Chlamydocin-hydroxamic acid analogues as histone deacetylase inhibitors

Chlamydocin-hydroxamic acid analogues were designed and synthesized as histone deacetylase (HDAC) inhibitors based on the structure and HDAC inhibitory activity of chlamydocin and trichostatin A. Chlamydocin is a cyclic tetrapeptide containing an epoxyketone moiety in the side chain that makes it an irreversible inhibitor of HDAC. We replaced the epoxyketone moiety of chlamydocin with hydroxamic acid to design potent and reversible inhibitors of HDAC. In addition, a number of amino-cycloalkanecarboxylic acids (Acc) are introduced instead of the simple amino-isobutric acid (Aib) for a variety of the series of chlamydocin analogues. The compounds synthesized were tested for HDAC inhibitory activity and the results showed that many of them are potent inhibitors of HDAC. The replacement of Aib residue of chlamydocin with an aromatic amino acid enhances the in vivo and in vitro inhibitory activity. We have carried out circular dichroism and molecular modeling studies on chlamydocin-hydroxamic acid analogue and compared it with the solution structure of chlamydocin.     

Synthesis and Structure–Activity Relationship of Dehydroxymethylepoxyquinomicin Analogues as Inhibitors of NF-κB Functions

We previously found dehydroxymethylepoxyquinomicin (DHMEQ) inhibited NF-κB activation and showed anti-inflammatory activity in vivo. Here we designed and synthesized analogues of DHMEQ and tested their biological activity as NF-κB inhibitors in human T cell leukemia Jurkat cells. The hydroxyl group at the 2-position of the benzamide moiety was found to be essential for the inhibitory activity. But etherification of this group did not diminish the activity completely. Thus, for further mechanistic studies the hydroxyl group at the 2-position may be useful for extension with a linker and biotin moiety.     

Molecular basis for the binding of competitive inhibitors of maize polyamine oxidase

Maize polyamine oxidase (MPAO), the only member of the polyamine oxidase (PAO) family whose three-dimensional structure is known, is characterized by a 30 A long U-shaped catalytic tunnel located between the substrate binding domain and the FAD. To shed light on the MPAO ligand binding mode, we studied the inhibition properties of linear diamines, agmatine, prenylagmatine (G3), G3 analogues, and guazatine, and analyzed the structural determinants of their biological activity. Linear diamines competitively inhibited MPAO, with the inhibitory activity increasing as a function of the number of methylene groups. With regard to the guanidino competitive inhibitors, including agmatine, G3, and G3 analogues, the presence of a hydrophobic substituent constitutes the principal factor influencing MPAO inhibition, as the addition of a hydrophobic substituent to the guanidino group of both G3 and G3 analogues greatly increases the inhibitory activity. Moreover, results obtained by a molecular modeling procedure indicated that in their preferred orientation, G3 analogues point the ammonium group toward the narrow entrance of the tunnel, while the terminal hydrophobic group is located within the large entrance. The high binding affinity for MPAO exhibited by G3 and G3 analogues bearing a prenyl group as a substituent on the guanidino moiety is in agreement with the observation that the prenyl group binds in a well-defined hydrophobic pocket, mainly formed by aromatic residues. Finally, docking simulations performed with the charged and uncharged forms of MPAO inhibitors indicate that the stereoelectronic properties of the MPAO active site are consistent with the binding of inhibitors in the protonated form.     

Synthesis and structure-activity relationship of dehydroxymethylepoxyquinomicin analogues as inhibitors of NF-kappaB functions

We previously found dehydroxymethylepoxyquinomicin (DHMEQ) inhibited NF-kappaB activation and showed anti-inflammatory activity in vivo. Here we designed and synthesized analogues of DHMEQ and tested their biological activity as NF-kappaB inhibitors in human T cell leukemia Jurkat cells. The hydroxyl group at the 2-position of the benzamide moiety was found to be essential for the inhibitory activity. But etherification of this group did not diminish the activity completely. Thus, for further mechanistic studies the hydroxyl group at the 2-position may be useful for extension with a linker and biotin moiety.     

Synthesis and biological evaluation of thielocin B1 analogues as protein-protein interaction inhibitors of PAC3 homodimer

The synthesis and biological evaluation of thielocin B1 analogues have been demonstrated. Fourteen analogues modified in the central core and terminal carboxylic acid moiety were concisely synthesized by simple esterification or etherification reaction. The evaluation of synthetic analogues as inhibitors of proteasome assembling chaperone (PAC) complexes (the PAC3 homodimer and PAC1/PAC2) revealed that the natural product-like bending structure and terminal carboxylic acid groups were crucial for its biological activity. Moreover, SAR and in silico docking studies indicated that all methyl groups on the diphenyl ether moiety of thielocin B1 contribute to the potent and selective inhibition of the PAC3 homodimer via hydrophobic interactions.     

Synthesis, anticonvulsant activity, and neuropathic pain-attenuating activity of N-benzyl 2-amino-2-(hetero)aromatic acetamides

N-Benzyl 2-acetamido-2-substituted acetamides, where the 2-substituent is a (hetero)aromatic moiety, are potent anticonvulsants. We report the synthesis and whole animal pharmacological evaluation of 16 analogues where the terminal 2-acetyl group was removed to give the corresponding primary amino acid derivatives (PAADs). Conversion to the PAAD structure led to a substantial drop in seizure protection in animal tests, demonstrating the importance of the N-acetyl moiety for anticonvulsant activity. However, several of the PAADs displayed notable pain-attenuating activities in a mouse model.     

Synthesis, induced-fit docking investigations, and in vitro aldose reductase inhibitory activity of non-carboxylic acid containing 2,4-thiazolidinedione derivatives

In continuation of our studies, we here report a series of non-carboxylic acid containing 2,4-thiazolidinedione derivatives, analogues of previously synthesized carboxylic acids which we had found to be very active in vitro aldose reductase (ALR2) inhibitors. Although the replacement of the carboxylic group with the carboxamide or N-hydroxycarboxamide one decreased the in vitro ALR2 inhibitory effect, this led to the identification of mainly non-ionized derivatives with micromolar ALR2 affinity. The 5-arylidene moiety deeply influenced the activity of these 2,4-thiazolidinediones. Our induced-fit docking studies suggested that 5-(4-hydroxybenzylidene)-substituted derivatives may bind the polar recognition region of the ALR2 active site by means of the deprotonated phenol group, while their acetic chain and carbonyl group at position 2 of the thiazolidinedione ring form a tight net of hydrogen bonds with amino acid residues of the lipophilic specificity pocket of the enzyme.     

Cytotoxicity, cellular uptake and DNA damage by daunorubicin and its new analogues with modified daunosamine moiety

Daunorubicin (DRB) and its two analogues containing a trisubstituted amidino group at the C-3′ position of the daunosamine moiety have been compared regarding their cytotoxic activity, cellular uptake, subcellular localization and DNA damaging properties. An analogue containing in the amidino group a morpholine moiety (DRBM) as well as an analogue with a hexamethyleneimine moiety (DRBH), tested against cultured L1210 cells, exhibited lower cytotoxicity then DRB. The decrease of cytotoxic activity was not related to cellular uptake and subcellular localization of drugs. Although all tested drugs were active in the induction of DNA breaks and DNA–protein crosslinks, they differed in the mechanism of induction of DNA lesions. DRB produced DNA breaks mediated solely by topoisomerase II, whereas DRBM and DRBH induced two types of DNA breaks by two separate processes. The first is related to the inhibition of topoisomerase II and the second presumably reflects a covalent binding of drug metabolites to DNA. It is hypothesized that the replacement of the primary amino group (–NH₂) at the C-3′ position of the daunosamine moiety by a trisubstituted amidino group (–N=CH–NRR) may be a route to the synthesis of anthracycline derivatives with enhanced ability to form covalent adducts to DNA.     

A general method for the synthesis of aryl [11C]methylsulfones: potential PET probes for imaging cyclooxygenase-2 expression

A general one-pot method has been developed for the conversion of an aryl thiol moiety masked as the butyrate ester to the corresponding 11C-labeled methylsulfone group. The potential of this methodology has been demonstrated by the successful radiosynthesis of carbon-11 analogues of several highly selective cyclooxygenase-2 (COX-2) inhibitors such as Rofecoxib, Etoricoxib, and 3-(4-methylsulfonylphenyl)-4-phenyl-5-trifluoromethyl isoxazole in high yield. The chemical and radiochemical purities obtained for the 11C-labeled COX-2 inhibitors are >99% with a specific activity >1000 Ci/mmol.     

Purification of gamma-glutamyltransferase by phenyl boronate affinity chromatography. Studies on the acceptor specificity of transpeptidation by rat kidney gamma-glutamyltransferase

gamma-Glutamyltransferase has been purified from rat kidney by a novel procedure using phenyl boronate affinity chromatography. The highly purified enzyme has been studied with respect to acceptor specificity for a number of amino acids, amino acid analogues, dipeptides and tripeptides. The acceptor activity is specific for L-amino acids. The amino acids and the majority of the essential amino acids are poor acceptors while the sulphur-containing amino acids are the best acceptors. The acceptor activity is modulated by the substitution of the amino acid side chain. Substitution of the side chain at the delta, gamma or beta positions results in a proportionally decreasing ability to act as acceptor. The carbonyl moiety of the gamma-carboxy group of the acceptor appears to be essential for acceptor activity, absence of an alpha-carboxy carbonyl group increases the Kappm of the acceptor approximately 100-fold.     

Structure activity relationship studies on the antimicrobial activity of novel edeine A and D analogues.

Edeines are pentapeptide amide antibiotics composed of four nonprotein amino acids, glycine, and polyamine. They exhibit antimicrobial and immunosuppressive activities and are universal inhibitors of translation. Moreover, it was proven that the free ionizable carboxy group in the (2R, 6S, 7R)-2,6-diamino-7-hydroxyazelaic acid moiety is not essential for biological activity of these compounds. In this paper we describe the synthesis of four novel edeine A and D analogues in which the above-mentioned acid residue was replaced with the (3R, 4S)- or (3S, 4S)-4,5-diamino-3-hydroxypentanoic acid moiety. In one compound we also introduced into molecule the 3-N,N-dimethyl derivative of (S)-2,3-diaminopropanoic acid to prevent the transpeptidation process, which results in the loss of biological activity of alpha-isomers of edeines. All peptides were synthesized applying the active ester and azide methods and on the basis of the coupling of suitable N-terminal tripeptides with proper C-terminal dipeptide amides. The activities of the newly obtained edeine analogues against selected strains of bacteria and fungi are also presented.     

Oxytocin analogues with amide groups substituted by tetrazole groups in position 4, 5 or 9

Eleven oxytocin analogues substituted in position 4, 5 or 9 by tetrazole analogues of amino acids were prepared using solid-phase peptide synthesis method and tested for rat uterotonic in vitro and pressor activities, as well as for their affinity to human oxytocin receptor. The tetrazolic group has been used as a bioisosteric substitution of carboxylic, ester or amide groups in structure-activity relationship studies of biologically active compounds. Replacement of the amide groups of Gln(4) and Asn(5) in oxytocin by tetrazole analogues of aspartic, glutamic and alpha-aminoadipic acids containing the tetrazole moiety in the side chains leads to analogues with decreased biological activities. Oxytocin analogues in which the glycine amide residue in position 9 was substituted by tetrazole analogues of glycine had diminished activities as well. The analysis of differences in rat uterotonic activity and in the affinity to human oxytocin receptors of analogues containing either an acidic 5-substituted tetrazolic group or a neutral 1,5- or 2,5-tetrazole nucleus makes it possible to draw some new conclusions concerning the role of the amide group of amino acids in positions 4, 5 and 9 of oxytocin for its activity. The data suggest that the interaction of the side chain of Gln(4) with the oxytocin receptor is influenced mainly by electronic effects and the hydrogen bonding capacity of the amide group. Steric effects of the side chain are minor. Substitution of Asn(5) by its tetrazole derivative gave an analogue of very low activity. The result suggests that in the interaction between the amide group of Asn(5) and the binding sites of oxytocic receptor hydrogen bonds are of less importance than the spatial requirements for this group.     

The DNA sequence selectivity of maltolato-containing cisplatin analogues in purified plasmid DNA and in intact human cells

Cisplatin analogues with an attached DNA binding moiety have a higher affinity for DNA, but often suffer from poor aqueous solubility. In this study we examined the DNA sequence specificity of more soluble cisplatin analogues containing the maltolato leaving group in both purified DNA and in intact human cells. In both environments the DNA sequence specificity of these analogues was very similar to cisplatin. However, in purified DNA a higher concentration of the two maltolato-containing analogues was needed to achieve a similar level of DNA damage as cisplatin. This difference in reactivity was not observed in intact cells as the two maltolato-containing complexes were capable of producing a similar level of damage as cisplatin at comparable concentrations. This was consistent with the IC₅₀ values obtained for both cisplatin and the maltolato compounds which were also similar. This study indicated that maltolato can be utilised as the leaving group to increase the aqueous solubility of cisplatin analogues without reducing their biological activity.     

Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfanyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC₅₀ of 200 nM with a therapeutic index of >1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. C_max and absolute bioavailability of 34S were higher and half-life and time above EC₉₅ were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.     

Influence of the amino acid moiety on deconjugation of bile acid amidates by cholylglycine hydrolase or human fecal cultures

The influence of the chemical structure of the amino acid (or amino acid analogue) moiety of a number of synthetic cholyl amidates on deconjugation by cholylglycine hydrolase from Clostridium perfringens was studied in vitro at pH 5.4. Conjugates with alkyl homologues of glycine were hydrolyzed more slowly as the number of methylene units increased (cholylglycine greater than cholyl-beta-alanine greater than cholyl-gamma-aminobutyrate). In contrast, for conjugates with the alkyl homologues of taurine, cholylaminopropane sulfonate was hydrolyzed slightly faster than cholyltaurine, whereas cholylaminomethane sulfonate was hydrolyzed much more slowly. When glycine was replaced by other neutral alpha-amino acids, rates of hydrolysis decreased with increasing steric hindrance near the amide bond (cholyl-L-alpha-alanine much much greater than cholyl-L-leucine much greater than cholyl-L-valine greater than cholyl-L-tyrosine much greater than cholyl-D-valine). Conjugation with acidic or basic amino acids also greatly reduced the rates of hydrolysis, as cholyl-L-aspartate, cholyl-L-cysteate, cholyl-L-lysine, and cholyl-L-histidine were all hydrolyzed at a rate less than one-tenth that of cholylglycine. Methyl esterification of the carboxylic group of the amino acid moiety reduced the hydrolysis, but such substrates (cholylglycine methyl ester and cholyl-beta-alanine methyl ester) were completely hydrolyzed after overnight incubation with excess of enzyme. In contrast, cholyl-cholamine was not hydrolyzed at all, suggesting that a negative charge at the end of the side chain is required for optimal hydrolysis. Despite the lack of specificity for the amino acid moiety, a bile salt moiety was required, as the cholylglycine hydrolase did not display general carboxypeptidase activity for other non-bile acid substrates containing a terminal amide bond: hippuryl-L-phenylalanine and hippuryl-L-arginine, as well as oleyltaurine and oleylglycine, were not hydrolyzed. Fecal bacterial cultures from healthy volunteers also hydrolyzed cholyl-L-valine and cholyl-D-valine more slowly than cholylglycine, suggesting that cholylglycine hydrolase from Clostridium perfringens has a substrate specificity similar to that of the deconjugating enzymes of the fecal flora. The results indicate that modification of the position of the amide bond, introduction of steric hindrance near the amide bond, or loss of a negative charge on the terminal group of the amino acid moiety of the bile acid conjugate greatly reduces the rate of bacterial deconjugation in vitro when compared to that of the naturally occurring glycine and taurine conjugates.     

Discovery of novel 2-(3-phenylpiperazin-1-yl)-pyrimidin-4-ones as glycogen synthase kinase-3β inhibitors

We herein describe the results of further evolution of glycogen synthase kinase (GSK)-3β inhibitors from our promising compounds containing a 2-phenylmorpholine moiety. Transformation of the morpholine moiety into a piperazine moiety resulted in potent GSK-3β inhibitors. SAR studies focused on the phenyl moiety revealed that a 4-fluoro-2-methoxy group afforded potent inhibitory activity toward GSK-3β. Based on docking studies, new hydrogen bonding between the nitrogen atom of the piperazine moiety and the oxygen atom of the main chain of Gln185 has been indicated, which may contribute to increased activity compared with that of the corresponding phenylmorpholine analogues. Effect of the stereochemistry of the phenylpiperazine moiety is also discussed.     

Structural requirement for the agonist activity of the TLR2 ligand Pam2Cys

Synthetic lipopeptides have demonstrated great potential as a vaccine strategy for eliciting cellular and humoral immunity. One of the most potent lipid moieties used is S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys). Pam2Cys binds to and activates dendritic cells by engagement of Toll like receptor 2 (TLR 2). In this study, we have investigated the structural requirement of the agonist activity of Pam2Cys by varying the three structural elements of the core structure S-(2,3-dihydroxypropyl)-cysteine namely (1) the α-amino group of the cysteine residue (2) the sulphur atom of the cysteine residue and (3) the 2,3-dihydroxypropyl moiety. Four novel analogues of Pam2Cys were made and each of these analogues were incorporated into vaccine constructs and examined for immunogenicity. Our results demonstrate that (1) the potency of the peptide vaccine is least affected by removal of the amino group (2) substitution of the sulphur atom with an amide bond leads to significant reduction of biological activity (3) removal of the amino group and at the same time substitution of the sulphur with an amide bond significantly decreases the biological activity (4) in the two analogues in which the sulphur atom is replaced with an amide bond the analogue containing the 1,3-dihydroxypropyl moiety demonstrates higher activity than the one which contains 2,3-dihydroxypropyl. In conclusion, the results demonstrate strict structural requirements for agonist activity of the TLR2 ligand Pam2Cys.     

Synthesis of daidzin analogues as potential agents for alcohol abuse

Daidzin, the active principle of an herbal remedy for 'alcohol addiction', has been shown to reduce alcohol consumption in all laboratory animals tested to date. Correlation studies using structural analogues of daidzin suggests that it acts by raising the monoamine oxidase (MAO)/mitochondrial aldehyde dehydrogenase (ALDH-2) activity ratio (J. Med. Chem. 2000, 43, 4169). Structure-activity relationship (SAR) studies on the 7-O-substituted analogues of daidzin have revealed structural features important for ALDH-2 and MAO inhibition (J. Med. Chem. 2001, 44, 3320). We here evaluated effects of substitutions at 2, 5, 6, 8, 3' and 4' positions of daidzin on its potencies for ALDH-2 and MAO inhibition. Results show that analogues with 4'-substituents that are small, polar and with hydrogen bonding capacities are most potent ALDH-2 inhibitors, whereas those that are non-polar and with electron withdrawing capacities are potent MAO inhibitors. Analogues with a 5-OH group are less potent ALDH-2 inhibitors but are more potent MAO inhibitors. All the 2-, 6-, 8- and 3'-substituted analogues tested so far do not inhibit ALDH-2 and/or have decreased potencies for MAO inhibition. This, together with the results obtained from previous studies, suggests that a potent antidipsotropic analogue would be a 4',7-disubstituted isoflavone. The 4'-substituent should be small, polar, and with hydrogen bonding capacities such as, -OH and -NH(2); whereas the 7-substituent should be a straight-chain alkyl with a terminal polar function such as -(CH(2))(n)-OH with 2< or =n < or =6, -(CH(2))(n)-COOH with 5< or =n < or =10, or -(CH(2))(n)-NH(2) with n > or =4.     

The synthesis and biological evaluation of some carbocyclic analogues of PUGNAc

The synthesis of some analogues of O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenylcarbamate, PUGNAc, an inhibitor of β-N-acetylglucosaminidases, is described. The analogues were tested against a range of β-N-acetylglucosaminidases to establish any biological activity. As well, the analogues were tested as inhibitors of a uridine diphosphate-N-acetyl-d-glucosamine: polypeptidyl transferase, OGT, a critical protein involved in the post-translational modification of nuclear and cytosolic proteins by N-acetyl-d-glucosamine.     

Inhibition of eukaryotic translation by analogues of messenger RNA 5'-cap: chemical and biological consequences of 5'-phosphate modifications of 7-methylguanosine 5'-monophosphate

New analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized with modified 5'-phosphate moieties by replacement of -O with -H, -CH3, or -NH2. Additional analogues were synthesized with 8-methyl- or 8-aminoguanine base substitutions or ring-opened ribose (2',3'-diol). These compounds were analyzed by 1H and 31P NMR for solution conformation. In addition, they were also analyzed for biological activity as analogues of mRNA 5'-caps by competition as inhibitors of translation in reticulocyte lysate. Substitution of oxygen on the 5'-monophosphate moiety by -H and -CH3 diminished the activity of the cap analogue as a competitive inhibitor; however, replacement by -NH2 did not diminish the activity of the analogue as an inhibitor. It was inferred from this result that cap binding proteins require a hydrogen bond acceptor as opposed to having an exclusive requirement for a second anionic group on the alpha-phosphate moiety. Inhibition results obtained with C8-substituted m7GMP analogues indicated that the 8-amino derivative was a better inhibitor than the 8-methyl derivative of m7GMP. The former is primarily anti whereas the latter is primarily syn with respect to glycosidic bond conformation. This result further supports the model that the anti conformation is the preferred form of the cap structure for interaction with cap binding proteins. The 2',3'-diol derivative of m7GMP was inactive as an inhibitor of translation.