U.S. patent number RE42,890 [Application Number 12/796,135] was granted by the patent office on 2011-11-01 for furazanobenzimidazoles.
This patent grant is currently assigned to Basilea Pharmaceutica AG. Invention is credited to Felix Bachmann, Martin Eberle, Subho Roy, Goutam Saha, Sudhir Srivastava, Alessandro Strebel.
United States Patent |
RE42,890 |
Eberle , et al. |
November 1, 2011 |
Furazanobenzimidazoles
Abstract
The invention relates to compounds of formula (I) wherein R
represents aryl or heteroaryl, X is oxygen, a carbonyl group, an
oxime derivative of a carbonyl group or an
.alpha.,.beta.-unsaturated carbonyl group, and the substituents
R.sup.1 to R.sup.6 have the meanings given in the specification,
for use as medicaments, to novel compounds of formula (I), to
methods of synthesis of such compounds, to pharmaceutical
compositions containing compounds of formula (I), to the use of a
compounds of formula (I) for the preparation of a pharmaceutical
composition for the treatment of neoplastic and autoimmune
diseases, and to methods of treatment of neoplastic and autoimmune
diseases using such compounds of formula (I) or of pharmaceutical
compositions containing same ##STR00001##
Inventors: |
Eberle; Martin (Bottmingen,
CH), Bachmann; Felix (Basel, CH), Strebel;
Alessandro (Vedano al Lambro, IT), Roy; Subho
(West Bengal, IN), Srivastava; Sudhir (West Bengal,
IN), Saha; Goutam (West Bengal, IN) |
Assignee: |
Basilea Pharmaceutica AG
(Basel, CH)
|
Family
ID: |
33462273 |
Appl.
No.: |
12/796,135 |
Filed: |
May 19, 2004 |
PCT
Filed: |
May 19, 2004 |
PCT No.: |
PCT/IB2004/001723 |
371(c)(1),(2),(4) Date: |
November 21, 2005 |
PCT
Pub. No.: |
WO2004/103994 |
PCT
Pub. Date: |
December 02, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
10557539 |
Nov 21, 2005 |
7385061 |
Jun 10, 2008 |
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Foreign Application Priority Data
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May 23, 2003 [EP] |
|
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03405365 |
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Current U.S.
Class: |
548/125 |
Current CPC
Class: |
A61P
37/02 (20180101); A61P 17/00 (20180101); A61P
9/10 (20180101); A61P 3/10 (20180101); A61P
35/00 (20180101); A61P 9/00 (20180101); A61P
25/28 (20180101); A61P 37/06 (20180101); A61P
15/00 (20180101); C07D 413/14 (20130101); A61P
19/02 (20180101); A61P 43/00 (20180101); A61P
29/00 (20180101); A61P 25/00 (20180101); A61P
37/00 (20180101); C07D 413/04 (20130101) |
Current International
Class: |
A61K
31/4245 (20060101); C07D 413/04 (20060101) |
Field of
Search: |
;548/125 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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3100771 |
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Aug 1982 |
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DE |
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03066629 |
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Aug 2003 |
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WO |
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WO03/066629 |
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Aug 2003 |
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WO |
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Other References
Database Chemcats Online Chemical Abstracts Service, Columbus,
Ohio, US, XP002257449, Apr. 30, 2003. cited by other .
Database Chemcats Online, Chemical Abstracts Service, Columbus,
Ohio, US, XP002257450, May 19, 2003. cited by other .
Database Chemcats Online, Chemical Abstracts Service, Columbus,
Ohio, US, XP002257451, Apr. 29, 2003. cited by other .
Sergievskii A V et al: "4-Aminofurazan-3-Carvoxylic Acid Iminoester
in Reactions with N,O-Nucelphiles" Russian Journal of Organic
Chemistry, Consultants Bureau, US, vol. 38, No. 6, 2002, pp.
872-874. cited by other .
Sergievskii A V et al: "Reactions of Methyl
4-Aminofurazan-3-Carboximidate With Nitrogen-Containing
Nucleophiles", Russian Journal of Organic Chemistry, Consultants
Bureau, US, vol. 37, No. 5, 2001, pp. 717-720. cited by other .
The European Search Report issued on Oct. 13, 2003 in related
European Application No. 03405365.2. cited by other .
The International Search Report and Written Opinion issued on Sep.
20, 2004 in related PCT Application No. PCT/IB2004/001723. cited by
other .
The Communication from the Examining Division issued on Aug. 24,
2006 in related European Application No. 04733874.4. cited by other
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The Communication from the Examining Division issued on Oct. 1,
2007 in related European Application No. 04733874.4. cited by other
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European Search Report dated Oct. 13, 2003. cited by other .
International Search Report dated Sep. 20, 2004. cited by other
.
DE 31 00 771 A (USV Pharma Corp) Aug. 26, 1982. cited by
other.
|
Primary Examiner: Stockton; Laura L.
Claims
The invention claimed is:
1. A compound of the formula ##STR00116## wherein R represents
phenyl, naphthyl, thienyl, pyridinyl or pyridazinyl ring, said
phenyl ring being optionally substituted by one or two substituents
independently selected from alkyl, halo-lower alkyl, hydroxy-lower
alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl,
hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy lower
alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino,
monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower
alkycarbonylamino, substituted amino wherein the two substituents
on nitrogen form together with the nitrogen a heterocyclcyl, lower
alkylcarbonyl, formyl, carboxy, lower alkoxycarbonyl, cyano,
halogen, and nitro; and wherein two adjacent substituents are
methylenedioxy; and said pyridinyl or pyridazinyl being optionally
substituted in one or two positions with lower alkoxy, amino, or
halogen; X is --O-- or >C.dbd.Y, wherein Y is oxygen; R.sup.1
represents hydrogen, hydroxy-lower alkyl, cyano-lower alkyl or
lower alkyl-carbonyl, and R.sup.2, R.sup.3, R.sup.4, R.sup.5 and
R.sup.6 is hydrogen; or a pharmaceutically acceptable salt
thereof.
2. The compound of claim 1 where X is >C.dbd.Y, wherein Y is
oxygen, or it's pharmaceutically acceptable salts.
3. The compound of claim 2, which compounds are selected from the
group consisting of the compounds 1, 5, 6, 11, 14, 15, 16, 19, 23,
29, 35, 41, 42, 44, 45, 46, 47, 48, 50, 52, 53, 54, 55, 56, 57, 58,
59, 61, 62, 64, 65, 66, 67, 68, 69, 70, 72, 74, 76, 77, 78 and 79,
which compounds are set forth according to the following table:
TABLE-US-00009 Compound R R.sup.1 1 ##STR00117## H 5 ##STR00118##
(CO)CH.sub.3 6 ##STR00119## CH.sub.2CH.sub.2CN 11 ##STR00120##
CH.sub.2CH.sub.2CH.sub.2OH 14 ##STR00121## H 15 ##STR00122## H 16
##STR00123## H 19 ##STR00124## H 23 ##STR00125## H 29 ##STR00126##
H 35 ##STR00127## H 41 ##STR00128## H 42 ##STR00129## H 44
##STR00130## H 45 ##STR00131## H 46 ##STR00132## CH.sub.2CH.sub.2CN
47 ##STR00133## CH.sub.2CH.sub.2CN 48 ##STR00134##
CH.sub.2CH.sub.2CN 50 ##STR00135## H 52 ##STR00136##
CH.sub.2CH.sub.2CH.sub.2OH 53 ##STR00137## H 54 ##STR00138##
CH.sub.2CH.sub.2CN 55 ##STR00139## H 56 ##STR00140##
CH.sub.2CH.sub.2CN 57 ##STR00141## CH.sub.2CH.sub.2CN 58
##STR00142## CH.sub.2CH.sub.2CN 59 ##STR00143## H 61 ##STR00144##
CH.sub.2CH.sub.2CN 62 ##STR00145## H 64 ##STR00146## H 65
##STR00147## H 66 ##STR00148## H 67 ##STR00149## H 68 ##STR00150##
H 69 ##STR00151## CH.sub.2CH.sub.2CN 70 ##STR00152## H 72
##STR00153## H 74 ##STR00154## H 76 ##STR00155## H 77 ##STR00156##
H 78 ##STR00157## H 79 ##STR00158## CH.sub.2CH.sub.2CN
or their pharmaceutically acceptable salts.
4. The compound of claim 2 wherein R.sup.1 represents hydrogen or
cyano-lower alkyl.
5. The compound of claim 4 wherein the compounds are selected from
the group consisting of the compounds 6, 15, 29, 42, 44, 45, 46,
47, 48, 50, 54, 56, 58, 61, 64, 65, 70, 78 and 79, which compounds
are set forth according to the following table: TABLE-US-00010
Compound R R.sup.1 6 ##STR00159## CH.sub.2CH.sub.2CN 15
##STR00160## H 29 ##STR00161## H 42 ##STR00162## H 44 ##STR00163##
H 45 ##STR00164## H 46 ##STR00165## CH.sub.2CH.sub.2CN 47
##STR00166## CH.sub.2CH.sub.2CN 48 ##STR00167## CH.sub.2CH.sub.2CN
50 ##STR00168## H 54 ##STR00169## CH.sub.2CH.sub.2CN 56
##STR00170## CH.sub.2CH.sub.2CN 58 ##STR00171## CH.sub.2CH.sub.2CN
61 ##STR00172## CH.sub.2CH.sub.2CN 64 ##STR00173## H 65
##STR00174## H 70 ##STR00175## H 78 ##STR00176## H 79 ##STR00177##
CH.sub.2CH.sub.2CN
or their pharmaceutically acceptable salts.
6. The compound of claim 2, wherein R is optionally substituted
phenyl.
7. The compound of claim 6 wherein said compound is
4-[1-(4-aminophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl-
)-amine or pharmaceutically acceptable salts thereof.
8. The compound of claim 4 where the compound has the formula
##STR00178## wherein R is pyridinyl optionally substituted in one
or two positions by lower alkoxy, amino, or halogen; X is
--C.dbd.Y; Y is oxygen; R.sup.1 is cyano-lower alkyl or hydrogen
and; R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 is hydrogen; or a
pharmaceutically acceptable salt thereof.
9. The compound of claim 8 wherein R.sup.1 is cyano-lower
alkyl.
10. The compound of claim 9 wherein said compound is
4-[1-(6-amino-3-pyridylcarbonylmethyl)-1H-benzimidazol-2-yl]-furazan-3-yl-
]-N-(2-cyanoethyl)-amine or its pharmaceutical acceptable
salts.
11. The compound of claim 8 wherein R.sup.1 is hydrogen.
12. The compound of claim 11 wherein said compound is
4-[1-(6-amino-3-pyridylcarbonylmethyl)-1H-benzimidazol-2-yl]-furazan-3-yl-
amine; or pharmaceutical acceptable salts thereof.
13. The compound of claim 6 where said compound has the formula
##STR00179## which compound is selected from the group consisting
of the compounds 7, 10, 88, 89, 92, 93, 94, 95, 96, 97, 101 and
103, which compounds are set forth according to the following
table: TABLE-US-00011 Compound R R.sup.1 7 ##STR00180## H 10
##STR00181## CH.sub.2CH.sub.2CN 88 ##STR00182## H 89 ##STR00183## H
92 ##STR00184## H 93 ##STR00185## CH.sub.2CH.sub.2CN 94
##STR00186## CH.sub.2CH.sub.2CN 95 ##STR00187## CH.sub.2CH.sub.2CN
96 ##STR00188## H 97 ##STR00189## H 101 ##STR00190## H 103
##STR00191## H
or pharmaceutically acceptable salts thereof.
14. The compound of claim 13, which compound is selected from the
group consisting of the compounds 89, 92, 94 and 101, which
compound are set forth according to the following table:
TABLE-US-00012 Compound R R.sup.1 89 ##STR00192## H 92 ##STR00193##
H 94 ##STR00194## CH.sub.2CH.sub.2CN 101 ##STR00195## H
or their pharmaceutically acceptable salts.
15. A compound of the formula (I) ##STR00196## wherein R represents
phenyl or pyridinyl wherein phenyl is optionally substituted by one
or two substituents independently selected from alkyl, halo-lower
alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower
alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower
alkoxy lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy,
amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino,
lower alkylcarbonylamino, substituted amino wherein the two
substituents on nitrogen form together with the nitrogen a
heterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl,
formyl, cyano, halogen, and nitro; and wherein two adjacent
substituents are methylenedioxy; and wherein pyridinyl is
optionally substituted by lower alkoxy, amino or halogen; X is
--C.dbd.Y and Y is nitrogen substituted by an alkoxy; R.sup.1
represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or
cyano-lower alkyl; R.sup.2, R.sup.3 and R.sup.6 represent hydrogen;
R.sup.4 and R.sup.5, independently of each other, represent
hydrogen, lower alkyl or lower alkoxy; or R.sup.4 and R.sup.5
together represent methylenedioxy; or pharmaceutically acceptable
salts thereof.
16. The compound of claim 15, which compound is selected from the
group consisting of the compounds 18 and 22, which compounds are
set forth according to the following table: TABLE-US-00013 Compound
R R.sup.1 18 ##STR00197## H 22 ##STR00198## H
or their pharmaceutically acceptable salts.
17. A compound selected from the group consisting of Compound 9 and
13, which compounds are as represented by the following formula and
are set forth according to the following table: ##STR00199##
wherein Y is oxygen TABLE-US-00014 Compound R R.sup.1 9
##STR00200## CH.sub.2CH.sub.2(CO)OCH.sub.3 13 ##STR00201##
CH.sub.2CH.sub.2(CO)OH
or their pharmaceutically acceptable salts.
.Iadd.18. A pro-drug of a compound selected from the group
consisting of a furazanobenzimidazole of the formula (I)
##STR00202## and a pharmaceutically acceptable salt thereof,
wherein R represents a phenyl, naphthyl, thienyl, pyridinyl or
pyridazinyl ring, said phenyl ring being optionally substituted by
one or two substituents independently selected from the group
consisting of alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower
alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy, lower
alkoxy, hydroxy-lower alkoxy, lower alkoxy lower alkoxy,
phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,
dialkylamino, lower alkoxycarhonylamino, lower alkycarhonylamino,
substituted amino wherein the two substituents on nitrogen form
together with the nitrogen hetercyclcyl, lower alkylcarbonyl,
formyl, carboxy, lower alkoxycarbonyl, cyano, halogen, and nitro;
and said pyridinyl or pyridazinyl being optionally substituted in
one or two positions with lower lower alkoxy, amino, or halogen; X
is --O-- or >C.dbd.Y, wherein Y is oxygen; R.sup.1 represents
hydrogen, hydroxy-lower alkyl, cyano-lower alkyl or lower
alkyl-carbonyl, and R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6
is hydrogen; and said furazanobenzimidazole of formula (I)
comprises a substituent selected from the group consisting of
hydroxyl, carboxy and amino and said pro-drug is selected from the
group consisting of an ester or an amide of said compound with a
natural occurring amino acid and an ester or amide of said compound
with a small peptide consisting of up to 5 amino acids, where said
esters are formed by reaction of an acid function of the said
natural occurring amino acid or the C terminal of said small
peptide with a hydroxyl substituent of said compound and said
amides are formed by reaction of an amino function of said natural
occurring amino acid or the N terminal of said small peptide with a
carboxy group of said compound or by reaction of an acid function
of said natural occurring amino acid or the C terminal of said
small peptide with an amino group of said compound..Iaddend.
.Iadd.19. A pro-drug of a compound selected from the group
consisting of a furazanobenzimidazole of the formula (I)
##STR00203## and a pharmaceutically acceptable salt thereof,
wherein R represents phenyl or pyridinyl wherein phenyl is
optionally substituted by one or two substituents independently
selected from the group consisting of alkyl, halo-lower alkyl,
hydroxy-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl,
phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy
lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino,
monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower
alkylcarbonylamino substituted amino wherein the two substituents
on nitrogen form together with the nitrogen a heterocyclyl, lower
alkylcarhonyl, carboxy, lower alkoxycarbonyl, formyl, cyano,
halogen, and nitro; and wherein pyridinyl is optionally substituted
by lower alkoxy, amino or halogen; X is --C.dbd.Y and Y is nitrogen
substituted by a alkoxy; R.sup.1 represents hydrogen, lower
alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R.sup.2,
R.sup.3 and R.sup.6 represent hydrogen; R.sup.4 and R.sup.5,
independently of each other, represent hydrogen, lower alkyl or
lower alkoxy; or R.sup.4 and R.sup.5 together represent
methylenedioxy; and said furazanobenzimidazole of formula (I)
comprises a substituent selected from hydroxyl, carboxy and amino;
and said pro-drug is selected from the group consisting of an ester
or an amide of said compound with a natural occurring amino acid
and an ester or amide of said compound with a small peptide
consisting of up to 5 amino acids, where said esters are formed by
reaction of an acid function of the said natural occurring amino
acid or the C terminal of said small peptide with a hydroxyl
substituent of said compound and said amides are formed by reaction
of an amino function of said natural occurring amino acid or the N
terminal of said small peptide with a carboxy group of said
compound of formula (I) by reaction of an acid function of said
natural occurring amino acid or the C terminal of said small
peptide with an amino group of said compound of formula
(I)..Iaddend.
.Iadd.20. The pro-drug according to claim 18, wherein X in formula
(I) is >C.dbd.Y and Y is oxygen..Iaddend.
.Iadd.21. The pro-drug according to claim 20, wherein said
furazanobenzimidazole of formula (I) is selected from the group
consisting of 1, 11, 14, 15, 16, 19, 23, 29, 35, 41, 42, 44, 45,
50, 52, 53, 55, 58, 59, 62, 64, 65, 66, 67, 68, 69, 70, 72, 74, 76,
77, 78 and 79 according to the following table:.Iaddend.
TABLE-US-00015 .Iadd. R R1 1 ##STR00204## H 11 ##STR00205##
CH.sub.2CH.sub.2CH.sub.2OH 14 ##STR00206## H 15 ##STR00207## H 16
##STR00208## H 19 ##STR00209## H 23 ##STR00210## H 29 ##STR00211##
H 35 ##STR00212## H 41 ##STR00213## H 42 ##STR00214## H 44
##STR00215## H 45 ##STR00216## H 50 ##STR00217## H 52 ##STR00218##
CH.sub.2CH.sub.2CH.sub.2OH 53 ##STR00219## H 55 ##STR00220## H 58
##STR00221## CH.sub.2CH.sub.2CN 59 ##STR00222## H 62 ##STR00223## H
64 ##STR00224## H 65 ##STR00225## H 66 ##STR00226## H 67
##STR00227## H 68 ##STR00228## H 69 ##STR00229## CH.sub.2CH.sub.2CN
70 ##STR00230## H 72 ##STR00231## H 74 ##STR00232## H 76
##STR00233## H 77 ##STR00234## H 78 ##STR00235## H 79 ##STR00236##
CH.sub.2CH.sub.2CN..Iaddend.
.Iadd.22. The pro-drug according to claim 20, wherein R.sup.1 in
formula (I) represents hydrogen or cyano-lower alkyl..Iaddend.
.Iadd.23. The pro-drug according to claim 22, wherein said
furazanobenzimidazole is selected from the group consisting of 15,
29, 42, 44, 45, 50, 58, 64, 65, 70, 78 and 79 according to the
following table:.Iaddend. TABLE-US-00016 .Iadd. R R1 15
##STR00237## H 29 ##STR00238## H 42 ##STR00239## H 44 ##STR00240##
H 45 ##STR00241## H 50 ##STR00242## H 58 ##STR00243##
CH.sub.2CH.sub.2CN 64 ##STR00244## H 65 ##STR00245## H 70
##STR00246## H 78 ##STR00247## H 79 ##STR00248##
CH.sub.2CH.sub.2CN..Iaddend.
.Iadd.24. The pro-drug according to claim 20, wherein R in formula
(I) is optionally substituted phenyl..Iaddend.
.Iadd.25. A pro-drug of an amino compound selected from the group
consisting of
4-[1-(4-aminophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl-
)-amine and a pharmaceutically acceptable salt thereof with a
naturally occurring amino acid, wherein said pro-drug is an amide
formed by the reaction of an acid group of said naturally occurring
amino acid with the amino group of said compound..Iaddend.
.Iadd.26. A pro-drug of an amino compound selected from the group
consisting of
4-[1-(4-aminophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl-
)-amine and a pharmaceutically acceptable salt thereof with a small
peptide having up to 5 amino acids, wherein said pro-drug is an
amide formed by reaction of said C terminal of said small peptide
with the amino group of said compound..Iaddend.
.Iadd.27. The pro-drug according to claim 18, wherein said pro-drug
is an amide of said compound with a natural occurring amino acid,
wherein said amide is formed by reaction of an acid function of
said natural occurring amino acid with an amino group of said
compound..Iaddend.
.Iadd.28. The pro-drug according to claim 27, wherein in formula
(I) R is pyridinyl optionally substituted in one or two positions
by lower alkoxy, amino, or halogen; X is --C.dbd.Y; Y is oxygen;
R.sup.1 is cyano-lower alkyl or hydrogen and; R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6 is hydrogen..Iaddend.
.Iadd.29. The pro-drug according to claim 28, wherein in formula
(I) R is pyridinyl substituted in one or two positions by amino and
R.sup.1 in formula (I) is cyano-lower alkyl..Iaddend.
.Iadd.30. A pro-drug of an amino compound selected from the group
consisting of
4-[1-(6-amino-3-pyridylcarbonylmethyl)-1H-benzimidazol-2-yl]-furazan-3-yl-
]-N-(2-cyanoethyl)-amine and a pharmaceutically acceptable salt
thereof with a naturally occurring amino acid, wherein said
pro-drug is an amide formed by the reaction of an acid function of
said naturally occurring amino acid with the amino group of said
compound..Iaddend.
.Iadd.31. A pro-drug of an amino compound selected from the group
consisting of
4-[1-(6-amino-3-pyridylcarbonylmethyl)-1H-benzimidazol-2-yl]-furazan-3-yl-
]-N-(2-cyanoethyl)-amine and a pharmaceutically acceptable salt
thereof with a small peptide having up to 5 amino acids, wherein
said pro-drug is an amide formed by reaction of said C terminal of
said small peptide with the amino group of said
compound..Iaddend.
.Iadd.32. The pro-drug according to claim 28, wherein R.sup.1 in
formula (I) is hydrogen..Iaddend.
.Iadd.33. A pro-drug of an amino compound selected from the group
consisting of
4-[1-(6-amino-3-pyridylcarbonylmethyl)-1H-benzimidazol-2-yl]-furazan-3-yl-
amine and a pharmaceutically acceptable salt thereof with a
naturally occurring amino acid, wherein said pro-drug is an amide
formed by the reaction of an acid function of said naturally
occurring amino acid with an amino group of said
compound..Iaddend.
.Iadd.34. A pro-drug of an amino compound selected from the group
consisting of
4-[1-(6-amino-3-pyridylcarbonylmethyl)-1H-benzimidazol-2-yl]-furazan-3-yl-
amine or of said pharmaceutically acceptable salt thereof with a
small peptide having up to 5 amino acids, wherein said pro-drug is
an amide formed by reaction of said C terminal of said small
peptide with an amino group of said compound..Iaddend.
.Iadd.35. A pro-drug of a compound selected from the group
consisting of a furazanobenzimidazole of formula (IB) ##STR00249##
and a pharmaceutically acceptable salt thereof, wherein the
furazanobenzimidazole is selected from the group consisting of
furazanobenzimidazoles 7, 88, 89, 92, 96, 97, 101 and 103, which
are set forth according to the following table: TABLE-US-00017
.Iadd. R R1 7 ##STR00250## H 88 ##STR00251## H 89 ##STR00252## H 92
##STR00253## H 96 ##STR00254## H 97 ##STR00255## H 101 ##STR00256##
H 103 ##STR00257## H.Iaddend.
wherein said pro-drug is selected from the group consisting of an
amide of said compound with a natural occurring amino acid and an
amide of said compound with a small peptide consisting of up to 5
amino acids, wherein said amide is formed by reaction of an acid
function of said natural occurring amino acid or the C terminal of
said small peptide with an amino group of said
compound..Iaddend.
.Iadd.36. The pro-drug according to claim 35, wherein the
furazanobenzimidazole of formula (IB) is selected from the group
consisting of furazanobenzimidazoles 89, 92, and 101, which are set
forth according to the following table:.Iaddend. TABLE-US-00018
.Iadd. R R1 89 ##STR00258## H 92 ##STR00259## H 101 ##STR00260##
H..Iaddend.
.Iadd.37. The pro-drug according to claim 19, wherein the
furazanobenzimidazole of formula (I) is selected from the group
consisting of furazanobenzimidazoles 18 and 22 which are set forth
according to the following table:.Iaddend. TABLE-US-00019 .Iadd. R
R1 18 ##STR00261## H 22 ##STR00262## H..Iaddend.
.Iadd.38. A pro-drug of a furazanobenzimidazole of the formula (I)
##STR00263## wherein R represents a phenyl, naphthyl, thienyl,
pyridinyl or pyridazinyl ring, said phenyl ring being optionally
substituted by one or two substituents independently selected from
the group consisting of alkyl, halo-lower alkyl, hydroxy-lower
alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl,
hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy lower
alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino,
monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower
alkycarbonylamino, substituted amino wherein the two substituents
on nitrogen form together with the nitrogen hetercyclcyl, lower
alkylcarbonyl, formyl, carboxy, lower alkoxycarbonyl, cyano,
halogen, and nitro; and said pyridinyl or pyridazinyl being
optionally substituted in one or two positions with lower lower
alkoxy, amino, or halogen; X is --O-- or >C.dbd.Y, wherein Y is
oxygen; R.sup.1 represents hydrogen, hydroxy-lower alkyl,
cyano-lower alkyl or lower alkyl-carbonyl, and R.sup.2, R.sup.3,
R.sup.4, R.sup.5 and R.sup.6 is hydrogen; and said
furazanobenzimidazole of formula (I) comprises a substituent
selected from the group consisting of hydroxyl, carboxy and amino
and said pro-drug is selected from the group consisting of an ester
or an amide of said compound of formula (I) with a natural
occurring amino acid, and an ester or amide of said
furazanobenzimidazole of formula (I) with a small peptide
consisting of up to 5 amino acids, where said esters are formed by
reaction of an acid function of the said natural occurring amino
acid or the C terminal of said small peptide with a hydroxyl
substituent of said compound of formula (I) and said amides are
formed by reaction of an amino function of said natural occurring
amino acid or the N terminal of said small peptide with a carboxy
group of said furazanobenzimidazole of formula (I) or by reaction
of an acid function of said natural occurring amino acid or the C
terminal of said small peptide with an amino group of said
furazanobenzimidazole of formula (I)..Iaddend.
.Iadd.39. A pro-drug according to claim 38 which is a pro-drug of
4-[1-(4-aminophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl-
)-amine with a naturally occurring amino acid, wherein said
pro-drug is an amide formed by the reaction of an acid group of
said naturally occurring amino acid with an amino group of said
compound..Iaddend.
Description
.Iadd.This application is a Reissue Application of U.S. Pat. No.
7,385,061, issued on Jun. 10, 2008 from U.S. application Ser. No.
10/557,539, filed on Nov. 21, 2005, which is a National Stage
Application under 35 U.S.C. 371 of PCT Application No.
PCT/IB2004/001723, filed on May 19, 2004..Iaddend.
The invention relates to novel substituted furazanobenzimidazoles,
processes for the preparation thereof, pharmaceutical compositions
containing same, the use thereof optionally in combination with one
or more other pharmaceutically active compounds for the therapy of
neoplastic diseases and autoimmune diseases, and a method for the
treatment of such a diseases.
BACKGROUND OF THE INVENTION
Cancer is one of the leading causes of death in humans. Although a
variety of drugs against neoplastic diseases have been developed
and techniques are available such as surgery and radiation therapy,
there is still a need for alternative and improved methods of
treatment of neoplastic diseases.
Autoimmune diseases are associated with abnormal
lymphoproliferation as a result of defects in the termination of
lymphocyte activation and growth. Often, such diseases are
associated with inflammation like rheumatoid arthritis, insulin
dependent diabetes mellitus, multiple sclerosis, systemic lupus
erythematosus and the like. The treatment of such diseases is
focused on anti-inflammatory and immunosuppressive drugs which in
numerous cases show severe side effects. Hence, there is a need for
alternative drugs with a new mode of action showing less side
effects.
Apoptosis is a term used to describe a series of cellular events
which occur to bring about programmed cell death. There are various
apoptotic pathways, some of which have been characterized, whereas
others remain to be elucidated. If the balance between cell
division and apoptosis is disturbed, life-threatening diseases
including cancer, autoimmune disorders, neurodegenerative and
cardiovascular diseases may occur.
In recent years it has become evident that programmed cell death
(apoptosis) is as important to the health of a multicellular
organism as cell division. By repeated cell division and
differentiation throughout development or tissue repair, surplus or
even harmful cells are generated. In order to maintain tissue
homeostasis these cells have to be removed or killed. The delicate
interplay between cell growth and apoptosis in an organism is
mirrored in the complex molecular balance that determines whether
an individual cell undergoes division, arrests in the cell cycle or
commits to programmed cell death.
Dysregulation of cell proliferation, or lack of appropriate cell
death, has wide ranging clinical implications. A number of diseases
associated with such dysregulation involve hyperproliferation,
inflammation, tissue remodeling and repair. Familiar indications in
this category include cancers, restenosis, neointimal hyperplasia,
angiogenesis, endometriosis, lymphoproliferative disorders,
transplantation related pathologies (graft rejection), polyposis,
loss of neural function in the case of tissue remodeling and the
like. Such cells may lose the normal regulatory control of cell
division, and may also fail to undergo appropriate cell death.
As apoptosis is inhibited or delayed in most types of
proliferative, neoplastic diseases, induction of apoptosis is an
option for treatment of cancer, especially in cancer types which
show resistance to classic chemotherapy, radiation and
immunotherapy (Apoptosis and Cancer Chemotherapy, Hickman and Dive,
eds., Blackwell Publishing, 1999). Also in autoimmune and
transplantation related diseases and pathologies compounds inducing
apoptosis may be used to restore normal cell death processes and
therefore can eradicate the symptoms and might cure the diseases.
Further applications of compounds inducing apoptosis may be in
restenosis, i.e. accumulation of vascular smooth muscle cells in
the walls of arteries, and in persistent infections caused by a
failure to eradicate bacteria- and virus-infected cells.
Furthermore, apoptosis can be induced or re-established in
epithelial cells, in endothelial cells, in muscle cells, and in
others which have lost contact with extracellular matrix. These
cells are potentially able to colonize other organs and therefore
can develop into pathologies like neoplasias, endometriosis and the
like.
Recently, a patent application was published disclosing a number of
structurally related compounds (WO 03/066629). These compounds are
described as being inhibitors of GSK-3 and LCK kinase, but have no
relevance in apoptosis and medical conditions connected
therewith.
SUMMARY OF THE INVENTION
Furazanobenzimidazoles of formula (I) are selectively inducing
apoptosis in cancer cells, and can be used for the treatment of
neoplastic and autoimmune diseases. The invention relates to
compounds of formula (I), in particular to such compounds for use
as medicaments, to methods of synthesis of such compounds, to
pharmaceutical compositions containing compounds of formula (I), to
the use of a compounds of formula (I) for the preparation of a
pharmaceutical composition for the treatment of neoplastic and
autoimmune diseases, and to methods of treatment of neoplastic and
autoimmune diseases using such compounds of formula (I) or of
pharmaceutical compositions containing same.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to compounds of formula (I)
##STR00002## wherein R represents aryl or heteroaryl optionally
substituted by up to four substituents independently selected from
alkyl, cycloalkyl, cycloalkyl-lower alkyl, halo-lower alkyl,
hydroxy-lower alkyl, lower alkoxy-lower alkyl, lower alkoxy-lower
alkoxy-lower alkyl, halo-lower alkoxy-lower alkyl, acyloxy-lower
alkyl, heterocyclyl, heterocyclyl-lower alkyl, optionally
substituted phenyl, optionally substituted phenyl-lower alkyl,
optionally substituted heteroaryl, optionally substituted
heteroaryl-lower alkyl, optionally substituted alkenyl, optionally
substituted alkinyl, hydroxy, lower alkoxy, optionally substituted
alkenyloxy, optionally substituted alkinyloxy, cycloalkoxy,
halo-lower alkoxy, cycloalkyl-lower alkoxy, hydroxy-lower alkoxy,
lower alkoxy-lower alkoxy, heterocyclyloxy, heterocyclyl-lower
alkoxy, optionally substituted phenyloxy, optionally substituted
phenyl-lower alkoxy, optionally substituted heteroaryloxy,
optionally substituted heteroaryl-lower alkoxy, sulfamoyloxy,
carbamoyloxy, lower alkylcarbonyloxy, amino, monoalkylamino,
dialkylamino, aminocarbonylamino wherein each of the two amino
groups is optionally substituted by alkyl, alkenyl, alkinyl or
alkoxy-lower alkyl, heterocyclylcarbonylamino wherein heterocyclyl
is bound via a nitrogen atom, aminosulfonylamino wherein each of
the two amino groups is optionally substituted by alkyl, alkenyl,
alkinyl or alkoxy-lower alkyl, heterocyclylsulfonylamino wherein
heterocyclyl is bound via a nitrogen atom, lower
alkoxycarbonylamino, lower alkylcarbonylamino wherein alkyl is
optionally substituted by one or two substituents selected from
optionally substituted phenyl, guanidyl, halogen, cyano, alkoxy,
optionally substituted phenoxy, alkylmercapto and optionally
substituted amino; lower alkenylcarbonylamino wherein alkenyl is
optionally substituted by one or two substituents selected from
lower alkyl, halo-lower alkyl, optionally substituted phenyl,
halogen, cyano, alkoxy and optionally substituted amino;
amino-lower alkyl or amino-lower alkylamino, wherein the nitrogen
atom is unsubstituted or substituted by one or two substitutents
selected from lower alkyl, cycloalkyl, cycloalkyl-lower alkyl,
hydroxy-lower alkyl, lower alkoxy-lower alkyl, optionally
substituted phenyl, optionally substituted phenyl-lower alkyl,
optionally substituted heteroaryl, optionally substituted
heteroaryl-lower alkyl and lower alkylcarbonyl, or wherein the two
substituents on nitrogen form together with the nitrogen
heterocyclyl, lower alkylcarbonyl, formyl, cycloalkylcarbonyl,
optionally substituted phenylcarbonyl, optionally substituted
heteroarylcarbonyl, heterocyclylcarbonyl, carboxy, lower
alkoxycarbonyl, hydroxy-lower alkoxycarbonyl, lower alkoxy-lower
alkoxycarbonyl, optionally substituted phenyl-lower alkoxycarbonyl,
cyano, lower alkylmercapto, optionally substituted phenylmercapto,
lower alkylsulfinyl, halo-lower alkylsulfinyl, optionally
substituted phenylsulfinyl, lower alkylsulfonyl, halo-lower
alkylsulfonyl, optionally substituted phenylsulfonyl,
aralkylsulfonyl, halogen, and nitro; and wherein two adjacent
substituents together with the atoms of aryl or heteroaryl may form
a 5 or 6 membered carbocyclic or heterocyclic ring; X represents
oxygen; a group C.dbd.Y, wherein Y stands for oxygen or nitrogen
substituted by hydroxy or alkoxy; or a group --CO--CH.dbd.CH--
wherein the C.dbd.C bond is connected to R; R.sup.1 and R.sup.2,
independently of each other, represent hydrogen, alkyl, cycloalkyl,
cycloalkyl-alkyl, optionally substituted arylalkyl, optionally
substituted heteroarylalkyl, hydroxyalkyl, alkoxyalkyl,
hydroxyalkoxyalkyl, alkoxyalkoxyalkyl, cyanoalkyl, optionally
substituted alkenyl, optionally substituted alkinyl, or lower
alkylcarbonyl wherein lower alkyl is optionally substituted by one
or two substitutents selected from aryl, optionally substituted
amino, alkoxy and aryloxy, R.sup.3, R.sup.4, R.sup.5 and R.sup.6,
independently of each other, represent hydrogen, lower alkyl,
halo-lower alkyl, cycloalkyl, cycloalkyl-lower alkyl, hydroxy-lower
alkyl, lower alkoxy-lower alkyl, lower alkoxy-lower alkoxy-lower
alkyl, halo-lower alkoxy-lower alkyl, heterocyclyl,
heterocyclyl-lower alkyl, optionally substituted phenyl, optionally
substituted phenyl-lower alkyl, optionally substituted heteroaryl,
optionally substituted heteroaryl-lower alkyl, optionally
substituted alkenyl, optionally substituted alkinyl, hydroxy, lower
alkoxy, halo-lower alkoxy, cycloalkoxy, cycloalkyl-lower alkoxy,
hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, heterocyclyloxy,
heterocyclyl-lower alkoxy, optionally substituted phenyloxy,
optionally substituted phenyl-lower alkoxy, optionally substituted
heteroaryloxy, optionally substituted heteroaryl-lower alkoxy,
amino, carbamoyl, sulfamoyl, amino-lower alkyl or amino-lower
alkylamino, wherein in each case the nitrogen atom is unsubstituted
or substituted by one or two substitutents selected from lower
alkyl, cycloalkyl, cycloalkyl-lower alkyl, hydroxy-lower alkyl,
lower alkoxy-lower alkyl, optionally substituted phenyl, optionally
substituted phenyl-lower alkyl, optionally substituted heteroaryl,
optionally substituted heteroaryl-lower alkyl and lower
alkylcarbonyl, or wherein the two substituents on nitrogen form
together with the nitrogen heterocyclyl, lower alkylcarbonyl,
cycloalkylcarbonyl, optionally substituted phenylcarbonyl,
optionally substituted heteroarylcarbonyl, heterocyclylcarbonyl,
carboxy, lower alkoxycarbonyl, hydroxy-lower alkoxycarbonyl, lower
alkoxy-lower alkoxycarbonyl, optionally substituted phenyl-lower
alkoxycarbonyl, cyano, lower alkylmercapto, optionally substituted
phenylmercapto, lower alkylsulfinyl, halo-lower alkylsulfinyl,
optionally substituted phenylsulfinyl, lower alkylsulfonyl,
halo-lower alkylsulfonyl, optionally substituted phenylsulfonyl,
aralkylsulfonyl, halogen, or nitro, or R.sup.3 and R.sup.4, R.sup.4
and R.sup.5, or R.sup.5 and R.sup.6 together with the atoms of the
phenyl ring form a 5 or 6 membered carbocyclic or heterocyclic
ring; and salts thereof.
The general terms used hereinbefore and hereinafter preferably have
within the context of this disclosure the following meanings,
unless otherwise indicated:
The prefix "lower" denotes a radical having up to and including a
maximum of 7, especially up to and including a maximum of 4 carbon
atoms, the radicals in question being either linear or branched
with single or multiple branching.
Where the plural form is used for compounds, salts, and the like,
this is taken to mean also a single compound, salt, or the
like.
Double bonds in principle can have E- or Z-configuration. The
compounds of this invention may therefore exist as isomeric
mixtures or single isomers. If not specified both isomeric forms
are intended.
Any asymmetric carbon atoms may be present in the (R)-, (S)- or
(R,S)-configuration, preferably in the (R)- or (S)-configuration.
The compounds may thus be present as mixtures of isomers or as pure
isomers, preferably as enantiomer-pure diastereomers.
The invention relates also to possible tautomers of the compounds
of formula (I).
Alkyl has from 1 to 12, preferably from 1 to 7 carbon atoms, and is
linear or branched. Alkyl is preferably lower alkyl.
Lower alkyl has 1 to 4 carbon atoms and is butyl, such as n-butyl,
sec-butyl, isobutyl, tert-butyl, propyl, such as n-propyl or
isopropyl, ethyl or methyl. Preferably lower alkyl is methyl or
ethyl.
Cycloalkyl has preferably 3 to 7 ring carbon atoms, and may be
unsubstituted or substituted, e.g. by lower alkyl or lower alkoxy.
Cycloalkyl is, for example, cyclohexyl, cyclopentyl, or
methylcyclopentyl.
Aryl stands for a mono- or bicyclic fused ring aromatic group with
5 to 10 carbon atoms, such as phenyl, 1-naphthyl or 2-naphthyl, or
also a partially saturated bicyclic fused ring comprising a phenyl
group, such as indanyl, dihydro- or tetrahydronaphthyl.
In optionally substituted phenyl, substituents are preferably lower
alkyl, lower alkoxy, lower alkoxy-lower alkoxy, methylenedioxy,
halo-lower alkyl, lower alkoxy-lower alkyl, halo, or nitro.
Heteroaryl represents an aromatic group containing at least one
heteroatom selected from nitrogen, oxygen and sulfur, and is mono-
or bicyclic. Monocyclic heteroaryl includes 5 or 6 membered
heteroaryl groups containing 1, 2, 3 or 4 heteroatoms selected from
nitrogen, sulfur and oxygen. Bicyclic heteroaryl includes 9 or 10
membered fused-ring heteroaryl groups. Examples of heteroaryl
include pyrrolyl, thienyl, furyl, pyrazolyl, imidazolyl, triazolyl,
oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,
thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, benzo
fused derivatives of such monocyclic heteroaryl groups, such as
indolyl, benzimidazolyl or benzofuryl, quinolinyl, isoquinolinyl,
quinazolinyl, or purinyl.
In optionally substituted heteroaryl, substituents are preferably
lower alkyl, lower alkoxy, lower alkoxy-lower alkoxy, amino,
optionally substituted by one or two substituents selected from
lower alkyl, lower alkenyl and alkylcarbonyl, halo-lower alkyl,
lower alkoxy-lower alkyl, halo, or nitro.
Alkenyl contains one or more, e.g. two or three, double bonds, and
is preferably lower alkenyl, such as 1- or 2-butenyl, 1-propenyl,
allyl or vinyl.
Alkinyl is preferably lower alkinyl, such as propargyl or
acetylenyl.
In optionally substituted alkenyl or alkinyl, substituents are
preferably lower alkyl, lower alkoxy, halo or di(lower alkyl)amino,
and are connected with a saturated carbon atom of alkenyl or
alkinyl or with an unsaturated carbon atom of alkenyl.
Heterocyclyl designates preferably a saturated, partially saturated
or unsaturated, mono- or bicyclic ring containing 4-10 atoms
comprising one, two or three heteroatoms selected from nitrogen,
oxygen and sulfur, which may, unless otherwise specified, be carbon
or nitrogen linked, wherein a ring nitrogen atom may optionally be
substituted by a group selected from lower alkyl, amino-lower
alkyl, aryl, aryl-lower alkyl and acyl, and a ring carbon atom may
be substituted by lower alkyl, amino-lower alkyl, aryl, aryl-lower
alkyl, heteroaryl, lower alkoxy, hydroxy or oxo. Examples of
heterocyclyl are pyrrolidinyl, oxazolidinyl, thiazolidinyl,
piperidinyl, morpholinyl, piperazinyl, dioxolanyl and
tetrahydropyranyl.
Acyl designates, for example, alkylcarbonyl, cyclohexylcarbonyl,
arylcarbonyl, aryl-lower alkylcarbonyl, or heteroarylcarbonyl.
Lower acyl is preferably lower alkylcarbonyl, in particular
propionyl or acetyl.
Hydroxyalkyl is especially hydroxy-lower alkyl, preferably
hydroxymethyl, 2-hydroxyethyl or 2-hydroxy-2-propyl.
Cyanoalkyl designates preferably cyanomethyl and cyanoethyl.
Haloalkyl is preferably fluoroalkyl, especially trifluoromethyl,
3,3,3-trifluoroethyl or pentafluoroethyl.
Halogen is fluorine, chlorine, bromine, or iodine.
Lower alkoxy is especially methoxy, ethoxy, isopropyloxy, or
tert-butyloxy.
Arylalkyl includes aryl and alkyl as defined hereinbefore, and is
e.g. benzyl, 1-phenethyl or 2-phenethyl.
Heteroarylalkyl includes heteroaryl and alkyl as defined
hereinbefore, and is e.g. 2-, 3- or 4-pyridylmethyl, 1- or
2-pyrrolylmethyl, 1-pyrazolylmethyl, 1-imidazolylmethyl,
2-(1-imidazolyl)ethyl or 3-(1-imidazolyl)propyl.
Two adjacent substituents which together with the atoms of aryl or
heteroaryl may form a 5 or 6 membered carbocyclic or heterocyclic
ring are, for example, propylene, 1- or 2-oxopropylene, 1- or
2-oxapropylene, 1-oxapropylidene, methylenedioxy,
difluoromethylenedioxy, 1- or 2-azapropylene, 1- or
2-azapropylidene, 1,2- or 1,3-diazapropylidene,
1,3-diaza-2-oxopropylene, butylene, 1- or 2-oxabutylene,
ethylenedioxy, 1- or 2-azabutylene, or 1- or 2-azabutadienylidene,
or such groups carrying further substituents as defined
hereinbefore.
In substituted amino, the substituents are preferably those
mentioned as substituents R.sup.1 and R.sup.2. In particular,
substituted amino is alkylamino, dialkylamino, optionally
substituted arylalkylamino, or lower alkylcarbonylamino, and also
lower alkoxycarbonylamino or optionally substituted
aminocarbonylamino.
When X represents a group C.dbd.Y, wherein Y stands for nitrogen
substituted by hydroxy, this corresponds to an oxime function.
Oximes and the corresponding oxime alkyl ethers (nitrogen
substituted by alkoxy) may be present in E or Z form, or as mixture
of isomers.
When X represents a group --CO--CH.dbd.CH-- wherein the C.dbd.C
bond is connected to R, R is connected to the terminal carbon atom
of the C.dbd.C bond. The C.dbd.C bond may be present in E or Z
form, preferably in E form.
Salts are especially the pharmaceutically acceptable salts of
compounds of formula (I).
Such salts are formed, for example, as acid addition salts,
preferably with organic or inorganic acids, from compounds of
formula (I) with a basic nitrogen atom, especially the
pharmaceutically acceptable salts. Suitable inorganic acids are,
for example, halogen acids, such as hydrochloric acid, sulfuric
acid, or phosphoric acid. Suitable organic acids are, for example,
carboxylic, phosphonic, sulfonic or sulfamic acids, for example
acetic acid, propionic acid, octanoic acid, decanoic acid,
dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic
acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic
acid, tartaric acid, citric acid, amino acids, such as glutamic
acid or aspartic acid, maleic acid, hydroxymaleic acid,
methylmaleic acid, cyclohexanecarboxylic acid,
adamantane-carboxylic acid, benzoic acid, salicylic acid,
4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic
acid, cinnamic acid, methane- or ethane-sulfonic acid,
2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,
benzenesulfonic acid, 2-naphthalenesulfonic acid,
1,5-naphthalene-disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic
acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric
acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or
N-propyl-sulfamic acid, or other organic protonic acids, such as
ascorbic acid.
For isolation or purification purposes it is also possible to use
pharmaceutically unacceptable salts, for example picrates or
perchlorates. For therapeutic use, only pharmaceutically acceptable
salts or free compounds are employed (where applicable in the form
of pharmaceutical preparations), and these are therefore
preferred.
In view of the close relationship between the novel compounds in
free form and those in the form of their salts, including those
salts that can be used as intermediates, for example in the
purification or identification of the novel compounds, any
reference to the free compounds hereinbefore and hereinafter is to
be understood as referring also to the corresponding salts, as
appropriate and expedient.
The compound of the formula (I) may be administered in the form of
a pro-drug which is broken down in the human or animal body to give
a compound of the formula (I). Examples of pro-drugs include in
vivo hydrolysable esters and amides of a compound of the formula
(I). Particular pro-drugs considered are ester and amides of
naturally occurring amino acids and ester or amides of small
peptides, in particular small peptides consisting of up to five,
preferably two or three amino acids as well as esters and amides of
pegylated hydroxy acids, preferably hydroxy acetic acid and lactic
acid. Pro-drug esters are formed from the acid function of the
amino acid or the C terminal of the peptide and suitable hydroxy
group(s) in the compound of formula (I). Pro-drug amides are formed
from the amino function of the amino acid or the N terminal of the
peptide and suitable carboxy group(s) in the compound of formula
(I), or from the acid function of the amino acid or the C terminal
of the peptide and suitable amino group(s) in the compound of
formula (I).
The compounds of formula (I) have valuable pharmacological
properties. The invention also relates to compounds of formula (I)
as defined hereinbefore for use as medicaments.
The efficacy of the compounds of the invention in inducing
apoptosis in tumor cells can be demonstrated as follows:
Relative fluorescent activities of suitable tumor cell lines
transfected with green fluorescent protein (GFP) are measured in
the presence of compounds of the invention and of standard tumor
drugs, using the method described in WO 99/35493. Suitable tumor
cell lines are A20.2J, a BALB/c B cell lymphoma, PB-3c, an IL-3
dependent, non tumorigenic mastocyte line isolated from the bone
marrow of a DBA/2 mouse, Jurkat, a human acute T cell leukemia cell
line, K562, a human chronic myelogenous leukemia cell line, HL60, a
human acute promyelocytic leukemia cell line, Ramos and Raji, human
B-cell lymphoma cell lines, H9 and Hut78, human T-cell lymphoma
cell lines, HeLa and KB, human squamous cell carcinoma cell lines,
MCF7, SK-BR-3, PC3, HBL-100, SW480, H460 and H1792, human
adenocarcinoma cell lines and HT-1080, a human fibrosarcoma cell
line.
Preferred standard drugs as compounds for comparisons are: a)
antimetabolites such as 5-fluorouracil (ICN), gemcitabine HCl
(Gemzar.TM., Eli Lilly), b) alkylating agents such as oxaliplatin
(Eloxantin.TM., Sanofi-Synthelabo), dacarbazin (Detimedac.TM.,
Medac), cyclophosphamide (Endoxan.TM., Asta) and carboplatin
(Paraplatin.TM., Bristol-Meyers Squibb), c) cell-cycle inhibitor
such as vinorelbine (Navelbine.TM., Robapharm), vinblastine
(Velbe.TM., Eli Lilly), docetaxel (Taxotere.TM., Aventis), d) DNA
breaker (topo-isomerase inhibitor, intercalator, strand breaker)
such as doxorubicin HCl (Adriblastin.TM., Pharmacia-Upjohn),
bleomycin (Asta-Medica), irinotecan (Campto.TM., Aventis),
etoposide phosphate (Etopophos.TM., Bristol-Meyers Squibb),
topotecan HCl, (Hycamtin.TM., GlaxoSmithKline), e) mixtures
thereof, f) compounds interfering with the signal transduction
pathway, such as caspase activity modifiers, agonists and
antagonists of cell death receptors, modifiers of nucleases,
phosphatases and kinases such as imatinib mesylate (Gleevec.TM.,
Novartis), dexamethasone, phorbol myristate acetate, cyclosporin A,
quercetin, tamoxifen (Alexis Corporation, Switzerland).
Apoptosis is determined in a primary screen using a fluorescence
plate reader and then in a secondary screen using FACS
(fluorescence activated cell scanning). Compounds causing apoptosis
without substantial cytotoxic side effects are chosen for further
testing and characterization by using a combination of the
following well established assays: A) Nuclear staining with Hoechst
33342 dye providing information about nuclear morphology and DNA
fragmentation which are hallmarks of apoptosis. B) MTS
proliferation assay measuring the metabolic activity of cells.
Viable cells are metabolically active whereas cells with
compromised respiratory chain show a reduced activity in this test.
C) AnnexinV binding assay which reflects the phosphatidylserine
content of the outer lipid bilayer of the plasma membrane. This
event is considered an early hallmark of apoptosis. D) PI staining
for cell cycle distribution which shows any alterations in the
distribution among the different phases of the cell cycle. Cell
cycle arresting points can be determined. E) Proliferation assay
monitoring DNA synthesis by incorporating bromodeoxyuridine (BrdU).
Inhibitory effects on growth/proliferation can be directly
determined. F) Cystein proteinase dependency, respectively caspase
dependency are determined by using specific inhibitors. This
provides information about possible involvement of specific
proteases in the mechanisms.
On the basis of these studies, a compound of formula (I) according
to the invention shows therapeutic efficacy especially against
neoplastic diseases and autoimmune diseases. In particular, the
compounds of the invention are active against malignancies, e.g.
epithelial neoplasms, squamous cell neoplasms, basal cell
neoplasms, transitional cell papillomas and carcinomas, adenomas
und adenocarcinomas, adnexal and skin appendage neoplasms,
mucoepidermoid neoplasms, cystic neoplasms, mucinous and serous
neoplasms, ductal-, lobular and medullary neoplasms, acinar cell
neoplasms, complex epithelial neoplasms, specialized gonadal
neoplasms, paragangliomas and glomus tumors, naevi and melanomas,
soft tissue tumors and sarcomas, fibromatous neoplasms, myxomatous
neoplasms, lipomatous neoplasms, myomatous neoplasms, complex mixed
and stromal neoplasms, fibroepithelial neoplasms, synovial like
neoplasms, mesothelial neoplasms, germ cell neoplasms,
trophoblastic neoplasms, mesonephromas, blood vessel tumors,
lymphatic vessel tumors, osseous and chondromatous neoplasms, giant
cell tumors, miscellaneous bone tumors, odontogenic tumors,
gliomas, neuroepitheliomatous neoplasms, meningiomas, nerve sheath
tumors, granular cell tumors and alveolar soft part sarcomas,
Hodgkin's and non Hodgkin's lymphomas, other lymphoreticular
neoplasms, plasma cell tumors, mast cell tumors,
immunoproliferative diseases, leukemias, miscellaneous
myeloproliferative disorders, lymphoproliferative disorders and
myelodysplastic syndromes.
In particular, a compound of formula (I) according to the invention
shows therapeutic efficacy especially against solid neoplastic
diseases, e.g. epithelial neoplasms, squamous cell neoplasms, basal
cell neoplasms, transitional cell papillomas and carcinomas,
adenomas und adenocarcinomas, adnexal and skin appendage neoplasms,
mucoepidermoid neoplasms, cystic neoplasms, mucinous and serous
neoplasms, ductal-, lobular and medullary neoplasms, acinar cell
neoplasms, complex epithelial neoplasms, specialized gonadal
neoplasms, paragangliomas and glomus tumors, naevi and melanomas,
soft tissue tumors and sarcomas, fibromatous neoplasms, myxomatous
neoplasms, lipomatous neoplasms, myomatous neoplasms, complex mixed
and stromal neoplasms, fibroepithelial neoplasms, synovial like
neoplasms, mesothelial neoplasms, germ cell neoplasms,
trophoblastic neoplasms, mesonephromas, blood vessel tumors,
lymphatic vessel tumors, osseous and chondromatous neoplasms, giant
cell tumors, miscellaneous bone tumors, odontogenic tumors,
gliomas, neuroepitheliomatous neoplasms, meningiomas, nerve sheath
tumors, granular cell tumors and alveolar soft part sarcomas.
The compounds of the invention are likewise active against
autoimmune diseases, e.g. against systemic, discoid or subacute
cutaneous lupus erythematosus, rheumatoid arthritis,
antiphospholipid syndrome, CREST, progressive systemic sclerosis,
mixed connective tissue disease (Sharp syndrome), Reiter's
syndrome, juvenile arthritis, cold agglutinin disease, essential
mixed cryoglobulinemia, rheumatic fever, ankylosing spondylitis,
chronic polyarthritis, myasthenia gravis, multiple sclerosis,
chronic inflammatory demyelinating polyneuropathy, Guillan-Barre
syndrome, dermatomyositis/polymyositis, autoimmune hemolytic
anemia, thrompocytopenic purpura, neutropenia, type I diabetes
mellitus, thyroiditis (including Hashimoto's and Grave' disease),
Addison's disease, polyglandular syndrome, pemphigus (vulgaris,
foliaceus, sebaceous and vegetans), bullous and cicatricial
pemphigoid, pemphigoid gestationis, epidermolysis bullosa
acquisita, linear IgA disease, lichen sclerosus et atrophicus,
morbus Duhring, psoriasis vulgaris, guttate, generalized pustular
and localized pustular psoriasis, vitiligo, alopecia greata,
primary biliary cirrhosis, autoimmune hepatitis, all forms of
glomerulo-nephritis, pulmonal hemorrhage (goodpasture syndrome),
IgA nephropathy, pernicious anemia and autoimmune gastritis,
inflammatory bowel diseases (including colitis ulcerosa and morbus
Crohn), Behcet's disease, Celic-Sprue disease, autoimmune uveitis,
autoimmune myocarditis, granulomatous orchitis, aspermatogenesis
without orchitis, idiopatic and secondary pulmonary fibrosis,
inflammatory diseases with a possibility of autoimmune
pathogensesis, such as pyoderma gangrensosum, lichen ruber,
sarcoidosis (including Lofgren and cutaneous/subcutaneous type),
granuloma anulare, allergic type I and type IV immunolgical
reaction, asthma bronchiale, pollinosis, atopic, contact and
airborne dermatitis, large vessel vasculitis (giant cell and
Takayasu's arteritis), medium sized vessel vasculitis
(polyarteritis nodosa, Kawasaki disease), small vessel vasculitis
(Wegener's granulomatosis, Churg Strauss syndrome, microscopic
polangiitis, Henoch-Schoenlein purpura, essential cryoglobulinemic
vasculitis, cutaneous leukoklastic angiitis), hypersensitivity
syndromes, toxic epidermal necrolysis (Stevens-Johnson syndrome,
erythema multiforme), diseases due to drug side effects, all forms
of cutaneous, organ-specific and systemic effects due to type I-VI
(Coombs classification) immunologic forms of reaction,
transplantation related pathologies, such as acute and chronic
graft versus host and host versus graft disease, involving all
organs (skin, heart, kidney, bone marrow, eye, liver, spleen, lung,
muscle, central and peripheral nerve system, connective tissue,
bone, blood and lymphatic vessel, genito-urinary system, ear,
cartillage, primary and secondary lymphatic system including bone
marrow, lymph node, thymus, gastrointestinal tract, including
oro-pharynx, esophageus, stomach, small intestine, colon, and
rectum, including parts of above mentioned organs down to single
cell level and substructures, e.g. stem cells).
A compound of formula (I) can be administered alone or in
combination with one or more other therapeutic agents, possible
combination therapy taking the form of fixed combinations, or the
administration of a compound of the invention and one or more other
therapeutic agents being staggered or given independently of one
another, or the combined administration of fixed combinations and
one or more other therapeutic agents. A compound of formula (I)
can, besides or in addition, be administered especially for tumor
therapy in combination with chemotherapy, radiotherapy,
immunotherapy, surgical intervention, or a combination of these.
Long-term therapy is equally possible as is adjuvant therapy in the
context of other treatment strategies, as described above. Other
possible treatments are therapy to maintain the patient's status
after tumor regression, or even chemopreventive therapy, for
example in patients at risk. Particularly preferred is the use of
compounds of formula (I) in combination with radiotherapy.
Therapeutic agents for possible combination are especially one or
more cytostatic or cytotoxic compounds, for example a
chemotherapeutic agent or several selected from the group
comprising indarubicin, cytarabine, interferon, hydroxyurea,
bisulfan, or an inhibitor of polyamine biosynthesis, an inhibitor
of protein kinase, especially of serine/threonine protein kinase,
such as protein kinase C, or of tyrosine protein kinase, such as
epidermal growth factor receptor tyrosine kinase, a cytokine, a
negative growth regulator, such as TGF-.beta. or IFN-.beta., an
aromatase inhibitor, a classical cytostatic, an inhibitor of the
interaction of an SH2 domain with a phosphorylated protein, an
inhibitor of Bcl-2 and modulators of the Bcl-2 family members such
as Bax, Bid, Bad, Bim, Nip3 and BH3-only proteins.
A compound according to the invention is not only for the
(prophylactic and preferably therapeutic) management of humans, but
also for the treatment of other warm-blooded animals, for example
of commercially useful animals, for example rodents, such as mice,
rabbits or rats, or guinea-pigs. Such a compound may also be used
as a reference standard in the test systems described above to
permit a comparison with other compounds.
With the groups of preferred compounds of formula (I) mentioned
hereinafter, definitions of substituents from the general
definitions mentioned hereinbefore may reasonably be used, for
example, to replace more general definitions with more specific
definitions or especially with definitions characterized as being
preferred.
In particular, the invention relates to compounds of formula (I)
wherein
R represents aryl or heteroaryl optionally substituted by up to
four substituents independently selected from
alkyl, cycloalkyl, cycloalkyl-lower alkyl, halo-lower alkyl,
hydroxy-lower alkyl, lower alkoxy-lower alkyl, lower alkoxy-lower
alkoxy-lower alkyl, halo-lower alkoxy-lower alkyl, acyloxy-lower
alkyl, heterocyclyl, heterocyclyl-lower alkyl, optionally
substituted phenyl, optionally substituted phenyl-lower alkyl,
optionally substituted heteroaryl, optionally substituted
heteroaryl-lower alkyl, optionally substituted alkenyl, optionally
substituted alkinyl, hydroxy, lower alkoxy, optionally substituted
alkenyloxy, optionally substituted alkinyloxy, cycloalkoxy,
halo-lower alkoxy, cycloalkyl-lower alkoxy, hydroxy-lower alkoxy,
lower alkoxy-lower alkoxy, heterocyclyloxy, heterocyclyl-lower
alkoxy, optionally substituted phenyloxy, optionally substituted
phenyl-lower alkoxy, optionally substituted heteroaryloxy,
optionally substituted heteroaryl-lower alkoxy, sulfamoyloxy,
carbamoyloxy, lower alkylcarbonyloxy, amino, monoalkylamino,
dialkylamino, aminocarbonylamino wherein each of the two amino
groups is optionally substituted by alkyl, alkenyl, alkinyl or
alkoxy-lower alkyl, heterocyclylcarbonylamino wherein heterocyclyl
is bound via a nitrogen atom, aminosulfonylamino wherein each of
the two amino groups is optionally substituted by alkyl, alkenyl,
alkinyl or alkoxy-lower alkyl, heterocyclylsulfonylamino wherein
heterocyclyl is bound via a nitrogen atom, lower
alkoxycarbonylamino, lower alkylcarbonylamino wherein alkyl is
optionally substituted by one or two substituents selected from
optionally substituted phenyl, guanidyl, halogen, cyano, alkoxy,
optionally substituted phenoxy, alkylmercapto and optionally
substituted amino; lower alkenylcarbonylamino wherein alkenyl is
optionally substituted by one or two substituents selected from
lower alkyl, halo-lower alkyl, optionally substituted phenyl,
halogen, cyano, alkoxy and optionally substituted amino;
amino-lower alkyl or amino-lower alkylamino, wherein the nitrogen
atom is unsubstituted or substituted by one or two substitutents
selected from lower alkyl, cycloalkyl, cycloalkyl-lower alkyl,
hydroxy-lower alkyl, lower alkoxy-lower alkyl, optionally
substituted phenyl, optionally substituted phenyl-lower alkyl,
optionally substituted heteroaryl, optionally substituted
heteroaryl-lower alkyl and lower alkylcarbonyl, or wherein the two
substituents on nitrogen form together with the nitrogen
heterocyclyl, lower alkylcarbonyl, cycloalkylcarbonyl, optionally
substituted phenylcarbonyl, optionally substituted
heteroarylcarbonyl, heterocyclylcarbonyl, carboxy, lower
alkoxycarbonyl, hydroxy-lower alkoxycarbonyl, lower alkoxy-lower
alkoxycarbonyl, optionally substituted phenyl-lower alkoxycarbonyl,
cyano, lower alkylmercapto, optionally substituted phenylmercapto,
lower alkylsulfinyl, halo-lower alkylsulfinyl, optionally
substituted phenylsulfinyl, lower alkylsulfonyl, halo-lower
alkylsulfonyl, optionally substituted phenylsulfonyl,
aralkylsulfonyl, halogen, and nitro; and wherein two adjacent
substituents together with the atoms of aryl or heteroaryl may form
a 5 or 6 membered carbocyclic or heterocyclic ring; X represents
oxygen; or a group C.dbd.Y, wherein Y stands for oxygen, nitrogen
substituted by hydroxy or alkoxy; R.sup.1 and R.sup.2,
independently of each other, represent hydrogen, alkyl, cycloalkyl,
cycloalkylalkyl, optionally substituted arylalkyl, optionally
substituted heteroarylalkyl, hydroxyalkyl, alkoxyalkyl,
hydroxyalkoxyalkyl, alkoxyalkoxyalkyl, cyanoalkyl, optionally
substituted alkenyl, optionally substituted alkinyl, or lower
alkylcarbonyl wherein lower alkyl is optionally substituted by one
or two substitutents selected from aryl, optionally substituted
amino, alkoxy and aryloxy, R.sup.3, R.sup.4, R.sup.5 and R.sup.6,
independently of each other, represent hydrogen, lower alkyl,
halo-lower alkyl, cycloalkyl, cycloalkyl-lower alkyl, hydroxy-lower
alkyl, lower alkoxy-lower alkyl, lower alkoxy-lower alkoxy-lower
alkyl, halo-lower alkoxy-lower alkyl, heterocyclyl,
heterocyclyl-lower alkyl, optionally substituted phenyl, optionally
substituted phenyl-lower alkyl, optionally substituted heteroaryl,
optionally substituted heteroaryl-lower alkyl, optionally
substituted alkenyl, optionally substituted alkinyl, hydroxy, lower
alkoxy, halo-lower alkoxy, cycloalkoxy, cycloalkyl-lower alkoxy,
hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, heterocyclyloxy,
heterocyclyl-lower alkoxy, optionally substituted phenyloxy,
optionally substituted phenyl-lower alkoxy, optionally substituted
heteroaryloxy, optionally substituted heteroaryl-lower alkoxy,
amino, carbamoyl, sulfamoyl, amino-lower alkyl or amino-lower
alkylamino, wherein in each case the nitrogen atom is unsubstituted
or substituted by one or two substitutents selected from lower
alkyl, cycloalkyl, cycloalkyl-lower alkyl, hydroxy-lower alkyl,
lower alkoxy-lower alkyl, optionally substituted phenyl, optionally
substituted phenyl-lower alkyl, optionally substituted heteroaryl,
optionally substituted heteroaryl-lower alkyl and lower
alkylcarbonyl, or wherein the two substituents on nitrogen form
together with the nitrogen heterocyclyl, lower alkylcarbonyl,
cycloalkylcarbonyl, optionally substituted phenylcarbonyl,
optionally substituted heteroarylcarbonyl, heterocyclylcarbonyl,
carboxy, lower alkoxycarbonyl, hydroxy-lower alkoxycarbonyl, lower
alkoxy-lower alkoxycarbonyl, optionally substituted phenyl-lower
alkoxycarbonyl, cyano, lower alkylmercapto, optionally substituted
phenylmercapto, lower alkylsulfinyl, halo-lower alkylsulfinyl,
optionally substituted phenylsulfinyl, lower alkylsulfonyl,
halo-lower alkylsulfonyl, optionally substituted phenylsulfonyl,
aralkylsulfonyl, halogen, or nitro, or R.sup.3 and R.sup.4, R.sup.4
and R.sup.5, or R.sup.5 and R.sup.6 together with the atoms of the
phenyl ring form a 5 or 6 membered carbocyclic or heterocyclic
ring; and salts thereof.
More particularly the invention relates to compounds of formula (I)
wherein
R represents phenyl, naphthyl, thienyl, furyl, thiazolyl,
oxadiazolyl, thiadiazolyl, imidazolyl, pyrazolyl, pyridinyl,
pyrimidinyl, benzothienyl, benzofuryl, indolyl, benzoisoxazolyl,
optionally substituted by up to four substituents independently
selected from alkyl, cycloalkyl, cycloalkyl-lower alkyl, halo-lower
alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, lower
alkoxy-lower alkoxy-lower alkyl, halo-lower alkoxy-lower alkyl,
acyloxy-lower alkyl, heterocyclyl, heterocyclyl-lower alkyl,
optionally substituted phenyl, optionally substituted phenyl-lower
alkyl, optionally substituted heteroaryl, optionally substituted
heteroaryl-lower alkyl, optionally substituted alkenyl, optionally
substituted alkinyl, hydroxy, lower alkoxy, optionally substituted
alkenyloxy, optionally substituted alkinyloxy, cycloalkoxy,
halo-lower alkoxy, cycloalkyl-lower alkoxy, hydroxy-lower alkoxy,
lower alkoxy-lower alkoxy, heterocyclyloxy, heterocyclyl-lower
alkoxy, optionally substituted phenyloxy, optionally substituted
phenyl-lower alkoxy, optionally substituted heteroaryloxy,
optionally substituted heteroaryl-lower alkoxy, sulfamoyloxy,
carbamoyloxy, lower alkylcarbonyloxy, amino, monoalkylamino,
dialkylamino, aminocarbonylamino wherein each of the two amino
groups is optionally substituted by alkyl, alkenyl, alkinyl or
alkoxy-lower alkyl, heterocyclylcarbonylamino wherein heterocyclyl
is bound via a nitrogen atom, aminosulfonylamino wherein each of
the two amino groups is optionally substituted by alkyl, alkenyl,
alkinyl or alkoxy-lower alkyl, heterocyclylsulfonylamino wherein
heterocyclyl is bound via a nitrogen atom, lower
alkoxycarbonylamino, lower alkylcarbonylamino wherein alkyl is
optionally substituted by one or two substituents selected from
optionally substituted phenyl, guanidyl, halogen, cyano, alkoxy,
optionally substituted phenoxy, alkylmercapto and optionally
substituted amino; lower alkenylcarbonylamino wherein alkenyl is
optionally substituted by one or two substituents selected from
lower alkyl, halo-lower alkyl, optionally substituted phenyl,
halogen, cyano, alkoxy and optionally substituted amino;
amino-lower alkyl or amino-lower alkylamino, wherein the nitrogen
atom is unsubstituted or substituted by one or two substitutents
selected from lower alkyl, cycloalkyl, cycloalkyl-lower alkyl,
hydroxy-lower alkyl, lower alkoxy-lower alkyl, optionally
substituted phenyl, optionally substituted phenyl-lower alkyl,
optionally substituted heteroaryl, optionally substituted
heteroaryl-lower alkyl and lower alkylcarbonyl, or wherein the two
substituents on nitrogen form together with the nitrogen
heterocyclyl, lower alkylcarbonyl, cycloalkylcarbonyl, optionally
substituted phenylcarbonyl, optionally substituted
heteroarylcarbonyl, heterocyclylcarbonyl, carboxy, lower
alkoxycarbonyl, hydroxy-lower alkoxycarbonyl, lower alkoxy-lower
alkoxycarbonyl, optionally substituted phenyl-lower alkoxycarbonyl,
cyano, lower alkylmercapto, optionally substituted phenylmercapto,
lower alkylsulfinyl, halo-lower alkylsulfinyl, optionally
substituted phenylsulfinyl, lower alkylsulfonyl, halo-lower
alkylsulfonyl, optionally substituted phenylsulfonyl,
aralkylsulfonyl, halogen, and nitro; and wherein two adjacent
substituents together with the atoms of aryl or heteroaryl may form
a 5 or 6 membered carbocyclic or heterocyclic ring; X represents
oxygen; or a group C.dbd.Y, wherein Y stands for oxygen or nitrogen
substituted by hydroxy or alkoxy; R.sup.1 and R.sup.2,
independently of each other, represent hydrogen, lower
alkylcarbonyl or optionally substituted phenylcarbonyl; R.sup.3,
R.sup.4, R.sup.5 and R.sup.6, independently of each other,
represent hydrogen, lower alkyl, halo-lower alkyl, alkoxy,
hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, amino, carbamoyl,
sulfamoyl, amino-lower alkyl or amino-lower alkylamino, wherein in
each case the nitrogen atom is unsubstituted or substituted by one
or two substitutents selected from lower alkyl, cycloalkyl,
cycloalkyl-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower
alkyl, optionally substituted phenyl, optionally substituted
phenyl-lower alkyl, optionally substituted heteroaryl, optionally
substituted heteroaryl-lower alkyl and lower alkylaminocarbonyl, or
wherein the two substituents on nitrogen form together with the
nitrogen heterocyclyl, carboxy, lower alkoxycarbonyl, hydroxy-lower
alkoxycarbonyl, lower alkoxy-lower alkoxycarbonyl, optionally
substituted phenyl-lower alkoxycarbonyl, cyano, lower
alkylmercapto, optionally substituted phenylmercapto, lower
alkylsulfinyl, halo-lower alkylsulfinyl, optionally substituted
phenylsulfinyl, lower alkylsulfonyl, halo-lower alkylsulfonyl,
optionally substituted phenylsulfonyl, aralkylsulfonyl, halogen, or
nitro, or R.sup.3 and R.sup.4, R.sup.4 and R.sup.5, or R.sup.5 and
R.sup.6 together represent methylenedioxy; and salts thereof.
Preferred are compounds of formula (I) wherein
R represents phenyl, naphthyl, thienyl, furyl, thiazolyl,
oxadiazolyl, thiadiazolyl, imidazolyl, pyrazolyl, pyridinyl,
pyrimidinyl, benzothienyl, benzofuryl, indolyl, benzoisoxazolyl,
optionally substituted by up to four substituents independently
selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower
alkoxy-lower alkyl, lower alkoxy-lower alkoxy-lower alkyl,
halo-lower alkoxy-lower alkyl, acyloxy-lower alkyl, heterocyclyl,
heterocylyl-lower alkyl, optionally substituted phenyl, optionally
substituted phenyl-lower alkyl, optionally substituted heteroaryl,
optionally substituted heteroaryl-lower alkyl, optionally
substituted alkenyl, optionally substituted alkinyl, hydroxy, lower
alkoxy, optionally substituted alkenyloxy, optionally substituted
alkinyloxy, cycloalkoxy, halo-lower alkoxy, cycloalkyl-lower
alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,
heterocyclyloxy, heterocyclyl-lower alkoxy, optionally substituted
phenyloxy, optionally substituted phenyl-lower alkoxy, optionally
substituted heteroaryloxy, optionally substituted heteroaryl-lower
alkoxy, sulfamoyloxy, carbamoyloxy, lower alkylcarbonyloxy, amino,
monoalkylamino, dialkylamino, aminocarbonylamino wherein each of
the two amino groups is optionally substituted by alkyl, alkenyl,
alkinyl or alkoxy-lower alkyl, heterocyclylcarbonylamino wherein
heterocyclyl is bound via a nitrogen atom, aminosulfonylamino
wherein each of the two amino groups is optionally substituted by
alkyl, alkenyl, alkinyl or alkoxy-lower alkyl,
heterocyclylsulfonylamino wherein heterocyclyl is bound via a
nitrogen atom, lower alkoxycarbonylamino, lower alkylcarbonylamino
wherein alkyl is optionally substituted by one or two substituents
selected from optionally substituted phenyl, guanidyl, halogen,
cyano, alkoxy, optionally substituted phenoxy, alkylmercapto and
optionally substituted amino; lower alkenylcarbonylamino wherein
alkenyl is optionally substituted by one or two substituents
selected from lower alkyl, halo-lower alkyl, optionally substituted
phenyl, halogen, cyano, alkoxy and optionally substituted amino;
amino-lower alkyl or amino-lower alkylamino, wherein the nitrogen
atom is unsubstituted or substituted by one or two substitutents
selected from lower alkyl, cycloalkyl, cycloalkyl-lower alkyl,
hydroxy-lower alkyl, lower alkoxy-lower alkyl, optionally
substituted phenyl, optionally substituted phenyl-lower alkyl,
optionally substituted heteroaryl, optionally substituted
heteroaryl-lower alkyl and lower alkylcarbonyl, or wherein the two
substituents on nitrogen form together with the nitrogen
heterocyclyl, lower alkylcarbonyl, cycloalkylcarbonyl, optionally
substituted phenylcarbonyl, optionally substituted
heteroarylcarbonyl, heterocyclylcarbonyl, carboxy, lower
alkoxycarbonyl, hydroxy-lower alkoxycarbonyl, lower alkoxy-lower
alkoxycarbonyl, optionally substituted phenyl-lower alkoxycarbonyl,
cyano, lower alkylmercapto, optionally substituted phenylmercapto,
lower alkylsulfinyl, halo-lower alkylsulfinyl, optionally
substituted phenylsulfinyl, lower alkylsulfonyl, halo-lower
alkylsulfonyl, optionally substituted phenylsulfonyl,
aralkylsulfonyl, halogen, and nitro; and wherein two adjacent
substituents together with the atoms of aryl or heteroaryl may form
a 5 or 6 membered carbocyclic or heterocyclic ring; X represents a
group C.dbd.Y, wherein Y stands for oxygen or nitrogen substituted
by hydroxy or alkoxy; R.sup.1 and R.sup.2, independently of each
other, represent hydrogen or lower alkylcarbonyl; R.sup.3, R.sup.4,
R.sup.5 and R.sup.6, independently of each other, represent
hydrogen, lower alkyl, halo-lower alkyl, hydroxy, lower alkoxy,
carboxy, lower alkoxycarbonyl, cyano, halogen or nitro; and salts
thereof.
Most preferably, the invention relates to the compounds of the
Examples and pharmaceutically acceptable salts, especially to the
compounds 4-(1-Phenacyl-1H-benzimidazol-2-yl)-furazan-3-ylamine;
4-(1-Phenacyl-1H-benzimidazol-2-yl)-furazan-3-ylamine oxime;
4-(1-Phenacyl-1H-benzimidazol-2-yl)-furazan-3-ylamine oxime methyl
ether;
4-[1-(4-Bromophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine;
4-[1-(4-Bromophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
oxime;
4-[1-(4-Bromophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
oxime methyl ether;
4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine;
4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
oxime;
4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
oxime methyl ether;
4-[1-(4-Methoxyphenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine;
4-[1-(4-Methoxyphenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
oxime;
4-[1-(3-Methoxyphenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine;
4-[1-(3-Methoxyphenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
oxime;
4-[1-(3-Methoxyphenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
oxime methyl ether;
4-[1-(4-Phenylphenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine;
4-[1-(4-Phenylphenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
oxime;
4-[1-(4-Phenylphenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
oxime methyl ether; and
4-[1-(2,4-Dichlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine;
and to pharmaceutically acceptable salts thereof.
In another embodiment, the invention relates to compounds of
formula (I) wherein
R represents phenyl, naphthyl, thienyl, furyl, thiazolyl,
oxadiazolyl, thiadiazolyl, imidazolyl, pyrazolyl, pyridinyl,
pyrimidinyl, benzothienyl, benzofuryl, indolyl, benzoisoxazolyl,
optionally substituted by up to four substituents independently
selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower
alkoxy-lower alkyl, lower alkoxy-lower alkoxy-lower alkyl,
halo-lower alkoxy-lower alkyl, acyloxy-lower alkyl, heterocyclyl,
heterocyclyl-lower alkyl, optionally substituted phenyl, optionally
substituted phenyl-lower alkyl, optionally substituted heteroaryl,
optionally substituted heteroaryl-lower alkyl, optionally
substituted alkenyl, optionally substituted alkinyl, hydroxy, lower
alkoxy, optionally substituted alkenyloxy, optionally substituted
alkinyloxy, cycloalkoxy, halo-lower alkoxy, cycloalkyl-lower
alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,
heterocyclyloxy, heterocyclyl-lower alkoxy, optionally substituted
phenyloxy, optionally substituted phenyl-lower alkoxy, optionally
substituted heteroaryloxy, optionally substituted heteroaryl-lower
alkoxy, sulfamoyloxy, carbamoyloxy, lower alkylcarbonyloxy, amino,
monoalkylamino, dialkylamino, aminocarbonylamino wherein each of
the two amino groups is optionally substituted by alkyl, alkenyl,
alkinyl or alkoxy-lower alkyl, heterocyclylcarbonylamino wherein
heterocyclyl is bound via a nitrogen atom, aminosulfonylamino
wherein each of the two amino groups is optionally substituted by
alkyl, alkenyl, alkinyl or alkoxy-lower alkyl,
heterocyclylsulfonylamino wherein heterocyclyl is bound via a
nitrogen atom, lower alkoxycarbonylamino, lower alkylcarbonylamino
wherein alkyl is optionally substituted by one or two substituents
selected from optionally substituted phenyl, guanidyl, halogen,
cyano, alkoxy, optionally substituted phenoxy, alkylmercapto and
optionally substituted amino; lower alkenylcarbonylamino wherein
alkenyl is optionally substituted by one or two substituents
selected from lower alkyl, halo-lower alkyl, optionally substituted
phenyl, halogen, cyano, alkoxy and optionally substituted amino;
amino-lower alkyl or amino-lower alkylamino, wherein the nitrogen
atom is unsubstituted or substituted by one or two substitutents
selected from lower alkyl, cycloalkyl, cycloalkyl-lower alkyl,
hydroxy-lower alkyl, lower alkoxy-lower alkyl, optionally
substituted phenyl, optionally substituted phenyl-lower alkyl,
optionally substituted heteroaryl, optionally substituted
heteroaryl-lower alkyl and lower alkylcarbonyl, or wherein the two
substituents on nitrogen form together with the nitrogen
heterocyclyl, lower alkylcarbonyl, cycloalkylcarbonyl, optionally
substituted phenylcarbonyl, optionally substituted
heteroarylcarbonyl, heterocyclylcarbonyl, carboxy, lower
alkoxycarbonyl, hydroxy-lower alkoxycarbonyl, lower alkoxy-lower
alkoxycarbonyl, optionally substituted phenyl-lower alkoxycarbonyl,
cyano, lower alkylmercapto, optionally substituted phenylmercapto,
lower alkylsulfinyl, halo-lower alkylsulfinyl, optionally
substituted phenylsulfinyl, lower alkylsulfonyl, halo-lower
alkylsulfonyl, optionally substituted phenylsulfonyl,
aralkylsulfonyl, halogen, and nitro; and wherein two adjacent
substituents together with the atoms of aryl or heteroaryl may form
a 5 or 6 membered carbocyclic or heterocyclic ring; X represents a
group C.dbd.Y, wherein Y stands for oxygen or nitrogen substituted
by hydroxy or alkoxy; R.sup.1 represents cyanoalkyl; R.sup.2
represents hydrogen; R.sup.3, R.sup.4, R.sup.5 and R.sup.6,
independently of each other, represent hydrogen, lower alkyl,
halo-lower alkyl, hydroxy, lower alkoxy, carboxy, lower
alkoxycarbonyl, cyano, halogen or nitro; and salts thereof.
Alternatively, the invention relates to compounds of formula (I)
wherein R, X and R.sup.2 to R.sup.6 are as defined in the preceding
paragraphs and R.sup.1 represents hydroxyalkyl, and salts
thereof.
In yet another embodiment, the invention relates to compounds of
formula (I) wherein
R represents phenyl, thienyl, pyridinyl or pyridazinyl, optionally
substituted by one or two substituents independently selected
from
alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower
alkyl, acyloxy-lower alkyl, phenyl,
hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower
alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy,
amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino,
lower alkylcarbonylamino, substituted amino wherein the two
substituents on nitrogen form together with the nitrogen
heterocyclyl,
lower alkylcarbonyl, formyl,
carboxy, lower alkoxycarbonyl, cyano,
halogen, and nitro;
and wherein two adjacent substituents are methylenedioxy;
X represents oxygen; a group C.dbd.Y, wherein Y stands for oxygen
or nitrogen substituted by hydroxy or alkoxy; or a group
--CO--CH.dbd.CH-- wherein the C.dbd.C bond is connected to R;
R.sup.1 represents hydrogen, lower alkylcarbonyl, hydroxy-lower
alkyl or cyano-lower alkyl;
R.sup.2, R.sup.3 and R.sup.6 represent hydrogen;
R.sup.4 and R.sup.5, independently of each other, represent
hydrogen, lower alkyl or lower alkoxy;
or R.sup.4 and R.sup.5 together represent methylenedioxy;
and salts thereof.
Preferred are compounds of formula (I) wherein
R represents phenyl, thienyl, pyridinyl or pyridazinyl,
wherein phenyl is optionally substituted by one or two substituents
independently selected from alkyl, halo-lower alkyl, hydroxy-lower
alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl,
hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower
alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino,
monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower
alkylcarbonylamino, substituted amino wherein the two substituents
on nitrogen form together with the nitrogen heterocyclyl, lower
alkylcarbonyl, formyl, carboxy, lower alkoxycarbonyl, cyano,
halogen, and nitro; and wherein two adjacent substituents are
methylenedioxy; and wherein pyridinyl or pyridazinyl are optionally
substituted by lower alkoxy, amino or halogen; X represents a group
C.dbd.Y, wherein Y stands for oxygen or nitrogen substituted by
hydroxy or lower alkoxy; R.sup.1 represents hydrogen, lower
alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R.sup.2,
R.sup.3 and R.sup.6 represent hydrogen; R.sup.4 and R.sup.5,
independently of each other, represent hydrogen, lower alkyl or
lower alkoxy; or R.sup.4 and R.sup.5 together represent
methylenedioxy; and pharmaceutically acceptable salts thereof.
More preferred are compounds of formula (I) wherein
R represents phenyl, thienyl or pyridinyl
wherein phenyl is optionally substituted by one or two substituents
independently selected from alkyl, halo-lower alkyl, hydroxy-lower
alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl,
hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower
alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino,
monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower
alkylcarbonylamino, substituted amino wherein the two substituents
on nitrogen form together with the nitrogen heterocyclyl, lower
alkylcarbonyl, carboxy, lower alkoxycarbonyl, cyano, halogen, and
nitro; and wherein two adjacent substituents are methylenedioxy;
and wherein pyridinyl is optionally substituted by lower alkoxy,
amino or halogen; X represents a group C.dbd.Y, wherein Y stands
for oxygen or nitrogen substituted by hydroxy or lower alkoxy;
R.sup.1 represents hydrogen, lower alkylcarbonyl, hydroxy-lower
alkyl or cyano-lower alkyl; R.sup.2, R.sup.3 and R.sup.6 represent
hydrogen; R.sup.4 and R.sup.5, independently of each other,
represent hydrogen, lower alkyl or lower alkoxy; or R.sup.4 and
R.sup.5 together represent methylenedioxy; and pharmaceutically
acceptable salts thereof.
Particularly preferred are compounds of formula (I) wherein
R represents phenyl, thienyl, pyridinyl or pyridazinyl,
wherein phenyl is optionally substituted by one or two substituents
independently selected from alkyl, halo-lower alkyl, hydroxy-lower
alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl,
hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower
alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino,
monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower
alkylcarbonylamino, substituted amino wherein the two substituents
on nitrogen form together with the nitrogen heterocyclyl, lower
alkylcarbonyl, formyl, carboxy, lower alkoxycarbonyl, cyano,
halogen, and nitro; and wherein two adjacent substituents are
methylenedioxy; and wherein pyridinyl or pyridazinyl are optionally
substituted by lower alkoxy, amino or halogen; X represents a group
C.dbd.Y, wherein Y stands for oxygen or nitrogen substituted by
hydroxy or lower alkoxy; R.sup.1 represents cyano-lower alkyl;
R.sup.2, R.sup.3 and R.sup.6 represent hydrogen; R.sup.4 and
R.sup.5, independently of each other, represent hydrogen, lower
alkyl or lower alkoxy; or R.sup.4 and R.sup.5 together represent
methylenedioxy; and pharmaceutically acceptable salts thereof.
Highly preferred are compounds of formula (I) wherein
R represents phenyl or pyridinyl
wherein phenyl is optionally substituted by one or two substituents
independently selected from alkyl, halo-lower alkyl, hydroxy-lower
alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl,
hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower
alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino,
monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower
alkylcarbonylamino, substituted amino wherein the two substituents
on nitrogen form together with the nitrogen heterocyclyl, lower
alkylcarbonyl, carboxy, lower alkoxycarbonyl, formyl, cyano,
halogen, and nitro; and wherein two adjacent substituents are
methylenedioxy; and wherein pyridinyl is optionally substituted by
lower alkoxy, amino or halogen; X represents oxygen; R.sup.1
represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or
cyano-lower alkyl; R.sup.2, R.sup.3 and R.sup.6 represent hydrogen;
R.sup.4 and R.sup.5, independently of each other, represent
hydrogen, lower alkyl or lower alkoxy; or R.sup.4 and R.sup.5
together represent methylenedioxy; and pharmaceutically acceptable
salts thereof.
Likewise preferred are compounds of formula (I) wherein R and
R.sup.1 to R.sup.6 are defined as in the preceding paragraphs, and
X represents nitrogen substituted by alkoxy, and pharmaceutically
acceptable salts thereof.
Other preferred compounds that come into consideration are
compounds of formula (I)
wherein
R represents phenyl or pyridinyl
wherein phenyl is optionally substituted by one or two substituents
independently selected from alkyl, halo-lower alkyl, hydroxy-lower
alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl,
hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower
alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino,
monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower
alkylcarbonylamino, substituted amino wherein the two substituents
on nitrogen form together with the nitrogen heterocyclyl, lower
alkylcarbonyl, carboxy, lower alkoxycarbonyl, formyl, cyano,
halogen, and nitro; and wherein two adjacent substituents are
methylenedioxy; and wherein pyridinyl is optionally substituted by
lower alkoxy, amino or halogen; X represents oxygen; R.sup.1
represents cyano-lower alkyl; R.sup.2, R.sup.3, R.sup.4, R.sup.5
and R.sup.6 represent hydrogen; and pharmaceutically acceptable
salts thereof.
More preferred are compounds of formula (I) wherein
R represents phenyl or pyridinyl
wherein phenyl is optionally substituted by one or two substituents
independently selected from alkyl, lower alkoxy-lower alkyl,
acyloxy-lower alkyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy,
lower alkoxy-lower alkoxy, amino, monoalkylamino, dialkylamino,
lower alkoxycarbonylamino, lower alkylcarbonylamino, substituted
amino wherein the two substituents on nitrogen form together with
the nitrogen heterocyclyl, halogen, and nitro; and wherein two
adjacent substituents are methylenedioxy; and wherein pyridinyl is
optionally substituted by lower alkoxy, amino or halogen; X
represents oxygen; R.sup.1 represents cyano-lower alkyl; R.sup.2,
R.sup.3, R.sup.4, R.sup.5 and R.sup.6 represent hydrogen; and
pharmaceutically acceptable salts thereof.
Particularly preferred are compounds of formula (I) according to
claim 8 wherein
R represents phenyl or pyridinyl
wherein phenyl is optionally substituted by one or two substituents
independently selected from alkyl, lower alkoxy-lower alkyl,
acyloxy-lower alkyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy,
lower alkoxy-lower alkoxy, amino, monoalkylamino, dialkylamino,
lower alkoxycarbonylamino, lower alkylcarbonylamino, substituted
amino wherein the two substituents on nitrogen form together with
the nitrogen heterocyclyl, halogen, and nitro; and wherein two
adjacent substituents are methylenedioxy; and wherein pyridinyl is
optionally substituted by lower alkoxy, amino or halogen; X
represents nitrogen substituted by alkoxy; R.sup.1 represents
cyano-lower alkyl; R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6
represent hydrogen; and pharmaceutically acceptable salts
thereof.
Other preferred compounds of formula (I) are those wherein
R represents phenyl optionally substituted by one or two
substituents independently selected from alkyl, halo-lower alkyl,
hydroxy-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl,
phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower
alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy,
amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino,
lower alkylcarbonylamino, substituted amino wherein the two
substituents on nitrogen form together with the nitrogen
heterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl,
cyano, halogen, and nitro; and wherein two adjacent substituents
are methylenedioxy; X represents a group --CO--CH.dbd.CH-- wherein
the C.dbd.C bond is connected to R; R.sup.1 represents cyano-lower
alkyl; R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 represent
hydrogen; and pharmaceutically acceptable salts thereof.
Most preferred are compounds selected from the group consisting of
4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethy-
l)-amine;
4-[1-(3-Methoxy-4-methoxymethoxy-phenacyl)-1H-benzimidazol-2-yl]-
-furazan-3-ylamine;
4-[1-(4-Bromophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl-
)-amine;
4-[1-(4-Aminophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cy-
anoethyl)-amine;
4-[1-(4-Methoxyphenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoeth-
yl)-amine;
4-[1-(3,4-Dimethylphenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl--
N-(2-cyanoethyl)-amine;
4-[1-(4-Ethylphenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl-
)-amine;
4-[1-(6-Chloro-3-pyridyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine-
;
4-[1-(6-Amino-3-pyridyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoet-
hyl)-amine; and
4-[1-(6-Amino-3-pyridyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine;
and pharmaceutically acceptable salts thereof.
Especially, the invention relates to compounds as described
hereinbefore for use as medicaments.
The invention also relates to the use of a compound of formula (I),
a prodrug or a pharmaceutically acceptable salt of such a compound
for the preparation of a pharmaceutical composition for the
treatment of a neoplastic disease, autoimmune disease,
transplantation related pathology and/or degenerative disease, in
particular for the treatment of a solid neoplastic disease.
Furthermore, the invention provides a method for the treatment of a
neoplastic disease, autoimmune disease, transplantation related
pathology and/or degenerative disease, in particular of a solid
neoplastic disease, which comprises administering a compound of
formula (I), a prodrug or a pharmaceutically acceptable salt
thereof, wherein the radicals and symbols have the meanings as
defined above, in a quantity effective against said disease, to a
warm-blooded animal requiring such treatment.
Method of Preparation
A compound of the invention may be prepared by processes that,
though not applied hitherto for the new compounds of the present
invention, are known per se, in particular
A) a process, wherein a compound of formula (II)
##STR00003## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5
and R.sup.6 are defined as for formula (I), or a derivative thereof
with functional groups in protected form and/or a salt thereof, is
alkylated with an alkylating agent of formula (III)
R--X--CH.sub.2-Z (III) wherein R is as defined for formula (I), X
is CO or --CO--CH.dbd.CH-- and Z is a nucleophilic leaving group;
or B) a process, wherein a compound of formula (II) wherein
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are defined
as for formula (I), or a derivative thereof with functional groups
in protected form and/or a salt thereof, is alkylated with a
mixture of a dihalomethane type compound of formula
Z.sup.1-CH.sub.2-Z.sup.2 (IV), wherein Z.sup.1 and Z.sup.2 are
leaving groups, and a compound of formula R--XH (V), wherein R is
as defined for formula (I) and X is oxygen; any protecting groups
in a protected derivative of a compound of the formula (I) are
removed; and, if so desired, an obtainable compound of formula (I)
is converted into another compound of formula (I), a free compound
of formula (I) is converted into a salt, an obtainable salt of a
compound of formula (I) is converted into the free compound or
another salt, and/or a mixture of isomeric compounds of formula (I)
is separated into the individual isomers.
Suitable nucleophilic leaving groups Z in an alkylating agent of
formula (III) are for example halides, e.g. chloride, bromide or
iodide, or sulfonates, e.g. aromatic sulfonic acid esters such as
benzenesulfonates, p-toluenesulfonates or p-nitrobenzenesulfonates,
or also methanesulfonate or trifluormethanesulfonate. Also other
customary leaving groups are considered, e.g. ammonium salts,
azides, diazonium salts, di(p-toluenesulfonyl)-amines, nitrates,
oxonium salts, sulfonium salts, or phosphonium salts. Suitable
nucleophilic leaving groups Z.sup.1 and Z.sup.2 in a dihalomethane
type compound formula (IV) are those mentioned above, in particular
chlorine, bromine and iodine.
Alkylation of a compound of formula (II) with an alkylating agent
of formula (III) is performed in a manner known per se, usually in
the presence of a suitable polar or dipolar aprotic solvent, with
cooling or heating, for example in a temperature range from
approximately -30.degree. C. to approximately +150.degree. C.,
especially approximately around 0.degree. C. to room temperature.
Optionally a suitable base is added, in particularly a tertiary
amine base such as triethylamine or diisopropylethylamine, or an
inorganic basic salt, e.g. potassium or sodium carbonate.
Mixed alkylation of a compound of formula (II) and of a compound of
formula (V) with a dihalomethane type compound of formula (IV) is
performed in the presence of a suitable polar or dipolar aprotic
solvent, with cooling or heating, for example in a temperature
range from approximately -30.degree. C. to approximately
+150.degree. C., especially approximately around 0.degree. C. to
room temperature. Suitable bases used in this reaction are for
example potassium and sodium carbonate.
If one or more other functional groups, for example carboxy,
hydroxy or amino, are or need to be protected in a compound of
formula (II), (III) or (V), because they should not take part in
the reaction, these are such protecting groups as are usually
applied in the synthesis of amides, in particular peptide
compounds, cephalosporins, penicillins, nucleic acid derivatives
and sugars.
The protecting-groups may already be present in precursors and
should protect the functional groups concerned against unwanted
secondary reactions, such as alkylations, acylations,
etherifications, esterifications, oxidations, solvolysis, and
similar reactions. It is a characteristic of protecting groups that
they lend themselves readily, i.e. without undesired secondary
reactions, to removal, typically by solvolysis, reduction,
photolysis or also by enzyme activity, for example under conditions
analogous to physiological conditions, and that they are not
present in the end products. The specialist knows, or can easily
establish, which protecting groups are suitable with the reactions
mentioned hereinabove and hereinafter.
The protection of such functional groups by such protecting groups,
the protecting groups themselves, and their removal reactions are
described for example in standard reference books for peptide
synthesis and in special books on protective groups such as J. F.
W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press,
London and New York 1973, in "Methoden der organischen Chemie"
(Methods of organic chemistry), Houben-Weyl, 4th edition, Volume
15/l, Georg Thieme Verlag, Stuttgart 1974, and in T. W. Greene,
"Protective Groups in Organic Synthesis", Wiley, New York.
In the additional process steps, carried out as desired, functional
groups of the starting compounds which should not take part in the
reaction may be present in unprotected form or may be protected for
example by one or more of the protecting groups mentioned
hereinabove under "protecting groups". The protecting groups are
then wholly or partly removed according to one of the methods
described there.
In the conversion of an obtainable compound of formula (I) into
another compound of formula (I), X with the meaning C.dbd.Y wherein
Y is oxygen may, for example, be reacted with an optionally
O-substituted hydroxylamine to give the corresponding oxime or
oxime ether of formula (I) wherein X is C.dbd.Y and Y is nitrogen
substituted by hydroxy or alkoxy.
An obtainable compound of formula (I), wherein R.sup.1 and/or
R.sup.2 is hydrogen, may be alkylated or acylated with a compound
of formula R.sup.1-Z or R.sup.2-Z, respectively, wherein Z is a
nucleophilic leaving group as described above, to give a compound
of formula (I), wherein R.sup.1 and/or R.sup.2 is different from
hydrogen. Preferred acylation conditions include the use of acid
anhydrides and acid chlorides at elevated temperatures, typically
in a range from approximately +30.degree. C. to approximately
+150.degree. C. An acidic or basic catalyst may be employed if
desired. A compound of formula (I) wherein R.sup.1 and/or R.sup.2
is alkyl may be obtained by alkylation of the parent compound of
formula (I). Typical reaction conditions allowing this
transformation include the combination of a strong base, such as a
metal hydride or a metal alcoholate and a compound of formula
R.sup.1-Z or R.sup.2-Z.
Further amino groups present in an aryl or heteroaryl group R or in
one of the substitutents R.sup.3, R.sup.4, R.sup.5 or R.sup.6 may
be transformed to other nitrogen containing substituents under
conditions known in the art. For example, alkylation at nitrogen
may be performed with an aldehyde under reducing conditions. For
acylation the corresponding acyl chloride (Z=Cl) is preferred.
Alternatively, an acid anhydride may be used, or acylation may be
accomplished with the free acid (Z=OH) under conditions used for
amide formation known per se in peptide chemistry, e.g. with
activating agents for the carboxy group, such as
1-hydroxybenzotriazole, optionally in the presence of suitable
catalysts or co-reagents.
Compounds of formula (I) wherein X.dbd.NOH may be alkylated
allowing access to the corresponding oxime ethers. The reaction
conditions leading to this transformation include combinations of
weak bases and alkylating agents. Typical bases include metal
carbonates or bicarbonates.
Reduction of a nitro group in an nitro-substituted aryl or
heteroaryl group R or in one of the substituents R.sup.3, R.sup.4,
R.sup.5 or R.sup.6 to give the corresponding amino group is done,
e.g., with iron powder in alcohol or with other reducing
agents.
A carboxy group in a carboxy-substituted aryl or heteroaryl group R
or in one of the substituents R.sup.3, R.sup.4, R.sup.5 or R.sup.6
may be amidated under conditions used for amide formation known per
se in peptide chemistry, e.g. with the corresponding amine and an
activating agent for the carboxy group, such as
1-hydroxybenzotriazole, optionally in the presence of suitable
catalysts or co-reagents.
A bromo or iodo substitutent in an aryl or heteroaryl group R or in
one of the substituents R.sup.3, R.sup.4, R.sup.5 or Re may be
replaced by phenyl or a phenyl derivative by reaction with a
suitable phenylboronic acid in a Suzuki reaction, preferably in a
dipolar aprotic solvent such as dimethyl formamide, or in a polar
ether, e.g. tetrahydrofuran or dimethoxyethane, in the presence of
a soluble palladium(0) or related metal catalyst, for example
tetrakis-(triphenylphosphine)palladium.
Salts of a compound of formula (I) with a salt-forming group may be
prepared in a manner known per se. Acid addition salts of compounds
of formula (I) may thus be obtained by treatment with an acid or
with a suitable anion exchange reagent.
Salts can usually be converted to free compounds, e.g. by treating
with suitable basic agents, for example with alkali metal
carbonates, alkali metal hydrogencarbonates, or alkali metal
hydroxides, typically potassium carbonate or sodium hydroxide.
It should be emphasized that reactions analogous to the conversions
mentioned in this chapter may also take place at the level of
appropriate intermediates.
All process steps described here can be carried out under known
reaction conditions, preferably under those specifically mentioned,
in the absence of or usually in the presence of solvents or
diluents, preferably such as are inert to the reagents used and
able to dissolve these, in the absence or presence of catalysts,
condensing agents or neutralising agents, for example ion
exchangers, typically cation exchangers, for example in the H.sup.+
form, depending on the type of reaction and/or reactants at
reduced, normal, or elevated temperature, for example in the range
from -100.degree. C. to about 190.degree. C., preferably from about
-80.degree. C. to about 150.degree. C., for example at -80 to
+60.degree. C., at -20 to +40.degree. C., at room temperature, or
at the boiling point of the solvent used, under atmospheric
pressure or in a closed vessel, where appropriate under pressure,
and/or in an inert atmosphere, for example under argon or
nitrogen.
Salts may be present in all starting compounds and transients, if
these contain salt-forming groups. Salts may also be present during
the reaction of such compounds, provided the reaction is not
thereby disturbed.
At all reaction stages, isomeric mixtures that occur can be
separated into their individual isomers, e.g. diastereomers or
enantiomers, or into any mixtures of isomers, e.g. racemates or
diastereomeric mixtures.
The invention relates also to those forms of the process in which
one starts from a compound obtainable at any stage as a transient
and carries out the missing steps, or breaks off the process at any
stage, or forms a starting material under the reaction conditions,
or uses said starting material in the form of a reactive derivative
or salt, or produces a compound obtainable by means of the process
according to the invention and further processes the said compound
in situ. In the preferred embodiment, one starts from those
starting materials which lead to the compounds described
hereinabove as preferred, particularly as especially preferred,
primarily preferred, and/or preferred above all.
In the preferred embodiment, a compound of formula (I) is prepared
according to or in analogy to the processes and process steps
defined in the Examples.
The compounds of formula (I), including their salts, are also
obtainable in the form of hydrates, or their crystals can include
for example the solvent used for crystallization, i.e. be present
as solvates.
New starting materials and/or intermediates, as well as processes
for the preparation thereof, are likewise the subject of this
invention. In the preferred embodiment, such starting materials are
used and reaction conditions so selected as to enable the preferred
compounds to be obtained.
Starting materials of formula (II), (III), (IV) and (V) are known,
commercially available, or can be synthesized in analogy to or
according to methods that are known in the art.
Pharmaceutical Preparations, Methods, and Uses
The present invention relates also to pharmaceutical compositions
that comprise a compound of formula (I) as active ingredient and
that can be used especially in the treatment of the diseases
mentioned at the beginning. Compositions for enteral
administration, such as nasal, buccal, rectal or, especially, oral
administration, and for parenteral administration, such as
intravenous, intramuscular or subcutaneous administration, to
warm-blooded animals, especially humans, are especially preferred.
The compositions comprise the active ingredient alone or,
preferably, together with a pharmaceutically acceptable carrier.
The dosage of the active ingredient depends upon the disease to be
treated and upon the species, its age, weight, and individual
condition, the individual pharmacokinetic data, and the mode of
administration.
The present invention relates especially to pharmaceutical
compositions that comprise a compound of formula (I), a tautomer, a
prodrug or a pharmaceutically acceptable salt, or a hydrate or
solvate thereof, and at least one pharmaceutically acceptable
carrier.
The invention relates also to pharmaceutical compositions for use
in a method for the prophylactic or especially therapeutic
management of the human or animal body, in particular in a method
of treating neoplastic disease, autoimmune disease, transplantation
related pathology and/or degenerative disease, especially those
mentioned hereinabove.
The invention relates also to processes and to the use of compounds
of formula (I) thereof for the preparation of pharmaceutical
preparations which comprise compounds of formula (I) as active
component (active ingredient).
A pharmaceutical composition for the prophylactic or especially
therapeutic management of a neoplastic disease, autoimmune disease,
transplantation related pathology and/or degenerative disease, of a
warm-blooded animal, especially a human or a commercially useful
mammal requiring such treatment, comprising a novel compound of
formula (I) as active ingredient in a quantity that is
prophylactically or especially therapeutically active against the
said diseases, is likewise preferred.
The pharmaceutical compositions comprise from approximately 1% to
approximately 95% active ingredient, single-dose administration
forms comprising in the preferred embodiment from approximately 20%
to approximately 90% active ingredient and forms that are not of
single-dose type comprising in the preferred embodiment from
approximately 5% to approximately 20% active ingredient. Unit dose
forms are, for example, coated and uncoated tablets, ampoules,
vials, suppositories, or capsules. Further dosage forms are, for
example, ointments, creams, pastes, foams, tinctures, lip-sticks,
drops, sprays, dispersions, etc. Examples are capsules containing
from about 0.05 g to about 1.0 g active ingredient.
The pharmaceutical compositions of the present invention are
prepared in a manner known per se, for example by means of
conventional mixing, granulating, coating, dissolving or
lyophilizing processes.
Preference is given to the use of solutions of the active
ingredient, and also suspensions or dispersions, especially
isotonic aqueous solutions, dispersions or suspensions which, for
example in the case of lyophilized compositions comprising the
active ingredient alone or together with a carrier, for example
mannitol, can be made up before use. The pharmaceutical
compositions may be sterilized and/or may comprise excipients, for
example preservatives, stabilizers, wetting agents and/or
emulsifiers, solubilizers, salts for regulating osmotic pressure
and/or buffers and are prepared in a manner known per se, for
example by means of conventional dissolving and lyophilizing
processes. The said solutions or suspensions may comprise
viscosity-increasing agents, typically sodium
carboxymethylcellulose, carboxymethylcellulose, dextran,
polyvinylpyrrolidone, or gelatins, or also solubilizers, e.g. Tween
80.RTM. (polyoxyethylene(20)sorbitan mono-oleate).
Suspensions in oil comprise as the oil component the vegetable,
synthetic, or semi-synthetic oils customary for injection purposes.
In respect of such, special mention may be made of liquid fatty
acid esters that contain as the acid component a long-chained fatty
acid having from 8 to 22, especially from 12 to 22, carbon atoms.
The alcohol component of these fatty acid esters has a maximum of 6
carbon atoms and is a monovalent or polyvalent, for example a
mono-, di- or trivalent, alcohol, especially glycol and glycerol.
As mixtures of fatty acid esters, vegetable oils such as cottonseed
oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and
groundnut oil are especially useful.
The manufacture of injectable preparations is usually carried out
under sterile conditions, as is the filling, for example, into
ampoules or vials, and the sealing of the containers.
Suitable carriers are especially fillers, such as sugars, for
example lactose, saccharose, mannitol or sorbitol, cellulose
preparations, and/or calcium phosphates, for example tricalcium
phosphate or calcium hydrogen phosphate, and also binders, such as
starches, for example corn, wheat, rice or potato starch,
methylcellulose, hydroxypropyl methylcellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if
desired, disintegrators, such as the above-mentioned starches, also
carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic
acid or a salt thereof, such as sodium alginate. Additional
excipients are especially flow conditioners and lubricants, for
example silicic acid, talc, stearic acid or salts thereof, such as
magnesium or calcium stearate, and/or polyethylene glycol, or
derivatives thereof.
Tablet cores can be provided with suitable, optionally enteric,
coatings through the use of, inter alia, concentrated sugar
solutions which may comprise gum arabic, talc,
polyvinyl-pyrrolidone, polyethylene glycol and/or titanium dioxide,
or coating solutions in suitable organic solvents or solvent
mixtures, or, for the preparation of enteric coatings, solutions of
suitable cellulose preparations, such as acetylcellulose phthalate
or hydroxypropyl-methylcellulose phthalate. Dyes or pigments may be
added to the tablets or tablet coatings, for example for
identification purposes or to indicate different doses of active
ingredient.
Pharmaceutical compositions for oral administration also include
hard capsules consisting of gelatin, and also soft, sealed capsules
consisting of gelatin and a plasticizer, such as glycerol or
sorbitol. The hard capsules may contain the active ingredient in
the form of granules, for example in admixture with fillers, such
as corn starch, binders, and/or glidants, such as talc or magnesium
stearate, and optionally stabilizers. In soft capsules, the active
ingredient is preferably dissolved or suspended in suitable liquid
excipients, such as fatty oils, paraffin oil or liquid polyethylene
glycols or fatty acid esters of ethylene or propylene glycol, to
which stabilizers and detergents, for example of the
polyoxyethylene sorbitan fatty acid ester type, may also be
added.
Pharmaceutical compositions suitable for rectal administration are,
for example, suppositories that consist of a combination of the
active ingredient and a suppository base. Suitable suppository
bases are, for example, natural or synthetic triglycerides,
paraffin hydrocarbons, polyethylene glycols or higher alkanols.
For parenteral administration, aqueous solutions of an active
ingredient in water-soluble form, for example of a water-soluble
salt, or aqueous injection suspensions that contain
viscosity-increasing substances, for example sodium
carboxymethylcellulose, sorbitol and/or dextran, and, if desired,
stabilizers, are especially suitable. The active ingredient,
optionally together with excipients, can also be in the form of a
lyophilizate and can be made into a solution before parenteral
administration by the addition of suitable solvents.
Solutions such as are used, for example, for parenteral
administration can also be employed as infusion solutions.
Preferred preservatives are, for example, antioxidants, such as
ascorbic acid, or microbicides, such as sorbic acid or benzoic
acid.
The present invention relates furthermore to a method for the
treatment of a neoplastic disease, autoimmune disease,
transplantation related pathology and/or degenerative disease,
which comprises administering a compound of formula (I) or a
pharmaceutically acceptable salt thereof, wherein the radicals and
symbols have the meanings as defined above for formula (I), in a
quantity effective against said disease, to a warm-blooded animal
requiring such treatment. The compounds of formula (I) can be
administered as such or especially in the form of pharmaceutical
compositions, prophylactically or therapeutically, preferably in an
amount effective against the said diseases, to a warm-blooded
animal, for example a human, requiring such treatment. In the case
of an individual having a bodyweight of about 70 kg the daily dose
administered is from approximately 0.05 g to approximately 5 g,
preferably from approximately 0.25 g to approximately 1.5 g, of a
compound of the present invention.
The present invention relates especially also to the use of a
compound of formula (I), or a pharmaceutically acceptable salt
thereof, especially a compound of formula (I) which is said to be
preferred, or a pharmaceutically acceptable salt thereof, as such
or in the form of a pharmaceutical formulation with at least one
pharmaceutically acceptable carrier for the therapeutic and also
prophylactic management of one or more of the diseases mentioned
hereinabove, in particular a neoplastic disease, autoimmune
disease, transplantation related pathology and/or degenerative
disease.
The preferred dose quantity, composition, and preparation of
pharmaceutical formulations (medicines) which are to be used in
each case are described above.
The following Examples serve to illustrate the invention without
limiting the invention in its scope.
EXAMPLES
Abbreviations: DMSO dimethyl=sulfoxide; THF=tetrahydrofuran,
DMAP=N,N-dimethylaminopyridine, DMF=N,N-dimethylformamide,
DIPEA=N,N-diisopropyl-N-ethylamine.
Example 1
4-(1-Phenacyl-1H-benzimidazol-2-yl)-furazan-3-ylamine
Phenacylbromid (0.1 g, 0.49 mmol) is added to an efficiently
stirred suspension of 4-(1H-benzimidazol-2-yl)-furazan-3-ylamine
(0.1 g, 0.4 mmol) [A. V. Sergievskii, O. A. Krasnoshek, S. F.
Mel'nikova, I. V. Tselinskii, Russian Journal of Organic Chemistry,
2002, 38, 915-917] and potassium carbonate (0.172 g, 1.24 mmol) in
dry DMF (5 ml) at room temperature. After 4 hours the reaction
mixture is diluted with ethyl acetate and the organic phase is
washed repeatedly with brine. Drying of the solvent, filtering and
evaporation of the solvent under reduced pressure gives the title
compound in crude form. The title compound is obtained in pure form
by chromatography over silicagel, m.p. 202-204.degree. C.
Example 2
4-[1-(4-Bromophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
oxime
A mixture of
4-[1-(4-bromophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
(0.083 g, 0.21 mmol, prepared according to Example 1), sodium
bicarbonate (0.021 g, 0.25 mmol) and hydroxylamine hydrochloride
(0.014 g, 0.21 mmol) in ethanol (5 ml) is refluxed for 20 hours.
Partitioning of the reaction mixture between ethyl acetate and
water, separation of the organic phase followed by drying and
evaporation of the solvent gives the crude product. Purification by
chromatography on silicagel yields the title compound as an
E/Z-mixture, m.p. 198-201.degree. C.
Example 3
Methyl-{4-[1-(4-chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl}-amine
A suspension of
4-[1-(4-chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
(0.10 g, 0.282 mmol, prepared according to Example 1), potassium
carbonate (0.233 g, 1.69 mmol) and dimethylsulfate (0.142 g, 1.12
mmol) in acetone (5 ml) is stirred at room temperature for 16
hours. Filtration of the solids, concentration of the filtrate
under reduced pressure and chromatography of the residue on
silicagel using hexane-ethyl acetate as eluent gives the title
compound as a colorless solid, m.p. 210-214.degree. C.
Example 4
Dimethyl-{4-[1-(4-chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl}-amin-
e
A suspension of
4-[1-4-chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
(0.10 g, 0.282 mmol), potassium carbonate (0.60 g, 4.22 mmol) and
dimethylsulfate (0.50 g, 2.82 mmol) in DMF (5 ml) is stirred at
60.degree. C. for 6 hours. The reaction mixture is diluted with
ethyl acetate, washed with water and dried over sodium sulphate.
Filtration of the sodium sulphate, concentration of the filtrate
under reduced pressure and chromatography of the residue on
silicagel using hexane-ethyl acetate as eluent gives the title
compound as a yellowish solid, m.p. 120-123.degree. C.
Example 5
N-{4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl}-acetamide
A solution of
4-[1-(4-chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
(0.05 g, 0.143 mmol), pyridine (0.022 g, 0.282 mmol), acetyl
chloride (0.013 g, 0.169 mmol) and a catalytic amount of DMAP in
DMF (5 ml) is stirred at 80.degree. C. for 16 hours. The reaction
mixture is diluted with ethyl acetate, washed with water and dried
over sodium sulphate. Filtration of the sodium sulphate,
concentration of the filtrate under reduced pressure and
chromatography of the residue on silicagel using hexane-ethyl
acetate as eluent gives the title compound as a yellowish solid,
m.p. 202-205.degree. C.
Example 6
4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl-
)-amine
A suspension of
4-(1H-benzimidazol-2-yl)-furazan-3-yl-N-(2-cyanoethyl)-amine (0.10
g, 0.39 mmol), potassium carbonate (0.08 g, 0.58 mmol) and
4-chlorophenacyl bromide (0.11 g, 0.47 mmol) in DMF (5 ml) is
stirred at room temperature for 16 hours. The reaction mixture is
diluted with ethyl acetate, washed with water and dried over sodium
sulphate. Filtration of the sodium sulphate, concentration of the
filtrate under reduced pressure and chromatography of the residue
on silicagel using hexane-ethyl acetate as eluent gives the title
compound as a yellowish solid, m.p. 191-192.degree. C.
Example 6a
4-(1H-Benzimidazol-2-yl)-furazan-3-yl-N-(2-cyanoethyl)-amine
To a solution of 4-(1H-benzimidazol-2-yl)-furazan-3-ylamine (0.10
g, 0.497 mmol) in pyridine (5 ml) sodium in methanol (0.02 g, 0.86
mmol in 1 ml) and acrylonitrile (0.03 g, 0.39 mmol) are added
sequentially at 0.degree. C. The mixture is stirred over night.
Evaporation of the solvent under reduced pressure and partitioning
of the resulting residue between water and ethyl acetate followed
by drying of the organic solution over sodium sulphate gives the
title compound in pure form. .sup.1H-NMR (400 MHz, d.sup.6-DMSO):
13.7 (s, 1H); 7.82 (d, 1H); 7.60 (d, 1H); 7.36 (m, 2H); 7.20 (t,
1H); 3.67 (q, 2H); 2.94 (t, 2H).
Example 7
4-(1-Phenoxymethyl-1H-benzimidazol-2-yl)-furazan-3-ylamine
To a solution of 4-(1H-benzimidazol-2-yl)-furazan-3-ylamine (0.10
g, 0.497 mmol) is added potassium carbonate (0.172 g, 1.24 mmol)
and iodomethoxy benzene (0.128 g, 0.546 mmol). The mixture is
stirred over night. Evaporation of the solvent under reduced
pressure and partitioning of the resulting residue between water
and ethyl acetate followed by drying of the organic solution over
sodium sulphate and chromatography of the residue gives the title
compound as a colorless solid, m.p. 171-173.degree. C.
Example 8
4-[1-(4-Fluorophenoxymethyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
To a solution of 4-(1H-benzimidazol-2-yl)-furazan-3-ylamine (0.10
g, 0.497 mmol) is added potassium carbonate (0.172 g, 1.24 mmol)
followed by 4-fluorophenol (0.0557 g, 0.497 mmol) and diiodomethane
(0.133 g, 0.497 mmol). The mixture is stirred over night.
Evaporation of the solvent under reduced pressure and partitioning
of the resulting residue between water and ethyl acetate followed
by drying of the organic solution over sodium sulphate and
chromatography of the residue gives the title compound as a
colorless solid, m.p. 155-158.degree. C.
Example 9
4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-methoxycar-
bonylethyl)-amine
A suspension of
4-(1H-benzimidazol-2-yl)-furazan-3-yl-N-(2-methoxycarbonylethyl)-amine
(0.052 g, 0.181 mmol), potassium carbonate (0.062 g, 0.452 mmol)
and 4-chlorophenacyl bromide (0.047 g, 0.199 mmol) in DMF (5 ml) is
stirred at room temperature for 16 hours. The reaction mixture is
diluted with ethyl acetate, washed with water and dried over sodium
sulphate. Filtration of the sodium sulphate, concentration of the
filtrate under reduced pressure and chromatography of the residue
on silicagel using hexane-ethyl acetate as eluent gives the title
compound as hygroscopic solid of undefined melting point.
.sup.1H-NMR (400 MHz, d.sup.6-DMSO): 8.14 (d, 2H); 7.87 (m, 2H);
7.41 (m, 2H); 7.32 (d, 2H); 7.29 (t, 1H); 6.37 (s, 2H); 3.63 (m,
2H); 3.61 (s, 3H); 2.76 (t, 2H).
Example 9a
4-[1-(1H-Benzimidazol-2-yl]-furazan-3-yl-N-(2-methoxycarbonylethyl)-amine
A solution of
4-(1H-benzimidazol-2-yl)-furazan-3-yl-N-(2-cyanoethyl)-amine (0.05
g, Example 6a) in methanol saturated with hydrochloric acid (5 ml)
is heated at reflux for 40 minutes. After addition of a drop of
water refluxing is continued for 2 hours. The mixture is
neutralized using sodium bicarbonate and then extracted repeatedly
using ethyl acetate. The combined organic extracts are dried and
evaporated to dryness under reduced pressure. Trituration with
hexane and filtering yields the title compound in pure form.
.sup.1H-NMR (400 MHz, d.sup.6-DMSO): 13.8 (s, 1H); 7.80 (m, 1H);
7.59 (m, 1H); 7.32 (m, 2H); 703 (m, 1H); 3.63 (m, 2H); 3.61 (s,
3H); 2.76 (t, 2H).
Example 10
4-[1-(3,4-Dimethylphenoxymethyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-c-
yanoethyl)-amine
A suspension of
4-(1H-benzimidazol-2-yl)-furazan-3-yl-N-(2-cyanoethyl)-amine (0.15
g, 0.59 mmol, Example 6a), potassium carbonate (0.325 g, 2.36
mmol), diiodomethane (0.16 g, 0.59 mmol) and 3,4-dimethylphenol
(0.072 g, 0.59 mmol) in DMF (5 ml) is stirred at room temperature
for 16 hours. The reaction mixture is diluted with ethyl acetate,
washed with water and dried over sodium sulphate. Filtration of the
sodium sulphate, concentration of the filtrate under reduced
pressure and chromatography of the residue on silicagel using
hexane-ethyl acetate as eluent gives the title compound as a
yellowish solid, m.p. 132-135.degree. C.
Example 11
4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(3-hydroxypro-
pyl)-amine
A suspension of
4-(1H-benzimidazol-2-yl)-furazan-3-yl-N-(3-hydroxypropyl)-amine
(0.70 g, 0.27 mmol), potassium carbonate (0.472 g, 3.42 mmol) and
4-chlorophenacyl bromide (0.069 g, 0.29 mmol) in DMF (5 ml) is
stirred at room temperature for 16 hours. The reaction mixture is
diluted with ethyl acetate, washed with water and dried over sodium
sulphate. Filtration of the sodium sulphate, concentration of the
filtrate under reduced pressure and chromatography of the residue
on silicagel using hexane-ethyl acetate as eluent gives the title
compound as a yellowish solid, m.p. 166-169.degree. C.
Example 11a
4-(1H-Benzimidazol-2-yl)-furazan-3-yl-N-(3-hydroxypropyl)-amine
A solution of
4-(1H-benzimidazol-2-yl)-furazan-3-yl-N-(2-cyanoethyl)-amine (0.272
g, 0.947 mmol, Example 6a) in THF (5 ml) is added dropwise at
0.degree. C. to an efficiently stirred suspension of LiAlH.sub.4
(0.054 g, 1.42 mmol) in THF (5 ml). After stirring for 16 hours at
room temperature the mixture is quenched by careful addition of
aqueous saturated solution of sodium sulphate. The suspension is
filtered and the filtrate evaporated to dryness. Crystallization by
addition of hexane yields the title compound in pure form.
.sup.1H-NMR (400 MHz, d.sup.6-DMSO): 13.6 (s, 1H); 7.66 (m, 2H);
7.31 (m, 2H); 6.92 (t, 1H); 4.61 (m, 1H); 3.51 (m, 2H); 3.39 (m,
2H); 1.81 (m, 2H).
Example 12
E-1-[2-(4-Aminofurazan-3-yl)-benzimidazol-1-yl]-4-phenyl-but-3-en-2-one
A suspension of 4-(1H-benzimidazol-2-yl)-furazan-3-ylamine (0.275
g, 1.37 mmol), potassium carbonate (0.472 g, 3.42 mmol) and
1-chloro-4-phenyl-but-3-en-2-one (0.297 g, 1.64 mmol) in DMF (5 ml)
is stirred at room temperature for 16 hours. The reaction mixture
is diluted with ethyl acetate, washed with water and dried over
sodium sulphate. Filtration of the sodium sulphate, concentration
of the filtrate under reduced pressure and chromatography of the
residue on silicagel using hexane-ethyl acetate as eluent gives the
title compound as a yellowish solid, m.p. 176-180.degree. C.
Example 12a
1-Chloro-4-phenyl-but-3-en-2-one
A mixture of 1-chloro-3-(triphenylphosphanylidene)-propane-2-one
(2.8 g, 7.9 mmol) and freshly distilled benzaldehyde (0.7 g, 6.6
mmol) in toluene (10 ml) is heated at reflux for 20 hours.
Evaporation to dryness yields a mixture containing the title
compound that is used in the subsequent step without
purification.
Example 12b
1-Chloro-3-(triphenylphosohanylidene)-propane-2-one
A mixture of triphenylphosphine (10.0 g, 38.1 mmol) and
1,3-dichloroacetone (4.84 g, 38.1 mmol) in THF (20 ml) is heated at
reflux for 4 hours. On cooling the resulting precipitate is
filtered, washed with THF and dried. To the efficiently stirred
precipitate in methanol (20 ml) a 20% aqueous solution of sodium
carbonate (2.02 g, 19 mmol) is added followed by additional water.
The resulting product is filtered and dried to yield the pure
product. .sup.1H-NMR (400 MHz, d.sup.6-DMSO): 7.61 (m, 15H); 4.07
(2s, 0.5H each); 3.98 (s, 2H).
Example 13
4-[1-(4-Aminophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-carboxyethy-
l)-amine
A solution of
4-[1-(4-acetaminophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoe-
thyl)-amine (0.061 g) in aqueous hydrochloric acid (5 ml, HCl
conc.) is heated at reflux for two hours. The mixture is diluted
with water and neutralized by addition of sodium bicarbonate.
Extraction with ethyl acetate, drying over sodium sulphate,
filtering and evaporation of the resulting filtrate to dryness
gives the title compound in pure form, m.p. 174-177.degree. C.
.sup.1H-NMR (400 MHz, d.sup.6-DMSO): 12.40 (s, 1H); 7.84 (m, 4H);
7.38 (m, 3H); 6.65 (m, 2H); 6.28 (S, 2H); 6.17 (s, 2H); 3.57 (m,
2H); 2.66 (t, 2H).
Example 14
4-[1-(3-Amino-4-chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
To a stirred solution of
4-[1-(4-chloro-3-nitrophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
(0.07 g, 0.175 mmol) in ethanol (6 ml) and water (1 ml) is added
two drops of concentrated hydrochloric acid and iron powder (0.1 g,
17.5 mmol). The reaction mixture is heated at 80.degree. C. for 8
hours. Filtration and evaporation at reduced pressure gives the
crude product. Purification by extraction with ethyl acetate and
chromatography on silicagel yields the title compound as colorless
solid, m.p. 228-230.degree. C.
Example 14a
4-[1-(4-Chloro-3-nitrophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine
A suspension of 4-(1H-benzimidazol-2-yl)-furazan-3-ylamine (0.228
g, 1.14 mmol), potassium carbonate (0.40 g, 2.85 mmol) and
4-chloro-3-nitrophenacyl bromide (0.35 g, 1.25 mmol) in DMF (5 ml)
is stirred at room temperature for 16 hours. The reaction mixture
is diluted with ethyl acetate, washed with water and dried over
sodium sulphate. Filtration of the sodium sulphate, concentration
of the filtrate under reduced pressure and chromatography of the
residue on silicagel using hexane-ethyl acetate as eluent gives the
title compound as a solid, m.p. 198-200.degree. C. (This compound
is also listed as Example 66 in Table 1.)
Example 15
4-[1-(3-Methoxy-4-methoxymethoxy-phenacyl)-1H-benzimidazol-2-yl]-furazan-3-
-ylamine
A mixture of
4-[1-(3-methoxy-4-hydroxyphenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamin-
e (0.10 g, 0.27 mmol), DIPEA and methoxymethyl chloride in dry DMF
is stirred at room temperature for 14 hours. The reaction mixture
is diluted with ethyl acetate, washed with water and dried over
sodium sulphate. Filtration of the sodium sulphate and
concentration of the filtrate under reduced pressure gives the
title compound as colorless, pure solid, m.p. 190.degree. C.
Example 15a
4-[1-(3-Methoxy-4-hydroxyphenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-amin-
e
To a solution of
4-[1-(3-methoxy-4-benzyloxyphenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylam-
ine (1.00 g) in THF (20 ml) is added palladium on carbon (10%, 0.2
g). The mixture is stirred under a hydrogen atmosphere for 1 hour.
The catalyst is filtered and the filtrate is evaporated to dryness
to give the title compound in pure form, m.p. 265.degree. C. (This
compound is also listed as Example 74 in Table 1.)
The following compounds are prepared in analogy to Examples
1-15:
TABLE-US-00001 TABLE I ##STR00004## Ex R Y R.sup.1 Salt Comment
m.p. 16 ##STR00005## O H -- 198-200.degree. C. 17 ##STR00006## NOH
H -- E/Z 210.degree. C. 18 ##STR00007## NOMe H -- E/Z
156-158.degree. C. 19 ##STR00008## O H -- 160-162.degree. C. 20
##STR00009## NOH H -- E/Z 176-178.degree. C. 21 ##STR00010## NOH H
-- E/Z 204-206.degree. C. 22 ##STR00011## NOMe H -- E/Z
206-208.degree. C. 23 ##STR00012## O H -- 181-184.degree. C. 24
##STR00013## NOH H -- E/Z 189-192.degree. C. 25 ##STR00014## NOMe H
-- E/Z 160.degree. C. 26 ##STR00015## O H -- 226-228.degree. C. 27
##STR00016## NOH H -- E/Z 196-198.degree. C. 28 ##STR00017## NOMe H
-- E/Z 164-166.degree. C. 29 ##STR00018## O H -- 212-214.degree. C.
30 ##STR00019## NOMe H -- E/Z 150-154.degree. C. 31 ##STR00020## O
H -- 200-201.degree. C. 32 ##STR00021## O H -- 207-209.degree. C.
33 ##STR00022## NOH H -- E/Z 160-163.degree. C. 34 ##STR00023##
NOMe H -- E/Z 138-140'' C. 35 ##STR00024## O H -- 192-194.degree.
C. 36 ##STR00025## NOH H -- E/Z 188-190.degree. C. 37 ##STR00026##
NOMe H -- E/Z 125-127.degree. C. 38 ##STR00027## NOMe H -- E/Z
110-113.degree. C. 39 ##STR00028## O H -- 202.degree. C. 40
##STR00029## O Ac -- 226.degree. C 41 ##STR00030## O H --
226.degree. C. 42 ##STR00031## O H -- 180-183.degree. C. 43
##STR00032## O H -- 215-218.degree. C. 44 ##STR00033## O H --
206-209.degree. C. 45 ##STR00034## O H -- 211-214.degree. C. 46
##STR00035## O CH.sub.2CH.sub.2CN -- 180-182.degree. C. 47
##STR00036## O CH.sub.2CH.sub.2CN -- 204.degree. C. 48 ##STR00037##
O CH.sub.2CH.sub.2CN -- 210.degree. C. 49 ##STR00038## O H --
224-227.degree. C. 50 ##STR00039## O H -- 92-96.degree. C. 51
##STR00040## O H HCl 227-230.degree. C. 52 ##STR00041## O
CH.sub.2CH.sub.2CH.sub.2OH -- 160-162.degree. C. 53 ##STR00042## O
H -- 191-194.degree. C. 54 ##STR00043## O CH.sub.2CH.sub.2CN --
175-178.degree. C. 55 ##STR00044## O H -- 208-210.degree. C. 56
##STR00045## O CH.sub.2CH.sub.2CN -- 180-183.degree. C. 57
##STR00046## O CH.sub.2CH.sub.2CN -- 178-181.degree. C. 58
##STR00047## O CH.sub.2CH.sub.2CN -- 196-199.degree. C. 59
##STR00048## O H -- 179.degree. C. 60 ##STR00049## O H --
250.degree. C. 61 ##STR00050## O CH.sub.2CH.sub.2CN (COOH).sub.2
137-140.degree. C. 62 ##STR00051## O H (COOH).sub.2 148-150.degree.
C. 63 ##STR00052## O H -- 242-246.degree. C. 64 ##STR00053## O H --
250-252.degree. C. 65 ##STR00054## O H -- >250.degree. C. 66
##STR00055## O H -- 198-200.degree. C. 67 ##STR00056## O H --
218-220.degree. C. 68 ##STR00057## O H -- 220-223.degree. C. 69
##STR00058## O CH.sub.2CH.sub.2CN -- 225-226.degree. C. 70
##STR00059## O H -- 190-192.degree. C. 71 ##STR00060## O H --
186-188.degree. C. 72 ##STR00061## O H -- 205.degree. C. 73
##STR00062## O H -- 208-210.degree. C. 74 ##STR00063## O H --
265.degree. C. 75 ##STR00064## O H -- 205-208.degree. C. 76
##STR00065## O H -- 244.degree. C. 77 ##STR00066## O H --
174.degree. C. 78 ##STR00067## O H -- >250.degree. C. 79
##STR00068## O CH.sub.2CH.sub.2CN -- 223.degree. C. 80 ##STR00069##
O CH.sub.2CH.sub.2CN -- 81 ##STR00070## O H -- 250.degree. C. 82
##STR00071## O CH.sub.2CH.sub.2CN -- 83 ##STR00072## O H -- oil 84
##STR00073## O CH.sub.2CH.sub.2CN -- 85 ##STR00074## O
CH.sub.2CH.sub.2CN HCl 86 ##STR00075## O CH.sub.2CH.sub.2CN -- 87
##STR00076## O CH.sub.2CH.sub.2CN HCl
TABLE-US-00002 TABLE 2 ##STR00077## Com- Ex R R.sup.1 Salt ment
m.p. 88 ##STR00078## H 162-165.degree. C. 89 ##STR00079## H
164-167.degree. C. 90 ##STR00080## H 156-159.degree. C. 91
##STR00081## H 148-150.degree. C. 92 ##STR00082## H 151-153.degree.
C. 93 ##STR00083## CH.sub.2CH.sub.2CN 162-165.degree. C. 94
##STR00084## CH.sub.2CH.sub.2CN 176-179.degree. C. 95 ##STR00085##
CH.sub.2CH.sub.2CN 175-178.degree. C. 96 ##STR00086## H
193-196.degree. C. 97 ##STR00087## H 98 ##STR00088## H
190-193.degree. C. 99 ##STR00089## H 110-113.degree. C. 100
##STR00090## H HCl 251-252.degree. C. 101 ##STR00091## H
142-145.degree. C. 102 ##STR00092## H 155-158.degree. C. 103
##STR00093## H 145-148.degree. C. 104 ##STR00094##
CH.sub.2CH.sub.2CN 105 ##STR00095## CH.sub.2CH.sub.2CH.sub.2OH 106
##STR00096## H 107 ##STR00097## H
TABLE-US-00003 TABLE 3 ##STR00098## Com- Ex R R.sup.4 R.sup.5 Salt
ment m.p. 108 ##STR00099## Me Me -- 250.degree. C. 109 ##STR00100##
Me Me -- 238.degree. C. 110 ##STR00101## Me Me -- 237.degree. C.
111 ##STR00102## Me Me -- 218-220.degree. C. 112 ##STR00103## Me Me
-- 228.degree. C. 113 ##STR00104## OMe OMe -- >250.degree. C.
114 ##STR00105## OMe OMe -- >250.degree. C. 115 ##STR00106## OMe
OMe -- >250.degree. C. 116 ##STR00107## OMe OMe --
>250.degree. C. 117 ##STR00108## OMe OMe -- 238.degree. C.
TABLE-US-00004 TABLE 4 ##STR00109## Ex R Salt Comment m.p. 118
##STR00110## -- E 194-196.degree. C. 119 ##STR00111## -- E
188-192.degree. C. 120 ##STR00112## -- E 194-195.degree. C.
The following compounds from WO 03/066629 are prepared for
comparison of their activity in analogy to the examples described
hereinbefore:
TABLE-US-00005 TABLE 5 ##STR00113## Ex R MH.sup.+ A ##STR00114##
244 B ##STR00115## 256
General methods for testing of compounds of the invention:
Example 121
Cell Cultures and Cell Lines
Cell lines are cultured in RPMI-1640 tissue culture medium
containing either 5% or 10% fetal calf serum, 0.05 mM
2-mercaptoethanol, 2 mM glutamine and penicillin/streptomycin 50
.mu.g/ml (complete medium) (Sigma, Buchs, Switzerland). General
growth conditions are 37.degree. C. and 7.5% CO.sub.2.
The following mouse cell lines (either EGFP transfected or not) are
being used: A20.2J (ATCC: TIB-208), MC57G (ATCC: CRL-2295).
The following human cell lines (either EGFP transfected or not) are
being used: HeLa (ATCC: CCL-2), KB (ATCC: CCL-17), MCF7 (ATCC:
HTB-22), SK-BR-3 (ATCC: HTB-30), SK-Mel 1 (ATCC: HTB-67), SK-Mel 28
(ATCC: HTB-72), PC-3 (ATCC: CRL-1435), SW 480 (ATCC: CCL-228),
NCl-H460 (ATCC: HTB-177), NCl-H1792 (ATCC: CRL-5895), HT1080 (ATCC:
CCL-21), Jurkat (ATCC: TIB-152), Ramos (ATCC: CRL-1596), Raji
(ATCC: CCL-86), H9 (ATCC: HTB-176), Hut78 (ATCC: TIB-161), K562
(ATCC: CCL 243), HL-60 (ATCC: CCL 240), U-87MG (ATCC: HTB-14),
HepG2 (ATCC: HB-8065), U-2 OS (ATCC: HTB-96), Saos-2 (ATCC:
HTB-85), U937 (ATCC: CRL 1593), Hs 578T (ATCC: HTB 126), HBL-100
(ATCC: HTB 124), Molt-4 (ATCC: CRL 1582).
Example 122
Primary Screening Setup
All the manipulations are performed under sterile conditions. The
assays are being performed in commercially available 96 or 384 well
flat bottom clear microtiter plates (Greiner, Germany)
respectively, which are suitable for tissue culture techniques. A
defined number of EGFP transfected adherent test cells (96 well
plates: 10.sup.4-10.sup.5, 384 well plates: 1500-2*10.sup.4) are
plated out 24 hours before treatment either in 75 .mu.l (96 well
plates) or 60 .mu.l (384 well plates) complete medium per well in
order to ensure appropriate cell spreading. For this purpose a
peristaltic pump (e.g. Multidrop by Thermo-Labsystems, Finland) or
another suitable device is used. Cells in suspension are plated out
according to the same procedure but 1 h prior to treatment. Between
seeding out and treatment or addition of compounds the cells are
incubated at 37.degree. C. under 7.5% CO.sub.2. Subsequently, the
compounds under investigation are added at defined concentrations
(40-80 .mu.M in either 25 .mu.l (96 well plates) or 20 .mu.l (384
well plates) complete medium containing max 4% DMSO) with an
appropriate device (e.g. liquid handling system, multi channel
pipette etc.) resulting in a final concentration in the test well
of 10-20 .mu.M compound in max 1% DMSO.
Immediately after the addition of the compounds to the cells the
zero fluorescence value (t=0 h) is determined by using a
fluorescence microplate reader in order to be able to normalize the
fluorescence activities. Afterwards, the test plates are further
incubated for a total of 48 h at 37.degree. C. under 7.5% CO.sub.2
and are shortly removed only for the purpose of measurement at 8 h,
24 h and 48 h, respectively.
Example 123
Measurement and Quantification of the Primary Screening
Relative fluorescence activities of EGFP in compound treated test
cells in relation to control cells and cells treated with standard
drugs are measured by using a BMG Fluostar microplate fluorescence
reader equipped with a filter pair for excitation/emission at 485
nm/520 nm. The optimum signal to noise ratio is detected by using
the time-resolved mode of measurement with a delay of 20 .mu.s and
an integration time over 1 ms. The gain is adjusted in such a way
that the control cells produce a fluorescence activity of 90% of
the maximum. Kinetics is performed by measuring the relative
fluorescence activities at t=0 h, 8 h, 24 h and 48 h. Crude
fluorescence activities are individually normalized for different
cell numbers and various optical activities of the test
compounds/plate-wells by dividing each value from t=8 h, 24 h and
48 h by the value of t=0 h resulting in E(8), E(24) and E(48)
values. Subsequently, the E(x) values are further processed by
forming the inverse (Q-value) of the products E(8)*E(24)*E(48)
which result in numbers >1 for apoptotic/necrotic activities of
the compounds and numbers <1 for proliferative activities of the
compounds. Controls (untreated) show values similar to 1. Compounds
producing Q values >2 are being considered relevant in terms of
apoptotic/necrotic activity and are subsequently tested in the
secondary screening setup.
Example 124
Secondary Screening Setup
All the manipulations are performed under sterile conditions. The
assays are being performed in case of adherent cells in
commercially available 24 well flat bottom tissue culture plates
(Greiner, Germany) and in case of suspension cells in polypropylene
tubes (P-tubes) 1.4 ml (Matrix, UK), respectively.
Adherent test cells: 2*10.sup.4-4*10.sup.4 of EGFP transfected
cells in 0.5 ml complete medium are plated out 24 h before
treatment. At t=0 the medium is removed and 450 .mu.l new complete
medium is added. Subsequently, 50 .mu.l complete medium containing
the test compound in max. 5% DMSO is added resulting in final
concentrations of 20 .mu.M, 10 .mu.M, 3 .mu.M, 1 .mu.M and 0.3
.mu.M of the test compounds, respectively. After 48 h incubation
the cells are harvested and analyzed with fluorescence activated
cell scanning device (FACS Calibur.TM., BD Biosciences) according
to standard procedures.
Suspension cells: 10.sup.5 test cells in 450 .mu.l complete medium
are pipetted into P-tubes. 50 .mu.l complete medium containing the
compounds (see adherent cells) is added immediately. After 48 h of
incubation the test cells are analyzed directly on a
FACSCalibur.TM..
Example 125
Quantification of the Secondary Screening
By monitoring the EGFP fluorescence activity in FL1 on a
FACSCalibur.TM., it is possible to distinguish between
proliferating cells, apoptotic cells and necrotic cells within the
same cell population. The proliferating cells show a high GFP
fluorescence activity, the apoptotic population shows an
intermediate fluorescence activity whereas the necrotic cells
demonstrate a residual fluorescence activity comparable to
mock-transfected cells. Within the CellQuest Software (BD
Biosciences) three regions are defined in the histogram: M1
comprising the proliferating cells, M2 comprising the apoptotic
cell population and M3 comprising the necrotic cell population. As
readout the relative abundance of the cells belonging either to M1,
M2 or M3 are expressed. Compounds inducing M2 values >50% and M3
values <30% are being considered relevant and are further tested
and characterized in the tertiary/advanced screening setup.
Example 126
Tertiary Screening Setup
A) Hoechst 33342 Nuclear Staining
This assay is performed in 96 well tissue culture plates.
Appropriate number of cells (adherent cells: 3-5*10.sup.3,
suspension cells: 8-10*10.sup.3) are being seeded out in 80 .mu.l
complete medium. Adherent cells are incubated for 24 h for proper
spreading out before addition of test compounds while suspension
cells are immediately treated with test compounds after seeding
out. The test compounds are added in 20 .mu.l complete medium
containing max 5% DMSO. The final compound concentrations in the
assays are 10 .mu.M, 3 .mu.M, 1 .mu.M and 0.3 .mu.M, respectively.
After 24 h or 48 h incubation at culture conditions, 10 .mu.l
medium containing Hoechst 33342 dye (Sigma B-2261) at 2-5 .mu.g/ml
are added to each well. The assay plates are then further incubated
for 30 minutes and subsequently analyzed with a standard inverted
fluorescence microscope.
The readout allows the determination of the fraction of apoptotic
nuclei as well as other morphological criteria specific for
apoptosis as a function of the treatment. Results are indicated in
Table 6. The following scores are used: 0 relating to no activity,
1 relating to weak activity comprising less than 70% of the cells
and score 2 relating to strong activity comprising more than 70% of
the cells.
TABLE-US-00006 TABLE 6 Hoechst 33342 nuclear staining Example conc
Jurkat Jily PBLs HeLa H460 MRC5 1 1 .mu.m 2 n.d. 0 2 2 1 0.1 .mu.m
0 n.d. 0 0 0 0 0.01 .mu.m 0 n.d. 0 0 0 0 2 1 .mu.m 0 n.d. 0 0 0 0
0.1 .mu.m 0 n.d. 0 0 0 0 0.01 .mu.m 0 n.d. 0 0 0 0 3 1 .mu.m 0 n.d.
0 0 n.d. 0 0.1 .mu.m 0 n.d. 0 0 n.d. 0 0.01 .mu.m 0 n.d. 0 0 n.d. 0
4 1 .mu.m 2 2 0 2 1 2 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0
5 1 .mu.m 2 n.d. 0 2 n.d. 0 0.1 .mu.m 0 n.d. 0 0 n.d. 0 0.01 .mu.m
0 n.d. 0 0 n.d. 0 6 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2 0.01
.mu.m 2 2 0 0 0 2 7 1 .mu.m 2 2 0 2 2 0 0.1 .mu.m 0 0 0 0 0 0 0.01
.mu.m 0 0 0 0 0 0 8 1 .mu.m 1 0 0 1 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01
.mu.m 0 0 0 0 0 0 9 1 .mu.m 2 2 2 2 0 2 0.1 .mu.m 0 0 0 0 0 0 0.01
.mu.m 0 0 0 0 0 0 10 1 .mu.m 2 2 0 2 2 1 0.1 .mu.m 2 1 0 0 1 0 0.01
.mu.m 0 0 0 0 0 0 11 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 1 0 1 0.01
.mu.m 0 0 0 0 0 0 12 1 .mu.m 2 2 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01
.mu.m 0 0 0 0 0 0 13 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 0 0 0 0 0 0 0.01
.mu.m 0 0 0 0 0 0 14 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 1 2 1 0.01
.mu.m 0 0 0 0 0 0 15 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 1 0.01
.mu.m 2 2 0 1 2 0 16 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2 0.01
.mu.m 0 0 0 0 0 0 17 1 .mu.m 0 0 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01
.mu.m 0 0 0 0 0 0 18 1 .mu.m 2 n.d. 0 2 1 1 0.1 .mu.m 0 n.d. 0 0 0
0 0.01 .mu.m 0 n.d. 0 0 0 0 19 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0
1 0 0 0.01 .mu.m 0 0 0 0 0 0 20 1 .mu.m 0 n.d. 0 0 0 0 0.1 .mu.m 0
n.d. 0 0 0 0 0.01 .mu.m 0 n.d. 0 0 0 0 21 1 .mu.m 0 n.d. 0 0 0 0
0.1 .mu.m 0 n.d. 0 0 0 0 0.01 .mu.m 0 n.d. 0 0 0 0 22 1 .mu.m 2
n.d. 0 2 0 0 0.1 .mu.m 0 n.d. 0 0 0 0 0.01 .mu.m 0 n.d. 0 0 0 0 23
1 .mu.m 2 n.d. 0 2 n.d. 0 0.1 .mu.m 0 n.d. 0 0 n.d. 0 0.01 .mu.m 0
n.d. 0 0 n.d. 0 24 1 .mu.m 0 0 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01
.mu.m 0 0 0 0 0 0 25 1 .mu.m 1 0 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01
.mu.m 0 0 0 0 0 0 26 1 .mu.m 0 0 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01
.mu.m 0 0 0 0 0 0 27 1 .mu.m 0 n.d. 0 0 0 0 0.1 .mu.m 0 n.d. 0 0 0
0 0.01 .mu.m 0 n.d. 0 0 0 0 28 1 .mu.m 0 0 0 0 0 0 0.1 .mu.m 0 0 0
0 0 0 0.01 .mu.m 0 0 0 0 0 0 29 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0
1 0 2 0.01 .mu.m 0 0 0 0 0 0 30 1 .mu.m 2 1 0 1 0 2 0.1 .mu.m 0 0 0
0 0 0 0.01 .mu.m 0 0 0 0 0 0 31 1 .mu.m 1 n.d. 0 0 0 0 0.1 .mu.m 0
n.d. 0 0 0 0 0.01 .mu.m 0 n.d. 0 0 0 0 32 1 .mu.m 2 0 0 0 0 0 0.1
.mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 33 1 .mu.m 0 0 0 0 0 0 0.1
.mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 34 1 .mu.m 0 0 0 0 0 0 0.1
.mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 35 1 .mu.m 2 2 0 2 2 2 0.1
.mu.m 2 1 0 0 0 2 0.01 .mu.m 0 0 0 0 0 0 36 1 .mu.m n.d. 0 0 0 0 0
0.1 .mu.m n.d. 0 0 0 0 0 0.01 .mu.m n.d. 0 0 0 0 0 37 1 .mu.m n.d.
2 0 0 0 1 0.1 .mu.m n.d. 0 0 0 0 0 0.01 .mu.m n.d. 0 0 0 0 0 38 1
.mu.m 0 0 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 39 1
.mu.m 0 0 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 40 1
.mu.m 0 1 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 41 1
.mu.m 2 2 1 2 n.d. 0 0.1 .mu.m 0 0 0 0 n.d. 0 0.01 .mu.m 0 0 0 0
n.d. 0 42 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 1 2 2 0.01 .mu.m 0 0
0 0 0 0 43 1 .mu.m 2 1 0 1 n.d. 1 0.1 .mu.m 0 0 0 0 n.d. 0 0.01
.mu.m 0 0 0 0 n.d. 0 44 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 1 2 0
0.01 .mu.m 0 0 0 0 0 0 45 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 1 2 0
0.01 .mu.m 0 0 0 0 0 0 46 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 47 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 2 0 0 0 0 0 48 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 0 2
0.01 .mu.m 0 0 0 0 0 0 49 1 .mu.m 2 1 0 1 0 0 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 50 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 2 2 0 0 1 0 51 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 1 0 0 0 0 0 52 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 1 2
0.01 .mu.m 0 0 0 0 0 0 53 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 2 0 0 0 0 1 54 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 1 1 0 0 0 0 55 1 .mu.m 2 2 0 2 2 0 0.1 .mu.m 1 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 56 1 .mu.m 2 2 0 2 2 1 0.1 .mu.m 2 2 0 1 0 1
0.01 .mu.m 0 0 0 0 0 0 57 1 .mu.m 2 2 0 2 0 2 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 58 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 2 2 0 2 2 1 59 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 60 1 .mu.m 2 2 0 1 0 2 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 61 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 0 0 0 0 0 0 62 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 0 0 0 0 0 0 63 1 .mu.m 0 0 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 64 1 .mu.m 2 2 0 2 2 1 0.1 .mu.m 2 2 0 2 2 1
0.01 .mu.m 2 2 0 0 0 0 65 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 2 2 0 0 2 0 66 1 .mu.m 2 2 0 2 2 1 0.1 .mu.m 1 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 67 1 .mu.m 2 2 0 2 2 1 0.1 .mu.m 2 2 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 68 1 .mu.m 2 1 0 2 2 1 0.1 .mu.m 2 1 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 69 1 .mu.m 2 2 0 2 2 1 0.1 .mu.m 1 1 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 70 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 2 2 0 0 1 1 71 1 .mu.m 2 1 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 72 1 .mu.m 2 2 0 2 2 0 0.1 .mu.m 1 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 73 1 .mu.m 2 1 0 1 2 1 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 74 1 .mu.m 2 2 0 2 2 1 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 75 1 .mu.m 2 2 0 1 2 1 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 76 1 .mu.m 2 2 0 2 2 I 0.1 .mu.m 2 2 0 0 1 0
0.01 .mu.m 0 0 0 0 0 0 77 1 .mu.m 2 2 0 2 2 1 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 78 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 2 2 0 2 2 1 79 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2
0.01 .mu.m 2 2 0 2 2 1 80 1 .mu.m 0.1 .mu.m 0.01 .mu.m 81 1 .mu.m
0.1 .mu.m 0.01 .mu.m 82 1 .mu.m 0.1 .mu.m
0.01 .mu.m 83 1 .mu.m 0.1 .mu.m 0.01 .mu.m 84 1 .mu.m 0.1 .mu.m
0.01 .mu.m 85 1 .mu.m 0.1 .mu.m 0.01 .mu.m 86 1 .mu.m 0.1 .mu.m
0.01 .mu.m 87 1 .mu.m 0.1 .mu.m 0.01 .mu.m 88 1 .mu.m 2 1 0 2 n.d.
0 0.1 .mu.m 0 0 0 0 n.d. 0 0.01 .mu.m 0 0 0 0 n.d. 0 89 1 .mu.m 2 2
0 2 2 2 0.1 .mu.m 2 2 0 2 2 2 0.01 .mu.m 0 0 0 0 0 0 90 1 .mu.m 2 1
0 1 n.d. 1 0.1 .mu.m 0 0 0 0 n.d. 0 0.01 .mu.m 0 0 0 0 n.d. 0 91 1
.mu.m 2 2 0 0 n.d. 0 0.1 .mu.m 0 0 0 0 n.d. 0 0.01 .mu.m 0 0 0 0
n.d. 0 92 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 2 2 2 0.01 .mu.m 0 0
0 0 0 0 93 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 0 1 0 0.01 .mu.m 0 0
0 0 0 0 94 1 .mu.m 2 2 0 2 2 2 0.1 .mu.m 2 2 0 1 2 0 0.01 .mu.m 0 0
0 0 0 0 95 1 .mu.m 2 1 0 1 n.d. 0 0.1 .mu.m 0 0 0 0 n.d. 0 0.01
.mu.m 0 0 0 0 n.d. 0 96 1 .mu.m 2 2 0 2 1 0 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 97 1 .mu.m 2 2 0 2 2 0 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 98 1 .mu.m 2 2 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 99 1 .mu.m 1 1 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0
0.01 .mu.m 0 0 0 0 0 0 100 1 .mu.m 1 1 0 0 0 0 0.1 .mu.m 0 0 0 0 0
0 0.01 .mu.m 0 0 0 0 0 0 101 1 .mu.m 2 2 0 2 2 1 0.1 .mu.m 2 1 0 1
1 1 0.01 .mu.m 0 0 0 0 0 0 102 1 .mu.m 2 1 0 0 1 0 0.1 .mu.m 0 0 0
0 0 0 0.01 .mu.m 0 0 0 0 0 0 103 1 .mu.m 2 2 0 2 2 1 0.1 .mu.m 1 0
0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 104 1 .mu.m 0.1 .mu.m 0.01 .mu.m 105
1 .mu.m 0.1 .mu.m 0.01 .mu.m 106 1 .mu.m 0.1 .mu.m 0.01 .mu.m 107 1
.mu.m 0.1 .mu.m 0.01 .mu.m 108 1 .mu.m 2 2 0 0 1 0 0.1 .mu.m 0 0 0
0 0 0 0.01 .mu.m 0 0 0 0 0 0 109 1 .mu.m 0 0 0 0 0 0 0.1 .mu.m 0 0
0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 110 1 .mu.m 0 0 0 0 0 0 0.1 .mu.m 0
0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 111 1 .mu.m 0 0 0 0 0 0 0.1 .mu.m
0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 112 1 .mu.m 0 0 0 0 0 0 0.1
.mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 113 1 .mu.m 0 0 0 0 0 0
0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 114 1 .mu.m 0 0 0 0 0
0 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 115 1 .mu.m 0 0 0 0
0 0 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 116 1 .mu.m 0 0 0
0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 117 1 .mu.m 0 0
0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 118 1 .mu.m 0
1 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 119 1 .mu.m
0 0 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 120 1
.mu.m 0 0 0 0 0 0 0.1 .mu.m 0 0 0 0 0 0 0.01 .mu.m 0 0 0 0 0 0 A 30
.mu.m 0 0 0 0 0 0 10 .mu.m 0 0 0 0 0 0 3 .mu.m 0 0 0 0 0 0 B 30
.mu.m 0 0 0 0 0 0 10 .mu.m 0 0 0 0 0 0 3 .mu.m 0 0 0 0 0 0 0: no
effect 1: weak effect 2: strong effect
B) MTS Proliferation Assay
The assay is performed in 96 well tissue culture plates. The cells
(range: 1.5*10.sup.3-10.sup.4) are seeded out in 80 .mu.l complete
medium 24 h prior to compound treatment. The test compounds are
added in 20 .mu.l complete medium containing max 5% DMSO. The final
compound concentrations in the assays are 10 .mu.M, 3 .mu.M, 1
.mu.M and 0.3 .mu.M, respectively. The assay plates are incubated
for 72 h at culture conditions. The MTS reagent is prepared
according to the manufacturer's protocol (Promega G1111). 20 .mu.l
MTS reagent are added to each well, the assay plates are quickly
spun and incubated for another 3 h at culture conditions.
Subsequently, the plates are shortly shaked and absorption measured
with a microplate-reader at 492 nm. IC.sub.50 values are determined
by graphical analysis and are indicated in the Table 7 in .mu.M
concentration.
TABLE-US-00007 TABLE 7 MTS proliferation assay IC 50 No Jurkat Jily
HT1080 HeLa MRC5 1 2 n.d. n.d. 1 0 2 0 n.d. n.d. 0 0 3 1 n.d. n.d.
n.d. 0 4 2 1 1 1 1 5 1 n.d. n.d. 1 1 6 3 2 2 2 1 7 1 1 1 1 1 8 1 1
1 1 1 9 1 1 1 1 1 10 n.d. 2 n.d. 1 n.d. 11 n.d. 2 n.d. 1 n.d. 12 1
1 0 0 0 13 1 1 1 1 1 14 2 2 2 2 2 15 3 3 3 2 3 16 1 2 2 1 2 17 0
n.d. n.d. 0 n.d. 18 1 n.d. n.d. 0 1 19 2 n.d. n.d. 1 1 20 1 n.d.
n.d. 0 0 21 0 n.d. n.d. 0 0 22 1 n.d. n.d. 1 1 23 1 n.d. n.d. 1 1
24 0 n.d. n.d. 0 0 25 1 n.d. n.d. 0 0 26 0 n.d. n.d. 0 0 27 0 n.d.
n.d. 0 0 28 0 n.d. n.d. 0 0 29 2 n.d. n.d. 2 2 30 1 n.d. n.d. 1 1
31 1 1 0 1 1 32 1 n.d. n.d. 1 0 33 0 n.d. n.d. 0 0 34 0 n.d. n.d. 0
0 35 2 2 1 2 1 36 2 2 1 2 1 37 1 n.d. n.d. 1 1 38 1 n.d. n.d. 1 0
39 0 n.d. n.d. 0 0 40 1 n.d. n.d. 1 0 41 1 1 1 1 1 42 2 2 2 2 1 43
1 1 1 1 1 44 3 2 2 2 2 45 2 2 2 2 1 46 3 2 2 2 1 47 3 3 2 2 1 48 3
2 2 2 2 49 1 1 0 0 0 50 2 2 2 2 2 51 2 2 2 2 2 52 n.d. 2 n.d. 2
n.d. 53 n.d. 2 n.d. 2 n.d. 54 n.d. 2 2 2 2 55 n.d. 2 1 1 1 56 n.d.
2 2 2 2 57 2 2 1 1 1 58 3 3 3 2 3 59 1 1 1 1 1 60 1 1 1 1 1 61 2 2
2 2 2 62 2 2 2 1 2 63 0 n.d. 0 0 0 64 2 2 2 2 2 65 3 3 3 2 3 66 2 1
1 1 1 67 1 2 1 1 1 68 2 2 1 1 1 69 2 1 1 1 1 70 3 3 3 3 3 71 n.d. 1
0 1 0 72 1 1 1 1 1 73 1 1 1 1 1 74 1 1 1 1 1 75 1 1 1 1 1 76 1 2 1
1 1 77 1 1 1 1 1 78 3 3 3 3 3 79 3 3 3 3 3 80 81 82 83 84 85 86 87
88 1 1 1 1 1 89 2 2 2 2 1 90 1 1 1 1 1 91 1 1 1 1 1 92 2 2 2 2 1 93
2 2 1 1 1 94 2 2 2 2 1 95 1 1 1 1 1 96 1 1 1 1 1 97 1 1 1 1 1 98 1
1 0 0 0 99 1 1 0 0 0 100 1 0 0 0 0 101 n.d. 2 2 2 2 102 n.d. 1 0 0
1 103 n.d. 2 1 1 1 104 105 106 107 108 1 1 1 1 1 109 1 n.d. n.d. 0
0 110 0 n.d. 0 0 0 111 0 n.d. 0 0 0 112 0 n.d. 0 0 0 113 0 n.d. 0 0
0 114 0 n.d. 0 0 0 115 0 n.d. 0 0 0 116 0 n.d. 0 0 0 117 0 n.d. 0 0
0 118 0 0 0 0 0 119 0 0 0 0 0 120 0 0 0 0 0 A 0 0 0 0 0 B 0 0 0 0 0
0 IC 50 > 1 .mu.M 1 0.1 .mu.M < IC 50 < 1 .mu.M 2 0.01
< IC 50 < 0.1 .mu.M 3 IC 50 < 0.01 .mu.M
C) Annexin V/7-AAD Staining
Adherent cells (1-2*10.sup.5) are 24 h prior to compound treatment
seeded into 24 well tissue culture plates. Suspension cells are
pipetted into P-tubes immediately before treatment. Test compounds
are added leading to a final concentrations of 10 .mu.M. After 24 h
Treatment cells are harvested (in case of adherent cells by
trypsinization) and transferred to FACS tubes (BD Biosciences).
After centrifugation and removal of the supernatant, 100 .mu.l
complete medium containing AnnexinV-GST (10 .mu.g) is added, mixed
and incubated at 4.degree. C. for 30 minutes. Subsequently, the
cells are washed once with medium and incubated with 100 .mu.l
anti-GST Alexa 488 (Molecular Probes A-11131) in medium diluted
1:500 for 30 minutes at 4.degree. C. Then, cells are washed once
and stained with 1 .mu.g/ml 7-aminoactino-mycin D (7-AAD)
(Molecular Probes A-1310) in 250 .mu.l medium and analyzed on the
FACSCalibur.TM.. AnnexinV is measured in FL1 whereas 7-AAD is
measured in FL3.
D) PI Staining for Cell Cycle Distribution
1-2*10.sup.5 cells are seeded into 24 well tissue culture plates
and incubated for 24 h prior to compound addition. Compounds are
added for 24 h in a final concentration of 3 .mu.M or 10 .mu.M.
Adherent cells are harvested by trypsinization. The cell
suspensions are fixed by adding 2 parts ice cold ethanol 100% while
vortexing. Then the samples are stored for >2 h at -20.degree.
C. Subsequently the cells are washed with PBS once and resuspended
in 250 .mu.l PBS containing 50 .mu.g/ml PI (Calbiochem # 537059),
then the samples are incubated at 37.degree. C. for 30 minutes and
subsequently analyzed on a FACSCalibur.TM. monitoring linear PI
fluorescence activity on FL2. The readout allows the detection of a
possible direct or indirect influence of the tested compounds on
the cell cycle. The following events can occur: a) Generation of a
subG1 peak indicative for DNA fragmentation, b) increase of the
cell population arrested in G2M phase. Both events are scored by 1
for weak and 2 for strong occurrence. 0 indicates no occurrence at
all. In Table 8 the influences of several tested compounds are
demonstrated.
TABLE-US-00008 TABLE 8 PI staining for cell cycle distribution
Jurkat 3 .mu.M HeLa 10 .mu.M No subG1 G2M subG1 G2M 16 0 0 0 2 19 0
0 0 2 29 0 0 0 2 30 0 0 0 2 58 0 0 0 2 0: no effect 1: weak effect
2: strong effect
E) BrdU Incorporation (Proliferation)
Adherent cells are seeded out at 2-4*10.sup.4 cells/well/ml in 24
well tissue culture plates 24 h prior to treatment. Suspension
cells are seeded out at 2*10.sup.5 cells/ml/well in 24 well plates.
Compounds are added leading to final concentrations of 3 .mu.M and
10 .mu.M, respectively. Subsequently, BrdU (Molecular Probes
#B-23151) at 10 .mu.M final concentration is added and the plates
are incubated for 48 h. After the incubation cells are processed
according to standard procedures. The detection of the incorporated
BrdU is done with the anti-bromodeoxyuridine Mab PRB-1, Alexa Fluor
660 conjugate (Molecular Probes #A-21306). The analysis is
performed on a FACSCalibur.TM. by monitoring the fluorescence
activity on FL3. The readout reflects DNA synthesis which is a
hallmark for proliferation.
F) Caspase Dependencies
Caspase dependencies are being evaluated by combining the compound
treatment with the pan-caspase inhibitor ZVAD or its control
peptide zFA (ICN Pharmaceuticals # FK009 and FK029, respectively).
Both peptides are being used at 20 .mu.M concentration. In case of
caspase dependencies a clear inhibition of the specific readout in
all apoptosis tests should be detected. By comparing the readout of
zVAD and zFA treated samples with the compound control it is
possible to detect caspase resp. cystein proteinase dependencies.
In case of inhibition by ZVAD but not by zFA a clear caspase
dependency is obvious. An inhibition by zVAD as well as by zFA
points towards the involvement of cystein proteinases In the
apoptotic cascade. Table 9 demonstrates the protease dependency of
the nuclear fragmentation visualized by Hoechst 33342 staining.
Example 127
Soft Capsules
5000 soft gelatin capsules, each comprising as active ingredient
0.05 g of one of the compounds of formula (I) mentioned in the
preceding Examples, are prepared as follows: 250 g pulverized
active ingredient is suspended in 2 liter Lauroglykol.RTM.
(propylene glycol laurate, Gattefosse S. A., Saint Priest, France)
and ground in a wet pulverizer to produce a particle size of about
1 to 3 .mu.m. 0.419 g portions of the mixture are then introduced
into soft gelatin capsules using a capsule-filling machine.
* * * * *