Development and validation of LC-MS/MS method for determination of DNDI-VL-2098 in mouse, rat, dog and hamster blood.

Aim: To develop a bioanalytical method to support pharmacokinetic evaluation of DNDI-VL-2098 in mouse, rat, dog and hamster following oral administration. Results & methodology: A robust LC-MS/MS bioanalytical method was developed to quantify DNDI-VL-2098. DNDI-VL-2098 showed time-dependent recovery loss in acetonitrile precipitated plasma in all species. Acid-lysed whole blood was identified as a matrix in which recovery was stable over time. A two-step extraction procedure was used, with protein precipitation followed by liquid-liquid extraction with methyl tert-butyl ether. The assay was validated in the dynamic range of 5-5000 ng/ml for mouse, rat and dog blood, and a fit-for-purpose method was developed for hamster. Conclusion: A specific LC-MS/MS assay for DNDI-VL-2098 was developed and validated in hemolyzed blood.

Design and synthesis of leucine-linked quinazoline-4(3H)-one-sulphonamide molecules distorting malarial reductase activity in the folate pathway.

Optimization of a modified Grimmel's method for N-heterocyclization of a leucine-linked sulfonamide side-arm at position 2 leading to 2,3-disustituted-4-quinazolin-(3H)-ones was accomplished. Further, 22 hybrid quinazolinone motifs (4a-v) were synthesized by N-heterocyclization reaction under microwave irradiation using the ionic liquid [Bmim][BF4 ]-H2 O as green solvent as well as the catalyst. The in vitro screening of the hybrid entities against the malarial species Plasmodium falciparum yielded five potent molecules 4l, 4n, 4o, 4t, and 4u owning antimalarial activity comparable to those of the reference drugs. In continuation, an in silico study was carried out to obtain a pharmacophoric model and quantitative structure-activity relationship. We also built a 3D-QSAR model to procure more information that could be applied to design new molecules with more potent Pf-DHFR inhibitory activity. The designed pharmacophore was recognized to be more potent for the selected molecules, exhibiting five pharmacophoric features. The active scaffolds were further evaluated for enzyme inhibition efficacy against alleged receptor Pf-DHFR computationally and in vitro, proving their candidature as lead dihydrofolate reductase inhibitors, and the selectivity of the test candidates was ascertained by toxicity study against Vero cells. Good oral bioavailability was also proved by studying pharmacokinetic properties.

Green synthesis, biological evaluation, molecular docking studies and 3D-QSAR analysis of novel phenylalanine linked quinazoline-4(3H)-one-sulphonamide hybrid entities distorting the malarial reductase activity in folate pathway.

A modified Grimmel's method for N-heterocyclization of phenylalanine linked sulphonamide side arm at position-2 was optimized leading to 2,3-disustituted-4-quinazolin-(3H)-ones. Further, [Bmim][BF4]-H2O (IL) was used as green solvent as well as catalyst for the synthesis of twenty two hybrid quinazolinone motifs (4a-4v) by N-heterocyclization reaction using microwave irradiation technique. The in vitro screening of the hybrid entities against the malarial species Plasmodium falciparum yielded five potent molecules 4l, 4n, 4r, 4t & 4u owing comparable antimalarial activity to the reference drugs. In continuation, anin silicostudy was carried out to obtain a pharmacophoric model and quantitative structure activity relationship. We also built a 3D-QSAR model to procure more information that could be applied to design new molecules with more potent Pf-DHFR inhibitory activity. The designed pharmacophore was recognized to be more potent for the selected molecules, exhibiting five pharmacophoric features. The active scaffolds were further evaluated for enzyme inhibition efficacy against alleged receptor Pf-DHFR computationally and in vitro, proving their candidature as lead dihydrofolate reductase inhibitors as well as the selectivity of the test candidates was ascertained by toxicity study against vero cells. The perception of good oral bioavailability was also proved by study of pharmacokinetic properties.

Development and validation of a chiral LC-ESI-MS/MS method for simultaneous determination of the enantiomeric metabolites isosorbide 2-mononitrate and isosorbide 5-mononitrate of isosorbide dinitrate in rat and human plasma.

A specific liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) assay was developed and validated for simultaneous determination of two active metabolites of isosorbide dinitrate (ISDN), namely, isosorbide 2-mononitrate (IS 2-MN) and isosorbide 5-mononitrate (IS 5-MN). A simple protein precipitation extraction technique was employed using 13C6 isosorbide 5-mononitrate as the internal standard. The two isomers were separated on a chiral column and mass detection was carried out by electrospray ionization (ESI) in negative multiple reaction monitoring (MRM) mode (ESI -ve). As neutral organic nitrates do not ionize well in ESI ion source, adduct formation of IS 2-MN and IS 5-MN were evaluated. Acetate adduct ions of IS 2-MN and IS 5-MN were well ionized and fragmentable in the negative mode by liquid chromatography electrospray ionization/tandem mass spectrometry. These acetates adduct ions, of IS 2-MN and IS 5-MN were selected as parent mass for quantitation. The method was developed and validated in rat and human plasma with K2EDTA as an anticoagulant. This simultaneous quantitation method was shown to be linear over a working range of 25.0 ng/mL to 5050 ng/mL and 12.4 ng/mL to 2500 ng/mL for IS 2-MN (r2 > 0.99) and IS 5-MN (r2 > 0.99), respectively, in rat and human plasma. Sensitivity was determined as 25.0 ng/mL for IS 2-MN and 12.4 ng/mL for IS 5-MN in rat and human plasma. Inter- and intra-day accuracy and precision were within ±15% in both method validations. This validated method was subsequently applied to a pharmacokinetic (PK) study of ISDN in rat after oral administration.

A simple and sensitive LC-MS/MS method for quantification of Bepridil in rat plasma and its application to pharmacokinetic studies.

Bepridil is potent inhibitor of Na+, K+ and Ca+ channel in cardiomyocytes. It has demonstrated strong antianginal effect with type I antiarrhythmic and with minimum antihypertensive therapeutic effect. Till date, a specific LC-MS/MS method to quantify Bepridil concentrations in biological matrix have not been reported yet. In current study, a highly sensitive, specific and simple LC-MS/MS method for quantification of antianginal drug Bepridil in rat plasma is presented. The LC-MS/MS method was validated in terms of selectivity, specificity, sensitivity, accuracy and precision, matrix effect, extraction recovery and stability as per USFDA's bioanalytical method validation guideline. The validated assay was applied for quantification of Bepridil from pharmacokinetic study in rats following oral and intravenous administration. The lower limit of quantification (LLOQ) of Bepridil was 1 ng/mL. The calibration curve ranges from 1 ng/mL to 1000 ng/mL with desirable linearity and r2 > 0.99. The method exhibited 10-fold dilution integrity. The intra-day and inter-day accuracy were within 101.32-96.80% and 102.87-95.35% with coefficient of variation 10.11-2.89% and 10.45-3.97% respectively. No significant interference observed by endogenous peak at the retention time of Bepridil and IS. The assay was free from any matrix effect, precise recovery across the calibration curve range and samples were stable under all experimental conditions. The validated assay was successfully applied to analyze plasma samples of pharmacokinetic study in rat to determine concentrations of Bepridil. In summary, a novel method for analyzing Bepridil in rat plasma has been successfully validated and is now being utilized for quantification of Bepridil from pre-clinical studies.

Ionic liquid mediated stereoselective synthesis of alanine linked hybrid quinazoline-4(3H)-one derivatives perturbing the malarial reductase activity in folate pathway.

Grimmel's method was optimized as well as modified leading to the cyclization and incorporation of alanine linked sulphonamide in 4-quinazolin-(3H)-ones. Further, the generation of heterocyclic motif at position-3 of 4-quinazolinones was explored by synthesis of imines, which unfortunately led to an isomeric mixture of stereoisomers. The hurdle of diastereomers encountered on the path was eminently rectified by development of new rapid and reproducible methodology involving the use of imidazolium based ionic liquid as solvents as well as catalyst for cyclization as well as synthesis of imines in situ at position-3 leading to procurement of single E-isomer as the target hybrid heterocyclic molecules. The purity and presence of single isomer was also confirmed by HPLC and spectroscopic techniques. Further, the synthesized sulphonamide linked 4-quinazolin-(3H)-ones hybrids were screened for their antimalarial potency rendering potent entities (4b, 4c, 4 l, 4 t and 4u). The active hybrids were progressively screened for enzyme inhibitory efficacy against presumed receptor Pf-DHFR and h-DHFR computationally as well as in vitro, proving their potency as dihydrofolate reductase inhibitors. The ADME properties of these active molecules were also predicted to enhance the knowhow of the oral bioavailability, indicating good bioavailability of the active entities.

Novel 2,3-disubstituted quinazoline-4(3H)-one molecules derived from amino acid linked sulphonamide as a potent malarial antifolates for DHFR inhibition.

An optimization of a modified Grimmel's method for N-heterocyclization of Leucine linked sulphonamide leading to 2,3-disustituted-4-quinazolin-(3H)-ones was accomplished. Further, nineteen hybrid quinazolinone motifs (5a-5s) were synthesized by N-heterocyclization reaction under microwave irradiation using TEAA (IL) as green solvent as well as catalyst. The in vitro screening of the hybrid entities against the plasmodium species P. falciparum yielded five antimalarial potent molecules 5g, 5l, 5m, 5n &5p owing comparable activity to the reference drugs. The active scaffolds were further evaluated for enzyme inhibition efficacy against alleged receptor Pf-DHFR computationally as well as in vitro, proving their candidature as lead dihydrofolate reductase inhibitors. The prediction of the ADMET properties of the potent molecules also indicated their good oral bioavailability.

Studies on kinetic properties of acid phosphatase from nuclei-free rat liver homogenate using different substrates.

Kinetic properties of rat liver acid phosphatase were evaluated using the conventional synthetic substrates sodium beta glycerophosphate (betaGP) and p-nitrophenyl phosphate (PNPP) and physiologically occurring phosphate esters of carbohydrates, vitamins and nucleotides. The extent of hydrolysis varied depending on the substrates; phosphate esters of vitamins and carbohydrates were in general poor substrates. Kinetic analysis revealed the presence of two components of the enzyme for all the substrates. Component I had low Km and low Vmas. Opposite was true for component II. The Km values were generally high for betaGP, PNPP and adenosine diphosphate (ADP). Amongst the nucleotides substrates AMP showed high affinity i.e. low Km. The increase in enzyme activity in general at high substrate concentration seems to be due to substrate binding and positive cooperativity. AMP which showed highest affinity was inhibitory at high concentration beyond 1 mM. The results suggest that in situ the nucleotides may be the preferred substrates for acid phosphatase.