Synthesis and Assessment of Antiplatelet and Antithrombotic Activity of 4-Amino-Substituted 5-Oxoproline Amides and Peptides

Venous thromboembolism is a serious problem because it significantly increases the risk of developing vascular complications in elderly patients with obesity or immobilization, cancer, and many other diseases. Thus, there is a need to study new therapeutic strategies, including new medicinal agents for the efficient and safe correction of thrombus disorders. In this work, we have synthesized a number of new amides and peptides of 4-amino-5-oxoprolines and studied their antiplatelet and antithrombotic activity in experiments in vitro and in vivo. It has been found that the newly obtained compounds slow down the process of thrombus formation in a model of arterial and venous thrombosis, without affecting plasma hemostasis parameters. (2S,4S)-4-Amino-1-(4-fluorophenyl)-5-oxoprolyl-(S)-phenylalanine proved to be the most efficient among the studied derivatives. The results obtained indicate the advisability of further studies on 5-oxoproline derivatives in order to design pharmaceutical agents for the prevention and treatment of the consequences of thrombosis.


Introduction
Thromboembolic complications remain a serious medical problem, representing one of the leading causes of mortality in cardiology, oncology, pulmonology, and traumatology patients.Among these complications, pulmonary embolism (PE) stands out as a particularly dangerous condition, for which postoperative mortality is 10-20% [1][2][3].In addition, PE is the third most common cardiovascular disease with a significant public health burden, affecting 250-300 thousand people annually in Europe and in the United States [4,5].
Moreover, venous thromboembolism (VTE) is emerging as another major issue, ranking third among all cardiovascular diseases, second only to myocardial infarction and ischemic stroke.The significance of VTE goes beyond its direct consequences, since VTE significantly increases the risk of developing vascular complications in elderly patients with obesity or immobilization, cancer, and many other diseases [6][7][8].
Thus, there is a need to study new therapeutic strategies, including new medicinal agents, for the efficient and safe correction of thrombus disorders [9,10].
It is known that, among the derivatives of 5-oxoproline (or pyroglutamic acid, Glp), there are compounds exhibiting antiplatelet activity, slowing down collagen-induced platelet aggregation in vitro and in vivo [11,12].Short peptides containing N-terminal 5-oxoproline have also been reported to exhibit antiplatelet activity: for example, Glp-Lys-GlyNH 2 inhibits ADP-induced platelet aggregation in vitro and in vivo [13], and Glp-Asn-Trp inhibits platelet aggregation induced by ADP, collagen, and the platelet activating factor in vitro [14] (Figure 1).
Molecules 2023, 28, x FOR PEER REVIEW 2 Thus, there is a need to study new therapeutic strategies, including new medic agents, for the efficient and safe correction of thrombus disorders [9,10].
It is known that, among the derivatives of 5-oxoproline (or pyroglutamic acid, G there are compounds exhibiting antiplatelet activity, slowing down collagen-indu platelet aggregation in vitro and in vivo [11,12].Short peptides containing N-termin oxoproline have also been reported to exhibit antiplatelet activity: for example, Glp-GlyNH2 inhibits ADP-induced platelet aggregation in vitro and in vivo [13], and Glp-A Trp inhibits platelet aggregation induced by ADP, collagen, and the platelet activa factor in vitro [14] (Figure 1).Selected literature examples of 5-oxoproline derivatives with antiplatelet activity.Am of N-substituted 5-oxoprolines as inhibitors of collagen-induced platelet aggregation [11,12] short peptides with N-terminal 5-oxoproline as inhibitors of platelet aggregation [13,14].
We have previously shown that 4-amino-1-aryl-5-oxoprolines and 4-(arylamino oxoprolines exhibit antiplatelet and antithrombotic activity in vitro and in vivo (Figur the best results in this type of activity were shown by (2S,4S)-4-amino-1-(4-fluorophen 5-oxoproline [15,16].Some derivatives of this series also demonstrated cerebroprotec and psychotropic activity in experiments in vivo [16,17].In this work, we synthesized a number of new amides and peptides of 4-aminostituted 5-oxoprolines and tested their antiplatelet and antithrombotic activity in bot vitro and in vivo experiments.
Thus, there is a need to study new therapeutic strategies, including new medicinal agents, for the efficient and safe correction of thrombus disorders [9,10].
It is known that, among the derivatives of 5-oxoproline (or pyroglutamic acid, Glp), there are compounds exhibiting antiplatelet activity, slowing down collagen-induced platelet aggregation in vitro and in vivo [11,12].Short peptides containing N-terminal 5oxoproline have also been reported to exhibit antiplatelet activity: for example, Glp-Lys-GlyNH2 inhibits ADP-induced platelet aggregation in vitro and in vivo [13], and Glp-Asn-Trp inhibits platelet aggregation induced by ADP, collagen, and the platelet activating factor in vitro [14] (Figure 1).Selected literature examples of 5-oxoproline derivatives with antiplatelet activity.Amides of N-substituted 5-oxoprolines as inhibitors of collagen-induced platelet aggregation [11,12] and short peptides with N-terminal 5-oxoproline as inhibitors of platelet aggregation [13,14].
We have previously shown that 4-amino-1-aryl-5-oxoprolines and 4-(arylamino)-5oxoprolines exhibit antiplatelet and antithrombotic activity in vitro and in vivo (Figure 2); the best results in this type of activity were shown by (2S,4S)-4-amino-1-(4-fluorophenyl)-5-oxoproline [15,16].Some derivatives of this series also demonstrated cerebroprotective and psychotropic activity in experiments in vivo [16,17].In this work, we synthesized a number of new amides and peptides of 4-amino-substituted 5-oxoprolines and tested their antiplatelet and antithrombotic activity in both in vitro and in vivo experiments.

Chemistry
(2S,4S)-4-Amino-1-phenyl-5-oxoproline (1a) [18] and (2S,4S)-4-amino-1-(4-fluorophenyl)-5-oxoproline (1b) [15] were chosen as the starting derivatives of 4-amino  Starting from the previously described (2S,4S)-4-[(methyl)(phenyl)amino]-5-oxoproline (11) [19], we synthesized amides with piperidine (12) and morpholine (13) (Scheme 3).The coupling to the amino component (piperidine or morpholine) was carried out under the action of HBTU in DMF. 12 and 13 are fine crystals (powders).The crystallization of compound 12 from a 10:1 n-hexane-ethyl acetate mixture in the presence of one equiv.AcOH made it possible to obtain single crystals of compound 14 in the form of a solvate with AcOH, which were suitable for an X-ray diffraction analysis.The coupling of 5-oxoproline 11 with tert-butyl (S)-phenylalaninate under the action of HBTU in DMF, followed by cleavage of the tert-butyl ester with TFA in compound 15 yielded dipeptide 16 (Scheme 3).Starting from the previously described (2S,4S)-4-[(methyl)(phenyl)amino]-5-o line (11) [19], we synthesized amides with piperidine (12) and morpholine (13) ( 3).The coupling to the amino component (piperidine or morpholine) was carried der the action of HBTU in DMF. 12 and 13 are fine crystals (powders).The crystal of compound 12 from a 10:1 n-hexane-ethyl acetate mixture in the presence of one AcOH made it possible to obtain single crystals of compound 14 in the form of a with AcOH, which were suitable for an X-ray diffraction analysis.The coupling oproline 11 with tert-butyl (S)-phenylalaninate under the action of HBTU in DM lowed by cleavage of the tert-butyl ester with TFA in compound 15 yielded dipep (Scheme 3).Starting from the previously described (2S,4S)-4-[(methyl)(phenyl)amino]-5oxoproline (11) [19], we synthesized amides with piperidine (12) and morpholine (13) (Scheme 3).The coupling to the amino component (piperidine or morpholine) was carried out under the action of HBTU in DMF. 12 and 13 are fine crystals (powders).The crystallization of compound 12 from a 10:1 n-hexane-ethyl acetate mixture in the presence of one equiv.AcOH made it possible to obtain single crystals of compound 14 in the form of a solvate with AcOH, which were suitable for an X-ray diffraction analysis.The coupling of 5-oxoproline 11 with tert-butyl (S)-phenylalaninate under the action of HBTU in DMF, followed by cleavage of the tert-butyl ester with TFA in compound 15 yielded dipeptide 16 (Scheme 3).According to the XRD data, solvate 14 is crystallized in the non-centrosym space group as a pure enantiomer (see the Supplementary Materials, Table S1) shows the general view of compound 14.The mean bond distances and angles in ecules are near the expected values.In particular, the N atoms of the CON-grou planar configuration with significant asymmetry in the N-C bonds.The N ato arylalkylamino group has a pyramidal configuration with a deviation of the N a the plane of the nearest C atoms on the 0.23 Å.The pyrrolidine moiety has, appro a planar configuration.It is notable that compound 12 is crystallized as solvate 14 with AcOH AcOH is placed near the lactam moiety C(5)O-NH and forms a dimeric intermol bond without transferring the H atoms toward the C(4)N nitrogen atom and wi formation of salt.This observation is confirmed by a strong asymmetry in the tances of the C-O(H) and C=O groups of the AcOH molecule.In the crystal, s forms the layers oriented in the plane (100) (Figure 4).According to the XRD data, solvate 14 is crystallized in the non-centrosymmetrical space group as a pure enantiomer (see the Supplementary Materials, Table S1).Figure 3 shows the general view of compound 14.The mean bond distances and angles in the molecules are near the expected values.In particular, the N atoms of the CON-group have a planar configuration with significant asymmetry in the N-C bonds.The N atom of the arylalkylamino group has a pyramidal configuration with a deviation of the N atom from the plane of the nearest C atoms on the 0.23 Å.The pyrrolidine moiety has, approximately, a planar configuration.According to the XRD data, solvate 14 is crystallized in the non-centros space group as a pure enantiomer (see the Supplementary Materials, Table S shows the general view of compound 14.The mean bond distances and angles ecules are near the expected values.In particular, the N atoms of the CON-g planar configuration with significant asymmetry in the N-C bonds.The N arylalkylamino group has a pyramidal configuration with a deviation of the N the plane of the nearest C atoms on the 0.23 Å.The pyrrolidine moiety has, app a planar configuration.It is notable that compound 12 is crystallized as solvate 14 with AcOH AcOH is placed near the lactam moiety C(5)O-NH and forms a dimeric interm bond without transferring the H atoms toward the C(4)N nitrogen atom and formation of salt.This observation is confirmed by a strong asymmetry in th tances of the C-O(H) and C=O groups of the AcOH molecule.In the crysta forms the layers oriented in the plane (100) (Figure 4).It is notable that compound 12 is crystallized as solvate 14 with AcOH (1:1).The AcOH is placed near the lactam moiety C(5)O-NH and forms a dimeric intermolecular H-bond without transferring the H atoms toward the C(4)N nitrogen atom and without the formation of salt.This observation is confirmed by a strong asymmetry in the bond distances of the C-O(H) and C=O groups of the AcOH molecule.In the crystal, solvate 14 forms the layers oriented in the plane (100) (Figure 4).
The structure of compounds 2b, 3-10, and 12-16 was confirmed using 1 H, 19 F, 13 C NMR spectroscopy and elemental analysis.For the NMR spectra of the compounds obtained, see the Supplementary Materials, Figures S1-S61.S1).
The structure of compounds 2b, 3-10, and 12-16 was confirmed usin NMR spectroscopy and elemental analysis.For the NMR spectra of the com tained, see the Supplementary Materials, Figures S1-S61.
The differentiation of the C(2)H and C(4)H signals in structure 8a is analysis of the HMBC spectra (Figure S20), in which there are cross peaks NH-Boc protons and the C(4) carbon, on the one hand, and NH-His and C(2) hand.In the absence of characteristic NH signals in the 1 H NMR spectra, as in 10a (solution in D2O) and 14, experiments based on nOe measurements wer ambiguously assign the H-2 and H-4 signals.In the 2D NOESY spectra for co there was a correlation between H-2 and the ortho protons of the phenyl sub 1; for compound 14, there were correlations between H-2 and the NCH2 of pi between the H-4 and the protons of the N-methyl group.
The differentiation of the C(2)H and C(4)H signals in structure 8a is based on the analysis of the HMBC spectra (Figure S20), in which there are cross peaks between the NH-Boc protons and the C(4) carbon, on the one hand, and NH-His and C(2), on the other hand.In the absence of characteristic NH signals in the 1 H NMR spectra, as in compounds 10a (solution in D 2 O) and 14, experiments based on nOe measurements were used to unambiguously assign the H-2 and H-4 signals.In the 2D NOESY spectra for compound 10a there was a correlation between H-2 and the ortho protons of the phenyl substituent at N-1; for compound 14, there were correlations between H-2 and the NCH 2 of piperidine and between the H-4 and the protons of the N-methyl group.
The significant difference in the chemical shifts of the nonequivalent protons of the 5-oxoproline ring C(3)H A H B , which is 0.5-1.1 ppm, indicates the cis-configuration of the synthesized compounds 2b, 3-10, 12-16, in accordance with the previously established pattern [15,18,19].It should be noted that compounds 9a and 9b are poorly soluble in DMSO-d 6 (0.3-0.5 mg/mL); therefore, their NMR spectra were recorded in a CF 3 CO 2 D solution.In the 13 C NMR spectra of compounds 9a (Figure S37) and 9b (Figure S39), a doubling of almost all signals was observed, which was likely due to the protonation of the N and O atoms and the formation of salt forms.
It should be also noted that, in the 1 H (or 19 F) NMR spectra of the Boc-protected intermediates 2b, 3a,b, 4a,b, 7a,b, and 8a,b, we observed double sets of some protons with a certain ratio of intensities.This is probably caused by the existence of conformers due to the restricted rotation around a partial double bond (HN-C(O)).

Antiplatelet Activity of the Studied Compounds In Vitro
The antiplatelet activity of compounds 5a,b, 6a,b, 9a,b, 10a,b, 12, 13, and 16 was studied on the model of adenosine-5-diphosphoric acid (ADP)-induced platelet aggregation using platelet-rich rat plasma in accordance with the method described in [20].The tested compounds and acetylsalicylic acid (a reference drug) were dissolved in distilled water and tested at different final concentrations: 10 −5 , 10 −6 , 10 −7 , and 10 −8 M (Table 1).After the incubation of the test compounds with the plasma, an ADP solution was added to the aggregometer cuvette, and the antiplatelet activity was evaluated.For each plasma sample, the degree of ADP-induced aggregation was first measured after their incubation with distilled water (control) and, subsequently, with the test compounds at four different dilutions in order to calculate the ∆% inhibition of platelet aggregation.From the data presented in Table 1, it can be seen that the amides with morpholine (compounds 6b and 13), as well as the peptides with (S)-phenylalanine 9a and 9b, most pronouncedly inhibited platelet aggregation.The antiplatelet activity of these compounds was comparable to the activity of ASA and persisted even with further dilution to a concentration of 10 −8 M.

Antithrombotic Activity of the Studied Compounds In Vivo
The antithrombotic activity of the studied compounds was assessed in vivo (Table 2), in models of arterial thrombosis and deep vein thrombosis, as well as under bleeding conditions.To evaluate the in vivo antithrombotic activity, the test compounds were administered at their most active equimolar doses (1/10 MW equivalent in mg/kg), determined at the previous stage of the study.In all three tests, the amides with morpholine 6b and 13, as well as the peptides with (S)-phenylalanine 9a and 9b, showed a pronounced antithrombotic activity.All the studied compounds did not significantly affect the parameters of plasma hemostasis in the experimental animals (thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen content), which indicates the key involvement of the platelet component of hemostasis in the implementation of the antithrombotic effect of these compounds (Table 3).

Assessment of Acute Toxicity
Acute toxicity was determined for the compounds that showed the greatest activity: 1b, 6a,b, 9a,b, 10a,b, and 13.Acute toxicity was expressed as a dose (LD 50 ) that caused the death of 50% of the animals (white outbred male mice) within 14 days after oral administration.The LD 50 values for compounds 9a and 9b were determined to be 2.5 g/kg, while for compounds 1b, 6a,b, 10a,b, and 13, the LD 50 exceeded 3 g/kg, which indicates the low toxicity of the most active compounds.
Noteworthy is the fact that there was a slight inhibition of platelet aggregation in the in vitro experiments (Table 1), while, in the in vivo experiments, compounds 9a and 9b showed a pronounced antithrombotic activity (Table 2).Unfortunately, at this stage of research, i.e., the screening stage, it is impossible to accurately determine the factors that caused such a difference.These factors may include complex interactions within biological systems, potential pharmacokinetic characteristics (including the possibility for the formation of active metabolites), and other factors affecting antithrombotic activity in vivo, such as the regulation of the blood vessel tone, endothelial function, or thrombus dissolution, i.e., the aspects which are not fully registered during in vitro platelet aggregation assays.
Therefore, the identified chemical structures with a noticeable activity and a low toxicity undoubtedly require further study.
A solution of compound 15 (1.080 g, 2.47 mmol) in CF 3 CO 2 H (15 mL) was stirred at room temperature for 2 h and evaporated to dryness under a reduced pressure.The residue was stirred with water (15 mL) for 30 min, evaporated again, dried in vacuo, then crystallized from ethyl acetate (30 mL).The resulting precipitate of the product contained ~30 mol.% of EtOAc (according to 1 H NMR); so, it was treated with water (40 mL) for 30 min in an ultrasonic bath (100 W), filtered off, washed with water (5 mL), and dried in vacuo to afford 0.725 g (76%) of compound 16 as a light gray powder with m.p. 121-123

X-ray Diffraction Analysis
The X-ray diffraction study of solvate 14 was carried out according to a standard procedure using an Xcalibur 3 diffractometer (Oxford Diffraction, London, UK) equipped with a CCD detector (λ(Mo-Kα) = 0.71073 Å, graphite monochromator, ω/2θ scan mode, scanning increment 1 • , T = 295(2) K).The empirical absorption correction was applied.The structures were solved with direct methods using the OLEX2 software package [22].The structures were solved using the method of the intrinsic phases in the ShelXT program and refined with ShelXL using the full-matrix least-squared method for non-hydrogen atoms [23].The hydrogen atoms of the C-H bonds were placed in the calculated positions and were refined in an isotropic approximation.The selected X-ray single-crystal data and structure refinement details are summarized in Table S1.The crystallographic data for solvate 14 have been deposited in the Cambridge Crystallographic Data Centre (CCDC No. 2290217).Copies of the data can be obtained, free of charge, by applying to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (https://www.ccdc.cam.ac.uk/structures/; accessed on 1 October 2023).

General Section
The study of the biological activity of the compounds was carried out in 239 outbred male rats weighing 250-270 g and on 127 outbred male mice (Stolbovaya Branch of the Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency, Moscow Region, Russia).The age of the animals from which blood was taken for the in vitro studies was 18-24 months.The male rats aged 6-8 months were used for the studies of antiplatelet and antithrombotic activity in vivo.The animals were kept under standard vivarium conditions (22-24 • C, 40-50% humidity, ambient light) during the study.All painful manipulations were performed under general anesthesia with a single intraperitoneal injection of zolazepam 20 mg/kg (Zoletil ® 100; Valdepharm, Val-de-Reuil, France) + xylazine 8 mg/kg (Xyla; Interchemie, Venray, The Netherlands).At the end of the experiment, the animals (except for the untreated animals used for blood collection only) were euthanized in a CO 2 chamber.

Study of the Antiplatelet Activity In Vitro
The antiplatelet activity of the compounds was determined with a 220LA two-channel laser platelet aggregation analyzer (Biola, Moscow, Russia) using an in vitro model of ADP-induced platelet aggregation, according to a previously described protocol [20].The activity of the compounds was assessed through the degree of platelet aggregation after the incubation of the platelet-rich plasma with the test compounds at the final concentrations of 1 × 10 −5 , 1 × 10 −6 , 1 × 10 −7 , and 1 × 10 −8 M. Acetylsalicylic acid (ASA; Sigma-Aldrich, St. Louis, MO, USA) was used as a reference drug [24].The tested compounds and ASA were dissolved in distilled water (30 µL).Distilled water in a similar volume was used as a control.The blood was collected from the rats with a puncture of their hypoglossal vein [25].Next, the blood was centrifuged for 15 min at 1000 rpm on a SM-6M centrifuge (Elmi, Riga, Latvia).After centrifugation, the platelet-rich plasma was immediately used.ADP (Sigma-Aldrich, St. Louis, MO, USA) was used as an aggregation inducer at a final concentration of 5 µM.The antiplatelet activity was evaluated with a degree of aggregation defined as the maximum increment of light transmission after the addition of the inducer.

Study of the Antithrombotic Activity In Vivo
The study of the antithrombotic activity of the compounds was carried out in vivo on a model of arterial thrombosis induced by applying 25 µL of a 50% FeCl 3 solution to the carotid artery of the rats, according to the method described in [26], 24 h and 1 h after the intraperitoneal administration of the compounds at their most active equimolar doses (1/10 MW).Zoletil and xylazine were used for anesthesia.The study was carried out using a Doppler ultrasound scanner (Mini-Max Doppler, St. Petersburg, Russia); the time until the complete occlusion of the vessel was recorded.
The ligation of the inferior vena cava was used as a model for deep vein thrombosis according to the method described in [27].In the rats under general anesthesia (zoletil and xylazine), the inferior vena cava was ligated 1 cm above the bifurcation site; the resulting thrombus was removed and weighed after 24 h.The test compounds at a dose of 1/10 MW (mg/kg) or the reference drug were administered 1 h before the ligation of the inferior vena cava.
The bleeding time was assessed in the adult male mice (20-22 g) after the intragastric administration of the test compounds.A scalpel was used to transect the tail tip (5 mm).
The bleeding tail stump was immersed in normal saline heated to 37 • C, and the time to stop the bleeding was recorded [28].The test compounds at their most active equimolar doses (1/10 MW) or the reference drug were administered 24 h and 1 h before the experiment.

Study of the Anticoagulant Activity In Vivo
The thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen content were determined chronometrically with an APG2-01 MINILAB 701 hemostasis analyzer (Unimed, Moscow, Russia) using reagent kits (Renam, Moscow, Russia).The test compounds at a dose of 1/10 MW (mg/kg) were administered intraperitoneally once a day for 3 days, after which the blood was collected from the hypoglossal vein.All the coagulation tests were performed on platelet-poor plasma, stabilized with a 3.8% sodium citrate solution in a ratio of 9:1, followed by centrifugation at 3000 rpm for 15 min, according to the method described in [28].

Study of Acute Toxicity
The acute toxicity of their most active compounds was assessed using the outbred mice.The mortality of the animals was observed over a two-week period after the intragastric administration of the compounds, followed by calculation of the LD 50 .

Statistical Analysis
Statistical data processing was carried out using the GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) and Microsoft Office Excel 2016 software packages.The normality of data distribution was tested using the Shapiro-Wilk test.A one-way ANOVA with Bonferroni's multiple comparisons test or a Kruskal-Wallis test with Dunn's multiple comparisons test was employed to compare the experimental groups with the control group.The data were presented as the mean ± standard error of the mean (M ± SEM).The statistical significance was set to p < 0.05.

Conclusions
In summary, the study of the antiplatelet and antithrombotic activity of the newly obtained amides and peptides of (2S,4S)-4-amino-1-phenyl-, (2S,4S)-4-amino-1-(4-fluorophenyl)-, and (2S,4S)-4-[(methyl)(phenyl)amino]-5-oxoprolines made it possible to identify a number of compounds that exhibit antiplatelet activity in vitro and in vivo.It was found that these compounds slow down the process of thrombus formation in models of arterial and venous thrombosis, without affecting plasma hemostasis parameters.(2S,4S)-4-Amino-1-(4fluorophenyl)-5-oxoprolyl-(S)-phenylalanine (9b) proved to be the most efficient among the studied derivatives.The results obtained indicate the advisability of further studies of 5-oxoproline derivatives in order to design pharmaceutical agents for the prevention and treatment of the consequences of thrombosis (cerebrovascular accident, myocardial infarction, etc.).
-5-oxoprolines (Scheme 1).During the first stage, the 4-amino group of 5-oxoprolines 1a,b was protected by introducing a tert-butoxycarbonyl (Boc) group through the action of Boc 2 O to afford acids 2a,b in high yields.The coupling reaction with piperidine or morpholine was carried out using EDC × HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) as a coupling agent in the presence of HOBt•(1-hydroxybenzotriazole hydrate) and DIEA (N,N-diisopropylethylamine).In the next stage, the Boc group was removed through the action of trifluoroacetic acid (TFA) in CH 2 Cl 2 and the target compounds 5a,b and 6a,b were isolated in moderate to high yields using alkalization with aqueous NaOH.For the synthesis of dipeptides, acids 2a,b were coupled to methyl (S)-phenylalaninate or (S)-histidinate under the action of HBTU (hexafluorophosphate benzotriazole tetramethyl uranium) or TBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate) as coupling agents to obtain compounds 7a,b or 8a,b, respectively (Scheme 2).The protecting groups in peptides 7-8a,b were removed using a sequential treatment with TFA and aqueous NaOH in order to form the corresponding peptides 9-10a,b.9a,b precipitated from an aqueous alkali solution upon acidification with 1M HCl to pH 3-4; the dipeptides 10a,b were isolated on an ion-exchange resin (Scheme 2).

Figure 4 .
Figure 4. Unit cell of compound 14; for edge dimensions a, b, and c, see the Supplem rials (TableS1).
C NMR spectra of compounds 9a (Figure S37) and 9b (Figure bling of almost all signals was observed, which was likely due to the protona and O atoms and the formation of salt forms.It should be also noted that, in the 1 H (or 19 F) NMR spectra of the Boctermediates 2b, 3a,b, 4a,b, 7a,b, and 8a,b, we observed double sets of some a certain ratio of intensities.This is probably caused by the existence of confo the restricted rotation around a partial double bond (HN-C(O)).

Figure 4 .
Figure 4. Unit cell of compound 14; for edge dimensions a, b, and c, see the Supplementary Materials (TableS1).

Table 1 .
Antiplatelet activity of the test compounds in ADP-induced platelet aggregation in vitro (M ± SEM, n = 8).
[15]data for compound 1b[15]are given for comparison.* Differences are statistically significant relative to the control group (H 2 O), p < 0.05.

Table 2 .
In vivo antithrombotic activity of the tested compounds (M ± SEM; in the control group n = 15; in other groups, n = 8).

Table 3 .
Effect of the studied compounds on the parameters of the coagulogram of rat blood in vivo, at a concentration of 1/10 MW in mg/kg (M ± SEM; in the control group n = 10; in other groups n = 8).aPTT, activated partial thromboplastin time; PT, prothrombin time; and TT, thrombin time.
Differences are statistically significant relative to the control group (H 2 O), p < 0.05.