Selective antagonists of mouse trace amine-associated receptor 1 (mTAAR1): discovery of EPPTB (RO5212773)

High throughput screening of the Roche compound library identified benzanilides such as 1 and 2 as antagonists of TAAR1. Optimisation of this hit series led to the first selective TAAR1 antagonist (N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide EPPTB (RO5212773, 9f) having IC₅₀ of 28 nM at mouse TAAR1.     

4-Aminophenyl acetamides and propanamides as potent transient receptor potential vanilloid 1 (TRPV1) ligands

A series of 2-(3,5-substituted 4-aminophenyl)acetamide and propanamide derivatives were investigated as human TRPV1 antagonists. The analysis of the structure-activity relationship indicated that 2-(3,5-dihalo 4-aminophenyl)acetamide analogues displayed excellent antagonism of hTRPV1 activation by capsaicin and showed improved potency compared to the corresponding propanamides. The most potent antagonist (36) exhibited potent and selective antagonism for hTRPV1 not only to capsaicin but also to NADA and elevated temperature; however, it only displayed weak antagonism to low pH. Further studies indicated that oral administration of antagonist 36 blocked the hypothermic effect of capsaicin in vivo but demonstrated hyperthermia at that dose. A docking study of 36 was performed in our established hTRPV1 homology model to understand its binding interactions with the receptor and to compare with that of previous antagonist 1.     

Discovery of ORN0829, a potent dual orexin 1/2 receptor antagonist for the treatment of insomnia

Here, we present the design, synthesis, and SAR of dual orexin 1 and 2 receptor antagonists, which were optimized by balancing the antagonistic activity for orexin receptors and lipophilicity. Based on the prototype compound 1, ring construction and the insertion of an additional heteroatom into the resulting ring led to the discovery of orexin 1 and 2 receptor antagonists, which were 3-benzoyl-1,3-oxazinane derivatives. Within these derivatives, (−)-3h enabled a high dual orexin receptor antagonistic activity and a low lipophilicity. Compound (−)-3h exhibited potent sleep-promoting effects at a po dose of 1 mg/kg in a rat polysomnogram study, and optimal PK properties with a rapid T_max and short half-lives in rats and dogs were observed, indicating a predicted human half-life of 0.9–2.0 h. Thus, (−)-3h (ORN0829; investigation code name, TS-142) was selected as a viable candidate and is currently in clinical development for the treatment of insomnia.     

Inverse Agonistic Action of 3-Iodothyronamine at the Human Trace Amine-Associated Receptor 5

ObjectiveApplication of 3-iodothyronamine (3-T₁AM) results in decreased body temperature and body weight in rodents. The trace amine-associated receptor (TAAR) 1, a family A G protein-coupled receptor, is a target of 3-T₁AM. However, 3-T₁AM effects still persist in mTaar1 knockout mice, which suggest so far unknown further receptor targets that are of physiological relevance. TAAR5 is a highly conserved TAAR subtype among mammals and we here tested TAAR5 as a potential 3-T₁AM target. First, we investigated mouse Taar5 (mTaar5) expression in several brain regions of the mouse in comparison to mTaar1. Secondly, to unravel the full spectrum of signaling capacities, we examined the distinct G_s-, G_i/o-, G_12/13-, G_q/11- and MAP kinase-mediated signaling pathways of mouse and human TAAR5 under ligand-independent conditions and after application of 3-T₁AM. We found overlapping localization of mTaar1 and mTaar5 in the amygdala and ventromedial hypothalamus of the mouse brain. Second, the murine and human TAAR5 (hTAAR5) display significant basal activity in the G_q/11 pathway but show differences in the basal activity in G_s and MAP kinase signaling. In contrast to mTaar5, 3-T₁AM application at hTAAR5 resulted in significant reduction in basal IP₃ formation and MAP kinase signaling. In conclusion, our data suggest that the human TAAR5 is a target for 3-T₁AM, exhibiting inhibitory effects on IP₃ formation and MAP kinase signaling pathways, but does not mediate G_s signaling effects as observed for TAAR1. This study also indicates differences between TAAR5 orthologs with respect to their signaling profile. In consequence, 3-T₁AM-mediated effects may differ between rodents and humans.     

One-step partial synthesis of (±)-asperteretone B and related hPTP1B1–400 inhibitors from butyrolactone I

Protein tyrosine phosphatase 1B (PTP1B) is a validated target for developing antiobesity, antidiabetic and anticancer drugs. Over the past years, several inhibitors of PTP1B have been discovered; however, none has been approved by the drug regulatory agencies. Interestingly, the research programs focused on discovering PTP1B inhibitors typically use truncated structures of the protein (PTP1B_1-300, 1–300 amino acids), leading to the loss of valuable information about the inhibition and selectivity of ligands and repeatedly misleading the optimization of putative drug leads. Up to date, only six inhibitors of the full-length protein (hPTP1B_1-400), with affinity constants ranging from 1.3 × 10⁴ to 3.3 × 10⁶ M⁻¹, have been reported. Towards the discovery of new ligands of the full-length human PTP1B (hPTP1B_1-400) from natural sources, herein we describe the isolation of a γ-lactone (1, butyrolactone I) from the fungus Aspergillus terreus, as well as the semisynthesis, inhibitory properties (in vitro and in silico), and the structure-activity relationship of a set of butyrolactone derivatives (1 and 2, and 6–12) as hPTP1B_1-400 inhibitors, as well as the affinity constant (k_a = 2.2 × 10⁵ M⁻¹) of the 1-hPTP1B₁–₄₀₀ complex, which was determined by fluorescence quenching experiments, after the inner filter effect correction.     

Synthesis and docking studies of novel antitumor benzimidazoles

In this work, the benzimidazole-pyrrole conjugates 6a–h and benzimidazole-tetracycles conjugates 12–14 were prepared. The cytotoxicity of the compounds 3, 4a–h, 6a–h, 8, 10 and 12–14 was tested against lung cancer cell line A549. Compound 6b exhibited higher activity than the bis-benzoxazole natural product (UK-1), the standard. The tested 4g,h, 6a–h, 10 and 12–14 exhibited remarkable cytotoxicity activity against breast cancer cell line MCF-7 with higher activity than tamoxifen. Furthermore, compound 4h was found to be also more potent than doxurubicin. The antitumor promotion activity of synthesized compounds 4g,h, 6a–h, 10 and 12–14 has been estimated by studying their possible inhibitory effects on EBV-EA activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among the studied compounds, the inhibitory activities of compounds 8, 13 and 14 demonstrated strong inhibitory effects on the Epstein–Barr virus early antigen (EBV-EA) activation without showing any cytotoxicity on the Raji cells and their effects being stronger than that of a representative control, oleanolic acid.Moreover, the molecular docking of the new compounds into plasminogen activator (uPA) receptor has been in correlation with the antitumor activity. All synthesized compounds 3, 4a–h, 6a–h, 8, 10 and 12–14 were docked into same groove of the binding site of the native co-crystalized (4-iodobenzo[b]thiophene-2-carboxamidine) ligand (PDB code:1c5x) for activity explaination. Compounds 4h, 6b and 13, giving the best docking results, were further studied to estimate their effect on the level of uPA using AssayMax human urokinase (uPA) ELISA kit. In case of A549 cell line, compound 6 exhibited similar activity to MMC, and for MCF-7 cell line, compound 4h exhibited similar activity to doxorubicin, in inhibiting the expression of uPA.     

Characterization of new PPARγ agonists: Benzimidazole derivatives—importance of positions 5 and 6, and computational studies on the binding mode

In this and previous studies we investigated the importance of partial structures of Telmisartan on PPARγ activation. The biphenyl-4-ylmethyl moiety at N1 and residues at C2 of the central benzimidazole were identified to be essential for receptor activation and potency of receptor binding. Now we focused our attention on positions 5 and 6 of the central benzimidazole and introduced bromine (3b–5/6, 3c), phenylcarbonyl (3d–5/6), hydroxy(phenyl)methyl (3g–5/6), hydroxymethyl (3h–5/6) and formyl (3i) groups. The selection of these moieties was inspired by the structure of Losartan and its metabolite EXP3179. In order to increase the hydrophobicity of the central part of the molecule, the benzimidazole was exchanged by a naphtho[2,3-d]imidazole (5). The compounds 3a–3i and 5 were tested in a differentiation assay using 3T3-L1 preadipocytes and a luciferase assay using COS-7 cells, transiently transfected with pGal4-hPPARγDEF, pGal5-TK-pGL3 and pRL-CMV, as established models for the assessment of cellular PPARγ activation. An enhanced effect on PPARγ activation could be observed if lipophilic moieties are introduced in these positions. 4′-[(2-Propyl-1H-naphtho[2,3-d]imidazol-1-yl)methyl]biphenyl-2-carboxylic acid (5) was identified as the most potent compound with an EC₅₀ of 0.26 μM and the profile of a full agonist.Together with compounds of the former structure–activity relationship study (position 2-substituted benzimidazole derivatives 4a–4j), the binding mode of Telmisartan and its derivatives have been analyzed in 3D pharmacophore-driven docking experiments.     

A new series of aryl sulfamate derivatives: Design,synthesis, and biological evaluation

Steroid sulfatase (STS) has recently emerged as a drug target for management of hormone-dependent malignancies. In the present study, a new series of twenty-one aryl amido-linked sulfamate derivatives 1a-u was designed and synthesized, based upon a cyclohexyl lead compound. All members were evaluated as STS inhibitors in a cell-free assay. Adamantyl derivatives 1h and 1p-r were the most active with more than 90% inhibition at 10 µM concentration and, for those with the greatest inhibitory activity, IC₅₀ values were determined. These compounds exhibited STS inhibition within the range of ca 25–110 nM. Amongst them, compound 1q possessing a o-chlorobenzene sulfamate moiety exhibited the most potent STS inhibitory activity with an IC₅₀ of 26 nM. Furthermore, to assure capability to pass through the cell lipid bilayer, compounds with low IC₅₀ values were tested against STS activity in JEG-3 whole-cell assays. Consequently, 1h and 1q demonstrated IC₅₀ values of ca 14 and 150 nM, respectively. Thus, compound 1h is 31 times more potent than the corresponding cyclohexyl lead (IC₅₀ value = 421 nM in a JEG-3 whole-cell assay). Furthermore, the most potent STS inhibitors (1h and 1p-r) were evaluated for their antiproliferative activity against the estrogen-dependent breast cancer cell line T-47D. They showed promising activity with single digit micromolar IC₅₀ values (ca 1–6 µM) and their potency against T-47D cells was comparable to that against STS enzyme. In conclusion, this new class of adamantyl-containing aryl sulfamate inhibitor has potential for further development against hormone-dependent tumours.     

TRPV1 antagonist with high analgesic efficacy: 2-Thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides

A series of 2-thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Among them, compound 24S showed stereospecific and excellent TRPV1 antagonism of capsaicin-induced activation. Further, it demonstrated strong anti-allodynic in a rat neuropathic pain model. Consistent with its action in vitro being through TRPV1, compound 24S blocked capsaicin-induced hypothermia in mice. Docking analysis of 24S with our hTRPV1 homology model was performed to identify its binding mode.     

A new class of pyrazolo[5,1-c][1,2,4]triazines as γ-aminobutyric type A (GABAA) receptor subtype ligand: synthesis and pharmacological evaluation

A comparison between compounds with pyrazolo[1,5-a]pyrimidine structure (series 4–6) and pyrazolo[5,1-c][1,2,4]triazine core (series 9) as ligands at GABA_A-receptor subtype, was evaluated. Moreover, for pyrazolotriazine derivatives having binding recognition, the interaction on recombinant rat α(1–3,5) GABA_A receptor subtypes, was performed. Among these latter, emerge compounds 9c, 9k, 9l, 9m and 9n as α1-selective and 9h as α2-selective ligands.     

Discovery of human lactate dehydrogenase 5 inhibitors (hLDH5) with anti-lung cancer activity through an in silico method and biological validation

Human lactate dehydrogenase 5 (hLDH5) is an important metabolic enzyme playing critical roles in the anaerobic glycolysis. Herein, we employed an in silico method and biological validation to identify a novel hLDH5 inhibitor with a promising cellular activity under hypoxia condition. The identified compound 9 bound to hLDH5 with a K_d value of 1.02 µM, and inhibited the enzyme with an EC₅₀ value of 0.7 µM. Compound 9 exhibited a weak potency against NCI-H1975 cell proliferation under normal condition (IC₅₀ = 36.5 µM), while dramatically increased to 5.7 µM under hypoxia condition. In line with the observation, hLDH5 expression in NCI-H1975 cell under hypoxia condition is much higher as compared to the normal oxygenated condition, indicating the hLDH5 inhibition may contribute to the cancer cell death.     

Genetic Polymorphisms Affect Mouse and Human Trace Amine-Associated Receptor 1 Function

Methamphetamine (MA) and neurotransmitter precursors and metabolites such as tyramine, octopamine, and β-phenethylamine stimulate the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 has been implicated in human conditions including obesity, schizophrenia, depression, fibromyalgia, migraine, and addiction. Additionally TAAR1 is expressed on lymphocytes and astrocytes involved in inflammation and response to infection. In brain, TAAR1 stimulation reduces synaptic dopamine availability and alters glutamatergic function. TAAR1 is also expressed at low levels in heart, and may regulate cardiovascular tone. Taar1 knockout mice orally self-administer more MA than wild type and are insensitive to its aversive effects. DBA/2J (D2) mice express a non-synonymous single nucleotide polymorphism (SNP) in Taar1 that does not respond to MA, and D2 mice are predisposed to high MA intake, compared to C57BL/6 (B6) mice. Here we demonstrate that endogenous agonists stimulate the recombinant B6 mouse TAAR1, but do not activate the D2 mouse receptor. Progeny of the B6XD2 (BxD) family of recombinant inbred (RI) strains have been used to characterize the genetic etiology of diseases, but contrary to expectations, BXDs derived 30–40 years ago express only the functional B6 Taar1 allele whereas some more recently derived BXD RI strains express the D2 allele. Data indicate that the D2 mutation arose subsequent to derivation of the original RIs. Finally, we demonstrate that SNPs in human TAAR1 alter its function, resulting in expressed, but functional, sub-functional and non-functional receptors. Our findings are important for identifying a predisposition to human diseases, as well as for developing personalized treatment options.     

Synthesis and pharmacological evaluation of novel 1,3,8- and 1,3,7,8-substituted xanthines as adenosine receptor antagonists

A number of novel xanthines bearing a variety of substituents at positions 1, 3, 7 and 8 were prepared and evaluated for their binding affinity to the human adenosine receptor A₁, A_2A, A_2B and A₃ subtypes. Several of the 1,3,8- and 1,3,7,8-substituted xanthines showed moderate-to-high affinity at human A_2B and A₁ receptors, with the most active compound (14q) having a pK_i of 7.57 nM for hA_2B receptors and a selectivity over hA_2A receptors of 8.1-fold and hA₁ receptors of 3.7-fold.     

Synthesis,antileishmanial and antitrypanosomal activities of N-substituted tetrahydro-β-carbolines

A series of N-substituted tetrahydro-β-carbolines were synthesized and screened for antileishmanial activity through an in vitro assay that involves promastigotes and axenic amastigotes of Leishmania donovani, the causative agent for visceral leishmaniasis. The thiophen-2-yl analogs 9b and 11f and naphthyl analog 11h were found to show significant activity against promastigotes with IC₅₀ values of 12.7, 9.1 and 22.1 μM, respectively. Analogs 9b and 11h were also effective against axenic amastigotes with IC₅₀ values of 62.8 and 87.6 μM, respectively. The antileishmanial activity of analogs was then tested in human macrophage cell line infected with L. donovani amastigotes and 2-naphthyl linked analog 11h was found to be effective with IC₅₀ value of 28.3 μM. Several analogs also displayed antitrypanosomal activity against Trypanosoma brucei, the causative agent for human African trypanosomiasis. Compounds 11e, 11f and 11h were more effective than others with IC₅₀ values of 1.0, 8.9 and 10.2 μM, respectively. All synthesized analogs were not cytotoxic towards mammalian cell lines including Vero (monkey kidney fibroblasts), HEPG2 (human hepatoma cells), LLC-PK₁ (pig kidney epithelial cells) and THP-1 (human macrophages).     

Sulfonate and sulfamate derivatives possessing benzofuran or benzothiophene nucleus as potent carbonic anhydrase II/IX/XII inhibitors

In the current work, we report the discovery of new sulfonate and sulfamate derivatives of benzofuran- and benzothiophene as potent inhibitors of human carbonic anhydrases (hCAs) II, IX and XII. A set of derivatives, 1a–t, having different substituents on the fused benzofuran and benzothiophene rings (R = alkyl, cyclohexyl, aryl, NH₂, NHMe, or NMe₂) was designed and synthesized. Most of the derivatives exhibited higher potency than acetazolamide as inhibitors of the purified hCAII, IX and XII isoforms. The most potent inhibitors for hCAII, hCAIX and hCAXII were 1g, 1b and 1d with an IC₅₀ ± SEM values of 0.14 ± 0.03, 0.13 ± 0.03 and 0.17 ± 0.06 µM, respectively. In addition, compounds 1d and 1n exerted preferential inhibitory effect against hCAXII isozyme with good potencies. Some selected compounds were docked within the active pocket of these isozymes and binding of the molecules revealed that sulfonate and sulfamate rings were located towards the active cavity and compounds coordinated to zinc ions.     

Discovery of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazoles as novel class of corticotropin releasing factor 1 receptor antagonists

A new class of corticotropin releasing factor 1 (CRF₁) receptor antagonists characterized by a tricyclic core ring was designed and synthesized. Novel tricyclic derivatives 2a–e were designed as CRF₁ receptor antagonists based on conformation analysis of our original 2-anilinobenzimidazole CRF₁ receptor antagonist. The synthesized tricyclic derivatives 2a–e showed CRF₁ receptor binding activity with IC₅₀ values of less than 400?nM, and the 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2e was selected as a lead compound with potent in vitro CRF₁ receptor binding activity (IC₅₀?=?7.1?nM). To optimize the pharmacokinetic profiles of lead compound 2e, we explored suitable substituents on the 1-position and 6-position, leading to the identification of compound 42c-R, which exhibited potent CRF₁ receptor binding activity (IC₅₀?=?58?nM) with good oral bioavailability (F?=?68% in rats). Compound 42c-R exhibited dose-dependent inhibition of [¹²⁵I]-CRF binding in the frontal cortex (5 and 10?mg/kg, p.o.) as well as suppression of locomotor activation induced by intracerebroventricular administration of CRF in rats (10?mg/kg, p.o.). These results suggest that compound 42c-R successfully binds CRF₁ receptors in the brain and exhibits the potential to be further examined for clinical studies.     

Computer-aided discovery and biological characterization of human lactate dehydrogenase 5 inhibitors with anti-osteosarcoma activity

Human lactate dehydrogenase 5 (hLDH5) is overexpressed in various tissues of human tumors, which could be a potential therapeutic target for cancer treatment. Herein, we describe the computer-aided discovery and biological characterizations of hLDH5 inhibitors with anti-osteosarcoma activity. Biochemical assay indicated that the identified compounds 3 and 9 strongly inhibited hLDH5 function with EC₅₀ values of 0.67 and 0.39?µM, respectively. The MTT assay revealed that most of the identified inhibitors had little effect on MG-63 cell proliferation at 4?µM, only 9 reduced the cancer cell proliferation at the same concentration, with an IC₅₀ value of 3.18?µM. Our data suggested that 9 could be a starting lead of developing potent hLDH5 inhibitor for the anti-osteosarcoma agents in cancer treatment.     

Novel 3,4-dihydro-4-oxoquinazoline-based acetohydrazides: Design,synthesis and evaluation of antitumor cytotoxicity and caspase activation activity

In search for novel small molecules with antitumor cytotoxicity via activating procaspase-3, we have designed and synthesized three series of novel (E)-N′-benzylidene-4-oxoquinazolin-3(4H)-yl)acetohydrazides (5a-j, 6a-h, and 7a-h). On the phenyl ring ò the benzylidene part, three different substituents, including 2-OH-4-OCH₃, 4-OCH₃, and 4-N(CH₃)₂, were introduced, respectively. Biological evaluation showed that the acetohydrazides in series 5a-j, in which the phenyl ring of the benzylidene part was substituted by 2-OH-4-OCH₃ substituent, exhibited potent cytotoxicity against three human cancer cell lines (SW620, colon; PC-3, prostate; NCI-H23, lung). Most of the compounds, in this series, especially compounds 5c, 5b and 5h, also significantly activated caspase-3 activity. Among these, compound 5c displayed 1.61-fold more potent than PAC-1 as caspase-3 activator. Cell cycle analysis showed that compounds 5b, 5c, and 5h significantly arrested the cell cycle in G1 phase. Further apoptotic studies also demonstrated compounds 5b, 5c, and 5h as strong apoptotic cell death inducers. The docking simulation studies showed that these compounds could activate procaspase via chelating Zn²⁺ ion bound to the allosteric site of the zymogen.     

Synthesis and DNA cleavage activity of 2-hydrazinyl-1,4,5,6-tetrahydropyrimidine containing hydroxy group

2-Hydrazinyl-1,4,5,6-tetrahydropyrimidin-5-ol dihydrochloride 2, as well as 2-hydrazinyl-4,5-dihydro-1H-imidazole dihydrochloride 1, was synthesized as metal-free DNA cleaving agent. Agarose gel electrophoresis was used to assess the plasmid pUC 19 DNA cleavage activities in the presence of 1 and 2. DNA cleavage efficiency of 2 exhibits remarkable increases compared with its corresponding non-hydroxy compound 1. Kinetic data of DNA cleavage promoted by 2 fit to the Michaelis–Menten-type equation with k_max of 0.0378 ± 0.0013 h^?1 giving 10⁶-fold rate acceleration over uncatalyzed DNA. The acceleration is driven by the spatial proximity of the nucleophilic hydroxy group and the electrophilic activation for the phosphodiester by the ammonium and/or guanidinium groups. In vitro cytotoxic activities toward Hela cells and human leukemia HL-60 cells were also examined, and 2 exhibits stronger cytotoxic activities than 1.