Discovery and optimization of novel 4-[(aminocarbonyl)amino]-N-[4-(2-aminoethyl)phenyl]benzenesulfonamide ghrelin receptor antagonists

This Letter describes optimization of ghrelin receptor antagonists and inverse agonists starting from a screening hit.     

Mutagenicity of urine of various species fed N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide or 2-amino-4-(5-nitro-2-furyl)thiazole

Rats, mice and hamsters, which are susceptible to the bladder carcinogenesis by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), and guinea pigs, which are not, were fed a diet containing 0.188% FANFT or 0.188% 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) for 1 week and their urine was then examined for mutagenicity for S. typhimurium TA100. The mutagenicities of the urine of these species fed FANFT were approximately equal. Similarly, that of the urine of these species fed ANFT were also approximately equal. However, the urine from FANFT-fed animals was approximately 10 times as mutagenic as that from ANFT-fed animals. ANFT was detected only in the urine of rats, mice or hamsters fed FANFT. A positive correlation between the susceptibility toward bladder carcinogenesis by FANFT and urinary ANFT excretion was demonstrated, although the correlation between this susceptibility and urine mutagenicity was lacking.     

Synthesis and evaluation of 1-[2-(4-[11C]methoxyphenyl)phenyl]piperazine for imaging of the serotonin 5-HT7 receptor in the rat brain

1-[2-(4-Methoxyphenyl)phenyl]piperazine (4) is a potent serotonin 5-HT₇ receptor antagonist (K_i = 2.6 nM) with a low binding affinity for the 5-HT_1A receptor (K_i = 476 nM). As a potential positron emission tomography (PET) radiotracer for the 5-HT₇ receptor, [¹¹C]4 was synthesized at high radiochemical yield and specific activity, by O-[¹¹C]methylation of 2′-(piperazin-1-yl)-[1,1′-biphenyl]-4-ol (6) with [¹¹C]methyl iodide. Autoradiography revealed that [¹¹C]4 showed in vitro specific binding with 5-HT₇ in the rat brain regions, such as the thalamus which is a region with high 5-HT₇ expression. Metabolite analysis indicated that intact [¹¹C]4 in the brain exceeded 90% of the radioactive components at 15 min after the radiotracer injection, although two radiolabeled metabolites were found in the rat plasma. The PET study of rats showed moderated uptake of [¹¹C]4 in the brain (1.2 SUV), but no significant regional difference in radioactivity in the brain. Pretreatment with 5-HT₇-selective antagonist SB269970 (3) did not decrease the uptake of [¹¹C]4 in the rat brain. Further studies are warranted that focus on the development of PET ligand candidates with higher binding affinity for 5-HT₇ and higher in vivo stability in brain than 4.     

Genotoxicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitro-biphenyl (ADDB) in goldfish (Carassius auratus) using the micronucleus test and the comet assay

2-[2-(Acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl (ADDB) are two compounds, which show strong mutagenicity toward bacteria, that have been identified as major mutagens in river water in Japan. In the present study, we examined the genotoxicity of PBTA-6 and ADDB in goldfish (Carassius auratus) by the micronucleus test and single-cell gel electrophoresis (comet assay). The frequencies of micronuclei in gill cells gradually increased until 96h after i.p. injection of PBTA-6 and ADDB at doses of 50mg/kg body weight, and then decreased 144h after injection. PBTA-6 induced micronuclei in gill cells dose-dependently at a dose range of 1-100mg/kg body weight, giving significantly high frequencies at doses of 50 and 100mg/kg body weight. On the other hand, no significant increase was observed in the peripheral erythrocytes of goldfish exposed to PBTA-6 or ADDB. In the comet assay, values of DNA tail moment and tail length in peripheral erythrocytes increased significantly until 6h after the i.p. injection of PBTA-6 (50mg/kg body weight), only to decrease by 9h after injection. Both the DNA tail moment and tail length were dose-dependently increased by injections of PBTA-6 at doses ranging from 1 to 50mg/kg. Significantly high values for tail moment and tail length were found in peripheral erythrocytes 3h after an i.p. injection of ADDB and persisted for up to 6h. These results show that both PBTA-6 and ADDB have genotoxic effects in goldfish.     

Mutagenicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and benzo[a]pyrene (BaP) in the gill and hepatopancreas of rpsL transgenic zebrafish

We examined the in vivo mutagenicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and benzo[a]pyrene (BaP) by using transgenic (Tg) zebrafish carrying the mutational target gene rpsL. PBTA-6 is one of the PBTA-type compounds that were recently identified in highly mutagenic river water in Japan. BaP is a well-known contaminant that is frequently found in polluted water. Both compounds are potent mutagens, as determined by using the Ames test employing S9 mix and Salmonella. Adult rpsL Tg zebrafish were exposed to 0, 7, or 10 mg/L PBTA-6 or 0, 1.5, or 3 mg/L BaP for 96 h in a water bath and the mutations in their gills and hepatopancreata were measured 2-4 weeks later. At 3 weeks after exposure, 3 mg/L BaP significantly increased the rpsL mutant frequency (MF) in the gill and hepatopancreas by 5- and 2.3-fold, respectively, as compared to control fish. Sequence analysis showed that BaP mainly induced G:C to T:A and G:C to C:G transversions, which is consistent with the known mutagenic effects of BaP. In contrast, despite its extremely high mutagenic potency in Salmonella strains, PBTA-6 did not significantly increase the MF in the zebrafish gill or hepatopancreas. Although PBTA-6 is 300 times more mutagenic than BaP in the Ames test [T. Watanabe, H. Nukaya, Y. Terao, Y. Takahashi, A. Tada, T. Takamura, H. Sawanishi, T. Ohe, T. Hirayama, T. Sugimura, K. Wakabayashi, Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5 and PBTA-6, and their detection in river water from Japan, Mutat. Res. 498 (2001) 107-115], calculation of the mutagenicity per mole of compound indicated that PBTA-6 was 33- and <3.7-fold less mutagenic in the zebrafish gill and hepatopancreas, respectively, than BaP.     

Antagonists of the human CCR5 receptor as anti-HIV-1 agents. Part 3: a proposed pharmacophore model for 1-[N-(methyl)-N-(phenylsulfonyl)amino]-2-(phenyl)-4-[4-(substituted)piperidin-1-yl]butanes.

Structure-activity relationship studies directed toward the optimization of (2S)-2-(3-chlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[4-(substituted)piperidin-1-yl]butanes as CCR5 antagonists resulted in the synthesis of the spiro-indanone derivative 8c (IC50=5 nM). These and previous results are summarized in a proposed pharmacophore model for this class of CCR5 antagonist.     

3-(4-(Benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl acetate induced Hep G2 cell apoptosis through a ROS-mediated pathway

3-(4-(Benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl acetate (DPB-5) is a synthetic benzothiazole derivative. In the present study, we revealed that DPB-5 had strong cytotoxicity to induce cell apoptosis, which was mediated by ROS. And DPB-5 was more cytotoxic toward hepatoma cells than toward normal hepatic cells, which was resulted from the greater susceptibility of the malignant cells to ROS. DBP-5 caused massive ROS accumulation and GSH decrease, which lead to MMP disruption, caspase activation and finally induced cell apoptosis. Additionally, rotenone, an inhibitor of mitochondria electron transport system, effectively blocked the ROS elevated effect of DPB-5, which suggested that DPB-5-induced ROS generated from the mitochondria. Further studies showed that DPB-5-induced cell apoptosis through caspases-cascade, but failed to activate caspase-9. Hence, we concluded that DPB-5-induced Hep G2 cells apoptosis via a ROS-mediated pathway which was caspase-dependent but did not rely on caspase-9.     

3-[3-Fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)phenyl]-4-methylpyridine: a highly potent and orally bioavailable metabotropic glutamate subtype 5 (mGlu5) receptor antagonist

Structure-activity relationship studies performed around 3-fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile for the purpose of developing novel mGlu5 receptor antagonists are described. Synthesis of a series of four-ring tetrazoles led to the discovery of 3-[3-fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)phenyl]-4-methylpyridine, a highly potent, brain penetrant, azole-based mGlu5 receptor antagonist.     

Synthesis and biological evaluation of 3-[4-(amino/methylsulfonyl)phenyl]methylene-indolin-2-one derivatives as novel COX-1/2 and 5-LOX inhibitors

Fourteen new 3-[4-(amino/methylsulfonyl)phenyl]methylene-indolin-2-one derivatives were synthesized. Six compounds displayed potent inhibitory activities against COX-1/2 and 5-LOX with IC(50) in the range of 0.10-9.87 μM. Particularly, 10f exhibited well balanced inhibitory action on these enzymes (IC(50)=0.10-0.56 μM). More importantly, 10f and several other compounds had comparable or stronger anti-inflammatory and analgesic activities, but better gastric tolerability in vivo, as compared with darbufelone mesilate and tenidap sodium. Therefore, our findings may aid in the design of new and safe anti-inflammatory reagents for the intervention of painful inflammatory diseases, such as rheumatoid arthritis at clinic.     

Antagonists of the human CCR5 receptor as anti-HIV-1 agents. Part 4: synthesis and structure-activity relationships for 1-[N-(methyl)-N-(phenylsulfonyl)amino]-2-(phenyl)-4-(4-(N-(alkyl)-N-(benzyloxycarbonyl)amino)piperidin-1-yl)butanes

(2S)-2-(3-Chlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[spiro(2,3-dihydrobenzthiophene-3,4'-piperidin-1'-yl)]butane S-oxide (1b) has been identified as a potent CCR5 antagonist having an IC50=10 nM. Herein, structure-activity relationship studies of non-spiro piperidines are described, which led to the discovery of 4-(N-(alkyl)-N-(benzyloxycarbonyl)amino)piperidine derivatives (3-5) as potent CCR5 antagonists.     

5-amino-1-(2,6-dichloro-4-trifluoromethyl-phenyl)-3-[3H3]-methylsulfanyl-1H-pyrazole-4-carbonitrile (CTOM): synthesis and characterization of a novel and selective insect GABA receptor radioligand

Pyrazole 2a is a novel, potent ligand for insect GABA receptors obtained from housefly head membrane preparations (K(i)=8 nM). It is 500-fold selective against the mammalian receptor (mouse brain preparations). Its specifically tritiated version (2b) was synthesized by reduction of disulfide 10 with NaBH(4) followed by alkylation with [3H(3)]-CH(3)I.     

AMPA receptor agonists: Resolution,configurational assignment,and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-isoxazol-4-yl]-propionic acid (2-Py-AMPA)

We have previously shown that whereas (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA) shows the characteristics of a partial agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, (S)-APPA is a full AMPA receptor agonist and (R)-APPA a weak competitive AMPA receptor antagonist. This observation led us to introduce the new pharmacological concept, functional partial agonism. Recently we have shown that the 2-pyridyl analogue of APPA, (RS)-2-amino-3-[3-hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA), is a potent and apparently full AMPA receptor agonist, and this compound has now been resolved into (+)- and (-)-2-Py-AMPA (ee ≥ 99.0%) by chiral HPLC using a Chirobiotic T column. The absolute stereochemistry of the enantiomers of APPA has previously been established by X-ray analysis, and on the basis of comparative studies of the circular dichroism spectra of the enantiomers of APPA and 2-Py-AMPA, (+)- and (-)-2-Py-AMPA were assigned the (S)- and (R)-configuration, respectively. In a series of receptor binding studies, neither enantiomer of 2-Py-AMPA showed detectable affinity for kainic acid receptor sites or different sites at the N-methyl-D-aspartic acid (NMDA) receptor complex. (+)-(S)-2-Py-AMPA was an effective inhibitor of [³H]AMPA binding (IC⁵⁰ = 0.19 ± 0.06 μM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC₅₀ = 4.5 ± 0.3 μM) comparable with AMPA (IC₅₀ = 0.040 ± 0.01 μM; EC₅₀ = 3.5 ± 0.2 μM), but much more potent than (+)-(S)-APPA (IC₅₀ = 5.5 ± 2.2 μM; EC₅₀ = 230 ± 12 μM). Like (-)-(R)-APPA (IC₅₀ > 100 μM), (-)-(R)-2-Py-AMPA (IC₅₀ > 100 μM) did not significantly affect [³H]AMPA binding, and both compounds were week AMPA receptor antagonists (K_i = 270 ± 50 and 290 ± 20 μM, respectively). Chirality 9:274–280, 1997. © 1997 Wiley-Liss, Inc.     

Synthesis and in vivo evaluation of [18F]2-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)-dione ([18F]FECUMI-101) as an imaging probe for 5-HT1A receptor agonist in nonhuman primates

The 5-HT_1AR partial agonist PET radiotracer, [¹¹C]CUMI-101, has advantages over an antagonist radiotracer as it binds preferentially to the high affinity state of the receptor and thereby provides more functionally meaningful information. The major drawback of C-11 tracers is the lack of cyclotron facility in many health care centers thereby limiting widespread clinical or research use. We identified the fluoroethyl derivative, 2-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione (FECUMI-101) (K_i = 0.1 nM; E_max = 77%; EC₅₀ = 0.65 nM) as a partial agonist 5-HT_1AR ligand of the parent ligand CUMI-101. FECUMI-101 is radiolabeled with F-18 by O-fluoroethylation of the corresponding desmethyl analogue (1) with [¹⁸F]fluoroethyltosylate in DMSO in the presence of 1.6 equiv of K₂CO₃ in 45 ± 5% yield (EOS). PET shows [¹⁸F]FECUMI-101 binds specifically to 5-HT_1AR enriched brain regions of baboon. The specificity of [¹⁸F]FECUMI-101 binding to 5-HT_1AR was confirmed by challenge studies with the known 5-HT_1AR ligand WAY100635. These findings indicate that [¹⁸F]FECUMI-101 can be a viable agonist ligand for the in vivo quantification of high affinity 5-HT_1AR with PET.     

Synthesis and biological data of 4-amino-1-(2-chloro-2-phenylethyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid ethyl esters, a new series of A1-adenosine receptor (A1AR) ligands.

The synthesis of a new family of A1-adenosine receptor (A1AR) ligands 3a-n has been performed in a straightforward way. Affinity data at A1AR, A2AAR and A3AR in bovine membranes show that these new compounds bind the A1AR in a selective way over A2AAR and A3AR and one of them (3j) presents a very high affinity, probably due to the phenethylamine substituent at C-4.