Synthesis and properties of chromophores of fluorescent proteins

We describe the existing approaches to the synthesis of 5-arylidene-3,5-dihydro-4 H-imidazol-4-ones—model chromophores of fluorescent proteins and their nonnatural analogs. We discuss in detail the chemical (acid-base and redox reactions, cis-trans isomery, etc.) and spectral properties of the chromophores and the influence of substitutes and the environment. The study of synthetic chromophores allows for modeling of the photophysical characteristics of fluorescent proteins.     

Synthesis of biosynthetic precursors of chromophores of red fluorescent proteins

A method for the synthesis of 5-arylidene-3,5-dihydro-4H-imidazol-4-ones corresponding to the chromophore of the green fluorescent protein (GFP) with acylaminoalkyl substituents at position 2 of the imidazolone core has been developed. These biomimetic model compounds are the precursors of the chromophores of red fluorescent proteins. The method is based on the masking of the dehydrotyrosine fragment of target compounds by the β-hydroxytyrosine moiety. The key stages of the synthesis include the condensa-tion of β-hydroxytyrosine with the appropriate N-acetylamino acid, the unmasking of dehydrotyrosine by O-acylation with subsequent elimination, and the cyclization of the resulting 3-acylaminocinnamic acid derivatives in basic medium.     

Optimization of diarylpentadienones as chemotherapeutics for prostate cancer

Our earlier studies indicate that (1E,4E)-1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones and (1E,4E)-1,5-bis(1-alkyl-1H-benzo[d]imidazol-2-yl)penta-1,4-diene-3-ones exhibit up to 121-fold greater antiproliferative potency than curcumin in human prostate cancer cell models, but only 2–10 fold increase in mouse plasma concentrations. The present study aims to further optimize them as anti-prostate cancer agents with both good potency and bioavailability. (1E,4E)-1,5-Bis(1H-imidazol-2-yl)penta-1,4-diene-3-one, the potential metabolic product of (1E,4E)-1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones, was synthesized and evaluated for its anti-proliferative activity. The promising potency of 1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones was completely abolished by removing the 1-alkyl group, suggesting the critical role of an appropriate group on the N1 position. We then envisioned that N-aryl substitution to exclude the C–H bond on the carbon adjacent to the N1 position (α-H) may increase the metabolic stability. Consequently, seven (1E,4E)-1,5-bis(1-aryl-1H-imidazol-2-yl)penta-1,4-dien-3-ones and three (1E,4E)-1,5-bis(1-aryl-1H-benzo[d]imidazol-2-yl)penta-1,4-dien-3-ones, as well as three (1E,4E)-1,5-bis(1-aryl-1H-pyrrolo[3,2-b]pyridine-2-yl)penta-1,4-dien-3-ones, were synthesized through a three-step transformation, including N-arylation via Ullmann condensation, formylation, and Horner-Wadsworth-Emmons reaction. Six optimal (1E,4E)-1,5-bis(1-aryl-1H-imidazol-2-yl)penta-1,4-dien-3-ones exhibit 24- to 375-fold improved potency as compared with curcumin. Replacement of the imidazole with bulkier benzoimidazole and 4-azaindole results in a substantial decrease in the potency. (1E,4E)-1,5-Bis(1-(2-methoxyphenyl)-1H-imidazol-2-yl)penta-1,4-dien-3-one (17d) was established as an optimal compound with both superior potency and good bioavailability that is sufficient to provide the therapeutic efficacy necessary to suppress in vivo tumor growth.     

The Sonogashira reaction as a new method for the modification of borated analogues of the green fluorescence protein chromophore

The Sonogashira reaction was used for modifications of borated green fluorescence protein chromophore derivatives, 4-(2-(difluoroboryl)benzylidene)-1H-imidazol-5(4H)-ones, for the development of new fluorescent dyes. The derivatives bearing an acetylene fragment and a difluoroboryl group were obtained in high yields. The modification resulted in a significant bathochromic shift of the absorption and emission maxima and is a promising method for the development of new fluorescent dyes.     

Novel N-quinonyl amino acids and their transformation to 3-substituted p-isoxazinones

Summary. Quinonyl amino acids are building blocks in the preparation of peptides which target the quinonic drug to cancer damaged area. Novel N-(3-chloro-1,4-dihydro-1,4-dioxonaphthalen-2-yl)-α-amino acids 1a–f were prepared by direct substitution of 2,3-dichloro-1,4-naphthoquinone. The quinonic moiety was reduced by NaBH₄ to yield the corresponding hydroquinones 2a–f, which in acidic conditions underwent internal cyclization to yield the 3,4-dihydro-2H-naphth[1,2-b]-1,4-oxazine-2-ones (six-membered azlactones) 3a–f. Received February 2, 2000 Accepted March 29, 2000     

Facile synthesis of SSR180575 and discovery of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[18F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide,a potent pyridazinoindole ligand for PET imaging of TSPO in cancer

A novel synthesis of the translocator protein (TSPO) ligand 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (SSR180575, 3) was achieved in four steps from commercially available starting materials. Focused structure–activity relationship development about the pyridazinoindole ring at the N3 position led to the discovery of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (14), a novel ligand of comparable affinity. Radiolabeling with fluorine-18 (¹⁸F) yielded 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[¹⁸F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide ([¹⁸F]-14) in high radiochemical yield and specific activity. In vivo studies of [¹⁸F]-14 revealed this agent as a promising probe for molecular imaging of glioma.     

Fluorescent labeling of proteins. A new methodology

A new reagent, 2-methoxy-2,4-diphenyl-3(2$\text{H}$)-furanone (MDPF) has been utilized for the fluorescent labeling of proteins. MDPF, which is nonfluorescent, reacts with primary amino groups to form fluorescent $\text{N}$-substituted 3,5-diphenyl-5-hydroxy-2-pyrrolin-4-ones. Antibodies labeled with MDPF afford intense immunofluorescent staining.     

A systematic study of ligand intermolecular interactions in crystals of copper(II) complexes of bidentate guanidino derivatives

The synthesis and X-ray structures of copper(II) complexes of the bidentate ligands, N-(4-oxo-5,5-diphenyl-4,5-dihydro-1H-imidazol-2-yl)-N′-phenylguanidino, 2-uanidinobenzimidazolo and N-(4-oxo-3-phenyl-1,3-diazaspiro[4.4]non-1-en-2-yl)guanidino, are reported. These complexes, which possess potential doublet (DA) or triplet (DAD) hydrogen bonding motifs, can form supramolecular structures based on synthons involving hydrogen bonding or phenyl embraces. The changes in supramolecular structure resulting from small changes in ligand structure, as well as from the use of different solvents for their crystallisation, are examined. The structures adopted are compared with others reported previously for complexes of related ligands.     

1-(Arylsulfonyl)-2,3-dihydro-1H-quinolin-4-one derivatives as 5-HT(6) serotonin receptor ligands

Piperazinyl derivatives of 1-(arylsulfonyl)-2,3-dihydro-1H-quinolin-4-ones have been identified with high binding affinities for 5-HT₆ receptor. In particular, 2-methyl-5-(N-methyl-piperazin-1-yl)-1-(naphthalene-2-sulfonyl)-2,3-dihydro-1H-quinolin-4-one (8g) exhibits high binding affinity toward 5-HT₆ (IC₅₀ = 8 nM) receptor with good selectivity over other serotonin and dopamine receptors.     

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pH_in established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pH_out = 7.8, we found that pH_in ≈ 5.4-5.7 in the state of photosynthetic control, and pH_in ≈ 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8-2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H⁺/ATP is n ≥ 4-4.7.     

2-Butyl-4-chloroimidazole based substituted piperazine-thiosemicarbazone hybrids as potent inhibitors of Mycobacterium tuberculosis

Here a series of 2-butyl-4-chloroimidazole based substituted piperazine-thiosemicarbazone hybrids were designed by combining three different pharmacophoric fragments in single molecular architecture. 2-Butyl-4-chloro-1-(3-(4-substituted)piperazin-1-yl)propyl)-1H-imidazole-5-carbaldehydes (4a–p) prepared by reacting carboxaldehyde 2 with N-alkyl piperazines 3a–p which were condensed with thiosemicarbazine to give desired compounds 5a–p in very good yields. Among all sixteen compounds screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (MTB), two compounds (E)-2-((2-butyl-4-chloro-1-(3-(4-(o-tolyl) piperazin-1-yl)propyl)-1H-imidazol-5-yl)methylene)hydrazinecarbothioamide 5e and (E)-2-((2-butyl-4-chloro-1-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-1H-imidazol-5-yl)methylene) hydrazine carbothioamide 5f were found to be the most potent antitubercular agents (MIC: 3.13 μg/mL) with low toxicity profile.     

Synthesis and antimicrobial activity of novel 1,4,5-triphenyl-1<Emphasis Type="Italic">H</Emphasis>-imidazol-[1,2,3]-triazole derivatives

Novel 1-phenyl-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole derivatives were synthesized by click chemistry reaction and screened for antimicrobial activity against grampositive and gram-negative bacterial and fungal species. All the compounds were characterized by ¹H and ¹³C NMR, IR, and mass spectral data. The results of antibacterial study indicated that 1-(4-nitrophenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole, 1-(4-(4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)ethanone, 1-(2,6-dichloro-4-nitrophenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole, and 1-(2-methoxy-4-nitrophenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole showed appreciable antibacterial activity while 1-(4-fluorophenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy) methyl)-1H-1,2,3-triazole, 1-(2,6-dichloro-4-nitrophenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole, and 1-(4-methoxyphenyl)-4-((4-(1,4,5-triphenyl-1H-imidazol-2-yl)phenoxy)methyl)-1H-1,2,3-triazole emerged as the most potential antifungal agents.     

Amine substituted N-(1H-benzimidazol-2ylmethyl)-5,6,7,8-tetrahydro-8-quinolinamines as CXCR4 antagonists with potent activity against HIV-1

Several novel amine substituted N-(1H-benzimidazol-2ylmethyl)-5,6,7,8-tetrahydro-8-quinolinamines were synthesized which had potent activity against HIV-1. The synthetic approaches adopted allowed for variation of the substitution pattern and resulting changes in antiviral activity are highlighted. This led to the identification of compounds with low and sub-nanomolar anti-HIV-1 activity.     

Microwave assisted synthesis and in vitro cytotoxicities of substituted (Z)-2-amino-5-(1-benzyl-1H-indol-3-yl)methylene-1-methyl-1H-imidazol-4(5H)-ones against human tumor cell lines

The synthesis of several novel substituted (Z)-2-amino-5-(1-benzyl-1H-indol-3-yl)methylene-1-methyl-1H-imidazol-4(5H)-ones structurally related to aplysinopsin have been carried out under microwave irradiation and conventional heating methods. The analogs 3a, 3b, 3d–3g, 3k and 3l were evaluated for their in vitro cytotoxic activity against an NCI 60 human tumor cell line panel. Compound 3f exhibited good growth inhibitory properties against all but four of the human cancer cell lines examined, and afforded LC₅₀ values <10 μM for 30% of the cell lines in the panel. Compound 3e was an effective inhibitor of leukemia, CNS, melanoma, and breast cancer cell growth, but generally less effective as a cytotoxic agent. Thus, the aplysinopsin analog 3f was regarded as a useful lead compound for further structural optimization.     

Phosphodiesterase inhibitors. Part 6: Design,synthesis, and structure–activity relationships of PDE4-inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory activity

We previously identified KCA-1490 [(?)-6-(7-methoxy-2-trifluoromethyl-pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone], a dual PDE3/4 inhibitor. In the present study, we found highly potent selective PDE4 inhibitors derived from the structure of KCA-1490. Among them, N-(3,5-dichloropyridin-4-yl)-7-methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridine-4-carboxamide (2a) had good anti-inflammatory effects in an animal model.     

Site selectivity in reactions of hydrazonoyl halides with heterocycles containing amino and thione groups leading to fused heterocycles of potential antimicrobial activity

Reaction of hydrazonoyl halides with 6-(benzylidenamino)-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one and 2,3-diaminoquinazolin-4-one site-selectively afforded 3-substituted-7-(benzylidenamino)-1-phenyl-[1,2,4]triazolo[4,3-a]-pyrimidin-5(1H)-ones, [1,2,4,5]tetrazino[6,1-b]quinazolin-6(4H)-one, and 3-methyl-2-(4-substituted-phenylhydrazo)-[1,2,4]triazino[3,2-b]quinazolin-10-ones in good yields. The structures of the newly synthesized compounds were elucidated by chemical evidence and their IR, ¹H, ¹³C NMR, and MS spectra. Furthermore, some of the products were screened against different strains of bacteria and fungi.     

Design,synthesis and antimicrobial evaluation of novel 1-benzyl 2-butyl-4-chloroimidazole embodied 4-azafluorenones via molecular hybridization approach

A series of novel 1-benzyl-2-butyl-4-chloroimidazole embodied 4-azafluorenone hybrids, designed via molecular hybridization approach, were synthesized in very good yields using one pot condensation of 1-benzyl-2-butyl-4-chloroimidazole-5-carboxaldehyde, 1,3-indanedione, aryl/heteroaryl methyl ketones and ammonium acetate. All the synthetic derivatives were fully characterized by spectral data and evaluated for antimicrobial activity by disc diffusion method against selected bacteria and fungal strains. Among the 15 new compounds screened, 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(furan-2-yl)-5H-indeno[1,2-b]pyridin-5-one(10k) has pronounced activity with higher zone of inhibition (ZoI) against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Aspergillus flavus and Candida albicans. Also 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(dibenzo[b,d]thiophen-2-yl)-5H-indeno [1,2-b]pyridin-5-one (10n) and 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(3-tosyl-3H-inden-1-yl)-5H-indeno[1,2-b]pyridin-5-one (10o) showed selective higher inhibitory activity against Aspergillus flavus and Candida albicans. The results demonstrated potential importance of molecular hybridization in the development of 10k as potential antimicrobial agent.     

Design,synthesis, and structure–activity relationships of pyrazole derivatives as potential FabH inhibitors

Fatty acid biosynthesis is essential for bacterial survival. FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and -negative bacteria. Fifty-six 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole derivatives were synthesized and developed as potent inhibitors of FabH. This inhibitor class demonstrates strong antibacterial activity. Escherichia coli FabH inhibitory assay and docking simulation indicated that the compounds 1-(5-(4-fluorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (12) and 1-(5-(4-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (13) were potent inhibitors of E. coli FabH.     

Biodegradation of nicotine by a novel nicotine-degrading bacterium, Pseudomonas plecoglossicida TND35 and its new biotransformation intermediates

Tobacco wastes that contain nicotine alkaloids are harmful to human health and the environment. In the investigation, a novel nicotine-biodegrading bacterium TND35 was isolated and identified as Pseudomonas plecoglossicida on the basis of phenotypic, biochemical characteristics and 16S rRNA sequence homology. We have studied the nicotine biodegradation potential of strain TND35 by detecting the intermediate metabolites using an array of approaches such as HPLC, GC–MS, NMR and FT-IR. Biotransformation metabolites, N-methylmyosmine, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and other three new intermediate metabolites namely, 3,5-bis (1-methylpyrrolidin-2-yl) pyridine, 2,3-dihydro-1-methyl-5-(pyridin-3-yl)-1H-pyrrol-2-ol and 5-(pyridin-3-yl)-1H-pyrrol-2(3H)-one have been identified. Interestingly, these intermediate metabolites suggest that the strain TND35 employs a novel nicotine biodegradation pathway, which is different from the reported pathways of Aspergillus oryzae 112822, Arthrobacter nicotinovorans pAO1, Agrobacterium tumefaciens S33 and other species of Pseudomonas. The metabolite, HPB reported in this study can also be used as biochemical marker for tobacco related cancer studies.     

Discovery of novel hepatoselective HMG-CoA reductase inhibitors for treating hypercholesterolemia: a bench-to-bedside case study on tissue selective drug distribution

The design of drugs with selective tissue distribution can be an effective strategy for enhancing efficacy and safety, but understanding the translation of preclinical tissue distribution data to the clinic remains an important challenge. As part of a discovery program to identify next generation liver selective HMG-CoA reductase inhibitors we report the identification of (3R,5R)-7-(4-((3-fluorobenzyl)carbamoyl)-5-cyclopropyl-2-(4-fluorophenyl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoic acid (26) as a candidate for treating hypercholesterlemia. Clinical evaluation of 26 (PF-03491165), as well as the previously reported 2 (PF-03052334), provided an opportunity for a case study comparison of the preclinical and clinical pharmacokinetics as well as pharmacodynamics of tissue targeted HMG-CoA reductase inhibitors.