Photochemistry of 7-azide-1-ethyl-3-carboxylate-6,8-difluoroquinolone: a novel reagent for photoaffinity labeling

A novel reagent for photoaffinity labeling, 7-azido-1-ethyl-3-carboxylate-6,8-difluoroquinolone, was obtained by nucleophilic substitution of 1-ethyl-3-carboxylate-6,7,8-trifluoroquinolone. Photochemical reaction of this azide with diethyl amine gave 7-hydrazino-derivative as the major product. This compound was generated by singlet nitrene N-H insertion. In addition, 7-amino-1-ethyl-3-carboxylate-6,8-difluoroquinolone was also obtained.     

AZIDOTETRAFLUOROPHENYL RETINAL ANALOGUE: SYNTHESIS AND BACTERIORHODOPSIN PIGMENT FORMATION

Abstract —The retinal derivative, all-truns-9–(4-azido-2,3,5,6-tetrafluorophenyl)-3,7-dimethyl-2,4,6,8-nonatetraenal, was synthesized by two routes as a potential photoactivatable cross-linking agent for studies in bacteriorhodopsin (BR) of the chromophore interaction with its apoprotein. The retinal analogue formed a stable, moderately functional BR pigment confirming that the ring cavity of the retinal binding site has a significant tolerance for derivatization on that portion of the molecule. Attempts to cross-link the azido chromophore to the protein by photoactivation were unsuccessful. The electron delocalization effect of the conjugated polyene side chain of the retinal appears to interfere with the formation or reactivity of the nitrene intermediate to the extent that photoactivated cross-linking is not achieved. These results demonstrate a limitation to the use of fluorinated aryl azides as photoaffinity reagents.     

A Thio-Staudinger Reaction: Thermolysis of a Vinyl Azide in the Presence of t -Butyl Mercaptan

The thermal decomposition of E-3-azido-3-hexene-2,5-dione ( 1 ) in protic media gave rise to several novel reactions of the azide 1 including adducts derived from keteneimine 11 . Reaction with t-butyl mercaptan yielded two products, keteneimine-derived mercaptan addition product 20 and a sulfenimine 6 . Trapping of a vinyl nitrene intermediate or a thio-Staudinger reaction was considered as a possible mechanism for the formation of sulfenimine 6 . The absence of the vinyl nitrene addition products 3 and 5 when the thermal decomposition was conducted in either methanol or t-butylamine suggests a thio-Staudinger reaction is operative.     

Studies in azide chemistry. Part IX [1]. Investigations involving fluorinated azidopyrimidines and 4-azido-3-chloro-2,5,6-trifluoropyridine

4-Azido-2,5,6-trifluoro- and 4,6-diazido-2,5-difluoro- pyrimidine were obtained by treating tetrafluoropyrimidine with sodium azide in acetonitrile; similar azidation of 5-chlorotrifluoropyrimidine gave 4-azido-5-chloro-2,6-difluoro- and 4,6-diazido-5-chloro-2-fluoro-pyrimidine. Each monoazide reacted with triphenylphosphine to yield the corresponding iminophosphorane (Staudinger reaction), and the trifluoro- compound gave cycloadducts when heated with phenylacetylene [→ 4-phenyl-1-(2,5,6-trifluoro-4-pyrimidinyl)-1,2,3- triazole] and acrylonitrile [→ 2-cyano-1-(2,5,6-trifluoro- 4-pyrimidinyl)aziridine]; attack on the trifluoro-azide by the sodium salt of pentafluoroaniline produced 4-azido-2,5- difluoro-6-(pentafluorophenylamino)pyrimidine and bis(4- azido-2,5-difluoro-6-pyrimidinyl)(pentafluorophenyl)amine. Attempts to intercept nitrenes during thermal decomposition of both mono-azides failed. Thermolysis of 4-azido-3-chloro- 2,5,6-trifluoropyridine in the presence of dimethyl sulphoxide, cyclohexane, or pentafluoroaniline gave products [py_FNS(O)Me₂, py_FNHC₆H₁₁, and py_FNNPh_F (PY_F = 3-chlorotrifluoro-4-pyridyl), respectively] compatible with release of the corresponding nitrene.     

Long-lived reactive intermediate photogenerated from N-(5-azido-2-nitrobenzoyl)-N'-(D-biotinyl)-1,2-diaminoethane as an affinity reagent to streptavidin

Irradiation of a complex between N-(5-azido-2-nitrobenzoyl)-N'-(D-biotinyl)-1,2-diaminoethane (I) and streptavidin with light of 313 nm led to the covalent attachment of the photobiotin analogue I to the protein. Streptavidin could also be labelled in the dark with prephotolyzed I. These results indicate that a long-lived reactive intermediate was formed upon irradiation. Moreover, after cleavage of labelled streptavidin with proteinase K this intermediate appears to be covalently attached to the same peptide as the one obtained by direct photoaffinity labelling. An iminosulfurane II derived from the reaction of biotin sulfur atom with aryl nitrene is responsible for the dark-labelling reaction. The photoproduct II converts in an aqueous solution almost completely into N-(5-amino-2-nitrobenzoyl)-N'-(D-(S-oxo)biotinyl)-1,2-diaminoethane (the half-life of II is 10 days).     

4-Azidocinnoline—Cinnoline-4-amine Pair as a New Fluorogenic and Fluorochromic Environment-Sensitive Probe

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide–amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.     

Mechanism of cobalt(II) porphyrin-catalyzed C-H amination with organic azides: radical nature and H-atom abstraction ability of the key cobalt(III)-nitrene intermediates

The mechanism of cobalt(II) porphyrin-catalyzed benzylic C-H bond amination of ethylbenzene, toluene, and 1,2,3,4-tetrahydronaphthalene (tetralin) using a series of different organic azides [N(3)C(O)OMe, N(3)SO(2)Ph, N(3)C(O)Ph, and N(3)P(O)(OMe)(2)] as nitrene sources was studied by means of density functional theory (DFT) calculations and electron paramagnetic resonance (EPR) spectroscopy. The DFT computational study revealed a stepwise radical process involving coordination of the azide to the metal center followed by elimination of dinitrogen to produce unusual "nitrene radical" intermediates (por)Co(III)-N(?)Y (4) [Y = -C(O)OMe, -SO(2)Ph, -C(O)Ph, -P(O)(OMe)(2)]. Formation of these nitrene radical ligand complexes is exothermic, predicting that the nitrene radical ligand complexes should be detectable species in the absence of other reacting substrates. In good agreement with the DFT calculations, isotropic solution EPR signals with g values characteristic of ligand-based radicals were detected experimentally from (por)Co complexes in the presence of excess organic azide in benzene. They are best described as nitrene radical anion ligand complexes (por)Co(III)-N(?)Y, which have their unpaired spin density located almost entirely on the nitrogen atom of the nitrene moiety. These key cobalt(III)-nitrene radical intermediates readily abstract a hydrogen atom from a benzylic position of the organic substrate to form the intermediate species 5, which are close-contact pairs of the thus-formed organic radicals R'(?) and the cobalt(III)-amido complexes (por)Co(III)-NHY ({R'(?)···(por)Co(III)-NHY}). These close-contact pairs readily collapse in a virtually barrierless fashion (via transition state TS3) to produce the cobalt(II)-amine complexes (por)Co(II)-NHYR', which dissociate to afford the desired amine products NHYR' (6) with regeneration of the (por)Co catalyst. Alternatively, the close-contact pairs {R'(?)···(por)Co(III)-NHY} 5 may undergo β-hydrogen-atom abstraction from the benzylic radical R'(?) by (por)Co(III)-NHY (via TS4) to form the corresponding olefin and (por)Co(III)-NH(2)Y, which dissociates to give Y-NH(2). This process for the formation of olefin and Y-NH(2) byproducts is also essentially barrierless and should compete with the collapse of 5 via TS3 to form the desired amine product. Alternative processes leading to the formation of side products and the influence of different porphyrin ligands with varying electronic properties on the catalytic activity of the cobalt(II) complexes have also been investigated.     

Some 1,3-dipolar cycloaddition reactions of perfluoro-(2-azido-4-isopropylpyridine) and 2-azido-3,5,6-trifluoro-4-methoxypyridine

In continuation with studies on perfluorinated aryl [1] and hetaryl [2] azides some 1,3-dipolar cycloaddition reactions of perfluoro-(2-azido-4-isopropylpyridine) are summarized in Scheme 1. The azide reacts with styrene under forcing thermal conditions to give a mixture of perfluoro-(2-amino-4-isopropylpyridine) and benzaldehyde involving a more unusal type of cleavage of the triazoline formed initially [2]. Perfluoro-(2-azido-4-isopropyl-pyridine) does not react with cyclopentene or cyclohexene upto 110 °C but when the reaction is carried out at the decomposition temperature of the azide i.e. 160 °C, a mixture of the amino compound and the corresponding cyclic ketone is obtained. It is believed that at 160 °C the reaction still proceeds $\text{via}$ 1,3-cycloaddition because no evidence of nitrene formation was found when the azide was decomposed thermally or photochemically in the presence of conventional nitrene traps e.g. dimethyl sulphoxide, cyclohexane etc. [2] and only polymeric material was obtained. Perfluoro-(2-azido-4-isopropylpridine) reacts smoothly with norbornene at room temperature with evolution of nitrogen and only the products of decomposition of initially formed triazoline are obtained.     

Polymer-bound 4-methylcoumarin/1-heptanoyl-5-fluorouracil photodimers: NMR elucidation of dimer structure

Heterodimers based on the polymer-bound chromophore 4-methylcoumarin and the prodrug 1-heptanoyl-5-fluorouracil, synthesized by photochemical [2 + 2]-cycloaddition are promising photoresponsive drug depots. Drug release experiments are one possibility to deliver proof of a successful reversible drug immobilization, whereas NMR spectroscopy is a potent tool for further structural characterization of these polymer-bound heterodimers. In case of the random copolymer poly(methyl methacrylate-co-7-(2'-methacryloyloxyethoxy)-4-methylcoumarin) three dimers have been identified of which the syn head-to-tail was the predominant one. In contrast, only the syn head-to-head dimer was formed in reasonable yield when the 4-methylcoumarin monofunctionalized pMMA was used as the base polymer. 1D and 2D NMR spectroscopic techniques combined with some theoretical calculations helped in successfully closing one major gap concerning polymer bound 4-methylcoumarin/1-heptanoyl-5-fluorouracil heterodimers that are of potential use in photoresponsive drug delivery devices.     

Synthesis of [D-alanine, 4'-azido-3',5'-ditritio-L-phenylalanine, norvaline4[alpha-melanotropin as a 'photoaffinity probe' for hormone-receptor interactions (author's transl)]

Synthesis of [D -alanine¹, 4′-azido-3′, 5′-ditritio-L -phenylalanine², norvaline⁴]α-melanotropin as a ‘photoaffinity probe’ for hormone-receptor interactions. The synthesis of an α-MSH derivative containing 4′-azido-3′,5′-ditritio-L -phenylalanine is described: Ac · D -Ala-Pap(³H₂)-Ser-Nva-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val · NH₂. This hormone analogue is being used for specific photoaffinity labelling of receptor molecules. The synthesis was performed in a way to minimize the number of radioactive steps and to introduce the radio-active and the photoaffinity label exclusively into position 2. The dipeptide N(α)-acetyl-D -alanyl- (4′-amino-3′,5′-diiodo)-L -phenylalanine was tritriated and transformed into the azido compound, N(α)-acetyl-D -alanyl-(4′-azido-3′,5′-ditritio)-L -phenylalanine which was then condensed with H · Ser-Nva-Glu(OtBu)-His-Phe-Arg-Trp-Gly-Lys(BOC)-Pro-Val · NH₂ to the tridecapeptide. The α-MSH analog displayed a specific activity of 11 Ci/mmol, and a biological activity of about 4 · 10⁹ U/mmol (10% of α-MSH).     

Synthesis and anticholinesterase activity of some new fluorogenic analogues of organophosphorus nerve agents

Eighteen new fluorogenic analogues of organophosphorus nerve agents were synthesised and characterised. They included analogues of tabun, sarin, cyclosarin, soman, VX, and Russian VX, with the 7-oxy-4-methylcoumarin or 7-oxy-4-(trifluoromethyl)coumarin leaving group. These analogues inhibited acetylcholinesterase (AChE) effectively in vitro and therefore have potential as tools for the identification of novel organophosphatases in biological systems. Analogues of VX and Russian VX with the 7-amino-4-methylcoumarin group, although poor AChE inhibitors, may have utility for screening enzyme libraries for phosphoramidases capable of cleaving P-N bonds.     

The chemistry of 4-hydrazino-7-phenylpyrazolo[1,5-a] -1,3,5-triazines

The reaction of 4-hydrazino-7-phenylpyrazolo[1,5-a]-1,3,5-triazine ( 4 ) with nitrous acid gave 8-phenyltetrazolo[1,5-e]pyrazolo[1,5-a]-1,3,5-triazine ( 5b ), which was determined by pmr and ir spectra to be in equilibrium with 4-azido-7-phenylpyrazolo[1,5-a]-1,3,5-triazine ( 5a ). The equilibrium between the tetrazolo ( 5b ) and azido ( 5a ) forms was studied by pmr and an attempt was made to determine if substituents in the pyrazole nucleus could sufficiently stabilize the tricyclic tetrazolo form ( 5b ) over the bicyclic azido form ( 5a ). Thermal degradation of 5 (a ? b) in an aprotic solvent gave 4-amino-7-phenylpyrazolo[1,5-a]-1,3,5-triazine ( 7 ), indicating the probability of a nitrene mechanism involved in the decomposition. Heating 5 in aqueous base gave both 7 and the “hydroxy” analog, 7-phenylpyrazolo[1,5-a]-1,3,5-triazin-4(3H)one ( 6 ), further substantiating the existence of a nitrene intermediate with a competing nucleophilic displacement of the azido group by a hydroxyl group. Cyclization of 4 with diethoxymethylacetate (DEMA) gave 8-phenyl-s-triazolo[4,3-e]pyrazolo[1,5-a]-1,3,5-triazine ( 8 ), which underwent thermal rearrangement to 8-phenyl-s-triazolo[2,3-e]pyrazolo[1,5-a]-1,3,5-triazine ( 9 ). Acid catalyzed ring opening of 9 with formic acid gave 3-N-formamido-5-phenyl-2(2-s-triazolyl)pyrazole ( 10 ). The failure of 10 to recyclize to 9 with the resultant loss of water, supported the theory that the rearrangement of 8 to 9 might occur simply as a concerted, thermally induced “anhydrous” rearrangement rather than via a covalently hydrated intermediate or a Dimroth type mechanism (in the base catalyzed rearrangement).     

Photolysis of 2-azidopyridines. the behavior of 1-(2-azido-6-chloropyrid-4-yl)-3-phenylurea,a photoaffinity labeling reagent for probing cytokinin-binding proteins

The photolysis of l-(2-azido-6-chloropyrid-4-yl)-3-phenylurea ( 1 ) was studied under various conditions. In alcohols or in hexane, complex mixtures of products were obtained. Methoxide anions or diethylamine gave rise in high yield to 1,3-diazepines resulting from ring enlargement of the intermediate nitrene with addition of one molecule of the nucleophile, and nucleophilic substitution of the chlorine atom. A similar reaction was observed in water, when Pyrex filtered light was used. However, with unfiltered light produced by a powerful lamp, the main reaction was photodechlorination. The reagent 1 is expected to bind covalently to cytokinin-binding proteins through different ways upon photolysis.     

Studies in azide chemistry. Part 13 [1]. Intermolecular insertion of azide-derived polyfluorinated aryl- and heteroaryl-nitrenes into ring CH bonds of 1,3, 5-trimethyl- and 1,3,5-trimethoxy-benzene

Thermolysis of perfluoroazidobenzene, perfluoro-4- azidotoluene, perfluoro-4-azidopyridine, 4-azido-3- chlorotrifluoropyridine, and 4-azido-3, 5-dichlorodifluoropyridine (Ar_FN₃) in the presence of a large excess ($\text{ca}$. 10 molar) of 1,3,5-trimethyl- or 1,3,5-trimethoxy-benzene (ArH) gave the diarylamines expected from nitrene ‘insertions’ at nuclear CH bonds (Ar_FN₃ + ArH→Ar_FNHAr + N₂); product yields in the cases of the perfluorinated azides are the highest ever recorded for this type of reaction. By contrast, no recognisable products were obtained when either perfluoro-(2-azido-4-isopropylpyridine) or 2-azido- 4-chlorotrifluoropyridine were decomposed thermally in 1,3,5-trimethylbenzene.     

Improved and rapid synthesis of new coumarinyl chalcone derivatives and their antiviral activity

Two closely structurally related coumarins, 4-hydroxy-8-isopropyl-5-methylcoumarin and 4-hydroxy-6-chloro-7-methylcoumarin were acylated at C-3 and further converted to the respective chalcones and two series of eighteen new compounds, which were evaluated for possible antiviral activity.     

Triplet-sensitized photoreactivity of a geminal diazidoalkane

Photolysis of 1 in chloroform yielded 2 as the major product and a small quantity of 3. Laser flash photolysis demonstrated that upon irradiation, the first excited triplet state of the ketone (T(1K)) of 1 is formed and decayed to form radical 4, which has a λ(max) at 380 nm (τ = 2 μs). Radical 4 expelled a nitrogen molecule to yield imine radical 5 (λ(max) at 300 nm). Density functional theory (DFT) calculations showed that the transition state barrier for the formation of 5 is approximately 4 kcal/mol. In comparison, photolysis of 1 in argon matrices resulted in triplet nitrene 6, which was further characterized with (15)N and D isotope labeling and DFT calculations. Prolonged irradiation of 6 yields triplet imine nitrene 7.     

Aromatic substitution by n-arylhydroxylamines—I : Formation of 8-amino-5,8′-iminobis(6-methoxyquinoline) by an intermolecular aromatic nitrene insertion reaction

Thermolysis of 8-hydroxylamino-6-methoxyquinoline at 65° in methanol gave 8-amino-5,8′-iminobis(6-methoxyquinoline), the same product being formed by thermolysis of 8-azido-6-methoxyquinoline as well as by deoxygenation of 6-methoxy-8-nitroquinoline with triethylphosphite in the presence of 8-amino-6-methoxyquinoline. Solvent effects were also consistent with the involvement of a nitrenoid species in these intermolecular aromatic substitutons. 8-Hydroxylaminoquinoline behaved in an analogous fashion but no iminobis compound was obtained from the corresponding 6-hydroxylaminoquinoline, indicating an internal interaction of the ring N atom with the 8-hydroxylamino function. Thermolysis of 8-hydroxylamino-6-methoxyquinoline in the presence of amines gave rise to o-diamines reconcilable with a nitrene intermediate.     

Design,Synthesis, Spectroscopic Characterisation and In Vitro Cytostatic Evaluation of Novel Bis(coumarin-1,2,3-triazolyl)benzenes and Hybrid Coumarin-1,2,3-triazolyl-aryl Derivatives

In this work, a series of novel 1,2,3-triazolyl-coumarin hybrid systems were designed as potential antitumour agents. The structural modification of the coumarin ring was carried out by Cu(I)-catalysed Huisgen 1,3-dipolar cycloaddition of 7-azido-4-methylcoumarin and terminal aromatic alkynes to obtain 1,4-disubstituted 1,2,3-triazolyl-coumarin conjugates 2a–g, bis(1,2,3-triazolyl-coumarin)benzenes 2h–i and coumarin-1,2,3-triazolyl-benzazole hybrids 4a–b. The newly synthesised hybrid molecules were investigated for in vitro antitumour activity against five human cancer cell lines, colon carcinoma HCT116, breast carcinoma MCF-7, lung carcinoma H 460, human T-lymphocyte cells CEM, cervix carcinoma cells HeLa, as well as human dermal microvascular endothelial cells (HMEC-1). Most of these compounds showed moderate to pronounced cytotoxic activity, especially towards MCF-7 cell lines with IC₅₀ = 0.3–32 μM. In addition, compounds 2a–i and 4a–b were studied by UV-Vis absorption and fluorescence spectroscopy and their basic photophysical parameters were determined.     

4-Azidoaniline,a Versatile Protein and Peptide Modifying Agent for Photo Affinity Labeling

A modified synthesis of 4-azidoaniline and the use of this compound as a protein and peptide modifying agent for photoaffinity labeling is described. 4-Azidoaniline was diazotized to 4-azidophenyldiazonium and coupled to N-acetyl-tyrosine-ethylester, and to a tyrosine containing analog of bradykinin. It is shown that the 4-azidoaniline reagent offers great advantages over other protein modifying agents for photoaffinity labeling, i.e., very high nitrene reactivity, possibility of tritium or iodine labeling, ligand-receptor complex can be cleaved after isolation. The biological activity of the modified bradykinin analog is measured on rabbit aorta strip in the dark and is similar to the unsubstituted analog. Under the influence of light the modified peptide is able to block partially the myotropic action of bradykinin.     

Synthesis of 4-azido-2-isopropylamino-6-ethylamino-s-triazine (ring UL14C)

Radioactive 4-azido-2-isopropylamino-6-ethylamino-s-triazine (¹⁴C triazine ring) was synthesized for use as a photoaffinity label to study the site of triazine action in the chloroplast. It was prepared from ¹⁴C 4-chloro-2-isopropylamino-6-ethylamino-s-triazine by forming the trimethylamine salt followed by treatment with sodium azide. ¹⁴C azidotrazine, 6.6 mg, was obtained in a yield of 64% based on ¹⁴C atrazine. The specific activity was 37.4 μCi/mg, and it assayed over 99.5% radiochemically pure after purification by TLC.