Light-induced coupling of aqueous-soluble proteins to liposomes formed from carbene-generating phospholipids.

A novel bilayer-forming phospholipid analogue with a photoactivatable carbene-generating head group was synthesized and characterized with respect to molecular structure and light-induced reactivity. N'-(1,2-Dimyristoyl-sn-glycero-3-phosphoethyl)-N-[m-[3- (trifluoromethyl)diazirin-3-yl]phenyl]thiourea (PED) was prepared by thiocarbamoylation of synthetic dimyristoylphosphatidylethanolamine with 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine. PED formed liposomes in aqueous media. Gel to liquid-crystalline transitions occurred at 10.5 degrees C. Neither PED- nor PED/dimyristoylphosphatidylcholine mixed liposomes underwent major structural changes when photoactivated. Liposome sizes, determined by electron microscopy, were not altered upon light exposure. PED combines the advantages of facile synthesis and timed carbene reactivity by photoactivation at wavelengths greater than or equal to 320 nm. Conditions used for PED photoactivation did not inactivate catalytically active or complex-forming proteins. Light-induced binding of aqueous-soluble proteins to PED containing liposomes was attained through photoactivation in the presence of myoglobin, streptavidin, or trypsin. The proteins mentioned were utilized to characterize carbene-initiated ligand coupling. Procedures described establish a new and versatile method for the formation of proteoliposomes.     

A carbene-yielding amino acid for incorporation into peptide photoaffinity reagents

3-[p-[3-(Trifluoromethyl)-3H-diazirin-3-yl]phenyl]alanine has been prepared in 10 steps from p-bromobenzyl alcohol. The alcohol was converted to 3-(alpha-iodo-p-tolyl)-3-(trifluoromethyl)-3H-diazirine, which was used to alkylate N-(diphenylmethylene)glycine ethyl ester. After deprotection the amino acid was obtained with an overall yield of 18%. The D- and L-isomers of 3-[p-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]alanine have also been resolved, and 3-[p-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]alanine labeled with tritium has been prepared. The advantages of using this amino acid as a building block for peptide photoaffinity reagents is discussed.     

Synthesis and anticonvulsant activity of new 1-[2-oxo-2-(4-phenylpiperazin-1-yl)ethyl]pyrrolidine-2,5-diones

Twenty-two new 1-[2-oxo-2-(4-phenylpiperazin-1-yl)ethyl]pyrrolidine-2,5-diones were synthesized and tested for anticonvulsant activity. Initial anticonvulsant screening was performed using standard maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) screens in mice. Several compounds were tested additionally in the 6-Hz psychomotor seizure model. The neurotoxicity was determined applying the rotarod test. Excluding one compound, all other molecules were found to be effective in at least one seizure model. The most active were 1-(2-oxo-2-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}ethyl)pyrrolidine-2,5-dione (14), 1-{2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}-3-methylpyrrolidine-2,5-dione (17), 1-{2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}-3,3-dimethylpyrrolidine-2,5-dione (23) and 3,3-dimethyl-1-(2-oxo-2-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}ethyl)pyrrolidine-2,5-dione (26). These compounds showed high activity in the 6-Hz psychomotor seizure test as well as were active in the maximal electroshock and subcutaneous pentylenetetrazole (14 and 23) screens. Initial SAR studies for anticonvulsant activity have been discussed.     

Trifluoromethyldiazirine-Containing dUTP: Synthesis and Application in DNA/Protein Crosslinking

Abstract

The 5-[N-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoyl)-3-aminoallyl]-2′-deoxyuridine-5′-triphosphate was synthesized via acylation of 5-aminoallyl-2′-deoxyuridine-5′-triphosphate with 4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate N-hydroxysuccinimide. It was used for the preparation of 30 bp ATFMD-DNA coding for promoter sequence. UV-Irradiation (365 nm) of the specific complex of this duplex and E. coli RNA polymerase leads to the effective crosslinking DNA with all protein subunits.     

Syntheses of phenylpyrazolodiazepin-7-ones as conformationally rigid analogs of aminopyrazole amide scaffold and their antiproliferative effects on cancer cells

Recently, we have reported the syntheses and antiproliferative activities of N-(5-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl amide derivatives on melanoma cells. As a continuous work for antiproliferative agents in melanoma, here we report the synthesis of conformationally rigid analogs, phenyl-6,8-dihydropyrazolo[3,4-b][1,4]diazepin-7(1H)-one derivatives 7a-g, 8a-o and their antiproliferative activities against A375P melanoma cell line and U937 hematopoietic cell line. Most compounds showed competitive antiproliferative activities to sorafenib, the reference standard. Among them, N-(3-(1-benzyl-7-oxo-1,6,7,8-tetrahydropyrazolo[3,4-b][1,4]diazepin-5-yl)phenyl)-4-chloro-3-(trifluoro methyl)benzamide-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-(3-(4-chloro-3-(trifluoromethyl) phenyl) ureido)-2-methylbenzamide (7b) exhibited potent activities (GI(50)=0.43 μM and 0.06 μM) on both cell lines. It has been further confirmed to be a potent and selective Raf kinases inhibitor and also mild inhibitor of PI3Kα.     

Mefloquine-oxazolidine derivatives, derived from mefloquine and arenecarbaldehydes: In vitro activity including against the multidrug-resistant tuberculosis strain T113

Ten new mefloquine-oxazolidine derivatives, 4-[(1S,8aR)-3-(aryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinoline (1: aryl=substituted phenyl) and 4-[(1S,8aR)-3-(heteroaryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinoline [2: heteroaryl=5-nitrothien-2-yl (2a); 5-nitrofuran-2-yl (2b) and 4H-imidazol-2-yl) (2c)], have been synthesized and evaluated against Mycobacterium tuberculosis. Compounds 1f (aryl=3-ethoxyphenyl), 1g (Ar=3,4,5-(MeO)(3)-C(6)H(2)) and 2c were slightly more active than mefloquine (MIC=33μM) with MICs=24.5, 22.5 and 27.4, respectively, whereas compounds 1e (aryl=3,4-(MeO)(2)-C(6)H(3)) and 2a (MICs=11.9 and 12.1μM, respectively) were ca. 2.7 times more active than mefloquine, with a better tuberculostatic activity than the first line tuberculostatic agent ethambutol (MIC=15.9). The compounds were also assayed against the MDR strain T113 and the same MICs were observed. Thus the new derivatives have advantages over such anti-TB drugs as isoniazid, rifampicin, ethambutol and ofloxacin, for which this strain is resistant. The most active compounds were not cytotoxic to Murine Macrophages Cells in a concentration near their MIC values.     

Synthesis of a new Photo-Cross-Linking Nucleoside Analogue Containing an Aryl(Trifluoromethyl)Diazirine Group: Application for Eco RII and Mva I Restriction-Modification Enzymes

Abstract

A new photo-cross-linking dU analog, 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2′-deoxyuridine, was synthesized and incorporated into the recognition site of Eco RII and Mva I restriction-modification enzymes. The resulting base-modified 14-mer substrate was tested for cross-linking to these enzymes. Cross-linking is effected by irradiation of the enzyme-substrate complexs at 366 nm.     

Discovery of AZD3514, a small-molecule androgen receptor downregulator for treatment of advanced prostate cancer

Removal of the basic piperazine nitrogen atom, introduction of a solubilising end group and partial reduction of the triazolopyridazine moiety in the previously-described lead androgen receptor downregulator 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (1) addressed hERG and physical property issues, and led to clinical candidate 6-(4-{4-[2-(4-acetylpiperazin-1-yl)ethoxy]phenyl}piperidin-1-yl)-3-(trifluoromethyl)-7,8-dihydro[1,2,4]triazolo[4,3-b]pyridazine (12), designated AZD3514, that is being evaluated in a Phase I clinical trial in patients with castrate-resistant prostate cancer.     

Dual neurokinin NK(1)/NK(2) antagonists: N-[(R,R)-(E)-1-arylmethyl-3-(2-oxo-azepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamides and 3-[N'-3,5-bis(trifluoromethyl)benzoyl-N-arylmethyl-N'-methylhydrazino]-N-[(R)-2-oxo-azepan-3-yl]propionamides.

Based on the structure of N-[(R,R)-(E)-1-(4-chlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (1), attempts to improve the NK(2) affinity have resulted in the discovery of N-[(R,R)-(E)-1-(3,4-dichlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (9, DNK333) exhibiting a 5-fold improved affinity to the NK(2) receptor in comparison to 1. Simplification of the structure via elimination of a chiral centre led to 3-[N'-3,5-bis(trifluoromethyl)benzoyl-N-(3,4-dichlorobenzyl)-N'-methylhydrazino]-N-[(R)-2-oxo-azepan-3-yl]propionamide (22), a potent and fairly balanced NK(1)/NK(2) antagonist.     

Design, structure-activity relationship, and highly efficient asymmetric synthesis of 3-phenyl-4-benzylaminopiperidine derivatives as novel neurokinin-1 receptor antagonists

We synthesized a series of novel 3-phenyl-4-benzylaminopiperidine derivatives that were identified as potent tachykinin NK(1) receptor antagonists by structural modification of the 3-benzhydrylpiperidone derivative through high-throughput screening. N-{2-[(3R,4S)-4-({2-Methoxy-5-[5-(trifluoromethyl)-1H-tetrazol-1-yl]benzyl}amino)-3-phenyl-1-piperidinyl]-2-oxoethyl}acetamide ((+)-39) was found to be one of the most potent tachykinin NK(1) receptor antagonists with high metabolic stability. Highly efficient asymmetric synthesis of (+)-39 was achieved via dynamic kinetic resolution.     

Amino substituted analogs of 1-phenyl-3-phenylimino-2-indolones with potent galanin Gal3 receptor binding affinity and improved solubility

A series of amino analogs of 1,3-dihydro-1-phenyl-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one (1) were synthesized to improve aqueous solubility, while retaining high affinity for the human galanin Gal3 receptor. A very potent analog (9e, 1,3-dihydro-1-[3-(2-pyrrolidinylethoxy)phenyl]-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one, Ki=5 nM) shows good selectivity and solubility of 48 microg/mL at pH 7.4.     

The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion

The interaction between influenza virus and target membrane lipids during membrane fusion was studied with hydrophobic photoactivatable probes. Two probes, the newly synthesized bisphospholipid diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine and the phospholipid analogue 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]-[2-3H]- undecanoyl]-sn-glycero-3-phosphocholine (Harter, C., B?chi, T., Semenza, G., and Brunner , J. (1988) Biochemistry 27, 1856-1864), were used. Both labeled the HA2 subunit of the virus at low pH. By measuring virus-liposome interactions at 0 degrees C, it could be demonstrated that HA2 was inserted into the target membrane prior to fusion. As we have recently demonstrated, at this temperature, exposure of the fusion peptide of HA2 takes place within 15 s after acidification, but fusion does not start for 4 min (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). HA2 was labeled at least 2 min before fusion. No labeling of the HA1 subunit was seen. These data indicate that fusion is triggered by a direct interaction of the HA2 subunit of a kinetic intermediate form of HA with the lipids of the target membrane. Most likely, it is the fusion peptide of HA2 that is inserted into the target membrane. Just before fusion, HA is thus an integral membrane protein in both membranes. In contrast, the bromelain-derived ectodomain of HA was labeled by 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]- [2-3H]undecanoyl)-sn-glycerol-3-phosphocholine at low pH but not by diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine. This indicates that insertion of the fusion peptide of the bromelain-derived ectodomain of HA into a membrane differs from that of viral HA during fusion.     

Synthesis of some N-[4-(benzothiazole-2yl) phenyl]-2-aryloxyacetamide derivatives and their anticancer activities

In this study, some N-[4-(Benzothiazole-2-yl) phenyl]-2-aryloxyacetamide derivatives were prepared by reacting N-[4-(benzothiazole-2yl)phenyl]-2-chloroacetamide and different substituent phenol or thiophenol derivatives. The anticancer activities of the compounds obtained were investigated. It was observed that some of the compounds, namely 25 and 38, showed notable anticancer activity.     

Synthesis of some novel pyrazolo[3,4-d]pyrimidine derivatives as potential antimicrobial agents

The reaction of 4-hydrazino-8-(trifluoromethyl)quinoline (2) with ethoxymethylenecyanoacetate afforded ethyl 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylate (3) and that with ethoxymethylenemalononitrile afforded 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carbonitrile (5). Compounds 3 and 5 were hydrolyzed to get 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylic acid and then reacted with acetic anhydride to afford 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]pyrazolo[3,4-d]oxazin-4-one (6), which was condensed with different aromatic amines to give a series of 5-substituted 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (7). Compounds 3 and 5 also reacted with formamide, urea, and thiourea affording the corresponding pyrazolo[3,4-d]pyrimidines (8-13), respectively. Structures of the products have been determined by chemical reactions and spectral studies. All compounds of the series have been screened for their antibacterial and antifungal activity studies. The results are summarized in Tables 1 and 2.     

Prostate cancer PET bioprobes: synthesis of [18F]-radiolabeled hydroxyflutamide derivatives

Approximately 80-90% of prostate cancers are androgen dependent at initial diagnosis. The androgen receptor (AR) is present in most advanced prostate cancer specimens and is believed to have a critical role in its development. Today, treatment of prostate cancer is done by inhibition of AR using antiandrogens such as flutamide (pro-drug of hydroxyflutamide), nilutamide, and bicalutamide. However, there is currently no noninvasive imaging modalities to detect, guide, and monitor specific treatment of AR-positive prostate cancer. (R)-3-Bromo-N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-propanamide [18F]-1 and N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide [18F]-2, derivatives of hydroxyflutamide, were synthesized as a fluorine-containing imaging agent candidates. A three-step fluorine-18 radiosynthesis route was developed, and the compounds were successfully labeled with a 10+/-3% decay corrected radiochemical yield, 95% radiochemical purity, and a specific activity of 1500+/-200 Ci/mmol end of bombardment (n = 10). These labeled biprobes not only may enable for the future quantitative molecular imaging of AR-positive prostate cancer using positron emission tomography but may also allow for image-guided treatment of prostate cancer.     

Synthesis of some N-[4-(benzothiazole-2yl) phenyl]-2-aryloxyacetamide derivatives and their anticancer activities

In this study, some N-[4-(Benzothiazole-2-yl) phenyl]-2-aryloxyacetamide derivatives were prepared by reacting N-[4-(benzothiazole-2yl)phenyl]-2-chloroacetamide and different substituent phenol or thiophenol derivatives. The anticancer activities of the compounds obtained were investigated. It was observed that some of the compounds, namely 25 and 38, showed notable anticancer activity.     

Evidence for a central apolipoprotein A-I domain loosely bound to lipids in discoidal lipoproteins that is capable of penetrating the bilayer of phospholipid vesicles

Previous evidence indicated that discoidal reconstituted high density lipoproteins (rHDL) of apolipoprotein A-I (apoA-I) can interact with lipid membranes (Tricerri, M. A., Córsico, B., Toledo, J. D., Garda, H. A., and Brenner, R. R. (1998) Biochim. Biophys. Acta 1391, 67-78). With the aim of studying this interaction, photoactivable reagents and protein cleavage with CNBr and hydroxylamine were used. The generic hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine gave information on the apoA-I regions in contact with the lipid phase in the rHDL discs. Two protein regions loosely bound to lipids were detected: a C-terminal domain and a central one located between residues 87 and 112. They consist of class Y amphipathic alpha-helices that have a different distribution of the charged residues in their polar faces by comparison with class A helices, which predominate in the rest of the apoA-I molecule. The phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoro-methyl-3-H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, which does not undergo significant exchange between membranes and lipoproteins, was used to identify the apoA-I domain directly involved in the interaction of rHDL discs with membranes. By incubating either rHDL or lipid-free apoA-I with lipid vesicles containing 125I-TID-PC, only the 87-112 apoA-I segment becomes labeled after photoactivation. These results indicate that the central domain formed by two type Y helices swings away from lipid contact in the discoidal lipoproteins and is able to insert into membrane bilayers, a process that may be of great importance for the mechanism of cholesterol exchange between high density lipoproteins and cell membranes.     

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.     

Novel vasopressin V2 receptor-selective antagonists, pyrrolo[2,1-a]quinoxaline and pyrrolo[2,1-c][1,4]benzodiazepine derivatives.

The intent of the work was to study the structure-activity relationships of AVP receptor antagonists bearing a chiral ring as a partial structure since such studies had been reported for only achiral compounds. In the present paper, we deal with compounds consisting of the chiral tricyclic hetero ring (1,2,3,3a,4,5-hexahydropyrrolo[1,2-a]quinoxaline and 1,2,3,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine) and 2-phenylbenzanilide analogues. These compounds exhibited a highly selective affinity for V2 receptor, and their stereochemical configuration had a great influence on V2 receptor binding. VP-343 (N-[4-[[(2S,3aR)-2-hydroxy-2,3,3a,4-tetrahydropyrrolo[1,2-a] quinoxalin-5(1H)-yl]carbonyl]phenyl]-4'-methyl[1,1'-biphenyl]-2-ca rboxamide), VP-365 (N-[4-[[(11aS)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benz odiazepin-10(5H)-yl]carbonyl]phenyl][1,1'-biphenyl-2-carboxamide) and VP-339 (N-[4-[[(11aS)-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]+ ++benzodiazepin-10(5H)-yl]carbonyl]phenyl][1,1'-biphenyl]-2-carboxami de) were the most potent compounds in vitro and in vivo. The IC50 values of VP-343, VP-365 and VP-339 against V2 receptor were 0.772, 1.18 and 0.216 nM, respectively. The ED300 values (dose required to increase three times the urine volume of the control rats; oral administration) of VP-343, VP-365 and VP-339 were 0.22, 0.31 and 0.78 mg/kg, respectively.