Synthesis of 2-Chloro-3-amino indenone derivatives and their evaluation as inhibitors of DNA dealkylation repair.

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.

High-Precision Sulfur Metabolomics Innovated by a New Specific Probe for Trapping Reactive Sulfur Species.

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 ß-(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.

An aza-nucleoside, fragment-like inhibitor of the DNA repair enzyme alkyladenine glycosylase (AAG).

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.

Reactive OFF-ON type alkylating agents for higher-ordered structures of nucleic acids.

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.

Nitrogen Mustards as Alkylating Agents: A Review on Chemistry, Mechanism of Action and Current USFDA Status of Drugs.

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.

Design, synthesis, nuclear localization, and biological activity of a fluorescent duocarmycin analog, HxTfA.

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.

Novel mitochondria-targeted, nitrogen mustard-based DNA alkylation agents with near infrared fluorescence emission.

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.

Design, synthesis, and mode of action studies of a mitomycin tetramer inducing double activations with a single probe.

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.

Synthesis and Performance of a Biomimetic Indicator for Alkylating Agents.

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.

Synthesis and DNA cleavage activity of Bis-3-chloropiperidines as 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.

Design and synthesis of 2-nitroimidazoles with variable alkylating and acylating functionality.

The synthesis of a small series of 2-nitroimidazoles in which the ß-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.

Specific alkylation of human telomere repeat sequences by a tandem-hairpin motif of pyrrole-imidazole polyamides with indole-seco-CBI.

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.

Synthesis and characterization of DNA minor groove binding alkylating agents.

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.

Studies on synthesis and activation mechanism of mitomycin dimers connected by 1,2-dithiolane and diol linkers.

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.

Synthesis of pyrrole-imidazole polyamide seco-1-chloromethyl-5-hydroxy-1,2-dihydro-3h-benz[e]indole conjugates with a vinyl linker recognizing a 7 bp DNA sequence.

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.

α-Amidoalkylating agents from N-acyl-α-amino acids: 1-(N-acylamino)alkyltriphenylphosphonium salts.

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.

Synthesis and mechanistic studies of a mitomycin dimer containing an eight-membered cyclic disulfide.

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.

A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA.

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.

Anticancer active illudins: recent developments of a potent alkylating compound class.

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.

Synthesis of the first spacer containing prodrug of a duocarmycin analogue and determination of its biological activity.

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.