[99mTc]Tc-labeled HYNIC conjugated chlorambucil as a tumor targeting Agent: Synthesis, characterization and ex-vivo evaluation.

Chlorambucil is an alkylating drug that finds application towards chemotherapy of different types of cancers. In order to explore the possibility of utilization of this drug as an imaging agent for early diagnosis of solid tumors, attempt was made to synthesize a 99mTc complex of chlorambucil and evaluate its potential in tumor bearing small animal model. HYNIC-chlorambucil was synthesized by conjugation of HYNIC with chlorambucil via an ethylenediamine linker. All the intermediates and final product were purified and characterized by standard spectroscopic techniques viz. FT-IR, 1H/13C-NMR as well as by mass spectrometry. HYNIC-chlorambucil conjugate was radiolabeled with [99mTc]Tc and found to be formed with > 95 % radiochemical purity via RP-HPLC studies. The partition coefficient (Log10Po/w) of the synthesized complex was found to be -0.78 ± 0.25 which indicated the moderate hydrophilic nature for the complex. Biological behaviour of [99mTc]Tc-HYNIC-chlorambucil, studied in fibrosarcoma bearing Swiss mice, revealed a tumor uptake of about 4.16 ± 1.52 %IA/g at 30 min post-administration, which declined to 1.91 ± 0.13 % IA/g and 1.42 ± 0.14 %IA/g at 1 h and 2 h post-administration, respectively. A comparison of different [99mTc]Tc-chlorambucil derivatives (reported in the contemporary literature) formulated using different methodologies revealed that tumor uptake and pharmacokinetics exhibited by these agents strongly depend on the lipophilicity/hydrophilicity of such agents, which in turn is dependent on the bifunctional chelators used for formulating the radiolabeled chlorambucils.

New Synthetic Analogs of Nitrogen Mustard DNA Interstrand Cross-Links and Their Use to Study Lesion Bypass by DNA Polymerases.

Nitrogen mustards are a widely used class of antitumor agents that exert their cytotoxic effects through the formation of DNA interstrand cross-links (ICLs). Despite being among the first antitumor agents used, the biological responses to NM ICLs remain only partially understood. We have previously reported the generation of NM ICL mimics by incorporation of ICL precursors into DNA using solid-phase synthesis at defined positions, followed by a double reductive amination reaction. However, the structure of these mimics deviated from the native NM ICLs. Using further development of our approach, we report a new class of NM ICL mimics that only differ from their native counterpart by substitution of dG with 7-deaza-dG at the ICL. Importantly, this approach allows for the synthesis of diverse NM ICLs, illustrated here with a mimic of the adduct formed by chlorambucil. We used the newly generated ICLs in reactions with replicative and translesion synthesis DNA polymerase to demonstrate their stability and utility for functional studies. These new NM ICLs will allow for the further characterization of the biological responses to this important class of antitumor agents.

Synthesis and evaluation of new dinitrobenzamide mustards in human prostate cancer.

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.

Targeting the mutant PIK3CA gene by DNA-alkylating pyrrole-imidazole polyamide in cervical cancer.

PIK3CA is the most frequently mutated oncogene in cervical cancer, and somatic mutations in the PIK3CA gene result in increased activity of PI3K. In cervical cancer, the E545K mutation in PIK3CA leads to elevated cell proliferation and reduced apoptosis. In the present study, we designed and synthesized a novel pyrrole-imidazole polyamide-seco-CBI conjugate, P3AE5K, to target the PIK3CA gene bearing the E545K mutation, rendered possible by nuclear access and the unique sequence specificity of pyrrole-imidazole polyamides. P3AE5K interacted with double-stranded DNA of the coding region containing the E545K mutation. When compared with conventional PI3K inhibitors, P3AE5K demonstrated strong cytotoxicity in E545K-positive cervical cancer cells at lower concentrations. PIK3CA mutant cells exposed to P3AE5K exhibited reduced expression levels of PIK3CA mRNA and protein, and subsequent apoptotic cell death. Moreover, P3AE5K significantly decreased the tumor growth in mouse xenograft models derived from PIK3CA mutant cells. Overall, the present data strongly suggest that the alkylating pyrrole-imidazole polyamide P3AE5K should be a promising new drug candidate targeting a constitutively activating mutation of PIK3CA in cervical cancer.

Development of a PAMAM Dendrimer for Sustained Release of Temozolomide against Experimental Murine Lymphoma: Assessment of Therapeutic Efficacy.

Enhanced drug localization at the tumor sites with minimal toxicity was demonstrated using dendrimer-conjugated temozolomide for treating experimental lymphoma, developed as a solid tumor. Herein, we have constructed a polyamidoamine (PAMAM) dendrimer conjugated with temozolomide to enhance the stability of the active drug metabolites, derived from the prodrug temozolomide. Our results suggest that the active drug (5-(3-methyltriazen-1-yl)imidazole-4-carboxamide) (MTIC) (derived from temozolomide) showed stable and sustained release from the dendrimer-temozolomide conjugate, suggesting the suitability of the construct for therapy. Besides growth inhibition and direct killing, the dendrimer-temozolomide construct induced extensive apoptosis not only in parental Dalton lymphoma tumor cells but also in the doxorubicin-resistant form of the tumor cells. Dendrimer-temozolomide conjugation significantly reduced the solid tumor growth and increased the lifespan with better prognosis, including improved histopathology of the treated mice, while untreated littermates developed extensive metastasis and succumbed to death.

Biodegradable reduction-responsive polymeric micelles for enhanced delivery of melphalan to retinoblastoma cells.

Melphalan (MEL) is an effective chemotherapeutic agent for treatment of retinoblastoma (Rb) which is the most common childhood malignancy. However, the inherent cardiopulmonary toxicity and hazardous integration limit its therapeutic effect on RB. N-Acetylheparosan (AH), a natural heparin-like polysaccharide in mammals with long circulation effect and good biocompatibility, was linked by d-α-tocopherol acid succinate (VES) via and cystamine (CYS) to synthesize reduction-responsive N-acetylheparosan-CYS-Vitamin E succinate (AHV) copolymers. In addition, CYS was replaced by adipic acid dihydrazide (ADH) to obtain a control of non-reduction-responsive polymers N-acetylheparosan-ADH-Vitamin E succinate (ADV). MEL-loaded AHV micelles (MEL/AHV) as well as ADV micelles (MEL/ADV) were prepared with small particle size and high drug loading content. In vitro drug release showed that MEL/AHV micelles presented obvious reduction-triggered release behavior compared with MEL/ADV. In vitro antitumor effects were investigated using WERI-Rb-1 retinoblastoma cells. Cytotoxicity experiments showed that the IC50 of MEL/AHV was significantly lower than that of free MEL and MEL/ADV, suggesting that MEL/AHV enhanced the cytotoxicity against retinoblastoma cells. Furthermore, MEL/AHV micelles were more easily uptaken by multiple pathways compared with MEL/ADV and free MEL. Therefore, MEL/AHV might be a potential delivery system for enhanced delivery of melphalan to Rb cells.

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.

Self-assembled angiopep-2 modified lipid-poly (hypoxic radiosensitized polyprodrug) nanoparticles delivery TMZ for glioma synergistic TMZ and RT therapy.

The addition of temozolomide (TMZ) to radiotherapy (RT) improves survival of patients with glioblastoma (GBM). However, TMZ + RT causes excess toxicity in patients. In this study, we prepared angiopep-2 (A2) modified lipid-poly (hypoxic radiosensitized polyprodrug) nanoparticles for TMZ delivery (A2-P(MIs)25/TMZ) to achieve synergistic effects against glioma. This A2-P(MIs)25/TMZ display highly promising advantages: (1) a hydrophobic P-(MIs)25 core where poorly water-soluble TMZ can be encapsulated; (2) nitro groups of the hydrophobic P-(MIs)25 core that are converted into hydrophilic amino groups (P(NH2s)25) under low oxygen conditions to mimic the oxygen-increased sensitization to RT; (3) a lipid monolayer at the interface of the core and the shell to modify the A2 (a specific ligand for low-density lipoprotein receptor-related protein-1 (LRP-1), which are expressed in the blood-brain barrier (BBB) and human glioma cells), thereby enhancing the drug encapsulation efficiency in glioma. These nanoparticles appear as a promising and robust nanoplatforms for TMZ and hypoxic cell radiosensitization delivery.

N-7-Guanine Adduct of the Active Monoepoxide of Prodrug Treosulfan: First Synthesis, Characterization, and Decomposition Profile Under Physiological Conditions.

(2S,3S)-1,2:3,4-diepoxybutane (DEB) cross-links DNA guanines by forming the intermediate epoxy-adduct ((2'S,3'S)-N-7-(3',4'-epoxy-2'-hydroxybut-1'-yl)guanine [EHBG]). This process is presently considered a primary mechanism for the action of treosulfan (TREO), the prodrug that transforms to DEB via the monoepoxide intermediate (2S,3S)-1,2-epoxybutane-3,4-diol 4-methanesulfonate (EBDM). In this article, the N-7-guanine adduct of EBDM ((2'S,3'S)-N-7-(2'3'-dihydroxy-4'-methylsulfonyloxybut-1'-yl)guanine [HMSBG]) was synthesized for the first time, and its stability was investigated at physiological in vitro conditions. To synthesize HMSBG, EBDM, formed in-situ from TREO, was treated with guanosine in glacial acetic acid at 60°C followed by ribose cleavage in 1 M HCl at 80°C. HMSBG was stable during the synthesis, which showed that a ß-hydroxy group protects the sulfonate moiety against hydrolysis in acid environment. At pH 7.2 and 37°C, HMSBG exclusively underwent first-order epoxidation to EHBG with a half-life of 5.0 h. EHBG further decomposed to trihydroxybutyl-guanine, chlorodihydroxybutyl-guanine (major products), phosphodihydroxy-guanine, and a structural isomer (minor products). The isomeric derivative was identified as guanine with a fused 7-membered ring, which provided a new insight into the EHBG stability. To conclude, the exclusive conversion of HMSBG to EHBG indicates that EBDM might contribute to DNA cross-linking independently from DEB and play a more important role in the TREO action than expected before.

Box-Behnken Design of Experiment Assisted Development and Optimization of Bendamustine HCl loaded Hydroxyapatite Nanoparticles.

BACKGROUND: Bendamustine HCl, an antineoplastic drug, has a very short life of about 40 minutes which necessitates administration of large doses which leads to increased side effects as well as costs. OBJECTIVE: The present work describes the fabrication, optimization, and evaluation of bioactive hydroxyapatite nanoparticles to achieve sustained delivery of bendamustine HCl. METHODS: Hydroxyapatite nanoparticles (NPs) were prepared by the wet chemical precipitation method by reacting a calcium and phosphate precursor and the reaction was optimized via Box-Behnken DOE. The drug was loaded on particles by physical adsorption. Various analytical studies were performed on the fabricated nanoparticles in addition to biodistribution studies to establish the physicochemical and biological characteristics of the designed formulation. RESULTS: pH of the reactant solution was found to have a more profound effect on the particle size and size distribution in comparison to reactant concentration. The particles were found to have a spherical morphology by SEM. Size of the blank and drug-loaded nanoparticles was found to be 130±20 nm by TEM. Energy Dispersive X-ray Spectroscopy (EDS) studies confirmed the presence of hydroxyapatite as the dominant phase while DSC studies indicated the presence of the drug in its amorphous form after its adsorption on NPs. Tissue distribution studies further suggested that the majority of drug concentration was released in blood rather than the other organs implying low organ toxicity. CONCLUSION: Bendamustine loaded hydroxyapatite nanoparticles were successfully optimized and fabricated. Favorable results were obtained in in vitro, in vivo, and analytical studies.

Aryl sulfonate based anticancer alkylating agents.

This research work revolves around synthesis of antineoplastic alkylating sulfonate esters with dual alkylating sites for crosslinking of the DNA strands. These molecules were evaluated as potential antineoplastic cross linking alkylating agents by reaction with the nucleoside of Guanine DNA nucleobase at both ends of the synthesized molecule. Synthesis of the alkylating molecules and the crosslinking with the guanosine nucleoside was monitored by MALDITOF mass spectroscopy. The synthesized molecule's crosslinking or adduct forming rate with the nucleoside was compared with that of 1,4 butane disulfonate (busulfan), in form of time taken for the appearance of [M+H]+. It was found that aryl sulfonate leaving group was causing higher rate of nucleophilic attack by the Lewis basic site of the nucleobase. Furthermore, the rate was also found to be a function of electron withdrawing or donating nature of the substituent on the aryl ring. Compound with strong electron withdrawing substituent on the para position of the ring reacted fastest. Hence, new alkylating agents were synthesized with optimized or desired reactivity.

Oxidative activation of leinamycin E1 triggers alkylation of guanine residues in double-stranded DNA.

It may be useful to develop prodrugs that are selectively activated by oxidative stress in cancer cells to release cell-killing reactive intermediates. However, relatively few chemical strategies exist for the activation of prodrugs under conditions of oxidative stress. Here we provide evidence for a novel process in which oxidation of a thiol residue in the natural product leinamycin E1 by H2O2 and other byproducts of cellular oxidative stress initiates generation of an episulfonium ion that selectively alkylates guanine residues in duplex DNA.

Glycoconjugated Site-Selective DNA-Methylating Agent Targeting Glucose Transporters on Glioma Cells.

DNA-alkylating drugs continue to remain an important weapon in the arsenal against cancers. However, they typically suffer from several shortcomings because of the indiscriminate DNA damage that they cause and their inability to specifically target cancer cells. We have developed a strategy for overcoming the deficiencies in current DNA-alkylating chemotherapy drugs by designing a site-specific DNA-methylating agent that can target cancer cells because of its selective uptake via glucose transporters, which are overexpressed in most cancers. The design features of the molecule, its synthesis, its reactivity with DNA, and its toxicity in human glioblastoma cells are reported here. In this molecule, a glucosamine unit, which can facilitate uptake via glucose transporters, is conjugated to one end of a bispyrrole triamide unit, which is known to bind to the minor groove of DNA at A/T-rich regions. A methyl sulfonate moiety is tethered to the other end of the bispyrrole unit to serve as a DNA-methylating agent. This molecule produces exclusively N3-methyladenine adducts upon reaction with DNA and is an order of magnitude more toxic to treatment resistant human glioblastoma cells than streptozotocin is, a Food and Drug Administration-approved, glycoconjugated DNA-methylating drug. Cellular uptake studies using a fluorescent analogue of our molecule provide evidence of uptake via glucose transporters and localization within the nucleus of cells. These results demonstrate the feasibility of our strategy for developing more potent anticancer chemotherapeutics, while minimizing common side effects resulting from off-target damage.

Targeting Gliomas: Can a New Alkylating Hybrid Compound Make a Difference?

Glioblastoma (GBM) is the most common and aggressive type of brain tumor in adults. The triazene Temozolomide (TMZ), an alkylating drug, is the classical chemotherapeutic agent for gliomas, but has been disappointing against the highly invasive and resistant nature of GBM. Hybrid compounds may open new horizons within this challenge. The multicomponent therapeutic strategy here used resides on a combination of two repurposing drugs acting by different but potentially synergistic mechanisms, improved efficacy, and lower resistance effects. We synthesized a new hybrid compound (HYBCOM) by covalently binding a TMZ analogue to valproic acid, a histone deacetylase inhibitor drug that was shown to sensitize TMZ-resistant glioma cells. Advantages of this new molecule as compared to TMZ, in terms of chemotherapeutic efficacy, were investigated. Our results evidenced that HYBCOM more efficiently decreased the viability and proliferation of the GL261 glioma cells, while showing to better target the tumor cells than the functionally normal astrocytes. Increased cytotoxicity by HYBCOM may be a consequence of the improved autophagic process observed. Additionally, HYBCOM changed the morphology of GL261 cells into a nonpolar, more rounded shape, impairing cell migration ability. Most interesting, and in opposite to TMZ, cells exposed to HYBCOM did not enhance the expression of drug resistance proteins, a major issue in the treatment of GBM. Overall, our studies indicate that HYBCOM has promising chemotherapeutic benefits over the classical TMZ, and future studies should assess if the treatment translates into efficacy in glioblastoma experimental models and reveal clinical benefits in GBM patients.

Effect of O6-Substituted Guanine Analogs on O6-methylguanine DNA-methyltransferase Expression and Glioblastoma Cells Viability.

BACKGROUND: Glioblastoma multiforme (GBM) is often associated with a poor survival prognostic for patients. The main reason seems to be the acquired or inherent resistance to the chemotherapeutic agent used to treat the tumor, temozolomide (TMZ). To this day, the most recognized pathway of resistance is the DNA Direct Repair pathway by the means of the protein O6- methylguanine DNA-methyltransferase (MGMT). OBJECTIVES: To design and synthesize a series of MGMT inhibitors that can sensitize GBM cells to TMZ. METHODS: Twenty-five O6-alkyl, O6-aryl and O6-substituted-aryl guanine analogs including nine novel compounds were synthesized, characterized, analyzed by molecular docking and tested on the T98G GBM cells viability. RESULTS: Following molecular modeling with MGMT, the newly designed compounds 19, 22, and 24 emerged as the most promising MGMT ligands and displayed modest cytotoxicity. Guanine analog (19), bearing a p-nitrobenzyl moiety, reduced considerably the O6-methylguanine DNAmethyltransferase expression level. When combined with TMZ (1), which is used as first line treatment for brain tumors, compounds 19, 22, and 24 decreased T98G cells proliferation by 32%, 68% and 50%, respectively. TMZ (1) displayed negligible effect on the proliferation of these cells further supporting the notion that this cell model is resistant to this alkylating agent. CONCLUSION: Overall, these results notably highlight a group of MGMT inhibitors that warrants further exploration in the development of therapeutic options to circumvent TMZ resistance in brain tumors.

The p53 Pathway: Origins, Inactivation in Cancer, and Emerging Therapeutic Approaches.

Inactivation of the transcription factor p53, through either direct mutation or aberrations in one of its many regulatory pathways, is a hallmark of virtually every tumor. In recent years, screening for p53 activators and a better understanding of the molecular mechanisms of oncogenic perturbations of p53 function have opened up a host of novel avenues for therapeutic intervention in cancer: from the structure-guided design of chemical chaperones to restore the function of conformationally unstable p53 cancer mutants, to the development of potent antagonists of the negative regulators MDM2 and MDMX and other modulators of the p53 pathway for the treatment of cancers with wild-type p53. Some of these compounds have now moved from proof-of-concept studies into clinical trials, with prospects for further, personalized anticancer medicines. We trace the structural evolution of the p53 pathway, from germ-line surveillance in simple multicellular organisms to its pluripotential role in humans.

The Alkylating-HDAC Inhibition Fusion Principle: Taking Chemotherapy to the Next Level with the First in Class Molecule EDO-S101.

Chemotherapy may still be an essential component to treat cancer in combination with new targeted therapies. But chemotherapy needs to get smarter in order to make those combination regimens more effective and also more tolerable, particularly for an aging population. We describe the first time the synthesis and pharmacological testing of a fusion molecule comprising of the alkylator bendamustine and the HDAC-inhibitor vorinostat. The drug was designed to allow for the exploitation of both mechanisms of action simultaneously with the goal to provide a molecule with superior efficacy over the single agents. The pharmacological testing confirms the full functional capacity of both moieties and encouraging pharmacological data raises the hope that the drug may turn out to be a great addition to the armentarium of anticancer agents.

Synthetic Development of New 3-(4-Arylmethylamino)butyl-5-arylidene-rhodanines under Microwave Irradiation and Their Effects on Tumor Cell Lines and against Protein Kinases.

A new route to 3-(4-arylmethylamino)butyl-5-arylidene-2-thioxo-1,3-thiazolidine-4-one 9 was developed in six steps from commercial 1,4-diaminobutane 1 as starting material. The key step of this multi-step synthesis involved a solution phase "one-pot two-steps" approach assisted by microwave dielectric from N-(arylmethyl)butane-1,4-diamine hydrochloride 6a-f (as source of the first point diversity) and commercial bis-(carboxymethyl)-trithiocarbonate reagent 7 for construction of the rhodanine platform. This platform was immediately functionalized by Knoevenagel condensation under microwave irradiation with a series of aromatic aldehydes 3 as second point of diversity. These new compounds were prepared in moderate to good yields and the fourteen synthetic products 9a-n have been obtained with a Z-geometry about their exocyclic double bond. These new 5-arylidene rhodanines derivatives 9a-n were tested for their kinase inhibitory potencies against four protein kinases: Human cyclin-dependent kinase 5-p25, HsCDK5-p25; porcine Glycogen Synthase Kinase-3, GSK-3α/ß; porcine Casein Kinase 1, SsCK1 and human HsHaspin. They have also been evaluated for their in vitro inhibition of cell proliferation (HuH7 D12, Caco 2, MDA-MB 231, HCT 116, PC3, NCI-H727, HaCat and fibroblasts). Among of all these compounds, 9j presented selective micromolar inhibition activity on SsCK1 and 9i exhibited antitumor activities in the HuH7 D12, MDA-MBD231 cell lines.

Initial development of a cytotoxic amino-seco-CBI warhead for delivery by prodrug systems.

Cyclopropabenzaindoles (CBIs) are exquisitely potent cytotoxins which bind and alkylate in the minor groove of DNA. They are not selective for cancer cells, so prodrugs are required. CBIs can be formed at physiological pH by Winstein cyclisation of 1-chloromethyl-3-substituted-5-hydroxy-2,3-dihydrobenzo[e]indoles (5-OH-seco-CBIs). Corresponding 5-NH2-seco-CBIs should also undergo Winstein cyclisation similarly. A key triply orthogonally protected intermediate on the route to 5-NH2-seco-CBIs has been synthesised, via selective monotrifluoroacetylation of naphthalene-1,3-diamine, Boc protection, electrophilic iodination, selective allylation at the trifluoroacetamide and 5-exo radical ring-closure with TEMPO. This intermediate has potential for introduction of peptide prodrug masking units (deactivating the Winstein cyclisation and cytotoxicity), addition of diverse indole-amide side-chains (enhancing non-covalent binding prior to alkylation) and use of different leaving groups (replacing the usual chlorine, allowing tuning of the rate of Winstein cyclisation). This key intermediate was elaborated into a simple model 5-NH2-seco-CBI with a dimethylaminoethoxyindole side-chain. Conversion to a bio-reactive entity and the bioactivity of this system were confirmed through DNA-melting studies (ΔTm=13°C) and cytotoxicity against LNCaP human prostate cancer cells (IC50=18nM).

Evaluation of novel imidazotetrazine analogues designed to overcome temozolomide resistance and glioblastoma regrowth.

The cellular responses to two new temozolomide (TMZ) analogues, DP68 and DP86, acting against glioblastoma multiforme (GBM) cell lines and primary culture models are reported. Dose-response analysis of cultured GBM cells revealed that DP68 is more potent than DP86 and TMZ and that DP68 was effective even in cell lines resistant to TMZ. On the basis of a serial neurosphere assay, DP68 inhibits repopulation of these cultures at low concentrations. The efficacy of these compounds was independent of MGMT and MMR functions. DP68-induced interstrand DNA cross-links were demonstrated with H2O2-treated cells. Furthermore, DP68 induced a distinct cell-cycle arrest with accumulation of cells in S phase that is not observed for TMZ. Consistent with this biologic response, DP68 induces a strong DNA damage response, including phosphorylation of ATM, Chk1 and Chk2 kinases, KAP1, and histone variant H2AX. Suppression of FANCD2 expression or ATR expression/kinase activity enhanced antiglioblastoma effects of DP68. Initial pharmacokinetic analysis revealed rapid elimination of these drugs from serum. Collectively, these data demonstrate that DP68 is a novel and potent antiglioblastoma compound that circumvents TMZ resistance, likely as a result of its independence from MGMT and mismatch repair and its capacity to cross-link strands of DNA.