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DIM360 Ver 2.4 (29 Nov 2007) - 2999001/0
20070329
0951281
CORRECTED EUROPEAN PATENT SPECIFICATION
B9
B1
20080220
W1
de
Ansprüche DE
en
Claims DE
fr
Revendications DE
de
Ansprüche EN
en
Claims EN
fr
Revendications EN
de
Ansprüche FR
en
Claims FR
fr
Revendications FR
EP
97954382.4
19971211
19990709
20020415
en
en
en
RM960867
19961213
IT
20080220
200808
19991027
199943
20060628
200626
20050422
20080220
200808
A61K 31/00 20060101AFI20050315BHEP
A61K 31/122 20060101ALI20050315BHEP
de
2-CYCLOPENTEN-1ONE UND SEINE DERIVATE ALS INHIBITOREN VON NF-kappa B
en
2-CYCLOPENTEN-1-ONE AND ITS DERIVATIVES AS INHIBITORS OF THE NF-kappa B FACTOR
fr
2-CYCLOPENTENE-1-ONE ET SES DERIVES UTILISES COMME INHIBITEURS DU FACTEUR NF-kappa B
EP-A- 0 131 441
ROSSI A ET AL: "2-Cyclopenten-1-one, a new inducer of heat shock protein 70 with antiviral activity." J BIOL CHEM, DEC 13 1996, 271 (50) P32192-6, UNITED STATES, XP002069381
ROSSI A ET AL: "Inhibition of nuclear factor kappa B by prostaglandin A1: an effect associated with heat shock transcription factor activation." PROC NATL ACAD SCI U S A, JAN 21 1997, 94 (2) P746-50, UNITED STATES, XP002069382
SANTORO GABRIELLA, M.: "Antiviral activity of cyclopentenone prostanoids" TRENDS IN MICROBIOLOGY, vol. 5, no. 7, July 1997, pages 276-281, XP002069383
DATABASE WPI Week 8528 Derwent Publications Ltd., London, GB; AN 85-168496 XP002069392 & JP 60 097 926 A (TEIJIN LTD) , 31 May 1985
DATABASE WPI Week 8616 Derwent Publications Ltd., London, GB; AN 86-103244 XP002069393 & JP 61 047 437 A (TEIJIN LTD) , 7 March 1986
DATABASE WPI Week 8819 Derwent Publications Ltd., London, GB; AN 88-129559 XP002069394 & JP 63 072 672 A (TEIJIN LTD) , 2 April 1988
DATABASE WPI Week 8716 Derwent Publications Ltd., London, GB; AN 87-112980 XP002069395 & JP 62 059 258 A (TEIJIN LTD) , 14 March 1987
PICA, F. ET AL: "Efect of combined alphaIFN and prostaglandin A1 treatment on vesicular stomatitis virus replication and heat shock protein synthesis in epithelial cells" ANTIVIRAL RESEARCH, vol. 29, no. 2,3, 1995, pages 187-198, XP002069384
SANTORO M.G.: "Heat shock proteins and virus replication: Hsp70s as mediators of the antiviral effects of prostaglandins" EXPERIENTIA, 1994, 50/11-12 (1039-1047), SWITZERLAND, XP002069385
D'ONOFRIO C ET AL: "Antiproliferative activity of cyclopentenone prostaglandins in early HTLV-1 infection is independent of IL-2 and is associated with HSP70 induction." LEUKEMIA, JUN 1994, 8 (6) P1045-56, ENGLAND, XP002069386
CONTI C ET AL: "Inhibition of poliovirus replication by prostaglandins A and J in human cells." ANTIMICROB AGENTS CHEMOTHER, FEB 1996, 40 (2) P367-72, UNITED STATES, XP002069387
AMICI C ET AL: "Selective inhibition of virus protein synthesis by prostaglandin A1: a translational block associated with HSP70 synthesis." J VIROL, NOV 1994, 68 (11) P6890-9, UNITED STATES, XP002069388
ROSSI, A. ET AL: "Induction by prostaglandin A1 of haem oxygenase in myoblastic cells: an effect independent of expression of the 70kDa heat shock protein" BIOCHEMICAL JOURNAL, vol. 308 , no. 2, 1995, pages 455-463, XP002069389
SASAKI H. ET AL: "Prostaglandins in the treatment of cancer" ANTI-CANCER DRUGS, 1994, 5/2 (131-138), UNITED KINGDOM, XP002069390
AMICI C. ET AL: "Antiproliferative prostaglandins activate heat shock transcription factor" PROC. NATL ACAD. SCI. U. S. A., 1992, 89/14 (6227-6231), USA, XP002069391
06116132.9
1704855
20060627
SANTORO, Maria, Gabriella
Via Vasto, 8
I-83100 Avellino
IT
ROSSI, Antonio
Via Orientale, 33
I-66010 Colledimacine
IT
ELIA, Giuliano
Riehenstrasse 163
CH-4058 Palestrina
CH
CONSIGLIO NAZIONALE DELLE RICERCHE
00480850
SVH/39878EP1
Piazzale Aldo Moro, 7
00198 Roma
IT
Jump, Timothy John Simon
et al
00055592
Venner Shipley LLP 20 Little Britain
London EC1A 7DH
GB
AT
BE
CH
DE
DK
ES
FI
FR
GB
GR
IE
IT
LI
LU
MC
NL
PT
SE
EP1997006930
19971211
en
WO1998025593
19980618
199824

FIELD OF INVENTION
The present invention relates to 2-cyclopenten-1-one and its derivatives as inhibitors of the transcription factor Nuclear Factor-kappaB (NF-kB). In particular the invention relates to 2-cyclopenten-1-one and its derivatives comprising the cyclopentenone nucleus as inhibitors of the NF-kB factor with anti-inflammatory, antiproliferative, immuno-suppressive, cytoprotective and antiviral activity.

BACKGROUND OF THE INVENTION
NF-kB (Nuclear Factor - kB) is an eucariotic transcription factor of the rel family, which is normally located in the cytoplasm in an inactive complex, whose predominant form is a heterodimer composed of p5o and p65 subunits, bound to inhibitory proteins of the IkB family, usually IkB-alpha (D. Thanos and T. Maniatis, and Cell 80:529-532, 1995).
NF-kB is activated in response to different stimuli, among which phorbol esters, inflammatory cytokines, UV radiation, bacterial and viral infections. Stimulation triggers the release of NF-kB from IkB in consequence of the phosphorylation and the following degradation of the IkB-alpha protein (P.A. Baeuerle and T. Henkel, Annu. Rev. Immunol. 12: 141-179, 1994). Once it is activated, NF-kB translocates to the nucleus where it binds to DNA at specific kB-sites and induces the transcription of a variety of genes encoding proteins involved in controlling the immune and inflammatory responses, among which a variety of interleukins, the tumor necrosis factor alpha, the NO synthase and the cyclo-oxigenase 2 (S. Grimm and P.A. Baeuerle, Biochem. J. 290: 297-308, 1993). Accordingly, NF-kB is considered an early mediator of the immune and inflammatory responses and it is involved in the control of cell proliferation and in the pathogenesis of various human diseases, among which rheumatoid arthritis (H. Beker et al., Clin. Exp. Immunol. 99: 325, 1995), ischemia (A. Salminen et al. Biochem. Biophys. Res. Comm. 212: 939, 1995), arteriosclerosis (A.S. Baldwin. Annals Rev. Immunol. 14: 649, 1996), as well as in the pathogenesis of the acquired immunodeficiency syndrome (AIDS), due to the enhanced human immunodeficiency virus (HIV-1) transcription in the presence of activated NF-kB. The increase of HIV-1 virus RNAs transcription by NF-kB is caused by the presence of kB-sites in the (LTR) (Long Terminal Repeats) sequences of the virus genome (M.J. Lenardo and D. Baltimore, Cell 58: 227-229, 1989).
It is also known that prostaglandins (PGs) are a class of naturally occurring cyclic 20-carbon fatty acids that are synthetized by various types of eukaryotic cells in response to external stimuli and play an important role in a variety of physiological responses. Since their discovery, PGs were shown to act as microenvironmental hormones and intracellular signal mediators and to control a large number of physiological and pathological processes, including cell proliferation and differentiation, the immune response, inflammation, cytoprotection and the febrile response. In particular, type A and J PGs, which possess a cyclopentenonic structure, are strong inhibitors of virus replication ("Stress Proteins: Induction and Function" Schlesinger MJ, Garaci E., Santoro M.G. ed.s, Springer-verlag, Heidelberg-Berlin, 27-44, 1990). Particularly, it has been recently demonstrated that cyclopentenonic prostaglandins inhibit HIV-1 virus replication, by blocking the viral RNAs transcription (C. Rozera et al. J. Clin. Invest. 97: 1795, 1996).
It is also known that the Heat Shock Proteins (HSPs), also called stress proteins (Proc. Natl. Acad. Sci. USA 86, 8407-8411. 1989), are a family of polypeptides synthetized by eukaryotic and prokaryotic cells in response to heat shock or other kinds of environmental stresses. The HSPs are encoded by a cellular subgroup of genes, identified as stress genes.
The authors have shown that the cyclopentenone prostaglandin PGA inhibits the activation of NF-kB in human cells by inhibiting the phosphorylation and degradation of the inhibitory IkB-alpha protein (A. Rossi, G. Elia and M.G. Santoro, Cold Spring Harbour, New York 1-5 May, 1996, Abstract p. 255).
The authors have also recently shown that inhibition of NF-kB activation is one of the molecular mechanisms used by cyclopentenonic prostaglandins to cause a selective and reversible block of HIV-1 virus RNAs transcription.

SUMMARY OF THE INVENTION
It has now been found that 2-cyclopenten-1-one, the structure constituting the center nucleus of PGA, possesses an activity which is analogous to PGA, that is, it is able to inhibit NF-kB activation, even though it does not contain the corresponding acid function and aliphatic lateral chains. Therefore it is found that the lateral chains, which are present in the PGA with their substituents and double bonds, in particular the acid function, which implies the fatty acid nature of prostaglandins, can be eliminated without substantially modifying the herein above described specific activity. It is also found that the alpha,β-unsatured carbonyl group in the cyclopentenone ring is the key structure necessary for NF-kB inhibition.
Furthermore it has been found that the inhibition of NF-kB by the cyclopentenone group is related to the ability to activate the HSF transcription factor (Heat Shock Transcription Factor), which is responsible for the synthesis of HSPs (Heat Shock Proteins).
In view of the fact that NF-kB inhibition is associated with HSF activation, it is evident that molecules containing the cyclopentenone nucleus, which is active in inhibiting NF-kB, will be inducers of the HSF factor and therefore they will be inducers of heat shock proteins. It is therefore an object of the present invention the 2-cyclopenten-1-one, and its substituted derivatives comprising the cyclopentenone nucleus, as inhibitors of NF-kB. the substituents being selected among the ones which do not affect the NF-kB inhibitory activity.
Another object of the present invention is the 2-cyclopenten-1-one and its pharmacologically acceptable derivatives as inhibitors of NF-kB.
Another object of the invention is the 2-cyclopenten-1-one and its derivatives as inhibitors of NF-kB with anti-inflammatory, antiproliferative, immuno-suppressive, cytoprotective and antiviral activity.
A further object of the invention are pharmaceutical compositions comprising 2-cyclopenten-1-one and/or its pharmaceutically acceptable derivatives to make medicaments with anti-inflammatory, antiproliferative, immuno-suppressive, cytoprotective and antiviral activity. In particular with antiviral activity against the HIV-1 virus and viruses whose transcription is controlled by NF-kB, including herpesviruses.

BRIEF DESCRIPTION OF FIGURES
- Fig. 1A shows the dose-dependent inhibition of NF-kB activation by 2-cyclopenten-1-one.
- Fig. 1B shows the activation of the HSF factor (Heat Shock Transcription Factor) by 2-cyclopenten-1-one in association with NF-kB inhibition.
- Fig. 1C shows the specificity of the chemical structure which is responsible for NF-kB inhibition.
- Fig. 1D shows the specificity of the chemical structure which is responsible for HSF activation.

DETAILED DESCRIPTION OF THE INVENTION
The 2-cyclopenten-1-one is a known product, which can be synthetized according to the process described in Beilstein (Daene, Eder, A. 539 [1939] 207, 211).
According to the present invention 2-cyclopente-1-one, preferably in concentrations ranging between 100 and 500 uM, is able to inhibit NF-kB activation in human cells (Fig. 1A) .
Inhibition tests have been carried out in type T lymphoid human cells (Jurkat cell line), as well as in other human cell lines. NF-kB activation was stimulated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA). 2-Cyclopenten-1-one was also effective in inhibiting NF-kB activation after other types of stimulation, including stimulation by tumor necrosis factor alpha or viral infection, and in different types of human cells (data not shown). It is demonstrated that NF-kB inhibition is associated with the activation of HSF factor (Fig. 1B).
It is also proved that the ability to inhibit the NF-kB factor is specific for 2-cyclopenten-1-one, whereas similar molecules, such as cyclopentanone and cyclopentene, do not inhibit NF-kB (Fig. 1C) and do not activate HSF (Fig. 1D).
Based on these results it is possible to use 2-cyclopenten-1-one, as well as its pharmaceutically acceptable derivatives, as active substances to produce medicaments, in particular medicaments having activity in inhibiting the NF-kB factor, and in particular:
- anti-inflammatory and immunosuppressive medicaments, in view of the role of NF-kB in stimulating the inflammatory and immune responses;
- cytoprotective medicaments, in view of the role of NF-kB in ischemia and oxidative damages;
- antiproliferative medicaments, in view of the role of NF-kB in cell proliferation;
- antiviral medicaments, in view of the role of NF-kB in activating the viral RNAs transcription.
The following examples are reported to illustrate the invention. They should be considered in any case non limiting the scope of the invention itself.
The reagents used in the examples, including 2-cyclopenten-1-one, cyclopentene, cyclopentanone and 12-o-tetradecanoyl-phorbol-13-acetate (TPA), were products of Sigma Aldrich. ^{32} P e ^{35}S were produced by AMERSHAM. Fetal calf serum and cellular culture media were produced by GIBCO.

EXAMPLE 1
The effect of the treatment with 2-cyclopenten-1-one on NF-kB activation by TPA has been tested in Jurkat cells by using the procedures described hereinbelow and shown in Fig. 1.

Dose-response effect
The cells were prepared according to the method described in C. Amici et al. (Cancer Research 55, 4452-4457, 1995).
The cells were treated with 2-cyclopenten-1-one at different concentrations (125-100uM) for 1 hour and then were stimulated with TPA (25 ng/ml).
After 3 hours the whole-cell extracts were prepared and subjected to EMSA (Electrophoretic Mobility Shift Assay) as described for NF-kB in U- Zabel et al. (J. Siol. Chem. 266: 242, 1991) and HSF in C. Amici et al. (Cancer Res. 55: 4452, 1995), to determine NF-kB and HSF activation respectively. The positions of the complex NF-kB-DNA (NF-kB) and the non-specific binding (ns) are indicated in Fig. 1A.
The positions of the complex HSF-DNA (HSF), the HSF-DNA constitutive activity (CHBA) and the proteins-DNA non-specific interactions (ns) are indicated in Fig. 1B. The line "control" indicates the non-TPA-stimulated cells as a control of non-activated NF-kB.
As evident, 2-cyclopenten-1-one is able to inhibit NF-kB activation by TPA even at the lower concentration of 125 uM. At the concentration of 500 um the NF-kB band is absent (Fig. A). In correlation with NF-kB inhibition, in the same samples it is evident the activation of HSF starting from the concentration of 125 uM (Fig. 1B).

Specificity of the inhibitory effect (Fig. 1C)
The cells were treated for 1 hour with the same concentration (500 uM) of: 2-cyclopenten-1-one (line 3), cyclopentanone (line 4) or cyclopentene (line 5), and then were stimulated with TPA (25 ng/ml). C represents the non-TPA-stimulated control. After 3 hours the whole-cell extracts were prepared and subjected to EMSA to verify the activation of NF-kB (Fig. 1C) and of HSF (Fig. 1D) respectively.
As evident, (i) TPA activates NF-kB (line 2); (ii) 2-cyclopenten-1-one inhibits TPA-induced NF-kB activation (line 3); cyclopentanone (line 4) and cyclopentene (line 5) do not inhibit NF-kB activation.
3 In addition, as shown in Fig. 1D, in the same samples inhibition of NF-kB, shown in Fig. 1C, is associated with activation of HSF. These results clearly show that the alpha,β-unsaturated carbonyl group is the key structure trigging HSF activation and its presence is necessary to inhibit NF-kB activation.
1. A composition comprising 2-cyclopenten-1-one or a substituted derivative thereof comprising a cyclopentenonic nucleus, with the proviso that the derivative is not a prostaglandin or a derivative presenting two adjacent aliphatic side chains in the cyclopentenonic nucleus, for use in treating a disorder that can be treated using an inhibitor of NF-KB.
2. A composition as claimed in claim 1, wherein the disorder is:
(a) a viral infectious disease;
(b) an inflammatory disorder;
(c) a disorder treatable with an immuno-suppresive agent;
(d) a disorder involving cell proliferation;
(e) a disorder treatable with a cytoprotective agent;
(f) ischemia;
(g) oxidative cell damage;
(h) arteriosclerosis;
(i) rheumatoid arthritis;
(j) AIDS; or
(k) a herpes virus infection.
3. A composition as claimed in either of the preceding claims, wherein the 2-cyclopenten-1-one or substituted derivative is at a concentration of between 100 and 500 µM.
4. Use of 2-cyclopenten-1-one or a substituted derivative thereof comprising a cyclopentenonic nucleus in the preparation of a medicament, with the proviso that the derivative is not a prostaglandin or a derivative presenting two adjacent aliphatic side chains in the cyclopentenonic nucleus, wherein the medicament is for treating a disorder that can be treated using an inhibitor of NF-KB, the disorder being:
(a) a viral infectious disease;
(b) an inflammatory disorder;
(c) a disorder treatable with an immuno-suppresive agent;
(d) a disorder involving cell proliferation;
(e) a disorder treatable with a cytoprotective agent;
(f) ischemia;
(g) oxidative cell damage;
(h) arteriosclerosis;
(i) rheumatoid arthritis;
(j) AIDS; or
(k) a herpes virus infection.
5. A use as claimed in claim4, wherein the 2-cyclopenten-1-one or substituted derivative is used at a concentration of between 100 and 500 µM.
1. Eine Mischung, die 2-Cyclopenten-1-one oder ein substituiertes Derivat davon mit einem Cyclopentenone-Kern enthält, mit der Bedingung, daß das Derivat nicht ein Prostaglandin oder ein Derivat mit einem Cyclopentenone-Kern mit zwei nebeneinander liegenden aliphatische Seitenketten ist, für den Gebrauch zur Behandlung einer Erkrankung, die mit einem Inhibitor von NF-KB behandelt werden kann.
2. Eine Mischung wie beansprucht in Anspruch 1, worin die Erkrankung:
(a) eine ansteckende Virenkrankheit;
(b) eine Entzündung;
(c) eine mit einem immunosuppressiven Mittel behandelbare Erkrankung;
(d) eine Erkrankung mit Zellenvermehrung;
(e) eine mit einem cytoprotectiven Mittel behandelbare Erkrankung;
(f) Ischemia;
(g) oxydierende Zellenbeschädigung;
(h) Arteriosklerose;
(i) rheumatische Arthritis;
(j) AIDS; oder
(k) eine Herpesvirusinfektion;
ist.
3. Eine Mischung wie beansprucht in einem der vorausgegangenen Ansprüche, worin das 2-Cyclopenten-1-one oder das substituierte Derivat in einer Konzentration von zwischen 100 und 500 µM vorliegt.
4. Gebrauch von 2-Cyclopenten-1-one oder einem substituierten Derivat davon mit einem Cyclopentenone-Kern, in der Herstellung eines Medikaments, mit der Bedingung, daß das Derivat nicht ein Prostaglandin oder ein Derivat mit einem Cyclopentenone-Kern mit zwei nebeneinander liegenden aliphatische Seitenketten ist, worin das Medikament für die Behandlung einer Erkrankung ist, die mit einem Inhibitor von NF-KB behandelt werden kann, worin die Erkrankung:
(a) eine ansteckende Virenkrankheit;
(b) eine Entzündung;
(c) eine mit einem immunosuppressiven Mittel behandelbare Erkrankung;
(d) eine Erkrankung mit Zellenvermehrung;
(e) eine mit einem cytoprotectiven Mittel behandelbare Erkrankung;
(f) Ischemia;
(g) oxydierende Zellenbeschädigung;
(h) Arteriosklerose;
(i) rheumatische Arthritis;
(j) AIDS; oder
(k) eine Herpesvirusinfektion;
ist.
5. Ein Gebrauch wie beansprucht in Anspruch 4, worin das 2-Cyclopenten-1-one oder das substituierte Derivat in einer Konzentration von zwischen 100 und 500 µM benutzt wird.
1. Composition qui comprend une 2-cyclopentène-1-one ou un dérivé substitué de celle-ci comportant un noyau cyclopentènone, à condition que ledit dérivé ne soit pas une prostaglandine ni un dérivé ayant deux chaînes aliphatiques latérales adjacentes dans le noyau cyclopentènone, ladite composition étant conçue pour être utilisée en traitement d'un état pathologique qui peut être traité par un inhibiteur du NF-KB.
2. Composition selon la revendication 1, ledit état pathologique étant :
(a) une maladie infectieuse virale ;
(b) une maladie inflammatoire ;
(c) une maladie qui peut être traitée par un immunosuppresseur;
(d) une maladie qui met en jeu une prolifération cellulaire ;
(e) une maladie traitable par un agent cytoprotecteur ;
(f) une ischémie ;
(g) des lésions cellulaires oxydatives ;
(h) l'artériosclérose ;
(i) la polyarthrite rhumatoïde ;
(j) le SIDA ; ou
(k) une infection à herpèsvirus.
3. Composition selon l'une ou l'autre des revendications précédentes, où la 2-cyclopentène-1-one ou le dérivé substitué de celle-ci est présent à une concentration comprise entre 100 et 500 µM.
4. Utilisation d'une 2-cyclopentène-1-one ou d'un dérivé substitué de celle-ci comportant un noyau cyclopentènone dans la préparation d'un médicament, à condition que ledit dérivé ne soit pas une prostaglandine ni un dérivé ayant deux chaînes aliphatiques latérales adjacentes dans le noyau cyclopentènone, où ledit médicament est conçu pour traiter un état pathologique qui peut être traité par un inhibiteur du NF-KB, ledit état pathologique étant :
(a) une maladie infectieuse virale ;
(b) une maladie inflammatoire ;
(c) une maladie qui peut être traitée par un immunosuppresseur;
(d) une maladie qui met en jeu une prolifération cellulaire ;
(e) une maladie traitable par un agent cytoprotecteur ;
(f) une ischémie ;
(g) des lésions cellulaires oxydatives ;
(h) l'artériosclérose ;
(i) la polyarthrite rhumatoïde ;
(j) le SIDA ; ou
(k) une infection à herpèsvirus.
5. Utilisation selon la revendication 4, où la 2-cyclopentèn-1-one ou le dérivé substitué de celle-ci est utilisé à une concentration comprise entre 100 et 500 µM.

REFERENCES CITED IN THE DESCRIPTION
This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

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