Dimeric calixarenes: a new family of major-groove binders.

A new class of potent DNA binding agents is presented. Dimeric calix[4]arenes with cationic groups at their upper rims and flexible alkyl bridges can be synthesized from triply acyl-protected calix[4]arene tetramines in relatively short synthetic sequences (3-5 steps). The compounds attach themselves to double-stranded nucleic acids in a noncovalent fashion, with micro- to nanomolar affinities. Guanidinium headgroups with their extended hydrogen-bonding "fingers" are more powerful than ammonium groups, and the benzylamine series is superior to the anilinium series (see below). The new ligands easily distinguish between RNA and various DNA types, and produce characteristic changes in UV/Vis, fluorescence, CD, as well as NMR spectra. Especially extended oligonucleotides of more than 100 base pairs are bound with affinities increasing from RNA (10 µM K(d))

Synthesis, X-ray crystal structure study and antitumoral evaluations of 5,6-disubstituted pyrimidine derivatives.

5,6-Disubstituted pyrimidine derivatives (3-20) were prepared by intramolecular cyclization reaction of alpha-(1-carbamyliminomethylene)-gamma-butyrolactone (2) with sodium ethoxide and subsequent chemical transformation of 2-hydroxy group in C-5 side chain as well as lithiation reaction for introduction of acyclic side chain at C-6. All compounds were characterized by (1)H NMR, (13)C NMR and mass spectra. Structures of compounds 4, 7 and 14 were unambiguously confirmed by X-ray crystal structural analysis. Supramolecular structures of these three compounds differ significantly. Two N-H...O and one C-H...O hydrogen bonds in 4 form three-dimensional network. One O-H...N hydrogen bond and one pi...pi interaction self-assemble the molecules of 7 into sheets. In supramolecular aggregation of 14, only pi...pi stacking interactions participate, so forming chains. The compounds were evaluated for their cytostatic activities against human malignant cell lines. Of all tested compounds, 2,4-dimethoxy-5-methoxytritylethylpyrimidine (9) and 2,4-dichloro-5-chloroethylpyrimidine (14) exhibited the most prominent inhibitory effects. Furthermore, compound 14 showed marked activity against human colon carcinoma (IC(50)=0.4microM).

The novel ketoprofen amides--synthesis and biological evaluation as antioxidants, lipoxygenase inhibitors and cytostatic agents.

The novel amides of ketoprofen and its reduced derivatives (5a-f, 4a-n, 6a-g) with aromatic and cycloalkyl amines or hydroxylamines were prepared and screened for their reducing and cytostatic activity as well as for their ability to inhibit soybean lipoxygenase and lipid peroxidation. 1,1-Diphenyl-picrylhydrazyl test for reducing ability revealed that ketoprofen amides were more potent antioxidants than the amides of the reduced ketoprofen derivatives. The most active compound was benzhydryl ketoprofen amide 5f. Lipoxygenase inhibition of the tested compounds varied from strong to very weak. The most potent compound was benzhydryl derivative 6f (IC(50) = 20.5 mum). Aromatic and cycloalkyl amides 4 and 5 were more potent lipoxygenase inhibitors than derivatives with carboxylic group. Aromatic amides of series 4 and 5 showed excellent lipid peroxidation inhibition (92.2-99.9%). On the other hand, the most pronounced cytostatic activity was exerted by O-benzyl derivative 4i, although in general all tested reduced and non-reduced lipophilic derivatives showed similar activity.

Novel amidino substituted 2-phenylbenzothiazoles: synthesis, antitumor evaluation in vitro and acute toxicity testing in vivo.

The efficient synthesis of new bis-substituted nitro-amidino, amino-amidino (10a, 10b-13a, 13b) and previously prepared diamidino 2-phenyl-benzothiazoles (9a, 9b) is described. The compounds 11a and 11b were prepared by recently developed methodology of the key precursors in zwitterionic form 8a and 8b with 4-nitrobenzoylchloride in a very good yield (70%). All compounds except diamidino-substituted 2-phenylbenzothiazole 9a show exceptionally prominent tumor cell-growth inhibitory activity and cytotoxicity, whereby the special selectivity of amino-amidine 2-phenylbenzothiazole 12a towards MCF-7 and H 460 cells makes this compound a prospective lead compound that should be further evaluated in animal models. All in vivo tested compounds (12a, 12b, 13a and 13b) are absorbed from mice gastrointestinal system. LD(50) are between 67.33 and 696.2mg/kg body weight (OECD/EPA toxicity categories 2-3).

Synthesis, structural studies and antitumoral evaluation of C-6 alkyl and alkenyl side chain pyrimidine derivatives.

The synthetic route for introduction of fluorophenylalkyl (compounds 5, 7, 14 and 15) and fluorophenylalkenyl (compounds 4E and 13) side chains at C-6 of the pyrimidine nucleus involved the lithiation of the pyrimidine derivatives 1, 2 and 11 and subsequent nucleophilic addition or substitution reactions of the organolithium intermediate thus obtained with 2-fluorophenylacetone, 4-fluoroacetophenone or ethyl 4-fluorobenzoate as electrophiles. The structures of novel compounds were confirmed by (1)H-, (19)F- and (13)C-NMR and MS. Compounds 8 and 10 containing unsaturated fluorophenylalkyl side chains showed better inhibitory effect than their saturated fluorophenylalkylated pyrimidine counterparts 7 and 9. A conformational study based on NOE enhancements showed the importance of the double bond and substitution in the side chain for the conformational preferences in relation to inhibitory activity. Among all tested compounds, C-5 furyl (12) and phenyl (13 and 15) substituted pyrimidine derivatives showed significant cytostatic activities against all tested tumor cell lines.

Novel side-chain glucosylated and adamantylated [Asp(2)/Glu(2)]enkephalin analogs: synthesis and in vitro growth inhibition of human tumor cells.

A series of new backbone-modified Leu- and Met-enkephalin analogs (13-20 a and b) were synthesized. Backbone manipulations involved the replacement of the Gly(2) residue in Tyr-Gly-Gly-Phe-Leu/Met with side-chain glucosylated or adamantylated D/L-aspartic or -glutamic acids. The in vitro antiproliferative activity of these compounds was evaluated for several cell lines and the results were compared with the effect of Met-enkephalin, the native opioid growth factor. The tested compounds modestly inhibited the growth of the tumor cells (20-50% inhibition at millimolar concentrations). Among the tested compounds, Tyr-D-Glu(AdNH)-Gly-Phe-Met (20b) showed significant antiproliferative activity, somewhat more pronounced on MCF-7 (breast carcinoma) and MOLT-4 (lymphoblastic leukemia) cells.

Synthesis of modified pyrimidine bases and positive impact of chemically reactive substituents on their in vitro antiproliferative activity.

The antiproliferative activity screening on human tumor cell lines of a series of modified uracil and cytosine bases as well as some corresponding acyclonucleosides, and comparison of structure-activity relationship revealed the importance of chemical reactivity of the substituent attached to the C5-position of uracil for the activity of studied compounds. Namely, the results obtained for the most active compounds, 5-(chloroacetylamino)uracil (2) and its acyclic sugar analogue 18, suggest that formation of a covalent bond between reactive substituent and several possible targets within the thymidylate synthase mechanism (sulphur of the cysteine residue, basic part of the enzyme, N,N-methylene tetrahydrofolate or its reactive iminium forms) is the most probable mode of action. In addition, novel C5-substituted uracil derivative 6 (5-[bis-(2-p-methoxybenzylthioethyl)amine]acetylaminouracil) exhibited high antiproliferative activity against HeLa and MiaPaCa-2 cell lines, by an as yet unknown mechanism.

Novel amidino-substituted thienyl- and furylvinylbenzimidazole: derivatives and their photochemical conversion into corresponding diazacyclopenta[c]fluorenes. synthesis, interactions with DNA and RNA, and antitumor evaluation. 4.

Synthesis of novel nonfused amidino-substituted thienyl- and furylvinylbenzimidazole: derivatives and their photochemical cyclization into corresponding diazacyclopenta[ c]fluorenes is described. All studied compounds showed prominent growth inhibitory effect. The fused compounds showed stronger activity than nonfused ones, whereby imidazolyl-substituted compound 11 proved to be the most active one. Besides, it induced strong G2/M arrest of the cell cycle followed by drastic apoptosis, which is in accordance with the DNA intercalative binding mode determined by the spectroscopic studies. Nonfused derivatives induced strong S phase arrest of the cell cycle followed by apoptosis that together with DNA minor groove binding mode pointed to topoisomerase I inhibition. In addition, all nonfused compounds revealed pronounced selectivity toward tumor cells in comparison with nontumor cells. On the basis of the presented results, both nonfused and fused thiophene-containing imidazolyl derivatives should be considered as promising lead compounds for further investigation.

Atypical cytostatic mechanism of N-1-sulfonylcytosine derivatives determined by in vitro screening and computational analysis.

We have previously shown that N-1-sulfonylpyrimidine derivatives have strong antiproliferative activity on human tumor cell lines, whereby 1-(p-toluenesulfonyl)cytosine showed good selectivity with regard to normal cells and was easily synthesized on a large scale. In the present work we have used an interdisciplinary approach to elucidate the compounds' mechanistic class. An augmented number of cell lines (11) has allowed a computational search for compounds with similar activity profiles and/or mechanistic class by integrating our data with the comprehensive DTP-NCI database. We applied supervised machine learning methodology (Random Forest classifier), which offers information complementary to unsupervised algorithms commonly used for analysis of cytostatic activity profiles, such as self-organizing maps. The computational results taken together with cell cycle perturbation and apoptosis analysis of the cell lines point to an unusual mechanism of cytostatic action, possibly a combination of nucleic acid antimetabolite activity and a novel molecular mechanism.

Novel cyano- and amidino-substituted derivatives of styryl-2-benzimidazoles and benzimidazo[1,2-a]quinolines. Synthesis, photochemical synthesis, DNA binding, and antitumor evaluation, part 3.

Synthesis of novel cyano- and amidino-substituted styryl-2-benzimidazoles and benzimidazo[1,2-a]quinolines by condensation reactions and photochemical dehydrocyclization and dehydrohalogenation cyclization is described. Thermal denaturation experiments reveal that cyclic derivatives considerably stabilize DNA double helix, while the effect of their acyclic analogues is negligible. According to the spectroscopic study of the interaction of cyclic derivative 19, we propose intercalation of benzimidazo[1,2-a]quinoline moiety into ct-DNA as a dominant interaction underlying biologically relevant effects of this compound, whereas for its acyclic derivative 11, we propose binding into the minor groove of DNA. All compounds show noticeable antiproliferative effect. Morpholino- and chloro-substituted compound 9 is the most active among all acyclic derivatives. All cyclic compounds were 2- to 10-fold more potent, which is correlated with their property to intercalate into DNA. The most active imidazolyl-substituted compound 19 inhibits topoisomerase II and induces strong G2/M cell cycle arrest, pointing to the impairment in mitotic progression. Its pronounced selectivity toward colon carcinoma cells encourages further development of this compound as a lead.

Synthesis and antiviral and cytostatic evaluations of the new C-5 substituted pyrimidine and furo[2,3-d]pyrimidine 4',5'-didehydro-L-ascorbic acid derivatives.

The novel C-5 alkynyl substituted pyrimidine (1-11) and furo[2,3-d]pyrimidine derivatives (12-22) of l-ascorbic acid were synthesized by coupling of 5-iodouracil-4',5'-didehydro-5',6'-dideoxy-l-ascorbic acid with terminal alkynes by using Sonogashira cross-coupling reaction conditions. The new compounds were evaluated for their cytostatic and antiviral activities. Among all evaluated compounds, the octynyl-substituted uracil derivative of l-ascorbic acid (3) exhibited the most pronounced cytostatic activities against all examined tumor cell lines (IC50 = 2-12 microM). Pyrimidine derivatives of l-ascorbic acid containing p-substituted phenylacetylene groups (8-11) displayed also a rather pronounced (IC50 = 3-37 microM) inhibitory effect toward all tumor cell lines. From the bicyclic series of compounds, 6-butylfuro[2,3-d]pyrimidine derivative (12) and 6-p-bromophenylfuro[2,3-d]pyrimidine derivative (19) showed the highest cytostatic activity (IC50 = 4.5-20 microM), particularly against malignant leukemia (L1210) and T-lymphocyte (Molt4/C8 and CEM) cells. Compounds 3 and 9 showed specific albeit moderate activity against cytomegalovirus (CMV, Davis strain, EC50 = 1.8 and 3.8 microM, respectively, for compounds 3 and 9) at a approximately 5-fold lower concentration than that required to show cytotoxicity.

Novel C-6 fluorinated acyclic side chain pyrimidine derivatives: synthesis, (1)H and (13)C NMR conformational studies, and antiviral and cytostatic evaluations.

The synthetic route for introduction of a fluoroalkyl (7-12, 14), fluoroalkenyl (15 and 16), fluorophenylalkyl (17, 19, 20, and 22), and fluorophenylalkenyl (18, 21) side chain at C-6 of the pyrimidine involved the lithiation of the pyrimidine derivatives 3 and 3a and subsequent nucleophilic addition or substitution reactions of the organolithium intermediate thus obtained with various electrophiles. Conformational properties of the novel fluorinated pyrimidine derivatives were assessed by the use of 1D difference NOE enhancements and C-F coupling constants. Compounds 4-22 were evaluated for their antiviral and cytostatic activities. Of all compounds evaluated, the 5-bromopyrimidine derivatives 5 and 6 showed the highest inhibitory activities. Among the series of fluoroalkylated pyrimidines, which is generally more active than the series of fluorophenylalkylated pyrimidines, compounds 8 and 14 displayed moderate cytostatic activities against the tested tumor cell lines. Moreover, compound 8 containing a 2-fluoromethylpropyl side chain expressed some but not highly specific activity against varicella-zoster virus (VZV). From C-6 fluorophenylalkylated pyrimidine derivatives, 17a and 21 showed a slight activity against cytomegalovirus (CMV), VZV, and Coxsackie B4 virus, respectively. Besides, compounds 17a and 21 showed no cytotoxic effect.

Synthesis, X-ray crystal structural study, antiviral and cytostatic evaluations of the novel unsaturated acyclic and epoxide nucleoside analogues.

A series of the novel purine and pyrimidine nucleoside analogues were synthesised in which the sugar moiety was replaced by the 4-amino-2-butenyl (2-6 and 10-18) and oxiranyl (8 and 20) spacer. The Z- (2-6) and E-isomers (10-18) of unsaturated acyclic nucleoside analogues were synthesized by condensation of 2- and 6-substituted purine and 5-substituted uracil bases with Z- (1) or E-phthalimide (9) precursors. The oxiranyl nucleoside analogues (8 and 20) were obtained by epoxidation of 1 and 9 with m-chloroperoxybenzoic acid and subsequent coupling with adenine. The new compounds were evaluated for their antiviral and antitumor cell activities. Among the olefinic nucleoside analogues, Z-isomer of adenine containing 4-amino-2-butenyl side chain (6) exhibited the best cytostatic activities, particularly against colon carcinoma (SW 620, IC50 = 26 microM). Its E-isomer 15 did not show any antiproliferative activity against malignant tumor cell lines, except for a slight inhibition of colon carcinoma (SW 620, IC50 = 56.5 microM) cells. In general, Z-isomers showed better cytostatic activities than the corresponding E-isomers. (Z)-4-Amino-2-butenyl-adenine nucleoside analogue 6 showed albeit modest but selective activity against HIV-1 (EC50 = 4.83 microg mL(-1)).

Synthesis, photochemical synthesis, DNA binding and antitumor evaluation of novel cyano- and amidino-substituted derivatives of naphtho-furans, naphtho-thiophenes, thieno-benzofurans, benzo-dithiophenes and their acyclic precursors.

As a part of the research on the improvement of an alternative to conventional photodynamic therapy by light-induced formation of intercalators, we synthesized a series of novel heterocyclic compounds and their acyclic precursors. We now report details about their synthesis/characterization in respect to their potential of photoinduced cyclization, interactions with DNA and inhibition of the tumor cell growth in vitro. Among studied compounds only amidino-furyl-substituted phenyl acrylates were efficiently converted to the corresponding naphthofuranes, while their thiophene analogues, all non-charged derivatives and amidino-phenyl-substituted analogues didn't show acceptable photoconversion. The significantly stronger antiproliferative activity of cyclic analogues could be correlated to the property of these molecules to intercalate into DNA. The acyclic molecules did not show any interaction with DNA, correlating with the inferior biological activity, except for one cyano-bearing molecule.

Novel cyano- and amidino-substituted derivatives of thieno[2,3-b]- and thieno[3,2-b]thiophene-2-carboxanilides and thieno[3',2':4,5]thieno- and thieno[2',3':4,5]thieno [2,3-c]quinolones: synthesis, photochemical synthesis, DNA binding, and antitumor evaluation.

A series of cyano- and amidino-substituted derivatives of thieno[2,3-b]- and thieno[3,2-b]thiophene-2-carboxanilides and their 'cyclic' derivatives (quinolones) were synthesized. 'Cyclic' compounds displayed a rather strong and differential antiproliferative effect on various cell lines, while the 'acyclic' amidino-substituted compounds were much more active, but showing mostly non-differential cytotoxicity, whereas cyano-substituted compounds (2a,b) produced a strikingly strong effect selectively on HeLa and Hep-2 cell lines. Antiproliferative activity of 'cyclic' derivatives is very likely caused by intercalation into DNA, while their 'acyclic' analogues use other target(s) and/or mechanisms of action.

Synthesis, structural studies, and cytostatic evaluation of 5,6-di-O-modified L-ascorbic acid derivatives.

The 5,6-di-O-tosylated derivative of l-ascorbic acid was synthesized by selective protection and deprotection of 2,3- and 5,6-dihydroxy functional groups involving 5,6-ditosylation in the final step, while the novel 6-acetoxy, 6-hydroxy, and 6-chloro derivatives of 4,5-didehydro-l-ascorbic acid were obtained by reaction of ditosylated compound with nucleophilic reagents. The analysis of 3JH-4-H-5 homonuclear coupling constants shows that all l-ascorbic acid derivatives except for epoxy and 4,5-didehydro compounds exist in high population as gauche conformers across C-4-C-5 bonds, while 3JC-3-H-5 heteronuclear coupling constants in 4,5-didehydro derivatives indicate cis geometry along C-4-C-5 double bond. The X-ray crystal structure analysis of 2,3-di-O-benzyl-5,6-epoxy- and 5,6-isopropylidene-l-ascorbic acid shows that the oxygen atoms attached at positions 2 and 3 of the lactone ring are disposed in a synperiplanar fashion. Besides that, the dioxolane ring adopts half-chair conformation. The molecules of epoxy derivative are joined into infinite chains by one weak hydrogen bond of C-H...O type. Two O-H...O, and C-H...O hydrogen bonds link the molecules of 5,6-di-O-isopropylidene compound into two-dimensional network. 6-Chloro derivative of 2,3-di-O-benzyl-l-ascorbic acid showed the best cytostatic effects against all tested malignant tumor cells (IC50: approximately 18 microM).

Novel cyano- and N-isopropylamidino-substituted derivatives of benzo[b]thiophene-2-carboxanilides and benzo[b]thieno[2,3-c]quinolones: synthesis, photochemical synthesis, crystal structure determination, and antitumor evaluation. 2.

Derivatives of 3-chlorobenzo[b]thiophene-2-carboxanilides and their "cyclic" analogues benzo[b]thieno[2,3-c]quinolones were synthesized. Spectroscopic study of the interactions of some representatives of "cyclic" derivatives and their "acyclic" precursors with ds-DNA/RNA supported strong intercalative binding of the former and weak nonintercalative binding of the latter group of compounds. All tested compounds showed a certain antiproliferative effect on a series of human tumor cells and on a normal cell line. Among the compounds, those with one amidino-substituent have shown the best effect. The most active benzo[b]thieno[2,3-c]quinolones induced apparent S and G2/M arrests of the cell cycle, which resulted in apoptosis. These results strongly suggest that the compounds may act as topoisimerase "poisons", which is in good agreement with their intercalative mode of binding to ds-DNA/RNA, in contrast to the studied "acyclic"group of derivatives. 6a and 6d showed the best selectivity by inhibiting the growth of tumor cells but not of normal fibroblasts.

Photoinduced switch of a DNA/RNA inactive molecule into a classical intercalator.

Spectroscopic titrations and thermal denaturation experiments show that "acyclic" analogue 1 does not bind to ds-DNA, but under same conditions "cyclic" 2 strongly interacts with ds-DNA and ds-RNA by intercalation into the double helix. Besides, 2 is significantly more effective in inhibition of the tumor cell growth in vitro than 1. We have shown that it is possible to efficiently and irreversibly convert "DNA inactive" compound 1 into "DNA active" compound 2 by light irradiation of the aqueous solutions of the former. This strategy offers a new and attractive approach to photoinduced anticancer therapy.

Radioprotective and antitumor activity evaluation of newly synthesized adamantyl tenocyclidines.

Several adamantyl derivatives of thienyl phencyclidine (tenocyclidine; TCP) were newly sythesized and characterized: adamantyl derivatives containing piperidine (TAPIP), pyrrolidine (TAPYR), and morpholine (TAMORPH) groups. Their biological activity was evaluated by in vitro testing of their effect on the proliferative and reproductive ability (cytotoxicity) of a human tumor cell strain and nonmalignant mouse fibroblasts in culture. We also tested them for their radioprotective effect after ionizing irradiation, and as anticancer agents on the same human tumor cell strain. Compared with TCP, adamantyl derivatives are less toxic and have outstanding radioprotective properties. These derivatives (especially TAMORPH) increase apoptotic death of human malignant cells. The radiation-modifying effect studied on C3Hf mice in vivo showed that the adamantyl derivatives of TCP have a more enhanced radioprotective effect and that they are less toxic than TCP itself. The present data are discussed and compared with those previously reported for structurally related phencyclidine derivatives.