Optimization of 4-phenylamino-3-quinolinecarbonitriles as potent inhibitors of Src kinase activity.

Subsequent to the discovery of 4-[(2,4-dichlorophenyl)amino]-6,7-dimethoxy-3-quinolinecarbonitrile (1a) as an inhibitor of Src kinase activity (IC(50) = 30 nM), several additional analogues were prepared. Optimization of the C-4 anilino group of 1a led to 1c, which contains a 2,4-dichloro-5-methoxy-substituted aniline. Replacement of the methoxy group at C-7 of 1c with a 3-(morpholin-4-yl)propoxy group provided 2c, resulting in increased inhibition of both Src kinase activity and Src-mediated cell proliferation. Analogues of 2c with other trisubstituted anilines at C-4 were also potent Src inhibitors, and the propoxy group of 2c was preferred over ethoxy, butoxy, or pentoxy. Replacement of the morpholine group of 2c with a 4-methylpiperazine group provided 31a, which had an IC(50) of 1.2 nM in the Src enzymatic assay, an IC(50) of 100 nM for the inhibition of Src-dependent cell proliferation and was selective for Src over non-Src family kinases. Compound 31a, which had higher 1 and 4 h plasma levels than 2c, effectively inhibited tumor growth in xenograft models.

Synthesis and Src kinase inhibitory activity of a series of 4-phenylamino-3-quinolinecarbonitriles.

Screening of a directed compound library in a yeast-based assay identified 4-[(2,4-dichlorophenyl)amino]-6,7-dimethoxy-3-quinolinecarbonitrile (2a) as a Src inhibitor. An enzymatic assay established that 2a was an ATP-competitive inhibitor of the kinase activity of Src. We present here SAR data for 2a which shows that the aniline group at C-4, the carbonitrile group at C-3, and the alkoxy groups at C-6 and C-7 of the quinoline are crucial for optimal activity. Increasing the size of the C-2 substituent of the aniline at C-4 of 2a from chloro to bromo to iodo resulted in a corresponding increase in Src inhibition. Furthermore, replacement of the 7-methoxy group of 2a with various 3-heteroalkylaminopropoxy groups provided increased inhibition of both Src enzymatic and cellular activity. Compound 25, which contains a 3-morpholinopropoxy group, had an IC(50) of 3.8 nM in the Src enzymatic assay and an IC(50) of 940 nM for the inhibition of Src-dependent cell proliferation.

Pyrido[2,3-d]pyrimidin-7-one inhibitors of cyclin-dependent kinases.

The identification of 8-ethyl-2-phenylamino-8H-pyrido[2, 3-d]pyrimidin-7-one (1) as an inhibitor of Cdk4 led to the initiation of a program to evaluate related pyrido[2, 3-d]pyrimidin-7-ones for inhibition of cyclin-dependent kinases (Cdks). Analysis of more than 60 analogues has identified some clear SAR trends that may be exploited in the design of more potent Cdk inhibitors. The most potent Cdk4 inhibitors reported in this study inhibit Cdk4 with IC(50) = 0.004 microM ([ATP] = 25 microM). X-ray crystallographic analysis of representative compounds bound to the related kinase, Cdk2, reveals that they occupy the ATP binding site. Modest selectivity between Cdks is exhibited by some compounds, and Cdk4-selective inhibitors block pRb(+) cells in the G(1)-phase of the cell division cycle.

Inhibitors of src tyrosine kinase: the preparation and structure-activity relationship of 4-anilino-3-cyanoquinolines and 4-anilinoquinazolines.

Src is a nonreceptor tyrosine kinase involved in signaling pathways that control proliferation, migration, and angiogenesis. Increased Src expression and activity are associated with an increase in tumor malignancy and poor prognosis. Several quinolines and quinazolines were identified as potent and selective inhibitors of Src kinase activity.

4-Anilino-6,7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor kinase and their bioisosteric relationship to the 4-anilino-6,7-dialkoxyquinazoline inhibitors.

The synthesis and SAR of a series of 4-anilino-6, 7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor (EGF-R) kinase are described. Condensation of 3, 4-dialkoxyanilines with ethyl (ethoxymethylene)cyanoacetate followed by thermal cyclization gave, regiospecifically, 6,7-dialkoxy-4-oxo-1, 4-dihydroquinoline-3-carbonitriles. Chlorination (POCl(3)) followed by the reaction with substituted anilines furnished the 4-anilino-6, 7-dialkoxyquinoline-3-carbonitrile inhibitors of EGF-R kinase. An alternate synthesis of these compounds starts with a methyl 3, 4-dialkoxybenzoate. Nitration followed by reduction (Fe, NH(4)Cl, MeOH-H(2)O) gave a methyl 2-amino-4,5-dialkoxybenzoate. Amidine formation using DMF-acetal followed by cyclization using LiCH(2)CN furnished a 6,7-dialkoxy-4-oxo-1,4-dihydroquinoline-3-carbonitrile, which was transformed as before. Compounds containing acid, ester, amide, carbinol, and aldehyde groups at the 3-position of the quinoline ring were also prepared for comparison, as were several 1-anilino-6,7-dimethoxyisoquinoline-4-carbonitriles. The compounds were evaluated for their ability to inhibit the autophosphorylation of the catalytic domain of EGF-R. The SAR of these inhibitors with respect to the nature of the 6,7-alkoxy groups, the aniline substituents, and the substituent at the 3-position was studied. The compounds were further evaluated for their ability to inhibit the growth of cell lines that overexpress EGF-R or HER-2. It was found that 4-anilinoquinoline-3-carbonitriles are effective inhibitors of EGF-R kinase with activity comparable to the 4-anilinoquinazoline-based inhibitors. A new homology model of EGF-R kinase was constructed based on the X-ray structures of Hck and FGF receptor-1 kinase. The model suggests that with the quinazoline-based inhibitors, the N3 atom is hydrogen-bonded to a water molecule which, in turn, interacts with Thr 830. It is proposed that the quinoline-3-carbonitriles bind in a similar manner where the water molecule is displaced by the cyano group which interacts with the same Thr residue.

Synthesis and tyrosine kinase inhibitory activity of a series of 2-amino-8H-pyrido[2,3-d]pyrimidines: identification of potent, selective platelet-derived growth factor receptor tyrosine kinase inhibitors.

Screening of a compound library led to the identification of 2-amino-6-(2,6-dichlorophenyl)-8-methylpyrido[2,3-d]pyrimidine (1) as a inhibitor of the platelet-derived growth factor receptor (PDGFr), fibroblast growth factor receptor (FGFr), and c-src tyrosine kinases (TKs). Replacement of the primary amino group at C-2 of 1 with a 4-(N,N-diethylaminoethoxy)phenylamino group yielded 2a, which had greatly increased activity against all three TKs. In the present work, variation of the aromatic group at C-6 and of the alkyl group at N-8 of the pyrido[2,3-d]pyrimidine core provided several analogues that retained potency, including derivatives that were biased toward inhibition of the TK activity of PDGFr. Analogues of 2a with a 3-thiophene or an unsubstituted phenyl group at C-6 were the most potent inhibitors. Compound 54, which had IC50 values of 31, 88, and 31 nM against PDGFr, FGFr, and c-src TK activity, respectively, was active in a variety of PDGF-dependent cellular assays and blocked the in vivo growth of three PDGF-dependent tumor lines.

2-Substituted aminopyrido[2,3-d]pyrimidin-7(8H)-ones. structure-activity relationships against selected tyrosine kinases and in vitro and in vivo anticancer activity.

While engaged in therapeutic intervention against a number of proliferative diseases, we have discovered the 2-aminopyrido[2, 3-d]pyrimidin-7(8H)-ones as a novel class of potent, broadly active tyrosine kinase (TK) inhibitors. An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. From the lead structure 2, a series of analogues bearing variable substituents at the C-2 position and methyl or ethyl at N-8 was made. Compounds of this series were competitive with ATP and displayed submicromolar to low nanomolar potency against a panel of TKs, including receptor (platelet-derived growth factor, PDGFr; fibroblast growth factor, FGFr; epidermal growth factor, EGFr) and nonreceptor (c-Src) classes. One of the more thoroughly evaluated members was 63 with IC50 values of 0.079 microM (PDGFr), 0.043 microM (bFGFr), 0.044 microM (EGFr), and 0.009 microM (c-Src). In cellular studies, 63 inhibited PDGF-mediated receptor autophosphorylation in a number of cell lines at IC50 values of 0.026-0.002 microM and proliferation of two PDGF-dependent lines at 0.3 microM. It also caused inhibition of soft agar colony formation in three cell lines that overexpress the c-Src TK, with IC50 values of 0.33-1.8 microM. In in vivo studies against a panel of seven xenograft tumor models with known and/or inferred dependence on the EGFr, PDGFr, and c-Src TKs, compound 63 produced a tumor growth delay of 10.6 days against the relatively refractory SK-OV-3 ovarian xenograft and also displayed activity against the HT-29 tumor. In rat oral bioavailability studies, compound 63 plasma concentrations declined in a biexponential manner, and systemic plasma clearance was high relative to liver blood flow. Finally, in rat metabolism studies, HPLC chromatography identified two metabolites of 63, which were proved by mass spectrometry and synthesis to be the primary amine (58) and N-oxide (66). Because of the excellent potency of 63 against selected TKs, in vitro and in vivo studies are underway for this compound in additional tumor models dependent upon PDGFr, FGFr, and c-Src to assess its potential for advancement to clinical trials.

Structure-activity relationships for 1-phenylbenzimidazoles as selective ATP site inhibitors of the platelet-derived growth factor receptor.

1-Phenylbenzimidazoles are shown to be a new class of ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR). Structure-activity relationships (SARs) are narrow, with closely related heterocycles being inactive. A systematic study of substituted 1-phenylbenzimidazoles showed clear SARs. Substituents at the 4'- and 3'-positions of the phenyl ring are tolerated but do not significantly improve activity, while substituents at the 2'-position abolish it. Substituents in the 2-, 4-, and 7-positions of the benzimidazole ring (with the exception of 4-OH) also abolish activity. Most substituents at the 5- and 6-positions maintain or increase activity, with the 5-OH, 5-OMe, 5-COMe, and 5-CO2Me analogues being >10-fold more potent than the parent 1-phenylbenzimidazole. The 5-OMe analogue was both the most potent inhibitor, and showed the highest selectivity (50-fold) between PDGFR and FGFR isolated enzymes, and also a moderately effective inhibitor (IC50 = 1.9 microM) of PDGF-stimulated PDGFR autophosphorylation in rat aorta smooth muscle cells.

Inhibition of adhesion molecule expression by N-alkylthiopyridine-benzo[b]thiophene-2-carboxamides.

The surface levels of ICAM-1 and E-selectin on activated endothelial cells can be reduced by 3-alkoxybenzo[b]thiophene-2-carboxamides. This property is shared by several N-alkylthiopyridine substituted imides. Combining structural elements of these two diverse series lead to a new class of small molecule inhibitors of adhesion molecule expression.

Inhibition of E-selectin-, ICAM-1-, and VCAM-1-mediated cell adhesion by benzo[b]thiophene-, benzofuran-, indole-, and naphthalene-2-carboxamides: identification of PD 144795 as an antiinflammatory agent.

It was previously reported that 3-alkoxybenzo[b]thiophene-2-carboxamides exemplified by 1, 5-methoxy-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide, decreased the adherence of neutrophils to activated endothelial cells by inhibiting the upregulation of the adhesion molecules E-selectin and ICAM-1 on the surface of the endothelium. This finding is extended here to a series of 3-thiobenzo[b]thiophene-2-carboxamides and also heterocyclic analogs of 1, including benzofurans, indoles, and napthalenes. The compounds that inhibited the expression of E-selectin and ICAM-1 had the same effect on the expression of VCAM-1. PD 144795, 5-methoxy-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide 1-oxide (44), the sulfoxide analog of 1, was orally active in several models of inflammation. The in vitro and in vivo activity of PD 144795 resided predominately in the S-enantiomer.

Inhibition of tumor necrosis factor induced human aortic endothelial cell adhesion molecule gene expression by an alkoxybenzo[b]thiophene-2-carboxamide.

The effects of a novel anti-inflammatory agent, 5-methoxy-3-(1-methyl-ethoxy)benzo[b]thiophene-2-carboxamide-1-oxide (PD 144795) on adhesion molecule expression in tumor necrosis factor (TNF) stimulated human aortic endothelial cells (HAEC) were examined. PD 144795 treatment markedly inhibited the TNF-induced cell expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein and mRNA. Gel shift assays using nuclear extracts from HAEC treated with PD 144795 failed to show a decrease in the activation of NFkB by this compound, whereas pyrrolidine dithiocarbamate (PDTC), an antioxidant, markedly inhibited the activation of this transcription factor. Thus, PD 144795 inhibits agonist-stimulated VCAM-1 and ICAM-1 expression likely via an NFkB independent mechanism, distinct from that of PDTC. Such agents may provide a novel approach for control of adhesion molecule gene expression in inflammation.

1,3,4-Oxadiazole, 1,3,4-thiadiazole, and 1,2,4-triazole analogs of the fenamates: in vitro inhibition of cyclooxygenase and 5-lipoxygenase activities.

N-Arylanthranilic acids, known generically as the fenamates, are nonsteroidal antiinflammatory drugs (NSAIDs) that block the metabolism of arachidonic acid by the enzyme cyclooxygenase (CO). Substitution of the carboxylic acid functionality of several fenamates with acidic heterocycles provided dual inhibitors of CO and 5-lipoxygenase (5-LO) activities when tested in an intact rat basophilic leukemia (RBL-1) cell line. Compound 5b (IC50 = 0.77 microM (5-LO), 0.27 microM (CO)) which contains an 1,3,4-oxadiazole-2-thione replacement and 10b (IC50 = 0.87 microM (5-LO), 0.85 microM (CO)) which contains a 1,3,4-thiadiazole-2-thione are the most potent inhibitors of 5-LO and CO activities from these series. Both of these heterocyclic analogs of flufenamic acid are also active in carageenin-induced rat footpad edema (CFE), a model of acute inflammation. When dosed orally the ID50s for 5b and 10b in CFE are 8.5 and 4.7 mg/kg, respectively.

Synthesis and biological properties of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid.

The synthesis of the antifolate 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF) has been modified. It is prepared from 2-acetamido-6-formyl-4(3H)-pyrido[2,3-b]pyrimidone and [P-(N-[1,3-bis(ethoxycarbonyl)propan-1-yl]aminocarbonyl)] phenylmethyl]-triphenylphosphonium bromide. The synthesis proceeds via a sodium hydride promoted Wittig condensation in 1-methyl-2-pyrrolidone followed by catalytic reduction, mild base hydrolysis, and acid precipitation of the product. Synthesis of DDATHF is achieved in a total of seven steps from commercially available reagents. DDATHF is transported effectively into CCRF-CEM cells and inhibits growth of both human (CEM) and murine (L1210) cells in culture. Studies reported here support the view that methotrexate and DDATHF are transported via a shared transport mechanism.