Inhibition of Plasmodium falciparum fatty acid biosynthesis: evaluation of FabG, FabZ, and FabI as drug targets for flavonoids

After the discovery of a potent natural flavonoid glucoside as a potent inhibitor of FabI, a large flavonoid library was screened against three important enzymes (i.e., FabG, FabZ, and FabI) involved in the fatty acid biosynthesis of P. falciparum. Although flavones with a simple hydroxylation pattern (compounds 4-9) showed moderate inhibitory activity toward the enzymes tested (IC50 10-100 microM), the more complex flavonoids (12-16) exhibited strong activity toward all three enzymes (IC50 0.5-8 microM). Isoflavonoids 26-28 showed moderate (IC50 7-30 microM) but selective activity against FabZ. The most active compounds were C-3 gallic acid esters of catechins (32, 33, 37, 38), which are strong inhibitors of all three enzymes (IC50 0.2-1.1 microM). Kinetic analysis using luteolin (12) and (-)-catechin gallate (37) as model compounds revealed that FabG was inhibited in a noncompetitive manner. FabZ was inhibited competitively, whereas both compounds behaved as tight-binding noncompetitive inhibitors of FabI. In addition, these polyphenols showed in vitro activity against chloroquine-sensitive (NF54) and -resistant (K1) P. falciparum strains in the low to submicromolar range.     

Non-nucleoside inhibitors of mitochondrial thymidine kinase (TK-2) differentially inhibit the closely related herpes simplex virus type 1 TK and Drosophila melanogaster multifunctional deoxynucleoside kinase

5'-O-Trityl derivatives of thymidine (dThd), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), and their acyclic analogs 1-[(Z)-4-triphenylmethoxy-2-butenyl]thymine (KIN-12) and (E)-5-(2-bromovinyl)-1-[(Z)-4-triphenylmethoxy-2-butenyl]uracil (KIN-52) have been synthesized and evaluated for their inhibitory activity against the amino acid sequence related mitochondrial dThd kinase (TK-2), herpes simplex virus type 1 (HSV-1) TK, and Drosophila melanogaster multifunctional 2'-deoxynucleoside kinase (Dm-dNK). Several compounds proved markedly inhibitory to these enzymes and represent a new generation of nucleoside kinase inhibitors. KIN-52 was the most potent and selective inhibitor of TK-2 (IC(50), 1.3 microM; K(i), 0.50 microM; K(i)/K(m), 0.37) but was not inhibitory against HSV-1 TK and Dm-dNK at 100 microM. As found for the alternative substrate BVDU, the tritylated compounds competitively inhibited the three enzymes with respect to dThd. However, whereas BVDU behaved as a noncompetitive inhibitor (alternative substrate) of TK-2 and HSV-1 TK with respect to ATP as the varying substrate, the novel tritylated enzyme inhibitors emerged as reversible purely uncompetitive inhibitors of these enzymes. Computer-assisted modeling studies are in agreement with these findings. The tritylated compounds do not act as alternative substrates and they showed a type of kinetics against the nucleoside kinases different from that of BVDU. KIN-12, and particularly KIN-52, are the very first non-nucleoside specific inhibitors of TK-2 reported and may be useful for studying the physiological role of the mitochondrial TK-2 enzyme.     

Selective inhibition of herpes simplex virus ribonucleoside diphosphate reductase by derivatives of 2-acetylpyridine thiosemicarbazone

The effects of thiosemicarbazone derivatives of 2-acetylpyridine on mammalian and viral ribonucleoside diphosphate reductases were investigated. The enzymes were partially purified from uninfected and herpes simplex virus type-1 (HSV-1)-infected KB cells by sequential salt fractionation with streptomycin sulfate and ammonium sulfate and by affinity chromatography on ATP-agarose. The five thiosemicarbazone derivatives investigated were all potent inhibitors of the virus-induced reductase. Fifty percent inhibitory concentrations (IC50 values) range from 2 to 13 microM. Four of the five derivatives also were inhibitors of the host cell reductase (IC50 values = 7-34 microM). A semicarbazone was inactive against the cellular enzyme and relatively weak as an inhibitor of the viral enzyme (IC50 = 340 microM). Four of six compounds were preferential inhibitors of the viral reductase based on a comparison of IC50 values (5- to greater than 85-fold difference). Kinetic experiments revealed that inhibition of the HSV-1 reductase by the thiosemicarbazones was noncompetitive with respect to CDP and dithiothreitol. A comparison of the inhibitory effects of 2-acetylpyridine thiosemicarbazone itself on viral reductase and on virus replication in vitro demonstrated a similarity in the dose-response relationships for the two parameters. This observation supports the hypothesis that the HSV-induced ribonucleoside diphosphate reductase is an important target for the design of antiviral drugs.     

Structure-activity relationships of phenothiazines and related drugs for inhibition of protein kinase C

Phenothiazines are known to inhibit the activity of protein kinase C. To identify structural features that determine inhibitory activity against the enzyme, we utilized a semiautomated assay [Anal. Biochem. 187:84-88 (1990)] to compare the potency of greater than 50 phenothiazines and related compounds. Potency was decreased by trifluoro substitution at position 2 on the phenothiazine nucleus and increased by quinoid structures on the nucleus. An alkyl bridge of at least three carbons connecting the terminal amine to the nucleus was required for activity. Primary amines and unsubstituted piperazines were the most potent amino side chains. We selected 7,8-dihydroxychlorpromazine (DHCP) (IC50 = 8.3 microM) and 2-chloro-9-(3-[1-piperazinyl]propylidene)thioxanthene (N751) (IC50 = 14 microM) for further study because of their potency and distinct structural features. Under standard (vesicle) assay conditions, DHCP was noncompetitive with respect to phosphatidylserine and a mixed-type inhibitor with respect to ATP. N751 was competitive with respect to phosphatidylserine and noncompetitive with respect to ATP. Using the mixed micelle assay, DHCP was a competitive inhibitor with respect to both phosphatidylserine and ATP. DHCP was selective for protein kinase C compared with cAMP-dependent protein kinase, calmodulin-dependent protein kinase type II, and casein kinase. N751 was more potent against protein kinase C compared with cAMP-dependent protein kinase and casein kinase but less potent against protein kinase C compared with calmodulin-dependent protein kinase type II. DHCP was analyzed for its ability to inhibit different isoenzymes of protein kinase C, and no significant isozyme selectivity was detected. These data provide important information for the rational design of more potent and selective inhibitors of protein kinase C.     

Chitin synthase 2 inhibitory activity of O-methyl pisiferic acid and 8,20-dihydroxy-9(11),13-abietadien-12-one, isolated from Chamaecyparis pisifera

In the course of search for potent chitin synthase inhibitors from plant extracts, the chitin synthase 2 inhibitors, O-methyl pisiferic acid and 8,20-dihydroxy-9(11),13-abietadien-12-one which have diterpene skeleton, were isolated from the leaves of Chamaecyparis pisifera. These compounds inhibited chitin synthase 2 of Saccharomyces cerevisiae with the IC50 values of 5.8 and 226.4 microM, respectively. Especially, O-methyl pisiferic acid showed 15.3-fold stronger inhibitory activity than polyoxin D (IC50=88.6 microM), a well-known chitin synthase inhibitor. These compounds exhibited weaker inhibitory activities against chitin synthase 1 than chitin synthase 2, whereas it showed no inhibitory activity for chitin synthase 3. The compound exhibited mixed competitive inhibition with respect to UDP-N-acetyl-D-glucosamine as substrate (Ki=5 microM). These results indicated that O-methyl pisiferic acid is a specific inhibitor of chitin synthase 2. The compound also inhibited chitin synthase 1 of Candida albicans, which represents analogues to chitin synthase 2 of S. cerevisiae, with an IC50 of 75.6 microM, which represents 1.8-fold weaker activity than that of polyoxin D. Although O-methyl pisiferic acid has been reported for antibacterial and insecticidal activities, the present study is the first report on its inhibitory activity against chitin synthase 2.     

Inhibition of herpes simplex virus thymidine kinases by 2-phenylamino-6-oxopurines and related compounds: structure-activity relationships and antiherpetic activity in vivo

Derivatives of the herpes simplex thymidine kinase inhibitor HBPG [2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine] have been synthesized and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [3H]thymidine by both enzymes, but potencies differed quantitatively from those of HBPG and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published X-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogues with bulky tertiary amino substituents were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with Ki values of 0.03 and 0.005 microM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [3H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.     

Identification of novel parasitic cysteine protease inhibitors by use of virtual screening. 2. The available chemical directory

The incidence of parasitic infections such as malaria, leishmaniasis, and trypanosomiasis has been steadily increasing. Since the existing chemotherapy of these diseases suffers from lack of safe and effective drugs and/or the presence of widespread drug resistance, there is an urgent need for development of potent, mechanism-based antiparasitic agents against these diseases. Cysteine proteases have been established as valid targets for this purpose. The Available Chemical Directory consisting of nearly 355,000 compounds was screened in silico against the homology models of plasmodial cysteine proteases, falcipain-2, and falcipain-3, to identify structurally diverse non-peptide inhibitors. The study led to identification of 22 inhibitors of parasitic cysteine proteases out of which 18 compounds were active against falcipain-2 and falcipain-3. Eight compounds exhibited dual activity against both enzymes. Additionally, four compounds were found to inhibit L. donovani cysteine protease. While one of the cysteine protease inhibitors also exhibited in vitro antiplasmodial activity with an IC50 value of 9.5 microM, others did not show noticeable antiplasmodial activity up to 20 microM. A model identifying important pharmacophoric features common to the structurally diverse falcipain-2 inhibitors has also been developed. Very few potent non-peptide inhibitors of the parasitic cysteine proteases have been reported so far, and identification of these novel and chemically diverse inhibitors should provide leads to be optimized into candidates to treat protozoal infections.     

Inhibition of viral proteases by Zingiberaceae extracts and flavones isolated from Kaempferia parviflora

In order to identify novel lead compounds with antiviral effect, methanol and aqueous extracts of eight medicinal plants in the Zingiberaceae family were screened for inhibition of proteases from human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV) and human cytomegalovirus (HCMV). In general, the methanol extracts inhibited the enzymes more effectively than the aqueous extracts. HIV-1 protease was strongly inhibited by the methanol extract of Alpinia galanga. This extract also inhibited HCV and HCMV proteases, but to a lower degree. HCV protease was most efficiently inhibited by the extracts from Zingiber officinale, with little difference between the aqueous and the methanol extracts. Many of the methanol extracts inhibited HCMV protease, but the aqueous extracts showed weak inhibition. In a first endeavor to identify the active constituents, eight flavones were isolated from the black rhizomes of Kaempferia parviflora. The most effective inhibitors, 5-hydroxy-7-methoxyflavone and 5,7-dimethoxyflavone, inhibited HIV-1 protease with IC50 values of 19 microM. Moreover, 5-hydroxy-3,7-dimethoxyflavone inhibited HCV protease and HCMV protease with IC50 values of 190 and 250 microM, respectively.     

Synthesis and antiviral activity of 3-formyl- and 3-cyano-2,5,6-trichloroindole nucleoside derivatives

A series of trichlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. The previously reported 3-formyl-2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole (FTCRI) and its 3-cyano homologue (CTCRI) were chemically modified at the 3-position. The formation of hydrazones and oximes of FTCRI was accomplished by a dehydrative addition of the appropriate hydrazine or hydroxylamine derivatives, respectively. A carboxamide oxime and imidate were synthesized from CTCRI by the addition of hydroxylamine or methanol, respectively, to the 3-nitrile substituent. Analogues synthesized from FTCRI generally had less antiviral activity than either FTCRI or CTCRI. However, the derivatives of CTCRI were potent and selective inhibitors of HCMV in vitro. The analogue 2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole-3-carboxamide oxime was especially selective (HCMV IC50 = 0.30 microM, CC50 > 100 microM). None of the analogues had significant activity against HSV-1.     

Phenoxazine derivatives inactivate human cytomegalovirus, herpes simplex virus-1, and herpes simplex virus-2 in vitro.

We examined whether phenoxazine derivatives, 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha-8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-amino-phenoxazine-3-one (Phx-3) may have antiviral activity against herpes family viruses: human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2). The antiviral activity was evaluated by the selectivity index (SI), which is the ratio of 50% cytotoxic concentration (CC(50)) and 50% antiviral concentration (IC(50)). Among these phenoxazines, Phx-2 exerted strong antiviral activity to HCMV with the SI of 200, while Phx-1 and Phx-3 exerted no marked anti-HCMV activity. Phx-2 also showed moderate inhibition of HSV-1 and HSV-2, with the SI of 6.7 and 17, respectively. In the time-of-addition experiments, inhibitory effect of Phx-2 against HCMV was active even when applied to cells at 100 h after HCMV infection, while ganciclovir (GCV) showed potent inhibition when applied to cells before 42-h post-infection, but its inhibitory effects disappeared thereafter. Attachment and penetration of HCMV was not affected by the presence of Phx-2. When HCMV was pretreated with Phx-2, concentration-dependent virucidal action was observed, suggesting that Phx-2 inactivates HCMV directly. From these data, it was found that Phx-2 might have a different anti-HCMV target from GCV.     

Selective inhibitors of the serine protease plasmin: probing the S3 and S3' subsites using a combinatorial library

A combinatorial library of 400 serine protease inhibitors with the general structure Cbz-X(aa)-Trp-cyclohexanone-Trp-Y(aa)-OH has been constructed. The library was synthesized on the solid phase using mix-and-split synthesis, where 20 different amino acids were incorporated at both the X(aa) and Y(aa) positions. These two positions correspond to the S3 and S3' subsites of the active site. Iterative deconvolution was used to identify hits from the library. The library was screened against four serine proteases: plasmin, kallikrein, thrombin, and trypsin. Seven inhibitors from the library that showed promising activities were resynthesized using solution-phase methods. Four of these compounds were good inhibitors of plasmin with IC(50) values in the range of 2.7-3.6 microM. The most potent of these inhibitors showed >150-fold selectivity for plasmin when compared to the other three serine proteases.     

Design, synthesis, and antiviral activity of certain 3-substituted 2,5,6-trichloroindole nucleosides

A series of trichlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. Modifications of the previously reported 2,5,6-trichloro-1-(beta-d-ribofuranosyl)indole at the 3-position of the heterocycle were designed in part to test our hypothesis that hydrogen bonding is required at that position for antiviral activity. Analogues were synthesized using electrophilic addition at the 3-position or by synthesis of modified indole heterocycles followed by glycosylation and modification of the sugar. Among the modifications at the 3-position, only those analogues with hydrogen-bond-accepting character were active against HCMV (e.g., 3-formyl-2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole, FTCRI, IC50 = 0.23 microM). Conversely, analogues with non-hydrogen-bonding substituents at the 3-position (e.g., 3-methyl-2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole) were much less active (IC50 = 32 microM) than those with the requisite hydrogen-bonding capacity. The 5'-O-acyl analogue of FTCRI was obtained as an intermediate and also found to be a potent inhibitor of HCMV (IC50 < 0.1 microM). The synthesis of some additional 5'-O-acylated analogues did not provide a compound with increased antiviral activity. None of the indole nucleosides had significant activity against HSV-1, and none were cytotoxic to uninfected cells in their antiviral dose range. Results obtained from the antiviral evaluations have validated our hypothesis that hydrogen bonding at the 3-position is required for antiviral activity in this series of chlorinated indole nucleosides.     

Synthesis and biological evaluation of the first N-alkyl cage dimeric 4-aryl-1,4-dihydropyridines as novel nonpeptidic HIV-1 protease inhibitors

A first series of novel NH and N-alkyl-substituted cage dimeric 4-aryl-1,4-dihydropyridines 3a-f has been synthesized and evaluated as HIV-1 protease inhibitors in in vitro assays. While the NH and N-methyl derivatives 3a,b,e,f were almost inactive with IC(50) values of about 200 microM, the N-Benzyl compounds exhibited stronger activity with an IC(50) value of 16.2 microM for the presently best compound 3c. The type of HIV-1 protease inhibition of these novel inhibitors was characterized as competitive. With the increase of observed activity from NH and N-methyl derivatives to N-benzyl compounds, respectively, the binding mode may correspond to that of cyclic and azacyclic ureas showing hydrophobic interactions of the four aromatic residues to the S1/S1' and S2/S2' regions of HIV-1 protease.     

Thiosemicarbazones of formyl benzoic acids as novel potent inhibitors of estrone sulfatase

Thiosemicarbazones of the microbial metabolite madurahydroxylactone, a polysubstituted benzo[a]naphthacenequinone, have been previously reported by us as potent nonsteroidal inhibitors of the enzyme estrone sulfatase (cyclohexylthiosemicarbazone 1, IC50 0.46 microM). The active pharmacophore of 1 has now been identified to be 2-formyl-6-hydroxybenzoic acid cyclohexylthiosemicarbazone (25, IC50 4.2 microM). The active partial structure was derivatized in the search for novel agents against hormone-dependent breast cancer. Further substantial increases in activity were achieved by reversal of functional groups leading to the cyclohexylthiosemicarbazones of 5-formylsalicylic acid (35, IC50 0.05 microM) and 3-formylsalicylic acid (34, IC50 0.15 microM) as the most potent analogues identified to date. Both compounds were shown to be noncompetitive inhibitors of estrone sulfatase with Ki values of 0.13 microM and 0.12 microM, respectively. The compounds showed low acute toxicity in the hen's fertile egg screening test.     

Novel small-molecule inhibitors of anthrax lethal factor identified by high-throughput screening

Anthrax lethal factor (LF) is a key virulence factor of anthrax lethal toxin. We screened a chemolibrary of 10,000 drug-like molecules for their ability to inhibit LF and identified 18 novel small molecules with potent LF inhibitory activity. Three additional LF inhibitors were identified through further structure-activity relationship (SAR) analysis. All 21 compounds inhibited LF with an IC50 range of 0.8 to 11 muM, utilizing mixed-mode competitive inhibition. An evaluation of inhibitory activity against a range of unrelated proteases showed relatively high specificity for LF. Furthermore, pharmacophore modeling of these compounds showed a high degree of similarity to the model published by Panchal et al. (Nat. Struct. Mol. Biol. 2004, 11, 67-72), indicating that the conformational features of these inhibitors are structurally compatible with the steric constraints of the substrate-binding pocket. These novel LF inhibitors and the structural scaffolds identified as important for inhibitory activity represent promising leads to pursue for further LF inhibitor development.     

Comparative study of the inhibition of metallo-beta-lactamases (IMP-1 and VIM-2) by thiol compounds that contain a hydrophobic group

For the purpose of screening of inhibitors that are effective for wide range of metallo-beta-lactamases, the inhibitory effect of two series of compounds, 2-omega-phenylalkyl-3-mercaptopropionic acid (PhenylCnSH (n=1-4)) and N-[(7-chloro-quinolin-4-ylamino)-alkyl]-3-mercapto-propionamide (QuinolineCnSH (n=2-6)), where n denotes the alkyl chain length, on metallo-beta-lactamases IMP-1 and VIM-2 was examined. These inhibitors contain a thiol group and a hydrophobic group linked by variable-length methylene chain. PhenylCnSH (n=1-4) was found to be a potent inhibitor of both IMP-1 and VIM-2. PhenylC4SH was the potent inhibitor of both IMP-1 (IC(50)=1.2 microM) and VIM-2 (IC(50)=1.1 microM) among this study. When the number of methylene units was varied, QuinolineC4SH showed the maximum inhibitory activity against IMP-1 and VIM-2 (IC(50)=2.5 microM and IC(50)=2.4 microM). The relationship between the inhibitory effect of the alkyl chain length was different for both series of inhibitors, suggesting that IMP-1 has a tighter binding site than VIM-2. QuinolineCnSH did not serve as a fluorescence reagent for metallo-beta-lactamases.     

Anti-plasmodial and anti-leishmanial activity of conformationally restricted pentamidine congeners

A library of 52 pentamidine congeners in which the flexible pentyldioxy linker in pentamidine was replaced with various restricted linkers was tested for in-vitro activity against two Plasmodium falciparum strains and Leishmania donovani. The tested compounds were generally more effective against P. falciparum than L. donovani. The most active compounds against the chloroquine-sensitive (D6, Sierra Leone) and -resistant (W2, Indochina) strains of P. falciparum were bisbenzamidines linked with a 1,4-piperazinediyl or 1, 4-homopiperazinediyl moiety, with IC50 values (50% inhibitory concentration, inhibiting parasite growth by 50% in relation to drug-free control) as low as 7 nM based on the parasite lactate dehydrogenase assay. Seven piperazine-linked bisbenzamidines substituted at the amidinium nitrogens with a linear alkyl group of 3-6 carbons (22, 25, 27, 31) or cycloalkyl group of 4, 6 or 7 carbons (26, 32, 34) were more potent (IC50<40 nM) than chloroquine or pentamidine as anti-plasmodial agents. The most active anti-leishmanial agents were 4,4'-[1,4-phenylenebis(methyleneoxy)]bisbenzenecarboximidamide (2, IC50 approximately 0.290 microM) and 1,4-bis[4-(1H-benzimidazol-2-yl)phenyl] piperazine (44, IC50 approximately 0.410 microM), which were 10- and 7-fold more potent than pentamidine (IC50 approximately 2.90 microM). Several of the more active anti-plasmodial agents (e.g. 2, 31, 33, 36-38) were also potent anti-leishmanial agents, indicating broad antiprotozoal properties. However, a number of analogues that showed potent anti-plasmodial activity (1, 18, 21, 22, 25-28, 32, 43, 45) were not significantly active against the Leishmania parasite. This indicates differential modes of anti-plasmodial and anti-leishmanial actions for this class of compounds. These compounds provide important structure-activity relationship data for the design of improved chemotherapeutic agents against parasitic infections.     

Synthesis and antiviral activity of novel 5-(1-cyanamido-2-haloethyl) and 5-(1-hydroxy(or methoxy)-2-azidoethyl) analogues of uracil nucleosides

A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes.     

Evaluation of Natural and Synthetic 1,4‐naphthoquinones as Inhibitors of Monoamine Oxidase

Previous reports have documented that 1,4‐naphthoquinones act as inhibitors of the monoamine oxidase (MAO) enzymes. In particular, fractionation of the extracts of cured tobacco leafs has led to the characterization of 2,3,6‐trimethyl‐1,4‐naphthoquinone, a non‐selective MAO inhibitor. To derive structure–activity relationships for MAO inhibition by the 1,4‐naphthoquinone class of compounds, this study investigates the human MAO inhibitory activities of fourteen structurally diverse 1,4‐naphthoquinones of natural and synthetic origin. Of these, 5,8‐dihydroxy‐1,4‐naphthoquinone was found to be the most potent inhibitor with an IC₅₀ value of 0.860 μm for the inhibition of MAO‐B. A related compound, shikonin, inhibits both the MAO‐A and MAO‐B isoforms with IC₅₀ values of 1.50 and 1.01 μm , respectively. It is further shown that MAO‐A and MAO‐B inhibition by these compounds is reversible by dialysis. In this respect, kinetic analysis suggests that the modes of MAO inhibition are competitive. This study contributes to the discovery of novel MAO inhibitors, which may be useful in the treatment for disorders such as Parkinson's disease, depressive illness, congestive heart failure and cancer.     

Synthesis and kinetic evaluation of peptide alpha-keto-beta-aldehyde-based inhibitors of trypsin-like serine proteases

New, synthetic peptide analogues bearing a C-terminal basic alpha-keto-beta-aldehyde moiety were prepared as novel inhibitors of the trypsin-like serine proteases. The compounds, Ac-Leu-Leu-Arg-COCHO, Ac-Arg-Gln-Arg-COCHO and Boc-Val-Leu-Lys-COCHO were evaluated kinetically against trypsin and three other trypsin-like serine proteases, tryptase, plasmin and thrombin, all of which are implicated as mediators of important disease processes. Results illustrate that alpha-keto-beta-aldehydes are potent inhibitors, with similar potency to comparable peptide aldehydes, and intriguingly, appearto act, in some instances, by a novel mechanism of action. Ac-Leu-Leu-Arg-COCHO, an analogue of the natural product leupeptin, is a potent, tight-binding inhibitor of trypsin (Ki(final) = 1.9 microM), plasmin (Ki(final) = 4.9 microM) and tryptase (Ki(final) = 1.2 microM) and an irreversible inactivator of thrombin (k2nd 4,500 M(-1).min(-1)). Boc-Val-Leu-Lys-COCHO was found to be a tight-binding inhibitor of its target protease plasmin (Ki(final) = 3.1 microM) and was inactive against thrombin. Ac-Arg-Gln-Arg-COCHO was a slow-binding inhibitor of tryptase (Ki(final) = 1.6 microM) and also irreversibly inactivated trypsin (k2nd = 8,920 M(-1) min(-1)). Peptides or peptidomimetics with a C-terminal basic alpha-keto-beta-aldehyde function thus provide a useful new molecular template for the development of new therapeutic agents against a wide range of disorders, such as coagulopathies and asthma, which may be mediated by the aberrant activity of trypsin-like serine proteases.