Technically Extended MultiParameter Optimization (TEMPO): An Advanced Robust Scoring Scheme To Calculate Central Nervous System Druggability and Monitor Lead Optimization.

At the discovery stage, it is important to understand the drug design concepts for a CNS drug compared to those for a non-CNS drug. Previously, we published on ideal CNS drug property space and defined in detail the physicochemical property distribution of CNS versus non-CNS oral drugs, the application of radar charting (a graphical representation of multiple physicochemical properties used during CNS lead optimization), and a recursive partition classification tree to differentiate between CNS- and non-CNS drugs. The objective of the present study was to further understand the differentiation of physicochemical properties between CNS and non-CNS oral drugs by the development and application of a new CNS scoring scheme: Technically Extended MultiParameter Optimization (TEMPO). In this multiparameter method, we identified eight key physicochemical properties critical for accurately assessing CNS druggability: (1) number of basic amines, (2) carbon-heteroatom (non-carbon, non-hydrogen) ratio, (3) number of aromatic rings, (4) number of chains, (5) number of rotatable bonds, (6) number of H-acceptors, (7) computed octanol/water partition coefficient (AlogP), and (8) number of nonconjugated C atoms in nonaromatic rings. Significant features of the CNS-TEMPO penalty score are the extension of the multiparameter approach to generate an accurate weight factor for each physicochemical property, the use of limits on both sides of the computed property space range during the penalty calculation, and the classification of CNS and non-CNS drug scores. CNS-TEMPO significantly outperformed CNS-MPO and the Schrödinger QikProp CNS parameter (QP_CNS) in evaluating CNS drugs and has been extensively applied in support of CNS lead optimization programs.

Discovery of Clinical Candidate CEP-37440, a Selective Inhibitor of Focal Adhesion Kinase (FAK) and Anaplastic Lymphoma Kinase (ALK).

Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein.

Discovery of an orally efficacious inhibitor of anaplastic lymphoma kinase.

Anaplastic lymphoma kinase (ALK) is a promising therapeutic target for the treatment of cancer, supported by considerable favorable preclinical and clinical activities over the past several years and culminating in the recent FDA approval of the ALK inhibitor crizotinib. Through a series of targeted modifications on an ALK inhibitor diaminopyrimidine scaffold, our research group has driven improvements in ALK potency, kinase selectivity, and overall pharmaceutical properties. Optimization of this scaffold has led to the identification of a potent and efficacious inhibitor of ALK, 25b. A striking feature of 25b over previously described ALK inhibitors is its >600-fold selectivity over insulin receptor (IR), a closely related kinase family member. Most importantly, 25b exhibited dose proportional escalation in rat compared to compound 3 which suffered dose limiting absorption preventing further advancement. Compound 25b exhibited significant in vivo antitumor efficacy when dosed orally in an ALK-positive ALCL tumor xenograft model in SCID mice, warranting further assessment in advanced preclinical models.

Knowledge-Based, Central Nervous System (CNS) Lead Selection and Lead Optimization for CNS Drug Discovery.

The central nervous system (CNS) is the major area that is affected by aging. Alzheimer's disease (AD), Parkinson's disease (PD), brain cancer, and stroke are the CNS diseases that will cost trillions of dollars for their treatment. Achievement of appropriate blood-brain barrier (BBB) penetration is often considered a significant hurdle in the CNS drug discovery process. On the other hand, BBB penetration may be a liability for many of the non-CNS drug targets, and a clear understanding of the physicochemical and structural differences between CNS and non-CNS drugs may assist both research areas. Because of the numerous and challenging issues in CNS drug discovery and the low success rates, pharmaceutical companies are beginning to deprioritize their drug discovery efforts in the CNS arena. Prompted by these challenges and to aid in the design of high-quality, efficacious CNS compounds, we analyzed the physicochemical property and the chemical structural profiles of 317 CNS and 626 non-CNS oral drugs. The conclusions derived provide an ideal property profile for lead selection and the property modification strategy during the lead optimization process. A list of substructural units that may be useful for CNS drug design was also provided here. A classification tree was also developed to differentiate between CNS drugs and non-CNS oral drugs. The combined analysis provided the following guidelines for designing high-quality CNS drugs: (i) topological molecular polar surface area of <76 Å(2) (25-60 Å(2)), (ii) at least one (one or two, including one aliphatic amine) nitrogen, (iii) fewer than seven (two to four) linear chains outside of rings, (iv) fewer than three (zero or one) polar hydrogen atoms, (v) volume of 740-970 Å(3), (vi) solvent accessible surface area of 460-580 Å(2), and (vii) positive QikProp parameter CNS. The ranges within parentheses may be used during lead optimization. One violation to this proposed profile may be acceptable. The chemoinformatics approaches for graphically analyzing multiple properties efficiently are presented.

Design, synthesis, and anaplastic lymphoma kinase (ALK) inhibitory activity for a novel series of 2,4,8,22-tetraazatetracyclo[14.3.1.1³,7.19,¹³]docosa-1(20),3(22),4,6,9(21),10,12,16,18-nonaene macrocycles.

A novel set of 2,4,8,22-tetraazatetracyclo[14.3.1.1(3,7).1(9,13)]docosa-1(20),3(22),4,6,9(21),10,12,16,18-nonaene macrocycles were prepared as potential anaplastic lymphoma kinase (ALK) inhibitors, designed to rigidly lock an energy-minimized bioactive conformation of the diaminopyrimidine (DAP) scaffold, a well-documented kinase platform. From 13 analogues prepared, macrocycle 2m showed the most promising in vitro ALK enzymatic (IC(50) = 0.5 nM) and cellular (IC(50) = 10 nM) activities. In addition, macrocycle 2m exhibited a favorable kinase selectivity preference for inhibition of ALK relative to the highly homologous insulin receptor (IR) kinase (IR/ALK ratio of 173). The inclusive in vitro biological results for this set of macrocycles validate this scaffold as a viable kinase template and further corroborate recent DAP/ALK solid state studies indicating that the inverted "U" shaped conformation of the acyclic DAPs is a preferred bioactive conformation.

Identification of 1-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea hydrochloride (CEP-32496), a highly potent and orally efficacious inhibitor of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF) V600E.

The Ras/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway plays a central role in the regulation of cell growth, differentiation, and survival. Expression of mutant BRAF(V600E) results in constitutive activation of the MAPK pathway, which can lead to uncontrolled cellular growth. Herein, we describe an SAR optimization campaign around a series of quinazoline derived BRAF(V600E) inhibitors. In particular, the bioisosteric replacement of a metabolically sensitive tert-butyl group with fluorinated alkyl moieties is described. This effort led directly to the identification of a clinical candidate, compound 40 (CEP-32496). Compound 40 exhibits high potency against several BRAF(V600E)-dependent cell lines and selective cytotoxicity for tumor cell lines expressing mutant BRAF(V600E) versus those containing wild-type BRAF. Compound 40 also exhibits an excellent PK profile across multiple preclinical species. In addition, significant oral efficacy was observed in a 14-day BRAF(V600E)-dependent human Colo-205 tumor xenograft mouse model, upon dosing at 30 and 100 mg/kg BID.

Pyrazolone-based anaplastic lymphoma kinase (ALK) inhibitors: control of selectivity by a benzyloxy group.

Anaplastic lymphoma kinase (ALK) is transmembrane receptor tyrosine kinase, with oncogenic variants that have been implicated in ALCL, NSCLC and other cancers. Screening of a VEGFR2-biased kinase library resulted in identification of 1 which showed cross-reactivity with ALK. SAR on the indole segment of 1 showed that a subtle structural modification (the ethoxy group of 1 changed to a benzyloxy to generate 5a) enhanced potency (ALK), selectivity for VEGFR2 and IR along with improvement in metabolic stability. From docking studies of ALK versus VEGFR2 kinase, we postulated that the loss of entropy of the VEGFR2 in the bound form with 5a might be the origin of the reduced activity against that protein. Modification of the heterocyclic segment showed that thiazole-bearing pyrazolones preserved enzyme potency, and enhanced inhibition of NPM-ALK autophosphorylation in ALK-positive ALCL cells (Karpas-299). SAR of the benzyloxy group resulted in compounds which demonstrated good cellular potency in Karpas-299 cells. Compound 8 showed best overall profile for the series with broad kinome selectivity and liver micorsome stability. Compound 8 showed reasonable iv PK in rat, but with little oral exposure.

2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazines: new variant of an old template and application to the discovery of anaplastic lymphoma kinase (ALK) inhibitors with in vivo antitumor activity.

A novel 2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazine scaffold has been designed as a new kinase inhibitor platform mimicking the bioactive conformation of the well-known diaminopyrimidine motif. The design, synthesis, and validation of this new pyrrolo[2,1-f][1,2,4]triazine scaffold will be described for inhibitors of anaplastic lymphoma kinase (ALK). Importantly, incorporation of appropriate potency and selectivity determinants has led to the discovery of several advanced leads that were orally efficacious in animal models of anaplastic large cell lymphoma (ALCL). A lead inhibitor (30) displaying superior efficacy was identified and in depth in vitro/in vivo characterization will be presented.

Synthesis and structure-activity relationships of 1,2,3,4-tetrahydropyrido[2,3-b]pyrazines as potent and selective inhibitors of the anaplastic lymphoma kinase.

Dysregulation of the anaplastic lymphoma kinase (ALK) is implicated in a variety of cancers. A series of tetrahydropyrido[2,3-b]pyrazines was constructed as ring-constrained analogs of a known aminopyridine kinase scaffold. Chemistry was developed to rapidly elaborate the SAR, structural elements impacting ALK inhibitory activity were exploited, and kinase selective analogs were identified that inhibit ALK with IC(50) values approximately 10 nM (enzyme) and approximately 150 nM (cell).

ALK mutants in the kinase domain exhibit altered kinase activity and differential sensitivity to small molecule ALK inhibitors.

Abnormal expression of constitutively active anaplastic lymphoma kinase (ALK) chimeric proteins in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well established. Recent studies with small molecule kinase inhibitors have provided solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK (+) ALCL cells. In this study, several missense mutants of ALK in the phosphate anchor and gatekeeper regions were generated and their kinase activity was measured. NPM-ALK L182M, L182V, and L256M mutants displayed kinase activity in cells comparable to or higher than that of NPM-ALK wild type (WT) and rendered BaF3 cells into IL-3-independent growth, while NPM-ALK L182R, L256R, L256V, L256P, and L256Q displayed much weaker or little kinase activity in cells. Similar kinase activities were obtained with corresponding GST-ALK mutants with in vitro kinase assays. With regard to inhibitor response, NPM-ALK L182M and L182V exhibited sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor comparable to that of NPM-ALK WT but were dramatically less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK L256M exhibited >30-fold lower sensitivity to both FP-derived and DAP-derived ALK inhibitors. The growth inhibition and cytotoxicity of BaF3/NPM-ALK mutant cells induced by ALK inhibitors were consistent with inhibition of cellular NPM-ALK autophosphorylation. In a mouse survival model, treatment with the orally bioavailable DAP-ALK inhibitor substantially extended the survival of the mice inoculated with BaF3/NPM-ALK WT cells but not those inoculated with BaF3/NPM-ALK L256M cells. Binding of ALK inhibitors to ALK WT and mutants was analyzed using ALK homology models. In summary, several potential active ALK mutants were identified, and our data indicate that some of these mutants are resistant to select small molecule ALK inhibitors. Further characterization of these mutants may help to identify and develop potent ALK inhibitors active against both WT and resistant mutants of ALK.

Knowledge-based chemoinformatic approaches to drug discovery.

The modern drug discovery process is steadily becoming more information driven. Structural, physicochemical and ADME-Tox property profiles of reference (successful) ligands, along with structural information of their target proteins, have been extremely useful for early-stage drug discovery. Recently, databases of known biologically active ligands (knowledge bases) have become more focused toward different protein-target classes. The number of new chemoinformatics tools used to analyze structures and properties of successful molecules has also increased enormously. Scientists in this area are exploring new physicochemical properties and appropriate drug sets to understand druglike properties. In this review, the various uses of the ligand knowledge bases in the drug discovery process have been critically reviewed.