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We describe the design, organic synthesis, and characterization, including X-ray crystallography, of a series of novel analogues of the clinically used antitumor agent temozolomide, together with their in vitro biological evaluation. The work has resulted in the discovery of a new series of anticancer imidazotetrazines that offer the potential to overcome the resistance mounted by tumors against temozolomide. The rationally designed compounds that incorporate a propargyl alkylating moiety and a thiazole ring as isosteric replacement for a carboxamide, are readily synthesized (gram-scale), exhibit defined solid-state structures, and enhanced growth-inhibitory activity against human tumor cell lines, including MGMT-expressing and MMR-deficient lines, molecular features that confer tumor resistance. The cell proliferation data were confirmed by clonogenic cell survival assays, and DNA flow cytometry analysis was undertaken to determine the effects of new analogues on cell cycle progression. Detailed 1 H NMR spectroscopic studies showed that the new agents are stable in solution, and confirmed their mechanism of action. The propargyl and thiazole substituents significantly improve potency and physicochemical, drug metabolism and permeability properties, suggesting that the thiazole 13 should be prioritized for further preclinical evaluation., Competing Interests: Declaration of competing interest The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

It is 40 years since the publication of the patent that announced the imidazotetrazines temozolomide and mitozolomide to the world and 30 since the discovery that they function as prodrugs of alkyldiazonium reactive intermediates. Temozolomide combined with radiation is established as the first-line treatment for glioma but despite the attentions of the inventors and others, further examples of this intriguing ring system have yet to enter the clinic., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The lack of clinical response to the alkylating agent temozolomide (TMZ) in pediatric diffuse midline/intrinsic pontine glioma (DIPG) has been associated with O 6 -methylguanine-DNA-methyltransferase (MGMT) expression and mismatch repair deficiency. Hence, a potent N(3)-propargyl analogue (N3P) was derived, which not only evades MGMT but also remains effective in mismatch repair deficient cells. Due to the poor pharmacokinetic profile of N3P ( t 1/2 < 1 h) and to bypass the blood-brain barrier, we proposed convection enhanced delivery (CED) as a method of administration to decrease dose and systemic toxicity. Moreover, to enhance N3P solubility, stability, and sustained distribution in vivo , either it was incorporated into an apoferritin (AFt) nanocage or its sulfobutyl ether β-cyclodextrin complex was loaded into nanoliposomes (Lip). The resultant AFt-N3P and Lip-N3P nanoparticles (NPs) had hydrodynamic diameters of 14 vs 93 nm, icosahedral vs spherical morphology, negative surface charge (-17 vs -34 mV), and encapsulating ∼630 vs ∼21000 N3P molecules per NP, respectively. Both NPs showed a sustained release profile and instant uptake within 1 h incubation in vitro . In comparison to the naked drug, N3P NPs demonstrated stronger anticancer efficacy against 2D TMZ-resistant DIPG cell cultures [IC 50 = 14.6 (Lip-N3P) vs 32.8 μM (N3P); DIPG-IV) and (IC 50 = 101.8 (AFt-N3P) vs 111.9 μM (N3P); DIPG-VI)]. Likewise, both N3P-NPs significantly ( P < 0.01) inhibited 3D spheroid growth compared to the native N3P in MGMT + DIPG-VI (100 μM) and mismatch repair deficient DIPG-XIX (50 μM) cultures. Interestingly, the potency of TMZ was remarkably enhanced when encapsulated in AFt NPs against DIPG-IV, -VI, and -XIX spheroid cultures. Dynamic PET scans of CED-administered zirconium-89 ( 89 Zr)-labeled AFt-NPs in rats also demonstrated substantial enhancement over free 89 Zr radionuclide in terms of localized distribution kinetics and retention within the brain parenchyma. Overall, both NP formulations of N3P represent promising approaches for treatment of TMZ-resistant DIPG and merit the next phase of preclinical evaluation.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma, which is the most aggressive form of brain tumor. The standard of care for this disease includes surgery, radiotherapy and temozolomide (TMZ) chemotherapy. Poor accumulation of TMZ at the tumor site, tumor resistance to drug, and dose-limiting bone marrow toxicity eventually reduce the success of this treatment. Herein, we have encapsulated >500 drug molecules of TMZ into the biocompatible protein nanocage, apoferritin (AFt), using a "nanoreactor" method (AFt-TMZ). AFt is internalized by transferrin receptor 1-mediated endocytosis and is therefore able to facilitate cancer cell uptake and enhance drug efficacy. Following encapsulation, the protein cage retained its morphological integrity and surface charge; hence, its cellular recognition and uptake are not affected by the presence of this cargo. Additional benefits of AFt include maintenance of TMZ stability at pH 5.5 and drug release under acidic pH conditions, encountered in lysosomal compartments. MTT assays revealed that the encapsulated agents displayed significantly increased antitumor activity in U373V (vector control) and, remarkably, the isogenic U373M (MGMT expressing TMZ-resistant) GBM cell lines, with GI 50 values <1.5 μM for AFt-TMZ, compared to 35 and 376 μM for unencapsulated TMZ against U373V and U373M, respectively. The enhanced potency of AFt-TMZ was further substantiated by clonogenic assays. Potentiated G2/M cell cycle arrest following exposure of cells to AFt-TMZ indicated an enhanced DNA damage burden. Indeed, increased O6 -methylguanine ( O6 -MeG) adducts in cells exposed to AFt-TMZ and subsequent generation of γH2AX foci support the hypothesis that AFt significantly enhances the delivery of TMZ to cancer cells in vitro, overwhelming the direct O6 -MeG repair conferred by MGMT. We have additionally encapsulated >500 molecules of the N3-propargyl imidazotetrazine analog (N3P), developed to combat TMZ resistance, and demonstrated significantly enhanced activity of AFt-N3P against GBM and colorectal carcinoma cell lines. These studies support the use of AFt as a promising nanodelivery system for targeted delivery, lysosomal drug release, and enhanced imidazotetrazine potency for treatment of GBM and wider-spectrum malignancies.

Stomach cancer is the fourth most common cancer worldwide. Identification of novel molecular therapeutic targets and development of novel treatments are critical. Against a panel of gastric carcinoma cell lines, the activity of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) was investigated. Adopting RT-PCR, Western blot and immunohistochemical techniques, we sought to determine molecular pharmacodynamic (PD) markers of sensitivity and investigate arylhydrocarbon (AhR) receptor-mediated signal transduction activation by 5F 203. Potent (IC 50  ≤ 0.09 μmol/L), selective (>250-fold) in vitro antitumour activity was observed in MKN-45 and AGS carcinoma cells. Exposure of MKN-45 cells to 5F 203 triggered cytosolic AhR translocation to nuclei, inducing CYP1A1 (>50-fold) and CYP2W1 (~20-fold) transcription and protein (CYP1A1 and CYP2W1) expression. G2/M arrest and γH2AX expression preceded apoptosis, evidenced by PARP cleavage. In vivo, significant (P < .01) 5F 203 efficacy was observed against MKN-45 and AGS xenografts. In mice-bearing 5F 203-sensitive MKN-45 and 5F 203-insensitive BGC-823 tumours in opposite flanks, CYP1A1, CYP2W1 and γH2AX protein in MKN-45 tumours only following treatment of mice with 5F 203 (5 mg/kg) revealed PD biomarkers of sensitivity. 5F 203 evokes potent, selective antitumour activity in vitro and in vivo in human gastric cancer models. It triggers AhR signal transduction, CYP-catalysed bioactivation to electrophilic species causing lethal DNA double-strand breaks exclusively in sensitive cells. 5F 203 represents a novel therapeutic agent with a mechanism of action distinct from current clinical drugs, exploiting novel molecular targets pertinent to gastric tumourigenesis: AhR, CYP1A1 and CYP2W1. PD markers of 5F 203 sensitivity that could guide patient selection have been identified., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Glioblastoma multiforme is the most common and lethal brain tumour-type. The current standard of care includes Temozolomide (TMZ) chemotherapy. However, inherent and acquired resistance to TMZ thwart successful treatment. The direct repair protein methylguanine DNA methyltransferase (MGMT) removes the cytotoxic O6 -methylguanine ( O 6-MeG) lesion delivered by TMZ and so its expression by tumours confers TMZ-resistance. DNA mismatch repair (MMR) is essential to process O 6-MeG adducts and MMR-deficiency leads to tolerance of lesions, resistance to TMZ and further DNA mutations. In this article, two strategies to overcome TMZ resistance are discussed: (1) synthesis of imidazotetrazine analogues - designed to retain activity in the presence of MGMT or loss of MMR; (2) preparation of imidazotetrazine-nanoparticles to deliver TMZ preferably to the brain and tumour site. Our promising results encourage belief in a future where better prognoses exist for patients diagnosed with this devastating disease., Competing Interests: All authors declared that there are no conflicts of interest., (© The Author(s) 2019.)

Temozolomide (TMZ) is the standard of care chemotherapeutic agent used in the treatment of glioblastoma multiforme. Cytotoxic O6 -methylguaine lesions formed by TMZ are repaired by O6 -methyl-guanine DNA methyltransferase (MGMT), a DNA repair protein that removes alkyl groups located at the O6 -position of guanine. Response to TMZ requires low MGMT expression and functional mismatch repair. Resistance to TMZ conferred by MGMT, and tolerance to O6 -methylguanine lesions conferred by deficient MMR severely limit TMZ clinical applications. Therefore, development of new TMZ derivatives that can overcome TMZ-resistance is urgent. In this study, we investigated the anti-tumor mechanism of action of two novel TMZ analogs: C8-imidazolyl (377) and C8-methylimidazole (465) tetrazines. We found that analogs 377 and 465 display good anticancer activity against MGMT-overexpressing glioma T98G and MMR deficient colorectal carcinoma HCT116 cell lines with IC 50 value of 62.50, 44.23, 33.09, and 25.37 μM, respectively. Analogs induce cell cycle arrest at G2/M, DNA double strand break damage and apoptosis irrespective of MGMT and MMR status. It was established that analog 377, similar to TMZ, is able to ring-open and hydrolyze under physiological conditions, and its intermediate product is more stable than MTIC. Moreover, DNA adducts of 377 with calf thymus DNA were identified: N7 -methylguanine, O6 -methylguanine, N3 -methyladenine, N3 -methylthymine, and N3 -methylcytidine deoxynucleotides. We conclude that C8 analogs of TMZ share a mechanism of action similar to TMZ and are able to methylate DNA generating O6 -methylguanine adducts, but unlike TMZ are able at least in part to thwart MGMT- and MMR-mediated resistance.

The efficacy of temozolomide (TMZ) treatment for cancers is currently limited by inherent or the development of resistance, particularly, but not exclusively, due to the expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) in a significant proportion of tumors. We have found that TMZ analog C8-methyl imidazole tetrazine (PMX 465) displayed good anticancer activity against the colorectal carcinoma HCT116 cells which are MGMT-overexpressing and mismatch repair (MMR)-deficient. In this study, we found that PMX 465 could downregulate the expression of MGMT in HCT116 cells at the protein and mRNA levels. We found that PMX 465 could reduce MGMT expression by increasing the binding of wild-type p53 to the MGMT promoter and reducing the binding of Sp1 to the MGMT promoter., (© 2018 Wiley Periodicals, Inc.)

A series of 3-(benzyl-substituted)-imidazo[5,1- d ]-1,2,3,5-tetrazines ( 13 ) and related derivatives with 3-heteromethyl groups has been synthesised and screened for growth-inhibitory activity in vitro against two pairs of glioma cell lines with temozolomide-sensitive and -resistant phenotypes dependent on the absence/presence of the DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT). In general the compounds had low inhibitory activity with GI 50 values >50 μM against both sets of cell lines. Two silicon-containing derivatives, the TMS-methylimidazotetrazine ( 9 ) and the SEM-analogue ( 10 ), showed interesting differences: compound ( 9 ) had a profile very similar to that of temozolomide with the MGMT+ cell lines being 5 to 10-fold more resistant than MGMT- isogenic partners; the SEM-substituted compound ( 10 ) showed potency across all cell lines irrespective of their MGMT status.

Background: CJM126, 2-(4-aminophenyl) benzothiazole, is a potent inhibitor of human-derived breast carcinoma cell lines. Previous studies have shown that CJM126 elicits concentration-dependent, biphasic growth inhibitory effects against a panel of estrogen receptor-positive and receptor-negative human mammary carcinoma cell lines by a mechanism which has not been fully elucidated., Materials and Methods: In an effort to understand the mechanism(s) of resistance to CJM126, the present study used cDNA microarrays (Clontech Laboratories, Inc.) representing 1,176 human cancer-related genes to analyze expression profile changes of two CJM126-resistant cell lines, MCF-7 10nM126 and MCF-7 10μM126 , previously created by exposing MCF-7 cells to 10 nM and 10 μM CJM126, respectively., Results: Expression changes in the CJM126-resistant MCF-7 cell lines were observed in genes involved in a variety of cell signaling pathways. Gene expression changes common to MCF-7 10nM126 and MCF-7 10μM126 cells, as compared to sensitive MCF-7 wt cells, were the shut-down of transcription factor Oct-2 and the up-regulation of the negative apoptosis regulator MCL-1, the G1-to-S-phase regulator ubiquitin carrier protein and the GTP-binding protein GST1-HS. These findings indicate the association of a CJM126-resistance phenotype with profound gene transcription dysregulation, decreased apoptotic activity and increased proliferation. Specific changes unique to each of the CJM126-resistant cell lines were also observed. Genes involved in the DNA mismatch-repair pathway, such as MSH2, DNA repair protein RAD51 and damage-specific DNA binding protein were down-regulated in MCF-710nM126, while genes involved in the nucleotide-excision repair pathway, such as ERCC1, RFC and PCNA were overexpressed in MCF-7 10μM126 Conclusion: The differential changes in the DNA-repair pathways between MCF-7 10nM126 and MCF-7 10μM126 cell lines indicate that different processes may have been employed to circumvent the growth inhibition produced by exposure to CJM126. This would also suggest that CJM126 may have concentration-dependent mechanisms of growth inhibition., (Copyright© 2004 International Institute of Anticaner Research (Dr. John G. Delinassios), All rights reserved.)