Synthesis and in vitro antibiotic activity of 16-membered 9-O-arylalkyloxime macrolides

A series of novel 9-O-arylalkyloxime analogs based on three different 16-membered macrolide scaffolds-5-O-mycaminosyltylonolide (OMT), tilmicosin, and 20-deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT-was synthesized. In vitro antibiotic activities were assayed against Gram-positive Streptococcus pneumoniae and Staphylococcus aureus and Gram-negative Haemophilus influenzae bacterial strains. Analogs derived from OMT (3-15) showed similar or better antibacterial activities against macrolide-susceptible strains and enhanced activities against macrolide-resistant strains compared with erythromycin A, tylosin, or OMT. Similar results were observed for tilmicosin 9-O-arylalkyloxime analogs (18-24). In contrast, most of the 20-deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT analogs (25-33) showed reduced antibacterial activities compared with OMT. Ribosome-binding studies were performed on compounds 12 (OMT derivative), 20 (tilmicosin derivative), and 29 [20-deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT derivative]. It was found that these compounds interacted with both domain V and domain II of the Escherichia coli 23S rRNA.     

Recognition of human telomeric G‐quadruplex DNA by berberine analogs: effect of substitution at the 9 and 13 positions of the isoquinoline moiety

G‐quadruplex forming sequences are widely distributed in human genome and serve as novel targets for regulating gene expression and chromosomal maintenance. They offer unique targets for anticancer drug development. Here, the interaction of berberine (BC) and two of its analogs bearing substitution at 9 and 13‐position with human telomeric G‐quadruplex DNA sequence has been investigated by biophysical techniques. Both the analogs exhibited several‐fold higher binding affinity than berberine. The Scatchard binding isotherms revealed non‐cooperative binding. 9‐ω‐amino hexyl ether analog (BC1) showed highest affinity (1.8 × 10⁶ M^?1) while the affinity of the 13‐phenylpropyl analog (BC2) was 1.09 × 10⁶ M^?1. Comparative fluorescence quenching and polarization anisotropy of the emission spectra gave evidence for a stronger stacking interaction of the analogs compared to berberine. The thiazole orange displacement assay has clearly established that the analogs were more effective in displacing the end stacked dye in comparison to berberine. However, the binding of the analogs did not induce any major structural perturbation in the G‐quadruplex structure, but led to higher thermal stability. Energetics of the binding indicated that the association of the analogs was exothermic and predominantly entropy driven phenomenon. Increasing the temperature resulted in weaker binding; the enthalpic contribution increased and the entropic contribution decreased. A small negative heat capacity change with significant enthalpy–entropy compensation established the involvement of multiple weak noncovalent interactions in the binding process. The 9‐ω‐amino hexyl ether analog stabilized the G‐quadruplex structure better than the 13‐phenyl alkyl analog. Copyright © 2015 John Wiley & Sons, Ltd.     

9-(Adenylyl)alkylcobalamins as inhibitors of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes

The behavior of two coenzyme analogs, [(5-aden-9-yl)methoxyethyl] cob (III) alamin and [(5-aden-9-yl)pentyl] cob (III) alamin modified at the nucleoside ligand sugar moiety was studied in the system of adenosyl-cobalamin-dependent glycerol dehydratase from Aerobacter aerogenes. It was shown that neither of the analogs possesses coenzyme properties and that both are strong competitive inhibitors for adenosylcobalamin (AdoCbl). The affinity of the two analogs for the apoenzyme is higher than that of AdoCbl. The data obtained are indicative of the essential role of the ribofuranoside fragment of AdoCbl in the manifestation of the coenzyme activity. The apoenzyme interaction with the analogs under study is discussed in terms of the Dreiding stereomodels for AdoCbl and its analogs.     

Novel non-classical C9-methyl-5-substituted-2,4-diaminopyrrolo[2,3-d]pyrimidines as potential inhibitors of dihydrofolate reductase and as anti-opportunistic agents

Six novel C9-methyl-5-substituted-2,4-diaminopyrrolo[2,3-d]pyrimidines 18-23 were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) and as anti-opportunistic agents. These compounds represent the only examples of 9-methyl substitution in the carbon-carbon bridge of 2,4-diaminopyrrolo[2,3-d]pyrimidines. The analogs 18-23 were synthesized in a concise eight-step procedure starting from the appropriate commercially available aromatic methyl ketones. The key step involved a Michael addition reaction of 2,4,6-triaminopyrimidine to the appropriate 1-nitroalkene, followed by ring closure of the nitro adducts via a Nef reaction. The compounds were evaluated as inhibitors of DHFR from Pneumocystis carinii (pc), Toxoplasma gondii (tg), Mycobacterium avium (ma) and rat liver (rl). The biological result indicated that some of these analogs are potent inhibitors of DHFR and some have selectivity for pathogen DHFR. Compound 23 was a two digit nanomolar inhibitor of tgDHFR with 9.6-fold selectivity for tgDHFR.     

11, 15, 19-trihydroxy-9-ketoprost-13-enoic acid and 11, 15, 19-trihydroxy-9-ketoprosta-5, 13-dienoic acid in human seminal fluid.

Two novel prostaglandins (PG) have been found in human seminal fluid which had been frozen immediately after collection. They were characterized by combined gas-liquid chromatography-mass spectrometry of various derivatives as 19-hydroxy prostaglandin E1 (11, 15, 19-trihydroxy-9-ketoprost-13-enoic acid) and 19-hydroxy prostaglandin E2 (11, 15, 19-trihydroxy-9-ketoprosta-5, 13-dienoic acid). They were present in three to five times the quantity of prostaglandins E1 and E2. Incubation of seminal fluid for 3 hr at 25 degrees C reduced levels of 190H-PGEs2.5-fold and PGE22-fold, while increasing levels of PGAs and PGBs 2-fold. No 190H PGA or 190H PGB was detected in extracts of unincubated fluid. The PGAs, PGBs and their 19-hydroxy analogs are probably artifacts arising metabolically or as a result of classical isolation techniques.     

9-Aminoacridine blocks NMDA and AMPA receptors by different mechanisms

Tricyclic mono- and dicationic compounds (derivatives of 9-aminoacridine) antagonize AMPA and NMDA glutamate receptors. The aim of the present study was to compare mechanisms of the 9-aminoacridine action on AMPA and NMDA receptors. Experiments were carried out by whole-cell patch-clamp technique on native receptors from rat brain neurons. An important peculiarity of the 9-aminoacridine action on NMDA receptors is the large slope of the concentration dependence, which suggests the binding of two molecules in the channel. AMPA receptors blockade also demonstrated interesting features. In contrast to the NMDA receptor channel block, inhibition of AMPA receptors is voltage-independent. 9-Aminoacridine and its dicationic analog demonstrated similar anti-AMPA activity. For classical AMPA-receptor channel blockers (derivatives of adamantane and phenylcyclohexyl) it was demonstrated that dicationic analogs are much more potent than monocationic analogs. We conclude that 9-aminoacridine binds to a specific site in AMPA receptors. This finding opens a possibility to develop a new family of non-competitive antagonists of AMPA receptors.     

Computational analysis of the receptor binding specificity of novel influenza A/H7N9 viruses

Background

Influenza viruses are undergoing continuous and rapid evolution. The fatal influenza A/H7N9 has drawn attention since the first wave of infections in March 2013, and raised more grave concerns with its increased potential to spread among humans. Experimental studies have revealed several host and virulence markers, indicating differential host binding preferences which can help estimate the potential of causing a pandemic. Here we systematically investigate the sequence pattern and structural characteristics of novel influenza A/H7N9 using computational approaches.

Results

The sequence analysis highlighted mutations in protein functional domains of influenza viruses. Molecular docking and molecular dynamics simulation revealed that the hemagglutinin (HA) of A/Taiwan/1/2017(H7N9) strain enhanced the binding with both avian and human receptor analogs, compared with the previous A/Shanghai/02/2013(H7N9) strain. The Molecular Mechanics - Poisson Boltzmann Surface Area (MM-PBSA) calculation revealed the change of residue-ligand interaction energy and detected the residues with conspicuous binding preference.

Conclusion

The results are novel and specific to the emerging influenza A/Taiwan/1/2017(H7N9) strain compared with A/Shanghai/02/2013(H7N9). Its enhanced ability to bind human receptor analogs, which are abundant in the human upper respiratory tract, may be responsible for the recent outbreak. Residues showing binding preference were detected, which could facilitate monitoring the circulating influenza viruses.

     

Inhibition of RNA polymerase and formyltetrahydrofolate synthetase activity by 6-chloro-8-aza-9-cyclopentylpurine. Structure-activity relationships

The structural requirements for inhibition of bacterial RNA polymerase and rabbit liver formyltetrahydrofolate synthetase activity by a series of purine nucleoside analogs related to 6-chloro-8-aza-9-cyclopentylpurine (689) were investigated. To achieve an inhibitory effect, preincubation of the enzyme preparations with the purine analogs, prior to assay of enzyme activity, was required. The greatest inhibition was produced by analogs containing all three alterations of the purine nucleoside structure: the 6-halo, 8-aza, and 9-cyclopentyl groups. It is suggested that 689 inhibits the activity of enzymes involved in nucleic acid synthesis by a site-directed alkylation.     

Activation of collagen IV gene expression in F9 teratocarcinoma cells by 3-deazaadenosine analogs. Indirect inhibitors of methylation.

3-Deazaadenosine analogs can function as inhibitors and also as alternative substrates of S-adenosylhomocysteine (AdoHcy) hydrolase. In cells treated with the analogs, AdoHcy invariably accumulates, leading to inhibition of cellular methylation. F9 teratocarcinoma cells, stably transfected with two collagen (IV) promoter-enhancer-CAT constructs and treated with 10 microM 3-deazaadenosine, 3-deaza-(+-)-aristeromycin or 3-deazaneplanocin, showed a strong induction of CAT activities without affecting differentiation. In comparison, the same 3-deaza analogs did not affect the CAT activity in F9 cells transfected with the beta-actin promoter-CAT construct. Furthermore, Northern blot analysis of endogenous mRNA from wild-type F9 cells treated with the 3-deaza nucleosides all showed an induction of the collagen alpha 1(IV) chain mRNA. Thus, the 3-deaza analogs most likely affect DNA methylation because their results are consistent with the previous observation that the integrated collagen alpha 1(IV) promoter-enhancer constructs were activated with 5-azacytidine.     

Ion-binding and pharmacological properties of Tyr6 and Tyr9 antamanide analogs.

In order to investigate the antiproliferative properties of antamanide, we have synthesized and studied two antamanide analogs where the phenylalanine residue in positions 6 or 9 is substituted by tyrosine, their corresponding linear forms and the cyclic and linear des Phe5,Phe6-Tyr9-analogs. Antamanide and its biologically active synthetic analogs are able to form highly stable complexes with metal ions, particularly Na+, K+ and Ca2+. We studied the ion-binding properties of the Tyr-antamanide analogs by CD and Tb3+ -mediated fluorescence in acetonitrile. In this medium the far-and near-UV CD spectra of the neat Tyr6-antamanide analog are very similar to that of the parent cyclic decapeptide. Substantial differences occur on the contrary in the CD spectra of the neat Tyr9-antamanide, particularly in the regions at 220 nm and 270-290 nm. In acetonitrile, as already found for antamanide, the interaction with the above-mentioned metal ions always produces evident changes in the far- and near-UV CD spectra of both analogs. On the contrary, the CD spectra of the linear deca- and octa- and of the cyclic octa-analogs are affected by the presence of metal ions only in the near-UV region. In the same solvent the Tb3+ -mediated fluorescence spectra of all the synthetic peptides are remarkably affected by the addition of ions. On the basis of the spectral total changes, by using either or both the spectroscopic techniques, it has been possible to determine the ion binding constants for all the linear and cyclic Tyr-antamanide analogs and to compare them with that of the parent peptide. The antitoxic and antiproliferative activities of these antamanide analogs have been tentatively correlated to their ion-binding properties. A preliminary account of this work was given in (1).     

Binding of the 9-O-N-aryl/arylalkyl Amino Carbonyl Methyl Substituted Berberine Analogs to tRNAphe

Background

Three new analogs of berberine with aryl/arylalkyl amino carbonyl methyl substituent at the 9-position of the isoquinoline chromophore along with berberrubine were studied for their binding to tRNA^phe by wide variety of biophysical techniques like spectrophotometry, spectrofluorimetry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry.

Methodology/Principal Findings

Scatchard binding isotherms revealed that the cooperative binding mode of berberine was propagated in the analogs also. Thermal melting studies showed that all the 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs stabilized the tRNA^phe more in comparison to berberine. Circular dichroism studies showed that these analogs perturbed the structure of tRNA^phe more in comparison to berberine. Ferrocyanide quenching studies and viscosity results proved the intercalative binding mode of these analogs into the helical organization of tRNA^phe. The binding was entropy driven for the analogs in sharp contrast to the enthalpy driven binding of berberine. The introduction of the aryl/arylalkyl amino carbonyl methyl substituent at the 9-position thus switched the enthalpy driven binding of berberine to entropy dominated binding. Salt and temperature dependent calorimetric studies established the involvement of multiple weak noncovalent interactions in the binding process.

Conclusions/Significance

The results showed that 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs exhibited almost ten folds higher binding affinity to tRNA^phe compared to berberine whereas the binding of berberrubine was dramatically reduced by about twenty fold in comparison to berberine. The spacer length of the substitution at the 9-position of the isoquinoline chromophore appears to be critical in modulating the binding affinities towards tRNA^phe.     

The effects of delta 9-tetrahydrocannabinol and other cannabinoids on cAMP accumulation in synaptosomes.

The effects of delta 9-THC and other cannabinoids on cAMP levels in synaptosomes from mouse brains were investigated in order to determine whether cannabinoids produced their behavioral effects through alterations in adenylate cyclase. delta 9-THC (0.01-10 microM) did not significantly alter basal cAMP levels, whereas delta 9-THC and other cannabinoids were able to alter forskolin-stimulated cAMP levels in synaptosomes. In general, three kinds of responses were observed. Some cannabinoids displayed a modest, concentration-dependent decrease in cAMP levels, producing significant inhibition between 1-10 microM. Other cannabinoids, including delta 9-THC and delta 8-THC, appeared to produce a biphasic effect in that inhibition of cAMP was observed only at a single concentration. Finally, some analogs were unable to significantly alter forskolin-stimulated cAMP. There was not a clear relationship between the ability of the cannabinoids to alter cAMP levels in synaptosomes and the behavioral effects observed in mice. However, it was demonstrated that the analogs which are the most potent in producing cannabimimetic effects in mice were the analogs which inhibited cAMP in a concentration-dependent manner. While cannabinoids were able to alter cAMP levels in synaptosomes, the ability to alter cAMP levels does not appear to be absolutely necessary for the production of cannabinoid effects in mice.     

Potent and prolonged melanotropic activities of the alpha-MSH fragment analog, Ac-[Nle4,D-Phe7]-alpha-MSH4-9-NH2

Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 and Ac-[Nle4]-alpha-MSH4-9-NH2, fragment analogs of the tridecapeptide, alpha-melanocyte stimulating hormone (alpha-MSH, alpha-melanotropin), were synthesized. The potency and prolonged activity of the analogs were compared to alpha-MSH in several melanotropin bioassays. The D-Phe-containing hexapeptide was 10 times more active than alpha-MSH in stimulating melanoma tyrosinase activity. This analog was also 10-fold more potent than alpha-MSH in the lizard skin bioassay and about 10-fold less active in the frog skin bioassay. The melanotropic activity of Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 was considerably prolonged compared to alpha-MSH in each of the bioassays. These results demonstrate that the structural requirements for superpotency and prolonged activity of [Nle4, D-Phe7]-substituted analogs reside within this hexapeptide sequence.     

Synthesis and Biological Assays of 9‐(Acylamino)homocamptothecins as DNA Topoisomerase I Inhibitors

In an effort to improve the stability of homocamptothecin and reduce the toxicity, novel homocamptothecin analogs with acylamino groups at C(9) were designed and synthesized. The cytotoxic activities of all the synthetic compounds against three cancer cell lines were evaluated by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay, and irinotecan was used as reference compound. Compound 7c with a piperidinylacetamido group and 10a with phenylacetamido group at C(9) showed potent activities both in vitro and in vivo. In addition, they also revealed remarkable topoisomerase I inhibitions which were exhibited with well‐established bonds with amino acid residues Arg364 and Asp533 in the active pocket. On the basis of the biological activities, 7c and 10a would be potential candidates for further studies.     

Design and synthesis of novel azapeptide activators of apoptosis mediated by caspase-9 in cancer cells

A set of azapeptides was designed based on the Ala-Val-Pro-Ile peptide (derived from Smac protein) to activate caspase-9 and induce apoptosis in breast cancer cells. The diversity-oriented synthesis of the aza-peptides 5–9 was accomplished by alkylation of the aza-residue of aza-Gly-Pro dipeptide 15 using potassium tert-butoxide and a range of different alkyl halides. The resulting protected aza-dipeptide building blocks were then introduced into mimics 5–9 using standard coupling conditions. Biological evaluation of 5–9 was performed in MDA-MB-231 breast cancer cells, and indicated that the aza-Gly and aza-Phe analogs 5 and 7 were most efficient in inducing cell death by a caspase-9 mediated apoptotic pathway. Revealing a relationship between azabicycloalkanone and aza peptide mimics, novel AVPI mimics were synthesized which exhibit utility for studying structure–activity relationships to develop leads for activating apoptosis in cancer cells.     

Preparation of human Melanocortin-4 receptor agonist libraries: linear peptides X-Y-DPhe7-Arg8-Trp(or 2-Nal)9-Z-NH2

Two libraries of hMC4R agonists, X-Y-DPhe⁷-Arg⁸-2-Nal⁹-Z-NH₂ and X-Y-DPhe⁷-Arg⁸-Trp⁹-Z-NH₂, totaling 185 peptides were prepared using Irori radiofrequency tagging technology and Argonaut Quest 210 Synthesizer, where X stands for N-caps, Y for His⁶ surrogates and Z for Gly¹⁰ surrogates. As a result of this study, His-modified pentapeptides with Trp were found to be more hMC4R potent than the corresponding 2-Nal analogs, novel N-caps and Gly surrogates were identified and 19 new peptides which are potent hMC4R agonists (EC₅₀ 1–15 nM) and selective against hMC1R were discovered.     

Activity-dependent neurotrophic factor, ADNF, determines the structure characteristics of Colivelin, a fusion protein of ADNF9 and Humanin analog.

A 24-amino acid long peptide, Humanin, protects neurons from Alzheimer's disease (AD)-related cell toxicities at sub-nM-uM concentrations. Activity-dependent neurotrophic factor (ADNF) is a glia-derived neurotrophic peptide, which protects neurons from tetrodoxin treatment and AD-related and amyotrophic lateral sclerosis-related insults at fM concentrations. An attempt was made to further improve the activity of Humanin by fusing this peptide to ADNF9, a 9-amino acid long core peptide of the ADNF. This fusion resulted in a novel molecule, termed Colivelin, with the neuroprotective activity at fM range, which is approximately 10(3)-10(7) fold higher than the activity of Humanin and Humanin analogs and follows the activity profile of fM-active ADNF9. We have characterized the structural properties of Colivelin and compared with those of ADNF9 and Humanin in water and phosphate-buffered saline (PBS). The secondary structure of Colivelin was similar to that of ADNF9, but not that of Humanin, and hence was not the average of the contributions of the two peptides fused. Colivelin was stable and monomeric in PBS, consistent with the monomeric property of ADNF9, while Humanin showed strong tendency to self-associate. Thus, it is evident that the structural properties of Colivelin resemble those of ADNF9, rather than those of Humanin.     

Synthesis and evaluation of 17α-triazolyl and 9α-cyano derivatives of estradiol

A variety of 17α-triazolyl and 9α-cyano derivatives of estradiol were prepared and evaluated for binding to human ERβ in both a TR-FRET assay, as well as ERβ and ERα agonism in cell-based functional assays. 9α-Cyanoestradiol (5) was nearly equipotent as estradiol as an agonist for both ERβ and ERα. The potency of the 17α-triazolylestradiol analogs is considerably more variable and depends on the nature of the 4-substituent of the triazole ring. While rigid protein docking simulations exhibited significant steric clashing, induced fit docking providing more protein flexibility revealed that the triazole linker of analogs 2d and 2e extends outside of the traditional ligand binding domain with the benzene ring located in the loop connecting helix 11 to helix 12.     

Structure-function studies of five natural, including catfish and dogfish, gonadotropin-releasing hormones and eight analogs on reproduction in Thai catfish (Clarias macrocephalus).

Five distinct forms of gonadotropin-releasing hormone (GnRH) and their analogs, six of which are newly designed, were used to study reproduction in Thai catfish, Clarias macrocephalus. Determination was made for the percentage of fish that ovulated within 16-18 h; the percentage of eggs fertilized; and the percentage of larva that hatched and survived for 7 days. The results show, firstly, that natural chicken GnRH-II, which is identical with catfish GnRH-II, was significantly more effective at a dose of 300 micrograms/kg than the control injection for the induction of ovulation. Dogfish GnRH at the same dose was also significantly more effective than the control, but was not significantly different from chicken (catfish) GnRH-II for ovulation induction. The novel catfish GnRH-I, mammalian GnRH and salmon GnRH were not effective at 100, 150 or 300 micrograms/kg in Thai catfish. Secondly, 5 of 8 analogs of GnRH at a dose of 20 micrograms/kg resulted in a significantly higher percentage of ovulating fish compared with the control fish. Among these five analogs, the most effective were the two analog forms of chicken GnRH-II (D-Arg6,Pro9 NEt and D-Nal6,Pro9 NEt), followed by the salmon GnRH analog (D-Arg6,Pro9 NEt), a dogfish GnRH analog (D-Arg6,Pro9 NEt) and the mammalian GnRH analog (D-Ala6,Pro9 NEt). Not significantly different from the controls were the two catfish GnRH-I analogs and one of the dogfish (D-Nal6,Pro9 NEt) analogs. The six new analogs had not been previously tested in any animal. Thirdly, the number of fish ovulating was the same whether GnRH was administered in one or two injections.