Synthesis and evaluation of 4,6-disubstituted pyrimidines as CNS penetrant pan-muscarinic antagonists with a novel chemotype

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M₄ antagonists, based on a 4,6-disubstituted core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at both human and rat M₄ (IC₅₀s < 300 nM), with no substantial species differences noted. Moreover, CNS penetration proved attractive for this series (brain:plasma K_p,uu = 0.87), while other DMPK attributes were addressed in the course of the optimization effort, providing low in vivo clearance in rat (CL_p = 5.37 mL/min/kg). Surprisingly, this series displayed pan-muscarinic antagonist activity across M_1–5, despite the absence of the prototypical basic or quaternary amine moiety, thus offering a new chemotype from which to develop a next generation of pan-muscarinic antagonist agents.     

Discovery of a novel,CNS penetrant M4 PAM chemotype based on a 6-fluoro-4-(piperidin-1-yl)quinoline-3-carbonitrile core

This Letter details the discovery and subsequent optimization of a novel M₄ PAM scaffold based on an 6-fluoro-4-(piperidin-1-yl)quinoline-3-carbonitrile core, which represents a distinct departure from the classical M₄ PAM chemotypes. Optimized compounds in this series demonstrated improved M₄ PAM potency on both human and rat M₄ (4 to 5-fold relative to HTS hit), and displayed attractive physicochemical and DMPK profiles, including good CNS penetration (rat brain:plasma K_p = 5.3, K_p,uu = 2.4; MDCK-MDR1 (P-gp) ER = 1.1).     

SAR inspired by aldehyde oxidase (AO) metabolism: Discovery of novel,CNS penetrant tricyclic M4 PAMs

This letter describes progress towards an M₄ PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M₄ PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.     

Discovery of a novel 2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide M4 positive allosteric modulator (PAM) chemotype

This Letter details our efforts to discover structurally unique M₄ PAMs containing 5,6-heteroaryl ring systems. In an attempt to improve the DMPK profiles of the 2,3-dimethyl-2H-indazole-5-carboxamide and 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide cores, we investigated a plethora of core replacements. This exercise identified a novel 2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide core that provided improved M₄ PAM activity and CNS penetration.     

Discovery and optimization of 4-oxo-2-thioxo-thiazolidinones as NOD-like receptor (NLR) family,pyrin domain-containing protein 3 (NLRP3) inhibitors

Aberrant activation of NLRP3 inflammasome is present in a subset of acute and chronic inflammatory diseases. The NLRP3 inflammasome has been recognized as an attractive therapeutic target for developing novel and specific anti-inflammatory inhibitors. Cellular structure-activity relationship-guided optimization resulted in the identification of 4-oxo-2-thioxo-thiazolidinone derivative 9 as a selective and direct small-molecule inhibitor of NLRP3 with IC₅₀ of 2.4 μM, possessing favorable ex vivo and in vivo pharmacokinetic properties. Compound 9 may represent a lead for the development of anti-inflammatory therapeutics for treating NLRP3-driven diseases.     

Discovery of a novel 3,4-dimethylcinnoline carboxamide M4 positive allosteric modulator (PAM) chemotype via scaffold hopping

This Letter details our efforts to replace the 2,4-dimethylquinoline carboxamide core of our previous M₄ PAM series, which suffered from high predicted hepatic clearance and protein binding. A scaffold hopping exercise identified a novel 3,4-dimethylcinnoline carboxamide core that provided good M₄ PAM activity and improved clearance and protein binding profiles.     

Discovery of a novel 2,4-dimethylquinoline-6-carboxamide M4 positive allosteric modulator (PAM) chemotype via scaffold hopping

This Letter details our efforts to replace the 3-amino moiety, an essential pharmacophore for M₄ PAM activity in most M₄ PAMs to date, within the thieno[2,3-b]pyridine core, as the β-amino carboxamide motif has been shown to engender poor solubility, varying degrees of P-gp efflux and represents a structural alert. A scaffold hopping exercise identified a novel 2,4-dimethylquinoline carboxamide core that provided M₄ PAM activity and good CNS penetration without an amino moiety. In addition, MacMillan photoredox catalysis chemistry was essential for construction of the 2,4-dimethylquinoline core.     

Discovery of structurally distinct tricyclic M4 positive allosteric modulator (PAM) chemotypes

This Letter details our efforts to develop new M₄ PAM scaffolds with improved pharmacological properties. This endeavor involved replacing the 3,4-dimethylpyridazine core with two novel cores: a 2,3-dimethyl-2H-indazole-5-carboxamide core or a 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide core. Due to shallow SAR, these cores were further evolved into two unique tricyclic cores: an 8,9-dimethyl-8H-pyrazolo[3,4-h]quinazoline core and an 1-methyl-1H-[1,2,3]triazolo[4,5-h]quinazoline core. Both tricyclic cores displayed low nanomolar potency against both human and rat M₄.     

Challenges in the development of an M4 PAM in vivo tool compound: The discovery of VU0467154 and unexpected DMPK profiles of close analogs

This letter describes the chemical optimization of a novel series of M₄ positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0467154 (5). This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0467154, and details all of the challenges faced in allosteric modulator programs (steep SAR, species differences in PAM pharmacology and subtle structural changes affecting CNS penetration).     

Continued optimization of the M5 NAM ML375: Discovery of VU6008667, an M5 NAM with high CNS penetration and a desired short half-life in rat for addiction studies

This letter describes the continued optimization of M₅ NAM ML375 (VU0483253). While a valuable in vivo tool compound, ML375 has an excessively long elimination half-life in rat (t_1/2 = 80 h), which can be problematic in certain rodent addiction paradigms (e.g., reinstatement). Thus, we required an M₅ NAM of comparable potency to ML375, but with a rat t_1/2 of less than 4 h. Steep SAR plagued this chemotype, and here we detail aniline replacements that offered some improvements over ML375, but failed to advance. Ultimately, incorporation of a single methyl group to the 9b-phenyl ring acted as a metabolic shunt, providing (S)-11 (VU6008667), an equipotent M₅ NAM, with high CNS penetration, excellent selectivity versus M_1–4 and the desired short half-life (t_1/2 = 2.3 h) in rat.     

Design,synthesis, and evaluation of N-(4-(4-phenyl piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamides as selective dopamine D3 receptor ligands

As part of our on-going effort to explore the role of dopamine receptors in drug addiction and identify potential novel therapies for this condition, we have a identified a series of N-(4-(4-phenyl piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide D₃ ligands. Members of this class are highly selective for D₃ versus D₂, and we have identified two compounds (13g and 13r) whose rat in vivo IV pharmacokinetic properties that indicate that they are suitable for assessment in in vivo efficacy models of substance use disorders.     

Design,synthesis, and evaluation of novel 4-amino-2-(4-benzylpiperazin-1-yl)methylbenzonitrile compounds as Zika inhibitors

The prevalence of Zika virus (ZIKV) has become widespread in recent years. ZIKV infection is associated with severe congenital CNS malformations in both newborns and adults. However, neither vaccines nor therapeutics are available to control ZIKV infection until now. We started by hit screening our in-house small molecule library, then designed, synthesized, and evaluated a new class of 1, 4-bibenzylsubstituted piperazine derivatives for their cytopathic effect (CPE) protection effect in a ZIKV-infected Vero E6 cellular assay. A preliminary structure–activity relationship study identified five novel 4-amino-2-(4-benzylpiperazin-1-yl)methylbenzonitrile analogs with obvious CPE reduction effects against ZIKV at micromolar concentrations. Moreover, compound 3p exerted a significant antiviral effect on both Zika RNA replication and virus protein expression in a dose-dependent manner at low micromolar concentrations. This study demonstrated the potential of a novel 4-amino-2-(4-benzylpiperazin-1-yl)methylbenzonitrile scaffold for the development of anti-ZIKV candidates.     

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one derivatives. Screening of alternative azole rings identified 1,3-dimethyl pyrazole 6a as a lead compound with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR binding activity while maintaining the agonistic response at the lower level. Among these compounds, 6-[1-(2,2-difluoro-3-hydroxypropyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone receptors, and good pharmacokinetic profiles. Oral administration of 37a in deoxycorticosterone acetate-salt hypertensive rats showed a significant blood pressure-lowering effect with no signs of antiandrogenic effects.     

Discovery of 6-[4-(6-nitroxyhexanoyl)piperazin-1-yl)]-9H-purine,as pharmacological post-conditioning agent

Novel purine analogues bearing nitrate esters were designed and synthesized in an effort to develop compounds triggering endogenous cardioprotective mechanisms such as ischemic preconditioning (IPC) or postconditioning (PostC). The majority of the compounds reduced infarct size compared to the control group in anesthetized rabbits, whereas administration of the most active analogue 16 at a dose of 3.8 μmol/kg resulted on a significant reduction of infarct size, compared to PostC group (13.4 ± 1.9% vs 26.4 ± 2.3%). These findings introduce a novel class of promising pharmacological compounds that could be used as mimics or enhancers of PostC.     

Discovery of N-(piperidin-3-yl)-N-(pyridin-2-yl)piperidine/piperazine-1-carboxamides as small molecule inhibitors of PCSK9

A series of N-(piperidin-3-yl)-N-(pyridin-2-yl)piperidine/piperazine-1-carboxamides were identified as small molecule PCSK9 mRNA translation inhibitors. Analogues from this new chemical series, such as 4d and 4g, exhibited improved PCSK9 potency, ADME properties, and in vitro safety profiles when compared to earlier lead structures.     

Design and synthesis of novel 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine derivatives as selective G-protein-coupled receptor kinase-2 and -5 inhibitors

G-protein-coupled receptor kinase (GRK)-2 and -5 are emerging therapeutic targets for the treatment of cardiovascular disease. In our efforts to discover novel small molecules to inhibit GRK-2 and -5, a class of compound based on 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine was identified as a novel hit by high throughput screening campaign. Structural modification of parent benzoxazole scaffolds by introducing substituents on phenyl displayed potent inhibitory activities toward GRK-2 and -5.     

Cytotoxicity and topoisomerase I/II inhibition activity of novel 4-aryl/alkyl-1-(piperidin-4-yl)-carbonylthiosemicarbazides and 4-benzoylthiosemicarbazides

Abstract

A series of eight thiosemicarbazide derivatives was examined for cytotoxicity in breast cancer cell cultures. Among them, 4-benzoylthiosemicarbazides proved to be only slightly less potent than chlorambucil in both MDA-MB-231 and MCF-7 lines. In contrast, 4-aryl/alkylthiosemicarbazides revealed significantly lower cytotoxicity effect. Subsequently, all titled compounds were tested as potential human topoisomerase I and II (topo I and topo II) inhibitors. Mechanistic studies revealed that tested thiosemicarbazides act as both topoisomerase I and topoisomerase II inhibitors. Among them, the best inhibitory activity was found for 4-benzoylthiosemicarbazides (1 and 2) with IC₅₀ at 50?µM against topo II.     

Discovery of novel histone lysine methyltransferase G9a/GLP (EHMT2/1) inhibitors: Design,synthesis, and structure-activity relationships of 2,4-diamino-6-methylpyrimidines

The discovery and optimization of a novel series of G9a/GLP (EHMT2/1) inhibitors are described. Starting from known G9a/GLP inhibitor 5, efforts to explore the structure-activity relationship and optimize drug properties led to a novel compound 13, the side chain of which was converted to tetrahydroazepine. Compound 13 showed increased G9a/GLP inhibitory activity compared with compound 5. In addition, compound 13 exhibited improved human ether-a-go-go related gene (hERG) inhibitory activity over compound 5 and also improved pharmacokinetic profile in mice (oral bioavailability: 17 to 40%). Finally, the co-crystal structure of G9a in complex with compound 13 provides the basis for the further development of tetrahydroazepine-based G9a/GLP inhibitors.     

N-(4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl,butenyl and butynyl)arylcarboxamides as novel dopamine D(3) receptor antagonists

The dopamine D(3) receptor subtype has been targeted as a potential neurochemical modulator of the behavioral actions of psychomotor stimulants, such as cocaine. Previous synthetic studies provided structural requirements for high affinity binding to D(3) receptors which included a 2,3-dichloro-phenylpiperazine linked to an arylamido function via a butyl chain. To reduce lipophilicity of these agents and further investigate optimal conformation, a second series of 15 novel ligands was designed that included heteroaromatic substitution and unsaturated alkyl linkers. These compounds were synthesized and evaluated for binding at rat D(3) and D(2) receptors stably expressed in Sf9 cells. D(3) binding affinities ranged from K(i)=0.6-1080 nM, with a broad range of D(3)/D(2) selectivities (2-97). The discovery of potent, selective and bioavailable D(3) receptor ligands will provide essential molecular probes to elucidate the role D(3) receptors play in the psychomotor stimulant and reinforcing effects of cocaine.     

Design of novel CXCR4 antagonists that are potent inhibitors of T-tropic (X4) HIV-1 replication

A novel series of CXCR4 antagonists were identified based on the substantial redesign of AMD070. These compounds possessed potent anti-HIV-1 activity and showed excellent pharmacokinetics in rat and dog.