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.

Hybrids of Steroid and Nitrogen Mustard as Antiproliferative Agents: Synthesis, In Vitro Evaluation and In Silico Inverse Screening.

Hybrids of 16E-arylidene steroids and nitrogen mustard have been synthesized and evaluated for their in vitro cytotoxic activity to develop tissue specific antineoplastic agents from steroids. These hybrids displayed specificity towards leukemia cell lines, however somewhat reduced potency was observed in comparison with the earlier reported 16E-arylidene steroids. The in silico reverse screening experiments were employed to find out the probable pharmacological mechanism of these hybrids. Molecular docking studies suggested glucocorticoid receptors as a probable target for the antileukemic action of these steroid-nitrogen mustard hybrids.

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.

Design, synthesis and biological evaluation of novel sesquiterpene mustards as potential anticancer agents.

Several novel series of sesquiterpene mustards (SMs) bearing nitrogen mustard and glutathione (GSH)-reactive α-methylene-γ-butyrolactone groups were successfully prepared for the first time and showed excellent antiproliferative activities in vitro. Among them, compounds 2e and 2g displayed the highest antiproliferative properties with IC50 values ranging from 2.5 to 8.7 µM. The selectivity of these two compounds was evaluated by SRB method against human cancer and normal hepatic cells (HepG2 and L02). The induction of apoptosis and effects on the cell cycle distribution with compounds 2e and 2g were investigated by Hoechst 33,258 staining and flow cytometry, which exhibited that they could induce selective cell apoptosis and cell cycle arrest in HepG2 and L02 cells. In addition, further investigation showed that compounds 2e and 2g could obviously inhibit the proliferation of HepG2 cells by inducing significant DNA cross-linking and depleting GSH in cell media. The good cytotoxicity and selectivity of compounds 2e and 2g pointed them as promising leads for anticancer drug design.

Synthesis and biological studies of the thiols-triggered anticancer prodrug for a more effective cancer therapy.

A novel anticancer prodrug compound 1, which was designed to be triggered by thiols and release the chemotherapeutic agent mechlorethamine, was successfully prepared and evaluated for the first time. The activation of compound 1 was determined by NMR analysis and denaturing alkaline agarose gel electrophoresis. A fluorescence image and comet assay indicated that the inducible reactivity of 1 could be accomplished in cell media. The anticancer activities are also discussed.

Functional vesicles formed by anticancer drug assembly.

In this Letter, a new type of nitrogen mustard conjugate vesicles is developed to improve the stability and efficiency of anticancer drug. Benzoic acid nitrogen mustard-peptide (AAAK) conjugate was designed and synthesized, which was found to self-assemble into vesicles in water. The formation of the vesicles was confirmed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and circular dichroism (CD). The degradation data revealed that the benzoic acid nitrogen mustard peptide (AAAK) conjugate vesicles are more stable than the parent drug in aqueous solution. Furthermore, MTT assay revealed that the free drug conjugate has similar antitumor activity against MCF-7, Hela, HepG-2 cell lines compared with the parent drug. The benzoic acid nitrogen mustard-peptide conjugate vesicles may have potential in the treatment of cancers.

Synthesis and in vitro antiproliferative activity of thiazole-based nitrogen mustards: the hydrogen bonding interaction between model systems and nucleobases.

Synthesis, characterization and investigation of antiproliferative activity of eight thiazole-based nitrogen mustard against human cancer cells lines (MV4-11, A549, HCT116 and MCF-7) and normal mouse fibroblast (BALB/3T3) are presented. Their structures were determined using NMR, FAB MS, HRMS and elemental analyses. Among the derivatives, 3a, 3b, 3e and 3h were found to exhibit high activity against human leukemia MV4-11 cells with IC50 values of 0.634-3.61 µg/ml. The cytotoxic activity of compound 3a against BALB/3T3 cells is up to 40 times lower than against cancer cell lines. Our data indicated also that compound 3e had very strong activity against MCF-7 and HCT116 with IC50 equal to 2.32 µg/ml and 2.81 µg/ml, respectively. Their activity was similar to the activity of cis-platin, which is clinically used as anticancer drug in the treatment of human solid tumours. We also perform quantum chemical calculation of interaction and binding energies in complexes of model systems and 3e with DNA bases. Interaction of real drug 3e with guanine is much stronger than with the remaining nucleobases, and the strongest among all investigated complexes. Computer simulations were performed with ATP-binding domain and DNA-binding site of hTopoII. Compounds 3a-h were recognized as potential inhibitors of hTopoII.

Synthesis and analysis of activity of a potential anti-melanoma prodrug with a hydrazine linker.

A potential anti-melanoma prodrug containing a phenolic activator, a hydrazine linker, and a nitrogen mustard effector - (N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N'-(4-hydroxybenzyl)hydrazine) has been synthesized in seven steps. Spectrophotometric measurements of its oxidation by tyrosinase showed a rapid increase of absorbance at 337 nm. HPLC analysis demonstrated that two major products were formed. However, during the reaction one of the products was converted into the other. The stable product with a maximum of absorption at 337 nm was isolated and identified as 5,6-dihydroxy-1H-indazol-1-yl 4-[bis-(2-chloroethyl)amino]benzoate. It was formed by a cyclization of the enzymatically generated o-quinone. This reaction was unexpected, since the acylated hydrazine nitrogen atom should not be sufficiently nucleophilic to attack the o-quinone ring. This cyclization prevented the effector release from the enzyme-activated prodrug. As a result, the prodrug showed only limited specificity for B16-F10 murine melanoma cells compared to reference cell lines. When applied in solid tumors in mice it showed slightly higher activity than the parent mustard drug (4-[bis-(2-chloroethyl)amino]benzoic cid), but significantly lower activity than melphalan, a commercial mustard drug with a structure resembling tyrosine, occasionally used in the treatment of melanoma.

Design and synthesis of novel quinazoline nitrogen mustard derivatives as potential therapeutic agents for cancer.

Thirteen novel quinazoline nitrogen mustard derivatives were designed, synthesized and evaluated for their anticancer activities in vitro and in vivo. Cytotoxicity assays were carried out in five cancer cell lines (HepG2, SH-SY5Y, DU145, MCF-7 and A549) and one normal human cell line (GES-1), in which compound 22b showed very low IC50 to HepG2 (the IC50 value is 3.06 µM), which was lower than Sorafenib. Compound 22b could inhibit cell cycle at S and G2/M phase and induce cell apoptosis. In the HepG2 xenograft model, 22b exhibited significant cancer growth inhibition with low host toxicity in vivo.

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.

Synthesis and antitumor activity of formononetin nitrogen mustard derivatives.

A series of formononetin nitrogen mustard derivatives were synthesized and evaluated in vitro for their cytotoxicity against five cancer cell lines (SH-SY5Y, HCT-116, DU-145, Hela and SGC-7901). The pharmacological results showed that many of the new derivatives displayed more potent cytotoxicity than alkeran. Furthermore, compounds 6d and 6n could induce cell cycle arrest at G2/M phase and cell apoptosis.

New thymine-based derivative of nitrogen mustards.

This work deals with the synthesis of a new nitrogen mustard derivative based on thymine. To introduce the bis(2-chloroethyl)amine group to position 4 of the pyrimidine base, many strategies were explored and the desired compound was finally obtained, thanks to a synthetic pathway in five steps.

Synthesis and cytotoxicity studies of bifunctional hybrids of nitrogen mustards with potential enzymes inhibitors based on melamine framework.

The new class of hybrid anticancer drugs were obtained by selective functionalization of the triazine scaffold. These were prepared by rearrangement of mono-, bis- and/or tris-(1,3,5-triazin-2-yl)-1,4-diazabicyclo[2.2.2]octanium chlorides leading to formation of 2-chloroethylamino fragments attached to 1,3,5-triazine via one, two or three piperazine rings respectively. Their inhibitory effect was found strongly dependent on the structure of substituents in triazine ring. The anti-proliferative activity of the hybrids evaluated in vitro by using mammalian tumour cells estimated as IC(50) was in the range 0.62-139,78 µM. Both cytotoxicity and alkylating activity depended on the substituents of triazine ring, however, also the mono-functional analogues of nitrogen mustards, which are unable to form liaisons between two DNA strands, induced apoptosis and necrosis in the tested cells.

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.

Recent outcome in the field of distamycin-derived minor groove binders.

DNA minor groove binders represent a class of cytotoxic antitumor agents whose DNA sequence specificity may lead to a high selectivity of action. Tallimustine, benzoyl nitrogen mustard derivative of distamycin A, showed excellent antitumor activity in the preclinical tests, but as other minor groove binders, showed severe myelotoxicity. Novel nitrogen mustard derivatives of distamycin showing improved activity profile, have been identified recently. Moreover, a series of alpha-halogenoacrylamido derivatives of distamycin-like frames, in which the typical amidino moiety has been replaced with other moieties, was found to show cytotoxic and antitumor activity and cytotoxicity/myelotoxicity ratio improved significantly in comparison to tallimustine. The structural features of the alkylating moieties and binding frames, of distamycin and distamycin-like derivatives disclosed recently are discussed.