Bronchodilator activity of a nonxanthine phosphodiesterase inhibitor; 2,4-diamino-5-cyano-6-bromopyridine (compound I)

The ability of 2,4-diamino-5-cyano-6-bromopyridine (compound 1) to inhibit bronchiolar smooth muscle constriction was examined in isolated rings of rabbit primary bronchi and intrapulmonary bronchioles. After carbachol-induced constriction these tissue were significantly relaxed by either compound I or 1-methyl-3-isobutylxanthine (MIX) in a similar dose-dependent manner with 50 to 80% relaxation occurring at 100 microM of either compound. Compound I also attenuated the constrictor response of bronchial rings to histamine and significantly reduced the tension generated by horseradish peroxidase in sensitized tissues responding to this antigen. In addition, both compound I and MIX were found to inhibit the soluble cyclic AMP phosphodiesterase activity of rabbit bronchioles. Finally, both compound I and MIX caused a nearly 2-fold, time-dependent increase in cyclic AMP levels in isolated rabbit intrapulmonary bronchioles. The similarities of both the in vitro tissue responses to these compounds and the phosphodiesterase inhibitory properties suggest that the ability of compound I to reduce constrictor-induced tension generation in bronchial smooth muscle is related to the inhibition of cyclic nucleotide phosphodiesterases and the consequent elevation of cyclic AMP.     

CGS 21680C, an A2 selective adenosine receptor agonist with preferential hypotensive activity

CGS 21680C (2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethyl-carboxamido adenosine) a 2-substituted analog of the riboside uronamide, 5'-N-ethylcarboxamido adenosine and the related analog CGS 21577 (2-phenethylamino-5'-N-ethylcarboxamido adenosine), have high in vitro affinity for brain striatal adenosine A2 receptors (IC50 values = 22 and 13 nM, respectively). Both compounds were considerably less active at A1 receptors with CGS 21577 and CGS 21680C having respective IC50 values of 0.76 and 3.1 microM. The former compound was thus 59-fold selective for A2 receptors whereas CGS 21680C was 140-fold selective. In contrast, the reference A2 selective ligand, CV 1808 (2-phenylaminoadenosine), showed only 8-fold selectivity as an A2 ligand, having an IC50 of 115 nM in the [3H]-5'N-ethylcarboxamide adenosine assay and an IC50 of 910 nM at the N6-[3H] cyclohexyladenosine site. Further examination of CGS 21680C showed that the compound was without effect on binding to 17 other putative neurotransmitter/neuromodulator sites indicating its selectivity as an adenosine receptor ligand. In an isolated perfused working rat heart model, CGS 21680C effectively increased coronary flow with an ED25 value of 1.8 nM. The corresponding value for CGS 21577 was 3 nM whereas that for CV 1808 was 110 nM. The EC25 for eliciting bradycardia for all three compounds was greater than 1000 nM. The effects of all three compounds could be reversed by treatment with the xanthine adenosine antagonist, xanthine amine congener.(ABSTRACT TRUNCATED AT 250 WORDS)     

2,4-Diamino-7-hydroxy-pteridines in biological fluids of patients on high-dose methotrexate

Concentrations of 2,4-diamino-7-hydroxy-pteridines in plasma and cerebrospinal fluid (CSF) are reported for the 0.5–8 g/m² dose range of methotrexate in children with acute lymphoblastic leukemia or non-Hodgkin's lymphoma. This experiment has revealed that: (1) there is a wide inter-individual but relatively narrow intra-individual variability of the maximal concentrations of2,4-diamino-7-hydroxy-pteridines in plasma during consecutive methotrexate cycles; (2) the increase in the level of the metabolites in plasma was related to increments of the methotrexate dose, but not above 5 g/m²: this can be explained by a saturable conversion of methotrexate to 2,4-diamino-7-hydroxy-pteridines; (3) significant correlations were found between simultaneous values of 2,4-diamino-7-hydroxy-pteridines in plasma and CSF; (4) the formation of 2,4-diamino-7-hydroxy-pteridines did not depend on the ages of patients receiving the same dose of methotrexate. The presence of these compounds in plasma and CSF in significant amounts creates the potential for a number of competitive interactions with pteridine-dependent metabolism which may open up new possibilities for understanding the metabolic side-effects of methotrexate therapy.     

Antiviral and cytostatic evaluation of the novel 6-acyclic chain substituted thymine derivatives

A series of the novel 5-methyl pyrimidine derivatives with an acyclic side chain at the C-6 position were synthesized using lithiation of a 2,4-dimethoxy-5,6-dimethyl pyrimidine and subsequent nucleophilic addition or substitution reactions of the organolithium intermediate thus obtained with acetaldehyde, epichlorhydrine, fluorinated ketones and fluorinated ester. The novel compounds were evaluated for their cytostatic and antiviral activities. Among all the compounds evaluated, two fluorinated acyclic pyrimidine derivatives showed the highest cytostatic activities. The compound containing a 2-hydroxy-3,3,3-trifluoro-1-propenyl side chain exhibited a pronounced effect against breast carcinoma (MCF-7, IC50=8.38 micorg/ml), while the compound with a 2-fluoromethyl-2-acetoxypropyl chain exhibited moderate effect against cervical carcinoma (HeLa, IC50=19.73 microg/ml).     

Structure-activity and structure-selectivity studies on diaminoquinazolines and other inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase.

Twenty-eight 2,4-diaminopteridines with alkyl and aralkyl groups at the 6- and 7-positions, five 1,3-diamino-7,8,9,10-tetrahydropyrimido [4,5-c]isoquinolines with an alkyl, alkylthio, or aryl group at the 6-position, and nine 4,6-diamino-1,2-dihydro-s-triazines with one or two alkyl groups at the 2-position and a substituted phenyl or naphthyl group at the 1-position were evaluated as inhibitors of dihydrofolate reductase enzymes from Pneumocystis carinii, Toxoplasma gondii, and rat liver. Halogen substitution at the 5- or 6-position of 2,4-diaminoquinazoline favored selective binding to the P. carinii enzyme but not the T. gondii enzyme. For example, the 50% inhibitory concentrations of 2,4-diamino-6-chloroquinazoline as an inhibitor of P. carinii, T. gondii, and rat liver dihydrofolate reductase were 3.6, 14 and 29 microM, respectively, corresponding to 12-fold selectivity for the P. carinii enzyme but only marginal selectivity for the T. gondii enzyme. Greater than fivefold selectivity for P. carinii but not T. gondii dihydrofolate reductase was also observed for the 2,4-diaminoquinazolines with 5-methyl, 5-fluoro, 5- and 6-bromo, 6-chloro, and 5-chloro-6-bromo substitution. In contrast, alkyl and aralkyl substitution at the 6- and 7-positions of 2,4-diaminopteridines was found to be a favorable feature for selective inhibition of the T. gondii enzyme and, in two cases, for both enzymes. Nine of the fifty-one compounds tested against P. carinii dihydrofolate reductase and four of the thirty compounds tested against T. gondii dihydrofolate reductase displayed fivefold or greater selectivity for the microbial enzyme versus the rat liver enzyme. The most selective against both enzymes was 2,4-diamino-6,7-bis(cyclohexylmethyl) pteridine, with a selectivity ratio 2 orders of magnitude greater than the value reported for trimetrexate and piritrexim. Since substitution at the 7-position is generally considered to be detrimental to the binding of 2,4-diaminop-teridines and related compounds to mammalian dihydrofolate reductase, the selectivity observed in this study with the 6,7-bis(cyclohexylmethyl) analog may represent a useful approach to enhancing selective inhibition of the enzyme from nonmammalian species.     

Susceptibilities of Mycobacterium tuberculosis and Mycobacterium avium complex to lipophilic deazapteridine derivatives, inhibitors of dihydrofolate reductase

Twelve lipophilic 2,4-diamino-5-methyl-5-deazapteridine derivatives and trimethoprim were evaluated for activity against Mycobacterium tuberculosis and Mycobacterium avium in vitro. Six of the compounds had MICs of < or =12.8 mg/L and < or =1.28 mg/L against M. tuberculosis and M. avium, respectively; trimethoprim MICs were >128 mg/L and >12.8 but < or =128 mg/L, respectively. Two compounds, with either a 2-methyl-5-methoxy phenyl or 2-methoxy-5-trifluoromethyl phenyl linked at the 6-position of the deazapteridine moiety by a CH2NH bridge, had MICs of < or =0.13 mg/L against M. avium; the two compounds also had apparent I50 values for dihydrofolate reductase of 2 and 8 nM, respectively, compared with an I50 of 400 nM with trimethoprim. Four of the compounds were selectively toxic to mycobacteria as compared with Vero cells. These results demonstrated that lipophilic antifolates can be synthesized which are more active against mycobacteria than trimethoprim and which possess selective toxicity.     

In vitro activity of expanded-spectrum pyridazinyl oxime ethers related to pirodavir: novel capsid-binding inhibitors with potent antipicornavirus activity

Picornaviruses (PV) include human rhinovirus (HRV), the primary cause of the common cold, and the enteroviruses (EV), which cause serious diseases such as poliomyelitis, meningoencephalitis, and systemic neonatal disease. Although no compounds for PV infections have been approved in the United States, pirodavir was one of the most promising capsid-binding compounds to show efficacy in human clinical trials for chemoprophylaxis of the common cold. Susceptibility to hydrolysis precluded its use as an oral agent. We have developed orally bioavailable pyridazinyl oxime ethers that are as potent as pirodavir. Compounds BTA39 and BTA188 inhibited a total of 56 HRV laboratory strains and three clinical isolates as determined by neutral red uptake assay. At concentrations of <100 nM, BTA39 inhibited 69% of the HRV serotypes and isolates evaluated, BTA188 inhibited 75%, and pirodavir inhibited 59% of the serotypes and isolates. The 50% inhibitory concentrations (IC(50)s) for the two compounds ranged from 0.5 nM to 6,701 nM. The compounds also inhibited EV, including coxsackie A and B viruses (IC(50) = 773 to 3,608 nM) and echoviruses (IC(50) = 193 to 5,155 nM). BTA39 only inhibited poliovirus strain WM-1 at 204 nM, and BTA188 only inhibited poliovirus strain Chat at 82 nM. EV 71 was inhibited by BTA39 and BTA188, with IC(50)s of 1 and 82 nM, respectively. Both compounds were relatively nontoxic in actively growing cells (50% cytotoxic doses, >/=4,588 nM). These data suggest that these oxime ethers warrant further investigation as potential agents for treating selected PV infections.     

In vitro antagonistic properties of a new angiotensin type 1 receptor blocker, azilsartan, in receptor binding and function studies

The angiotensin II (AII) antagonistic action of azilsartan (AZL) [2-ethoxy-1-{[2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylic acid] was investigated in radioligand binding and function studies. AZL inhibited the specific binding of 12?I-Sar1-Ile?-AII to human angiotensin type 1 receptors with an IC?? of 2.6 nM. The inhibitory effect of AZL persisted after washout of the free compound (IC(50) value of 7.4 nM). Olmesartan, telmisartan, valsartan, and irbesartan also inhibited the specific binding with IC?? values of 6.7, 5.1, 44.9, and 15.8 nM, respectively. However, their inhibitory effects were markedly attenuated with washout (IC?? values of 242.5, 191.6, >10,000, and >10,000 nM). AZL also inhibited the accumulation of AII-induced inositol 1-phosphate (IP1) in the cell-based assay with an IC?? value of 9.2 nmol; this effect was resistant to washout (IC?? value of 81.3 nM). Olmesartan and valsartan inhibited IP1 accumulation with IC?? values of 12.2 and 59.8 nM, respectively. The activities of these compounds were markedly reduced after washout (IC?? value of 908.5 and 22,664.4 nM). AZL was defined as an inverse agonist in an experiment by using a constitutively active mutant of human angiotensin type 1 receptors. In isolated rabbit aortic strips, AZL reduced the maximal contractile response to AII with a pD'? value of 9.9. The inhibitory effects of AZL on contractile responses induced by AII persisted after the strips were washed; these inhibitory effects were more potent than those of olmesartan. These results suggest that AZL is a highly potent and slowly dissociating AII receptor blocker. Its tight receptor binding might be expected to produce potent and long-lasting antihypertensive effects in preclinical and clinical settings.     

2,4-diaminopteridine-based compounds as precursors for de novo synthesis of antifolates: a novel class of antimalarials

We have tested the hypothesis that 2,4-diamino-6-hydroxymethyl-pteridine (DAP), 2,4-diaminopteroic acid (DAPA), and 2,4 diamino-N10-methyl-pteroic acid (DAMPA) could be converted into aminopterin (from DAP and DAPA) and methotrexate (from DAMPA), both of which are potent inhibitors of dihydrofolate reductase, a proven drug target for Plasmodium falciparum. DAP, DAPA, and DAMPA inhibited parasite growth in the micromolar range; DAMPA was the most active, with 50% inhibitory concentrations in vitro of 446 ng/ml against the antifolate-sensitive strain and 812 ng/ml against the highly resistant strain under physiological folate conditions. DAMPA potentiates the activity of the sulfone dapsone, an inhibitor of dihydropteroate synthase, but not that of chlorcycloguanil, a known inhibitor of dihydrofolate reductase (DHFR). Experiments with a Saccharomyces cerevisiae strain dependent upon the P. falciparum DHFR enzyme showed that DHFR is a target of DAMPA in that system. We hypothesize that DAMPA is converted to methotrexate by the parasite dihydrofolate synthase, which explains the synergy of DAMPA with dapsone but not with chlorcycloguanil. This de novo synthesis will not occur in the host, since it lacks the complete folate pathway. If this hypothesis holds true, the de novo synthesis of the toxic compounds could be used as a framework for the search for novel potent antimalarial antifolates.     

3H]2-phenylaminoadenosine ([3H]CV 1808) labels a novel adenosine receptor in rat brain.

Earlier studies have demonstrated that the vasoactive compound CV 1808 displays 10-fold selectivity for the adenosine A2 receptor, and as such, was the first reported A2-selective agonist. After the radiolabeling of CV 1808, its binding characteristics were evaluated in rat striatal, cortical and hippocampal membranes. Using 5 nM [3H]CV 1808, unlabeled CV 1808 produced shallow inhibition curves in all three brain areas, with 61 to 75% of the binding displaying IC50 values of 16 to 24 nM, whereas the remaining 28 to 37% of binding had lower affinity (IC50 595-1130 nM). The A2-selective agonist CGS 21680 and the nonselective adenosine agonist 5'-N-ethylcarboxamidoadenosine displayed very low affinity (IC50 > 10 microM). The A1-selective compound N6-cyclopentyladenosine inhibited only 28 to 44% of specific binding, with IC50 of 272-1750 nM. In contrast, the nonselective adenosine antagonist CGS 15943A inhibited specific binding by 48 to 64% (at 1 microM) with IC50 ranging from 106 to 295 nM. Additionally, several novel adenosine analogs fully inhibited specific binding, producing multicomponent inhibition curves. Electrophysiological studies in porcine coronary artery cells demonstrated that CV 1808, but not CGS 21680, 5'-N-ethylcarboxamidoadenosine and N6-cyclopentyladenosine, activated potassium channels. Further, the CV 1808-induced activation was blocked by CGS 15943A. These results indicate that [3H]CV 1808 binding consists of two components in rat brain: a low-affinity site with A1-like characteristics, and a novel high-affinity site, designated as the A4 receptor, where potassium channel activation appears to be a functional correlate.     

Inhibition of gene markers of fibrosis with a novel inhibitor of transforming growth factor-beta type I receptor kinase in puromycin-induced nephritis

SB-525334 (6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline) has been characterized as a potent and selective inhibitor of the transforming growth factor-beta1 (TGF-beta1) receptor, activin receptor-like kinase (ALK5). The compound inhibited ALK5 kinase activity with an IC(50) of 14.3 nM and was approximately 4-fold less potent as an inhibitor of ALK4 (IC(50) = 58.5 nM). SB-525334 was inactive as an inhibitor of ALK2, ALK3, and ALK6 (IC(50) > 10,000 nM). In cell-based assays, SB-525334 (1 microM) blocked TGF-beta1-induced phosphorylation and nuclear translocation of Smad2/3 in renal proximal tubule cells and inhibited TGF-beta1-induced increases in plasminogen activator inhibitor-1 (PAI-1) and procollagen alpha1(I) mRNA expression in A498 renal epithelial carcinoma cells. In view of this profile, SB-525334 was used to investigate the role of TGF-beta1 in the acute puromycin aminonucleoside (PAN) rat model of renal disease, a model of nephritis-induced renal fibrosis. Orally administered doses of 1, 3, or 10 mg/kg/day SB-525334 for 11 days produced statistically significant reductions in renal PAI-1 mRNA. Also, the compound produced dose-dependent decreases in renal procollagen alpha1(I) and procollagen alpha1(III) mRNA, which reached statistical significance at the 10-mg/kg/day dose when compared with vehicle-treated PAN controls. Furthermore, PAN-induced proteinuria was significantly inhibited at the 10-mg/kg/day dose level. These results provide further evidence for the involvement of TGF-beta1 in the profibrotic changes that occur in the PAN model and for the first time, demonstrate the ability of a small molecule inhibitor of ALK5 to block several of the markers that are predictive of fibrosis and renal injury in this model.     

Structure-activity relationships of Bacillus cereus and Bacillus anthracis dihydrofolate reductase: toward the identification of new potent drug leads

New and improved therapeutics are needed for Bacillus anthracis, the etiological agent of anthrax. To date, antimicrobial agents have not been developed against the well-validated target dihydrofolate reductase (DHFR). In order to address whether DHFR inhibitors could have potential use as clinical agents against Bacillus, 27 compounds were screened against this enzyme from Bacillus cereus, which is identical to the enzyme from B. anthracis at the active site. Several 2,4-diamino-5-deazapteridine compounds exhibit submicromolar 50% inhibitory concentrations (IC(50)s). Four of the inhibitors displaying potency in vitro were tested in vivo and showed a marked growth inhibition of B. cereus; the most potent of these has MIC(50) and minimum bactericidal concentrations at which 50% are killed of 1.6 mug/ml and 0.09 mug/ml, respectively. In order to illustrate structure-activity relationships for the classes of inhibitors tested, each of the 27 inhibitors was docked into homology models of the B. cereus and B. anthracis DHFR proteins, allowing the development of a rationale for the inhibition profiles. A combination of favorable interactions with the diaminopyrimidine and substituted phenyl rings explains the low IC(50) values of potent inhibitors; steric interactions explain higher IC(50) values. These experiments show that DHFR is a reasonable antimicrobial target for Bacillus anthracis and that there is a class of inhibitors that possess sufficient potency and antibacterial activity to suggest further development.     

Gastrointestinal serotonin: depletion due to tetrahydrobiopterin deficiency induced by 2,4-diamino-6-hydroxypyrimidine administration.

To study the role of serotonin in gastrointestinal function in mice, a peripheral deficiency was induced by administration of 2,4-diamino-6-hydroxypyrimidine, an inhibitor of tetrahydrobiopterin (6-[dihydroxypropyl-(L-erthro)-5,6,7,8,-tetrahydropterin) (BH4) biosynthesis. BH4 is an essential cofactor for tryptophan hydroxylase, the rate limiting enzyme in the biosynthesis of serotonin. When 2,4-diamino-6-hydroxypyrimidine (DAHP) was administered for 4 days at the dose of 3 g/kg/day, serotonin decreased in the duodenum and colon to approximately 46 and 40% of the control levels, respectively. These mice showed visual signs of gastrointestinal dysfunction in addition to a remarkable decrease in uptake of a p.o. glucose load. The decrease in serotonin was prevented and prevention of the apparent dysfunction of digestive tract was observed by simultaneous administration of (6R)BH4. Serotonin levels in the brain and blood were also decreased by treatment with p-chlorophenylalanine, an irreversible inhibitor of tryptophan hydroxylase. In contrast, DAHP had no effect on brain serotonin levels, presumably due to poor brain penetration. DAHP caused a rapid decrease (T1/2 of less than 12 hr) in the BH4 levels in all tissues examined, except in the brain. The BH4 level returned to within the normal range less than 24 hr after cessation of the administration of DAHP, a finding which suggests that the BH4 level is in a dynamic steady state maintained by rapid local biosynthesis. Thus, the local biosynthesis of BH4 supports normal digestive functions, presumably by controlling normal serotonin biosynthesis.     

Design,synthesis, and biological evaluation of new 6,N2-diaryl-1,3,5-triazine-2,4-diamines as anticancer agents selectively targeting triple negative breast cancer cells

New 6,N²-diaryl-1,3,5-triazine-2,4-diamines were designed using the 3D-QSAR model developed earlier. These compounds were prepared and their antiproliferative activity was evaluated against three breast cancer cell lines (MDA-MB231, SKBR-3 and MCF-7) and non-cancerous MCF-10A epithelial breast cells. The synthesized compounds demonstrated selective antiproliferative activity against triple negative MDA-MB231 breast cancer cells. The most active compound in the series inhibited MDA-MB231 breast cancer cell growth with a GI₅₀ value of 1 nM. None of the tested compounds significantly affected the growth of the normal breast cells. The time-dependent cytotoxic effect, observed when cytotoxicity was assessed at different time intervals after the treatment, and morphological features, observed in the fluorescence microscopy and live cell imaging experiments, suggested apoptosis as the main pathway for the antiproliferative activity of these compounds against MDA-MB231 cells.

New highly potent and selective 6,N²-diaryl-1,3,5-triazine-2,4-diamines were designed and prepared using the 3D-QSAR model developed earlier.     

GSK256066, an exceptionally high-affinity and selective inhibitor of phosphodiesterase 4 suitable for administration by inhalation: in vitro, kinetic, and in vivo characterization

Oral phosphodiesterase (PDE) 4 inhibitors such as roflumilast have established the potential of PDE4 inhibition for the treatment of respiratory diseases. However, PDE4 inhibitor efficacy is limited by mechanism-related side effects such as emesis and nausea. Delivering the inhibitor by the inhaled route may improve therapeutic index, and we describe 6-({3-[(dimethylamino)carbonyl]phenyl}sulfonyl)-8-methyl-4-{[3-methyloxy) phenyl]amino}-3-quinolinecarboxamide (GSK256066), an exceptionally high-affinity inhibitor of PDE4 designed for inhaled administration. GSK256066 is a slow and tight binding inhibitor of PDE4B (apparent IC(50) 3.2 pM; steady-state IC(50) <0.5 pM), which is more potent than any previously documented compound, for example, roflumilast (IC(50) 390 pM), tofimilast (IC(50) 1.6 nM), and cilomilast (IC(50) 74 nM). Consistent with this, GSK256066 inhibited tumor necrosis factor α production by lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes with 0.01 nM IC(50) (compared with IC(50) values of 5, 22, and 389 nM for roflumilast, tofimilast, and cilomilast, respectively) and by LPS-stimulated whole blood with 126 pM IC(50). GSK256066 was highly selective for PDE4 (>380,000-fold versus PDE1, PDE2, PDE3, PDE5, and PDE6 and >2500-fold against PDE7), inhibited PDE4 isoforms A-D with equal affinity, and had a substantial high-affinity rolipram binding site ratio (>17). When administered intratracheally to rats, GSK256066 inhibited LPS-induced pulmonary neutrophilia with ED(50) values of 1.1 μg/kg (aqueous suspension) and 2.9 μg/kg (dry powder formulation) and was more potent than an aqueous suspension of the corticosteroid fluticasone propionate (ED(50) 9.3 μg/kg). Thus, GSK256066 has been demonstrated to have exceptional potency in vitro and in vivo and is being clinically investigated as a treatment for chronic obstructive pulmonary disease.     

(-)-2''-deoxy-3''-thiacytidine is a potent, highly selective inhibitor of human immunodeficiency virus type 1 and type 2 replication in vitro.

The (-)-enantiomer of 2'-deoxy-3'-thiacytidine (3TC) was found to be a potent and selective inhibitor of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) in vitro. We determined its antiviral activity against a number of laboratory strains of HIV-1 and HIV-2 in a range of CD4-bearing lymphocyte cell lines (mean 50% inhibitory concentration [IC50] range, 4 nM to 0.67 microM). 3TC was also active against a range of HIV-1 strains in peripheral blood lymphocytes (mean IC50 range, 2.5 to 90 nM). The IC50 for cytotoxicity in seven lymphocyte cell cultures, including human peripheral blood lymphocytes, ranged from 0.5 to 6 mM. 3TC had no detectable antiviral activity against a range of other viruses or in cells chronically infected with HIV-1 or HIV-2. The effects of time of addition of the compound and varying the multiplicity of infection on the antiviral activity of 3TC were determined. The results showed that 3TC is a potent and selective inhibitor of HIV-1 and HIV-2 replication in vitro.     

Specific inhibition of leukotriene B4-induced neutrophil activation by LY223982.

LY223982, (E)-5-(3-carboxybenzoyl)-2-((6-(4-methoxyphenyl)-5- hexenyl)oxy)benzenepropanoic acid, is a newly discovered potent inhibitor of specific binding of leukotriene B4 (LTB4) to its receptor on human neutrophils. This study demonstrated that the compound is also a specific antagonist of LTB4-induced neutrophil activation under both in vitro and in vivo conditions. LY223982 was found to be 189-fold more effective in displacing [3H]LTB4 than 35S-N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) from their corresponding receptors on human neutrophils. The concentration inhibiting 50% of response (IC50) for displacement of [3H]LTB4 (13.2 nM) was only 6.8-fold higher than the value for nonradioactive LTB4. The compound inhibited the aggregation of guinea pig neutrophils caused by LTB4 more strongly than FMLP or platelet-activating factor. The IC50 for inhibition of LTB4-induced responses (74 nM) was 93- and > 135-fold lower than the IC50 for inhibition of the corresponding FMLP and platelet-activating factor-induced effects. LY223982 was also a potent antagonist of the aggregation of human neutrophils by LTB4 (IC50, 100 nM). Chemotaxis of human neutrophils induced by LTB4 was only modestly inhibited by the compound (IC50 = 6 microM) but it had even less effect on cell movement caused by FMLP. LY223982 inhibited transient leukopenia induced in rabbits with LTB4 (ED50, 3 mg/kg) but not with FMLP. It had no agonist activity in any of the test systems. In summary, the results indicate that LY223982 is a potent specific antagonist of LTB4-induced neutrophil activation.     

Triazine Inhibits Toxoplasma gondii tachyzoites in vitro and in vivo

The triazine WR99210 [4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5-trichlorophenoxypropyloxy)-1,3,5 triazine] inhibits Toxoplasma gondii in vitro at nanomolar levels (P < 0.05). The 50% inhibitory concentration (IC(50)) was approximately 50 nM. It is a potent inhibitor in vitro and is also effective in vivo. Administration of WR99210 parenterally (i.e., intraperitoneally) reduced the mean number of RH strain tachyzoites present in peritoneal fluid substantially 4 days after intraperitoneal infection of mice. There was a mean of approximately 35 million parasites in control mice as contrasted with approximately 2 million parasites in mice treated with 1.25 mg WR99210/kg of body weight in a representative experiment (P < 0.05). In addition the prodrug PS-15 N'-[3-(2,4, 5-trichlorophenoxy)propyloxy]-N9-(1-methylethyl) imidocarbonimidicdiamide is converted to 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5-trichlorophenoxypropyloxy)-1,3,5 triazine in vivo when the prodrug is administered orally. PS-15 administered by gavage also reduced intraperitoneal RH strain T. gondii tachyzoite numbers. WR99210 has high efficacy and relatively low toxicity because of its substantial effect on T. gondii dihydrofolate reductase (DHFR) but not the mammalian host DHFR. Amino acid sequences of T. gondii, Plasmodium falciparum, and Homo sapiens DHFRs were compared. It is of interest that of the DHFR amino acids considered to be interacting with WR99210 in P. falciparum within interatomic distances within 3 to 5 A, four of eight were shared with T. gondii DHFR. H. sapiens also shared four amino acids thought to be interacting with WR99210. Efficacy of intraperitoneal administration of WR99210 and peroral administration of PS-15 demonstrate the potential usefulness of this class of compounds in treatment of toxoplasmosis administered either parenterally or perorally. The recent development program for this class of antimicrobials as antimalarials makes our proof of principle of improved efficacy of triazines (compared with the gold standard treatment, pyrimethamine) against T. gondii especially promising.     

SR147778 [5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide], a new potent and selective antagonist of the CB1 cannabinoid receptor: biochemical and pharmacological characterization

Based on binding, functional, and pharmacological data, this study introduces SR147778 [5-(4-bromophenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide] as a highly potent, selective, and orally active antagonist for the CB1 receptor. This compound displays nanomolar affinity (Ki = 0.56 and 3.5 nM) for both the rat brain and human CB1 recombinant receptors, respectively. It has low affinity (Ki = 400 nM) for both the rat spleen and human CB2 receptors. Furthermore, it shows no affinity for any of the over 100 targets investigated (IC50 > 1 microM). In vitro, SR147778 antagonizes the inhibitory effects of CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol] on both the mouse vas deferens contractions (pA2 value = 8.1) and on forskolin-stimulated adenylyl cyclase activity in the U373 MG cell lines (pA2 value = 8.2) but not in Chinese hamster ovary (CHO) cells permanently expressing the human peripheral cannabinoid receptor (hCB2). SR147778 is able to block the mitogen-activated protein kinase activity induced by CP 55,940 in the CHO cell line expressing human brain cannabinoid receptor (IC50 = 9.6 nM) but was inactive in cells expressing hCB2. After oral administration, SR147778 displaced the ex vivo [3H]-CP 55,940 binding to mouse brain membranes (ED50 = 3.8 mg/kg) with a long duration of action, whereas it did not interact with the CB2 receptor expressed in the mouse spleen. Using different routes of administration, SR147778 (0.3-3 mg/kg) is shown to antagonize pharmacological effects (hypothermia, analgesia, and gastrointestinal transit) induced by R-(+)-(2,3-dihydro-5-methyl-3-[[4-morpholinyl]methyl] pyrol [1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone in mice. Finally, per se, SR147778 (0.3-10 mg/kg) is able to reduce ethanol or sucrose consumption in mice and rats and food intake in fasted and nondeprived rats.