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Na v 1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 ( 1 ) and GDC-0310 ( 2 ), selective Na v 1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3 . This resulted in the discovery of GDC-0276 ( 1 ), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Na v 1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 ( 2 ), which possesses improved Na v selectivity and pharmacokinetic profile over 1 .

Herein, we report the discovery and optimization of a series of orally bioavailable acyl sulfonamide Na V 1.7 inhibitors that are selective for Na V 1.7 over Na V 1.5 and highly efficacious in in vivo models of pain and hNa V 1.7 target engagement. An analysis of the physicochemical properties of literature Na V 1.7 inhibitors suggested that acyl sulfonamides with high f sp3 could overcome some of the pharmacokinetic (PK) and efficacy challenges seen with existing series. Parallel library syntheses lead to the identification of analogue 7, which exhibited moderate potency against Na V 1.7 and an acceptable PK profile in rodents, but relatively poor stability in human liver microsomes. Further, design strategy then focused on the optimization of potency against hNa V 1.7 and improvement of human metabolic stability, utilizing induced fit docking in our previously disclosed X-ray cocrystal of the Na V 1.7 voltage sensing domain. These investigations culminated in the discovery of tool compound 33, one of the most potent and efficacious Na V 1.7 inhibitors reported to date.

The sodium channel Na V 1.7 has emerged as a promising target for the treatment of pain based on strong genetic validation of its role in nociception. In recent years, a number of aryl and acyl sulfonamides have been reported as potent inhibitors of Na V 1.7, with high selectivity over the cardiac isoform Na V 1.5. Herein, we report on the discovery of a novel series of N-([1,2,4]triazolo[4,3- a]pyridin-3-yl)methanesulfonamides as selective Na V 1.7 inhibitors. Starting with the crystal structure of an acyl sulfonamide, we rationalized that cyclization to form a fused heterocycle would improve physicochemical properties, in particular lipophilicity. Our design strategy focused on optimization of potency for block of Na V 1.7 and human metabolic stability. Lead compounds 10, 13 (GNE-131), and 25 showed excellent potency, good in vitro metabolic stability, and low in vivo clearance in mouse, rat, and dog. Compound 13 also displayed excellent efficacy in a transgenic mouse model of induced pain.

We report on a novel series of aryl sulfonamides that act as nanomolar potent, isoform-selective inhibitors of the human sodium channel hNaV1.7. The optimization of these inhibitors is described. We aimed to improve potency against hNaV1.7 while minimizing off-target safety concerns and generated compound 3. This agent displayed significant analgesic effects in rodent models of acute and inflammatory pain and demonstrated that binding to the voltage sensor domain 4 site of NaV1.7 leads to an analgesic effect in vivo. Our findings corroborate the importance of hNaV1.7 as a drug target for the treatment of pain.

DNA (cytosine-5)-methyltransferases (DNMTs) catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 5-position of cytosine residues and thereby silence transcription of regulated genes. DNMTs are important epigenetic targets. However, isolated DNMTs are weak catalysts and are difficult to assay. We report an ultrasensitive luciferase-linked continuous assay that converts the S-adenosyl-L-homocysteine product of DNA methylation to a quantifiable luminescent signal. Results with this assay are compared with the commonly used DNA labeling from [methyl-(3)H]AdoMet. A 5'-methylthioadenosine-adenosylhomocysteine nucleosidase is used to hydrolyze AdoHcy to adenine. Adenine phosphoribosyl transferase converts adenine to AMP and pyruvate orthophosphate dikinase converts AMP to ATP. Firefly luciferase gives a stable luminescent signal that results from continuous AMP recycling to ATP. This assay exhibits a broad dynamic range (0.1-1000 pmol of AdoHcy). The rapid response time permits continuous assays of DNA methylation detected by light output. The assay is suitable for high-throughput screening of chemical libraries for DNMT inhibition activity. The kinetic properties of human and bacterial CpG methyltransferases are characterized using this assay. Human catalytic domain DNMT3b activation by DNMT3L is shown to involve two distinct kinetic states that alter k(cat) but not K(m) for AdoMet. The assay is shown to be robust in the presence of high concentrations of the pyrimidine analogues 5-azacytidine and 5-azacytosine.

The S-adenosylmethionine (AdoMet) salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5'-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2(-/-)γC(-/-) and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP(-/-) and H358 MTAP(+/+) tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥ 3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.

The synthesis of 4-deoxy-4-nitrosialic acid (3,4,5-trideoxy-4-nitro-D-glycero-beta-D-galacto-non-2-ulopyranosonic acid, 5), was completed in seven steps starting from D-arabinose. Coupling of the 6-carbon fragment, 2-acetamido-1,2-dideoxy-1-nitro-D-mannitol (6) with ethyl alpha-(bromomethyl)acrylate afforded a 2 : 1 mixture of ethyl 5-acetamido-2,3,4,5-tetradeoxy-2-methylene-4-nitro-D-glycero-D-galacto-nononate (9a-S) and ethyl 5-acetamido-2,3,4,5-tetradeoxy-2-methylene-4-nitro-D-glycero-D-talo-nononate (9a-R). This mixture of enones was subjected to ozonolysis, and following reduction of the ozonide, the resultant products cyclised to the pyranosides. The target compound, ethyl 4-deoxy-4-nitrosialate (11a) was isolated by fractional crystallisation. Hydrolysis of the ethyl ester proved problematic; thus, the synthesis was modified by using tert-butyl alpha-(bromomethyl)acrylate. Following ozonolysis of the corresponding tert-butyl enoate esters and diastereomer separation, the tert-butyl ester of 4-nitrosialic acid (11b) could be deprotected under acidic conditions to afford . The target compound is a useful intermediate for synthesis of a variety of C-4 substituted sialic acid derivatives, and it is synthesised by a modular route.

Silylstannanes can be regioselectively added across terminal alkynes in a quantitative fashion in the presence of a palladium(0) catalyst immobilised in the [bmim]PF6 ionic liquid which can be recycled without loss of activity.