Coupling of isoprenoid triflates with organoboron nucleophiles: synthesis and biological evaluation of geranylgeranyl diphosphate analogues

The Suzuki coupling reaction has been used to introduce a methyl group derived from commercially available methylboronic acid into a vinyl triflate. This has led to a concise synthesis of all-trans-geranylgeraniol, with the key step being the palladium-catalyzed, silver-mediated methylation of triflate to give ethyl geranylgeranoate. This coupling protocol has also been used to produce the novel geranylgeranyl diphosphate (GGPP) analogue 3-phenyl-3-desmethylgeranylgeranyl diphosphate (3-PhGGPP, ). Our previously developed organocuprate coupling protocol has been used to introduce the cyclopropyl and tert-butyl moieties into the 3-position of vinyl triflate. The four GGPP analogues 3-vinyl-3-desmethylgeranylgeranyl diphosphate (3-vGGPP, ), 3-cyclopropyl-3-desmethylgeranylgeranyl diphosphate (3-cpGGPP, ), 3-tert-butyl-3-desmethyl-geranylgeranyl diphosphate (3-tbGGPP, ), and were then evaluated as potential inhibitors of recombinant yeast protein-geranylgeranyl transferase I (PGGTase I). The potential mechanism-based inhibitors 3-vGGPP and 3-cpGGPP did not exhibit time-dependent inactivation of PGGTase I. Instead, both analogues were alternative substrates, in accord with the interaction of the corresponding farnesyl analogues 3-vFPP and 3-cpFPP with PFTase. The tert-butyl and phenyl analogues were not substrates, but were instead competitive inhibitors of PGGTase I. Note that all four of the GGPP analogues were bound less tightly by the enzyme than the natural substrate, in contrast to the behavior of the 3-substituted FPP analogues.     

Studies on hydroperoxide-dependent folic acid degradation by hemin

Hemin (ferric protoporphyrin IX chloride) in the presence of hydrogen peroxide or tert-butyl hydroperoxide was found to cleave folic acid at the C9-N10 bond. The ferrous form of hemin was not involved in hydroperoxide-dependent folic acid degradation, as indicated by the lack of inhibition by carbon monoxide. Molecular oxygen was not required for the degradation. GSH-Mn(II) or NAD(P)H in the presence of molecular oxygen did not support hemin-mediated folic acid degradation. The degradation increased as the temperature was elevated from 10 to 70 degrees C. Ascorbic acid and azide were potent inhibitors. Superoxide dismutase and hydroxyl radical quenchers, such as ethanol, mannitol, benzoate, and dimethyl sulfoxide did not inhibit the reaction. Catalase inhibited hydrogen peroxide-supported degradation but not the tert-butyl hydroperoxide-dependent one. Thiol compounds, such as thioglycolic acid, thiourea, glutathione, cysteine, and 2-mercaptoethanol, inhibited the hydrogen peroxide-dependent degradation but supported the tert-butyl hydroperoxide-mediated one. N5-formyl tetrahydrofolic acid, but not N10-formyl folic acid, was degraded by hemin in the presence of H2O2 or TBHP. The data obtained are suggestive of a mechanism similar to N-demethylation reactions catalyzed by cytochrome P-450 and some peroxidases.     

Synthesis and biological evaluation of 2',3'-didehydro-2',3'-dideoxy-9-deazaguanosine, a monophosphate prodrug and two analogues, 2',3'-dideoxy-9-deazaguanosine and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine

2',3'-Didehydro-2',3'-dideoxy-9-deazaguanosine (1), its monophosphate prodrug (2), and two analogues, 2',3'-dideoxy-9-deazaguanosine (3) and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine (4), have been synthesized from benzoylated 9-deazaguanosine (5). Basic hydrolysis of 5, selective protection of the 2-amino and 5'-hydroxy functions with isobutyryl and silyl groups, respectively, followed by reaction with thiocarbonyldiimidazole gave the cyclic thiocarbonate, which, upon reaction with triethyl phosphite, followed by deprotection, afforded 1. Treatment of 1 with phenyl methoxyalaninylphosphochloridate and N-methylimidazole gave 2. Catalytic hydrogenation of 1 gave 3. Hydrodediazoniation of 1 with tert-butyl nitrite and tris(trimethylsilyl)silane gave 4. Compounds 1-4 were found to be inactive against the human immunodeficiency virus and exhibited minimal to no cytotoxic activity against the L1210 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma in vitro.     

Interaction of tert-butyl hydroperoxide with hypochlorous acid. A spin trapping and chemiluminescence study

The formation of radical species during the reaction of ter-tbutyl hydroperoxide and hypochlorous acid has been investigated by spin trapping and chemiluminescence. A superposition of two signals appeared incubating tert-butyl hydroperoxide with hypochlorous acid in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The first signal (aN = 1.537 mT, aH beta = 0.148 mT) was an oxidation product of POBN caused by the action of hypochlorous acid. The second spin adduct (aN = 1.484 mT, aH beta = 0.233 mT) was derived from a radical species that was formed in the result of reaction of tert-butyl hydroperoxide with hypochlorous acid. Similarly, a superposition of two signals was also obtained using the spin trap N-tert-butyl-alpha-phenylnitrone (PBN). tert-Butyl hydroperoxide was also treated with Fe2+ or Ce4+ in the presence of POBN. Using Fe2+ a spin adduct with a N = 1.633 mT and aH beta = 0.276 mT was observed. The major spin adduct formed with Ce4+ was characterised by a N = 1.480 mT and aH beta = 0.233 mT. The reaction of tert-butyl hydroperoxide with hypochlorous acid was accompanied by a light emission, that time profile and intensity were identical to those emission using Ce4+. The addition of Fe2+ to tert-butyl hydroperoxide yielded a much smaller chemiluminescence. Thus, tert-butyl hydroperoxide yielded in its reaction with hypochlorous acid or Ce4+ the same spin adduct and the same luminescence profile. Because Ce4+ is known to oxidize organic hydroperoxides to peroxyl radical species, it can be concluded that a similar reaction takes place in the case of hypochlorous acid.     

Sulfamide antifolates inhibiting thymidylate synthase: synthesis,enzyme inhibition and cytotoxicity

Synthesis and biological evaluation are described of seven new analogues (3-9) of two potent thymidylate synthase inhibitors, 10-propargyl-5,8-dideazafolate (1) and its 2-methyl-2-deamino congener ICI 198583 (2). While the new compunds 3 and 4 were analogues of 1 and 2, respectively, containing a p-aminobenzenesulfonyl residue in place of the p-aminobenzoic acid residue, the remaining 5 new compounds were analogues of 4 with the L-glutamic acid residue replaced by glycine (5), L-valine (6), L-alanine (7), L-phenylglycine (8) or L-norvaline (9). The new analogues were tested as inhibitors of thymidylate synthases isolated from tumour (Ehrlich carcinoma), parasite (Hymenolepis diminuta) and normal tissue (regenerating rat liver) and found to be weaker inhibitors than the parent 10-propargyl-5,8-dideazafolic acid. Selected new analogues, tested as inhibitors of growth of mouse leukemia L 5178Y cells, were less potent than the parent 10-propargyl-5,8-dideazafolic acid. Substitution of the glutamyl residue in compound 4 with L-norvaline (9) resulted in only a 5-fold stronger thymidylate synthase inhibitor, but a 40-fold weaker cell growth inhibitor.     

Synthesis of enantiopure non-natural alpha-amino acids using tert-butyl (2S)-2-[bis-(tert-butoxycarbonyl)amino]-5-oxopentanoate as key-intermediate:the first synthesis of(S)-2-amino-oleic acid.

A general method for the synthesis of enantiopure non-natural alpha-amino acids is described. The key intermediate tert-butyl (2S)-2-[bis(tert-butoxycarbonyl)amino]-5-oxopentanoate was obtained from l-glutamic acid after suitable protection and selective reduction of the gamma-methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of delta,epsilon-unsaturated alpha-amino acids. This methodology was applied to the synthesis of (S)-2-amino-oleic acid.     

Synthesis of the mitogenic S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylpentapeptide from Escherichia coli lipoprotein

The N-terminal pentapeptide of the lipoprotein from the outer membrane of Escherichia coli was obtained by coupling S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine to O-tert-butylseryl-O-tert-butyl-seryl-asparaginyl-alanine tert-butyl ester followed by deprotection with trifluoroacetic acid. The tetrapeptide was built up from alanine tert-butyl ester with N-9-fluorenylmethyloxycarbonyl protected amino acids. S-[2,3-Bis(palmitoyloxy)propyl]-N-palmitoylcysteine was obtained from N,N'-dipalmitoylcystine di-tert-butyl ester via reduction to the thiol, and S-alkylation with racemic 3-bromo-1,2-propanediol followed by esterification with palmitic acid in the presence of dicyclohexylcarbodiimide/dimethylaminopyridine and deprotection with trifluoroacetic acid. The compounds were characterized unequivocally by 13C-NMR and mass spectra. The diastereomers of S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylcysteine tert-butyl ester with opposite configuration at the propyl-C-2 atom could be separated on a silica-gel column.     

Solid-phase synthesis of new saphenamycin analogues with antimicrobial activity.

An array of 12 new saphenamycin analogues modified at the benzoate moiety was synthesized on solid support. Synthesis commenced with a chemoselective anchoring of saphenic acid through the carboxyl group to a 2-chlorotrityl functionalized polystyrene resin. The secondary alcohol was acylated in parallel with a series of differently substituted benzoic acid derivatives. Treatment with TFA-CH(2)Cl(2) (5:995) released the expected saphenamycin analogues into solution. These new analogues were purified, characterized and screened for antimicrobial activity against Bacillus subtilis and Proteus mirabilis. Eight analogues exhibited MIC values against B. subtilis ranging from 0.07 to 3.93 microg/mL, comparable to the activities of previously reported saphenamycin analogues.     

Enantioselective synthesis of tranylcypromine analogues as lysine demethylase (LSD1) inhibitors

Asymmetric cyclopropanation of styrenes by tert-butyl diazoacetate followed by ester hydrolysis and Curtius rearrangement gave a series of tranylcypromine analogues as single enantiomers. The o,- m- and p-bromo analogues were all more active than tranylcypromine in a LSD1 enzyme assay. The m- and p-bromo analogues were micromolar growth inhibitors of the LNCaP prostate cancer cell line as were the corresponding biphenyl analogues prepared from the bromide by Suzuki crosscoupling.     

Bioactive N-terminal undecapeptides derived from parathyroid hormone: the role of alpha-helicity.

The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N-terminal fragments presenting the principal activating domain such as PTH(1-11) and PTH(1-14) with helicity-enhancing substitutions yield potent analogues with PTH(1-34)-like activity. To further investigate the role of alpha-helicity on biological potency, we designed and synthesized by solid-phase methodology the following hPTH(1-11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix-promoting C(alpha)-tetrasubstituted alpha-amino acids alpha-amino isobutyric acid (Aib), 1-aminocyclopentane-1-carboxylic acid (Ac(5)c) and 1-aminocyclohexane-1-carboxylic acid (Ac(6)c): Ac(5)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (I); Aib-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (II); Ac(6)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (III); Aib-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (IV); Aib-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (V); S-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (VI), S-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (VII); Ac(5)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (VIII); Ac(6)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (IX); Ac(5)c-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (X); Ac(6)c-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (XI). All analogues were biologically evaluated and conformationally characterized in 2,2,2-trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I-V, which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand-stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III, VI and VII are very weakly active and analogues IV, V, VIII-XI are inactive. The most potent analogue, I exhibits biological activity 3500-fold higher than that of the native PTH(1-11) and only 15-fold weaker than that of the native sequence hPTH(1-34). Remarkably, the two most potent analogues, I and II, and the very weakly active analogues, VI and VII, exhibit similar helix contents. These results indicate that the presence of a stable N-terminal helical sequence is an important but not sufficient condition for biological activity.     

Formation of some monopositive and dipositive carbonium ions in chlorosulphuric acid. Part XV

Tetraphenyl-p-xylene-glycol, tetraphenyl-phthalein and Dipheno(3-10′)thiazinyl are shown to form dipositive carbonium ions, but triphenyl acetic acid, 2-3-5-6 tetramethyl benzoic acid, 2-3-4-5-6 pentamethyl benzoic acid, tert-butyl alcohol, triphenyl carbinol, tri-p-tolyl carbinol, tri-o-tolyl carbinol, tri-p-chlorophenyl carbinol and tri-p-nitrophenyl carbinol form monopositive carbonium ions in chlorosulphuric acid, as revealed by conductometric and u.v. spectral studies. Oxalyl chloride decomposes while ethylene glycol is sulphonated in chlorosulphuric acid. Dichloroethane behaves as a non-electrolyte but dibromomethane disproportionates in this medium.     

Synthesis of 4-epi-2-deoxy-2-Heq-N-acetylneuraminic acid and 2,4-dideoxy-2-Heq N-acetylneuraminic acid

We have continued our work to develop novel analogues of sialic acid [1–4] that may specifically modulate the interaction between endogenous sialic acid and influenza virus haemagglutinin [3,5,6]. Functional groups of sialic acid that have been implicated for this virus-host recongnition are the glycerol side chain, N-acetyl group and the axially oriented carboxylic acid function In this report we describe the synthesis of two analogues, namely, 4-epi-2-deoxy-2-H_eq-N-acetylneuraminic acid (4-epi-2-d-2-H_eq-Neu5Ac) and 2,4-dideoxy-2-H_eq-N-acetylneuraminic acid (2,4-d₂-2-H_eq-Neu5Ac).     

Synthesis of CMP-9'-modified-sialic acids as donor substrate analogues for mammalian and bacterial sialyltransferases

Cytidine-5'-monophospho-sialic acid (CMP-Neu5Ac) derivatives bearing a phenyl group in which the tether length between the phenyl group and the 9-position of Neu5Ac varied were synthesized and evaluated as substrates for sialyltransferases. In the synthesis of the compounds, a coupling reaction between methyl 5-acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-beta-D-glycero-D-galacto-2-nonulopyranosonate and 2-cyanoethyl 2',3'-O,N4, triacetylcytidine-5'-yl N,N-diisopropylphosphoramidite was carried out and the phosphite derivative thus obtained was oxidized and then deprotected to yield CMP-9'-azido-Neu5Ac. Modification of the 9-amino group prepared by reduction of the azido groups was performed by the use of several phenyl-substituted alkylcarboxylic acid derivatives. Using these CMP-9'-modified-Neu5Ac analogues bearing the phenyl-substituted alkyl-amide group, sialyltransferase assays were performed with both rat liver alpha-(2-->6)-sialyltransferase and Photobacterium alpha-(2-->6)-sialyltransferase. These 9-modified analogues could be transferred to disaccharide acceptors, and a practical enzymatic synthesis using CMP-9'-modified-Neu5Ac yielded sialoside analogues and sialylglycoproteins in good yield. These experiments demonstrate that the Photobacterium sialyltransferase can be used in the synthesis of sialoside analogues having a large substituent at the 9-position of Neu5Ac.     

Stimulation of Ca2(+)-independent catecholamine secretion from digitonin-permeabilized bovine adrenal chromaffin cells by guanine nucleotide analogues. Relationship to arachidonate release.

The effect of GTP analogues on catecholamine secretion and [3H]arachidonic acid release from digitonin-permeabilized adrenal chromaffin cells was examined. Several GTP analogues stimulated Ca2(+)-independent exocytosis, with the order of efficacy being XTP greater than ITP greater than guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The stimulatory effect of the GTP analogues appeared to be due to activation of a conventional GTP-binding protein, as it was inhibited by guanosine 5'-[beta-thio]diphosphate (GDP[S]). In contrast, Ca2(+)-dependent exocytosis was only partially inhibited by high doses of GDP[S]. GTP did not stimulate Ca2(+)-independent exocytosis, but instead was found to inhibit secretion caused by micromolar Ca2+. Arachidonic acid (100 microM) also stimulated Ca2(+)-independent catecholamine secretion. Determination of the effect of GTP analogues on release of free [3H]arachidonic acid into the medium showed that it was stimulated by GTP[S] but inhibited by GTP, p[NH]ppG, ITP and XTP. The inhibition of [3H]arachidonic acid release by XTP was not prevented by GDP[S]. These results demonstrate that activation of a GTP-binding protein by certain GTP analogues can induce Ca2(+)-independent secretion in adrenal chromaffin cells and that the effect of GTP analogues on Ca2(+)-independent secretion can be dissociated from generation of arachidonic acid.     

Synthesis and Potent Anti-HCMV Activity of 2′,5′,5′-trifluoro-3′-hydroxy-apiosyl Nucleoside Phosphonic Acid Analogues

As antiviral nucleosides containing a fluorine atom at 2′-position are endowed with increased stabilization of glycosyl bond, it was of interest to investigate the influence of three fluorine atoms at 2′- and 5′-positions of apiosyl nucleoside phosphonate analogues. Various pyrimidine and purine 2′,5′,5′-trifluoro-3′-hydroxy-apiose nucleoside phosphonic acid analogues were synthesized from 1,3-dihydroxyacetone. Electrophilic fluorination of lactone was performed using N-fluorodibenzenesulfonimide. Difluorophosphonation was performed by direct displacement of triflate intermediate with diethyl(lithiodifluoromethyl) phosphonate to give the corresponding (α,α-difluoroalkyl) phosphonate. Condensation successfully proceeded from a glycosyl donor with persilylated bases to yield nucleoside phosphonate analogues. Deprotection of diethyl phosphonates provided the final phosphonic acid sodium salts. The synthesized nucleoside analogues were subjected to antiviral screening against various viruses.     

Synthesis and biological activities of linear and cyclic enkephalin analogues containing a psi (E,CH = CH) or psi (CH2CH2) isosteric replacement.

The peptide CO-NH function was replaced by a trans carbon-carbon double bond or by a CH2-CH2 isostere in enkephalin analogues of DADLE, DCDCE-NH2 or DPDPE. In DADLE the 2-3 and the 3-4 peptide bond was modified, whereas in the cyclic analogues the Gly3-Phe4 bond was replaced by the isosteres Gly psi (E,CH = CH)Phe [5-amino-2-(phenylmethyl)-3(E)-pentenoic acid] or Gly psi (CH2CH2)Phe [5-amino-2-(phenylmethyl)pentanoic acid]. In general, the modification results in a drop in potency which is the largest for the flexible CH2-CH2 replacement. The Gly3 psi (E,CH = CH)Phe4 DCDCE-NH2 analogue retains considerable potency. These results confirm the importance of the peptide function at the 2-3 and 3-4 position in enkephalin analogues for biological potency.     

Stereoconvergent approach for synthesizing enantiopure 5, 6-dialkylpipecolic acids

Investigating a general route for synthesizing pipecolic acid ) piperidine-2-carboxylic acid ( derivatives with substituents at the 3-, 4-, 5- and 6-position, we discovered a stereoconvergent process that provides an effective means for making 5, 6-dialkyl-epsilonpipecolate (Scheme 1, PhF = 9-phenylfluoren-9-yl). Hydrogenation of diastereomeric mixtures of gamma-oxo gamma-hydroxy and gamma-acetoxy alpha-N-(PhF)amino tert-butyl esters causes the eventual loss of the gamma-substituent to furnish an azadiene intermediate that can reduce diastereoselectively to 5, 6-dialkylpipecolate having the all cis relative stereoconfiguration. Five enantiopure (>94% ee) 5,6-dialkylpipecolic acids were synthesized, employing aspartic acid as an inexpensive chiral educt in this process.     

Induction of vascular relaxation by hydroperoxides

Hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, and 3-chloroperoxybenzoic acid (CPB) and 15-HPETE relaxed, in a concentration dependent manner rat aortic rings contracted with PGF2 alpha (1 X 10(-5)). Relaxation is not inhibited by either indomethacin (2 X 10(-5) M), a cyclo-oxygenase inhibitor or eicosatetraynoic acid (1 X 10(-5) M), a dual cyclo-oxygenase and lipoxygenase inhibitor. Rings with intact endothelium relaxed to a greater degree on exposure to CPB and 15-HPETE. Methylene blue, a soluble guanylate cyclase inhibitor (1 X 10(-5) M) blocked the relaxation elicited by the five peroxides, whereas both superoxide dismutase (scavenger of superoxide anion) and mannitol (scavenger of hydroxyl radical) have no effect. We conclude that relaxation of vascular smooth muscle is a general property of peroxides and that the endothelium may in some instances facilitate this effect.     

Synthesis of two biologically active fluorescent probes of thymopentin

This study reports on the synthesis of two fluorescent analogues of thymopentin (TP-5; Arg-Lys-Asp-Val-Tyr). A fluorescein isothiocyanate labeled analogue (FITC-TP-5) and a stilbene isothiocyanate labeled analogue (SITS-TP-5) were extensively purified by ion-exchange and gel filtration chromatography. Characterization of the coupling site through amino acid analysis, dansylation and N-terminal cleavage of the fluorescent amino acid yielded results which indicated that both were mono-labeled analogues derivatized at the N-terminal. These analogues were shown to be TP-5-like in nature by their ability to induce the expression of the Thy 1.2 surface marker on nude mouse prothymocytes in both in vivo and in vitro assays. In addition, these analogues were able to inhibit the specific binding of radiolabeled TP-5 to human lymphocytes. Initial studies describing the interaction of FITC-TP-5 with human lymphocytes are shown.     

Synthesis of new class dipeptide analogues with improved permeability and antithrombotic activity

3-(S)-1,2,3,4-Tetrahydro-beta-carboline-3-carboxylic acid isolated from A. Chinese G. Don was found to possess moderate anti-aggregation activity, but with poor bioavailability. To improve its pharmacological property, we designed and synthesized a series of novel dipeptide analogues by incorporating tetrahydro-beta-carboline-3-carboxylic acid skeleton as an amino acid surrogate (*Trp). It turned out these dipeptide analogues exhibited good membrane permeability based on in vitro Caco-2 cell monolayers permeability assay. As a result, the overall biological properties of these molecules were significantly improved depending on the nature of the amino acid residues introduced onto the 3-position of the tetrahydro-beta-carboline moiety. It was very interesting to notice that these dipeptide analogues (5b,c,h,i,n,o,p,q) displayed a remarkable dual antiaggregatory activity in both of ADP- and PAF-induced platelet aggregation assay, and their aggregation response was significantly higher than that of aspirin (p<0.01). In addition, these dipeptide analogues were observed for the dose-dependent antithrombotic effect using in vivo rat arterial thrombosis model. The potency of antithrombotic activity of 5h,i,n,p was significantly higher than that of aspirin (n=12, p<0.01) at equal dose (5 micromol/kg).