97/04011 and WO 97/49733:
##STR00009##
[0044] A compound of formula III, IIIa or IIIb comprising greater
than 70% by weight of the R or S enantiomer may alternatively be
obtained by removing the hydrolysable groups present in R.sub.2' in
a compound of formula IV, IVa or IVb comprising greater than 70% by
weight of the R or S enantiomer:
##STR00010##
wherein m, n, X, R.sub.1, R.sub.6 and R.sub.6' are as defined above
and R.sub.2' is
##STR00011##
wherein each of R.sub.3' and R.sub.4' is a hydrolysable group.
[0045] Preferably R.sub.3' and R.sub.4' are identical and have the
significance of e.g. phenyl or benzyl or form together a cyclic
system such as in 1,5-dihydro-2,4,3-benzodioxaphosphepin.
[0046] A compound of formula IV, IVa or IVb comprising greater than
70% by weight of the R or S enantiomer may be obtained by
separating the S enantiomer from the R enantiomer in a racemic
mixture of a compound of formula IV, IVa or IVb, e.g. as described
above for the separation of enantiomers of compounds of formula
III, IIIa or IIIb.
[0047] A compound of formula IV, IVa or IVb, e.g. a racemic mixture
thereof, wherein X is O may be obtained by reacting a compound of
formula V, Va or Vb:
##STR00012##
wherein m, n, R.sub.1, R.sub.6 and R.sub.6' are as defined above,
with a phosphorylating agent, e.g. a phosphorochloridate, e.g.
diphenylchlorophosphate or dibenzylchlorophosphate,
cyanoethylphosphate, a phosphoramidate such as N-phenyl
phosphoramidate,
3-(diethylamino)-1,5-dihydro-2,4,3-benzodioxaphosphepin and the
like. The reaction may be carried out according to methods known in
the art, e.g. as disclosed in J. Org. Chem. supra. In the compounds
of formula IIIa the amino group is preferably in protected form, as
R'.sub.4 when R.sub.4 is other than acyl.
[0048] A compound of formula IV, IVa or IVb, e.g. a racemic mixture
thereof, wherein X is a direct bond may be obtained by reacting a
compound of formula V', Va' or Vb':
##STR00013##
wherein m, n, R.sub.1, R.sub.6 and R.sub.6' are as defined above,
and Y is a leaving group, e.g. Br, with a phosphorylating agent,
e.g. diethyl phosphite under reducing conditions, e.g. in the
presence of NaH. The reaction may be performed in accordance with
methods known in the art.
[0049] Alternatively the chiral separation may be performed at an
earlier stage in the process. Thus a compound of formula IV, IVa or
IVb comprising greater than 70% by weight of the R or S enantiomer
may be obtained by reacting a compound of formula V, Va, Vb, V',
Va' or Vb' comprising greater than 70% by weight of the R or S
enantiomer with a phosphorylating agent. A compound of formula V,
Va, Vb, V', Va' or Vb' comprising greater than 70% by weight of the
R or S enantiomer may be obtained by separating the S enantiomer
from the R enantiomer in a racemic mixture of a compound of formula
V, Va, Vb, V', Va' or Vb', e.g. using HPLC or simulated moving bed
(multi-column) chromatography with a polysaccharide-based chiral
stationary phase as described above for separation of the
enantiomers of a compound of formula IV, IVa or IVb.
[0050] A compound of formula V or V' may be prepared by reacting a
compound of formula VI:
##STR00014##
wherein m, n and R.sub.1 are as defined above and R.sub.9 is OH or
a leaving group, e.g. Br, with an amino protecting group donor
compound. A compound of formula Va, Va', Vb or Vb' may be prepared
by reacting a compound of formula VI with an OH and amino
protecting compound, e.g the OH and amino protection may be
performed simultaneously by reacting the free aminoalcohol or
aminodiol of formula VI in order to obtain a cyclic residue, e.g. a
5 to 7-membered heterocyclic ring, e.g oxazolidin-2-one or
2-methyl-4,5-dihydro-oxazole, e.g. by reaction with Cbo-Cl,
Boc-anhydride, triethylortho acetate and acetonitrile, or phosgene
under basic conditions.
[0051] At any stage in the process, R.sub.1 in any of the formulae
above may optionally be converted to an alternative R.sub.1 group
using known methods. For example, a compound of formula Vb wherein
R.sub.1 is (4-benzyloxy-phenyl)-ethyl may be converted to an
alternative compound of formula Vb wherein R.sub.1 is
[4-(6-fluoro-hexyloxy)-phenyl]-ethyl by (a) removal of a benzyl
group from R.sub.1 by hydrogenation to leave a
(4-hydroxy-phenyl)-ethyl residue, followed by (b) reaction with
1-bromo-6-fluorohexane. The alternative compound of formula Vb may
then undergo chiral separation or be converted to a compound of
formula IVb as described above.
[0052] The compounds of formulae III, IIIa, IIIb, IV, IVa and IVb
comprising greater than 70% by weight of the R or S enantiomer used
as starting materials, and salts thereof are also novel and form
part of the present invention.
[0053] In a further alternative aspect, a compound of formula II
wherein greater than 70% by weight of the compound is in the form
of the R or S enantiomer, may be obtained by deprotecting and
hydrolyzing a compound of formula VII, wherein greater than 70% by
weight of the compound is in the form of the R or S enantiomer:
##STR00015##
wherein m, n, X and R.sub.1 are as defined above, and R.sub.2''
is
##STR00016##
wherein each of R.sub.3' and R.sub.4' is a hydrolysable group, e.g.
tert-butyl, and R.sub.7 is an amino protecting group, e.g.
benzyloxycarbonyl.
[0054] A compound of formula VII, wherein greater than 70% by
weight of the compound is in the form of the R or S enantiomer, may
be obtained by reacting a compound of formula VIII, wherein greater
than 70% by weight of the compound is in the form of the R or S
enantiomer,
##STR00017##
with a phosphorylating agent, e.g. as described above.
[0055] Insofar as the production of the starting materials is not
particularly described, the compounds are known or may be prepared
analogously to methods known in the art or as disclosed in the
Examples hereinafter.
[0056] The following Examples are illustrative of the
invention.
RT=room temperature CBO=benzyloxycarbonyl
FTY720=2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol
EDTA=ethylenediaminetetraacetic acid OPA=ortho-phthalaldehyde
(benzene-1,2-dicarbaldehyde)
EXAMPLE 1
Phosphoric acid
mono-[(R/S)-2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoin-
dol-2-yl)-butyl]ester
##STR00018##
[0057] a)
(R/S)-4-Hydroxymethyl-4-[2-(4-octyl-phenyl)-ethyl]-oxazolidin-2--
one
[0058] Benzyl chloroformate (0.45 ml; 3.2 mmol) is added to a
suspension of FTY720.HCl (1.03 g, 3 mmol) in 2N NaOH (20 ml). The
reaction is kept at RT over night and in order to complete the
reaction further benzyl chloroformate (0.9 ml; 6.4 mmol) is added.
After 2 days at RT the reaction is acidified with 1N HCl, extracted
with methylenehloride and purified on a silica gel column using
methylenehloride/methanol/acetic acid.sub.50% (9/1/0.125) as mobile
phase.
[0059] [M+H].sup.+: 334 (ESI-MS)
b)
(R/S)-4-[2-(4-Octyl-phenyl)-ethyl]-4-(3-oxo-1,5-dihydro-3lambda*5*-benz-
o[e][1,3,2]dioxaphosphepin-3-yloxymethyl)-oxazolidin-2-one
[0060] To a solution of the endproduct of a) (2.4 g; 7.2 mmol) in
methylenehloride/THF 1/1 (100 ml) at 0.degree. C. is added
tetrazole (recrystallized; 2.52 g; 36 mmol) and
3-(diethylamino)-1,5-dihydro-2,4,3-benzodioxaphosphepintriphenyl-phosphit-
e (5.17 g; 21.6 mmol). After 18 hours at RT, H.sub.2O.sub.2 (8.2 ml
[30% in water]; 72 mmol) is added (cooling) to the solution and
kept at RT for additional 90 minutes. After quenching with
saturated Na.sub.2S.sub.2O.sub.3 solution (100 ml) the reaction is
extracted with ethylacetate (three times). The organic layer is
dried over Na.sub.2SO.sub.4 and the compound is purified on silica
gel using cyclohexane/ethylacetate 1/1 as mobile phase.
c) Phosphoric acid
mono-(R/S)-4-[2-(4-octyl-phenyl)-ethyl]-2-oxo-oxazolidin-4-ylmethyl)
ester
[0061] The endproduct of step b) (1.03 g; 2 mmol) is hydrogenated
at normal pressure (Pd/C.sub.10%; 50 mg) over a period of 90
minutes. After filtration the reaction is concentrated and used in
step d) without further purification.
d) Phosphoric acid
mono-[(R/S)-2-amino-2-hydroxymethyl-4-(4-octyl-phenyl)-butyl]ester
((R/S)-FTY720-phosphate)
[0062] To a solution of the endproduct of step c) in ethanol (20
ml) LiOH (20 ml; 10% solution in water) is added. After 24 hours at
reflux the reaction is neutralized with HCl (1N in water) and
concentrated. The residue is treated with glacial acetic acid (5
ml) and precipitation of the endproduct occurs after addition water
(50 ml). After filtering, washing (water) and drying pure
endproduct is obtained without any further purification.
e) Phosphoric acid
mono-[(R/S)-2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoin-
dol-2-yl)-butyl]ester (OPA-derivatization)
[0063] The endproduct of step d) ((R/S)-FTY720-phosphate) (50 mg;
0.125 mmol) is suspended in a solution of EDTA (0.5 ml; 10 mM in
water) and aqueous boric acid (0.5 ml; 3% in water; adjusted to pH
10.5 with aqueous KOH.sub.10%). After addition of OPA (33 mg, 0.25
mmol), dissolved in ethanol (0.5 ml), the reaction is kept at RT
for 1 hour (ultrasound). After that the pH is adjusted to 3.5
(aqueous HCl; 1N) and extracted with ethylacetate (three times).
The organic layer is dried over Na.sub.2SO.sub.4 and the compound
is purified on silica gel using methylenechloride/methanol
(95/5.fwdarw.0/100) as mobile phase.
[0064] [M-H].sup.-: 502.5 (ESI-MS)
EXAMPLE 2
Phosphoric acid
mono-[(R)-2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoindo-
l-2-yl)-butyl]ester
##STR00019##
[0066] Example 2 is obtained after separation of the final product
of step b) by HPLC on CHIRALPAK AS column at a preparative scale
(ethanol/n-hexane 40/60 as mobile phase), or by simulated moving
bed chromatography on HPLC columns packed with immobilized amylose
tris[(S)-.alpha.-methylbenzyl carbamate coated on silica gel
(n-hexane/ethanol/chloroform 60/20/20 as the mobile phase; feed
concentration, 1%) and applying steps c), d) and e) as described
for example 1.
EXAMPLE 3
Phosphoric acid
mono-[(S)-2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoindo-
l-2-yl)-butyl]ester
##STR00020##
[0068] Example 3 is obtained after separation of the final product
of step b) by HPLC on CHIRALPAK AS column at a preparative scale
(ethanol/n-hexane 40/60 as mobile phase), or by simulated moving
bed chromatography on HPLC columns packed with immobilized amylose
tris[(S)-.alpha.-methylbenzyl carbamate coated on silica gel
(n-hexane/ethanol/chloroform 60/20/20 as the mobile phase; feed
concentration, 1%) and applying steps c), d) and e) as described
for example 1.
EXAMPLE 4
((R/S)-4-{2-[4-(6-Fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-oxa-
zol-4-yl)-methanol
[0069] a) To a solution of 4-(2-hydroxy-ethyl)-phenol (50 g, 0.36
mol) in ethanol (400 ml) is added potassium carbonate (75 g, 0.54
mol, 1.5 eq) and benzyl bromide (47.2 ml, 0.39 mol, 1.1 eq), the
reaction mixture is stirred at RT overnight. The reaction mixture
is then filtered off through celite and concentrated under vacuum.
2-(4-Benzyloxy-phenyl)-ethanol is isolated after crystallization
with diethyl ether. b) To a solution of
2-(4-benzyloxy-phenyl)-ethanol (78.72 g, 0.34 mol) in methylene
chloride (400 ml) is added triethylamine (67.3 ml, 0.44 mol, 1.4
eq), then at 0.degree. C. is added mesylchloride (34.8 ml, 0.44
mol, 1.3 eq). The reaction mixture is stirred at 0.degree. C. for
30 minutes and allowed to rise to RT. The reaction mixture is
extracted with methylene chloride (2.times.300 ml), the combined
organic layers are then washed with brine (2.times.300 ml) and
concentrated under vacuum. c) To the crude product in solution in
ethyl acetate (600 ml) is added sodium iodide (67.2 g, 0.44 mol,
1.3 eq) and the reaction mixture is stirred under reflux for 6
hours. After filtration, the organic layer is washed with brine
(3.times.400 ml), dried with Na.sub.2SO.sub.4, filtered and
concentrated under vacuum. 1-Benzyloxy-4-(2-iodo-ethyl)-benzene is
isolated after crystallization with diethyl ether. d) To a solution
of acetamidomalonate (59.4 g, 0.27 mol, 2 eq) in dry
dimethylformamide (400 ml) is added at 0.degree. C. under inert
atmosphere sodium hydride (60% in oil) (9.94 g, 0.49 mol, 1.8 eq),
the reaction mixture is stirred for 3 hours at 0.degree. C.
1-Benzyloxy-4-(2-iodo-ethyl)-benzene (46.8 g, 0.13 mol, 1 eq) in
solution in dry dimethylformamide (250 ml) is then slowly added at
0.degree. C. and the reaction mixture is stirred at RT overnight.
The reaction mixture is quenched with few drops of methanol and
concentrated almost to dryness under vacuum, then extracted with
ethyl acetate and washed subsequently with 1N HCl (2.times.500 ml),
saturated solution of NaHCO.sub.3 (2.times.500 ml) and brine
(2.times.500 ml), dried with Na.sub.2SO.sub.4, filtered and
concentrated under vacuum.
2-Acetylamino-2-[2-(4-benzyloxy-phenyl)ethyl]-malonic acid diethyl
ester is isolated after multiple crystallization using diethyl
ether. e) To a solution of
2-acetylamino-2-[2-(4-benzyloxy-phenyl)-ethyl]-malonic acid diethyl
ester (44.1 g, 0.1 mol) in ethanol water (2/1) (285 ml/285 ml) is
added CaCl.sub.2 (28.5 g, 0.26 mol, 2.5 eq) and NaBH.sub.4 by
portion (19.4 g, 0.52 mol, 5.0 eq), the reaction mixture is stirred
overnight at RT. At 0.degree. C. the reaction mixture is carefully
quenched with drop wise methanol (10 ml) and concentrated to almost
dryness under vacuum. The crude mixture is extracted with ethyl
acetate (4.times.500 ml) and washed subsequently with 1N HCl
(2.times.300 ml), saturated solution of NaHCO.sub.3 (2.times.300
ml) and brine (2.times.300 ml). The combined organic layers are
then dried with Na.sub.2SO.sub.4, filtered and concentrated under
vacuum.
N-[3-(4-Benzyloxy-phenyl)-1,1-bis-hydroxymethyl-propyl]acetamide is
carried on without further purification. f) To a solution of crude
N-[3-(4-benzyloxy-phenyl)-1,1-bis-hydroxymethyl-propyl]-acetamide
in a mixture of tetrahydrofuran, methanol, water (1/2/2) (450
ml/900 ml/900 ml) is added at RT lithium hydroxide (32.7 g, 1.36
mol, 8.0 eq). The reaction mixture is stirred at 55.degree. C. for
5 hours, then extracted with ethyl acetate (500 ml) and washed with
brine (2.times.300 ml), the combined organic layers are then dried
with Na.sub.2SO.sub.4, filtered and concentrated under vacuum.
2-Amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diol is
isolated after crystallization using ethyl acetate. g) To a
solution of
2-amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diol (31.1 g,
0.10 mol) in acetonitrile (2.381) is added triethylortho acetate
(17.1 ml, 0.12 mol, 1.2 eq) and acetic acid (5.48 ml, 0.11 mol, 1.1
eq), the reaction mixture is then stirred at 80.degree. C. for 5
hours. The reaction mixture is then concentrated under vacuum,
{4-[2-(4-benzyloxy-phenyl)-ethyl]-2-methyl-4,5-dihydro-oxazol-4-yl}-metha-
nol is isolated after crystallization with ethyl acetate. h) To a
solution of
{4-[2-(4-benzyloxy-phenyl)-ethyl]-2-methyl-4,5-dihydro-oxazol-4-yl}-me-
thanol (26.1 g, 0.08 mol) in methanol (800 ml) is added palladium
on charcoal (2.6 g, 10% wt), and the reaction mixture is stirred
under hydrogen atmosphere at RT for 5 hours. The reaction mixture
is then filtered through celite and concentrated under vacuum.
(R/S)-4-[2-(4-Hydroxymethyl-2-methyl-4,5-dihydro-oxazol-4-yl)-ethyl]-phen-
ol is isolated in quantitative yield after crystallization with
ethyl acetate and hexanes. i) To a solution of
(R/S)-4-[2-(4-hydroxymethyl-2-methyl-4,5-dihydro-oxazol-4-yl)-ethyl]-phen-
ol (500 mg, 2.12 mmol) in dry DMF (8 ml) is added under inert
atmosphere Cs.sub.2CO.sub.3 (901 mg, 2.76 mmol, 1.3 eq.) and
1-bromo-6-fluoro-hexane (464.1 mg, 2.55 mmol, 1.2 eq.). The
reaction mixture is stirred under inert atmosphere at 85.degree. C.
overnight. A saturated solution of NaHCO.sub.3 (20 ml) and ethyl
acetate (40 ml) are then added. The organic layer is separated and
the aqueous phase is extracted with ethyl acetate (3.times.40 ml).
The combined organic extracts are washed with brine and 1M HCl,
dried over MgSO.sub.4, and evaporated to dryness. Purification by
flash chromatography (cy Hexane/ethyl acetate (9/1) to (1/1) and
(0/1)) affords
(R/S)-(4-{2-[4-(6-fluoro-hexyloxy)-phenyl]ethyl}-2-methyl-4,5-dih-
ydro-oxazol-4-yl)-methanol as colorless oil.
[0070] Separation of enantiomers of
((R/S)-4-{2-[4-(6-Fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-ox-
azol-4-yl)-methanol is performed by HPLC on CHIRALPAK AD column at
a preparative scale (n-hexane/2-propanol 96/6 as the mobile
phase).
##STR00021##
EXAMPLE 5
Phosphoric acid
mono-{(S)-2-amino-4-[4-(6-fluoro-hexyloxy)-phenyl]-2-hydroxymethyl-butyl}-
ester
##STR00022##
[0071] a) To a solution of chiral
((S)-4-{2-[4-(6-fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-oxaz-
ol-4-yl)-methanol (300 mg, 0.80 mmol) and tetrazole (337.4 mg, 4.82
mmol, 6 eq., recrystallized from toluene) in dry THF (6 ml) at
-25.degree. C. is added
3-diethylamino-1,5-dihydro-benzo[e][1,3,2]dioxaphosphepine (433.5
.mu.L, 1.56 mol, 1.95 eq.). The reaction mixture is stirred under
argon at -25.degree. C. for 3 h, then allowed to come back to RT.
Then, H.sub.2O.sub.2 (30%, 75 .mu.L, 4.0 mmol, 5 eq.) is injected
at 0.degree. C. with vigorous stirring. The reaction mixture is
stirred for further 30 min, followed by addition of saturated
sodium thiosulfate solution (1 ml). The organic layer is separated
and the aqueous phase is extracted with ether (3.times.20 ml). The
combined organic extracts are washed with brine, dried over
MgSO.sub.4, and evaporated to dryness. Purification by flash
chromatography (ethyl acetate) affords phosphoric acid
di-tert-butyl ester
(S)-4-{2-[4-(6-fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-oxazo-
l-4-ylmethyl ester as colorless oil. b) To a solution of phosphoric
acid di-tert-butyl ester
(S)-4-{2-[4-(6-fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-oxazo-
l-4-ylmethyl ester (33 mg, 0.050 mmol) in ethanol (2 ml) is added
conc. HCl (2 ml). The reaction mixture is stirred at 85.degree. C.
for 2 hours, then concentrated to dryness. The residue is
re-dissolved in ethyl acetate and precipitated with hexanes. The
solid is filtered off, washed with dry ether and dried under vacuum
to afford phosphoric acid
mono-{(S)-2-amino-4-[4-(6-fluoro-hexyloxy)-phenyl]-2-hydroxymethyl-butyl}-
ester as a colorless powder.
EXAMPLE 6
Phosphoric acid
mono-{(R)-2-amino-4-[4-(6-fluoro-hexyloxy)-phenyl]-2-hydroxymethyl-butyl}-
ester
##STR00023##
[0073] The synthesis is performed applying the chemistry described
for example 5.
EXAMPLE 7
Phosphoric acid
mono-[(S)-4-[4-(6-fluoro-hexyloxy)-phenyl]-2-hydroxymethyl-2-(1-oxo-1,3-d-
ihydro-isoindol-2-yl)-butyl]ester
##STR00024##
[0075] OPA-derivatization is performed according to the procedure
given in example 1, step e.
EXAMPLE 8
Phosphoric acid
mono-[(R)-4-[4-(6-fluoro-hexyloxy)-phenyl]-2-hydroxymethyl-2-(1-oxo-1,3-d-
ihydro-isoindol-2-yl)-butyl]ester
##STR00025##
[0077] OPA-derivatization is performed according to the procedure
given in example 1, step e.
EXAMPLE 9
Phosphoric acid
mono-((R)-2-amino-4-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-2-hydroxymet-
hyl-butyl) ester
##STR00026##
[0079] To a solution of 4-(2-hydroxy-ethyl)-phenol (50 g, 0.36 mol)
in ethanol (400 ml) is added potassium carbonate (75 g, 0.54 mol,
1.5 eq) and benzyl bromide (47.2 ml, 0.39 mol, 1.1 eq), the
reaction mixture is stirred at room temperature overnight. The
reaction mixture is then filtered off trough celite and
concentrated under vacuum. 2-(4-Benzyloxy-phenyl)-ethanol is
isolated after crystallization with diethyl ether (82.6 g,
95%).
[0080] To a solution of 2-(4-benzyloxy-phenyl)-ethanol (78.72 g,
0.34 mol) in methylene chloride (400 ml) is added triethylamine
(67.3 ml, 0.44 mol, 1.4 eq), then at 0.degree. C. is added
mesylchloride (34.8 ml, 0.44 mol, 1.3 eq). The reaction mixture is
stirred at 0.degree. C. for 30 minutes and allowed to rise to room
temperature. The reaction mixture is extracted with methylene
chloride (2.times.300 ml), the combined organic layers are then
washed with brine (2.times.300 ml) and concentrated under vacuum.
To the crude product in solution in ethyl acetate (600 ml) is added
sodium iodide (67.2 g, 0.44 mol, 1.3 eq) and the reaction mixture
is stirred under reflux for 6 hours. After filtration, the organic
layer is washed with brine (3.times.400 ml), dried with
Na.sub.2SO.sub.4, filtered and concentrated under vacuum.
1-Benzyloxy-4-(2-iodo-ethyl)-benzene is isolated after
crystallization with diethyl ether (116.5 g, 86%).
[0081] To a solution of acetamidomalonate (59.4 g, 0.27 mol, 2 eq)
in dry dimethylformamide (400 ml) is added at 0.degree. C. under
inert atmosphere sodium hydride (60% in oil) (9.94 g, 0.49 mol, 1.8
eq), the reaction mixture is stirred for 3 hours at 0.degree. C.
1-Benzyloxy-4-(2-iodo-ethyl)-benzene (46.8 g, 0.13 mol, 1 eq) in
solution in dry dimethylformamide (250 ml) is then slowly added at
0.degree. C. and the reaction mixture is stirred at room
temperature overnight. The reaction mixture is quenched with few
drops of methanol and concentrated almost to dryness under vacuum,
then extracted with ethyl acetate and washed subsequently with 1N
HCl (2.times.500 ml), saturated solution of NaHCO.sub.3
(2.times.500 ml) and brine (2.times.500 ml), dried with
Na.sub.2SO.sub.4, filtered and concentrated under vacuum.
2-Acetylamino-2-[2-(4-benzyloxy-phenyl)-ethyl]-malonic acid diethyl
ester is isolated after multiple crystallization using diethyl
ether (47.3 g, 80%).
[0082] To a solution of
2-acetylamino-2-[2-(4-benzyloxy-phenyl)-ethyl]-malonic acid diethyl
ester (44.1 g, 0.1 mol) in ethanol water (2/1) (285 ml/285 ml) is
added CaCl.sub.2 (28.5 g, 0.26 mol, 2.5 eq) and NaBH.sub.4 by
portion (19.4 g, 0.52 mol, 5.0 eq), the reaction mixture is stirred
overnight at room temperature. At 0.degree. C. the reaction mixture
is carefully quenched with drop wise methanol (10 ml) and
concentrated to almost dryness under vacuum. The crude mixture is
extracted with ethyl acetate (4.times.500 ml) and washed
subsequently with 1N HCl (2.times.300 ml), saturated solution of
NaHCO.sub.3 (2.times.300 ml) and brine (2.times.300 ml). The
combined organic layers are then dried with Na.sub.2SO.sub.4,
filtered and concentrated under vacuum.
N-[3-(4-Benzyloxy-phenyl)-1,1-bis-hydroxymethyl-propyl]-acetamide
is carried on without further purification.
[0083] To a solution of crude
N-[3-(4-benzyloxy-phenyl)-1,1-bis-hydroxymethyl-propyl]-acetamide
in a mixture of tetrahydrofuran, methanol, water (1/2/2) (450
ml/900 ml/900 ml) is added at room temperature lithium hydroxide
(32.7 g, 1.36 mol, 8.0 eq). The reaction mixture is stirred at
55.degree. C. for 5 hours, then extracted with ethyl acetate (500
ml) and washed with brine (2.times.300 ml), the combined organic
layers are then dried with Na.sub.2SO.sub.4, filtered and
concentrated under vacuum.
2-Amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diol is
isolated after crystallization using ethyl acetate (28.8 g,
97%).
[0084] To a solution of
2-amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diol (200 mg,
0.66 mmol) in dioxane (10 ml) is added a solution of 1M NaOH (0.73
ml, 0.73 mmol, 1.1 eq) and Boc anhydride (217 mg, 0.99 mmol, 1.5
eq). The reaction mixture is then stirred overnight at room
temperature. Extraction with ethyl acetate (80 ml) and washed with
brine (2.times.50 ml), the combined organic layers are then dried
with Na.sub.2SO.sub.4, filtered and concentrated under vacuum.
2-Amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diol is
isolated as a white solid after flash chromatography (Ethyl
acetate/Hexane (3/1)) and crystallization with diethyl ether (204
mg, 87%).
[0085] To a solution of
2-amino-2-[2-(4-benzyloxy-phenyl)ethyl]-propane-1,3-diol (6.31 g,
0.16 mol) in dichloromethane (120 ml) and pyridine (1.26 ml, 0.16
mol) is added at room temperature o-nitrobenzoylchloride (2.27 ml.
0.017 mol). The reaction mixture is then stirred overnight at room
temperature. Extraction with dichloromethane (300 ml) and washed
with brine (2.times.200 ml), the combined organic layers are then
dried with Na.sub.2SO.sub.4, filtered and concentrated under
vacuum. 2-Nitro-benzoic acid
4-(4-benzyloxy-phenyl)-2-tert-butoxycarbonylamino-2-hydroxymethyl-bu-
tyl ester is isolated as a white solid after flash chromatography
(Ethyl acetate/Hexane (1/1)) and crystallization with diethyl ether
(6.55 mg, 76%). 1.40 g of starting material could be isolated.
[0086] To a solution of 2-nitro-benzoic acid
4-(4-benzyloxy-phenyl)-2-tert-butoxycarbonylamino-2-hydroxymethyl-butyl
ester (105 mg, 0.19 mmol) in toluene (2 ml) is added 2,2
dimethoxypropane (0.06 ml, 0.57 mmol, 3 eq) and a catalytic amount
of p-toluene sulfonic acid. The reaction mixture is stirred at
95.degree. C. for 6 hours, then concentrated to dryness under
negative pressure.
4-[2-(4-Benzyloxy-phenyl)-ethyl]-2,2-dimethyl-4-(2-nitro-benzoyloxy-methy-
l)-oxazolidine-3-carboxylic acid tert-butyl ester is isolated as an
oil (88 mg, 78%) after purification by flash chromatography (Ethyl
acetate/Hexane (1/3)).
[0087] To a solution of
4-[2-(4-benzyloxy-phenyl)-ethyl]-2,2-dimethyl-4-(2-nitro-benzoyloxymethyl-
)-oxazolidine-3-carboxylic acid tert-butyl ester (80 mg, 0.13 mmol)
in methanol (1 ml) and THF (1 ml) is added K.sub.2CO.sub.3 (1.5 mg,
0.076 eq), the reaction mixture is stirred overnight at room
temperature. The reaction mixture is concentrated to dryness and
4-[2-(4-benzyloxy-phenyl)-ethyl]-4-hydroxymethyl-2,2-dimethyl-oxazolidine-
-3-carboxylic acid tert-butyl ester (51 mg, 85%) is isolated as a
white solid after flash chromatography (Ethyl acetate/Hexane
(1/4)).
[0088] To a solution of
4-[2-(4-benzyloxy-phenyl)-ethyl]-4-hydroxymethyl-2,2-dimethyl-oxazolidine-
-3-carboxylic acid tert-butyl ester (25 mg, 0.05 mmol) in methanol
(10 ml) is added a catalytic amount of paladium on charcoal (10%
wt). The reaction mixture is stirred under H.sub.2 atmosphere at
room temperature for 2 hours.
4-Hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidine-3-
-carboxylic acid tert-butyl ester is isolated as a white solid
after filtration of the suspension through celite and concentration
to dryness.
[0089] To a solution of
(S)-4-hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidi-
ne-3-carboxylic acid tert-butyl ester (100 mg, 0.28 mmol) in DMF (5
ml) is added CsCO.sub.3 (120.5 mg, 0.37 mmol, 1.3 eq) and
1-(2-bromo-ethoxy)-3-fluoro-benzene (80.7 mg, 0.37 mmol, 1.3 eq).
The reaction mixture is stirred at 85.degree. C. for 4 hours. Ethyl
acetate and water are then added, the organic layer is separated
and the aqueous phase is extracted with ethylacetate (3.times.50
ml). The combined organic extracts are washed with brine, dried
over MgSO.sub.4, and evaporated to dryness. Purification by flash
chromatography (AcOEt/Hx 9:1) affords
(S)-4-(2-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-ethyl)-4-hydroxymethyl--
2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester as
colorless oil.
[0090] To a solution of
(S)-4-(2-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-ethyl)-4-hydroxymethyl--
2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester (70 mg,
0.14 mmol) and tetrazole (49 mg, 0.7 mmol, 5 eq., recrystallized
from toluene) in dry THF (5 ml) is added
di-tBu-N,N-diisopropylphosphoramide (155 mg, 0.56 mmol, 4 eq.).
After stirring under argon at RT for 3 h, H.sub.2O.sub.2 (30%, 10
eq.) is slowly added at 0.degree. C. with vigorous stirring. The
reaction mixture is stirred for further 30 min, followed by
addition of saturated sodium thiosulfate solution (5 ml). The
organic layer is separated and the aqueous phase is extracted with
ether (3.times.20 ml). The combined organic extracts are washed
with brine, dried over MgSO.sub.4, and evaporated to dryness.
Purification by flash chromatography (AcOEt/Hx 1:1) affords
(R)-4-(di-tert-butoxy-phosphoryloxymethyl)-4-(2-{4-[2-(3-fluoro-phenoxy)--
ethoxy]-phenyl}-ethyl)-2,2-dimethyl-oxazolidine-3-carboxylic acid
tert-butyl ester as colorless crystals.
[0091] Finally, a solution of
(R)-4-(di-tert-butoxy-phosphoryloxymethyl)-4-(2-{4-[2-(3-fluoro-phenoxy)--
ethoxy]-phenyl}-ethyl)-2,2-dimethyl-oxazolidine-3-carboxylic acid
tert-butyl ester (70 mg, 0.10 mmol) in conc. HCl (2 ml). is stirred
at room temperature for one hour and is then heated to 95.degree.
C. for 2 hours, then concentrated to dryness. The residue is
re-dissolved in ethyl acetate and precipitated with hexanes. The
solid is filtered off, washed with dry ether and dried in vacuo to
afford phosphoric acid
mono-((R)-2-amino-4-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-2-hydroxymet-
hyl-butyl) ester as a colorless powder.
EXAMPLE 10
Phosphoric acid
mono-((S)-2-amino-4-{4-[2-(3-fluoro-phenoxy)-ethoxy]phenyl}-2-hydroxymeth-
yl-butyl) ester
##STR00027##
[0093] Phosphoric acid
mono-((S)-2-amino-4-{(4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-2-hydroxyme-
thyl-butyl) ester is prepared as described in example 9 using
(R)-4-hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidi-
ne-3-carboxylic acid tert-butyl ester instead of
(S)-4-hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidi-
ne-3-carboxylic acid tert-butyl ester.
EXAMPLE 11
Phosphoric acid
mono-{(R)-2-amino-2-hydroxymethyl-4-[4-(2-m-tolyloxy-ethoxy)-phenyl]-buty-
l}ester
##STR00028##
[0095] Phosphoric acid
mono-{(R)-2-amino-2-hydroxymethyl-4-[4-(2-m-tolyloxy-ethoxy)-phenyl]-buty-
l}ester is prepared using an analogous method to that described in
example 9.
EXAMPLE 12
Phosphoric acid
mono-((R)-2-amino-2-hydroxymethyl-4-{4-[2-(4-methoxy-phenyl)-ethoxy]-phen-
yl}-butyl) ester
##STR00029##
[0097] Phosphoric acid
mono-((R)-2-amino-2-hydroxymethyl-4-{4-[2-(4-methoxy-phenyl)-ethoxy]-phen-
yl}-butyl) ester is prepared using an analogous method to that
described in example 9.
EXAMPLE 13
Phosphoric acid
mono-{(R)-2-amino-2-hydroxymethyl-4-[4-(2-p-tolyl-ethoxy)-phenyl]butyl}es-
ter
##STR00030##
[0099] Phosphoric acid
mono-{(R)-2-amino-2-hydroxymethyl-4-[4-(2-p-tolyl-ethoxy)-phenyl]-butyl}e-
ster is prepared using an analogous method to that described in
example 9.
EXAMPLE 14
Phosphoric acid
mono-((R)-2-amino-2-hydroxymethyl-4-{4-[2-(4-trifluoromethyl-phenyl)-etho-
xy]-phenyl}-butyl) ester
##STR00031##
[0101] Phosphoric acid
mono-((R)-2-amino-2-hydroxymethyl-4-{4-[2-(4-trifluoromethyl-phenyl)-etho-
xy]-phenyl}-butyl) ester is prepared using an analogous method to
that described in example 9.
EXAMPLE 15
Phosphoric acid diethyl ester
4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-ethyl)-2-methyl-4,-
5-dihydro-oxazol-4-ylmethyl ester
##STR00032##
[0103] To a solution of phosphoric acid
mono-(2-amino-4-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-2-hydro-
xymethyl-butyl) ester (100 mg, 0.22 mmol) in triethyl orthoacetate
(5 ml) is added acetic acid (13 ul, 0.22 mmol). The reaction
mixture is stirred at 80.degree. C. for 2 hours and concentrated to
dryness. Purification by flash chromatography (AcOEt/Hx 1:4)
affords phosphoric acid diethyl ester
4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-ethyl)-2-methyl-4,-
5-dihydro-oxazol-4-ylmethyl ester as colorless oil.
EXAMPLE 16
Phosphoric acid diethyl ester
(S)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethyl
ester
##STR00033##
[0105] Phosphoric acid diethyl ester
(S)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethyl
ester is prepared as described in example 15 using of phosphoric
acid
mono-(2-amino-4-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy}-phenyl]-2-hydro-
xymethyl-butyl) ester.
EXAMPLE 17
Phosphoric acid diethyl ester
(R)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethyl
ester
##STR00034##
[0107] Phosphoric acid diethyl ester
(R)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethyl
ester is prepared as described in example 15 using phosphoric acid
mono-(2-amino-4-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]phenyl}-2-hydrox-
ymethyl-butyl) ester.
EXAMPLE 18
Chromatographic resolution of racemic
4-hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidine-3-
-carboxylic acid tert-butyl ester
[0108] 10 micro-litre of a 0.1% ethanol solution of racemic
4-hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidine-3-
-carboxylic acid tert-butyl ester are injected on a Chiralcel OD-H
column (0.46.times.25 cm; commercially available from Chiral
Technologies). The chromatographic separation is achieved at room
temperature and at a flow rate of 1 ml/min using a mixture of
n-hexane/ethanol 90/10 (volume) containing 0.1% trifluoroacetic
acid (TFA) as the mobile phase. Detection is performed by UV at 210
nm. The enantiomers elute respectively after 7.23 min and 9.39 min
(Separation factor .alpha.: 1.52)
EXAMPLE 19
Chromatographic resolution of racemic Phosphoric acid diethyl ester
4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-ethyl)-2-methyl-4,-
5-dihydro-oxazol-4-ylmethyl ester.
[0109] 10 micro-litre of a 0.1% ethanol solution of racemic
phosphoric acid diethyl ester
4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-ethyl)-2-methyl-4,-
5-dihydro-oxazol-4-ylmethyl ester are injected on a Chiralcel OD-H
column (0.46.times.25 cm; commercially available from Chiral
Technologies). The chromatographic separation is achieved at room
temperature and at a flow rate of 1 ml/min using a mixture of
n-hexane/ethanol 95/5 (volume) as the mobile phase. Detection is
performed by UV at 210 nm. The enantiomers elute respectively after
31.17 min and 35.44 min (Separation factor .alpha.: 1.15)
EXAMPLE 20
Chromatographic separation of the enantiomers phosphoric acid
diethyl ester
4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-ethyl)-2-met-
hyl-4,5-dihydro-oxazol-4-ylmethyl ester
[0110] 10 micro-litre of a 0.1% ethanol solution of a mixture of
the enantiomers phosphoric acid diethyl ester
4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-ethyl)-2-methyl-4,-
5-dihydro-oxazol-4-ylmethyl ester are injected on a Chiralcel OJ-H
column (0.46.times.25 cm; commercially available from Chiral
Technologies). The chromatographic separation is achieved at room
temperature and at a flow rate of 1 ml/min using a mixture of
n-hexane/2-propanol 75/25 (volume) as the mobile phase. Detection
is performed by UV at 210 nm. The enantiomers elute respectively
after 1189 min and 16.77 min (Separation factor .alpha.: 1.27)
EXAMPLE 21
Chromatographic separation of the enantiomers phosphoric acid
diethyl ester
(S)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylme-
thyl ester
[0111] 10 micro-litre of a 0.1% ethanol solution of a mixture of
the enantiomers phosphoric acid diethyl ester
(S)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethyl
ester are injected on a Chiralpak AD-H column (0.46.times.25 cm;
commercially available from Chiral Technologies). The
chromatographic separation is achieved at room temperature and at a
flow rate of 1 ml/min using a mixture of n-hexane/ethanol 95/5
(volume) as the mobile phase. Detection is performed by UV at 210
nm. The enantiomers elute respectively after 10.35 min and 12.32
min (Separation factor .alpha.: 1.27).
[0112] The compounds of formula II comprising greater than 70% of
the R or S enantiomer, especially the S enantiomer, in free form or
in pharmaceutically acceptable salt form, (hereinafter referred to
as the compounds of the invention) exhibit valuable pharmacological
properties, e.g. lymphocyte recirculation modulating properties,
e.g. as indicated in in vitro and in vivo tests and are therefore
indicated for therapy.
A. In vitro
[0113] The compounds of the invention have binding affinity to
individual human S1P receptors as determined in following
assays:
Transient Transfection of Human S1P Receptors into HEK293 Cells
[0114] S1P receptors and G.sub.i proteins are cloned, and equal
amounts of 4 cDNAs for the S1P receptor, G.sub.i-.alpha.,
G.sub.i-.beta. and G.sub.i-.gamma. are mixed and used to transfect
monolayers of HEK293 cells using the calcium phosphate precipitate
method (M. Wigler et al., Cell. 1977; 11; 223 and DS. Im et al.,
Mol. Pharmacol. 2000; 57; 753). Briefly, a DNA mixture containing
25 .mu.g of DNA and 0.25 M CaCl is added to HEPES-buffered 2 mM
Na.sub.2HPO.sub.4. Subconfluent monolayers of HEK293 cells are
poisoned with 25 mM chloroquine, and the DNA precipitate is then
applied to the cells. After 4 h, the monolayers are washed with
phosphate-buffered saline and refed media (90% 1:1 Dulbecco's
modified essential media (DMEM):F-12+10% fetal bovine serum). The
cells are harvested 48-72 h after addition of the DNA by scraping
in HME buffer (in mM: 20 HEPES, 5 MgCl.sub.2, 1 EDTA, pH 7.4)
containing 10% sucrose on ice, and disrupted using a Dounce
homogenizer. After centrifugation at 800.times.g, the supernatant
is diluted with HME without sucrose and centrifuged at
100,000.times.g for 1 h. The resulting pellet is rehomogenized and
centrifuged a second hour at 100,000.times.g. This crude membrane
pellet is resuspended in HME with sucrose, aliquoted, and
snap-frozen by immersion in liquid nitrogen. The membranes are
stored at 70.degree. C. Protein concentration is determined
spectroscopically by Bradford protein assay.
GTP.gamma.S Binding Assay Using S1P Receptor/HEK293 Membrane
Preparations
[0115] GTP.gamma.S binding experiments are performed as described
by DS. Im et al., Mol. Pharmacol. 2000; 57:753. Ligand-mediated
GTP.gamma.S binding to G-proteins is measured in GTP binding buffer
(in mM: 50 HEPES, 100 NaCl, 10 MgCl.sub.2, pH 7.5) using 25 .mu.g
of a membrane preparation from transiently transfected HEK293
cells. Ligand is added to membranes in the presence of 10 .mu.M GDP
and 0.1 nM [.sup.35S]GTP.gamma.S (1200 Ci/mmol) and incubated at
30.degree. C. for 30 min. Bound GTP.gamma.S is separated from
unbound using the Brandel harvester (Gaithersburg, Md.) and counted
with a liquid scintillation counter.
[0116] In these assays, the compounds of the invention have binding
affinities to S1P receptors in the sub-microM range.
[0117] In particular, the EC.sub.50 values in nM for the following
compounds at various S1P receptors are shown in the table
below:
TABLE-US-00001 S1P-1 S1P-3 S1P-2 S1P-4 S1P-5 EC.sub.50 EC.sub.50
EC.sub.50 EC.sub.50 EC.sub.50 nM nM nM nM nM (R)-FTY720-P 191.15
28.91 >10000 80.13 >10000 (S)-FTY720-P 0.33 3.21 >10000
0.75 0.33 Ex. 14 0.4 140 >10000 6.6 7.7
wherein (R)-FTY720-P is (R)-FTY720-phosphate, (S)-FTY720-P is
(S)-FTY720-phosphate, In contrast to (R)-FTY720-phosphate, which
shows agonistic effects on S1P receptors only at very high
concentrations, (S)-FTY720-phosphate is a full agonist on S1P1 and
S1P3 and a partial agonist on S1P4 and S1P5 in the low nanomolar
range. B. In vivo: Blood Lymphocyte Depletion
[0118] A compound of the invention or the vehicle is administered
orally by gavage to rats. Tail blood for hematological monitoring
is obtained on day -1 to give the baseline individual values, and
at 2, 6, 24, 48 and 72 hours after drug application. In this assay,
the compounds of the invention deplete peripheral blood lymphocytes
when administered at a dose of 0.03 to 3 mg/kg.
[0119] The compounds of the invention are, therefore, useful in the
treatment and/or prevention of diseases or disorders mediated by
lymphocytes interactions, e.g. in transplantation, such as acute or
chronic rejection of cell, tissue or organ allo- or xenografts or
delayed graft function, graft versus host disease, autoimmune
diseases, e.g. rheumatoid arthritis, systemic lupus erythematosus,
hashimoto's thyroidis, multiple sclerosis, myasthenia gravis,
diabetes type I or II and the disorders associated therewith,
vasculitis, pernicious anemia, Sjoegren syndrome, uveitis,
psoriasis, Graves ophthalmopathy, alopecia areata and others,
allergic diseases, e.g. allergic asthma, atopic dermatitis,
allergic rhinitis/conjunctivitis, allergic contact dermatitis,
inflammatory diseases optionally with underlying aberrant
reactions, e.g. inflammatory bowel disease, Crohn's disease or
ulcerative colitis, intrinsic asthma, inflammatory lung injury,
inflammatory liver injury, inflammatory glomerular injury,
atherosclerosis, osteoarthritis, irritant contact dermatitis and
further eczematous dermatitises, seborrhoeic dermatitis, cutaneous
manifestations of immunologically-mediated disorders, inflammatory
eye disease, keratoconjunctivitis, myocarditis or hepatitis,
ischemia/reperfusion injury, e.g. myocardial infarction, stroke,
gut ischemia, renal failure or hemorrhage shock, traumatic shock,
others, cancer, e.g. T cell lymphomas or T cell leukemias,
infectious diseases, e.g. toxic shock (e.g. superantigen induced),
septic shock, adult respiratory distress syndrome or viral
infections, e.g. AIDS, viral hepatitis or chronic bacterial
infection. Examples of cell, tissue or solid organ transplants
include e.g. pancreatic islets, stem cells, bone marrow, corneal
tissue, neuronal tissue, heart, lung, combined heart-lung, kidney,
liver, bowel, pancreas, trachea or oesophagus.
[0120] For the above uses the required dosage will of course vary
depending on the mode of administration, the particular condition
to be treated and the effect desired. In general, satisfactory
results are indicated to be obtained systemically at daily dosages
of from about 0.03 to 2.5 mg/kg per body weight. An indicated daily
dosage in the larger mammal, e.g. humans, is in the range from
about 0.5 mg to about 100 mg, conveniently administered, for
example, in divided doses up to four times a day or in retard form.
Suitable unit dosage forms for oral administration comprise from
ca. 1 to 50 mg active ingredient.
[0121] The compounds of the invention may be administered by any
conventional route, in particular enterally, e.g. orally, e.g. in
the form of tablets or capsules, or parenterally, e.g. in the form
of injectable solutions or suspensions, topically, e.g. in the form
of lotions, gels, ointments or creams, or in a nasal or a
suppository form. Pharmaceutical compositions comprising a compound
of the invention in free form or in pharmaceutically acceptable
salt form in association with at least one pharmaceutical
acceptable carrier or diluent may be manufactured in conventional
manner by mixing with a pharmaceutically acceptable carrier or
diluent.
[0122] The compounds of the invention may be administered in free
form or in pharmaceutically acceptable salt form e.g. as indicated
above. Such salts may be prepared in conventional manner and
exhibit the same order of activity as the free compounds.
[0123] In accordance with the foregoing the present invention
further provides: [0124] 1.1 A method for preventing or treating
disorders or diseases mediated by lymphocytes, e.g. such as
indicated above, in a subject in need of such treatment, which
method comprises administering to said subject an effective amount
of a compound of formula II comprising greater than 70% by weight
of the R or S enantiomer, or a pharmaceutically acceptable salt
thereof; [0125] 1.2 A method for preventing or treating acute or
chronic transplant rejection or T-cell mediated inflammatory or
autoimmune diseases, e.g. as indicated above, in a subject in need
of such treatment, which method comprises administering to said
subject an effective amount of a compound of formula II comprising
greater than 70% by weight of the R or S enantiomer, or a
pharmaceutically acceptable salt thereof; [0126] 2. A compound of
formula II comprising greater than 70% by weight of the R or S
enantiomer, in free form or in a pharmaceutically acceptable salt
form for use as a pharmaceutical, e.g. in any of the methods as
indicated under 1.1 or 1.2 above. [0127] 3. A pharmaceutical
composition, e.g. for use in any of the methods as in 1.1 or 1.2
above comprising a compound of formula II comprising greater than
70% by weight of the R or S enantiomer in free form or
pharmaceutically acceptable salt form in association with a
pharmaceutically acceptable diluent or carrier therefor. [0128] 4.
A compound of formula II comprising greater than 70% by weight of
the R or S enantiomer or a pharmaceutically acceptable salt thereof
for use in the preparation of a pharmaceutical composition for use
in any of the method as in 1.1 or 1.2 above.
[0129] The compounds of the invention may be administered as the
sole active ingredient or in conjunction with, e.g. as an adjuvant
to, other drugs e.g. immunosuppressive or immunomodulating agents
or other anti-inflammatory agents, e.g. for the treatment or
prevention of allo- or xenograft acute or chronic rejection or
inflammatory or autoimmune disorders, or a chemotherapeutic agent,
e.g a malignant cell anti-proliferative agent. For example, the
compounds of the invention may be used in combination with a
calcineurin inhibitor, e.g. cyclosporin A or FK 506; a mTOR
inhibitor, e.g. rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779
or ABT578; an ascomycin having immunosuppressive properties, e.g.
ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide;
azathioprene; methotrexate; leflunomide; mizoribine; mycophenolic
acid; mycophenolate mofetil; 15-deoxyspergualine or an
immunosuppressive homologue, analogue or derivative thereof;
immunosuppressive monoclonal antibodies, e.g., monoclonal
antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD7,
CD8, CD25, CD28, CD40. CD45, CD58, CD80, CD86 or their ligands;
other immunomodulatory compounds, e.g. a recombinant binding
molecule having at least a portion of the extracellular domain of
CTLA4 or a mutant thereof, e.g. an at least extracellular portion
of CTLA4 or a mutant thereof joined to a non-CTLA4 protein
sequence, e.g. CTLA4Ig (for ex. designated ATCC 68629) or a mutant
thereof, e.g. LEA29Y; adhesion molecule inhibitors, e.g. LFA-1
antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4
antagonists; or a chemotherapeutic agent, e.g. paclitaxel,
gemcitabine, cisplatinum, doxorubicin or 5-fluorouracil.
[0130] Where the compounds of the invention are administered in
conjunction with other immunosuppressive/immunomodulatory,
anti-inflammatory or chemotherapeutic therapy, dosages of the
co-administered immunosuppressant, immunomodulatory,
anti-inflammatory or chemotherapeutic compound will of course vary
depending on the type of co-drug employed, e.g. whether it is a
steroid or a calcineurin inhibitor, on the specific drug employed,
on the condition being treated and so forth. In accordance with the
foregoing the present invention provides in a yet further aspect:
[0131] 5. A method as defined above comprising co-administration,
e.g. concomitantly or in sequence, of a therapeutically effective
non-toxic amount of a compound of the invention and at least a
second drug substance, e.g. an immunosuppressant, immunomodulatory,
anti-inflammatory or chemotherapeutic drug, e.g. as indicated
above. [0132] 6. A pharmaceutical combination, e.g. a kit,
comprising a) a first agent which is a compound of the invention as
disclosed herein, in free form or in pharmaceutically acceptable
salt form, and b) at least one co-agent, e.g. an immunosuppressant,
immunomodulatory, anti-inflammatory or chemotherapeutic drug. The
kit may comprise instructions for its administration.
[0133] The terms "co-administration" or "combined administration"
or the like as utilized herein are meant to encompass
administration of the selected therapeutic agents to a single
patient, and are intended to include treatment regimens in which
the agents are not necessarily administered by the same route of
administration or at the same time.
[0134] The term "pharmaceutical combination" as used herein means a
product that results from the mixing or combining of more than one
active ingredient and includes both fixed and non-fixed
combinations of the active ingredients. The term "fixed
combination" means that the active ingredients, e.g. a compound of
the invention and a co-agent, are both administered to a patient
simultaneously in the form of a single entity or dosage. The term
"non-fixed combination" means that the active ingredients, e.g. a
compound of the invention and a co-agent, are both administered to
a patient as separate entities either simultaneously, concurrently
or sequentially with no specific time limits, wherein such
administration provides therapeutically effective levels of the 2
compounds in the body of the patient. The latter also applies to
cocktail therapy, e.g. the administration of 3 or more active
ingredients.
[0135] In a further aspect, the present invention provides a method
of separating the R and S enantiomers of a compound of formula I,
comprising separating the enantiomers by high performance liquid
chromatography (HPLC) using a chiral ion-exchange phase e.g. based
on quinine carbamate or quinidine carbamate as described above.
[0136] In a further aspect, the present invention provides a method
of determining the amount of the R and/or S isomers of a compound
of formula II present in a sample, comprising (a) reacting the
compound of formula II present in the sample with
benzene-1,2-dicarbaldehyde to form a compound of formula I, and
separating the R and S isomers of the compound of formula I by
HPLC. The HPLC is preferably performed as described above, using a
chiral ion-exchange phase e.g. based on quinine carbamate or
quinidine carbamate. The compounds of formula I are useful as
intermediates in such a method. The sample may be e.g. a sample
derived from a bodily fluid e.g. blood, plasma, saliva, or urine,
and may be first subjected to one or more separation steps, (e.g.
extraction with methanol and/or non-chiral HPLC) in order to
separate the compound of formula II from the fluid. By such a
method the amount of the active R or S enantiomer in the sample may
be determined. Thus the method may be useful e.g. for monitoring
the blood concentration of the active S enantiomer of a compound of
formula II in a subject, following administration of a compound of
formula II (e.g. a racemic mixture thereof) or following
administration of a precursor of a compound of formula II (forming
the compound of formula II as a metabolite in the body) to the
subject.
[0137] For example, achiral 14C-labeled FTY720 is administered to
rats either orally (7.5 mg/kg) or by intravenous infusion (4
mg/kg). Chiral 14C-labeled FTY720-phosphate is formed in the body
of the rat as a metabolite of 14C-labeled FTY720. Blood samples are
taken at different times (e.g. 3 or 72 hours) after dosing. Each
blood sample is extracted with methanol and [14C]FTY720-phosphate
is isolated by non-chiral HPLC. The isolated [14C]FTY720-phosphate
is derivatized with OPA. The derivative is spiked with unlabelled
OPA-derivatized R and S FTY720-phosphate as retention time markers
(monitored by UV at 215 nm) and subjected to chiral HPLC separation
using a ProntoSIL Chiral AX QN-1 column. The [14C]FTY720-phosphate
in all blood samples represents exclusively the pharmacologically
active S-enantiomer. The inactive R-enantiomer is not
detectable.
* * * * *