Synthesis and Hemostatic Activity of New Amide Derivatives

Eight dipeptides containing antifibrinolytic agents (tranexamic acid, aminocaproic acid, 4-(aminomethyl)benzoic acid, and glycine—natural amino acids) were synthesized in a three-step process with good or very good yields. DMT/NMM/TsO− (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate) was used as a coupling reagent. Hemolysis tests were used to study the effects of the dipeptides on blood components. Blood plasma clotting tests were used to examine their effects on thrombin time (TT), prothrombin time (PT), and the activated partial thromboplastin time (aPTT). The level of hemolysis did not exceed 1%. In clotting tests, TT, PT, and aPTT did not differentiate any of the compounds. The prothrombin times for all amides 1–8 were similar. The obtained results in the presence of amides 1–4 and 8 were slightly lower than for the other compounds and the positive control, and they were similar to the results obtained for TA. In the case of amide 3, a significantly decreased aPTT was observed. The aPTTs observed for plasma treated with amide 3 and TA were comparable. In the case of amide 6 and 8, TT values significantly lower than for the other compounds were found. The clot formation and fibrinolysis (CFF) assay was used to assess the influence of the dipeptides on the blood plasma coagulation cascade and the fibrinolytic efficiency of the blood plasma. In the clot formation and fibrinolysis assay, amides 5 and 7 were among the most active compounds. The cytotoxicity and genotoxicity of the synthesized dipeptides were evaluated on the monocyte/macrophage peripheral blood cell line. The dipeptides did not cause hemolysis at any concentrations. They exhibited no significant cytotoxic effect on SC cells and did not induce significant DNA damage.


Introduction
Hemostasis involves a complex network of interactions between blood components and the blood vessel wall, which is responsible for inhibiting bleeding after injuries to the blood vessels and for maintaining the fluidity of circulating blood. It requires an appropriate balance between the coagulation cascade, which leads to the formation of fibrin clot, and the fibrinolytic system, which removes fibrin clots [1]. The critical point in the Figure 1. Key steps in the blood plasma coagulation cascade. The extrinsic pathway is activated by the tissue factor, which is abundantly expressed in the subendothelial tissue but becomes available to blood plasma proteins after vascular injury. Induction of the intrinsic coagulation pathway requires activation of the coagulation factor XII and the presence of a high-molecular (HMW) kininogen and prekallikrein. Independently on the starting point, both pathways trigger the tenase and prothrombinase enzymatic complexes, leading to the generation of the thrombin enzyme. The thrombin-catalyzed removal of short peptides from the fibrinogen molecule activates fibrinogen polymerization and the formation of fibrin monomers and polymers. This complex cascade of reactions results in the conversion of soluble fibrinogen into an insoluble fibrin clot.
The main physiological role of the fibrinolytic system is to remove intravascular fibrin deposits and restore blood flow. Fibrinolysis is initiated by plasminogen activators, which convert the proenzyme plasminogen into the active serine protease-plasmin. Analogously to the coagulation cascade, activation of the fibrinolytic system may also be associated with the activity of components of the intrinsic pathway (prekallikrein, kininogen, and factor XII). However, an essential role in the stimulation of fibrinolytic activity plays the proteolytic activation of plasminogen to plasmin by the tissue-type plasminogen activator (t-PA) and the urokinase-type plasminogen activator (u-PA). The main activator of intravascular fibrinolysis is t-PA. The u-PA activator is involved in extravascular proteolysis, including tissue remodeling and repair. The fibrinolytic system can be inhibited on two levels. The first level is the plasminogen activation process, which is controlled by plasminogen activator inhibitors (PAIs). The main regulator of plasmin generation is the plasminogen activator inhibitor 1 (PAI-1), which is capable of inhibiting both t-PA and u-PA. Plasminogen activator inhibitor 2 (PAI-2) is of minor importance. It selectively inactivates u-PA. The second level of the fibrinolytic system regulation is the inactivation of plasmin, which is mediated by α 2 -antiplasmina and α 2 -macroglobulin. The main components of the fibrinolytic system are presented in Figure 2. Due to the increased risk of thromboembolic complications during many civilization diseases such as atherosclerosis, obesity, metabolic syndrome, and cancer, current therapeutic strategies have been mostly focused on anticoagulant drugs. However, disorders of fibrinolytic activity of blood plasma are also a serious medicinal challenge, especially in the context of the risk of uncontrolled bleedings during different surgical procedures. The number of fibrinolysis modulators available to patients is limited. Currently used antifibrinolytic agents include aprotinin-a naturally occurring competitive inhibitor of serine proteinases as well as synthetic lysine analogs such as tranexamic acid, aminocaproic acid, and 4-(aminomethyl)benzoic acid ( Figure 3) [2]. Lysine itself displays weak antifibrinolytic activity.  The molecular mechanism of action by the main clinically used antifibrinolytic drugs belonging to the group of lysine analogs is the blockage of lysine-binding sites (LBS) in the plasminogen molecule. Lysine-binding sites are responsible for interactions between plasminogen and the fibrin surface, which is essential for its t-PA-mediated activation of plasmin. As a consequence, the blockage of LBS inhibits fibrin degradation. Tranexamic acid binds with plasminogen 6-to 10-fold more than aminocaproic acid in Due to the increased risk of thromboembolic complications during many civilization diseases such as atherosclerosis, obesity, metabolic syndrome, and cancer, current therapeutic strategies have been mostly focused on anticoagulant drugs. However, disorders of fibrinolytic activity of blood plasma are also a serious medicinal challenge, especially in the context of the risk of uncontrolled bleedings during different surgical procedures. The number of fibrinolysis modulators available to patients is limited. Currently used antifibrinolytic agents include aprotinin-a naturally occurring competitive inhibitor of serine proteinases as well as synthetic lysine analogs such as tranexamic acid, aminocaproic acid, and 4-(aminomethyl)benzoic acid ( Figure 3) [2]. Lysine itself displays weak antifibrinolytic activity. Due to the increased risk of thromboembolic complications during many civilization diseases such as atherosclerosis, obesity, metabolic syndrome, and cancer, current therapeutic strategies have been mostly focused on anticoagulant drugs. However, disorders of fibrinolytic activity of blood plasma are also a serious medicinal challenge, especially in the context of the risk of uncontrolled bleedings during different surgical procedures. The number of fibrinolysis modulators available to patients is limited. Currently used antifibrinolytic agents include aprotinin-a naturally occurring competitive inhibitor of serine proteinases as well as synthetic lysine analogs such as tranexamic acid, aminocaproic acid, and 4-(aminomethyl)benzoic acid ( Figure 3)   The molecular mechanism of action by the main clinically used antifibrinolytic drugs belonging to the group of lysine analogs is the blockage of lysine-binding sites (LBS) in the plasminogen molecule. Lysine-binding sites are responsible for interactions between plasminogen and the fibrin surface, which is essential for its t-PA-mediated activation of plasmin. As a consequence, the blockage of LBS inhibits fibrin degradation. Tranexamic acid binds with plasminogen 6-to 10-fold more than aminocaproic acid in The molecular mechanism of action by the main clinically used antifibrinolytic drugs belonging to the group of lysine analogs is the blockage of lysine-binding sites (LBS) in the plasminogen molecule. Lysine-binding sites are responsible for interactions between plasminogen and the fibrin surface, which is essential for its t-PA-mediated activation of plasmin. As a consequence, the blockage of LBS inhibits fibrin degradation. Tranexamic acid binds with plasminogen 6-to 10-fold more than aminocaproic acid in fibrinolytic test  [3]. Synthetic lysine analogs [2] (tranexamic acid and aminocaproic acid) almost completely block the binding of plasminogen to fibrin.
Similar to TA, aminocaproic acid (ε-aminocaproic acid, 6-aminocaproic acid, 6-aminohexanoic acid, EACA) is a competitive inhibitor of plasminogen activation. The recommended dose of EACA is 150 mg/kg as an intravenous bolus before surgery, which is followed by an infusion of 15 mg/kg/h during the operation [33]. It is used in cardiac surgery [34,35] and orthotopic liver transplantation [36]. The most common side effect of rapid intravenous administration of EACA is hypotension. Occasionally, during longer-term administration, patients can suffer from rashes, nausea, vomiting, weakness, retrograde ejaculation, myopathy, and rhabdomyolysis [37].
4-(Aminomethyl)benzoic acid (para-(aminomethyl)benzoic acid, PAMBA) is another antifibrinolytic agent but a weak competitive inhibitor of plasminogen activation. It is a type II antifibrinolytic agent that is used for the treatment of fibrotic skin problems, such as Peyronie's disease [38,39].
This study aimed to synthesize eight new dipeptides 1-8 derivatives containing tranexamic acid, aminocaproic acid, and 4-(aminomethyl)benzoic acid (synthetic analogs of lysine) composed exclusively of unnatural amino acids or their combination with glycine ( Figure 4). As a result of the known hemostatic properties of synthetic lysine analogs, the designed derivatives were expected to have antifibrinolytic properties. Therefore, we evaluated the effects of the synthesized dipeptide derivatives on the hemostatic activity of blood plasma and their activity toward blood cells. fibrinolytic test systems [3]. Synthetic lysine analogs [2] (tranexamic acid and aminocaproic acid) almost completely block the binding of plasminogen to fibrin. Tranexamic acid (trans-4-(aminomethyl)cyclohexane carboxylic acid, TA) is a synthetic derivative of lysine [1,[3][4][5][6][7][8]. It is an antifibrinolytic agent that blocks LBS in plasminogen. For local fibrinolysis, the recommended dosage is between 500 mg and 1 g administered by slow intravenous injection three times daily or between 1 and 1.5 g administered orally two to three times daily. For general fibrinolysis, a single dose of 1 g or 10 mg/kg administered by slow intravenous injection is recommended [5]. The indications for which TA is approved vary in different countries. The range of indications is limited in some (e.g., the US [9]) and broad in others (e.g., Japan [10] and the UK [11]). Tranexamic acid is used in cardiac surgery [12][13][14][15][16][17][18][19], in orthopedic surgery [20][21][22], in spinal and cranial surgery [23], in hepatic surgery [24], in prostate surgery [25], in gynecology for heavy menstrual bleeding [26,27], in postpartum hemorrhage [28,29], in acute upper gastrointestinal bleeding [30,31], and in oral surgery [32]. After oral administration of TA, gastrointestinal side effects have been reported, including nausea, diarrhea, and abdominal cramping. These adverse effects have been not reported with intravenous administration. Rapid intravenous administration may cause hypotension [2].
Similar to TA, aminocaproic acid (ε-aminocaproic acid, 6-aminocaproic acid, 6-aminohexanoic acid, EACA) is a competitive inhibitor of plasminogen activation. The recommended dose of EACA is 150 mg/kg as an intravenous bolus before surgery, which is followed by an infusion of 15 mg/kg/h during the operation [33]. It is used in cardiac surgery [34,35] and orthotopic liver transplantation [36]. The most common side effect of rapid intravenous administration of EACA is hypotension. Occasionally, during longer-term administration, patients can suffer from rashes, nausea, vomiting, weakness, retrograde ejaculation, myopathy, and rhabdomyolysis [37].
4-(Aminomethyl)benzoic acid (para-(aminomethyl)benzoic acid, PAMBA) is another antifibrinolytic agent but a weak competitive inhibitor of plasminogen activation. It is a type II antifibrinolytic agent that is used for the treatment of fibrotic skin problems, such as Peyronie's disease [38,39].
This study aimed to synthesize eight new dipeptides 1-8 derivatives containing tranexamic acid, aminocaproic acid, and 4-(aminomethyl)benzoic acid (synthetic analogs of lysine) composed exclusively of unnatural amino acids or their combination with glycine ( Figure 4). As a result of the known hemostatic properties of synthetic lysine analogs, the designed derivatives were expected to have antifibrinolytic properties. Therefore, we evaluated the effects of the synthesized dipeptide derivatives on the hemostatic activity of blood plasma and their activity toward blood cells.

Chemistry
The first step was the synthesis of N-Boc alkyl (tert-butyl or ethyl) esters of amides 18-25. The synthesis was carried out in solution (DCM as a solvent). The N-protected unnatural amino acid 6-(N-Boc-amino)caproic acid (9) and the N-protected natural amino acid N-Boc-glycine (10) were used as the main carboxylic substrates. In the presence of
In the case of amides 18 and 22 with a tert-butyl moiety both in the ester group and in the Boc group, deprotection and hydrolysis were performed in one step using 4  dioxane. The final amides 2-4 and 6-8 were isolated after crystallization with diethyl ether, with more than 90% yield ( Figure 6). In the case of amides 18 and 22 with a tert-butyl moiety both in the ester group and in the Boc group, deprotection and hydrolysis were performed in one step using 4 M HCl in dioxane. Final hydrochlorides of amides 1 and 5 were isolated after crystallization with Et2O with almost quantitative yields ( Figure 6).

Biological Activity
Due to the prevalence to prothrombotic complications in many diseases, numerous studies devoted to substances with anticoagulant activity have been executed [42,43]. In contrast to advances in the antithrombotic therapy development, research on the modulation of fibrinolytic proteins is less advanced. Currently available antifibrinolytic therapies have several limitations [44]. Aprotinin, a Kunitz-type serine protease inhibitor, is the only antifibrinolytic drug approved for the direct inhibition of plasmin activity. However, its clinical use is limited by several serious side effects. Research on new, safer plasmin inhibitors has not yet provided satisfactory results. Studies on the interactions between plasmin active sites and potential inhibitors (including peptide-type substances) are only at the preliminary stages of in vitro tests or in silico prediction [45]. Another trend in recent research on new natural and synthetic substances with antifibrinolytic agents is the allosteric modulation of plasmin activity [46]. For example, synthetic sulfated small molecules have been found to have plasmin-inhibitory activity in vitro, in a

Biological Activity
Due to the prevalence to prothrombotic complications in many diseases, numerous studies devoted to substances with anticoagulant activity have been executed [42,43]. In contrast to advances in the antithrombotic therapy development, research on the modulation of fibrinolytic proteins is less advanced. Currently available antifibrinolytic therapies have several limitations [44]. Aprotinin, a Kunitz-type serine protease inhibitor, is the only antifibrinolytic drug approved for the direct inhibition of plasmin activity. However, its clinical use is limited by several serious side effects. Research on new, safer plasmin inhibitors has not yet provided satisfactory results. Studies on the interactions between plasmin active sites and potential inhibitors (including peptide-type substances) are only at the preliminary stages of in vitro tests or in silico prediction [45]. Another trend in recent research on new natural and synthetic substances with antifibrinolytic agents is the allosteric modulation of plasmin activity [46]. For example, synthetic sulfated small molecules have been found to have plasmin-inhibitory activity in vitro, in a study in which 55 compounds were screened. A pentasulfated flavonoid-quinazolinone dimer was the most effective plasmin inhibitor (IC 50 = 45 µM) [47]. Our previous studies showed that natural compounds belonging to the bufadienolide group can also act as uncompetitive inhibitors of plasmin in vitro [48].
In the present study, we synthesized new amide derivatives containing well-known antifibrinolytic compounds. We conducted hemolytic as well as genotoxic tests on the new amide derivatives and evaluated their cellular safety and antifibrinolytic potential in vitro.

Hemolysis
To study the interactions of the synthesized amides 1-8 with blood cells, we examined their impact on erythrocytes. None of the synthesized compounds 1-8 caused hemolysis at any concentrations (5, 50, or 500 mg/L), even after 4 h. Hemolysis did not exceed 1% with any of the compounds. Even at a concentration of 500 mg/L, hemolysis was less than 1% in the initial incubation period up to 4 h. Figure 7a-c compare results from the hemolysis tests for all synthetized amides 1-8 at concentrations 5, 50, and 500 mg/L. As can be seen, there is no difference in the hemolysis parameter between blood cells samples exposed to the amides. Other graphs showing results of the hemolysis tests for all single amides 1-8 are given in the Supporting Information (Figures S33-S40).

Clotting Assays and Determination of Fibrinolytic Efficiency
The hemostatic properties of the synthesized amides were evaluated based on a comprehensive study on their effects on both the human blood plasma coagulation process and its physiological counterpart, i.e., fibrinolysis. The efficiency of the examined amides was tested using blood clotting times, well-known diagnostic parameters, and the clot formation and fibrinolysis (CFF) assay, which provides data not only on the plasma coagulation process but also on the rate of fibrinolysis. The hemostatic activity of the examined amides was established in comparison with an antifibrinolytic drug, tranexamic acid. In our tests, this compound slightly shortened the prothrombin time (PT) and the activated partial thromboplastin time (aPTT), with no effects on the thrombin time TT. All synthesized amides 1-8 were evaluated in clotting tests, including the prothrombin time (PT), the activated partial thromboplastin time (aPTT), and the (TT). The compounds were tested at three concentrations: 10, 25, and 50 mg/L. TA was used as a reference compound, and untreated blood plasma was used as a control sample. The results for all clotting times are presented as a percentage of the control.

Prothrombin Time (PT)
The prothrombin times for untreated plasma and plasma treated with amides ranged from 14.1 to 15.2 s (see Supporting Information, Figure S41). The results are presented as a percentage of the control, which was plasma without amides and TA ( Figure 8). Figure 8 shows that prothrombin time was slightly but statistically significantly reduced in plasma with amides 1-4 and 8, as well as with tranexamic acid. Amide 1 at all concentrations resulted in a significantly decreased prothrombin time, as did amides 2 and 4 at two higher concentrations (25 and 50 mg/L). Amide 3 and TA at two lower concentrations (10 and 25 mg/L) decreased the prothrombin time, which fell only slightly with amide 8 at the lowest concentration (10 mg/L). The prothrombin time was most effectively lowered by amide 8 at a concentration of 10 mg/L.

Clotting Assays and Determination of Fibrinolytic Efficiency
The hemostatic properties of the synthesized amides were evaluated based on a comprehensive study on their effects on both the human blood plasma coagulation process and its physiological counterpart, i.e., fibrinolysis. The efficiency of the examined amides was tested using blood clotting times, well-known diagnostic parameters, and the clot formation and fibrinolysis (CFF) assay, which provides data not only on the plasma coagulation process but also on the rate of fibrinolysis. The hemostatic activity of the examined amides was established in comparison with an antifibrinolytic drug, tranexamic acid. In our tests, this compound slightly shortened the prothrombin time (PT) and the activated partial thromboplastin time (aPTT), with no effects on the thrombin time TT. All synthesized amides 1-8 were evaluated in clotting tests, including the prothrombin time (PT), the activated partial thromboplastin time (aPTT), and the (TT). The compounds were tested at three concentrations: 10, 25, and 50 mg/L. TA was used as a reference compound, and untreated blood plasma was used as a control sample. The results for all clotting times are presented as a percentage of the control.

Prothrombin Time (PT)
The prothrombin times for untreated plasma and plasma treated with amides ranged from 14.1 to 15.2 s (see Supporting Information, Figure S41). The results are presented as a percentage of the control, which was plasma without amides and TA ( Figure 8). Figure 8 shows that prothrombin time was slightly but statistically significantly reduced in plasma with amides 1-4 and 8, as well as with tranexamic acid. Amide 1 at all concentrations resulted in a significantly decreased prothrombin time, as did amides 2 and 4 at two higher concentrations (25 and 50 mg/L). Amide 3 and TA at two lower concentrations (10 and 25 mg/L) decreased the prothrombin time, which fell only slightly with amide 8 at the lowest concentration (10 mg/L). The prothrombin time was most effectively lowered by amide 8 at a concentration of 10 mg/L.  Figure S42). These results are presented as a percentage of the control in the Figure 9. Only two of the tested substances (amid 3 and TA) significantly decreased the aPTT time. The other compounds did not  Figure S42). These results are presented as a percentage of the control in the Figure 9. Only two of the tested substances (amid 3 and TA) significantly decreased the aPTT time. The other compounds did not cause statistically significant changes in aPTT times. Amide 1 appears to increase aPTT time, but the changes are not statistically significant.
cause statistically significant changes in aPTT times. Amide 1 appears to increase aPTT time, but the changes are not statistically significant.

Thrombin Time (TT)
The thrombin time (TT) for all synthesized amides 1-8 was between 14.6 and 16.9 s, and for the control sample TT, it was 15.7 s (see Supporting Information, Figure S43). The results are presented as a percentage of the control in Figure 10. Only amide 6 at all used concentrations and amide 8 (c= 25, and 50 mg/l) had shorter TT than the control sample. There were no significant changes in TT for the other tested amides.

Clot Formation and Fibrinolysis (CFF) Assay
The CFF assay enables analysis of both the blood plasma coagulation process and fibrinolysis efficiency. The thrombin enzyme in the reagent mixture initiates blood plasma fibrinogen polymerization, leading to an increase in absorbance in the sample

Thrombin Time (TT)
The thrombin time (TT) for all synthesized amides 1-8 was between 14.6 and 16.9 s, and for the control sample TT, it was 15.7 s (see Supporting Information, Figure S43). The results are presented as a percentage of the control in Figure 10. Only amide 6 at all used concentrations and amide 8 (c = 25, and 50 mg/L) had shorter TT than the control sample. There were no significant changes in TT for the other tested amides.
Molecules 2022, 27, x FOR PEER REVIEW 12 of 28 cause statistically significant changes in aPTT times. Amide 1 appears to increase aPTT time, but the changes are not statistically significant. Thrombin Time (TT) The thrombin time (TT) for all synthesized amides 1-8 was between 14.6 and 16.9 s, and for the control sample TT, it was 15.7 s (see Supporting Information, Figure S43). The results are presented as a percentage of the control in Figure 10. Only amide 6 at all used concentrations and amide 8 (c= 25, and 50 mg/l) had shorter TT than the control sample. There were no significant changes in TT for the other tested amides.

Clot Formation and Fibrinolysis (CFF) Assay
The CFF assay enables analysis of both the blood plasma coagulation process and fibrinolysis efficiency. The thrombin enzyme in the reagent mixture initiates blood plasma fibrinogen polymerization, leading to an increase in absorbance in the sample

Clot Formation and Fibrinolysis (CFF) Assay
The CFF assay enables analysis of both the blood plasma coagulation process and fibrinolysis efficiency. The thrombin enzyme in the reagent mixture initiates blood plasma fibrinogen polymerization, leading to an increase in absorbance in the sample until the maximal absorbance (A max ) is attained. The fibrinolytic activity of blood plasma is induced by the tissue-type plasminogen activator (t-PA). The activation of fibrinolysis in the tested samples leads to fibrin clot degradation and decreases the absorbance to the baseline. An exemplary plot obtained in the assay is presented in Figure 11. until the maximal absorbance (Amax) is attained. The fibrinolytic activity of blood plasma is induced by the tissue-type plasminogen activator (t-PA). The activation of fibrinolysis in the tested samples leads to fibrin clot degradation and decreases the absorbance to the baseline. An exemplary plot obtained in the assay is presented in Figure 11. Figure 11. An exemplary curve of blood plasma coagulation and clot lysis recorded during the clot formation and fibrinolysis (CFF) assay. The first step in the CFF test involves activation of the blood plasma coagulation cascade and fibrin clot formation (1). It is characterized by the maximal velocity of the fibrin polymerization parameter (VmaxC). The maximal absorbance (Amax) peak corresponds to the fibrin stabilization phase and is an indicator of fibrin clot thickness (2). The third step of the CFF assay covers the activation of fibrinolytic mechanisms and fibrin clot degradation (3) and is described by the maximal velocity of clot lysis (VmaxF).
In the CFF assay, some of the examined compounds showed pro-coagulant and antifibrinolytic activity (Table 1). Mild pro-coagulant effects (measured as VmaxC) were observed mainly for 5, 1, and 2, while 8 and TA (a reference antifibrinolytic drug) did not influence the blood plasma polymerization process. Most of the examined substances showed significant changes in the Amax parameter, indicating that their presence may enhance fibrin clot thickness. For 5 and 7, the Amax parameter was significantly higher across the full range of tested concentrations (10-50 μg/mL). Fibrinolytic activity was diminished by most of the examined compounds at a concentration of 50 μg/mL, except for 1, which had no statistically significant effect (p > 0.05). The most effective reducers of blood plasma fibrinolytic activity were 4 and 7, which decreased VmaxF by over 30-40%. Figure 11. An exemplary curve of blood plasma coagulation and clot lysis recorded during the clot formation and fibrinolysis (CFF) assay. The first step in the CFF test involves activation of the blood plasma coagulation cascade and fibrin clot formation (1). It is characterized by the maximal velocity of the fibrin polymerization parameter (V maxC ). The maximal absorbance (A max ) peak corresponds to the fibrin stabilization phase and is an indicator of fibrin clot thickness (2). The third step of the CFF assay covers the activation of fibrinolytic mechanisms and fibrin clot degradation (3) and is described by the maximal velocity of clot lysis (V maxF ).
In the CFF assay, some of the examined compounds showed pro-coagulant and antifibrinolytic activity (Table 1). Mild pro-coagulant effects (measured as V maxC ) were observed mainly for 5, 1, and 2, while 8 and TA (a reference antifibrinolytic drug) did not influence the blood plasma polymerization process. Most of the examined substances showed significant changes in the A max parameter, indicating that their presence may enhance fibrin clot thickness. For 5 and 7, the A max parameter was significantly higher across the full range of tested concentrations (10-50 µg/mL). Fibrinolytic activity was diminished by most of the examined compounds at a concentration of 50 µg/mL, except for 1, which had no statistically significant effect (p > 0.05). The most effective reducers of blood plasma fibrinolytic activity were 4 and 7, which decreased V maxF by over 30-40%. Table 1. Effects of the examined amides on the hemostatic properties of human blood plasma. The effects of each type of amide were determined in the CFF assay, based on the maximal velocity of fibrin polymerization/clotting (V maxC ), the maximum of fibrin clot absorbance at 360 nm (A max ), and the maximal velocity of fibrin clot lysis (V maxF ). The hemostatic activity of the control plasma was assumed as 100% (of V maxC , V maxF , and A max ). The results are presented as the mean ± SD; n = number of independent experiments, * p < 0.05, ** p < 0.001; *** p < 0.001; n = 5-7.

Cytotoxicity and Genotoxicity Analysis
The cytotoxicity and genotoxicity of all the synthesized amides 1-8 were evaluated, in the range from 50 to 1.5 µM, on a commercially available monocyte/macrophage peripheral blood cell line (SC). Untreated cells cultured in a complete medium were used as a negative control. SC cells treated with 100% DMSO were used as a positive control. The results are presented in Figures 12 and 13. As can be seen in Figure 12, none of the investigated amides 1-8 had any significant cytotoxic effect on SC cells at any concentration. The results for the cells incubated with the tested compounds are very similar to those obtained for the negative control. The cytotoxicity of TA was similar to the cytotoxicity of the synthesized amides 1-8.  The alkaline version of the comet assay was used to assess the level of DNA damage caused by the tested compounds. The results show that after 48 h incubation, none of the synthesized amides 1-8 had caused significant DNA damage in the SC cells at any of the tested concentrations ( Figure 13). The genotoxicity of TA was similar to the genotoxicity of amides 1-8.

Materials and Methods
NMR spectra were measured on a Bruker Avance II Plus (Bruker Corporation, Billerica, MA, USA) spectrometer (700 MHz for 1 H-NMR and 176 MHz for 13 C-NMR) in CDCl3 solution. 1 H and 13 C-NMR spectra were referenced according to the residual peak of the solvent based on literature data. Chemical shifts (d) were reported in ppm and coupling constants (J) were reported in Hz. 13 C-NMR spectra were proton-decoupled. Flash chromatography was performed using a glass column packed with Baker silica gel (30-60 μm). For TLC, silica gel was used with a 254 nm indicator on Al foils (Sigma-Aldrich, St. Louis, MO, USA). All reagents and solvents were purchased and used as obtained from Sigma-Aldrich (Poznan, Poland). Melting points were obtained using a Büchi SMP-20 apparatus. Mass spectrometry analysis was performed on a Bruker mi-croOTOF-QIII (Bruker Corporation, Billerica, MA, USA) equipped with electrospray ionization mode and a time-of-flight detector (TOF). IR spectra were measured on an FT-IR Alpha Bruker (ATR) instrument in cm −1 . Figure 13. Genotoxicity of the tested compounds. Statistical analysis was based on the results of three independent tests. The differences were statistically significant as follows: *** p < 0.001 versus the negative control.
The alkaline version of the comet assay was used to assess the level of DNA damage caused by the tested compounds. The results show that after 48 h incubation, none of the synthesized amides 1-8 had caused significant DNA damage in the SC cells at any of the tested concentrations ( Figure 13). The genotoxicity of TA was similar to the genotoxicity of amides 1-8.

Materials and Methods
NMR spectra were measured on a Bruker Avance II Plus (Bruker Corporation, Billerica, MA, USA) spectrometer (700 MHz for 1 H-NMR and 176 MHz for 13 C-NMR) in CDCl 3 solution. 1 H and 13 C-NMR spectra were referenced according to the residual peak of the solvent based on literature data. Chemical shifts (d) were reported in ppm and coupling constants (J) were reported in Hz. 13 C-NMR spectra were proton-decoupled. Flash chromatography was performed using a glass column packed with Baker silica gel (30-60 µm). For TLC, silica gel was used with a 254 nm indicator on Al foils (Sigma-Aldrich, St. Louis, MO, USA). All reagents and solvents were purchased and used as obtained from Sigma-Aldrich (Poznan, Poland). Melting points were obtained using a Büchi SMP-20 apparatus. Mass spectrometry analysis was performed on a Bruker microOTOF-QIII (Bruker Corporation, Billerica, MA, USA) equipped with electrospray ionization mode and a time-of-flight detector (TOF). IR spectra were measured on an FT-IR Alpha Bruker (ATR) instrument in cm −1 .

General Procedures for the Synthesis of N-Boc Amides 18-25
6-(Boc-amino)caproic acid (9) (0.231 g, 1 mmol, 1 equiv.) or N-Boc glycine (10) (0.175 g, 1 mmol, 1 equiv.) were dissolved in dichloromethane (5 mL) in a 25 mL round-bottom flask. The solution was mixed and cooled in an ice-water bath. Then, DMT/NMM/TsO − (11) (0.414 g, 1 mmol, 1 equiv.) and NMM (0.11 mL, 1 mmol, 1 equiv.) were added. In the case of compound 9, the reaction was controlled by TLC (DCM/acetone 10:1). For compound 10, the reaction was mixed for 30 min. After that, the appropriate hydrochloride (glycine tert-butyl ester hydrochloride (14) (0.167 g, 1 mmol, 1 equiv.), ethyl ester of 4-(aminomethyl)benzoic acid hydrochloride (15)  Heparinized whole blood was used from a healthy volunteer (male, 37 years old). The whole blood (10 mL) was diluted with saline solution (90 mL). For each UV-Vis measurement, 2.5 mL of diluted blood was used. For each blood sample, 1 mL of a saline solution containing hydrochlorides 1-8 at concentrations of 5, 50, and 500 mg/L was added. As a control were used blood samples with the addition of 1% solution of DMSO in saline and 1% solution of SDS in saline. In all cases, the measurements were repeated twice for each concentration. The samples of blood were incubated for 45 min at 37 • C. The cells were centrifuged, and the absorbance of the supernatant, which contained plasma and lysed erythrocytes, was measured at 540 nm. The percentage of lysis was calculated from a standard curve of lysed erythrocytes treated with SDS [52].

Blood Plasma Clotting Assays
Human blood plasma was isolated from fresh buffy coats (collected onto the citratedextrose solution-ACD) purchased from the Regional Centre of Blood Donation and Blood Treatment in Lodz, Poland. In the isolation procedure, the buffy coats (60 mL units) were divided into volumes of 10 mL and centrifuged (4000 rpm/15 min) in 15 mL tubes. Next, 1 mL solutions of each amide 1-8 were prepared in distilled water at concentrations of 10, 25, and 50 mg/L. Then, 450 µL of plasma and 50 µL of each amide at the tested concentrations were placed into 1.5 mL vials. The vials were vortexed (Vortex Biosan V-1 plus), incubated at 37 • C on a thermoblock (Dry Block Thermostat Biosan TDB-120), and analyzed using a Kselmed K-3002 OPTIC coagulometer (Grudziądz, Poland), based on the manufacturer's protocols (described below). The control sample was native blood plasma, untreated with any of the examined substances. Both 100 µL of control plasma and plasma samples treated with the amides were transferred to measuring cuvettes, which were placed in the thermoblock module (37 • C) of the coagulometer (KSELMED K-3002 Optic). Then, 100 µL of thrombin solution (4.5 U/mL, in 0.9% NaCl; Biomed, Lublin, Poland) was added to the cuvette. Measurements were started immediately.
Measurements were performed using a kinetic model, as described in our previous work [53]. Briefly, blood plasma was preincubated with the examined substances (at final concentrations of 10, 25, and 50 mg/L) for 15 min at 37 • C. Then, 100 µL of blood plasma was added to microtiter plate wells, which was followed by 200 µL of the reagent mixture (0.75 U/mL thrombin, 225 ng/mL t-PA and 7.5 mM CaCl 2 ) suspended in tris-buffered saline (0.05 M TBS, 0.9% NaCl; pH 7.4). Measurements of absorbance changes were started immediately and continued for 60 min at 37 • C, λ = 360 nm. The following parameters were used to determine the effects of the examined substances on the coagulation and fibrinolytic properties of the blood plasma: the maximal velocity of fibrinogen polymerization (V maxC ), which is an indicator of the plasma coagulation efficiency; the maximal absorbance (A max ), which is an indicator of the fibrin clot stabilization and its thickness; the maximal velocity of clot lysis (V maxF ), which is an indicator of blood plasma fibrinolytic activity. statistical analysis of the two groups was performed using the Mann-Whitney rank-sum test. Each of the statistical analyses was based on the results of three independent tests. Statistical significance in the CFF assay was established using the Wilcoxon test. In the figures and tables, the differences are statistically significant as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Conclusions
Eight hydrochloride dipeptides 1-8 containing antifibrinolytic agents (tranexamic acid, aminocaproic acid, and 4-(aminomethyl)benzoic acid) and a natural amino acid (glycine) were synthesized with very good yields, using DMT/NMM/TsO − (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate) as a coupling reagent. The compounds were tested for their hemostatic properties and submitted to blood plasma clotting tests. None of the synthesized compounds 1-8 caused hemolysis at any concentration. The level of hemolysis did not exceed 1%. In clotting tests, thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT) did not differentiate any of the compounds. The prothrombin times for all amides 1-8 were similar. However, the results for amides 1-4 and 8 were slightly lower than for the other compounds and the positive control. They were also similar to the results obtained for TA. The activity of the other compounds did not statistically affect aPTT except in the case of amide 3, which significantly decreased aPTT. The aPTTs recorded for plasma treated with amide 3 and TA were similar. Thrombin time (TT) for amides 1-8 was mostly comparable to the control sample. Only in the case of amide 6 and 8 was TT significantly lower than for the other compounds. In the clot formation and fibrinolysis (CFF) assay, amides 5 and 7 were among the most active compounds. The maximal velocity of fibrin polymerization (V maxC ) and the maximal absorbance (A max ) of the fibrin clot indicate that among the tested compounds, amide 5 had the most evident procoagulant activity. Measurements of the maximal velocity of clot lysis (V maxF ) indicate that amides 4 and 7 were the most active inhibitors of the fibrinolytic activity of blood plasma. The synthesized amides 1-8 did not exhibit significant cytotoxic effects toward SC cells and did not induce significant DNA damage. The results of hemostatic properties of amides 1-8 (hemolysis, blood plasma clotting assays: PT, aPTT, TT) are summarized in Table 2.  Research on the design and synthesis of new lysine analogs with hemostatic activity will be continued.