Synthesis, antiproliferative and antiplasmodial evaluation of new chloroquine and mefloquine-based harmiquins.

Here we present the synthesis and evaluation of the biological activity of new hybrid compounds, ureido-type (UT) harmiquins, based on chloroquine (CQ) or mefloquine (MQ) scaffolds and ß-carboline alkaloid harmine against cancer cell lines and Plasmodium falciparum. The hybrids were prepared from the corresponding amines by 1,1'-carbonyldiimidazole (CDI)-mediated synthesis. In vitro evaluation of the biological activity of the title compounds revealed two hit compounds. Testing of the antiproliferative activity of the new UT harmiquins, and previously prepared triazole-(TT) and amide-type (AT) CQ-based harmiquins, against a panel of human cell lines, revealed TT harmiquine 16 as the most promising compound, as it showed pronounced and selective activity against the tumor cell line HepG2 (IC 50 = 5.48 ± 3.35 µmol L-1). Screening of the antiplasmodial activities of UT harmiquins against erythrocytic stages of the Plasmodium life cycle identified CQ-based UT harmiquine 12 as a novel antiplasmodial hit because it displayed low IC 50 values in the submicromolar range against CQ-sensitive and resistant strains (IC 50 0.06 ± 0.01, and 0.19 ± 0.02 µmol L-1, respectively), and exhibited high selectivity against Plasmodium, compared to mammalian cells (SI = 92).

Design, synthesis and antiplasmodial evaluation of new amide-, carbamate-, and ureido-type harmicines.

Malaria, one of the oldest parasitic diseases, remains a global health threat, and the increasing resistance of the malaria parasite to current antimalarials is forcing the discovery of new, effective drugs. Harmicines, hybrid compounds in which harmine/ß-carboline alkaloids and cinnamic acid derivatives are linked via an amide bond or a triazole ring, represent new antiplasmodial agents. In this work, we used a multiple linear regression technique to build a linear quantitative structure-activity relationship (QSAR) model, based on a group of 40 previously prepared amide-type (AT) harmicines and their antiplasmodial activities against erythrocytic stage of chloroquine-sensitive strain of P. falciparum (Pf3D7). After analysing the QSAR model, new harmicines were designed and synthesized: six amide-type, eleven carbamate-type and two ureido-type harmicines at the N-9 position of the ß-carboline core. Subsequently, we evaluated the antiplasmodial activity of the new harmicines against the erythrocytic and hepatic stages of the Plasmodium life cycle in vitro and their antiproliferative activity against HepG2 cells. UT harmicine (E)-1-(2-(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)ethyl)-3-(3-(3-(trifluoromethyl)phenyl)allyl)urea at the N-9 position of the ß-carboline ring exhibited pronounced antiplasmodial activity against both the erythrocytic and the hepatic stages of the Plasmodium life cycle, accompanied by good selectivity towards Plasmodium.

Synthesis and Biological Evaluation of New Quinoline and Anthranilic Acid Derivatives as Potential Quorum Sensing Inhibitors.

Inhibiting quorum sensing (QS), a central communication system, is a promising strategy to combat bacterial pathogens without antibiotics. Here, we designed novel hybrid compounds targeting the PQS (Pseudomonas quinolone signal)-dependent quorum sensing (QS) of Pseudomonas aeruginosa that is one of the multidrug-resistant and highly virulent pathogens with urgent need of new antibacterial strategies. We synthesized 12 compounds using standard procedures to combine halogen-substituted anthranilic acids with 4-(2-aminoethyl/4-aminobuthyl)amino-7-chloroquinoline, linked via 1,3,4-oxadiazole. Their antibiofilm activities were first pre-screened using Gram-negative Chromobacterium violaceum-based reporter, which identified compounds 15-19 and 23 with the highest anti-QS and minimal bactericidal effects in a single experiment. These five compounds were then evaluated against P. aeruginosa PAO1 to assess their ability to prevent biofilm formation, eradicate pre-formed biofilms, and inhibit virulence using pyocyanin as a representative marker. Compound 15 displayed the most potent antibiofilm effect, reducing biofilm formation by nearly 50% and pre-formed biofilm masses by 25%. On the other hand, compound 23 exhibited the most significant antivirulence effect, reducing pyocyanin synthesis by over 70%. Thus, our study highlights the potential of 1,3,4-oxadiazoles 15 and 23 as promising scaffolds to combat P. aeruginosa. Additionally, interactive QS systems should be considered to achieve maximal anti-QS activity against this clinically relevant species.

Harmicens, Novel Harmine and Ferrocene Hybrids: Design, Synthesis and Biological Activity.

Cancer and malaria are both global health threats. Due to the increase in the resistance to the known drugs, research on new active substances is a priority. Here, we present the design, synthesis, and evaluation of the biological activity of harmicens, hybrids composed of covalently bound harmine/ß-carboline and ferrocene scaffolds. Structural diversity was achieved by varying the type and length of the linker between the ß-carboline ring and ferrocene, as well as its position on the ß-carboline ring. Triazole-type harmicens were prepared using Cu(I)-catalyzed azide-alkyne cycloaddition, while the synthesis of amide-type harmicens was carried out by applying a standard coupling reaction. The results of in vitro biological assays showed that the harmicens exerted moderate antiplasmodial activity against the erythrocytic stage of P. falciparum (IC50 in submicromolar and low micromolar range) and significant and selective antiproliferative activity against the MCF-7 and HCT116 cell lines (IC50 in the single-digit micromolar range, SI > 5.9). Cell localization experiments showed different localizations of nonselective harmicene 36 and HCT116-selective compound 28, which clearly entered the nucleus. A cell cycle analysis revealed that selective harmicene 28 had already induced G1 cell cycle arrest after 24 h, followed by G2/M arrest with a concomitant drastic reduction in the percentage of cells in the S phase, whereas the effect of nonselective compound 36 on the cell cycle was much less pronounced, which agreed with their different localizations within the cell.

Design and synthesis of harmiquins, harmine and chloroquine hybrids as potent antiplasmodial agents.

Malaria remains one of the major health problems worldwide. The lack of an effective vaccine and the increasing resistance of Plasmodium to the approved antimalarial drugs demands the development of novel antiplasmodial agents that can effectively prevent and/or treat this disease. Harmiquins represent hybrids that combine two moieties with different mechanisms of antiplasmodial activity in one molecule, i.e., a chloroquine (CQ) scaffold, known to inhibit heme polymerization and a ß-carboline ring capable of binding to P. falciparum heat shock protein 90 (PfHsp90). Here we present their synthesis, evaluation of biological activity and potential mechanism of action. The synthesized hybrids differed in the type of linker employed (triazole ring or amide bond) and in the position of the substitution on the ß-carboline core of harmine. The antiplasmodial activity of harmiquins was evaluated against the erythrocytic stage of the Plasmodium life cycle, and their cytotoxic effect was tested on HepG2 cells. The results showed that harmiquins exerted remarkable activity against both CQ-sensitive (Pf3D7) and CQ-resistant (PfDd2, PfK1, and Pf7G8). P. falciparum strains. The most active compound, harmiquine 32, displayed single-digit nanomolar IC50 value against Pf3D7 (IC50 = 2.0 ± 0.3 nM). Importantly, it also showed significantly higher activity than CQ against the resistant Plasmodium strains and had a very high selectivity index (4450). Harmiquins may act through the inhibition of heme polymerization and binding to the ATP binding site of the PfHsp90, which would explain their increased activity against the CQ-resistant Plasmodium strains. These results establish harmiquins as valuable antiplasmodial hits for future optimization.

Synthesis and Biological Evaluation of Harmirins, Novel Harmine-Coumarin Hybrids as Potential Anticancer Agents.

As cancer remains one of the major health burdens worldwide, novel agents, due to the development of resistance, are needed. In this work, we designed and synthesized harmirins, which are hybrid compounds comprising harmine and coumarin scaffolds, evaluated their antiproliferative activity, and conducted cell localization and cell cycle analysis experiments. Harmirins were prepared from the corresponding alkynes and azides under mild reaction conditions using Cu(I) catalyzed azide-alkyne cycloaddition, leading to the formation of the 1H-1,2,3-triazole ring. Antiproliferative activity of harmirins was evaluated in vitro against four human cancer cell lines (MCF-7, HCT116, SW620, and HepG2) and one human non-cancer cell line (HEK293T). The most pronounced activities were exerted against MCF-7 and HCT116 cell lines (IC50 in the single-digit micromolar range), while the most selective harmirins were 5b and 12b, substituted at C-3 and O-7 of the ß-carboline core and bearing methyl substituent at position 6 of the coumarin ring (SIs > 7.2). Further experiments demonstrated that harmirin 12b is localized exclusively in the cytoplasm. In addition, it induced a strong G1 arrest and reduced the percentage of cells in the S phase, suggesting that it might exert its antiproliferative activity through inhibition of DNA synthesis, rather than DNA damage. In conclusion, harmirin 12b is a novel harmine and coumarin hybrid with significant antiproliferative activity and warrants further evaluation as a potential anticancer agent.

Further investigation of harmicines as novel antiplasmodial agents: Synthesis, structure-activity relationship and insight into the mechanism of action.

The rise of the resistance of the malaria parasite to the currently approved therapy urges the discovery and development of new efficient agents. Previously we have demonstrated that harmicines, hybrid compounds composed from ß-carboline alkaloid harmine and cinnamic acid derivatives, linked via either triazole or amide bond, exert significant antiplasmodial activity. In this paper, we report synthesis, antiplasmodial activity and cytotoxicity of expanded series of novel triazole- and amide-type harmicines. Structure-activity relationship analysis revealed that amide-type harmicines 27, prepared at N-9 of the ß-carboline core, exhibit superior potency against both erythrocytic stage of P. falciparum and hepatic stages of P. berghei. Notably, harmicine 27a, m-(trifluoromethyl)cinnamic acid derivative, exhibited the most favourable selectivity index (SI = 1105). Molecular dynamics simulations revealed the ATP binding site of P. falciparum heat shock protein 90 as a druggable binding location, confirmed the usefulness of the harmine's N-9 substitution and identified favourable N-H … π interactions involving Lys45 and the aromatic phenyl unit in the attached cinnamic acid fragment as crucial for the enhanced biological activity. Thus, those compounds were identified as promising and valuable leads for further derivatization in the search of novel, more efficient antiplasmodial agents.

Novel Harmicines with Improved Potency against Plasmodium.

Harmicines represent hybrid compounds composed of ß-carboline alkaloid harmine and cinnamic acid derivatives (CADs). In this paper we report the synthesis of amide-type harmicines and the evaluation of their biological activity. N-harmicines 5a-f and O-harmicines 6a-h were prepared by a straightforward synthetic procedure, from harmine-based amines and CADs using standard coupling conditions, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA). Amide-type harmicines exerted remarkable activity against the erythrocytic stage of P. falciparum, in low submicromolar concentrations, which was significantly more pronounced compared to their antiplasmodial activity against the hepatic stages of P. berghei. Furthermore, a cytotoxicity assay against the human liver hepatocellular carcinoma cell line (HepG2) revealed favorable selectivity indices of the most active harmicines. Molecular dynamics simulations demonstrated the binding of ligands within the ATP binding site of PfHsp90, while the calculated binding free energies confirmed higher activity of N-harmicines 5 over their O-substituted analogues 6. Amino acids predominantly affecting the binding were identified, which provided guidelines for the further derivatization of the harmine framework towards more efficient agents.

Harmicines - harmine and cinnamic acid hybrids as novel antiplasmodial hits.

Harmicines constitute novel hybrid compounds that combine two agents with reported antiplasmodial properties, namely ß-carboline harmine and a cinnamic acid derivative (CAD). Cu(I) catalyzed azide-alkyne cycloaddition was employed for the preparation of three classes of hybrid molecules: N-harmicines 6a-i, O-harmicines 7a-i and N,O-bis-harmicines 8a-g,i. In vitro antiplasmodial activities of harmicines against the erythrocytic stage of Plasmodium falciparum (chloroquine-sensitive Pf3D7 and chloroquine-resistant PfDd2 strains) and hepatic stage of P. berghei, as well as cytotoxicity against human liver hepatocellular carcinoma cell line (HepG2), were evaluated. Remarkably, most of the compounds exerted significant activities against both stages of the Plasmodium life cycle. The conjugation of various CADs to harmine resulted in the increased antiplasmodial activity relative to harmine. In general, O-harmicines 7 exhibited the highest activity against the erythrocytic stage of both P. falciparum strains, whereas N,O-bis harmicines 8 showed the most pronounced activity against P. berghei hepatic stages. For the latter compound, molecular dynamics simulations confirmed binding within the ATP binding site of PfHsp90, while the weaker binders, namely 6b and harmine, were found to be positioned away from this structural element. In addition, decomposition of the computed binding free energies into contributions from individual residues suggested guidelines for further derivatization of harmine towards more efficient compounds. Cytotoxicity screening revealed N-harmicines 6 as the least, and O-harmicines 7 as the most toxic compounds. Harmicines 6g, 8b and 6d exerted the most selective action towards Plasmodium over human cells, respectively. These results establish harmicines as hits for future optimisation and development of novel antiplasmodial agents.

Antiproliferative evaluation of various aminoquinoline derivatives.

Four classes of aminoquinoline derivatives were prepared: primaquine ureas 1a-f, primaquine bis-ureas 2a-f, chloroquine fumardiamides 3a-f and mefloquine fumardiamides 4a-f. Their antiproliferative activities against breast adeno-carcinoma (MCF-7), lung carcinoma (H460) and colon carcinoma (HCT 116 and SW620) cell lines were evaluated in vitro, using MTT cell proliferation assay. The results revealed a low activity of primaquine urea and bis-urea derivatives and high activity of all fumardiamides, with IC50 values in low micromolar range against all tested cancer cell lines.

Primaquine derivatives: Modifications of the terminal amino group.

Numerous modifications of the well-known antimalarial drug primaquine, both at the quinoline ring and at the primary amino group, have been reported, mostly to obtain antimalarial agents with improved bioavailability, reduced toxicity and/or prolonged activity. Modifications of the terminal amino group were made with the main idea to prevent the metabolic pathway leading to inactive and toxic carboxyprimaquine (follow-on strategy), but also to get compounds with different activity (repurposing strategy). The modifications undertaken until 2009 were included in a review published in the same year. The present review covers various classes of primaquine N-derivatives with diverse biological profiles, prepared in the last decade by our research group as well as the others. We have summarized the synthetic procedures applied for their preparation and discussed the main biological results. Several hits for the development of novel antiplasmodial, anticancer, antimycobacterial and antibiofilm agents were identified.

Primaquine and Chloroquine Fumardiamides as Promising Antiplasmodial Agents.

This paper describes a continuation of our efforts in the pursuit of novel antiplasmodial agents with optimized properties. Following our previous discovery of biologically potent asymmetric primaquine (PQ) and halogenaniline fumardiamides (1-6), we now report their significant in vitro activity against the hepatic stages of Plasmodium parasites. Furthermore, we successfully prepared chloroquine (CQ) analogue derivatives (11-16) and evaluated their activity against both the hepatic and erythrocytic stages of Plasmodium. Our results have shown that PQ fumardiamides (1-6) exert both higher activity against P. berghei hepatic stages and lower toxicity against human hepatoma cells than the parent drug and CQ derivatives (11-16). The favourable cytotoxicity profile of the most active compounds, 5 and 6, was corroborated by assays performed on human cells (human breast adenocarcinoma (MCF-7) and non-tumour embryonic kidney cells (HEK293T)), even when glucose-6-phosphate dehydrogenase (G6PD) was inhibited. The activity of CQ fumardiamides on P. falciparum erythrocytic stages was higher than that of PQ derivatives, comparable to CQ against CQ-resistant strain PfDd2, but lower than CQ when tested on the CQ-sensitive strain Pf3D7. In addition, both sets of compounds showed favourable drug-like properties. Hence, quinoline fumardiamides could serve as a starting point towards the development of safer and more effective antiplasmodial agents.

Primaquine homodimers as potential antiplasmodial and anticancer agents.

Primaquine homodimers, e.g. symmetric PQ-diamides of dicarboxylic acids containing 4 to 8 carbon atoms, were evaluated against Plasmodium berghei hepatic stages and P. falciparum blood stages, as well as against three cancer cell lines. Novel PQ-homodimers exerted much higher activity against hepatic stages, but less pronounced activity against blood stages in comparison to the parent drug. The submicromolar activity of succinic, fumaric and maleic derivatives against P. berghei was determined (IC50 values: 726.2, 198.1 and 358.4 nM, respectively). Our results indicated that the length and type of spacer between two PQ moieties highly modified the antiproliferative activities of PQ-homodimers. The general antiproliferative activity of the adipic and mesaconic derivatives against three cancer cell lines (MCF-7, HCT116, H 460) was observed (GI50 = 1.78-13.7 and 2.36-4.31 µM, respectively), but adipic derivative was less toxic to human embryonic kidney cells (HEK 293). High selectivity of fumaric and suberic derivatives against breast adenocarcinoma cell line MCF-7 was detected. These two compounds have shown no antiproliferative activity against other tumor cells and HEK 293.

Synthesis and antiplasmodial evaluation of novel mefloquine-based fumardiamides.

The paper is focused on the synthesis and screening of the antiplasmodial activity of novel fumardiamides 5-10 with the mefloquine pharmacophore and a Michael acceptor motif. Multi-step reactions leading to the title compounds included two amide bond formations. The first amide bond was achieved by the reaction of (E)-ethyl 4-chloro-4-oxobut-2-enoate (1) and N1-(2,8-bis(trifluoromethyl)quinolin-4-yl) butane-1,4-diamine (2). The obtained ester 3 was hydrolyzed and gave acid 4, which then reacted with the selected halogenanilines in the presence of HATU/DIEA and formed products 5-10. Title compounds showed marked, dose dependent activity in vitro against hepatic stages of Plasmodium berghei. IC50 values of the most active compounds 5, 7 and 9 bearing 3-fluoro, 3-chloro and 3-trifluoromethyl substituents were 3.04-4.16 µmol L-1, respectively. On the other hand, the compounds exerted only weak activity against the erythrocytic stages of two P. falciparum strains (Pf3D7 and PfDd2) in vitro, with the exception of compound 5 (IC50 = 2.9 µmol L-1).

Second generation of primaquine ureas and bis-ureas as potential antimycobacterial agents.

Here, we describe design and synthesis of twelve novel compounds bearing primaquine motif and hydroxy- or halogenamine linked by an urea or bis-urea spacer. Preparation of ureas 3a-f started with the conversion of primaquine to benzotriazolide 2 and aminolysis of the later compound by 4-(2-aminoethyl)phenol or amino alcohols bearing fluorine atom, cycloalkyl or trifluoromethyl group under microwave irradiation. The four-step sequence leading to bis-ureas 6a-f included preparation of benzotriazolide 2 and two intermediates, semicarbazide 4 and benzotriazole bis-urea 5, which upon aminolysis with the same aminophenol or amino alcohols gave the title compounds. Antimycobacterial screening detected three active compounds against Mycobacterium marinum and M. tuberculosis, namely 3b, 3f and 6f, derived from cyclobutyl amino alcohol or amino phenol.

Inhibition of glioblastoma cell proliferation, invasion, and mechanism of action of a novel hydroxamic acid hybrid molecule.

Glioblastoma multiforme is one of the most aggressive brain tumors and current therapies with temozolomide or suberoylanilide hydroxamic acid (SAHA, vorinostat) show considerable limitations. SAHA is a histone deacetylase (HDAC) inhibitor that can cause undesirable side effects due to the lack of selectivity. We show here properties of a novel hybrid molecule, sahaquine, which selectively inhibits cytoplasmic HDAC6 at nanomolar concentrations without markedly suppressing class I HDACs. Inhibition of HDAC6 leads to significant α-tubulin acetylation, thereby impairing cytoskeletal organization in glioblastoma cells. The primaquine moiety of sahaquine reduced the activity of P-glycoprotein, which contributes to glioblastoma multiforme drug resistance. We propose the mechanism of action of sahaquine to implicate HDAC6 inhibition together with suppression of epidermal growth factor receptor and downstream kinase activity, which are prominent therapeutic targets in glioblastoma multiforme. Sahaquine significantly reduces the viability and invasiveness of glioblastoma tumoroids, as well as brain tumor stem cells, which are key to tumor survival and recurrence. These effects are augmented with the combination of sahaquine with temozolomide, the natural compound quercetin or buthionine sulfoximine, an inhibitor of glutathione biosynthesis. Thus, a combination of agents disrupting glioblastoma and brain tumor stem cell homeostasis provides an effective anti-cancer intervention.

SAHAquines, Novel Hybrids Based on SAHA and Primaquine Motifs, as Potential Cytostatic and Antiplasmodial Agents.

We report the synthesis of SAHAquines and related primaquine (PQ) derivatives. SAHAquines are novel hybrid compounds that combine moieties of suberoylanilide hydroxamic acid (SAHA), an anticancer agent with weak antiplasmodial activity, and PQ, an antimalarial drug with low antiproliferative activity. The preparation of SAHAquines is simple, cheap, and high yielding. It includes the following steps: coupling reaction between primaquine and a dicarboxylic acid monoester, hydrolysis, a new coupling reaction with O-protected hydroxylamine, and deprotection. SAHAquines 5 a-d showed significant reduction in cell viability. Among the three human cancer cell lines (U2OS, HepG2, and MCF-7), the most responsive were the MCF-7 cells. The antibodies against acetylated histone H3K9/H3K14 in MCF-7 cells revealed a significant enhancement following treatment with N-hydroxy-N'-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}pentanediamide (5 b). Ethyl (2E)-3-({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)prop-2-enoate (2 b) and SAHAquines were the most active compounds against both the hepatic and erythrocytic stages of Plasmodium parasites, some of them at sub-micromolar concentrations. The results of our research suggest that SAHAquines are promising leads for new anticancer and antimalarial agents.

Asymmetric Primaquine and Halogenaniline Fumardiamides as Novel Biologically Active Michael Acceptors.

Novel primaquine (PQ) and halogenaniline asymmetric fumardiamides 4a⁻f, potential Michael acceptors, and their reduced analogues succindiamides 5a⁻f were prepared by simple three-step reactions: coupling reaction between PQ and mono-ethyl fumarate (1a) or mono-methyl succinate (1b), hydrolysis of PQ-dicarboxylic acid mono-ester conjugates 2a,b to corresponding acids 3a,b, and a coupling reaction with halogenanilines. 1-[bis(Dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) was used as a coupling reagent along with Hünig's base. Compounds 4 and 5 were evaluated against a panel of bacteria, several Mycobacterium strains, fungi, a set of viruses, and nine different human tumor cell lines. p-Chlorofumardiamide 4d showed significant activity against Staphylococcus aureus,Streptococcus pneumoniae and Acinetobacter baumannii, but also against Candida albicans (minimum inhibitory concentration (MIC) 6.1⁻12.5 µg/mL). Together with p-fluoro and p-CF3 fumardiamides 4b,f, compound 4d showed activity against Mycobacterium marinum and 4b,f against M. tuberculosis. In biofilm eradication assay, most of the bacteria, particularly S. aureus, showed susceptibility to fumardiamides. m-CF3 and m-chloroaniline fumardiamides 4e and 4c showed significant antiviral activity against reovirus-1, sindbis virus and Punta Toro virus (EC50 = 3.1⁻5.5 µM), while 4e was active against coxsackie virus B4 (EC50 = 3.1 µM). m-Fluoro derivative 4a exerted significant cytostatic activity (IC50 = 5.7⁻31.2 µM). Acute lymphoblastic leukemia cells were highly susceptible towards m-substituted derivatives 4a,c,e (IC50 = 6.7⁻8.9 µM). Biological evaluations revealed that fumardiamides 4 were more active than succindiamides 5 indicating importance of Michael conjugated system.

Challenges encountered during development of Mn porphyrin-based, potent redox-active drug and superoxide dismutase mimic, MnTnBuOE-2-PyP5+, and its alkoxyalkyl analogues.

We disclose here the studies that preceded and guided the preparation of the metal-based, redox-active therapeutic Mn(III) meso-tetrakis(N-n-butoxyethylpyridyl)porphyrin, MnTnBuOE-2-PyP5+ (BMX-001), which is currently in Phase I/II Clinical Trials at Duke University (USA) as a radioprotector of normal tissues in cancer patients. N-substituted pyridylporphyrins are ligands for Mn(III) complexes that are among the most potent superoxide dismutase mimics thus far synthesized. To advance their design, thereby improving their physical and chemical properties and bioavailability/toxicity profiles, we undertook a systematic study on placing oxygen atoms into N-alkylpyridyl chains via alkoxyalkylation reaction. For the first time we show here the unforeseen structural rearrangement that happens during the alkoxyalkylation reaction by the corresponding tosylates. Comprehensive experimental and computational approaches were employed to solve the rearrangement mechanism involved in quaternization of pyridyl nitrogens, which, instead of a single product, led to a variety of mixed N-alkoxyalkylated and N-alkylated pyridylporphyrins. The rearrangement mechanism involves the formation of an intermediate alkyl oxonium cation in a chain-length-dependent manner, which subsequently drives differential kinetics and thermodynamics of competing N-alkoxyalkylation versus in situ N-alkylation. The use of alkoxyalkyl tosylates, of different length of alkyl fragments adjacent to oxygen atom, allowed us to identify the set of alkyl fragments that would result in the synthesis of a single compound of high purity and excellent therapeutic potential.

Radioprotection of the brain white matter by Mn(III) n-Butoxyethylpyridylporphyrin-based superoxide dismutase mimic MnTnBuOE-2-PyP5+.

Cranial irradiation is a standard therapy for primary and metastatic brain tumors. A major drawback of radiotherapy (RT), however, is long-term cognitive loss that affects quality of life. Radiation-induced oxidative stress in normal brain tissue is thought to contribute to cognitive decline. We evaluated the effectiveness of a novel mimic of superoxide dismutase enzyme (SOD), MnTnBuOE-2-PyP(5+)(Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin), to provide long-term neuroprotection following 8 Gy of whole brain irradiation. Long-term RT damage can only be assessed by brain imaging and neurocognitive studies. C57BL/6J mice were treated with MnTnBuOE-2-PyP(5+) before and after RT and evaluated three months later. At this time point, drug concentration in the brain was 25 nmol/L. Mice treated with MnTnBuOE-2-PyP(5+)/RT exhibited MRI evidence for myelin preservation in the corpus callosum compared with saline/RT treatment. Corpus callosum histology demonstrated a significant loss of axons in the saline/RT group that was rescued in the MnTnBuOE-2-PyP(5+)/RT group. In addition, the saline/RT groups exhibited deficits in motor proficiency as assessed by the rotorod test and running wheel tests. These deficits were ameliorated in groups treated with MnTnBuOE-2-PyP(5+)/RT. Our data demonstrate that MnTnBuOE-2-PyP(5+) is neuroprotective for oxidative stress damage caused by radiation exposure. In addition, glioblastoma cells were not protected by MnTnBuOE-2-PyP(5+) combination with radiation in vitro. Likewise, the combination of MnTnBuOE-2-PyP(5+) with radiation inhibited tumor growth more than RT alone in flank tumors. In summary, MnTnBuOE-2-PyP(5+) has dual activity as a neuroprotector and a tumor radiosensitizer. Thus, it is an attractive candidate for adjuvant therapy with RT in future studies with patients with brain cancer.