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[Image: see text] Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase involved in the regulation of transcription elongation. An inhibition of CDK9 downregulates a number of short-lived proteins responsible for tumor maintenance and survival, including the antiapoptotic BCL-2 family member MCL-1. As pan-CDK inhibitors under development have faced dosing and toxicity challenges in the clinical setting, we generated selective CDK9 inhibitors that could be amenable to an oral administration. Here, we report the lead optimization of a series of azaindole-based inhibitors. To overcome early challenges with promiscuity and cardiovascular toxicity, carboxylates were introduced into the pharmacophore en route to compounds such as 14 and 16. These CDK9 inhibitors demonstrated a reduced toxicity, adequate pharmacokinetic properties, and a robust in vivo efficacy in mice upon oral dosing.

Aided by molecular modeling, compounds with a pyrimidine-based tricyclic scaffold were designed and confirmed to inhibit Wee1 kinase. Structure-activity studies identified key pharmacophores at the aminoaryl and halo-benzene regions responsible for binding affinity with sub-nM K i values. The potent inhibitors demonstrated sub-μM activities in both functional and mechanism-based cellular assays and also possessed desirable pharmacokinetic profiles. The lead molecule, 31, showed oral efficacy in potentiating the antiproliferative activity of irinotecan, a cytotoxic agent, in a NCI-H1299 mouse xenograft model.

To investigate the role played by the unique pre-DFG residue Val 195 of Cdc7 kinase on the potency of azaindole-chloropyridines (1), a series of novel analogues with various chloro replacements were synthesized and evaluated for their inhibitory activity against Cdc7. X-ray cocrystallization using a surrogate protein, GSK3β, and modeling studies confirmed the azaindole motif as the hinge binder. Weaker hydrophobic interactions with Met 134 and Val 195 by certain chloro replacements (e.g., H, methyl) led to reduced Cdc7 inhibition. Meanwhile, data from other replacements (e.g., F, O) indicated that loss of such hydrophobic interaction could be compensated by enhanced hydrogen bonding to Lys 90. Our findings not only provide an in-depth understanding of the pre-DFG residue as another viable position impacting kinase inhibition, they also expand the existing knowledge of ligand-Cdc7 binding.

We have investigated the SAR of a series of pyrimidinone-containing Cdc7 kinase inhibitors. A wide range of amine substitutions give potent compounds with activities (K(i)) less than 1nM. Kinase selectivity is reasonable and cytotoxicity corresponds to inhibition of MCM2 phosphorylation.

Small molecule inhibitors of PARP-1 have been pursued by various organizations as potential therapeutic agents either capable of sensitizing cytotoxic treatments or acting as stand-alone agents to combat cancer. As one of the strategies to expand our portfolio of PARP-1 inhibitors, we pursued unsaturated heterocycles to replace the saturated cyclic amine derivatives appended to the benzimidazole core. Not only did a variety of these new generation compounds maintain high enzymatic potency, many of them also displayed robust cellular activity. For example, the enzymatic IC(50) and cellular EC(50) values were as low as 1 nM or below. Compounds 24 (EC(50) = 3.7 nM) and 44 (EC(50) = 7.8 nM), featuring an oxadiazole and a pyridine moiety, respectively, demonstrated balanced potency and PK profiles. In addition, these two molecules exhibited potent oral in vivo efficacy in potentiating the cytotoxic agent temozolomide in a B16F10 murine melanoma model.

A variety of macrocyclic urea compounds were prepared as potent Chk1 inhibitors by modifying the C5 position of the benzene ring of the macrocyclic urea with ether moieties, aliphatic carbon chains, amide and halides. Enzymatic activity less than 20 nM was observed in 29 of 40 compounds. Compounds 14 , 46d , and 48j provided the best overall results in the cellular assays as they abrogated doxorubicin-induced cell cycle arrest (IC 50 = 3.31, 3.08, and 3.13 μM) and enhanced doxorubicin cytotoxicity (IC 50 = 0.54, 1.27, and 0.96 μM) while displaying no single agent activity, respectively.

A series of 1,4-dihydroindeno[1,2-c]pyrazole compounds with a cyanopyridine moiety at the 3-position of the tricyclic pyrazole core was explored as potent CHK-1 inhibitors. The impact of substitutions at the 6 and/or 7-position of the core on pharmacokinetic properties was studied in detail. Compounds carrying a side chain with an ether linker at the 7-position and a terminal morpholino group, such as 29 and 30, exhibited much-improved oral biovailability in mice as compared to earlier generation inhibitors. These compounds also possessed desirable cellular activity in potentiating doxorubicin and will serve as valuable tool compounds for in vivo evaluation of CHK-1 inhibitors to sensitize DNA-damaging agents.

A study on substitutions at the four open positions on the phenyl ring of the 1,4-dihydroindeno[1,2-c]pyrazoles as potent CHK-1 inhibitors is described. Bis-substitution at both the 6- and 7-positions led to inhibitors with IC(50) values below 0.3nM. The compound with the best overall activities (36) was able to potentiate the anti-proliferative effect of doxorubicin in HeLa cells by at least 47-fold. Physicochemical, metabolic, and pharmacokinetic properties of selected inhibitors are also disclosed.

A new class of checkpoint kinase 1 (CHK-1) inhibitors bearing a 1,4-dihydroindeno[1,2-c]pyrazole core was developed after initial hits from high throughput screening. The efficient hit-to-lead process was facilitated by X-ray crystallography and led to potent inhibitors (

A pyridyl moiety was introduced into a previously developed series of farnesyltransferase inhibitors containing imidazole and cyanophenyl (such as 4), resulting in potent inhibitors with improved pharmacokinetics.

Two constrained analogs of the tripeptide hormone thyroliberin (TRH) have been synthesized. In both, the HisPro peptide bond of the hormone has been ‘locked’ into a cis -conformation. In the first analog, the constraint used was identical to the constraint used in an earlier trans -peptide bond analog that was a potent agonist for the TRH endocrine receptor TRH-R 1 . The second analog was built in order to take advantage of a tetrazole ring as the cis -peptide bond constraint. Neither analog showed agonist or antagonist behavior toward TRH-R 1 suggesting that the cis -peptide bond conformation may not play a role in the affinity of TRH for TRH-R 1 .

A series of isoxazolo[3,4-b]quinoline-3,4(1H,9H)-diones were synthesized as potent inhibitors against Pim-1 and Pim-2 kinases. The structure–activity-relationship studies started from a high-throughput screening hit and was guided by molecular modeling of inhibitors in the active site of Pim-1 kinase. Installing a hydroxyl group on the benzene ring of the core has the potential to form a key hydrogen bond interaction to the hinge region of the binding pocket and thus resulted in the most potent inhibitor, 19, with Ki values at 2.5 and 43.5 nM against Pim-1 and Pim-2, respectively. Compound 19 also exhibited an activity profile with a high degree of kinase selectivity.

An extensive structure-activity relationship study of the 3-position of a series of tricyclic pyrazole-based Chk1 inhibitors is described. As a result, 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles (4) and 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-2'-carbonitriles (29) emerged as new lead series. Compared with the original lead compound 2, these new leads fully retain the biological activity in both enzymatic inhibition and cell-based assays. More importantly, the new leads 4 and 29 exhibit favorable physicochemical properties such as lower molecular weight, lower Clog P, and the absence of a hydroxyl group. Furthermore, structure-activity relationship studies were performed at the 6- and 7-positions of 4, which led to the identification of ideal Chk1 inhibitors 49, 50, 51, and 55. These compounds not only potently inhibit Chk1 in an enzymatic assay but also significantly potentiate the cytotoxicity of DNA-damaging agents in cell-based assays while they show little single agent activity. A cell cycle analysis by FACS confirmed that these Chk1 inhibitors efficiently abrogate the G2/M and S checkpoints induced by DNA-damaging agent. The current work paved the way to the identification of several potent Chk1 inhibitors with good pharmacokinetics that are suitable for in vivo study with oral dosing.

A new series of potent tricyclic pyrazole-based Chk1 inhibitors are described. Analogues disubstituted on the 6- and 7-positions show improved Chk1 inhibition potency compared with analogues with a single substituent on either the 6- or 7-position. Based on the lead compound 4'-(6,7-dimethoxy-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol (2), detailed SAR studies on the 6- and 7-positions were performed. 3'-morpholin-4'-yl-propoxy, pyridin-4'-ylmethoxy, pyridin-3'-ylmethoxy, 2'-(5''-ethyl-pyridin-2''-yl)-ethoxy, pyridin-2'-ylethoxy, (6'-methyl-pyridin-2'-yl)-propoxyethoxy, 2',3'-dihydroxyl-1'-yl-propoxy, and tetrahydro-furan-3'-yloxy have been identified as the best groups on the 6-position when the 7-position is substituted with methoxyl group. Pyridin-2'-ylmethoxy and pyridin-3'-ylmethoxy have been identified as the best substituents at the 7-position while the 6-position bearing methoxyl group. These compounds significantly potentiate the cytotoxicity of DNA-damaging antitumor agents in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and the camptothecin-induced S checkpoints, suggesting that their potent biological activities are mechanism-based through Chk1 inhibition.

All authors are employees of AbbVie and participated in the design, conduct, and interpretation of these studies. AbbVie and Genentech provided financial support for these studies and participated in the review and approval of this publication. The BCL-2-selective inhibitor ABT-199 has demonstrated efficacy in numerous preclinical models of hematologic malignancies without causing thrombocytopenia, a dose-limiting toxicity associated with the BCL-2/BCL-XL inhibitor navitoclax (Souers et al. 2013. Nat. Med. 19, 202-208). ABT-199 has also demonstrated clinical activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) (Seymour et al. 2014. J. Clin. Oncol. 32, 448s; Davids et al. 2014. J. Clin. Oncol. 32, 544s). Despite these encouraging early clinical data, some subjects do not respond to ABT-199 or progress while on treatment. Pre-clinical models indicate that both intrinsic and acquired resistance may be a consequence of MCL-1 expression. Consequently, we have explored potent and selective small molecule inhibitors of CDK9, a kinase known to maintain the expression of MCL-1 through its role in p-TEFb-mediated transcription. Inhibition of CDK9 resulted in the rapid loss in RNA polymerase II phosphorylation (Serine 5) and MCL-1 expression that was closely followed by the induction of apoptosis in MCL-1-dependent cell lines, a cellular response that could be rescued by overexpression of BCL-2. Substantial synergy was observed between CDK9 inhibitors and ABT-199 in a number of hematologic cell lines with intrinsic or acquired resistance to ABT-199. Direct inhibition of MCL-1 with the small molecule BH3 mimetic A-1210477 was also highly synergistic with ABT-199, further validating the utility of co-inhibiting MCL-1 and BCL-2 function simultaneously in ABT-199 resistant tumors. Importantly, the CDK9 inhibitor-ABT-199 combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in xenograft models of non-Hodgkin’s lymphoma (NHL) and acute myelogenous leukemia (AML). These data indicate that CDK9 inhibitors may be highly efficacious when used in combination with ABT-199 for the treatment of hematologic malignancies. Disclosures Chen: Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Tapang:abbvie: Employment, Equity Ownership. Tahir:abbvie: Employment, Equity Ownership. Smith:abbvie: Employment, Equity Ownership. Xue:abbvie: Employment, Equity Ownership. Zhang:abbvie: Employment, Equity Ownership. Gao:abbvie: Employment, Equity Ownership. Tong:abbvie: Employment, Equity Ownership. Clark:abbvie: Employment, Equity Ownership. Ricker:abbvie: Employment, Equity Ownership. Penning:abbvie: Employment, Equity Ownership. Albert:abbvie: Employment, Equity Ownership. Phillips:abbvie: Employment, Equity Ownership. Souers:abbvie: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership.

Three substance P analogs with conformation constraints in the Phe7-Phe8 region have been prepared in connection with an effort to differentiate two families of potential conformations for the binding of substance P to its NK1 receptor. While the analogs did not bind the NK1 receptor with high affinity, the synthesis of the analogs demonstrated the utility of a general method for constructing piperazinone based peptidomimetics.