CN1275964C - 7-oxo pyridopyrimidines as inhibitors of a cellular proliferation - Google Patents

Abstract

Compounds of formula (I) wherein R<1> is hydrogen or alkyl; R<2> is substituted cycloalkyl, heterosubstituted cycloalkyl, heteroalkylsubstituted cycloalkyl, heterosubstituted cycloalkyl-aryl, heterocyclyl, heterocyclyl spiro cycloalkyl, aralkoxy, alkoxy, -alkylene-S(O)n-alkyl(where n is 1 or 2) or -SO2Ar<2>; R<3> is hydrogen, amino, monoalkylamino, dialkylamino, acylamino, -NR<a>-C(=O)-R<b> (where R<a> is hydrogen or alkyl and R<b> is hetericyclyl or heteroalkyl) alkyl, cycloalkyl, aryl, aralkyl, haloalkyl, heteroalkyl, cyanoalkyl, -alkylene-C(O)-R (where R is hydrogen, alkyl, hydroxy, alkoxy, amino, monoalkylamino or dialkylamino) acyl, or phthalimidoalkyl; and each of Ar<1> and Ar<2> is independently aryl, and their use for treatment of disorders selected from the group consisting of arthritis, Crohns disease, Alzheimer's disease, irritable bowel syndrome, adult respiratory distress syndrome, and chronic obstructive pulmonary disease.

Description

7-oxopyridopyrimidines as inhibitors of cell proliferation

The present invention relates to 7-oxo-pyridopyrimidines. Specifically, the present invention provides 2,6-disubstituted 7-oxo-pyrido[2,3-d]pyrimidines, processes for their preparation, pharmaceutical formulations containing them, and methods of using them.

Mitogen-activated protein kinases (MAPs) belong to the family of proline-directed serine/threonine kinases that activate their substrates by dual phosphorylation. This kinase is activated by a number of signals including nutrient and osmotic stress, UV light, growth factors, endotoxins and inflammatory cytokines. One group of MAP kinases is the p38 kinase group comprising multiple isoforms (eg, p38α, p39β, p38γ and p38δ). This p38 kinase is responsible for phosphorylating and activating transcription factors and other kinases, which are activated by physical and chemical stress, pro-inflammatory cytokines and bacterial lipopolysaccharide.

More importantly, products of p38 phosphorylation have been shown to mediate the production of inflammatory cytokines including TNF and IL-1, as well as the production of cyclooxygenase-2. Each of these cytokines is involved in a variety of disorders. For example, TNF-α is a cytokine mainly produced by activated monocytes and macrophages. Its excessive or uncontrolled production plays an etiological role in the pathology of rheumatoid arthritis. Recently, inhibition of TNF production has been shown to have broad application in the treatment of inflammation, inflammatory bowel disease, multiple sclerosis and asthma.

TNF is also involved in viral infections such as HIV, influenza virus, and viruses including herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), cytomegalovirus (CMV), varicella-zoster virus ( VZV), Epstein-Barr virus, human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV-7), herpesviruses including human herpesvirus-8 (HHV-8), pseudorabies and nasal Tracheitis, etc.

Likewise, IL-1 is also produced by activated monocytes and macrophages and plays a role in many pathophysiological responses including rheumatoid arthritis, fever, and impairment of bone resorption.

In addition, the involvement of p38 may be related to stroke, Alzheimer's disease, osteoarthritis, lung injury, septic shock, angiogenesis, dermatitis, psoriasis and atopic dermatitis, see, eg, J.Exp.Opin.Ther . Patents, (2000) 10(1).

WO 96/34867 discloses certain pyrido[2,3-d]pyrimidines as inhibitors of protein tyrosine kinase mediated cell proliferation.

Inhibition of these cytokines through inhibition of p38 kinase would be beneficial in controlling, reducing and alleviating the symptoms of many of these diseases.

In one aspect, the invention provides compounds represented by the formula:

in:

R ¹ is hydrogen or alkyl;

R ² substituted cycloalkyl, heterosubstituted cycloalkyl, heteroalkyl substituted cycloalkyl, heterosubstituted cycloalkyl-alkyl, heterocyclyl, heterocyclylspirocycloalkyl, aralkoxy , alkoxy, alkyl-S(O) _n -alkylene-(wherein n is 1 or 2), or SO ₂ Ar ² ;

R ³ is hydrogen, amino, monoalkylamino, dialkylamino, acylamino, -NRa ^- C(=O) ^-Rb (wherein ^Ra is hydrogen or alkyl and ^Rb is heterocyclyl or heterocyclyl Alkyl), alkyl, cycloalkyl, aryl, aralkyl, haloalkyl, heteroalkyl, cyanoalkyl, -alkylene-C(O)-R (wherein R is hydrogen, alkyl, hydroxy, alkoxy, amino, mono- or di-alkylamino), acyl, or phthalimidoalkyl;

and each Ar ¹ and Ar ² are independently aryl;

or a pharmaceutically acceptable salt or prodrug thereof.

The compounds of formula I and their aforementioned salts are inhibitors of protein kinases and exhibit potent in vivo anti-p38 activity. Accordingly, such compounds are useful in the treatment of diseases mediated by pro-inflammatory cytokines such as TNF and IL-1.

Accordingly, another aspect of the present invention provides a method for the treatment of a p38-mediated disease or condition, wherein a therapeutically effective amount of a compound of formula I is administered to a patient in need of such treatment.

Another aspect of the present invention provides methods for preparing the above compounds.

Another aspect of the invention provides a method of preparing a medicament for the treatment of a p38-mediated disease or condition.

Unless otherwise stated, the following terms used in the specification and claims have the following meanings:

"Acyl" means the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl, where alkyl, cycloalkyl, cycloalkane Alkyl and phenylalkyl are as defined herein. Representative examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.

"Acylamino" means the group -NR'C(O)R, wherein R' is hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkane wherein alkyl, cycloalkyl, cycloalkylalkyl and phenylalkyl are as defined herein. Representative examples include, but are not limited to, formylamino, acetylamino, cyclohexylcarbonylamino, cyclohexylmethyl-carbonylamino, benzoylamino, benzylcarbonylamino, and the like.

"Alkoxy" refers to a group -OR wherein R is alkyl as defined herein, eg, methoxy, ethoxy, propoxy, butoxy, and the like.

"Alkyl" refers to a straight-chain saturated monovalent hydrocarbon group of 1-6 carbon atoms or a branched saturated monovalent hydrocarbon group of 3-6 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n -Butyl, iso-butyl, tert-butyl, pentyl, etc.

"Alkylene" refers to a straight-chain saturated divalent hydrocarbon group of 1-6 carbon atoms or a branched saturated divalent hydrocarbon group of 3-6 carbon atoms, such as methylene, ethylene, 2,2-dimethyl Ethylene, propylene, 2-methylpropylene, butylene, pentylene, etc.

"Alkylsulfonyl-alkyl" refers to the group R ^a -S(O) ₂ -R ^b -, wherein R ^a is alkyl and R ^b is alkylene, as defined herein.

"Alkylthio" means a group -SR where R is alkyl as defined above, eg, methylthio, ethylthio, propylthio, butylthio, and the like.

"Aralkoxy" refers to the group -OR ^a R ^b , where R ^a is alkylene as defined above, and R ^b is aryl as defined above.

"Aryl" refers to a monovalent monocyclic or bicyclic aromatic hydrocarbon group, which is optionally substituted independently by one or more substituents, preferably one, two or three substituents, the substituents are preferably selected from alkyl groups, Hydroxy, Alkoxy, Haloalkyl, Haloalkoxy, Heteroalkyl, Halo, Nitro, Cyano, Amino, Monoalkylamino, Dialkylamino, Methylenedioxy, Ethylenedioxy and acyl. More specifically, the term aryl includes, but is not limited to, phenyl, chlorophenyl, methoxyphenyl, 1-naphthyl, 2-naphthyl and derivatives thereof.

"Cycloalkyl" means a saturated monovalent cyclic hydrocarbon group of 3-7 ring carbon atoms, such as cyclopropyl, cyclobutyl, cyclohexyl, 4-methylcyclohexyl and the like.

"Cycloalkylalkyl" refers to the group ^-RaRb , wherein ^Ra is alkylene as defined herein and ^Rb is cycloalkyl as defined herein, e.g. cyclohexylmethyl, ^etc. wait.

"Substituted cycloalkyl" means a cycloalkyl group as defined herein, wherein one, two or three (preferably one) ring hydrogen atoms are independently replaced by cyano or -Y-C(O)R (where Y is not present or substituted by an alkylene group, and R is hydrogen, alkyl, haloalkyl, hydroxy, alkoxy, amino, monoalkylamino, dialkylamino, or optionally substituted phenyl).

"Dialkylamino" refers to the group -NRR', wherein R and R' independently represent an alkyl, hydroxyalkyl, cycloalkyl, or cycloalkylalkyl group as defined herein. Representative examples include, but are not limited to, dimethylamino, methylethylamino, bis(1-methylethyl)amino, (methyl)(hydroxymethyl)amino, (cyclohexyl)(methyl) Amino, (cyclohexyl)(ethyl)amino, (cyclohexyl)(propyl)amino, (cyclohexylmethyl)(methyl)amino, (cyclohexylmethyl)(ethyl)amino and the like.

"Halogen" means fluorine, chlorine, bromine, or iodine, preferably fluorine and chlorine.

"Haloalkyl" refers to an alkyl group substituted by one or more of the same or different halogen atoms, such as -CH ₂ Cl, -CF ₃ , -CH ₂ CF ₃ , -CH ₂ CCl ₃ and the like.

"Heteroalkyl" means an alkyl group as defined herein, wherein one, two or three hydrogen atoms are independently selected from -OR ^a , -NR ^b R ^c , and -S(O) _n R ^d Substituent substituted (where n is an integer of 0-2), it is understood that the point of attachment of the heteroalkyl group is through a carbon atom, wherein R ^a is hydrogen, acyl, alkyl, cycloalkyl, or cycloalkylalkane R ^b and R ^c independently of each other are hydrogen, acyl, alkyl, cycloalkyl, or cycloalkylalkyl, and when n is 0, R ^d is hydrogen, alkyl, cycloalkyl, or cycloalkane R is alkyl, and when n is 1 or 2, R ^is alkyl, cycloalkyl, cycloalkylalkyl, amino, acylamino, monoalkylamino, or dialkylamino. Representative examples include, but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxypropyl, 1-hydroxymethylethyl, 3-hydroxybutyl, 2,3-dihydroxybutyl, 2-hydroxy-1-methylpropyl, 2-aminoethyl, 3-aminopropyl, 2-methylsulfonylethyl, aminosulfonyl Methyl, aminosulfonylethyl, aminosulfonylpropyl, methylaminosulfonylmethyl, methylaminosulfonylethyl, methylaminosulfonylpropyl and the like.

"Heteroalkyl substituted cycloalkyl" means a cycloalkyl group as defined herein, wherein one, two or three hydrogen atoms in the cycloalkyl group have been replaced by a heteroalkyl group, it being understood that the heteroalkyl The group is attached to the cycloalkyl group by a carbon-carbon bond. Representative examples include, but are not limited to, 1-hydroxymethylcyclopentyl, 2-hydroxymethylcyclohexyl, and the like.

"Heterosubstituted cycloalkyl" means a cycloalkyl group as defined herein, wherein one, two or three hydrogen atoms of the cycloalkyl group have been replaced by substituents independently selected from the group consisting of: hydroxyl, Alkoxy, amino, acylamino, monoalkylamino, dialkylamino, oxo (C=O), imino, oximino (=NOH), NR'SO ₂ R ^d (wherein R' is hydrogen or Alkyl, and R ^d is alkyl, cycloalkyl, amino, monoalkylamino or dialkylamino), -XC(O)R (wherein X is O or NR', R is hydrogen, alkyl, haloalkane radical, alkoxy, amino, monoalkylamino, dialkylamino, or optionally substituted phenyl, and R' is H or alkyl), or -S(O) _n R (wherein n is 0- 2), wherein when n is 0, R is hydrogen, alkyl, cycloalkyl, or cycloalkylalkyl, and when n is 1 or 2, R is alkyl, cycloalkyl, cycloalkylalkane radical, amino, acylamino, monoalkylamino or dialkylamino. Representative examples include, but are not limited to, 2-, 3-, or 4-hydroxycyclohexyl, 2-, 3-, or 4-aminocyclohexyl, 2-, 3-, or 4-methanesulfonylamino ( methanesulfonamido)-cyclohexyl and the like, preferably 4-hydroxycyclohexyl, 2-aminocyclohexyl, or 4-methanesulfonamido-cyclohexyl.

"Heterosubstituted cycloalkyl-alkyl" refers to the group Ra ^{R b} -, wherein ^Ra is a heterosubstituted cycloalkyl group, and R ^b is an alkylene group.

"Heterocyclic group" refers to a saturated or unsaturated non-aromatic ring group of 3-8 ring atoms, wherein one or two ring atoms are selected from N, O or S(O) _n (wherein n is 0-2 integer of ), the remaining ring atoms are carbon, one or two of which may be optionally substituted by a carbonyl group. The heterocyclyl ring may be optionally substituted independently by one, two or three substituents selected from the group consisting of alkyl, haloalkyl, heteroalkyl, halogen, nitro, cyano, cyanoalkyl, Hydroxy, alkoxy, amino, monoalkylamino, dialkylamino, aralkyl, -(X) _n -C(O)R (wherein X is O or NR', n is 0 or 1, R is hydrogen, alkyl, haloalkyl, hydroxy (when n is 0), alkoxy, amino, monoalkylamino, dialkylamino or optionally substituted phenyl, and R' is H or alkyl), - Alkylene-C(O)R (where R is OR or NR'R", and R is hydrogen, alkyl or haloalkyl, and R' and R" are independently hydrogen or alkyl, or R' and R "together with the nitrogen atoms to which they are attached to form a ring), or -S(O) _n R (wherein n is an integer from 0 to 2), wherein when n is 0, R is hydrogen, alkyl, cycloalkyl or ring alkylalkyl, and when n is 1 or 2, R is alkyl, cycloalkyl, cycloalkylalkyl, amino, acylamino, monoalkylamino or dialkylamino. More specifically, the term hetero Cyclic groups include but are not limited to tetrahydropyranyl, piperidino, N-methylpiperidin-3-yl, piperazino, N-methylpyrrolidin-3-yl, 3-pyrrolidino , morpholino, thiomorpholino, thiomorpholino-1-oxide, thiomorpholino-1,1-dioxide, pyrrolinyl, imidazolinyl, N-methylsulfonyl- Piperidin-4-yl, and derivatives thereof.

In a further definition, the term "heterocyclyl" has the same meaning as previously defined, however, the heterocyclyl may additionally be substituted by 4 substituents, wherein the substituents are as previously defined. In addition, the substituent -alkylene-C(O)R may also have the meaning R is NR'R", such that R' and R" together with the nitrogen atom to which they are attached form a ring, in the substituent -S(O) _n In R, wherein n is 1 or 2, R may also have the meaning of haloalkyl. More specifically, the term heterocyclyl may have the following meanings in addition to the above-mentioned meanings: tetrahydrothiopyranyl, tetrahydrothiofuranyl and tetrahydrofuranyl, even more specifically piperidin-4-yl, piperidine Pyridin-1-yl, tetrahydropyran-4-yl, pyrrolidin-3-yl, morpholino, piperazin-1-yl, tetrahydrothiopyran-4-yl, tetrahydrothiopyran -3-yl or tetrahydrofuran-3-yl, preferably piperidin-4-yl, wherein each of the above specifically named heterocyclyl substituents themselves can also be defined as above, preferably exemplified in Table 1 and specific examples substituted by any specific substituent.

"Heterocyclylalkyl" refers to the group -R ^a R ^b , wherein R ^a is an alkylene group and R ^b is a heterocyclyl group as defined above, it being understood that R ^b passes through the carbon of the heterocyclyl ring Atom is attached to ^Ra , eg tetrahydropyran-2-ylmethyl, 2- or 3-piperidinylmethyl and the like.

"Heterocyclylspirocycloalkyl" refers to a spirocyclic group consisting of a cycloalkyl ring and a heterocyclic ring, wherein each ring has 5-8 ring atoms, and the two rings have only one carbon atom in common, It is understood that the point of attachment of the heterocyclylspirocycloalkyl is through the cycloalkyl ring. When two hydrogen atoms on the same carbon atom of a cycloalkyl group are replaced by a heterocyclyl group as defined herein, a spirocyclic group is formed, which may optionally be replaced by alkyl, hydroxy, hydroxyalkyl, or oxygen substitution. For example, their examples include but are not limited to 1,4-dioxaspiro[4.5]decane-8-yl, 1,3-diazaspiro[4.5]decane-8-yl, 2,4-di Ketone (dione)-1,3-diazaspiro[4.5]decane-8-yl, 1,5-dioxaspiro[5.5]undecane-9-yl, (3-hydroxymethyl-3 -methyl)-1,5-dioxaspiro[5.5]undec-9-yl and the like, preferably 1,4-dioxaspiro[4.5]decane-8-yl and 1,5-di Oxaspiro[5.5]undec-9-yl.

"Hydroxyalkyl" means an alkyl group as defined herein, substituted by one or more, preferably one, two or three hydroxy groups, provided that the same carbon atom does not bear more than one hydroxy group group. Representative examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl Base, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxy Butyl and 2-(hydroxymethyl)-3-hydroxypropyl, preferably 2-hydroxyethyl, 2,3-dihydroxypropyl and 1-(hydroxymethyl)-2-hydroxyethyl. Accordingly, the term "hydroxyalkyl" is used herein to define a subset of heteroalkyl groups.

"Leaving group" has the conventional definition in synthetic organic chemistry, that is, an atom or group capable of being displaced by a nucleophile, including halogens (such as chlorine, bromine, and iodine), alkanesulfonyloxy, arenesulfonyl Oxygen, alkylcarbonyloxy (such as acetoxy), arylcarbonyloxy, methanesulfonyloxy, tosyloxy, trifluoromethanesulfonyloxy, aryloxy (such as 2,4-dinitro phenoxy), methoxy, N, O-dimethylhydroxylamino and the like.

"Monoalkylamino" refers to the group -NHR, wherein R is an alkyl, hydroxyalkyl, cycloalkyl, or cycloalkylalkyl group as defined above, such as methylamino, (1-methylethyl base) amino, hydroxymethylamino, cyclohexylamino, cyclohexylmethylamino, cyclohexylethylamino and the like.

"Optionally substituted phenyl" refers to a phenyl ring optionally substituted independently by one or more, preferably one or two, substituents selected from the group consisting of alkyl, hydroxy, alkoxy, haloalkyl, haloalkyl Oxy, heteroalkyl, halogen, nitro, cyano, amino, methylenedioxy, ethylenedioxy, and acyl.

"Pharmaceutical excipient" refers to an excipient used in the preparation of a pharmaceutical composition that is generally safe, non-toxic and has neither biological nor other adverse effects, and includes veterinary and human pharmaceutically acceptable excipients. A "pharmaceutically acceptable excipient" as used in the specification and claims includes one or more than one excipient.

A "pharmaceutically acceptable salt" of a compound refers to a compound that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed with organic acids Salt formation, organic acids such as acetic acid, propionic acid, caproic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, lemon acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, Benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucose Heptonic Acid, 3-Phenylpropionic Acid, Trimethylacetic Acid, Tert-Butylacetic Acid, Lauryl Sulfate, Gluconic Acid, Glutamic Acid, Hydroxynaphthoic Acid, Salicylic Acid, Stearic Acid, Muconic Acid etc.; or (2) salts formed when the acidic protons present in the parent compound are replaced by metal ions, such as alkali metal ions, alkaline earth metal ions or aluminum ions; or formed when coordinated with an organic base The salt of the organic base is, for example, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc.

As used herein, "prodrug" and "pro-drug" are used interchangeably and refer to any drug that releases the active parent drug of formula (I) in vivo when such prodrug is administered to a mammalian subject. compound. Prodrugs of compounds of formula (I) are prepared by modifying one or more functional groups present in compounds of formula (I) in such a way that the modifications can be cleaved in vivo to release the parent compound. Prodrugs include compounds of formula (I), wherein the hydroxyl, amino, mercapto, carboxyl or carbonyl groups in the compound of formula (I) are combined with any group that can be cleaved in vivo to regenerate a free hydroxyl, amino or mercapto group, respectively. Examples of prodrugs include, but are not limited to, esters (e.g., acetate, dialkylglycinate, formate, phosphate, sulfate, and benzoate derivatives) of the hydroxyl functional group in compounds of formula (I). ) and carbamates (e.g. N,N-dimethylaminocarbonyl), ester groups of carboxyl functional groups (e.g. ethyl ester, morpholinoethanol ester), N-acyl derivatives of amino functional groups (e.g. N -Acetyl)N-Mannich bases, Schiff bases and enamines, oximes, acetals, ketals, enol esters of ketone and aldehyde functional groups, etc., see Bundegaard, H. "Design of Prodrugs" p 1-92, Elesevier, NewYork-Oxford (1985).

"Protecting group" refers to a group of atoms that, when attached to a reactive group in a molecule, masks, reduces or prevents that reactivity. Examples of protecting groups can be found in T.W. Green and P.G. Futs, Protecting Groups in Organic Chemistry (Wiley, 2nd ed. 1991), and Harrison and Harrison et al., Compendium of Synthetic Organic Methods, vol. 1-8 (John Wiley and Sons, 1971-1996). Representative amino protecting groups include formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (Boe), trimethylsilyl (TMS) , 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC) , Nitro-veratryloxycarbonyl (NVOC) and so on. Representative hydroxyl protecting groups include hydroxyl or acylated or alkylated groups such as benzyl and trityl ethers and alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl groups ethers and allyl ethers.

"Treatment" or "therapy" of a disease includes: (1) preventing the disease, that is, preventing the clinical symptoms of the disease from developing in mammals that may have been exposed to or are susceptible to the disease but have not Experiencing or manifesting such symptoms of a disease, (2) inhibiting the disease, that is, arresting or lessening the development of the disease or its clinical symptoms, or (3) ameliorating the disease, that is, causing a decrease in the disease or its clinical symptoms.

A "therapeutically effective amount" refers to an amount of a compound which, when administered to a mammal to treat a disease, is sufficient to effect treatment of the disease. A "therapeutically effective amount" will vary depending on the compound, the disease and its severity, the body weight of the mammal being treated, and the like.

If in the drawings of the structural formulas in this specification, "N" shows only one or two bonds connected to the remaining structures, or "O" shows one bond connected to the remaining structures, those of ordinary skill in the art will understand that in the formula In , two or one "H" atoms are present respectively in the case of "N" and one "H" atom in the case of "O", but are not shown by computer programs used to draw structural formulas, such as ISIS diagrams. Thus, "-N" means " _-NH2 ", "-N-" means "-NH-", and "O" means "-OH".

In one aspect, the invention provides compounds represented by the formula:

in:

R ¹ is hydrogen or alkyl;

^R is substituted cycloalkyl, heterosubstituted cycloalkyl, heteroalkyl substituted cycloalkyl, heterosubstituted cycloalkyl-alkyl, heterocyclyl, heterocyclylspirocycloalkyl, aralkoxy Base, alkoxy, alkyl-S(O) _n -alkylene-(wherein n is 1 or 2), or SO ₂ Ar ² ;

R ³ is hydrogen, amino, monoalkylamino, dialkylamino, acylamino, -NRa ^- C(=O) ^-Rb (wherein ^Ra is hydrogen or alkyl and ^Rb is heterocyclyl or heterocyclyl Alkyl), alkyl, cycloalkyl, aryl, aralkyl, haloalkyl, heteroalkyl, cyanoalkyl, -alkylene-C(O)-R (wherein R is hydrogen, alkyl, hydroxy, alkoxy, amino, mono- or di-alkylamino), acyl, or phthalimidoalkyl;

and each Ar ¹ and Ar ² is independently an aryl group.

Particularly preferred compounds of formula I, wherein ^Ar is optionally substituted phenyl, are preferably compounds represented by formula II:

wherein n is 1 or 2, preferably 1, and X is hydrogen, alkyl, halogen, nitro, cyano or methoxy, especially halogen, alkyl or methoxy, preferably substituted at the 2-position (fluorine, chlorine, methyl and methoxy).

More preferred compounds of formula I are those represented by formula III:

Regarding compounds of formula I, II or III and/or their preferred compounds above:

Preferably R ¹ is hydrogen or alkyl, eg methyl. More preferably ^R1 is hydrogen.

Preferably, R ^is alkylsulfonyl-alkyl, aralkoxy, e.g. benzyloxy, alkoxy, hetero-substituted cycloalkyl, heteroalkyl-substituted cycloalkyl, hetero-substituted cycloalkyl - alkyl, or heterocyclyl. More preferably ^R is heterosubstituted cycloalkyl, preferably 4-heterosubstituted cyclohexyl, such as 4-hydroxycyclohexyl or heterocyclyl, preferably substituted piperidinyl, morpholino, optionally substituted piperazinyl , tetrahydrofuryl, tetrahydrothiofuranyl, S-oxo-tetrahydrothiofuryl, S, S-dioxo-tetrahydrothiofuranyl, tetrahydrothiopyranyl, S-oxygen-tetrahydro Thiopyranyl, S,S-dioxo-tetrahydrothiopyranyl or tetrahydropyranyl, more preferably substituted piperidinyl, such as N-methylsulfonyl-piperidin-4-yl, or 4-Tetrahydropyranyl, whereby more particularly preferred substituents are those indicated in the tables and examples for the specific examples. More preferably, R can ^also be alkyl-sulfonylalkyl, such as (1,1-dimethyl-2-methylsulfonyl)ethyl or (1,1-dimethyl-3-methyl Sulfonyl) propyl.

Preferably, ^R3 is hydrogen, amino, monoalkylamino, dialkylamino, acylamino, -NRa ^- C(=O) ^-Rb (wherein ^Ra is hydrogen or alkyl, and ^Rb is hetero cycloyl or heteroalkyl), alkyl, haloalkyl, cycloalkyl, cyanomethyl, heteroalkyl, aryl, aralkyl or -alkylene-C(O)-R (wherein R is hydrogen , alkyl, hydroxyl, alkoxy, amino, monoalkylamino or dialkylamino). Most preferably ^R3 is hydrogen, amino, dimethylamino, isopropylamino, (morpholinoformyl)amino (ie -NR ^a -C(=O)-R ^b , where R ^a is hydrogen and R ^b morpholino), methyl, 2,2,2-trifluoroethyl, cyclopropyl, cyanomethyl, 2-hydroxyethyl, 4-fluorophenyl, benzyl, carboxymethyl or methoxy ylcarbonylmethyl, ethyl, 2-fluoroethyl, 2-hydroxy-2-methylpropyl, or 2-phthalimidopropyl. More preferably, ^R3 is hydrogen or methyl.

It should be understood that it is also the object of the present invention to combine the preferred schemes of each substituent R ¹ , R ² and R ³ with each other, and the compounds of formulas I, II and III and the above-mentioned preferred schemes.

It should be understood that when ^R3 is hydrogen, the compound may exist in the following tautomeric configurations.

Therefore, in addition to the compounds described above and below, the present invention also includes all tautomers. Furthermore, the present invention also includes all pharmaceutically acceptable salts of these compounds, as well as prodrug forms and all stereoisomers of the compounds, whether in pure chiral form or in racemic or other mixtures.

In another aspect, the present invention provides compounds represented by the formula:

in

R ¹ is hydrogen or alkyl;

^R is substituted cycloalkyl, heterosubstituted cycloalkyl, ^heteroalkyl substituted cycloalkyl, heterosubstituted cycloalkyl-alkyl, heterocyclyl, heterocyclic spirocycloalkyl or _SO2Ar2 ;

R ^is hydrogen, alkyl, cycloalkyl, aryl, aralkyl, haloalkyl, heteroalkyl, cyanoalkyl, -alkylene-C(O)-R (wherein R is hydrogen, alkyl , hydroxy, alkoxy, amino, mono- or di-alkylamino), or acyl;

and each Ar ¹ and Ar ² is independently an aryl group.

According to a further aspect of the present invention, particularly preferred compounds of formula I are represented by formula II below:

wherein n is 1 or 2, and X is hydrogen, alkyl, halogen, nitro, cyano or methoxy, especially halogen, alkyl or methoxy, more preferably substituted at the 2-position.

More preferred compounds of formula I are represented by formula III below:

According to a deeper aspect of the present invention, in the compound of formula I:

Preferably, R ¹ is hydrogen or alkyl. More preferably, R ¹ is hydrogen.

Preferably, R is hetero ^- substituted cycloalkyl, heteroalkyl-substituted cycloalkyl, hetero-substituted cycloalkyl-alkyl or heterocyclyl. More preferably, R ^is heterosubstituted cycloalkyl or heterocyclyl, such as 4-substituted cyclohexyl, substituted piperidinyl, or tetrahydropyranyl.

Preferably, ^R of the compound of formula I is hydrogen, alkyl, haloalkyl, cycloalkyl, cyanomethyl, heteroalkyl, aryl, aralkyl or -alkylene-C(O)-R( wherein R is hydrogen, alkyl, hydroxy, alkoxy, amino, monoalkylamino or dialkylamino). Most preferably ^R is hydrogen, methyl, 2,2,2-trifluoroethyl, cyclopropyl, cyanomethyl, 2-hydroxyethyl, 4-fluorophenyl, benzyl, carboxymethyl, or Methoxycarbonylmethyl. Even more preferably, ^R3 is hydrogen or methyl.

In another aspect of the present invention there is provided a compound as defined below:

(i). Compounds of the formula:

or its medicinal salts,

in:

R ¹ is hydrogen or alkyl;

^R is substituted cycloalkyl, heterosubstituted cycloalkyl, heteroalkyl-substituted cycloalkyl, heterosubstituted cycloalkyl-alkyl, heterocyclyl, heterocyclylspirocycloalkyl, or -SO ₂ Ar ² ;

R ^is hydrogen, alkyl, cycloalkyl, aryl, aralkyl, haloalkyl, heteroalkyl, cyanoalkyl, -alkylene-C(O)-R (wherein R is hydrogen, alkyl , hydroxy, alkoxy, amino, mono- or di-alkylamino) or acyl; and

Each Ar ¹ and Ar ² is independently an aryl group.

(ii). A compound as defined in (i), wherein Ar ¹ is optionally substituted phenyl.

(iii). A compound as defined in (ii), wherein Ar ¹ is phenyl independently substituted by one or two halogen, alkyl or methoxy groups.

(iv). A compound as defined in (iii), wherein Ar ¹ is 2-chlorophenyl, 2-methylphenyl or 2-methoxyphenyl.

(v). Compounds of formula as defined in (v):

(vi). Compounds as defined in (i), wherein ^R is hydrogen, alkyl, haloalkyl, cycloalkyl, cyanomethyl, heteroalkyl, aryl, aralkyl or -alkylene-C (O)-R.

(vii). Compounds as defined in (vi), wherein R is hydrogen, ^methyl , 2,2,2-trifluoroethyl, cyclopropyl, cyanomethyl, 2-hydroxyethyl, 4-fluoro Phenyl, benzyl, carboxymethyl, or methoxycarbonylmethyl.

(viii). A compound as defined in (vii), wherein R ³ is hydrogen or methyl.

(ix). A compound as defined in (i), wherein R ³ is hydrogen or methyl, and Ar ¹ is phenyl independently substituted by one or two halogen, alkyl or methoxy groups.

(x). A compound as defined in (i), wherein R ¹ is hydrogen or methyl.

(xi). Compounds as defined in (x), wherein R ¹ is hydrogen.

(xii). A compound as defined in (x), wherein R ² is heterosubstituted cycloalkyl, heteroalkyl substituted cycloalkyl or heterocyclyl.

(xiii). A compound as defined in (xii), wherein R ² is heterosubstituted cycloalkyl or heterocyclyl.

(xiv). A compound as defined in (xiii), wherein R ² is 4-heterosubstituted cyclohexyl.

(xv). A compound as defined in (xiv), wherein R ² is 4-hydroxycyclohexyl.

(xvi). A compound as defined in (xiii), wherein R ² is heterocyclyl.

(xvii). A compound as defined in (xvi), wherein R ² is substituted piperidinyl.

(xviii). A compound as defined in (xvi), wherein R ² is N-methylsulfonyl-piperidin-4-yl.

(xix). A compound as defined in (xvi), wherein R ² is a 4-tetrahydropyranyl group.

(xx). A compound as defined in (i), wherein ^R is heterosubstituted cycloalkyl or heterocyclyl, and ^Ar is independently substituted by one or two halogen, alkyl or methoxy groups phenyl group.

(xxi). Compounds as defined in (xx), wherein R ³ is hydrogen.

(xxii). Compounds as defined in (xx), wherein R ³ is methyl.

With reference to the broadest scope of the present invention, some representative formula I compounds are shown in Table 1 below:

Table 1. Representative Compounds of Formula I

In an in vitro p38 assay, formula I has an _IC50 of less than 10 μM, preferably less than 5 μM, more preferably less than 3 μM, most preferably less than 1 μM. Specifically, the IC ₅₀ of the compound of formula I in Table I in the in vitro p38 assay ranges from about 4.76 μM to 0.0003 μM.

The compounds of the present invention can exist in unsolvated as well as solvated (including hydrated) forms. In general, the solvated forms, including hydrated forms, are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Furthermore, as stated above, the present invention also includes all pharmaceutically acceptable salts of these compounds, as well as prodrug forms of the compounds and all stereoisomers, whether in pure chiral form or racemic mixtures or other mixture of forms.

The compounds of the present invention are further capable of forming pharmaceutically acceptable acid addition salts. All such forms are included within the scope of the present invention.

Pharmaceutically acceptable acid addition salts of compounds of formula I include salts derived from inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc.; and salts derived from organic acids, such as aliphatic Mono- or dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedicarboxylic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. These salts include sulfates, pyrosulfates, bisulfates , sulfite, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, propionate, Caprylate, Isobutyrate, Oxalate, Malonate, Succinate, Suberate, Sebacate, Fumarate, Maleate, Mandelate, Benzoate , chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, Lactate, Maleate, Tartrate, Methanesulfonate, etc. Also included are salts of amino acids such as arginine salts, etc., gluconate, galacturonic acid (see, for example, Berge et al. "Pharmaceutical salts", J. of Pharmaceutical Science, 1977, 66, 1-19) .

Preparation of Acid Addition Salts of Basic Compounds Salts may be prepared by contacting the free base with a sufficient amount of the desired acid in the conventional manner. The free base form can be regenerated by contacting the salt form with a base in the conventional manner and isolating the free base. The free base forms differ somewhat from their respective salts in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free bases for the purposes of the present invention.

Basic pharmaceutical addition salts can be formed with metal ions or amines, such as alkali and alkaline earth metal ions or organic amines. Examples of metal ions used as cations include sodium, potassium, magnesium, calcium and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine (see for example Berge et al. "Pharmaceutical Salts", J. of Pharmaceutical Science, 1977, 66, 1-19).

Preparation of Base Addition Salts of Acidic Compounds The salts may be prepared by bringing the free acid into contact with a sufficient amount of the desired base in the conventional manner. The free acid form can be regenerated by contacting the salt form with an acid in the conventional manner and isolating the free acid. The free acid forms differ somewhat from their respective salts in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acids for the purposes of the present invention.

The compounds of the present invention can be prepared in a number of ways using techniques well known to those of ordinary skill in the art. In one aspect of the present invention, the method for preparing the compound of formula I is as shown in the following reaction scheme 1:

Reaction scheme 1

Treatment of compounds of formula Ia (whose preparation is familiar to the person of ordinary skill in the art and/or purchased from commercial sources where he can purchase them) with a primary amine ( ^R3 - _NH2 ) affords compounds of formula Ib. The reaction is usually carried out in a solvent which is inert under the reaction conditions, preferably halogenated aliphatic hydrocarbons, especially dichloromethane, optionally halogenated aromatic hydrocarbons, open-chain or cyclic ethers (such as tetrahydrofuran), formamide or lower alkanols. The reaction is conveniently carried out at a temperature of from about -20°C to about 120°C.

Reduction of compounds of formula Ib affords alcohols of formula Ic. The reduction is usually carried out with lithium aluminum hydride (e.g. in a solvent which is inert under reducing conditions, preferably open-chain or cyclic ethers, especially tetrahydrofuran, at about -20°C to about 70°C, preferably about 0°C to about room temperature).

In a next step, the alcohol of formula Ic is oxidized to give carboxaldehyde of formula Id. This oxidation is typically performed using manganese dioxide, although a variety of other methods can be used (see, for example, Advanced Organic Chemistry, 4th Edition, March, John Wiley & Sons. New York (1992)). Depending on the oxidizing agent used, the reaction is generally carried out in a solvent which is inert under the particular oxidation conditions, preferably a halogenated aliphatic hydrocarbon, especially dichloromethane, or an optionally halogenated aromatic hydrocarbon. The reaction is conveniently carried out at a temperature of from about 0°C to about 60°C.

Carboxaldehyde of formula Id reacts with aryl-substituted acetate Ar ¹ -CH ₂ -CO ₂ R (wherein R is an alkyl group) in the presence of a base to give a compound of formula Ie. Any relatively non-nucleophilic base can be used, including carbonates such as potassium carbonate, lithium carbonate, sodium carbonate; bicarbonates such as potassium bicarbonate, lithium bicarbonate, sodium bicarbonate; amines such as secondary and tertiary amines and resin-bound amines, such as 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine. Conveniently, the reaction is carried out in a relatively polar, but inert solvent under the reaction conditions, preferably amides, such as dimethylformamide, N-substituted pyrrolidones, especially 1-methyl-2-pyrrolidone, the reaction The temperature is in the range of about 70°C to about 150°C, especially at or near the reflux temperature of the solvent, to facilitate the azeotropic removal of water.

Oxidation of Ie using oxidizing agents such as peracids such as 3-chloroperbenzoic acid (ie MCPBA) and potassium persulfate (Oxone(R)) can provide sulfones (If) which can be converted to a variety of target compounds. The oxidation of Ie is usually carried out in a solvent which is inert under the oxidizing conditions. For example, when using MCPBA as the oxidizing agent, the solvent is preferably a halogenated aliphatic hydrocarbon, especially chloroform. When potassium persulfate preparation (Oxone(R)) is used as the oxidizing agent, the solvent can be water, methanol or a mixture of organic solvents (such as methanol, acetonitrile or tetrahydrofuran) and water. The reaction temperature is determined according to the solvent used. For organic solvents, the reaction temperature is usually about -20°C to about 50°C, preferably about 0°C to about room temperature. When water is used as a solvent, the reaction temperature is usually about 0°C to about 50°C, preferably about 0°C to about room temperature. Alternatively, oxidation can be performed under catalytic conditions using rhenium/peroxide based reagents. See for example "Oxidation of Sulfoxides Using Hydrogen Peroxide Catalyzed by Methyltrioxyrhenium(VII)", Lahti, David W.; Espenson, James H, Inorganic Chemistry 2000, 39(10) pp.2164-2167 ; "Rhenium Oxygen Complexes in Catalytic Oxidation", Catal.Today, 2000, 55(4), pp317-363; and "A Simple and Efficient Process for the Preparation of Pyridine N-Oxides", Coperet, Christophe; Adolfsson, Hans; Khuong, Tinh-Alfredo V.; Yudin, Andrei K.; Sharpless, K.Barry, Journal of Organic Chemistry (J.Org.Chem.), 1998, 63(5), pp1740-1741, which are in This article is cited as a reference.

Reaction of the If compound with an amine (R ₂ —NH ₂ ) provides a compound of Formula I′ (ie compound I, wherein R ¹ is hydrogen). I' is then further alkylated to provide compounds of formula I wherein ^R1 is other than hydrogen. This reaction can be performed in the presence or absence of a solvent. Conveniently, the reaction is carried out at a temperature of from about 0°C to about 200°C, preferably from about room temperature to about 150°C. Alternatively, instead of using the sulfone If, the sulfide Ie or the corresponding sulfoxide can be reacted directly with an amine (R ¹ —NH ₂ ) under certain conditions to provide compounds of formula I′. Alternatively, alkylation of If with _the amine ^R1R2NH2 , wherein ^R1 and ^R2 are as described above, directly provides compounds of formula ^I.

Accordingly, the present invention provides a process for the preparation of compounds of formula I by treating a compound of general formula Ie or If with an amine (R ¹ -NH ₂ ) and optionally reacting the resulting product with R ¹ -L, wherein ^R1 is as defined above, excluding hydrogen, and L is a leaving group.

Alternatively, formaldehyde compounds of formula Ie can be prepared as shown in Scheme II below, which does not require ester reduction and alcohol oxidation of Scheme I.

Reaction Scheme II

A compound of formula II-a (wherein each R ^a is independently an alkyl group) is treated with an alkyl formate (for example methyl formate) in the presence of a base; a person of ordinary skill in the art is familiar with its preparation and/or can purchase it from purchased from commercial sources) to give compounds of formula II-b (wherein M is a metal). The reaction is generally carried out at a temperature in the range of about 0°C to about 100°C. Ethers, such as THF, and other solvents that are inert under the reaction conditions can generally be used. Suitable bases include alkoxides, such as metal tert-butoxide, and other relatively non-nucleophilic bases capable of deprotonating compounds of formula II-a.

Cyclization of compounds of formula II-b with thiourea in the presence of base affords pyrimidines of formula II-c. Typically, the cyclization reaction is carried out in an alcoholic solvent under reflux conditions using the corresponding alkoxide as the base.

Alkylation of the compound of formula II-c with an alkylating agent ^RX1 (wherein R is an alkyl group and ^X1 is a leaving group such as a halide) in the presence of a base yields a compound of formula II-d . Suitable bases include relatively non-nucleophilic bases, including carbonates, such as potassium carbonate, lithium carbonate, and sodium carbonate; bicarbonates, such as potassium bicarbonate, lithium bicarbonate, and sodium bicarbonate. Conveniently, the reaction is carried out in a relatively polar solvent that is inert under the reaction conditions, preferably acetone, dimethylformamide (DMF) or methylpyrrolidone (MP).

Compounds of formula II-d are reacted with aryl-substituted acetates Ar ¹ -CH ₂ -CO ₂ R (where R is an alkyl group) under conditions similar to those described above for the preparation of compounds of formula Ie in Scheme I, Compounds of formula II-e are provided. Although the alkylation of the compound of formula II-c is usually carried out prior to the reaction with the aryl-substituted acetate, the order of the two reactions is not critical and may be reversed. Therefore, the compound of formula II-c can be reacted with aryl-substituted acetate Ar ¹ -CH ₂ -CO ₂ R, and the obtained product can be alkylated with an alkylating agent RX ¹ to obtain the compound of formula II-e.

The amine group of the compound of formula II-e is then alkylated using an alkylating agent ^R3 - ^X2 (wherein ^R3 is as defined above and ^X2 is a leaving group such as a halide) to provide compound Ie, which can be obtained as Scheme I depicts further conversions to compounds of formula I'.

Accordingly, another aspect of the present invention provides a process for the preparation of pyrimidine compounds of formula II-c by reacting an acetal of formula IIa with an alkyl formate and then reacting the resulting product with thiourea.

Another aspect of the present invention provides a method for the preparation of a compound of formula II-e by reacting a compound of formula II-c with an alkylating agent or an aryl-substituted acetate, and then reacting the obtained product with aryl-substituted Acetate or alkylating agent reaction.

Those of ordinary skill in the art will understand that some changes can be made to the above reaction schemes, but they are also within the scope of the present invention. For example, certain procedures involve the use of protecting groups for functional groups that are incompatible with the particular reaction conditions.

The compounds of formula I and the pharmaceutically acceptable salts of basic compounds of formula I and acids according to the invention can be used as medicaments, for example in the form of pharmaceutical preparations. The pharmaceutical preparation can be administered enterally, e.g. orally in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions, e.g. as a nasal spray Intranasal administration in the form of, for example, rectal administration in the form of suppositories. However, they can also be administered parenterally, eg in the form of injection solutions.

The compounds of the formula I and the aforementioned pharmaceutically acceptable salts thereof can be processed together with pharmaceutically inert organic or inorganic carriers for the production of pharmaceutical preparations. For example, lactose, corn starch or derivatives thereof, talc, or stearic acid or its salts etc. can be used as carriers for tablets, coated tablets, dragees, hard or soft gelatin capsules. Suitable carriers for soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols and the like; however, depending on the nature of the active ingredient, there may be cases where soft gelatin capsules do not require any excipients at all. Suitable carriers for preparing solutions and syrups include, for example, water, polyols, sugars, invert sugar and glucose, and the like. Suitable carriers for suppositories include, for example, natural or hardened oils, waxes, fats, semi-solid or liquid polyols and the like.

The pharmaceutical composition may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. Besides the compounds of the formula I and their pharmaceutically acceptable salts, they may also contain other therapeutically valuable active agents.

A medicament comprising a compound of the formula I and a pharmaceutically acceptable salt of a basic compound of the formula I with an acid and a compatible pharmaceutically acceptable carrier material is also an object of the present invention, and likewise also a process for the preparation of the medicament comprising the addition of a One or more of these compounds or their salts, and, if desired, one or more other therapeutically valuable substances, are formulated with a compatible pharmaceutical carrier in a galenical dosage form.

As mentioned above, according to the present invention, the compounds of formula I and the above-mentioned pharmaceutically acceptable salts thereof can be used as therapeutically active agents, especially as anti-inflammatory agents or drugs for preventing transplant rejection after transplant surgery. The dosage will vary widely and will, of course, be suited to the individual requirements of each particular case. In general, if administered to an adult, a convenient daily dosage should be in the range of about 0.1 mg/kg to about 100 mg/kg, preferably about 0.5 mg/kg to about 5 mg/kg. The daily dose may be administered in a single dose or in divided doses, and the above-mentioned upper dose limits may be exceeded when indicated.

Finally, another object of the present invention is the use of the compound of formula I and its above-mentioned pharmaceutically acceptable salts for the production of medicaments, especially for the treatment or prevention of inflammation, immune diseases, neoplastic diseases, bronchopulmonary diseases, skin diseases and Cardiovascular disease, for the treatment of asthma, central nervous system disease, or complications of diabetes, or to prevent transplant rejection following transplant surgery.

Compounds of formula I will be useful, but not limited to, in the treatment of any disease or condition in a human or other mammal which is exacerbated or caused by excessive or uncontrolled production of TNF or p38 kinase by the mammal. Accordingly, the present invention provides a method of treating a cytokine-mediated disease comprising administering an amount of a compound of formula I, or a pharmaceutically acceptable salt or tautomer thereof, effective to interfere with the cytokine.

Compounds of formula I will be useful, but not limited to, in the treatment of inflammation in a subject, and as antipyretic agents in the treatment of fever. The compounds of the present invention will be used in the treatment of arthritis, including but not limited to, rheumatoid arthritis, spondyloarthropathy, gouty arthritis, osteoarthritis, systemic lupus erythematosus and juvenile arthritis, osteoarthritis, gouty Arthritis, and other arthritic conditions. The compound will be used in the treatment of lung disease or pneumonia, including adult respiratory distress syndrome, pulmonary sarcoidosis, asthma, silicosis, and chronic pneumonia disease. The compound is also indicated for the treatment of viral and bacterial infections, including sepsis, septic shock, Gram-negative sepsis, malaria, meningitis, infections or malignancies secondary to cachexia, acquired immunodeficiency syndrome secondary to cachexia (AIDS), AIDS, ARC (AIDS-related complex), pneumonia, and herpes virus. The compounds are also useful in the treatment of bone resorption disorders such as osteoporosis, endotoxic shock, toxic shock syndrome, reperfusion injury, autoimmune diseases including graft versus host reactions and allograft rejection , cardiovascular disease, including atherosclerosis, thrombosis, congestive heart failure, and cardiac reperfusion injury, renal reperfusion injury, liver disease and nephritis, and myalgia due to infection.

The compound is also used in the treatment of Alzheimer's disease, influenza, multiple sclerosis, cancer, diabetes, systemic lupus erythematosus (SLE), skin-related diseases such as psoriasis, eczema, burns, dermatitis, scarring, and scar tissue form. The compounds of the invention will also be useful in the treatment of gastrointestinal disorders such as inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, ulcerative colitis. The compounds will also be useful in the treatment of eye diseases such as retinitis, retinopathy, uveitis, photophobia, and acute damage to ocular tissue. The compounds of the present invention will also be useful in the treatment of angiogenesis, including neoplasia, metastasis, ophthalmic diseases such as corneal transplant rejection, ocular neovascularization, retinal neovascularization, including neovascularization following injury or infection, diabetic retinopathy, Retrolentic fibroplasia and neovascular glaucoma; ulcerative disease, such as gastric ulcer; pathological, but nonmalignant conditions, such as hemangiomas, including neonatal hemangioma, nasopharyngeal angiofibroma, and avascular necrosis of bone; diabetes nephropathy and cardiomyopathy; and reproductive system diseases in women such as endometriosis. The compounds of the invention can also be used to prevent the production of cyclooxygenase-2.

In addition to being useful in the treatment of humans, these compounds are also useful in the veterinary treatment of companion animals including mammals, rodents, etc., exotic animals and livestock. More preferred animals include horses, dogs, and cats.

The compounds of the invention are also used in co-therapy, partially or completely replacing other conventional anti-inflammatory drugs, for example with steroids, cyclooxygenase-2 inhibitors, NSAIDs, DMARDS, immunosuppressants, 5-lipoxygenase inhibitors , LTB4 antagonists and LTA4 hydrolase inhibitors co-administered.

As used herein, the term "TNF-mediated disease" refers to TNF through the control of TNF itself, or through the release of another monokine (such as but not limited to IL-1, IL-6 or IL-8) caused by TNF. Any and all diseases and conditions that play a role. For example, a condition in which IL-1 is a major component whose production or action is exacerbated or secreted in response to TNF is therefore considered a TNF-mediated disease.

As used herein, the term "p38-mediated disease" refers to a disease that acts through the control of p38 itself, or through the release of another factor (such as but not limited to, IL-1, IL-6 or IL-8) caused by p38 any and all diseases and conditions. For example, a condition in which IL-1 is a major component, whose production or action is exacerbated or secreted in response to p38, is therefore considered a p38-mediated disease.

Because TNF-β shares close structural homology with TNF-α (also known as cachectin), and because they both induce similar biological responses and bind to the same cellular receptors, TNF-α and TNF The synthesis of -beta is inhibited by the compounds of the present invention and is therefore collectively referred to herein as "TNF" unless specifically stated otherwise.

Example

All temperatures, including melting points (ie, Mpts.), are in degrees Celsius (° C.) unless otherwise indicated.

Example 1:

Sulfone 1

This example illustrates the preparation of sulfone 1 from ethyl 4-chloro-2-methylthiopyrimidine-5-carboxylate.

Step 1 Preparation of 4-methylamino-2-methylthiopyrimidine-5-carboxylic acid ethyl ester

A solution of 20 g (86 mmol) of ethyl 4-chloro-2-methylthiopyrimidine-5-carboxylate (Aldrich Chemical Co., Milwaukee, Wisconsin, USA) in 250 mL of dichloromethane was cooled to 0 °C, and 35 mL of (281 mmol) 33% solution of methylamine in ethanol. After stirring for 30 minutes, 150 mL of water was added and the phases were separated. The organic phase was dried over magnesium sulfate and filtered. The filtrate was evaporated under reduced pressure to afford 19 g (97%) of ethyl 4-methylamino-2-methylthiopyrimidine-5-carboxylate as a white solid.

Step 2 Preparation of 4-methylamino-2-methylthiopyrimidine-5-methanol

Lithium aluminum hydride (8.2g, 215mmol) was suspended in 300mL of anhydrous tetrahydrofuran cooled in an ice bath, to which was added 46g of ethyl 4-methylamino-2-methylthio-pyrimidine-5-carboxylate in 450mL of anhydrous Solution in THF (215 mmol). The reaction mixture was stirred for 15 minutes and quenched by adding 18 mL of water. 8.5 mL of 15% sodium hydroxide solution was added dropwise, followed by 25.5 mL of water. The resulting suspension was stirred at room temperature for 7 hours, then filtered. The filter residue was washed twice with 100 mL tetrahydrofuran. The combined filtrates were concentrated under reduced pressure. The residue was suspended in 200 mL of ethyl acetate/hexane (1:2), the solid therein was filtered, and dried to obtain 32.7 g (82%) of 4-methylamino-2-methylthiopyrimidine-5-methanol, which It is a yellow solid.

Step 3 Preparation of 4-methylamino-2-methylthiopyrimidine-5-carbaldehyde

To a solution of 4-methylamino-2-methylthiopyrimidine-5-methanol (20 g, 108 mmol) in 1 L of dichloromethane was added 87 g (1 mol) of manganese dioxide. The resulting mixture was stirred for 24 hours, then filtered through filter aid. The filter residue was washed with 100 mL of dichloromethane. The combined filtrates were concentrated under reduced pressure to afford 15.8 g (80%) of 4-methylamino-2methylthiopyrimidine-5-carbaldehyde as a white solid.

step 4

To the mixture of 3.3g (18.1mmol) 4-methylamino-2-methylthiopyrimidine-5-carbaldehyde, 4.0g (20.1mmol) ethyl 2-chlorophenylacetate in 30mL NMP, add 1.5g resin , polymers incorporating 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine. The reaction mixture was stirred at 120°C for 48 hours, cooled to room temperature, filtered, washed with NMP and ethyl acetate, and suspended in water. The product was isolated by filtration. Extraction of the filtrate with ethyl acetate afforded some additional product. The combined product was washed with 5% aqueous HCl and water, and dried to obtain 4.0 g of sulfide (mass spectrum MH ⁺ =318.Mpt. 193.0-193.4°C).

step 5

A solution of 13.5 g (42.5 mmol) of the sulfide in chloroform was cooled in an ice bath and treated with 20.5 g (91 mmol) of 3-chloroperbenzoic acid. The mixture was stirred at room temperature for 16 hours, then washed with saturated aqueous sodium bicarbonate and the phases were separated. The organic phase was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The product was stirred in diethyl ether, filtered and dried to obtain 13.1 g of sulfone 1 (mass spectrum MH ⁺ =350.MP = 232.6-232.8°C).

Example 2

Sulfone 2

This example illustrates the preparation of 6-(2-chlorophenyl)-2-methylsulfonyl-pyrido[2,3- d] Method for pyrimidin-7-ol (sulfone 2).

Step 2.1 Preparation of ethyl 4-amino-2-methylthiopyrimidine-5-carboxylate

Ethyl 4-chloro-2-methylthiopyrimidine-5-carboxylate (25.4 g, 106 mmol, Aldrich Chemical Co., Milwaukee, Wisconsin, USA) was treated with 50 mL of triethylamine and 40 mL of aqueous ammonium hydroxide in 300 mL of tetrahydrofuran The solution. After stirring for 4 hours, 300 mL of water were added and the phases were separated. The organic layer was washed with 300 mL of brine, concentrated in vacuo, dissolved in dichloromethane, dried over sodium sulfate, filtered and concentrated in vacuo to give 16.5 g (95%) of ethyl 4-amino-2-methylthiopyrimidine-5-carboxylate ester as a white solid.

Step 2.2 Preparation of 4-amino-2-methylthiopyrimidine-5-methanol

A solution of 4-amino-2-methylthiopyrimidine-5-carboxylate (34.7 g, 163 mmol) in 500 mL of anhydrous THF was added to a solution of lithium aluminum hydride (175 mmol) in 175 mL of diethyl ether over 1.5 h. ℃ solution. The reaction mixture was slowly warmed to ambient temperature, then cooled to 0 °C before quenching the reaction with 7 mL of water, 7 mL of 2 M sodium hydroxide solution, followed by 14 mL of water. The resulting suspension was filtered, and the filter residue was washed with 2 x 300 mL of ethyl acetate. The filtrates were combined and concentrated to afford 23.0 g (83%) of 4-amino-2-methylthiopyrimidine-5-methanol as a white solid.

Step 2.3 Preparation of 4-amino-2-methylthiopyrimidine-5-carbaldehyde

A suspension of 4-amino-2-methylthiopyrimidine-5-methanol (21.8 g, 128 mmol) in 800 mL of dichloromethane was treated with activated manganese oxide powder (63.0 g, 725 mmol). The reaction mixture was stirred for 18 hours, then filtered through celite. The filter residue was washed repeatedly with hot dichloromethane and methanol solutions. The filtrates were combined and concentrated to afford 17.5 g (81%) of 4-amino-2-methylthiopyrimidine-5-carbaldehyde as a white solid.

Step 2.4 Preparation of 6-(2-chlorophenyl)-2-methylthio-pyrido[2,3-d]pyrimidin-7-ol

To 4-amino-2-methylthiopyrimidine-5-carbaldehyde (21.7g, 128mmol) and ethyl 2-chlorophenylacetate (31.3g, 158mmol) in 250mL of anhydrous 1-methyl-2-pyrrolidone Potassium carbonate (63.0 g, 491 mmol) was added to the solution. The reaction mixture was stirred at 95 °C for 16 h, monitored by TLC (20:80, ethyl acetate/hexane). Then 12.0 g (60 mmol) of ethyl 2-chlorophenylacetate was added, and the reaction mixture was placed at 95° C. and stirred for another 16 hours. The cooled reaction mixture was filtered, and the resulting solid was washed with ethyl acetate. The combined filtrates were diluted with 400 mL of water and 300 mL of ethyl acetate. The organic phase was separated, washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo until a yellow precipitate formed. The resulting solid was washed with ethyl acetate and dried to yield a small amount of product. Most of the product remained in the aqueous layer and slowly precipitated out on standing. The precipitate was filtered, washed with water and ethyl acetate. This process was repeated 6 times, and a total of 31.9 g (82%) of 6-(2-chlorophenyl)-2-methanesulfonyl-pyrido[2,3-d]pyrimidin-7-ol was produced (mass spectrum M ⁺ =303 , mp=234.5-235.3°C).

Step 2.5 Preparation of 6-(2-chlorophenyl)-2-methylsulfonyl-pyrido[2,3-d]pyrimidin-7-ol

To a solution of 6-(2-chlorophenyl)-2-methylthio-pyrido[2,3-d]pyrimidin-7-ol (25.2 g, 82.9 mmol) in 700 mL THF was added potassium persulfate formulation (105 g, 171 mmol) as a slurry in 200 mL of water. The reaction mixture was stirred for 5 hours, filtered, and concentrated in vacuo. The resulting slurry was filtered and the collected solids were washed with water and dried to give 23.2 g (83%) of 6-(2-chlorophenyl)-2methanesulfonyl-pyrido[2,3-d]pyrimidin-7-ol ( Sulfone 2) as a pale yellow solid. (Mass spectrum MH ⁺ =336, mp=215.1-221.1°C)

Example 3

step 1

3.1 g (8.9 mmol) of sulfone 1 and 2.8 g (17.7 mmol) of 1,4-dioxo-spiro[4,5]dec-8-ylamine (see WO 99/01452 for its preparation) were stirred at 80°C in 15 mL The mixture in NMP for 16 hours was diluted with water, stirred, filtered and dried to yield 3.7 g of the ketal intermediate as a yellow solid.

step 2

A solution of 3.8 g (8.9 mmol) of the ketal in 40 mL of 80% aqueous acetic acid was stirred at 65° C. for 4 hours, cooled, poured into water, and extracted with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate and brine, dried over sodium sulfate. Concentrate under reduced pressure. The residue was purified on flash chromatography (gradient elution: 20-30% acetone/hexanes) to afford 2.4 g of a white solid (mass spectrum MH ⁺ = 383).

Example 4

A mixture of 0.4 g (1.05 mmol) of compound 3, 0.23 ml (3.2 mmol) of 1,3-propanediol and 0.25 g (1.3 mmol) of pTsOH.H ₂ O in 15 mL of toluene was heated to reflux through a Dean-Stark trap . After 16 hours, the reaction solution was cooled to room temperature, poured into aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The extract was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash chromatography (gradient elution: 2-3% acetone/hexanes) to give 0.3 g of a white solid (mass spectrum MH ⁺ = 441.Mpt. 196.1-196.5°C).

Example 5

A mixture of 0.3g (0.78mmol) of compound 3, 0.29g (2.4mmol) of 1,1,1-tris(hydroxymethyl)ethanol and 0.19g (1mmol) of p-TsOH.H ₂ O in 10mL of toluene was refluxed After 16 hours, it was cooled to room temperature, poured into aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The extract was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with ethyl acetate) and the product was converted to the hydrochloride salt using HCl/ethyl acetate to give 0.2 g of a white solid (mass spectrum M ⁺ = 485.Mpt. 180.3-181.2°C).

Example 6

A mixture of 0.1g (0.26mmol) of compound 3, 0.09g (0.81mmol) of 2-(hydroxymethyl)-1,3-propanediol and 0.065g (0.34mmol) of p-TsOH.H ₂ O in 5mL of toluene Reflux for 16 hours, cool to room temperature, pour into aqueous sodium bicarbonate solution, and extract with ethyl acetate. The extract was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with ethyl acetate) and the product was converted to the HCl salt using HCl/ether to give 0.08 g of a white solid (mass spectrum M ⁺ = 471.Mpt. 146-150°C).

Example 7

A mixture of 0.2 g (0.5 mmol) of compound 3 and 0.16 g (2.4 mmol) of hydroxylamine hydrochloride in 5 mL of pyridine was heated at 65°C. After 2 hours, the reaction solution was cooled to room temperature and poured into water. The product was filtered, washed with water, and dried to give a white solid, 0.21 g (mass spectrum MH ⁺ = 398.Mpt. > 300°C).

Example 8

A mixture of 0.26 g (0.79 mmol) of compound 3, 0.077 g (1.2 mmol) of KCN and 0.23 g (2.4 mmol) of ammonium carbonate in 10 mL of 50% aqueous ethanol was heated to 65°C. After 16 hours, the reaction mixture was diluted with 10 mL of water and heated to reflux. After 15 minutes, the reaction was cooled and the product was isolated by filtration. The product was suspended in methanol, and excess hydrochloric acid/ether was added. The solution was concentrated under reduced pressure, the residue was stirred with diethyl ether, filtered and dried to give 0.24 g of product as a white solid (mass spectrum MH ⁺ = 453. Mpt. > 300°C).

Example 9

step 1

0.1 g (0.3 mmol) of sulfone 2 and 0.94 g (0.6 mmol) of 1,4-dioxa-spiro[4,5]dec-8-ylamine were stirred at 80 °C (see WO 99/01452 for preparation) The mixture in 5 mL of NMP was cooled to room temperature for 16 hours, poured into water, diluted with saturated aqueous sodium bicarbonate, and extracted with ethyl acetate. The extract was washed with water and brine, then dried over sodium sulfate. The solution was concentrated under reduced pressure and then purified by flash chromatography (gradient elution: 40-80% ethyl acetate/hexanes) to afford 0.1 g of the ketal intermediate as a white solid. (Mass spectrum MH ⁺ = 413).

step 2

A solution of 2.35 g (5.7 mmol) of the ketal in 40 mL of 80% aqueous acetic acid was stirred at 65 °C for 3 hours, cooled, poured into water, filtered and dried to give 0.8 g of the ketone (MH ⁺ = 369 in mass spectrum) which was yellow solid. The sample was converted to the HCl salt with HCl/ether (mass spectrum MH ⁺ = 369.Mpt. = 265.5-269.9°C).

Example 10

A mixture of 0.2g (0.54mmol) of compound 9, 0.2g (1.7mmol) of 1,1,1-tris(hydroxymethyl)ethanol and 0.13g (0.7mmol) of p-TsOH.H ₂ O in 30mL of toluene Reflux for 16 hours, cool to room temperature, and filter. The isolated white solid product was p-toluenesulfonate (0.19g) (mass spectrum MH ⁺ =470.Mpt.=223-226.5°C).

Example 11

To a solution of sulfone 2 (0.5 g, 1.3 mmol) in 3 mL of 1-methyl-2-pyrrolidone was added trans-4-aminocyclohexanol (0.54 g, 4.7 mmol). The reaction mixture was stirred at 70°C for 2 hours, cooled and diluted with ethyl acetate until a precipitate started to form. The resulting suspension was filtered, the solid was washed with water and ethyl acetate, and dried to afford 0.40 g (78%) of the title compound as a bright yellow solid (mass spectrum M+H ⁺ = 371, mp = 191.1-194.3°C). The free base was taken up in ethyl acetate and then treated with 1M HCl/ _Et2O solution to form 2-(trans-4-hydroxycyclohexylamino)-6-(2-chlorophenyl)pyrido[2, 3-d] The hydrochloride salt of pyrimidin-7-ol (11), which is a white powder with a melting point of 263.2-264.0°C.

Example 12

To a solution of sulfone 2 (1.27 g, 3.78 mmol) in 15 mL of 1-methyl-2-pyrrolidone was added trans-1,4-diaminocyclohexane (3.75 g, 32.8 mmol). The reaction mixture was stirred at 65°C for 1.5 hours, cooled, and diluted with 80 mL of ethyl acetate. The resulting suspension was filtered and washed several times with ethyl acetate and methanol. The solid was dried to afford 1.31 g (94%) of 2-(trans-4-aminocyclohexylamino)-6-(2-chlorophenyl)-pyrido[2,3-d]pyrimidin-7-ol, which It is off-white powder (mass spectrum M+H ⁺ =370). The free base was taken up in ethyl acetate and then treated with 1M HCl/ _Et2O solution to form the dihydrochloride salt of the title compound as a white powder, mp &gt;300&lt;0&gt;C.

Example 13

To a solution of sulfone 1 (0.36 g, 1.0 mmol) in 10 mL of 1-methyl-2-pyrrolidone was added trans-1,4-diaminocyclohexane (1.05 g, 9.20 mmol). The reaction mixture was stirred at 80°C for 20 minutes, cooled and diluted with ethyl acetate. A small precipitate formed and the suspension was filtered. Water was added to the organic layer and extracted with 3 x 60 mL ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated to give a yellow liquid. Purification by column chromatography (50:50, methanol/dichloromethane) afforded 0.25 g (62%) of 2-(trans-4-aminocyclohexylamino)-6-(2-chlorophenyl)-8-methanol yl-pyrido[2,3-d]pyrimidin-7-one as a yellow foam (mass spectrum M+H ⁺ =384, mp=190.3-191.4°C). The free base was taken up in ethyl acetate and then treated with 1M HCl/ _Et2O solution to form the dihydrochloride salt of the title compound as a white powder, mp 224.0-228.5°C.

Example 14

To a solution of amine 13 (70 mg, 0.18 mmol) in 10 mL of dichloromethane was added triethylamine (0.040 mL, 0.29 mmol) and methanesulfonic anhydride (50 mg, 0.29 mmol). The reaction mixture was stirred for 1 hour and concentrated in vacuo. Purification by column chromatography (3:97, methanol/dichloromethane) afforded 78 mg (93%) of 2-(trans-4-methanesulfonylaminocyclohexylamino)-6-(2-chlorophenyl)-8- Methyl-pyrido[2,3-d]pyrimidin-7-one as a white solid (mass spectrum M+H ⁺ =462, mp=260.9-261.1°C). The free base was taken up in ethyl acetate and then treated with 1M HCl/ _Et2O solution to form the hydrochloride salt of the title compound as a white powder, mp 250.8-252.2°C.

Example 15

To a solution of amine 13 (116 mg, 0.302 mmol) in 15 mL of dichloromethane was added triethylamine (0.050 mL, 0.36 mmol) and dimethylsulfamoyl chloride (0.075 mL, 0.696 mmol). The reaction mixture was refluxed for 1 hour. Then add 0.075mL (0.696mmol) dimethylsulfamoyl chloride, reflux the reaction mixture for 16 hours, cool, concentrate in vacuo, and purify by column chromatography (3:97, methanol/dichloromethane) to obtain 90mg (61% ) 2-(trans-4-N, N-dimethylsulfonylaminocyclohexylamino)-6-(2-chlorophenyl)-8-methyl-pyrido[2,3-d]pyrimidine- 7-Kone, which is a white solid (mass spectrum M+H ⁺ =491, mp=226.5-229.0°C). The free base was dissolved in dichloromethane and then treated with 1M HCl/ _Et2O solution to form the hydrochloride salt of the title compound as a pale yellow powder, mp 160.0-168.0°C.

Example 16

This example illustrates the synthesis of 2-(trans-4-methanesulfonyl Acylamidocyclohexylamino)-6-(2-chlorophenyl)-pyrido[2,3-d]pyrimidin-7-one.

To a solution of sulfone 2 (1.84 g, 5.48 mmol) in 18 mL of anhydrous 1-methyl-2-pyrrolidone was added sodium hydride (0.31 g, 7.75 mmol, 60.% dispersion in mineral oil). The reaction mixture was stirred for 10 minutes until no gas evolved, then 2-(trimethylsilyl)ethoxymethyl chloride (0.10 mL, 5.7 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred for 1.5 hours, after which time a solution of trans-1,4-diaminocyclohexane (6.24 g, 54.6 mmol) in 20 mL of dry 1-methyl-2-pyrrolidone was added to the reaction mixture. The resulting reaction mixture was stirred for 30 minutes and poured into 60 mL of water. The resulting cloudy mixture was extracted with 2 x 100 mL ethyl acetate. The combined organic layers were washed with 2 x 150 mL of brine, dried over sodium sulfate, and concentrated in vacuo to give a crude brown liquid. Purification by column chromatography (5-50:95-50, methanol/dichloromethane) gave 1.66 g (61%) of product 16A as a yellow foam (mass spectrum M+H ⁺ = 500, mp = 96.5-105.0 °C)

To a solution of amine 16A (0.46 g, 0.91 mmol) in 20 mL of dichloromethane was added triethylamine (0.15 mL, 1.1 mmol) and methanesulfonic anhydride (0.18 g, 1.1 mmol). The reaction mixture was stirred for 19 hours, then concentrated under vacuum. Purification by column chromatography (3:97, methanol/dichloromethane) afforded 0.34 g (65%) of product 16B as a white solid (mass spectrum M+H ⁺ 578, mp = 231-232.5°C).

SEM-protected pyridone 16B (320 mg, 0.553 mmol) was suspended in 20 mL of methanol and treated with 10 mL of 10% hydrochloric acid. The reaction mixture was refluxed for 26 hours, cooled, then concentrated under vacuum until a precipitate started to form. The resulting suspension was filtered, washed with water and ethyl acetate, and dried to give a white solid. Another portion of solid was obtained by reconcentrating the mother liquor, which was filtered to give a total of 221 mg (85%) of 2-(trans-4-methanesulfonylaminocyclohexylamino)-6-(2-chlorophenyl)-pyrido[2, 3-d] Pyrimidin-7-one hydrochloride (16) as a white solid (mass spectrum M+H ⁺ =448, mp>300°C).

Example 17

This example illustrates the synthesis of 2-(trans-4-N,N-dimethylsulfonyl-aminocyclohexylamino)-6-(2-chlorophenyl)-pyrido from a SEM-protected pyridone [2,3-d]pyrimidin-7-one.

To a solution of amine 16A (448 mg, 0.889 mmol) in 20 mL of dichloromethane was added triethylamine (0.15 mL, 1.1 mmol) and dimethylsulfamoyl chloride (0.22 mL, 2.1 mmol). The reaction mixture was refluxed for 17 hours. The cooled reaction mixture was concentrated under vacuum and purified by column chromatography (3:97, methanol/dichloromethane) to give 290 mg (54%) of product 17A as a white solid (mass spectrum M+H ⁺ =607, mp=104.3- 114.4°C).

SEM-protected compound 17A (270 mg, 0.445 mmol) was suspended in 30 mL of methanol and treated with 20 mL of 10% hydrochloric acid. The reaction mixture was refluxed for 2 days, cooled, then concentrated under vacuum until a precipitate started to form. The resulting suspension was filtered, washed with water and ethyl acetate, and dried to give 205 mg (90%) of 2-(trans-4-dimethylsulfonylaminocyclohexyl-amino)-6-(2-chlorobenzene yl)-pyrido[2,3-d]pyrimidin-7-one hydrochloride (17) as a white solid (mass spectrum M+H ⁺ =477, mp=262.2-262.5°C)

Example 18

A mixture of compound 11 (300 mg, 0.8 mmol) and 96% formic acid (3 mL) was stirred at 60°C for 3 hours, then at 25°C overnight. The reaction mixture was concentrated under reduced pressure, triturated with ether, filtered, and dried to give 320 mg of 6-(2-chlorophenyl)-2-(4-trans-formyloxy-cyclohexylamino)-8H-pyridine And[2,3-d]pyrimidin-7-one (mass spectrum MH ⁺ =399, mpt.>300°C).

Example 19

A suspension of compound 11 (300 mg, 0.8 mmol), acetic anhydride (0.23 mL, 2.4 mmol) and pyridine (0.26 mL, 3.2 mmol) in dichloromethane (3 mL) was refluxed for 3 days. The reaction was filtered and the precipitate was purified by column chromatography on silica gel with 97:3 _CH2Cl2 /MeOH to give 158 mg _of purified product. Addition of hydrochloric acid (1.0M/ _Et2O , 2.0eq) gave the salt, which was filtered and dried to give 155 mg of 6-(2-chlorophenyl)-2-(4-trans-acetyloxycyclohexylamino) -8H-pyrido[2,3-d]pyrimidin-7-one (19). (Mass spectrum MH ⁺ = 413, mpt. > 300°C).

Example 20

A suspension of compound 11 (250 mg, 0.68 mmol) and 4-(dimethylamino)pyridine (DMAP) (6.8 mg, 0.06 mmol) in THF (5 mL) was cooled in an ice bath and dimethyl pyrocarbonate was added ester (0.7 mL, 6.8 mmol), and the mixture was stirred at 25°C for 18 hours. The reaction was filtered and the precipitate was purified by column chromatography on silica gel eluting with 98: _{2 CH2Cl2} /MeOH to yield 33 mg of purified product. Addition of hydrochloric acid (1.0M/ _Et2O , 2.0 equiv) gave the salt, which was filtered and dried to give 34 mg of 6-(2-chlorophenyl)-2-(4-transmethoxycarbonyloxy-cyclohexylamino )-8H-pyrido[2,3-d]pyrimidin-7-one (20). (Mass spectrum MH+ = 429).

Example 21

A suspension of compound 11 (308 mg, 0.83 mmol) in dichloromethane (5 mL) was cooled in an ice bath, and chlorosulfonyl isocyanate (0.08 mL, 0.87 mmol) was added. The mixture was stirred at 25°C for 18 hours, then treated with water (0.5 mL). The biphasic mixture was stirred for 8 hours, filtered and the precipitate was purified by column chromatography on silica gel with 97:3 _CH2Cl2 /MeOH to _give 108 mg of purified product. Addition of hydrochloric acid (1.0M/ _Et2O , 2.0 equiv) gave the salt, which was filtered and dried to give 94 mg of 6-(2-chlorophenyl)-2-(4-carbamoyloxycyclohexylamino)-8- Pyrido[2,3-d]pyrimidin-7-ones (21). (Mass spectrum MH+ = 414, mpt. 291.0-293.5°C).

Example 22

A suspension of compound 38, see Example 38, (190 mg, 0.49 mmol) in dichloromethane (5 mL) was cooled in an ice bath, and chlorosulfonyl isocyanate (0.05 mL, 1.01 mmol) was added. The mixture was stirred at 25°C for 18 hours, then treated with water (0.5 mL). The biphasic mixture was stirred for 8 hours, filtered and the _precipitate purified by column chromatography on silica gel eluting with 97:3 _CH2Cl2 /MeOH to give 108 mg of purified product. Addition of hydrochloric acid (1.0M/ _Et2O , 2.0 equiv) gave the salt, which was filtered and dried to yield 94 mg of product 22. (Mass spectrum MH+ = 428, mpt. 234.7-235.4°C).

Example 23

Sulfone 2 (500 mg, 1.5 mmol) was combined with 1-hydroxymethylcyclopentylamino (686 mg, 5.96 mmol) and 1-methyl-2-pyrrolidone (1 mL). The mixture was heated to 100°C for 3 hours and then cooled to room temperature. Methanol (3 mL) was added. The suspension was stirred for 10 minutes, filtered, the precipitate washed thoroughly with methanol, dried and suspended in ethyl acetate. Addition of hydrochloric acid (1.0M/ _Et2O , 2.0 equiv) gave the salt, which was filtered and dried to yield 235mg of product 23. (Mass spectrum MH+ = 371, mpt. 290-293°C).

Example 24

Sulfone 2 (250 mg, 0.71 mmol) was mixed with 4-aminomethylcyclohexanol (630 mg, 3.4 mmol) and 1-methyl-2-pyrrolidone (0.5 mL). The mixture was heated to 95°C for 3 hours and then cooled to room temperature. The mixture was purified by silica gel column chromatography eluting with 96: _{4 CH2Cl2} . The column fractions containing product were combined, concentrated to a solid, which was suspended in ethyl acetate. Addition of hydrochloric acid (1.0M/ _Et2O , 2.0 equiv) gave the salt, which was filtered and dried to afford 85 mg of product 24. (Mass spectrum MH+ = 385, mpt. 205-213°C).

Example 25

Sulfone 1 (500 mg, 1.42 mmol) was mixed with 4-aminomethylcyclohexanol (630 mg, 3.4 mmol) and 1-methyl-2-pyrrolidone (0.5 mL). The mixture was heated to 95°C for 3 hours and then cooled to room temperature. The mixture was purified by column chromatography on silica gel eluting with 96: _{4 CH2Cl2} . The column fractions containing product were combined, concentrated to a solid, which was suspended in ethyl acetate. Addition of hydrochloric acid (1.0M/ _Et2O , 2.0 equiv) gave the salt, which was filtered and dried to yield 79 mg of product 25. (Mass spectrum MH+ = 399, mpt. 148-151.5°C).

Example 26

A suspension of compound 38 (350 mg, 0.9 mmol) and 4-(dimethylamino)pyridine (DMAP) (55.5 mg, 0.45 mmol) in THF (5 mL) was cooled in an ice bath and dimethyl pyrocarbonate was added Ester (0.3 mL, 2.8 mmol), and the resulting mixture was stirred at 25°C for 18 hours. Filtration and purification of the precipitate by silica gel column _{chromatography} eluting with 99:1 _CH2Cl2 /MeOH afforded 205 mg of purified product. Addition of hydrochloric acid (1.0M/ _Et2O , 2.0 equiv) gave the salt, which was filtered and dried to give 200 mg of product 26. (Mass spectrum MH+ = 443: mpt 213.9-214.4°C).

Example 27

To a solution of compound 38 (0.36 g, 0.94 mmol) in 10 mL of dichloromethane was added pyridine (0.60 g, 7.6 mmol) and acetic anhydride (0.58 g, 5.6 mmol) sequentially at room temperature. The resulting mixture was stirred at room temperature for 3 days, then concentrated under reduced pressure. The resulting residue was purified by column chromatography to yield 250 mg of a gray solid as pure product (mass spectrum MH ⁺ = 427, mpt. 200.9-201.2°C).

Example 28

A solution of compound 38 (0.34 g, 0.89 mmol) in 6 mL of formic acid was stirred at 60 °C for 3 days. The resulting mixture was concentrated under reduced pressure. The resulting foam was diluted with hexanes and concentrated to give a solid. Purification by column chromatography afforded 340 mg of product 28 as a powdery, brown solid. (Mass spectrum MH ⁺ = 413, mpt. 196.1-196.6°C).

Example 29

1,4-Dioxa-spiro[4,5]dec-8-ylamine (see WO 99/01452 for preparation) (0.157 g, 1.00 mmole), N-methylpyrrolidone (1 mL) and sulfone 1 (0.350 g, 1.00 mmole) mixture was stirred at 100°C for 2 hours. The reaction mixture was cooled, poured into water, extracted with ethyl acetate, washed with saturated brine, dried over magnesium sulfate, and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (0-3% methanol/dichloromethane). Column fractions containing product were combined and concentrated in vacuo. The concentrated fraction (0.100 g) was taken up in methanol, treated with hydrochloric acid (1.0M/ _Et2O , 1.0 equiv), evaporated to dryness, washed with diethyl ether, filtered, and dried to give 0.070 g of the hydrochloride salt of compound 29 ( M ⁺ = 427).

To the remaining free base (0.167 g, 3.56 mmole) in 5 mL THF was added 2N aqueous hydrochloric acid (1 mL). After 6 hours, the reaction mixture was poured into saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, dried over magnesium sulfate, and evaporated in vacuo. The residue was purified by flash chromatography on silica gel (20-30% acetone/hexane). Column fractions containing product were combined and concentrated in vacuo. The product was dissolved in methanol, treated with hydrochloric acid (1.0M/ _Et2O , 1.0 equiv), evaporated to dryness, washed with ether, filtered and dried to give 0.102g of the compound's hydrochloride (M ⁺ =382).

Example 30

A solution of sulfone 1 (0.500 g, 1.43 mmole) in ethyl 4-amino-1-piperidinecarboxylate (1.0 mL, 5.83 mmole) was stirred at 120 °C for 3 hours. The reaction mixture was poured into water and extracted with dichloromethane. The combined organic layers were dried over magnesium sulfate and evaporated in vacuo. The residue was purified by flash chromatography on silica gel (20-30% acetone/hexane). Column fractions containing product were combined and concentrated in vacuo.

The purified ethyl carbamate (0.617 g, 1.40 mmole) was added to a solution of potassium hydroxide in hot ethanol (6.58 g, 117 mmole; 37 mL EtOH) and stirred at 80 °C for 3 hours. The reaction mixture was then cooled in an ice bath and quenched with aqueous citric acid (6.6 g, 31.4 mmole; 37 mL _H2O ). The resulting solution was concentrated in vacuo to give a viscous aqueous solution which was extracted with ethyl acetate. The combined organic layers were dried over sodium carbonate and concentrated in vacuo. The residue was purified by flash chromatography on a silica gel column (10-30% methanol/dichloromethane). Column fractions containing product were combined and concentrated in vacuo to afford 30B.

A portion of the piperidine product was dissolved in methanol and treated with 1 eq 1N HCl/ether. The solution was concentrated under vacuum. The dried residue was washed with ether and dried to give the hydrochloride salt of compound 30B (mp>300.0°C).

A solution of compound 30B (0.100 g, 0.270 mmole) and bromoacetamide (0.100 g, 0.724 mmole) in 2 mL of DMF was heated to 120 °C for several hours. The reaction mixture was cooled and purified by flash chromatography on silica gel (3-5, 10% [1:9 ammonium hydroxide/methanol]/dichloromethane). The column fractions containing product were combined, concentrated in vacuo, dissolved in methanol, and treated with hydrochloric acid (1.0M/ _Et2O , 1.0 equiv). The solution was concentrated, washed with ether, filtered and dried to give 0.117 g of the hydrochloride salt of compound 30A (mp 76.3-1 36.5; M+.427).

Example 31

A mixture of compound 30B (0.100 g, 0.270 mmole), bromoacetonitrile (0.091 mL, 1.35 mmole) and sodium carbonate (0.114 g, 1.35 mmole) in 1 mL of DMF was stirred at room temperature. The mixture was then purified by flash chromatography on silica gel (2-5 [1:9 ammonium hydroxide/methanol]/dichloromethane). The column fractions containing product were combined and concentrated in vacuo to give 0.086 g of the desired product. A portion of the free base (0.016 g) was dissolved in methanol and treated with hydrochloric acid (1.0M/ _Et2O , 1.0 equiv). The solution was concentrated, washed with ether, filtered and dried to give 0.012 g of the hydrochloride salt of compound 31 (mp 228.3-228.9; M+.409).

Example 32

A mixture of compound 30B (0.050 g, 0.135 mmole) and methyl bromoacetate (0.030 mL, 0.317 mmole) in 1 mL of DMF was stirred at room temperature. The mixture was purified by silica gel flash chromatography (10-40% methanol/dichloromethane + 1% ammonium hydroxide). Column fractions containing product were combined and concentrated in vacuo. The residue was dissolved in methanol and treated with hydrochloric acid (1.0M/ _Et2O , 1.0 equiv). The mixture was concentrated, washed with diethyl ether, filtered and dried to give 0.013 g of the hydrochloride salt of compound 32 (mp 150-168; M+.442).

Example 33

Compound 30B (0.050 g, 0.135 mmole) was taken in 1 mL THF with trimethylsilyl isocyanate (0.021 mL, 0.149 mmole), stirred at room temperature for 3 hours, then evaporated under vacuum. The residue was purified by flash chromatography on silica gel (10% [1:9 ammonium hydroxide/methanol]/dichloromethane). Column fractions containing product were combined and concentrated in vacuo. The final product was placed in methanol, treated with hydrochloric acid (1.0M/Et ₂ O, 1.0 equivalents), evaporated to dryness, washed with ether, filtered, and dried to obtain 0.057g of compound 33 hydrochloride (mp 192.0-202.0; M+.413).

Example 34

Imidazole (0.28 g, 4.06 mmole) and sodium carbonate (0.43 g, 4.06 mmole) were combined with DMF (0.63 mL, 8.11 mmole), and chlorotrimethylsilane (0.63 mL, 4.06 mmole) was added dropwise thereto. The slurry was stirred at room temperature for 20 minutes before adding dry powdered compound 30B (0.50 g, 1.35 mmole). Stir at room temperature for 2 hours, then pour into water and extract with dichloromethane and ethyl acetate. The combined organic extracts were washed with saturated brine, dried over magnesium sulfate, and evaporated under vacuum. The residue was purified by flash chromatography (2-3% methanol/dichloromethane) and the column fractions containing product were combined and concentrated in vacuo. The final product was placed in methanol, treated with hydrochloric acid (1.0M/Et ₂ O, 1.0 equiv), evaporated to dryness, then washed with ether, filtered, and dried to obtain 0.259 g of the hydrochloride salt of compound 34 (mp 105.0-115.0 C, M+.427).

Example 35

The urethane was prepared from the corresponding benzyl sulfone as described and cleaved with iodotrimethylsilane to afford the piperidine intermediate 35A (mp > 300.0).

A portion of the free base of compound 35A (0.030 g, 0.084 mmole) was placed in 1 mL of DMF with bromoacetonitrile (0.009 mL, 0.126 mmole) and sodium carbonate (0.015 g, 0.141 mmole), stirred at room temperature for 3 h, then Purified by flash chromatography on silica gel (1-10% [1:9 ammonium hydroxide/methanol]/dichloromethane). Column fractions containing product were combined and concentrated in vacuo. The final product was placed in methanol, treated with hydrochloric acid (1.0M/Et ₂ O, 1.0 equiv), evaporated to dryness, then washed with ether, filtered, and dried to give 0.006 g of the hydrochloride salt of compound 35B (M+.395) .

Example 36

A portion of the free base of compound 35A (0.050 g, 0.141 mmole) was prepared with 2-chloro-N,N-dimethylacetamide (0.026 g, 0.211 mmole) and N,N-diisopropylethylamine (0.037 mL, 0.211 mmole) in 1 mL of DMF, stirred at 60°C for 3 hours and at 120°C for 2 hours, then purified by silica gel flash chromatography (3-20% [1:9 ammonium hydroxide/methanol]/ dichloromethane). Column fractions containing product were combined and concentrated in vacuo. The final product was placed in methanol, treated with hydrochloric acid (1.0M/Et ₂ O, 1.0 equiv), evaporated to dryness, washed with ether, filtered, and dried to obtain 0.006 g of the hydrochloride salt of compound 36 (mp103.0- 120.0°C).

Example 37

Compound 11 was taken in 5 mL tetrahydrofuran with potassium tert-butoxide (0.95 g, 8.42 mmole) and methyl iodide (0.11 mL, 1.68 mmole). After stirring at room temperature (rt) for 4 hours, the reaction mixture was purified by flash chromatography (1-3% methanol/dichloromethane; 25-35% acetone/hexanes) to afford the two products described above (i.e. compounds 37A and 37B ). They were individually placed in methanol, treated with hydrochloric acid (1.0M/ _Et2O , 1.0 equiv), evaporated to dryness, washed with ether, filtered, and dried to give 0.157 g of the di-methylated product (compound 37A) ( mp 182.3-183.1°C) and 0.069 g of the mono-methylated product (compound 37B) (mp 218.2-218.5°C).

Example 38

Sulfide 1a (317 mg, 1.0 mmole) was dissolved in NMP (0.3 mL), and trans-4-aminocyclohexanol (570 mg, 5 mmole) was added to the solution. The reaction was heated at 120°C for 12 hours. The reaction was cooled to room temperature, water (50 mL) was added, and extracted with ethyl acetate. The organic layers were combined, washed twice with water, and concentrated in vacuo. The residue _was purified by column chromatography on silica gel using 10:90 MeOH/ _CH2Cl2 . Column fractions containing the desired product were combined and concentrated to a foam. The foam was suspended in MeOH, then hydrochloric acid (1.0M/ _Et2O , 1.0 equiv) was added to give compound 38 as a salt. The solvent was evaporated. The resulting solid was diluted with a mixture of _Et2O /MeOH, filtered and dried. Yield 282 mg. Mp 163.4-171.1 °C. MS (M+H) ⁺ 385.

Example 39

Sulfone 1 (350 mg, 1.0 mmole) was mixed with 1-amino-1-cyclopentanemethanol (345 mg, 3.0 mmole) and NMP (0.3 mL), and stirred at 120° C. for 1 hour. The reaction mixture was added to water, and extracted with ethyl acetate. The organic layers were combined, washed twice with water, and concentrated in vacuo to give the crude product. The residue _was purified by column chromatography on silica gel eluting with 10:90 MeOH/ _CH2Cl2 . Fractions containing the desired product were combined and evaporated in vacuo to give the desired product as a foam. The residue was suspended in MeOH, hydrochloric acid (1.0M/Et ₂ O, 1 eq) was added, stirred for 20 minutes, and concentrated under reduced pressure. The residue was stirred with a mixture of MeOH/ _Et2O for 1 h and filtered to give the product 39 as a white solid. Yield 263mg.Mp 213.7-214.6°C, MS(M+H) ⁺³⁸⁵ .

Example 40

Sulfone 1 (350 mg, 1.0 mmole) was mixed with 1-amino-1-cyclopentanemethanol (387 mg, 3.0 mmole) and NMP (0.3 mL), and stirred at 120° C. for 90 minutes. The reaction mixture was cooled to room temperature, added to water, and extracted with ethyl acetate. The organic layers were combined, dried over magnesium sulfate, and concentrated under reduced pressure. The residue _was purified by column chromatography on silica gel eluting with 10:90 MeOH/ _CH2Cl2 . Fractions containing the desired product were combined and evaporated under reduced pressure to give the desired product as a foam. The foam was suspended in MeOH and hydrochloric acid (1.0M/ _Et2O , 1 equiv) was added. The mixture was stirred for 20 minutes, evaporated in vacuo, stirred with a mixture of MeOH/ _Et2O for 2 hours, filtered and dried to give the product 40 as a white solid. Yield 242 mg. Mp 185-188.6°C, MS(M+H) ⁺ 399.

Example 41

Sulfone 1 (350 mg, 1.0 mmole) and ethyl cis/trans aminocyclohexanecarboxylate (520 mg, 3 mmole) were mixed and stirred at 100°C for 30 minutes. The resulting solution was cooled to room temperature and purified by column chromatography on silica gel using 65:35 hexane/acetone. Fractions containing the desired product were combined and concentrated to a foam. The residue was suspended in MeOH and a solution of hydrochloric acid (1.0M/ _Et2O , 1 equiv) was added. The resulting mixture was stirred for 30 minutes and the solution was evaporated. The resulting residue was stirred with a mixture of MeOH/ _Et2O for 1 h and filtered to give the product 41 as a white solid. Yield 414mg. Mp 192.1-198.3°C, MS (M+H) ⁺⁴⁴¹ .

Example 42

To a solution of compound 41 (300 mg, 0.67 mmole) in EtOH was added concentrated aqueous hydrochloric acid (3.0 mL). The resulting solution was refluxed for 24 hours. After addition of hydrochloric acid (1.0 mL), the solution was refluxed for an additional 24 hours. The reaction mixture was evaporated to dryness under reduced pressure and dried under high vacuum to give the hydrochloride salt of compound 42 as a white solid. Yield: 225 mg. Mp: 263.6-264.2°C, MS: (M+H) ⁺⁴¹³ .

Example 43

Sulfone 1 (350 mg, 1.0 mmole) was mixed with 4-aminocyclohexylmethanol (1:1 cis/trans) (Chem. Ber: 96; 1963; 2377-2386) (400 mg, 3.3 mmole) and NMP (0.4 ml) and stirred at 100°C for 1 hour. The reaction mixture was cooled to room temperature, added with water, and extracted with ethyl acetate. The organic layers were combined, washed 5 times with water, dried over magnesium sulfate, and concentrated to give the crude product as an oil. The residue was purified by column chromatography on silica gel eluting with 70:30 hexane/acetone. Fractions containing the desired product were combined and concentrated under reduced pressure to give the product as a foam. The material was suspended in MeOH, acidified by adding hydrochloric acid (1.0M/Et ₂ O, 1 eq), stirred at room temperature for 30 minutes, and concentrated under reduced pressure. The resulting residue was stirred with a mixture of MeOH/ _Et2O for 2 hours and filtered to afford the hydrochloride salt of compound 43 as a white solid. Yield 335 mg. Mp 153.6-157°C, MS (M+H) ⁺ 339.

Example 44

step 1

To a suspension of compound 11 (3.0 g, 8.1 mmol) in NMP (35 mL) was added tert-butyldimethylsilyl chloride (1.7 g, 11.3 mmol) and imidazole (1.2 g, 17.6 mmol Moore). The reaction mixture was stirred at 50 °C for 12 hours, cooled to room temperature, and water (150-200 mL) was added. The mixture was stirred for 1 hour and filtered to afford compound 44A as a white solid. Yield 3.71. Mp 289.9-291.4°C MS (M+H) ⁺ 485.

Step 2: Alkylation

To a suspension of silyl-protected alcohol 44A (483 mg, 1.0 mmole) in NMP was added sodium hydride (44 mg, 1.1 mmole (60% oil suspension). The reaction mixture was stirred for 25 minutes, after which 2 -(iodoethoxy)tri-isopropylsilane (328 mg, 1.0 mmole), the resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was added to water and extracted with ethyl acetate. The combined organic layers were washed with water 5 times, dried over magnesium sulfate, and concentrated to give silyl-protected intermediate 44B as an oil. The residue was purified by column chromatography on silica gel using 25:75 acetone/hexanes as eluent to give product 44B, which As an oil. Yield 650 mg. MS (M+H) ⁺ 685.

Step 3: Deprotect

To a solution of compound 44B (650 mg, 0.94 mmole) in THF (15 mL) was added tetrabutylammonium fluoride (1.0 M in THF, 1.1 equiv). The resulting solution was stirred at room temperature for 12 hours, added with water, and extracted with ethyl acetate. The organic layers were combined, dried over magnesium sulfate, and concentrated to give the product as an oil. The residue was purified by silica gel column _{chromatography} with 90:10 _CH2Cl2 /MeOH to afford product 44 as an oil. It was suspended in MeOH, acidified with HCl (1.0M/ _Et2O , 1 eq), stirred for 20 min, concentrated, diluted again with a mixture of _Et2O /MeOH, and stirred for 2 h. The solution was filtered to afford the hydrochloride salt of compound 44 as a white solid. Yield 270 mg. MP 210.5-212.0°C. MS(M+H) ⁺⁴¹⁵ .

Example 45

Step 1: Alkylation

To a suspension of silyl-protected alcohol 44A (483 mg, 1.0 mmole) in NMP was added sodium hydride (44 mg, 1.1 mmole (60% oil dispersion). The reaction mixture was stirred at room temperature for 25 minutes. To the Iodoacetonitrile (0.167 mg, 1.0 mmole) was added in the solution, and the resulting solution was stirred at room temperature for 4 hours. The reaction mixture was added to water and extracted with ethyl acetate. The organic layers were combined, washed with water 5 times, passed through magnesium sulfate Drying and concentration gave an oil. The residue was purified by column chromatography on silica gel using 25:75 acetone/hexanes as eluent to give the product as a white solid. Yield 480 mg. MS (M+H) ⁺ 524.

Step 2: Deprotect

To a solution of the product obtained in the above step 1 (480 mg, 0.92 mmole) in THF was added tetrabutylammonium fluoride (1.0 M/THF, 1.1 eq) and stirred at room temperature for 12 hours. The reaction mixture was added to water, and extracted with ethyl acetate. The organic layers were combined, dried over magnesium sulfate, and concentrated to give the product as an oil. The residue was purified by column chromatography on silica gel using 90:10 _CH2Cl2 /MeOH as eluent to afford product 45 as a white _solid . Yield 260 mg. MP 245.8-246.8 °C. MS (M+H) ⁺ 410.

The product 45 was suspended in MeOH, and a solution of hydrochloric acid (1.0M/ _Et2O , 1 equiv) was added. The mixture was stirred at room temperature for 30 minutes and concentrated. The residue was stirred in a mixture of Et ₂ O/MeOH for 2 hours, and filtered to obtain the hydrochloride salt of compound 45 as a white solid. Yield 255 mg. Mp 232.1-233.4°C. MS (M+H) ⁺ 410.

Example 46

Step 1: Alkylation

To a suspension of silyl-protected alcohol 44A (483 mg, 1.0 mmole) in NMP was added sodium hydride (44 mg, 1.1 mmole (60% oil dispersion). The resulting solution was stirred at room temperature for 30 minutes. To Benzyl bromide (0.12ml, 1.0mmole) was added in the reaction mixture, and the resulting solution was stirred at room temperature for 12 hours, then water was added to the reaction mixture, and extracted with ethyl acetate. The combined organic layers were washed with water for 5 times , dried over magnesium sulfate, concentrated, and purified by silica gel column chromatography using 25:75 acetone/hexanes as eluent to give the product as a white solid. Yield 502 mg. Mp 188.8-191.0 °C MS (M+H ) ⁺ 575

Step 2: Deprotect

To a solution of the product obtained in Step 1 above (480 mg, 0.84 mmole) in THF was added tetrabutylammonium fluoride (1.0 M/THF, 1.1 equiv). The resulting reaction mixture was stirred at room temperature for 12 hours, added to water, and extracted with ethyl acetate. The organic layers were combined, dried over magnesium sulfate, concentrated, and purified by silica gel column chromatography using 10:90 MeOH/ _CH2Cl2 as _eluent to give product 46 as a white solid. Yield 320mg. Mp 119-131°C. MS (M+H) ⁺⁴⁶¹ .

A solution of the above product in MeOH was added to a solution of hydrochloric acid (1.0 M/ _Et2O , 1 equiv). The resulting solution was stirred at room temperature for 20 minutes and concentrated to a foam. The foam was dissolved in a mixture of _Et2O /MeOH, stirred for 2 hours, and filtered to obtain the hydrochloride salt of compound 46 as a white solid. Yield 305 mg. Mp 202-206°C. MS (M+H) ⁺ 461.

Example 47

Step 1: Alkylation

To a suspension of silyl-protected alcohol 44A in NMP was added sodium hydride (44 mg, 1.1 mmole (60% dispersion in oil). The resulting solution was stirred at room temperature for 25 minutes. To the reaction mixture was added bromine Methyl acetate (0.095ml, 1.0mmole) was stirred at room temperature for 12 hours. The reaction mixture was added to water and extracted with ethyl acetate. The organic layers were combined, washed 5 times with water, dried over magnesium sulfate, concentrated, and passed through Purification by silica gel column chromatography using 25:75 acetone/hexane as eluent gave the product as a white solid. Yield 480 mg. Mp 182-187°C. MS (M+H) ⁺ = 557.

Step 2: Deprotect

To a solution of the product obtained in step 1 above (480 mg, 0.86 mmole) in tetrahydrofuran (15 mL) was added tetrabutylammonium fluoride (1.0 M/THF, 1.1 equiv). The resulting reaction mixture was stirred at room temperature for 12 hours, added to water, and extracted with ethyl acetate. The organic layers were combined, dried over magnesium sulfate, and concentrated to give product 47 as a foam. Yield 225 mg. Mp 118-130°C. MS (M+H) ⁺ 443.

Example 48

To a suspension of compound 47 (210 mg, 0.475 mmole) in MeOH (4.0 mL) was added aqueous sodium hydroxide (0.477 mL of 1.0 N NaOH). The resulting suspension was stirred at room temperature for 5 days and evaporated to dryness. The residue was diluted with _Et2O , stirred for 2 hours, filtered and dried to give product 48 as a light yellow solid. Yield 205 mg. Mp 260-265°C. MS (M+H) ⁺ 429.

Example 49

To a solution of 4-methoxybenzenesulfonamide (0.417 g, 2.23 mmol) in 3 mL of N-methylpyrrolidone was added 60% sodium hydride (107 mg, 2.68 mmol) at room temperature. The mixture was stirred for 10 minutes. Sulfone 1 (189 mg) was then added and the resulting mixture was stirred at 130°C for 6 hours. The reaction mixture was cooled to room temperature and water (50 mL) was added. The resulting solid was filtered, washed successively with water, ethyl acetate and methanol, and dried under vacuum to give product 49 (190 mg), mp>300°C; ms 457 (M+H) ⁺ .

Example 50 6-(2-chlorophenyl)-2-(N-2-cyanoethylpiperidinyl-4-amino)-pyrido[2,3-d]-pyrimidin-7-ol

Step 1: Preparation of 4-trifluoroacetylamino-1-benzylpiperidine

To a cold (5° C.) solution of 25 g (131 mmol) 4-amino-1-benzylpiperidine and 15.36 g (152 mmol) triethylamine in 130 mL of dichloromethane was added dropwise 28 g ( 18.8 mL, 133 mmol) of trifluoroacetic anhydride, the rate was adjusted to maintain the reaction temperature between 5-10 °C. After the addition was complete, the reaction mixture was stirred for an additional 15 minutes. Solvent and volatiles were removed under reduced pressure. The residue was dissolved in 100 mL of ethyl acetate, then diluted with 100 mL of hexane. The solution was filtered through a pad of silica gel (50 g), washing with 1 L of 50% ethyl acetate/hexanes. The filtrate was concentrated under reduced pressure to afford 37 g (quantitative yield) of the desired 4-trifluoroacetylamino-1-benzylpiperidine, (pale yellow solid).

Step 2: Preparation of 4-trifluoroacetylaminopiperidine

Into a 500 mL, three-neck round bottom flask was added 4 g of 10% Palladium on Carbon and 100 mL of methanol under a nitrogen atmosphere. The suspension was cooled to 5°C and 10 g of ammonium formate was added in one portion (gas evolution was observed). A solution of 10 g (35 mmol) 4-trifluoroacetylamino-1-benzylpiperidine in 100 mL methanol was then gradually added dropwise. The resulting reaction mixture was refluxed for 2.5 hr., then at room temperature overnight. Under a nitrogen blanket, the catalyst was filtered off through celite and washed with ethanol. The filtrate was concentrated under reduced pressure to afford 6.9 g (quantitative yield) of the title compound as a light yellow solid.

Step 3: Preparation of N-(2-cyanoethyl)-4-aminopiperidine

A mixture of 3 g (15.3 mmol) 4-trifluoroacetamidopiperidine, 3 g (4.25 mL, 30.6 mmol) triethylamine, 3 mL (45.9 mmol) acrylonitrile in 10 mL THF was stirred at room temperature for 17 hours. Solvent and volatiles were removed under reduced pressure. The residue was dissolved in 20 mL of methanol and 4 mL of 30% _NH4OH , and the solution was refluxed for 5 hours. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography on silica gel, washing with initially 500 mL dichloromethane, then 500 mL 0.5% methanol/dichloromethane, followed by 1000 mL 9:1:0.1 dichloromethane/methanol/ammonium hydroxide Removal gave 0.7 g (30% yield) of N-(2-cyanoethyl)-4-aminopiperidine.

Step 4: Preparation of 6-(2-chlorophenyl)-2-(N-2-cyanoethylpiperidinyl-4-amino)-pyrido[2,3-d]-pyrimidin-7-ol.

With 0.2g (0.58mmol) sulfone 2,6-(2-chlorophenyl)-2-methanesulfonyl-pyrido[2,3-d]pyrimidin-7-ol, 0.18g (1.16mmol)N-( A mixture of 2-cyanoethyl)-4-aminopiperidine in 0.5 mL of NMP was heated in a 60°C oil bath for 2.5 hr., cooled, and diluted with 5 mL of dichloromethane. The solution was purified by column chromatography on 40 g of silica gel using 200 mL of dichloromethane to elute NMP followed by 5% methanol/dichloromethane to elute the product. Fractions containing product were combined and concentrated. Ether was added to the product and after trituration the solid was filtered, washed with ether and dried to give 165 mg (70% yield) of the desired product. Approximately 50 mg of this solid was dissolved in 3 mL of dichloromethane. 1 mL of 1M HCl/ether solution was added, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure to obtain 54 mg of compound 50. (Mass spectrum MH ⁺ = 408, mpt. 217.5-219.5°C).

Example 51

2g (5.8mmol) 6-(2-chlorophenyl)-2-methanesulfonylpyrido[2,3-d]pyrimidin-7-ol, 1.97g (11.4mmol) 4-amino-1-piperidine A mixture of ethyl carboxylate in 2 mL of NMP was heated in a 60°C oil bath for 1.5 hr. The suspension quickly turned into a clear brown solution. The mixture was partitioned between 150 mL water and 150 mL ethyl acetate. The aqueous layer was extracted one or more times with 150 mL of ethyl acetate. The combined ethyl acetate solutions were washed with water, 2 x 150 mL, then brine, dried, filtered, and the filtrate was concentrated under reduced pressure. After trituration in diethyl ether, the solvent was removed under reduced pressure and 2.5 g (quantitative yield) of compound 51 were isolated. About 0.13 g of this material was dissolved in 1.5 mL of ethyl acetate and 1 mL of 1M HCl (in ether) was added. The suspension was stirred at room temperature for 1 hour, diluted with ether, filtered and washed with ether to give 0.12 g of compound 51 hydrochloride. (Mass spectrum M ⁺ =441.9, mpt.190-191°C)

Example 52

0.3g (0.8mmol) 6-(2-chlorophenyl)-2-(piperidinyl-4-amino)-pyrido[2,3-d]pyrimidin-7-ol, 0.3g(2.4mmol) A mixture of 2-bromoethanol, 0.22 mL (1.6 mmol) triethylamine, 1.5 mL NMP and 10 mL toluene was heated in an oil bath at 100 °C for 2.5 hr. The mixture was diluted with 75 mL ethyl acetate and 100 mL water. The phases were separated and the aqueous layer was extracted with ethyl acetate (2 x 75 mL). The combined organic extracts were then washed with brine, filtered and concentrated under reduced pressure. The residue was dissolved in a small amount of methanol and purified by 40 g silica gel column chromatography using 8.5:1.5:0.2 ethyl acetate/methanol/ammonium hydroxide as eluent to give 0.115 g of white solid (35% yield). This was dissolved in 2 mL of EtOAc and 2 mL of 1M HCl (in ether) was added. The suspension was stirred at room temperature for 30 minutes. Filtration and drying yielded 0.13 g of compound 52. (Mass spectrum MH ⁺ = 414, mpt. 265.9-266.6°C).

Example 53

0.2g (0.54mmol) 6-(2-chlorophenyl)-2-(piperidinyl-4-amino)-pyrido[2,3-d]pyrimidin-7-ol, 0.11g(1.6mmol) A mixture of methyl vinyl sulfone, 0.5 mL triethylamine in 5 mL THF was stirred at room temperature for 17 hours. Triethylamine and THF were evaporated under reduced pressure. The residue was partitioned between 75 mL of water and 100 mL of ethyl acetate. After phase separation, the aqueous layer was extracted again with 75 mL of ethyl acetate. The combined organic solutions were washed with water, brine, dried and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography on 40 g silica gel using 90:10:5 dichloromethane/ethyl acetate/ammonium hydroxide as eluent. The recovered product was dissolved in 1.5 mL of ethyl acetate and 2 mL of 1M HCl (in ether) was added. The suspension was stirred for 1 hour, filtered and washed with diethyl ether to give 165 mg of compound 53 hydrochloride (hydrochloride). (Mass spectrum MH ⁺ = 476, mpt. 204.6-205.6°C).

Example 54

Preparation of 4-amino-1-(2,2,2,-trifluoroethyl)piperidine, 54A

4-N-BOC-aminopiperidine (commercially purchased) (5g, 24.96mmol), 2,2,2-trifluoroethyl trichloromethanesulfonate (7.03g, 1eq) and potassium carbonate (4.1g, A solution of 1.2 eq) in acetone (80 mL) was heated to reflux for 17 hours. The solvent was removed under reduced pressure at 40°C, and ethyl acetate (250 mL) and water (150 mL) were added to the residue. The organic layer was separated, washed successively with water (1 x 150 mL) and brine (1 x 200 mL), dried over magnesium sulfate, filtered and concentrated to give a dark solid. Purification by silica gel chromatography using 15% ethyl acetate/hexanes as eluent afforded 4-NBOC-amino-1-(2,2,2-trifluoroethyl)-piperidine (4.45 g) as Off-white powder, MP99.2-99.8°C, (M+H) ⁺ =283. The amine was then taken up in dioxane (80 mL) and HCl gas was bubbled through the solution for 10 minutes (a precipitate formed immediately). Tighten the cap of the reaction vessel, stir for 1.5 hours, and concentrate to obtain a white powder. This hydrochloride was dissolved in 42 mL of 0.5M sodium methoxide/methanol solution, and stirred at room temperature for 3 hours. The solution was filtered through a medium frit, and the filtrate was concentrated. The residue was dissolved in ethyl acetate (100 mL), the solid was crushed, and the mixture was filtered. The filtrate was concentrated to give 4-amino-1-(2,2,2-trifluoroethyl)-piperidine, 54A, as a dark oil (1.0 g), (M+H) ⁺ =183.

Sulfone 2 (200 mg, 0.614 mmol), compound 54A (224 mg, 2 eq) and N-methylpyrrolidone (0.3 mL) were mixed in a 10 mL flask and stirred at 110° C. for 10 minutes. The reaction was cooled, then methanol (10 mL) was added and the solid was crushed, filtered and collected to give an off-white powder. The free amine was dried under high vacuum at 56°C for 2 hours, mp=267.4-267.6°C, (M+H) ⁺ =438. The free amine was taken up in dioxane (20 mL) at room temperature and a solution of 1 M HCl in diethyl ether (0.5 mL, 1.3 eq) was added with stirring. The resulting mixture was stirred at room temperature for 1 hour, filtered, and dried under high vacuum at 56 °C for 8 hours to afford compound 54 as an off-white powder. Mp = 260.0-265.0 °C, (M+H) ⁺ = 438 (free amine).

Example 55

Preparation 55B

To a solution of sulfone 2 (1.485 g, 4.56 mmol) in dimethylformamide (25 mL) at 0°C was added 60% sodium hydride solution (200 mg, 1.1 eq). The resulting mixture was stirred vigorously at 0°C for 15 minutes, after which 2,2,2-trifluoroethyltrichloromethanesulfonate 55A (4.2 g, 3.3 eq) in dimethylformamide (15 mL) was added middle). The resulting mixture was stirred at 0 °C to room temperature for 4 days. The reaction mixture was diluted with ethyl acetate (300 mL) and water (100 mL). The organic layer was separated, washed with water (100 mL) and brine (2 x 100 mL), dried over magnesium sulfate, filtered and concentrated. The residue was washed several times with ether/hexane, decanting the supernatant each time. Concentration and drying of the residue under high vacuum afforded compound 55B as a semi-solid in quantitative yield.

Preparation 55

Compound 55B (0.9 g, 2.15 mmol), compound 55C (743 mg, 3 eq) and N-methylpyrrolidone were added into a 10 mL flask and stirred at 110° C. for 25 minutes. The reaction mixture was cooled, diluted with 10 mL of methanol, and concentrated. The residue was dissolved in ethyl acetate (35 mL), washed with water (7 x 25 mL) and brine (1 x 25 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated. Purification by preparative thin layer chromatography 3 (20 x 40 cm, 1000 μΜ) plate using 5% methanol/dichloromethane as eluent afforded 55 as off-white powder, mp = 227.2-230.4, (M+H) ⁺ = 453.

Example 56

Compound 3 (0.13 g, 0.34 mmol) was dissolved in anhydrous tetrahydrofuran and stirred in an ice bath. Methylmagnesium chloride (3M in THF, 0.25 mL, 0.75 mmol) was added dropwise. The reaction was stirred at room temperature for 1 hour. The reaction was cooled in an ice bath and a saturated solution of ammonium chloride (2 mL) was added followed by water. The reaction mixture was extracted twice with ethyl acetate, and the combined ethyl acetate layers were dried over sodium sulfate. After concentration, the reaction mixture was purified and the isomers were separated by silica gel chromatography using 5% methanol/dichloromethane. The less polar product fraction was assigned the trans-isomer (ie compound 56A) and was found to be pure by reverse phase HPLC. The more polar product fraction was assigned the cis-isomer (ie compound 56B), which was found to be cis:trans 98:2 by reverse phase HPLC. Each product fraction was dissolved separately in dichloromethane, treated with 1 equivalent of 1M HCl/ether, and evaporated to a foamy residue. 13 mg of the trans isomer compound 56A hydrochloride (mass spectrum MH ⁺ = 399, melting point = 155-168 ° C), and 26 mg of cis isomer, compound 56B hydrochloride (mass spectrum MH ⁺ = 399, melting point = 156-169°C).

Example 57

This example illustrates the preparation of 6-(2-chlorophenyl)-8-cyclopropyl-2-(4-trans-hydroxy-cyclohexylamino)-8H from 4-chloro-2-methylthiopyrimidine -pyrido[2,3-d]pyrimidin-7-one.

Step 1 Preparation of 4-fluoro-2-methylthiopyrimidine

This compound was prepared by the method of Ple et al., J. Het. Chem., 31, 1311 (1994). Using this method, 7.6 g of the product 4-fluoro-2-methylthiopyrimidine were prepared from 11.5 g of 4-chloro-2-methylthiopyrimidine (71.7 mmol).

Step 2 Preparation of 4-cyclopropylamino-5-formyl-2-methylthiopyrimidine

Butyllithium (10.2 mL, 2.5 M solution in hexanes) was added to 55 mL THF (distilled from sodium/benzophenone) at -30 °C. The butyllithium solution was removed from the ice bath, and diisopropylamine (4 mL, 28.9 mmol) was added dropwise. The reaction was stirred for 30 minutes, then cooled to approximately -80°C in a dry ice/ether bath. A solution of 4-fluoro-2-methylthiopyrimidine (1.6 g, 11.1 mmol) in 4 mL THF was added dropwise and the resulting mixture was stirred for 2 hours. Ethyl formate (2.1 mL, 22.2 _mmol , treated with _K2CO3 and _distilled from _P2O5 ) was then added dropwise, and the resulting reaction mixture was stirred for a further 1 h. 0.1 eq ethyl formate was added and the reaction mixture was stirred for a further 1 hour. Cyclopropylamine (1.5 mL, 22.2 mmol, Aldrich Chemical) and water were added and the reaction was stirred for 90 minutes. Approximately 50 mL of 1M HCl/ether was added and the reaction mixture was stirred to room temperature. More water was added and THF was removed by evaporation. The residue was extracted twice with dichloromethane. The combined dichloromethane layers were dried over sodium sulfate and evaporated to a syrup. The residue was purified by silica gel chromatography using 7% methanol/dichloromethane as eluent.

Fractions containing the imine were combined, concentrated, treated with 10N 3M aqueous hydrochloric acid (in THF) for 3 hours, neutralized with sodium bicarbonate solution, concentrated, and extracted with dichloromethane. The dichloromethane solution was washed with water and bicarbonate solution, dried over sodium sulfate and concentrated. The resulting material was combined with the previously obtained pure material to give 1.8 g of 4-cyclopropylamino-5-formyl-2-methylthiopyrimidine.

step 3

4-Cyclopropylamino-5-formyl-2-methylthiopyrimidine (1.8 g, 8.6 mmol) was dissolved in 17 mL of 1-methyl-2-pyrrolidone, ethyl 2-chlorophenylacetate (2.1 g, 10.75 mmol) and potassium carbonate, and the mixture was stirred overnight at 95°C. A total of more than 0.8 g of ester was added and the reaction was stirred overnight again. The reaction was partitioned between 120 mL ethyl acetate and 100 mL water. After separation, the aqueous layer was extracted one more time with ethyl acetate. The combined ethyl acetate layers were washed with brine, dried over sodium sulfate, concentrated and purified by chromatography on silica gel 60 using 20-25% ethyl acetate/hexanes as eluent to afford 1.78 g of product.

step 4

The product from step 3 (1.66 g, 4.82 mmol) was dissolved in 20 mL THF and cooled in an ice bath. A solution of potassium persulfate formulation (5.9 g, 9.64 mmol) in water was added dropwise. The ice bath was removed and the reaction was stirred overnight. The reaction was filtered, approximately 40 mL of water was added, and the mixture was stirred for 30 minutes. The solid was filtered, resuspended in water and stirred for an additional 60 minutes. The mixture was filtered, rinsed with water and 20% ether/hexanes, and dried to give 1.08 g of product.

step 5

The product from Step 4 (0.35 g, 0.93 mmol), trans-4-aminocyclohexanol (0.322 g, 2.79 mmol, TCI America) and 0.5 mL of 1-methyl-2-pyrrolidone were combined and heated to 90°C. After 1 hour, the reaction was cooled to room temperature and ethyl acetate and water were added. The phases were separated and the aqueous layer was extracted again with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, concentrated and purified over silica gel 60 using 5% methanol/dichloromethane as eluent. After evaporation of the product fractions, the residue was redissolved in methanol/dichloromethane and treated with 1 equivalent of 1M HCl/ether. The solution was evaporated and the residue was triturated in 20% ether/hexane and dried to give 366 mg of product (hydrochloride salt of compound 57). Mass spectrum MH ⁺ = 411, melting point = 241.7-242.3°C.

Example 58

Compound 30B (0.300 g, 0.811 mmole), acetyl chloride (0.061 mL, 0.852 mmole) and sodium carbonate (0.090 g, 0.852 mmole) were placed in 5 mL of dichloromethane and stirred overnight at room temperature. After 18 hours, the reaction was purified by flash chromatography (3-5% (1:9 ammonium hydroxide/methanol)/dichloromethane) and column fractions containing product 58 were combined and concentrated in vacuo. The final product was placed in methanol, treated with hydrochloric acid (1.0M/Et ₂ O, 1.0 equiv), evaporated to dryness, washed with ether, filtered, and dried to obtain 0.288 g of the hydrochloride salt of compound 58 (mp 215.3-218.6 °C, M+.427).

Example 59

Compound 30B (0.300 g, 0.811 mmole), methanesulfonyl chloride (0.066 mL, 0.852 mmole) and sodium carbonate (0.090 g, 0.852 mmole) were placed in 5 mL of dichloromethane and stirred overnight at room temperature. After 18 hours, the reaction was purified by flash chromatography (3-5% (1:9 ammonium hydroxide/methanol)/dichloromethane) and column fractions containing product 59 were combined and concentrated in vacuo. The final product was placed in methanol, treated with hydrochloric acid (1.0M/Et ₂ O, 1.0 equiv), evaporated to dryness, washed with ether, filtered, and dried to obtain 0.326 g of the hydrochloride salt of compound 59 (mp 185.0-194.0 °C, M+.427).

Example 60

Compound 35A (0.086 g, 0.242 mmole), sodium carbonate (0.026 g, 0.242 mmole) and bromoacetonitrile (0.016 mL, 0.242 mmole) were placed in 2 mL of DMF. After 30 minutes at room temperature, the initial starting material was consumed. Sodium hydride (60% in oil, 0.011 g, 0.266 mmole) was added and stirred for an additional 30 minutes, then an additional 1 eq of bromoacetonitrile (0.016 mL, 0.242 mmole) was added and stirred overnight at room temperature. The reaction was purified by flash chromatography (1-5% (1:9 ammonium hydroxide/methanol)/dichloromethane) and the column fractions containing product 60 were combined and concentrated in vacuo. The final product was placed in methanol, treated with hydrochloric acid (1.0M/Et ₂ O, 1.0 equiv), evaporated to dryness, washed with ether, filtered, and dried to give 0.054 g of the hydrochloride salt of compound 60 (mp 143.0-158.5 ℃).

Example 61

Compound 30B (0.709 g, 1.92 mmole), methyl chloroformate (0.16 mL, 2.01 mmole) and sodium carbonate (0.213 g, 2.01 mmole) were placed in 5 mL of dichloromethane and stirred overnight at room temperature. After 18 hours, the reaction was purified by flash chromatography (2-10% (1:9 ammonium hydroxide/methanol)/dichloromethane) and column fractions containing product 61 were combined and concentrated in vacuo. The final product was placed in methanol, treated with hydrochloric acid (1.0M/Et ₂ O, 1.0 equiv), evaporated to dryness, then washed with ether, filtered, and dried to obtain 0.063 g of the hydrochloride salt of compound 61 (mp 133.5-136.5 ℃).

Example 62

Compound 30B (1.0 g, 2.81 mmole), sodium carbonate (0.447 g, 4.22 mmole) and 2-chloro-N-methylacetamide (0.453 g, 4.22 mmole) were placed in 5 mL DMF and stirred overnight at room temperature. After 20 hours, the reaction was purified by flash chromatography (3-5% (1:9 ammonium hydroxide/methanol)/dichloromethane) and column fractions containing product 62 were combined and concentrated in vacuo. The final product was placed in methanol, treated with hydrochloric acid (1.0M/Et ₂ O, 1.0 equiv), evaporated to dryness, washed with ether, filtered, and dried to obtain 0.708 g of the hydrochloride salt of compound 62 (mp 64.0-102.6 ℃).

Example 63

This example illustrates an alternative method for the synthesis of compound 30B.

This methyl sulfide (15g, 49.4mmole) was placed in 70mL NMP, stirred at room temperature; To this solution, a solution of N-chlorosuccinimide (7.6g, 56.8mmole) was added in 6mL of NMP, and Water (0.87 mL, 48.1 mmole) was added. The initial slurry quickly dissolved to a clear yellow solution. After 20 min at room temperature, the sulfoxide 30C was then treated with ethyl 4-amino-1-piperidinecarboxylate (12.7 mL, 74.1 mmole). The reaction darkened rapidly, warmed to contact temperature, and stirred at room temperature for 4 days. Upon completion, the opaque yellow suspension was transferred to a large Erlenmeyer flask with 250 mL of water and stirred in an ice bath for 4 hours. The suspension was then filtered to collect a yellow cake which was rinsed with water and hexanes and dried under vacuum. This urethane intermediate showed an excess weight which, despite prolonged drying, did not decrease below 31.6 g.

Potassium hydroxide (111 g, 1.98 mol) was placed in 400 mL of ethanol, heated until dissolved, then cooled slowly, after which the above carbamate intermediate (theoretically 49.4 mmole) was added. The solution was stirred at reflux for 3 hours, then cooled to room temperature, after which it was placed in an ice bath and neutralized with aqueous citric acid (111 g, 577 mmole, dissolved in 400 mL of water). The solution was stirred for 30 minutes, then evaporated under vacuum to a syrupy aqueous solution. The slurry was extracted with dichloromethane (x3); the combined extracts were washed with saturated brine, dried over sodium carbonate and evaporated in vacuo to give 12 g of a yellow foam (32.4 mmole).

A portion (500 mg) of this foam was purified by flash chromatography (5-20% methanol/dichloromethane + 1% ammonium hydroxide) and the column fractions containing product were combined and concentrated in vacuo. The purified product was taken up in methanol, treated with 2 equivalents of 1N HCl/Et ₂ O, and evaporated in vacuo; the resulting solid was washed with diethyl ether and collected to give 520 mg of the dihydrochloride salt of compound 30B (mp > 300 °C, (M+ H) ⁺ 370).

This methyl sulfide (15g, 49.4mmole) was placed in 70mL NMP, stirred at room temperature; N-chlorosuccinimide (7.6g, 56.8mmole) was added in 6mL NMP and water (0.87mL , a solution in 48.1 mmole). The initial slurry was rapidly dissolved to a clear yellow solution. After 20 minutes at room temperature, ethyl 4-amino-1-piperidinecarboxylate (12.7 mL, 74.1 mmole) was added to the solution and stirred at room temperature for 4 days. The opaque yellow suspension was transferred to a large Erlenmeyer flask with 250 mL of water and stirred in an ice bath for 4 hours. The suspension was then filtered to collect a yellow cake which was rinsed with water and hexanes and dried under vacuum. This urethane intermediate showed an excess weight which, despite prolonged drying, did not decrease below 31.6 g.

Potassium hydroxide (111 g, 1.98 mol) was placed in 400 mL of ethanol, heated until dissolved, then cooled slowly, after which the above carbamate intermediate (theoretically 49.4 mmole) was added. The solution was stirred at reflux for 3 hours, then cooled to room temperature, after which it was placed in an ice bath and neutralized with aqueous citric acid (111 g, 577 mmole, dissolved in 400 mL of water). The solution was stirred for 30 minutes, then evaporated under vacuum to a syrupy aqueous solution. The slurry was extracted with dichloromethane (x3); the combined extracts were washed with saturated brine, dried over sodium carbonate and evaporated in vacuo to give 12 g of a yellow foam (32.4 mmole).

A portion (500 mg) of this foam was purified by flash chromatography (5-20% methanol/dichloromethane + 1% ammonium hydroxide) and the column fractions containing product were combined and concentrated in vacuo. The purified product was taken up in methanol, treated with 2 equivalents of 1N HCl/Et ₂ O, and evaporated in vacuo; the resulting solid was washed with diethyl ether and collected to give 520 mg of the dihydrochloride salt of compound 30B (mp > 300 °C, (M+ H) ⁺ 370).

Example 64

To a solution of 0.25 g (0.68 mmol) of compound 30B and 0.11 g (0.74 mmol) of dimethylsulfamoyl chloride in 20 mL of dichloromethane was added 0.11 mL (0.81 mmol) of triethylamine. The mixture was heated to reflux for 6 hr, the solvent was removed under reduced pressure, and the residue was purified by 40 g silica gel column chromatography using 5% methanol/ethyl acetate as eluent to obtain 200 mg of white powder. It was dissolved in 2 mL of ethyl acetate and 0.75 mL of 1M HCl/ether was added. It was stirred for 1 hour and evaporated under reduced pressure to give the hydrochloride salt of compound 64 (200 mg). Mass spectrum MH ⁺ =477, mp 207.2-208°C).

Example 65

Within 30 minutes, 0.914 g (2.7 mmol) 6-(chlorophenyl) 2-methanesulfonyl-pyrido[2,3-d]pyrimidin-7-ol, 1.041 g (5.4 mmol) N-(2 -Hydroxyethyl)-phthalimide, a cold (5°C) suspension of 1.43 g (5.4 mmol) of triphenylphosphine in 15 mL of 1,4-dioxane was added dropwise to 0.95 g (0.86 mL, 5.4 mmol) in a solution of diethylazodicarboxylate in 5 mL of 1,4-dioxane. After the addition was complete, the ice-water cooling bath was removed. The suspension quickly turned into a clear light brown solution. It was allowed to stir overnight at room temperature. The reaction mixture was concentrated under reduced pressure and purified by column chromatography on 120 g of silica gel using 90:10:0.5 ethyl acetate/dichloromethane/ammonium hydroxide as eluent to give 0.38 g of N-ethylphthaloyl Imine intermediates. A mixture of 0.18 g (0.35 mmol) of this intermediate and 0.05 g (0.42 mmol) of trans-4-aminocyclohexanol in 1 mL of NMP was heated at 120° C. for 30 minutes. The reaction mixture was cooled, diluted with 75 mL of water, and extracted with ethyl acetate (3 x 75 mL). The combined ethyl acetate solutions were washed with water (3 x 75 mL), brine, dried, filtered and concentrated. This material was purified by column chromatography on 40 g of silica gel using 5% methanol/dichloromethane as eluent to afford 45 mg of compound 65. (Mass spectrum MH ⁺ = 544, mp 208-210°C).

Example 66

At room temperature, under a nitrogen atmosphere, to 0.179g (0.4mmol) 6-(2-chlorophenyl)-2-(N-ethoxypiperidinyl-4-amino)pyrido[2,3-d]- To the suspension of pyrimidin-7-ol in 1 mL of DMF, 0.012 g (0.46 mmol) of sodium hydride (95%) was added all at once. After 5 minutes, gas evolution was observed. The suspension turned into a clear yellow solution. This was stirred at room temperature for 30 minutes, then 0.068 g (0.05 mL, 0.4 mmol) of benzyl bromide was added via syringe. The reaction mixture was stirred overnight, quenched with concentrated aqueous ammonium chloride and extracted with ethyl acetate (3 x 60 mL). The combined ethyl acetate solutions were washed with water (3 x 60 mL), brine, dried, filtered and concentrated. This material was purified by column chromatography on 40 g of silica gel using 20% ethyl acetate/dichloromethane as eluent to afford 75.6 mg of compound 66. It was dissolved in 2 mL of ethyl acetate and 1 mL of 1M HCl/ether was added. The suspension was stirred for 1 hour and concentrated to give 74 mg of compound 66 hydrochloride. (Mass spectrum MH ⁺ = 518, mp. 162-173°C).

Example 67

A mixture of 0.350 g (1.0 mmol) sulfone and 0.303 g (3.0 mmol) 4-amino-tetrahydropyran in 0.7 mL NMP was heated at 100°C. After 1 hour, the reaction was cooled, poured into water, and extracted with ethyl acetate. The organic fraction was washed 5 times with water, 1 time with brine, dried over magnesium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (5% methanol/dichloromethane) to afford compound 67 as a foam. The residue was suspended in MeOH, and hydrochloric acid (1.0M/ _Et2O , 1 equiv) was added. The resulting mixture was stirred for 20 minutes, and concentrated under reduced pressure. The residue was stirred with a mixture of MeOH/ _Et2O for 1 h and the hydrochloride salt of compound 67 was filtered off as a white solid. Yield 330 mg. Mp 217.2-218.9°C. MS (M+H) ⁺ 371.

Example 68

The sulfone (350 mg, 1.04 mmol) was combined with 4-aminotetrahydropyran (303 mg, 3.0 mmol) and 0.7 mL of NMP and heated at 100° C. for 30 minutes. Cool to room temperature, add water, and filter to obtain compound 68 as a brown solid. The solution was dissolved in MeOH, made acidic with hydrochloric acid (1.0M/ _Et2O ), and evaporated. The residue _was purified over silica gel using 10:90 MeOH/ _CH2Cl2 as eluent. Fractions containing compound 68 hydrochloride were combined and evaporated under reduced pressure to give compound 68 hydrochloride as a white foam. The residue was suspended in MeOH, hydrochloric acid (1.0M/ _Et2O , 1 eq) was added, stirred for 20 min, and concentrated under reduced pressure. The residue was stirred with a mixture of MeOH/ _Et2O for 1 h and the product was filtered off as a white solid. Yield 228 mg. Mp > 300°C. MS (M+H) ⁺ 357.

Example 69

This example illustrates the synthesis of 2-(trans-4-methoxyamido-cyclohexylamino)-6-(2-chlorophenyl)-8H-pyrido[2, 3-d]pyrimidin-7-one

step 1

To a solution of the amine (218 mg, 0.436 mmol) in 10 mL of anhydrous tetrahydrofuran at 0° C. was added dimethyl pyrocarbonate (0.080 mL, 0.746 mmol), dimethylaminopyridine (2 mg, 0.016 mmol). The reaction mixture was stirred at 0 °C for 15 minutes, then slowly warmed to room temperature overnight. The crude reaction mixture was concentrated under vacuum and purified by column chromatography (5:95, methanol/dichloromethane) to give 230 mg (95%) of the product as a white solid (mass spectrum M+H ⁺ =558, mp=110.0 -112.0°C).

step 2

The SEM-protected pyridone obtained in step 1 (320 mg, 0.553 mmol) was suspended in 10 mL of methanol and treated with 10 mL of 10% hydrochloric acid. The reaction mixture was refluxed for 24 hours, cooled, then concentrated under vacuum until a precipitate started to form. The resulting suspension was filtered, washed with water and ethyl acetate, then dried to give 95 mg (53%) of 2-(trans-4-methoxyamido-cyclohexylamino)-6-(2-chlorophenyl) -8H-pyrido[2,3-d]pyrimidin-7-one 69, which is a white solid (mass spectrum M+H ⁺ =428, mp=294.6-296.8°C).

Example 70

The amine (0.49 mg, 1.3 mmol) was suspended in 6 mL of acetic anhydride and stirred at ambient temperature for 16 hours. The reaction mixture was diluted with ethyl acetate and filtered. The collected solid was washed with water and ethyl acetate, then dried to give 2-(trans-4-methylamidocyclohexylamino)-6-(2-chlorophenyl)-pyrido[2,3-d]pyrimidine -7-one as a white solid. The free base was suspended in ethyl acetate and treated with 1M HCl/ether solution to form 0.51 g (87%) of the hydrochloride salt of compound 70 as a white powder (mass spectrum M+H ⁺ =412, mp> 300°C).

Example 71

This example illustrates the synthesis of 2-(trans-4-amidoamidocyclohexylamino)-6-(2-chlorophenyl)-8H-pyrido[2, 3-d] pyrimidin-7-one.

step 1

To a solution of the amine (0.20 g, 0.39 mmol) in 15 mL of dichloromethane was added trimethylsilyl isocyanate (0.12 mL, 0.73 mmol) and stirred at room temperature for 5 hours. The reaction mixture was quenched with 5 mL of methanol, concentrated in vacuo, and purified by column chromatography (5:95, methanol/dichloromethane) to afford 0.14 g (66%) of the product as a white solid (mass spectrum M+H ⁺ = 543, mp = 182.2-188.9°C).

step 2

The SEM-protected compound obtained in step 1 (130 mg, 0.245 mmol) was suspended in 5 mL of methanol and treated with 5 mL of 10% hydrochloric acid. The reaction mixture was refluxed for 18 hours. The resulting suspension was filtered, the collected solid was washed with water, ethyl acetate, and dried to give 68 mg (62%) of 2-(trans-4-amidoamidocyclohexylamino)-6-(2-chlorophenyl )-8H-pyrido[2,3-dl pyrimidin-7-one (71) hydrochloride as a white solid (mass spectrum M+H ⁺ =413, mp>300°C).

Example 72

This example illustrates the synthesis of 2-(cis-4-methyl Sulfonylaminocyclohexylamino)-6-(2-chlorophenyl)-pyrido[2,3-d]pyrimidin-7-one.

step 1

To a solution of sulfone (1.93 g, 5.75 mmol) in 15 mL of anhydrous 1-methyl-2-pyrrolidone at 0° C. was added sodium hydride (0.160 g, 6.33 mmol, 95% dry powder). The reaction mixture was stirred for 10 minutes until no more gas had evaporated, then 2-(trimethylsilyl)ethoxymethyl chloride (1.10 mL, 6.22 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred for 30 minutes, then a mixture containing cis-1,4-diaminocyclohexane (6.27 g, 54.9 mmol, TCI America, Portland, OR, mixture of cis and trans isomers) was added over 0 °C cooled shake flask. The reaction mixture was stirred at room temperature for 3 hours. 100 mL of water and 100 mL of ethyl acetate were added, the phases were separated, and the aqueous layer was extracted with 100 mL of ethyl acetate. The combined organic layers were washed with 4 x 200 mL of brine, dried over sodium sulfate, and concentrated in vacuo to give a crude yellow liquid. Purification by column chromatography (30-40:70-60, methanol/dichloromethane and ammonium hydroxide) afforded 1.62 g (56%) of the product as a bright yellow foam (mass spectrum M+H ⁺ =500, mp=79.0-81.5 ℃.

step 2

To a solution of the amine obtained in step 1 (0.26 g, 0.53 mmol) in 12 mL of dichloromethane was added triethylamine (0.11 mL, 0.79 mmol) and methanesulfonic anhydride (0.18 g, 1.0 mmol). The reaction mixture was stirred for 3 hours, then concentrated in vacuo. Purification by column chromatography (3:97, methanol/dichloromethane) afforded 0.30 g (98%) of the product as a white foam (mass spectrum M+H ⁺ = 578, mp = 117.0-144.0°C). The isolated product was a mixture of cis and trans stereoisomers in a ratio of 82:18 as determined by ¹ H NMR chromatography.

step 3

The SEM-protected pyridone obtained in step 2 (0.29 g, 0.50 mmol) was suspended in 10 mL of methanol and treated with 10 mL of 10% hydrochloric acid. The reaction mixture was refluxed for 5 hours, cooled, then concentrated in vacuo until a precipitate started to form. The resulting suspension was filtered, washed with water and ethyl acetate, then dried to give 0.17 g (70%) of 2-(cis-4-methanesulfonylamino-cyclohexylamino)-6-(2-chlorophenyl) - The hydrochloride salt of 8H-pyrido[2,3-d]pyrimidin-7-one (72) as a white solid (mp>300°C). The isolated product was a mixture of cis and trans stereoisomers in a ratio of 82:18 as determined by ¹ H NMR chromatography.

Example 73

To a solution of sulfone (0.20 g, 0.57 mmol) and triethylamine (0.24 mL, 1.7 mmol) in 25 mL of tetrahydrofuran was added cis-4-aminocyclohexanol (Aust. J. Chem., 1961, 14, 610) . The reaction mixture was stirred at 80°C for 48 hours, cooled, and then 1N HCl was added. Extraction with chloroform, washing with sodium bicarbonate solution and brine, drying over magnesium sulfate. Filtration and concentration afforded 0.398 g of a crude solid, which was purified by column chromatography (4% methanol/dichloromethane) to yield 0.118 g (54% ) 2-(cis-4-hydroxycyclohexylamino)-6-(2-chlorophenyl)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one (73), It was a white solid (mass spectrum M+H ⁺ = 385, mp = 209.5-216.5°C).

Example 74

Preparation 74B:

Compound 74A (800 mg, 1.91 mmol), 1,4-dioxa-spiro[4,5]dec-8-ylamine (prepared see WO 99/001452) and N-methylpyrrolidone (0.5 mL) were mixed together , heated to 110°C with stirring. After 35 minutes, the reaction was cooled to room temperature, diluted with ethyl acetate (25 mL)/water (25 mL), partitioned, the phases were separated, and the organic layers were combined, washed with water (2 x 25 mL) and brine (1 x 25 mL), and passed through Drying over magnesium sulfate, filtration and concentration afforded compound 74B as an off-white powder (990 mg, (M+H) ⁺ = 495, MP = 200.0-206.5 °C).

Preparation of compound 74:

Compound 74B (990 mg) was placed in 10 mL of 80% acetic acid (aq) and heated to 65° C. for 4 hours with stirring. The reaction mixture was extracted with ethyl acetate (1 x 70 mL). The organic layer was washed with saturated sodium bicarbonate (4 x 50 mL) until pH = 8 and brine (1 x 50 mL), dried over magnesium sulfate, filtered and concentrated to afford 800 mg of crude product. Purification by preparative TLC using 70% ethyl acetate/hexane as eluent afforded 349 mg of compound 74, (M+H) ⁺ = 451, MP = 251.2-252°C).

Example 75

Compound 74A (2 g, 4.79 mmol), ethyl 4-amino-1-piperidinecarboxylate (Aldrich, 2.5 g, 3 eq) and N-methylpyrrolidone (1.5 mL) were mixed together and stirred at 110° C. for 3.5 hours . The reaction was cooled to room temperature and diluted with ethyl acetate (80 mL) and water (20 mL). The organic layer was separated, washed with water (2 x 25 mL) and brine (1 x 25 mL), dried over magnesium sulfate, filtered and concentrated. The crude product was purified by gradient elution on silica gel using 25% to 50% ethyl acetate in hexane as eluent to give 1.023 g of free amine ((M+H) ⁺ =510, MP=194.2- 221.4°C). 200 mg of the free amine was dissolved in ethyl acetate (15 mL), then 1.0 M HCl (in diethyl ether) (0.5 mL, 1.25 eq) was added and stirred for 1 hour. The solvent was then removed under reduced pressure at 50°C. 20 mL of diethyl ether was added to the residue and the solid was crushed. The resulting suspension was stirred for 30 minutes, filtered, and the off-white powder was dried under high vacuum at 56 °C for 2 hours to obtain 65 mg of compound 75 ((M+H) ⁺ =510, MP = 107.0-110.0 °C).

Example 76

Compound 74A (365mg, 0.87mmol), 4-aminotetrahydropyran, 76A, (265mg, 3eq) and N-methylpyrrolidone (0.3mL) were mixed and heated at 110°C for 30 minutes. The reaction solution was cooled to room temperature, diluted with ethyl acetate (30 mL) and water (25 mL). The organic layer was separated, washed with water (2 x 25 mL) and brine (1 x 25 mL), dried over magnesium sulfate, filtered and concentrated to afford 440 mg of crude product. Purification by preparative TLC eluting with 70% ethyl acetate (in hexanes) afforded the free amine (405 mg) as an off-white powder ((M+H) ⁺ = 439, MP = 200.9-202.1 °C ). The free amine was dissolved in ethyl acetate (25 mL), then 1.0 M HCI (in diethyl ether) (1.4 mL, 1.5 eq) was added and the resulting mixture was stirred for 1 hour. Then the solvent was removed under reduced pressure at 50°C, and the obtained residue was dried under high vacuum at 56°C to obtain 339 mg of compound 76 hydrochloride ((M+H) ⁺ =439, MP=198.2-201°C).

Example 77

Preparation of Compound 77B

Compound 77A (TCI Chemicals, 5 g, 26 mmol) and potassium cyanate (10.1 g, 5 eq) were dissolved in methanol (60 mL) and water (60 mL). The resulting mixture was heated to 80°C with stirring for 4 hours, then stirred at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate (350 mL). The organic layer was separated, washed with brine (5 x 150 mL) and diluted HCl/brine (1 x 150 mL), and concentrated to about 50 mL until solids started to appear. These solids were filtered and dried to obtain compound 77B (2 g, (M+H) ⁺ =230, MP = 170.4-173.5°C).

Preparation of Compound 77C:

Compound 77B (2 g) was placed in dioxane (125 mL), HCl (g) was bubbled through the heterogeneous mixture for 15 minutes, then the vessel was tightly sealed and stirred for 5 hours. The solvent was removed under reduced pressure at 50 °C. The crude product was taken up in methanol (250 mL) and 2.5 mL of sodium methoxide (in methanol) (25 wt%, 1 eq) was added. The resulting mixture was stirred for 1 hour, then concentrated to about 75 mL and filtered through a medium frit. The filtrate was concentrated and dried to obtain compound 77C (1.76 g, (M+H) ⁺ =130).

Preparation of compound 77:

Compound 74A (500mg, 1.2mmol), compound 77C (660mg, 3eq) 4-aminotetrahydropyran, 76A, (265mg, 3eq) and N-methylpyrrolidone (0.8mL) were mixed together, stirred at 110°C Heat for 30 minutes. The reaction solution was cooled to room temperature, diluted with ethyl acetate (80 mL) and water (40 mL). The organic layer was separated, washed with water (2 x 40 mL) and brine (1 x 40 mL), dried over magnesium sulfate, filtered and concentrated to give crude product. Purification by preparative TLC using pure ethyl acetate as eluent afforded compound 77 (120 mg) as free amine. The free amine was then dissolved in ethyl acetate (25 mL), then 1.0 M HCI (in diethyl ether) (0.4 mL, 1.5 eq) was added and the resulting mixture was stirred for 1 hour. The solvent was then removed under reduced pressure at 50 °C, and the resulting residue was dried under high vacuum at 56 °C to obtain the hydrochloride salt of compound 77 as an off-white powder (126 mg, (M+H) ⁺ =467, MP = 164.5-168.0°C).

Example 78 2-(4-tetrahydrothiopyranylamino)-6-(2-chlorophenyl)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one

step 1

A mixture of 5 g (43 mmol) tetrahydrothiopyran-4-one, 29.26 g (215 mmol) sodium acetate trihydrate and 14.94 g (215 mmol) hydroxylamine hydrochloride in 200 mL ethanol was refluxed for 6 hours. The mixture was diluted with 400 mL of ice-water mixture and extracted with ethyl acetate (2 x 150 mL). The extract was washed with brine, dried, filtered and concentrated under reduced pressure to afford 5.6 g (quantitative yield) of oxime (white solid). 2 g (15 mmol) of the above oxime (in 30 mL THF) was added dropwise to 76 mL (76 mmol) of 1M LAH/THF solution at room temperature. After the dropwise addition, the mixture was refluxed for 7 hours and then cooled to 5°C. 2.9 mL of water was carefully added dropwise to the reaction mixture, followed by dropwise addition of 2.9 mL of 15% aqueous NaOH and 8.7 mL of water. The suspension was stirred for 30 minutes, filtered through celite, washing with 300 mL of ethyl acetate. The filtrate was removed under reduced pressure (<50°C) to afford 1.62 g of 4-aminothiopyran, (92.3% yield).

step 2

A mixture of 1 g (2.9 mmol) of sulfone 1 and 0.67 g (5.7 mmol) of 4-aminothiopyran in 1.5 mL of NMP was heated in an oil bath at 85° C. for 1.5 hours until the reaction was complete. The reaction mixture was cooled, diluted with 100 mL of water, and extracted with ethyl acetate (2 x 75 mL). The combined ethyl acetate solution was washed again with water (2 x 75 mL) and brine, dried, filtered and concentrated under reduced pressure. Purification by column chromatography on silica gel (100 g) using 5% methanol in dichloromethane as eluent afforded 0.77 g (77% yield). 0.2 g of the above sulfide (in 2 mL of dichloromethane) was stirred with 0.62 mL (1.2 eq) of 1M HCI/ether for 30 minutes. Evaporation of the solvent gave 206 mg of the desired 2-(4-tetrahydrothiopyranylamino)-6-(2-chlorophenyl)-8-methyl-8H-pyrido[2,3-dpyrimidine HCl salt of -7-one, (mass spectrum MH ⁺ =387, mp 232.1-233.1°C).

Example 79

2-(S-Oxo-4-tetrahydrothiopyranylamino)-6-(2-chlorophenyl)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one

2-(S,S-Dioxy-4-tetrahydrothiopyranylamino)-6-(2-chlorophenyl)-8-methyl-8H-pyrido[2,3-d]pyrimidine- 7-keto

To a cooled solution (5° C.) of 0.2 g (0.5 mmol) of this sulfide in 50 mL of dichloromethane was added 0.13 g (0.57 mmol) of 77% 3-chloroperbenzoic acid. After 15 minutes, TLC analysis of a portion of the reaction showed complete conversion of the sulfide to sulfoxide (n=1) and traces of sulfone (n=2). An additional amount of 0.04 g of 77% 3-chloroperbenzoic acid was added and the reaction was stirred for an additional 30 minutes to convert more sulfoxide to sulfone. The mixture was poured into 10% aqueous sodium bisulfite and extracted with dichloromethane. The organic layer was washed with 10% aqueous sodium bicarbonate, then brine, dried, filtered and concentrated. Purification and separation of sulfoxide and sulfone were performed three times on 20 x 40 cm preparative TLC _SiO2 plates eluting with 5% methanol in dichloromethane. 68 mg of sulfoxide (racemate) was recovered and dissolved in 3 mL of dichloromethane. 0.25 mL of 1M HCI/ether was added and the suspension was stirred for 30 minutes. The solvent was evaporated under reduced pressure to give 70 mg of the desired hydrochloride of the sulfoxide (mass spectrum MH ⁺ = 403, mp 205.6-207.3°C).

0.15 g sulfone (higher Rf) was also recovered from the preparative TLC plate. It was dissolved in 3 mL of dichloromethane and 0.54 mL of 1M HCl/ether was added. The suspension was stirred for 30 minutes, filtered and washed with ether to afford 122 mg of the desired sulfone hydrochloride (mass spectrum MH ⁺ = 419, mp).

Example 80

This example illustrates the preparation of 6-(2-chlorophenyl)-8-methyl-2-methylthio-8-hydropyrido[2,3-d]pyrimidin-7-one (VI). method of choice

Preparation of 3,3-diethoxy-2-formylpropionitrile potassium salt (11)

To a stirred solution of 3,3-diethoxypropionitrile (1, 283.80 g, 1.98 mol) and methyl formate (148.80 g, 2.48 mol) in anhydrous THF (1.1 L) at 10°C, was added 1.0 M Potassium tert-butoxide in THF (2.2 L, 2.2 mol). The temperature was maintained at 10-15°C throughout the 45 minute addition period. After the addition, the resulting slurry was stirred at ambient temperature for 2 hours. Hexane (400 mL) was then added and stirring was continued for another 20 minutes. The slurry was filtered, the filter cake was washed with 1/1 hexane/THF, and dried overnight in a vacuum oven at 60°C. Yield of beige powder was 302.5 g (73.0%). ¹ H-NMR (CD ₃ OD) showed agreement with the desired structure II.

Preparation of 4-amino-2-sulfanylpyrimidine-5-carbaldehyde (III)

A slurry of thiourea (92.8 g, 1.22 mol) in ethanol (90 mL) was heated at reflux with vigorous stirring. To this slurry was added 3,3-diethoxy-2-formylpropionitrile potassium salt II (222.20 g, 1.06 mol) in 25% sodium methoxide/methanol (85.5 mL, 0.37 mole) and ethanol (285 mL) while maintaining reflux conditions (alternatively for addition, the slurry can be heated to 50°C to obtain a homogeneous solution). Another portion of ethanol (150 mL) was added to facilitate stirring. After the addition, the viscous slurry turned bright yellow and was held at reflux for an additional 1 hour. The mixture was then cooled and evaporated to near dryness on a rotary evaporator. The residue was dissolved in water (940 mL). The crude product was precipitated from solution by adding 30% acetic acid (280 mL) and then isolated by filtration using a sintered glass filter funnel. The filter cake was washed with water (800 mL). Purification by trituration in hot water (1 L) for 30 minutes followed by cooling and filtration afforded 118.9 g (72.3%) of product as a bright yellow solid after drying overnight in a vacuum oven at 60°C (subsequent preparations showed that development is optional). HPLC gave a purity of 98.67%. ¹ H-NMR (DMSO-d ₆ ) showed agreement with the desired structure III.

Preparation of 4-amino-2-methylthiopyrimidine-5-carbaldehyde (IV)

In 20 minutes, under gentle cooling, 4-amino-2-sulfanyl-pyrimidine-5-carbaldehyde III (100.00 g, 644.4 mmol) and potassium carbonate (178.10 g, 1.29 mol) passing through a 325 mesh sieve were added to the To a solution in acetone (1.5 L), iodomethane (128.10 g, 902.2 mmol) was added dropwise. The mixture was stirred at ambient temperature over weekend. TLC showed III still remaining, another portion of iodomethane (8 mL) was added and stirring continued overnight. TLC still showed some III remaining, another portion of iodomethane (8 mL) was added and stirring was continued for another 24 hours. HPLC showed 95.9% S-alkylated product and 3.7% compound III. The reaction mixture was stripped to near dryness via a rotary evaporator. Water (1 L) was added to the residue and the product was collected by filter and washed with water (200 mL). The product was dried overnight in a vacuum oven at 60°C. The yield was 103.37 g (94.8%), HPLC showed 95.8% IV and 4.2% III.

Preparation of 6-(2-chlorophenyl)-2-methylthio-8-hydropyrido[2,3-d]pyrimidin-7-one (V)

Mix IV (10.00g, 59.1mmol), ethyl 2-(2-chlorophenyl)acetate (14.40g, 71.8mmol), NMP (115mL) and potassium carbonate (29.00g, 209.8mM mol) was heated at 90°C overnight. The reaction mixture was cooled and diluted with water (800 mL). The resulting slurry was stirred overnight and product (V) was isolated by filtration. The filter cake was washed with water and dried overnight in a vacuum oven at 60°C. Isolated yield 14.9 g (83.0%) as a dark brown solid. HPLC analysis showed 98.3% purity.

Preparation of 6-(2-chlorophenyl)-8-methyl-2-methylthio-8-hydropyrido[2,3d]pyrimidin-7-one (VI)

A mixture of V (0.25 g, 0.82 mmole), NMP (5 mL), potassium carbonate (0.11 g, 0.82 mmole) and iodomethane (0.14 g, 0.96 mmole) was stirred overnight at ambient temperature under nitrogen atmosphere. Water (15 mL) was added and stirring continued for 24 hours. The slurry was filtered and the filter cake was washed with water (10 mL). HPLC analysis showed 97.8% purity.

Example 81

To pyridone 68 (1.6 g, 4.5 mmol) in NMP (10 mL) was added sodium hydride (188 mg, 4.7 mmol), the mixture was stirred for 45 minutes, then (2-iodoethoxy)triisopropylsilane ( 1.62 g, 5 mmol). After 12 hours, the mixture was poured into water, extracted with ethyl acetate, dried over sodium sulfate, concentrated in vacuo and the residue was purified by column chromatography on silica gel using 95:5 dichloromethane/methanol as eluent. The column fractions containing product were combined, concentrated in vacuo to a solid, which was suspended in ethyl acetate. Addition of hydrochloric acid (1.0M/ _Et2O , 1.2 equiv) gave the salt which was filtered and dried to give 262 mg of the desired product. Mass spectrum MH ⁺ = 401, mpt. 217-220°C.

Example 82

To pyridone 68 (500 mg, 1.4 mmol) in NMP (5 mL) was added sodium hydride (56 mg, 1.4 mmol), the mixture was stirred for 45 minutes, then iodoacetonitrile (0.11 mL, 1.54 mmol) was added. After 12 hours, the mixture was poured into water, extracted with ethyl acetate, dried over sodium sulfate, concentrated in vacuo and the residue was purified by column chromatography on silica gel using 97:3 dichloromethane/methanol as eluent. The column fractions containing product were combined, concentrated in vacuo to a solid, which was suspended in ethyl acetate. Addition of hydrochloric acid (1.0M/ _Et2O , 1.2 equiv) gave the salt which was filtered and dried to give 62 mg of the desired product. Mass spectrum MH ⁺ = 395, mpt. 230.2-230.4°C.

Example 83

Add 4-amino-2,2,6,6-tetramethylpiperidine (0.74 mg, 4.7 mmol) to sulfone 1 (0.4 g, 1.2 mmol) in NMP (1 mL), and stir the mixture at 75° C. 20 minutes. The mixture was diluted with water (30 mL), and the suspension was stirred for 30 minutes. The solid was filtered and washed with 100 mL of water and 50 mL of ether. After air drying for 30 minutes, 0.376 g of the desired product was obtained. To 0.27 g of this solid in 2 mL of dichloromethane was added hydrochloric acid (1.0 M/ _Et2O , 1.2 eq) and the resulting salt was filtered and dried to give 264 mg of the desired product. Mass spectrum MH ⁺ = 426, mpt. > 300°C.

Example 84

To sulfone 1 (0.16 g, 0.47 mmol) was added tetrahydro-3-thienamine 1,1-dioxide (0.19 g, 1.4 mmol) in NMP (1 mL), and the mixture was stirred at 70° C. for 30 minutes. The mixture was diluted with water, and the solid was filtered, washed with water and ether. The crude solid was purified by column chromatography on silica gel using 5:95:0.01 methanol/dichloromethane/ _NH4OH as eluent. The obtained solid was suspended in dichloromethane, and hydrochloric acid (1.0M/Et ₂ O, 1.2 equiv) was added. The resulting salt was filtered and dried to give 150 mg of the desired product. Mass spectrum MH ⁺ = 405, mpt. 256-260°C.

Example 85

A mixture of sulfone 1 (105 mg, 0.3 mmol) and 4-aminomorpholine (0.3 mL, 3.0 mmol) was stirred at 60 °C overnight. The reaction mixture was cooled, then purified by silica gel chromatography eluting with dichloromethane to give a white powder (91 mg, (M+H) ⁺ = 372, MP = 243.3-244.0 °C).

Example 86

step 1

S-3-(N-tritylamino) tetrahydrofuran (prepared according to the method reported in the literature, Barlos, Kleomenis; Papaioannou, Dionysios; Patrianakou, Stella; Sanida, Chariklia; Tsegenidis, Theodoros; J.Chem.Soc.Chem .Commun.; EN; 6; 1987; 474-475) (5.12g, 0.0155mol) and concentrated hydrochloric acid (5mL) was heated to reflux in ethanol (60mL) for 15 minutes and concentrated. The residue was stirred in hot diethyl ether (100 mL), filtered, washed with diethyl ether, and dried in vacuo to give (S)-3-aminotetrahydrofuran hydrochloride.

step 2

A mixture of sulfone 1 (1.877 g), (S)-3-aminotetrahydrofuran hydrochloride (0.66 g) and di-isopropylamine (3.73 mL) was refluxed in anhydrous acetonitrile (20 mL) until TLC showed sulfone Has reacted completely. The solvent was removed and the residue was phase separated between ethyl acetate and brine. The organic layer was separated, dried and concentrated. The crude product was purified by column chromatography on silica gel (1% MeOH/ _CH2Cl2 ) to give the desired product (0.6 _g ). The product was dissolved in MeOH/ _CH2Cl2 , treated with 2 mL 1N HCl (in ether), evaporated and dried to give the hydrochloride salt, MP 171.9-173°C, MS 357 (M+H ₎ .

Example 87

step 1

A solution of _the above sulfide 2.4 (8.2 g) in anhydrous DMF was stirred with _K2CO3 (4.1 g) and ethyl iodide (5 mL) at room temperature overnight. The mixture was stirred in EtOAc (300 mL) and brine (200 mL). The organic layer was separated, washed with brine, dried and concentrated to give crude product.

step 2

To a solution of the above product in THF (170 mL) was added potassium persulfate formulation (41 g) in water (170 mL) at 0-5°C. The mixture was stirred at room temperature for 4 hours, diluted with EtOAc (600 mL) and water (200 mL). The organic layer was separated, washed with brine (3X), dried and concentrated to give the sulfone.

step 3

A mixture of the above sulfone (2.23 g) and 4-amino-tetrahydropyran (1.17 g) in NMP (0.4 mL) was stirred at 120°C for 1 hour and then cooled to room temperature. Methanol (5 mL) was added and the mixture was stirred for 10 minutes. The solid was filtered and washed with ice methanol. The resulting solid was dissolved in _CH2Cl2 and treated with 5 _mL of 1-2N HCl in methanol. After evaporation of the solvent, the residue was recrystallized from isopropanol/ethyl acetate to give the final product (1.45 g). MP 185.3-190.1°C.

Example 88

step 1

A mixture of the sulfide (5.0 g), isobutyren oxide (3 mL) and potassium carbonate (2.6 g) in DMF (20 mL) was stirred overnight at 80°C. An additional amount of isobutane oxide (1.0 mL) was added and the mixture was stirred for an additional 8 hours. Aqueous work-up with EtOAc and brine gave 5.8 g of crude product.

step 2

To a 0-5°C solution of the above sulfide (5.8 g) in THF (150 mL) was added a solution of potassium persulfate formulation (3.5 g) in water (150 mL). The mixture was warmed to room temperature and stirred for 4 hours. EtOAC (400 mL) was added and the phases were separated. The organic layer was washed with brine (3 x 200 mL), dried over sodium sulfate, filtered and concentrated to give the desired sulfone (6.0 g), which was used in the next step without purification.

step 3

A mixture of sulfone (5.0 g) and 4-aminothiopyran (2.4 g) in NMP (4 mL) was heated at 100° C. for 1 hour. Aqueous work-up with EtOAc and brine gave the crude product, which was purified by column chromatography (silica gel, 30-35% EtOAc/hexanes) to give 2.3 g of a solid. MP 105-108.5°C, MS 445 (M+H).

step 4

To a solution of the above compound (2.3 g) in 90 mL of dichloromethane at 0-5°C was added a solution of MCPBA (2.6 g) in 70 mL of dichloromethane. The mixture was stirred overnight at room temperature and concentrated. The residue was partitioned between EtOAc and brine. The organic layer was separated, washed with saturated _NaHCO3 (5x), dried, concentrated, and purified by column chromatography (silica gel, 2-3% MeOH/dichloromethane). The product (1.3 g) was dissolved in dichloromethane/EtOAc and treated with 3 mL of 1M HCl in ether. The resulting solid was filtered, washed with ether and recrystallized from MeOH/EtOH to give the hydrochloride salt (0.65g), MP 223.72-230.2°C, MS 477 (M+H).

Example 89 Sulfone 89

step 1

To a solution of ethyl 4-chloro-2-methylthiopyrimidine-5-carboxylate (Aldrich, 24 g, 103 mmol) in 250 mL of acetonitrile was added 4-fluoroaniline (22.75 g, 205 mmol) at room temperature. After stirring for 2 days, the mixture was heated at 60 °C for 4 hours. The resulting solid was filtered and extracted with EtOAc. The filtrate was concentrated, diluted with EtOAc (300 L), washed with brine, dried (MgSO4), filtered and concentrated to give crude product. The crude product was stirred with hexanes (400 mL) and filtered to afford 23 g of ethyl 4-(4-fluorophenyl)amino-2-methylthiopyrimidine-5-carboxylate as a white solid.

step 2

At 5°C, lithium aluminum hydride (3.0 g) was stirred in anhydrous tetrahydrofuran (300 mL), and ethyl 4-(4-fluorophenyl)amino-2-methylthio-pyrimidine-5-carboxylate was added dropwise A solution of the ester (22.5 g) in anhydrous tetrahydrofuran (250 mL) was worked up. The reaction mixture was stirred overnight at room temperature. A further 1.0 M solution of lithium aluminum hydride in THF (55 mL) was added at 5°C, and the mixture was stirred at room temperature for 3 hours. Water was added dropwise and the resulting mixture was stirred for 30 min, after which aqueous sodium hydroxide (2M, 9 mL) was added, followed by water (12 mL). The resulting suspension was stirred at room temperature for 17 hours, then filtered. The filtered residue was washed with tetrahydrofuran (2X, 100 mL), and the combined filtrate and washings were concentrated. The residue was suspended in ethyl acetate/hexane-1/2 (200 mL), the solid was filtered and dried to obtain 14 g of 4-(4-fluorophenyl)amino-2-methylthiopyrimidine-5-methanol, which was yellow solid.

step 3

A solution of 4-(4-fluorophenyl)amino-2-methylthiopyrimidine-5-methanol (14.5 g) in 260 mL of dichloromethane was treated with manganese dioxide (58 g). The resulting suspension was stirred for 5 hours and filtered through celite. The filtered residue was washed with dichloromethane (100 mL), and the combined filtrate and washings were concentrated to give a solid. The solid was stirred with diethyl ether (100 mL) to afford 8.6 g of 4-(4-fluorophenyl)amino-2-methylthiopyrimidine-5-carbaldehyde as a white solid.

step 4

The aldehyde (8.6g, 0.033mol), ethyl o-chlorophenylacetate (8g) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2a]pyrimidine combined The polymer (base from Aldrich, 2.5 g) was mixed in NMP (60 mL) and stirred at 120° C. for 4 days. A further 1.4 g of base were added and the mixture was stirred at 120°C for a further 3 days. The reaction mixture was cooled to room temperature, filtered through a pad of celite, washing with NMP (10 mL). The filtrate was poured into water (600 mL), extracted with EtOAc (3 x 500 mL). The organic layers were combined, washed with brine (3x), dried over sodium sulfate, filtered and concentrated. The resulting solid was triturated with hot ether to give the product (6.5g).

step 5

To a solution of the above sulfide (6.4 g) in THF (70 mL) was added a solution of potassium persulfate formulation (27.3 g) in water (90 mL) at 0-5°C. The mixture was stirred at room temperature for 5 hours, diluted with EtOAC (600 mL) and water (250 mL). The organic layer was separated, washed with brine (3x), dried over sodium sulfate, filtered and concentrated to give sulfone 89 (5.5 g), MP 115.5-117 °C, MS 430 (M+H).

Example 90

step 1

To a solution of 2-fluoroethanol (13.85 g, 216.2 mmol, Aldrich Chemicals) and pyridine (50 mL) in dichloromethane (50 mL) was added tosyl chloride (61.9 g, 1.5 equiv) with stirring. The resulting mixture was stirred at 0 °C for 5 hours and then placed in the refrigerator overnight. The next day, 150 mL of ice water was added to quench the reaction, dichloromethane and pyridine were removed under reduced pressure at 0 °C, and diluted with ethyl acetate (600 mL). The organic layer was separated and extracted with ethyl acetate (1 x 200 mL). The combined organic layers were washed with 0.5N HCl (3 x 200 mL) until pH = 2, washed with saturated sodium bicarbonate (2 x 200 mL) and brine (1 x 200 mL), dried over magnesium sulfate, filtered and concentrated to give The color oil was crystallized upon standing to give tosylate (46.5 g, M=218) as a white powder.

step 2

To a solution of sulfide 2.4 (4.06 g, 13.4 mmol) in DMF (200 mL) at 0 °C was added a 60% NaH suspension in oil (0.59 g, 1.1 equiv). The resulting mixture was stirred at 0°C for 30 min, and a solution of the above tosylate (3.8 g, 1.3 equiv) in DMF (25 mL) was added dropwise. The resulting mixture was stirred overnight at 0°C to room temperature, then diluted with ethyl acetate (600 mL) and water (200 mL). The organic layer was separated, washed with water (3 x 200 mL), saturated sodium bicarbonate (1 x 200 mL), brine (2 x 200 mL), dried over magnesium sulfate, filtered and concentrated to give a semi-solid material (9.16 g). Hexane (400 mL) was added to the crude product and stirred for 15 minutes. The solvent was decanted, and the residue was concentrated to obtain N-monofluoroethyl compound (4.92 g, (M+H) ⁺ =350, MP = 114.7-118.1°C) as a tan powder.

step 3

To a 0° C. solution of the above N-monofluoroethyl compound (4.8 g, 13.4 mmol) in tetrahydrofuran (75 mL), a solution of potassium persulfate preparation (20.6 g, 2.5 equivalents) in water (75 mL) was added dropwise. solution. After the addition was complete, the ice bath was removed and the resulting mixture was stirred at room temperature for 7 hours and kept at 0°C overnight. The reaction was stirred at room temperature for an additional 5 hours, then diluted with ethyl acetate (600 mL) and water (250 mL). The organic layer was separated, washed with water (3 x 250 mL) and brine (1 x 250 mL), dried over magnesium sulfate, filtered and concentrated to give the desired sulfone (5.4 g, (M+H) ⁺ = 382, MP = 156.0- 168.0°C), which is a brown-yellow powder.

step 4

A mixture of the above sulfone (2.7 g, 7.07 mmol), 4-aminotetrahydropyran (2.15 g, 3 equiv) and NMP (2.7 mL) was stirred at 110 °C for 3.5 hours. Heating and stirring were then discontinued and the mixture was left overnight. The next day, ethyl acetate (180 mL) and water (65 mL) were added and the layers were partitioned and separated. The organic layer was washed with water (2 x 65 mL) and brine (1 x 65 mL), dried over magnesium sulfate, filtered and concentrated to afford 2.6 g of crude product. Purification by silica gel column chromatography using 25% ethyl acetate/hexane as eluent afforded the free amine (1.267 g, (M+H) ⁺ = 403). The free amine was taken up in dichloromethane (50 mL) and 1M HCl (in diethyl ether) (4.5 mL, 1.5 equiv) was added with stirring. The resulting mixture was stirred for 5 minutes, then the solvent was removed under reduced pressure at 55°C. Drying under high vacuum at 56°C for 24 hours afforded the desired compound (1.247 g, (M+H) ⁺ =403) as the hydrochloride salt.

Example 91

Sulfide hydrazide 91

To a solution of sulfide 2.4 (2.10 g, 6.59 mmol) in DMF (60 mL) at 0°C was added a 60% suspension of sodium hydride in oil (266 mg, 1.0 equiv). The resulting mixture was stirred at 0° C. for 30 minutes, then diphenylphosphinyl-O-hydroxylamine (Tetrahedron Let., vol 23, No. 37, 3835-3836, 1982) (1.854 g, 1.26 eq. ). After 1 min, a large amount of precipitate appeared and an additional 100 mL of DMF was added with stirring. After stirring for 1 hour, the reaction was poured into a mixture of ethyl acetate (700 mL) and water (200 mL). The aqueous phase was separated and extracted with ethyl acetate (125 mL). The combined ethyl acetate layers were washed with water (5 x 150 mL) and brine (1 x 150 mL), dried over magnesium sulfate, filtered and concentrated to give the sulfide hydrazide (2.3 g, MP = 183.4-184.2 °C, (M+H ) ⁺ =319), which is an off-white powder.

Example 92

The sulfide hydrazide 91 (250mg, 0.78mmol) and 4-aminotetrahydropyran (397mg, 5eq) were combined and stirred at 150°C for 10 hours. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (35 mL) and water (25 mL). The organic layer was separated, washed with water (2 x 25 mL) and brine (1 x 25 mL), dried over magnesium sulfate, filtered and concentrated to give the crude product (300 mg). Purification by preparative TLC eluting with 5% methanol/dichloromethane afforded the amine (209 mg, MP = 117.4-121.3°C, (M+H) ⁺ = 372.1). The free amine (200 mg, 0.538 mmol) was taken up in dichloromethane (5 mL) and ethyl acetate (20 mL). To this solution was added 1M HCl/diethyl ether (0.8 mL, 1.5 eq) and stirred for 2 hours. The solvent was removed under reduced pressure at 55°C and dried under vacuum at 56°C for 24 hours to obtain the desired compound (171 mg, MP = 207.1-215.9°C, (M+H) ⁺ =372) as an off-white powder.

Example 93

Step A: Preparation of Benzyl 1-Benzylpiperidin-4-ylcarbamate

A 0 °C solution of 4-amino-1-benzylpiperidine (41.2 g, 216.5 mmol) and triethylamine (51.3 mL, 369 mmol) in 600 mL THF was added dropwise to benzyl chloroformate over 30-45 minutes base ester (31 mL, 217 mmol), the rate was adjusted to maintain the reaction temperature between 5-10 °C. After the addition was complete, the reaction mixture was warmed to room temperature and stirred for 12 hours. Solvent and volatiles were removed under reduced pressure. Water (500 mL) and ethyl acetate (1.2 L) were then added and the phases were separated. The organic layer was washed with saturated aqueous sodium bicarbonate (2X, 150 mL) and brine, dried over magnesium sulfate, filtered and concentrated to give a tan liquid which was purified by column chromatography (Silica, EtOAc/Hexane - 30/70 to EtOAc-100), yielding 27.8 g of the amine as a white solid (mass spectrum M+ = 324, MP = 79.1-79.6°C).

Step B: Preparation of benzyl piperidin-4-ylcarbamate

To a solution of benzylamine (27.8 g, 85.7 mmol) in 400 mL of dichloromethane was added dropwise 1-chloroethyl chloroformate (25.4 g, 178 mmol) in 50 mL of dichloromethane at room temperature. solution in methane. After the addition was complete, the reaction mixture was stirred at room temperature for 3 hours. Solvent and volatiles were removed under reduced pressure and methanol (500 mL) was added. The reaction mixture was heated to reflux with stirring for 1 hour, cooled to room temperature, and concentrated to give 26.3 g of piperidine as an off-white solid (mass spectrum M+1 = 235, MP = 190.7-192.2 °C).

Step C: Preparation of benzyl 1-(methylsulfonyl)piperidin-4-ylcarbamate

The protected piperidine (10 g, 42.7 mmol) and triethylamine (12 mL, 86.7 mmol) were dissolved in 500 mL of dichloromethane at room temperature. Methanesulfonyl chloride (4.3 mL, 55.5 mmol) in 20 mL of dichloromethane was added dropwise via addition funnel. The reaction mixture was stirred at room temperature for 3 hours. The solvent and volatiles were removed under reduced pressure, ethyl acetate (500 mL) and aqueous hydrochloric acid (0.5M, 350 mL) were added to the reaction mixture, and the two phases were separated. The organic layer was washed with aqueous hydrochloric acid (0.5M, 2 x 100 mL), saturated aqueous sodium bicarbonate (3 x 100 mL), brine, dried over magnesium sulfate, filtered and concentrated to give methanesulfonamide (MP = 148.6-152.8 °C).

Step D: Preparation of 1-(methylsulfonyl)piperidin-4-amine

To a solution of methanesulfonamide (9.2 g, 29.5 mmol) in 200 mL of tetrahydrofuran in a 500 mL round bottom flask was added palladium on carbon (10%, 2-3 g) at room temperature under nitrogen atmosphere. The reaction vessel was flushed with hydrogen (3X), the reaction flask was filled with hydrogen via a hydrogen balloon, and the solution was stirred for 15 hours (more catalyst was added and balloon filled with hydrogen if necessary). Dichloromethane (100 mL) was added to the reaction and filtered through a pad of celite. Concentration of the filtrate afforded 4.63 g of the desired amine (mass spectrum M+1 = 179, MP = 65.3-65.7°C).

Step E:

A mixture of sulfide hydrazide 91 (255 mg, 0.8 mmol), 4-amino-1-methanesulfonylpiperidine (200 mg, 1.4 eq) and NMP (0.3 mL) was stirred at 150 °C for 2 days. The resulting mixture was diluted with methanol (8 mL), ethyl acetate (180 mL) and water (65 mL). The organic layer was separated, washed with water (2 x 60 mL) and brine (1 x 60 mL), and concentrated to give 640 mg of crude product. Purification by preparative TLC eluting with 8% methanol/dichloromethane afforded the free amine (180 mg, MP = 180.0-194.0°C, (M+H) ⁺ = 449).

To a solution of the free amine (180 mg, 0.40 mmol) in ethyl acetate (150 mL), dichloromethane (20 mL) and methanol (3 mL) was added 1M HCl (in diethyl ether) (0.6 mL, 1.5 equiv). The resulting mixture was stirred for 2 hours, then the solvent was removed under reduced pressure. The residue was dried under high vacuum at 56°C for 18 hours to afford the desired compound (168 mg, MP = 180.7-213.2°C, (M+H) ⁺ = 449) as an off-white powder.

Example 94 1,1-dioxo-4-aminothiopyran

step 1

To a 0° C. mixture of 1.0 N NaOH (25 mL, 25 mmol) and 4-aminotetrahydrothiopyran (2.34 g, 20 mmol) was added dropwise benzyl chloroformate, 3.14 mL (22 mmol). The reaction mixture was stirred at room temperature for 1 hour, filtered, washed twice with water, stirred with hexane, filtered, and dried in a vacuum oven to yield 4.4 g of the carbamate as a white solid. (M+H) ⁺ 252.

step 2

To a solution of the carbamate (40 g, 159 mmol) in dichloromethane was added 3-chloroperbenzoic acid (75%, 75 g, 320 mmol) in one portion over 1 hour. The reaction mixture was stirred for 12 hours, filtered, washed twice with 10% sodium sulfite solution, three times with 10% sodium bicarbonate solution, dried over magnesium sulfate and concentrated under reduced pressure to give the sulfone (37.5 g). M ⁺ =283.

step 3

To a mixture of sulfone (3.0 g, 10.6 mmol) in 50 mL of ethanol was added 5% palladium on carbon (300 mg). The reaction mixture was hydrogenated using a Paar hydrogenator at 40 psi for 8 hours. The resulting solution was filtered through celite, and the filtrate was concentrated to give 1.5 g of the amine. (M+H) ⁺ =150.

Example 95

A mixture of sulfide hydrazide 91 (660 mg, 2.07 mmol), aminosulfone (500 mg, 2 equiv), and NMP (0.5 mL) was stirred at 150 °C for 3 days. TLC showed that a large amount of starting material was still present, so an additional amount of amino-sulfone (200 mg, 1.34 mmol) was added and the mixture was stirred for another 1 day. By TLC analysis, no starting material remained. The reaction mixture was cooled to room temperature, diluted with methanol (230 mL) and dichloromethane (150 mL), and 10 g of silica gel was added. The mixture was concentrated and the residue was loaded onto a flash column (silica gel, 20 g) eluting with 1% methanol/dichloromethane to afford 623 mg of impure product. The mixture was further purified on preparative TLC eluting with neat ethyl acetate to give the free amine (70 mg, (M+H) ⁺ = 420). The free amine (70 mg, 0.17 mmol) was taken up in dichloromethane (10 mL) and methanol (10 mL), then 1M HCl (in diethyl ether) (0.3 mL, 1.5 equiv) was added. The resulting mixture was stirred for 5 minutes, concentrated and dried under high vacuum at 56°C to afford the desired product (62 mg, MP = 240.0-243.0°C).

Example 96

step 1

To a solution of sulfide hydrazide 91 (291 mg, 0.913 mmol) in methanol (30 mL) and acetic acid (10 mL) was added isobutyraldehyde (0.11 mL, 1.3 equiv) followed by sodium cyanoborohydride (58 mg, 1 equiv ). The resulting mixture was stirred at room temperature for 40 minutes, then diluted with ethyl acetate (175 mL). The organic layer was separated, washed with saturated sodium bicarbonate solution (4 x 60 mL) until basic, washed with brine (1 x 60 mL), dried over magnesium sulfate, filtered and concentrated to give 400 mg of crude product. Purification by preparative TLC eluting with 20% ethyl acetate/hexanes afforded N-alkylated sulfide hydrazide (319 mg, (M+H) ⁺ = 375) as an off-white foamy powder.

step 2

To a solution of N-alkylated sulfide hydrazide (319 mg, 0.85 mmol) in tetrahydrofuran (15 mL) at 0° C. was added potassium persulfate (523 mg, 1 equiv) in water (15 mL) dropwise. The resulting mixture was gradually warmed to room temperature over 4 hours, then diluted with ethyl acetate (300 mL). The organic layer was separated, washed with water (4 x 150 mL) and brine (1 x 150 mL), dried over magnesium sulfate, filtered and concentrated to afford the desired compound (308 mg, (M+H) ⁺ = 391 ) as an off-white foam.

step 3

A mixture of N-alkylated sulfoxide hydrazide (300 mg, 0.768 mmol), 4-amino-tetrahydropyran (233 mg, 3 equiv) and NMP was stirred at 80°C for 35 minutes. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (90 mL). The organic layer was separated, washed with water (3 x 25 mL) and brine (1 x 25 mL), dried over magnesium sulfate, filtered and concentrated to afford 327 mg of crude product. Purification by preparative TLC eluting with 5% methanol/dichloromethane afforded the free amine. The free base was then dissolved in dichloromethane (20 mL) and 1M HCl (in diethyl ether) (1.15 mL, 1.5 equiv) was added at room temperature. The resulting mixture was stirred for 2 minutes, then the solvent was removed under reduced pressure. Drying under high vacuum at 56°C for 24 hours afforded the desired product (239 mg, MP = 111.3-117.5°C, (M+H) ⁺ = 428) as an off-white powder.

Example 97

step 1

A mixture of sulfide hydrazide 91 (500 mg, 1.57 mmol) and 4-morpholinecarbonyl chloride (0.5 mL, 2.6 equiv) in pyridine (40 mL) was stirred at 90 °C for 7 hours. Pyridine was removed under reduced pressure at 50°C, and the residue was diluted with ethyl acetate (175 mL). The organic layer was washed with HCl/brine (3 x 75 mL) and brine (75 mL), dried over magnesium sulfate, filtered and concentrated to give the N-acylated sulfide hydrazide in quantitative yield ((M+H) ⁺ = 432).

step 2

To a solution of N-acylated sulfide hydrazide (0.785 mmol) in tetrahydrofuran (15 mL) at 0° C. was added potassium persulfate (483 mg, 1 equiv) in water (15 mL) dropwise. The resulting solution was gradually warmed to room temperature over 4 hours, then diluted with ethyl acetate (175 mL) and water (50 mL). The organic layer was separated, washed with water (3 x 50 mL) and brine (1 x 50 mL), dried over magnesium sulfate, filtered and concentrated to give N-acylated sulfoxide hydrazide (209 mg, (M+H) ⁺ =448).

step 3

A mixture of N-acylated sulfoxide hydrazide (209 mg, 0.0468 mmol), 4-amino-tetrahydropyran (142 mg, 3 equivalents) and NMP was stirred at 90°C for 1 hour, cooled to room temperature, and washed with ethyl acetate Dilute the ester (35 mL) and water (25 mL). The organic layer was separated, washed with water (2 x 25 mL) and brine (1 x 25 mL), dried over magnesium sulfate, filtered and concentrated to afford 293 mg of crude product. Purification by preparative TLC eluting with 5% methanol/dichloromethane afforded the free amine (36 mg, (M+H) ⁺ = 485). The free base was then dissolved in dichloromethane, then 1.0M HCl (in diethyl ether) (0.11 mL, 1.5 equiv) was added and the resulting mixture was stirred for 2 hours. The solvent was removed under reduced pressure at 55°C, followed by drying under high vacuum at 56°C for 24 hours to obtain the desired compound (38 mg, (M+H) ⁺ =485) as an off-white powder.

Example 98

step 1

To a solution of sulfide hydrazide 91 (500 mg, 1.57 mmol) in acetonitrile (10 mL) was added 37% formaldehyde (aq) (0.65 mL, 5 equiv) followed by sodium cyanoborohydride (155 mg, 1.6 equiv) . The resulting mixture was stirred for 15 minutes, then acetic acid was added to maintain neutral pH. The resulting mixture was stirred for 2 hours with occasional addition of acetic acid to maintain neutral pH. The reaction mixture was diluted with ethyl acetate (300 mL) and water (150 mL). The organic layer was separated, washed with saturated sodium bicarbonate (3 x 150 mL) and brine (1 x 150 mL), dried over magnesium sulfate, filtered and concentrated to afford 651 mg of crude product. Purification by flash column chromatography eluting with 15% ethyl acetate/hexane afforded N,N-dialkylated sulfide hydrazide (37 mg, (M+H) ⁺ = 347).

step 2

To a solution of N,N-dialkylated sulfide hydrazide (37 mg, 0.107 mmol) in tetrahydrofuran (3 mL) at 0 °C was added dropwise potassium persulfate formulation (66 mg, 1 equivalent). The resulting mixture was gradually warmed to room temperature over 4 hours, stored at 0°C overnight, and diluted with ethyl acetate (35 mL) and water (20 mL). The organic layer was separated, washed with water (2 x 20 mL) and brine (1 x 20 mL), dried over magnesium sulfate, filtered and concentrated to give the N,N-dialkylated sulfoxide hydrazide (35 mg, (M+H) ⁺ = 363).

step 3

A mixture of N,N-dialkylated sulfoxide hydrazide (35mg, 0.0965mmol), 4-amino-tetrahydropyran (39mg, 4eq) and NMP was stirred at 80°C for 35 minutes. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (35 mL) and water (25 mL). The organic layer was separated, washed with water (2 x 25 mL) and brine (1 x 25 mL), dried over magnesium sulfate, filtered and concentrated to give 39 mg of crude product. Purification by preparative TLC eluting with 5% methanol/dichloromethane afforded the free amine (29 mg). The free base was then dissolved in dichloromethane (5 mL) and 1M HCl (in diethyl ether) (0.1 mL, 1.5 equiv) was added. The resulting mixture was stirred for 2 minutes, then the solvent was removed under reduced pressure. Drying under high vacuum at 56°C for 24 hours afforded the desired product (29 mg, (M+H) ⁺ =400) as an off-white powder.

Example 99

A mixture of sulfone 89 (354 mg, 0.823 mmol), 4-amino-tetrahydropyran (250 mg, 3 eq) and NMP (0.3 mL) was stirred at 110 °C for 35 min. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (170 mL) and water (70 mL). The organic layer was separated, washed with water (2 x 70 mL) and brine (1 x 70 mL), dried over magnesium sulfate, filtered and concentrated to afford 393 mg of crude product. Purification by preparative TLC eluting with 65% ethyl acetate/hexane afforded the free amine (261 mg, (M+H) ⁺ = 451, MP = 281.7-283.4°C). The amine was dissolved in dichloromethane (15 mL) and methanol (2 mL), and 1M HCl (in diethyl ether) (0.8 mL, 1.5 eq) was added. The resulting mixture was stirred for 1 hour, then the solvent was removed under reduced pressure. The material was dried under high vacuum at 56°C to give the desired product (252 mg, (M+H) ⁺ = 451, MP = 150.0-154.0°C).

Example 100

step 1

To a solution of benzyl sulfide (5 g, 12.1 mmol) in DMF (50 mL) was added potassium carbonate (1.05 equiv, 1.76 g), followed by 2-iodoethanol (1.42 mL, 1.5 equiv), and the resulting mixture was heated at room temperature Stir overnight. The reaction mixture was diluted with ethyl acetate (300 mL) and water (150 mL). The organic layer was separated and concentrated without heating to a volume of approximately 150 mL. The white solid was collected by vacuum filtration and air dried on a frit for 2 hours. The solid was further dried under vacuum to give N-alkylated hydroxyethyl sulfide (4.139 g, MP = 155.6-156.2 °C, (M+H) ⁺ = 424).

step 2

To a solution of N-alkylated hydroxyethyl sulfide (4.13 g, 9.06 mmol) in tetrahydrofuran (200 mL) at 0 °C was added dropwise potassium persulfate formulation (11.13 g, 2 equiv) in water (200 mL) The solution. After the addition was complete, the ice bath was removed and the resulting mixture was stirred at room temperature overnight. The reaction mixture was poured into a mixture of ethyl acetate (700 mL) and water (400 mL). The organic layer was separated, washed with water (7 x 500 mL) and brine (5 x 500 mL), dried over magnesium sulfate, filtered and concentrated to give 2.2 g of N-alkylated hydroxyethyl sulfone. Some of this compound had crystallized when dried over magnesium sulfate and in a separatory funnel. The aqueous layers were combined and further extracted with dichloromethane (3 x 300 mL), magnesium sulfate dried powder from the first organic phase was added and the slurry was stirred overnight. Filtration and concentration of the mixture afforded another 2 g of desired product (MP = 197.1-198.7°C, (M+H) ⁺ = 456).

step 3

A mixture of N-alkylated hydroxyethylsulfone (1 g, 2.19 mmol), 4-amino-tetrahydrothiopyran (385 mg, 1.5 equiv) and NMP (0.5 mL) was stirred at 100 °C for 2.5 hours. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (50 mL) and water (40 mL), and stirred for 20 minutes. The organic layer was separated, washed with water (2 x 45 mL) and brine (1 x 45 mL), dried over magnesium sulfate, filtered and concentrated to afford 1 g of crude product. Purification by flash column chromatography on silica gel eluting with 20% ethyl acetate/dichloromethane afforded the desired compound (519 mg, M.P.=195.1-195.4C, (M+H)+=417) as off-white powder.

step 4

To a solution of the sulfide free amine (500 mg, 1.2 mmol) in dichloromethane (50 mL) at 0 °C was added a solution of MCPBA (724 mg, 3.5 eq) in dichloromethane (50 mL). The resulting mixture was gradually warmed to room temperature. The reaction mixture was concentrated under reduced pressure at 55°C, and the obtained residue was diluted with ethyl acetate (180 mL) and saturated sodium bicarbonate solution (60 mL). The organic layer was separated, washed with saturated sodium bicarbonate solution (3 x 60 mL) and brine (1 x 60 mL), dried over magnesium sulfate, filtered and concentrated to give 528 mg of the crude sulfone free amine. Elution by flash column chromatography on silica gel using a gradient elution from 1% methanol/dichloromethane to 2% methanol/dichloromethane afforded the sulfone free amine (279 mg, MP=155.0-155.9°C, (M+H) ⁺ =449 ), which is off-white powder. The free amine (278 mg, 0.62 mmol) was taken up in ethyl acetate (50 mL) followed by the addition of 1M HCl (in diethyl ether) (1 mL, 1.5 eq). The resulting mixture was stirred for 1 hour, then the solvent was removed under reduced pressure. The residue was dried under high vacuum at 56 °C to give the desired product (242 mg, MP = 182.0-186.0 °C, (M+H) ⁺ = 449) as an off-white powder.

Example 101

A mixture of silver nitrate (1.10 g, 6.50 mmole) and sodium hydroxide (0.52 g, 13.0 mmole) in 5 mL of water was stirred at room temperature for 15 minutes. The synthesized silver oxide was collected by vacuum filtration, washed with water, dried in vacuo, then a solution of trans-aminocyclohexanol adduct 38 (0.500 g, 1.30 mmole) in 2 mL of THF was added, and methyl iodide was added to it (0.16 mL, 2.60 mmole). The reaction mixture was stirred overnight at 40°C, then warmed to 60°C for 5 days, and methyl iodide (1.30 mmole, 0.08 mL) was added on the first day at 60°C.

Purification by flash chromatography (10-50% acetone/hexanes) yielded the desired product shown previously, as well as the N-methylated isomer. Each product was separately taken up in methanol, treated with hydrochloric acid (1.0M/ _Et2O , 1.0 equiv), reevaporated to dryness, then washed with ether, filtered, and dried to give 0.157 g of the desired O-methylated product ( mp 189.0-192.0°C) and 0.069 g of N-methylated product (mp 130.3-130.5°C).

Example 102

Step A: Preparation of 4-ethylsulfanyl-butan-2-one:

To a solution of ethanethiol (6.2 g, 7.4 mL, 0.1 mol) at 5 °C, 3 drops of DBU (in 50 mL THF) was added methyl vinyl ketone (7.3 g, 8.45 mL, 0.105 mol) dropwise. The mixture was stirred overnight at ambient temperature. The mixture was then concentrated under vacuum to afford 13.6 g of the desired ketone.

Step B: Preparation of 4-ethylsulfanyl-butan-2-one oxime:

4-Ethylsulfanyl-butan-2-one (13.6g, 0.1mol), sodium acetate trihydrate (68g, 0.5mol) and hydroxylamine hydrochloride (34.7g, 0.5mol) in 500mL ethanol The mixture was heated to reflux for 3 hours. The mixture was cooled and concentrated in vacuo. The residue was diluted with water and extracted with ethyl acetate (2 x 200 mol). The organic layers were combined, washed with brine, dried, filtered, and concentrated in vacuo to afford 14.7 g of the oxime.

Step C: Preparation of 2-amino-4-ethanesulfanyl-butane:

To a solution of lithium aluminum hydride (1M, 120 mL, 0.12 mol) in tetrahydrofuran was added 4-ethylsulfanyl-butan-2-one oxime (6 g, 0.04 mol) dropwise in 30 mL tetrahydrofuran. After complete addition, the mixture was stirred at reflux for 4 hours. The suspension was cooled with an ice-water bath, and water (4.6 mL) and 20 mL of tetrahydrofuran were added dropwise, followed by aqueous sodium hydroxide (15%, 4.6 mL). Additional water (13.8 mL) was then added and the reaction mixture was stirred for 30 minutes and filtered through a pad of celite, rinsing with ethyl acetate (300 mL). The filtrate was dried (brine, magnesium sulfate) and evaporated under reduced pressure to give 3.43 g of 2-amino-4-ethanesulfanyl-butane (mass spectrum M+1=134).

Step D:

A solution of sulfone 1 (0.55 g, 1.6 mmol) and 2-amino-4-ethanesulfanyl-butane (0.63 g, 4.8 mmol) in 10 mL of tetrahydrofuran was refluxed for 1 hour. The solution was cooled, concentrated in vacuo, and the product was purified by column chromatography on silica gel eluting with 5% ethyl acetate (in dichloromethane) to give 421 mg of a racemic mixture of 102A (mass spectrum M+1=403), and 31 mg of aziridine Pyridine compound 102B (mass spectrum M+1 = 401, MP = 160-167°C).

Example 103

This example describes an assay protocol for determining inhibition of p-38(MAP) kinase in vitro.

The compound of the present invention inhibits the activity of p-38 MAP kinase in vitro, which is determined by measuring the transfer of γ-phosphate in γ- ³³ P-ATP to myelin basic protein (MBP) by p-38 kinase, using Ahn, NG; et al. J.Biol.Chem.Vol.266(7), 4220-4227, (1991) the method described in the minor improvement method.

The phosphorylated form of recombinant p38 MAP kinase was expressed in E. coli using SEK-1 and MEKK, and then purified by affinity chromatography using a nickel column.

Phosphorylated p38 MAP kinase in kinase buffer (20 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2, 25 mM β-glycerol phosphate, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N, N , N', N'-tetraacetic acid, 1mM sodium vanadate, 1mM dithiothreitol, 40mM magnesium chloride). Test compounds dissolved in DMSO or DMSO alone (control) were added and samples were incubated at 30°C for 10 minutes. Kinase reactions are initiated by the addition of a substrate mix containing MBP and γ- ^33P -ATP. After further incubation at 30°C for 20 minutes, the reaction was terminated by the addition of 0.75% phosphoric acid. Phosphorylated MBP was separated from residual [gamma] ^-33P -ATP using a phosphocellulose membrane (Millipore, Bedford, MA) and quantified using a scintillation counter (Packard, Meriden, CT).

Compounds of the invention are active in this assay. The p-38 inhibitory activity (expressed as _IC50 , the concentration resulting in 50% inhibition of the tested p-38 enzyme) of some compounds of the invention is: CPD# (from Table 1) _IC50 , M 1 2.05×10 ^-8 2 4.19×10 ^-8 11 4.80×10 ^-9 twenty four 3.55×10 ^-8 29 4.15×10 ^-8 37 3.05×10 ^-8 38 6.2× ^10-9 41 5.1×10 ^-9 44 7.8× ^10-9 63 7.30×10 ^-9 73 2.09×10 ^-8 126 3.00×10 ^-10 128 6.00×10 ^-10 136 8.20×10 ^-9

Example 104

This example describes an in vitro assay evaluating the inhibitory effect on LPS-induced TNF-[alpha] production in THP1 cells.

The ability of the compounds of the invention to inhibit TNF-[alpha] release was determined using a minor modification of the method described by Blifeld, et al. Transplantation, 51(2), 498-503 (1991).

(a) Induction of TNF biosynthesis

THP-1 cells were suspended in culture medium [RPMI (Gibco-BRL, Gailthersburg, MD) containing 15% fetal calf serum, 0.02mM 2-mercaptoethanol] to a concentration of 2.5×10 ⁶ cells/ml, and then plated on In 96-well plates (0.2 ml aliquots in each well). The test compound was dissolved in DMSO, and thereafter diluted with the medium so that the final concentration of DMSO was 5%. A 25 [mu]l aliquot of test solution or medium containing DMSO alone (control) was added to each well. Cells were incubated at 37°C for 30 minutes. LPS (Sigma, St. Louis, MO) was added to the wells to a final concentration of 0.5 [mu]g/ml, and the cells were incubated for an additional 2 hours. At the end of the culture period, culture supernatants were collected and the amount of TNF-[alpha] present was determined using the ELISA assay described below.

(b) ELISA test:

Human TNF-α was measured by the specific capture ELISA assay described by Reimund, J.M. et al. The amount of α present.

Polystyrene 96-well plates were coated with 50 μl/well of antibody 2TNF-H12 (10 μg/ml) in PBS and incubated overnight at 4°C in a humid incubator. Plates were washed with PBS, then mounted with 5% non-fat dry milk in PBS for 1 hour at room temperature, and washed with 0.1% BSA (bovine serum albumin) in PBS.

TNF standards were prepared from stock solutions of human recombinant TNF-α (R&D Systems, Minneapolis, MN). Concentrations of standards in this assay started at 10 ng/ml, followed by 6 semi-log serial dilutions.

25 μl aliquots of the above culture supernatant or TNF standard or media alone (control) with 25 μl aliquots of biotinylated monoclonal antibody 2TNF-H34 (2 μg/ml in PBS containing 0.1% BSA) Mix before adding to each well. Samples were incubated for 2 hours at room temperature with gentle shaking and subsequently washed 3 times with PBS containing 0.1% BSA. 50 μl of peroxidase-streptavidin (Zymed, S. San Francisco, CA) solution containing 0.416 μg/ml peroxidase-streptavidin in PBS and 0.1% BSA. Samples were incubated for an additional 1 hour at room temperature and subsequently washed 4 times with PBS containing 0.1% BSA. 50 μl of O-phenylenediamine solution (lug/ml O-phenylenediamine and 0.03% hydrogen peroxide in 0.2 M citrate buffer pH 4.5) was added to each well, and the samples were incubated in the dark at room temperature. Conditioned for 30 minutes. Read the optical densities at 450nm and 650nm for the sample and reference, respectively. TNF-[alpha] levels were determined using a plot of optical density at 450 nm versus the concentration used.

_IC50 values are defined as the concentration of test compound corresponding to a half-maximum decrease in absorbance at 450 nm.

Example 105

This example describes an in vivo assay to evaluate the inhibition of LPS-induced TNF-[alpha] production in mice (or rats).

Using Zanetti. et al. J. Immunol., 148, 1890, (1992) and Sekut et al. J. Lab. Clin. Med., 124, 813, (1994) described the method slightly modified method, the compound of the present invention is determined to inhibit in vivo Ability to release TNF-α.

Female BALB/c mice (Chales River, Hollister, CA) weighing 18-21 g were acclimatized for 1 week. Each group comprising 8 mice was orally administered suspended or dissolved in an aqueous vehicle containing 0.9% sodium chloride, 0.5% sodium carboxymethylcellulose, 0.4% Tween 80, 0.9% benzyl alcohol (CMC vehicle) The test compound or only the vehicle (control group) was administered. Thirty minutes later, mice were injected intraperitoneally with 20 μg of LPS (Sigma, St. Louis, MO). After 1.5 h, mice were sacrificed by _CO inhalation, and blood was collected by cardiac puncture. The blood was sedimented by centrifugation at 15,600×g for 5 minutes, the serum was transferred to a clean tube and frozen at −20° C. until TNF-αα was determined by ELISA assay according to the manufacturer’s protocol (Biosource International, Camarillo, CA, USA) .

Claims (24)

1. Compounds of the following formula:

or a pharmaceutically acceptable salt or prodrug thereof, in R ¹ is hydrogen or alkyl; ^R is substituted cycloalkyl, heterosubstituted cycloalkyl, heteroalkyl substituted cycloalkyl, heterosubstituted cycloalkyl-alkyl, heterocyclyl, heterocyclylspirocycloalkyl, alkyl- S(O) _n -alkylene-, or SO ₂ Ar ² , where n is 1 or 2; R is ^hydrogen , amino, monoalkylamino, dialkylamino, acylamino, -NRa ^- C(=O) ^-Rb , alkyl, cycloalkyl, aryl, aralkyl, haloalkyl, Heteroalkyl, cyanoalkyl, -alkylene-C(O)-R, acyl, or phthalimidoalkyl, wherein ^R is hydrogen or alkyl, ^and R is heterocyclyl or hetero Alkyl, R is hydrogen, alkyl, hydroxy, alkoxy, amino, monoalkylamino or dialkylamino; And Ar ¹ and Ar ² are phenyl or phenyl independently substituted by one or more substituents selected from the group consisting of alkyl, alkoxy, and halogen; in Substituted cycloalkyl refers to a cycloalkyl group in which one, two or three ring hydrogen atoms are independently substituted by cyano or -Y-C(O)R, wherein Y is absent or is an alkylene group, and R is hydrogen, alkyl, haloalkyl, hydroxy, alkoxy, amino, monoalkylamino, dialkylamino, or phenyl; Heteroalkyl-substituted cycloalkyl means a cycloalkyl group in which one, two or three of the hydrogen atoms in the cycloalkyl group have been replaced by a heteroalkyl group, provided that the heteroalkyl group is attached to on cycloalkyl groups; Heterosubstituted cycloalkyl refers to cycloalkyl in which one, two or three hydrogen atoms in the cycloalkyl have been replaced by substituents independently selected from the group consisting of hydroxy, alkoxy, amino, acylamino , monoalkylamino, dialkylamino, oxo, imino, oxime, NR′SO ₂ R ^d , where R′ is hydrogen or alkyl, and R ^d is alkyl, cycloalkyl, amino, mono Alkylamino or dialkylamino; -XC(O)R, wherein X is O or NR', R is hydrogen, alkyl, haloalkyl, alkoxy, amino, monoalkylamino, dialkylamino, or phenyl, and R' is H or alkyl; or -S(O) _n R, wherein n is an integer from 0 to 2, wherein when n is 0, R is hydrogen, alkyl, cycloalkyl, or ring Alkylalkyl, and when n is 1 or 2, R is alkyl, cycloalkyl, cycloalkylalkyl, amino, acylamino, monoalkylamino or dialkylamino; and The heterocyclic group is piperidinyl, or tetrahydropyranyl. 2. The compound according to claim 1, wherein ^Ar1 is a phenyl group independently substituted by one or two halogen, alkyl or methoxy groups. 3. The compound according to claim 2, wherein ^Ar1 is 2-chlorophenyl, 2-fluorophenyl, 2-methylphenyl, or 2-methoxyphenyl. 4. A compound of the formula according to claim 3:

5. According to the compound according to any one of claims 1-4, wherein R ³ is hydrogen, amino, monoalkylamino, dialkylamino, acylamino, -NR ^a -C(=O)-R ^b , wherein R ^a is hydrogen or alkyl, and R ^b is heterocyclyl or heteroalkyl, alkyl, haloalkyl, cycloalkyl, cyanomethyl, heteroalkyl, aryl, aralkyl, or -alkylene Base-C(O)-R; wherein the heterocyclic group is piperidinyl, or tetrahydropyranyl. 6. according to the compound of claim 5, wherein R ³ is hydrogen, amino, dimethylamino, isopropylamino, (morpholinoformyl) amino, methyl, 2,2,2-trifluoroethyl, Cyclopropyl, cyanomethyl, 2-hydroxyethyl, 4-fluorophenyl, benzyl, carboxymethyl or methoxycarbonylmethyl, ethyl, 2-fluoroethyl, 2-hydroxy-2- Methylpropyl, or 2-phthalimidopropyl. 7. A compound according to claim 6, wherein ^R3 is hydrogen. 8. A compound according to claim 6, wherein ^R3 is methyl. 9. The compound according to claim 1, wherein ^R1 is hydrogen. 10. The compound according to claim 1, wherein ^R1 is methyl. 11. according to the compound of claim 1, wherein ^R is heterosubstituted cycloalkyl, heteroalkyl substituted cycloalkyl, aralkoxy, alkoxy, alkylsulfonyl-alkyl, heterocyclyl, Or heterosubstituted cycloalkyl-alkyl; wherein the heterocyclyl is piperidinyl, or tetrahydropyranyl. 12. The compound according to claim 11, wherein ^R2 is heterosubstituted cycloalkyl or heterocyclyl. 13. The compound according to claim 12, wherein ^R2 is 4-heterosubstituted cyclohexyl. 14. A compound according to claim 13, wherein ^R2 is 4-hydroxycyclohexyl. 15. The compound according to claim 12, wherein ^R2 is piperidinyl, or tetrahydropyranyl. 16. The compound according to claim 15, wherein ^R2 is a piperidinyl group substituted by N-methylsulfonyl. 17. The compound according to claim 16, wherein ^R2 is N-methylsulfonyl-piperidin-4-yl. 18. A compound according to claim 15, wherein ^R2 is a 4-tetrahydropyranyl group. 19. The compound according to claim 11, wherein ^R2 is alkylsulfonylalkyl. 20. according to the compound of claim 19, wherein ^R Be (1,1-dimethyl-2-methylsulfonyl) ethyl or (1,1-dimethyl-3-methylsulfonyl) propyl . 21. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound as claimed in any one of claims 1-20. 22. A method for preparing a compound of claim 1, comprising the steps of: under conditions sufficient to prepare a compound of formula I, Compound of formula Ig

contact with an amine of formula R ¹ R ² NH, Wherein R ¹ , R ² , R ³ and Ar ¹ are as defined in claim 1; n is an integer from 0 to 2; and R ⁶ is an alkyl group. 23. Use of a compound according to any one of claims 1-20 for the manufacture of a medicament for the treatment of p38-mediated diseases. 24. Use according to claim 23, wherein said p38-mediated disease is selected from arthritis, Crohn's disease, Alzheimer's disease, irritable bowel syndrome, adult respiratory distress syndrome or chronic obstructive pulmonary disease .