Preparation of Sustained Release Tablet with Minimized Usage of Glyceryl Behenate Using Post-Heating Method.

The purpose of this study was to prepare sustained release (SR) matrix tablets using a direct compression incorporated with a post-heating process. Allopurinol was selected due to the water-soluble property and Compritol 888 ATO® (also known as glyceryl behenate) was used as an SR matrix-forming agent. The API, SR material, microcrystalline cellulose, and magnesium stearate (lubricant) were mixed and prepared into a tablet by a direct compression method. The compressed tablets were stored in a dry oven at four temperatures (60, 70, 80, and 90°C) and for three time periods (15, 30, 45 min). The DSC and PXRD data indicated that the crystallinity of the API was not altered by the post-heating method. However, SEM images demonstrated that Compritol 888 ATO® was melted by the post-heating method, and that the melted Compritol 888 ATO® could form a strong matrix. This strong matrix led to the significant sustained release behavior of hydrophilic APIs. As little as 3 mg of Compritol 888 ATO® (0.65% of total tablet weight), when heated at 80°C for 15 min, showed sustained release over 10 h. The post-heating method exerted a significant influence on lipid-based matrix tablets and allowed a reduction in the amount of material required for a water-soluble drug. This will also provide a valuable insight into lipid-based SR tablets and will allow their application to higher quality products and easier processing procedures.

Xanthine oxidase inhibitors beyond allopurinol and febuxostat; an overview and selection of potential leads based on in silico calculated physico-chemical properties, predicted pharmacokinetics and toxicity.

Xanthine oxidase (XO), a versatile metalloflavoprotein enzyme, catalyzes the oxidative hydroxylation of hypoxanthine and xanthine to uric acid in purine catabolism while simultaneously producing reactive oxygen species. Both lead to the gout-causing hyperuricemia and oxidative damage of the tissues where overactivity of XO is present. Over the past years, significant progress and efforts towards the discovery and development of new XO inhibitors have been made and we believe that not only experts in the field, but also general readership would benefit from a review that addresses this topic. Accordingly, the aim of this article was to overview and select the most potent recently reported XO inhibitors and to compare their structures, mechanisms of action, potency and effectiveness of their inhibitory activity, in silico calculated physico-chemical properties as well as predicted pharmacokinetics and toxicity. Derivatives of imidazole, 1,3-thiazole and pyrimidine proved to be more potent than febuxostat while also displaying/possessing favorable predicted physico-chemical, pharmacokinetic and toxicological properties. Although being structurally similar to febuxostat, these optimized inhibitors bear some structural freshness and could be adopted as hits for hit-to-lead development and further evaluation by in vivo studies towards novel drug candidates, and represent valuable model structures for design of novel XO inhibitors.

Discovery of Novel Allopurinol Derivatives with Anticancer Activity and Attenuated Xanthine Oxidase Inhibition.

A series of pyrazolo[3,4-d]pyrimidine derivatives related to allopurinol has been synthesized and evaluated for its cytotoxicity against a panel of three cancer cell lines as well as its xanthine oxidase (XOD) inhibitory activities. Among them, compound 4 showed potent cytotoxicity with IC50 values of 25.5 and 35.2 µM against human hepatoma carcinoma cell lines, BEL-7402 and SMMC-7221, respectively. The anticancer activity of 4 was comparable to that of Tanespimycin (17-N-allylamino-17-demethoxy geldanamycin, 17-AAG) that inhibited the growth of BEL-7402 and SMMC-7221 cells at IC50 values of 12.4 and 9.85 µM, respectively. However, unlike allopurinol, which is also a strong inhibitor of XOD, compound 4 is a much weaker XOD inhibitor, suggesting that the anticancer activities of the allopurinol derivatives may not be associated with XOD inhibition. Moreover, the cytotoxicity of 4 toward normal cells is significantly lower than that of 17-AAG, making 4 a promising lead compound for further optimization of structure-activity relationships that may lead to anticancer agents of clinical utility.

Synthesis and pharmaceutical properties of N-acyloxymethyl prodrugs of Allop with potential anti-trypanosomal activity.

We report herein the synthesis, and the physicochemical and pharmacokinetic properties of N-acyloxymethyl prodrugs of allopurinol (Allop) (2a-f). Allop is a compound with activity against Trypanosoma cruzi, a causative agent of Chagas disease. Its pathology leads to a huge number of infections and deaths per year, because in addition to many sufferers only having limited access to health services only an inefficient chemotherapy is available. Relevant pharmaceutical properties (pKa, stability, solubility, lipophilicity, in vitro permeability, binding protein, xanthine oxidase binding) were also determined. The results obtained showed that derivatives behave as prodrugs of Allop, since they exhibit improved physicochemical and pharmacokinetic properties relative to their precursor. This behavior turns these compounds into active reservoirs of Allop, and reduces its unfavorable characteristics, so 2a-f compounds are excellent candidates for the treatment of Chagas disease. This work is therefore an important contribution leading to the suppression of Chagas disease.

Synthesis, physicochemical properties of allopurinol derivatives and their biological activity against Trypanosoma cruzi.

Chagas disease is caused by Trypanosoma cruzi (T. cruzi) leading to a huge number of infections and deaths per year, because in addition to many sufferers only having limited access to health services only an inefficient chemotherapy is available using drugs such as benznidazole and nifurtimox. Here, C6-alkyl (2a-c) and N1-acyl (3a-c) derivatives of Allopurinol (Allop, compound with activity against T. cruzi) were synthesized in good yields and their structures were unambiguously characterized. Only 2a, 2b and 3c showed inhibitory activity against the proliferative stages of the parasite when tested at 1 µg mL(-1) with the 3c derivative exhibiting an IC50 value similar to that of Allop and not being toxic for mammalian cells. Relevant pharmaceutical physicochemical properties (pKa, stability, solubility, lipophilicity) were also determined as well by using Lipinski's rule, polar surface area and molecular rigidity. Taken together, the results demonstrated that the studied derivatives had optimal properties for bioavailability and oral absorption. For the stability studies, Micellar Liquid Chromatography was used as the analytical method which was fully validated according to the FDA guidelines and shown to be a suitable, sensitive and simple method for routine analysis of these Allop derivatives.

Discovery of novel xanthone derivatives as xanthine oxidase inhibitors.

Xanthine oxidase is the key enzyme that catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. In this study, a series of xanthone derivatives were synthesized as effective and a new class of xanthine oxidase inhibitor. Compounds 8a, 8c, 8i, 8g and 8r showed good inhibition against xanthine oxidase. The presence of a cyano group at the para position of benzyl moiety turned out to be the preferred substitution pattern. Molecular modeling studies were performed to gain an insight into its binding mode with xanthine oxidase, and to provide the basis for further structure-guided design of new non-purine xanthine oxidase inhibitors associated with the xanthone framework.

Preparation and preservation of hypoxia UW solution.

In order to explore the method to prepare hypoxia UW solution and the stability and preservation of hypoxia UW solution, UW solution was purged by argon or air for 15 min or 60 at a flow rate of 0.8 or 2 L/min, and the oxygen partial pressure of UW solution was detected. The hypoxia UW solution was exposed to the air or sealed up to preserve by using different methods, and the changes of oxygen partial pressure was tested. The results showed that oxygen partial presure of 50 mL UW solution, purged by argon for 15 min at a flow rate of 2 L/min, was declined from 242+/-6 mmHg to 83+/-10 mmHg. After exposure to the air, oxygen partial pressure of hypoxia UW solution was gradually increased to 160+/-7 mmHg at 48 h. After sealed up by the centrifuge tube and plastic bad filled with argon, oxygen partial pressure of hypoxia UW solution was stable, about 88+/-13 mmHg at 72 h. It was concluded that oxygen of UW solution could be purged by argon efficiently. Sealed up by the centrifuge tube and plastic bag filled with argon, oxygen partial pressure of UW solution could be stabilized.

Symmetrically and unsymmetrically bridged methylenebis(allopurinols): synthesis of dimeric potential anti-gout drugs.

Liquid-liquid phase transfer alkylation of 4-methoxy-pyrazolo[3,4-d]-pyrimidine (1a) with a dichloromethane/dibromomethane mixture (3:1, v/v) gave the regioisomeric methylenebis(heterocycles) 3a-5a. These were converted by dilute aqueous sodium hydroxide containing dimethylsulfoxide (DMSO) at concentrations between 0 and 60 vol-% into the methylenebis(allopurinols) 3b-5b by nucleophilic SNAr reactions at C(4). The effect of DMSO on the reaction kinetics was investigated.

The screening and characterization of 6-aminopurine-based xanthine oxidase inhibitors.

Xanthine oxidase (XO) is a key enzyme which can catalyze xanthine to uric acid causing hyperuricemia in humans. By using the fractionation technique and inhibitory activity assay, an active compound that prevents XO from reacting with xanthine was isolated from wheat leaf. It was identified by the Mass and NMR as 6-aminopurine (adenine). A structure-activity study based on 6-aminopurine was conducted. The inhibition of XO activity by 6-aminopurine (IC(50)=10.89+/-0.13 microM) and its analogues was compared with that by allopurinol (IC(50)=7.82+/-0.12 microM). Among these analogues, 2-chloro-6(methylamino)purine (IC(50)=10.19+/-0.10 microM) and 4-aminopyrazolo[3,4-d] pyrimidine (IC(50)=30.26+/-0.23 microM) were found to be potent inhibitors of XO. Kinetics study showed that 2-chloro-6(methylamino)purine is non-competitive, while 4-aminopyrazolo[3,4-d]pyrimidine is competitive against XO.

Effect of hexacosanol on the characteristics of novel sustained-release allopurinol solid lipospheres (SLS): factorial design application and product evaluation.

This investigation involved the evaluation of the effect of hexacosanol (HC, ceryl alcohol), a new hydrophobic wax modifier (WM) in comparison with conventional modifiers, on the development of sustained-release allopurinol (AP) solid lipospheres (SLS) intended for use in a suspension formulation and other oral dosage forms. Various beeswax (BW)/WM blends (composition ratio 1:1) were thus used to prepare SLS by a modified oil-in-water emulsion meltable disperse-phase (MDP) encapsulation method without using organic solvents and the influence of these blends on the drug encapsulation efficiency (EE), size distribution and the time for 50% of the drug to be released (t50%) was investigated. Results indicated that incorporation of HC in wall matrix of SLS provided the means to enhance the EE of AP and to modulate the rate of drug release into dissolution media (simulated gastric fluid (S.G.F.: pH 1.2) and simulated intestinal fluid (S.I.F.: pH 7.4). The effects of the process variables; HC concentration, dispersant (pluronic F-68: PF-68) concentration and drug:wax ratio were also studied on the properties of AP-loaded SLS by a 2(3) factorial design. The EE values were in the range of 80.8-92.67%. The only significant parameter affecting (P<0.01) the size and size distribution of the SLS formulations was the amount of the PF-68, whereas the factor with the biggest influence (P<0.05) on the drug EE was the initial loading of AP (in terms of the drug:wax ratio). The amount of HC blended with wax and the initial drug loading significantly (P<0.01) affected the t50% values of all of the formulations. The release of AP was more extended (t50% values (S.I.F.; pH 7.4)=9.91-25.36 h, depending on the drug:wax ratio) and surface morphology of SLS was improved with higher HC content (15%, w/w) formulations. The release patterns fitted the Baker-Lonsdale dissolution kinetics for spherical matrices. A significant decrease of plasma uric acid levels (P<0.05) and hepatic impairment in male rats was observed after oral administration of a SLS (mean size: 120 microm) suspensions of the optimum formulation, compared to suspensions of pure AP.

Solid-phase parallel synthesis of 4-beta-D-ribofuranosylpyrazolo[4,3-d]pyrimidine nucleosides.

The synthesis of pyrazolo[4,3-d]pyrimidine nucleoside library using solid-phase parallel synthesis methodology is described. Glycosylation of the trimethylsilyl (TMS) derivative of 1- and 2-(methyl)-1H and 2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H, 6H)-dione (5) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of TMS triflate provided two novel protected nucleosides 6 and 7. The structures of 6 and 7 were assigned by 1H and 2D NMR experiments. Nucleosides 6 and 7 were then transformed to the key intermediates 12 and 15 respectively. Reaction of 12 and 15 with MMTCl resin in the presence of 2,6-lutidine afforded the necessary scaffolds B and C. Different amines (96) were introduced selectively by nucleophilic substitution on scaffolds B and C using solid-phase parallel semi-automated synthesizer. Cleavage of the products from the solid support with 30% HFIP in a parallel fashion yielded nucleoside libraries simultaneously, and they were analyzed and characterized by high-throughput LC-MS.

Antileishmanial agents: synthesis of allopurinol prodrugs for oral administration

Synthesis of six prodrugs of allopurinol (4-hydroxypyrazolo-(3,4-d)pyrimidine) is described. These compounds were given orally to male and female BALB/C (sensitive) and C57BL/6 (partially resistant) strains of mice, wich had been previously infected with Leishmania mexicana. The results show that at end of the treatment there was weak regression of lesions confirmed through measurement of nodule diameter in the infected animals

Characterization of a novel potent and specific inhibitor of type V phosphodiesterase.

Guanosine cyclic 3':5'-monophosphate (cGMP) plays a crucial role in regulating vascular smooth muscle contractile state. In rat aortic smooth muscle cells (RSMC) three isozymes of phosphodiesterase (PDE) may be involved in the degradation of cGMP, namely PDE I, PDE III, and PDE V. To study the effective contribution of PDE V to the control of intracellular cGMP levels, a specific and potent PDE V inhibitor 1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazolo[3, 4d]- pyrimidin-4-(5H)-one (DMPPO) was synthesized. DMPPO is a competitive inhibitor with respect to cGMP (Ki = 3 nM) and displayed high selectivity for PDE V as compared to other PDE isozymes. DMPPO strongly potentiated the cGMP response of atrial natriuretic peptide- or sodium nitroprusside-treated RSMC (EC50 = 0.5 microM). In addition, similar intracellular cGMP levels were obtained in the presence of a saturating concentration of DMPPO or 3-isobutyl-1-methylxanthine, a nonspecific PDE inhibitor, suggesting that cGMP is almost exclusively hydrolyzed by PDE V in RSMC. Stimulation of RSMC with atrial natriuretic factor resulted in accumulation of cGMP in the extracellular media. This egression was shown to be proportional to the intracellular level of cGMP and a first-order rate constant of 0.04 min-1 was determined for the egression process. DMPPO did not interfere with the efflux and allowed us to show that intracellular cGMP levels are mainly controlled by PDE V, rather than by egression in RSMC. DMPPO is, therefore, a useful tool for determining the role of PDE V in the control of cGMP levels in living cells and tissues.

C4-substituted 1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidines as adenosine agonist analogues.

The synthesis of four novel C4-substituted 1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidines is reported, and the compounds were examined as adenosine receptor agonist analogues. Neither receptor affinity nor biological activity was as potent as the purine counterparts. Adenosine agonists appear to be sensitive to modification of the purine base, with a nitrogen atom in the 7 position necessary for efficacy.

A convenient synthesis of 6-amino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4-one and related 4,6-disubstituted pyrazolopyrimidine nucleosides.

The glycosylation of 4,6-dichloropyrazolo[3,4-d]pyrimidine and 4-chloro-6-methylthiopyrazolo[3,4-d]pyrimidine via the corresponding trimethylsilyl intermediate and tetra-O-acetyl-beta-D-ribofuranose in the presence of trimethylsilyl triflate as a catalyst, gave selective glycosylation at N1 as the only nucleoside product. The intermediates 4,6-dichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo [3,4-d]pyrimidine 7 and 4-chloro-6-methylthio-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo [3,4-d]pyrimidine 13 gave new and convenient synthetic routes to the inosine analog 1, the guanosine analog 2, the adenosine analog 3, and the isoguanosine analog 16. Glycosylation of the trimethylsilyl derivative of 6-chloropyrazolo[3,4-d]pyrimidine-4-one unexpectedly gave the N2-glycosyl isomer 20 as the major product. A number of new 4,6-disubstituted pyrazolo[3,4-d]pyrimidine nucleosides were prepared from these glycosyl intermediates.