Synthesis and biological evaluation of prodigiosene conjugates of porphyrin,estrone and 4-hydroxytamoxifen

To generate the first series of prodigiosene conjugates, the tripyrrolic skeleton was appended to estrone, tamoxifen and porphyrin frameworks by way of ester linkers and various hydrocarbon chain lengths. The ability of the conjugates to inhibit various types of cancer cells was evaluated in vitro. The porphyrin conjugates did not exhibit significant activity. The estrone conjugates exhibited modest activity, for the most part. However, significantly greater growth inhibition activity against certain breast, colon, lung, leukemia, melanoma and prostate cell lines was noted. This unusual effect for this first generation model class of compound warrants further investigation and comparison to cases where estrogens are linked to prodigiosenes via connection points that do not feature in estrogen receptor binding. The 4-hydroxytamoxifen conjugates exhibit nanomolar range activity against the MCF-7 breast cancer cell line, paving the way to expand the scope and connectivity of prodigiosene–tamoxifen conjugates.     

Design and studies of novel polyoxysterol-based porphyrin conjugates

New types of steroid-porphyrin conjugates derived from 20-hydroxyecdysone (20E) and 24-epibrassinolide (EBl) were synthesized. An exceptional regioselectivity in the reaction of both steroids with porphyrin boronic acids was found to give side-chain-conjugated boronic esters as sole products. UV-Vis-, fluorescence and NMR spectroscopy yielded similar data for all the studied compounds confirming the solvent driven supramolecular assembly with formation of J-aggregates. CD measurements of water diluted solutions showed a clear difference between 20E and EBl conjugates. The latter showed a strong supramolecular chirality, whereas 20E J-aggregates did not.     

New porphyrin amino acid conjugates: Synthesis and photodynamic effect in human epithelial cells

The efficacy of new porphyrin amino acid conjugates as photosensitizers for photodynamic therapy (PDT) were assayed in vitro on tumoral (HeLa) and on non tumoral (HaCaT) human cell lines. The conjugates stable in liposomes are able to penetrate efficiently in the cytoplasm of cultured cancer and normal cells. No dark cytotoxicity is observed at the same concentration used for PDT cell treatment and during long incubation time (24 h). The cell survival after the PDT treatment with visible light is dependent upon light exposure level and compound concentration. The tested compounds show higher photocytotoxicity in tumoral HeLa cells than in no tumoral HaCaT cells. The results suggest that these amino acid porphyrin conjugates are potential photosensitizers for PDT.     

Synthesis of aminoglycoside-DNA conjugates

Development of aminoglycoside-nucleic acid conjugates is presented. Synthesis of a DNA dimer covalently linked to kanamycin and neomycin isothiocyanates has been carried out. The development of such conjugates will help couple the sequence specificity of nucleic acids to the electrostatic/shape complementarity of aminoglycoside antibiotics in binding nucleic acid targets.     

Synthesis of pyochelin-norfloxacin conjugates

Using synthetic functionalized analogues of pyochelin, a siderophore common to several pathogenic Pseudomonas and Burkholderia species, four fluoroquinolone-pyochelin conjugates were efficiently synthesized and evaluated for their biological activities.     

Synthesis of carotenoid-cysteine conjugates

Isozeaxanthin under acidic conditions forms an allylic cation which reacts readily with thiol nucleophiles. With N-acetylcysteine as a nucleophile the products obtained are carotenoid-cysteine conjugates in which the amino acid moiety is attached to the carotenoid via sulphur in position 4. The water solubility of the products can be increased by deprotection of the amino group. The antioxidant activity of the products were examined on human liver cells under conditions of hydrogen-peroxide induced oxidative stress.     

Synthesis and characterization of digoxin-phospholipid conjugates

The preparation of immunoreactive derivatives of digoxin for analytical applications is most often carried out by periodate cleavage of the terminal sugar ring (digitoxose) followed by reaction with an enzyme, protein, carrier, or related biological molecules. Here we report an improved and more efficient synthesis which was developed to provide digoxin-phospholipid conjugates useful for liposome immunoassay. The approach used involved the linking of the cleaved digitoxose through a carboxymethyl oxime functionality, which provides much improved yields of readily purified products. The synthetic modification should be applicable to the preparation of analogous phospholipid conjugates involving linkage through a sugar ring (digitoxin, ouabain, and related cardiac glycosides) or to those involving steroids (i.e., 3-digoxigenone) which can be modified to form oxime derivatives remote from key functionalities important for immunorecognition by specific antibody. The characterization of the digoxin-phospholipid conjugates with high-resolution NMR and fast atom bombardment mass spectrophotometry will also be discussed.     

Synthesis and characterization of insulin-transferrin conjugates

Receptor-mediated endocytosis can be exploited for improving the transcellular delivery of therapeutic proteins. Insulin conjugated to transferrin by forming disulfide bonds has been shown to improve insulin oral bioavailability in diabetic rats. We are developing a combination strategy involving complexation hydrogels as delivery vehicles for insulin-transferrin conjugates. The complexation hydrogels developed in our laboratory have been shown to be promising carriers for oral delivery of proteins and peptides. Integrating the strategies based on the complexation hydrogels and insulin-transferrin conjugates may prove to be a novel approach for oral delivery of insulin and other therapeutic proteins. In this work, electrospray ionization mass spectrometry (ESI-MS) was used to study the modification of insulin during its reaction with transferrin. The stability of the conjugated insulin to enzymatic degradation was also studied. ESI-MS studies confirmed the site-specific modifications of insulin. The transferrin conjugation of insulin was also shown to increase the stability of insulin to enzymatic degradation.     

Synthesis and applications of oligonucleotide-carbohydrate conjugates

Nowadays, oligonucleotide-carbohydrate conjugates are used in antisense biotechnology and in the study of glycosylated DNA functioning in vitro. The application of mono- and disaccharide phosphoramidites, solid-phase supports with immobilized carbohydrates, glycosylated nucleoside phosphoramidites, and postsynthetic conjugation of reactive sugar derivatives with oligonucleotides for preparation of oligonucleotide-carbohydrate conjugates have been systematically studied. The advantages and disadvantages of these approaches are considered. Possible strategies for synthesis of glycoclusters with different topologies conjugated to DNA are discussed. Applications of oligonucleotide-carbohydrate conjugates are highlighted. Studies of interactions of glycosylated oligonucleotides with proteins and effective cell-specific delivery of oligonucleotide-carbohydrate conjugates are discussed.     

Synthesis and electrochemistry of anthraquinone-oligodeoxynucleotide conjugates

Electroactive oligodeoxynucleotides (ODNs) with specific base sequences have a potential application as electrical sensors for DNA molecules. To this end, a phosphoramidite that bears a 9, 10-anthraquinone (AQ) group tethered to the 2'-O of the uridine via a hexylamino linker, 2'-O-[6-[2-oxo(9, 10-anthraquinon-2-yl)amino]hexyl]-5'-O-(4,4'-dimethoxytrityl)uridi ne 3'-[2-(cyanoethyl)bis(1-methylethyl)phosphoramidite] (3), has been synthesized and used to prepare three ODNs with tethered AQs using standard phosphoramidite chemistry. The synthetic methodology thus allows the synthesis of ODNs with electroactive tags attached to given locations in the base sequence. Cyclic voltammetric behavior of these AQ-ODN conjugates was examined in aqueous buffer solutions at a hanging mercury drop electrode. At slow sweep rates, nearly reversible two-electron waves characteristic of an adsorbed anthraquinone/hydroquinone redox couple was observed for all of the AQ-ODN conjugates. Approximate Langmuirian isotherms were found for the AQ-ODNs with molecular footprints, calculated from the saturation coverages, that scaled with molecular size. The cyclic voltammetric response of the duplexes formed from the AQ-ODNs and their complementary ODN was complicated by the competitive adsorption of the individual ODNs and possibly the duplex species as well.     

Synthesis of radiolabeled cytarabine conjugates

N4-Modified, novel Ara-C conjugate capable of radiolabeling with gamma ray-emitting (^99mTc) as well as positron emitting (¹⁸F) radionuclides, that is, N4-hydrazine derivative was synthesized. The radiolabeling of N4-(hydrazinonicotinyl)-1-β-arabinofuranosyl cytosine (HAra-C) with ^99mTc was performed with over 95% labeling yield. To label HAra-C with ¹⁸F, 4-fluoro(¹⁸F)-benzaldehyde was synthesized from 4-formyl-N,N,N-trimethylanilinium triflate in 30% radiochemical yield; it quantitatively formed hydrazone derivative with HAra-C within 45 min. The radiolabeled conjugates were analyzed by radio-UV-RP-HPLC. The cold precursors were characterized by ¹H, ¹³C NMR. Additionally, HAra-C was evaluated for cytotoxicity in lung adenocarcinoma (H441) cells and found to be comparable in cell killing efficiency to that of Ara-C. Uptake of ^99mTc-HAra-C in cultures of H441 cells and sensitive pancreatic cancer cells (MIAPaCa-2) was inhibited by nucleoside transporter inhibitor nitrobenzylthioinosine. The results suggest that ^99mTc-labeled HAra-C is a substrate for the membrane nucleoside transporters, and that it may be used in molecular imaging of nucleoside transporter expression for the verification of potential anticancer efficacy of nucleoside drugs, such as Ara-C and gemcitabine.     

Synthesis of site-specific methotrexate-IgG conjugates

Summary With a view to increasing drug incorporation without loss of antibody activity, tritium-labeled methotrexate (MTX) was covalently linked to a polyclonal rabbit IgG antibody against bovine serum albumin and a monoclonal mouse IgG antibody against human renal cancer (Dal K20) by a site-specific method based on hydrazone bond formation between MTX hydrazide and the aldehyde groups generated by periodate oxidation of carbohydrate moieties in IgG (which are uncommon in the antigen-binding region). These conjugates were compared with the corresponding non-site-specific MTX-IgG conjugates produced by the N-hydroxysuccinimide active-ester method with regard to synthesis, stability, retention of antibody activity, inhibition of the target enzyme dihydrofolate reductase and antitumor effect. Incorporation levels achieved with the hydrazide method were no greater than with the active-ester method, typically 6–7 mol MTX/mol IgG. Approximately the same dihydrofolate-reductase-inhibitory capacity was observed for MTX bound by either method. Hydrazide conjugates lost bound drug more rapidly than active-ester conjugates on freezing and thawing, on incubation at 37° C and 51° C, and in the presence of serum or rat liver homogenates. Exposure to rat liver homogenates at 37° C, pH 4.6, for 24 h led to the loss of 50%–60% of the bound drug from hydrazide conjugates compared to 20%–30% from the active ester conjugates. Bio-Gel P-2 chromatography of low-molecular-mass fractions, obtained after exposure of each of the conjugates to liver homogenates, revealed the presence of a compound that had the same elution volume and R _F on thin-layer chromatography as free MTX. Enzyme-linked immunosorbent assay showed loss of antibody activity of both types of conjugates at 51° C and on freezing and thawing. In a clonogenic assay, the active-ester conjugate of Dal K20 appeared to be equally effective or slightly better as a tumor inhibitor than the corresponding hydrazide conjugate. The hydrazide method may be useful in linking MTX to those monoclonal antibodies that tend to denature when subjected to the active-ester method of linkage. Abbreviations used: aBSA, rabbit anti-(bovine serum albumin) IgG; EDCI, 3-ethyl-1-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbant assay; IC₅₀, concentration giving 50% inhibition; MTX, methotrexate; MTXAE, N-hydroxy-succinimide-based active ester of MTX; MTXAE-IgG, MTX-IgG conjugate prepared by the active-ester method; MTXH, methotrexate hydrazide; MTXH-IgG, MTX-IgG conjugate prepared by the hydrazide method; PBS, 0.01 M sodium phosphate, pH 7.1, containing 0.145 M sodium chloride; TLC, thin-layer chromatography     

Synthesis of uniform protein-polymer conjugates

We have developed a novel technique to synthesize near-uniform protein-polymer conjugates by initiating atom transfer radical polymerization of monomethoxy poly(ethylene glycol)-methacrylate from 2-bromoisobutyramide derivatives of chymotrypsin (a protein-initiator). Polymerization initiated from the monosubstituted protein-initiator resulted in the conjugate containing a single, near-monodisperse polymer chain per protein molecule with polydispersity index 1.05. Increasing the number of conjugated 2-bromoisobutyramide initiators per molecule of protein increased the molecular weights and polydispersity indices of the final protein-polymer conjugates. The generic nature of this technique was demonstrated by initiating polymerization of nonionic, cationic, and anionic monomers from the protein-initiator. Protein-polymer conjugates synthesized by this novel technique retained 50-86% of the original enzyme activity. The technique described herein should be useful in synthesizing well-defined protein-polymer conjugates exhibiting a wide range of physical and chemical properties.     

Synthesis and biological activities of nucleoside-estradiol conjugates

Nucleosides were coupled to estradiol via a 17alpha-ethynyl spacer group using Pd(II) as a catalyst. The conjugates were evaluated in vitro for estrogen receptor (ER) binding affinity and cytotoxicity against cell lines with and without ER. The highest receptor binding affinities (RBA approximately 3) were observed with conjugates coupled via a relative long spacer group, while none of the conjugates exhibited cytotoxicity against either cell lines.     

Synthesis and cellular studies of porphyrin-cobaltacarborane conjugates

The total syntheses of five new porphyrin-cobaltacarborane conjugates (1-5) have been achieved in 88-98% yields in a single-step reaction between a nucleophilic meso-pyridyl-containing porphyrin and zwitterionic cobaltacarborane [3,3'-Co(8-C(4)H(8)O(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))]. These unique zwitterionic compounds have one to four cobaltabisdicarbollide anions conjugated to the porphyrin macrocycle via (CH(2)CH(2)O)(2) chains. The X-ray structure of one of these conjugates (1) is presented and discussed. The cellular uptake, cytotoxicity, and subcellular localization of cobaltacarboraneporphyrins 1-5 were investigated in human HEp2 cells. The number and distribution of cobaltacarborane residues linked to the porphyrin macrocycle has a significant effect on the cellular uptake of the conjugates.     

Synthesis and biological evaluation of PEG-tirofiban conjugates

We have conjugated tirofiban, an antagonist of the GPIIb/IIIa integrin receptor, to PEG, and shown that these polymers effectively inhibit platelet aggregation. This inhibition decreased with the size of the polymer. Our goal was to develop new cryoprotective agents to store frozen platelets. Surprisingly, tirofiban-conjugated PEG did not exhibit any protection.     

Synthesis and anticancer activity of open-resorcinarene conjugates

The first example of conjugation of open-resorcinarenes with chlorambucil, ibuprofen, naproxen and indomethacin are presented. The cytotoxic properties of the obtained conjugates were tested against the cancer cell lines U-251, PC-3, K-562, HCT-15, MCF-7 and SKLU-1. It was found that the conjugate with chlorambucil, naproxen or indomethacin (having 8 moieties) was toxic towards cancer cell lines U-251 and K-562, with no activity against non-cancerous COS-7 cells. The conjugates with naproxen and indomethacin showed high selectivity towards U-251 tumor cells.     

Synthesis and biological activities of phthalocyanine-estradiol conjugates

Phthalocyanine-based photosensitizers, coupled via a 17alpha-ethynyl group to estradiol using Pd(II) as a catalyst, were synthesized and evaluated for their estrogen receptor binding affinity and in vitro photocytotoxicity. The highest receptor binding affinities (RBA=8-13) were observed with lipophilic conjugates coupled via a relative long spacer group while the sulfonated analogues showed little binding affinities (RBA <2). The highest photocytotoxicity was observed with the sulfonated conjugates, the nature of the spacer group did not have a pronounced effect.     

苯并异噻唑啉酮类化合物的生物活性研究

对合成的5种苯并异噻唑啉酮类化合物进行了生物活性研究。抑菌试验结果表明,化合物a与化合物b对大肠杆菌最小抑菌浓度为12.5mg/L。对金黄色葡萄球菌都表现出良好的抑菌活性。对亚心形扁藻,蛋白核小球藻,球等鞭金藻,化合物都有抑制其生长的活性,亚心形扁藻耐受性最高,球等鞭金藻的耐受性最低,蛋白核小球藻的耐受性居中。     

Synthesis of bioadhesive lectin-HPMA copolymer-cyclosporin conjugates

An amino group containing cyclosporin A (CsA) derivative has been synthesized and conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via an aromatic azo bond, which can be specifically cleaved by azoreductase activity in colon to release the drug for the treatment of colon diseases. Lectins, peanut (Arachis hypogea) agglutinin (PNA) and wheat germ agglutinin (WGA), have been conjugated to HPMA copolymer-CsA derivative conjugates (PCsA), respectively, to give bioadhesive conjugates. The PNA and WGA are the targeting proteins that can bind to diseased colon tissue and healthy tissue, respectively. There were on average four P(CsA) copolymer chains attached on one WGA molecule with a drug content of 16.0 wt % and five P(CsA) copolymer chains attached on one PNA molecule with a drug content of 11.5 wt %. The incubation of a P(CsA) copolymer with the rat cecal contents resulted in the cleavage of the azo bond and release of the cyclosporin derivative. The biological evaluation of the conjugates is under way.