Synthesis of a novel series of amino acid prodrugs based on thienopyridine scaffolds and evaluation of their antiplatelet activity

Article abstract of DOI:10.3390/molecules23051041

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.

Full text of DOI:10.3390/molecules23051041

molecules
Article
Synthesis of a Novel Series of Amino Acid Prodrugs
Based on Thienopyridine Scaffolds and Evaluation of
Their Antiplatelet Activity
Nan Lu 1,2 , Lingjun Li , Xuemin Zheng , Shijun Zhang , Yuquan Li , Jing Yuan ,
ID
2
2
2
2
2
2
1,
2, ID
Qunchao Wei , Youjun Xu * and Fancui Meng *
1
School of Pharmaceutical Engineering, and Key Laboratory of Structure-Based Drug Design &
Discovery (Ministry of Education), Shenyang Pharmaceutical University, Shenyang 110016, China;
lunantj@126.com
Tianjin Key Laboratory of Molecular Design and Drug Discovery, Tianjin Institute of Pharmaceutical
2
Research, Tianjin 300193, China; lilj@tjipr.com (L.L.); zhengxm@tjipr.com (X.Z.); zhangsj@tjipr.com (S.Z.);
liyq@tjipr.com(Y.L.); yuanj@tjipr.com (J.Y.); weiqc@tjipr.com (Q.W.)
*
Correspondence: xuyoujun@syphu.edu.cn (Y.X.); mengfc@tjipr.com (F.M.); Tel.: +86-24-4352-0223(Y.X.);
86-22-2300-6833 (F.M.)
+
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 15 March 2018; Accepted: 26 April 2018; Published: 28 April 2018
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.
Keywords: thienopyridines; P2Y12 receptor antagonist; antiplatelet; amino acid prodrugs;
ADP-induced platelet aggregation
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 [
After platelet activation, ADP is released from intracellular storage granules and then further
activates platelets, enlarging the activation and thus aggregation processes [ ]. It was shown
that the ADP response was due to activation of two receptors, the G -coupled P2Y receptor,
which induces a calcium response and shape change of the blood platelet and the G -coupled
1].
2,3
q
1
i
P2Y R, which decreases the intracellular adenylyl cyclase activity and prolongs intracellular calcium
12
signaling, thereby stabilizing the formed platelet aggregates [
4–7]. Consequently, blocking the
P2Y R is a valid strategy to antiplatelet therapy, as demonstrated by the thienopyridine class
12
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) [
therapy with aspirin and clopidogrel has remained the treatment of choice for patients with ACS
and for those undergoing percutaneous coronary interventions [ 12]. However, up to 30% of
8]. Until now, dual antiplatelet
9–
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
Molecules 2018, 23, 1054; doi:10.3390/molecules23051054
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1054
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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 [15–18]. All these considerations reinforce the strong need
of novel and safe P2Y12 antagonists.
Figure 1. The representative chemical structures of drugs of ADP receptor antagonist.
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
3
b
(Scheme 1) which are, respectively, the thiolactone of clopidogrel and prasugrel, as a parent part of
the target compounds.
O
N
O
R
R
P450
N
P450
HOOC
N
H
CO2Me
O
S
N
S
CYP2C19
HS
active metabolite
2
HO C
Cl
HS
Clopidogrel
thiolactone
Cl
6
a (active unit thienopyridine
Prodrug design
from clopidogrel)
O
O
O
O
Esterase
N
F
R
N
S
N
HO2C
O
O
R1
S
HS
F
Prasugrel
6
b(active unit thienopyridine
from prasugrel )
Figure 2. Simplified biosynthetic mechanism of the active units of thienopyridines and prodrug design.
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.

Molecules 2018, 23, 1054
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2
. Results
2
.1. Chemistry
The synthesis of the molecules was designed and carried out as shown in Scheme 1.
The thienopyridine hydrochloride was reacted with substituted intermediates 2a in the absence of
potassium bicarbonate to afford the N-alkylated thiolactones 3a , 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
1
–b
–
b
–
,
–
salts after treating with hydrochloric acid in ethyl acetate for the removal of the Boc-group. All the
structures are outlined in Table 1.
Scheme 1. Synthetic route of compounds 5a–5p. Reagents and conditions: (i) K CO , CH CN,
2 3 3
◦
r.t. overnight (60–65% yield); (ii) N-Boc-L-amino acid, EDCI, DMAP, DCM, 0 C-r.t., 4 h (71–86%
yield); (iii) hydrochloric ethyl acetate, r.t., 5 h (84–93% yield).
Table 1. Structures of target compounds.
R or R4 a
R or R
4
1
a
3
3
Compound
Compound
(
R =COOCH , R =Cl)
(R =COCH(CH ) , R =F)
1
3
2
2 2
2
H C
H C
5
5
a
3
5i
5j
3
b
5
5
c
5k
5l
d
5
e
5m
5
f
5n
5
5
g
h
5o
5p
a
R4 was only used for compounds 5g–5h and 5o–5p.

Molecules 2018, 23, 1054
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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 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.
Table 2. Inhibitory effect of target compounds on ADP-induced platelet aggregation in rats at a dose of
3
mg/kg and 1mg/kg.
Inhibition Ration (%)
Compounds
3
mg/kg
1 mg/kg
5
a
55.1
36.8
98.4
68.9
43.3
63.8
64.2
51.6
93.4
86.3
89.2
96.4
100
35.1
19.4
39.6
16.2
15.8
37.2
35.5
27.2
17.5
52.6
66.2
89.8
47.6
46.4
30.7
35.1
–
5
b
5
c
5
5
d
e
5
f
5
g
5
h
5
5
i
j
5
k
5
l
5
5
m
n
99.3
93.6
69.5
34.4
100
5
o
5
p
Clopidogrel
Prasugrel
91.0
3
mg/kg
1 mg/kg
1
00
100
80
8
6
4
2
0
0
0
0
0
6
4
2
0
0
0
0
Compounds
Compounds
Figure 3. Inhibition ratios of all compounds at doses of 3 mg/kg and 1 mg/kg.
As shown in the 1 mg/kg level results, 5l approached prasugrel in inhibition behavior and 5c
5m and 5n presented slightly less potency than prasugrel at the 1 mg/kg level. Although these
23],
, 5j,
5
k,
compounds should covert to the same activate metabolites 6a or 6b to behave efficaciously [22
,
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.

Molecules 2018, 23, 1054
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2
.2.2. Determination of ED50 and BT of 5c and 5l
2
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 summarizes the ED50 and BT values for the tested
2
compounds. The BT was defined as the dose that doubled the vehicle bleeding time. The ED
2
50
and BT values of clopidogrel and prasugrel were in good agreement with those reported [24,25].
2
In addition, the ratios of ED50 to BT indicated the tested compounds have a similar benefit/bleeding
2
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.
5
c
5l
1
00
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
*
*
*
*
**
2
vehicle
0.5
1
vehicle
1
2
4
Dose (mg/kg)
Dose (mg/kg)
Prasugrel
Clopidogrel
1
00
1
00
8
6
4
0
0
0
80
60
40
20
0
**
**
20
0
**
1
**
2
vehicle
0.5
vehicle
2.5
5
10
-20
-
20
Dose (mg/kg)
Dose (mg/kg)
Figure 4. The platelet aggregation of 5c, 5k, clopidogrel and prasugrel at different doses. ** p < 0.01 vs.
vehicle. Data are the mean ± SD, n = 5).

Molecules 2018, 23, 1054
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5c
5l
6
5
4
3
2
1
0
0
0
0
0
0
0
60
50
40
30
20
10
0
**
*
vehicle
0.96
1.92
2.4
vehicle
0.24
0.48
0.96
Dose (mg/kg)
Dose (mg/kg)
Clopidogrel
Prasugrel
***
***
6
0
60
*
5
4
3
2
1
0
0
0
0
0
0
50
40
30
20
10
0
vehicle
1.5
3
6
vehicle
0.24
0.48
0.96
Dose (mg/kg)
Dose (mg/kg)
Figure 5. The bleeding time of 5c and 5k at different doses. * p < 0.05, ** p < 0.01, *** p < 0.001 vs.
vehicle. Data are the mean ± SD, n = 5).
Table 3. Comparison of antiplatelet antihaemostatic effects of potent compounds.
Compounds
ED50 (mg/kg)
BT (mg/kg)
ED50/BT2
2
Clopidogrel
Prasugrel
3.96
0.50
2.16
0.74
3.82
0.51
2.32
0.70
1.04
0.98
0.93
1.06
5
c
l
5
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
1
13
University, Tianjin, China). H-NMR and C-NMR spectra were recorded for DMSO-d solutions
6
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).

Molecules 2018, 23, 1054
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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 CH CN (500 mL) were
3
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
◦
1
afford a white solid (20.9 g, 62% yield). m.p.: 118–119 C. H-NMR(400 MHz, DMSO-d ):
δ
7.50–7.46
6
(
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,
+
1
1H), 1.62–1.53 (m, 1H). ESI-MS (m/z) = 338.16 [M + H] . The H-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
Compound 3b was synthesized from 2b and compound according to the procedure described for
the preparation of 3a. White solid product (20.0 g, 65% yield), m.p. 122–124 C. H-NMR (400 MHz,
DMSO-d6): 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),
.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),
(3b).
1
◦
1
δ
3
+
1
1
.04–1.01 (m, 2H), 0.90–0.78 (m, 2H). ESI-MS (m/z) = 332.19 [M + H] . The H-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-car
bonyl)amino)propanoate (4a). EDCI (1.2 g, 6 mmol) was slowly added to an ice-cooled mixture of
(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).
3
a
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 Na CO , and brine. It was dried over Na SO and
2
3
2
4
1
vacuum evaporated to give 4a (1.2 g, 81.2% yield) as a yellow oil. H-NMR (400 MHz, DMSO-d ):
6
7
3
.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),
.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
of 4a.
–4p were synthesized according to the procedure described for the preparation
5
-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-
1
carbonyl)-amino)-3-methylbutanoate (4b). Yellow oil. H-NMR (400 MHz, DMSO-d ):
δ 7.55 (d, 1H,
6
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,
+
6
H). 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-car
1
bonyl)amino)-4-methylpentanoate (4c). Yellow oil. H-NMR (400 MHz, DMSO-d ): 7.55 (d, 1H,
6
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,
3
5
H), 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) =
+
+
+
51.05 [M + H] , HRMS(ESI) calcd. for C H ClN O S : [M + H] m/z: 551.1977, found: 551.1968.
27
36
2
6
5
-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-car
1
bonyl)amino)-3-methylpentanoate (4d). Yellow oil. H-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] .

Molecules 2018, 23, 1054
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5
-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tert-butoxy-car
1
bonyl)amino)-3-phenylpropanoate (4e). Yellow oil. H-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-car
1
bonyl)amino)-3-(1H-indol-2-yl)propanoate (4f). Yellow oil. H-NMR (400 MHz, DMSO-d ):
δ 10.86 (s,
6
1
H), 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)
1
pyrrolidine-1,2-dicarboxylate (4g). Yellow oil. H-NMR (400 MHz, DMSO-d ):
δ 7.56 (t, 1H, J = 2.0 Hz
6
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
5
-tert-Butyl 2-(5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl)
-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-
1
carbonyl)amino)propanoate (4i). Yellow oil. H-NMR (400 MHz, DMSO-d ): 7.52–7.49 (m, 1H), 7.40–7.38
6
(
2
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),
.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-car
◦
1
bonyl)amino)-3-methylbutanoate (4j). White solid. m.p. 106–108 C. H-NMR (400 MHz, DMSO-d ):
6
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 C H FN O S : [M + H]
28 36 2 5
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-
1
carbonyl)amino)-4-methylpentanoate (4k). Yellow oil. H-NMR (400 MHz, DMSO-d ): 7.52–7.48 (m, 2H),
6
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-car
◦
1
bonyl)amino)-3-methylpentanoate (4l). White solid. m.p.: 80–82 C. H-NMR (400 MHz, DMSO-d ):
6
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 C H FN O S :
29 38 2 5
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-car
◦
1
bonyl)amino)-3-phenylpropanoate (4m). White solid. m.p.: 110–112 C. H-NMR (400 MHz, DMSO-d ):
6
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
(
1
[
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),
+
+
.31 (s, 9H), 0.87–0.76 (m, 4H). ESI-MS (m/z) = 579.17 [M+H] , HRMS(ESI) calcd. for C H FN O S :
32 36 2 5
+
M + H] m/z: 579.2323, found: 579.2315.

Molecules 2018, 23, 1054
9 of 13
5
-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-((tertbutoxy-
1
carbonyl)amino)-3-(1H-indol-2-yl)propanoate (4n). Yellow oil. H-NMR (400 MHz, DMSO-d ):
δ
10.86 (s,
6
1
7
2
H), 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),
.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),
+
.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)
1
pyrrolidine-1,2-dicarboxylate (4o). Yellow oil. H-NMR (400 MHz, DMSO-d ):
δ 7.50, (t, 1H, J = 7.2 Hz),
6
7
3
9
.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),
.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,
+
H), 0.88–0.75 (m, 4H). ESI-MS (m/z) = 529.10 [M + H] .
1
5
-tert-Butyl 2-(5-(2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl)
1
-oxopyrrolidine-1,2-dicarboxylate (4p). Yellow oil. H-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
4
.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,
H). 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
9
.0 M) at r.t. for 5.0 h. The formed precipitate was filtered and dried to give compound 5a (0.82 g,
◦
151 C. H-NMR (400 MHz, DMSO-d ): δ 8.80 (s, 3H), 7.57–7.55 (m,
6
1
2.1%) as a white solid. m.p.: 149
−
1
4
H), 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,
H), 1.52 (d, 3H, J = 8.0 Hz). ESI-MS (m/z) = 408.98 [M + H] , HRMS(ESI) calcd. for C H ClN O S :
+
+
19
22
2
4
+
[
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-
◦
1
butanoate hydrochloride (5b). White solid. m.p.: 165–167 C. H-NMR (400 MHz, DMSO-d ):
δ
8.93 (s,
H), 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,
H), 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 +
6
3
2
+
+
+
H] , HRMS(ESI) calcd. for C H ClN O S : [M + H] m/z: 437.1296, found: 437.1333.
21
26
2
4
5
-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-4-methyl-
◦
1
pentanoate hydrochloride (5c). White solid. m.p.: 133–135 C. H-NMR (400 MHz, DMSO-d ):
3
3
δ
8.82 (s,
H), 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),
.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).
6
+
+
+
ESI-MS (m/z) = 451.03 [M + H] , HRMS(ESI) calcd. for C H ClN O S : [M + H] m/z: 451.1453,
22
28
2
4
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-
◦
1
pentanoate hydrochloride (5d). White solid. m.p.: 150–153 C. H-NMR (400 MHz, DMSO-d ):
δ
8.91 (s,
6
3
H), 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 C H ClN O S : [M + H]
22 28 2 4
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-
◦
1
propanoate hydrochloride (5e). White solid. m.p.: 166–168 C. H-NMR (400 MHz, DMSO-d ):
δ
9.01 (s,
6
3
H), 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 C H ClN O S : [M + H] m/z: 485.1296, found: 485.1296.
25
26
2
4

Molecules 2018, 23, 1054
10 of 13
5
2
δ
-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-(1H-indol-
◦
1
-yl)propanoate hydrochloride (5f). White solid. m.p.: 173–175 C. H-NMR (400 MHz, DMSO-d ):
6
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,
1
2
H), 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,
H), 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 C H ClN O S : [M + H] m/z: 524.1405, found: 524.1408.
27
27
3
4
5
-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-pyrrolidine-2-car
◦
1
boxylate hydrochloride (5g). White solid. m.p.: 102–104 C. H-NMR (400 MHz, DMSO-d ):
δ
10.47
6
(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 C H ClN O S : [M + H] m/z: 435.1140, found: 435.1147.
21
24
2
4
5
-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-5-oxopyrrolidine-2-car
◦
1
boxylate hydrochloride (5h). White solid. m.p.: 88–90 C. H-NMR (400 MHz, DMSO-d ):
δ
8.10 (s,
6
1
1
H), 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,
+
H), 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 C H ClN O S : [M + H] m/z: 449.0932, found: 449.0932.
21
22
2
5
5
-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-propanoate
◦
1
hydrochloride (5i). White solid. m.p.: 148–150 C. H-NMR (400 MHz, DMSO-d ):
δ
8.90 (s, 3H),
.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)
6
7
(
+
+
+
=
403.05 [M + H] , HRMS(ESI) calcd. for C H FN O S : [M + H] m/z: 403.1486, found: 403.1491.
21 24 2 3
5
-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-methyl
◦
1
butanoate hydrochloride (5j). White solid. m.p.: 147–148 C. H-NMR (400 MHz, DMSO-d ):
3
2
δ
8.90 (s,
H), 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,
H), 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).
6
+
+
+
ESI-MS (m/z) = 431.09 [M + H] , HRMS(ESI) calcd. for C H FN O S : [M + H] m/z: 431.1799,
23
28
2
3
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-methyl
◦
1
pentanoate hydrochloride (5k). White solid. m.p.: 141–143 C. H-NMR (400 MHz, DMSO-d ):
δ
8.64
6
(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 C H FN O S : [M + H] m/z:
24 30 2 3
4
45.1956, 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-methyl
◦
1
pentanoate hydrochloride (5l). White solid. m.p.: 142–144 C. H-NMR (400 MHz, DMSO-d ):
δ
8.90
6
(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,
2
H), 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 C H FN O S : [M + H] m/z: 445.1956,
24
30
2
3
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-phenyl
◦
1
propanoate hydrochloride (5m). White solid. m.p.: 156–158 C. H-NMR (400 MHz, DMSO-d ):
3
3
δ
9.06 (s,
H), 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),
.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 +
6
+
+
+
H] , HRMS(ESI) calcd. for C H FN O S : [M + H] m/z: 479.1799, found: 479.1798.
27
28
2
3
5
-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-2-amino-3-(1H-
◦
1
indol-2-yl)propanoate hydrochloride (5n). White solid. m.p.: 166–168 C. H-NMR (400 MHz, DMSO-d6):

Molecules 2018, 23, 1054
11 of 13
δ
6
3
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),
.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),
+
.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 C H FN O S : [M + H] m/z: 518.1908, found: 518.1912.
29
29
3
3
5
-(2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-pyrrolidine-2-car
◦
1
boxylate hydrochloride (5o). White solid. m.p.: 96–98 C. H-NMR (400 MHz, DMSO-d ):
δ
10.74 (s, 1H),
.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).
6
9
(
+
+
+
ESI-MS (m/z) = 429.08 [M + H] , HRMS(ESI) calcd. for C H FN O S : [M + H] m/z: 429.1643,
23
26
2
3
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-
◦
1
2
1
2
-carboxylate hydrochloride (5p). White solid. m.p.: 98–101 C. H-NMR (400 MHz, DMSO-d ):
δ
8.11 (s,
H), 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),
.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 +
6
+
+
+
H] , HRMS(ESI) calcd. for C H FN O S : [M + H] m/z: 443.1435, found: 443.1433.
23
24
2
4
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
8
was 5 × 10 /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 (%) = (MPAvehicle − MPAcompound)/MPAvehicle × 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
6
0 min were regarded as 60 min for the purpose of statistical analysis. BT values were calculated from
2
linear-regression analysis.

Molecules 2018, 23, 1054
12 of 13
5
. Conclusions
In summary, we designed and synthesized a series of amino acid prodrugs based on
thienopyridine scaffolds as novel potent P2Y R inhibitors. Inhibition of ADP-induced platelet
12
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.
Author Contributions: Nan Lu and Lingjun Li conceived, designed and performed the synthetic experiment part;
Yuquan Li and Jing Yuan analyzed the data; Xuemin Zheng, Shijun Zhang and Qunchao Wei conceived, designed
and performed the pharmacological test part; Nan Lu wrote the paper. Youjun Xu and Fancui Meng assisted
paper revision.
Acknowledgments: This project was supported by Tianjin Natural Science Foundation-Young Foundation (Nos.
1
7JCQNJC13700 and 17JCQNJC13100) and Innovative Research Team in SYPHU by the supporting Fund for
Universities from the Chinese Central Government (51150039). And we also thank the innovative research team
of the Ministry of Education and program for Liaoning innovative research team in university.
Conflicts of Interest: The authors declare no conflict of interest.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(