WO2008132138A1

WO2008132138A1 - Derivatives of 4,6-disubstituted aminopyrimidines

Info

Publication number: WO2008132138A1
Application number: PCT/EP2008/055015
Authority: WO
Inventors: Axel Choidas; Bert Klebl; Gerhard Müller; Wilfried Schwab
Original assignee: Ingenium Pharmaceuticals Gmbh
Priority date: 2007-04-25
Filing date: 2008-04-24
Publication date: 2008-11-06
Also published as: WO2008132138A9

Abstract

The present invention relates to methylenesulfone and methylenesulfonamide derivatives of 4,6-disubstituted aminopyrimidines having the general formula (II) as given below and/or pharmaceutically acceptable salts thereof, the use of these derivatives as pharmaceutically active agents, especially for the prophylaxis and/or the treatment of cell proliferative diseases, inflammation, cardiac hypertrophy and infectious diseases, especially viral infections such as HIV. Furthermore, the present invention is directed towards pharmaceutical composition containing at least one of the methylenesulfone or methylenesulfonamide derivatives of 4,6-disubstituted aminopyrimidines and/or pharmaceutically acceptable salts thereof.

Description

Derivatives of 4,6-disubstituted aminopyrimidines

The present invention relates to methylenesulfone and methylenesulfonamide derivatives of 4,6-disubstituted aminopyrimidines and/or pharmaceutically acceptable salts thereof, the use of these derivatives as pharmaceutically active agents, in particular for the prophylaxis and/or the treatment of cell proliferative disease, pain, inflammation, cardiovascular disease, or infectious disease. Furthermore, the present invention is directed towards pharmaceutical composition containing at least one of the methylenesulfone or methylenesulfonamide derivatives of 4,6-disubstituted aminopyrimidines and/or pharmaceutically acceptable salts thereof.

One of the most important and fundamental processes in biology is the division of cells during the cell cycle. This process ensures the controlled production of subsequent generations of cells with defined biological function. It is a highly regulated phenomenon and responds to a diverse set of cellular signals both within the cell and from external sources. Cyclin dependent kinases (CDKs) play a key role in regulating the cell cycle machinery. These complexes consist of two components: a catalytic subunit (the kinase) and a regulatory subunit (the cyclin). To date, thirteen kinase subunits have been identified in humans (Chen et al., Biochem Biophys Res Commun. 2007, 354, 735-40; S. Mani et al., Exp. Opin. Invest. Drugs 2000, 9(8), 1849 - 1870, J.C. Sergere et al., Biochem. Biophys. Res. Commun. 2000, 276, 271 - 277, D. Hu et al, J. Biochem. Chem. 2003, 278(10), 8623 - 8629).

It is known, that CDKs play a role in the regulation of cellular proliferation. Therefore, CDK inhibitors could be useful in the treatment of cell proliferative disorders such as cancer, neuro-fibromatosis, psoriasis, fungal infections, endotoxic shock, transplantation rejection, vascular smooth cell proliferation associated with artheroscelerosis, pulmonary fibrosis, arthritis, glomerulonephritis and post-surgical stenosis and restenosis (U.S. Patent No. 6,114,365).

CDKs are also known to play a role in apoptosis. Therefore CDK inhibitors could be useful in the treatment of cancer; autoimmune diseases, for example systemic lupus, erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes; neurodegenerative diseases for example Alzheimer^'s disease, AIDS-related dementia, Parkinson^'s disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration; myelodysplastic syndromes, aplastic anemia, ischemic injury associated with myocardial infarctions, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol related liver diseases; hematological diseases, for example, chronic anemia and aplastic anemia; degenerative diseases of the musculoskeletal system, for example, osteoporosis and arthritis, cystic fibrosis, multiple sclerosis, kidney diseases and cancer pain and for the treatment of cardiovascular diseases (U.S. patent No. 6,107,305 and WO 02/100401 ).

Further it is known, that CDK inhibitors could be used for the treatment of virally induced infectious diseases, such as EBV, HBV, HCV and HIV (WO 02/100401 ). Recently, it was described, that HIV-1 replication could be affected by inhibiting CDKs (C. de Ia Fuenta, Current HIV research, 2003, 1 (2), 131 - 152; Y.K. Kim et al., Molecular and Cellular Biology, 2002, 22(13), 4622-4637). Especially CDK9 is reported to be essential for the HIV-1 replication (H. S. Mancebo et al, Genes Dev. 1997, 11 (20): 2633-44, O. Flores et al., Proc Natl. Acad. Sci. U S A. 1999, 96(13):7208-13).

CDK9 is also involved in the development of pain: both cyclin T1 and CDK9 stimulate the basal promoter activity of TNFα, a pro-inflammatory cytokine and pain mediator that controls expression of inflammatory genetic networks. For mediation of cellular TNF receptor responses, the nuclear factor-KB (NFKB) pathway is crucial. TNFα triggers its recruitment to cytokine genes while NFKB interacts with the p-TEFb complex for stimulation of gene transcription (Barbohc M. et al., NFKB Binds P- TEFb to Stimulate Transcriptional Elongation by RNA Polymerase II. Molecular Cell, 2001 , Vol. 8, 327-337).

Additionally, it has been shown that CDK9 is a binding partner of TRAF2, a member of the TNFa receptor complex (MacLachlan T. K. et al., Binding of CDK9 to TRAF2. J Cell Biochem, 1998, 71 (4), 467-478), while GP130, a subunit of the pro-inflammatory IL6 receptor complex has recently been identified as another potential binding partner of CDK9 (Falco G. D. et al., CDK9, a member of the cdc2- like family of kinases, binds to gp130, the receptor of the IL-6 family of cytokines. Oncogene, 2002, 21 (49), 7464-7470). As a key player in TNFα and interleukin signaling as well as NFKB mediated expression of several genes (e.g. cytokines as pain mediators), CDK9 can thus be considered as a central target for the treatment of inflammatory pain.

There also exists a strong link between CDK9 and caridac hypertrophy (reviewed in Sano & Schneider, Circulation Research, 2004, 95, 867) and inhibitors of CDK9 are expected to be effective in the treatment of cardiovascular diseases, such as caridac hypertrophy.

Most of the known CDK inhibitors, such as olomoucine, roscovitine (CYC202), purvalanols, indolinones, paullones and 7-hydroxy-staurosporine are focusing on the inhibiton of CDK1 and CDK2 with the goal of antitumor activity (Current

Opinion in Pharmacalogy, 2003, 3, 1 -9). A summary of the known CDK inhibitors is given by M. Huwe et al. (A. Huwe et al., Angew Chem lnt Ed Engl. 2003; 42(19):

2122-38). Flavopihdol is described as a low-molecular, but unselective inhibitor of CDKs, including CDK9 (W. Filgueira de Azevedo et al., Biochem.and Biophys.

Res. Commun. 2002, 293(1 ), 566-571 ). Other compounds that were shown to inhibit CDKs are staurosporine, fascaplysin and hymenialdisine.

The use of 4-aminopyrimidine derivatives as neuroprotective agents is described in WO 02/12198. These compounds generally contain as a basic residue a substituted amine in para position of the anilino part of the molecule, and it is stated that these compounds did not inhibit MEK1/2 kinase activity in P19 neurons. US Patent No. 3,950,525 describes the use of 4-amino-6-aryl- pyrimidines as platelet aggregation inhibitors and bronchodilators. US Patent No. 3,478,030 describes the synthesis of benzamide substituted anilino aminopyrimidine derivatives. These compounds are used as potent dilators of coronary arteries. WO 02/79197 describes the use of aryl-substituted 2- aminopyrimidine derivatives as protein kinase inhibitors, for example as inhibitor of JNK, GSK-3, Src, Lck or CDK2.

Certain 4,6-disubstituted aminopyrimidines are described in WO 05/026129. WO 05/026129 describes derivatives which are useful as pharmaceutically active agents, especially for the prophylaxis and/or treatment of infectious diseases, including opportunistic diseases, prion diseases, immunological diseases, autoimmune diseases, bipolar and clinical disorders, cardiovascular diseases, cell proliferative diseases, diabetes, inflammation, transplant rejections, erectile dysfunction, neurodegenerative diseases and stroke. However, the 4,6- disubstituted aminopyrimidines described in WO 05/026129 are structurally different to those described in the present application.

WO 06/125616 describes the use of CDK inhibitors, including CDK9 inhibitors, such as the 4,6-disubstituted aminopyrimidines disclosed in WO 05/026129, for the treatment of pain and inflammatory diseases.

There is a high unmet medical need to develop CDK inhibitors, useful in treating various medical conditions or diseases associated with CDK activation, in particular those conditions or diseases concerning CDK9 kinase activity, which is associated with cell proliferative diseases, pain, inflammation, cardiovascular diseases, and infectious diseases.

It is an object of the present invention to provide compounds and/or pharmaceutically acceptable salts thereof which can be used as pharmaceutically active agents, in particular for prophylaxis and/ or treatment of one or more disease or medical condition selected from: infectious disease, such as retroviral infectious disease, including HIV; pain, such as inflammatory pain and neuropathic pain; cardiovascular disease, such as cardiac hypertrophy; cell proliferative disease, such as cancer; and inflammation, methods to treat said disease, as well as compositions comprising at least one of those compounds and/or pharmaceutically acceptable salts thereof as pharmaceutically active ingredients.

This object is solved by the compounds and/or their pharmaceutically acceptable salts, the compounds of the present invention for use as a pharmaceutically active agents, the use of the compounds of the present invention for the preparation of a pharmaceutical composition for the prophylaxis and/or treatment of infectious disease, cell proliferative disease, pain, or inflammation, the use of compounds according to the present invention as inhibitors of a protein kinase and the pharmaceutical compositions as described herein.

Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the examples and the drawings.

The novel 4,6-disubstituted aminopyhmidine compounds according to the present invention are defined by the general formula (I)

wherein

R¹ is selected from the group comprising: hydrogen, linear or branched Ci-C₆ substituted or unsubstituted alkyl, linear or branched C₂-C₆ alkenyl or linear or branched C₂-C₆ alkynyl;

R² and R⁴ are independently selected from the group consisting of: hydrogen, linear or branched substituted or unsubstituted Ci-C₆ alkyl, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, -F, -Cl, -Br, -I, -CN, - NH₂ Or -NO₂;

R³ and R⁵ are independently selected from substituted or unsubstituted phenyl or pyridine, wherein each substituent is independently selected from the group consisting of linear or branched Ci-C₆ substituted or unsubstituted alkyl, linear or branched substituted or unsubstituted Ci-C₆ alkoxy, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, -F, -Cl, -Br, -I, -CN, -NH₂, -NO₂, - NR₂₀R₂I, -CO-R₂₀ or -CO-NR₂₀R₂i, wherein R₂₀ and R₂i are independently of each other selected from hydrogen, linear or branched substituted or unsubstituted Ci- C₆ alkyl, acetyl, or substituted or unsubstituted amino;

R⁶ is selected from hydrogen, linear or branched substituted or unsubstituted Ci- Cs alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C₃-C₈ cycloalkyl or -(CH₂)_q-A, wherein q is an integer selected from O to 5 and A is selected from hydrogen, -F, -Cl, -Br, -I, -CN, -NH₂, -NO₂, linear or branched substituted or unsubstituted Ci-C₆ alkyl, linear or branched substituted or unsubstituted Ci-C₆ alkoxy, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted cycloalkyl or carboxamido substituted with one or two Ci-C₆ alkyl; or R⁶, when M is -NR⁴⁰-, can form a heterocyclic structure when taken together with the nitrogen of M and R 40. L is - CR⁵⁰R⁵¹ -SO₂-M-, wherein R⁵⁰ and R⁵¹ are independently selected from the group consisting of hydrogen, linear C₁-C₃ alkyl and fluorine, wherein M is a bond or -NR⁴⁰ -;

R⁴⁰ is selected from hydrogen, linear or branched substituted or unsubstituted Ci- C₈ alkyl, and substituted or unsubstituted C₃-C₈ cycloalkyl;

and/or tautomeric forms and/or pharmaceutically acceptable salts thereof.

In particular embodiments R¹ is selected from methyl and hydrogen. In more particular embodiments R¹ is hydrogen.

In particular embodiments R² and R⁴ are independently selected from methyl, amino and hydrogen. In other particular embodiments R² is -NH₂ or hydrogen and R⁴ is hydrogen. In other particular embodiments R² is hydrogen. In other particular embodiments R⁴ is hydrogen. In even other particular embodiments R² and R⁴ are both hydrogen.

In particular embodiments of the invention R³ is substituted or unsubstituted phenyl. In other particular embodiments R³ is substituted or unsubstituted pyridine. In certain embodiments R³ is substituted with linear or branched Ci-Cβ alkoxy, particularly linear or branched Ci-C₄ alkoxy, more particularly methoxy. In certain particular embodiments R³ is phenyl substituted with linear or branched Ci-Cβ alkoxy, particularly linear or branched Ci-C₄ alkoxy, more particularly methoxy. In other particular embodiments R³ is pyridine substituted with linear or branched Ci-C₆ alkoxy, particularly linear or branched Ci-C₄ alkoxy, more particularly methoxy. In certain embodiments R³ is substituted in the o/f/70-position. In certain particular embodiments R³ is phenyl substituted in the o/t/70-position. In other particular embodiments R³ is phenyl substituted with methoxy in the o/f/70-position. Where R³ is pyridine, the nitrogen atom of said pyridine may be in any position of the pyridine group. In particular embodiments R³ is o/?/7O-pyridine. In other embodiments R³ is mefa-pyhdine. In yet other embodiments R³ is para-pyhdine. In other particular embodiments R³ is substituted with one or more residues R⁴¹.

In particular embodiments R⁵ is substituted or unsubstituted phenyl. In certain particular embodiments R⁵ is substituted phenyl. In other particular embodiments R⁵ is unsubstituted phenyl. In other particular embodiments, R⁵ is substituted or unsubstituted phenyl and L is linked to said phenyl group in mefa-position to -NRr.

In other particular embodiments R⁵ is substituted or unsubstituted pyridine. In certain particular embodiments R⁵ is substituted pyridine. In other particular embodiments R⁵ is unsubstituted pyridine. Where R⁵ is pyridine, the nitrogen atom of said pyridine may be in any position of the pyridine group. In particular embodiments R⁵ is pyrid-2-yl. In other embodiments R⁵ is pyrid-3-yl. In yet other embodiments R⁵ is pyrid-4-yl.

The optional substituents of R⁵ are in addition to the residue -L-R⁶ depicted in formula (I), which is compulsory. In particular embodiments the substituent of R⁵ is selected from methyl, methoxy, thfluoromethyl, isopropyl, ethyl, ethoxy, -NMe2, - NHAc, -NMeAc, -CO-Me, -CO-NH₂, -CO-NH-Me and -CO-NMe₂. In particular embodiments such optional substituent of R⁵ is linear or branched substituted or unsubstituted Ci-C₄ alkyl. In yet other particular embodiments the substituent of R⁵ is methyl, ethyl and isopropyl. In particular embodiments, R⁵ is linear or branched Ci-C₄ alkoxy. In yet other particular embodiments R⁵ is methoxy or ethoxy Substituent on R⁵ may be in ortho-, meta-, or para-position to -NRr. In particular embodiments R⁵ is in orfrjo-position. In other particular embodiments R⁵ is in mefa-position. In yet other particular embodiments R⁵ is in the para-position. In certain alternative embodiments, R⁵ is unsubstituted.

In particular aspects of the invention, R⁶ is selected from hydrogen, linear or branched substituted or unsubstituted Ci-Cs alkyl, and -(CH₂)_q-A, wherein q is an integer selected from O to 5 and A is selected from linear or branched substituted or unsubstituted Ci-Cβ alkoxy, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroaryl and carboxamido substituted with one or two Ci-Cβ alkyl. In certain particular aspects of the invention, R⁶ is hydrogen. In other particular aspects of the invention R⁶ is linear or branched unsubstituted Ci-

C₅ alkyl. In certain particular aspects of the invention R⁶ is methyl, isopropyl or 3- methyl-butyl. In some particular aspects of the invention R⁶ is methyl.

In certain aspects of the invention R⁶ is -(CH₂)_q-A, wherein q is an integer selected from O to 3 and A is linear, unsubstituted Ci-Cβ alkoxy. In certain particular aspects of the invention q is 2 and A is methoxy. In other aspects of the invention R⁶ is -(CH₂)_q-A, wherein q is an integer selected from 0 to 3 and A is carboxamido substituted with two C1-C3 alkyl groups. In certain particular aspects of the invention q is 2 and A is carboxamido substituted with two methyl groups. In other aspects of the invention R⁶ is-(CH₂)_q-A, wherein q is an integer selected from 0 to 3 and A is substituted or unsubstituted heterocyclyl. In certain particular aspects of the invention said substituted or unsubstituted heterocyclyl is substituted or unsubstituted pyrrolidine or piperidine. In other aspects of the invention R⁶ is-(CH₂)_q-A, wherein q is 0 and A is unsubstituted heteroaryl. In certain particular aspects of the invention said unsubstituted heteroaryl is thiazole or oxazole.

In other particular aspects of the invention M is -NR⁴⁰- and R⁶ is selected from

hydrogen

In certain aspects of the present invention M is -NR⁴⁰- and R⁶ taken together with tthhee nniittrrooggeenn ooff MM aanndd RR⁴⁴⁰⁰ ffoorrmmss aa hheetteerrooccyycclliicc ssttrruuccttuurre. In particular aspects of the invention said heterocyclic structure is selected from:

In particular embodiments R ,40 is selected from hydrogen and linear Ci-C₄ alkyl or C₃-C₄ cycloalkyl. In particular embodiments R⁴⁰ is selected from hydrogen, methyl, ethyl and isopropyl. In certain particular embodiments wherein R 40 is hydrogen. In other particular embodiments R⁴⁰ is methyl.

L is - CR⁵⁰R⁵¹ -SO₂-M-,

Wherein R⁵⁰ and R⁵¹ are independently selected from the group consi hydrogen, linear C₁-C₃ alkyl and fluorine_, wherein M is a bond or -NR⁴⁰ -.

For the avoidance of doubt it is noted that the - CR⁵⁰R⁵¹ - group of L, and not the -M- group of L, is linked to R⁵ of a compound of formula (I) or the phenyl group of a compound of formula (II). L can be linked to R⁵ of a compound of formula (I) or the phenyl group of a compound of formula (II) in ortho-, meta- or para-position. In particular embodiments L is linked to R⁵ in mefa-position.

In particular embodiments R⁵⁰ is hydrogen. In other particular embodiments R⁵¹ is hydrogen. In other particular embodiments R⁵⁰ and R⁵¹ are both hydrogen. In yet other embodiments R⁵⁰ and R⁵¹ are independently selected from the group consisting of hydrogen, methyl and fluorine. In particular embodiments, both R 50 and R⁵¹ are fluorine.

M is a bond or -NR⁴⁰-. In particular embodiments M is -NR⁴⁰-. In other particular embodiments R⁵⁰ and R⁵¹ are both hydrogen and M is -NR⁴⁰-. In other particular embodiments of the invention M is a bond.

In particular embodiments the novel 4,6-disubstituted aminopyrimidine compounds according to the present invention are compounds of formula (II),

wherein R >1 , D R2 , n R4 , n R6 , and L have the same meaning as defined above;

m is an integer selected from 0 to 5; n is an integer selected from 0 to 4;

each R⁴¹ and R⁴² is independently selected from the group consisting of linear or branched Ci-C₆ substituted or unsubstituted alkyl, linear or branched substituted or unsubstituted Ci-C₆ alkoxy, linear or branched C₂-C₆ alkenyl, linear or branched C₂-C₆ alkynyl, -F, -Cl, -Br, -I, -CN, -NH₂ or -NO₂;

and/or tautomeric forms and/or pharmaceutically acceptable salts thereof.

In other particular embodiments, L is linked to the phenyl group of a compound of formula (II) in mefa-position.

In other particular embodiments, L is -CH₂-SO₂-NR₄₀-. In other particular embodiments L is -CH₂-SO₂-NH-.

In other particular embodiments, m is 1 or 2. In yet other particular embodiments m is 1 .

In other particular embodiments, n is 0 or 1 . In yet other particular embodiments n is 0.

In particular embodiments at least one R⁴¹ is linear or branched Ci-C₆ alkoxy. In other particular embodiments at least one R⁴¹ is linear Ci-C₆ alkoxy. In yet other particular embodiments at least one R⁴¹ is methoxy. In particular embodiments the substitution of at least one residue R⁴¹ is in the o/ffto-position. In other particular embodiments at least one residue R⁴¹ is methoxy and the substitution of said methoxy residue is in the o/ffto-position.

In other particular embodiments of the invention, the compound is selected from the group of compounds consisting of:

Compound 1 : C-{3-[6-(2-Methoxy-phenyl)-pyrimidin-4-ylamino]-phenyl}-

N,N-dimethyl-methanesulfonamide,

Compound 2: C-{3-[6-(2-Methoxy-phenyl)-pyrimidin-4-ylamino]-phenyl}- methanesulfonamide,

Compound 3: C-{3-[6-(2-Methoxy-phenyl)-pyrimidin-4-ylamino]-phenyl}- N-methyl-methanesulfonamide, and Compound 4: N-(2-Methoxy-ethyl)-C-{3-[6-(2-methoxy-phenyl)- pyrimidin-4-ylamino]-phenyl}-methanesulfonamide.

In the context of the present invention, it is intended to include all stereoisomeric forms of the compounds of the present invention, as well as their quaternary amine, N-oxide, salt, polymorph, solvate, prodrug and derivative forms. The term "stereoisomer" as used herein includes all possible stereoisomeric forms, including all chiral, diastereomehc, racemic forms and all geometric isomeric forms of a structure, unless the specific stereochemistry or isomer form is specifically indicated. Where the compounds of the present invention contain one or more chiral centers, all possible enantiomeric and diastereomeric forms, as well as the racemate, are included. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. All processes used to prepare compounds of the present invention and intermediates made therein are considered to be part of the present invention.

The term "tautomer" as used herein includes all possible tautomeric forms of the structures used to show the compounds of the present invention, as well as their stereoisomeric, quaternary amine, N-oxide, salt, polymorph, solvate, and prodrug forms.

In certain embodiments, a compound of the invention, a salt form thereof, including a pharmaceutically acceptable salt, or a solvate form thereof, including a hydrate, is isolated. In certain embodiments, a compound of the invention, or a pharmaceutically acceptable salt thereof, is purified, e.g., to have a purity of at least 80%, preferably at least 90%, more preferably at least 95%, such as at least 97%, at least 98% or even at least 99%. Purity, as used herein, can refer to either absolute or relative purity. Absolute purity refers to the amount of compound of interest in relation to the total amount of a composition including such compound. Relative purity refers to the amount of a compound of interest in a composition relative to the amount of one or more other substances included in such composition, e.g. one or more impurities such as by-products, degradation products (e.g., metabolites, products of oxidation or hydrolysis, etc.) and/or compounds that degrade to form the compound of the invention (e.g., precursors or prodrugs). Such other substance(s) may, for example, be present in the product of a synthetic chemistry scheme for such compound of interest. Thus, absolute purity refers to the amount of the compound of interest relative to all others components of a composition including such compound, while relative purity is mainly used to describe purity with regard to closely related substances, and thus is unaffected by the addition of unrelated compounds, such as excipients, stabilizers, or other medicaments for conjoint administration. Purity can be assessed based upon weight, volume or molar ratios of one compound relative to others. Purity can be measured by a variety of analytical techniques, including elemental abundance, UV-visible spectrometry, HPLC, GC-MS, NMR, mass spectrometry, and thin layer chromatography, preferably by HPLC, GC-MS, or NMR.

In certain embodiments, a compound of the invention, or a salt thereof, is synthetically produced. The term "synthetically produced" refers to the generation of a compound using synthesis techniques well known to the skilled artisan with the aim of obtaining such compound.

In certain embodiments, a compound of the invention, a salt form thereof, including a pharmaceutically acceptable salt, or a solvate form thereof, including a hydrate, is in amorphous form.

In certain embodiments, a compound of the invention, a salt form thereof, including a pharmaceutically acceptable salt, or a solvate form thereof, including a hydrate, is in crystalline form.

The term "alkyl" refers to optionally substituted straight- or branched-chain saturated hydrocarbon groups. These groups may or may not be branched. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n- butyl, s-butyl, t-butyl, n-pentyl, and s-pentyl. In addition, the term is intended to include both unsubstituted and substituted alkyl groups, the latter referring to alkyl moieties having one or more hydrogen atoms replaced by, but not limited to halogen, hydroxyl, carbonyl, alkoxy, ester, ether, cyano, phosphoryl, amino, imino, amido, sulfhydryl, alkylthio, thioester, sulfonyl, nitro, heterocyclo, aryl or heteroaryl. It will also be understood by those skilled in the art that the substituted moieties themselves can be substituted as well when appropriate. In certain embodiments alkyl groups are unsubstituted.

The terms "alkenyl" and "alkynyl" herein refer to alkenyl and alkynyl groups, respectively. The terms "alkenyl" and "alkynyl" refer to monovalent groups. The terms "alkenylene" and "alkynylene" refer to the corresponding divalent groups. These groups may or may not be branched. At least one of the bonds of an alkenyl or an alkenylene group is a double bond, other, additional, bonds may be single bonds or double bonds. At least one of the bonds of an alkynyl or an alkynylene group is a triple bond, other, additional, bonds may be single bonds, double bonds or triple bonds. Examples of such alkenyl (and alkenylene) groups include ethenyl (ethenylene), 1-propenyl (1 -propenylene), 2-propenyl (2- propenylene), 1-butenyl (1-butenylene), 2-butenyl (2-butenylene), 3-butenyl (3- butenylene), 2-methyl-1-propenyl (2-methyl-1 -propenylene), 2-methyl-2-propenyl (2-methyl-2-propenylene), and the like. Examples of such alkynyl (and alkynylene) groups include ethynyl (ethynylene), 1 -propynyl (1 -propynylene), 2-propynyl (2- propynylene), and so forth. In addition, the terms are intended to include both unsubstituted and substituted alkenyl and alkenylene groups. Substituted alkenyl and alkenylene groups refer to alkenyl and alkenylene moieties having one or more hydrogen atoms replaced by, but not limited to halogen, hydroxyl, carbonyl, alkoxy, ester, ether, cyano, phosphoryl, amino, imino, amido, sulfhydryl, alkylthio, thioester, sulfonyl, nitro, heterocyclo, aryl or heteroaryl. The same applies for alkynyl and alkynylene groups, which may be unsubstituted or substituted with the substituents indicated above. In certain embodiments alkenyl, alkynyl, alkenylene and alkynylene groups are unsubstituted.

The term "aryl" is intended to mean any stable and optionally substituted monocyclic or polycyclic aromatic moiety, which may contain 3 to about 12 members per ring. This includes optionally substituted benzene rings or optionally substituted benzene ring systems fused to one or more optionally substituted benzene rings, to form, e.g., anthracene, phenanthrene, or naphthalene ring systems, or fused to heteroaryl rings. Aryl moieties may be optionally substituted with between 0 to about 10 substituents, and in certain embodiments greater than 10 substituents. Such substituents may be selected from a group consisting of d- C₆ alkyl, CrC₆ alkoxy, CrC₆ haloalkyl, CrC₆ haloalkoxy, CrC₆ alkylsulfanyl, d- C₆ alkylsulfenyl, CrC₆ alkylsulfonyl, Ci-C₆ alkylsulfonylamino, arylsulfonylamino, alkylcarboxy, alkylcarboxamido, oxo, hydroxy, mercapto, amino (optionally substituted by alkyl, aryl, or heteroaryl), carboxy, tetrazolyl, carboxamido, carbamoyl (optionally substituted by alkyl, aryl, or heteroaryl), aminosulfonyl, acyl, aroyl, aroylamino, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halo, heteroaryl, heterocyclyl, aryl, ureido, arylureido, alkylureido, cycloalkylureido, alkylthioureido, aryloxy, aralkoxy, or -O(CH₂)_nCOOH, - (CH₂)_nCOOH, -C(O)O(CH₂)_nR, -(CH2)_nN(H)C(O)OR, or -N(R')S(0)₂R wherein n is 1 -4 and R is -H, alkyl, aryl or heteroaryl, multiple degrees of substitution being allowed. Examples of "heteroaryl" groups used herein include furanyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, triazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxo-pyridyl, thiadiazolyl, isothiazolyl, pyridyl, pyridazyl, pyrazinyl, pyrimidyl, quinazolinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiophenyl, indolyl, indazolyl, and substituted versions thereof. Examples of aryl groups include phenyl, p-tolyl, 4-methoxyphenyl, 4-tert-butoxyphenyl, 3- methylmethoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 3-nitrophenyl, 3- aminophenyl, 3-acetamidophenyl, 4-acetamidophenyl, 2-methyl acetamidophenyl, 2-methyl aminophenyl, 3-methyl aminophenyl, 2-amino methylphenyl, 2,4-dimethyl aminophenyl, 4-hydroxyphenyl, 3-methyl hydroxyphenyl, 1 -naphthyl, 2-naphthyl, 3- amino-l-naphthyl, 2-methyl amino naphthyl, 6-amino naphthyl, 4,6-dimethoxy naphthyl and the like. In certain embodiments aryl moieties are unsubstituted. Aryl moieties that do not contain any heteroatoms are designated "homoaryl" moieties. Heteroaryl moieties and homoaryl moieties are aryl moieties.

As used herein, the term "heteroaryl" refers to any stable and optionally substituted mono- or polycyclic aromatic moiety containing one or more nitrogen, sulfur, and/or oxygen heteroatoms, where N-oxides and sulfur oxides and dioxides are permissible heteroatom substitutions. Heteroaryl moieties may contain 3 to about 12 members per ring and may be optionally substituted with between 0 to about 10 substituents, and in certain embodiments greater than 10 substituents. Such substituents may be selected from a group consisting of CrC₆ alkyl, CrC₆ alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 alkylsulfanyl, C1-C6 alkylsulfenyl, CrC₆ alkylsulfonyl, Ci-C₆ alkylsulfonylamino, arylsulfonoamino, alkylcarboxy, alkylcarboxyamido, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, aryl, or heteroaryl, carboxy, tetrazolyl, carboxamido, carbamoyl optionally substituted by alkyl, aryl, or heteroaryl, aminosulfonyl, acyl, aroyl, aroylamino, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halo, heteroaryl, heterocyclyl, aryl, ureido, arylureido, alkylureido, cycloalkylureido, alkylthioureido, aryloxy, aralkoxy, or -O(CH₂)_nCOOH, -(CH₂)_nCOOH, - C(O)O(CH₂)_nR, -(CH₂)_nN(H)C(O)OR, or -N(R')S(0)₂R wherein n is 1 -4 and R is -H, alkyl, aryl or heteroaryl, multiple degrees of substitution being allowed. Examples of "heteroaryl" groups used herein include furanyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxo- pyridyl, thiadiazolyl, isothiazolyl, pyridyl, pyridazyl, pyrazinyl, pyrimidyl, quinazolinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiophenyl, indolyl, indazolyl, and substituted versions thereof. In certain embodiments heteroaryl moieties are unsubstituted.

In a further aspect of the present invention, the compounds of the present invention are used as pharmaceutically active agents.

Further aspects of the present invention relate to the use of the compounds of the present invention for the preparation of a pharmaceutical composition useful for prophylaxis and/or treatment of a disease selected from: cell proliferative disease, such as cancer; pain, such as inflammatory pain and neuropathic pain; inflammation; cardiovascular diseases, such as cardiac hypertrophy; and infectious disease, such as viral infections including HIV.

Other aspects of the present invention relate to methods for the prophylaxis and/or treatment of a disease selected from: cell proliferative disease, such as cancer; pain, such as inflammatory pain and neuropathic pain; inflammation; cardiovascular disease, such as cardiac hypertrophy; and infectious disease, such as viral infections including HIV; comprising administering to an individual a compound according to the present invention. In certain embodiments of these aspects of the invention, the disease for prophylaxis and/or treatment can be found in an individual, such as a patient in need of such prophylaxis and/or treatment. An "individual" means a multi-cellular organism, for example an animal such as a mammal, including a primate. In addition to primates, such as humans, a variety of other mammals can be treated according to a method that utilizes one or more compounds of the present invention. For example, other mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rabbits, rats, mice or other bovine, ovine, equine, canine, feline, or rodent species can be used. In one particular such embodiment of these aspects, the individual is a human.

Infectious diseases including opportunistic infections

In yet another aspect of the present invention, the compounds of the present invention can be used for the preparation of a pharmaceutical composition for the prophylaxis and/or treatment of infectious disease, including opportunistic diseases and opportunistic infections. The term infectious disease comprises infections caused by viruses, bacteria, prions, fungi, and/or parasites.

Virally induced infectious diseases, including opportunistic diseases are addressed in particular aspects of the invention. In a particular embodiment of this aspect, the virally induced infectious diseases, including opportunistic diseases, are caused by retroviruses, human endogenous retroviruses (HERVs), hepadnaviruses, herpesviruses, flaviviridae, and/or adenoviruses. In certain embodiments, the retroviruses are selected from lentiviruses or oncoretroviruses, wherein the lentivirus can be selected from the group comprising: HIV-1 , HIV-2, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), sivian immunodeficiency viruses (SIVs), chimeras of HIV and SIV (SHIV), caprine arthritis encephalitis virus (CAEV), visna/maedi virus (VMV) or equine infectious anemia virus (EIAV), preferably HIV-1 and HIV-2, and the oncoretrovirus is preferably selected from HTLV-I, HTLV-II or bovine leukemia virus (BLV), preferably HTLV-I and HTLV-II. In particular embodiments, the hepadnavirus is selected from HBV, ground squirrel hepatitis virus (GSHV) or woodchuck hepatitis virus (WHV), and in certain embodiments the hepadnavirus is HBV. In other particular embodiments, the herpesvirus is selected from the group comprising: Herpes simplex virus I (HSV I), herpes simplex virus Il (HSV II), Epstein-Barr virus (EBV), varicella zoster virus (VZV), human cytomegalovirus (HCMV) or human herpesvirus 8 (HHV-8), and in certain embodiments the herpesvirus is HCMV. In other particular embodiments, the flaviviridae is selected from HCV, West Nile or Yellow Fever.

Hence, in a further aspect of the present invention, a method for preventing and/or treating infectious disease, including opportunistic disease, in a mammal, including a human, is provided, which method comprises administering to the mammal an amount of at least one compound of the present invention, effective to prevent and/or treat said infectious disease, including a opportunistic disease. In a particular embodiment of this method, the infectious disease, including opportunistic disease, includes virally induced infectious diseases. The virally induced infectious diseases, including opportunistic diseases, are caused by retroviruses, hepadnaviruses, herpesviruses, flaviviridae, and/or adenoviruses. In a further particular embodiment of this method, the retroviruses are selected from lentiviruses or oncoretroviruses, wherein the lentivirus is selected from the group comprising: HIV-1 , HIV-2, FIV, BIV, SIVs, SHIV, CAEV, VMV or EIAV, including certain embodiments where the lentivirus is HIV-1 or HIV-2, or wherein the oncoretrovirus is selected from the group consisting of: HTLV-I, HTLV-II or BLV. In a further particular embodiment of this method, the hepadnavirus is selected from HBV, GSHV or WHV, including certain embodiments where the hepadnavirus is HBV, or wherein the herpesivirus is selected from the group comprising: HSV I, HSV II, EBV, VZV, HCMV or HHV 8, including certain embodiments where the herpesivirus is HCMV, or wherein the flaviviridae is selected from HCV, West Nile or Yellow Fever.

Cell proliferative disease

As used herein, a "cell proliferative disease" includes a disease or disorder that affects a cellular growth, differentiation, or proliferation process. As used herein, a "cellular growth, differentiation or proliferation process" is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus. A cellular growth, differentiation, or proliferation process includes amino acid transport and degradation and other metabolic processes of a cell. A cellular proliferation disorder may be characterized by aberrantly regulated cellular growth, proliferation, differentiation, or migration. Cellular proliferation disorders include tumorigenic diseases or disorders. As used herein, a "tumorigenic disease or disorder" includes a disease or disorder characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, or migration, which may result in the production of or tendency to produce tumors. As used herein, a "tumor" includes a benign or malignant mass of tissue. Examples of cellular growth or proliferation disorders include, but are not limited to tumors, cancer, autoimmune diseases, viral diseases, fungal diseases, neurodegenerative disorders and cardiovascular diseases.

In certain embodiments, tumors may be solid tumors, which are cancer of body tissues other than blood, bone marrow, or the lymphatic system. In other embodiments tumors may be hematological tumors, such as leukemia and lymphomas. Leukemia is a collective term for malignant diseases characterized by a proliferation of malignantly changed white blood cells. Diseases arising from lymphatic tissue are called lymphomas.

Solid tumors may be selected from: liver cancer, stomach cancer, colon cancer, breast cancer, pancreas cancer, prostate cancer, skin cancer, renal cancer, bone cancer, thyroid cancer, skin cancer, including squamous cell carcinoma, esophagus cancer, kidney cancer, bladder cancer, gall cancer, cervical cancer, ovarian cancer, lung cancer, bronchial, small and non-small-cell lung cancer, gastric, and head and neck cancer.

Hematological tumors may be leukemia, such as Acute Myelogenous Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Acute Lymphocytic Leukemia, Acute Leukemia, Acute Promyelocytic Leukemia, Chronic Granulocytic Leukemia (CGL), Chronic Leukemia, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myelomonocytic Leukemia, Common-type Acute Lymphoblastic Leukemia, Eosinophilic Leukemia, Erythroleukemia, Extranodal Lymphoma, Follicular Lymphoma, Hairy Cell Leukemia, Monocytic Leukemia, Prolymphocytic Leukemia.

Hematological tumors may also be lymphoma, such as B Cell Lymphomas, Burkitt Lymphoma, Cutaneous T Cell Lymphoma, High-Grade Lymphoma, Hodgkin

Lymphoma, Non-Hodgkin Lymphoma, Low-grade Lymphoma, Lymphoblastic

Lymphoma, Mantle Cell Lymphoma, Marginal Zone Lymphoma, Mucosa-

Associated Lymphoid Tissue (MALT) Lymphomas, T Cell Lymphomas, peripheral

T cell lymphoma, multiple myeloma, Essential Thrombocythemia, Hairy Cell Lymphoma, Extramedullar myeloma, Granulocytic Sarcomae.

Hematological tumors may also be tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome, and promyelocytic leukaemia.

Tumors may also be of mesenchymal origin, such as fibrosarcoma and rhabdomyosarcoma. Furthermore, tumors may be tumors of the central and peripheral nervous system, such as astrocytoma, neuroblastoma, glioma, and schwannomas; and tumors may be other tumors, such as melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer, and Kaposi's sarcoma.

Compounds of the present invention may induce or inhibit apoptosis. The apoptotic response is aberrant in a variety of human diseases. Compounds described herein, as modulators of apoptosis, will be useful in the treatment of cancer (including but not limited to those types mentioned hereinabove), viral infections (including but not limited to herpesvirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus), prevention of AIDS development in HIV-infected individuals, autoimmune diseases (including but not limited to systemic lupus, erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus), neurodegenerative disorders (including but not limited to Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration), myelodysplastic syndromes, aplastic anemia, ischemic injury associated with myocardial infarctions, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol related liver diseases, hematological diseases (including but not limited to chronic anemia and aplastic anemia), degenerative diseases of the musculoskeletal system (including but not limited to osteoporosis and arthritis) aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis, kidney diseases and cancer pain.

As described above, in certain embodiments of the invention, the compounds of the present invention are pharmaceutically active agents for prophylaxis and/or treatment of cell proliferative disease, including cancer. Thus, these compounds can be used for the manufacture of a pharmaceutical formulation for prophylaxis and/or treatment of cell proliferative disease, including such disease in a mammal such as a human.

Compounds of the present invention may also be useful in the chemoprevention of cancer. Chemoprevention is defined as inhibiting the development of invasive cancer by either blocking the initiating mutagenic event or by blocking the progression of pre-malignant cells, such as by blocking growth of the tumor, that have already suffered an insult or inhibiting tumor relapse.

Compounds disclosed herein may also be useful in inhibiting tumor angiogenesis and metastasis.

Inflammation

As described above, in certain embodiments of the invention, the compounds of the present invention are pharmaceutically active agents for prophylaxis and/or treatment of inflammatory diseases. Thus, these compounds can be used for the manufacture of a pharmaceutical formulation for prophylaxis and/or treatment of inflammation and inflammatory disease in mammals, including humans.

In yet another particular embodiment, said inflammation is mediated preferably by the cytokines TNF-α, IL-1 β, GM-CSF, IL-6 and/or IL-8. Inflammatory diseases can emanate from infectious and non-infectious inflammatory conditions which may result from infection by an invading organism or from irritative, traumatic, metabolic, allergic, autoimmune, or idiopathic causes as shown in the following list.

I. Acute infections

A. Viral B. Bacterial

II. Noninfectious causes

III. Chronic (granulomatous) diseases

A. Bacterial B. Spirochetal

C. Mycotic (Fungal) D. Idiopathic

IV. Allergic, immune, and idiopathic disorders

A. Hypersensitivity reactions

B. Immune and idiopathic disorders

V. Miscellaneous inflammatory conditions A. Parasitic infections

B. Inhalation causes: - Acute (thermal) injury

- Pollution and inhalant allergy

- Carcinogens

C. Radiation injury: - Radionecrosis

Thus, the compounds of the present invention can be used for prophylaxis and/or treatment of inflammation caused by invading organisms such as viruses, bacteria, prions, and parasites as well as for prophylaxis and/or treatment of inflammation caused by irritative, traumatic, metabolic, allergic, autoimmune, or idiopathic reasons.

Consequently, the compounds of the present invention can be used for prophylaxis and/or treatment of inflammatory disease which is initiated or caused by viruses, parasites, and bacteria which are connected to or involved in inflammations.

The following bacteria are known to cause inflammatory diseases: mycoplasma pulmonis (causes e.g. chronic lung diseases (CLD), murine chronic respiratory disease), ureaplasma urealyticum (causes pneumonia in newborns), mycoplasma pneumoniae and chlamydia pneumoniae (cause chronic asthma), C. pneumoniae (causes atherosclerosis, pharyngitis to pneumonia with empyema, human coronary heart disease), Helicobacter pylori (human coronary heart disease, stomach ulcers).

The following viruses are known to cause inflammatory diseases: herpesviruses especially cytomegalovirus (causes human coronary heart disease).

The compounds of the present invention can be used for prophylaxis and/or treatment of inflammatory disease caused and/or induced and/or initiated and/or enhanced by any of the afore-mentioned bacteria or viruses.

Furthermore, the compounds of the present invention can be used for prophylaxis and/or treatment of a disease selected from: inflammatory disease of the central nervous system (CNS), inflammatory rheumatic disease, inflammatory disease of blood vessels, inflammatory disease of the middle ear, inflammatory bowel disease, inflammatory disease of the skin, inflammatory disease uveitis, inflammatory disease of the larynx, including such disease in a mammal, such as a human.

Examples for inflammatory diseases of the central nervous system (CNS) are algal disorders, protothecosis, bacterial disorders, abscessation, bacterial meningitis, idiopathic inflammatory disorders, eosinophilic meningoencephalitis, feline polioencephalomyelitis, granulomatous meningoencephalomyelitis, meningitis, steroid responsive meningitis-arteritis, miscellaneous meningitis / meningoencephalitis, meningoencephalitis in greyhounds, necrotizing encephalitis, pug dog encephalitis, pyogranulomatous meningoencephalomyelitis, shaker dog disease, mycotic diseases of the CNS, parasitic encephalomyelitis, prion protein induced diseases, feline spongiform encephalopathy, protozoal encephalitis-encephalomyelitis, toxoplasmosis, neosporosis, sarcocystosis, encephalitozoonosis, trypanosomiasis, acanthamebiasis, babesiosis, leishmaniasis, rickettsial disorders, rocky mountain spotted fever, canine ehrlichiosis, salmon poisoning, viral disorders, aujeszky's disease, borna disease, canine herpes virus encephalomyelitis, canine distemper encephalomyelitis, canine distemper encephalomyelitis in immature animals, multifocal distemper encephalomyelitis in mature animals, old dog encephalitis, chronic relapsing encephalomyelitis, post-vaccinal canine distemper encephalitis, feline immunodeficiency virus, feline infectious peritonitis, feline leukemia virus, infectious canine hepatitis, La Crosse virus encephalitis, parvovirus encephalitis, rabies, post-vaccinal rabies, tick-borne encephalitis in dogs.

Examples for inflammatory rheumatic diseases are rheumatoid arthritis, scleroderma, lupus, polymyositis, dermatomyositis, psoriatic arthritis, ankylosing spondylitis, Reiters's syndrome, juvenile rheumatoid arthritis, bursitis, tendinitis (tendonitis), and fibromyositis.

Examples for inflammatory diseases of blood vessels are vasculitis, autoantibodies in vasculitis, microscopic polyangiitis, giant cell arteritis,

Takayasu's arteritis, vasculitis of the central nervous system, thromboangiitis obliterans (Buerger's Disease), vasculitis secondary to bacterial, fungal, and parasitic infection, vasculitis and rheumatoid arthritis, vasculitis in systemic lupus erythematosus, vasculitis in the idiopathic inflammatory myopathies, relapsing polychondritis, systemic vasculitis in sarcoidosis, vasculitis and malignancy, and drug-induced vasculitis.

Examples for inflammatory diseases of the middle ear are acute suppurative otitis media, bullous myringitis, granular myringitis, and chronic suppurative otitis media, which can manifest as mucosal disease, cholesteatoma, or both.

Examples for inflammatory bowel diseases are ulcerative colitis, Crohn's disease.

Examples for inflammatory diseases of the skin are acute inflammatory dermatoses, urticaria (hives), spongiotic dermatitis, allergic contact dermatitis, irritant contact dermatitis, atopic dermatitis, erythemal multiforme (EM minor), Stevens-Johnson syndrome (SJS, EM major), toxic epidermal necrolysis (TEN), chronic inflammatory dermatoses, psoriasis, lichen planus, discoid lupus erythematosus, and acne vulgaris

Uveitis is an inflammation located in and/or on the eye and may be associated with inflammation elsewhere in the body. In most circumstances, patients who have uveitis as part of a disease elsewhere in the body are aware of that illness. The majority of patients with uveitis do not have an apparent associated systemic illness. Causes of uveitis can be infectious causes, masquerade syndromes, suspected immune-mediated diseases, and/or syndromes confined primarily to the eye. The following viruses are associated with inflammations: human immunodeficiency virus-l, herpes simplex virus, herpes zoster virus, and cytomegalovirus.

Bacterial or spirochetal caused, induced, initiated and/or enhanced inflammations are tuberculosis, leprosy, proprionobacterium, syphilis, Whipple's disease, leptospirosis, brucellosis, and lyme disease.

Parasitic (protozoan or helminthic) caused, induced, initiated and/or enhanced inflammations are toxoplasmosis, acanthameba, toxocariasis, cysticercosis, onchocerciasis.

Examples of inflammatory diseases caused, induced, initiated and/or enhanced by fungi are histoplasmosis, coccidioidomycosis, candidiasis, aspergillosis, sporotrichosis, blastomycosis, and cryptococcosis.

Masquerade syndromes are, for instance, leukemia, lymphoma, retinitis pigmentosa, and retinoblastoma.

Suspected immune-mediated diseases can be selected from the group comprising ankylosing spondylitis, Behcet's disease, Crohn's disease, drug or hypersensitivity reaction, interstitial nephritis, juvenile rheumatoid arthritis, Kawasaki's disease, multiple sclerosis, psoriatic arthritis, Reiter's syndrome, relapsing polychondritis, sarcoidosis, Sjogren's syndrome, systemic lupus erythematosus, ulcerative colitis, vasculitis, vitiligo, Vogt Koyanagi Harada syndrome.

Syndromes confined primarily to the eye are, for instance, acute multifocal placoid pigmentary epitheliopathy, acute retinal necrosis, birdshot choroidopathy, Fuch's heterochromic cyclitis, glaucomatocyclitic crisis, lens-induced uveitis, multifocal choroiditis, pars planitis, serpiginous choroiditis, sympathetic ophthalmia, and trauma.

Examples for inflammatory diseases of the larynx are gastroesophageal (laryngopharyngeal) reflux disease, pediatric laryngitis, acute laryngeal infections of adults, chronic (granulomatous) diseases, allergic, immune, and idiopathic disorders and miscellaneous inflammatory conditions.

Pediatric laryngitis is known as acute (viral or bacterial) infection such as laryngotracheitis (croup), supraglottitis (epiglottitis), diphtheria, and noninfectious causes are for example spasmodic croup and traumatic laryngitis. Acute laryngeal infections of adults are, for instance, viral laryngitis, common upper respiratory infection, laryngotracheitis, herpes simplex, bacterial laryngitis, supraglottitis, laryngeal abscess, and gonorrhea.

Chronic (granulomatous) diseases can be selected from the group comprising bacterial diseases, tuberculosis, leprosy, scleroma, actinomycosis, tularemia, glanders, spirochetal (syphilis) diseases, mycotic (fungal) diseases, candidiasis, blastomycosis, histoplasmosis, coccidiomycosis, aspergillosis, idiopathic diseases, sarcoidosis, and Wegener's granulomatosis.

Allergic, immune, and idiopathic disorders are, for example, hypersensitivity reactions, angioedema, Stevens-Johnson syndrome, immune and idiopathic disorders, infections of the immunocompromised host, rheumatoid arthritis, systemic lupus erythematosus, cicatricial pemphigoid, relapsing polychondritis, Sjogren's syndrome, and amyloidosis.

Miscellaneous inflammatory conditions are, for instance, parasitic infections, trichinosis, leishmaniasis, schistosomiasis, syngamus laryngeus, inhalation laryngitis, acute (thermal) injury, pollution and inhalant allergy, carcinogens, radiation injury, radiation laryngitis, radionecrosis, vocal abuse, vocal-cord hemorrhage, muscle tension dysphonias, and contact ulcer and granuloma.

Cardiovascular diseases

As described above, in certain embodiments of the invention, the compounds of the present invention can be used for prophylaxis and/or treatment of cardiovascular disease such as a cardiovascular diseases selected from: adult congenital heart disease, aneurysm, stable angina, unstable angina, angina pectoris, angioneurotic edema, aortic valve stenosis, aortic aneurysm, arrhythmia, arrhythmogenic right ventricular dysplasia, arteriosclerosis, arteriovenous malformations, atrial fibrillation, Behcet syndrome, bradycardia, cardiac tamponade, cardiomegaly, congestive cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, cardiovascular disease prevention, carotid stenosis, cerebral hemorrhage, Churg-Strauss syndrome, diabetes, Ebstein's Anomaly, Eisenmenger complex, cholesterol embolism, bacterial endocarditis, fibromuscular dysplasia, congenital heart defects, heart diseases, congestive heart failure, heart valve diseases, heart attack, epidural hematoma, hematoma, subdural, Hippel-Lindau disease, hyperemia, hypertension, pulmonary hypertension, hypertrophic growth, left ventricular hypertrophy, right ventricular hypertrophy, hypoplastic left heart syndrome, hypotension, intermittent claudication, ischemic heart disease, Klippel-Trenaunay-Weber syndrome, lateral medullary syndrome, long QT syndrome mitral valve prolapse, moyamoya disease, mucocutaneous lymph node syndrome, myocardial infarction, myocardial ischemia, myocarditis, pericarditis, peripheral vascular diseases, phlebitis, polyarteritis nodosa, pulmonary atresia, Raynaud disease, restenosis, Sneddon syndrome, stenosis, superior vena cava syndrome, syndrome X, tachycardia, Takayasu's arteritis, hereditary hemorrhagic telangiectasia, telangiectasis, temporal arteritis, tetralogy of fallot, thromboangiitis obliterans, thrombosis, thromboembolism, tricuspid atresia, varicose veins, vascular diseases, vasculitis, vasospasm, ventricular fibrillation, Williams syndrome, peripheral vascular disease, varicose veins and leg ulcers, deep vein thrombosis, Wolff-Parkinson- White syndrome, including such disease in a mammal, such as a human.

In particular embodiments the compounds of the present invention can be used for prophylaxis and/or treatment of a cardiovascular disease selected from: adult congenital heart disease, aneurysms, angina, angina pectoris, arrhythmias, cardiovascular disease prevention, cardiomyopathies, congestive heart failure, myocardial infarction, pulmonary hypertension, hypertrophic growth, restenosis, stenosis, thrombosis and arteriosclerosis, including such disease in a mammal, such as a human.

In other particular embodiments the compounds of the present invention can be used for prophylaxis and/or treatment of cardiac hypertrophy, including such disease in a mammal, such as a human.

Cardiac hypertrophy is the heart's response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress. While hypertrophy can eventually normalize wall tension, it is associated with an unfavorable outcome and threatens affected patients with sudden death or progression to overt heart failure. Accumulating evidence from studies in human patients and animal models suggests that in most instances hypertrophy is not a compensatory response to the change in mechanical load, but rather is a maladaptive process. Cardiac hypertrophy, or thickening, of the heart muscle (myocardium) occurs in response to increased stress on the heart. It typically involves one of the bottom chambers of the heart, which are known as the ventricles. The right ventricle pumps blood to the lungs and the left ventricle pumps blood to the body. The most common causes of hypertrophy are related to increased blood pressure in either the lungs or the body. The extra work of pumping blood against the increased pressure causes the ventricle to thicken over time, the same way a body muscle increases in mass in response to weightlifting.

High blood pressure, or hypertension, is the most frequent cause of left ventricular hypertrophy (LVH). Stenosis of the aortic valve - a condition in which, for a variety of reasons, this heart valve cannot open fully - is another common cause of LVH. Hypertrophic cardiomyopathy (a disease previously known as idiopathic hypertrophic subaortic stenosis or IHSS), and the ongoing use of cocaine round out the list of most common causes of LVH. Hypertrophic cardiomyopathy is a genetic disease related to weakness of the individual muscle fibers of the heart. These fibers need to work harder to pump blood and become thickened over time. Hypertrophic cardiomyopathy occurs in 1 in 500 people and is the most common cardiac cause of sudden death in young athletes.

The most common causes of right ventricle hypertrophy (RVH) are diseases that damage the lung like emphysema and cystic fibrosis. These diseases destroy blood vessels in the lung, causing increased pressure in the remaining vessels. Conditions that decrease oxygen levels, such as chronic bronchitis and sleep apnea, also lead to RVH. Stenosis of the pulmonic heart valve, repeated blood clots to the lungs (chronic pulmonary embolism), and primary pulmonary hypertension are a few of the remaining causes of RVH.

CDK9 is known to be involved in cardiac hypertrophy (reviewed in Sano & Schneider, Circulation Research, 2004, 95, 867). Activation of CDK9 to pathophysiological levels leads to mitochondrial dysfunction, apoptosis, and heart failure via suppression of PGC-1 , an essential co-activator for the transcription of nuclear and mitochondrial genes that encode mitochondrial proteins (Sano et al.,

EMBO J., 2004, 23, 3559-3569), and hence blockade of Cdk9 activity is an accepted strategy expected to aid in the treatment of cardiac hypertrophy.

Pain

As described above, in certain embodiments of the invention, the compounds of the present invention may also be used to treat one or more of any type of pain, including those referenced herein, including such pain in a mammal, such as a human. In particular such embodiments said pain comprises inflammatory pain and/or neuropathic pain. Debilitating acute or chronic pain is a constant backdrop to daily life for many people. Current estimates suggest that 1 in 10 adults suffer from chronic pain at some point in their lives. In terms of both lost productivity and treatment, the cost to society in the US alone surpasses 100 billion dollars annually. Unfortunately, current treatments for pain are only partially effective, and many also cause debilitating or dangerous side effects. For example, many of the traditional analgesics used to treat severe pain induce debilitating side effects such as nausea, dizziness, constipation, respiratory depression, and cognitive dysfunction (Brower, New paths to pain relief, Nat Biotechnol, 2000, 18(4), 387- 39).

Although there is already a broad panel of approved pain medications like nonnarcotic analgesics, opioid analgesics, calcium channel blockers, muscle relaxants, and systemic corticosteroids available, said treatments remain merely empirical and, while they may relieve the symptoms of pain, they do not lead to complete relief in most cases. This is also due to fact that the mechanisms underlying the development of the different types of pain are still only poorly understood. Researchers are only just beginning to appreciate the complexity and diversity of the signaling systems used to relay nerve impulses for each type of pain.

Generally, pain is defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage, according to the International Association for the Study of Pain (IASP). Specifically, pain may occur as acute or chronic pain.

Acute pain occurs for brief periods of time, typically less than 1 month and is associated with temporary disorders. It is a natural body response to let the host be aware of physiological or biochemical alteration that could result in further damage within a short period of time. It is felt when noxious stimuli activate high threshold mechanical and/or thermal nociceptors in peripheral nerve endings and the evoked action potentials in thinly myelinated (AS) and/or unmyelinated (C) afferent fibres reach a conscious brain. Said noxious stimuli may be provided by injury, surgery, illness, trauma or painful medical procedures. Acute pain usually disappears when the underlying cause has been treated or has healed. Unrelieved acute pain, however, may lead to chronic pain problems that may result in long hospital stays, rehospitalizations, visits to outpatient clinics and emergency departments, and increased health care costs. In contrast to acute pain, chronic pain persists long after the initial injury has healed and often spreads to other parts of the body, with diverse pathological and psychiatric consequences. Chronic somatic pain results from inflammatory responses to trauma in peripheral tissues (e.g., nerve entrapment, surgical procedures, cancer, or' arthritis), which leads to oversensitization of nociceptors and intense searing pain responses to normally non-noxious stimuli (hyperalgesia). Chronic pain is continuous and recurrent and its intensity will vary from mild to severe disabling pain that may significantly reduce quality of life. Chronic pain is currently treated with conventional analgesics such as Non steroidal anti- inflammatory drugs (NSAIDs such as Ibuprofen, Naproxen), Cox-2 inhibitors (Celecoxib, Valdecoxib, Rofecoxib) and opiates (codeine, morphine, thebaine, papaverine, noscapine). For a significant number of patients however, these drugs provide insufficient pain relief.

Another subtype of pain, inflammatory pain, can occur as acute as well as chronic pain. Resulting injuries of tissue and neurons must not but may develop into long- lasting chronic neuropathic pain effects in succession to such inflammatory events. Inflammatory pain is mediated by noxious stimuli like e.g. inflammatory mediators (e.g. cytokines, such as TNFα, prostaglandins, substance P, bradykinin, purines, histamine, and serotonine), which are released following tissue injury, disease, or inflammation and other noxious stimuli (e.g. thermal, mechanical, or chemical stimuli). In addition, cytokines and growth factors can influence neuronal phenotype and function (Besson J. M., The neurobiology of pain, Lancet, 1999, 353(9164), 1610-1615). These mediators are detected by nociceptors (sensory receptors) that are distributed throughout the periphery of the tissue. Said nociceptors are sensitive to noxious stimuli (e.g. mechanical, thermal, or chemical), which would damage tissue if prolonged (Koltzenburg M, Neural mechanisms of cutaneous nociceptive pain, Clin J Pain, 2000, 16(3 Suppl), 131 - 138). A particular class of so called C-nociceptors represent a class of "silent" nociceptors that do not respond to any level of mechanical or thermal stimuli but are activated in presence of inflammation only.

Current approaches for the treatment of especially inflammatory pain aim at cytokine inhibition (e.g. 1 L1 β) and suppression of pro- inflammatory TNFα. Current approved anticytokine / antiTNFα treatments are based on chimeric antibodies such as Infliximab and Etanercept which reduce TNFα circulation in the bloodstream. TNFα is one of the most important inflammatory mediators which induces synthesis of important enzymes such as COX-2, MMP, iNOS, cPLa2 and others. The main drawbacks of "biologicals" such as chimeric antibodies, however, reside in their immunogenic potential with attendant loss of efficacy and their kinetics, leading to a more or less digital all-or-nothing reduction of circulating TNFα. The latter can result in severe immune suppressive side effects.

A distinct form of chronic pain, neuropathic (or neurogenic) pain, arises as a result of peripheral or central nerve dysfunction and includes a variety of conditions that differ in aetiology as well as location. Generally, the causes of neuropathic pain are diverse, but share the common symptom of damage to the peripheral nerves or components of central pathways. Without being bound by theory, the causative factors of neuropathic pain may be metabolic, viral or a mechanical nerve lesion. Neuropathic pain is believed to be sustained by aberrant somatosensory processes in the peripheral nervous system, the CNS, or both. Neuropathic pain is not directly linked to stimulation of nociceptors, but instead, is thought to arise e.g. from oversensitization of glutamate receptors on postsynaptic neurons in the gray matter (dorsal horn) of the spinal cord. Neuropathic pain is associated with conditions such as nerve degeneration in diabetes and postherpetic neuralgia (shingles). Neuropathic pain conditions are the consequence of a number of diseases and conditions, including diabetes, AIDS, multiple sclerosis, stump and phantom pain after amputation, cancer-related neuropathy, post-herpetic neuralgia, traumatic nerve injury, ischemic neuropathy, nerve compression, stroke and spinal cord injury.

Management of neuropathic pain remains a major clinical challenge, partly due to an inadequate understanding of the mechanisms involved in the development and maintenance of neuropathic pain. Many existing analgesics are ineffective in treating neuropathic pain and most of current narcotic and non-narcotic drugs do not provide control of neuropathic pain. Current clinical practice includes the use of a number of drug classes for the management of neuropathic pain, for example anticonvulsants, tricyclic antidepressants, and systemic local anaesthetics. However, the usual outcome of such treatment is merely partial or unsatisfactory pain relief, and in some cases the adverse effects of these drugs outweigh their clinical usefulness. Classic analgesics are widely believed to be poorly effective or ineffective in the treatment of neuropathic pain. Few clinical studies on the use of non steroidal anti- inflammatory drugs (NSAIDs) or opiates in the treatment of neuropathic pain have been conducted, but in those which have, the results appear to indicate that NSAIDs are poorly effective or ineffective and opiates only work at high doses. A review analysing the controlled clinical data for peripheral neuropathic pain (PNP) (Pain 1997 73(2), 123-39) reported that NSAIDs were probably ineffective as analgesics for PNP and that there was no long-term data supporting the analgesic effectiveness of any drug.

Summarizing, available analgesic drugs often only produce insufficient pain relief. Although tricyclic antidepressants and some antiepileptic drugs, for example gabapentine, lamothgine and carbamazepine, are efficient in some patients, there remains a large unmet need for efficient drugs for the treatment of these conditions. In conclusion, there is a high unmet need for safe and effective methods of treating one or more of any type of pain, in particular chronic inflammatory and/or neuropathic pain.

One aspect of the invention relates to methods and compositions for treating one or more of any type of pain, including those referenced herein, comprising administering an effective amount of at least one compound according to the present invention to a subject in need thereof, including where such subject is a mammal such as a human.

The term "pain" as used herein generally relates to any type of pain and broadly encompasses types of pain such as acute pain, chronic pain, inflammatory and neuropathic pain.

One aspect of the present invention relates to a pharmaceutical composition comprising at least one compound according to the present invention as an active ingredient, together with at least one pharmaceutically acceptable carrier, excipient and/or diluent, in combination with an analgesic agent, wherein said analgesic agent has a mechanism of action other than inhibition of a CDK.

In a particular embodiment of the present invention, pain comprises neuropathic pain and associated conditions. The pain may be chronic, allodynia (the perception of pain from a normally innocuous stimulus), hyperalgesia (an exaggerated response to any given pain stimulus) and an expansion of the receptive field (i.e. the area that is "painful" when a stimulus is applied), phantom pain or inflammatory pain.

Acute pain types comprise, but are not limited to pain associated with tissue damage, postoperative pain, pain after trauma, pain caused by burns, pain caused by local or systemic infection, visceral pain associated with diseases comprising: pancreatitis, intestinal cystitis, dysmenorrhea, Irritable Bowel syndrome, Crohn's disease, ureteral colic and myocardial infarction.

Furthermore, the term "pain" comprises pain associated with CNS disorders comprising: multiple sclerosis, spinal cord injury, traumatic brain injury, Parkinson's disease and stroke.

In a particular embodiment, "pain" relates to chronic pain types comprising headache (for example migraine disorders, episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania), low back pain, cancer pain, osteoarthritis pain and neuropathic pain, but is not limited thereto.

Inflammatory pain (pain in response to tissue injury and the resulting inflammatory process) as defined herein relates to inflammatory pain associated with diseases comprising connective tissue diseases, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and arthritis, but is not limited thereto.

Neuropathic pain (pain resulting from damage to the peripheral nerves or to the central nervous system itself) may comprise painful diabetic peripheral neuropathy, post- herpetic neuralgia, trigeminal neuralgia, cranial neuralgia, post- stroke neuropathic pain, multiple sclerosis- associated neuropathic pain, postsurgical neuropathic pain, neuropathy- associated pain such as in idiopathic or post-traumatic neuropathy and mononeuritis, HIV/AIDS-associated neuropathic pain, cancer-associated neuropathic pain, carpal tunnel- associated neuropathic pain, spinal cord injury-associated pain, complex regional pain syndrome, fibromyalgia-associated neuropathic pain, lumbar and cervical pain, reflex sympathic dystrophy, phantom limb syndrome or peripheral nerve or spinal cord trauma, entrapment neuropathy, nerve transection including surgery, Lissauer tract section, limb amputation and stump pain, neuroma/tumour compression, arteriovenous malformation, Vitamin B 12 deficiency, diabetic neuropathy, alcoholic neuropathy, pain caused by the side effects of anti-cancer and anti-AIDS therapies, pain associated with inflammation or infection of a tooth (toothache), visceral pain, pain caused by chemical bums, pain caused by local or systemic infection, or pain caused by connective tissue disease. The connective tissue disease may be one of: rheumatoid arthritis, Wallenberg's syndrome, systemic lupus erythematosus, multiple sclerosis, or polyarteritis nodosa. The neuropathy can be classified as radiculopathy, mononeuropathy, mononeuropathy multiplex, polyneuropathy or plexopathy. Diseases in this class can be caused by a variety of nerve- damaging conditions or procedures, including, without limitation, trauma, stroke, demyelinating diseases, abscess, surgery, amputation, inflammatory diseases of the nerves, causalgia, diabetes, collagen vascular diseases, trigeminal neuralgia, rheumatoid arthritis, toxins, cancer, chronic alcoholism, herpes infection, AIDS and chemotherapy. Nerve damage causing hyperalgesia can be in peripheral or CNS nerves.

The term "allodynia" denotes pain arising from stimuli which are normally not painful. Allodynic pain may occur other than in the area stimulated. The terms "hyperalgesia'V'hyperalgesic" denote an increased sensitivity to a painful stimulus. The terms "hypoalgesia'V'hypoalgesic" denote a decreased sensitivity to a painful stimulus.

One aspect of the present invention relates to a method for treating one or more of any type of pain, such as the types of pain referenced herein, and associated conditions, wherein the term "treating" comprises the prevention, amelioration or treatment of any type of pain and associated conditions. Specifically, the one aspect of the invention relates to a method for the treatment of neuropathic and/or inflammatory pain, comprising administering an effective amount of at least one compound according to the present invention to a subject in need thereof, including where such subject is a mammal such as a human.

Without being bound by theory, the role of CDK9 in the development of pain could be based on the following mechanism of action: Both cyclin T1 and CDK9 stimulate the basal promoter activity of TNFα. TNFα is a pro-inflammatory cytokine and pain mediator that controls expression of inflammatory genetic networks. For mediation of cellular TNF receptor responses, the nuclear factor-KB (NFKB) pathway is crucial. TNFα triggers its recruitment to cytokine genes while NFKB interacts with the p-TEFb complex for stimulation of gene transcription (Barbohc M. et al., NFKB Binds P- TEFb to Stimulate Transcriptional Elongation by RNA Polymerase II. Molecular Cell, 2001 , Vol. 8, 327-337).

Additionally, it has been shown that CDK9 is a binding partner of TRAF2, a member of the TNFα receptor complex (MacLachlan T.K. et al., Binding of CDK9 to TRAF2. J Cell Biochem, 1998, 71 (4), 467-478), while GP130, a subunit of the pro-inflammatory IL6 receptor complex has recently been identified as another potential binding partner of CDK9 (Falco G. D. et al., CDK9, a member of the cdc2- like family of kinases, binds to gp130, the receptor of the IL-6 family of cytokines. Oncogene, 2002, 21 (49), 7464-7470). As a key player in TNFα and interleukin signaling as well as NFKB mediated expression of several genes (e.g. cytokines as pain mediators), CDK9 can thus be considered as a central target for the treatment of inflammatory pain.

One aspect of the present invention relates to methods to down-regulate NFKB by administering a compound according to the present invention to a patient in need thereof, including where such patient is a mammal such as a human.

For the treatment of neuropathic pain, pharmacological action has to take place beyond blood-brain-barrier (BBB) in the central nervous system (CNS). Microglial cells as the principal immune cells in the CNS, release, if activated, a variety of noxious factors such as cytokines (TNFα, IL1 β, IL6) and other pro-inflammatory molecules (Huwe et al., Small molecules as inhibitors of cyclin-dependent kinases. Angew Chem lnt Ed Engl, 2003, 42(19), 2122-2138). Microglia is activated by stimulation of TNFα receptor or Toll-like receptor and signal is mediated via IK kinase (IKK) and NFKB leading to transcriptional activation of the cytokines described above. Microglial contribution has been discussed as instrumental in chronic CNS diseases and may contribute to pain perception (Watkins L. R. et al., Glial proinflammatory cytokines mediate exaggerated pain states: implications for clinical pain. Adv Exp Med Biol., 2003, 521 , 1 -21 ).

Recently it has been shown that NFKB regulates expression of Cyclooxygenase-2 (COX-2) via Interleukin 1 β (IL1 β) in the spinal cord (Lee K.M. et al., Spinal NFKB activation induces COX-2 upregulation and contributes to inflammatory pain hypersensitivity. European Journal of Neuroscience, 2004, Vol. 19, 3375-3381 ). As the major contributor to elevation of spinal prostaglandin E2, the pain mediator COX-2 is already known as a target for a variety of anti-nociceptive/anti- inflammatory drugs. NFKB inhibitors have proven their ability to reduce COX-2 levels and mechanical allodynia as well as thermal hyperalgesia in animal models significantly.

In contrast to inhibition of Cox-2, inhibition of CDK9 action could lead to suppression of a variety of pain mediators instead of just a single one. Thereby, anti-nociceptive action of CDK9 inhibitors might be improved in comparison to e.g. COX-2 inhibitors. Due to its relevance for NFKB mediated gene transcription, inhibition of CDK9 may therefore be a reasonable approach not only for the treatment of acute inflammatory pain, but also for the treatment of chronic pain.

The present invention is also directed to pharmaceutical compositions comprising at least one compound according to the present invention as an active ingredient together with at least one pharmaceutically acceptable (i. e. non-toxic) carrier, excipient and/or diluent for administration to a subject in need thereof, including where such subject is a mammal such as a human.

Furthermore, the invention also comprises compositions combining at least two inhibitors of CDK, wherein at least one of said inhibitors of said CDK is a compound according to the present invention, and/or pharmaceutically acceptable salts thereof. Said at least two inhibitors may inhibit the same cyclin- dependent kinase or may also inhibit different types of cyclin-dependent kinases, e.g. one inhibitor in the composition may inhibit CDK9 while the other inhibitor is capable of inhibiting CDK2, for example.

In a further particular embodiment, the invention is directed to compositions comprising at least one compound according to the present invention in combination with one or more additional pain- reducing agents and to a method of administering such a composition.

An individual pain medication often provides only partially effective pain alleviation because it interferes with just one pain- transducing pathway out of many. Thus, it is also intended to administer a compound according to the present invention in combination with a pain-reducing (analgesic) agent that acts at a different point in the pain perception process. An "analgesic agent" comprises a molecule or combination of molecules that causes a reduction in pain. An analgesic agent employs a mechanism of action other than inhibition of CDK.

One class of analgesics, such as nonsteroidal anti-inflammatory drugs (NSAIDs), down- regulates the chemical messengers of the stimuli that are detected by the nociceptors and another class of drugs, such as opioids, alters the processing of nociceptive information in the CNS. Other analgesics are local anesthetics, anticonvulsants and antidepressants such as tricyclic antidepressants. Administering one or more classes of drug in addition to CDK inhibitors can provide even more effective amelioration of pain. Particular NSAIDs for use in the methods and compositions of the present invention include aspirin, acetaminophen, ibuprofen, and indomethacine. Furthermore, cyclooxygenase-2 (COX- 2) inhibitors, such as specific COX- 2 inhibitors (e.g. celecoxib, COX189, and rofecoxib) may also be used as an analgesic agent in the methods or compositions of the present invention. Particular tricyclic antidepressants are selected from the group consisting of Clomipramine, Amoxapine, Nortriptyline, Amitriptyline, Imipramine, Desipramine, Doxepine, Trimipramine, Protriptyline, and Imipramine pamoate. Furthermore, the use of anticonvulsants (e.g. gabapentine), GABAB agonists (e.g. L-baclofen), opioids, vanniloid receptor antagonists and cannabinoid (CB) receptor agonists, e.g. CB1 receptor agonists as analgesic is also preferred in the methods and compositions in the present invention.

Other uses

In another aspect of the present invention, the compounds of the present invention or pharmaceutically acceptable salts thereof can be used as an inhibitor for a protein kinase, preferably as an inhibitor for a cellular protein kinase.

In a particular embodiment of these aspects said cellular protein kinase is a cyclin- dependent protein kinase (CDK). The cyclin-dependent protein kinase can be selected from the group comprising: CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11 , CDK12, CDK13, CrkRS (Crk7, CDC2- related protein kinase 7), CDKL1 (cyclin-dependent kinase-like 1 ); KKIALRE, CDKL2 (cyclin-dependent kinase-like 2), KKIAMRE, CDKL3 (cyclin-dependent kinase-like 3), NKIAMRE, CDKL4, similar to cyclin-dependent kinase-like 1 , CDC2L1 (cell division cycle 2-like 1 ), PITSLRE B, CDC2L1 (cell division cycle 2- like 1 ), PITSLRE A, CDC2L5 (cell division cycle 2-like 5), PCTK1 (PCTAIRE protein kinase 1 ), PCTK2 (PCTAIRE protein kinase 2), PCTK3 (PCTAIRE protein kinase 3) or PFTK1 (PFTAIRE protein kinase 1 ). In particular such embodiments, said cyclin-dependent protein kinase is CDK9.

In other aspects the present invention provides a method to inhibit a cyclin- dependent protein kinase, including a cyclin-dependent protein kinase in a cell, such as a cell in a patient in need thereof, including where patient is a mammal such as a human. In certain such aspects the present invention provides a method to inhibit CDK9, including in a patient in need thereof. In a further aspect the present invention provides methods for prophylaxis and/or treatment of a disease selected from: cell proliferative disease, such as cancer; pain, such as inflammatory pain or neuropathic pain; inflammation; cardiovascular disease, such as cardiac hypertrophy; and infectious diseases, such as viral infections including HIV, comprising administering to an individual, such as a mammal an amount of at least one compound according to the present invention and/or pharmaceutically acceptable salts thereof, effective to prevent and/or treat such disease. In certain such aspects of the invention, said mammal is a human.

Formulations, Dosages, Packages and Applications

The compositions of this invention can be formulated and administered to treat individuals in need by any means that produces contact of the active ingredient with the agent's site of action in the body of a mammal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The pharmaceutical compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1 ) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam. In certain embodiments, the pharmaceutical preparations may be non-pyrogenic, i.e., do not elevate the body temperature of a patient.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1 ) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene

(BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of inhibitor which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous (i.m., i.v., i.p., and i.e. respectively). The phrases "systemic administration," "administered systemically,"

"peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

For injection, the pharmaceutical compositions of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the pharmaceutical compositions may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, sachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in- oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. An inhibitor of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Gelatin capsules contain the active ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. Solid compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. A particular formulation is a solution or suspension in an oil, for example olive oil, Miglyol, or Capmul, in a soft gelatin capsule. Antioxidants may be added to prevent long-term degradation as appropriate.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered inhibitor moistened with an inert liquid diluent. The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulations so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active inhibitor(s) of the present invention, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

For buccal administration the therapeutic compositions may take the form of tablets or lozenges formulated in a conventional manner.

For administration by inhalation, the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, thchlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic agents and a suitable powder base such as lactose or starch.

The pharmaceutical compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more inhibitors of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In addition to the formulations described previously, the pharmaceutical compositions may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic compositions may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the compositions of the invention are formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing.

In some cases, in order to prolong the therapeutic effect of an inhibitor, it is desirable to slow the absorption of the inhibitor from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the inhibitor then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered inhibitor form is accomplished by dissolving or suspending the inhibitor in an oil vehicle.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active inhibitor.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to a compound of the invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing an inhibitor of the present invention in the proper medium. Absorption enhancers can also be used to increase the flux of the drug across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound of the present invention in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. In other embodiments, the pack or dispenser may be further packaged in an outer carton.

A pharmaceutical composition of the present invention can also be formulated as a sustained and/or timed release formulation. Such sustained and/or timed release formulations may be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference. The pharmaceutical compositions of the present invention can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions. Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, may be readily selected for use with the pharmaceutical compositions of the invention. Thus, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelcaps, caplets, powders, and the like, that are adapted for sustained release are encompassed by the present invention.

Injectable depot forms are made by forming microencapsuled matrices of the subject inhibitors in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydhdes). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

When the compounds of the present invention are administered as pharmaceuticals, to individuals, such as humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (in certain embodiments, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

The present invention provides new methods of treating proliferative, degenerative and other disorders or diseases, including cancer, by administering a therapeutically effective amount of at least one of the compounds disclosed herein or an isomeric, prodrug, tautomeric, pharmaceutically acceptable salt, N-oxide or stereoisomer^ form thereof. The present invention further provides methods of treating proliferative, degenerative or other disorders or diseases, including cancer, by administering a therapeutically effective combination of at least one of these compounds and another anti-cancer or anti-proliferative agent.

The term "prodrug", as used herein, refers to an agent which is converted into a pharmacologically active parent drug in vivo, such as a compound as defined herein. The term "prodrug" includes any covalently bonded carriers which release an active parent drug of the present invention in vivo when such prodrug is administered to an individual. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (i.e., solubility, bioavailability, manufacturing, transport, pharmacodynamics etc.) the compounds of the present invention may be delivered in prodrug form. Prodrugs, for instance, may be bioavailable by oral administration whereas the parent drug is not. Thus, the present invention is intended to cover prodrugs of the presently claimed compounds, methods of delivering the same, and compositions containing the same. Prodrugs of the present invention are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Prodrugs include compounds of the present invention wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when the prodrug of the present invention is administered to a mammalian subject, it cleaves to form a free hydroxyl, free amino, or free sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups in the compounds of the present invention. Generally speaking, prodrugs are derivatives of per se drugs, which after administration undergo conversion to the physiologically active species. The conversion may be spontaneous, such as hydrolysis in the physiological environment, or may be enzyme catalyzed. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, and/or dephosphorylated to produce the active compound.

From among the voluminous scientific literature devoted to prodrugs in general, the foregoing examples are cited: Gangwar et al., "Prodrug, molecular structure and percutaneous delivery", Des. Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21. (1977); Nathwani and Wood, "Penicillins: a current review of their clinical pharmacology and therapeutic use", Drugs 45(6): 866-94 (1993); Sinhababu and Thakker, "Prodrugs of anticancer agents", Adv. Drug Delivery Rev. 19(2): 241 -273 (1996); Stella et al., "Prodrugs. Do they have advantages in clinical practice?", Drugs 29(5): 455-73 (1985); Tan et al. "Development and optimization of anti-HIV nucleoside analogs and prodrugs: A review of their cellular pharmacology, structure-activity relationships and pharmacokinetics", Adv. Drug Delivery Rev. 39(1-3): 117-151 (1999); Design of Prodrugs (Bundgaard H. ed.) 1985 Elsevier Science Publishers B. V. (Biomedical Division), Chapter 1 ; Design of Prodrugs: Bioreversible derivatives for various functional groups and chemical entities (Hans Bundgaard); Bundgaard et al. Int. J. of Pharmaceutics 22 (1984) 45 - 56 (Elsevier); Bundgaard et al. Int. J. of Pharmaceutics 29 (1986) 19 - 28 (Elsevier); Bundgaard et al. J. Med. Chem. 32 (1989) 2503 - 2507 Chem. Abstracts 93, 137935y {Bundgaard et al.); Chem. Abstracts 95, 138493f {Bundgaard et al.); Chem. Abstracts 95, 138592n {Bundgaard et al.); Chem. Abstracts 110, 57664p {Alminger et al.); Chem. Abstracts 115, 64029s {Buur et al.); Chem. Abstracts 115, 189582y {Hansen et al.); Chem. Abstracts 117, 14347q {Bundgaard et al.); Chem. Abstracts 117, 5579Ox {Jensen et al.); and Chem. Abstracts 123, 17593b {Thomsen et al.).

An active compound may be administered as a salt or prodrug that, upon administration to the individual, is capable of providing directly or indirectly the parent compound, such as a compound as defined herein, or that exhibits activity itself. Nonlimiting examples include a pharmaceutically-acceptable salt, alternatively referred to as a "physiologically acceptable salt". In addition, modifications made to a compound can affect its biologic activity, in some cases increasing the activity over the parent compound. This activity can be assessed by preparing a salt or prodrug form of the compound, and testing its activity by using methods described herein or other methods known to those of skill in the art.

As will be apparent to a person skilled in the art, through the use of a prodrug of a given subject compound, an individual treated with such prodrug will be exposed to, and hence indirectly administered with, the subject compound. Such a procedure may expose those cells associated with a disease, such as a proliferative disease or disorder including cancer, to the subject compound.

The present invention is intended to include all isotopes of atoms occurring on the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include ¹²C and ¹⁴C.

The term "metabolite", as used herein, refers to any substance produced by the metabolism or by a metabolic process. Metabolism, as used herein, refers to the various physical/chemical/biochemical/phamacological reactions involved in the transformation of molecules or chemical compounds occurring in the cell, tissue, system, body, animal, individual, patient or human therein.

Another aspect of the invention relates to a packaged pharmaceutical comprising a pharmaceutical composition of a compound according to the present invention and/or stereoisomeric form and/or pharmaceutically acceptable salts thereof wherein said packaged pharmaceutical further comprises instructions to administer an effective amount of the pharmaceutical composition to an individual suffering from a disease selected from a cell proliferative diseases, such as cancer, pain, inflammation, a cardiovascular diseases, such as cardiac hypertrophy, and a infectious diseases, especially viral infections such as HIV. Another aspect of the invention relates to a method to treat a disease amenable to the inhibition of CDK9 by administering a compound according to the present invention to a patient suffering from said disease.

Another aspect of the invention relates to a method to inhibit CDK9 with a compound according to the present invention. Said inhibition of CDK9 may be in vivo or in vitro. Inhibition of CDK9 in vivo includes inhibition of CDK9 in a patient suffering from a disease amenable to the inhibition of CDK9.

Description of figures:

Figure 1 : Figure 1 schematically depicts the spared nerve injury model (SNI model, as developed by Decosterd and Woolf (Decosterd and Woolf, Pain 2000; 87:149-158), which is characterized by ligation and section of two branches of the sciatic nerve (namely tibial and common peroneal nerves) leaving the sural nerve intact.

Figure 2: Figure 2 schematically depicts a possible role of CDK9 as a target in the development of pain.

EXAMPLES

1. Syntheses of compounds:

1.1. General synthesis schemes:

The synthesis of the inventive methylenesulfone and methylenesulfonamide derivatives of 4,6-disubstituted pyrimidines according to the present invention was preferably carried out according to the general synthetic sequence, shown in Scheme 1 , involving in a first step amination of the pyrimidine ring followed by Suzuki reaction or an inverse order of the reaction steps. Synthesis of the compounds given in 2.2. below ("Preparation of specific compounds") was according to the anticlockwise route.

Scheme 1

R⁴ R⁴

Hal represents -Cl, -Br or -I.

R² and R⁴ have independently of each other the meanings as defined above, preferably R² and R⁴ are hydrogen and L has the meanings as defined herein. In the case protecting groups have been used, a final deprotection step may follow.

Introduction of the amine moiety can be performed by known methods (J. E. Arrowsmith et al., Journal of Medicinal Chemistry 1989, 32(3), 562-568, J. R. Porter et al, Bioorganic Medicinal Chemistry Letters 2002, 12(12), 1595-1598): For example, as outlined in Scheme 1 , amination is performed by reacting equimolar quantities of 4, 6-dihalogenated pyrimidine and an amino compound in a polar solvent, and in the presence of an organic base or an organic or inorganic acid at temperatures in the range of 50 to 120⁰C. Preferably, the polar solvent is N-methyl-2-pyrrolidinone (NMP) or a lower alcohol, such as isopropanol or butanol, the organic base is selected for instance from N,N-diisopropylethylamine (DIPEA), N-methyl-piperidine or NEt₃, the acid can be selected for instance from HCI, H₂SO₄, CH₃COOH and the reaction is carried out at a temperature in the range of 60 to 110⁰C, preferably in the range of 70 to 100°C. It is to be understood, that the reaction temperature depends on the reactivity of the amino compound: For less reactive amino compounds a reaction temperature in the range of 80 to 110⁰C is preferred and in these cases a higher boiling solvent such as butanol or NMP affords the desired compounds in good yields.

The introduction of R³ into the pyrimidine scaffold as outlined in Scheme 1 , is performed preferably via Suzuki coupling at temperatures in the range of 60 to 110°C, preferably at temperatures in the range of 70 to 100°C, more preferably between 75 to 90⁰C. (I. Minoru, K. Machiko, T. Masanao, Synthesis 1984, 936- 938; J. P. Wolfe, R. A. Singer, B. H. Yang and S. L. Buchwald, Journal of the American Chemical Society 1999, 121 , 9550-9561 ).

The reaction is carried out in organic solvents, such as DME, DMF, THF, dioxane or methanol or this reaction is carried out in a mixture of an organic solvent and water, such as DMF/water, DME/water or THF/water, in the presence of a base, such as NaHCO₃, NaOH, TIOH, NaOMe, K₂CO₃, K₃PO₄, NEt₃, Cs₂CO₃ or TI₂CO₃ and in the presence of a catalyst, such as PdCI₂(dppf) {[1 ,1 -bis- (diphenylphosphino)ferrocene]dichloropalladium II}, Pd(PPh₃)₄ or PdCI₂(PPh₃)₂ or a catalyst/ligand system, such as Pd(OAc)₂/PPh₃, Pd(OAc)₂/ 2- (Dicyclohexylphosphino)-biphenyl or Pd(OAc)₂/tris(2,4,6-thmethoxyphenyl) phosphine.

The R³ containing boron compound used for this reaction is selected from the group comprising:

R₃B(OH)₂, R₃B(OPr')₂, R₃-9-BBN

R₃-B, (9-BBN = 9-borabicyclo[3.3.1]nonanyl) or The chemistry described above can be done in either order and further dehvatisation can be carried out after amination and before/after subsequent Suzuki cross coupling. Other suitable methods will be apparent to the chemist skilled in the art as will be the methods for preparing the starting materials and intermediates. When protecting groups have been used, optionally a final deprotecting step can be carried out according to general deprotecting reactions known to a person skilled in the art.

For example, inventive compounds according to the present invention can be prepared from suitably functionalised anilines on reaction with the appropriate reagents. Introduction and removal of protective groups (PG) may be necessary for several synthetic steps. This includes for example the use of t-butylcarbamate (BOC) protection for amino acids with standard conditions for introduction and removal.

The compounds H₂N-R⁵-L-R⁶ of the present invention can be synthesized as follows:

c) Amine, DCM d) Fe, EtOH, HCI, 80⁰C 2 hours

Alternatively the compounds H₂N-R⁵-L-R⁶ of the present invention can be synthesized as depicted in Scheme 2:

Scheme 2

Commercially available Ingold; Ingold; Shaw J. Chem. Soc. 1927, 818. Wyrick, S.D.; Hall, I.H.; Dubey, A. J. Pharm. Sci. 1984, 73(3), 374-377.

The residue R in Scheme 2 may be amino, methyl or substituted amino or substituted methyl as described herein.

Alternatively, the compounds of the present invention can be synthesized as depicted in Scheme 3.

Scheme 3

Commercially available Lutter Chem. Ber. 1897, 1065. Sammond, D. M. et al. Sammond, D. M. et al. Bioorg. Med. Chem. Lett. Bioorg. Med. Chem. L 2005, 15(15), 3519-3523. 2005, 15(15), 3519-352

When protecting groups have been used, optionally a final deprotecting step can be carried out according to general deprotecting reactions known to a person skilled in the art.

1.2. Preparation of specific compounds:

Synthesis of C-{3-[6-(2-methoxy-phenyl)-pyrimidin-4-ylamino1-phenyl)-N,N- dimethyl-methanesulfonamide (compound #1 )

a) ArB(OH)₂, PdCI₂ (dppf), DME, 85°C, 16 hours b) ArNH₂, PrOH, HCI, 85°C, 16 hours

Step a) 4-Chloro-6-(2-methoxy-phenyl)-pyrimidine

A mixture of 4,6-dichloropyrimdine (0.98 g), 2 -methoxy - benzeneboronic acid (1.0 g),

[1 ,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (11 ) (0.54 g), 1 ,2- dimethoxyethane (60 ml), sodium hydrogen carbonate (1.1 g) and water (20 ml) were heated together at 85⁰C for 16 hours. The mixture was cooled to room temperature, diluted with ethyl acetate and the organic phase separated, dried over magnesium sulphate, filtered, evaporated, then purified by column chromatography (silica gel, ethyl acetate/petroleum ether), to give 4-Chloro-6-(2-methoxy-phenyl)-pyrimidine,

1.18 g

¹H NMR CDCI₃, 3.94 (3H, s, CH₃), 7.04 (1 H, d, ArH), 7.14 (1 H, m, ArH), 7.47 (1 H, m,

ArH), 8.06 (2H, s, ArH), 9.05 (1 H, s, ArH)

Step b) C-{3-[6-(2-nnethoxy-phenyl)-pynnnidin-4-ylannino1-phenyl)-N,N-dinnethyl- methanesulfonamide (compound #1 )

A solution of 4-Chloro-6-(2-methoxy-phenyl)-pyrimidine (0.1 g), (3-Amino-phenyl)- N,N-dimethyl-methanesulfonamide (0.1 g) concentrated hydrochloric acid (3 drops) in propan-2-ol (10 ml) were heated together at 85°C for 16 hours, then cooled and evaporated. The residue was dissolved in ethyl acetate and sodium hydrogen carbonate solution. The organic phases separated, over magnesium sulphate, filtered, evaporated, and then purified by column chromatography (silica gel, ethyl acetate/petroleum ether), to give C-{3-[6-(2-methoxy-phenyl)-pyrimidin-4- ylamino]-phenyl}-N,N-dimethyl-methanesulfonamide (Compound #1 ), 0.12 g, ¹H NMR DMSOd6 2.79 (6H, s, 2 x NCH₃), 3.94 (3H, s, OCH₃), 4.50 (ZH, s, CH₂), 7.21 (1 H, t, ArH), 7.31 (2H, m, ArH), 7.39 (1 H, s, ArH), 7.49 (1 H, t, ArH), 7.65 (1 H, t, ArH), 7.75 (2H, m, ArH), 7.80 (1 H, m, ArH), 8.94 (1 H, s, ArH), 11.21 (1 H, br s, NH) LCMS (ammonium bicarbonate) Rτ = 3.17 mins, 100%, MH⁺399 Synthesis of C-{3-[6-(2-methoxy-phenyl)-pyrimidin-4-ylamino1-phenyl)- methanesulfonamide (compound #2)

C-{3-[6-(2-Methoxy-phenyl)-pyrinnidin-4-ylannino]-phenyl}-nnethanesulfonannide was prepared similarly: 1H NMR DMSOd6, 3.94 (3H, s, OCH₃), 4.35 (2H, s, CH₂), 6.95 (2H, t, NH₂), 7.22 (2H, m, ArH), 7.31 (1 H, d, ArH), 7.39 (1 H, s, ArH), 7.49 (1 H, t, ArH), 7.64 (2H, m, ArH), 7.73 (1 H; d; ArH); 7;79 (1 H, m, ArH), 8.93 (1 H, s, ArH), 11.15 (1 H, br s, NH) LCMS (ammonium bicarbonate) Rτ = 2.75 mins, 100%, MH⁺371

Synthesis of C-{3-r6-(2-methoxy-phenyl)-pyrimidin-4-ylamino1-phenyl)-N-methyl- methanesulfonamide (compound #3)

C-{3-[6-(2-Methoxy-phenyl)-pyrimidin-4-ylamino]-phenyl}-N-methyl- methanesulfonamide was prepared similarly:

¹H NMR DMSOd6, 2.62 (3H, d, NCH₃), 3.94 (3H, s, OCH₃), 4.41 (2H, s, CH₂), 7.03 (1 H, m, NH), 7.22 (2H, m, ArH), 7.31 (1 H, d, ArH), 7.41 (1 H, s, ArH), 7.49 (1 H, t, ArH), 7.67 (2H, m, ArH), 7.73 (1 H, d, ArH), 7.80 (1 H, m, ArH), 8.94 (1 H, s, ArH), 11.25 (1 H, br s, NH) LCMS (ammomium bicarbonate) Rτ = 2.94 mins, 100%, MH⁺385 Synthesis of N-(2-methoxy-ethyl)-C-{3-r6-(2-methoxy-phenyl)-pyπmidin-4-ylamino1- phenvD-methanesulfonamide (compound #4)

N-(2-Methoxy-ethyl)-C-{3-[6-(2-methoxy-phenyl)-pyhnnidin-4-ylannino]-phenyl}- methanesulfonamide was prepared similarly:

¹H NMR DMSOd6, 3.11 (2H, q, CH₂), 3.29 (3H, s, OCH₃), 3.40 (2H, m, CH₂), 3.94 (3H, s, CH₃), 4.42 (2H, s, CH₂), 7.50 (4H, m, ArH), 7.40 (1 H, s, ArH), 7.70 (4H, m, NH + ArH), 8.93 (1 H, s, ArH), 11.22 (1 H, br s, NH) LCMS (ammonium bicarbonate) Rτ = 3.00 mins, 97.66%, MH⁺429

Preparation of intermediates

c) Amine, DCM d) Fe, EtOH, HCI, 80⁰C 2 hours

Intermediates of step c):

N,N-Dimethyl-C-(3-nitro-phenyl)-methanesulfonamide

To a solution of (3-Nitrophenyl)methanesulfonyl chloride (1.0 g, 0.0042 mol) in dry dichloromethane (25 mis) at room temperature dimethylamine (2M in THF, 5.3 mis, 0.105 mol) was added dropwise. The mixture was stirred for 3 hours then filtered through silica, then evaporated to give N,N-Dimethyl-C-(3-nitro-phenyl)- methanesulfonamide, 1.0 g, ¹H NMR CDCI₃ 2.84 (6H, s, 2 x CH₃), 4.29 (2H, s, CH₂), 7.60 (1 H, m, ArH), 7.79 (1 H, d, ArH), 8.25 (2H, m, ArH)

Similarly were prepared:

N-Methyl-C-(3-nitro-phenyl)-methanesulfonamide

¹H NMR DMSO 2.64 (3H, d, NHCH₃), 4.60 (2H, s, CH₂), 7.05 (1 H, q, NH), 7.74 (1 H, m, ArH), 7.87 (1 H d, ArH), 8.27 (1 H, m, ArH), 8.31 (1 H, s, ArH)

N,-2-methoxyethyl-C-(3-nitro-phenyl)-methanesulfonamide

¹H NMR CDCI₃ 3.24 (2H, t, CH₂), 3.34 (3H, S, OCH₃), 3.46 (2H, t, CH₂), 4.41 (2H, S, CH₂), 7.57 (1 H, t, ArH), 7.79 (1 H, d, ArH), 8.20 (1 H, d, ArH), 8.30 (1 H, s, ArH)

Intermediates of step d):

C-(3-Amino-phenyl)-N,N-dimethyl-methanesulfonamide

To a stirred mixture of iron powder (0.5 g), ethanol (20 mis), water (1 ml) and concentrated hydrochloric acid (5 drops) at reflux was added N,N-Dimethyl-C-(3- nitro-phenyl)-methanesulfonamide (0.5 g). The mixture was then heated under reflux for a further 2 hours then basified with excess sodium carbonate solution, cooled to 6O⁰C, then filtered. The filter cake washed with ethanol and the filtrate evaporated. The crude product was partitioned between ethyl acetate and water, separation and evaporation of the organic phase gave C-(3-Amino-phenyl)-N,N-dimethyl- methanesulfonamide, 0.4 g, sufficiently pure to use.

¹H NMR CDCI₃, 7.75 (6H, s, N(CH₃) ₂, 3.72 (2H, br s, NH₂), 4.15 (2H, s, CH₂), 6.68 (1 H, d,ArH), 6.75 (2H, m, ArH), 7.15 (1 H, t, ArH)

Similarly were prepared: C-(3-Amino-phenyl)-N-methyl-methanesulfonamide

¹H NMR CDCI₃2.73 (3H, d, CH₃), 3.75 (2H, br s, NH₂), 4.05 (1 H, br s, NH), 4.18 (2H, s, CH₂, 6.72 (3H, m, ArH), 7.18 (1 H, t, ArH)

C-(3-Amino-phenyl)-N-(2-methoxy-ethyl)-methanesulfonamide 1H NMR CDCI₃ 3.15 (2H, m, CH₂), 3.31 (3H, s, CH₃), 3.40 (2H, m, CH₂), 3.75 (2H, br s, NH₂), 4.18 (2H, s, CH₂), 4.56 (1 H, br t, NH), 6.67 (1 H, d, ArH), 6.76 (2H, m, ArH), 7.15 (1 H, t, ArH)

C-(3-Amino-phenyl)-methanesulfonamide, is commercially available.

2. Materials and Methods - Biological Experiments:

2.1. Cloning and purification of CDK9 and Cvclin T1 :

Both cDNA fragments were cloned by PCR into pDONR201 vectors using the gateway recombination system (Invitrogen) according to the manufacturer's recommendations. The fragments were subcloned into a gateway-adapted shuttle vector (pPM7) for production of recombinant adenovirus. All plasmids were verified by restriction digests and sequencing analysis. Expression and purification of CDK9/Cyclin T1 proteins was in principle performed as described by Cotten et al. (M. Cotten et al., Nucleic acids research, 2003, 31 (28), 128).

Kinase assays using CDK9/Cyclin T1 were performed in principle as described by Cotten et al. (M. Cotten et al., Nucleic acids research, 2003, 31 (28), 128).

Results:

CDK9/CyclinT1 complexes from HEK293 cells (ATCC number: CRL-1573) were completely solubilised. CDK9/CyclinT1 proteins were almost completely precipitated by and eluted from streptavidin beads (data not shown). Enrichment was verified from blots stained with PonceauS. CDK9/CyclinT1 proteins can be seen in the eluate whereas they are not visible within the cells or extract. Probing nitrocellulose with antibodies against CDK2 and CDK4 revealed that those kinases do not contaminate the purifications (data not shown).

Increasing amounts of CDK9 wt proteins incubated with substrates (ATP and GST-CTDII) resulted in incorporation of radioactive phosphate. As expected, mutation of critical kinase domain residues (K48R and D167N) within CDK9 revealed no phosphate incorporation, confirming that these mutations render the kinase inactive. Additionally, EDTA pre-incubation completely inhibited activity.

These results show that purification of CDK9/CyclinT1 proteins using adenovirus leads to an active and pure enzyme. A putative contamination with other protein kinases can be ruled out because purification of mutated CDK9 resulted in negligible kinase activity.

2.2. Kinase assays:

Kinase assays determining CDK2/CyclinA and CDK5/p35 activity were performed as described by the manufacturers recommendations (ProQinase (Freiburg, Germany) for CDK2/CyclinA and Upstate for CDK5/p35). General kinase assay:

The inhibitory effect of compounds according to the present invention on the activity of protein kinases can be measured according to the following protocol:

Reaction Volume: 40μl Reaction Time: 60min

Reaction Temperature: room temperature

Assay Plate: 96 well U bottom plate (Greiner, 650161 )

MultiScreen-PH Plate: 96 well MAPH Filter Plates (Millipore, MAPHNOB50)

Filter Washing Solution: 0.75% H₃PO₄ Scintillation Liquid: Supermix Liquid Scintillator (PerkinElmer, 1200-439)

Controls:

Negative Control (C-): 10OmM EDTA (Ethylenediaminetetraacetic acid), no

Inhibitor

Positive Control (C+): no Inhibitor

Reaction Buffer:

2OmM Tris (Ths(hydroxymethyl)aminomethane hydrochloride), pH 7.5

1 OmM MgCI₂

1 mM DTT Final Assay Concentrations:

Kinase: Use kinase cone, yielding 10% ATP turn over.

ATP: 1 μM

Adenosine 5'-[γ-³³P]triphosphate: 12.5 μCi/ml (Amersham Biosciences, BF1000) Substrate: Myelin Basic Protein 10 μM (Invitrogen, 13228-010)

Pipetting Sequence:

1 ) Add 10 μl 4 fold concentrated Substrate + 4 fold concentrated ATP in 3 fold concentrated Reaction Buffer to each well of Assay Plate 2) Add 10 μl 4 fold concentrated inhibitor in 4% DMSO in H₂O to each well except to C- and C+ wells

3) Add 10 μl 4% DMSO in H₂O to C- and C+ wells

4) Add 10 μl 50OmM EDTA in H₂O to C- wells

5) Add 10 μl 50 μCi/ml Adenosine 5'-[γ-³³P]thphosphate in H₂O to each well 6) Add 10 μl 4 fold concentrated kinase in Reaction Buffer to each well

7) Incubate 1 hr at room temperature

8) Add 10 μl 5OmM EDTA in H₂O to each well except to C- wells

9) Prepare MAPH plates by adding 200 μl 0.75% H₃PO₄ to each well

10) Exhaust 0.75% H₃PO₄ using Millipore vacuum station 11 ) Add 60 μl 0.75% H₃PO₄ to each well of MAPH Filter Plate

12) Transfer 30 μl sample per well from Assay Plate to corresponding well of MAPH Filter Plate

13) Incubate 30 min at room temperature

14) Wash each well of MAPH Filter Plates 3x with 200 μl 0.75% H₃PO₄ using Millipore vacuum station.

15) Add 20 μl Scintillation Liquid to each well of MAPH Filter Plate

16) Seal MAPH Filter Plate

17) Store MAPH Filter Plate 30 min in darkness

18) Quantify radioactivity

Results:

IC50S were calculated for each kinase based on serial dilutions. Table 1 shows the inhibitory effect of selected compounds according to the present invention on the activity of certain protein kinases. . Table 1 : IC₅oS of selected compounds according to the present invention (all data in μM; n. a. =not available).

These data show that compounds according to the present invention, do have an inhibitory effect on the protein kinase activity of various protein kinases, such as CDKI/CycB, CDK2/CycA, CDK4/CycD1 , CDK5/p35, CDK6/CycD3 and CDK9/CycT1.

2.3. Determination of RNA Polymerase Il C-terminal domain phosphorylation:

The phosphorylation status of RNA polymerase Il C-terminal domain is determined by western blot techniques. PM1 cells (obtainable from the National Institute of Allergy and Infectious Diseases; Division of AIDS via the NIH AIDS Research & Reference Reagent Program), are seeded in 6-well plates at a density of about 5x10⁵ per well. After over night incubation cells are treated with compounds of the present invention. Cells are pelleted and lysed with 300μl_ 3x Laemmli buffer followed by 30min denaturing at 65°C. After separation of equal lysate volumes by SDS-PAGE the proteins are transferred to nitrocellulose membranes (Schleicher&Schuell) and probed with anti-SER2 (H5), anti-SER5 (H14) or RNA Poll ll-antibodies purchased from Eurogentec and Santa Cruz, respectively. The amount of reactive protein is visualized by ECL detection methods (Amersham).

Results: In order to see, if the compounds of the present invention do have the intrinsic capacity to penetrate cells and act against cellular target proteins, such as CDK9, the effect of the compounds of the present invention on CDK9-dependent phosphorylation of RNA-polymerase Il is investigated. Probing blots with antibodies against the phosphorylated forms of RNA polymerase Il show, that specifically serine 2 phosphorylation is decreased, whereas antibodies recognizing serine 5 phosphorylation do not show any differences. These results indicate that kinases being responsible for the phosphorylation of this site, for example CDK7 are not touched. Additionally, a reduction in the molecular weight of RNA polymerase Il is observed indicating that phosphorylation is decreased.

2.4. Growth assay using Alamar Blue™:

Cells were seeded in 384-well plates (white, Greiner: 781080) at a density of

30,000-40,000 cells per well in medium (RPMI + 10% FCS).

Cells were incubated in 25 μl medium/well and were grown over night at 37°C, 6% CO2. On the following day compounds were added in DMSO in serial fold dilutions in fresh medium. Negative control wells were incubated without cells. Positive control wells were incubated with cells but without inhibitors (DMSO only). These wells served as data points to determine relative growth (given in % of the DMSO control [= 100%]). The cell number was determined by addition of 5μl_ Alamar Blue™ (Biosource DAL1100) to each well. Fluorescence was measures with an Analyst GT machine (Molecular Devices) at an excitation wavelength of 560 nm and an emission wavelength of 590 nm.

The following cell lines were used: A2780 (ECACC order number 93112519; human ovarian carcinoma; Semin Oncol (1984) 11 :285; Cancer Res (1987) 47:414), B16F1 (ATCC order number CRL-6323; melanoma; Nat. New Biol. 242: 148-149, 1973); HCT116 (ATCC order number CCL-247; Colorectal carcinoma; Cancer Res (1981 ) 41 :1751 ; Cancer (1995) 76:201 ), HT29 (ATTC order number HTB-38; colorectal adenocarcinoma; J. Biol. Chem. 271 : 9490-9496, 1996) HepG2 (ATCC order number HB-8065; hepatocellular carcinoma; J. Biol. Chem. 271 : 10073-10078, 1996), J774 (ATCC order number TIB-67; reticulum cell sarcoma; J. Biol. Chem. 271 : 18431 -18436, 1996); MCF7 (ATCC order number HTB-22; Breast carcinoma; J Natl Cancer Inst (1973) 51 :1409; Cancer Res (1993) 53:5193), PM1 (obtainable from the National Institute of Allergy and Infectious Diseases; Division of AIDS via the NIH AIDS Research & Reference Reagent Program; Lusso P et al (1995), J Virol 69: 3712-3720) and U373-MAGI-CCR5 (obtainable from the National Institute of Allergy and Infectious Diseases; Division of AIDS via the NIH AIDS Research & Reference Reagent Program; Kensinger RD et al (2004), Antimicrob Agents Chemother 48: 1614-1623).

Results:

IC50S were calculated for each cell line based on serial dilutions. Table 2 shows the IC50S of selected compounds according to the present invention against various cell lines.

Table 2: IC50S of selected compounds according to the present invention against various cell lines (all data in μM; n.a.=not available).

These data show, that compounds according to the present invention exhibit growth inhibitory activity against all cell lines tested.

2.5. HIV replication assay: PM1 cells are seeded in 12-well plates at a density of about 1.5x10⁵ per well with

RPMI 1640 containing 10% FCS, 1 % L-Glutamine and 1 % Na-Pyruvate (Sigma).

Cells were previously infected with HIV-1 BaL for 3h at a concentration of about 5x10⁸ μg p24/cell. After addition of the compounds of the present invention cells are incubated for 6 to 10 days. During this incubation the cells are passaged and compound-containing medium is renewed. The concentration of p24 in the cellular supernatants is determined at each of this time points using a previously described

ELISA assay (Bevec et al., Proceedings of the National Academy of Sciences U.S.A. 1992, 89(20), 9870 - 9874).

Results:

The growth of PM1 cells is not generally affected by compounds of the present invention. No correlation between CDK9 inhibition and toxicity is observed. Compounds of to the present invention are potent inhibitors of HIV replication.

2.6. NFκB-dependent transcriptional activity:

The used NIH 3T3 75E11/300D8 cell line is described elsewhere (J. Eickhoff et al., Journal of Biological Chemistry, 2004, 279(10), 9642 - 9652). Results:

It is known, that CDK9 regulates NFκB-dependent transcriptional activity. The compounds of the present invention are able to affect TNF-α stimulated NFKB- dependent promotor activity. Under non-stimulated conditions no inhibition is observed.

2.7. HBV-replication:

To test the anti-HBV-activity of compounds of the present invention the HBV- producing cell line HepG2-2.2.15 (M.A. Sells, PNAS 1987, 84, 1005-1009) is used. About 1.0x10⁴CeIIs are seeded in 96-well microtiter plates in DMEM medium supplemented with 10% FCS. After incubation at 37°C in 5%CO2 atmosphere for 24 hours the medium is replaced with fresh medium containing the appropriately diluted compounds of the present invention. 3 days later medium is replaced by freshly prepared compound-containing medium and the cells are incubated for further 3 days. Subsequently 200μl lysis buffer (5OmM Tris-CI 7.5; 1 mM EDTA 8.0; 0.5% NP40) per well is added. To remove cell debris and nucleic acids, lysate is centrifuged (15000rpm, 10min, 4°C). Cellular and viral RNA is removed by addition of 2μl of RNase. 10Oμl of the samples are spotted onto an uncharged nylon membrane pre-wetted with PBS (phosphate-buffered saline) using a 96well- blotting chamber (MINIfold Dot-Blot, Schleicher&Schϋll). After further washing with 200μl PBS per well the membrane is treated twice with 0.5M NaOH, 1.5M NaCI (2min) and 4 times with 0.5M Tris 7.5, 3M NaCI (1 min). The nucleic acids are fixed by UV-treatment and is used for hybridisation with a radioactive HBV-fragment prepared from the overlength HBV genome plasmid pT-HBV1.3 (L. G. Guidotti et al., Journal of Virology 1995, 69(10), 6158 - 6169). The fixed membrane is pre-hybhdized in a standard hybridisation buffer (50% formamide, 5xSSPE, 10xDenhards, 1 % SDS, 100μg/ml salmon sperm DNA) for at least 3 hours at 42°C and hybridised overnight against the labelled HBV-fragment. The preparation of the HBV-fragment with the "Random primers DNA labelling system" (Invitrogen) is done according to the manufacturer's instructions. Hybridized filter are washed at room temperature with 2xSSC, at 62°C with 2xSSC, 0.5%SDS and at 62°C with O.δxSSC, 0.5%SDS. Each washing step is carried out twice. The intensity of the HBV-DNA is quantified using a phosphoimager (Fuji). To test the cell viability 0.5x10⁴ HepG2-2.2.15-cells are seeded in 96-well-microtiter plates in DMEM medium supplemented with 10% fetal bovine serum. After incubation at 37°C for 24 hours the medium is replaced by fresh compound-containing medium. 3 days later medium is replaced again by freshly prepared medium containing the compounds of the present invention and the cells are incubated for further 3 days at 37°C. After the incubation period 1/10 volume of Alamar Blue (Serotec) solution containing a growth dependant indicator is added and the cells are incubated for 3 h at 37°C. Absorbance is monitored at 570nm and 600nm wavelength.

Results:

Compounds according to the present invention are tested in a HBV replication assay. Some compounds of the present invention inhibit HBV replication without affecting viability in those cells. Some compounds of the present invention are inactive in those assays indicating that other protein kinase targets than CDK9 (especially further CDKs) might be important for HBV replication. This is underlined by flavopiridol, which inhibits replication, but is known to be a more or less unspecific inhibitor of CDKs.

2.8. HCMV replication: Human foreskin fibroblasts (HFF) cell culture is grown in DMEM containing 10% FCS. For HCMV-replication assays, HFF cells are infected with HCMV strain AD169 producing EGFP (HCMV AD169-GFP; 27). 1 h post infection, medium is changed with medium containing compounds of the present invention. After incubation of 7 days cells are lysed (in 25mM Tris, pH 7.5, 2mM DTT, 1 % Triton X- 100 and 10% glycerol) and analysed for EGFP content in a Wallac Victor fluorescence detector.

Results: Compounds of the present invention are identified as potent inhibitors of HCMV replication in cell culture: some compounds according to the present invention show inhibition of HCMV replication (using strain AD 169 in HFF cells).

2.9. HCV replicon assay:

Compounds of the present invention are tested for activity in the HCV replicon system described by Bartenschlager and coworkers (Lohmann et al, Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110, 1999).

2.10. Pain models:

Behavioral animal models for the analysis of inflammatory and neuropathic pain

Several animal models for the analysis of inflammatory and neuropathic pain are known. Said models share the common feature that after e.g., induction of a nerve lesion (e.g., spared nerve injury, SNI) or after exposing experimental animals to a noxious stimulus (e.g., injection of formalin or carrageenan), the signs of pain as induced by said interventions are measured by quantifiable behavioral components such as, e.g., paw withdrawal threshold to mechanical stimulation with von Frey hairs (or to thermal stimulation using a laser source or licking behaviour). These reactions are interpreted as being equivalent to mechanical and thermal allodynia (hypersensitivity to mechanical stimuli) or hyperalgesia in humans.

The spared nerve injury model (SNI model, Decosterd and Woolf, Pain 2000; 87:149-158, see Figure 1 ) is characterized by the induction of clinically relevant nerve lesions and after surgical intervention, subsequent behavioral experiments (e.g., von Frey Assay). Said model constitutes a common nerve injury model which consists of ligation and section of two branches of the sciatic nerve (namely tibial and common peroneal nerves) leaving the sural nerve intact. The SNI model results in early (less than 24 hours), prolonged and substantial changes in mechanical and cold sensitivity that closely mimic the features of clinical neuropathic pain. Animals with these types of nerve injury have been shown to develop abnormal pain sensations and hypersensitivity to mechanical stimuli (allodynia) similar to those reported by neuropathic pain patients. Alternatively, the formalin assay in mice is a valid and reliable behavioral model of nociception in inflammatory and neuropathic pain. It is sensitive to various classes of analgesic drugs (Hunskaar S & Hole K, The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 1987, 103-114). The noxious stimulus consists of an injection of 10 μl diluted formalin (2% in saline) under the skin of the dorsal surface of the left hind paw (subcutaneous or interplantar into the left hindpaw). The response is licking and flinching of the injected paw.

An additional assay, the so-called carrageenan assay, comprises a subcutaneous injection of 25 μl of 1 % carrageenan (in saline) into a single hind paw (ipsi-lateral paw) of mice.

Subsequent inflammation results in long lasting swelling and hypersensitivity (against mechanical and thermal stimuli) of the paw. The carrageenan assay is a standard laboratory assay used to predict anti- inflammatory activity of test compounds. Paw edema measurements and Hargreaves Assay (which measures withdrawal of paws due to thermal stimulation via a light source) are used for read out.

In the present invention, the effect of administration of cyclin- dependent kinase (CDK)-inhibiting compounds on the development of inflammatory and neuropathic pain are assayed in an SNI model, in a carrageenan and in a formalin assay. The experimental procedure and results are described in detail below.

Example 2.10.1

Spared nerve injury (SNI) - Model of chronic neuropathic pain

As outlined above, the spared nerve injury (SNI) model (see Figure 1 ) involves a lesion of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) of experimental animals, leaving the sural nerve intact. SNI results in mechanical and thermal allodynia in the non-injured sural nerve skin territory (Decosterd & Woolf, Pain 2000; 87:149-158; Tsujino et al., MoI. CeI. Neurosci. 2000; 15:170-182).

1. Induction of spared nerve injury (nerve lesion) in wild type mice

Wild type mice (strain C3HeB/FeJ) (age, sex and weight matched) are anesthetized with Hypnorm (0.3 15 mg/ml fentanyl citrate + 10 mg/ml fluanisone; Janssen)/Hypnovel (5 mg/ml midazolam; Roche Applied Sciences)/water at a ratio of 1 :1 :2 at 4 μl/g prior to surgical preparation.

Subsequently, an incision is made under aseptic precautions in the ipsi- lateral right hind leg of all mice just above the level of the knee, exposing the three terminal branches of the sciatic nerve: the common peroneal, tibial, and sural nerves. The common peroneal and tibial nerves are ligated tightly with 7/0 silk and sectioned distal to the ligation removing about 2 mm of distal nerve stump. The sural branch remains untouched during the procedure (denoted herein "SNI ipsi"). The overlying muscle and skin is sutured, and the animals are allowed to recover and to permit wound healing. In the same mice, the sciatic nerve branches of the contra-lateral left hind leg are exposed but not lesioned (denoted herein "SNI contra- lateral"). Mice that undergo spared nerve injury are hereinafter denoted "SNI mice".

2. Administration of compounds according to the present invention to SNI mice

After recovery from surgery and wound healing, SNI mice receive intraperitoneal (i.p.) injections of compounds according to the present invention. Compounds of the present invention, for example at 30mg/kg, dissolved in DMA/Labrafil (10:90), are administered by a single i.p. injection 30 min prior to von Frey measurements (mechanical allodynia). As a negative control, the same amount (200μl) of DMA/Labrafil (10:90) vehicle is administered by a single i.p. injection 30 min prior to von Frey measurements.

Injection of inhibitor or vehicle, and subsequent measurements of paw withdrawal threshold upon mechanical stimulation in von Frey assays are performed at day 0, day 7, day 21 and day 28 post SNI. Reflex nociceptive responses to mechanical stimulation are measured in a von Frey assay 30 min after each injection. An additional von Frey measurement is performed at day 14, but without prior injection of compound or vehicle. The effect of administration of compounds of the present invention to SNI mice on the development of mechanical allodynia is analyzed in a von Frey assay, as described below.

3. Behavioral testing of SNI mice after administration of compounds of the present invention (von Frey assay)

Mice which undergo SNI and subsequent administration of compounds of the present invention are tested for signs of mechanical allodynia post nerve injury and post administration in a von Frey assay (Decosterd and Woolf, Pain 2000; 87:149-158). This assay determines the mechanical threshold upon which a stimulus, which normally is not painful, is recognized by an animal as uncomfortable or painful. SNI ipsi and SNI contra baselines, respectively, are established. Mechanical thresholds of SNI mice are quantified using the up- down method based on Chaplan et al. (1994) and Malmberg and Basbaum (1998) (Chaplan SR, Bach FW, Pogrel JW, Chung JM, and Yaksh, TL. (1994) Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53: 55- 63; Malmberg AB and Basbaum Al. (1998) Partial sciatic nerve injury in the mouse as a model of neuropathic pain: behavioral and neuroanatomical correlates. Pain 76: 215-2): Mice are placed in plexiglass cylinders of about 9.5 cm in diameter, 14 cm high with four vent holes towards the top and a Plexiglas lid. The cylinders are placed on an elevated mesh surface (7x7mm squares). Prior to the day of testing, the mice are adapted to the testing cylinders for 1 -2 hours. On the day of testing the mice are adapted to the cylinders for about an hour, wherein the adaptation time depends on factors such as the strain of the mouse and the number of times they have been tested previously. In general, testing may begin once the mice are calm and stop exploring the new environment. For testing mice, filaments 2.44, 2.83, 3.22, 3.61 , 3.84, 4.08, and 4.31 (force range = 0.04 to 2.0 g) are used. The 3.61 mN filament is applied first. Said filament is gently applied to the plantar surface of one paw, allowed to bend, and held in position for 2 - 4 seconds.

Whenever a positive response to the stimulus (flexion reaction) occurs, the next weaker von Frey filament is applied; whenever a negative response (no reaction) occurred, the next stronger force is applied. The test is continued until the response to 4 more stimuli after the first change in response is obtained. The highest force tested is 4.31. The cut-off threshold is 2g. The series of scores (i.e., "flexion reaction" and "no reaction") and the force of the last filament applied are used to determine the mechanical threshold as described in Chaplan et al., Journal of Neuroscience Methods, 53(1 ):55-63, 1994. The threshold determined is that to which the animal would be expected to respond to 50% of the time. Mice are sacrificed after von Frey measurements were accomplished.

4. Compounds of the present invention have a hypoalgesic effect in SNI mice

Compounds of the present invention are administered to SNI mice as described above. Von Frey measurements are performed as described above. It is shown that the compounds of the present invention have a hypoalgesic effect on SNI mice. Von Frey measurements are performed at ipsi-lateral and contra- lateral paws of the animals at 4 different time points (day 0, day 7, day 14, day 21 and day 28 after surgery). Day 0 represents baseline behavior prior to SNI surgery. The "day 7" group receives treatment (30 mg/kg compound i.p. or vehicle) at day 7, 21 and 28, the "day 21 " group receives the same treatment at day 21 only while the "vehicle" group receives vehicle only (10% DMA; 90% Labrafil) at day 7, 21 and day 28.

At day 7, treatment with compound of the present invention does not show any effect in the von Frey assay. All three groups display high sensitivity (= low threshold) against the mechanical stimulus. Low thresholds are also seen at day 14 without any prior treatment. At day 21 , however, i.p. treatment with compounds according to the present invention, for example with 30 mg/kg, 30 minutes prior to measurements does show an effect. While the vehicle control group remains sensitive with low thresholds similar to measurements on day 7 and 14, animals treated with compounds display a significant increase of threshold values, indicating reduced sensitivity to mechanical stimuli (reduced allodynia). At day 28, the "day 7" group is treated again at midnight and 8.00 a.m. with compounds according to the present invention each, e.g. at 30 mg/kg, and thresholds are measured at 8.30 a.m., whereas the vehicle control group receives solvent at the same time. Again, significant increases in thresholds of animals treated with compounds of the present invention is observed. In comparison, animals treated with vehicle alone display low thresholds and high allodynia. The threshold value of the ipsi-lateral paw of vehicle-treated mice shows that these animals are very sensitive at day 21 and 28, whereas the ipsi- lateral paws of mice treated with compounds of the present invention are less sensitive. The contra-lateral paw of vehicle-treated mice is similar to baseline sensitivity at day 0, whereas mice treated with compounds of the present invention show a reduced threshold at day 21 and 28.

These findings indicate that the compounds of the present invention are effective as hypoalgesic drugs in models of chronic neuropathic pain.

Example 2.10.2

Formalin Assay - Model of Inflammatory / Chronic Neuropathic Pain

The formalin assay in mice is a valid and reliable behavioral model of nociception and is sensitive to various classes of analgesic drugs (Hunskaar S & Hole K, Pain. 1987, 30(1 ):103-14). The noxious stimulus consists of a subcutaneous or an intraplantar injection of 10 μl diluted formalin (2% in saline) into the left hind paw. The response is licking and flinching of the injected paw. The response shows two phases, which reflect different parts of the inflammatory process (Abbott, F. V., Franklin, K. B., and Westbrook, R. F. (1995). The formalin test: scoring properties of the first and second phases of the pain response in rats. Pain 60, 91-102), an early/acute phase 0-5 min post-injection, and a late/chronic phase 5-30 min post- injection.

1. Injection of formalin and administration of compounds of the present invention

Age, sex and weight matched wild type mice (C3HeB/FeJ) are used in this assay. Prior to formalin injection, the animals are randomly subdivided into experimental groups of 10 animals each. Thirty minutes prior to formalin injection, compounds of the present invention (e.g. at 30mg/kg dissolved in DMA/Labrafil (10:90)) are administered by i.p. injection. Similarly, IK Kinase (IKK) inhibitor (30 mg/kg) in DMA/Labrafil (positive control), or vehicle alone (DMA/Labrafil, 10:90) (negative control) are administered by i.p. injection 30 mm before formalin injection. For formalin injection, the mouse is held with a paper towel in order to avoid disturbance of the injection by movements. The injected hind paw is held between thumb and forefinger and 10 μl of Formalin (2%) is injected subcutaneously (s.c.) between the two front toes into the plantar hind paw using a Hamilton syringe. The behavior of the mice treated with formalin and with compounds of the present invention is analyzed as described below. 2. Behavioral analysis of mice after injection of formalin and after administration of compounds of the present invention

The behaviour of the formalin- treated mice, i.e. licking and flinching, is monitored by an automated tracking system (Ethovision 3.0 Color Pro, Noldus, Wageningen, Netherlands) over a defined period of time: measurement is initiated 5 min after formalin injection and terminated 30 mm after formalin injection. This time frame covers phase Il of formalin- induced nociception (pain), which is hyperalgesia. Two different fluorescent dyes are used for topically marking the injected hind paw (yellow dye) (Lumogenyellow; BASF Pigment, Cologne, Germany) and the contralateral paw (blue dye) (Lumogenviolet; Kremer Pigmente, Aichstetten, Germany), respectively. To determine licking behaviour, mice are monitored with a CCD camera. After monitoring and recording, the video is analyzed using the EthoVision software (Ethovision 3.0 Color Pro, Noldus, Wageningen, Netherlands) or by manual analysis. Fluorescent dot sizes and fluorescence intensities are measured and reduction of fluorescent dot size through licking and biting is calculated. The overall licking time intensity is automatically calculated by comparison of dot size reduction of treated versus untreated paws. As another possibility of assay read-out, the licking behaviour of the individual animals is tracked manually based on video files. Licking times are recorded over 30 minutes after formalin injection and subdivided for three different licking zones (dorsum, plantar, toes).

Overall licking times can be calculated for each animal as well as each experimental group and can be used as a parameter for determination of compound efficacy.

Mice receiving vehicle treatment prior to formalin injection (negative control) display a prolonged licking time and a significant reduction of fluorescent dot size at the formalin- treated paw, thus indicating a high sensitivity of the mice to the noxious stimulus. In contrast, a reduction in licking time and consequently, no significant reduction of fluorescent dot size of the formalin-treated paw is observed in mice treated with compounds of the present invention/formalin, demonstrating reduced sensitivity of said mice to the noxious stimulus. The same effect, i.e. a reduction in licking time and a minor change in fluorescent dot size, is observed in control mice treated with IK kinase inhibitor (IKK; for function of IKK see Fig. 2, positive control).

This finding is indicative of reduced inflammatory / chronic inflammatory pain perception in mice treated with compounds of the present invention and for a hypoalgesic effect of the compounds of the present invention. 3. Administration of compounds of the present invention has a hypoalgesic effect in formalin-treated mice

Total licking time is significantly reduced in mice treated with compounds of the present invention, comprising all three parts of the paw observed: dorsal side, plantar side and toes (of injected paws, compared to the vehicle control. This observation is indicative for reduced inflammatory / chronic inflammatory pain perception in mice treated with compounds of the present invention and for a hypoalgesic effect of the compounds of the present invention.

Example 2.10.3

Carrageenan Assay - Model of Inflammatory Pain

The model of carrageenan-induced paw edema constitutes a standard laboratory assay used to predict anti-inflammatory activity of therapeutically active compounds and reduction of inflammation-induced pain perception achieved by administration of therapeutically active compounds.

The basic measurement constitutes in the measurement of edema size and of mechanical as well as thermal hypersensitivity in response to irritants, such as carrageenan.

Inflammation and subsequent inflammatory pain is induced by subcutaneous injection of 25 μl of 1 % carrageenan (in saline) into the hind paw (ipsi- lateral paw) of mice. Groups of 10 mice each receive a compound of the present invention (e.g. 30 mg/kg body weight), vehicle (DMA/Labrafil; 10:90) and saline (physiol. NaCI) by i.p. injection 30 min prior to carrageenan injection. Contra-lateral paws did not receive carrageenan injection.

1. Effects of administration of compounds of the present invention on carrageenan-treated mice

Paw edema induced by carrageenan injection is detected by increased paw size measured from dorsal to plantar at the metatarsus region of the injected (ipsi- lateral) paws. Sizes of ipsi- and contra- lateral paws serve as surrogate markers for inflammation and are measured at several time points after carrageenan injection: 1 h before injection (-1 ), immediately after injection, 1 h (1 ), 2h (2), 4h (4) , 5h (5), 6h (6), 7h (7), 24h (24), 48h (48), 72h (72), 96h (96), and 98h (98) after injection.

The paw size of all mice increases within the first hour after carrageenan injection, irrespective of the type of injection 30 minutes prior to carrageenan. During the time course, mice which receive compounds of the present invention prior to carrageenan injection display a reduction of the edema until 24h after carrageenan injection: the increase in paw size drops. In contrast, the paw size of the control mice increases at this time point. After 24h post carrageenan injection, the size of all paws treated with carrageenan increases to reach their maximum at 96h after injection.

2. Effects of administration of compounds of the present invention on carrageenan-treated mice as measured by Hargreaves Assay

As a read-out of the carrageenan assay, a Hargreaves Assay may be performed, wherein said assay allows the measuring of thermal sensitivity to radiant heat. The Hargreaves assay (Hargreaves, K, Pain 32(1 ) (1988) 77- 88) measures nociceptive sensitivity in a freely moving animal by focusing a radiant heat source on the plantar surface of an animal's hindpaw as it stands in a plexiglass chamber. Specifically, the lower side of a paw is exposed to a luminous source, generating a temperature of, e.g. 55°C. Thermal sensitivity is measured as latency between start of exposure and lifting/pulling the exposed paw. Mice treated with compounds of the present invention, e.g. with 30 mg/kg, and carrageenan, or with Naproxen and carrageenan, or with solvent and carrageenan, respectively, are subjected to a Hargreaves assay. Compounds of the present invention are administered as an i. p. injection 30 min prior to administration of carrageenan. Mice treated with compounds of the present invention display a longer latency, thus indicating that intraperitoneal treatment with compound of the present invention 30 minutes before carrageenan application results in significant reduction of thermal hyperalgesia detected by the Hargreaves assay comparable to 50mg/kg Naproxen.

Example 2.10.4

In order to validate the hypothesis that TNFα stimulation is regulated by an autocrine loop (i.e., TNFα induces expression of TNFα; see Figure 2), a microglial cell line (EOC20; ATCC number: CRL-2469) of human origin is stimulated in vitro by addition of TNFα (20ng/ml). Microglial cells which did not undergo TNFα stimulation are used as controls. 1 hour prior to addition of TNFα, stimulated or unstimulated cells, respectively, are treated with DMSO (vehicle control) or compounds of the present invention. Cells are harvested 48 h after addition of TNFα. RNA is isolated from the samples and the amount of amplicon of TNFα (and of other cytokines) and of several housekeeping genes is measured by light cycler-based Real-Time PCR. Samples from TNFα-stimulated cells show a clear increase of TNFα amplicon in comparison to the unstimulated cells. Addition of compounds of the present invention effect a dose-dependent reduction of said inductive effect. A reduction of TNFα message is even detected in unstimulated cells that have been treated with compounds of the present invention. These findings show that compounds of the present invention exert a suppressive effect on expression of TNFα.

References:

Prenzei N, Fischer GM, Streit S, Hart S, Uiirich A.

The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr Relat Cancer. 2001 Mar;8(1 ):11 -31. Review.

M§D_.Q_jng_..G_j..Whyte DB, _..Martinez R_Λ Hunter T. Sudarsana

The protein kinase complement of the human genome. Science. 2002 Dec

6;298(5600):1912-34. Review.

Biume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature. 2001 May 17;411 (6835):355-65. Review.

Flgres.Q,, Lee G₁, KesslerJ. M_ιiiie_ιr_ιM_ιLSch_ιi_ιief_ιW, IQ₁QIa₁SSiO₁J₁₁J,,, Hazyda P.,

Host-cell positive transcription elongation factor b kinase activity is essential and limiting for HIV type 1 replication. Proc Natl Acad Sci U S A. 1999 Jun

22;96(13):7208-13. Mapcebo HS₁, Lee_,,,G, Fi^gare_,,,J_,,,_,,,,Tgmass_,io_,i_,,,,J, Lyu P₁₁₁Zh₁U₁₁₁Y₁, Pe₁Q₁Q₁₁₁₁J₈ B₁Ia₁U C₁

Hazyda.D₁..Prlce.D₁..Fjores.O.,

P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro.

Genes Dev. 1997 Oct 15;1 1 (20):2633-44.

ZhU₁₁₁Y, Pe'ejxX, Pe₁Qg₁₁₁₁J₈ Rjmanatnan.Y, Marsha]] N, Marshals L_,,,Amepdt B₁ Mathews MB, Price DH.

Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. Genes Dev. 1997 Oct 15;1 1 (20):2622-32.

Shim EY, Walker AK, Shi Y, Biackweii TK.

CDK-9/cyclin T (P-TEFb) is required in two postinitiation pathways for transcription in the C. elegans embryo. Genes Dev. 2002 Aug 15;16(16):2135-46. fjampsey M_t Reinberg D.

Tails of intrigue: phosphorylation of RNA polymerase Il mediates histone methylation.

Cell. 2003 May 16;113(4):429-32. Review Bieniasz PD, Grdina TA, Bogerd HP, Cuilen BR.

Recruitment of cyclin T1 /P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription. Proc Natl Acad Sci U S A. 1999 JuI 6;96(14):7791 -6

Inhibition of human immunodeficiency virus type 1 replication in human T cells by retroviral-mediated gene transfer of a dominant-negative Rev trans-activator. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9870-4. SeMs ■ MA. ZeSent AZ, _..Shyartsman M, Acs G. Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions. J Virol. 1988 Aug;62(8):2836-44.

Sells MA, Chen ML, Acs G (1987): Production of hepatitis B virus particles in

HepG2 cells transfected with the cloned hepatitis B virus DNA, PNAS, 84, p.1005- 1009.

Guidotti LG, Matzke B, Schalier H. Chisari FV.

High-level hepatitis B virus replication in transgenic mice. J Virol. 1995

Oct;69(10):6158-69.

Briqnoia PS, Lackey K. Kadweli SH, Hoffman C. Home E, Carter HL. Stuart JD, Blackburn K, Moyer MB, Aiiigood KJ, Knight VVB, Wood ER.

Comparison of the biochemical and kinetic properties of the type 1 receptor tyrosine kinase intracellular domains. Demonstration of differential sensitivity to kinase inhibitors. J Biol Chem. 2002 Jan 1 1 ;277(2):1576-85. Epub 2001 Nov 05. hjuwe A_Mazjtschek_..R_1..Giannis A,. Small molecules as inhibitors of cycl in-dependent kinases. Angew Chem lnt Ed

Engl. 2003 May 16;42(19):2122-38.

Claims

New PCT Application Our Ref.: GPC12273PCT Applicant: GPC Biotech AGCLAIMS

Compounds having the general formula (II)

wherein

R¹ and R⁴ are hydrogen;

R² is -NH₂ or hydrogen;

R⁶ is selected from hydrogen, linear or branched substituted or unsubstituted Ci-Cs alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C3-C8 cycloalkyl or -(CH₂)_q-A, wherein q is an integer selected from 0 to 5 and A is selected from hydrogen, -F, -Cl, -Br, -I, -CN, -NH₂, -NO₂, linear or branched substituted or unsubstituted Ci-Cβ alkyl, linear or branched substituted or unsubstituted C1-C3 alkoxy, linear or branched C₂-Cs alkenyl, linear or branched C₂-Cs alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted cycloalkyl or carboxamido substituted with one or two C1-C3 alkyl; or R⁶, when M is -NR⁴⁰-, can form a heterocyclic structure when taken together with the nitrogen of M and R⁴⁰;

L is - CR⁵⁰R⁵¹ -SO₂-M-,

Wherein R⁵⁰ and R⁵¹ are independently selected from the group consisting of hydrogen, linear C1-C3 alkyl and fluorine, and wherein M is a bond, or

-NR⁴⁰ -; R⁴⁰ is selected from hydrogen and linear or branched substituted or unsubstituted Ci-C₈ alkyl, and substituted or unsubstituted C₃-C₈ cycloalkyl;

m is an integer selected from 0 to 5;

n is an integer selected from 0 to 4;

each R⁴¹ and R⁴² is independently selected from the group consisting of linear or branched Ci-C₆ substituted or unsubstituted alkyl, linear or branched substituted or unsubstituted Ci-C₆ alkoxy, linear or branched C₂- C₆ alkenyl, linear or branched C₂-C₆ alkynyl, -F, -Cl, -Br, -I, -CN, -NH₂ or -NO₂;

and/or tautomeric forms and/or pharmaceutically acceptable salts thereof.

2. A compound according to claim 1 , wherein R² is hydrogen.

3. A compound according to claim 1 or 2, wherein at least one R⁴¹ is linear or branched Ci-C₆ alkoxy.

4. A compound according to claim 3, wherein said linear or branched Ci-C₆ alkoxy is methoxy.

5. A compound according to any one of claims 1 to 4, wherein at least one R⁴¹ is in the orffto-position.

6. A compound according to claim 5, wherein said R⁴¹ in the o/f/70-position is methoxy.

7. A compound according to any one of claims 1 to 6, wherein m is 1.

8. A compound according to any one of claims 1 to 7, wherein n is 0.

9. A compound according to any one of claims 1 -8, wherein R⁵⁰ and R⁵¹ are both fluorine.

10. A compound according to any one of claims 1 -8, wherein R⁵⁰ is hydrogen.

11. A compound according to claim 10, wherein R⁵¹ is hydrogen.

12. A compound according to any one of claims 1 -11 , wherein M is -NR 40 - and R⁴⁰ is selected from hydrogen, methyl, ethyl and isopropyl.

13. A compound according to claim 12, wherein R⁴⁰ is hydrogen.

14. A compound according to claim 12, wherein R⁴⁰ is methyl.

15. A compound according to any one of claims 1 -14, wherein R⁶ is selected from hydrogen, linear or branched substituted or unsubstituted Ci-Cs alkyl, and - (CH₂)_q-A, wherein q is an integer selected from 0 to 5 and A is selected from linear or branched substituted or unsubstituted Ci-Cβ alkoxy, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroaryl and carboxamido substituted with one or two Ci-Cβ alkyl.

16. A compound according to claim 15, wherein R⁶ is hydrogen.

17. A compound according to claim 15, wherein R⁶ is linear or branched unsubstituted Ci-C₅ alkyl.

18. A compound according to claim 17, wherein R⁶ is selected from: methyl, isopropyl and 3-methyl-butyl.

19. A compound according to claim 18, wherein R⁶ is methyl.

20. A compound according to claim 15, wherein R⁶ is -(CH₂)_q-A, wherein q is an integer selected from 0 to 3 and A is linear, unsubstituted Ci-Cβ alkoxy.

21. A compound according to claim 20, wherein wherein q is 2 and A is methoxy.

22. A compound according to claim 15, wherein R⁶ is -(CH₂)_q-A, wherein q is an integer selected from 0 to 3 and A is carboxamido substituted with two C1-C3 alkyl groups.

23. A compound according to claim 22, wherein q is 2 and A is carboxamido substituted with two methyl groups.

24. A compound according to claim 15, wherein R⁶ is -(CH₂)_q-A, wherein q is an integer selected from 0 to 3 and A is substituted or unsubstituted heterocyclyl.

25. A compound according to claim 24, wherein said substituted or unsubstituted heterocyclyl is substituted or unsubstituted pyrrolidine or piperidine.

26. A compound according to claim 15, wherein R⁶ is -(CH₂)_q-A, wherein q is 0 and A is unsubstituted heteroaryl.

27. A compound according to claim 26, wherein said unsubstituted heteroaryl is thiazole or oxazole.

28. A compound according to any one of claims 1 -14, wherein M is -NR⁴⁰- and R⁶ taken together with the nitrogen of M and R⁴⁰ forms a heterocyclic structure.

29. A compound according to claim 28, wherein said heterocyclic structure is selected from:

30. A compound according to any one of the preceding claims, wherein L is linked to the phenyl group of a compound of formula (II) in mefa-position.

31. A compound according claim 1 , wherein the compound is C-{3-[6-(2- methoxy-phenyl)-pyhmidin-4-ylamino]-phenyl}-N,N-dimethyl-methanesulfonamide.

32. A compound according claim 1 , wherein the compound is C-{3-[6-(2- methoxy-phenyl)-pyhmidin-4-ylamino]-phenyl}-methanesulfonamide.

33. A compound according claim 1 , wherein the compound is C-{3-[6-(2- methoxy-phenyl)-pyhmidin-4-ylamino]-phenyl}-N-methyl-methanesulfonamide.

34. A compound according claim 1 , wherein the compound is N-(2-methoxy- ethyl)-C-{3-[6-(2-methoxy-phenyl)-pyhmidin-4-ylamino]-phenyl}- methanesulfonamide.

35. A compound according to any one of claims 1 to 34 for use as pharmaceutically active agent.

36. Use of at least one compound according to any one of claims 1 to 34 for the preparation of a pharmaceutical composition for the prophylaxis and / or treatment of a disease selected from: cell proliferative disease, pain, inflammation, cardiovascular disease and infectious disease.

37. Use according to claim 36, wherein said disease is a cell proliferative disease.

38. Use according to claim 37, wherein said cell proliferative disease is cancer.

39. Use according to claim 36, wherein said disease is an infectious disease.

40. Use according to claim 39, wherein said infectious disease is a viral infection.

41. Use according to claim 40, wherein said viral infection is an infection with HIV.

42. Use according to claim 36, wherein said disease is a cardiovascular disease.

43. Use according to claim 42, wherein cardiovascular disease is cardiac hypertrophy.

44. Use according to claim 36, wherein said disease is inflammation.

45. Use according to claim 36, wherein said disease is pain.

46. Use according to claim 45, wherein said pain comprises inflammatory pain and/or neuropathic pain.

47. Pharmaceutical composition comprising at least one compound according to any one of claims 1 to 34 as an active ingredient, together with at least one pharmaceutically acceptable carrier, excipient and/or diluent.

48. Pharmaceutical composition according to claim 47 for use in combination with an analgesic agent, wherein said analgesic agent has a mechanism of action other than inhibition of a CDK.

49. Pharmaceutical composition according to claim 47 further comprising an analgesic agent, wherein said analgesic agent has a mechanism of action other than inhibition of a CDK.

50. The use of any one of claims 36 to 46, wherein said pharmaceutical composition is for administration to a human.