beta-Lactam derivatives as enzyme inhibitors: 1-peptidyl derivatives of 4-phenylazetidin-2-one as inhibitors of elastase and papain

N-Peptidyl substituted azetidin-2-ones were synthesized and evaluated as inhibitors of the serine protease elastase, and the cysteine protease papain. All compounds were synthesized from 4-phenylazetidin-2-one, either from the racemate or from the pure enantiomers. The (S)-enantiomer was prepared by enantioselective synthesis from (S)-beta-phenyl-beta-alanine, while the (R)-enantiomer was obtained by enzymatic resolution with alpha-chymotrypsin. N-Alkylation with bromoacetates introduced a spacer group which, after hydrolysis to the free acid, was acylated with amino acid esters or di- or tripeptide esters. The enzymatic assays proved some derivatives to be effective inhibitors of PPE and/or papain. N-BOC protected amino acid derivatives without a spacer group inhibited PPE reversibly, while derivatives with spacer group showed either weak or no inhibitory properties. On the other hand, papain was inactivated irreversibly by ethyl (RS)-2-oxo-4-phenylazetidin-1-acetate. The highest inhibitory activity against papain was found for the diastereomers of N-(2-oxo-4-phenylazetidin-1-acetyl)-L-alanyl-L-valine benzyl ester, a compound with a spacer group.     

S-alkylated homocysteine derivatives: new inhibitors of human betaine-homocysteine S-methyltransferase

A series of S-alkylated derivatives of homocysteine were synthesized and characterized as inhibitors of human recombinant betaine-homocysteine S-methyltransferase (BHMT). Some of these compounds inhibit BHMT with IC50 values in the nanomolar range. BHMT is very sensitive to the structure of substituents on the sulfur atom of homocysteine. The S-carboxybutyl and S-carboxypentyl derivatives make the most potent inhibitors, and an additional sulfur atom in the alkyl chain is well tolerated. The respective (R,S)-5-(3-amino-3-carboxy-propylsulfanyl)-pentanoic, (R,S)-6-(3-amino-3-carboxy-propylsulfanyl)-hexanoic, and (R,S)-2-amino-4-(2-carboxymethylsulfanyl-ethylsulfanyl)-butyric acids are very potent inhibitors and are the strongest ever reported. We determined that (R,S)-5-(3-amino-3-carboxy-propylsulfanyl)-pentanoic acid displays competitive inhibition with respect to betaine binding with a Kappi of 12 nM. Some of these compounds are currently being tested in mice to study the influence of BHMT on the metabolism of sulfur amino acids in vivo.     

Protease inhibitors. Part 7. Inhibition of Clostridium histolyticum collagenase with sulfonylated derivatives of L-valine hydroxamate.

Sulfonylated L-valine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with the title amino acid, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group. Other derivatives were obtained by reaction of N-benzyl-L-valine with arylisocyanates, arylsulfonylisocyanates or benzoylisothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety, with hydroxylamine in the presence of carbodiimides. The obtained compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen. The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized derivatives, substitution patterns leading to best ChC inhibitors were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethylphenylsulfonyl; or 1- and 2-naphthyl among others. Similarly to the matrix metalloproteinase hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P(2') and P(3') subsites, in order to achieve tight binding to the enzyme. Such compounds might lead to drugs useful in the treatment corneal bacterial keratitis.     

Cysteine derivatives as inhibitors for carboxypeptidase A: synthesis and structure-activity relationships

A series of cysteine (Cys) derivatives having an alkyl or arylalkyl moiety on the alpha-amino group of the amino acid have been synthesized as a novel type of inhibitor for carboxypeptidase A. These compounds are readily prepared starting with Cys in an optically active form. The structure-activity relationship study revealed that the inhibitors prepared from D-Cys are much more potent than the corresponding inhibitors obtained from L-Cys, and the most potent inhibitor in the series, (S)-1j with a K(i) value of 55 +/- 4 nM, is obtained by introducing a phenethyl moiety on the amino group of D-Cys. In comparison, the most active inhibitor in the series of 2-substituted 3-mercaptopropanoic acid is found to be 20, in which the phenyl ring is linked to the mercaptocarboxylic acid at the alpha-position with a methylene unit. A proposal that accounts for the different structural requirement for the maximum activity between the two series of inhibitors is provided.     

Arginine binding motifs: design and synthesis of galactose-derived arginine tweezers as galectin-3 inhibitors

Anionic O2 derivatives of methyl 3-deoxy-3-(4-methylbenzamido)-1-thio-beta-D-galactopyranoside have been synthesized as inhibitors against galectin-3. The sulfate, H-phosphonate, and benzyl phosphate derivatives showed an increased affinity as compared to the parent unsubstituted galactopyranoside. Modeling revealed arginine-144 being pinched by the C3 benzamide and O2 anionic substituents in that the benzamide stacked face-to-face and the anionic O2 substituent ion-paired with the guanidinium moiety.     

5'-deoxy-5'-methylthioadenosine phosphorylase--V. Acycloadenosine derivatives as inhibitors of the enzyme

Various adenosine acyclonucleoside derivatives were tested as inhibitors of 5'-deoxy-5'-methylthioadenosine (MeSAdo) phosphorylase, an enzyme involved in the salvage of adenine and methionine from MeSAdo. The 2-halogenated derivatives of acyloadenosine [9-(2-hydroxyethoxy-methyl)adenine], including the chloro-, bromo- and iodo-congeners, all inhibited murine Sarcoma 180 (S180) MeSAdo phosphorylase, with Ki values in the range of 10(-6) to 10(-5) M. Halogenated derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)adenine, which more closely resemble the natural substrate, were substantially more potent inhibitors of the enzyme, with Ki values in the range of 2-7 x 10(-7) M. 5'-Methylthio and 5'-halogenated analogs of 2'-deoxy-1',2'-seco-adenosine were weak inhibitors, with Ki values of 10(-4) M or greater. 9-[(1-Hydroxy-3-iodo-2-proxy)methyl]adenine. (HIPA), the derivative with the lowest Ki values among these analogs, was a competitive inhibitor of S180 MeSAdo phosphorylase. In preliminary studies, HIPA inhibited MeSAdo phosphorylase in intact HL-60 human promyelocytic leukemia cells, as it limited the incorporation of [8-14C]MeSAdo into cellular adenine nucleotide pools. In addition, 9-(phosphonoalkyl)adenines, representing potential multisubstrate inhibitors of MeSADo phosphorylase, were synthesized. Of these the heptyl derivative was the most potent inhibitor, with a Ki of 1.5 x 10(-5) M at low (3.5 mM) phosphate concentrations. The inhibitory effects of these analogs could be ablated at high phosphate concentrations (50 mM), suggesting that they interact with the phosphate binding site on the enzyme. Some of these novel MeSAdo phosphorylase inhibitors may have a role in cancer chemotherapy as potentiators of agents that block purine de novo synthesis, e.g. antifolates and 6-methylmercaptopurine ribonucleoside.     

Structure-activity relationships of 17alpha-derivatives of estradiol as inhibitors of steroid sulfatase

The steroid sulfatase or steryl sulfatase is a microsomal enzyme widely distributed in human tissues that catalyzes the hydrolysis of sulfated 3-hydroxy steroids to the corresponding free active 3-hydroxy steroids. Since androgens and estrogens may be synthesized inside the cancerous cells starting from dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E(1)S) available in blood circulation, the use of therapeutic agents that inhibit steroid sulfatase activity may be a rewarding approach to the treatment of androgeno-sensitive and estrogeno-sensitive diseases. In the present study, we report the chemical synthesis and biological evaluation of a new family of steroid sulfatase inhibitors. The inhibitors were designed by adding an alkyl, a phenyl, a benzyl, or a benzyl substituted at position 17alpha of estradiol (E(2)), a C18-steroid, and enzymatic assays were performed using the steroid sulfatase of homogenized JEG-3 cells or transfected in HEK-293 cells. We observed that a hydrophobic substituent induces powerful inhibition of steroid sulfatase while a hydrophilic one was weak. Although a hydrophobic group at the 17alpha-position increased the inhibitory activity, the steric factors contribute to the opposite effect. As exemplified by 17alpha-decyl-E(2) and 17alpha-dodecyl-E(2), a long flexible side chain prevents adequate fitting into the enzyme catalytic site, thus decreasing capacity to inhibit the steroid sulfatase activity. In the alkyl series, the best compromise between hydrophobicity and steric hindrance was obtained with the octyl group (IC(50) = 440 nM), but judicious branching of side chain could improve this further. Benzyl substituted derivatives of estradiol were better inhibitors than alkyl analogues. Among the series of 17alpha-(benzyl substituted)-E(2) derivatives studied, the 3'-bromobenzyl, 4'-tert-butylbenzyl, 4'-butylbenzyl, and 4'-benzyloxybenzyl groups provided the most potent inhibition of steroid sulfatase transformation of E(1)S into E(1) (IC(50) = 24, 28, 25, and 22 nM, respectively). As an example, the tert-butylbenzyl group increases the ability of the E(2) nucleus to inhibit the steroid sulfatase by 3000-fold, and it also inhibits similarly the steroid sulfatase transformations of both natural substrates, E(1)S and DHEAS. Interestingly, the newly reported family of steroid sulfatase inhibitors acts by a reversible mechanism of action that is different from the irreversible mechanism of the known inhibitor estrone sulfamate (EMATE).     

New potent indole derivatives as hyaluronidase inhibitors

Because of the physiologic importance of hyaluronidases, the identification of potent and selective inhibitors of hyaluronidases has become increasingly important. A variety of assay methods have been used for such a purpose, i.e. classical turbidimetric, viscometric and colorimetric. In this study, a modified enzymatic assay has been used to obtain a microtiter plate-based sensitive activity screening. All inhibitors were tested in a stains-all assay at pH 7 and in a Morgan-Elson assay at pH 3.5. Among the tested compounds, 1, 2, 3, 6, 7, 8, 16, 17 and 18 showed good inhibition of more than 50%, so the IC(50) values of these derivatives were determined in the range of 25-41 microm. The IC(50) value of the most active hyaluronidase inhibitor Vcpal (6-palmitoyl-L-ascorbic acid) was measured as 8.36 microm. All inhibitors including Vcpal showed twofold less activity at pH 3.5 in a Morgan-Elson assay. Examination of substituent effects on the activity showed that para-positions of benzamide needs to be chlorinated or fluorinated to obtain good inhibitory effect. It was found that the introduction of a p-fluoro benzyl ring in the indole nitrogen has a positive effect for the inhibitory effects of both indole-2- and 3-carboxamide derivatives.     

Synthesis and pharmacological evaluation of 2,4-dinitroaryldithiocarbamate derivatives as novel monoacylglycerol lipase inhibitors

Monoacylglycerol lipase (MAGL) is responsible for signal termination of 2-arachidonoylglycerol (2-AG), an endocannabinoid neurotransmitter endowed with several physiological effects. Previously, we showed that the arylthioamide scaffold represents a privileged template for designing MAGL inhibitors. A series of 37 compounds resulting from pharmacomodulations around the arylthioamide template were synthesized and tested to evaluate their inhibitory potential on MAGL activity as well as their selectivity over fatty acid amide hydrolase (FAAH), another endocannabinoid-hydrolyzing enzyme. We have identified 2,4-dinitroaryldithiocarbamate derivatives as a novel class of MAGL inhibitors. Among the synthesized compounds, we identified [2,4-dinitrophenyl-4-(4-tert-butylbenzyl)piperazine-1-carbodithioate] (CK37), as the most potent MAGL inhibitor within this series (IC(50) = 154 nM). We have also identified [2,4-dinitrophenyl-4-benzhydrylpiperazine-1-carbodithioate] (CK16) as a selective MAGL inhibitor. These compounds are irreversible MAGL inhibitors that probably act by interacting with Cys208 or Cys242 and Ser122 residues of the enzyme. Moreover, CK37 is able to raise 2-arachidonoylglycerol (2-AG) levels in intact cells.     

Aza-peptide epoxides: a new class of inhibitors selective for clan CD cysteine proteases

Aza-peptide epoxides, a new class of irreversible protease inhibitors, are specific for the clan CD cysteine proteases. The inhibitors have second-order rate constants up to 10(5) M(-1) s(-1), with the most potent epoxides having the S,S stereochemistry. The aza-Asn derivatives are effective legumain inhibitors, while the aza-Asp epoxides were specific for caspases. The inhibitors have little or no inhibition with other proteases such as chymotrypsin, papain, or cathepsin B.     

Novel heterobivalent tacrine derivatives as cholinesterase inhibitors with notable selectivity toward butyrylcholinesterase

Two series of novel heterobivalent tacrine derivatives were synthesized. A trimethoxy substituted benzene was linked to the tacrine moiety by a hydrazide-based linker. The compounds were evaluated as cholinesterase inhibitors, and trimethoxybenzoic acid derivatives with 11- or 12-atom spacers were the most potent inhibitors of human acetylcholinesterase. The inhibitors showed a surprising selectivity toward human butyrylcholinesterase, where several trimethoxyphenylpropionic acid derivatives had IC(50) values less than 250 pM.     

Pyrido[1,2-a]pyrimidin-4-one derivatives as a novel class of selective aldose reductase inhibitors exhibiting antioxidant activity

2-Phenyl-pyrido[1,2-a]pyrimidin-4-one derivatives bearing a phenol or a catechol moiety in position 2 were tested as aldose reductase (ALR2) inhibitors and exhibited activity levels in the micromolar/submicromolar range. Introduction of a hydroxy group in position 6 or 9 gave an enhancement of the inhibitory potency (compare 18, 19, 28, and 29 vs 13 and 14). Lengthening of the 2-side chain to benzyl determined a general reduction in activity. The lack or the methylation of the phenol or catechol hydroxyls gave inactive (10-12, 21, 22, 25-27) or scarcely active (15, 17, 20) compounds, thus demonstrating that the phenol or catechol hydroxyls are involved in the enzyme pharmacophoric recognition. Moreover, all the pyridopyrimidinones displayed significant antioxidant properties, with the best activity shown by the catechol derivatives. The theoretical binding mode of the most active compounds obtained by docking simulations into the ALR2 crystal structure was fully consistent with the structure-activity relationships in the pyrido[1,2-a]pyrimidin-4-one series.     

Synthesis and evaluation of mono-, di-, and trifluoroethenyl-GABA derivatives as GABA-T inhibitors

The syntheses of four derivatives of gamma-vinyl-GABA, in which vinylic hydrogen atoms were replaced by fluorine, are described. With use of 5-ethenyl-2-pyrrolidinone as starting material, the E and Z isomers of 4-amino-6-fluoro-5-hexenoic acid were prepared. The 6,6-difluoro and 5,6,6-trifluoro analogues could be synthesized from 4-oxobutanoic acid tert-butyl ester and (2,2-difluoroethenyl)- and (trifluoroethenyl)lithium correspondingly. The compounds were tested as inhibitors of GABA-T, and their in vitro and in vivo biochemistry is reported. The most active derivative was (Z)-4-amino-6-fluoro-5-hexenoic acid; the structure-activity relationship in the series is discussed.     

Benzothiadiazine dioxide dibenzyl derivatives as potent human cytomegalovirus inhibitors: synthesis and comparative molecular field analysis

The benzothiadiazine dioxide (BTD) derivatives are potent nonnucleoside human cytomegalovirus (HCMV) inhibitors. As part of our comprehensive structure-activity relationship study of these compounds, we have now synthesized N,N- and N,O-dibenzyl derivatives with different para-substituents (alkyl, phenyl, electron-donating, electron-withdrawing) in the phenyl ring of the benzyl moieties. The antiviral activity against HCMV (AD-169 strain) was also experimentally measured showing IC(50) values between 2.5 and 50 microM. Comparative molecular field analysis (CoMFA) was employed to generate a model, based upon 32 diverse BTD derivatives, to delineate structural and electrostatic features important for enhanced activity against HCMV. The steric (van der Waals) interactions with the receptor majoritary describes the variation in antiviral activity among the inhibitors. Finally, the CoMFA model was used to design two sets of novel BTD derivatives. Synthesis and subsequent anti-HCMV evaluation of these compounds enabled us to maintain the activity of this new kind of HCMV inhibitors.     

Antitumor agents. 120. New 4-substituted benzylamine and benzyl ether derivatives of 4'-O-demethylepipodophyllotoxin as potent inhibitors of human DNA topoisomerase II.

A number of new 4'-O-demethylepipodophyllotoxin derivatives possessing various 4 beta-N- or 4 beta-O-benzyl groups have been synthesized and evaluated for their inhibitory activity against the human DNA topoisomerase II as well as for their activity in causing cellular protein-linked DNA breakage. The 4 beta-N-benzyl derivatives 9-22 are, in general, as active or more active than etoposide (1). The most active compounds are 14, 16, and 17, which are more than 2-fold more potent than 1. The results indicated that a basic unsubstituted 4 beta-benzylamino moiety is structurally required for the enhanced activity. Replacement of the benzyl nitrogen with oxygen gave compounds (23 and 24) which are inactive. The ability of these compounds to inhibit human DNA topoisomerase II and to cause protein-linked DNA breakage appears to have no direct correlation with cytotoxicity in KB cells.     

Novel 2-thiopyrimidine derivatives as CDK2 inhibitors: molecular modeling, synthesis, and anti-tumor activity evaluation

A novel series of pyrimidine-benzenesulfonamide derivatives as potential cyclin-dependent kinase 2 inhibitors were designed depending upon the molecular docking simulation study. This study was preceded by modification and optimization of the lead compound 4-(2-amino-4-methylthiazol-5-yl)-N-(3-nitrophenyl) pyrimidin-2-amine. The target proposed compounds were synthesized using the derivative 6-(3,4-dimethoxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1) as a key starting compound. Some of the synthesized derivatives were selected as representative examples to evaluate their anti-proliferative activity against cultured human Hela cell line using doxorubicin as a reference drug and the results obtained were correlated with the data of molecular modeling simulation study. The structures of the novel derivatives were confirmed on the bases of micro-analytical and spectral data.     

Synthesis and three-dimensional qualitative structure selectivity relationship of 3,5-disubstituted-2,4-thiazolidinedione derivatives as COX2 inhibitors

In our effort for synthesis of selective COX2 inhibitors, certain new 2,4-thiazolidinedione derivatives were synthesized. It necessitates preparation of potassium salt of 2,4-thiazolidinedione 2, which condensed with intermediate 4a. The resulting 3-[2-(4-methylphenyl)-2-oxo-l-phenylethyl]-2,4-thiazolidinedione 8 was condensed with appropriate aldehyde to afford compounds 10a, 10i-l, 10o and 10p. Compounds (9a-l, 10a-n, 10p, 11 and 12) were obtained through the preparation of 5-arylmethylidene-2,4-thiazolidinediones 6a-p and reaction of its potassium salt 7a-p with compounds 4a, 4b, and 5. Some compounds displayed significant analgesic activity as compared to reference standards. The anti-inflammatory activity of the synthesized compounds revealed that intermediate 8 and compounds 9c, 10c and 10d showed good results. Compound 10c produced no significant mucosal injury. HipHop methodology of Catalyst program was used to build up hypothetical model of selective COX2 inhibitors followed by fitting the synthesized compounds to this model. Compounds 10c and 10d were suspected to be promising selective COX2 inhibitors. Also, compounds (6c, 8, 9a,c,d,k, 10a,c,d,k, 11 and 12) were docked into COX1 and COX2 X-ray structures, using DOCK6 program. Docking results suggested that several of these derivatives are active COX inhibitors with a significant preference for COX2.     

Quaternary and tertiary quinuclidine derivatives as inhibitors of choline uptake.

The uptake of choline into cholinergic neurons for acetylcholine (ACh) synthesis is by a specific, high-affinity, sodium- and temperature-dependent transport mechanism (HAChU). Of several quaternary quinuclidinol derivatives tested, the N-allyl derivative proved to be most potent. Though the methyl, ethyl, and isopropyl derivatives were less potent at comparable concentrations, at higher concentrations they also maximally inhibited HAChU. The benzyl, hydroxyethyl, and methoxyethyl derivatives failed to inhibit HAChU by greater than 50% at concentrations up to 100 microM. N-Allyl-3-quinuclidinol (NAQ) proved to be a specific inhibitor of HAChU (IC50 = 0.9 microM) and a poor inhibitor of both sodium-independent transport (IC50 = 680 microM) and choline acetyltransferase activity (Ki = 200 microM). The NAQ exhibited noncompetitive type inhibition compared with N-methyl-3-quinuclidinol, a competitive inhibitor of HAChU. Thus, substitution at the N-functional group not only alters potency, but may change the mechanism by which inhibition is produced. The optical isomers of NAQ and several derivatives were prepared and employed to examine the stereochemical selectivity for inhibition of choline uptake. The S(+)-isomer of NAQ (IC50 = 0.1 microM) had approximately 100-fold greater inhibitory activity for HAChU than the corresponding R(-)-isomer (IC50 = 10 microM). With all other quinuclidinols tested, the S(+)-isomers were also more potent than the corresponding R(-)-isomers. In an effort to obtain a tertiary inhibitor of HAChU that would be expected to cross the blood-brain barrier following peripheral administration, 3-biphenyl-3-quinuclidinol (BHQ) and 3-naphthyl-3-quinuclidinol (NHQ) were synthesized and evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,3-Dihydroxyacridone derivatives as inhibitors of herpes virus replication

The nuclear enzyme DNA topoisomerase II is a candidate pharmacological target for treating herpes virus infections and the novel catalytic inhibitors, 7-chloro-1,3-dihydroxyacridone (compound 1), and 1,3,7-trihydroxyacridone (2) are potential lead compounds [Bastow, K.F., Itoigawa, M., Furukawa, H., Kashiwada, Y., Bori, I.D., Ballas, L.M., Lee, K.-H., 1994. Antiproliferative actions of 7-substituted 1,3-dihydroxyacridones; possible involvement of DNA topoisomerase II and protein kinase C as biochemical targets. Bioorg. Med. Chem. 2, 1403-1411; Vance, J.R., Bastow, K.F., 1999. Inhibition of DNA topoisomerase II catalytic activity by the antiviral agents 7-chloro,1,3-dihydroxyacridone and 1,3,7-trihydroxyacridone. Biochem. Pharmacol. 58, 703-708]. In this report, four new 1,3-dihydroxyacridone analogs with functional groups at either the 5-, 6- or 8-positions (compounds 3-6) were synthesized. Target compounds, three other analogs including compounds 1 and 2 and three anticancer drugs that inhibit DNA topoisomerase II (etoposide, amsacrine and aclarubicin) were then evaluated as selective inhibitors of herpes simplex virus (HSV) replication in cell culture and as enzyme inhibitors in vitro. Etoposide and amsacrine inhibited HSV but acted non-selectively. In general, the activities of 1,3-dihydroxyacridone derivatives as selective anti-HSV agents and as enzyme inhibitors varied inversely suggesting that DNA topoisomerase II probably is not the critical antiviral target. The 5-Cl congener (compound 3) was the most selective agent (about 26-fold under a stringent assay condition) but was not an enzyme inhibitor. Results of exploratory mechanistic studies with compounds 1 and 3 show that HSV replication was blocked at a stage after DNA and late protein synthesis. The acridone derivatives were also tested against human cytomegalovirus (HCMV) replication but none of them were active.