1,4-Benzoxazine-3(4H)-ones as Potent Inhibitors of Platelet Aggregation: Design, Synthesis and Structure–Activity Relations

Abstract

A series of novel potentially platelet aggregation-inhibiting 1,4-benzoxazine-3(4H)-one derivatives was designed and synthesized through Smiles rearrangement, reduction and acetylation reactions. The antiaggregatory activities of the target molecules on arterial blood samples from rabbits, expressed by IC₅₀ values (μM), were then evaluated in vitro against ADP induced platelet aggregation. The favorable IC₅₀ values of compound 8c (IC₅₀=8.99 µM) and 8d (IC₅₀=8.94 µM) indicated that these two compounds were the most potent molecules among all the synthesized compounds. A detailed molecular docking study to explore the interaction of compounds 8c and 8d with GP IIb/IIIa receptor showed that they these two compounds were docked into the active site of GPIIb/IIIa receptor. These results suggest that the 1,4-benzoxazine-3(4H)-one derivatives are promising lead compounds to develop new platelet aggregation inhibitors.

Thrombotic disease represents one of the most important causes of morbidity and mortality.¹⁾ Anti-thrombotic therapy agents, mainly aiming at treating thrombin platelet and thrombosis, are divided into 1) anti-platelet drugs, to prevent the coronary and cerebrovascular thrombosis from forming, such as aspirin and ticlopidine²⁾; 2) drugs against thrombin,^3,4) known as anticoagulants, including heparin and warfarin; 3) thrombolytic drugs such as fibrinolytic drugs.⁵⁾ Currently the most widely clinically applicable anti-platelet drugs are cyclooxygenase inhibitor, ADP receptor antagonists, phosphodiesterase inhibitor and GPIIb/IIIa receptor antagonist.⁶⁾ It has been found that there are a large number of GPIIb/IIIa receptors on the surface of platelets.⁷⁾ After activation, GPIIb/IIIa receptor is exposed, accelerating the Arg-Gly-Asp (RGD) sequence of fibrinogen to combine with platelet receptors.⁸⁾ A fibrinogen molecule could bind to several platelets and one platelet might also be combined with a plurality of fibrinogen, so by “bridging effect” platelet aggregation occures.⁹⁾ Ultimately, the burden of thrombus overwhelms the luminal area of the vessel, potentially resulting in myocardial ischaemia and necrosis. Activation of the platelet surface receptor GPIIb/IIIa is the final common pathway of platelet aggregation, regardless of the initiating stimulus, thus blocking the platelet GPIIb/IIIa receptor could effectively suppress platelet aggregation.

Most GPIIb/IIIa inhibitors are available in the intravenous form and are only limited in hospital applications. Moreover, several compounds designed as oral GPIIb/IIIa antagonists have been discontinued because of a lack of efficacy and increased mortality.^10,11) Therefore, the low efficacy and high side effects of these existing drugs allowed us to rapidly search for more novel ones.^12,13) Considering that low molecular weight inhibitors to be a major goal in scientists’ research these years, the GPIIb/IIIa inhibitors with low molecular weight remain an important target in the discovery of novel antithrombotic compounds.³⁾

On the other hand, it has been reported that 1,4-benzoxazine-3(4H)-one derivatives owned a wide range of biological activities.¹⁴⁾ As shown in Fig. 1, compound 1 was designed as a small molecule rennin inhibitor¹⁵⁾ and compound 2 (PHRL0010) was considered as a cardiotonic agent.¹⁶⁾ Besides, 1,4-benzoxazine-3(4H)-one derivatives have also been discovered to possess a good affinity with GPIIb/IIIa receptor, which in turn met with the mechanism as platelet membrane GPIIb/IIIa receptor antagonists.⁶⁾ In our laboratory, a set of 1,4-benzoxazine-3(4H)-one molecules 3 have been synthesized to be the inhibitors of platelet aggregation.¹⁷⁾

Fig. 1. Bioactive 1,4-Benzoxazinone Derivatives

In our continuous study on the synthesis of 1,4-benzoxazine-3(4H)-ones and their biological activities, we developed a variety of new benzoxazinone derivatives, through the attachment of diverse substituents to the 7-position and alkyl groups on the 4-position of the benzoxazinone core (Chart 1) and assayed their platelet aggregation inhibition activities, to explore their structure–activity relationships (SAR). Besides, a docking study was performed to investigate the plausible binding mode between these synthesized molecules and the receptor.

Chart 1. The Synthetic Route for the Target Molecule 8

Reagents and conditions: a) chloroacetyl chloride, CH₂Cl₂, K₂CO₃, 0–5°C, b) 2-chloro-4-nitrophenol, DMF, NaH, 150°C, c) H₂, 5–10% Pd/C, MeOH, r.t., d) substituted acyl chloride, CH₂Cl₂, K₂CO₃, 0–5°C

Results and Discussion

Chemistry

The synthetic route for the synthesis of 1,4-benzoxazine-3(4H)-one is shown in Chart 1. Firstly, the starting material alkylamine 4 was converted to the intermediate 2-chloro-N-substituted acetamide 5 through acetylation reaction with chloroacetyl chloride at 0–5°C in dichloromethane. Then the obtained 2-chloro-N-substituted acetamide 5 was reacted with 2-chloro-4-nitrophenol to construct the benzoxazinone skeleton 6 via Smiles rearrangement, in the system of NaH/N,N-dimethylformamide (DMF).¹⁷⁾ The reduction of the nitro group on the benzene ring gave 7-amino-4-substituted-2H-benzo[b][1,4]oxazin-3(4H)-one 7 through the use of hydrogen catalyzed by Pd/C, followed by the amidation reaction with substituted acyl chloride at room temperature, to afford the final product 8.

Notably, considering the stability of the intermediate and the total yield of the reactions, at first we tried to attach the substituted acyl group to the amine moiety at the 7-position, which was obtained by the reduction of the nitro group of the O-alkylated product of 2-chloro-4-nitrophenol by the compound 5, and then cyclized to construct the benzoxazinone core through Smiles rearrangement in the final step. However, due to the multi byproducts and extremely low yield of the reactions, we then turned to beginning with the reaction between 2-chloro-4-nitrophenol and 2-chloro-N-substituted acetamide 5 to give benzoxazinone, which was then reduced and acylated to afford the target molecules. In addition, the previous method for the synthesis of benzoxazinone via two steps, Chart 2, at first the reaction of 2-chloro-4-nitrophenol with 2-chloro-N-substituted acetamide to get an O-alkylated intermediate in K₂CO₃/CH₃CN system, followed by initiating Smiles rearrangement after separation and purification, was difficultly handled and time consuming. In contrast, the one-pot reaction in refluxing NaH/DMF system, which was screened from NaOH, Cs₂CO₃ and K₂CO₃ in CH₂Cl₂, MeCN and DMF, respectively, greatly improved the total yield of the reactions and dramatically shortened the reaction time, shown in Chart 3. The structures of the target molecules were characterized by ¹H-NMR, ¹³C-NMR, high resolution-mass spectrum (HR-MS) spectra and compound 8g was further confirmed by X-ray single crystal diffraction (Fig. 2).

Chart 2. The Previous Method via Two Steps for the Synthesis of 1,4-Benzoxazine-3(4H)-one

Chart 3. The Present Method: One-Pot Synthesis of 1,4-Benzoxazine-3(4H)-ones

Fig. 2. The Crystal Structure of Compound 8g

Biological Activity

The antiaggregatory activities of the target molecules 8 on the artery blood sample from rabbits, were assayed against ADP-induced platelet aggregation.¹⁸⁾ Aspirin and ticlopidine were used as the positive control and the inhibitory activities of all the compounds, expressed by IC₅₀ values (concentration required to inhibit platelet aggregation by 50%), were illustrated in Table 1.

Table 1. The Anti-platelet Aggregation Activities of the Novel 1,4-Benzoxazine-3(4H)-ones

Compound	R1	R2	Yielda) (%)	IC50 (μM)b)
8a	CH3CH2	CH3	61	15.11
8b	CH3CH2	CH3CH2	56	12.16
8c	CH3CH2	Ph	82	8.99
8d	CH3CH2	ClCH2CH2	52	8.94
8e	CH3CH2CH2	CH3	62	17.09
8f	CH3CH2CH2	CH3CH2	62	12.42
8g	CH3CH2CH2	Ph	78	9.76
8h	CH3CH2CH2	ClCH2CH2	50	10.06
8i	CH3CH2CH2CH2	CH3	61	18.96
8j	CH3CH2CH2CH2	CH3CH2	54	13.71
8k	CH3CH2CH2CH2	Ph	71	14.49
8l	CH3CH2CH2CH2	ClCH2CH2	48	10.56
Aspirin				6.81
Ticlopidine				3.33

a) Isolated yield of the final step. b) Inhibitory activity was assayed by testing the changes in optical density of ADP-induced platelet rich plasma.

As depicted in Table 1, the IC₅₀ values of all the synthesized compounds ranged from 8.94 to 18.96 µM, among which the preferable values 8.99 and 8.94 µM belonged to compound 8c and 8d, respectively. These compounds exhibited stronger inhibition activities against platelet aggregation than those without containing an amide group,¹⁷⁾ illustrated by the generally lower IC₅₀ values. This trend suggested that the amide moiety formed additional interactions with the surface of the GPIIb/IIIa receptor. The shorter alkyl group ethyl on the 4-position of the oxazine ring, contributed to the stronger inhibition activity, compared with the compounds containing substituent n-propyl and n-butyl moieties, due to the sterically hindered effect, which is consistent with our previous results. On the other hand, the compounds containing substituent phenyl and chloroethyl group in R², behaved better biological activities than those with a methyl and ethyl moiety, namely longer branched substituent or ring structure on C-7 was more conducive to bind the receptor.

Molecular Docking

Besides, to further study the specific binding mode between the synthesized molecules and the GPIIb/IIIa receptor, the two molecules performing the most potent activities in the anti-platelet aggregation experiments were docked into the binding site of the GPIIb/IIIa receptor, as depicted in Fig. 3. As we could see, the two molecules 8c and 8d were both docked into the same cavity which was enclosed by the two chains of the GPIIb/IIIa. The two carbonyl groups in molecule 8c might have two hydrogen bonds with the amino acid residues ALA-217 and TYR-166 respectively in the blue chain and the green chain of GPIIb/IIIa receptor in Fig. 3a, and the derivative 8d might have hydrophobic bond with TYR-190 which was positioned green chain in Fig. 3b. Furthermore, the molecular configuration of compound 8c was more twisted and folded, compared to the compound 8d.

Fig. 3. Molecular Docking of Compounds 8c and 8d in to the Binding Site of GPIIb/IIIa Receptor (PDB: 2VDM)

(a) Compound 8c docked into the active site of GPIIb/IIIa receptor; (b) Compound 8d docked into the active site of GPIIb/IIIa receptor.

Conclusion

In summary, as our continuous exploration for the potent biologically active compounds, we have successfully designed and synthesized a series of 1,4-benzoxazine-3(4H)-one derivatives, whose activities as inhibitors of platelet aggregation were further investigated. The experimental results displayed that this series of compounds possessed potent anti-platelet aggregation abilities, among which compounds 8c and 8d showed the most potent activities. It was inferred that the benzoxazinone with an amide bond at C-7 position, which was linked to a long branched chain or a cyclic structure, exhibited stronger platelet aggregation inhibition activity, after the SAR analysis. The shorter alkyl chain linked to the nitrogen atom of the oxazinone ring, for less steric effect reason, contributed to stronger inhibition activity. Furthermore, a docking study was performed to confirm the specific binding mode between the compounds 8c and 8d and the GPIIb/IIIa receptor and it was shown that they were both docked into the binding site of the GPIIb/IIIa receptor. These results suggested that the 1,4-benzoxazine-3(4H)-one derivatives are promising lead compounds to develop a new class of platelet aggregation inhibitors.

Experimental

Chemistry

All of the reagents were obtained from commercial sources. Solvents were dried and purified with known conventional methods. Melting points (uncorrected) were determined on a micro melting point apparatus (Shanghai Shenguang Instrument Co., Ltd., China). ¹H- and ¹³C- nuclear magnetic resonance (NMR) spectra (at 400 MHz and 100 MHz, respectively) were recorded in CDCl₃ with tetramethylsilane as internal reference on a Bruker Advance 500 FT spectrometer. Chemical shifts were reported in parts per million. Mass spectra (MS) were measured by the ESI method on an Agilent 6510 Q-TOF mass spectrometer. CDCl₃ was used as delivered from Adamas Co., Ltd. (Shanghai, China). Silica gel (70–230 mesh) was used for flash column chromatography. All reactions were monitored by thin-layer chromatography (TLC) using 0.25 mm silica gel plates with UV indicator (Shanghai Jiapeng Technology Co., Ltd., China). Unless otherwise noted, other reagents were obtained from commercial suppliers and used without further purification.

General Procedure for the Synthesis of 8a–l

To a magnetically stirred solution of substituted amine 4 (50.0 mmol, 1.0 equiv) and K₂CO₃ (75.0 mmol, 1.5 equiv) in CH₂Cl₂ (100 mL), cooled in an ice bath, the chloroacetyl chloride (60.0 mmol, 1.2 equiv) was added dropwise slowly. The reaction mixture was stirred at room temperature and monitored by TLC (iodine as streak reagent). After the reaction was completed, the solvent was removed under vacuum and ice water (50 mL) was added into the residue. The mixture was then extracted with ethyl acetate (3×50 mL). The organic layers were combined, dried over anhydrous MgSO₄, and evaporated under vacuum to give the crude product 5 without further purification. The solution of N-substituted-2-chloroacetamide 5 (5.0 mmol, 1.0 equiv) and 2-chloro-4-nitro-phenol (5.0 mmol, 1.0 equiv) was heated at 150°C and generated the desired product by the slowly addition of appropriate amounts NaH (12.5 mmol, 2.5 equiv) in DMF (40 mL). The reaction was monitored by TLC and pH indicator (control value 8.0). After completion of the reaction, the solvent was removed under vacuum and water (40 mL) was added into the residue. The mixture was then extracted with ethyl acetate (4×40 mL). The organic layers were combined, dried over anhydrous MgSO₄, and evaporated under vacuum to give the crude product. The residue obtained was purified by silica gel column chromatography (elute : ethyl acetate : petroleum=1 : 10) to obtain the corresponding compound 6. And then to a stirred solution of 7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one 6 (5.0 mmol, 1.0 equiv) in methanol (30 mL), 10% Pd–C (0.4 mmol, 0.08 equiv) was added to the reaction mixture at room temperature. Hydrogen was bubbled into the round bottomed flask connecting with a sealing system at atmospheric pressure at about 1 mL/min. The air in the sealing system was replaced by H₂ 4–6 times after the system was closed and tested. After hydrogenation was terminated, the brown solid 7 formed were isolated by filtration and the filtrate was distilled at vacuum without further purification. To the magnetically stirred solution of substituted 7-amino-2H-benzo[b][1,4]oxazin-3(4H)-one 7 (5.0 mmol, 1.0 equiv) and K₂CO₃ (7.5 mmol, 1.5 equiv) in CH₂Cl₂ (30 mL), cooled in an ice bath, the chloroacetyl chloride (6.0 mmol, 1.2 equiv) was added dropwise slowly. The reaction mixture was stirred at room temperature and monitored by TLC. After the reaction was completed, the solvent was removed under vacuum and ice water (30 mL) was added into the residue. The mixture was then extracted with ethyl acetate (3×30 mL). The organic layers were combined, dried over anhydrous MgSO₄, and evaporated under vacuum to give the crude product 8 without further purification.

N-(4-Ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)acetamide (8a)

White solid. mp 164.2–165.2°C. ¹H-NMR (CDCl₃) δ: 7.77 (1H, s), 7.31–7.18 (2H, m), 6.93 (1H, d, J=8.6 Hz), 4.58 (2H, s), 3.97 (2H, q, J=7.1 Hz), 2.18 (3H, s), 1.27 (3H, t, J=7.1 Hz). ¹³C-NMR (CDCl₃) δ: 12.5, 24.5, 36.2, 67.6, 109.3, 114.2, 114.8, 124.6, 134.1, 145.5, 163.6, 168.6. Electrospray ionization (ESI)-HR-MS m/z: 235.1225 (Calcd for C₁₂H₁₄N₂O₃: 235.1004 [M+H]⁺).

N-(4-Ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)propionamide (8b)

Milky white solid, mp 152.1–152.4°C. ¹H-NMR (CDCl₃) δ: 7.65 (1H, s), 7.29–7.21 (2H, m), 6.92 (1H, d, J=8.6 Hz), 4.58 (2H, s), 3.97 (2H, q, J=7.2 Hz), 2.40 (2H, q, J=7.6 Hz), 1.29–1.23 (6H, m). ¹³C-NMR (CDCl₃) δ: 9.7, 12.5, 30.6, 36.2, 67.7, 109.2, 114.1, 114.8, 124.4, 134.2, 145.6, 163.5, 172.3. ESI-HR-MS m/z: 249.1227 (Calcd for C₁₃H₁₆N₂O₃: 249.1161 [M+H]⁺).

N-(4-Ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)benzamide (8c)

White solid, mp 118.8–119.2°C. ¹H-NMR (CDCl₃) δ: 7.99 (1H, s), 7.89–7.87 (2H, m), 7.59–7.55 (1H, m), 7.51–7.47 (2H, m), 7.41–7.27 (2H, m), 6.98 (1H, d, J=8.8 Hz), 4.60 (2H, s), 4.00 (2H, q, J=7.2 Hz), 1.30 (3H, t, J=7.2 Hz). ¹³C-NMR (CDCl₃) δ: 12.5, 36.2, 67.7, 109.6, 114.6, 114.9, 124.9, 127.0, 128.8, 132.0, 134.0, 134.7, 145.6, 163.5, 165.8. ESI-HR-MS m/z: 297.1233 (Calcd for C₁₇H₁₆N₂O₃: 297.1161 [M+H]⁺).

3-Chloro-N-(4-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)propanamide (8d)

White solid, mp 119.4–119.5°C. ¹H-NMR (CDCl₃) δ: 7.32 (1H, s), 7.29–7.22 (2H, m), 6.96 (1H, d, J=8.7 Hz), 4.61 (2H, s), 4.00 (2H, q, J=7.1 Hz), 3.91 (2H, t, J=6.3 Hz), 2.83 (2H, t, J=6.2 Hz), 1.29 (3H, t, J=7.1 Hz). ¹³C-NMR (DMSO) δ: 12.5, 36.2, 39.8, 40.5, 67.7, 109.4, 114.3, 114.8, 125.0, 133.4, 145.6, 163.5, 167.6. ESI-HR-MS m/z: 283.0847 (Calcd for C₁₃H₁₅N₂O₃Cl: 283.0771 [M+H]⁺).

N-(3-Oxo-4-propyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)acetamide (8e)

White solid, mp 96.8–97.5°C. ¹H-NMR (CDCl₃) δ: 7.73 (1H, s), 7.31–7.16 (2H, m), 6.91 (1H, d, J=8.7 Hz), 4.59 (2H, s), 3.88 (2H, t, J=7.6 Hz), 2.18 (3H, s), 1.77–1.62 (2H, m), 0.97 (3H, t, J=7.4 Hz). ¹³C-NMR (CDCl₃) δ: 11.2, 20.4, 24.5, 42.6, 67.6, 109.3, 114.2, 115.0, 124.8, 134.0, 145.6, 163.8, 168.6. ESI-HR-MS m/z: 249.1234 (Calcd for C₁₃H₁₆N₂O₃: 249.1161 [M+H]⁺).

N-(3-Oxo-4-propyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)propionamide (8f)

Light yellow solid, mp 86.8–87.5°C. ¹H-NMR (CDCl₃) δ: 7.73 (1H, s), 7.30–7.20 (2H, m), 6.90 (1H, d, J=8.7 Hz), 4.57 (2H, s), 3.91–3.83 (2H, m), 2.40 (2H, q, J=7.6 Hz), 1.75–1.59 (2H, m), 1.24 (3H, t, J=7.6 Hz), 0.97 (3H, t, J=7.4 Hz). ¹³C-NMR (CDCl₃) δ: 9.7, 11.2, 20.4, 30.6, 42.6, 67.6, 109.2, 114.1, 115.0, 124.6, 134.2, 145.6, 163.8, 172.3. ESI-HR-MS m/z: 263.1388 (Calcd for C₁₄H₁₈N₂O₃: 263.1317 [M+H]⁺).

N-(3-Oxo-4-propyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)benzamide (8g)

White solid, mp 193.0–193.7°C. ¹H-NMR (CDCl₃) δ: 8.07 (1H, s), 7.91–7.84 (2H, m), 7.58–7.54 (1H, m), 7.48 (2H, t, J=7.5 Hz), 7.39–7.28 (2H, m), 6.95 (1H, d, J=8.7 Hz), 4.59 (2H, s), 3.94–3.84 (2H, m), 1.80–1.61 (2H, m), 0.99 (3H, t, J=7.4 Hz). ¹³C-NMR (CDCl₃) δ: 11.2, 20.4, 42.6, 67.7, 109.7, 114.6, 115.1, 125.0, 127.1, 128.8, 132.0, 134.0, 134.7, 145.7, 163.8, 165.8. ESI-HR-MS m/z: 311.1380 (Calcd for C₁₈H₁₈N₂O₃: 311.1317 [M+H]⁺).

3-Chloro-N-(3-oxo-4-propyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)propanamide (8h)

White solid, mp 97.7–97.9°C. ¹H-NMR (CDCl₃) δ: 7.74 (1H, s), 7.31–7.20 (2H, m), 6.92 (1H, d, J=8.7 Hz), 4.60 (2H, s), 3.95–3.84 (4H, m), 2.83 (2H, t, J=6.4 Hz), 1.77–1.61 (2H, m), 0.98 (3H, t, J=7.4 Hz). ¹³C-NMR (CDCl₃) δ: 11.2, 20.4, 39.9, 40.4, 42.7, 67.6, 109.5, 114.3, 115.1, 125.1, 133.5, 145.6, 163.9, 167.9. ESI-HR-MS m/z: 297.1002 (Calcd for C₁₄H₁₇N₂O₃Cl: 297.0928 [M+H]⁺).

N-(4-Butyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)acetamide (8i)

Light yellow solid, mp 129.8–130.9°C. ¹H-NMR (CDCl₃) δ: 7.94 (1H, s), 7.31–7.16 (2H, m), 6.91 (1H, d, J=8.7 Hz), 4.58 (2H, s), 3.95–3.86 (2H, m), 2.18 (3H, s), 1.68–1.58 (2H, m), 1.46–1.33 (2H, m), 0.96 (3H, t, J=7.3 Hz). ¹³C-NMR (CDCl₃) δ: 13.8, 20.1, 24.4, 29.1, 40.9, 67.6, 109.3, 114.2, 115.0, 124.7, 134.1, 145.6, 163.8, 168.7. ESI-HR- MS m/z: 263.1389 (Calcd for C₁₄H₁₈N₂O₃: 263.1317 [M+H]⁺).

N-(4-Butyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]-oxazin-7-yl)propionamide (8j)

Milky white solid, mp 142.8–144.6°C. ¹H-NMR (CDCl₃) δ: 7.52 (1H, s), 7.30–7.20 (2H, m), 6.91 (1H, d, J=8.6 Hz), 4.58 (2H, s), 3.91 (2H, t, J=6.0 Hz), 2.40 (2H, q, J=7.6 Hz), 1.68–1.60 (2H, m), 1.45–1.33 (2H, m), 1.25 (3H, t, J=7.6 Hz), 0.96 (3H, t, J=7.3 Hz). ¹³C-NMR (CDCl₃) δ: 9.7, 13.8, 20.1, 29.1, 30.6, 40.9, 67.7, 109.2, 114.0, 115.0, 124.6, 134.1, 145.6, 163.8, 172.2. ESI-HR-MS m/z: 277.1547 (Calcd for C₁₅H₂₀N₂O₃: 277.1474 [M+H]⁺).

N-(4-Butyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)benzamide (8k)

White solid, mp 163.0–163.5°C. ¹H-NMR (CDCl₃) δ: 8.07 (1H, s), 7.88–7.86 (2H, m), 7.58–7.54 (1H, m), 7.50–7.47 (2H, m), 7.41–7.24 (2H, m), 6.96 (1H, d, J=8.7 Hz), 4.59 (2H, s), 3.93 (2H, t, J=7.6 Hz), 1.69–1.62 (2H, m), 1.49–1.35 (2H, m), 0.98 (3H, t, J=7.4 Hz). ¹³C-NMR (CDCl₃) δ: 13.8, 20.1, 29.1, 40.9, 67.7, 109.6, 114.6, 115.1, 125.0, 127.1, 128.8, 132.0, 134.0, 134.7, 145.7, 163.8, 165.8. ESI-HR-MS m/z: 325.1536 (Calcd for C₁₉H₂₀N₂O₃: 325.1474 [M+H]⁺).

N-(4-Butyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-3-chloropropanamide (8l)

White solid, mp 121.7–122.1°C. ¹H-NMR (CDCl₃) δ: 7.78 (1H, s), 7.33–7.19 (2H, m), 6.93 (1H, d, J=8.6 Hz), 4.59 (2H, s), 3.91–3.89 (4H, m), 2.83 (2H, t, J=6.4 Hz), 1.70–1.58 (2H, m), 1.46–1.35 (2H, m), 0.96 (3H, t, J=7.3 Hz). ¹³C-NMR (CDCl₃) δ: 13.8, 20.1, 29.1, 39.9, 40.4, 40.9, 67.6, 109.5, 114.3, 115.1, 125.1, 133.5, 145.6, 163.8, 167.9. ESI-HR-MS m/z: 311.1152 (Calcd for C₁₅H₁₉N₂O₃Cl: 311.1084 [M+H]⁺).

Platelet Aggregation Inhibition Activity Assay

According to the method of Tian et al.,¹⁷⁾ the artery blood samples from New Zealand white rabbits was collected and placed to the vacuum vessel (including 3.8% sodium citrate as an anticoagulant). Then the blood was centrifuged at room temperature for 5 min at 800×g to give platelet-rich plasma (PRP). The remaining blood was further centrifuged for 15 min at 3000 ×g to give platelet-poor plasma (PPP). Under the microscope, the number of platelet in PRP was regulated to 250±25×10⁶/mL by PPP. Compounds and control drugs were prepared into 200, 100, 50, 25 µmol/L through dimethyl sulfoxide (DMSO) (20% aqueous solution). Add 170 µL of platelet rich plasma (PRP) to the 96-well plates, and the optical density (OD) values were measured at 630 nm by microplate reader. The aggregation was initiated by adding 10 µL ADP (4 µmol/L) in each well of 96-well plates, respectively. The tested compounds, control drugs and blank solvent were incubated in oscillating table for 5 min at 37°C, and their OD values were measured after 1, 2, 4, 6 min of the addition. All the experiments were performed in triplicate.

The platelet aggregation rate was expressed as PAR (%) and was calculated as follows:

  

Here, OD₀ and OD₁ indicated the optical density before the ADP was added and after the ADP was added, respectively.

The following equation was used to calculate the inhibition rate of platelet aggregation (IR).

  

Here, PAR_test and PAR_blank represented the PAR of the tested compounds and the blank solvent, respectively.

Molecular Docking Study

Docking study was performed using FlexX program in SYBYL1.1 software package in the catalytic binding site of the X-ray structure of GPIIb/IIIa receptor, with a resolution of 2.9 Å (PDB: 2VDM).¹⁹⁾ Molecules 8c and 8d were built using SYBYL1.1 molecular modeling package and minimized using Powell method. Acceptor protein was prepared by removing water molecules and adding hydrogens using Prepare Protein Structure tool in SYBYL Biopolymer module. Docking calculations were carried out using FlexX program and 20 top ranked docking poses were saved for each docking run.

Acknowledgment

We would like to thank the National Training Programs of Innovation for Undergraduates (No. 201210635077), National Natural Science Foundation of China (21002081) and the Fundamental Research Funds for the Central Universities, P. R. China (XDJK2012B012), for financial support and.

References

• 1) Lopez A. D., Mathers C. D., Ezzati M., Jamison D. T., Murray C. J., Lancet, 367, 1747–1757 (2006).
• 2) Van De Graaff E., Steinhubl S. R., Ann. Med., 32, 561–571 (2000).
• 3) Schwienhorst A., Cell. Mol. Life Sci., 63, 2773–2791 (2006).
• 4) Srivastava S., Goswami L. N., Dikshit D. K., Med. Res. Rev., 25, 66–92 (2005).
• 5) Urmey W. F., Rowlingson J., Reg. Anesth. Pain Med., 23 (Suppl. 2), 146–151 (1998).
• 6) Andronati S. A., Karaseva T. L., Krysko A. A., Curr. Med. Chem., 11, 1183–1211 (2004).
• 7) Wagner C. L., Mascelli M. A., Neblock D. S., Weisman H. F., Coller B. S., Jordan R. E., Blood, 88, 907–914 (1996).
• 8) Zablocki J. A., Rico J. G., Garland R. B., Rogers T. E., Williams K., Schretzman L. A., Rao S. A., Bovy P. R., Tjoeng F. S., J. Med. Chem., 38, 2378–2394 (1995).
• 9) Lefkovits J., Plow E. F., Topol E. J., N. Engl. J. Med., 332, 1553–1559 (1995).
• 10) Leclerc J. R., Crit. Care Med., 30 (Suppl.), S332–S340 (2002).
• 11) Quinn M. J., Byzova T. V., Qin J., Topol E. J., Plow E. F., Arterioscler. Thromb. Vasc. Biol., 23, 945–952 (2003).
• 12) Strong S. H., Halperin J. L., Geriatrics, 62, 22–27 (2007).
• 13) Ruef J., Katus H. A., Expert Opin. Investig. Drugs, 12, 781–797 (2003).
• 14) Ilić M., Ilaš J., Dunkel P., Mátyus P., Boháč A., Liekens S., Kikelj D., Eur. J. Med. Chem., 58, 160–170 (2012).
• 15) Powell N. A., Ciske F. L., Cai C., Holsworth D. D., Mennen K., Van Huis C. A., Jalaie M., Day J., Mastronardi M., McConnell P., Mochalkin I., Zhang E., Ryan M. J., Bryant J., Collard W., Ferreira S., Gu C., Collins R., Edmunds J. J., Bioorg. Med. Chem., 15, 5912–5949 (2007).
• 16) Yang K., Sun L.-P., Liu J.-Y., Cui X., Piao H.-R., Bioorg. Med. Chem. Lett., 20, 4464–4467 (2010).
• 17) Tian X., Wang L.-Y., Xia S., Li Z.-B., Liu X.-H., Yuan Y., Fang L., Zuo H., Bioorg. Med. Chem. Lett., 22, 204–206 (2012).
• 18) Born G. V. R., Nature (London), 194, 927–929 (1962).
• 19) Xiao T., Takagi J., Coller B. S., Wang J.-H., Springer T. A., Nature (London), 432, 59–67 (2004).