U.S. patent application number 09/757142 was filed with the patent office on 2001-07-26 for antineoplastic peptides.
This patent application is currently assigned to BASF Aktiengesellschaft Germany. Invention is credited to Amberg, Wilhelm, Barlozzari, Teresa, Bernard, Harald, Buschmann, Ernst, Haupt, Andreas, Hege, Hans-Guenther, Janssen, Bernd, Kling, Andreas, Lietz, Helmut, Ritter, Kurt, Ullrich, Martina, Weymann, Jurgen, Zierke, Thomas.
Application Number | 20010009901 09/757142 |
Document ID | / |
Family ID | 22261867 |
Filed Date | 2001-07-26 |
United States Patent
Application |
20010009901 |
Kind Code |
A1 |
Amberg, Wilhelm ; et
al. |
July 26, 2001 |
Antineoplastic peptides
Abstract
The present invention provides antineoplastic peptides of
formula I, R.sup.1R.sup.2N-CHX-CO-A-B-D-E-(G).sub.s-K I wherein
R.sup.1, R.sup.2, X, A, B, D, E, G, K and s have the meanings
stated in the description. The compounds have antineoplastic
activity.
Inventors: |
Amberg, Wilhelm;
(Friedrichsdorf, DE) ; Barlozzari, Teresa;
(Wellesley, MA) ; Bernard, Harald; (Bad Durkheim,
DE) ; Buschmann, Ernst; (Ludwigshafen, DE) ;
Haupt, Andreas; (Northborough, MA) ; Hege,
Hans-Guenther; (Neustadt, DE) ; Janssen, Bernd;
(Marlborough, MA) ; Kling, Andreas; (Mannheim,
DE) ; Lietz, Helmut; (Neustadt, DE) ; Ritter,
Kurt; (Newton, MA) ; Ullrich, Martina;
(Schriesheim, DE) ; Weymann, Jurgen; (Bad
Durkheim, DE) ; Zierke, Thomas; (Bohl-Iggelheim,
DE) |
Correspondence
Address: |
Carolyn S. Elmore
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
Two Militia Drive
Lexington
MA
02421-4799
US
|
Assignee: |
BASF Aktiengesellschaft
Germany
|
Family ID: |
22261867 |
Appl. No.: |
09/757142 |
Filed: |
January 9, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09757142 |
Jan 9, 2001 |
|
|
|
09097184 |
Jun 12, 1998 |
|
|
|
09097184 |
Jun 12, 1998 |
|
|
|
PCT/EP96/05518 |
Dec 11, 1996 |
|
|
|
Current U.S.
Class: |
530/330 ;
514/19.2; 514/334; 514/338; 530/331; 530/333 |
Current CPC
Class: |
C07K 7/06 20130101; C07K
7/02 20130101; A61P 35/00 20180101; A61K 38/00 20130101; C07K 5/101
20130101 |
Class at
Publication: |
514/18 ; 530/331;
514/17; 530/330; 530/333; 514/334; 514/338 |
International
Class: |
A61K 038/06; C07K
005/083 |
Claims
What is claimed is:
1. Novel peptides of the formula I
R.sup.1R.sup.2N-CHX-CO-A-B-D-E-(G).sub.- s-K I where R.sup.1 is
hydrogen, methyl; or ethyl; R.sup.2 is methyl; or ethyl; or
R.sup.1-N-R.sup.2 together are a pyrrolidine ring; A is a valyl,
isoleucyl, allo-isoleucyl, 2-tert-butylglycyl, 2-ethylglycyl,
norleucyl or norvalyl residue; B is a N-methyl-valyl,
N-methyl-norvalyl, N-methyl-leucyl, N-methyl-isoleucyl,
N-methyl-2-tert-butylglycyl, N-methyl-2-ethylglycyl, or
N-methyl-norleucyl residue; D is a prolyl, homoprolyl,
hydroxyprolyl, or thiazolidine-4-carbonyl residue; E is a prolyl,
homoprolyl, hydroxyprolyl, thiazolidine-4-carbonyl,
trans-4-fluoro-L-prolyl, cis-4-fluoro-L-prolyl,
trans-4-chloro-L-prolyl or cis-4-chloro-L-prolyl residue; X is
ethyl, propyl, butyl, isopropyl, sec. butyl, tert.-butyl,
cyclopropyl, or cyclopentyl; G is a L-2-tert.butylglycyl,
D-2-terr.butylglycyl, D-valyl, D-isoleucyl, D-leucyl, D-norvalyl,
1-aminopentyl-1-carbonyl, or 2,2-dimethylglycyl residue; s is 0 or
1; K is --NH-C.sub.1-8-alkyl, --NH-C.sub.3-8-alkenyl,
--NH-C.sub.3-8-alkinyl, --NH-C.sub.6-8-cycloalkyl,
--NH-C.sub.1-4-alkene-C.sub.3-8-cycloalkyl,
C.sub.1-4-alkyl-N-C.sub.1-6-a- lkyl, in which residues one CH.sub.2
group may be replaced by O or S, one H by phenyl or cyano, or 1, 2
or 3 H by F, except the N-methoxy-N-methylamino, N-benzylamino, or
N-methyl-N-benzylamino residue, or K is 107and the salts thereof
with physiologically tolerated acids.
2. Novel peptides of the formula I
R.sup.1R.sup.2N-CHX-CO-A-B-D-E-(G).sub.- s-K I where R.sup.1 is
hydrogen, methyl; or ethyl; R.sup.2 is methyl; or ethyl; or
R.sup.1-N-R.sup.2 together are a pyrrolidine ring; A is a valyl,
isoleucyl, allo-isoleucyl, 2-tert-butylglycyl, 2-ethylglycyl,
norleucyl or norvalyl residue; B is a N-methyl-valyl,
N-methyl-norvalyl, N-methyl-leucyl, N-methyl-isoleucyl,
N-methyl-2-tert-butylglycyl, N-methyl-2-ethylglycyl, or
N-methyl-norleucyl residue; D is a prolyl, homoprolyl,
hydroxyprolyl, or thiazolidine-4-carbonyl residue; E is a prolyl,
homoprolyl, hydroxyprolyl, thiazolidine-4-carbonyl,
trans-4-fluoro-L-prolyl, cis-4-fluoro-L-prolyl,
trans-4-chloro-L-prolyl or cis-4-chloro-L-prolyl residue; X is
ethyl, propyl, butyl, isopropyl, sec. butyl, tert.butyl,
cyclopropyl, or cyclopentyl; G is a L-2-tert.butylglycyl,
D-2-terr.butylglycyl, D-valyl, D-isoleucyl, D-leucyl, D-norvalyl,
1-aminopentyl-1-carbonyl, or 2,2-dimethylglycyl residue; s is 0 or
1; K --NHCH.sub.3, --NHCH.sub.2CH.sub.3,
--NH(CH.sub.2).sub.2CH.sub.3, --NH(CH.sub.2).sub.3CH.sub.3,
--NH(CH.sub.2).sub.4CH.sub.3, --NH(CH.sub.2).sub.5CH.sub.3,
--NH(CH.sub.2).sub.6CH.sub.3, --NHCH(CH.sub.2).sub.7CH.sub.3,
--NHCH(CH.sub.3).sub.2, --NHCH(CH.sub.3)CH.sub.2CH.sub.3,
--NHCH(CH.sub.2CH.sub.3).sub.2,
--NHCH(CH.sub.2CH.sub.2CH.sub.3).sub.2, --NHC(CH.sub.3).sub.3,
--NHCH(CH.sub.2CH.sub.3)CH.sub.2CH.sub.2CH.sub.3,
--NHCH(CH.sub.3)CH(CH.sub.3).sub.2,
--NHCH(CH.sub.2CH.sub.3)CH(CH.sub.3).- sub.2,
--NHCH(CH.sub.3)C(CH.sub.3).sub.3, --NH-cyclohexyl,
--NH-cycloheptyl, --NH-cyclooctyl, --N(CH.sub.3)OCH.sub.2CH.sub.3,
--N(CH.sub.3)OCH.sub.2CH.sub.2CH.sub.3,
--N(CH.sub.3)OCH(CH.sub.3).sub.2,
--N(CH.sub.3)O(CH.sub.2).sub.3CH.sub.3,
--N(CH.sub.3)OCH.sub.2C.sub.6H.su- b.5,
--NH(CH.sub.2).sub.2C.sub.6H.sub.5,
--NH(CH.sub.2).sub.3C.sub.6H.sub.- 5,
--NHCH(CH.sub.3)C.sub.6H.sub.5,
--NHC(CH.sub.3).sub.2C.sub.6H.sub.5,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.3,
--NHC(CH.sub.3)(CH.sub.2CH.sub.3).- sub.2,
--NHCH[CH(CH.sub.3).sub.2].sub.2, --NHC(CH.sub.3).sub.2CN,
--NHCH(CH.sub.3)CH(OH)C.sub.6H.sub.5, --NHCH.sub.2-cyclohexyl,
--NHCH.sub.2C(CH.sub.3).sub.3, --NHCH.sub.2CH(CH.sub.3).sub.2,
--NHCH.sub.2CF.sub.3, --NHCH(CH.sub.2F).sub.2,
--NHCH.sub.2CH.sub.2F, --NHCH.sub.2CH.sub.2OCH.sub.3,
--NHCH.sub.2CH.sub.2SCH.sub.3, --NHCH.sub.2CHCH.sub.2,
--NH-C(CH.sub.3).sub.2CH.dbd.CH.sub.2,
--NHC(CH.sub.3).sub.2C.ident.CH,
--NHC(CH.sub.2CH.sub.3).sub.2C.ident.CH,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.2OH,
--NH(CH.sub.2CH.sub.2O).sub.2CH.- sub.2CH.sub.3,
--NHC(CH.sub.3).sub.2CH(CH.sub.3).sub.2,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3,
--NHC(CH.sub.3).sub.2CH.su- b.2C.sub.6H.sub.5,
--N(OCH.sub.3)CH(CH.sub.3).sub.2, --N(OCH.sub.3)CH.sub.2CH.sub.3,
--N(OCH.sub.3)CH.sub.2CH.sub.2CH.sub.3,
--N(OCH.sub.3)CH.sub.2C.sub.6H.sub.5, --N(OCH.sub.3)C.sub.6H.sub.5,
--N(CH.sub.3)OC.sub.6H.sub.5, --NHCH[CH(CH.sub.3).sub.2].sub.2,
--N(OCH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3, or K is 108And the
salts thereof with physiologically tolerated acids.
3. Novel peptides of the formula I
R.sup.1R.sup.2N-CHX-CO-A-B-D-E-(G).sub.- s-K I where R.sup.1 is
hydrogen, methyl; or ethyl; R.sup.2 is methyl; or ethyl; A is a
valyl, isoleucyl, 2-tert-butylglycyl, 2-ethylglycyl, norleucyl or
norvalyl residue; B is a N-methyl-valyl, N-methyl-norvalyl,
N-methyl-isoleucyl, N-methyl-2-tert-butylglycyl,
N-methyl-2-ethylglycyl, or N-methyl-norleucyl residue; D is a
prolyl, or thiazolidine-4-carbonyl residue; E is a prolyl,
homoprolyl, thiazolidine-4-carbonyl, trans-4-fluoro-L-prolyl,
cis-4-fluoro-L-prolyl, trans-4-chloro-L-prolyl or
cis-4-chloro-L-prolyl residue; X is ethyl, propyl, isopropyl, sec.
butyl, tert.-butyl, or cyclopropyl; G is a L-2-tert.butylglycyl,
D-2-terr.butylglycyl, D-valyl, D-isoleucyl, D-leucyl, or
2,2-dimethylglycyl residue; s is 0 or 1; K is --NH-C.sub.1-8-alkyl,
--NH-C.sub.6-8-cycloalkyl, --NH-CH.sub.2-cyclohexyl,
C.sub.1-4-alkyl-N-C.sub.1-6-alkyl, in which residues one CH.sub.2
group may be replaced by O, one H by phenyl or 1 or 2 H by F,
except the N-methoxy-N-methylamino, N-benzylamino or
N-methyl-N-benzylamino residue, or K is 109
4. Novel peptides of the formula I
R.sup.1R.sup.2N-CHX-CO-A-B-D-E-(G).sub.- s-K I where R.sup.1 is
methyl; R.sup.2 is methyl; A is a valyl, isoleucyl,
2-tert-butylglycyl, or 2-ethylglycyl; B is a N-methyl-valyl,
N-methyl-isoleucyl, N-methyl-2-tert-butylglycyl,
N-methyl-2-ethylglycyl, or N-methyl-norleucyl residue; D is a
prolyl, or thiazolidine-4-carbonyl residue; E is a prolyl,
trans-4-fluoro-L-prolyl, cis-4-fluoro-L-prolyl,
trans-4-chloro-L-prolyl or cis-4-chloro-L-prolyl residue; X is
ethyl, isopropyl, sec. butyl, or tert.butyl; G is a
L-2-tert.butylglycyl, D-2-terr.butylglycyl, D-valyl, D-isoleucyl,
D-leucyl, or 2,2-dimethylglycyl residue; s is 0 or 1; K is
--NH-C.sub.1-8-alkyl, --NH-C.sub.6-8-cycloalkyl,
--NH-CH.sub.2-cyclohexyl, C.sub.1-4-alkyl-N-C.sub.1-6-alkyl, in
which residues one CH.sub.2 group may be replaced by O, one H by
phenyl or 1 or 2 H by F, except the N-methoxy-N-methylamino,
N-benzylamino or N-methyl-N-benzylamino residue, or K is 110
5. Novel peptides of the formula I
R.sup.1R.sup.2N-CHX-CO-A-B-D-E-(G).sub.- s-K I where R.sup.1 is
methyl; R.sup.2 is methyl; A is a valyl, isoleucyl, or
2-tert-butylglycyl residue; B is a N-methyl-valyl,
N-methyl-isoleucyl, or N-methyl-2-tert-butylglycyl residue; D is a
prolyl, or thiazolidine-4-carbonyl residue; E is a prolyl,
cis-4-fluoro-L-prolyl or cis-4-chloro-L-prolyl residue; X is
isopropyl, sec. butyl, or tert.-butyl; s is 0 or 1; K is
--NHC(CH.sub.3).sub.3, --NHCH(CH.sub.2CH.sub.2)CH(CH.sub.3).sub. 2,
--NHCH(CH.sub.3)C(CH.sub.3).- sub.3,
--N(CH.sub.3)OCH.sub.2CH.sub.3,
--N(CH.sub.3)OCH.sub.2CH.sub.2CH.su- b.3,
--N(CH.sub.3)OCH(CH.sub.3).sub.2,
--N(CH.sub.3)O(CH.sub.2).sub.3CH.su- b.3,
--N(CH.sub.3)OCH.sub.2C.sub.6H.sub. 5,
--NHC(CH.sub.3).sub.2C.sub.6H.- sub.5,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.3,
--NHC(CH.sub.3)(CH.sub.2CH.s- ub.3).sub.2,
--NHCH[CH(CH.sub.3).sub.2].sub.2, --NHC(CH.sub.3).sub.2CN,
--NHCH(CH.sub.3)CH(OH)C.sub.6H.sub.5,
--NH-C(CH.sub.3).sub.2CH.ident.CH.s- ub.2,
--NHC(CH.sub.3).sub.2C.ident.CH,
--NHC(CH.sub.2CH.sub.3).sub.2C.iden- t.CH,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.2OH,
--NHC(CH.sub.3).sub.2CH(CH.- sub.3).sub.2, --NHC(CH.sub.3).sub.
2CH.sub.2CH.sub.2CH.sub.3,
--NHC(CH.sub.3).sub.2CH.sub.2C.sub.6H.sub.5,
--N(OCH.sub.3)CH(CH.sub. 3).sub.2, --N(OCH.sub.3)CH.sub.2CH.sub.3,
--N(OCH.sub.3)CH.sub.2CH.sub.2C- H.sub.3,
--N(OCH.sub.3)CH.sub.2C.sub.6H.sub.5, --N(OCH.sub.3)C.sub.6H.sub.-
5, --N(CH.sub.3)OC.sub.6H.sub.5,
--N(OCH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.- sub.3, or K is 111and
the salts therof with physiologically tolerated acids.
6. Novel peptides of the formula I
R.sup.1R.sup.2N-CHX-CO-A-B-D-E-(G).sub.- s-K I where R.sup.1 is
methyl; R.sup.2 is methyl; A is a valyl residue; B is a
N-methyl-valyl residue; D is a prolyl residue; E is a prolyl
residue; X is isopropyl; s is 0 or 1; K is --NHC(CH.sub.3).sub.3,
--NHCH(CH.sub.2CH.sub.2)CH(CH.sub.3).sub.2,
--NHCH(CH.sub.3)C(CH.sub.3).s- ub.3,
--N(CH.sub.3)OCH.sub.2CH.sub.3,
--N(CH.sub.3)OCH.sub.2CH.sub.2CH.sub- .3,
--N(CH.sub.3)OCH(CH.sub.3).sub.2,
--N(CH.sub.3)O(CH.sub.2).sub.3CH.sub- .3,
--N(CH.sub.3)OCH.sub.2C.sub.6H.sub.5,
--NHC(CH.sub.3).sub.2C.sub.6H.su- b.5,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.3,
--NHC(CH.sub.3)(CH.sub.2CH.sub- .3).sub.2,
--NHCH[CH(CH.sub.3).sub.2].sub.2, --NHC(CH.sub.3).sub.2CN,
--NHCH(CH.sub.3)CH(OH)C.sub.6H.sub.5,
--NH-C(CH.sub.3).sub.2CH.dbd.CH.sub- .2,
--NHC(CH.sub.3).sub.2C.ident.CH,
--NHC(CH.sub.2CH.sub.3).sub.2C.ident.- CH,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.2OH,
--NHC(CH.sub.3).sub.2CH(CH.su- b.3).sub.2,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3,
--NHC(CH.sub.3).sub.2CH.sub.2C.sub.6H.sub.5,
--N(OCH.sub.3)CH(CH.sub. 3).sub.2, --N(OCH.sub.3)CH.sub.2CH.sub.3,
--N(OCH.sub.3)CH.sub.2CH.sub.2C- H.sub.3,
--N(OCH.sub.3)CH.sub.2C.sub.6H.sub.5, --N(OCH.sub.3)C.sub.6H.sub.-
5, --N(CH.sub.3)OC.sub.6H.sub.5,
--N(OCH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.- sub.3, or K is 112and
the salts thereof with physiologically tolerated acids.
7. Novel peptides of the formula I
R.sup.1R.sup.2N-CHX-CO-A-B-D-E-(G).sub.- s-K I where R.sup.1 is
methyl; R.sup.2 is methyl; A is a valyl, isoleucyl, or
2-tert-butylglycyl residue; B is a N-methyl-valyl,
N-methyl-isoleucyl, or N-methyl-2-tert-butylglycyl residue; D is a
prolyl, or thiazolidine-4-carbonyl residue; E is a prolyl residue;
X is isopropyl, sec. butyl, or tert.-butyl; G is a
D-2-tert.butylglycyl, D-isoleucyl, 2,2-dimethylglycyl residue,
D-valyl or L-2-tert.butylglycyl; s is 1; K is --NHCH.sub.3,
--NHCH.sub.2CH.sub.3, --NH(CH.sub.2).sub.2CH.s- ub.3,
--NH(CH.sub.2).sub.3CH.sub.3, --NH(CH.sub.2).sub.4CH.sub.3,
--NH(CH.sub.2).sub.5CH.sub.3, --NHCH(CH.sub.3).sub.2,
--NHCH(CH.sub.3)CH.sub.2CH.sub.3, --NHCH(CH.sub.2CH.sub.3).sub.2,
--NHC(CH.sub.3).sub.3, --NH-cyclohexyl, --NHC(CH.sub.3).sub.2CN,
--NCH(CH.sub.3).sub.2C.ident.CH or --NHC(CH.sub.3).sub.2CONH.sub.2;
or K is 113and the salts thereof with physiologically tolerated
acids.
8. Compounds of formula I or salts thereof for use in treating
diseases.
9. The method or preparing compounds of formula I according to
claim 1 characterized in that they are prepared according to known
methods of peptide chemistry.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/097,184, filed Jun. 12, 1998, which is a continuation-in-part of
International Application No. PCT/EP96/05518, filed Dec. 11, 1996,
which designated the United States, published in English, which
claims priority to U.S. patent application No. 08/573,422, filed
Dec. 15, 1995, now abandoned. This application also claims benefit
of U.S. Provisional Application entitled "Antineoplastic Peptides",
which resulted from the conversion of U.S. Ser. No. 08/573,422.
[0002] The entire teachings of the above application(s) are
incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention described herein provides novel peptides and
derivatives thereof which offer potentially improved therapeutic
utilities for the treatment of neoplastic diseases as compared to
dolastatin -10 and -15 (U.S. Pat. Nos. 4,879,276 and 4,816,444) and
the compounds desribed in WO 93/23424.
SUMMARY OF THE INVENTION
[0004] Compounds of this invention include novel peptides of the
formula I
R.sup.1R.sup.2N-CHX-CO-A-B-D-E-(G).sub.s-K I
[0005] where
[0006] R.sup.1 is hydrogen, methyl, or ethyl;
[0007] R.sup.2 is methyl; or ethyl; or
[0008] R.sup.1-N-R.sup.2 together are a pyrrolidine ring;
[0009] A is a valyl, isoleucyl, allo-isoleucyl, 2-tert-butylglycyl,
2-ethylglycyl, norleucyl or norvalyl residue;
[0010] B is a N-methyl-valyl, N-methyl-norvalyl, N-methyl-leucyl,
N-methyl-isoleucyl, N-methyl-2-tert-butylglycyl,
N-methyl-2-ethylglycyl, or N-methyl-norleucyl residue;
[0011] D is a prolyl, homoprolyl, hydroxyprolyl, or
thiazolidine-4-carbonyl residue;
[0012] E is a prolyl, homoprolyl, hydroxyprolyl,
thiazolidine-4-carbonyl, trans-4-fluoro-L-prolyl,
cis-4-fluoro-L-prolyl, trans-4-chloro-L-prolyl or
cis-4-chloro-L-prolyl residue;
[0013] X is ethyl, propyl, butyl, isopropyl, sec. butyl,
tert.-butyl, cyclopropyl, or cyclopentyl;
[0014] G is a L-2-tert.butylglycyl, D-2-terr.butylglycyl, D-valyl,
D-isoleucyl, D-leucyl, D-norvalyl, 1-aminopentyl-1-carbonyl, or
2,2-dimethylglycyl residue;
[0015] s is 0 or 1;
[0016] K is --NH-C.sub.1-8-alkyl, --NH-C.sub.3-8-alkenyl,
--NH-C.sub.3-8-alkinyl, --NH-C.sub.6-8-cycloalkyl,
--NH-C.sub.1-4-alkene-C.sub.3-8-cycloalkyl,
C.sub.1-4-alkyl-N-C.sub. 1-6-alkyl, in which residues one CH.sub.2
group may be replaced by O or S, one H by phenyl or cyano, or 1, 2
or 3 H by F, except the N-methoxy-N-methylamino, N-benzylamino, or
N-methyl-N-benzylamino residue, or K is 1
[0017] and the salts thereof with physiologically tolerated
acids.
DETAILED DESCRIPTION OF THE INVENTION
[0018] In specific embodiments of the compounds of formula I, K may
be --NHCH.sub.3, --NHCH.sub.2CH.sub.3,
--NH(CH.sub.2).sub.2CH.sub.3, --NH(CH.sub.2).sub.3CH.sub.3,
--NH(CH.sub.2).sub.4CH.sub.3, --NH(CH.sub.2).sub.5CH.sub.3,
--NH(CH.sub.2).sub.6CH.sub.3, --NHCH(CH.sub.2).sub.7CH.sub.3,
--NHCH(CH.sub.3).sub.2, --NHCH(CH.sub.3)CH.sub.2CH.sub.3,
--NHCH(CH.sub.2CH.sub.3).sub.2,
--NHCH(CH.sub.2CH.sub.2CH.sub.3).sub. 2, --NHC(CH.sub.3).sub.3,
--NHCH(CH.sub.2CH.sub.3)CH.sub.2CH.sub.2CH.sub.3,
--NHCH(CH.sub.3)CH(CH.s- ub.3).sub.2, --NHCH(CH.sub.
2CH.sub.3)CH(CH.sub.3).sub.2, --NHCH(CH.sub.3)C(CH.sub.3).sub.3,
--NH-cyclohexyl, --NH-cycloheptyl, --NH-cyclooctyl,
--N(CH.sub.3)OCH.sub.2CH.sub.3, --N(CH.sub.3)OCH.sub.2CH-
.sub.2CH.sub.3, --N(CH.sub.3)OCH(CH.sub.3).sub.2,
--N(CH.sub.3)O(CH.sub.2)- .sub.3CH.sub.3,
--N(CH.sub.3)OCH.sub.2C.sub.6H.sub.5,
--NH(CH.sub.2).sub.2C.sub.6H.sub.5,
--NH(CH.sub.2).sub.3C.sub.6H.sub.5, --NHCH(CH.sub.3)C.sub.6H.sub.5,
--NHC(CH.sub.3).sub.2C.sub.6H.sub.5,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.3,
--NHC(CH.sub.3)(CH.sub.2CH.sub.3).- sub.2,
--NHCH[CH(CH.sub.3).sub.2].sub.2, --NHC(CH.sub.3).sub.2CN,
--NHCH(CH.sub.3)CH(OH)C.sub.6H.sub.5, --NHCH.sub.2-cyclohexyl,
--NHCH.sub.2C(CH.sub.3).sub.3, --NHCH.sub.2CH(CH.sub.3).sub.2,
--N(CH.sub.3).sub.2, --N(CH.sub.2CH.sub.3).sub.2,
--N(CH.sub.2CH.sub.2CH.- sub.3).sub.2, --NHCH.sub.2CF.sub.3,
--NHCH(CH.sub.2F).sub.2, --NHCH.sub.2CH.sub.2F,
--NHCH.sub.2CH.sub.2OCH.sub.3, --NHCH.sub.2CH.sub.2SCH.sub.3,
--NHCH.sub.2CHCH.sub.2, --NH-C(CH.sub.3).sub.2CH.dbd.CH.sub.2,
--NHC(CH.sub.3).sub.2C.ident.CH,
--NHC(CH.sub.2CH.sub.3).sub.2C.ident.CH,
--NHC(CH.sub.3).sub.2CH.sub.2CH.- sub.2OH,
--NH(CH.sub.2CH.sub.2O).sub.2CH.sub.2CH.sub.3,
--NHC(CH.sub.3).sub.2CH(CH.sub.3).sub.2,
--NHC(CH.sub.3).sub.2CH.sub.2CH.- sub.2CH.sub.3,
--NHC(CH.sub.3).sub. 2CH.sub.2C.sub.6H.sub.5,
--N(OCH.sub.3)CH(CH.sub.3).sub.2, --N(OCH.sub.3)CH.sub.2CH.sub.3,
--N(OCH.sub.3)CH.sub.2CH.sub.2CH.sub.3,
--N(OCH.sub.3)CH.sub.2C.sub.6H.su- b.5,
--N(OCH.sub.3)C.sub.6H.sub.5, --N(CH.sub.3)OC.sub.6H.sub.5,
--NHCH[CH(CH.sub.3).sub.2].sub.2,
--N(OCH.sub.3)CH.sub.2CH.sub.2CH.sub.2C- H.sub.3, or the special
ring systems mentioned above.
[0019] In one embodiment of the compounds of formula I described
above, s is 0 and E is homoprolyl or hydroxyprolyl.
[0020] Preferred are compounds of the formula I where the
substituents R.sup.1, R.sup.2, A, B, D, E, X, G and s have the
following meanings:
[0021] R.sup.1 hydrogen, methyl, or ethyl, especially methyl;
[0022] R.sup.2 methyl or ethyl, especially methyl;
[0023] A valyl, isoleucyl, 2-tert-butylglycyl, 2-ethylglycyl,
norleucyl or norvalyl, especially valyl, isoleucyl,
2-tert-butylglycyl, 2-ethylglycyl,
[0024] B N-methyl-valyl, N-methyl-norvalyl, N-methyl-isoleucyl,
N-methyl-2-tert-butylglycyl, N-methyl-2-ethylglycyl, or
N-methyl-norleucyl, especially N-methyl-valyl,
N-methyl-2-ethylglycyl, N-methyl-norleucyl, N-methyl-isoleucyl, or
N-methyl-2-tert.butyl-glycyl;
[0025] D prolyl, homoprolyl or thiazolidine-4-carbonyl, especially
prolyl or thiazolidine-4-carbonyl;
[0026] E prolyl, homoprolyl, thiazolidine-4-carbonyl,
trans-4-fluoro-L-prolyl, cis-4-fluoro-L-prolyl,
trans-4-chloro-L-prolyl or cis-4-chloro-L-prolyl, especially
prolyl, trans-4-fluoro-prolyl, cis-4-fluoro-prolyl,
trans-4-chloro-prolyl, or cis-4-chloro-prolyl;
[0027] X ethyl, propyl, isopropyl, sec.butyl, tert.butyl or
cyclo-propyl, especially ethyl, isopropyl, sec.butyl or
tert.butyl;
[0028] G L-2-tert.butylglycyl, D-2-tert.butylglycyl, D-valyl,
D-isoleucyl, D-leucyl or 2,2-dimethylglycyl residue;
[0029] s 0 or 1.
[0030] Preferred meanings for K are:
[0031] --NH-C.sub.1-8-alkyl, 'NH-C.sub.6-8-cycloalkyl,
--NH-CH.sub.2-cyclohexyl, C.sub.1-4-alkyl-N-C.sub.1-6-alkyl, in
which residues one CH.sub.2 group may be replaced by O, one H by
phenyl or 1 or 2 H by F, except the N-methoxy-N-methylamino,
N-benzylamino, or N-methyl-N-benzylamino residue, or K is 2
[0032] More preferred K is --NHCH.sub.3, --NHCH.sub.2CH.sub.3,
--NH(CH.sub.2).sub.2CH.sub.3, --NH(CH.sub.2).sub.3CH.sub.3,
--NH(CH.sub.2).sub.4CH.sub.3, --NH(CH.sub.2).sub.5CH.sub.3,
--NH(CH.sub.2).sub.6CH.sub.3, -- NH(CH.sub.2).sub.7CH.sub.3,
--NHCH(CH.sub.3).sub.2, --NHCH(CH.sub.3)CH.sub.2CH.sub.3,
--NHCH(CH.sub.2CH.sub.3).sub.2,
--NHCH(CH.sub.2CH.sub.2CH.sub.3).sub.2, --NHC(CH.sub.3).sub.3,
--NHCH(CH.sub.2CH.sub.3)CH.sub.2CH.sub.2CH.sub.3,
--NHCH(CH.sub.3)CH(CH.sub.3).sub.2,
--NHCH(CH.sub.2CH.sub.3)CH(CH.sub.3).- sub.2,
--NHCH(CH.sub.3)C(CH.sub.3).sub.3, --NH-cyclohexyl,
--NH-cycloheptyl, --NH-cyclooctyl, --N(CH.sub.3)OCH.sub.2CH.sub.3,
--N(CH.sub.3)OCH.sub.2CH.sub.2CH.sub.3,
--N(CH.sub.3)OCH(CH.sub.3).sub.2, --N(OCH.sub.3)CH(CH.sub.3).sub.2,
--N(CH.sub.3)OCH.sub.2C.sub.6H.sub.5,
--NH(CH.sub.2).sub.2C.sub.6H.sub.5,
--NH(CH.sub.2).sub.3C.sub.6H.sub.5, --NHCH(CH.sub.3)C.sub.6H.sub.5,
--NHC(CH.sub.3).sub.2C.sub.6H.sub.5,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.3,
--NHC(CH.sub.3)(CH.sub.2CH.sub.3).- sub.2,
--NHCH(CH.sub.3)CH(OH)C.sub.6H.sub.5, --NHCH.sub.2-cyclohexyl,
--N(CH.sub.3).sub.2, --N(CH.sub.2CH.sub.3).sub.2,
--N(CH.sub.2CH.sub.2CH.- sub.3).sub.2, --NHCH(CH.sub.2F).sub.2,
--NHC(CH.sub.3)CH.ident.CH.sub.2, --NHC(CH.sub.3).sub.2CN,
--NHC(CH.sub.3).sub.2C.dbd.CH, --NHC(CH.sub.3).sub.2CONH.sub.2,
--NHCH[CH(CH.sub.3).sub.2].sub.2,
--N(OCH.sub.3)CH.sub.2C.sub.6H.sub.5,
--N(OCH.sub.3)CH.sub.2CH.sub.3,
--N(OCH.sub.3)CH.sub.2CH.sub.2CH.sub.3,
--N(OCH.sub.3)CH.sub.2CH.sub.2CH.- sub.2CH.sub.3, 3
[0033] In one embodiment of the preferred compounds of formula I
described above, s is 0 and E is homoprolyl or hydroxyprolyl.
[0034] Especially preferred are compounds of the formula I
where
[0035] R.sup.1 and R.sup.2 are methyl,
[0036] A is a valyl, isoleucyl, 2-tert.-butylglycyl residue
[0037] B is a N-methylvalyl, N-methyl-isoleucyl,
N-methyl-2-tert.-butylgly- cyl residue,
[0038] D is a prolyl or thiazolidine-4-carbonyl residue,
[0039] E is a prolyl, cis-4-fluoro-L-prolyl, or
cis-4-chloro-L-prolyl residue,
[0040] X is a isopropyl, sec.-butyl, or tert.-butyl residue,
[0041] s is 0, and
[0042] K is --NHCH(CH.sub.3).sub.2,
--NHCH(CH.sub.3)CH.sub.2CH.sub.3, --NHCH(CH.sub.2CH.sub.3).sub.2,
--NHCH(CH.sub.2CH.sub.2CH.sub.3).sub.2, --NHC(CH.sub.3).sub.3,
--NHCH(CH.sub.2CH.sub.3)CH.sub.2CH.sub.2CH.sub.3,
--NHCH(CH.sub.3)CH(CH.sub.3).sub.2,
--NHCH(CH.sub.2CH.sub.3)CH(CH.sub.3).- sub.2,
--NHCH(CH.sub.3)C(CH.sub.3).sub.3, --NH-cycloheptyl,
--NH-cyclooctyl, --N(CH.sub.3)OCH.sub.2CH.sub.3,
--N(CH.sub.3)OCH.sub.2CH- .sub.2CH.sub.3,
--N(CH.sub.3)OCH(CH.sub.3).sub.2, --N(OCH.sub.3)CH(CH.sub.-
3).sub.2, --N(CH.sub.3)OCH.sub.2C.sub.6H.sub.5,
--NH(CH.sub.2).sub.2C.sub.- 6H.sub.5,
--NH(CH.sub.2).sub.3C.sub.6H.sub.5, --NHCH(CH.sub.3)C.sub.6H.sub-
.5, --NHC(CH.sub.3).sub.2C.sub.6H.sub.5,
--NHC(CH.sub.3).sub.2CH.sub.2CH.s- ub.3,
--NHC(CH.sub.3)(CH.sub.2CH.sub.3).sub.2,
--NHCH(CH.sub.3)CH(OH)C.sub- .6H.sub.5, --NHCH(CH.sub.2F).sub.2,
--NHC(CH.sub.3).sub.2CH.sub.2CH.sub.2O- H,
--NH(CH.sub.2CH.sub.2O).sub.2CH.sub.2CH.sub.3,
--NHC(CH.sub.3).sub.2CH.- dbd.CH.sub.2,
--NHC(CH.sub.3).sub.2CH(CH.sub.3).sub.2,
--N(OCH.sub.3)CH.sub.2CH.sub.3,
--N(OCH.sub.3)CH.sub.2CH.sub.2CH.sub.3,
--N(OCH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3,
--NHC(Ch.sub.3).sub.2CN, --NHC(CH.sub.3).sub.2C.ident.CH,
--NHCH[CH(CH.sub.3).sub.2].sub.2, --NHC(CH.sub.3).sub.2CONH.sub.2,
--NHC(CH.sub.3).sub.2CH.sub.2C.sub.6H.su- b.5,
--N(OCH.sub.3)C.sub.6H.sub.5, --N(OCH.sub.3)CH.sub.2C.sub.6H.sub.5,
4
[0043] This invention also provides methods for preparing the
compounds of formula I, pharmaceutical compositions containing such
compounds together with a pharmaceutically acceptable carrier and
methods for using same for treating cancer in mammals.
[0044] The new compounds may be present as salts with
physiologically tolerated acids such as: hydrochloric acid, citric
acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic
acid, acetic acid, formic acid, maleic acid, fumaric acid, malic
acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid,
L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, glucuronic
acid, oxalic acid, ascorbic acid and acetylglycine.
[0045] The novel compounds can be prepared by known methods of
peptide chemistry. Thus, the peptides can be assembled sequentially
from amino acids or by linking suitable small peptide fragments. In
the sequential assemblage, starting at the C terminus the peptide
chain is extended stepwise by one amino acid each time. In fragment
coupling it is possible to link together fragments of different
lengths, and the fragments in turn can be obtained by sequential
assemblage from amino acids or themselves by fragment-coupling.
[0046] Both in the sequential assemblage and in the fragment
coupling it is necessary to link the units by forming an amide
linkage. Enzymatic and chemical methods are suitable for this.
[0047] Chemical methods for forming the amide linkage are described
in detail by Mueller, Methoden der organischen Chemie Vol. XV/2, pp
1 to 264, Thieme Verlag, Stuttgart, 1974; Stewart, Young, Solid
Phase Peptide Synthesis, pp 31 to 34, 71 to 82, Pierce Chemical
Company, Rockford, 1984; Bodanszky, Klausner, Ondetti, Peptide
Synthesis, pp 85 to 128, John Wiley & Sons, New York, 1976; The
Practice of Peptide Synthesis, M. Bodanszky, A. Bodanszky,
Springer-Verlag, 1994, and other standard works on peptide
chemistry. Particular preference is given to the azide method, the
symmetric and mixed anhydride method, in situ generated or
preformed active esters, the use of urethane protected N-carboxy
anhydrides of amino acids and the formation of the amide linkage
using coupling reagents, especially dicyclohexylcarbodiimide (DCC),
diisopropylcarbodiimide (DIC),
1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquino- line (EEDQ),
pivaloylchloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimi- de
hydrochloride (EDCI), n-propanephosphonic anhydride (PPA),
N,N-bis(2-oxo-3-oxazolodinyl)-amidophosphoryl chloride (BOP-C1),
bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBrop),
diphenylphosphoryl azide (DPPA), Castro's reagent (BOP, PyBop),
O-benzotriazolyl-N,N,N',N'-tetramethyluronium salts (HBTU),
O-azabenzotriazolyl-N,N,N',N'-tetramethyluronium salts (HATU),
diethylphosphoryl cyanide (DEPCN),
2,5-diphenyl-2,3-dihydro-3-oxo-4-hydro- xythiophene dioxide
(Steglich's reagent; HOTDO) and 1,1'-carbonyldiimidazole (CDI). The
coupling reagents can be employed alone or in combination with
additives such as N,N-dimethyl-4-aminopyridi- ne (DMAP),
N-hydroxy-benzotriazole (HOBt), N-hydroxybenzotriazine (HOOBt),
Azabenzotriazole, N-hydroxysuccinimide (HOSu) or
2-hydroxypyridine.
[0048] Whereas it is normally possible to dispense with protective
groups in enzymatic peptide synthesis, reversible protection of
reactive groups not involved in formation of the amide linkage is
necessary for both reactants in chemical synthesis. Three
conventional protective group techniques are preferred for the
chemical peptide synthesis: the benzyloxycarbonyl (Z), the
t-butoxycarbonyl (Boc) and the 9-fluorenylmethoxycarbonyl (Fmoc)
techniques.
[0049] Identified in each case is the protective group on the
alpha-amino group of the chain-extending unit. A detailed review of
amino-acid protective groups is given by Mueller, Methoden der
organischem Chemie vol. XV/1, pp 20 to 906, Thieme Verlag,
Stuttgart, 1974. The units employed for assembling the peptide
chain can be reacted in solution, in suspension or by a method
similar to that described by Merrifield in J. Amer. Chem. Soc. 85
(1963) 2149.
[0050] Suitable for peptide synthesis in solution are all solvents
which are inert under the reaction conditions, especially water,
N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),
acetonitrile, dichloromethane (DCM), ethyl acetate, 1,4-dioxane,
tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and mixtures of
the said solvents.
[0051] Peptide synthesis on the polymeric support can be carried
out in all inert organic solvents in which the amino-acid
derivatives used are soluble. However, preferred solvents
additionally have resin-swelling properties, such as DMF, DCM, NMP,
acetonitrile and DMSO, and mixtures of these solvents. After
synthesis is complete, the peptide is cleaved off the polymeric
support. The conditions under which cleavage off the various resin
types is possible are disclosed in the literature. The cleavage
reactions most commonly used are acid- and palladium-catalyzed,
especially cleavage in liquid anhydrous hydrogen fluoride, in
anhydrous trifluoromethanesulfonic acid, in dilute or concentrated
trifluoroacetic acid, palladium-catalyzed cleavage in THF or
THF-DCM mixtures in the presence of a weak base such as morpholine
or cleavage in acetic acid/dichloromethane/trifluoroethanol
mixtures. Depending on the chosen protective groups, these may be
retained or likewise cleaved off under the cleavage conditions.
[0052] Partial deprotection of the peptide may also be worthwhile
when certain derivatization reactions are to be carried out.
[0053] Peptides dialkylated at the N-terminus can be prepared
either by coupling on the appropriate N,N-di-alkylamino acids in
solution or on the polymeric support, by reductive alkylation of
the resin-bound peptide in DMF/1% acetic acid with NaCNBH.sub.3 and
the appropriate aldehydes, by hydrogenation of the peptide in
solution in the presence of aldehyde or ketone and Pd/C.
[0054] The various non-naturally occurring amino acids as well as
the various non-amino acid moieties disclosed herein may be
obtained from commercial sources or synthesized from
commercially-available materials using methods known in the art.
For example, amino acids building blocks with R.sup.1 and R.sup.2
moieties can be prepared according to E. Wuensch, Houben Weyl,
Meth. d. Org. Chemie, Bd. XV, 1, p. 306 following, Thieme Verlag
Stuttgart 1974 and Literature cited therein.
[0055] The compounds of this invention may be used to inhibit or
otherwise treat solid tumors (e.g. tumors of the lung, breast,
colon, prostate, bladder, rectum, or endometrial tumors) or
hematological malignancies (e.g. leukemias, lymphomas) by
administration of the compound to the mammal.
[0056] It is a special advantage of the new compounds that they are
very resistant to enzymatic degradation and can also be
administered orally.
[0057] Administration may be by any of the means which are
conventional for pharmaceutical, preferably oncological, agents,
including oral and parenteral means such as subcutaneously,
intravenously, intramuscularly and intraperitoneally.
[0058] The compounds may be administered alone or in the form of
pharmaceutical compositions containing a compound of formula I
together with a pharmaceutically accepted carrier appropriate for
the desired route of administration. Such pharmaceutical
compositions may be combination products, i.e., may also contain
other therapeutically active ingredients.
[0059] The dosage to be administered to the mammal with contain an
effective tumor-inhibiting amount of active ingredient which will
depend upon conventional factors including the biological activity
of the particular compound employed; the means of administration;
the age, health and body weight of the recipient; the nature and
extent of the symptoms; the frequency of treatment; the
administration of other therapies; and the effect desired. A
typical daily dose will be about 0.05 to 50 milligrams per kilogram
of body weight on oral administration and about 0.01 to 20
milligrams per kilogram of body weight on parenteral
administration.
[0060] The novel compounds can be administered in conventional
solid or liquid pharmaceutical administration forms, e.g. uncoated
or (film-)coated tablets, capsules, powders, granules,
suppositories or solutions. These are produced in a conventional
manner. The active substances can for this purpose be processed
with conventional pharmaceutical aids such as tablet binders,
fillers preservatives, tablet disintegrants, flow regulators,
plasticizers, wetting agents, dispersants, emulsifiers, solvents,
sustained release compositions, antioxidants and/or propellant
gases (cf. H. Sucker et al.: Pharmazeutische Technologie,
Thieme-Verlag, Stuttgart, 1978). The administration forms obtained
in this way normally contain 1-90% by weight of the active
substance.
[0061] The following examples are intended to illustrate the
invention. The proteinogenous amino acids are abbreviated in the
examples using the known three-letter code. Other abbreviations
used: Me.sub.2Val=N,N-dimethylvaline, MeVal= N-methylvaline.
EXAMPLES
[0062] A. General Procedures
[0063] I. The peptides claimed in claim 1 are either synthesized by
classical solution synthesis using standard Z- and Boc-methodology
as described above or by standard methods of solid-phase synthesis
using Boc and Fmoc protective group techniques.
[0064] In the case of solid phase synthesis, the N,N-dialkylpenta-
or hexapeptide acids are liberated from the solid support and
further coupled with the corresponding C-terminal amines in
solution. BOP-C1 and PyBrop were used as reagents for coupling of
the amino acid following the N-methylamino acids. The reaction
times were correspondingly increased. For reductive alkylation of
the N-terminus, the peptide-resin was deprotected at the N terminus
and then reacted with a 3-fold molar excess of aldehyde or ketone
in DMF/1% acetic acid with addition of 3 equivalents of
NaCNBH.sub.3. After the reaction was complete (negative Kaisertest)
the resin was washed several times with water, isopropanol, DMF and
dichloromethane.
[0065] In solution synthesis, the use of either Boc-protected amino
acid NCAs (N-tert.-butyloxycarbonyl-amino
acid-N-carboxy-anhydrides), Z-protected amino acid NCAs
(N-benzyloxycarbonyl-amino acid-N-carboxy-anhydrides), or the use
of pivaloylchloride as condensing agent respectively is most
advantageous for coupling of the amino acid following the
N-methylamino acids. Reductive alkylation of the N terminus can
e.g. be achieved by reaction of the N-terminally deprotected
peptides or amino acids with the corresponding aldehydes or ketones
using NaCNBH.sub.3 or hydrogen, Pd/C.
[0066] II. Purification and characterization of the peptides
[0067] Purification was carried out by gel chromatography (SEPHADEX
G-10, G-15/10% HOAc, SEPHADEX LH20/MeOH), medium pressure
chromatography (stationary phase: HD-SIL C-18, 20-45 mikron, 100
Angstrom; mobile phase: gradient with A=0.1% TFA/MeOH, B=0.1%
TFA/water), or preparative HPLC (stationary phase: Waters Delta-Pak
C-18, 15 mikron, 100 Angstrom; mobile phase: gradient with A=0.1%
TFA/MeOH, 3=0.1% TFA/water).
[0068] The purity of the resulting products was determined by
analytical HPLC (stationary phase: 100 2.1 mm VYDAC C-18, 5 1, 300
A; mobile phase: acetonitrile-water gradient, buffered with 0.1%
TFA, 40.degree. C.).
[0069] Characterization was by amino-acid analysis and fast atom
bombardment mass spectroscopy.
[0070] B. Specific Procedures
Example 1 (SEQ ID NO: 1)
Me.sub.2Val-Val-MeVal-Pro-Pro-NHCH(CH.sub.3).sub.2
[0071] a) Z-MeVal-Pro-OME
[0072] 66.25 g (250 mmol) Z-MeVal-OH were dissolved in 250 ml dry
dichloromethane. After addition of 36.41 ml (262.5 mmol)
triethylamine, the reaction mixture was cooled to -25.degree. C.
and 32.27 ml (262.5 mmol) pivaloyl chloride were added. After
stirring for 2,5 h, 41.89 g (250 mmol) H-Pro-OMe.times.Chl in 250
ml dichloromethane, neutralized with 36.41 ml (262.5 mmol)
triethylamine at 0.degree. C., were added to the reaction mixture.
Stirring continued for 2 h at -25.degree. C. and overnight at room
temperature. The reaction mixture was diluted with dichloromethane
and thoroughly washed with saturated aqueous NaHCO.sub.3 solution
(3x), water (1x), 5% citric acid (3x) and saturated NaCl solution.
The organic phase was dried over sodium sulfate and evaporated to
dryness. The residue (91.24 g) was stirred with petroleum ether
overnight and filtered. 62.3 g of product were obtained.
[0073] b) H-MeVal-Pro-OMe
[0074] 48.9 g (130 mmol) Z-MeVal-Pro-OMe were dissolved in 490 ml
methanol. After addition of 10.9 ml (130 mmol) concentrated
hydrochloric acid and 2.32 g 10% Palladium/charcoal, the reaction
mixture was hydrogenated. Filtration and evaporation to dryness
yielded 36.32 g of the product.
[0075] c) Z-Val-MeVal-Pro-OMe
[0076] 18.1 g (65 mmol) H-MeVal-Pro-OMe, 21.6 g (78 mmol)
Z-Val-N-carboxyanhydride and 22.8 ml (130 mmol)
diisopropylethylamine were stirred in 110 ml DMF at 40.degree. C.
for 2 d. After evaporation of DMF, dichloromethane was added and
the organic phase washed with saturated aqueous NaHCO.sub.3
solution (3x), water (1x), 4% citric acid (3X) and saturated NaCl
solution. The organic phase was dried over sodium sulfate and
evaporated to dryness. The product (29.3 g) was obtained as a
viscous oil.
[0077] d) H-Val-MeVal-Pro-OMe
[0078] 29.3 g (61.6 mmol) of Z-Val-MeVal-Pro-OMe were dissolved in
230 ml methanol. After addition of 1.15 g 10% Palladium/charcoal,
the reaction mixture was hydrogenated. Filtration and evaporation
to dryness yielded 21.96 g of the product.
[0079] e) Z-Val-Val-MeVal-Pro-OMe
[0080] 15.29 g (61 mmol) Z-Val-OH and 21.96 g (61 mmol)
H-Val-MeVal-Pro-OMe were dissolved in 610 ml dichloromethane and
cooled to 0.degree. C. After addition of 8.16 ml (73.2 mmol)
N-Methylmorpholine, 2.77 g (20.3 mmol) HOBt and 11.73 g (61 mmol)
EDCI, the reaction mixture was stirred overnight at room
temperature, diluted with dichloromethane and thoroughly washed
with saturated aqueous NaHCO.sub.3 solution (3x), water (1x), 5%
citric acid (3X) and saturated NaCl solution. The organic phase was
dried over sodium sulfate and evaporated to dryness to yield 31.96
g of the product.
[0081] f) Z-Val-Val-MeVal-Pro-OH
[0082] 31.96 g (57 mmol) Z-Val-Val-MeVal-Pro-OMe were dissolved in
250 ml methanol. 102.6 ml of a 1 N LiOH solution was added and the
mixture stirred overnight at room temperature. After addition of
500 ml water, the aqueous phase was washed three times with ethyl
acetate, adjusted to pH 2 at 0.degree. C. and extracted three times
with ethyl acetate. The organic phase was dried over sodium sulfate
and evaporated to dryness yielding 30.62 g of the desired product
as a white solid.
[0083] g) Z-Val-Val-MeVal-Pro-Pro-NHCH(CH.sub.3).sub.2
[0084] 2 g (3.35 mmol) Z-Val-Val-MeVal-Pro-OH and 0.664 g (3.35
mmol) H-Pro-NHCH(CH.sub.3).sub.2 were dissolved in 34 ml of dry
dichloromethane. After cooling to 0.degree. C., 1.35 ml (12.1 mmol)
N-methyhnorpholine, 0.114 g (0.84 mmol) HOBt and 0.645 g (3.35
mmol) EDCI were added and the reaction mixture stirred overnight at
room temperature. 80 ml dichloromethane were added and the organic
phase thoroughly washed with saturated aqueous NaHCO.sub.3 solution
(3x), water (1x), 5% citric acid (3x) and saturated NaCl solution
(1x). The organic phase was dried over sodium sulfate and
evaporated to dryness to yield 1.96 g of the product which was used
in the next reaction without further purification.
[0085] h) Me.sub.2Val-Val-MeVal-Pro-Pro-NHCH(CH.sub.3).sub.2
[0086] 1.96 g Z-Val-Val-MeVal-Pro-Pro-NHCH(CH.sub.3).sub.2 were
dissolved in 11 ml methanol. 0.054 g 10% Pd/C were added under
nitrogen atmosphere and the reaction mixture hydrogenated at room
temperature for 4 h. After addition of 0.86 ml (11.24 mmol) of a
37% aqueous formaldehyde solution and 0.281 g 10% Pd/C,
hydrogenation was continued for 5 h. Filtration and evaporation of
the solvent gave rise to 2.77 g of crude product. Further
purification was achieved by dissolving the peptide in water,
adjusting the pH to 2 and extracting the aqueous phase three times
with ethyl acetate. The aqueous phase was then adjusted to pH 8-9
and extrcted four times with dichloromethane. The organic phase was
dried over sodium sulfate to yhield 1.37 g of purified product as a
white foam. The compound was further purified using medium pressure
liquid chromatography (10-50% A in 10 min.; 50-90% A in 320 min.).
Fractions containing the product were combined, lyophilized,
redissolved in water and the pH adjusted to 9 with 1 N LiOH. After
extraction with dichloromethane, the organic phase was dried over
sodium sulfate and evaporated to dryness. Lyophilization led to 500
mg of pure product, which was characterized by fast atom
bombardment mass spectrometry ([M+H].sup.+=593).
Example 2 (SEQ ID NO: 1)
Me.sub.2Val-Val-MeVal-Pro-Pro-NHC(CH.sub.3).sub.3
[0087] i) Z-Val-Val-MeVal-Pro-Pro-NHC(CH.sub.3).sub.3
[0088] 2 g (3.35 mmol) Z-Val-Val-MeVal-Pro-OH and 0.692 g (3.35
mmol) H-Pro-NHC (CH.sub.3).sub.3 were dissolved in 34 ml of dry
dichloromethane. After cooling to 0.degree. C., 1.35 ml (12.1 mmol)
N-methylmorpholine, 0.114 g (0.84 mmol) HOBt and 0.645 g (3.35
mmol) EDCI were added and the reaction mixture stirred overnight at
room temperature. 80 ml dichloromethane were added and the organic
phase thoroughly washed with saturated aqueous NaHCO.sub.3 solution
(3x), water (1x), 5% citric acid (3x) and saturated NaCl solution
(1x). The organic phase was dried over sodium sulfate and
evaporated to dryness to yield 1.8 g of the product which was used
in the next reaction without further purification.
[0089] k) Me.sub.2Val-Val-MeVal-Pro-Pro-NHC(CH.sub.3).sub.3
[0090] 1.8 g Z-Val-Val-MeVal-Pro-Pro-NHC(CH.sub.3).sub.3 were
dissolved in 10 ml methanol. 0.049 g 10% Pd/C were added under
nitrogen atmosphere and the reaction mixture hydrogenated at room
temperature for 4 h. After addition of 0.86 ml (11.24 mmol) of a
37% aqueous formaldehyde solution and 0.252 g 10% Pd/C,
hydrogenation was continued for 5 h. Filtration and evaporation of
the solvent gave rise to 1.82 g of crude product. The compound was
further purified using medium pressure liquid chromatography
910-50% A in 10 min.; 50-90% A in 320 min.). Fractions containing
the product were combined, lyophilized, redissolved in water and
the pH adjusted to 9 with 1 N LiOH. After extraction with
dichloromethane, the organic phase was dried over sodium sulfate
and evaporated to dryness. Lyophilization led to 547 mg of pure
product, which was characterized by fast atom bombardment mass
spectrometry ([M+H].sup.+32 607).
[0091] The following compounds were prepared or can be prepared
according to examples 1 and 2:
[0092] 3. Xaa Val Xab Pro Xac
[0093] 4. Xaa Val Xab Pro Xad
[0094] 5. Xaa Val Xab Pro Xae
[0095] 6. Xaa Val Xab Pro Xaf
[0096] 7. Xaa Val Xab Pro Xag
[0097] 8. Xaa Val Xab Pro Xah
[0098] 9. Xaa Val Xab Pro Xai
[0099] 10. Xaa Val Xab Pro Xak
[0100] 11. Xaa Val Xab Pro Xal
[0101] 12. Xaa Val Xab Pro Xam
[0102] 13. Xaa Val Xab Pro Xan
[0103] 14. Xaa Val Xab Pro Xao
[0104] 15. Xaa Val Xab Pro Xap
[0105] 16. Xaa Val Xab Pro Xaq
[0106] 17. Xaa Val Xab Pro Xar
[0107] 18. Xaa Val Xab Pro Xas
[0108] 19. Xaa Val Xab Pro Xat
[0109] 20. Xaa Val Xab Pro Xau
[0110] 21. Xaa Val Xab Pro Xav
[0111] 22. Xaa Val Xab Pro Xaw
[0112] 23. Xaa Val Xab Pro Xax
[0113] 24. Xdd Val Xab Pro Xay
[0114] 25. Xaa Val Xab Pro Xaz
[0115] 26. Xaa Val Xab Pro Xba
[0116] 27. Xaa Val Xab Pro Xbb
[0117] 28. Xaa Val Xbc Pro Xay
[0118] 29. Xaa Val Xab Pro Xbd
[0119] 30. Xaa Val Xab Pro Xbe
[0120] 31. Xaa Val Xab Pro Xbf
[0121] 32. Xaa Val Xab Pro Xbg
[0122] 33. Xaa Val Xab Pro Xbh
[0123] 34. Xaa Val Xab Pro Xbi
[0124] 35. Xaa Val Xab Pro Xbk
[0125] 36. Xaa Val Xab Pro Xbl
[0126] 37. Xaa Val Xab Pro Xbm
[0127] 38. Xaa Val Xab Pro Xbn
[0128] 39. Xaa Val Xab Pro Xbo
[0129] 40. Xaa Val Xab Pro Xbp
[0130] 41. Xaa Val Xab Pro Xbq
[0131] 42. Xaa Val Xab Pro Xbr
[0132] 43. Xaa Val Xab Pro Xbs
[0133] 44. Xaa Val Xab Pro Xbt
[0134] 45. Xaa Val Xab Pro Xbu
[0135] 46. Xaa Val Xab Pro Xbv
[0136] 47. Xaa Val Xab Pro Xbw
[0137] 48. Xaa Val Xab Pro Xbx
[0138] 49. Xaa Val Xab Pro Xby
[0139] 50. Xaa Val Xab Pro Xbz
[0140] 51. Xaa Val Xab Pro Xca
[0141] 52. Xaa Val Xab Pro Xcb
[0142] 53. Xaa Val Xab Pro Xcc
[0143] 54. Xaa Val Xab Pro Xcd
[0144] 55. Xaa Val Xab Pro Xce
[0145] 56. Xaa Val Xab Pro Xcf
[0146] 57. Xaa Xdf Xab Pro Xay
[0147] 58. Xaa Val Xab Pro Xch
[0148] 59. Xaa Val Xab Pro Xci
[0149] 60. Xaa Val Xab Pro Xck
[0150] 61. Xaa Val Xab Pro Xcl
[0151] 62. Xaa Val Xab Pro Xcm
[0152] 63. Xaa Val Xab Pro Xcn
[0153] 64. Xaa Val Xab Pro Xco
[0154] 65. Xaa Val Xab Pro Xcp
[0155] 66. Xaa Val Xab Pro Xcq
[0156] 67. Xaa Val Xab Pro Xcr
[0157] 68. Xaa Val Xab Pro Xcs
[0158] 69. Xaa Val Xab Pro Xct
[0159] 70. Xaa Val Xab Pro Xcu
[0160] 71. Xcw Val Xab Pro Xcv
[0161] 72. Xcx Val Xab Pro Xcv
[0162] 73. Xaa Val Xab Pro Pro Xcy
[0163] 74. Xaa Val Xab Pro Pro Xcz
[0164] 75. Xaa Val Xda Pro Xcv
[0165] 76. Xaa Xdb Xab Pro Xcv
[0166] 77. Xdc Val Xab Pro Xcv
[0167] 78. Xaa Ile Xab Pro Xcv
[0168] 79. Xdd Val Xab Pro Xcv
[0169] 80. Xde Val Xab Pro Xcv
[0170] 81. Xaa Xdf Xab Pro Xcv
[0171] 82. Xaa Val Xab Pro Xcg
[0172] 83. Xaa Val Xab Pro Pro Xdg
[0173] 84. Xaa Val Xab Pro Pro Xdh
[0174] 85. Xaa Val Xab Pro Pro Xdi
[0175] 86. Xaa Val Xab Pro Pro Xdk
[0176] 87. Xaa Val Xdl Pro Xcv
[0177] 88. Xde Val Xab Pro Xay
[0178] 89. Xaa Val Xdl Pro Xay
[0179] 90. Xaa Val Xab Pro Xdm
[0180] 91. Xaa Val Xab Pro Xdn
[0181] 92. Xaa Val Xab Pro Xdo
[0182] 93. Xaa Val Xab Pro Xdp
[0183] 94. Xaa Val Xab Pro Xdq
[0184] 95. Xaa Val Xab Pro Pro Xdr
[0185] 96. Xaa Val Xab Pro Xds
[0186] 97. Xaa Val Xbc Pro Xcv
[0187] 98. Xaa Ile Xab Pro Xay
[0188] 99. Xcw Val Xab Pro Xay
[0189] 100. Xaa Val Xbc Pro Xal
[0190] 101. Xaa Val Xdl Pro Xal
[0191] 102. Xaa Xdf Xab Pro Xal
[0192] 103. Xaa Ile Xab Pro Xal
[0193] 104. Xdd Val Xab Pro Xal
[0194] 105. Xde Val Xab Pro Xal
[0195] 106. Xcx Val Xab Pro Xcy
[0196] 107. Xcw Val Xab Pro Xal
[0197] 108. Xcx Val Xab Pro Xal
[0198] 109. Xcw Val Xab Pro Xav
[0199] 110. Xcx Val Xab Pro Xav
[0200] 111. Xcw Val Xab Pro Xaw
[0201] 112. Xcx Val Xab Pro Xaw
[0202] 113. Xab Val Xab Pro Xay
[0203] 114. Xab Val Xab Pro Xcv
[0204] 115. Xab Val Xab Pro Xal
[0205] 116. Xab Val Xab Pro Xam
[0206] 117. Xab Val Xab Pro Xan
[0207] 118. Xab Val Xab Pro Xao
[0208] 119. Xab Val Xab Pro Xav
[0209] 120. Xab Val Xab Pro Xaw
[0210] 121. Xab Val Xab Pro Xat
[0211] 122. Xab Val Xab Pro Xau
[0212] 123. Xab Val Xab Pro Xbf
[0213] 124. Xab Val Xab Pro Xbm
[0214] 125. Xab Val Xab Pro Xbn
[0215] 126. Xab Val Xab Pro Xbo
[0216] 127. Xab Val Xab Pro Xch
[0217] 128. Xaa Val Xab Pro Xdt
[0218] 129. Xaa Val Xab Pro Xdu
[0219] 130. Xaa Val Xab Pro Xdv
[0220] 131. Xaa Val Xab Pro Xdw
[0221] 132. Xaa Val Xab Pro Xdx
[0222] 133. Xaa Val Xab Pro Xdy
[0223] 134. Xaa Val Xab Pro Xdz
[0224] 135. Xaa Val Xab Pro Xea
[0225] 136. Xaa Val Xab Pro Xeb
[0226] 137. Xaa Val Xab Pro Xec
[0227] 138. Xaa Val Xab Pro Xed
[0228] 139. Xaa Val Xab Pro Xef
[0229] 140. Xaa Val Xab Pro Xeg
[0230] 141. Xaa Val Xab Pro Xeh
[0231] 142. Xaa Val Xab Pro Xei
[0232] 143. Xaa Val Xab Pro Xek
[0233] 144. Xaa Val Xab Pro Xel
[0234] 145. Xaa Val Xab Pro Xem
[0235] 146. Xaa Val Xab Pro Xen
[0236] 147. Xaa Val Xab Pro Xeo
[0237] 148. Xaa Val Xab Pro Xep
[0238] 149. Xaa Val Xab Pro Xeq
[0239] 150. Xaa Val Xab Pro Xer
[0240] 151. Xaa Val Xab Pro Xcg
[0241] Examples for the MS-characterization of the synthesized
novel compounds are given in the following table.
1 EXAMPLE Fast atom bombardment MS analysis. [NO.] [Mol.-Weight
(measured)] 3. 565 4. 579 5. 593 6. 607 7. 621 8. 635 11. 607 12.
607 13. 621 14. 649 15. 635 16. 635 17. 635 18. 635 19. 621 20. 621
21. 635 22. 635 25. 633 26. 647 27. 661 31. 623 32. 671 33. 667 34.
631 35. 655 36. 655 37. 669 38. 621 39. 635 41. 649 42. 621 43. 633
44. 667 45. 607 46. 647 47. 668 48. 655 49. 669 50. 685 51. 629 52.
625 53. 721 55. 579 58. 623 61. 597 62. 621 63. 609 64. 625 65. 635
66. 591 67. 715 68. 685 69. 685 70. 591 71. 607 72. 621 74. 706 75.
579 76. 579 77. 579 78. 607 79. 607 80. 607 81. 607 82. 637 83. 692
84. 706 85. 706 86. 706 87. 607 90. 635 92. 659 93. 617 94. 636 95.
678 128. 671 131. 625 139. 625 151. 637
[0242]
2 Compound Number(s) Sequence ID Number 1-56, 58-72, 75, 77, 79,
80, 82, 1 87-94, 96, 97, 99-101, 104-151 73, 74, 83-86, 95, 2 57,
76, 81, 102 3 78, 98, 103 4
[0243]
3 Xaa: N,N-Dimethylvaline Xab: N-Methylvaline Xac: 5 Xad: 6 Xae: 7
Xaf: 8 Xag: 9 Xah: 10 Xai: 11 Xak: 12 Xal: 13 Xam: 14 Xan: 15 Xao:
16 Xap: 17 Xaq: 18 Xar: 19 Xas: 20 Xat: 21 Xau: 22 Xav: 23 Xaw: 24
Xax: 25 Xay: 26 Xaz: 27 Xba: 28 Xbb: 29 Xbc: N-Methyl-isoleucine
Xbd: 30 Xbe: 31 Xbf: 32 Xbg: 33 Xbh: 34 Xbi: 35 Xbk: 36 Xbl: 37
Xbm: 38 Xbn: 39 Xbo: 40 Xbp: 41 Xbq: 42 Xbr: 43 Xbs: 44 Xbt: 45
Xbu: 46 Xbv: 47 Xbw 48 Xbx: 49 Xby: 50 Xbz: 51 Xca: 52 Xcb: 53 Xcc:
Proline adamantyl(1)amide Xcd: 54 Xce: 55 Xcf: 56 Xcg: 57 Xch: 58
Xci: 59 Xck: 60 Xcl: 61 Xcm: 62 Xcn: 63 Xco: 64 Xcp: 65 Xcq: 66
Xcr: 67 Xcs: 68 Xct: 69 Xcu: 70 Xcv: 71 Xcw:
N-Methyl-N-ethyl-valine Xcx: N,N-Diethylvaline Xcy: 72 Xcz: 73 Xda:
N-Methyl-2-aminobutyroyl Xdb: 2-aminobutyroyl Xdc:
N,N-Dimethyl-2-aminobutyroyl Xdd: N,N-Dimethyl-2-tert.butylgl-
ycine Xde: N,N-Dimethyl-isoleucine Xdf: 2-tert.butylglycine Xdg: 74
Xdh: 75 Xdi: 76 Xdk: 77 Xdl: N-Methyl-2-tert.butylglycine Xdm: 78
Xdn: 79 Xdo: 80 Xdp: 81 Xdr: 82 Xds: 83 Xdt: 84 Xdu: 85 Xdv: 86
Xdw: 87 Xdx: 88 Xdy: 89 Xdz: 90 Xaa: 91 Xab: 92 Xec: 93 Xed: 94
Xee: 95 Xef: 96 Xeg: 97 Xeh: 98 Xei: 99 Xek: 100 Xel: 101 Xem: 102
Xen: 103 Xeo: 104 Xep: 105 Xeq: 106
[0244]
[0245] Compounds of this invention may be assayed for anti-cancer
activity by conventional methods, including for example, the
methods described below.
[0246] A. In Vitro Methodology
[0247] Cytotoxicity was measured using a standard methodology for
adherent cell lines such as the microculture tetrazolium assay
(MTT). Details of this assay have been published (Alley, M C et al,
Cancer Research 48:589-601, 1988). Exponentially growing cultures
of tumor cells such as the HT-29 colon carcinoma or LX-1 lung tumor
are used to make microtiter plate cultures. Cells are seeded at
3000 cells per well in 96-well plates (in 150 .mu.l or media), and
grown overnight at 37.degree. C. Test compounds are added, in
10-fold dilutions varying from 10.sup.-4M to 10.sup.-10M. Cells are
then incubated for 72 hours. To determine the number of viable
cells in each well, the MTT dye is added (50 .mu.l or 3 mg/ml
solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide in saline). This mixture is incubated at 37.degree. C. for
5 hours, and then 50 .mu.l of 25% SDS, pH2 is added to each well.
After an overnight incubation, the absorbance of each well at 550
nm is read using an ELISA reader. The values for the mean +/-SD of
data from replicated wells are calculated, using the formula % T/C
(% viable cells treated/control). 1 OD of treated cells OD of
control cells .times. 100 + % T / C
[0248] The concentration of test compound which gives a T/C of 50%
growth inhibition was designated as the IC.sub.50 value.
[0249] B. In Vivo Methodology
[0250] Compounds of this invention were further tested in
pre-clinical assay for in vivo activity which is indicative of
clinical utility. Such assays were conducted with nude mice into
which tumor tissue, preferably of human origin, had been
transplanted (xenografted), as is well known in this field. Test
compounds were evaluated for their anti-tumor efficacy following
administration to the xenograft-bearing mice.
[0251] More specifically, human breast tumors (MX-1) which had been
grown in athymic nude mice were transplanted into new recipient
mice, using tumor fragments which were about 50 mg in size. The day
of transplantation was designated as day 0. Six to ten days later,
mice were treated with the test compounds given as an intravenous
injection or orally, in groups of 5-10 mice at each dose. Compounds
were given every other day, for 3 weeks, at doses from 1-200 mg/kg
body weight.
[0252] Tumor diameters and body weights were measured twice weekly.
Tumor volumes were calculated using the diameters measured with
Vernier calipers, and the formula
(Length.times.width.sup.2)/2=mm.sup.3 of tumor volume
[0253] Mean tumor volumes are calculated for each treatment group,
and T/C values determined for each group relative to the untreated
control tumors.
[0254] The new compounds possess good tumor inhibiting
properties.
* * * * *