A quantum chemical study of the formation of 2-hydroperoxy-coelenterazine in the Ca<Superscript>2+</Superscript>-regulated photoprotein obelin

The Ca²⁺-regulated photoprotein obelin determines the luminescence of the marine hydroid Obelia longissima. Bioluminescence is initiated by calcium and appears as a result of the oxidative decarboxylation related to the coelenterazine substrate. The luciferase of the luminescent marine coral Renilla muelleri (RM) also uses coelenterazine as a substrate. However, three proteins are involved in the in vivo bioluminescence of these animals: luciferase, green fluorescent protein, and Ca²⁺-regulated coelenterazine-binding protein (CBP). In fact, CBP that contains one strongly bound coelenterazine molecule is the RM luciferase substrate in the in vivo bioluminescent reaction. Coelenterazine becomes available for oxygen and the reaction with luciferase only after binding CBP with calcium ions. Unlike Ca²⁺-regulated photoproteins, the coelenterazine molecule is not activated by oxygen in the CBP molecule. In this work, by means of quantum chemical methods the behavior of substrates in these proteins is analyzed. It is shown that coelenterazine can form different tautomers: CLZ(2H) and CLZ(7H). The formation of 2-hydroperoxy-coelenterazine is studied. According to the obtained data, these proteins use different forms of the substrates for the reaction. In obelin, the substrate is in the CLZ(2H) form that affords hydrogen peroxide. In RM, coelenterazine is in the CLZ(7H) form, and therefore, CBP is not activated by oxygen.     

Bioluminescent properties of obelin and aequorin with novel coelenterazine analogues

The main analytical use of Ca²⁺-regulated photoproteins from luminous coelenterates is for real-time non-invasive visualization of intracellular calcium concentration ([Ca²⁺]_i) dynamics in cells and whole organisms. A limitation of this approach for in vivo deep tissue imaging is the fact that blue light emitted by the photoprotein is highly absorbed by tissue. Seven novel coelenterazine analogues were synthesized and their effects on the bioluminescent properties of recombinant obelin from Obelia longissima and aequorin from Aequorea victoria were evaluated. Only analogues having electron-donating groups (m-OCH₃ and m-OH) on the C6 phenol moiety or an extended resonance system at the C8 position (1-naphthyl and α-styryl analogues) showed a significant red shift of light emission. Of these, only the α-styryl analogue displayed a sufficiently high light intensity to allow eventual tissue penetration. The possible suitability of this compound for in vivo assays was corroborated by studies with aequorin which allowed the monitoring of [Ca²⁺]_i dynamics in cultured CHO cells and in hippocampal brain slices. Thus, the α-styryl coelenterazine analogue might be potentially useful for non-invasive, in vivo bioluminescence imaging in deep tissues of small animals.     

Efficient synthesis and antioxidant activity of coelenterazine analogues

A convenient and highly convergent method for the synthesis of new imidazo[1,2-a]pyridine-based coelenterazine analogues is reported. The imidazo[1,2-a]pyridine core was constructed through a condensation between 2-aminopyridine analogues and arylglyoxals. Additionally, a new approach to the synthesis of benzylglyoxals was introduced. The imidazo[1,2-a]pyridines display moderate antioxidant activities at a low micromolar level in 2,2-diphenyl-1-picrylhydrazyl (DPPH).     

Purine analog inhibitors of xanthine oxidase - structure activity relationships and proposed binding of the molybdenum cofactor

A number of new hypoxanthine analogs have been prepared as substrate inhibitors of xanthine oxidase. Most noteworthy inhibitory new hypoxanthine analogs are 3-(m-tolyl)pyrazolo[1,5-a]pyrimidin-7-one ( 47 ), ID₅₀ 0.06 μM and 3-phenylpyrazolo[1,5-a]pyrimidin-7-one ( 46 ), ID₅₀ 0.40 μM. 5-(p-Chlorophenyl)pyrazolo[1,5-a]pyrimidin-7-one ( 63 ) and the corresponding 5-nitrophenyl derivative 64 exhibited an ID₅₀ of 0.21 and 0.23 μM, respectively. 7-Phenylpyrazolo[1,5-a]-s-triazin-4-one ( 40 ) is shown to exhibit an ID₅₀ of 0.047 μM. The structure-activity relationships of these new phenyl substituted hypoxanthine analogs are discussed and compared with the xanthine analogs 3-m-tolyl- and 3-phenyl-7-hydroxypyrazolo[1,5-a]pyrimidin-5-ones ( 90 ) and ( 91 ), previously reported from our laboratory to have ID₅₀ of 0.025 and 0.038 μM, respectively. The presence of the phenyl and substitutedphenyl groups contribute directly to the substrate binding of these potent inhibitors. This work presents an updated study of structure-activity relationships and binding to xanthine oxidase. In view of the recent elucidation of the pterin cofactor and the proposed binding of this factor to the molybdenum ion in xanthine oxidase, a detailed mechanism of xanthine oxidase oxidation of hypoxanthine and xanthine is proposed. Three types of substrate binding are viewed for xanthine oxidase. The binding of xanthine to xanthine oxidase is termed Type I binding. The binding of hypoxanthine is termed Type II binding and the specific binding of alloxanthine is assigned as Type III binding. These three types of substrate binding are analyzed relative to the most potent compounds known to inhibit xanthine oxidase and these inhibitors have been classified as to the type of inhibitor binding most likely to be associated with specific enzyme inhibition. The structural requirements for each type of binding can be clearly seen to correlate with the inhibitory activity observed. The chemical syntheses of the new 3-phenyl- and 3-substituted phenylpyrazolo[1,5-a]pyrimidines with various substituents are reported. The syntheses of various 8-phenyl-2-substituted pyrazolo-[1,5-a]-s-triazines, certain s-triazolo[1,5-a]-s-triazines and s-triazolo[1,5-a]pyrimidine derivatives prepared in connection with the present study are also described.     

Photochemistry of 3H-furo[3,4-c]pyrazoles and 3H-thieno[3,4-c]pyrazoles

The first examples of 3H-furo[3,4-c]pyrazoles and 3H-thieno[3,4-c]pyrazoles were prepared in an attempt to isolate examples of cyclopropane-fused thiophenes and furans. Adducts of acetylenic ketones and 2-diazopropane were reacted with 10-camphorsulfonic acid or phosphorus pentasulfide to produce the title compounds. Photolysis of these compounds did not give the cyclopropane-fused products rather an unreported photochemical fragmentation was observed.     

New red-shifted coelenterazine analogues with an extended electronic conjugation

A new promising approach to the development of red-shifted coelenterazine analogues was described. In order to alter the photochemical properties of native coelenterazine, we have designed and synthesized analogues bearing a new electron-rich structure. The spectroscopic results obtained, in the presence of the target enzyme (Renilla Luciferase), show a bathochromic emission shift of the entire class of new derivatives. Among them, the 2-benzyl-8-(4-chlorophenylthio)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one (8) shows an emission at 510 nm and uncommon slow kinetic decay.     

Regioselective synthesis of 3-deazacarbovir and its 3-deaza-adenosine analogues

We herein report the hitherto unknown synthesis of 3-deazacarbovir and its adenosine analogue. The major highlight in the synthesis of adenosine analogs is to use 6-N,N-diboc protected 3-deazapurines 9 and 11 for regioselective Mitsunobu coupling as well as unexplored palladium catalyzed coupling with these substrates. Synthesis of 3-deazacarbovir 1 has been accomplished by the regioselective palladium catalyzed coupling of 6-N,N-diphenylcarbamoyl protected 3-deazaguanine base 18 with dicarbonate 14. All the target nucleosides were screened for anti-HIV-1 activity and none of them have significant activity as well as toxicity up to 100 μM.     

The first 6′-O-sulfated phenylanthraquinones: isolation from Bulbine frutescens, structural elucidation, enantiomeric purity, and partial synthesis

From the roots of Bulbine frutescens, the first sulfated phenylanthraquinones were isolated, together with their known sulfate-free analogs. Their structures were elucidated by spectroscopic and chiroptical methods, by acid hydrolysis or by partial synthesis. The new compounds have the usual stereo-orientation at the biaryl axis (i.e., with the acetyl portion above the anthraquinone plane) except for sodium ent-knipholone 6′-O-sulfate (and thus, also its hydrolysis product, ent-knipholone), which exhibit an opposite axial configuration. We also describe the first stereoanalysis of natural phenylanthraquinones, some of which were found to be not enantiomerically pure, some even near-racemic. We furthermore, report on the first X-ray structure analysis of a phenylanthraquinone, viz. 4′-O-demethylknipholone.     

Synthesis of novel 8-(het)aryl-6H-pyrano[4′,3′:4,5]thieno[3,2-b]pyridines by 6-endo-dig cyclization of Sonogashira products and halolactonizations with Cu salts/NXS. Preliminary antitumor evaluation

Novel 8-(het)aryl-6H-pyrano[4′,3′:4,5]thieno[3,2-b]pyridines were prepared in good to high yields by a tandem one-pot procedure of Sonogashira coupling and 6-endo-dig lactonization from 3-bromothieno[3,2-b]pyridine-2-carboxylic acid and (het)arylalkynes. Sonogashira coupling products were also prepared from the corresponding methyl ester giving in the same reaction the corresponding 6-endo-dig compounds as minor products. The Sonogashira phenyl ester product gave cyclization with electrophiles only in low to moderate yields. Nevertheless, halolactonizations using Cu(I) or (II) salts/N-halosuccinimides (NXS) from either the phenyl ester or the carboxylic acid derivatives occurred in good to high yields. The growth inhibition potential of the compounds was evaluated using human tumor cell lines, HCT-15 (colorectal adenocarcinoma) and NCI-H460 (non-small cell lung cancer) and studies of apoptosis induction were performed for the three most promising compounds in HCT-15?cells. Two of them caused almost 40% of cell death by apoptosis when tested at their 1.5?×?GI₅₀ concentrations. The tricyclic lactone with a F atom in the meta position showed to be the most promising one.     

Luminescence Activity Decreases When v-coelenterazine Replaces Coelenterazine in Calcium-Regulated Photoprotein—A Theoretical and Experimental Study

Calcium-regulated photoproteins are found in at least five phyla of organisms. The light emitted by those photoproteins can be tuned by mutating the photoprotein and/or by modifying the substrate coelenterazine (CTZ). Thirty years ago, Shimomura observed that the luminescence activity of aequorin was dramatically reduced when the substrate CTZ was replaced by its analog v-CTZ. The latter is formed by adding a phenyl ring to the π-conjugated moiety of CTZ. The decrease in luminescence activity has not been understood until now. In this paper, through combined quantum mechanics and molecular mechanics calculations as well as molecular dynamics simulations, we discovered the reason for this observation. Modification of the substrate changes the conformation of nearby aromatic residues and enhances the π-π stacking interactions between the conjugated moiety of v-CTZ and the residues, which weakens the charge transfer to form light emitter and leads to a lower luminescence activity. The microenvironments of CTZ in obelin and in aequorin are very similar, so we predicted that the luminescence activity of obelin will also dramatically decrease when CTZ is replaced by v-CTZ. This prediction has received strong evidence from currently theoretical calculations and has been verified by experiments.     

Synthesis of 1-thio-phytosphingolipid analogs by microwave promoted reactions of thiols and aziridine derivatives

A practical and versatile method for the synthesis of 1-thio-phytosphingolipid analogs through regioselective nucleophilic ring-opening reactions of phytosphingosine aziridine derivatives with thiols is described. The reactions were carried out with N-acylaziridines and a variety of thiol compounds. Microwave irradiation highly improved the yield of the ring-opening reaction and the intermediate N-acyl adducts were converted into 1-S-phytosphingolipid analogs, such as phytoceramide and phytosphingosine derivatives.     

A rapid and versatile synthesis of novel pyrimido[5,4-b]carbazoles

A one-pot synthesis of 2-dialkylamino-5,11-dimethyl-6H-pyrimido[5,4-b]carbazol-4(3H)-ones, as new ellipticine analogs, starting from aminocarbazole derivatives is reported. This method allowed us to prepare a library of potentially useful compounds in the pharmaceutical field.     

TIPS-Ethynylated Naphthodiquinoline and Naphthodiacridine: Novel Diazabisacenes

The synthesis of two diazabisacenes is reported. A bisboronated naphthalene was Suzuki-coupled to substituted ethyl nicotinates, then cyclized by intramolecular Friedel-Crafts acylation. The resulting diketones were alkynylated and reduced to give the title compounds, bis(TIPS-ethynyl)-substituted naphtha[1,8-gh:5,4-g′h′]diquinoline and naphtho[1,8-bc:5,4-b′c′]diacridine. Nitrogen incorporation stabilizes the bisacenes with respect to oxidation compared to their consanguine nonaza analogs.     

Stereoselectivity in the anionic oxy-Cope rearrangement of acyclic vinyl sulfides

Novel anionic oxy-Cope (AOC) rearrangements of substituted 5-alkylthio-1,5-dien-3-ols were studied. Aqueous quench gave new access to compounds with a nucleophilic vinyl sulfide and an electrophilic aldehyde in a 1,5 relationship. 3,4-Anti and 3,4-syn 1E,5Z-5-hexylthio-4-methyl-1-phenylhepta-1,5-dien-3-ols were synthesised separately by stereoselective aldol reaction, thioesterification and alkylidenation of the resulting thioesters to give Z-vinyl sulfides with ?90% stereoselectivity. AOC rearrangement of the 3,4-syn substrate gave predominantly 3,4-syn Z-vinyl sulfide while the 3,4-anti substrate gave mostly 3,4-syn E-vinyl sulfide, via chair-like transition states with the oxyanion pseudo-equatorial. Stereochemistry was assigned by NOE taking advantage of the conformational stability of the products.     

Synthesis of 11-aryl-11H-indeno[1′,2′:4,5]imidazo[1,2-a]pyridines via dehydrative cyclization of aryl(2-arylimidazo[1,2-a]pyridin-3-yl)methanols

A series of 11-aryl-11H-indeno[1′,2′:4,5]imidazo[1,2-a]pyridines was obtained via AlCl₃-promoted, dehydrative cyclization of the corresponding aryl(2-arylimidazo[1,2-a]pyridin-3-yl)methanols in moderate to good yields. The synthesized compounds can be considered as conformationally restricted, privileged aza-heterocyclic scaffold bearing triarylmethane analogs.     

New disaccharide blocks for OSW-1 and its analogs

A new disaccharide block for OSW-1 natural steroidal antitumor agent was described. Regioisomeric 2- and 3-O-p-methoxybenzoyl derivatives of phenyl 1-thio-??-d-xylopyranoside and phenyl 2-O-acetyl-1-thio-??-l-arabinopyranoside derivatives blocked at positions 3 and 4 by R₃Si groups were synthesized with a view to use them in the preparation of OSW-1 analogs modified at the disaccharide fragment.     

Reactions of phenylenedioxytrihalophosphoranes with arylacetylenes: XIII. Reaction of 5-tert-butyl-2,2,2-trihalo-1,3,2λ5-benzodioxaphospholes with acetylenes

Reactions of 5-tert-butyl-2,2,2-trichloro-, 2,2,2-tribromo-5-tert-butyl-, and 2,2-dibromo-5-tert-butyl-2-fluoro-1,3,2λ⁵-benzodioxaphospholes with aryl- and alkylacetylenes lead to quantitative formation of 2-halo-1,2λ⁵-benzoxaphosphinine 2-oxides which may be regarded as phosphorus analogs of natural heterocyclic compounds, coumarin and chromene. The major products (>70%) are 4-aryl-7-tert-butyl-2,6-dichloro-, 4-aryl-2-bromo-7-tert-butyl-, and 4-aryl-7-tert-butyl-2-fluoro-1,2λ⁵-benzoxaphosphinine 2-oxides. Hydrolysis of these compounds and their treatment with amines gives the corresponding 2-hydroxy and 2-amino derivatives, as well as ammonium salts. The structure of some compounds was proved by X-ray analysis.     

Palladium-benzimidazolium salt catalyst systems for Suzuki coupling: development of a practical and highly active palladium catalyst system for coupling of aromatic halides with arylboronic acids

Palladium-benzimidazolium salt catalyst systems have been studied for the Suzuki coupling. A different substitutent effect has been uncovered with respect to nitrogen substituents in the benzimidazolium salts from the palladium-imidazolium salt analogs. A practical and highly active palladium catalyst system, PdCl₂/N,N′-dibenzylbenzimidazolium chloride 2, has been identified for the Suzuki coupling of aromatic halides with arylboronic acids. The coupling of a wide array of aromatic halides with arylboronic acids with the PdCl₂-2 catalyst system gave good to excellent yields. The effective palladium loading could be as low as 0.0001 mol% and 0.01-0.1 mol% for iodide and bromide substrates, respectively. The coupling of unactivated aromatic chlorides with arylboronic acids also gave good results using Cs₂CO₃ as base with a 2 mol% palladium loading. The electronic factors from aromatic halides exert a significant influence on the Suzuki coupling catalyzed by the PdCl₂-2 system while the electronic effect from the arylboronic counterparts is negligible. The aromatic halides with modest steric hindrance could also couple smoothly with phenylboronic acids using the PdCl₂-2 catalyst system.     

Total synthesis of 4-epi-atpenin A5 as a potent nematode complex II inhibitor

It is clear that atpenins and their analogs are useful chemical tools for elucidation of complex II functionality and that they could act as lead compounds for the development of novel helminth complex II-specific inhibitors. Recently, we discovered 4-epi-atpenin A5 as a potent nematode complex II inhibitor during our SAR studies of atpenin A5. This result led us to embark on a concise total synthesis of 4-epi-atpenin A5. In this study, we describe the total synthesis of 4-epi-atpenin A5. Importantly, this was more concise and practical synthesis than our previous total synthesis of atpenin A5.     

Comparative toxicity of [3]ferrocenophane and ferrocene moieties on breast cancer cells

Recent results suggest that [3]ferrocenophane may be an interesting motif in the development of cytotoxic anti-cancer agents. We here report the synthesis of three such compounds based on the 1-[(p-R-phenyl)-phenyl-methylidenyl)]-[3]ferrocenophane skeleton with R = OH, NH₂ and NHC(O)CH₃ substitution on one of the phenyl rings. Cytotoxicity studies show that these compounds are up to four times more powerful against hormone-independent breast cancer cells than their corresponding ferrocene analogs.