CRAMP analogues having potent antibiotic activity against bacterial, fungal, and tumor cells without hemolytic activity

CRAMP-18 (GEKLKKIGQKIKNFFQKL) is the antibacterial sequence derived from CRMAP, a member of cathelicidin-derived antimicrobial peptides. To develop the novel antibiotic peptides useful as therapeutic drugs requires strong antibiotic activity against bacterial and fungal cells without hemolytic effect. To this goal, the analogues were designed to increase only net positively charge by Lys-substitution of positions 2, 9, 13, or 16 at the hydrophilic helix face of CRAMP-18 without any change at the hydrophobic helix face. In particular, Lys-substitution (K(2)-CRAMP-18) of position 2 in CRAMP-18 induced the enhanced antibiotic activity without any increase in hemolysis. Thus, this peptide may provide a useful template for the design novel antibiotic peptides for the treatment of infectious diseases. Additional CD spectra studies suggested that the alpha-helical structure of the peptides plays an important role in killing bacterial and fungal cells, but the increase of alpha-helical content is less connected with the enhanced antibiotic activity.     

Synthesis and antitumor activity of goniofufurone analogues.

Synthesis and antitumor activity of goniofufurone analogues 15, 16, 17, 33, and 46 is reported. Key step in the synthesis is Pd (II) mediated oxidative cyclisation of vinyl-(hydroxy) furans 18, 19 to the corresponding lactols 32, 43. Cytotoxicities of 15, 16, 17, 33, and 46 tested against six human cancer cell lines are reported. Change of stereochemistry at C-5, C-6 and C-7 position of goniofufurone (1) did not enhance the cytotoxicities significantly.     

Leishmanicidal activity of combretastatin analogues and heteroanalogues.

We have investigated the in vitro leishmanicidal activity of representative members from series II-V of combretastatin analogues and heteroanalogues. Most of them exhibited different degrees of activity against various strains of Leishmania spp. The diaryl(heteroaryl)ethane system or the more complex fused heterocyclic stilbenoids, constitute useful skeletal bases to support some kind of antiparasitic activity. Particularly, the incorporation of 2-furyl substituents led to potent antileishmanial compounds, which have been selected for in vivo testing on murine models.     

Main-chain length control of conformation, membrane activity, and antibiotic properties of lipopeptaibol sequential analogues

To investigate the effect of backbone length and amphiphilicity on the 3D structure, membrane permeability, and antibacterial properties of trichogins, a subclass of lipopeptaibols, we prepared, by the segment condensation approach in solution and chemically characterized, a set of N(alpha)-1-octanoylated -X-(GLUG)(n)-I-L- ( X=G or U where U=Aib; n=1-4) sequential peptide esters. In parallel, the 12-mer (UGGL)(3) aneurism peptide, an analogue of the 11-mer sequential peptide (n=2) with an amino acid insertion was also synthesized and studied. By FT-IR absorption technique, we clearly showed that, in CDCl(3) solution, all peptides essentially populate intramolecularly H-bonded, helical conformations. Moreover, CD spectroscopy indicates that all peptides, with the single exception of the shortest oligomer (the heptamer), adopt mixed 3(10)-/alpha-helical structures, to an extent approximately correlating with main-chain length, in MeOH solution and sodium dodecylsulfate (SDS) micelles. Significant membrane permeability properties were found only for the three longest GLUG-based peptides, with the 15-mer oligomer (n=4) resulting the most active. The lack of activity exhibited by the aneurism peptide in this experiment strongly suggests a relevant role for the sequence amphiphilicity. In addition, antibacterial activity and selectivity were highlighted and demonstrated to be dependent on peptide main-chain length and amphiphilicity, in the sense that the two shortest GLUG-based homologues are active against Gram-positive strains, whereas the two longest homologues are able to penetrate the membranes of the Gram-negative strains, and the UGGL-based aneurism peptide is inactive.     

Mixed lineage kinase activity of indolocarbazole analogues.

The MLK1-3 activity for a series of analogues of the indolocarbazole K-252a is reported. Addition of 3,9-bis-alkylthiomethyl groups to K-252a results in potent and selective MLK inhibitors. The in vitro and in vivo survival promoting activity of bis-isopropylthiomethyl-K-252a (16, CEP-11004/KT-8138) is reported.     

Preparation of coenzyme M analogues and their activity in the methyl coenzyme M reductase system of Methanobacterium thermoautotrophicum.

A number of 2-(methylthio)ethanesulfonate (methyl-coenzyme M) analogues were synthesized and investigated as substrates for methyl-coenzyme M reductase, an enzyme system found in extracts of Methanobacterterium thermoautotrophicum. Replacement of the methyl moiety by an ethyl group yielded an analogue which served as a precursor for ethane formation. Propyl-coenzyme M, however, was not converted to propane. Analogues which contained additional methylene carbons such as 3-(methylthio)propanesulfonate or 4-(methylthio)butanesulfonate or analogues modified at the sulfide or sulfonate position, N-methyltaurine and 2-(methylthio)ethanol, were inactive. These analogues, in addition to a number of commercially available compounds, also were tested for their ability to inhibit the reduction of methyl-coenzyme M to methane. Bromoethanesulfonate and chloroethanesulfonate proved to be potent inhibitors of the reductase, resulting in 50% inhibition at 7.9 X 10(6) M and 7.5 X 10(5) M. Analogues to coenzyme M which contained modifications to other regions were evaluated also and found to be weak inhibitors of methane biosynthesis.     

The synthesis and biological activity of alkyloxy prostaglandin analogues.

Prostaglandin analogues in which the alkyl chain attached to C-15 in the natural compounds is replaced by an alkyoxyalkyl group have been synthesised. Compounds of the 17-oxa series are particularly potent luteolytic agents and are selective in the sense that they are less effective than PGE-2alpha in causing isotonic contractions of isolated uterus muscle.     

Synthesis and anti-HSV-1 activity of quinolonic acyclovir analogues.

Several 1-[(2-hydroxy-ethoxy)methyl]-3-carbethoxy-4(1H)quinolones (2a-l) and l-[(2-hydroxy-ethoxy)methyl]-4(1H)quinolone-3-carboxylic acids (3a-j and 3l) were synthesized and 2a-j, 2l and 3a-j, 3l were evaluated against herpes simplex virus type 1 (HSV-1), employing a one-pot reaction: silylation of the desired quinolone (BSTFA 1% TMCS) followed by equimolar amount addition of 1,3-dioxolane, chlorotrimethylsilane and KI, at room temperature. The acyclonucleosides 2a-l were obtained in 40-77% yields. The esters 2a-j and 2l were subsequently converted into the corresponding hydroxyacids 3 in 40-70% yields. Attempts of hydrolysis of 2k produced only a mixture of degradation products. Antiviral activity of 2 and 3 on HSV-1 virus infection was assessed by the virus yield assay. Except for compounds 2i and 3e, the acyclonucleosides were found to reduce the virus yield by 70-99% at the concentration of 50 microM, being the acids, in general, more effective inhibitors than their corresponding esters. Compounds 3j and 2d exhibited antiviral activity against HSV-1 virus with EC50 of 0.7+/-0.04 and 0.8+/-0.09 microM, respectively. Both compounds were not toxic towards the Vero cell line.     

The reversible DNA-alkylating activity of duocarmycin and its analogues.

Intact drugs with spirocyclopropylhexadienone moieties can be regenerated from the covalent DNA adducts induced by antitumor antibiotics duocarmycin (DUM) A, SA and some DUMA analogues in neutral aqueous solution. We detected the reversible nature of DUMs by determination of the antimicrobial activity and cytotoxicity of DUM-DNA adducts. All of the adducts selectively inhibited the growth of a sensitive strain of Bacillus but not that of the wild type strain, a property of parent DUM and its analogues. Most of the DNA adducts were also cytotoxic to HeLa S3. These results suggested that active drugs can be released from their covalent DNA adducts under these biological assay conditions. Regeneration of intact drugs was quantitatively analyzed by HPLC and the amount of free drug released from DNA adducts revealed that the rate and efficiency of this reversal were dependent on structural variables among the drugs. The differences in rates of reversibility were correlated with the biological activity of DUMs. The effect of pH, temperature and salt concentration on the regeneration of drugs from their DNA adducts suggest a catalytic role of double-helical DNA on the reversal pathway.     

Synthesis and activity of 5'-uridinyl dipeptide analogues mimicking the amino terminal peptide chain of nucleoside antibiotic mureidomycin A

A series of 5'-uridinyl dipeptides were synthesised which mimic the amino terminal chain of nucleoside antibiotic mureido omycin A. Aminoacyl-beta-alanyl- and aminoacyl-N-methyl-beta-alanyl- dipeptides were attached either via an ester linkage to the 5'-hydroxyl of uridine, or via an amide linkage to 5'-amino-5'-deoxyuridine. The most active inhibitor of Escherichia coli phospho-MurNAc-pentapeptide translocase (MraY) was 5'-O-(L-Ala-N-methyl-beta-alanyl)-uridine (13l), which also showed 97% enzyme inhibition at 2.35 mM concentration, and showed antibacterial activity at 100 microg/mL concentration against Pseudomonas putida. Both the central N-methyl amide linkage and a 5' uridine ester linkage were required for highest biological activity. Enzyme inhibition was shown to be competitive with Mg(2+). It is proposed that the primary amino terminus of the inhibitor binds in place of the Mg(2+) cofactor at the MraY active site, positioned via a cis-N-methyl amide linkage.     

A solid-phase approach to analogues of the antibiotic mureidomycin

A library of 80 analogues of the antibacterial compound mureidomycin was prepared using solid-phase chemistry techniques.     

New indolicidin analogues with potent antibacterial activity.

Indolicidin is a 13-residue antimicrobial peptide amide, ILPWKWPWWPWRR-NH2, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two-fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR-NH2 in which Trp-residues 4,6,8,9,11 were replaced in all positions with X = a single non-natural building block; N-substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non-natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N-terminus with a hydrophobic non-natural building block. We prepared 22 analogues of indolicidin and [Phe(4,6,8,9,11)] indolicidin, 11 of each, carrying a hydrophobic non-natural building block attached to the N-terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non-natural building blocks, are promising lead structures for developing future therapeutics.     

1-Aza-sugars from D-glucose. Preparation of 1-deoxy-5-dehydroxymethyl-nojirimycin, its analogues and evaluation of glycosidase inhibitory activity

D-glucose derived pentodialdoses 11a-c on reduction followed by tosylation, azide displacement, hydrogenation and protection with -Cbz group gave N-Cbz protected compounds 14a-c, respectively, which on removal of 1,2-acetonide functionality and hydrogenation afforded corresponding 1-aza-sugars 3, 9 and 10 in good overall yields. The glycosidase inhibition activity of these 1-aza-sugars was tested with sweet almond as a rich source of different glycosidases.     

Synthesis and cytotoxicity of analogues of the antibiotic BE 10988 inhibitors of DNA topoisomerase II.

Indolequinone derivatives of the antitumour antibiotic BE 10988 were synthesized and evaluated for their cytotoxicity and action mechanism. The quinone system is essential to biological activity and the thiazole ring plays a major role in the poisoning of topoisomerase II.     

Linear and cyclic beta-casomorphin analogues with high analgesic activity.

We investigated the antinociceptive efficacy of casomorphin (CM) derivatives using the vocalization test. Male Wistar rats received chronic microcannulae into the right lateral ventricle. One week later we examined the analgesic effect of CM derivatives 10, 30, 60, and 90 min after intraventricular injection (5 microliters). The analgesic effect was calculated as the individual percent increase in the pain threshold and was compared to controls (saline treatment). The substitution of D-lysine and D-ornithine in position 2 in connection with a cyclization through ring closure of the 2 position side chain amino group to the C-terminal glycine-COOH group resulted in high analgesic potency. The substitution of D-Pro4 was without any effect in the ineffective linear derivatives and decreased the effectiveness in the highly effective cyclic derivatives. The cyclic [D-Orn2]CM-5 and the cyclic [D-Lys2]CM-5 are the CM derivatives with the highest antinociceptive activity. The cyclic [D-Orn2]CM-5 is greater than 1000 times more effective than morphine. We conclude, on the basis of studies of receptor binding and in vitro investigations, that mu receptor activity alone is not responsible for the analgesic activity. The delta receptor and possibly also the kappa receptor could modulate the nociceptive effectiveness.