One of the strategies in the modern rational efforts aimed at the discovery of new biologically active compounds is molecular hybridization, which relies on the combination of several pharmacophoric moieties into new molecular structures.1,2,3,4,5,6,7,8 The pharmacophoric moieties are typically selected from privileged substructures that are part of already known active pharmaceutical preparations, providing a reliable avenue to the development and synthesis of new physiologically active compounds.9, 10 It is known that pyrimidine ring, which is the key structural feature of pyrimidine bases, thiamine, and orotic acid, plays an important role in medicinal chemistry. Certain pyrimidine derivatives show antiviral,11 antitumor,12,13,14 cardioprotective, 15 antihypertensive,16 and antithrombotic17, 18 activity.

The main direction in the creation of effective oral anticoagulants is the search for inhibitors of factor Xa (the serine protease factor), linking the external and internal blood clotting pathways.19,20,21,22,23,24 Some of these drugs have been already approved for clinical use.25,26,27 In turn, the developed inhibitors for blood coagulation factor XIа are currently only in preclinical and clinical trials.28,29,30,31,32,33,34 The range of compounds showing strong inhibitory activity against the blood coagulation factors Xa and XIa includes derivatives of dihydroquinolones27, 35 and tetrahydroquinolines. 29, 30, 36,37,38 Besides that, some coumarin derivatives have been also identified among the inhibitors of this btype.34, 36, 39 However, due to their drawbacks associated with the risk of uncontrollable hemorrhage, as well as the similar binding sites of serine protease factor and thrombin,40, 41 the search for new selective inhibitors of factors Ха and XIa still remains an important task.

Therefore, on the basis of molecular hybridization principles, it was considered worthwhile to search for new inhibitors of factors Xa and XIa by assembling a pyrimidine-substituted 2H-pyrano[3,2-g]quinolinone system that combines coumarin and hydroquinoline rings (Fig. 1).

Figure 1.
figure 1

The hybrid structure of 2H-pyrano[3,2-g]quinolinone.

Full size image

It has been demonstrated42 that during the condensation of a salicylic aldehyde derivative with dimethyl 3-oxopentanedioate (dimethyl 1,3-acetonedicarboxylate), the reaction of two ester and methylene groups led to the formation of dicoumarinyl ketone. Subsequent studies43,44,45,46,47,48,49,50 have shown that this reaction is applicable to the preparation of 3-oxo-3-(2-oxo-2H-chromen-3-yl)propanoates. By studying the condensation of 7-hydroxy-1,2,2,4-tetramethylhydroquinoline-6-carbaldehydes 1, 2 with dimethyl 3-oxopentanedioate (3), we found that the use of compound 3 in a threefold excess provided relatively good yields of synthetically promising products: methyl 3-oxo-3-(6,8,8,9-tetramethyl-2-oxo-8,9-dihydro-2H-pyrano[3,2-g]-quinolin-3-yl)propanoate (4) and methyl 3-oxo-3-(6,8,8,9-tetramethyl-2-oxo-6,7,8,9-tetrahydro-2H-pyrano[3,2-g]-quinolin-3-yl)propanoate (5) (58 and 62%, respectively) (Scheme 1).

scheme 1

Scheme 1

The proposed route for the reaction described above (Scheme 2) includes the formation of Knoevenagel adduct A, which undergoes dehydratation to arylidene derivative B that is susceptible to intramolecular cyclization leading to the final products 4, 5 (Scheme 2).

scheme 2

Scheme 2

It is known that compounds containing β-keto ester group have been employed as important building blocks for the synthesis of pyrimidines.51,52,53,54,55,56 Besides that, a series of 3-oxo-3-(2-oxo-2H-chromen-3-yl)propanoates have been studied in three-component reactions with aldehydes and urea, providing a route for the assembly of tetrahydropyrimidine ring at position 3 of the 2-oxo-2H-chromene system.50 As a confirmation of this, we established that the interaction of (2H-pyrano[3,2-g]quinolin-3-yl)propanoates 4, 5 with various carboximidamides 6a–h in refluxing DMF provided access to a series of new, potentially biologically active 6,8,8,9-tetramethyl-3-(6-oxo-1,6-dihydropyrimidin-4-yl)-2H-pyrano[3,2-g]quinolin-2-ones 7 and 8 a–e (Scheme 3). 2H-Pyrano[3,2-g]quinolin-2-ones 7 and 8 a–e were obtained in 66–85% yields.

scheme 3

Scheme 3

All of the synthesized compounds were isolated as yellow crystals, the structure of which was determined by 1H and 13С NMR spectroscopy and high-resolution mass spectrometry.

1H NMR spectra of compounds 4 and 7а–е, acquired in DMSO-d6, featured singlets with integrated intensity equal to 6 protons, which were assigned to the protons of two equivalent methyl groups at position 8 of dihydropyridine ring in the tricyclic 6,8,8,9-tetramethyl-2H-pyrano[3,2-g]-quinolin-2-one system, with the chemical shifts of 1.36 ppm (for compound 4) and 1.34–1.35 ppm (for compounds 7а–е). The three methyl group protons at position 6 of the dihydropyridine ring in the tricyclic system gave a signal at 1.92 ppm (for compound 4) and in the range of 1.94–1.98 ppm (for compounds 7а–е). The singlet signal arising from the three protons of NCH3 group at position 9 of dihydropyridine ring of the tricyclic system was observed with a chemical shift of 2.92 ppm (for compound 4) and in the range of 2.87–2.89 ppm (for compounds 7а–е). The singlet from one proton at position 7 was observed at 5.50 ppm (for compound 4) and over the range of 5.45–5.48 ppm (for compounds 7а–е). The singlet due to one proton at position 10 of the tricyclic system appeared over the range of 6.37–6.40 ppm (for compounds 4 and 7а–е). Thus, the electronic character of pyrimidine substituent at position 3 of the tricyclic 6,8,8,9-tetramethyl-2H-pyrano-[3,2-g]quinolin-2-one system had little effect on the chemical shift values of all aforementioned protons. Analogous situation was observed for the signal assigned to the proton located at position 5 of the tricyclic 6,8,8,9-tetramethyl-2Hpyrano[3,2-g]quinolin-2-one system. Thus, its singlet had a chemical shift of 7.42 ppm in the spectrum of compound 4, while the spectra of compounds 7а–е contained this signal in the region of 7.38–7.42 ppm.

1H NMR spectrum of compound 4 also contained two singlets with chemical shifts of 3.60 and 3.94 ppm. The integrals of these signals were equal to 3 and 2 protons, respectively. These singlet signals were assigned to the protons of carboxymethyl and methylene groups in the β-keto ester moiety at position 3 of 6,8,8,9-tetramethyl-2Hpyrano[3,2-g]quinolin-2-one 4.

Common features in 1H NMR spectra of compounds 7а–е were the proton signals arising from the pyrimidin-6-one rings – two singlets with integrated intensity of one proton each, observed in the regions of 6.70–6.86 and 10.85–11.24 ppm. The former of these were assigned to the СН group proton, while the latter – to the NH proton in the pyrimidin-6-one rings of compounds 7а–е.

1H NMR spectra of compounds 7а–е also contained proton signals due to substituents at position 2 of the pyrimidin-6-one ring of these molecules. These signals were assigned to the cyclic aliphatic secondary amines present in the structure of the aforementioned substituents. For compounds 7d,e, additional characteristic signals were those of the aromatic protons in the phenyl and benzyl moieties, respectively.

13С NMR spectra of compounds 4 and 7а–е, acquired for samples in DMSO-d6 solutions, contained the signals of sp3-hybridized carbon atoms of the tricyclic 6,8,8,9-tetramethyl-8,9-dihydro-2H-pyrano[3,2-g]quinolin-2-one system in the range of 12.4–66.4 ppm. The signals of sp3-hybridized carbon atoms of substituents at position 2 of pyrimidin-6-one ring in the molecules of compounds 7а–е and the sp3-hybridized carbon atoms of β-keto ester group at position 3 of the tricyclic 6,8,8,9-tetramethyl-2H-pyrano-[3,2-g]quinolin-2-one system in compound 4 also appeared in this region of spectrum. The signals of sp2-hybridized carbon atoms in the tricyclic 6,8,8,9-tetramethyl-8,9-dihydro-2H-pyrano[3,2-g]quinolin-2-one system, the pyrimidin-6-one ring in the molecules of compounds 7а–е, as well as the phenyl and benzyl groups in compounds 7d,е appeared in the range of 95.6–189.8 ppm. The signals of the sp2-hybridized carbon atoms in С=О groups of the pyranone part in the tricyclic system of compounds 4 and 7а–е and the pyrimidinone ring in the case of compounds 7а–е appeared in the region characteristic for carbonyl derivatives of coumarins and pyrimidinones. The signals of sp2-hybridized carbon atoms of ester and ketone carbonyl groups of the β-keto ester moiety at position 3 of the tricyclic 6,8,8,9-tetramethyl-2H-pyrano[3,2-g]quinolin-2-one system in the spectrum of compound 4 showed chemical shifts of 168.6 and 189.5 ppm, respectively.

The general appearance of 1H NMR spectra acquired for samples of compounds 5 and 8а–e in DMSO-d6 solutions was largely similar to the respective spectra of compounds 4 and 7а–е that were described above. However, the replacement of dihydropyridine ring in the tricyclic 6,8,8,9-tetramethyl-2H-pyrano[3,2-g]quinolin-2-one system with a tetrahydropyridine ring somewhat complicated the aliphatic region in 1H NMR spectra of compounds 5 and 8а–e. For example, the protons of methyl groups at position 8 of the tetrahydropyridine ring in the tricyclic system lost their equivalence and gave two singlet signals with integrated intensity of three protons each, with chemical shifts of 1.24–1.25 and 1.30 ppm, respectively. The proton signals of methyl groups at position 6 of the tricyclic system in spectra of compounds 8а–e split due to the interaction with protons bonded to the carbon atom at position 6, and appeared as doublets in the region of 1.32–1.35 ppm, with integrated intensity equal to three protons and the spin-spin coupling constant of 6.4–6.5 Hz. In the spectrum of compound 5, these signals were shifted upfield and overlapped with the proton signal of one of the methyl groups at position 8 of the tricyclic system. Two protons at position 7 formed a three-spin AMX system with the proton at position 6, where the proton Х was additionally coupled to the protons of methyl group at position 6 of the tricyclic system. This system was manifested as a triplet having an integrated intensity of one proton with a spinspin coupling constant of 12.7–13.2 Hz and a chemical shift of 1.41 ppm (1.40 ppm in the case of compound 4) for proton А and a double doublet having integrated intensity of one proton with a spin-spin coupling constant of 12.7–13.2 Hz and chemical shift of 1.86–1.87 ppm for the proton М. The signal of proton Х (the proton at position 6 of the tricyclic system) appeared as a weakly split septet having an integrated intensity of one proton, with unresolved spin-spin coupling constant and chemical shift of 2.78– 2.79 ppm for compounds 8а–e (2.76 ppm for compound 4).

The signals assigned to protons at positions 4, 5, and 10 of the tricyclic 6,8,8,9-tetramethyl-2H-pyrano[3,2-g]-quinolin-2-one system in spectra of compounds 5 and 8а–e matched the analogous signals in the spectra of compounds 4 and 7а–е. The difference between the chemical shifts of these signals in the spectra of compounds containing the same substituents did not exceed 0.1 ppm. The same situation was also observed for the proton signals of pyrimidin-6-one rings and the proton signals of substituents at position 2 of the pyrimidin-6-one ring in the spectra of molecules 7 and 8. The proton signals of carboxymethyl and methylene groups in the β-keto ester substituent at position 3 of the tricyclic 6,8,8,9-tetramethyl-2H-pyrano-[3,2-g]quinolin-2-one system were identical in the case of compounds 4 and 5.

In 13С NMR spectra of compounds 5 and 8а–e that were acquired in DMSO-d6, three new signals appeared in the region typical for sp3-hybridized carbon atoms. The appearance of two of them could be explained by the replacement of dihydropyridine ring of the tricyclic 6,8,8,9-tetramethyl-2H-pyrano[3,2-g]quinolin-2-one system with a tetrahydropyridine ring. These signals could be assigned to the carbon atoms at positions 6 and 7 of the tetrahydropyridine ring in the tricyclic system. The third signal (26.6–26.8 ppm) belonged to the carbon atom of one of the methyl groups at position 8 of the tricyclic system. The same ring replacement also resulted in the loss of two sp2- hybridized carbon signals at 120 and 130 ppm, which were assigned to the carbon atoms at positions 6 and 7 of the tetrahydropyridine ring of the tricyclic system in the spectra of compounds 4 and 7а–е. Otherwise, 13С NMR spectra of compounds 5 and 8а–f were similar to the respective spectra of compounds 4 and 7а–е.

The synthesized compounds 7b and 8a–e were submitted for primary in vitro screening at the laboratory of the Center for Theoretical Problems of Physico-Chemical Pharmacology, Russian Academy of Sciences, in order to identify potential lead compounds and to determine their inhibitory activity against the factors Xa and XIa. The reference compound used in these studies was rivaroxaban, a drug approved for clinical use that selectively inhibits the factor Xa and practically does not affect the factor XIa. The highest inhibitory activity against factor XIa was observed in the case of compound 8c, which also exhibited moderate inhibitory activity against the factor Xa. In addition, 8,9- dihydro-2H-pyrano[3,2-g]quinolin-2-one (7b) had equal inhibitory effect on factors Xa and XIa (Table 1).

Table 1. The activity of factors Xa and XIa in the presence of the obtained compounds 7b, 8a–e, compared to the activity in their absence, %
Full size table

On the basis of the obtained data, further studies can be proposed for exploring the applicability of molecular hybridization methodology in the search for highly effective and selective inhibitors of blood coagulation factors among hydroquinoline derivatives.

Experimental

1H and 13C NMR spectra were acquired on an Agilent MR 400+ instrument (400 and 100 МHz, respectively) for samples in DMSO-d6 solutions, using the residual solvent signals as internal standards (2.50 ppm for 1Н nuclei, 39.5 ppm for 13С nuclei). HPLC-HRMS analysis was performed on an Agilent Technologies 1260 Infinity chromatograph equipped with an Agilent 6230 TOF LC/MS detector (high resolution time-of-flight detector), using double electrospray ionization. The signals were detected and recorded in positive ion mode; the nebulizer gas (N2) pressure was 20 psi, the drying gas (N2) flow was 6 ml/min, temperature 325°C; the mass detection range was 50–2000 Da. The capillary voltage was 4.0 kV, fragmenter +191 V, skimmer +66 V, OctRF 750 V. The following chromatographic conditions were used: Poroshell 120 EC-C18 column (4.6 × 50 mm, 2.7 μm). Gradient elution: MeCN/H2O (0.1 % HCOOH), flow rate 0.4 ml/min. The results were processed using the MassHunter Workstation / Data Acquisition V.06.00 software. Melting points were determined on a Stuart SMP30 apparatus. The reaction progress was controlled and identification of the reactants and the obtained products were performed with TLC using Merck TLC Silica gel 60 F254 plates, eluents: CHCl3, MeOH, and their mixtures in various ratios, with visualization under UV light and with iodine vapor.

7-Hydroxy-1,2,2,4-tetramethylhydroquinoline-6-carbaldehydes 1, 2 were obtained according to published procedures,57, 58 the starting carboximidamides 6a–h were supplied by Alinda Chemical Ltd., while dimethyl 3-oxopentadioate was purchased from Acros Organics.

Synthesis of (2 H -pyrano[3,2- g ]quinolin-3-yl)propanoates 4, 5 (General method). A mixture of the appropriate 7-hydroxy-1,2,2,4-tetramethylquinoline-6-carbaldehyde 1 or 2 (0.05 mol), dimethyl 3-oxopentanedioate (3) (21.1 g, 0.15 mol), piperidine (1 ml), and EtOH (40 ml) was refluxed for 2 h. The precipitate that formed upon cooling of the reaction mixture was filtered off and recrystallized from i-PrOH.

Methyl 3-oxo-3-(6,8,8,9-tetramethyl-2-oxo-8,9-dihydro-2 H -pyrano[3,2- g ]quinolin-3-yl)propanoate (4). Yield 10.31 g (58%), yellow crystals, mp 130–132°С. 1H NMR spectrum, δ, ppm: 1.36 (6H, s, 8-CH3); 1.92 (3H, s, 6-CH3); 2.92 (3H, s, NCH3); 3.60 (3H, s, COOCH3); 3.94 (2H, s, CH2); 5.50 (1H, s, 7-CH); 6.39 (1H, s, H-10); 7.42 (1H, s, H-5); 8.50 (1H, s, H-4). 13C NMR spectrum, δ, ppm: 18.6; 29.2; 32.5; 48.5; 52.1; 58.8; 95.6; 108.2; 113.8; 120.4; 125.2; 125.5; 130.7; 148.6; 151.8; 159.2; 160.4; 168.6; 189.5. Found, m/z: 356.1490 [M+H]+. C20H22NO5. Calculated, m/z: 356.1494.

Methyl 3-oxo-3-(6,8,8,9-tetramethyl-2-oxo-6,7,8,9-tetrahydro-2 H -pyrano[3,2- g ]quinolin-3-yl)propanoate (5). Yield 11.08 g (62%), yellow crystals, mp 123–125°С. 1H NMR spectrum, δ, ppm (J, Hz): 1.25 (3H, s, 8-CH3); 1.30 (6H, d, 6,8-CH3); 1.40 (1H, t, J = 13.2, 7-CH2); 1.87 (1H, dd, J = 13.2, J = 2.9, 7-CH2); 2.71–2.79 (1H, m, 6-CH); 2.93 (3H, s, NCH3); 3.60 (3H, s, COOCH3); 3.94 (2H, s, CH2); 6.43 (1H, s, H-10); 7.53 (1H, s, H-5); 8.53 (1H, s, H-4). 13C NMR spectrum, δ, ppm: 19.3; 25.5; 26.6; 28.9; 32.9; 45.1; 48.5; 52.1; 56.6; 96.2; 108.0; 113.7; 127.3; 127.4; 148.7; 153.0; 157.8; 160.7; 169.9; 189.6. Found, m/z: 358.1651 [M+H]+. C20H24NO5. Calculated, m/z: 356.1650.

Synthesis of 6,8,8,9-tetramethyl-3-(6-oxo-1,6-dihydropyrimidin-4-yl)-2 H -pyrano[3,2-g]quinolin-2-ones 7, 8a–e (General method). A mixture of (2H-pyrano[3,2-g]-quinolin-3-yl)propanoate 4 or 5 (2 mmol) with the appropriate carboximidamide 6a–h (2 mmol) in DMF (5 ml) was refluxed for 1 h. The precipitate that formed from the cooled reaction mixture was filtered off and recrystallized from i-PrOH.

6,8,8,9-Tetramethyl-3-[6-oxo-2-(pyrrolidin-1-yl)-1,6-dihydropyrimidin-4-yl]-8,9-dihydro-2 H -pyrano[3,2- g ]-quinolin-2-one (7a). Yield 0.60 g (72%), yellow crystals, mp >300°С. 1H NMR spectrum, δ, ppm: 1.34 (6H, s, 8-CH3); 1.91 (4H, br. s, CH2 pyrrolidine); 1.96 (3H, s, 6-CH3); 2.88 (3H, s, NCH3); 3.52 (4H, br. s, CH2 pyrrolidine); 5.46 (1H, s, 7-CH); 6.38 (1H, s, H-10); 6.70 (1H, s, CH pyrimidine); 7.38 (1H, s, H-5); 8.81 (1H, s, H-4); 10.85 (1H, s, NH pyrimidine). 13C NMR spectrum, δ, ppm: 18.8; 25.2; 29.0; 32.0; 47.0; 54.0; 58.1; 95.6; 108.5; 112.2; 114.8; 120.0; 121.3; 121.9; 123.3; 123.9; 126.0; 130.5; 136.0; 139.1; 144.7; 150.0; 157.3. Found, m/z: 419.2079 [M+H]+. C24H27N4O3. Calculated, m/z: 419.2079.

6,8,8,9-Tetramethyl-3-[2-(4-methylpiperidin-1-yl)-6-oxo-1,6-dihydropyrimidin-4-yl]-8,9-dihydro-2 H -pyrano[3,2- g ]-quinolin-2-one (7b). Yield 0.69 g (77%), yellow crystals, mp 289–291°С. 1H NMR spectrum, δ, ppm (J, Hz): 0.92 (3H, d, J = 6.2, СН3 piperidine); 1.05–1.13 (2H, m, CH2 piperidine); 1.35 (6H, s, 8-CH3); 1.58–1.69 (3H, m, CH2 piperidine); 1.97 (3H, s, 6-CH3); 2.84–2.91 (5H, m, CH2 piperidine, NCH3); 4.49 (2H, br. s, CH2 piperidine); 5.46 (1H, s, 7-CH); 6.39 (1H, s, H-10); 6.75 (1H, s, CH pyrimidine); 7.42 (1H, s, H-5); 8.77 (1H, s, H-4); 11.00 (1H, s, NH pyrimidine). 13C NMR spectrum, δ, ppm: 19.4; 24.1; 25.5; 26.7; 29.0; 32.7; 44.2; 45.3; 56.2; 66.4; 96.5; 107.5; 117.3; 121.4; 126.5; 126.9; 148.7; 149.1; 152.2; 155.9; 157.2; 159.2; 159.8; 167.9; 189.8. Found, m/z: 447.2390 [M+H]+. C26H31N4O3. Calculated, m/z: 447.2392.

3-[2-(4-Ethylpiperazin-1-yl)-6-oxo-1,6-dihydropyrimidin-4-yl]-6,8,8,9-tetramethyl-8,9-dihydro-2 H -pyrano[3,2- g ]-quinolin-2-one (7c). Yield 0.62 g (67%), yellow crystals, mp 285–287°С. 1H NMR spectrum, δ, ppm (J, Hz): 1.02 (3H, t, J = 7.1, CH3СН2); 1.35 (6H, s, 8-CH3); 1.97 (3H, s, 6-CH3); 2.58 (2H, q, J = 7.1, CH3CH2); 2.42 (4H, br. s, CH2 piperazine); 2.88 (3H, s, NCH3); 3.69 (4H, br. s, CH2 piperazine); 5.46 (1H, s, 7-CH); 6.38 (1H, s, H-10); 6.81 (1H, s, CH pyrimidine); 7.41 (1H, s, H-5); 8.79 (1H, s, H-4); 11.10 (1H, s, NH pyrimidine). 13C NMR spectrum, δ, ppm: 12.4; 18.6; 29.0; 32.1; 44.5; 52.0; 52.5; 58.1; 95.6; 108.5; 110.5; 114.5; 120.0; 124.1; 125.9; 130.5; 144.6; 149.1; 150.1; 155.8; 157.1; 160.0; 161.3. Found, m/z: 462.2501 [M+H]+. C26H32N5O3. Calculated, m/z: 462.2501.

3-[2-(4-Benzylpiperazin-1-yl)-6-oxo-1,6-dihydropyrimidin-4-yl]-6,8,8,9-tetramethyl-8,9-dihydro-2 H -pyrano-[3,2- g ]quinolin-2-one (7d). Yield 0.69 g (66%), yellow crystals, mp >300°С. 1H NMR spectrum, δ, ppm: 1.34 (6H, s, 8-CH3); 1.94 (3H, s, 6-CH3); 2.43 (4H, br. s, CH2 piperazine); 2.87 (3H, s, NCH3); 3.51 (2H, s, CH2 benzyl); 3.70 (4H, br. s, CH2 piperazine); 5.45 (1H, s, 7-CH); 6.37 (1H, s, H-10); 6.82 (1H, s, CH pyrimidine); 7.26–7.35 (5H, m, Н Ph); 7.39 (1H, s, H-5); 8.79 (1H, s, H-4); 11.12 (1H, s, NH pyrimidine). 13C NMR spectrum, δ, ppm: 18.8; 29.0; 32.0; 44.6; 52.7; 58.1; 62.3; 95.6; 108.5; 114.5; 117.0; 120.0; 122.5; 124.0; 125.9; 127.5; 128.7; 129.4; 130.4; 138.3; 143.6; 144.7; 150.1; 157.1; 155.9; 156.0; 156.1; 159.2; 160.0. Found, m/z: 524.2655 [M+H]+. C31H34N5O3. Calculated, m/z: 524.2658.

6,8,8,9-Tetramethyl-3-[6-oxo-2-(4-phenylpiperazin-1-yl)-1,6-dihydropyrimidin-4-yl]-8,9-dihydro-2 H -pyrano[3,2- g ]-quinolin-2-one (7e). Yield 0.85 g (83%), yellow crystals, mp >300°С. 1H NMR spectrum, δ, ppm (J, Hz): 1.35 (6H, s, 8-CH3); 1.98 (3H, s, 6-CH3); 2.89 (3H, s, NCH3); 3.22 (4H, br. s, CH2 piperazine); 3.86 (4H, br. s, CH2 piperazine); 5.48 (1H, s, 7-CH); 6.40 (1H, s, H-10); 6.78–6.86 (2H, m, CH pyrimidine, H-4 Ph); 6.98 (2H, d, J = 7.1, H-2,6 Ph); 7.23 (2H, t, J = 7.1, H-3,5 Ph); 7.39 (1H, s, H-5); 8.85 (1H, s, H-4); 11.24 (1H, s, NH pyrimidine). 13C NMR spectrum, δ, ppm: 18.9; 29.0; 32.1; 39.6; 44.5; 48.6; 58.2; 95.6; 108.5; 114.3; 116.3; 119.7; 120.0; 124.0. 125.9; 129.5; 130.5; 138.9; 142.1; 145.2; 150.1; 155.0; 156.9; 157.2; 157.3; 158.2; 160.0. Found, m/z: 510.2500 [M+H]+. C30H32N5O3. Calculated, m/z: 510.2501.

6,8,8,9-Tetramethyl-3-(6-oxo-2-phenyl-1,6-dihydropyrimidin-4-yl)-6,7,8,9-tetrahydro-2 H -pyrano[3,2- g ]-quinolin-2-one (8a). Yield 0.68 g (80%), yellow crystals, mp >300°С. 1H NMR spectrum, δ, ppm (J, Hz): 1.25 (3H, s, 8-CH3); 1.30 (3H, s, 8-CH3); 1.35 (3H, d, J = 6.4, 6-CH3); 1.41 (1H, t, J = 13.2, 7-CH2); 1.87 (1H, dd, J = 13.2, J = 2.6, 7-CH2); 2.76–2.84 (1H, m, 6-CH); 2.90 (3H, s, NCH3); 6.44 (1H, s, H-10); 7.37 (1H, s, H-5); 7.53–7.61 (4H, m, СH pyrimidine, H-3–5 Ph); 8.28 (2H, d, J = 6.8, H-2,6 Ph); 9.07 (1H, s, H-4); 12.61 (1H, s, NH pyrimidine). 13C NMR spectrum, δ, ppm: 19.5; 25.6; 26.8, 29.0; 32.6; 45.5; 56.0; 96.1; 108.3; 113.3; 126.2; 126.9; 128.4; 129.1; 129.2; 132.2; 145.5; 148.9; 151.5; 155.9; 159.3; 160.3; 166.8. Found, m/z: 428.1968 [M+H]+. C26H26N3O3. Calculated, m/z: 428.1970.

3-[2-(4-Benzylpiperazin-1-yl)-6-oxo-1,6-dihydropyrimidin-4-yl]-6,8,8,9-tetramethyl-6,7,8,9-tetrahydro-2 H pyrano[3,2- g ]quinolin-2-one (8b). Yield 0.88 g (84%),yellow crystals, mp >300°С. 1H NMR spectrum, δ, ppm (J, Hz): 1.24 (3H, s, 8-CH3); 1.30 (3H, s, 8-CH3); 1.32 (3H, d, J = 6.5, 6-CH3); 1.41 (1H, t, J = 13.0, 7-CH2); 1.86 (1H, dd, J = 13.0, J = 2.6, 7-CH2); 2.44 (4H, br. s, CH2 piperazine); 2.73–2.79 (1H, m, 6-CH); 2,89 (3H, s, NCH3); 3.52 (2H, s, CH2 benzyl); 3.70 (4H, br. s, CH2 piperazine); 6.42 (1H, s, H-10); 6.82 (1H, s, CH pyrimidine); 7.23–7.28 (1H, m, H-4 Ph); 7.31–7.34 (4H, m, H-2,3,5,6 Ph); 7.46 (1H, s, H-5); 8.81 (1H, s, H-4); 11.07 (1H, s, NH pyrimidine). 13C NMR spectrum, δ, ppm: 19.6; 25.5; 26.8; 29.0; 32.5; 44.6; 45.6; 52.7; 55.9; 62.4; 96.1; 108.2; 114.2; 125.9; 126.7; 127.5; 128.7; 129.4; 138.4; 144.8; 151.2; 155.8; 157.8; 158.3; 159.7; 160.2. Found, m/z: 526.2812 [M+H]+. C31H36N5O3. Calculated, m/z: 526.2814.

6,8,8,9-Tetramethyl-3-[6-oxo-2-(4-phenylpiperazin-1-yl)-1,6-dihydropyrimidin-4-yl)]-6,7,8,9-tetrahydro-2 H pyrano[3,2- g ]quinolin-2-one (8c). Yield 0.87 g (85%), yellow crystals, mp >300°С. 1H NMR spectrum, δ, ppm (J, Hz): 1.24 (3H, s, 8-CH3); 1.30 (3H, s, 8-CH3); 1.35 (3H, d, J = 6.4, 6-CH3); 1.41 (1H, t, J = 13.1, 7-CH2); 1.87 (1H, dd, J = 13.1, J = 2.6, 7-CH2); 2.74–2.81 (1H, m, 6-CH); 2,89 (3H, s, NCH3); 3.22 (4H, br. s, CH2 piperazine); 3.86 (4H, br. s, CH2 piperazine); 6.43 (1H, s, H-10); 6.80 (1H, t, J = 7.1, H-4 Ph); 6.86 (1H, s, CH pyrimidine); 6.97 (2H, d, J = 8.0, H-2,6 Ph); 7.23 (2H, t, J = 8.0, H-3,5 Ph); 7.50 (1H, s, H-5); 8.86 (1H, s, H-4); 11.23 (1H, s, NH pyrimidine). 13C NMR spectrum, δ, ppm: 19.6; 25.6; 26.8; 29.0; 32.5; 45.6; 48.6; 55.7; 64.7; 96.2; 103.8; 108.1; 114.2; 116.3; 119.7; 125.9; 126.8; 129.4; 151.2; 151.3; 155.7; 158.1; 160.3; 166.4; 190.9. Found, m/z: 512.2654 [M+H]+. C30H34N5O3. Calculated, m/z: 512.2658.

3-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]-6,8,8,9-tetramethyl-6-oxo-1,6-dihydropyrimidin-4-yl}-6,7,8,9-tetrahydro-2 H -pyrano[3,2- g ]quinolin-2-one (8d). Yield 0.88 g (81%), yellow crystals, mp >300°С. 1H NMR spectrum, δ, ppm (J, Hz): 1.24 (3H, s, 8-CH3); 1.30 (3H, s, 8-CH3); 1.34 (3H, d, J = 6.4, 6-CH3); 1.41 (1H, dd, J = 13.2, J = 2.6, 7-CH2); 1.87 (1H, dd, J = 13.2, J = 2.6, 7-CH2); 2.74–2.81 (1H, m, 6-CH); 2.89 (3H, s, NCH3); 3.03 (4H, br. s, CH2 piperazine); 3.80 (3H, s, OCH3); 3.85 (4H, br. s, CH2 piperazine); 6.42 (1H, s, H-10); 6.84–7.00 (5H, m, H Ar, CH pyrimidine); 7.52 (1H, s, H-5); 8.87 (1H, s, H-4); 11.17 (1H, s, NH pyrimidine). 13C NMR spectrum, δ, ppm: 19.5; 25.5; 26.8; 29.0; 32.5; 44.8; 45.6; 50.4; 55.8; 55.9; 96.1; 108.2; 112.3; 114.2; 118.6; 121.3; 123.6; 125.9; 126.7; 141.3; 114.8; 137.9; 144.9; 151.2; 152.4; 155.8; 156.0; 159.3; 160.3. Found, m/z: 542.2760 [M+H]+. С31H36N5O4. Calculated, m/z: 542.2764.

6,8,8,9-Tetramethyl-3-[2-(morpholin-4-yl)-6-oxo-1,6-dihydropyrimidin-4-yl]-6,7,8,9-tetrahydro-2 H -pyrano-[3,2- g ] quinolin-2-one (8e). Yield 0.70 g (80%), yellow crystals, mp 292–294°С. 1H NMR spectrum, δ, ppm (J, Hz): 1.24 (3H, s, 8-CH3); 1.30 (3H, s, 8-CH3); 1.34 (3H, d, J = 6.5, 6-CH3); 1.41 (1H, t, J = 13.1, 7-CH2); 1.87 (1H, dd, J = 13.1, J = 2.6, 7-CH2); 2.73–2.81 (1H, m, 6-CH); 2.89 (3H, s, NCH3); 3.67 (8H, br. s, CH2 morpholine); 6.42 (1H, s, H-10); 6.85 (1H, s, CH pyrimidine); 7.49 (1H, s, H-5); 8.83 (1H, s, H-4); 11.13 (1H, s, NH pyrimidine). 13C NMR spectrum, δ, ppm: 19.5; 25.5; 26.8; 29.0; 32.5; 45.0; 45.6; 55.9; 66.3; 96.1; 108.2; 110.0; 125.9; 126.8; 144.8; 150.1; 151.2; 156.0; 156.1; 158.8; 160.2. Found, m/z: 437.2185 [M+H]+. C24H29N4O4. Calculated, m/z: 437.2185.

Testing of compounds 7b, 8a–e for inhibitory activity against the blood coagulation factors Xa and XIa. The inhibition of factors Ха and XIa by compounds 7b, 8a–e was determined by measuring the hydrolysis reaction kinetics of substrates specific to each of these enzymes in the presence of the test compounds. In the case of factor Xa, a specific low molecular mass chromogenic substrate S2765 was used (Z-D-Arg-Gly-Arg-pNA, 2HCl, a Chromogenix product by Instrumentation Laboratory), while substrate S2366 was used for factor XIa (pyroGlu-Pro-Arg-pNA·HCl, a Chromogenix product by Instrumentation Laboratory).

The wells of a 96-well plate were charged with a рН 8.0 buffer solution containing 140 mM NaCl, 20 mM HEPES, 0.1% PEG (6000), then the factor Ха was added to the final concentration of 2.5 nM or factor XIa – to the final concentration of 0.8 nM, followed by substrate S2765 (final concentration – 200 μM) or S2366 (final concentration – 200 μM), respectively, as well as the test compounds at the concentration of 30 μM and DMSO at an amount not exceeding 2%. The kinetics of p-nitroaniline (pNA) formation were measured with a THERMOmax Microplate Reader (Molecular Devices Corporation) by the absorbance of the obtained solution at 405 nm wavelength. The initial rate of the substrate cleavage reaction was determined by the starting slope of the graph showing the formation of pNA. The ratio of substrate cleavage rate by enzyme in the presence or absence of test compound was calculated. The results were processed with GraphPad Prism59 (GraphPad) and OriginPro 860 (OriginLab Corporation) software.

Supplementary information file containing 1H and 13С NMR spectra of all synthesized compounds is available at the journal website at http://springerlink.bibliotecabuap.elogim.com/journal/10593.

This study was funded by a grant from the Russian Science Foundation (project No. 18-74-10097).