U.S. patent application number 14/804693 was filed with the patent office on 2015-11-05 for methods for the synthesis of dicarba bridges in organic compounds.
The applicant listed for this patent is SYNGENE LIMITED. Invention is credited to Jomana Elaridi, William Roy Jackson, Jim Patel, Andrea Robinson.
Application Number | 20150315243 14/804693 |
Document ID | / |
Family ID | 38371126 |
Filed Date | 2015-11-05 |
United States Patent
Application |
20150315243 |
Kind Code |
A1 |
Robinson; Andrea ; et
al. |
November 5, 2015 |
METHODS FOR THE SYNTHESIS OF DICARBA BRIDGES IN ORGANIC
COMPOUNDS
Abstract
The present invention relates to methods for forming dicarba
bridges in organic compounds. This involves the use of a pair of
complementary metathesisable groups on the organic compound, and
subjecting the compound to cross-metathesis under microwave
radiation conditions. In an alternative, the compounds contain a
turn-inducing group between the pair of cross-metathesisable groups
to facilitate the cross-metathesis.
Inventors: |
Robinson; Andrea; (St.
Kilda, AU) ; Jackson; William Roy; (Camberwell,
AU) ; Patel; Jim; (Parkdale, AU) ; Elaridi;
Jomana; (Endeavour Hills, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SYNGENE LIMITED |
South Yarra |
|
AU |
|
|
Family ID: |
38371126 |
Appl. No.: |
14/804693 |
Filed: |
July 21, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12279383 |
Mar 13, 2009 |
9102708 |
|
|
PCT/AU2007/000176 |
Feb 16, 2007 |
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14804693 |
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Current U.S.
Class: |
530/317 |
Current CPC
Class: |
C07K 1/006 20130101;
C07B 37/04 20130101; C07C 231/12 20130101; C07C 231/12 20130101;
C07K 1/1077 20130101; C07K 7/54 20130101; C07C 2/42 20130101; C07C
231/12 20130101; C07K 1/06 20130101; C07C 231/12 20130101; C07C
2/52 20130101; C07C 2603/18 20170501; C07C 271/22 20130101; C07C
233/47 20130101; C07C 231/12 20130101; C07C 233/82 20130101; C07C
233/85 20130101; C07C 233/83 20130101 |
International
Class: |
C07K 7/54 20060101
C07K007/54 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 17, 2006 |
AU |
2006900799 |
Claims
1. A method for the synthesis of an organic compound with a dicarba
bridge, comprising: providing a reactable organic compound having a
pair of unblocked complementary metathesisable groups, or two or
more reactable organic compounds having between them a pair of
unblocked complementary metathesisable groups, and subjecting the
reactable organic compound or compounds to cross-metathesis under
microwave radiation conditions to form an organic compound with an
unsaturated dicarba bridge.
2. The method of claim 1, further comprising: hydrogenating the
unsaturated dicarba bridge.
3. The method of claim 1 or claim 2, wherein the complementary
metathesisable groups of the first pair of complementary
metathesisable groups are each independently selected from the
group consisting of olefins comprising the portion .dbd.CH.sub.2,
and monosubstituted olefins comprising the group .dbd.CHR.sub.5, in
which R.sub.5 is alkyl or an alkyl substituted by a polar
functional group.
4. The method of any one of claims 1 to 3, wherein the reactable
organic compound, or one of the reactable organic compounds, is
attached to a solid support during the cross-metathesis of the
complementary metathesisable groups.
5. The method of claim 4, wherein the loading of the reactable
organic compound on the solid support is at least 0.2 mmol/g.
6. The method of any one of claims 4 to 5, wherein each
cross-metathesis is performed using a cross-metathesis catalyst,
and each cross-metathesis is performed in a solvent combination of
a resin-swelling solvent, with a co-ordinating solvent for the
catalyst.
7. The method of claim 6, wherein the co-ordinating solvent is an
alcohol.
8. The method of claim 6 or claim 7, wherein the co-ordinating
solvent is used in an amount of about 1-30% by volume, with respect
to the total solvent combination.
9. The method of any on eof claims 1 to 8, wherein the reactable
organic compound, or the two or more reactable organic compounds,
between them contain a second pair of complementary metathesisable
groups which are blocked, and can be unblocked by an unblocking
reaction specific to the second pair of complementary
metathesisable groups, and the method further comprises unblocking
the blocked second pair of complementary metathesisable groups,
followed by cross-metathesis of the second pair of
cross-methatesisable groups.
10. The method of claim 9, wherein the blocked second pair of
complementary metathesisable groups comprise dialkyl-blocked
olefins.
11. The method of claim 9 or claim 10, wherein the blocked second
pair of complementary metathesisable groups are unblocked by
cross-methatiesis with a disposable olefin, which is
1,3-butadiene-free.
12. The method of claim 11, wherein the disposable olefin is a
1,3-butadiene-free olefin or olefin mixture of one or more of the
following: ##STR00228## wherein X and Y are each independently
selected from the group consisting of H, alkyl and substituted
alkyl, wherein the substituent of the substituted alkyl is selected
from one or more of halo, hydroxy, alkoxy, nitrile, acid and
ester.
13. The method of any one of claims 9 to 12, wherein the reactable
organic compound comprises a third pair of complementary
methathesisable groups, which are blocked and can be unblocked by
an unblocking reaction or series of reactions specific to the third
pair, and the method comprises subjecting the third pair of
complementary metathesisable groups to unblocking reactions
specific to those pairs, followed by cross metathesis.
14. The method of claim 13, wherein the blocking group of the third
pair of complementary metathesisable groups comprises an electronic
blocking group, and the unblocking reactions comprise conversion of
the electronic steric blocking group to a group that is
cross-metathesisable.
15. The method of claim 14, wherein the electronic blocking group
comprises a diblocked conjugated diene, and the unblocking reaction
comprises subjecting the blocked third pair of complementary
metathesisable groups to asymmetric hydrogenation to
regioselectively, stereoselectively and chemoselectively
hydrogenate one of the diene double bonds to leave a
sterically-blocked double bond, followed by cross-metathesis with a
disposable olefin to effect removal of the steric blocking groups
on the remaining double bond, to yield unblocked complementary
metathesisable groups.
16. The method of claim 14, wherein the electronic blocking group
comprises .dbd.CH--CH.dbd.CR.sub.3R.sub.4, in which R.sub.3 and
R.sub.4 are each alkyl, and the unblocking reaction comprises
hydrogenation of this group to .dbd.CH--CH.sub.2--CHR.sub.3R.sub.4,
followed by cross-metathesis with a disposable olefin to yield the
unblocked group .dbd.CHR.sub.5, in which R.sub.5 is alkyl or an
alkyl substituted by a polar functional group.
17. The method of any one of claims 1 to 16, wherein the reactable
organic compound is a peptide.
18. The method of claim 17, wherein the organic compound with two
or more dicarba bridges prepared by the method is a
peptidomimetic.
19. The method of claim 17, wherein the organic compound with two
or more dicarba bridges prepared by the method is a
pseudopeptide.
20. The method of any one of claims 1 to 19, wherein the reactable
organic compound comprises a peptide comprising a series of amino
acids supported on a solid support, wherein two of the amino acids
comprise sidechains with a first pair of complementary
metathesisable groups.
21. The method of claim 20, wherein the peptide contains at least
one turn-inducing amino acid between the amino acids that comprise
the first pair of complementary metathesisable groups.
22. The method of claim 21, wherein the turn-inducing amino acids
are selected from one or more of .psi.serine, .psi.threonine and
.psi.cysteine.
23. The method of claim 22, further comprising cleaving the protein
from the solid support and converting any .psi.serine,
.psi.threonine and .psi.cysteine residues present into serine,
threonine and cysteine, respectively.
24. A method for the synthesis of an organic compound with a
dicarba bridge, comprising: synthesising a reactable organic
compound to contain a pair of unblocked complementary
metathesisable groups, and a turn-inducing group in between the
pair of complementary metathesisable groups, and subjecting the
reactable organic compound to cross-metathesis to form a compound
with an unsaturated dicarba bridge.
25. The method of claim 24, further comprising hydrogenating the
unsaturated dicarba bridge to form an organic compound with a
saturated dicarba bridge.
26. The method of any one of claims 24 to 25, wherein the reactable
organic compound comprises a peptide comprising a series of amino
acids supported on a solid support, wherein two of the amino acids
comprise sidechains with a first pair of complementary
metathesisable groups.
27. The method of claim 26, wherein the peptide contains at least
one turn-inducing amino acid between the amino acids that comprise
the first pair of complementary metathesisable groups.
28. The method of claim 27, wherein the turn-inducing amino acids
are selected from one or more of .psi.serine, .psi.threonine and
.psi.cysteine.
29. The method of any one of claims 26 to 28, wherein the
complementary metathesisable groups of the first pair of
complementary metathesisable groups are each independently selected
from the group consisting of olefins comprising the portion
.dbd.CH.sub.2, and monosubstituted olefins comprising the group
.dbd.CHR.sub.5, in which R.sub.5 is alkyl or an alkyl substituted
by a polar functional group.
30. The method of any one of claims 26 to 29, wherein the loading
of the reactable organic compound on the solid support is at least
0.2 mmol/g.
31. The method of any one of claims 26 to 30, wherein each
cross-metathesis is performed using a cross-metathesis catalyst,
and each cross-metathesis is performed in a solvent combination of
a resin-swelling solvent, with a co-ordinating solvent for the
catalyst.
32. The method of claim 31, wherein the co-ordinating solvent is an
alcohol.
33. The method of claim 31 or claim 32, wherein the co-ordinating
solvent is used in an amount of about 1-30% by volume, with respect
to the total solvent combination.
34. An organic compound or a peptide containing a dicarba bridge
when produced by the method of any one of claims 1 to 33.
35. Fmoc-protected prenyl glycine.
36. A method for the synthesis of Fmoc-protected prenyl glycine,
the method comprising cross-metathesis of Fmoc-protected allyl
glycine with 2-alkyl-2-butylene in the presence of a
cross-metathesis catalyst.
Description
1.1 FIELD OF THE INVENTION
[0001] The present application broadly relates to methods for
forming dicarba bridges in organic compounds, and compounds such as
peptides containing dicarba bridges.
1.2 BACKGROUND TO THE INVENTION
[0002] Cystine (--S--S--) bridges are common structural motifs in
naturally occurring cyclic peptides. In some cases, these disulfide
bridges act as reactive functional groups. In many other cases
however, the cystine bridge serves only a skeletal, structural
role, maintaining secondary and tertiary structure. Disulfide bonds
in peptides and other compounds are highly reactive under
broad-ranging conditions, and therefore useful peptides containing
disulfide bonds which have a structural role are at risk of
denaturation, resulting in loss of properties. There is accordingly
some interest in developing methods for creating more robust
bridges in such compounds--such as dicarba (--C--C--) containing
bridges, which are not as reactive, so as to produce compounds
having the activity of, or similar activity to, the
disulfide-containing polypeptides, but with better
biostability.
[0003] Once a suitably strategy for forming such dicarba bridges is
established, it is of additional interest to be able to form
multiple dicarba bridges--selectively. By way of explanation, a
peptide possessing four cysteine residues, and two cystine bridges,
has three topoisomers--the [1,3],[2,4]-isomer (globule), the
[1,4],[2,3]-isomer (ribbon) and the [1,2],[3,4]-isomer (bead). It
would be useful to be able to selectively form one of these
isomers, without any of the other two topoisomers. It is also of
interest to be able to form one or more dicarba bridges using
chemistry that does not destroy any disulfide bridges that are
present, so that dicarba-disulfide containing compounds can
additionally be formed. It is of further interest to have a dicarba
bridge forming method that can take place despite the presence of
disulfide, which could otherwise interfere with dicarba
bridge-forming reactions.
[0004] Once this is achievable, it is of interest to be able to
form dicarba-containing analogues of a range of
disulfide-containing peptides, such as conotoxins. It is also of
interest to form peptide and non-peptide compounds containing one
or more intramolecular dicarba bridge, and an olefin-handle
enabling reaction to other moieties.
2.0 SUMMARY OF THE INVENTION
[0005] According to the present invention, there is provided a
range of methods for forming dicarba bridges, as well as new
compounds containing dicarba bridges and a range of new compounds
that facilitate the construction of these bridges.
[0006] According to a first aspect, there is provided a method for
the synthesis of an organic compound with a dicarba bridge,
comprising: [0007] providing a reactable organic compound having a
pair of unblocked complementary metathesisable groups, or two or
more reactable organic compounds having between them a pair of
unblocked complementary, metathesisable groups, and [0008]
subjecting the reactable organic compound or compounds to
cross-metathesis under microwave radiation conditions to form an
organic compound with an unsaturated dicarba bridge.
[0009] As explained in further detail below, cross-metathesis
involves the formation of an unsaturated dicarba bridge (inter- or
intramolecular, depending on whether there are one or two reactable
organic compounds) from two unblocked metathesisable olefinic
groups. It has been surprisingly found that for many situations
where the reaction will not proceed under normal conditions, the
performance of this reaction under microwave radiation conditions
overcomes this problem and enables this reaction to proceed.
Another strategy for improving the performance of the
cross-metathesis which does not rely on microwave is outlined
below. The advantages of the use of microwave irradiation applies
particularly to the situation where the method is performed on a
single reactable organic compound having a pair of unblocked
complementary metathesisable groups, for the formation of an
intramolecular dicarba bridge. In other cases, microwave radiation
overcomes inefficient metathesis reactions that do not otherwise go
to completion. Other details relating to the types of compounds
that this method is particularly suited to are outlined in the
detailed description.
[0010] In a related aspect, in which it is desired to form a
saturated dicarba bridge, the process involves a following step of
subjecting the unsaturated dicarba bridge to hydrogenation
(suitably homogeneous hydrogenation). Accordingly, in total, this
second aspect provides a method for the synthesis of an organic
compound with a saturated dicarba bridge, comprising: [0011]
providing a reactable organic compound having a pair of unblocked
complementary metathesisable groups, or two or more reactable
organic compounds having between them a pair of unblocked
complementary metathesisable groups, [0012] subjecting the
reactable organic compound or compounds to cross-metathesis under
microwave radiation conditions to form an organic compound with
unsaturated dicarba bridge, and [0013] subjecting the unsaturated
dicarba bridge to hydrogenation.
[0014] The hydrogenation step is suitably homogeneous
hydrogenation.
[0015] According to one particularly preferred embodiment, the
process enables the selective formation of multiple dicarba
bridges. According to this embodiment, there is provided a method
for the synthesis of an organic compound with a plurality of
dicarba bridges, comprising: [0016] providing one or more reactable
organic compounds having within the single compound, or between the
multiple compounds, a first pair of complementary metathesisable
groups which are unblocked, a second pair of complementary
metathesisable groups, which are blocked and can be unblocked by an
unblocking reaction or series of reactions specific to that second
pair, and optionally further pairs of complementary methathesisable
groups, which are blocked and can be unblocked by an unblocking
reaction or series of reactions specific to each further pair,
[0017] subjecting the reactable organic compound or compounds to
cross-metathesis to form an organic compound with an unsaturated
dicarba bridge across the first pair of complementary
metathesisable groups, without cross-metathesis between the pair or
pairs of blocked complementary metathesisable groups, [0018]
subjecting the second pair of complementary metathesisable groups
to the unblocking reaction or series of reactions specific to the
second pair, [0019] subjecting the second pair of complementary
metathesisable groups to cross-metathesis to form an organic
compound with an unsaturated dicarba bridge across the second pair
of complementary metathesisable groups, without cross-methathesis
between any pair or pairs of complementary methathesisable groups
that remain blocked, and [0020] if any complementary metathesisable
groups remain, subjecting those groups to unblocking reactions
specific to those pairs, followed by cross metathesis, wherein at
least one of the cross-metathesis reactions is conducted under
microwave radiation conditions.
[0021] Preferably, the unblocking reaction specific to the second
pair comprises cross-metathesis with a butadiene-free disposable
olefin. 1,3-butadiene acts as a poison in the unblocking reaction,
if it is present in the disposable olefin composition used in this
reaction.
[0022] In many circumstances it will be desirable to subject some
or all of the unsaturated dicarba bridges formed by
cross-metathesis to hydrogenation. This can be completed in stages
following each cross-metathesis, or it may be conducted as a single
hydrogenation step for converting all unsaturated dicarba bridges
present at that point into saturated dicarba bridges (following two
or more cross-metathesis reactions). By convenient selection of the
appropriate time at which to perform the hydrogenation(s), it is
possible for selected dicarba bridges to be saturated and for other
dicarba bridges to remain unsaturated. The hydrogenation step(s)
is/are suitably homogeneous hydrogenation.
[0023] Thus, where all dicarba bridges are desired to be saturated,
the process described above may comprise the further steps of:
[0024] subjecting the unsaturated dicarba bridge formed between the
first pair of complementary metathesisable groups to hydrogenation,
and [0025] subjecting the unsaturated dicarba bridge formed between
the second pair of complementary metathesisable groups to
hydrogenation, wherein each homogenous hydrogenation is performed
either separately or in the one step.
[0026] According to one embodiment, the hydrogenation of the
complementary methathesisable groups takes place immediately after
cross-metathesis of that pair of complementary metathesisable
groups. The hydrogenation is suitably a homogeneous
hydrogenation.
[0027] It is an option to perform each intramolecular
cross-metathesis reaction under microwave radiation conditions.
[0028] In the detailed description, a particularly suitable series
of reactions appropriate to the formation of two and three dicarba
bridges is described.
[0029] The method of the present invention is particularly suited
to the formation of peptides with dicarba bridges. In this event,
the reactable compound, or one of the reactable compounds, is
attached to a solid support. Suitable conditions for performing the
reaction, taking into account the difficulties that are introduced
as a result of conducting the reaction on a solid support, are
described in the detailed description. It is noted however that
compounds other than peptides can also suitably be prepared through
a reactable compound which is attached to a solid support, using
the microwave cross-metathesis reaction conditions.
[0030] A strategy that is an alternative to microwave irradiation
has been devised for improving the performance of a
cross-metathesis between two complementary metathesisable groups
(olefins) in the one organic compound.
[0031] According to this embodiment, there is provided a method for
the synthesis of an organic compound with a dicarba bridge,
comprising: [0032] synthesising a reactable organic compound to
contain a pair of unblocked complementary metathesisable groups,
and a turn-inducing group in between the pair of complementary
metathesisable groups, and [0033] subjecting the reactable organic
compound to cross-metathesis to form a compound with an unsaturated
dicarba bridge.
[0034] If the target organic compound is to contain a saturated
dicarba bridge, the compound is subjected to hydrogenation
(suitably homogeneous hydrogenation).
[0035] This method is particularly suited to the synthesis of
peptides with dicarba bridges.
[0036] The present invention also provides for a compound produced
by the method of the invention. The compound may be a peptide with
at least one dicarba bridge, or may be any other organic compound
with a dicarba bridge.
2.1 BRIEF DESCRIPTION OF THE FIGURES
[0037] FIG. 1 is a reaction scheme demonstrating some possible
locations for the complementary metathesisable groups, in a
peptide.
[0038] FIG. 2 is a .sup.1H n.m.r. spectrum for assessing binding
between dienamide 57 and catalyst, forming a
ruthenium-vinylalkylidene complex 73 (spectrum a), a new species 74
(spectrum b) after 60 minutes, and complex 74 (spectrum c) after 18
hours.
[0039] FIG. 3 is a .sup.1H n.m.r. spectrum of compounds 83, 19 and
82, showing separation of characteristic peaks for each.
[0040] FIG. 4 is a graph of catecholamine release from
dicarba-conotoxims 118 and 119.
[0041] FIG. 5 is a gas chromatogram trace for commercial trans
2-butene, showing trans 2-butene (A), cis 2-butene (B) and catalyst
poison 1,3-butadiene.
3.0 DETAILED DESCRIPTION
[0042] As described above, this application relates to the
formation of organic compounds containing dicarba bridges.
3.1 Types of Compounds and Groups
[0043] The term organic compound is used in its broadest sense to
refer to organic, carbon-containing compounds, as opposed to
inorganic compounds that are not based on carbon. To the extent
that the method can be used to prepare organic ligands for
organometallic compounds, this is also encompassed. Specific
examples of organic compounds that the invention is particularly
suited to are peptides.
[0044] The term "peptide" is used in this specification in its
broadest sense to refer to oligomers of two or more amino acids.
The term "side chain" is used in the usual sense to refer to the
side chain on the amino acid, and the backbone to the
H.sub.2N--(C).sub.x--CO.sub.2H (where x=1, 2 or 3) component, in
which the carbon in bold text bears the side chain (the side chain
being possibly linked to the amino nitrogen, as in the case of
proline).
[0045] One class of peptides of interest are the
peptidomimetics--that is, a peptide that has a series of amino
acids that mimics identically or closely a naturally occurring
peptide, but with at least one dicarba bridge, and optionally one
or more further differences, such as the removal of a cystine
bridge, a change by up to 20% of the amino acids in the sequence,
as non-limiting examples. Of particular interest are dicarba
analogues of naturally-occurring disulfide-containing peptides, in
which one or more of the disulfide bonds are replaced with dicarba
bridges. These may also be classed as pseudo-peptides.
[0046] The term "amino acid" is used in its broadest sense and
refers to L- and D-amino acids including the 20 common amino acids
such as alanine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine and valine (illustrated in the Appendix); and
the less common amino acid derivatives such as homo-amino acids,
N-alkyl amino acids, dehydroamino acids, aromatic amino acids and
.alpha.,.alpha.-di substituted amino acids, for example, cystine,
5-hydroxylysine, 4-hydroxyproline, .alpha.-aminoadipic acid,
.alpha.-amino-n-butyric acid, 3,4-dihydroxyphenylalanine,
homoserine, .alpha.-methylserine, ornithine, pipecolic acid, ortho,
meta or para-aminobenzoic acid, citrulline, canavanine, norleucine,
.delta.-glutamic acid, aminobutyric acid, L-fluorenylalanine,
L-3-benzothienylalanine and thyroxine; .beta.-amino acids (as
compared with the typical .alpha.-amino acids) and any amino acid
having a molecular weight less than about 500. The term also
encompasses amino acids in which the side chain of the amino acid
comprises a metathesisable group, as described herein. Further, the
amino acid may be a pseudoproline (.psi.Pro).
[0047] The amino acids may be optionally protected. The term
"optionally protected" is used herein in its broadest sense and
refers to an introduced functionality which renders a particular
functional group, such as a hydroxy, amino, carbonyl or carboxyl
group, unreactive under selected conditions and which may later be
optionally removed to unmask the functional group. A protected
amino acid is one in which the reactive substituents of the amino
acid, or the amino group or carboxyl group of the amino acid are
protected. Suitable protecting groups are known in the art and
include those disclosed in Greene, T. W., "Protective Groups in
Organic Synthesis" John Wiley & Sons, New York 1999, (the
contents of which are incorporated herein by reference) as are
methods for their installation and removal.
[0048] Preferably the N-protecting group is a carbamate such as,
9-fluorenylmethyl carbamate (Fmoc), 2,2,2-trichloroethyl carbamate
(Troc), t-butyl carbamate (Boc), allyl carbamate (Alloc),
2-trimethylsilylethyl (Teoc) and benzyl carbamate (Cbz), more
preferably Fmoc.
[0049] The carboxyl protecting group is preferably an ester such as
an alkyl ester, for example, methyl ester, ethyl ester, t-Bu ester
or a benzyl ester.
[0050] The amino acids may be protected, for example, the carboxyl
groups of aspartic acid, glutamic acid and .alpha.-aminoadipic acid
may be esterified (for example as a C.sub.1-C.sub.6 alkyl ester),
the amino groups of lysine, ornithine and 5-hydroxylysine, may be
converted to carbamates (for example as a C(.dbd.O)OC.sub.1-C.sub.6
alkyl or C(.dbd.O)OCH.sub.2Ph carbamate) or imides such as
thalimide or succinimide, the hydroxyl groups of 5-hydroxylysine,
4-hydroxyproline, serine, threonine, tyrosine,
3,4-dihydroxyphenylalanine, homoserine, .alpha.-methylserine and
thyroxine may be converted to ethers (for example a C.sub.1-C.sub.6
alkyl or a (C.sub.1-C.sub.6 alkyl)phenyl ether) or esters (for
example a C.dbd.OC.sub.1-C.sub.6 alkyl ester) and the thiol group
of cysteine may be converted to thioethers (for example a C1-C6
alkyl thioether) or thioesters (for example a
C(.dbd.O)C.sub.1-C.sub.6 alkyl thioester).
[0051] The term "dicarba bridge" is used broadly, unless the
context indicates otherwise, to refer to a bridging group that
includes the sequence --C--C--. This encompasses both the
unsaturated (--C.dbd.C--) and saturated (--C--C--) dicarba
sequence. The atoms directly attached to the carbon atoms of the
dicarba sequence (--C--C--) are typically H, although further or
alternative reactions can be performed to introduce substituents
other than hydrogen onto the carbon atoms of the dicarba sequence
of the dicarba bridge. Hydrogenated dicarba bridge refers to the
specific case where the dicarba bridge is --CH.sub.2--CH.sub.2--.
The term unsaturated hydrogen dicarba bridge is used to refer to
--CH.dbd.CH--. This may be cis- or trans-in geometry.
[0052] In addition to the dicarba sequence, the dicarba bridge may
include any other series of atoms, typically selected from C, N, O,
and P, although the atoms to either side of the dicarba sequence
are preferably carbon, and with the proviso that the nitrogen atoms
present in the compound during metathesis are not free amines
(protected amines, such as carbamates, are acceptable). Thus, the
dicarba bridge encompasses the following possible bridges, as
illustrative examples:
##STR00001##
[0053] In IV, R.sub.1 and R.sub.2 are each independently selected
from any divalent linking group. Such divalent linking groups
should not be groups that poison the metathesis catalyst. Most free
amines poison metathesis catalysts and therefore are preferably
protected or avoided during methathesis.
[0054] The dicarba bridge may form a bridge between two separate
reactable organic compounds, to form an intermolecular bridge, or
it may form a bridge between two points in a single reactable
organic compound, so as to form an intramolecular bridge, otherwise
known as a ring. It is particularly difficult to form
intramolecular bridges, due to steric hindrance, and the need to
bring the reactable (metathesisable) groups together. The use of
microwave radiation in the cross-metathesis step has enabled this
to occur, or occur more efficiently.
[0055] "Reactable organic compound" is a term used to refer to the
organic compound that is subjected to the reaction, as distinct
from the target organic compound, to facilitate understanding of
which "organic compound" is being referred to in the process. The
"reactable" organic compound is therefore any compound that can be
subjected to the reaction described, and using other terminology
may be considered to be a starting material, an intermediate, a
reagent or otherwise.
[0056] In this specification, including the claims which follow,
except where the context requires otherwise due to express language
or necessary implication, the word "comprising" or variations such
as "comprise" or "comprises" is used in the inclusive sense, to
specify the presence of the stated features or steps but not to
preclude the presence or addition of further features or steps.
[0057] As used in the specification, the words "a", "an" and "the"
include the plural equivalents, unless the context clearly
indicates otherwise. Thus, for example, reference to "an amino
acid" includes one or more amino acids.
[0058] The method for the formation of dicarba bridges involves the
use of complementary pairs of metathesisable groups on a
compound.
3.2 Cross-Metathesis
[0059] Cross-metathesis is a type of metathesis reaction involving
the formation of a single olefin bond across two unblocked, or
reactive olefins, to form a new olefinic bridge spanning across the
two reactive olefins. In a general sense, metathesis can be
described as the mutual intermolecular exchange of alkylidene (or
carbene) fragments between two olefins promoted by metal-carbene
complexes. The cross-metathesis is conducted with a metathesis
catalyst. There are many metathesis catalysts known in the art.
Examples of suitable catalysts are the ruthenium catalysts, such as
Grubbs' catalyst--first and second generation. For details of other
suitable cross-metathesis catalysts, reference is made to Grubbs,
R.H. Handbook of Metathesis; Wiley-VCH: New York, 2003; 1204 pages,
3 volumes, the entirety of which is incorporated by reference. New
catalysts are being developed all the time, and any of these new
cross-metathesis catalysts can be used. For additional information
on this reaction, and appropriate conditions and catalysts for the
performance of the reaction, reference is also made to Chatterjee
et al, J. Am, Chem, Soc, 2003, 125, 11360-11370, the entirety of
which is incorporated herein by reference.
[0060] Ring-closing metathesis is a particular example of
cross-metathesis where the two reactive olefins are on the one
compound, so as to form an intramolecular bridge, or ring.
3.3 Blocking and Activation
[0061] For metathesis to occur between two alkylidenes (olefins),
the alkylidenes must not be blocked by any steric or electronic
blocking groups. A steric blocking group is any bulky group that
sterically prevents the metathesis from taking place in the
presence of a cross-metathesis catalyst. Examples of steric
blocking groups on an olefin are alkyl. Prenylglycine is an example
of an amino acid containing a dialkyl-blocked olefin side chain
(specifically, dimethyl-blocked). Removal of one or both of the
blocking groups unblocks the olefin, and enables the
cross-metathesis to take place. It is noted that the pair of
metathesisable groups that remain after unblocking need not be
identical--a mono-substituted olefin (such as a mono-methylated
olefin) and an unsubstituted olefin (being unsubstituted at the
open olefinic end) can form a suitable pair of cross-metathesisable
groups. The term "complementary" is used to indicate that the pair
of unblocked metathesisable groups are not necessarily identical,
but are merely complementary in the sense that cross-metathesis can
take place across the two olefinic groups.
[0062] Electronic blocking refers to the presence of a group on the
reactable organic compound or compounds that modifies the
electronic nature of the olefin group of the reactable organic
compound (which would otherwise undergo cross-metathesis), so as to
prevent that olefin group from undergoing cross-metathesis. An
example of an electronic blocking group is a conjugated double
bond--that is, a double bond located in an .alpha.-.beta.
relationship to the olefinic group that would otherwise undergo
cross-metathesis. The .alpha.-.beta.-unsaturation withdraws
electrons away from the olefinic cross-metathesisable group, to
cause electronic blocking preventing cross-metathesis from taking
place.
[0063] By using a combination of blocking mechanisms, a series of
pairs of cross-metathesisable olefinic groups in the reactable
organic compound or compounds can be designed, with different
reaction conditions to effect selective unblocking of particular
pairs. In this way, it becomes possible to regioselectively
synthesise multiple dicarba bridges (inter and/or intramolecular)
in compounds.
3.4.1 Microwave Reaction Conditions
[0064] It has been found that when the cross-metathesis reaction is
performed under microwave reaction conditions, the reaction may
take place in situations where the reaction would not otherwise
take place--for instance, when the metathesisable groups are
unblocked, but the arrangement, length or spatial orientation of
the reactable organic compound prevents the metathesisable groups
from being close enough to one another to enable the reaction to
proceed. An alternative strategy is described in Section 3.4.2.
[0065] The microwave reaction conditions involve applying microwave
radiation to the reactable organic compounds in the presence of the
cross-metathesis catalyst for at least part of the reaction,
usually for the duration of the reaction. The microwave or
microwave reactor may be of any type known in the art, operated at
any suitable frequency. Typical frequencies in commercially
available microwave reactors are 2.45 GHz, at a power of up to 500
W, usually of up to 300 W. The temperature of the reaction is
preferably at elevated temperature, as a consequence of the
microwave radiation, preferably at reflux, or around 100.degree.
C., as is appropriate in the case. The reaction is preferably
performed in a period of not more than 5 hours, suitably for up to
about 2 hours.
3.4.2 Turn-Inducing Groups
[0066] There is a strategy that is an alternative to microwave
irradiation that has been devised for improving the performance of
a cross-metathesis between two complementary metathesisable groups
(olefins) in the one organic compound.
[0067] According to this embodiment, there is provided a method for
the synthesis of an organic compound with a dicarba bridge,
comprising: [0068] synthesising a reactable organic compound to
contain a pair of unblocked complementary metathesisable groups,
and a turn-inducing group in between the pair of complementary
metathesisable groups, and [0069] subjecting the reactable organic
compound to cross-metathesis to form a compound with an unsaturated
dicarba bridge.
[0070] If the target organic compound is to contain a saturated
dicarba bridge, the compound is subjected to hydrogenation
(suitably homogeneous hydrogenation).
[0071] This method is particularly suited to the synthesis of
peptides with dicarba bridges.
[0072] Peptides are generally quite linear, as the component amino
acids (especially when these are the 20 common amino acids, with
exception of proline) and the backbone of the peptide, is linear.
Proline, with the ring structure linking to the amino nitrogen
atom, induces a turn or a bend in an otherwise linear peptide. This
is a naturally-occurring turn-inducing group. This embodiment is
particularly suited to those peptides that do not contain a
naturally-occurring turn-inducing amino acid. In this case, a
synthetic (non-naturally occurring) turn-inducing group is located
in the compound--or in the amino acid sequence.
[0073] Preferably the turn-inducing group is a turn-inducting amino
acid or protein, and is preferably synthetic (non-naturally
occurring). Examples of suitable synthetic turn-inducing amino
acids are the pseudoprolines, including derivatives of serine,
threonine and cysteine which have been derivatised to contain a
cyclic group between the amino acid sidechain (via the --OH or --SH
group), and the amino nitrogen atom. A typical derivatising agent
is CH.sub.3--C(.dbd.O)--CH.sub.3, such that the turn-inducing amino
acids are:
##STR00002##
[0074] After cross-metathesis, the pseudoproline(s) are converted
back to the underivatised amino acid (serine, threonine or
cysteine) by removal of the derivatiseing agent. The conditions for
cleavage from a solid support will achieve this.
[0075] According to the present invention, there is provided a
method for the synthesis of a peptide with an intramolecular
dicarba bridge, the method comprising: [0076] synthesising a
peptide comprising a series of amino acids supported on a solid
support, wherein two amino acids comprise a first pair of
complementary metathesisable groups, and one amino acid between
said amino acids comprising the first pair of complementary
metathesisable groups in the series which is a turn-inducing amino
acid, and [0077] subjecting the peptide to cross-metathesis to form
a peptide with an unsaturated dicarba bridge between the amino
acids bearing the metathesisable groups.
[0078] The method may further comprise one or more of the following
additional steps: [0079] subjecting the unsaturated discarba bridge
to hydrogenation to form a peptide with a saturated intramolecular
dicarba bridge; [0080] cleaving the peptide from the solid
support.
[0081] If the turn-inducing amino acid is one of pseudo-serine,
pseudo-proline or pseudo-cysteine, then the method may further
comprise the step of converting the pseudo-serine, pseudo-proline
or pseudo-cysteine to serine, proline or cysteine,
respectively.
[0082] The process can be combined with the other preferred
features described herein.
3.5 Solvents
[0083] Particularly for reactions conducted with the (or one of
the) reactable organic compound(s) attached to a solid support such
as a resin, the cross-metathesis is preferably performed in a
solvent combination of a resin-swelling solvent, with a
co-ordinating solvent for the catalyst. In resin-supported
reactions, swelling of the resin is required to avoid "clumping",
but such solvents are not generally compatible with
cross-metathesis catalysts. For example, polystyrene-based resins
show optimal swelling in chlorinated solvents such as
dichloromethane, however these solvents are not compatible with
hydrogenation catalysts. The solvents react with such catalysts to
compromise catalyst function--which in turn reduces the catalytic
cycle (or turn-over number--TON), resulting in incomplete
conversion. It was found that the addition of a small amount of a
co-ordinating solvent for the catalyst, such as an alcohol
(methanol, isopropanol, etc) which can co-ordinate into a vacant
site of the catalyst to facilitate stability, overcame this
problem. The co-ordinating solvent is suitably used in an amount of
about 1-30%, for example constituting 10% of the solvent, by
volume. The resin swelling agent may be any polar solvent known to
swell the resin, such as dichloromethane. Other suitable solvents
for a range of resins are as set out in Santini, R., Griffith, M.
C. and Qi, M., Tet. Lett., 1998, 39, 8951-8954, the entirety of
which is incorporated herein by reference.
3.6 Solid Supports
[0084] The (or one of the) reactable organic compound(s) is
preferably attached to a solid support--especially in the case of
peptide reactable organic compound(s). A plethora of solid supports
are known and available in the art, and include pins, crowns,
lanterns and resins. Examples are polystyrene-based resins
(sometimes referred to as solid supports), including cross-linked
polystyrene containing some divinylbenzene (eg 1%), functionalised
with linkers (or handles) to provide a reversible linkage between
the reactable organic compound (which may be a peptide sequence
containing side-chains with cross-metathesisable groups) and the
resin. Examples are the Wang resin, Rink amide resin,
BHA-Gly-Gly-HMBA resin and 2-chlorotrityl chloride resin, which are
all polystyrene-based. Other forms of solid supports that may not
necessarily be characterised as resins can also be used.
[0085] It has been surprisingly found that using the microwave
reaction conditions, it is possible to have a higher solid support
loading than is conventionally used in peptide synthesis on solid
supports. Typical solid support loadings are at the 0.1 mmol/g
level, but microwave radiation (optionally combined with solvent
choice, as described above) overcomes the aggregation problems at
higher solid support loadings, so that solid support loading at
around 0.9 mmol/g (nine times higher) is achievable. As a
consequence, one embodiment of the invention relates to the
performance of the reaction at high solid support loadings--that
is, at loadings of 0.2 mmol/g and above, such as 0.5 mmol/g and
above.
3.7 Hydrogenation
[0086] The product of the cross-metathesis reaction is a compound
with an unsaturated dicarba bridge. If the target organic compound
is to contain a saturated dicarba bridge, the process further
comprises the step of subjecting the unsaturated dicarba bridge to
hydrogenation (suitably homogeneous hydrogenation).
[0087] Hydrogenation of the dicarba bridge is performed with a
catalyst that is chemoselective for unblocked non-conjugated
carbon-carbon double bonds, as distinct from other double bonds
(such as carbon-oxygen double bonds in carbonyl groups and
carboxylic acids, and blocked conjugated double bonds). One notable
example of a suitable catalyst is Wilkinson's catalyst. Wilkinson's
catalyst and catalysts like it are not asymmetric hydrogenation
catalysts but however as this type of hydrogenation does not form a
new chiral centre this is acceptable for this form of hydrogenation
reaction. Although the use of asymmetric hydrogenation catalyst is
not necessary in the hydrogenation of the dicarba bridge,
asymmetric hydrogenation catalysts can nevertheless be used.
Suitable catalysts are well known in the art, and include the range
of catalysts described for this purpose in Ojima, I. Catalytic
Asymmetric Synthesis; Wiley-VCH: New York, 2000; Second Edition,
Chapter 1, 1-110, the entirety of which is incorporated by
reference. New catalysts having such properties are developed from
time to time, and these may also be used. Further examples of
suitable asymmetric hydrogenation catalysts are the chiral
phosphine catalysts, including chiral phospholane Rh(I) catalysts.
Catalysts in this class are described in U.S. Pat. No. 5,856,525.
Such homogenous hydrogenation catalysts are tolerant of sulfide,
and disulfide bonds, so that the presence of disulfide bonds and
the like will not interfere with the synthetic strategy. The
hydrogenation can be conducted at any temperature, such as room
temperature or at elevated temperature. The reaction is typically
conduced at elevated pressure, although if slower reaction times
can be tolerated, the reaction can be performed at atmospheric
pressure.
[0088] In other stages of the process in which hydrogenation is
used as a strategy for unblocking complimentary methasisable
groups, it may be beneficial for the hydrogenation catalyst used in
those reactions to be asymmetric to stereoselectively form a new
chiral centre. Nevertheless, if a racemic mixture can be tolerated,
a catalyst such as Wilkinson's catalyst could be used.
[0089] Homogeneous hydrogenation is used in its broadest sense to
refer to catalytic hydrogenations conducted in one phase such as a
liquid phase, where the liquid phase contains the substrate
molecule/s and solvent. More than one solvent, such as
organic/aqueous solvent combinations, or fluorous solvent
combinations, non-aqueous ionic pairs, supercritical fluids, or
systems with soluble polymers may also be employed. This is
distinct from heterogeneous reactions, which involve more than one
phase--as in the case of hydrogenations performed with
solid-supported catalysts in a liquid reaction medium.
3.8 Regioselective Formation of Multiple Dicarba Bridges
[0090] The strategy for the formation of a dicarba bridge described
above can be combined with other reaction steps for the formation
of an organic compound with a dicarba bridge and a disulfide
bridge, or for the formation of organic compounds with multiple
dicarba bridges, optionally with disulfide bridges.
[0091] To form a plurality of (i.e. two or more) dicarba bridges,
it is necessary to include at appropriate locations in the reactive
organic compound or compounds pairs of complementary metathesisable
groups which are blocked or deactivated for the times when
different pairs of metathesisable groups are being linked together,
and unblocked or "activated" to enable reaction to occur between
those pairs. Accordingly, for each bridge-forming pair, there needs
to be an unblocking reaction available that will selectively
unblock the required pairs.
[0092] The first pair to be subjected to the cross metathesis and
hydrogenation to form a saturated dicarba bridge need not be
blocked during synthesis of the reactive organic compound or
compounds. The compound with this pair of unblocked complementary
metathesisable groups is then subjected to the reactions described
above to form a dicarba bridge (saturated or unsaturated). Suitable
groups for forming the first pair of complementary methathesisable
groups which are not blocked are --CH.dbd.CH.sub.2-- containing
organic moieties, and --CH.dbd.CH--CH.sub.3-- containing moieties.
In the case of peptide synthesis, this may be provided by any amino
acid containing the side chain --CH.dbd.CH.sub.2, optionally with
any divalent linking group linking the carbon at the "open" end
(the --CH.dbd. carbon atom) to the amino acid backbone, such as an
-alkylene-, -alkylene-carbonyl-, and so forth. Examples of
--CH.dbd.CH.sub.2-- containing amino acids and
--CH.dbd.CH--CH.sub.3-- containing amino acids are allyl glycine
and crotyl glycine, respectively. Each of these amino acids
contains the divalent linking group --CH.sub.2-- between the
alkylene and the amino acid (peptide) backbone.
[0093] At the completion of that reaction, (and optionally after
hydrogenation of the first dicarba bridge) the blocked second pair
of complementary metathesisable groups, can be subjected to an
unblocking reaction. This unblocking reaction involves
cross-metathesis with a disposable olefin--which replaces the
steric blocking groups on the olefin (metathesisable group) with
.dbd.CHR.sub.5, described further below.
[0094] Suitable functional groups for forming the second pair of
complementary metathesisable groups are di-blocked alkylenes, such
as the group --CH.dbd.CR.sub.3R.sub.4, in which R.sub.3 and R.sub.4
are each independently selected from any blocking groups, such as
alkyl, for example methyl. Again, there may be a divalent linking
group between the --CH.dbd. carbon atom, and the amino acid
backbone, such as an alkylene group, for instance --CH.sub.2--. An
example of an amino acid containing this group is prenyl glycine,
or protected prenyl glycine.
[0095] The unblocking reaction, or activation reaction, to convert
the pair of di-blocked alkylenes into an unblocked alkylenes
involves subjecting the blocked second pair of complementary
metathesisable groups to cross-metathesis with a disposable olefin,
to effect removal of the blocking groups (such as R.sub.3 and
R.sub.4 in the example shown above).
[0096] It will be understood that in this case, cross metathesis is
used to replace the portion .dbd.CR.sub.3H.sub.4 with another
unblocked portion .dbd.CH.sub.2 or .dbd.CHR.sub.5, (in which
R.sub.5 may be --H, functionalised alkyl or alkyl for instance)
which is then "activated" or "unblocked" and ready for being
subjected to cross-metathesis for the formation of a dicarba
bridge, using the same techniques described above.
[0097] The conditions for this activation-type of cross-metathesis
are the same as described above for the dicarba bridge forming
metathesis. It can be performed under microwave conditions,
although it need not be, as the disposable olefin is a smaller
molecule and less subject to the spatial constraints as larger
reactable organic compounds and single reactable organic compounds
in which intramolecular bridges are to be formed.
[0098] The "disposable olefin" is suitably a mono-substituted
ethylene (such as monoalkylated ethylene--such as propene, which is
a mono-methylated ethylene), or a 1,2-disubstituted ethylene such
as high purity 2-butene (cis, trans or a mixture). Previously,
commercial 2-butene has been attempted to be used as the disposable
olefin in this unblocking reaction, and the reaction is thus
sometimes referred to as "butenolysis". However, until now
commercially available 2-butene (which is a mixture of cis- and
trans-2-butene) has inexplicably not enabled the reaction to
proceed. As detailed further below, a method has been found for
overcoming this problematic reaction.
[0099] The substituents of the substituted ethylene disposable
olefin are substituents that do not participate in the reaction.
Examples are alkyl or a functionalised (substituted) alkyl. The
functional group of the functionalised alkyl is suitably a polar
functional group, to assist with swelling of the solid support, and
solubility. Examples are hydroxy, alkoxy, halo, nitrile and
carboxylic acids/esters. One specific example is the di-ester
functionalised disposable olefin 1,4-diacetoxy-2-butene.
[0100] Thus the disposable olefin is suitable a 1,3-butadiene-free
disposable olefin, or a 1,3-butadiene-free mixture of disposable
olefin and is preferably 1,3-butadiene-free olefin or olefin
mixture of one or more of the following olefins:
##STR00003##
wherein X and Y are each independently selected from the group
consisting of --H, alkyl and alkyl substituted with one or more
substituents selected from halo, hydroxy, alkoxy, nitrile, acid and
ester.
[0101] Preferably, at least one of X and Y is not H.
[0102] Preferably, in the case of the alkyl substituents, the
substituent is preferably on the carbon atom. Preferably the
substituted alkyl is a substituted methyl. According to one
embodiment, at least one of X and Y is a substituted alkyl, such as
a substituted methyl. X and Y may be the same or different. The
olefins may be cis or trans, or mixtures of both.
3.9 Peptide Synthesis
[0103] For the synthesis of a peptide with an intramolecular
dicarba bridge, the method may comprise: [0104] providing a peptide
comprising a series of amino acids supported on a resin, wherein
two amino acids comprise sidechains with a first pair of
complementary metathesisable groups which may be blocked or
unblocked; [0105] unblocking the first pair of complementary
metathesisable groups, if said groups are blocked; and [0106]
subjecting the peptide to cross-metathesis under microwave
radiation conditions to form a peptide with an unsaturated dicarba
bridge between the amino acids bearing the metathesisable
groups.
[0107] The method may further comprise the step of: [0108]
subjecting the unsaturated dicarba bridge to hydrogenation
(suitably homogeneous hydrogenation), to form a peptide with a
saturated intramolecular dicarba bridge.
[0109] Generally, the peptide will be a protected peptide (such as
Fmoc protected). The amino acids can be any of the amino acids
described earlier, but it is convenient for the synthesis of
peptidomimetics for the amino acids to be selected from the 20
naturally-occurring amino acids, .gamma.- and .beta.-amino acids
and from any cross-metathesisable group-bearing analogues thereof.
An example of a cross-metathesisable group-bearing analogue is
allyl glycine.
[0110] The peptide may also be formed so as to have a disulfide
bridge in addition to one or more dicarba bridges. According to
this embodiment, the reactable peptide comprises two protected
cysteine residues, and the method comprises deprotecting the
cysteine residues and oxidising the cysteine residues to form a
disulfide bridge. This may be performed at any stage, such as
before the formation of the dicarba bridge(s) or after. This step
can be combined with the processes described in the following for
the formation of two or three dicarba bridges and a disulfide
bridge. It is noted that the cysteine residues may be located on
the first peptide, on the second peptide (when present) or on a
third peptide.
[0111] For the synthesis of a peptide with two intramolecular
bridges, the method comprises: [0112] providing a first peptide
comprising a series of amino acids supported on a resin, wherein
two amino acids comprise sidechains with a first pair of
complementary metathesisable groups and two amino acids comprise
sidechains with a second pair of blocked complementary
metathesisable groups, [0113] subjecting the peptide to
cross-metathesis under microwave radiation conditions to form a
peptide with an unsaturated dicarba bridge between the amino acids
that bore the first pair of complementary metathesisable groups,
[0114] unblocking the second pair of complementary metathesisable
groups, and [0115] subjecting the peptide to cross-metathesis to
form a peptide with an unsaturated dicarba bridge between the amino
acids that bore the second pair of complementary metathesisable
groups.
[0116] As described previously, one or both unsaturated dicarba
bridges formed between the amino acids that bore the first and
second pairs of complementary metathesisable groups may be
subjected to homogenous hydrogenation, separately or at the same
time.
[0117] For the synthesis of a peptide with the intramolecular
bridge, and a second bridge which is an intermolecular, the method
comprises: [0118] providing a first peptide comprising a series of
amino acids supported on a resin, wherein two amino acids comprise
sidechains with a first pair of complementary metathesisable groups
which may be blocked or unblocked, and one amino acid comprises a
sidechain with a second metathesisable group which may be blocked
or unblocked, with the proviso that the metathesisable groups out
of at least one of the first or the second metathesisable groups
are blocked; [0119] (a) unblocking the first pair of complementary
metathesisable groups, if said groups are blocked; [0120]
subjecting the peptide to cross-metathesis under microwave
radiation conditions to form a peptide with an unsaturated dicarba
bridge between the amino acids bearing the first pair of
complementary metathesisable groups, to form a peptide with an
intramolecular dicarba bridge, and [0121] (b) contacting the first
peptide with a second peptide comprising one amino acid with a
metathesisable group complementary to the second metathesisable
group on the first peptide; [0122] unblocking the second
complementary metathesisable groups, if the second metathesisable
groups are blocked; [0123] subjecting the peptide to
cross-metathesis to form a peptide with an unsaturated dicarba
bridge between the amino acids bearing the second pair of
complementary metathesisable groups, to form a dicarba bridge
between the amino acids that bore the second metathesisable groups,
wherein steps (a) and (b) are performed in either order, so as to
form a peptide with an intermolecular bridge and an intramolecular
bridge.
[0124] The method may further comprise the step or steps of
subjecting one or both of the products of step (a) and step (b) to
hydrogenation (suitably homogeneous hydrogenation) to form a
peptide with a saturated intramolecular dicarba bridge and/or a
saturated intermolecular dicarba bridge.
[0125] These methods may be combined with a third stage of
bridge-formation, to form a peptide with three bridges, one two or
three of which are intramolecular. This is achieved by providing a
third pair of metathesisable groups in the first peptide, or one in
the first peptide and one in the second or in a third peptide to be
coupled to the first peptide through an intermolecular bridge, and
then subjecting the third pair of metathesisable groups to
unblocking to form the compound. In another alternative, a
complimentary metathesisable group can be "added" to the first or
second peptide through the addition of an amino acid or peptide
fragment bearing the metathesisable group. This is illustrated in
FIG. 1.
[0126] For the formation of a peptide with three intramolecular
bridges, the method comprises: [0127] providing a first peptide
comprising a series of amino acids supported on a resin, wherein
two amino acids comprise sidechains with a first pair of
complementary metathesisable groups, two amino acids comprise
sidechains with a second pair of blocked complementary
metathesisable groups and two amino acids comprise sidechains with
a third pair of blocked complementary metathesisable groups, [0128]
subjecting the peptide to cross-metathesis under microwave
radiation conditions to form a peptide with an unsaturated dicarba
bridge between the amino acids bearing the first pair of
complementary metathesisable groups, [0129] optionally subjecting
the unsaturated dicarba bridge to hydrogenation, [0130] unblocking
the second pair of complementary metathesisable groups, [0131]
subjecting the peptide to cross-metathesis to form a peptide with
an unsaturated dicarba bridge between the amino acids that bore the
second pair of complementary metathesisable groups, [0132]
optionally subjecting the unsaturated dicarba bridge to
hydrogenation, [0133] unblocking the third pair of complementary
metathesisable groups, [0134] subjecting the peptide to
cross-metathesis to form a peptide with an unsaturated dicarba
bridge between the amino acids that bore the third pair of
complementary metathesisable groups, and [0135] optionally
subjecting the unsaturated dicarba bridge to hydrogenation.
[0136] Each of these techniques for the synthesis of peptides with
one or more intramolecular bridges may be combined with additional
steps for the formation of one or more intramolecular disulfide
bridges.
[0137] In each of these techniques it is also preferred that the
unblocking reaction specific to the second pair of complementary
metathesisable groups comprise cross-metathesis with a
1,3-butadiene free disposable olefin.
[0138] It will be appreciated that if a peptide sequence is added
later through an intermolecular bridge, the corresponding
metathesisable groups on that peptide need not be blocked--as they
can be added to the reaction at the time of cross-metathesis, after
the unblocking of the groups on the resin-supported peptide.
3.10 Products of Methods
[0139] The present invention also provides for a compound produced
by the method of the invention. The compound may be a peptide with
at least one dicarba bridge, or may be any other organic compound
with a dicarba bridge. Salts, solvates, derivatives, isomers and
tautomers are encompassed in this context.
[0140] Possible products include the dicarba analogues of
cystine-containing peptides. Dicarba analogues refers to peptides
contain the same amino acid sequence as the native peptide, but
with one or more of the bridged cysteine-amino acid residue pairs
substituted with amino acids bearing a dicarba bridge. "Native" is
a term used to refer to the natural or synthetic analogue of a
natural peptide--to be distinguished from the dicarba analogue
being synthesised. Bis- and higher dicarba analogues are of
particular interest, in view of the difficulty in synthesising such
compounds. Examples are the dicarba analogues of Conotoxin ImI
presented in FIG. 6.4. These include the fully dicarba-substituted
analogues (the final three compounds in that figure) and the
partial dicarba analogues (identified as "hybrids" in FIG. 6.4).
Other suitable terminology is the mono-dicarba analogues (in which
one disulfide bridge is replaced with a dicarba bridge), and the
bis-dicarba analogues (two replaced). Thus, the present application
also relates to dicarba analogues of Conotoxin, including the
bis-dicarba, cystino-dicarba and higher-dicarba analogues.
[0141] In FIG. 6.4, the residue between the bridges is represented
as "Hag"--based on its synthesis via this amino acid, although the
double bond of Hag is no longer present. In some cases the new
bridge is unsaturated and bears a new double bond; in other cases
the bridge is saturated. If the peptide was synthesised via another
amino acid, such as crotyl glycine (Crt), Crt would appear in place
of Hag. In fact, the peptides are identical irrespective of whether
they were synthesised via one of these amino acids or the other, as
the dicarba bridge is all that remains from those starting amino
acids. Accordingly, the amino acid indicated in the formula for the
peptide should not be read as limiting the peptide to one made
specifically through that amino acid. Sub (representing the amino
acid suberic acid, which has the cyclised side chain
--(CH.sub.2).sub.4--) could also have been used to represent the
same peptide.
[0142] "Conotoxin" is used in its broadest sense to refer to the
peptides or peptide fragments that are present in the venom of cone
snails of the genus Conus (Conidae). All species which are
encompassed within this genus [class] are contemplated, including
the species Conus imperialis, Conus geographus, Conus textile,
Conus amadis, Conus tulipa, Conus marmoreus, Conus lynceus, Conus
armadillo, Conus geographus and so forth. The peptides within this
class include natural and synthetic peptides, and derivatives of
the naturally-occurring peptides and peptide fragments. Conotoxins
are classified according to their receptor subtype specificity and
the arrangement of disulfide bonds and resultant loop sizes. The
paralytic components of the venom (the conotoxins) that have been
the focus of recent investigation are the .alpha.-, .omega.- and
.mu.-conotoxins. All of these conotoxins act by preventing neuronal
communication, but each targets a different aspect of the process
to achieve this. The .alpha.-conotoxins target nicotinic ligand
gated channels, and the .mu.-conotoxins target the voltage-gated
sodium channels and the .omega.-conotoxins target the voltage-gated
calcium channels. Of particular interest here are the .alpha.-,
.chi.- and .omega.-conotoxins, which contain two or three disulfide
bridges, although .mu.-conotoxins, .delta.-conotoxins,
.kappa.-conotoxins .pi.-conotoxins and conatokins are also
relevant. The conotoxins are generally between 12 and 30 amino acid
residues in length.
[0143] The salts of compounds are preferably pharmaceutically
acceptable, but it will be appreciated that non-pharmaceutically
acceptable salts also fall within the scope of the present
invention, since these are useful as intermediates in the
preparation of pharmaceutically acceptable salts. Examples of
pharmaceutically acceptable salts include salts of pharmaceutically
acceptable cations such as sodium, potassium, lithium, calcium,
magnesium, ammonium and alkylammonium; acid addition salts of
pharmaceutically acceptable inorganic acids such as hydrochloric,
orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric,
sulfamic and hydrobromic acids; or salts of pharmaceutically
acceptable organic acids such as acetic, propionic, butyric,
tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic,
gluconic, benzoic, succinic, oxalic, phenylacetic,
methanesulphonic, trihalomethanesulphonic, toluenesulphonic,
benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic,
edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic,
ascorbic and valeric acids.
[0144] In addition, some of the compounds may form solvates with
water or common organic solvents. Such solvates are encompassed
within the scope of the invention.
[0145] By "derivative" is meant any salt, hydrate, protected form,
ester, amide, active metabolite, analogue, residue or any other
compound which is not biologically or otherwise undesirable and
induces the desired pharmacological and/or physiological effect.
Preferably the derivative is pharmaceutically acceptable.
[0146] The term "tautomer" is used in its broadest sense to include
compounds which are capable of existing in a state of equilibrium
between two isomeric forms. Such compounds may differ in the bond
connecting two atoms or groups and the position of these atoms or
groups in the compound.
[0147] The term "isomer" is used in its broadest sense and includes
structural, geometric and stereo isomers. As the compounds that may
be synthesised by these techniques may have one or more chiral
centres, it is capable of existing in enantiomeric forms.
3.11 New Reagents to Facilitate Production of Dicarba Bridge
Containing Peptides
[0148] The present applicant has synthesised amino acids that are
particularly useful as they enable the formation of a dicarba
bridge when included in a peptide sequence. These amino acids
include prenyl glycine in which the amino group is protected with a
base-removable carbamate-protecting group. A particular example of
this compound is Fmoc-protected prenyl glycine. Fmoc-protected
prenyl glycine is a protected, blocked olefin-containing amino
acid, suitable for forming the "second" of the dicarba bridges in a
peptide, and its synthesis is achieved through the use of a
specific reagent.
[0149] Fmoc-protected prenyl glycine requires preparation through
the cross-metathesis of Fmoc-protected allyl glycine with
2-alkyl-2-butylene (such as 2-methyl-2-butylene) in the presence of
a cross-metathesis catalyst. The reaction is not complete when
isobutylene is used as the olefin in the reaction. The reaction is
suitably conducted at a pressure above 5 psi--preferably at 8 psi
or above--for instance at about 10 psi.
4.0 CONTROLLED SYNTHESIS OF (S,S)-2,7-DIAMINOSUBERIC ACID
A Method for the Regioselective Construction of Dicarba Analogues
of Dicystine-Containing Peptides
[0150] This section describes a solution phase model study for the
development of a methodology that enables the regioselective
formation of dicarba isosteres of cystine bonds. We investigated a
sequence of ruthenium-catalysed metathesis and rhodium-catalysed
hydrogenation reactions of non-proteinaceous allylglycine
derivatives to achieve high yielding and unambiguous formation of
two dicarba bridges. This theory can also be applied to the
synthesis of non-peptide compounds with 2 or 3 dicarba bridges.
4.1 Initial Strategy
[0151] Our initial strategy planned to capitalise on the use of
.alpha.-N-acyl-dienamide 57, a masked precursor to allylglycine
derivatives..sup.118,119 We devised a strategy involving a double
metathesis-hydrogenation sequence (Scheme 4.1). This required a
selective ring closing metathesis of allylglycine units in the
presence of dienamide functionalities. Grubbs et al. have
previously reported that selective cross metathesis can be
accomplished with olefins of varying reactivity..sup.130,182
Terminal olefins such as allylglycine undergo rapid
homodimerisation with both Grubbs' catalyst.sup.120 and second
generation Grubbs' catalyst,.sup.121 whereas the electron-deficient
.alpha.-N-acyl-dienamide 57 should be considerably less reactive.
Subsequent asymmetric hydrogenation of the dienamide moieties would
lead to reactive allylglycine units which could undergo ring
closing metathesis to produce the second carbocycle. The final step
in this catalytic sequence involves hydrogenation of the
unsaturated carbocycles, if required, to afford the saturated
cystine isosteres.
##STR00004##
[0152] In order to validate the proposed strategy, we needed to
show that: i) the dienamide 57 would not react under conditions
required for the ring closing metathesis of allylglycine residues,
ii) asymmetric hydrogenation of the dienamide 57 would proceed in a
highly regioselective and stereoselective manner, iii) ring closing
metathesis of the resulting allylglycine units would proceed in the
presence of an unsaturated carbocycle (without resulting in mixed
cross metathesis products), and iv) the unsaturated carbocycles
could be reduced to afford saturated dicarba bridges. We therefore
conducted a series of independent experiments that would serve as a
model to the peptide system.
4.1.1 Synthesis of Olefinic Moieties
[0153] The dienamide 57 was synthesised according to a literature
procedure reported by Teoh et al..sup.119 from a Homer-Emmons
olefination of a phosphonate ester 39 and an
.alpha.,.beta.-unsaturated aldehyde 58 (Scheme 4.2).
##STR00005##
[0154] The phosphonate, methyl
2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 39, was prepared in
three steps from commercially available acetamide 34 and glyoxylic
acid 41.
##STR00006##
[0155] A mixture of commercially available acetamide 34 and
glyoxylic acid 41 was heated at reflux in acetone to give pure
N-acetyl-2-hydroxyglycine 42 as a viscous yellow oil in
quantitative yield. The .sup.1H n.m.r. spectrum supported formation
of the .alpha.-hydroxyglycine derivative 42 with the appearance of
a methine (H2) doublet and broad amide (NH) doublet at .delta. 5.39
and .delta. 8.65 respectively. Spectroscopic data were in agreement
with those reported in the literature..sup.195
[0156] Treatment of N-acetyl-2-hydroxyglycine 42 with a catalytic
amount of concentrated sulfuric acid in methanol furnished methyl
N-acetyl-2-hydroxyglycinate 43 in 60% yield. These reaction
conditions converted the carboxylic acid to the methyl ester and
the hydroxyl functional group to methyl ether.
[0157] Modification of the reported work-up procedure led to a
significantly improved yield to that reported in the literature
(32%)..sup.196 The presence of two new methoxyl peaks in the
.sup.13C n.m.r. spectrum at .delta. 53.0 and .delta. 56.8 and the
corresponding methyl singlets in the .sup.1H n.m.r. spectrum at
.delta. 3.47 and .delta. 3.82 supported formation of the desired
product 43. Spectroscopic data were also in agreement with those
reported in the literature..sup.196
[0158] The final step in the synthesis of methyl
2-N-acetylamino-2-(dimethoxyphosphinyl)-acetate 39 involved
reaction of methyl N-acetyl-2-hydroxyglycinate 43 with phosphorous
trichloride to produce the intermediate .alpha.-chloro ester.
Nucleophilic attack of the newly introduced chlorine substituent
with trimethyl phosphite gave phosphonate ester 39 as a colourless
solid in low yield (14%). The high solubility of the ester in water
initially led to poor mass recovery. The use of continuous
extraction partially overcame this problem and led to isolation of
the product in satisfactory yield (44%).
[0159] The .sup.1H n.m.r. spectrum confirmed formation of the
target compound 39 with the appearance of a doublet of doublets
attributed to the methine (H2) proton coupling to the phosphorous
(J=22.2 Hz) and amide proton (J=8.8 Hz). The .sup.13C n.m.r.
spectrum displayed similar behaviour with the methine (C2) peak
appearing as a doublet with large coupling to the vicinal
phosphorous atom (J=146.8 Hz). The melting point of the isolated
solid (89-91.degree. C.) was consistent with that reported in the
literature (88-89.degree. C.)..sup.197
[0160] (2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 57 was
synthesised by Homer-Emmons olefination of methyl
2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 39 with commercially
available acrolein 58 in the presence of tetramethylguanidine (TMG)
(Scheme 4.4). Hydroquinone was added to prevent polymerisation of
acrolein 58 and was found to be critical to the success of this
reaction. The reaction requires the addition of base to a solution
of phosphonate 39 in tetrahydrofuran to generate the carbanion 45,
which was then reacted with aldehyde 58 to afford the dienoate 57
as an off-white solid in 85% yield (Scheme 4.4).
##STR00007##
[0161] The .sup.1H n.m.r. spectrum of the product supported
formation of the dienamide 57 with the appearance of signals
corresponding to a new terminal olefin. Doublets at .delta. 5.49
and .delta. 5.61 for H5-E and H5-Z respectively, and an olefinic
methine (H4) multiplet at .delta. 6.47 were consistent with
formation of dienamide 57. The melting point of the isolated solid
(60-62.degree. C.) was also in agreement with that reported in the
literature (61-63.degree. C.)..sup.119
[0162] Our group have demonstrated that high regioselectivity and
enantioselectivity can be achieved in the asymmetric hydrogenation
of dienamide esters. In this case, hydrogenation of dienamide 57
was effected with Rh(I)--(S,S)-Et-DuPHOS under 30 psi of hydrogen
in benzene for 3 hours (Scheme 4.5). Over-reduction of the terminal
olefinic bond was minimal (<3% 59) under these mild conditions.
The (S)-configuration was determined based on literature assignment
for the same transformation.sup.118 and a comparative optical
rotation sign to that reported in the literature for (2S)-methyl
2-N-acetylaminopent-4-enoate 21a..sup.208
##STR00008##
[0163] Asymmetric hydrogenation of dienamide 57 was also performed
with Rh(I)--(R,R)-Et-DuPHOS to facilitate enantiomeric excess
assessment. The reaction proceeded in quantitative conversion and
<5% over-reduced product was detected. Chiral GC indicated the
reactions proceeded with excellent enantioselectivity (95%
e.e.).
[0164] .sup.1H n.m.r. spectroscopy showed the replacement of an
olefinic methine (H3) doublet at .delta. 7.05 with a methylene (H3)
multiplet at .delta. 2.43-2.62. The .sup.13C n.m.r. spectrum also
displayed new methine (C2) and methylene (C3) peaks at .delta. 51.8
and .delta. 36.5 respectively. Spectroscopic data were in agreement
with those reported in the literature..sup.119
4.1.2 Cross Metathesis: Homodimerisation
[0165] Homodimerisation is a type of cross metathesis in which an
olefin self-couples. Conveniently, the only byproduct is a low
molecular weight volatile olefin which is most commonly ethylene
(Scheme 4.6).
##STR00009##
[0166] The mechanism involves an intermolecular exchange of
alkylidene fragments between the metal-carbene catalyst and the
reacting olefin. An unstable metallocyclobutane intermediate then
decomposes to release the homodimer and a volatile olefinic
byproduct (Scheme 4.7).
##STR00010##
[0167] Quantitative homodimerisation of allylglycine derivative 21a
was achieved using Grubbs' catalyst in dichloromethane heated at
reflux (Scheme 4.8). Purification of the crude product by flash
chromatography gave the target compound, (2S,7S)-dimethyl
2,7-N,N'-diacetylaminooct-4-enedioate 60, as a brown oil in 88%
yield.
##STR00011##
[0168] High resolution mass spectrometry confirmed formation of the
desired product 60 with the appearance of a molecular ion plus
sodium peak at m/z 337.1375 for the expected molecular formula
(C.sub.14H.sub.22N.sub.2NaO.sub.6). In addition, the .sup.13C
n.m.r. spectrum displayed a new olefinic methine (C4, 5) peak at
.delta. 128.8, whilst the terminal and methine olefinic (C5 and C4)
peaks of the starting material 21a were absent.
[0169] The solution phase dimerisation of the allylglycine unit 21a
is analogous to ring closing metathesis of allylglycine sidechains
in a peptide (Step 1, Scheme 4.1). In order to regioselectively
construct multiple dicarba bonds within a peptide, via the strategy
shown in Scheme 4.1, the dienamide 57 must not react under the
conditions used for cross metathesis of allylglycine units 21a
(Scheme 4.8).
[0170] The dienamide 57 was therefore subjected to analogous
dimerisation conditions to those described above for allylglycine
21a. .sup.1H n.m.r. spectroscopy confirmed complete recovery of the
starting olefin 57 with no evidence of the dimerised dienoate 61
(Scheme 4.9). These results were very encouraging and supported our
postulate that the dienamide 57 would be electronically compromised
and therefore inert to metathesis (Step 1, Scheme 4.1).
##STR00012##
[0171] Subsequent asymmetric hydrogenation of the dienoate 57 would
activate the olefin to metathesis by producing a reactive
allylglycine unit 21a (Step 2, Scheme 4.1). This hydrogenation
proceeds with excellent stereoselectivity (>95% e.e.) and
regioselectivity (<3% over-reduction) (Section 4.1.1) as it
relies on chelation of the asymmetric Rh(I)-catalyst to the enamide
olefin and amide carbonyl group. The terminal C.dbd.C bond does not
chelate to the catalyst and is therefore not reduced under these
conditions. Similarly, the newly formed C.dbd.C bond, generated via
cross metathesis in Step 1, would be inert to this catalyst.
4.1.3 Dimerisation of an Allylglycine Unit in the Presence of an
Unsaturated Dimer
[0172] In our strategy, the next step involved ring closing
metathesis of allylglycine units in the presence of an unsaturated
carbocycle (Step 3, Scheme 4.1).The solution phase model study
therefore required the dimerisation of allylglycine in the presence
of an unsaturated dimer (Scheme 4.10). A differentially protected
allylglycine derivative 62 was synthesised to facilitate
unambiguous assessment of cross metathesis selectivity.
##STR00013##
4.1.3.1 Synthesis of (2S)-Methyl 2-N-Benzoylaminopent-4-enoate
62
[0173] The benzoyl-protected allylglycine derivative 62 was
prepared via catalytic asymmetric hydrogenation of the dienamide
63. The hydrogenation precursor 63 was synthesised by Horner-Emmons
olefination of the phosphonate ester 64 which was isolated in three
steps from commercially available benzamide 35 and glyoxylic acid
41 (Scheme 4.11).
##STR00014##
[0174] A mixture of commercially available benzamide 35 and
glyoxylic acid 41 was heated at reflux in acetone to give pure
N-benzoyl-2-hydroxyglycine 65 as a colourless solid in quantitative
yield (Scheme 4.12). The .sup.1H n.m.r. spectrum supported
formation of the .alpha.-hydroxyglycine derivative 65 with the
appearance of a methine (H2) doublet and broad amide (NH) doublet
at .delta. 5.60 and .delta. 9.26 respectively. Spectroscopic data
were in agreement with those reported in the
literature..sup.209
##STR00015##
[0175] Treatment of N-benzoyl-2-hydroxyglycine 65 with a catalytic
amount of concentrated sulfuric acid in methanol furnished methyl
N-benzoyl-2-methoxyglycinate 66 in 87% yield (Scheme 4.13). These
reaction conditions converted the carboxylic acid to the methyl
ester and the hydroxyl functional group to the methyl ether.
##STR00016##
[0176] The presence of two new methoxyl peaks in the .sup.13C
n.m.r. spectrum at .delta. 53.2 and .delta. 57.0 and the
corresponding methyl singlets in the .sup.1H n.m.r. spectrum at
.delta. 3.54 and .delta. 3.85 supported formation of the desired
product 66. Spectroscopic data were in agreement with those
reported in the literature..sup.209
[0177] Reaction of methyl N-benzoyl-2-methoxyglycinate 66 with
phosphorous trichloride and trimethyl phosphite in toluene at
70.degree. C. gave the phosphonate ester 64 in 76% yield (Scheme
4.14). The appearance of a methine doublet of doublets (H2) at
.delta. 5.47 was consistent with vicinal phosphorous coupling and
was in agreement with data reported in the literature..sup.210
##STR00017##
[0178] (2Z)-Methyl 2-N-benzoylaminopenta-2,4-dienoate 63 was
synthesised by Horner-Emmons olefination of methyl
2-N-benzoylamino-2-(dimethoxyphosphinyl)acetate 64 with
commercially available acrolein 58 in the presence of
tetramethylguanidine (TMG) (Scheme 4.15). The reaction proceeded
through a nucleophilic intermediate 67 which reacted with acrolein
58 to afford the dienoate 63 as colourless needles in 80%
yield.
[0179] The .sup.1H n.m.r. spectrum displayed a new terminal olefin
doublet of doublets at .delta. 5.50 and .delta. 5.64 corresponding
to H5-E and H5-Z respectively in addition to a well-defined methine
(H4) doublet of doublet of doublets at .delta. 6.56. Spectroscopic
data were in agreement with those reported in the
literature..sup.211
##STR00018##
[0180] The final step in the synthesis of (2S)-methyl
2-N-benzoylaminopent-4-enoate 62 involved asymmetric hydrogenation
of the dienamide 63..sup..dagger. Use of Rh(I)--(S,S)-Et-DuPHOS
under 30 psi H.sub.2 in benzene gave the allylglycine derivative 62
with excellent enantioselectivit.sup..dagger-dbl. (100% e.e.,
Scheme 4.16). Approximately 7% of the over-reduced product 68 was
obtained under these conditions and attempts to separate
allylglycine 62 from 68 were unsuccessful. The contaminated sample
of allylglycine 62 was used in subsequent reactions as the presence
of the inert impurity 68 would not interfere in the catalytic
strategy. .sup..dagger. The benzoyl-protected olefin 62 can also be
prepared in two steps from commercially available L-allylglycine
((2S)-2-aminopent-4-enoic acid)..sup..dagger-dbl. Asymmetric
hydrogenation of the dienamide 63 with Rh(I)--(R,R)-Et-DuPHOS was
performed in order to facilitate enantiomeric excess determination.
Chiral GC confirmed that the (R)- and (S)-allylglycine derivatives
62 were produced in 100% e.e.
[0181] Formation of allylglycine 62 was supported by .sup.13C
n.m.r. spectroscopy which showed the replacement of an olefinic
methine (C3) peak with a new methylene signal at .delta. 36.8 and a
methine (C2) peak at .delta. 52.1. Spectroscopic data were in
agreement with those reported in the literature..sup.212
##STR00019##
4.1.3.2 Dimerisation of (2S)-Methyl 2-N-Benzoylaminopent-4-enoate
62
[0182] The benzoyl-protected allylglycine unit 62 was
quantitatively homodimerised under general cross metathesis
conditions using Grubbs' catalyst (Scheme 4.17). The loss of
ethylene drives the metathesis reaction to completion.
##STR00020##
[0183] Purification by flash chromatography furnished
(2S,7S)-dimethyl 2,7-N,N'-dibenzoylaminooct-4-enedioate 69 as a
pale brown solid in 82% yield. .sup.1H n.m.r. spectroscopy
supported synthesis of the dimer 69 with the replacement of
terminal olefin peaks by a new methine (H4, 5) triplet at .delta.
5.49. The accurate mass spectrum also displayed a molecular ion
plus sodium peak at m/z 461.1695 which is consistent with that
expected for the molecular formula
C.sub.24H.sub.26N.sub.2NaO.sub.6.
[0184] With the benzoyl-protected allylglycine 62 in hand and
characterisation of its dimer 69 complete, we attempted the cross
metathesis of allylglycine 62 in the presence of the unsaturated
N-acetyl-protected allylgycine dimer 60 (Step 3, Scheme 4.4).
4.1.3.3 Dimerisation of (2S)-Methyl 2-N-Benzoylaminopent-4-enoate
62 in the presence of (2S,7S)-Dimethyl
2,7-N,N'-Diacetylaminooct-4-enedioate 60
[0185] Cross metathesis of allylglycine derivative 62 in the
presence of unsaturated dimer 60 proceeded with Grubbs' catalyst to
afford dimer 69 (Scheme 4.18). No mixed cross metathesis product 70
was observed. However, use of the more reactive metathesis
catalyst, second generation Grubbs' catalyst, did lead to a mixture
of cross metathesis products, 69, 70 and recovered dimer 60. The
complicated .sup.1H n.m.r. spectrum did not allow the distribution
of products to be quantified but mass spectrometry confirmed the
presence of homodimers 60 and 69 and the mixed cross metathesis
product 70.
##STR00021##
[0186] These results indicated that in a peptide application of
this strategy (Step 3, Scheme 4.1), selective cyclisation of the
allylglycine units will only be successful in the presence of
Grubbs' catalyst and the use of the more reactive second generation
Grubbs' catalyst must be avoided. With successful completion of
Step 3, we moved to the last step of the strategy (Step 4, Scheme
4.1).
4.1.4 Wilkinson's Hydrogenation of Unsaturated Dimers
[0187] The final step in the model sequence involved reduction of
the unsaturated dimers 60 and 69 to give the corresponding
saturated dicarba bridges 71 and 72. Homogeneous hydrogenation of
dimers 60 and 69 with Wilkinson's catalyst,
Rh(I)(PPh.sub.3).sub.3Cl, under mild experimental conditions, gave
the saturated diaminosuberic acid derivatives 71 and 72 in
excellent yields (>99%) (Scheme 4.19). We employed a homogeneous
catalyst in order to facilitate the on-resin application of this
hydrogenation which would otherwise be complicated by the more
commonly employed heterogeneous systems such as palladium on
charcoal.
##STR00022##
[0188] Formation of the saturated dimers 71 and 72 was supported by
spectroscopic analysis which displayed new methylene proton (H3, 4)
and carbon (C3, 4) signals in the .sup.1H and .sup.13C n.m.r.
spectra respectively.
4.1.5 Dimerisation of Allylglycine 21a in the presence of
(2Z)-Methyl 2-N-Acetylaminopenta-2,4-dienoate 57
[0189] These results looked very promising: We had successfully
completed all four steps in our devised synthesis (Scheme 4.1).
However, our attempts to dimerise allylglycine 21a in the presence
of dienamide 57 were unsuccessful with both first and second
generation Grubbs' catalysts (Scheme 4.20). The inclusion of
dienamide 57 also hampered dimerisation of benzoyl-protected
allylglycine 62 and led to complete recovery of the starting
dienoate 57 and allylglycine unit 62.
##STR00023##
[0190] .sup.1H n.m.r. binding studies between the catalyst and
dienamide 57 (ratio of 1:1) showed that the dienamide 57 rapidly
coordinated to the ruthenium centre forming a
ruthenium-vinylalkylidene complex 73 (spectrum a in FIG. 2). Within
60 minutes, complex 73 had diminished and a new ruthenium species
74 was generated (spectrum b in FIG. 2). The second species is
postulated to involve coordination of the ester carbonyl group to
the ruthenium centre and liberation of a tricyclohexylphosphine
ligand 75. Formation of complex 74 was complete within 2 hours and
was stable and unreactive over 18 hours (spectrum c in FIG.
2)..sup.213 Unfortunately, attempts to isolate this complex 74 were
unsuccessful.
##STR00024##
[0191] Furthermore, attempts to regenerate the dienamide 57 from
the ruthenium-carbonyl chelate 74 via reaction with ethyl vinyl
ether and formation of the Fischer-type carbene complex,.sup.214
failed due to conjugate addition of liberated
tricyclohexylphosphine 75 to the dienamide substrate 57. This
highlighted the sensitivity of acrylate 57 to N- and P-based
nucleophiles and potential problems that could arise during peptide
synthesis, where piperidine is routinely used to facilitate
Fmoc-cleavage from residues prior to coupling.
[0192] Although the dienamide 57 was unexpectedly reactive to
Grubbs' catalyst, the proposed strategy showed potential. Solution
phase experiments with Steps 2-4 (Scheme 4.21) were not problematic
and indicated that multiple dicarba bond formation was indeed
feasible via a modified strategy. The first step, however, required
revision. We postulated that the presence of a substituent at the
olefinic terminus of the dienamide substrate might impede binding
to the metathesis catalyst and therefore allow the ring closing
metathesis of the more reactive allylglycine sidechains to
proceed.
##STR00025##
4.2 Revised Strategy
[0193] A revised strategy was investigated centering on the use of
non-proteinaceous, terminally functionalised allylglycine units.
This modified route involved: i) metathesis of allylglycine units
in the presence of a terminal-phenyl substituted dienamide 76, and
ii) subsequent hydrogenation of the dienamide 76 to yield a more
reactive olefin 77 for the second ring closing metathesis (Scheme
4.22). We postulated that the presence of a phenyl substituent at
the olefin terminus might impede binding of the metathesis catalyst
and circumvent the problems experienced in the first strategy. The
solution phase model studies of this revised strategy therefore
commenced with the synthesis of the phenyl-substituted dienamide
76.
##STR00026##
4.2.1 Synthesis of (2Z)-Methyl
2-N-Acetylamino-5-phenylpenta-2,4-dienoate
[0194] The dienamide 76 was prepared according to a procedure by
Burk et al..sup.117 from a Horner-Emmons olefination of methyl
2-N-acetylamino-2-(dimethoxyphosphinyl)-acetate 39 and commercially
available trans-cinnamaldehyde 78 (Scheme 4.23). The phosphonate 39
was prepared in three steps from commercially available acetamide
34 and glyoxylic acid 41.
[0195] The dienamide 76 was isolated as an off-white solid in 74%
yield. Mass spectrometry supported formation of the dienoate 76
with a molecular ion plus proton peak at m/z 246.2 which is
consistent with that expected for molecular formula
C.sub.14H.sub.16NO.sub.3. Spectroscopic data were in agreement with
those reported in the literature..sup.117
##STR00027##
4.2.2 Solution Phase Reactions with Dienamide 76
[0196] .sup.1H n.m.r. binding studies of a 1:1 mixture of Grubbs'
catalyst and dienamide 76 showed no ruthenium-vinylalkylidene
formation. Hence, this suggested that the poor chelating properties
of the modified dienamide 76 to Grubbs' catalyst should now
facilitate high yielding homodimerisation of allylglycine 21a into
its dimer 60 (Scheme 4.24).
[0197] Surprisingly, homodimerisation of 21a to 60 was found to
proceed but with poor conversion (28%). This suggested that the
dienamide 76 was still capable of influencing the metathesis cycle.
Hopeful that this would later be rectified through modification of
metathesis conditions, we continued to investigate subsequent steps
of the proposed strategy.
##STR00028##
[0198] Rh(I)-DuPHOS-catalysed asymmetric hydrogenation of dienamide
76 under mild conditions (75 psi H.sub.2) gave (2S)-methyl
2-N-acetylamino-5-phenylpent-4-enoate 77 in 99% e.e. (Scheme 4.25).
Formation of the desired phenyl-substituted enamide 77 was
confirmed by spectroscopic analysis which was in agreement with
literature data..sup.117
##STR00029##
[0199] Disappointingly, cross metathesis of 77 using Grubbs'
catalyst was unsuccessful. After 13 hours, .sup.1H n.m.r.
spectroscopy showed no conversion to the desired homodimer 60.
Conditions to facilitate the required cross metathesis were found,
however, using a 5 mol % solution of second generation Grubbs'
catalyst in dichloromethane (Scheme 4.26). A modest conversion
(44%) to the expected homodimer 60 was achieved. In spite of this
promising result, this chemistry was not investigated further since
the requirement for the more reactive second generation Grubbs'
catalyst would render the previously formed unsaturated carbocycle
vulnerable to further cross metathesis. Mixed cross metathesis
products would therefore result (Section 4.1.3.3).
##STR00030##
[0200] Selective reduction of the first-formed unsaturated
carbocycle prior to the second metathesis reaction would, however,
eliminate the chance of mixed cross metathesis (Step 2, Scheme
4.27). We therefore subjected the phenyl substituted diene 76 to
the hydrogenation conditions previously developed for the
hydrogenation of the unsaturated dimer 60. Unfortunately, these
conditions resulted in a 1:4 mixture of olefin 77: saturated
derivative 79 (Scheme 4.28).
##STR00031##
##STR00032##
[0201] This disappointing result is not without literature
precedent. The rate of olefin reduction by Wilkinson's catalyst is
profoundly influenced by steric hindrance about the C.dbd.C double
bond, but related reductions involving styrene have previously
shown that electronic effects override these steric effects and
that the aromatic substituent enhances the rate of
reduction..sup.215,216
4.3 Final Strategy
[0202] The failure of this second strategy led to a final revision
and the discovery of a strategy which would enable the selective
hydrogenation of an unsaturated carbocycle in the presence of a
deactivated but potentially metathesis-active olefin. We decided to
capitalise on the slow reactivity of trisubstituted olefins to
Wilkinson's hydrogenation and their reduced reactivity to
metathesis. 1,1-Disubstituted olefins, for example, do not undergo
homodimerisation and only react with more reactive
olefins..sup.130,182 This differential reactivity would therefore
facilitate the cross metathesis of allylglycine units and
subsequent hydrogenation without interference from the
1,1-disubstituted olefin residues. A simple transformation then
renders the trisubstituted olefin more reactive to metathesis and
facilitates the formation of the second carbocycle (Scheme
4.29).
##STR00033##
4.3.1 Synthesis of (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate
19
[0203] The prenyl olefin 19 was prepared via asymmetric
hydrogenation of the corresponding dienamide 20. The prenylglycine
derivative 19 was isolated in quantitative yield and excellent
enantioselectivity (Scheme 4.30).
##STR00034##
4.3.2 Reactions with (2S)-Methyl
2-N-Acetylamino-5-methylhex-4-enoate 19
[0204] This prenylglycine unit 19 was subjected to the
hydrogenation conditions that quantitatively reduce the dimer 60 to
the saturated analogue 71 (Scheme 4.19) and encouragingly, 94% of
the starting enamide 19 was recovered (Scheme 4.31). This was a
very promising result which prompted us to further investigate
cross metathesis reactions involving this substrate 19.
Furthermore, we envisaged that incorporation of this unit 19 into a
peptide via solid phase peptide synthesis (SPPS) would be
straightforward.
##STR00035##
[0205] Cross metathesis of allylglycine unit 21a into dimer 60 in
the presence of the prenyl enamide 19 proceeded smoothly with
quantitative conversion (Scheme 4.32); the starting prenyl enamide
19 was recovered unchanged.
##STR00036##
[0206] The reduced reactivity of prenylglycine 19 enabled the
dimerisation of allylglycine 21a and the selective hydrogenation of
the resultant homodimer 60. The next step in the strategy involves
activation of the dormant olefin 19 (Step 3, Scheme 4.29). This can
be achieved by cross metathesis with ethylene via a more active
ruthenium alkylidene.
[0207] The prenyl compound 19 was subjected to ethenolysis to
convert it to the more reactive allylglycine derivative 21a (Scheme
4.33). Exposure of 19 to 20 mol % of Grubbs' catalyst under an
atmosphere of ethylene resulted in the recovery of the starting
olefin 19. Use of the more reactive 2.sup.nd generation Grubbs'
catalyst at higher reaction temperature (50.degree. C.) and
ethylene pressure (60 psi) still led to only poor conversions to
21a (<32%).
##STR00037##
[0208] We postulated that this result may be due to the unstable
nature of the in situ generated ruthenium-methylidene intermediate
48 at elevated temperature (50.degree. C.),202-204 or unfavourable
competition between the rising concentration of terminal olefins
and 21a for binding to the ruthenium catalyst..sup.217
##STR00038##
[0209] In order to circumvent this problem, the prenyl enamide 19
was instead exposed to an atmosphere of cis-2-butene (15 psi)
thereby facilitating the catalysis via the more stable
ruthenium-ethylidene complex 49. Butenolysis of 19 in the presence
of 5 mol % second generation Grubbs' catalyst gave the expected
crotylglycine derivative 81 with quantitative conversion (Scheme
4.33).
[0210] (2S)-Methyl 2-N-acetylaminohex-4-enoate 81 was isolated as a
brown oil in 84% yield after flash chromatography. The .sup.1H
n.m.r. spectrum showed the replacement of the olefinic methine (H4)
triplet in the starting prenyl compound 19 with new olefinic
methine (H4, 5) multiplets at .delta. 5.49 and .delta. 5.24
respectively. Spectroscopic data were also in agreement with those
reported in the literature..sup.117,119
[0211] Interestingly, the purity of the 2-butene was found to be
critical to the success of the cross metathesis reaction. When
butenolysis reactions were conducted with a less expensive,
commercially available mixture of cis- and trans-2-butene, only a
trace of the butenolysis product was detected. Gas chromatographic
analysis of the isomeric butene mixture showed that it was
contaminated with 2.6% butadiene while none was detected in the
pure cis-2-butene..sup.218 The addition of butadiene (2%) to
cis-2-butene inhibited formation of the butenolysis product while a
cis+trans mixture (30:70) of 2-butene, free of
butadiene,.sup..dagger. led to quantitative conversion to the
expected cross metathesis product. These results strongly suggested
that butadiene was poisoning the metathesis catalyst. Grubbs et al.
have previously reported that butadiene can react with the
ruthenium-benzylidene catalyst to produce a vinyl alkylidene which
is inactive for acyclic metathesis reactions..sup.219 .sup..dagger.
The cis+trans-2-butene mixture (30:70)free of butadiene was
obtained by isomerisation of cis-2-butene with
benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dic-
hloro[bis(3-bromo-pyridine)]ruthenium at -5.degree. C..sup.218
[0212] This activated crotylglycine derivative 81 was readily cross
metathesised to the expected homodimer 60 with 5 mol % of second
generation Grubbs' catalyst in dichloromethane (Scheme 4.34).
Spectroscopic data were in agreement with those previously reported
(Section 4.1.2).
##STR00039##
4.3.3 Reaction Sequence
[0213] Finally, an equimolar mixture of olefins 62 and 19 was
exposed to a tandem sequence of the previously described five
homogeneous catalytic reactions: i) dimerisation of allylglycine
62, ii) hydrogenation of the resultant homodimer 69, iii)
activation of prenylglycine 19, iv) dimerisation of the activated
crotylglycine derivative 81 and v) hydrogenation of the resultant
homodimer 60. Solvent removal and subsequent .sup.1H n.m.r.
analysis was performed on the crude product mixture after each
transformation. The catalytic sequence resulted in quantitative
conversion of the reactive substrate in each step and ultimately
yielded diaminosuberic acid derivatives 71 and 72 as the only
isolated products in 84 and 70% yield respectively (Scheme
4.35).
##STR00040##
4.4 Summary
[0214] In conclusion, these model studies demonstrate that through
the combination of homogeneous catalysis and judicious selection of
non-proteinaceous allylglycine residues of varying reactivity, a
highly efficient, unambiguous and regioselective synthesis of
dicarba analogues of multi-cystine containing peptides may be
achievable. The methodology is also amenable to natural product and
polymer synthesis or wherever selective carbon-carbon bond
formation is required. Section 6 investigates the application of
this methodology to synthetic and naturally occurring peptides.
5.0 A TANDEM METATHESIS-HYDROGENATION STRATEGY FOR THE SELECTIVE
FORMATION OF THREE CARBON-CARBON BONDS
[0215] The selective formation of multiple dicarba bonds in complex
molecules is a significant synthetic challenge. In section 4, we
devised a strategy for a solution phase regioselective synthesis of
two dicarba bridges. This chapter describes a catalytic strategy
for the regioselective construction of three dicarba bridges in
solution by selective and successive metathesis-hydrogenation
transformations.
5.1 Proposed Strategy
[0216] In the preceding chapter we achieved regioselective C--C
bond formation through the use of olefinic substrates possessing
tuneable reactivity and highly chemo- and stereoselective
catalysts. The varying reactivity of allylglycine and prenylglycine
units towards metathesis and hydrogenation has been previously
described (Chapter 4). We postulated that the steric and
particularly electronic effects of a prenylglycine dienoate 82
would render it inert to metathesis and Wilkinson's hydrogenation.
Two dicarba bridges can therefore be constructed in the presence of
this inert olefin (Scheme 5.1). The diene can then be activated in
two simple steps, the first of which involves a catalytic
asymmetric hydrogenation to give optically pure prenylglycine. We
have already demonstrated the facile activation of the prenyl
sidechain by cross metathesis with either ethylene or cis-2-butene
to give the corresponding allyl- or crotylglycine' derivative
respectively. The resultant activated olefin can readily undergo
homodimerisation to give an unsaturated dimer which can be reduced
to afford the saturated dicarba bridge. The final product mixture
would ultimately contain three different diaminosuberic acid
derivatives where the selective C--C bond formation would represent
the formation of a dicarba analogue of a tricystine-containing
peptide (Scheme 5.1). In order to validate the proposed strategy we
conducted a series of solution phase reactions.
##STR00041##
5.2 Solution Phase Model Study
[0217] A metathesis triplet 83, 19 and 82 was developed to
facilitate the controlled formation of three diaminosuberic acid
derivatives (Table 5.1). The differing olefin substitution in the
molecules provides tuneable reactivity towards homogeneous
metathesis.sup.120,121 and hydrogenation catalysts..sup.33,215
TABLE-US-00001 TABLE 5.1 Reaction Sequence for the Construction of
Three Dicarba Bridges.sup.a ##STR00042## Step 1: CM-H Grubbs'
catalyst Step 2: Wilkason's Hydrogena- tion Step 3: CM 2.sup.nd
gen. Grubbs' catalyst Step 4: CM-H 2.sup.nd gen. Grubbs' catalyst
Step 5: Wilkinson's Hydrogena- tion Step 6: Rh(I)- DuPHOS
Hydrogena- tion Step 7: CM 2.sup.ndgen. Grubbs' catalyst Step 8:
CM-H 2.sup.nd gen. Grubbs' catalyst Step 9: Wilkinson's Hydrogena-
tion ##STR00043## Substrates C.dbd.C C--C Act C.dbd.C C--C Act Act
C.dbd.C C--C Products ##STR00044## Sidechain Reactivity Summary of
Activity ##STR00045## -- -- -- -- -- -- -- Terminal allylic olefin.
No activation required. ##STR00046## X X -- -- -- -- Trisubstituted
olefin. Activated via CM with 2-butene. ##STR00047## X X X X X
Hindered extended acrylamide olefin. Activated via i) asymmetric
hydrogenation and ii) CM with 2-butene. .sup.a = Reactive olefin, X
= Unreactive olefin, -- = Unreactive dicarba bridge, Act = Olefin
activation step, CM-H = Cross metathesis-homodimerisation, CM =
Cross metathesis
[0218] Three different N-acyl protecting groups were employed to
facilitate unambiguous assessment of cross metathesis selectivity.
A mixture of a p-nitrobenzoyl-protected allylglycine derivative 83,
an acetyl-protected prenylglycine unit 19 and a benzoyl-protected
prenylglycine dienamide 82 gave adequate separation of
characteristic peaks in the .sup.1H n.m.r. spectrum (FIG. 3) to
enable reaction monitoring of Steps 1-9. Importantly, the
protecting groups on the amino group do not affect the mechanistic
course of the reaction sequence.
[0219] The solution phase studies therefore commenced with
preliminary experiments on the diene 82 to ensure it was inert to
metathesis and Wilkinson's hydrogenation.
5.2.1 Synthesis of (2Z)-Methyl
2-N-Benzoylamino-5-methylhexa-2,4-dienoate
[0220] The dienamide 82 was synthesised by Horner-Emmons
olefination of methyl
2-N-benzoylamino-2-(dimethoxyphosphinyl)acetate 64 with
commercially available 3-methyl-2-butenal 40 and
tetramethylguanidine (TMG) (Scheme 5.2), as described for several
dienamides in this application
##STR00048##
[0221] Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 was
isolated as an off-white solid in 73% yield. Formation of the
prenylglycine dienamide 82 was supported by .sup.13C n.m.r.
spectroscopy which displayed new olefinic methyl peaks at .delta.
19.3 and .delta. 27.1 respectively, in addition to characteristic
olefinic methine (C3, 4) and quaternary (C2, 5) peaks. A molecular
ion plus proton peak at m/z 260.1282 in the accurate mass spectrum
was consistent with the molecular formula C.sub.15H.sub.18NO.sub.3
and also supported formation of the dienamide 82.
5.2.2 Reactivity of (2Z)-Methyl
2-N-Benzoylamino-5-methylhexa-2,4-dienoate 82 toward Metathesis and
Hydrogenation
[0222] The dienamide 82 was subjected to homodimerisation
conditions with second generation Grubbs' catalyst (Scheme 5.3).
.sup.1H n.m.r. spectroscopy confirmed complete recovery of the
starting olefin 82 with no evidence of the dimerised dienoate 84.
This result supported our postulate that diene 82 is electronically
and sterically compromised and therefore inert to metathesis.
##STR00049##
[0223] Our proposed reaction sequence then required the reduction
of an unsaturated dicarba bridge in the presence of a diene moiety
(Step 2, Scheme 5.1). The dienamide 82 was therefore subjected to
the hydrogenation conditions that quantitatively reduce unsaturated
dimers to their saturated analogues (Wilkinson's catalyst, 50 psi
H.sub.2). Encouragingly, the reduced prenyl compound 85 was not
observed and the starting olefin 82 was recovered unchanged (Scheme
5.3).
[0224] Finally, the diene 82 was exposed to metathesis conditions
used to activate prenylglycine 19 by conversion to the crotyl
derivative 81 (cis-2-butene, second generation Grubbs' catalyst,
Scheme 5.3). Again .sup.1H n.m.r. spectroscopy indicated that the
dienamide 82 was inert to these conditions. The starting olefin 82
was recovered unchanged with no evidence of the potential cross
metathesis product 86.
5.2.3 Activation of (2Z)-Methyl
2-N-Benzoylamino-5-methylhexa-2,4-dienoate
[0225] Activation of the dienamide 82 was initiated with a
Rh(I)-Et-DuPHOS-catalysed asymmetric hydrogenation to give the
prenylglycine derivative 87 in excellent yield and
enantioselectivity (100% e.e.) (Scheme 5.4).
[0226] The replacement of olefinic methine (H3, 4) proton peaks in
the .sup.1H n.m.r. spectrum with new methylene (H3) and olefinic
(H4) multiplets at .delta. 2.52-2.76 and .delta. 5.08 confirmed
formation of the prenylglycine residue 87. Over-reduction of the
terminal double bond was not observed under these conditions.
##STR00050##
[0227] The second activation step involved treatment of the prenyl
olefin 87 with 5 mol % second generation Grubbs' catalyst and
cis-2-butene (15 psi) to yield the crotylglycine derivative 88
(Scheme 5.4). The reaction proceeded with quantitative conversion
as indicated by .sup.1H n.m.r. and .sup.13C n.m.r. spectroscopic
analysis. The accurate mass spectrum also displayed a molecular ion
plus proton peak at m/z 248.1284 which is consistent with that
expected for the molecular formula C.sub.14H.sub.18NO.sub.3.
5.2.4 Reactions with (2S)-Methyl
2-N-(p-Nitrobenzoyl)aminopent-4-enoate 83
[0228] The third olefin in the metathesis triplet is the
allylglycine derivative 83. Reaction of the hydrochloride salt of
allylglycine methyl ester 51 with p-nitrobenzoyl chloride 89 and
triethylamine in a mixture of dichloromethane:diethyl ether gave
the protected allylglycine residue 83 in 99% yield (Scheme
5.5).
[0229] The .sup.1H n.m.r. and .sup.13C n.m.r. spectra supported
formation of the protected allylglycine 83 with the downfield shift
of the methine (H2) doublet of triplets at .delta. 4.90 and the
introduction of aromatic resonances at .delta. 7.95 (H2',6') and
.delta. 8.30 (H3',5').
##STR00051##
[0230] The allylglycine derivative 83 was quantitatively dimerised
with Grubbs' catalyst in dichloromethane heated at reflux (Scheme
5.6). Formation of the dimer 90 was supported by .sup.1H and
.sup.13C n.m.r. spectroscopic analysis which displayed signals due
to the new olefinic methine proton (H4, .delta. 5.49-5.53) and
carbon (C4, 128.8) respectively.
[0231] The unsaturated dimer 90 was subjected to the previously
described Wilkinson's hydrogenation conditions (50 psi H.sub.2,
benzene, 4 hours). Unfortunately, under these conditions, the
aromatic nitro substituents were reduced, thus providing a
potential mechanism for poisoning of the metathesis catalyst.
Fortunately, Jourdant et al. recently reported the selective
reduction of an olefin in the presence of an aromatic nitro
group..sup.220 Homogeneous hydrogenation under 15 psi H.sub.2 in a
mixture of tetrahydrofuran:tert-butanol (1:1) led to the selective
reduction of the unsaturated dimer 90 without concomitant reduction
of pendant aromatic nitro groups (Scheme 5.6).
##STR00052##
[0232] (2S,7S)-Dimethyl
2,7-N,N'-di(p-nitrobenzoyl)aminooctanedioate 91 was isolated as an
off-white solid in 67% yield. The replacement of olefinic peaks in
the .sup.1H n.m.r. spectrum with new methylene (H4, 6 and H3, 5)
multiplets at .delta. 1.39-1.54 and .delta. 1.74-2.04 respectively
confirmed formation of the saturated dimer 91.
5.2.5 Reaction Sequence
[0233] An equimolar mixture of olefins 83, 19 and 82 was subjected
to the catalytic sequence outlined in Scheme 5.7. Solvent removal
and subsequent .sup.1H n.m.r. and mass spectral analysis was
performed on the crude product mixture after each transformation.
Exposure of the olefinic mixture 83, 19 and 82 to Grubbs' catalyst
in dichloromethane led to homodimerisation of allylglycine 83 to
form an unsaturated dicarba bridge 90. Predictably, the more
sterically hindered olefin 19 and the electronically compromised
olefin 82 were unreactive under these reaction conditions. The
resultant alkene 90 was then selectively hydrogenated in a mixture
of tert-butanol:tetrahydrofuran (1:1) with Wilkinson's catalyst to
afford the saturated dicarba bridge 91. Again, olefins 19 and 82
were inert to these conditions. Both the metathesis and
hydrogenation reactions proceeded under mild experimental
conditions with quantitative, unambiguous conversion to give the
first suberic acid derivative 91 as shown by n.m.r. and MS
analysis.
##STR00053##
[0234] The next reaction in this catalytic sequence involved the
activation of the dormant prenyl olefin 19 via cross metathesis
with cis-2-butene (butenolysis) to generate a more reactive
crotylglycine derivative (Section 4.3.2). The mixture of 91, 19 and
82 was exposed to an atmosphere of cis-2-butene (15 psi) in the
presence of 5 mol % second generation Grubbs' catalyst to afford
the expected crotylglycine derivative 81 and a trace of the
corresponding homodimer 60. The activated olefin 81 was then
quantitatively homodimerised to the expected unsaturated dimer 60
with 5 mol % of second generation Grubbs' catalyst. Exposure of the
newly formed olefin 60 to a hydrogen atmosphere and Wilkinson's
catalyst resulted in quantitative conversion to the saturated
dicarba bridge 71 (Section 4.1.4). Once again, the sterically and
electronically compromised olefin 82 remained a spectator over the
three reactions used to form the second diaminosuberic acid
derivative 71.
[0235] The remaining acrylate-type olefin 82 was then used to form
the final dicarba bridge. A double activation sequence was employed
to render this remaining olefin reactive to homodimerisation.
Homogeneous hydrogenation of dienamide 82 using chiral
Rh(I)--(S,S)-Et-DuPHOS catalyst gave (S)-configured prenylglycine
derivative 87 in excellent enantioselectivity (100% e.e.),
chemoselectivity and conversion. No evidence of over-reduction of
the C4 carbon-carbon double bond was observed. The resulting prenyl
olefin 87 was then converted to the crotylglycine analogue 88 via
butenolysis. Exposure of this olefin to the previously described
cross-metathesis and hydrogenation conditions then led to the
formation of the final dicarba bond and the third diaminosuberic
acid derivative 72 via alkene intermediate 69. The
metathesis-hydrogenation sequence led to generation of three
diamidosuberic acid esters 91, 71 and 72 in 67, 81 and 70% yields
respectively. Significantly, residual catalyst and/or decomposition
products did not compromise subsequent transformations and no other
byproducts were isolated. This demonstrates the high
chemoselectivity exhibited by each catalytic step.
5.3 Summary
[0236] A combination of homogeneous hydrogenation and metathesis
reactions has enabled the highly efficient, stepwise chemo- and
stereoselective formation of three identical dicarba C--C bonds in
three different 2,7-diaminosuberic acid derivatives without
purification of intermediates. This homogeneous catalytic
methodology can be used widely in peptidomimetics and total product
synthesis where multiple (preferably 3) C--C bonds and/or rings
need to be selectively constructed.
6.0 Synthesis of Dicarba Cyclic Peptides Via Regioselective Cross
Metathesis
[0237] This section describes the application of the regioselective
strategy developed in section 4 to a series of peptides. A model
synthetic pentapeptide was initially investigated. The results from
this substrate led to the production of dicarba analogues of
conotoxin ImI.
6.1 Solid Phase Peptide Synthesis (SPPS)
[0238] Linear peptides were synthesised via standard solid phase
peptide synthesis (SPPS) methodology..sup.221 This procedure
involves the attachment of an N-Fmoc-protected amino acid to a
solid support and the construction of the sequence from the C- to
N-terminus Scheme 6.1. Peptide construction requires: i)
Fmoc-deprotection of the resin-tethered amino acid under basic
conditions, ii) activation of the incoming Fmoc-protected amino
acid and iii) its subsequent coupling to the resin-tethered amino
acid. The process is repeated until the desired peptide sequence is
constructed. Conveniently, the use of orthogonally protected amino
acids enables sequential Fmoc-deprotection and coupling without
loss of acid-sensitive sidechain protecting groups.
##STR00054##
[0239] The choice of resin plays an important role in peptide
synthesis. A plethora of polystyrene-based supports are
commercially available. These resins are typically cross-linked
polystyrene (PS) containing 1% divinylbenzene and are
functionalised with linkers (or handles) to provide a reversible
linkage between the synthetic peptide chain and the solid
support..sup.221 Several linkers commonly utilised in Fmoc-SPPS are
presented in. Diagram 6.1. With the target peptide in mind, the
appropriate resin-linker can be chosen to functionalise the
C-terminus as a carboxylic acid, carboxamide, ester or alcohol. In
addition, peptides can be cleaved under acidic or basic conditions
where acid sensitive sidechain protecting groups can be retained or
simultaneously deprotected during peptide cleavage. Importantly,
the resin-linkers must be inert to metathesis and hydrogenation
catalysis conditions.
##STR00055##
[0240] Construction of the linear peptides via solid phase
methodology provides two options for the construction of dicarba
bridges: The complete linear sequence can be cleaved from the resin
and then subjected to metathesis and hydrogenation in solution.
Alternatively, the regioselective catalytic sequence can be
performed entirely on the resin-bound peptides.
[0241] We have conducted an on-resin metathesis-hydrogenation
sequence for the preparation of carbocyclic analogues of
cystine-containing peptides. This strategy involves conventional
solid phase peptide synthesis followed by on-resin
ruthenium-catalysed ring closing metathesis and on-resin
homogeneous rhodium-catalysed hydrogenation of the resultant
unsaturated bridge (Scheme 6.2).
##STR00056##
[0242] The on-resin strategy, however, is compromised by decreased
activity of the metathesis and hydrogenation catalysts in the
heterogeneous system. Previous studies have shown that higher
catalyst loadings and longer reaction times are required to achieve
quantitative conversion on resin-bound substrates..sup.141,142In
addition, ring closing metathesis of peptidic substrates is highly
sequence dependent due to the involvement of aggregation phenomena.
We have found that peptide aggregation, resulting from interchain
secondary structures, can lead to poor solvation of the
peptidyl-resin, reduced reagent penetration and ultimately low
reaction yields. Strategies had to be developed to address these
problems.
6.2 Ring Closing Metathesis Reactions of Synthetic
Pentapeptides
[0243] We have investigated the synthesis of bis-dicarba analogues
of bicyclic peptides possessing two disulfide bonds. To achieve
this aim we required the use of complimentary pairs of both allyl-
and prenylglycine residues (although variations described above can
be used). In order to transfer the solution phase methodology
across to the solid phase, we needed to demonstrate that
Fmoc-protected prenylglycine 92 could be i) synthesised and
incorporated into a peptide sequence using standard SPPS protocol;
that it was stable to peptide coupling and deprotection conditions,
and iii) that it possessed analogous reactivity to its solution
phase congener in the catalysis steps. We therefore decided to
synthesise model peptides based on naturally occurring conotoxin
peptides..sup.171,172,225 Conotoxin ImI 93 (Ctx ImI) is a small
dodecapeptide possessing two cystine bonds..sup.173,174,226 A
truncated sequence 94 of the Cys8-Ala9-Trp10-Arg11-Cys12 Ctx ImI
domain was initially investigated. This sequence possesses two
allylglycine residues which undergo ring closing metathesis to
yield an unsaturated carbocycle 95. After establishing optimum
conditions for the formation of the first dicarba bond, the
sequence was modified to include a prenylglycine residue to
facilitate the formation of a second dicarba linkage.
##STR00057##
[0244] The pentapeptide 94 was synthesised on Rink amide resin, a
polystyrene-based solid support bearing an amine linker that
generates a C-terminal carboxamide upon resin cleavage. (Diagram
6.1). Prior to attachment of the first amino acid, the resin was
swollen in dichloromethane to increase surface availability of
resin active sites towards the incoming C-terminal Fmoc-protected
amino acid. Peptide construction began with attachment of
non-proteinaceous Fmoc-L-allylglycine (Fmoc-Hag-OH) 96 to Rink
amide resin (A, Diagram 6.1) and remaining resin active sites were
capped with acetic anhydride. Fmoc-deprotection of resin-tethered
allylglycine followed by coupling of the successive amino acid and
repetition of these steps (B and C, Diagram 6.1) enabled chain
elongation. After coupling the last amino acid, a small aliquot of
peptidyl-resin was exposed to trifluoroacetic acid cleavage
solution (D, Diagram 6.1) to liberate the peptide 94 as a
colourless solid. The mass spectrum displayed a molecular ion peak
at m/z 847.1 (M+H).sup.+ which was consistent with the formation of
the pentapeptide 94.
##STR00058##
[0245] After confirming that pentapeptide synthesis had been
successful, ring closing metathesis of the fully-protected
resin-tethered sample 94a was performed with 20 mol % Grubbs'
catalyst in dichloromethane and 10% lithium chloride in
dimethylformamide. Mass spectral analysis of a cleaved aliquot of
peptide indicated that these conditions resulted in complete
recovery of the linear peptide 94. Use of the more active second
generation Grubbs' catalyst did, however, lead to unsaturated
carbocycle 95 but cyclisation failed to go to completion (Scheme
6.3). The presence of molecular ion peaks at m/z 819.2 (M+H).sup.+
and m/z 847.2 (M+Na).sup.+ were consistent with the presence of the
unsaturated carbocycle 95 and the linear peptide 94
respectively.
##STR00059##
[0246] We postulated that the peptide sequence itself may be
responsible for the reduced ring closing metathesis yield.
Pentapeptide 94 lacks a proline residue between the two
allylglycine sidechains and hence the predominance of transoid
peptide bonds would disfavour a close arrangement of the reacting
terminal olefins. The inclusion of turn inducers in a peptide
sequence can reduce peptide aggregation via the formation of
cisoidal amide bonds..sup.227-229 In addition, the resultant turn
can position the reactive allylglycine sidechains in close
proximity to each other and thus facilitate cyclisation. The
peptide was therefore reconstructed to incorporate proline, a
naturally occurring turn-inducing amino acid.
[0247] The pentapeptide 97 was synthesised on Rink amide resin via
the general SPPS methodology previously described. The peptide
possessed an Ala.fwdarw.Pro replacement adjacent to the N-terminal
allylglycine residue. Formation of the pentapeptide 97 was
confirmed by mass spectrometry with the appearance of a molecular
ion peak at m/z 873.2 (M+H).sup.+.
##STR00060##
[0248] Ring closing metathesis of the fully protected resin-bound
peptide 97a with Grubbs' catalyst (20 mol %) in dichloromethane and
10% lithium chloride in dimethylformamide led to recovery of the
starting peptide 97 with only a trace of product 98 evident in the
mass spectrum. Use of second generation Grubbs' catalyst (20 mol
%), however, led to complete cyclisation (Scheme 6.4). The
appearance of molecular ion peaks at m/z 845.1 (M+H).sup.+ and m/z
867.1 (M+Na).sup.+ in the mass spectrum confirmed formation of the
unsaturated carbocycle 98. This result clearly demonstrates the
influence of the turn-inducing proline residue on peptide
conformation and reactivity.
##STR00061##
[0249] In conjunction with this study, we simultaneously assessed
the role of the catalytic cycle in affecting ring closing
metathesis yield. We postulated that the incomplete cyclisation of
linear sequence 94 could be due to thermal decomposition of the
ruthenium-methylidene intermediate 48. We therefore investigated
synthesis of the crotylglycine-containing peptide,
Fmoc-Crt-Ala-Trp-Arg-Crt-NH.sub.2 99, for which metathesis proceeds
through the more stable ruthenium-ethylidene species 49.
[0250] This initially required the synthesis of the crotylglycine
derivative 100. Acid-promoted hydrolysis of (2S)-methyl
2-N-acetylaminohex-4-enoate 81 gave (2S)-2-aminohex-4-enoic acid
hydrochloride salt 101. Fmoc-protection of amino acid 101 was
performed according to the procedure described by Paquet et al.
using N-fluorenylmethoxycarbonylamino-suecinimide (Fmoc-OSu) in
aqueous sodium carbonate and acetone (Scheme 6.5)..sup.230
##STR00062##
[0251] .sup.1H n.m.r. and .sup.13C n.m.r. spectral analysis of the
product confirmed the formation of
(2S)-2-N-fluorenylmethoxycarbonylaminohex-4-enoic acid
(Fmoc-Crt-OH) 100 with the downfield shift of the methine proton
(H2) peak (.delta. 4.55) and the corresponding carbon signal
(.delta. 52.3). In addition, the appearance of aromatic signals
characteristic of the Fmoc-group supported product formation.
Spectroscopic data were also in agreement with those reported in
the literature..sup.146
[0252] With the Fmoc-protected crotylglycine derivative 100 in
hand, we synthesised the linear peptide,
Fmoc-Crt-Ala-Trp-Arg-Crt-NH.sub.2 99, on Rink amide resin using the
SPPS methodology previously described. The mass spectrum displayed
a molecular ion peak at tn/z 875.2 (M+H).sup.+ corresponding to the
linear peptide 99.
##STR00063##
[0253] Ring closing metathesis of the linear resin-tethered peptide
99a with second generation Grubbs' catalyst (20 mol %) in
dichloromethane and 10% lithium chloride in dimethylformamide led
to quantitative formation of the unsaturated carbocycle 95.sup.\
(Scheme 6.6). Note: RCM of the crotylglyeine-containing peptide 99
leads to the same unsaturated carbocycle 95 resulting from
cyclisation of the allylglycine-containing sequence 94, i.e.
Fmoc-c[Hag-Ala-Trp-Arg-Hag]-OH is identical to
Fmoc-c[Crt-Ala-Trp-Arg-Crt]-OH.
##STR00064##
[0254] These studies revealed two successful strategies for the
synthesis of a dicarba cyclic peptide: i) the inclusion of proline
residues to induce a turn in the peptide backbone and ii) the use
of crotylglycine to avoid a ruthenium-methylidene intermediate in
the catalytic cycle. Many naturally occurring cyclic peptides
possess proline residues in their primary sequences and this could
be used to advantage in RCM reactions. On the other hand, if the
target peptide does not possess a proline residue (or a residue
which can temporarily act as a pseudo-proline), incorporation of a
non-native proline residue to enhance RCM yield is likely to have
significant structural and biological impact on the final peptide.
In this case, the use of crotylglycine residues would be
beneficial.
6.2.1 Synthesis of Dicarba-AOD Using Pseudoproline Residues
[0255] The Melbourne-based pharmaceutical company Metabolic have a
peptidic agent, AOD9604, currently undergoing clinical trials.
AOD9604 143 is a peptide fragment derived from the C-terminus of
human growth hormone (hGH) and is believed to be responsible for
the lipolytic activity of hGH..sup.267 This 16-residue peptide was
derived from the parent anti-obesity drug AOD9401 144 by addition
of a terminal tyrosine residue, and is known to induce lipolysis
and fat oxidation in vitro in adipose tissue..sup.267 Ng et al.
report the synthesis of both of these peptides using standard solid
phase peptide synthesis techniques..sup.267,268
[0256] The x-ray crystal structure of native hGH shows that the
region of interest (residues 177-191) contains a disulphide bridge
between residues 182 and 189. An alanine scan of AOD9401 showed
that when cysteine was replaced by alanine a dramatic reduction in
antilipogenic activity was observed..sup.268 This suggests that the
cystine bridge and the cyclic conformation of the peptide are vital
for the activity of AOD9401 and related peptide analogues..sup.268
Thus, we were interested in synthesising the dicarba analogue of
AOD9604 using the technology developed and described herein to
provide analogues with increased biological stability.
##STR00065##
6.2.1.1 Synthesis of Linear Hag.sup.6-Hag.sup.13 AOD9401 and
AOD9604
[0257] The linear derivative of the carbocyclic analogue of AOD9401
was initially synthesised utilising natural amino acids, as well as
the non-proteinaceous residue allylglycine in place of cysteine.
Upon synthesis of the linear peptide 145, an aliquot was subjected
to cleavage conditions to assess the success of the synthesis. Mass
spectral analysis indicated the synthesis of linear Fmoc-protected
AOD9401.
##STR00066##
[0258] At this point, it was established that AOD9604 would be a
more suitable target molecule, and the additional amino acid
residue was coupled to the parent AOD9401 molecule already
synthesised. The presence of the linear Hag.sup.6-Hag.sup.13
containing derivative 146 was confirmed by mass spectral
analysis.
##STR00067##
6.2.1.2 Synthesis of Dicarba AOD9604 147
[0259] Ring closing metathesis, catalysed by second generation
Grubbs' catalyst was employed to achieve cyclisation leading to the
synthesis of unsaturated dicarba AOD9604 147. Initially, standard
metathesis conditions were used, as perfected in the synthesis of
somatostatin analogues. Lithium chloride was employed to decrease
aggregation and 20 mol % catalyst loading was used to initiate the
metathesis reaction. Mass spectral analysis post-TFA cleavage
indicated the failure of cyclisation, with the only peaks
indicative of linear Fmoc-protected starting material 146. This
reaction was repeated a number or times, including with a higher
boiling solvent, however, all attempts yielded solely uncyclised
starting material.
[0260] Deleterious hydrogen bonding in the linear peptide was
suspected as the cause of this failed ring closure under standard
metathesis conditions. Hence, microwave-accelerated ring closing
metathesis of the same resin-tethered peptide was attempted.
Similar catalytic conditions to previous attempts were employed,
with dichloromethane as the solvent. The temperature was increased
from 40.degree. C. to 100.degree. C. and the time decreased to just
10 h. Again, mass spectral analysis of cleaved material indicated
the failure of the reaction.
[0261] Attention was turned to the primary sequence of the peptide
itself. It was identified that residues such as proline and glycine
can induce turns in peptides, and thus facilitate N.fwdarw.C
cyclisation of peptides. N-alkylated residues and D-amino acids can
also achieve this. There is a lack of any turn-inducing amino acid
residues (peptides) in the sequence of AOD9604, a potential
contributing factor in the failure to cyclise.
6.2.1.3 Incorporation of a Turn-Inducing Pseudoproline Residue
[0262] Proline is the only naturally occurring amino acid which is
known to induce cis/trans isomerisation about a peptide bond, a
feature known to induce a turn in the peptide backbone, often
resulting in a reversal of the direction of the backbone. This has
led researchers to develop alternatives to native proline, and
numerous mimetics which produce proline-like cis-peptide bonds and
reverse turns have been investigated..sup.269
[0263] Pseudoproline (.psi.Pro) residues derived from naturally
occurring serine, threonine and cysteine residues have gained
popularity in recent years. Their formation is reversible; they are
synthesised by a cyclocondensation reaction with an aldehyde or
ketone and upon exposure to acidic conditions they revert to the
parent amino acid.
[0264] The incorporation of pseudoproline residues into peptide
sequences increase the rate and yield of head to tail cyclisation
(macrolactamisation). It was decided to incorporate a pseudoproline
residue in the synthesis of the linear AOD analogue and to conduct
the metathesis under microwave irradiation conditions. There are
two serine residues in the sequence, and serine 13 was chosen to be
replaced by a pseudoproline residue. The incorporation of a
pseudoproline residue is highly dependent on the adjacent residue
attached to the amine of the pseudoproline. Pseudoprolines are
incorporated into the peptide sequence as a dipeptide due to the
ease of synthesis and stability. Adjacent to serine 9 is an
arginine residue; this pseudoproline is not commercially available
and is highly difficult to synthesise due to the bulky side chain
and equally bulky protecting group necessary for peptide
synthesis.
[0265] The linear peptide was again synthesised, this time with the
dipeptide sequence -Ser(.sup.tBu)-Gly-replaced with the
commercially available pseudoproline analogue. This residue reverts
to the required dipeptide upon exposure to the acidic cleavage
solution after the cyclisation step.
[0266] The microwave-accelerated metathesis reaction was repeated
using the resin-tethered, pseudoproline-containing peptide 146a.
After 1 h, an aliquot of resin was exposed to cleavage conditions.
Mass spectral analysis indicated the reaction had been successful,
with the presence of a peak at m/z 1000.1 corresponding to the
doubly charged adduct of the unsaturated dicarba product 147. This
example clearly illustrates the importance of using turn-inducing
residues when the metathesisable groups are not naturally proximate
to facilitate high yielding ring closure.
##STR00068##
[0267] Finally, the carbocyclic peptide 147 was obtained with a 75%
conversion from the linear parent moiety 146a. A large aliquot of
resin was exposed to cleavage conditions, and purification via
preparative HPLC yielded the desired peptide in 6% yield. The low
yield was attributed to purification difficulties caused by
lingering catalyst, despite treatment with DMSO prior to cleavage,
a technique thought to destroy interaction between the catalyst and
resin.
##STR00069##
[0268] Catalytic hydrogneation of the unsaturated AOD peptide 147
proved to be difficult. Exposure of the peptide to Wilkinson's
catalyst and 90 psi of hydrogen for 4 days failed to achieve
complete reduction of the peptide to the saturated AOD derivative
148. The two dicarba analogues 147 and 148, however, were readily
separated from each other using preparative HPLC.
6.3 Regioselective Synthesis of an Intra- and Intermolecular
Dicarba Bridge in a Synthetic Pentapeptide
[0269] Capitalising on the findings of the previous study (Section
6.2) we constructed another model peptide,
Fmoc-Hag-Pro-Pre-Arg-Hag-OH 102, with a strategically placed
proline residue. The synthetic pentapeptide 102 contains two types
of metathesis active groups: Two allylglycine (Hag) residues and a
less reactive prenylglycine unit (Pre). This linear sequence
facilitates the regioselective construction of two dicarba bonds:
An intramolecular metathesis reaction (RCM) of the allylglycine
residues generates a carbocyclic ring and the remaining
prenylglycine can be used to form an intermolecular dicarba bridge
via cross metathesis (CM) with a second unsaturated molecule
(Scheme 6.7).
##STR00070##
[0270] This second dicarba linkage could be used to attach the
carbocyclic peptide to another peptide chain, a drug molecule, a
solid support or a chelating heterocycle for the generation of
radiopharmaceuticals.
##STR00071##
[0271] Synthesis of the peptide 102 firstly required the
preparation of the Fmoc-protected prenylglycine derivative
(Fmoc-Pre-OH) 92. Cross metathesis of Fmoc-protected allylglycine
96 with 2-methyl-2-butene in the presence of 5 mol % second
generation Grubbs' catalyst gave the target
(2S)-2-N-fluorenylmethoxycarbonylamino-5-methylhex-4-enoic acid 92
with quantitative conversion (Scheme 6.8).
##STR00072##
[0272] .sup.1H n.m.r. spectroscopy confirmed formation of the
trisubstituted olefinic amino acid 92 by the replacement of
terminal olefinic peaks with a new methine multiplet (H4) at
.delta. 5.11 and two methyl singlets at .delta. 1.63 and .delta.
1.73. These signals are consistent with the generation of a prenyl
group. The accurate mass spectrum also displayed a molecular ion
peak at m/z 388.1525 (M+Na).sup.+ which was consistent with that
required for 92. Unfortunately, purification of the product 92 from
residual catalyst was difficult. We later found, however, that the
crude amino acid 92 could be used without affecting subsequent SPPS
procedures.
[0273] The peptide 102 was synthesised on inexpensive, readily
available Wang resin, a polystyrene-based solid support bearing a
benzylic alcohol linker (FIG. 6.1). The non-proteinaceous
prenylglycine residue 92 was incorporated into the peptide sequence
without complication. Formation of the pentapeptide 102 was
confirmed by mass spectral analysis with the appearance of a
molecular ion peak at m/z 813.5 (M+H).sup.+ and an additional peak
at m/z 831.5 (M+H.sub.2O+H).sup.+. The latter peak was due to the
acid-promoted hydration of the prenyl sidechain during peptide
cleavage, leading to the alcohol 103. The hydration of the prenyl
group under acidic conditions was not unexpected. During the
acid-catalysed cyclisation of the simple prenylglycine derivative
19 to pseudo-proline 18, acid-mediated hydration yielded alcohol 47
as a minor byproduct.
##STR00073##
[0274] After confirming the synthesis of the pentapeptide 102, the
peptidyl-resin was subjected to the regioselective catalytic
strategy outlined in section 4. This is presented in Scheme
6.7.
[0275] The first step involved selective RCM of the allylglycine
residues in the presence of the less reactive prenyl sidechain. RCM
of the resin-tethered pentapeptide 102a was performed with 40 mol %
second generation Grubbs' catalyst in dichloromethane and 10%
lithium chloride in dimethylformamide and, as expected,
incorporation of prenylglycine did not hinder cyclisation (Scheme
6.9). Mass spectral analysis of a cleaved aliquot of peptide
confirmed formation of the unsaturated carbocycle 104 with the
appearance of a molecular ion peak at m/z 785.4 (M+H).sup.+. A peak
at m/z 803.4 (M+H.sub.2O+H).sup.+, corresponding to a hydrated
prenyl sidechain in the cyclic product, was also evident.
Importantly, prenylglycine remained inert to the metathesis
conditions and no mixed cross metathesis products were
observed.
##STR00074##
[0276] Attempts to decrease reaction time and catalyst loading led
to incomplete reaction. We therefore decided that the high catalyst
loading and extended reaction times could be tolerated in order to
avoid the time consuming and poor yielding HPLC purification of
mixtures resulting from non-quantitative cyclisation reactions.
Decreasing peptide loading on the resin (from 0.9 to 0.3
mmolg.sup.-1) did, however, enable complete RCM with 10 mol % of
second generation Grubbs' catalyst. This is probably due to the
fact that the use of low substitution resins decreases the density
of peptide chains on the solid phase and minimises aggregation. The
reduced loading enhances resin solvation and reagent access and
ultimately leads to improved reaction yields.
[0277] Selective hydrogenation of the resin-bound unsaturated
carbocycle 104a was performed under 80 psi of hydrogen with
homogeneous Wilkinson's catalyst, Rh(I)(PPh.sub.3).sub.3Cl, in a
mixture of dichloromethane:methanol (9:1) (Scheme 6.10). This
solvent system served a dual function in maintaining a swollen
resin (dichloromethane) and participating in the catalytic cycle
(methanol). After 22 hours, a small aliquot of peptide was cleaved
and analysed by mass spectrometry. The appearance of peaks at m/z
787.3 (M+H).sup.+ and m/z 805.4 (M+H.sub.2O+H).sup.+ were
consistent with formation of the saturated carbocycle 105.
Importantly, the prenyl group remained stable to these reducing
conditions which was consistent with the observed reactivity of
prenylglycine in the solution phase model studies.
##STR00075##
[0278] So far, the application of the solution phase methodology to
resin-bound peptide substrates was proceeding as expected. A need
for longer reaction times and catalyst loadings was apparent,
however, and highlighted the subtle differences between the two
approaches. After selective ring closing metathesis, the remaining
prenylglycine residue was employed for the formation of the second
dicarba bond.
[0279] Activation of the prenyl group was achieved via butenolysis
of the resin-bound pentapeptide 105a. The peptide was exposed to an
atmosphere of cis-2-butene (15 psi) and 40 mol % second generation
Grubbs' catalyst in dichloromethane for 42 hours. This led to a
mixture of the desired product 106 and the starting peptide 105.
The reaction was unexpectedly and inexplicably slow compared to the
analogous solution phase activation step. The recovered
resin-peptide was therefore re-subjected to analogous butenolysis
conditions which led to the formation of the target
crotylglycine-containing peptide 106 (Scheme 6.11). Mass spectral
analysis of the cleaved peptide displayed the product molecular ion
peak at m/z 773.2 (M+H).sup.+ and no evidence of the starting
prenylglycine-containing peptide 105 was observed.
##STR00076##
2-Butene
[0280] High purity 2-butene was found to be critical for high
turnovers in butenolysis reactions (when butane is the disposable
olefin). For example, when butenolysis reactions were conducted on
an unsaturated triglyceride (triolein) with commercially available
and less expensive cis+trans-2-butene mixtures only traces of
butenolysis products were detected, even with high catalyst
loadings. GC analysis of the isomeric 2-butene mixture showed that
it was contaminated with 2.6% of butadiene, while none of this
impurity was found in the commercially available cis-2-butene. The
addition of 1,3-butadiene (2%) to pure cis-2-butene gave a mixture
that did not give cross-metathesis products with triolein while a
cis+trans-2-butene mixture (30:70) free of 1,3-butadiene was found
to give the same activity in butenolysis reactions as pure
cis-2-butene. These results suggested that the 1,3-butadiene was
acting as a poison in reactions employing commercial grade
cis+trans-2-butene. This discovery is significant and previously
unreported; a GC trace of commercially available trans 2-butene is
contaminated with 1,3-butadiene (FIG. 5), GC traces of
cis+trans-2-butene mixtures show the same impurities. In
conclusion, cis-, trans- and mixtures of cis+trans-2-butene can all
be used in butenolysis (unblocking reactions with a disposable
olefin) reactions but all must be 1,3-butadiene free. Later work
with other disposable olefins shows that functionalisation of the
C1 or C4 carbon atoms of 2-butene further improves turnover,
especially for resin-based peptides.
[0281] A cross metathesis reaction between the activated-resin
bound peptide 106a and crotylglycine derivative 81 was then
performed. We decided to investigate microwave technology as a
means of decreasing reaction time in the solid-phase approach.
Microwave irradiation of a mixture of resin-tethered peptide 106a
with 40 mol % second generation Grubbs' catalyst, excess
crotylglycine 81 (.about.50 equiv) in dichloromethane and 10%
lithium chloride in dimethylformamide resulted in formation of the
desired intermolecular dicarba linkage (Scheme 6.12). Mass
spectrometry confirmed product formation 107 with the appearance of
a molecular ion peak at m/z 902.3 (M+H).sup.+.
##STR00077##
[0282] Wilkinson's hydrogenation of the unsaturated intermolecular
bridge was achieved under conditions previously established (80 psi
H.sub.2, dichloromethane:methanol (9:1), room temperature, 22
hours) to give the target peptide 108 containing two
regioselectively constructed dicarba bridges (Scheme 6.13).
##STR00078##
[0283] The successful application of the solution phase methodology
(section 4) to a resin-bound pentapeptide 102a led to selective
construction of an intramolecular and an intermolecular dicarba
bridge. Several important biologically active peptides, such as
those within the insulin superfamily (insulin and relaxin), possess
metabolically unstable inter- and intramolecular cystine bonds.
This methodology can be applied to the regioselective construction
of stable dicarba analogues of these peptides. We next examined the
extension of this strategy to the construction of bicyclic
peptides--as cystino-dicarba analogues and bis-dicarba analogues.
The latter analogues require the formation of two intramolecular
dicarba bridges via sequential ring closing metathesis
reactions.
[0284] Liskamp et al. recently reported the synthesis of a crossed
alkene-bridge of the complex DE-bisthioether ring system of nisin,
a lantibiotic that possesses five thioether bridges (as distinct to
disulfide bridges--which are less stable) (Diagram
6.2)..sup.157
##STR00079##
[0285] A linear precursor 109 containing four identical
allylglycine residues was subjected to a solution phase double ring
closing metathesis reaction. The first cyclisation reaction yielded
four out of a possible six mono-cyclic peptides. Successive ring
closing metathesis under similar conditions ultimately yielded the
target 1-4, 3-6-carbocyclic peptide 110 (72%) and a contaminating
1-3, 4-6-bicycle 111 (19%) (Scheme 6.14).
##STR00080##
[0286] These results suggest favourable pre-organisation of the
linear peptide for generation of the target regioisomer..sup.157
The selective synthesis of multiple bridges, however, is rarely so
fortuitous..sup.129,225,231,232 Indeed, in the synthesis of native
conotoxin sequences, several topoisomers (ribbon, globule and
beads) are obtained after oxidative folding..sup.225,231,233
Multiple cystine formation usually requires an orthogonal
protection strategy and sequential oxidation of cysteine
residues..sup.225 For this reason, we investigated the
regioselective methodology developed in section 4 for the synthesis
of dicarba analogues of a native conotoxin sequence, Ctx ImI
93.
6.4 Synthesis of Dicarba Analogues of Conotoxin ImI
[0287] Conotoxins are venom components of cone snails (Conidae) and
represent a group of small disulfide-rich peptides that act as
potent and highly specific antagonists for different receptor
targets..sup.171-174 Conotoxins derive their receptor subtype
specificity from the arrangement of their disulfide bonds and
resultant loop sizes. For example, .alpha.-conotoxins, which
contain two disulfide bonds in a 1-3, 2-4 arrangement (Diagram
6.3), target nicotinic acetylcholine receptors of
vertebrates..sup.234 .chi.-Conotoxins, on the other hand, possess a
1-4, 2-3 disulfide bond arrangement and are selective for
noradrenaline transporters.
##STR00081##
[0288] The small size (typically between 10-40 amino acids),
selectivity and potency of conotoxins make them ideal therapeutic
candidates for clinical conditions such as pain, epilepsy, stroke
and cancer..sup.171,234 Recently Ziconotide, an .omega.-conotoxin,
completed Phase III clinical trials for neuropathic pain whilst two
new conotoxin analogues (.omega.-Ctx CVID and .chi.-Ctx MrIA) are
in clinical trials for chronic pain.
[0289] Conotoxins possess a rich diversity of amino acid residues
and this, coupled with their potential as pharmaceutical agents,
makes them challenging and interesting targets. We chose to examine
.alpha.-conotoxin ImI 93 (Ctx ImI), a small cysteine rich
dodecapeptide isolated from the vermivorous conus species Conus
imperialis..sup.173,174,226 Its two intramolecular disulfide bonds
form the hydrophobic core of the molecule and generate a
constrained two loop structure which, together with a central
proline residue, arrange three essential residues (Asp5, Arg7,
Trp10) for selective interaction with complementary residues within
the .alpha.7 neuronal nicotinic acetylcholine receptor.
##STR00082##
[0290] Interestingly, the structural and functional role of the
disulfide bonds in these natural products is yet to be elucidated.
Generation of dicarba-cystino hybrids of conotoxin ImI and
ultimately bis-dicarba analogues allows the importance of the
constituent bridges on the structure and activity of the peptide to
be elucidated. We therefore investigated the application of the
on-resin metathesis-hydrogenation sequence to generate a library of
dicarba analogues of conotoxin ImI (Diagram 6.4).
##STR00083##
[0291] Metathesis catalysts display high functional group tolerance
and homogeneous rhodium-based catalysts, unlike their heterogeneous
counterparts, are not poisoned by sulfur-containing functionality.
We decided to initiate our study with the synthesis of
dicarba-cystino hybrids of Ctx ImI.
6.4.1 Cystino-Dicarba Hybrids of Conotoxin ImI
[0292] Native .alpha.-conotoxins are amidated at their C-termini.
Rink amide resin was therefore chosen to facilitate linear peptide
construction and generate the required C-terminal carboxamide upon
resin cleavage. The low loading (0.52 mmolg.sup.-1) of the Rink
amide linker helps to reduce crowding and aggregation of peptide
chains and reduces the likelihood of homodimerisation in the
subsequent metathesis reaction. Standard SPPS using HATU-NMM
activation and Fmoc-protected amino acids was used to construct the
two linear peptides: [2,8]-Hag-[3,12]-Cys conotoxin ImI 112 and
[2,8]-Cys-[3,12]-Hag conotoxin ImI 113. Both of these sequences
possess two strategically placed non-proteinaceous L-allylglycine
(Hag) residues to facilitate construction of the dicarba bridge.
Intermediates were carried through without purification or
characterisation up to the dodecapeptides 112 and 113. A sample of
each linear peptide was obtained by cleavage from the resin and
determined to be of >95% purity by reverse-phase-HPLC. Mass
spectral analysis gave the molecular ion peak at m/z 1565.7
(M+H).sup.+ and the corresponding doubly charged ion peak at m/z
783.5 [1/2(M+2H)].sup.+. Both ions are consistent with the
structures of the isomeric sidechain deprotected linear peptides
112 and 113.
##STR00084##
[0293] Ring closing metathesis was performed on resin-attached
linear peptides to eliminate any potential problems arising from
dimerisation and/or poor peptide solubility. Exposure of [2,8]-Hag
Ctx ImI 112a to first generation Grubbs' catalyst (50 mol %) in
dichloromethane at 50.degree. C. for 72 hours gave only trace
amounts (<10%) of cyclised product 114. The more reactive second
generation Grubbs' catalyst was then used to improve the
cyclisation yield (Scheme 6.15). While RCM progressed further
(.about.70%) with this catalyst, conditions could not be found to
effect full cyclisation to 114. Change in solvent, concentration,
catalyst loading, and reaction time had no positive effect on
conversion. The addition of a chaotropic salt (lithium chloride in
dimethylformamide) to the reaction mixture also had no effect on
RCM yield. Similarly, RCM of a dicrotylglycine analogue of the
primary sequence of 112, which avoids catalytic cycling through an
unstable ruthenium-methylidene species, also failed to achieve
complete conversion to the cyclic target 114.
##STR00085##
[0294] Construction of the isomeric [3,12]-unsaturated carbocyclic
Ctx ImI 115 was found to be even more problematic. Exposure of the
resin-bound peptide 113a to both first and second generation
Grubbs' catalysts under a variety of experimental conditions failed
to yield the unsaturated carbocycle 115. Possible reasons for the
poor reactivity of this isomer 113 included the diminished
influence of the proline residue in assisting formation of the
larger carbocycle (28-membered ring) and the close proximity of the
C-terminal allylglycine residue to the bulky Rink amide linker. The
sequence was therefore reconstructed on BHA resin bearing a linear
HMBA-Gly-Gly-linker. Cyclisation of the BHA resin-bound peptide was
attempted in the presence of 20 mol % second generation Grubbs'
catalyst and chaotropic salts. Unfortunately, mass spectral
analysis of the product mixture again showed only the starting
peptide 113.
[0295] Microwave-assisted ring closing metathesis of isomeric
linear peptides 112a and 113a provided both of the target
carbocycles 114 and 115. In our study, a microwave reactor emitting
a focused irradiation at 2.45 GHz with a maximum power of 300 W was
used. Irradiation of a mixture of Rink amide-bound
[2,8]-Hag-[3,12]-Cys Ctx ImI 112a and second generation Grubbs'
catalyst (10 mol %) in dichloromethane containing 10% lithium
chloride in dimethylformamide resulted in complete ring closure in
only one hour (Scheme 6.16). Decreasing the catalyst loading (5 mol
%) also led to quantitative conversion to the unsaturated
carbocycle 114 after just two hours of microwave irradiation. Mass
spectral analysis of the product mixture showed the required
molecular ion peak at m/z 1537.7 (M+H).sup.+ and the corresponding
doubly charged ion at m/z 769.5 [1/2(M+2H)].sup.+ for the
unsaturated carbocyclic peptide 114 and no starting linear peptide
112.
##STR00086##
[0296] Similar reaction conditions also resulted in complete
cyclisation of 113a to the isomeric [3,12]-dicarba analogue 115
(Scheme 6.17). Although a higher catalyst loading (20 mol %) was
required, the reaction went to completion in one hour. The
enhancement in RCM yield via this microwave-assisted approach is
remarkable in light of the poor results obtained using conventional
heating methods. It is considered that the results must be
attributed to something beyond just more efficient heating. It has
been postulated that another possible factor is that microwave
radiation causes highly efficient disruption of peptide aggregation
on the solid support. It is noted that the reaction of scheme 6.17
does not proceed without microwave irradiation.
##STR00087##
[0297] On-resin Fmoc-deprotection of the unsaturated carbocycles
114a and 115a followed by acid-mediated cleavage yielded the fully
deprotected peptides 116 and 117. Aerial oxidation of 116 and 117
in 5% dimethylsulfoxide/aqueous ammonium carbonate (0.1 M, pH 8)
then afforded the unsaturated cystino-dicarba Ctx ImI analogues 118
and 119 respectively (Scheme 6.18, Scheme 6.19). Each peptide was
purified by reverse-phase HPLC (>99% purity) and isolated in 5%
yield. These dicarba-analogues, and others based on native
conotoxin sequences of pharmaceutical significance (e.g.
dicarba-analogues of conotoxins extracted from Conus regius and
Conus victoriae (ACV1)), see experimental section) are predicted to
be biologically active due to their similarities to the disulfide,
and are predicted to have in vivo stability due to the presence of
the dicarba-bond.
[0298] It is important to note that the isolation and purification
of native conotoxin sequences from cone snail venom is a low
yielding and tedious process..sup.247 Recently, 200 mg of venom
extract from five cone snails (Conus textile) was purified to yield
1.1 mg (560 nmol) of conotoxin .epsilon.-TxIX..sup.248 Most
references detailing the isolation of conotoxin molecules from
venom, however, do not cite isolation yields. Synthesis of
conotoxin molecules can also be low yielding where oxidative
folding leads to several topoisomers..sup.225,231,233,249-251
Extensive chromatography must be employed to isolate pure samples
of the target peptide. Although the final purified yields of our
dicarba-cystino conotoxin analogues 118 and 119 were low,
separation conditions were not optimised and the scale of the
reactions could be easily increased to afford larger quantities of
pure peptide.
##STR00088##
##STR00089##
[0299] Hydrogenation of resin-bound unsaturated carbocyclic
peptides 114a and 115a was performed with Wilkinson's catalyst.
This homogeneous catalyst is ideal for this transformation as it
allows reduction to be performed on the resin, operates under mild
reaction conditions and is highly tolerant of sulfur-containing
functionality. Hence, rhodium-catalysed hydrogenation of
resin-bound carbocycles 114a and 115a in dichloromethane:methanol
(9:1) effected quantitative reduction of the olefin at room
temperature and low hydrogen pressure (80 psi) (Scheme 6.20).
Interestingly, the crude product from each of these reactions was
obtained as a mixture of the cystine reduced (120, 121) and
oxidised (122, 123) forms. It is important to note that the final
targets 122 and 123 are isomeric with the unsaturated deprotected
precursor peptides, 116 and 117 respectively. An analogous
hydrogenation experiment spiked with linear diallyl conotoxin
sequence 112, the precursor to the unsaturated carbocycle 114,
showed a molecular ion consistent with the formation of the
dipropyl sidechain-containing peptide 124..sup.235 This mass
spectral data strongly suggests that the catalyst is not poisoned
by the trityl-protected cysteine residues and that the
hydrogenation conditions needed for olefin reduction are
uncompromised. Hence, the species contributing to the peak at m/z
769.5 [1/2(M+2H)].sup.+ are likely to be the final isomeric
cystino-dicarba Ctx ImI peptides 122 and 123.
##STR00090##
[0300] Further support for this hypothesis comes from the LC-MS
traces of the product mixtures. In each case, a signal at
t.sub.R=6.01 min (122) and t.sub.R=7.02 min (123) was observed. In
comparison, the retention times for the isomeric unsaturated
carbocycles 116 and 117, under identical chromatographic
conditions, are 5.66 min and 6.64 min respectively. The saturated
cystino-dicarba .alpha.-conotoxin analogues 122 and 123 are
currently undergoing chromatographic purification and are being
assessed for biological activity and in vivo stability. NMR
spectroscopy will also be used to further confirm the structures of
the isomeric conotoxin analogues 122 and 123.
##STR00091##
6.4.2 Bis-Dicarba Conotoxin Analogues
[0301] The regioselective on-resin methodology described in section
4 was also applied to the synthesis of fully carbocyclic conotoxin
ImI analogues. The catalytic sequence involves the selective RCM of
reactive allylglycine units in the presence of dormant
prenylglycine residues followed by selective hydrogenation of the
resultant unsaturated carbocycle. Activation of the prenyl groups
via butenolysis gives the active crotyl sidechains which can
undergo the RCM-hydrogenation process to afford the target bicycles
125 and 126 (Scheme 6.21).
##STR00092##
[0302] This study commenced with the construction of the linear
isomeric conotoxin analogues, [2,8]-Hag-[3,12]-Pre conotoxin ImI
127 and [2,8]-Pre-[3,12]-Hag conotoxin ImI 128. Standard SPPS
techniques employing Rink amide resin, HATU-NMM activation and
Fmoc-protected amino acids facilitated synthesis of the peptides
127 and 128. Both of these sequences possess two strategically
placed non-proteinaceous L-allylglycine (Hag) residues to
facilitate the selective construction of the first carbocycle. The
incorporation of two less reactive prenylglycine residues later
enables the selective formation of the second carbocycle. During
construction of the linear peptides, intermediates were carried
through without purification or characterisation up to the
dodecapeptides 127 and 128. As expected, the prenylglycine residues
were incorporated without complication and mass spectral analysis
gave doubly charged molecular ion peaks at m/z 805.6
[1/2(M+2H)].sup.+ and 816.6 [1/2(M+Na+H)].sup.+ which are
consistent with the structures of the isomeric sidechain
deprotected linear peptides 127 and 128. An additional peak at m/z
814.6 [1/2(M+H.sub.2O+2H)].sup.+, corresponding to the
acid-promoted hydration of a prenyl group, was also apparent in the
spectrum.
##STR00093##
[0303] After confirming the successful synthesis of the linear
peptides 127 and 128, ring closing metathesis of the resin-tethered
peptides was performed using conventional heating methods. Exposure
of peptide 127a to second generation Grubbs' catalyst (40 mol %) in
dichloromethane and 10% lithium chloride in dimethylformamide at
50.degree. C. for 40 hours gave the unsaturated carbocycle 129
(Scheme 6.22).
##STR00094##
[0304] Analogous RCM conditions for 128a, however, led to complete
recovery of the linear peptide. These results highlight the
influence of the peptide sequence on RCM success when microwave is
not used. A derivative of the problematic sequence 128 was
therefore constructed to elucidate the effect of a turn-inducer. A
new peptide sequence 130 was synthesised possessing a Pro9 residue
rather than the native Ala9 residue. Interestingly, the resultant
solid-supported peptide 130a cyclised under the previously
unsuccessful metathesis conditions (without microwave radiation) to
give 131, but the RCM did not go to completion (Scheme 6.23).
Unfortunately, LC-MS analysis did not enable separation of the
linear 130 and cyclic 131 peptide and hence an estimation of
reaction conversion could not be made.
##STR00095##
[0305] Microwave-assisted ring closing metathesis, however,
provided expedient syntheses for both the target carbocycles 129
and 132. Microwave irradiation of a solution of Rink amide
bound-peptide 127a and second generation Grubbs' catalyst (10 mol
%) in dichloromethane containing 10% lithium chloride in
dimethylformamide at 100.degree. C. resulted in complete ring
closure in only one hour (Scheme 6.24). Mass spectral analysis of
the product mixture showed the required molecular ion with m/z
791.4 [1/2(M+2H)].sup.+ for the unsaturated dicarba peptide 129 and
no starting linear peptide 127.
[0306] The resin-bound isomeric dicarba analogue 128a also
completely cyclised in one hour with 20 mol % second generation
Grubbs' catalyst using the same solvent system at 100.degree. C.
(Scheme 6.25).
##STR00096##
##STR00097##
[0307] These results were very exciting and demonstrated the power
of microwave energy to yield carbocycles that were unattainable by
conventional heating methods. In addition, the prenyl sidechains
remained inert to the microwave-accelerated metathesis conditions
and no cross metathesis products were observed.
[0308] Rhodium-catalysed hydrogenation of the resin-bound
carbocycles 129a and 132a in dichloromethane:methanol (9:1)
effected quantitative reduction of the unsaturated carbocycle at
room temperature and low hydrogen pressure (80 psi) (Scheme 6.26
and Scheme 6.27). The mass spectra of cleaved peptides from both
reactions displayed doubly charged molecular ion peaks at m/z 792.5
[1/2(M+2H)].sup.+ and m/z 801.5 [1/2(M+H.sub.2O+2H)].sup.+
confirming formation of the isomeric products 133 and 134.
Importantly, the prenyl groups resisted hydrogenation and were now
available for activation to facilitate construction of the second
carbocycle.
##STR00098##
##STR00099##
[0309] Activation of the prenyl sidechains involved butenolysis of
the solid-supported peptides 133a and 134a. The peptide 133a was
exposed to an atmosphere of cis-2-butene (15 psi) and a mixture of
40 mol % second generation Grubbs' catalyst and benzoquinone in
dichloromethane for 38 hours (Scheme 6.28). Benzoquinone was added
to the reaction mixture to reduce or eliminate the potential for
olefin isomerisation. Mass spectral analysis of a cleaved aliquot
of peptide confirmed formation of the target
dicrotylglycine-containing peptide 135 with the appearance of a
peak at m/z 778.4 [1/2(M+2H)].sup.+. No starting prenyl-containing
peptide 133 was observed, however, mass spectral data revealed low
intensity, doubly charged higher homologue species separated by
m/z+7 units. Under the above described metathesis conditions, the
generated crotyl sidechain can isomerise to a terminal butenyl
chain and then undergo secondary cross metathesis with cis-2-butene
(Scheme 6.29). The products arising from this process of
isomerisation-cross metathesis are consistent with the observed
mass spectral data.
##STR00100##
##STR00101##
[0310] Reaction conditions were modified to minimise this competing
isomerisation reaction. These changes involved the addition of
chaotropic salts and variation of catalyst loading and reaction
time. This aim was realised, although to a small extend this was
still accompanied by partially metathesised peptide 136 and
starting material 133.
##STR00102##
[0311] Microwave-accelerated ring closing metathesis of the
resin-tethered peptide 135a using second generation Grubbs'
catalyst (20 mol %) in dichloromethane and 10% lithium chloride in
dimethylformamide afforded the target peptide 140 in only one hour
(Scheme 6.30). Preliminary LC-MS analysis was encouraging with the
appearance of the required doubly charged molecular ion peak at m/z
750.4 [(M+2H)].sup.+, corresponding to the bicyclic peptide 140.
Interestingly, a very low intensity peak at m/z 764.4 was also
evident which corresponded to the cyclic product of a contaminating
isomerisation-butenolysis adduct. The Fmoc-deprotected product 125
is being purified and submitted for biological testing.
##STR00103##
[0312] Rhodium-catalysed hydrogenation of the resin-bound bicycle
140a was performed in dichloromethane:methanol (9:1) at room
temperature under low hydrogen pressure (80 psi) (Scheme 6.31).
Preliminary mass spectral and LC-MS data of the isolated residue
confirm the formation of the saturated bicycle 126.
##STR00104##
[0313] The problem of isomerization experienced during activation
of the diprenyl-conotoxin sequence 133a (see FIG. 6.5) was
subsequently reinvestigated. It was postulated that the highly
non-polar ethylene and 2-butene, used for activation of the prenyl
groups, could be incompatible with the polystyrene resin support.
Resins, such as Wang and Rink amide, swell well in polar solvents;
exposure to non polar solvents results in resin shrinkage, poor
accessibility of reagents (ie catalysts) to reactive functionality
and consequently poor conversion. A more polar derivative of
ethylene and 2-butene, however, would achieve better resin swell
and higher activation yields. Conseqeuntly, an investigation using
1,4-diacetoxy-cis-2-butene was initiated. This molecule has the
advantage of being a liquid at ambient temperature while exhibiting
greater polarity than either ethylene or butene.
[0314] Before 1,4-diacetoxy-cis-2-butene, or other like analogues
(such as 1,4-dichloro-2-butene), could be used to activate
prenyl-containing resin-tethered peptides, (i) its reactivity and
ability to activate hindered olefins; (ii) compatibility with
resin-tethered substrates; and (iii) its ability to form a reactive
intermediate (i.e. allylic acetate) capable of a subsequent CM with
a reactive type I olefin (i.e. to form subsequent intra/inter
dicarba bonds) needed to be investigated. Steps (i) and (iii) were
investigated using a simple small molecule derived from
commercially available racemic allylglycine in three steps (Scheme
6.32).
##STR00105##
[0315] To generate the required type III olefin, the
benzoyl-protected methyl ester of allylglycine 62 underwent a cross
metathesis reaction with 2-methyl-2-butene (Scheme 6.32). The
reaction proceeded at 50.degree. C. in dichloromethane for 72 h in
a pressurised vessel giving the prenylglycine derivative 87 in
quantitative conversion after chromatographic purification.
[0316] Next, the fully protected prenylglycine analogue 87 was
subjected to standard cross metathesis conditions using an excess
of 1,4-diacetoxy-cis-2-butene, in the presence of second generation
Grubbs' catalyst (Scheme 6.32). After stirring at 50.degree. C.
overnight, the reaction was complete, as indicated by t.l.c. Column
chromatography of the crude material yielded the activated molecule
141 in 57% yield.
[0317] With activation of the prenylglycine derivative complete,
the reactivity of the resultant molecule was assessed. Initially,
homodimerisation was attempted; the molecule was subjected to
standard cross metathesis conditions, in the presence of second
generation Grubbs' catalyst at 50.degree. C. (Scheme 6.33). Mass
spectral analysis indicated both the desired product and starting
material. Gas chromatographic analysis indicated the expected
equilibrium statistical mixture of the desired homodimer 69,
1,4-diacetoxy-2-butene and starting material 141.
##STR00106##
[0318] To assess the reactivity of the activated moiety 141, cross
metathesis with a type I olefin was investigated (Scheme 6.31).
Standard cross metathesis conditions were applied, with a 6-fold
excess of the type I olefin to avoid the statistical distribution
of products and increase the yield of the desired peptide. The
desired cross metathesis product 142 was obtained as a brown oil in
81% yield following purification via column chromatography.
Spectroscopic analysis confirmed the presence of both the E- and
Z-isomers, though individual NMR signals could not be assigned to a
specific geometry.
[0319] To assess the compatibility of 1,4-diacetoxy-cis-2-butene
with resin-tethered substrates, a simple prenylglycine-containing
dipeptide was subjected to a cross metathesis reaction with
1,4-diacetoxy-cis-2-butene (Scheme 6.34). This reaction showed
complete conversion of the resin-tethered type II olefin to the
corresponding type I olefin, indicating complete compatibility of
1,4-diacetoxy-cis-2-butene with resin-bound substrates. No
isomerization of the double bond was observed leading to the
conclusion that the extended reaction times needed during
activation reactions using non-polar olefins is responsible for the
competing isomerisation pathway.
##STR00107##
6.5 Stability
[0320] Despite the known activity of conotoxins as therapeutics,
their multiple disulfide bond frameworks are known to be unstable
under reducing conditions. Reduction or framework scrambling by
thiol containing molecules such as glutathione or serum albumin in
intracellular or extracellular environments such as blood plasma
can decrease their effectiveness as drugs.
[0321] Incubation of native-Ctx ImI in human blood plasma has been
shown to produce significant rearrangement of the disulfide
framework (i.e. scrambling). Similarly, treatment of Ctx-IMI with
glutathione, a reducing enzyme commonly found in blood plasma,
results in complete scrambling of the disulfide framework in
.about.6 hours. See for instance Armishaw, C. J., Daly, N. L.,
Nevin, S. T., Adams, D. J., Craik, D. J., Alewood, P. F., J. Biol.
Chem., 2006, in press. Such scrambling or reduction is not possible
with dicarba-Ctx IMI analogues; the dicarba linkage is completely
inert to such reductants.
6.6 Summary
[0322] In conclusion, the strategy developed can be used for the
regioselective construction of multi-dicarba bond-containing
peptides. The methodology was successfully applied to a model
synthetic pentapeptide 102 and led to the regioselective
construction of an intramolecular and intermolecular dicarba
bridge. Similarly, a tandem metathesis-hydrogenation sequence
provided dicarba-cystino analogues of naturally occurring conotoxin
ImI 93. Here, a microwave accelerated ring closing metathesis
provided cyclic peptides that were unattainable via conventional
heating methods. A fully carbocyclic analogue of conotoxin ImI 140
was also synthesised. Although activation of the prenylglycine
units with non-polar olefins (such as 2-butene and ethene) was
significantly retarded on the resin-bound peptide 133a, butenolysis
did lead to the desired activated crotyl sidechains. The
selectivity of the methodology was maintained when investigated in
the heterogeneous system. An intramolecular dicarba bridge was
selectively constructed from allylglycine units in the presence of
two prenyl olefins. A microwave-promoted RCM of the resin-bound
crotyl-containing peptide ultimately afforded the desired bicycle
140, and reduction lead to conotoxin 126. The use on polar 2-butene
analogues, such as 1,4-diacteoxy-2-butene, was found to be more
compatible with polystyrene supports and led to improved resin
swelling and activation yields. This was illustrated in both
solution phase model studies and on solid-supported peptide
substrates.
7.0 EXAMPLES
7.1 Instrumentation
[0323] Microwave reactions were carried out on a Personal Chemistry
(now Biotage) Smith Synthesiser. The instrument produces a
continuous focussed beam of microwave irradiation at 2.45 GHz with
a maximum power delivery of 300 W, which reaches and maintains a
selected temperature (100.degree. C.). Reactions were performed in
high pressure quartz microwave vessels fitted with self-sealing
Teflon septa as a pressure relief device, that were crimped in
place. The vessels contained magnetic stirrer beads and the
pressure and temperature of each reaction was monitored
continuously with an in built pressure transducer (located in the
lid) and infrared pyrometer respectively. Reaction times were
measured from the time the microwave began heating until the
reaction period had elapsed (cooling periods were not
inclusive).
7.2 Intentionally Left Blank
7.3 Peptide Procedures
7.3.1 Materials and Reagents
[0324] Peptides were synthesised in polypropylene Terumo syringes
(10 mL) fitted with a polyethylene porous (20 .mu.m) filter. Solid
phase peptide synthesis (SPPS) was performed using a Visprep.TM.
SPE DL 24-port model vacuum manifold supplied by Supelco. Coupling
reactions and cleavage mixtures were shaken on a KS 125 basic KA
elliptical shaker supplied by Labortechnik at 400 motions per
minute. Cleaved peptides were centrifuged on a HermLe Z200A
centrifuge supplied by Medos at a speed of 4500 cycles per
minute.
[0325] N,N'-Dimethylformamide (DMF) was supplied by Auspep and
stored over 4 .ANG. molecular sieves. Dichloromethane (DCM) was
supplied by BDH and stored over 4 .ANG. molecular sieves. Wang
resin, Rink amide resin, piperidine and trifluoroacetic acid (TFA)
were used as supplied by Auspep. Phenol was used as supplied by
BDH. Diisopropylcarbodiimide (DIC), diisopropylethylamine (DIPEA),
4-(N,N'-dimethylamino)pyridine (DMAP), ethanedithiol (EDT),
Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid),
N-methylmorpholine (NMM), and thioanisole were used as supplied by
Aldrich. (2S)-2-Aminopent-4-enoic acid (L-allylglycine, Hag) was
used as supplied by Peptech.
N-Fluorenylmethoxycarbonylaminosuccinimide (Fmoc-OSu),
O-(7-azabenzo-triazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluoro-phosphate (HATU) and sidechain-protected Fmoc-amino
acids were used as supplied by GL Biochem unless otherwise
specified.
7.3.2 Peptide Synthesis Procedure
[0326] Peptides were prepared using general Fmoc-SPPS
methodology..sup.142,221 Manual SPPS was carried out using fritted
plastic syringes, allowing filtration of solution without the loss
of resin. The tap fitted syringes were attached to a vacuum tank
and all washings were removed in vacuo. This involved washing (or
swelling) the resin in the required solvent for a reported period
of time, followed by evacuation which allowed the removal of excess
reagents before subsequent coupling reactions.
7.3.2.1 Wang Resin
[0327] In a fitted syringe, Wang resin was swollen with DCM (7 mL,
3.times.1 min, 1.times.60 min) and DMF (7 mL, 3.times.1 min,
1.times.30 min). DIC (3 equiv.) was added to a solution of
protected amino acid, Fmoc-L-Xaa-OH, (3 equiv.) in DMF (3 mL). The
activated amino acid solution was added to the swelled resin and
shaken gently for 1 min. A solution of DMAP (0.3 equiv.) in DMF (1
mL) was added to the resin and the reaction mixture was shaken
gently for the reported period of time. The mixture was then
filtered and the resin-tethered amino acid was washed with DMF (7
mL, 3.times.1 min) to ensure excess reagents were removed. In order
to prevent formation of deletion products, remaining resin active
sites were capped with an anhydride solution (5% acetic anhydride,
1% NMM, 94% DMF) for 1 h. The mixture was filtered and the resin
was washed with DMF (7 mL, 3.times.1 min) and deprotected with 20%
piperidine in DMF (7 mL, 1.times.1 min, 2.times.10 min). After this
deprotection step, the resin was washed with DMF (7 mL, 5.times.1
min) to remove traces of base prior to coupling the next amino
acid.
[0328] Subsequent amino acids were coupled using the following
procedure:
[0329] NMM (6 equiv.) was added to a solution of protected amino
acid, Fmoc-L-Xaa-OH (3 equiv.) and HATU (2 equiv.) in DMF (3 mL)
and shaken gently for 1 min. The activated amino acid solution was
added to the resin-tethered amino acid and shaken gently for the
reported period of time. The peptidyl-resin was then washed with
DMF (7 mL, 3.times.1 min) and the Kaiser test.sup.255 was performed
to monitor coupling success. Any incomplete reactions were repeated
with extended reaction times (indicated in brackets). Upon negative
test results for the presence of free amine, the resin-peptide was
deprotected with 20% piperidine in DMF (7 mL, 1.times.1 min,
2.times.10 min) and washed again with DMF (7 mL, 5.times.1 min) to
remove traces of base prior to coupling the next amino acid.
[0330] The above procedure was repeated until the desired peptide
sequence was constructed. Once complete, the resin was washed with
DMF (7 mL, 3.times.1 min), DCM (7 mL, 3.times.1 min), MeOH (7 mL,
3.times.1 min), DCM (7 mL, 3.times.1 min), MeOH (7 mL, 3.times.1
min) and dried in vacuo for 1 h. A small aliquot of resin-tethered
peptide was then exposed to a TFA cleavage solution (Section
7.3.3).
7.3.2.2 Rink Amide Resin
[0331] In a fitted syringe, Rink amide resin was swollen with DCM
(7 mL, 3.times.1 min, 1.times.60 min) and DMF (7 mL, 3.times.1 min,
1.times.30 min) and deprotected with 20% piperidine in DMF (7 mL,
1.times.1 min, 2.times.10 min) and washed again with DMF (7 mL,
5.times.1 min). NMM (6 equiv.) was added to a solution of a
protected amino acid, Fmoc-L-Xaa-OH (3 equiv.) and HATU (2 equiv.)
in DMF (3 mL) and shaken gently for 1 min. The activated amino acid
solution was added to the resin and shaken gently for the reported
period of time. The peptidyl-resin was washed with DMF (7 mL,
3.times.1 min) to ensure excess reagents were removed. Kaiser tests
were performed to monitor coupling success and any incomplete
reactions were repeated with extended reaction times (indicated in
brackets). Upon negative test results for the presence of free
amine, the resin-peptide was deprotected with 20% piperidine in DMF
(7 mL, 1.times.1 min, 2.times.10 min) and washed again with DMF (7
mL, 5.times.1 min). This coupling procedure was repeated until the
desired peptide sequence was constructed.
[0332] The above procedure was repeated until the desired peptide
sequence was constructed. Once complete, the resin was washed with
DMF (7 mL, 3.times.1 min), DCM (7 mL, 3.times.1 min), MeOH (7 mL,
3.times.1 min), DCM (7 mL, 3.times.1 min), MeOH (7 mL, 3.times.1
min) and dried in vacuo for 1 h. A small aliquot of resin-tethered
peptide was then exposed to a TFA cleavage solution (Section
7.3.3).
Kaiser Test
[0333] The Kaiser test was performed in order to monitor coupling
success by detecting the presence of resin-bound free
amines..sup.221,255 Two drops of 5% ninhydrin in EtOH, 80% phenol
in EtOH and 2% v/v 0.001 M potassium cyanide in pyridine were added
to pre-washed (EtOH) resin beads in a tube and the mixture was
subsequently heated at 120.degree. C. for 3-5 min. Blue colouration
of the beads indicate the presence of free amines and provide
evidence for an incomplete coupling reaction. It should be noted
that this test cannot be performed after coupling asparagine,
aspartic acid, serine and proline..sup.221,256
7.3.3 Peptide Cleavage: TFA-Mediated Cleavage Procedure
[0334] A small aliquot of the resin-peptide (.about.1 mg) was
suspended in a cleavage solution 2 mL):90% TFA:5% thioanisole:2.5%
EDT 2.5% water and phenol (1.6 g/5 mL of cleavage solution) and
shaken gently for 1.5 h. The mixture was then filtered and the
resin beads were rinsed with TFA (2.times.0.5 mL). The filtrate was
concentrated with a constant stream of air to yield an oil. The
peptide was precipitated with ice-cold Et.sub.2O (2 mL) and
collected by centrifugation (3.times.10 min). The supernatant
liquid was decanted and the resultant residue was collected and
analysed by mass spectrometry.
7.3.4 Ellman's Test
[0335] The Ellman's test was performed in order to monitor reaction
progress during thiol oxidation (cystine formation) by detecting
the presence of free sulfhydryl groups..sup.257 200 of a solution
of Ellman's reagent in aqueous (NH.sub.4).sub.2CO.sub.3 buffer (4
mg mL.sup.-1 in 0.1 M buffer) was added to 200 .mu.L of the
reacting peptide solution. An intense yellow colouration of the
solution indicates the presence of free thiol groups and provides
evidence for an incomplete oxidation reaction.
7.3.5 Automated Peptide Synthesis
[0336] Peptide synthesis was also performed on a CEM Liberty
Peptide Synthesiser.TM. with a CEM Microwave Discover System.TM..
Both systems were operated with the use of PepDriver software. The
desired peptide sequence and methods were installed on PepDriver.
The resin was weighed directly into a 50 mL centrifuge tube, DMF (5
mL) added, then the tube was screwed into position on the Liberty
resin manifold. Amino acid solutions (0.2M in DMF) were prepared
and installed onto the Liberty amino acid manifold. External
reagents were prepared as described: A 0.45M solution of HOBt and
HBTU in DMF was prepared as the activator reagent. A 20% v/v
solution of piperidine in DMF was used at the deactivation reagent.
Activator base reagent was prepared by making a 2M solution of DIEA
in NMP. Delivery volumes of all external reagents were calibrated
on the Liberty Peptide Synthesizer.TM. prior to use. The
temperature of the Discover System.TM. was maintained via a fiber
optic sensor located below the microwave cavity.
[0337] For all automated synthesis, "B.01 Initial Deprotection"
followed by "B.01 Extended Deprotection" cycles were used in the
method. These deprotection cycles consisted of washing with DMF
(1.times.7 mL), addition of the deprotection reagent (20%
piperidine in DMF, 10 mL), followed by the "B.01 Initial
Deprotection" microwave program. The peptidyl-resin was exposed to
a temperature of 37.degree. C. at a power of 37 watts for 2 min.
The resin was then washed with DMF (12 mL) and a further 10 mL of
the deprotection reagent was added followed by the "B.01 Extended
Deprotection" cycle. The peptidyl-resin was exposed to a
temperature of 75.degree. C. at a power of 45 watts for 10 min. The
resin was then washed with DMF (3.times.7 mL). Amino acid coupling
cycles varied for each type of peptide and these are specified in
each automated peptide synthesis description.
7.4 Hydrogenation Procedures
7.4.1 Catalysts and Materials
Catalysts:
[0338] Palladium on charcoal (Pd/C) with 10% Pd concentration was
used as supplied by Aldrich and stored in a desiccator.
Tris(triphenylphosphine)rhodium(I) chloride (Wilkinson's catalyst,
Rh(I)(PPh.sub.3).sub.3Cl]) was used as supplied by Aldrich and
stored under argon in a dry box. Asymmetric catalysts:
(+)-1,2-Bis[(2S,5S)-2,5-diethylphospholano]benzene(1,5-cyclooctadiene)rho-
dium(I) trifluoromethane-sulfonate
([(COD)Rh(I)--(S,S)-Et-DuPHOS]OTf, Rh(I)--(S,S)-Et-DuPHOS),
(-)-1,2-bis[(2R,5R)-2,5-diethylphospholano]benzene(1,5-cyclooctadiene)rho-
dium(I) tetra-fluoroborate ([(COD)Rh(I)--(R,R)-Et-DuPHOS]BF.sub.4,
Rh(I)--(R,R)-Et-DuPHOS), (+)-1,2-bis
[(2S,5S)-2,5-dimethylphospholano]benzene
(1,5-cyclooctadiene)rhodium(I) trifluoromethanesulfonate
([(COD)Rh(I)--(S,S)-Me-DuPHOS] OTf, Rh(I)--(S,S)-Me-DuPHOS),
(-)-1,2-bis[(2R,5R)-2,5-dimethylphospholano]benzene(1,5-cyclooctadiene)rh-
odium(I)tetrafluoroborate ([(COD)Rh(I)--(R,R)-Me-DuPHOS]BF.sub.4,
Rh(I)--(R,R)-Me-DuPHOS), and (+)-1,2-bis
[(2R,5R)-2,5-dimethylphospholano]ethane(1,5-cyclooctadiene)rhodium(I)
trifluoromethanesulfonate ([(COD)Rh(I)--(R,R)-Me-BPE]OTf,
Rh(I)--(R,R)-Me-BPE) and
bis(carboxylato)[2,2'-bis(diphenylphosphino)-(R)-1,1-binapthyl]ruthenium(-
II) ((S)--Ru--BINAP) were used as supplied by Strem Chemicals and
stored under argon.
Gases:
[0339] Argon and hydrogen were supplied by BOC gases and were of
high purity (<10 ppm oxygen). Additional purification was
achieved by passage of the gases through water, oxygen and
hydrocarbon traps.
Solvents:
[0340] Benzene, MeOH, DCM, .sup.tBuOH and THF used in
metal-catalysed hydrogenation reactions were degassed with high
purity argon prior to use.
Reaction Vessels:
[0341] Fischer-Porter shielded aerosol pressure reactors (100 mL)
fitted with pressure gauge heads and stirrer beads were employed
for hydrogenation reactions.
7.4.2 Pd/C Hydrogenation Procedure.sup.36,37
[0342] A Fischer-Porter tube was charged with substrate, catalyst
(substrate:catalyst, 50:1) and solvent (5-10 mL). The reaction
vessel was connected to the hydrogenation manifold, evacuated and
flushed with argon gas before being charged with hydrogen gas to
the reported pressure. The reaction was stirred at the specified
temperature for the reported period of time. The hydrogen gas was
then vented, the catalyst removed via filtration through a Celite
pad and the solvent evaporated under reduced pressure.
7.4.3 Asymmetric Hydrogenation Procedure.sup.36,37,119
[0343] In a dry box, a Fischer-Porter tube was charged with
substrate, catalyst (substrate:catalyst, 100:1) and dry
deoxygenated solvent (4-10 mL). The reaction vessel was assembled
and tightly sealed within the dry box. The apparatus was connected
to the hydrogenation manifold and purged three times using a vacuum
and argon flushing cycle before being pressurised with hydrogen gas
to the reported pressure. The reaction was then stirred at the
specified temperature for the reported period of time. The hydrogen
gas was vented and the solvent was evaporated under reduced
pressure. Purification was achieved by flash chromatography
(silica, EtOAc).
Freeze-Pump-Thaw Procedure
[0344] For liquid substrates, a freeze-pump-thaw cycle was applied
and the solution was transferred into a dry box and loaded into a
Fischer-Porter tube as described above. The substrate was dissolved
in MeOH or benzene in a Teflon-sealed vessel. The solution was
frozen upon immersion in liquid nitrogen and opened to a vacuum
source (high vacuum line .about.0.05 mm) to remove gases. The
vessel was re-sealed and the solution was allowed to thaw before
being frozen with liquid nitrogen again. This cycle was repeated
until gas evolution was no longer observed during the thaw
cycle.
7.4.4 Wilkinson's Hydrogenation Procedure
[0345] In a dry box, a Fischer-Porter tube was charged with
substrate, Wilkinson's catalyst (substrate:catalyst, 50:1) and dry
deoxygenated solvent (4-10 mL). The apparatus was connected to the
hydrogenation manifold and purged three times using a vacuum and
argon flushing cycle before being pressurised with hydrogen gas to
the reported pressure. The reaction was then stirred at ambient
temperature for the reported reaction time. The hydrogen gas was
vented and the solvent was evaporated under reduced pressure.
Purification was achieved by flash chromatography (silica,
EtOAc).
[0346] Hydrogenation experiments are described in the following
format: substrate (mg), solvent (mL), catalyst, hydrogen pressure
(psi), reaction temperature (.degree. C.), reaction time (h),
isolated yield (%), retention time (t.sub.R, GC/HPLC conditions)
and enantiomeric excess (e.e.).
7.5 Metathesis Procedures
7.5.1 Catalysts and Materials
Catalysts:
[0347] Bis(tricyclohexylphosphine)(benzylidene)ruthenium(II)
dichloride (Grubbs' catalyst),
tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-
-2-ylidene](benzylidene)ruthenium(II) dichloride (second generation
Grubbs' catalyst) and
1,3-bis(2,4,6-trimethylphenyl)-2-(imidazolidinylidene)dichloro-(o-iso-pro-
poxyphenylmethylene)ruthenium(II) dichloride (second generation
Hoveyda-Grubbs' second generation catalyst) were used as supplied
by Aldrich and stored under nitrogen.
Volatile Olefins:
[0348] Cis-2-butene (99%), cis+trans-2-butene (99%) 2-methylpropene
(iso-butylene) and 2-methyl-2-butene were used as supplied by
Aldrich. Ethylene was used as supplied by BOC gases.
Solvents:
[0349] DCM and a solution of lithium chloride in DMF (0.4 M
LiCl/DMF) used in metal-catalysed metathesis reactions were
degassed with high purity argon prior to use.
Reaction Vessels:
[0350] Schlenk tubes and microwave reactor vessels fitted with
stirrer beads were employed for ring closing and cross metathesis
reactions involving the use of solid or liquid (non-volatile)
reactants. Fischer-Porter shielded aerosol pressure reactors (100
mL) fitted with pressure gauge heads and stirrer beads were
employed for cross metathesis reactions involving gaseous
(ethylene, cis-2-butene, iso-butylene) or volatile
(2-methyl-2-butene) reactants.
7.5.2 Conventional Ring Closing and Cross Metathesis
Procedure.sup.116,142,152
[0351] A Schlenk tube was charged with substrate(s), catalyst (5-40
mol %) and deoxygenated solvent (.about.5 mL) under an inert
(nitrogen or argon) atmosphere. The reaction mixture was stirred at
50.degree. C. for the specified period of time. Metathesis
reactions were terminated upon exposure to oxygen and volatile
species were removed under reduced pressure. The crude product was
purified by flash chromatography.
7.5.3 Microwave-Accelerated Ring Closing and Cross Metathesis
Procedure
[0352] A high pressure quartz microwave vessel was loaded with
resin-tethered peptide, catalyst (5-40 mol %) and deoxygenated
solvent (.about.3-5 mL) under an inert (nitrogen and argon)
atmosphere. The reaction mixture was irradiated with microwaves and
stirred at 100.degree. C. for the reported period of time. The
mixtures were then filtered and washed with DMF (3 mL, 3.times.1
min), DCM (3 mL, 3.times.1 min), MeOH (3 mL, 3.times.1 min) and
dried on the SPPS manifold for 1 h. A small aliquot of
resin-peptide (.about.1 mg) was then subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). The isolated peptide was
analysed by mass spectrometry.
[0353] Microwave-accelerated reactions were also performed on a CEM
Discover System.TM.. The instrument produces a continuous focused
beam of microwave irradiation at a power delivery of 40 W. The
temperature on the Discover System.TM. was monitored via an
infra-red sensor located below the microwave cavity. Reactions were
performed in a 10 mL high-pressure quartz vessel fitted with a
self-sealing Teflon septa. The vessel was charged with the
peptidyl-resin, degassed solvent (5 mL DCM and 0.2 mL 2M LiCl in
DMF), 2.sup.nd generation Grubb's catalyst (20 mol %) in an inert
environment. The reaction mixture was irradiated with microwave
energy whilst being stirred at 100.degree. C. for 1 hr, cooled to
room temperature, then terminated upon exposure to oxygen. The
peptidyl-resin was filtered through a fritted syringe and washed
with DMF (5 mL, 3.times.1 min), DCM (5 mL, 3.times.1 min), DMF (5
mL, 3.times.1 min) then MeOH (5 mL, 3.times.1 min) and dried in
vacuo for 30 min prior to cleavage and analysis.
7.5.4 Conventional Cross Metathesis Procedure (Gaseous
Reactant)
[0354] In a dry box, a Fischer-Porter tube was charged with
substrate, catalyst (5-50 mol %) and deoxygenated solvent (.about.5
mL). The reaction vessel was then evacuated and purged with
ethylene, cis-2-butene or iso-butylene to the reported pressure.
The reaction mixture was stirred at 50.degree. C. for the specified
period of time. Metathesis reactions were terminated upon exposure
to oxygen and volatile species were removed under reduced pressure.
The crude product was purified by flash chromatography.
7.5.5 Conventional Cross Metathesis Procedure (Volatile
Reactant)
[0355] A Fischer-Porter tube was charged with substrate, catalyst
(5 mol %), deoxygenated solvent (.about.5 mL) and
2-methyl-2-butene. The reaction mixture was stirred at 50.degree.
C. for the specified period of time. Metathesis reactions were
terminated upon exposure to oxygen and volatile species were
removed under reduced pressure. The crude product was purified by
flash chromatography.
[0356] Metathesis experiments are described using the following
format: substrate (mg), solvent (mL), catalyst (mg), reacting
olefin (in the case of cross metathesis) reaction temperature
(.degree. C.), reaction time (h), percent conversion (%).
Chromatographic purification conditions (isolated yield, %) are
also listed.
[0357] Hydrogenation and metathesis experiments performed on-resin
were subjected to the conditions described above. Resin-based
metathesis reactions were quenched with ethyl vinyl ether (0.5 mL,
5 min). The mixtures were then filtered, washed with DCM (3 mL,
3.times.1 min), MeOH (3 mL, 3.times.1 min) and dried on the SPPS
manifold for 1 h. A small aliquot of resin-peptide (.about.1 mg)
was subjected to the TFA-mediated cleavage procedure (Section
7.33). The isolated peptide was analysed by mass spectrometry.
Experimental for Section 4
7.6 Synthesis of 5,5-Dimethylproline Precursors
7.6.1 N-Acetyl-2-hydroxyglycine 42
##STR00108##
[0359] The titled compound 42 was prepared according to a procedure
described by Williams et al..sup.195 A solution of acetamide 34
(6.10 g, 0.10 mol) and glyoxylic acid monohydrate 41 (10.60 g, 0.14
mol) in anhydrous acetone (150 mL) was heated at reflux for 18 h.
The reaction mixture was evaporated under reduced pressure to
afford the desired product 42 as a viscous yellow oil (13.75 g,
100%). Spectroscopic data indicated the crude product 42 did not
require purification and was used directly in the subsequent
reaction (Section 7.9.2). .nu..sub.max (neat): 3317bs, 2974w,
1732s, 1668s, 1538s, 1379m, 1234w, 1112w, 1048m, 880m cm.sup.-1.
.sup.1H n.m.r. (300 MHz, D.sub.6-DMSO): .delta. 1.84 (s, 3H,
CH.sub.3CO), 5.39 (d, J=8.7 Hz, 1H, H2), (8.65, bd, J=8.4 Hz, 1H,
NH), two exchangeable protons (OH) not observed. .sup.13C n.m.r.
(75 MHz, D.sub.6-DMSO): .delta. 22.5 (CH.sub.3CO), 70.9 (C2), 169.4
(CONH), 171.5 (C1). Mass Spectrum (ESI.sup.+, MeOH): m/z 134.2
(M+H).sup.+, C.sub.4H.sub.8NO.sub.4 requires 134.1. Spectroscopic
data were in agreement with those reported in the
literature..sup.195
7.6.2 Methyl N-Acetyl-2-methoxyglycinate 43
##STR00109##
[0361] The methyl ester 43 was prepared according to a procedure
described by Legall et al..sup.196 Concentrated H.sub.2SO.sub.4
(4.5 nit) was added to an ice-cooled solution of
N-acetyl-2-hydroxyglycine 42 (13.69 g, 0.10 mol) in MeOH (150 mL).
The solution was stirred for 2 d at room temperature then poured
into an ice-cooled saturated NaHCO.sub.3 solution (400 mL). The
mixture was extracted with EtOAc (3.times.250 mL) and the combined
organic extract was dried (MgSO.sub.4) and evaporated under reduced
pressure to yield the titled compound 43 as a yellow oil (9.57 g,
60%). Spectroscopic data indicated the crude product 43 did not
require purification and was used directly in the subsequent
reaction (Section 7.9.3). .nu..sub.max (neat): 3334bm, 2955w,
1753s, 1671s, 1528m, 1439m, 1375m, 1221m, 1088m, 783w cm.sup.-1.
.sup.1H n.m.r. (300 MHz, CDCl.sub.3): .delta. 2.10 (s, 3H,
CH.sub.3CO), 3.47 (s, 3H, OCH.sub.3), 3.82 (s, 3H, COOCH.sub.3),
5.55 (d, J=9.3 Hz, 1H, H2), 6.72 (bd, J=8.1 Hz, 1H, NH). .sup.13C
n.m.r. (75 MHz, CDCl.sub.3): .delta. 23.3 (CH.sub.3CO), 53.0
(OCH.sub.3), 56.8 (COOCH.sub.3), 78.3 (C2), 168.7 (CONH), 170.8
(C1). Mass Spectrum (ESI.sup.+, MeOH): m/z 184.1 (M+Na).sup.+,
C.sub.6H.sub.11NNaO.sub.4 requires 184.2. Spectroscopic data were
in agreement with those reported in the literature..sup.196
7.6.3 Methyl 2-N-Acetylamino-2-(dimethoxyphosphinyl)acetate 39
##STR00110##
[0363] The phosphinyl compound 39 was prepared according to a
procedure described by Schmidt et al..sup.197 Phosphorus (III)
chloride (3.91 mL, 44.6 mmol) was added to a solution of methyl
N-acetyl-2-methoxyglycinate 43 (7.19 g, 44.6 mmol) in toluene (100
mL) at 70.degree. C. and the mixture was stirred at this
temperature for 17 h. Trimethyl phosphite (5.27 mL, 44.7 mmol) was
then added dropwise and the reaction mixture was left to stir for 2
h at 70.degree. C. The mixture was evaporated under reduced
pressure and the resultant oil was re-dissolved in DCM (100 mL) and
washed with saturated NaHCO.sub.3 solution (3.times.100 mL). The
organic extract was dried (MgSO.sub.4) and evaporated under reduced
pressure to afford the product 39 as a colourless solid (1.46 g,
14%). The combined aqueous layers were extracted in a continuous
extractor with DCM (150 mL) for 3 d. The organic layer was then
evaporated under reduced pressure to give the product 39 as a
colourless solid (3.21 g, 30%) (44% combined yield), m.p.
89-91.degree. C. (lit..sup.197 88-89.degree. C.). Spectroscopic
data indicated the crude product 39 did not require purification
and was used directly in the subsequent reaction (Section 7.94).
.nu..sub.max (KBr): 3281m, 3050w, 2852w, 1749m, 1673m, 1540m,
1309m, 1287w, 1232w, 1133m, 1061m, 1028m cm.sup.-1. .sup.1H n.m.r.
(300 MHz, CDCl.sub.3): .delta. 2.08 (s, 3H, CH.sub.3CO), 3.80-3.85
(m, 9H, COOCH.sub.3, 2.times.P--OCH.sub.3), 5.23 (dd, J=22.2, 8.9
Hz, 1H, H2), 6.42 (d, J=8.8 Hz, 1H, NH). .sup.13C n.m.r. (75 MHz,
CDCl.sub.3): .delta. 22.7 (CH.sub.3CO), 50.0 (d, J=146.8 Hz, C2),
53.3 (COOCH.sub.3), 54.1 (d, J=6.8 Hz, P--OCH.sub.3), 54.2 (d,
J=6.5 Hz, P--OCH.sub.3), 167.0 (d, J=2.2 Hz, CONH), 169.0 (d, J=6.0
Hz, C1). Mass Spectrum (ESI.sup.+, MeOH): m/z 262.1 (M+Na).sup.+,
C.sub.6H.sub.11NNaO.sub.4 requires 262.2.
7.7 Synthesis of Olefinic Substrates
7.7.1 (2Z)-Methyl 2-N-Acetylaminopenta-2,4-dienoate 57
##STR00111##
[0365] The dienamide 57 was prepared according to a procedure
described by Teoh et al..sup.119 Tetramethylguanidine (3.22 mL,
25.7 mmol) and hydroquinone (10.0 mg) were added to a solution of
methyl 2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 39 (4.63 g,
19.4 mmol) in THF (60 mL) at -78.degree. C. After 15 min, acrolein
58 (1.55 mL, 23.2 mmol) was added and the mixture was stirred at
-78.degree. C. for 2 h and then warmed to 25.degree. C. and allowed
to react an additional 2 h. The reaction mixture was diluted with
DCM (100 mL) and washed with dilute HCl solution (1 M, 2.times.80
mL), CuSO.sub.4 solution (1 M, 2.times.80 mL), saturated
NaHCO.sub.3 solution (2.times.80 mL) and saturated NaCl solution
(1.times.80 mL). The organic layer was dried (MgSO.sub.4) and
evaporated under reduced pressure to give the desired dienamide 57
as an off-white solid (2.78 g, 85%), m.p. 60-62.degree. C.
(lit..sup.119 61-63.degree. C.). Spectroscopic data indicated the
crude product 57 did not require purification and was used directly
in the subsequent reaction (Section 7.11.2). .nu..sub.max (KBr):
3277m, 3011m, 2955m, 1733s, 1655s, 1594m, 1518s, 1438m, 1377w,
1350w, 1250w, 1113s, 1016m, 994m, 950s, 768m cm.sup.-1. .sup.1H
n.m.r. (300 MHz, CDCl.sub.3): .delta. 2.16 (s, 3H, CH.sub.3CO),
3.81 (s, 3H, OCH.sub.3), 5.49 (d, J=9.9 Hz, 1H, H5-E), 5.61 (d,
J=17.1 Hz, 1H, H5-Z), 6.47 (m, 1H, H4), 7.05 (d, J=11.1 Hz, 1H,
H3), 7.07 (bs, 1H, NH). .sup.13C n.m.r. (75 MHz, CDCl.sub.3):
.delta. 23.6 (CH.sub.3CO), 52.7 (OCH.sub.3), 123.7 (C2), 125.2
(C5), 132.0 (C4), 132.9 (C3), 165.5, 168.9 (C1, CONH). Mass
Spectrum (ESI.sup.+, MeOH): m/z 170.2 (M+1V,
C.sub.8H.sub.12NO.sub.3 requires 170.2. Spectroscopic data were in
agreement with those reported in the literature..sup.119
7.7.2 (2S)-Methyl 2-N-Acetylaminopent-4-enoate 21a
##STR00112##
[0367] The dienamide 57 was subjected to the general asymmetric
hydrogenation procedure (Section 7.4.3) under the following
conditions: (2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 57 (108
mg, 0.64 mmol), benzene (7 mL), Rh(I)--(S,S)-Et-DuPHOS, 30 psi,
22.degree. C., 3 h. At the end of the reaction period, the solvent
was evaporated under reduced pressure and the residue was purified
by flash chromatography (SiO.sub.2, EtOAc) to give a yellow oil
(106 mg, 97%). .sup.1H n.m.r. spectroscopy confirmed formation of
the desired product 21a and the fully saturated compound,
(2S)-methyl 2-N-acetylaminopentanoate 59 (.delta. 0.93 (t, J=7.3
Hz, 3H, H5), 1.25-1.44 (m, 4H, H3, 4)), in a 97:3 ratio
respectively. GC: (2S)-21a t.sub.R=18.6 min (GC chiral column 50
CP2/XE60-SVALSAPEA, 100.degree. C. for 1 min, 5.degree. C.
min.sup.-1 to 280.degree. C. for 9 min), 95% e.e.
[.alpha.].sub.D.sup.22+45.0.degree. (c=0.76, CHCl.sub.3) containing
3% of 59 (lit..sup.208 for (S)-21a
[.alpha.].sub.D.sup.22+45.4.degree. (c=3.57, CHCl.sub.3)).
.nu..sub.max (neat): 3278s, 3079w, 2955w, 1744s, 1657s, 1546m,
1438m, 1375m, 1275w, 1226w, 1151m, 997w, 924w cm.sup.-1. .sup.1H
n.m.r. (300 MHz, CDCl.sub.3): .delta. 2.00 (s, 3H, CH.sub.3CO),
2.43-2.62 (m, 2H, H3), 3.73 (s, 3H, OCH.sub.3), 4.67 (dt, J=11.6,
5.8 Hz, 1H, H2), 5.08 (m, 1H, H5-E), 5.14 (m, 1H, H5-Z), 5.67 (m,
1H, H4), 6.06 (bs, 1H, NH). .sup.13C n.m.r. (100 MHz, CDCl.sub.3):
.delta. 23.1 (CH.sub.3CO), 36.5 (C3), 51.8 (C2), 52.4 (OCH.sub.3),
119.2 (C5), 132.3 (C4), 169.9, 172.4 (C1, CONH). Mass Spectrum
(EST.sup.+, MeOH): m/z 194.1 (M+Na).sup.+,
C.sub.8H.sub.13NNaO.sub.3 requires 194.2. Spectroscopic data were
in agreement with those reported in the literature..sup.119
(2R)-Methyl 2-N-acetylaminopent-4-enoate 21a
##STR00113##
[0369] The dienamide 57 was subjected to the general asymmetric
hydrogenation procedure (Section 7.4.3) under the following
conditions: (2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 57 (40.0
mg, 0.24 mmol), benzene (5 mL), Rh(I)--(R,R)-Et-DuPHOS, 30 psi,
22.degree. C., 3 h. At the end of the reaction period, the solvent
was evaporated under reduced pressure and the residue was purified
by flash chromatography (SiO.sub.2, EtOAc) to give as a yellow oil
(36.0 mg, 88%). .sup.1H n.m.r. spectroscopy confirmed formation of
the desired product 21a and the fully saturated compound,
(2R)-methyl 2-N-acetylaminopentanoate 59 in a 95:5 ratio
respectively GC: (2R)-21a t.sub.R=18.2 min (GC chiral column 50
CP2/XE60-SVALSAPEA, 100.degree. C. for 1 min, 5.degree. C.
min.sup.-1 to 280.degree. C. for 9 min), 95% e.e.
[.alpha.].sub.D.sup.22-43.0.degree. (c=0.47, CHCl.sub.3) containing
5% of 59. Spectroscopic data were in agreement with those
previously reported for the (S)-enantiomer.
7.7.3 N-Benzoyl-2-hydroxyglycine 65
##STR00114##
[0371] The titled compound 65 was prepared according to a procedure
described by Zoller et al..sup.209 A mixture of benzamide 35 (5.00
g, 41.3 mmol) and glyoxylic acid monohydrate 41 (4.32 g, 46.9 mmol)
in anhydrous acetone (70 mL) was heated at reflux for 19 h. The
reaction mixture was evaporated under reduced pressure to afford
the desired product 65 as a colourless solid (8.06 g, 100%), m.p.
198-200.degree. C. (lit..sup.209 200-201.degree. C. (dec))
Spectroscopic data indicated the crude product 65 did not require
purification and was used directly in the subsequent reaction
(Section 7.11.4). .nu..sub.max (KBr): 3310bs, 3058w, 1728s, 1646s,
1602w, 1582w, 1535s, 1491w, 1452w, 1315m, 1292w, 1254m, 1161m,
1097s, 1040m, 1002w, 957m, 805w, 770w, 728m, 692m, 654m, 609w,
515m, 483w cm.sup.-1. .sup.1H n.m.r. (300 MHz, D.sub.6-DMSO):
.delta. 5.60 (d, J=7.8 Hz, 1H, H2), 7.41-7.49 (m, 2H, H3', 5), 7.55
(m, 1H, H4'), 7.86-7.92 (m, 2H, H2', 6'), 9.26 (d, J=7.8 Hz, 1H,
NH), two exchangeable OH protons not observed. .sup.13C n.m.r. (75
MHz, D.sub.6-DMSO): .delta. 71.7 (C2), 127.6, 128.3, 131.7 (Arom
CH), 133.7 (C1'), 166.0, 171.5 (C1, CONH). Mass Spectrum
(ESI.sup.+, MeOH): m/z 218.2 (M+Na).sup.+, C.sub.9H.sub.9NNaO.sub.4
requires 218.2.
7.7.4 Methyl N-Benzoyl-2-methoxyglycinate 66
##STR00115##
[0373] The methyl ester 66 was prepared according to a procedure
described by Zoller et al..sup.209 Concentrated H.sub.2SO.sub.4
(2.0 mL) was added to an ice-cooled solution of
N-benzoyl-2-hydroxyglycine 65 (8.05 g, 41.3 mmol) in MeOH (65 mL).
The solution was stirred for 48 h at ambient temperature then
poured into an ice-cooled saturated NaHCO.sub.3 solution (100 mL).
The mixture was extracted with EtOAc (3.times.100 mL) and the
combined organic extract was dried (MgSO.sub.4) and evaporated
under reduced pressure to yield the titled compound 66 as a yellow
oil (8.00 g, 87%). Spectroscopic data indicated the crude product
66 did not require purification and was used directly in the
subsequent reaction (Section 7.11.5). .nu..sub.max (neat): 3310bm,
2955m, 2837w, 1760s, 1651s, 1603w, 1580w, 1525s, 1488m, 1439m,
1338w, 1286w, 1226w, 1198w, 1147w, 1108m, 1022w, 924m, 850w, 803m,
778m, 692m cm.sup.-1. .sup.1H n.m.r. (300 MHz, CDCl.sub.3): .delta.
3.54 (s, 3H, OCH.sub.3), 3.85 (s, 3H, COOCH.sub.3), 5.78 (d, J=9.0
Hz, 1H, H2), 7.22 (bd, J=9.0 Hz, 1H, NH), 7.42-7.51 (m, 2H, H3',
5), 7.56 (m, 1H, H4'), 7.80-7.88 (m, 2H, H2', 6'). .sup.13C n.m.r.
(75 MHz, CDCl.sub.3): .delta. 53.2 (OCH.sub.3), 57.0 (COOCH.sub.3),
78.8 (C2), 127.4, 128.9, 132.5 (Arom CH), 133.2 (C1'), 167.6, 168.7
(C1, CONH). Mass Spectrum (ESI.sup.+, MeOH): m/z 224.2 (M+H).sup.+,
C.sub.11H.sub.14NO.sub.4 requires 224.2; m/z 246.3 (M+Na).sup.+,
C.sub.11H.sub.13NNaO.sub.4 requires 246.2. Spectroscopic data were
in agreement with those reported in the literature..sup.209
7.7.5 Methyl 2-N-Benzoylamino-2-(dimethoxyphosphinyl)acetate 64
##STR00116##
[0375] The phosphinyl compound 64 was prepared according to a
procedure described by Teoh et al..sup.119 Phosphorus (III)
chloride (3.15 mL, 36.0 mmol) was added to a solution of methyl
N-benzoyl-2-methoxyglycinate 66 (8.00 g, 35.9 mmol) in toluene (70
mL) at 70.degree. C. and the mixture was stirred at this
temperature for 14 h. Trimethyl phosphite (4.25 mL, 36.0 mmol) was
added dropwise and the reaction mixture was left to stir for 2 h at
70.degree. C. At the end of the reaction period, the mixture was
evaporated under reduced pressure and the resultant oil was
re-dissolved in DCM (100 mL) and washed with saturated NaHCO.sub.3
solution (3.times.70 mL). The organic extract was isolated, dried
(MgSO.sub.4) and evaporated under reduced pressure to afford the
titled compound 64 as a colourless solid (8.21 g, 76%), m.p.
110-112.degree. C. (lit..sup.210 112-114.degree. C.). Spectroscopic
data indicated the crude product 64 did not require purification
and was used directly in the subsequent reaction (Section 7.11.6).
.nu..sub.max (KBr): 3300m, 3248m, 3059w, 2958m, 2852w, 1737s,
1671s, 1638m, 1618w, 1602w, 1581w, 1541s, 1492m, 1432w, 1292s,
1235s, 1188w, 1152w, 1044s, 915m, 881w, 832m, 812w, 791w, 780w,
758m, 708m, 616w, 562m cm.sup.-1. .sup.1H n.m.r. (300 MHz,
CDCl.sub.3): .delta. 3.82-3.90 (m, 9H, COOCH.sub.3,
2.times.P--OCH.sub.3), 5.47 (dd, J=21.9, 9.0 Hz, 1H, H2), 6.97 (bd,
J=7.8 Hz, 1H, NH), 7.43-7.49 (m, 2H, H3', 5'), 7.56 (m, 1H, H4'),
7.82-7.87 (m, 2H, H2', 6'). .sup.13C n.m.r. (75 MHz, CDCl.sub.3):
.delta. 50.6 (d, J=147.1 Hz, C2), 53.5 (COOCH.sub.3), 54.2 (d,
J=6.8 Hz, P--OCH.sub.3), 127.4, 128.8, 132.3 (Arom CH), 133.1
(C1'), 166.9 (d, J=5.4 Hz, C1), 167.3 (d, J=2.0 Hz, CONH). Mass
Spectrum (ESI.sup.+, MeOH): m/z 302.2 (M+H).sup.+,
C.sub.12H.sub.17NO.sub.6P requires 302.2; m/z 324.2 (M+Na).sup.+,
C.sub.12H.sub.16NNaO.sub.6P requires 324.2.
7.7.6 (2Z)-Methyl 2-N-Benzoylaminopenta-2,4-dienoate 63
##STR00117##
[0377] The dienamide 63 was prepared according to a procedure
described by Teoh et al..sup.119 Tetramethylguanidine (4.35 mL,
34.7 mmol) and hydroquinone (12.0 mg) were added to a solution of
methyl 2-N-benzoylamino-2-(dimethoxyphosphinyl)acetate 64 (7.79 g,
25.9 mmol) in THF (120 mL) at -78.degree. C. After 30 min, acrolein
58 (2.10 mL, 31.4 mmol) was added and the mixture was stirred at
-78.degree. C. for 2 h and then warmed to 25.degree. C. and allowed
to react an additional 2 h. The reaction mixture was diluted with
DCM (150 mL) and washed with dilute HCl solution (1 M, 2.times.100
mL), CuSO.sub.4 solution (1 M, 2.times.100 mL), saturated
NaHCO.sub.3 solution (2.times.100 mL) and saturated NaCl solution
(1.times.100 mL). The organic extract was dried (MgSO.sub.4) and
evaporated under reduced pressure to give the crude product 63 as a
waxy-brown solid. Purification by flash chromatography (SiO.sub.2,
light petroleum:EtOAc:DCM, 3:2:2) furnished the pure enamide 63 as
colourless needles (4.78 g, 80%), m.p. 138-141.degree. C. (dec).
(KBr): 3288bm, 2952m, 2361w, 1727s, 1652s, 1602w, 1580w, 1514s,
1484s, 1436w, 1257s, 1196w, 1074w, 1028w, 991w, 931w, 800w, 737m,
710m cm.sup.-1. .sup.1H n.m.r. (300 MHz, CDCl.sub.3): .delta. 3.83
(s, 3H, OCH.sub.3), 5.50 (dd, J=10.0, 1.7 Hz, 1H, H5-K), 5.64 (dd,
J=16.8, 1.7 Hz, 1H, H5-Z), 6.56 (ddd, J=17.1, 11.4, 10.2 Hz, 1H,
H4), 7.14 (d, J=11.2 Hz, 1H, H3), 7.45-7.51 (m, 2H, H3', 5'), 7.56
(m, 1H, H4'), 7.78 (bs, 1H, NH), 7.88-7.91 (m, 2H, H2', 6').
.sup.13C n.m.r. (75 MHz, CDCl.sub.3): .delta. 52.8 (OCH.sub.3),
123.6 (C2), 125.2 (C5), 127.6 (CT, 6'), 128.9 (C3', 5'), 132.2,
132.2, 132.3 (C3, 4, 4'), 133.9 (C1'), 165.6, 165.8 (C1, CONH).
Mass Spectrum (ESI.sup.+, MeOH): m/z 232.1 (M+H).sup.+,
C.sub.13H.sub.14NO.sub.3 requires 232.3; m/z 254.2 (M+Na).sup.+,
C.sub.13H.sub.13NNaO.sub.3 requires 254.2. Spectroscopic data were
in agreement with those reported in the literature..sup.211
7.7.7 (2S)-Methyl 2-N-Benzoylaminopent-4-enoate 62
##STR00118##
[0379] The dienamide 63 was subjected to the general asymmetric
hydrogenation procedure (Section 7.4.3) under the following
conditions: (2Z)-Methyl 2-N-benzoylaminopenta-2,4-dienoate 63 (100
mg, 0.43 mmol), benzene (8 mL), Rh(I)--(S,5)-Et-DuPHOS, 30 psi,
22.degree. C., 3 h. At the end of the reaction period, the solvent
was evaporated under reduced pressure and the residue was purified
by flash chromatography (SiO.sub.2, EtOAc) to give a pale yellow
oil (100 mg, 99%). .sup.1H n.m.r. spectroscopy confirmed formation
of the desired product 62 and the fully saturated compound,
(2S)-methyl 2-N-benzoylaminopentanoate 68 (.delta. 0.95 (t, J=7.3
Hz, 3H, H5), 1.36-1.50 (m, 2H, H4), 1.90-1.96 (m, 2H, H3)), in a
93:7 ratio respectively. GC: (2S)-62 t.sub.R=27.0 min (GC chiral
column 50 CP2/XE60-SVALSAPEA, 180.degree. C. for 1 min, 2.degree.
C. min.sup.-1 to 210.degree. C. for 20 min), 100% e.e.
[.alpha.].sub.D.sup.22+49.3.degree. (c=1.12, CHCl.sub.3) containing
7% of 68. .nu..sub.max (neat): 3325bw, 3062w, 2955w, 2360w, 1743s,
1644s, 1603w, 1580w, 1538m, 1489m, 1438w, 1360w, 1268w, 1225w,
1159w, 1075w, 1028w, 925m, 802w, 714w, 668w cm.sup.-1. .sup.1H
n.m.r. (400 MHz, CDCl.sub.3): .delta. 2.63-2.73 (m, 2H, H3), 3.79
(s, 3H, OCH.sub.3), 4.91 (m, 1H, H2), 5.15 (m, 1H, H5-E), 5.18 (m,
1H, H5-Z), 5.75 (m, 1H, H4), 6.67 (bd, J=7.0 Hz, 1H, NH), 7.42-7.46
(m, 2H, H3', 5'), 7.52 (m, 1H, H4'), 7.78-7.81 (m, 2H, H2', 6').
.sup.13C n.m.r. (100 MHz, CDCl.sub.3): .delta. 36.8 (C3), 52.1
(C2), 52.6 (OCH.sub.3), 119.5 (C5), 127.2 (C2', 6'), 128.8 (C3',
5'), 131.9 (C4), 132.4 (C4'), 134.1 (C1'), 167.0, 172.4 (C1, CONH).
Mass Spectrum (ESI.sup.+, MeOH): m/z 234.3 (M+H).sup.+,
C.sub.13H.sub.16NO.sub.3 requires 234.3; m/z 256.2 (M+Na).sup.+,
C.sub.13H.sub.15NNaO.sub.3 requires 256.3. Spectroscopic data were
in agreement with those reported in the literature..sup.212
(2R)-Methyl 2-N-benzoylaminopent-4-enoate 62
##STR00119##
[0381] The dienamide 63 was subjected to the general asymmetric
hydrogenation procedure (Section 7.4.3) under the following
conditions: (2Z)-Methyl 2-N-benzoylaminopenta-2,4-dienoate 63 (100
mg, 0.43 mmol), benzene (8 mL), Rh(I)--(R,R)-Et-DuPHOS, 30 psi,
22.degree. C., 3 h. At the end of the reaction period, the solvent
was evaporated under reduced pressure and the residue was purified
by flash chromatography (SiO.sub.2, EtOAc) to give a yellow oil
(93.8 mg, 93%). .sup.1H n.m.r. spectroscopy confirmed formation of
the desired product 62 and the fully saturated compound,
(2R)-methyl 2-N-benzoylaminopentanoate 68, in a 91:9 ratio
respectively. GC: (2R)-62 t.sub.R=26.4 min (GC chiral column 50
CP2/XE60-SVALSAPEA, 180.degree. C. for 1 min, 2.degree. C.
min.sup.-1 to 210.degree. C. for 20 min), 100% e.e.
[.alpha.].sub.D.sup.22-49.7.degree. (c=0.64, CHCl.sub.3) containing
9% of 68. Spectroscopic data were in agreement with those
previously reported for the (S)-enantiomer.
7.7.8 (2Z)-Methyl 2-N-Acetylamino-5-phenylpenta-2,4-dienoate 76
##STR00120##
[0383] The dienamide 76 was prepared according to a procedure
described by Burk et al..sup.117 Tetramethylguanidine (0.70 mL,
5.58 mmol) was added to a solution of methyl
2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 64 (1.00 g, 4.18
mmol) in THF (50 mL) at -78.degree. C. After 15 min,
trans-cinnamaldehyde 78 (0.63 mL, 5.00 mmol) was added and the
mixture was stirred at -78.degree. C. for 2 h, warmed to 25.degree.
C. and allowed to react an additional 2 h. The reaction mixture was
diluted with DCM (100 mL) and washed with dilute HCl solution (1 M,
2.times.75 mL), CuSO.sub.4 solution (1 M, 2.times.75 mL), saturated
NaHCO.sub.3 solution (2.times.75 mL) and saturated NaCl solution
(1.times.75 mL). The organic layer was dried (MgSO.sub.4) and
evaporated under reduced pressure to give the crude product 76 as a
waxy solid (0.87 g). Purification by recrystallisation from a
mixture of light petroleum, EtOAc and DCM furnished the pure
dienamide 76 as an off-white solid (0.76 g, 74%), m.p.
180-181.degree. C. (lit..sup.117 179-180.degree. C.). .nu..sub.max
(KBr): 3263w, 1721s, 1662s, 1517s, 1439m, 1368m, 1286m, 1229s,
1192w, 1116m, 993m, 769w, 752m, 728w, 692m, 600w cm.sup.-1. .sup.1H
n.m.r. (300 MHz, CDCl.sub.3): .delta. 2.20 (s, 3H, CH.sub.3CO),
3.82 (s, 3H, OCH.sub.3), 6.89-6.91 (m, 2H, H3, 4), 7.01 (m, 1H,
H5), 7.22 (bd, J obscured by residual CHCl.sub.3 peak, 1H, NH),
7.29-7.37 (m, 3H, H3', 4', 5'), 7.45-7.48 (m, 2H, H2', 6').
.sup.13C n.m.r. (100 MHz, CDCl.sub.3): .delta. 23.9, (CH.sub.3CO),
52.7 (OCH.sub.3), 123.0 (C2), 124.0, 127.5, 128.9, 129.2, 132.8,
140.2 (Arom CH, C3, 4, 5), 136.5 (C1'), 165.6, 168.7 (C1, CONH).
Mass Spectrum (ESI.sup.+, MeOH): m/z 246.2 (M+H).sup.+,
C.sub.14H.sub.16NO.sub.3 requires 246.3. Spectroscopic data were in
agreement with those reported in the literature..sup.117
7.7.9 (2S)-Methyl 2-N-Acetylamino-5-phenylpent-4-enoate 77
##STR00121##
[0385] The dienamide 76 was subjected to the general asymmetric
hydrogenation procedure (Section 7.4.3) under the following
conditions: (2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 76 (28.0
mg, 0.11 mmol), MeOH (7 mL), Rh(I)--(S,5)-Et-DuPHOS, 90 psi,
22.degree. C., 2 h. At the end of the reaction period, the solvent
was evaporated under reduced pressure and the residue was purified
by flash chromatography (SiO.sub.2, EtOAc) to give a yellow oil
(27.2 mg, 96%). .sup.1H n.m.r. spectroscopy confirmed formation of
the desired product 77 and the fully saturated compound,
(2S)-methyl 2-N-acetylamino-5-phenylpentanoate 79 (.delta.
1.60-1.87 (m, 4H, H3, 4), 2.48-2.53 (m, 2H, H5), 3.72 (s, 3H,
OCH.sub.3), 4.65 (m, 1H, H2)), in a 91:9 ratio respectively.
[.alpha.].sub.D.sup.22+90.0.degree. (c=0.64, CHCl.sub.3) containing
9% of 79. .nu..sub.max (neat): 3280bw, 3070m, 2960w, 2350w, 1745s,
1648s, 1605w, 1575w, 1550m, 1478m, 1440w, 1369w, 1270w, 1225w,
1153w, 1075w, 1028w, 925m, 805w, 720w cm.sup.-1. .sup.1H n.m.r.
(400 MHz, CDCl.sub.3): .delta. 2.02 (s, 3H, CH.sub.3CO), 2.64-2.78
(m, 2H, H3), 3.76 (s, 3H, OCH.sub.3), 4.77 (m, 1H, H2), 6.00-6.09
(m, 214, H4, NH), 6.45 (d, J=15.8 Hz, 1H, H5), 7.20-7.34 (m, 5H,
Arom CH). .sup.13C n.m.r. (100 MHz, CDCl.sub.3): .delta. 23.3
(CH.sub.3CO), 36.0 (C3), 52.1, 52.6 (C2, OCH.sub.3), 123.6, 126.4,
127.8, 128.7, 134.3 (Arom CH, C4, 5), 136.9 (C1'), 171.5, 172.5
(C1, CONH). Mass Spectrum (ESI.sup.+, MeOH): m/z 248.1 (M+H).sup.+,
C.sub.14H.sub.18NO.sub.3 requires 248.2. Spectroscopic data were in
agreement with those reported in the literature..sup.117
(2R)-Methyl 2-N-Acetylamino-5-phenylpent-4-enoate 77
##STR00122##
[0387] The dienamide 76 was subjected to the general asymmetric
hydrogenation procedure (Section 7.4.3) under the following
conditions: (2Z)-Methyl 2-N-acetylamino-5-phenylpenta-2,4-dienoate
76 (27.4 mg, 0.11 mmol), MeOH (5 mL), Rh(I)--(R,R)-Et-DuPHOS, 90
psi, 22.degree. C., 2 h. At the end of the reaction period, the
solvent was evaporated under reduced pressure and the residue was
purified by flash chromatography (SiO.sub.2, EtOAc) to give a
yellow oil (25.7 mg, 93%). .sup.1H n.m.r. spectroscopy confirmed
formation of the desired product 77 and the fully saturated
compound, (2R)-methyl 2-N-acetylamino-5-phenylpentanoate 79 in a
87:13 ratio respectively. [.alpha.].sub.D.sup.22-89.8.degree.
(c=1.03, CHCl.sub.3) containing 13% of 79. Spectroscopic data were
in agreement with those previously reported for the
(S)-enantiomer.
7.7.10 (2Z)-Methyl 2-N-Acetylamino-5-methylhexa-2,4-dienoate 20
##STR00123##
[0389] The preparation of (2Z)-methyl
2-N-acetylamino-5-methylhexa-2,4-dienoate 20 from the phosophonate
39 has been previously described (Section 7.9.4).
7.7.11 (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate 19
##STR00124##
[0391] The preparation of (2S)-methyl
2-N-acetylamino-5-methylhex-4-enoate 19 via asymmetric
hydrogenation of dienoate 20 has been previously described (Section
7.9.5).
Metathesis Reactions with Olefinic Substrates
7.8.1 (2S,7S)-Dimethyl 2,7-N,N'-Diacetylaminooet-4-enedioate 60
##STR00125##
[0393] The dimer 60 was prepared via the conventional cross
metathesis procedure (Section 7.5.2) under the following
conditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a (95.0 mg,
0.56 mmol), DCM (4 mL), Grubbs' catalyst (91.0 mg, 0.11 mmol, 20
mol %), 50.degree. C., 20 h, 100% conversion into 60. Purification
by flash chromatography (SiO.sub.2, DCM:light petroleum:EtOAc,
1:1:1.fwdarw.10% MeOH:DCM) furnished pure dimer 60 as a brown oil
(76.7 mg, 88%). GC: t.sub.R (E/Z)=12.7 min, 12.8 min (GC column
30QC5/BPX5, 150.degree. C. for 1 min, 10.degree. C. min.sup.-1 to
280.degree. C. for 6 min). [.alpha.].sub.D.sup.22+92.0.degree.
(c=0.004, CHCl.sub.3). .nu..sub.max (neat): 3286bm, 2956m, 2931m,
2856w, 1742s, 1659s, 1542m, 1438m, 1375m, 1267m, 1220m, 1138w,
1017w cm.sup.-1. .sup.1H n.m.r. (300 MHz, CDCl.sub.3): .delta. 2.04
(s, 6H, CH.sub.3CO), 2.40-2.50 (m, 4H, H3, 6), 3.74 (s, 6H,
OCH.sub.3), 4.64-4.70 (m, 2H, H2, 7), 5.36-5.40 (m, 2H, H4, 5),
6.34 (bd, J=7.2 Hz, 2H, NH). .sup.13C n.m.r. (100 MHz, CDCl.sub.3):
.delta. 23.1 (CH.sub.3CO), 35.1 (C3, 6), 51.7 (C2, 7), 52.6
(OCH.sub.3), 128.8 (C4, 5), 170.3, 172.6 (C1, 8, CONH). HRMS
(ESI.sup.+, MeOH). Found: m/z 337.1375 (M+Na).sup.+,
C.sub.14H.sub.22N.sub.2NaO.sub.6 requires 337.1376. Spectroscopic
data were in agreement with those reported in the
literature..sup.264
7.8.2 (2S,7S)-Dimethyl 2,7-N,N'-Dibenzoylaminooct-4-enedioate
69
##STR00126##
[0394] Method 1:
[0395] The dimer 69 was prepared via the conventional cross
metathesis procedure (Section 7.5.2) under the following
conditions: (2S)-Methyl 2-N-benzoylaminopent-4-enoate 62 (49.0 mg,
0.21 mmol), DCM (5 mL), Grubbs' catalyst (34.6 mg, 42.1 .mu.mol, 20
mol %), 50.degree. C., 18 h, 100% conversion into 69. Purification
by flash chromatography (SiO.sub.2, DCM:light petroleum:EtOAc,
1:1:1) gave pure dimer 69 as a pale brown solid (37.8 mg, 82%),
m.p. 140-142.degree. C. GC: t.sub.R (E/Z)=13.5, 13.9 min (GC column
30QC5/BPX5, 150.degree. C. for 1 min, 10.degree. C. min.sup.-1 to
280.degree. C. for 6 min).
[.alpha.].sub.D.sup.22+56.4.degree.=0.27, CHCl.sub.3). .nu..sub.max
(KBr): 3322bm, 2953m, 2358w, 1742s, 1644s, 1603w, 1580w, 1538m,
1488m, 1436m, 1267w, 1218m, 1027w, 973w, 802w, 736m cm.sup.-1.
.sup.1H n.m.r. (300 MHz, CDCl.sub.3): .delta. 2.57-2.69 (m, 4H, H3,
6), 3.67 (s, 6H, OCH.sub.3), 4.85-4.98 (m, 2H, H2, 7), 5.49 (t,
J=4.1 Hz, 2H, H4, 5), 6.86 (bd, J=7.4 Hz, 2H, NH), 7.40-7.44 (m,
4H, H3', 5'), 7.48-7.52 (m, 2H, H4'), 7.81-7.83 (m, 4H, H2', 6').
.sup.13C n.m.r. (100 MHz, CDCl.sub.3): .delta. 35.2 (C3, 6), 52.5
(C2, 7), 52.6 (OCH.sub.3), 127.2 (C2', 6'), 128.7 (C3', 5'), 128.8
(C4, 5), 131.9 (C4'), 133.9 (C1'), 167.1, 172.4 (C1, 8, CONH). HRMS
(ESI.sup.+, MeOH). Found: m/z 461.1695 (M+Na).sup.+,
C.sub.24H.sub.26N.sub.2NaO.sub.6 requires 461.1689.
Method 2:
[0396] The dimer 69 was also prepared via the conventional cross
metathesis procedure (Section 7.5.2) under the following
conditions: (2S)-Methyl 2-N-benzoylaminopent-4-enoate 142, DCM (5
mL), Grubbs' catalyst (20 mol %), 50.degree. C., 20 h, 100%
conversion into 69.
7.8.3 Dimerisation of (2S)-Methyl 2-N-Benzoylaminopent-4-enoate 62
in the presence of (2S,7S)-Dimethyl
2,7-N,N'-Diacetylaminooct-4-enedioate 60
##STR00127##
[0398] The olefinic mixture 62 and 60 was subjected to the
conventional cross metathesis procedure (Section 7.5.2) under the
following conditions:
Method A:
[0399] (2S)-Methyl 2-N-benzoylaminopent-4-enoate 62 (37.0 mg, 0.16
mmol), (2S,7S)-dimethyl 2,7-N,N'-diacetylaminooct-4-enedioate 60
(29.5 mg, 93.9 .mu.mol), DCM (3 mL), 2.sup.nd generation Grubbs'
catalyst (13.5 mg, 15.9 .mu.mol, 10 mol %), 50.degree. C., 15 h.
Spectroscopic data indicated the presence of the starting
acetyl-allylglycine dimer 60, the benzoyl-allylglycine dimer 69 and
additional peaks which mass spectrometry indicated could be
attributed to the "mixed" cross metathesis product,
(2S,7S)-dimethyl 2-N-acetylamino-7-N-benzoylaminooct-4-enedioate
70. .sup.1H n.m.r. spectroscopic data for the homodimers 60 and 69
were in agreement with those previously reported (Section 7.12.1
and Section 7.12.2). The heterodimer 70 was detected by mass
spectrometry. Mass spectrum (ESI.sup.+, MeOH): m/z 337.2
(M+Na).sup.+.sub.60, C.sub.14H.sub.22N.sub.2NaO.sub.6; m/z 399.3
(M+Na).sup.+.sub.70, C.sub.19H.sub.24N.sub.2NaO.sub.6; m/z 461.2
(M+Na).sup.+.sub.69, C.sub.24H.sub.26N.sub.2NaO.sub.6 requires
461.1689.
Method B:
[0400] (2S)-Methyl 2-N-benzoylaminopent-4-enoate 62 (37.0 mg, 0.16
mmol), (2S,7S)-dimethyl 2,7-N,N'-diacetylaminooct-4-enedioate 60
(30.0 mg, 95.5 .mu.mol), DCM (4 mL), Grubbs' catalyst (26.1 mg,
31.7 .mu.mol, 20 mol %), 50.degree. C., 18 h, 100% conversion of 62
into dimer 69. Dimer 60 was recovered unchanged. Spectroscopic data
for dimers 60 and 69 were in agreement with those previously
reported (Section 7.12.1 and Section 7.12.2). No "mixed" cross
metathesis product, heterodimer 70, was observed.
7.8.4 Attempted Dimerisation of (2Z)-Methyl
2-N-Acetylaminopenta-2,4-dienoate 57
##STR00128##
[0402] The dienamide 57 was subjected to the conventional cross
metathesis procedure (Section 7.5.2) under the following
conditions: (2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 57 (33.0
mg, 0.20 mmol), DCM (3 mL), Grubbs' catalyst (34.0 mg, 41.3
.mu.mol, 20 mol %), 50.degree. C., 15 h, 0% conversion into dimer
61. The dienamide 57 did not react under these conditions. .sup.1H
n.m.r. spectroscopic data for the recovered dienamide 57 were in
agreement with those previously reported (Section 7.11.1).
7.8.5 Attempted Dimerisation of (2S)-Methyl
2-N-Acetylaminopent-4-enoate 21a in the presence of (2Z)-Methyl
2-N-Acetylaminopenta-2,4-dienoate 57
##STR00129##
[0404] The mixture of olefins 21a and 57 was subjected to the
conventional cross metathesis procedure (Section 7.5.2) under the
following conditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a
(34.0 mg, 0.20 mmol), (2Z)-methyl 2-N-acetylaminopenta-2,4-dienoate
57 (33.6 mg, 0.20 mmol), DCM (4 mL), Grubbs' catalyst (16.3 mg,
19.8 .mu.mol, 10 mol %), 50.degree. C., 18 h. The .sup.1H n.m.r.
spectrum displayed peaks characteristic of the starting
allylglycine derivative 21a and dienamide 57 but no peaks
characteristic of expected dimer 60. The mass spectrum displayed
peaks attributed to the allylglycine derivative 21a and the
tricyclohexylphosphine-dienamide conjugate addition adduct,
(2S)-methyl 2-N-acetylamino-5-tricyclohexylphosphinylpent-2-enoate
143. Mass Spectrum (ESI.sup.+, DCM/MeOH): m/z 194.1
(M+Na).sup.+.sub.21a, C.sub.8H.sub.13NNaO.sub.3; m/z 450.4
(M.sup.+).sub.143, C.sub.26H.sub.45NO.sub.3P.sup.+.
##STR00130##
7.8.6 Attempted Dimerisation of (2S)-Methyl
2-N-Benzoylaminopent-4-enoate 62 in the presence of (2Z)-Methyl
2-N-Acetylaminopenta-2,4-dienoate 57
##STR00131##
[0406] The mixture of olefins 57 and 62 was subjected to the
conventional cross metathesis procedure (Section 7.5.2) under the
following conditions: (2S)-Methyl 2-N-benzoylaminopent-4-enoate 62
(46.0 mg, 0.20 mmol), (2Z)-methyl 2-N-acetylaminopenta-2,4-dienoate
57 (33.4 mg, 0.20 mmol), DCM (4 mL), 2.sup.nd generation Grubbs'
catalyst (16.8 mg, 19.8 .mu.mol, 10 mol %), 50.degree. C., 4.5 h.
The reaction mixture was evaporated under reduced pressure to
afford a dark brown oil (97.9 mg). The .sup.1H n.m.r. spectrum
displayed peaks characteristic of the starting allylglycine
derivative 62, dienamide 57, traces of the target allylglycine
dimer 69 and additional peaks which were difficult to characterise.
Mass spectrometry displayed peaks attributed to the allylglycine
derivative 62, dienamide 57, allylglycine dimer 69, dienamide dimer
(2S,7S)-dimethyl 2,7-N,N'-diacetylaminooct-2,4,6-trienedioate 61,
"mixed" dienamide-allylglycine dimer (2S,7S)-dimethyl
2-N-acetylamino-7-N-benzoyl-aminoocta-2,4-dienedioate 144 and the
tricyclohexylphosphine-dienamide conjugate addition adduct 143.
##STR00132##
[0407] Mass Spectrum (ESI.sup.+, DCM/MeOH): m/z 256.1
(M+Na).sup.+.sub.62, C.sub.13H.sub.15NNaO.sub.3; m/z 337.3
(M+Na).sup.+.sub.61, C.sub.14H.sub.18N.sub.2NaO.sub.6; m/z 397.3
(M+Na).sup.+.sub.144, C.sub.19H.sub.22N.sub.2NaO.sub.6; m/z 450.4
(M).sup.+.sub.143, C.sub.26H.sub.45NO.sub.3P.sup.+; m/z 461.3
(M+Na).sup.+.sub.69, C.sub.24H.sub.26N.sub.2NaO.sub.6 requires
461.5.
7.8.7 NMR Study of Grubbs' Catalyst with Dienamide 57
##STR00133##
[0409] In a dry box, a Teflon-sealed n.m.r. tube was charged with
(2S)-methyl 2-N-acetylaminopenta-2,4-dienoate 57 (10.8 mg, 63.9
.mu.mol), Grubbs' catalyst (50.7 mg, 61.6 .mu.mol) and degassed
deuterated DCM (CD.sub.2Cl.sub.2, 0.8 mL) at room temperature. The
n.m.r. tube was shaken gently and reaction progress was monitored
by .sup.1H and .sup.31P n.m.r. spectroscopy. Compounds were
identified by the following diagnostic resonances: .sup.1H n.m.r.
(300 MHz, CD.sub.2Cl.sub.2): After 15 min: Grubbs' catalyst:
.delta. 8.61 (d, J=7.6 Hz, 2H, ortho-Arom CH), 20.05 (s, 1H,
[Ru].dbd.CHPh); Ruthenium-dienamide complex 73: .delta. 7.96 (d,
J=11.0 Hz, 1H, [Ru].dbd.CH.dbd.CH), 20.11 (d, J=11.0 Hz, 1H,
[Ru].dbd.CH); Ruthenium-dienamide chelate 74 (trace): .delta. 15.20
(d, J=4.2 Hz, 1H, [Ru].dbd.CH); Ratio of ruthenium complexes
[Ru].dbd.CHPh:73:74=1.0:1.0:<0.1. After 60 min: Grubbs'
catalyst: .delta. 8.45 (d, J=7.6 Hz, 2H, ortho-Arom CH), 20.04 (s,
1H, [Ru].dbd.CHPh); Ruthenium-dienamide complex 73: .delta. 7.96
(d, J=11.0 Hz, 1H, [Ru].dbd.CH.dbd.CH), 20.10 (d, J=11.0 Hz, 1H,
[Ru].dbd.CH); Ruthenium-dienamide chelate 74: .delta. 6.73 (d,
J=3.0 Hz, 1H, [Ru].dbd.CH.dbd.CH), 15.19 (d, J=4.2 Hz, 1H,
[Ru].dbd.CH); Ratio of ruthenium complexes
[Ru].dbd.CHPh:73:74=3:1:1. After 120 min (no change after 18 h):
Ruthenium-dienamide chelate 74: .delta. 6.71 (d, J=3.0 Hz, 1H,
[Ru].dbd.CH.dbd.CH), 15.19 (d, J=4.0 Hz, 1H, [Ru].dbd.CH). .sup.31P
n.m.r. (300 MHz, CDCl.sub.3): .delta. Ruthenium-dienamide chelate
74:35.0; Grubbs' catalyst: 37.0; Ruthenium-dienamide complex 73:
38.8; Tricyclohexylphosphine oxide: 46.5.
7.8.8 NMR Study of Grubbs' Catalyst with Dienamide 76
##STR00134##
[0411] In a dry box, a Teflon-sealed n.m.r. tube was charged with
(2S)-methyl 2-N-acetylamino-5-phenylpenta-2,4-dienoate 76 (10.0 mg,
40.8 .mu.mol), Grubbs' catalyst (33.6 mg, 40.9 .mu.mol) and
degassed CD.sub.2Cl.sub.2 (0.8 mL) at room temperature. The n.m.r.
tube was shaken gently and reaction progress was monitored by
.sup.1H n.m.r. spectroscopy. After 4 h, ruthenium-vinylalkylidene
formation was not observed and only peaks corresponding to Grubbs'
catalyst and the starting dienamide 76 were present.
7.8.9 Dimerisation of (2S)-Methyl 2-N-Acetylamino-pent-4-enoate 21a
in the presence of (2Z)-Methyl
2-N-Acetylamino-5-phenylpenta-2,4-dienoate 76
##STR00135##
[0413] The mixture of olefins 21a and 76 was subjected to the
conventional cross metathesis procedure (Section 7.5.2) under the
following conditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a
(18.1 mg, 0.11 mmol), (2Z)-methyl
2-N-acetylamino-5-phenylpenta-2,4-dienoate 76 (26.1 mg, 0.11 mmol),
DCM (4.0 mL), Grubbs' catalyst (8.7 mg, 10.6 .mu.mol, 10 mol %),
50.degree. C., 18 h, 28% conversion of allylglycine 21a into 60.
Dienamide 76 did not react under these conditions. n.m.r.
spectroscopic data for dienamide 76, dimer 60 and recovered
allylglycine derivative 21a were in agreement with those previously
reported (Section 7.11.8, Section 7.12.1 and Section 7.11.2
respectively).
7.8.10 Dimerisation of (2S)-Methyl
2-N-Acetylamino-5-phenylpent-4-enoate 77
##STR00136##
[0415] The enamide 77 was subjected to the conventional cross
metathesis procedure (Section 7.5.2) under the following
conditions:
Method A:
[0416] (2S)-Methyl 2-N-acetylamino-5-phenylpent-4-enoate 77 (59.3
mg, 0.24 mmol), DCM (10 mL), Grubbs' catalyst (19.8 mg, 24.1
.mu.mol, 10 mol %), 50.degree. C., 13 h, 0% conversion into dimer
60. The starting enamide 77 was recovered. .sup.1H n.m.r.
spectroscopic data for olefin 77 were in agreement with those
previously reported (Section 7.11.9).
Method B:
[0417] (2S)-Methyl 2-N-acetylamino-5-phenylpent-4-enoate 77 (59.3
mg, 0.24 mmol), DCM (7 mL), 2.sup.nd generation Grubbs' catalyst
(10.2 mg, 12.0 .mu.mol, 5 mol %), 50.degree. C., 20 h, 44%
conversion into dimer 60. .sup.1H n.m.r, spectroscopic data for
dimer 60 were in agreement with those previously reported (Section
7.12.1). The stilbene byproduct 145 was observed in the .sup.1H
n.m.r. spectrum. .sup.1H n.m.r. (300 MHz, CDCl.sub.3): 7.15 (s, 2H,
CH.dbd.), 7.40 (m, 4H, Arom CH), 7.55 (m, 4H, Arom CH), ortho-Arom
CH peaks masked by starting olefin 77. .sup.1H n.m.r. spectroscopic
data for stilbene 145 were in agreement with those reported in the
literature..sup.265
7.8.11 Dimerisation of (2S)-Methyl 2-N-Acetylaminopent-4-enoate 21a
in the presence of (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate
19
##STR00137##
[0419] The mixture of olefins 21a and 19 was subjected to the
conventional cross metathesis procedure (Section 7.5.2) under the
following conditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a
(12.7 mg, 74.2 .mu.mol), (2S)-methyl
2-N-acetylamino-5-methylhex-4-enoate 19 (14.5 mg, 72.9 .mu.mol),
DCM (4 mL), Grubbs' catalyst (11.5 mg, 14.0 .mu.mol, 20 mol %),
50.degree. C., 18 h, 100% conversion of 21a into dimer 60. .sup.1H
n.m.r. spectroscopic data for dimer 60 were in agreement with those
previously reported (Section 7.12.1). The prenylglycine derivative
19 was recovered unchanged.
7.8.12 Ethenolysis of (2S)-Methyl
2-N-Acetylamino-5-methylhex-4-enoate 19
##STR00138##
[0421] The prenylglycine derivative 19 was subjected to the
conventional cross metathesis procedure (Section 7.5.4) with
ethylene under the following conditions:
Method A:
[0422] (2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (11.0
mg, 55.2 .mu.mol), ethylene (atmospheric pressure), Grubbs'
catalyst (11.0 mg, 13.4 .mu.mol, 20 mol %), DCM (4 mL), 22.degree.
C., 17h, 0% conversion into 21a. .sup.1H n.m.r. spectroscopy
indicated the starting hex-4-enoate 19 was recovered.
Method B:
[0423] (2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (10.8
mg, 54.2 .mu.mol, ethylene (60 psi), 2.sup.nd generation Grubbs'
catalyst (9.3 mg, 11 .mu.mol, 20 mol %), DCM (4 mL), 22.degree. C.,
19 h, 9% conversion into 21a.
Method C:
[0424] (2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (24.3
mg, 0.12 mmol), ethylene (60 psi), 2.sup.nd generation Grubbs'
catalyst (31.1 mg, 36.6 .mu.mol, 30 mol %), DCM (5 mL), 50.degree.
C., 38 h, 32% conversion into 21a. Spectroscopic data for 21a and
the recovered prenylglycine derivative 19 were in agreement with
those previously reported (Section 7.11.2 and Section 7.9.5
respectively).
7.8.13 Butenolysis of (2S)-Methyl
2-N-Acetylamino-5-methylhex-4-enoate 19
##STR00139##
[0426] The prenylglycine derivative 19 was subjected to the
conventional cross metathesis procedure (Section 7.5.4) with
cis-2-butene under the following conditions: (2S)-Methyl
2-N-acetylamino-5-methylhex-4-enoate 19 (16.2 mg, 81.4 .mu.mol),
DCM (5 mL), 2.sup.nd generation Grubbs' catalyst (3.5 mg, 4.1
.mu.mol, 5 mol %), cis-2-butene (5 psi), 50.degree. C., 14 h, 100%
conversion into 81. Purification by flash chromatography
(SiO.sub.2, light petroleum:DCM EtOAc:MeOH, 1:2:1:0.2) gave
(2S)-methyl 2-N-acetylaminohex-4-enoate 81 as a brown oil (12.6 mg,
84%). GC: t.sub.R (E/Z)=4.2 min, 4.4 min (GC column 30QC5/BPX5,
150.degree. C. for 1 min, 10.degree. C. min.sup.-1 to 280.degree.
C. for 6 min). .nu..sub.max(neat): 3284s, 2966w, 2954m, 2856w,
1747s, 1658s, 1547s, 1437s, 1375s, 1217m, 1142m, 1072w, 1016w,
968m, 848m cm.sup.-1. .sup.1H n.m.r. (300 MHz, CDCl.sub.3): .delta.
1.60 (dd, J=6.3, 1.2 Hz, 3H, H6), 1.95 (s, 3H, CH.sub.3CO),
2.36-2.44 (m, 2H, H3), 3.67 (s, 3H, OCH.sub.3), 4.55 (dt, J=7.8 Hz,
5.9 Hz, 1H, H2), 5.24 (m, 1H, H5), 5.49 (m, 1H, H4), 6.17 (bd,
J=6.4 Hz, 1H, NH). .sup.13C n.m.r. (100 MHz, CDCl.sub.3): .delta.
18.1 (C6), 23.3 (CH.sub.3CO), 35.4 (C3), 52.1, 52.4 (C2,
OCH.sub.3), 124.6, 130.2 (C4, 5), 169.7, 172.6 (C1, CONH). Mass
Spectrum (ESI.sup.+, MeOH): m/z 208.1 (M+Na).sup.+,
C.sub.9H.sub.15NNaO.sub.3 requires 208.1. Spectroscopic data were
in agreement with those reported in the literature..sup.117,119
[0427] An analogous cross metathesis reaction was performed using a
mixture of cis+trans-2-butene under the following conditions:
(2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (12.8 mg, 64.3
.mu.mol), DCM (5 mL), 2.sup.nd generation Grubbs' catalyst (2.8 mg,
3.3 .mu.mol, 5 mol %), trans+cis-2-butene (10 psi), 50.degree. C.,
16 h, <10% conversion into crotylglycine 81.
7.8.14 Dimerisation of (2S)-Methyl 2-N-Acetylaminohex-4-enoate
81
##STR00140##
[0429] The crotylglycine derivative 81 was subjected to the
conventional cross metathesis procedure (Section 7.5.2) under the
following conditions: (2S)-Methyl 2-N-acetylaminohex-4-enoate 81
(17.0 mg, 91.9 .mu.mol), DCM (4 mL), 2.sup.nd generation Grubbs'
catalyst (4.2 mg, 5.0 .mu.mol, 5 mol %), 50.degree. C., 17 h, 100%
conversion into dimer 60. The solvent was evaporated under reduced
pressure to give the homodimer 60 as a brown oil (21.5 mg, 100%
crude yield). Spectroscopic data for dimer 60 were in agreement
with those previously reported (Section 7.12.1).
7.8.15 Activation of (2S)-Methyl
2-N-Benzoylamino-5-methylhex-4-enoate 87
##STR00141##
[0431] The prenylglycine derivative 87 was subjected to the
conventional cross metathesis procedure (Section 7.5.5) with
cis-1,4-diacetoxy-2-butene 141 under the following conditions:
(2S)-Methyl 2-N-benzoylamino-5-methylhex-4-enoate 87 (170 mg, 0.65
mmol), DCM (10 mL), 2.sup.nd generation Grubbs' catalyst (16.5 mg,
0.03 mmol, 5 mol %), cis-1,4-diacetoxy-2-butene (5 psi), 50.degree.
C., 20 h, 100% conversion into 142. Purification by flash
chromatography (SiO.sub.2, light petroleum:EtOAc, 1:1) gave
(2S)-6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 142 as a
dark brown oil (113 mg, 57%). .nu..sub.max (neat): 3333.3s;
3056.4w; 3015.4w; 2943.6s; 1738.5s; 1661.5m; 1641.0s; 1605.1m;
1574.4m; 1533.3s; 1487.2m; 1435.9m; 1364.1, m; 1235.9, s; 1153.8,
w; 1071.6, w; 1025.6, m; 969.2, m; 800.8, w; 717.9, m; 692.3,w
cm.sup.-1. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 2.00, s, 3H,
CH.sub.3; 2.67, m, 2H, H3; 3.77, s, 3H, OCH.sub.3; 4.49, d, J 4.7
Hz, 2H, H6; 4.89, q, J 5.8 Hz, 1H, H2; 5.68, t, J 5.2 Hz, 2H, H4,
5; 6.75, d, J 7.4 Hz, 2H, H4, 5; 7.42, t, J 7.2 Hz, 2H, H4', 6';
7.50, t, J 6.4 Hz, 1H, H5'; 7.78, d, J 7.1 Hz, 2H, H3', 7'.
.sup.13C NMR (125 MHz, CDCl.sub.3): .delta. 20.92, CH.sub.3; 35.22,
C3; 52.09, OCH.sub.3; 52.65, C2; 64.52, C6; 127.17, C3', 7';
128.70, C4', 6'; 128.93, C5; 129.14, C4; 131.93, C5'; 133.93, C2';
167.07, C1'; 170.80, C1''; 172.27, C1. Mass Spectrum (ESP,
CH.sub.3CN): m/z 328.1 (M+Na.sup.+) C.sub.16H.sub.19NO.sub.5Na.
7.8.16 Synthesis of (2S,7S)-dimethyl
2-N-acetylamino-7-N-benzoylamino-octa-4-enedioate 143
##STR00142##
[0433] 2-Acetylamino-7-benzoylamino-oct-4-enedioic acid dimethyl
ester 143 was synthesised using standard solution-phase metathesis
conditions (refer to Section 7.5.2):
6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 142 (50 mg,
0.16 mmol), dichloromethane (10 mL), second generation Grubbs'
catalyst (5 mol %, 7 mg, 8 .mu.mol),
methyl-2-acetylamino-4-pentenoate 21a (168 mg, 0.98 mmol),
50.degree. C., 18 h. The desired compound was obtained as a brown
oil and purified via column chromatography (SiO.sub.2;
EtOAc:hexane; 2:1). .sup.1H NMR (500 MHz, CDCl.sub.3, mixture of
isomers (1:1.2)): .delta. 1.95, s (major isomer) and 1.97, s (minor
isomer), 3H, CH.sub.3; 2.42-2.70, m, 4H, H3, 6; 3.62, s (minor
isomer), 3.64, s (major isomer), 3.78, s (minor isomer) and 3.79, s
(major isomer), 6H, 2.times.OCH.sub.3; 4.63-4.66, m, 1H, H2;
4.85-4.91, m, 1H, H7; 5.35-5.49, m, 2H, 114, 5; 6.20, d, J 7.7 Hz
(major isomer) and 6.34, d, J 7.5 Hz, 1H, NH (minor isomer); 6.87,
t, J 7.55 Hz, 1H, NH; 7.44, t, J 7.1 Hz, 2H, H4', 6'; 7.50, t, J
6.9 Hz, 1H, H5'; 7.84, t, J 7.9 Hz, 2H, H3', 7'. .sup.13C NMR (75
MHz, CDCl.sub.3, mixture of isomers (1:1.2)): .delta. 22.83,
CH.sub.3; 34.84, 35.05, 35.38 and 35.73, C3, 6; 51.51 and 51.55,
C2; 52.35, 52.46, 52.53, 52.60 and 52.66, C7, 2.times.OCH.sub.3;
127.18 and 127.22, C3', 7'; 128.57 and 128.62, C4', 6'; 128.88 and
129.00, C4, 5; 131.86 and 131.91, C5'; 133.71, C2'; 167.06, COPh;
170.03 and 170.11, COMe; 172.20, 172.21, 172.24 and 172.43,
2.times.COOMe. Mass Spectrum (ESI.sup.+, CH.sub.3OH): m/z 399.2
(M+Na.sup.+) C.sub.19H.sub.24N.sub.2O.sub.6Na.
7.9 Wilkinson's Hydrogenation of Olefinic Substrates
7.9.1 (2S,7S)-Dimethyl 2,7-N,N'-Diacetylaminooctanedioate 71
##STR00143##
[0435] (2S,7S)-Dimethyl 2,7-N,N'-diacetylaminooct-4-enedioate 60
was subjected to the general Wilkinson's hydrogenation procedure
(Section 7.4.4) under the following conditions: Dimer 60 (25.0 mg,
79.6 .mu.mol), benzene (5 mL), Wilkinson's catalyst, 60 psi,
22.degree. C., 4 h. At the end of the reaction period, the solvent
was evaporated under reduced pressure and the resulting oil was
purified by flash chromatography (SiO.sub.2, EtOAc) to afford the
saturated product 71 as a brown oil (25.0 mg, 99%). GC:
t.sub.R=14.4 min (GC column 30QC5/BPX5, 150.degree. C. for 1 min,
10.degree. C. min.sup.-1 to 280.degree. C. for 6 min). .nu..sub.max
(neat): 3426bm, 3055w, 2932m, 2857w, 2360w, 1741s, 1666s, 1543w,
1438m, 1375w, 1266s, 1177w, 1120w, 896w, 738w, 702w cm.sup.-1.
.sup.1H n.m.r. (400 MHz, CDCl.sub.3): .delta. 1.30-1.40 (m, 4H, H4,
5), 1.82-1.90 (m, 4H, H3, 6), 2.02 (s, 6H, CH.sub.3CO), 3.74 (s,
6H, OCH.sub.3), 4.56-4.63 (m, 2H, H2, 7), 6.16 (bd, J=7.5 Hz, 2H,
NH). .sup.13C n.m.r. (100 MHz, CDCl.sub.3): .delta. 23.3
(CH.sub.3CO), 24.7 (C4, 5), 32.3 (C3, 6), 52.0 (C2, 7), 52.5
(OCH.sub.3), 170.0, 173.1 (C1, 8, CONH). HRMS MeOH). Found: m/z
339.1531 (M+Na).sup.+, C.sub.14H.sub.24N.sub.2NaO.sub.6 requires
339.1532.
7.9.2 (2S,7S)-Dimethyl 2,7-N,N'-Dibenzoylaminooctanedioate 72
##STR00144##
[0437] (2S,7S)-Dimethyl 2,7-N,N'-dibenzoylaminoocta-4-enedioate 69
was subjected to the general Wilkinson's hydrogenation procedure
(Section 7.4.4) under the following conditions: Dimer 69 (20.0 mg,
45.7 .mu.mol, benzene (5 mL), Wilkinson's catalyst, 60 psi,
22.degree. C., 4 h. At the end of the reaction period, the solvent
was evaporated under reduced pressure and the resulting oil was
purified by flash chromatography (SiO.sub.2, EtOAc) to afford the
saturated product 72 as a brown oil (20.0 mg, 100%). GC: t.sub.R
17.2 min (GC column 30QC5/BPX5, 150.degree. C. for 1 min,
10.degree. C. min.sup.-1 to 280.degree. C. for 6 min). .nu..sub.max
(neat): 3055m, 2986w, 2955w, 1741s, 1662s, 1603w, 1580w, 1518m,
1486m, 1438s, 1359w, 1286s, 1182m, 1120m, 1028w, 896m cm.sup.-1.
.sup.1H n.m.r. (400 MHz, CDCl.sub.3): .delta. 1.35-1.53 (m, 4H, H4,
5), 1.80-2.02 (m, 4H, H3, 6), 3.78 (s, 6H, OCH.sub.3), 4.82 (dt,
J=7.3, 5.4 Hz, 2H, H2, 7), 6.73 (bd, J=7.4 Hz, 2H, NH), 7.40-7.49
(m, 6H, H3', 4', 5'), 7.78-7.82 (m, 4H, H2', 6'). .sup.13C n.m.r.
(100 MHz, CDCl.sub.3): .delta. 24.9 (C4, 5), 32.6 (C3, 6), 52.5,
52.7 (C2, OCH.sub.3), 127.2 (C2', 6'), 128.6 (C3', 5'), 131.9
(C4'), 134.1 (C1'), 167.2, 173.2 (C1, 8, CONH). HRMS (ESI.sup.+,
MeOH). Found: m/z 463.1842 (M+Na).sup.+,
C.sub.24H.sub.28N.sub.2NaO.sub.6 requires 463.1845.
7.9.3 Wilkinson's Hydrogenation of (2Z)-Methyl
2-N-Acetylamino-5-phenylpenta-2,4-dienoate 76
##STR00145##
[0439] The dienamide 76 was subjected to the general Wilkinson's
hydrogenation procedure (Section 7.4.4) under the following
conditions: (2Z)-Methyl 2-N-acetylamino-5-phenylpenta-2,4-dienoate
76 (11.5 mg, 46.9 .mu.mol), benzene (5 mL), Wilkinson's catalyst,
50 psi H.sub.2, 22.degree. C., 4 h, 99% yield (mass recovery) of a
1:4 mixture of 77:79 as a brown oil. GC: t.sub.R=10.8 min 79, 13.9
min 77 (GC column 30QC5/BPX5, 150.degree. C. for 1 min, 10.degree.
C. min.sup.-1 to 280.degree. C. for 6 min). .sup.1H n.m.r.
spectroscopic datafor olefin 77 were in agreement with those
previously reported (Section 7.11.9). Hydrogenation of the mixture
using identical conditions led to 100% conversion into 79 (41.2 mg,
100% crude yield). .nu..sub.max (neat): 3262w, 3054m, 2956m, 1736s,
1676s, 1509m, 1438s, 1372w, 1265s, 1174w, 1120m, 1028w, 738s, 700w
cm.sup.-1. .sup.1H n.m.r. (300 MHz, CDCl.sub.3): .delta. 1.53-1.65
(m, 4H, H3, 4), 1.94 (s, 3H, CH.sub.3CO), 2.52-2.59 (rn, 2H, H5),
3.65 (s, 3H, OCH.sub.3), 4.59 (m, 1H, H2), 5.90 (bd, J=7.2 Hz, 1H,
NH), 7.07-7.29 (m, 5H, Arom CH). .sup.13C n.m.r. (75 MHz,
CDCl.sub.3): .delta. 23.3 (CH.sub.3CO), 27.2 (C4), 32.3 (C3), 35.5
(C5), 52.1, 52.5 (C2, OCH.sub.3), 126.1, 128.5, 132.2 (Arom CH),
141.7, (Arom C), 169.9, 173.2 (C1, CONH). Mass Spectrum (ESI.sup.+,
MeOH): m/z 272.2 (M+Na).sup.+, C.sub.14H.sub.19NNaO.sub.3 requires
272.1.
7.9.4 Wilkinson's Hydrogenation of (2S)-Methyl
2-N-Acetylamino-5-methylhex-4-enoate 19
##STR00146##
[0441] (2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 was
subjected to the general Wilkinson's hydrogenation procedure
(Section 7.4.4) under the following conditions: Hex-4-enoate
derivative 19 (11.3 mg, 56.8 .mu.mol), benzene (4 mL), Wilkinson's
catalyst, 50 psi, 22.degree. C., 4 h. At the end of the reaction
period, the solvent was evaporated under reduced pressure to afford
a brown oil (12.5 mg). .sup.1H n.m.r. spectroscopy indicated the
reaction gave only 6% conversion into the saturated product 80; 94%
of the starting prenylglycine derivative 19 was recovered. .sup.1H
n.m.r. (300 MHz, CDCl.sub.3): Hexanoate 80: .delta. 0.87 (d, J=6.6
Hz, 6H, H6), 1.09-1.28 (m, 2H, H4), 1.54 (h, J=6.7 Hz, 1H, H5),
1.61-1.71 (m, 2H, H3), 2.03 (s, 3H, CH.sub.3CO), 3.75 (s, 3H,
OCH.sub.3), 4.60 (dt, J=7.8 Hz, 5.5 Hz, 1H, H2), 5.96 (bd, J=7.8
Hz, 1H, NH).
Experimental for Chapter 5
7.10 Synthesis of Olefinic Substrates
7.10.1 (2S)-Methyl 2-N-(p-Nitrobenzoyl)aminopent-4-enoate 83
##STR00147##
[0443] A solution of p-nitrobenzoyl chloride 89 (1.21 g, 6.54 mmol)
in a mixture of DCM:Et.sub.2O (2:1, 15 mL) was added dropwise to a
stirred solution of methyl 2-aminopent-4-enoate hydrochloride 51
(0.98 g, 5.94 mmol) and Et.sub.3N (1.80 mL, 13.0 mmol) in a mixture
of DCM:Et.sub.2O (2:1, 15 mL) at 0.degree. C. The reaction mixture
was allowed to warm to room temperature and stirred for 20 h. The
mixture was acidified with dilute HCl solution (1 M, pH 2) and
extracted with DCM (3.times.20 mL). The combined organic extract
was washed with distilled water (20 mL), dried (MgSO.sub.4) and
evaporated under reduced pressure to afford the titled compound 83
as an off-white solid (1.63 g, 99%), m.p. 99-100.degree. C.
Spectroscopic data indicated the crude product 83 did not require
purification and was used directly in the subsequent reaction
(Section 7.15.1). GC: t.sub.R=12.20 min (GC column 30QC5/BPX5,
150.degree. C. for 1 min, 10.degree. C. min.sup.-1 to 280.degree.
C. for 6 min). [.alpha.].sub.D.sup.22+29.9.degree. (c=0.37,
CHCl.sub.3). .nu..sub.max (neat): 3293w, 2954m, 2839m, 1725s,
1641m, 1602w, 1538w, 1529w, 1456s, 1377s, 1256m, 1160m, 1118w,
1066w, 998m, 972m, 941w, 841m cm.sup.-1. .sup.1H n.m.r. (300 MHz,
CDCl.sub.3): .delta. 2.65-2.76 (m, 2H, H3), 3.82 (s, 3H,
OCH.sub.3), 4.90 (dt, J=5.6, 7.5 Hz, 1H, H2), 5.14-5.30 (m, 2H,
H5), 5.75 (m, 1H, H4), 6.73 (bd, J=6.6 Hz, 1H, NH), 7.95 (d, J=7.7
Hz, 2H, H2', 6'), 8.30 (d, J=7.6 Hz, 2H, H3', 5'). .sup.13C n.m.r.
(75 MHz, CDCl.sub.3): .delta. 36.6 (C3), 52.4, 52.9 (C2,
OCH.sub.3), 119.9 (C5), 124.0 (C3', 5'), 128.4 (C2', 6'), 132.0
(C4), 139.6 (C1'), 150.0 (C4'), 165.1 (C1), 172.1 (CONH). HRMS
(ESI.sup.+, MeOH). Found: m/z 279.0977 (M+H).sup.+,
C.sub.13H.sub.15N.sub.2O.sub.5 requires 279.0981; m/z 301.0798
(M+Na).sup.+, C.sub.13H.sub.14N.sub.2NaO.sub.5 requires
301.0800.
7.10.2 (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate 19
##STR00148##
[0445] The preparation of (2S)-methyl
2-N-acetylamino-5-methylhex-4-enoate 19 via asymmetric
hydrogenation of the dienoate 20 has been previously described
(Section 7.9.5).
7.10.3 (2Z)-Methyl 2-N-Benzoylamino-5-methylhexa-2,4-dienoate
82
##STR00149##
[0447] The dienamide 82 was prepared according to a procedure
described by Teoh et al..sup.118,119 Tetramethylguanidine (3.40 mL,
27.1 mmol) and hydroquinone (3.0 mg) were added to a solution of
methyl 2-N-benzoylamino-2-(dimethoxyphosphinyl)-acetate 64 (6.10 g,
20.3 mmol) in THF (120 mL) at -78.degree. C. After 30 min,
3-methyl-2-butenal 40 (2.40 mL, 24.9 mmol) was added and the
mixture was stirred at -78.degree. C. for 2 h, warmed to 25.degree.
C. and allowed to react an additional 16 h. The mixture was diluted
with DCM (150 mL) and washed with dilute HCl solution (1 M,
2.times.70 mL), CuSO.sub.4 solution (1 M, 2.times.70 mL), saturated
NaHCO.sub.3 solution (2.times.70 mL) and saturated NaCl solution
(1.times.70 mL). The organic extract was dried (MgSO.sub.4) and
evaporated under reduced pressure to give the crude product 82 as a
yellow oil (5.37 g). Purification by flash chromatography
(SiO.sub.2, light petroleum:EtOAc, 2:1) furnished the pure
dienamide 82 as an off-white solid (3.84 g, 73%), m.p.
98-99.degree. C. GC: t.sub.R=11.00 min (GC column 30QC5/BPX5,
150.degree. C. for 1 min, 10.degree. C. min.sup.-1 to 280.degree.
C. for 6 min). .nu..sub.max (KBr): 3286m, 2991w, 2948w, 1716s,
1649s, 1601w, 1579w, 1524s, 1489s, 1436m, 1389w, 1331m, 1286m,
1254s, 1207m, 1190w, 1162w, 1135w, 1087s, 1048w, 996w, 977w, 931w,
864m, 802m, 760m, 739m, 710s, 688w, 677w, 630w, 614w, 585w
cm.sup.-1. .sup.1H n.m.r. (400 MHz, CDCl.sub.3): .delta. 1.87 (s,
3H, H6), 1.91 (d, J=0.7 Hz, 3H, CH.sub.3C--), 3.78 (s, 3H,
OCH.sub.3), 6.03 (d with fine splitting, J=11.9 Hz, 1H, H4), 7.41
(d, J=11.8 Hz, 1H, H3), 7.43-7.47 (m, 2H, H3', 5'), 7.53 (m, 1H,
H4'), 7.63 (bs, 1H, NH), 7.89-7.90 (m, 2H, H2', 6'). .sup.13C
n.m.r. (100 MHz, CDCl.sub.3): .delta. 19.3 (CH.sub.3C.dbd.), 27.1
(C6), 52.5 (OCH.sub.3), 121.0 (C4), 121.2 (C5), 127.6 (C2', 6'),
128.9 (C3', 55, 129.9 (C3), 132.0 (C4'), 134.1 (C2), 147.2 (C1'),
166.0, 166.1 (C1, CONH). HRMS (ESI.sup.+, MeOH). Found: m/z
260.1282 (M+H).sup.+, C.sub.15H.sub.18NO.sub.3 requires 260.1287;
m/z 282.1099 (M+Na).sup.+, C.sub.15H.sub.17NNaO.sub.3 requires
282.1106.
7.11 Metathesis Reactions with Olefinic Substrates
7.11.1 (2S,7S)-Dimethyl
2,7-N,N'-Di[(p-nitrobenzoyl)amino]oct-4-enedioate 90
##STR00150##
[0448] Method A:
[0449] The dimer 90 was prepared via the conventional cross
metathesis procedure (Section 7.5.2) under the following
conditions: (2S)-Methyl 2-N-(p-nitrobenzoyl)aminopent-4-enoate 83
(43.5 mg, 0.16 mmol), DCM (3 mL), Grubbs' catalyst (26.0 mg, 31.6
.mu.mol, 20 mol %), 50.degree. C., 14 h, 100% conversion into dimer
90. The reaction mixture was evaporated under reduced pressure to
give the homodimer 90 as a brown oil (69.7 mg, 100% crude
yield).
Method B:
[0450] The dimer 90 was also prepared and purified from an
analogous reaction using 2.sup.nd generation Grubbs' catalyst under
the following conditions: (2S)-Methyl
2-N-(p-nitrobenzoyl)aminopent-4-enoate 83 (86.3 mg, 0.31 mmol), DCM
(4 mL), 2.sup.nd generation Grubbs' catalyst (13.6 mg, 16.0
.mu.mol, 5 mol %), 50.degree. C., 12 h, 100% conversion into dimer
90. Purification by flash chromatography (SiO.sub.2, light
petroleum:EtOAc:DCM, 4:2:1) gave the pure dimer 90 as a pale brown
solid (74.0 mg, 90%), m.p. 90-92.degree. C. GC: t.sub.R (E/Z)=16.1
min, 16.2 min (GC column 30QC5/BPX5, 150.degree. C. for 1 min,
10.degree. C. min.sup.-1 to 280.degree. C. for 6 min).
[.alpha.].sub.D.sup.22+20.0.degree. (c=0.21, CHCl.sub.3).
.nu..sub.max (neat): 3365m, 3057w, 2957w, 2854w, 1728s, 1667s,
1602m, 1525s, 1487m, 1437m, 1348s, 1267m, 1227m, 1174w, 1157w,
1110w, 1014m, 974m, 869m, 874m, 737s, 718s em'. .sup.1H n.m.r. (400
MHz, CDCl.sub.3): .delta. 2.60-2.64 (m, 4H, H3, 6), 3.70 (s, 6H,
OCH.sub.3), 4.88 (apparent q, J=5.9 Hz, 2H, H2, 7), 5.49-5.53 (m,
2H, H4, 5), 7.11 (bd, J=7.4 Hz, 2H, NH), 8.02 (d, J=8.7 Hz, 4H,
H2', 6'), 8.21-8.29 (m, 4H, H3', 5'). .sup.13C n.m.r. (100 MHz,
CDCl.sub.3): .delta. 35.0 (C3, 6), 52.8, 52.8 (C2, 7, OCH.sub.3),
123.8 (C3', 5'), 128.8, 128.9 (C2', 6', C4, 5), 139.2 (C1'), 149.9
(C4'), 165.2 (C1, 8), 172.1 (CONH). HRMS (ESI.sup.+, MeOH). Found:
m/z 529.1560 (M+H).sup.+, C.sub.24H.sub.25N.sub.4O.sub.10 requires
529.1571; m/z 551.1379 (M+Na).sup.+,
C.sub.24H.sub.24N.sub.4NaO.sub.10 requires 551.1390.
7.11.2 Attempted Dimerisation of (2Z)-Methyl
2-N-Benzoylamino-5-methylhexa-2,4-dienoate 82
##STR00151##
[0452] The dienamide 82 was subjected to the conventional cross
metathesis procedure (Section 7.5.2) under the following
conditions: (2Z)-Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate
82 (30.7 mg, 0.12 mmol), DCM (5 mL), 2.sup.nd generation Grubbs'
catalyst (5.1 mg, 6.0 .mu.mol, 5 mol %), 50.degree. C., 15 h, 0%
conversion into dimer 84. The dienamide 82 was recovered unchanged.
.sup.1H n.m.r. spectroscopic data for the recovered dienamide 82
were in agreement with those previously reported (Section
7.14.3).
7.11.3 Attempted Butenolysis of (2Z)-Methyl
2-N-Benzoylamino-5-methylhexa-2,4-dienoate 82
##STR00152##
[0454] The dienamide 82 was subjected to the conventional cross
metathesis procedure (Section 7.5.4) with cis-2-butene under the
following conditions: (2Z)-Methyl
2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 (39.3 mg, 0.15 mmol),
DCM (5 mL), cis-2-butene (15 psi), 2.sup.nd generation Grubbs'
catalyst (6.6 mg, 7.8 .mu.mol, 5 mol %), 50.degree. C., 14 h, 0%
conversion into 86. The dienamide 82 was recovered unchanged.
.sup.1H n.m.r. spectroscopic data for the recovered dienamide 82
were in agreement with those previously reported (Section
7.14.3).
7.12 Activation of Dormant Olefins
7.12.1 Butenolysis of (2Z)-Methyl
2-N-Acetylamino-5-methylhex-4-enoate 19
##STR00153##
[0456] The activation of prenylglycine 19 via butenolysis (Section
7.5.4) to give the crotylglycine derivative 81 has been previously
described (Section 7.12.13).
7.12.2 (2S)-Methyl 2-N-Benzoylamino-5-methylhex-4-enoate 87
##STR00154##
[0458] The dienamide 82 was subjected to the general asymmetric
hydrogenation procedure (Section 7.4.3) under the following
conditions: (2Z)-Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate
82 (26.1 mg, 0.10 mmol), MeOH (5 mL), Rh(I)--(S,S)-Et-DuPHOS, 75
psi, 22.degree. C., 3 h. At the end of the reaction period, the
solvent was evaporated under reduced pressure and the residue was
purified by flash chromatography (SiO.sub.2, EtOAc) to give the
prenylglycine derivative 87 as a pale yellow oil (23.9 mg, 91%).
HPLC: t.sub.R=6.20 min (Chiralcel OJ column, 1.0 mL min.sup.-1,
detection at 254 nm, 5% EtOH:95% hexane).
[.alpha.].sub.D.sup.22+53.0.degree. (c=1.19, CHCl.sub.3).
.nu..sub.max (neat): 3334m, 2953w, 1744s, 1645s, 1603w, 1580w,
1538s, 1489m, 1437m, 1353w, 1274w, 1211w, 1175w, 1095w, 1031w,
736w, 714w, 693w cm.sup.-1. .sup.1H n.m.r. (300 MHz, CDCl.sub.3):
.delta. 1.61 (d, J=0.5 Hz, 3H, CH.sub.3C.dbd.), 1.71 (d, J=1.0 Hz,
3H, H6), 2.52-2.76 (m, 2H, H3), 3.77 (s, 3H, OCH.sub.3), 4.85 (dt,
J=7.7, 5.5 Hz, 1H. H2), 5.08 (m, 1H, H4), 6.65 (bd, J=6.9 Hz, 1H
NH), 7.41-7.47 (m, 2H, H3', 5), 7.51 (m, 1H, H4'), 7.76-7.79 (m,
2H, H2', 6'). .sup.13C n.m.r. (75 MHz, CDCl.sub.3): .delta. 18.0
(CH.sub.3C.dbd.), 26.0 (C6), 30.9 (C3), 52.5, 52.6 (C2, OCH.sub.3),
117.6 (C4), 127.2 (C2', 6'), 128.7 (C3', 5'), 131.8 (C4'), 134.3
(C5), 136.8 (C1'), 167.0, 172.8 (C1, CONH). HRMS (ESI.sup.+, MeOH).
Found: m/z 262.1441 (M+H).sup.+, C.sub.15H.sub.20NO.sub.3 requires
262.1443; m/z 284.1256 (M+Na).sup.+, C.sub.15H.sub.19NNaO.sub.3
requires 284.1263.
(2R)-Methyl 2-N-benzoylamino-5-methylhex-4-enoate 87
##STR00155##
[0460] The dienamide 82 was subjected, to the general asymmetric
hydrogenation procedure (Section 7.4.3) under the following
conditions: (2Z)-Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate
82 (80.1 mg, 0.31 mmol), MeOH (7 mL), Rh(I)--(R,R)-Et-DuPHOS, 75
psi, 22.degree. C., 3 h. At the end of the reaction period, the
solvent was evaporated under reduced pressure and the residue was
purified by flash chromatography (SiO.sub.2, EtOAc) to give the
prenylglycine derivative 87 as a yellow oil (78.2 mg, 97%). HPLC:
t.sub.R=5.90 min (Chiralcel OJ column, 1.0 mL min.sup.-I, detection
at 254 nm, 5% EtOH:95% hexane). [.alpha.].sub.D.sup.22-53.4.degree.
(c=0.98, CHCl.sub.3). Spectroscopic data were in agreement with
those previously reported for the (S)-enantiomer.
7.12.3 (2S)-Methyl 2-N-Benzoylaminohex-4-enoate 88
##STR00156##
[0462] The enamide 87 was subjected to the conventional cross
metathesis procedure (Section 7.5.5) with cis-2-butene under the
following conditions: (2S)-Methyl
2-N-benzoylamino-5-methylhex-4-enoate 87 (90.0 mg, 0.35 mmol), DCM
(5 mL), cis-2-butene (15 psi), 2.sup.nd generation Grubbs' catalyst
(14.6 mg, 17.2 .mu.mol, 5 mol %), 50.degree. C., 12 h, 100%
conversion into 88. The reaction mixture was evaporated under
reduced pressure to give the crotylglycine derivative 88 as a brown
oil (101 mg, 100% crude yield). GC: t.sub.R (E/Z)=9.68 min, 9.93
min (GC Column 30QC5/BPX5, 150.degree. C. for 1 min, 10.degree. C.
min.sup.-1 to 280.degree. C. for 6 min). .nu..sub.max (neat):
3337bm, 3057w, 2954m, 2856w, 1743s, 1652s, 1603w, 1580w, 1532s,
1488m, 1438m, 1360w, 1266s, 1217w, 1180w, 1116w, 1031m, 969w, 896w,
801w, 738s, 638w cm.sup.-1. .sup.1H n.m.r. (300 MHz, CDCl.sub.3):
.delta. 1.66 (dd, J=6.4, 1.4 Hz, 3H, H6), 2.52-2.66 (m, 2H, H3),
3.77 (s, 3H, OCH.sub.3), 4.82 (apparent dd, J=7.6, 5.7 Hz, 1H, H2),
5.33 (m, 1H, H5), 5.63 (m, 1H, H4), 6.66 (bd, J=7.0 Hz, 1H, NH),
7.43 (t, J=7.0 Hz, 2H, H3', 5'), 7.50 (m, 1H, H4'), 7.78 (d, J=7.1
Hz, 2H, H2', 6'). .sup.13C n.m.r. (100 MHz, CDCl.sub.3): .delta.
18.0 (C6), 35.4 (C3), 52.4, 52.5 (C2, OCH.sub.3), 124.5 (C5), 127.1
(CT, 6'), 128.6 (C3', 5'), 130.2 (C4), 131.7 (C4'), 134.1 (C1'),
166.9, 172.5 (C1, CONH). HRMS (ESI.sup.+, MeOH). Found: m/z
248.1284 (M+H).sup.+, C.sub.14H.sub.18NO.sub.3 requires 248.1287;
m/z 270.1098 (M+Na).sup.+, C.sub.14H.sub.17NNaO.sub.3 requires
270.1106.
7.12.4 Dimerisation of (2S)-Methyl 2-N-Acetylaminohex-4-enoate
81
##STR00157##
[0464] The dimerisation of crotylglycine 81 using the conventional
cross metathesis procedure has been previously described (Section
7.12.14).
7.12.5 Dimerisation of (2S)-Methyl 2-N-Benzoylaminohex-4-enoate
88
##STR00158##
[0466] The enamide 88 was subjected to the conventional cross
metathesis procedure under the following conditions: (2S)-Methyl
2-N-benzoylaminohex-4-enoate 88 (89.6 mg, 0.36 mmol), DCM (5 mL),
2.sup.nd generation Grubbs' catalyst (15.3 mg, 18.0 .mu.mol, 5 mol
%), 50.degree. C., 17 h, 100% conversion into dimer 69. The
reaction mixture was evaporated under reduced pressure to afford
the homodimer 69 as a brown oil (106 mg, 100% crude yield).
Spectroscopic data for dimer 69 were in agreement with those
previously reported (Section 7.12.2).
7.13 Wilkinson's Hydrogenation Reactions
7.13.1 Wilkinson's Hydrogenation of (2Z)-Methyl
2-N-Benzoylamino-5-methylhexa-2,4-dienoate 82
##STR00159##
[0467] Method A:
[0468] The dienamide 82 was subjected to the general Wilkinson's
hydrogenation procedure (Section 7.4.4) under the following
conditions: (2Z)-Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate
82 (47.0 mg, 0.18 mmol), benzene (5 mL), Wilkinson's catalyst, 50
psi, 22.degree. C., 4 h. The dienamide 82 was recovered unchanged.
.sup.1H n.m.r. spectroscopic data for the recovered dienamide 83
were in agreement with those previously reported (Section
7.14.3).
7.13.2 (2S,7S)-Dimethyl
2,7-N,N'-Di(p-nitrobenzoyl)aminooctanedioate 91
##STR00160##
[0470] (2S,7S)-Dimethyl
2,7-N,N'-di(p-nitrobenzoyl)aminoocta-4-enedioate 90 was subjected
to the general Wilkinson's hydrogenation procedure (Section 7.4.4)
under the following conditions: Dimer 90 (20.6 mg, 0.04 mmol),
THF:.sup.tBuOH (1:1, 5 mL), Wilkinson's catalyst, 15 psi H.sub.2,
22.degree. C., 14 h. At the end of the reaction period, the solvent
was evaporated under reduced pressure to afford the product 91 as a
brown oil. Purification by flash chromatography (SiO.sub.2, light
petroleum:EtOAc:DCM, 1:1:1) gave the pure dimer 91 as an off-white
solid (13.8 mg, 67%), m.p. 117-119.degree. C. GC: t.sub.R=16.8 min
(GC column 30QC5/BPX5, 150.degree. C. for 1 min, 10.degree. C.
min.sup.-1 to 280.degree. C. for 6 min). .nu..sub.max (neat):
3304w, 2932w, 1740s, 1637s, 1603m, 1528s, 1438w, 1348m, 1265s,
1109w cm.sup.-1. .sup.1H n.m.r. (400 MHz, CDCl.sub.3): .delta.
1.39-1.54 (m, 4H, H4, 5), 1.74-2.04 (m, 4H, H3, 6), 3.81 (s, 6H,
OCH.sub.3), 4.82 (dt, J=7.3, 5.4 Hz, 2H, H2, 7), 6.85 (bd, J=7.4
Hz, 2H, NH), 7.96 (d, J=8.8 Hz, 4H, H2', 6'), 8.28 (d, J=8.7 Hz,
4H, H3', 5'). .sup.13C n.m.r. (100 MHz, CDCl.sub.3): .delta. 24.7
(C4, 5), 32.4 (C3, 6), 52.7, 52.9 (C2, OCH.sub.3), 124.0 (C2', 6'),
128.5 (C3', 5'), 139.5 (C1'), 150.0 (C4'), 165.3, 172.8 (C1, 8,
CONH). HRMS (ESI.sup.+, MeOH). Found: m/z 553.1550 (M+Na).sup.+,
C.sub.24H.sub.26N.sub.4NaO.sub.10 requires 553.1547.
Experimental for Section 6
7.14 Synthesis of Non-Proteinaceous Fmoc-Amino Acids
[0471] Peptide sequences are represented by structural diagrams and
three-letter codes of constituent amino acids. Synthetic amino
acids allylglycine, crotylglycine and prenylglycine are represented
by Hag, Crt and Pre respectively. Procedures for the preparation of
the Fmoc-protected olefinic amino acids:
(2S)-2-N-Fluorenylmethoxy-carbonylaminopent-4-enoic acid
(Fmoc-L-Hag-OH) 96,
(2S)-2-N-fluorenylmethoxy-carbonylaminohex-4-enoic acid
(Fmoc-L-Crt-OH) 100 and
(2S)-2-N-fluorenyl-methoxycarbonylamino-5-methylhex-4-enoic acid
(Fmoc-L-Pre-OH) 92, are detailed below.
7.14.1 2-N-Fluorenylmethoxycarbonylaminopent-4-enoic acid 96
(Fmoc-Hag-OH)
##STR00161##
[0473] The allylglycine derivative 96 was prepared according to the
procedure described by Paquet..sup.230 Fmoc-OSu (14.60 g, 43.3
mmol) was added to stirred solution of L-allylglycine (5.00 g, 43.5
mmol) and NaHCO.sub.3 (18.20 g, 0.22 mol) in a mixture of
acetone:water (200 mL). The resultant white suspension was stirred
at room temperature and after 20 h, t.l.c. analysis (SiO.sub.2,
light petroleum:EtOAc; 1:1) showed the absence of starting
material. The reaction mixture was acidified with concentrated HCl
(pH 2) and the acetone was removed under reduced pressure. The
resultant suspension was extracted into DCM (3.times.75 mL) and the
combined organic extract was washed with dilute HCl solution (1 M,
2.times.50 mL), water (2.times.50 mL), dried (MgSO.sub.4) and
evaporated under reduced pressure to afford the titled Fmoc-amino
acid 96 as a colourless solid (14.01 g, 96%), m.p. 137-138.degree.
C. (lit..sup.266 134-136.degree. C.). .nu..sub.max (KBr): 3484s,
3198bs, 3085m, 2967m, 2923m, 1723s, 1644m, 1525s, 1478w, 1449s,
1396m, 1340m, 1233s, 1189s, 1099m, 1048s, 998w, 966w, 943m, 924w,
850m, 781m, 761s, 740m, 648w, 623m, 582m, 560w, 540m, 424w
cm.sup.-1. .sup.1H n.m.r. (400 MHz, CDCl.sub.3): .delta. 2.52-2.70
(2.34-2.49) (m, 2H, H3), 4.23 (t, J=6.9 Hz, 1H, H9'), 4.42 (4.30)
(d, J=6.9 Hz, 2H, CH.sub.2O), 4.52 (m, 1H, H2), 5.13-5.23 (m, 2H,
H5), 5.31 (5.87) (bd, J=7.8 Hz, 1H, NH), 5.75 (m, 1H, H4), 6.63
(bs, 1H, OH), 7.31 (td, J=7.4, 0.8 Hz, 2H, H2', 7'), 7.38 (t, J=7.4
Hz, 2H, H3', 6'), 7.52-7.63 (m, 2H, H1', 8'), 7.76 (d, J=7.5 Hz,
2H, H4', 5'), one exchangeable proton (OH) not observed. .sup.13C
n.m.r. (100 MHz, CDCl.sub.3): .delta. 36.7 (C3), 47.5 (C9'), 53.4
(C2), 68.1 (CH.sub.2O), 122.0 (C5), 120.1 (C2', 7'), 125.4 (C3',
6'), 127.9 (C1', 8'), 128.0 (C4', 5'), 131.1 (C4), 141.7 (C8'a,
9'a), 144.0 (C4'a, 4'b), 156.3 (OCONH), 176.4 (C1). Mass Spectrum
(ESI+, MeOH): m/z 338.4 (M+H).sup.+, C.sub.20H.sub.20NO.sub.4
requires 338.1; 360.3 (M+Na).sup.+, C.sub.20H.sub.19NNaO.sub.4
requires 360.1. Spectroscopic data were in agreement with those
reported in the literature..sup.266
7.14.2 2-N-Fluorenylmethoxycarbonylaminohex-4-enoic acid 100
(Fmoc-Crt-OH)
##STR00162##
[0475] A solution of (2S)-methyl 2-N-acetylaminohex-4-enoate 81
(1.30 g, 7.05 mmol) in dilute HCl (1 M, 8 mL) was heated at reflux
for 21 h. The reaction mixture was evaporated under reduced
pressure to give 2-aminohex-4-enoic acid hydrochloride salt
(L-crotylglycine.HCl) 101 as a pale brown solid (1.17 g, 100%),
m.p. 212-214.degree. C. .nu..sub.max (KBr): 3500bs, 2965m, 2358s,
1731s, 1651m, 1455m, 901m cm.sup.-1. .sup.1H n.m.r. (300 MHz,
CD.sub.3OD): .delta. 1.69 (d, J=5.3 Hz, 3H, H6), 2.51-2.74 (m, 2H,
H3), 3.99 (m, 1H, H2), 5.42 (m, 1H, H5), 5.73 (m, 1H, H4),
exchangeable protons (NH and OH) not observed. .sup.13C n.m.r. (75
MHz, CD.sub.3OD): .delta. 18.7 (C6), 35.1 (C3), 48.7 (C2), 124.6
(C5), 133.6 (C4), 174.3 (C1). Mass Spectrum (ESI+, MeOH): m/z 130.1
(M+H).sup.+, C.sub.6H.sub.12NO.sub.2 requires 130.1.
[0476] 2-N-Fluorenylmethoxycarbonylaminohex-4-enoic acid 100 was
prepared according to the procedure described by Paquet..sup.230
Fmoc-OSu (2.36 g, 7.00 mmol) was added to a stirred suspension of
L-crotylglycine.HCl 101 (1.16 g, 7.03 mmol) and NaHCO.sub.3 (2.95
g, 35.0 mmol) in a mixture of acetone:water (1:1, 30 mL) The
resultant suspension was stirred at room temperature for 15 h. The
reaction mixture was then acidified with concentrated HCl (pH 2)
and the acetone was removed under reduced pressure. The resultant
suspension was extracted into DCM (3.times.25 mL) and the combined
organic extract was washed with dilute HCl solution (1 M,
2.times.25 mL), water (2.times.25 mL), dried (MgSO.sub.4) and
evaporated under reduced pressure to afford the titled Fmoc-amino
acid 100 as colourless solid (1.91 g, 78%), m.p. 119-121.degree. C.
(KBr): 3390bm, 3033m, 2961s, 2357w, 1730s, 1651w, 1505w, 1450w,
1395w, 850w cm.sup.-1. .sup.1H n.m.r. (300 MHz, CDCl.sub.3):
.delta. 1.67 (d, J=6.2 Hz, 3H, H6), 2.37-2.69 (m, 2H, H3), 4.23 (t,
J=6.8 Hz, 1H, H9'), 4.42-4.48 (m, 3H, CH.sub.2O, H2), 5.30-5.37 (m,
2H, H5, NH), 5.61 (m, 1H, H4), 7.31 (td, J=7.2, 1.3 Hz, 2H, H2',
7'), 7.34 (td, J=7.4, 1.5 Hz, 2H, H3', 6'), 7.60 (d, J=7.3 Hz, 2H,
H1', 8'), 7.74 (d, J=7.0 Hz, 2H, H4', 5'), one exchangeable proton
(OH) not observed. .sup.13C n.m.r. (75 MHz, CDCl.sub.3): .delta.
16.7 (C6), 34.1 (C3), 46.2 (C9'), 52.3 (C2), 66.2 (CH.sub.2O),
118.9 (C5), 123.0 (C2', 7'), 124.6 (C3', 6'), 125.2 (C1', 8'),
127.5 (C4', 5'), 129.7 (C4), 140.3 (C8'a, 9'a), 142.7 (C4'a, 4'b),
154.9 (OCONH), 175.0 (C1). Mass Spectrum (ESI+, MeOH): m/z 352.1
(M+H).sup.+, C.sub.21H.sub.22NO.sub.4 requires 352.2, Spectroscopic
data were in agreement with those reported in the
literature..sup.146
7.14.3 2-N-Fluorenylmethoxycarbonylamino-5-methylhex-4-enoic acid
92 (Fmoc-Pre-OH)
##STR00163##
[0478] The allylglycine derivative 96 was subjected to the
conventional cross metathesis procedure with 2-methyl-2-butene
(Section 0) under the following conditions:
2-N-Fluorenylmethoxycarbonylaminopent-4-enoic acid 96 (200 mg, 0.59
mmol), DCM (7 mL), 2' generation Grubbs' catalyst (26.0 mg, 30.6
.mu.mol, 5 mol %), 2-methyl-2-butene (1 mL, 10 psi), 50.degree. C.,
12 h, 100% conversion into 92. The reaction mixture was evaporated
under reduced pressure to give the prenylglycine derivative 92 as a
brown oil (245 mg, 100% crude yield). .nu..sub.max (neat): 3426w,
3324w, 3066w, 2932m, 1716s, 1514m, 1478w, 1450m, 1378w, 1338m,
1265m, 1220w, 1106w, 1057m, 910m, 855w, 759w, 738s, 704w, 648w, 621
w cm.sup.-1. .sup.1H n.m.r. (400 MHz, CDCl.sub.3): .delta. 1.63 (s,
3H, H6), 1.73 (s, 3H, CH.sub.3), 2.49-2.65 (m, 2H, H3), 4.23 (t,
J=6.7 Hz, 1H, H9'), 4.40 (d, J=6.7 Hz, 2H, CH.sub.2O), 5.11 (m, 1H,
H4), 5.41 (bd, J=7.5 Hz, 1H, NH), 7.31 (t, J=7.3 Hz, 2H, H2', 7'),
7.40 (t, J=7.3 Hz, 2H, H3', 6'), 7.58-7.66 (m, 2H, H1', 8'), 7.76
(d, J=7.4 Hz, 2H, H4', 5'), 9.22 (bs, 1H, OH). .sup.13C n.m.r. (100
MHz, CDCl.sub.3): .delta. 18.1 (C6), 26.0 (CH.sub.3C.dbd.), 30.8
(C3), 47.3 (C9'), 53.8 (C2), 67.2 (CH.sub.2O), 117.5 (C4), 120.1
(C2', 7'), 125.2 (C3', 6'), 127.2 (Cr, 8'), 127.8 (C4', 5'), 136.9
(C5), 141.4, 143.9 (Arom C), 156.1 (CONH), 176.2 (C1). HRMS
(ESI.sup.+, MeOH). Found: m/z 388.1522 (M+Na).sup.+,
C.sub.22H.sub.23NNaO.sub.4 requires 388.1525. The product later
crystallised on standing to give a pale brown solid, m.p.
109-111.degree. C.
7.15 Pentapeptide Transformations
7.15.1 Linear: Fmoc-Hag-Ala-Trp-Arg-Hag-NH.sub.2 94
##STR00164##
[0480] The procedure described in Section 73.2.2 was used for the
attachment of the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide
resin. Quantities of the resin and coupling reagents HATU and NMM
are presented in Table 7.1. The first coupling reaction was shaken
for 14 h.
TABLE-US-00002 TABLE 7.1 Quantities of Reagents used in the
Synthesis of Peptide 94 Reagent Mass (mg) or Volume (.mu.l) Mole
(mmol) Rink Amide Resin 155 mg 0.11 Fmoc-L-Hag-OH 110 mg 0.33 HATU
83.0 mg 0.22 NMM 71.8 .mu.l 0.65
[0481] The procedure outlined in Section 7.3.2.2 was also utilised
for subsequent coupling reactions in the synthesis of the
pentapeptide 94. Quantities of the coupling agents HATU and NMM
remained constant throughout the synthesis. The quantities of
successive amino acids and their reaction durations are detailed in
Table 7.2.
TABLE-US-00003 TABLE 7.1 Quantities of Amino Acids used in the
Synthesis of Peptide 94 Amino Acid Mass (mg) Mote (mmol) Reaction
Time (h)* Fmoc-L-Arg(Pbf)-OH 211 0.33 5 Fmoc-L-Trp(Boc)-OH 171 0.32
3 Fmoc-L-Ala-OH 102 0.33 4.5 Fmoc-L-Hag-OH 110 0.33 20 *Note:
Reaction times have not been optimised.
[0482] After the final amino acid coupling, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the pentapeptide 94. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 847.1 (M+H).sup.+,
C.sub.45H.sub.55N.sub.10O.sub.7 requires 847.4.
7.15.2 Linear: Fmoc-Crt-Ala-Trp-Arg-Crt-NH.sub.2 99
##STR00165##
[0484] The procedure described in Section 7.3.2.2 was used for the
attachment of the first amino acid, Fmoc-L-Crt-OH 100, to Rink
amide resin. Quantities of the resin and coupling reagents HATU and
NMM are presented in Table 7.3. The first coupling reaction was
shaken for 3 h.
TABLE-US-00004 TABLE 7.3 Quantities of Reagents used in the
Synthesis of Peptide 99 Reagent Mass (mg) or Volume (.mu.l) Mole
(mmol) Rink Amide Resin 110 mg 0.08 Fmoc-L-Crt-OH 81.5 mg 0.23 HATU
58.6 mg 0.15 NMM 51.0 .mu.l 0.46
[0485] The procedure outlined in Section 7.3.2.2 was also utilised
for subsequent coupling reactions in the synthesis of the
pentapeptide 99. Quantities of the coupling agents HATU and NMM
remained constant throughout the synthesis. The quantities of
successive amino acids and their reaction durations are detailed in
Table 7.4.
TABLE-US-00005 TABLE 7.4 Quantities of Amino Acids used in the
Synthesis of Peptide 99 Amino Acid Mass (mg) Mole (mmol) Reaction
Time (h)* Fmoc-L-Arg(Pbf)-OH 150 0.23 20 Fmoc-L-Trp(Boc)-OH 122
0.23 4 Fmoc-L-Ala-OH 72.0 0.23 2 Fmoc-L-Crt-OH 81.5 0.23 12 *Note:
Reaction times have not been optimised.
[0486] After the final amino acid coupling, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the pentapeptide 99. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 875.2 (M+H).sup.+,
C.sub.47H.sub.59N.sub.10O.sub.7 requires 875.4.
7.15.3 Linear: Fmoc-Hag-Pro-Trp-Arg-Hag-NH.sub.2 97
##STR00166##
[0488] The procedure described in Section 7.3.2.2 was used for the
attachment of the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide
resin. Quantities of the resin and coupling reagents HATU and NMM
are presented in Table 7.5. The first coupling reaction was shaken
for 14 h.
TABLE-US-00006 TABLE 7.5 Quantities of Reagents used in the
Synthesis of Peptide 97 Reagent Mass (mg) or Volume (.mu.l) Mole
(mmol) Rink Amide Resin 154 mg 0.11 Fmoc-L-Hag-OH 109 mg 0.32 HATU
82 mg 0.22 NMM 71.4 .mu.l 0.65
[0489] The procedure outlined in Section 7.3.2.2 was also utilised
for subsequent coupling reactions in the synthesis of the
pentapeptide 97. Quantities of the coupling agents HATU and NMM
remained constant throughout the synthesis. The quantities of
successive amino acids and their reaction durations are detailed in
Table 7.6
TABLE-US-00007 TABLE 0.2 Quantities of Amino Acids used in the
Synthesis of Peptide 97 Amino Acid Mass (mg) Mole (mmol) Reaction
Time (h)* Fmoc-L-Arg(Pbf)-OH 210 0.32 5 Fmoc-L-Trp(Boc)-OH 170 0.32
3 Fmoc-L-Pro-OH 110 0.33 4.5 Fmoc-L-Hag-OH 109 0.32 22 *Note:
Reaction times have not been optimised.
[0490] After the final amino acid coupling, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the pentapeptide 97. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 873.2 (M+H).sup.+,
C.sub.47H.sub.57N.sub.10O.sub.7 requires 873.4; 895.1 (M+Na).sup.+,
C.sub.47H.sub.56N.sub.10NaO.sub.7 requires 895.4.
7.15.4 Unsaturated Cyclic: Fmoc-c[Hag-Ala-Trp-Arg-Hag]-NH.sub.2
95
##STR00167##
[0491] Method A:
[0492] The resin-bound peptide 94a was subjected to the
conventional RCM procedure (Section 7.5.2) under the following
conditions: Resin-peptide 94a (20.0 mg, 14.0 .mu.mol), DCM (3 mL),
LiCl/DMF (0.4 M, 0.3 mL), Grubbs' catalyst (2.3 mg, 2.8 .mu.mol, 20
mol %), 50.degree. C., 41 h. At the end of the reaction period, a
small aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue indicated recovery of the starting linear peptide
94. Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 847.2
(M+H).sup.+.sub.linear, C.sub.45H.sub.55N.sub.10O.sub.7.
Method B:
[0493] The resin-bound peptide 94a was subjected to the
conventional RCM procedure (Section 7.5.2) under the following
conditions: Resin-peptide 94a (37.0 mg, 25.9 .mu.mol), DCM (3 mL),
LiCl/DMF (0.4 M, 0.3 mL), 2.sup.nd generation Grubbs' catalyst (4.4
mg, 5.2 .mu.mol, 20 mol %), 50.degree. C., 41 h. At the end of the
reaction period, a small aliquot of peptidyl-resin was subjected to
the TFA-mediated cleavage procedure (Section 0). Mass spectral
analysis of the isolated residue confirmed the presence of both
cyclic 95 and linear 94 peptides. Mass spectrum (ESI.sup.+,
MeCN/H.sub.2O): m/z 819.2 (M+H).sup.+.sub.cyclic,
C.sub.43H.sub.51N.sub.10O.sub.7 requires 819.4; m/z 847.2
(M+H).sup.+.sub.linear, C.sub.45H.sub.55N.sub.10O.sub.7.
Method C:
[0494] The resin-bound peptide 99a was subjected to the
conventional RCM procedure (Section 7.5.2) under the following
conditions: Resin-peptide 99a (32.8 mg, 23.0 .mu.mol), DCM (5 mL),
LiCl/DMF (0.4 M, 0.5 mL), 2.sup.nd generation Grubbs' catalyst (4.0
mg, 4.7 .mu.mol, 20 mol %), 50.degree. C., 41 h, 100% conversion
into 95. At the end of the reaction period, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the cyclic peptide 95..sup..dagger. Mass
spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 819.2
(M+H).sup.+.sub.cyclic, C.sub.43H.sub.51N.sub.10O.sub.7 requires
819.4. .sup..dagger. RCM of the crotylglycine-containing peptide 99
leads to the same unsaturated carbocycle 95 resulting from
cyclisation of the allylglycine-containing sequence 94, i.e.
Fmoc-c[Hag-Ala-Trp-Arg-Hag]-OH is identical to
Fmoc-c[Crt-Ala-Trp-Arg-Crt]-OH.
7.15.5 Unsaturated Cyclic: Fmoc-c[Hag-Pro-Trp-Arg-Hag]-NH.sub.2
98
##STR00168##
[0495] Method A:
[0496] The resin-bound peptide 97a was subjected to the
conventional RCM procedure (Section 7.5.2) under the following
conditions: Resin-peptide 97a (26.4 mg, 18.5 DCM (5 mL), LiCl/DMF
(0.4 M, 0.5 mL), Grubbs' catalyst (6.1 mg, 7.4 .mu.mol, 20 mol %),
50.degree. C., 41 h. At the end of the reaction period, a small
aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue indicated recovery of the starting linear peptide
97. Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 873.2
(M+H).sup.+.sub.linear, C.sub.47H.sub.57N.sub.10O.sub.7.
Method B:
[0497] The resin-bound peptide 97a was subjected to the
conventional RCM procedure (Section 7.5.2) under the following
conditions: Resin-peptide 97a (36.0 mg, 25.2 .mu.mol), DCM (3 mL),
LiCl/DMF (0.4 M, 0.3 mL), 2.sup.nd generation Grubbs' catalyst (4.4
mg, 5.2 .mu.mol, 20 mol %), 50.degree. C., 41 h, 100% conversion
into 98. At the end of the reaction period, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the cyclic peptide 98. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 845.1 (M+H).sup.+,
C.sub.45H.sub.53N.sub.10O.sub.7 requires 845.4; 867.1
(M+Na).sup.+,C.sub.45H.sub.52N.sub.10NaO.sub.7 requires 867.4.
7.15.6 Linear: Fmoc-Hag-Pro-Pre-Arg-Hag-OH 102
##STR00169##
[0499] The procedure outlined in Section 7.3.2.1 was used for the
attachment of the first amino acid, Fmoc-Hag-OH 96, to Wang resin.
Quantities of the resin and coupling reagents are presented in
Table 7.7. The first coupling reaction was shaken for 14 h.
TABLE-US-00008 TABLE 7.7 Quantities of Reagents used in the
Synthesis of Peptide 102 Reagent Mass (mg) or Volume (.mu.l) Mole
(mmol) Wang Resin 212 mg 0.19 Fmoc-L-Hag-OH 195 mg 0.58 DIC 90.6
.mu.l 0.58 DMAP 7.1 mg 0.06
[0500] The procedure outlined in Section 7.3.2.1 was also utilised
for subsequent coupling reactions in the synthesis of the
pentapeptide 102. Quantities of the coupling reagents HATU and NMM
are tabulated (Table 7.8) and remained constant throughout the
synthesis. The quantities of successive amino acids and their
reaction durations are detailed in Table 7.9.
TABLE-US-00009 TABLE 7.8 Quantities of Coupling Reagents used in
the Synthesis of Peptide 102 Coupling Reagent Mass (mg) or Volume
(mL) Mole (mmol) HATU 147 mg 0.39 NMM 128 .mu.l 1.16
TABLE-US-00010 TABLE 7.9 Quantities of Amino Acids used in the
Synthesis of Peptide 102 Amino Acid Mass (mg) Mole (mmol) Reaction
Time (h) Fmoc-L-Arg(Pbf)-OH 376 0.58 2 Fmoc-L-Pre-OH 211 0.58 3
Fmoc-L-Pro-OH 196 0.58 6 Fmoc-L-Hag-OH 195 0.58 2 (1) *Note:
Reaction times have not been optimised.
[0501] After the final amino acid coupling, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the pentapeptide 102. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 813.6 (M+H).sup.+,
C.sub.43H.sub.57N.sub.8O.sub.8 requires 813.4; m/z 831.5
(M+H.sub.2O+H).sup.+.sub.103, C.sub.43H.sub.59N.sub.8O.sub.9
requires 831.4; m/z 927.6 (M+TFA+H).sup.+,
C.sub.45H.sub.58F.sub.3N.sub.8O.sub.10 requires 927.4.
[0502] The pentapeptide 102 was also synthesised on Wang resin (590
mg) with reduced loading (0.3 mmol g.sup.-1) using the procedured
described above. The relative quantities of the Fmoc-amino acids
and coupling agents remained constant throughout the synthesis:
Wang resin:DIC:DMAP:Fmoc-amino acid:HATU:NMM, 1:3:0.3:3:2:6
equiv.
7.15.7 Unsaturated Cyclic: Fmoc-c[Hag-Pro-Pre-Arg-Hag]-OH 104
##STR00170##
[0504] The resin-bound peptide 102a was subjected to the
conventional RCM procedure (Section 7.5.2) under the following
conditions: Resin-peptide 102a (70.0 mg, 63.7 .mu.mol), DCM (5 mL),
LiCl/DMF (0.4 M, 0.5 mL), 2.sup.nd generation Grubbs' catalyst
(21.6 mg, 25.4 .mu.mol, 40 mol %), 50.degree. C., 42 h, 100%
conversion into 104. At the end of the reaction period, a small
aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue confirmed formation of the cyclic peptide 104.
Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 785.4 (M+H).sup.+,
C.sub.41H.sub.53N.sub.8O.sub.8 requires 785.4; m/z 803.3
(M+H.sub.2O+H).sup.+, C.sub.41H.sub.55N.sub.8O.sub.9 requires
803.4; m/z 899.4 (M+TFA+H).sup.+,
C.sub.43H.sub.54F.sub.3N.sub.8O.sub.10 requires 899.4.
[0505] The resin-bound peptide 102a (synthesised on reduced loading
Wang resin) was subjected to the conventional RCM procedure
(Section 7.5.2) under the following conditions: Resin-peptide 102a
(97.0 mg, 29.1 .mu.mol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL),
2.sup.nd generation Grubbs' catalyst (2.5 mg, 2.9 .mu.mol, 10 mol
%), 50.degree. C., 42 h, 100% conversion into 104. Mass spectral
data of the isolated residue confirmed formation of the cyclic
peptide 104 and were in agreement with those reported above.
7.15.8 Saturated Cyclic: Fmoc-c [Hag-Pro-Pre-Arg-Hag]-OH 105
##STR00171##
[0507] The resin-bound peptide 104a was subjected to the general
Wilkinson's hydrogenation procedure (Section 7.4.4) under the
following conditions: Resin-peptide 104a (350 mg, 0.32 mmol),
DCM:MeOH (9:1, 8 mL), Wilkinson's catalyst, 80 psi H.sub.2,
22.degree. C., 22 h, 100% conversion into 105. At the end of the
reaction period, a small aliquot of peptidyl-resin was subjected to
the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral
analysis of the isolated residue confirmed formation of the
saturated cyclic pentapeptide 105. Mass spectrum (ESI.sup.+,
MeCN/H.sub.2O): m/z 787.2 (M+H).sup.+,
C.sub.41H.sub.55N.sub.8O.sub.8 requires 787.4; m/z 805.2
(M+H.sub.2O+H).sup.+, C.sub.41H.sub.57N.sub.8O.sub.9 requires
803.4; m/z 901.3 (M+TFA+H).sup.+,
C.sub.43H.sub.56F.sub.3N.sub.8O.sub.10 requires 901.4.
7.15.9 Olefin Activation: Saturated Cyclic:
Fmoc-c[Hag-Pro-Crt-Arg-Hag]-OH 106
##STR00172##
[0509] The resin-bound peptide 105a was subjected to the general
cross metathesis procedure (Section 7.5.4) with cis-2-butene under
the following conditions: Resin-peptide 105a (212 mg, 0.19 mmol),
ICM (8 mL), 2.sup.nd generation Grubbs' catalyst (82 mg, 9.7
.mu.mol, 50 mol %), cis-2-butene (15 psi), 50.degree. C., 42 h. At
the end of the reaction period, a small aliquot of peptidyl-resin
was subjected to the TFA-mediated cleavage procedure (Section
7.3.3). Mass spectral analysis of the isolated residue indicated
the presence of the starting peptide 105 and the desired
butenolysis product 106. The recovered resin-peptide was subjected
to the same butenolysis conditions in order to drive the reaction
to completion. After 42 h, a small aliquot of peptidyl-resin was
subjected to the TFA-mediated cleavage procedure (Section 7.3.3).
Mass spectral analysis of the isolated residue confirmed
quantitative conversion to the activated peptide 106. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 773.2 (M+H).sup.+,
C.sub.40H.sub.53N.sub.8O.sub.8 requires 773.4.
7.15.10 Cross Metathesis of Activated Olefin: Saturated
CyclicFmoc-c[Hag-Pro-Sub-Arg-Hag]-OH 107
##STR00173##
[0511] The resin-bound peptide 106a was subjected to the general
microwave-accelerated cross metathesis procedure (Section 7.5.3)
under the following conditions: Resin-peptide 106a (20.0 mg, 18.0
.mu.mol), DCM (4 mL), LiCl/DMF (0.4 M, 0.4 mL), 2.sup.nd generation
Grubbs' catalyst (6.2 mg, 7.3 .mu.mol, 40 mol %), (2S)-methyl
2-N-acetylaminohex-4-enoate 81 (70.0 mg, 0.38 mmol), 100.degree.
C., 2 h. At the end of the reaction period, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the cross metathesis product 107. Mass
spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 902.4 (M+H).sup.+,
C.sub.45H.sub.60N.sub.9O.sub.11 requires 902.4.
7.15.11 Wilkinson's Hydrogenation of Saturated Cyclic 107:
Fmoc-c[Hag-Pro-sat(Sub)-Arg-Hag]-OH 108
##STR00174##
[0513] The resin-bound peptide 107a was subjected to the general
Wilkinson's hydrogenation procedure (Section 7.4.4) under the
following conditions: Resin-peptide 107a (15.0 mg, 13.5 .mu.mol),
DCM:MeOH (9:1, 5 mL), Wilkinson's catalyst, 80 psi H.sub.2,
22.degree. C., 22 h. At the end of the reaction period, a small
aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue confirmed formation of the reduced cyclic
pentapeptide 108. Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z
904.4 (M+H).sup.+, C.sub.45H.sub.62N.sub.9O.sub.11 requires
904.5.
7.15.12 Olefin Activation: Synthesis of
Fmoc-Gly(CH.sub.2CH.dbd.CHCH.sub.2OAc)-Phe-OH 145
##STR00175##
[0515] The resin-bound peptide Fmoc-Pre-Phe-Wang 144 was subjected
to the microwave-assisted cross metathesis procedure (Section
7.5.3) with cis-1,4-diacetoxy-2-butene 141 under the following
conditions: Resin (Wang)-peptide 144 (180 mg, 0.09 mmol), DCM (10
mL), 2.sup.nd generation Grubbs' catalyst (16 mg, 20 mol %),
cis-1,4-diacetoxy-2-butene (96 mg, 0.56 mmol, 15 psi), 100.degree.
C., 1 h. At the end of the reaction period, the peptidyl-resin was
subjected to the TFA-mediated cleavage procedure (Section 7.3.3).
Mass spectral analysis of the isolated residue indicated the
presence of the desired dipeptide product 145 and no starting
material. Mass spectral analysis of the isolated residue confirmed
quantitative conversion to the activated peptide 145. Mass spectrum
(ESI.sup.+, CH.sub.3OH): m/z 579.0 (M+Na)
C.sub.32H.sub.32N.sub.2O.sub.7Na.
7.16 [2,8]-Dicarba-[3,12]-Cystino Conotoxin Transformations
7.16.1 Linear [2,8]-Hag-[3,12]-Cys Conotoxin ImI:
Fmoc-Gly-Hag-Cys-Ser-Asp-Pro-Arg-Hag-Ala-Trp-Arg-Cys-NH.sub.2
112
##STR00176##
[0517] The procedure described in Section 7.3.2 was used for the
attachment of the first amino acid, Fmoc-L-Cys(Trt)-OH, to Rink
amide resin. Quantities of the resin and coupling reagents HATU and
NMM are presented in Table 7.10. The first coupling reaction was
shaken for 14 h.
TABLE-US-00011 TABLE 7.10 Quantities of Reagents used in the
Synthesis of Peptide 112 Reagent Mass (mg) or Volume (.mu.l) Mole
(mmol) Rink Amide Resin 740 mg 0.39 Fmoc-L-Cys(Trt)-OH 676 mg 1.15
HATU 293 mg 0.77 NMM 255 .mu.l 2.31
[0518] The procedure outlined in Section 7.3.2.2 was also utilised
for subsequent coupling reactions in the synthesis of the
dodecapeptide 112. Quantities of the coupling agents HATU and NMM
remained constant throughout the synthesis. The quantities of
successive amino acids and their reaction durations are detailed in
Table 7.11.
TABLE-US-00012 TABLE 7.11 Quantities of Amino Acids used in the
Synthesis of Peptide 112 Amino Acid Mass (mg) Mole (mmol) Reaction
Time (h)* Fmoc-L-Arg(Pbf)-OH 749 1.15 2.5 Fmoc-L-Trp(Boc)-OH 608
1.15 2.5 Fmoc-L-Ala-OH 360 1.16 14 Fmoc-L-Hag-OH 390 1.16 2.5
Fmoc-L-Arg(Pbf)-OH 750 1.16 2.5 Fmoc-L-Pro-OH 390 1.16 1
Fmoc-L-Asp(.sup.tBu)--OH 475 1.15 14 Fmoc-L-Ser(.sup.tBu)--OH 443
1.16 2.5 Fmoc-L-Cys(Trt)-OH 676 1.15 2.5 Fmoc-L-Hag-OH 390 1.16 2.5
Fmoc-L-Gly-OH 343 1.15 14 *Note: Reaction times have not been
optimised.
[0519] After the final amino acid coupling, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the dodecapeptide 112. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 783.5 [1/2(M+2H)].sup.+,
1/2(C.sub.71H.sub.98N.sub.20O.sub.17S.sub.2) requires 783.3; m/z
1565.7 (M+H).sup.+, C.sub.71H.sub.97N.sub.20O.sub.17S.sub.2
requires 1565.7. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic
acid): t.sub.R=8.86 min.
7.16.2 [2,8]-Dicarba-[3,12]-Cys Conotoxin ImI:
Fmoc-Gly-c[Hag-Cys-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Cys-NH.sub.2
114
##STR00177##
[0521] The resin-bound peptide was subjected to the general
microwave-accelerated RCM procedure (Section 7.5.3) under the
following conditions: Resin-peptide 112a (158 mg, 82.2 .mu.mol),
DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), 2.sup.nd generation Grubbs'
catalyst (7.0 mg, 8.2 .mu.mol, 10 mol %), 100.degree. C., 1 h, 100%
conversion into 114. At the end of the reaction period, a small
aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue confirmed formation of the cyclic peptide 114.
Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 769.4
[1/2(M+2H)].sup.+, 1/2(C.sub.69H.sub.94N.sub.20O.sub.17S.sub.2)
requires 769.3; m/z 1537.7 (M+H).sup.+,
C.sub.69H.sub.93N.sub.20O.sub.17S.sub.2 requires 1537.6. LC-MS
(Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): t.sub.R=8.59
min.
[0522] An analogous microwave-accelerated RCM reaction using 5 mol
% 2.sup.nd generation Grubbs' catalyst was performed: Resin-peptide
112a (80.2 mg, 42 .mu.mol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL),
2.sup.nd generation Grubbs' catalyst (7.0 mg, 2.1 .mu.mol, 5 mol
%), 100.degree. C., 2 h, 100% conversion into 114. Mass spectral
data were in agreement with those reported above.
7.16.3 [2,8]-Dicarba-[3,12]-Cystino Conotoxin ImI:
NH.sub.2-Gly-c[Hag-Cys-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Cys)-NH.sub.2
118
##STR00178##
[0524] The Rink-amide bound peptide 114a (100 mg, 52.0 .mu.mol) was
swollen with DCM (3.times.1 min, 1.times.30 min) and DMF (3.times.1
min, 1.times.30 min) and deprotected with 20% piperidine/DMF
(1.times.1 min, 2.times.20 min). The resin was then washed with DMF
(5.times.1 min), DCM (3.times.1 min), MeOH (3.times.1 min) and
dried on the SPPS manifold for 1 h. The Fmoc-deprotected
peptidyl-resin (47.0 mg, 24.4 .mu.mol) was subjected to the
TFA-mediated cleavage procedure (Section 0). The residue was then
lyophilised for 18 h to give the fully deprotected carbocyclic
peptide 116 as a colourless solid (20.0 mg, 15.2 .mu.mol). Mass
spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 658.4 [1/2(M+2H)].sup.+,
1/2(C.sub.54H.sub.84N.sub.20O.sub.15S.sub.2) requires 658.3; m/z
1315.6 (M+H).sup.+, C.sub.54H.sub.83N.sub.20O.sub.15S.sub.2
requires 1315.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic
acid): t.sub.R=5.63 min
##STR00179##
[0525] A sample of lyophilised peptide (10.1 mg, 7.7 .mu.mol) was
dissolved in an aqueous solution of (NH.sub.4).sub.2CO.sub.3 (0.1
M, 80 mL) containing 5% DMSO (4 mL). The reaction was stirred at
room temperature and monitored by the Ellman's test (Section
7.3.4). After 3 d, the reaction mixture was lyophilised and mass
spectral analysis of the isolated residue confirmed formation of
the cystine-oxidised peptide 118. The peptide was purified by
RP-HPLC (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid) and the
unsaturated [2,8]-dicarba-[3,12]-cystino conotoxin hybrid 118 was
isolated as a colourless solid (1.8 mg, 5%) in >99% purity. Mass
spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 657.4 [1/2(M+2H)].sup.+,
1/2(C.sub.54H.sub.82N.sub.20O.sub.15S.sub.2) requires 657.3; m/z
668.3 [1/2(M+H+Na)].sup.+,
1/2(C54H.sub.81N.sub.20NaO.sub.15S.sub.2) requires 668.3; m/z
1313.5 (M+H).sup.+, C.sub.54H.sub.81N.sub.20O.sub.15S.sub.2
requires 1313.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic
acid): t.sub.R=5.50 min.
7.16.4 [2,8]-Saturated Dicarba-[3,12]-Cystino Conotoxin ImI:
NH.sub.2-Gly-c[Hag-Cys-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Cys-NH.sub.2
122
##STR00180##
[0527] The resin-bound peptide 114a was subjected to the general
Wilkinson's hydrogenation procedure (Section 7.4.4) under the
following conditions: Resin-peptide 114a (285 mg, 0.15 mmol),
DCM:MeOH (9:1, 5 mL), Wilkinson's catalyst, 80 psi H.sub.2,
22.degree. C., 22 h. At the end of the reaction period, a small
aliquot of peptidyl-resin was Fmoc-deprotected (20% piperidine/DMF,
1.times.1 min, 2.times.10 min) and washed with DMF (5.times.1 min),
DCM (5.times.1 min), MeOH (5.times.1 min) and dried on the SPPS
manifold for 1 h. The Fmoc-deprotected peptidyl-resin was then
subjected to the TFA-mediated cleavage procedure (Section 7.3.3).
Mass spectral analysis of the isolated residue indicated the
presence of a mixture of the cystine-oxidised 122 and reduced 120
form of the saturated product. Mass spectrum (ESI.sup.+,
MeCN/H.sub.2O): m/z 658.6 [1/2(M+2H)].sup.+.sub.oxidised,
1/2(C.sub.54H.sub.84N.sub.20O.sub.15S.sub.2) requires 658.3; m/z
1315.7 (M+H).sup.+.sub.oxidised,
C.sub.54H.sub.83N.sub.20O.sub.15S.sub.2 requires 1315.6; m/z 659.4
[1/2(M+2H)].sup.+.sub.reduced,
1/2(C.sub.54H.sub.86N.sub.20O.sub.15S.sub.2) requires 659.3; m/z
1317.8 (M+H).sup.+.sub.reduced,
C.sub.54H.sub.85N.sub.20O.sub.15S.sub.2 requires 1317.6. LC-MS
(Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): t.sub.R
(122)=6.01 min.
7.17 [3,12]-Dicarba-[2,8]-Cystino Conotoxin Transformations
7.17.1 Linear [2,8]-Cys-[3,12]-Hag Conotoxin ImI:
Fmoc-Gly-Cys-Hag-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Hag-NH.sub.2
113
##STR00181##
[0529] The procedure described in Section 7.3.2.2 was used for the
attachment of the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide
resin. Quantities of the resin and coupling reagents HATU and NMM
are presented in Table 7.12. The first coupling reaction was shaken
for 14 h.
TABLE-US-00013 TABLE 7.12 Quantities of Reagents used in the
Synthesis of Peptide 113 Reagent Mass (mg) or Volume (.mu.l) Mole
(mmol) Rink Amide Resin 730 mg 0.38 Fmoc-L-Hag-OH 384 mg 1.14 HATU
289 mg 0.76 NMM 250 .mu.l 2.27
[0530] The procedure outlined in Section 7.3.2.2 was also utilised
for subsequent coupling reactions in the synthesis of the
dodecapeptide 113. Quantities of the coupling agents HATU and NMM
remained constant throughout the synthesis. The quantities of
successive amino acids and their reaction durations are detailed in
Table 7.13.
TABLE-US-00014 TABLE 7.13 Quantities of Amino Acids used in the
Synthesis of Peptide 113 Amino Acid Mass (mg) mole (mmol) Reaction
Time (h)* Fmoc-L-Arg(Pbf)-OH 739 1.14 2.5 Fmoc-L-Trp(Boc)-OH 600
1.14 2.5 Fmoc-L-Ala-OH 355 1.14 14 Fmoc-L-Cys(Trt)-OH 667 1.14 2.5
Fmoc-L-Arg(Pbf)-OH 740 1.14 2.5 Fmoc-L-Pro-OH 385 1.14 1
Fmoc-L-Asp(.sup.tBu)--OH 470 1.14 14 Fmoc-L-Ser(.sup.tBu)--OH 437
1.14 2.5 Fmoc-L-Hag-OH 385 1.14 2.5 Fmoc-L-Cys(Trt)-OH 667 1.14 2.5
Fmoc-L-Gly-OH 340 1.14 14 *Note: Reaction times have not been
optimised.
[0531] After the final amino acid coupling, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the dodecapeptide 113. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 783.5 [1/2(M+2H)].sup.+,
1/2(C.sub.71H.sub.98N.sub.20O.sub.17S.sub.2) requires 783.3; m/z
1565.7 (M+H).sup.+, C.sub.71H.sub.97N.sub.20O.sub.17S.sub.2
requires 1565.7. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic
acid): t.sub.R=9.13 min.
7.17.2 [2,8]-Cys-[3,12]-Dicarba Conotoxin ImI:
Fmoc-Gly-Cys-c[Hag-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Hag]-NH.sub.2
115
##STR00182##
[0533] The resin-bound peptide 113a was subjected to the general
microwave-accelerated RCM procedure (Section 7.5.3) under the
following conditions: Resin-peptide 113a (840 mg, 0.44 mmol), DCM
(5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2.sup.nd generation Grubbs'
catalyst (74.3 mg, 87.5 .mu.mol, 20 mol %), 100.degree. C., 1 h,
100% conversion into 115. At the end of the reaction period, a
small aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue confirmed formation of the cyclic peptide 115.
Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 769.4
[1/2(M+2H)].sup.+, 1/2(C.sub.69H.sub.94N.sub.20O.sub.17S.sub.2)
requires 769.3; m/z 1537.7 (M+H).sup.+,
C.sub.69H.sub.93N.sub.20O.sub.17S.sub.2 requires 1537.6. LC-MS
(Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): t.sub.R=8.99
min.
7.17.3 [2,8]-Cystino-[3,12]-Dicarba Conotoxin ImI:
NH.sub.2-Gly-Cys-c[Hag-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Hag]-NH.sub.2
119
##STR00183##
[0535] The resin-bound peptide 115a (100 mg, 52.0 .mu.mol) was
swollen with DCM (3.times.1 min, 1.times.30 min) and DMF (3.times.1
min, 1.times.30 min) and deprotected with 20% piperidine/DMF
(1.times.1 min, 2.times.20 min). The resin was then washed with DMF
(5.times.1 min), DCM (3.times.1 min), MeOH (3.times.1 min) and
dried on the SPPS manifold for 1 h. The Fmoc-deprotected
peptidyl-resin (61.7 mg, 32.1 .mu.mol) was subjected to the
TFA-mediated cleavage procedure (Section 7.3.3). The residue was
then lyophilised for 18 h to give the fully deprotected carbocyclic
peptide 117 as a colourless solid (15.1 mg, 11.5 .mu.mol). Mass
spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 658.4 [1/2(M+2H)].sup.+,
1/2(C.sub.54H.sub.84N.sub.20O.sub.15S.sub.2) requires 658.3; m/z
669.4 [1/2(M+H+Na)].sup.+,
1/2(C.sub.54H.sub.84N.sub.20O.sub.15S.sub.2) requires 669.4; m/z
1315.6 (M+H).sup.+, C.sub.54H.sub.83N.sub.20O.sub.15S.sub.2
requires 1315.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic
acid): t.sub.R=6.62 min.
##STR00184##
[0536] A sample of lyophilised peptide (11.2 mg, 8.5 .mu.mol) was
dissolved in an aqueous solution of (NH.sub.4).sub.2CO.sub.3 (0.1
M, 80 mL) containing 5% DMSO (4 mL). The reaction was stirred at
room temperature and monitored by the Ellman's test. After 3 d, the
reaction mixture was lyophilised and mass spectral analysis of the
isolated residue confirmed formation of the cystine-oxidised
peptide 119. The peptide was purified by RP-HPLC (Luna C8
RP-column, 10-60% MeOH, 0.1% formic acid) and the unsaturated
[2,8]-cystino-[3,12]-dicarba conotoxin hybrid 119 was isolated as a
colourless solid (2.3 mg, 5%) in >99% purity. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 657.3 [1/2(M+2H)].sup.+,
1/2(C.sub.54H.sub.82N.sub.20O.sub.15S.sub.2) requires 657.3; m/z
668.3 [1/2(M+H+Na)].sup.+,
1/2(C.sub.54H.sub.81N.sub.20NaO.sub.15S.sub.2) requires 668.3; m/z
1313.6 (M+H).sup.+, C.sub.54H.sub.31N.sub.20O.sub.15S.sub.2
requires 1313.6. LC-MS (Luna C8 RP-column, 10-60% MeOH, 0.1% formic
acid): t.sub.R=4.46 min.
7.17.4 [2,8]-Cystino[3,12]-Saturated Dicarba Conotoxin ImI:
NH.sub.2-Gly-Cys-c[Hag-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Hag]-NH.sub.2
123
##STR00185##
[0538] The resin-bound peptide 119a was subjected to the general
Wilkinson's hydrogenation procedure (Section 7.4.4) under the
following conditions: Resin-peptide 119a (320 mg, 0.17 mmol),
DCM:MeOH (9:1, 5 mL), Wilkinson's catalyst, 80 psi H.sub.2,
22.degree. C., 22 h. At the end of the reaction period, a small
aliquot of peptidyl-resin was Fmoc-deprotected (20% piperidine/DMF,
1.times.1 min, 2.times.10 min) and washed with DMF (5.times.1 min),
DCM (5.times.1 min), MeOH (5.times.1 min) and dried on the SPPS
manifold for 1 h. The Fmoc-deprotected peptidyl-resin was then
subjected to the TFA-mediated cleavage procedure (Section 7.3.3).
Mass spectral analysis of the isolated residue indicated the
presence of a mixture of the cystine-oxidised 123 and reduced 121
form of the saturated product. Mass spectrum (ESI.sup.+,
MeCN/H.sub.2O): m/z 658.5 [1/2(M+.sup.2H)].sup.+.sub.oxidised,
1/2(C.sub.54H.sub.84N.sub.20O.sub.15S.sub.2) requires 658.3; m/z
1315.7 (M+H).sup.+.sub.oxidised,
C.sub.54H.sub.83N.sub.20O.sub.15S.sub.2 requires 1315.6; m/z 659.3
[1/2(M+2H)].sup.+.sub.reduced,
1/2(C.sub.54H.sub.86N.sub.20O.sub.15S.sub.2) requires 659.3; m/z
1317.6 (M+H).sup.+.sub.reduced,
C.sub.54H.sub.85N.sub.20O.sub.15S.sub.2 requires 1317.6. LC-MS
(Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): t.sub.R
(123)=7.02 min.
7.17.5 Linear [2,8]-Hag-[3,16]-Cystino Conotoxin Vc1.1 (ACV1)
[0539]
Gly-Hag-Cys-Ser-Asp-Pro-Arg-Hag-Asn-Tyr-Asp-His-Pro-Glu-Ile-Cys-NH-
.sub.2
[0540] The procedure described in Section 7.3.5 was used for the
synthesis of Vc1.1 on Rink Amide resin (loading 0.52 mmol/g).
Quantities of the resin, coupling reagents and amino acids are
tabulated below:
TABLE-US-00015 Mole Quantity (mmol)/ Compound (mL/g) Volume Conc
(M) Cycle Name Rink Amide 0.481 g 5 mL DMF 0.25 mmol -- DIPEA 7.7
mL 22 mL NMP 2M -- HBTU 6.827 g 36 mL DMF 0.45M -- HOBt 2.432 g
Fmoc-Arg- 0.389 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Asn-
0.358 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Asp- 0.494 6 mL DMF
0.2M B0.25-Single (ext.) OH Fmoc-Cys- 0.703 6 mL DMF 0.2M
B0.25-Single (ext.) OH Fmoc-Glu- 0.255 3 mL DMF 0.2M B0.25-Single
(ext.) OH Fmoc-Gly- 0.178 3 mL DMF 0.2M B0.25-Single (ext.) OH
Fmoc-His- 0.372 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Ile-
0.212 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Pro- 0.405 6 mL DMF
0.2M B0.25-Single (ext.) OH Fmoc-Ser- 0.230 3 mL DMF 0.2M
B0.25-Single (ext.) OH Fmoc-Hag- 0.405 6 mL DMF 0.2M B0.25-Single
(ext.) OH Fmoc-Tyr- 0.276 3 mL DMF 0.2M B0.25-Single (ext.) OH
[0541] Resin washings and deprotection cycles were performed as
described in Section 7.3.5. The amino acid, activator and activator
base solutions were added to the resin, followed by the "B.01
Extended Coupling" cycle. The peptidyl-resin was exposed to a
temperature of 75.degree. C. with no power (0 watts) for 2 min,
then at a temperature of 75.degree. C., power at 25 watts for 10
min. The peptidyl-resin was then washed with DMF (3.times.10
mL).
[0542] Following the final amino acid coupling, a small aliquot of
the resin bound peptide was cleaved as described in Section 7.3.3
for mass spec analysis. Mass spectrum (ESI.sup.+, MeOH/H.sub.2O):
m/z 592.8 (M+3H/3), m/z 1011.1 (M+2H/2), m/z 1039.5
((M+tBu)+3H/3).
7.17.6 [2,8]-Unsaturated-[3,16]-Cystino Conotoxin Vc1.1 (ACV1)
##STR00186##
[0544] The resin bound linear peptide was subjected to microwave
RCM procedure outlined in Section 7.5.3. Peptidyl-resin (0.4810 mg,
0.25 mmol) and 2.sup.nd generation Grubb's catalyst (42.4 mg, 0.05
mmol) was weighted into a glass vial loaded with stirrer bar. In a
drybox, DCM (5 mL) and LiCl/DMF (0.2 mL) were added and the vial
was sealed. The reaction vessel was placed in the microwave for 1
hr at 100 C. A small aliquot of the resin bound peptide was
subjected to TFA cleavage and analysed by mass spectroscopy. Mass
spectrum (ESI.sup.+, MeOH/H.sub.2O): m/z 997.2 (M+2H/2), m/z 1011.0
(SM+2H/2). The same procedure was followed for ring closure of
linear .alpha.-RgIA.
7.17.7 Linear [2,8]-Hag-[3,12]-Cystino Conotoxin .alpha.-RgIA from
Conus regius
[0545] Gly-Hag-Cys-Ser-Asp-Pro-Arg-Hag-Arg-Tyr-Arg-Cys-Arg-N
H.sub.2
[0546] The procedure described in Section 7.3.5 was used for the
synthesis of RgIA on Rink Amide resin (loading 0.52 mmol/g).
Quantities of the resin, coupling reagents and amino acids are
tabulated in the Table below:
TABLE-US-00016 Mole Quantity (mmol)/ Compound (mL/g) Volume Conc
(M) Cycle Name Rink Amide 0.192 g 5 mL DMF 0.10 mmol -- DIPEA 3.8
mL 11 mL 2M -- NMP HBTU 2.655 g 18 mL 0.45M -- HOBt 0.946 g DMF
Fmoc-Arg- 1.427 g 11 mL 0.2M B0.25-Single (ext.) OH DMF Fmoc-Asp-
0.247 g 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Cys- 0.703 g 6 mL
DMF 0.2M B0.25-Single (cys/ OH his ext.) Fmoc-Gly- 0.178 g 3 mL DMF
0.2M B0.25-Single (ext.) OH Fmoc-Pro- 0.202 g 3 mL DMF 0.2M
B0.25-Single (ext.) OH Fmoc-Ser- 0.230 g 3 mL DMF 0.2M B0.25-Single
(ext.) OH Fmoc-Hag- 0.405 g 6 mL DMF 0.2M B0.25-Single (ext.) OH
Fmoc-Tyr- 0.276 g 3 mL DMF 0.2M B0.25-Single (ext.) OH
[0547] Resin washings and deprotection cycles were performed as
described in Section 7.3.5. The amino acid, activator and activator
base solutions were added to the resin, followed by the "B.01
Extended Coupling" cycle. The peptidyl-resin was exposed to a
temperature of 75.degree. C. with no power (0 watts) for 2 min,
then at a temperature of 75.degree. C., power at 25 watts for 10
min. The peptidyl-resin was then washed with DMF (3.times.10 mL).
However cysteine residues in RgIA have been known to be susceptible
to racemisation at 75.degree. C., therefore a different cycle was
used for the coupling of this amino acid. Following the
deprotection cycles, "B.01 Single Cys/His Extended" coupling cycle
was included in the method for the coupling of cysteine. This
involves exposure to a temperature of 50.degree. C. with no power
(0 watts) for 2 min, then at a temp of 50.degree. C., power at 25
watts for 10 min. The peptidyl resin was then washed with DMF
(3.times.10 mL).
[0548] Following the final amino acid coupling, a small aliquot of
the resin bound peptide was cleaved as described in Section 7.3.3
for mass spec analysis. Mass spectrum (ESI.sup.+, MeOH/H.sub.2O):
m/z 595.5 (M+3H/3), m/z 614.3 ((M+tBu)+2H/2), m/z 892.6
(M+2H/2).
7.18 [2,8][3,12]-Dicarba Conotoxin Transformations
7.18.1 Linear [2,8]-Hag-[3,12]-Pre Conotoxin ImI:
Fmoc-Gly-Hag-Pre-Ser-Asp-Pro-Arg-Hag-Ala-Trp-Arg-Pre-NH.sub.2
127
##STR00187##
[0550] The procedure described in Section 7.3.2.2 was used for the
attachment of the first amino acid, Fmoc-L-Pre-OH 92, to Rink amide
resin. Quantities of the resin and coupling reagents HATU and NMM
are presented in Table 7.14. The first coupling reaction was shaken
for 4 h.
TABLE-US-00017 TABLE 7.14 Quantities of Reagents used in the
Synthesis of Peptide 127 Regent Mass (mg) or Volume (.mu.l) Mole
(mmol) Rink Amide Resin 610 mg 0.32 Fmoc-L-Pre-OH 350 0.96 HATU 241
mg 0.63 NMM 210 .mu.l 1.91
[0551] The procedure outlined in Section 7.3.2.2 was also utilised
for subsequent coupling reactions in the synthesis of the
dodecapeptide 127. The quantities of successive amino acids and
their reaction durations are detailed in Table 7.15.
TABLE-US-00018 TABLE 7.15 Quantities of Amino Acids used in the
Synthesis of Peptide 127 Amino Acid Mass (mg) Mole (mmol) Reaction
Time (h)* Fmoc-L-Arg(Pbf)-OH 617 0.95 12 Fmoc-L-Trp(Boc)-OH 502
0.95 2.5 Fmoc-L-Ala-OH 297 0.95 2.5 Fmoc-L-Hag-OH 321 0.95 2.5
Fmoc-L-Arg(Pbf)-OH 617 0.95 14 Fmoc-L-Pro-OH 321 0.95 4
Fmoc-L-Asp(.sup.tBu)--OH 392 0.95 2.5 Fmoc-L-Ser(.sup.tBu)--OH 365
0.95 2.5 Fmoc-L-Pre-OH 350 0.96 12 Fmoc-L-Hag-OH 322 0.95 2.5
Fmoc-L-Gly-OH 285 0.96 2.5 (1) *Note: Reaction times have not been
optimised.
After the final amino acid coupling, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed fonnation of the dodecapeptide 127. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 805.6 [1/2(M+2H)].sup.+,
1/2(C.sub.79H.sub.110N.sub.20O.sub.17) requires 805.4; m/z 814.6
[1/2(M+H.sub.2O+2H)].sup.+, 1/2(C.sub.79H.sub.112N.sub.20O.sub.18)
requires 814.4.
7.18.2 [2,8]-Dicarba-[3,12]-Pre Conotoxin ImI:
Fmoc-Gly-c[Hag-Pre-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Pre-NH.sub.2
129
##STR00188##
[0552] Method A:
[0553] The Rink amide-bound peptide 127a was subjected to the
conventional RCM procedure (Section 7.5.2) under the following
conditions: Resin-peptide 127a (165 mg, 0.12 mmol), DCM (5 mL),
LiCl/DMF (0.4 M, 0.5 mL), 2.sup.nd generation Grubbs' catalyst
(39.9 mg, 47.0 .mu.mol, 40 mol %), 50.degree. C., 40 h, 100%
conversion into 129. At the end of the reaction period, a small
aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue confirmed formation of the cyclic peptide 129.
Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 791.4
[1/2(M+2H)].sup.+, 1/2(C.sub.77H.sub.106N.sub.20O.sub.17) requires
791.4. m/z 800.5 [1/2(M+H.sub.2O+2H)].sup.+,
1/2(C.sub.77H.sub.108N.sub.20O.sub.18) requires 800.4.
Method B:
[0554] The Rink amide-bound peptide 127a was subjected to the
general microwave-accelerated RCM procedure (Section 7.5.3) under
the following conditions: Resin-peptide 127a (127 mg, 66.0
.mu.mol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2.sup.nd generation
Grubbs' catalyst (5.6 mg, 6.6 .mu.mol, 10 mol %), 100.degree. C., 1
h, 100% conversion into 129. At the end of the reaction period, a
small aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue confirmed formation of the cyclic peptide 129.
Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 791.5
[1/2(M+2H)].sup.+, 1/2(C.sub.77H.sub.106N.sub.20O.sub.17) requires
791.4.
7.18.3 [2,8]-Saturated Dicarba-[3,12]-Pre Conotoxin ImI:
Fmoc-Gly-c[Hag-Pre-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Pre-NH.sub.2
133
##STR00189##
[0556] The resin-bound peptide 129a was subjected to the general
Wilkinson's hydrogenation procedure (Section 7.4.4) under the
following conditions: Resin-peptide 129a (130 mg, 91.0 .mu.mol),
DCM:MeOH (9:1, 6.5 mL), Wilkinson's catalyst, 80 psi H.sub.2,
22.degree. C., 24 h. At the end of the reaction period, a small
aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue confirmed formation of the selectively
hydrogenated cyclic dodecapeptide 133. Mass spectrum (ESI.sup.+,
MeCN/H.sub.2O): m/z 792.5 [1/2(M+2H)].sup.+,
%(C.sub.77H.sub.108N.sub.20O.sub.17) requires 792.4; m/z 801.4
[1/2(M+H.sub.2O+2H)].sup.+, 1/2(C.sub.77H.sub.110N.sub.20O.sub.18)
requires 801.4.
7.18.4 Olefin Activation: [2,8]-Saturated
Dicarba-[3,12]-Act.sup..dagger. Conotoxin ImI:
Fmoc-Gly-c[Hag-Act-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Act-NH.sub.2
[0557] .sup..dagger. Act=Activated sidechain, i.e, crotylglycine
(Crt) or allylglycine (Hag)
##STR00190##
[0558] The resin-bound peptide 133a was subjected to the general
cross metathesis procedure with cis-2-butene (Section 7.5.4) under
the following conditions:
Method A:
[0559] Resin-peptide 133a (76.0 mg, 55.5 .mu.mol), DCM (5 mL),
2.sup.nd generation Grubbs' catalyst (24.0 mg, 28.3 .mu.mol, 50 mol
%), cis-2-butene (15 psi), benzoquinone (6.2 mg, 57.4 mol),
50.degree. C., 38 h. At the end of the reaction period, a small
aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue indicated the presence of the desired butenolysis
product 135. No starting material was evident, however, low
intensity doubly charged higher homologue species separated by
m/z+7 units were observed.
[0560] Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 778.4
[1/2(M+2H)].sup.+.sub.product,
1/2(C.sub.75H.sub.104N.sub.20O.sub.17) requires 778.4. Low
intensity higher homologue species at m/z 785.4, 793.5, 800.4; Very
low intensity peaks at m/z 807.8, 814.3, 821.3, 828.1, 835.7,
842.6, 849.8, 856.1, 863.6.
Method B:
[0561] Resin-peptide 133a (20.0 mg, 10.4 .mu.mol), DCM (3 mL),
LiCl/DMF (0.3 mL), 2.sup.nd generation Grubbs' catalyst (1.0 mg,
1.2 .mu.mol, 10 mol %), cis-2-butene (20 psi), 50.degree. C., 62 h.
At the end of the reaction period, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
indicated the presence of the desired product 135, a partially
metathesised peptide (mono-crotylglycine containing peptide) 136
and the starting peptide 133. Mass spectrum (ESI.sup.+,
MeCN/H.sub.2O): m/z 778.4 [1/2(M+2H)].sup.+.sub.product,
1/2(C.sub.75H.sub.104N.sub.20O.sub.17) requires 778.4. m/z 785.5
[1/2(M+2H)].sup.+.sub.136, 1/2(C.sub.76H.sub.106N.sub.20O.sub.17);
m/z 792.4 [1/2(M+2H)].sup.+.sub.133,
1/2(C.sub.77H.sub.108N.sub.20O.sub.17); m/z 801.4
[1/2(M+H.sub.2O+2H)].sup.+.sub.133,
1/2(C.sub.77H.sub.110N.sub.20O.sub.18).
Method C:
[0562] Resin-peptide 133a (35.0 mg, 18 .mu.mol), DCM (5 mL),
LiCl/DMF (0.5 mL), 2.sup.nd generation Grubbs' catalyst (6.2 mg,
7.3 .mu.mol, 40 mol %), cis-2-butene (20 psi), 50.degree. C., 24 h.
At the end of the reaction period, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
indicated the presence of the desired product 135, a partially
metathesised peptide (mono-crotylglycine containing peptide) 136
and the starting peptide 133. Mass spectral data were consistent
with those reported above (Method B).
[0563] An analogous reaction was performed for 62 h under the
following conditions: Resin-peptide 133a (30.2 mg, 15.7 .mu.mol),
DCM (5 mL), LiCl/DMF (0.5 mL), 2.sup.nd generation Grubbs' catalyst
(6.2 mg, 7.3 .mu.mol, 40 mol %), cis-2-butene (20 psi), 50.degree.
C., 62 h. At the end of the reaction period, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
indicated the presence of the desired product 135, a partially
metathesised peptide 136 and the starting peptide 133. Mass
spectral data were consistent with those previously reported
(Method B).
[0564] The resin-bound peptide 133a was subjected to the general
cross metathesis procedure with ethylene (Section 7.5.4) under the
following conditions:
Method D:
[0565] Resin-peptide 133a (42.0 mg, 21.8 mop, DCM (5 mL), LiCl/DMF
(0.5 mL), 2.sup.nd generation Grubbs' catalyst (7.5 mg, 8.8
.mu.mol, 40 mol %), ethylene (60 psi), 50.degree. C., 62 h. At the
end of the reaction period, a small aliquot of peptidyl-resin was
subjected to the TFA-mediated cleavage procedure (Section 7.3.3).
Mass spectral analysis of the isolated residue indicated the
presence of the starting peptide 133 and a partially metathesised
peptide (mono-allylglycine containing peptide) 137. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 778.4 [1/2(M+2H)].sup.+.sub.137,
1/2(C.sub.75H.sub.104N.sub.20O.sub.17); m/z 792.4
[1/2(M+2H)].sup.+.sub.133.
[0566] An analogous reaction was performed in the absence of the
chaotropic salt (LiCl) under the following conditions:
Resin-peptide 133a (68.0 mg, 35.4 .mu.mol), DCM (5 mL), 2.sup.nd
generation Grubbs' catalyst (12.0 mg, 14.1 .mu.mol, 40 mol %),
ethylene (60 psi), 50.degree. C., 62 h. At the end of the reaction
period, a small aliquot of peptidyl-resin was subjected to the
TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral
analysis of the isolated residue indicated the presence of the
starting peptide 133 and a partially metathesised peptide
(mono-allylglycine containing peptide) 137. Mass spectral data were
consistent with those reported above.
7.18.5 [2,8]-Saturated Dicarba-[3,12]-Dicarba Conotoxin ImI:
Fmoc-Gly-c[Hag-c(Crt-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Crt)-NH.sub.2
140
##STR00191##
[0568] The Rink amide-bound peptide 135a was subjected to the
general microwave-accelerated RCM procedure (Section 7.5.3) under
the following conditions: Resin-peptide 135a (20.0 mg, 14.0
.mu.mol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2.sup.nd generation
Grubbs' catalyst (2.4 mg, 2.8 .mu.mol, 20 mol %), 100.degree. C., 1
h. At the end of the reaction period, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). LC-MS analysis of the isolated residue supported
formation of the bicyclic peptide 140. LC-MS (Luna C8 RP-column,
10-60% MeOH, 0.1% formic acid): t.sub.R=9.18 min, m/z 750.4
[1/2(M+2H)].sup.+, 1/2(C.sub.71H.sub.94N.sub.20O.sub.17) requires
750.4.
7.18.6 Attempted Synthesis of [2,8]-[3,12]-Saturated Bis-Dicarba
Conotoxin ImI:
NH.sub.2-c[Hag-c(Crt-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Crt)-NH.sub.2
126
##STR00192##
[0570] The resin-bound peptide 140a was subjected to the general
Wilkinson's hydrogenation procedure (Section 7.4.4) under the
following conditions: Resin-peptide 140a (12.2 mg, 8.5 .mu.mol),
DCM:MeOH (9:1, 6.5 mL), Wilkinson's catalyst, 80 psi H.sub.2,
22.degree. C., 24 h. At the end of the reaction period, a small
aliquot of peptidyl-resin was Fmoc-deprotected with 20%
piperidine/DMF (1.times.1 min, 2.times.20 min). The resin was then
washed with DMF (5.times.1 min), DCM (3.times.1 min), MeOH
(3.times.1 min) and dried on the SPPS manifold for 1 h. The
Fmoc-deprotected peptidyl-resin was then subjected to the
TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral and
LC-MS data of the isolated residue were inconclusive. The mass
spectrum and LC-traces did not display peaks due to the fully
deprotected starting peptide 125 and the target saturated bicycle
126. Lack of material and time constraints did not allow us to
investigate this chemistry further.
7.19 [2,8]-[3,12]-Dicarba Conotoxin Transformations
7.19.1 Linear [2,8]-Pre-[3,12]-Hag Conotoxin ImI:
Fmoc-Gly-Pre-Hag-Ser-Asp-Pro-Arg-Pre-Ala-Trp-Arg-Hag-NH.sub.2
128
##STR00193##
[0572] The procedure described in Section 7.3.2.2 was used for the
attachment of the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide
resin. Quantities of the resin and coupling reagents HATU and NMM
are presented in Table 7.16. The first coupling reaction was shaken
for 12 h.
TABLE-US-00019 TABLE 7.16 Quantities of Reagents used in the
Synthesis of Peptide 128 Reagent Mass (mg) or Volume (.mu.l) Mole
(mmol) Rink Amide Resin 705 mg 0.37 Fmoc-L-Hag-OH 371 mg 1.10 HATU
280 mg 0.74 NMM 245 .mu.l 2.22
[0573] The procedure outlined in Section 7.3.2.2 was also utilised
for subsequent coupling reactions in the synthesis of the
dodecapeptide 128. The quantities of successive amino acids and
their reaction durations are detailed in Table 7.17.
TABLE-US-00020 TABLE 7.17 Quantities of Amino Acids used in the
Synthesis of Peptide 128 Amino Acid Mass (mg) Mole (mmol) Reaction
Time (h)* Fmoc-L-Arg(Pbf)-OH 715 1.10 2.5 Fmoc-L-Trp(Boc)--OH 580
1.10 2.5 Fmoc-L-Ala-OH 343 1.10 2.5 Fmoc-L-Pre-OH 402 1.10 12
Fmoc-L-Arg(Pbf)-OH 715 1.10 2.5 Fmoc-L-Pro-OH 371 1.10 2.5
Fmoc-L-Asp(.sup.tBu)--OH 453 1.10 2.5 Fmoc-L-Ser(.sup.tBu)--OH 422
1.10 12 Fmoc-L-Hag-OH 371 1.10 2.5 Fmoc-L-Pre-OH 402 1.10 2.5
Fmoc-L-Gly-OH 328 1.10 2 (12) *Note: Reaction times have not been
optimised.
[0574] After the final amino acid coupling, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectra analysis of the isolated residue
confirmed formation of the dodecapeptide 128. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 805.6 [1/2(M+2H)].sup.+,
1/2(C.sub.79H.sub.110N.sub.20O.sub.17) requires 805.4; m/z 816.6
[1/2(M+Na+H)].sup.+, 1/2(C.sub.79H.sub.111N.sub.20NaO.sub.18)
requires 816.4.
7.19.2 [2,8]-Pre-[3,12]-Dicarba Conotoxin
Fmoc-Gly-Pre-c[Hag-Ser-Asp-Pro-Arg-Pre-Ala-Trp-Arg-Hag]-NH.sub.2
132
##STR00194##
[0575] Method A:
[0576] The resin-bound peptide 128a was subjected to the
conventional RCM procedure (Section 7.5.2) under the following
conditions: Resin-peptide 128a (19.7 mg, 10.2 .mu.mol), DCM (3 mL),
LiCl/DMF (0.4 M, 0.3 mL), 2.sup.nd generation Grubbs' catalyst (3.5
mg, 4.1 .mu.mol, 40 mol %), 50.degree. C., 40 h. At the end of the
reaction period, a small aliquot of peptidyl-resin was subjected to
the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral
analysis of the isolated residue indicated recovery of the linear
peptide 132. Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 805.6
[1/2(M+2H)].sup.+.sub.linear,
1/2(C.sub.79H.sub.110N.sub.20O.sub.17).
Method B:
[0577] The resin-bound peptide 128a was subjected to the general
microwave-accelerated RCM procedure (Section 7.5.3) under the
following conditions: Resin-peptide 128a (550 mg, 0.29 mmol), DCM
(5 mL), LiCl/DMF (0.4 M, 0.5 mL), generation Grubbs' catalyst (49.0
mg, 57.7 .mu.mol, 20 mol %), 100.degree. C., 1 h, 100% conversion
into 132. At the end of the reaction period, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the cyclic peptide 132. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 791.4 [1/2(M+2H)].sup.+,
1/2(C.sub.77H.sub.106N.sub.20O.sub.17) requires 791.4.
7.19.3 [3,12]-Pre-[2,8]-Saturated Dicarba Conotoxin:
Fmoc-Gly-Pre-c[Hag-Ser-Asp-Pro-Arg-Pre-Ala-Trp-Arg-Hag]-NH.sub.2
134
##STR00195##
[0579] The resin-bound peptide 132a was subjected to the general
Wilkinson's hydrogenation procedure (Section 7.4.4) under the
following conditions: Resin-peptide 132a (365 mg, 0.19 mmol),
DCM:MeOH (9:1, 5 mL), Wilkinson's catalyst, 80 psi H.sub.2,
22.degree. C., 19 h. At the end of the reaction period, a small
aliquot of peptidyl-resin was subjected to the TFA-mediated
cleavage procedure (Section 7.3.3). Mass spectral analysis of the
isolated residue confirmed formation of the selectively
hydrogenated cyclic peptide 134. Mass spectrum (ESI.sup.+,
MeCN/H.sub.2O): m/z 792.5 [1/2(M+2H)].sup.+,
1/2(C.sub.77H.sub.108N.sub.20O.sub.17) requires 792.4; m/z 801.4
[1/2(M+H.sub.2O+2H)].sup.+, 1/2(C.sub.77H.sub.110N.sub.20O.sub.18)
requires 801.4.
7.19.4 Attempted Synthesis of
Fmoc-Gly-Crt-c[Hag-Ser-Asp-Pro-Arg-Crt-Ala-Trp-Arg-Hag]-NH.sub.2
138
##STR00196##
[0581] The resin-bound peptide 134a was subjected to the
conventional cross metathesis procedure with cis-2-butene (Section
7.5.4) under the following conditions:
Method A:
[0582] Resin-peptide 134a (78.5 mg, 41 .mu.mol), DCM (5 mL),
2.sup.nd generation Grubbs' catalyst (13.9 mg, 16 .mu.mol, 40 mol
%), cis-2-butene (15 psi), 50.degree. C., 62 h. At the end of the
reaction period, a small aliquot of peptidyl-resin was subjected to
the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral
analysis of the isolated residue indicated the presence of a
mixture of peptides: the starting peptide 134, the desired product
138 and a partially metathesised peptide (mono-butenolysis product)
139. Mass spectrum (ESI.sup.+, MeCN/H.sub.2O): m/z 778.5
[1/2(M+2H)].sup.+.sub.product,
1/2(C.sub.75H.sub.104N.sub.20O.sub.17) requires 778.4; m/z 785.5
[1/2(M+2H)].sup.+.sub.139, 1/2(C.sub.76H.sub.106N.sub.20O.sub.17);
m/z 792.5 [1/2(M+2H)].sup.+.sub.134,
1/2(C.sub.77H.sub.108N.sub.20O.sub.17).
[0583] An analogous reaction in the presence of a chaotropic salt
(LiCl) was performed under the following conditions: Resin-peptide
134a (60.1 mg, 31 .mu.mol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL),
2.sup.nd generation Grubbs' catalyst (10.6 mg, 12 .mu.mol, 40 mol
%), cis-2-butene (15 psi), 50.degree. C., 62 h. At the end of the
reaction period, a small aliquot of peptidyl-resin was subjected to
the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectral
analysis of the isolated residue indicated the presence of a
mixture of peptides: the starting peptide 134a, the desired product
138 and a partially metathesised peptide 139. Mass spectral data
were consistent with those reported above.
7.19.5 Linear [2,8]-Pre-[3,12]-Hag Conotoxin ImI (Ala9.fwdarw.Pro9
Replacement):
Fmoc-Gly-Pre-Hag-Ser-Asp-Pro-Arg-Pre-Ala-Trp-Arg-Hag-NH.sub.2
130
##STR00197##
[0585] The procedure described in Section 7.3.2.2 was used for the
attachment of the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide
resin. Quantities of the resin and coupling reagents HATU and NMM
are presented in Table 7.18. The first coupling reaction was shaken
for 5 h.
TABLE-US-00021 TABLE 7.18 Quantities of Reagents used in the
Synthesis of Peptide 130 Reagent Mass (mg) or Volume (.mu.l) Mole
(mmol) Rink Amide Resin 400 mg 0.29 Fmoc-L-Hag-OH 295 mg 0.88 HATU
223 mg 0.59 NMM 193 .mu.l 1.75
[0586] The procedure outlined in Section 7.3.2.2 was also utilised
for subsequent coupling reactions in the synthesis of the
dodecapeptide 130. The quantities of successive amino acids and
their reaction durations are detailed in Table 7.19.
TABLE-US-00022 TABLE 7.19 Quantities of Amino Acids used in the
Synthesis of Peptide 130 Amino Acid Mass (mg) Mole (mmol) Reaction
Time (h)* Fmoc-L-Arg(Pbf)-OH 570 0.88 12 Fmoc-L-Trp(Boc)--OH 462
0.88 2 (1) Fmoc-L-Pro-OH 296 0.88 1 (12) Fmoc-L-Pre-OH 328 0.90 4
Fmoc-L-Arg(Pbf)-OH 570 0.88 2 (12) Fmoc-L-Pro-OH 296 0.88 2 (2)
Fmoc-L-Asp(.sup.tBu)--OH 361 0.88 2 (2) Fmoc-L-Ser(.sup.tBu)--OH
336 0.88 2 (12) Fmoc-L-Hag-OH 296 0.88 4 Fmoc-L-Pre-OH 330 0.90 1
(2) Fmoc-L-Gly-OH 262 0.88 2 (1) *Note: Reaction times have not
been optimised.
[0587] After the final amino acid coupling, a small aliquot of
peptidyl-resin was subjected to the TFA-mediated cleavage procedure
(Section 7.3.3). Mass spectral analysis of the isolated residue
confirmed formation of the dodecapeptide 130. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 818.6 [1/2(M+2H)].sup.+,
1/2(C.sub.81H.sub.112N.sub.20O.sub.17) requires 818.4; m/z 827.6
[1/2(M+H.sub.2O+2H)].sup.+, 1/2(C.sub.81H.sub.114N.sub.20O.sub.18)
requires 827.4.
7.19.6 [2,8]-Pre-[3,12]-Dicarba Conotoxin ImI (Ala9.fwdarw.Pro9
replacement):
Fmoc-Gly-Pre-c[Hag-Ser-Asp-Pro-Arg-Pre-Ala-Trp-Arg-Hag]-NH.sub.2
131
##STR00198##
[0589] The resin-bound peptide 130a was subjected to the
conventional RCM procedure (Section 7.5.2) under the following
conditions: Resin-peptide 130a (97.0 mg, 70.8 .mu.mol), DCM (5 mL),
LiCl/DMF (0.4 M, 0.5 mL), 2.sup.nd generation Grubbs' catalyst
(24.1 mg, 28.4 .mu.mol, 40 mol %), 50.degree. C., 40 h. At the end
of the reaction period, a small aliquot of peptidyl-resin was
subjected to the TFA-mediated cleavage procedure (Section 7.3.3).
Mass spectral analysis of the isolated residue confirmed the
presence of both cyclic 131 and linear 130 peptides. Mass spectrum
(ESI.sup.+, MeCN/H.sub.2O): m/z 804.5
[1/2(M+.sup.2H)].sup.+.sub.cyclic,
1/2(C.sub.79H.sub.108N.sub.20O.sub.17) requires 804.4; m/z 813.8
[1/2(M+H.sub.2O+2H)].sup.+.sub.cyclic,
1/2(C.sub.79H.sub.110N.sub.20O.sub.18) requires 813.4; m/z 818.7
[1/2(M+2H)].sup.+.sub.linear,
1/2(C.sub.81H.sub.112N.sub.20O.sub.17); m/z 827.3
[1/2(M+H.sub.2O+2H)].sup.+.sub.linear,
1/2(C.sub.81H.sub.114N.sub.20O.sub.18).
7.20 Activation Studies
7.20.1 6-Acetoxy-2-benzamido-4-hexenoic acid methyl ester 141
##STR00199##
[0591] Standard solution phase metathesis conditions (see section
7.5) were employed to synthesise
6-acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 141 from the
cross metathesis of the corresponding prenyl derivative 87 and
1,4-diacetoxy-cis-2-butene. The desired product was obtained as a
dark brown oil following by column chromatography (SiO.sub.2;
EtOAc:Hexane, 1:1).
[0592] N-Bzl-O-Me-prenylglycine (170 mg, 0.65 mmol),
dichloromethane (10 mL), second generation Grubbs' catalyst (16.5
mg, 5 mol %, 0.03 mmol), 1,4-diacetoxy-cis-2-butene (671.5 mg, 3.9
mmol), 50.degree. C., 20 h; 112.5 mg, 57%.
[0593] GC: t.sub.R (E/Z)=12.96, 13.06 min (GC column 30QC5/BPX5,
150.degree. C. for 1 min, 10.degree. C. min.sup.-1 to 280.degree.
C. for 6 min.)
[0594] IR (film): 3333s; 3056w; 3015w; 2944s; 1739s; 1662m; 1641s;
1605m; 1574m; 1533s; 1487m; 1436m; 1364m; 1236s; 1154w; 1072w;
1026m; 969m; 801w; 718m; 692w cm.sup.-1.
[0595] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 2.00, s, 3H,
CH.sub.3; 2.67, m, 2H, H3; 3.77, s, 3H, OCH.sub.3; 4.49, d, J 4.7
Hz, 2H, H6; 4.89, q, J 5.8 Hz, 1H, H2; 5.68, t, J 5.2 Hz, 2H, H4,
5; 6.75, d, J 7.4 Hz, 2H, H4, 5; 7.42, t, J 7.2 Hz, 2H, H4', 6';
7.50, t, J 6.4 Hz, 1H, H5'; 7.78, d, J 7.1 Hz, 2H, H3', 7'.
[0596] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta. 20.9, CH.sub.3;
35.2, C3; 52.1, OCH.sub.3; 52.7, C2; 64.5, C6; 127.2, C3', 7;
128.7, C4', 6'; 128.9, C5; 129.1, C4; 131.9, C5'; 133.9, CT; 167.1,
C1'; 170.8, C1''; 172.3, C1.
[0597] Mass Spectrum (ES CH.sub.3CN): m/z 328.1 (M+Na.sup.+)
C.sub.16H.sub.19NO.sub.5Na.
[0598] HRMS (EI, CH.sub.3OH). found m/z 305.1263,
C.sub.16H.sub.19NO.sub.5 requires 305.1263.
7.20.2 2,7-Bis-benzamido-oct-4-enedioic acid dimethyl ester 69
##STR00200##
[0600] 2,7-Bis-benzoylamino-4-octenedioic acid dimethyl ester was
synthesised using standard solution phase metathesis conditions
(refer to section 7.5). Due to the equilibrium generated in the
reaction, a mixture of the homodimer 69 and the starting material
141 was obtained.
[0601] 6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 141
(53.5 mg, 0.18 mmol), dichloromethane (10 mL), second generation
Grubbs' catalyst (5 mol %, 7.4 mg, 8.8 .mu.mol), 50.degree. C., 18
h.
[0602] GC: t.sub.R (1,4-diacetoxy-cis-2-butene)=3.28; (product
E/Z)=13.18, 13.31 min (GC column 30QC5/BPX5, 150.degree. C. for 1
min, 10.degree. C. min.sup.-1 to 280.degree. C. for 6 min.). The
mass spectrum was consistent with that previously described for
this compound.
7.20.3 2-Acetylamino-7-benzoylamino-4-octenedioic acid dimethyl
ester 142
##STR00201##
[0604] 2-Acetylamino-7-benzoylamino-4-octenedioic acid dimethyl
ester 142 was synthesised using standard solution-phase metathesis
conditions (refer to section 7.5) from
6-acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 141 and
methyl-2-acetylamino-4-pentenoate 121a. The desired compound was
obtained as a brown oil, and purified via column chromatography
(SiO.sub.2; EtOAc:Hexane; 2:1).
[0605] 6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 141
(50 mg, 0.16 mmol), dichloromethane (10 mL), second generation
Grubbs' catalyst (7 mg, 5 mol %, 8 .mu.mol),
methyl-2-acetylamino-4-pentenoate 142 (168 mg, 0.98 mmol),
50.degree. C., 18 h, 48.6 mg, 81%.
[0606] GC: t.sub.R (E/Z)=14.30, 14.50 min (GC column 30QC5/BPX5,
150.degree. C. for 1 min, 10.degree. C. min.sup.-1 to 280.degree.
C. for 6 min.)
[0607] .sup.1H NMR (500 MHz, CDCl.sub.3, mixture of isomers
(1:1.2)): .delta. 1.95, s (major isomer) and 1.97, s (minor
isomer), 3H, CH.sub.3; 2.42-2.70, m, 4H, H3, 6; 3.62, s (minor
isomer), 3.64, s (major isomer), 3.78, s (minor isomer) and 3.79, s
(major isomer), 6H, 2.times.OCH.sub.3; 4.63-4.66, m, 1H, H2;
4.85-4.91, m, 1H, H7; 5.35-5.49, m, 2H, H4, 5; 6.20, d, J 7.7 Hz
(major isomer) and 6.34, d, J 7.5 Hz, 1H, NH (minor isomer); 6.87,
t, J 7.55 Hz, 1H, NH; 7.44, t, J 7.1 Hz, 2H, H4', 6'; 7.50, t, J
6.9 Hz, 1H, H5'; 7.84, t, J 7.9 Hz, 2H, H3', 7'.
[0608] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 22.8, CH.sub.3;
34.8, 35.1, 35.4 and 35.7, C3, 6; 51.5 and 51.6, C2; 52.4, 52.5,
52.5, 52.6 and 52.7, C7, 2.times.OCH.sub.3; 127.2 and 127.2, C3',
7'; 128.6 and 128.6, C4', 6'; 128.9 and 129.0, C4, 5; 131.9 and
131.9, C5'; 133.7, C2; 167.1, COPh; 170.0 and 170.1, COMe; 172.2,
172.3, 172.3 and 172.4, 2.times.COOMe.
[0609] Mass Spectrum (ESI.sup.+, CH.sub.3OH): m/z 399.2
(M+Na.sup.+) C.sub.19H.sub.24N.sub.2O.sub.6Na.
7.20.4 Synthesis of Fmoc-Pre-Phe-OH on Wang Resin
##STR00202##
[0611] The dipeptide, Fmoc-Pre-Phe-OH, was synthesised on
pre-functionalised Fmoc-Phe-Wang resin (250 mg, 0.13 mmol)
according to standard SPPS techniques (see section 7.3.2).
Fmoc-prenylglycine (138 mg, 0.38 mmol) was coupled using HATU
(144.5 mg, 0.38 mmol) and NMM (83.6 mL, 0.76 mmol). An aliquot of
resin was subjected to cleavage condtions (see section 7.3.3) to
assess reaction success.
[0612] Mass Spectrum (ESI+, CH.sub.3OH): m/z 513.2 (M+H.sup.+), +),
C.sub.31H.sub.33N.sub.2O.sub.5; 535.1 (M+Na+),
C.sub.31H.sub.32N.sub.2O.sub.5Na.
7.20.5 Activation of Resin-Bound Prenylglycine
##STR00203##
[0614] The resin-tethered dipeptide was subjected to
microwave-assisted cross metathesis conditions (see section 7.3.2)
with 1,4-diacetoxy-cis-2-butene. An aliquot of resin was subjected
to cleavage condtions (see section 7.3.3) to assess reaction
success.
[0615] Resin-tethered dipeptide (180 mg, 0.09 mmol), second
generation Grubbs' catalyst (15.3 mg, 20 mol %, 0.018 mmol),
1,4-diacetoxy-cis-2-butene (97 mg, 0.56 mmol), dichloromethane (10
mL), 100.degree. C., 1 h, 100% conversion.
[0616] Mass Spectrum (ESI+, CH.sub.3OH): m/z 557.2 (M+H.sup.+), +),
C.sub.32H.sub.33N.sub.2O.sub.7; 579.2 (M+Na+),
C.sub.32H.sub.32N.sub.2O.sub.7Na.
7.21 Dicarba AOD Studies
7.21.1 Manual Synthesis of Linear AOD9604 146
[0617] The manual peptide synthesis procedure described in Section
7.3.2 was used for the synthesis of AOD9604 on Wang-Phe-Fmoc resin.
Quantities of the resin and coupling reagents HATU and NMM are
tabulated below. The quantities of successive amino acids are
summarized below:
TABLE-US-00023 Mole Compound Quantity MW or Loading (mmol)
Equivalents Wang-Phe-Fmoc 500 mg 0.52 mmol/g 0.25 1 Resin HATU 198
mg 380.23 0.52 2 NMM 172 .mu.L 101.15 1.56 6 Quantity Mole Reaction
Compound (mg) MW (mmol)/Eq. Time (hr) Fmoc-Gly-OH 232 297.14 0.78/3
2 Fmoc-Hag-OH 263 337.37 0.78/3 16 Fmoc-Gly-Ser(.psi.Pro)- 331
424.5 0.78/3 2 OH Fmoc-Glu-OH 332 425.5 0.78/3 2 Fmoc-Val-OH 265
339.22 0.78/3 2 Fmoc-Ser-OH 299 383.4 0.78/3 2 Fmoc-Arg-OH 534
684.4 0.78/3 16 Fmoc-Hag-OH 263 337.37 0.78/3 2 Fmoc-Gln-OH 476
610.7 0.78/3 2 Fmoc-Val-OH 265 339.22 0.78/3 2 Fmoc-Ile-OH 276
353.24 0.78/3 2 Fmoc-Arg-OH 534 684.8 0.78/3 16 Fmoc-Leu-OH 276
353.24 0.78/3 2 Fmoc-Tyr-OH 358 459.5 0.78/3 2
[0618] Following the final amino acid coupling, a small aliquot of
the resin bound peptide was cleaved as described in Section 7.3.3
for mass spec analysis. Mass spectrum (ESI.sup.+, MeOH/H.sub.2O):
m/z 676.5 (M+3H/3), m/z 1014.6 (M+2H/2).
7.21.2 Automated Synthesis of Linear AOD9604 146
[0619] The procedure described in Section 7.5.3 was used for the
synthesis of AOD9604 on Wang-Phe-Fmoc resin. Quantities of the
resin, coupling reagents and amino acids are tabulated below:
TABLE-US-00024 Mole Quantity (mmol)/ Compound (mL/g) Volume Conc
(M) Cycle Name Wang-Phe- 0.481 g 5 mL DMF 0.25 mmol -- Fmoc DIPEA
7.7 mL 22 mL NMP 2M -- HBTU 6.827 g 36 mL DMF 0.45M -- HOBt 2.432 g
Fmoc-Arg- 1.427 g 11 mL DMF 0.2M B0.25-Double OH (ext.) Fmoc-Hag-
1.289 g 11 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Gln- 0.737 g 6
mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Glu- 0.425 g 5 mL DMF 0.2M
B0.25-Single (ext.) OH Fmoc-Gly- 0.357 g 6 mL DMF 0.2M B0.25-Single
(ext.) OH Fmoc-Ile- 0.424 g 6 mL DMF 0.2M B0.25-Single (ext.) OH
Fmoc-Leu- 0.353 g 5 mL DMF 0.2M B0.25-Single (ext.) OH
Fmoc-.psi.Pro- 0.562 g 6 mL DMF 0.2M B0.25-Single (ext.) OH
Fmoc-Ser- 0.460 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Tyr-
0.551 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Val- 0.747 g 11
mL DMF 0.2M B0.25-Single (ext.) OH
[0620] Resin washings and deprotection cycles were performed as
described in Section 7.5.3. The amino acid, activator and activator
base solutions were added to the resin, followed by the "B.01
Extended Coupling" cycle. The peptidyl-resin was exposed to a
temperature of 75.degree. C. with no power (0 watts) for 2 min,
then at a temperature of 75.degree. C., power at 25 watts for 10
min. The peptidyl-resin was then washed with DMF (3.times.10 mL).
Most amino acids are programmed with the "B0.25-Single (Extended)"
coupling cycle, which concludes at this point. However arginine
requires and "B0.25-Double (Extended)" coupling cycle as several
AOD deletion products have been produced in the past. This involves
two "B0.1 Extended Coupling" cycle programs.
[0621] Following the final amino acid coupling, a small aliquot of
the resin bound peptide was cleaved as described in Section 7.3.3
for mass spec analysis. Mass spectrum (ESI.sup.+, MeOH/H.sub.2O):
m/z 689.1 (M+3H/3), m/z 1014.2 (M+2H/2).
7.21.3 Ring Closing Metathesis of Linear AOD9604 146
[0622] The resin bound peptide 146 was subjected to microwave RCM
procedure outlined in section 7.5.3. Peptidyl-resin (0.9088 mg,
0.475 mmol) and 2.sup.nd generation Grubb's catalyst (80 mg, 0.095
mmol) was weighted into a glass vial loaded with stirrer bar. In a
drybox, DCM (5 mL) and LiCl/DMF (0.2 mL) were added and the vial
was sealed. The reaction vessel was placed in the microwave for 1
hr at 100 C. A small aliquot of the resin bound peptide was
subjected to TFA cleavage and analysed by mass spec to show the
target unsaturated AOD 147. Mass spectrum (ESI.sup.+,
MeOH/H.sub.2O): m/z 1000.3 (SM+2H/2), m/z 1014.2 (M+2H/2). The
crude peptide was purified by reverse phase HPLC.
7.21.4 Hydrogenation of Unsaturated Cyclic AOD 147
[0623] A 150 mL glass hydrogenation vessel with plastic shield was
loaded with the peptidyl-resin (0.9168 mg, 0.476 mmol) and stirrer
bar. In an inert atmosphere, Wilkinson's catalyst (22 mg, 0.024
mmol) and 10 mL solvent (DCM:MeOH, 9:1) was added. The vessel was
sealed with a rubber 0 ring and fitted with a pressure regulator.
The vessel was purged with argon then hydrogen to a pressure of 90
psi and reacted at r.t. for 4 days. The reaction was terminated
upon exposure to oxygen and the resin was washed with DCM (5 mL,
3.times.1 min), DMF (5 mL, 3.times.1 min) then MeOH (5 mL,
3.times.1 min) and dried in vacuo for 30 min prior to cleavage and
mass spec analysis. Mass spec analysis showed conversion to the
saturated cyclic peptide 148 (ESI.sup.+, MeOH/H.sub.2O): m/z 1000.9
(M+2H/2), m/z 1015.6 (SM+2H/2). The crude peptide was purified by
reverse phase HPLC.
8.0 BIOLOGICAL TESTING
[0624] Bovine adrenal chromaffin cells can be used to test for the
activity of .alpha.-CTX ImI at neuronal-type nicotinic receptors.
These cells are of two types, adrenaline- and
noradrenaline-containing, and possess the neuronal-type nicotinic
receptor subtypes .alpha.3.beta.4 and .alpha.7. When stimulated
with nicotine, these cells release adrenaline and noradrenaline
which can be measured. Native .alpha.-CTX ImI peptides inhibit the
nicotine-stimulated release of these neurotransmitters by
interacting with the .alpha.3.beta.4-receptor subtype.
[0625] Dicarba-conotoxins 118 and 119 were assayed in quadruplicate
in multiwells (6.times.4) containing monolayer cultures of bovine
adrenal chromaffin cells as described by Broxton et al. (Loughnan,
M., Bond, T., Atkins, A., Cuevas, J., Adams, D. J., Broxton, N.M.,
Livett, B. G., Down, J. G., Jones, A., Alewood, P. F., Lewis, R. J.
J. Biol. Chem., 1998, 273 (25), 15667-15674.).sup.X The response of
the cells to these peptides was tested at two peptide
concentrations 1 uM and 5 uM. The cells were stimulated with
nicotine (4 uM) for 5 min at room temp (23C) and the amount of
catecholamine release (noradrenaline and adrenaline) was measured
over a 5 period and expressed as a % of the initial cellular
content of these amines. This was performed in the presence and
absence (control) of dicarba-conotoxin peptides. The chromaffin
cells also leak small amounts of catecholamine over the measurement
period, so a `basal release` (no nicotine added) measurement is
also recorded.
[0626] FIG. 4 shows catecholamine release from dicarba-conotoxins
118 and 119. Basal release was measured at 0.72% (and is subtracted
from other measurements). Nicotine stimulation alone released
(6.85-0.72)=6.13% of the noradrenaline in the cells, but only
(4.15-0.72)=3.43% in the presence of 5 uM of
[2,8]-cystino-[3,12]-dicarba conotoxin 119. This represents 55.97%
of the release produced by nicotine alone, or a 44% inhibition of
release (see Table). The % inhibition of release of adrenaline
(41%) was similar to that for noradrenaline (44%). This inhibition
was found to be concentration related: A 1 uM sample of
dicarba-conotoxin 119 produced only a 21.6% inhibition of
noradrenaline release and a 16.5% inhibition of release of
adrenaline.
[0627] Data for [2,8]-dicarba-[3,12]-cystino conotoxin 118 is also
shown in FIG. 4 and Table 8.1. This data shows that these
dicarba-analogues are biologically active and possess activity
profiles analogous to the native conotoxin sequences.
TABLE-US-00025 TABLE 8.1 Catecholamine release for
dicarba-conotoxins 118 and 119 1st 2nd 3rd 4th mean SEM n % control
% inhibition Noradrenaline Release BASE 0.68 0.61 0.66 0.93 0.72
0.07 4 NICOTINE (4 uM) 6.62 6.89 6.99 6.89 6.85 0.08 4 100.000 119
(1 uM) 4.89 5.75 5.73 5.73 5.52 0.21 4 78.400 21.600 119 (5 uM)
3.22 4.58 3.99 4.81 4.15 0.35 4 55.971 44.029 118 (1 uM) 4.52 6.29
7.00 7.34 6.29 0.63 4 90.859 9.141 118 (5 uM) 4.19 5.86 4.32 5.51
4.97 0.42 4 69.384 30.616 Adrenaline Release BASE 0.38 0.28 0.31
0.34 0.33 0.02 4 NICOTINE (4 uM) 4.31 4.52 4.52 4.48 4.46 0.05 4
100.000 119 (1 uM) 3.31 3.67 4.14 3.98 3.77 0.18 4 83.420 16.580
119 (5 uM) 2.32 2.95 2.76 3.02 2.76 0.16 4 58.888 41.112 118 (1 uM)
2.94 4.40 4.46 4.09 3.97 0.35 4 88.260 11.740 118 (5 uM) 2.55 3.63
2.62 3.31 3.03 0.26 4 65.306 34.694
9.0 STABILITY
9.1 Thiol Stability
[0628] Peptides samples (0.25 mM) were dissolved in a solution
containing either 0.25 mM reduced glutathione, 12.3 .mu.M reduced
thioredoxin (Promega, Madison, Wis.) or 0.5 mM human serum albumin
(Sigma, Madison, Wis.) in 100 mM phosphate buffer+1 mM EDTA, pH 7.4
(300 .mu.L) and incubated at 37 C. Thioredoxin was reduced by
treating the oxidised form with 0.9 equivalents of dithiothreitol
for 15 minutes immediately prior to use. Aliquots (30 .mu.L) were
taken at various time intervals, quenched with extraction buffer
consisting of 50% aqueous acetonitrile, 100 mM NaCl, and 1% TFA (30
.mu.L) and analysed by reverse-phase-HPLC. The ratio of the
degradation product to the tested peptide sample was determined by
measuring the peak height, and compared against the peak height
results for the HPLC of the corresponding natural or native
peptide. The product was considered to have improved stability if
the comparative HPLC test results showed less degradation product
after 6 hours of contact with one of the agents (reduced
glutathione, reduced thioredoxin or human serum albumin).
9.2 Human Blood Plasma Stability
[0629] Whole human blood containing 1% EDTA was centrifuged at
14,000 rpm for 30 minutes. The supernatant was then transferred to
an Eppendorf tube and centrifuged for an additional 30 min at
14,000 rpm. Peptide samples were dissolved in plasma (200 .mu.L) to
an initial peptide concentration of approximately 0.25 mM. Aliquots
(30 .mu.L) were removed at various time intervals and quenched with
extraction buffer (30 .mu.L). The aliquot was then vortexed,
diluted with additional water (60 .mu.L) and chilled in an ice bath
for 5 minutes prior to centrifuging at 14,000 rpm for 15 minutes.
The supernatant was then analysed by RP-HPLC. The stability of the
peptide sample was assessed by comparing the ratio of the peak
heights representing the tested peptide, and the degradation
products, against a sample of the corresponding natural or native
peptide not containing the dicarba bridge or bridges. The product
was considered to have improved stability in human blood plasma if
the comparative HPLC test results showed less degradation product
after 6 hours of contact.
[0630] It will be understood to persons skilled in the art of the
invention that many modifications may be made without departing
from the spirit and scope of the invention.
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TABLE-US-00026 [0899] A.1 The Amino Acids One Three letter letter
Amino acid code code Structure Alanine A Ala ##STR00204##
Allylglycine* -- Hag ##STR00205## Arginine R Arg ##STR00206##
Asparagine N Asn ##STR00207## Aspartic acid D Asp ##STR00208##
Crotylglycine* -- Crt ##STR00209## Cysteine C Cys ##STR00210##
5,5-Dimethylproline* -- dmP ##STR00211## Glutamic acid E Glu
##STR00212## Glutamine Q Gln ##STR00213## Glycine G Gly
##STR00214## Histidine H His ##STR00215## Isoleucine I Ile
##STR00216## Leucine L Leu ##STR00217## Lysine K Lys ##STR00218##
Methionine M Met ##STR00219## Phenylalanine F Phe ##STR00220##
Prenylglycine* -- Pre ##STR00221## Proline P Pro ##STR00222##
Serine S Ser ##STR00223## Threonine T Thr ##STR00224## Tryptophan W
Trp ##STR00225## Tyrosine Y Tyr ##STR00226## Valine V Val
##STR00227## *Synthetic amino acids.
Sequence CWU 1
1
5315PRTArtificial SequenceSynthetic Polypeptide 1Cys Ala Trp Arg
Cys 1 5 25PRTArtificial SequenceSynthetic Polypeptide 2Xaa Ala Trp
Arg Xaa 1 5 35PRTArtificial SequenceSynthetic Polypeptide 3Xaa Ala
Trp Arg Xaa 1 5 412PRTConus imperialisDISULFID(2)..(8)Cysteines in
positions 2 and 8 linked via disulfide bridge 4Gly Cys Cys Ser Asp
Pro Arg Cys Ala Trp Arg Cys 1 5 10 55PRTArtificial
SequenceSynthetic Polypeptide 5Xaa Pro Trp Arg Xaa 1 5
65PRTArtificial SequenceSynthetic Polypeptide 6Xaa Pro Trp Arg Xaa
1 5 75PRTArtificial SequenceSynthetic Polypeptide 7Xaa Ala Trp Arg
Xaa 1 5 85PRTArtificial SequenceSynthetic Polypeptide 8Xaa Ala Trp
Arg Xaa 1 5 915PRTHomo sapiensDISULFID(6)..(13)Linked via disulfide
bridge 9Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe
1 5 10 15 1016PRTHomo sapiensDISULFID(7)..(14)Linked via disulfide
bridge 10Tyr Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys
Gly Phe 1 5 10 15 115PRTArtificial SequenceSynthetic Polypeptide
11Xaa Pro Xaa Arg Xaa 1 5 125PRTArtificial SequenceSynthetic
Polypeptide 12Xaa Pro Xaa Arg Xaa 1 5 135PRTArtificial
SequenceSynthetic Polypeptide 13Xaa Pro Xaa Arg Xaa 1 5
145PRTArtificial SequenceSynthetic Polypeptide 14Xaa Pro Xaa Arg
Xaa 1 5 155PRTArtificial SequenceSynthetic Polypeptide 15Xaa Pro
Xaa Arg Xaa 1 5 1634PRTArtificial SequenceSynthetic Polypeptide
16Ile Xaa Ala Ile Xaa Leu Ala Xaa Pro Gly Ala Lys Xaa Gly Ala Leu 1
5 10 15 Met Gly Ala Asn Met Lys Xaa Ala Xaa Ala Asn Ala Ser Ile His
Val 20 25 30 Xaa Lys 176PRTArtificial SequenceSynthetic Polypeptide
17Xaa Ala Xaa Xaa Asn Xaa 1 5 186PRTArtificial SequenceSynthetic
Polypeptide 18Xaa Ala Xaa Xaa Asn Xaa 1 5 196PRTArtificial
SequenceSynthetic Polypeptide 19Xaa Ala Xaa Xaa Asn Xaa 1 5
206PRTArtificial SequenceSynthetic Polypeptide 20Xaa Ala Xaa Xaa
Asn Xaa 1 5 2112PRTConus imperialisDISULFID(2)..(8)Cysteines in
positions 2 and 8 linked via disulfide bridge 21Gly Cys Cys Ser Asp
Pro Arg Cys Ala Trp Arg Cys 1 5 10 2212PRTArtificial
SequenceSynthetic Polypeptide 22Gly Xaa Cys Ser Asp Pro Arg Xaa Ala
Trp Arg Cys 1 5 10 2312PRTArtificial SequenceSynthetic Polypeptide
23Gly Cys Xaa Ser Asp Pro Arg Cys Ala Trp Arg Xaa 1 5 10
2412PRTArtificial SequenceSytnthetic Polypeptide 24Gly Xaa Cys Ser
Asp Pro Arg Xaa Ala Trp Arg Cys 1 5 10 2512PRTArtificial
SequenceSynthetic Polypeptide 25Gly Cys Xaa Ser Asp Pro Arg Cys Ala
Trp Arg Xaa 1 5 10 2612PRTArtificial SequenceSynthetic Polypeptide
26Gly Xaa Xaa Ser Asp Pro Arg Xaa Ala Trp Arg Xaa 1 5 10
2712PRTArtificial SequenceSynthetic Polypeptide 27Gly Xaa Xaa Ser
Asp Pro Arg Xaa Ala Trp Arg Xaa 1 5 10 2812PRTArtificial
SequenceSynthetic Polypeptide 28Gly Xaa Xaa Ser Asp Pro Arg Xaa Ala
Trp Arg Xaa 1 5 10 2912PRTArtificial SequenceSynthetic Polypeptide
29Gly Xaa Cys Ser Asp Pro Arg Xaa Ala Trp Arg Cys 1 5 10
3012PRTArtificial SequenceSynthetic Polypeptide 30Gly Cys Xaa Ser
Asp Pro Arg Cys Ala Trp Arg Xaa 1 5 10 3112PRTArtificial
SequenceSynthetic Polypeptide 31Gly Xaa Cys Ser Asp Pro Arg Xaa Ala
Trp Arg Cys 1 5 10 3212PRTArtificial SequenceSynthetic Polypeptide
32Gly Cys Xaa Ser Asp Pro Arg Cys Ala Trp Arg Xaa 1 5 10
3312PRTArtificial SequenceSynthetic Polypeptide 33Gly Xaa Cys Ser
Asp Pro Arg Xaa Ala Trp Arg Cys 1 5 10 3412PRTArtificial
SequenceSynthetic Polypeptide 34Gly Cys Xaa Ser Asp Pro Arg Cys Ala
Trp Arg Xaa 1 5 10 3512PRTArtificial SequenceSynthetic Polypeptide
35Gly Xaa Xaa Ser Asp Pro Arg Xaa Ala Trp Arg Xaa 1 5 10
3612PRTArtificial SequenceSynthetic Polypeptide 36Gly Xaa Xaa Ser
Asp Pro Arg Xaa Ala Trp Arg Xaa 1 5 10 3712PRTArtificial
SequenceSynthetic Polypeptide 37Gly Xaa Xaa Ser Asp Pro Arg Xaa Ala
Trp Arg Xaa 1 5 10 3812PRTArtificial SequenceSynthetic Polypeptide
38Gly Xaa Xaa Ser Asp Pro Arg Xaa Pro Trp Arg Xaa 1 5 10
3912PRTArtificial SequenceSynthetic Polypeptide 39Gly Xaa Xaa Ser
Asp Pro Arg Xaa Pro Trp Arg Xaa 1 5 10 4012PRTArtificial
SequenceSynthetic Polypeptide 40Gly Xaa Xaa Ser Asp Pro Arg Xaa Ala
Trp Arg Xaa 1 5 10 4112PRTArtificial SequenceSynthetic Polypeptide
41Gly Xaa Xaa Ser Asp Pro Arg Xaa Ala Trp Arg Xaa 1 5 10
4212PRTArtificial SequenceSynthetic Polypeptide 42Gly Xaa Xaa Ser
Asp Pro Arg Xaa Ala Trp Arg Xaa 1 5 10 4312PRTArtificial
SequenceSynthetic Polypeptide 43Gly Xaa Xaa Ser Asp Pro Arg Xaa Ala
Trp Arg Xaa 1 5 10 4412PRTArtificial SequenceSynthetic Polypeptide
44Gly Xaa Xaa Ser Asp Pro Arg Xaa Ala Trp Arg Xaa 1 5 10
4512PRTArtificial SequenceSynthetic Polypeptide 45Gly Xaa Xaa Ser
Asp Pro Arg Xaa Ala Trp Arg Xaa 1 5 10 4612PRTArtificial
SequenceSynthetic Polypeptide 46Gly Xaa Xaa Ser Asp Pro Arg Xaa Ala
Trp Arg Xaa 1 5 10 4712PRTArtificial SequenceSynthetic Polypeptide
47Gly Xaa Cys Ser Asp Pro Arg Xaa Ala Trp Arg Cys 1 5 10
4812PRTArtificial SequenceSynthetic Polypeptide 48Gly Cys Xaa Xaa
Asp Pro Arg Cys Ala Trp Arg Xaa 1 5 10 4916PRTArtificial
SequenceSynthetic Polypeptide 49Gly Xaa Cys Ser Asp Pro Arg Xaa Asn
Tyr Asp His Pro Glu Ile Cys 1 5 10 15 5016PRTArtificial
SequenceSynthetic Polypeptide 50Gly Xaa Cys Ser Asp Pro Arg Xaa Asn
Tyr Asp His Pro Glu Ile Cys 1 5 10 15 5113PRTArtificial
SequenceSynthetic Polypeptide 51Gly Xaa Cys Ser Asp Pro Arg Xaa Arg
Tyr Arg Cys Arg 1 5 10 5212PRTArtificial SequenceSynthetic
Polypeptide 52Gly Xaa Xaa Ser Asp Pro Arg Xaa Ala Trp Arg Xaa 1 5
10 535PRTArtificial SequenceSynthetic Polypeptide 53Xaa Pro Xaa Arg
Xaa 1 5
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