Nitrogen Mustard Agents Research Articles

ANALYSIS OF CYCLOPHOSPHAMIDE-INDUCED N5-NITROGEN MUSTARD FORMAMIDOPYRIMIDINE ADDUCT IN DRIED BLOOD SPOT USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Cyclophosphamide is one of the nitrogen mustard agents that is frequently used in cancer chemotherapy. Nevertheless, long-term use of high dosage cyclophosphamide has been proven to increase the risk of secondary cancer. This can be traced by the mutagenic DNA adduct formation, for instance, N5-nitrogen mustard formamidopyrimidine (NM-Fapy-G). Therefore, it may serve as one of the secondary cancer biomarkers in patients receiving cyclophosphamide. Several NM-Fapy-G analysis methods employ Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) developed by experts. However, it was discovered not applicative for patients because cells and tissues were utilized as the biospecimens. Therefore, this summary is presented to emphasize the idea of adopting Dried Blood Spot (DBS) as the blood bio sampling method, DNA extraction and hydrolysis method that is suitable for enriching NM-Fapy-G adduct; and method that is proper for NM-Fapy-G analysis. Based on the literature study, DBS has been proven beneficial for this analysis; DNA can be extracted from the DBS cards using the QIAamp DNA Mini Kit. Therefore, research with a little bit of adjustment can be applied for NM-Fapy-G analysis.

Therapeutic Potential of Nitrogen Mustard Based Hybrid Molecules.

As medicine advances, cancer is still among one of the major health problems, posing significant threats to human health. New anticancer agents features with novel scaffolds and/or unique mechanisms of action are highly desirable for the treatment of cancers, especially those highly aggressive and drug-resistant ones. Nitrogen mustard has been widely used as an anticancer drug since the discovery of its antitumor effect in the 1942. However, the lack of selectivity to cancer cells restricts the wide usage of a mass of nitrogen mustard agents to achieve further clinical significance. Discovery of antitumor hybrids using nitrogen mustards as key functional groups has exhibited enormous potential in the drug development. Introduction of nitrogen mustards resulted in improvement in the activity, selectivity, targetability, safety, pharmacokinetics and pharmacodynamics properties of corresponding lead compounds or agents. Herein, the recently developed nitrogen mustard based hybrids have been introduced in the cancer therapy.

Design, synthesis and in vitro cytotoxicity study of benzodiazepine-mustard conjugates as potential brain anticancer agents

The combination of two pharmacological entities in a single compound has been utilized as a promising drug design strategy for site-specificity. So two nitrogen mustard agents were synthesized by conjugating mustard with the benzodiazepine nucleus in the hope to obtain central nervous system (CNS) antitumor agents. The benzodiazepine part is aimed to serve as a CNS active carrier enabling the alkylating moiety to cross the BBB by altering its physicochemical properties. Structures of all the synthesized compounds were confirmed by IR, NMR (1H &13C), mass spectral and elemental studies. The benzodiazepine-mustard conjugates are oily at room temperature and quite stable when stored in refrigerator for 2months. Both compounds when evaluated for NBP alkylating activity against chlorambucil, proved to be active alkylating agents. The compounds were markedly active when subjected to in vitro biological evaluation using an MTT colorimetric assay against four human cancer cell lines (A-549, COLO 205, U-87 MG and IMR-32). The physicochemical ADME studies were also analyzed using Qikprop 2.5 tools of Schodinger software which further indicates that both compounds can be potential candidates for the treatment of brain tumor.

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 DNA Crosslinking Agent SJG-136 (SG2000, BN2629) Is Highly Potent in Acute Myeloid Leukaemia and Is Synergistic with Cytarabine

Standard treatment of acute myeloid leukemia (AML) results in a median survival of approximately 1 year. However, increased understanding of the complex biology of AML has led to the introduction of more targeted therapies, including gemtuzumab ozogamycin and a number of FLT3 inhibitors, in the expectation that they will improve outcomes in this condition. The future is likely to see combinations of new targeted drugs together with more standard therapies. The challenge will be to identify the optimal combinations for individual patients based on their specific disease markers. SJG-136 (SG2000, BN2629) is a pyrrolo[2,1-c][1,4]benzodiazepine dimer which binds into the minor grove of DNA with a preference for 5′Œ-GATC-3′Œ sequences. The crosslinks that SJG-136 forms are more persistent than those formed by nitrogen mustard agents like melphalan due to their slow rate of repair. We have previously shown that SJG-136 is very potent against primary chronic lymphocytic leukaemia (CLL) cells so we set out to investigate its effect on AML cell lines and primary AML. The AML cell lines, NB4 and HL60, had LD50 values of 5.75nM and 12.10nM respectively following exposure to SJG-136 for 48 hours. Apoptosis induction was dose-dependent, as evidenced by Annexin V/propidium iodide assay and caspase-3 activation assay. Subsequently, primary AML cells from 37 diagnostic samples were isolated and treated with SJG-136 and cytarabine yielding an average LD50 of 19.4nM (±22.8) and 5.7mM (±5.6) respectively. SJG-136 was >100 times more potent than cytarabine in samples derived from the same patients (P< 0.0001). We next investigated the potential for synergy between cytarabine and SJG-136 in the AML cell lines (cytarabine:SJG-136, 100:1). NB4 cells demonstrated a combination index (CI) for the two agents of 0.37 and HL60 cells showed a CI of 0.69 indicating that these two agents were synergistic in the two cells lines under these conditions. The primary AML samples were also tested in vitro for synergy between SJG-136 and cytarabine at the same ratio. In these samples the mean CI was 0.56 (± 0.49) with synergy being demonstrated in 29/37 patient samples. Closer examination of these results showed that the 8 patient samples with a CI greater than 1 were all particularly sensitive to cytarabine as a single agent. Therefore, the increased sensitivity of these patient samples to cytarabine may account for their apparent lack of synergy as calculated by the median effect method. These results indicate that SJG-136 has significant potential as a therapy for AML both as a single agent and in combination with cytarabine.

Design and analysis of small-molecule antineoplastic agents targeting brain tumors by utilizing pattern recognition methods and in silico optimized pharmaceutical properties

The purpose of this work is the in silico design and characterization of small-molecule anticancer agents that target the central nervous system. All agents are cytotoxic nitrogen mustard agents, a class of drugs widely used in the clinical treatment of cancer. Molecular properties such as polar surface area, log P, and formula weight have previously been shown to be highly useful properties to utilize in optimizing drug design for penetration of the blood–brain barrier and subsequently the central nervous system. These properties are rigorously applied in this work for compiling the actual molecular structures of the anticancer agents. In silico molecular modeling and pattern recognition methods such as cluster analysis, non-metric multidimensional scaling, and K-means cluster analysis are applied to determine the importance of various pharmaceutical descriptors and interrelationships. Discriminant analysis of the molecular properties of 17 nitrogen mustard agents designed in this work determined the extent of dissimilarity among members of this group. Utilizing analysis of similarity (ANOSIM) on the ten pharmaceutical descriptors provides a discrete numerical result that indicates overall dissimilarity among these agents. K-means cluster analysis distinguished these agents into clusters of small, moderate, and high molecular weight. Discriminant analysis and cluster analysis generally discerned association of these 17 compounds on a basis of higher and lower formula weight criteria. Another pattern recognition method known as non-metric multidimensional scaling discerned finer associations that fell within categorizations by low, moderate, and higher formula weight. ANOSIM resulted in R = 0.5551, which indicates that there is a moderate level of dissimilarity within these brain-penetrating small anticancer agents. The use of in silico assisted drug design and multivariate analysis of these compounds discerned discrete interrelationships and information for evaluating potential clinical application.

Applying pattern recognition methods to analyze the molecular properties of a homologous series of nitrogen mustard agents

The purpose of this research was to analyze the pharmacological properties of a homologous series of nitrogen mustard (N-mustard) agents formed after inserting 1 to 9 methylene groups (-CH2-) between 2 -N(CH2CH2Cl)2 groups. These compounds were shown to have significant correlations and associations in their properties after analysis by pattern recognition methods including hierarchical classification, cluster analysis, nonmetric multi-dimensional scaling (MDS), detrended correspondence analysis, K-means cluster analysis, discriminant analysis, and self-organizing tree algorithm (SOTA) analysis. Detrended correspondence analysis showed a linear-like association of the 9 homologs, and hierarchical classification showed that each homolog had great similarity to at least one other member of the series-as did cluster analysis using paired-group distance measure. Nonmetric multi-dimensional scaling was able to discriminate homologs 2 and 3 (by number of methylene groups) from homologs 4, 5, and 6 as a group, and from homologs 7, 8, and 9 as a group. Discriminant analysis, K-means cluster analysis, and hierarchical classification distinguished the high molecular weight homologs from low molecular weight homologs. As the number of methylene groups increased the aqueous solubility decreased, dermal permeation coefficient increased, Log P increased, molar volume increased, parachor increased, and index of refraction decreased. Application of pattern recognition methods discerned useful interrelationships within the homologous series that will determine specific and beneficial clinical applications for each homolog and methods of administration.

Synthesis and Alkylation Activity of a Nitrogen Mustard Agent to Penetrate the Blood-Brain Barrier

Nitrogen mustard agents are widely used for the clinical treatment of cancers. A nitrogen mustard (N-mustard) agent was synthesized utilizing nicotinic acid as the carrier of the alkylating substituent (—OCH2CH2N(CH2CH2Cl)2) that forms an ester group (R—C(O)—OR) on a heterocyclic ring. The N-mustard agent is a solid at room temperature and is stable for more than 6 weeks when stored at −10°C. To determine the kinetics of alkylation activity a nucleophilic primary amine compound (4-chloroaniline) was placed in aqueous solution with the mustard agent at physiological pH 7.4 (pH of blood) and 37°C. The alkylation reaction was found to be second-order with rate equation: rate = k2[N-mustard][Nu], where Nu = nucleophile and k2 = 0.0415 L/(mol · min). Pharmacological descriptors calculated showed values indicating a strong potential of penetrating the blood-brain barrier. The partition coefficient (Log P) of the mustard agent is 1.95 compared with 0.58 for nicotinic acid. Values of descriptors such as dipole, polar surface area, Log BB, molar refractivity, parachor, and violations of Rule of 5 were found to be 5.057 Debye, 42.44 A2, 0.662, 72.7 cm3, 607.7 cm3, and 0.0 for the N-mustard agent. Value of polar surface area for the mustard agent (42.44 A2) predicts that > 90% of any amount present in the intestinal tract will be absorbed.

Two Identical Twin Nitrogen Mustard Agents that Express Rapid Alkylation Activity at Physiological pH 7.4 and 37&amp;#176;C

Two nitrogen mustard (N-mustard ) agents were synthesized utilizing ethylene diamine and hexane diamine as the parent compounds. These N-mustard agents were solids at 25 o C and stable while stored dry at -10 o C. The two N-mustards assumed the configuration of identical twin drugs which when placed in aqueous solution were highly reactive. Both N-mustards were soluble in aqueous NaHCO3 buffer and expressed alkylation activity directed towards a nucleophilic primary amine target (4-chloroaniline) at blood pH 7.4 and 37 o C. Utilizing the fluorescent probe fluorescamine, which is highly specific for primary amines, the quantity of unreacted nucleophilic 4-chloroaniline remaining after a known time period was determined. This enabled the calculation of rate constants and the determination of rate equation for alkylation to be first-order for N,N,N',N'-tetrakis(2-chloroethyl)ethane-1,2-diamine and zero-order for N,N,N',N'- tetrakis(2-chloroethyl)hexane-1,6-diamine. Molecular property descriptors such as Log P, parachor, molar volume, molar refractivity, dipole, molecular volume & area, and polar surface area were calculated for comparison. Zero violations of the Rule of 5 indicates these two mustard agents will have good bioavailability and good bioactivity.

Role of glutathione in cellular resistance to alkylating agents

Both elevated glutathione levels and increased activity of the enzyme glutathione S-transferase have been associated with the resistance of cells to alkylating agents. We have demonstrated that one mechanism of this resistance is the inactivation of the alkylating agents by conjugation with glutathione. This conjugation can be catalyzed by glutathione S-transferase. For the nitrogen mustard agents we have studied, both the spontaneous and enzyme catalyzed reactions proceed through the aziridinium intermediates of the alkylating agents, and the α isoenzymes of GST are involved. In a study of cyclophosphamide resistant medulloblastoma cell lines elevated cellular concentrations of glutathione correlated well with the resistance of the cell lines.