Massimiliano Fonsi

Discovery of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide (MK-4827): a novel oral poly(ADP-ribose)polymerase (PARP) inhibitor efficacious in BRCA-1 and -2 mutant tumors.

We disclose the development of a novel series of 2-phenyl-2H-indazole-7-carboxamides as poly(ADP-ribose)polymerase (PARP) 1 and 2 inhibitors. This series was optimized to improve enzyme and cellular activity, and the resulting PARP inhibitors display antiproliferation activities against BRCA-1 and BRCA-2 deficient cancer cells, with high selectivity over BRCA proficient cells. Extrahepatic oxidation by CYP450 1A1 and 1A2 was identified as a metabolic concern, and strategies to improve pharmacokinetic properties are reported. These efforts culminated in the identification of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide 56 (MK-4827), which displays good pharmacokinetic properties and is currently in phase I clinical trials. This compound displays excellent PARP 1 and 2 inhibition with IC(50) = 3.8 and 2.1 nM, respectively, and in a whole cell assay, it inhibited PARP activity with EC(50) = 4 nM and inhibited proliferation of cancer cells with mutant BRCA-1 and BRCA-2 with CC(50) in the 10-100 nM range. Compound 56 was well tolerated in vivo and demonstrated efficacy as a single agent in a xenograft model of BRCA-1 deficient cancer.

Design and synthesis of bicyclic pyrimidinones as potent and orally bioavailable HIV-1 integrase inhibitors.

HIV integrase is one of the three enzymes encoded by HIV genome and is essential for viral replication, but integrase inhibitors as marketed drugs have just very recently started to emerge. In this study, we show the evolution from the N-methylpyrimidinone structure to bicyclic pyrimidinones. Introduction of a suitably substituted amino moiety modulated the physical-chemical properties of the molecules and conferred nanomolar activity in the inhibition of spread of HIV-1 infection in cell culture. An extensive SAR study led to sulfamide (R)- 22b, which inhibited the strand transfer with an IC50 of 7 nM and HIV infection in MT4 cells with a CIC95 of 44 nM, and ketoamide (S)- 28c that inhibited strand transfer with an IC50 of 12 nM and the HIV infection in MT4 cells with a CIC95 of 13 nM and exhibited a good pharmacokinetic profile when dosed orally to preclinical species.

Discovery of a Potent Class I Selective Ketone Histone Deacetylase Inhibitor with Antitumor Activity in Vivo and Optimized Pharmacokinetic Properties

The optimization of a potent, class I selective ketone HDAC inhibitor is shown. It possesses optimized pharmacokinetic properties in preclinical species, has a clean off-target profile, and is negative in a microbial mutagenicity (Ames) test. In a mouse xenograft model it shows efficacy comparable to that of vorinostat at a 10-fold reduced dose.

High-Throughput Microsomal Stability Assay for Screening New Chemical Entities in Drug Discovery

In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities. (Journal of Biomolecular Screening 2008:862-869)

N-(2-alkylaminoethyl)-4-(1,2,4-oxadiazol-5-yl)piperazine-1-carboxamides as highly potent smoothened antagonists.

Smoothened (Smo) antagonists are emerging as new therapies for the treatment of neoplasias with aberrantly reactivated hedgehog (Hh) signaling pathway. A novel series of 4-[3-(quinolin-2-yl)-1,2,4-oxadiazol-5-yl]piperazinyl ureas as smoothened antagonists was recently described, herein the series has been further optimized through the incorporation of a basic amine into the urea. This development resulted in identification of some exceptionally potent smoothened antagonists with low serum shifts, however, reductive ring opening on the 1,2,4-oxadiazole in rats limits the applicability of these compounds in in vivo studies.

The metabolism and disposition of a potent inhibitor of hepatitis C virus NS3/4A protease

Compound A ((1aR,5S,8S,10R,22aR)-5-tert-butyl-N-{(1R,2S)-1-[(cyclopropylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-14-methoxy-3,6-dioxo-1,1a,3,4,5,6,9,10,18,19,20,21,22,22a-tetradecahydro-8H-7,10-methanocyclopropa[18,19][1,10,3,6]dioxadiazacyclononadecino[12,11-b]quinoline-8-carboxamide) is a prototype of a series of subnanomolar inhibitors of genotypes 1, 2, and 3 hepatitis C virus (HCV) NS3/4A proteases. HCV NS3/4A protease inhibitors have demonstrated high antiviral effects in patients with chronic HCV infection and are likely to form a key component of future HCV therapy. Compound A showed excellent liver exposure in rats, which is essential for compounds intended to treat HCV. The compound was mainly eliminated intact in bile and showed greater than dose proportional systemic exposure in rats. Compound A demonstrated time- and temperature-dependent uptake into rat and human hepatocytes and proved to be a substrate for rat hepatic uptake transporter Oatp1b2 and for human hepatic uptake transporters OATP1B1 and OATP1B3. The liver selectivity observed for this compound is likely to be due to transporter-mediated hepatic uptake together with moderate passive permeability. Metabolism was mainly CYP3A-mediated and generated a reactive epoxide on the vinylcyclopropyl sulfonamide moiety that could be quenched by glutathione. Similar metabolic profiles of Compound A were obtained in liver microsomes of rats and humans. The oral bioavailability at 5 mg/kg was low due to extensive hepatic first-pass effect but clearly the intestinal absorption was enough to deliver a high amount of the compound to the liver. The metabolism and disposition properties of Compound A are particularly attractive to support its evaluation as a drug candidate for the treatment of hepatitis C.

Optimization of a series of potent and selective ketone histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors offer a promising strategy for cancer therapy and the first generation HDAC inhibitors are currently in the clinic. Herein we describe the optimization of a series of ketone small molecule HDAC inhibitors leading to potent and selective class I HDAC inhibitors with good dog PK.

Metabolism-related liabilities of a potent histone deacetylase (HDAC) inhibitor and relevance of the route of administration on its metabolic fate

Compound A [1-methyl-N-{(1S)-1-[5-(2-naphthyl)-1H-imidazol-2-yl]-7-oxooctyl}piperidine-4-carboxamide is a potent class I histone deacetylase (HDAC) inhibitor that demonstrated good antiproliferative activity against human tumour cell lines of different origin. This compound showed high in vivo clearance in rats (160 ml min−1 kg−1) due to metabolism. The main metabolite detected in urine after intravenous dosing was characterized as a dihydrohydroxy S-mercapturic acid conjugate. Following oral dosing, however, the mercapturic acid derivative was no longer the main metabolite but the major metabolites were mono- and di-glucuronide conjugates of oxidized species having a mass shift of +34 m/z with respect to the parent. Comparison of plasma concentration after intra-arterial infusion and intravenous infusion and incubation with microsomes from different tissues (liver, kidney, small intestine and lung) in the presence of β-nicotinamide adenine dinucleotide phosphate (NADPH) indicated that the compound was highly cleared by the lung. Oxidation of the naphthalene moiety was demonstrated to be the cause of the high in vivo clearance of compound A and the potential for bioactivation of this group was flagged.