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A series of new analogs of nitrogen mustards ( 4a - 4 h ) containing the 1,3,5-triazine ring substituted with dipeptide residue were synthesized and evaluated for the inhibition of both acetylcholinesterase (AChE) and β -secretase (BACE1) enzymes. The AChE inhibitory activity studies were carried out using Ellman's colorimetric method, and the BACE1 inhibitory activity studies were carried out using fluorescence resonance energy transfer (FRET). All compounds displayed considerable AChE and BACE1 inhibition. The most active against both AChE and BACE1 enzymes were compounds A and 4a , with an inhibitory concentration of AChE IC 50 = 0.051 µM; 0.055 µM and BACE1 IC 50 = 9.00 µM; 11.09 µM, respectively.

In previous studies, we demonstrated that esters of bendamustine containing a basic moiety are far more cytotoxic anticancer agents than their parent compound and that the substitution of the labile ester moiety by a branched ester or an amide markedly increases stability in the blood plasma. In the current study, we showed that this substitution was bioisosteric. Aiming at increased cytotoxicity, we introduced the same modification to related nitrogen mustards: 6-isobendamustine, chlorambucil, and melphalan. The synthesis was accomplished using the coupling reagents N,N'-dicyclohexylcarbodiimide or 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate. Cytotoxicity against a panel of diverse cancer cells (carcinoma, sarcoma, and malignant melanoma) was assessed in a kinetic chemosensitivity assay. The target compounds showed cytotoxic or cytocidal effects at concentrations above 1 µM: a striking enhancement over bendamustine and 6-isobendamustine, both ineffective against the selected cancer cells at concentrations up to 50 µM, and a considerable improvement over chlorambucil, showing some potency only against the sarcoma cells. Melphalan was almost as effective as the target compounds-derivatization only provided a small improvement. The novel cytostatics are of interest as model compounds for analyzing a correlation between cytotoxicity and membrane transport and for the treatment of malignancies., (© 2020 The Authors. Archiv der Pharmazie published by Wiley-VCH Verlag GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Tumor hypoxia has been widely explored over the years as a diagnostic and therapeutic marker. Herein, we have reported the design and synthesis of a series of dinitrobenzamide mustards (DNBM) based on the PR-104A hypoxia-selective prodrug. Specifically, we explored the impact of various leaving groups and the introduction of a carboxylic acid group on the biological performance of the DNBM constructs. Once in hand, the Log D values, cytotoxicity in PC-3 and DU-145 human prostate cancer cells lines and the hypoxia selectivities of the DNBM analogs were examined. Overall, the DNBM constructs were found to be tolerant to modifications with none of the explored modifications substantially degrading the cytotoxic potential of the constructs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Several novel flavonoids nitrogen mustard derivatives were synthesized and evaluated for antiproliferative activity against seven human cancer cell lines (HeLa, A549, HepG2, MCF7, SH-SY5Y, PC-3, DU145) by the MTT assay in vitro. The resulting IC 50 showed that most compounds exhibited better inhibitory activity against seven cell lines. IC 50 values of some compounds were lower than well-known melphalan. In particular, compound 8b was the most promising compound which inhibited HeLa cells with IC 50 value of 1.43 μM. It showed excellent antitumor activity against these seven cell lines. Besides, it could arrest cell cycle of HeLa in G2/M phase and induce cell apoptosis. The loss of mitochondrial membrane potential may be an apoptotic mediating factor., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

One of the greatest challenges of modern medicine is to find cheaper and easier ways to produce transporters for biologically active substances, which will provide selective and efficient drug delivery to the target cells, while causing low toxicity towards healthy cells. Currently, metal-based nanoparticles are considered a successful and viable solution to this problem. In this work, we propose the use of novel synthesis method of platinum nanoparticles (PtNPs) connected with their precise biophysical characterization and assessment of their potential toxicity. To work as an efficient nanodelivery platform, nanoparticles should interact with the desired active compounds spontaneously and non-covalently. We investigated possible direct interactions of PtNPs with ICR-191, a model acridine mutagen with well-established biophysical properties and mutagenic activity, by Dynamic Light Scattering, fluorescence spectroscopy, and Isothermal Titration Calorimetry. Moreover, to determine the biological activity of ICR-191-PtNPs aggregates, we employed Ames mutagenicity test, eukaryotic cell line analysis and toxicity test against the model organism Caenorhabditis elegans. PtNPs' interesting physicochemical properties associated to the lack of toxicity in a tested range of concentrations, as well as their ability to modulate ICR-191 biological activity, suggest that these particles successfully work as potential delivery platforms for different biologically active substances.

Nitrogen mustards, a family of DNA alkylating agents, marked the start of cancer pharmacotherapy. While traditionally characterized by their dose-limiting toxic effects, nitrogen mustards have been the subject of intense research efforts, which have led to safer and more effective agents. Even though the alkylating prodrug mustards were first developed decades ago, active research on ways to improve their selectivity and cytotoxic efficacy is a currently active topic of research. This review addresses the historical development of the nitrogen mustards, outlining their mechanism of action, and discussing the improvements on their therapeutic profile made through rational structure modifications. A special emphasis is made on discussing the nitrogen mustard prodrug category, with Cyclophosphamide (CPA) serving as the main highlight. Selected insights on the latest developments on nitrogen mustards are then provided, limiting such information to agents that preserve the original nitrogen mustard mechanism as their primary mode of action. Additionally, future trends that might follow in the quest to optimize these invaluable chemotherapeutic medications are succinctly suggested., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background & Objective: Nitrogen mustard derivatives form one of the major classes of anti-cancer agents in USFDA approved drugs list. These are polyfunctional alkylating agents which are distinguished by a unique mechanism of adduct formation with DNA involving cross-linking between guanine N-7 of one strand of DNA with the other. The generated cross-linking is irreversible and leads to cell apoptosis. Hence it is of great interest to explore this class of anticancer alkylating agents., Methods: An exhaustive list of reviews, research articles, patents, books, patient information leaflets, and orange book is presented and the contents related to nitrogen mustard anti-cancer agents have been reviewed. Attempts are made to present synthesis schemes in a simplified manner. The mechanism of action of the drugs and their side effects are also systematically elaborated., Results: This review provides a platform for understanding all aspects of such drugs right from synthesis to their mechanism of action and side effects, and lists USFDA approved ANDA players among alkylating anticancer agents in the current market., Conclusion: Perusing this article, generic scientists will be able to access literature information in this domain easily to gain insight into the nitrogen mustard alkylating agents for further ANDA development. It will help the scientific and research community to continue their pursuit for the design of newer and novel heterocyclic alkylating agents of this class in the coming future., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In this report, the design of two novel nitrogen mustard-based DNA cross-linking agents with fluorophores incorporated into the structure (TN-A and TN-B) is disclosed. The results indicate that TN-A and TN-B can serve directly as both reporting and imaging agents for flow cytometry and gel electrophoresis without the necessity of another fluorescent tagging agents. TN-A and TN-B both selectively locate in the mitochondria and exhibit good antitumor activity. Notably, TN-A is the first DNA crosslinking agent with near infrared fluorescence emission properties., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A series of novel nitrogen mustard sophoridinic acid derivatives were designed, synthesized and evaluated for their cytotoxicity. Of the newly synthesized compounds, compound 6 exhibited a potent effect against hepatocellular carcinoma in vitro and in vivo. SAR analysis indicated that introduction of a nitrogen mustard group to the structure of sophoridinic acid significantly enhance the antitumor activity. Moreover, molecular docking study exhibited benzyl group introduced to the nitrogen atom at the 12-position and aryl nitrogen mustard group at the 4'-carboxyl region for compound 6 were beneficial for the higher anticancer activity. This work provides useful information for further structural modifications of these compounds and for the synthesis of new, potent antitumor agents., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A series of hydroxyanthraquinones having an alkylating N-mustard pharmacophore at 1'-position were synthesized via a bioisostere approach to evaluate their cytotoxicity against four tumor cell lines (MDA-MB-231, HeLa, MCF-7 and A549). These compounds displayed significant in vitro cytotoxicity against MDA-MB-231 and MCF-7 cells, reflecting the excellent selectivity for the human breast cancer. Among them, compound 5k was the most cytotoxic with IC50 value of 0.263 nM and is more potent than DXR (IC50 = 0.294 nM) in inhibiting the growth of MCF-7 cells. The excellent cytotoxicity and good selectivity of compound 5k suggest that it could be a promising lead for further design and development of anticancer agents, especially for breast cancer., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Nitrogen mustards are an important class of bifunctional alkylating agents routinely used in chemotherapy. They react with DNA as electrophiles through the formation of highly reactive aziridinium ion intermediates. The antibiotic 593A, with potential antitumor activity, can be considered a naturally occurring piperidine mustard containing a unique 3-chloropiperidine ring. However, the total synthesis of this antibiotic proved to be rather challenging. With the aim of designing simplified analogues of this natural product, we developed an efficient bidirectional synthetic route to bis-3-chloropiperidines joined by flexible, conformationally restricted, or rigid diamine linkers. The key step involves an iodide-catalyzed double cyclization of unsaturated bis-N-chloroamines to simultaneously generate both piperidine rings. Herein we describe the synthesis and subsequent evaluation of a series of novel nitrogen-bridged bis-3-chloropiperidines, enabling the study of the impact of the linker structure on DNA alkylation properties. Our studies reveal that the synthesized compounds possess DNA alkylating abilities and induce strand cleavage, with a strong preference for guanine residues., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To explore novel coumarin derivatives with more potent anti-proliferative activity, a series of novel compounds were designed and synthesized by linking Schiff base and N, N-bis (2-chloroethyl) amine pharmacophore of nitrogen mustards to the coumarin's framework. Their structures were confirmed by 1H NMR, MS and element analysis techniques. In vitro anti-proliferative activities were evaluated against HepG2, DU145 and MCF7 cell lines by the standard MTT assay. The results showed that some of the target compounds exhibited strong anti-proliferative activities against selected tumor cells, and compounds 7c, 7f, 7g, 7h and 7q were better than or equal to the activities of positive control, they deserved further development.

A series of nitrogen mustard-linked chalcones were synthesized and evaluated for their antitumor activity in vitro against the K562 and HepG2 cell lines. The aldol condensation of [N,N-bis(chloroethyl)-3-amino]-acetophenone (2) with aromatic aldehydes afforded the nitrogen mustard-linked chalcones. Among the analogs tested, compounds 5e and 5k exhibited significant anti-proliferation activities against K562 cells with IC50 values of 2.55 and 0.61 µM, respectively, which revealed higher cell toxicity than the standard drugs cisplatin (IC50>200 µM) and adriamycin (IC50=14.88 µM). The methoxyl and N,N-dimethyl groups on the B-ring of the chalcone frame enhanced the inhibitory activities against both the K562 and HepG2 cell lines. The structure-activity relationship study indicated that the inhibitory activities significantly varied with the position(s) and species of the substituted group(s)., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

On the basis of the results of in-silico predictions and in an effort to extend our structure-activity relationship studies, the aromatic nitrogen mustard 2-[4-N,N-bis(2-chloroethyl) amino-phenyl]butanoic acid (2-PHE-BU) was synthesized and conjugated with various steroidal alcohols. The resulting steroidal esters were evaluated for their in-vivo toxicity and antileukemic activity in P388-leukemia-bearing mice. The new derivatives showed significantly reduced toxicity and marginally improved antileukemic activity compared with free 2-PHE-BU. Nevertheless, they did not prove to be superior either to the template steroidal ester used for in-silico predictions or to previously synthesized steroidal esters of aromatic nitrogen mustards. The results obtained indicate that in-silico design predictions may guide the design and synthesis of new bioactive steroidal esters, but further parameters should be considered aiming at the discovery of compounds with optimum activity.

Hepatocellular carcinoma (HCC) is one of the main causes of death in cancer. Some naphthalimide derivatives exert high anti-proliferative effects on HCC. In this study, it is confirmed that 3-nitro-naphthalimide and nitrogen mustard conjugate (NNM-25), a novel compound conjugated by NNM-25, displayed more potent therapeutic action on HCC, both in vivo and in vitro, than amonafide, a naphthalimide drug in clinical trials. More importantly, preliminary toxicological evaluation also supported that NNM-25 exhibited less systemic toxicity than amonafide at the therapeutic dose. The antitumor mechanism of conjugates of naphthalimides with nitrogen mustard remains poorly understood up to now. Here, we first reported that apoptosis might be the terminal fate of cancer cells treated with NNM-25. Inhibition of p53 by siRNA resulted in a significant decrease of NNM-25-induced apoptosis, which corroborated that p53 played a vital role in the cell apoptosis triggered by NNM-25. NNM-25 inhibited the PARP-1 activity, AKT phosphorylation, up-regulated the protein expression of p53, Bad, and mTOR as well as down-regulating the protein expression of Bcl-2 and decreasing mitochondrial membrane potential. It also facilitated cytochrome c release from mitochondria to cytoplasm, activated caspase 8, caspase 9, and caspase 3 in HepG2 cells in vitro, as also authenticated in H22 tumor-bearing mice in vivo. Collectively, the conjugation of naphthalimides with nitrogen mustard provides favorable biological activity and thus is a valuable strategy for future drug design in HCC therapy.

A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The alkylating agents bendamustine and melphalan are currently used in the treatment of various tumoral diseases. In order to increase their antitumor potency and tumor selectivity both compounds were integrated in structure-activity relationship studies including new drug carrier systems. Here we describe the synthesis and the cytotoxicity of new bivalent bendamustine and melphalan derivatives. Two molecules each esterified with N-(2-hydroxyethyl)maleimide were connected by diamines with various chain lengths (n=6, 7, 8, 12). It was supposed that these conjugates (5a-d, 10a-d, 11a-d) cause cytotoxic effects preferred as bivalent drug. Indeed the cytotoxicity of the new compounds increased compared to bendamustine and melphalan as determined in concentration-dependent in vitro assays using the human MCF-7 and MDA-MB-231 breast cancer cell lines., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Two series of cyclic nitrogen mustards structurally related to L-carnitine have been prepared. The cytotoxic activity of these compounds was evaluated by using Chlorambucil as a reference. In accordance with earlier report, the cytotoxicity is in direct correlation with the lipophilicity of the introduced alkyl chains. Among the cyclic nitrogen mustards synthesized, the most cytotoxic compounds were the one acylated with a palmitoyl side chain, which showed activities comparable to that of Chlorambucil., (2010 Elsevier Masson SAS. All rights reserved.)

This article reported the synthesis and bioevaluation of two [(18)F] labeled benzimidazole derivatives, 4-(5-(2-[(18)F] fluoro-4-nitrobenzamido)-1-methyl-1H-benzimidazol-2-yl) butanoic acid ([(18)F] FNBMBBA, [(18)F]a1) and 3-(2-fluoroethyl)-7-methyl-2-propyl-3H-benzimidazole-5-carboxylic acid ([(18)F] FEMPBBA, [(18)F]b1) for PET tumor imaging. The preparation [(18)F] FEMPBBA was completed in 1h with overall radiochemical yield of 50-60% (without decay corrected). Biodistribution assay in S180 tumor bearing mice of both compounds were carried out, and the results are both meaningful. [(18)F] FEMPBBA which can be taken as a revision of [(18)F] FNBMBBA got an excellent result, and has significant advantages in some aspects compared with L-[(18)F] FET and [(18)F]-FDG in the same animal model, especially in tumor/brain uptake ratio. The tumor/brain uptake ratio of [(18)F] FEMPBBA gets to 4.81, 7.15, and 9.8 at 30min, 60min and 120min, and is much higher than that of L-[(18)F] FET (2.54, 2.92 and 2.95) and [(18)F]-FDG (0.61, 1.02, 1.33) at the same time point. The tumor/muscle and tumor/blood uptake ratio of [(18)F] FEMPBBA is also higher than that of L-[(18)F] FET at 30min and 60min. This result indicates compound [(18)F] FEMPBBA is a promising radiotracer for PET tumor imaging., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A series of amide-coupled benzoic nitrogen mustard derivatives as potential EGFR and HER-2 kinase inhibitors were synthesized and reported for the first time. Some of them exhibited significant EGFR and HER-2 inhibitory activity. Of all the studied compounds, compounds 5b and 5t exhibited the most potent inhibitory activity, which was comparable to the positive control erlotinib. Docking simulation was performed to position compounds 5b and 5t into the EGFR active site to determine the probable binding model. Antiproliferative assay results indicated that some of the benzoic nitrogen mustard derivatives possessed high antiproliferative activity against MCF-7. In particular, compounds 5b and 5t with potent inhibitory activity in tumor growth inhibition may function as potential antitumor agents., (Copyright 2009 Elsevier Ltd. All rights reserved.)

In this study, we have investigated the genotoxic, cytostatic, antineoplastic and apoptotic effects of three newly synthesized modified steroidal esters, having as alkylating agent p-N,N-bis(2-chloroethyl) aminophenyl butyrate (CHL) or p-N,N-bis(2-chloroethyl) aminophenyl acetate (PHE) esterified with the steroidal nucleus modified in the B- and D-ring. The genotoxic and cytotoxic effects of the compounds were investigated both in vitro, in lymphocyte cultures obtained from blood samples of healthy donors and in vivo, in ascites cells of P388 leukemia obtained from the peritoneal cavity of DBA/2 mice. Preparations were scored for sister-chromatid exchange (SCE) and proliferation-rate indices (PRI). The newly synthesized compounds were also studied for antineoplastic activity against lymphocytic P388 and lymphoid L1210 leukemias in mice, by calculating the mean of the median survival of the drug-treated animals (T) versus the untreated control (C) (T/C%). The activity of caspase-2 and caspase-3, indicators of apoptosis, was assessed biochemically in primary cultures of human lymphocytes. Our results show that the newly synthesized compounds caused severe genotoxic effects by significantly increasing the frequency of SCE and decreasing the PRI values in cultures of peripheral lymphocytes in vitro and in ascites cells of lymphocytic P388 leukemia in vivo. A significant correlation was also observed in both the in vitro and in vivo experiments: the higher the SCE frequency the lower the PRI value (r=-0.65, P<0.001 and r=-0.99, P<0.01, respectively). The measured antileukemic potency was statistically increased by all test compounds in both types of tumours, while the activity of caspase-2 and caspase-3 showed a statistically significant increase after two periods of exposure. The genotoxic (increase of SCE), cytostatic/cytotoxic (decrease of PRI) and antileukemic effects (increase of T/C%) in combination with the induction of apoptosis (activation of caspase-2 and caspase-3) caused by the newly synthesized compounds, lead us to propose them as agents with potentially antineoplastic properties.

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.

To study the effect of isoprenoid and aliphatic saturated alcohols as modificator on benzoic nitrogen mustard, the intermediate 4-[N,N-bis(2-chloroethyl) amino] benzoic acid 4 was prepared in four steps utilizing p-amino benzoic acid as the starting material. Target compounds were synthesized by the catalytic esterification of DCC/DMAP and the structures of the six new esters were characterized by elemental analysis, 1H NMR, 13C NMR and MS. Antitumor activities were evaluated in vitro using MTT assay. The result showed that some derivatives were more potent than the intermediate 4, and compound 5c modified with dodecanol exhibited similar activity to the commercial drug melphalan.

To enhance the potency of "combi-molecules", we designed 6a-d and 18 to release an inhibitor of EGFR TK and a bifunctional alkylator. The combi-molecules blocked EGFR TK with potency increasing with the basicity of the mustard moiety. They selectively killed cells transfected with EGFR and were potent against the DU145 prostate cancer cells. Combi-molecule 6a blocked EGFR phosphorylation in an irreversible manner, induced DNA-cross-links, and arrested the cells in mid-S.

A series of 2,4-dinitrobenzamide mustards were prepared from 5-chloro-2,4-dinitrobenzoic acid or the corresponding 5-dimesylate mustard as potential prodrugs for gene-directed enzyme prodrug therapy (GDEPT) with the E. coli nfsB nitroreductase (NTR). The compounds, including 32 new examples, were evaluated in four pairs of NTR+ve/-ve cell lines for selective cytotoxicity (IC50 and IC50 ratios), in multicellular layer (MCL) cultures for bystander effects, and for in vivo activity against tumors grown from stably NTR transfected EMT6 and WiDr cells in nude mice. Multivariate regression analysis of the IC50 results was undertaken using a partial least-squares projection to latent structures model. In NTR-ve lines, cytotoxicity correlated positively with logP, negatively with hydrogen bond acceptors (HA) and donors (HD) in the amide side chain, and positively with the reactivity of the less-reactive leaving group of the mustard function, likely reflecting toxicity due to DNA monoadducts. Potency and selectivity for NTR+ve lines was increased by logP and HD, decreased by HA, and was positively correlated with the leaving group efficiency of the more-reactive group, likely reflecting DNA crosslinking. NTR selectivity was greatest for asymmetric chloro/mesylate and bromo/mesylate mustards. Bystander effects in the MCL assay also correlated positively with logP and negatively with leaving group reactivity, presumably reflecting the transcellular diffusion/reaction properties of the activated metabolites. A total of 18 of 22 mustards showed equal or greater bystander efficiencies in MCLs than the aziridinylbenzamide CB 1954, which is currently in clinical trial for NTR-GDEPT. The dibromo and bromomesylate mustards were surprisingly well tolerated in mice. High MTD/IC50 (NTR+ve) ratios translated into curative activity of several compounds against NTR+ve tumors. A bromomesylate mustard showed superior activity against WiDr tumors grown from 1:9 mixtures of NTR+ve and NTR-ve cells, indicating a strong bystander effect in vivo.

A series of 9-anilinoacridine and acridine derivatives bearing an alkylating N-mustard residue at C4 of the acridine chromophore were synthesized. The N-mustard pharmacophore was linked to the C4 of the acridine ring with an O-ethyl (O-C(2)), O-propyl (O-C(3)), or O-butyl (O-C(4)) spacer. It revealed that all newly synthesized compounds were very potent cytotoxic agents against human leukemia and various solid tumors in vitro. These agents did not exhibit cross-resistance against vinblastine-resistant (CCRF-CEM/VBL) or taxol-resistant (CCRF-CEM/taxol) cells. It also showed that these agents were DNA cross-linking agents rather than topoisomerase II inhibitors. Of these agents, compounds 27a and 27c were shown to have potent antitumor activity in nude mice bearing the human breast carcinoma MX-1 xenograft. The therapeutic efficacies of these two agents are comparable to that of taxol.

Aim: To develop a rationally designed new nitrogen mustard namely Fluorenhymustine (compound 2), where N,N'-bis(2chloro-ethyl)amino group, the established anticancer functionality, is attached to the 2-ethyl fluorenone hydantoin moiety., Materials and Methods: Starting from fluorenone hydantoin, a 3-step synthetic procedure was followed to obtain the title compound. 4-(4-Nitrobenzyl)pyridine was used to assess its chemical alkylating activity. Murine tumors (Ehrlich ascites carcinoma (EAC) and Sarcoma-180 (S-180)) were used to assess its in vivo activity. Its cytotoxicity was determined in vitro in MCF-7 human breast tumor cell line, toxicity - in vivo in normal and EAC bearing mice. 3H-Thymidine and 3H-Uridine were employed to study its inhibitory effect on DNA and RNA synthesis respectively in S-180 tumor cells in vitro., Results: Alkylating activity of fluorenmustine exceeded that of N-di(2-chloroethyl)amine used as a standard alkylating compound. It has displayed an excellent and reproducible antitumor activity in vivo against EAC and S-180 comparable to that of 5-fluorouracil judging by the increase in median survival times of treated animals. It also significantly increased the life span of mice bearing advanced tumors for 6 days before the drug challenge. However in vitro screening in MCF-7 did not reveal any significant cytotoxicity. The compound did not adversely affect hematopoiesis at its optimum dose. Drug-induced hepatotoxicity and nephrotoxicity were also not detected. It inhibited the synthesis of DNA and RNA in S-180 tumor cells at 8 microM concentration., Conclusion: Results indicated promising chemotherapeutic potential of Fluorenhymustine.

The esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers have been prepared and their cytogenetic studies of structure-biological activity relationship were evaluated. The cytogenetic effects (sister chromatid exchanges (SCEs) induction and proliferation rate indices (PRIs) depression) by o-, m- and p-[N,N-bis(2-chloroethyl)amino] cinnamic acid were also investigated. Among the above compounds tested, those of the m-[N,N-bis(2-chloroethyl)amino] cinnamic acid and of the o-[N,N-bis(2-chloroethyl)amino] cinnamic acid ester of aza-homo-Hecogenin were more active in comparison to the others.

The evaluation of an alkylating nitrogen mustard agent that utilizes D-alanine as a drug carrier for three chloroethyl substituents (CICH2CH2-) 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 (CICH2CH2-) substituents. A nitrogen mustard 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 degrees 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.

The synthesis of 6-(2-chloroethylamino)-4-anilinoquinazolines ZR2002 and ZR2003 designed to block EGFR tyrosine kinase and to damage genomic DNA is described. These compounds present fluorescence properties that permitted the quantitation of their subcellular uptake by flow cytometry. Fluorescence intensities increased with increasing levels of EGFR in a panel of isogenic and established cell lines.

Poly-[N-(2-hydroxyethyl)-L-glutamine] (PHEG) and poly(ethylene glycol) (PEG)-grafted PHEG conjugates of N,N-di(2-chloroethyl)-4-phenylenediamine mustard (PDM) were synthetised. A collagenase-sensitive oligopeptide spacer was selected to link the cytotoxic agent PDM onto the polymeric carrier. First, the oligopeptide-drug conjugate, L-pro-L-leu-gly-L-pro-gly-PDM, was prepared. In a second step, the low molecular weight PDM derivative and PEG-NH(2) were coupled to a N,N-disuccinimidylcarbonate activated PHEG. Dynamic laser light scattering measurements indicated the formation of aggregates. The presence of human serum albumin had no significant effect on the diameter of the conjugates. The hydrolytic stability of the conjugates was investigated in buffer solutions. The conjugates showed an improved stability compared to the parent nitrogen mustard. The enzymatic degradation studies of the polymeric conjugates were performed in the presence of collagenase type IV (Clostridiopeptidase A; EC 3.4.24.3), cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5) and tritosomes. Only the bacterial collagenase type IV was able to cleave the spacer releasing free PDM and its peptidyl derivative, gly-L-pro-gly-PDM. The in vitro cytotoxicity of the conjugates was evaluated against HT1080 fibrosarcoma cells and MDA adenocarcinoma cells. All conjugates showed low toxicity towards these cell lines.

Tumor hypoxia provides a key difference between healthy and cancerous cells. It can be exploited to produce drug selectivity, offering a reductase-rich environment for prodrug activation. Nitrogen mustard drugs are cytotoxic, but usually unselective. Polyamine mustards are candidates for conversion into hypoxia-selective prodrugs via complexation with metals. Reduction to a less stable complex can free the active drug. The novel Cu(II) complexes of N-mustard derivatives of 1,4,7-triazacyclononane (tacn), 1,4,7,10-tetraazacyclododecane (cyclen), and 1,4,8,11-tetraazacyclotetradecane (cyclam) were assessed in vitro as hypoxia-selective cytotoxins. The cyclen mustard complex showed 24-fold selectivity as a hypoxia-selective bioreductive prodrug, with an IC50 value of 2 microM against the lung tumor cell line A549. Reversible redox behavior and stability of the cyclen-Cu(II) complex in aqueous solution correlated with good hypoxia selectivity. The two other related complexes showed irreversible redox behavior and low aqueous stability and were not hypoxia-selective. The use of macrocyclic nitrogen mustard complexes represents a promising new strategy in the design of hypoxia-selective cytotoxins.

A series of 9-anilinoacridine N-mustard derivatives, in which the alkylating N-mustard residue was linked to the C-3' or C-4' position of the anilino ring with an O-ethylene spacer, was synthesized and evaluated for cytotoxicity against human lymphoblastic leukemic cells (CCRF-CEM) in culture. The results showed that all of the new compounds exhibited potent cytotoxicity with IC(50) values ranging from 0.002 to 0.7 microM, which were as potent or significantly more potent than 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA). Compound 9 did not exhibit cross-resistance against both vinblastine-resistant (CCRF-CEM/VBL) and taxol-resistant (CCRF-CEM/taxol) cells. Additionally, compound 9 demonstrated potent antitumor effect in nude mice bearing human breast carcinoma MX-1 xenografts, resulting in complete tumor remission in two out of three mice at the maximal dose of 1-2mg/kg (Q3Dx7) or 3mg/kg (Q4Dx5) via intravenous injection.

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.

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.

Nine new nitrogen mustard compounds derived from 2,6-difluoro-4-hydroxy- (3a-e) and 2,6-difluoro-4-amino- (4a-d) aniline were synthesized as potential prodrugs. They were designed to be activated to their corresponding 3,5-difluorophenol and -aniline (4)-nitrogen mustards by the enzyme carboxypeptidase G2 (CPG2) in gene-directed enzyme prodrug therapy (GDEPT) models. The compounds were tested for cytotoxicity in the MDA MB-361 breast adenocarcinoma. The cell line was engineered to express stably either CPG2 tethered to the cell surface stCPG2-(Q)3 or beta-galactosidase (beta-Gal) as control. The cytotoxicity differentials were calculated between CPG 2-expressing and -nonexpressing cells and yielded different results for the two series of prodrugs despite their structural similarities. While the phenol compounds are ineffective as prodrugs, their aniline counterparts exhibit outstanding activity in the tumor cell lines expressing CPG2. [3,5-Difluoro-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl-l-glutamic acid gave a differential of >227 in MDA MB361 cells as compared with 19 exhibited by 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-l-glutamic acid, 1a, which has been in clinical trials.

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 degrees 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 degrees 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 x 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.

The paper studies the coupling reaction, through ester-type covalent bonds, of an oxazolone derived from the N-(m-nitrobenzoyl)-L-asparagic acid, the cycle of which is opened with an N-mustard derivative, on xanthan (a polysaccharide of microbian synthesis), in conditions of activation with dicyclohexyl carbodiimide. The coupling product has been characterized through elemental analysis and IR spectroscopy. For the establishment of the capacity of the active principle's controlled release by the polymer-active principle system thus obtained, active principle's release kinetics from the polysaccharide support, in conditions of basic hydrolysis, is studied. In vivo tests realized on mice proved the antitumoral activity of the compounds resulted by chemical bonding of the N-mustard derivative on xanthan.

Naphthalmustine, 2-[2-[bis-(2-chloroethyl)amino]ethyl]-1H-benz[de]isoquinoline-1,3-dione (Compound 1) has been synthesized as a rationally designed new anticancer agent from N-(2-bromoethyl)naphthalimide. Its chemical alkylating activity exceeded that of nor-HN2 used as standard compound for comparison. Its antitumour efficacy was assessed in vivo in two murine ascites tumours namely Sarcoma-180 (S-180) and Ehrlich ascites carcinoma (EAC) by measuring the increase in median survival times (MST) of drug treated (T) over untreated control (C) mice. The clinical drug cyclophosphamide and the experimental compound mitonafide were used as positive controls for comparison. Compound 1 has displayed substantial and reproducible antitumoural activity in these tumours since very high remission times of treated animals were observed. Significant increase in the life span of mice bearing highly advanced tumour for 10 days before the drug challenge was also noted after its treatment. Its LD50 value was 200 mg/Kg by single i.p. injection. Its toxicity was also assessed in vivo in normal and in S-180 bearing mice by measuring drug-induced changes in hematological parameters, femoral bone marrow and splenic cellularity sequentially on days 9, 15 and 21 following drug treatment at the optimum dose of 12 mg/kg from day 1 to 7. The results indicated that the compound did not adversely affect hematopoiesis. Drug-induced hepatotoxicity and nephrotoxicity were also evaluated on those days but no such toxicities were detected. Naphthalmustine inhibits the synthesis of DNA and RNA in S-180 tumour cells. It was further screened in vitro in 4 different human tumour cell lines but no significant activity was observed in those lines.

Bromonapmustine 4a and chloronapmustine 4b, two new nitrogen mustards of substituted naphthalimides, have been synthesized as mixed-function anticancer compounds from 4-bromo- and 4-chloro-N-(2-hydroxyethyl)-naphthalimide respectively following a three-step process. Their chemical alkylating activity exceeded that of nor-HN2. Their antitumour efficacy were assessed in vivo in two murine ascites tumours, namely Ehrlich ascites carcinoma (EAC) and Sarcoma-180 (S-180) by measuring the increase in median survival times (MST) of drug treated (T) over untreated control (C) mice. Two standard clinical drugs, namely endoxan (cyclophosphamide) and 5-fluorouracil (5-FU) were used as positive controls for comparison. Both of them have displayed substantial and reproducible antitumoural activity in these tumours comparable with 5-FU. These compounds inhibit the synthesis of DNA and RNA in S-180 tumour cells. These were further screened in vitro in 3 different human tumour cell lines but no significant activity was observed in those lines.

Ibuprofen and aspirin are two common non-steroidal anti-inflammatory drugs (NSAIDs). Both NSAIDs have a carbonyl carbon [-C(O)-], which was utilized to attach a nitrogen mustard (N-mustard) ester group or a tripeptide group. The tripeptide consisted of a L-Gly-D-Ala-D-Ala sequence, where D-Ala-D-Ala is the reactive site for antibacterial activity and L-Gly serves as a linker to the NSAID carrier drug. The aspirin tripeptide and N-mustard show significant antibacterial activity at >or=5.0 x 10(-5) M against penicillin-susceptible or -resistant Escherichia coli. The partition coefficients (log Kow)log P of aspirin and ibuprofen tripeptide drugs were -1.05 and 2.23, respectively. The NSAIDs served as carrier drugs of the N-mustard group which expressed alkylation activity directed towards the nucleophilic primary amine of p -chloroaniline. Hydrolysis of the N-mustard agents yielded the parent structure of aspirin (or ibuprofen) and an N-mustard moiety, 2-[bis(2-chloroethyl)amino]ethanol. The (log Kow)log P for the N-mustard structures of aspirin and ibuprofen were 2.61 and 5.63, respectively. The (log Kow)log P value of 2-[bis(2-chloroethyl)amino]ethanol was 0.56. Fluorescamine was utilized to determine unreacted p -chloroaniline at known time intervals, which permitted calculation of rate constants and rate equations. The aspirin N-mustard agent expressed strong antibacterial activity against a penicillin-resistant bacteria and first-order alkylation kinetics. The ibuprofen N-mustard and 2-[bis(2-chloroethyl)amino]ethanol followed second-order alkylation kinetics. All N-mustard and tripeptide compounds showed zero violations of the Rule of 5. Values of TPSA (molecular polar surface area), C log P and molecular dipoles were calculated.

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 microM, while compounds 8 and 9 exhibit the lowest activity (IC(50)=2-12 microM). 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.

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.

Enantiopure nitrogen mustards which mimic (L)-carnitine framework are prepared by a multi-step synthesis from the (R)-di-tert-butyl malate and their antitumor properties evaluated.

Three newly synthesised steroidal esteric derivatives of nitrogen mustard (compounds 1-3) were comparatively studied on a molar basis regarding their ability to induce sister chromatid exchanges (SCEs) in normal human lymphocytes in vitro and therapeutic effects on leukemia P388 bearing mice. Compounds 1 and 3 are modified steroidal esters of p-methyl-m-N,N-bis(2-chloroethyl)amino benzoic acid, and compound 2 is a modified steroidal ester of chlorambucil. All compounds induced statistically significant increases in SCEs and decreases in proliferation rate indices (PRIs) of cultured human lymphocytes and significantly increased the life span of P388 bearing mice. In this study, the doses applied for therapeutic purposes upon leukemia P388 bearing mice in vivo were derived from cytogenetic observations in normal human lymphocytes in vitro. A substantially better therapeutic effect was obtained compared to the effect achieved after the use of quite higher doses related with LD(10) values. We have demonstrated that the order of anti-tumour effectiveness of the treatment schedules of the three newly synthesised compounds tested (at doses derived from cytogenetic observations) coincides with the order of the cytogenetic effects they induce. The SCE assay appears to have an application in the clinical prediction of tumour sensitivity to potential chemotherapeutics., (Copyright 2002 Elsevier Science B.V.)

The strategy of using small peptides for effective targeting of tumor cells in chemotherapy has proven beneficial. Recently we showed that J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester), an alkylating nitrogen mustard-containing dipeptide, exhibited strong cytotoxic activity in fresh human tumor samples in addition to rapid and pronounced inhibition of macromolecular syntheses and cellular respiration in the human tumor lymphoma cell line U-937 GTB. In this study, an additional series of 17 nitrogen mustard-containing dipeptides has been synthesized and analyzed for cytotoxic activity in a panel of 10 human tumor cell lines. The results were compared to the single amino acid mustard derivative melphalan and its ethyl and isopropyl esters. Also P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester), a tripeptide that previously has shown impressive effects in human tumor cells, was used as reference. The tested compounds displayed various activities in the different cell lines but also showed a high correlation, indicating a similar mechanism of action. Factors like amino acid composition, amino acid sequence, modifications of the C- and N-termini, and to a minor extent the lipophilicity of the dipeptide derivatives appear to influence the in vitro activity. The results indicate that the activity of these compounds not only relies on their chemical reactivity, but also on active biological interactions such as transport across membranes and/or enzymatic liberation of reactive molecular entities.

Barbituric acid is the parent compound of a large family of hypnotic barbiturates. A nitrogen mustard (N-mustard) group (-CH2CH2N[CH2CH2Cl]2) was placed onto the two nitrogen atoms at positions 1 and 3 of the pyrimidine ring. This N-mustard agent is a solid at 25 degrees C, stable at -10 degrees C for >10 weeks, and soluble in aqueous solvent at 37 degrees C and 25 degrees C. The partition coefficients miLog P and CLog P were calculated to be -0.93 and -1.441 for barbituric acid. The miLog P and CLog P for the N-mustard agent were 1.82 and 2.707, respectively. The N-mustard substituents significantly increased solubility in lipid by-layers. The N-mustard agent alkylated a nucleophilic primary amine (p-chloroaniline) at physiological conditions of pH 7.4 and 37 degrees C. Aliquots of reaction mixtures were withdrawn at known time periods to react with fluorescamine for determination of unreacted p-chloroaniline and calculation of rate constants. The alkylation of the primary amine was second order with rate = k2[Nu]2, (Nu is nucleophile) and rate constant k2 = 0.01358 L/(mole.min). The molecular dipole of barbituric acid and the N-mustard agent was calculated by SPARTAN software (wavefunction, Irvine, CA) to be 0.681 and 2.153 Debye, respectively. The brain/blood partition coefficient (Log BB) of the N-mustard agent was -0.399. Values of molecular polar surface area (TPSA) for barbituric acid and the N-mustard agent was 75.27 and 64.17, respectively. TPSA values indicate an expected intestinal absorbance to be 79% and 90%, respectively. The N-mustard agent showed zero violations of the Rule of 5, indicating good bioavailability.