Synthesis and transdermal permeation-enhancing activity of carbonate and carbamate analogs of Transkarbam 12

Transkarbam 12 (5-(dodecyloxycarbonyl)pentylammonium-5-(dodecyloxycarbonyl)pentylcarbamate, T12) is a highly effective skin permeation enhancer. In this study, ester groups in the molecule of T12 were replaced by carbonate and carbamate ones, respectively. The in vitro permeation-enhancing activities were evaluated using porcine skin and compared with those of T12 and previously prepared series of amide, ketone, and alkyl analogs. According to the activities and behavior of the compounds in donor samples, ester group is essential for the activity of T12; its replacement not only decreases the enhancing potency, but is likely to change the mechanism of action.     

Relationship between temporary inhibition and structure of disulfide-linkage analogs of marinostatin, a natural ester-linked protein protease inhibitor.

A 12-residue marinostatin [MST(1-12): (1)FATMRYPSDSDE(12)] which contains two ester linkages of Thr(3)-Asp(9) and Ser(8)-Asp(11) strongly inhibits subtilisin. In order to study the relationship between the inhibitory activity, structure, and stability of MST, MST analogs were prepared by changing ester linkages to a disulfide linkages. The analogs without the disulfide linkage between 3 and 9 positions lost their inhibitory activity. The K(i) value of 1SS(C(3)-C(9)) ((1)FACMRYPSCSDE(12)), which has a single disulfide linkage of Cys(3)-Cys(9) was comparable with those of MST(1-12) and MST-2SS ((1)FACMRYPCCSCE(12)), a doubly linked analog of Cys(3)-Cys(9) and Cys(8)-Cys(11). However, 1SS(C(3)-C(9)) and MST-2SS showed temporary inhibition, but not MST(1-12): These analogs were inactivated after incubation with subtilisin for 30 min, and were specifically hydrolyzed at the reactive site. (1)H NMR study showed that 1SS(C(3)-C(9)) has two conformations, which contain a cis- (70%) or trans- (30%) Pro residue, while MST-2SS as well as MST(1-12) takes a single conformation containing only a cis-Pro residue. Hydrogen-deuterium exchange rate of the Arg(5) (P1') NH proton of the MST analogs was about 100 times faster than that of MST(1-12). These results indicate that the linkage between the positions 8 and 11 plays a role for fixing the cis-conformation of the Pro(7) residue, and that the linkage between 3 and 9 is indispensable for the inhibition, but not enough for stable protease-inhibitor complex.     

Suppression of phorbol ester-enhanced radiation-induced malignancy in vitro by protease inhibitors is independent of protein kinase C

X-irradiation and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act in a synergistic manner to increase the yield of transformed C3H10T1/2 cells in vitro. TPA modulated both translocation from the cytosol to the plasma membrane, and down regulation of protein kinase C (PKC) after prolonged (48 h) TPA exposure. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), antipain, and soybean-derived Bowman-Birk inhibitor, protease inhibitors that suppress transformation of C3H10T1/2 cells, had no effect on these TPA-mediated alterations of PKC activity, suggesting that protease inhibitors suppress TPA-stimulated promotion in vitro via a PKC-independent pathway. Several experiments were performed to determine whether non-toxic concentrations of the PKC inhibitors, N-p-tosyl-L-lysine chloromethyl ketone (TLCK), TPCK, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), or 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine (H-7), modulated the movement of cells from a quiescent state into the cell cycle. TPCK and the combination of H-7 and W-7 lowered DNA synthesis when cells were stimulated to divide by TPA. Because other protease inhibitors that slow transformation in vitro did not have the same suppressive effect on DNA synthesis, the inhibitory pathway that suppresses carcinogenic activity is likely to be different from the suppression of DNA synthesis.     

Discovery of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. Part 1: Structure-activity relationships of the 1- and 3-positions

1-Benzoyl-3-cyanopyrrolo[1,2-a]quinoline (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a had good activity against several breast cancer cell lines but was much less active against several other cancer cell lines. SAR studies of 2a found that substitution at the 4-position of the 1-benzoyl group was important for activity. Replacing the 3-cyano group by an ester or ketone group led to inactive compounds. Interestingly, 4-substituted analogs such as 1-(4-(1H-imidazol-1-yl)benzoyl)-3-cyanopyrrolo[1,2-a]quinoline (2k) were found to be broadly and highly active in the caspase activation assay as well as in the cell growth inhibition assay with low nM EC(50) and GI(50) values in human breast cancer cells T47D, human colon cancer cells HCT116, and hepatocellular carcinoma cancer cells SNU398. Compound 2a was found not to inhibit tubulin polymerization up to 50 microM, while 2k was found to inhibit tubulin polymerization with an IC(50) value of 5 microM, indicating that certain substituents at the 4-position of the 1-benzoyl group can change the mechanism of action.     

Conformationally constrained analogues of diacylglycerol (DAG). Effect on protein kinase C (PK-C) binding by the isosteric replacement of sn-1 and sn-2 esters in DAG-lactones

In order to determine the importance of the two ester pharmacophores in high affinity, conformationally constrained DAG-lactones (Lac-1-5) as PK-C ligands, we have independently replaced the sn-1 and sn-2 carbonyl esters in these compounds by ketone (2, 10, 11), amide (3, 25-28), and hydroxyl (12, 13) isosteres. Although the ketone analogue of the sn-1 ester (2) exhibited comparable activity to the parent Lac-1 when taking into account the difference in lipophilicities, the other isosteres were significantly poorer PK-C alpha ligands compared to the parent DAG-lactones. This study demonstrates that the ester functionality in DAG-lactone plays an important role in the ligand's capacity to form a strong hydrogen bond with Gly253 at the active site. The discrete K(i) analysis from the sn-1 and sn-2 isosteres further confirms that the DAG-lactones bind preferentially to the C1-domain in the sn-2 binding mode, as previously suggested.     

The First Synthesis of 4′-Branched 5′-Deoxycarbocyclic 9-Deazaadenosine and Phosphonic Acids as Antiviral Agents

The first synthetic route to 4′-trifluoromethylated 5′-deoxycarbocyclic-9-deazaadenosine analog and its phosphonic acid derivatives was described from α-trifluoromethyl-α,β-unsaturated ester. The C–C bond connection between cyclopentane and base moiety was accomplished using Knoevenagel type condensation from ketone derivative 11. Synthesized nucleoside and phosphonic acid analogs were tested for anti-HIV activity as well as cytotoxicity.     

Structural determinants of blockade of eosinophil activation, adhesion and secretion by synthetic analogs of phomactin

We examined the structural determinants of phomactin analogs to assess their efficacy as antagonist of PAF. Six analogs of phomactin were synthesized to determine their inhibitory effects on adhesion, superoxide release, leukotriene C4 (LTC4) synthesis and [3H]PAF binding in human eosinophils. Phomactin analogs inhibited both PAF- and IL-5-induced eosinophil adhesion. Analog A, which bears an alkene moiety between C-1 and C-14, a ketone at the C-2 position, and an alkyne moiety between C-3 and C-4, had the greatest anti-adhesive effect. Change of the alkene between C-1 and C-14 to an alkane (analog I) decreased the anti-adhesive effect by 2.5-4 fold, while substitution of ketone by hydroxyl (analog G) at the C-2 position caused an 11-fold decrease in the anti-adhesive effect. Substitution of the alkyne moiety between C-3 and C-4 by an alkene (B and E) or alkane (D) blocked completely the anti-adhesive effect. Analogs A and I completely blocked superoxide release from eosinophils caused by phorbol-12-myristate-13-acetate or PAF and LTC4-release caused by fMLP plus cytochalasin B. Change of the alkyne moiety between C-3 and C-4 to an alkene (B and E) or alkane (D) blocked completely these inhibitory effects of phomactin. Analog A decreased the maximal binding of [3H]PAF binding to eosinophils without change of the apparent dissociation constant. We conclude that phomactin analogs are specific non-competitive PAF antagonists and have exceptional efficacy in inhibiting adhesion, metabolic activity and leukotriene secretion in human eosinophils. We further define the structural alterations in the phomactin molecule that regulate its inhibitory functions.     

Stereospecific recognition of inositol 1,4,5-trisphosphate analogs by the phosphatase, kinase, and binding proteins

A series of DL-inositol 1,4,5-trisphosphate (IP3) analogs, with a bulky substitutent on the 2nd carbon of the inositol ring, has been synthesized. These compounds exert biological activities with only minor reduction in potency, in several assay systems (Hirata, M., Watanabe, Y., Ishimatsu, T., Ikebe, T., Kimura, Y., Yamaguchi, K., Ozaki, S., and Koga, T. (1989) J. Biol. Chem. 264, 20303-20308). Two analogs with aminocyclohexanecarbonyl (designated as analog 206) or aminobenzoyl group (analog 209) were separated into individual optical isomers and examined for stereospecificity in recognition by IP3-5-phosphatase, IP3-3-kinase and IP3 binding activity. IP3-5-phosphatase activity of erythrocyte ghosts was competitively inhibited by L-209 with a lower Ki value than D-IP3, but with a higher Ki value by L-206. D-Isomers of both analogs at 100 microM failed to inhibit the hydrolysis of D-[3H]IP3. On the other hand, D-isomers but not L-isomers of both analogs were as potent as D-IP3 in the recognition by IP3-3-kinase of rat brain cytosol and only the D-isomer of analog 206 could serve as substrate for the kinase. Also D-isomers of both analogs were equipotent to D-IP3 in displacing [3H]IP3 binding to rat cerebellum microsomes. These observations suggest that the IP3 analogs we synthesized are stereospecifically recognized by three IP3-recognizable proteins, but the phosphatase recognizes opposite isomers. Such being the case, the second hydroxyl group of D-IP3 may be involved in the recognition by IP3-5-phosphatase, but not by IP3-3-kinase and binding sites.     

Synthesis and evaluation of novel dipeptidyl benzoyloxymethyl ketones as caspase inhibitors

We describe novel peptide-based caspase inhibitors. Potent and comparatively selective compounds containing a dipeptide scaffold and a substituted oxymethyl ketone as a warhead were developed. The newly synthesized compounds were tested for inhibition in in vitro enzymatic assays of caspases-1, -3, -6, -8, and -9. The benzyloxycarbonyl-phenylglycyl-aspartyl benzoyloxymethyl ketone (Z-Phg-Asp-CH2OCO-Ph, coded as HU44) was the most potent inhibitor of caspase-1 and caspase-3. Of several analogs of HU44 that were made, the beta-Asp methyl ester (2) is an effective inhibitor against caspase-3 and caspase-8, and less effective against caspase-1. These compounds did not inhibit caspase-6 and caspase-9 significantly.     

Inhibition of human thrombin assessed with different substrates and inhibitors. Characterization of fibrinopeptide binding interaction.

The activity of human thrombin has been assessed with fibrinogen, N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide, N-alpha-benzoyl-arginine-p-nitroanilide, N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and p-nitrophenylacetate: increased rates of hydrolysis were found for N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide compared to N-alpha-benzoyl-arginine-p-nitroanilide and p-nitrophenylacetate. Phenylmethyl sulfonyl fluoride and N-alpha-tosyl-L-lysine chloromethyl ketone inhibited, to the same degree, the activity toward each substrate. Inclusion of N-alpha-tosyl-arginine methyl ester in the phenylmethyl sulfonyl fluoride reaction mixtures protected the enzyme from inhibition as shown with N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. N-Acetylimidazole inhibited the activity towards fibrinogen, N-alphrosine-p-nitrophenyl ester to varying degrees. Inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide was completely reversible with neutral hydroxylamine, whereas coagulant activity towards fibrinogen was only partially regained. Human fibrinopeptide A inhibited activity toward N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. The mode of inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide by fibrinopeptide A was non=competitive (K1 = 3.02.10(-5) M), whereas N-alpha-toysyl-arginine methyl ester was a competitive inhibitor of this substrate (K1 = 2.6.10(-5) M). These studies demonstrate more than one binding domain for fibrinogen on human thrombin.     

Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based HTS assay. Part 5: modifications of the 2- and 3-positions

As a continuation of our efforts to discover and develop apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored modifications at the 2- and 3-positions. It was found that replacement of the 3-cyano group by an ester, including methyl and ethyl ester, resulted in >200-fold reduction of activity. Conversion of the 2-amino group into an amide or urea resulted in 4- to 10-fold drop of activity. Similarly, converting the 2-amino group into a hydrogen resulted in 4- to 10-fold reduction of activity. Compound 3d was highly active with an EC(50) value of 29 nM and a GI(50) value of 6 nM in T47D cells. Importantly, the 2-H analog 3d was found to be much more stable under acidic conditions compared to the 2-NH(2) analog 3b, suggesting that 2-H analogs might have better bioavailability than the 2-NH(2) analogs.     

Comparison of vasodilatory properties of 14,15-EET analogs: structural requirements for dilation

Epoxyeicosatrienoic acids (EETs) are endothelium-derived eicosanoids that activate potassium channels, hyperpolarize the membrane, and cause relaxation. We tested 19 analogs of 14,15-EET on vascular tone to determine the structural features required for activity. 14,15-EET relaxed bovine coronary arterial rings in a concentration-related manner (ED(50) = 10(-6) M). Changing the carboxyl to an alcohol eliminated dilator activity, whereas 14,15-EET-methyl ester and 14,15-EET-methylsulfonimide retained full activity. Shortening the distance between the carboxyl and epoxy groups reduced the agonist potency and activity. Removal of all three double bonds decreased potency. An analog with a Delta8 double bond had full activity and potency. However, the analogs with only a Delta5 or Delta11 double bond had reduced potency. Conversion of the epoxy oxygen to a sulfur or nitrogen resulted in loss of activity. 14(S),15(R)-EET was more potent than 14(R),15(S)-EET, and 14,15-(cis)-EET was more potent than 14,15-(trans)-EET. These studies indicate that the structural features of 14,15-EET required for relaxation of the bovine coronary artery include a carbon-1 acidic group, a Delta8 double bond, and a 14(S),15(R)-(cis)-epoxy group.     

Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide.

A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-D-arginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us. Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide. The D-BAPA-hydrolyzing enzymes (D-BAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B. cereus T were compared. These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels. The D-BAPAases of B. subtilis 168 and B. cereus T differed in the effect of inhibitors on enzymatic activity. While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B. cereus T was inhibited by p-chloromercuribenzene sulfonic acid. The D-BAPAases of B. subtilis and B. cereus T had a Michaelis constant for D-BAPA of 2.9 x 10(-5) M and 1.4 x 10(-4) M, respectively. D-BAPAase is an intracellular enzyme localized in the protoplast (80 to 90% in soluble form in the cytoplasm). The ability to cleave D-BAPA is suggested as an additional chemotaxonomic characteristic of sporeforming bacteria of the genera Bacillus and Clostridium.     

Effect of Age on Brain Cortical Protein Kinase C and Its Mediation of 5-Hydroxytryptamine Release

The effects of age on the activity and translocation of protein kinase C (PKC) and on the facilitation of 5-hydroxytryptamine (5-HT, serotonin) release induced by PKC activation with the phorbol ester phorbol 12-myristate 13-acetate were investigated. The activities of cortical PKC and its translocation in response to K+ depolarization and phorbol ester stimulation were reduced during aging in Fischer-344 rats. Parietal cortical brain slices from 6-, 12-, and 24-month-old animals were preloaded with [3H]5-HT and release was evoked by 65 mM K+ or the calcium ionophore A23187. 5-HT release induced by either K+ or A23187 was found to be reduced in 12- and 24-month-old as compared to 6-month-old animals. This decrease was not reversed by high extracellular Ca2+. Activation of PKC resulted in a facilitated transmitter release in tissue from 6- and 12-month-old animals but reduced [3H]5-HT release in slices from 24-month-old animals. These responses were prevented by the putative PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), but not by increasing extracellular or intracellular Ca2+. The results demonstrate an age-related change (1) in brain PKC activity and translocation and (2) in a physiological response to PKC stimulation. These results may have implications for other PKC-mediated functions that are altered during senescence.     

Design, synthesis, and antiviral evaluation of some polyhalogenated indole C-nucleosides

2,5, 6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB), 2-bromo-5, 6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB) and 2-benzylthio-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BTDCRB) are benzimidazole nucleosides that exhibit strong and selective anti-HCMV activity. Polyhalogenated indole C-nucleosides were prepared as 1-deaza analogs of the benzimidazole nucleosides TCRB and BDCRB. A mild Knoevenagel coupling reaction between an indol-2-thione and a ribofuranose derivative was developed for the synthesis of 2-benzylthio-5, 6-dichloro-3-(beta-D-ribofuranosyl)indole (12). 3-(beta-D-ribofuranosyl)-2,5,6-trichloroindole (16) was prepared from 12 in 4 steps. A Lewis acid-mediated glycosylation method was then developed to prepare the targeted 2-haloindole C-nucleoside 16 stereoselectively in four steps from the corresponding 2-haloindole aglycons. Only 12 was active against HCMV but it also was somewhat cytotoxic.     

The monomer of pyruvate kinase, subtype M1, is both a kinase and a cytosolic thyroid hormone binding protein

Using a T7 expression system, the monomer of rat pituitary pyruvate kinase, subtype M1 (PKM1), was overexpressed in Escherichia coli and purified to homogeneity. The monomeric p58-M1 has intrinsic enzymatic activity with a Vmax of 79 +/- 20 units/mg and Km's for ADP and PEP of 1.43 +/- 0.76 and 0.14 +/- 0.07 mM, respectively. The monomer binds 3,3',5-triiodo-L-thyronine (T3) with Ka = 1.5 x 10(7) M-1. The order of analog specificity is L-T3 greater than L-thyroxine greater than D-T3 greater than 3'-isopropyl-3,5-diiodo-L-thyronine greater than or equal to 3',5',3-triiodo-L-thyronine. In contrast, tetrameric PKM1 lacks T3 binding activity. The kinase activity of p58-M1 is inhibited by T3 and its analogs in a concentration-dependent manner with the order of inhibitory activity similar to that of binding activity. This inhibition, however, is reversed by the addition of fructose 1,6-bisphosphate. p58-M1 is the second PK isoenzyme monomer to be identified as having thyroid hormone binding activity.     

Woody pretzels: spirocycles from vetiver to patchouli and Georgywood

This review, including new experimental results, is the summary of a talk at the RSC/SCI conference 'flavours & fragrances 2007' in London, Imperial College, 24-26 September, 2007. Though the third dimension of the receptor models of J. E. Amoore rarely was exceeding 4 A, the world of woody odorants such as (+)-cedrol (3; cedarwood), (-)-khusimone (4; vetiver), and (-)-patchoulol (5; patchouli) is anything but flat. Any tricyclic skeleton with a zero-bridge contains a spirocyclic ring system determining its 3D structure, so spirocycles (spira, Lat. pretzel) are the fastest access to the third dimension. In the vetiver family, a spirocyclic mimic 9 of (-)-khusimone (4) was first discovered by chance by Büchi in 1976, and also by chance, we obtained another system, 12, with a characteristic vetiver smell by tandem-Rupe-Nazarov reaction of alkyne diols. A 5-A distance between a quaternary C-atom and a carbonyl group (or alternative HB acceptor) with an alpha-methyl or methylene branching is proposed to be the key to their vetiver odor. Upon scale-up of one of these odorants, 24, we discovered a very powerful (0.067 ng/l) impurity with a most typical patchouli scent: the spirocyclic, sterically crowded hydroxy ketone 33--a most unusual structure for a patchouli odorant. Several spirocyclic hydroxy ketone analogs, also with inverted ring systems such as in 70 and 84, provided new insights into the structure-odor correlation of this family. A superposition analysis indicated the carbonyl function of the hydroxy ketone to overlay on the geminal dimethyl motive of (-)-patchoulol. And indeed, the corresponding hydroxy ketone of patchoulol, 59, synthesized in 13 steps from Cyclal C (63), also emanated a patchouli odor. Finally, the synthesis and olfactory properties of twelve rigid spirocyclic analogs, 95-97, 99-102, and 106-110, of Georgywood (91) are presented that highlight stereochemical requirements for woody odorants and raise doubts about an alpha-helical binding motive postulated by Hong and Corey.     

Solution structure of marinostatin, a natural ester-linked protein protease inhibitor

Marinostatin is a unique protein protease inhibitor containing two ester linkages. We have purified a 12-residue marinostatin [MST(1-12), (1)FATMRYPSDSDE(12)] and determined the residues involved in the formation of the ester linkages and the solution structure by (1)H NMR spectroscopy and restrained molecular dynamics calculation. The two ester linkages of MST(1-12) are formed between hydroxyl and carboxyl groups, Thr(3)-Asp(9) and Ser(8)-Asp(11), indicating that MST(1-12) has two cyclic regions which are fused at the residues of Ser(8) and Asp(9). A strong NOE cross-peak between Tyr(6) H(alpha) and Pro(7) H(alpha) was observed, indicating that the Pro(7) residue takes a cis-conformation. Well-converged structures and hydrogen-deuterium experiments of MST(1-12) showed that the backbone NH proton of the P1'residue, Arg(5), is hydrogen-bonded to the carbonyl oxygen of the ester linkage between Thr(3) and Asp(9). To reveal the significance of the ester linkages, a marinostatin analogue, MST-2SS ((1)FACMRYPCCSCE(12)) with two disulfide bridges of Cys(3)-Cys(9) and Cys(8)-Cys(11), was also synthesized. The inhibitory activity of MST-2SS was as strong as that of MST(1-12), and the Pro(7) residue of MST-2SS also takes a cis-conformation. However, the exchange rate of the Arg(5) NH proton of MST-2SS was about 100 times faster than that of MST(1-12), and the structure calculation of MST-2SS was not converged on account of the small number of NOEs, indicating that MST-2SS takes a more flexible structure. The hydrogen acceptability of the ester linkage formed by the P2 position residue, Thr(3), is crucial for suppressing the fluctuation of the reactive site and sustaining the inhibitory activity, which enables marinostatin to be one of the smallest protease inhibitors in nature.     

Synthesis, antimalarial, antileishmanial, antimicrobial, cytotoxicity, and methemoglobin (MetHB) formation activities of new 8-quinolinamines

We report the synthesis, in vitro antiprotozoal (against Plasmodium and Leishmania), antimicrobial, cytotoxicity (Vero and MetHb-producing properties), and in vivo antimalarial activities of two series of 8-quinolinamines. N1-{4-[2-(tert-Butyl)-6-methoxy-8-quinolylamino]pentyl}-(2S/2R)-2-aminosubstitutedamides (21-33) and N1-[4-(4-ethyl-6-methoxy-5-pentyloxy-8-quinolylamino)pentyl]-(2S/2R)-2-aminosubstitutedamides (51-63) were synthesized in six steps from 6-methoxy-8-nitroquinoline and 4-methoxy-2-nitro-5-pentyloxyaniline, respectively. Several analogs displayed promising antimalarial activity in vitro against Plasmodium falciparum D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) clones with high selectivity indices versus mammalian cells. The most promising analogs (21-24) also displayed potent antimalarial activity in vivo in a Plasmodium berghei-infected mouse model. Most interestingly, many analogs exhibited promising in vitro antileishmanial activity against Leishmania donovani promastigotes, and antimicrobial activities against a panel of pathogenic bacteria and fungi. Several analogs, notably 21-24, 26-32, and 60, showed less MetHb formation compared to primaquine indicating the potential of these compounds in 8-quinolinamine-based antimalarial drug development.     

Characterization of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)-induced protein phosphorylation in rabbit platelets: inhibitory effects of AGEPC analogs

1-O-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) induced phosphorylation of two proteins having molecular masses of approximately 20- and 40-kDa in washed rabbit platelets in a concentration- and time-dependent manner. Sequential stimulation with AGEPC did not induce additional protein phosphorylation, supporting the concept of desensitization of the AGEPC receptors responsible for biological activity. AGEPC analogs 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-6'-trimethylammonium hexyl ester and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-10'-trimethylammonium decyl ester (U66985 and U66982), containing polar head groups with methylene chain lengths of C6 and C10, did not cause protein phosphorylation, but they did inhibit the AGEPC-induced events. Thus protein phosphorylation is closely associated with the receptor-mediated stimulation of platelets and is a useful indicator of the signaling process initiated through the receptors. Other synthetic analogs of AGEPC such as rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate and 1-(N-n-pentadecylcarbamoyloxy)-2-methoxy-rac-glycero-3-phosphochol ine (CV3988 and U68043) were also shown to be inhibitors of the AGEPC-induced protein phosphorylation. Inhibition by these analogs was specific for AGEPC since there was no observed effect of thrombin, ADP, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and arachidonic acid-induced changes. The extent of inhibition was dependent on the concentration of AGEPC and its analogs and did not change with time after the addition of AGEPC. In platelets incubated with AGEPC analogs before and simultaneously with the addition of AGEPC, protein phosphorylation was prevented; however, addition of AGEPC to platelets shortly before the addition of these analogs showed a high response. In experiments where platelets were previously incubated with AGEPC analogs and washed with buffer containing 0.5% bovine serum albumin, AGEPC-induced protein phosphorylation was recovered to a level of 80%. These observations support the conclusion that AGEPC stimulates platelets through its specific receptor, and that the AGEPC analogs bind to the AGEPC receptor and block that pathway sensitive to AGEPC stimulation but not because of the desensitization of its receptor. On the other hand, in platelets where phosphorylation of the 40-kDa protein was induced by a 2-min preincubation with 3 X 10(-10) M TPA, 5 X 10(-10) M AGEPC-induced serotonin release decreased by 51% compared to a control value.(ABSTRACT TRUNCATED AT 400 WORDS)