GB2390090A - Pharmaceutically active 6-carbamoylpyrimido[4,5-b]indoles - Google Patents

Abstract

Carbamoyl-pyrimido [4,5-6] indoles of the formula: <EMI ID=1.1 HE=30 WI=75 LX=603 LY=1991 TI=CF> <PC>wherein the symbols are as defined in the specification, and salts thereof have MKK7 and MKK4 inhibitory activity. Corresponding intermediates in which the -CONH2 group is replaced by -CN or -CO2(C1-6 alkyl) are also novel compounds.

Description

- 1 6-CARBAMOYL9YRIMIDO14,5-bllNDOLE Derivatives 3. Detailed Description

of Invention

5 Technical Field

The present invention relates to a 6-carbamoylpyrimido[4,5-b]indoles which are useful as an active ingredient of pharmaceutical preparations. The 6-carbamoyl pyrimido[4,5-b]indoles of the present invention have MKK7 and MKK4 [MAPK 10 (mitogen activated protein kinase) kinase 7 and 4] inhibitory activity, and can be used for the prophylaxis and treatment of diseases associated with MKK7 and MKK4 activity. More specifically, the 6carbamoylpyrimido[4,5-b]indoles derivatives of the present 15 invention are useful for treatment and prophylaxis of diseases as follows: inflam matory and immunoregulatory disorders, such as asthma, atopic dermatitis, rhinitis, allergic rhinitis, allergic diseases, COPD, septic shock, arthritis, joint diseases and myocardial injuries, as well as autoirnmune pathologies such as rheumatoid arthritis, Grave's disease, and atherosclerosis.

The compounds of the present invention are also useful for treatment of ischemia, myocardial injury, pulmonary hypertension, renal failure, Huntington's chorea and cardiac hypertrophy, as well as neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and focal ischemia, since the diseases also relate to 25 MKK7 and/or MKK4.

Background Art

The mitogen-activated protein kineses (MAPKs) are a family of serine/threonine 30 kineses involved in the transduction of signals from the cell membrane to the nucleus in response to of various types of stimuli such as lipopolysaccharide (LPS), tumor

- 2 ^ necrosis factor-a (TNF-a), interleukins, CD40 and others. These kineses participate in a wide variety of signaling cascades controlling cellular events such as cell growth, differentiation, activation, apoptosis, stress responses, and transformation. Three subgroups of MAPK currently are known as extracellular-regulated kineses (ERK), 5 p-38 MAPK, and stress-activated/cjun N-terminal kinase (SAPK/INK) (Pouyssegur J. An arresting start for MAPK. Science, 290: 1515-1518, 2000). SAPK/JNKs are activated in response to cellular "stress" such as changes in osmolarity or metabolism, ischemia, heat shock, shear stress, ceramide or inflammatory cytokines (TNF-a, ILK). Once activated, JNKs control gene activity via phosphorylation of a 10 variety of transcriptional factors including c-dun, JunD, nuclear factor of activated T cells (NFAT)4, or Elk-1, all present in immune cells and known to regulate the transcription of many genes during an inflammatory response. Thus, among other functions such as induction of pro-inflammatory cytokines and Thl/Th2 differentiation, SAPK/JNKs regulate the activation and proliferation of T and B 15 lymphocytes, activation of mast cells [Sasaki T., Wada T., Kishimoto, H., Irie-Sasaki J., Matsumoto G., Goto T., Yao Z. et al., The stress kinase mitogen-activated protein kinase kinase (MKK)7 is a negative regulator of antigen receptor and growth factor receptor- induced proliferation in hematopoietic cells. J. Exp Med. 194:1-14, 2001].

20 To become activated, MAPKs themselves require dual phosphorylation of both threonine and tyrosine at their so-called Thr-X-Tyr motif, which is brought upon by the upstream regulators MAPK kineses (MKKs). MKK1-MKK7 (MEKI, MEK2, MKK3, MKK4, MEK5, MKIC6, and MKK7) are known to date with MKK7 being the most recently identified (Tournier C., Whitmarsh J., Cavanagh J., Barrett T., 25 Davis RJ. Mitogen-activated protein kinase kinase 7 is an activator of the c-dun NH2 terrninal kinase. Proc Natl Acad Sci USA, 94: 7337-7342, 1997), (Moriguchi T., Toyoshima F., Masuyama N., Hanatusa H., Gotoh Y., Nishida E. A novel SAPK/JNK kinase, MKK7, stimulated by TNFa and cellular stresses. EMBO J. 16: 7045-7053, 197) and (Lu X., Nemoto S., Lin A. Identification of c-dun NH2-terminal 30 protein kinase (JNK)-activating kinase 2 as an activator of JNK but not p38. J Biol Chem, 272: 24751-24754, 1997). Among this family of kineses MKK4 and MKK7

- 3 are the only ones capable of phosphorylating SAPK/JNKs. Overexpression of dominant negative forms of these MKKs and the use of cells from mice lacking MKK4 or MKK7 have clearly shown their implication in the regulation of many inflammatory responses. Whereas MKK7 is believed to exclusively use SAPK/JNKs 5 as substrates, MKK4 is also capable of phosphorylating p-38 MAP kineses. p-38 kineses are also involved in the control of inflammatory gene expression, especially after stimulation of cells with lipopolysaccharide and cytokines (Han J., Lee JD., Jiang Y., Li Z., Feng L., Ulevitch I<J. A MAP kinase targeted by endotoxin in mammalian cells. Science, 265: 808-811, 1994.), (Lee JC., Laydon IT., McDonnell 10 PC., Gallagher TF., Kumar S., Green D., McNulty D., Blumenthal MJ., Heys JR., Landvatter SW., Strickler JE., McLaughlin MM., Siemens JR., Fisher SM., Livi GP., White JR., Adams JL., Young PR. A protein kinase involved in the regulation of inflammatory cytokine synthesis. Nature, 372: 739-746, 1994),. In T cells, p38 controls the release of IL-12 and IFNy and in B cells, CD40 cross-linking leads to 15 rapid p38 activation and thus controls proliferation, and a&esion molecule expression. In addition, p38 MAPK are activated by hypoxia and, by controlling the transcription factor ATF2, play a role in neuronal development and survival (Lee JC., Kumar S., Griswold DE., Underwood DC., Votta EJ., Adams JL. Inhibition of p38 MAP kinase as a therapeutic strategy. Immunopharmacology, 47: 185-201, 2000).

A specific inhibitor of MKK7 and/or of MKK4, which is expected to block the synthesis of pro-inflammatory cytokines and the activation of various immune cells, should have a broad anti-inflamnatory profile with potential for the treatment of inflammatory and immunoregulatory disorders and diseases, including asthma, 25 rhinitis, allergic diseases, septic shock, joint diseases and myocardial injuries, as well as autoimmune pathologies such as rheumatoid arthritis, Grave's disease, and atherosclerosis. By interfering with apoptotic pathways, such inhibitors should also have therapeutic 30 potential for the treatment of renal failure, Huntington's chorea, cardiac hypertrophy and neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and

focal ischemia (Xia XG., Harding T., Weller M., Bieneman A., Uney JB., Schulz JB.

Gene transfer of the INK interacting protein-1 protects dopaminergic neurons in the MPTP model of Parkinson's disease. Proc Natl Acad Sci USA, 98: 10433-10438, 2001).

WO 97/02266 discloses anti-hyperproliferative disease agents represented by the general formula: fR)n' (: (CHR6)q N 2p wherein 10 e R1 P. R2P, R6', q and n' are defined in the application.

WO 98/43973 also discloses anti-proliferative disease agents represented by the general formula: {R3d)m. (: (CHR4')q N 2d W/R.d ' wherein Rld, R2d, R3d, R4', q and m' are defined in the application.

However, none of the references and other reference discloses 6-carbamoyl; pyrimido[4,5-b]indoles derivatives having MKK7 and/or MKK4 inhibitory activity.

- s - The development of a compound, which has effective MKK7 and/or MKK4 inhibitory activity and can be used for the prophylaxis and treatment of diseases associated with MKK7 and/or MKK4 activity has been desired.

Summarv of the invention As the result of extensive studies on chemical modification of 6-carbarnoy!-

pyrimido[4,5-b]indoles derivatives, the present inventors have found that the 10 compounds of the structure related to the present invention have unexpectedly excellent MKK7 andlor inhibitory activity. The present invention has been accomplished based on these findings. This invention is to provide a novel 6 carbamoylpyrimido[4,5-b]indole derivative of the formula (I), its tautomeric or stereoisomeric form, or a salt thereof R2: R3 NH2

wherein Ri is hydrogen, amino, (Cl)alkyl, (C2 6)alkenyl, (C2 6)alkynyl, halogen 20 substituted (Cry) aLkyl, cyano, cyano(C 6)alkyl, (C3 a) cycloalkyl, nitro, nitro(CI 6)alkyl or fluoro; and R2 and R3 independently represent hydrogen, (C2 6)alkenyl, (C26)alkynyl, (C3) cycloalkyl, (Cal 6)alkylcarbonyl, (Cat 6)alkylsulfonyl, arylsulfonyl, 25 heteroarylsulfonyl, or (C, 6)alkyl,

- 6 which (Cl 6)alkyl is optionally substituted by hydroxy, (Clb)alkoxy, (Cl 6)alkoxycarbonyl, (C3 8)cycloalkyl, carbamoyl, (Clb)alkyl carbamoyl, di(C'b)alkylcarbamoyl, cyano, amino, (Clb)alkylamino, di(Cb)alkylamino, arylamino, heteroarylamino, diarylamino, di 5 heteroarylamino, carboxyl, nitro, trifuluoromethyl, piperidinyl, (C'b) alkylpiperidinyl, (Cl.6) alkylcarbonylpiperidinyl, (C' 6)alkoxyearbonyl piperidinyl, carbamoylpiperidinyl, ((Cl 6)alkylcarbamoyl)piperidinyl, (di(C' 6) alkylcarbamoyl)piperidinyl, arylpiperidinyl, hetero arylpiperidinyl, aryloxycarbonylpiperidinyl, heteroaryloxycarbonyl 10 piperidinyl, (C) alkylsulfonylpiperidinyl, arylsulfonylpiperidinyl, heteroarylsulfonylpiperidinyl, pyrrolidyl, (C' 6)alkylpyrrolidyl, (C'b) alkylcarbonylpyrrolidyl, (C, 6)alkoxycarbonyl-pyrrolidyl, carbamoyl pylTolidyl, ((Cb)alkylcarbarnoyl)pyrrolidyl, (di(C,b)alkylcarbamoyl) pyrrolidyl, arylpyrrolidyl, heteroarylpyrrolidyl, aryloxycarbonyl 15 pyrrolidyl, heteroaryloxycarbonylpyrrolidyl, (C')alkylsulfonyl pyrrolidyl, arylsulfonylpyrrolidyl, or, heteroarylsulfonylpyrrolidyl, or 20 R2 and R3 are taken together with the attached nitrogen atom to form a 5-7 membered saturated ring optionally interrupted by one or two atoms selected from the group consisting of oxygen, sulfur and nitrogen, which saturated ring is optionally substituted by (C2 6)alkenyl, (C26)alkynyl, (C3)cycloalkyl, (Ci 6)alkylcarbonyl, (C, 6)alkyl 25 sulfonyl, arylsulfonyl, (C, 6)alkyl, hydroxy(C 6)alkyl, (C, 6)alkoxy(C 6)alkyl, (Ct 6)alkoxycarbonyl(C 6)alkyl, (C3,7)cycloalkyl(C 6)aLkyl, carbamoyl(C, 6) alkyl, (C,)alkylcarbamoyl(C, 6)alkyl, di(C, 6)alkylcarbamoyl(C, 6)alkyl, cyano(C')alkyl, amino (C,)alkyl, N (C' 6)alkylamino(C,b)alkyl, N,N-(C, 6) dialkylamino (C' 6)alkyl, 30 carboxyl(C' 6)alkyl, nitro (C, 6)alkyl, or trifuluoromethyl(C, 6)alkyl,

- l - which saturated ring is optionally fused by benzene or 5-7 membered saturated ring having O to 3 N atoms.

The Alkyl per se and "alk" and "alkyl" in alkoxy, alkanoyl, alkylamino, aLkyl 5 aminocarbonyl, alkylaTninosulphonyl, alkylsulphonylamino, alkoxycarbonyl, alkoxy carbonylamino and alkanoylamino represent a linear or branched alkyl radical having generally 1 to 6, preferably 1 to 4 and particularly preferably 1 to 3 carbon atoms, representing illustratively and preferably methyl, ethyl, n-propyl, isopropyl, tert butyl, n-pentyl and n-hexyl.

Alkoxy illustratively and preferably represents methoxy, ethoxy, npropoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.

Alkanoyl illustratively and preferably represents acetyl and propanoyl.

Alkylamino represents an alkylamino radical having one or two (independently selected) alkyl substituents, illustratively and preferably representing methylamino, ethylamino, n-propylamino, isopropylamino, tert-butylamino, n-pentylamino, n hexyl-amino, N,N- dimethylamino, N,N-diethylamino, N-ethyl-N-methylamino, N 20 methyl-N-n- propylamino, N-isopropyl-N-n-propylamino, N-t-butyl-N-methylarnino, Nethyl-N-n-pentylamino and N-n-hexyl-N-methylamino.

Alkylaminocarbonyl represents an alkylaminocarbonyl radical having one or tsvo (independently selected) alkyl substituents, illustratively and preferably representing 25 methylaminocarbonyl, ethylaminocarbonyl, npropylaminocarbonyl, isopropylamino carbonyl, tert-butylaminocarbonyl, npentylaminocarbonyl, n-hexylaminocarbonyl, N,N-dimethylaminocarbonyl, N,Ndiethylaminocarbonyl, N-ethyl-N-methylamino carbonyl, N-methyl-N-npropylaminocarbonyl, N-isopropyl-N-n-propylarnino carbonyl, N-t-butyl-Nmethylaminocarbonyl, N-ethyl-N-n-pentylamino-carbonyl and 30 N-n-hexyl-Nmethylaminocarbonyl.

- 8 Alkylaminosulphonyl represents an alkylaminosulphonyl radical having one or two (independently selected) alkyl substitutents, illustratively and preferably representing methylaminosulphonyl, ethylaminosulphonyl, npropylaminosulphonyl, isopropyl aminosulphonyl, tert-butylaminosulphonyl, n-pentylaminosulphonyl, n-hexyl S aminosulphonyl, N,Ndimethylaminosulphonyl, N,N-diethylaminosulphonyl, N ethyl-N-methylaminosulphonyl, N-methyl-N-n-propylaminosulphonyl, N-isopropyl N-npropylaminosulphonyl, N-t-butyl-N-methylaminosulphonyl, N-ethyl-N-n pentylaminosulphonyl and N-n-hexyl-N-methylaminosulphonyl.

10 Alkylsulphonylarnino illustratively and preferably represents methylsulphonylamino, ethyl sulphonyl am ino, n-propylsulphonylamino, i sopropylsulphonyl amino, tert-butyl sulphonylamino, npentylsulphonylamino and n-hexylsulphonylamino.

Alkoxycarbonyl illustratively and preferably represents methoxycarbonyl, 15 ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, tertbutoxycarbonyl, n pentoxycarbonyl and n-hexoxycarbonyl. Alkoxycarbonylamino illustratively and preferably represents methoxycarbonylarnino, ethoxycarbonylamino, n-propoxy carbonylamino, isopropoxycarbonylamino, tert-butoxycarbonylarnino, n-pentoxy carbonylamino and n-hexoxycarbonylamino.

Alkanoylamino illustratively and preferably represents acetylamino and ethyl carbonylarnino. Cycloalkyl per se and in cycloalkylamino and in cycloalkylcarbonyl represents a 25 cycloalkyl group having generally 3 to 8 and preferably 5 to 7 carbon atoms, illustratively and preferably representing cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

Cycloalkylarnino represents a cycloalkylarnino radical having one or two 30 (independently selected) cycloalkyl substituents, illustratively and preferably

representing cyclopropylamino, cyclobutylanino, cyclopentylamino, cyclohexyl amino and cycloheptylamino.

Cycloalkylcarbonyl illustratively and preferably represents cyclopropylcarbonyl, 5 cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl and cycloheptyl carbonyl. Aryl per se and in arylamino and in arylcarbonyl represents a mono- to tricyclic aromatic carbocyclic radical having generally 6 to 14 carbon atoms, illustratively and 10 preferably representing phenyl, naphthyl and phenanthrenyl.

Arylamino represents an arylanino radical having one or two (independently selected) aryl substituents, illustratively and preferably representing phenylamino, diphenylamino and naphthylamino.

Arylcarbonyl illustratively and preferably represents phenylcarbonyl and naphthyl carbonyl. Heteroaryl per se and in heteroarylcarbonyl represents an aromatic mono- or bicyclic 20 radical having generally 5 to lO and preferably 5 or 6 ring atoms and up to 5 and preferably up to 4 hetero atoms selected from the group consisting of S. O and N. illustratively and preferably representing thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, imidazolyl, pyridyl, pyrimidyl, pyridazinyl, indolyl, indazolyl, benzofuranyl, benzothiophenyl, quinolinyl, isoquinolinyl.

Heteroarylcarbonyl illustratively and preferably represents thienylcarbonyl, furylcarbonyl, pyrrolylcarbonyl, thiazolylcarbonyl, oxazolylcarbonyl, imidazolyl carbonyl, pyridylcarbonyl, pyrimidylcarbonyl, pyridazinylcarbonyl, indolylcarbonyl, indazolylcarbonyl, benzofuranylcarbonyl, benzothiophenylcarbonyl, quinolinyl 30 carbonyl, isoquinolinylcarbonyl.

- 10 Heterocyclyl per se and in heterocyclylcarbonyl represents a mono- or polycyclic, preferably mono- or bicyclic, nonaromatic heterocyclic radical having generally 4 to 10 and preferably 5 to 8 ring atoms and up to 3 and preferably up to 2 hetero atoms and/or hetero groups selected from the group consisting of N. O. S. SO and S02. The 5 heterocyclyl radicals can be saturated or partially unsaturated. Preference is given to 5- to 8-membered monocyclic saturated heterocyclyl radicals having up to two hetero atoms selected from the group consisting of 0, N and S. such as illustratively and preferably tetrahydrofuran-2-yl, pyrrolidin-2- yl, pyrrolidin-3-yl, pyrrolinyl, piperidinyl, morpholinyl, perhydroazepinyl.

Heterocyclylcarbonyl illustratively and preferably represents tetrahydrofuran-2 carbonyl, pyrrolidine-2-carbonyl, pyrrolidine-3carbonyl, pyrrolinecarbonyl, piperidinecarbonyl, morpholinecarbonyl, perhydroazepinecarbonyl.

15 Halogen represents fluorine, chlorine, bromine and iodine.

The compounds of the present invention surprisingly show excellent MKK7 and/or MKK4 inhibitory activity. They are, therefore, suitable for the production of medicament or medical composition, which may be useful to treat MKK7 and/or 20 MKK4 related diseases.

More specifically, since the 6-carbamoylpyrimido[4,5-bindoles derivatives of the present invention inhibit MKK7 and/or MKK4, they are useful for treatment and prophylaxis of diseases as follows: inflmnmatory and immunoregulatory disorders, such as asthma, atopic dermatitis, rhinitis, allergic rhinitis, allergic diseases, COPD, septic shock, arthritis, joint diseases and myocardial injuries, as well as autoinnune pathologies such as rheumatoid arthritis, Grave's disease, and atherosclerosis.

- 11 Therefore, MKK7 and/or MKK4 is an important target and inhibition of MKK7 and/or MKK4 is likely to be effective in the treatment of such inflammatory and immunoregulatory disorders and diseases.

5 The compounds of the present invention are also useful for treatment of ischernia, myocardial injury, pulmonary hypertension, renal failure, Huntington's chorea and cardiac hypertrophy, as well as neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and focal ischemia, since the diseases also relate to MKK7 and/or MKK4.

In one embodiment, the compounds of formula (I) are those wherein: Ret is hydrogen or amino.

15 In another embodiment, the compounds of formula (I) are those wherein: R2 and R3 independently represent hydrogen or (C3)cycloalkyl, or R2 and R3 are taken together with the attached nitrogen atom to form a saturated heterocyclic ring selected from the group consisting of pyrrolidino, piperidino, piperazino, homopiperidino, and morpholino and which hetero cyclic ring is optionally substituted by hydroxy, benzyl, bromo, Can alkyl, 25 hydroxy C,-6 alkyl, carboxy, or pyrrolidino or which heterocyclic ring is optionally fused by benzene.

In yet another embodiment, the compounds of formula (I) are those wherein: 30 R is hydrogen or amino, and

- 12 R2 and R3 independently represent hydrogen or (C3 7)cycloalkyl, or 5 R2 and R3 are taken together with the attached nitrogen atom to form a saturated heterocyclic ring selected from the group consisting of pyrrolidino, piperidino, piperazino, homopiperidino, and morpholino and which hetero cyclic ring is optionally substituted by hydroxy, benzyl, bromo, Can alkyl, hydroxy C,-6 alkyl, carboxy, or pyrrolidino or which heterocyclic ring is 10 optionally fused by benzene.

In addition, the present invention also provide a pyrimido [4,5-b]indole derivative of the formula (II), its tautomeric or stereoisomeric form, or salt thereof: R'': (

wherein Rat R2, and R3 are the same as defined above and 20 R4 is cyano or (Cl-6) alkoxycarbonyl. The pyrimido [4,5-b]indole denvatives of the formula (II) can be used as a intermediate to produce the 6carbarnoylpyrimido[4,5-b]indole derivatives of the formula (I).

Further, the present invention provides a medicament which include one of the 25 compounds described above and optionally pharmacoutically acceptable excipient.

- 13 EMBODIMENT OF THE INVENTION

The compound of the formula (I) of the present invention can be, but not limited to be, prepared by combining various known methods. In some embodiments, one or 5 more of the substituents, such as amino group, carboxyl group, and hydroxyl group of the compounds used as starting materials or intermediates are advantageously protected by a protecting group known to those skilled in the art. Examples of the protecting groups are described in "Protective Groups in Organic Synthesis (3rd Edition)" by Greene and Wuts, John Wiley and Sons, New York 1999.

The compound of the formula (I) of the present invention can be, but not limited to be, prepared by the methods [A] or [B] below.

Method [A] R: R3,=40 R:,R3 NH2

1 R1 scat, (1) \ R2,R3 =0

1 -N (lla-2) IS The compound of formula (I) (wherein Ri, R2 and R3 are the same as defined above) can be prepared by the reaction of the compound of formula (IIa-l)(wherein R', R2 and R3 are the same as defined above, and X represents (C')alkyl) and ammonia.

- 14 The reaction can be carried out without solvent or in a solvent including, for instance, alcohols such as methanol and ethanol, 1propanol, isopropanol and tert-butanol; water; ethers such as diethyl ether, isopropyl ether, dioxane and tetrahydrofuran (THF)and 1,2dimethoxyethane; aromatic hydrocarbons such as benzene, toluene and 5 xylene; amides such as N. N-dimethylformamide (DMF), N. Ndimethylacetamide; sulfoxides such as dimethyl sulfoxide, and others. Optionally, two or more of the solvents selected from the listed above can be mixed and used.

The reaction temperature can be optionally set depending on the compounds to be 10 reacted. The reaction temperature is usually, but not limited to, about 0 C to 60 C.

The reaction may be conducted for, usually, 30 minutes to 48 hours and preferably 1 to 24 hours.

The compound of formula (I) can also be prepared by the reaction of ammonia and 15 the compound (IIa-2).

The reaction can be carried out without solvent or in a solvent including, for instance, alcohols such as methanol and ethanol, 1-propanol, isopropanol and tert-butanol; water; ethers such as diethyl ether, isopropyl ether, dioxane and tetrahydrofuran 20 (THF)and 1,2dimethoxyethane; aromatic hydrocarbons such as benzene, toluene and xylene; amides such as N. N-dimethylformamide (DMF), N. Ndimethylacetamide; sulfoxides such as dimethyl sulfoxide, and others. Optionally, two or more of the solvents selected hom the listed above can be mixed and used.

25 The reaction can be advantageously carried out using coupling agent including, for instance, carbodiimides such as N. Ndicyclohexylcarbodiimide and 1-(3-dimethyl aminopropyl)-3ethylcarbodiimide; carbonyldiazoles such as 1,1'-carbonyldi(1,3 imiazole) (CDI) and 1,1'-carbonyldi(1,2,4-triazole)(CDT), and others.

30 The reaction temperature can be optionally set depending on the compounds to be reacted. The reaction temperature is usually, but not limited to, about 0 C to 60 C.

- 15 The reaction may be conducted for, usually, 30 minutes to 48 hours and preferably 1 to 24 hours.

The compound of formula (IIa-2)(wherein Rat, R2 and R3 are the same as defined 5 above) can be prepared by the hydrolysis of compound (IIa-1). The reaction may be carried out in a solvent including, for instance, halogenated hydrocarbons such as dichloromethane, chloroform and 1,2dichloroethane; ethers such as diethyl ether, isopropyl ether, dioxane and tetrabydrofuran (THF) and 1,2-dimethoxyethane; aromatic hydrocarbons such as benzene, toluene and xylene; amides such as N. N 10 dimethylformamide (DMF), N. N-dimethylacetamide and N- methylpyrrolidone; sulfoxides such as dimethylsulfoxide (DMSO); alcohols such as methanol, ethanol, I-propanol, isopropanol and tert-butanol,; water, and others. Optionally, two or more of the solvents selected from the listed above can be mixed and used. The reaction temperature can be optionally set depending on the compounds to be reacted. The 15 reaction temperature is usually, but not limited to, about 20 C to 100 C. The reaction may be conducted for, usually, 30 minutes to 48 hours and preferably 1 to 24 hours.

The reaction can be advantageously carried out in the presence of a base including, for instance, an alkali metal alkoxide such as sodium methoxide, sodium ethoxide 20 and potassium tert-butoxide; alkali metal hydroxide such as sodium hydroxide, lithium hydroxide ard potassium hydroxide; and others.

Method [B] CN, NH:

(lib) (1)

- 16 Alternatively, the compound of formula (I) (wherein R', R2 and R3 are the same as defined above) can be prepared by the hydrolysis of the compound of formula (IIb)(wherein R., R2 and R3 are the same as defined above).

The reaction can be carried out in a solvent including, for instance, alcohols such as methanol and ethanol, 1-propanol, isopropanol, n-butanol and tert-butanol; water; ketone such as acetone; ethers such as diethyl ether, isopropyl ether, dioxane and tetrahydrofuran (THF)and 1,2dimethoxyethane; aromatic hydrocarbons such AS 10 benzene, toluene and xylene; amides such as N. N-dimethylformamide (DMF), N. N dimethylacetamide, and others. Optionally, two or more of the solvents selected from the listed above can be mixed and used.

The reaction temperature can be optionally set depending on the compounds to be 15 reacted. The reaction temperature is usually, but not limited to, about 0 C to 60 C.

The reaction may be conducted for, usually, 30 minutes to 48 hours and preferably 1 to 24 hours.

The reaction can be advantageously conducted in the presence of a base, including, 20 for instance, an alkali metal alkoxide such as sodium methoxide, sodium ethoxide and potassium tert-butoxide; alkali metal hydroxide such as sodium hydroxide and potassium hydroxide; alkali metal carbonates such as sodium carbonate and potassium carbonate; alkali metal hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; alkali metal phosphate such as sodium 25 phosphate, and others.

When base is used, the reaction can be advantageously conducted in the presence of oxydating agent, for instance, hydrogen peroxide, manganese dioxide, dimethyl dioxirane, sodium percarbonate, sodium perforate, oxone, and the others.

- 17 The reaction can be advantageously conducted in the presence of an acid including, for instance, trifluoroacetic acid, hydrochloric acid and sulfonic acid, and others.

Preparation of intermediates s The compound of formula (it) (wherein R', R2 and R3 are the same as defined above and R4 represents COOX or CN, wherein X represents hydrogen or (Cal 6)allcyl) can be prepared by the following procedures.

CH3 CH3

R4 4 reducing,R4 L' NC-CH2COOEt agent O2N 1 O2N H2N H

2 3 R''CN for 4a 4 /R4 (R1'=amin0.,OHJR t alkyl, cycloalkyl. etc.,) HCOONH4 / '.1 J R' N

HCONH2 for 4b R N H 5 H 4a (R.'= amino, alkyl, cycloalkyl, etc.,) 4b (R1'= H) R: R3 R' 44

Thus, Compounds 2 can be prepared by the reaction of Compound 1 (wherein L' is a leaving group, such as halogen atom e.g., chlorine, bromine, or iodine atom, C6,0 arylsulfonyloxy group e.g., benzenesulfonyloxy, or ptoluenesulfonyloxy; and C' 15 alkylsulfonyloxy group, e.g., trifluoromethanesulfonyloxy, methanesulfonyloxy and

- 18 the like, and R is the same as defined above) with ethyl cyanoacetate using a base, for instance, sodium hydride.

The reaction may be carried out in a solvent including, for instance, ethers such as 5 diethyl ether, isopropyl ether, dioxane and tetrahydrofuran (THF)and 1,2-dimethoxy ethane; aromatic hydrocarbons such as benzene, toluene and xylene; amides such as N. N-dimethylformamide (DMF), N. N-dimethylacetamide and N-methylpyrrolidone; sulfoxides such as dimethylsulfoxide (DMSO); alcohols such as methanol, ethanol, 1-propanol, isopropanol and tert-butanol,; and others. Optionally, two or more of the 10 solvents selected from the listed above can be mixed and used. The reaction may be carried out, usually, at room temperature to 100 C for 4h to 1 2h.

Compound I and ethyl cyanoacetate are commercially available or can be synthe sized by conventional methods.

Compounds 3 (R is the same as defined above) can be prepared by reducing nitro group of compound 2 using agent including, for instance, metals such as zinc and iron in the presence of acid including, for instance, hydrochloric acid and acetic acid.

The reaction can be carried out without solvent or in a solvent including, for instance; 20 aromatic hydrocarbons such as benzene, toluene and xylene, and others. The reaction may be camed out, usually, at room temperature to 100 C for 30 minutes to 12h.

Compounds 4a (wherein R' means amino, (C.6)alkyl, (C2.6)alkenyl, (C2 6)alkynyl, halogen substituted (C 6) alkyl, cyano, cyano(C)alkyl, (C3x) cycloalkyl, nitro 25 (C')alkyl or fluoro and R4 is the same as defined above) can be prepared by the reaction of compound 3 with cyanamide, alkyl cyanide or cycloalkylcyanide. The reaction can be carried out in a solvent including, for instance, alcohols such as methanol, ethanol, 1- propanol, isopropanol and tert-butanol; ethers such as diethyl ether, isopropyl ether, dioxane and tetrahydrofitran (THF)and 1,2- dimethoxyethane; 30 aromatic hydrocarbons such as benzene, toluene and xylene, and others. The reaction may be carried out, usually, at 40 C to 1 80 C for 2hrs to two days.

- 19 Cyanamide, alkyl cyanide or cycloalkylcyanide are commercially available or can be synthesized by conventional methods.

Compounds 4b (wherein R' is hydrogen and R4 is the same as defined above) can be prepared by the reaction of compound 3 with ammonium formate in a solvent such as forrnamide. The reaction may be carried out, usually, at 40 C to 180 C for 2hrs to two days. If desired, the resulting 4b can be further modify to introduce nitro group at the position of R'.

Ammonium formats and formamide are commercially available or can be synthesized by conventional methods.

The compound 5 (wherein Ri and R4 are the same as defined above and L represents 15 leaving group including, for instance, halogen atom such as chlorine, bromine, or iodine atom or Call arylsulfonyloxy group such as benzenesulfonyloxy, or p toluenesulfonyloxy; and C1-4 alkylsulfonyloxy group such as trifluoromethane sulfonyloxy, methanesulfonyloxy, and the like.) can be prepared for instance, by the reaction of compound 4 with appropriate halogenating reagent including, for 20 instance, POCK, PC15, SOCl2, and the like; or can be prepared, for instance, by the reaction of compound 4 with the corresponding sulfonyl chloride.

The reaction may be carried out without solvent or in a solvent including, for instance, halogenated hydrocarbons such as dichloromethane, chloroform and 1,2 25 dichloroethane;such as ethers such as dioxane and tetrahydrofuran (THF)and 1,2 dimethoxyethane; aromatic hydrocarbons such as benzene, toluene and xylene, and others. Optionally, two or more of the solvents selected from the listed above can be mixed and used. The reaction can be advantageously conducted in the presence of a base, including, for instance, such as pyridine, triethylarnine and N,Ndiisopropyl 30 ethylamine, dimethylaniline, diethylaniline, and others. The reaction may be carried out, usually, at 40 C to 1 80 C for 2hrs to 12hrs.

- 20 The halogenating reagents are commercially available or can be synthesized by conventional methods.

5 Alkyl- or arylsulfonyl chlorides are commercially available or can be synthesized by conventional methods.

The compound of formula (II) (wherein R', R2 and R3 are the same as defined above and R4 represents COOX or CN, wherein X represents hydrogen or (C1-C6)alkyl) 10 can be prepared by the reaction of compound 5 (wherein Rt, R4 and L are the same as defined above) with amine 6 (wherein R2 and R3 are the same as defined above).

The reaction may be carried out without solvent or in a solvent including, for instance, such as ethers such as diethyl ether, isopropyl ether, dioxane and tetra 15 hydrofuran (THF)and 1,2-dimethoxyethane; aromatic hydrocarbons such as benzene, toluene and xylene, amides such as N. Ndimethylformamide (DMF), N. N-dimethyl acetamide and N-methylpyrrolidone; sulfoxides such as dimethylsulfoxide (DMSO); alcohols such as methanol, ethanol, 1-propanol, isopropanol and tert-butanol, and others. Optionally, two or more of the solvents selected from the listed above can be 20 mixed and used.

The reaction temperature can be optionally set depending on the compounds to be reacted. The reaction temperature is usually, but not limited tO, about 20 C to 1 50 C.

The reaction may be conducted for, usually, 30 minutes to 48 hours and preferably 1 25 to 24 hours.

The amine are commercially available or can be synthesized by conventional methods. 30 If required, R' and/or R4 can be optionally protected during the reaction and deprotected afterward.

- 21 When the compound shown by the formula (I) or a salt thereof has an asymmetric carbon in the structure, their optically active compounds and racemic mixtures are also included in the scope of the present invention.

Typical salts of the compound shown by the formula (I) include salts prepared by reaction of the compounds of the present invention with a mineral or organic acid, or an organic or inorganic base. Such salts are known as acid addition and base addition salts, successively.

Acids to Loran salts include inorganic acids such as, without limitation, sulfuric acid, phosphoric acid, hydrochloric acid, hydrobrornic acid, hydriodic acid and the like, and organic acids, such as, without limitation, p-toluenesulfonic acid, methane sulfonic acid, oxalic acid, pbromophenylsulfonic acid, succinic acid, citric acid, 15 benzoic acid, acetic acid, and the like.

Base addition salts include those derived from inorganic bases, such as, without limitation, ammonium hydroxide, alkaline metal hydroxide, alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, and organic bases, such as, 20 without limitation, ethanolamine, triethylamine, tris(hydroxymethyl)aminomethane, and the like. Examples of inorganic bases include, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

25 The compound of the present invention or a salts thereof, depending on its substituents, may be modified to form lower alkylesters or known other esters; and/or hydrates or other solvates. Those esters, hydrates, and solvates are included in the scope of the present invention.

30 The compound of the present invention may be administered in oral forms, such as, without limitation normal and enteric coated tablets, capsules, pills, powders,

- 22 granules, elixirs, tinctures, solution, suspensions, syrups, solid and liquid aerosols and emulsions. They may also be administered in parenteral forms, such as, without limitation, intravenous intraperitoneal, subcutaneous, intramuscular, and the like forms, wellknown to those of ordinary skill in the pharmaceutical arts. The 5 compounds of the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using transdermal delivery systems well-known to those of ordinary skilled in the art.

The dosage regimen with the use of the compounds of the present invention is 10 selected by one of ordinary skill in the arts, in view of a variety of factors, including, without limitation, age, weight, sex, and medical condition of the recipient, the severity of the condition to be treated, the route of administration, the level of metabolic and excretory function of the recipient, the dosage form employed, the particular compound and salt thereof employed.

The compounds of the present invention are preferably formulated prior to administration together with one or more pharmaceutically-acceptable excipients.

Excipients are inert substances such as, without limitation carriers, diluents, flavoring agents, sweeteners, lubricants, solubilizers, suspending agents, binders, tablet 20 disintegrating agents and encapsulating material.

Yet another embodiment of the present invention is pharmaceutical formulation comprising a compound of the invention and one or more pharmaceutically acceptable excipients that are compatible with the other ingredients of the 25 formulation and not deleterious to the recipient thereof. Pharmaceutical formulations of the invention are prepared by combining a therapeutically effective amount of the compounds of the invention together with one or more pharmaceutically-acceptable excipients. In making the compositions of the present invention, the active ingredient may be mixed with a diluent, or enclosed within a carrier, which may be in 30 the form of a capsule, sachet, paper, or other container. The carrier may serve as a diluent, which may be solid, semi-solid, or liquid material which acts as a vehicle, or

- 23 can be in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

s For oral administration, the active ingredient may be combined with an oral, and non toxic, pharmaceutically-acceptable carrier, such as, without limitation, lactose, starch, sucrose, glucose, sodium carbonate, mannitol, sorbitol, calcium carbonate, calcium phosphate, calcium sulfate, methyl cellulose, and the like; together with, optionally, 10 disintegrating agents, such as, without limitation, maize, starch, methyl cellulose, agar bentonite, xanthan gum, alginic acid, and the like; and optionally, binding agents, for example, without limitation, gelatin, natural sugars, beta-lactose, corn sweeteners, natural and synthetic gums, acacia, tragacanth, sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like; and, optionally, 15 lubricating agents, for example, without limitation, magnesium stearate, sodium stearate, stearic acid, sodium oleate, sodium benzoate, sodium acetate, sodium chloride, talc, and the like.

In powder forms, the carrier may be a finely divided solid which is in admixture with 20 the finely divided active ingredient. The active ingredient may be mixed with a carrier having binding properties in suitable proportions and compacted in the shape and size desired to produce tablets. The powders and tablets preferably contain from about 1 to about 99 weight percent of the active ingredient which is the novel composition of the present invention. Suitable solid carriers are magnesium 25 carboxymethyl cellulose, low melting waxes, and cocoa butter.

Sterile liquid formulations include suspensions, emulsions, syrups and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable earner, such as sterile water, sterile organic solvent, or a mixture of both sterile water 30 and sterile organic solvent.

- 24 The active ingredient can also be dissolved in a suitable organic solvent, for example, aqueous propylene glycol. Other compositions can be made by dispersing the finely divided active ingredient in aqueous starch or sodium carboxymethyl cellulose solution or in a suitable oil.

s The formulation may be in unit dosage form, which is a physically discrete unit containing a unit dose, suitable for administration in human or other mammals. A unit dosage form can be a capsule or tablets, or a number of capsules or tablets. A "unit dose" is a predetermined quantity of the active compound of the present 10 invention, calculated to produce the desired therapeutic effect, in association With one or more excipients. The quantity of active ingredient in a unit dose may be varied or adjusted from about 0.1 to about 1000 milligrams or more according to the particular treatment involved.

15 Typical oral dosages of the present invention, when used for the indicated effects, will range from about 0.01mg /kg/day to about 100 mg/lg/day, preferably from 0.1 mg/kg/day to 30 mg/kg/day, and most preferably from about 0.5 mg/kg/day to about 10 mg/kg/day. In the case of parenteral administration, it has generally proven advantageous to administer quantities of about 0.001 to 100mg /kg/day, preferably 20 from 0.01 mg/lcg/day to I mg/kg/day. The compounds of the present invention may be administered in a single daily dose, or the total daily dose may be administered in divided doses, two, three, or more times per day. Where delivery is via kansdermal forms, of course, administration is continuous.

- 25 Examples

The present invention will be described in detail below in the form of examples, but they should by no means be construed as defining the metes and bounds of the 5 present invention.

In the examples below, all quantitative data, if not stated otherwise, relate to percentages by weight.

10 Melting points are uncorrected. Liquid Chromatography - Mass spectroscopy (LC MS) data were recorded on a Micromass Platform LC with Shimadzu Phenomenex ODS column(4.6 mm X 30 mm) flushing a mixture of acetonitrile-water (9:1 to 1:9) at 1 ml/min of the flow rate. Mass spectra were obtained using electrospray (ES) ionization techniques (micromass Platform LC). TLC was performed on a precoated 15 silica gel plate (Merck silica gel 60 F-254). Silica gel (WAKO-gel C-200 (7S 150 m)) was used for all column chromatography separations. All chemicals were reagent grade and were purchased from Sigma-Aldrich, Wako pure chemical industries, Ltd., Tokyo kasei kogyo Co., Ltd., Nacalai tesque, Inc., Watanabe Chemical d. Ltd., Maybridge plc, Lancaster Synthesis Ltd., Merck KgaA, Kanto 20 Chemical Co.,Ltd.

The effect of the present compounds were examined by the following assays and pharmacological tests.

25 1The measurement of MKK7 kinase activiq (1) Preparation of MKK7 protein A plasmid containing human MKK7 open reading frame was cloned into a 30 pGEM-T Easy vector (Promega, Madison, WI) and further into a pGEX-6P-2 vector (Pharmacia) to construct human GST(Glutathione-S- transferase)

- 26 MKK7 fusion protein. This construct was coexpressed with human MEKKc (catalytic domain of MEKK (MEK (Map kinase kinase) kinase) on plasmid pBB131, in E.coli (BL21(DE3)pLysS).

5 The resulting GST-MKK7 was purified with the use of a glutathione column (Amersham Phannacia Biotech AB, Uppsala, Sweden) according to the manufacturer's instructions.

(2) A construct containing the MKK7 substrate rat GST-KN-SAPKa (GST + I O kinase negative rat SAPKa2) was inserted into a pGEX-SAP plasmid (Arnersham Phannacia Biotech AB, Uppsala, Sweden) and transformed into E. coli BL21(DE3)pLysS. Using this expression strain, GST-KN-SAPKO was purified with the use of glutathione column (Amersham Phannacia Biotech AB, Uppsala, Sweden) according to the manufacturer's instruction.

15 The purity of the protein was confirmed to be more than 90% by SDSPAGE.

Biotinylation of the substrate protein was done using sulfo-NHS-LC Biotin according to the manufacturer's instructions (Pierce, Rockford, US) (3) The measurement of MKK7 kinase activity All Test compounds (2.5 pi) at various concentrations (in 1% DMSO) were added to 15 ill of reaction buffer (20mM HEPES, O.1M NaCI, 0.1 mM Na3VO4, lOmM MgC12, lmM DTT, lmg/ml BSA, pH 7.5)) containing 0.5 1lg/ml GST-MKK7 and 0.8 1M SAPKa (biotinylated GST-KN-SAPKa 25 fusion protein). The kinase reaction was started by the addition of 12.5 1ul of 12 M ATP. After a one hour incubation period at room temperature, the reaction was stopped by the addition of 40 Ill stop solution (O.lM EDTA, pH 8.0).

30 60 p1 of this reaction mixture were transferred to a well of the streptavidine coated detection plate GA-plate, Steffens: 08114E14.FWD) and 40 1ul Tris

- 27 buffered saline (TBS, 50 mM Tris-HC1 (pH8.0), 20 mM EDTA, 1 % BSA, 1 M NaCl, 0.05% tween 20) were added. This mixture was incubated for 30 min and washed 3 times with 0.05% tween20 in (TBS), before 100 pi of Eu labeled anti-phosphothreonine-proline antibody (LANCE) was added. Alter 5 incubation for 30 min., plates were again washed 3 times win TBS, and 100 pi of the enhancement solution (Amersharn Pharmacia Biotech) was added. One hour later, time-resolved fluorescence was measured by a multi label counter (ARVO, Wallac Oy, Finland) using 340 mn for excitation and 615 nm for emission with 400 me of delay and 400 ms of window.

[The measurement of MKK4 kinase activity] (1) Preparation of MKK4 protein 15 A plasmid containing human MKK4 open reading frame was cloned into a pGEX-2T vector (Pharmacia) to construct human GST(Glutathione-S transferase)-MKK4 fusion protein. This construct was coexpressed with human MEKKc (catalytic domain of MEKK on plasmid pBB131) in E.coli (BL2 1 (DE3)pLysS).

The resulting GST-MKK4 was purified with the use of glutathione column (Amersharn Pharmacia Biotech AB, Uppsala, Sweden) according to the manufacturer's instruction. The purity of the protein was confirmed to be more than 90% by SDS-PAGE.

(2) A construct containing the MKK4 substrate rat GST-KN-SAPKa (GST + kinase negative rat SAPKa2) was inserted into a pGEX-SAP p1asmid (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and transformed into E. coli BL21(DE3)pLysS. Using this expression strain, GST-KN-SAPKa was 30 purified with the use of glutathione column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to the manufacturer's instruction. The purity

- 28 of the protein was confirmed to be more than 90% by SDS-PAGE.

Biotinylation of the substrate protein was done using sulfo-NHS-LC Biotin according to the manufacturer's instructions (Pierce, Rockford, US) 5 (3) The measurement of MKK4 kinase activity All Test compounds (5 pi) at various concentrations (in 1% DMSO) were added to 30 pi of reaction buffer (20mM HEPES, O.1M NaCl, 0.1 mM Na3VO4, lOmM MgC12, lmM DTT, lmg/ml BSA, pH 7.5)) containing 10 0.5 1lg/ml GST-MKK4 and 6,uM ATP. The kinase reaction was started by the addition of 25 Ill assay buffer containing 0. 48 EM SAPKO (biotinylated GST KN-SAPKor fusion protein). After a two hours incubation period at room temperature, the reaction was stopped by the addition of 80 pI stop solution (0.1 MEDTA,pH8.0).

120 pi of this reaction mixture were transferred to a well of the streptavidine coated detection plate GA-plate, Steffens: 08114E14.FWD) and 40 pI Tris buffered saline (TBS, 50 mM Tris-HCI (pH8.0), 20 mM EDTA, 1 % BSA, 1 M NaCI, 0.05% tween 20) were added. This mixture was incubated for 30 20 min and washed 3 times with 0.05% tween20 in (TBS), before 100 Ill of Eu labeled anti-phosphotyrosine antibody (5 ng/well;4G10, Upstate Bio technology, Lake Placid, NY, US)) was added. After incubation for 30 min., plates were again washed 3 times with TBS, and 100 Ill of the enhancement solution (Amersham Phannacia Biotech) was added. One hour later, time 25 resolved fluorescence was measured by a multi-label counter (ARVO, Wallac Oy, Finland) using 340 rim for excitation and 615 rim for emission with 400 ms of delay and 400 ms of window.

- 29 [Cell-based assaysl IL-2 and IFN-Y release in human PBMC 5 Human peripheral blood mononucleated cells (huPBMC) isolated using mono-poly resolving medium (Dainippon Seiyaku, Osaka, Japan) were incubated with test compounds (various concentrations in 0.1% DMSO) for 1 hour in a 37 C C02 incubator. Cells were then plated on 96 well plates (1x105 cell per well in 200 Ill RPMI1640 cell culture medium) pre-coated for 3 hrs with 100 1ul anti-CD3 antibody 10 (NU-T3: Nichirei) (4 g/ml)) or without any coating (unstimulated controls).

Solution was removed and plates were washed three times with 200 Ill/well phosphate buffered saline (PBS). Anti-CD28 antibody (KOLT-2: Nichirei, Tolyo, Japan) and goat anti mouse kappa antibody (Bethyl Laboratories, Inc., Montgomery, Texas, US) was added to the wells at final concentrations of 1.5 1lg/ml and 2 1lg/ml, 15 respectively. Plates were incubated for 20 hours in the incubator. Supernatant was removed and stored at -30 C in aliquots until further use. The amount of interleukin 2 (IL-2) and interferon-y (IFN- released from huPBMC was determined by commercially available ELISA (Genzyme Tech., Minneapolis, US) according to the manufacturer's instructions.

TNF-ct and IL-12 release in human PBMC and human dendritic cells Human peripheral blood mononucleated cells (huPBMC) isolated using mono-poly resolving medium were either directly used for experiments (1x105 cells per well in 25 200 pI medium) or differentiated to dendritic cells (DC) in the presence of GM-SCF (Pepro Tech., New Jersey, US, 25 ng/ml) + IL-4 (Pepro Tech., New Jersey, US, 10 ng/ml) over 7 days, then collected, counted and plated at a density of 2x104 cells per well per 200 Ill). Cells were incubated with test compounds (various concen trations in 0.1% DMSO) for I hour in a 37 C C02 incubator and then plated on 96 30 well plates (1x105 cell per well in 200 pI RPMI1640 cell culture medium). Induction of TNF-a or IL-12 was induced by stimulation with LPS (B8, Sigma, Missouri, US)

- 30 10 nB/ml). 20 hr later supernatant was removed and stored at -30 C in aliquots until further use.

The amounts of TNF-a and IL 12 released from cell cultures were determined by 5 commercially available ELISA (Genzyme Tech., Minneapolis, US) according to the manufacturer's instructions.

1Systemic inflammatory response syndrome in micel -= 10 Male Balb/c mice (20-2S g body weight) were in injected with agonistic anti-CD3 Ab (Pharmingen, San Diego, US; 10 g/mouse; clone 145-2C11) i.v. 5 rain. after compound application (i.v. in 10 %. 2 hrs post-challenge, mice were sacrificed and the serum cytokines IL-2, IL-4 and IFN-y were determined by ELISA (Genzyrne Tech., Minneapolis, US) according to the manufacturer's instruction. Data represent 15 mean values SD of 5-6 animals each. * p<0.05, ** pa 0.01 vs. vehicle control (V); Dunnett's test was used to detect differences among groups and statistics were performed using one-sided ANOVA or Student's t-test if applicable.

Results of MKK 7 kinase assay (MKK7) and MKK4 kinase assay (MKK4) are 20 shown in Examples and tables of the Examples below. The data corresponds to the compounds as yielded by solid phase synthesis and thus to levels of purity of about 40 to 90%. For practical reasons, the compounds are grouped in three classes of activity as follows: IC50=A lMcB 10pMcC The compounds of the present invention also show excellent selectivity, and strong activity in viva assays.

- 31 Examnle 1-1 (1) 3-Fluoro-4-nitrobenzyl bromide O2N NBS O2N,, ITCH cat. (PhC00)2 Br i To a solution of 3-fluoro-4-nitrotoluene (4.83 g) in carbon tetrachloride (50 ml) were added N-bromosuccinimide (NBS, 12.6 g) and benzoyl peroxide (0.45 g). The mixture was reflexed overnight and additional NBS (6.3 g) and 10 benzoyl peroxide (0.15 g) were added to reflex for another 10 h. After cooling, the reaction mixture was passed through a filter paper to remove resulting precipitates, that were washed with chloroform (50 ml). The filtrates were combined and washed with saturated sodium thiosulfite water solution and brine, successively. The organiz layer was dried over sodium sulfate and 15 evaporated in vacua. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 15/1 to 5/1) to obtain 3-fluoro-4 nitrobenzyl bromide as a yellow oil (3.43 g, 47%).

(2) 3-Fluoro-4-nitrobenzyl alcohol O2N^ CaCO3 O2N Water/Dioxane = 1t1 Br OH A mixture of 3-fluoro-4-nitrobenzyl bromide (3.43 g) and calcium carbonate (7.63 g) in a mixture of water (40 ml) and 1,4-dioxane (40 ml) was reflexed

- 32 overnight. After cooling to room temperature, the reaction mixture was passed through a paper filter to remove insoluble materials, that were washed with 1,4-dioxane (20 ml). The filtrates were combined and evaporated in vacua. The residue was dissolved in ethyl acetate (40 ml) and washed win IN 5 hydrochloric acid, saturated sodium bicarbonate water solution and bone, successively, to be dried over sodium sulfate. When the solvent was removed in vacuo and the residue was triturated with hexane, 3-fluoro-4-nitrobenzyl alcohol was obtained as colorless powders (2.07 g, 83%).

10 (3) 3-Fluoro-4-nitrobenzoic acid - OzN- Jones reagent O2N, j'-COOH OH To a solution of 3-fluoro-4-nitrobenzyl alcohol (2.97 g) in acetone (60 ml) was added Jones reagent (13 ml), prepared fiom chromic acid (26.7 g) and IS sulfuric acid (23 ml) in water (100 ml), dropwise at 0 C. The mixture was stirred on a ice-bath for 0.5 h and quenched with isopropanol (20 ml) to be concentrated in vacua. The residue was dissolved in ethyl acetate (30 ml) and washed with water (30 ml X 3), and brine (30 ml X 1), successively. The organic layer was dried over sodium sulfate and concentrated in vacua to 20 obtain a yellow solid, that was triturated with hexane to give 3-fluoro -

nitrobenzoic acid as a pale yellow solid (2.94 g, 92%).

- 33 (4) Methyl 3-fluoro-4-nitrobenzoate O2N MeOH / H2SO4 O2N: COOH COOMe 5 To a solution of 3-fluoro-4-nitrobenzoic acid (5.00 g) in methanol (50 ml) was added 98% sulfuric acid (1 ml). The mixture was reflexed overnight and concentrated in vacuo. The residue was dissolved in ethyl acetate (30 ml) and washed with saturated sodium bicarbonate water solution (30 ml) and brine (30 ml), successively. The organic layer was dried over sodium sulfate and 10 evaporated in vacua. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = S/1) to give methyl 3-fluoro-4 nitrobenzoate (5.0 g, 93%) as a colorless solid.

(S) Ethyl a-cyano-5 -methoxycarbonyl-2-nitrophenylacetate F NCCOOEt O2N[ tM CCH,COOEt O2N 63COOMe To a suspension of 60% sodiD hydride (2.01 g) in N,N-dimethylformamide (DMF, 15 ml) was added a solution of ethyl cyancacetate (5.68 g) in DMF 20 (5 ml) at 0 C. The mixture was stirred at room temperature for 0.5 h and a solution of methyl 3-fluoro-4nitrobenzoate (5.00 g) in DMF (5 ml) was added. The mixture was stirred at room temperature for another 3 h and poured into a mixture of ethyl acetate (100 ml) and 1N hydrochloric acid (200 ml). The organic layer was separated and washed with water (100 ml X 25 2) and brine (100 ml), successively, to be dried over sodium sulfate. The

- 34 solvent was removed in vacua to obtain a blown solid, that was purified by silica gel column chromatography (hexane/ethyl acetate = 5/1 to 2tl) to give ethyl a-cyano-5-methoxycarbonyl-2-nitrophenylacetate as a pale yellow solid (5.74 g, 78%).

(6) Methyl 2-Amino-3-ethoxycarbonyl-lH-indole-5-carboxylate COOMe COOMe 10 Ethyl a-cyano-5-methoxycarbonyl-2-nitrophenylacetate (3.10 g) was dis solved in glacial acetic acid (30 ml) at 80 C. Keeping the temperature, zinc powder (5.55 g) was added portionwise. The mixture was stirred at 90 C to 100 C for 1 h. After cooling to room temperature, the reaction mixture was passed through a filter paper to remove insoluble materials and washed with 15 glacial acetic acid (10 ml). The filtrates were combined and evaporated in vacua. The residue was dissolved in ethyl acetate (30 ml) and washed with saturated sodium bicarbonate water solution (30 ml X 3) and brine (30 ml), successively. The organic layer was dried over sodium sulfate and evaporated in vacua. The residue was triturated with isopropyl ether to give methyl 2 20 amino-3-emoxycarbonyllH-indole-5-carboxylate as a colorless solid (2.37 g, 85%).

- 3 5 (7) Methyl 4-Hydroxy-9H-pyrimido[4,5-b]indole-6-carboxylate H2N COOEt if)-OH )=( HCOONH4)=

\ HCONH2

COOMe COOMe 5 A mixture of methyl 2-amino-3-ethoxycarbonyl-lH-indole-Scarboxylate (0.80 g) and ammonium formate (0.20 g) in formamide (4 ml) was stirred at 175 C under the atmosphere of argon gas stream overnight. After cooling to room temperature, the reaction mixture was diluted with water (40 ml) to give precipitates, that were washed with methanol (20 ml) to obtain pure methyl 4 10 hydroxy-9H-pyrimido[4,5-b]indole-6- carboxylate (0.413 g, 56%).

(8) Methyl 4-Chloro-9H-pyrimido[4,5-b]indole-6-carboxylate No POCI3 CI HNCOOVm HN:COOMe 15 To a solution of methyl 4-hydroxy-9H-pyrimido[4,5-b] indole-6-carboxylate (0.35 g) and N,N-dimethylaniline (0.52 g) in 1,4dioxane (2 ml) was added phosphoryl chloride (1.4 ml). The mixture was stirred at 100 C for 6 h and, after cooling to room temperature, poured into crushed ice. When this quenching was completed, precipitates resulted in to be collected by a paper 20 filter and dried at 80 C in vacuo for 5 h. Resulting solid was suspended in methanol and passed through a filter paper to obtain methyl 4-chloro-9H pyrimido[4,5-b]indole- 6-carboxylate as a pale yellow solid (0.298 g, 79%).

- 36 (9) Methyl 4-(1-piperidinyl)-9H-pyrimido[4,5-b]indole-6-carboxylate HWOO WCOOMe WCOOMe s A mixture of methyl 4-chloro-9H-pyrimido[4,5-b] indole-6-carboxylate (0.29 g) and piperidine (0.5 ml) in 1,4-dioxane (1 ml) was stirred at 100 C for 0.5 h and poured into water (10 ml). Resulting mixture was extracted with chlorofonn (10 ml X 3). The organic layer was washed with water (10 ml X 10 3) and dried over sodium sulfate. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography (chloroform/ methanol = 20/1) and trituarated with isopropylether to give methyl 4-(1 piperidinyl)-9H-pyrimido[4,5-b] indole-6-carboxylate as a colorless solid (0.224 g, 64%).

Mp 224-227 C; LC-MS (ESI): Retention time: 2.4 min (YMC-Pack Pro C18, 2.0 mix 35 mm, acetonitrile-water (9:1 to 1:9), 1 ml/min), Calcd [M+1]: 311, Found: m/z 311.

Molecular weight: 310.36

- 37 (10) 4-(1-Piperidinyl)-9H-pyrimido[4,5-b]indole-6-carboxylic acid :60 4N NaOH COOMe POOH 5 To a solution of methyl 4-(1-piperidinyl)-9Hpyimido[4,5-b]indole-6 carboxylate (0.20 g) in methanol (2 ml) was added 4N NaOH aqueous solution (I ml). The mixture was reflexed for 2.5 h and acidified with acetic acid (0.3 ml). When the solution was diluted with water (30 ml), precipitates were given to be collected to give 4-(1piperidinyl)-9H-pyrimido[4,5 10 b]indole-6-carboxylic acid (0.172 g, 90%).

Mp >300 C; LC-MS (ESI): Retention time: 1.0 min (YMC-Pack Pro C18, 2.0mmx 35 mm, acetonitrile-water (9:1 to 1:9), 1 mVmin), Calcd [M+1]: 297, Found: m/z 297.

Molecular weight: 296.33 (11) 6-Carbamoyl-4-(1 -piperidinyl)-9Hpyrimido[4,5-b]indole )/ 1)CDI N:

HNg: 2) NH4OH ó3 20 COOH CONH2

4-(1-Piperidinyl)-9H-pyrimido[4,5-b]indole-6-carboxylic acid (0.05 g) was dissolved in DMF (2 ml) and carbonyldiimidazole (0.03 g) was added at 0 C.

- 38 The mixture was stirred at room temperature for 0.5 h and 28% ammonium aqueous solution (0.5 ml) was added to be stirred at room temperature for 0.5 h. The reaction mixture was extracted with ethyl acetate (5 ml X 3) and the organic layer was washed with brine (10 ml) to be dried over sodium sulfate and evaporated. The residue was triturated with isopropyl ether to give 6-carbamoyl-4-(1-piperidinyl)-9H-pyrimido[4, 5-b]indole (0.028 g, 56%) as a colorless powder.

Mp 293-294 C, LC-MS (ESI): Retention time: 3.I min. Calcd [M+1]: 296, Found: m/z 296.

Molecular weight: 295.35 Activity grade MKK7: A (12) 6-Carbamoyl-4-(1piperidinyl)-9H-pyrimido[4,5-b]indoIe Hydrochloride Salt To a suspension of 6-carbamoyl-4-(1-piperidinyl)-9H-pyrimido[4,5-b]indole (0.082 g) in I, 4-dioxane (I ml) and methanol (1 ml) was added 4N 10 hydrochloride 1,4dioxane solution (0.15 ml). Me mixture was allowed to stand for 5 min and water (0.5 ml) was added. When the mixture was warmed by heat gun, the clouded reaction mixture turned to a clear solution, that was concentrated in vacua. The residue was recrystallized from a mixture of ethanol and water to obtain 6-carbamoyl-4-(1-piperidinyl)-9H-pyrimido[4,5 15 b]indole hydrochloride Salt as colorless crystals (94 ma, >99%).

Mp 278 C (dec.); LC-MS (ESI): Retention time: 2.4 min (YMC-Pack Pro C18, 2.0 rnmx35mm,acetonitrile-water(9:1 to I:9), 1 ml/min)min,Calcd [M+1]: 296, Found: m/z 296.

Molecular weight: 331.81 Activity grade MKK7: A

- 39 Example 1-2

(1) 6-Cyano-4-(1-morphoryl)-9H-pyrimido[4,5-b]indole _ HI According to the procedure for the synthesis of methyl 4-(1-piperidinyl)-9H pynmido[4, 5-b]indole-6-carboxylate from methyl 3-fluoro-4-nitrobenzoate, that was described in Example 1-1, 6-cyano-4-(1-piperidinyl)-9H-pyrimido 10 [4,5-b] indole was prepared from 3-bromo-4-nitrobenzonitrile.

Mp >300 C (dec.); LC-MS (ESI): Retention time: 3.70 run, Calcd [M+l]: 280, Found: m/z 280.

15 Molecular weight: 279.30 (2) 6-Carbamoyl-4-(l-morphoryl)-9H-pyrimido[4, 5-b]indole Nils Intro HI 30% H202 HI

20 To a solution of 6-cyano-4-(1-morpholino)pyrimido[4,5-b]indole (mb) in the mixture of 30% hydrogen peroxide (2 ml) and methanol (2 ml) was added IN

- 40 sodium hydroxide (1 ml) at 0 C. The mixture was stirred at room temperature for 1 h and extracted with dichloromethane (5 ml X 3). The organic layer was washed with brine and dried over sodium sulfate to be concentrated in vacua.

The residue was applied onto preparative silica gel TLC (dichloromethane / 5 methanol- 95/5) to obtain 6-carbamoyl-4-(1-morphoryl)-9H-pyrimido[4,5 b]indole (0.015 g, 28%) as a pale yellow solid Mp >300 C (dec.); LC-MS (ESI): Retention time: 3.06 min. Calcd tM+13: 298, Found: m/z 298.

10 Molecular weight: 279.32 Activity grade MKK7: A a similar method according to the Example 1-2 above, the compounds in Example 15 I-3 to 11 I were synthesized.

- 41 E Emote N STR UCTUR e TV MKK! M P 1-4: A 282

1 A 27

::' A 5

1 7 H3c4H' A 236 - 239 1-8 H,C: A 267

1 A 29Z

1-10 -, A 238 z 1 1 1 A >300

- 42 ExamnIe 1-12 (1) 2-Amino-6-cyano-4-hydroxy-9H-pyrimido[4,5-b]indole Hydrochloride Salt s H2N\ HzNCOOEt -OH _ NH2CN)=.

HN:: 31 HCI in 14-Dioxane \ HCI The starting material, ethyl 2-amino-6cyanoindole-3-carboxylate was pre pared according to the procedure described in Example 1-2. To a suspension 10 of ethyl 2-amino-6cyanoindole-3-carboxylate (0.96 g) and cyanamide (0.88 g) in 1,4-dioxane (50 ml) was added 36% hydrochrolic acid (0.84 ml).

The mixture was reflexed for 2 days and, after cooling to room temperature, concentrated in vacua. The residue was washed with diethylether and triturated with methanol to give precipitates, that was collected by a paper 15 filter and washed with methanol. The collected solid was dried at 85 C in vacua to give a colorless solid (0.72 g, 66%).

(2) 2-Arnino-4-chloro-6-cyano-9H-pyrimido[4,5-b]indole Hydrochloride Salt H2\ H2N

POOH \CI

)y POCK)( HE HCI HE; HCI

- 43 To a solution of 2-amino-6-cyano-4-hydroxy-9H-pyrimido[4,5-b]indole (0.49 g) and N,N'-dimethylaniline (0.8 ml) in l,4-dioxane (0.5 ml) was added phosphoryl chloride (3 ml) dropwide. The reaction mixture was stirred at 1 00 C overnight and, after cooling to room temperature, poured into crushed 5 ice to give yellow precipitates, that was collected by a filter paper and dried at 80 C in vacua to give a yellow solid (0.42 g, 69%).

(3) 2-Amino-6-cyano4-(1-piperidinyl)-9H-pyrimido[4,5-b]indole H2N H2N

H HI HI

A mixture of 2-amino-4-chloro-6-cyanopyrimido[4,5-b]pyrimido[4,5-b]indole (0.42 g) and piperidine (4 ml) was stirred at 100 C for 4 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo and the 15 residue was dissoved in dichloromethane (10 ml) and washed with brine. The organic layer was dried over sodium sulfate and concentrated in vacua. The residue was purified by silica gel column chromatography (dichloromethane/ methanol = 10/0 to 98/2) to obtain a yellow solid (0.13 g, 29%).

20 (4) 2-Amino-6-carbamoyl-4-(1-piperidinyl)-9H-pyrimido[4,5-b]indole

- 44 H2\ H2\

:0 30% H2O2 >60

IN WCONH2

To a solution of 2-amino-6-cyano-4-(1-piperidinyl)-9H-pyrimido[4,5-b] indole (0.08 g) in ethanol (3 ml) were added 4N sodium hydroxide water solution 5 (0.6 ml) and 30% hydroperoxide (3 ml), successively. The mixture was stirred at room temperature for 2 h and poured into water (30 ml) to give yellow precipitates, that were collected by a filter paper and washed with diethylether and methanol, successively, to obtain 2amino-6-carbarnoyl-4-(1-piperidinyl) 9H-pyomido[4,5-b]indole as a yellow solid (0.07 ma, 80%).

Mp >184 C, LC-MS (ESI): Retention time: 3.45 min. Calcd [M+1]: 311, Found: m/z 31 1.

Molecular weight: 310.36 Activity grade MKK7: A

Claims (1)

1. - 45 Claims

   (1) A carbamoylpyrimido[4,5-b]indole derivative of the formula (I), its tauto meric or stereoisomeric form, or a salt thereof: s R:,R3 i=:NH2 S: wherein Rl is hydrogen, amino, (C 6)alkyl, (C2)alkenyl, (C2 6)alkynyl, halogen 10 substituted (C) alkyl, cyano, cyano(C)alkyl, (C3 g)cycloalkyl, nitro, nitro(C,)alkyl or fluoro, and R2 and R3 independently represent hydrogen, (C2 6)alkenyl, (C2)alkynyl, (C3 a)cycloalkyl, (C)alkylcarbonyl, (C)alkylsulfonyl, arylsulfonyl, 15 heteroarylsulfonyl, or (C 6)alkyl, which (C' 6)alkyl is optionally substituted by hydroxy, (C 6)alkoxy, (C, 6)alkoxycarbonyl, (C3 ')cycloalkyl, carbamoyl, (C)alkyl carbamoyl, di(C) alkylcarbarnoyl, cyano, amino, (C 6)alkylamino, 20 di(C)alkylamino, arylamino, heteroarylamino, diarylamino, dibeteroarylarnino, carboxyl, nitro, trifuluoromethyl, piperidinyl, (C 6)alkylpiperidinyl, (C,.6) alkylcarbonylpiperidinyl, (C)alkoxy carbonylpiperidinyl, carbamoylpiperidinyl, ((C)alkylcarbarnoyl) piperidinyl, (di(C 6) alkylcarbarnoyl)piperidinyl, arylpiperidinyl, 25 heteroarylpiperidinyl, aryloxycarbonylpiperidinyl, heteroaryl oxycarbonylpiperidinyl, (C') alkylsulfonylpiperidinyl, arylsulfonyl

   - 46 piperidinyl, heteroarylsulfonylpiperidinyl, pyrrolidyl, (C, 6)alkyl pyrrolidyl, (C, 6)alkylcarbonylpyrrolidyl, (Cl.6)alkoxycarbonyl pyrrolidyl, carbamoylpyrrolidyl, ((Cl 6)alkylcarbamoyl)pyrrolidyl, (di(C, 6)alkylcarbamoyl)pyrrolidyl, arylpyrrolidyl, heteroaryl 5 pyrrolidyl, aryloxycarbonylpyrrolidyl, heteroaryloxycarbonyl pyrrolidyl, (C, 6) alkylsulfonylpyrrolidyl, arylsulfonylpyrrolidyl, or, heteroarylsulfonylpyrrolidyl, or R2 and R3 are taken together with the attached nitrogen atom to form a 5-7 membered saturated ring optionally interrupted by one or two atoms selected from the group consisting of oxygen, sulfur and nitrogen, which saturated ring is optionally substituted by (C2 6)alkenyl, 15 (C2)alkynyl, (C3 s)cycloalkyl, (C' 6) alkylcarbonyl, (Ci 6)alkyl sulfonyl, arylsulfonyl, (C)alkyl, hydroxy(C'-) alkyl, (Cal 6)alkoxy (CI)alkyl, (CI 6)alkoxycarbonyl(C' 6)alkyl, (C3 7) cycloalkyl (Cl)alkyl, carbarnoyl(C)alkyl, (Cal 6)alkylcarbamoyl(C)alkyl, di(C, 6)alkylcarbamoyl(C, 6)alkyl, cyano(C, 6)alkyl, amino (C)alkyl, 20 N(C' 6)alkylamino(C 6)alkyl, N,N-(C, 6)dialkylamino (C.6)alkyl, carboxyl(C, )alkyl, nitro (C,)alkyl, or trifuluoromethyl(C,)allyl, which saturated ring is optionally fused by benzene or 5-7 membered saturated ring having O to 3 N atoms.

   (2) The 6-carbamoylpyrimido[4,5-bindole derivative, its tautomeric or stereo isomeric form, or a salt thereof as claimed in claim 1, wherein R is hydrogen or amino.

   - 47 (3) The 6-carbamoylpyrimido[4,5-b]indole derivative, its tautomeric or stereo isomeric form, or a salt thereof as claimed in claim 1, 5 wherein R2 and R3 independently represent hydrogen or (C3 s)cycloalkyl, or R2 and R3 are taken together with the attached nitrogen atom to form a saturated heterocyclic ring selected from the group consisting of pyrrolidino, pipendino, piperazino, homopiperidino, and morpholino and which heterocyclic ring is optionally substituted by hydroxy, 15 benzyl, bromo, Cat alkyl, hydroxy Can alkyl, carboxy, or pyrrolidino or which heterocyclic ring is optionally fused by benzene.

   (4) The 6-carbamoylpyrimido[4,5-b]indole derivative, its tautomeric or stereo isomeric form, or a salt thereof as claimed in claim l, wherein R is hydrogen or amino, and 25 R2 and R3 independently represent hydrogen or (C3,)cycloalkyl, or R2 and R3 are taken together with the attached nitrogen atom to form a 30 saturated heterocyclic ring selected from the group consisting of pyrrolidino, piperidino, piperazino, homopiperidino, and morpholino

   - 48 and which heterocyclic ring is optionally substituted by hydroxy, benzyl, bromo, Clue alkyl, hydroxy Cry alkyl, carboxy, or pyrrolidino or which heterocyclic ring is optionally fused by benzene.

   5 (5) The 6-carbamoylpyrimido[4,5-b]indole derivative, its tautomeric or stereo isomeric form, or a salt thereof as claimed in claim 1, wherein said derivative is selected from the group consisting of the following compounds: 6-Carbamoyl-4-( 1 -piperidinyl)-9H-pyrimido[4,5-b]indole; 10 6Carbamoyl-4-(1-morphoryl)-9H-pyrimido[4,5-b]indole; 6-Carbarnoyl-4-(4hydroxy- 1 -piperidinyl)-9H-pyrimido[4,5-b]indole; 6-Carbamoyl-4(cycloheptylamino)-9H-pyrimido[4,5-b]indole; 6-Carbamoyl-4-(1 piperidinyl)-9H-pyrimido[4,5-b]indole; 6-Carbamoyl-4-(4-methyl- 1 piperidinyl)-9H-pyrimido[4,5-b]indole; 1 S 6-Carbamoyl-4-(3-methyl- 1 piperidinyl)-9H-pyrimido[4,5-b]indole; 6-Carbamoyl-4-(3,5-dimethyl- 1 piperidinyl)-9H-pyrimido[4,5-b]indole; 6-Carbamoyl-4-(4-morpholinyl)-9Hpyrimido[4,5-b]indole; 6-Carbamoyl-4-[2-(hydroxymethyl)- 1 -piperidinyl]9H-pyrimido[4,5-b]indole; 6-Carbamoyl-4-(cyclopentylamino)-9H-pyrimido[4, 5-b]indole; 20 2-Amino-6-carbamoyl-4-(1-piperidinyl)-9H-pyrimido[4,5-b] indole; and their tautomeric and stereoisomeric form, arid salts thereof.

   (6) A medicament comprising the 6-carbamoylpyrimido[4,5-b]indole derivative, 25 its tautomeric or stereoisomeric form, or a physiologically acceptable salt thereof as claimed in claim 1 as an active ingredient.

   (7) The medicament as claimed in claim 6, further comprising one or more pharmaceutically acceptable excipients.

   - 49 (8) The medicament as claimed in claim 6, wherein the 6-carbamoyl-

   pyrimido[4,5-b]indole derivative, its tautomeric or stereoisomeric form, or a physiologically acceptable salt thereof is a MKK7 inhibitor.

   5 (9) The medicament as claimed in claim 6, wherein the 6carbamoylpyrimido [4,5-b]indole derivative, its tautomeric or stereoisomeric form, or a physiologically acceptable salt thereof is a MKK4 inhibitor.

   (10) An agent to treat or prevent a inflammatory or immunoregulatory disorder; 10 comprising the 6-carbarnoylpyrimido[4,5-b]indole derivative, its tautomeric or stereoisomeric form, or a physiologically acceptable salt thereof as claimed in claim 1 as an active ingredient.

   (11) An agent to treat a neurodegenerative disorders, Alzheimer's disease, or focal 15 ischemia; comprising the 6-carbamoylpyrimido[4,5-b]indole derivative, its tautomeric or stereoisomeric form, or a physiologically acceptable salt thereof as claimed in claim 1 as an active ingredient.

   (12) An agent to treat a disease selected from the group consisting of ischernia, 20 myocardial injury, pulmonary hypertension, renal failure, Huntington's chorea and cardiac hypertrophy; comprising the 6carbamoylpyrimidol4,5-b]indole derivative, its tautomeric or stereoisomeric form, or a physiologically acceptable salt thereof as claimed in claim 1 as an active ingredient.

   25 (13) Use of 6-carbamoylpyrimido [4,5-b]indole derivative of the formula (I) according to claim 1 for the preparation of medicaments.

   (14) A pyrimido [4,5-b]indole derivative of the formula (II), its tautomeric or stereoisomeric form, or salt thereof:

   - 50 R1 where) Rl is hydrogen, amino, (C' 6)alkyl, (C2 6)alkenyl, (C2 6) alkynyl, halogen 5 substituted (Cl 6) alkyl, cyano, cyano(Cb)alkyl, (C38) cycloalkyl, nitro, nitro(C, 6)alkyl or fluoro; and R2 and R3 independently represent hydrogen, (C2)alkenyl, (C2)alkynyl, (C3 g) cycloalkyl, (Cl 6)alkylcarbonyl, (C 6)alkylsulfonyl, arylsulfonyl, 10 heteroarylsulfonyl, or (C')alkyl, which (Cl 6)alkyl is optionally substituted by hydroxy, (C, 6)alkoxy, (C, 6)alkoxycarbonyl, (C3 8) cycloalkyl, carbarnoyl, (Cl)aLlcyl carbamoyl, di(CI 6)alkylcarbamoyl, cyano, amino, (C')alkylarnino, 15 di(CI-)alkylarnino, arylamino, heteroarylamino, diarylamino, di heteroarylamino, carboxyl, nitro, trifuluoromethyl, piperidinyl, (Cl 6) alkylpiperidinyl, (Cl 6) alkylcarbonylpiperidinyl, (Cb)alkoxy carbonylpiperidinyl, carbamoylpiperidinyl, ((Cl-)alkylcarbamoyl) piperidinyl, (di(C' 6) alkylcarbamoyl)piperidinyl, arylpiperidinyl, 20 heteroarylpiperidinyl, aryloxycarbonylpiperidinyl, heteroaryloxy carbonylpiperidinyl, (C') alkylsulfonylpiperidinyl, arylsulfonyl piperidinyl, heteroarylsulfonylpiperidinyl, pyrrolidyl, (Cl)alkyl pyrrolidyl, (C, 6) alky]carbonylpyrrolidyl, (Cl 6)alkoxycarbonyl pyrrolidyl, carbarnoylpyrrolidyl, ((Cl 6)alkylcarbamoyl)pyrrolidyl, 25 (di(C, 6) aLkylcarbamoyl)pyrrolidyl, arylpyrrolidyl, heteroaryl pyrrolidyl, aryloxycarbonylpyrrolidyl, heteroaryloxycarbonyl

   - 51 pyrrolidyl, (Cl)alkylsulfonylpyrrolidyl, arylsulfonylpyrrolidyl, or, heteroarylsulfonylpyrrolidyl, or R2 and R3 are taken together with the attached nitrogen atom to form a 5-7 membered saturated ring optionally interrupted by one or two atoms selected from the group consisting of oxygen, sulfur and nitrogen, which saturated ring is optionally substituted by (Cz 6)allenyl, 10 (C2 6)alkynyl, (C3 e)cycloalkyl, (C) alkylcarbonyl, (C)alkyl sulfonyl, arylsulfonyl, (C,.6)alkyl, hydroxy(C 6) alkyl, (C, 6)alkoxy (C! 6)alkyl, (Cat 6)alkoxycarbonyl(C, 6)alkyl, (C3.7) cycloalkyl (C, 6)alkyl, carbamoyl(C, 6)alkyl, (C, 6)alkylcarbamoyl(C) aLkyl, ding 6)alkylcarbamoyl(C)aLkyl, cyano(C 6)alkyl, amino (C,.6)aLkyl, 15 N-(C')alkylamino(C.6)alkyl, N,N-(C,.6)dialkylamino (C,)alkyl, carboxyl(C' 6)alkyl, nitro (C, 6)alkyl, or trifuluoromethyl(C)alkyl, which saturated ring is optionally fused by benzene or 5-7 membered saturated ring having O to 3 N atoms; and R4 is cyano or (C1-6) alkoxycarbonyl.

   (15) The pyrimido[4,5-b]indole derivative, its tautomeric or stereoisomeric form, or a salt thereof as claimed in claim 14, wherein R' is hydrogen or amino.

   30 (16) The pyrimido[4,5-b]indole derivative, its tautomeric or stereoisomeric form, or a salt thereof as claimed in claim 14,

   - 52 wherein R2 and R3 independently represent hydrogen or (C3 ') cycloalkyl, 5 or R2 and R3 are taken together with the attached nitrogen atom to form a saturated heterocyclic ring selected from the group consisting of pyrrolidino, pip eridino, piperazi no, homopip eri di no, and morpholino and which 10 heterocyclic ring is optionally substituted by hydroxy, benzyl, bromo, Cal aLkyl, hydroxy Cry alkyl, carboxy, or pyrrolidino or which heterocyclic ring is optionally fused by benzene.

   (17) The pyrimido[4,5-b]indole derivative, its tautomeric or stereoisomeric form, 15 or a salt thereof as claimed in claim 14, wherein R' is hydrogen or amino, and R2 and R3 independently represent hydrogen or (C3 7)cycloalkyl, or 25 R2 and R3 are taken together with the attached nitrogen atom to form a saturated heterocyclic ring selected from the group consisting of pyrrolidino, piperidino, piperazino, homopiperidino, and morpholino and which heterocyclic ring is optionally substituted by hydroxy, benzyl, bromo, C' alkyl, hydroxy C' alkyl, carboxy, or pyrrolidino 30 or which heterocyclic ring is optionally fused by benzene.

   - 53 18. Process for controlling urological disorders in humans and animals by administration of a PGI2-antagonisticly effective amount of at least one compound according to any of Claims 1 to 3.