Optimization of M4 positive allosteric modulators (PAMs): The discovery of VU0476406, a non-human primate in vivo tool compound for translational pharmacology

This letter describes the further chemical optimization of the 5-amino-thieno[2,3-c]pyridazine series (VU0467154/VU0467485) of M₄ positive allosteric modulators (PAMs), developed via iterative parallel synthesis, culminating in the discovery of the non-human primate (NHP) in vivo tool compound, VU0476406 (8p). VU0476406 is an important in vivo tool compound to enable translation of pharmacodynamics from rodent to NHP, and while data related to a Parkinson’s disease model has been reported with 8p, this is the first disclosure of the optimization and discovery of VU0476406, as well as detailed pharmacology and DMPK properties.     

Design,synthesis, and biological evaluation of spiroindolines as novel inducers of oligodendrocyte progenitor cell differentiation—Use of a conformation-based hypothesis to facilitate compound design

Inducing oligodendrocyte progenitor cell (OPC) differentiation is a novel therapeutic strategy for the treatment of demyelinating diseases such as multiple sclerosis (MS). In the preceding article, we detailed the discovery of compound 1, a potent inducer of OPC differentiation possessing a characteristic spiroindoline structure. Also, we found that N-methylation and des-carbonyl compound 1 (4) led to a loss in potency. Herein, we describe our investigations of a conformation-based hypothesis for OPC differentiation activity based on the preferred conformation of the spiro core, and further structure–activity relationship (SAR) exploration led to the identification of 6-CF₃ derivative 8, which was more potent compared to compound 1.     

Inhibitory effects in vitro of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-glutathione,a proposed metabolite of l-histidine,on γ-glutamyltransferase activity

A transfer of the γ-glutamyl moiety of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (I), an adduct of glutathione and l-histidine metabolite urocanic acid, has been investigated by using γ-glutamyltransferase preparation from bovine kidney. When an equimolar mixture of two diastereomers of compound I in a phosphate buffer was allowed to react with glycylglycine in the presence of the transferase, two diastereomers of N-{S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteinyl}glycine (II) were formed in the same yield with each other and this was accompanied by a formation of γ-glutamylglycylglycine. Kinetics of compound I with the transferase indicated high affinity between the two materials, while the maximal reaction velocity of the γ-glutamyl transfer was low. Effects of compound I in vitro on the transfer of γ-glutamyl moiety of γ-glutamyl-p-nitroanilide to glycylglycine with the transferase were also studied, and the results indicated that the transfer was suppressed by compound I based on its competitive and non-competitive inhibitions. These results suggest that little variation in reactivities of two diastereomers of compound I as the substrate is given by the difference in stereomerism of their asymmetric carbon atoms and that inhibitory effects of compound I on the catalytic action of the transferase is of sufficient physiological importance to decrease the degradation of natural γ-glutamyl compounds, such as glutathione and its analogs.     

Synthesis,antiplatelet and antithrombotic activities of resveratrol derivatives with NO-donor properties

Resveratrol (RVT) is a stilbene with a protective effect on the cardiovascular system; however, drawbacks including low bioavailability and fast metabolism limit its efficacy. In this work we described new resveratrol derivatives with nitric oxide (NO) release properties, ability to inhibit platelet aggregation and in vivo antithrombotic effect. Compounds (4a–f) were able to release NO in vitro, at levels ranging from 24.1% to 27.4%. All compounds (2a–f and 4a–f) have exhibited platelet aggregation inhibition using as agonists ADP, collagen and arachidonic acid. The most active compound (4f) showed reduced bleeding time compared to acetylsalicylic acid (ASA) and protected up to 80% against in vivo thromboembolic events. These findings suggest that hybrid resveratrol-furoxan (4f) is a novel lead compound able to prevent platelet aggregation and thromboembolic events.     

Discovery of 1-(1,3,5-triazin-2-yl)piperidine-4-carboxamides as inhibitors of soluble epoxide hydrolase

1-(1,3,5-Triazin-yl)piperidine-4-carboxamide inhibitors of soluble epoxide hydrolase were identified from high through-put screening using encoded library technology. The triazine heterocycle proved to be a critical functional group, essential for high potency and P450 selectivity. Phenyl group substitution was important for reducing clearance, and establishing good oral exposure. Based on this lead optimization work, 1-[4-methyl-6-(methylamino)-1,3,5-triazin-2-yl]-N-{[[4-bromo-2-(trifluoromethoxy)]-phenyl]methyl}-4-piperidinecarboxamide (27) was identified as a useful tool compound for in vivo investigation. Robust effects on a serum biomarker, 9, 10-epoxyoctadec-12(Z)-enoic acid (the epoxide derived from linoleic acid) were observed, which provided evidence of robust in vivo target engagement and the suitability of 27 as a tool compound for study in various disease models.     

Synthetic strategy for increasing solubility of potential FLT3 inhibitor thieno[2,3-d]pyrimidine derivatives through structural modifications at the C2 and C6 positions

Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic progenitor cell. In AML, a mutation in FLT3 is commonly occurs and is associated with poor prognosis. We have previously reported that thieno[2,3-d]pyrimidine derivative compound 1 exhibited better antiproliferative activity against MV4-11 cells which harbor mutant FLT3 than AC220, which is a well-known FLT3 inhibitor, and has good microsomal stability. However, compound 1 had poor solubility. We then carried out further structural modification at the C₂ and the C₆ positions of thieno[2,3-d]pyrimidine scaffold. Compound 13b, which possesses a thiazole moiety at the C₂ position, exhibited better antiproliferative activity than compound 1 and showed increased solubility and moderate microsomal stability. These results indicate that compound 13b could be a promising potential FLT inhibitor for AML chemotherapy.     

Discovery of novel 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid derivatives as HIF prolyl hydroxylase inhibitors for treatment of renal anemia

Prolyl hydroxylase domain-containing protein (PHD) inhibitors are useful as orally administered agents for the treatment of renal anemia. Based on the common structures of known PHD inhibitors, we found novel PHD inhibitor 1 with a 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid motif. The PHD2-inhibitory activity, lipophilicity (CLogP), and PK profiles (hepatocyte metabolism, protein binding, and/or elimination half-life) of this inhibitor were used as the evaluation index to optimize the structure and eventually discovered clinical candidate 42 as the suitable compound. Compound 42 was demonstrated to promote the production of erythropoietin (EPO) following oral administration in mice and rats. The predicted half-life of this compound in humans was 1.3–5.6?h, therefore, this drug may be expected to administer once daily with few adverse effects caused by excessive EPO production.     

Synthesis and evaluation of an orally available “Y”-shaped biaryl peroxisome proliferator-activated receptor δ agonist

In this study, we designed and synthesized several novel “Y”-shaped biaryl PPARδ agonists. Structure-activity relationship (SAR) studies demonstrated that compound 3a was the most active agonist with an EC₅₀ of 2.6?nM. We also synthesized and evaluated enantiospecific R and S isomers of compound 3a to confirm that R isomer (EC₅₀?=?0.7?nM) shows much more potent activity than S isomer (EC₅₀?=?6.1?nM). Molecular docking studies between the PPAR ligand binding domain and enantiospecific R and S isomers of compound 3a were performed. In vitro absorption, distribution, metabolism, excretion, and toxicity (ADMET) and in vivo PK profiles show that compound 3a possesses superior drug-like properties including good bioavailability. Our overall results clearly demonstrate that this orally administrable PPARδ agonist 3a is a viable drug candidate for the treatment of various PPARδ-related disorders.     

11a-N-tosyl-5-carbapterocarpans: Synthesis,antineoplastic evaluation and in silico prediction of ADMETox properties

11a-N-tosyl-5-carbapterocarpans (5a–c and 6a–c), 9-N-tosyl-4,4a,9,9a-tetrahydro-3H-carbazole (7), 11a-N-tosyl-5-carbapterocarpen (8) analogues of LQB-223 (4a), were synthesized through palladium catalyzed azaarylation of substituted dihydronaphtalenes (14a–c) and cyclohexadiene (15), respectively, with N-tosyl-o-iodoaniline (11). In order to understand the role of the N-tosyl moiety for the pharmacological activity, the azacarbapterocarpen (9) was also synthesized by Fischer indol reaction. The structural requirements at the A and D-rings for the antineoplastic activity toward human leukemias and breast cancer cells were evaluated as well. Substitutions on the A-ring of 4a and analogues alter the effect on different breast cancer subtypes. On the other hand, A-ring is not essential for antileukemic activity since compound 7, which does not contain the A-ring, showed efficacy with high selectivity indices for drug-resistant leukemias. On the other hand, substitutions on the D-ring of 4a for fluorine or iodine did not improve the antileukemic activity. In silico studies concerning Lipinskís rule of five, ADMET properties and drug scores of those compounds were performed, indicating good physicochemical properties for all compounds, in special for compound 7.     

Design and synthesis of 6,7-methylenedioxy-4-substituted phenylquinolin-2(1H)-one derivatives as novel anticancer agents that induce apoptosis with cell cycle arrest at G2/M phase

Novel 6,7-methylenedioxy-4-substituted phenylquinolin-2(1H)-one derivatives 12a–n were designed and prepared through an intramolecular cyclization reaction and evaluated for in vitro anticancer activity. Among the synthesized compounds, 6,7-methylenedioxy-4-(2,4-dimethoxyphenyl)quinolin-2(1H)-one (12e) displayed potent cytotoxicity against several different tumor cell lines at a sub-micromolar level. Furthermore, results of fluorescence-activated cell sorting (FACS) analysis suggested that 12e induced cell cycle arrest in the G2/M phase accompanied by apoptosis in HL-60 and H460 cells. This action was confirmed by Hoechst staining and caspase-3 activation. Due to their easy synthesis and remarkable biological activities, 4-phenylquinolin-2(1H)-one analogs (4-PQs) are promising new anticancer leads based on the quinoline scaffold. Accordingly, compound 12e was identified as a new lead compound that merits further optimization and development as an anticancer candidate.     

Design and synthesis of a novel 1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative as an orally available ACC1 inhibitor

We initiated our structure-activity relationship (SAR) studies for novel ACC1 inhibitors from 1a as a lead compound. Our initial SAR studies of 1H-Pyrrolo[3,2-b]pyridine-3-carboxamide scaffold revealed the participation of HBD and HBA for ACC1 inhibitory potency and identified 1-methyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative 1c as a potent ACC1 inhibitor. Although compound 1c had physicochemical and pharmacokinetic (PK) issues, we investigated the 1H-pyrrolo[3,2-b]pyridine core scaffold to address these issues. Accordingly, this led us to discover a novel 1-isopropyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative 1k as a promising ACC1 inhibitor, which showed potent ACC1 inhibition as well as sufficient cellular potency. Since compound 1k displayed favorable bioavailability in mouse cassette dosing PK study, we conducted in vivo Pharmacodynamics (PD) studies of this compound. Oral administration of 1k significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at a dose of 100 mg/kg. Accordingly, our novel series of potent ACC1 inhibitors represent useful orally-available research tools, as well as potential therapeutic agents for cancer and fatty acid related diseases.     

Discovery and synthesis of tetrahydropyrimidinedione-4-carboxamides as endothelial lipase inhibitors

Endothelial lipase (EL) inhibitors have been shown to elevate HDL-C levels in pre-clinical murine models and have potential benefit in prevention and treatment of cardiovascular diseases. Modification of the 1-ethyl-3-hydroxy-1,5-dihydro-2H-pyrrol-2-one (DHP) lead, 1, led to the discovery of a series of potent tetrahydropyrimidinedione (THP) EL inhibitors. Synthesis and SAR studies including modification of the amide group, together with changes on the pyrimidinone core led to a series of arylcycloalkyl, indanyl, and tetralinyl substituted 5-amino or 5-hydroxypyrimidinedione-4-carboxamides. Several compounds were advanced to PK evaluation. Among them, compound 4a was one of the most potent with measurable EL_HDL hSerum potency and compound 3g demonstrated the best overall pharmacokinetic parameters.     

Benzoxazine derivatives of phytophenols show anti-plasmodial activity via sodium homeostasis disruption

Development of new class of anti-malarial drugs is an essential requirement for the elimination of malaria. Bioactive components present in medicinal plants and their chemically modified derivatives could be a way forward towards the discovery of effective anti-malarial drugs. Herein, we describe a new class of compounds, 1,3-benzoxazine derivatives of pharmacologically active phytophenols eugenol (compound 3) and isoeugenol (compound 4) synthesised on the principles of green chemistry, as anti-malarials. Compound 4, showed highest anti-malarial activity with no cytotoxicity towards mammalian cells. Compound 4 induced alterations in the intracellular Na⁺ levels and mitochondrial depolarisation in intraerythrocytic Plasmodium falciparum leading to cell death. Knowing P-type cation ATPase PfATP4 is a regulator for sodium homeostasis, binding of compound 3, compound 4 and eugenol to PfATP4 was analysed by molecular docking studies. Compounds showed binding to the catalytic pocket of PfATP4, however compound 4 showed stronger binding due to the presence of propylene functionality, which corroborates its higher anti-malarial activity. Furthermore, anti-malarial half maximal effective concentration of compound 4 was reduced to 490?nM from 17.54?µM with nanomaterial graphene oxide. Altogether, this study presents anti-plasmodial potential of benzoxazine derivatives of phytophenols and establishes disruption of parasite sodium homeostasis as their mechanism of action.     

Discovery of triazole-based uracil derivatives bearing amide moieties as novel dipeptidyl peptidase-IV inhibitors

Dipeptidyl peptidase-IV (DPP-4) is a validated target for T2DM treatment. We previously reported a novel series of triazole-based uracil derivatives bearing aliphatic carboxylic acids with potent DPP-4 inhibitory activities in vitro, but these compounds showed poor hypoglycemic effects in vivo. Herein we further optimized the triazole moiety by amidation of the carboxylic acid to improve in vivo activities. Two series of compounds 3a-f and 4a-g were designed and synthesized. By screening in DPP-4, compound 4c was identified as a potent DPP-4 inhibitor with the IC₅₀ value of 28.62 nM. Docking study revealed compound 4c has a favorable binding mode and interpreted the SAR of these analogs. DPP-8 and DPP-9 tests indicated compound 4c had excellent selectivity over DPP-8 and DPP-9. Further in vivo evaluations revealed that compound 4c showed more potent hypoglycemic activity than its corresponding carboxylic acid in ICR mice and dose-dependently reduced glucose levels in type 2 diabetic C57BL/6 mice. The overall results have shown that compound 4c could be a promising lead for further development of novel DPP-4 agents treating T2DM.     

Synthesis,nitric oxide release,and dipeptidyl peptidase-4 inhibition of sitagliptin derivatives as new multifunctional antidiabetic agents

Nitric oxide (NO) dysfunction has been found to be an important factor in both the development and progression of diabetic complications due to its many roles in the vascular system. Multifunctional compounds with hypoglycemic and endothelial protective action will be promising agents for the treatment of diabetes and its complications. In this study, a series of novel NO-donating sitagliptin derivatives and relevant metabolites were synthesized and evaluated as potential multifunctional hypoglycemic agents. All of synthetic compounds shown remarkable inhibitory activity against dipeptidyl peptidase IV (DPP-IV) in vitro and demonstrated excellent hypoglycemic activities in diabetic mice, similar to the activity of sitagliptin, and compounds T1-T4 shown different extents of NO-releasing abilities and potent antioxidant abilities in vivo. By screening in DPP-4, compound T4 was recognized as a potent DPP-4 inhibitor with the IC₅₀ value of 0.060?μM. Docking study revealed compound T4 has a favorable binding mode. Furthermore, compounds T1-T4 exhibited different extents of NO-releasing abilities and excellent anti-platelet aggregation in vitro. The overall results suggested that T4 could help to the amelioration of endothelial dysfunction by reducing blood glucose, lessening oxidative stress and raising NO levels as well as inhibiting platelet aggregation. Based on this research, compound T4 deserves further investigation as potential new multifunctional anti-diabetic agent with antioxidant, anti-platelet aggregation and endothelial protective properties.     

One-dimensional ladder like and two-dimensional polymorphs of heterometallic thiocyanate bridged copper(II) and mercury(II) coordination polymer: Syntheses, structural, vibration, luminescence and EPR studies

The 1D triclinic {CuHg(en)(μ-NCS-N,S)₄}_n (1) and 2D monoclinic {CuHg(en)(μ-NCS-N,S)₃(SCN)}_n (2) (en = ethylenediamine) heterometallic coordination polymers, as the two polymorphs of the {CuHg(en)(NCS)₄}_n, were synthesized at room temperature by the reaction of mercuric thiocyanate, potassium thiocyanate, copper(II) malonate and ethylenediamine using different reagent ratios. XRD on single crystals shows that the compound 1 consists of 1D ladder like zigzag ribbons extended along the b axis, whereas compound 2 shows a 2D wavy polymeric structure running parallel to the ab plane. In the crystal packing of the both polymorphs, the polymeric structures are further interlinked to each other via weak interactions and hydrogen bonding to afford a 3D network. Diagnostic ligand and metal-ligand bands in the IR, far-IR and Raman spectra are assigned for the studied compounds. While compound 1 shows no significant emission upon excitation at any wavelength in the UV-Vis region, compound 2 exhibits intense emission at around 410 nm. Moreover, the room temperature X-band EPR spectrum of a powdered sample of 1, shows a signal of rhombic symmetry with g₁ = 2.2637, g₂ = 2.0765 and g₃ = 2.0483. In contrast to this, 2 reveals an axial signal with g_⊥ = 2.0742; however, the g_|| is unresolved.     

Design,stereoselective synthesis,and biological evaluation of novel tri-cyclic compounds as inhibitor of apoptosis proteins (IAP) antagonists

We recently reported the discovery of octahydropyrrolo[1,2-a]pyrazine A as a lead compound for an inhibitor of apoptosis proteins (IAP) antagonist. To develop IAP antagonists with favorable PK profiles, we designed novel tri-cyclic compounds, octahydro-1H-cyclopropa[4,5]pyrrolo[1,2-a]pyrazines 1 and 2 based on co-crystal structural analysis of A with cellular IAP-1 (cIAP-1). The additional cyclopropane moiety was used to block the predicted metabolic site of compound A without detriment to the binding affinity for cIAP. Compounds 1 and 2 were stereoselectively synthesized via intermediates 4a and 5b′, which were obtained by Simmons?Smith cyclopropanation of ethylester 3a and silyl ether 3b′. Compounds 1 and 2 showed strong growth inhibition in MDA-MB-231 breast cancer cells and improved metabolic stability in comparison to A. Compound 2 exhibited significant in vivo PD effects to increase tumor necrosis factor-alpha mRNA in a dose dependent manner.     

Structure-based design of a new series of N-(piperidin-3-yl)pyrimidine-5-carboxamides as renin inhibitors

The action of the aspartyl protease renin is the rate-limiting initial step of the renin-angiotensin-aldosterone system. Therefore, renin is a particularly promising target for blood pressure as well as onset and progression of cardiovascular and renal diseases. New pyrimidine derivatives 5–14 were designed in an attempt to enhance the renin inhibitory activity of compound 3 identified by our previous fragment-based drug design approach. Introduction of a basic amine essential for interaction with the two aspartic acids in the catalytic site and optimization of the S1/S3 binding elements including an induced-fit structural change of Leu114 (‘Leu-in’ to ‘Leu-out’) by a rational structure-based drug design approach led to the discovery of N-(piperidin-3-yl)pyrimidine-5-carboxamide 14, a 65,000-fold more potent renin inhibitor than compound 3. Surprisingly, this remarkable enhancement in the inhibitory activity of compound 14 has been achieved by the overall addition of only seven heavy atoms to compound 3. Compound 14 demonstrated excellent selectivity over other aspartyl proteases and moderate oral bioavailability in rats.     

Inhibitory effects of novel synthetic methimazole derivatives on mushroom tyrosinase and melanogenesis

In this study, we synthesized 4 methimazole (2-mercapto-1-methylimidazole, MMI) derivatives. The kinetics of inhibition on mushroom tyrosinase by methimazole and its derivatives were investigated. The results indicated that tert-butyl 3-methyl-2-sulfanylidene-2,3-dihydro-1H-imidazole-1-carboxylate (compound 3; 3), 2-mercaptoimidazole (MI; compound 1; 1) and MMI (compound 2; 2) significantly inhibited tyrosinase activity in a dose-dependent manner, exhibiting an IC₅₀ value of 1.50 mM, 4.11 mM, and 1.43 mM. However, compound 4 (4), compound 5 (5), and compound 6 (6) exerted no inhibitory effect on mushroom tyrosinase activity. Kinetic analysis indicated that 3 was a noncompetitive tyrosinase inhibitor, whereas both 1 and 2 were exhibited as mixed-type tyrosinase inhibitors. Furthermore, 3 exerted a potent inhibitory effect on intracellular melanin formation in the B16/F10 murine melanoma cells and did not cause cytotoxicity, as 1 and 2 did.     

Discovery of N-(2,3,5-triazoyl)mycophenolic amide and mycophenolic epoxyketone as novel inhibitors of human IMPDH

Syntheses of ten derivatives of mycophenolic acid (MPA) at C-6′ position, and structure–activity relationship study among these derivatives, MPA and mycophenolic hydroxamic acid (MPHA) led to discovery of N-(2,3,5-triazolyl)mycophenolic amide 4, (7′S) mycophenolic epoxyketone 9 and (7′R) mycophenolic epoxyketone 10 having potent inhibitory activity against human inosine-5′-monophosphate dehydrogenase (IMPDH) type I and II as well as antiproliferative activity on human leukemia K562 cells. Compounds 4, 9, and 10 showed induction activity of erythroid differentiation in K562 cells. Inhibitory effects of 4 and 10 against IMPDH were attenuated by supplemental guanosine in K562 cells. In contrast, attenuation effect by supplemental guanosine was not significant in the case of 9. Compound 9 weakly inhibited the enzyme activity of HDAC in the nuclear lysate of K562 cells at 10 μM. These observations suggest that the primary target of 4, 9, and 10 is IMPDH, whereas compound 9 partially inhibits a certain type of HDAC.