Russell C. Petter

The resurgence of covalent drugs

Covalent drugs haveproved to be successful therapies for various indications, but largely owing to safety concerns, they are rarely considered when initiating a target-directed drug discovery project. There is a need to reassess this important class of drugs, and to reconcile the discordance between the historic success of covalent drugs and the reluctance of most drug discovery teams to include them in their armamentarium. This Review surveys the prevalence and pharmacological advantages of covalent drugs, discusses how potential risks and challenges may be addressed through innovative design, and presents the broad opportunities provided by targeted covalent inhibitors.

Selective irreversible inhibition of a protease by targeting a noncatalytic cysteine

Designing selective inhibitors of proteases has proven problematic, in part because pharmacophores that confer potency exploit the conserved catalytic apparatus. We developed a fundamentally different approach by designing irreversible inhibitors that target noncatalytic cysteines that are structurally unique to a target in a protein family. We have successfully applied this approach to the important therapeutic target HCV protease, which has broad implications for the design of other selective protease inhibitors.

Piperazine derivatives of [1,2,4]triazolo[1,5-a][1,3,5]triazine as potent and selective adenosine A2a receptor antagonists.

The [1,2,4]triazolo[1,5-a]triazine derivative 3, more commonly known in the field of adenosine research as ZM-241385, has previously been demonstrated to be a potent and selective adenosine A2a receptor antagonist, although with limited oral bioavailability. This [1,2,4]triazolo[1,5-a]triazine core structure has now been improved by incorporating various piperazine derivatives. With some preliminary optimization, the A2a binding affinity of some of the best piperazine derivatives is almost as good as that of compound 3. The selectivity level over the adenosine A1 receptor subtype for some of the more active analogues is also fairly high, > 400-fold in some cases. Many compounds within this piperazine series of [1,2,4]triazolo[1,5-a]triazine have now been shown to have good oral bioavailability in the rat, with some as high as 89% (compound 35). More significantly, some piperazines derivatives of [1,2,4]triazolo[1,5-a]triazine also possessed good oral efficacy in rodent models of Parkinsons disease. For instance, compound 34 was orally active in the rat catalepsy model at 3 mg/kg. In the 6-hydroxydopamine-lesioned rat model, this compound was also quite effective, with a minimum effective dose of 3 mg/kg po.

Transition states and energetics of nucleophilic additions of thiols to substituted α,β-unsaturated ketones: Substituent effects involve enone stabilization, product branching, and solvation

CBS-QB3 enthalpies of reaction have been computed for the conjugate additions of MeSH to six α,β-unsaturated ketones. Compared with addition to methyl vinyl ketone, the reaction becomes 1-3 kcal mol(-1) less exothermic when an α-Me, β-Me, or β-Ph substituent is present on the C=C bond. The lower exothermicity for the substituted enones occurs because the substituted reactant is stabilized more by hyperconjugation or conjugation than the product is stabilized by branching. Substituent effects on the activation energies for the rate-determining step of the thiol addition (reaction of the enone with MeS(-)) were also computed. Loss of reactant stabilization, and not steric hindrance, is the main factor responsible for controlling the relative activation energies in the gas phase. The substituent effects are further magnified in solution; in water (simulated by CPCM calculations), the addition of MeS(-) to an enone is disfavored by 2-6 kcal mol(-1) when one or two methyl groups are present on the C=C bond (ΔΔG(‡)). The use of CBS-QB3 gas-phase energies in conjunction with CPCM solvation corrections provides kinetic data in good agreement with experimental substituent effects. When the energetics of the thiol additions were calculated with several popular density functional theory and ab initio methods (B3LYP, MPW1PW91, B1B95, PBE0, B2PLYP, and MP2), some substantial inaccuracies were noted. However, M06-2X (with a large basis set), B2PLYP-D, and SCS-MP2 gave results within 1 kcal mol(-1) of the CBS-QB3 benchmark values.

Discovery of a potent and isoform-selective targeted covalent inhibitor of the lipid kinase PI3Kα.

PI3Kα has been identified as an oncogene in human tumors. By use of rational drug design, a targeted covalent inhibitor 3 (CNX-1351) was created that potently and specifically inhibits PI3Kα. We demonstrate, using mass spectrometry and X-ray crystallography, that the selective inhibitor covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ, and -δ are not covalently modified. 3 is able to potently (EC(50) < 100 nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell lines, and this leads to a potent antiproliferative effect (GI(50) < 100 nM). A covalent probe, 8 (CNX-1220), which selectively bonds to PI3Kα, was used to investigate the duration of occupancy of 3 with PI3Kα in vivo. This is the first report of a PI3Kα-selective inhibitor, and these data demonstrate the biological impact of selectively targeting PI3Kα.

Lewis-acid catalysis of the asymmetric Diels–Alder reaction of dimenthyl fumarate and cyclopentadiene

Abstract The effects of Lewis acid catalysts, solvent, catalyst loading, and temperature on the diastereomeric excess in the Diels–Alder reaction of (+)-dimenthyl fumarate and cyclopentadiene were investigated.

Abstract 1745: Translational medicine enables rapid early clinical development of AVL-292, a highly selective, orally available inhibitor of Bruton's tyrosine kinase, in a phase 1b clinical trial

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Brutons tyrosine kinase (Btk) plays a key role in the signaling pathway from the B cell receptor (BCR) that contributes to the proliferation and survival of malignancies of B cell origin. AVL-292 is an orally available and highly selective covalent inhibitor of Btk that is currently undergoing Phase 1b clinical development for CLL and B-NHL. Using a novel covalent probe that enables precise and quantitative assessment of Btk molecular target engagement by AVL-292, we have pursued an innovative early clinical development approach in healthy adult volunteers based on this translational capability. 5 cohorts of up to 8 subjects (6 active, 2 placebo) were enrolled in a single ascending dose, placebo controlled clinical study. The objectives of this study were to determine safety, pharmacokinetics, and to determine the minimum dose and plasma concentration of AVL-292 required to achieve complete Btk engagement. Single oral doses of 0.5-7.0 mg/kg were administered to up to 6 subjects per cohort. Samples for AVL-292 PK and Btk occupancy in peripheral blood mononuclear cells enriched for B cells were collected immediately prior to dosing and at several time points after dosing to determine the PK-PD relationship. AVL-292 was found to be safe and well tolerated following oral administration at all dose levels. All subjects that received an oral dose of 1.0 mg/kg of AVL-292 achieved >80% Btk occupancy. 5 of the 6 subjects administered AVL-292 at 2.0 mg/kg achieved complete Btk occupancy that was sustained through 24 hours even after plasma levels of AVL-292 had declined. This prolonged duration of activity demonstrates that covalent inhibition of Btk with AVL-292 enables continued activity without requiring persistence of circulating drug. The minimum dose that achieved complete target occupancy in this study (125 mg) was carried forward as the starting dose in a Phase 1b study of hematological malignancies of B cell origin. Subjects diagnosed with B cell malignancies received single daily oral doses of AVL-292 in continuous 28 day cycles. Total Btk content was determined in peripheral blood mononuclear cells at pre-dose, and Btk occupancy was determined 4 and 24 hours post dose on Day 1 and Day 28. Btk occupancy in this Phase 1b clinical study confirmed observations in healthy volunteer subjects. The relationship between inhibition of Btk activity and clinical response will be determined in the current and future clinical studies. Performing dose escalation in healthy volunteers enables rapid determination of an active dose level for Btk occupancy and inhibition by AVL-292. This innovative clinical trial design precludes the administration of sub-therapeutic dose levels to patients with high medical need and rapidly identified the therapeutic dose range and schedule for AVL-292 for use in treatment of patients with B cell malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1745. doi:1538-7445.AM2012-1745

Abstract C189: CO-1686, an orally available, mutant-selective inhibitor of the epidermal growth factor receptor (EGFR), causes tumor shrinkage in non-small cell lung cancer (NSCLC) with T790M mutations.

Introduction: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond well to EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by the development of resistance. The most common mechanism of resistance is a second site mutation within exon 20 of EGFR (T790M), observed in ∼50% of cases. Our goal was to develop a mutant-selective EGFR inhibitor that potently inhibits activating EGFR mutations as well as the T790M resistance mutation while sparing wild-type EGFR for the treatment of NSCLC patients. Such a drug has the potential to effectively treat first- and second-line NSCLC patients with EGFR mutations without causing the dose limiting toxicities associated with EGFR kinase inhibitors currently in clinical development. Experimental procedures: Using structure-based drug design, we identified CO-1686, a covalent, irreversible small molecule, which selectively inhibits mutant EGFR. We assessed antitumor activity of CO-1686 both in vitro and in vivo in two NSCLC cell lines harboring EGFR mutations: H1975 (EGFR L858R/T790M) and HCC827 (EGFR delE746-A750). We evaluated inhibition of EGFR phosphorylation and downstream signaling by immunoblot analysis in cells and tissue samples. IHC staining on skin samples was performed to address effects on wild-type EGFR. Results: CO-1686 is a potent inhibitor of cell proliferation and EGFR signaling in NSCLC cells harboring the single activating mutation EGFR delE746-A750 as well as the double mutation EGFR L858R/T790M. When administered orally, CO-1686 (3 − 100 mg/kg) significantly suppresses tumor growth of H1975 cells (L858R/T790M) in a dose-dependent manner causing tumor regressions at the highest dose (100 mg/kg) without affecting body weight. Erlotinib at the same dose exhibits no effect against H1975 xenografts. In HCC827 (delE746-A750) xenografts, both agents cause tumor shrinkage. In both NSCLC mouse models, inhibition of EGFR phosphorylation in tumors correlate with the observed anti-tumor activity, while no effect on EGFR signaling is observed in normal lung or skin tissues with CO-1686 treatment, confirming that CO-1686 does not inhibt wild-type EGFR. Conclusions: Our results establish CO-1686 as a mutant-selective, wild-type sparing EGFR inhibitor with in vivo efficacy against tumors with activating EGFR mutations as well as the resistance mutation T790M. These data suggest that treatment with CO-1686 as a single agent can overcome T790M-mediated drug resistance in NSCLC. This hypothesis will be tested clinically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C189.

Convenient One‐Pot Preparation of Dimethyl Bicyclo[2.2.2]octane‐1,4‐dicarbolylate, a Key Intermediate for a Novel Adenosine A1 Receptor Antagonist

Abstract Dimethyl bicyclo[2.2.2]octane‐1,4‐dicarboxylate (4), a key starting material for a novel adenosine A1 receptor antagonist, was prepared in a one‐pot reaction with convenient workup and improved yield.

Farnesyl chain modification of squalene synthase inhibitor benzylfarnesylamine: Conversion to the terminal bis(trifluoromethyl) derivative

Abstract Potent squalene synthase inhibitor 1 was converted to the bis(trifluoromethyl) analog 14 in 11% overall yield for 9 steps. The amine nitrogen of 1 was protected with the 2-(trimethylsilyl)ethoxycarbonyl (TEOC) protecting group. The 10,11 olefin was selectively epoxidized, cleaved and converted to the phosphonium salt 6. The ylid from 6 underwent a Wittig condensation with hexafluoroacetone to give the TEOC containing olefin 8. Tetrabutylammonium fluoride or HF could not remove the TEOC group without isomerizing the 10,11 olefin of the farnesyl chain to the E-9,10 olefin. The bis(trifluoromethyl) olefin of 8 is very sensitive to either acidic or basic conditions. However, it was found that BF3·Et2O could remove the TEOC group without the undesired isomerization to give 14.