Stimulatory and protective effects of alkylating agents applied in ultra-low concentrations

Alkylating drugs belonging to the nitrogen mustard family are known as cytostatic and immunosuppressive agents. Ultra-low doses of these drugs may demonstrate pharmacological effects unlike this category of drugs. In the case of a gradual dose decrease, the number of targets for alkylation is also reduced and the drug switches from cytostatic to cell growth modifier. We postulate that application of ultra-low doses of alkylating drugs may result in a beneficial effect in the therapy of diseases associated with chronic inflammation of the mucosa, especially with the signs of epithelial atrophy.     

The discovery of a new potential anticancer drug: a case history

DNA minor groove binders (MGB) represent a class of anticancer agents whose DNA sequence specificity was hypothesized to lead to high selectivity of action. Tallimustine (TAM), a benzoyl nitrogen mustard derivative of distamycin A (DST), showed excellent antitumor activity in preclinical tests, but also a severe myelotoxicity. Novel nitrogen mustard, nitrogen half-mustard and sulfur mustard derivatives of DST showing excellent activity were recently identified and SAR reported. In particular nitrogen half-mustard and sulfur mustard derivatives, as one-arm alkylating agents, represent interesting structural novelties. A further new class of cytotoxic anticancer agents is that of alpha-halogenoacrylamido derivatives of DST-like oligopeptides, which show an activity profile substantially improved in comparison to TAM. In particular brostallicin (PNU-166196), alpha-bromo-acrylamido tetra-pyrrole derivative ending with a guanidino moiety, showed high cytotoxic potency and myelotoxicity dramatically reduced in comparison to TAM and other MGB. Brostallicin binds to the minor groove but appears unreactive in classical in vitro DNA alkylation assays. About the apparent lack of DNA alkylation we speculated that an intracellular nucleophile, e.g. glutathione (GSH), could activate the reactivity of the compound leading to alkylation of DNA in vivo. Evidence of both covalent interaction of brostallicin with plasmidic DNA in the presence of GSH and of enhanced cytotoxicity in cancer cells characterized by high levels of GSH were obtained. Brostallicin was selected for clinical development and is now undergoing Phase II studies.     

A new class of cytotoxic DNA minor groove binders: alpha-halogenoacrylic derivatives of pyrrolecarbamoyl oligomers

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 preclinical tests but also a severe myelotoxicity. Novel nitrogen mustard derivatives of distamycin showing improved activity profile were recently identified. In particular, cinnamic nitrogen mustard and cinnamic ethyl half-mustard analogs of tallimustine showed increased potency and more favorable cytotoxicity/myelotoxicity ratio. However a series of alpha-halogenoacrylamido derivatives of distamycin-like frames showed an activity profile substantially improved in comparison to tallimustine. In particular PNU-166196, alpha-bromo-acrylamido derivative of four pyrrole distamycin-like frame ending with a guanidino moiety, showed high cytotoxic potency even on tumor cell lines resistant to alkylating agents and camptothecin, broad antitumor activity and myelotoxicity dramatically reduced in comparison to tallimustine. This compound was found to bind to minor groove TA-rich sequences but appeared unreactive in classical in vitro DNA alkylation assays. With respect to the apparent lack of DNA alkylation we speculated that an intracellular reactive nucleophilic species, e.g. glutathione (GSH), could activate the reactivity of the compound leading to alkylation of DNA in vivo. Recent evidence of both covalent interaction of PNU-166196 with plasmidic DNA in the presence of GSH and of enhanced cytotoxicity in tumor cells characterized by high levels of GSH were obtained. PNU-166196, in view of its excellent activity profile and its outstanding favorable cytotoxicity/myelotoxicity ratio, was selected for clinical development and is undergoing phase I studies.     

Covalent sequestration of phosphoramide mustard by metallothionein--an in vitro study.

Acquired drug resistance is one of the most important problems in cancer chemotherapy. One of the proposed mechanisms for these phenomena is the sequestration of alkylating agents by metallothionein in vivo. This research shows that metallothionein can covalently sequester phosphoramide mustard, the active form of cyclophosphamide in vitro. On-line electrospray mass spectrometry reveals that it is phosphoramide, not nornitrogen mustard that alkylates metallothionein, although the metallothionein/nornitrogen mustard adduct was isolated as the major adduct. Tandem mass spectrometric experiments were performed on an isolated drug-modified tryptic peptide. The alkylation occurred predominantly at Cys48 of metallothionein. These results provide further evidence that overexpression of metallothionein can detoxify the active form of the drugs.     

Synthesis in vitro/in vivo evaluation and in silico physicochemical study of prodrug approach for brain targeting of alkylating agent

Every year, there are more than two lakhs of population affected with CNS tumor. Nitrogen mustard class of alkylating drugs, used clinically against various types of tumor, is too polar to cross the BBB. The redox chemical drug delivery prodrug approach is one of the most interesting procedures for delivering drugs in a sustained and specific manner to the CNS. The objective of the present study is to investigate the redox drug delivery system for the delivery of bis(2-chloroethyl)amine (nor mustard) as alkylating cytotoxic agent to the brain. Various redox derivatives of CDS-M (4a–d) were synthesized incorporating different alkyl/aryl moieties at ring nitrogen and subjected to in silico physicochemical parameters determination required for CNS activity through computational, online, and QikProp 3.2 software. The results of stability study, in vitro chemical (silver nitrate), and biological oxidation studies in human blood, rat blood, and brain homogenate for all CDS-M (4a–d) have been promising and suggest that brain targeting could be possible with more stable CDS-M (4d). The in vivo study showed that CDS-M (4d) was able to cross the BBB at detectable concentrations, and in vitro NBP alkylating activity of its quaternary salt (3d) was comparable to the known drug chlorambucil among all the synthesized derivatives.     

Different uptake of two alkylating substances by ascites tumor cells

Considerable quantitative differences have been observed between alkylating agents with respect to their effect on cell multiplication of tumor cells. The ethyleneimine derivative triaziquone is approximately a thousand times more effective than the biologically active conversion products of the nitrogen mustard derivative cyclophosphamide. The main difference may be directly related to the extent to which each of the two substances is able to pass into the cells. After a 60-min incubation period approximately 1% of the cyclophosphamide conversion products have been bound by ascites tumor cells. The uptake of the more lipophilic triaziquone, however, is nearly 50% under identical conditions.     

Chemical factors in the action of phosphoramidic mustard alkylating anticancer drugs: roles for computational chemistry.

The nitrogen mustard based DNA alkylating agents were the first effective anticancer agents and remain important drugs against many forms of cancer. More than fifty years of research on the nitrogen mustards has yielded a broad range of therapeutically useful compounds and a detailed knowledge of the biochemical mechanism of these drugs. Nevertheless, there is much ongoing research on the phosphosphoramidic and other nitrogen mustards to increase their potency and reduce their toxic and mutagenic side effects. To understand the existing nitrogen mustards, and to design the next generation of these drugs, more knowledge is needed about the effects of chemical modifications on their activation and selectivity. Because of the existing knowledge of these drugs, atomic-level chemical modeling can play an important role in the understanding of the phosphoramidic mustard compounds; however, it has not proved straight forward to directly relate the activity of these mustards with simple chemical properties such as bond lengths or atomic charges. Instead, quantum chemical simulations will be required to simulate the activation and alkylation reactions of these compounds, which will require the newest generation of quantum chemical and solvent modeling methods. Additionally, molecular dynamics simulations of the adducted DNA can provide data on the factors favoring crosslinking and its structural consequences. This review summarizes the extensive literature on the metabolism, activation, and action of the phosphoramidic mustards, with an emphasis on the roles that chemical modeling has and will play in the development of this important class of drugs.     

Synthesis and antitumor activity of new benzoheterocyclic derivatives of distamycin A

The design, synthesis, and in vivo and in vitro antileukemic activity of a novel series of compounds (13-22 and 34), in which different benzoheterocyclic rings, bearing a nitrogen mustard or a benzoyl nitrogen mustard or an alpha-bromoacryloyl group as alkylating moieties, are tethered to a distamycin frame, are reported, and structure-activity relationships are discussed. The new derivatives were prepared by coupling nitrogen mustard-substituted, benzoyl nitrogen mustard-substituted, or alpha-bromoacryloyl-substituted benzoheterocyclic carboxylic acids 23-32 with desformyldistamycin (33) or in one case with its two-pyrrole analogue 35. With very few exceptions, the activities of compounds bearing the same alkylating moiety are slightly affected by the kind of the heteroatom present on the benzoheterocyclic ring. All novel compounds, with one exception, showed in vitro activity against L1210 murine leukemia cell line comparable to or better than that of tallimustine. The compounds in which the nitrogen mustard and the alpha-bromoacryloyl moieties are directly linked to benzoheterocyclic ring showed potent cytotoxic activities (IC(50) ranging from 2 to 14 nM), while benzoyl nitrogen mustard derivatives of benzoheterocycles showed reduced cytotoxic activities, and one compound (16) of this cluster was the sole derivative devoid of significant activity. Compound 18, a 5-nitrogen mustard N-methylindole derivative of distamycin, showed the best antileukemic activity in vivo, with a very long survival time (%T/C = 457), significantly increased in comparison to tallimustine (%T/C = 133), and was selected for further extensive evaluation. Arrested polymerase chain reaction and direct DNA fragmentation assays were performed for compound 18 and the structurally related compounds 13-17 and 19. The results obtained have shown that both alkylating groups and oligopeptide frames play a crucial role in the sequence selectivity of these compounds.     

Novel dynamic flow cytoenzymological determination of intracellular esterase inhibition by BCNU and related isocyanates

We present a novel dynamic flow cytoenzymological demonstration of the potent inhibition by the antitumour chloroethylnitrosourea BCNU of the intracellular hydrolysis of fluorescein diacetate by esterases of viable, intact murine and human tumour cells in vitro. The BCNU metabonate chloroethyl isocyanate and the related compound n-butyl isocyanate were also potent inhibitors. I50 values were in the range 4.2 X 10(-5)-2.0 X 10(-4) M. Generally similar quantitative results were obtained for intact cells and sonicates by conventional spectrofluorimetry, and inhibition of purified porcine liver carboxyl esterase (EC 3.1.1.1) was demonstrated. Little or no inhibitory activity was seen with the alkylating agents methyl methane-sulphonate, melphalan and nitrogen mustard. The results are consistent with carbamoylation of the esterase molecules by isocyanates, and this may provide a basis for the flow cytometric determination of intracellular carbamoylation in discrete subpopulations of heterogeneous samples.     

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.     

Alkylation activity and molecular properties of two antineoplastic agents that utilise indometacin and a conjugate of aspirin with 2-aminonicotinic acid to transport nitrogen mustard groups

BACKGROUND: Nitrogen mustard (N-mustard) compounds are considered important anticancer drugs. Various transporting agents have been utilised to carry N-mustard groups including coumarins, amides, polyaromatic molecules and cycloalkyl structures. N-mustards act as bifunctional alkylating agents that induce cross-linking within DNA strands and cytotoxic activity. Compounds that transport the N-mustard group in vivo can also express drug-likeness that can have advantages in clinical application. This study presents data on two anticancer drugs with N-mustard groups covalently attached to NSAIDs. METHODS: Two alkylating compounds were synthesised by covalently attaching a single N-mustard group to 2-2-acetoxybenzoylaminonicotinic acid (for compound I) and indometacin (for compound II). Molecular properties such as aqueous solubility, 1-octanol/water partitioning coefficient (log Kow), molar volume, polar surface area, 1-octanol/water partitioning at pH values other than human blood (log D) and the dermal permeability coefficient (Kp) were determined. The rate-order of reaction and rate constant of alkylation were determined by reacting compound I and compound II with a target compound having a primary amine group in buffered aqueous solution at blood pH 7.4 and 37 masculineC, and monitoring absorbance at 400nm. RESULTS AND C onclusion: Compounds I and II were stable at room temperature, soluble in water and effectively alkylated a nucleophilic primary amine target at physiological temperature and pH. The water solubility of compound I was considerably greater than that of compound II. Both compounds showed second-order rate order of alkylation and effectively alkylated a nucleophilic target under aqueous physiological conditions of pH 7.4 and 37 masculineC. Kp values for compounds I and II were determined to be 0.000786 cm/h and 0.024 cm/h, respectively. Both compound I and compound II had zero percent ionisation at pH 7.4, and compound I showed zero violations of the Rule of 5. Log P values for compounds I and II were 3.27 and 5.08, respectively. This study describes the benefits of antineoplastic agents with NSAID substituents that provide favourable pharmacological properties.     

Reichardt's dye and its reactions with the alkylating agents 4‐chloro‐1‐butanol,ethyl methanesulfonate, 1‐bromobutane and Fast Red B ‐ a potentially useful reagent for the detection of genotoxic impurities in pharmaceuticals

Objectives Alkylating agents are potentially genotoxic impurities that may be present in drug products. These impurities occur in pharmaceuticals as by‐products from the synthetic steps involved in drug production, as impurities in starting materials or from in‐situ reactions that take place in the final drug product. Currently, analysis for genotoxic impurities is typically carried out using either HPLC/MS or GC/MS. These techniques require specialist expertise, have long analysis times and often use sample clean‐up procedures. Reichardt's dye is well known for its solvatochromic properties. In this paper the dye's ability to undergo alkylation is reported. Methods The reaction between Reichardt's dye and alkylating agents such as 4‐chloro‐1‐butanol and ethyl methanesulfonate was monitored spectrophotometrically at 618 nm in acetonitrile and 624 nm in N,N‐dimethylformamide. Key findings Changes in absorption were observed using low levels of alkylating agent (5–10 parts per million). Alkylation of the dye with 4‐chloro‐1‐butanol and ethyl methanesulfonate was confirmed. Reichardt's dye, and its changing UV absorption, was examined in the presence of paracetamol (10 and 100 mg/ml). Whilst the alkylation‐induced changes in UV absorption were not as pronounced as with standard solutions, detection of alkylation was still possible. Conclusions Using standard solutions and in the presence of a drug matrix, Reichardt's dye shows promise as a reagent for detection of low levels of industrially important alkylating agents.     

Nucleic acid adducts of chemical carcinogens and mutagens

The literature on the covalent nucleic acid, nucleotide, nucleoside, and base adducts of chemical carcinogens and mutagens was reviewed. The adducts of 25 simple alkylating agents, 10 aromatic amines, six polycyclic aromatic hydrocarbons, 20 epoxide derivatives, seven cytostatic drugs, and 25 miscellaneous compounds were presented in the tables. The relevance of covalent modifications and, particularly, of site-specific modifications in DNA is discussed in relation to genotoxic hazards.     

Molecular dynamics simulation of metallothionein-drug complexes.

The intermolecular interactions of metallothionein with nitrogen mustard drugs were studied by molecular dynamics simulations. Previous laboratory experiments have defined selective alkylation of two cysteine residues, and selective binding was proposed to precede alkylation. The present study provides information about accessibility to cysteines based on evaluating the intermolecular energies and distances in the first few ps of dynamics simulations. A series of dynamics simulations was performed with three drug molecules positioned at the eight most solvent accessible cysteine residues of the dimeric form of the protein. Sites proximal to the sulfhydryl groups of Cys-33 and Cys-48 were found to be the most favorable for complexing the aziridinium forms of chlorambucil, melphalan, and mechlorethamine. The sites for preferential binding are in qualitative agreement with the sites of selective alkylation defined experimentally.     

Utilizing a D-amino acid as a drug carrier for antineoplastic nitrogen mustard groups

The evaluation of an alkylating nitrogen mustard agent that utilizes D-alanine as a drug carrier for three chloroethyl substituents (ClCH₂CH₂-) is shown. Various important pharmacological properties were determined including polar surface area, partition coefficient, molar volume, polarizability, numbers of -OH and -NH₂ groups, and aqueous solubility. The synthetic approach utilizes 1,2-dichloroethane reaction with the primary amine of D-alanine resulting in chloroethyl (ClCH₂CH₂-) substituents. A nitrogen mus-tard group results, and this agent showed alkylation activity in aqueous solution directed toward a nucleophilic primary amine group. Kinetics of alkylation activity is determined by placing p-chloroaniline and the nitrogen mustard agent in sodium bicarbonate buffered aqueous solution at physiological pH 7.4 and 37°C. Samples taken from the test solution are injected with fluorescamine that reacts specifically with primary amine functional groups. Absorbance measurements obtained at 400 nm by UV-Vis spectrometer indicates the relative amounts of nonalkylated p-chloroaniline in the test solution. The D-alanine nitrogen mustard agent effectively alkylated the nucleophilic primary amine of p-chloroaniline with zero-order kinetics. The rate constant was determined to be 7.445E-04 mol/l/min. Formula weight, polar surface area, Log P, molar volume, violations of Rule of 5 for D-alanine mustard agent are 276.59, 29.543 angstroms², 1.605, 222.3 cm³, and zero violations, respectively. Cluster analysis of molecular properties showed D-alanine mustard agent to be quite similar to cyclophosphamide. This agent showed good druglikeness and zero violations of the Rule of 5, indicating good bioavailability. Molecular properties calculated for the mustard agent are numerically comparable to some clinical anticancer drugs.     

A comparison of the antifertility effects of alkylating agents and vinca alkaloids in male rats.

1 The anti-fertility effects of cyclophosphamide, nitrogen mustard, vincristine and vinblastine were studied and compared in male rats. 2 The effects of the drugs on body weight and haematological values were used to monitor the pharmacological actions of the drugs. 3 All four drugs impaired fertility, the severity of the impairment depending on dose and duration of treatment. 4 Testicular size and histological appearances remained mostly normal, even in infertile animals, but seminiferous tubules were fewer in number and maturation arrest at the spermatid level was evident in some sections. 5 Recovery of drug-induced infertility occurred in 64% of treated animals, 9 to 40 weeks after cessation of treatment. 6 Morbidity and mortality were much higher with alkylating agents than with vinca alkaloids for approximately similar degrees of impairment in fertility.     

Attempted use of zinc in vivo to protect against nitrogen mustard toxicity in tumor-free and in L1210 leukemia-bearing female B6D2F1 mice.

The use of alkylating agents in treating cancer is limited by their toxicity to both normal and tumor tissue. Early in vitro studies indicated that zinc might be effective in mitigating this toxicity to normal tissue. The present studies were done to determine the capability of zinc to induce in vivo a protective response to an alkylating agent without also contributing to mortality. Tumor-free and L1210 leukemia-bearing female B6D2F1 mice were treated with zinc before administration of the alkylating agent nitrogen mustard. Protocols for administration route and frequency as well as the chemical formulation of the zinc were varied. The effect of a phytate-free diet was studied. Two parameters were used to determine the effectiveness of zinc in protecting animals from the toxicity of nitrogen mustard: the number of tumor-free mice that survived and an increase in the median life span of the tumor-bearing mice. The zinc-induction protocols used in these studies provided a limited degree of protection against nitrogen mustard toxicity in tumor-free female mice, but in tumor-bearing animals the protective response elicited with the protocols examined did not provide an appreciable therapeutic benefit.     

Modulation of membrane transport of alkylating agents and amino acids by an analog of vasopressin in murine L5178Y lymphoblasts in vitro

The synthetic vasopressin analog 1-deamino-8-D-arginine vasopressin (dDAVP) has been shown to influence a wide range of cell-membrane-related events. Accordingly, the effect of dDAVP on membrane transport of various alkylating agents and amino acids was evaluated in L5178Y lymphoblasts in vitro. dDAVP stimulated melphalan uptake but conversely inhibited uptake of nitrogen mustard, choline (the natural transport substrate for the nitrogen mustard carrier), and leucine. No effect on the uptake of cyclophosphamide or glutamine was observed. Increased melphalan uptake was due to effects on both substrate influx and efflux. The effect of dDAVP on melphalan influx was particularly complex: dDAVP stimulated melphalan influx by amino acid transport system ASC but inhibited influx by system L, resulting in a net increase in unidirectional drug influx. Melphalan efflux was inhibited by dDAVP. Decreased uptake of nitrogen mustard, choline and leucine was due, at least in part, to decreased substrate influx. However, the mechanisms of inhibition were dissimilar: inhibition of substrate influx was non-competitive for choline but competitive for leucine. In conclusion, dDAVP induced diverse but apparently specific effects on membrane transport of several alkylating agents and amino acids. Since the accumulation of alkylating agents such as melphalan within tumor cells is a major determinant of cytotoxicity, dDAVP may have a role as a biological response modifier.