U.S. patent application number 10/459071 was filed with the patent office on 2003-11-13 for peptides.
Invention is credited to Seebach, Dieter.
Application Number | 20030212250 10/459071 |
Document ID | / |
Family ID | 26309509 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030212250 |
Kind Code |
A1 |
Seebach, Dieter |
November 13, 2003 |
Peptides
Abstract
The invention provides novel .beta.-peptides comprising 2 or
more different .beta.-amino acid residues, preferably compounds of
formula I 1 wherein the the R residues, X and n are as defined.
Compounds of the invention having as few as 5 or 6 .beta.-amino
acid residues exhibit stable structures in solution and the
compounds generally exhibit good resistance to proteolytic
degradation. The compounds of the invention provide a valuable new
source of structural diversity for synthesis of biologically active
compounds, e.g. for pharmaceutical uses.
Inventors: |
Seebach, Dieter; (Zuerich,
CH) |
Correspondence
Address: |
THOMAS HOXIE
NOVARTIS, CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 430/2
EAST HANOVER
NJ
07936-1080
US
|
Family ID: |
26309509 |
Appl. No.: |
10/459071 |
Filed: |
June 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10459071 |
Jun 11, 2003 |
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09202098 |
Dec 9, 1998 |
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6617425 |
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09202098 |
Dec 9, 1998 |
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PCT/EP97/03077 |
Jun 12, 1997 |
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Current U.S.
Class: |
530/320 ;
435/7.1; 436/518; 562/553 |
Current CPC
Class: |
C07C 237/22 20130101;
A61P 43/00 20180101; C07C 271/22 20130101; A61K 31/435
20130101 |
Class at
Publication: |
530/320 ;
435/7.1; 436/518; 562/553 |
International
Class: |
G01N 033/53; C07K
007/08; C07K 007/06; G01N 033/543; C07C 229/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 1996 |
GB |
9612402.9 |
Oct 15, 1996 |
GB |
9621496.0 |
Claims
1. A .beta.-peptide comprising 2 or more different .beta.-amino
acid residues.
2. A .beta.-peptide according to claim 1, comprising from 2 to 11
.beta.-amino acid residues.
3. A compound of formula I 11wherein each R is H, --R.sub.5,
--OR.sub.5, --C(O)R.sub.5, --R.sub.5C(O)R.sub.6, --C(O)OR.sub.5,
--R.sub.5C(O)OR.sub.6, --R.sub.5OC(O)R.sub.6,
--R.sub.5OC(O)OR.sub.6, --R.sub.5NR.sub.6C(O)R.sub.7,
--R.sub.5C(O)NR.sub.6R.sub.7, --C(O)NR.sub.6R.sub.7,
--R.sub.5OC(O)NR.sub.6R.sub.7,
--R.sub.5NR.sub.6C(O)NR.sub.7R.sub.8,
--R.sub.5NR.sub.6C(O)OR.sub.7, --R.sub.5--O--R.sub.6,
--R.sub.5--NR.sub.6R.sub.7, --R.sub.5--S--R.sub.6,
--R.sub.5--SO.sub.m--R.sub.6, --R.sub.5OR--O--R.sub.7,
--R.sub.5NR.sub.6R.sub.7--O--R.sub.8,
--R.sub.5SO.sub.mR.sub.6--O--R.sub.- 7, --C(O)R.sub.5--O--R.sub.6,
--C(O)OR.sub.5--O--R.sub.6, --R.sub.5C(O)R.sub.6--O--R.sub.7,
--R.sub.5C(O)OR.sub.6--O--R.sub.7,
--R.sub.5OC(O)R.sub.6--O--R.sub.7,
--R.sub.5OC(O)OR.sub.6--O--R.sub.7,
--R.sub.5NR.sub.6C(O)R.sub.7--O--R.sub.8,
--C(O)NR.sub.5R.sub.6--O--R.sub- .7,
--R.sub.5C(O)NR.sub.6R.sub.7--O--R.sub.8,
--R.sub.5OC(O)NR.sub.6R.sub.- 7--O--R.sub.8,
--R.sub.5NR.sub.6C(O)NR.sub.7R.sub.8--O--R.sub.9,
--R.sub.5NR.sub.6C(O)OR.sub.7--O--R.sub.8,
--R.sub.5OR.sub.6--S--R.sub.7,
--R.sub.5NR.sub.6R.sub.7--S--R.sub.8,
--R.sub.5SO.sub.mR.sub.6--S--R.sub.- 7, --C(O)R.sub.5--S--R.sub.6,
--C(O)OR.sub.5--S--R.sub.6, --R.sub.5C(O)R.sub.6--S--R.sub.7,
--R.sub.5C(O)OR.sub.6--S--R.sub.7,
--R.sub.5OC(O)R.sub.6--S--R.sub.7,
--R.sub.5OC(O)OR.sub.6--S--R.sub.7,
--R.sub.5NR.sub.6C(O)R.sub.7--S--R.sub.8,
--C(O)NR.sub.5R.sub.6--S--R.sub- .7,
--R.sub.5C(O)NR.sub.6R.sub.7--S--R.sub.8,
--R.sub.5OC(O)NR.sub.6R.sub.- 7--S--R.sub.8,
--R.sub.5NR.sub.6C(O)NR.sub.7R.sub.8--S--R.sub.9,
--R.sub.5NR.sub.6C(O)OR.sub.7--S--R.sub.8,
--R.sub.5OR.sub.6--NR.sub.7R.s- ub.8,
--R.sub.5NR.sub.6R.sub.7--NR.sub.8R.sub.9,
--R.sub.5SO.sub.mR.sub.6-- -NR.sub.7R.sub.8,
--C(O)R.sub.5--NR.sub.6R.sub.8, --C(O)OR.sub.5--NR.sub.6- R.sub.8,
--R.sub.5C(O)R.sub.6--NR.sub.7R.sub.8, --R.sub.5C(O)OR.sub.6--NR.-
sub.7R.sub.8, --R.sub.5OC(O)R.sub.613 NR.sub.7R.sub.8,
--R.sub.5OC(O)OR.sub.6--NR.sub.7R.sub.8,
--R.sub.5NR.sub.6C(O)R.sub.7--NR- .sub.8R.sub.9,
--C(O)NR.sub.5R.sub.6--NR.sub.7R.sub.8,
--R.sub.5C(O)NR.sub.6R.sub.7--NR.sub.8R.sub.9,
--R.sub.5OC(O)NR.sub.6R.su- b.7--NR.sub.8R.sub.9,
--R.sub.5NR.sub.6C(O)NR.sub.7R.sub.8--NHR.sub.9, or
--R.sub.5NR.sub.6C(O)OR.sub.7--NR.sub.8R.sub.9', where R.sub.5,
R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are each independently
C.sub.1-10alkyl, C.sub.1-10alkenyl, C.sub.1-10alkynyl,
C.sub.6-10aryl, C.sub.6-14aralkyl, C.sub.6-14aralkenyl or
C.sub.6-14aralkynyl and m is 1,2,3 or 4; and where R.sub.5,
R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are each unsubstituted or
substituted with up to 6 substituents selected from halo, NO.sub.2,
--OH, C.sub.1-4alkyl, --SH, --SO.sub.3, --NH.sub.2, C.sub.1-4acyl,
C.sub.1-4acyloxy, C.sub.1-4alkylamino, C.sub.1-4dialkylamino,
trihalomethyl, --CN, C.sub.1-4alkylthio, C.sub.1-4alkylsulfinyl, or
C.sub.1-4alkylsulfonyl, provided that all the R substituents are
not identical, or R forms a cyclic structure, e.g. a carbocyclic or
heterocyclic ring, with itself, with R.sub.3 or with the carbonyl
group attached to the immediately adjacent nitrogen atom; R.sub.1
and R.sub.2, which may be the same or different, are H, an
N-protecting group or as defined above for R, or R.sub.1 and
R.sub.2 are linked together in a 3 to 7 membered heterocyclic ring
structure, or either R.sub.1 or R.sub.2 together with OX signify an
amide bond; each R.sub.3, which may be the same or different, is as
defined above for R, or R.sub.3 forms a cyclic structure, e.g. a
carbocyclic or heterocyclic ring, with itself, with R or with the
nitrogen atom of its .beta.-amino acid residue, provided that R and
R.sub.3 are not both H; X is H, an O-protecting group or as defined
above for R, except that X is not --OR.sub.5, or OX together with
R.sub.1 or R.sub.2 signify an amide bond; n is 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10.
4. A compound according to claim 3 in which n is 5 or 6.
5. A compound according to claim 3 of formula IV 12wherein R,
R.sub.1, R.sub.2, X and n are as defined in claim 3.
6. A compound according to claim 3 of formula V 13wherein R.sub.1,
R.sub.2, R.sub.3, X and n are as defined in claim 3.
7. A compound according to claim 1 or 3 which is a .beta.-turn
mimetic.
8. A discrete compound collection (typically comprising from 2 to
about 1000 compounds) or a library of compounds (typically
comprising from 20 to 100 compounds up to many thousands of
compounds, e.g. 100,000 compounds or more) comprising a plurality
of compounds according to claim 1 or 3.
9. A pharmaceutical composition comprising an effective amount of a
compound according to claim 1 or 3 together with a pharmaceutically
acceptable diluent or carrier.
10. The use of a compound according to claim 1 or 3 for the
manufacture of a medicament for the treatment of any disease
associated with any of the assays identified above.
11. A process for the preparation of a compound according to claim
1 or 3 comprising forming an amide bond between the .beta.-amino
group of a first .beta.-amino acid residue or a precursor thereof
and the .alpha.-carboxyl group of a second .beta.-amino acid
residue or a precursor thereof and repeating the amide bond forming
step as required.
12 A process for the preparation of a compound according to claim 3
comprising Arndt-Eisert homologation of an N-protected
.alpha.-amino acid of formula VI 14wherein Pg, is an amine
protecting group and R and R.sub.2 are as defined in claim 3, by
reaction with diazomethane, e.g. in the presence of
triethylamine/ethylchloroformate to yield a diazo ketone
intermediate of formula VII 15
13. A process for the preparation of a compound of formula I as
defined in claim 3 comprising coupling of a diazo ketone of formula
VII 16wherein Pg is an amine protecting group and R and R.sub.2 are
as defined in claim 3, with a .beta.-amino acid of formula VIII
17wherein R and R.sub.3 are as defined in claim 3 and Y is an
O-protecting group, or with a .beta.-amino acid or .beta.-peptide
residue of formula IX 18wherein R, R.sub.3, are as defined in claim
3, P'g is an O-protecting group and p is 1, 2, 3, 4, 5, 6, 7, 8 or
9.
Description
[0001] This invention relates to peptides and in particular to
.beta.-peptides, i.e. peptides of .beta.-amino acids.
[0002] Many naturally occurring, biologically active compounds are
proteins or peptides based upon .alpha.-amino acids (i.e. sequences
of .alpha.-amino acids in which the .alpha.-carboxyl group of one
amino acid is joined by an amide bond to the .alpha.-amino group of
the adjacent amino acid). In recent years an approach to the
discovery of new pharmaceutically active drugs has been to
synthesise libraries of peptides and then to assay for compounds
within the library which have a desired activity, such as a desired
binding activity. However, .alpha.-amino acid peptides are not
altogether satisfactory for pharmaceutical uses, in particular
because they are often poorly absorbed and subject to proteolytic
degradation in vivo.
[0003] We have now synthesised new peptides based on .beta.-amino
acids and have found that these peptides have unexpected and
desirable properties.
[0004] Accordingly the present invention provides a .beta.-peptide
comprising 2 or more different .beta.-amino acid residues.
[0005] For the purposes of the present description a .beta.-peptide
comprises a sequence of 2 or more .beta.-amino acid residues in
which the .alpha.-carboxyl group of one amino acid is joined by an
amide bond to the .beta.-amino group of the adjacent amino acid.
The .beta.-peptides of the invention may comprise any number of
amino acid residues, though conveniently comprise from 2 to 11,
preferably from 4 to 7, especially 5 or 6, .beta.-amino acid
residues.
[0006] The .beta.-amino acid residues of the .beta.-peptides of the
invention are characteristically .beta.-amino-n-propionic acid
derivatives, typically further substituted at the .alpha.- and/or
.beta.-carbon atoms and may be further substituted, e.g. at the
N-terminal amino nitrogen atom. The .alpha.-, .beta.- and amino
substituents may include substituents containing from 1 to 43
carbon atoms optionally interrupted by up to 4 hetero atoms,
selected from O, N or S, optionally containing a carbonyl (i.e.
--C(O)--) group, and optionally further substituted by up to 6
substituents selected from halo, NO.sub.2, --OH, C.sub.1-4alkyl,
--SH, --SO.sub.3, --NH.sub.2, C.sub.1-4acyl, C.sub.1-4acyloxy,
C.sub.1-4alkylamino, C.sub.1-4dialkylamino, trihalomethyl, --CN,
C.sub.1-4alkylthio, C.sub.1-4alkylsulfinyl, or
C.sub.1-4alkylsulfonyl. Conveniently, the .alpha.-, .beta.- and
amino substituents may include any of those substituents defined
below as the "R" substituents of formula I. For example, the
.alpha.- and .beta.-substituents may be selected from the group
comprising the substituents which are present on the .alpha.-carbon
atoms of .alpha.-amino acids, e.g. --H, --CH.sub.3,
--CH(CH.sub.3).sub.2, --CH.sub.2--CH(CH.sub.3).sub.2,
--CH(CH.sub.3)CH.sub.2CH.sub.3, --CH.sub.2-phenyl,
CH.sub.2--pOH-phenyl, --CH.sub.2-indole, --CH.sub.2--SH,
--CH.sub.2--CH.sub.2--S--CH.sub.3, --CH.sub.2OH, --CHOH--CH.sub.3,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH.sub.2,
--CH.sub.2--CH.sub.2--CH.sub.2--NH--C(NH)NH.sub.2,
--CH.sub.2-imidazole, --CH.sub.2--COOH, --CH.sub.2--CH.sub.2--COOH,
--CH.sub.2--CONH.sub.2, --CH.sub.2--CH.sub.2--CONH.sub.2 or
together with an adjacent --NH group forms a proline amino acid
residue.
[0007] The .beta.-peptides of the invention preferably comprise
only .beta.-amino acid residues. However, the invention includes
.beta.-peptides, for instance longer .beta.-peptides, e.g.
comprising 7 or more acid residues, which contain one or a few,
e.g. 1 or 2, acid residues which are not .beta.-amino acids, e.g.
.alpha.- or .gamma.-amino acids, or an .alpha.-, .beta.- or
.gamma.-hydroxycarboxlic acids. For example, a non-.beta.-amino
acid may be present in the .beta.-peptide at a junction or
transition region between two sequences of .beta.-amino acid
residues.
[0008] Characteristically substitutents present on the .alpha.-
and/or .beta.-carbon atoms of the .beta.-amino acid residues are in
the radial orientation with respect to the peptide chain.
[0009] Preferably the present invention provides a compound of
formula I 2
[0010] wherein
[0011] each R is H, --R.sub.5, --OR.sub.5, --C(O)R.sub.5,
--R.sub.5C(O)R.sub.6, --C(O)OR.sub.5, --R.sub.5C(O)OR.sub.6,
--R.sub.5OC(O)R.sub.6, --R.sub.5OC(O)OR.sub.6,
--R.sub.5NR.sub.6C(O)R.sub- .7, --R.sub.5C(O)NR.sub.6R.sub.7,
--C(O)NR.sub.6R.sub.7, --R.sub.5OC(O)NR.sub.6R.sub.7,
--R.sub.5NR.sub.6C(O)NR.sub.7R.sub.8,
--R.sub.5NR.sub.6C(O)OR.sub.7, --R.sub.5--O--R.sub.6,
--R.sub.5--NR.sub.6R.sub.7, --R.sub.5--S--R.sub.6,
--R.sub.5--SO.sub.m--R.sub.6, --R.sub.5OR.sub.6--O--R.sub.7,
--R.sub.5NR.sub.6R.sub.7--O--R.sub.8,
--R.sub.5SO.sub.mR.sub.6--O--R.sub.- 7, --C(O)R.sub.5--O--R.sub.6,
--C(O)OR.sub.5--O--R.sub.6, --R.sub.5C(O)R.sub.6--O--R.sub.7,
--R.sub.5C(O)OR.sub.6--O--R.sub.7,
--R.sub.5OC(O)R.sub.6--O--R.sub.7,
--R.sub.5OC(O)OR.sub.6--O--R.sub.7,
--R.sub.5NR.sub.6C(O)R.sub.7--O--R.sub.8,
--C(O)NR.sub.5R.sub.6--O--R.sub- .7,
--R.sub.5C(O)NR.sub.6R.sub.7--O--R.sub.8,
--R.sub.5OC(O)NR.sub.6R.sub.- 7--O--R.sub.8,
--R.sub.5NR.sub.6C(O)NR.sub.7R.sub.8--O--R.sub.9,
--R.sub.5NR.sub.6C(O)OR.sub.7--O--R.sub.8,
--R.sub.5OR.sub.6--S--R.sub.7,
--R.sub.5NR.sub.6R.sub.7--S--R.sub.8,
--R.sub.5SO.sub.mR.sub.6--S--R.sub.- 7, --C(O)R.sub.5--S--R.sub.6,
--C(O)OR.sub.5--S--R.sub.6, --R.sub.5C(O)R.sub.6--S--R.sub.7,
--R.sub.5C(O)OR.sub.6--S--R.sub.7,
--R.sub.5OC(O)R.sub.6--S--R.sub.7,
--R.sub.5OC(O)OR.sub.6--S--R.sub.7,
--R.sub.5NR.sub.6C(O)R.sub.7--S--R.sub.8,
--C(O)NR.sub.5R.sub.6--S--R.sub- .7,
--R.sub.5C(O)NR.sub.6R.sub.7--S--R.sub.8,
--R.sub.5OC(O)NR.sub.6R.sub.- 7--S--R.sub.8,
--R.sub.5NR.sub.6C(O)NR.sub.7R.sub.8--S--R.sub.9,
--R.sub.5NR.sub.6C(O)OR.sub.7--S--R.sub.8,
--R.sub.5OR.sub.6--NR.sub.7R.s- ub.8,
--R.sub.5NR.sub.6R.sub.7--NR.sub.8R.sub.9,
--R.sub.5SO.sub.mR.sub.6-- -NR.sub.7R.sub.8,
--C(O)R.sub.5--NR.sub.6R.sub.8, --C(O)OR.sub.5--NR.sub.6- R.sub.8,
--R.sub.5C(O)R.sub.6--NR.sub.7R.sub.8, --R.sub.5C(O)OR.sub.6--NR.-
sub.7R.sub.8, --R.sub.5OC(O)R.sub.6--NR.sub.7R.sub.8,
--R.sub.5OC(O)OR.sub.6--NR.sub.7R.sub.8,
--R.sub.5NR.sub.6C(O)R.sub.7--NR- .sub.8R.sub.9,
--C(O)NR.sub.5R.sub.6--NR.sub.7R.sub.8,
--R.sub.5C(O)NR.sub.6R.sub.7--NR.sub.8R.sub.9,
--R.sub.5OC(O)NR.sub.6R.su- b.7--NR.sub.8R.sub.9,
--R.sub.5NR.sub.6C(O)NR.sub.7R.sub.8--NHR.sub.9 or
--R.sub.5NR.sub.6C(O)OR.sub.7--NR.sub.8R.sub.9,
[0012] where R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are
each independently C.sub.1-10alkyl, C.sub.1-10alkenyl,
C.sub.1-10alkynyl, C.sub.6-10aryl, C.sub.6-14aralkyl,
C.sub.6-14aralkenyl or C.sub.6-14aralkynyl and m is 1,2,3 or 4; and
where R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are each
unsubstituted or substituted with up to 6 substituents selected
from halo, NO.sub.2, --OH, C.sub.1-4alkyl, --SH, --SO.sub.3,
--NH.sub.2, C.sub.1-4acyl, C.sub.1-4acyloxy, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, trihalomethyl, --CN, C.sub.1-4alkylthio,
C.sub.1-4alkylsulfinyl, or C.sub.1-4alkylsulfonyl, provided that
all the R substituents are not identical, 35
[0013] or R forms a cyclic structure , e.g. a carbocyclic or
heterocyclic ring, with itself, with R.sub.3 or with the carbonyl
group attached to the immediately adjacent nitrogen atom;
[0014] R.sub.1 and R.sub.2, which may be the same or different, are
H, an N-protecting group or as defined above for R, or R.sub.1 and
R.sub.2 are linked together in a 3 to 7 membered heterocyclic ring
structure, or either R.sub.1 or R.sub.2 together with OX signify an
amide bond;
[0015] each R.sub.3, which may be the same or different, is as
defined above for R, or R.sub.3 forms a cyclic structure, e.g. a
carbocyclic or heterocyclic ring, with itself, with R or with the
nitrogen atom of its .beta.-amino acid residue, provided that R and
R.sub.3 are not both H;
[0016] X is H, an O-protecting group or as defined above for R,
except that X is not --OR.sub.5, or OX together with R.sub.1 or
R.sub.2 signify an amide bond;
[0017] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0018] The .beta.-peptides of the invention, and in particular the
compounds of formula I, are hereinafter referred to as the
compounds of the invention.
[0019] Characteristically when R and R.sub.3 are not H, R and
R.sub.3 are in the radial orientation with respect to the peptide
chain.
[0020] Typically when R or R.sub.3 form a cyclic strtucture, the
cyclic structure is a 3 to 7 membered cyclic structure.
[0021] Preferably n is 4, 5, 6 or 7 or more preferably n is 5 or
6.
[0022] Thus in preferred embodiments the invention provides a
compound of formula II or a compound of formula III 3
[0023] wherein R, R.sub.1, R.sub.2, R.sub.3 and X are as defined
above.
[0024] When R.sub.1 and R.sub.2 are linked together in a ring
structure the structure is optionally substituted as for R.sub.5
etc. above and may be fused with one or more rings such as phenyl
rings. For example the R.sub.1 and R.sub.2 ring system may be a
piperidine or pyridine ring system.
[0025] When either R.sub.1 or R.sub.2 together with OX signify an
amide bond, i.e. when the compounds of formula I are cyclic
compounds, n is preferably at least 2.
[0026] In the present description unless otherwise indicated terms
such as "compounds of the invention" embrace the compounds in salt
form as well as in free base form and also when the compounds are
attached to a solid phase. Where a basic substituent such as an
amine substituent is present, the salt form may be a double acid
addition salt, for example a dihydrochloride.
[0027] The term halogen includes F, Cl, Br and I, preferably F and
Cl.
[0028] Suitable N-protecting groups as R.sub.1 or R.sub.2 include
groups of formula H(CH.sub.2CH.sub.2O).sub.p-- where p=3-30;
R.sub.10CO--; R.sub.11OCO-- or R.sub.12SO.sub.2--,
[0029] in which R.sub.10 is C.sub.1-4 alkyl or
H(CH.sub.2--O--CH.sub.2).su- b.p wherein p is defined as above;
[0030] R.sub.11 is C.sub.1-6 alkyl, phenyl, benzyl or naphthyl; and
R.sub.12 is phenyl, naphthyl, or
[0031] C.sub.1-4 alkylphenyl;
[0032] of which R.sub.11OCO-- is particularly preferred, especially
when R.sub.11 is tert. butyl (i.e. Boc as the protecting group), or
when R.sub.11 is benzyl (i.e. the protecting group customarily
designated as Z).
[0033] Suitable O-protecting groups as X include are alkyl, e.g.
C.sub.1-4 alkyl, groups or aromatic groups, e.g. benzyl, including
substituted phenyl groups such pentafluorophenyl.
[0034] In a particular embodiment the compounds of formula I are
peptides of .beta.-amino acids which are unsubstituted at the
.alpha.-carbon atom, i.e. compounds of formula IV 4
[0035] wherein R, R.sub.1, R.sub.2, X and n are as defined
above.
[0036] .beta.-amino acids which are unsubstituted at the
.alpha.-carbon atom and the corresponding peptides of formula IV
are conveniently referred to as .beta..sup.3 amino acids and 62
.sup.3 peptides respectively.
[0037] In a further particular embodiment the compounds of formula
I are peptides of .beta.-amino acids which are unsubstituted, apart
from amino substituted, at the .beta.-carbon atom, i.e. compounds
of formula V 5
[0038] wherein R.sub.1, R.sub.2, R.sub.3, X and n are as defined
above.
[0039] .beta.-amino acids which are unsubstituted, apart from the
amino substitituent, at the .beta.-carbon atom and the
corresponding peptides of formula V are conveniently referred to as
.beta..sup.2 amino acids and .beta..sup.2 peptides
respectively.
[0040] In further particular embodiments the compounds of the
invention comprise a mixture of .beta..sup.2- and
.beta..sup.3-amino acids; for instance, as alternating
.beta..sup.2- and .beta..sup.3-amino acids or as discrete stretches
of .beta..sup.2-amino acids linked to discrete stretches of
.beta..sup.3-amino acids.
[0041] The substituents R, R.sub.1, R.sub.2, R.sub.3 and X have
many possible significations as indicated above. Conveniently,
however, in particular embodiments the substituents R, R.sub.1,
R.sub.2, R.sub.3 and X may be selected from the group comprising
the substituents which are present on the .alpha.-carbon atoms of
.alpha.-amino acids, e.g. --H, --CH.sub.3, --CH(CH.sub.3).sub.2,
--CH.sub.2--CH(CH.sub.3).sub.2, --CH(CH.sub.3)CH.sub.2CH.sub.3,
--CH.sub.2-phenyl, CH.sub.2-pOH-phenyl, --CH.sub.2-indole,
--CH.sub.2--SH, --CH.sub.2--CH.sub.2--S--CH.sub.3, --CH.sub.2OH,
--CHOH--CH.sub.3, --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--
-NH.sub.2, --CH.sub.2--CH.sub.2--CH.sub.2--NH--C(NH)NH.sub.2,
--CH.sub.2-imidazole, --CH.sub.2--COOH, --CH.sub.2--CH.sub.2--COOH,
--CH.sub.2--CONH.sub.2, --CH.sub.2--CH.sub.2--CONH.sub.2 or
together with an adjacent --NH group forms a proline amino acid
residue.
[0042] In accordance with the present invention it has been found
that the compounds of the invention have desirable properties. For
example, compounds of formula I in which n is 5 or 6 are able to
form stable helix structures in solution, in contrast to
.alpha.-peptides for which distinct secondary structures are
observed in solution only when the peptide comprises at least 15 to
20 amino acid residues. Thus it has been found that representative
compounds of formula I comprising L-.beta.-amino acids form
compact, left-handed or (M) 3.sub.1 helices of 5 .ANG. pitch.
Representative compounds of formula I comprising D-.beta.-amino
acids form corresponding compact, right-handed helices. The
corresponding helices formed by .alpha.-peptides of L-amino acids
are right-handed 3.6.sub.13 .alpha.-helices with a pitch of 5.6
.ANG.. Other structures of the compounds of the invention are
discussed hereinafter and in the references referred to
hereinafter.
[0043] In view of their structure and in preferred embodiments
.beta.-peptides of the invention, especially .beta.-hexapeptides,
may be used as .beta.-turn mimetics.
[0044] Generally also the compounds of the invention have much
greater stability to the action of peptidases, such as pepsin, than
.alpha.-peptides. As such the compounds of formula may conveniently
exhibit correspondingly longer half lives, e.g. serum half lives,
in vivo than than .alpha.-peptides.
[0045] In formula I above the carbon atoms marked with an asterisk
(*) may be optically active centres, i.e. when R and R.sub.3 are
not H, and may be in the R or the S configuration. Thus the
invention includes the compounds of the invention in pure isomeric
form, e.g. consisting of at least 90%, preferably at least 95% of a
single isomeric form, as well as mixtures of these forms. Thus the
compounds of the invention may be in the form of individual
enantiomers or may be in the form of racemates or diastereoisomeric
mixtures or any other mixture of the possible isomers (e.g. as
derived from a racemic building block).
[0046] The compounds of the invention may be prepared by synthetic
chemical procedures, including procedures similar to those which
may be used for the synthesis of .alpha.-amino acid peptides. Such
procedures include both solution and solid phase procedures, e.g.
using both Boc and Fmoc methodologies. Thus the compounds of
formula I may be prepared by successive amide bond forming
procedures in which amide bonds are formed between the .beta.-amino
group of a first .beta.-amino acid residue or a precursor thereof
and the .alpha.-carboxyl group of a second .beta.-amino acid
residue or a precursor thereof. The amide bond forming step may be
repeated as many times, and with specific .beta.-amino acid
residues or precursors thereof, as required to give the desired
.beta.-peptide. Also .beta.-peptides comprising 2, 3 or more
.beta.-amino acid residues may be joined together to yield larger
.beta.-peptides. Cyclic compounds may be prepared by forming
peptide bonds between the N-terminal and C-terminal ends of a
previously synthesised linear .beta.-peptide.
[0047] .beta.-amino acids may be produced enantioselectively from
corresponding .alpha.-amino acids; for instance, by Arndt-Eisert
homologation of N-protected .alpha.-amino acids. Conveniently such
homologation may be followed by coupling of the reactive diazo
ketone intermediate of the Wolff rearrangement with a .beta.-amino
acid residue.
[0048] Thus the invention includes a process for the preparation of
a compound of formula I comprising Arndt-Eisert homologation of an
N-protected .alpha.-amino acid of formula VI 6
[0049] wherein Pg, is an amine protecting group and R and R.sub.2
are as defined above, by reaction with diazomethane, e.g. in the
presence of triethylamine/ethylchloroformate, to yield a diazo
ketone intermediate of formula VII 7
[0050] wherein Pg, and R and R.sub.3 are as defined above.
[0051] The invention also includes a process for the preparation of
a compound of formula I comprising coupling of a diazo ketone of
formula VII 8
[0052] with a .beta.-amino acid of formula VIII 9
[0053] wherein R and R.sub.3 are as defined above and Y is an
O-protecting group, or with a .beta.-amino acid or .beta.-peptide
residue of formula IX 10
[0054] wherein R and R.sub.3, are as defined above, P'g is an
O-protecting group and p is 1, 2, 3, 4, 5, 6, 7, 8 or 9.
[0055] Any suitable reaction conditions or procedure may be used
for the coupling reaction between the compounds of formulae VIII
and IX. For example, the coupling reaction may be carried out in
the presence of a silver catalyst, e.g. Ag benzoate in Et.sub.3N,
with cooling, e.g. at a temperature of 0.degree. C. to -40.degree.
C., in the dark under an inert atmosphere. Alternatively, for
example, the coupling reaction may be catalysed by light, e.g.
irradiation of the reaction mixture with a low pressure mercury
lamp.
[0056] The compounds of formulae of the invention may be used to
establish discrete compound collections or libraries of compounds
for use in screening for compounds having desirable activities, in
particular biological activities indicative of particular
pharmaceutical uses.
[0057] Thus the invention also includes discrete compound
collections (typically comprising from 2 to about 1000 compounds)
and libraries of compounds (typically comprising from 20 to 100
compounds up to many thousands of compounds, e.g. 100,000 compounds
or more) comprising pluralities of the compounds of the
invention.
[0058] As will be appreciated the particular identity of the
substituents R, R.sub.1, R.sub.2, R.sub.3 and X depends upon the
choice of building blocks and reaction steps used for synthesis of
the compounds of formula I. The substituents R, R.sub.1, R.sub.2,
R.sub.3 and X may be chosen as desired to provide groups of related
compounds having particular structural themes or to provide
unrelated, structurally diverse compounds.
[0059] Compounds having desired biological activities may be
identified using appropriate screening assays. For example the
following screening assays may be used to screen for particular
biological activities indicative of corresponding pharmaceutical
uses.
[0060] Antiinflammatory and immunosuppressive activities of
compounds of formula I are determined by means of the following and
similar assays: the IL-1.beta. secretion inhibition, LPS fever,
cytokine release from THP-1 cells, and functional IL-1 antagonist
assays and the assay of carrageenan induced paw edema in the rat
(as described in EP 0606044 and EP 0618223); the macrophilin
binding, Mixed Lymphocyte Reaction (MLR), IL-6 mediated
proliferation, localised graft-versus-host (GvH) reaction, kidney
allograft reaction in the rat, experimentally induced allergic
encephalomyelitis (EAE) in the rat, Freund's adjuvant arthritis,
FKBP binding, steroid potentiation and Mip and Mip-like factor
inhibition assays (as described in WO 94/09010, EP 0296123 and EP
0296122).
[0061] Central Nervous System (CNS) activity of compounds of
formula I is determined by means of the following and similar
assays: serotonin 1D (5HT 10) receptor agonist assays including the
method of Weber et al.(Schmiedeberg's Arch. Pharmacol. 337, 595-601
(1988)) (and as described in EP 0641787)); 5HT.sub.3 receptor
agonist assays (as described in GB 2240476 and EP 0189002); assays
for activity in treatment of psychotic disorders and Parkinson's
Disease, such as the apomorphine induced gnawing in the rat assay
and dopamine receptor (D1 and D2) binding assays (as described in
GB 20206115 B); assays for dopamine receptor antagonist activity
(in relation to schizophrenia and related diseases, as described in
EP 0483063 and EP 0544240); assays for activity in relation to
senile dementia and Alzheimer's Disease (as described in EP
0534904); assays for activity in relation to cerebral ischaemia (as
described in EP 0433239), and assays in relation to
gastrointestinal motility such as the peristaltic reflex in
isolated guinea pig ileum and assays of antiserotoninergic effects
(specifically at the 5-HT.sub.4 receptors) (as described in EP
0505322)
[0062] Activity of the compounds of formula I in relation to bone
and calcium metabolism is determined by assays as or similar to
those described in WO 94/02510, GB 2218102B and WO 89/09786.
[0063] Activity of the compounds of formula I in relation to asthma
and other allergic and inflammatory conditions is determined by the
following assay procedures: the PDE isoenzyme inhibition,
inhibition of eosinophil activation by formyl Met Leu Phe (f MLP),
inhibition of TNF.alpha. secretion, inhibition of SRS-A production,
bacterial endotoxin (LPS) induced lethality in the guinea pig,
arachidonic acid induced irritant dermatitis in the mouse,
relaxation of the human bronchus, suppression of SRS-A induced
bronchoconstriction, suppression of bombesin induced
bronchoconstriction, suppression of methacholine (MeCH) induced
bronchoconstriction in the rhesus monkey and suppression of airways
hyperactivity in the guinea pig assays (as described in European
patent application No. 94810628.1 [EP 0664289], WO 94/12493 and GB
2213482).
[0064] Serine protease, e.g. Thrombin, inhibition activity of the
compounds of formula I is determined using assays such as those
described in WO 94/20526. Glycoprotein IIb/IIIa antagonist,
activity of the compounds of formula I is determined using the
assay procedures described by Cook et al. (Thrombosis and
Haemostasis, 70(3), 531-539 (1993) and Thrombosis and Haemostasis,
70(5), 838-847 (1993)) Muiller et al. (J. Biol. Chem., Vol. 268,
No. 9, 6800-6808 (1993)).
[0065] Anticancer activity of the compounds of formula I is
determined by the anti tumour activity assay as described in EP
0296122 or by trial procedures, for instance as described in GB
2239178. Multidrug resistance (MDR) reversing activity is
determined by the assays described in EP 0296122.
[0066] The relevant teachings of the patent documents and other
publications referred to above is incorporated herein by reference.
Compounds of formula I which have appropriate levels of activity in
these assays are useful as pharmaceuticals in relation to the
corresponding therapies or disease states.
[0067] Thus the invention includes compounds of formula I for use
as pharmaceuticals and the use of a compound of formula I for the
manufacture of a medicament for the treatment of any disease
associated with any of the assays hereinbefore described. The
invention also includes the use of a compound of formula I as a
pharmaceutical, and pharmaceutical compositions comprising an
effective amount of a compound of formula I together with a
pharmaceutically acceptable diluent or carrier.
[0068] The invention is further described, by way of illustration
only, in the following description and Examples which refer to the
accompanying Figures in which
[0069] FIG. 1a is a CD spectra overlay of 1. Boc-b-HAla-b-HLeu-OMe,
2. Boc-b-HVal-b-HAla-b-HLeu-OMe, 3.
H-(b-HVal-b-HAla-b-HLeu).sub.2-OMe and 4. the a-hexapeptide
TFA.H-Val-Ala-Leu-Val-Ala-Leu-OMe in MeOH at 0.2 mM
concentration;
[0070] FIG. 1b is a CD spectra overlay of
H-(.beta.-HVal-.beta.-HAla-.beta- .-HLeu).sub.2-OMe at 0.2 mM
concentration in 1. MeOH, 2. trifluoroethanol (TFE) and 3.
hexafluoro-2-propanol (HFIP);
[0071] FIG. 1c is a CD spectra overlay of
H-(.beta.-HVal-.beta.-HAla-.beta- .-HLeu).sub.2-OH.TFA in MeOH at
1. 0.2 mM, 2. 0.04 mM and 3. 0.02 mM and 4. in Aqueous
H.sub.2SO.sub.4 at pH<1;
[0072] FIG. 1d is a CD spectra overlay of 1. the cyclic
.beta.-tripeptide cyclo(-.beta.-HVal-.beta.-HAla-.beta.-HLeu-) in
TFE and 2. the cyclic .beta.-hexapeptide
Cyclo(-.beta.-HVal-.beta.-HAla-.beta.-HLeu-.beta.-HVal-
-.beta.-HAla-.beta.-HLeu-) at 0.2 mM concentration;
[0073] FIG. 2 shows a comparison of the 3.sub.1 helical structures
of a) the left-handed helix of
H-(.beta.-HVal-.beta.-HAla-.beta.-HLeu).sub.2-OH- .TFA as
determined by NMR spectroscopy and b) the predicted right-handed
helical secondary structure of
(R)-Aminomethylvalyl-(R)-aminomethylalanyl-
-(R)-aminomethylleucyl-(R)-aminomethylvalyl-(R)-aminomethylalanyl-(R)-amin-
omethyl leucinate Trifluoroacetate, and
[0074] FIG. 3 is a CD spectra overlay of
H-(.beta.-HVal-.beta.-HAla-.beta.- -HLeu).sub.2-OH.TFA (curve A)
and (R)-Aminomethylvalyl-(R)-aminomethylalan-
yl-(R)-aminomethylleucyl-(R)-aminomethylvalyl-(R)-aminomethylalanyl-(R)-am-
inomethyl leucinate Trifluoroacetate (curve B) in MeOH at a
concentration of 2.times.10.sup.-4, molar ellipeicity [Q] in 10 deg
cm.sup.2 mol.sup.-1.
[0075] The synthesis and analysis of compounds of formula I is
described in detail in recent publications by the inventor and
co-workers, including the following
[0076] 1. Seebach et al. Helvetica Chimica Acta (1996), Vol. 79,
pages 913-941;
[0077] 2. Seebach et al. Helvetica Chimica Acta (1996), Vol 79,
pages 2043-2066;
[0078] 3. T. Hintermann and D. Seebach, Synlett (1997), pages
437-438;
[0079] 4. Seebach et al. Helvetica Chimica Acta (1997), Vol. 80,
pages 173-182.
[0080] Thus the synthesis, analysis and properties of the following
compounds of formula I are descibed in detail in publication No. 1
above:
[0081] 1 i) Methyl
(3S)-3-(tert-Butoxycarbonylamino)-5-methylhexanoate;
[0082] 1 ii)
Methyl-[(3S)-3-(tert-butoxycarbonylamino)-butanoyl]-(3S)-3-am-
ino-5-methylhexanoate (Boc-.beta.-HAla-.beta.-HLeu-OMe);
[0083] 1 iii )
Methyl-[(3S)-3-(tert-butoxycarbonylamino)-4-methylpentanoyl-
)-(3S)-3aminobutanoyl]-(3S )-3-amino-5-methyl-hexanoate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-OMe);
[0084] 1 iv) [(3S
)-3-(tert-butoxycarbonylamino)-4-methylpentanoyl)-(3S)-3-
-aminobutanoyl](3S)-3-amino-5-methylhexanoic acid
(Boc-.beta.-HVal-.beta.-- HAla-.beta.-HLeu-OH);
[0085] 1 v)
N-(tert-Butoxycarbonyl-.beta.-homo-valine-.beta.-homo-alanine--
.beta.-homo-leucine-.beta.--homo-valine-.beta.-homo-alanine-.beta.-homo-le-
ucine-methylester
(Boc-(.beta.-HVal-.beta.HAla-.beta.-HLeu).sub.2-OMe).
[0086] 1 vi)
.beta.-Homo-valine-.beta.-homo-alanine-.beta.-homo-leucine-.b-
eta.-homo-valine-.beta.-homo-alanine-.beta.-homo
-leucine-methylester-trif- luoroacetate
(H-(.beta.-HVal-.beta.-HAla-.beta.-HLeu).sub.2-OMe TFA);
[0087] 1 vii)
N-(tert-Butoxycarbonyl)-.beta.-homo-valine-.beta.-homo-alani-
ne-.beta.-homo-leucine-.beta.-homo-valine-.beta.-homo-alanine-.beta.-homo--
leucine (Boc-(.beta.-HVal-.beta.-HAla-.beta.-HLeu).sub.2-OH);
[0088] 1 viii)
.beta.-Homo-valine-.beta.-homo-alanine-.beta.-homo-leucine--
.beta.-homo-valine-.beta.-homo-alanine-.beta.-homo-leucine-trifluoroacetat-
e (H-(.beta.-HVal-.beta.-HAla-.beta.-HLeu).sub.2-OH-TFA);
[0089] 1 ix)
Pentafluorophenyl[(3S)-3-(tert-butoxycarbonylaminol-4-methylp-
entanoyl)-(3S)-3-aminobutanoyl]-(3S)-3-amino-5-methyl-hexanoate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-OPFP);
[0090] 1 x) Cyclo(-.beta.-HVal-.beta.-HAla-.beta.-HLeu);
[0091] 1 xi)
N-(tert-Butoxycarbony)-.beta.-homo-valine-.beta.-homo-alanine-
-.beta.-homo-leucine-.beta.-homo-valine-.beta.-homo-alanine-.beta.-homo-le-
ucine-pentafluorophenylester(Boc(.beta.-HVal-.beta.-HAla-.beta.-HLeu).sub.-
2-OPFP);
[0092] 1 xii)
Cyclo(-.beta.-HVal-.beta.-HAla-.beta.-HLeu-.beta.-HVal-.beta-
.-HAla-.beta.-HLeu-);
[0093] Thus the synthesis, analysis and properties of the
additional following compounds of formula I are described in detail
in publication No. 2 above:
[0094] 2 i)
Methyl-N-{(tert.-butoxy)carbonyl}-(S)-alanyl-(R)-.beta.-homova-
lyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-Ala-.beta.-HVal-.b- eta.-HAla-.beta.-HLeu-OMe);
[0095] 2 ii)
Methyl-[2-{(tert-butoxy)carbonylamino)}-2-methylpropanoyl]-(R-
)-.beta.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-Aib-.beta.-HVal-.beta.-HAla-.beta.-HLeu-OMe);
[0096] 2 iii)
Methyl-[3-{(tert-butoxy)carbonylamino}-propanoyl]-(R)-.beta.-
-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.HGly-.beta.-HVal-.beta.-HAla-.beta.-HLeu-OMe);
[0097] 2 iv)
Methyl-N-{(tert-butoxy)carbonyl}-(S)-.beta.-homoalanyl-(R)-.b-
eta.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.HAla-.beta.-HVal-.beta.-HAla-.beta.-HLeu-OMe);
[0098] 2 v)
Methyl-N-{(tert-butoxy)carbonyl}-(R)-.beta.-homoalanyl-(R)-.be-
ta.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-(R)-.beta.HAla-.beta.-HVal-.beta.-HAla-.beta.-HLeu-OMe);
[0099] 2 vi)
Methyl-[3-{(tert-butoxy)carbonylamino}-3-methylbutanoyl]-(R)--
.beta.-homoalanyl-(R)-.beta.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-ho-
moleucinate
(Boc-.beta.HAib-.beta.-HVal-.beta.-HAla-.beta.-HLeu-OMe);
[0100] 2 vii)
Methyl-N-{(tert-butoxy)carbonyl}-(2S,2R)-2-methyl-.beta.-hom-
oalanyl-(R)-.beta.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinat-
e
(Boc-(S,S)-.beta.-HAla(.alpha.Me)-.beta.-HVal-.beta.-HAla-.beta.-HLeu-OM-
e);
[0101] 2 viii)
Methyl-N-{(tert-butoxy)carbonyl}-(2R,3S)-2-methyl-.beta.-ho-
moalanyl-(R)-.beta.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucina-
te
(Boc-(R,S)-.beta.-HAla(.alpha.Me)-.beta.-HVal-.beta.-HAla-.beta.-HLeu-O-
Me);
[0102] 2 ix)
Methyl-N-{(tert-butoxy)carbonyl}-(S)-N-methyl-.beta.-homoalan-
yl-(R)-.beta.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc(Me)-.beta.-HAla-.beta.-HVal-.beta.-HAla-.beta.-HLeu-OMe);
[0103] 2 x)
Methyl-N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(S)-.bet-
a.-homoalanyl-(S)-.beta.-homoleucyl-(2-amino-2-methylpropanoyl)-(R)-.beta.-
-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-Aib-.beta.-HVal-.beta.-HAla-.bet-
a.-HLeu-OMe);
[0104] 2 xi)
Methyl-N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(S)-.be-
ta.-homoalanyl-(S)-.beta.-homoleucyl-(3-aminopropanoyl)-(R)-.beta.-homoval-
yl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.-HVal-.beta.--
HAla-.beta.-HLeu-.beta.-HGly-.beta.-HVal-.beta.-HAla-.beta.-HLeu-OMe);
[0105] 2 xii)
Methyl-N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(S)-.b-
eta.-homoalanyl-(S)-.beta.-homoleucyl-(S)-.beta.-homoalanyl-(R)-.beta.-hom-
ovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-.beta.-HAla-.beta.-HVal-.beta.-H-
Ala-.beta.-HLeu-OMe);
[0106] 2 xiii)
Methyl-N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(S)-.-
beta.-homoalanyl-(S)-.beta.-homoleucyl-(2S,3S)-2-methyl-.beta.-homoalanyl--
(R)-.beta.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-(S,S)-.beta.-HAla(.alpha.Me)-.be-
ta.-HVal-.beta.-HAla-.beta.-HLeu-OMe);
[0107] 2 xiv)
(R)-.beta.-Homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homole-
ucyl-(2S,3S)-2-methyl-.beta.-homoalanyl-(R)-.beta.-homovalyl-(S)-.beta.-ho-
moalanyl-(S)-.beta.-homoleucine Trifluoroacetate
(.beta.-HVal-.beta.-HAla--
.beta.-HLeu-(S,S)-.beta.-HAla(.alpha.Me)-.beta.-HVal-.beta.-HAla-.beta.-HL-
eu-OH.CF.sub.3COOH);
[0108] 2 xv)
Benzyl-(3S)-[N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(-
S)-.beta.-homoalanyl-(S)-.beta.-homoleucyloxy]-butanoate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-(S)-3HB-OBn);
[0109] 2 xvi)
(3S)-[N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(S)-.be-
ta.-homoalanyl-(S)-.beta.-homoleucyloxy]-butanoic acid
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-(S)-3HB-OH);
[0110] 2 xvii)
Methyl-N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(S)-.-
beta.-homoalanyl-(S)-.beta.-homoleucyl-{(S)-3-hydroxy-butanoyl}-(R)-.beta.-
-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-(S)-3HB-.beta.-HVal-.beta.-HAla--
.beta.-HLeu-OMe);
[0111] Also as described in publication No. 2. the following
.beta.-heptapeptides are synthesised for comparison purposes:
[0112] 2 xviii)
Methyl-N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(S)--
.beta.-homoalanyl-(S)-.beta.-homoleucyl-(R)-.beta.-homoalanyl-(R)-.beta.-h-
omovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-(R)-.beta.-HAla-.beta.-HVal-.bet-
a.-HAla-.beta.-HLeu-OMe);
[0113] 2 xix)
Methyl-N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(S)-.b-
eta.-homoalanyl-(S)-.beta.-homoleucyl-(3-amono-3-methylbutanoyl)-(R)-.beta-
.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-.beta.-HAib-.beta.-HVal-.beta.-H-
Ala-.beta.-HLeu-OMe);
[0114] 2 xx)
Methyl-N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(S)-.be-
ta.-homoalanyl-(S)-.beta.-homoleucyl-(2R,3S)-2-methyl-.beta.-homoalanyl-(R-
)-.beta.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-(R,S)-.beta.-HAla(.alpha.Me)-.be-
ta.-HVal-.beta.-HAla-.beta.-HLeu-OMe);
[0115] 2 xxi)
Methyl-N-{(tert-butoxy)carbonyl}-(R)-.beta.-homovalyl-(S)-.b-
eta.-homoalanyl-(S)-.beta.-homoleucyl-(S)-N-methyl-.beta.-homoalanyl-(R)-.-
beta.-homovalyl-(S)-.beta.-homoalanyl-(S)-.beta.-homoleucinate
(Boc-.beta.-HVal-.beta.-HAla-.beta.-HLeu-Me-.beta.-HAla-.beta.-HVal-.beta-
.-HAla-.beta.-HLeu-OMe);
[0116] These latter compounds have "non-allowed" .beta.-amino acid
residues (i.e. residues having substituents which are in the axial
orientation) at their central positions and exhibit CD spectroscopy
results which indicate an at least partial loss of secondary
structure.
[0117] The above compounds are .beta..sup.3 peptides, i.e. peptides
having sidechains in the .beta. position. The preparation of
.beta..sup.2 peptides of the invention, i.e peptides having
substuents in the .alpha. position. is described in the T.
Hintermann and D. Seebach publication identified above as
publication No. 3, and also as described in the examples below.
[0118] The abbreviations, procedures and equipment used in these
examples are identified below. the synthesis and analysis
procedures used are essentially as described in publications Nos. 1
and 2 above.
[0119] EDC (1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride), HOBt (1-hydroxy-1H-benzotriazole), h.v. (high
vacuum, 0.01-0.1 Torr), NMM (N-methyl-morpholine).
[0120] General: Flash chromatography (FC): SiO.sub.2 60
(0.04-0.063, Fluka). IR: Perkin Elmer 1600 FTIR, v in cm.sup.-1.
NMR Spectra: Bruker AMX 500, Gemini 300 or Gemini 200. .delta. in
ppm rel. to SiMe.sub.4 (=0 ppm), J in Hz; Carbon multiplicities
were assigned by DEPT techniques. MS: VG Tribrid spectrometer (E1),
VG ZAB2-SEQ with 3nitrobenzyl alcohol (FAB, 3-NOBA).
EXAMPLE 1
Preparation of Benzyl
(2R)-2-{(2R)-3-[(tert-Butoxy)carbonylamino]-2methylp- ropanoyl
amino methyl}-4-methylpentanoate
[0121] A solution of
(2R)-3-(tert-butoxy)carbonylamino-2-methylpropanoate (0.81 g, 4.0
mmol) in THF (20 ml) was cooled under Ar to -15.degree. and
successively treated with NMM (0.46 ml, 4.2 mmol), isobutyl
chloroformate (0.55 ml, 4.2 mmol) and, after stirring for 5 min, a
precooled solution of benzyl (2R)-2-aminomethyl-4methylpentanoate
toluenesulfonate ((2),1.79 g, 4.4 mmol) and NMM (0.48 ml, 4.4 mmol)
in DMF (10 ml). The mixture was allowed to warm to r.t. over a
period of 2 h and stirring was continued for further 2 h. The
solvent was removed under reduced pressure, the residue dissolved
in AcOEt and washed with 10% aq. citric acid (3.times.), sat. aq.
K.sub.2CO.sub.3 (3.times.), H.sub.2O and sat. aq. NaCI soln. The
org. phase was dried (MgSO.sub.4) and evaporated. Purification by
FC (hexane/AcOEt 2:1) yielded The title compound (1.46 g, 87%):
White solid. .sup.1H-NMR (200 MHz, CDCl.sub.3): 0.92 (d, J=5.5, 6H,
CH(CH.sub.3).sub.2); 1.05 (d, J=6.5, 3H, CHRCH.sub.3); 1.23-1.40
(m,1 H, CH(CH.sub.3).sub.2); 1.44 (s, 9H, C(CH.sub.3).sub.3);
1.52-1.77 (m, 2H, CH.sub.2CH(CH.sub.3).sub.2); 2.30-2.50 (m,1 H,
COCHR); 2.68-2.85 (m,1 H, COCHR); 3.08-3.42 (m, 3H, 3 NCH);
3.45-3.60 (m,1 H, NCH); 5.04 (br.,1 H, NHCOCHR); 5.16 (s, 2H,
CH.sub.2Ph); 5.90 (br., 1H, NHCOOtBu); 7.10-7.28 (m, 5H,
H-arom.).
EXAMPLE 2
Preparation of Benzyl
(2R)-2-.about.(2R)-2-{(2R)-2-[(tert-Butoxy)carbonyla-
mino]methyl-3methyl
butanoylamino}-2-methylpropanoylaminomethyl.about.-4-m-
ethylpentanoate:
[0122] The Boc-protected dipeptide Benzyl
(2R)-2-{(2R)-3-[(tert-Butoxy)car-
bonylamino]-2-methylpropanoylamino methyl}-4-methylpentanoate (243
mg, 0.58 mmol) was dissolved in sat. HCl/dioxane (1 ml) and stirred
for 2 h at r.t. The solvent was evaporated, the residue dried under
h.v. and dissolved in THF (5 ml). The solution was successively
treated with (2R)-2-(tert-butoxy)carbonylaminomethyl-3-methyl
butanoate (148 mg, 0.64 mmol), NMM (0.20 ml,1.8 mmol) and HOBt (95
mg, 0.70 mmol), cooled to 0.degree. and treated with EDC (123 mg.
0.64 mmol). The mixture-was stirred at 0.degree. for 1 h, then at
r.t. for 6 h, diluted with AcOEt and washed with 10% aq. citric
acid (3.times.), sat. aq. K.sub.2CO.sub.3 (3.times.), H.sub.2O and
sat. aq. NaCI soln. (2.times.). The org. phase was dried
(MgSO.sub.4) and evaporated. Purification by FC (Et.sub.2O) yielded
the title compound (231 mg, 74%): White solid. .sup.1H-NMR (300
MHz, CDCl.sub.3): 0.85-0.92 (m, 12H, 2 CH(CH.sub.3).sub.2); 1.00
(d, J=4.5, 3H, CHRCH.sub.3); 1.14-1.30 (m, 1H,
CH.sub.2CH(CH.sub.3).sub.2); 1.38 (s, 9H, C(CH.sub.3).sub.3);
1.49-1.62 (m, 2H, CH.sub.2CH(CH.sub.3).sub.2); 1.74-1.88 (m, 1H,
CHRCH(CH.sub.3).sub.2); 2.02-2.13 (m, 1H, COCHR); 2.35-2.50 (m, 1H,
COCHR); 2.66-2.77 (m, 1H, COCHR); 3.10-3.54 (m, 6H, 3 NCH.sub.2);
5.08 (d, J=12.3,1H, CH.sub.2Ph); 5.11 (br., 1H, NHBoc); 5.13 (d,
J=12.3. CH.sub.2Ph): 6.32 (br., 1 H, CONH); 6.46 (br., 1 H, CONH);
7.25-7.40 (m, 5H, H-arom.). .sup.13C-NMR (75 MHz, CDCl.sub.3):
15.7; 20.1; 20.9; 22.3; 22.6; 26.0; 28.4; 28.7; 38.9; 40.5; 40.8;
41.0; 42.2: 43.8: 54.0; 66.7; 79.3; 128.5; 128.7; 128.9; 136.0;
156.4; 174.8; 175.5; 175.6.
EXAMPLE3
Preparation of
(2R)-2-{(2R)-2-{(2R)-2-[(tert-Butoxy)carbonylamino]methyl-3-
-methyl
butanoylamino}-2-methylpropanoylaminomethyl}-4-methylpentanoate:
[0123] To a solution of the benzylester, Benzyl
(2R)-2-{(2R)-2-{(2R)-2-[(t- ert-Butoxy)carbonyl
amino]methyl-3-methylbutanoylamino}-2-methylpropanoyla-
minomethyl}-4-methyl pentanoate (177 mg, 0.33 mmol) in EtOH/DMF (5
ml/2.5 ml) under Ar, was added 10% Pd/C (15 mg). The Ar atmosphere
was replaced by H.sub.2, the suspension stirred for 18 h at r.t.,
the catalyst removed by filtration over Celite, and the filtrate
evaporated. The title free acid obtained (152 mg) was used without
further purification.
EXAMPLE 4
Preparation of Benzyl
N-[(tert-Butoxy)carbonyl]-(R)-aminomethylvalyl-(R)-a-
minomethylalanyl-(R)aminomethylleucyl-(R)-aminomethylvalyl-(R)-aminomethyl
alanyl-(R)-aminomethyl leucinate:
[0124] The Boc-protected tripeptide, Benzyl
(2R)-2-.about.(2R)-2-{(2R)-2-[-
(tert-Butoxy)carbonylamino]methyl-3methylbutanoylamino}-2-methylpropanoyla-
minomethyl.about.-4-methylpentanoate (107 mg, 0.20 mmol) was
dissolved in sat. HCI/dioxane (1 ml) and stirred for 2 h at r.t.
The solvent was evaporated, the residue dried under h.v. and
dissolved in THF (8 ml). The acid,
(2R)-2-{(2R)-2-.about.(2R)-2-[(tert-Butoxy)carbonylamino]methyl-3-m-
ethylbutanoylamino}--2-methyl
propanoylaminomethyl}-4-methylpentanoate (89 mg, 0.20 mmol) was
added, the solution cooled to 0.degree. and successively treated
with NMM (0.067 ml, 0.60 mmol), HOBt (34 mg, 0.25 mmol) and EDC (42
mg, 0.22 mmol). The mixture was stirred at r.t. for 24 h, then
diluted with CHCI.sub.3 (75 ml) and washed with 10% aq. citric acid
(3.times.), sat. aq. K.sub.2CO.sub.3 (3.times.), H.sub.2O and sat.
aq. NaCI soln. (2.times.). The org. phase was dried (MgSO.sub.4)
and evaporated. Purification by FC (CH.sub.2Cl.sub.2/MeOH 19:1)
yielded the title compound (119 mg, 69%): White solid.
[.alpha.]D=-110.2 (c=0.95, CHCl.sub.3). IR (CHCl.sub.3): 3445m,
3307m, 3008m, 2965s, 2873m 1702m, 1654s, 1522s, 1469m, 1368m,
1170s. 1H-NMR (500 MHz, CDCl.sub.3): 0.88-0.93 (m, 18H, 6
CH.sub.3); 0.96 (d, J=6.6, 6H, 2 CH.sub.3); 1.03 (d, J=7.0, 3H,
CH.sub.3); 1.16 (d, J=7.1, 3H, CH.sub.3); 1.24-1.29 (m, 2H, 2
CH.sub.2CH(CH.sub.3).sub.2); 1.43 (s, 9H, C(CH.sub.3).sub.3);
1.52-1.63 (m, 4H, 2 CH.sub.2CH(CH.sub.3).sub.2); 1.801.85 (m, 1H,
CHRCH(CH.sub.3).sub.2); 1.89-1.92 (m, 1H, CHRCH(CH.sub.3).sub.2);
1.99-2.05 (m, 1H, COCHR); 2.06-2.11 (m, 1H, COCHR); 2.42-2.52 (m,
3H, 3 COCHR); 2.71-2.76 (m, 1H, COCHR); 3.05-3.75 (m, 12H, 6
NCH.sub.2); 5.10 (d, J=12.3, 1H, CH.sub.2Ph); 5.16 (d, J=12.2, 1H,
CH.sub.2Ph); 5.54 (t, J=5.9, 1H, NHBoc); 6.80 (br., 1H, NHCO); 7.02
(br., 1H, NHCO); 7.09 (br., 1H, NHCO); 7.15 (br., 1H, NHCO);
7.31-7.37 (m, 6H, 5 H-arom./NHCO). .sup.13C-NMR (125 MHz,
CDCl.sub.3): 15.4; 15.5; 20.3; 20.4; 21.0; 21.1; 22.3; 22.6; 23.1
(CH.sub.3); 25.9; 26.0; 28.4; 28.4 (CH); 28.5 (CH.sub.3); 38.8;
38.8; 39.8 (CH.sub.2); 40.8 (CH); 41.0; 41.2 (CH.sub.2); 42.0;
42.3; 42.6; 42.8; 44.3; 45.3; 54.8; 55.1 (CH); 66.7 (CH.sub.2);
79.0 (C); 128.2; 128.3; 128.6 (CH); 135.9; 156.3; 174.6; 174.6;
174.9; 175.2; 175.3; 175.4 (C). MS (FAB): 860 (18, [M+1]+), 859
(30, M+), 764 (10), 761 (20), 760 (59), 759 (100), 91 (21).
EXAPMLE 5
Preparation of
(R)-Aminomethylvalyl-(R)-aminomethylalanyl-(R)-aminomethyll-
eucyl-(R)-Aminomethylvalyl-(R)-aminomethylalanyl-(R)-aminomethylleucinate
Trifluoroacetate:
[0125] The Boc-protected hexapeptide, Benzyl
N-[(tert-Butoxy)carbonyl]-(R)- -aminomethyl
valyl-(R)-aminomethylalanyl-(R)aminomethylleucyl-(R)-aminomet-
hylvalyl-(R)-amino methylalanyl-(R)-aminomethyl leucinate (98 mg,
0.11 mmol) was dissolved in CH.sub.2Cl.sub.2/CF.sub.3COOH 1:1 (1
ml) and stirred for 4.5 h at r.t. The solvent was evaporated, the
residue dried under h.v. and dissolved in MeOH (5 ml). To this
solution 10% Pd/C (15 mg) was added under Ar. The Ar atmosphere was
replaced by H.sub.2, the suspension stirred for 24 h at r.t.. the
catalyst removed by filtration over Celite, and the filtrate
evaporated. The colorless oil obtained was purified by FC
(CH.sub.2Cl.sub.2/MeOH 15:1), some CF.sub.3COOH was added, the
solvent evaporated and the colorless oil dried under h.v. to give
the title compound (83 mg, 96%): glass. .sup.1H-NMR (300 MHz,
CD.sub.30D): 0.85-1.03 (m, 24H, 4 CH(CH.sub.3).sub.2); 1.09-116(m,
6H, 2 CHRCH.sub.3); 1.16-1.31 (m, 2H, 2
CH.sub.2CH(CH.sub.3).sub.2); 1.40-1.71 (m, 4H, 2
CH.sub.2CH(CH.sub.3).sub.2); 1.72-1.95 (m, 2H, 2
CHRCH(CH.sub.3).sub.2); 2.22-2.32 (m, 1 H, COCHR); 2.45-2.54 (m,1
H, COCHR); 2.55-2.84 (m, 4H, 4 COCHR); 3.06-3.43 (m,1 OH,10 NCH);
3.45-3.66 (m, 2H, 2 NCH). .sup.13C-NMR (75 MHz, CD.sub.30D): 16.2;
16.7; 20.2; 20.8; 21.2; 22.6; 23.4; 23.7; 27.2; 27.4; 30.5; 30.9;
40.7; 40.7; 41.2; 41.8; 42.0; 42.6; 42.8; 43.5; 45.0; 45.7; 51.9;
54.1; 174.6; 176.1; 177.1; 177.6; 177.9; 179.0.
[0126] Compounds of formula I in which R and R.sub.3 are both not
H, i.e. .alpha.,.beta. disubstuited compounds, may be prepared by
alkylation of Li.sub.2 derivatives generated from
N-acyl-.beta.-amino esters (D. Seebach, H. Estermann, Tetrahedron
Lett. (1987), 28, 3103 & D. Seebach, H. Estermann, Helv. Chim.
Acta.(1988), 71, 1824).
[0127] Solid Phase Synthesis
[0128] The compounds of the invention may be synthesised using
solid phase synthesis techniques.
[0129] Thus Fmoc-N-Protected .beta.-amino acids of
(S)-configuration bearing the side chains of Ala, Val, Leu and Phe
in the 3- or 2-position are used to synthesise the
.beta.-heptapeptides H-.beta..sup.3-HVal-.beta-
..sup.3-HAla-.beta..sup.3-HLeu-.beta..sup.3-HPhe-.beta..sup.3-HVal-.beta..-
sup.3-HAla-.beta..sup.3-HLeu-OH and
H-.beta..sup.2-HVal-.beta..sup.2-HAla--
.beta..sup.2HLeu-.beta..sup.2-HPhe-.beta..sup.2-HVal-.beta..sup.2-HAla-.be-
ta..sup.2-HLeu-OH by conventional manual solid-phase synthesis
procedures under standard conditions on ortho-chloro-trityl
chloride resin.
[0130] Esterification of Fmoc-.beta..sup.3-HLeu-OH and
Fmoc-.beta..sup.2-HLeu-OH with the the ortho-chloro-trityl resin is
performed according to the method of Barlos et al. (Tetrahedron
Lett. (1989), 30, 3943). The resin (150 mg, 1.05 mmol Cl.sup.31/g)
is swelled in 2 ml CH.sub.2Cl.sub.2 for 10 min. A solution of
Fmoc-.beta..sup.3-HLeu-OH or Fmoc-.beta..sup.2-HLeu-OH (41 mg, 112
.mu.mol) in CH.sub.2Cl.sub.2 (2 ml) and iPr.sub.2EtN (77 .mu.l, 448
.mu.mol) are then added successively and the suspension is mixed
under Argon for 4 h. Subsequently, the resin is filtered and washed
with CH.sub.2Cl.sub.2/MeOH/iPr.sub.2EtN (17:2:1, 3.times.3 min),
CH.sub.2Cl.sub.2 (3.times.3 min), DMF (2.times.3 min),
CH.sub.2Cl.sub.2 (3.times.3 min), MeOH (2.times.3 min). The
substitution of the resin determined on a 3 mg sample by measuring
the absorbance of the dibenzofulvene adduct at 300 nm, is 0.42
(74%) and 0.43 (75%) for Fmoc-.beta..sup.3-HLeu-OH and
Fmoc-.beta..sup.2-HLeu-OH respectively. The Fmoc group is removed
using 20% piperidine in DMF (4 ml, 2.times.20 min) under Ar
bubbling. The resin is then filtered and washed with DMF (6.times.3
min). For each coupling step, a solution of the .beta..sup.2- or
.beta..sup.3-amino acid (3 equiv.), BOP (3 equiv.) and HOBT (3
equiv.) in DMF (2 ml) and iPr.sub.2EtN (9 eq) are added
successively to the resin and the suspension is mixed for 1 h under
Ar. Monitoring of the coupling reaction is performed with
2,4,6-trinitrobenzene-sulphonic acid (TNBS) (W. S. Hancock and J.
E. Battersby, Anal. Biochem. (1976), 71, 260) instead of the
classical ninhydrin test (Fmoc deprotected .beta..sup.3-peptide
resin fails to give a blue colour with ninhydrin). In the case of a
positive TNBS test (indicating incomplete coupling), the suspension
is allowed to react for a further 1 h. The resin is then filtered
and washed with DMF (3.times.3 min) prior to the following Fmoc
deprotection step. After the removal of the last Fmoc protecting
group, the resin is washed with DMF (6.times.3 min),
CH.sub.2Cl.sub.2 (3.times.3 min), Et.sub.2O (3.times.3 min) and
dried under h.v. for 3 h. Finally the peptides are cleaved from the
resin using 2% TFA in CH.sub.2Cl.sub.2 (2 ml, 5.times.15 min) under
Ar. The solvent is removed and the oily residues are triturated in
ether to give the crude heptapeptides
H-.beta..sup.3-HVal-.beta..sup.3-HAla-.beta..sup.3-HLeu-.beta..sup.3-HPhe-
-.beta..sup.3-HVal-.beta..sup.3-HAla-.beta..sup.3-HLeu-OH (68 mg,
89%) and
H-.beta..sup.2-HVal-.beta..sup.2-HAla-.beta..sup.2-HLeu-.beta..sup.2-HPhe-
-.beta..sup.2-HVal-.beta..sup.2-HAla-.beta..sup.2-HLeu-OH (60 mg,
79%) as white solids. The compounds are further purified by
HPLC.
[0131] Peptides Comprising .beta..sup.2- and .beta..sup.3-amino
acids
[0132] Peptides comprising a mixture of .beta..sup.2- and
.beta..sup.3-amino acids are prepared essentially as described
above for preparation of .beta..sup.2- and .beta..sup.3-peptides
from appropriate .beta..sup.2- and .beta..sup.3-amino acid starting
materials. For example the following compounds are prepared:
[0133] 3 i) (2S
)-2-{(3S)-(3-tert-butoxycarbonylamino-butyrylamino)-methyl-
}-4-methyl-pentanoic acid benzylester; Smp: 113.0-113.5.degree. C.,
R.sub.f (Pentane/EE 2:1)=0.25, [.alpha.].sub.D.sup.RT=-23.0
(c=0.85, CHCl.sub.3)
[0134] 3 ii)
(3S)-3-(3-tert-butoxycarbonylamino-(2R)-2-methyl-propionylami-
no)-5-methyl-hexanoic acid benzylester; Smp: 113.5-114.0.degree.
C., R.sub.f (Pentane/EE 1:1)=4.5, [.alpha.].sub.D.sup.RT =+7.9
(c=0.97, CHCl.sub.3)
[0135] 3 iii) (2S
)-2-({3-[(2S)-2-(tert-butoxycarbonylamino-methyl)-(3S)-3-
-methyl-butyrylamino]-butyrylamino}-methyl)-4-methyl-pentanoic acid
benzylester; Smp: 167.0-168.0.degree. C., R.sub.f
(CH.sub.2Cl.sub.2/MeOH 97:3)=0.30, [.alpha.].sub.D.sup.RT=+10.6
(c=0.95, CHCl.sub.3)
[0136] 3 iv)
(3S)-3-[(3R)-3-(3-tert-butoxycarbonylamino-4-methyl-pentanoyl-
amino)-(2S)-2-methyl-propionylamino]-5-methyl-hexanoic acid
benzylester; Smp: 126.0-127.0.degree. C., R.sub.f
(CH.sub.2Cl.sub.2/MeOH 97:3)=0.23, [.alpha.].sub.D.sup.RT=+65.75
(c=0.71, CHCl.sub.3)
[0137] 3 v)
(3S)-3-(3-[3-[2-({3-[2-tert-butyloxycarbonylamino-methyl)-3-me-
thyl-(2S)butyrylamino]-(3S)-butyrylamino)-4-methyl-(2S)-pentanoylamino]-4--
methyl-(3R)-pentanoylamino}-(2S)-2-methyl-proionylamino)-5-methyl-hexanoic-
acid benzylester; Smp: 209.0-210.0.degree. C., R.sub.f
(CH.sub.2Cl.sub.2/MeOH 96:4)=0.23, [.alpha.].sub.D.sup.RT=+141.10
(c=0.80, CHCl.sub.3)
[0138] 3 vi) (3S
)-3-(3-[3-[2-({3-[2-aminomethyl-3-methyl-(2S)butyrylamino-
]-(3S)-butyrylamino)-4-methyl-(2S)-pentanoylamino]-4-methyl-(3R)-pentanoyl-
amino}-(2S)-2-methyl-proionylamino)-5-methyl-hexanoic acid
hydrochloride; characterised by NMR and MS(FAB) spectroscopy
[0139] 3 vii)
BocNH.beta..sup.2-HVal-.beta..sup.3-HAla-.beta..sup.2-HLeu-.-
beta..sup.3-HVal-.beta..sup.2-HAla-.beta..sup.3-HLeu-.beta..sup.2-HVal-.be-
ta..sup.3-HAla-.beta..sup.2-HLeu-.beta..sup.3-HVal-.beta..sup.2-HAla-.beta-
..sup.3-HLeu-OBn; Smp: 220.0-221.0.degree. C., R.sub.f
(CH.sub.2Cl.sub.2/Et.sub.2O/MeOH 48:48:4)=0.66,
[.alpha.].sub.D.sup.RT=+2- 16.38 (c=0.81, CHCl.sub.3)
[0140] 3 viii) TFA.
H.sub.2N-.beta..sup.2-HVal-.beta..sup.3-HAla-.beta..su-
p.2-HLeu-.beta..sup.3-HVal-.beta..sup.2-HAla-.beta..sup.3-HLeu-.beta..sup.-
2-HVal-.beta..sup.3-HAla-.beta..sup.2-HLeu-.beta..sup.3-HVal-.beta..sup.2--
HAla-.beta..sup.3-HLeu-OH; characterised by NMR and MS(FAB)
spectroscopy
[0141] 3 ix) Benzyl
(2R)-2-{(2R)-3-[(tert-butoxy)carbonylamino]-2-methylpr-
opanoyl-amonomethyl}-4-methylpentanoate;
[.alpha.].sub.D.sup.RT=+53.3 (c=1.07, CHCl.sub.3)
[0142] 3 x) (3S )-3-((3S
)-3-tert-butoxycarbonylamino-butyrylamino)-5-meth-
yl-hexanoicacidbezylester; Smp: 122.5-123.5.degree. C., R.sub.f
(Pentane/EE 3:1)=0.08, [.alpha.].sub.D.sup.RT=-34.70 (c=0.98,
CHCl.sub.3)
[0143] 3 xi) Benzyl
(2R)-2-{2R)-2-{(2R)-2-[(tert-butoxy)carbonylamino]meth-
yl-3-methyl-butanoylamino}-2-methylpropanoylaminomethyl}-4-methylpentanoat-
e; [.alpha.].sub.D.sup.RT=69.62 (c=0.88, CHCl.sub.3)
[0144] 3 xii)
(3S)-3-[(3R)-2-{(2R)-2-[(tert-butoxycarbonylamino-4-methyl-p-
entanoylamino)-(3S)-butyrylamino]-5-methyl-hexanoic acid
benzylester; Smp: 178.5-179.5.degree. C., R.sub.f (CHCl.sub.3/MeOH
97:3)=0.37, [.alpha.].sub.D.sup.RT=-35.03 (c=0.99, CHCl.sub.3)
[0145] 3 xiii) (3S
)-3-(3-{3-[2-({3-[2-(tert-butoxycarbonylamino-methyl)-3-
-methyl-(2S)-butyrylamino]-2-methyl-(2S)-propionylamino}-methyl)-4-methyl--
(2S)-pentanoylamino]-4-methyl-(3R)-pentanoylamino}-(3S)-butyrylamino)-5-me-
thyl-hexanoic acid benzylester; Smp: 221.0-222.0.degree. C.,
R.sub.f (CHCl.sub.3/Et.sub.2O/MeOH 75:23:4)=0.12,
[.alpha.].sub.D.sup.RT=+88.98 (c=0.57, CHCl.sub.3)
[0146] 3 xiv) (3S
)-3-(3-{3-[2-({3-[2-aminomethyl-3-methyl-(2S)-butyrylami-
no]-2-methyl-(2S)-propionylamino}-methyl)-4-methyl-(2S)-pentanoylamino]-4--
methyl-(3R)-pentanoylamino}-(3S)-butyrylamino)-5-methyl-hexanoic
acid trifluoroacetate; characterised by NMR and MS (FAB)
spectroscopy
[0147] Struture Determinations, Results and Analysis
[0148] Methods, analysis and results obtained for structural
determinations for the compounds of the invention are described in
detail in publications Nos. 1 and 2 above; including circular
dichroism (CD) spectroscopy, NMR and X-ray crystallography
determinations. These publications also include description of the
methods used and results obtained in studies of the stability of
the .beta.-peptides of the invention to enzymatic degradation by
pepsin.
Comparison of a Compound of Formula IV and a Compound of Formula
V
[0149] The CD spectra and stability to enzymatic cleavage of the
product
[(R)-Aminomethylvalyl-(R)-aminomethylalanyl-(R)-aminomethylleucyl-(R)-ami-
nomethylvalyl-(R)-aminomethylalanyl-(R)-aminomethylleucinate
Trifluoroacetate] and the product
[H-(.beta.-HVal-.beta.-HAla-.beta.-HLeu- ).sub.2-OH-TFA] are
compared.
[0150] As discussed in Seebach et al. publications Nos. 1 and 2
identified above, .beta.-peptides such as
H-(.beta.-HVal-.beta.-HAla-.beta.-HLeu).su- b.2-OH-TFA, built from
L-configuration .beta.-amino acids which are unsubstituted at the
.alpha.-carbon atom, form (M) 3.sub.1 helices in MeOH solution
(FIG. 2a). A .beta.-peptide built from .beta.-amino acids which are
unsubstituted at the .beta.-carbon atom may be predicted to have
the opposite (P) helicity as shown in FIG. 2b. As with
(.alpha.-peptides, the presence of a helical secondary structure of
a .beta.-peptide may be corroborated by optical measurements. As
discussed in Seebach et al. publications Nos. 1 and 2 identified
above, characteristic CD spectra have been observed for
.beta.-peptides such as
H-(.beta.-HVal-.beta.-HAla-.beta.-HLeu).sub.2-OH-TFA, with a
typical pattern of a trough at ca. 216 and a peak at ca. 197 nm
(see curve A in FIG. 3 and also FIGS. 1a to 1c). These
characteristics are consistent with the (M) 3.sub.1 helical
structure as shown in FIG. 3a. The CD spectrum in methanol of
(R)-Aminomethylvalyl-(R)-aminomethylalanyl-(R)-am-
inomethylleucyl-(R)-Aminomethylvalyl-(R)-aminomethylalanyl-(R)-aminomethyl
leucinate Trifluoroacetate measured at room temperature is shown by
curve B in FIG. 3. Indeed, the pattern of the spectrum of this
latter compound is similar to that of
H-(.beta.-HVal-.beta.-HAla-.beta.-HLeu).sub.2-OH-TF- A, but the
sign of the cotton effect (CE) is positive at 216 nm (molar
ellipticity .THETA.[10 deg cm.sup.2 mol.sup.-1]=3.3.times.10.sup.4
vs -4.9.times.10.sup.4) and negative at 198 nm
(.THETA.=-7.9.times.10.sup.4 vs 9.1.times.10.sup.4); such mirror
image-type spectra result from the two isomers. This is consistent
with (R)-Aminomethylvalyl-(R)-aminomethyl- alanyl-(R)-amino
methylleucyl-(R)-aminomethylvalyl-(R)-aminomethylalanyl-(-
R)-aminomethylleucinate Trifluoroacetate forming a right-handed or
(P) helix, the backbone of which has a mirror-image relationship
with that of H-(.beta.-HVal-.beta.-HAla-.beta.-HLeu).sub.2-OH-TFA
(see FIG. 2). The less intense CE of the former compared to the
latter may be indicative of a less stable structure. In order to
examine the stability, the CD spectrum of the former is measured at
-20.degree. C. with the finding of a 40% increase in the molar
ellipticity of the 216 nm CE. More dramatic alterations are found
on change of solvents. Whereas the molar ellipticity at 216 nm
decreases in water from 3.3.times.10.sup.4 (methanol) to
1.7.times.10.sup.4, it increases in acetonitrile to
5.9.times.10.sup.4. Thus it appears that less polar solvents favour
the formation of the secondary structure of
(R)-Aminomethylvalyl-(R)-aminomet-
hylalanyl-(R)-aminomethylleucyl-(R)-aminomethyl
valyl-(R)-aminomethylalany- l-(R)-aminomethylleucinate
Trifluoroacetate.
[0151]
(R)-Aminomethylvalyl-(R)-aminomethylalanyl-(R)-aminomethylleucyl-(R-
)-aminomethylvalyl-(R)-aminomethylalanyl-(R)-aminomethylleucinate
Trifluoroacetate is tested as a substrate for pepsin (as described
for the .beta..sup.3-peptides in Seebach et al. publications Nos. 1
and 2 identified above) and, as for the .beta..sup.3-peptides, is
found to be fully stable for several days under conditions for
which the .alpha.-peptide H-(Val-Ala-Leu).sub.2-OH is degraded
within minutes.
[0152] Peptides comprising .beta..sup.2- and .beta..sup.3-amino
acids
[0153] From structure determinations, peptides comprising alternate
.beta..sup.2- and .beta..sup.3-amino acids, such as the hexapeptide
identified above as compound 3 vi), appear to exhibit a stable
structure, though this structure does not appear to be a 3.sub.1
helical structure. However, compounds stretches of .beta..sup.2-
and .beta..sup.3-amino acids, such as the hexapeptide identified
above a compound 3 xiv), do appear to exhibit 3.sub.1 helical
structure.
[0154] Bioavailability
[0155] The oral bioavailability of a model .beta..sup.3-peptide
according to the invention,
.beta.-HAla-.beta.-HLys-.beta.-HAla-.beta.-HLeu-.beta.--
HLys-.beta.-HAla-.beta.-HLeu, TFA salt is determined in the rat
using standard procedures. The absolute oral bioavailabilty is
found to be about 1%. It is to be expected that greater oral
bioavailablity may be seeen with other compounds of the invention,
having substituents selected to improve this property.
[0156] The above compound is also administered intravenously to
rats and found to have a relatively long terminal half life in
blood of about 10 hours, with a sytemic clearance of about 15
ml/min/kg and a volume of distribution of about 2 l/kg. It appears
therefore that the relatively low oral bioavailability of the
compound is due to low absorption from the gastrointestinal
tract.
[0157] In view of the stable structures which .beta.-peptides
exhibit in solution, their stability to enzymatic degradation and
their encouraging pharmacokinetic properties, the compounds of the
invention have the potential to provide useful pharmaceutical
products.
* * * * *