Synthesis of a convenient thymidine glycol phosphoramidite monomer and its site-specific incorporation into DNA fragments

An original phosphoramidite building block of the thymidine glycol lesion has been prepared taking into account the additional diol function and the high lability of this oxidatively induced nucleobase damage. Then the modified nucleoside was site-specifically inserted into DNA fragments by solid support assembling followed by a "one-step" mild final deprotection treatment.     

Synthesis of monomers bearing at the 2'-position cyanomethoxycarbonyl group for phosphoramidite oligonucleotide synthesis

A novel series of phosphoroamidites for the synthesis of 2-modified oligonucleotides was designed and synthesized on the base of 2'-amino uridine and 2'-amino arabinoadenosine. The amino groups in these compounds were acidified by bis-cyanomethyl esters of different dicarbonic acids. Generated reactive linker groups containing cyanomethoxycarbonyl groups are stable under conditions of oligonucleotide synthesis but could be easily functionalised in post-synthetic stage by treatment with compounds bearing primary amino groups.     

Design,synthesis and evaluation of novel,potent DNA alkylating agents and their antibody-drug conjugates (ADCs)

Antibody-drug conjugates (ADCs) incorporating potent indolinobenzodiazepine (IGN) DNA alkylators as the cytotoxic payload are currently undergoing clinical evaluation. The optimized design of these payloads consists of an unsymmetrical dimer possessing both an imine and an amine effectively eliminating DNA crosslinking and demonstrating improved tolerability in mice. Here we present an alternate approach to generating DNA alkylating ADCs by linking the IGN monomer with a biaryl system which has a high DNA binding affinity to potentially enhance tolerability. These BIA ADCs were found to be highly cytotoxic in vitro and demonstrated potent antitumor activity in vivo.     

Chemical synthesis and characterization of conjugates of a novel catechol-O-methyltransferase inhibitor

BIA 3-202, 1-(3,4-dihydroxy-5-nitrophenyl)-2-phenylethanone 3, is a novel, reversible, and tight-binding peripheral inhibitor of the enzyme catechol-O-methyltransferase (COMT), which is currently under clinical evaluation for the treatment of Parkinson's disease as an adjunct to current L-Dopa/peripheral decarboxylase inhibitor therapy. Chemically pure, well-characterized reference standards of conjugates of 3 were required for investigation of the routes of metabolism in several animal species (including humans) and for pharmacokinetic studies. The lack of suitable literature precedents for efficient, regioselective synthesis of nitrocatechol conjugate metabolites prompted us to develop efficient and highly selective chemical preparations of O-glucuronide and O-sulfate conjugates of 3 such that multigram quantities of excellent purity can now be conveniently synthesized. It is anticipated that these procedures could be applied to the synthesis of conjugates of other COMT inhibitors, also based on the 3-nitrocatechol pharmacophore.     

Design and synthesis of novel antibacterial peptide-resin conjugates

We synthesized a novel peptide-resin conjugate by immobilizing beta-sheet antibacterial peptide on PEG-PS resin. The peptide-resin conjugate, similar to cationic antimicrobial peptides, demonstrated unique properties such as potent antibacterial activity, no hemolytic activity, lipid membrane perturbation activity, and potent synergism with vancomycin. Specially, the peptide-resin conjugate showed a more increased lipid membrane perturbation activity in comparison to unbound beta-sheet antibacterial peptide.     

A novel approach for the solid phase synthesis of DNA-peptide conjugates

The strategy of this study involves automated synthesis of oligonucleotides on a CPG support using standard cyanoethyl phosphoramidite chemistry followed by covalent linkage to peptide fragments bearing a free terminal alpha-amino group and residues with protected side chains. Conjugation was formed through an alkyldiisocyanate linker. Conjugates were isolated by cleavage from the solid support and deprotection in one step.     

A convenient solid-phase method for the synthesis of novel oligonucleotide-folate conjugates

We describe the preparation of two batches of a polymer support for the incorporation of folic acid into oligonucleotides. The method permits the regioselective attachment of a target nucleic acid sequence through its 3'-end to either the alpha-or gamma-carboxyl group of L-glutamic acid, respectively. The supports have been tested in solid-phase synthesis of oligonucleotide-folate conjugates for cell delivery studies.     

Effect of thiol pendant conjugates on plasmid DNA binding, release, and stability of polymeric delivery vectors

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release.     

A novel versatile phosphoramidite building block for the synthesis of 5'- and 3'-hydrazide modified oligonucleotides

We introduce a novel versatile phosphoramidite building block for the modification of oligonucleotides (ONs) with acyl hydrazides on the 5'- or 3'-terminus, or both. The reaction of these hydrazide functionalized ONs with 4-methoxyphenylaldehyde is demonstrated for solution derivatization. Hydrazides are considered nowadays as promising reactants, which show enhanced reactivity at neutral and slightly acidic conditions and higher stability of yielding products as compared to the aliphatic amines, which are broadly used for ONs derivatization. Our method to introduce hydrazides into ONs employs a phosphoramidite modifier designed to split, during ammonia or lithium hydroxide treatment, into two hydrazides via beta-elimination of a central bis-2-carbonylethoxysulfone unit. It allows the creation of ONs derivatized with a hydrazide moiety at the 5'-, 3'- and both 5'- and 3'-termini, as well as two different hydrazide containing ONs at the same time, viz. in one sequence on the same solid support In latter case one can, for example, synthesize two hydrazide containing ONs, where one is 5'-modified and second one is 3'-modified.     

Alpha-hydroxybenzylphosphonate modified oligonucleotides: synthesis, properties, and a novel route via monomer building blocks

In general, alpha-hydroxybenzylphosphonate modified 2'-deoxyadenosine-thymidine dimer building blocks 1, 2 are utilized for the incorporation into alpha-hydroxybenzylphosphonate pro-oligonucleotides. For a universal application of our pro-oligonucleotide concept on biologically relevant oligonucleotides a route for the synthesis of modified monomer building blocks 3 was developed and is presented herein.     

Synthesis and cytotoxicity of novel fatty acid-nucleoside conjugates

N(3)-Hydroxyethyltegafur (3) was synthesized in 91.0% yield under a new condition. A series of novel fatty acid esters of 3 were synthesized. These fatty acid-nucleoside conjugates have shown cytotoxicities against Ec9706 cells and A549 cells, and the structure activity relationship was discussed.     

Synthesis and biological evaluation of novel daunorubicin-estrogen conjugates

The synthesis of two novel daunorubicin-estrogen conjugates with a steroidal and a non-steroidal ligand was undertaken in an attempt to target the cytotoxicity of anthracycline to estrogen-receptor positive cells. These conjugates (3 and 4), in contrast to their corresponding ligands, displayed weak binding affinities of 0.079 and 0.851 for the estrogen receptor. Conjugate 3 was consistently more cytotoxic than 4, which however showed some selectivity to estrogen receptor positive cell lines.     

A novel thymidine phosphoramidite synthon for incorporation of internucleoside phosphate linkers during automated oligodeoxynucleotide synthesis

A novel thymidine phosphoramidite synthon was synthesized and successfully used for incorporation of primary amino groups, attached through a triethylene glycol linker to the internucleoside phosphates, at desired locations during automated oligodeoxynucleotide synthesis. The synthesized amino-linker bearing oligonucleotides are stable under deprotection conditions and exhibit Watson-Crick base-pairing properties. Covalent labeling of oligonucleotides with carbocyanine near-infrared fluorochromes resulted in 2.5 times higher labeling yields when compared with oligonucleotides containing base-attached aminolinkers. We anticipate that the developed synthetic approach will be useful for nucleotide sequence-specific attachment of single or multiple ligands or reporter molecules.     

Some novel approaches to the design and synthesis of peptide-catecholamine conjugates

A series of novel, functionalized catecholamines (congeners) has been synthesized in which, formalistically, the N-isopropyl group of isoproterenol has been extended by a linear alkyl chain of varying length, terminated by a carboxyl group. Model amide derivatives have also been prepared in order to optimize the biological activity of these derivatives and also to aid in the design of appropriate peptides for the synthesis of conjugates. As a result of these studies, a series of amino acid and monodisperse peptide carriers, containing p-aminophenylalanine as the point of attachment for the drug, was prepared, together with the corresponding conjugates. In vitro and in vivo evaluation of the congeners, model amides, and conjugates has demonstrated that the biological activity of these derivatives is extremely sensitive to structural modifications at a point far-removed from the pharmacophore, in both the congener amide and conjugate series. A number of the model amides and conjugates have proven to be highly active when tested in both in vitro and in vivo test systems. The implications of these results in terms of a novel structure–activity approach to drug design are discussed.     

Synthesis,antimalarial properties and 2D-QSAR studies of novel triazole-quinine conjugates

Click chemistry technique led to novel 1,2,3-triazole-quinine conjugates 8a–g, 10a–o, 11a–h and 13 utilizing benzotriazole-mediated synthetic approach with excellent yields. Some of the synthesized analogs (11a, 11d–h) exhibited antimalarial properties against Plasmodium falciparum strain 3D7 with potency higher than that of quinine (standard reference used) through in vitro standard procedure bio-assay. Statistically significant BMLR-QSAR model describes the bio-properties, validates the observed biological observations and identifies the most important parameters governing bio-activity.     

Design, synthesis and inhibition activity of a novel cyclic enediyne amino acid conjugates against MPtpA

In course of studies towards the discovery of selective inhibitors of MPtpA, a novel cyclic endiyne malonamic acid has been designed and synthesized. The synthesis involves a crucial intramolecular Knoevenagel reaction. The compound displayed a reversible non-competitive inhibition against MPtpA with inhibition constant K(i) of 22.5 μM. The enediyne acts as a recognition framework in inducing the inhibition and not as a reactive functional moiety. This was confirmed by comparing the inhibiting activity with that of the corresponding saturated cyclic non-enediyne analogue.     

Synthesis and application of a novel sunscreen-antioxidant

Background information on the inefficacy of sunscreens to provide free radical protection in skin, despite their usefulness in preventing sunburn/erythema, prompted us to synthesize a compound which would display in the same molecule both UV-absorbing and antioxidant capacities. For this purpose, the UVB absorber, 2-ethylhexyl-4-methoxycinnamate (OMC) was combined with the piperidine nitroxide TEMPOL, which has antioxidant properties. The spectral properties of the new nitroxide-based sunscreen (MC-NO) as well as its efficacy to prevent photo-oxidative damage to lipids induced by UVA, natural sunlight and 4-tert-butyl-4-methoxydibenzoylmethane (BMDBM), a photo-unstable sunscreen which generates free radicals upon UV radiation, was studied. The results obtained demonstrate that MC-NO: (a) absorbs in the UVB region even after UVA irradiation; (b) acts as free radical scavenger as demonstrated by EPR experiments; (c) strongly reduces both UVA-, sunlight- and BMDBM-induced lipid peroxidation in liposomes, measured as reduced TBARS levels; and (d) has comparable antioxidant activity to that of commonly used vitamin E and BHT in skin care formulations. These results suggest that the use of the novel sunscreen-antioxidant or of other nitroxide-based sunscreens in formulations aimed at reducing photoinduced skin damage may be envisaged.     

Synthesis and antiproliferative activity of novel steroidal dendrimer conjugates

We describe the synthesis of steroidal dendrimer conjugates of first and second generation with tetramethylene core and 5-hydroxy-isophtalic acid dimethyl ester as branching unit modified to incorporate ethynylestradiol or 17α-estradiol as terminal units. The steroidal dendrimer conjugates, the free drug (steroids) and dendrimer were tested against a panel of cancer cell lines (CEM, MCF7, HeLa) and normal human fibroblast (BJ). The steroidal dendrimer conjugates of first generation exhibited cytotoxic activity and induced apoptosis in chronic leukemia (CEM) as resultant activation of caspase cascade which is mainly provoked in G2/M arrested cells.     

Synthesis and biological activities of novel dexibuprofen tetraacetylriboflavin conjugates

A series of novel dexibuprofen derivatives covalently linked via alkylene spacers of variable length to tetraacetylated riboflavin have been developed. The target compounds became accessible by reaction of the chloromethyl ester of dexibuprofen with tetraacetylriboflavin (compound 7) or by synthesis of the appropriate N3-(omega-iodoalkyl)-2',3',4',5'-Tetraacetylriboflavin followed by treatment with dexibuprofen (derivatives 8-11), respectively. Biological screening revealed that the target compounds exhibit antiproliferative effects on MCF-7 breast cancer and HT-29 colon carcinoma cells with IC50 values in the range of 8-15 microM. Enzymatic studies on human platelets indicated significant COX-1 inhibitory activities of the target compounds.     

Design and synthesis of releasable folate-drug conjugates using a novel heterobifunctional disulfide-containing linker

Cellular uptake of vitamin folic acid occurs via folate-receptor mediated endocytosis. Many types of cancer cells express high levels of folate receptors as they need continuous supply of this vitamin for their proliferation. With an objective to use folic acid as a 'Trojan Horse' to transport anticancer drugs into cancer cells, a novel heterobifunctional disulfide-containing linker was synthesized and utilized to covalently link an amino- and hydroxyl-containing anticancer drug, and an appropriately functionalized folic acid to create novel targetable folate-drug conjugates that are shown to release free drugs under biologically relevant pH via sulfhydryl-assisted cleavage of the self-immolative disulfide-containing linker.