Optimization of 5-vinylaryl-3-pyridinecarbonitriles as PKCtheta inhibitors.

Analog 8, a 3-pyridinecarbonitrile with an (E)-2-[6-[(4-methylpiperazin-1-yl)methyl]pyridin-2-yl]vinyl group at C-5, had an IC(50) value of 1.1 nM for the inhibition of PKCtheta and potently blocked the production of IL-2 in both stimulated murine T cells (IC(50)=34 nM) and human whole blood (IC(50)=500 nM).

Optimization of 7-alkene-3-quinolinecarbonitriles as Src kinase inhibitors.

The 7-alkene-3-quinolinecarbonitrile 20, a potent inhibitor of Src enzymatic and cellular activity with IC(50) values of 2.1 and 58 nM, respectively, had comparable efficacy to bosutinib in a colon tumor xenograft study.

4-Anilino-7-pyridyl-3-quinolinecarbonitriles as Src kinase inhibitors.

A series of 4-anilino-7-pyridyl-3-quinolinecarbonitriles was prepared as Src kinase inhibitors. A systematic SAR study of substitutions on both the pyridine ring and the 3-quinolinecarbonitrile core established the requirements for optimal activity. The lead compound, 17, showed potent activity in both the Src enzyme assay and cell assays, and demonstrated in vivo anti-tumor activity in a xenograft model.

Second generation 4-(4-methyl-1H-indol-5-ylamino)-2-phenylthieno[2,3-b]pyridine-5-carbonitrile PKCtheta inhibitors.

Thieno[2,3-b]pyridine-5-carbonitrile 16 with a 4-methyl-5-indolylamine at C-4 and a 5-methoxy-2-(dimethylamino)-methylphenyl group at C-2 had an IC(50) value of 16 nM for the inhibition of PKCtheta. While moderate inhibition of PKCdelta was also observed (IC(50)=130 nM), 16 had IC(50) values of greater than 5 microM against Lyn and other members of the Src kinase family.

C-5 Substituted heteroaryl 3-pyridinecarbonitriles as PKCtheta inhibitors: Part I.

We earlier reported that 3-pyridinecarbonitriiles with a 4-methylindolyl-5-amino group at C-4 and a phenyl group at C-5 were inhibitors of PKCtheta. Keeping the group at C-4 of the pyridine core constant, we varied the water solubilizing group on the phenyl ring at C-5 and then replaced the C-5 phenyl ring with several monocyclic heteroaryl rings, including furan, thiophene and pyridine. Analog 6e with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-2-furyl group C-5 had an IC50 value of 4.5 nM for the inhibition of PKCtheta.

Optimization of 5-phenyl-3-pyridinecarbonitriles as PKCtheta inhibitors.

The key intermediate, 4-chloro-5-iodo-3-pyridinecarbonitrile, allowed for ready optimization of the PKCtheta inhibitory activity of a series of 3-pyridinecarbonitriles. Analog 13b with a 4-methylindol-5-ylamino group at C-4 and a 4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl group at C-5 had an IC(50) value of 7.4nM for the inhibition of PKCtheta.

First generation 5-vinyl-3-pyridinecarbonitrile PKCtheta inhibitors.

A series of 5-vinyl-3-pyridinecarbonitriles were synthesized and evaluated as PKCtheta inhibitors. The systematic optimization of 4-[(4-methyl-1H-indol-5-yl)amino]-5-[(E)-2-phenylvinyl]-3-pyridinecarbonitrile 3 resulted in the identification of compound 23e as a potent PKCtheta inhibitor with good selectivity over PKCdelta.

C-5 substituted heteroaryl-3-pyridinecarbonitriles as PKCtheta inhibitors: part II.

We previously reported that a 3-pyridinecarbonitrile analog with a furan substituent at C-5 and a 4-methylindol-5-ylamino substituent at C-4, 1, was a potent inhibitor of PKCtheta (IC50=4.5 nM). Replacement of the C-5 furan ring of 1 with bicyclic heteroaryl rings, led to compounds with significantly improved potency against PKCtheta. Analog 6b with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-1-benzofuran-2-yl group at C-5 had an IC50 value of 0.28 nM for the inhibition of PKCtheta.

Lead identification to generate 3-cyanoquinoline inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment.

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.

Synthesis, SAR study and biological evaluation of novel pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides as anti-proliferative agents.

Checkpoint deficiency of malignant cells can be exploited in cancer drug discovery. Compounds that selectively kill checkpoint-deficient cells versus checkpoint-proficient cells can be utilized to preferentially target tumor cells, while sparing normal cells. The protein p21(Wafl/Cipl/Sdi1) (hereafter referred to as p21) inhibits progression of the cell cycle by inhibiting the activity of G1 kinases (cyclin D/cdk4 and cyclin E-cdk2) and the G2 kinase (cyclin B/cdkl) in response to DNA damage or abnormal DNA content. The expression of p21 is often low in human cancer cells due to frequent loss of the upstream activator, p53, and is associated with poor prognosis in some cancer patients. Using an isogenic pair of cell lines, HCT116 (p21+/+) and 80S14 (p21-/-), we have disclosed previously a novel series of pyrazolo[1,5-a]pyrimidines that preferentially kill the p21-deficient cells. We will present the synthesis, biological activities and SAR study of a series of pyrazolo[1,5-a]pyrimidines with an optimized phenyl amide moiety at the C-7 position. The mechanism of action of these compounds will also be discussed.

Synthesis and PKCtheta inhibitory activity of a series of 4-(indol-5-ylamino)thieno[2,3-b]pyridine-5-carbonitriles.

The thieno[2,3-b]pyridine-5-carbonitrile with a 5-indolylamine at C-4 and a phenyl group at C-2 had a moderate activity against PKCtheta. Optimization of the groups at C-4 and C-2 led to analog 29, which has an IC(50) value of 7.5nM for the inhibition of PKCtheta.

Facile preparation of new 4-phenylamino-3-quinolinecarbonitrile Src kinase inhibitors via 7-fluoro intermediates: identification of potent 7-amino analogs.

A more efficient preparation of 4-[(2,4-dichloro-5-methoxyphenyl)amino]-7-fluoro-6-methoxy-3-quinolinecarbonitrile (2), the penultimate intermediate in the synthesis of bosutinib (1a), was developed. New 7-alkoxy-4-phenylamino-3-quinolinecarbonitrile Src inhibitors were prepared from 5 and 9, the 6-ethoxy and 6-hydrogen analogs of 2. In addition, the fluoro group of 2 was readily displaced by primary and secondary amines to give 7-amino analogs. Two of these 7-amino analogs, 15 and 18, were potent Src inhibitors with in vivo activity.

Synthesis and Src kinase inhibitory activity of a series of 4-[(2,4-dichloro-5-methoxyphenyl)amino]-7-furyl-3-quinolinecarbonitriles.

Compound 1 (SKI-606, bosutinib), a 7-alkoxy-4-[(2,4-dichloro-5-methoxyphenyl)amino]-3-quinolinecarbonitrile, is a potent inhibitor of Src kinase activity. We previously reported that analogs of 1 with thiophene groups at C-7 retained the Src activity of the parent compound. The corresponding C-7 furan analogs were prepared and it was found that the 3,5-substituted furan analog had increased activity compared to that of the 2,5-substituted furan isomer. Addition of a methoxy group at C-6 decreased the Src inhibitory activity of the C-7 2,5-substituted furan analog but increased the activity of the C-7 3,5-substituted furan isomer. This compound, 10, was a more potent Src inhibitor than 1 in both enzymatic and cell-based assays. The kinase selectivity profile of 10 was similar to that of 1, with 10 also inhibiting the activity of Abl and Lck. When tested in a solid tumor xenograft model, 10 had comparable oral activity to that of 1.

Synthesis and Src kinase inhibitory activity of 2-phenyl- and 2-thienyl-7-phenylaminothieno[3,2-b]pyridine-6-carbonitriles.

2-phenyl-7-phenylaminothieno[3,2-b]pyridine-6-carbonitriles were recently reported to be inhibitors of Src kinase activity. In this study we present structure-activity relationships for additional thieno[3,2-b]pyridine-6-carbonitriles, modifying the substituents on the C-2 phenyl and C-7 phenylamino groups. Derivatives with various aminomethyl and aminoethyl substituents on the para position of the C-2 phenyl group retained the activity of the initial analogues. However, direct attachment of an amino group led to decreased activity. A 2,4-dichloro-5-methoxyphenylamino group at C-7 provided superior inhibition of Src enzymatic activity. Replacement of the C-2 phenyl group with a 3,5-substituted thiophene led to improved Src inhibitory activity compared to the parent compound, but other thiophene isomers were less active. One of the analogues reported here exhibited in vivo activity comparable to that of SKI-606, a related 3-quinolinecarbonitrile currently in clinical trials.

Identification of 4-anilino-3-quinolinecarbonitrile inhibitors of mitogen-activated protein/extracellular signal-regulated kinase 1 kinase.

A high-throughput screen for Ras-mitogen-activated protein kinase (MAPK) signaling inhibitors identified two series (class 1 and 2) of substituted 4-anilino-3-quinolinecarbonitriles as potent (IC(50)s <10 nmol/L) mitogen-activated protein/extracellular signal-regulated kinase 1 (MEK1) kinase inhibitors. These compounds had cyanoquinoline cores, but differed in their respective aniline groups [1a, 1b: 4-phenoxyphenylaniline; 2a, 2b: 3-chloro-4-(1-methylimidazol-2-sulfanyl)aniline]. These compounds were competitive inhibitors of ATP binding by MEK1 kinase, and they had minimal or no effect on Raf, epidermal growth factor receptor (EGFR), Akt, cyclin-dependent kinase 4 (CDK4), or MK2 kinases at concentrations >100-fold higher than those that inhibited MEK1 kinase. Both class 1 and 2 compounds inhibited in vitro growth of human tumor cell lines. A class 2 compound (2b) was the most potent inhibitor of human tumor cell growth in vitro, and this effect was linked to distinct suppression of MAPK phosphorylation in cells. Compound 2b did not affect phosphorylation status of other kinases, such as EGFR, Akt, and stress-activated protein (SAP)/c-jun-NH kinase (Jnk); nor did it affect overall tyrosine phosphorylation level in cells. However, compound 2b did inhibit MEK1 phosphorylation in cells. Inhibition of MEK1 phosphorylation by 2b was not due to a major effect on Raf kinase activity, because enzyme assays showed minimal Raf kinase inhibition. We believe compound 2b inhibits kinase activity upstream of Raf, and thereby affects MEK1 phosphorylation in cells. Even with the dual effect of 2b on MEK and MAPK phosphorylation, this compound was well tolerated and significantly inhibited growth of the human colon tumor cell line LoVo (at 50 and 100 mg/kg BID, i.p.) in a nude mouse xenograft model.

Identification of 7-phenylaminothieno- [3,2-b]pyridine-6-carbonitriles as a new class of Src kinase inhibitors.

We disclose here a new class of kinase inhibitors, obtained by replacing the phenyl ring of a 3-quinolinecarbonitrile system with a thiophene ring. When suitably substituted, the resultant 7-phenylaminothieno[3,2-b]pyridine-6-carbonitrile analogues show potent inhibition of Src kinase activity.

7-Alkoxy-4-phenylamino-3-quinolinecar-bonitriles as dual inhibitors of Src and Abl kinases.

We previously reported that several 7-alkoxy-4-phenylamino-3-quinolinecarbonitriles were potent inhibitors of Src kinase activity. We disclose here a new highly efficient and versatile route to these compounds, which are also potent inhibitors of Abl kinase.

7-(aryl/heteroaryl-2-ylethynyl)-4-phenylamino-3-quinolinecarbonitriles as new Src kinase inhibitors: addition of water solubilizing groups.

New 4-phenylamino-3-quinolinecarbonitriles with a 7-ethynyl group substituted by a pyridine, phenyl or thiophene ring containing basic water solubilizing groups were prepared and evaluated as Src kinase inhibitors. Of these new analogs, potent activity was observed with compounds having a (2,4-dichloro-5-methoxyphenyl)amino group at C-4, a methoxy or ethoxy group at C-6, and a pyridyl group bearing a dimethylamine or N-methylpiperazine on the ethynyl group at C-7.

Further studies on ethenyl and ethynyl-4-phenylamino-3-quinolinecarbonitriles: identification of a subnanomolar Src kinase inhibitor.

Several new ethynyl- and ethenyl-4-phenylamino-3-quinolinecarbonitriles were synthesized and tested for Src inhibition. Derivatives bearing an ethenyl or ethynyl substituent at C-6 showed decreased Src inhibitory activity. Incorporation of an ethenylpyridine N-oxide group at C-7 provided 20b, a 0.6 nM inhibitor of Src enzymatic activity with excellent cellular potency.

Inhibition of Src kinase activity by 7-[(2,4-dichloro-5-methoxyphenyl)amino]-2-heteroaryl-thieno[3,2-b]pyridine-6-carbonitriles.

7-[(2,4-Dichloro-5-methoxyphenyl)amino]thieno[3,2-b]pyridine-6-carbonitriles with various heteroaryl groups at C-2 are inhibitors of Src kinase activity. Of these new analogs, compounds substituted at C-2 by a 3,5-furan or a 2,5-pyridine had the best activity in the Src enzyme and cell assays.