Synthesis of a Novel Series of Amino Acid Prodrugs Based on Thienopyridine Scaffolds and Evaluation of Their Antiplatelet Activity

Abstract

The thienopyridines class of drugs used as P2Y₁₂ receptor antagonists plays a vital role in antiplatelet therapy. To further optimized this compound class, we designed and synthesized a series of amino acid prodrugs of 2-hydroxytetrahydrothienopyridine. All compounds were then evaluated for their inhibitory effect on ADP-induced platelet aggregation in rats and then ED₅₀ and bleeding time of the most potent compounds were compared with commercial drugs. The results showed compound 5c could be a potent and safe candidate for further research.

1. Introduction

Platelets play a critical role in the development of acute coronary syndromes (ACS) and contribute to cerebrovascular events through adhesion, aggregation and subsequent thrombus formation [1]. After platelet activation, ADP is released from intracellular storage granules and then further activates platelets, enlarging the activation and thus aggregation processes [2,3]. It was shown that the ADP response was due to activation of two receptors, the G_q-coupled P2Y₁ receptor, which induces a calcium response and shape change of the blood platelet and the G_i-coupled P2Y₁₂R, which decreases the intracellular adenylyl cyclase activity and prolongs intracellular calcium signaling, thereby stabilizing the formed platelet aggregates [4,5,6,7]. Consequently, blocking the P2Y₁₂R is a valid strategy to antiplatelet therapy, as demonstrated by the thienopyridine class of drugs, including clopidogrel and prasugrel (Figure 1). Thienopyridines are prodrugs those are converted into their respective active metabolites (AMs) through thiolactone intermediates. Clopidogrel is oxidized by cytochrome P450 (CYP) isoforms to its thiolactone, while prasugrel is rapidly hydrolysed by esterases to its thiolactone intermediate (Figure 2) [8]. Until now, dual antiplatelet therapy with aspirin and clopidogrel has remained the treatment of choice for patients with ACS and for those undergoing percutaneous coronary interventions [9,10,11,12]. However, up to 30% of Caucasian patients carry CYP2C19 loss-of-function alleles. They cannot complete the oxidative biotransformation after receiving clopidogrel and thus are prone to suffer a high rate of subsequent cardiovascular events [13,14]. Prasugrel achieved more pronounced inhibition of platelet aggregation and lower interindividual variability of pharmacological response, but the improved efficacy was associated with an increased bleeding risk [15]. Ticagrelor, unlike the thienopyridines, is an oral cyclopentyl-triazolopyrimidine (CPTP) that is a direct and reversible inhibitor of the P2Y₁₂ receptor and does not require CYP450-mediated activation. Although it effectively reduces the ischaemic events and mortality rates of cardiovascular patients, high nonlethal bleeding rates are observed and obvious undesirable side-effect appear [16]. The TRITON and PLATO clinical trials have both accounted for the long-standing hypothesis of more potent platelet inhibition translating into reduced atherothrombotic events at the expense of increased bleeding [15,16,17,18]. All these considerations reinforce the strong need of novel and safe P2Y₁₂ antagonists.

In this study, we mimicked the metabolic pattern of prasugrel and aimed to find a drug candidate overcoming the drawbacks of clopidogrel and prasugrel, assuming that ester prodrugs might be readily converted to thiolactones by esterase-mediated hydrolysis and subsequently to the active metabolite through only one CYP-dependent step. We chose the most potential intermediates 3a and 3b (Scheme 1) which are, respectively, the thiolactone of clopidogrel and prasugrel, as a parent part of the target compounds.

Furthermore, amino acid prodrug design has been approached into our work as an attractive strategy. There are some good reasons for amino acid prodrugs: (1) they can be conveniently hydrolysed to their parent drug and amino acid part by enzymes in vivo. Amino acids are generally regarded as safe because they building blocks for proteins; (2) it is also proven that they can improve oral delivery and sustained release [19].

Taking the above information into account, we introduced several amino acids into the thiolactone moiety and synthesized a series of amino acid prodrugs of 2-hydroxytetra-hydrothienopyridine as novel antiplatelet agents. We also describe the inhibition of ADP-induced platelet aggregation in rats. Moreover, the potent compounds were tested for most ED₅₀ and bleeding time.

2. Results

2.1. Chemistry

The synthesis of the molecules was designed and carried out as shown in Scheme 1. The thienopyridine hydrochloride 1 was reacted with substituted intermediates 2a–b in the absence of potassium bicarbonate to afford the N-alkylated thiolactones 3a–b, which were converted to 4a–4p with N-boc-l-amino acids via EDCI and DMAP [20,21]. We chose several aliphatic and aromatic amino acids, especially including the l-proline and l-2-pyrrolidone-5-carboxylic acid with an imine and amide structure, respectively. Finally, the target amino acid esters 5a–5p were obtained as hydrochloride salts after treating with hydrochloric acid in ethyl acetate for the removal of the Boc-group. All the structures are outlined in

Table 1. Structures of target compounds.

Compound	R3 or R4 a (R1=COOCH3, R2=Cl)	Compound	R3 or R4 a (R1=COCH(CH2)2, R2=F)
5a		5i
5b		5j
5c		5k
5d		5l
5e		5m
5f		5n
5g		5o
5h		5p

^a R₄ was only used for compounds 5g–5h and 5o–5p.

.

2.2. Biological Activity Evaluation

2.2.1. Inhibition of ADP-induced Platelet Aggregation in Rats at a Dose of 3 mg/kg and 1 mg/kg

All the targeted analogues were evaluated for their inhibitory effect on ADP-induced platelet aggregation in rats, with clopidogrel and prasugrel as positive controls. The assay results are summarized in

Table 2. Inhibitory effect of target compounds on ADP-induced platelet aggregation in rats at a dose of 3 mg/kg and 1mg/kg.

Compounds	Inhibition Ration (%)
	3 mg/kg	1 mg/kg
5a	55.1	35.1
5b	36.8	19.4
5c	98.4	39.6
5d	68.9	16.2
5e	43.3	15.8
5f	63.8	37.2
5g	64.2	35.5
5h	51.6	27.2
5i	93.4	17.5
5j	86.3	52.6
5k	89.2	66.2
5l	96.4	89.8
5m	100	47.6
5n	99.3	46.4
5o	93.6	30.7
5p	69.5	35.1
Clopidogrel	34.4	--
Prasugrel	100	91.0

and Figure 3. Considering that prasugrel exhibited strong potency at a dose of 3 mg/kg, while clopidogrel was almost inactive, we initiated the screening at a dose of 3 mg/kg for all compounds. As we expected, almost all the first screening round compounds presented outstanding inhibitory effect on platelet aggregation that was more potent than clopidogrel. In particular, 5c and 5i–5p showed almost equal activity to that of prasugrel at 3 mg/kg dose. To make better option for further study, we attempted to increase the difference behavior among the targeted compounds on platelet aggregation via carrying out a secondary screening of all compounds at a reduced dose of 1 mg/kg.

As shown in the 1 mg/kg level results, 5l approached prasugrel in inhibition behavior and 5c, 5j, 5k, 5m and 5n presented slightly less potency than prasugrel at the 1 mg/kg level. Although these compounds should covert to the same activate metabolites 6a or 6b to behave efficaciously [22,23], the hindrance of hydrolysis at the 2-ester moiety might have a significant impact on the potency. It was notable that 5c, based on the 2-oxoclopidogrel structure, was superior to clopidogrel and approach to prasugrel on inhibitory of platelet aggregation.

2.2.2. Determination of ED₅₀ and BT₂ of 5c and 5l

Aiming to find a candidate which has a balance between antiplatelet effects and bleeding complications, we selected 5c and 5l to further test for ED₅₀ value and bleeding risk, because 5c and 5l performed the best in the aggregation assays among their own series of backbone structures which included 3a (2-oxoclopidogrel) and 3b (thiolactone of prasugrel), respectively. Firstly, we executed above bio-assay at doses of 0.5–4 mg/kg to determine the ED₅₀ values of compounds 5c and 5l (Figure 4). The results showed that 5c (ED₅₀ = 2.16 mg/kg) and 5l (ED₅₀ = 0.74 mg/kg) had a moderate value between clopidogrel (ED₅₀ = 3.96 mg/kg) and prasugrel (ED₅₀ = 0.50 mg/kg). Moreover, in the tail bleeding test (Figure 5), 5c induced much shorter bleeding time in rats, while 5l showed a slightly shorter bleeding time than prasugrel.

Table 3. Comparison of antiplatelet antihaemostatic effects of potent compounds.

Compounds	ED50 (mg/kg)	BT2 (mg/kg)	ED50/BT2
Clopidogrel	3.96	3.82	1.04
Prasugrel	0.50	0.51	0.98
5c	2.16	2.32	0.93
5l	0.74	0.70	1.06

summarizes the ED₅₀ and BT₂ values for the tested compounds. The BT₂ was defined as the dose that doubled the vehicle bleeding time. The ED₅₀ and BT₂ values of clopidogrel and prasugrel were in good agreement with those reported [24,25]. In addition, the ratios of ED₅₀ to BT₂ indicated the tested compounds have a similar benefit/bleeding ratio risk. It should be noted that the ratio of 5c was the lowest one, although its efficacy was lower than that of prasugrel. Taken together, these results suggest that 5c is a potent antiplatelet agent with relatively moderate antiheamostatic potency, but this remains to be proven in future clinical studies.

3. Discussion

It has been reported that, at same dose, the activite metabolite of prasugrel (compound 6b) is more potent at inhibiting platelet aggregation than that of clopidogrel (compound 6a) [26]. This very important point is exactly conformed by our study results wherebyt compounds 5i–5p generally were more effective than 5a–5h, because of the generation of their respective AM after administration at the same dose. It is also indicated that 5a–5h can easier and more directly transform into 3a than clopidogrel via the evidence that 5a–5h are superior to clopidogrel while 5i–5p are not so to prasugrel. Taking all this into account, we believe that the compounds we designed and synthesized, especially 5a–5h, are metabolized to 3a via a one-esterase hydrolysis-step and one-P450-step in vivo, which exactly overcomes the drawback of clopidogrel resistance. Further studies will focus on the pharmacokinetics of 5c.

4. Materials and Methods

4.1. General Information

All reagents and solvents were purchased from commercial suppliers and used without further purification. Reactions were monitored by thin layer chromatography. The melting points (m.p.) of the compounds were determined on a YRT-3 Melting Point Tester (Precision Instrument of Tianjin University, Tianjin, China). ¹H-NMR and ¹³C-NMR spectra were recorded for DMSO-d₆ solutions on a 400 MHz Bruker spectrometer (Bruker, Billerica, MA, USA). MS were measured on a Finnigan LCQ Mass (Thermo Fisher Scientific, Waltham, MA, USA), HRMS were measured on a TOF LC/MS instrument (Agilent Technologies, Santa Clara, CA, USA). Blood sample were handled with a low speed table centrifuge (LD5-2A, Beijing, China) and an aggregometer (LBY-NJ4, Beijing, China). The ED₅₀ was calculated using the SPSS software (IBM, North Castle, NY, USA).

4.2. Chemistry

Methyl 2-(2-chlorophenyl)-2-(2-oxo-7,7a-dihydrothieno[3,2-c]pyridin-5(2H,4H,6H)-yl)acetate (3a). To a stirred solution of methyl 2-bromo-2-(2-chlorophenyl)acetate (2a, 26.3 g, 0.1 mol) in CH₃CN (500 mL) were added 5,6,7,7a-tetrahydrothieno[3,2-c]pyridin-2(4H)-one hydrochloride (1, 20.9 g, 0.11 mol) and potassium bicarbonate (30.0 g, 0.3 mol). The reaction was stirred at room temperature overnight. The reaction mixture was filtered and the liquid was concentrated under reduced pressure. The residue was purified by column chromatography to give a yellow oil, which was recrystallized from EtOH to afford a white solid (20.9 g, 62% yield). m.p.: 118–119 °C. ¹H-NMR(400 MHz, DMSO-d₆): δ 7.50–7.46 (m, 2H), 7.41–7.35 (m, 2H), 6.20 (s, 1H), 4.85 (s, 1H), 4.50 (q, 1H, J = 6.0 Hz), 3.86 (dd, 1H, J = 1.2 Hz), 3.65 (s, 3H), 3.22 (d, 1H, J = 12.0 Hz), 2.92 (d, 1H, J = 12.0 Hz), 2.58 (t, 1H, J = 12.0 Hz), 2.40–2.35 (m, 1H), 1.62–1.53 (m, 1H). ESI-MS (m/z) = 338.16 [M + H]⁺. The ¹H-NMR and MS data were in good agreement with those reported [27].

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-5,6,7,7a-tetrahydrothieno[3,2-c]pyridin-2(4H)-one (3b). Compound 3b was synthesized from 2b and compound 1 according to the procedure described for the preparation of 3a. White solid product (20.0 g, 65% yield), m.p. 122–124 °C. ¹H-NMR (400 MHz, DMSO-d6): δ 7.37–7.31 (m, 2H), 7.24–7.10 (m, 2H), 6.03 (s, 1H), 4.82 (s, 1H), 4.07 (q, 1H, J = 4.8 Hz), 3.94 (dd, 1H, J = 12.0 Hz), 3.08–3.05 (m, 2H), 2.35–2.30 (m, 2H), 2.12–2.06 (m, 1H), 1.93–1.83 (m, 1H), 1.04–1.01 (m, 2H), 0.90–0.78 (m, 2H). ESI-MS (m/z) = 332.19 [M + H]⁺. The ¹H-NMR and MS data were in good agreement with those reported [20].

5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-carbonyl)amino)propanoate (4a). EDCI (1.2 g, 6 mmol) was slowly added to an ice-cooled mixture of 3a (1.0 g, 3 mmol), l-N-Boc-alanine (0.7 g, 3.6 mmol), DMAP (40 mg, 0.3 mmol) in DCM (15 mL). The mixture was gradually warmed to room temperature and stirred for additional 4.0 h until the completion of the reaction detected by TLC. Then it was poured into ice-water (500 mL). The organic was separated and the aqueous was extracted with DCM. The combined organic was successively washed with cold 1.0 M aq HCl, saturated aq Na₂CO₃, and brine. It was dried over Na₂SO₄ and vacuum evaporated to give 4a (1.2 g, 81.2% yield) as a yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): 7.55 (d, 1H, J =7.8 Hz), 7.50–7.45 (m, 2H), 7.38–7.32 (m, 2H), 6.40 (s, 1H), 4.82 (s, 1H), 4.21–4.18 (m, 1H), 3.63 (s, 3H), 3.52 (s, 2H), 2.83–2.74 (m, 2H), 2.66 (s, 2H), 1.45–1.30 (m, 12H). ESI-MS (m/z) = 509.08 [M + H]⁺.

Compounds 4b–4p were synthesized according to the procedure described for the preparation of 4a.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy- carbonyl)-amino)-3-methylbutanoate (4b). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.55 (d, 1H, J = 7.2 Hz), 7.45 (t, 2H, J = 8.0 Hz and 9.2 Hz), 7.38–7.31 (m, 2H), 6.40 (s, 1H), 4.81 (s, 1H), 3.99 (t, 1H, J = 6.8 Hz), 3.63 (s, 3H), 3.52 (s, 2H), 2.83–2.66 (m, 4H), 2.10–2.05 (m, 1H), 1.37 (s, 9H), 0.92–0.89 (m, 6H). ESI-MS (m/z) = 537.04 [M + H]⁺.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-carbonyl)amino)-4-methylpentanoate (4c). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): 7.55 (d, 1H, J = 6.8 Hz), 7.47 (d, 2H, J = 7.2 Hz), 7.39–7.32 (m, 2H), 6.40 (s, 1H), 4.83 (s, 1H), 4.16–4.10 (m, 1H), 3.63 (s, 3H), 3.52 (s, 2H), 2.83–2.60 (m, 4H), 1.65–1.40 (m, 3H), 1.36 (s, 9H), 0.88–0.84 (m, 6H). ESI-MS (m/z) = 551.05 [M + H]⁺, HRMS(ESI) calcd. for C₂₇H₃₆ClN₂O₆S⁺: [M + H]⁺ m/z: 551.1977, found: 551.1968.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-carbonyl)amino)-3-methylpentanoate (4d). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.57 (d, 1H, J = 9.2 Hz), 7.48–7.34 (m, 4H), 6.42 (s, 1H), 4.83 (s, 1H), 4.08–4.04 (m, 1H), 3.65 (s, 3H), 3.53 (s, 2H), 2.85–2.75 (m, 2H), 2.65–2.63 (m, 2H), 1.88–1.80 (m, 1H), 1.38–1.18 (m, 11H), 0.88–0.73 (m, 6H). ESI-MS (m/z) = 551.10 [M + H]⁺.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-carbonyl)amino)-3-phenylpropanoate (4e). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.38 (t, 2H, J = 6.4 Hz), 7.31–7.29 (m, 1H), 7.22–7.16 (m, 2H), 7.12–7.03 (m, 5H), 6.16 (s, 1H), 4.66 (s, 1H), 4.23–4.14 (m, 1H), 3.47 (s, 3H), 3.35 (s, 2H), 3.26–3.10 (m, 2H), 2.92–2.80 (m, 2H), 2.67–2.57 (m, 2H), 1.16 (s, 9H). ESI-MS (m/z) = 585.11 [M + H]⁺.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-carbonyl)amino)-3-(1H-indol-2-yl)propanoate (4f). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): δ 10.86 (s, 1H), 7.59 (d, 1H, J = 8.0 Hz), 7.53–7.48 (m, 3H), 7.39–7.33 (m, 2H), 7.18 (s, 1H), 7.06 (t, 1H, J = 8.0 Hz), 6.98 (t, 1H, J = 8.0 Hz), 6.26 (s, 1H), 4.83 (s, 1H), 4.42–4.37 (m, 1H), 3.65 (s, 3H), 3.52 (s, 2H), 3.19–3.12 (m, 2H), 2.86–2.75 (m, 2H), 2.66–2.58 (m, 2H), 1.35 (s, 9H). ESI-MS (m/z) = 624.17 [M + H]⁺.

1-tert-Butyl 2-(5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl) pyrrolidine-1,2-dicarboxylate (4g). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.56 (t, 1H, J = 2.0 Hz and 6.8 Hz), 7.46 (t, 1H, J = 13.6 Hz), 7.38–7.32 (m, 2H), 6.44 (s, 1H), 4.82 (s, 1H), 4.37–4.34 (m, 1H), 3.63 (s, 3H), 3.52 (s, 2H), 3.41–3.27 (m, 2H), 2.80–2.74 (m, 2H), 2.66 (s, 2H), 2.33–2.26 (m, 1H), 1.98–1.94 (m, 1H), 1.86–1.80 (m, 2H), 1.35 (s, 9H). ESI-MS (m/z) = 535.05 [M + H]⁺.

1-tert-Butyl 2-(5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl) 5-oxopyrrolidine-1,2-dicarboxylate (4h). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.35 (dd, 1H, J = 7.2 Hz), 7.28–7.25 (m, 1H), 7.17–7.14 (m, 2H), 6.30 (s, 1H), 4.66–4.62 (m, 2H), 3.43 (s, 3H), 3.22 (s, 2H), 2.60–2.47 (m, 4H), 2.30–2.18 (m, 3H), 1.85–1.76 (m, 1H), 1.17 (s, 9H). ESI-MS (m/z) = 549.05 [M + H]⁺.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy- carbonyl)amino)propanoate (4i). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): 7.52–7.49 (m, 1H), 7.40–7.38 (m, 2H), 7.27–7.22 (m, 2H), 6.43 (s, 1H), 4.78 (s, 1H), 4.08–4.06 (m, 1H), 3.48 (s, 2H), 2.89–2.72 (m, 4H), 2.27–2.25 (m, 1H), 1.47–1.15 (m, 12H), 0.88–0.80 (m, 4H). ESI-MS (m/z) = 503.11 [M + H]⁺.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-carbonyl)amino)-3-methylbutanoate (4j). White solid. m.p. 106–108 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.52–7.45 (m, 2H), 7.42–7.37 (m, 1H), 7.26–7.22 (m, 2H), 6.44 (s, 1H), 4.78 (s, 1H), 4.00 (t, 1H, J = 6.4 Hz), 3.44 (t, 2H, J = 16.0 Hz), 2.80–2.69 (m, 4H), 2.38–2.36 (m, 1H), 2.11–2.06 (m, 1H), 1.38 (s, 9H), 0.94–0.78 (m, 10H). ESI-MS (m/z) = 531.22 [M + H]⁺, HRMS(ESI) calcd. for C₂₈H₃₆FN₂O₅S⁺: [M + H]⁺ m/z: 531.2323, found: 531.2314.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy- carbonyl)amino)-4-methylpentanoate (4k). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): 7.52–7.48 (m, 2H), 7.40 (dd, 1H, J = 13.6 Hz), 7.26–7.21 (m, 2H), 6.43 (s, 1H), 4.78 (s, 1H), 4.17–4.12 (m, 1H), 3.44 (t, 2H, J = 6.4 Hz), 2.80–2.70 (m, 4H), 2.37–2.35 (m, 1H), 1.66–1.42 (m, 3H), 1.37 (s, 9H), 0.90–0.80 (m, 10H). ESI-MS (m/z) = 545.14 [M + H]⁺.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-carbonyl)amino)-3-methylpentanoate (4l). White solid. m.p.: 80–82 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.52–7.45 (m, 2H), 7.40 (dd, 1H, J = 12.8 Hz), 7.26–7.21 (m, 2H), 6.43 (s, 1H), 4.77 (s, 1H), 4.05 (t, 1H, J = 6.8 Hz), 3.44 (t, 2H, J = 15.2Hz), 2.80–2.69 (m, 4H), 2.39–2.35 (m, 1H), 1.86–1.82 (m, 1H), 1.38–1.20 (m, 11H), 0.88–0.73 (m, 10H). ESI-MS (m/z) = 545.10 [M + H]⁺, HRMS(ESI) calcd. for C₂₉H₃₈FN₂O₅S⁺: [M + H]⁺ m/z: 545.2480, found: 545.2467.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-carbonyl)amino)-3-phenylpropanoate (4m). White solid. m.p.: 110–112 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.54 (d, 1H, J = 7.2 Hz), 7.48 (t, 1H, J = 7.2 Hz), 7.40–7.36 (m, 1H), 7.28–7.20 (m, 7 H), 6.32 (s, 1H), 4.76 (s, 1H), 4.35–4.14 (m, 1H), 3.45–3.36 (m, 2H), 3.05–3.00 (m, 2H), 2.79–2.66 (m, 4H), 2.36–2.34 (m, 1H), 1.31 (s, 9H), 0.87–0.76 (m, 4H). ESI-MS (m/z) = 579.17 [M+H]⁺, HRMS(ESI) calcd. for C₃₂H₃₆FN₂O₅S⁺: [M + H]⁺ m/z: 579.2323, found: 579.2315.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tertbutoxy- carbonyl)amino)-3-(1H-indol-2-yl)propanoate (4n). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): δ 10.86 (s, 1H), 7.52–7.49 (m, 3H), 7.40 (dd, 1H, J = 13.2Hz), 7.33 (d, 1H, J = 8.0 Hz), 7.27–7.22 (m, 2H), 7.18 (s, 1H), 7.08–6.95 (m, 2H), 6.26 (s, 1H), 4.78 (s, 1H), 4.40–4.36 (m, 1H), 3.45–3.37 (m, 2H), 3.18–3.14 (m, 2H), 2.80–2.68 (m, 4H), 2.38–2.36 (m, 1H), 1.34 (s, 9H), 0.88–0.80 (m, 4H). ESI-MS (m/z) = 618.23 [M + H]⁺.

1-tert-Butyl 2-(5-(2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl) pyrrolidine-1,2-dicarboxylate (4o). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.50, (t, 1H, J = 7.2 Hz), 7.40 (dd, 1H, J = 13.6 Hz), 7.28–7.22 (m, 2H), 6.47 (s, 1H), 4.78 (s, 1H), 4.42–4.36 (m, 1H), 3.65 (m, 1H), 3.48–3.33 (m, 4H), 3.03–2.79 (m, 4H), 2.37–2.24 (m, 2H), 2.02–2.00 (m, 1H), 1.98–1.87 (m, 2H), 1.39 (s, 9H), 0.88–0.75 (m, 4H). ESI-MS (m/z) = 529.10 [M + H]⁺.

1-tert-Butyl 2-(5-(2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl) 5-oxopyrrolidine-1,2-dicarboxylate (4p). Yellow oil. ¹H-NMR (400 MHz, DMSO-d₆): δ 7.50 (t, 1H, J = 7.2 Hz), 7.40 (dd, 1H, J = 13.2Hz), 7.27–7.22 (m, 2H), 6.53 (s, 1H), 4.87(q, 1H, J = 5.6 HZ), 4.79 (s, 1H), 3.49–3.41 (m, 2H), 2.82–2.69 (m, 4H), 2.56–2.34 (m, 4H), 2.08–2.01 (m, 1H), 1.39 (s, 9H), 0.88–0.84 (m, 4H). ESI-MS (m/z) = 543.15 [M + H]⁺.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl 2-aminopropanoate hydrochloride (5a). Compound 4a (1.0 g, 2.0 mmol) was stirred with hydrochloric ethyl acetate (10 mL, 2.0 M) at r.t. for 5.0 h. The formed precipitate was filtered and dried to give compound 5a (0.82 g, 92.1%) as a white solid. m.p.: 149−151 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.80 (s, 3H), 7.57–7.55 (m, 1H), 7.52–7.40 (m, 3H), 6.62 (s, 1H), 5.30 (m, 1H), 4.38 (s, 1H), 3.93 (s, 2H), 3.70 (s, 3H), 3.20–2.88 (m, 4H), 1.52 (d, 3H, J = 8.0 Hz). ESI-MS (m/z) = 408.98 [M + H]⁺, HRMS(ESI) calcd. for C₁₉H₂₂ClN₂O₄S⁺: [M + H]⁺ m/z: 409.0983, found: 409.0986.

Compounds 5b–5p were synthesized according to the procedure described for the preparation of 5a.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-methyl-butanoate hydrochloride (5b). White solid. m.p.: 165–167 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.93 (s, 3H), 7.77 (s, 1H), 7.63–7.58 (m, 1H), 7.52–7.46 (m, 2H), 6.64 (s, 1H), 5.34 (m, 1H), 4.15 (s, 1H), 3.93 (s, 2H), 3.70 (s, 3H), 3.23–2.92 (m, 4H), 2.34–2.29 (m, 1H), 1.05–0.98 (m, 6 H). ESI-MS (m/z) = 437.02 [M + H]⁺, HRMS(ESI) calcd. for C₂₁H₂₆ClN₂O₄S⁺: [M + H]⁺ m/z: 437.1296, found: 437.1333.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-4-methyl-pentanoate hydrochloride (5c). White solid. m.p.: 133–135 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.82 (s, 3H), 7.69–7.64 (m, 1H), 7.62–7.59 (m, 1H), 7.51–7.40 (m, 2H), 6.62 (s, 1H), 5.20 (m, 1H), 4.26 (s, 1H), 3.89 (s, 2H), 3.70 (s, 3H), 3.12–2.86 (m, 4H), 2.70–2.66 (m, 2H), 1.63–1.59 (m, 1H), 0.92–0.90 (m, 6 H). ESI-MS (m/z) = 451.03 [M + H]⁺, HRMS(ESI) calcd. for C₂₂H₂₈ClN₂O₄S⁺: [M + H]⁺ m/z: 451.1453, found: 451.1455. HPLC purity: 94.00%.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-methyl-pentanoate hydrochloride (5d). White solid. m.p.: 150–153 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.91 (s, 3H), 7.75–7.72 (m, 1H), 7.64–7.62 (m, 1H), 7.56–7.46 (m, 2H), 6.63 (s, 1H), 5.30–5.26 (m, 1H), 4.21 (s, 1H), 3.87 (s, 2H), 3.70 (s, 3H), 3.17–2.88 (m, 4H), 2.08–1.99 (m, 1H), 1.56–1.49 (m, 1H), 1.36–1.29 (m, 1H), 0.94–0.87 (m, 6 H). ESI-MS (m/z) = 451.04 [M + H]⁺, HRMS(ESI) calcd. for C₂₂H₂₈ClN₂O₄S⁺: [M + H]⁺ m/z: 451.1453, found: 451.1459.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-phenyl-propanoate hydrochloride (5e). White solid. m.p.: 166–168 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 9.01 (s, 3H), 7.65–7.57 (m, 3H), 7.46 (d, 2H, J = 6.4 Hz), 7.33–7.27 (m, 4H), 6.43 (s, 1H), 5.30–5.25 (m, 1H), 4.54 (s, 1H), 3.89 (s, 2H), 3.70 (s, 3H), 3.39–3.16 (m, 2H), 3.14–2.87 (m, 4H). ESI-MS (m/z) = 485.03 [M + H]⁺, HRMS(ESI) calcd. for C₂₅H₂₆ClN₂O₄S⁺: [M + H]⁺ m/z: 485.1296, found: 485.1296.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-(1H-indol-2-yl)propanoate hydrochloride (5f). White solid. m.p.: 173–175 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 11.10 (s, 1H), 8.92 (s, 3H), 7.57 (d, 2H, J = 7.2 Hz), 7.52–7.50 (m, 3H), 7.38 (d, 1H, J = 6.4 Hz), 7.25 (s, 1H), 7.08 (t, 1H, J = 12.0 Hz), 6.98 (t, 1H, J = 16.0 Hz), 6.36 (s, 1H), 5.28–5.24 (m, 1H), 4.50 (s, 1H), 3.93 (s, 2H), 3.70 (s, 3H), 3.51–3.46 (m, 1H), 3.38–3.33 (m, 1H), 3.20–2.88 (m, 4H). ESI-MS (m/z) = 524.09 [M + H]⁺, HRMS(ESI) calcd. for C₂₇H₂₇ClN₃O₄S⁺: [M + H]⁺ m/z: 524.1405, found: 524.1408.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-pyrrolidine-2-carboxylate hydrochloride (5g). White solid. m.p.: 102–104 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 10.47 (s, 1H), 9.47 (s, 1H), 7.72 (d, 1H, J = 8.8 Hz), 7.62–7.52 (m, 1H), 7.41–7.39 (m, 2H), 6.63 (s, 1H), 5.29–5.24 (m, 1H), 4.64 (s, 1H), 3.88 (s, 2H), 3.70 (s, 3H), 3.23–3.20 (m, 4H), 2.90–2.88 (m, 2H), 2.38–2.29 (m, 1H), 2.17–2.10 (m,1H), 1.97–1.90 (m, 2H). ESI-MS (m/z) = 434.96 [M + H]⁺, HRMS(ESI) calcd. for C₂₁H₂₄ClN₂O₄S⁺: [M + H]⁺ m/z: 435.1140, found: 435.1147.

5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-5-oxopyrrolidine-2-carboxylate hydrochloride (5h). White solid. m.p.: 88–90 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.10 (s, 1H), 7.76 (s, 1H), 7.61–7.59 (m, 1H), 7.51–7.39 (m, 3H), 6.59 (s, 1H), 5.42–5.37 (m, 1H), 4.49–4.46 (m, 1H), 3.71 (s, 3H), 3.67 (s, 2H), 3.04–2.90 (m, 4H), 2.47–2.10 (m, 4H). ESI-MS (m/z) = 449.00 [M + H]⁺, HRMS(ESI) calcd. for C₂₁H₂₂ClN₂O₅S⁺: [M + H]⁺ m/z: 449.0932, found: 449.0932.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-propanoate hydrochloride (5i). White solid. m.p.: 148–150 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.90 (s, 3H), 7.66–7.55 (m, 2H), 7.50–7.33 (m, 2H), 6.67 (s, 1H), 6.08–6.03 (m, 1H), 4.38 (s, 1H), 3.99 (s, 2H), 3.61–3.57 (m, 2H), 3.10–2.91 (m, 2H), 1.98–1.89 (m, 1H), 1.50 (d, 3H, J = 17.2 Hz), 1.07–0.93 (m, 4H). ESI-MS (m/z) = 403.05 [M + H]⁺, HRMS(ESI) calcd. for C₂₁H₂₄FN₂O₃S⁺: [M + H]⁺ m/z: 403.1486, found: 403.1491.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-methylbutanoate hydrochloride (5j). White solid. m.p.: 147–148 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.90 (s, 3H), 7.62 (d, 2H, J =6.4 Hz), 7.45–7.36 (m, 2H), 6.69 (s, 1H), 6.01–5.97 (m, 1H), 4.16 (s, 1H), 3.96 (s, 2H), 3.65–3.49 (m, 2H), 3.04–3.00 (m, 2H), 2.34–2.26 (m, 1H), 1.97–1.93 (m, 1H), 1.16–0.90 (m, 10H). ESI-MS (m/z) = 431.09 [M + H]⁺, HRMS(ESI) calcd. for C₂₃H₂₈FN₂O₃S⁺: [M + H]⁺ m/z: 431.1799, found: 431.1798.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-4-methylpentanoate hydrochloride (5k). White solid. m.p.: 141–143 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.64 (s, 3H), 7.70–7.62 (m, 2H), 7.47–7.37 (m, 2H), 6.68 (s, 1H), 6.08–6.01 (m, 1H), 4.23 (s, 1H), 3.98 (s, 2H), 3.61–3.48 (m, 2H), 3.05–3.00 (m, 2H), 2.76–2.71 (m, 2H), 1.97–1.87 (m, 1H), 1.65–1.63 (m, 1H), 0.91–0.90 (m, 10H). ESI-MS (m/z) = 445.08 [M + H]⁺, HRMS(ESI) calcd. for C₂₄H₃₀FN₂O₃S⁺: [M + H]⁺ m/z: 445.1956, found: 445.1958.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-methylpentanoate hydrochloride (5l). White solid. m.p.: 142–144 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.90 (s, 3H), 7.62–7.60 (m, 2H), 7.45–7.36 (m, 2H), 6.68 (s, 1H), 6.08–6.03 (m, 1H), 4.22 (s, 1H), 3.85 (s, 2H), 3.15–2.92 (m, 4H), 2.06–1.88 (m, 2H), 1.54–1.49 (m, 1H),1.36–1.32 (m, 1H), 1.03–0.87 (m, 10H). ESI-MS (m/z) = 445.07 [M + H]⁺, HRMS(ESI) calcd. for C₂₄H₃₀FN₂O₃S⁺: [M + H]⁺ m/z: 445.1956, found: 445.1957.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-phenylpropanoate hydrochloride (5m). White solid. m.p.: 156–158 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 9.06 (s, 3H), 7.65–7.63 (m, 2H), 7.43–7.28 (m, 7 H), 6.48 (s, 1H), 6.06–6.02 (m, 1H), 4.53 (s, 1H), 3.88 (s, 2H), 3.39–3.15 (m, 2H), 3.14–2.87 (m, 4H), 1.99–1.95 (m, 1H), 0.91–0.89 (m, 4H). ESI-MS (m/z) = 479.05 [M + H]⁺, HRMS(ESI) calcd. for C₂₇H₂₈FN₂O₃S⁺: [M + H]⁺ m/z: 479.1799, found: 479.1798.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-(1H-indol-2-yl)propanoate hydrochloride (5n). White solid. m.p.: 166–168 °C. ¹H-NMR (400 MHz, DMSO-d6): δ 11.11 (s, 1H), 8.93 (s, 3H), 7.61–7.56 (m, 3H), 7.46–7.30 (m, 3H), 7.25 (s, 1H), 7.07 (t, 1H, J = 7.6 Hz), 6.99 (t, 1H, J = 7.2 Hz), 6.43 (s, 1H), 4.97–4.95 (m, 1H), 4.50 (s, 1H), 4.00 (s, 2H), 3.51–3.23 (m, 2H), 3.20–2.88 (m, 4H), 1.91–1.89 (m, 1H), 1.04–0.89 (m, 4H). ESI-MS (m/z) = 518.09 [M + H]⁺, HRMS(ESI) calcd. for C₂₉H₂₉FN₃O₃S⁺: [M + H]⁺ m/z: 518.1908, found: 518.1912.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-pyrrolidine-2-carboxylate hydrochloride (5o). White solid. m.p.: 96–98 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 10.74 (s, 1H), 9.72 (s, 1H), 7.69–7.62 (m, 2H), 7.47–7.37 (m, 2H), 6.69 (s, 1H), 6.10–6.03 (m, 1H), 4.63 (s, 1H), 4.23–4.19 (s, 2H), 3.57–3.47 (m, 2H), 3.28–3.06 (m, 4H), 2.34–2.10 (m,2H), 1.97–1.90 (m, 3H), 1.07–0.90 (m, 4H). ESI-MS (m/z) = 429.08 [M + H]⁺, HRMS(ESI) calcd. for C₂₃H₂₆FN₂O₃S⁺: [M + H]⁺ m/z: 429.1643, found: 429.1640.

5-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-5-oxo-pyrrolidine-2-carboxylate hydrochloride (5p). White solid. m.p.: 98–101 °C. ¹H-NMR (400 MHz, DMSO-d₆): δ 8.11 (s, 1H), 7.64–7.39 (m, 5 H), 6.63 (s, 1H), 6.09–6.03 (m, 1H), 4.48–4.47 (m, 1H), 3.44 (s, 2H), 3.04 (m, 2H), 2.44–2.40 (m, 1H), 2.20–2.12 (m, 3H), 1.97–1.90 (m, 3H), 1.05–0.90 (m, 4H). ESI-MS (m/z) = 443.06 [M + H]⁺, HRMS(ESI) calcd. for C₂₃H₂₄FN₂O₄S⁺: [M + H]⁺ m/z: 443.1435, found: 443.1433.

4.3. Inhibition of ADP-Induced Platelet Aggregation in Rats

ADP-induced platelet aggregation was determined by Born’s method [28]. Male SD rats (200–300 g, 5 in each group) were orally gavaged at random with vehicle control, target compounds, clopidogrel and prasugrel. The volume of target compounds and positive control was 10 mL/kg × body weight, while the vehicle group was equal volume as experimental group instead of 0.5% CMC-Na. Two hours after administration, the animals were anesthetized (with 0.7% chloral hydrate intraperitoneal injection) and bloods were collected via aorta ventralis puncture into anticoagulant solution (3.8% sodium citrate). Platelet rich plasma (PRP) was centrifuged at 230 rpm for 15 mins and then adjusted by platelet poor plasma (PPP, centrifuged at 2000 rpm for 10 mins). Platelet count was 5 × 10⁸/mL. Aggregation was induced by ADP (20 μM) and measured using an aggregometer (LBY-NJ4, Beijing, China ). The platelet aggregation was observed maximum platelet aggregation (MPA) from the aggregometer. The percentage of inhibition of platelet aggregation (IPA) was calculated from the observed MPA by the following equation:

IPA (%) = (MPA_vehicle − MPA_compound)/MPA_vehicle × 100%

Inhibition rats at different doses were calculated ED₅₀ by the software named Statistical Product and Service Solutions (SPSS). The animal laboratory got animal use certificate issued by Science and Technology department of Tianjin (SYXK(jin)2016-0013).

4.4. Determination of Bleeding Time

The tail transection bleeding time was determined by the method of Dejana et al. [29]. Male SD rats (200–300 g, 5 in each group) were orally gavaged at random with vehicle control, target compounds, clopidogrel and prasugrel. The volume of target compounds and positive control was 10 mL/kg × body weight, while the vehicle group was equal volume as experimental group instead of 0.5% CMC-Na. The test drugs were orally administered 1H before the tail transection. Under anaesthesia with urethane (5 mL/kg), the rat tail was transected at 5 mm from the tip by a scalpel, and the tail was immediately immersed into warmed (37 °C) saline until blood flow stopped. Bleeding time was assessed as the time from the tail transection to the termination of blood flow. Bleeding times beyond 60 min were regarded as 60 min for the purpose of statistical analysis. BT₂ values were calculated from linear-regression analysis.

5. Conclusions

In summary, we designed and synthesized a series of amino acid prodrugs based on thienopyridine scaffolds as novel potent P2Y₁₂R inhibitors. Inhibition of ADP-induced platelet aggregation in rat assays showed that compounds 5k, 5l, 5c, 5j, 5m, and 5n displayed good activity and compounds 5c and 5l have moderate ED₅₀ values between those of clopidogrel and prasugrel. In the tail bleeding test, 5c induced much shorter bleeding time in rats and 5l behaved slightly better than prasugrel. Based on their ratio of benefit/bleeding risk, we will take 5c as a drug candidate for further research.