Functionalized azirine based scaffolds as endothelin inhibitors for the selective anti-angiogenic activity.

Anti-angiogenic therapy has long been used as an adjunct therapy for the resolution of tumor burden. The current findings describe the synthesis of novel marine-based azirine-containing compounds that exhibit anti-angiogenic mediated anti-tumor activity. Azirine-2-carboxylate inhibited HUVEC-mediated tubulogenesis without causing cell death in a dose-dependent manner. Ex-vivo CAM, in-vivo Matrigel implantation, and ear angiogenesis experiments have all shown that azirine-2-carboxylate effectively inhibits angiogenesis. Furthermore, azirine-2-carboxylate inhibits the migration of ECs without disrupting the preformed tubule network. Azirine-2-carboxylate had adequate intramuscular systemic exposure and inhibited tumor growth in a xenograft mouse model. DARTS analysis, competitive binding assay, and gene expression investigations revealed that azirine-2-carboxylate inhibits endothelin-1-mediated angiogenesis. Overall, the discovery of azirine-2-carboxylate demonstrated a potent inhibition of angiogenesis targeting ET1 and a possible application in anti-angiogenic therapy.

Novel diaryl-2H-azirines: Antitumor hybrids for dual-targeting tubulin and DNA.

Multiple-target drugs may achieve better therapeutic effect via different pathways than single-target ones, especially for complex diseases. Tubulin and DNA are well-characterized molecular targets for anti-cancer drug development. A novel class of diaryl substituted 2H-azirines were designed based on combination of pharmacophores from Combretastatin A-4 (CA-4) and aziridine-type alkylating agents, which are known tubulin polymerization inhibitor and DNA damaging agents, respectively. The antitumor activities of these compounds were evaluated in vitro and 6h showed the most potent activities against four cancer cell lines with IC50 values ranging from 0.16 to 1.40 µM. Further mechanistic studies revealed that 6h worked as a bifunctional agent targeting both tubulin and DNA. In the nude mice xenograft model, 6h significantly inhibited the tumor growth with low toxicity, demonstrating the promising potential for further developing novel cancer therapy with a unique mechanism.

Synthesis of Novel Diaziridinyl Quinone Isoxazole Hybrids and Evaluation of Their Anti-Cancer Activity as Potential Tubulin-Targeting Agents.

Two series of diaziridinyl quinone isoxazole derivatives were prepared and evaluated for their cytotoxic activity against MCF7, HeLa, BT549, A549 and HEK293 cell lines and interaction with tubulin. Compounds (6A-M: ) showed promising activity against all the 5 human cancer cell lines. Compounds 6A: , 6E: and 6 M: were potent [IC50 ranging between 2.21 µg to 2.87 µg] on ER-positive MCF7 cell line similar to the commercially available drug molecule Doxorubicin. The results from docking models are in consistent with the experimental values which demonstrated the favourable binding modes of compounds 6A-M: to the interface of α- and ß-tubulin dimer.

Synthesis and preliminary biological evaluation of radiolabeled 5-BDBD analogs as new candidate PET radioligands for P2X4 receptor.

P2X4 receptor has become an interesting molecular target for treatment and PET imaging of neuroinflammation and associated brain diseases such as Alzheimer's disease. This study reports the first design, synthesis, radiolabeling and biological evaluation of new candidate PET P2X4 receptor radioligands using 5-BDBD, a specific P2X4 receptor antagonist, as a scaffold. 5-(3-Hydroxyphenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD analog, [11C]9) and 5-(3-Bromophenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD, [11C]8c) were prepared from their corresponding desmethylated precursors with [11C]CH3OTf through N-[11C]methylation and isolated by HPLC combined with SPE in 30-50% decay corrected radiochemical yields with 370-1110GBq/µmol specific activity at EOB. 5-(3-[18F]Fluorophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]F-5-BDBD, [18F]5a) and 5-(3-(2-[18F]fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]FE-5-BDBD, [18F]11) were prepared from their corresponding nitro- and tosylated precursors by nucleophilic substitution with K[18F]F/Kryptofix 2.2.2 and isolated by HPLC-SPE in 5-25% decay corrected radiochemical yields with 111-740GBq/µmol specific activity at EOB. The preliminary biological evaluation of radiolabeled 5-BDBD analogs indicated these new radioligands have similar biological activity with their parent compound 5-BDBD.

Synthesis and evaluation of a photoactive probe with a multivalent carbohydrate for capturing carbohydrate-lectin interactions.

Lectins are ubiquitous carbohydrate-binding proteins of nonimmune origin that are characterized by their specific recognition of defined monosaccharide or oligosaccharide structures. However, the use of carbohydrates to study lectin has been restricted by the weak binding affinity and noncovalent character of the interaction between carbohydrates and lectin. In this report, we designed and synthesized a multifunctional photoaffinity reagent composed of a trialkyne chain, a masked latent amine group, and a photoreactive 3-trifluoromethyl-3-phenyl-diazirine group in high overall yield. Two well-defined chemistries, Huisgen-Sharpless click chemistry and amide bond coupling, were the key steps for installing the multivalent character and tag in our designed photoaffinity probe. The photolabeling results demonstrated that the designed probe selectively labeled the target lectin, RCA120 ( Ricinus communis Agglutinin), in an E. coli lysate and an asialoglycoprotein receptor (ASGP-R) on intact HepG2 cell membranes. Moreover, the probe also enabled the detection of weak protein-protein interactions between RCA120 and ovalbumin (OVA).

Novel chiral ferrocenylpyrazolo[1,5-a][1,4]diazepin-4-one derivatives--synthesis, characterization and inhibition against lung cancer cells.

A series of novel 2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one derivatives with optical activity (2) was synthesized in the microwave-assisted condition and characterized by means of IR, (1)H NMR and mass spectroscopy, and furthermore confirmed by X-ray analysis of a representative compound (R)-2a. Preliminary biological evaluation showed that some compounds could suppress the growth of A549, H322 and H1299 lung cancer cells. Among the tested compounds, 2b-d were more effective and might perform their action through cell cycle arrest for A549 cell. Whereas these compounds inhibited growth of H1299 and H322 cells by inducing apoptosis. The anti-tumor activities of these compounds were related to the nature of substituents in benzene moiety. In addition, the results indicated also that compounds 2b-d possessed notable cytotoxicity and selectivity for A549 vs H1299 and H322 lung cancer cells.

Selective inhibition of BET bromodomains.

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

Fragmentation of carbohydrate anomeric alkoxyl radicals. A new synthesis of chiral beta-iodo azides, vinyl azides, and 2H-azirines.

[reaction: see text] The reaction of 3-azido-2,3-dideoxy-hexopyranose compounds from the d-gluco, d-galacto, d-lacto, and l-arabino carbohydrate series, with (diacetoxyiodo)benzene and iodine, generated 2-azido-1,2-dideoxy-1-iodo-alditols with one carbon less than the starting carbohydrate. These beta-iodo azides could be transformed by dehydroiodination into vinyl azides, which in turn afforded 3-monosubstituted 2H-azirines under thermal conditions. These beta-iodo azides and 2H-azirines may be interesting chiral synthons for the preparation of more complex heterocyclic systems.

Synthesis and evaluation of oligodeoxyribonucleotides containing an aryl(trifluoromethyl)diazirine moiety as the cross-linking probe: photoaffinity labeling of mammalian DNA polymerase beta.

Photolabile 2'-deoxy- E -5-[4-(3-trifluoromethyl-3 H-diazirin-3-yl)styryl]uridine and its protected phosphoramidite derivatives have been synthesized and introduced into DNA oligomers through solid-phase DNA synthesis. The (trifluoromethyldiazirinyl)stylyl moiety of this nucleoside was found to be sufficiently stable for automated DNA synthesis. In addition, this moiety was found to be stable at 60 degrees C in aqueous solution under the annealing conditions for duplex formation with complementary strands, since >95% of the photolabile nucleoside remained after heating for 1 h. The oligo(dT) 15mer analog bearing the photolabile residue was activated/decomposed by near-UV irradiation. In photoaffinity cross-linking experiments with recombinant rat DNA polymerasebeta, constituted from a 40 kDa polypeptide, using oligo(dT) 15mer analogs bearing the photolabile residue near the 3'-terminus, a covalently bound complex of 45 kDa was obtained in the presence of complementary templates. Thus it was demonstrated that our method for synthesis of photolabile oligodeoxyribonucleotides may be useful for studies of DNA-related enzymes and DNA binding proteins.

Evidence for H(+)-induced insertion of influenza hemagglutinin HA2 N-terminal segment into viral membrane.

Fusion of influenza virus with target membranes is induced by acid and involves complex changes in the viral fusion protein hemagglutinin. At 0 degree C, in a first kinetically resolvable step, the hemagglutinin polypeptide 2 (HA2) N-terminal segment (fusion peptide) is exposed and inserts into the target membrane (Tsurudome, M., Glück, R., Graf, R., Falchetto, R., Schaller, U., and Brunner, J. (1992) J. Biol. Chem. 267, 20225-20232). We now report studies of the changes taking place at pH 5.0 and 37 degrees C, conditions that result in fusion or, in the absence of a target membrane, in inactivation of the virus' fusion capacity. To this end, we synthesized the new photosensitive phospholipid, 1-palmitoyl-2-[decanedioyl mono-[2-(125I)iodo-4-(3-trifluoromethyl-3H-diazirin-3-yl)-benzyl]e ster]- sn-glycero-3-phosphocholine (specific radioactivity, > 2000 Ci/mmol), and worked out a protocol to incorporate this lipid into the viral membrane. Subsequent photoactivation of the reagent resulted in selective labeling of the C-terminal portion of the HA2 polypeptide chain, in agreement with the membrane topology of hemagglutinin. When, however, prior to reagent activation, the viruses were exposed at pH 5.0, 37 degrees C, both the HA2 C-terminal and the N-terminal regions were labeled, suggesting that the HA2 N-terminal segment (fusion peptide) inserted into the viral membrane. Possible implications for fusion and virus inactivation are discussed.

Hexestrol diazirine photoaffinity labeling reagent for the estrogen receptor.

3-Azibutyl (2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentyl sulfide (1), a photoaffinity labeling reagent for the estrogen receptor (ER), has been prepared in unlabeled and in high specific activity tritium-labeled form (32 Ci/mmol) and has been shown to undergo selective and efficient photocovalent attachment to rat uterine ER. Diazirine 1 demonstrates high binding affinity for ER, as determined by both a competitive binding assay and a direct binding assay (relative binding: estradiol = 100; (1) = 17. Kd: estradiol = 0.19 nM; (1) = 0.98 nM, respectively). It is efficient in site-specific photoinactivation of ER, reaching the level of 31% after 5 min of irradiation at > 315 nm. The tritium-labeled diazirine [3H]-1 undergoes specific photocovalent attachment to ER with an attachment efficiency of 29% and a selectivity of 90%. Both of these values are quite high for a photoaffinity reagent. SDS-polyacrylamide gel electrophoretic analysis of the photolabeled proteins shows specific labeling of a major species at M(r) 65,000, the same species that is labeled by [3H]tamoxifen aziridine, a well-characterized affinity label for ER. Hexestrol diazirine 1 is the first carbene-generating photoaffinity label that covalently labels ER with high efficiency and selectivity, and it should be useful in further studies on the hormone-binding domain of ER.

Oriented and covalent immobilization of target molecules to solid supports: synthesis and application of a light-activatable and thiol-reactive cross-linking reagent.

Light-dependent oriented and covalent immobilization of target molecules has been achieved by combining two modification procedures: light-dependent coupling of target molecules to inert surfaces and thiol-selective reactions occurring at macromolecule or substrate surfaces. For immobilization purposes the heterobifunctional reagent N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide was synthesized and chemically characterized. The photosensitivity of the carbene-generating reagent and its reactivity toward thiols were ascertained. Light-induced cross-linking properties of the reagent were documented (i) by reacting first the maleimide function with a thiolated surface, followed by carbene insertion into applied target molecules, (ii) by photochemical coupling of the reagent to an inert support followed by thermochemical reactions with thiol functions, and (iii) by thermochemical modification of target molecules prior to carbene-mediated insertion into surface materials. Procedures mentioned led to light-dependent covalent immobilization of target molecules including amino acids, a synthetic peptide, and antibody-derived F(ab') fragments. Topically selective, light-dependent immobilization was attained with the bifunctional reagent by irradiation of coated surfaces through patterned masks. Glass and polystyrene served as substrates. Molecular orientation is asserted by inherently available or selectively introduced terminal thiol functions in F(ab') fragments and synthetic polypeptides, respectively.

Synthesis of 2,3-diaziridinyl-1,4-naphthoquinone sulfonate derivatives as potential antineoplastic agents.

A new class of 2,3-diaziridinyl-1,4-naphthoquinone sulfonates (27 compounds) has been synthesized and evaluated as potential antineoplastic agents. The most active compounds, benzenesulfonate 4, p-toluenesulfonate 5, p-methoxybenzenesulfonate 7,8-quinolinesulfonate 17, and 2-thiophenesulfonate 20, in the aromatic sulfonate series, at their optimum daily dosage level of 25 mg/kg X 6, produced 100%, 90%, 75%, 80%, and 100% 50-day survivors, respectively, of L1210 tumor-bearing mice. At a lower optimum daily dosage level of 20 mg/kg X 6, treatments with p-fluorobenzenesulfonate 11 and p-nitrobenzenesulfonate 15 resulted in 100% and 80% 50-day survivors. In the aliphatic sulfonate series, methanesulfonate 21 produced 80% 50-day survivors at a 10 mg/kg daily dosage level X 6. Benzenesulfonate 4, p-fluorobenzenesulfonate 11, 8-quinolinesulfonate 17, and 2-thiophenesulfonate 20 derivatives were also tested in mice bearing the B16 melanoma; these agents gave T/C X 100 values of 180, 182, 219, and 161, respectively, in this neoplastic cell system. Structure-activity relationships of compounds of this class are discussed.

Hydrophobic photolabeling identifies BHA2 as the subunit mediating the interaction of bromelain-solubilized influenza virus hemagglutinin with liposomes at low pH.

To investigate the molecular basis of the low-pH-mediated interaction of the bromelain-solubilized ectodomain of influenza virus hemagglutinin (BHA) with membranes, we have photolabeled BHA in the presence of liposomes with the two carbene-generating, membrane-directed reagents 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) and a new analogue of a phospholipid, 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl][2-3H] undecanoyl]-sn-glycero-3-phosphocholine ([3H]-PTPC/11). With the latter reagent, BHA was labeled in a strictly pH-dependent manner, i.e., at pH 5 only, whereas with [125I]TID, labeling was seen also at pH 7. In all experiments, the label was selectively incorporated into the BHA2 polypeptide, demonstrating that the interaction of BHA with membranes is mediated through this subunit, possibly via its hydrophobic N-terminal segment. Similar experiments with a number of other water-soluble proteins (ovalbumin, carbonic anhydrase, alpha-lactalbumin, trypsin, and soybean trypsin inhibitor) indicate that the ability to interact with liposomes at low pH is not a property specific for BHA but is observed with other, perhaps most, proteins.

Synthesis and antineoplastic activity of 1a-formyl and 1a-thioformyl derivatives of mitomycin C and 2-methylaziridine.

A select number of 1-formyl- and 1-thioformyl-2-methylaziridine derivatives and the corresponding 1a-substituted mitomycin C analogues were synthesized and tested for antineoplastic activity by using an in vivo test with murine P388 leukemia. Select compounds were also tested in vivo with murine melanoma B16. Several of the mitomycin C derivatives displayed activity and some of the mitomycin C analogues were comparable in activity to the parent compound.

Desmethylnafoxidine aziridine: an electrophilic affinity label for the estrogen receptor with high efficiency and selectivity.

Desmethylnafoxidine aziridine (Naf-Az), an affinity label for the estrogen receptor based structurally on the antiestrogen nafoxidine, has been prepared in unlabeled and in high specific activity, tritium-labeled form and has been evaluated for its apparent competitive binding, and time-dependent irreversible, covalent attachment to the estrogen receptor. Naf-Az was synthesized through a key 1,2-diaryl-3,4-dihydronaphthalene intermediate that was prepared from 6-methoxy-1-tetralone by two routes involving alternate strategies for arylation. Conversion of the diaryldihydronaphthalene to Naf-Az through a series of deprotection-activation reactions culminated in ethyleneimine displacement of a methanesulfonate. The tritium-labeled material was prepared by tritium-iodine exchange on an iodinated methanesulfonate precursor, followed by ethyleneimine displacement. Compared to our previously-prepared reagent tamoxifen aziridine (Tam-Az), Naf-Az has a higher apparent competitive binding affinity, and it reacts with the estrogen receptor in cytosol preparations and in intact MCF-7 breast cancer cells rapidly and with at least comparable efficiency and selectivity. SDS-polyacrylamide gel electrophoretic analysis confirms its selective labeling of the Mr 66,000 estrogen receptor. Naf-Az should prove to be useful in studies aimed at characterizing the properties and structure of estrogen receptors.

In the search for new anticancer drugs. 19. A predictive design of N,N:N',N':N'',N''-tri-1,2-ethanediylphosphorothioic triamide (TEPA) analogues.

The nitroxyl-labeled analogues of N,N:N',N':N",N"-tri-1,2-ethanediylphosphoric triamide (TEPA), N,N:N',N'-bis(1,2-ethanediyl)-N"-[[(2,2,6,6-tetramethyl-1-oxypiperidi n-4- yl)amino]carbonyl]phosphoric triamide (5a) and N,N:N',N'-bis(1,2-ethanediyl)-N"-[[(2,2,5,5-tetramethyl-1-oxypyrrolid in-3- yl)amino]carbonyl]phosphoric triamide (11a), possess therapeutic indexes that are 8-12 times higher than those of thio-TEPA (1) and TEPA (2). The introduction of methyl groups into the aziridine ring, or the replacement of the nitroxyl moiety with hydroxylamine or amine derivatives, or with an adamantane moiety, results in compounds of lesser activity. An attempt is made to rationalize these results using a lipophilicity scale. A predictive design pattern is established.

Preparation and antitumor activity of 7-substituted 1,2-aziridinomitosenes.

7-Methoxy-1,2-aziridinomitosenes were prepared from mitomycin A and its N-methyl homologue by catalytic reduction followed by air oxidation. Treatment of these products with amines, including ammonia, ethylenimine, 2-methylethylenimine, propargylamine, and furfurylamine gave the corresponding 7-(substituted amino) derivatives. Screening of these compounds against P-388 leukemia in mice revealed some good activities. The more easily reduced compounds gave prolongation of life span comparable to that of mitomycin C, but their optimal doses were higher. Among these compounds, a methyl group on the aziridine nitrogen increased potency. The 7-amino derivatives, which were difficult to reduce to hydroquinones, were essentially inactive. The aziridinomitosenes were subjected to a Hansch-type analysis, but no statistically significant correlation was found.

Isolation, synthesis, and antitumor evaluation of spirohydantoin aziridine, a mutagenic metabolite of spirohydantoin mustard.

Spirohydantoin mustard (SHM), a central nervous system directed nitrogen mustard with anticancer activity, was metabolized in the presence of mouse liver postmitochondrial supernatant (9000g fraction) to a nonpolar alkylating metabolite. The metabolite was isolated by thin-layer chromatography of chloroform or ethyl acetate extracts of incubation mixtures, and its structure was established by mass spectral analysis, synthesis, and cochromatography. The metabolite, spirohydantoin aziridine, was mutagenic for Salmonella typhimurium TA1535 in the Ames assay but inactive as an antitumor agent against P388 leukemia in vivo.

In the search for new anticancer drugs. X. N,N; N',N'-bis(1,2-ethanediyl)-N''-(1-oxyl-2,2,6,6-tetramethyl- 4-piperidinylaminocarbonyl) phosphoric triamide--a new potential anticancer drug of high activity and low toxicity.

A new nitroxyl labeled TEPA derivative 5 containing the urea bridge between the phosphorus and the nitroxyl moiety, and the congeners containing the NOH and NH groups instead of the nitroxyl function were synthesized, and tested in vivo on CD2F1 mice for anticancer activity against P388 and L1210. The nitroxyl compound is more active than the reduced forms. The nitroxyl compound 5 elicits 170% ILS at 90 mg/kg after 30 days and 439% ILSmax after 60 days against P388, and has a higher therapeutic ratio (26.4) than the clinically used Thio-TEPA (2.75). The LD50 of 5 is 270 mg/kg, while that of Thio-TEPA is 18 mg/kg. Consequently, the nitroxyl compound 5 is a promising new anticancer drug.