U.S. patent application number 12/197657 was filed with the patent office on 2008-12-18 for process for the synthesis of peptides containing a 4-hydroxy-proline substructure.
Invention is credited to Bernhard Erb, Werner Pachinger, Walter Prikoszovich, Bernhard Wietfeld.
Application Number | 20080312456 12/197657 |
Document ID | / |
Family ID | 32510405 |
Filed Date | 2008-12-18 |
United States Patent
Application |
20080312456 |
Kind Code |
A1 |
Wietfeld; Bernhard ; et
al. |
December 18, 2008 |
PROCESS FOR THE SYNTHESIS OF PEPTIDES CONTAINING A
4-HYDROXY-PROLINE SUBSTRUCTURE
Abstract
The present invention relates to processes for preparing
peptides and to intermediates involved in such processes, e.g. a
process for preparing a compound of formula VIII ##STR00001##
wherein R.sub.12 and R.sub.13 are as defined herein.
Inventors: |
Wietfeld; Bernhard;
(Efringen-Kirchen, DE) ; Prikoszovich; Walter;
(Schoenenbuch, CH) ; Erb; Bernhard;
(Gipf-Oberfrick, CH) ; Pachinger; Werner; (Basel,
CH) |
Correspondence
Address: |
NOVARTIS;CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 104/3
EAST HANOVER
NJ
07936-1080
US
|
Family ID: |
32510405 |
Appl. No.: |
12/197657 |
Filed: |
August 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11839565 |
Aug 16, 2007 |
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12197657 |
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10538028 |
Mar 1, 2006 |
7294722 |
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PCT/EP03/14082 |
Dec 11, 2003 |
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11839565 |
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Current U.S.
Class: |
548/532 |
Current CPC
Class: |
C07C 51/15 20130101;
Y02P 20/55 20151101; C07C 51/15 20130101; C07C 65/01 20130101 |
Class at
Publication: |
548/532 |
International
Class: |
C07D 207/16 20060101
C07D207/16 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 2002 |
GB |
0229020.3 |
Dec 16, 2002 |
GB |
0229280.3 |
Claims
1-4. (canceled)
5. A compound of formula VI in free or salt form ##STR00018##
wherein R.sub.3, R.sub.4 and R.sub.5 are each independently
hydrogen or one or more substituents attached to each benzene ring,
and are selected from hydroxy, amino, C.sub.1-10-alkyl,
C.sub.1-10-alkoxy, C.sub.1-10-alkylamino, di-C.sub.1-10-alkylamino,
carbamoyl, C.sub.1-10-alkylcarbamoyl, di-C.sub.1-10-alkylcarbamoyl,
halo-C.sub.1-10-alkyl, halogeno or nitro, optionally protected by a
removable protecting group; R.sub.9 is --OH, --OM or --OMX, where M
is metal and X is a nucleophilic substituent; and R.sub.10 is -M or
-MX, where M is metal and X is a nucleophilic substituent.
6-10. (canceled)
Description
[0001] The present invention relates to processes for preparing
peptides and intermediates involved in such processes.
[0002] In one aspect, the invention relates to:
[0003] (A) a process for preparing a compound of formula I
##STR00002##
wherein R.sub.1 is a reactive substituent or an attachment to a
solid phase;
[0004] R.sub.2 is a reactive substituent; and
[0005] R.sub.3, R.sub.4 and R.sub.5 are each independently hydrogen
or one or more substituents attached to each benzene ring and
selected from hydroxy, amino, C.sub.1-10-alkyl, C.sub.1-10-alkoxy,
C.sub.1-10-alkylamino, di-C.sub.1-10-alkylamino, carbamoyl,
C.sub.1-10-alkylcarbamoyl, di-C.sub.1-10-alkylcarbamoyl,
halo-C.sub.1-10-alkyl, halogeno and nitro;
[0006] in free or salt form; comprising
[0007] (a) reacting a compound of formula VI with an
electrophile:
##STR00003##
wherein R.sub.3, R.sub.4 and R.sub.5 are as defined above;
[0008] R.sub.9 is --OH, --OM or --OMX, where M is metal and X is a
nucleophilic substituent;
[0009] R.sub.10 is -M or -MX, where M is metal and X is a
nucleophilic substituent; in free or salt form;
[0010] and hydrolyzing the resulting compound to form a compound of
formula I wherein R.sub.2 is hydroxy;
[0011] (b) optionally converting a compound of formula I wherein
R.sub.2 is hydroxy to a compound of formula I wherein R.sub.2 is
other than hydroxy;
[0012] (c) optionally converting R.sub.1 in a compound of formula I
to an alternative R.sub.1 group;
[0013] (d) optionally deprotecting a compound of formula I in
protected form; and
[0014] (e) where required, converting a compound of formula I
obtained in free form into the desired salt form, or vice
versa;
[0015] (B) a process for the preparation of a solid phase support
system, comprising preparing a compound of formula I by a process
as defined above, and coupling the compound with a suitably
derivatised or functionalised solid phase material;
[0016] (C) a compound of formula V in free or salt form
##STR00004##
wherein R.sub.3, R.sub.4, R.sub.5, and R.sub.9 are as defined
above; and
[0017] R.sub.7 is a nucleophilic substituent;
[0018] (D) a compound of formula VI in free or salt form
##STR00005##
wherein R.sub.3, R.sub.4, R.sub.5 and R.sub.9 are as defined above;
and
[0019] R.sub.10 is -M or -MX, where M is metal and X is a
nucleophilic substituent.
[0020] The present invention provides a simple route for the
preparation of compounds of formula I, which are useful for solid
phase chemical synthesis. The process of the invention may directly
produce a compound of formula I attached to a solid phase, or where
R.sub.1 is a reactive substituent, the compound of formula I can
easily be coupled to a solid phase at a later stage. The presence
of the reactive substituent R.sub.2 permits the use of the
compounds of formula I as linkers in the synthesis of oligomers and
polymers, such as glycopeptides, nucleotides and proteins,
especially in the solid phase synthesis of peptides. The compounds
of formula I, particularly where R.sub.2 is halogeno, may also be
used as protecting agents for protecting functional groups, e.g.
amino or hydroxy groups, in chemical synthesis.
[0021] The compounds of formula V and VI are useful as intermediate
compounds in the preparation of compounds of formula I.
[0022] A compound of formula VI may be prepared by reacting a
compound of formula V with a metal or organometallic compound:
##STR00006##
wherein R.sub.3, R.sub.4, R.sub.6 and R.sub.9 are as defined above;
and
[0023] R.sub.7 is a nucleophilic substituent.
[0024] A compound of formula V may be prepared by:
[0025] (i) reacting a compound of formula II with a metal or
organometallic compound
##STR00007##
wherein R.sub.6 and R.sub.7 are each a nucleophilic substituent and
R.sub.3 is as defined above and is protected if necessary by a
removable protecting group; and
[0026] (ii) reacting the compound obtained in (i) with a compound
of formula III
##STR00008##
wherein R.sub.4 and R.sub.5 are as defined above and are protected
if necessary by a removable protecting group.
[0027] The process of the present invention may suitably be
performed in a single reaction vessel without intermediary
isolation.
[0028] Terms used in the specification have the following
meanings:
[0029] "Alkyl" may be straight or branched. Preferably alkyl is
C.sub.1-4alkyl.
[0030] "Alkoxy" may be straight or branched alkoxy. Preferably
alkoxy is C.sub.1-4alkoxy.
[0031] "Acylamino" denotes a group of formula --NH--C(O)--R where R
is straight chain or branched C.sub.1-10-alkyl, cycloalkyl or aryl.
Preferably R is C.sub.1-4alkyl.
[0032] "Acyloxy" denotes a group of formula --O--C(O)--R where R is
as defined above.
[0033] "Aryl" is preferably C.sub.6-10 aryl, e.g. phenyl.
[0034] "Halogeno" means fluoro, chloro, bromo or iodo.
[0035] "Haloalkyl" means straight chain or branched
C.sub.1-40-alkyl, substituted by one or more, for example one, two
or three halogen atoms, preferably fluorine or chlorine atoms.
Preferably haloalkyl is C.sub.1-4-alkyl substituted by one, two or
three fluorine or chlorine atoms.
[0036] "Organometallic compound" denotes a compound in which a
carbon atom of an organic group is bound to a metal. The
organometallic compound is preferably an alkylmetallic compound,
for example an alkyllithium, e.g. a straight or branched chain
C.sub.1-10 alkyllithium compound or may alternatively be an
arylmetallic compound, for example an arylithium.
[0037] More preferably the alkyllithium compound is a C.sub.3-6
alkyllithium compound, such as butyllithium or hexyllithium.
[0038] Alternatively, the organometallic compound may be an
organomagnesium compound, for example a straight or branched chain
alkylmagnesium or arylmagnesium compound, preferably a C.sub.1-6
alkylmagnesium compound. Organomagnesium compounds are commonly
known as Grignard reagents. The organomagnesium compound is
preferably an organomagnesium halide, especially an iodide or
bromide.
[0039] In further alternative embodiments, the organometallic
compound may be an alkyl- or arylzinc compound, for example a
C.sub.1-6-alkylzinc compound, or an C.sub.1-4-alkyl- or aryltin
compound.
[0040] M is preferably lithium or magnesium.
[0041] R.sub.1 may be a reactive substituent suitable for linking
the compound to a solid phase. R.sub.1 may suitably be --C(O)R',
--C(O)--OR', --C(O)--NR'R'', --R.sub.12--NR'R'', --R.sub.12--OR',
--NR'R'', or --C(O)X, wherein R' and R'' are each independently
hydrogen or straight or branched C.sub.1-10-alkyl, e.g.
C.sub.1-4-alkyl, R.sub.12 is straight or branched C.sub.1-10-alkyl,
e.g. C.sub.1-4-alkyl, and X is a nucleophilic substituent,
preferably halogeno, e.g. chloro. R.sub.1 may suitably be in the
para, ortho or meta position, preferably in the para position.
[0042] Alternatively R.sub.1 may be an attachment to a solid phase
material, e.g. polystyrene. Preferably the attachment is of the
formula --C(O)--P, --C(O)--OP, --C(O)--NR'--P, --R.sub.12--NR'--P,
--R.sub.12--OP, --NR'--P, --C(O)--R.sub.12--P,
--C(O)--OR.sub.12--P, --C(O)--NR'--R.sub.12--P,
--R.sub.12--NR'--R.sub.12--P, --R.sub.12--OR.sub.12--P,
--NR'--R.sub.12--P or --R.sub.12--P, wherein R', R'' and R.sub.12
are as defined above and P is a solid phase material. More
preferably R.sub.1 is --C(O)--OP, --C(O)--OR.sub.12--P,
--C(O)--NH--P, --C(O)--NH--R.sub.12--P, --NH--R.sub.12--P or
--R.sub.12--P, wherein R.sub.12 is methyl, e.g. --CH.sub.2--P.
[0043] R.sub.2 is preferably a reactive substituent suitable for
linking the compound to a biological oligomer or polymer, or a
monomer unit thereof, e.g. an amino acid or polypeptide. R.sub.2
may suitably be hydroxy, acylamino, acyloxy, amino, halogeno,
sulfhydryl, C.sub.1-10-alkoxy or C.sub.6-40-aryloxy, preferably
halogeno.
[0044] Each benzene ring shown in formulae I to VII may be
substituted by one or more groups. For example R.sub.3 may
designate one to four substituent groups, preferably one or two
substituent groups, attached to the benzene ring shown in formulae
I, II and IV to VII. R.sub.4 and R.sub.6 may designate one to five
substituent groups, preferably one to three substituent groups,
attached to each of the benzene rings shown in formulae I, II and
IV to VII. Each substituent group may be present at any suitable
position on the benzene rings to which they are attached. More
preferably R.sub.4 and/or R.sub.5 is a substituent group at the
ortho or para position on the benzene ring to which it is
attached.
[0045] Each of R.sub.3, R.sub.4 and R.sub.5 may be protected by a
removable protecting group if necessary, e.g. when it contains an
--OH or --NH.sub.2 group which does not participate in the
reaction. Protecting groups, their introduction and removal are
described, for example, In "Protective Groups in Organic
Synthesis", T. W. Greene et al., John Wiley & Sons Inc., Second
Edition 1991. Preferably each of R.sub.3, R.sub.4 or R.sub.5 is a
group which does not require protection, e.g any of the groups
listed above other than hydroxy, amino or nitro.
[0046] When R.sub.3, R.sub.4 or R.sub.5 is halogeno, it is
preferably fluoro or chloro. When R.sub.3, R.sub.4 or R.sub.5 is
haloalkyl it is preferably trifluoromethyl. Preferably R.sub.3 is
C.sub.1-4-alkyl, halogeno, or hydrogen. Preferably R.sub.4 and
R.sub.5 are each independently C.sub.1-4-alkylcarbamoyl,
dl-C.sub.1-4-alkylcarbamoyl, carbamoyl, trifluoromethyl, fluoro or
chloro. Preferably R.sub.4 and R.sub.5 are the same.
[0047] Preferably the nucleophilic substituents R.sub.6 and R.sub.7
are each independently halogeno, more preferably bromo or iodo, and
most preferably R.sub.6 and R.sub.7 are each bromo. R.sub.7 may
suitably be in the para, ortho or meta position, preferably in the
para position.
[0048] In one embodiment of the invention, the compound of formula
II is first reacted with the metal or organometallic compound to
form a compound of formula IV:
##STR00009##
wherein R.sub.3 and R.sub.7 are as defined above and R.sub.8 is -M
or -MX, where M is metal and X is a nucleophilic substituent,
preferably halogeno.
[0049] Where the metal is lithium or the organometallic compound is
an organolithium compound, R.sub.8 is --Li. Where the metal is
magnesium or the organometallic compound is a Grignard reagent,
R.sub.8 is --MgX, and X is preferably halogeno. The compound of
formula IV is then reacted with a compound of formula III to form a
compound of formula V.
[0050] The compounds of formulae IV and V need not be separated or
isolated but may be prepared in situ.
[0051] Suitable electrophiles for use in the process include carbon
dioxide, isocyanates, nitrites, acyl halides (such as phosgene),
leading to the formation of, for example, compounds of formula I
wherein R.sub.1 is carboxy, carbamoyl, alkylcarbamoyl or acyl.
Alternatively the electrophile may be a derivatised solid phase
material, e.g. a Merrifield polymer, enabling direct coupling of
the compound of formula VI to a solid phase. In one embodiment the
electrophile is a compound of formula X'--(CH.sub.2).sub.n--P,
wherein X' is a nucleophilic substituent e.g. halogeno or tosyloxy,
n is an Integer between 1 and 4, preferably 1, and P is a solid
phase material.
[0052] Where the electrophile is carbon dioxide, the process
preferably comprises first reacting the compound of formula V with
a metal or organometallic compound to form a compound of formula VI
as defined above and reacting, preferably in situ, the compound of
formula VI with carbon dioxide.
[0053] Where the electrophile is carbon dioxide, preferably a
compound of formula VII is formed:
##STR00010##
wherein R.sub.3, R.sub.4, R.sub.5 and R.sub.9 are as defined above;
and
[0054] R.sub.11 is --OH, --OM or --OMX, where M is metal and X is a
nucleophilic substituent, preferably halogeno, in salt or free
form.
[0055] Alternatively, the carboxylation step comprises reacting a
compound of formula V with carbon dioxide in the presence of a
metal or organometallic compound, to form a compound of formula
VII.
[0056] The hydrolysis step preferably comprises reacting a compound
of formula VII wherein R.sub.11 is --OM or --OMX and/or R.sub.9 is
--OM or --OMX with water or an acid yielding a compound of formula
I wherein R.sub.1 is carboxy and R.sub.2 is hydroxy, in salt or
free form. Suitable acids include ammonium chloride, acetic acid,
sulphuric acid and hydrochloric acid. A pH-buffered solution may
also be used. Preferably a weak acid is used and/or the step may be
carried out at a pH of 4 to 7. The reaction temperature may
conveniently be -50 to 50.degree. C., preferably -10 to 10.degree.
C.
[0057] Alternatively the compound of formula VII where R.sub.11 is
--OM or --OMX may be reacted with a a nucleophile, e.g. an amine or
halide, to form a compound of formula I wherein R.sub.1 is
--C(O)--NR'R'' or --(O)--X and R', R'' and X are as defined
above.
[0058] The process of the invention may conveniently be carried out
in an inert organic solvent, preferably an ether solvent, for
example diethyl ether, tetrahydrofuran or tert-butyl methyl ether.
Alternatively a hydrocarbon solvent may be used. The reaction
temperature for step (a) is conveniently -30 to +10.degree. C.,
preferably -5 to -0.degree. C. The reaction may, for example, be
carried out using 0.5 to 2 equivalents, preferably 0.8 to 1.2
equivalents and most preferably about 1 equivalent of the metal or
organometallic compound per equivalent of the compound of formula
II. 0.5 to 2 equivalents, preferably 0.8 to 1.2 equivalents of the
compound of formula III may be used per equivalent of the compound
of formula II.
[0059] The temperature during the reaction of a compound of formula
V with a metal or organometallic compound may conveniently be 0 to
+50.degree. C., preferably +20 to +30.degree. C. The reaction
temperature for the reaction with an electrophile (e.g. CO.sub.2)
may conveniently be 0 to -30.degree. C., preferably -5 to
-10.degree. C. The hydrolysis step, e.g. with acid, may
conveniently be performed at -10.degree. C. to +10.degree. C., e.g.
0 to +5.degree. C. Preferably 0.5 to 2 equivalents, more preferably
0.8 to 1.2 equivalents of a metal or organometallic compound per
equivalent of the compound of formula V are used.
[0060] The groups R.sub.1 and R.sub.2 may be converted to
alternative R.sub.1 and R.sub.2 groups specified above by standard
processes, such as by esterification, amidation or nucleophilic
substitution. For example, a compound of formula I wherein R.sub.2
is hydroxy may be converted to a compound of formula I wherein
R.sub.2 is halogeno by reaction with an acyl halide, e.g. acyl
chloride.
[0061] Preferably the compound of formula I is in free form. The
compounds in free or salt form can be obtained in the form of
hydrates or solvates containing a solvent used for
crystallization.
[0062] Compounds of formula I can be recovered from the reaction
mixture and purified in a conventional manner.
[0063] The starting compounds of formula II or formula III are
known or may be prepared by methods analogous to those known in the
art. Organometallic compounds may be prepared by standard
processes, for example by reaction of an alkyl or aryl halide with
a metal, for example lithium or magnesium, suspended in diethyl
ether or tetrahydrofuran. The organometallic compound is preferably
prepared and used in an inert (oxygen-free) anhydrous atmosphere,
for instance under nitrogen.
[0064] The process according to the invention may suitably include
a further step of coupling the compound of formula I wherein
R.sub.1 is a reactive substituent to a solid phase material.
Suitable solid phase materials are disclosed, for example, in DE
4306839 A1, and include naturally occurring or synthetic organic or
inorganic polymers in particulate form, e.g. as beads, or
preferably as a surface layer on a suitable inert substrate
material. Examples of suitable polymer materials include
crosslinked polystyrene, e.g. polystyrene pins, Gly-HMD-MA/DMA pins
and HEMA pins. The compound of formula I may conveniently be
coupled to a solid phase material by reacting a group present on
the solid phase with R.sub.1. Thus the solid phase material
preferably comprises reactive groups, such as amino groups.
Preferably a compound of formula I, wherein R.sub.1 is a carboxy
group or an activated carboxy group, e.g. by reaction with
diisopropylcarbodiimide, is reacted with a polymer bearing free
amino groups.
[0065] A compound of formula I may be used as a linker. Thus the
process according to the invention may also suitably include a
further step of coupling the compound of formula I, optionally
bound to a solid phase material, to a biological oligomer or
polymer, or a monomer unit thereof. The compound may conveniently
be coupled to the biological molecule, e.g. an amino acid or
polypeptide, by reacting a group present on the biological molecule
with R.sub.2. For example, where R.sub.2 is hydroxy and the
biological molecule is a polypeptide or amino acid, the terminal
carboxylic acid group of the biological molecule can be esterified
by the R.sub.2 hydroxy group, optionally via initial reaction of
the compound of formula I with an acyl halide leading to in situ
substitution of hydroxy by halogeno.
[0066] In a further aspect, the present invention provides:
[0067] (E) a process for preparing a compound of formula VIII
##STR00011##
wherein R.sub.12 and R.sub.13 are each a removable protecting group
and R.sub.12 and R.sub.13 are different; comprising reacting a
compound of formula IX
##STR00012##
with a suitable R.sub.12 donor compound;
[0068] (F) intermediates useful in the above process, defined by
the general formula XIV
##STR00013##
wherein R.sub.16 is a removable protecting group other than
fluorenylmethoxycarbonyl, and is different to R.sub.18;
[0069] R.sub.17 is hydrogen or a blocking group removable by
hydrolysis or hydrogenolysis; and
[0070] R.sub.18 is hydrogen or a removable protecting group other
than fluorenylmethoxycarbonyl.
[0071] The present invention provides a simple and efficient route
for the preparation of compounds of formula VIII, which are useful
in the synthesis of peptides, for example as described in WO
02/10192. The compounds of formula XIV are useful as intermediate
compounds in the preparation of compounds of formula VIII.
[0072] The compound of formula IX may be prepared from a compound
of formula X
##STR00014##
wherein R.sub.13 is as defined above,
[0073] R.sub.14 is a removable protecting group and R.sub.14 is
different to R.sub.12 and R.sub.13, and
[0074] R.sub.15 is a blocking group removable by hydrolysis or
hydrogenolysis.
[0075] Protecting groups, their introduction and removal are
described, for example, in "Protective Groups in Organic
Synthesis", T. W. Greene et al., John Wiley & Sons Inc., Second
Edition 1991. Suitable protecting group donor compounds, e.g. amino
group protecting agents, are well-known to a skilled person, e.g.
anhydrides, halides, carbamates or N-hydroxysuccinimides which
provide one of the protecting groups below.
[0076] The protecting group R.sub.12 is preferably
fluorenylmethoxycarbonyl. R.sub.13 or R.sub.16 is preferably a
protecting group other than fluorenylmethoxycarbonyl, and is
preferably more resistant to removal by hydrolysis (for example
base-catalysed hydrolysis) and/or hydrogenolysis than R.sub.12
and/or R.sub.14, e.g. more resistant than fluorenylmethoxycarbonyl
and/or benzyloxycarbonyl. More preferably R.sub.13 or R.sub.16 is
tert-butoxycarbonyl.
[0077] The protecting group R.sub.14 or R.sub.18 is preferably more
resistant to removal by hydrolysis than R.sub.12, e.g. more
resistant than fluorenylmethoxycarbonyl. R.sub.14 or R.sub.18 is
preferably removable by hydrogenolysis. Suitable R.sub.14 or
R.sub.18 substituents include benzyloxycarbonyl,
1,1,-dimethylpropynyloxycarbonyl, vinyloxycarbonyl,
N-hydroxypiperidinyloxycarbonyl, 9-anthrylmethyloxycarbonyl and
phenylaminothiocarbonyl, allyl, nitrobenzyl, triphenylmethyl,
(p-methoxyphenyl)diphenylmethyl, diphenyl-4-pyridylmethyl or
benzylsulfonyl. Preferably R.sub.14 or R.sub.18 is an
oxycarbonyl-containing protecting group, e.g. benzyloxycarbonyl
(carbobenzoxy).
[0078] R.sub.15 or R.sub.17 may suitably be:
[0079] (i) C.sub.1-10-alkyl, e.g. C.sub.1-4-alkyl, preferably
methyl, ethyl, propyl or butyl other than tert-butyl, more
preferably methyl.
[0080] (ii) C.sub.3-8-cycloalkyl, optionally substituted by one or
more C.sub.1-4 alkyl, e.g. methyl. Preferably cycloalkyl is
C.sub.3-6-cycloalkyl.
[0081] (iii) C.sub.6-10-aryl, optionally substituted by one or more
stabilising substitutents, e.g halogeno or nitro. Preferably aryl
is phenyl, optionally substituted by one, two or three halogeno,
e.g. chloro.
[0082] (iv) (C.sub.6-10-aryl).sub.1-3-C.sub.1-10-alkyl, optionally
substituted on the aryl group by (I) one or more stabilising
substituents, e.g halogeno or nitro, or (ii) by two substituents
which together with the ring carbon atoms to which they are
attached form a 5- or 6-membered ring, optionally containing one or
two nitrogen or oxygen atoms.
(C.sub.6-10-aryl).sub.1-3-C.sub.1-10-alkyl is preferably (I)
(phenyl).sub.1-3-C.sub.1-4-alkyl, more preferably benzyl,
diphenylmethyl or triphenylmethyl, optionally substituted on each
benzene ring by one, two or three halogeno, e.g chloro, (ii)
anthrylmethyl, e.g. 9-anthrylmethyl, or (iii) piperonyl.
[0083] (v) C.sub.6-10-aryl-C.sub.1-4alkoxy-C.sub.1-4-alkyl,
preferably benzyloxymethyl.
[0084] (vi) C.sub.6-10-aryl-carbonyl-C.sub.1-4-alkyl, preferably
phenacyl.
[0085] Preferably R.sub.15 or R.sub.17 is a group which is
removable by hydrogenolysis, such as benzyl, benzyloxymethyl,
phenacyl, triphenylmethyl, piperonyl or 9-anthrylmethyl, preferably
benzyl.
[0086] The compound of formula IX may be prepared by (i)
hydrolysing the ester compound of formula X to obtain the
corresponding carboxylic acid and (II) removing the protecting
group R.sub.14. Preferably the hydrolysis step is performed before
removal of the protecting group R.sub.14. The protecting group
R.sub.14 may conveniently be removed by reductive hydrogenation
(hydrogenolysis). This route, involving a hydrolysis step, is
suitably followed when R.sub.15 is not removable by hydrogenolysis.
The hydrolysis step is preferably a base-catalysed hydrolysis, for
example using sodium hydroxide and may suitably be performed in a
polar solvent, e.g. methanol.
[0087] Alternatively, a compound of formula IX may conveniently be
prepared by hydrogenation (hydrogenolysis) of a compound of formula
X wherein R.sub.15 is a group which is removable by hydrogenolysis,
e.g. benzyl. The hydrogenation step may conveniently be performed
using a suitable catalytic agent, for instance
palladium-on-charcoal.
[0088] Compound of formula X may be prepared by reacting a compound
of formula XI
##STR00015##
wherein X is a nucleophilic substituent and R.sub.14 and R.sub.15
are as defined above, with a compound of formula XII
##STR00016##
wherein R.sub.13 is as defined above. This step may be performed in
any suitable organic solvent, preferably in a hydrocarbon solvent,
more preferably toluene.
[0089] The compound of formula XII is protected ethylenediamine,
wherein one amino group has been protected with a removable
protecting group. The nucleophilic substituent X in formula XI is
preferably halogeno, such as fluoro, chloro, bromo or iodo, more
preferably chloro. The compound of formula XI wherein X is halogeno
may be formed by reaction of a compound of formula XIII
##STR00017##
with an acyl halide, for instance phosgene, tri-phosgene,
phenylchloroformate or 4-nitrophenylchloroformate, preferably
4-nitrophenylchloroformate. This step may suitably be performed in
the presence of an organic base, e.g. dimethylaminopyridine, in a
non-polar solvent, e.g. toluene.
[0090] The compound of formula XIII may be commercially available,
e.g. when R.sub.15 is methyl or may be formed by esterification of
4-hydroxy-proline according to methods known in the art, for
instance by reaction with benzyl alcohol or methanol. The resulting
ester is then protected by reaction with a suitable R.sub.14 donor
compound, e.g. benzyloxycarbonyl-N-hydroxysuccinimide.
[0091] The compound of formula XI need not be separated or
isolated, as the compound of formula XIII may be reacted with an
acyl halide and the product of this reaction subsequently reacted
with a compound of formula XII in the same vessel.
[0092] The addition of the protecting group R.sub.12 to the
compound of formula IX may suitably be performed in the presence of
sodium carbonate/acetonitrile.
[0093] Compounds of formula VIII can be recovered from the reaction
mixture and purified in a conventional manner.
[0094] In the compounds of formulae VIII-XI and XIII above, the oxy
substituent on the proline may be in position cis or trans,
preferably trans. The cis or trans isomers may be individually
prepared, using the corresponding cis or trans hydroxyproline as
starting material.
[0095] 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 described
thereafter.
[0096] In a further aspect, the present invention relates to a
process for producing a compound of formula VIII, wherein R.sub.12
is fluorenylmethoxycarbonyl and R.sub.13 is a removable protecting
group other than fluorenylmethoxycarbonyl, comprising reacting a
compound of formula VIII with a fluorenylmethoxycarbonyl donor
compound, e.g. fluorenylmethoxycarbonyl-N-hydroxysuccinimide.
[0097] The invention will now be described with reference to the
following specific embodiments, in which the following
abbreviations are used:
TABLE-US-00001 Fmoc = fluorenylmethoxycarbonyl Boc =
tert-butoxycarbonyl Cbo = carbobenzoxy (benzyloxycarbonyl) OSu =
N-hydroxysuccinlmide HPTF = Heptane fraction JT = Jacket
temperature HPLC = High performance liquid chromatography THF =
Tetrahydrofuran TBME = Tert-butyl methyl ether DMF =
Dimethylformamide
EXAMPLE 1
Preparation of 4-(diphenyl-hydroxy-methyl)-benzoic acid
[0098] 1,4-dibromobenzene (47.2 g, 0.2 M) is added to THF (240 ml).
The clear solution is cooled to -65.degree. C. A butyllithium
solution (0.22 M, 94 ml of a 20% solution in CHX) is added over 30
minutes.
[0099] After 5 minutes of stirring a solution of benzophenone (36.4
g, 0.2 M in 180 ml THF) is added over 30 minutes (exothermic). The
mixture is stirred for a further 30 minutes at -65.degree. C. Then
over 30 minutes the temperature is raised to -10.degree. C. and the
solution is stirred at this temperature for one hour.
[0100] The reaction mixture is then re-cooled to -65.degree. C.
Over 30 minutes a butyllithium solution (0.22 M, 94 ml of a 20%
solution in cyclohexane) is added.
[0101] The resulting suspension is diluted with 200 ml THF. Then
carbon dioxide gas is introduced over 90 minutes at 65.degree. C.
The temperature is raised to 20.degree. C. and the mixture stirred
overnight. The mixture is then cooled to 0.degree. C. and an
aqueous solution of ammonium chloride (120 ml of a 10% solution) is
added over 30 minutes. 4-(diphenyl-hydroxy-methyl)-benzoic acid is
formed at this stage.
[0102] The mixture is evaporated at 45.degree. C. under a vacuum.
The residue is adjusted to pH 4 with acetic acid and mixed with 400
ml H.sub.2O. Extraction is performed with 2.times.150 ml ethyl
acetate. The organic phases are extracted again with 100 ml water.
The combined EST-phases are shaken with a 10% aqueous potassium
hydroxide solution (2.times.120 ml). The combined aqueous phases
are adjusted to pH 1-2 with hydrochloric acid at 20.degree. C. and
then extracted with 2.times.150 ml TBME. The combined TBME phases
are mixed with 50 ml water and 50 ml saturated Na.sub.2SO.sub.4,
dried with magnesium sulphate and evaporated at 45.degree. C. under
vacuum to obtain a crude product.
[0103] 38.3 g crude product is dissolved in TBME (300 ml) at
40.degree. C. The clear yellow solution is concentrated in a volume
of 60 ml (240 ml TBME distilled off). The mixture is stirred for
one hour at 40.degree. C. (crystallisation). 50 ml HPTF is added,
the mixture is cooled to 0.degree. C. and stirred at 0.degree. C.
for 1 hour. Evaporating and washing with 2.times.15 ml heptane
fraction and drying overnight at 45.degree. C. under vacuum gives
white crystals.
[0104] Attachment of 4(diphenyl-hydroxy-methyl)benzoic acid to a
Solid Phase
[0105] 15 g 4-(diphenyl-hydroxy-methyl)-benzoic acid with 7.54 g
hydroxybenzotriazole is dissolved in 140 ml DMF by stirring for 15
min. 15.3 ml di-isopropylcarbo-di-imide is added and the solution
kept at room temperature for 30 min. The solution is then stirred
overnight at room temperature in the presence of aminomethylated
polystyrene. After washing with DMF, methanol and THF the linker
derivatised support is dried under vacuum.
EXAMPLE 2
Preparation of 4-(diphenyl-hydroxy-methyl)-benzoic acid
(Alternative Method)
[0106] To 12 It TBME in a well dried 100 It Hastelloy-Reactor, 3.0
kg n-butyllithium (20% in cyclohexane; 9.37 mol) is added during a
period of 20 min, keeping the temperature at -5.degree. C. (clear
solution). During a period of 30 min 2.00 kg 1,4-dibromobenzene
(8.48 mol) dissolved in 16 I TBME is added keeping the temperature
between -50 and 0.degree. C. The addition container is rinsed with
3 I TBME.
[0107] After 30 min stirring at -5.degree. C. a solution of 1.55 kg
benzophenone (8.50 mol) in 8 It TBME is added during a period of 20
min keeping the temperature between -5.degree. and 0.degree. C. The
addition container is rinsed with 3 It TBME. A very small amount of
a white solid is formed. After 15 min stirring at -5.degree. C. a
process control sample (HPLC 1) is taken. The reaction mixture is
stirred for further 25 min at -5.degree. C. and warmed up to
+25.degree. C.
[0108] 3.2 kg n-butyllithium (20% in cyclohexane; 10.00 mol) is
added during a period of 25 min keeping the temperature between +25
and 27.degree. C. The addition is slightly exothermic and the
colour turned to slightly green. Some precipitate and froth are
formed. After 20 min stirring a process control sample is taken
(HPLC 2). Depending on the result of HPLC 2 further 0.3 kg
n-butyllithium is added after stirring at +25.degree. C. for 35
min. After 15 min stirring a sample for process control is taken
(HPLC 3). The lines of the n-butyllithium addition are rinsed with
1.5 It TBME and the reaction is cooled to -10.degree. C.
[0109] 1.99 kg dry ice (solid CO.sub.2) is added portionwise during
a period of 20 min keeping the temperature between -10 and
-5.degree. C. The reaction is exothermic and a slightly yellow
precipitate is formed. After 15 min stirring at -10.degree. C. 11 I
TBME are added and the reaction mixture is warmed to 0.degree. C. 5
I 18% aqueous hydrochloric acid is added during a period of 15 min
keeping the temperature between 0.degree. and +5.degree. C. The
addition is exothermic and the precipitate is dissolved
(pH.ltoreq.1).
[0110] The clear solution is transferred to a separation tank and
the reactor is rinsed with 5 It TBME. After separation of the
aqueous phase the organic phase is washed with 20 it water. After
separation of the two layers the organic phase is extracted with 13
It 5% aqueous KOH solution. The basic water phase is separated and
the organic layer is extracted again with 13 It 5% aqueous KOH
solution. The combined basic aqueous layers are transferred to the
100 It Hastelloy-Reactor. 22 I of TBME and 6 I aqueous 18%
hydrochloric acid are added over a period of 20 min at a
temperature between 0.degree. and +5.degree. C. The addition is
exothermic and a white precipitate is formed but it dissolves again
at a low pH-value (pH.ltoreq.1 after HCl-addition). The mixture is
stirred during 10 min and transferred to a separation tank. The
layers are separated and the aqueous phase is extracted again with
16 I TBME. After separation of the layers the combined organic
phases are concentrated at 500 mbar/45.degree. C. JT to a volume of
4-5 I (32 I TBME are destined off) and seed crystals are added. The
temperature is raised up to 50.degree. C. and 20 I HPTF are added
slowly with good stirring. The white precipitate is stirred for 2 h
at 50.degree. C. JT. The jacked temperature is regulated at
0.degree. C. and stirring is continued over night (16 h) letting
cool down the suspension to 0.degree. C. The white suspension is
filtered off and the reactor is rinsed 5 times with 5 It of the
mother liquor. The residue is dried at 45.degree. C. JT under
vacuum (.gtoreq.10 mbar) to constant weight (over night).
EXAMPLE 3
Preparation of
Fmoc-(2S,4R)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OH starting
from Cbo-(2S,4R)-Pro(4-OH)--OMe
[0111] 1. Dimethylaminopyridine (30.5 g, 250 mmol) and
Cbo-(2S,4R)-Pro(4-OH)--OMe (34.9 g, 125 mmol) are dissolved in
toluene (870 ml). A solution of 4-nitrophenylchloroformate (31.5 g,
157 mmol) in toluene (206 ml) is added dropwise to this solution at
0.degree. C. to 5.degree. C. over 20 minutes and stirred for an
additional 2 hours. This is followed by addition of a solution of
Boc-ethylenediamine (80.1 g, 500 mmol) in toluene (205 ml) and
stirring at ambient temperature for 12 hours. A solution of
concentrated sulfuric acid (43.7 g, 450 mmol) in water (873 ml) is
then added while maintaining a temperature of 20.degree. C. to
25.degree. C. The white suspension is filtered by suction and
washed with toluene (30 ml). The toluene phase is washed with water
(450 ml), sodium carbonate (10% w/w, 450 ml) and three times with
water (450 ml each). The toluene phase is azeotropically dried by
distilling off 300 ml, which is continuously replaced by dry
toluene (2.times.300 ml). Heptane (130 ml) is added to the dry
toluene solution at 50.degree. C. and cooled to 0.degree. C. over
two hours. The precipitated product is filtered, washed two times
with toluene/heptane 1:2 v/v (70 ml), and dried at 50.degree. C.
under vacuum to leave
Cbo-(2S,4R)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OMe as a
white solid.
[0112] 2.
Cbo-(2S,4R)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OMe (20.0 g,
43.0 mmol) is dissolved in a 1:1 mixture of tetrahydrofuran and
methanol (380 ml). A 1 M sodium hydroxide solution (51.6 ml) is
added and the resulting mixture stirred for 4 hours at ambient
temperature. The mixture is adjusted to pH 3 by adding sulfuric
acid (50 ml, 1 M). Tetrahydrofuran and methanol are distilled off
at 50.degree. C. and 50 mbar until no further solvents distil. The
remaining milky solution is diluted with isopropyl acetate (113 ml)
and water (57 ml), the phases are separated and the isopropyl
acetate phase is washed with sodium chloride solution (10%, 113
ml). The solvent is distilled off (50.degree. C., 50 mbar) to yield
a foam of Cbo-(2S,4R)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OH
(19.8 g), which was used without further purification in the next
reaction.
[0113] 3. Palladium on charcoal (10%, 1.94 g, 0042 mmol) is added
to a solution of
Cbo-(2S,4R)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OH (19.4 g,
43.0 mmol) in isopropanol (350 ml) and water (37 ml). Hydrogen is
bubbled through this mixture for 4 hours, the catalyst is filtered
off, and the residue is washed with a mixture of isopropanol (50
ml) and water (50 ml). The isopropanol/water phase is
azeotropically dried by distilling off 2/3 of the volume, which is
continuously replaced by a toluene/isopropanol mixture (1:1 v/v).
The remaining dry solution is concentrated in vacuo to dryness
(50.degree. C., 200 mbar) to leave
(2S,4R)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OH as a brownish
solid, which was used without further purification.
[0114] 4. (2S,4R)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OH
(5.0 g, 15 mmol) is dissolved in a mixture of water (25 ml) and
triethylamine (1.5 g, 15 mmol) at 40.degree. C. A solution of
Fmoc-OSu (4.65 g, 14 mmol) in acetonitrile (25 ml) is added to the
clear solution over 30 minutes and stirred for 2 hours. Then the
reaction mixture is adjusted to pH 3 with hydrochloric acid (1 m,
13 ml) and stirred for a further hour. Acetonitrile is distilled
off (40.degree. C., 80 mbar) and replaced by isopropyl acetate,
affording a two-phase mixture. The lower aqueous phase is separated
off, whilst the remaining organic layer is washed with water and
distilled two times with replacement with isopropylacetate and then
concentrated to a brownish foam. This foam is dissolved in
isopropylacetate (25 ml) and added dropwise to heptane (200 ml)
whereby the product is precipitated. The solid is filtered, washed
with isopropylacetate/heptane and dried in vacuo at 40.degree. C.
to leave
Fmoc-(2S,4R)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OH.
EXAMPLE 4
Preparation of
Fmoc-(2S,4R)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OH Starting
from Cbo-(2S,4R)-Pro(4-OH)--OBzl
[0115] The synthesis of Cbo-(2S,4R)-Pro(4-OH)--OBzl is described in
T. Makoto, H. Guoxia, V. J. Hruby, J. Org. Chem. 2001, 66,
1038-1042. The process of example 3 is repeated, but using
Cbo-(2S,4R)-Pro(4-OH)--OBzl in place of Cbo-(2S,4R)-Pro(4-OH)--OMe
and performing steps 1, 3 and 4 only (omitting step 2).
EXAMPLE 5
Preparation of
Fmoc-(2R,4R)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OH
[0116] The process of example 3 or example 4 is repeated but using
Cbo-(2R,4R)-Pro(4-OH)--OMe or Cbo-(2R,4R)-Pro(4-OH)--OBzl in place
of Cbo-(2S,4R)-Pro(4-OH)--OMe or Cbo-(2S,4R)-Pro(4-OH)--OBzl.
EXAMPLE 6
Preparation of
Fmoc-(2S,4S)-Pro(4-OCO--NH--CH.sub.2--CH.sub.2--NH-Boc)-OH
[0117] The process of example 3 or example 4 is repeated but using
Cbo-(2S,4S)-Pro(4-OH)--OMe or Cbo-(2S,4S)-Pro(4-OH)--OBzl in place
of Cbo-(2S,4R)-Pro(4-OH)--OMe or Cbo-(2S,4R)-Pro(4-OH)--OBzl.
* * * * *