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

Qualitative and quantitative methods for analysis of three new amino-acidic anticonvulsants of low neurotoxicity: pyrazole-3,5-dicarboxylic acid benzylamine (1), pyrazine-2-carboxylic acid benzylamine (2) and (R, S) N-acetyl-2-pyrrolidone-5-carboxylic acid benzylamide (3) have been developed. Qualitative analysis included characteristic reactions, chromatographic (TLC) investigations, IR and UV spectra interpretation and quantitative analysis involved spectrophotometric, chromatographic (HPLC) and acidimetric methods.

Amino acid derivatives with anticonvulsant activity.

A series of benzylamides of N-alkylated, N-acylated or free nine cyclic and one linear amino acids as potential anticonvulsants have been synthesized. The structures of the obtained compounds were designed on the basis of the previously determined structure and physicochemical properties/anticvonvulsant activity relationship of the formerly synthesized compounds of this type. The obtained compounds were evaluated in mice after intraperitoneal (i.p.) administration, by maximal electroshock seizure test (MES test), subcutaneous (s.c.) pentylenetetrazol test (s.c. PTZ test) and by the rotarod neurotoxicity test (Tox test). The results were the basis for their classification into one of three classes of the Anticonvulsant Screening Project (ASP) of the Antiepileptic Drug Development Program (ADDP) of the NIH. Three selected compounds were tested quantitatively in rats after oral administration. The MES ED50, s.c. PTZ ED50, Tox TD50 were determined and their protective index (PI) values were calculated. Anticonvulsant activity of the most promising compound (15) was examined in different seizure models. The respective ED50 and PI values of this compound were as follows: against bicuculline, 73 and 1.4; against PTZ, 47 and 2.2; against strychnine, 73 and 1.4; against pilocarpine 156, and 0.7; against kainic acid (2-carboxy-4-isopropenyl-3-pyrrolidineacetic acid), 39 and 2.6; against AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), 10 and 10.3 and against NMDA (N-methyl-D-Aspartic acid), 114 and 0.9.

Gravimetric and spectrophotometric determination of some phenothiazine and imidazole derivatives in coated tablets and tablets.

Two drugs in the form of coated tablets: Promazin (promazine hydrochloride) (1) and Thioridazin (thioridazine hydrochloride) (2), and tablets Clotrimazolum (clotrimazole) (3) were assayed gravimetrically and spectrophotometrically in the same process using complexes with ammonium molybdate. Stoichiometry of these complexes was established by elemental analysis and analysis of the incineration residue (MoO3). The complexes were subsequently characterized using their IR and UV spectra and melting points. The active substances of the complexes were also determined spectrophotometrically. Using this method Beers Law was found to hold for the concentration ranges of 5-40 microg/ml (complex of 1), 5-60 (microg/ml (complex of 2) and 2-10 microg/ml (complex of 3). The method was validated in terms of precision, linearity, detection limit and quantification limit. The two methods of drug determination, used in a single analytical process verify each other.

Gravimetric and spectrophotometric determination of some drugs--tertiary amine hydrochlorides in coated tablets.

Four drugs in the form of coated tablets: Amitryptylinum (Amitryptyline) (1), Imipramin (Imipramine) (2), Chloropyribenzaminum (Chloropyramine) (3), and Phenazolinum (Antazoline) (4) were determined gravimetrically and spectrophotometrically in the same process by using complexes with ammonium molybdate. Stoichiometry of the complexes was established by elemental analysis and analysis of the incineration residue (MoO3). The complexes were characterized by IR and UV spectra and melting points. Contents of the drugs in the complexes were also determined spectrophotometrically. In this method Beer's law was found to obey over the concentration ranges 10-80 micrograms/ml (complex of 1), 10-100 micrograms/ml (complexes of 2 and 3), and 10-60 micrograms/ml (complex of 4). This method was validated in terms of precision, linearity, limit of detection and limit of quantitation. The two methods of the drug determination used in a single process of analysis, verify each other.

Synthesis and anticonvulsant activity of some amino acid derivatives. Part 3: Derivatives of Ala, Arg, Tzl, Gly and chi Abu.

Ten amino acid derivatives as antagonists of the excitatory amino acid (EAA) receptor and anticonvulsant activity have been designed. Five of these compounds supposed to show rather strong and five of them rather weak action as it was expected on the base of their hydrophobicity. All the compounds were synthesized and then evaluated in mice in the maximal electroshock seizure (MES) test, the subcutaneous Metrasol seizure threshold (scMet) test and the rotorod neurotoxicity (Tox) test. Four of the obtained compounds have shown high activity (three received class I and one class II) and six were classified in class III according to the classification of the Anticonvulsant Screening Project (ASP) of the Antiepileptic Drug Development Program (ADDP) of NINDS. One of the compounds classified in class I (10) was tested quantitatively following i.p. administration in mice. It has a MES ED50 = 29.05 b.w. and protective index (PI) of 3.77.

New renin inhibitors with hydrophilic C-terminus.

Four new peptide-based renin inhibitors, Boc-Phe(4-OMe)-MePhe-AHPPA-epsilon Ahx-EA (11), Boc-Phe(4-OMe)-MeLeu-AHP-PA-epsilon Ahx-EA (15), Boc-Phe(4-OMe)-MePhe-Sta-epsilon Ahx-EA (20) and Boc-Phe(4-OMe)-MeLeu-Sta-epsilon Ahx-EA (21) have been synthesized in search of structures with improved biological properties. They were designed as compounds with moderate hydrophobicity (5.28, 4.79, 4.79 and 4.30), respectively. All synthesized inhibitors were resistant to chymotrypsin activity, all were poorly soluble in buffers pH 2.0 and pH 7.4. The inhibitory potency of renin activity in vitro of 11, 15, 20 and 21 expressed as IC50 was 7.0 x 10(-4), 7.5 x 10(-5), 6.0 x 10(-4) and 2.5 x 10(-4) M/l, respectively.

Potential anxiolytic-like activity of some amino acid derivatives.

Three derivatives of amino acids: (R)-Ac-Pro-BZA (1), Ac-beta Ala-BZA (2) and (S)-Ac-Arg(NO2)-BZA (3) were evaluated as potential anxiolytics. In the conflict drinking test in rats used as a model of anxiety, the tested compounds exhibited anxiolytic-like activity. Compounds 1 and 2 administered in doses of 50-100 mg/kg produced an anticonflict effect in a dose-dependent manner. A distinct effect is observed after dose of 100 mg/kg of 1 and 2, comparable to those induced by 10 mg/kg of diazepam. The anticonflict effect of 3 was weak and dose independent. It was revealed only in one mean dose (50 mg/kg) but not in the higher doses.

Synthesis and anticonvulsant activity of some amino acid derivatives. Part 2: Derivatives of Gly, Ala, Leu, Pro, Trp, Phe(4 Cl), Ala(alpha-Me).

Fourteen amides of N-substituted natural and anatural amino acids have been designed and synthesized as potential anticonvulsants. They were evaluated in the maximal electroshock seizure (MES) test, the subcutaneous Metrazol seizure threshold (sc Met) test and the rotorod neurotoxicity (Tox) test. According to the classification of the Anticonvulsant Screening Project (ASP) of the Antiepileptic Drug Development Program (ADDP) eight of synthesized compounds received class I, two class II and four class III. One of the compounds classified in class I (18) was tested quantitatively after i.p. administration in mice. It showed MES ED50 = 67.41 mg/kg and protective index (PI) = 4.5. Conformational models of the synthesized compounds were compared to one another, as well as to models of some standard compounds.

Synthesis and anticonvulsant activity of some amino acid derivatives. Part 1: alanine derivatives.

Fifteen amides of N-substituted D-Ala, DL-Ala and beta Ala have been designed and synthesized as potential anticonvulsants. All obtained amides as well as one intermediate (8) were evaluated in the maximal electroshock seizure (MES) test, the subcutaneous Metrazol seizure threshold (sc Met) test and the rotorod neurotoxicity (Tox) test in mice. According to the classification of the Anticonvulsant Screening Project (ASP) of the Antiepileptic Drug Development Program (ADDP) eight compounds received class I, three class II and five class III designations. Two of the most active compounds (20, 24) were tested quantitatively. They exhibited, after i.p. administration in mice, a large protective index (PI) 3.2 for 20 and 4.3 for 24 and after oral administration in rat PI > 18 for 20 and > 14 for 24.

Renin inhibitors containing nicotinic or isonicotinic acid at the N-terminus. Part V.

Four new compounds: Nic-Phe/4-OMe/-MePhe-Sta-epsilonAhx-OMe/23/,Nic-Phe/4-OMe/-MePhe- Sta-epsilonAhx-Iaa/24/,iNic-Phe/4-OMe/-MeLeu-Sta-ep silonAhx-OMe/29/ and iNic-Phe/4-OMe/-MeLeu-Sta-epsilonAhx-Iaa/30/ have been synthesized in search after renin inhibitors of improved biological properties. Their stability against chymotrypsin activity, solubility in water at pH 7.4, 6.9 and 2.0, partition coefficient and activity in vitro were determined. All synthesized inhibitors are resistant to enzymatic degradation, all are very good soluble in water at pH 2.0, poorly soluble at pH 6.9 and insoluble at pH 7.4. Partition coefficients go up together with increase of pH worth of buffer. IC50 of obtained inhibitors 23,24,29 and 30 is 3 x 10(-4),7.5 x 10(-4),4 x 10(5) and 4 x 10(-3)M/1 respectively.

Enzymatically stable renin inhibitors containing statine and 6 aminohexanoic acid. Part IV.

Eight new peptide renin inhibitors: Boc-Phe/4-OMe/His-Sta-epsilonAhx-Iaa(13), Boc-Phe/4-OMe/-His-Sta-episilonAhx-OMe,(21),Boc-Phe/4-OMe/-MePhe-S ta-epsilonAhx-Iaa(27),Boc-Phe/4-OMe/-MePhe-Sta-epsilonAhx-++ +epsilonAhx-Iaa(32),Boc-Phe/4-OMe/-MePhe-Sta-Val-epsilonAhx- OMe (38),Boc-Phe/4-OMe/-MeVal-Sta-Val-Iaa(48),Boc-Phe/4-OMe/-Me Val-Sta-Iaa(51), Boc-Phe/4-OMe/-MeLeu-Sta-epsilonAhx-Iaa (57) have been synthesized in search after compounds of improved biological properties. All peptides were obtained by carbodimide method in solution by stepwise elongation of the peptide chain or by fragment condensation. Their potency was assayed in vitro by a spectrofluorometric method/assay of Leu-Val-Tyr-Ser released from N-acetyltetradecapeptide substrate by renin in the presence of an inhibitor/. Their resistance to enzymatic degradation was assayed by determination of stability to chymotrypsin activity. The most potent inhibitor was (13):IC50 = 7 x 10(-8)M/1. All inhibitors were stable to chymotrypsin.

Renin inhibitors containing statine and 6-aminohexanoic acid. Part III.

Five peptide renin inhibitors containing the sequence: Phe-His-Sta-epsilon Ahx (Sta = 4(S)-amino-3(S)-hydroxy-6-methylheptanoic acid, epsilon Ahx = 6-aminohexanoic acid) were synthesized and their potency was assayed in vitro by a spectrofluorometric method (assay of Leu-Val-Tyr-Ser released from N-acetyltetradecapeptide substrate by renin in the presence of an inhibitor). Their stability was tested by assay of Phe and Pro-Phe released after incubation with chymotrypsin. The most potent inhibitor was Boc-Phe-His-Sta-epsilon Ahx-OMe (IC50 = 5 x 10(-9) M/l), the most stable--Boc-Pro-Phe-His-Sta-epsilon Ahx-OMe (resistant to incubation with chymotrypsin for 4 h).

Synthesis of enkephalin analogs. Part VI. N,N-disubstituted derivatives.

Synthesis of four new N,N-disubstituted derivatives of enkephalin analogs: All2Tyr-DMet-Gly-Phe-epsilon Ahx-OMe 5, Bu2Tyr-DMet-Gly-Phe-epsilon Ahx-OMe 6, All2Tyr-DMet-Gly-Phe-epsilon Ahx-epsilon Ahx-OMe 11 and Bu2Tyr-DMet-Gly-Phe-epsilon Ahx-epsilon Ahx-OMe 12 is reported. they were tested for agonistic and antagonistic activity. Compound 5 is a little more potent agonist (IC50 = 1.9 x 10(-7) M/l, GPI) than compound 6(IC50 = 7.2 x 10(-7) M/l, GPI). They both are highly selective to mu receptor, because they show no trace of activity to delta receptor in concentration up to 10(-5) M/l. Compound 11 and 12 are less active and not selective as agonists. None of these compounds showed antagonistic activity.

Synthesis of enkephalin analogs. Part V. N-monosubstituted derivatives.

Synthesis of four new derivatives of enkephalin analogs: H-BuTyr-DMet-Gly- Phe-epsilon Ahx-OH 9, H-BzlTyr-DMet-Gly-Phe-epsilon Ahx-OH 12, H-Butyr-DMet-Gly- -Phe-epsilon Ahx-epsilon Ahx-OH 15 and H-BzlTyr-DMet-Gly-Phe-epsilon Ahx-OH is reported. They were examined for agonistic, antagonistic and analgesic activity. Compound 12 is the most potent among investigated peptides. Its agonistic activity in vitro is 7.85 x 10(-8) M/l (GPI) and 9.5 x 10(-7) M/l (MVD). None of the peptides showed antagonistic activity. Only compound 12 showed weak, not dose-dependent analgesic activity in rats.

Synthesis and antienzymic activity of two new angiotensin converting enzyme inhibitors.

Two new analogs of captopril were synthesized. Inhibition of angiotensin converting enzyme (ACE) by these compounds in vitro and in vivo was determined using fluorimetric method. The obtained compounds were much weaker ACE inhibitors than captopril.

Synthesis of two new angiotensin II analogs.

The synthesis of two new angiotensin II analogs: [Des-Arg2, epsilon Ahx1]-angiotensin (II (1) and [Des-His0)-angiotensin (II (2) is reported. Rather strong agonist activity shows analog (1)-78% and weak-analog (2)-30% of the contractile activity of angiotensinamide. None of the synthesized peptides has antagonist activity.

Effects of some new angiotensin II analogs on the reactivity of small arterioles in the mesentery of rat intestine.

The influence of nine new angiotensin II analogs: Asp-Arg-Val-Tyr-Gly-His-Pro-Phe (1-RP), Asp-Arg-Val-DL-p-ClPhe-Ile-His-Pro-Phe (2-RP), epsilon Ahx-Arg-Val-Tyr-Ile-His-Pro-Phe (3-RP), Asp-Arg-Val-Tyr-beta Ala-His-Pro-Ile (4-RP), Asp-Arg-Val-Tyr-Ile-Pro-Phe (5-RP), beta Ala-Arg-Val-Tyr-D-Val-His-Pro-Lac (6-RP), beta Ala-Arg-Val-Tyr-Ile-His-Pro-Lac (7-RP), epsilon Ahx-Val-Tyr-Ile-His-Pro-Phe (8-RP) and Arg-Val-Tyr-Ile-His-Pro-Thr (9-RP) on the contractility of small arterioles in the rat intestinal mesentery was investigated. All the peptides had weaker contractile activity than angiotensinamide. The most potent new peptides presented 56% (1-RP), 67% (3-RP) and 78% (8-RP) of the agonistic activity of angiotensinamide. None of the new angiotensin II analogs had antagonistic activity. Compounds 1-RP and 3-RP presented the tendency to cause tachyphylaxis.