Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 65 No. 3 pp. 377ñ381, 2008
ISSN 0001-6837 Polish Pharmaceutical Society
BIOLOGICAL ACTIVITY OF AMIDE DERIVATIVES OF LYSINE KRYSTYNA MIDURA-NOWACZEK*, IZABELA LEPIETUSZKO, IRENA BRUZGO and AGNIESZKA MARKOWSKA Department of Organic Chemistry, Medical University of Bia≥ystok, 1 KiliÒskiego Str., Poland Abstract: 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. Keywords: lysine derivatives, fibrinolytic activity, anticlotting activity.
of dipeptide derivatives with C-terminal lysine cyclohexyl, benzyl and hexyl amides (6, 7), as well as dipeptides and tripeptides with C-terminal unsubstituted amide or carboxyl groups (8). Compounds containing cadaverine residue connected with the lysine were also tested (6, 7). Some of these compounds inhibited amidolytic and fibrinolytic activities of plasmin. However, in some cases, at the highest examined concentration (0.02 M), the clot formation was not observed although the examined dipeptides did not inhibit amidolytic activity of thrombin. In order to explain this unexpected biological activity, and in the search of new low molecular plasmin inhibitors, we synthesized five lysine derivatives with the general formula: A-Lys(X)-NHY, where X = acetyl or ethoxycarbonyl; Y = cyclohexyl, benzyl, hexyl or cadaverine residue (Table 1.) and examined their effect on the fibrinolytic activity of plasmin, the clotting activity of thrombin and amidolytic activities of both enzymes. The obtained results were compared with the literature value of Ntosyllysine-p-nitroanilide (Tos-Lys-pNA) activity. This compound is the inhibitor of the fibrinolytic and amidolytic activities of plasmin (2).
Plasmin, a key enzyme for fibrynolysis, also plays an important role in a variety of biological processes like wound healing, tissue repair, cell migration, as well as in pathological phenomena such as inflammation, tumor cell growth and metastasis. At present, ε-aminocaproic acid (EACA) and transaminomethylcyclohexanecarboxylic acid (AMCHA) are clinically used as plasmin inhibitors. These ωamino acids inhibit the fibrinolytic activity of plasmin by blocking the lysine binding sites of the enzyme. Their activity towards plasmin with respect to fibrinogen, other proteins and synthetic substrates is much weaker than towards fibrin. The synthesis of the active center directed inhibitor of plasmin is a research field with an aim to obtain an inhibitor of this enzyme in order to influence not only fibrinolysis but also amidolysis and proteolysis. The inhibitor controlling such plasmin activity will be very useful in defining the physiological and the pathological function of this enzyme and treating plasmin-associated disorders. Plasmin has P1 preference for lysine (1). Derivatives of this amino acid are widely examined as potential synthetic substrates and inhibitors of the enzyme. Substituted anilides of lysine and short peptides inhibited the amidolytic and fibrinolytic activities of plasmin (2) but heptyl amides of tripeptides: H-Ala-Phe-Lys-NHC7H15 (3) and H-DVal-Leu-Lys-NHC7H15 (4) showed only the antifibrinolytic activity. N-Substituted derivatives of lysine are potent inhibitors of proteolytic and fibrinolytic activities of plasmin but sometimes also slight activation was observed (5). During our earlier investigations at active centre directed plasmin inhibitors, we examined a series
EXPERIMENTAL Synthesis of the compounds The examined compounds were obtained from respective Boc derivatives (6, 7). The t-butoxycarbonyl group was removed with the use of HCl saturated solvents. Acetyl derivatives were obtained in reaction with acetic anhydride and ethoxycarbonyl derivative in reaction with ethyl chloroformate in the
* Corresponding author: e-mail: [email protected]
KRYSTYNA MIDURA-NOWACZEK et al.
presence of triethylamine. The benzyloxycarbonyl group was removed by catalytic hydrogenation. Organic solutions were dried over anhydrous MgSO4. Homogeneity of the products was examined on silica gel plates (Kiesegel 60 F254, Merck) using the following solvent systems: 1: benzene/methanol/acetic acid (12:5:1, v/v/v), 2: ethanol/water/25% ammonia solution (18:0.5:0.5, v/v/v), 3: chloroform/methanol/acetic acid (17:2:1, v/v/v). Spots were visualized with iodine and ninhydrin The melting points were determined on Boetius block and are uncorrected. The specific optical rotation was measured with a polarimeter (Optical Activity LTD AA-10R). 1H NMR spectra were recorded with 200 MHz Bruker AC 200F spectrometer. Elemental analyses were performed on PerkinElmer analyzer and results were within ± 0.4 % of theoretical values. Physicochemical data are given in Tables 2 and 3. Enzymatic investigation General Plasmin, S-2251 (H-D-Val-L-Leu-L-LyspNA∑2HCl) and S-2238 (H-D-Phe-Pip-Arg-pNA ◊ 2HCl) (Chromogenix), thrombin and bovine fibrinogen (Lubelska WytwÛrnia Szczepionek, Lublin, Poland). Effects of the obtained compounds on clotting and amidolytic activities of thrombin and fibrinolytic and amidolytic activities of plasmin were examined with the use of standard methods. Detailed descriptions of the methods are given below. The results are presented in Table 4. Every value represents the average of triplicate determinations ± SD.
Plasmin activity Fibrinolytic activity All examined substances were dissolved in 0.15 M NaCl solution, other reagents were dissolved in Palitsch buffer pH 7.4. 0.1 cm3 of examined compound (in the control ñ 0.1 cm3 of 0.15 M NaCl) and 0.1 cm3 of fibrinogen (0.5%) was added to 0.1 cm3 of thrombin (40 units/ cm3). After clot formation, 0.2 cm3 of plasmin (0.2%) was added and the time of fibrinolysis was measured at 37OC. IC50 value was considered as the concentration of examined compound, which decreased the time of fibrinolysis by 50%, compared with the value measured under the same condition without the examined compound. The results are given in Table 4. Amidolytic activity determined with the use of synthetic substrate S-2251. 0.2 cm3 of examined compounds (in control 0.2 3 cm of 0.15 M NaCl) was added to the mixture of 0.1 cm3 plasmin solution (0.05 units / cm3) and 0.5 cm3 of Tris buffer (pH 7.4). After preincubation for 3 min at 37OC, 0.2 cm3 of S-2251 solution (3 mM) was added. The mixture was incubated for 15 min at 37OC, then the reaction was stopped by the addition of 0.1 cm3 of 50% acetic acid and absorbance at 405 nm was measured. IC50 value was considered as the concentration of inhibitor, which decreased the absorbance at 405 nm by 50%, compared with the absorbance measured under the same condition without inhibitor. The results are given in Table 4.
Table 1. Structure of the obtained lysine amide derivatives A-Lys(Y)-NH-X.
Ac = acetyl, Ec = ethoxycarbonyl, C6H11 = cyclohexyl, Bzl = benzyl C6H13 = hexyl, (C5H10)-NH2 = cadaverine
Biological activity of amide derivatives of lysine
Table 2. Physicochemical data of protected lysine amide derivatives.
(1) 0.63 (3) 0.59
(1) 0.6 (3) 0.54
(1) 0.62 (3) 0.58
(1) 0.61 (3) 0.55
(1) 0.77 (3) 0.78
-25.3 210-214 (c =1, MeOH)
-16.4 174-176 (c =1, MeOH)
-19.2 169-172 (c =1, MeOH)
-15.6 121-126 (c =1, MeOH)
-17.8 129-133 (c =1, MeOH)
Thrombin activity Clotting activity determined with the use of fibrinogen. All examined substances were dissolved in 0.15 M NaCl solution, other reagents were dissolved in Palitsch buffer pH 7.4. 0.1 cm3 of examined compound (in the control ñ 0.1 cm3 of 0.15 M NaCl) was added to 0.3 cm3 of fibrinogen (0,5%). After 1 min preincubation at 37OC, 0.1 cm3 of thrombin solution was
H NMR (DMSO-d6) [ppm]
8.44-8.38 (t, 1H, NH), 8.01-7.96 (d, 1H, NH), 7.38-7.21 (m, 6H ZC6H5, NH), 5.03-4.95 (s, 2H, ZCH2), 4.28-4.21 (m, 2H, cyclohexyl CH, Lys α CH), 2.97-2.91 (m, 2H, Lys ε CH2,), 1,85-1,82 (s, 3H, acetyl CH3), 1.62-1.23 (m, 16H, Lys βCH2, γCH2, δCH2, cyclohexyl 5x CH2) 8.44-8.38 (t, 1H, NH), 8.01-7.79 (d, 1H, NH), 7.34-7.21 (m, 11H, Z C6H5, Bzl C6H5, NH), 5.02-5.0 (s, 2H, Z CH2), 4.27-4.21 (m, 3H, BzlCH2, Lys α CH), 2..97-2..91 (m, 2H, Lys ε CH2,), 1,85-1,82 (s, 3H, acetyl CH3), 1.62-1.27 (m, 6H, Lys β CH2, γCH2, δCH2) 7.88 -7.77 (m, 2H, 2×NH), 7.367.18 (m, 6H, Z C6H5, NH), 5.085.01 (s, 2H, Z CH2), 4.16-4.09 (m, 1H, Lys α CH), 3.04-2.91(m, 4H, Lys ε CH2, hexyl, NCH2 ), 1.831.56- (s, 3H, acetyl CH3) 1.56-1.23 (m, 14H, Lys βCH2, γCH2, δCH2, hexyl 4×CH2), 0.88-0.82 (t, 3H, hexyl CH3) 7.94-7.86 (t, 2H, NH), 7.42-7.20 (m, 12H Z C6H5, 2×NH), 5.09-5.01(s, 4H, Z CH2), 4.20-4.09 (m, 1H, Lys αCH), 2.98-2.95 (m, 6H, Lys ε CH2, cadaverine 2×CH2 N), 1.831.77 (s, 3H, Ac CH3), 1.60-1.14 (m, 12H, Lys βCH2, γCH2, δCH2, cadaverine 3×CH2) 7.76-7.71 (t, 1H, NH), 7.36-7.30 (m, 5H, Z C6H5,), 7.10-7.05 (t, 1H, NH), 7.03- 6.99 (d,1H, NH), 5.085.01 (s, 2H, Z CH2), 4.0-3.84 (m, 3H, Lys α CH, ethoxycarbonyl CH2), 3.04-2.91 (m, 4H, Lys ε CH2, hexyl CH2N ), 1.52-1.11 (m, 17 H, Lys βCH2, γCH2, δCH2, hexyl 4×CH2, ethoxycarbonyl CH3), 0.88-0.82 (t, 3H, hexyl CH3)
added and clotting time was measured. IC50 value was determined by the Kawasaki method (9). From the standard curve of thrombin amount against clotting time, the clotting time of each specimen was converted to the thrombin amount and then the percent inhibition was calculated. Concentration of examined compound was then plotted against percent inhibition and the IC50 value, concentration of each examined compound exhibiting 50% inhibition, was estimated. The results are presented in Table 4.
KRYSTYNA MIDURA-NOWACZEK et al.
Table 3. Lysine amide derivatives.
C14H27N3O2 × 1.5 CH3COOH
C15H23N3O2 × 1.5 CH3COOH
C14H29N3O2 × CH3COOH
C15H31N3O3 × 2 CH3COOH
(2) 0.17 (3) 0.29
(2) 0.17 (3) 0.3
(2) 0.19 (3) 0.22
(2) 0.1 (3) 0.2
(2) 0.21 (3) 0.28
-24.3 185-187 (c =1, MeOH)
-26.2 127-129 (c =1, MeOH)
-20.8 (c =1, MeOH)
-2.4 amorph. (c =1, MeOH)
-16.6 (c =1, MeOH)
H NMR (DMSO-d6) [ppm]
7.96-7.92 (d, 1H, NH), 7.81-7.77 (d, 1H, NH), 5.44 (s, 3H NH3+), 4,24-4,13 (m, 1H, Lys 〈CH), 3.36-3.66 (m, 1H, cyclohexyl CHN), 2.64-2.48 (m, 2H, Lys εCH2), 1.83 (s, 3H, acetyl CH3), 1.77-1.2 (m,16H, cyclohexyl 5◊CH2, Lys βCH2, γCH2, δCH2) 8.56-8,48 (t, 1H, NH), 8.13-8.09 (d, 1H, NH), 7.35-7.21 (m, 5H, C6H5), 5.09 (s, 3H, NH3+), 4.28-4.22 (m, 3H, CH2 benzyl, Lys αCH), 1.86 (s, 3H, acetyl CH3), 1.68-1.22 (m, 6H, Lys βCH2, γCH2, δCH2) 8.03 (d, 1H, NH), 7.96 (t, 1H, NH), 5.87 (s, 3H, NH3+), 4.21-4.01 (m, 1H, Lys αCH), 3.04-2.98 (m, 2H, hexyl CH2-N ), 2.59-2.48 (m, 2H, Lys εCH2), 1.83 (s, 3H, acetyl CH3), 1.771.24 (m, 14H, hexyl, 4◊CH2, Lys βCH2,γCH2, δCH2), 0.89-0.82 (t, 3H, hexyl CH3) 8.34-8.12 (m, 1H, NH), 8.12-8.01 (m, 7H, NH, 2◊NH3+), 3.9-4.2 (m, 1H, Lys αCH), 3.03-3.0 (m, 2H, Lys ε CH2), 2.78-2.6 (m, 4H, cadaverine 2x CH2N), 1.85 (s, 3H, acetyl CH3), 1.581.2 (m,12H, Lys βCH2, γCH2, δCH2, cadaverine 3◊CH2) 7.93 (t, 1H, NH), 7.17 (d, 1H, NH), 5,62 (s, 3H, NH3+), 4.01-3.85 (m, 3H, ethoxycarbonyl CH2, Lys αCH), 3.043.01 (m, 2H, Lys ε CH2 ), 2.76-2.56 (m, 2H, hexyl CH2-N), 1.51-1.11 (m, 17H, ethoxycarbonyl CH3, Lys βCH2, γCH2, δCH2, hexyl 4◊CH2), 0.85 (t, 3H, hexyl CH3)
Table 4. Biological activity of lysine amide derivatives.
Amidolytic activity of plasmin (S-2251)
0.78 ± 0.05
1.4 ± 0.09
10 ± 0.7
0.44 ± 0.02
1.0 ± 0.06
9 ± 0.56
0.38 ± 0.02
1.6 ± 0.1
6 ± 0.42
0.58 ± 0.04
0.8 ± 0.05
8 ± 0.56
0.39 ± 0.02
1.8 ± 0.1
12 ± 0.84
* antifibrinolytic activity (IC50 = 0.78 mM, this value was taken as the concentration of the inhibitor which prolonged the complete lysis time twofold in comparison with that in the case without inhibitor)a a lit ( 2)
Biological activity of amide derivatives of lysine
Amidolytic activity determined with the use of synthetic substrate S-2238. 0.2 cm3 of examined compound (in the control ñ 0.2 cm3 of 0.15 M NaCl) was added to the mixture of 0.1 cm3 of thrombin solution (5 units / cm3) and 0.5 cm3 of Tris buffer (pH 8.4). After preincubation for 3 min at 37OC, 0.2 ml of S-2238 solution (0.75 mM) was added. The mixture was incubated for 15 min at 37OC, then the reaction was stopped by the addition of 0.1 cm3 of 50% acetic acid and absorbance at 405 nm was measured. The tested compounds at concentrations: 1 ñ 100 mM did not influence the thrombin amidolytic activity. RESULTS AND DISCUSSION All the examined compounds did not infuence the amidolytic activity of thrombin but they were poor inhibitors of the amidolytic plasmin activity similar to Tos-Lys-pNA (2). On the contrary to the reference compound, the examined lysine derivatives were not antifibrinolytics. The tested amides shortened the time of fibrinolysis in the fibrinolytic test. They also prolonged the thrombin clotting time determined with the use of fibrinogen. According to the obtained results, the examined amide derivatives are weak active centre inhibitors of plasmin. They did not block the thrombin active site, as the hydrolysis of synthetic substrate was not inhibited. It is also rather difficult to state if the tested lysine amides can interact with the thrombin exosites because they require compounds with acidic residues (10). Their anticlotting and fibrinolytic activity should rather be connected with the interaction with fibrinogen or fibrin monomers. The presence of C-terminal benzyl or
alkyl amide of lysine in the earlier examined dipeptide structures can be responsible for the absence of clot formation (6, 7). The change of the acetyl group into the ethoxycarbonyl group helped in the crystallization of the obtained compound but it did not influence its biological activity. REFERENCES 1. Backes B.J., Harris J.L., Leonetti F., Craik C.S., Ellman J.A.: Nature Biotech. 18, 187 (2000). 2. Okada Y., Tsuda Y., Teno N., Wanaka K., Boghaki M., Hijikata-Okunomija A., Naito T., Okamoto S.: Chem. Pharm. Bull. 36, 1289 (1988). 3. Midura-Nowaczek K, Bruzgo I., RoszkowskaJakimiec W., Worowski K.: Acta Pol. Pharm. Drug Res. 47, 39 (1990). 4. Fareed J., Messmore H.L., Kindel G., Balis J.U.; Ann. N.Y. Acad. Sci. 370, 765 (1981). 5. Nagamatsu A., Okuma T., Watanabe M., Yamamura Y.: J. Biochem. 54, 491 (1963). 6. Midura-Nowaczek K., Roszkowska-Jakimiec W., Lepietuszko I., Bruzgo I.: Pharmazie 58, 687 (2003). 7. Midura-Nowaczek K., Lepietuszko I., Bruzgo I.: Acta Pol. Pharm. Drug Res. 63, 33 (2006). 8. Markowska A., Midura-Nowaczek K., Bruzgo I.: Acta Pol. Pharm. Drug Res. 64, 355 (2007). 9. Maruyama S., Nonaka I., Tanaka H.: Biochim. Biophys. Acta 1164, 215 (1993). 10. Lane D.A., Philippou H., Huntington J.: Blood 106, 2605 (2005). Received: 31. 12. 2007