U.S. patent application number 13/187230 was filed with the patent office on 2012-07-26 for asymmetric synthesis of peptides.
This patent application is currently assigned to University of Reading. Invention is credited to Laurence M. Harwood, Ran Yan.
Application Number | 20120190816 13/187230 |
Document ID | / |
Family ID | 35221406 |
Filed Date | 2012-07-26 |
United States Patent
Application |
20120190816 |
Kind Code |
A9 |
Harwood; Laurence M. ; et
al. |
July 26, 2012 |
ASYMMETRIC SYNTHESIS OF PEPTIDES
Abstract
The present invention provides a process comprising substitution
of an acceptor molecule comprising a group --XC(O)-- wherein X is
O, S or NR.sup.8, where R.sup.8 is C.sub.1-6 alkyl, C.sub.6-12 aryl
or hydrogen, with a nucleophile, wherein the acceptor molecule is
cyclised such that said nucleophilic substitution at --XC (O)--
occurs without racemisation. This process has particular
application for the production of a peptide by extension from the
activated carboxy-terminus of an acyl amino acid residue without
epimerisation.
Inventors: |
Harwood; Laurence M.;
(Berkshire, GB) ; Yan; Ran; (Berkshire,
GB) |
Assignee: |
University of Reading
|
Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20110275784 A1 |
November 10, 2011 |
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Family ID: |
35221406 |
Appl. No.: |
13/187230 |
Filed: |
July 20, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12052383 |
Mar 20, 2008 |
8044173 |
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13187230 |
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12066727 |
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PCT/GB2005/003797 |
Sep 30, 2005 |
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12052383 |
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Current U.S.
Class: |
530/321 ;
530/331; 544/162; 548/228; 560/39 |
Current CPC
Class: |
C07K 5/06052 20130101;
C07K 5/06026 20130101; C07D 265/32 20130101; C07K 5/0806 20130101;
C07K 5/1008 20130101 |
Class at
Publication: |
530/321 ;
544/162; 530/331; 560/39; 548/228 |
International
Class: |
C07K 7/64 20060101
C07K007/64; C07K 5/083 20060101 C07K005/083; C07C 231/10 20060101
C07C231/10; C07D 263/18 20060101 C07D263/18; C07D 265/32 20060101
C07D265/32; C07K 1/00 20060101 C07K001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 13, 2005 |
GB |
0518667.1 |
Claims
1. A process for nucleophilic substitution comprising substitution
of an acceptor molecule comprising a group --X--C(O)-- wherein X is
O, S or NR.sup.8 with a nucleophile, wherein the acceptor molecule
is cyclised, such that said nucleophilic substitution at
--X--C(O)-- occurs without racemisation, wherein the acceptor
molecule is a compound of formula (II) ##STR00063## wherein X is O,
S, or NR.sup.8, where R.sup.8 is hydrogen, an aliphatic group or
aromatic group; R.sup.2 is independently selected from an aliphatic
group or an aromatic group; R.sup.3 is as defined for R.sup.2 or is
hydrogen, or a group ##STR00064## or a group
--C(R.sup.1')(R.sup.9)--N(R.sup.10)(R.sup.11); wherein R.sup.1' is
independently selected from an aliphatic or aromatic group; wherein
when Y is NR.sup.8, R.sup.8 and R.sup.1' can together form an
optionally substituted 4 to 7 membered ring, wherein said ring can
be fully, partially or unsaturated, and wherein the ring may
contain one or more additional heteroatoms selected from O, S or N;
R.sup.12 is hydrogen, an aliphatic group or aromatic group; and
R.sup.4' is a carboxyl protecting group or hydrogen; R.sup.9 and
R.sup.10 are independently hydrogen or a group as defined for
R.sup.1'; or R.sup.9 and R.sup.10 or R.sup.10 and R.sup.11 can
together form an optionally substituted 4 to 7 membered ring,
wherein said ring can be fully, partially or unsaturated, and
wherein the ring may contain one or more heteroatoms selected from
O, S or N R.sup.11 is hydrogen or an amino protecting group; Y is
O, S or NR.sup.8, where R.sup.8 is as defined above; R.sup.5 is an
aliphatic group, an aromatic group, a linker for attachment of
formula (II) to a resin or a linked resin; n is 1, 2 or 3 and m is
a value selected from 1-100.
2. A process as claimed in claim 1 wherein the nucleophilic
substitution occurs without epimerisation.
3. A process as claimed in claim 2, wherein said process is carboxy
terminal extension of the acceptor molecule.
4. A process for the production of a compound of formula (I)
##STR00065## comprising reaction of a compound of formula (II)
##STR00066## with a compound of formula (III) HY--R.sup.7 (III)
wherein X is O, S, or NR.sup.8, where R.sup.8 is C.sub.1-16 alkyl,
C.sub.6-12 aryl or hydrogen; Y is O, S or NH; R.sup.2 is
independently selected from a C.sub.1-10 branched or straight chain
alkyl group, C.sub.5-12 heteroaryl group or C.sub.6-12 aryl group,
optionally substituted with OR.sup.13, SR.sup.13,
N(R.sup.13).sub.2, CO.sub.2R.sup.13, CON(R.sup.13).sub.2,
SO.sub.2R.sup.12, SO.sub.3R.sup.12, phenyl, imidazolyl, indolyl,
hydroxyphenyl or NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2;
R.sup.3 is as defined for R.sup.2 or is hydrogen, or a group
##STR00067## or a group
--C(R.sup.1')(R.sup.9)--N(R.sup.10)(R.sup.11); wherein R.sup.1' is
hydrogen or as defined from R.sup.1 below; Y is as defined above
and R.sup.4' is as defined for R.sup.4 below; R.sup.12 is hydrogen,
C.sub.1-6 alkyl, C.sub.6-12 aryl or N(R.sup.13).sub.2, wherein each
occurrence of R.sup.13 is independently hydrogen, C.sub.1-6 alkyl
or C.sub.6-12 aryl, R.sup.9 and R.sup.10 are independently hydrogen
or a group as defined for R.sup.1'; or R.sup.9 and R.sup.10 can
together form a 4 to 7 membered ring, optionally substituted with
CO.sub.2R.sup.13, OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2, C.sub.1-10 alkyl or
C.sub.6-12 aryl, wherein said ring can be fully, partially or
unsaturated, and wherein the ring may contain one or more
heteroatoms selected from O, S or N; R.sup.11 is hydrogen or an
amino protecting group preferably selected from a benzyloxycarbonyl
group, a t-butoxycarbonyl group, a
2-(4-biphenylyl)-isopropoxycarbonyl group, a
fluorenylmethoxycarbonyl group, a triphenylmethyl group and/or a
2-nitrophenylsulphenyl group; R.sup.5 is a C.sub.5-12 aryl,
C.sub.5-12 heteroaryl or C.sub.1-8 branched or straight chain alkyl
optionally substituted with OR.sup.13, SR.sup.13,
N(R.sup.13).sub.2, CO.sub.2R.sup.13, CO N(R.sup.13).sub.2, phenyl,
imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2 or a linker for
attachment of formula (II) to a resin; R.sup.6 is hydrogen or
##STR00068## wherein R.sup.5 and X are as defined above; R.sup.7 is
a group ##STR00069## or is independently selected from a C.sub.1-10
branched or straight chain alkyl group or a C.sub.6-12 aryl group,
optionally substituted with OR.sup.13, SR.sup.13,
N(R.sup.13).sub.2, CO.sub.2R.sup.13, CON(R.sup.13).sub.2,
SO.sub.2R.sup.12, SO.sub.3R.sup.12, phenyl, imidazolyl, indolyl,
hydroxyphenyl or NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2 or
wherein R.sup.7 and Y together form a 4 to 7 membered ring,
optionally substituted with OR.sup.13, SR.sup.13,
N(R.sup.13).sub.2, CO.sub.2R.sup.13, CON(R.sup.13).sub.2,
SO.sub.2R.sup.12, SO.sub.3R.sup.12, phenyl, imidazolyl, indolyl,
hydroxyphenyl or NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2,
wherein said ring can be fully, partially or unsaturated, and
wherein the ring may contain one or more heteroatoms in addition to
Y, selected from O, S or N; wherein R.sup.1 is independently
selected from a C.sub.1-10 branched or straight chain alkyl group,
C.sub.5-12 heteroaryl group or C.sub.6-12 aryl group optionally
substituted with OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2, SO.sub.2R.sup.12,
SO.sub.3R.sup.12, phenyl, imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2; and R.sup.4 is a
carboxyl protecting group or hydrogen; n is 1, 2 or 3 and m is a
value selected from 1-100.
5. A process as claimed in claim 4 wherein R.sup.1 and R.sup.2 are
independently selected from C.sub.1 alkyl optionally substituted
with OH, SH, CO.sub.2H, CONH.sub.2, phenyl, imidazolyl, indolyl or
hydroxyphenyl; C.sub.2 alkyl optionally substituted with OH,
CO.sub.2H, CONH.sub.2 or SCH.sub.3; C.sub.3 alkyl
NHC(.dbd.NH)NH.sub.2 or C.sub.4 alkyl optionally substituted with
NH.sub.2.
6. A compound of formula (II) ##STR00070## R.sup.2 is independently
selected from a C.sub.1-10 branched or straight chain alkyl group,
C.sub.5-12 heteroaryl group or C.sub.6-12 aryl group, optionally
substituted with OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2, SO.sub.2R.sup.12,
SO.sub.3R.sup.12, phenyl, imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2; R.sup.3 is as defined
for R.sup.2 or is hydrogen, or a group ##STR00071## or a group
--[Y--C(.dbd.R.sup.1')--C(O)].sub.m--; or a group
--C(R.sup.1')(R.sup.9)--N(R.sup.10)(R.sup.11); or a group
--C(.dbd.R.sup.1')--N(R.sup.10)(R.sup.11); wherein R.sup.1' is
independently selected from a C.sub.1-10 branched or straight chain
alkyl group, C.sub.5-12 heteroaryl group or C.sub.6-12 aryl group
optionally substituted with OR.sup.13, SR.sup.13,
N(R.sup.13).sub.2, CO.sub.2R.sup.13, CON(R.sup.13).sub.2,
SO.sub.2R.sup.12, SO.sub.3R.sup.12, phenyl, imidazolyl, indolyl,
hydroxyphenyl or NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2;
wherein when Y is NR.sup.8, where R.sup.8 is C.sub.1-6 alkyl,
C.sub.6-12 aryl or hydrogen, R.sup.8 and R.sup.1' can together form
a 4 to 7 membered ring, optionally substituted with
CO.sub.2R.sup.13, OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2, C.sub.1-10 alkyl or
C.sub.6-12 aryl, wherein said ring can be fully, partially or
unsaturated, and wherein the ring may contain one or more
heteroatoms selected from O, S or N; R.sup.12 is hydrogen,
C.sub.1-6 alkyl, C.sub.6-12 aryl or N(R.sup.13).sub.2, wherein each
occurrence of R.sup.13 is independently hydrogen, C.sub.1-6 alkyl
or C.sub.6-12 aryl, R.sup.9 and R.sup.10 are independently hydrogen
or a group as defined for R.sup.1'; or R.sup.9 and R.sup.10 can
together form a 4 to 7 membered ring, optionally substituted with
CO.sub.2R.sup.13, OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2, C.sub.1-10 alkyl or
C.sub.6-12 aryl, wherein said ring can be fully, partially or
unsaturated, and wherein the ring may contain one or more
heteroatoms selected from O, S or N; R.sup.11 is hydrogen or an
amino protecting group preferably selected from a benzyloxycarbonyl
group, a t-butoxycarbonyl group, a
2-(4-biphenylyl)-isopropoxycarbonyl group, a
fluorenylmethoxycarbonyl group, a triphenylmethyl group and/or a
2-nitrophenylsulphenyl group; R.sup.1' is independently selected
from C.sub.1-10 branched or straight chain alkyl optionally
substituted with OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CO N(R.sup.13).sub.2, phenyl, imidazoyl, indolyl,
hydroxyphenyl or NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2 and
R.sup.4' is a carboxyl protecting group or hydrogen, m is 1-100;
and R.sup.5 is a linker for attachment of formula (II) to a resin
or C.sub.6-12 aryl, C.sub.5-12 heteroaryl or C.sub.1-8 branched or
straight chain alkyl optionally substituted with OR.sup.13,
SR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13, CO
N(R.sup.13).sub.2, phenyl, imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2; wherein when X is O or
S, n is 1, 2 or 3; or when X is NR.sup.8, where R.sup.8 is
C.sub.1-6 alkyl, C.sub.6-12 aryl or hydrogen, n is 2 or 3; and
wherein when X.dbd.O, and R.sup.5 is phenyl, n is not 1.
7. A process for the formation of a compound of formula (II), as
defined in claim 6, ##STR00072## by the reaction of a compound of
formula (IV) ##STR00073## with a compound of formula (V)
##STR00074## wherein Z is any substituent capable of being involved
in peptide bond formation and R.sup.2, R.sup.3, R.sup.5, X and n
are as defined above in claim 6.
8. A process for the production of a compound of formula (VI) as
defined in claim 7, ##STR00075## from formula (I) ##STR00076## by
the removal of R.sup.6 wherein X, Y, R.sup.2, R.sup.3, R.sup.6 and
R.sup.7 are as defined above in claim 7.
9. A process as claimed in claim 8 wherein R.sup.5 is a linked
resin and the process is carried out on solid phase.
10. A process as claimed in claim 8 wherein the process is carried
out in solution.
11. A compound of formula (VII); ##STR00077## wherein m is an
integer of 1 to 50, and R.sup.1', R.sup.2, R.sup.5, R.sup.9,
R.sup.10, R.sup.11, X and n are as defined in claim 4.
12. A compound of formula (VIII); ##STR00078## wherein R.sup.1',
R.sup.2, R.sup.5, R.sup.9, X, m and n are as defined in claim
4.
13. A compound of formula (XI) ##STR00079## wherein R.sup.1',
R.sup.2, R.sup.9, X and m are as defined in claim 4.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 12/066,727, filed Mar. 13, 2008, which is the
U.S. national stage of International Application No.
PCT/GB05/03797, which designated the United States and was filed on
Sep. 30, 2005, published in English, which claims priority under 35
U.S.C. .sctn.119 or 365 to United Kingdom, Application No.
0518667.1, filed 13 Sep. 2005. The entire teachings of the above
applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to a novel process for peptide
synthesis, by the addition of amino acids to the activated
C-terminus of a peptide chain. Peptide synthesis is central to the
manufacture of many drugs and medicaments. Peptides or derivatives
thereof are used for the treatment of many disorders from
antibiotics to anticancer agents. Therefore improving peptide
synthesis and the yield of peptide produced by chemical synthesis
has been the focus of much research in recent years.
[0003] Chemical synthesis of proteins or peptides has been a
particular focus in the art. The chemical synthesis of proteins or
peptides allows the production of purified peptides of specific
amino acid sequence. It also allows the production of truncated
sequences of amino acids and allows the introduction of non-natural
amino acid derivatives.
[0004] Proteins are produced in nature by the stepwise condensation
of amino acid monomers on a ribosome. Synthesis of the protein
begins from the N-terminal residue and grows towards the
C-terminus. The conventional approach to peptide synthesis has
concentrated on extension at the N-terminus of a growing peptide.
This approach forms the basis of conventional solid phase peptide
synthesis. Peptide synthesis solely by extension from the
N-terminus is limiting as it renders any peptide synthesis linear
in nature, rather than convergent. This can severely increase
overall length of synthesis, increase operational time and decrease
overall yield with consequent possibilities for the loss of
stereochemical fidelity.
[0005] To overcome the problems associated with N-terminal
extension of a peptide, it could be envisaged that the synthesis
could instead provide extension of a peptide from the C-terminal.
However, attempts at peptide synthesis in the N to C direction have
been generally unsuccessful due to epimerisation of the
carboxy-terminal amino acid residue. This is due to the tendency of
carboxy-terminal-activated acylamino acids and peptides to form
oxazolones. As illustrated below, the formation of the oxazolone
allows rapid racemisation of the alpha-position of the terminal
amino acid residue of the acyl amino acid or peptide.
##STR00001##
[0006] This racemisation prevents the production of
stereochemically homogeneous peptides by C-terminus extension.
[0007] It will be appreciated that the production of isomerically
pure compounds is a particular requirement in the art. Any method
which results in the production of a mixture of isomers will
require the use of time consuming and expensive purification steps
to separate the isomers. Chiral compounds which are administered to
humans or animals are usually required in enantiomerically pure
form. The presence of unwanted isomers even in low concentrations
can reduce the potency of the compound and can produce unwanted and
in some cases disastrous side effects. The incorporation of an
unwanted enantiomer into a peptide chain (for example the
incorporation of a D-amino acid into a peptide composed of L-amino
acids) may disrupt the folding and/or 3D shape of the peptide, thus
resulting in a peptide which may have unpredictable binding
activities and/or biological properties. The production of
enantiomerically pure peptides is therefore of paramount
importance.
[0008] Various attempts have been made to overcome this problem.
Iorga, B and Campagne, J-M (2004, Synlett 10, 1826-1828) attempted
to reduce the degree of epimerisation by improving the rate of
peptide bond formation over the rate of oxazolone formation, so
that peptide bond formation was the predominant reaction. However,
this method does not entirely prevent the formation of the
oxazolone and therefore epimerisation at the carboxy-terminal
activated amino acid residue occurs. Native chemical ligation has
been developed to overcome problems of carboxy-terminal extension
but is ordinarily restricted to couplings in which the
amino-terminal partner is an assisting cysteine residue and is not
applicable to general techniques of automated solid phase peptide
synthesis. The application of native chemical ligation to other
amino-terminal amino acids has had very limited success.
[0009] The present invention provides a new method of producing a
peptide by extension from the activated carboxy-terminus of an acyl
amino acid residue. This new method overcomes the problems of
epimerisation of the terminal amino acid residue during the
coupling step. The present invention therefore allows the
production of peptides by a convergent approach and provides a new
method for the production of potentially biologically important
compounds instead of the linear repetitive amino terminal extension
approach currently used.
[0010] The first aspect of the present invention provides a process
comprising substitution of an acceptor molecule comprising a group
--XC(X)-- (preferably --X(CO)--) wherein each X is independently O,
S or NR.sup.8, where R.sup.8 is hydrogen, aliphatic group or an
aromatic group, preferably hydrogen, C.sub.1-6 alkyl, C.sub.6-12
aryl, with a nucleophile, wherein the acceptor molecule is cyclised
such that said nucleophilic substitution at --XC(X)-- occurs
without racemisation. The acceptor molecule is preferably a
cyclised amino acid or derivative thereof. In particular, the
acceptor molecule is a compound of formula (II):
##STR00002##
wherein each X is O, S, or NR.sup.8, where R.sup.8 is as defined
above; R.sup.2 is independently selected from an aliphatic group,
such as a C.sub.1-10 branched or straight chain alkyl group, or an
aromatic group, such as C.sub.5-12 heteroaryl group or C.sub.6-12
aryl group, each optionally substituted with a group including, for
example, OR.sup.13, SR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13,
CON(R.sup.13).sub.2, SO.sub.2R.sup.12, SO.sub.3R.sup.12, phenyl,
imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2; R.sup.3 is as defined
for R.sup.2 or is hydrogen,
##STR00003##
or a group --C(R.sup.1') (R.sup.9)--N(R.sup.10)(R.sup.11); wherein
R.sup.1' is independently selected from an aliphatic group such as
C.sub.1-10 branched or straight chain alkyl group, an aromatic
group, such as C.sub.5-12 heteroaryl group or C.sub.6-12 aryl
group, each optionally substituted with a group such as OR.sup.13,
SR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13,
CON(R.sup.13).sub.2, SO.sub.2R.sup.12, SO.sub.3R.sub.12, phenyl,
imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2; wherein when Y is
NR.sup.8, R.sup.8 and R.sup.1' can together form a 4 to 7 membered
ring, optionally substituted with a group such as CO.sub.2R.sup.13,
OR.sup.13, SR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13,
CON(R.sup.13).sub.2, C.sub.1-10 alkyl or C.sub.6-12 aryl, wherein
said ring can be fully, partially or unsaturated, and wherein the
ring may contain one or more additional heteroatoms selected from
O, S or N; R.sup.12 is hydrogen, C.sub.1-6 alkyl, C.sub.6-12 aryl
or N(R.sup.13).sub.2, wherein each occurrence of R.sup.13 is
independently hydrogen, C.sub.1-6 alkyl or C.sub.6-12 aryl, and
R.sup.4' is a carboxyl protecting group or hydrogen; R.sup.9 and
R.sup.10 are independently hydrogen or a group as defined for
R.sup.1'; R.sup.11 is hydrogen or an amino protecting group
preferably selected from a benzyloxycarbonyl group, a
t-butoxycarbonyl group, a 2-(4-biphenylyl)-isopropoxycarbonyl
group, a fluorenylmethoxycarbonyl group, a triphenylmethyl group
and/or a 2-nitrophenylsulphenyl group; or R.sup.9 and R.sup.10 or
R.sup.10 and R.sup.11 or R.sup.1' and R.sup.10 or two R.sup.13 can
together form a 4 to 7 membered ring, optionally substituted with a
group such as CO.sub.2R.sup.13, OR.sup.13, SR.sup.13,
N(R.sup.13).sub.2, CO.sub.2R.sup.13, CON(R.sup.13).sub.2,
C.sub.1-10 alkyl or C.sub.6-12 aryl, wherein said ring can be
fully, partially or unsaturated, and wherein the ring may contain
one or more additional heteroatoms selected from O, S or N; Y is O,
S or NR.sup.8, where R.sup.8 is as defined above;
or YR.sup.4' is R.sup.3;
[0011] R.sup.5 is an aromatic group such as C.sub.6-12 aryl,
C.sub.5-12 heteroalkyl or an aliphatic group such as C.sub.1-8
branched or straight chain alkyl optionally substituted with a
group such as OR.sup.13, SR.sup.13N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2, phenyl, imidazolyl, indolyl,
hydroxyphenyl or NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13), or a linker
for attachment of formula (II) to a resin or a linked resin; n is
0, 1, 2 or 3 and m is an integer, such as an integer selected from
1-100.
[0012] The nucleophilic substitution of the acceptor molecule
preferably occurs without epimerisation.
[0013] Preferably, the process is carboxy terminal extension of an
acceptor molecule, for example an amino acid or peptide. The
invention therefore provides a process for the synthesis of a
peptide or a peptide analog by carboxy terminal extension, by the
addition of a nucleophile to an acceptor molecule, such as a
compound of formula (II).
[0014] There is further provided a process for the production of a
compound of formula (1)
##STR00004##
comprising reaction of a compound of formula (II) or (II') (above)
with a compound of formula (III)
HY--R.sup.7 (III)
wherein the variables are defined as above; preferably X is O, S,
or NR.sup.8, where R.sup.8 is as defined above; Y is O, S or NH;
R.sup.2 is independently selected from a C.sub.1-10 branched or
straight chain alkyl group, C.sub.5-12 heteroaryl group or
C.sub.6-12 aryl group, optionally substituted with OR.sup.13,
SR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13,
CON(R.sup.13).sub.2, SO.sub.2R.sup.12, SO.sub.3R.sup.12, phenyl,
imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2; R.sup.3 is as defined
for R.sup.2 or is hydrogen, or a group
##STR00005##
or a group --C(R.sup.1')(R.sup.9)--N(R.sup.10)(R.sup.11); wherein R
is hydrogen or as defined from R.sup.1 below; Y is as defined above
and R.sup.4 is as defined for R.sup.4 below; R.sup.12 is hydrogen,
C.sub.1-6 alkyl, C.sub.6-12 aryl or N(R.sup.13).sub.2, wherein each
occurrence of R.sup.13 is independently hydrogen, C.sub.1-6 alkyl
or C.sub.6-12 aryl, R.sup.9 and R.sup.10 are independently hydrogen
or a group as defined for R.sup.1': or R.sup.9 and R.sup.10 can
together form a 4 to 7 membered ring, optionally substituted with
CO.sub.2R.sup.13, OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2, C.sub.1-10 alkyl or
C.sub.6-12 aryl, wherein said ring can be fully, partially or
unsaturated, and wherein the ring may contain one or more
additional heteroatoms selected from O, S or N, R.sup.11 is
hydrogen or an amino protecting group preferably selected from a
benzyloxycarbonyl group, a t-butoxycarbonyl group, a
2-(4-biphenylyl)-isopropoxycarbonyl group, a
fluorenylmethoxycarbonyl group, a triphenylmethyl group and/or a
2-nitrophenylsulphenyl group; R.sup.5 is an aromatic group, such as
C.sub.5-12 aryl, C.sub.5-12 heteroalkyl or an aliphatic group, such
as C.sub.1-8 branched or straight chain alkyl optionally
substituted with OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CO N(R.sup.13).sub.2, phenyl, imidazolyl,
indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2 or a linker for
attachment of formula (II) to a resin or a linked resin; R.sup.6 is
hydrogen or
##STR00006##
wherein R.sup.5 and X are as defined above; R.sup.7 is a chiral,
substituted methylene, such as a group
##STR00007##
or is independently selected from an aliphatic group such as a
C.sub.1-10 branched or straight chain alkyl group or an aromatic
group, such as a C.sub.6-12 aryl group, optionally substituted with
OR.sup.13, SR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13,
CON(R.sup.13).sub.2, SO.sub.2R.sup.12, SO.sub.3R.sup.12, phenyl,
imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2, or wherein R.sup.7 and
Y together form a 4 to 7 membered ring, optionally substituted with
a group such as OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2, SO.sub.2R.sup.12,
SO.sub.3R.sup.12, phenyl, imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2, wherein said ring can
be fully, partially or unsaturated, and wherein the ring may
contain one or more heteroatoms in addition to Y, selected from O,
S or N; wherein R.sup.1 is R.sup.1' or is independently selected
from an aliphatic group such as C.sub.1-10 branched or straight
chain alkyl group, or an aromatic group such as C.sub.5-12
heteroaryl group or C.sub.6-12 aryl group optionally substituted
with a group such as OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2, SO.sub.2R.sup.12,
SO.sub.3R.sup.12, phenyl, imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2, and R.sup.4 is
R.sup.4' or a carboxyl protecting group or hydrogen; n is 0, 1, 2
or 3 and m is an integer such as a value selected from 1-100 and
when n=0, R.sup.6 is H.
[0015] The inventors have surprisingly found that activation of an
amino acid or peptide via a cyclic compound as exemplified in
formula (II) prevents the formation of an oxazolone thereby
allowing the condensation of a compound of formula (III) without
concommitant epimerisation. The invention therefore provides
peptides via C-terminus extension, said peptides being produced in
an enantiomerically and diastereochemically pure form.
[0016] The use of activated cyclic N-acyl amino acids, peptides or
derivatives thereof eliminates oxazolone formation and associated
epimerisation. The use of cyclic activated intermediates in the
present invention provides an improved method of peptide synthesis
via carboxy-terminal extension.
[0017] Therefore, rather than merely reducing the probability of
epimerisation occurring, as has been attempted in the prior art,
the process of the present invention does not permit epimerisation
and therefore guarantees the production of a peptide of correct
stereochemistry as the activated carboxyl terminus is held in a
cyclic template such that the adjacent amide cannot form the
oxazolone.
[0018] In accordance with usual practice, * denotes a stereocenter
(asymmetric center). Where a compound contains a stereocenter
(whether marked in the present application with * or not) the
stereochemistry of the asymmetric centers may be in the R or S
configuration. The compounds of the present application can be
provided in enantiomerically pure form or as a mixture of isomers
(including a racemic mixture). Preferably, the compounds of the
present inventions are provided in an enantiomerically pure form.
The present invention allows maintenance of the desired
stereochemistry throughout the synthetic pathway. Thus wherein Y is
NH, the amino acids to be attached may be of U or D configuration
as required.
[0019] Preferably R.sup.1 and R.sup.2 are independently selected
from C.sub.1-4 branched or straight chain alkyl optionally
substituted with OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2, phenyl, imidazolyl, indolyl,
hydroxyphenyl or NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2,
preferably optionally substituted with OH, SH, NH.sub.2, CO.sub.2H,
CONH.sub.2, phenyl, imidazolyl, indolyl, hydroxyphenyl or
NH(C.dbd.NH)NH.sub.2.
[0020] More preferably, R and R are independently selected from
C.sub.1 alkyl optionally substituted with OH, SH, CO.sub.2H,
CONH.sub.2, phenyl, imidazolyl, indolyl or hydroxyphenyl; C.sub.2
alkyl optionally substituted with OH, CO.sub.2H, CONH.sub.2 or
SCH.sub.3; C.sub.3 alkyl optionally substituted with
NHC(.dbd.NH)NH.sub.2 or C.sub.4 alkyl optionally substituted with
NH.sub.2.
[0021] The integer, m is preferably 1-50, more preferably 1 to 30,
most preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 or 20. The integer n is preferably 0 or 1.
[0022] When X is NR, R is preferably Ci.sub.-4 alkyl, more
preferably methyl, ethyl, n-propyl, iso-propyl, n-butyl or
tert-butyl, phenyl, naphthyl, anthracenyl or phenanthracenyl, more
preferably phenyl or hydrogen.
[0023] R.sup.3 may also be substituted pipecolic acid or derivative
thereof, .alpha.-alkoxy-.alpha.-amino acids, cc, .alpha.-diamino
acids, .beta.-substituted dehydroamino acids, canavanine,
cysteinesulphonamide, homocysteinesulphonamide,
.gamma.,.delta.-unsaturated amino acids. substituted
4-hydroxyprolines, 4-hydroxtyornithines, imino sugars, Fmoc
-BPC--OH, Fmoc-TPG-OH and Fmoc-CAA-OH, or
(5)-3,5-dihydroxyphenylglycine.
[0024] It will be appreciated by a person skilled in the art that
amino acids, hydroxy acids and derivatives thereof contain
functional groups which require protection. In particular it is
known in the art to protect the amino terminus, the carboxyl
terminus and/or the side chains of an amino acid or peptide (for
example wherein R.sup.1 or R.sup.2 is C.sub.2CO.sub.2H or
CH.sub.2CH.sub.2OH). Examples of such protection are well known in
the art. In particular the amino terminus of an amino acid may be
protected by one or more of a benzyloxycarbonyl group, a
t-butoxycarbonyl group, a 2-(4-biphenylyl)-isopropoxycarbonyl
group, a fluorenylmethoxycarbonyl group, a triphenylmethyl group
and/or a 2-nitrophenylsulphenyl group. The carboxyl group can be
protected by one or more of an ester group especially a methyl,
ethyl, benzyl, t-butyl or phenyl ester. Thus R.sup.4 is preferably
methyl, ethyl, benzyl, t-butyl or phenyl.
[0025] Conditions for the removal of the protecting groups
discussed above are well known in the art. The protecting groups
may be removed after each coupling reaction (for example, the
carboxyl protection) or alternatively at the end of the synthesis
(for example, the side chain protection and/or the N-terminal
group).
[0026] In a particular feature of the first aspect, the invention
provides a process for production of a compound of formula (Ia)
##STR00008##
comprising reacting a compound of formula (IIa) (above) with a
compound of formula (III) HY--R.sup.7: Wherein the groups Y, X,
R.sup.2, R.sup.3, R.sup.5, and R.sup.7 are as defined above.
[0027] In an alternative feature of the first aspect, the invention
provides a process for production of a compound of formula (Ib)
##STR00009##
comprising reacting a compound of formula (IIb)
##STR00010##
with a compound of formula (III) HY--R.sup.7 wherein the groups Y,
X, R.sup.2, R.sup.3, R.sup.5 and R.sup.7 are as defined above for
compounds (I), (II) and (III).
[0028] The N and the terminal ester of formula (I), (Ia) or (Ib)
can be unmasked by processes known in the art. for example, sodium
liquid ammonia in the presence of an alcohol when R.sup.1=phenyl
and R.sup.4=t-butyl. Alternatively, the ester can be further
derivatized, including for example, amidation.
[0029] The nucleophilic substitution of the acceptor molecule of
the first aspect of the invention can be carried out using reaction
conditions known in the art. In some circumstances, for example
where the nucleophile and/or the acceptor molecule are sterically
hindered it may be necessary for example to use high pressure such
as around 19-20 bar, and/or longer reaction times such as 12-72
hours, preferably 24-48 hours. Alternatively, the reaction can be
carried out in the presence of a reagent such as AlMe.sub.3. When
the substitution is carried out on the solid phase, the reaction
can be promoted by the use of an excess of nucleophile.
[0030] The invention further relates to a process for the
production of a compound of formula (II) (above) by the reaction of
a compound of formula (IV)
##STR00011##
with a compound of formula (V) Z--CO--R.sup.3 wherein Z is any
substituent capable of being involved in peptide bond formation
preferably hydroxide, halide or azide, and R.sup.2, R.sup.3,
R.sup.5, X and n are as defined above. It will be appreciated that
when R.sup.3 is a protected peptide, subsequent N-terminus
extension may be carried out using peptide synthesis methods known
in the art, such as deprotection and further peptide bond
formation.
[0031] The process of the present invention can particularly be
used for the production of cyclic compounds, for example cyclic
peptides.
[0032] It will be appreciated that when R.sup.3 is
C(R.sup.1')(R.sup.9)--N(R.sup.10)(R.sup.11), the compound of
formula (II) can be reacted with one or more compounds of formula
(V) in a stepwise direction.
[0033] The present invention therefore encompasses a compound of
formula (VII);
##STR00012##
wherein the variables are described above, preferably m is an
integer of 1 to 50, preferably 1 to 30, more preferably 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18, 19 or 20; and
R.sup.1', R.sup.2, R.sup.5, R.sup.9, R.sup.10, R.sup.11, X and n
are as defined above.
[0034] The compound of formula (VII) can be used in a process for
the formation of a compound of formula (I) (illustrated below as
(Ic));
##STR00013##
when R.sup.14 is
--[C(O)--C(R.sup.9)(R.sup.1')--N(R.sup.10)--].sub.m--(R.sup.11) and
R.sup.1', R.sup.2, R.sup.6, R.sup.7, R.sup.9, R.sup.10, R.sup.11,
m, X and Y are as described above, comprising reaction of a
compound of formula (VII) with a compound of formula (III) as
described above. The compound of formula (I) can then be converted
into a compound of formula (VI) by removal of the group R.sup.6 as
described below. For the compound of formula (VII) and compounds of
formula (I) or (VI) obtained therefrom, the substituents R.sup.1
and R.sup.9 can be replaced by a group (.dbd.R.sup.1) wherein
R.sup.1 is as described above.
[0035] It will be appreciated that when m is 3 or more, and
R.sup.10 and R.sup.11 are hydrogen, condensation can occur at the
X--C(O)-- functionality of the compound of formula (VII) to form a
cyclised compound of formula (VIII);
##STR00014##
wherein the variables are as defined above.
[0036] The present invention therefore provides a compound of
formula (VIII). In addition, the invention provides a process for
the production of a compound of formula (VIII) comprising
cyclisation of a compound of formula (VII) wherein m is 3 or more.
Reaction of the compound of formula (VIII) under reducing
conditions (for example in the presence of lithium and liquid
ammonia) results in the formation of a compound of formula
(IX);
##STR00015##
wherein R.sup.1', R.sup.2, R.sup.9, X and m are as defined
above.
[0037] The present invention therefore provides a compound of
formula (IX) and a process for the production of a compound of
formula (IX) comprising the reduction of a compound of formula
(VIII).
[0038] It will be appreciated that the process of the present
invention can be carried out in solution. Alternatively, a compound
of formula II may be attached to a resin via the group R.sup.5 and
the peptide synthesis carried out via solid phase peptide
synthesis. When R.sup.5 is a linker it can be a group OR.sup.13,
N(R.sup.13).sub.2, CO.sub.2R.sup.13 or Se, or an alkyl group having
1 to 4 carbons or a C.sub.6-12 aryl group, said alkyl and aryl
groups being optionally substituted with OR.sup.13,
N(R.sup.13).sub.2, CO.sub.2R.sup.13 or SR.sup.13. Alternatively,
part of the synthesis may be carried out on the solid phase and
part in solution.
[0039] The compound of formula II can be attached to and removed
from a resin using methods known in the art.
[0040] Solid phase peptide synthesis using the process of the
present invention may be carried out by using procedures attaching
the carboxy-terminal to any resin known in the art. Examples of
suitable resins include Wang, Merrifield, polyimide,
2-chlorotrityl, Rink, Knorr, DCHD, PAL and any other known in the
art. Solid phase coupling partners such as BOP, PyBOP and DCC may
be used, as well as any other suitable coupling partners known in
the art.
[0041] A further feature of the first aspect is a process for the
production of a compound of formula (VI)
##STR00016##
from formula (I)
##STR00017##
by the removal of R.sup.6 by any method known in the art, wherein
X, Y, R.sup.2, R.sup.3, R.sup.6 and R.sup.7 are as defined above.
It will be appreciated that when R.sup.6 is hydrogen, the compound
of formula (I) corresponds to the compound of formula (VI).
[0042] In particular, the removal of the group R.sup.6 may be
carried out under reducing conditions such as under Birch
conditions (i.e. with lithium and liquid ammonia). As it will be
appreciated by the skilled person, the peptide produced by the
process of the first aspect may be post modified by any suitable
method known in the art. A second aspect of the present invention
relates to the compounds described herein, including a compound of
formula (II)
##STR00018##
wherein X is O, S or NR.sup.8, where R.sup.8 is C.sub.1-6 alkyl,
C.sub.6-12 aryl or hydrogen R.sup.2 is independently selected from
a C.sub.1-10 branched or straight chain alkyl group, C.sub.5-12
heteroaryl group or C.sub.6-12 aryl group, optionally substituted
with OR.sup.13, SR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13,
CON(R.sup.13).sub.2, SO.sub.2R.sup.12, SO.sub.3R.sup.12, phenyl,
imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2; R.sup.3 is as defined
for R.sup.2 or is hydrogen, or a group
##STR00019##
or a group --C(R.sup.1')(R.sup.9)--N(R.sup.10)(R.sup.11) wherein
R.sup.1 is independently selected from a C.sub.1-10 branched or
straight chain alkyl group, C.sub.5-12 heteroaryl group or
C.sub.6-12 aryl group optionally substituted with OR.sup.13,
SR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13,
CON(R.sup.13).sub.2, SO.sub.2R.sup.12, SO.sub.3R.sup.12, phenyl,
imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2 wherein when Y is
NR.sup.8, R.sup.8 and R.sup.1' can together form a 4 to 7 membered
ring, optionally substituted with CO.sub.2R.sup.13, OR.sup.13,
SR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13,
CON(R.sup.13).sub.2, C.sub.1-10 alkyl or C.sub.6-12 aryl, wherein
said ring can be fully, partially or unsaturated, and wherein the
ring may contain one or more heteroatoms selected from O, S or N;
R.sup.12 is hydrogen, C.sub.1-6 alkyl, C.sub.6-12 aryl or
N(R.sup.13).sub.2, wherein each occurrence of R.sup.13 is
independently hydrogen, C.sub.1-6 alkyl or C.sub.6-12 aryl, R.sup.9
and R.sup.10 are independently hydrogen or a group as defined for
R.sup.1; or R.sup.9 and R.sup.10 can together form a 4 to 7
membered ring, optionally substituted with CO.sub.2R.sup.13,
OR.sup.13, SR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13,
CON(R.sup.13).sub.2, C.sub.1-10 alkyl or C.sub.6-12 aryl, wherein
said ring can be fully, partially or unsaturated, and wherein the
ring may contain one or more heteroatoms selected from O, S or
NR.sup.11 is hydrogen or an amino protecting group preferably
selected from a benzyloxycarbonyl group, a t-butoxycarbonyl group,
a 2-(4-biphenylyl)-isopropoxycarbonyl group, a
fluorenylmethoxycarbonyl group, a triphenylmethyl group and/or a
2-nitrophenylsulphenyl group; R.sup.1 is independently selected
from C.sub.1-10 branched or straight chain alkyl optionally
substituted with OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CO.sub.2R.sup.13, CO N(R.sup.13).sub.2, phenyl, imidazoyl, indolyl,
hydroxyphenyl or NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2 and
R.sup.4' is a carboxyl protecting group or hydrogen and n is 0, 1,
2 or 3, m is 1-100; and R.sup.5 is a linker for attachment of
formula (II) to a resin, a linked resin, or C.sub.6-12 aryl,
C.sub.5-12 heteroalkyl or C.sub.1-8 branched or straight chain
alkyl optionally substituted with OR.sup.13, SR.sup.13,
N(R.sup.13).sub.2, CO.sub.2R.sup.13, CON(R.sup.13).sub.2, phenyl,
imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2; wherein when X.dbd.O,
and R.sup.5 is phenyl, n is not 0 or 1.
[0043] Preferably R.sup.1 and R.sup.2 are independently selected
from C.sub.1-4 branched or straight chain alkyl optionally
substituted with OR.sup.13, SR.sup.13, N(R.sup.13).sub.2,
CON(R.sup.13).sub.2, phenyl, imidazolyl, indolyl, hydroxyphenyl or
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2. More preferably
R.sup.1 and R.sup.2 are independently selected from C.sub.1 alkyl
optionally substituted with OR.sup.13, SR.sup.13, CO.sub.2R.sup.13,
CO N(R.sup.13).sub.2, phenyl, imidazolyl, indolyl or hydroxyphenyl;
C.sub.2 alkyl optionally substituted with OR.sup.13,
CO.sub.2R.sup.13, CON(R.sup.13).sub.2 or SCH.sub.3; C.sub.3 alkyl
NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2 or C.sub.4 alkyl
optionally substituted with N(R.sup.13).sub.2.
[0044] As set out above, R.sup.4 is a carboxyl protecting group,
such as an ester group. In particular R.sup.4 is preferably methyl,
ethyl, benzyl, t-butyl or phenyl. When R.sup.5 is a linker it can
be OR.sup.13, N(R.sup.13).sub.2. CO.sub.2R.sup.13 or SR.sup.13 or
an alkyl group having 1 to 4 carbons or a C.sub.6-12 aryl group,
wherein the alkyl group and/or aryl group can be substituted with
one or more of OR.sup.13, N(R.sup.13).sub.2, CO.sub.2R.sup.13 or
SR.sup.13.
[0045] The integer, m is preferably 1-50, more preferably 1 to 30,
most preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 or 20. The integer n is preferably 0 or 1.
[0046] When X is NR.sup.8, R.sup.8 is preferably C.sub.1-4 alkyl,
more preferably methyl, ethyl, n-propyl, iso-propyl, n-butyl or
tert-butyl, phenyl, naphthyl, anthracenyl or phenanthracenyl, more
preferably phenyl or hydrogen.
[0047] R.sup.13 is preferably hydrogen or C.sub.1-4 alkyl, more
preferably methyl, ethyl, n-propyl, iso-propyl, n-butyl or
tert-butyl.
[0048] For the purposes of this invention, alkyl relates to both
straight chain and branched, saturated or unsaturated alkyl
radicals having, for example, 1 to 10 carbon atoms, preferably 1 to
8 carbon atoms and most preferably 1 to 4 carbon atoms including
but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, isobutyl, tert-butyl n-pentyl, n-hexyl, n-heptyl,
n-octyl. Alkyl therefore relates to a group having 1, 2, 3, 4, 5,
6, 7, 8, 9 or 10 or more carbon atoms. The term alkyl also
encompasses cycloalkyl radicals of 3 to 12 carbon atoms, preferably
4 to 8 carbon atoms, and most preferably 5 to 6 carbon atoms
including but not limited to cyclopropyl, cyclobutyl,
CH.sub.2-cyclopropyl, CH.sub.2-cyclobutyl, cyclopentyl or
cyclohexyl. Cycloalkyl groups may be optionally substituted or
fused to one or more carbocyclyl or heterocyclyl group. Haloalkyl
relates to an alkyl radical preferably having 1 to 8 carbon atoms,
preferably 1 to 4 carbon atoms substituted with one or more halide
atoms for example CH.sub.2CH.sub.2Br, CF.sub.3 or CCl.sub.3. An
alkyl group may be optionally interrupted by one or more O, S or NH
groups, preferably one or more O atoms to form an alkoxy group. An
alkyl group may be optionally interrupted by one or more double or
triple bonds to form a group including but not limited to ethylene,
n-propyl-1-ene, n-propyl-2-ene, isopropylene, ethynyl,
2-methylethynyl etc.
[0049] "Aryl" means an aromatic 6 to 12 membered hydrocarbon or
heteroaryl containing one ring or being fused to one or more
saturated or unsaturated rings including but not limited to phenyl,
naphthyl, anthracenyl or phenanthracenyl. "Heteroaryl" means an
aromatic 5 to 12 membered aryl containing one or more heteroatoms
selected from N, O or S and containing one ring or being fused to
one or more saturated or unsaturated rings including but not
limited to furan, imidazole, indole, oxazole, purine, pyran,
pyridine, pyrimidine, pyrrole, tetrahydrofuran, thiophene and
triazole. The aryl and heteroaryl groups can be fully saturated,
partially saturated or unsaturated.
[0050] Halogen means F, CI, Br or I, preferably F.
[0051] A third aspect of the invention relates to the use of a
compound of formula (II) as defined in the first and/or second
aspects of the invention in asymmetric synthesis.
[0052] All preferred features of each of the aspects of the
invention apply to all other aspects mutatis mutandis.
[0053] The present invention will now be illustrated by reference
to one or more of the following non-limiting examples:
EXAMPLES
[0054] Example of a method for the production of a cyclic
peptide.
##STR00020##
[0055] Example of a method for N-terminal extension of a compound
of formula (II).
##STR00021##
[0056] Example of a method for use of the compound of formula (II)
in solid phase synthesis
##STR00022##
[0057] Example of a method to produce a thiomorpholinone template
of formula (II)
##STR00023##
[0058] Examples of peptide synthesis
(5R)-3-Methyl-5-.rho.henyl-5,6-dihydro-2H-1,4-oxazin-2-one
##STR00024##
[0059] (R)-2-phenylglycinol (3.00 g, 21.9 mmol, 1.0 equiv.) and
ethyl pyruvate (2.67 mL, 24.1 mmol, 1.1 equiv.) were refluxed in
tritluoroethanol (50 mL) over activated 4 molecular sieves (8.00 g)
for 24 hours. Filtration through a short pad of Celite.RTM. and
removed of solvent from the filtrate in vacuo generated the crude
product which was purified by flash column chromatography on
silica, eluting with petrol and diethyl ether (7:3) to furnish the
title compound as a white solid (1.70 g, 41%); mp 71.0-72.0.degree.
C. (lit 71.0-72.0.degree. C.); v.sub.(max) (KBr) 3001 (C--H), 1734
(C.dbd.O), 1642 (C.dbd.N) cm.sup.''1; .delta..sub.H (250 MHz,
CDCl.sub.3) 7.42-7.32 (5H, m, Ph), 4.89-4.80 (1H, m, PhCH), 4.56
(1H, dd, /4.49 Hz, T 11.55 Hz, 6.beta.-H), 4.25 (1H, dd J 10.97 Hz,
r 11.51 Hz, 6.alpha.-H), 2.40 (3H, S, CH.sub.3); .delta..sub.c
(62.5 MHz, CDCl.sub.3) 160.7, 155.9, 137.2, 129.4, 128.7, 127.5,
71.9, 60.1, 22.2; .sup.mI.sub.z (CL, NH.sub.3), 189 (M.sup.+, 25%),
159 (12%), 130 (24%), 104 (100%), 90 (21%), and 78 (6%); HRMS for
C.sub.11H.sub.11NO.sub.2 requires 189.0787 found 189.0782.
[.alpha.].sub.D.sup.20-256.0 (c 1.11 CHCl.sub.3) (lit.
[.alpha.].sub.D.sup.20-237.1 (c 1.11 CHCl.sub.3)).
(3S,5R)-3-Methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
##STR00025##
[0061] To a solution of
(5R)-3-methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one (1.70 g, 9.0
mmol, 1.0 equiv.) in anhydrous dichloromethane (60 mL) under an
atmosphere of nitrogen was added PtO.sub.2 (170 mg, 0.1 equiv.).
The mixture was consecutively degassed and purged three times with
hydrogen and then stirred for 5 hours under an atmosphere of
hydrogen. Filtration through a short pad of Celite.RTM. and removal
of solvent from the filtrate in vacuo yield the crude product which
was purified by recrystallisation dichloromethane diethyl diethyl
ether and hexane to furnish the title compound as a white needles
(1.37 g, 80%); m.p. 82.0-83.0.degree. C. (lit. m.p.
81.0-82.0.degree. C.); v.sub.(max) (KBr) 3314 (N--H), 2981 (C--H),
1739 (C.dbd.O), cm.sup.''1; .delta..sub.H (250 MHz, CDCl.sub.3)
7.44-7.33 (5H, m, Ph),; 4.42-4.23 (3H, m, CHCH.sub.2), 3.88 (1H, q,
/6.76 Hz, CHCH.sub.3), 1.86 (1H, br, NH), 1.50 (3H, d, J 16.16 Hz,
CH.sub.3); .delta..sub.c (62.5 MHz, CDCl.sub.3) 170.7, 138.0,
129.3, 127.5, 127.2, 75.4, 58.2, 55.4, 19.0; .sup.mI.sub.z (CL,
NH.sub.3), 191 (M.sup.+, 7%), 147 (20%), 131 (65%), 104 (100%), 91
(20%), and 77 (12%); FIRMS for C.sub.11H.sub.13NO.sub.2 requires
191.0943 found 191.0940. [.alpha.].sub.D.sup.20-92.9 (c 1.02
CHCl.sub.3) (lit. for the enantiomer [.alpha.].sub.D.sup.20+92.3 (c
0.84 CHCl.sub.3)).
(3S,5R)-4-N-Acetyl-3-methyl-5-.rho.henyl-3,4,5,6,-tetrahydro-2H-1,4-oxazin-
-2-one
##STR00026##
[0063] To a vigorously stirred mixture of
(3S,5R)-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(300 mg, 1.56 mmol), Na.sub.2CO.sub.3 (500 mg, 4.68 mmol, 3.0
equiv.) in anhydrous dichloromethane (30 mL) was added acetyl
chloride (0.17 mL, 2.34 mmol, 1.5 equiv.) dropwise over 1 min. The
resulting solution was stirred for 15 minutes under an atmosphere
of nitrogen. The reaction was quenched by the addition of saturated
Na.sub.2CO.sub.3 (20 mL), the aqueous phase was extracted with
diethyl diethyl ether (3.times.10 mL) and the combined extracts
were dried over MgSO.sub.4. The solvents were removed in vacuo and
the crude material was purified by flash column chromatography on
silica, eluting with diethyl ether and dichloromethane (9:1) to
furnish the title compound as fine colourless needles (270 mg,
74%); m.p. 82-85.degree. C.; V.sub.(max) (KBr) 2943 (C--H), 1733
(C.dbd.O, lactone), 1647 (C.dbd.O, amide) cm.sup.''1; .delta..sub.H
(250 MHz, DMSO-d) 7.42-7.36 (5H, m, Ph), 5.49 (0.4H,
PhCH.times.0.4) 5.33 (0.6H, PhCH.times.0.6), 5.08-5.05 (1H, m,
CH.sub.3CH), 4.65 (2H, d, J=6.10 Hz, CH.sub.2); 2.13 (1H, s,
CH.sub.3CON.times.1), 1.86 (2H, s, CH.sub.3CON.times.2), 1.39 (3H,
d, J=12.40 Hz CH.sub.3CH); .delta..sub.c (62.5 MHz, DMSO--O 170.5,
169.8, 137.8, 129.4, 128.4, 127.1, 68.6, 55.5, 50.7, 49.7, 22.7,
19.0; .sup.mI.sub..tau. (CL, NH.sub.3), 234 (MH.sup.+, 8%), 233 (M,
13%), 220 (4%), and 219 (100%); HRMS for C.sub.13H.sub.16NO.sub.3
requires 234.1126. found 234.1130; [.alpha.].sub.D.sup.20-29.8 (c
1.16 CHCl.sub.3).
[0064] N-Fmoc-L-alanine Acid Chloride
##STR00027##
[0065] To a solution of N-Fmoc-L-alanine (2.00 g, 6.4 mmol, 1.0
equiv.) in anhydrous dichloromethane (40 mL) was added thionyl
dichloride (4.70 mL, 64 mmol, 10 equiv.). The resulting mixture was
refluxed for 2 hours under an atmosphere of nitrogen. The solvent
and excess of thionyl chloride were removed in vacuo and the crude
N-Fmoc-L-alanine acid chloride was partially purified by
recrystallization from dichloromethane and hexane (1.74 g, 86%);
m.p. 88-90.degree. C. (lit. m.p. 112-114.degree. C.); v.sub.(max)
(KBr) 3328 (N--H). 3040 (C--H), 1778 (C.dbd.O, chloride), 1694
(C.dbd.O,carbamate), cm.sup.1; .delta..sub.H (250 MHz. CDCl.sub.3)
7.79-7.29 (8H, m. Fmoc). 5.22 (1H, d. J 8.0 Hz, NH), 4.67-4.38 (3H,
m, CH.sub.3CH.times.1, CHCH.sub.2.times.2), 4.23 (1H, t, /6.52 Hz,
CHCH.sub.2); 1.55 (3H, d, J=7.28 Hz, CH.sub.3CH); 8, (62.5 MHz,
CDCl.sub.3) 176.8, 158.2, 143.9, 141.7, 128.2, 127.5, 125.3, 120.5,
67.7, 59.1, 47.5, 17.7; .sup.mI.sub.z (CL, NH.sub.3), 330
(MH.sup.+, 48%), 258 (20%), 197 (65%), 154 (100%), 95 (20%), and 72
(12%); HRMS for C.sub.18H.sub.17ClNO.sub.3 requires 330.1595 found
330.1592. [.alpha.].sub.D.sup.20+8.50 (c 1.30 CHCl.sub.3) (lit.
[.alpha.].sub.D.sup.24+4.03 (c 1.00 CH.sub.2Cl.sub.2)).
(3S,5R)--N--[N-Fmoc-(S)alanyl]-3-methyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,-
4-oxazin-2-one
##STR00028##
[0067] To a vigorously stirred mixture of
(3S,5R)-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(600 mg, 3.14 mmol), Na.sub.2CO.sub.3 (1.70 g. 15.70 mmol, 5.0
equiv.) in 1:1 dichloromethane and water (40 mL) was added
N-Fmoc-L-alanine acid chloride (1.26 g, 3.84 mmol, 1.2 equiv.) in
dichloromethane (10 mL) dropwise over 5 min. The resulting solution
was stirred for 2 hours. The aqueous phase was extracted with
dichloromethane (3.times.15 mL). The combined extracts were washed
with saturated Na.sub.2CO.sub.3 (50 mL), water (2.times.30 mL),
brine (50 mL) and dried over MgSO.sub.4. The solvents were removed
in vacuo and the crude material was purified by flash column
chromatography on silica, eluting with petrol and diethyl ether
(1:4) to furnish the title compound as fine colorless needles (1.21
g, 80%); m.p. 89-90.degree. C.; V.sub.(max) (KBr) 3321 (N--H).sub.5
2983 (C--H), 1741 (C.dbd.O, lactone), 1718 (C.dbd.O, carbamate),
1654 (C.dbd.O, amide) cm.sup.''1; .delta..sub.H (250 MHz, DMSO-d)
7.91-7.27 (13H, m, Fmoc.times.8, Ph.times.5), 5.50 (1H, br, NH),
5.07 (1H, m, PhCH), 4.90 (1H, m, NCHCH.sub.3); 4.77 (1H, d, 77.0
Hz, Cc-PhCHCH.sub.2), 4.57 (2H, m, CHCH.sub.3NH.times.1,
.beta.-PhCHCH.sub.2.times.1), 4.26 (1H, m, OCH.sub.2CH), 4.19 (2H,
m, OCH.sub.2CH), 1.28 (3H, d, /4.O Hz.sub.5 NCHCH.sub.3), 1.10 (3H,
d, /4.0 Hz, CHCH.sub.3NH); .delta..sub.c (62.5 MHz, DMSO-d) 172.0,
170.0, 156.1, 144.2, 141.1, 136.2, 128.9, 128.3, 127.9, 127.4,
126.7, 125.7, 120.4, 66.0, 65.3, 53.0, 50.7, 47.8, 46.9, 18.6,
17.8; (Cl, NH.sub.3), 508 (MNa.sup.+, 6%), 502 (MNH.sub.4.sup.+,
45%), 487 (4%), and 485 (MH.sup.+, 100%); HRMS for
C.sub.29H.sub.29N.sub.2O.sub.5 requires 485.2069. found 485.2060;
[.alpha.].sub.D.sup.20-13.1 (c1.06 CHCl.sub.3).
(5S)-3-Methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one
##STR00029##
[0069] (S)-2-phenylglycinol (3.00 g, 21.9 mmol, 1.0 equiv.) and
ethyl pyruvate (2.67 mL, 24.1 mmol, 1.1 equiv.) were refluxed in
trifluoroethanol (50 mL) over activated 4 molecular sieves (8.00 g)
for 24 hours. Filtration through a short pad of Celite.RTM. and
removal of solvent from the filtrate in vacuo generated the crude
product which was purified by flash column chromatography on
silica, eluting with petrol and diethyl ether (7:3) to furnish the
title compound as a white solid (1.83 g, 44%); m.p.
70.0-71.0.degree. C. (lit 71.0-72.0.degree. C.); v.sub.(max) (KBr)
3007 (C--H), 1735 (C.dbd.O), 1640 (C.dbd.N) cm.sup.''1;
.delta..sub.H(250 MHz, CDCl.sub.3) 7.45-7.32 (5H, m, Ph), 4.88-4.81
(1H, m, PhCH), 4.56 (1H, dd, J=4.49 Hz, T 9.48 Hz, 6.beta.-H), 4.25
(1H, dd/13.01 Hz, JT 14.99 Hz, 6.alpha.-H), 2.41 (3H, s, CH.sub.3);
.delta..sub.c (62.5 MHz, CDCl.sub.3) 160.7, 155.9, 137.2, 129.4,
128.7, 127.5, 71.9, 60.1, 22.2; % (CL, NH.sub.3), 189 (M.sup.+,
25%), 159 (12%), 130 (24%), 104 (100%), 90 (21%), ! and 78 (6%);
HRMS for C.sub.11H.sub.nNO.sub.2 requires 189.0787 found 189.0782.
[.alpha.].sub.D.sup.20 253.0 (c 0.98 CHCl.sub.3) (the enantiomer
lit. [.alpha.].sub.D.sup.20-237.1 (c 1.11 CHCl.sub.3)).
(3R,5S)-3-Methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
##STR00030##
[0071] To a solution of
(5S)-3-methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one (1.70 g, 9.0
mmol, 1.0 equiv.) in anhydrous dichloromethane (60 mL) under an
atmosphere of nitrogen was added PtO.sub.2 (170 mg, 0.1 equiv.).
The mixture was consecutively degassed and purged three times with
hydrogen and then stirred for 5 hours under an atmosphere of
hydrogen. Filtration through a short pad of Celite.RTM. and removal
of solvent from the filtrate in vacuo yielded the crude product
which was purified by recrystallization dichloromethane. diethyl
diethyl ether and hexane to furnish the title compound as a white
needles (1.26 g, 74%); m.p. 81.0-82.0.degree. C. (lit. m.p.
81.0-82.0.degree. C.); v.sub.(max) (KBr) 3314 (N--H), 2981 (C--H),
1736 (C.dbd.O), cm.sup.''1; .delta..sub.H (250 MHz, CDCl.sub.3)
7.43-7.26 (5H, m, Ph),; 4.42-4.23 (3H, m, CHCH.sub.2), 3.88 (1H, q,
/6.76 Hz, CHCH.sub.3). 1.80 (1H, br, NH), 1.50 (3H, d, J 6.76 Hz,
CH.sub.3); .delta..sub.c (62.5 MHz, CDCl.sub.3) 170.7, 138.1,
129.3, 129.1, 127.5, 75.4, 58.2, 55.4, 19.0; .sup.mI.sub.z (CL,
NH.sub.3), 192 (MH.sup.+, 30%). 147 (68%), 132 (64%), 104 (100%),
and 91 (10%); HRMS for C.sub.11H.sub.13NO.sub.2 requires 192.1025
found 192.1019. [.alpha.].sub.D.sup.20+88.8 (c 0.96 CHCl.sub.3)
(lit. [.alpha.].sub.D.sup.20+92.3 (c 0.84 CHCl.sub.3)).
(3R,5S)--N--[N-Fmoc(S)alanyl]-3-methyl-5-phenyl-3,4,5,6tetrahydro-2H-1,4
oxazin-2-one
##STR00031##
[0073] To a vigorously stirred mixture of
(3R,5S)-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(500 mg, 2.62 mmol), Na.sub.2CO.sub.3 (1.40 g, 13.3 mmol, 5.0
equiv.) in 1:1 dichloromethane and water (40 mL) was added
N-Fmoc-L-alanine acid chloride (1.04 g, 3.14 mmol. 1.2 equiv.) in
dichloromethane (10 mL) dropwise over 5 min. The resulting solution
was stirred for 2 hours. The aqueous phase was extracted with
dichloromethane (3.times.15 mL). The combined extracts were washed
with saturated Na.sub.2CO.sub.3 (50 mL), water (2.times.30 mL brine
(50 mL) and dried over MgSO.sub.4. The solvents were removed in
vacuo and the crude material was purified by flash column
chromatography on silica, eluting with petrol and diethyl ether
(1:4) to furnish the title compound as fine colourless needles
(1.02 g, 80%); m.p. 87-88.degree. C.; v.sub.(max) (KBr) 3323
(N--H), 2982 (C--H), 1761 (C.dbd.O, lactone), 1717 (C.dbd.O,
carbamate), 1656 (C.dbd.O, amide) cm.sup.''1; .delta..sub.H (400
MHz, DMSO-d, 110.degree. C.) 7.84-7.29 (13H, m, Fmoc.times.8,
Ph.times.5), 7.08 (1H, br, NH), 5.54 (1H, t, /5.88 Hz, PhCH), 4.96
(1H, q, /7.11 Hz, NCHCH.sub.3); 4.68-4.60 (2H, m, PhCHCH.sub.2),
4.41-4.32 (3H, m, CHCH.sub.3NH.times.1, OCH.sub.2CH.times.2), 4.22
(1H, t, /6.72 Hz OCH.sub.2CH), 1.45 (3H, d, /7.15 Hz, NCHCH.sub.3),
1.08 (3H, d, /6.71 Hz, CHCH.sub.3NH); 6, (62.5 MHz, OMSO-d) 174.0,
172.8, 170.4, 169.6, 156.3, 144.1, 141.1, 137.7, 129.4, 128.7,
128.0, 127.4, 127.3, 127.1, 125.6, 120.5, 68.8, 66.0, 55.5, 55.3,
52.2, 51.4, 50.0, 47.2, 20.5, 18.6, 17.7, 17.1; .sup.mI.sub.z (C.I.
NH.sub.3), 485 (MH.sup.+, 12%), 431 (8%), 381 (7%), 281 (15%) and
149 (100%); HRMS for C.sub.29H.sub.29N.sub.2O.sub.5 requires
485.2069. found 485.2076; [.alpha.].sub.D.sup.20+22.2 (c 0.94
CHCl.sub.3).
(5R)-3-Isopropyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one
##STR00032##
[0075] (R)-2-phenylglycinol (4) (2.00 g, 14.6 mmol, 1.0 equiv.) and
ethyl 3-methyl-2-oxobtayrate (2.20 mL, 14.6 mmol, 1.0 equiv.) were
refluxed in trifluoroethanol (30 mL) over activated 4 molecular
sieves (7.0 g) for 24 hours. Filtration through a short pad of
Celite.RTM. and removal of solvent from the filtrate in vacuo
generated the crude product which was purified by flash column
chromatography on silica, eluting with petrol and diethyl ether
(4:1) to furnish the title compound as colourless oil (1.13 g,
36%); v.sub.(max) (film) 2965 (C--H), 1740 (C.dbd.O), 1638
(C.dbd.N) cm.sup.''1; .delta..sub.H (250 MHz, CDCl.sub.3) 7.45-7.32
(5H, m, Ph), 4.90-4.83 (1H, m, PhCH), 4.55 (1H, dd, J=4.4 Hz, T
11.5 Hz, 6.beta.-H), 4.13 (1H, dd/10.9 Hz, r 11.4 Hz, 6.alpha.-H),
3.32 (1H, m, CH(CH.sub.3).sub.2), 1.25 (3H, d, J=5.0 Hz,
CH(CH.sub.3).sub.2.times.3), 1.22 (3H, d, /5.0 Hz,
CH(CH.sub.3).sub.2.times.3); .delta..sub.c (62.5 MHz, CDCl.sub.3)
167.7, 155.7, 137.5, 129.3, 128.6, 127.4, 71.7, 59.7, 32.7, 20.7,
19.9; % (CL, NH.sub.3). 220 (100%), 218 (MH.sup.+, 17%), 217 (27%),
and 216 (25%); HRMS for C.sub.13H.sub.16NO.sub.2 requires 218.1177
found 218.1181. [.alpha.].sub.D.sup.20-207.5 (c 1.17
CHCl.sub.3).
(3S,5R)-3-Isopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(39)
##STR00033##
[0077] To a solution of
(5R)-3-isopropyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one (38)
(1.03 g, 4.74 mmol, 1.0 equiv.) in anhydrous dichloromethane (50
mL) under an atmosphere of nitrogen was added PtO.sub.2 (103 mg,
0.1 equiv.). The mixture was consecutively degassed and purged
three times with hydrogen and then stirred for 5 hours under an
atmosphere of hydrogen. Filtration through a short pad of
Celite.RTM. and removal of solvent from the filtrate in vacuo
yielded the crude product which was purified by flash column
chromatography on silica eluting with petrol and diethyl ether
(4:1) to furnish the title product as a waxy solid (662 mg, 64%);
m.p. 61.5-62.5.degree. C.; v.sub.(max) (KBr) 3326 (N--H), 2960
(C--H), 1733 (C.dbd.O), cm.sup.''1; .delta..sub.H (250 MHz,
CDCl.sub.3) 7.47-7.35 (5H, m, Ph),; 4.33-4.18 (3H, m,
PhCHCH.sub.2), 3.81 (1H, m, NHCH), 2.49, (1H, m,
CH(CH.sub.3).sub.2), 1.68 (1H, br, NH), 1.09 (3H, dd, J=6.75 Hz,
CH(CH.sub.3).sub.2.times.3), 1.05 (3H, dd, J 6.75,
CH(CH.sub.3).sub.2.times.3); .delta..sub.c (62.5 MHz, CDCl.sub.3)
170.3, 138.5, 129.3, 129.1, 127.6, 74.9, 64.2, 57.3, 32.0, 19.4,
17.4; % (CL, NH.sub.3), 237 (MNH.sub.4.sup.+, 5%), 220 (100%,
MH.sup.+), 219 (22%), and 216 (6%); HRMS for
Cl.sub.3H.sub.8NO.sub.2 requires 220.1333 found 220.1338.
[.alpha.].sub.D.sup.20-92.1 (c 1.45 CHCl.sub.3).
(35,5R)--N--[N-Fmoc-(S)alanyl]-3-isopropyl-5-.rho.henyl-3,4,5,6,-tetrahydr-
o-2H-1,4-oxazin-2-one
##STR00034##
[0078] Method 1:
[0079] To a solution of
(3S,5R)-3-isopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(150 mg, 0.68 mmol) in anhydrous dichloromethane (10 mL) was added
N-Fmoc-L-alanine acid chloride (276 mg, 0.82 mmol, 1.2 equiv.) in
anhydrous dichloromethane (5 mL). The resulting solution was
stirred for 24 hours. The solvent was removed in vacuo and the
crude material was purified by flash column chromatography on
silica, eluting with petrol and diethyl ether (2:3) to furnish the
title product as colourless fine needles (95 ma, 27%).
Method 2:
[0080] To a vigorously stirred solution of
(3S,5R)-3-w.sigma.propyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(200 mg. 0.91 mmol) and Na.sub.2CO.sub.3 (415 mg, 2.73 mmol, 3.0
equiv.) in anhydrous dichloromethane (15 mL) was added
N-Fmoc-L-alanine acid chloride (428 mg. 1.36 mmol, 1.5 equiv.) in
anhydrous dichloromethane (10 mL). The resulting mixture was
stirred under nitrogen for 1 hour. Filtration through a short pad
of Celite.RTM. and removal of solvent from the filtrate in vacuo
furnished the crude material which was purified by flash column
chromatography on silica, eluting with petrol and diethyl ether
(3:7) to furnish the title product as colourless fine needles (380
mg, 82%); m.p. 72-75.degree. C.; v.sub.(max) (KBr) 3402 (N--H),
2969 (C--H), 1760 (C.dbd.O, lactone), 1718 (C.dbd.O, carbamate),
1654 (C.dbd.O, amide) cm.sup.''1; .delta..sub.H (250 MHz,
CDCl.sub.3) 7.65-7.15 (13H, m, Fmoc.times.8, Ph.times.5), 5.46 (1H,
br, NH), 5.03-4.98 (1H, m, Phal), 4.75 (1H, d, /10.0 Hz
CHCH(CHs).sub.2), 4.32-4.13 (3H, m, PhCHCH, and OCH.sub.2CH),
4.07-4.02 (1H, m. CHCH.sub.3), 3.89-3.79 (2H, m, OCH.sub.2CH),
2.18-2.00 (1H, m, CH(CHg).sub.2), 1.21 (3H, d. J 5.5 Hz,
CH(CH.sub.3).sub.2.times.3), 1.19 (3H, d, /5.0 Hz, CHCH.sub.3),
1.02 (3H, d, J=6.5 Hz, CH(CH.sub.3).sub.2.times.3); 8, (62.5 MHz,
CDCl.sub.3) 174.1, 167.1, 153.7, 142.8, 140.2, 134.9, 128.8, 128.2,
127.2, 124.9, 124.1, 118.9, 66.3, 65.7, 55.2, 52.5, 47.5, 45.0,
31.6 20.1, 18.2, 17.8; .sup.mI.sub.z {CI., NH.sub.3), 535
(MNa.sup.+, 73%), 513 (MH.sup.+, 100%), 334 (11%), 333 (53%), 328
(23%), and 311 (19%); (CI.) FIRMS for
C.sub.31H.sub.33N.sub.2O.sub.5 requires 513.2381. found 513.2378.
[.alpha.].sub.D.sup.20-22.1 (c 1.05 CHCl.sub.3).
[0081] N-Fmoc-L-valine Acid Chloride
##STR00035##
[0082] To a solution of N-Fmoc-L-alanine (3.00 g, 8.7 mmol, 1.0
equiv.) in anhydrous dichloromethane (40 mL) was added thionyl
chloride (6.5 mL, 87 mmol, 10.0 equiv.). The resulting mixture was
refluxed for 2 hours under an atmosphere of nitrogen. The solvent
and excess of thionyl dichloride were removed in vacuo and the
crude N-Fmoc-L-alanine acid chloride was partially purified by
recrystallization from dichloromethane and hexane (2.50 g, 80%);
m.p. 75-79.degree. C. (lit. m.p. 111-112.degree. C.); v.sub.(max)
(K Br) 3317 (N--H), 2969 (C--H), 1788 (C.dbd.O, acid chloride),
1696 (C.dbd.O, carbamate), cm.sup.''1; .delta..sub.H (250 MHz,
CDCl.sub.3) 7.78-7.29 (8H, m, Fmoc), 5.20 (1H, d, J=9.5 Hz, NH),
4.55-4.33 (3H, m, CH.sub.3CH.times.1, CHCH.sub.2.times.2), 4.23
(1H, t, J=6.5 Hz, CHCH.sub.2); 2.40 (1H, m, CH(CH.sub.3).sub.2),
1.05 (3H, d, J 7.0 Hz, CH(CHg).sub.2.times.3), 0.95 (3H, d, /7.0
Hz, CH(CHb).sub.2.times.3); .delta..sub.c (62.5 MHz, CDCl.sub.3)
175.4, 157.1, 143.9, 141.8, 128.2, 127.5, 125.3, 120.5, 68.2, 67.7,
47.5, 30.3 19.7, 17.4; [.alpha.].sub.D.sup.20+13.2 (c 1.05
CHCl.sub.3) (lit. [.alpha.].sub.D.sup.24+5.5 (c 1.00
CH.sub.2Cl.sub.2)).
(3S,5R)--N--[N-Fmoc-(S)valinyl]-3-isopropyl-5-.rho.henyl-3,4,5,6,-tetrahyd-
ro-2H-1,4-oxazin-2-one
##STR00036##
[0083] Method 1:
[0084] To a solution of
(3S,5R)-3-wopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(200 mg, 0.91 mmol) in anhydrous dichloromethane (20 mL) was added
N-Fmoc-L-valine acid chloride (391 mg. 1.10 mmol. 1.2 equiv.) in
anhydrous dichloromethane (5 mL). The resulting solution was
stirred for 24 hours. The solvent was removed in vacuo and the
crude material was purified by flash column chromatography on
silica, eluting with petrol and diethyl ether (2:3) to furnish the
title product as colourless fine needles (80 mg, 16%).
Method 2:
[0085] To a vigorously stirred solution of
(3,S,5R)-3-isopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(200 mg, 0.91 mmol) and Na.sub.2CO.sub.3 (415 mg, 2.73 mmol, 3.0
equiv.) in anhydrous dichloromethane (15 mL) was added
N-Fmoc-L-valine acid chloride (533 mg, 1.36 mmol, 1.5 equiv.) in
anhydrous dichloromethane (10 mL). The resulting mixture was
stirred under nitrogen for 6 hours. Filtration through a short pad
of Celite.RTM. and removal of solvent from the filtrate in vacuo
furnished the crude material which was purified by flash column
chromatography on silica, eluting with petrol and diethyl ether
(1:1) to furnish the title product as colourless fine needles (260
mg, 52%); m.p. 75-78.degree. C.; v.sub.(max) (KBr) 3421 (N--H),
2966 (C--H), 1763 (C.dbd.O, lactone), 1718 (C.dbd.O, carbamate),
1654 (C.dbd.O, amide) cm.sup.''1; .delta..sub.H (400 MHz,
DMSO-J.sub.6, 120.degree. C.) 7.83-7.25 (13H, m, Fmoc.times.8,
Ph.times.5), 6.81 (1H, br, NH), 5.34 (1H, dd, /6.2 Hz, /' 10.8 Hz,
PhCH), 4.77 (1H, d, /9.5 Hz, NCH), 4.58 (1H, dd, J=6.2 Hz, /' 12.5
Hz, PhCHCH.sub.2.times.1), 4.47 (1H, dd, J=10.8 Hz, J' 12.4 Hz,
PhCHCH.sub.2.times.1), 4.32-4.28 (1H, m, CH.sub.3CH), 4.24-4.15
(3H, m. OCH.sub.2CH), 2.22-2.05 (2H, m,
CH(CH.sub.3).sub.2.times.2), 1.18 (3H, d, J 6.5 Hz,
CH(CH.sub.3).sub.2.times.3), 0.96 (3H, d, J=6.5 Hz, CHCH.sub.3),
0.84 (6H, t, J=7.0 Hz, CH(CH.sub.3).sub.2.times.6); .delta..sub.c
(62.5 MHz, CDCl.sub.3) 174.5, 168.5, 155.5, 144.2, 141.7, 130.2,
129.6, 128.1, 127.4, 126.7, 126.4, 125.5, 120.4, 68.0, 67.1, 61.3,
57.7 57.0, 47.4, 33.2, 32.6, 21.8, 20.1, 19.7, 18.4, 17.5; % (CL),
614 (100%). 576 (50%), 564 (9%), 541 (MH.sup.+), 519 (22%), and 503
(16%); HRMS for C.sub.33H.sub.37N.sub.2O.sub.5 requires 541.2692
found 541.2709. [.alpha.].sub.D.sup.20-28.5 (c 0.56
CHCl.sub.3).
[0086] Phenacyl N-tert-butoxycarboxyl-(S)-phenylalanate
##STR00037##
[0087] To a solution of potassium hydroxide (0.64 g, 11.31 mmol) in
methanol (15 mL) was added Boc-L-phenylalanine (3.00 g, 11.31
mmol). The resulting solution was stirred at room temperature for 3
hours. The solvent was removed and the crude material was dried in
vacuo to yield white powder which was subsequently added in
anhydrous N,N-dimethylformamide (15 mL) and treated with
2-bromoacetophenone (2.07 g, 13.57 mmol, 1.2 equiv.). The resulting
solution was stirred at room temperature under nitrogen for 24
hours and was quenched by addition of water (20 mL). The
precipitate can be either used as crude in next step or purified by
recrystallization from diethyl ether yield the title compound as
white fine needles (3.55 g, 82%); nip 140.0-141.0.degree. C.;
v.sub.(max) (KBr) 3395 (N--H), 2973 (C--H), 1757 (C.dbd.O, ester),
1715 (C.dbd.O, ketone), 1692 (C.dbd.O, carbamate) cm.sup.1;
.delta..sub.H (250 MHz, CDCl.sub.3) 7.93-7.24 (1OH, m, Ph), 5.50
(1H, d, /16.4 Hz, OCH.sub.2.times.1), 5.31 (1H, d, /16.4 Hz,
OCH.sub.2.times.1), 4.97 (1H, d, 8.1 Hz, NH), 4.75 (1H, dd, /7.2
Hz, r 6.1 Hz, CH), 3.36 (1H, dd, /5.4 Hz, r 14.1 Hz, PhCH, X 1),
3.14 (1H, dd, /7.1 Hz, /.sup.v 14.0 Hz, PhCH.sub.2.times.1), 1.40
(9H, s, f-butyl); .delta..sub.c (62.5 MHz, CDCl.sub.3) 191.9 172.0,
155.6, 136.5, 134.4, 129.9, 129.3, 129.0, 128.2, 127.4, 80.4, 66.8,
54.7, 38.6, 28.7. [.alpha.].sub.D.sup.20-7.9 (c 1.01
CHCl.sub.3).
(3S)-3-Benzyl-5-phenyl-3,6-dihydro-2H-1,4-oxazin-2-one
##STR00038##
[0089] To a suspension of phenacyl
N-tert-butoxycarboxyl-(S)-phenylalanate (3.51 g. 9.16 mmol) in
diethyl ether (150 mL) was added hydrogen bromide in acetic acid
(33% w/w, 4.8 mL, 27.5 mmol, 3.0 equiv.). The resulting mixture was
stirred under nitrogen for 3 hours during which time another
portion of diethyl ether (100 mL) was added. The solid was
separated by filtration through a sinter, washed with diethyl ether
(2.times.30 mL) and dried in vacuo to furnish the amino ester
hydrobromide which was subsequently dissolved in pH 5 acetate
buffer (100 mL, 0.2 M, prepared from 70 parts 0.2 M aqueous sodium
acetate and 30 parts 0.2 M aqueous acetic acid). The resulting
mixture was stirred under nitrogen for 12 hours during which time
yellow oil was formed. The precipitate can be either used as crude
in next step or purified by flash column chromatography on silica,
eluting with petrol and diethyl ether (3:2) to furnish the title
compound as white solid (1.56 g, 65%); v.sub.(max) (KBr) 2932
(C--H), 1751 (C.dbd.O), cm.sup.''1; m.p. 57.0-59.0.degree. C. (lit.
58.0-60.0.degree. C.).sup.63; .delta..sub.H (250 MHz, CDCl.sub.3)
7.90-7.18 (10H, m, Ph), 5.01 (1H, d, /14.2 Hz,
CHCH.sub.2O.times.1), 4.83 (H, m, CHCH.sub.2Ph), 4.06 (1H, m,
CHCH.sub.2), 4.11 (1H, d, J=14.2 Hz, CHCH.sub.2O.times.1), 3.46
(1H, dd, /5.45 Hz, /13.5 Hz, CHCH.sub.2Ph.times.1), 3.32 (1H, dd,
/5.45 Hz, /13.5 Hz, CHCH.sub.2Ph.times.1); .delta..sub.c (62.5 MHz,
CDCl.sub.3) 169.0, 162.8, 136.6, 134.7, 131.7, 130.6, 129.2, 128.8,
127.6, 126.3, 67.8, 61.0, 39.4; % (CL, NH.sub.3), 266 (32%), 265
(M.sup.+, 100%), 264 (42%), 263 (5%) and 262 (3%); HRMS for
C.sub.17H.sub.15NO.sub.2 requires 265.1103. found 265.1108.
[.alpha.].sub.D.sup.20+85.1 (c 1.04 CHCl.sub.3) (lit.
[.alpha.].sub.D.sup.20+85.7 (c 2.00 CHCl.sub.3)).
(3S,5R)-3-benzyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
##STR00039##
[0091] To a solution of (3.English
Pound.)-3-Benzyl-5-.rho.henyl-3,6-dihydro-2H-1,4-oxazin-2-one (1.56
g, 5.88 mmol, 1.0 equiv.) in anhydrous methanol (40 mL) under an
atmosphere of nitrogen was added palladium on activated carbon (156
mg, 0.1 equiv. by mass). The mixture was consecutively degassed and
purged three times with hydrogen and then stirred for 5 hours under
an atmosphere of hydrogen. Filtration through a short pad of
Celite.RTM. and removal of solvent from the filtrate in vacuo
yielded the crude product which was purified by flash column
chromatography on silica, eluting with petrol and diethyl ether
(4:1) to furnish the title product as a white solid (514 mg, 33%);
m.p. 75.0-76.0.degree. C. (lit. 76.0-78.0.degree. C.).sup.5;
v.sub.(max) (KBr) 3321 (N--H), 2949 (C--H), 1731 (C.dbd.O),
cm.sup.1; .delta..sub.H (250 MHz, CDCl.sub.3) 7.36-7.21 (1OH, m,
Ph), 4.34-4.1.5 (3H, m, PhCHCH.sub.2), 3.97 (1H, dd, J 3.2, r 10.0
Hz, NHCH), 3.56, (1H, dd, J 3.2, J.sup.v 13.6 Hz, CH Ph.times.1),
3.00, (1H, dd, /10.0, T 13.6 Hz, CH.sub.2Ph.times.1), 1.84 (1H, br,
NH); .delta..sub.c (62.5 MHz, CDCl.sub.3) 169.6, 138.0, 137.8,
129.8, 129.3, 129.2, 129.1, 127.5, 127.4, 75.3, 60.6, 57.9, 39.4;
.sup.mI.sub.z (CL, NH.sub.3), 268 (MH.sup.+, 40%), 267 (80%,
M.sup.+), 223 (100%), and 209 (57%); HRMS for
C.sub.17H.sub.17NO.sub.2 requires 267.1259 found 267.1263.
[.alpha.].sub.D.sup.20-156.4 (c 1.02 CHCl.sub.3) (lit.
[.alpha.].sub.D.sup.20-157.9 (c 2.00 CHCl.sub.3)).
(3S,5R)--N--[N-Fmoc-(S)-alanyl]-3-benzyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1-
,4-oxazin-2-one
##STR00040##
[0093] To a vigorously stirred solution of
(3S,5R)-3-benzyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(200 mg, 0.75 mmol) and Na.sub.3CO.sub.3 (239 mg, 2.25 mmol, 3.0
equiv.) in anhydrous dichloromethane (20 mL) was added
N-Fmoc-L-alanine acid chloride (354 mg, 1.12 mmol, 1.5 equiv.) in
anhydrous dichloromethane (6 mL). The resulting mixture was stirred
under nitrogen for 4 hours. Filtration through a short pad of
Celite.RTM. and removal of solvent from the filtrate in vacuo
furnished the crude product which was purified by flash column
chromatography on silica, eluting with petrol and diethyl ether
(2:3) to furnish the title product as colourless fine needles (150
mg, 36%); m.p. 87-89.degree. C.; v.sub.(max) (KBr) 3413 (N--H),
2981 (C--H), 1760 (C.dbd.O, lactone), 1718 (C.dbd.O, carbamate),
1656 (C.dbd.O, amide) cm.sup.-1; .delta..sub.H (400 MHz, DMSO-J,
100.degree. C.) 7.90-7.27 (18H, m, Fmoc.times.8, Ph.times.10), 7.07
(1H, br, NH), 5.47 (1H, dd, /4.8 Hz, /' 7.6 Hz, PhCH), 5.32 (1H, t,
J 7.2 Hz, NCH), 4.82 (1H, dd, /7.6 Hz, J' 12.4 Hz,
PhCHCH.sub.2.times.1). 4.71 (1H, dd, J 12 Hz, J' 12.4 Hz,
PhCHCH.sub.2.times.1). 4.41 (1H, q, J 6.4 Hz, CH.sub.3CH),
4.37-4.21 (3H, m, OCH.sub.2CH), 3.15 (2H, d, /6.8 Hz, CH.sub.2Ph),
1.21 (3H, d, /6.8 Hz, CH.sub.3); 6, (62.5 MHz, CDCl.sub.3) 174.8,
170.8, 168.8, 159.6, 155.0, 142.7, 140.2, 128.9, 128.5, 127.9,
127.6, 127.4, 126.9, 126.6, 126.1, 124.2, 119.0, 662, 62.1, 61.0,
52.5, 47.8, 45.9, 33.8, 15.6; .sup.mI.sub.z (CL), 560 (M.sup.+),
502 (100%), 464 (43%), 426 (19%), and 414 (83%); HRMS for
C.sub.35H.sub.32N.sub.2O.sub.5 requires 560.2303 found 560.2293.
[.alpha.].sub.D.sup.20-56.2 (c 1.01 CHCl.sub.3).
(3S,5R)--N--[N-Fmoc-(S)-alanyl]-3-tert-butyl-5-phenyl-3,4,5,6,-tetrahydro-
-2H-1,4-oxazin-2-one
##STR00041##
[0094] To a vigorously stirred solution of
(3S,5R)-3-fe/t-butyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(150 mg, 0.63 mmol) in anhydrous dichloromethane (15 mL) and
Na.sub.2CO.sub.3 (341 mg, 3.22 mmol, 5.0 equiv.) was added
N-Fmoc-L-alanine acid chloride (298 mg, 0.95 mmol, 1.5 equiv.). The
resulting mixture was stirred under an atmosphere of nitrogen for
18 hours. Filtration through a short pad of Celite.RTM. and removal
of solvent from the filtrate in vacuo gave the crude material which
was purified by flash column chromatography on silica, eluting with
petrol and diethyl ether (1:1) to furnish the title product as
colourless fine needles (170 mg, 51%); m.p. 74-75.degree. C.;
v.sub.(max) (KBr) 2976 (C--H). 1752 (C.dbd.O, lactone), 1724
(C.dbd.O, carbamate), 1662 (C.dbd.O, amide) cm.sup.''1;
.delta..sub.H (250 MHz, DMSO- .sub.6) 7.90-7.29 (13H, m,
Fmoc.times.8. Ph.times.5), 5.39 (1H, br, PhCH), 4.62 (2H, br,
CH.sub.2O), 4.33 (1H, br, CH.sub.3CH), 4.18 (3H, br, OCH.sub.2CH),
1.23 (3H, d, /6.70 Hz, CHCH.sub.3), 0.88 (9H, s, QCH );
.delta..sub.c (62.5 MHz, DMSO- .sub.6) 175.9, 168.1, 155.8, 144.2,
144.1, 141.0, 137.1, 129.0, 128.0, 127.4, 125.8, 125.7, 120.4,
66.0, 62.2, 55.3, 53.0, 48.7, 46.9, 37.0, 28.6, 17.4; .sup.mI.sub.z
(CL); 528 (15%), 527 (MH.sup.+, 34%), 526 (M, 100%), 470 (62%), and
414 (74%); FIRMS for C.sub.32H.sub.34N.sub.2O.sub.5 requires
526.2459 found 526.2457. [.alpha.].sub.D.sup.20-10.9 (c 1.20
CHCl.sub.3).
[0095]
(3S,5R)--N--[N-Fmoc-(S)valinyl]-3-tert-butyl-5-phenyl-3,4,5,6,-tetr-
ahydro-2H-1, 4-oxazin-2-one
##STR00042##
[0096] To a vigorously stirred solution of
(3S,5R)-3-tert-butyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(75 mg, 0.32 mmol) in anhydrous dichloromethane (7 mL) and
Na.sub.2CO.sub.3 (170 mg, 1.61 mmol, 5.0 equiv.) was added
N-Fmoc-L- valine acid chloride (170 mg, 0.48 mmol, 1.5 equiv.). The
resulting mixture was stirred under an atmosphere of nitrogen for
48 hours. Filtration through a short pad of Celite.RTM. and removal
of solvent from the filtrate in vacuo gave the crude material which
was purified by flash column chromatography on silica, eluting with
petrol and diethyl ether (1:1) to furnish the title product as
colourless fine needles (32 mg, 18%); m.p. 71-73.degree. C.;
v.sub.(max) (KBr) 2964 (C--H), 1752 (C.dbd.O, lactone), 1734
(C.dbd.O, carbamate), 1655 (C.dbd.O, amide) cm.sup.''1;
.delta..sub.H (250 MHz, CDCl.sub.3) 7.77-7.25 (13H, m,
Fmoc.times.8, Ph.times.5), 5.30 (1H, br, (CH.sub.3).sub.2CHCH),
5.13 (1H, t, J 10.0 Hz, PhCH), 4.52-4.38 (2H, m, PhCHCH.sub.2O),
4.29-4.21 (1H, m, OCH.sub.2CH), 4.15 (3H, br, OCH.sub.2CH.times.2
and CHC(CH.sub.3).sub.3), 1.99-1.96 (1H, m, (CH.sub.3).sub.2CH),
1.22 (9H, s, C(CH.sub.3).sub.3); 0.97-0.84 (6H, m,
(CH.sub.3).sub.2CH); .delta..sub.c (62.5 MHz, CDCl.sub.3) 175.9,
168.4, 155.4, 144.2, 141.7, 136.5, 130.2, 129.2, 128.1, 127.6,
126.4, 125.5, 120.4, 68.3, 67.1, 64.3, 57.6, 56.5, 47.5, 38.0,
32.7, 30.7, 20.2, 17.4; .sup.m/.sub.z (CL), 554 (MH.sup.+, 14%),
502 (100%), 464 (40%), 426 (20%), and 414 (74%); FIRMS for
C.sub.34H.sub.38N.sub.2O.sub.5 requires 554.2781 found 554.2790.
[.alpha.].sub.D.sup.20-17.5 (c 1.48 CHCl.sub.3).
[0097]
N-t-Boc-(S)-alanyl-N-(1-phenyl-2-hydroxylethyl)glycyl-(S)-alanine
tert-butyl ester
##STR00043##
[0098] To a suspension of L-alanine i-butyl ester hydrochloride
(408 mg, 2.3 mmol) and
(5S)-4-[N-?-Boc-(S)-alanyl]-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-o-
ne (0.78 g, 2.3 mmol) in anhydrous dioxane (20 mL) was added
distilled triethylamine (0.32 mL, 2.3 mmol). The resulting mixture
was stirred at room temperature vigorously for 5 days. Water (20
mL) was added and the mixture was extracted with diethyl ether
(3.times.25 mL). The combined organic extracts were washed with
brine (50 mL) and dried over MgSO.sub.4. The solvents were removed
in vacuo and the crude product was purified by flash column
chromatography on silica, eluting with petrol, diethyl ether, and
methanol (10:10:1) to furnish the title compound as fine colourless
needles (0.67 g, 59%); mp 69.0-70.0.degree. C.; v.sub.(max) (KBr)
3312 (O--H), 2980 (C--H), 1738 (C.dbd.O, lactone), 1701 (C.dbd.O,
carbamate), 1654 (C.dbd.O, amide) cm.sup.''1; .delta..sub.H (250
MHz, DMSO--O 8.69 (1H, d, J 7.0 Hz, NH), 7.39 (1H, d, J 7.0 Hz,
NH), 7.40-7.26 (5H, m, Ph), 5.80-5.74 (1H, m, PhCH), 5.07-5.02 (1H,
m, OH), 4.69 (1H, d, J 14.0 Hz, NCH.sub.2.times.1), 4.13-4.04 (2H,
m, CH.sub.3CH.times.2), 3.96-3.91 (1H, m, HOCH.sub.2), 3.77-3.74
(1H, m, HOCH.sub.2), 3.63 (1H, d, J 18.0 Hz, NCH.sub.2.times.1),
1.43 (18H, s, f-butyl.times.2), 1.23 (3H, d, J 13.0 Hz, CH.sub.3),
1.12 (3H, d, J 13.0 Hz.sub.5CH.sub.3); .delta..sub.c (62.5 MHz,
DMSO-J) 176.2, 172.0, 170.7, 156.1, 138.3, 129.0, 128.5, 127.5,
80.9, 78.6, 60.2, 57.5, 49.0, 46.7, 28.5, 27.9, 17.4, 16.9; % (CL,
NH.sub.3), 494 (MH.sup.+, 8%), 476 (6%), 376 (35%), 293 (21%), 249
(46%), and 44 (100%); HRMS for C.sub.25H.sub.40N.sub.3O.sub.7
requires 494.2856. found 494.2872. [.alpha.].sub.D.sup.20+24.3 (c
1.09 CHCl.sub.3).
[0099] L-Alanine tert-butyl ester
##STR00044##
[0100] To a mixture of sodium carbonate (875 mg, 8.25 mmol, 5.0
equiv.) in deioned water (10 mL) and diethyl ether (10 mL) was
added L-alanine tert-butyl ester hydrochloride (300 mg, 1.65 mmol,
1.0 equiv.). The resulting solution was stirred under an atmosphere
of nitrogen for 1 hour. The aqueous phase was extracted with
diethyl ether (3.times.10 mL) and the combined extracts were dried
over MgSO.sub.4. The solvent was removed in vacuo to settle the
title compounds as a colourless oil (200 mg, 84%); v.sub.(max)
(film) 3377 (N--H), 2978 (C--H), 1729 (C.dbd.O) cm.sup.''1;
.delta..sub.H(250 MHz, CDCl.sub.3) 3.42 (1H, q, J 7.02 Hz CH). 1.63
(2H, br, NH.sub.2), 1.46 (9H, s, (CH.sub.3).sub.3), 1.29 (3H, d, J
7.02 Hz, CH.sub.3CH); .delta..sub.c (62.5 MHz, CDCl.sub.3) 177.1,
81.2, 51.0, 28.4, 21.2; [.alpha.].sub.D.sup.20+3.7 (c 0.97
CHCl.sub.3), (lit. [.alpha.].sub.D.sup.24+2.3 (c 1.00
CHCl.sub.3)).
N-Fmoc-(S)-alanyl-N-((1R)-.rho.henyl-2-hydroxylethyl)-(S)-alanyl-(S)-alani-
ne tert-butyl ester
##STR00045##
[0101] Method 1:
[0102]
(3S,5R)--N--[N-Fmoc-(S)alanyl]-3-methyl-5-phenyl-3,4,5,6,-tetrahydr-
o-2H-1,4-oxazin-2-one (300 mg, 0.62 mmol) and L-alanine tert-butyl
ester (135 mg, 0.93 mmol, 1.5 equiv.) were dissolved in anhydrous
dichloromethane (14 mL) in a doubly sealed PTFE cylinder and
subjected to ultra-high pressure of 19 kbar for 48 hours. After
releasing the pressure, the solution was filtered through a short
pad of Celite.RTM. and removal of solvent from the filtrate in
vacuo yielded the crude product which was purified by flash column
chromatography on silica, eluting with diethyl ether and
dichloromethane (4:1) to furnish the starting material (33) (40 mg,
13%) and the title product as fine colourless needles (122 mg,
31%).
Method 2:
[0103] To a solution of L-alanine tert-butyl ester (466 mg, 3.21
mmol, 3.0 equvi.) in anhydrous dichloromethane (30 mL) was added
trimethyl aluminium (1.87 mL, 3.75 mmol, 2 M in hexane, 3.5 equiv.)
under an atmosphere of nitrogen. After 15 minutes,
(3S,5R)--N--[N-Fmoc-(S)alanyl]-3-methyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1-
,4-oxazin-2-one (520 mg, 1.07 mmol) in anhydrous dichloromethane
(10 mL) was added. The resulting solution was stirred at room
temperature for 24 hours. The reaction was quenched by the addition
of water (10 mL) and the organic phase was then washed with
saturated copper sulphate (20 mL). The aqueous phase was extracted
with diethyl ether (3.times.20 mL) and the combined organic
extracts were washed with brine (50 mL) and dried over MgSO.sub.4.
The solvents were removed in vacuo and the crude product was
purified by flash column chromatography on silica, eluting with
diethyl ether and dichloromethane (4:1) to furnish the title
compound as fine colourless needles (497 mg, 74%); m.p.
69-71.degree. C.; v.sub.(max) (KBr) 3409 (O--H), 2979 (C--H), 1733
(C.dbd.O, lactone), 1718 (C.dbd.O, carbamate), 1646 (C.dbd.O,
amide) cm.sup.''1; .delta..sub.H (250 MHz, OMSO-d) 7.96-7.34 (13H,
m, Fmoc.times.8 and Ph.times.5), 7.90 (1H, d, /7.41 Hz, NH), 6.10
(1H, s, OH), 5.41-5.33 (1H, m, PhCH), 4.86 (1H, q, /6.76 Hz,
CHCH.sub.3), 4.40-4.24 (3H, m, OCH.sub.2CH), 4.16-4.07 (2H, m,
CH.sub.2OH), 3.79 (1H, q, /6.95 Hz, CHCH.sub.3), 3.63 (1171, q,
/6.65 Hz, CHCH.sub.3), 1.44 (3H, d, /6.12 Hz, CHCH.sub.3), 1.40
(3H, d, /7.19 Hz, CHCH.sub.3), 1.27 (9H, s, C(CHg).sub.3), 0.90
(3H, d, /7.42 Hz, CHCH.sub.3); .delta..sub.c (62.5 MHz, OMSO-d)
173.5, 171.3, 170.3, 156.6, 144.1, 141.1, 137.6, 129.1, 128.7,
128.0, 127.6, 125.6, 121.7, 120.5, 80.2, 66.2, 60.9, 60.2, 48.7,
47.4, 46.9, 27.8, 17.4, 17.1, 14.9; % (CL, NH.sub.3), 775 (100%),
630 (MH.sup.+, 25%), 485 (34%), and 408 (17%); HRMS for
C.sub.36H.sub.44N.sub.3O.sub.7 requires 630.3170. found 630.3166;
[.alpha.].sub.D.sup.20-41.2 (c 1.08 CHCl.sub.3).
N-Acetyl-N-(1-phenyl-2-hydroxylethyl)alanyl-(S)-alanine tert-butyl
ester
##STR00046##
[0105]
(3S,5R)-4-N-acetyl-3-methyl-5-.rho.henyl-3,4,5,6,-tetrahydro-2H-1,4-
-oxazin-2-one (68) (220 mg, 0.94 mmol) and L-alanine tert-butyl
ester (66) (205 mg, 1.42 mmol, 1.5 equiv.) were dissolved in
anhydrous dichloromethane (14 mL) in a doubly sealed PTFE cylinder
and subjected to ultra-high pressure of 19 kbar for 48 hours. After
releasing the pressure, the solution was filtered through a short
pad of Celite.RTM. and removal of solvent from the filtrate in
vacuo yielded the crude product which was purified by flash column
chromatography on silica eluting with diethyl ether and
dichloromethane (4:1) to furnish the title product as fine
colourless needles (120 mg, 34%); m.p. 38-40.degree. C.;
v.sub.(max) (KBr) 3411 (O--H), 2980 (C--H), 1735 (C.dbd.O,
lactone), 1646 (C.dbd.O, amide) cm.sup.''1; .delta..sub.H(250 MHz,
OMSO-d) 8.06 (0.5H, d, /6.95 Hz, NH.times.0.5), 7.55-7.24 (5H, m,
Ph), 6.54 (0.5H, d, J 6.38 Hz, NH.times.0.5), 5.82 (0.5H, t, /4.88
Hz, OH.times.0.5), 5.14-5.06 (1H, m, PhCH), 4.83 (0.5H, t, J 6.56
Hz, OH.times.0.5), 4.35 (0.5H, q, J 6.99 Hz, CH.sub.3CH.times.0.5),
4.08-4.03 (2H, m, CH.sub.2OH), 3.98-3.76 (1.5H, m,
CHCH.sub.3.times.0.5 and CHCH.sub.3.times.1), 2.23 (1.5H, s,
COCH.sub.3), 2.14 (1.5H, s, COCH.sub.3), 1.46-1.39 (12H, m,
CHCH.sub.3.times.3 and C(CH.sub.3).sub.3.times.9), 1.19 (1.5H, d,
/7.15 Hz, CHCH.sub.3.times.1.5), 0.97 (1.5H, d, J 7.23 Hz,
CHCH.sub.3.times.1.5); .delta..sub.c (62.5 MHz, DMSO-d) 171.8,
171.7, 171.0, 170.8, 170.2, 140.0, 138.2, 129.1, 128.2, 128.0,
126.8, 80.7, 62.7, 62.5, 60.8, 56.3, 53.4, 48.8, 48.7, 27.9, 17.5,
16.9, 16.7, 15.2; % (CL, NH.sub.3), 379 (MH.sup.+, 70%), 361 (18%),
305 (20%), 249 (38%), 234 (100%), and 219 (40%); FIRMS for
C.sub.20H.sub.31N.sub.2O.sub.5 requires 379.2225 found 379.2240;
[.alpha.].sub.D.sup.20-37.0 (c 1.06 CHCl.sub.3).
N-Fmoc-(S)-alanyl-N-((1S)-.rho.henyl-2-hydroxylethyl)-(R)-alanyl-(S)-alani-
ne tent-butyl ester
##STR00047##
[0107] To a solution of L-alanine tert-butyl ester (251 mg, 1.73
mmol, 3.0 equiv.) in anhydrous dichloromethane (25 mL) was added
trimethyl aluminium (2.03 mL, 2.03 mmol, 2 M in hexane, 3.5 equiv.)
under an atmosphere of nitrogen. After 15 minutes,
(3R,5S)--N--[N-Fmoc-(S)alanyl]-3-methyl-5-.rho.henyl.about.3,4,5,6,-tetra-
hydro-2H-1,4-oxazin-2-one (280 mg, 0.58 mmol) in anhydrous
dichloromethane (8 mL) was added. The resulting solution was
stirred at room temperature for 24 hours. The reaction was quenched
by the addition of water (7 mL) and the organic phase was then
washed with saturated copper sulphate (15 mL). The aqueous phase
was extracted with diethyl ether (3.times.20 mL) and the combined
organic extracts were dried over MgSO.sub.4. The solvent was
removed in vacuo and the crude product was purified by flash column
chromatography on silica, eluting with petrol and diethyl ether
(1:4) to furnish the title compound as fine colourless needles (226
mg, 62%); m.p. 87-88.degree. C.; v.sub.(max) (KBr) 3410 (O--H),
2980 (C--H), 1727 (C.dbd.O). 1654 (C.dbd.O) cm.sup.''1;
.delta..sub.H (250 MHz, DMSO-d), 8.35 (0.5H, d, /6.18 Hz, NH)
7.91-7.12 (13H, m, Fmoc.times.8 and Ph.times.5), 7.65 (0.51-1, d,
/7.14 Hz, NH), 7.55 (0.5H, d, /7.32 Hz, NH). 6.39 (0.5H, d, /7.45
Hz, NH), 5.21-5.18 (0.5H, m, PhCH), 5.03-5.01 (0.5H, m, PhCH),
4.94-4.92 (0.5H, m, NHCHCH.sub.3), 4.80-4.78 (0.5H, m,
NHCHCH.sub.3), 4.65-4.62 (0.5H, m, NCHCH.sub.3), 4.27-4.18 (3.5H,
m, OCH.sub.2CH, and Cl.sub.2O.times.0.5), 4.09-3.93 (2.51-1, in,
CH.sub.2OH.times.1.5 and NHCHCH.sub.3), 3.62-3.60 (0.5H, in,
NCHCH.sub.3.times.1), 1.44-1.36 (13.5H, s, (CEb).sub.3.times.9,
CHCH.sub.3.times.4.5), 1.23 (1.5H, d, /6.63 Hz, CHCH.sub.3), 1.13
(1.51-1, d, /7.35 Hz, CHCH.sub.3), 0.92 (1.5H, d, /7.07 Hz,
CHCH.sub.3); .delta..sub.c (62.5 MHz, OMSO-d) 173.4, 172.9, 171.9,
171.6, 169.7, 156.0, 144.2, 144.1, 141.1, 137.2, 129.0, 128.3,
128.0, 127.7, 127.4, 127.3, 125.7, 120.5, 81.2, 80.8, 66.1, 61.6,
55.3, 53.6, 48.6, 48.3, 47.5, 47.0, 27.9, 18.5, 18.2, 17.7, 17.1,
15.4; .sup.mI.sub.z (CL, NH.sub.3), 775 (100%), 629 (M.sup.+, 36%),
457 (58%), 345 (27%), 231 (33%) and 178 (100%); HRMS for
C.sub.36H.sub.43N.sub.3O.sub.7 requires 629.3092. found 629.3093;
[.alpha.].sub.D.sup.20+14.20 (c 1.15 CHCl.sub.3).
[0108] L-Valine tort-butyl ester
##STR00048##
[0109] To a mixture of sodium carbonate (1.20 g, 9.54 mmol, 5.0
equiv.) in deioned water (20 mL) and diethyl ether (20 mL) was
added L-valine tert-butyl ester hydrochloride (400 mg, 1.71 mmol,
1.0 equiv.). The resulting solution was stirred for 2 hours under
an atmosphere of nitrogen. The aqueous phase was extracted with
diethyl ether (3.times.15 mL) and the combined extracts were dried
over MgSO.sub.4. The solvent was removed in vacuo to settle the
title compound as colourless oil (320 mg, 97%); v.sub.(max) (film)
3393 (N--H), 2967 (C--H), 1728 (C.dbd.O) cm.sup.''1; .delta..sub.H
(250 MHz, CDCl.sub.3) 3.16 (1H, d, J 4.79 Hz, CHNH.sub.2),
2.04-1.97 (1H, m, CH(CH.sub.3).sub.2) 1.47 (9H, s,
(CH.sub.3).sub.3), 1.38 (2H, s, NH.sub.2), 0.97 (3H, d, /6.88 Hz,
(CH)CH.times.3), 0.90 (3H, d, J 6.86 Hz,
(CH.sub.3).sub.2CH.times.3); .delta..sub.c (62.5 MHz, CDCl.sub.3)
175.3, 81.2, 60.7 32.6, 28.5, 19.7, 17.4;
[.alpha.].sub.D.sup.20+26.4 (c CHCl.sub.3), (lit.
[.alpha.].sub.D.sup.24+25.3 (c 1.00 CHCl.sub.3)).
N-Fmoc-(S)-a\any\-N-(1-phenyl-2-hydroxylethyl)-(5)-alanyl-(5)-valine
tert-butyl ester (74)
##STR00049##
[0111] To a solution of L-valine tert-huiyl ester (161 mg, 0.93
mmol, 3.0 equiv.) in anhydrous dichloromethane (10 mL) was added
trimethyl aluminium (0.55 mL, 1.09 mmol, 2 M in hexane, 3.5 equiv.)
under an atmosphere of nitrogen. After 15 minutes,
(3S,5R)--N(N-Fmoc-(S)alanyl]-3-methyl-5-.rho.henyl-3,4,5,6,-tetrahydro-2H-
-1,4-oxazin-2-one (150 mg, 0.31 mmol) in anhydrous dichloromethane
(5 mL) was added. The resulting solution was stirred at room
temperature for 24 hours. The reaction was quenched by the addition
of saturated copper sulphate (20 mL) and the mixture was extracted
with diethyl ether (3.times.20 mL). The combined organic extracts
were dried over MgSO.sub.4. The solvents were removed in vacuo and
the crude product was purified by flash column chromatography on
silica, eluting with diethyl ether and dichloromethane (9:1) to
furnish the title compound as fine colourless needles (128 mg,
63%); m.p. 75.0-76.0.degree. C.; v.sub.9max) (KBr) 3411 (O--H),
2973 (C--H), 1718 (C.dbd.O). 1654 (C.dbd.O) cm.sup.1; .delta..sub.H
(250 MHz, DMSO--O 7.96 d, /7.4 Hz, NH), 7.97-7.29 (13H, m,
Fmoc.times.8 and Ph.times.5), 5.43 (1H, br, OH), 5.35 (1H, t, /4.2
Hz, PhCH), 4.91 (1H, t, /6.8 Hz, CHCH.sub.3), 4.43-4.40 (1H, m,
OCH.sub.2CH), 4.31-4.21 (2H, m, OCH.sub.2CH), 4.20-4.05 (2H, m,
HOCH.sub.2), 3.79-3.71 (1H, m, CHCH(CH.sub.3).sub.2), 3.70-3.68
(1H, m, CHCH.sub.3), 1.81-1.76 (1H, m, CH(CH.sub.3).sub.2),
1.46-1.34 (6H, m, CHCH.sub.3.times.2) 1.31 (9H, s, f-butyl),
0.68-0.64 (6H, m, CH(CHg).sub.2); .delta..sub.c (62.5 MHz, OMSO-d)
173.1, 170.1, 170.0, 156.4, 144.2, 141.1, 137.6, 129.1, 128.7,
128.3, 128.0, 127.4, 125.7, 124.5, 80.5, 66.4, 63.2, 61.2, 58.1,
53.4, 47.1, 30.1, 27.9, 18.8, 18.4, 17.8, 15.1; .sup.mI.sub.z (CL,
NH.sub.3), 657 (M.sup.+, 34%), 579 (40%), 427 (59%), 363 (26%), and
244 (100%); FIRMS for C.sub.38H.sub.47N.sub.3O.sub.7 requires
657.3404. found 657.3398; [.alpha.].sub.D.sup.20-38.7 (c 0.96
CHCl.sub.3).
t-Butyl
N--[(S)-2-hydroxy-1-phenylethyl]-(5)-valinyl-(5)-alanate
##STR00050##
[0113] To a solution of L-alanine tert-butyl ester (180 mg, 1.24
mmol, 3.0 equiv.) in anhydrous dichloromethane (10 mL) was added
trimethyl aluminium (0.56 mL, 1.12 mmol, 2 M in hexane, 2.7 equiv.)
under an atmosphere of nitrogen. After 15 minutes.
(3S,5R)-3-wopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(91 mg, 0.41 mmol) in anhydrous dichloromethane (5 mL) was added.
The resulting solution was stirred at room temperature for 24
hours. The reaction was quenched by the addition of saturated
copper sulphate (15 mL) and the mixture extracted with diethyl
ether (3.times.15 mL). The combined organic extracts were dried
over MgSO.sub.4. The solvents were removed in vacuo and the crude
product was purified by flash column chromatography on silica,
eluting with petrol and diethyl ether (9:1 then 1:20) to furnish
the starting material (60 mg, 60%) and the title compound as light
yellow oil (50 mg, 33%); v.sub.(max) (film) 3328 (O--H), 2977
(C--H), 1733 (C.dbd.O, ester), 1651 (C.dbd.O, amide) cm.sup.''1;
.delta..sub.H (250 MHz, CDCl.sub.3) 7.73 (1H, d, J 8.6 Hz, NH),
7.30-7.18 (5H, m, Ph), 4.54-4.78 (1H, m, CHCH.sub.3), 3.74-3.68
(1H, m, PhCH), 3.59-3.50 (2H, m, HOCH.sub.2), 2.79 (1H, d, 74.8 Hz,
CHCH(CH.sub.3).sub.2), 1.97-1.93 (1H, m, CH(CH.sub.3).sub.2), 1.41
(9H, s, t-butyl), 1.29 (3H, d, /8.7 Hz, CH.sub.3), 0.81 (31-1, d,
14.4 Hz, CH(CH.sub.3).sub.2), 0.78 (3H, d, /4.4 Hz,
CH(CH.sub.3).sub.2); 6, (62.5 MHz, CDCl.sub.3) 174.0, 140.8, 128.8,
128.1, 127.7, 125.9, 82.7, 67.6, 66.9, 64.6, 48.4, 31.8, 28.4,
20.0, 19.2, 18.2; .sup.mI.sub.z (CL, NH.sub.3), 365 (MH.sup.+,
52%), 339 (29%). 333 (76%), 291 (28%), 277 (100%) and 263 (10%);
HRMS for C.sub.20H.sub.33N.sub.2O.sub.4 requires 365.2432. found
365.2430. [.alpha.].sub.D.sup.20-80.2 (c 0.98 CHCl.sub.3).
[0114] Potassium N-benzylidenyl-(S)-valinate
##STR00051##
[0115] To a solution of potassium hydroxide (958 mg, 17.10 mmol) in
methanol (15 mL) was added L-valine (2.00 g, 17.10 mmol). The
resulting solution was stirred under nitrogen for 3 hours. The
solvent was removed in vacuo before benzaldehyde (2.61 mL, 25.70
mmol 2.0 equiv.) in anhydrous pentane (30 mL) was added and the
mixture was azeotropic distillated under nitrogen for 8 hours. The
precipitate was collected and dried in high vacuo to furnish the
title compound as a white solid (3.85 g, 93%), V(m.sub.ax) (KBr)
2956 (C--H), 1594 (C.dbd.O) cm.sup.''1; .delta..sub.H(250 MHz,
OMSO-d). 8.19 (1H, s, CH.dbd.N), 7.75-7.71 (2H, m, Ph.times.2),
7.44-7.40 (3H, m, Ph.times.3), 3.26 (1H, d, J 7.24,
CHCH(CH.sub.3).sub.2), 2.21 (1H, m, CHCH(CH.sub.3).sub.2), 0.86
(3H, d, J 6.72 Hz, CHCH(CH.sub.3).sub.2.times.3), 0.79 (31-1, d,
/6.72 Hz, CHCH(CH.sub.3).sub.2.times.3); 6, (62.5 MHz, DMSO-d)
174.7, 159.0, 137.1, 130.3, 128.8, 128.1, 84.8, 31.4, 20.8,
19.5.
N--[N-Fmoc-(S)-alanyl)]-3(S')-isopropyl-5(R,S)-phenyl
oxazolidinone
##STR00052##
[0117] To the stirring suspension of potassium
N-benzylidenyl-5-valinate (122 mg, 0.50 mmol) in anhydrous
dichloromethane (20 mL) was added Fmoc-L-alanine acid chloride (166
mg, 0.50 mmol) under nitrogen at 0.degree. C. The resulting mixture
was stirred for 4 hours at 0.degree. C. and for another 12 hours at
room temperature. The solvent was removed in vacuo and the crude
product was purified by flash column chromatography on silica,
eluting with petrol:diethyl ether (1:1) to furnish the title
compound as fine colourless needles (174 mg, 70%); m.p.
69.0-71.5.degree. C.; v.sub.(maX) (KBr) 3400 (N--H), 2966 (C--H),
1802 (C.dbd.O, lactone), 1718 (C.dbd.O, carbamate), 1674 (C.dbd.O,
amide) cm.sup.1; .delta..sub.H (250 MHz, OMSO-d) 7.70-7.17 (13H, m,
Fmoc.times.8 and Ph.times.5), 6.45-6.43 (0.3H, m, PhCH.times.0.3),
5.36-5.28 (0.7H, m, PhCH.times.0.7), 4.64-4.47 (1H, m,
CHCH.sub.3.times.0.7 and CHCH(CH.sub.3).sub.2.times.0.3), 4.47-4.09
(3H, m, CHCH.sub.2), 4.01-3.82 (1H, m, CHCH.sub.3.times.0.3 and
CHCH(CH.sub.3).sub.2.times.0.7), 2.83-2.55 (0.3H, m,
CH(C.sub.1-13).sub.2.times.0.3), 2.20-2.05 (0.7H, m,
CH(CH.sub.3).sub.2.times.0.7), 1.43-1.40 (3H, m, CHCH.sub.3),
1.26-1.13 (3H, m, CH(CHs).sub.2.times.3) 1.02-0.91 (3H, m,
CH(CH.sub.3).sub.2.times.3), 0.87-0.80 (3H, m, CHCH.sub.3);
.delta..sub.c (62.5 MHz, CDCl.sub.3) 177.5, 171.3, 169.5, 166.1,
156.0, 144.1, 141.7, 136.3, 131.7, 130.6, 130.2, 129.1, 128.2,
127.5, 127.0, 125.5, 120.4, 91.5, 70.2, 67.5, 61.8, 49.3, 47.5,
46.2, 34.2, 31.1, 30.7, 19.3, 18.9, 18.3, 17.7, 16.7; % (Cl,
NH.sub.3), 498 (M.sup.+, 35%), 476 (47%), 463 (44%), 425 (23%), and
413 (100%); HRMS for C.sub.30H.sub.30N.sub.2O.sub.5 requires
498.2155. found 498.2151.
t-Butyl Fmoc-(S)-ala-(S)-val-(S)-alanate (79)
##STR00053##
[0119] To a solution of L-alanine tert-butyl ester (114 mg, 0.78
mmol, 3.0 equiv.) in anhydrous dichloromethane (10 mL) was added
trimethyl aluminium (0.46 mL, 0.91 mmol, 2 M in hexane, 3.5 equiv.)
under an atmosphere of nitrogen. After 15 minutes,
N--[N-Fmoc-(S)-alanyl)]-3(S)-wopropyl-5(R,S)-phenyl oxazolidinone
(130 mg, 0.26 mmol) in anhydrous dichloromethane (5 mL) was added.
The resulting solution was stirred at room temperature for 24
hours. The reaction was quenched by the addition of saturated
copper sulphate (20 mL) and the mixture was extracted with diethyl
ether (3.times.20 mL). The combined organic extracts were dried
over MgSO.sub.4. The solvents were removed in vacuo and the crude
product was purified by flash column chromatography on silica,
eluting with petrol:diethyl ether (1:9) to furnish the title
compound as fine colourless needles (76 mg. 54%); mp
153.0-155.0.degree. C.; v.sub.(max) (KBr) 3292 (N--H), 2974 (C--H),
1734 (C.dbd.O, ester), 1696 (C.dbd.O, carbamate), 1645 (C.dbd.O,
amide) cm.sup.''1; .delta..sub.H (250 MHz, DMSO-d) 8.37 (0.5H, d,
/6.42 Hz, NH), 8.18 (0.5H, d, /6.81 Hz, NH), 7.95 (0.5H, d, J 7.56
Hz, NH.times.0.5), 7.63 (0.5H, d, /7.48 Hz, NH.times.0.5),
7.96-7.35 (8H, m, Fmoc), 4.31-4.13 (5H, m, CHCH.sub.3.times.2 and
CHCH.sub.2), 2.03 (1H, q, /6.57 Hz, CH(CH.sub.3).sub.2), 1.42 (9H,
s, f-butyl), 1.28-1.25 (6H, m, 2.times.CH.sub.3) 0.94-0.85 (6H, m,
CH(CHb).sub.2); .delta..sub.c (62.5 MHz, DMSO-J) 173.1, 172.6,
171.9, 170.8, 156.1, 144.1 141.1, 128.0, 127.4, 125.6, 120.5, 80.7,
66.0, 57.4, 57.0, 50.4, 48.7, 47.0, 31.5, 31.0, 30.8, 27.9, 19.5,
18.7, 18.3, 17.9, 17.4, 17.2; .sup.mI.sub.z (CL. NH.sub.3). 537
(M.sup.+, 12%), 502 (100%), 464 (50%), 426 (21%), and 414 (86%);
FIRMS for C.sub.30H.sub.39N.sub.3O.sub.6 requires 537.2829. found
537.2836. [.alpha.].sub.D.sup.20-6.2 (c 0.57 CHCl.sub.3).
N--[N-Fmoc-(S)-valinyl)]-3 (S)-isopropyl-5 (R,S)-phenyl
oxazolidinone
##STR00054##
[0121] To the stirring suspension of potassium
N-benzylidenyl-(5)-valinate (123 mg, 0.46 mmol) in anhydrous
dichloromethane (20 mL) was added Fmoc-L-valine acid chloride (164
mg, 0.46 mmol) under nitrogen at 0.degree. C. The resulting mixture
was stirred for 4 hours at 0.degree. C. and for another 12 hours at
room temperature. The solvent was removed in vacuo and the crude
product was purified by flash column chromatography on silica,
eluting with petrol:diethyl ether (3:2) to furnish the title
compound as fine colourless needles (183 mg. 75%); mp
76.0-78.0.degree. C.; v.sub.(max) (KBr) 3326 (N--H), 2966 (C--H),
1802 (C.dbd.O, lactone), 1722 (C.dbd.O, carbamate), 1663 (C.dbd.O,
amide) cm.sup.''1; .delta..sub.H (250 MHz, DMSO-d) 7.81-7.11 (14H,
m. NH.times.1, Fmoc.times.8 and Ph.times.5), 6.4 (0.5H, s,
PhCH.times.0.5), 4.98 (0.5H, s. PhCH.times.0.5), 4.48-4.76 (5H, m,
CHCH(CH.sub.3).sub.2.times.2 and CHCH.sub.2.times.3), 2.57-2.54
(1H, m, CH(CH.sub.3).sub.2), 2.24-2.10 (1H, m, CH(CHs).sub.2),
1.28-0.63 (12H, m, CH(CH.sub.3).sub.2.times.2); .delta..sub.c (62.5
MHz, DMSO-d) 170.3, 170, 169.7, 156.9, 144.1, 141.0, 137.5, 130.9,
129.9, 129.5, 128.5, 128.0, 127.7, 127.4, 125.8, 120.5, 90.6, 66.2
61.3, 60.9, 58.3, 46.9, 34.7, 32.6, 30.8, 30.3, 19.7, 18.7, 18.3,
17.2, 16.7, 16.4; % (CL, NH.sub.3), 526 (M.sup.+, 10%), 331 (40%),
230 (7%) and 178 (100%); HRMS for C.sub.32H.sub.34N.sub.2O.sub.5
requires 526.2459. found 526.2466.
[0122] Tri-L-alanine
##STR00055##
[0123] To a solution of
N-Fmoc-(S)-alanyl-N-((1R)-phenyl-2-hydroxylethyl)-(S)-alanyl-(S)-alanine
tert-butyl ester (235 mg, 0.37 mmol) and tert-butanol (0.12 mL,
1.20 mmol, 3.0 equiv.) in liquid ammonia (15 mL) and anhydrous
tetrahydrofuran (10 mL) was added lithium (23 mg, 3.70 mmol, 10.0
equiv.) at -78.degree. C. under an atmosphere of nitrogen. The
resulting solution was stirred until the blue colour was
disappeared and then warmed to room temperature to evaporate all
the liquid ammonia. A mixture of water (15 mL) and diethyl ether
(10 mL) was added and the aqueous phase was extracted with diethyl
ether (3.times.10 mL). Water was removed in vacuo and the crude
product purified first by acidic ion exchange chromatography and
then by flash column chromatography on silica, eluting with
methanol and water (7:3) to furnish the title compound as fine
colourless needles (71 mg, 84%), v.sub.(max) (KBr) 3276 (N--H),
2985 (C--H), 1645 (C.dbd.O), 1592 (C.dbd.O), 1531 (C.dbd.O)
cm.sup.''1; .delta..sub.H (250 MHz, D.sub.2O), 4.53 (1H, q, /9.37,
CH), 4.03 (2H, q, /7.22 Hz, CH.times.2), 1.23 (3H, d, J 7.14,
CH.sub.3), 1.09 (3H, d, J 7.21, CH.sub.3), 1.02 (3H, d, /7.24 Hz,
CH.sub.3); .delta..sub.c (62.5 MHz, D.sub.2O) 180.1, 173.9, 170.9,
51.3, 50.1, 49.2, 17.7, 16.9, 16.7; .sup.mI.sub.z (CL, NH.sub.3),
231 (M.sup.+, 40%), 230 (100%), and 228 (35%); HRMS for
C.sub.9H.sub.17H.sub.3O.sub.4 requires 231.1215 found 231.1209;
[.alpha.].sub.D.sup.20-72.8 (c 1.01 H.sub.2O) (commercial one
[.alpha.].sub.D2.sup.j0.sup.n-73.2 (c 1.02 H.sub.2O)).
(5S)-3-Methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one(35).sup.61
##STR00056##
[0125] (S)-2-phenylglycinol (3.00 g, 21.9 mmol, 1.0 equiv.) (34)
and ethyl pyruvate (2.67 mL, 24.1 mmol, 1.1 equiv.) were refluxed
in trifluoroethanol (50 mL) over activated 4 molecular sieves (8.00
g) for 24 hours. Filtration through a short pad of Celite.RTM. and
removal of solvent from the filtrate in vacuo delivered the crude
product which was purified by flash column chromatography on
silica, eluting with petrol and diethyl ether (7:3) to furnish the
title compound as a white solid (1.83 g, 44%); m.p.
70.0-71.0.degree. C. (lit 71.0-72.0.degree. C.).sup.64;
v.sub.(max)(KBr) 3007 (C--H), 1735 (C.dbd.O), 1640 (C.dbd.N) cm
.sup.1; .delta..sub.H (250 MHz, CDCl.sub.3) 7.45-7.32 (5H, m, Ph),
4.88-4.81 (1H, m, PhCH), 4.56 (1H, dd, J 4.49 Hz, T 9.48 Hz,
6.beta.-H), 4.25 (1H, dd 13.01 Hz, r 14.99 Hz, 6.alpha.-H), 2.41
(3H, s, CH.sub.3); .delta..sub.c (62.5 MHz, CDCl.sub.3) 160.7,
155.9, 137.2, 129.4, 128.7, 127.5, 71.9, 60.1, 22.2; % (CL,
NH.sub.3), 189 (M.sup.+, 25%), 159 (12%), 130 (24%), 104 (100%), 90
(21%), and 78 (6%); HRMS for C.sub.11H.sub.11NO.sub.2 requires
189.0787 found 189.0782. [.alpha.].sub.D.sup.20+253.0 (c 0.98
CHCl.sub.3) (the enantiomer lit. [.alpha.].sub.D.sup.20-237.1 (c
1.11 CHCl.sub.3)).sup.61.
(3R,5.English
Pound.)-3-Methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4.about.oxazin-2-one
(36).sup.61
##STR00057##
[0127] To a solution of
(5S)-3-methyl-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one (35) (1.70
g, 9.0 mmol, 1.0 equiv.) in anhydrous dichloromethane (60 mL) under
an atmosphere of nitrogen was added PtO.sub.2 (170 mg, 0.1 equiv.).
The mixture was consecutively degassed and purged three times with
hydrogen and then stirred for 5 hours under an atmosphere of
hydrogen. Filtration through a short pad of Celite.RTM. and removal
of solvent from the filtrate in vacuo yielded the crude product
which was purified by recrystallization from dichloromethane,
diethyl ether and hexane to furnish the title compound as a
colourless needles (1.26 g, 74%); m.p. 81.0-82.0.degree. C. (lit.
m.p. 81.0-82.0.degree. C.).sup.61; V.sub.(max) (KBr) 3314 (N--H),
2981 (C--H), 1736 (C.dbd.O), cm.sup.''1; .delta..sub.H (250 MHz,
CDCl.sub.3) 7.43-7.26 (5H, m, Ph),; 4.42-4.23 (3H, m, CHCH.sub.2),
3.88 (1H, q, /6.76 Hz, CHCH.sub.3), 1.80 (1H, br, NH), 1.50 (3H, d,
J 6.76 Hz, CH.sub.3); 8, (62.5 MHz, CDCl.sub.3) 170.7, 138.1,
129.3, 129.1, 127.5, 75.4, 58.2, 55.4, 19.0; .sup.mI.sub.z (CL,
NH.sub.3), 192 (MH.sup.+, 30%), 147 (68%), 132 (64%), 104 (100%),
and 91 (10%); HRMS for C.sub.11H.sub.13NO.sub.2 requires 192.1025
found 192.1019. [.alpha.].sub.D.sup.20+88.8 (c 0.96 CHCl.sub.3)
(lit. .sup.[.alpha.].sub.D.sup.20+92.3 (c 0.84
CHCl.sub.3)).sup.61.
(3R,5S)--N--[N-Fmoc-(S)alanyl]-3-methyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,-
4-oxazin-2-one (37)
##STR00058##
[0129] To a vigorously stirred mixture of
(3R,5S)-3-methyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,4-oxazin-2-one
(36) (500 mg. 2.62 mmol), Na.sub.2CO.sub.3 (1.40 g, 13.3 mmol, 5.0
equiv.) in 1:1 dichloromethane and water (40 mL) was added
N-Fmoc-L-alanine acid chloride (1.04 g, 3.14 mmol, 1.2 equiv.) in
dichloromethane (10 mL) dropwise over 5 min. The resulting solution
was stirred for 2 hours. The aqueous phase was extracted with
dichloromethane (3.times.15 mL). The combined extracts were washed
with saturated Na.sub.2CO.sub.3 (50 mL), water (2.times.30 mL),
brine (50 mL) and dried over MgSO.sub.4. The solvents were removed
in vacuo and the crude material was purified by flash column
chromatography on silica, eluting with petrol and diethyl ether
(1:4) to furnish the title compound as fine colourless needles
(1.02 g, 80%); m.p. 87-88.degree. C.; v.sub.(max) (KBr) 3323
(N--H), 2982 (C--H), 1761 (C.dbd.O, lactone), 1717 (C.dbd.O,
carbamate), 1656 (C.dbd.O, amide) cm.sup.''1; .delta..sub.H (400
MHz, DMSO-J, 110.degree. C.) 7.84-7.29 (13H, m, Fmoc.times.8,
Ph.times.5), 7.08 (1H, br, NH), 5.54 (1H, t, /5.88 Hz, PhCH), 4.96
(1H, q, /7.11 Hz, NCHCH.sub.3); 4.68-4.60 (2H, m, PhCHCH.sub.2),
4.41-4.32 (3H, m, CHCH.sub.3NH.times.1, OCH.sub.2CH.times.2), 4.22
(1H, t, /6.72 Hz OCH.sub.2CH), 1.45 (3H, d, /7.15 Hz.sub.5
NCHCH.sub.3), 1.08 (3H, d, /6.71 Hz, CHCH.sub.3NH); 8, (62.5 MHz,
OMSO-d) 174.0, 172.8, 170.4, 169.6, 156.3, 144.1, 141.1, 137.7,
129.4, 128.7, 128.0, 127.4, 127.3, 127.1, 125.6, 120.5, 68.8, 66.0,
55.5, 55.3, 52.2, 51.4, 50.0, 47.2, 20.5, 18.6, 17.7, 17.1;
'7.sub.Z (C.I., NH.sub.3), 485 (MH.sup.+, 12%), 431 (8%), 381 (7%),
281 (15%) and 149 (100%); HRMS for C.sub.29H.sub.29N.sub.2O.sub.5
requires 485.2069. found 485.2076; [.alpha.].sub.D.sup.20+22.2 (c
0.94 CHCl.sub.3).
N-Fmoc-(S)-alanyl-N-((1S)-phenyl-2-hydroxylethyl)-(R)-alanyl-(S)-alanine
tert-butyl ester (71).sup.48
##STR00059##
[0131] To a solution of L-alanine tert-butyl ester (66) (251 mg,
1.73 mmol, 3.0 equiv.) in anhydrous dichloromethane (25 mL) was
added trimethyl aluminium (2.03 mL, 2.03 mmol, 2 M in hexane. 3.5
equiv.) under an atmosphere of nitrogen. After 15 minutes,
(3R,5S)-[N4N-Fmoc-(S)alanyl]-3-methyl-5-phenyl-3,4,5,6,-tetrahydro-2H-1,4-
-oxazin-2-one (37) (280 mg, 0.58 mmol) in anhydrous dichloromethane
(8 mL) was added. The resulting solution was stirred at room
temperature for 24 hours. The reaction was quenched by the addition
of water (7 mL) and the organic phase was then washed with
saturated copper sulphate (15 mL). The aqueous phase was extracted
with diethyl ether (3.times.20 mL) and the combined organic
extracts were dried over MgSO.sub.4. The solvent was removed in
vacuo and the crude product was purified by flash column
chromatography on silica, eluting with petrol and diethyl ether
(1:4) to furnish the title compound as fine colourless needles (226
mg, 62%); m.p. 87-88.degree. C.; v.sub.(max) (KBr) 3410 (O--H),
2980 (C--H), 1727 (C.dbd.O), 1654 (C.dbd.O) cm.sup.1; .delta..sub.H
(250 MHz, OMSO-d), 8.35 (0.5H, d, /6.18 Hz, NH) 7.91-7.12 (13H, m,
Fmoc.times.8 and Ph.times.5). 7.65 (0.5H, d, /7.14 Hz, NH), 7.55
(0.5171, d, /7.32 Hz, NH), 6.39 (0.5H, d, /7.45 Hz, NH), 5.21-5.18
(0.5H, m, PhCH), 5.03-5.01 (0.5H, m. PhCH), 4.94-4.92 (0.5H, m,
NHCHCH.sub.3), 4.80-4.78 (0.5H, m, NHCHCH.sub.3), 4.65-4.62 (0.5H,
m, NCHCH.sub.3), 4.27-4.18 (3.5H, m, OCH.sub.2CH, and
CH.sub.2OH.times.0.5), 4.09-3.93 (2.5H, m, CH.sub.2OH.times.1.5 and
NHCHCH.sub.3), 3.62-3.60 (0.5H, m, NCHCH.sub.3.times.1), 1.44-1.36
(13.5H, s, (CHa).sub.3.times.9, CHCH.sub.3.times.4.5), 1.23 (1.5H,
d, /6.63 Hz, CHCH.sub.3), 1.13 (1.5H, d, /7.35 Hz, CHCH.sub.3),
0.92 (1.5H, d, /7.07 Hz, CHCH.sub.3); .delta..sub.c (62.5 MHz,
OMSO-d) 173.4, 172.9, 171.9, 171.6, 169.7, 156.0, 144.2, 144.1,
141.1, 137.2, 129.0, 128.3, 128.0, 127.7, 127.4, 127.3, 125.7,
120.5, 81.2, 80.8, 66.1, 61.6, 55.3, 53.6, 48.6, 48.3, 47.5, 47.0,
27.9, 18.5, 18.2, 17.7, 17.1, 15.4; % (CL, NH.sub.3), 775 (100%),
629 (M.sup.+, 36%), 457 (58%), 345 (27%), 231 (33%) and 178 (100%);
HRMS for C.sub.36H.sub.43N.sub.3O.sub.7 requires 629.3092. found
629.3093; [.alpha.].sub.D .sup.20+14.20 (c 1.15 CHCl.sub.3).
[0132] LDL-alanine
##STR00060##
[0133] To a solution of
N-Fmoc-(S)-alanyl-NOR)-phenyl-2-hydroxylethyl)-(R)-alanyl-(S)-alanine
tert-butyl ester (235 mg. 0.37 mmol) and tert-butanol (0.12 mL,
1.20 mmol, 3.0 equiv.) in liquid ammonia (15 mL) and anhydrous
tetrahydrofuran (10 mL) was added lithium (23 mg. 3.70 mmol, 10.0
equiv.) at -78.degree. C.; under an atmosphere of nitrogen. The
resulting solution was stirred until the blue colour disappeared
and then allowed to warm to room temperature to evaporate off the
liquid ammonia. A mixture of water (15 mL) and diethyl ether (10
mL) was added and the aqueous phase was extracted with diethyl
ether (3.times.10 mL). Water was removed in vacuo and the crude
product purified first by acidic ion exchange chromatography and
then by flash column chromatography on silica, eluting with
methanol and water (7:3) to furnish the title compound as fine
colourless needles.
##STR00061##
[0134] To a solution of
(3S,5R)--N--[N-Fmoc-(S)alanyl]-3-methyl-5-.rho.henyl-3,4,5,6,-tetrahydro--
2H-1,4-oxazin-2-one (33) (1.00 g, 2.06 mmol, 1.0 equiv.) in
anhydrous tetrahydrofuran (40 mL) was added
1,8-diazabicyclo[5.4.0]-undec-7-ene (0.03 mL, 0.21 mmol, 0.1
equiv.) under an atmosphere of nitrogen. The resulting solution was
stirred at room temperature for 3.5 hours before
--N.sub.5N-diisopropylethylamine (0.40 mL, 2.26 mmol, 1.1 equiv.),
N-Fmoc-L-alanine (0.76 g, 2.46 mmol, 1.2 equiv.) and
bromotripyrrolidinophosphonium hexafluorophosphate (1.21 g, 2.46
mmol, 1.2 equiv.) were added. The resulting solution was stirred
for another 18 hours during which time a white precipitate was
formed. The mixture was filtered through a short pad of Celite.RTM.
and removal of solvent from the filtrate in vacuo yielded the crude
product which was purified by flash column chromatography on
silica, eluting with dichloromethane and diethyl ether (3:7) to
furnish the title product as a white powder (0.96 g, 86%);
100.0-101.0.degree. C.; v.sub.(max) (KBr) 3311 (N--H), 2981 (C--H),
1741 (C.dbd.O), 1718 (C.dbd.O), 1647 (C.dbd.O) cm-.sup.1;
.delta..sub.H (250 MHz, OMSO-d) 7.79 (1H, /6.70 Hz, NH), 7.68-7.07
(13H, m, Fmoc.times.8. Ph.times.5), 7.32 (1H, J 7.85 Hz, NH), 5.27
(1H, br, PhCH), 5.16-4.96 (1H, m, PhCHCH.sub.2.times.1), 5.16-4.96
(1H, m, PhCHCH.sub.2.times.1), 4.64-4.62 (1H, m, CHCH.sub.3),
4.55-4.51 (1H, m, CHCH.sub.3), 4.35-4.32 (1H, m, OCH.sub.2CH), 4.00
(2H, m, OCH.sub.2CH), 3.89-3.80 (1H, m, CHCH.sub.3), 0.98 (3H, d, J
3.92 Hz, CHCH.sub.3). 0.93 (3H, br, CHCH.sub.3), 0.62 (3H, d, /6.89
Hz, CHCH.sub.3); 6, (62.5 MHz, DMSO-d) 172.8, 171.6, 170.0, 156.0,
144.2, 141.1, 136.3, 128.9, 128.3, 128.0, 127.4, 126.8, 125.7,
120.4, 69.1, 66.0, 52.9, 50.6, 49.9, 47.0, 30.8, 20.9, 18.5, 17.8;
% (CL, NH.sub.3). 231 (M.sup.+, 40%), 230 (100%), and 228 (35%);
HRMS for C.sub.32H.sub.33N.sub.3O.sub.6 requires 555.2369 found
231.1209; [.alpha.].sub.D.sup.20-31.3 (c 0.90 CHCl.sub.3).
##STR00062##
[0135] To a solution of
(3S,5R)--N--[N-Fmoc-(S)alanyl-(S)-alanine]-3-methyl-5-phenyl-3,4,5,6-tetr-
ahydro-2H-1,4-oxazin-2-one (49) (114 mg. 0.206 mmol. 1.0 equiv.) in
anhydrous tetrahydrofuran (5 mL) was added
1,8-diazabicyclo[5.4.0]-undec-7-ene (0.015 mL, 0.103 mmol, 0.5
equiv.) under an atmosphere of nitrogen. The resulting solution was
stirred at room temperature for 5 hours before
N,N-diwcpropylethylamine (0.026 mL, 0.144 mmol, 0.7 equiv.),
N-Fmoc-L-alanine (76 mg, 0.246 mmol, 1.2 equiv.) and
bromotripyrrolidinophosphonium hexafluorophosphate (0.121 g, 0.246
mmol, 1.2 equiv.) were added. The resulting solution was stirred
for another 19 hours during which time a white precipitate was
formed. The mixture was filtered through a short pad of Celite.RTM.
and removal of solvent from the filtrate in vacuo yielded the crude
product which was purified by flash column chromatography on
silica, eluting with dichloromethane and acetone (7:3) to furnish
the title product as a white powder. (89 mg, 68%); m.p.
118.0-120.0.degree. C.; v.sub.(max) (KBr) 3316 (N--H), 2985 (C--H),
1738 (C.dbd.O), 1708 (C.dbd.O), 1647 (C.dbd.O) Cm''.sup.1;
.delta..sub.H (250 MHz, DMSO--O 8.00 (1H, d, /7.28 Hz, NH),
7.91-7.30 (13H, m. Fmoc.times.8, Ph.times.5), 7.52 (1H, d, /7.58
Hz, NH), 5.48 (1H, br, PhCH). 5.32-5.02 (1H, m, PhCHCH.sub.2),
4.88-4.85 (1H, m, CHCH.sub.3), 4.74-4.69 (1H, m, CHCH.sub.3), 4.56
(1H, br, OCH.sub.2CH), 4.26-4.27 (2H, m, OCH.sub.2CH), 4.03 (1H, t,
/7.25 Hz, CHCH.sub.3), 3.63-3.58 (0.5H, m, CHCH.sub.3), 3.14-3.12
(0.5H, m, CHCH.sub.3), 1.26-0.97 (12H, m, CHCH.sub.3.times.4); 8,
(62.5 MHz, DMSO-d) 172.5, 172.2, 171.6, 170.0, 156.0, 144.2, 141.1,
136.5, 128.9, 128.3, 128.0, 127.4, 126.7, 125.7, 120.5, 65.9, 56.2,
53.9, 53.0, 47.9, 46.0, 42.2, 32.5, 31.0, 29.9, 18.5, 17.8, 17.1,
12.9; % (CL, NH.sub.3); FIRMS for C.sub.35H.sub.38N.sub.4O.sub.7
requires 626.2741 found; [.alpha.].sub.D.sup.2u-27.2 (c 1.04
acetone).
[0136] .sup.1H-NMR data obtained for Tri-L-alanine made according
to the invention with commercial tri-L-alanine was compared with
excellent results.
[0137] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *