Synthesis and antipicornavirus activity of (R)- and (S)-1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one

The new pyridyl imidazolidinone derivative, 1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one (+/-)-1a, was synthesized and found to have an excellent antiviral activity against EV71 (IC50 = 0.009 microM). Therefore, both the enantiomers, (S)-(+)-1a and (R)-(-)-1a, have been prepared starting from readily available monomethyl (R)-3-methylglutarate (7) as a useful chiral building block and their antiviral activity was evaluated in a plaque reduction assay. Interestingly, we observed that the enantiomer (S)-(+)-1a was 10-fold more active against enterovirus71 (EV71) (IC50 = 0.003 microM) than the corresponding enantiomer (R)-(-)-1a (IC50 = 0.033 microM). Similar results were found against all five strains (1743, 2086, 2231, 4643, and BrCr) of EV71 tested. This demonstrated that the absolute configuration of the chiral carbon atom at the 3-position of the alkyl linker considerably influenced the anti-EV71 activity of these pyridyl imidazolidinones.     

N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea and N'-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]-thiourea as potent inhibitors of multidrug-resistant human immunodeficiency virus-1.

We have replaced the pyridyl ring of trovirdine with an alicyclic cyclohexenyl, adamantyl or cis-myrtanyl ring. Only the cyclohexenyl-containing thiourea compound N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]- thiourea (HI-346) (as well as its chlorine-substituted derivative N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]- thiourea/HI-445) showed RT inhibitory activity. HI-346 and HI-445 effectively inhibited recombinant RT with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cell-free RT inhibition assays was: HI-346 (IC50 = 0.4 microM) > HI-445 (IC50 = 0.5 microM) > trovirdine (IC50 = 0.8 microM) > MKC-442 (IC5 = 0.8 microM) = delavirdine (IC50 = 1.5 microM) > nevirapine (IC50 = 23 microM). In accord with this data, both compounds inhibited the replication of the drug-sensitive HIV-1 strain HTLV(IIIB) with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cellular HIV-1 inhibition assays was: HI-445 = HI-346 (IC50 = 3 nM) > MKC-442 (IC50 = 4 nM) = AZT (IC50 = 4 nM) > trovirdine (IC50 = 7 nM) > delavirdine (IC50 = 9 nM) > nevirapine (IC50 = 34 nM). Surprisingly, the lead compounds HI-346 and HI-445 were 3-times more effective against the multidrug resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74V,41L, and 215Y) than they were against HTLV(IIIB) with wild-type RT. HI-346 and HI-445 were 20-times more potent than trovirdine, 200-times more potent than AZT, 300-times more potent than MKC-442, 400-times more potent than delavirdine, and 5000-times more potent than nevirapine against the multidrug resistant HIV-1 strain RT-MDR. HI-445 was also tested against the RT Y181C mutant A17 strain of HIV-1 and found to be >7-fold more effective than trovirdine and >1,400-fold more effective than nevirapine or delavirdine. Similarly, both HI-346 and HI-445 were more effective than trovirdine, nevirapine, and delavirdine against the problematic NNI-resistant HIV-1 strain A17-variant with both Y181C and K103N mutations in RT, although their activity was markedly reduced against this strain. Neither compound exhibited significant cytotoxicity at effective concentrations (CC50 >100 microM). These findings establish the lead compounds HI-346 and HI-445 as potent inhibitors of drug-sensitive as well as multidrug-resistant stains of HIV-1.     

Resolution, absolute stereochemistry, and enantiopharmacology of the GluR1-4 and GluR5 antagonist 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid

The phosphono amino acid, (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propio nic acid (ATPO), is a structural hybrid between the NMDA antagonist (RS)-2-amino-7-phosphonoheptanoic acid (AP7) and the AMPA and GluR5 agonist, (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA). ATPO has been resolved into (S)-ATPO and (R)-ATPO using chiral HPLC, and the absolute stereochemistry of the two enantiomers was established by an X-ray crystallographic analysis of (R)-ATPO. (S)-ATPO and (R)-ATPO were characterized pharmacologically using rat brain membrane binding and electrophysiologically using the cortical wedge preparation as well as homo- or heteromeric GluR1-4, GluR5-6, and KA2 receptors expressed in Xenopus oocytes. (R)-ATPO was essentially inactive as an agonist or antagonist in all test systems. (S)-ATPO was an inhibitor of the binding of [(3)H]AMPA (IC(50) = 16 +/- 1 microM) and of [(3)H]-6-cyano-7-nitroquinoxaline-2,3-dione ([(3)H]CNQX) (IC(50) = 1.8 +/- 0.2 microM), but was inactive in the [(3)H]kainic acid and the [(3)H]-(RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([(3)H]CPP) binding assays. (S)-ATPO did not show detectable agonist effects at any of the receptors under study, but antagonized AMPA-induced depolarization in the cortical wedge preparation (IC(50) = 15 +/- 1 microM). (S)-ATPO also blocked kainic acid agonist effects at GluR1 (K(i) = 2.0 microM), GluR1+2 (K(i) = 3.6 microM), GluR3 (K(i) = 3.6 microM), GluR4 (K(i) = 6.7 microM), and GluR5 (K(i) = 23 microM), but was inactive at GluR6 and GluR6+KA2. Thus, although ATPO is a structural analog of AP7 neither (S)-ATPO nor (R)-ATPO are recognized by NMDA receptor sites.     

Homonojirimycin analogues and their glucosides from Lobelia sessilifolia and Adenophora spp. (Campanulaceae)

2,6-Dideoxy-7-O-(beta-D-glucopyranosyl) 2,6-imino-D-glycero-L-gulo- heptitol (7-O-beta-D-glucopyranosyl-alpha-homonojirimycin, 1) was isolated from the 50% methanol extract of the whole plant of Lobelia sessilifolia (Campanulaceae), which was found to potently inhibit rice alpha-glucosidase. Adenophorae radix, roots of Adenophora spp. (Campanulaceae), yielded new homonojirimycin derivatives, adenophorine (2), 1-deoxyadenophorine (3), 5-deoxyadenophorine (4), 1-C-(5-amino-5-deoxy-beta-D-galactopyranosyl)butane (beta-1-C-butyl-deoxygalactonojirimycin, 5), and the 1-O-beta-D-glucosides of 2 (6) and 4 (7), in addition to the recently discovered alpha-1-C-ethylfagomine (8) and the known 1-deoxymannojirimycin (9) and 2R,5R-bis(hydroxymethyl)-3R,4R- dihydroxypyrrolidine (DMDP, 10). Compound 4 is a potent inhibitor of coffee bean alpha-galactosidase (IC50 = 6.4 microM) and a reasonably good inhibitor of bovine liver beta-galactosidase (IC50 = 34 microM). Compound 5 is a very specific and potent inhibitor of coffee bean alpha-galactosidase (IC50 = 0.71 microM). The glucosides 1 and 7 were potent inhibitors of various alpha-glucosidases, with IC50 values ranging from 1 to 0.1 microM. Furthermore, 1 potently inhibited porcine kidney trehalase (IC50 = 0.013 microM) but failed to inhibit alpha-galactosidase, whereas 7 was a potent inhibitor of alpha-galactosidase (IC50 = 1.7 microM) without trehalase inhibitory activity.     

Design, synthesis, and biological evaluation of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides as cyclooxygenase isozyme inhibitors

A group of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides, possessing either a F or a substituted-phenyl ring substituent (4-F, 2,4-F2, 4-SO2Me, 4-OCHMe2) attached to its C-4 or C-6 position, was prepared using a palladium-catalyzed Suzuki cross-coupling reaction for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. Although N-acetyl-3-carboxymethyl-6-fluorobenzenesulfonamide [14, COX-1 IC50 = 2.26 microM; COX-2 IC50 = 0.012 microM; COX-2 selectivity index (SI) = 188] and N-acetyl-3-carboxymethyl-6-(4-isopropoxyphenyl)benzenesulfonamide (20c, COX-1 IC50 >100 microM; COX-2 IC50 = 0.15 microM; COX-2 SI >667) exhibited potent in vitro COX-2 inhibitory activity and high COX-2 selectivity, both compounds were inactive anti-inflammatory agents in a carrageenan-induced rat paw edema assay. In contrast, the less potent and less selective COX-2 inhibitors N-acetyl-2-carboxymethyl-4-fluorobenzenesulfonamide (12, COX-1 IC50 = 4.25 microM; COX-2 IC50 = 0.978 microM; COX-2 SI = 4.3), N-acetyl-2-carboxymethyl-4-(2,4-difluorophenyl)benzenesulfonamide (17c, COX-1 IC50 = 1.02 microM; COX-2 IC50 = 1.00 microM; COX-2 SI = 1.02), and N-acetyl-3-carboxymethyl-6-(4-methanesulfonylphenyl)benzenesulfonamide (20e, COX-1 IC50 = 0.109 microM; COX-2 IC50 = 1.14 microM; COX-2 SI = 0.095) exhibited moderate anti-inflammatory activity where a 75 mg/kg oral dose reduced inflammation 26%, 14%, and 20%, respectively, at 3 h postdrug administration relative to the reference drug aspirin where a 50 mg/kg oral dose reduced inflammation by 25% at 3 h postdrug administration.     

Resolution, configurational assignment, and enantiopharmacology at glutamate receptors of 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) and demethyl-ACPA

We have previously described (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) as a potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of (S)-glutamic acid (Glu) receptors. We now report the chromatographic resolution of ACPA and (RS)-2-amino-3-(3-carboxy-4-isoxazolyl)propionic acid (demethyl-ACPA) using a Sumichiral OA-5000 column. The configuration of the enantiomers of both compounds have been assigned based on X-ray crystallographic analyses, supported by circular dichroism spectra and elution orders on chiral HPLC columns. Furthermore, the enantiopharmacology of ACPA and demethyl-ACPA was investigated using radioligand binding and cortical wedge electrophysiological assay systems and cloned metabotropic Glu receptors. (S)-ACPA showed high affinity in AMPA binding (IC(50) = 0.025 microM), low affinity in kainic acid binding (IC(50) = 3.6 microM), and potent AMPA receptor agonist activity on cortical neurons (EC(50) = 0.25 microM), whereas (R)-ACPA was essentially inactive. Like (S)-ACPA, (S)-demethyl-ACPA displayed high AMPA receptor affinity (IC(50) = 0.039 microM), but was found to be a relatively weak AMPA receptor agonist (EC(50) = 12 microM). The stereoselectivity observed for demethyl-ACPA was high when based on AMPA receptor affinity (eudismic ratio = 250), but low when based on electrophysiological activity (eudismic ratio = 10). (R)-Demethyl-ACPA also possessed a weak NMDA receptor antagonist activity (IC(50) = 220 microM). Among the enantiomers tested, only (S)-demethyl-ACPA showed activity at metabotropic receptors, being a weak antagonist at the mGlu(2) receptor subtype (K(B) = 148 microM).     

Polyhydroxylated pyrrolidine and piperidine alkaloids from Adenophora triphylla var. japonica (Campanulaceae)

Adenophora triphylla var. japonica (Campanulaceae) yielded two new alkaloids, the 6-C-butyl derivative of 2R,5R-bis(hydroxymethyl)-3R,4R-dihydroxypyrrolidine (DMDP) and alpha-1-C-ethyl-fagomine, together with the known alkaloids 1,4-dideoxy-1,4-imino-D-arabinitol, 1-deoxynojirimycin, and 1-deoxymannojirimycin. 6-C-Butyl-DMDP showed inhibitory activity toward almond beta-glucosidase (IC50 = 68 microM), whereas alpha-1-C-ethyl-fagomine inhibited bovine liver beta-galactosidase (IC50 = 29 microM).     

2-Amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid: resolution, absolute stereochemistry and enantiopharmacology at glutamate receptors

In order to identify new subtype-selective (S)-glutamate (Glu) receptor ligands we have synthesized (RS)-2-amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid [(RS)-TDPA]. Resolution of (RS)-TDPA by chiral chromatography was performed using a Crownpac CR(+) column affording (R)- and (S)-TDPA of high enantiomeric purity (enantiomeric excess=99.9%). An X-ray crystallographic analysis revealed that the early eluting enantiomer has R-configuration. Both enantiomers showed high affinity as well as high agonist activity at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, determined using a [(3)H]AMPA binding assay and an electrophysiological model, respectively. The affinities and agonist activities obtained for (R)-TDPA (IC(50)=0.265 microM and EC(50)=6.6 microM, respectively) and (S)-TDPA (IC(50)=0.065 microM and EC(50)=20 microM, respectively) revealed a remarkably low AMPA receptor stereoselectivity, (S)-TDPA showing the highest affinity and (R)-TDPA the most potent agonist activity. In addition, (S)-TDPA was shown to interact with synaptosomal Glu uptake sites displacing [(3)H](R)-aspartic acid (IC(50 ) approximately 390 microM). An enantiospecific and subtype-selective agonist activity was observed for (S)-TDPA at group I metabotropic Glu (mGlu) receptors (EC(50)=13 microM at mGlu(5) and EC(50)=95 microM at mGlu(1)).     

Design, synthesis, and biological evaluation of linear 1-(4-, 3- or 2-methylsulfonylphenyl)-2-phenylacetylenes: a novel class of cyclooxygenase-2 inhibitors

A group of regioisomeric 1-(methylsulfonylphenyl)-2-phenylacetylenes possessing a COX-2 SO(2)Me pharmacophore at the para-, meta- or ortho-position of the C-1 phenyl ring, in conjunction with a C-2 phenyl or substituted-phenyl ring substituent (3-F, 3-OMe, 3-OH, 3-OAc, 4-Me), were designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. These target linear 1,2-diarylacetylenes were synthesized via a palladium-catalyzed Sonogashira cross-coupling reaction followed by oxidation of the respective 1-(methylthiophenyl)-2-phenylacetylene intermediate. In vitro COX-1/COX-2 isozyme inhibition structure-activity studies identified 1-(3-methylsulfonylphenyl)-2-(4-methylphenyl)acetylene (12d) as a potent COX-2 inhibitor (IC(50) = 0.32 microM) with a high COX-2 selectivity index (SI > 320) comparable to the reference compound rofecoxib (COX-2 IC(50) = 0.50 microM; COX-2 SI > 200). A molecular modeling study where (12d) was docked in the binding site of COX-2 showed that the MeSO(2) COX-2 pharmacophore was positioned in the vicinity of the secondary COX-2 binding site near Val(523). The 1-(4-methylsulfonylphenyl)-2-(3-acetoxyphenyl)acetylene (11f, COX-1 IC(50) = 1.00 microM; COX-2 IC(50) = 0.06 microM; COX-2 SI = 16.7) and 1-(3-methylsulfonylphenyl)-2-(3-acetoxyphenyl)acetylene (12f, COX-1 IC(50) = 6.5 microM; COX-2 IC(50) = 0.05 microM; COX-2 SI = 130) regioisomers exhibited comparable COX-2 inhibition, and moderately lower selective COX-2 selectivity, relative to the reference drug celecoxib (COX-1 IC(50) = 33.1 microM; COX-2 IC(50) = 0.07 microM; COX-2 SI = 472). The most potent anti-inflammatory agent 1-(3-methylsulfonylphenyl)-2-(4-methylphenyl)acetylene (12d) exhibited moderate oral anti-inflammatory activity (ED(50)= 129 mg/kg) at 3 h postdrug administration relative to the reference drug celecoxib (ED(50) = 10.8 mg/kg) in a carrageenan-induced rat paw edema assay. The structure-activity data acquired indicate that the acetylene moiety constitutes a suitable scaffold (template) to design novel acyclic 1,2-diarylacetylenes with selective COX-2, or dual COX-1/COX-2, inhibitory activities.     

Synthesis of N-substituted piperidine-4-(benzylidene-4-carboxylic acids) and evaluation as inhibitors of steroid-5alpha-reductase type 1 and 2

The synthesis of N-substituted piperidine-4-(benzylidene-4-carboxylic acids) is described [benzoyl (1), benzyl (2), adamantanoyl (3), cyclohexanoyl (4), cyclohexylacetyl (5), diphenylacetyl (6), dicyclohexylacetyl (7), 2-propylpentanoyl (8), diphenylcarbamoyl (9), trimethylacetyl (10), 3,3-dimethylacryloyl (11), dicyclohexylacetyl derivative of the benzyl compound (12)]. Compounds were tested for inhibitory activity toward 5alpha-reductase isozymes 1 and 2 in human and rat. The test compounds inhibited 5alpha-reductase, showing a broad range of inhibitory potencies. In rat, compounds 6 (IC50 = 3.44 and 0.37 microM for type 1 and 2, respectively) and 9 (IC50=0.54 and 0.69 microM for type 1 and 2, respectively) displayed the best inhibition toward both isozymes. Compound 7 showed a strong inhibition toward type 2 human and rat enzyme (IC50 = 60 and 80 nM) but only a moderate activity versus type 1 enzyme (IC50 approximately 10 microM for rat and human enzyme). In vivo, selected compounds reduced prostate weights in castrated testosterone treated rats.     

Prostaglandin-H-synthase (PGHS)-1 and -2 microtiter assays for the testing of herbal drugs and in vitro inhibition of PGHS-isoenzyms by polyunsaturated fatty acids from Platycodi radix

In order to test inhibition of prostaglandin-H-synthase-1 and -2 (PGHS-1 and -2) by plant extracts, we have established two enzyme based in vitro assays with enzyme immunoassay (EIA) evaluation. The assays have been evaluated with known synthetic inhibitors and with plant extracts. In a screening of traditionally used Chinese herbs for anti-inflammatory activity, a series of n-hexane and dichloromethane extracts showed significant inhibitory effect in comparison with the known specific PGHS-2 inhibitors NS-398 (IC(50) = 2.6 microM) and nimesulide (IC(50) = 36 microM). The lipophilic extracts of the Chinese drug Jiengeng, the dried roots of Platycodon grandiflorum (Jacq.) A. DC. (Campanulaceae), showed good inhibitory activity against both PGHS isoenzymes. The directly prepared DCM-extract exhibited better activity against PGHS-2 (IC(50) = 4.0 microg/ml) than against PGHS-1 (IC(50) = 17.6 microg/ml). We identified fatty acids as main active constituents and quantified them. Linoleic acid showed the highest content (ca. 20% of the dried extract) and a high and preferential PGHS-2 inhibitory activity (IC(50) (PGHS-1) = 20 microM; IC(50) (PGHS-2) = 2 microM). The comparison of the concentration of linoleic acid and the inhibitory activity of the direct DCM-extract showed, that linoleic acid is mainly responsible for the in vitro activity of the extract on PGHS-2.     

Design, synthesis, and biological evaluation of (E)-3-(4-methanesulfonylphenyl)-2-(aryl)acrylic acids as dual inhibitors of cyclooxygenases and lipoxygenases

A group of (E)-3-(4-methanesulfonylphenyl)acrylic acids possessing a substituted-phenyl ring (4-H, 4-Br, 3-Br, 4-F, 4-OH, 4-OMe, 4-OAc, and 4-NHAc) attached to the acrylic acid C-2 position were prepared using a stereospecific Perkin condensation reaction. A related group of compounds having 4- and 3-(4-isopropyloxyphenyl)phenyl, 4- and 3-(2,4-difluorophenyl)phenyl and 4- and 3-(4-methanesulfonylphenyl)phenyl substituents attached to the acrylic acid C-2 position were also synthesized, using a palladium-catalyzed Suzuki cross-coupling reaction, for evaluation as dual cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitors. (E)-2-(3-Bromophenyl)-3-(4-methanesulfonylphenyl)acrylic acid (9h), and compounds having 4-(4-isopropyloxyphenyl-, 2,4-difluorophenyl-, or 4-methylsulfonylphenyl)phenyl moieties at the acrylic acid C-2 position (11a,b,d), were particularly potent COX-2 inhibitors with a high COX-2 selectivity index (COX-2 IC50 approximately 0.32 microM, SI > 316) similar to the reference drug rofecoxib (COX-2 IC50 = 0.5 microM, SI > 200). Acrylic acid analogs with a C-2 4-hydoxyphenyl (9d, IC50 = 0.56 microM), or 4-acetamidophenyl (9g, IC50 = 0.11 microM), substituent were particularly potent 5-LOX inhibitors that may participate in an additional specific hydrogen-bonding interaction. A number of compounds possessing a C-2 substituted-phenyl moiety (4-Br, 4-F, and 4-OH), or a 4- or 3-(2,4-difluorophenyl)phenyl moiety, showed potent 15-LOX inhibitory activity (IC50 values in the 0.31-0.49 microM range) relative to the reference drug luteolin (IC50 = 3.2 microM). Compounds having a C-2 4-acetylaminophenyl, or 4-(2,4-difluorophenyl)phenyl, moiety exhibited anti-inflammatory activities that were equipotent to aspirin, but less than that of celecoxib. The structure-activity data acquired indicate the acrylic acid moiety constitutes a suitable scaffold (template) to design novel acyclic dual inhibitors of the COX and LOX isozymes.     

Design and synthesis of (Z)-1,2-diphenyl-1-(4-methanesulfonamidophenyl)alk-1-enes and (Z)-1-(4-azidophenyl)-1,2-diphenylalk-1-enes: novel inhibitors of cyclooxygenase-2 (COX-2) with anti-inflammatory and analgesic activity

A group of novel (Z)-1,2-diphenyl-1-(4-methanesulfonamidophenyl)alk-1-enes was designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1/COX-2 enzyme inhibition studies identified (Z)-1,2-diphenyl-1-(4-methanesulfonamidophenyl)oct-1-ene (8d) as a highly potent (IC50=0.03 microM), and an extremely selective [COX-2 SI (selectivity index)>3,333], COX-2 inhibitor that showed good anti-inflammatory (AI) activity (ID50=2.8 mg/kg). A molecular modeling (docking) study showed that the p-MeSO2NH group present in (Z)-8d inserts deep inside the 2 degrees-pocket of the COX-2 binding site, it undergoes a hydrophobic interaction with Ala516 and Gly519, and one of the O-atoms of the MeSO2 group participates in a weak hydrogen bonding interaction with the NH2 of Arg513 (distance= 3.85 angstroms). Similar in vitro COX-1/COX-2 enzyme inhibition studies showed that the azido compound 1-(4-azidophenyl)-1,2-diphenyloct-1-ene (9c) is also a potent and selective COX-2 inhibitor (COX-2 IC50=0.11 microM: SI>909) that exhibits good AI activity (ID50=5.0 mg/kg). A docking experiment to determine the orientation of (Z)-9c within the COX-2 binding site showed that the linear p-N3 group inserts into the COX-2 2 degrees-pocket, where it undergoes an ion-ion (electrostatic) interaction with Arg513. Structure-activity data acquired indicate that an olefin having either a C-1 p-MeSO2NH-phenyl, or a p-N3-phenyl, substituent, that is, cis to a C-2 unsubstituted phenyl substituent, in conjunction with C-1 unsubstituted phenyl and C-2 alkyl substituents, provides a novel template to design acyclic olefinic COX-2 inhibitors.     

Resolution, configurational assignment, and enantiopharmacology of 2-amino-3-[3-hydroxy-5-(2-methyl-2H- tetrazol-5-yl)isoxazol-4-yl]propionic acid, a potent GluR3- and GluR4-preferring AMPA receptor agonist

We have previously shown that (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol -4-yl] propionic acid (2-Me-Tet-AMPA) is a selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, markedly more potent than AMPA itself, whereas the isomeric compound 1-Me-Tet-AMPA is essentially inactive. We here report the enantiopharmacology of 2-Me-Tet-AMPA in radioligand binding and cortical wedge electrophysiological assay systems, and using cloned AMPA (GluR1-4) and kainic acid (KA) (GluR5, 6, and KA2) receptor subtypes expressed in Xenopus oocytes. 2-Me-Tet-AMPA was resolved using preparative chiral HPLC. Zwitterion (-)-2-Me-Tet-AMPA was assigned the (R)-configuration based on an X-ray crystallographic analysis supported by the elution order of (-)- and (+)-2-Me-Tet-AMPA using four different chiral HPLC columns and by circular dichroism spectra. None of the compounds tested showed detectable affinity for N-methyl-D-aspartic acid (NMDA) receptor sites, and (R)-2-Me-Tet-AMPA was essentially inactive in all of the test systems used. Whereas (S)-2-Me-Tet-AMPA showed low affinity (IC(50) = 11 microM) in the [(3)H]KA binding assay, it was significantly more potent (IC(50) = 0.009 microM) than AMPA (IC(50) = 0.039 microM) in the [(3)H]AMPA binding assay, and in agreement with these findings, (S)-2-Me-Tet-AMPA (EC(50) = 0.11 microM) was markedly more potent than AMPA (EC(50) = 3.5 microM) in the electrophysiological cortical wedge model. In contrast to AMPA, which showed comparable potencies (EC(50) = 1.3-3.5 microM) at receptors formed by the AMPA receptor subunits (GluR1-4) in Xenopus oocytes, more potent effects and a substantially higher degree of subunit selectivity were observed for (S)-2-Me-Tet-AMPA: GluR1o (EC(50) = 0.16 microM), GluR1o/GluR2i (EC(50) = 0.12 microM), GluR3o (EC(50) = 0.014 microM) and GluR4o (EC(50) = 0.009 microM). At the KA-preferring receptors GluR5 and GluR6/KA2, (S)-2-Me-Tet-AMPA showed much weaker agonist effects (EC(50) = 8.7 and 15.3 microM, respectively). It is concluded that (S)-2-Me-Tet-AMPA is a subunit-selective and highly potent AMPA receptor agonist and a potentially useful tool for studies of physiological AMPA receptor subtypes.     

Studies of the inhibitory activity of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol- 2-yl]-2,2-dimethyl propanoic acid) on arachidonic acid metabolism in human phagocytes.

We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-i ndol-2- yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or formyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its omega-oxidation products, and 6-trans-isomers with IC50 values of 2.8-4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 microM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its omega-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5-11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3-0.9, 3.7-5.3, and 8.5-17.3 nM, respectively. In neutrophils primed with granulocyte--macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7-8.7 nM. At the concentration of 1 microM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.     

Pharmacological evaluation of both enantiomers of (R,S)-BM-591 as thromboxane A2 receptor antagonists and thromboxane synthase inhibitors

The aim of this work is to evaluate the anti-thromboxane activity of two pure enantiomers of (R,S)-BM-591, a nitrobenzene sulfonylurea chemically related to torasemide, a loop diuretic. The drug affinity for thromboxane A2 receptor (TP) of human washed platelets has been determined. In these experiments, (R)-BM-591 (IC50 = 2.4+/-0.1 nM) exhibited a significant higher affinity than (S)-BM-591 (IC50 = 4.2+/-0.15 nM) for human washed platelets TP receptors. Both enantiomers were stronger ligands than SQ-29548 (IC50 = 21.0+/-1.0 nM) and sulotroban (IC50 = 930+/-42 nM), two reference TXA2 receptor antagonists. Pharmacological characterisations of (S)-BM-591 and (R)-BM-591 were compared in several models. Thus, (R)-BM-591 strongly prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) and U-46619 (1 microM) while (S)-BM-591 showed a lower activity. On isolated tissues pre-contracted by U-46619, a stable TXA2 agonist, (S)-BM-591 was more potent in relaxing guinea-pig trachea (EC50 = 0.272+/-0.054 microM) and rat aorta (EC50 = 0.190+/-0.002 microM) than (R)-BM-591 (EC50 of 9.60+/-0.63 microM and 0.390+/-0.052 microM, respectively). Moreover, at 1 microM, (R)-BM-591 totally inhibited TXA2 synthase activity, expressed as TXB2 production from human platelets, while at the same concentration, (S)-BM-591 poorly reduced the TXB2 synthesis (22%). Finally, in rats, both enantiomers lost the diuretic activity of torasemide. In conclusion, (R)-BM-591 exhibits a higher affinity and antagonism on human platelet TP receptors than (S)-BM-591 as well as a better thromboxane synthase inhibitory potency. In contrast, (S)-BM-591 is more active than the (R)-enantiomer in relaxing smooth muscle contraction of rat aorta and trachea guinea pig. Consequently, (R)-BM-591 represents the best candidate for further development in the field of thrombosis disorders.     

Anti-inflammatory 5-(11'Z-heptadecenyl)- and 5-(8'Z,11'Z-heptadecadienyl)-resorcinols from mango (Mangifera indica L.) peels

Bioassay directed extraction and purification of mango peels revealed the 5-(11'Z-heptadecenyl)-resorcinol (1) and the known 5-(8'Z,11'Z-heptadecadienyl)-resorcinol (2) previously not described in Mangifera indica L. The structures of both compounds were determined by extensive 1D and 2D NMR studies and MS. Both compounds exhibited potent cyclooxygenase (COX)-1 and COX-2 inhibitory activity with IC(50) values ranging from 1.9 (2) to 3.5 microM (1) and from 3.5 (2) to 4.4 (1) microM, respectively, coming close to the IC(50) values of reference drugs. 5-Lipoxygenase (5-LOX) catalyzed leukotriene formation was only slightly inhibited. Structure-activity studies by referring to synthetic saturated homologues indicated that the degree of unsaturation in the alkyl chain plays a key role for COX inhibitory activity, whereas the influence of chain length was less significant.     

Asymmetric synthesis and stereochemical structure-activity relationship of (R)- and (S)-8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester, a potent inhibitor of allene oxide synthase

The preparation of both enantiomers of 8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester (JM-8686), a potent inhibitor of allene oxide synthase, has been achieved using 2,4-dichlorophenacyl bromide as a starting material. The key step was the asymmetric reduction of 1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethanone with chiral BINAL-H. The products were purified by chiral high-performance liquid chromatography (HPLC) to afford pure (R)-JM-8686 and (S)-JM-8686. The inhibitory activities and binding affinities of these enantiomers toward allene oxide synthase were determined. We found that the inhibition potency of (R)-JM-8686 is approximately 200 times greater than that of (S)-JM-8686, with IC(50) values of approximately 5+/-0.2 nM and 950+/-18 nM, respectively. The dissociation constants of (R)-JM-8686 and (S)-JM-8686 with respect to the recombinant allene oxide synthase were approximately 1.4+/-0.3 microM and 4.8+/-0.6 microM, respectively.     

Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone A hybrid compounds and evaluation of their molting hormone activity

Two stereoisomers of a castasterone/ponasterone A hybrid compound, the (20R,22R) and (20R,22S)-isomers of 2alpha,3alpha,20,22-tetrahydroxy-5alpha-cholestan-6-one, were synthesized stereoselectively and their binding activity to the ecdysteroid receptor was determined. From the concentration-response curve for the inhibition of the incorporation of tritiated ponasterone A into ecdysteroid receptor containing insect cells, the concentration (IC50) required to inhibit 50% of the incorporation of radioactivity into cells was evaluated. The IC50 values of the (22R)- and (22S)-isomers were determined to be 0.30 and 38.9 microM against Kc cells, respectively, indicating that the (22R)-isomer is about 100 times more potent than the corresponding (22S)-isomer. IC50 values of these compounds against lepidopteran Sf-9 cells were determined to be 0.36 and 12.9 microM, respectively. The molting hormonal effect was examined in a Chilo suppressalis integument system and the 50% effective concentration for the stimulation of N-acetylglucosamine incorporation into the cultured integument was determined to be 2.7 microM for the (22R)-isomer, while the (22S)-isomer was inactive. On the other hand, both isomers did not show brassinolide-like activity in the rice lamina inclination assay.     

Non-nucleoside HIV-1 reverse-transcriptase inhibitors. Part 10. Synthesis and anti-HIV activity of 5-alkyl-6-(1-naphthylmethyl)pyrimidin-4(3H)-ones with a mono- or disubstituted 2-amino function as novel 'dihydro-alkoxy-benzyl-oxopyrimidine' (DABO) analogues

A series of structurally related 2,5-disubstituted 6-(1-naphthylmethyl)-pyrimidin-4(3H)-ones, compounds 6a-6r, were synthesized and evaluated for their in vitro anti-HIV activities in MT-4 cells. Most of the new compounds investigated showed moderate-to-good activities against wild-type HIV-1, with IC(50) values in the range 5.64-0.21 microM. Compound 6d was the most potent congener (IC(50)=0.21 microM, SI=724) in inhibiting HIV-1 replication, which is ca. 25 times more effective than the reference compound 2',3'-dideoxyinosine (DDI). Preliminary structure-activity relationship (SAR) studies revealed that both modulation of the amino function at C(2) and of the alkyl group at C(5) of the pyrimidine ring are crucial for high anti-HIV-1 activity.