New Synthetic Analogs of Nitrogen Mustard DNA Interstrand Cross-Links and Their Use to Study Lesion Bypass by DNA Polymerases.

Nitrogen mustards are a widely used class of antitumor agents that exert their cytotoxic effects through the formation of DNA interstrand cross-links (ICLs). Despite being among the first antitumor agents used, the biological responses to NM ICLs remain only partially understood. We have previously reported the generation of NM ICL mimics by incorporation of ICL precursors into DNA using solid-phase synthesis at defined positions, followed by a double reductive amination reaction. However, the structure of these mimics deviated from the native NM ICLs. Using further development of our approach, we report a new class of NM ICL mimics that only differ from their native counterpart by substitution of dG with 7-deaza-dG at the ICL. Importantly, this approach allows for the synthesis of diverse NM ICLs, illustrated here with a mimic of the adduct formed by chlorambucil. We used the newly generated ICLs in reactions with replicative and translesion synthesis DNA polymerase to demonstrate their stability and utility for functional studies. These new NM ICLs will allow for the further characterization of the biological responses to this important class of antitumor agents.

A trans-dichloridoplatinum(II) complex of a monodentate nitrogen mustard: Synthesis, stability and cytotoxicity studies.

A trans-dichloridoplatinum(II) complex, trans-[PtIICl2(L)(DMSO)] (1) of a monodentate nitrogen mustard, bis(2-chloroethyl)amine (L), was synthesized by the reaction of cis-[PtIICl2(DMSO)2] &L.HCl in presence of Et3N. 1 was characterised by NMR, FT-IR and elemental analysis. L is unstable in aqueous solution while 1 displayed moderate stability. In aqueous buffer solution of pD 7.4, 1 starts to loose L slowly upon dissolution and even after 48 h there is still intact/aquated complex present in solution. 1 interacts with the model nucleobase 9-ethyl guanine. The ligand L was non-toxic against MCF-7, A549, HepG2 & MIA PaCa-2 up to 200 µM. In contrast, the Pt(II) complex 1 showed an excellent IC50 (ca. 600 nM) against MIA PaCa-2 and also displayed good IC50 value (3-7 µM) against the other cancer cell lines probed. The in vitro cytotoxicity of 1 is better than cisplatin against each of the treated cancer cell lines and it is not affected by hypoxia as per the in vitro studies. Complex 1 displays higher cellular accumulation than cisplatin and arrests the cell cycle in both S & G2/M phase inducing apoptotic cell death. The G2/M phase arrest is dominant at higher concentrations. The depolarisation of mitochondria by 1 combined with activation of caspase-7 indicates apoptotic cell death. Complex 1 induces low hemolysis of human blood signifying excellent blood compatibility.

The evolving role of DNA inter-strand crosslinks in chemotherapy.

DNA crosslinking agents make up a broad class of chemotherapy agents that target rapidly dividing cancer cells by disrupting DNA synthesis. These drugs differ widely in both chemical structure and biological effect. In cells, crosslinking agents can form multiple types of DNA lesions with varying efficiencies. Inter-strand crosslinks (ICLs) are considered to be the most cytotoxic lesion, creating a covalent roadblock to replication and transcription. Despite over 50 years in the clinic, the use of crosslinking agents that specialize in the formation of ICLs remains limited, largely due to high toxicity in patients. Current ICL-based therapeutics have focused on late-stage and drug-resistant tumors, or localized treatments that limit exposure. In this article, we review the development of clinical crosslinking agents, our understanding of how cells respond to different lesions, and the potential to improve ICL-based chemotherapeutics in the future.

Novel 19 F-MRS ß-galactosidase reporter molecules incorporated nitrogen mustard analogues.

In this study, we propose a novel molecular platform-integrated fluorinated antitumor nitrogen mustards for 19 F-MRS assay of ß-galactosidase (ß-gal) activity. Following this idea, we have designed, synthesized, and characterized 2-fluoro-4-[bis(2'-chloroethyl)amino]phenyl ß-D-galactopyranoside 5, 2-fluoro-4-{bis[2'-O-(ß-D-galactopyranosyl)ethyl]amino}phenyl ß-D-galactopyranoside 8, 2-fluoro-4-{bis[[1″-(ß-D-galactopyranosyl)-1″, 2″, 3″-triazol-4″-yl]methyl] amino}phenyl ß-D-galactopyranoside 14 and 2-fluoro-4-{bis[[1″-(ß-D-glucopyranosyl)-1″, 2″, 3″-triazol-4″-yl]methyl]amino}phenyl ß-D-galactopyranoside 15 through glycosylation and click reaction strategies, and their structures were confirmed by NMR and HRMS or elemental analysis data. Among them, 2-fluoro-4-[bis(2'-chloroethyl)amino]phenyl ß-D-galacto-pyranoside 5 was found very sensitive to ß-gal (E801A) in PBS at 37°C with big ΔδF response. Here, we demonstrated the feasibility of this platform for assessing ß-gal activity in solution, and in vitro with lacZ-transfected human MCF7 breast and PC3 prostate tumor cells, by the characterization of ß-gal-responsive 19 F-chemical shift changes ΔδF and hydrolytic kinetics.

Synthesis and Biological Evaluations of Click-Generated Nitrogen Mustards.

This paper describes the synthesis of new click-generated nitrogen mustards and their biological evaluation. By using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, we managed to synthesize eight new nitrogen mustards. This strategy paves the way for the synthesis of a new family of nitrogen mustard, with an important structural variability. Furthermore, we studied the biological activity of synthesized compounds by testing their cytotoxicity on four representative cancer cell lines A431, JURKAT, K562, and U266. One structure, 1-benzyl-4-(N,N-di-2-chloroethylaminomethyl)-1H-[1,2,3]triazole, showed an interesting cytotoxic effect.

Characterization of an array of Love-wave gas sensors developed using electrospinning technique to deposit nanofibers as sensitive layers.

The electrospinning technique has allowed that very different materials are deposited as sensitive layers on Love-wave devices forming a low cost and successful sensor array. Their excellent sensitivity, good linearity and short response time are reported in this paper. Several materials have been used to produce the nanofibers: polymers as Polyvinyl alcohol (PVA), Polyvinylpyrrolidone (PVP) and Polystirene (PS); composites with polymers as PVA+SnCl4; combined polymers as PS+Poly(styrene-alt-maleic anhydride) (PS+PSMA) and metal oxides (SnO2). In order to test the array, well-known chemical warfare agent simulants (CWAs) have been chosen among the volatile organic compounds due to their importance in the security field. Very low concentrations of these compounds have been detected by the array, such as 0.2 ppm of DMMP, a simulant of sarin nerve gas, and 1 ppm of DPGME, a simulant of nitrogen mustard. Additionally, the CWA simulants used in the experiment have been discriminated and classified using pattern recognition techniques, such as principal component analysis and artificial neural networks.

Synthesis and biological evaluation of nitrogen mustard derivatives of purine bases.

This paper deals with the synthesis of nitrogen mustard analogs, derivatives of purine bases. Alkylation in position N-9 and diethanolamine fixation on position 6 were managed by microwave irradiations. Chlorination of these dihydroxylated intermediates led to a cyclization, giving tricyclic purine base analogs bearing a chloroethyl chain. Finally, MTT assays on obtained compounds do not show cytotoxicity on four different cancer cell lines.

The influence of chromatin structure on DNA damage induced by nitrogen mustard and cisplatin analogues.

The interaction of anti-tumour drugs with reconstituted chromatin has been investigated using defined nucleosomal complexes. This allowed the effect of nucleosome cores on drug-induced DNA damage to be assessed for four nitrogen mustard analogues, dimethylsulphate and three cisplatin analogues. A defined nucleosomal complex was employed that contained two precisely positioned nucleosome cores. The construct was then subjected to drug treatment, and the resulting DNA damage was quantitatively analysed using a Taq DNA polymerase stop assay. At the sites of damage, densitometric comparisons between purified and reconstituted DNA were used to evaluate the influence of nucleosomal core proteins on specific drug-DNA interactions. Results were combined with previous data obtained for other DNA-damaging drugs investigated using the same nucleosomal construct. For most of the DNA-damaging agents studied, this method revealed protection at the positioned nucleosome cores and indicated that the preferred site of DNA binding for these compounds was in the linker region of the construct. Statistical analyses confirmed the significant level of damage protection conferred by the nucleosome cores and revealed differences between the examined compounds. Larger compounds generally displayed a greater tendency to target the linker region of the nucleosomal DNA and were impeded from damaging nucleosomal core DNA. In contrast, smaller molecules had greater access to nucleosomal core DNA.

Synthesis and cytotoxic activity of novel amidine analogues of bis(2-chloroethyl)amine.

Novel nitrogen mustard agents 7-12 involving 4-(N,N-bis(2-chloroethyl)aminophenyl)propylamine linked to a 5-(4-N-alkylamidinophenyl)-2-furancarboxylic acid moiety by the formation of an amide bond have been synthesized, characterized, and evaluated for their in-vitro cytotoxic activity against MDA-MB-231 and MCF-7 human breast cancer cells. Evaluation of the cytotoxicity of 7-12 employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA demonstrated that these compounds exhibit remarkable cytotoxic effects in comparison with 4-[bis(2-chloroethyl)amino]benzenebutanoic acid. Compounds 7 and 9, which possess a cationic amidine and 4,5-dihydro-1H-imidazol function moiety are approximately ten times more potent than 4-[bis(2-chloroethyl)amino]benzenebutanoic acid. The new compounds were evaluated as DNA topoisomerase II inhibitors. The cytotoxicity of the compounds 7-12 correlates with their DNA-binding affinities and their relative potency as topoisomerase II inhibitors.

The new analogues of nitrogen mustard with one, two or three 2-chloroethylamino fragments. Reactions with nucleophiles.

1,3,5-Triazines substituted with mono-, di, and tri-[4-(2-chloroethyl)piperazin-l-yl] groups gave products of substitution of chlorine atom when treated with ethanol, phenol, butylamine, toluidine,or thiophenol under mild reaction conditions.

Cytokinetics and mechanism of action of AKO4: a novel nitrogen mustard targeted to bcr-abl.

The "combi-targeting" concept seeks to design molecules to not only block tyrosine kinase (TK) activity but also to induce DNA damage. Here we design AK04, a molecule that combines the pharmacophore chlorambucil with that of STI-571 (Gleevec). The results showed that although a less potent abl TK inhibitor than STI571, AK04 was capable of significantly blocking bcr-abl phosphorylation not only in a purified abl assay but also in the bcr-abl+ K562 cells. In contrast to STI571 and like chlorambucil, it induced a dose-dependent increase in DNA damage in these cells. More importantly, AK04 was 12-32-fold more potent than chlorambucil in all bcr-abl+ cells of our cell panel. In the isogenic human megakaryocytic Mo7e and Mo7/bcr-abl cells, AK04 selectively killed the bcr-abl transfectants. Flow cytometry revealed that despite being a five-fold less potent inhibitor of bcr-abl than STI-571, it induced a significant dose-dependent increase in levels of cell death by apoptosis in KU812 cells 24 h post-treatment. Under these conditions, chlorambucil did not induce any significant level of apoptosis. These results suggest that AK04 is a nitrogen mustard with binary bcr-abl/DNA targeting effects, a property that may account for its superior potency when compared with the classical mustard chlorambucil.

Sulfoxide-containing aromatic nitrogen mustards as hypoxia-directed bioreductive cytotoxins.

A series of diaryl and alkylaryl sulfoxide-containing nitrogen mustards were synthesized and evaluated for their hypoxia-selective cytotoxicity against V-79 cells in vitro as well as for their metabolism profiles with the rat S-9 fractions. In general, the diaryl sulfoxides (4, 5, and 7-9) showed much greater hypoxia selectivity (11-27-fold) than the alkylaryl sulfoxides (approximately 3-fold) (1 and 3). The fused diphenyl sulfoxides (10 and 11), on the other hand, showed very low hypoxia selectivity (1.3-3-fold). Compound 10 was highly cytotoxic under both aerobic and anaerobic conditions, while 11 showed low cytotoxicity under both conditions. The bioreduction of 8 by the rat S-9 fraction under anaerobic conditions was inhibited by menadione and enhanced by benzaldehyde, acetaldehyde, or 2-hydroxypyrimidine suggesting the involvement of aldehyde oxidase in the reduction of the sulfoxides. Bioreductive metabolism studies of selected model sulfoxides suggested that diaryl sulfoxides are better substrates for aldehyde oxidase than alkylaryl sulfoxides.

Protection from cytotoxic effects induced by the nitrogen mustard mechlorethamine on human bronchial epithelial cells in vitro.

The present study was undertaken to find potent molecules against the toxicity of nitrogen mustard mechlorethamine (HN2) on respiratory epithelial cells, using a human bronchial epithelial cell line (16HBE14o-) as an in vitro model. The compounds examined included inhibitors of poly(ADP-ribose) polymerase (PARP), sulfhydryl-group donors as nucleophiles, and iron chelators and inhibitors of lipid peroxidation as antioxidants. Their effectiveness was determined upon observance of metabolic dysfunction induced by HN2 following a 4-h exposure, using (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and ATP-level assays as indicators. Moreover, the fluorescent probe, monobromobimane (mBBr), and 2',7'-dichlorofluorescin-diacetate (H2DCF-DA) were used to assess intracellular sulfhydryl and peroxide level modifications by flow cytometry, respectively, following a 3-h exposure. At last, cell death was assessed by flow cytometry using the propidium iodide (PI)-dye-exclusion assay following 24-h exposure. PARP inhibitors (niacinamide, 3-aminobenzamide, 6(5H)-phenanthridinone), and two sulfhydryl-group donors (N-acetylcysteine, WR-1065) were found to be effective in preventing HN2-induced metabolic dysfunction when added in immediate or delayed treatment with HN2. Only N-acetylcysteine, however, was found to prevent cell death induced by HN2, though it must be present at the time of the HN2 challenge. Flow cytometric measurements of intracellular sulfhydryl levels strongly suggested that N-acetylcysteine and WR-1065 are preventive in alkylation of cellular compounds, mainly by direct extracellular interaction with HN2. PARP inhibitors prevent secondary deleterious effects induced by HN2, considering metabolism dysfunction as the endpoint. Elsewhere, the oxidative stress appears to be a side effect in HN2 toxicity only upon considering the inefficiency of several antioxidants.

Protein-DNA footprinting of the human epsilon-globin promoter in human intact cells using nitrogen mustard analogues and other DNA-damaging agents.

Nitrogen mustard analogues, bleomycin and dimethyl sulphate (DMS) have been used as probes of protein-DNA interactions in intact human cells. The sites of damage have been determined at base pair resolution in the single copy epsilon-globin gene promoter in erythroid K562 cells, non-erythroid HeLa cells and purified DNA. Exponential amplification of gene-specific damage fragments was achieved using the ligation-mediated polymerase chain reaction (LMPCR) technique and analysed on DNA sequencing gels. A comparison of the relative damage band intensities between purified DNA and intact cells revealed several significant differences - both protection (footprint) and enhancement. These differences occurred at putative transcription factor binding sites and hence are thought to be due to protein-DNA interactions. A major feature of the band intensity ratio plots was the footprint observed at the CCAAT box binding motif as revealed by nitrogen mustard analogues. Enhanced band intensity (hypersensitivity) was displayed at the 5'- and 3'-ends of the CCAAT box in K562 cells - this feature was absent in HeLa cells and in vitro reconstitutions. A footprint was found at the GATA-1 motif in K562 cells that was also absent in non-expressing HeLa cells. Footprints were also evident at the TATA box, CACC box and the epsilonF1 DNA binding motif in K562 cells.

Self-immolative nitrogen mustard prodrugs for suicide gene therapy.

Four new potential self-immolative prodrugs derived from phenol and aniline nitrogen mustards, four model compounds derived from their corresponding fluoroethyl analogues and two new self-immolative linkers were designed and synthesized for use in the suicide gene therapy termed GDEPT (gene-directed enzyme prodrug therapy). The self-immolative prodrugs were designed to be activated by the enzyme carboxypeptidase G2 (CPG2) releasing an active drug by a 1, 6-elimination mechanism via an unstable intermediate. Thus, N-[(4-¿[4-(bis¿2-chloroethyl¿amino)phenoxycarbonyloxy]methyl¿pheny l)c arbamoyl]-L-glutamic acid (23), N-[(4-¿[4-(bis¿2-chloroethyl¿amino)phenoxycarbonyloxy]methyl¿pheno xy) carbonyl]-L-glutamic acid (30), N-[(4-¿[N-(4-¿bis[2-chloroethyl]amino¿phenyl)carbamoyloxy]methyl¿+ ++phen oxy)carbonyl]-L-glutamic acid (37), and N-[(4-¿[N-(4-¿bis[2-chloroethyl]amino¿phenyl)carbamoyloxy]methyl¿+ ++phen yl)carbamoyl]-L-glutamic acid (40) were synthesized. They are bifunctional alkylating agents in which the activating effects of the phenolic hydroxyl or amino functions are masked through an oxycarbonyl or a carbamoyl bond to a benzylic spacer which is itself linked to a glutamic acid by an oxycarbonyl or a carbamoyl bond. The corresponding fluoroethyl compounds 25, 32, 42, and 44 were also synthesized. The rationale was to obtain model compounds with greatly reduced alkylating abilities that would be much less reactive with nucleophiles compared to the corresponding chloroethyl derivatives. This enabled studies of these model compounds as substrates for CPG2, without incurring the rapid and complicated decomposition pathways of the chloroethyl derivatives. The prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs by CPG2 for use in GDEPT. The synthesis of the analogous novel parent drugs (21b, 51) is also described. A colorectal cell line was engineered to express CPG2 tethered to the outer cell surface. The phenylenediamine compounds were found to behave as prodrugs, yielding IC50 prodrug/IC50 drug ratios between 20- and 33-fold (for 37 and 40) and differentials of 12-14-fold between CPG2-expressing and control LacZ-expressing clones. The drugs released are up to 70-fold more potent than 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoic acid that results from the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA) which has been used previously for GDEPT. These data demonstrate the viability of this strategy and indicate that self-immolative prodrugs can be synthesized to release potent mustard drugs selectively by cells expressing CPG2 tethered to the cell surface in GDEPT.

Mono- and dysfunctional nitrogen mustard analogues of the DNA minor groove binder pibenzimol. Synthesis, cytotoxicity and interaction with DNA.

Two series of mono- and dysfunctional aniline mustards linked to a bisbenzimidazole minor groove binder have been prepared using a new method (polyphosphate ester-mediated direct coupling of appropriate mustard acids with a preformed advanced phenylenediamine intermediate). As the linker chain attaching the mustard was lengthened the binding site size of the compounds to calf thymus DNA remained essentially constant at 2.6 nucleotides, but reversible binding strength declined by a factor of 2. Analogues with longer linker chains alkylated DNA much more rapidly than those with shorter chains, consistent with the electronic factors. The short chain analogues also failed to alkylate a 120 bp HindIII to Bg/II fragment of the gpt gene, as measured by gel electrophoresis cleavage assays. The longer chain analogues (both mono- and dysfunctional mustards) showed patterns of DNA alkylation that varied with chain length. In particular, while most compounds showed substantial N7 alkylation at many guanine residues, the analogue with a (CH2)3 linker chain showed strong alkylation at adenine sites in poly-AT regions. For the longer chain analogues, the bifunctional mustards were substantially (10- to 20-fold) more cytotoxic than the corresponding monofunctional analogues.

Mustard prodrugs for activation by Escherichia coli nitroreductase in gene-directed enzyme prodrug therapy.

Twenty nitrogen mustard analogues derived from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954, 1) were evaluated as candidate prodrugs for gene-directed enzyme prodrug therapy (GDEPT) in Chinese hamster V79 cell lines engineered to express Escherichia coli nitroreductase (NR). Structural variations within the series included the use of N-dihydroxypropyl and (N-dimethylamino)ethyl carboxamide side chains, the use of chloro, bromo, mesyl, and iodo leaving groups on the mustards, and regioisomeric changes. The compounds were assayed for cytotoxicity (IC50) with the NR-expressing and controls of non-NR-expressing cell lines. The proportion of NR-expressing cells required in a mixture for nonexpressing cells to experience 50% of their cytotoxicity (termed the TE50) was used to assess the compounds' ability to induce a bystander effect. This study suggests that 5-[N,N-bis(2-bromoethyl)amino]-2,4-dinitrobenzamide (8), 5-[N,N-bis(2-iodoethyl)amino]-2,4-dinitrobenzamide (9), 2-[N,N-bis(2-bromoethyl)-amino]-3,5-dinitrobenzamide (13), and 2-[N,N-bis(2-iodoethyl)amino]-3,5-dinitrobenzamide (14) showed considerable improvements over 1, exhibiting greater potency, higher IC50 ratios, and lower TE50s, and are thus superior prodrugs to 1 for GDEPT.

Improved cytotoxicity of antitumor compounds deliverable by the LDL pathway.

The concept of LDL-based chemotherapy of cancer is based on the fact that many tumors have high LDL requirements. A series of compounds has been synthesized, some of which meet all criteria for such therapy, i.e., they can be reconstituted with LDL, they do not leak out of the reconstituted LDL (rLDL), and they are potent enough to kill cells exclusively via the LDL receptor pathway. Two of these compounds are significantly superior to the best one from our earlier study [Firestone et al. (1984) J. Med. Chem. 27, 1037-1043], being cytotoxic in rLDL at concentrations reasonably attainable in vivo.

Evaluation of phthalmustine, a new anticancer compound. I. Effect on Dalton's ascitic lymphoma in mice.

The anticancer property of phthalmustine, a hitherto unknown compound containing N-mustard attached to the phthalimide ethyl chain was evaluated using a murine tumor model. The results indicate that the compound was effective in significantly restraining tumor growth. This was accompanied by marked improvement in host survival. No toxic reactions were apparent as reflected in skin and hair texture, body weight and behavioral pattern (food and water intake and activity). Blood picture showed a shift towards the normal following treatment. DNA synthesis in tumor cells was found to be affected as revealed by radioactive thymidine incorporation.