Novel, potent THC/anandamide (hybrid) analogs

The structure–activity relationship (SAR) of the end pentyl chain in anandamide (AEA) has been established to be very similar to that of Δ⁹-tetrahydrocannabinol (Δ⁹-THC). In order to broaden our understanding of the structural similarities between AEA and THC, hybrid structures 1–3 were designed. In these hybrids the aromatic ring of THC–DMH was linked to the AEA moiety through an ether linkage with the oxygen of the phenol of THC. Hybrid 1 (O-2220) was found to have very high binding affinity to CB1 receptors (K_i = 8.5 nM), and it is interesting to note that the orientation of the side chain with respect to the oxygen in the phenol is the same as in THCs. To further explore the SAR in this series the terminal carbon of the side chain was modified by adding different substituents. Several such analogs were synthesized and tested for their CB1 and CB2 binding affinities and in vivo activity (tetrad tests). The details of the synthesis and the biological activity of these compounds are described.     

The anandamide membrane transporter. Structure-activity relationships of anandamide and oleoylethanolamine analogs with phenyl rings in the polar head group region

A new series of anandamide and N-oleoylethanolamine analogs, most of which with aromatic moieties in the head group region, has been synthesized and evaluated as inhibitors of anandamide uptake. Some of them efficaciously inhibit the uptake process with K(i) values in the low micromolar range (2.4-21.2microM). Strict structural requisites are needed to observe a significant inhibition and in no case inhibition of fatty acid amidohydrolase overlaps with inhibition of anandamide uptake.     

Binding mode prediction of conformationally restricted anandamide analogs within the CB1 receptor

Background

CB₁ cannabinoid receptors are G-protein coupled receptors for endocannabinoids including anandamide and 2-arachidonoylglycerol. Because these arachidonic acid metabolites possess a 20-carbon polyene chain as the alkyl terminal moiety, they are highly flexible with the potential to adopt multiple biologically relevant conformations, particularly those in a bent form. To better understand the molecular interactions associated with binding and steric trigger mechanisms of receptor activation, a series of conformationally-restricted anandamide analogs having a wide range of affinity and efficacy were evaluated.

Results

A CB₁ receptor model was constructed to include the extracellular loops, particularly extracellular loop 2 which possesses an internal disulfide linkage. Using both Glide (Schrödinger) and Affinity (Accelrys) docking programs, binding conformations of six anandamide analogs were identified that conform to rules applicable to the potent, efficacious and stereoselective non-classical cannabinoid CP55244. Calculated binding energies of the optimum structures from both procedures correlated well with the reported binding affinity values. The most potent and efficacious of the ligands adopted conformations characterized by interactions with both the helix-3 lysine and hydrophobic residues that interact with CP55244. The other five compounds formed fewer or less energetically favorable interactions with these critical residues. The flexibility of the tested anandamide analogs, measured by torsion angles around the benzene as well as the stretch between side chain moieties, could contribute to the differences in ability to interact with the CB₁ receptor.

Conclusion

Analyses of multiple poses of conformationally-restricted anandamide analogs permitted identification of favored amino acid interactions within the CB₁ receptor binding pocket. A ligand possessing both high affinity and cannabinoid agonist efficacy was able to interact with both polar and hydrophobic interaction sites utilized by the potent and efficacious non-classical cannabinoid CP55940. In contrast, other analogs characterized by reduced affinity or efficacy exhibited less favorable interactions with those key residues.     

Enzymatic synthesis of novel glutathione analogs

A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques has been immobilized in a carrageenan matrix and used for the synthesis of various types of isotopically labeled glutathione (L-gamma-glutamyl-L-cysteinyl-glycine) (K. Murata, W. A. Abbott, R. J. Bridges, and A. Meister (1985) Anal. Biochem. 150, 235-237). In the present work, this E. coli matrix was used as the basis of a method for the synthesis of glutathione analogs. Thus, amino acid analogs were used in place of the corresponding amino acid constituents of glutathione (e.g., 4-fluoroglutamate was substituted for glutamate) in the reaction mixtures. Using this method we have synthesized several analogs of glutathione including L-gamma-glutamyl-(beta-chloro)-L-alanyl-glycine, (R,S)-4-fluoro-DL-gamma-glutamyl-L-cysteinyl-glycine, D-gamma-glutamyl-L-cysteinyl-glycine, and L-gamma-glutamyl-L-homocysteinyl-glycine. This method may also be used for the synthesis of a number of L- and D-gamma-glutamyl amino acids. The analogs are purified by gel-filtration and ion-exchange chromatography. The analogs are used to examine the substrate specificity and mechanisms of action of glutathione-utilizing enzymes and for studies on glutathione metabolism and function. Fluorine-containing analogs may be used for NMR studies. The enzymatically prepared compounds may also be used as intermediates in the chemical synthesis of other analogs of glutathione and glutathione disulfide.     

Synthesis and evaluation of novel podophyllotoxin analogs

Because prior studies have shown inconsistency between structure-activity relationships for podophyllotoxin derivatives as topoisomerase II inhibitors and cytotoxic agents, eight novel podophyllotoxin analogs were synthesized to further explore the effects of structural variations on both A and D rings on activity. The new compounds contain a 4,5-dimethoxy substituted A ring and opened D-ring variants and were prepared by appropriate functional and stereochemical operations at the methylenedioxy group, C7, C8, and C8'. Four compounds (15, 18, 21 and 22) demonstrated noticeable inhibitory activity against A549, DU145, KB and KBvin tumor cells, and the most active compound 18 showed IC(50) values less than 10 μg/mL.     

Characterization of biotin-anandamide, a novel tool for the visualization of anandamide accumulation

Anandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist of both cannabinoid and vanilloid receptors. During the last two decades, its metabolic pathways and biological activity have been investigated extensively and relatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that has the same lipophilicity of the parent compound. In addition, by means of biochemical assays and fluorescence microscopy, we show that b-AEA is accumulated inside the cells in a way superimposable on that of AEA. Conversely, b-AEA does not interact or interfere with the other components of the endocannabinoid system, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEA hydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEA could be a very useful probe for visualizing the accumulation and intracellular distribution of this endocannabinoid.     

Conformational studies of novel antiretroviral analogs of zidovudine

Conformational properties of three novel zidovudine analogs, namely 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso, 2), (-)-trans-(5S,6S)-5-bromo-6, 5'-epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (3) and (+)-trans-(5R,6R)-5-bromo-6,5'-epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (4), have been investigated by AM1 calculations and NMR studies, and compared with those of the parent nucleoside (AZT, 1). Based on the results obtained the following correlation may be established, a) AZT and AZT-Iso exhibit a conformational behavior analog to other pyrimidinic nucleosides, displaying a dynamic equilibrium in solution where the two conformers (North and South) undergo a constant transformation. b) Compounds 3 and 4 show a different conformational profile. The estimate of the pseudorotation phase angle reveals the rigid structures of the latter compounds, which do not evidence conformational equilibrium in solution; the azide group being the only group free to rotate. c) Diastereoisomers 3 and 4 exhibit an extra conformational parameter compared with other pyrimidinic nucleosides: the chair or boat conformation in the third ring formed between the sugar and the base. In all cases, a reasonable correlation was obtained between theoretical and NMR spectroscopic data.     

Optimization of globomycin analogs as novel gram-negative antibiotics

Discovery of novel classes of Gram-negative antibiotics with activity against multi-drug resistant infections is a critical unmet need. As an essential member of the lipoprotein biosynthetic pathway, lipoprotein signal peptidase II (LspA) is an attractive target for antibacterial drug discovery, with the natural product inhibitor globomycin offering a modestly-active starting point. Informed by structure-based design, the globomycin depsipeptide was optimized to improve activity against E. coli. Backbone modifications, together with adjustment of physicochemical properties, afforded potent compounds with good in vivo pharmacokinetic profiles. Optimized compounds such as 51 (E. coli MIC 3.1 μM) and 61 (E. coli MIC 0.78 μM) demonstrate broad spectrum activity against gram-negative pathogens and may provide opportunities for future antibiotic discovery.     

Production of novel rapamycin analogs by precursor-directed biosynthesis

The natural product rapamycin, produced during fermentation by Streptomyces hygroscopicus, is known for its potent antifungal, immunosuppressive, and anticancer activities. During rapamycin biosynthesis, the amino acid l-pipecolate is incorporated into the rapamycin molecule. We investigated the use of precursor-directed biosynthesis to create new rapamycin analogs by substitution of unusual l-pipecolate analogs in place of the normal amino acid. Our results suggest that the l-pipecolate analog (+/-)-nipecotic acid inhibits the biosynthesis of l-pipecolate, thereby limiting the availability of this molecule for rapamycin biosynthesis. We used (+/-)-nipecotic acid in our precursor-directed biosynthesis studies to reduce l-pipecolate availability and thereby enhance the incorporation of other pipecolate analogs into the rapamycin molecule. We describe here the use of this method for production of two new sulfur-containing rapamycin analogs, 20-thiarapamycin and 15-deoxo-19-sulfoxylrapamycin, and report measurement of their binding to FKBP12.     

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric determination of anandamide and its analogs in rat brain and peripheral tissues

A simple and selective method for the determination of anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand, and its analogs with liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) was developed. The calibration curve for standard anandamide was linear over the range 625 fmol-125 pmol per injection (r=0.999) with a precision of 1.0% (C.V.) at 25 pmol. The detection limit attained was 200 fmol per injection at a signal-to-noise ration of 2. Anandamide and its analogs were extracted from rat brain and peripheral tissues according to the method of Folch, and the recovery of anandamide from rat brain homogenates was 67.0–72.6%. The method was applied to their determination in rat brain and peripheral tissues.     

Cardiovascular pharmacology of anandamide

The fatty acid amide anandamide produces hypotension and a decrease in systemic vascular resistance in vivo. A drop in blood pressure is also seen with synthetic cannabinoid (CB) receptor agonists. The hypotensive responses to anandamide and synthetic cannabinoids are absent in CB1 receptor gene knockout mice. In isolated arteries and perfused vascular beds, anandamide induces vasodilator responses, which cannot be mimicked by synthetic cannabinoids. Instead, vanilloid receptors on perivascular sensory nerves play a key role in these effects of anandamide. Activation of vanilloid receptors by anandamide triggers the release of sensory neuropeptides such as the vasodilator calcitonin gene-related peptide (CGRP). Anandamide is detected in blood and in many cells of the cardiovascular system, and macrophage-derived anandamide may be involved in several hypotensive clinical conditions. Interestingly, cannabinoid and vanilloid receptors display an overlap in ligand recognition properties, and the frequently used CB1 receptor antagonist SR141716A also inhibits vanilloid receptor-mediated responses. The presence of anandamide in endothelial cells, neurones and activated macrophages (monocytes), and its ability to activate CB and vanilloid receptors make this lipid a potential bioregulator in the cardiovascular system.     

Oxidative metabolism of anandamide

In addition to the well studied hydrolytic metabolism of anandamide, a number of oxidative processes are also possible. Several routes somewhat analogous to the metabolism of free arachidonic acid have been reported. These involve mediation by various lipoxygenases and COX-2 and lead to ethanolamide analogs of the prostaglandins and HETES. The physiological significance of these products is not well understood at this time. There are also preliminary data suggesting a pathway involving oxidation of the hydroxy group of anandamide to a putative metabolite, N-arachidonyl glycine (AA-gly). This molecule displays activities in experimental models that suggest that it may play a role in some of the activities attributed to its precursor, anandamide.     

Nucleic acid components and their analogs: Design and synthesis of novel cytosine thioglycoside analogs

The synthesis of a new category of novel cytosine 4-thioglycoside analogs has been first accomplished. The main step of this strategy is the synthesis of sodium pyrimidine-4-thiolate through the condensation of 2-cyano-N-arylacetamides with sodium cyanocarbonimidodithioate, followed by coupling with α-bromo-sugars to afford the corresponding cytosine 4-thioglycoside analogs. The free thioglycosides were also prepared. Subsequent studies on the application of this strategy for the preparation of other potent pyrimidine thioglycosides are reported.     

Two novel dATP analogs for DNA photoaffinity labeling

Two new photoreactive dATP analogs, N⁶-[4-azidobenzoyl–(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (AB-dATP) and N⁶-[4-[3-(trifluoromethyl)-diazirin-3-yl]benzoyl-(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (DB-dATP), were synthesized from 2′-deoxyadenosine-5′-monophosphate in a six step procedure. Synthesis starts with aminoethylation of dAMP and continues with rearrangement of N¹-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate to N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate (N⁶-dAMP). Next, N⁶-dAMP is converted into the triphosphate form by first protecting the N-6 primary amino group before coupling the pyrophosphate. After pyrophosphorylation, the material is deprotected to yield N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-triphosphate (N⁶-dATP). The N-6 amino group is subsequently used to attach either a phenylazide or phenyldiazirine and the photoreactive nucleotide is then enzymatically incorporated into DNA. N⁶-dATP and its photoreactive analogs AB-dATP and DB-dATP were successfully incorporated into DNA using the exonuclease-free Klenow fragment of DNA polymerase I in a primer extension reaction. UV irradiation of the primer extension reaction with AB-dATP or DB-dATP showed specific photocrosslinking of DNA polymerase I to DNA.     

Synthesis and antimitotic activity of novel 2-methoxyestradiol analogs

The syntheses and antimitotic activity of several novel 2-methoxyestradiol analogs are described. Structural modifications investigated include introduction of additional unsaturation in rings B and D; inversion at C-13; and substitution at the C-2, C-15, C-16, and C-7 alpha positions. Of 15 analogs synthesized, 2 have demonstrated superior biological activities compared to 2-methoxyestradiol.     

Structure and function relationships of three novel hGHRH-GGC analogs

Growth hormone releasing hormone (GHRH) is one of the hypothalamus hormones. For its potential applications in agriculture and medicine, GHRH analog with higher activity and longer half-life has been looked for. By using the fusion expression with unique acid labile linker Asp-Pro and biochemical purification, the three novel GHRH peptides, Pro-Pro-hGHRH(1–44)-Gly-Gly-Cys, Pro-hGHRH(1–44)-Gly-Gly-Cys, and ¹Pro-GHRH(2–44)-Gly-Gly-Cys, were obtained. The peptide molecular weight with 5,455, 5,373 or 5,210 Da measured by EIS-MS is coincident with the actual values. The peptides at 0.1–10 μg/ml increased rat pituitary GH releases in a dose-dependent manner and at 5 μg/ml increased human pituitary GH releases. The activity comparisons showed that at 10 μg/ml there were significant between ¹Pro-hGHRH(2–44)-Gly-Gly-Cys and Pro-Pro-hGHRH(1–44)-Gly-Gly-Cys or Pro-hGHRH(1–44)-Gly-Gly-Cys, ¹Pro-hGHRH(2–44) (P < 0.05). The ¹Pro-hGHRH(2–44)-Gly-Gly-Cys showed the highest GH release from rat pituitary. The activity results showed that the N-terminal Pro modulations and the C-terminal Gly-Gly-Cys extension regulate GH release from pituitary. The results showed that the three peptides had good GH release, function-selectivity and species specificity.     

Synthesis and anti-inflammatory effects of novel pimarane diterpenoid analogs

Syntheses and excellent anti-inflammatory effects of a series of novel acanthoic acid analogs are reported. In particular, the mechanistic basis for their anti-inflammatory effects is also described.     

Synthesis and antibacterial activity of novel water-soluble nocathiacin analogs

Semi-synthetic water-soluble analogs were synthesized from nocathiacin I through the formation of a versatile intermediate nocathiacin amine 5, and subsequent transformation via reductive amination, acylation or urea formation. Several of the novel analogs displayed much improved aqueous solubility over 1, while retained antibacterial activity. Compound 15 and 16 from the amide series, demonstrated excellent in vitro and in vivo antibacterial activity.     

Antiparasitic activities of novel,orally available fumagillin analogs

Fumagillin, an irreversible inhibitor of MetAP2, has been shown to potently inhibit growth of malaria parasites in vitro. Here, we demonstrate activity of fumagillin analogs with an improved pharmacokinetic profile against malaria parasites, trypanosomes, and amoebas. A subset of the compounds showed efficacy in a murine malaria model. The observed SAR forms a basis for further optimization of fumagillin based inhibitors against parasitic targets by inhibition of MetAP2.