Δ9-Tetrahydrocannabinolic acid A, the precursor to Δ9-tetrahydrocannabinol (THC).

While Δ9-tetrahydrocannabinolic acid A (THCA-A) has been reported to be difficult to crystallize and/or amorphous, we have obtained THCA-A in a pure crystalline form by extraction of marijuana and selective fractionation with liquid CO2. THCA-A (systematic name: 1-hydroxy-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]isochromene-2-carboxylic acid, C22H30O4) crystallizes in the orthorhombic space group P212121, with Z = 8 and Z' = 2. The two independent molecules are related by a pseudo-twofold axis centered between the two -CO2H groups, but the conformations of the two -C5H11 chains are quite different (tgt and ttg; t is trans and g is gauche). The carboxylate groups form an intermolecular R22(8) hydrogen-bonded ring; the two C2O2 carboxylate planes are twisted out of the planes of the attached arene rings in opposite directions by 13.59 (8) and 18.92 (8)°, respectively, with a resultant interplanar angle of 28.89 (8)°. Each molecule also has an intramolecular S(6) hydrogen-bond motif between the ortho -OH group and the dihydropyran-ring O atom. Other conformational aspects of the two independent molecules are quite similar to those found in the previously determined structure of THCA-B. THCA-A has shown promise in a number of medical applications. Demonstration of the crystallinity and details of the crystal structure are expected to provide a standard point of departure for chemical and medical experiments.

Antiviral Efficacy and Host Innate Immunity Associated with SB 9200 Treatment in the Woodchuck Model of Chronic Hepatitis B.

SB 9200, an oral prodrug of the dinucleotide SB 9000, is being developed for the treatment of chronic hepatitis B virus (HBV) infection and represents a novel class of antivirals. SB 9200 is thought to activate the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) resulting in interferon (IFN) mediated antiviral immune responses in virus-infected cells. Additionally, the binding of SB 9200 to these sensor proteins could also sterically block the ability of the viral polymerase to access pre-genomic RNA for nucleic acid synthesis. The immune stimulating and direct antiviral properties of SB 9200 were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) by daily, oral dosing at 15 and 30 mg/kg for 12 weeks. Prolonged treatment resulted in 2.2 and 3.7 log10 reductions in serum WHV DNA and in 0.5 and 1.6 log10 declines in serum WHV surface antigen from pretreatment level with the lower or higher dose of SB 9200, respectively. SB 9200 treatment also resulted in lower hepatic levels of WHV nucleic acids and antigen and reduced liver inflammation. Following treatment cessation, recrudescence of viral replication was observed but with dose-dependent delays in viral relapse. The antiviral effects were associated with dose-dependent and long-lasting induction of IFN-α, IFN-ß and IFN-stimulated genes in blood and liver, which correlated with the prolonged activation of the RIG-I/NOD2 pathway and hepatic presence of elevated RIG-I protein levels. These results suggest that in addition to a direct antiviral activity, SB 9200 induces antiviral immunity during chronic hepadnaviral infection via activation of the viral sensor pathway.

Evaluation of 3-O-methylfluorescein as a selective fluorometric substrate for CYP2C19 in human liver microsomes.

Cytochrome P450 (P450) fluorometric high-throughput inhibition assays have been widely used for drug-drug interaction screening particularly at the preclinical drug discovery stages. Many fluorometric substrates have been investigated for their selectivity, but most are found to be catalyzed by multiple P450 isozymes, limiting their utility. In this study, 3-O-methylfluorescein (OMF) was examined as a selective fluorescence substrate for CYP2C19 in human liver microsomes (HLMs). The kinetic studies of OMF O-demethylation in HLMs using a liquid chromatography/mass spectrometry method exhibited two-enzyme kinetics with apparent K(m) and V(max) values of 1.14 +/- 0.90 microM and 11.3 +/- 4.6 pmol/mg/min, respectively, for the high affinity component(s) and 57.0 +/- 6.4 microM and 258 +/- 6 pmol/mg/min, respectively, for the low affinity component(s). Studies utilizing cDNA-expressed individual P450 isoforms and P450-selective chemical inhibitors showed that OMF O-demethylation to fluorescein was selective for CYP2C19 at substrate concentrations < or =1 microM. At substrate concentrations > or =10 microM, other P450 isozymes were found to catalyze OMF O-demethylation. In HLMs, analysis of the two-enzyme kinetics in the presence of P450 isozyme-selective chemical inhibitors (ticlopidine for CYP2C19, sulfaphenazole for CYP2C9, and furafylline for CYP1A2) indicated that CYP2C19 was the high affinity component and CYP2C9 was the low affinity component. Based on these findings, a fluorometric assay was developed using 1 microM OMF and 2 microM sulfaphenazole for probing CYP2C19-mediated inhibition in HLMs. The IC(50) data of 13 substrates obtained from the fluorometric assay developed in this study correlated well with that reported in the literature using nonfluorescence assays.

Optimization of purine based PDE1/PDE5 inhibitors to a potent and selective PDE5 inhibitor for the treatment of male ED.

In search of a PDE5 inhibitor for erectile dysfunction, an SAR was developed from a PDE1/PDE5 purine series of leads, which had modest PDE5 potency and poor isozyme selectivity. A compound (41) with PDE5 inhibition and in vivo activity similar to sildenafil was discovered from this effort. In addition, purine 41 demonstrated superior overall PDE isozyme selectivity when compared to the approved PDE5 inhibitors sildenafil, vardenafil, and tadalafil, which may result in a more favorable side-effect profile.

Design and synthesis of xanthine analogues as potent and selective PDE5 inhibitors.

We have discovered potent and selective xanthine PDE5 inhibitors. Compound 25 (PDE5 IC(50)=0.6 nM, PDE6/PDE5=101) demonstrated similar functional efficacy and PK profile to Sildenafil (PDE5 IC(50)=3.5 nM, PDE6/PDE5=7).