Michael P. Zawistoski

Trifluoromethylpyrimidine-based inhibitors of proline-rich tyrosine kinase 2 (PYK2): structure-activity relationships and strategies for the elimination of reactive metabolite formation.

The synthesis and SAR for a series of diaminopyrimidines as PYK2 inhibitors are described. Using a combination of library and traditional medicinal chemistry techniques, a FAK-selective chemical series was transformed into compounds possessing good PYK2 potency and 10- to 20-fold selectivity against FAK. Subsequent studies found that the majority of the compounds were positive in a reactive metabolite assay, an indicator for potential toxicological liabilities. Based on the proposed mechanism for bioactivation, as well as a combination of structure-based drug design and traditional medicinal chemistry techniques, a follow-up series of PYK2 inhibitors was identified that maintained PYK2 potency, FAK selectivity and HLM stability, yet were negative in the RM assay.

Sulfoximine-substituted trifluoromethylpyrimidine analogs as inhibitors of proline-rich tyrosine kinase 2 (PYK2) show reduced hERG activity

The synthesis, in vitro properties, and in vivo pharmacokinetics for a series of sulfoximine-substituted trifluoromethylpyrimidines as inhibitors of proline-rich tyrosine kinase, a target for the possible treatment of osteoporosis, are described. These compounds were prepared as surrogates of the corresponding sulfone compound 1. Sulfone 1 was an attractive PYK2 lead compound; however, subsequent studies determined this compound possessed high dofetilide binding, which is an early indicator of cardiovascular safety. Surprisingly, the corresponding sulfoximine analogs displayed significantly lower dofetilide binding, which, for N-methylsulfoximine (S)-14a, translated to lower activity in a patch clamp hERG K(+) ion channel screen. In addition, compound (S)-14a shows good oral exposure in a rat pharmacokinetic model.

Pyrazolinone-piperidine dipeptide growth hormone secretagogues (GHSs) : Discovery of capromorelin

Novel pyrazolinone-piperidine dipeptide derivatives were synthesized and evaluated as growth hormone secretagogues (GHSs). Two analogues, capromorelin (5, CP-424391-18, hGHS-R1a K(i)=7 nM, rat pituicyte EC(50)=3 nM) and the des-methyl analogue 5c (hGHS-R1a K(i)=17 nM, rat pituicyte EC(50)=3 nM), increased plasma GH levels in an anesthesized rat model, with ED(50) values less than 0.05 mg/kg iv. Capromorelin showed enhanced intestinal absorption in rodent models and exhibited superior pharmacokinetic properties, including high bioavailabilities in two animal species [F(rat)=65%, F(dog)=44%]. This short-duration GHS was orally active in canine models and was selected as a development candidate for the treatment of musculoskeletal frailty in elderly adults.

Discovery and biological characterization of capromorelin analogues with extended half-lives

New tert-butyl, picolyl and fluorinated analogues of capromorelin (3), a short-acting growth hormone secretagogue (GHS), were prepared as part of a program to identify long-acting GHSs that increase 24-h plasma IGF-1 levels. Compounds 4c and 4d (ACD LogD values >or=2.9) displayed extended plasma elimination half-lives in dogs, primarily due to high volumes of distribution, but showed weak GH secretagogue activities in rats (ED(50)s>10 mg/kg). A less lipophilic derivative 4 (ACD LogD=1.6) exhibited a shorter canine half-life, but stimulated GH secretion in two animal species. Repeat oral dosing of 4 in dogs for 29 days (6 mg/kg) resulted in a significant down-regulation of the post dose GH response and a 60 and 40% increase in IGF-1 levels relative to pre-dose levels at the 8- and 24-h post dose time points. Compound 4 (CP-464709-18) has been selected as a development candidate for the treatment of frailty.

Discovery Tactics To Mitigate Toxicity Risks Due to Reactive Metabolite Formation with 2-(2-Hydroxyaryl)-5-(trifluoromethyl)pyrido[4,3-d]pyrimidin-4(3H)-one Derivatives, Potent Calcium-Sensing Receptor Antagonists and Clinical Candidate(s) for the Treatment of Osteoporosis

The synthesis and structure-activity relationship studies on 5-trifluoromethylpyrido[4,3-d]pyrimidin-4(3H)-ones as antagonists of the human calcium receptor (CaSR) have been recently disclosed [ Didiuk et al. ( 2009 ) Bioorg. Med. Chem. Lett. 19 , 4555 - 4559 ). On the basis of its pharmacology and disposition attributes, (R)-2-(2-hydroxyphenyl)-3-(1-phenylpropan-2-yl)-5-(trifluoromethyl)pyrido[4,3-d]pyrimidin-4(3H)-one (1) was considered for rapid advancement to first-in-human (FIH) trials to mitigate uncertainty surrounding the pharmacokinetic/pharmacodynamic (PK/PD) predictions for a short-acting bone anabolic agent. During the course of metabolic profiling, however, glutathione (GSH) conjugates of 1 were detected in human liver microsomes in an NADPH-dependent fashion. Characterization of the GSH conjugate structures allowed insight(s) into the bioactivation pathway, which involved CYP3A4-mediated phenol ring oxidation to the catechol, followed by further oxidation to the electrophilic ortho-quinone species. While the reactive metabolite (RM) liability raised concerns around the likelihood of a potential toxicological outcome, a more immediate program goal was establishing confidence in human PK predictions in the FIH study. Furthermore, the availability of a clinical biomarker (serum parathyroid hormone) meant that PD could be assessed side by side with PK, an ideal scenario for a relatively unprecedented pharmacologic target. Consequently, progressing 1 into the clinic was given a high priority, provided the compound demonstrated an adequate safety profile to support FIH studies. Despite forming identical RMs in rat liver microsomes, no clinical or histopathological signs prototypical of target organ toxicity were observed with 1 in in vivo safety assessments in rats. Compound 1 was also devoid of metabolism-based mutagenicity in in vitro (e.g., Salmonella Ames) and in vivo assessments (micronuclei induction in bone marrow) in rats. Likewise, metabolism-based studies (e.g., evaluation of detoxicating routes of clearance and exhaustive PK/PD studies in animals to prospectively predict the likelihood of a low human efficacious dose) were also conducted, which mitigated the risks of idiosyncratic toxicity to a large degree. In parallel, medicinal chemistry efforts were initiated to identify additional compounds with a complementary range of human PK predictions, which would maximize the likelihood of achieving the desired PD effect in the clinic. The back-up strategy also incorporated an overarching goal of reducing/eliminating reactive metabolite formation observed with 1. Herein, the collective findings from our discovery efforts in the CaSR program, which include the incorporation of appropriate derisking steps when dealing with RM issues are summarized.

Discovery of CP-533536: an EP2 receptor selective prostaglandin E2 (PGE2) agonist that induces local bone formation.

Sulfonamides, exemplified by 3a, were identified as highly selective EP(2) agonists. Lead optimization led to the identification of CP-533536, 7f, a potent and selective EP(2) agonist. CP-533536 demonstrated the ability to heal fractures when administered locally as a single dose in rat models of fracture healing.

Short-acting 5-(trifluoromethyl)pyrido[4,3-d]pyrimidin-4(3H)-one derivatives as orally-active calcium-sensing receptor antagonists.

Synthesis and structure-activity relationship (SAR) studies on 5-trifluoromethylpyrido[4,3-d]pyrimidin-4(3H)-ones, a novel class of calcium receptor antagonists is described with particular emphasis on optimization of the pharmacokinetic/pharmacodynamic parameters required for a short duration of action compound. Orally-active compounds were identified which displayed the desired animal pharmacology (rapid and transient stimulation of parathyroid hormone) essential for bone anabolic effects.

Differences in CYP3A4 catalyzed bioactivation of 5-aminooxindole and 5-aminobenzsultam scaffolds in proline-rich tyrosine kinase 2 (PYK2) inhibitors: retrospective analysis by CYP3A4 molecular docking, quantum chemical calculations and glutathione adduct detection using linear ion trap/orbitrap mass spectrometry.

Previous studies have demonstrated the CYP3A4 mediated oxidation of the 5-aminooxindole motif, present in the trifluoromethylpyrimidine class of PYK-2 inhibitors, to a reactive bis-imine species, which can be trapped with glutathione (GSH) in human liver microsomal incubations. The corresponding 5-aminobenzsultam derivatives, which should possess a similar oxidative liability, do not form GSH conjugates in microsomal incubations. In the current study, we conducted a retrospective analysis on representative 5-aminooxindole and 5-aminobenzsultam PYK-2 inhibitors utilizing CYP3A4 molecular docking and quantum chemical calculations to rationalize the bioactivation differences. Our analysis revealed key differences in (a) active site binding and (b) two-electron oxidation rates, which correlate with GSH adduct formation with the two moieties. The value of linear ion/orbitrap mass spectrometry to detect GSH conjugates with greater sensitivity, compared with conventional triple quadrupole mass spectrometry approaches, was also demonstrated in the course of these studies.

Identification of novel series of pyrazole and indole-urea based DFG-out PYK2 inhibitors.

Previous drug discovery efforts identified classical PYK2 kinase inhibitors such as 2 and 3 that possess selectivity for PYK2 over its intra-family isoform FAK. Efforts to identify more kinome-selective chemical matter that stabilize a DFG-out conformation of the enzyme are described herein. Two sub-series of PYK2 inhibitors, an indole carboxamide-urea and a pyrazole-urea have been identified and found to have different binding interactions with the hinge region of PYK2. These leads proved to be more selective than the original classical inhibitors.

Synthesis of [2,2,3α,4,4-D5]CP-88,818 (tiqueside), an internal standard for a quantitative HPLC/MS assay system

Abstract A polydeuterated form of CP-88,818 ( 1 , tiqueside) was needed as an internal standard for a quantitative HPLC/MS assay system. [2,2,3α,4,4-D 5 ]CP-88,818 ( 11 ) with undetectable D 0 content was synthesized in five steps from tigogenin ( 2 ). The low D 0 content was achieved through two sequential incorporation procedures which gave results superior to those achieved through a single incorporation procedure. A preparatively useful procedure for removing spirostane impurities found in naturally occurring tigogenin was also discovered.