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).

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.

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.

New potential renin inhibitors with dipeptide replacements in the molecule.

A series of eight non-peptidic potential renin inhibitors have been designed and synthesized. All of them contain dipeptide replacement: (3S,4S)-4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA) in their molecules. Four among them comprise two additional analogs of dipeptide: (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA) and (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (statine, Sta). All of the synthesized compounds contain also hydrophobic portions to receive a moderate lipophilicity of the molecules. Inhibitory activity of the compounds was measured in vitro by I-IPLC determination of Leu-Val-Tyr-Ser released from the N-acetyltetradecapeptide substrate by renin in the presence of the inhibitor. Asp-alpha(OEt)-(S,S)-ACHPA-epsilonAhx-Iaa (23) shows inhibitory activity (7%) at the concentration of 1.0 x 10(-2) M. The other synthesized compounds show no inhibitory activity up to this concentration.

New renin inhibitors with pseudodipeptidic units in P(1)-P(1') and P(2')-P(3') positions.

A series of four new potential renin inhibitors has been synthesized. The structure of the compounds was designed in such a way as to produce agents resistant to enzymatic degradation, metabolically stable, possibly potent and with improved oral absorption. All positions of the 8-13 fragment of the human angiotensinogen were occupied by unnatural units (two unnatural amino acids in positions P(3) and P(2) and two pseudodipeptides in positions P(1)-P(1') and P(2')-P(3')). Both N- and C-terminal functions of the inhibitors were blocked with tert-Boc and ethyl ester groups. Their hydrophobicity evaluated as a log P value, calculated by a computer method, was 6.57 and 6.08 respectively. All peptides were obtained by the carbodiimide method in solution and purified by chromatography on the SiO(2) column. Their resistance to enzymatic degradation was assayed by determination of stability against chymotrypsin activity. The potency was measured in vitro by a spectrofluorimetric method (assay of Leu-Val-Tyr-Ser released from the N-acetyltetradecapeptide substrate by renin in the presence of the inhibitor). All inhibitors were stable to chymotrypsin. Their IC(50) (M/l) values were: 9.6 x 10(-4) (12), 1.6 x 10(-5) (17), 1.0 x 10(-5) (22) and 1.0 x 10(-5) (23) respectively.