Mustard Derivatives Research Articles

Formulation and pharmacokinetics of lipid nanoparticles of a chemically sensitive nitrogen mustard derivative: Chlorambucil

Lipid nanoparticles of the cancer drug Chlorambucil (CLB) were prepared by ultrasonication, using stearic acid as the core lipid. Four types of lipid nanoparticle formulations were studied: (i) stearic acid solid lipid nanoparticles (SLN); (ii) sterically stabilized SLN with pegylated phospholipids as stabilizer; (iii) nanostructured lipid complexes with oleic acid as adjunct lipid; (iv) lipid nanocomplexes with dimethyl dioctadecyl ammonium bromide (DDAB) as surface modifier (LN). Lipid nanoparticles were characterized for particle size, assay and encapsulation efficiency, particle morphology and physico-chemical stability over 90 days. All of the formulations were physically stable, with an average particle size of 147 (±10) nm. The drug encapsulation efficiency (DEE) of all the formulations except LN decreased significantly over time ( p < 0.05), probably due to the expulsion of CLB upon crystallization. This indicated that the presence of DDAB in stearic acid nanoparticles increases DEE, preventing CLB degradation in the aqueous disperse phase. Pharmacokinetic studies of the intravenous LN formulation revealed plasma clearance kinetics were comparable to that of CLB solution ( p > 0.01), indicating electrostatic charge mediated clearance, as reported earlier. In tissue and tumor distribution studies, lower AUC values of CLB were observed for LN compared to CLB solution in liver, kidneys, heart and lungs. However, higher AUC values of LN formulation as compared to CLB solution ( p < 0.01) in tumors suggested that the presence of DDAB on the lipid nanoparticles resulted in greater accumulation of the drug in tumors.

NSC 290205-based therapy in colon 38 adenocarcinoma in combination with adriamycin (ADR)

15104 Background: NSC 290205 (A) is a hybrid synthetic antitumor ester which combines a D-lactam derivative of androsterone and nitrogen mustard. In this study cyclophosphamide in the standard CHOP chemotherapeutic regimen was replaced with NSC 290205 (AHOP) and the efficacy of these regimens against murine colon 38 adenocarcinoma was compared. Methods: Colon 38 adenocarcinoma was used in this study. It was purchased from NCI (USA). Tumor was grown in C57 Bl mice and was transplanted subcutaneously with puncture in the inguinal region. The amount of the transplanted graft was 40–50 mg fragment. The acute toxicity of the compounds was determined and the lethal dose LD10 was used as a therapeutic dose. The antitumor activity was assessed from the inhibition of tumor growth by volume in cm3 and T/C % oncostatic parameter, according to the protocol of the experimental evaluation of anticancer drugs of the NCI. Treatment was given as an intermitted dose on days 1, 5, 9. Results: Results show that treatment with steroidal derivative (A) or cyclophosphamide (C) produced almost equal borderline activity. Moreover, both CHOP and AHOP regimens showed significant and comparable antitumor effect. AHOP caused the maximum effect inhibiting tumor growth by 68.5 % and producing T/C values of 211%. CHOP was less effective producing 47.1% inhibition of tumor growth and T/C 174%. Conclusions: Although the treatment of colon adenocarcinoma with cyclophosphamide or NSC 290205 yielded equivalent results, AHOP showed higher antitumor potency than CHOP. It is very likely that the D-lactamic steroid (androstan) alkylator for A, containing the amide group -NH-CO-, combined with adriamycin which intercalates between DNA base-pairs, is the explanation for the higher activity of AHOP as compared to CHOP. Preclinical research supports that the aza-steroidal alkylator NSC 290205 demonstrates favorable acute and sub acute toxicity, as well as superior antitumor activity which in combination with adriamycin against colon 38 adenocarcinoma justifies further clinical studies. No significant financial relationships to disclose.

Synthesis and Biological Activity of Mustard Derivatives of Thymine

The synthesis and biological activity of a novel DNA cross-linking antitumor agent is presented. The new alkylating agent significantly inhibited cell proliferation, migration and invasion as tested in vitro on the A431 vulvar epidermal carcinoma cell line.

Investigation of the site of action of McN‐A‐343 at the M1 muscarinic receptor based on the binding profile of its nitrogen mustard analogue.

To date, the precise location of the binding site for McN‐A‐343 on the M1 muscarinic receptor has yet to be determined, although prior studies on the M2 receptor suggest binding to both the orthosteric and allosteric sites. To address this question, we investigated the mode of interaction of a nitrogen mustard derivative of McN‐A343 (BR384; 4‐[(2‐bromoethyl)methyl‐amino]‐2‐butynyl N‐(3‐chlorophenyl)carbamate) with the M1 receptor using radioligand binding methods. The modulatory effects of known competitive and allosteric muscarinic ligands on the irreversible alkylation of the receptor were investigated in stably transfected CHO‐K1 cells and homogenized tissue. In both brain tissue homogenates and CHO‐K1 M1 cells gallamine exhibited allosteric inhibition of M1 receptor alkylation by BR384, whereas N‐methylscopolamine, atropine and Mc‐N‐A‐343 competitively inhibited alkylation. WIN 62,777 had no significant effect on the rate of M1 receptor alkylation by BR384. Our data suggest that both BR384 and McN‐A‐343 bind to the orthosteric site of the M1 receptor.Supported by NIH grant 69829 (Frederick J. Ehlert) and Pre‐Doctoral Fellowship from The PhRMA Foundation (Katherine W. Figueroa).

Use of 4‐[(2‐bromoethyl)methyl‐amino]‐2‐butynyl N‐(3‐chlorophenyl)carbamate (BR 384), to study allosteric interactions at M2 muscarinic receptor

We explored the interaction of a nitrogen mustard derivative of McN‐A‐343 (BR 384) with the human M2 muscarinic receptor expressed in CHO cells using the muscarinic radioligand, [3H]N‐methylscopolamine ([3H]NMS). BR 384 caused a concentration‐dependent, first order loss of [3H]NMS binding at 37°C, with the half maximal rate constant occurring at 8.6 μM BR 384. We examined the effects of various ligands on the rate of alkylation of M2 receptors by BR 384. NMS competitively slowed the rate of alkylation, whereas the inhibition by gallamine and McN‐A‐343 reached a plateau at high concentrations, indicating an allosteric inhibition. In contrast, WIN51708 had no effect. We also measured the inhibition of [3H]NMS binding by BR 384 at 0°C, conditions under which covalent binding is reduced. In these experiments, the dissociation constants of NMS, gallamine, McN‐A‐343 and the aziridinium ion of BR 384 for their binding sites were 0.0001, 0.64, 18 and 3.7 μM, respectively. In contrast, the parent mustard and alcoholic hydrolysis product of BR 384 were without effect. Our results show that the aziridinium ion of BR 384 binds covalently to the orthosteric site of the M2 receptor, whereas McN‐A‐343 binds to an allosteric site. Measurement of the effects of ligands on the rate of inactivation of muscarinic receptors by a covalent orthosteric ligand is a powerful method for detecting allosterism.Supported by a NIH Grant GM 69829.

Epigenetic perturbations in the pathogenesis of mustard toxicity; hypothesis and preliminary results

Among the most readily available chemical warfare agents, sulfur mustard (SM), also known as mustard gas, has been the most widely used chemical weapon. SM causes debilitating effects that can leave an exposed individual incapacitated for days to months; therefore delayed SM toxicity is of much greater importance than its ability to cause lethality. Although not fully understood, acute toxicity of SM is related to reactive oxygen and nitrogen species, oxidative stress, DNA damage, poly(ADP-ribose) polymerase (PARP) activation and energy depletion within the affected cell. Therefore several antioxidants and PARP inhibitors show beneficial effects against acute SM toxicity. The delayed toxicity of SM however, currently has no clear mechanistic explanation. One third of the 100,000 Iranian casualties are still suffering from the detrimental effects of SM in spite of the extensive treatment. We, therefore, made an attempt whether epigenetic aberrations may contribute to pathogenesis of mustard poisoning. Preliminary evidence reveals that mechlorethamine (a nitrogen mustard derivative) exposure may not only cause oxidative stress, DNA damage, but epigenetic perturbations as well. Epigenetic refers to the study of changes that influence the phenotype without causing alteration of the genotype. It involves changes in the properties of a cell that are inherited but do not involve a change in DNA sequence. It is now known that in addition to mutations, epimutations contribute to a variety of human diseases. Under light of preliminary results, the current hypothesis will focus on epigenetic regulations to clarify mustard toxicity and the use of drugs to correct possible epigenetic defects.

Design and Synthesis of Glycosylated Aromatic Nitrogen Mustard Derivatives

Antibody-directed enzyme prodrug therapy(ADEPT) is a new strategy for the treatment of cancer that has arisen in recent twenty years, the main merits of which are that it can improve the selectivity of anticancer drugs and reduce the side effects in remote tissue. In the present study, two prodrugs-glycosylated aromatic nitrogen mustard derivatives were synthesized. Glucose and lactose were converted into glycosyl donors-trichloroacetimidate; the obtained glycosyl donors were glycosylated with p-nitrophenol(glycosyl donors) to form β-glucosyl p-nitrobenzene and β-lactosyl p-nitrobenzene that were protected by acetyl in a stereoselective manner; the two products were reduced by zinc dust and then treated with ethylene oxide, afforded two glycosylated nitrogen mustard derivatives that were protected by acetyl; the last step was to deacetylate and then afforded the two target compounds that could be used as prodrugs of ADEPT for further Anti-tumor research.

Synthesis and Preliminary Antitumor Activity of Distamycin Nitrogen Mustards

Distamycin nitrogen mustard derivatives with different substituents at the amidino moiety located at the C-terminal of the peptide were synthesized. The in vitro antitumor activity was determined against human chronic leukemia K562 cells. Compound 3, bearing a terminal ethylamido group, had the best antitumor activity with an IC50 value of 0.72 μM. Compound 5, bearing a terminal dimethylamino group, had an IC50 value of 2.0 μM. This result suggests that a terminal positive charge is not essential for optimal antitumor activity. Keywords: Distamycin, Nitrogen mustard, DNA alkylation

A preclinical survey on the efficacy of NSC-290205 in combination with doxorubicin (ADR) in Lewis lung carcinoma (LLC)

17131 Background: NSC-290205 (A) is a hybrid synthetic antitumor ester, which combines a D-lactam derivative of androsterone and nitrogen mustard. Studies on modified steroidal esters of carboxylic derivatives of N,N-bis(2-chloroethyl)aniline, have shown that they exhibit reduced toxicity and increased antitumor activity and specificity. In this study we investigated the antitumor activity of compound A in combination with ADR (AHOP) in comparison with standard CHOP regimen. Methods: C57Bl mice were used for the antitumor evaluation of AHOP/CHOP. Experiments were initiated by implanting the tumor. LLC cells (purchased by NCI, Bethesda, USA) were implanted intramuscularly into the right hind leg as a suspension of 7 × 106 cells in 0.1 ml. The antitumor activity was assessed from the inhibition of tumor growth by volume in cm3 and the oncostatic parameter T/C % according to the protocol of experimental evaluation of antitumor drugs of the NCI, USA. Treatments were given as a single dose (D) on day 1, intermitted dose (D/2 × 3) on days 1, 5, 9 or consecutive dose (D/4 × 9) on days 1 through 9. Results: Treatment with A or cyclophosphamide produced almost equal borderline activity. Moreover, both CHOP and AHOP regimens showed significant and comparable antitumor effect (p &lt; 0.05 by the Wilkoxon test). AHOP caused the maximum effect inhibiting the tumor growth by 67.7% and T/C values of 270%. CHOP was less effective producing 54.8% tumor inhibition and T/C values of 238%. Conclusions: It is very likely that the D-lactamic steroid (androstan) alkylator for A, containing the -NHCO group, combined with ADR, which intercalates between base-pairs, is the explanation for higher activity of AHOP vs. CHOP. This significant effect of NSC-290205 with the anthracycline adriamycin on LLC adds to NSC-290205 advantage for further clinical development. No significant financial relationships to disclose.

Synthesis and biological activity of mustard derivatives of combretastatins

A series of chimeric compounds bearing the combretastatin and the nitrogen mustard cores were synthesized. All the compounds were cytotoxic and inhibited tubulin polymerization. When combretastatin was joined to chlorambucil via an ester linkage, the resultant compound proved to be significantly more potent than the two compounds put together. When combretastatin was joined to nitrogen mustard via an ether linkage or when a true hybrid was synthesized, loss of potency was observed. Nonetheless, these latter compounds appeared to be more efficacious and surprisingly were able to inhibit tubulin depolymerization at high concentrations.

The nitrogen mustard melphalan activates mitogen-activated phosphorylated kinases (MAPK), nuclear factor-κB and inflammatory response in lung epithelial cells

To investigate how respiratory epithelial cells react to an alkylating agent, we exposed human bronchial (BEAS-2B) and alveolar (A549) cells to the nitrogen mustard derivative melphalan. The BEAS-2B cells were highly sensitive to melphalan, as shown by a reduced viability after a 10-min incubation with 300 microM melphalan. The A549 cells were less sensitive and required several hours of exposure to reduce significantly in viability. However, exposure to melphalan also induces activation of intracellular signal transduction pathways, as indicated by phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38 (proteins belonging to the family of stress-induced mitogen-activated phosphorylated kinases, MAPK) within 5 min, as well as translocation of the transcription factor nuclear factor (NF)-kappaB to the nucleus within 45 min. This early activation was followed by elevated levels of tumor necrosis factor (TNF)-alpha mRNA within 2 h. We also observed increased expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of both cell lines 18 h after exposure to 25 microM melphalan and an increased adhesion of monocytes to the epithelial cells in vitro.In conclusion, we have demonstrated that alkylating compounds not only cause cell death of lung epithelial cells but also activate stress-associated MAPK signal transduction pathways and induce expression of mediators known to participate in the recruitment of inflammatory cells.

Evaluation of Protective Ointments Used against Dermal Effects of Nitrogen Mustard, a Vesicant Warfare Agent

Mustard, a vesicant warfare agent, has cytotoxic, mutagenic, and cytostatic effects via alkylation of DNA and inhibition of DNA replication. Since symptoms appear following a latent period, it can cause some subacute and chronic effects to occur and delay in the treatment. Therefore, the main approach should be the use of protective preparation to reduce the skin toxicity. Thus, this study was conducted in guinea pigs (350-400 g) shaved in areas of 10 x 10 cm. Mechlorethamine HCl (100 mg), a nitrogen mustard derivative, in ethanol was applied by spraying on hairless regions where previously prepared pharmaceutical topical formulations were medicated before. The experimental regions of the animals were kept preserved from environmental factors. Forty-eight hours after the application of the protective ointments and mechlorethamine consecutively, skin-damaging effects were macroscopically evaluated in terms of erythema formation, ulceration, necrosis, and inflammation occurrences. Then, punch biopsy was performed from these damaged sites for histopathological evaluation. Although numerous topical formulations were prepared and tested for protection, according to microscopic examination of the pathologic sections, tissue specimen treated with the ointment containing the mixture of zinc oxide, zinc chloride, dimethylpolysiloxane in a base of petroleum jelly was determined as being the most effective protective against skin injury caused by the vesicant agent.

Design and synthesis of a nitrogen mustard derivative stabilized by apo-neocarzinostatin.

Neocarzinostatin (NCS) is an antitumor antibiotic comprising a 1:1 protein-chromophore complex and exhibits cytotoxic action through DNA cleavage via H-abstraction. Cytotoxic activity resides with the chromophore 1 alone, while the protein (apoNCS) protects and transports labile 1. The naphthoate portion (2) of NCS chromophore (1) is important for binding to apoNCS and DNA intercalation. In this paper we describe our attempts to use apoNCS to improve the hydrolytic stability of novel bifunctional DNA alkylating agents. The nitrogen mustards, melphalan and chlorambucil, were both conjugated to 2, and the biological activities of these conjugates were assessed. Chlorambucil did not benefit from conjugation. The melphalan conjugate (6) formed covalent DNA adducts at guanine bases and exhibited greater in vitro cytotoxic activity than unmodified melphalan. Fluorescence and NMR spectroscopy showed that 6 binds to apoNCS. Binding to apoNCS-protected 6 reduced the extent of hydrolysis of the conjugate. This novel approach demonstrates for the first time that an enediyne apo-protein can be used to improve the stability of substances that are of potential interest in cancer chemotherapy.

Cinnamoyl nitrogen mustard derivatives of pyrazole analogues of tallimustine modified at the amidino moiety: design, synthesis, molecular modeling and antitumor activity studies

The design, synthesis and in vitro activities of a series of cinnamoyl nitrogen mustard pyrazole analogues of tallimustine 8– 13, in which the amidino moiety has been replaced by moieties of different physico-chemical features are described, and the structure–activity relationships are discussed. In spite of the relevance of these modifications on the amidino moiety, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. A selected series of compounds have been evaluated for their sequence selective alkylating properties and cytotoxicity against human K562 leukemia cells. Therefore, the presence of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity. Our preliminary results indicated that the compounds of this series have a pattern of alkylation similar to that of tallimustine, but they seem to be less reactive overall in alkylating naked DNA.

The Discovery of a New Potential Anticancer Drug: A Case History.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis and Preliminary Cytotoxicity of Nitrogen Mustard Derivatives of Distamycin A.

Distamycin and nitrogen mustard conjugates, in which the nitrogen mustard unit was coupled to the C-terminus of the pyrrole, were synthesized. The switching of the nitrogen mustard unit from the N-terminus to the C-terminus did not compromise the compound's cytotoxicity. Compound 3, bearing three pyrrole units, was highly toxic to human K562 leukemia cells in vitro with an IC(50) value of 0.03 microM. Addition of a trans double bond to the molecule had little effects on cytotoxicity.

Benzoyl nitrogen mustard derivatives of benzoheterocyclic analogues of netropsin: Synthesis and biological activity

Synthesis, DNA binding properties and biological activity of a series of bis-benzoheterocycle derivatives 5– 11, structurally related to the natural dipyrrole antitumor agent netropsin, and tethered to a benzoyl nitrogen mustard (BAM) as alkylating moiety is reported and structure–activity relationships determined. These compounds 5– 11 have been evaluated for sequence selective alkylating properties and cytotoxicity against murine L1210 and human K562 leukaemia cells. Using as target sequence a portion of the long terminal repeat of the type-1 human immunodeficiency virus, we found that these compounds induce similar patterns of DNA fragmentation. In addition, the results obtained indicate that all synthesized compounds retain a good antiproliferative activity in the submicromolar range, and generally are more active against L1210 than K562 cells. With respect to both these cell lines, compounds 6, 7, 10 and 11 showed the greatest potency, ranging from 0.3 to 1 μM, while compounds 8 and 9 exhibit the lowest activity (IC 50=2–12 μM). Among compounds 5– 11, the derivative 11 was found to be the most potent member of this class and it is 5 and 10-fold less active than the bis-pyrrole counterpart 2 against K562 and L1210 cell lines, respectively. For compound 11, the substitution of the C-terminus benzofurane with N-methylindole and indole (to give the compounds 5 and 6, respectively) led to a decrease in cytotoxicity, which is more evident against the K562 cell line. Finally, differences were found among compounds 5– 11 in induction of K562 differentiation. Some of them (compounds 7, 8 and 9) are potent inducers of erythroid differentiation of K562 cells, and could be proposed for differentiation anti-cancer therapy.

Chlornaphazine and chlorambucil induce dominant lethal mutations in male mice.

Two antineoplastic agents, chlornaphazine (CN) and chlorambucil (CHL), were tested for the induction of dominant lethal mutations in male mice. Both compounds are nitrogen mustard derivatives and have been shown to be genotoxic in a variety of organisms. CN was administered intraperitoneally to DBA/2J male mice at a dosage of 0, 500, 1000, or 1500 mg/kg body weight (bw). Immediately following treatment, each male was mated at 4-day intervals to two virgin C57BL/6J females. CHL was administered intraperitoneally to C3H/HeJ and DBA/2J males at a dosage of 0, 2.5, or 5.0 mg/kg bw. These males were mated at weekly intervals to two virgin T-stock females. CN and CHL clearly induced dominant lethal mutations. CN induced dominant lethal effects in all post-meiotic germ-cell stages of treated DBA males, with a clear dose-response relationship. The results with CHL-treated DBA males indicated that all post-meiotic germ-cell stages, except late-spermatids, were affected by CHL treatment, while in C3H males, CHL induced dominant lethal effects in all post-meiotic germ-cell stages. A dose-response relationship was also observed with CHL in C3H male mice. In the present experiments, regardless of the agent or the mouse strain used, spermatids appeared to be the germ-cell stage most sensitive to dominant lethal induction.

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 α-halogenoacrylamido derivatives of DST-like oligopeptides, which show an activity profile substantially improved in comparison to TAM. In particular brostallicin (PNU-166196), α-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.

Synthesis and preliminary cytotoxicity of nitrogen mustard derivatives of distamycin A

Distamycin and nitrogen mustard conjugates, in which the nitrogen mustard unit was coupled to the C-terminus of the pyrrole, were synthesized. The switching of the nitrogen mustard unit from the N-terminus to the C-terminus did not compromise the compound's cytotoxicity. Compound 3, bearing three pyrrole units, was highly toxic to human K562 leukemia cells in vitro with an IC 50 value of 0.03 μM. Addition of a trans double bond to the molecule had little effects on cytotoxicity.