Investigation of biaryl heterocycles as inhibitors of Wee1 kinase.

In continuation of our previous research towards the discovery of potent, selective and drug-like Wee1 inhibitors, 2 novel series of biaryl heterocycles were designed, synthesized and evaluated. The new biaryl cores were designed to enable structure-activity exploration of substituents at C-8 or N-8 which were used for tuning compound properties and to improve compound profiles. The lead molecule 33 demonstrated a desirable pharmacokinetic profile and potentiated the anti-proliferative activity of irinotecan in vivo when dosed orally in the human breast MX-1 xenograft model.

Aminopyrimidinone cdc7 kinase inhibitors.

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.

Hit to Lead optimization of a novel class of squarate-containing polo-like kinases inhibitors.

A high throughput screening (HTS) hit, 1 (Plk1 K(i)=2.2 µM) was optimized and evaluated for the enzymatic inhibition of Plk-1 kinase. Molecular modeling suggested the importance of adding a hydrophobic aromatic amine side chain in order to improve the potency by a classic kinase H-donor-acceptor binding mode. Extensive SAR studies led to the discovery of 49 (Plk1 K(i)=5 nM; EC(50)=1.05 µM), which demonstrated moderate efficacy at 100 mpk in a MiaPaCa tumor model, with no overt toxicity.

Discovery and SAR of orally efficacious tetrahydropyridopyridazinone PARP inhibitors for the treatment of cancer.

PARP-1, the most abundant member of the PARP superfamily of nuclear enzymes, has emerged as a promising molecular target in the past decade particularly for the treatment of cancer. A number of PARP-1 inhibitors, including veliparab discovered at Abbott, have advanced into different stages of clinical trials. Herein we describe the development of a new tetrahydropyridopyridazinone series of PARP-1 inhibitors. Many compounds in this class, such as 20w, displayed excellent potency against the PARP-1 enzyme with a K(i) value of <1nM and an EC(50) value of 1nM in a C41 whole cell assay. The presence of the NH in the tetrahydropyridyl ring of the tetrahydropyridopyridazinone scaffold improved the pharmacokinetic properties over similar carbon based analogs. Compounds 8c and 20u are orally available, and have demonstrated significant efficacy in a B16 murine xenograft model, potentiating the efficacy of temozolomide (TMZ).

PARP inhibitors attenuate chemotherapy-induced painful neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major toxicity of chemotherapy treatment for which no therapy is approved. Poly(ADP-ribose) polymerase (PARP)1/2 are nuclear enzymes activated upon DNA damage, and PARP1/2 inhibition provides resistance against DNA damage. A role for PARP inhibition in sensory neurotransmission has also been established. PARP inhibitors attenuate pain-like behaviors and neuropathy-associated decreased peripheral nerve function in diabetic models. The hypothesis tested was that PARP inhibition protects against painful neuropathy. The objective of this study was to investigate whether the novel, selective PARP1/2 inhibitors (ABT-888 and related analogues) would attenuate development of mechanical allodynia in vincristine-treated rats. PARP inhibitors were dosed for 2 days, and then co-administered with vincristine for 12 days. Mechanical allodynia was observed in rats treated with vincristine. PARP1/2 inhibition significantly attenuated development of mechanical allodynia and reduced poly ADP-ribose (PAR) activation in rat skin. The data presented here show that PARP inhibition attenuates vincristine-induced mechanical allodynia in rats, and supports that PARP inhibition may represent a novel therapeutic approach for CIPN.

Balancing Properties with Carboxylates: A Lead Optimization Campaign for Selective and Orally Active CDK9 Inhibitors.

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.

Discovery and SAR of substituted 3-oxoisoindoline-4-carboxamides as potent inhibitors of poly(ADP-ribose) polymerase (PARP) for the treatment of cancer.

Through conformational restriction of a benzamide by formation of a seven-membered hydrogen-bond with an oxindole carbonyl group, a series of PARP inhibitors was designed for appropriate orientation for binding to the PARP surface. This series of compounds with a 3-oxoisoindoline-4-carboxamide core structure, displayed modest to good activity against PARP-1 in both intrinsic and cellular assays. SAR studies at the lactam nitrogen of the pharmacophore have suggested that a secondary or tertiary amine is important for cellular potency. An X-ray structure of compound 1e bound to the protein confirmed the formation of a seven-membered intramolecular hydrogen bond. Though revealed previously in peptides, this type of seven-membered intramolecular hydrogen bond is rarely observed in small molecules. Largely due to the formation of the intramolecular hydrogen bond, the 3-oxoisoindoline-4-carboxamide core structure appears to be planar in the X-ray structure. An additional hydrogen bond interaction of the piperidine nitrogen to Gly-888 also contributes to the binding affinity of 1e to PARP-1.

A novel CDK9 inhibitor increases the efficacy of venetoclax (ABT-199) in multiple models of hematologic malignancies.

MCL-1 is one of the most frequently amplified genes in cancer, facilitating tumor initiation and maintenance and enabling resistance to anti-tumorigenic agents including the BCL-2 selective inhibitor venetoclax. The expression of MCL-1 is maintained via P-TEFb-mediated transcription, where the kinase CDK9 is a critical component. Consequently, we developed a series of potent small-molecule inhibitors of CDK9, exemplified by the orally active A-1592668, with CDK selectivity profiles that are distinct from related molecules that have been extensively studied clinically. Short-term treatment with A-1592668 rapidly downregulates RNA pol-II (Ser 2) phosphorylation resulting in the loss of MCL-1 protein and apoptosis in MCL-1-dependent hematologic tumor cell lines. This cell death could be attenuated by either inhibiting caspases or overexpressing BCL-2 protein. Synergistic cell killing was also observed between A-1592668 or the related analog A-1467729, and venetoclax in a number of hematologic cell lines and primary NHL patient samples. Importantly, the CDK9 inhibitor plus venetoclax combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in mouse models of lymphoma and AML. These data indicate that CDK9 inhibitors could be highly efficacious in tumors that depend on MCL-1 for survival or when used in combination with venetoclax in malignancies dependent on MCL-1 and BCL-2.

Potentiation of temozolomide cytotoxicity by poly(ADP)ribose polymerase inhibitor ABT-888 requires a conversion of single-stranded DNA damages to double-stranded DNA breaks.

Poly(ADP-ribose) polymerase (PARP) senses DNA breaks and facilitates DNA repair via the polyADP-ribosylation of various DNA binding and repair proteins. We explored the mechanism of potentiation of temozolomide cytotoxicity by the PARP inhibitor ABT-888. We showed that cells treated with temozolomide need to be exposed to ABT-888 for at least 17 to 24 hours to achieve maximal cytotoxicity. The extent of cytotoxicity correlates with the level of double-stranded DNA breaks as indicated by gammaH2AX levels. In synchronized cells, damaging DNA with temozolomide in the presence of ABT-888 during the S phase generated high levels of double-stranded breaks, presumably because the single-stranded DNA breaks resulting from the cleavage of the methylated nucleotides were converted into double-stranded breaks through DNA replication. As a result, treatment of temozolomide and ABT-888 during the S phase leads to higher levels of cytotoxicity. ABT-888 inhibits poly(ADP-ribose) formation in vivo and enhances tumor growth inhibition by temozolomide in multiple models. ABT-888 is well tolerated in animal models. ABT-888 is currently in clinical trials in combination with temozolomide.

Synthesis and SAR of novel tricyclic quinoxalinone inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1).

Based on screening hit 1, a series of tricyclic quinoxalinones have been designed and evaluated for inhibition of PARP-1. Substitutions at the 7- and 8-positions of the quinoxalinone ring led to a number of compounds with good enzymatic and cellular potency. The tricyclic quinoxalinone class is sensitive to modifications of both the amine substituent and the tricyclic core. The synthesis and structure-activity relationship studies are presented.

Isoxazolo[3,4-b]quinoline-3,4(1H,9H)-diones as unique, potent and selective inhibitors for Pim-1 and Pim-2 kinases: chemistry, biological activities, and molecular modeling.

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 K(i) 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.

Synthesis and SAR of novel, potent and orally bioavailable benzimidazole inhibitors of poly(ADP-ribose) polymerase (PARP) with a quaternary methylene-amino substituent.

Poly(ADP-ribose) polymerases (PARPs) play significant roles in various cellular functions including DNA repair and control of RNA transcription. PARP inhibitors have been demonstrated to potentiate the effect of cytotoxic agents or radiation in a number of animal tumor models. Utilizing a benzimidazole carboxamide scaffold in which the amide forms a key intramolecular hydrogen bond for optimal interaction with the enzyme, we have identified a novel series of PARP inhibitors containing a quaternary methylene-amino substituent at the C-2 position of the benzimidazole. Geminal dimethyl analogs at the methylene-amino substituent were typically more potent than mono-methyl derivatives in both intrinsic and cellular assays. Smaller cycloalkanes such as cyclopropyl or cyclobutyl were tolerated at the quaternary carbon while larger rings were detrimental to potency. In vivo efficacy data in a B16F10 murine flank melanoma model in combination with temozolomide (TMZ) are described for two optimized analogs.

The PARP inhibitor, ABT-888 potentiates temozolomide: correlation with drug levels and reduction in PARP activity in vivo.

ABT-888 is a potent, orally bioavailable PARP-1/2 inhibitor shown to potentiate DNA damaging agents. The ability to potentiate temozolomide (TMZ) and develop a biological marker for PARP inhibition was evaluated in vivo. Doses/schedules that achieve TMZ potentiation in the B16F10 syngeneic melanoma model were utilized to develop an ELISA to detect a pharmacodynamic marker, ADP ribose polymers (pADPr), after ABT 888 treatment. ABT-888 enhanced TMZ antitumor activity, in a dose-proportional manner with no observed toxicity (44-75% tumor growth inhibition vs. TMZ monotherapy), but did not show single agent activity. Extended ABT-888 dosing schedules showed no advantage compared to simultaneous TMZ administration. Efficacy correlated with plasma/tumor drug concentrations. Intratumor drug levels correlated with a dose-proportional/time-dependent reduction in pADPr. Potentiation of TMZ activity by ABT-888 correlated with drug levels and inhibition of PARP activity in vivo. ABT-888 is in Phase 1 trials using a validated ELISA based on the assay developed here to assess pharmacological effect.

Discovery and SAR of 2-(1-propylpiperidin-4-yl)-1H-benzimidazole-4-carboxamide: A potent inhibitor of poly(ADP-ribose) polymerase (PARP) for the treatment of cancer.

We have developed a series of cyclic amine-containing benzimidazole carboxamide poly(ADP-ribose)polymerase (PARP) inhibitors, with good PARP-1 enzyme potency, as well as cellular potency. These efforts led to the identification of a lead preclinical candidate, 10b, 2-(1-propylpiperidin-4-yl)-1H-benzimidazole-4-carboxamide (A-620223). 10b displayed very good potency against both the PARP-1 enzyme with a K(i) of 8nM and in a whole cell assay with an EC(50) of 3nM. 10b is aqueous soluble, orally bioavailable across multiple species, and demonstrated good in vivo efficacy in a B16F10 subcutaneous murine melanoma model in combination with temozolomide (TMZ) and in an MX-1 breast xenograph model in combination with cisplatin.

ABT-888, an orally active poly(ADP-ribose) polymerase inhibitor that potentiates DNA-damaging agents in preclinical tumor models.

PURPOSE: To evaluate the preclinical pharmacokinetics and antitumor efficacy of a novel orally bioavailable poly(ADP-ribose) polymerase (PARP) inhibitor, ABT-888. EXPERIMENTAL DESIGN: In vitro potency was determined in a PARP-1 and PARP-2 enzyme assay. In vivo efficacy was evaluated in syngeneic and xenograft models in combination with temozolomide, platinums, cyclophosphamide, and ionizing radiation. RESULTS: ABT-888 is a potent inhibitor of both PARP-1 and PARP-2 with K(i)s of 5.2 and 2.9 nmol/L, respectively. The compound has good oral bioavailability and crosses the blood-brain barrier. ABT-888 strongly potentiated temozolomide in the B16F10 s.c. murine melanoma model. PARP inhibition dramatically increased the efficacy of temozolomide at ABT-888 doses as low as 3.1 mg/kg/d and a maximal efficacy achieved at 25 mg/kg/d. In the 9L orthotopic rat glioma model, temozolomide alone exhibited minimal efficacy, whereas ABT-888, when combined with temozolomide, significantly slowed tumor progression. In the MX-1 breast xenograft model (BRCA1 deletion and BRCA2 mutation), ABT-888 potentiated cisplatin, carboplatin, and cyclophosphamide, causing regression of established tumors, whereas with comparable doses of cytotoxic agents alone, only modest tumor inhibition was exhibited. Finally, ABT-888 potentiated radiation (2 Gy/d x 10) in an HCT-116 colon carcinoma model. In each model, ABT-888 did not display single-agent activity. CONCLUSIONS: ABT-888 is a potent inhibitor of PARP, has good oral bioavailability, can cross the blood-brain barrier, and potentiates temozolomide, platinums, cyclophosphamide, and radiation in syngeneic and xenograft tumor models. This broad spectrum of chemopotentiation and radiopotentiation makes this compound an attractive candidate for clinical evaluation.

Syntheses of potent, selective, and orally bioavailable indazole-pyridine series of protein kinase B/Akt inhibitors with reduced hypotension.

Compound 7 was identified as a potent (IC50 = 14 nM), selective, and orally bioavailable (F = 70% in mouse) inhibitor of protein kinase B/Akt. While promising efficacy was observed in vivo, this compound showed effects on depolarization of Purkinje fibers in an in vitro assay and CV hypotension in vivo. Guided by an X-ray structure of 7 bound to protein kinase A, which has 80% homology with Akt in the kinase domain, our efforts have focused on structure-activity relationship (SAR) studies of the phenyl moiety, in an attempt to address the cardiovascular liability and further improve the Akt potency. A novel and efficient synthetic route toward diversely substituted phenyl derivatives of 7 was developed utilizing a copper-mediated aziridine ring-opening reaction as the key step. To improve the selectivity of these Akt inhibitors over other protein kinases, a nitrogen atom was incorporated into selected phenyl analogues of 7 at the C-6 position of the methyl indazole scaffold. These modifications resulted in the discovery of inhibitor 37c with greater potency (IC50 = 0.6 nM vs Akt), selectivity, and improved cardiovascular safety profile. The SARs, pharmacokinetic profile, and CV safety of selected Akt inhibitors will be discussed.

Pyrimidine-based tricyclic molecules as potent and orally efficacious inhibitors of wee1 kinase.

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.

Synthesis of potent leukotriene A(4) hydrolase inhibitors. Identification of 3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid.

Leukotriene B(4) (LTB(4)) is a potent, proinflammatory mediator involved in the pathogenesis of a number of diseases including inflammatory bowel disease, psoriasis, rheumatoid arthritis, and asthma. The enzyme LTA(4) hydrolase represents an attractive target for pharmacological intervention in these disease states, since the action of this enzyme is the rate-limiting step in the production of LTB(4). Our previous efforts focused on the exploration of a series of analogues related to screening hit SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) and resulted in the identification of potent, orally active inhibitors such as 2. Additional structure-activity relationship studies around this structural class resulted in the identification of a series of alpha-, beta-, and gamma-amino acid analogues that are potent inhibitors of the LTA(4) hydrolase enzyme and demonstrated good oral activity in a mouse ex vivo whole blood LTB(4) production assay. The efforts leading to the identification of clinical candidate SC-57461A (8d, 3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid) are described.

Azaindole-Based Inhibitors of Cdc7 Kinase: Impact of the Pre-DFG Residue, Val 195.

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, GSK3ß, 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.