Effectiveness of an O-Alkyl Hydroxamate in Dogs with Naturally Acquired Canine Leishmaniosis: An Exploratory Clinical Trial.

Canine leishmaniosis is a challenge in veterinary medicine and no drug to date has achieved parasite clearance in dogs. Histone deacetylase inhibitors are a drug class widely used in cancer chemotherapy. We have successfully used O-alkyl hydroxamates (vorinostat derivatives) in the treatment of a laboratory model of visceral leishmaniasis without showing toxicity. In order to test the effectiveness of a particular compound, MTC-305, a parallel-group, randomized, single-centre, exploratory study was designed in naturally infected dogs. In this clinical trial, 18 dogs were allocated into 3 groups and were treated with either meglumine antimoniate (104 mg SbV/kg), MTC-305 (3.75 mg/kg) or a combination of both using a lower MTC-305 dose (1.5 mg/kg) through a subcutaneous route for 2 treatment courses of 30 days, separated by a 30-day rest period. After treatment, a follow-up time of 4 months was established. Parasite burden in bone marrow, lymph node and peripheral blood were quantified through qPCR. Antibody titres were determined through an immunofluorescence antibody test, and cytokine expression values were calculated through RT-qPCR. Treatment safety was evaluated through the assessment of haematological and biochemical parameters in blood, weight, and gastrointestinal alterations. Assessment was carried out before, between and after treatment series. Treatment with MTC-305 was effective at reducing parasite burdens and improving the animals' clinical picture. Dogs treated with this compound did not present significant toxicity signs. These results were superior to those obtained using the reference drug, meglumine antimoniate, in monotherapy. These results would support a broader clinical trial, optimised dosage, and an expanded follow-up stage to confirm the efficacy of this drug.

Lactate dehydrogenase A inhibitors with a 2,8-dioxabicyclo[3.3.1]nonane scaffold: A contribution to molecular therapies for primary hyperoxalurias.

Human lactate dehydrogenase A (hLDHA) is one of the main enzymes involved in the pathway of oxalate synthesis in human liver and seems to contribute to the pathogenesis of disorders with endogenous oxalate overproduction, such as primary hyperoxaluria (PH), a rare life-threatening genetic disease. Recent published results on the knockdown of LDHA gene expression as a safe strategy to ameliorate oxalate build-up in PH patients are encouraging for an approach of hLDHA inhibition by small molecules as a potential pharmacological treatment. Thus, we now report on the synthesis and hLDHA inhibitory activity of a new family of compounds with 2,8-dioxabicyclo[3.3.1]nonane core (23-42), a series of twenty analogues to A-type proanthocyanidin natural products. Nine of them (25-27, 29-34) have shown IC50 values in the range of 8.7-26.7 µM, based on a UV spectrophotometric assay, where the hLDHA inhibition is measured according to the decrease in absorbance of the cofactor ß-NADH (340 nm). Compounds 25, 29, and 31 were the most active hLDHA inhibitors. In addition, the inhibitory activities of those nine compounds against the hLDHB isoform were also evaluated, finding that all of them were more selective inhibitors of hLDHA versus hLDHB. Among them, compounds 32 and 34 showed the highest selectivity. Moreover, the most active hLDHA inhibitors (25, 29, 31) were evaluated for their ability to decrease the oxalate production by hyperoxaluric mouse hepatocytes (PH1, PH2 and PH3) in vitro, and the relative oxalate output at 24 h was 16% and 19 % for compounds 25 and 31, respectively, in Hoga1-/- mouse primary hepatocyte cells (a model for PH3). These values improve those of the reference compound used (stiripentol). Compounds 25 and 31 have in common the presence of two hydroxyl groups at rings B and D and an electron-withdrawing group (NO2 or Br) at ring A, pointing to the structural features to be taken into account in future structural optimization.

New salicylic acid derivatives, double inhibitors of glycolate oxidase and lactate dehydrogenase, as effective agents decreasing oxalate production.

The synthesis and biological evaluation of double glycolate oxidase/lactate dehydrogenase inhibitors containing a salicylic acid moiety is described. The target compounds are obtained in an easily scalable two-step synthetic procedure. These compounds showed low micromolar IC50 values against the two key enzymes in the metabolism of glyoxylate. Mechanistically they behave as noncompetitive inhibitors against both enzymes and this fact is supported by docking studies. The biological evaluation also includes in vitro and in vivo assays in hyperoxaluric mice. The compounds are active against the three types of primary hyperoxalurias. Also, possible causes of adverse effects, such as cyclooxygenase inhibition or renal toxicity, have been studied and discarded. Altogether, this makes this chemotype with drug-like structure a good candidate for the treatment of primary hyperoxalurias.

Small Molecule-Based Enzyme Inhibitors in the Treatment of Primary Hyperoxalurias.

Primary hyperoxalurias (PHs) are a group of inherited alterations of the hepatic glyoxylate metabolism. PHs classification based on gene mutations parallel a variety of enzymatic defects, and all involve the harmful accumulation of calcium oxalate crystals that produce systemic damage. These geographically widespread rare diseases have a deep impact in the life quality of the patients. Until recently, treatments were limited to palliative measures and kidney/liver transplants in the most severe forms. Efforts made to develop pharmacological treatments succeeded with the biotechnological agent lumasiran, a siRNA product against glycolate oxidase, which has become the first effective therapy to treat PH1. However, small molecule drugs have classically been preferred since they benefit from experience and have better pharmacological properties. The development of small molecule inhibitors designed against key enzymes of glyoxylate metabolism is on the focus of research. Enzyme inhibitors are successful and widely used in several diseases and their pharmacokinetic advantages are well known. In PHs, effective enzymatic targets have been determined and characterized for drug design and interesting inhibitory activities have been achieved both in vitro and in vivo. This review describes the most recent advances towards the development of small molecule enzyme inhibitors in the treatment of PHs, introducing the multi-target approach as a more effective and safe therapeutic option.

O-Alkyl Hydroxamates Display Potent and Selective Antileishmanial Activity.

Leishmania (L.) infantum causes visceral, cutaneous, and mucosal leishmaniasis in humans and canine leishmaniasis in dogs. Herein, we describe that O-alkyl hydroxamate derivatives displayed potent and selective in vitro activity against the amastigote stage of L. infantum while no activity was observed against promastigotes. Compound 5 showed potent in vivo activity against L. infantum. Moreover, the combination of compound 5 supported on gold nanoparticles and meglumine antimoniate was also effective in vivo and improved the activity of these compounds compared to that of the individual treatment. Docking studies showed that compound 5 did not reach highly conserved pocket C and established interactions with the semiconserved residues V44, A45, R242, and E243 in pocket A of LiSIR2rp1. The surface space determined by these four amino acids is not conserved in human sirtuins. Compound 5 represents a new class of selective ligands with antileishmanial activity.

A nanodelivered Vorinostat derivative is a promising oral compound for the treatment of visceral leishmaniasis.

There is currently no satisfactory treatment for visceral leishmaniasis; the disease is thus in desperate need of novel drugs. The ideal candidate should be effective, safe, affordable, and administered via the oral route. Histone deacetylases (HDACs) are involved in silencing critical regulatory pathways, including pro-apoptotic programs, and represent potential therapeutic targets for pharmacological interventions. O-alkyl hydroxamates have traditionally been considered to exert no effect on mammal HDACs. The aim of this study was to evaluate the effect of MDG, a SAHA derivative of the O-alkyl hydroxamate family with no activity on human histone deacetylase enzymes, on the visceral leishmaniasis causative agents and in a murine model of the disease. The effects of vorinostat, tubacin and valproic acid (well-known mammal HDAC inhibitors) on the parasite were also evaluated. MDG was found to be highly active against Leishmania infantum and L. donovani intracellular amastigotes in vitro but not against the promastigote stage. In contrast, vorinostat, tubacin and valproic acid showed no activity against the parasite. Assays investigating hERG and Cav1.2 channels in vitro found no evidence of MDG-driven cardiotoxicity. MDG showed neither hepatotoxicity nor mutagenicity, nor did it exert activity on cytochrome P450 enzymes. MDG was adsorbed onto gold nanoparticles for the in vivo experiments, performed on infected Balb/c mice. MDG was effective at reducing the parasite load in major target tissues (bone marrow, spleen and liver) in more than 70% at 25 mg/kg through both the oral and intraperitoneal route, proving more active than the reference compounds (meglumine antimoniate, MA) without showing toxicity. In addition, the combination of MDG and MA was very effective.

Salicylic Acid Derivatives Inhibit Oxalate Production in Mouse Hepatocytes with Primary Hyperoxaluria Type 1.

Primary hyperoxaluria type 1 (PH1) is a rare life-threatening genetic disease related to glyoxylate metabolism and characterized by accumulation of calcium oxalate crystals. Current therapies involve hepatic and/or renal transplantation, procedures that have significant morbidity and mortality and require long-term immunosuppression. Thus, a pharmacological treatment is urgently needed. We introduce here an unprecedented activity of salicylic acid derivatives as agents capable of decreasing oxalate output in hyperoxaluric hepatocytes at the low micromolar range, which means a potential use in the treatment of PH1. Though correlation of this phenotypic activity with glycolate oxidase (GO) inhibition is still to be verified, most of the salicylic acids described here are GO inhibitors with IC50 values down to 3 µM. Binding mode of salicylic acids inside GO has been studied using in silico methods, and preliminary structure-activity relationships have been established. The drug-like structure and ease of synthesis of our compounds make them promising hits for structural optimization.

Two-directional synthesis as a tool for diversity-oriented synthesis: Synthesis of alkaloid scaffolds.

Two-directional synthesis represents an ideal strategy for the rapid elaboration of simple starting materials and their subsequent transformation into complex molecular architectures. As such, it is becoming recognised as an enabling technology for diversity-oriented synthesis. Herein, we provide a thorough account of our work combining two-directional synthesis with diversity-oriented synthesis, with particular reference to the synthesis of polycyclic alkaloid scaffolds.

The selective cytotoxic activity in breast cancer cells by an anthranilic alcohol-derived acyclic 5-fluorouracil O,N-acetal is mediated by endoplasmic reticulum stress-induced apoptosis.

Advance in the knowledge of molecular biology has thrown light on many aspects of apoptosis regulation mechanisms. This has allowed a change in anti-cancer therapy trends, from classic cytotoxic strategies to the development of new non-harmful therapies which target the apoptosis response selectively only in tumour cells. We have selected an anthranilic alcohol-derived acyclic 5-fluorouracil O,N-acetal (5) to carry out the anti-cancer studies. This compound shows activity as a potent growth inhibitor of the tumour cell line MCF-7 at a very low concentration. Moreover, when this compound was administered to the non-neoplastic cell line, MCF-10A displayed less toxicity resulting in lower rates of apoptosis. Further studies by microarray hybridization, real-time PCR and western blot showed that when administered to human breast cancer cells, MCF-7, 5 had no activity against classic pro-apoptotic genes such as p53, and even induced the down-regulation of anti-apoptotic genes such as Bcl-2. In contrast, several pro-apoptotic genes related with the endoplasmic reticulum (ER)-stress-induced apoptosis, such as BBC3 and Noxa, appeared up-regulated. These results seem to show that the mechanism of action and selectivity of 5 was via the activation of the ER stress-induced apoptosis. The selective activity of this compound against tumour cells via the ER stress-induced apoptosis supposes a great advantage for future therapeutic use.

Anticancer activity and cDNA microarray studies of a (RS)-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl]-6-chloro-9H-purine, and an acyclic (RS)-O,N-acetalic 6-chloro-7H-purine.

Completing a SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H or 9H-purines was previously prepared. The most potent antiproliferative agent against the MCF-7 adenocarcinoma cell line that belongs to the benzoxazepine O,N-acetalic family is (RS)-9-[1-(9H-fluorenyl-9-methoxycarbonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl]-6-chloro-9H-purine (16, IC(50) = 0.67 ± 0.18 µM), whilst (RS)-7-{2-(N-hydroxymethylphenyl)-2-nitrobenzenesulfonamido]-1-methoxyethyl}-6-chloro-7H-purine (37) shows the lowest IC(50) value between the family of acyclic O,N-acetals (IC(50) = 3.25 ± 0.23 µM). Moreover, 16 showed the better in vitro Therapeutic Index in breast cell lines (3.19), whilst 37 was found to be 3.69-fold more active against HT-29 human colon cancer cell line than versus IEC-6 normal rat intestinal epithelial cell line. The global apoptotic cells caused by 16 and 37 against MCF-7 were 80.08% and 54.85% of cell population after 48 h, respectively. cDNA microarray technology reveals potential drug targets, which are mainly centred on positive apoptosis regulatory pathway genes, and the repression of genes involved in carcinogenesis, proliferation and tumour invasion.

Diversity-oriented synthesis of bicyclic and tricyclic alkaloids.

A diversity-oriented synthesis involving a cascade sequence, taking linear aminoalkenes to polycyclic scaffolds reminiscent of natural alkaloids, is presented.

Synthesis of unprecedented scaffold diversity.

Building a better library: The synthesis of a library of natural-product-like small molecules with unprecedented scaffold diversity has been reported (see scheme; Ns = nosylate). The library represents a significant advancement in the capability of synthetic chemists to generate structurally diverse and complex small molecules in a rapid manner.

Oligonucleotide-polyamine conjugates: influence of length and position of 2'-attached polyamines on duplex stability and antisense effect.

Tethering cationic ligands to oligonucleotides results in zwitterionic molecules with often improved target affinity and better cell membrane permeation. Due to the ideal distance between cationic groups, polyamines are perfect counter ions for oligonucleotides. Using an easy and versatile procedure for attaching ligands to the 2'-position, polyamines were conjugated to distinct terminal and internal positions of oligonucleotides. With polyamines attached to terminal nucleosides, the affinity to complementary DNA or RNA strands increased with growing number of cationic amines. Tethering polyamines to an internal nucleoside of wild type DNA oligonucleotides resulted in a considerable decrease in duplex stability, but in phosphorothioates, no significant decrease was detected. Conjugates exhibited progressively higher target downregulation ability with increasing polyamine chain length in a human melanoma cell culture assay.

Design, synthesis and anticancer activity against the MCF-7 cell line of benzo-fused 1,4-dihetero seven- and six-membered tethered pyrimidines and purines.

Having previously reported the synthesis and anticancer activities of cyclic 5-fluorouracil (5-FU) O,N-acetalic compounds, the decision was made to change 5-FU for uracil (U), with the prospect of finding an antiproliferative agent endowed with a new mechanism of action. The use of a reverse transcription-PCR-based assay decreased cyclin D1 mRNA, suggesting that this cyclic U O,N-acetalic compound exerts its regulatory action on cyclin D1 at the level of transcription. Following the ongoing Anticancer Drug Programme we planned the synthesis of compounds bearing a natural pyrimidine base and also, the oxygen atom at position 1 of the seven-membered cycle was replaced by its isosteric sulfur atom, and its oxidized states. Next, the pyrimidine base was substituted for the purine one, with the objective of increasing both the lipophilicity and the structural diversity of the target molecules. If the previously described compounds were not prodrugs, it would not be necessary to maintain the O,N-acetalic characteristic. Therefore, molecules were designed in which both structural entities (such as the benzoheterocyclic ring and the purine base) were linked by a heteroatom-C-C-N bond. A series of (RS)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purine derivatives was obtained and the anticancer activity for the most active compounds was correlated with their capability to induce apoptosis. Finally, completing a SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H- or 9H-purines was prepared. The studies by microarray technology showed that the main molecular targets of some of these compounds are pro-apoptotic genes with protein kinase activity such as GP132, ERN1 or RAC1, which prevent the metastatic progression.

Diversity-oriented synthesis.

Diversity-oriented synthesis (DOS), which describes the synthesis of structurally diverse collections of small molecules, was developed, in part, to address combinatorial chemistry's shortfalls. In this paper, we hope to give an indication of what can be achieved using the DOS approach to library generation. We describe some of the most successful strategies utilized in DOS, with special focus on our own area of interest; DOS from simple, pluripotent starting materials.

Anticancer activity of (1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-pyrimidines and -purines against the MCF-7 cell line: Preliminary cDNA microarray studies.

Completing a SAR study, a series of (RS)-1- or 3-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-pyrimidines and (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H- or 9H-purines have been prepared. Their antiproliferative activities on MCF-7 cells are here presented and discussed. (RS)-6-Chloro-9-[1-(9H-9-fluorenylmethoxycarbonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl]-9H-purine (28) is the most active (IC(50)=0.67+/-0.18 microM) of the series so far described. cDNA microarray technology reveals potential drug targets, which are mainly centred on apoptosis regulatory pathway genes.

From 5-fluorouracil acyclonucleosides to benzo-fused six- and seven-membered rings linked to pyrimidine and purine bases: the shift from differentiating anticancer agents to apoptotic inducers.

BACKGROUND: (RS)-1-{[3-(2-Hydroxyethoxy)-1-isopropoxy]propyl}-5-fluorouracil proved to be a good differentiating agent against rhabdomyosarcoma cells. OBJECTIVE: As lipophilicity is known to affect the anticancer activity, the synthesis of a wide range of 5-fluorouracil derivatives linked to benzo-fused seven-membered rings was undertaken. METHODS: The decision was then made to change 5-fluorouracil for uracil, with the prospect of finding an antiproliferative agent endowed with a new mechanism of action. Later on, pyrimidines were substituted for purines. Completing a structure-activity relationship study, a series of isosteric seven-membered derivatives were prepared. RESULTS/CONCLUSION: Finally, molecules were designed in which both structural entities (such as the benzoheterocycle and the purine) were linked through a strong C-C bond. The anticancer activity for the most active compounds was correlated with their capability to induce apoptosis.

Synthesis and anticancer activity of (R,S)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purines.

A series of eleven 2- and 6-substituted (R,S)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purine derivatives was obtained by applying a standard Mitsunobu protocol that led to a six-membered ring contraction from (R,S)-3,4-dihydro-2H-1,5-benzoxathiepin-3-ol via an episulfonium intermediate. The signal approximately delta=151 ppm, which corresponds to the C4' carbon atom, is unequivocal proof of the N9' regioisomer. The potential of the target molecules as anticancer agents is reflected in their activity against the MCF-7 cancer cell line. The most active compounds have IC(50) values of (6.18+/-1.70) and (8.97+/-0.83) microM. The results indicate that the anticancer activity for the most active compounds is correlated with their capacity to induce apoptosis.

A synthetic uracil derivative with antitumor activity through decreasing cyclin D1 and Cdk1, and increasing p21 and p27 in MCF-7 cells.

The anticarcinogenic potential of (RS)-1-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)uracil (DBDU), with the naturally occurring pyrimidine base uracil, is reported against the MCF-7 cancer cell line. The arrest in the G0/G1 and G2/M cell cycle phases was accounted for by decrease in the expression of the cyclin D1 and Cdk1 proteins, and increase in p21 and p27 proteins. Using a reverse transcription-polymerase chain reaction-based assay at a dose of 5 muM of DBDU cyclin D1 mRNA was decreased, suggesting that DBDU exerts its regulatory action on cyclin D1 at the level of transcription. DNA fragmentation was performed and demonstrated that apoptosis occurred in the tumor cell line treated with DBDU. The G0/G1 arrest is an irreversible process and the cells undergo apoptosis in a p53-independent manner. DBDU administered intravenously twice a week (50 mg/kg dose each time) induced neither toxicity nor death in mice for 5 weeks.

Study of the factors that control the ratio of the products between 5-fluorouracil, uracil, and tetrahydrobenzoxazepine O,O-acetals bearing electron-withdrawing groups on the nitrogen atom.

(RS)-1-(2-Nitrobenzenesulfonyl)- and (RS)-1-(4-nitrobenzenesulfonyl)-3-methoxy-1,2,3,5-tetrahydro-4,1-benzoxazepines are better substrates than 1-acyl-3-methoxy-1,2,3,5-tetrahydro-4,1-benzoxazepine derivatives for the Lewis acid mediated condensation reaction with pyrimidine bases to give O,N-acetals. Acetonitrile, stannic chloride, 50 degrees C, and a reaction time higher than 48 h are the optimum conditions for such condensation reactions. Under these conditions, 5-fluorouracil preferably links to the aminalic carbon through its N-1" position, while the attachment of the uracil fragment is through N-3" or N-1" of the cyclic or acyclic products, respectively. The causes that influence the course of the reactions are analyzed and discussed. Examination of the (1)H NMR spectra revealed the presence of a single form for the secondary amine 11 and of two conformers for the tertiary sulfonamides 7a,b, 9a,b, and 10b and for the amides 7d and 13, with the following distribution: 7a, 59/41; 7b, 53/47; 9a, 52/48; 9b, 59/41; 10b, 56/44; 7d, 50/50; 13, 80/20. On increasing the temperature, the (1)H NMR spectrum (DMSO-d(6)) of 7b showed coalescence at 110 degrees C. The torsional barrier determined [DeltaG(c)++ value of 19.0 +/- 0.2 kcal.mol(-1) (79.1 +/- 1.0 kJ.mol(-1))] proved to be the highest ever observed for sulfonamide moieties.