NEW RENIN INHIBITORS - STABILITY AND ACTIVITY DETERMINATION. PART IV.

A series of new seven potential renin inhibitors containing pseudodipeptides were synthesized. Stability for all compounds (1-7) in homogenates of liver, kidney, lung and in serum, gastric, intestinal juice and in the presence of α-chymotrypsin was determined. Compound 1 was unstable in all determined mediums. Compounds 2, 4, 5 and 7 were unstable, compound 3 was stable, compound 6 was unstable only in α-chy-motrypsin solution. Inhibitory activity of the compounds was measured in vitro by HPLC determination of low-ering concentration of substrate (angiotensinogen) in the presence of renin and the potential renin inhibitor (compounds 1-7). Compounds 1, 2, 4, 5, 6 and 7 showed inhibitory activity (1.12 x 10⁻7, 0.96 x 10⁻6, 1.58 x10⁻7,1.68 x 10⁻6, 1.30 x 10⁻6, 0.96 x 10⁻7M, respectively).

SIMULTANEOUS ASSAY OF ARIPIPRAZOLE AND ITS ACTIVE METABOLITE IN SERUM BY HPLC.

A rapid, convenient, precise HPLC method was developed for the simultaneous determination of aripiprazole and its active metabolite dehydroaripiprazole in blood serum. The separation was carried out by RP HPLC on Symmetry C18 column (150 x 4.6 mm; 5 µm). The mobile phase was composed of acetonitrile: water (30 : 70, v/v), pH 3.0 adjusted with o-phosphoric acid. The detection was monitored at 220 nm.

NEW RENIN-INHIBITORS--STABILITY AND ACTIVITY DETERMINATION. PART III.

A series of new four potential renin inhibitors containing pseudodipeptides were synthesized. Stability for all compounds (1-4) in homogenates of liver, kidney, lung and in serum, gastric, intestinal juice and in the presence of α-chymotrypsin was determined. Compound 1 was unstable, compounds 2, 3 were stable, compound 4 was partly unstable, (liver and kidney homogenates, (α-chymotrypsin solution). Inhibitory activity of the compounds was measured in vitro by HPLC determination of lowering concentration of substrate (angiotensinogen) in the presence of renin and the potential renin inhibitor (compounds 1-4). Compound 1, 2, 3 and 4 showed inhibitory activity (1.7 x 10(-6), 9.6 x 10(-7), 1.05 x 10(-9) and 1.31 x 10(-7)M, respectively).

Novel renin inhibitors containing derivatives of N-alkylleucyl-ß-hydroxy-γ-amino acids.

In search for new drugs lowering arterial blood pressure, which could be applied in anti-hypertensive therapy, research concerning agents blocking of renin-angiotensin-aldosteron system has been conducted. Despite many years of research conducted at many research centers around the world, aliskiren is the only one renin inhibitor, which is used up to now. Four novel potential renin inhibitors, having structure based on the peptide fragment 8-13 of human angiotensinogen, a natural substrate for renin, were designed and synthesized. All these inhibitors contain unnatural moieties that are derivatives of N-methylleucyl-ß-hydroxy-γ-amino acids at the P2-P1' position: 4-[N-(N-methylleucyl)-amino]-3-hydroxy-7-(3-nitroguanidino)-heptanoic acid (AHGHA), 4-[N-(N-methylleucyl)-amino]-3-hydroxy-5-phenyl-pentanoic acid (AHPPA) or 4-[N-(N-methylleucyl)-amino]-8-benzyloxycarbonylamino-3-hydroxyoctanoic acid (AAHOA). The previously listed synthetic ß-hydroxy-γ-amino acids constitute pseudodipeptidic units that correspond to the P1-P1' position of the inhibitor molecule. An unnatural amino acid, 4-methoxyphenylalanin (Phe(4-OMe)), was introduced at the P3 position of the obtained compounds. Three of these compounds contain isoamylamide of 6-aminohexanoic acid (ε-Ahx-Iaa) at the P2'-P3' position. The proposed modifications of the selected human angiotensinogen fragment are intended to increase bioactivity, bioavailability, and stability of the inhibitor molecule in body fluids and tissues. The inhibitor Boc-Phe(4-OMe)-MeLeu-AHGHA-OEt was obtained in the form of an ethyl ester. The hydrophobicity coefficient, expressed as log P varied between 3.95 and 8.17. In vitro renin inhibitory activity of all obtained compounds was contained within the range 10(-6)-10(-9) M. The compound Boc-Phe(4-OMe)-MeLeu-AHPPA-Ahx-Iaa proved to be the most active (IC50 = 1.05 × 10(-9) M). The compounds Boc-Phe(4-OMe)-MeLeu-AHGHA-Ahx-Iaa and Boc-Phe(4-OMe)-MeLeu-AHPPA-Ahx-Iaa are resistant to chymotrypsin.

Stability of new anticonvulsant derivatives of picolinic, nicotinic, cyclocarboxylic acids in body fluids and tissues.

The stability of new compounds with established anticonvulsant activity: picolinic acid 4-pyridyl-methylamide (Pic-4-PMA), cyclopentanecarboxylic acid benzylamide (Cpc-BZA), cycloheptanecarboxylic acid benzylamide (Chc-BZA), picolinic acid 2-fluoro-3-trifluoromethylbenzylamide (Pic-2F-3TFM-BZA), 2-chloronicotinic acid benzylamide (2-Cl-Na-BZA), 6-chloronicotinic acid benzylamide (6-Cl-Na-BZA) and 6-trifluoromethylnicotinic acid benzylamide (6-TFM-Na-BZA) in homogenates of body organs and in body fluids was determined after incubation. It was found that three compounds were stable against enzymes present in body fluids and organs and two were found to decompose in liver and kidney homogenates and two decomposed only in liver homogenate.

New renin inhibitors--stability and activity determination. Part I.

A series of new six potential renin inhibitors containing pseudodipeptides were synthesized. Stability for all compounds (1-6) in homogenates of liver, kidney, lung and in serum, gastric, intestinal juice and in the presence of alpha-chymotrypsin was determined. Compound 5 was unstable, compound 6 was stable, other compounds were partly unstable, compound 2 was stable except kidney homogenate and compound 4 was stable except liver homogenate. Inhibitory activity of the compounds was measured in vitro by HPLC determination of lowering concentration of substrate (angiotensinogen) in the presence of renin and the potential renin inhibitor (compounds 1-6). Compound 2, 4 and 6 showed inhibitory activity (1.4 x 10(-6), 5.2 x 10(-6), 1.5 x 10(-7) M, respectively). Other compounds (1, 3, 5) showed no inhibitory activity up to 10(-5) M.

New renin inhibitors--stability and activity determination. Part II.

A series of new six pseudodipeptic potential renin inhibitors were synthesized. Enzymatic stability for all compounds (1-6) in homogenates (liver, kidney, lung) and body fluids (serum, gastric, intestinal juice) and alpha-chymotrypsin was determined. Compounds 4 was stable, compound 5 was unstable and compounds 1, 2, 3, 6 were partly unstable. Inhibitory activity of the compounds was measured in vitro by HPLC determination of lowering concentration of substrate (angiotensinogen) in the presence of renin and the potential renin inhibitor (compounds 1-6). Compound 4, 5, 6 showed inhibitory activity (0.9 x 10(-6), 1.3 x 10(-8), 2.2 x 10(-6) M, respectively). Other compounds showed no inhibitory activity up to 10(-5) M.

New renin inhibitors containing phenylalanylhistidyl-gamma-amino acid derivatives in P3 - P1' position.

Five potential inhibitors of renin have been designed and obtained. In the molecule position P3 - P1', crucial for indicating inhibitory activity, all contain phenylalanylhistidylaminoalcanoyl group, ready for interaction with the hydrophobic pocket S3 - S1 of renin molecule. The aminoalcanoyl fragment consists of pseudo-dipeptidic units derivative of gamma-amino acids: of 4-amino-3-hydroxybutanoic acid (AHBA) [26], 4-amino-5-(4-ethoxyphenyl)-3-hydroxypentanoic acid (AEPHPA) [13], 4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA) (1) and 4-amino-3-hydroxynonanoic acid (AHNA) [21]. At the P3 - P2 position of obtained compounds an unnatural fragment, derivative of Phe-His dipeptide, was placed ande isoamyl amid of 6-amino-hexanoic acid was attached at the end of the molecule (epsilonAhx-Iaa). The preliminary in vitro tests indicated that all compounds were inactive. However, they provided valuable information on P3 - P2 fragment possible structure modification able to produce a resonable renin activity inhibition. All synthesized inhibitors were chymotrypsin-resistant.

Metabolic stability of new anticonvulsants in body fluids and organ homogenates.

The stability as a function of time of compounds with established anticonvulsant activity: picolinic acid benzylamide (Pic-BZA), picolinic acid 2-fluorobenzylamide (Pic-2-F-BZA), picolinic acid 3-fluorobenzylamide (Pic-3-F-BZA), picolinic acid 4-fluorobenzylamide (Pic-4-F-BZA) and picolinic acid 2-methylbenzylamide (Pic-2-Me-BZA) in body fluids and homogenates of body organs were determined after incubation. It was found that they decompose relatively rapidly in liver and kidney and are stable against enzymes present in body fluids and some organs. These results are consistent with the bond strength expressed as total energy of amide bonds (calculated by quantum chemical methods) in the studied anticonvulsants. The calculated values of the amide bond energy are: 199.4 kcal/mol, 200.2 kcal/mol, 207.5 kcal/mol, 208.4 kcal/mol and 198.2 kcal/mol, respectively. The strength of the amide bonds in the studied anticonvulsants correctly reflects their stability in liver or kidney.

Stability of two new anticonvulsants in body fluids and tissues.

Stability of two compounds with established anticonvulsant activity, picolinic acid benzylamide (Pic-BZA) and nicotinic acid benzylamide (Nic-BZA), incubated in homogenates of body organs and in body fluids was determined at different time points. Pic-BZA was found to decompose fairly rapidly in the liver and the kidney, while Nic-BZA demonstrated stability against enzymes present in these organs and fluids.

Qualitative and quantitative analysis of three amino acid derivatives of anticonvulsant activity.

Qualitative and quantitative methods of analysis of three new amino-acid derivatives potential anticonvulsants of low neurotoxicity: picolinic acid benzylamide (1), nicotinic acid benzylamide (2) and isonicotinic acid benzylamide (3) were developed. Qualitative analysis includes characteristic reactions, chromatographic (TLC) investigations, IR and UV spectra interpretation. The quantitative analysis involves spectophotometric, chromatographic (HPLC) and acidimetric methods.