The bisnaphthalimides as new active lead compounds against Plasmodium falciparum

The bisquaternary bisnaphthalimides are a versatile class of compounds being active against the malaria parasite Plasmodium falciparum in the lower nanomolar range of concentration combined with no cytotoxicity. The series of compounds is designed as choline analogues and interfering agents of the phosphatidylcholine biosynthesis. The qualitative analysis of the structure–activity relationships (SAR) revealed the importance of a long methylene middle chain of at least 8 methylene groups between the two bisquaternary naphthalimides or a monoquaternary naphthalimide consisting of a long alkyl chain attached to the positively charged nitrogen atom. Since the SARs are different from reported biscationic antimalarial drugs the mode of action remains to be elucidated.     

A Structure-Activity Study with Aryl Acylamidases

We examined the relationship between chemical structure and biodegradability of acylanilide herbicides by using a set of model compounds. Four bacterial isolates (one gram-negative and three gram-positive) that grew on acetanilide were used. These soil isolates cleaved the amide bond of acetanilide via an aryl acylamidase reaction, producing aniline and the organic acid acetate. A series of acetanilide analogs with alkyl substitutions on the nitrogen atom or the aromatic ring were tested for their ability to induce aryl acylamidase activity and act as substrates for the enzyme. The substrate range, in general, was limited to those analogs not disubstituted in the ortho position of the benzene ring or which did not contain an alkyl group on the nitrogen atom. These same N-substituted compounds did not induce enzyme activity either, whereas the ortho-substituted compounds could in some cases.     

Cis-Amide isosteric replacement in thienobenzoxepin inhibitors of PI3-kinase

Substructural class effects surrounding replacement of a ‘cis’ N-methyl aniline amide within potent and selective thienobenzoxepin PI3-kinase inhibitors are disclosed. While a simple aryl to alkyl switch was not tolerated due to differences in preferred amide conformation, heterocyclic amide isosteres with maintained aryl substitution improved potency and metabolic stability at the cost of physical properties. These gains in potency allowed lipophilic deconstruction of the arene to simple branched alkyl substituents. As such, overall lipophilicity-neutral, MW decreases were realized relative to the aniline amide series. The improved properties for lead compound 21 resulted in high permeability, solubility and bioavailability.     

Structure–activity relationship studies of manzamine A: Amidation of positions 6 and 8 of the β-carboline moiety

Twenty manzamine amides were synthesized and evaluated for in vitro antimalarial and antimicrobial activities. The amides of manzamine A (1) showed significantly reduced cytotoxicity against Vero cells, although were less active than 1. The structure–activity analysis showed that linear, short alkyl groups adjacent to the amide carbonyl at position 8 are favored for antimalarial activity, while bulky and cyclic groups at position 6 provided the most active amides. Most of the amides showed potent activity against Mycobacterium intracellulare. The antimicrobial activity profile for position 8 series was similar to that for antimalarial activity profile, in which linear, slightly short alkyl groups adjacent to the amide carbonyl showed improved activity. Two amides 14 and 21, which showed potent antimalarial activity in vitro against Plasmodium falciparum were further evaluated in vivo in Plasmodium berghei infected mice. Oral administration of 14 and 21 at the dose of 30 mg/kg (once daily for three days) caused parasitemia suppression of 24% and 62%, respectively, with no apparent toxicity.     

Structure-Activity Relationships of Novel Salicylaldehyde Isonicotinoyl Hydrazone (SIH) Analogs: Iron Chelation,Anti-Oxidant and Cytotoxic Properties

Salicylaldehyde isonicotinoyl hydrazone (SIH) is a lipophilic, tridentate iron chelator with marked anti-oxidant and modest cytotoxic activity against neoplastic cells. However, it has poor stability in an aqueous environment due to the rapid hydrolysis of its hydrazone bond. In this study, we synthesized a series of new SIH analogs (based on previously described aromatic ketones with improved hydrolytic stability). Their structure-activity relationships were assessed with respect to their stability in plasma, iron chelation efficacy, redox effects and cytotoxic activity against MCF-7 breast adenocarcinoma cells. Furthermore, studies assessed the cytotoxicity of these chelators and their ability to afford protection against hydrogen peroxide-induced oxidative injury in H9c2 cardiomyoblasts. The ligands with a reduced hydrazone bond, or the presence of bulky alkyl substituents near the hydrazone bond, showed severely limited biological activity. The introduction of a bromine substituent increased ligand-induced cytotoxicity to both cancer cells and H9c2 cardiomyoblasts. A similar effect was observed when the phenolic ring was exchanged with pyridine (i.e., changing the ligating site from O, N, O to N, N, O), which led to pro-oxidative effects. In contrast, compounds with long, flexible alkyl chains adjacent to the hydrazone bond exhibited specific cytotoxic effects against MCF-7 breast adenocarcinoma cells and low toxicity against H9c2 cardiomyoblasts. Hence, this study highlights important structure-activity relationships and provides insight into the further development of aroylhydrazone iron chelators with more potent and selective anti-neoplastic effects.     

Unexpected selectivity in electrophilic attack on (PNP)RuN

Protonation of (PNP)RuN, where PNP is (^tBu₂PCH₂SiMe₂)₂N, with HCl occurs at the amide nitrogen, with coordination of chloride to Ru^IV, while triflic acid protonates the same nitrogen, but has triflate anion hydrogen-bonded to the proton on the PNP amide nitrogen, not triflate coordinated to the metal. Methyl triflate however alkylates the nitride nitrogen, to give a C_2v symmetric product. DFT calculations show that the thermodyamic preference is for proton on amide nitrogen while alkyl favors nitride alkylation, even without the need for a hydrogen bond to reverse the H vs. alkyl preference. Alkylation at the amide nitrogen leads to nearly complete loss of the PN(R)P Ru/N bond in this unobserved isomer. These preferences among nucleophilic sites on (PNP)RuN are rationalized based on the frontier orbitals of this molecule.     

Synthesis,anti-plasmodial and cytotoxic evaluation of 1H-1,2,3-triazole/acyl hydrazide integrated tetrahydro-β-carboline-4-aminoquinoline conjugates

A series of 1H-1,2,3-triazole/acylhydrazide-tethered tetrahydro-β-carboline-4-aminoquinoline conjugates was synthesized with an aim to study their anti-plasmodial structure-activity relationship. The presence of 1H-1,2,3-triazole-core along with a flexible alkyl chain length on aminoquinoline core has favourable influence on the anti-plasmodial activity against Chloroquine-resistant W2 strain of P. falciparum while the introduction of hydrazine core not only diminished the activities but also resulted in increased cytotoxicity against mammalian Vero cells.     

Synthesis and study of antiproliferative,antitopoisomerase II,DNA-intercalating and DNA-damaging activities of arylnaphthalimides

A series of arylnaphthalimides were designed and synthesized to overcome the dose-limiting cytotoxicity of N-acetylated metabolites arising from amonafide, the prototypical antitumour naphthalimide whose biomedical properties have been related to its ability to intercalate the DNA and poison the enzyme Topoisomerase II. Thus, these arylnaphthalimides were first evaluated for their antiproliferative activity against two tumour cell lines and for their antitopoisomerase II in vitro activities, together with their ability to intercalate the DNA in vitro and also through docking modelization. Then, the well-known DNA damage response in Saccharomyces cerevisiae was employed to critically evaluate whether these novel compounds can damage the DNA in vivo. By performing all these assays we conclude that the 5-arylsubstituted naphthalimides not only keep but also improve amonafide’s biological activities.     

A series of cationic sterol lipids with gene transfer and bactericidal activity

A family of cationic lipids was synthesized via direct amide coupling of spermine to the C-24 position of cholic acid analogs. Four monosubstituted spermines and a bis-substituted spermine were evaluated as plasmid transfection reagents, as bacteriostatic agents, and as bactericidal agents. The incorporation of a double bond in the sterol moiety enhanced transfection efficiency significantly and produced two compounds with little cytotoxicity and transfection potency comparable to Lipofectamine2000^?. Inclusion of the double bond had no effect on the general trend of increasing bactericidal activity with increasing sterol hydrophobicity. Co-formulation of the most hydrophilic of the compounds with its bis-substituted analogue led to enhancement in transfection activity. The bis-substituted compound, when tested alone, emerged as the most bacteriostatic compound in the family with minimum inhibitory concentrations (MIC) of 4 μM against Bacillus subtilis and 16 μM against Escherichia coli and therapeutic indexes (minimum hemolytic concentration/minimum inhibitory concentration) of 61 and 15, respectively. Cationic lipids can be optimized for both gene delivery and antibacterial applications by similar modifications.     

Investigations on the use of fluorescence dyes for labeling dendrimers: cytotoxicity, accumulation kinetics, and intracellular distribution

Fluorescent dyes (e.g., dansyl, fluoresceine isothiocyanate, or naphthalimide groups) are widely used as markers to study biological properties of drugs. In order to evaluate possible mediated cytotoxicity, we attached three molecules each to 1,3,5-tris(3-propylamino)benzene initially synthesized as core molecule for the design of dendrimers. Cytotoxic effects were only observed for the NO(2)-substituted naphthalimide conjugate. The intracellular distribution was visualized via confocal fluorescence microscopy and pointed to an accumulation in the endosome or nucleus, dependent on the cell line used.     

The importance of the amide bond nearest the thiol group in enzymatic reactions of coenzyme A

Analogues of coenzyme A (CoA) and of CoA thioesters have been prepared in which the amide bond nearest the thiol group has been modified. An analogue of acetyl-CoA in which this amide bond is replaced with an ester linkage was a good substrate for the enzymes carnitine acetyltransferase, chloramphenicol acetyltransferase, and citrate synthase, with K(m) values 2- to 8-fold higher than those of acetyl-CoA and V(max) values from 14 to >80% those of the natural substrate. An analogue in which an extra methylene group was inserted between the amide bond and the thiol group showed less than 4-fold diminished binding to the three enzymes but exhibited less than 1% activity relative to acetyl-CoA with carnitine acetyltransferase and no measurable activity with the other two enzymes. Analogues of several CoA thioesters in which the amide bond was replaced with a hemithioacetal linkage exhibited no measurable activity with the appropriate enzymes. The results indicate that some aspects of the amide bond and proper distance between this amide and the thiol/thioester moiety are critical for activity of CoA ester-utilizing enzymes.     

Ostreol A: A new cytotoxic compound isolated from the epiphytic dinoflagellate Ostreopsis cf. ovata from the coastal waters of Jeju Island,Korea

Ostreol A was isolated from cultures of the epiphytic dinoflagellate Ostreopsis cf. ovata from the coastal waters of Jeju Island, Korea. The compound, a non-palytoxin derivative, has a polyhydroxy chain ending with the primary amino group and contains an amide bond, along with two tetrahydropyran rings in the chain. Its chemical structure was elucidated by nuclear magnetic resonance (NMR) spectroscopy methods and confirmed by mass analysis. The compound exhibited significant cytotoxicity in the brine shrimp lethality test at a concentration of 0.9 μg/mL.     

Anti-malarial activity of new N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) derivatives against Plasmodium falciparum

Malaria is the most common of the parasitic diseases in tropical and subtropical regions. Adverse side effects of anti-malarial drugs have precluded them as a potential clinical drug. In this study, novel derivatives of N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) based on a variety of dipeptidyl α,β-unsaturated amides containing lysine as a part were synthesized and evaluated. Lower toxicity was achieved by reducing or eliminating the tendency of forming chemically reactive and toxic intermediates and metabolites. The synthesized compounds were evaluated for anti-malarial efficacy against Plasmodium falciparum and cytotoxicity in human epitheloid carcinoma cervix (HeLa cells) by estimating the therapeutic index (TI). N-Methyl amide with N′-Boc protection among them exhibited strong anti-malarial activity and N-methyl amide with N′-m-methylbenzyl amide showed excellent anti-malarial activity with much lower toxicity than the ALLN. Therefore, the two chemicals, as well as the underlying design rationale, could be useful in the discovery and development of new anti-malarial drugs.     

Design and synthesis of indole, 2,3-dihydro-indole,and 3,4-dihydro-2H-quinoline-1-carbothioic acid amide derivatives as novel HCV inhibitors

An efficient synthetic methodology to provide indole, 2,3-dihydro-indole, and 3,4-dihydro-2H-quinoline-1-carbothioic acid amide derivatives is described. These conformationally restricted heterobicyclic scaffolds were evaluated as a novel class of HCV inhibitors. Introduction of an acyl group at the NH₂ of the thiourea moiety has been found to enhance inhibitory activity. The chain length and the position of the alkyl group on the indoline aromatic ring markedly influenced anti-HCV activity. The indoline scaffold was more potent than the corresponding indole and tetrahydroquinoline scaffolds and analogue 31 displayed excellent activity (EC₅₀ = 510 nM) against HCV without significant cytotoxicity (CC₅₀ >50 μM).     

Efficient synthesis of anacardic acid analogues and their antibacterial activities

Anacardic acid derivatives exhibit a broad range of biological activities. In this report, an efficient method for the synthesis of anacardic acid derivatives was explored, and a small set of salicylic acid variants synthesised retaining a constant hydrophobic element (a naphthyl tail). The naphthyl side chain was introduced via Wittig reaction and the aldehyde installed using directed ortho-metalation reaction of the substituted o-anisic acids. The failure of ortho-metalation using unprotected carboxylic acid group compelled us to use directed ortho-metalation in which a tertiary amide was used as a strong ortho-directing group. In the initial route, tertiary amide cleavage during final step was challenging, but cleaving the tertiary amide before Wittig reaction was beneficial. The Wittig reaction with protected carboxylic group (methyl ester) resulted in side-products whereas using sodium salt resulted in higher yields. The novel compounds were screened for antibacterial activity and cytotoxicity. Although substitution on the salicylic head group enhanced antibacterial activities they also enhanced cytotoxicity.     

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.     

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: A novel assay

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (Δ? = 860 m^?1 cm^?1) and by ninhydrin colorimetry (substrate I, ?₅₇₀ = 2.31 × 10⁴m^?1 cm^?1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5–6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.     

The effect of replacing the ester bond with an amide bond and of overall stereochemistry on the activity of daptomycin

Daptomycin, a cyclic lipodepsipeptide antibiotic, has been used clinically since 2003 to treat serious infections caused by Gram-positive bacteria. Although 37?years have passed since daptomycin’s discovery, its mechanism of action is still debated. In this report, the effect of replacing the ester bond with an amide bond, and overall stereochemistry, on daptomycin’s biological activity was examined. Two peptides were prepared in which the threonine4 residue in the active daptomycin analog, Dap-K6-E12-W13, was replaced with (2S,3R)-diaminobutyric acid ((2S,3R)-DABA) or its epimer (2S,3S-DABA) converting the ring-closing ester bond to an amide bond. Both of these peptides were found to be considerably less active than Dap-K6-E12-W13. These results, along with our previous studies on other daptomycin analogs, enabled us to conclude that the ester bond is crucial to daptomycin’s activity. ent-Dap-K6-E12-W13 was found to be at least 133-fold less active than Dap-K6-E12-W13, indicating that a chiral interaction with a chiral target is essential to daptomycin’s activity. Studies examining the binding of Dap-K6-E12-W13 and ent-Dap-K6-E12-W13 to model liposomes consisting of phosphatidylglycerol (PG) and phosphatidylcholine suggest that the stereochemistry of PG plays a crucial role in daptomycin-membrane interactions.     

A substrate variant as a high-affinity,reversible inhibitor: insight from the X-ray structure of cilastatin bound to membrane dipeptidase

An analysis of the X-ray structure of cilastatin bound to membrane dipeptidase, together with docking studies, is presented here to reveal how a simple amide may act as a high-affinity, reversible, amidase inhibitor. Cilastatin binds as a normal substrate and is orientated in a perfect near-attack conformer for formation of a tetrahedral intermediate with the zinc-bound water/hydroxide. This intermediate is fated, however, only to revert to its starting components as scission of the amide bond is prevented by the precise fit of cilastatin within the active site. The cilastatin alkyl end groups that are tightly buttressed against amino acid residues on opposite sides of the active site, are aligned along the C-N reaction coordinate axis thereby preventing collapse of the intermediate via rupture of the C-N bond. Such a feature could have more general applicability in the explicit design of substrate variants as selective, tight-binding, and reversible inhibitors.     

Enzymatic synthesis of arginine-based cationic surfactants

A novel enzymatic approach for the synthesis of arginine N-alkyl amide and ester derivatives is reported. Papain deposited onto solid support materials was used as catalyst for the amide and ester bond formation between Z-Arg-OMe and various long-chain alkyl amines and alcohols (H2N-Cn2, HO-Cn; n = 8-16) in organic media. Changes in enzymatic activity and product yield were studied for the following variables: organic solvent, aqueous buffer content, support for the enzyme deposition, presence of additives, enzyme loading, substrate concentration, and reaction temperature. The best yields (81-89%) of arginine N-alkyl amide derivatives were obtained at 25 degrees C in acetonitrile with an aqueous buffer content ranging from 0 to 1% (v/v) depending on the substrate concentration. The synthesis of arginine alkyl ester derivatives was carried out in solvent-free systems at 50 or 65 degrees C depending on the fatty alcohol chain length. In this case, product yields ranging from 86 to 89% were obtained with a molar ratio Z-Arg-OMe/fatty alcohol of 0.01. Papain deposited onto polyamide gave, in all cases, both the highest enzymatic activities and yields. Under the best reaction conditions the syntheses were scaled up to the production of 2 g of final product. The overall yields, which include reaction, Nalpha-benzyloxycarbonyl group (Z) deprotection and purification, varied from 53 to 77% of pure (99.9% by HPLC) product.