Identification of highly potent and orally available free fatty acid receptor 1 agonists bearing isoxazole scaffold

The free fatty acid receptor 1 (FFA1) is being considered to be a novel anti-diabetic target based on its role in amplifying insulin secretion. We have previously identified several series of FFA1 agonists with different heterocyclic scaffolds. Herein, we describe the structural exploration of other heterocyclic scaffolds directed by drug-like physicochemical properties. Further structure-based design and chiral resolution provided the most potent compound 11 (EC₅₀?=?7.9?nM), which exhibited improved lipophilicity (LogD_7.4: 1.93), ligand efficiency (LE?=?0.32) and ligand lipophilicity efficiency (LLE?=?6.2). Moreover, compound 11 revealed an even better pharmacokinetic property than that of TAK-875 in terms of plasma clearance, maximum concentration, and plasma exposure. Although robust agonistic activity and PK profiles for compound 11, the glucose-lowering effects in vivo is not ideal, and the exact reason for in vitro/in vivo difference was worthy for further exploration.     

Discovery of novel free fatty acid receptor 1 agonists bearing triazole core via click chemistry

The free fatty acid receptor 1 (FFA1/GPR40) is a novel antidiabetic target based on particular mechanism in enhancing glucose-stimulated insulin secretion. Most of reported FFA1 agonists, however, have been suffered from relatively high lipophilicity and molecular weight. Aiming to develop potent agonists with improved physicochemical property, 25 compounds containing triazole scaffold and various carboxylic acid fragments were synthesized via the click chemistry. Among them, the optimal lead compound 26 with relatively low lipophicity (Log D_7.4 = 1.95) and molecular weight (Mw = 391.78) exhibited a considerable FFA1 agonistic activity (36.15%). In addition, compound 26 revealed a significant improvement in the glucose tolerance with a 21.4% and 14.2% reduction of glucose AUC_0–2h in normal ICR mice and type 2 diabetic C57BL/6 mice, respectively. All of these results demonstrated that compound 26 was considered to be a promising lead compound suitable for further optimization.     

Discovery of a novel series of indoline carbamate and indolinylpyrimidine derivatives as potent GPR119 agonists

GPR119 has emerged as an attractive target for anti-diabetic agents. We identified a structurally novel GPR119 agonist 22c that carries a 5-(methylsulfonyl)indoline motif as an early lead compound. To generate more potent compounds of this series, structural modifications were performed mainly to the central alkylene spacer. Installation of a carbonyl group and a methyl group on this spacer significantly enhanced agonistic activity, resulting in the identification of 2-[1-(5-ethylpyrimidin-2-yl)piperidin-4-yl]propyl 7-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-indole-1-carboxylate (20). To further expand the chemical series of indoline-based GPR119 agonists, several heterocyclic core systems were introduced as surrogates of the carbamate spacer that mimic the presumed active conformation. This approach successfully produced an indolinylpyrimidine derivative 37, 5-(methylsulfonyl)-1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indole, which has potent GPR119 agonist activity. In rat oral glucose tolerance tests, these two indoline-based compounds effectively lowered plasma glucose excursion and glucose-dependent insulin secretion after oral administration.     

Design,synthesis, and biological evaluation of deuterated phenylpropionic acid derivatives as potent and long-acting free fatty acid receptor 1 agonists

The free fatty acid receptor 1 (FFA1) is a potential target due to its function in enhancement of glucose-stimulated insulin secretion. Takeda’s compound 1 has robustly in vitro activity for FFA1, but it has been suffered from poor pharmacokinetic (PK) profiles because the phenylpropanoic acid is vulnerable to β-oxidation. To identify orally available agonists, we tried to interdict the metabolically labile group by incorporating two deuterium atoms at the α-position of phenylpropionic acid. Interestingly, the differences of physicochemical properties between hydrogen and deuterium are quite small, but there are many differences in the structure-activity relationship between phenylpropionic acid series and present deuterated series. Further optimizations of deuterated series led to the discovery of compound 18, which exhibited a superior balance in terms of in vitro activity, lipophilicity, and solubility. Better still, compound 18 revealed a lower clearance (CL = 0.44 L/h/kg), higher maximum concentration (C_max = 7584.27 μg/L), and longer half-life (T_1/2 = 4.16 h), resulting in a >23-fold exposure than compound 1. In subsequent in vivo pharmacodynamic studies, compound 18 showed a robustly glucose-lowering effect in rodent without the risk of hypoglycemia.     

Fluorescent retinoid X receptor ligands for fluorescence polarization assay

Retinoid X receptor (RXR) agonists are candidate agents for the treatment of metabolic syndrome and type 2 diabetes via activation of peroxisome proliferator-activated receptor (PPAR)/RXR or liver X receptor (LXR)/RXR-heterodimers, which control lipid and glucose metabolism. Reporter gene assays or binding assays with radiolabeled compounds are available for RXR ligand screening, but are unsuitable for high-throughput screening. Therefore, as a first step towards stabilizing a fluorescence polarization (FP) assay system for high-throughput RXR ligand screening, we synthesized fluorescent RXR ligands by modification of the lipophilic domain of RXR ligands with a carbostyril fluorophore, and selected the fluorescent RXR agonist 6-[ethyl(1-isobutyl-2-oxo-4-trifluoromethyl-1,2-dihydroquinolin-7-yl)amino]nicotinic acid 8d for further characterization. Compound 8d showed FP in the presence of RXR and the FP was decreased in the presence of the RXR agonist LGD1069 (2). This compound should be a lead compound for use in high-throughput assay systems for screening RXR ligands.     

Discovery of HWL-088: A highly potent FFA1/GPR40 agonist bearing a phenoxyacetic acid scaffold

Based on a previously reported phenoxyacetic acid scaffold, compound 7 (HWL-088) has been identified as a superior free fatty acid receptor 1 (FFA1) agonist by comprehensive structure-activity relationship study. Our results indicated that the introduction of ortho-fluoro greatly increased the activity of phenoxyacetic acid series, and the unique structure-activity relationship in biphenyl moiety is different from previously reported FFA1 agonists. Moreover, the modeling study was also performed to better understand the binding mode of present series. Compound 7 significantly improved glucose tolerance both in normal and diabetic models, and even exerted greater potential on glucose control than that of TAK-875. These findings provided a novel candidate HWL-088, which is currently in preclinical study to evaluate its potential for the treatment of diabetes.     

Pharmacological properties and predicted binding mode of arylmethylene quinuclidine-like derivatives at the α3β4 nicotinic acetylcholine receptor (nAChR)

We have carried out a pharmacological evaluation of arylmethylene quinuclidine derivatives interactions with human α3β4 nAChRs subtype, using cell-based receptor binding, calcium-influx, electrophysiological patch-clamp assays and molecular modeling techniques. We have found that the compounds bind competitively to the α3β4 receptor with micromolar affinities and some of the compounds behave as non-competitive antagonists (compounds 1, 2 and 3), displaying submicromolar IC₅₀ values. These evidences suggest a mixed mode of action for these compounds, having interactions at the orthosteric site and more pronounced interactions at an allosteric site to block agonist effects. One of the compounds, 1-benzyl-3-(diphenylmethylene)-1-azoniabicyclo[2.2.2]octane chloride (compound 3), exhibited poorly reversible use-dependent block of α3β4 channels. We also found that removal of a phenyl group from compound 1 confers a partial agonism to the derived analog (compound 6). Introducing a hydrogen-bond acceptor into the 3-benzylidene quinuclidine derivative (compound 7) increases agonism potency at the α3β4 receptor subtype. Docking into the orthosteric binding site of a α3β4 protein structure derived by comparative modeling accurately predicted the experimentally-observed trend in binding affinity. Results supported the notion that binding requires a hydrogen bond formation between the ligand basic nitrogen and the backbone carbonyl oxygen atom of the conserved Trp-149.     

Chiral analogues of (+)-cyclazosin as potent α1B-adrenoceptor selective antagonist

(+)-Cyclazosin [(+)-1] is one of most selective antagonists of the α_1B-adrenoceptor subtype (selectivity ratios, α_1B/α_1A?=?13, α_1B/α_1D?=?38–39). To improve the selectivity, we synthesized and pharmacologically studied the blocking activity against α₁-adrenoceptors of several homochiral analogues of (+)-cyclazosin featuring different substituents on the carbonyl or amine groups, namely (?)-2, (+)-3, (?)-4–(?)-8, (+)-9. Moreover, we studied the activity of some their opposite enantiomers, namely (?)-1, (?)-3, (+)-6, and (?)-9, to evaluate the influence of stereochemistry on selectivity. The benzyloxycarbonyl and methyl (4aS,8aR) analogues (+)-3 and (?)-6 improved in a significant way the α_1B selectivity of the progenitor compound: 4 and 14 time vs. the α_1D subtype and 35 and 77 times vs. the α_1A subtype, respectively. The study confirmed the importance of the hydrophobic cis-octahydroquinoxaline moiety of these molecules for the establishment of interactions with the α₁-adrenoceptors as well that of their (4aS,8aR) stereochemistry to grant selectivity for the α_1B subtype. Hypotheses on the mode of interaction of these compounds were advanced on the basis of molecular modeling studies performed on compound (+)-3.     

Mode of interaction of 1,4-dioxane agonists at the M2 and M3 muscarinic receptor orthosteric sites

The methyl group in cis stereochemical relationship with the basic chain of all pentatomic cyclic analogues of ACh is crucial for the agonist activity at mAChR. Among these only cevimeline (1) is employed in the treatment of xerostomia associated with Sjögren’s syndrome. Here we demonstrated that, unlike 1,3-dioxolane derivatives, in the 1,4-dioxane series the methyl group is not essential for the activation of mAChR subtypes. Docking studies, using the crystal structures of human M₂ and rat M₃ receptors, demonstrated that the 5-methylene group of the 1,4-dioxane nucleus of compound 10 occupies the same lipophilic pocket as the methyl group of the 1,3-dioxolane 4.     

Specific spectral assignments for acetoxyl-group resonances in proton magnetic resonance spectra of methyl β-D-glucopyranoside tetraacetate and related derivatives

Methyl β-D-glucopyranoside tetraacetates (1) having a trideuterioacetyl group at O-2 (1a), O-3, (1b), O-4 (1c), and O-6 (1d) were synthesized by unambiguous routes to permit assignment of each individual acetoxyl-group signal in the p.m.r. spectrum of 1. The 6-acetoxyl resonance appears at lower field than signals of the other acetoxyl groups in carbon tetrachloride, chloroform-d, and methyl sulfoxide-d₆, but in pyridine-d₅ and benzene-d₆, the 2-acetoxyl-group signal appears at lower field. The acetoxyl resonances of methyl 2,3,4-tri-O-acetyl-6-O-trityl-β-D-glucopyranoside (2), methyl 2,3,4-tri-O-acetyl-β-D-glucopyranoside (3), methyl 2,3-di-O-acetyl-4,6-O-benzylidene-β-D-glucopyranoside (5), methyl 2,3-di-O-acetyl-β-D-glucopyranoside (6), methyl 2,3,6-tri-O-acetyl-β-D-glucopyranoside (7), and methyl 2,3-di-O-acetyl-6-O-trityl-β-D-glucopyranoside (12) were assigned similarly after synthesis of the 2-(trideuterioacetyl) (2a, 3a, 5a, 6a, 7a, and 12a), 3-(trideuterioacetyl) (2b, 3b, 5b, 6b, 7b, and 12b), 4-(trideuterioacetyl) (2c and 3c), and 6-(trideuterioacetyl) (7c) analogues. In chloroform-d and benzene-d₆, the 4-acetoxyl resonance appeared at about 0.3 p.p.m. to higher field than the other acetoxyl-group signals of 2. In chloroform-d and methyl sulfoxide-d₆, the 3-acetoxyl resonance is observed at highest field in compounds 1, 3, and 5. In all of these instances, the 4-hydroxyl group is substituted by an acetyl or benzylidene group. When no 4-substituent is present (compounds 6, 7, and 12), the 3-acetoxyl group resonates at lower field than the other acetoxyl groups.     

Properly substituted 1,4-dioxane nucleus favours the selective M3 muscarinic receptor activation

Novel analogues of cis-N,N,N-trimethyl-(6-methyl-1,4-dioxan-2-yl)methanaminium iodide (2a) were synthesized by inserting methyl groups alternatively or simultaneously in positions 5 and 6 of the 1,4-dioxane nucleus in all combinations. Their biological profile was assessed by receptor binding assays at human muscarinic M₁–M₅ receptors stably expressed in CHO cells and by functional studies performed on classical isolated organ preparations, namely, rabbit electrically stimulated vas deferens, and guinea pig electrically stimulated left atrium, ileum, and lung strips. The results showed that the simultaneous presence of one methyl group in both positions 5 and 6 with a trans stereochemical relationship with each other (diastereomers 4 and 5) or the geminal dimethylation in position 6 (compound 8) favour the selective activation of M₃ receptors. Compounds 4, 5, and 8 might be valuable tools in the characterization of the M₃ receptor, as well as provide useful information for the design and development of novel selective M₃ antagonists.     

Discovery of novel selective GPR120 agonists with potent anti-diabetic activity by hybrid design

GPR120 is an attractive target for the treatment of type 2 diabetes. In this study, a series of biphenyl derivatives were designed, synthesized by hybrid design. The selected compound 6a exhibited potent GPR120 agonist activity (EC₅₀?=?93?nM) and high selectivity over GPR40. The results of oral glucose tolerance test (OGTT) demonstrated that 6a exhibited significant glucose-lowering effect in glucose-loaded ICR male mice. Analysis of the structure–activity relationship is also presented. Compound 6a deserves further biological evaluation and structural modifications.     

Discovery of 3-aryl-3-ethoxypropanoic acids as orally active GPR40 agonists

The G protein-coupled receptor 40 (GPR40) mediates enhancement of glucose-stimulated insulin secretion in pancreatic β cells. The GPR40 agonist has been attracting attention as a novel insulin secretagogue with glucose dependency for the treatment of type 2 diabetes. The optimization study of compound 1 led to a potent and bioavailable GPR40 agonist 24, which showed insulin secretion and glucose lowering effects in rat OGTT. Compound 24 is a potential lead compound for a novel insulin secretagogue with a low risk of hypoglycemia.     

Synthesis and evaluation of isoleucine derived angiotensin II AT2 receptor ligands

Sixteen new C-terminally modified analogues of 2, a previously described potent and selective AT₂R ligand, were designed, synthesized and evaluated for their affinity to the AT₂R receptor. The introduction of large, hydrophobic substituents was shown to be beneficial and the most active compound (17, K_i = 8.5 μM) was over 12-times more potent than the lead compound 2.     

Design and synthesis of 1-(1-benzothiophen-7-yl)-1H-pyrazole,a novel series of G protein-coupled receptor 52 (GPR52) agonists

G-protein-coupled receptor 52 (GPR52) is classified as an orphan Gs-coupled G-protein-coupled receptor. GPR52 cancels dopamine D2 receptor signaling and activates dopamine D1/N-methyl-d-aspartate receptors via intracellular cAMP accumulation. Therefore, GPR52 agonists are expected to alleviate symptoms of psychotic disorders. A novel series of 1-(benzothiophen-7-yl)-1H-pyrazole as GPR52 agonists was designed and synthesized based on compound 1b. Compound 1b has been reported by our group as the first orally active GPR52 agonist, but high lipophilicity and poor aqueous solubility still remained as issues for candidate selection. To resolve these issues, replacement of the benzene ring at the 7-positon of compound 1b with heterocylic rings, such as pyrazole and pyridine, was greatly expected to reduce lipophilicity to levels for which calculated logD values were lower than that of compound 1b. While evaluating the pyrazole derivatives, introduction of a methyl substituent at the 3-position of the pyrazole ring led to increased GPR52 agonistic activity. Moreover, additional methyl substituent at the 5-position of the pyrazole and further introduction of hydroxy group to lower logD led to significant improvement of solubility while maintaining the activity. As a result, we identified 3-methyl-5-hydroxymethyl-1H-pyrazole derivative 17 (GPR52 EC₅₀?=?21?nM, E_max?=?103%, logD?=?2.21, Solubility at pH 6.8?=?21?μg/mL) with potent GPR52 agonistic activity and good solubility compared to compound 1b. Furthermore, this compound 17 dose-dependently suppressed methamphetamine-induced hyperlocomotion in mice.     

The discovery and optimization of a series of 2-aminobenzoxazole derivatives as ChemR23 inhibitors

A structural class of 2-aminobenzoxazole derivatives possessing biphenyltetrazole was discovered to be potent human ChemR23 inhibitors. We initially tried to improve the potency of compound 1, which was found through in-house screening using the human plasmacytoid dendritic cell (pDC)-like cell line CAL-1. The introduction of a chiral methyl moiety at a benzylic position in a center of compound 1 showed a large impact on the inhibitory activity against calcium signaling of ChemR23 induced by the natural ligand chemerin. As a result of further investigations at the benzylic position, (R)-isomer 6b was found to show a 30-fold increased potency over desmethyl compound 1. In addition, an extensive structure-activity relationship study on the benzoxazole moiety successfully led to a further increase in the potency. The antagonistic effect of the compounds was based on the induction of ChemR23 internalization. In addition, we observed that compound 31, which contained an amide moiety on benzoxazole, inhibited chemotaxis of CAL-1 cells induced by chemerin in vitro. These results suggest that our ChemR23 inhibitors are attractive compounds for the treatment of pDC-related autoimmune diseases, such as systemic lupus erythematosus and psoriasis.     

Synthesis and biological evaluation of C-glucosides with azulene rings as selective SGLT2 inhibitors for the treatment of type 2 diabetes mellitus: Discovery of YM543

Here, a series of C-glucosides with azulene rings in the aglycon moiety was synthesized and the inhibitory activities toward hSGLT1 and hSGLT2 were evaluated. Starting from the azulene derivative 7 which had relatively good SGLT2 inhibitory activity, compound 8a which has a 3-[(azulen-2-yl)methyl]phenyl group was identified as a lead compound for further optimization. Introduction of a phenolic hydroxyl group onto the central benzene ring afforded a potent and selective SGLT2 inhibitor 8e, which reduced blood glucose levels in a dose-dependent manner in rodent diabetic models. A mono choline salt of 8e (YM543) was selected as a clinical candidate for use in treating type 2 diabetes mellitus.     

Synthesis and biological evaluation of novel potent FFA1 agonists containing 2,3-dihydrobenzo[b][1,4]dioxine

FFA1 (free fatty acid receptor 1) has emerged as an attractive antidiabetic target due to its role in mediating the enhancement of glucose-stimulated insulin secretion in pancreatic β cells with a low risk of hypoglycemia. Many reported FFA1 agonists possessed somewhat pharmacokinetic and/or safety issues. Herein, we describe the identification of 2,3-dihydrobenzo[b][1,4]dioxine as a novel scaffold for FFA1 agonists. Comprehensive structure-activity relationship study based on this scaffold led to the discovery of (S)-3-(4-(((S)-7-(4-methoxyphenyl)-2,3-dihydrobenzo [b][1,4]dioxin-2-yl)methoxy) phenyl)hex-4-ynoic acid (26k), which displayed a potent FFA1 agonistic activity and good pharmacokinetic profiles. Subsequent in vivo studies demonstrated that compound 26k significantly improved the glucose tolerance in ICR mice. In summary, compound 26k is a promising drug candidate for further investigation.     

Discovery of N-sulfonyl-7-azaindoline derivatives as potent,orally available and selective M4 muscarinic acetylcholine receptor agonists

We designed and synthesized novel N-sulfonyl-7-azaindoline derivatives as selective M₄ muscarinic acetylcholine receptor agonists. Modification of the N-carbethoxy piperidine moiety of compound 2, an M₄ muscarinic acetylcholine receptor (mAChR)-preferring agonist, led to compound 1, a selective M₄ mAChR agonist. Compound 1 showed a highly selective M₄ mAChR agonistic activity with weak hERG inhibition in vitro. A pharmacokinetic study of compound 1 in vivo revealed good bioavailability and brain penetration in rats. Compound 1 reversed methamphetamine-induced locomotor hyperactivity in rats (1–10 mg/kg, po).     

N-Thiazolylamide-based free fatty-acid 2 receptor agonists: Discovery,lead optimization and demonstration of off-target effect in a diabetes model

Free fatty acid-2 (FFA2) receptor is a G-protein coupled receptor of interest in the development of therapeutics in metabolic and inflammatory disease areas. The discovery and optimization of an N-thiazolylamide carboxylic acid FFA2 agonist scaffold is described. Dual key objectives were to i) evaluate the potential of this scaffold for lead optimization in particular with respect to safety de-risking physicochemical properties, i.e. lipophilicity and aromatic content, and ii) to demonstrate the utility of selected lead analogues from this scaffold in a pertinent in vivo model such as oral glucose tolerance test (OGTT). As such, a concomitant improvement in bioactivity together with lipophilic ligand efficiency (LLE) and fraction sp³ content (Fsp³) parameters guided these efforts. Compound 10 was advanced into studies in mice on the basis of its optimized profile vs initial lead 1 (ΔLLE?=?0.3, ΔFsp³?=?0.24). Although active in OGTT, 10 also displayed similar activity in the FFA2-knockout mice. Given this off-target OGTT effect, we discontinued development of this FFA2 agonist scaffold.