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Aims: Persulfides and other reactive sulfur species are endogenously produced in large amounts in vivo and participate in multiple cellular functions underlying physiological and pathological conditions. In the current study, we aimed to develop an ideal alkylating agent for use in sulfur metabolomics, particularly targeting persulfides and other reactive sulfur species, with minimal artifactual decomposition. Results: We synthesized a tyrosine-based iodoacetamide derivative, N -iodoacetyl l-tyrosine methyl ester (TME-IAM), which reacts with the thiol residue of cysteine identically to that of β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM), a commercially available reagent. Our previous study revealed that although various electrophilic alkylating agents readily decomposed polysulfides, HPE-IAM exceptionally stabilized the polysulfides by inhibiting their alkaline hydrolysis. The newly synthesized TME-IAM stabilizes oxidized glutathione tetrasulfide more efficiently than other alkylating agents, including HPE-IAM, iodoacetamide, and monobromobimane. In fact, our quantitative sulfur-related metabolome analysis showed that TME-IAM is a more efficient trapping agent for endogenous persulfides/polysulfides containing a larger number of sulfur atoms in mouse liver and brain tissues compared with HPE-IAM. Innovation and Conclusions: We developed a novel iodoacetamide derivative, which is the most ideal reagent developed to date for detecting endogenous persulfides/polysulfides formed in biological samples, such as cultured cells, tissues, and plasma. This new probe may be useful for investigating the unique chemical properties of reactive persulfides, thereby enabling identification of novel reactive sulfur metabolites that remain unidentified because of their instability, and thus can be applied in high-precision sulfur metabolomics in redox biology and medicine. We did not perform any clinical experiments in this study. Antioxid. Redox Signal . 34, 1407-1419.

DNA alkylation damage, emanating from the exposure to environmental alkylating agents or produced by certain endogenous metabolic processes, affects cell viability and genomic stability. Fe(II)/2-oxoglutarate-dependent dioxygenase enzymes, such as Escherichia coli AlkB, are involved in protecting DNA from alkylation damage. Inspired by the natural product indenone derivatives reported to inhibit this class of enzymes, and a set of 2-chloro-3-amino indenone derivatives was synthesized and screened for their inhibitory properties against AlkB. The synthesis of 2-chloro-3-amino indenone derivatives was achieved from 2,3-dichloro indenones through addition-elimination method using alkyl/aryl amines under catalyst-free conditions. Using an in vitro reconstituted DNA repair assay, we have identified a 2-chloro-3-amino indenone compound 3o to be an inhibitor of AlkB. We have determined the binding affinity, mode of interaction, and kinetic parameters of inhibition of 3o and tested its ability to sensitize cells to methyl methanesulfonate that mainly produce DNA alkylation damage. This study established the potential of indenone-derived compounds as inhibitors of Fe(II)/2-oxoglutarate-dependent dioxygenase AlkB., (© 2021 John Wiley & Sons Ltd.)

The DNA repair enzyme AAG has been shown in mice to promote tissue necrosis in response to ischaemic reperfusion or treatment with alkylating agents. A chemical probe inhibitor is required for investigations of the biological mechanism causing this phenomenon and as a lead for drugs that are potentially protective against tissue damage from organ failure and transplantation, and alkylative chemotherapy. Herein, we describe the rationale behind the choice of arylmethylpyrrolidines as appropriate aza-nucleoside mimics for an inhibitor followed by their synthesis and the first use of a microplate-based assay for quantification of their inhibition of AAG. We finally report the discovery of an imidazol-4-ylmethylpyrrolidine as a fragment-sized, weak inhibitor of AAG., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Higher-ordered structure motifs of nucleic acids, such as the G-quadruplex (G-4), mismatched and bulge structures, are significant research targets because these structures are involved in genetic control and diseases. Selective alkylation of these higher-order structures is challenging due to the chemical instability of the alkylating agent and side-reactions with the single- or double-strand DNA and RNA. We now report the reactive OFF-ON type alkylating agents, vinyl-quinazolinone (VQ) precursors with a sulfoxide, thiophenyl or thiomethyl group for the OFF-ON control of the vinyl reactivity. The stable VQ precursors conjugated with aminoacridine, which bind to the G-4 DNA, selectively reacted with a T base on the G-4 DNA in contrast to the single- and double-strand DNA. Additionally, the VQ precursor reacted with the T or U base in the AP-site, G-4 RNA and T-T mismatch structures. These VQ precursors would be a new candidate for the T or U specific alkylation in the higher-ordered structures of nucleic acids., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.)

HxTfA 4 is a fluorescent analog of a potent cytotoxic and antimalarial agent, TfA 3, which is currently being investigated for the development of an antimalarial vaccine, PlasProtect®. HxTfA contains a p-anisylbenzimidazole or Hx moiety, which is endowed with a blue emission upon excitation at 318 nm; thus enabling it to be used as a surrogate for probing the cellular fate of TfA using confocal microscopy, and addressing the question of nuclear localization. HxTfA exhibits similar selectivity to TfA for A-tract sequences of DNA, alkylating adenine-N3, albeit at 10-fold higher concentrations. It also possesses in vitro cytotoxicity against A549 human lung carcinoma cells and Plasmodium falciparum. Confocal microscopy studies showed for the first time that HxTfA, and by inference TfA, entered A549 cells and localized in the nucleus to exert its biological activity. At biologically relevant concentrations, HxTfA elicits DNA damage response as evidenced by a marked increase in the levels of γH2AX observed by confocal microscopy and immunoblotting studies, and ultimately induces apoptosis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.)

We report design, synthesis, and mechanistic studies of a new mitomycin tetramer 9 along with a new mitomycin dimer 10. Mitomycin 9 is a tetramer connected by the disulfide linker 11, and easily undergoes disulfide cleavage to provide two dimeric structures 9r that each contains a single thiol probe for activations. So, tetramer 9 as a precursor of 9r was specifically targeted to undergo double activations with a single probe. A tetramer 9 was synthesized using 1 and key intermediate 11, and a dimer 10 was synthesized from 1 and diamine 12. Activation studies revealed that 9 underwent effective double activations with a single probe by nucleophiles while the reference 10 did not. Evaluations of DNA ISC formations showed that 9 generated substantial levels of DNA ISC by nucleophilic activation while the references 10 and 2 did not. The effectiveness of 9 in activation and formation of DNA ISC per probe was verified by comparing with dimers 5-8 of double activations with two probes. These findings highlighted the role of a single thiol in 9r and demonstrated the intended double activations with a single probe, which marks the first case in mitomycin studies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

4-(4-Nitrobenzyl)pyridine (NBP) is a colorimetric indicator compound for many types of carcinogenic alkylating agents. Because of the similar reactivity of NBP and guanine in DNA, NBP serves as a DNA model. NBP assays are used in the toxicological screening of pharmaceutical compounds, detection of chemical warfare agents, environmental hygiene technology, preliminary toxicology tests, mutagenicity of medicinal compounds, and other chemical analyses. Nevertheless, the use of NBP as a DNA model suffers from the compound's low water solubility, its lack of reactive oxygen sites, and dissimilar steric encumbrance compared to DNA. We report herein the design and synthesis of NBP derivatives that address some of these issues. These derivatives have been tested in solution and found to be superior in the colorimetric assay of the alkylating anticancer drug cyclophosphamide. The derivatives have also been integrated into a polymeric silica material which changes color upon the exposure to dangerous alkylating agents, such as iodomethane vapor, without the need for an exogenous base. This material modernizes the NBP assay from a time-consuming laboratory analysis to a real-time solid state sensor, which requires neither solvent nor additional reagents and can detect both gas- and solution-phase alkylating agents.

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.)

We designed and synthesized a tandem-hairpin motif of pyrrole (P)-imidazole (I) polyamide 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) conjugates (1) that targets the human telomere repeat sequence 5'-d(CCCTAA)n-3'. As a control, conjugate 2 (hairpin PI polyamide with seco-CBI), which also targets the human telomere repeat sequence, was synthesized. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) using 5' Texas Red-labeled 219-bp DNA fragments revealed the outstandingly high sequence selectivity of 1, with no mismatch alkylation. Furthermore, an evaluation performed in human cancer cell lines demonstrated that conjugate 1 has low cytotoxicity compared with conjugate 2. In addition, a cell-staining analysis indicated that conjugate 1 induced apoptosis moderately by DNA damage. This study demonstrated that conjugate 1 can be used as an effective alkylator for telomere repeat sequences or as an apoptotic inducer.

The synthesis of a small series of 2-nitroimidazoles in which the β-amino alcohol side chain was amidated with a range of alkylating/acylating functionality is described. Synthetic methodologies were developed that generally provided for selective N-acyl versus N,O-bisacyl products. In vitro, target analogs showed minimal radiosensitization activity, with only a few exhibiting a sensitizer enhancement ratio (SER) >2.0 and C(1.6) values comparable to reference agents RB-6145 and RSU-1069. In an assay to determine potential to alkylate biomolecules, representative analogs showed <1% of the alkylating activity of RSU-1069. In vivo, one analog showed an enhancement ratio of 1.6 relative to vehicle control when tested in B6C3F1 mice with an implanted KHT sarcoma. The data reinforce prior findings that there is a correlation between alkylation potential and in vivo activity.

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

We report the synthetic and mechanistic studies on a new cyclic disulfide mitomycin dimer, 7-N,7'-N'-(1″,2″-dithiolanyl-3″,5″-dimethylenyl)bismitomycin C (8), and a diol mitomycin dimer, 7-N,7'-N'-(2″,4″-dihydroxy-1″,5″-pentanediyl)bismitomycin C (9). Mitomycin 8 is a dimer connected by a 1,2-dithiolane (a five-membered cyclic disulfide) linker, and was specifically designed to undergo nucleophilic activation and double DNA alkylations leading to efficient production of DNA interstrand cross-link (DNA ISC) adducts. Disulfide cleavage in 8 would generate two thiol groups that could serve as probes to activate two mitomycin rings. At first, the target mitomycin 8 was synthesized using mitomycin A (1) and the key intermediate, cyclic disulfide (10), which was prepared through a seven-step synthetic sequence. Diol mitomycin 9 was also synthesized from 1 and diamine salt 13. Next, kinetic studies using solvolysis reaction revealed that the activation rates of 8 were much higher than those of 9 and mitomycin C (2) under nucleophilic conditions provided by Et(3)P presumably due to the presence of a cyclic disulfide unit in 8. These findings led us to propose a nucleophilic activation pathway for 8. Then, DNA ISC experiments further revealed that the levels of DNA ISC caused by 8 in the presence of Et(3)P were much higher (97%) than those by 9 (5%) and 2 (4%). More importantly, mitomycin 8 underwent much faster activation and produced slightly higher levels of DNA ISC than the previously reported mitomycins 5-7. Overall, we concluded that 8 was highly efficient for both nucleophilic activation and corresponding DNA ISC formation, and that this differentiation came from the crucial function of the cyclic disulfide unit in 8., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Convergent synthetic routes for N-methylpyrrole (P) and N-methylimidazole (I) seco-1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole (CBI) conjugates with a vinyl linker were developed. New hairpin polyamide-seco-CBI conjugates, compounds 16-19, were synthesized, and their DNA sequence-specific alkylating activities were evaluated via high-resolution denaturing gel electrophoresis and high-performance liquid chromatography (HPLC) product analysis. The new synthetic route for PI conjugates with a vinyl linker consisted of the introduction of a vinylpyrrole unit (8-11) into the C terminal of a PI polyamide synthesized by (fluorenylmethoxy)carbonyl solid-phase peptide synthesis (SPPS), followed by liquid-phase coupling with seco-CBI. The yield of the conjugates was significantly improved compared with that of the method reported previously, which allows us to synthesize various substituted conjugates containing a vinyl linker. Conjugates 16-19 were designed to investigate the substituent effect of the vinyl linker, and conjugate 16S was synthesized to evaluate the reactivity between racemic and S enantiomers of the seco-CBI derivative. The results of high-resolution denaturing gel electrophoresis using 208 bp DNA fragments indicated that alkylation by compounds 16 and 17, in which the H of the vinyl linker of compound 16 was replaced with F, occurred predominantly at the A of the 5'-TTTGTCA-3' sequence at nanomolar concentrations. In clear contrast, compounds 18 and 19, which were methyl or Br derivatives of compound 16, did not exhibit any DNA alkylating activity. Moreover, HPLC product analysis using synthetic oligonucleotides demonstrated that alkylation occurred between the N3 of the adenine of the oligomer and the cyclopropane ring of 16S. Density functional calculation of substituted vinylpyrrole seco-CBI units indicated that methyl and Br substituents led to a significantly distorted geometry of the vinyl group with the pyrrole ring compared with H and F derivatives. Molecular modeling studies offered the additional information that steric hindrance reduced the DNA alkylating activity of these derivatives.

N-Acyl-α-amino acids were efficiently transformed in a two-step procedure into 1-N-(acylamino)alkyltriphenylphosphonium salts, new powerful α-amidoalkylating agents. The effect of the α-amino acid structure, the base used [MeONa or a silica gel-supported piperidine (SiO(2)-Pip)], and the main electrolysis parameters (current density, charge consumption) on the yield and selectivity of the electrochemical decarboxylative α-methoxylation of N-acyl-α-amino acids (Hofer-Moest reaction) was investigated. For most proteinogenic and all studied unproteinogenic α-amino acids, very good results were obtained using a substoichiometric amount of SiO(2)-Pip as the base. Only in the cases of N-acylated cysteine, methionine, and tryptophan, attempts to carry out the Hofer-Moest reaction in the applied conditions failed, probably because of the susceptibility of these α-amino acids to an electrochemical oxidation on the side chain. The methoxy group of N-(1-methoxyalkyl)amides was effectively displaced with the triphenylphosphonium group by dissolving an equimolar amount of N-(1-methoxyalkyl)amide and triphenylphosphonium tetrafluoroborate in CH(2)Cl(2) at room temperature for 30 min, followed by the precipitation of 1-N-(acylamino)alkyltriphenylphosphonium salt with Et(2)O.

A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320 nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as demonstrated with tRNA and RNA fragments from the MS2 phage and the HIV genome. Alternatively, the azide function can be used for further derivatization by click-chemistry. This allows e.g. the introduction of an additional fluorophore for excitation with visible light.

Dimeric DNA alkylating agents have drawn significant interest because these compounds are expected to provide at least two reactive sites and as a result, generate enhanced levels of DNA interstrand cross-link (DNA ISC) adducts compared to their monomeric agents. We report the synthesis and mechanistic studies of a novel mitomycin dimer, 7-N,7'-N'-(1″,2″-dithiocanyl-3″,8″-dimethylenyl)bismitomycin C (8) connected by an eight-membered cyclic disulfide. Mitomycins require prior activation (i.e., transformation to a good electrophile) for DNA adduction and therefore, 8 was aimed to undergo facile nucleophilic activation and produce enhanced levels of DNA ISC. At the core of this function lies a cyclic disulfide in 8. It was expected that disulfide cleavage by an appropriate nucleophile would successively produce two thiols that may trigger activation of two mitomycin rings in a dimer through intramolecular cyclization to quinine rings. Compound 8 was synthesized from mitomycin A (1) and the key intermediate, cyclic disulfide (11), along with the reference diol mitomycin 7-N,7'-N'-(2″,7″-dihydroxy-1″,8″-octanediyl)bismitomycin C (23) which does not contain the disulfide unit. We found that 8 underwent significantly enhanced nucleophilic activation in the presence of Et(3)P compared with 23, and that the disulfide unit in 8 played a key role for the nucleophilic activation. Based on these findings, we proposed a mechanism for nucleophilic activation of 8. We further demonstrated that 8 generated much higher levels of DNA ISC (94%) compared with 23 (4%) and 2 (3%) in the presence of Et(3)P (and L-DTT) leading to the conclusion that 8 is more efficient for DNA ISC processes than 23 and 2 due to the role of disulfide unit., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

An overview of anticancer active spirocyclopropanes of the illudin class is provided. After a short introduction on the history and general chemistry of illudins M and S, new discoveries concerning their mode of action and metabolism are reported as well as new synthetic endeavors towards derivatives with improved selectivity for and efficacy against cancer cells. In addition, common and recently tapped biological sources and isolation procedures for known and new illudins are discussed. Pertinent literature is covered up to 2010.

The synthesis and evaluation of a key series of analogs of duocarmycin SA, bearing a single substituent at the C5' position of the DNA binding subunit, are described., (2010 Elsevier Ltd. All rights reserved.)

The synthesis of the first spacer containing, duocarmycin analogue prodrug was realised, its biological properties evaluated and compared to its counterpart prodrug without a spacer unit. The synthesis comprises the manufacture of the new acetylated derivatives and of two double spacer systems, their activation and coupling to the pharmacophoric seco-drug (+)-. Unprecedented biological results were found as the new prodrug showed a fairly low QIC(50) value of 20, but on the other hand a high stability and very low DNA alkylation efficiency. These findings indicate a changed cytostatic mode of action induced by the self-immolative spacer moiety which was employed.

The design, synthesis and evaluation of methyl 1,2,8,8a-tetrahydrocyclopropa[c]oxazolo[2,3-e]indol-4-one-6-carboxylate (COI) derivatives are detailed representing analogs of duocarmycin SA containing an oxazole replacement for the fused pyrrole found in the alkylation subunit., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Alkylating agents are simple and reactive molecules that are commonly used in many and diverse fields such as organic synthesis, medicine, and agriculture. Some highly reactive alkylating species are also being used as blister chemical-warfare agents. The detection and identification of alkylating agents is not a trivial issue because of their high reactivity and simple structure. Herein, we report on a new multispot luminescence-based approach to the detection and identification of alkylating agents. In order to demonstrate the potential of the approach, seven pi-conjugated oligomers and polymers bearing nucleophilic pyridine groups, 1-7, were adsorbed onto a solid support and exposed to vapors of alkylators 8-15. The alkylation-induced color-shift patterns of the seven-spot array allow clear discrimination of the different alkylators. The spots are sensitive to minute concentrations of alkylators and, because the detection is based on the formation of new covalent bonds, these spots saturate at about 50 ppb.

Alkylating agents have been shown to be very promising for the radiolabelling of oligonucleotides with fluorine-18. In this report we describe the fully automated synthesis of 2-bromo-N-[3-(2-[(18)F]fluoropyridin-3-yloxy)propyl]acetamide ([(18)F]FPyBrA) utilizing a modular synthesis unit. Reaction conditions for the coupling of this pyridine-based alkylating agent at the 5' end of a fully phosphorothioated random 20-mer DNA sequence were optimized to achieve very high radiochemical yields (>90%) and a maximum specific activity of 5-6 GBq/micromoL. The potential for rapid purification by solid phase extraction without need of chromatographic isolation of the radiolabelled oligonucleotide presents an overall benefit for the application of oligonucleotides in preclinical studies and potential clinical applications.

Structure-functional characterization of vitamin D receptor (VDR) requires identification of structurally distinct areas of VDR-ligand-binding domain (VDR-LBD) important for biological properties of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). We hypothesized that covalent attachment of the ligand into VDR-LBD might alter 'surface structure' of that area influencing biological activity of the ligand. We compared anti-proliferative activity of three affinity alkylating derivatives of 1,25(OH)(2)D(3) containing an alkylating probe at 1,3 and 11 positions. These compounds possessed high-affinity binding for VDR; and affinity labeled VDR-LBD. But, only the analog with probe at 3-position significantly altered growth in keratinocytes, compared with 1,25(OH)(2)D(3). Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)(2)D(3)-1-BE), Cys288 (beta-hairpin region: 1,25(OH)(2)D(3)-3-BE,) and Tyr295 (helix-6: 1,25(OH)(2)D(3)-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the beta-hairpin region (modified by 1,25(OH)(2)D(3)-3-BE) is most important for growth inhibition by 1,25(OH)(2)D(3), while helices 3 and 6 are less important for such activity.

A series of 3-[alpha-(acylamino)acyl]-1-aryl-3-methyltriazenes 6a-l, potential cytotoxic triazene prodrugs, were synthesised by coupling 1-aryl-3-methyltriazenes to N-acylamino acids. Their hydrolysis was studied in isotonic pH 7.4 phosphate buffer and in human plasma, while hydrolysis of the derivative 6a was studied in more depth across a range of pH values. Prodrugs 6a-l hydrolyse by cleavage of the triazene acyl group to afford the corresponding monomethyltriazenes. Studies in human plasma demonstrate that acylation of the alpha-amino group of the amino acid carrier is an effective means of reducing the chemical reactivity of the alpha-aminoacyl derivatives while retaining a rapid rate of enzymatic hydrolysis. These derivatives displayed logP values that suggest they should be well absorbed through biological membranes.

Irradiation of 2-diazo-3-oxochlorins (200 W, lambda > or = 345 nm, 10 degrees C) in the presence of nucleophilic and biomimetic substrates 1-butanol, tosylhydrazine, or tetrahydrofurfuryl alcohol generates Wolff-rearranged, pyrrole ring-contracted azeteoporphyrinoids in 11-34% yield, with the corresponding hydroxyporphyrins in up to 55% yield. For metalated diazo-oxochlorins, these products compete with intramolecular exocyclic ring formation by meso-phenyl ring addition, which occurs in up to 76% yield in the absence of substrate. The dependence of product distribution on substrate is established by photolysis in neat dichlorometane. Under these conditions, formation of the Wolff-rearranged product is inhibited and the phenyl addition product dominates (76%) due to the absence of a good nucleophile. A conceptually analogous dependence is also observed for the free-base derivative, with the exocyclic ring-containing dimerization product isolated in 42% yield. The third reaction pathway, formation of the hydroxyporphyrin, is enhanced by the presence of non-nucleophilic, oxidizable substrates such as 1,4-cyclohexadiene (M = Cu; 55%); however, in the presence of the bulky and oxidatively more stable tert-butyl alcohol, intramolecular exocyclic ring-quenching is observed in 51% yield with no detection of the hydroxyporphyrin. X-ray structure characterization of the azeteoporphyrinoids reveals a planar macrocycle, illustrating the strong influence of periphery contraction. Specifically, the copper-containing azeteoporphyrinoids show remarkably short Cu-N(azete) distances of 1.88-1.90 A. All porphyrinoid photoproducts possess intense absorption bands throughout the visible spectral region, indicating that ring-contracted substrate adducts, as well as phenyl ring addition products, maintain porphyrinoid aromaticity. Overall, the ability of these chromophores to photochemically react under substrate control may make unimolecular porphyrinoid photoreagents such as these useful for applications in photobiology or O2-independent photodynamic therapy.

Research, developed at the Laboratory of Organic Pharmaceutical Chemistry of the School of Pharmacy, UMR-CNRS 6517, is centred on the synthesis of novel therapeutic compounds using monoelectronic transfer reactions. Tetrakis(dimethylamino)ethylene (TDAE) is a powerful electron donor which has the specific property of activating the carbon-halogen bond leading to the formation of a stable electrophilic radical and a stable neutrophilic anion. Since 2002, our team has developed a program using monoelectronic transfer reactions initiated by TDAE of nitroaromatic, nitroheterocyclic and quinonic bioreducible alkylating agents. The goal is to synthesize new therapeutic compounds for use as anti-infectious agents, anticancer agents, and agents active on the central nervous system. In this context, we present the first pharmacochemical tools obtained with this strategy during reactions between diverse electrophilic compounds (aldehydes, ketones, alpha-keto-esters, ketomalonates, alpha-ketolactames, ...) and benzylic anions formed in situ by the action of TDAE. We illustrate the usefulness of this strategy by describing the preparation of new compounds of biological interest and the associated pharmacomodulation work.

Properties of benzimidazole and its derivatives have been studied for over hundred years; special interest of researchers triggered by the fact that 5,6-dimethylbenzimidazole is a component of naturally occurring vitamin B(12). Several antihelminthic, antacid and antibacterial drugs are known which have benzimidazole moiety as their essential constituent. Additionally, several bis- and tris-benzimidazole based systems are well-known for their interaction with DNA and interference with several DNA associated processes. This account gives an overview of the benzimidazole based systems and their relevance in medicinal chemistry.

Mild activation of water-soluble naphthalene diimides (NDIs) as bisalkylating agents has been achieved by base catalysis and by chemical and electrochemical reductions. NDI activation by a single electron reduction represents a novelty in the field of activatable electrophiles. Under mild reduction, induced by S2O4(2-) in aqueous solution, the resulting NDI radical anion (NDI*-) undergoes a monomolecular fragmentation to yield a new transient species, where the NDI radical anion is tethered to a quinone methide moiety. The latter still retains electrophilic properties, reacting with amines, thiols, and ethyl vinyl ether. Owing to the NDI recognition properties, these results represent the first step toward selective and bioactivatable cross-linking agents.

AS-I-145 is a novel achiral seco-amino-cyclopropylbenz[e]indolone (seco-amino-CBI) analogue of duocarmycin that has evolved from an alternative strategy of designing CC-1065/duocarmycin agents lacking the characteristic chiral center of the natural agents. The sequence specificity of this compound was assessed by a Taq polymerase stop assay, identifying the sites of covalent modification on plasmid DNA. The adenine-N3 adducts were confirmed at AT-rich sequences using a thermally induced strand cleavage assay. These studies reveal that this compound retains the inherent sequence selectivity of the related natural compounds. The AS-I-145 sensitivity of yeast mutants deficient in excision and post-replication repair (PRR) pathways was assessed. The sensitivity profile suggests that the sequence-specific adenine-N3 adducts are substrates for nucleotide excision repair (NER) but not base excision repair (BER). Single-strand ligation PCR was employed to follow the induction and repair of the lesions at nucleotide resolution in yeast cells. Sequence specificity was preserved in intact cells, and adduct elimination occurred in a transcription-coupled manner and was dependent on a functional NER pathway and Rad18. The involvement of NER as the predominant excision pathway was confirmed in mammalian DNA repair mutant cells. AS-I-145 showed good in vivo antitumor activity in the National Cancer Institute standard hollow fiber assay and was active against the human breast MDA-MD-435 xenograft when administered i.v. or p.o. Its novel structure and in vivo activity renders AS-I-145 a new paradigm in the design of novel achiral analogues of CC-1065 and the duocarmycins.

Novel palladium(II) complexes (4-6) have been synthesized from the reaction of (E)-3{[(diethoxythiophosphoryl)-methylhydrazone]-methylene}-4-hydroxy-2H-1-benzopyran-2-one (1), E)-3-{[(dimethoxythiophosphoryl)-methylhydrazine]-methylidene}-3,4-dihydro-2H-1- benzopyran-2,4-dione (2) and (E)-3-{[(diethoxythiophosphoryl)-hydrazone]-methylene}- 4H-1-benzopyran-4-one (3), respectively, with PdCl2(PhCN)2. These complexes have been characterized by elemental analysis and spectroscopic studies. The alkylating activity of the complexes was examined in vitro toward 4-(4'-nitrobenzyl)pyridine (NBP test). The palladium(lI) complexes were found to possess alkylating activity. The compounds (4-6) have been screened for their antibacterial properties and displayed low activity.

Hairpin polyamide-chlorambucil conjugates containing an alpha-diaminobutyric acid (alpha-DABA) turn moiety are compared to their constitutional isomers containing the well-characterized gamma-DABA turn. Although the DNA-binding properties of unconjugated polyamides are similar, the alpha-DABA conjugates display increased alkylation specificity and decreased rate of reaction. Treatment of a human colon carcinoma cell line with alpha-DABA versus gamma-DABA hairpin conjugates shows only slight differences in toxicities while producing similar effects on cell morphology and G2/M stage cell cycle arrest. However, striking differences in animal toxicity between the two classes are observed. Although mice treated with an alpha-DABA hairpin polyamide do not differ significantly from control mice, the analogous gamma-DABA hairpin is lethal. This dramatic difference from a subtle structural change would not have been predicted.

Complementary to studies that provided the first yatakemycin total synthesis resulting in its structure revision and absolute stereochemistry assignment, a second-generation asymmetric total synthesis is disclosed herein. Since the individual yatakemycin subunits are identical to those of duocarmycin SA (alkylation subunit) or CC-1065 (central and right-hand subunits), the studies also provide an improvement in our earlier total synthesis of CC-1065 and, as detailed herein, have been extended to an asymmetric total synthesis of (+)-duocarmycin SA. Further extensions of the studies provided key yatakemycin partial structures and analogues for comparative assessments. This included the definition of the DNA selectivity (adenine central to a five-base-pair AT sequence, e.g., 5'-AAAAA), efficiency, relative rate, and reversibility of ent-(-)-yatakemycin and its comparison with the natural enantiomer (identical selectivity and efficiency), structural characterization of the adenine N3 adduct confirming the nature of the DNA reaction, and comparisons of the cytotoxic activity of the natural product (L1210, IC50 = 5 pM) with those of its unnatural enantiomer (IC50 = 5 pM) and a series of key partial structures including those that probe the role of the C-terminus thiomethyl ester. The only distinguishing features between the enantiomers is that ent-(-)-yatakemycin alkylates DNA at a slower rate (krel = 0.13) and is reversible, whereas (+)-yatakemycin is not. Nonetheless, even ent-(-)-yatakemycin alkylates DNA at a faster rate and with a greater thermodynamic stability than (+)-duocarmycin SA, illustrating the unique characteristics of such "sandwiched" agents.

Aryl free-radicals generated at the C-7 position of ethyl indole-2-carboxylates bearing N-allyl and propargylic groups triggered intramolecular cyclizations to furnish a new class of Duocarmycin analogues, formal ethyl pyrrolo[3,2,1-ij]quinoline-2- carboxylate derivatives, through the less favorable 6-endo-trig cyclization mode.

The incorporation of anticancer prodrugs into polyacrylamide conjugates has been shown to improve tumor targeting via the so-called "enhanced permeability and retention" effect. This strategy has now been expanded to include two different classes of glutathione (GSH)-activated antitumor agents prepared by radical polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-methacryloyloxy-methyl-2-cyclohexenone (7) and/or with S-(N-4-chlorophenyl-N-hydroxycarbamoyl-thioethyl)methacrylamide (8), followed by treatment with 3-chloroperoxybenzoic acid, to give the HPMA copolymers of 7 and the 8-sulfoxide, respectively. In aqueous-buffered solution at pH 6.5, GSH reacts rapidly with poly-HPMA-8-sulfoxide (k approximately 2.3 mM(-1) min(-1)) to give S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (1), a tight-binding transition state analogue inhibitor of the antitumor target enzyme glyoxalase I (K(i) = 46 nM), or with poly-HPMA-7 (k approximately 0.02 mM(-1) min(-1)) to give the electrophilic antitumor agent 3-glutathio-2-methylenecyclohexenone (4). Indeed, B16 melanotic melanoma in culture is inhibited by poly-HPMA-8-sulfoxide and by poly-HPMA-7 with IC(50) values of 168 +/- 8 and 284 +/- 5 microM, respectively. These values are significantly greater than those of the unpolymerized prodrugs suggesting that the cytotoxicity of the polymer prodrugs might be limited by slow cellular uptake via pinocytosis. This prodrug strategy should be applicable to a range of different GSH-based antitumor agents.

Induced DNA interstrand cross-links by chemical agents or photoactivation play very important roles for cancer therapy. Several important clinical drugs (e.g. cisplatin, psoralens, and mitomycin C) are known to induce DNA ISC formation, which can disrupt cell maintenance and replication. Among these anti-tumor agents, one mechanism was involved in quinone methide intermediate. Quinone methide derivative has played important roles in organic syntheses as well as in chemical and biological processes. This review is concerned with current efforts of quinone methide derivatives to DNA alkylation and DNA cross-links. The latest advances in this field will be reviewed in this article. The chemical and physical properties of quinone methide derivatives, the interactions between nucleobases and quinone methide derivatives, the reactions with phosphodiester, DNA alkylation and cross-link via quinone methide intermediate action will be discussed.

The photogeneration and detection of new binol quinone methides undergoing mono- and bisalkylation of free nucleophiles was investigated by product distribution analysis and laser flash photolysis in water solution using binol quaternary ammonium derivatives 2 and 12 as photoactivated precursors. The alkylation processes of N and S nucleophiles are strongly competitive with the hydration reaction. DNA cross-linking potency of the water-soluble binol quaternary ammonium salt 2 was investigated as a pH function and compared to that of other quaternary ammonium salts capable of benzo-QM (QM = quinone methide) photogeneration by gel electrophoresis. DFT calculations in the gas phase and in water bulk on the binol and benzo quaternary ammonium salts 2 and 4 evidence structural and electrostatic features of the binol derivative which might offer a rationalization of its promising high photo-cross-linking efficiency.

S-Adenosyl-L-methionine (AdoMet) is one of Nature's most diverse metabolites, used not only in a large number of biological reactions but amenable to several different modes of reactivity. The types of transformations in which it is involved include decarboxylation, electrophilic addition to any of the three carbons bonded to the central sulfur atom, proton removal at carbons adjacent to the sulfonium, and reductive cleavage to generate 5'-deoxyadenosyl 5'-radical intermediates. At physiological pH and temperature, AdoMet is subject to three spontaneous degradation pathways, the first of which is racemization of the chiral sulfonium group, which takes place in a pH-independent manner. The two remaining pathways are pH-dependent and include (1) intramolecular attack of the alpha-carboxylate group onto the gamma-carbon, affording L-homoserine lactone (HSL) and 5'-methylthioadenosine (MTA), and (2) deprotonation at C-5', initiating a cascade that results in formation of adenine and S-ribosylmethionine. Herein, we describe pH-dependent stability studies of AdoMet and its selenium and tellurium analogues, Se-adenosyl-L-selenomethionine and Te-adenosyl-L-telluromethionine (SeAdoMet and TeAdoMet, respectively), at 37 degrees C and constant ionic strength, which we use as a probe of their relative intrinsic reactivities. We find that with AdoMet intramolecular nucleophilic attack to afford HSL and MTA exhibits a pH-rate profile having two titratable groups with apparent pK(a) values of 1.2 +/- 0.4 and 8.2 +/- 0.05 and displaying first-order rate constants of <0.7 x 10(-6) s(-1) at pH values less than 0.5, approximately 3 x 10(-6) s(-1) at pH values between 2 and 7, and approximately 15 x 10(-6) s(-1) at pH values greater than 9. Degradation via deprotonation at C-5' follows a pH-rate profile having one titratable group with an apparent pK(a) value of approximately 11.5. The selenium analogue decays significantly faster via intramolecular nucleophilic attack, also exhibiting a pH-rate profile with two titratable groups with pK(a) values of approximately 0.86 and 8.0 +/- 0.1 with first-order rate constants of <7 x 10(-6) s(-1) at pH values less than 0.9, approximately 32 x 10(-6) s(-1) at pH values between 2 and 7, and approximately 170 x 10(-6) s(-1) at pH values greater than 9. Degradation via deprotonation at C-5' proceeds with one titratable group displaying an apparent pK(a) value of approximately 14.1. Unexpectedly, TeAdoMet did not decay at an observable rate via either of these two pathways. Last, enzymatically synthesized AdoMet was found to racemize at rates that were consistent with earlier studies (Hoffman, J. L. (1986) Biochemistry 25, 4444-4449); however, SeAdoMet and TeAdoMet did not racemize at detectable rates. In the accompanying paper, we use the information obtained in these model studies to probe the mechanism of cyclopropane fatty acid synthase via use of the onium chalcogens of AdoMet as methyl donors.

A new class of potent electrophilic "R(+)" alkylating agents has been developed using weakly nucleophilic carborane anions as leaving groups. These reagents, R(CHB(11)Me(5)X(6)) (R = Me, Et, and i-Pr; X = Cl, Br), are prepared via metathesis reactions with conventional alkylating agents such as alkyl triflates, using the high oxophilicity of silylium ion-like species, Et(3)Si(carborane), as the driving force to obtain increased alkyl electrophilicity. The crystal structure of the isopropyl reagent, i-Pr(CHB(11)Me(5)Br(6)), has been determined, revealing covalence in the alkyl-carborane bonding. This contrasts with the free i-Pr(+) carbocation observed when the anion is less coordinating (e.g. Sb(2)F(11)(-)) or with tertiary alkyl centers, as in [tert-butyl][carborane] salts. In solution, the reagents exist as equilibrating isomers with the alkyl group at the 7-11 or 12 halide positions of the CB(11) icosahedral carborane anion. These alkylating agents are so electrophilic that they (a) react with alkanes at or below room temperature via hydride extraction to produce carbenium ions, (b) alkylate benzene without a Friedel-Crafts catalyst to give arenium ions, and (c) alkylate electron-deficient phosphorus compounds that are otherwise inert to conventional alkylating agents such as methyl triflate.

The development of high-load, soluble oligomeric sulfonate esters, generated via ROM polymerization, and their utility in the facile benzylation of an array of amines is reported. These polymeric sulfonate esters exist as free-flowing powders, are stable at refrigerated temperatures, and are readily dissolved in CH(2)Cl(2). Following the benzylation event, purification is attained via simple filtration, followed by solvent removal to deliver the desired benzylated product in good to excellent yield and high purity. [structure: see text]

Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized a cross-link that bridges the N(3) positions of a mismatched thymidine base pair. This cross-link, which is similar to the N(3)C-alkyl-N(3)C cross-link that has been observed between mismatched cytosine base pairs, was introduced by first incorporating a cross-linked phosphoramidite unit at the 5'-end of an oligonucleotide chain. Fully cross-linked duplexes were then synthesized using an orthogonal approach to selectively remove protecting groups, thus allowing construction of the cross-linked duplex via conventional solid-phase oligonucleotide synthesis. Short DNA duplexes with alkyl cross-links of various lengths (two, four, and seven methylene units) were prepared, and their physical properties were studied via UV thermal denaturation and circular dichroism spectroscopy. These linkers were found to stabilize the duplexes by 37, 31, and 16 degrees C for the two-, four-, and seven-carbon linkers, respectively, relative to a non-cross-linked duplex. Circular dichroism spectra suggested that these lesions induce very little deviation in the global structure relative to the non-cross-linked duplex DNA control. Molecular models show that the two-carbon cross-link spans the distance between the N(3) atoms of the T-T mismatch without perturbing the helix structure, whereas the longer linkers, particularly the seven-carbon linker, tend to push the thymines apart, creating a local distortion. This perturbation may account for the lower thermal stability of the seven-carbon versus two-carbon cross-linked duplex.

A designed molecule with capacity to alkylate DNA bulges has been prepared from readily available starting materials. The spirocyclic template utilized was designed on the basis of established architectures, and equipped with a mustard alkylating group. Preliminary studies confirm alkylation of specific bulged sequences, paving the way for second generation substrates with higher affinity.

A new synthesis of chiral acyclic nucleoside and nucleotide analogues starting from d(-)- or l(+)-riboses was proposed. Antiviral properties of the synthesized compounds towards the pox virus family were evaluated.

Whereas nitrosation of secondary amines produces nitrosamines, amino acids with primary amino groups and glycine ethyl ester were reported to react with nitrite to give unidentified agents that alkylated 4-(p-nitrobenzyl)pyridine to produce purple dyes and be direct mutagens in the Ames test. We report here that treatment of glycine ethyl ester at 37 degrees C with excess nitrite acidified with HCl, followed by ether extraction, gave 30-40% yields of a product identified as ethyl chloro(hydroximino)acetate [ClC(=NOH)COOEt, ECHA] and a 9% yield of ethyl chloroacetate. The ECHA was identical to that synthesized by a known method from ethyl acetoacetate, strongly alkylated nitrobenzylpyridine, and may have arisen by N-nitrosation of glycine ethyl ester to give ethyl diazoacetate, which was C-nitrosated and reacted with chloride to give ECHA. Nitrosation of ethyl diazoacetate also yielded ECHA. Ethyl nitroacetate was not an intermediate as its nitrosation did not produce ECHA. ECHA reacted with aniline to give ethyl (hydroxamino)(phenylimino)acetate [PhN=C(NHOH)CO2Et]. This product was different from ethyl [(phenylamino)carbonyl]carbamate [PhNHC(=O)NHCO2Et], which was synthesized by reacting ethyl isocyanatoformate (OCN.CO2Et) with aniline. ECHA reacted with guanosine to give a derivative, which may have been a guanine-C(=NOH)CO2Et derivative. ECHA showed moderate toxicity and weak but significant mutagenicity without activation in Salmonella typhimurium TA-100 (mean, 1.31 x control value for 12-18 microg/plats) and for V79 mammalian cells (1.5-1.7 x control value for 60-100 microM). In conclusion, gastric nitrosation of glycine derivatives such as peptides with a N-terminal glycine might produce ECHA analogues that alkylate bases of gastric mucosal DNA and thereby initiate gastric cancer.

Two novel DNA-direct alkylating agents, consisting of aniline mustard linked to an angular 3H-pyrrolo[3,2-f]quinoline nucleus, were synthetized and assayed for their in vitro antiproliferative activity. Simple convergent synthesis, consisting of separate preparation of 9-chloro-3H-pyrrolo[3,2-f]quinoline and p-amino-aniline derivatives, and following their linkage by substitution reactions 8a, b and 10, yielded the corresponding diol derivatives 7b and 9. Biological properties were evaluated with respect to cell growth inhibition, ability to form cross-links with DNA, and capacity to give rise to a molecular complex with the macromolecule for 7b, 8b, 9 and 10.

Enzyme prodrug monotherapy takes advantage of the selectivity and specificity of enzymes that are differentially active in the immediate environment of tumor cells. Matrix metalloproteinases-2 and -9 (MMP-2 and -9, respectively) are cell-surface Zn-dependent endoproteases associated with diverse processes throughout tumor formation and progression. These enzymes have demonstrated high ratios of tumor- to nontumor-associated activity and may represent candidates for antitumor prodrug activation. Our MMP targeting strategy was to prepare and evaluate two classes of enzyme prodrugs, peptides and sequence-similar peptidomimetics, and determine which would be substrates for the enzymes and thus suitable for further in vitro and in vivo evaluation. We selected representatives of three structurally and mechanistically distinct classes of compounds for delivery, doxorubicin, several auristatins (novel synthetic members of the dolastatin class of tubulin polymerization inhibitors), and CBI-TMI (a duocarmycin class minor groove binder). The drugs were acylated on available amines with the broadly recognized MMP substrate P3-P1' sequence acetyl L-prolyl-L-leucyl-glycyl-L-leucine, or with a peptidomimetic analogue. From a panel of four peptides and four peptidomimetics, two compounds, both peptides, were found to be substrates, with specific activities in the range of 1-20 nmol min(-1) mg(-1). For MMP-9, complete conversion took place in 4-16 h; proteolysis by MMP-2 was considerably slower. Cleavage occurred, as predicted, at the Gly-Leu bond to liberate a leucyl drug, and no other intermediates or cleavage products were observed. Although the MMP-9 proteolysis products were equipotent with the parent leucyl drugs, the prodrugs were not differentially active against MMP-2 or -9-expressing versus nonexpressing cell lines during a 4 h exposure. Our data can be interpreted in light of the current understanding of the structural and mechanistic factors governing MMP-2 and -9 proteolysis.