U.S. patent application number 10/358674 was filed with the patent office on 2003-10-09 for substituted nitrogen heterocyclic derivatives and pharmaceutical use thereof.
This patent application is currently assigned to USTAV EXPERIMENTALNI BOTANIKY AV CR. Invention is credited to Hajduch, Marian, Hanus, Jan, Krystof, Vladimir, Strnad, Miroslav, Vesely, Jaroslav.
Application Number | 20030191086 10/358674 |
Document ID | / |
Family ID | 5461478 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030191086 |
Kind Code |
A1 |
Hanus, Jan ; et al. |
October 9, 2003 |
Substituted nitrogen heterocyclic derivatives and pharmaceutical
use thereof
Abstract
Various substituted nitrogen heterocyclic derivatives and their
pharmaceutically acceptable salt derivatives are provided for use
as medicaments, and particularly, as antimitotic, anti-viral,
anti-cancer, anti-degenerative, immunosuppressive, and
anti-microbial drugs or, vaccines. These heterocyclic derivatives
can be used as an active agent in a pharmaceutical, as well as a
diagnostic utility. To this end, several families of heterocyclic
derivatives are provided including pyrrolopyrimidines,
pyrazolopyrimidines, purines, and imidazopyridines. In particular,
certain tri-substituted and tetra-substituted purines and
pyrazolopyrimidines and their deaza analogues are provided for
inhibiting cyclin-dependent kinase ("cdk") proteins, viruses, and
immunostimulation.
Inventors: |
Hanus, Jan; (Praha, CZ)
; Krystof, Vladimir; (Ostrava, CZ) ; Hajduch,
Marian; (Olomouc, CZ) ; Vesely, Jaroslav;
(Bohunovice, CA) ; Strnad, Miroslav; (Olomouc,
CZ) |
Correspondence
Address: |
NOTARO & MICHALOS P.C.
Suite 110
100 Dutch Hill Road
Orangeburg
NY
10962-2100
US
|
Assignee: |
USTAV EXPERIMENTALNI BOTANIKY AV
CR
|
Family ID: |
5461478 |
Appl. No.: |
10/358674 |
Filed: |
February 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10358674 |
Feb 5, 2003 |
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09889176 |
Jul 12, 2001 |
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6552192 |
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Current U.S.
Class: |
514/46 ;
514/263.2; 514/263.23; 514/263.38; 514/303; 514/81; 536/27.3;
544/269; 544/276; 544/277; 546/118 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 35/00 20180101; C07D 473/16 20130101; C07D 487/04 20130101;
A61P 35/04 20180101; A61P 37/06 20180101; C07D 473/40 20130101;
C07D 473/34 20130101; C07D 473/32 20130101; A61P 31/00
20180101 |
Class at
Publication: |
514/46 ;
514/263.23; 514/81; 514/263.2; 514/263.38; 536/27.3; 544/269;
544/276; 544/277; 514/303; 546/118 |
International
Class: |
A61K 031/7076; A61K
031/675; C07H 019/16; C07D 471/02; C07D 473/18; C07D 473/16; A61K
031/522; A61K 031/52 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 26, 1999 |
CZ |
PV 273-99 |
Claims
What is claimed is:
1. Substituted nitrogen heterocyclic derivatives of the formula I--
38wherein, A is a divalent group 39Z is N; R2 and R6 are
independent of one another, represent H, halogen, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkyl
alkyl, arylalkyl, heteroalkyl, heteroarylalkyl, heterocycloalkyl
alkyl or R6'-X wherein X is an --NH--, --N(C.sub.1-C.sub.6-alkyl)-,
--O-- or --S-- moiety; R6' is H, alkyl, substituted alkyl, acyl,
amido, sulpho, cycloalkyl, substituted cycloalkyl, aryl,
substituted aryl, heterocycle, heteroaryl, substituted heteroaryl,
arylalkyl, heterocycloalkyl, substituted heterocycloalkyl,
heteroarylalkyl, heteroalkyl, cycloalkyl alkyl and heterocycloalkyl
alkyl; R8 is halogen, hydroxyl, amino, carboxyl, cyano, nitro,
amido, sulpho, sulphamino, carbamino, alkyl, substituted alkyl,
cycloalkyl, substituted cycloalkyl, arylalkyl, heteroalkyl,
heteroarylalkyl, cycloalkyl alkyl, heterocycloalkyl alkyl or R8'-X
wherein X is --NH--, --N(alkyl)--, --O-- or --S-- moiety, and R8'
is according to any one of the substituents defined above for R2'
or R6'; R9 is alkyl, substituted alkyl, acyl, carboxyl, amido,
sulphamino, cycloalkyl, substituted cycloalkyl, cycloalkyl alkyl,
heteroalkylcycloalkyl alkyl, heterocycloalkyl, substituted
heterocycloalkyl, aryl, substituted aryl, heterocycle, heteroaryl,
substituted heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl or
--B-R9' wherein B is --CH.sub.2--, --(CH.sub.2).sub.2--,
--CH(CH.sub.3)CH.sub.2--, --CH(CH.sub.2F)CH.sub.2--- ,
--CH(CH.sub.2OH)CH.sub.2, or the groups of the following structure,
40wherein the left hand bond is linked to nitrogen of 5-membered
ring of compounds of the formula I; R4 and R5, that are independent
of one another, represent hydrogen, hydroxyl, halogen, amino,
acyloxy substituent having 1-5 carbon atoms, alkoxy substituent
having 1-5 carbon atoms, alkylmercapto substituent having 1-5
carbon atoms, alkylamino substituent having 1-5 carbon atoms and
dialkylamino in which each alkyl substituent has 1-5 carbon atoms;
R7 and R10, that are independent of one another, represent H or
alkyl substituent having 1-10 carbon atoms; or R9 is
--(CH.sub.2).sub.n-R9', wherein n=1-2 and the R9' is
-X(CH.sub.2).sub.mY wherein X is --O--, --S--, --NH-- or --N
(alkyl)- substituent having 1-6 carbon atoms; m=1-2; Y is carboxyl,
amido, sulpho, sulphamino, hydroxyl, carboxyl, mercapto,
carbylmercapto, amino, alkylamino, carbamino --PO(OH).sub.2,
--PO(O-C.sub.1-C.sub.6-alkyl).sub.2- ,
--PO(NH-C.sub.1-C.sub.6-alkyl).sub.2, --PO(O-C.sub.1-C.sub.6-alkyl)
(NH-C.sub.1-C.sub.6-alkyl) --PO(OH) (O-C.sub.1-C.sub.6-alkyl),
--PO(OH)(NH-C.sub.1-C.sub.6-alkyl) or --(CH.sub.2CHD)-R9', wherein
D is alkyl, substituted alkyl, --PO(OH).sub.2, --PO(OH)
(O-C.sub.1-C.sub.6-alkyl), --PO(OH) (NH-C.sub.1-C.sub.6-alkyl)
wherein: "halogen" is fluorine, bromine, chlorine, and iodine
atoms; "alkyl" is: a) a branched or unbranched alkyl group having
1-6 carbon atoms, b) a branched or unbranched alkenyl group having
2-6 carbon atoms, and c) a branched or unbranched alkinyl group
having 2-6 carbon atoms; "substituted alkyl" is a branched or
unbranched alkyl, alkenyl or alkinyl group having 1-6 carbon atoms
and having substituted by one or more substituents selected from
the group consisting of hydroxyl, mercapto, carbylmercapto,
halogen, carbyloxy, amino, amido, carboxyl, cycloalkyl, sulpho or
acyl; "carbyloxy" is the group --OR.sub.a, where R.sub.a is alkyl,
substituted alkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl or
substituted heterocycloalkyl whereas these generic groups have
meanings being identical with definitions of the corresponding
groups as defined in this legend; "carbylmercapto" is the group
-SR.sub.b where R.sub.b is alkyl, substituted alkyl, aryl,
substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl,
substituted cycloalkyl, heterocycloalkyl or substituted
heterocycloalkyl whereas these general groups have meanings being
identical with definitions of the corresponding groups as defined
in this legend; "sulpho" is the group --SO.sub.3R.sub.c where
R.sub.c is: a) hydrogen, b) a branched or unbranched alkyl group
having 1-6 carbon atoms, c) a branched or unbranched alkenyl group
having 2-6 carbon atoms, d) a branched or unbranched alkinyl group
having 2-6 carbon atoms, and e) a branched or unbranched alkyl,
alkenyl or alkinyl group having 1-6 carbon atoms and being
substituted by one or more substituents selected from the group
consisting of hydroxyl, mercapto, carbylmercapto, halogen,
carbyloxy, amino, amido, carboxyl, cycloalkyl, sulpho or acyl,
whereas these generic groups have meanings being identical with the
definitions of the corresponding groups as defined in this legend;
"sulphamino" is the group --NHSO.sub.3R.sub.d wherein R.sub.d is:
a) hydrogen, b) a branched or unbranched alkyl group having 1-6
carbon atoms, c) a branched or unbranched alkenyl group having 2-6
carbon atoms, d) a branched or unbranched alkinyl group having 2-6
carbon atoms, and e) a branched or unbranched alkyl, alkenyl or
alkinyl group having 1-6 carbon atoms and being substituted by one
or more substituents selected from the group consisting of
hydroxyl, mercapto, carbylmercapto, halogen, carbyloxy, amino,
amido, carboxyl, cycloalkyl, sulpho or acyl, whereas these generic
substituents have meanings being identical with definitions of the
corresponding groups as defined in this legend; "acyl" is the group
--C(O)R.sub.e where R.sub.e is hydrogen, alkyl, substituted alkyl,
aryl, substituted aryl, arylalkyl, substituted arylalkyl,
cycloalkyl, substituted cycloalkyl whereas these generic groups
have meanings being identical with definitions of the corresponding
groups as defined in this legend; "aryloxy" is the group --OAr,
where Ar is an aryl, substituted aryl, heteroaryl or substituted
heteroaryl whereas these generic groups have meanings being
identical with definitions of the corresponding groups as defined
in this legend; "alkylamino" is the group --NR.sub.fR.sub.g where
R.sub.f and R.sub.g, that are independent of one another, represent
hydrogen, alkyl, substituted alkyl, provided that R.sub.f and
R.sub.g are not both hydrogens; "amido" is the group
--C(O)NR.sub.hR.sub.i', where R.sub.h and R.sub.i' may
independently be hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl, heteroaryl, substituted heteroaryl whereas these
generic groups have meanings being identical with definitions of
the corresponding groups as defined in this legend; "carboxyl" is
the group --C(O)OR.sub.j where R.sub.j is hydrogen, alkyl,
substituted alkyl, aryl, substituted aryl, heteroaryl or
substituted heteroaryl; "carbamino" is the group --NHCOR.sub.k
where R.sub.k may be hydrogen, alkyl, substituted alkyl,
heterocycle, aryl, substituted aryl, heteroaryl and substituted
heteroaryl whereas these generic groups have meanings being
identical with definitions of the corresponding groups as defined
in this legend; "aryl" is an aromatic carbocyclic group having from
6 to 18 carbon atoms and being composed of at least one aromatic or
multiple condensed rings in which at least one of which being
aromatic; "substituted aryl" is an aromatic carbocyclic group
having from 6 to 18 carbon atoms and being composed of at least one
aromatic ring or of multiple condensed rings at least one of which
being aromatic; The ring(s) are optionally substituted with one or
more substituents selected from the group consisting of halogen,
alkyl, hydroxyl, carbylmercapto, alkylamino, carbyloxy, amino,
amido, carboxyl, nitro, mercapto or sulpho; "heterocycle" is a
heterocyclic group having from 4 to 9 carbon atoms and at least one
heteroatom selected from the group consisting of N, O or S;
"heteroaryl" is a heterocyclic group having from 4 to 9 carbon
atoms and at least one heteroatom selected from the group
consisting of N, O or S with at least one ring of this group being
aromatic; "substituted heteroaryl" is a heterocyclic group having
from 4 to 9 carbon atoms and at least one heteroatom selected from
the group consisting of N, O or S with at least one ring of this
group being aromatic and this group being substituted with one or
more substituents selected from the group consisting of halogen,
alkyl, carbyloxy, carbylmercapto, alkylamino, amido, carboxyl,
hydroxyl, nitro, mercapto or sulpho; "arylalkyl" is the group
--R.sub.1--Ar wherein R.sub.1 is: a) a branched or unbranched alkyl
group having 1-6 carbon atoms, b) a branched or unbranched alkenyl
group having 2-6 carbon atoms, c) a branched or unbranched alkinyl
group having 2-6 carbon atoms; "Ar" is an aromatic carbocyclic
group having from 6-18 carbon atoms and being composed of at least
one aromatic ring or of multiple condensed rings at least one of
which being aromatic and the group being optionally substituted
with one or more substituents selected from the group consisting of
halogen, alkyl, hydroxyl, carbylmercapto, alkylamino, carbyloxy,
amino, amido, carboxyl, nitro, mercapto or sulpho; "heteroalkyl" is
the group -R.sub.m-L wherein R.sub.m is: a) a branched or
unbranched alkyl group having 1-6 carbon atoms, b) a branched or
unbranched alkenyl group having 2-6 carbon atoms, c) a branched or
unbranched alkinyl group having 2-6 carbon atoms, d) a branched or
unbranched alkyl, alkenyl or alkinyl group having 1-6 carbon atoms
and being substituted by one or more substituents selected from the
group consisting of hydroxyl, mercapto, carbylmercapto, halogen,
carbyloxy, amino, amido, carboxyl, cycloalkyl, sulpho or acyl; and
L is a heterocyclic group having from 4 to 9 carbon atoms and at
least one heteroatom selected from the group consisting of N, O or
S and the group being unsubstituted or substituted with one or more
substituents selected from the group consisting of halogen, alkyl,
alkoxy, alkylmercapto, alkylamino, amido, carboxyl, hydroxy, nitro,
mercapto or sulpho; "heteroarylalkyl" is the group -R.sub.n-G
wherein R.sub.n is a) a branched or unbranched alkyl group having
1-6 carbon atoms, b) a branched or unbranched alkenyl group having
2-6 carbon atoms, c) a branched or unbranched alkinyl group having
2-6 carbon atoms, d) a branched or unbranched alkyl, alkenyl or
alkinyl group having 1-6 carbon atoms and being substituted by one
or more substituents selected from the group consisting of
hydroxyl, mercapto, carbylmercapto, halogen, carbyloxy, amino,
amido, carboxyl, cycloalkyl, sulpho or acyl; and G is a
heterocyclic group having from 4 to 9 carbon atoms and at least one
heteroatom selected from the group consisting of N, O or S with at
least one ring of which being aromatic and the group being
optionally substituted with one or more substituents selected from
the group consisting of halogen, alkyl, carbyloxy, carbylmercapto,
alkylamino, amido, carboxyl, hydroxyl, nitro, mercapto or sulpho;
"cycloalkyl" is a monocyclic or polycyclic alkyl group containing 3
to 15 carbon atoms; "substituted cycloalkyl" is a monocyclic or
polycyclic alkyl group containing 3 to 15 carbon atoms and being
substituted by one or more substituents selected from the group
consisting of halogen, alkyl, substituted alkyl, carbyloxy,
carbylmercapto, aryl, nitro, mercapto or sulpho; "heterocycloalkyl"
is a monocyclic or polycyclic alkyl group containing 3 to 15 carbon
atoms which at least one ring carbon atom of its cyclic structure
being replaced with a heteroatom selected from the group consisting
of N, O, S or P; "substituted heterocycloalkyl" is a monocyclic or
polycyclic alkyl group containing 3 to 15 carbon atoms which at
least one ring carbon atom of its cyclic structure being replaced
with a heteroatom selected from the group consisting of N, O, S or
P and the group is containing one or more substituents selected
from the group consisting of halogen, alkyl, substituted alkyl,
carbyloxy, carbylmercapto, aryl, nitro, mercapto or sulpho;
"cycloalkyl alkyl" is the group -R.sub.o-J where R.sub.o is: a) a
branched or unbranched alkyl group having 1-6 carbon atoms, b) a
branched or unbranched alkenyl group having 2-6 carbon atoms, c) a
branched or unbranched alkinyl group having 2-6 carbon atoms, d) a
branched or unbranched alkyl, alkenyl or alkinyl group having 1-6
carbon atoms and being substituted by one or more substituents
selected from the group consisting of hydroxyl, mercapto,
carbylmercapto, halogen, carbyloxy, amino, amido, carboxyl,
cycloalkyl, sulpho or acyl, whereas these generic substituent group
have meanings being identical with definitions of the corresponding
groups as defined in this legend; and J is: a) a monocyclic or
polycyclic alkyl group containing 3 to 15 carbon atoms; and b) a
monocyclic or polycyclic alkyl group containing 3 to 15 carbon
atoms which contains one or more substituents selected from the
group consisting of halogen, alkyl, substituted alkyl, carbyloxy,
carbylmercapto, aryl, nitro, mercapto or sulpho;
"heterocycloalkylalkyl" is the group -R.sub.pV where R.sub.p is: a)
a branched or unbranched alkyl group having 1-6 carbon atoms, b) a
branched or unbranched alkenyl group having 2-6 carbon atoms, c) a
branched or unbranched alkinyl group having 2-6 carbon atoms, and
d) a branched or unbranched alkyl, alkenyl or alkinyl group having
1-6 carbon atoms and being substituted by one or more substituents
selected from the group consisting of hydroxyl, mercapto,
carbylmercapto, halogen, carbyloxy, amino, amido, carboxyl,
cycloalkyl, sulpho or acyl; and V is: a) a monocyclic or polycyclic
alkyl group containing 3 to 15 carbon atoms with at least one being
replaced with a heteroatom selected from the group consisting of N,
O, S or P; and b) a monocyclic or polycyclic alkyl group containing
3 to 15 carbon atoms with at least one being replaced with a
heteroatom selected from the group consisting of N, O, S or P and
the group contains one or more substituents selected from the group
consisting of halogen, alkyl, substituted alkyl, carbyloxy,
carbylmercapto, aryl, nitro, mercapto or sulpho.
2. Substituted nitrogen heterocyclic derivatives of the formula I
according to claim 1, wherein R6.dbd.H.
3. Substituted nitrogen heterocyclic derivatives of the formula I
according to claim 1, wherein R2.dbd.H and R6, R8 and R9 are as
defined in claim 1.
4. Substituted nitrogen heterocyclic derivatives according to claim
1, of the formula Ib, 41wherein R2, R6, R8 and R9 are as defined in
claim 1.
5. Substituted nitrogen heterocyclic derivatives of the formula Ib
according to claim 4, wherein R6.dbd.H and R2, R8 and R9 are
defined as in claim 1.
6. Substituted nitrogen heterocyclic derivatives of the formula Ib
according to claim 4, wherein R2.dbd.H and R6, R8 and R9 are as
defined in claim 1.
7. Substituted nitrogen heterocyclic derivatives of the formula I
according to claim 1, selected from the group consisting of a)
2-(l-hydroxymethylpropylamino)-6-benzylamino-8-Q-9-isopropylpurine;
b) 2-(2-aminopropylamino)-6-benzylamino-8-Q-9-isopropylpurine; c)
2-(2-hydroxypropylamino)-6-benzylamino-8-Q-9-isopropylpurine; d)
2-diethylamino-6-(4-methoxybenzylamino)-8-Q-9-isopropylpurine; e)
2-(2-hydroxypropylamino)-6-(3-chloro-4-carboxyanilino)-8-Q-9-isopropylpur-
ine; f)
2-(R)-(2-hydroxypyrrolidin-1-yl)-6-benzylamino-8-Q-9-isopropylpuri-
ne; g)
2-(R)-(l-isopropyl-2-hydroxyethylamino)-6(3-chloro-4-carboxyanilino-
)-8-Q-9-isopropylpurine; h)
2-(R)-(1-isopropyl-2-hydroxyethylamino)-6-benz-
ylamino-8-Q-9-isopropylpurine; i)
2-(R)-(1-isopropyl-2-hydroxyethylamino)--
6-(3-chloroanilino)-8-Q-9-isopropylpurine; j)
2-alkylamino-6-dimethylamino- -8-Q
-9-(R)-(2-phosphonomethoxypropyl)purine; k)
2-alkylamino-6-diethylami-
no-8-Q-9(R)-(2-phosphonomethoxypropyl)purine; l) 2-alkylamino-6
-butylamino-8-Q-9-(R)-(2-phosphonomethoxypropyl) purine; m)
2-alkylamino-6-(2-butylamino)-8-Q-9-(R)-(2-phosphonomethoxypropyl)purine;
n)
2-alkylamino6-cyclopropylamino-8-Q-9-(R)-(2-phosphonomethoxypropyl)pur-
ine; o)
2-amino-6-cyclohexylamino-8-Q-9-(R)-(2-phosphonomethoxypropyl)puri-
ne; p)
2-alkylamino-6-(pyrrolidin-1-yl)-8-Q-9-(R)-(2-phosphonomethoxypropy-
l)purine; q)
2-alkylamino-6-(morpholin-1-yl)-8-Q-9-(R)-(2-phosphonomethoxy-
propyl)purine; wherein Q is chloro, hydroxy, bromo, fluoro, amino,
amido, carboxy, cyano, methylamino, thio, methylthio,
.omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy, or
propinyl.
8. Substituted nitrogen heterocyclic derivatives of the formula I
according to claim 1, selected from the group consisting of:
2-(2-hydroxypropylamino)-6-(3-chloroanilino)-8-S-9-isopropylpurine;
wherein S is chloro, hydroxy, bromo, amino, C.sub.1-C.sub.6 alkyl,
methyl, ethyl, propyl, isopropyl, vinyl, allyl, propargyl.
9. A method for preparing substituted nitrogen heterocyclic
derivatives of formula I according to claim 1, wherein R2, R6, R8
and R9 are as defined in claim 1, A is a group of formula 42and Z
is N, characterized in that a trisubstituted derivative of formula
XI, 43wherein R2, R6 and R9 are as defined in claim 1, is
brominated by using a brominating system selected from a group
consisting of bromine/acetic acid, bromine/chloroform,
bromine/acetate buffer, bromine/water,
N-bromosuccinimide/dimethylformamide and
bromoacetamide/dimethylformamide to obtain a derivative of formula
XIa, 44wherein R2, R6 and R9 are as defined in claim 1, and a
bromine atom in position 8 of the derivative of formula XIa is then
optionally subjected to a substitution in order to replace said
bromine atom by another substituent R8 as defined in claim 1.
10. A method for preparing substituted nitrogen heterocyclic
derivatives of the formula I according to claim 1, wherein R2, R6,
R8 and R9 are as defined in claim 1, A is a group of formula 45and
Z is N, characterized in that a trisubstituted derivative of
formula XIb, 46wherein R6 and R9 are as defined in claim 1, is
brominated by using a brominating system selected from a group
consisting of bromine/acetic acid, bromine/chloroform,
bromine/acetate buffer, bromine/water,
N-bromosuccinimide/dimethylformamide and
bromoacetamide/dimethylformamide to obtain a derivative of formula
XIc, 47wherein R6 and R9 are as defined in claim 1, and a chlorine
atom in position 2 and a bromine atom in position 8 of the
derivative of formula XIc are optionally, either progressively or
simultaneously, subjected to a nucleophilic substitution in order
to replace said chlorine and bromine atoms by other substituents R2
and R8 as defined in claim 1.
11. A method for preparing substituted nitrogen heterocyclic
derivatives of the formula I according to claim 1, wherein R2, R6
and R8 are as defined in claim 1, R9 is alkyl as defined in claim
1, A is a group of formula 48and Z is N, characterized in that, a
trisubstituted derivative of formula XIIa, 49wherein R6 is as
defined in claim 1, and said derivative of formula XIIa is
alkylated in position 9 by using an alkylating agent in a system
selected from a group consisting of K.sub.2CO.sub.3/
dimethylformamide, Cs.sub.2CO.sub.3/dimethylformamide,
t-BuOK/dimethylformamide, t-BuOK/dimethylsulphoxide and
NaH/dimethylformamide or under conditions of Mitsunobu reaction to
obtain a derivative of formula XIIb 50wherein R6 and alkyl are as
defined in claim 1, and chlorine atoms in positions 2 and 8 of the
derivative of formula XIIb are optionally, either progressively or
simultaneously, subjected to a substitution in order to replace
said chlorine atoms by other substituents R2 and R8, as defined in
claim 1.
12. A method for preparing substituted nitrogen heterocyclic
derivatives of the formula I according to claim 1, wherein R2, R6
and R8 are as defined in claim 1, R9 is alkyl as defined in claim
1, A is a group of formula 51and Z is N, characterized in that a
trisubstituted derivative of formula XIIa, 52wherein R6 is as
defined in claim 1, is protected by reacting with
2-dihydropyrane/H.sup.+ to obtain a derivative of formula XIIc,
53wherein R6 is as defined in claim 1, and the group R6 and/or a
chlorine atom in position 2 and/or a chlorine atom in position 8
are optionally converted to other substituents R2, R6 and R8, as
defined in claim 1, and in such optionally modified derivative of
formula XIIc, the 2-tetrahydropyranyl group is split off to obtain
a corresponding derivative of formula XIIc wherein R9 is hydrogen,
and so obtained derivative of formula XIIc is then alkylated in
position 9 by using an alkylating agent in a system selected from a
group consisting of K.sub.2CO.sub.3/dimethylformamide,
Cs.sub.2CO.sub.3/dimethylformamide, t-BuOK/dimethylformamide,
t-BuOK/dimethylsulphoxide and NaH/dimethylformamide or under
conditions of Mitsunobu reaction to obtain a tetrasubstituted
derivative of formula XIIc, wherein R2,R6,R8 and R9 are as defined
in claim 1.
13. A method for preparing substituted nitrogen heterocyclic
derivatives of the formula I according to claim 1, wherein R2, R6
and R8 are as defined in claim 1, R9 is alkyl as defined in claim
1, A is a group 54and Z is N, characterized in that,
2,6,8-trichloropurine is subjected to a nucleophilic substitution
in position 6 in order to replace a chlorine atom in position 6 by
another substituent R6 as defined in claim 1 to obtain a derivative
of formula XIIa, 55wherein R6 is as defined in claim 1, and said
derivative of formula XIIa is then alkylated in position 9 by using
an alkylating agent in a system selected from a group consisting of
K.sub.2CO.sub.3/dimethylformamide, Cs.sub.2CO.sub.3/dimethy-
lformamide, t-BuOK/dimethylformamide, t-BuOK/dimethylsulphoxide and
NaH/dimethylformamide or under conditions of Mitsunobu reaction to
obtain a derivative of formula XIIb, 56wherein R6 and alkyl are as
defined in claim 1, and chlorine atoms in positions 2 and 8 of the
derivative of formula XIIb are optionally, either progressively or
simultaneously, subjected to a nucleophilic substitution in order
to replace said chlorine atoms by other substituents R2 and R8 as
defined in claim 1.
14. A method for preparing substituted nitrogen heterocyclic
derivatives of the formula I according to claim 1, wherein R2 is
hydrogen, R6 and R8 are as defined in claim 1, R9 is alkyl as
defined in claim 1, A is a group of formula 57and Z is N,
characterized in that 6,8-dichloropurine is subjected to a
nucleophilic substitution in position 6 in order to replace a
chlorine atom in position 6 by another substituent R6 to obtain a
derivative of formula XVI, 58wherein R6 is as defined in claim 1,
and said derivative of formula XVI is alkylated in position 9 by
using an alkylating agent in a system selected from a group
consisting of K.sub.2CO.sub.3/dimethylformamide,
Cs.sub.2CO.sub.3/dimethylformamide, t-BuOK/dimethylformamide,
t-BuOK/dimethylsulphoxide and NaH/dimethylformamide or under
conditions of Mitsunobu reaction to obtain a derivative of formula
XVIa, 59wherein R6 and alkyl are as defined in claim 1, and a
chlorine atom in position 8 of the derivative of formula XVIa is
optionally subjected to a substitution in order to replace said
chlorine atom by another substituent R8 as defined in claim 1.
15. A method for preparing substituted nitrogen heterocyclic
derivatives of the formula I according to claim 1, wherein R2, R6
and R8 are as defined in claim 1, R9 is alkyl as defined in claim
1, A is a group of formula 60and Z is N, characterized in that
2,6-dichloropurine is subjected to a nucleophilic substitution in
position 6 in order to replace a chlorine atom in position 6 by
another substituent R6, to obtain a derivative of formula XVII,
61wherein R6 is as defined in claim 1, and said derivative of
formula XVII is alkylated in position 9 by using either methyl
acrylate or acrylonitrile or oxirane to obtain a derivative of
formula XVIIa 62wherein R6 and alkyl are as defined in claim 1, and
said derivative of formula XVIIa is then brominated in position 8
by using a brominating system selected from a group consisting of
bromine/acetic acid, bromine/chloroform, bromine/acetate buffer,
bromine/water, N-bromosuccinimide/dimethylformamide and
bromoacetamide/dimethylformamide to obtain a derivative of formula
XVIIb, 63wherein R6 and alkyl are as defined in claim 1; and said
derivative of formula XVIIb is then optionally, either
progressively or simultaneously, subjected to a nucleophilic
substitution in positions 2 and 8 in order to replace a chlorine
atom in position 2 and a bromine atom in position 8 by other
substituents R2 and R8 as defined in claim 1.
16. A method for preparing substituted nitrogen heterocyclic
derivatives of the formula I according to claim 1, wherein R6 is a
halogen or hydrogen, R2, R8 and R9 are as defined in claim 1, A is
a group of formula 64and Z is N, characterized in that a derivative
of formula XVIII, 65wherein R2, R8 and R9 are defined in claim 1,
is halogenated via diazotation by using
amylnitrite/CH.sub.2Br.sub.2 or amylnitratite/CHI.sub.3, and so an
obtained derivative where R6 is halogen is optionally
hydrogenolyzed by using H.sub.2/Pd catalyst to obtain a
corresponding derivative where R6 is hydrogen.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a divisional application of U.S.
application No. 09/889,176 filed on Jul. 12, 2001, from which
priority is claimed, the entirety of which is hereby incorporated
by reference.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to new purine and
pyrazolopyrimidine derivatives and their deaza analogues and to
their use in suitable utilities, especially diagnostic and
therapeutic methods.
[0003] The present invention relates, in particular, to purine
derivatives and their inhibitory effect with respect to
cyclin-dependent kinase proteins, abbreviated cdks and also with an
inhibitory effect with respect to viruses and immunostimulation.
Purine analogues as cdk inhibitors are disclosed for example in WO
97/16452, WO 98/05335 and WO97/20842. The teaching of these patents
includes 2,6,9-trisubstituted and less substituted purine
derivatives only.
[0004] Tetrasubstituted purines are disclosed in WO 98/01448 in
which substituents are short hydrocarbonyl chains, usually
represented by hydrogen. Substituents at C6 represents hydrogen or
amine optionally substituted by one or two hydrocarbon groups;
substituents at C8 are hydroxy, mercapto, acyloxy or oxycarbonyl
substituted by aliphatic alkyl only. Nucleotide analogues
containing phosphonate groups are disclosed for example in U.S.
Pat. Nos. 4,659,825; 4,724,233; 5,124,051; 5,302,585; 5,208,221;
5,352,786; 5,356,886; 5,142,051; in EP publication numbers 269,947;
481,214; 630,381; 369,409; 454,427; 618,214; 398,231; 454,427;
468,119; 481,119; 481,214; 434,450 and in WO 95/07920; WO 94/03467;
and WO96/33200. Typical purine base is adenine, 2,6-diaminopurine
and guanine. The purine bases may include the deaza analogues
thereof, 6,9-substituted and 2,6,9-trisubstituted purines and
related analogues are disclosed in WO 96/33200. 2,8,9-,
6,8,9-trisubstituted and 2,6,8,9-tetrasubstituted purines and tri-
and tetrasubstituted pyrazolopyrimidines and their deaza analogues
have not yet been described.
SUMMARY OF THE INVENTION
[0005] It is an object of this invention to provide anticancer,
antiviral, neurodepressive and immunosuppressive compounds with
improved selectivity and efficiency index, i.e. that are less toxic
yet more efficacious than analogues known heretofore.
[0006] The various features of novelty which characterize the
invention are pointed out with particularity in the claims annexed
to and forming a part of this disclosure. For a better
understanding of the invention, its operating advantages and
specific objects attained by its uses, reference is made to the
accompanying descriptive matter in which a preferred embodiment of
the invention is illustrated.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0007] The invention concerns substituted nitrogen heterocyclic
derivatives of the formula I-- 1
[0008] wherein,
[0009] A is a divalent group selected from the ensemble consisting
of 2
[0010] Z is N or CH, provided that at least one Z being N;
[0011] R2 and R6 are independent of one another, represent H,
halogen, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, cycloalkyl alkyl, arylalkyl, heteroalkyl,
heteroarylalkyl, heterocycloalkyl alkyl or R6'-X wherein
[0012] X is an --NH--, --N(C.sub.1-C.sub.6-alkyl)-, --O-- or --S--
moiety;
[0013] R6' is H, alkyl, substituted alkyl, acyl, amido, sulpho,
cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,
heterocycle, heteroaryl, substituted heteroaryl, arylalkyl,
heterocycloalkyl, substituted heterocycloalkyl, heteroarylalkyl,
heteroalkyl, cycloalkyl alkyl and heterocycloalkyl alkyl;
[0014] R8 is halogen, hydroxyl, amino, carboxyl, cyano, nitro,
amido, sulpho, sulphamino, carbamino, alkyl, substituted alkyl,
cycloalkyl, substituted cycloalkyl, arylalkyl, heteroalkyl,
heteroarylalkyl, cycloalkyl alkyl, heterocycloalkyl alkyl or R8'-X
wherein
[0015] X is --NH--, --N(alkyl)-, --O-- or --S-- moiety, and
[0016] R8' is according to any one of the substituents defined
above for R2' or R6'.
[0017] R9 is alkyl, substituted alkyl, acyl, carboxyl, amido,
sulphamino, cycloalkyl, substituted cycloalkyl, cycloalkyl alkyl,
heteroalkylcycloalkyl alkyl, heterocycloalkyl, substituted
heterocycloalkyl, aryl, substituted aryl, heterocycle, heteroaryl,
substituted heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl or
--B-R9' wherein
[0018] B is --CH.sub.2--, --(CH.sub.2).sub.2--,
--CH(CH.sub.3)CH.sub.2--, --CH(CH.sub.2F)CH.sub.2--,
--CH(CH.sub.2OH)CH.sub.2, or the groups of the following structure,
3
[0019] wherein the left hand bond is linked to nitrogen of
5-membered ring of compounds of the formula I;
[0020] R4 and R5, that are independent of one another, represent
hydrogen, hydroxyl, halogen, amino, acyloxy substituent having 1-5
carbon atoms, alkoxy substituent having 1-5 carbon atoms,
alkylmercapto substituent having 1-5 carbon atoms, alkylamino
substituent having 1-5 carbon atoms and dialkylamino in which each
alkyl substituent has 1-5 carbon atoms;
[0021] R7 and R10, that are independent of one another, represent H
or alkyl substituent having 1-10 carbon atoms;
[0022] or R9 is --(CH.sub.2).sub.n-R9', wherein n=1-2 and the
[0023] R9' is -X(CH.sub.2).sub.mY wherein
[0024] X is --O--, --S--, --NH-- or --N (alkyl)- substituent having
1-6 carbon atoms;
[0025] m=1-2;
[0026] Y is carboxyl, amido, sulpho, sulphamino, hydroxyl,
carboxyl, mercapto, carbylmercapto, amino, alkylamino, carbamino
--PO(OH).sub.2, --PO(O-C.sub.1-C.sub.6-alkyl).sub.2,
--PO(NH-C.sub.1-C.sub.6-alkyl) --PO(O-C.sub.1-C.sub.6-alkyl)
(NH-C.sub.1-C.sub.6-alkyl), --PO(OH) (O-C.sub.1-C.sub.6-alkyl);
[0027] --PO(OH) (NH-C.sub.1-C.sub.6-alkyl) or --(CH.sub.2CHD)-R9',
wherein
[0028] R9', X, m and Y are as defined above and
[0029] D is alkyl, substituted alkyl, --PO(OH).sub.2, --PO (OH)
(O-C.sub.1-C.sub.6-alkyl), --PO(OH)(NH-C.sub.1-C.sub.6-alkyl).
[0030] The above not yet defined generic groups having meanings as
introduced in the following legend.
[0031] "Halogen" refers to fluorine, bromine, chlorine, and iodine
atoms.
[0032] "Alkyl" refers to
[0033] a branched or unbranched alkyl group having 1-6 carbon
atoms,
[0034] a branched or unbranched alkenyl group having 2-6 carbon
atoms,
[0035] a branched or unbranched alkinyl group having 2-6 carbon
atoms.
[0036] "Substituted alkyl" refers to a branched or unbranched
alkyl, alkenyl or alkinyl group having 1-6 carbon atoms and having
substituted by one or more substituents selected from the group
consisting of hydroxyl, mercapto, carbylmercapto, halogen,
carbyloxy, amino, amido, carboxyl, cycloalkyl, sulpho or acyl.
These substituent generic groups having the meanings being
identical with the definitions of the corresponding groups as
defined in this legend.
[0037] "Carbyloxy" denotes the group --OR.sub.a, where R.sub.a is
alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,
substituted arylalkyl, cycloalkyl, substituted cycloalkyl,
heterocycloalkyl or substituted heterocycloalkyl whereas these
generic groups have meanings which are identical with definitions
of the corresponding groups as defined in this legend.
[0038] "Carbylmercapto" denotes the group -SR.sub.b, where R.sub.b
is alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,
substituted arylalkyl, cycloalkyl, substituted cycloalkyl,
heterocycloalkyl or substituted heterocycloalkyl whereas these
generic groups have meanings which are identical with definitions
of the corresponding groups as defined in this legend.
[0039] "Sulpho" denotes the group --SO.sub.3R.sub.c, where R.sub.c
is
[0040] hydrogen,
[0041] a branched or unbranched alkyl group having 1-6 carbon
atoms,
[0042] a branched or unbranched alkenyl group having 2-6 carbon
atoms,
[0043] a branched or unbranched alkinyl group having 2-6 carbon
atoms,
[0044] a branched or unbranched alkyl, alkenyl or alkinyl group
having 1-6 carbon atoms and being substituted by one or more
substituents selected from the group consisting of hydroxyl,
mercapto, carbylmercapto, halogen, carbyloxy, amino, amido,
carboxyl, cycloalkyl, sulpho or acyl, whereas these generic groups
have meanings which are identical with the definitions of the
corresponding groups as defined in this legend.
[0045] "Sulphamino" denotes the group --NHSO.sub.3R.sub.d, wherein
R.sub.d is
[0046] hydrogen,
[0047] a branched or unbranched alkyl group having 1-6 carbon
atoms,
[0048] a branched or unbranched alkenyl group having 2-6 carbon
atoms,
[0049] a branched or unbranched alkinyl group having 2-6 carbon
atoms,
[0050] a branched or unbranched alkyl, alkenyl or alkinyl group
having 1-6 carbon atoms and being substituted by one or more
substituents selected from the group consisting of hydroxyl,
mercapto, carbylmercapto, halogen, carbyloxy, amino, amido,
carboxyl, cycloalkyl, sulpho or acyl, whereas these generic
substituents have meanings which are identical with definitions of
the corresponding groups as defined in this legend.
[0051] "Acyl" denotes the group --C(O)R.sub.e, where R.sub.e is
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
arylalkyl, substituted arylalkyl, cycloalkyl, substituted
cycloalkyl whereas these generic groups have meanings which are
identical with definitions of the corresponding groups as defined
in this legend.
[0052] "Aryloxy" denotes the group --OAr, where Ar is an aryl,
substituted aryl, heteroaryl or substituted heteroaryl whereas
these generic groups have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0053] "Alkylamino" denotes the group --NR.sub.fR.sub.g, where
R.sub.f and R.sub.g, that are independent of one another, represent
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl or substituted heteroaryl, provided that R.sub.f and
R.sub.g are not both hydrogens, whereas these generic substituents
have meanings which are identical with definitions of the
corresponding groups defined herein.
[0054] "Amido" denotes the group -.sub.c(O)NR.sub.hR.sub.i', where
R.sub.h and R.sub.i' may independently be hydrogen, alkyl,
substituted alkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl whereas these generic groups have meanings which are
identical with definitions of the corresponding groups as defined
in this legend.
[0055] "Carboxyl" denotes the group --C(O)OR.sub.j, where R.sub.j
is hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl or substituted heteroaryl, whereas these generic
substituents have meanings which are identical with definitions of
the corresponding groups defined herein.
[0056] "Carbamino" denotes the group --NHCOR.sub.k, where R.sub.k
may be hydrogen, alkyl, substituted alkyl, heterocycle, aryl,
substituted aryl, heteroaryl and substituted heteroaryl whereas
these generic groups have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0057] "Aryl" refers to an aromatic carbocyclic group having from 6
to 18 carbon atoms and being composed of at least one aromatic or
multiple condensed rings in which at least one of which being
aromatic.
[0058] "Substituted aryl" refers to an aromatic carbocyclic group
having from 6 to 18 carbon atoms and being composed of at least one
aromatic ring or of multiple condensed rings at least one of which
being aromatic. The ring(s) are optionally substituted with one or
more substituents selected from the group consisting of halogen,
alkyl, hydroxyl, carbylmercapto, alkylamino, carbyloxy, amino,
amido, carboxyl, nitro, mercapto or sulpho, whereas these generic
substituent group have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0059] "Heterocycle" refers to a heterocyclic group having from 4
to 9 carbon atoms and at least one heteroatom selected from the
group consisting of N, O or S.
[0060] "Heteroaryl" refers to a heterocyclic group having from 4 to
9 carbon atoms and at least one heteroatom selected from the group
consisting of N, O or S with at least one ring of this group being
aromatic.
[0061] "Substituted heteroaryl" refers to a heterocyclic group
having from 4 to 9 carbon atoms and at least one heteroatom
selected from the group consisting of N, O or S with at least one
ring of this group being aromatic and this group being substituted
with one or more substituents selected from the group consisting of
halogen, alkyl, carbyloxy, carbylmercapto, alkylamino, amido,
carboxyl, hydroxyl, nitro, mercapto or sulpho, whereas these
generic substituent group have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0062] "Arylalkyl" denotes the group -R.sub.1--Ar where R.sub.i
is
[0063] a branched or unbranched alkyl group having 1-6 carbon
atoms,
[0064] a branched or unbranched alkenyl group having 2-6 carbon
atoms,
[0065] a branched or unbranched alkinyl group having 2-6 carbon
atoms,
[0066] and Ar is an aromatic carbocyclic group having from 6 to 18
carbon atoms and being composed of at least one aromatic ring or of
multiple condensed rings at least one of which being aromatic and
the group being optionally substituted with one or more
substituents selected from the group consisting of halogen, alkyl,
hydroxyl, carbylmercapto, alkylamino, carbyloxy, amino, amido,
carboxyl, nitro, mercapto or sulpho, whereas these generic
substituent group have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0067] "Heteroalkyl" denotes the group -R.sub.m-L where R.sub.m
is
[0068] a branched or unbranched alkyl group having 1-6 carbon
atoms,
[0069] a branched or unbranched alkenyl group having 2-6 carbon
atoms,
[0070] a branched or unbranched alkinyl group having 2-6 carbon
atoms,
[0071] a branched or unbranched alkyl, alkenyl or alkinyl group
having 1-6 carbon atoms and being substituted by one or more
substituents selected from the group consisting of hydroxyl,
mercapto, carbylmercapto, halogen, carbyloxy, amino, amido,
carboxyl, cycloalkyl, sulpho or acyl, whereas these generic
substituent group have meanings which are identical with
definitions of the corresponding groups as defined in this
legend;
[0072] and L is a heterocyclic group having from 4 to 9 carbon
atoms and at least one heteroatom selected from the group
consisting of N, O or S and the group being unsubstituted or
substituted with one or more substituents selected from the group
consisting of halogen, alkyl, alkoxy, alkylmercapto, alkylamino,
amido, carboxyl, hydroxy, nitro, mercapto or sulpho, whereas these
generic substituents have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0073] "Heteroarylalkyl" denotes the group -R.sub.n-G where R.sub.n
is
[0074] a branched or unbranched alkyl group having 1-6 carbon
atoms,
[0075] a branched or unbranched alkenyl group having 2-6 carbon
atoms,
[0076] a branched or unbranched alkinyl group having 2-6 carbon
atoms,
[0077] a branched or unbranched alkyl, alkenyl or alkinyl group
having 1-6 carbon atoms and being substituted by one or more
substituents selected from the group consisting of hydroxyl,
mercapto, carbylmercapto, halogen, carbyloxy, amino, amido,
carboxyl, cycloalkyl, sulpho or acyl, whereas these generic
substituent group have meanings which are identical with
definitions of the corresponding groups as defined in this
legend;
[0078] and G is
[0079] a heterocyclic group having from 4 to 9 carbon atoms and at
least one heteroatom selected from the group consisting of N, O or
S with at least one ring of which being aromatic and the group
being optionally substituted with one or more substituents selected
from the group consisting of halogen, alkyl, carbyloxy,
carbylmercapto, alkylamino, amido, carboxyl, hydroxyl, nitro,
mercapto or sulpho, whereas these generic substituent group have
meanings which are identical with definitions of the corresponding
groups as defined in this legend.
[0080] "Cycloalkyl" refers to a monocyclic or polycyclic alkyl
group containing 3 to 15 carbon atoms.
[0081] "Substituted cycloalkyl" refers to a monocyclic or
polycyclic alkyl group containing 3 to 15 carbon atoms and being
substituted by one or more substituents selected from the group
consisting of halogen, alkyl, substituted alkyl, carbyloxy,
carbylmercapto, aryl, nitro, mercapto or sulpho, whereas these
generic substituent groups have meanings which are identical with
definitions of the corresponding groups as defined in this
legend.
[0082] "Heterocycloalkyl" refers to a monocyclic or polycyclic
alkyl group containing 3 to 15 carbon atoms which at least one ring
carbon atom of its cyclic structure being replaced with a
heteroatom selected from the group consisting of N, O, S or P.
[0083] "Substituted heterocycloalkyl" refers to a monocyclic or
polycyclic alkyl group containing 3 to 15 carbon atoms which at
least one ring carbon atom of its cyclic structure being replaced
with a heteroatom selected from the group consisting of N, O, S or
P and the group is containing one or more substituents selected
from the group consisting of halogen, alkyl, substituted alkyl,
carbyloxy, carbylmercapto, aryl, nitro, mercapto or sulpho, whereas
these generic substituent group have meanings which are identical
with definitions of the corresponding groups as defined in this
legend.
[0084] "Cycloalkyl alkyl" denotes the group -R.sub.o-J where
R.sub.o is
[0085] a branched or unbranched alkyl group having 1-6 carbon
atoms,
[0086] a branched or unbranched alkenyl group having 2-6 carbon
atoms,
[0087] a branched or unbranched alkinyl group having 2-6 carbon
atoms,
[0088] a branched or unbranched alkyl, alkenyl or alkinyl group
having 1-6 carbon atoms and being substituted by one or more
substituents selected from the group consisting of hydroxyl,
mercapto, carbylmercapto, halogen, carbyloxy, amino, amido,
carboxyl, cycloalkyl, sulpho or acyl, whereas these generic
substituent group have meanings which are identical with
definitions of the corresponding groups as defined in this
legend;
[0089] and J is
[0090] a monocyclic or polycyclic alkyl group containing 3 to 15
carbon atoms;
[0091] a monocyclic or polycyclic alkyl group containing 3 to 15
carbon atoms which contains one or more substituents selected from
the group consisting of halogen, alkyl, substituted alkyl,
carbyloxy, carbylmercapto, aryl, nitro, mercapto or sulpho, whereas
these generic substituent groups have meanings which are identical
with definitions of the corresponding groups as defined in this
legend.
[0092] "Heterocycloalkylalkyl" denotes the group -R.sub.pV where
R.sub.p is
[0093] a branched or unbranched alkyl group having 1-6 carbon
atoms,
[0094] a branched or unbranched alkenyl group having 2-6 carbon
atoms,
[0095] a branched or unbranched alkinyl group having 2-6 carbon
atoms,
[0096] a branched or unbranched alkyl, alkenyl or alkinyl group
having 1-6 carbon atoms and being substituted by one or more
substituents selected from the group consisting of hydroxyl,
mercapto, carbylmercapto, halogen, carbyloxy, amino, amido,
carboxyl, cycloalkyl, sulpho or acyl, whereas these generic
substituent groups have meanings which are identical with
definitions of the corresponding groups as defined in this
legend;
[0097] and V is
[0098] a monocyclic or polycyclic alkyl group containing 3 to 15
carbon atoms with at least one being replaced with a heteroatom
selected from the group consisting of N, O, S or P;
[0099] a monocyclic or polycyclic alkyl group containing 3 to 15
carbon atoms with at least one being replaced with a heteroatom
selected from the group consisting of N, O, S or P and the group
contains one or more substituents selected from the group
consisting of halogen, alkyl, substituted alkyl, carbyloxy,
carbylmercapto, aryl, nitro, mercapto or sulpho, whereas these
generic substituent groups have meanings which are identical with
definitions of the corresponding groups as defined in this legend,
and the pharmaceutically acceptable acid salts, racemates and
optical isomers thereof.
[0100] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula I, wherein R6.dbd.H and R2,
R8 and R9 have above mentioned meanings.
[0101] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula I, wherein R2.dbd.H and R6,
R8 and R9 have above mentioned meanings.
[0102] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ia, 4
[0103] wherein R2, R6, R8 and R9 have above mentioned meanings.
[0104] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ia, wherein R6.dbd.H and
R2, R8 and R9 have above mentioned meanings.
[0105] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ia, wherein R2.dbd.H and
R6, R8 and R9 have above mentioned meanings.
[0106] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ib, 5
[0107] wherein R2, R6, R8 and R9 have above mentioned meanings.
[0108] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ib,
[0109] wherein R6.dbd.H and R2, R8 and R9 have above mentioned
meanings.
[0110] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ib, wherein R2.dbd.H and
R6, R8 and R9 have above mentioned meanings.
[0111] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ic, 6
[0112] wherein R2, R6, R8 and R9 have above mentioned meanings.
[0113] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ic, wherein R6.dbd.H and
R2, R8 and R9 have above mentioned meanings.
[0114] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ic, wherein R2.dbd.H and
R6, R8 and R9 have above mentioned meanings.
[0115] The inventions concerns further substituted nitrogen
heterocyclic derivatives of the formula Id 7
[0116] wherein R2, R6, R8 and R9 have above mentioned meanings.
[0117] The inventions concerns further substituted nitrogen
heterocyclic derivatives of the formula Id, wherein R6.dbd.H and
R2, R8 and R9 have above mentioned meanings.
[0118] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Id, wherein R2.dbd.H and
R6, R8 and R9 have above mentioned meanings.
[0119] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ie, 8
[0120] wherein R2, R6, R8 and R9 have above mentioned meanings.
[0121] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ie, wherein R6.dbd.H and
R2, R8 and R9 have above mentioned meanings.
[0122] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ie, wherein R2.dbd.H and
R6, R8 and R9 have above mentioned meanings.
[0123] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula If, 9
[0124] wherein R2, R6, R8 and R9 have above mentioned meanings.
[0125] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula If, wherein R6.dbd.H and
R2, R8 and R9 have above mentioned meanings.
[0126] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula If, wherein R2.dbd.H and
R6, R8 and R9 have above mentioned meanings.
[0127] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ig, 10
[0128] wherein R2, R6, R8 and R9 have above mentioned meanings.
[0129] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ig, wherein R6.dbd.H and
R2, R8 and R9 have above mentioned meanings.
[0130] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula Ig, wherein R2.dbd.H and
R6, R8 and R9 have above mentioned meanings.
[0131] The invention concerns further substituted nitrogen
heterocyclic derivatives of the formula I selected from the group
consisting of 2-(1-hydroxymethylpropylamino)-6-benzylamino-8-chloro
(or hydroxy, bromo, fluoro, amino, amido, carboxy, cyano,
methylamino, thio, methylthio, .omega.-hydroxyalkylamino,
.omega.-hydroxyalkyloxy, .omega.-carboxyalkylamino,
.omega.-aminoalkylamino, .omega.-fosfonoalkylamino,
.omega.-fosfonoalkyloxy, propinyl)-9-isopropylpurine,
2-(2-aminopropylamino)-6-benzylamino-8-chlor- o (or hydroxy, bromo,
fluoro, amino, amido, carboxy, cyano, methylamino, thio,
methylthio, .omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9-isopropylpurine,
2-(2-hydroxypropylamino)-6-benzylamino-8-chl- oro (or hydroxy,
bromo, fluoro, amino, amido, carboxy, cyano, methylamino, thio,
methylthio, .omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9-isopropylpurine,
2-diethylamino-6-(4-methoxybenzylamino)-8-ch- loro (or hydroxy,
bromo, fluoro, amino, amido, carboxy, cyano, methylamino, thio,
methylthio, .omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9-isopropylpurine,
2-(2-hydroxypropylamino)-6-(3-chloroanilino)-8-chloro (or hydroxy,
bromo, amino, C.sub.1-C.sub.6 alkyl, methyl, ethyl, propyl,
isopropyl, vinyl, allyl, propargyl)-9-isopropylpurine,
2-(2-hydroxypropylamino)-6-(3-chloro- -4-carboxyanilino)-8-chloro
(or hydroxy, bromo, fluoro, amino, amido, carboxy, cyano,
methylamino, thio, methylthio, .omega.-hydroxyalkylamino,
.omega.-hydroxyalkyloxy, .omega.-carboxyalkylamino,
.omega.-aminoalkylamino, .omega.-fosfonoalkylamino,
.omega.-fosfonoalkyloxy, propinyl)-9-isopropylpurine,
2-(R)-(2-hydroxypyrrolidin-1-yl) -6-benzylamino-8-chloro (or
hydroxy, bromo, fluoro, amino, amido, carboxy, cyano, methylamino,
thio, methylthio, .omega.-hydroxyalkylamino,
.omega.-hydroxyalkyloxy, .omega.-carboxyalkylamino,
.omega.-aminoalkylamino, .omega.-fosfonoalkylamino,
.omega.-fosfonoalkyloxy, propinyl)-9-isopropylpurine,
2-(R)-(1-isopropyl-2-hydroxyethylamino)-6(3--
chloro-4-carboxyanilino)-8-chloro (or hydroxy, bromo, fluoro,
amino, amido, carboxy, cyano, methylamino, thio, methylthio,
.omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9-isopropylpurine,
2-(R)-(1-isopropyl-2-hydroxyethylamino)-6-be- nzylamino-8-chloro
(or hydroxy, bromo, fluoro, amino, amido, carboxy, cyano,
methylamino, thio, methylthio, .omega.-hydroxyalkylamino,
.omega.-hydroxyalkyloxy, .omega.-carboxyalkylamino,
.omega.-aminoalkylamino, .omega.-fosfonoalkylamino,
.omega.-fosfonoalkyloxy, propinyl)-9-isopropylpurine,
2-(R)-(1-isopropyl-2-hydroxyethylamino)-6-(3-chloroanilino)-8-chloro
(or hydroxy, bromo, fluoro, amino, amido, carboxy, cyano,
methylamino, thio, methylthio, .omega.-hydroxyalkylamino,
.omega.-hydroxyalkyloxy, .omega.-carboxyalkylamino,
.omega.-aminoalkylamino, .omega.-fosfonoalkylamino,
.omega.-fosfonoalkyloxy, propinyl)-9-isopropylpurine,
2-alkylamino-6-dimethylamino-8-chloro (or hydroxy, bromo, fluoro,
amino, amido, carboxy, cyano, methylamino, thio, methylthio,
.omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9-(R)-(2-phosphonomethoxypropyl) purine,
2-alkylamino-6-diethylamino-8-chloro (or hydroxy, bromo, fluoro,
amino, amido, carboxy, cyano, methylamino, thio, methylthio,
.omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9(R)-(2-phosphonomethoxypropyl)purine,
2-alkylamino-6-butylamin- o-8-chloro (or hydroxy, bromo, fluoro,
amino, amido, carboxy, cyano, methylamino, thio, methylthio,
.omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9-(R)-(2-phosphonomethoxypropyl)purine- , 2-alkylamino-6-
(2-butylamino)-8-chloro (or hydroxy, bromo, fluoro, amino, amido,
carboxy, cyano, methylamino, thio, methylthio,
.omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9-(R)-(2-phosphonomethoxypropyl)purine,
2-alkylamino-6-cyclopro- pylamino-8-chloro (or hydroxy, bromo,
fluoro, amino, amido, carboxy, cyano, methylamino, thio,
methylthio, .omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9-(R)-(2-phosphonomethoxypropyl)purine- ,
2-amino-6-cyclohexylamino-8-chloro (or hydroxy, bromo, fluoro,
amino, amido, carboxy, cyano, methylamino, thio, methylthio,
.omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy
propinyl)-9-(R)-(2-phosphonomethoxypropyl)purine,
2-alkylamino-6-(pyrroli- din-1-yl)-8-chloro (or hydroxy, bromo,
fluoro, amino, amido, carboxy, cyano, methylamino, thio,
methylthio, .omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9-(R)-(2-phosphonomethoxypropyl) purine,
2-alkylamino-6-(morpholin-1-yl)-8-chloro (or hydroxy, bromo,
fluoro, amino, amido, carboxy, cyano, methylamino, thio,
methylthio, .omega.-hydroxyalkylamino, .omega.-hydroxyalkyloxy,
.omega.-carboxyalkylamino, .omega.-aminoalkylamino,
.omega.-fosfonoalkylamino, .omega.-fosfonoalkyloxy,
propinyl)-9-(R)-(2-phosphonomethoxypropyl) purine.
[0132] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives of formula I, wherein
R2, R6, R8 and R9 have above mentioned meanings, A is a group of
formula 11
[0133] and Z is N, characterized in that a trisubstituted
derivative of formula XI, 12
[0134] wherein R2, R6 and R9 have above mentioned meanings, is
brominated with using a brominating system selected from a group
consisting of bromine/acetic acid, bromine/chloroform,
bromine/acetate buffer, bromine/water,
N-bromosuccinimide/dimethylformamide and
bromoacetamide/dimethylformamide to obtain a derivative of formula
XIa, 13
[0135] wherein R2, R6 and R9 have above mentioned meanings, and the
bromine atom in position 8 of the derivative of formula XIa is then
optionally subjected to a substitution in order to replace it by
another substituent R8, which has above mentioned meanings.
[0136] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives of formula I, wherein
R2, R6, R8 and R9 have above mentioned meanings, A is a group of
formula 14
[0137] and Z is N, characterized in that a trisubstituted
derivative of formula XIb, 15
[0138] wherein R6 and R9 have above mentioned meanings, is
brominated with using a brominating system selected from a group
consisting of bromine/acetic acid, bromine/chloroform,
bromine/acetate buffer, bromine/water,
N-bromosuccinimide/dimethylformamide and
bromoacetamide/dimethylformamide to obtain a derivative of formula
XIc, 16
[0139] wherein R6 and R9 have above mentioned meanings, and the
chlorine atom in position 2 and the bromine atom in position 8 of
the derivative of formula XIc are optionally, either progressively
or simultaneously, subjected to a nucleophilic substitution in
order to replace them by other substituents R2 and R8, that have
above mentioned meanings.
[0140] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives of formula I, wherein
R2, R6 and R8 have above mentioned meanings, R9 is alkyl as defined
above, A is a group of formula 17
[0141] and Z is N, characterized in that, a trisubstituted
derivative of formula XIIa, 18
[0142] wherein R6 is as defined above, is alkylated in position 9
with using an appropriate alkylating agent in a system selected
from a group consisting of K.sub.2CO.sub.3/dimethylformamide,
Cs.sub.2CO.sub.3/dimethy- lformamide, t-BuOK/dimethylformamide,
t-BuOK/dimethylsulphoxide and NaH/dimethylformamide or under
conditions of Mitsunobu reaction to obtain a derivative of formula
XIIb 19
[0143] wherein R6 and alkyl are as defined above, and the chlorine
atoms in positions 2 and 8 of the derivative of formula XIIb are
optionally, either progressively or simultaneously, subjected to a
substitution in order to replace them by another substituents R2
and R8, that have above mentioned meanings.
[0144] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives, wherein R2, R6 and
R8 have above mentioned meanings, R9 is alkyl as defined above, A
is a group of formula 20
[0145] and Z is N, characterized in that a trisubstituted
derivative of formula XIIa, 21
[0146] wherein R6 has above mentioned meanings, is protected by
reacting, for example, with 2-dihydropyrane/H.sup.+ to obtain a
derivative of formula XIIc, 22
[0147] wherein R6 has above mentioned meanings, and a group R6
and/or the chlorine atom in position 2 and/or the chlorine atom in
position 8 are optionally converted to another substituents R2, R6
and R8, that have above mentioned meanings and in such optionally
modificated product of formula XIIc, the 2-tetrahydropyranyl group
is split off to obtain a corresponding derivative of formula XIIc
wherein R9 is hydrogen, and so obtained product is then alkylated
in position 9 with using an appropriate alkylating agent in a
system selected from a group consisting of
K.sub.2CO.sub.3/dimethylformamide,
Cs.sub.2CO.sub.3/dimethylformamide, t-BuOK/dimethylformamide,
t-BuOK/dimethylsulphoxide and NaH/dimethylformamide or under
conditions of Mitsunobu reaction to obtain a tetrasubstituted
derivative of formula XIIc, wherein R2,R6,R8 and R9 have above
mentioned meanings.
[0148] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives of formula I, wherein
R2, R6 and R8 have above mentioned meanings, R9 is alkyl as defined
above, A is a group 23
[0149] and Z is N, characterized in that, 2,6,8-trichloropurine is
subjected to a nucleophilic substitution in position 6 in order to
replace the chlorine atom in position 6 by another substituent R6
as defined above to obtain a derivative of formula XIIa, 24
[0150] wherein R6 has above mentioned meanings, and so obtained
product is then alkylated in position 9 with using an appropriate
alkylating agent in a system selected from a group consisting of
K.sub.2CO.sub.3/dimethylf- ormamide,
Cs.sub.2CO.sub.3/dimethylformamide, t-BuOK/dimethylformamide,
t-BuOK/dimethylsulphoxide and NaH/dimethylformamide or under
conditions of Mitsunobu reaction to obtain a derivative of formula
XIIb, 25
[0151] wherein R6 and alkyl have above mentioned meanings, and the
chlorine atoms in positions 2 and 8 of the derivative of formula
XIIb are optionally, either progressively or simultaneously,
subjected to a nucleophilic substitution in order to replace them
by another substituents R2 and R8 that have above mentioned
meanings.
[0152] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives of formula I, wherein
R2 is hydrogen, R6 and R8 have above mentioned meanings, R9 is
alkyl as defined above, A is a group of formula 26
[0153] and Z is N, characterized in that 6,8-dichloropurine is
subjected to a substitution in position 6 in order to replace the
chlorine atom in position 6 by another substituent R6 which has
above mentioned meanings to obtain a derivative of formula XVI,
27
[0154] wherein R6 has above mentioned meanings, and so obtained
derivative is alkylated in position 9 with using an appropriate
alkylating agent in a system selected from a group consisting of
K.sub.2CO.sub.3/dimethylform- amide,
Cs.sub.2CO.sub.3/dimethylformamide, t-BuOK/dimethylformamide,
t-BuOK/dimethylsulphoxide and NaH/dimethylformamide or under
conditions of Mitsunobu reaction to obtain a derivative of formula
XVIa, 28
[0155] wherein R6 and alkyl have above mentioned meanings, and the
chlorine atom in position 8 of the derivative of formula XVIa is
optionally subjected to a substitution in order to replace it by
another substituent R8 which has above mentioned meanings.
[0156] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives of formula I, wherein
R2, R6 and R8 have above mentioned meanings, R9 is alkyl as defined
above, A is a group of formula 29
[0157] and Z is N, characterized in that 2,6-dichloropurine is
subjected to a nucleophilic substitution in position 6 in order to
replace the chlorine atom in position 6 by another substituent R6
as defined above, and so obtained derivative of formula XVII,
30
[0158] wherein R6 is as defined above, is alkylated in position 9
with using either methyl acrylate or acrylonitrile or oxirane to
obtain a derivative of formula XVIIa 31
[0159] wherein R6 and alkyl have above mentioned meanings, which
product is then brominated in position 8 with using a brominating
system selected from a group consisting of bromine/acetic acid,
bromine/chloroform, bromine/acetate buffer, bromine/water,
N-bromosuccinimide/dimethylformami- de and
bromoacetamide/dimethylformamide to obtain a derivative of formula
XVIIb, 32
[0160] wherein R6 and alkyl have above mentioned meanings, and so
obtained product is then optionally, either progressively or
simultaneously, subjected to a nucleophilic substitution in
positions 2 and 8 in order to replace the chlorine atom in position
2 and the bromine atom in position 8 by another substituents R2 and
R8 that have above mentioned meanings.
[0161] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives of formula I, wherein
R6 is a halogen or hydrogen, R2, R8 and R9 have above mentioned
meanings, A is a group of formula 33
[0162] and Z is N, characterized in that a derivative of formula
XVIII, 34
[0163] wherein R2, R8 and R9 have above mentioned meanings, is
halogenated via diazotation with using, for example,
amylnitrite/CH.sub.2Br.sub.2 or amylnitrite/CHI.sub.3, and so
obtained derivative wherein R6 is halogen is optionally
hydrogenolyzed with using H.sub.2/Pd catalyst to obtain a
corresponding derivative wherein R6 is hydrogen.
[0164] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives of formula I, wherein
R9 has above mentioned meanings and one substituent of R2, R6 or R8
is alkinyl or alkenyl containing 2 to 6 carbon atoms whereas two
other substituents of R2, R6 and R8 have above mentioned meanings,
A is a group of formula 35
[0165] and Z is N or CH, characterized in that a derivative of
formula I, wherein R9 has above mentioned meanings and one
substituent of R2, R6 and R8 is a halogen whereas two other
substituents of R2, R6 and R8 have above mentioned meanings, is
alkinylated in the position of the halogen with using
alkine/triphenylphosphinePdCl.sub.2/CuI/triethylamine system and so
obtained 2-, 6- or 8-alkinyl derivative is optionally converted to
2-, 6- or 8-alkenyl derivative with using a Lindlar catalyst.
[0166] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives of formula I, wherein
R2, R6, R8 and R9 have above mentioned meanings, a is a group of
formula 36
[0167] and Z is N, characterized in that
2,5-dialkyl-3-alkoxycarbonyl-4-am- inopyrazole, wherein alkyls and
alkoxy have above mentioned meanings, is reacted with formamidine
acetate/triethylamine system to obtain
1,3-dialkyl-7-hydroxypyrazolo[4,3-d]pyrimidine and so obtained
7-hydroxy derivative is then optionally transferred to a
corresponding 7-chloro derivative by reaction with thionylchloride,
which product is optionally substituted to obtain a corresponding
R6-derivative, wherein R6 has above mentioned meanings.
[0168] The invention also relates to a method for preparing
substituted nitrogen heterocyclic derivatives of formula I, wherein
R2, R6, R8 and R9 have above mentioned meanings, A is a group of
formula 37
[0169] and Z is N, characterized in that 3,7-dialkylpyrazolo
[4,3-d]pyrimidine, wherein alkyls have above mentioned meanings, is
alkylated with using an appropriate alkylating agent in a system
selected from a group consisting of
K.sub.2CO.sub.3/dimethylformamide and
Cs.sub.2CO.sub.3/dimethylformamide to obtain a mixture of
trialkylated and tetraalkylated products and the trialkylated
product is separated by means of the column chromatography.
[0170] The invention also relates to substituted nitrogen
heterocyclic derivatives of formula I and pharmaceutically
acceptable salts thereof for use as medicaments.
[0171] The invention also relates to substituted nitrogen
heterocyclic derivatives of formula I for use as means for
preparing affinity adsorption matrices, immobilized enzymes for
process control, immunoassay reagents, diagnostic samples,
.sup.14C-, .sup.3H-, avidin- or biotin-labelled compounds,
oligonucleotides and diagnostic samples.
[0172] The invention also relates to substituted nitrogen
heterocyclic derivatives of formula I for use as antimitotic drugs,
in particular drugs for elimination or reduction of viral spread or
growth in tissue culture systems during the production of
biopharmaceutical or other products such as proteins and vaccines,
drugs for elimination or reduction of viral spread and growth in
clinical samples such as blood, and drugs for stopping of growth of
tissue culture cells while leaving the cells to carry on with
protein production.
[0173] The invention also relates to a pharmaceutical composition
with cytostatic, anticancer, antimitotic, antineurodegenerative,
immunosuppressive and antimicrobial activity, characterised in that
it comprises, in addition to auxiliary pharmaceutical matters, at
least one substituted nitrogen heterocyclic derivative of formula
I.
[0174] The invention also relates to a combined pharmaceutical
composition with cytostatic effect, characterised in that it
comprises, in addition to auxiliary pharmaceutical matters, a
combination of at least one substituted nitrogen heterocyclic
derivative of formula I and a cytostatic agent selected from a
group consisting of mitoxantron, cis- and carbo-platin,
methotrexate, taxol and doxorubicin.
[0175] The invention also relates to a use of substituted nitrogen
heterocyclic derivatives of formula I for preparing medicaments
destined for treating tumours, cancers, psoriasis, rheumatoid
arthritis, lupus, type I diabetes, multiple sclerosis, restenosis,
polycyclic kidney disease, host graft disease and gout,
parasitoses, such as those caused by fungi or protists, or
Alzheimer's disease, or for preparing antineurodegenerative drugs
and suppress immunostimulation agents.
[0176] The novel compounds of this invention have a wide variety of
diagnostic, therapeutic and industrial utilities.
[0177] The compounds of this invention are suitable as
intermediates for use in the preparation of affinity absorption
matrices. For example, the phosphonate groups in matrix bound form
are useful in the chromatographic separation of positively charged
molecules. Other immobilised examples of the compounds herein are
useful in purifying proteins, e.g., cell cycle enzymes (cdk's),
enzymes involved in recognition of the compound of this invention,
e.g. transport proteins. Suitable methods of incorporation of the
compounds of this invention into polymeric resins will be readily
apparent to the skilled artisan, for instance the compounds are
incorporated by cross-linking hydroxyl groups of the phosphonate or
hydroxymethyl substituents using cross-linking agents heretofore
known. Linking through a group other than the heterocyclic base
will produce a resin useful in hydrophobic affinity chromatography.
Other suitable linking methods are described in Cihlar (supra).
[0178] The compounds of the formula I and their pharmaceutically
acceptable salts inhibit selectively the enzyme p34.sup.cdc2/cyclin
B kinase and related cdks (cdk2, cdk5, cdk7, erk1, erk2).
[0179] In another embodiment, this invention is a method for
inhibiting cdks and cell proliferation in mammals comprising
administering a therapeutically effective amount of the composition
of claim 1 to the mammal. The cdk inhibiting molecules are useful
for treating cell proliferation disorders such as rheumatoid
arthritis, lupus, type I diabetes, multiple sclerosis, cancer,
restenosis, Alzheimer's disease, growth of parasites (animal,
protists), host graft disease, and gout.
[0180] In another embodiment, this invention is a composition
useful for treating fungal infections (fungi) in humans, animal,
and in plants.
[0181] 2,6,8,9-tetrasubstituted and 6,8,9- or 2,8,9-trisubstituted
adenine derivatives exhibit extremely high potency against DNA
viruses on the part of the defined compounds. Such compounds
otherwise have been considered to have little or no activity
against DNA viruses. Moreover, surprisingly the chirally enriched
or pure (S)-enantiomer is antivirally active. Heretofore, only the
(R)-enantiomer was notably antivirally active, and then only
against retroviruses.
[0182] In addition to other cdc2-related kinases, this kinase
controls certain steps of cell division cycles, in particular the
transition from GI phase into the S phase and in particular the
transition from the G2 phase into the M phase. Out the basis of
this findings, it can be expected that the compounds of the formula
I, II and their pharmaceutically acceptable salts can be used as
antimitotic compounds or for treatment of hypoproliferative
diseases, such as cancer, restenosis and Alzheimer's disease. Thus
in very low concentration (micromolar and lower), they are capable
of inhibiting cell cycle transitions (G1/S, G2/M,
M-phase/metaphase) carried out on the different animal bodies and
embryos. Furthermore, the compounds are useful in treating
autoimune diseases, e.g. rheumatoidal arthritis, lupus, type I
diabetes, multiple sclerosis, etc., in treating of cancer,
cardiovascular disease such as restenosis, host vs graft disease,
gout, polycystic kidney disease and other proliferative diseases
whose pathogenesis involves abnormal cell proliferation.
[0183] This invention also concerns novel compounds that have been
discovered to be potent and specific inhibitors of
I.kappa.B-.alpha. kinase which prevents signal induced NF-.kappa.B
activation and cytokine synthesis in vitro and in vivo. Such
inhibitors are expected to inhibit synthesis of cytokines and
adhesion proteins whose synthesis is transcriptionally regulated by
NF-.kappa.B. Pro-inflammatory cytokines such as IL-1, IL-6, TNF and
adhesion proteins (e.g. ICAM, VCAM and selections) belong to this
class of molecules and have implicated in the pathogenesis of
inflammatory diseases. Thus a potent inhibitor of I.kappa.B-.alpha.
kinase is useful in the clinical management of diseases where the
NF-.kappa.B activation is required for disease induction.
[0184] It also relates to novel compounds activating p53, the
mammal cell's own natural brake gene for stopping uncontrolled cell
proliferation (cancer), thus being able to switch off the cancer.
p53 as well as retinoblastoma (Rb) are two well-characterised
tumour suppressors whose inactivation may led to uncontrolled cell
proliferation and malignancy. Phosphorylation of these two
proteins, which are involved in the cell cycle regulatory
mechanisms, is known to modulate their function. Thus a potent cdk
inhibitor represent a good toll for treatment of cancers due to
induction of wild type p53 protein in cancers expressing mutant
p53.
[0185] In addition, studies carried out on the derivatives of the
invention have demonstrated strong effect on apoptosis of many
cancer cell lines. It has been seen that apoptosis can be induced
at stage G1 or G2 and following damage of the DNA, some cells stop
at stage G1 and p53-dependent apoptotic pathway is then induced. In
other situations, it seems that cells stop at G2/M stage in
response to damage caused to the DNA, and activation of an
independent p53 apoptotic path is observed. This path has proved to
be particularly significant in the therapy of tumours in which a
less of active p53 is observed. The interest is therefore assessed
by application of the derivatives of the invention for stimulating
on of p53-independent apoptosis in the cells, which have stopped at
stage G2 through damage to the DNA using agents such as
mitoxantrone or cis-platinum. The cdk inhibitors of this invention
can thus increase the therapeutic potential of the anti-tumour
agents currently used.
[0186] The compounds of this invention will generally be terminally
incorporated into the oligonucleotide. If they do not contain
phosphonyl group attached to the hydroxyl group, they optionally
are incorporated internally into the sequence of the
oligonucleotide. Terminally incorporated diphosphonyl compounds of
this invention which contain no free hydroxyl capable of
participating in chain elongation also are useful in DNA sequencing
in essentially the same manner as deoxyNTPs have been used in the
past (see example 8 of U.S. Pat. No. 5,276,143). The nucleotide
analogues of the invention (when diphosphorylated) are useful as
chain terminators for dideoxynucleotide-type DNA sequencing
protocols, provided that the nucleotide analogue lacks a free
hydroxyl group suitable for polymerase mediated chain elongation.
These compounds will not have R=hydroxymethyl and do not possess a
cyclic structure incorporating the phosphorus atom (although
compounds having such excluded structures can be intermediates).
The nucleotide analogue is included in a kit with other reagents
(such as Klenow polymerase or T4 polymerase, dNTPs, etc) needed for
DNA sequencing (Otvos, et al. "Nucl. Acids. Res." 15:1763-1777
(1987).
[0187] If the oligonucleotide-incorporated compound of this
invention is bindingcompetent for its complementary sequence, i.e.,
if it is capable of base pairing, then this nucleotide monomer will
participate in hybridisation. It is not necessary, however, that
the incorporated nucleotide analogue of this invention base pair or
otherwise participate in hybridisation. If it is located at the
terminus of the oligonucleotide it will be useful as an
immunological recognition site, or haptenic recognition site, to
facilitate detection of the oligonucleotide by an antibody capable
of binding the compound of this invention.
[0188] The compounds of this invention also are useful as linkers
or spacers in preparation affinity absorption matrices (as opposed
to functioning as affinity moieties per se as noted above),
immobilised enzymes for process control, or immunoassay reagents.
The compounds herein contain a multiplicity of functional groups
that are suitable as sites for cross-linking desired substances.
For example, it is conventional to link affinity reagents such as
hormones, peptides, antibodies, drugs, and the like to insoluble
substrates. These insolubilised reagents are employed in known
fashion to absorb binding partners for the affinity reagents from
manufactured preparations, diagnostic samples and other impure
mixture. Similarly, immobilised enzymes are used to perform
catalytic conversions with facile recovery of enzyme. Bifunctional
compounds are commonly used to link analytes to detectable groups
in preparing diagnostic reagents.
[0189] Many functional groups present in the compounds of this
invention are suitable for use in cross-linking. For example, the
phosphonic acid is used to form esters with alcohols or amides with
amines. The R groups substituted with OH, azido (which is reduced
to amino if desired before cross-linking) or vinyl are exemplary
suitable sites. Similarly, the amino, halo, acyl and other reactive
sites found on group B are suitable. Suitable protection of
reactive groups will be used where necessary while assembling the
cross-linked reagent. In general, the compounds here are used by
linking them through phosphonic acid or amino group to the hydroxyl
or amino groups of the linking partner in the same fashion as shown
herein, and covalently bonded to the other binding partner through
an R group. For example a first binding partner such as a steroid
hormone is esterified and then this conjugate is cross-linked
through hydroxymethyl R to cyanogen bromide activated Sepharose,
whereby immobilised steroid is obtained. Other chemistries for
conjugation are well known. See for example Maggio,
"EnzymeImmunoassay" (CRC, 1988, pp 71-135) and references cited
therein.
[0190] The oligonucleotides of this invention are labeled with any
conventional detectable label, e.g. a fluorescent moiety such a
fluorescein, radioisotopes such as .sup.14C or .sup.3H, stable free
radicals, avidin, biotin and the like all of which previously have
been used as labels for immunoassays or diagnostic probes. The
label will be present on the oligonucleotide or on the residue of
an analogue of this invention. Suitable labelling methods are well
known and are readily used with reactive groups such as hydroxyl,
allyl and the like. A simple method is to label the compound of
this invention with H.sub.3 by proton exchange. The compounds also
are biotinylated using conventional methods. See for instance U.S.
Pat. No. 5,276,143 for analogous structures. However, the compounds
of this invention also are useful directly in diagnostic probe
assays without an exogenous detectable label. In one embodiment of
this alternative, antibodies are raised against the compounds of
this invention. Such antibodies (which in turn are labelled or used
in a double antibody configuration) bind to the analogue of this
invention and thereby are useful in detecting its presence as label
for a protein or oligonucleotide.
[0191] The compounds of the invention are useful for treatment of
microbial infections, for treatment of tumours or for other
indications described below. Microbial infections treatable by the
compounds of this invention include viruses, parasites, yeast and
fungi, but it is believed that the compounds are most effective
against viruses, which constitutes the preferred utility. Exemplary
viral infections include infections caused by DNA or RNA viruses
including herpes viruses (CMV, HSV 1, HSV 2, EBV, varicella zoster
virus (VZV), bovid herpesvirus type 1, equid herpesvirus type 1,
HVV-6, papillomaviruses (HPV types 1-55 including carcinogenic
HPV), flaviviruses (including yellow fever virus, African swine
fever virus and Japanese encephalitis virus), togaviruses
(including Venezuelan equine encephalomyelitis virus), influenza
viruses (types A-C), retroviruses (HIV-1, HIV-2, HTLV-I, HTLV-II,
SIV, FeLV, FIV, MoMSV), adenoviruses (types 1-8), poxviruses
(vaccinia virus), enteroviruses (poliovirus types 1-3, Coxsackie,
hepatitis A virus, and ECHO virus), gastroenteritis viruses
(Norwalk viruses, rotaviruses), hantaviruses (Hantaan virus),
polyomavirus, papovaviruses, rhinoviruses, parainfluenza virus
types 1-4, rabies virus, respiratory synctial virus (RSV),
hepatitis viruses A, B, C and E, and the like.
[0192] The antiviral activity of individual compounds is determined
by routine assay of antiviral (or other antimicrobial) activity
using enzyme inhibition assays, tissue culture assays, animal model
assays and the like as will be understood by those skilled in the
art.
[0193] Protozoan parasite infections are treated using the
compounds of the invention. The term protozoa include those members
of the subphyla Sarcomastigophora and Sporozoa of the phylum
Protozoa. More particularly, the term protozoa as used herein
include genera of parasitic protozoa, which are important to man,
because they either cause disease in man or in his domestic
animals. These genera for the most part are classified in the
superclass Mastigophora of the subphylum Sarcomastigophora and the
class Telesporea of the subphylum Sporozoa in the classification
according to Baker (1969). Illustrative genera of these parasitic
protozoa include Histomonas, Pneumocystis, Trypanosoma, Giardia,
Trichomonas, Eimeria, Isopora, Leishmania, Entamoeba, Toxoplasma
and Plasmodium. Parasitic protozoans include Plasmodium falciparum,
Plasmodium berghei, Plasmodium malariae, Plasmodium vivax,
Leishmania braziliensis, Leishmania donovani, Trypanosoma cruzi,
Trypanosoma brucei, Trypanosoma rhodesiense, Pneumocystis carinii,
Entamoeba histolytica, Trichomonas vaginalis and the like (de
Vries, E., et al, "Mol. Biochem. Parasitol" 47:43-50 (1991) and
trypanosomes (Kaminsky et al. "J.Parasitol." 80(6): 1026-30 (1994).
The compounds in which R is CH.sub.2OH and B is 3-deazaadenine are
particularly interesting in the treatment of malarial
parasites.
[0194] Compounds of the invention are used to treat yeast or fungal
infections caused by Candida glabrata, Candida ropicalis, Candida
albicans, and other Candida species, Cryptococcus species including
Cryptococcus neoformans, Blastomyces species including Blastomyces
dermatidis, Torulopsis species including Torulopsis glabrata,
Coccidioides species including Coccidioides immitis, Aspergillus
species and the like.
[0195] The compounds of the invention can also be (1) applied to
tissue culture systems to eliminate or reduce viral spread or
growth during the production of biopharmaceutical or other products
(such as proteins or vaccines), (2) used to eliminate or reduce
viral spread or growth in clinical sample (such as blood), and (3)
used to stop growth of tissue culture cells while leaving the cells
to carry on with protein production.
[0196] The compounds herein have been found to suppress
immunostimulation. Accordingly, they can suppress metabolic
activities of T-lymphocytes stimulated by diverse agents, e.g.
concavalin A, they principally will find application in the
treatment of autoimmune diseases, e.g. arthritis, or in suppression
of transplant rejection. Their therapeutically active
concentrations are in the range of 1 mg/kg to 50 mg/kg of body
weight.
[0197] Therapeutic administration
[0198] Suitable routes for administration include oral, rectal,
vassal, topical (including ocular, buccal and sublingual), vaginal
and parental (including subcutaneous, intramuscular, intravitreous,
intravenous, intradermal, intrathecal and epidural). The preferred
route of administration will depend upon the condition of the
patient, the toxicity of the compound and the site of infection,
among other considerations known to the clinician.
[0199] The therapeutical composition comprise about 1% to about 95%
of the active ingredient, single-dose forms of administration
preferably comprising about 20% to about 90% of the active
ingredient and administration forms which are not single-dose
preferably comprising about 5% to about 20% of the active
ingredient. Unit dose forms are, for example, coated tablets,
tablets, ampoules, vials, suppositories or capsules. Other forms of
administration are, for example, ointments, creams, pastes, foams,
tinctures, lipsticks, drops, sprays, dispersions-and the like.
Examples are capsules containing from about 0.05 g to about 1.0 g
of the active ingredient.
[0200] The pharmaceutical compositions of the present invention are
prepared in a manner known per se, for example by means of
convential mixing, granulating, coating, dissolving or lyophilising
processes.
[0201] Preferably, solutions of the active ingredient, and in
addition also suspensions or dispersions, especially isotonic
aqueous solutions, dispersions or suspensions, are used, it being
possible for these to be prepared before use, for example in the
case of lyophilised compositions which comprise the active
substance by itself or together with a carrier, for example
mannitol. The pharmaceutical compositions can be sterilised and/or
comprise excipients, for example preservatives, stabilisers,
wetting agents and/or emulsifiers, solubilizing agents, salts for
regulating the osmotic pressure and/or buffers, and they are
prepared in a manner known per se, for example by means of
convential dissolving or lyophilising processes. The solutions or
suspensions mentioned can comprise viscosity-increasing substances,
such as sodium carboxymethylcellulose, carboxymethylcellulose,
dextran, polyvinylpyrrolidone or gelatin.
[0202] Suspensions in oil comprise, as the oily component, the
vegetable, synthetic or semisynthetic oils customary for injection
purposes. Oils which may be mentioned are, in particular, liquid
fatty acid esters which contain, as the acid component, a
long-chain fatty acid having 8-22, in particular 12-22, carbon
atoms, for example lauric acid, tridecylic acid, myristic acid,
pentadecylic acid, palmitic acid, margaric acid, stearic acid,
arachidinic acid, behenic acid or corresponding unsaturated acids,
for example oleic acid, elaidic acid, euric acid, brasidic acid or
linoleic acid, if appropriate with the addition of antioxidants,
for example vitamin E, (.beta.-carotene or 3,5-di-tert-butyl-4
hydroxytoluene. The alcohol component of these fatty acid esters
has not more than 6 carbon atoms and is mono- or polyhydric, for
example mono-, di- or trihydric alcohol, for example methanol,
ethanol, propanol, butanol, or pentanol, or isomers thereof, but in
particular glycol and glycerol. Fatty acid esters are therefore,
for example: ethyl oleate, isopropyl myristate, isopropyl
palmitate, "Labrafil M 2375" (polyoxyethylene glycerol trioleate
from Gattefosee, Paris), "Labrafil M 1944 CS" (unsaturated
polyglycolated glycerides prepared by an alcoholysis of apricot
kernel oil and made up of glycerides and polyethylene glycol
esters; from Gattefosee, Paris), "Labrasol" (saturated
polyglycolated glycerides prepared by an alcoholysis of TCM and
made up of glycerides and polyethylene glycol esters; from
Gattefosee, Paris) and/or "Miglyol 812" (triglyceride of saturated
fatty acids of chain length C.sub.8 to C.sub.12 from Hills AG,
Germany), and in particular vegetable oils, such as cottonseed oil,
almond oil, olive oil, castor oil, sesame oil, soybean oil and, in
particular, groundnut oil.
[0203] The preparation of the injection compositions is carried out
in the customary manner under sterile conditions, as are bottling,
for example in ampoules or vials, and closing of the
containers.
[0204] For example, pharmaceutical compositions for oral use can be
obtained by combining the active ingredient with one or more solid
carriers, if appropriate granulating the resulting mixture, and, if
desired, processing the mixture or granules to tablets or coated
tablet cores, if appropriate by addition of additional
excipients.
[0205] Suitable carriers are, in particular, fillers, such as
sugars, for example lactose, sucrose, mannitol or sorbitol,
cellulose preparations and/or calcium phosphates, for example
tricalcium phosphate, or calcium hydrogen phosphate, and
furthermore binders, such as starches, for example maize, wheat,
rice or potato starch, methylcellulose,
hydroxypropyl-methylcellulose, sodium carboxymethylcellulose and/or
polyvinylpyrrolidine, and/or, if desired, desintegrators, such as
the above mentioned starches, and furthermore carboxymethyl-starch,
crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof,
such as sodium alginate. Additional excipients are, in particular,
flow regulators and lubricants, for example salicylic acid, talc,
stearic acid or salts thereof, such as magnesium stearate or
calcium stearate, and/or polyethylene glycol, or derivatives
thereof.
[0206] Coated tablet cores ca be provided with suitable coatings
which, if appropriate, are resistant to gastric juice, the coatings
used being, inter alia, concentrated sugar solutions, which, if
appropriate, comprise gum arabic, talc, polyvinylpyrrolidine,
polyethylene glycol and/or titanium dioxide, coating solutions in
suitable organic solvents or solvent mixtures or, for the
preparation of coatings which are resistant to gastric juice,
solutions of suitable cellulose preparations, such as
acetylcellulose phthalate or hydroxypropylmethylcellulose
phthalate. Dyes or pigments can be admixed to the tablets or coated
tablet coatings, for example for identification or characterisation
of different doses of active ingredient.
[0207] Pharmaceutical compositions, which can be used orally, are
also hard capsules of gelatin and soft, closed capsules of gelatin
and a plasticiser, such as glycerol or sorbitol. The hard capsules
can contain the active ingredient in the form of granules, mixed
for example with fillers, such as maize starch, binders and/or
lubricants, such as talc or magnesium stearate, and stabilisers if
appropriate. In soft capsules, the active ingredient is preferably
dissolved or suspended in suitable liquid excipients, such as
greasy oils, parrafin oil or liquid polyethylene glycols or fatty
acid esters of ethylene glycol or propylene glycol, it being
likewise possible to add stabilisers and detergents, for example of
the polyethylene sorbitan fatty acid ester type.
[0208] Other oral forms of administration are, for example, syrups
prepared in the customary manner, which comprise the active
ingredient, for example, in suspended form and in a concentration
of about 5% to 20%, preferably about 10% or in a similar
concentration which results in a suitable individual dose, for
example, when 5 or 10 ml are measured out. Other forms are, for
example, also pulverulent or liquid concentrates for preparing of
shakes, for example in milk. Such concentrates can also be packed
in unit dose quantities.
[0209] Pharmaceutical compositions, which can be used rectally,
are, for example, suppositories that comprise a combination of the
active ingredient with a suppository base. Suitable suppository
bases are, for example, naturally occurring or synthetic
triglycerides, paraffin hydrocarbons, polyethylene glycols or
higher alkanols.
[0210] Compositions which are suitable for parenteral
administration are aqueous solutions of an active ingredient in
water-soluble form, for example of water-soluble salt, or aqueous
injection suspensions, which comprise viscosity-increasing
substances, for example sodium carboxymethylcellulose, sorbitol
and/or dextran, and if appropriate stabilisers. The active
ingredient can also be present here in the form of a lyophilisate,
if appropriate together with excipients, and be dissolved before
parenteral administration by addition of suitable solvents.
Solutions such as are used, for example, for parental
administration can also be used as infusion solutions. Preferred
preservatives are, for example. Antioxidants, such as ascorbic
acid, or microbicides, such as sorbic or benzoic acid.
[0211] Ointments are oil-in-water emulsions, which comprise not
more than 70%, but preferably 20-50% of water or aqueous phase. The
fatty phase consists, in particular, hydrocarbons, for example
vaseline, paraffin oil or hard paraffin's, which preferably
comprise suitable hydroxy compounds, such as fatty alcohol's or
esters thereof, for example cetyl alcohol or wool wax alcohols,
such as wool wax, to improve the waterbinding capacity. Emulsifiers
are corresponding lipophilic substances, such as sorbitan fatty
acid esters (Spans), for example sorbitan oleate and/or sorbitan
isostearate. Additives to the aqueous phase are, for example,
humectants, such as polyalcohols, for example glycerol, propylene
glycol, sorbitol and/or polyethylene glycol, or preservatives and
odoriferous substances.
[0212] Fatty ointments are anhydrous and comprise, as the base, in
particular, hydrocarbons, for example paraffin, vaseline or
paraffin oil, and furthermore naturally occurring or semisynthetic
fats, for example hydrogenated coconut-fatty acid triglycerides,
or, preferably, hydrogenated oils, for example hydrogenated
groundnut or castor oil, and furthermore fatty acid partial esters
of glycerol, for example glycerol mono- and/or distearate, and for
example, the fatty alcohols. They also contain emulsifiers and/or
additives mentioned in connection with the ointments which increase
uptake of water.
[0213] Creams are oil-in-water emulsions, which comprise more than
50% of water. Oily bases used are, in particular, fatty alcohols,
for example lauryl, cetyl or stearyl alcohols, fatty acids, for
example palmitic or stearic acid, liquid to solid waxes, for
example isopropyl myristate, wool wax or beeswax, and/or
hydrocarbons, for example vaseline (petrolatum) or paraffin oil.
Emulsifiers are surface-active substances with predominantly
hydrophilic properties, such as corresponding nonionic emulsifiers,
for example fatty acid esters of polyalcohols or ethyleneoxy
adducts thereof, such as polyglyceric acid fatty acid esters or
polyethylene sorbitan fatty esters (Tweens), and furthermore
polyoxyethylene fatty alcohol ethers or polyoxyethylene fatty acid
esters, or corresponding ionic emulsifiers, such as alkali metal
salts of fatty alcohol sulfates, for example sodium lauryl sulfate,
sodium cetyl sulfate or sodium stearyl sulfate, which are usually
used in the presence of fatty alcohols, for example cetyl stearyl
alcohol or stearyl alcohol. Additives to the aqueous phase are,
inter alia, agents which prevent the creams from drying out, for
example polyalcohols, such as glycerol, sorbitol, propylene glycol
and/or polyethylene glycols, and furthermore preservatives and
odoriferous substances.
[0214] Pastes are creams and ointments having secretion-absorbing
powder constituents, such as metal oxides, for example titanium
oxide or zinc oxide, and furthermore talc and/or aluminium
silicates, which have the task of binding the moisture or
secretions present.
[0215] Foams are administered from pressurised containers and they
are liquid oil-inwater emulsions present in aerosol for. As the
propellant gases, halogenated hydrocarbons, such as
chlorofluoro-lower alkanes, for example dichlorofluoromethane and
dichlorotetrafluoroethane, or, preferably, non-halogenated gaseous
hydrocarbons, air, N.sub.2O, or carbon dioxide are used. The oily
phases used are, inter alia, those mentioned above for ointments
and creams, and the additives mentioned there are likewise
used.
[0216] Tinctures and solutions usually comprise an
aqueous-ethanolic base to which, humectants for reducing
evaporation, such as polyalcohols, for example glycerol, glycols
and/or polyethylene glycol, and re-oiling substances, such as fatty
acid esters with lower polyethylene glycols, i.e. lipophilic
substances soluble in the aqueous mixture to substitute the fatty
substances removed from the skin with the ethanol, and, if
necessary, other excipients and additives, are admixed.
[0217] The present invention further provides veterinary
compositions comprising at least one active ingredient as above
defined together with a veterinary carrier therefor. Veterinary
carriers are materials for administering the composition and may be
solid, liquid or gaseous materials, which are inert or acceptable
in the veterinary art and are compatible with the active
ingredient. These veterinary compositions may be administered
orally, parenterally or by any other desired route.
[0218] The invention also relates to a process or method for
treatment of the disease states mentioned above. The compounds can
be administered prophylactically or therapeutically as such or in
the form of pharmaceutical compositions, preferably in an amount,
which is effective against the diseases mentioned. With a
warm-blooded animal, for example a human, requiring such treatment,
the compounds are used, in particular, in the form of
pharmaceutical composition. A daily dose of about 0.1 to about 5 g,
preferably 0.5 g to about 2 g, of a compound of the present
invention is administered here for a body weight of about 70
kg.
EXAMPLES OF THE INVENTION
EXAMPLE 1
2-(3-hydroxypropylamino)-6-benzylamino-8-bromo-9-isopropypurine.
[0219] 1 mmol of
2-(3-hydroxypropylamino)-6-benzylamino-9-isopropylpurine was
dissolved in chloroform and 1.1 mmol of bromine was added. The
8-bromo derivative hydrobromide was removed by filtration and
crystallized from n-propanol--ether. M.p. 156-161.degree. C. Yield
90%. TLC: chloroform-methanol (96:4), single spot. MS-ESI(+) m/z:
420, 422 [M+H].sup.+
1TABLE 1 Compounds Prepared by the Method of Example 1 SUBSTITUENT
C2 N6 C8 N9 2-hydroxyethylamino benzylamino bromo methyl
3-hydroxypropylamino benzylamino bromo isopropyl
(R)-1-(hydroxymethyl) propylamino benzylamino bromo isopropyl
(R)-1-(hydroxymethyl) propylamino 3-iodobenzylamino bromo isopropyl
diethanolamino 3-chloroanilino bromo isopropyl
(R)-1-(hydroxymethyl) isobutylamino 4-methoxybenzylamino bromo
isopropyl (R)-1-(hydroxymethyl) isobutylamino 3-chloroanilino bromo
isopropyl
EXAMPLE 2
2-(3-hydroxypropylamino)-6-benzylamino-8-methylthio-9-isopropylpurine.
[0220]
2-(3-Hydroxypropylamino)-6-benzylamino-8-bromo-9-isopropylpurine
(0.15 mmol), prepared as described in Example 1, was dissolved in 2
mL dimethylformamide, 0.9 mmol of CH.sub.3SNa was added and the
mixture stirred at 40.degree. C. for 1 hour. The solvent was
removed in vacuo and the rest partitioned between water-EtOAc. The
organic layer was dried, evaporated and the rest purified by column
chromatography (silica gel, chloroform-heptane (8:2)). Yield 75%.
M.p. 64-67.degree. C. .sup.1H NMR (300 MHz, CDCl.sub.3): 7.28-7.40
m, 5.55 t, 4.85 t, 4.75 d, 4.65 m, 3.62 m, 2.62 s, 1.74 dd, 1.60 d.
MS-ESI(+) m/z: 387 [M+H].sup.+
2TABLE 2 Compounds Prepared by the Method of Example 2 SUBSTITUENT
C2 N6 C8 N9 2-hydroxyethylamino benzylamino methyl methyl
2-hydroxyethylamino benzylamino mercapto methyl 2-hydroxyethylamino
benzylamino hydroxy methyl 2-hydroxyethylamino benzylamino amino
methyl 2-hydroxyethylamino benzylamino 2-hydroxyethylamino methyl
2-hydroxyethylamino benzylamino aminomethylamino methyl
3-hydroxypropylamino benzylamino methyl isopropyl
3-hydroxypropylamino benzylamino mercapto isopropyl
3-hydroxypropylamino benzylamino hydroxy isopropyl
3-hydroxypropylamino benzylamino amino isopropyl
3-hydroxypropylamino benzylamino 2-hydroxyethylamino isopropyl
3-hydroxypropylamino benzylamino aminomethylamino isopropyl
(R)-1-(hydroxymethyl) benzylamino methyl isopropyl propylamino
(R)-1-(hydroxymethyl) benzylamino mercapto isopropyl propylamino
(R)-1-(hydroxymethyl) benzylamino hydroxy isopropyl propylamino
(R)-1-(hydroxymethyl) benzylamino amino isopropyl propylamino
(R)-I-(hydroxymethyl) benzylamino 2-hydroxyethylamino isopropyl
propylamino (R)-1-(hydroxymethyl) benzylamino aminomethylamino
isopropyl propylamino (R)-1-(hydroxymethyl) 3-iodobenzylamino
methyl isopropyl propylamino (R)-1-(hydroxymethyl)
3-iodobenzylamino mercapto isopropyl propylamino
(R)-1-(hydroxymethyl) 3-iodobenzylamino hydroxy isopropyl
propylamino (R)-1-(hydroxymethyl) 3-iodobenzylamino amino isopropyl
propylamino (R)-1-(hydroxymethyl) 3-iodobenzylamino
2-hydroxyethylamino isopropyl propylamino (R)-1-(hydroxymethyl)
3-iodobenzylamino aminomethylamino isopropyl propylamino
diethanolamino 3-chloroanilino methyl isopropyl diethanolamino
3-chloroanilino mercapto isopropyl diethanolamino 3-chloroanilino
hydroxy isopropyl diethanolamino 3-chloroanilino amino isopropyl
diethanolamino 3-chloroanilino 2-hydroxyethylamino isopropyl
diethanolamino 3-chloroanilino aminomethylamino isopropyl
(R)-1-(hydroxymethyl) 4-methoxybenzylamino mercapto isopropyl
isobutylamino (R)-1-(hydroxymethyl) 4-methoxybenzylamino hydroxy
isopropyl isobutylamino (R)-1-(hydroxymethyl) 4-methoxybenzylamino
amino isopropyl isobutylamino (R)-1-(hydroxymethyl)
4-methoxybenzylamino 2-hydroxyethylamino isopropyl isobutylamino
(R)-1-(hydroxymethyl) 4-methoxybenzylamino aminomethylamino
isopropyl isobutylamino (R)-1-(hydroxymethyl) 3-chloroanilino
methyl isopropyl isobutylamino (R)-1-(hydroxymethyl)
3-chloroanilino mercapto isopropyl isobutylamino
(R)-1-(hydroxymethyl) 3-chloroanilino hydroxy isopropyl
isobutylamino (R)-1-(hydroxymethyl) 3-chloroanilino amino isopropyl
isobutylamino (R)-1-(hydroxymethyl) 3-chloroanilino
2-hydroxyethylamino isopropyl isobutylamino (R)-1-(hydroxymethyl)
3-chloroanilino aminomethylamino isopropyl isobutylamino
EXAMPLE 3
6-(3-Methoxybenzylamino)-2-chloro-8-bromo-9-isopropylpurine.
[0221] 1 mmol of
6-(3-methoxybenzylamino)-2-chloro-9-isopropylpurine was brominated
with 1.1 mmol of bromine as described in Example 1. The crude
2,8-dihalogeno derivative prepared was alkalinized with
NH.sub.3/MeOH and extracted between water--EtOAc. The product
crystallizes after partial evaporation of ethylacetate.
M.p.123-125.degree. C., yield 88%. MS-ESI (+) m/z 409.5, 411.4,
413.3 [M+H].sup.+
EXAMPLE 4
6-Phenylamino-2-(3-aminopropylamino)-9-isopropyl-8-aminopurine.
[0222]
6-Phenylamino-2-(3-aminopropylamino)-9-isopropyl-9-benzylaminopurin-
e, prepared from appropriate 8-bromo derivative, was dissolved in
glacial acetic acid. To this solution, 50 mg Pd/BaSO.sub.4 (10%)
was added and the mixture hydrogenated to constant consumption of
hydrogen (2 hours). The catalyst was removed by centrifugation and
acetic acid was evaporated in vacuo. The rest was purified by
column chromatography (silica gel, chloroform-MeOH-conc. NH.sub.4OH
(95:5:1). Yield 75%. MS-ESI (+) m/z 340.5 [M+H].sup.+
EXAMPLE 5
6-(4-Methoxybenzylamino)-2,8-bis(3-hydroxypropylamino)-9-isopropylpurine.
[0223] 6-(4-Methoxybenzylamino)-2,8-bis(3-hydroxypropylamino)purine
(0.07 mmol) was stirred in 2 mL dimethylformamide with 0.14 mmol of
allylbromide and with 0.36 mmol of dry potassium carbonate at
ambient temperature (6 hours). The solvent was evaporated to
dryness and the rest extracted between ethylacetate--water. The
organic layer was dried, ethylacetate was evaporated and the rest
purified by column chromatography (silica gel, chloroform). Yield
55%. MS-ESI(+) m/z 441.4 [M+H].sup.+
3TABLE 3 Compounds Prepared by the Method of Example 5 SUBSTITUENT
C2 N6 C8 N9 2-hydroxyethylamino benzylamino 2-hydroxyethylamino
allyl 3-hydroxypropylamino 4-methoxybenzylamino
3-hydroxypropylamino allyl 2-aminoethylamino benzylamino
2-aminoethylamino isopropyl 3-aminopropylamino benzylamino
3-aminopropylamino isopropyl aminomethylamino benzylamino
aminomethylamino isopropyl diethanolamino benzylamino
diethanolamino isopropyl hydroxymethylamino benzylamino
hydroxymethylamino isopropyl 3-aminopropylamino benzylamino
3-aminopropylamino isopropyl 2-hydroxyethylamino 3-chloroanilino
2-hydroxyethylamino isopropyl 3-hydroxypropylamino 3-chloroanilino
3-hydroxypropylamino isopropyl 2-aminoethylamino 3-chloroanilino
2-aminoethylamino isopropyl 3-aminopropylamino 3-chloroanilino
3-aminopropylamino isopropyl diethanolamino benzylamino
diethanolamino isopropyl hydroxymethylamino benzylamino
hydroxymethylamino isopropyl (R)-1-(hydroxymethyl) 3-chloroanilino
(R)-1-(hydroxymethyl)- isopropyl isobutylamino isobutylamino
EXAMPLE 6
6-Benzylamino-8-hydroxyethylamino-9-isopropylpurine
[0224] 1 mmol of 6-chloropurine was alkylated with isopropylbromide
in DMF as described in Example 5. The product,
6-chloro-9-isopropylpurine was brominated in acetic acid similarly
as described in Example 1. After purification by column
chromatography, 6-benzylamino-9-isopropyl-8-bromop- urine was
treated with 2-aminoethanol to give 6-benzylamino-8-hydroxyethyl-
amino-9-isopropylpurine. Yield 40%. MS-ESI(+) m/z 326
[M+H].sup.+
EXAMPLE 7
2-(3-Hydroxypropylamino)-9-methyl-8-(3-hydroxypropyloxy)purine.
[0225]
6-Amino-2-(3-hydroxypropylamino)-9-methyl-8-(3-hydroxypropyloxy)pur-
ine was brominated in the position 6 with
amylnitrite/CH.sub.2Br.sub.2. The 6-bromo derivative was then
hydrogenolyzed with PdO/BaSO.sub.4 in strong alkaline solution to
give 2,8,9-trisubstituted purine. The crude product was purified by
column chromatography (silica gel, chloroform-MeOH-conc. NH.sub.4OH
(8:2:0.2)). The total yield was 35%. MS-ESI(+) 281[M+H].sup.+
EXAMPLE 8
7-Hydroxy-1-methyl-3-isopropylpyrazolo[4,3]pyrimidine.
[0226] 0.02 mmol of
2-Methyl-4-amino-3-alkoxycarbonyl-5-isopropylpyrazole (prepared
from appropriate 4-nitro derivative by catalytic hydrogenation) was
dissolved in 30 mL of 2-ethoxyethanol and 0.02 mmol of formamidin
acetate and 5.2 mL of triethylamine were added. The mixture was
heated to 90.degree. C. for 2 hours. The solution was concentrated
in vacuo, dissolved in chloroform to give crystalline product.
After recrystallization from ethanol m.p. 295-298.degree. C.
MS-ESI(+) m/z 193 [M+H].sup.+
EXAMPLE 9
7-(2-Hydroxybenzylamino)-1-methyl-3-isopropylpyrazolo
[4,3]pyrimidine.
[0227] 1.1 mmol of 7-Hydroxy-l-methyl-3-isopropyl
pyrazolo[4,3]pyrimidine (Example 8) was dissolved in the mixture of
0.12 mL of dimethylformamide and 5 mL dry chloroform. 11 mmol of
thionylchloride (0.81 mL) was added and the solution heated to
80.degree. C. for 1 hour. A new portion of 0.8 mL of
thionylchloride and more chloroformdimethylformamide were then
added and heating continued for 3 hours. The solvents were
evaporated in vacuo and the rest partitioned between
water-chloroform. The organic extract was dried and used in next
reaction without purification. The chloroform solution was heated
with excess of 2-hydroxybenzylamine and N-ethyldiisopropylamine for
1 hour. The product was purified by column chromatography (silica
gel, chloroform MeOH-AcOH (20:0.4:0.1)). M.p.205-210.degree. C.
Yield 40%. MS-ESI(+) m/z 297 [M+H].sup.+
EXAMPLE 10
6-(Benzylamino)-2-[(1-hydroxymethyl)propylamino]-8-chloro-9-isopropylpurin-
e.
[0228] 6-(Benzylamino)-2,8-dichloropurine, prepared from
2,6,8-trichloropurine, m.p.220-223.degree. C., 50% yield, was
dissolved in 2-aminobutanol and heated to 150.degree. C. for 12
hours. After evaporation of the solvent in vacuo the product,
6-benzylamino-2-[(1-hydr- oxymethyl) propylamino]-8-chloropurine
was purified by column chromatography (silica gel,
chloroform--MeOH--conc. NH.sub.4OH 9:1:0.1) with total yield 36%.
MS-ESI(+) m/z 348 [M+H].sup.+. The product was subsequently
alkylated with isopropyliodide/K.sub.2CO.sub.3/dimethylforma- mide
to give, after preparative column chromatography (silica gel,
chloroform) 70% yield of the desired compound. MS (ESI+): 388.4,
390.6 (M+H).sup.+.
EXAMPLE 11
Preparation of Affinity Sorbent
Preparation of
2-(2-aminopropylamino)-6-(3-hydroxypropylamino)-8-bromo-9-i-
sopropylpurine Epoxy activated Sepharose 6B Affinity Matrix.
[0229] Freeze-dried epoxy activated Sepharose 6B (Pharmacia LKB,
Piscataway, N.J.) was chosen for the coupling reaction due to its
ability to form an ether bond between a hydroxyl-containing ligand
and the epoxide group on the Sepharose. The gel was swollen
according to the manufacturer's instructions, (100 mg) of any one
of the compound defined by Formula I (preferably with
R6=aminooctylamino, 3- or 4-benzylamino, etc.) was dissolved in 1
ml coupling solution (1.2:1, v/v, DMF, 0.1N NaOH) and mixed with
0.5 ml of swollen gel at pH 10-11 for 72 h at room temperature with
gentle agitation. Excess reactive groups were blocked with 1M
ethanolamine for 4 hours at 50.degree. C. and the gel slurry was
poured into 1-ml syringe column. The resin was activated with three
alternating cycles of twenty column volumes each of pH 4.0 (0.1 M
acetate, 0.5 M NaCl) and pH 8.0 (0.1 M tris-HCI, 0.5 M NaCl)
buffers followed by twenty column volumes of reaction buffer (20 mM
HEPES, pH 7.3, 10 mM MgCl.sub.2, 15 mM glycerophosphate, 0.5 mM
sodium orthovanadate, 0.5 mM EGTA). The column was stored at
4.degree. C in the reaction buffer containing 0.1% sodium azide and
regenerated prior to each use with alternating cycles of low and
high pH as described above.
[0230] The Sf9 insect cell lysate (500 .mu.g protein in 1-ml
reaction buffer) was passed over the affinity column matrix
sequentially five times and the flow through was saved, (unbound
material). The matrix was then washed three times with 1 ml
reaction buffer (wash 1-3) then three times each reaction buffer
containing 0.5M NaCl (eluate 1-3). The coupled proteins were eluted
at low pH (pH 4.0, 0.1M acetate, 0.5M NaCl) as described above and
aliquots (20.mu. from 1 ml) of each sample were assayed for their
ability to phosphorylate histone H1 and other substrate proteins as
described in Example 12. The presence of CDK complexes was also
determined by SDS-PAGE.
EXAMPLE 12
CDK Inhibition Assays
[0231] Proteins
[0232] Cyclin-dependent kinases (p34.sup.cdc2, p33.sup.cdk2,
p33.sup.cdk4) and cyclins (cyclin B, E and D1) are produced in Sf9
insect cells coinfected with appropriate baculoviral constructs.
The cells are harvested 68-72 hrs post infection in lysis buffer
for 30 min on ice and the soluble fraction is recovered by
centrifugation at 14.000 g for 10 min. The protein extract is
stored at -80.degree. C.
[0233] Rb-GST is produced using an E. coli expression system,
containing sequence encoding C terminus of retinoblastoma protein
(aminoacids 773-928), which is known to be phosphorylated by
p33.sup.cdk4 kinase. The fusion protein is purified on
glutathioneagarose beads.
[0234] Lysis buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 20
mM NaF, 1% Tween, 1 mM DTT, 0.1 mM PMSF, leupeptine,
aprotonine.
[0235] Enzyme inhibition assays
[0236] To carry out experiments on kinetics under linear
conditions, the final point test system for kinase activity
measurement is used. The kinase is added to reaction mixture in
such a way as to obtain linear activity with respect to the
concentration of enzyme and with respect to time.
[0237] The p34.sup.cdc2 and p33.sup.cdk2 kinase inhibition
determination involves the use of 1 mg/ml histone H1 (Sigma, type
III-S) in the presence of 15 .mu.M [.gamma.-.sup.32P]ATP (500-100
cpm/pmol) (Amersham) in a final volume of 20 .mu.l, inhibition of
p33.sup.cdk4 kinase is determined with Rb-GST (0.2 mg/ml) as a
substrate. Kinase activity is determined at 30.degree. C. in the
kinase buffer.
[0238] Tested compounds are usually dissolved to 100 mM solutions
in DMSO, final concentration of DMSO in reaction mixture never
exceeds 1%. The controls contain suitable dilutions of DMSO.
[0239] After 10 min, addition 3.times. SDS sample buffer stops the
incubations. Phosphorylated proteins are separated
electrophoretically using 12.5% SDS polyacrylamide gel. The
measurement of kinase activity is done using digital image
analysis.
[0240] The kinase activity is expressed as a percentage of maximum
activity. The apparent inhibition constants are determined by
graphic analysis. Kinase buffer: 50 mM Hepes pH 7.4. 10 mM
MgCl.sub.2, 5 mM EGTA. 10 mM 2-glycerolphosphate, 1 mM NaF, 1 mM
DTT
4TABLE 4 Kinase Inhibitory Activity of 2,6,8,9-Tetrasubstituted
Purine Derivatives CDC2 IKB-.alpha. SUBSTITUENT IC.sub.50 IC.sub.50
C2 N6 C8 N9 (.mu.M) (.mu.M) 2-hydroxyethylamino benzylamino --
methyl 7 15.4 OLOMOUCINE 2-hydroxyethylamino benzylamino fluoro
methyl 4.2 7.8 2-hydroxyethylamino benzylamino bromo methyl 16.2
22.8 2-hydroxyethylamino benzylamino mercapto methyl 22.4 35.7
2-hydroxyethylamino benzylamino hydroxy methyl 13.5 16.2
2-hydroxyethylamino benzylamino amino methyl 14.2 17.1
2-hydroxyethylamino benzylamino 2-hydroxy- methyl 125.2 153.8
ethylamino 2-hydroxyethylamino benzylamino aminomethyl methyl 114.3
126.5 amino 3-hydroxypropylamino benzylamino -- isopropyl 1 3.2
BOHEMINE 3-hydroxypropylamino benzylamino bromo isopropyl 12.93
24.5 3-hydroxypropylamino benzylamino mercapto isopropyl 13.1 20.9
3-hydroxypropylamino benzylamino hydroxy isopropyl 6.48 12.6
3-hydroxypropylamino benzylamino amino isopropyl 8.52 12.8
3-hydroxypropylamino benzylamino 2-hydroxy- isopropyl 143.8 152.6
ethylamino 3-hydroxypropylamino benzylamino aminomethyl isopropyl
123.5 138.7 amino (R)-1-(hydroxymethyl) benzylamino isopropyl 0.45
1.4 propylamino ROSCOVITINE (R)-1-(hydroxymethyl) benzylamino
fluoro isopropyl 0.38 1.2 propylamino (R)-1-(hydroxymethyl)
benzylamino bromo isopropyl 10.38 11.2 propylamino
(R)-1-(hydroxymethyl) benzylamino mercapto isopropyl 41.11 43.8
propylamino (R)-1-(hydroxylethyl) benzylamino hydroxy isopropyl
10.15 10.94 propylamino (R)-1-(hydroxylethyl) benzylamino amino
isopropyl 20.17 20.85 propylamino (R)-1-(hydroxymethyl)-
benzylamino 2-hydroxy- isopropyl 151.93 164.95 propylamino
ethylamino (R)-1-(hydroxymethyl)- benzylamino aminomethyl-
isopropyl 181.05 193.28 propylamino amino (R)-1-(hydroxymethyl)
3-chloroanilino isopropyl 4 nM 11.2 nM isobutylamino PURVALANOL A
(R)-1-(hydroxymethyl) 3-chloroanilino fluoro isopropyl 3.8 nM 11 nM
isobutylamino (R)-1-(hydroxymethyl) 3-chloroanilino bromo isopropyl
43.8 51 nM isobutylamino nM (R)-1-(hydroxymethyl) 3-chloroanilino
mercapto isopropyl 83.7 105.6 isobutylamino nM nM
(R)-1-(hydroxymethyl) 3-chloroanilino hydroxy isopropyl 72.1 89.5
nM isobutylamino nM (R)-1-(hydroxymethyl) 3-chloroanilino amino
isopropyl 91.5 92.8 nM isobutylamino nM (R)-1-(hydroxymethyl)-
3-chloroanilino 2-hydroxy- isopropyl 1529.6 1726.8 isobutylamino
ethylamino nM nM (R)-1-(hydroxymethyl)- 3-chloroanilino
aminomethyl- isopropyl 2521.3 3287.2 isobutylamino amino nM nM
[0241] Table 4 shows the results of inhibitory activity of novel
compounds against CDC2 and I.kappa.B-.alpha. in comparison with the
data on the prototype compounds. Most of the
2,6,8,9-tetrasubstituted purine derivatives showed marked
inhibitory activity in in vitro kinase assays. Modification of
2,6,8-trisubstituted purines by small substituent in R8 position
led usually to increase in cdk inhibitory activity of the tested
compound.
EXAMPLE 13
Antimitotic activities of CDK inhibitors
[0242] Metaphase-arrested Xenopus egg extracts were prepared as
described previously by Blow "J.Cell Biol." 122:993 (1993) and
stored in liquid nitrogen. Demembranated Xenopus sperm nuclei were
prepared as described by Blow & Laskey "Cell" 47:577 (1986).
After thawing, extracts were supplemented with 25 mM
phosphocreatine, 5 .mu.g/ml creatine phosphokinase, 250 .mu.g/ml
cycloheximide, [.alpha.-.sup.32P]dATP (for DNA synthesis assays).
Demembranated sperm nuclei were added to a final sperm
concentration of 3 ng/.mu.l DNA extract and CDK inhibitor tested
was then added at different concentrations. M-phase promoting
factor inhibition by different CDK inhibitors was monitored 1.5 h
after addition by assessing the amount of sperm nuclei that had
been assembled into interphase nuclei, possessing a complete
phase-dense nuclear envelope. DNA synthesis was assessed by
releasing extract into interphase by the addition of 0.3 mM
CaCl.sub.2 and measuring the total amount of [.alpha.-.sup.32P]dATP
incorporation after 3 h by TCA co-precipitation.
[0243] At concentrations of CDK inhibitors (see Table 5) ranging
from 0.1-2 .mu.M, chromosomes remained highly condensed and no
nuclear envelope was visible. At 4-6 .mu.M and higher
concentrations, interphase nuclei appeared with partially
decondensed chromatin and an intact nuclear envelope. Replication
was significantly inhibited at 1-5 .mu.M CDK inhibitors tested. For
the inhibition effect to become detectable, the first 15-min
incubation of the interphase extract is probably sufficient.
5TABLE 5 Antimitotic Activities of 2,6,8,9-Tetrasubstituted Purine
Derivatives Inhibition of MPF Inhibition activity of DNA
SUBSTITUENT IC.sub.50 synthesis C2 N6 C8 N9 (.mu.M) IC.sub.50
(.mu.M) hydroxyethylamino benzylamino -- methyl 12 15 OLOMOUCINE
hydroxypropylamino benzylamino fluoro isopropyl 2.6 3.2
(R)-1-(hydroxyethyl) benzylamino fluoro isopropyl 1.5 2.4
propylamino (R)-1-(hydroxyethyl) 3-chloroanilino fluoro isopropyl
0.8 1.5 propylamino (R)-1-(hydroxyethyl) 3-chloro-4-carboxy- fluoro
isopropyl 0.1 0.25 propylamino anilino (R)-1-(hydroxymethyl)
3-chloroanilino amino isopropyl 3.12 3.3 propylamino
(R)-1-(hydroxymethyl) 3-chloroanilino hydroxy isopropyl 4.03 4.2
propylamino 2-hydroxyethylamino 3-chloroanilino 2- isopropyl 41.7
63.5 hydroxy ethyl- amino (R)-1-(hydroxymethyl)- 3-chloroanilino
bromo isopropyl 31.6 52.8 propylamino
EXAMPLE 14
In vitro Cytotoxic Activity of Novel Compounds
[0244] One of the parameters used, as the basis for colorimetric
assays, is the metabolic activity of viable cells. For example, a
microtiter assay, which uses the tetrazolium salt MTT, is now
widely used to quantitate cell proliferation and cytotoxicity. For
instance, this assay is used in drug screening programs and in
chemosensitivity testing. Because only metabolically active cells
cleave tetrazolium salts, these assays detect viable cells
exclusively. In the case of MTT assay, yellow soluble tetrazolium
salt is reduced to coloured water-insoluble formazan salt. After it
is solubilized, the formazan formed can easily and rapidly be
quantified in a conventional ELISA plate reader at 570 nm (maximum
absorbance). The quantity of reduced formazan corresponds to number
of vital cells in the culture.
[0245] Human T-lymphoblastic leukemia cell line CEM; promyelocytic
HL-60 and monocytic U937 leukemias; breast carcinoma cell lines
MCF-7, BT549, MDA-MB-231; glioblastoma U87MG cells; cervical
carcinoma cells HELA; sarcoma cells U20S and Saos2; hepatocellular
carcinoma HepG2; mouse fibroblasts NIH3T3; mouse immortalized bone
marrow macrophages B2.4 and B10A.4; P388D1 and L1210 leukemia; B16
and B16F10 melanomas were used for routine screening of compounds.
The cells were maintained in Nunc/Corning 80 cm2 plastic tissue
culture flasks and cultured in cell culture medium (DMEM with 5 g/l
glucose, 2 mM glutamine, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, 10% fetal calf serum and sodium bicarbonate).
[0246] The cell suspensions that were prepared and diluted
according to the particular cell type and the expected target cell
density (2.500-30.000 cells per well based on cell growth
characteristics) were added by pipette (80 .mu.l) into 96/well
microtiter plates. Inoculates were allowed a pre-incubation period
of 24 hours at 37.degree. C. and 5% CO.sub.2 for stabilisation.
Four-fold dilutions of the intended test concentration were added
at time zero in 20 .mu.l aliquots to the microtiter plate wells.
Usually, test compound was evaluated at six 4-fold dilution. In
routine testing, the highest well concentration was 266.7 .mu.M,
but it can be the matter of change dependent on the agent. All drug
concentrations were examined in duplicates. Incubations of cells
with the test compounds lasted for 72 hours at 37.degree. C., in 5%
CO.sub.2 atmosphere and 100% humidity. At the end of incubation
period, the cells were assayed by using the MTT. Ten microliters of
the MTT stock solution were pipetted into each well and incubated
further for 1-4 hours. After this incubation period, formazan was
solubilized by addition of 100 .mu.l/well of 10% SDS in water
(pH=5.5) followed by further incubation at 37.degree. C. overnight.
The optical density (OD) was measured at 540 nm with the Labsystem
iEMS Reader MF (UK). The tumour cell survival (TCS) was calculated
using the following equitation: TCS=(OD.sub.drug exposed well/mean
OD.sub.control wells).times.100%. The TCS.sub.50 value, the drug
concentration lethal to 50% of the tumour cells, was calculated
from the obtained dose response curves.
[0247] Cytoxicity of novel compounds was tested on panel of cell
lines of different histogenetic and species origin (Table 6). We
show here that equal activities were found in all tumour cell lines
tested, however, the non-malignant cells, e.g. NIH3T3 fibroblasts
and normal human lymphocytes, were resistant to synthetic CDK
inhibitors induced cytotoxicity. As demonstrated in Table 6,
IC.sub.50 for NIH3T3 fibroblasts and normal human lymphocytes was
always higher than 250 .mu.M. Effective novel derivatives killed
tumour cells in concentrations close to 1-5 .mu.M. Notably, the
identical effectiveness of purine derivatives was also found in
cell lines bearing various mutations or deletions in cell cycle
associated proteins, e.g. HL-60, BT549, Hela, U20S, MDA-MB231, and
Saos2 (Table 6). It indicates that these substances should be
equally effective in tumours with various alterations of tumour
suppresser genes, namely p53, Rb, etc. Importantly, this
observation distinguishes novel compounds from flavopiridol and
related compounds, as their biological activity is dependent on p53
status.
6TABLE 6 Cytotoxicity of Novel Compounds for Different Cancer Cells
CEM B16 SUBSTITUENT IC.sub.50 IC.sub.50 C2 N6 C8 N9 (.mu.M) (.mu.M)
2-hydroxyethylamino benzylamino -- methyl 70 85.4 OLOMOUCINE
2-hydroxyethylamino benzylamino fluoro methyl 45.2 62.8
2-hydroxyethylamino benzylamino bromo methyl 165.2 172.8
2-hydroxyethylamino benzylamino mercapto methyl 92.4 85.7
2-hydroxyethylamino benzylamino hydroxy methyl 45.5 56.2
2-hydroxyethylamino benzylamino amino methyl 84.2 67.1
2-hydroxyethylamino benzylamino 2-hydroxy- methyl >166.7
>166.7 ethylamino 2-hydroxyethylamino benzylamino aminomethyl-
methyl 114.3 126.5 amino 3-hydroxypropylamino benzylamino --
isopropyl 4.1 7.2 BOHEMINE 3-hydroxypropylamino benzylamino fluoro
isopropyl 3.93 5.5 3-hydroxypropylamino benzylamino bromo isopropyl
116.9 76.4 3-hydroxypropylamino benzylamino methylthio isopropyl
68.4 60.6 3-hydroxypropylamino benzylamino hydroxy isopropyl 7.48
5.6 3-hydroxypropylamino benzylamino amino isopropyl 8.52 6.8
3-hydroxypropylamino benzylamino 2-hydroxy- isopropyl >166.7
>166.7 ethylamino 3-hydroxypropylamino benzylamino 2-aminoethyl
isopropyl 29.9 2.5 amino (R)-1-(hydroxymethyl)- benzylamino
isopropyl 3.45 4.4 propylamino ROSCOVITINE (R)-1-(hydroxymethyl)-
benzylamino fluoro isopropyl 3.38 3.2 propylamino
(R)-1-(hydroxymethyl)- -- bromo isopropyl 48.4 59.2 propylamino
benzylamino (R)-1-(hydroxymethyl)- benzylamino mercapto isopropyl
30.7 125.6 propylamino (R)-1-(hydroxymethyl)- benzylamino hydroxy
isopropyl 12.15 12.94 propylamino (R)-1-(hydroxymethyl)-
benzylamino amino isopropyl 12.17 12.85 propylamino
(R)-1-(hydroxymethyl)- benzylamino 2-hydroxy- isopropyl 140.2
124.95 propylamino ethylethoxy (R)-1-(hydroxymethyl)- benzylamino
1,2- isopropyl 128.2 147.5 propylamino dihydroxy- propylamino
(R)-1-(hydroxymethyl)- benzylamino 2-aminoethyl isopropyl 96.1
>166.7 propylamino amino (R)-1-(hydroxymethyl)- benzylamino
hydrazido isopropyl 58 61.5 propylamino (R)-1-(hydroxymethyl)-
3-chloroanilino isopropyl 2.4 3.5 isobutylamino PURVALANOL A
(R)-1-(hydroxymethyl)- 3-chloroanilino fluoro isopropyl 2.5 3.5
isobutylamino (R)-1-(hydroxymethyl)- 3-chloroanilino bromo
isopropyl 22.5 23.5 isobutylamino (R)-1-(hydroxymethyl)-
3-chloroanilino mercapto isopropyl 25.8 47.6 isobutylamino
(R)-1-(hydroxymethyl)- 3-chloroanilino hydroxy isopropyl 11.8 11.3
isobutylamino (R)-1-(hydroxymethyl)- 3-chloroanilino amino
isopropyl 11.5 11.8 isobutylamino (R)-1-(hydroxymethyl)-
3-chloroanilino 2- isopropyl 78 150 isobutylamino hydroxyethyl
amino (R)-1-(hydroxymethyl)- 3-chloroanilino 2- isopropyl 56 130
isobutylamino aminomethyl- amino
EXAMPLE 15
Novel Compounds Induce Apoptosis in Tumour Cells.
[0248] To analyse the mechanisms of induced cytotoxicity by novel
compounds, it is important to distinguish apoptosis from the other
major form of cell death, necrosis. First, at the tissue level,
apoptosis produces little or no inflammation, since the
neighbouring cells, especially macrophages, rather than being
released into the extracellular fluid, engulf shrunken portions of
the cell. In contract, in necrosis, cellular contents are released
into the extracellular fluid, and thus have an irritant affect on
the nearby cells, causing inflammation. Second, at the cellular
level, apoptotic cells exhibit shrinkage and blebbing of the
cytoplasm, preservation of structure of cellular organelles
including the mitochondria, condensation and margination of
chromatin, fragmentation of nuclei, and formation of apoptotic
bodies, thought not all of these are seen in all cell types. Third,
at the molecular level, a number of biochemical processes take an
important role in induction of apoptosis. However, majority of them
is not well understood, and they result in activation of proteases
and nucleases, which finally destruct key biological
macromolecules-proteins and DNA. For detection of apoptotic versus
necrotic mode of cell death, two independent methods were employed:
assessment of morphology by electron microscopy and analysis of DNA
fragmentation by flow-cytometry.
[0249] HL-60 cell line was cultured in 6-well culture plates with
or without 70 .mu.M concentration of novel derivatives at
37.degree. C. and 5% CO.sub.2 for 3-24 hours. Following the
incubation, cells were pelleted, washed in Hank's buffered salt
solution and processed as described below.
[0250] Cells were suspended in 2% glutaraldehyde/PBS, fixed
overnight at 4.degree. C., pelleted and embedded into 1% agar (Agar
Noble, Difco) thereafter. Agar block containing fixed cells was
epoxide polymerised, ultrathin sectioned, osmium tetraoxide
postfixed, uranium acetate contrasted and examined under electron
microscope.
[0251] Initial phase contrast microscopy examinations indicated
that treated HL-60 line exhibit typical morphological features of
apoptotic cells, and it was later confirmed by electron microscopy.
Typical morphological criteria of apoptosis were identified in
cells treated with tetrasubstituted purine derivatives: chromatin
condensation, nuclear fragmentation, cytoplasmatic blebbing, and
formation of apoptotic bodies.
EXAMPLE 16
Immunosuppressive activity
[0252] One of the most important parameters of specific cellular
immunity is proliferative response of lymphocytes to antigens or
polyclonal mitogens. Majority of normal mammalian peripheral
lymphocytes is resting cells. Antigens or nonspecific--polyclonal
mitogens have capacity to activate lymphoid cells and it is
accompanied with dramatic changes of intracellular metabolism
(mitochondrial activity, protein synthesis, nucleic acids
synthesis, formation of blastic cells and cellular proliferation).
Compounds with ability to selectively inhibit lymphocyte
proliferation are potent immunosuppressants. Variety of in vitro
assays were developed to measure proliferative response of
lymphocytes. The most commonly used is .sup.3H-thymidine
incorporation method.
[0253] During cell proliferation, the DNA has to be replicated
before the cell is divided into two daughter cells. This close
association between cell doublings and DNA synthesis is very
attractive for assessing cell proliferation. If labeled DNA
precursors are added to the cell culture, cells that are about to
divide incorporate the labeled nucleotide into their DNA.
Traditionally, those assays usually involve the use of radiolabeled
nucleosides, particularly tritiated thymidine ([.sup.3H]-TdR). The
amount of [.sup.3H]-TdR incorporated into the cellular DNA is
quantified by liquid scintillation counting.
[0254] Human heparinized peripheral blood was obtained from healthy
volunteers by cubital vein punction. The blood was diluted in PBS
(1:3) and mononuclear cells were separated by centrifugation in
Ficoll-Hypaque density gradient (Pharmacia, 1.077 g/ml) at 2200 g
for 30 minutes. Following centrifugation, lymphocytes were washed
in PBS and resuspended in cell culture medium (RMPI 1640, 2 mM
glutamine, 100 U/ml penicillin, 100 .mu.g/ml streptomycin, 10%
fetal calf serum and sodium bicarbonate).
[0255] The cells were diluted at target density of 1.100.000
cells/ml were added by pipet (180 .mu.l) into 96/well microtiter
plates. Four-fold dilutions of the intended test concentration were
added at time zero in 20 .mu.l aliquots to the microtiter plate
wells. Usually, test compound was evaluated at six 4-fold
dilutions. In routine testing, the highest well concentration was
266.7 .mu.M. All drug concentrations were examined in duplicates.
All wells with exception of unstimulated controls were activated
with 50 .mu.l of concanavalin A (25 .mu.g/ml). Incubations of cells
with test compounds lasted for 72 hours at 37.degree. C., in 5% CO2
atmosphere and 100% humidity. At the end of incubation period, the
cells were assayed by using the [.sup.3H]-TdR:
[0256] Cell cultures were incubated with 0.5 .mu.Ci (20 .mu.l of
stock solution 500 .mu.Ci/ml) per well for 6 hours at 37.degree. C.
and 5% CO.sub.2. The automated cell harvester was used to lyse
cells in water and adsorb the DNA onto glass-fiber filters in the
format of microtiter plate. The DNA incorporated [.sup.3H]-TdR was
retained on the filter while unincorporated material passes
through. The filters were dried at room temperature overnight,
sealed into a sample bag with 10-12 ml of scintillant. The amount
of [.sup.3H]-TdR present in each filter (in CCPM) was determined by
scintillation counting in a Betaplate liquid scintillation counter.
The effective dose of immunosuppressant (ED) was calculated using
the following equotation: ED=(CCPM.sub.drug exposed well/mean
CCPM.sub.controlwells).times.100%. The ED.sub.50 value, the drug
concentration inhibiting proliferation of 50% of lymphocytes, was
calculated from the obtained dose response curves.
[0257] To evaluate immunosuppressive activity of tetrasubstituted
adenines, their ability to inhibit polyclonal mitogen induced
proliferation of normal human lymphocytes was analyzed (Table 7).
Our data demonstrate that these compounds have only marginal
activity on .sup.3H-thymidine incorporation, nonetheless, they
efficiently inhibit proliferation of activated lymphocytes.
Effective immunosuppressive dose of tetrasubstituted derivatives
under in vitro conditions was close to 1-20 .mu.M.
7TABLE 7 Immunosuppressive effects of compounds on spontaneous and
mitogen activated proliferation of lymphocytes spont- mitogen
SUBSTITUENT aneous activated C2 N6 C8 N9 IC.sub.50 (.mu.M)
IC.sub.50 (.mu.M) hydroxyethylamino benzylamino -- methyl 245 72.1
OLOMOUCIN 2-hydroxyethylamino benzylamino bromo methyl 228 68.3
2-hydroxyethylamino benzylamino mercapto methyl 250 83.5
2-hydroxyethylamino benzylamino hydroxy methyl 201 48.6
2-hydroxyethylamino benzylamino amino methyl 170.3 26.7
2-hydroxyethylamino benzylamino 2-hydroxy- methyl 250 169.3
ethylamino 2-hydroxyethylamino benzylamino aminomethyl- methyl 250
113.5 amino 3-hydroxypropylamino benzylamino -- isopropyl 172 5.7
BOHEMIN 3-hydroxypropylamino benzylamino bromo isopropyl 178 5.9
3-hydroxypropylamino benzylamino mercapto isopropyl 197 18.4
3-hydroxypropylamino benzylamino hydroxy isopropyl 163 2.1
3-hydroxypropylamino benzylamino amino isopropyl 143 1.59
3-hydroxypropylamino benzylamino 2-hydroxy- isopropyl 181 29.3
ethylamino 3-hydroxypropylamino benzylamino aminomethyl- isopropyl
174 26.8 amino (R)-1-(hydroxymethyl)- benzylamino isopropyl 164 6.4
propylamino ROSCOVITIN (R)-1-(hydroxymethyl)- benzylamino bromo
isopropyl 168 17.1 propylamino (R)-1-(hydroxymethyl)- benzylamino
mercapto isopropyl 174 18.5 propylamino (R)-1-(hydroxymethyl)-
benzylamino hydroxy isopropyl 153 12.8 propylamino
(R)-1-(hydroxymethyl)- benzylamino amino isopropyl 149 13.3
propylamino (R)-1-(hydroxymethyl)- benzylamino 2-hydroxy- isopropyl
182 31.5 propylamino ethylamino (R)-1-(hydroxymethyl)- benzylamino
aminomethyl- isopropyl 177 25.8 propylamino amino
(R)-1-(hydroxymethyl)- 3-chloroanilino isopropyl 158 5.7
isobutylamino PURVALANOL A (R)-1-(hydroxymethyl)- 3-chloroanilino
bromo isopropyl 142 13.7 isobutylamino (R)-1-(hydroxymethyl)-
3-chloroanilino mercapto isopropyl 164 26.9 isobutylamino
(R)-1-(hydroxymethyl)- 3-chloroanilino hydroxy isopropyl 126 2.98
isobutylamino (R)1-(hydroxymethyl)- 3-chloroanilino amino isopropyl
103 3.1 isobutylamino (R)-1-(hydroxymethyl)- 3-chloroanilino
2-hydroxy- isopropyl 205 150 isobutylamino ethylamino
(R)-1-(hydroxymethyl)- 3-chloroanilino aminomethyl- isopropyl 230
138 isobutylamino amino
EXAMPLE 17
Antiviral Activity
[0258] The activity of the compounds against HIV-1 and HIV-2
induced cytopathicity was examined in human lymphocyte MT-4 cells.
The cells (300 000 cells/ml) were infected with 100 CCID.sub.50 (1
CCID.sub.50 is a virus quantity which causes cytopathicity effect
in 50% of the cells under the experimental conditions) of HIV-1 or
HIV-2 and added to 200 .mu.l wells of a microtiter plate containing
different dilutions of the tested compounds. The infected cell
cultures were incubated at 37.degree. C. for 5 days in a humidified
CO.sub.2 incubator. The cytopathicity of the virus was examined by
determination of MT-4 cell viability by trypan blue dye staining.
The results are summarised in Table 8 with comparison on the
prototype compounds.
[0259] Table 8 also shows the results of activity testing of novel
compounds against MSV-induced transformation in murine embryo
fibroblast C3H/3T3 cells. The cells were seeded in 1 ml wells of
48-well plates and exposed to 80 PFU (plague forming units) for
60-90 min. The virus was subsequently removed and culture medium
containing appropriate concentrations of the tested compounds was
added (1 ml per well). At day 6-post infection, MSV-induced
transformation of the cell culture was examined
microscopically.
8TABLE 8 Antiretroviral Activity of Purine Compounds, wherein R9 is
either PMP (2-phosphonomethoxypropyl)grou- p or PME
(2-phosphonomethoxyethyl)group. (.mu.g/ml), (R2 = NH.sub.2). HIV-1
HIV-2 R6 R8 MSV MT-4 CEM MT-4 CEM PME-derivatives amino 0.6 2.67
6.9 ND ND amino fluoro 0.57 3.12 7.2 ND ND amino mercapto 1.23 5.87
10.8 ND ND amino hydroxy 0.42 1.95 5.2 ND ND amino amino 0.58 1.98
4.8 ND ND amino 2-hydroxyethyl- 1.54 8.33 10.8 ND ND amino amino
aminomethyl- 132 7.54 9.3 ND ND amino cyclohexylamino 0.26 5.7
>20 4.8 >20 cyclohexylamino fluoro 0.24 6.3 >20 4.6 >20
cyclohexylamino mercapto 0.35 8.9 >20 8.5 >20 cyclohexylamino
hydroxy 0.21 4.3 >20 2.1 >20 cyclohexylamino amino 0.19 3.5
>20 1.6 >20 cyclohexylamino 2-hydroxyethyl- 0.45 12.7 >20
12.7 >20 amino cyclohexylamino aminomethyl- 0.43 10.8 >20 9.5
>20 amino benzylamino 1.5 50 >20 49 >20 benzylamino fluoro
1.3 47 >20 45 >20 benzylamino mercapto 1.8 56 >20 57
>20 benzylamino hydroxy 0.9 45 >20 32 >20 benzylamino
amino 0.8 48 >20 31 >20 benzylamino 2-hydroxyethyl- 1.7 67
>20 48 >20 amino benzylamino aminomethyl- 1.4- 55 >20 52
>20 amino PMP-derivatives amino 0.07 0.29 10 0.24 10 amino
fluoro 0.06 3.12 3.54 ND amino mercapto 0.15 4.18 ND 4.15 ND amino
hydroxy 0.05 0.25 ND 0.21 ND amino amino 0.06 0.19 ND 0.20 ND amino
2-hydroxyethyl- 0.35 5.16 ND 4.87 ND amino amino aminomethyl- 0.42
4.58 ND 4.65 ND amino cyclohexylamino 3.78 3.4 4.5 5.8 8.5
cyclohexylamino fluoro 2.54 3.2 4.1 4.6 8.3 cyclohexylamino
mercapto 6.32 10.1 >20 11.2 >20 cyclohexylamino hydroxy 1.37
2.1 5.2 3.2 7.8 cyclohexylamino amino 1.25 1.8 4.7 2.8 8.1
cyclohexylamino 2-hydroxyethyl- 5.42 12.7 >20 25.1 >20 amino
cyclohexylamino aminomethyl- 4.98 9.8 >20 18.6 >20 amino
benzylamino 0.3 10.3 11.6 8.3 12.5 benzylamino fluoro 0.27 8.5 12.5
6.4 11.1 benzylamino mercapto 1.35 21.8 >20 >20 >20
benzylamino hydroxy 0.21 3.7 10.3 4.3 11.1 benzylamino amino 0.18
2.9 10.7 3.8 11.9 benzylamino 2-hydroxyethyl- 2.17 19.5 >20
>20 >20 amino benzylamino aminomethyl- 1.89 16.8 >20
>20 >20 amino
[0260] Most of the PMP (9-(2-phosphonomethoxypropyl) derivative)
and PME (9-(2phosphonomethoxyethyl) derivative) compounds of the
formula I showed marked antiHIV activity in vitro. HIV-1 and HIV-2
did not differ in their sensitivity to the test compounds. (R)-PMP
compounds were markedly inhibitory to retroviruses at 2-3 .mu.g/ml
and not toxic to the cells at 100 .mu.g/ml. Its selectivity index
(ratio cytotoxic dose/antivirally active dose) proved superior over
that of the prototype compound PME. The (S)-enantiomer of PME was
devoid of marked antiretroviral activity. (R)-PMPD were exquisitely
inhibitory to retrovirus replication (EC.sub.50 0.01-0.1 .mu.g/ml)
and not toxic to the cells at 100 .mu.g/ml. It proved superior over
PMEA and other prototype compounds in terms of both antiviral
activity and lack of toxicity. Its selectivity index was higher
than 2000 for HIV-1 and HIV-2.
EXAMPLE 18
Dry Capsules
[0261] 5000 capsules, each of which contain 0.25 g of one of the
compounds of the formula I mentioned in the preceding Examples as
active ingredient, are prepared as follows:
[0262] Composition:
[0263] Active ingredient 1250 g
[0264] Talc 180 g
[0265] Wheat starch 120 g
[0266] Magnesium stearate 80 g
[0267] Lactose 20 g
[0268] Preparation process: The powdered substances mentioned are
pressed through a sieve of mesh width 0.6 mm. Portions of 0.33 g of
the mixture are transferred to gelatine capsules with the aid of a
capsule-filling machine.
EXAMPLE 19
Soft Capsules
[0269] 5000 soft gelatine capsules, each of which contain 0.05 g of
one of the compounds of the formula I mentioned in the preceding
Examples as active ingredient, are prepared as follows:
[0270] Composition:
[0271] Active ingredient 250 g
[0272] Lauroglycol 2 liters
[0273] Preparation process: The powdered active ingredient is
suspended in Lauroglycol.RTM. (propylene glycol laurate, Gattefosse
S. A., Saint Priest, France) and ground in a wet-pulveriser to a
particle size of about 1 to 3 .mu.m. Portions of in each case 0.419
g of the mixture are then transferred to soft gelatine capsules by
means of a capsule-filling machine.
EXAMPLE 20
Soft Capsules
[0274] 5000 soft gelatine capsules, each of which contain 0.05 g of
one of the compounds of the formula I mentioned in the preceding or
following Examples as active ingredient, are prepared as
follows:
[0275] Composition
[0276] Active ingredient 250 g
[0277] PEG 400 1 liter
[0278] Tween 80 1 liter
[0279] Preparation process: The powdered active ingredient is
suspended in PEG 400 (polyethylene glycol of Mr between 380 and
about 420, Sigma, Fluka, Aldrich, USA) and Tween.RTM. 80
(polyoxyethylene sorbitan monolaurate, Atlas Chem. Inc., USA,
supplied by Sigma, Fluka, Aldrich) and ground in a wet-pulveriser
to a particle size of about 1 to 3 mm. Portions of in each case
0.43 g. of the mixture are then transferred to soft gelatine
capsules by means of a capsule-filling machine.
[0280] While a specific embodiment of the invention has been shown
and described in detail to illustrate the application of the
principles of the invention, it will be understood that the
invention may be embodied otherwise without departing from such
principles.
* * * * *