Toxic blister agents: Chemistry, mode of their action and effective treatment strategies.

Since their use during the First World War, Blister agents have posed a major threat to the individuals and have caused around two million casualties. Major incidents occurred not only due to their use as chemical warfare agents but also because of occupational hazards. Therefore, a clear understanding of these agents and their mode of action is essential to develop effective decontamination and therapeutic strategies. The blister agents have been categorised on the basis of their chemistry and the biological interactions that entail post contamination. These compounds have been known to majorly cause blisters/bullae along with alkylation of the contaminated DNA. However, due to the high toxicity and restricted use, very little research has been conducted and a lot remains to be clearly understood about these compounds. Various decontamination solutions and detection technologies have been developed, which have proven to be effective for their timely mitigation. But a major hurdle seems to be the lack of proper understanding of the toxicological mechanism of action of these compounds. Current review is about the detailed and updated information on physical, chemical and biological aspects of various blister agents. It also illustrates the mechanism of their action, toxicological effects, detection technologies and possible decontamination strategies.

Mustards-Derived Terpyridine-Platinum Complexes as Anticancer Agents: DNA Alkylation vs Coordination.

The development of bifunctional platinum complexes with the ability to interact with DNA via different binding modes is of interest in anticancer metallodrug research. Therefore, we report platinum(II) terpyridine complexes to target DNA by coordination and/or through a tethered alkylating moiety. The platinum complexes were evaluated for their in vitro antiproliferative properties against the human cancer cell lines HCT116 (colorectal), SW480 (colon), NCI-H460 (non-small cell lung), and SiHa (cervix) and generally exhibited potent antiproliferative activity although lower than their respective terpyridine ligands. 1H NMR spectroscopy and/or ESI-MS studies on the aqueous stability and reactivity with various small biomolecules, acting as protein and DNA model compounds, were used to establish potential modes of action for these complexes. These investigations indicated rapid binding of complex PtL3 to the biomolecules through coordination to the Pt center, while PtL4 in addition alkylated 9-ethylguanine. PtL3 was investigated for its reactivity to the model protein hen egg white lysozyme (HEWL) by protein crystallography which allowed identification of the Nδ1 atom of His15 as the binding site.

Adduct of the blistering warfare agent sesquimustard with human serum albumin and its mass spectrometric identification for biomedical verification of exposure.

Apart from the well-known sulfur mustard (SM), additional sulfur-containing blistering chemical warfare agents exist. Sesquimustard (Q) is one of them and five times more blistering than SM. It is a common impurity in mustard mixtures and regularly found in old munitions but can also be used in pure form. Compared to the extensive literature on SM, very little experimental data is available on Q and no protein biomarkers of exposure have been reported. We herein report for the first time the adduct of Q with the nucleophilic Cys34 residue of human serum albumin (HSA) formed in vitro and introduce two novel bioanalytical procedures for detection. After proteolysis of this HSA adduct catalyzed either by pronase or by proteinase K, two biomarkers were identified by high-resolution tandem mass spectrometry (MS/HR MS), namely a dipeptide and a tripeptide, both alkylated at their Cys residue, which we refer to as HETETE-CP and HETETE-CPF. HETETE represents the Q-derived thio-alkyl moiety bearing a terminal hydroxyl group: "hydroxyethylthioethylthioethyl." Targeting both peptide markers from plasma, a micro liquid chromatography-electrospray ionization tandem mass spectrometry method working in the selected reaction monitoring mode (µLC-ESI MS/MS SRM) was developed and validated as well suited for the verification of exposure to Q. Fulfilling the quality criteria defined by the Organisation for the Prohibition of Chemical Weapons, the novel methods enable the detection of exposure to Q alone or in mixtures with SM. We further report on the relative reactivity of Q compared to SM. Based on experiments making use of partially deuterated Q as the alkylating agent, we rule out a major role for six-membered ring sulfonium ions as relevant reactive species in the alkylation of Cys34. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys34 allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts. Graphical abstract.

A quantitative method to detect human exposure to sulfur and nitrogen mustards via protein adducts.

Sulfur and nitrogen mustards are internationally banned vesicants listed as Schedule 1 chemical agents in the Chemical Weapons Convention. These compounds are highly reactive electrophiles that form stable adducts to a variety of available amino acid residues on proteins upon exposure. We present a quantitative exposure assay that simultaneously measures agent specific protein adducts to cysteine for sulfur mustard (HD) and three nitrogen mustards (HN1, HN2, and HN3). Proteinase K was added to a serum or plasma sample to digest protein adducts and form the target analyte, the blister agent bound to the tripeptide cysteine-proline-phenylalanine (CPF). The mustard adducted-tripeptide was purified by solid phase extraction and analyzed using isotope dilution LC-MS/MS. Product ion structures were identified using high-resolution product ion scan data for HD-CPF, HN1-CPF, HN2-CPF, and HN3-CPF. Thorough matrix comparison, analyte recovery, ruggedness, and stability studies were incorporated during method validation to produce a robust method. The method demonstrated long term-stability, precision (RSD < 15%), and intra- and inter-day accuracies > 85% across the reportable range of 3.00-200 ng/mL for each analyte. Compared to previously published assays, this method quantitates both sulfur and nitrogen mustard exposure biomarkers, requires only 10 µL of sample volume, and can use either a liquid sample or dried sample spot.

A Low-Toxicity DNA-Alkylating N-Mustard-Quinoline Conjugate with Preferential Sequence Specificity Exerts Potent Antitumor Activity Against Colorectal Cancer.

Efficacy and safety are fundamental prerequisites for anticancer drug development. In the present study, we explored the anti-colorectal cancer (CRC) activity of SL-1, a DNA-directed N-mustard-quinoline conjugate. The N-mustard moiety in SL-1 induced DNA strand breaks, interstrand cross-links (ICLs), G2/M arrest, and apoptosis, whereas its quinoline moiety preferentially directed SL-1 to target the selective guanine sequence 5'-G-G/C-N-G-C/T-3'. Notably, SL-1 was highly cytotoxic to various CRC cell lines. Experiments using xenograft models revealed that SL-1 was more potent than 5-fluorouracil (5-FU) and oxaliplatin for suppressing the growth of RKO and RKO-E6 (oxaliplatin-resistant subline) cells as well as metastatic SW620 cells. In addition, SL-1 combined with 5-FU was more effective than oxaliplatin and 5-FU for suppressing RKO or SW620 cell growth in mice. Significantly, compared with cisplatin, oxaliplatin, or 5-FU, SL-1 alone or in combination with 5-FU did not cause obvious kidney or liver toxicity in ICR mice. In summary, SL-1, a DNA-directed alkylating agent, is established as an anti-CRC agent with high efficacy and low toxicity and thus warrants further development for the treatment of CRC patients.

Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA.

Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds were designed, synthesized and biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topoisomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid had a mean GI(50) in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the N-mustard compounds from which they were derived. These results supported the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA.

Design and synthesis of potent antitumor water-soluble phenyl N-mustard-benzenealkylamide conjugates via a bioisostere approach.

A series of new, water-soluble phenyl N-mustard-benzenealkylamide conjugates containing hydrophilic ω-dialkylaminoalkylamide or ω-cyclic aminoalkylamide moieties were synthesized via a bioisostere approach. These compounds have a broad spectrum of antitumor activity against a panel of human tumor cell lines. Of these derivatives, compound 18b effectively suppressed the growth of colon cancer (HCT-116), prostate cancer (PC3), and lung cancer (H460) xenografts. The growth of HCT-116 xenografts was almost completely suppressed when co-treated with compound 18b and 5-fluorouracil. Furthermore, compound 18b can induce DNA cross-linking and cell-cycle arrest at the G2/M phase. Early preclinical studies, including pharmacokinetics in rats, inhibition of the hERG, and 14 days of acute intravenous injection toxicity, suggest that compound 18b is a promising candidate for further preclinical studies.

An essential type I nitroreductase from Leishmania major can be used to activate leishmanicidal prodrugs.

Nitroaromatic prodrugs are used to treat a range of microbial infections with selectivity achieved by specific activation reactions. For trypanosomatid parasites, this is mediated by type I nitroreductases. Here, we demonstrate that the causative agent of leishmaniasis, Leishmania major, expresses an FMN-containing nitroreductase (LmNTR) that metabolizes a wide range of substrates, and based on electron donor and acceptor preferences, it may function as an NADH:quinone oxidoreductase. Using gene deletion approaches, we demonstrate that this activity is essential to L. major promastigotes, the parasite forms found in the insect vector. Intriguingly, LmNTR(+/-) heterozygote promastigote parasites could readily differentiate into infectious metacyclic cells but these were unable to establish infections in cultured mammalian cells and caused delayed pathology in mice. Furthermore, we exploit the LmNTR activity evaluating a library of nitrobenzylphosphoramide mustards using biochemical and phenotypic screens. We identify a subset of compounds that display significant growth inhibitory properties against the intracellular parasite form found in the mammalian hosts. The leishmanicidal activity was shown to be LmNTR-specific as the LmNTR(+/-) heterozygote promastigotes displayed resistance to the most potent mustards. We conclude that LmNTR can be targeted for drug development by exploiting its prodrug activating property or by designing specific inhibitors to block its endogenous function.

Synthesis of an azide-bearing N-mustard analogue of S-adenosyl-L-methionine.

The synthesis of an azide-bearing N-mustard S-adenosyl-L-methionine (SAM) analogue, 8-azido-5'-(diaminobutyric acid)-N-iodoethyl-5'-deoxyadenosine, has been accomplished in 10 steps from commercially available 2',3'-isopropylidene adenosine. Critical to this success was executing C8 azidation prior to derivatizing the 5'-position of the ribose sugar and the late stage alkylation of the 5' amino group with bromoethanol, which was necessitated by the reactivity of the aryl azide moiety. The azide-bearing N-mustard is envisioned as a useful biochemical tool by which to probe DNA and protein methylation patterns.

Design, synthesis, and biological evaluation of novel water-soluble N-mustards as potential anticancer agents.

A series of novel water-soluble N-mustard-benzene conjugates bearing a urea linker were synthesized. The benzene moiety contains various hydrophilic side chains are linked to the meta- or para-position of the urea linker via a carboxamide or an ether linkage. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and therapeutic efficacy against human tumor xenografts in vivo. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft and significant suppression against prostate adenocarcinoma PC3 xenograft were achieved by treating with compound 9aa' at the maximum tolerable dose with relatively low toxicity. We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay. A pharmacokinetic profile of the representative 9aa' in rats was also investigated. The current studies suggest that this agent is a promising candidate for preclinical studies.

Synthesis and in vitro cytotoxicity of 9-anilinoacridines bearing N-mustard residue on both anilino and acridine rings.

A series of 9-anilinoacridines having an alkylating N-mustard pharmacophore on both anilino (C-3' or C-4') and acridine (C-4) rings with O-ethyl (O-C(2)) or O-butyl (O-C(4)) spacer were synthesized to evaluate their cytotoxicity against human lymphoblastic leukemia (CCRF-CEM) cell growth in vitro. It was revealed that these conjugates exhibited significant in vitro cytotoxicity. Among these agents, compound 13 was the most cytotoxic with IC(50) value of 1.3 nM and is as potent as taxol (IC(50)=1.1 nM). The structure-activity relationship study showed that the length of the spacer and the position of the substituent do affect their cytotoxicity.

Molecular mechanisms for the activity of PX-478, an antitumor inhibitor of the hypoxia-inducible factor-1alpha.

We have reported previously that PX-478 (S-2-amino-3-[4'-N,N,-bis(chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) has potent antitumor activity against a variety of human tumor xenografts associated with the levels of the hypoxia-inducible factor-1alpha (HIF-1alpha) within the tumor. We now report that PX-478 inhibits HIF-1alpha protein levels and transactivation in a variety of cancer cell lines. Hypoxia-induced vascular endothelial growth factor formation was inhibited by PX-478, whereas baseline levels of vascular endothelial growth factor in normoxia were unaffected. Studies of the mechanism of PX-478 action showed that HIF-1alpha inhibition occurs in both normoxia and hypoxia and does not require pVHL or p53. In addition, PX-478 decreases levels of HIF-1alpha mRNA and inhibits translation as determined by 35S labeling experiments and reporter assays using the 5' untranslated region of HIF-1alpha. Moreover, to a lesser extent, PX-478 also inhibits HIF-1alpha deubiquitination resulting in increased levels of polyubiquitinated HIF-1alpha. The inhibitory effect of PX-478 on HIF-1alpha levels is primarily due to its inhibition of translation because HIF-1alpha translation continues in hypoxia when translation of most proteins is decreased. We conclude that PX-478 inhibits HIF-1alpha at multiple levels that together or individually may contribute to its antitumor activity against HIF-1alpha-expressing tumors.

Influence of mustard group structure on pathways of in vitro metabolism of anticancer N-(2-hydroxyethyl)-3,5-dinitrobenzamide 2-mustard prodrugs.

The dinitrobenzamide mustards are a class of bioreductive nitro-aromatic anticancer prodrugs, of which a phosphorylated analog (PR-104) is currently in clinical development. They are bioactivated by tumor reductases to form DNA cross-linking cytotoxins. However, their biotransformation in normal tissues has not been examined. Here we report the aerobic in vitro metabolism of three N-(2 hydroxyethyl)-3,5-dinitrobenzamide 2-mustards and the corresponding nonmustard analog in human, mouse, rat, and dog hepatic S9 preparations. These compounds have a range of mustard structures (-N(CH(2)CH(2)X)(2) where X = H, Cl, Br, or OSO(2)Me). Four metabolic routes were identified: reduction of either nitro group, N-dealkylation of the mustard, plus O-acetylation, and O-glucuronidation of the hydroxyethyl side chain. Reduction of the nitro group ortho to the mustard resulted in intramolecular alkylation and is considered to be an inactivation pathway, whereas reduction of the nitro group para to the mustard generated potential DNA cross-linking cytotoxins. N-Dealkylation inactivated the mustard moiety but may result in the formation of toxic acetaldehyde derivatives. Increasing the size of the nitrogen mustard leaving group abrogated the ortho-nitroreduction and N-dealkylation routes and thereby improved overall metabolic stability but had little effect on aerobic para-nitroreduction. All four compounds underwent O-glucuronidation of the hydroxyethyl side chain and further studies to elucidate the relative importance of this pathway in vivo are in progress.

Reactions of {4-[bis(2-chloroethyl)amino]phenyl}acetic acid (phenylacetic acid mustard) with 2'-deoxyribonucleosides.

Phenylacetic acid mustard (PAM; 2), a major metabolite of the anticancer agent chlorambucil (CLB; 1), was allowed to react with 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), 2'-deoxy-5-methylcytidine (dMeC), and thymidine (T) at physiological pH (cacodylic acid, 50% base). The reactions were followed by HPLC and analyzed by HPLC/MS and/or (1)H-NMR techniques. Although the predominant reaction observed was hydrolysis of PAM, 2 also reacted with various heteroatoms of the nucleosides to give a series of products: compounds 5-31. PAM (2) was found to be hydrolytically slightly more stable than CLB (1). The principal reaction sites of 2 with dA, dG, and with all pyrimidine nucleosides were N(1), N(7), and N(3), resp. Also, several other adducts were detected and characterized. There was no significant difference in the reactivity of 1 and 2 with dG, dA or T, but the N(3) dC-PAM adduct was deaminated easier than the corresponding CLB derivative. The role of PAM-2'-deoxyribonucleoside adducts on the cytotoxic and mutagenic properties of CLB (1) is discussed.

Novel fluorinated prodrugs for activation by carboxypeptidase G2 showing good in vivo antitumor activity in gene-directed enzyme prodrug therapy.

Sixteen novel polyfluorinated benzoic acid mustards have been synthesized for use in gene-directed enzyme prodrug therapy (GDEPT). Eight of these were benzoic acid L-glutamate mustards for evaluation as prodrugs and the other eight were the active drugs formed by the action of the bacterial enzyme carboxypeptidase G2 (CPG2). All of the di- and trifluorinated prodrugs were efficiently cleaved by the enzyme. In contrast, the tetrafluorinated prodrugs were found to be competitive inhibitors of CPG2, the first such inhibitors to have been described. The di- and trifluorinated prodrugs were differentially cytotoxic to human breast carcinoma cells (MDA MB 361) expressing CPG2, compared to control cells that did not express the enzyme. The difluorinated prodrug {4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoyl}-L-glutamic acid and its iodoethylamino analogue were effective substrates for the enzyme and showed excellent therapeutic activity in CPG2-expressing MDA MB 361 xenografts, either curing or greatly inhibiting tumor growth and extending the life of the animals.

Nucleophilic substitution by grignard reagents on sulfur mustards.

With proper activation of the leaving group, sulfur mustards react with Grignard reagents with neighboring group participation of the sulfur atom. 2,6-Dichloro-9-thiabicyclo[3.3.1]nonane is especially useful in this regard, providing clean reactivity with organomagnesium nucleophiles on a topologically constrained scaffold.

Conjugation of a hairpin pyrrole-imidazole polyamide to a quinone methide for control of DNA cross-linking.

A series of quinone methide precursors designed for DNA cross-linking were prepared and conjugated to a pyrrole-imidazole polyamide for selective association to the minor groove. Although reaction was only observed for DNA containing the predicted recognition sequence, yields of strand alkylation were low. Interstrand cross-linking was more efficient than alkylation but still quite modest and equivalent to that generated by a comparable conjugate containing the N-mustard chlorambucil. Varying the length of the linker connecting the polyamide and quinone methide derivative did not greatly affect the yield of DNA cross-linking. Instead, intramolecular trapping of the quinone methide intermediate by nucleophiles of the attached polyamide appears to be the major determinant that limits its reaction with DNA. Self-adducts of the quinone methide conjugate form readily and irreversibly as detected by a combination of chromatography and mass spectroscopy. This result is unlike comparable self-adducts observed for oligonucleotide conjugates that form more slowly and remain reversible. Equivalent intramolecular alkylation of a polyamide by its attached chlorambucil mustard was not observed under similar condition. The presence of DNA, however, did facilitate hydrolysis of this mustard conjugate.

Chemical weapons: documented use and compounds on the horizon.

Man's inhumanity to man is expressed through a plethora of tools of modern warfare and terror. The use of chemical and biological weapons with the goals of assault, demoralisation and lethality has been documented in recent history, both on the battlefield and in urban terror against civilians. A general review of a few of the currently employed chemical weapons and biological toxins, along with a look at potential chemical weapons and tools of counter-terrorism, follows. While these weapons are fearsome elements, the dangers should be viewed in the context of the widespread availability and efficacy of conventional weapons.

Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-inducible factor-1alpha.

The hypoxia-inducible factor-1 (HIF-1) transcription factor is an important regulator of tumor response to hypoxia that include increased angiogenesis, glycolytic metabolism, and resistance to apoptosis. HIF-1 activity is regulated by the availability of the HIF-1alpha subunit, the levels of which increase under hypoxic conditions. PX-478 (S-2-amino-3-[4'-N,N,-bis(2-chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) is an inhibitor of constitutive and hypoxia-induced HIF-1alpha levels and thus HIF-1 activity. We report that PX-478 given to mice suppresses HIF-1alpha levels in HT-29 human colon cancer xenografts and inhibits the expression of HIF-1 target genes including vascular endothelial growth factor and the glucose transporter-1. PX-478 shows antitumor activity against established (0.15-0.40 cm(3)) human tumor xenografts with cures of SHP-77 small cell lung cancer and log cell kills up to 3.0 for other tumors including HT-29 colon, PC-3 prostate, DU-145 prostate, MCF-7 breast, Caki-1 renal, and Panc-1 pancreatic cancers. Large (0.83 cm(3)) PC-3 prostate tumors showed 64% regression, which was greater than for smaller tumors. The antitumor response to PX-478 was positively correlated with tumor HIF-1alpha levels (P < 0.02) and was accompanied by massive apoptosis. The results show that PX-478 is an inhibitor of HIF-1alpha and HIF-1 transcription factor activity in human tumor xenografts and has marked antitumor activity against even large tumor xenografts, which correlates positively with HIF-1alpha levels.

Distamycin A as stem of DNA minor groove alkylating agents.

Analogues of naturally occurring antitumor agents, such as distamycin A, which bind in the minor groove of DNA, represent a new class of anticancer compounds currently under investigation. Distamycin A has driven researcher's attention not only for their biological activity, but also for its non intercalative binding to the minor groove of double-stranded B-DNA, where it forms strong reversible complex preferentially at the nucleotide sequences consisting of 4-5 adjacent AT base pairs. The pyrrole-amide skeleton of distamycin A has been also used as DNA sequence selective vehicles for the delivery of alkylating functions to DNA targets, leading to a sharp increase of its cytotoxicity, in comparison to that, very weak, of distamycin itself. In the last few years, several hybrid compounds, in which known antitumor derivatives or simple active moieties of known antitumor agents have been tethered to distamycin frames, have been designed, synthesized and tested. Several efforts have been made to modify DNA sequence selectivity and stability of the distamycin and the structural modifications have been based on replacement of pyrrole by other heterocycles and/or benzoheterocycles obtaining a novel class of minor groove binding molecules called lexitropsins. The role of the amidino moiety, by means of the substitution with various groups, which includes ionizable, acid or basic, and non-ionizable groups, has been also studied. The synthesis of a hybrid deriving among the combination of the distamycin A and naturally occurring alkylating agent has been also reported. Several classes of distamycin derivatives that have been reported in the published literature have been described in this review article.