Design and optimization of (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones as potent PLK4 inhibitors with oral antitumor efficacy.

Previous efforts from our laboratory demonstrated that (E)-3-((3-(E)-vinylaryl)-1H-indazol-6-yl)methylene)-indolin-2-ones are potent PLK4 inhibitors with in vivo anticancer efficacy upon IP dosing. As part of a continued effort to develop selective and orally efficacious inhibitors, we examined variations on this theme wherein 'directly-linked' aromatics, pendant from the indazole core, replace the arylvinyl moiety. Herein, we describe the design and optimization of this series which was ultimately superseded by (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones. The latter compounds are potent and selective inhibitors of PLK4 with oral exposure in rodents and in vivo anticancer activity. Compound 13b, in particular, has a bioavailability of 22% and achieved a 96% tumor growth inhibition in an MDA-MB-468 xenograft study.

Discovery of Pyrazolo[1,5-a]pyrimidine TTK Inhibitors: CFI-402257 is a Potent, Selective, Bioavailable Anticancer Agent.

This work describes a scaffold hopping exercise that begins with known imidazo[1,2-a]pyrazines, briefly explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK inhibitors. An X-ray structure of a representative compound is consistent with 1(1)/2 type inhibition and provides structural insight to aid subsequent optimization of in vitro activity and physicochemical and pharmacokinetic properties. Incorporation of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the identification of a potent (TTK K i = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND enabling studies.

Discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as novel, highly potent and selective, orally bioavailable inhibitors of Tyrosine Threonine Kinase, TTK.

TTK/Mps1 is a key kinase controlling progression of cell division via participation in the mitotic spindle assembly checkpoint and is overexpressed in a number of human cancers. Herein we report the discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as a potent, novel class of TTK inhibitors. The series was identified by means of bioisosteric replacement of the related imidazopyrazine and imidazopyridazine scaffolds. Optimization led to the identification of compounds with excellent potency (Ki=0.8nM) and exceptional kinase selectivity. The SAR indicates a strong dependence of activity on the presence of the N-cyclopropyl-2-methylbenzamide moiety delineating the geometry for 1½ type kinase inhibitor. Molecular modeling indicates the extensive and optimal contacts, mediated through H-bonds and hydrophobic interactions, are responsible for the selectivity and potency of the inhibitors. The compounds demonstrate a strong anti-proliferative activity in a panel of human cancer cell lines (HCT116 GI50<15nM) and good rodent pharmacokinetics (oral %F 97%).

The Discovery of Orally Bioavailable Tyrosine Threonine Kinase (TTK) Inhibitors: 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides as Anticancer Agents.

The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors. However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharmacokinetic parameters, are not drug-like. By eliminating the polar 3-sulfonamide group and grafting a heterocycle at the 4 position of the phenyl ring, potent inhibitors with oral exposure were obtained. An X-ray cocrystal structure and a refined binding model allowed for a structure guided approach. Systematic optimization resulted in novel TTK inhibitors, namely 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides. Compounds incorporating the 3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl bicyclic system were potent (TTK IC50 < 10 nM, HCT116 GI50 < 0.1 µM), displayed low off-target activity (>500×), and microsomal stability (T(1/2) > 30 min). A subset was tested in rodent PK and mouse xenograft models of human cancer. Compound 75 (CFI-401870) recapitulated the phenotype of TTK RNAi, demonstrated in vivo tumor growth inhibition upon oral dosing, and was selected for preclinical evaluation.

The discovery of Polo-like kinase 4 inhibitors: identification of (1R,2S).2-(3-((E).4-(((cis).2,6-dimethylmorpholino)methyl)styryl). 1H.indazol-6-yl)-5'-methoxyspiro[cyclopropane-1,3'-indolin]-2'-one (CFI-400945) as a potent, orally active antitumor agent.

Previous publications from our laboratory have introduced novel inhibitors of Polo-like kinase 4 (PLK4), a mitotic kinase identified as a potential target for cancer therapy. The search for potent and selective PLK4 inhibitors yielded (E)-3-((1Hindazol-6-yl)methylene)indolin-2-ones, which were superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones, e.g., 3. The later scaffold confers improved drug-like properties and incorporates two stereogenic centers. This work reports the discovery of a novel one-pot double SN2 displacement reaction for the stereoselective installation of the desired asymmetric centers and confirms the stereochemistry of the most potent stereoisomer, e.g., 44. Subsequent work keys on the optimization of the oral exposure of nanomolar PLK4 inhibitors with potent cancer cell growth inhibitory activity. A short list of compounds with superior potency and pharmacokinetic properties in rodents and dogs was studied in mouse models of tumor growth. We conclude with the identification of compound 48 (designated CFI-400945) as a novel clinical candidate for cancer therapy.

The discovery of Polo-like kinase 4 inhibitors: design and optimization of spiro[cyclopropane-1,3'[3H]indol]-2'(1'H).ones as orally bioavailable antitumor agents.

Polo-like kinase 4 (PLK4), a unique member of the polo-like kinase family of serine-threonine kinases, is a master regulator of centriole duplication that is important for maintaining genome integrity. Overexpression of PLK4 is found in several human cancers and is linked with a predisposition to tumorigenesis. Previous efforts to identify potent and efficacious PLK4 inhibitors resulted in the discovery of (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones, which are superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones reported herein. Optimization of this new cyclopropane-linked series was based on a computational model of a PLK4 X-ray structure and SAR attained from the analogous alkenelinked series. The racemic cyclopropane-linked compounds showed PLK4 affinity and antiproliferative activity comparable to their alkene-linked congeners with improved hysicochemical, ADME, and pharmacokinetic properties. Positive xenograft results from the MDA-MB-468 human breast cancer xenograft model for compound 18 support the investigation of PLK4 inhibitors as anticancer therapeutics. A PLK4 X-ray co-structure with racemate 18 revealed preferential binding of the 1R,2S enantiomer to the PLK4 kinase domain.

Discovery of inhibitors of the mitotic kinase TTK based on N-(3-(3-sulfamoylphenyl)-1H-indazol-5-yl)-acetamides and carboxamides.

TTK kinase was identified by in-house siRNA screen and pursued as a tractable, novel target for cancer treatment. A screening campaign and systematic optimization, supported by computer modeling led to an indazole core with key sulfamoylphenyl and acetamido moieties at positions 3 and 5, respectively, establishing a novel chemical class culminating in identification of 72 (CFI-400936). This potent inhibitor of TTK (IC50=3.6nM) demonstrated good activity in cell based assay and selectivity against a panel of human kinases. A co-complex TTK X-ray crystal structure and results of a xenograft study with TTK inhibitors from this class are described.

The discovery of PLK4 inhibitors: (E)-3-((1H-Indazol-6-yl)methylene)indolin-2-ones as novel antiproliferative agents.

The family of Polo-like kinases is important in the regulation of mitotic progression; this work keys on one member, namely Polo-like kinase 4 (PLK4). PLK4 has been identified as a candidate anticancer target which prompted a search for potent and selective inhibitors of PLK4. The body of the paper describes lead generation and optimization work which yielded nanomolar PLK4 inhibitors. Lead generation began with directed virtual screening, using a ligand-based focused library and a PLK4 homology model. Validated hits were used as starting points for the design and discovery of PLK4 inhibitors of novel structure, namely (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones. Computational models, based on a published X-ray structure (PLK4 kinase domain), were used to understand and optimize the in vitro activity of the series; potent antiproliferative activity was obtained. The kinase selectivity profile and cell cycle analysis of selected inhibitors are described. The results of a xenograft study with an optimized compound 50 (designated CFI-400437) support the potential of these novel PLK4 inhibitors for cancer therapy.

In vitro activity (MICs and rate of kill) of AFN-1252, a novel FabI inhibitor, in the presence of serum and in combination with other antibiotics.

AFN-1252 is a novel inhibitor of FabI, an essential enzyme in fatty acid biosynthesis in Staphylococcus spp. AFN-1252 exhibits typical MIC(90) values of ≤0·015 µg/ml against diverse clinical isolates of S. aureus, oral absorption, long elimination half-live and efficacy in animal models. We now report high binding (∼95%) to serum proteins of mouse, rat, dog and humans, associated with an eight-fold increase in minimal inhibitory concentration (MIC) and which may be responsible for the long elimination half-lives on pharmacokinetic studies. Unlike daptomycin, AFN-1252 activity is not reduced in the presence of lung surfactant. AFN-1252 exhibits a short post-antibiotic effect of 1·1 hours against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) following a 4-hour exposure period. The AFN-1252 unique spectrum of activity is not compromised by interactions with major antibiotic classes, but demonstrates synergy with low concentrations of gentamicin against MSSA and MRSA. These studies support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.

Mode of action, in vitro activity, and in vivo efficacy of AFN-1252, a selective antistaphylococcal FabI inhibitor.

The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252-FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a ≥2-log(10) reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC(90), 0.015 µg/ml) and coagulase-negative staphylococci (MIC(90), 0.12 µg/ml), regardless of their drug resistance, hospital- or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.

Dimerization of ß-tryptase inhibitors, does it work for both basic and neutral P1 groups?

The tetrameric folding of ß-tryptase and the pair-wise distribution of its substrate binding sites offer a unique opportunity for development of inhibitors that span two adjacent binding sites. A series of dimeric inhibitors with two basic P1 moieties was discovered using this design strategy and exhibited tight-binder characteristics. Using the same strategy, an attempt was made to design and synthesize dimeric inhibitors with two neutral-P1 groups in hope to exploit the dimeric binding mode to achieve a starting point for further optimization. The unsuccessful attempt, however, demonstrated the important role played by Ala190 in neutral-P1 binding and casted further doubt on the possibility of developing neutral-P1 inhibitors for ß-tryptase.

Structure-based library design and the discovery of a potent and selective mast cell ß-tryptase inhibitor as an oral therapeutic agent.

A solid phase combinatorial library was designed based on X-ray structures and in-silico models to explore an inducible S4+ pocket, which is formed by a simple side-chain rotation of Tyr95. This inducible S4+ pocket is unique to ß-tryptase and does not exist for other trypsin-like serine proteases of interest. Therefore, inhibitors utilizing this pocket have inherent advantages for being selective against other proteases in the same family. A member of this library was found to be a potent and selective ß-tryptase inhibitor with a suitable pharmacokinetic profile for further clinical evaluation.

2,3,4,5-Tetrahydro-1H-pyrido[2,3-b and e][1,4]diazepines as inhibitors of the bacterial enoyl ACP reductase, FabI.

In the search for new antibacterial agents, the enzyme FabI has been identified as an attractive target. Employing a structure guided approach, the previously reported ene-amide series of FabI inhibitors were expanded to include 2,3,4,5-tetrahydro-1H-pyrido[2,3-b and e][1,4]diazepines. These novel series incorporate additional H-bonding functions and can be more water soluble than their naphthyridinone progenitors; diazepine 16c is shown to be efficacious in a mouse infection model.

Spiro-naphthyridinone piperidines as inhibitors of S. aureus and E. coli enoyl-ACP reductase (FabI).

Spiropiperidine naphthyridinone inhibitors of Staphylococcus aureus and Escherichia coli FabI have been prepared. Compounds 14a and 14c were identified as having sub-nanomolar E. coli FabI activity and are among the most potent FabI inhibitors yet described. The structural model of 14a bound to E. coli FabI is shown.

Design, synthesis, and biological activity of potent and selective inhibitors of mast cell tryptase.

A new series of novel mast cell tryptase inhibitors is reported, which features the use of an indole structure as the hydrophobic substituent on a m-benzylaminepiperidine template. The best members of this series display good in vitro activity and excellent selectivity against other serine proteases.

Structure based design of 4-(3-aminomethylphenyl)piperidinyl-1-amides: novel, potent, selective, and orally bioavailable inhibitors of betaII tryptase.

Tryptase is a serine protease found almost exclusively in mast cells. It has trypsin-like specificity, favoring cleavage of substrates with an arginine (or lysine) at the P1 position, and has optimal catalytic activity at neutral pH. Current evidence suggests tryptase beta is the most important form released during mast cell activation in allergic diseases. It is shown to have numerous pro-inflammatory cellular activities in vitro, and in animal models tryptase provokes broncho-constriction and induces a cellular inflammatory infiltrate characteristic of human asthma. Screening of in-house inhibitors of factor Xa (a closely related serine protease) identified beta-amidoester benzamidines as potent inhibitors of recombinant human betaII tryptase. X-ray structure driven template modification and exchange of the benzamidine to optimize potency and pharmacokinetic properties gave selective, potent and orally bioavailable 4-(3-aminomethyl phenyl)piperidinyl-1-amides.

Novel pyrazinone inhibitors of mast cell tryptase: synthesis and SAR evaluation.

In this manuscript, the synthesis and SAR evaluation of a novel pyrazinone class of tryptase inhibitors is described. Chemical optimization of the P1 and P4 groups led to the identification of 7p (K(i)=93 nM) as a potent inhibitor of mast cell tryptase.

Discovery of an orally efficacious inhibitor of coagulation factor Xa which incorporates a neutral P1 ligand.

The discovery and SAR of ketopiperazino methylazaindole factor Xa inhibitors are described. Structure-activity data suggesting that this class of inhibitors does not bind in the canonical mode were confirmed by an X-ray crystal structure showing the neutral haloaromatic bound in the S(1) subsite. The most potent azaindole, 33 (RPR209685), is selective against related serine proteases and attains higher levels of exposure upon oral dosing than comparable benzamidines and benzamidine isosteres. Compound 33 was efficacious in the canine AV model of thrombosis.

Molecular structures of human factor Xa complexed with ketopiperazine inhibitors: preference for a neutral group in the S1 pocket.

The structures of the noncovalent complex of human factor Xa (fXa) with four non-peptide inhibitors containing a central sulfonylpiperazinone scaffold have been determined to about 2.1 A resolution. Highly potent fXa inhibitors containing both neutral groups such as chlorobenzothiophene or chlorothiophene and basic groups such as benzamidine were shown to interact in the S1 pocket through the neutral group whereas the S4 pocket is occupied by the basic moiety. The scaffold comprising the sulfonyl keto piperazine moiety might play a pivotal role in the orientation of substituents, since there is a strong hydrogen bond between Gly219 of fXa and the carbonyl oxygen of the piperazine. This unique "reverse" binding mode is heretofore unreported in fXa and shows that electrostatic interactions in the S1 subsite are not an absolute requirement to maintain high affinity. Selectivity against other serine proteases can be readily explained in light of these structural results. It has opened up new prospects for designing fXa inhibitors with increased oral bioavailability.

Optimization of the beta-aminoester class of factor Xa inhibitors. Part 1: P(4) and side-chain modifications for improved in vitro potency.

A systematic modification of the C(3) side-chain of the beta-aminoester class of factor Xa inhibitors and a survey of P(4) variations is described. These changes have resulted in the identification of sub-nanomolar inhibitors with improved selectivity versus related proteases. Coagulation parameters (i.e., APTT doubling concentrations) are also improved.