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KTTKS is a matrikine that originates from the proteolytic hydrolysis of collagen. This peptide stimulates ECM production and types I and III collagen expression in vitro. A more stable form of KTTKS is pal-KTTKS, known as Matrixyl ® or palmitoyl pentapeptide-3. A series of novel pentapeptides, analogues of KTTKS with the general formula X-KTTKS-OH(NH 2 ), where X = acetyl, lipoyl, palmitoyl residues, was designed and synthesized. Their effect on amidolytic activity of urokinase, thrombin, trypsin, plasmin, t -PA, and kallikrein were tested. Cytotoxic tests on fibroblasts, as well as collagen and DNA biosynthesis tests for selected peptides, were also carried out. The test results showed that the most active plasmin inhibitors were palmitoyl peptides, whether in acid or amide form. No biological effects of lysine modification to arginine in the synthesized peptides were found. None of the synthesized peptides was not cytotoxic on fibroblasts, and three of them showed cell growth. These three compounds showed no concentration-activity relationship in the collagen and DNA biosynthesis assays.

Fifteen new peptide derivatives of ɛ-aminocaproic acid (EACA) containing the known fragment -Ala-Phe-Lys- with an affinity for plasmin were synthesised in the present study. The synthesis was carried out a solid phase. The following compounds were synthesised: H-Phe-Lys-EACA-X, H-d-Ala-Phe-Lys-EACA-X, H-Ala-Phe-Lys-EACA-X, H-d-Ala-Phe-EACA-X and H-Ala-Phe-EACA-X, where X = OH, NH 2 and NH-(CH 2 ) 5 -NH 2 . All peptides, except for those containing the sequence H-Ala-Phe-EACA-X, displayed higher inhibitory activity against plasmin than EACA. The most active and selective inhibitor of plasmin was the compound H-d-Ala-Phe-Lys-EACA-NH 2 which inhibited the amidolytic activity of plasmin (IC 50  = 0.02 mM), with the antifibrinolytic activity weaker than EACA. The resulting peptides did not affect the viability of fibroblast cells, colon cancer cell line DLD-1, breast MCF-7 and MDA-MB-231 cell lines.

The amino analogues of pentamidine with a polymethylene (n = 3 - 6) chain and their chlorambucil derivatives were synthesized. The obtained compounds revealed cytotoxic effect on MCF-7 human breast cancer cell line (IC50 = 22 - 95 +/- 2 pM), mainly by the induction of apoptosis. The topoisomerase I/II inhibition assay and the ethidium displacement assay with the use of pBR322 plasmid DNA were used to the study of mechanism by which the obtained compounds could act. All the compounds are able to bind with DNA and interfere in vitro with the activity of topoisomerase (I and II). The determination of association constants with the use of calf thymus DNA, T4 coliphage DNA, poly(dA-dT)2 and poly(dG-dC)2 showed that the tested compounds bind within minor groove of B-DNA, but not selectively. The alkylating activity of chlorambucil derivatives determined in vitro using a Preussmann test was similar to the activity of chlorambucil. The influence of all the compounds on the amidolytic activity of plasmin and trypsin was also examined. The plasmin activity was inhibited by pentamidine, chlorambucil and aromatic bis-amines (IC50 = 0.1 - 8 mM), whereas the trypsin activity was influenced only by pentamidine.

The biological evaluation of carbocyclic minor groove binders 1-6 is described. The cytotoxicity of the obtained compounds was tested on MDA-MB-231 breast cancer cells. The mechanism of action of compounds 1-6 was studied employing the topoisomerase I/II inhibition assay and ethidium displacement assay using pBR322. Determination of association constants was done using calf thymus DNA, T4 coliphage DNA, poly(dA-dT)(2), and poly(dG-dC)(2). The effect of compounds 1-6 on the amidolytic activity of plasmin, trypsin, thrombin, and urokinase was also examined.

Twelve peptides of the general X-SO(2)-D-Ser-Ala-Arg-OH formula (where X = methyl, phenyl, α-tolyl, p-tolyl, 4-methylbenzyl, 1-naphtyl, 2-naphtyl, 4-chlorophenyl, 4-bromophenyl, 2-mesityl, 2,4,6-triisopropylphenyl, 4-acetamidophenyl) were obtained and tested for their effect on the amidolytic activities of urokinase, thrombin, trypsin, plasmin, t-PA and kallikrein. 2,4,6-triisopropylphenyl-SO(2)-D-Ser-Ala-Arg-OH was the most selective inhibitor of urokinase and α-tolyl-SO(2)-D-Ser-Ala-Arg-OH was the most active inhibitor of uPA with K(i) value 24 µM. The compounds were tested for their in vitro antitumour activity in the following human breast cancer cells: standard MCF-7 and estrogen-independent MDA-MB-231. Four of the synthesized peptides showed cytotoxic effects against MDA-MB-231 cell lines in the range from 2.9 to 8.5 µM. The examined compound did not influence to MCF-7 cancer cells. The synthesized peptides were nontoxic to pig's erythrocytes.

Eleven peptides of the general formula H-d-Ser-Ala-Arg-NH-X, where X = (CH2)n-NH2, n = 2-9, (CH2)m-OH, m = 2-4, were obtained and tested for their effect on the amidolytic activities of urokinase, thrombin, trypsin, plasmin, t-PA, and kallikrein. H-D-Ser-Ala-Arg-NH-(CH2)5-NH2 inhibited urokinase with a Ki value of 6.3 microM.

Eight short peptides containing L-lysine and epsilon-aminocaproic acid were obtained and their effect on the amidolytic activities of plasmin, thrombin and trypsin was examined. Tripeptide amide Boc-EACA-L-Lys-EACA-NH2 was the most effective and specific plasmin inhibitor.

The effect of epsilon-aminocaproyl-S-benzyl-L-cysteine on the activation of plasminogen by t-PA. streptokinase and urokinase has been examined using fibrinolytic method. The obtained results have been compared with the obtained results for epsilon-aminocaproic acid and trans-4-(aminomethyl)cyclohexanecarboxylic acid. The inhibition of the plasminogen activation determined with the use of epsilon-aminocaproyl-S-benzyl-L-cysteine was weaker than the inhibition determined by using antifibrinolytic aminoacids.

Five substituted amides of lysine with the general formula: X-Lys-NH-Y, where X= acetyl or ethoxycarbonyl, Y= cyclohexyl, benzyl, hexyl or cadaverine residue were synthesised and their effects on fibrinolytic activity of plasmin, clotting activity of thrombin and amidolytic activities of both enzymes were examined.

Inhibitory effects of nine carbocyclic DNA minor groove binders on amidolytic activities of plasmin, trypsin and urokinase were examined. Some of the studied compounds affected plasmin or trypsin activity, but not urokinase activity. One of the pentamidine analogues (5) and two bis-netropsin like compounds (6, 8) were potent inhibitors of plasmin (IC50 equals 90 and 100 microM), whereas an analogue of netropsin (2) was trypsin inhibitor (IC50 = 100 microM).

Ten peptides of the general formula A-Phe-Lys-X where A = H, H-D-Val, H-L-Val, H-D-Ala, H-L-Ala and X = OH, NH2 were obtained and tested for their antiplasmin activity with the use of amidolytic test.

A series of peptide methylketones with generalformula Y-Phe-Lys-CH3 was prepared as potential inhibitors of plasmin.

Effect of three epsilon-aminocaproylamino acids with significant antifibrinolytic activity on polymerization of fibrin monomer, clot retraction, fibrin structure, prothrombin consumption and thrombin activity was examined. epsilon-Aminocaproyl-L-norleucine and epsilon-aminocaproyl-L-leucine were weak inhibitors of thrombin activity and epsilon-aminocaproyl-L-norleucine slightly inhibited polymerization of fibrin monomers.

Four dipeptide alkylamides with general formula H-D-Phe-L-Lys-NH-X, where X = cyclohexyl, - (CH2)5NH2, -(CH2)2-OH and hexyl were obtained. Effect of these compounds on amidolytic and fibrinolytic activity of plasmin was examined.

Effect of three epsilon-aminocaproylaminoacids with a significant antifibrinolytic activity on amidolytic activity of tissue plasminogen activator (t-PA), urokinase and kallikrein was examined. epsilon-Aminocaproyl-S-benzyl)-L-cysteine and epsilon-aminocaproyl-L-norleucine were weak inhibitors of kallikrein. Weak activation of t-PA activity was observed at high concentration of the tested compounds. Only one of the examined dipeptides was a weak inhibitor of amidolytic activity of urokinase.

Four benzylamides of dipeptides with the general formula: X-L-Lys-NH-CH2-C6H5, where X = L-or D-Leu and L-or D-Phe were prepared as potential inhibitors of plasmin. All of them influenced on the fibrynolytic activity of plasmin, but only D-Leu-L-Lys-NH-CH2-C6H5 inhibited the amidolytic activity of this enzyme. None of the tested compounds was an inhibitor of thrombin in an amidolytic test.

Effect of three epsilon-aminocaproylaminoacids with significant antifibrinolytic activity on chymotrypsin, trypsin, cathepsin B, cathepsin C and cathepsin D activities was examined. Slight inhibition of trypsin and chymotrypsin activity was observed only at high concentrations of these compounds. All tested dipeptides did not influence activities of cathepsin B, cathepsin C and cathepsin D.

Influence of four epsilon-aminocaproylaminoacids on prothrombin activation and thrombin activity was examined. Only epsilon-aminocaproylnorleucine markedly inhibited the prothrombin activation in an extrinsic system.

Derivatives of epsilon-aminocaproic acid with antifibrinolytic activity, at low concentration, do not influence the anticoagulant activity of heparin under the heparin-thrombin test conditions. At concentrations higher than 0.002 M tested compounds slightly enhance the anticoagulant action of heparin.

A series of tripeptide methylketones with C-terminal lysine was obtained via Dakin-West method and tested for their antiplasmin activity with the use of antifibrinolytic and antiamidolytic tests. The tripeptide methylketones has been found to inhibit plasmin, however with much lower potency than respective aldehydes.

Five new derivatives of epsilon-aminocaproyl-L-norleucine were synthesized. Antifibrinolytic and anticaseinolytic activities were tested. All tested compounds were inhibitors of plasmin.

Amino acids containing sulphur, dipeptide derivatives of methionine and S-substituted derivatives of cysteine are potent antifibrinolytic agents. The structural moiety of the substances responsible for the effect on the clot formation is not known. Present study was undertaken in order to evaluate the effect of some analogues of dipeptides containing S-substituted derivatives of cysteine with the formula A-Cys(S-X)-Y (where A-amino acid, X-benzyl, butyl, hexyl, nonyl and Y-OH or OMe) on clot dissolution under the antifibrinolytic test conditions. It has been found that dipeptide derivatives of S-substituted cysteine (except benzyl derivative) at low concentration evoke antifibrynolytic activity, while at high concentration they prevent clot formation. The results suggest that antifibrinolytic activity of tested compounds at low concentration may be due to the formation of antifibrinolitycally active conformation, while high concentration overcome the effect.

Four new dipeptides containing epsilon-aminocaproic acid were synthesized. Antibrinolytic, antiacaseinolytic activity and influence on activation of plasminogen by streptokinase were tested.

Five new dipeptides containing epsilon-aminocaproic acid were synthesized. All dipeptides markedly inhibit fibrinolytic activity of plasmin but only in low concentration (0.0002M). In higher concentration fibrinolytic activity was observed. Activity of dipeptides containing epsilon-aminocaproic acid, which antifibrinolytic activity is known, was tested on plasmin, thrombin, urokinase and euglobulin fraction using synthetic substrates.

Three new dipeptides of E-aminocaproic acid were synthesized and their antifibrinolytic activity was examined. H-EACA-L-Trp-OH possess stronger antifibrinolytic activity than H-EACA-L-Tyr-OH and H-EACA-L-Leu-OH. Antifibrinolytic activity of H-EACA-L-Gly-OH was smallest.

Seven peptides of the general formula A-Phe-Lys-X or A-Phe-NH-(CH2)5-NH2 where A = Glp or Ala, X = -OH, -NHC7H15 or -OC7H15 were obtained. The peptide H-Ala-Phe-Lys-NHC7H15 markedly inhibits the fibrinolytic activity of plasmin. The antifibrinolytic activity of the peptide Glp-Phe-Lys-OC7H15 is similar to EACA. The both peptides only slightly activate the caseinolytic activity of plasmin.

New peptides containing E-aminocaproic acid were synthetized. Some conclusions on structure-activity relationship were made.