U.S. patent application number 10/793941 was filed with the patent office on 2004-11-25 for purine derivatives having, in particular, anti-proliferative properties and their biological uses.
This patent application is currently assigned to Centre National De La Recherche Scientifique (C.N.R.S.). Invention is credited to Bisagni, Emile, Legraverend, Michel, Meijer, Laurent, Strnad, Miroslav.
Application Number | 20040235868 10/793941 |
Document ID | / |
Family ID | 9485066 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040235868 |
Kind Code |
A1 |
Meijer, Laurent ; et
al. |
November 25, 2004 |
Purine derivatives having, in particular, anti-proliferative
properties and their biological uses
Abstract
This invention provides 2-, 6, and 9-substituted purine
derivatives, particularly 2(1-R
hydroxymethylpropylamino)-6-benzylamino-9-isopropyl purine, having,
in particular, antiproliferative properties, and suitable for use
as pharmaceutical compositions and herbicidal compositions. Also
provided are pharmaceutical compositions and herbicidal
compositions comprising the 2-, 6, and 9-substituted purine
derivatives, and methods of treatment using the 2-, 6, and
9-substituted purine derivatives.
Inventors: |
Meijer, Laurent; (Roscoff,
FR) ; Bisagni, Emile; (Orsay, FR) ;
Legraverend, Michel; (Antony, FR) ; Strnad,
Miroslav; (Olomouc, CZ) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Centre National De La Recherche
Scientifique (C.N.R.S.)
Institute of Experimental Botany
Academy of Science of the Czech Republic
|
Family ID: |
9485066 |
Appl. No.: |
10/793941 |
Filed: |
March 8, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10793941 |
Mar 8, 2004 |
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10372670 |
Feb 25, 2003 |
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10372670 |
Feb 25, 2003 |
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09986329 |
Nov 8, 2001 |
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09986329 |
Nov 8, 2001 |
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09077470 |
Aug 31, 1998 |
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6316456 |
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09077470 |
Aug 31, 1998 |
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PCT/FR96/01905 |
Nov 29, 1996 |
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Current U.S.
Class: |
514/263.4 ;
544/277 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 25/28 20180101; A61P 31/00 20180101; A61P 35/00 20180101; C07D
473/16 20130101; A61P 17/06 20180101; A61P 33/00 20180101; A61P
31/10 20180101 |
Class at
Publication: |
514/263.4 ;
544/277 |
International
Class: |
A61K 031/52; C07D
473/14 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 1, 1995 |
FR |
95/14237 |
Claims
1. A compound of formula I: 3wherein: R.sub.2 is R--NH, wherein R
is saturated or unsaturated alkyl with a straight or branched
chain; R.sub.6 is R--NH wherein R is aryl; R.sub.9 is saturated or
unsaturated alkyl with a straight or branched chain; each
optionally substituted by one or more hydroxy, halogen, amino or
alkyl groups; with the proviso that when R.sub.2 is
hydroxyethylamino, R.sub.6 is not benzylamino or
3-hydroxybenzylamino.
2-28. (Cancelled)
Description
[0001] This application is a continuation of U.S. Ser. No.
09/077,470 filed Aug. 31, 1998, now U.S. Pat. No. 6,316,456, which
is a national stage application of International Application No.
PCT/FR96/01905, filed on Nov. 29, 1996, which claims the priority
benefit of French Application No. 95/14237, filed on Dec. 1, 1995.
The contents of these prior applications are hereby incorporated by
reference.
[0002] The invention relates to new purine derivatives having
anti-proliferative properties and to their biological uses.
[0003] It relates in particular to purine derivatives having an
inhibiting effect with respect to cyclin-dependent kinase proteins,
or cdk for short.
[0004] The study of the molecular mechanisms which control the cell
cycle has demonstrated the regulatory role of cdk These proteins
are made up of at least two sub-units, a catalytic sub-unit (of
which cdc2 is the prototype) and a regulatory sub-unit (cyclin).
Eight cdk have been described, cdk1 (=cdc2), cdk2-cdk8.
[0005] With the exception of cdk3, for which no associated cyclin
is known, the cdk are regulated by transitory combination with a
member of the cyclin family: cyclin A (cdc2, cdk2), cyclin B1-B3
(cdc2), cyclin C (cdk8), cyclin D1-D3 (cdk2-cdk4-cdk5-cdk6), cyclin
E (cdk2), cyclin H (cdk7),
[0006] Each of these complexes is involved in a phase of the cell
cycle. The activity of the cdk is regulated by post-translational
modification, by transitory combinations with other proteins and by
modification of their intracellular location. Regulators of the cdk
include activators (cyclins, cdk7/cyclin H, cdc25 phosphatases),
the sub-units p9.sup.CKS and p15.sup.cdk-BP and the inhibitory
proteins (p16.sup.INK4A, p15.sup.INK4B, p21.sup.Cip1, p18,
p27.sup.Kip1).
[0007] In parallel with purely fundamental research into the
regulatory mechanisms of cell division, the importance of
dysregulations of cyclin-dependent kinases in the development of
human tumours has been demonstrated by several studies.
Over-expression of cyclins D and E in several tumours,
over-expression of cdc2, the oncogenic properties of cyclins D and
A, abnormal temporary expression of cyclin-dependent kinases and
major dysregulation of protein inhibitors (mutations, deletions)
have thus been found.
[0008] The regulators of the cell division cycle are the subject of
a large number of clinical studies (use as indicating markers for
treatment).
[0009] These results greatly encourage efforts for detailed
comprehension of the regulatory mechanisms of the cell cycle. They
also lead to the search, by screening, for molecules which inhibit
cyclin-dependent kinases.
[0010] Several kinase inhibitors have been described, such as
butyrolactone, flavopinidol and
2-(2-hydroxyethylamino)-6-benzylamino-9-m- ethylpurine, called
olomoucine. Works relating to olomoucine are reported by Vesely et
al. in the article carrying the reference (1) in the list of
bibliographic references given at the end of the description.
[0011] This cdc2 inhibitor of high efficacy (its IC.sub.50 is 7
.mu.M) and high selectivity (more than 35 kinases have been tested)
corresponds to the formula: 1
[0012] The works of the inventors in this field have led them to
develop new molecules of particular interest which inhibit cdc2 in
low doses, while maintaining the enzymatic specificity of
olomoucine.
[0013] The object of the invention is therefore to provide new
purine derivatives having, in particular, anti-proliferative
properties.
[0014] The invention also relates to a process for obtaining these
derivatives by synthesis, which enables them to be prepared on an
industrial scale.
[0015] It also relates to their therapeutic use and their use as a
herbicide.
[0016] The purine derivatives of the invention are characterized in
that they correspond to the formula I 2
[0017] in which
[0018] R2, R6 and R9, which are identical to or different from one
another, represent a halogen atom or an R--NH--, R--NH--NH--,
NH.sub.2--R'--NH-- or R--NH--R'--NH-- radical, in which R
represents a straight- or branched-chain, saturated or unsaturated
alkyl radical, an aryl or cycloalkyl radical or a heterocyclic ring
and R' represents a straight- or branched-chain, saturated or
unsaturated alkylene group or an arylene or cycloalkylene group, R
and R' each containing 1 to 8 carbon atoms and being substituted,
where appropriate, by one or more --OH, halogen, amino or alkyl
groups,
[0019] R2 can also represent a heterocyclic ring carrying, where
appropriate, a straight- or branched-chain, saturated or
unsaturated alkyl radical, an aryl or cycloaryl radical or a
heterocyclic ring, optionally substituted by one or more --OH,
halogen, amino or alkyl groups,
[0020] R9 can also represent a straight- or branched-chain,
saturated or unsaturated alkyl radical or an aryl or cycloalkyl
radical,
[0021] R2 and R9 can also represent a hydrogen atom, with the
exception of the derivatives in which the said substituents have,
respectively, the following meanings:
[0022] R6 and R9--a benzylamino and methyl group,
[0023] R2 and R6--a 2-hydroxyethylamino and benzylamino group,
[0024] R2, R6 and R9--an amino, benzylamino and methyl, or chloro,
amino and methyl, or chloro, benzylamino and methyl, or chloro,
3-hydroxybenzylamino and methyl, or chloro, 5-hydroxypentylamino
and methyl, or 2-hydroxyethylamino, benzylamino and isopropyl, or
2-hydroxyethylamino, amino and methyl, or 2-hydroxyethylamino,
isopentenyl and methyl, or 2-hydroxyethylamino, isopentenylamino
and methyl, or 2-hydroxyethylamino, benzylamino and methyl, or
2-hydroxyethylamino, benzylamino and 2-hydroxyethyl, or
2-hydroxyethylamino, benzylamino and isopropyl, or
2-hydroxyethylamino, (3-hydroxybenzyl)amino and methyl, or
2-hydroxyethylamino, (3-hydroxybenzyl)amino and isopropyl, or
2-hydroxyisobutylamino, 6-benzylamino and methyl, or
2-hydroxyethylamino, isopentenylamino and isopropyl, or
(2-hydroxyethyl)amino, (4-methoxybenzyl)amino and isopropylamino
group,
[0025] and the purine derivatives of the invention are furthermore
characterized in that they have an IC.sub.50 less than or equal to
about 5 .mu.M for cdc2/cyclin B.
[0026] The abovementioned derivatives which are excluded from the
invention are described in reference (1).
[0027] In general, the derivatives of the invention are kinase
protein inhibitors of great interest.
[0028] Preferably, the halogen atom is chosen from chlorine,
bromine or fluorine, the alkyl radical is chosen from the methyl,
ethyl, propyl, isopropyl, butyl and isobutyl, pentyl, hexyl and
heptyl radicals, the alkylene radical is chosen from the methylene,
ethylene, propylene, isopropylene, butylene, isobutylene, pentene
or isopentene radicals, the aryl radical is a benzyl group, the
cycloalkyl radical is a cyclohexyl group, the arylene radical is a
benzylene group, the cycloalkylene radical is a cyclohexylene group
and the heterocyclic ring is a nitrogen-containing and/or
oxygen-containing heterocyclic ring, such as an imidazole, an
oxadiazole, a pyridine, a pyridazine or a pyrimidine, or also a
pyrrolidine.
[0029] According to one embodiment of the invention, R2 is chosen
from the radicals which are capable of bonding in a cdk2/ATP
complex to a region of the bonding domain of ATP occupied by
ribose. These are advantageously radicals chosen from a chlorine
atom, an amino, methylamino, ethylamino, n-heptylamino,
aminoethylamino, aminopropylamino, dimethylaminoethylamino- ,
hydroxyethylamino, hydroxy-propylamino, hydroxyisobutylamino,
hydroxypentylamino, dimethylhydrazino or hydroxymethylpropylamino,
[(2R)-2-hydroxymethyl-pyrrolidin-1-yl], N-benzyl-aminoethanol,
(R,S)-amino-hexanol, (S)-amino-2-phenylethanol,
(R)-amino-2-phenylethanol- , (R)-amino-3-phenylpropanol,
(R,S)-amino-pentanol, (R)-amino-propanol, (S)-amino-propanol and
(R)-N-pyrrolidine-methanol radical.
[0030] Particularly preferred radicals contain a hydroxypropylamino
radical as the group R2.
[0031] According to another embodiment of the invention, R6 is
chosen from an amino, isopentenylamino, hydroxypentylamino,
4-hydroxy-3-methyl-trans-- 2-butenylamino, benzylamino,
hydroxybenzylamino, hydroxyethylbenzylamino, cyclohexylmethylamino,
isopentene, benzylamino or (3-iodo)-benzylamino group.
[0032] R6 preferably comprises a hydrophobic radical, such as
benzyl, hydroxybenzyl or isopentenyl.
[0033] Preferably, R2 is chosen from the group consisting of
[1-D,L-hydroxymethylpropylamino],
[(2R)-2-hydroxymethyl-pyrrolidin-1-yl] and
[(R)-N-pyrrolidine-methanol] and R6 is benzylamino.
[0034] According to yet another embodiment of the invention, the
substituent R9 is chosen from a hydrogen atom and a methyl,
isopropyl or hydroxyethyl radical.
[0035] R9 is advantageously a hydrophobic group, in particular the
isopropyl group.
[0036] Preferred purine derivatives of the invention are chosen
from the compounds in which R2, R6 and R9 are as indicated in the
following table 1:
1TABLE 1 IC.sub.50 .mu.M cdc2/cyclin R2 R6 R9 B
3-hydroxypropylamino benzylamino isopropyl 1 2-hydroxypropylamino
benzylamino isopropyl 0.9 1-D,L- benzylamino isopropyl 0.65
hydroxymethylpropylamino aminoethylamino benzylamino isopropyl 1
2-hydroxypropylamino isopentenyl isopropyl 1.2 2-hydroxypropylamino
cyclohexylmethylamino methyl 4 chloro isopentenylamino isopropyl
2.5 (2R)-2-hydroxymethyl-pyrrolidin- benzylamino isopropyl-(9H)
0.45 1-yl N-benzylaminoethanol benzylamino isopropyl-(9H) 2.5
(R,S)-amino-hexanol benzylamino isopropyl-(9H) 2.5
(S)-amino-2-phenylethanol benzylamino isopropyl-(9H) 4.3
(R)-amino-2-phenylethanol benzylamino isopropyl-(9H) 1
(R)-amino-3-phenylpropanol benzylamino isopropyl-(9H) 2.7
(R,S)-amino-pentanol benzylamino isopropyl-(9H) 0.9
(R)-amino-propanol benzylamino isopropyl-(9H) 0.85
(S)-amino-propanol benzylamino isopropyl-(9H) 1
(R)-N-pyrrolidine-methanol (3-iodo)-benzylamino isopropyl-(9H) 0.45
.mu.M (R)-N-pyrrolidine-methanol benzylamino cyclopentyl-(9H)
0.7
[0037] The following derivatives are particularly preferred, that
is to say:
2-(1-D,L-hydroxymethylpropylamino)-6-benzylamino-9-isopropylpurine,
non-crystalline
6-benzylamino-2-[(2R)-2-hydroxymethyl-pyrrolidin-1-yl]-9--
isopropyl-(9H)-purine,
2-(R)-[6-benzylamino-9-isopropyl-(9H)-purin-2-yl]-a-
mino-2-phenylethanol,
2-(R,S)-[6-benzylamino-9-isopropyl-(9H)-purin-2-yl]--
amino-pentanol,
2-(R)-[6-benzylamino-9-isopropyl-(9H)-purin-2-yl]-amino-pr- opanol,
2-(S)-[6-benzylamino-9-isopropyl-(9H)-purin-2-yl]-amino-propanol,
2-(R)-(-)-[6-(3-iodo)-benzylamino-9-isopropyl-(9H)-purin-2-yl]-N-pyrrolid-
ine-methanol and
2-(R)-(-)-[6-benzylamino-9-cyclopentyl-(9H)-purin-2-yl]-N-
-pyrrolidine-methanol.
[0038] The invention also relates to the optical isomers and the
racemic mixtures and, where appropriate, the geometric isomers of
the derivatives defined above, in particular the R isomer of
(2-[6-benzylamino-9-isopropy-
l-(9H)-purin-2-yl]-amino-2-phenylethanol and of
2-[6-benzylamino-9-isoprop- yl-(9H)-purin-2-yl]-amino-propanol.
[0039] The derivatives defined above are obtained by the
conventional methods of organic synthesis. A starting purine
derivative of which the substitutions allow introduction of the
desired groups is used.
[0040] For example, using a 2-chloro-6-benzylamino derivative of
purine, it is possible to introduce an alkyl group in position 9 by
reaction with, for example, the corresponding alkyl halide.
[0041] Reaction with an aminoalcohol then allows introduction of an
alkylhydroxyalkylamino group in position 2, in place of the chloro
group.
[0042] According to an aspect of great interest, the derivatives of
the invention have inhibitory properties on kinases of high
selectivity. These inhibitory effects are reversible.
[0043] The cdk play a central role in the initiation, development
and achievement of the events of the cell cycle, and the inhibitory
molecules of cdk are capable of limiting undesirable cell
proliferation, such as cancer, psoriasis and growth of fungi and
parasites (animals, protists), and also of plants (herbicides), and
of intervening in the regulation of neurodegenerative diseases,
such as neuronal apoptosis and Alzheimer's disease.
[0044] The kinases which are more specifically sensitive to the
inhibitory effects of these derivative are the cdc2, the cdk2 and
the cdk5.
[0045] Their inhibition is obtained with very low doses of purine
derivatives.
[0046] An IC.sub.50 with respect to cdc2 of less than 50 .mu.M, and
even than that of olomoucine (7 .mu.M), which is regarded, however,
as a potent inhibitor, has thus been observed most generally.
[0047] The invention particularly relates to purine derivatives
having an IC.sub.50 which does not exceed 5 .mu.M, and especially
2-(1-D-L-hydroxymethylpropylamino)-6-benzylamino-9-isopropylpurine,
also called roscovitine below, the IC.sub.50 of which is 0.65
.mu.M, non-crystalline
6-benzylamino-2-[(2R)-2-hydroxymethyl-pyrrolidin-1-yl]-9--
isopropyl-(9H)-purine and
2-(R)-(-)-[6-(3-iodo)-benzylamino-9-isopropyl-(9-
H)-purin-2-yl]-N-pyrrolidine-methanol.
[0048] This derivative, which is an inhibitor of high efficacy and
high selectivity with respect to the cdk, cdc2, cdk2 and cdk5,
unexpectedly has in return effects on the kinases erk 1 and erk 2
similar to those of olomoucine. The selectivity is thus clearly
superior with respect to cyclin-dependent kinases. This advantage,
which is found with the other purine derivatives of the invention,
allows elimination of interferences with the transduction routes of
signals further upstream which involve the kinases erk 1 and erk 2
in several cell responses other than cell division.
[0049] The invention also relates to complexes of purine
derivatives with the cdk, and especially to the crystallized form
of the complex of cdk2 and roscovitine.
[0050] Studies carried out on the derivatives of the invention have
shown, in addition to their specific inhibitory properties on
kinases, cell effects and effects on apoptosis of great
interest.
[0051] At a very low concentration (micromolar for roscovitine and
a large number of derivatives), they are thus capable of inhibiting
prophase/metaphase transition, as shown by experiments carried out
on ovocytes of starfish and sea urchin embryos, which are reported
in the examples.
[0052] On acellular Xenopus extracts, they are capable of
inhibiting both the promoter factor of the M phase and DNA
synthesis.
[0053] These cell effects are advantageously obtained at very low
concentrations of derivatives.
[0054] It is known that various works relate to the relationships
which exist between the cell cycle and apoptosis. Various routes
lead to apoptosis of cells, some of which are dependent on kinases
and others of which, in contrast, do not seem to require these
enzymes. It has been demonstrated that apoptosis can be induced at
the G1 or G2 stage, and that following damage to the DNA, some
cells stop at the G1 stage and an apoptotic route dependent on p53
is thus induced.
[0055] In other situations, it seems that the cells stop at the
G2/M stage, in response to damage caused to the DNA, and activation
of an apoptotic p53-independent route is observed.
[0056] This route proves to be particularly important for treatment
of tumours in which a loss of active p53 is found.
[0057] The benefit of having available, with the derivatives of the
invention, means for stimulating a p53-independent apoptosis in
cells which have stopped at the G2 stage by damage to the DNA with
the aid of agents such as mitoxantrone or cis-platin is thus
estimated.
[0058] The cdc2 inhibitors of the invention can thus increase the
therapeutic effects of the anti-tumoral agents currently used.
[0059] As cdk5 inhibitors, the derivatives of the invention can
also play a role in reducing abnormal hyperphosphorylation of tau
observed during Alzheimer's disease.
[0060] To these various advantageous properties is added the
benefit of absence of cytotoxicity of the derivatives of the
invention.
[0061] The invention thus relates to the utilization of the
properties of these derivatives, in particular their antimitotic
and antineurodegenerative properties, for formulation of
pharmaceutical compositions.
[0062] The pharmaceutical compositions of the invention are
characterized in that they comprise an effective amount of at least
one purine derivative as described above, in combination with an
inert pharmaceutical vehicle.
[0063] The compositions of the invention are particularly suitable
as antimitotic medicaments, in particular for chemotherapy of
cancers, or also for treatment of psoriasis, parasitoses, such as
those caused by protists or fungi, or Alzheimer's disease, or
neuronal apoptosis.
[0064] These compositions comprise, where appropriate, active
principles of other medicaments. There may be mentioned, in
particular, their combination with antimitotic medicaments, such as
those based on taxol, cis-platin, agents for intercalation of DNA
and others.
[0065] The conditioning with respect to sale, in particular
labelling and instructions for use, and advantageously packaging,
are formulated as a function of the intended therapeutic use.
[0066] The pharmaceutical compositions of the invention can be
administered in various forms, more specifically by an oral or
injectable route.
[0067] For administration by the oral route, compressed tablets,
pills, tablets, capsules and drops are used in particular. These
compositions advantageously comprise 1 to 100 mg of active
principle per dose unit, preferably 10 to 40 mg.
[0068] Other forms of administration include injectable solutions
for the intravenous, subcutaneous or intramuscular route,
formulated from sterile or sterilizable solutions. They can also be
suspensions or emulsions.
[0069] These injectable forms comprise 1 to 50 mg of active
principle, preferably 10 to 30 mg, per dose unit.
[0070] By way of indication, the dosage which can be used in man
corresponds to the following doses: for example, 10 to 50 mg/day
are thus administered to the patient in one or more doses for
treatment of tumours or to treat psoriasis or parasitoses.
[0071] The invention also relates to herbicidal compositions
comprising at least one purine derivative as defined above,
optionally in combination with other phytopharmaceutical
agents.
[0072] The invention also relates to biological reagents, the
active principles of which consist of the purine derivatives
defined above.
[0073] These reagents can be used as references or standards in
studies of cell division.
[0074] Other characteristics and advantages of the invention are
described in the examples which follow with reference to FIGS. 1 to
8, in which
[0075] FIG. 1 shows the results of kinetics under linear conditions
from tests relating to the activity of p34.sup.cdc2/cyclin B at
various concentrations of roscovitine,
[0076] FIG. 2 shows the percentage breakdown of the germinal
vesicle of ovocytes of the starfish as a function of the
concentration of roscovitine,
[0077] FIGS. 3 and 4 show, respectively, the effects of roscovitine
on maturation of ovocytes of the starfish and dephosphorylation of
p34.sup.cdc2 tyrosine in vivo,
[0078] FIG. 4 shows the effects of roscovitine on the mitotic cycle
of sea urchin embryos,
[0079] FIG. 5 shows these embryos stopped at the late prophase
stage and
[0080] FIG. 6 shows the effects of roscovitine on the synthesis of
DNA in vitro and the MPF activity,
[0081] FIG. 7 shows the effects of roscovitine on the inhibition of
the growth of L1210 cells and the halting of their cell cycle at
G2/M, in FIG. 7A the growth of L1210 cells after exposure to
various concentrations of roscovitine (mean .+-. standard deviation
in relation to the growth of the untreated control cell) is shown,
and in FIG. 7B the means (.+-. standard deviation) of the
distribution over the cycle of cells which have first been cultured
for 48 hours in the presence or absence of 60 .mu.M roscovitine are
shown,
[0082] FIG. 8 shows the inhibitory effect of roscovitine on in vivo
phosphorylation of vimentine at sites specific to cdc2.
MATERIAL AND METHODS
[0083] Chemical products
[0084] Sodium orthovanadate, 1-methyladenine (1MeAde), EGTA, EDTA,
MOPS, .beta.-glycerophosphate, dithiothreitol (DTT), sodium
fluoride, nitrophenyl phosphate, leupeptin, aprotinin, soya trypsin
inhibitor, benzamidine, histone H1 (type III-S), basic myelin
protein, casein, protamine sulphate, isopropyl
.beta.-D-thiogalactopyranoside (IPTG), Sepharose 4B activated with
CNBr, LB medium, glutathione and glutathione-Sepharose beads; all
these products are such as those marketed by Sigma Chemicals.
[0085] The purine analogues are generally dissolved such that stock
solutions of 100 mM in DMSO are available. The final concentration
in DMSO in the reaction mixture is less than 1% (v/v).
[0086] [.gamma.-.sup.32P]-ATP is a product of Amersham.
[0087] The GST-retinoblastoma protein is expressed in bacteria and
purified over glutathione-Sepharose beads as described previously
in (1) and (2).
[0088] Buffers
[0089] Homogenization buffer:
[0090] 60 mM .beta.-glycerophosphate, 15 mM p-nitrophenyl
phosphate, 25 mM MOPS (pH 7.2), 15 mM EGTA, 15 mM MgCl.sub.2, 1 mM
DTT, 1 mM sodium vanadate, 1 mM NaF, 1 mM phenyl phosphate, 10
.mu.g leupeptin/ml, 10 .mu.g aprotinin/ml, 10 .mu.g soya trypsin
inhibitor/ml and 100 .mu.M benzamidine.
[0091] Buffer C: Composition of the homogenization buffer but with
5 mM EGTA, without NaF and without protease inhibitors.
[0092] Preparation of extracts of starfish ovocytes in phase M
[0093] To obtain preparations of ovocyte extracts on a large scale,
the gonads of mature Marthasterias glacialis are removed and are
incubated with 10 .mu.M 1-MeAde in natural sea-water filtered over
Millipore, until the eggs are laid. The ovocytes have thus all
entered phase M. They are separated off from the incubation medium
by centrifugation, frozen directly in liquid nitrogen and kept at
-80.degree. C. (see (1) and (3)).
[0094] The ovocytes in phase M are homogenized in the
homogenization buffer in an amount of 2 ml/g ovocytes.
[0095] After centrifugation for 45 minutes at 100,000 g, the
supernatant is recovered and used directly for purification of the
p34.sup.cdc2/cyclin B kinase by affinity chromatography over
p9.sup.CKShs1-Sepharose beads (see (1) and (4)).
[0096] Enzymes
[0097] The activities of the kinases are determined at 30.degree.
C. in buffer C by means of counter-indication. The blank values are
subtracted from the data and the activities are calculated in pmol
of phosphate incorporated in the protein acceptor for an incubation
of 10 minutes.
[0098] The controls are used with suitable dilutions in DMSO.
[0099] The phosphorylation of the substrate is determined, where
appropriate, by autoradiography after SDS-PAGE.
[0100] p34.sup.cdc2/cyclin B is purified from the phase M ovocytes
of the starfish by affinity chromatography over
p9.sup.CKShs1-Sepharose, where they are eluted with the aid of
p9.sup.CKShs1 as described above (see (2), (3), and (5)).
[0101] For the determination, 1 mg histone H1 (Sigma type III-S)/ml
in the presence of 15 .mu.M [.gamma.-.sup.32P]-ATP (3.000 Ci/mmol,
1 mCi/ml) in a final volume of 30 .mu.l is used (see (1) and
(6)).
[0102] After an incubation time of 10 minutes at 30.degree. C.,
aliquots of 25 .mu.l of supernatant are deposited on Whatman P81
phosphocellulose paper and, after 20 seconds, the filters are
washed 5 times (for at least 5 minutes each time) in a solution of
10 ml phosphoric acid per litre of water.
[0103] The damp filters are transferred to 6 ml plastic
scintillation ampoules, 5 ml SCS scintillation liquid (Amersham)
are then added and the radioactivity is measured in a Packard
counter.
[0104] The kinase activity is expressed in pmol of phosphate
incorporated into histone H1 for an incubation of 10 minutes or in
per cent of the maximum activity.
[0105] To carry out the kinetic experiments under linear
conditions, the test system up to the final point for the
p34.sup.cdc2 kinase is used, as described, but, on the basis of
preliminary tests, suitable unsaturated concentrations of substrate
are used.
[0106] The p34.sup.cdc2/cyclin B kinase is added to obtain a linear
activity with respect to the concentration of enzyme and to
time.
[0107] In the majority of cases, this requires a 3- to 10-fold
enzymatic dilution in buffer C.
[0108] The rate data are expressed in pmol incorporated into the
substrate per second per amount of enzyme added. The apparent
inhibition constants are determined by analysis by graph.
[0109] p33 .sup.cdc2/cyclin A and p33 .sup.cdk2/cyclin E are
reconstituted from extracts of sf9 insect cells infected with
various baculoviruses.
[0110] Cyclins A and E are fusion proteins of GST-cyclins and the
complexes are purified over glutathione-Sepharose beads.
[0111] The kinase activities are determined with 1 mg/ml-histone H1
(Sigma, type IIIS) in the presence of 15 .mu.M
[.gamma.-.sup.32p]-ATP for 10 minutes in a final volume of 30
.mu.l, as described for the p34.sup.cdc2/cyclin B kinase.
[0112] p33 .sup.cdk5/p25 is purified from the bovine brain (7), but
the Mono S chromatography stage is not used.
[0113] The active fractions recovered from the Superose 12 column
are combined and concentrated to a final concentration of about 25
.mu.g enzyme/ml.
[0114] The determination of the kinase is carried out with 1 mg/ml
histone H1 (Sigma, type IIIS) in the presence of 15 .mu.M
[.gamma.-.sup.32P]-ATP over 10 minutes in a final volume of 30
.mu.l, as described for p34.sup.cdc2/cyclin B.
[0115] 33 .sup.cd4/cyclin D1 is obtained from lysates of insect
cells. cdk4 is a GST-cdk4 construction product and the active
complex is purified over glutathione-Sepharose beads.
[0116] Its kinase activity is determined with a purified
GST-retinoblastoma protein in the presence of 15 .mu.M
[.gamma.-.sup.32P]-ATP in a final volume of 30 .mu.l.
[0117] After incubation for 15 minutes, Laemmli buffer (2.times.30
.mu.l) is added.
[0118] The phosphorylated substrate is resolved by 10% SDS-PAGE and
analysed by autoradiography by exposure to MP Hyperfilin for about
14 hours and densitometry.
[0119] p33 .sup.cdk6/cyclin D2 is obtained from lysates of insect
cells (8). For the tests, the procedure is as indicated above for
the p33 .sup.cdk4/cyclin D1 protein.
[0120] The MAP kinases: GST-erk1 (9) cloned from a human HepG2 bank
is expressed in bacteria, purified over glutathione-Sepharose beads
and tested with 1 mg basic myelin protein per ml in the presence of
15 .mu.M [.gamma.-.sup.32P]-ATP as described above for the
p34.sup.cdc2/cyclin B kinase.
[0121] The erk1 and erk2 proteins marked with the aid of histone
are activated in vitro by MAPKK, purified (affinity-Ni and Mono Q)
and tested as described above for 10 minutes in a final volume of
30 .mu.l.
[0122] The catalytic sub-unit of the cAMP-dependent kinase,
purified from the bovine heart, is tested with 1 mg histone H1 per
ml in the presence of 15 .mu.M [.gamma.-.sup.32P]-ATP as described
above for p34.sup.cdc2/cyclin B.
[0123] The cGMP-dependent kinase (10), purified to homogeneity from
the smooth muscle of the trachea of bovine origin, is tested with 1
mg histone H1 per ml in the presence of 15 .mu.M
[.gamma.-.sup.32P]-ATP as described above for p34.sup.cdc2/cyclin
B.
[0124] The casein 2 kinase is isolated from cytosol of the liver of
the rat (11) and tested with 1 mg casein per ml and 15 .mu.M
[.gamma.-.sup.32P]-ATP. The substrate is deposited on Whatman 3MM
filters and washed with 10% TCA (w/v).
[0125] The short-chain myosin kinase purified from chicken gizzards
(12) is tested in the presence of 100 nM calmodulin, 100 .mu.M
CaCl.sub.2, 50 mM HEPES, 5 mM MgCl.sub.2, 1 mM DTT and 0.1 mg
BSA/ml at pH 7.5 using a synthetic peptide on the basis of the
phosphorylation site of the light chain of myosin of the smooth
muscle (KKRPQRATSNVFAM, 50 .mu.M) and in the presence of 15 .mu.M
[.gamma.-.sup.32P]-ATP in a final volume of 50 .mu.l.
[0126] The incorporation of radioactive phosphate is checked on
phosphocellulose filters as described above.
[0127] The homologous ASK-.gamma. in the GSK-3 plant is expressed
as GST fusion protein in E. coli (13) and purified over
glutathione-Sepharose. The activity of the ASK-.gamma. kinase is
determined, over 10 minutes at 30.degree. C., with 5 .mu.gm basic
myelin protein in the presence of 15 .mu.M [.gamma.-.sup.32P]-ATP
in a final volume of 30 .mu.l. The basic phosphorylated myelin
protein is recovered on Whatman P81 phosphocellulose paper as
described above for p34.sup.cdc2/cyclin B.
[0128] The kinasic tyrosine domain of the insulin receptor (14) is
over-expressed in a baculovirus system and purified to homogeneity.
Its kinase activity is determined over 10 minutes at 30.degree. C.
with 5 .mu.g Raytide (Oncogene Sciences) in the presence of 15
.mu.M [.gamma.-.sup.32P]-ATP in a final volume of 30 .mu.l. The
phosphorylated Raytide product is recovered on Whatman P81
phosphocellulose paper as described above for p34.sup.cdc2/cyclin
B.
EXAMPLE 1
Synthesis of Roscovitine
[0129] The synthesis is carried out in 3 stages and comprises the
preparation 1) first of 6-benzylamino-2-chloropurine, then 2) of
6-benzylamino-2-chloro-9-isopropylpurine, and 3) of
6-benzylamino-2-R-(1-ethyl-2-hydroxyethylamino)-9-isopropylpurine.
[0130] 1) Synthesis of 6-benzylamino-2-chloropurine:
[0131] The procedure is as described by Hocart in Phytochemistry
1991, 30, 2477-2486.
[0132] 2) Synthesis of 6-benzylamino-2-chloro-9-isopropylpurine
(I):
[0133] A mixture of 6-benzylamino-2-chloropurine (3.7 g; 14.2
mmol), potassium carbonate (11 g; 80 mmol) and isopropyl bromide
(8.2 ml; 87 mmol) in 100 ml absolute DMSO is stirred at room
temperature for three days. The absence of
6-benzylamino-2-chloropurine is confirmed by thin layer
chromatography [CHCl.sub.3-MeOH (98:2)]. The DMSO and the excess
isopropyl bromide are removed by distillation in vacuo at below
50.degree. C. The residue is partitioned between water and ethyl
acetate. The organic phase is dried over Na.sub.2SO.sub.4 and
evaporated in vacuo.
[0134] Crystallization in MeOH gives 3.51 g (82%) of product; m.p.
181-182.degree. C.; UV (MeOH): .lambda..sub.max 273.5; IR (Nicolet
205, KBr, DRIFT cm 1713, 1626, 1572, 1537, 1497, 1471, 1456, 1425,
1398, 1355, 1314, 1292, 1255, 1228, 1202.
[0135] 3) Synthesis of
6-benzylamino-2-R-(1-ethyl-2-hydroxyethylamino)-9-i- sopropylpurine
(II), racemic derivative:
[0136] A sealed ampoule, in which a vacuum has been established,
containing 2.7 g (8.95 mmol) I and 17 ml (0.18 mol)
R(-)-2-amino-1-butanol (Fluka 90%, R:S>9:1) is heated in an oven
at 160-165.degree. C. for 3 h 30 min. The excess amine is
evaporated off at a temperature below 50.degree. C. and the product
II is purified over a chromatography column using increasing
amounts of MeOH in CHCl.sub.3, that is to say 0, then 2, and
3%.
[0137] Crystallization in ethyl acetate gives 2.2 g II (69%); m.p.
132-134.degree. C., [.alpha.]=+35.1 (c=0.29, CHCl.sub.3). Mass
spectrometry (Finnigam MAT 90, BE geometry 70 eV, temperature of
the source 250.degree. C., emission current 1 mA, acceleration
voltage 5 keV, direct entry, DIP temperature between
190-220.degree. C.]. HRMS was carried out by the method of
overlapping peaks using Ultramark 1600 F (PCR Inc.; Fl.: USA) as
the standard] 354.2167 (M.sup.+, C.sub.19H.sub.26N.sub.6O calc.
354.2168, 27%), 325 (7%), 324 (29%), 232 (100%), 295 (3%), 282
(7%), 281 (3%), 217 (6%), 185 (5%) 134 (3%), 91 (34%). FTIR
(Nicolet 205, KBr, DRIFT, cm.sup.-1): 1622, 1610, 1547, 1540, 1452,
1389, 1370, 1261, 1068.
EXAMPLE 2
Study of the Inhibitory Properties on Kinases of Roscovitine and
its Effects on the Cell Cycle
[0138] a) Study of the inhibitory properties on kinases.
[0139] The enzyme activities shown in the following table were
measured after addition of roscovitine or olomoucine at increasing
concentrations. These activities were measured with suitable
substrates (histone H1, basic myelin protein, casein etc. . . )
with 15 .mu.M ATP.
[0140] The IC.sub.50 were calculated from the dose/response curves
obtained. The symbol (--) indicates that no inhibitory effect was
observed. The highest concentration tested is given in
parentheses.
2 TABLE 2 IC.sub.50 (.mu.M) Enzyme Roscovitine Olomoucine
cdc2/cyclin B 0.65 7 cdc2/cyclin A 0.7 7 cdc2/cyclin E 0.7 7
cdc4/cyclin D1 >1000 >1000 cdk5/p35 0.16 3 cdk5/cyclin D3
>500 >250 GST-erk1 30 30 erk1 34 50 erk2 14 40 cAMP-dependent
PK >1000 >2000 cGMP-dependent PK -(1000) >2000 Light chain
myosin kinase 90 >1000 Casein 2 kinase -(1000) >2000
ASK-.gamma. (GSK-3 plant) 220 130 Insulin receptor tyrosine kinase
70 4000 c-src 250 -- v-abl >1000 --
[0141] Inhibition of cdc2, cdk2 and cdk5.
[0142] Examination of these results shows that roscovitine has an
activity which is 10 times higher than olomoucine with respect to
the targets cdc2 and cdk2 and 20 times higher with respect to
cdk5.
[0143] By comparison, its effect seems limited, as observed with
olomoucine, on the cdk4/cyclin D1 and cdk6/cyclin D2 kinases (the
IC.sub.50 are greater than 100 .mu.M). This absence of an effect
was confirmed with cdk4 originating from various sources. Working
under identical conditions, GST-p16.sup.INK4A inhibits cdk4/cyclin
D1.
[0144] Specificity of the inhibitory effect
[0145] As can be seen, the majority of the kinases are inhibited
weakly or not at all.
[0146] Although roscovitine has an efficacy at least 10 times
greater than that of olomoucine with respect to its cdk targets,
its inhibitory effect is very similar to that of olomoucine with
respect to erk1 and erk2. A 40-fold higher concentration of
roscovitine thus seems necessary to inhibit erk1 (20-fold for erk2)
in a manner similar to the inhibition of cdc2.
[0147] b) Effect on ATP
[0148] To study the action mechanism of roscovitine, kinetic
experiments were carried out in the presence of increasing
concentrations of roscovitine varying the levels of ATP (from 0.1
to 0.5 mM), the concentration of histone H1 being kept constant at
0.7 mg/ml.
[0149] The results are shown on FIG. 1.
[0150] These results demonstrate that roscovitine acts as a
competitive inhibitor for ATP. Taking account of the linearity of
the slopes as a function of the concentrations of roscovitine, it
is called a linear inhibitor. The apparent inhibition constant Ki
is 1.2 .mu.M.
[0151] Analysis of the structure of the co-crystal of roscovitine
and cdk2 confirms that, like olomoucine, roscovitine bonds to ATP
in the bonding pocket and that its purine ring is orientated in the
same way as that of olomoucine, that is to say in a totally
different manner to the purine ring of ATP.
[0152] c) Study of the effect on the synthesis of DNA and the MPF
activity.
[0153] The results of experiments carried out on several cell types
are described.
[0154] Effect on the maturation of ovocytes of the starfish and on
the dephosphorylation of p34.sup.cdc2 tyrosine in vivo.
[0155] The ovocytes of starfish, stopped at the prophase, are
treated for 15 minutes with increasing concentrations of
roscovitine before addition of the hormone 1-MeAde (1 .mu.M). After
30 minutes, the % breakdown of the germinal vesicle (GVBD) is
recorded. These values are shown on FIG. 2 as a function of the
concentration of roscovitine (in .mu.M). Roscovitine inhibits
breakdown of the nuclear envelope with an IC.sub.50 of 5 .mu.M (the
IC.sub.50 of olomoucine, working under the same conditions, is 30
.mu.M). These results are given on FIG. 2.
[0156] As already observed with olomoucine, roscovitine reduces,
but does not inhibit, the dephosphorylation of p34.sup.cdc2
tyrosine in vivo. The ovocytes are treated with 10 .mu.M
roscovitine for 15 minutes before addition of 1 .mu.M 1-MeAde at
time 0. The extracts are prepared at various times and introduced
on to a column of p9ckshs1-Sepharose beads.
[0157] The proteins bonded to the beads are resolved by SDS-PAGE
before carrying out a western blot with anti-PSTAIRE antibodies. A
photograph of the western blot is shown on FIG. 3. The
phosphorylated forms of p34.sup.cdc2 appear in the upper part and
the dephosphorylated forms appear in the lower part.
[0158] Roscovitine therefore inhibits not the activation of cdc2
but its activity. The dephosphorylation of p34.sup.cdc2 tyrosine is
catalysed by cdc25 and normally precedes the activation of the cdc2
kinase at the G2/M transition. Furthermore, the cdc2 kinase
phosphorylates and over-activates cdc25 phosphatase. Roscovitine
has thus been able to cause interruption at the cdc2 kinase level,
bringing about a reduction in the dephosphorylation.
[0159] Effects on the mitotic cycle of sea urchin embryos.
[0160] Roscovitine is added 60 minutes after fertilization. The
percentage of embryos which have divided is recorded 120 minutes
after fertilization. The results are given on FIG. 4.
[0161] It is found that it causes a dose-dependent halt at the late
prophase stage.
[0162] The IC.sub.50 is 10 .mu.M. (Even at 100 .mu.M, olomoucine
causes only a slowing down of the prephase/metaphase transition,
but does not stop the cells at the prophase).
[0163] A large nucleus is observed in the eggs stopped in this way
by roscovitine, as shown on FIG. 5.
[0164] This halt proves to be totally reversible. In fact, after
several washing with sea-water, the eggs enter the mitotic cycles
again and develop into normal pluteus larvae. These results are
obtained even at elevated concentrations of roscovitine of 100
.mu.M.
[0165] Effects on the synthesis of DNA in vitro and the MPF
activity in extracts of Xenopus eggs.
[0166] The tests are carried out in accordance with (15), working
as described in (1) for olomoucine.
[0167] The extracts of Xenopus stopped at the metaphase stage are
incubated with roscovitine and sperm chromatin.
[0168] With concentrations of roscovitine ranging from 0 to 5
.mu.M, the chromosomes remain highly condensed and no nuclear
envelope is visible. At a concentration of 10 .mu.M and above,
interphase nuclei appear, with the chromatin partly decondensed and
an intact nuclear envelope, showing that the MPF activity has been
inhibited (the IC.sub.50 is 5 .mu.M).
[0169] The inhibition of DNA synthesis has also been studied,
proceeding as described in (1) for olomoucine.
[0170] Roscovitine and sperm chromatin were thus added to an
extract of eggs which had been stopped at the metaphase stage.
[0171] The extract was then abandoned at the interphase stage by
addition of CaCl.sub.2 (15) and (16). The synthesis of total DNA
was measured 3 h later by incorporation of [.gamma.-.sup.32P]-dATP
into material which can be precipitated with TCA.
[0172] As shown in FIG. 6, the replication is inhibited by
roscovitine with an IC.sub.50 of 15 .mu.M.
[0173] The invention thus provides new purines having inhibitory
properties on cdc2/cyclin B of high specificity.
EXAMPLE 3
Biochemical Properties and Effects of Roscovitine on Mammalian
Cells
[0174] Method
[0175] In vitro screening of human tumoral cells
[0176] Sixty cell lines of human tumours comprising nine types of
tumour were cultured for 24 hours prior to continuous exposure for
48 hours to 0.01-100 .mu.M roscovitine. To estimate the
cytotoxicity, a sulphorhodamine B protein test was used.
[0177] Culture of the L1210 cell
[0178] L1210 cells sampled from cultures in exponential growth on
RPMI-1640 medium supplemented with 10% foetal calf serum,
penicillin and streptomycin were counted with the aid of a
haemocytometer, placed in an amount of 5.times.10.sup.4 cells per
millilitre in 96-well tissue culture plates in the presence or in
the absence of various concentrations of roscovitine or olomoucine,
and then incubated at 37.degree. C. under 5% CO.sub.2. To reverse
the effect of roscovitine, L1210 cells cultured for two days in the
presence or absence of roscovitine were washed in PBS to remove any
trace of active product, counted and placed again in fresh medium
containing no active product (roscovitine or olomoucine). The cell
growth was measured daily using the tetrazolium microculture test.
The analysis of the cell cycle was carried out on cells fixed in
ethanol, treated with 100 .mu.g/ml RNase and stained with propidium
iodide. Data acquisition was achieved with the aid of a Coulter
flow cytometer (Hialeah, Fla., USA) EPICS Elite (registered
trademark), and these data were analysed with the aid of Multicycle
software (Phoenix Flow Systems, San Diego, Calif., USA) (registered
trademark). All the tests were carried out with three repeats and
all the experiments were repeated at least twice.
[0179] In vivo phosphorylation of vimentine
[0180] To study the in vivo phosphorylation of vimentine by the
cdc2 kinase, the cells were either not treated or treated with 60
.mu.M roscovitine for 48 hours prior to exposure to 10 ng/ml
colcemide for an additional 2 hours. The cell extracts were then
placed on a 10% SDS-PAGE gel for migration, transferred by western
blots and incubated with 4A4 antibodies. These antibodies undergo a
cross-reaction with vimentine phosphorylated by cdc2, but react
neither with vimentine phosphorylated by other kinases
(cAMP-dependent kinase protein, C kinase protein,
Ca.sup.2+-calmodulin-dependent kinase protein), nor with
non-phosphorylated vimentine; the 4A4 antibodies specifically
recognize vimentine which is phosphorylated at its Ser-55 residue
by the cdc2 kinase when the cell enters mitosis.
[0181] Results
[0182] Roscovitine (0.01-100 .mu.M; exposure for 48 hours) was
tested on 60 human tumoral cell lines comprising nine types of
tumours (leukaemia, cancer of larger cells of the lungs, cancer of
the colon, cancer of the central nervous system, melanoma, cancer
of the ovaries, cancer of the kidney, cancer of the prostate and
cancer of the breast). All the cell lines had an equivalent
sensitivity to roscovitine. The mean IC.sub.50 value is 16 .mu.M
(whereas it is 60.3 .mu.M for olomoucine). No correlation was found
between the sensitivity of the cell lines to roscovitine and the
presence of wild or muted p53. The method of comparison analysis
showed that the effects of roscovitine and of flavopiridol are
comparable.
[0183] As regards the effects of roscovitine on the growth of the
cell line L1210, a very clear dose-dependent inhibition of the
growth was found, as shown on FIG. 7A, where the cell growth is
shown as a function of the concentration of roscovitine or
olomoucine. The curves are largely identical after two and three
days of culture, as found with the abovementioned tumoral cells.
Roscovitine is approximately four times more effective than
olomoucine in inhibiting cell growth (IC.sub.50 of 40 .mu.M for
roscovitine and 160 .mu.M for olomoucine). Although the majority of
cells are viable (96.+-.2% by Trypan blue exclusion) after a
treatment with 60 .mu.M roscovitine for 48 hours, they remain
irreversibly stopped, even after extensive washings. The cells
exposed to 120 .mu.M roscovitine die rapidly. The effects of
roscovitine on the distribution of the cell cycle were then studied
by flow cytometry. At 60 .mu.M roscovitine, the cells remain
stopped at G1 and accumulate in G2 as shown in FIG. 7B, where the
proportions (%) of each phase of the cell cycle observed (G1, S,
G2/M) in the presence or absence of roscovitine are shown.
[0184] 4A4 antibodies were used with the aim of identifying the
molecular target of roscovitine in vivo. The results are
illustrated in FIG. 8, where the total proteins extracted from
cells treated (+) or not treated (-) with roscovitine and then
resolved on SDS-PAGE before western transfer with the 4A4
antibodies are shown. The non-treated cells stop in the metaphase
and accumulate vimentine phosphorylated by cdc2. The cells treated
with roscovitine, on the other hand, do not have vimentine
phosphorylated by cdc2, which shows that cdc2 has in fact been
inhibited in vivo and that the cells were stopped before the
metaphase.
[0185] Roscovitine also helps to reduce the hyperphosphorylation of
tau observed during Alzheimer's disease: a cdk specific to the
brain (cdk5/p35) which phosphorylates certain sites of tau is
particularly sensitive to roscovitine.
Bibliographic References
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[0190] 5--Richardson et al. (1990) Genes and Developments 4,
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[0191] 6--Meijer et al. (1991) EMBO J. 10, 1545-1554.
[0192] 7--Lew et al. (1992) J. Biol. Chem. 267, 13383-13390.
[0193] 8--Meyerson et al. (1994) Molecul. Cellul. Biol. 14,
2077-2086.
[0194] 9--Charest et al. (1993) Molecul. Cellul. Biol. 13,
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[0195] 10--Landgraf et al. (1989) Eur. J. Biochem. 181,
643-650.
[0196] 11--Pinna, L. A. (1990) Biochim. Biophys. Acta 1054,
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[0197] 12--Craig et al (1987) J. Biol. Chem. 262, 3278-3284.
[0198] 13--Bianchi et al. (1994) Mol. Gen. Genet. 242, 337-345.
[0199] 14--Kallen et al. (1990) Biochem.-Biophys. Res. Comm. 168,
616-624.
[0200] 15.--Blow et al. (1986), Cell 47, 577-587
[0201] 16.--Blow (1993) J. Cell-Biol. 122, 993-1002
* * * * *