Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D475/00—Heterocyclic compounds containing pteridine ring systems
  • C07D475/06—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4
  • C07D475/10—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4 with an aromatic or hetero-aromatic ring directly attached in position 2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems

Definitions

• the invention relates to treating various disorders associated with enhanced activity of transforming growth factor beta (TGF- ⁇ ). More specifically, it concerns compounds that are related to quinazoline as useful in these methods
• TGF- ⁇ Transforming growth factor-beta receptor kinase (TGF- ⁇ ) denotes a family of proteins, TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3, which are pleiotropic modulators of cell growth and differentiation, embryonic and bone development, extracellular matrix formation, hematopoiesis, immune and inflammatory responses (Roberts and Sporn Handbook of Experimental Pharmacology (1990) 95:419-58; Massague et al. Ann Rev Cell Biol (1990) 6:597-646). Other members of this superfamily include activin, inhibin, bone morphogenic protein, and Mullerian inhibiting substance.
• TGF- ⁇ initiates an intracellular signaling pathway leading ultimately to the expression of genes that regulate the cell cycle, control proliferative responses, or relate to extracellular matrix proteins that mediate outside-in cell signaling, cell adhesion, migration and intercellular communication.
• inhibitors of the TGF- ⁇ intracellular signaling pathway are useful treatments for fibroproliferative diseases.
• fibroproliferative diseases include kidney disorders associated with unregulated TGF- ⁇ activity and excessive fibrosis including glomerulonephritis (GN), such as mesangial proliferative GN, immune GN, and crescentic GN.
• GN glomerulonephritis
• renal conditions include diabetic nephropathy, renal interstitial fibrosis, renal fibrosis in transplant patients receiving cyclosporin, and HIV-associated nephropathy.
• Collagen vascular disorders include progressive systemic sclerosis, polymyositis, scleroderma, dermatomyositis, eosinophilic fascitis, morphea, or those associated with the occurrence of Raynaud's syndrome.
• Lung fibroses resulting from excessive TGF- ⁇ activity include adult respiratory distress syndrome, idiopathic pulmonary fibrosis, and interstitial pulmonary fibrosis often associated with autoimmune disorders, such as systemic lupus erythematosus and scleroderma, chemical contact, or allergies.
• rheumatoid arthritis Another autoimmune disorder associated with fibroproliferative characteristics is rheumatoid arthritis.
• Eye diseases associated with a fibroproliferative condition include retinal reattachment surgery accompanying proliferative vitreoretinopathy, cataract extraction with intraocular lens implantation, and post glaucoma drainage surgery.
• PCT applications WO98/06715, WO98/07425, and WO 96/40143 all of which are incorporated herein by reference, describe compounds which are either imidazoles or are indoles substituted at the 3- or 4-position with a piperazine ring linked through a carboxamide linkage.
• 4,480,883 describes compounds that exhibit tyrosine kinase inhibition activity wherein the heterocyclic portion of a quinazoline or other fused ring nitrogen-containing aromatic system is substituted only once with an aromatic moiety, again optionally coupled through a linker.
• U.S. Patent No. 5,616,582 assigned to Zeneca describes tyrosine kinase inhibitors which are quinazolines linked through an amino group at the 4-position to a substituted or unsubstituted phenyl. These compounds contain no substituents at position 2.
• U.S. Patent No. 5,475,001 also assigned to Zeneca describes similar compounds with the same activity.
• These compounds include an N-containing substituent at the 1 -position that links the quinazoline or derivative thereof to an optionally substituted phenyl, pyridyl, indolyl, or pyrimidinyl group.
• PCT application WO 00/12497 also assigned to the assignee of the present application, Scios Inc., describes compounds and methods of inhibiting p38-a and TGF- ⁇ using substituted quinazolines or quinazoline derivatives wherein up to two N's replace up to two C's at the 5-position to the 8-position in the quinazoline backbone.
• These compounds include a linker at the 1 -position that links the quinazoline or derivative thereof to an optionally substituted cyclic aliphatic cyclic heteroaliphatic, aromatic or heteroaromatic group. These compounds also include an optionally substituted non-interfering substituent at the 2-position.
• a linker at the 1 -position that links the quinazoline or derivative thereof to an optionally substituted cyclic aliphatic cyclic heteroaliphatic, aromatic or heteroaromatic group.
• These compounds also include an optionally substituted non-interfering substituent at the 2-position.
• the invention is directed to methods and compounds useful in treating conditions that are characterized by enhanced TGF- ⁇ activity, preferably selective activity for TGF- ⁇ inhibition over p38 kinase, and which preferably have suppressed EGFR activity. These conditions include fibro-proliferative diseases, various cancers, and certain cardiovascular disorders as further described below. [0013] Compounds of the invention have been found to inhibit TGF- ⁇ and are thus useful in treating diseases mediated by these activities.
• the compounds of the invention are of the formula
• the invention is directed to methods of treating proliferative conditions using these compounds.
• the invention is also directed to treating conditions associated with cardiac diseases using the invention compounds.
• the compounds of formula (1) are useful in treating conditions which are characterized by enhanced activity of TGF- ⁇ .
• Conditions "characterized by enhanced TGF- ⁇ activity” include those wherein TGF- ⁇ synthesis is stimulated so that TGF- ⁇ is present in enhanced amount or wherein TGF- ⁇ latent protein is undesirably activated or converted to active TGF- ⁇ protein or wherein TGF- ⁇ receptors are upregulated or wherein the TGF- ⁇ protein shows enhanced binding to cells or extracellular matrix in the location of the disease.
• enhanced activity refers to any condition wherein the effectiveness of this protein is undesirably high, regardless of the cause.
• the compounds of the invention are useful in conditions where TGF- ⁇ shows enhanced activity since these compounds inhibit the activities of this protein. These conditions are those in which fibrosis and organ sclerosis are caused by, or accompanied by, 'inflammation, oxidation injury, hypoxia, altered temperature or extracellular osmolarity, conditions causing cellular stress, apoptosis or necrosis.
• TGF- ⁇ ischemia-reperfusion injury
• congestive heart failure progressive pulmonary and bronchial fibrosis
• hepatitis arthritis
• inflammatory bowel disease glomerular sclerosis
• interstitial renal fibrosis chronic scarring diseases of the eyes, bladder and reproductive tract
• bone marrow dysplasia chronic infectious or autoimmune states and traumatic or surgical wounds.
• the compounds useful in the invention are derivatives of quinazoline and related compounds containing mandatory substituents at positions corresponding to the 2- and 4-positions of the quinazoline.
• the compounds of the invention include a pteridine or pyridopyrimidine nucleus.
• Pteridine and 8-pyrido pyrimidine nuclei are preferred.
• Z and Z are N
• Z and Z are CH.
• at least one of each of Z 5 -Z 8 ust be N.
• the position that corresponds to the 2-position of the quinazoline contains a mandatory phenyl substituent, having 0-4 substituents, namely (R') m , where m is 0-4..
• the position that corresponds to the 4-position of the quinazoline contains a mandatory -NR 3 -4' -pyridyl substituent that may optionally contain 0-4 substituents, namely (R 2 ) n , wherein n is 0-4 .
• the pyridyl group is unsubstituted, i.e., n is 0.
• n is 1, and R 2 is positioned at the 3' position of the pyridyl moiety.
• n is 1, and R 2 is methyl, F, or CONH 2 , preferably, at the 1 ' or 2' position.
• the R 1 substituent(s) on the phenyl moiety preferably include minimally bulky groups.
• R is halo, lower alkyl, lower alkoxy, and lower alkyl halide groups.
• such groups include one or more halo, such as CI, F, Br, and I which may be the same or different if more than two halo groups are present.
• R 1 is an alkyl halide containing 1-3 halides, preferably methyl halide and even more preferably trifluoro methyl.
• R is an O-alkylaryl group such as O-benzyl.
• R is a lower alkyl, preferably Cl-6, more preferably Cl-3 alkyl, and even more preferably, methyl, ethyl, propyl or isopropyl.
• Two adjacent R groups may join to make an aliphatic or hetero aliphatic ring fused to the 2-phenyl.
• a fused ring if a fused ring is present it has 5 or 6 members, preferably 5 members and contains 1 or more heteroatoms such as N, S or O, and preferably 1 or 2 O.
• the fused ring is 1 , 3 dioxolane fused to phenyl at the 4 and 5 position of the phenyl ring.
• R 1 groups are F, CI, Br, I, CH , OCH 3 , CF 3 , OCH(CH 3 ) 2 , OCH 2 CH 3 , or O-benzyl.
• m is 0, 1 or 2.
• the R 1 group or groups that are bound to the 2-phenyl group may be bound at any available position of the phenyl ring.
• the R 1 group is bound at the position meta relative to the phenyl's attachment point on the quinazoline derivative nucleus.
• the groups are bound at the ortho and meta positions relative to the phenyl's attachment to the quinazoline derivative, more preferably at non-adjacent ortho and meta positions.
• Other embodiments include such groups at the ortho or para positions.
• a phenyl substituted at both meta positions or adjacent ortho and meta positions are contemplated if two groups are present.
• two groups may form a fused ring preferably attached at the meta and para positions relative to the phenyl's attachment to the quinazoline derivative.
• the phenyl is unsubstituted.
• the phenyl when the 6- or 7- isomers thereof are present, i.e. the nitrogen is in position 6 or 7 of pyridopyrimidine, the phenyl preferably is unsubstituted, or preferably contains one halo substituent, preferably chlorine, and preferably attached at the meta position relative to the phenyl's attachment to the pyridopyrimidine moiety.
• the phenyl is substituted, preferably with halo, more preferably one or two halos, and even more preferably chloro at the meta or para positions relative to the phenyl's attachment to the pyridopyrimidine moiety or dichloro at both meta positions; or more preferably substituted with fluoro, preferably difluoro, preferably at the ortho and meta positions relative to the phenyl's attachment to the pyridopyrimidine moiety; or more preferably bromo, preferably at the meta position relative to the phenyl's attachment to the pyridopyrimidine moiety; or more preferably iodo, preferably at the meta position relative to the phenyl's attachment to the pyridopyrimidine moiety.
• the phenyl group is substituted with two or more different halo substituents, preferably disubstituted, and preferably contains fluoro and chloro, and more preferably disubstituted at the non-adjacent ortho and meta positions relative to the phenyl's attachment to the pyridopyrimidine moiety, more preferably where fluoro is at the ortho position and chloro is at the meta position relative to the phenyl's attachment to the pyridopyrimidine moiety; or preferably is disubstituted with fluoro and bromo, preferably at the non-adjacent ortho and meta positions relative to the phenyl's attachment to the pyridopyrimidine moiety, more preferably where fluoro is at the ortho position and bromo is at the meta position relative to the phenyl's attachment to the pyridopyrimidine moiety [0028] In
• the phenyl is preferably substituted with alkyl, preferably methyl, and preferably at the meta position relative to the phenyl's attachment to the pyridopyrimidine moiety.
• two or more R 1 substituents may join to form a fused ring.
• the fused ring is a dioxolane ring, more preferably a 1,3-dioxolane ring, fused to the phenyl ring at the meta and para positions relative to the phenyl's attachment to the pyridopyrimidine moiety.
• the phenyl group is substituted with two or more different substituents, preferably disubstituted, and preferably chloro and methoxy, and preferably disubstituted at the non-adjacent ortho and meta positions relative to the phenyl's attachment to the pyridopyrimidine moiety, more preferably where methoxy is at the ortho position and chloro is at the meta position relative to the phenyl's attachment to the pyridopyrimidine moiety; or preferably is disubstituted with fluoro and methoxy, preferably at the adjacent ortho and meta positions relative to the phenyl's attachment to the pyridopyrimidine moiety, more preferably where fluoro is at the ortho position and methoxy is at the meta position relative to the phenyl's attachment to the pyridopyrimidine moiety.
• the phenyl group preferably contains at least one halo substituent at the ortho, meta or para positions relative to the phenyl's attachment to the pteridine moiety.
• the phenyl group contains one chloro group at the ortho or meta positions relative to the phenyl's attachment to the pteridine moiety; one fluoro group at the ortho, meta or para positions relative to the phenyl's attachment to the pteridine moiety; or one bromo or iodo at the meta position relative to the phenyl's attachment to the pteridine moiety.
• the phenyl group contains two halo groups preferably difluoro, preferably disubstituted at the non-adjacent ortho and meta positions relative to the phenyl's attachment to the pteridine moiety.
• the two halo groups are preferably dichloro, preferably disubstituted at the adjacent ortho and meta positions relative to the phenyl's attachment to the pteridine moiety.
• the two halo groups are preferably fluoro and chloro, preferably disubstituted at the adjacent or non-adjacent ortho and meta positions relative to the phenyl's attachment to the pteridine moiety, preferably where the fluoro is at the ortho position, and the chloro is at either meta position, and even more preferably where the chloro is at the non-adjacent meta position.
• the two halo groups are preferably fluoro and bromo preferably substituted at the non-adjacent ortho and meta positions relative to the phenyl's attachment to the pteridine moiety, preferably where the fluoro is at the ortho position, and the bromo is at the non-adjacent meta position.
• the phenyl group is substituted, preferably at one or more positions, preferably one position, and more preferably with alkoxy, even more preferably with methoxy, and preferably at the ortho or meta position relative to the phenyl's attachment to the pteridine moiety.
• the phenyl is preferably substituted with haloalkyl, preferably trifluoromethyl, and preferably at the meta position relative to the phenyl's attachment to the pteridine moiety.
• the phenyl group is substituted with two or more different substituents, preferably two substituents, and preferably disubstituted with halo and haloalkyl, more preferably fluoro and trifluoromethyl, and preferably disubstituted at the non-adjacent ortho and meta positions relative to the phenyl's attachment to the pteridine moiety, more preferably where fluoro is at the ortho position and trifluoromethyl is at the meta position relative to the phenyl's attachment to the pteridine moiety.
• the pyridyl moiety may be substituted with 0-3 R groups.
• n O; that is, the pyridyl moiety is unsubstituted.
• the following provisos apply to the inventive compounds: when Z 5 -Z 7 are CH and Z is N, when R is H, and when the pyridyl is unsubstituted, then m is 1-3 (i.e., the phenyl is substituted) and if m is 1, then R 1 is not 2-fluoro or 2-chloro; when Z 5 and Z 8 are N and Z 6 and Z 7 are CH, when R 3 is H, and when the pyridyl is unsubstituted, then the phenyl is substituted; and when Z 5 is N and Z 6 -Z 8 are CH, when R 3 is H, and when the pyridyl is unsubstituted, then the phenyl is substituted; and when Z 5 is N and Z 6 -Z 8 are CH, when R 3 is H, and when the pyridyl is unsubstituted
• hydrocarbyl residue refers to a residue which contains only carbon and hydrogen.
• the residue may be aliphatic or aromatic, straight-chain, cyclic, branched, saturated or unsaturated.
• the hydrocarbyl residue when indicated, may contain heteroatoms over and above the carbon and hydrogen members of the substituent residue.
• the hydrocarbyl residue may also contain carbonyl groups, amino groups, hydroxyl groups and the like, or contain heteroatoms within the "backbone" of the hydrocarbyl residue.
• alkyl straight- and branched-chain and cyclic monovalent substituents. Examples include methyl, ethyl, isobutyl, isopropyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. Typically, the alkyl, alkenyl and alkynyl substituents contain 1-12C (alkyl) or 2-12C (alkenyl or alkynyl).
• acyl encompasses the definitions of alkyl, alkenyl, alkynyl and the related hetero-forms which are coupled to an additional residue through a carbonyl group.
• Aromatic moiety refers to a monocyclic or fused bicyclic moiety such as phenyl or naphthyl; “heteroaromatic” also refers to monocyclic or fused bicyclic ring systems containing one ore more heteroatoms selected from O, S and N. The inclusion of a heteroatom permits inclusion of 5-membered rings as well as 6-membered rings.
• typical aromatic systems include pyridyl, pyrirnidinyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, imidazolyl and the like.
• Any monocyclic or fused ring bicyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system is included in this definition.
• the ring systems typically contain 5-12 ring member atoms.
• arylalkyl and heteroalkyl refer to aromatic and heteroaromatic systems which are coupled to another residue through a carbon chain, including substituted or unsubstituted, saturated or unsaturated, carbon chains, typically of 1-12C or preferably 1-6C. These carbon chains may also include a carbonyl group, thus making them able to provide substituents as an acyl moiety.
• halo include the chloro, fluoro, bromo and iodo, and may refer to one or more halides on a particular moiety.
• R-halide where R is alkyl, may refer to CFH 2 , CF 2 H or CF .
• the pyridyl moiety may also comprise two substituents which, when together, form a 5-7 membered carbocyclic or heterocyclic aliphatic ring.
• the compound has the formula:
• each of Z 5 , Z 6 and Z 7 is N or CH and wherein one or two Z 5 , Z 6 and Z 7 are N and wherein two adjacent Z positions cannot be N; wherein R 1 is F, CI, Br, I or CH 3 ; wherein R 1 is Br, F or CI; and wherein R is not present, is CH 3 or is an electron-withdrawing group.
• An electron-withdrawing group is a term known in the art, and encompasses a group that is more electronegative than carbon, and thus has an electron-withdrawing inductive effect. Examples of such groups are F, CI, OCF 2 H, OCF 3 , CF 3 , CONHR or CONH 2.
• the compounds of formula (1) may be supplied in the form of their pharmaceutically acceptable acid-addition salts including salts of inorganic acids such as hydrochloric, sulfuric, hydrobromic, or phosphoric acid or salts of organic acids such as acetic, tartaric, succinic, benzoic, salicylic, and the like.
• the compound of formula (1) may also be supplied as a salt with a pharmaceutically acceptable cation, or may be supplied as an ester or free base.
• Administration and Use [0045]
• the compounds of the invention are useful among other indications in treating proliferative conditions.
• the compounds of formula (1) or their pharmaceutically acceptable salts are used in the manufacture of a medicament for prophylactic or therapeutic treatment of mammals, including humans, in respect of conditions characterized by excessive production of TGF- ⁇ or enhanced sensitivity to TGF- ⁇ .
• the TGF- ⁇ inhibition activity is useful in treating fibroproliferative diseases, treating collagen vascular disorders, treating eye diseases associated with a fibroproliferative condition, venting excessive scarring, treating neurological conditions and other conditions that are targets for TGF- ⁇ inhibitors and in preventing excessive scarring that elicits and accompanies restenosis following coronary angioplasty, cardiac fibrosis occurring after infarction and progressive heart failure, and in hypertensive vasculopathy, and keloid formation or hypertrophic scars occurring during the healing of wounds including surgical wounds and traumatic lacerations.
• Neurological conditions characterized by TGF- ⁇ production include CNS injury after traumatic and hypoxic insults, Alzheimer's disease, and Parkinson's disease.
• TGF- ⁇ inhibitors include myelofibrosis, tissue thickening resulting from radiation treatment, nasal polyposis, polyp surgery, liver cirrhosis, and osteoporosis.
• Diseases benefited by TGF- ⁇ inhibition include cardiovascular diseases such as congestive heart failure, dilated cardiomyopathy, myocarditis, or vascular stenosis associated with atherosclerosis, angioplasty treatment, or surgical incisions or mechanical trauma; kidney diseases associated with fibrosis and/or sclerosis, including glomerulonephritis of all etiologies, diabetic nephropathy, and all causes of renal interstitial fibrosis, including hypertension, complications of drug exposure, such as cyclosporin, HIV-associated nephropathy, transplant nephropathy, chronic ureteral obstruction; hepatic diseases associated with excessive scarring and progressive sclerosis, including cirrhosis due to all etiologies,
• TGF- ⁇ The modulation of the immune and inflammation systems by TGF- ⁇ (Wahl et al. Immunol Today (1989) 10:258-61) includes stimulation of leukocyte recruitment, cytokine production, and lymphocyte effector function, and inhibition of T-cell subset proliferation, B-cell proliferation, antibody formation, and monocytic respiratory burst.
• TGF- ⁇ is a stimulator for the excess production of extracellular matrix proteins, including f ⁇ bronectin and collagen. It also inhibits the production of enzymes that degrade these matrix proteins. The net effect is the accumulation of fibrous tissue which is the hallmark of fibroproliferative diseases.
• TGF- ⁇ is active as a homodimer, but is synthesized and secreted from cells as an inactive latent complex of the mature homodimer and proregions, called latency associated protein (LAP). These proteins bind to each other through noncovalent interactions (Lyons and Moses Eur J Biochem (1990) 187:467). LAP is often disulfide-linked to separate gene products, called latent TGF- ⁇ binding proteins or LTBPs. These latent forms provide stability for the mature cytokine and a means for targeting it to the extracellular matrix and cell surfaces (Lawrence Eur Cytokine Network (1996) 7:363-74).
• Activation of the latent complex occurs after secretion from cells and is believed to result from the action of proteases, such as plasmin (Munger et al. Kidney Intl (1997) 51 : 1376-82), on LAP, thrombospondin-1 binding (Crawford et al. Cell (1998) 93: 1 159-70), and binding to the integrin v6 (Munger et al. Cell (1999) 319-28).
• proteases such as plasmin (Munger et al. Kidney Intl (1997) 51 : 1376-82), on LAP, thrombospondin-1 binding (Crawford et al. Cell (1998) 93: 1 159-70), and binding to the integrin v6 (Munger et al. Cell (1999) 319-28).
• proteases such as plasmin (Munger et al. Kidney Intl (1997) 51 : 1376-82), on LAP, thrombo
• Type IV is present only in the pituitary gland while the others are ubiquitous.
• the binding affinities among the three isoforms for the type I and II receptors differ such that these two receptors bind TGF- ⁇ 1 and TGF- ⁇ 3 more tightly than TGF- ⁇ 2 (Massague Cell (1992) 69: 1067-70).
• the type IV receptor or endoglin has a similar isoform binding profile in contrast to the type III receptor, betaglycan, which binds equally well to all three isoforms (Wang et al. Cell (1991) 67:797-805; Lopez-Casillas Cell (1991) 67:785-95).
• the type V receptor binds to IGFBP-3 and is thought to have an active kinase domain similar to the type I and II receptors. Cloning of the type I and type II receptors demonstrated the existence of cytoplasmic serine/threonine kinase domains (Wrana et al. Cell (1992) 71:1003-14; Lin et al. Cell (1992) 68:775-85; Ibid. 71:1069; Massague Cell (1992) 69:1067-70). Initiation of the TGF- ⁇ signaling pathway results from the binding of the TGF- ⁇ ligand to the extracellular domain of the type II receptor (Massague Ann Rev Biochem (1998) 67:753-91).
• the bound receptor then recruits type I receptor into a multimeric membrane complex, whereupon the constitutively active type II receptor kinase phosphorylates and activates type I receptor kinase.
• the function of the type I receptor kinase is to phosphorylate a receptor-associated co-transcription factor, smad-2/3, thereby releasing it into the cytoplasm where it binds to smad-4.
• This smad complex translocates into the nucleus, associates with a DNA-binding cofactor, such as Fast-1, binds to enhancer regions of specific genes, and activates transcription.
• the expression of these genes leads to the synthesis of cell cycle regulators that control proliferative responses or extracellular matrix proteins that mediate outside-in cell signaling, cell adhesion, migration, and intercellular communication.
• the manner of administration and formulation of the compounds useful in the invention and their related compounds will depend on the nature of the condition, the severity of the condition, the particular subject to be treated, and the judgement of the practitioner; fo ⁇ nulation will depend on mode of administration.
• the compounds of the invention are small molecules, they are conveniently administered by oral administration by compounding them with suitable pharmaceutical excipients so as to provide tablets, capsules, syrups, and the like.
• suitable formulations for oral administration may also include minor components such as buffers, flavoring agents and the like.
• the amount of active ingredient in the formulations will be in the range of 5%-95% of the total formulation, but wide variation is permitted depending on the carrier.
• Suitable carriers include sucrose, pectin, magnesium stearate, lactose, peanut oil, olive oil, water, and the like.
• the compounds useful in the invention may also be administered through suppositories or other transmucosal vehicles. Typically, such formulations will include excipients that facilitate the passage of the compound through the mucosa such as pharmaceutically acceptable detergents.
• the compounds may also be administered topically, for topical conditions such as psoriasis, or in formulation intended to penetrate the skin.
• the compounds may also be administered by injection, including intravenous, intramuscular, subcutaneous or intraperitoneal injection. Typical formulations for such use are liquid formulations in isotonic vehicles such as Hank's solution or Ringer's solution.
• Alternative formulations include nasal sprays, liposomal formulations, slow-release formulations, and the like, as are known in the art. [0059J Any suitable formulation may be used. A compendium of art-known formulations is found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Company, Easton, PA. Reference to this manual is routine in the art.
• the dosages of the compounds of the invention will depend on a number of factors which will vary from patient to patient. However, it is believed that generally, the daily oral dosage will utilize 0.001-100 mg/kg total body weight, preferably from 0.01-50 mg/kg and more preferably about 0.01 mg/kg- 10 mg/kg. The dose regimen will vary, however, depending on the conditions being treated and the judgment of the practitioner. [0061] It should be noted that the compounds of formula (1) can be administered as individual active ingredients, or as mixtures of several embodiments of this formula. In addition, the inhibitors of TGF- ⁇ , can be used as single therapeutic agents or in combination with other therapeutic agents.
• Drugs that could be usefully combined with these compounds include natural or synthetic corticosteroids, particularly prednisone and its derivatives, monoclonal antibodies targeting cells of the immune system, antibodies or soluble receptors or receptor fusion proteins targeting immune or non-immune cytokines, and small molecule inhibitors of cell division, protein synthesis, or mRNA transcription or translation, or inhibitors of immune cell differentiation or activation.
• corticosteroids particularly prednisone and its derivatives
• monoclonal antibodies targeting cells of the immune system antibodies or soluble receptors or receptor fusion proteins targeting immune or non-immune cytokines
• small molecule inhibitors of cell division, protein synthesis, or mRNA transcription or translation or inhibitors of immune cell differentiation or activation.
• the compounds of the invention may be used in humans, they are also available for veterinary use in treating animal subjects.
• the compounds of the invention having a pteridine nucleus may be synthesized from methyl 3-amino-2-pyrazine carboxylate as described in Reaction Scheme I.
• compounds of the invention having a pyridopyrimidine nucleus having a nitrogen at the 7 position may be synthesized from ethyl l-benzyl-3-oxo-piperidine carboxylate-hydrochloride and benzamidine hydrochloride or analog thereof as illustrated in Reaction Scheme II.
• Compounds of the invention having a pyridopyrimidine nucleus where there is an N in the 6 position of the nucleus may be prepared from methyl l-benzyl-oxo-3-piperidine carboxylate hydrochloride and benzamidine hydrochloride as shown in Reaction Scheme III.
• Compounds of the invention having a pyridopyrimidine nucleus having an N at the 8 position may be synthesized from 2-amino nicotinic acid methyl ester.
• An alternate procedure for the final step in Reaction Scheme IV is illustrated in Reaction Scheme V.
• Compounds of the invention include those in Table 1.
• Example 1 [0069] Examples 1-4 illustrates Reaction Scheme 1 where the substituents are as listed in the Table below. The compounds in bold are found in the Reaction Schemes.
• lid [0088] He (446mg, 2 mmole) was suspended in phosphorous oxychloride (8ml) and heated to reflux for 15 minutes. Removed excess phosphorous oxychloride under vacuum. Residue was treated with ice water, extracted with chloroform. Combined chloroform extracts were washed with water, dried over sodium sulfate (anh.) and solvent removed under vacuum to give 494mg product.
• Illb [0092] Ilia (3.5g, 11.03mmole) was dissolved in 80ml acetic acid, added 400mg 10%Pd/C and hydrogenated at 40psi at 50°C for 5 hours. Filtered mixture over celite to remove catalyst. Evaporated acetic acid to give product as acetate salt. 3.7g (97% yield).
• nib (3.5g) was dissolved in water, added ethyl acetate, basified with 1M NaOH to pH 9. Free base of Illb was extracted with ethyl acetate, then dichloromethane, combined extracts dried over sodium sulfate (anh.). Removed solvent to give the free base of Illb. This was suspended in m-xylene (60ml) added 10% Pd/C (500mg) and refluxed for 1.5 days. Removed catalyst by filtration over celite. Removed xylene under vacuum to give product (700mg).
• reaction mixture was heated to 1 lOoC for 15h.
• the reaction mixture was cooled to r.t. and filtered through Celite® and the crude material was purified by silica gel flash column chromatography (95% to 5% gradient CH2C12/EtOAc) to afford [2-(5-Chloro-2-fluoro-phenyl)- pyrido [2 ,3 -d]pyrimidin-4-yl] -(3 -fluoro-pyridin-4-yl)-amine (27.3mg) .
• This assay can be conducted as follows: Compound dilutions and reagents are prepared fresh daily. Compounds are diluted from DMSO stock solutions to 2 times the desired assay concentration, keeping final DMSO concentration in the assay less than or equal to 1%. TGF ⁇ l kinase is diluted to 4 times the desired assay concentration in buffer + DTT. ATP is diluted into 4x reaction buffer, and gamma-33P-ATP is added at 60uCi/mL. [0118] The assay is performed by adding lOul of the enzyme to 20ul of the compound solution. The reaction is initiated by the addition of lOul of ATP mix.
• Final assay conditions include lOuM ATP, 170nM TGF ⁇ l kinase, and IM DTT in 20mM MOPS, pH7.
• the reactions are then incubated at room temperature for 20 minutes afterwhich they are stopped by transferring 23ul of reaction mixture onto a phosphocellulose 96-well filter plate, ( pre-wetted with 15ul of 0.25M H3P04 per well). After 5 minutes, the wells are washed 4x with 75mM H3P04 and once with 95% ethanol. The plate is dried, scintillation cocktail is added to each well, and the wells are counted in a Packard TopCount microplate scintillation counter. The ability of a compound to inhibit the enzyme is determined by comparing the counts obtained in the presence of compound to those of the positive control (in the absence of compound) and the negative control (in the absence of enzyme).
• Example 14 Assay for TGF- ⁇ Inhibition
• Invention compounds can also be evaluated by measuring their abilities to inhibit the phosphorylation of the substrate casein.
• the assay can be conducted as follows: Compound dilutions and reagents are prepared fresh daily. Compounds are diluted from DMSO stock solutions to 2 times the desired assay concentration, keeping final DMSO concentration in the assay less than or equal to 1%. TGF ⁇ l kinase is diluted to 4 times the desired assay concentration in buffer + DTT. ATP and casein are diluted into 4x reaction buffer, and gamma- 33 P- ATP is added at 50uCi/mL.
• the assay is performed by adding lOul of the enzyme to 20ul of the compound solution.
• the reaction is initiated by the addition of lOul of the casein/ ATP mix.
• Final assay conditions included 2.5uM ATP, lOOuM casein, 6.4nM TGF ⁇ l kinase, and IM DTT in 20mM Tris buffer, pH 7.5.
• the reactions are incubated at room temperature for 45 minutes.
• the reactions are then stopped by transferring 23ul of reaction mixture onto a phosphocellulose 96- well filter plate, which had been pre-wetted with 15ul of 0.25M H3P04 per well. After 5 minutes, the wells are washed 4x with 75mM H3P04 and once with 95% ethanol.

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