Part 3: Design and synthesis of proline-derived α2δ ligands

A potent series of substituted (2S,4S)-benzylproline α₂δ ligands have been designed from the readily available starting material (2S,4R)-hydroxy-l-proline. The ligands have improved pharmacokinetic profile over the (4S)-phenoxyproline derivatives described previously and have potential for development as oral agents for the treatment of neuropathic pain. Compound 16 has been progressed to clinical development.     

Design and synthesis of 3-pyrrol-3-yl-3H-isobenzofuran-1-ones as inhibitors of human cytosolic phospholipase A2α

A series of 3-pyrrol-3-yl-3H-isobenzofuran-1-ones was synthesized and assessed for the ability to inhibit cytosolic phospholipase A₂α (cPLA₂α). Several of these compounds were found to be active in both a cell based assay and an isolated enzyme assay. The most potent inhibitor was the thiazolidine-2,4-dione substituted derivative 35. With IC₅₀-values of 0.7 μM and 7.3 μM in the cellular and isolated enzyme assay, respectively, it possesses similar inhibitory potency as the known cPLA₂α inhibitor arachidonyltrifluoromethyl ketone (AACOCF₃). Structure–activity relationship studies revealed that the evaluated isobenzofuran-1-ones seem to exert their cellular activities not only by a direct interaction with the enzyme but also by other as yet unknown mechanisms.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Stereoselective synthesis of spiro and condensed pyrazolines of steroidal α,β-unsaturated ketones and nitrilimines by 1,3-dipolar cycloaddition

Effective syntheses of endo- and exocyclic α,β-unsaturated ketones as CC dipolarophiles were carried out in the 13α-estrone series. The 1,3-dipolar cycloadditions of 15,16α,β-unsaturated ketones of 13α-estrone 3-methyl and 3-benzyl ether with nitrilimines stereoselectively furnished two regioisomers of new condensed pyrazolines in a ratio of 2:1. The main product was the isomer obtained by the attack of the N-terminus of the 1,3-dipole on the carbon atom β to the carbonyl group of the dipolarophile. The nitrilimine cycloadditions to the 16-methylene-17-ketones of 13α-estrone 3-methyl and 3-benzyl ether stereo- and regioselectively furnished spiropyrazolines. The attack of the N-terminus of the dipole occurred on the α-carbon of the α,β-unsaturated ketones. The reactions were performed under both homogeneous and heterogeneous conditions. Silver acetate as a base proved more effective than its triethylamine counterpart. Changes in regio- and stereoselectivities were not observed on variation of the conditions of the cycloaddition reactions. The structures of the new products were determined by NMR (one- and two-dimensional) and MALDI TOF MS techniques, with C₇₀ fullerenes as matrix in the latter case.     

Part 2: Design, synthesis and evaluation of hydroxyproline-derived α2δ ligands

Conformational constraint has been used to design a potent series of α₂δ ligands derived from the readily available starting material (2S,4R)-hydroxy-l-proline. The ligands have improved physicochemistry and potency compared to their linear counterparts (described in our earlier publication) and the lead compound has been progressed to clinical development.     

An efficient synthesis of γ-hydroxy-α,β-unsaturated aldehydic esters of 2-lysophosphatidylcholine

The diverse biological activities of ??-hydroxyalkenal phospholipids and their involvement in disease are the subject of intense study. Phospholipid aldehydes, such as the 4-hydroxy-7-oxohept-5-enoic acid ester of 2-lyso-phosphatidylcholine (HOHA-PC), the 5-hydroxy-8-oxo-6-octenoic acid ester of 2-lyso-PC (HOOA-PC), and the 9-hydroxy-12-oxododec-10-enoic acid ester of 2-lyso-PC (HODA-PC), are generated by oxidative cleavage of polyunsaturated fatty acyl phospholipids. To facilitate investigations of their chemistry and biology, we now report efficient total synthesis of HOOA, HODA, and HOHA phospholipids. Because the target ??-hydroxyalkenals readily decompose through oxidation of the aldehyde group to a carboxylic acid or through cyclization to furans, these synthesis generate the sensitive functional array of the target phospholipids under mild conditions from acetal derivatives that are suitable for long-term storage.     

Synthesis and biological evaluation of 3-tetrazolo steroidal analogs: Novel class of 5α-reductase inhibitors

In the present study, a series of steroidal tetrazole derivatives of androstane and pregnane have been prepared in which the tetrazole moiety was appended at C-3 and 17a-aza locations. 3-Tetrazolo-3,5-androstadien-17-one (6), 3-tetrazolo-19-nor-3,5-androstadien-17-one (10), 3-tetrazolo-3,5-pregnadien-20-one (14), 17a-substituted 3-tetrazolo-17a-aza-d-homo-3,5-androstadien-17-one (26–31) and 3-(2-acetyltetrazolo)-17a-aza-d-homo-3,5-androstadien-17-one (32) were synthesized from dehydroepiandrosterone acetate (1) through multiple synthetic steps. Some of the synthesized compounds were evaluated for their in vitro 5α-reductase (5AR) inhibitory activity by measuring the conversion of [³H] androstenedione in human embryonic kidney (HEK) cells. In vivo 5α-reductase inhibitory activity also showed a significant reduction (p <0.05) in rat prostate weight. The most potent compound 14 showed 5AR-2 inhibition with IC₅₀ being 15.6 nM as compared to clinically used drug finasteride (40 nM). There was also a significant inhibition of 5AR-1 with IC₅₀ 547 nM compared to finasteride (453 nM).     

Synthesis of 4-hydroxy-β3-homoprolines and their insertion in α/β/α-tripeptides

The stereoselective syntheses of 2-cyclopropyl- and (2S)-2-hydroxymethyl-(3R,4S)-4-hydroxy-β³-homoproline are described. The reported amino acids were constructed through 1,3-dipolar cycloaddition of strained alkylidenecyclopropanes with enantiopure pyrroline N-oxides derived from malic acid followed by thermal rearrangement of the adducts in the presence of trifluoroacetic acid. The two-step sequence afforded the homoprolines suitably protected to be directly used as building blocks in peptidomimetic synthesis as proved by the synthesis of the two model mixed α/β/α tripeptides Phe-β³-HPro-Val.     

Proteases as catalysts of enantioselective α-amino ester hydrolysis: 2. Pronase-catalysed hydrolysis of tryptophan methyl ester

The influence of pH, temperature, EDTA and metal ions on the rate of hydrolysis of methyl esters of d- and l-tryptophan by pronase from Streptomyces griseus has been studied. pH, Ca²⁺ and Co²⁺ ions have different effects on the hydrolysis of d- and l-substrates, which permits the enantioselectivity of the reaction to be regulated. Maximum enantioselectivity was observed at pH 7 in the presence of 10^?3–10^?2m Co²⁺. The data obtained are compared with the literature data for the hydrolysis of other low molecular weight substrates by pronase. It is suggested that leucine aminopeptidase makes a considerable contribution to the observed esterase activity of pronase.     

Novel glycoside of vanillyl alcohol, 4-hydroxy-3-methoxybenzyl-α-d-glucopyranoside: study of enzymatic synthesis, in vitro digestion and antioxidant activity

Novel glucoside of physiological active vanillyl alcohol was synthesized for the first time using maltase from Saccharomyces cerevisiae as catalyst, and established its structure as 4-hydroxy-3-methoxybenzyl-α-D: -glucopyranoside. The key reaction factors for this transglucosylation reaction were optimized using response surface methodology and the highest yield so far in maltase catalyzed transglucosylation reaction was obtained. It was found out that optimum temperature of reaction was 37 °C, optimal maltose concentration was 60% (w/v), optimal pH was 6.6, and optimal concentration of vanillyl alcohol was 158 mM. Under these conditions, yield of glucoside was 90 mM with no by product formation. It was shown that this compound posses good antioxidant activity as well as stability in gastrointestinal tract. It was demonstrated that it is hydrolyzed on brush border membrane of enterocytes, so it can serve in protecting gastrointestinal system from oxidation, as well as source of anticonvulsive drug after the hydrolysis of glucoside on brush border membrane of small intestine.     

Chemoenzymatic synthesis of C8-modified sialic acids and related α2-3- and α2-6-linked sialosides

Naturally occurring 8-O-methylated sialic acids, including 8-O-methyl-N-acetylneuraminic acid and 8-O-methyl-N-glycolylneuraminic acid, along with 8-O-methyl-2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid (Kdn8Me) and 8-deoxy-Kdn were synthesized from corresponding 5-O-modified six-carbon monosaccharides and pyruvate using a sialic acid aldolase cloned from Pasteurella multocida strain P-1059 (PmNanA). In addition, α2-3- and α2-6-linked sialyltrisaccharides containing Neu5Ac8Me and Kdn8Deoxy were also synthesized using a one-pot multienzyme approach. The strategy reported here provides an efficient approach to produce glycans containing various C8-modified sialic acids for biological evaluations.     

New CD1d agonists: synthesis and biological activity of 6″-triazole-substituted α-galactosyl ceramides

Huisgen [3+2] dipolar cycloaddition of 6″-azido-6″-deoxy-α-galactosyl ceramide 11 with a range of alkynes (or a benzyne precursor) yielded a series of triazole-containing α-galactosyl ceramide (α-GalCer) analogues in high yield. These α-GalCer analogues and the precursor azide 11 were tested for their ability to activate iNKT cells and stimulate IL-2 cytokine secretion in vitro, and IFN-γ and IL-4 cytokine secretion in vivo. Some of these analogues, specifically 11, 12b, 12f and 13, were more potent IL-2 stimulators than the prototypical CD1d agonist, α-GalCer 1. In terms of any cytokine bias, most of the triazole-containing analogues exhibited a small Th2 cytokine-biasing response relative to that shown by α-GalCer 1. In contrast, the cycloaddition precursor, namely azide 11, provided a small Th1 cytokine-biasing response.     

Metabolism of 3β-hydroxy-5α- and 3β-hydroxy-5β-pregnan-20-one by leaf homogenates

Leaf homogenates of Cheiranthus cheiri, Nerium oleander, Strophanthus kombé, Digitalis purpurea, and Corchorus capsularis were ex     

Purification and properties of a nad: 3α-hydroxy-5α-pregnan-20-one-oxidoreductase from rat liver microsomes

From rat liver microsorties a NAD: 3α-hydroxy-5α-pregnan-20-one oxidoreductase was isolated and purified up to a specific activity of 73 nmol/min.mg by affinity chromatography and DEAE-cellulose chromatography. Various Km-values have been determined. The enzyme exhibits highest affinity for 5α-pregnane-3,20-dione and NADH. The 3-oxo group of 5α-dihydrocortisone (17, 21-dihydroxy-5α-pregnane-3,11,20-trione) was not reduced by the purified enzyme preparation and NADH and no dehydrogenation with NAD was observed of 3α, 11β, 17, 21-tetrahydroxy-5α-pregnan-20-one. The optimal pH for the hydrogenation of the 3-oxo group was at pH 5.3 and for the dehydrogenation at pH 8.9. Disc gel electrophoresis in presence of 0.1% sodium dodecylsulfate yielded a homogeneous preparation.     

Metabolism of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane in the rabbit

The neutral urinary excretion products of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4α-position and one in the 6a-position with the cyclopropane ring intact. The fifth substance, 17β-hydroxy-3β-methyl-5α-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4?C-6>C-2.     

Studies on the hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution A synthesis of 3-methyl-2′-deoxyuridine

The hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution has been investigated. Varying proportions of 3-methylcytosine, 3-methyluracil and 3-methyl-2′-deoxyuridine are formed depending upon conditions of pH and temperature. All three hydrolytic products are formed at pH 6.8 and 90°C. At pH 2, depyrimidination of 3-methylcytosine occurs as the only hydrolysis product. When the pH is increased to 12, 3-methyl-2′-deoxycytidine on heating at 90°C is completely deaminated to 3-methyl-2′-deoxyuridine with few side products formed. This reaction serves as the basis for a convenient synthesis of 3-methyl-2′-deoxyuridine. The 300 MHz spectra of 3-methyl-2′-deoxycytidine and 3-methyl-2′-deoxyuridine indicate that the sugar ring in these compounds is predominantly in ²E conformation.     

Styrene-spaced copolymers including anthraquinone and β-O-4 lignin model units: synthesis, characterization and reactivity under alkaline pulping conditions

A series of random copoly(styrene)s has been synthesized via radical polymerization of functionalized anthraquinone (AQ) and β-O-4 lignin model monomers. The copolymers were designed to have a different number of styrene spacer groups between the AQ and β-O-4 lignin side chains aiming at investigating the distance effects on AQ/β-O-4 electron transfer mechanisms. A detailed molecular characterization, including techniques such as size exclusion chromatography, MALDI-TOF mass spectrometry, and (1)H, (13)C, (31)P NMR and UV-vis spectroscopies, afforded quantitative information about the composition of the copolymers as well as the average distribution of the AQ and β-O-4 groups in the macromolecular structures. TGA and DSC thermal analysis have indicated that the copolymers were thermally stable under regular pulping conditions, revealing the inertness of the styrene polymer backbone in the investigation of electron transfer mechanisms. Alkaline pulping experiments showed that close contact between the redox active side chains in the copolymers was fundamental for an efficient degradation of the β-O-4 lignin model units, highlighting the importance of electron transfer reactions in the lignin degradation mechanisms catalyzed by AQ. In the absence of glucose, AQ units oxidized phenolic β-O-4 lignin model parts, mainly by electron transfer leading to vanillin as major product. By contrast, in presence of glucose, anthrahydroquinone units (formed by reduction of AQ) reduced the quinone-methide units (issued by dehydration of phenolic β-O-4 lignin model part) mainly by electron transfer leading to guaiacol as major product. Both processes were distance dependent.     

New steroidal lactones as 5α-reductase inhibitors and antagonists for the androgen receptor

This study reports the synthesis of several new steroidal lactones: 5α,6β-dibromo-17a-oxa-D-homoandrostane-3β-yl-3'-oxapentanoate (11), 5α,6β-dibromo-17a-oxa-D-homoandrostane-3β-yl-propanoate (12), 5α,6β-dibromo-17a-oxa-D-homoandrostane-3β-yl-butanoate (13), 5α,6β-dibromo-17a-oxa-D-homoandrostane-3β-yl-pentanoate (14), 5α,6β-dibromo-17a-oxa-D-homoandrostane-3β-yl-hexanoate (15), 17a-oxa-D-homoandrost-5-en-17-one-3β-yl-3'-oxapentanoate (16), 17a-oxa-D-homoandrost-5-en-17-one-3β-yl-propanoate (17), 17a-oxa-D-homoandrost-5-en-17-one-3β-yl-butanoate (18), 17a-oxa-D-homoandrost-5-en-17-one-3β-yl-pentanoate (19) and 17a-oxa-D-homoandrost-5-en-17-one-3β-yl-hexanoate (20) with a therapeutic potential as antiandrogens. The biological effect of these steroids was demonstrated in in vivo as well as in vitro experiments. In the in vivo experiments, we measured the activity of ten new steroidal derivatives on the weight of the prostate and seminal vesicle glands of gonadectomized hamsters treated with testosterone. For the in vitro studies, we determined the IC(50) values by measuring the concentration of the steroidal derivatives that inhibits 50% of the activity of the 5α-reductase enzyme present in human prostate and also its binding capacity to the androgen receptors (AR) obtained from rat's prostate cytosol. The results from these experiments indicated that compounds 11-20, significantly decreased the weight of the prostate and seminal vesicles as compared to testosterone treated animals; this reduction of the weight of these glands was comparable to that produced by Finasteride. On the other hand, compounds 11-20 inhibited the enzyme 5α-reductase, with compounds 14-19 (IC(50) values of 4.2 ± 0.95, 0.025 ± 0.003, 1.2 ± 0.45, 1.2 ± 0.1, 0.028 ± 0.003, and 0.069 ± 0.005 nM, respectively) showing the highest inhibitory activity. The results from the in vitro experiments indicated that only 15-17 bind to the AR.     

Design and synthesis of novel sulfonamide-containing bradykinin hB(2) receptor antagonists. Synthesis and structure-relationships of α,α-tetrahydropyranylglycine

A series of α,α-cycloalkylglycine sulfonamide compounds of general formula 1 has previously been identified by our group as selective human B(2)(hB(2)) receptor antagonists. Here we report the in vitro and in vivo BK antagonist activity of a further evolution of the series, consisting in compounds of the general formula 2, containing either an alkyl piperazine or a 4-alkyl piperidine ring bearing various positively charged groups (R'). These studies unexpectedly revealed quite a flat nanomolar/subnanomolar SAR for the binding affinity, while differences were seen in the in vitro functional activities. We propose that variations in the residence time may explain these results.     

Asymmetric acetalation of α,α-trehalose: Synthesis of α-d-galactopyranosyl α-d-glucopyranoside and 6-deoxy-6-fluoro-α-d-glucopyranosyl α-d-glucopyranoside

Selective acetalation of α,α-trehalose with ethyl or methyl isopropenyl ether and toluene-p-sulphonic acid in N,N-dimethylformamide gave the 4,6-isopropylidene acetal as the major product, isolated as its hexa-acetate 1 (38%). The gluco-galacto analogue 6 of α,α-trehalose was synthesized from 1 by the sequence: hydrolysis of the isopropylidene group with trifluoroacetic acid, mesylation of the resulting diol, benzoate displacement, and saponification of the product. Deacetylation of 1 followed by benzylation and hydrolysis of the acetal group furnished a hexa-O-benzyl derivative 9. Tosylation of the primary hydroxyl group in 9, treatment of the product with tetrabutylammonium fluoride in acetonitrile, and subsequent catalytic hydrogenolysis of the benzyl groups gave 6-deoxy-6-fluoro-α,α-trehalose (12). Compounds 6 and 12 and 6-deoxy-6-iodo-α,α-trehalose are substrates for cockchafer trehalase, but have very low V_max values.