Thieno[2,3-b]pyridine derivatives are potent anti-platelet drugs, inhibiting platelet activation, aggregation and showing synergy with aspirin

Article abstract of DOI:10.1016/j.ejmech.2017.11.014

Drugs which inhibit platelet function are commonly used to prevent blood clot formation in patients with Acute Coronary Syndromes (ACS) or those at risk of stroke. The thieno[3,2-c]pyridine class of therapeutic agents, of which clopidogrel is the most com

Full text of DOI:10.1016/j.ejmech.2017.11.014

Accepted Manuscript
Thieno[2,3-b]pyridine derivatives are potent anti-platelet drugs, inhibiting platelet
activation, aggregation and showing synergy with aspirin
Naif K. Binsaleh, Catherine A. Wigley, Kathryn A. Whitehead, Michelle van Rensburg,
Johannes Reynisson, Lisa I. Pilkington, David Barker, Sarah Jones, Nina C.
Dempsey-Hibbert
PII:
S0223-5234(17)30907-8
10.1016/j.ejmech.2017.11.014
EJMECH 9889
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 October 2017
Revised Date: 19 October 2017
Accepted Date: 4 November 2017
Please cite this article as: N.K. Binsaleh, C.A. Wigley, K.A. Whitehead, M. van Rensburg, J. Reynisson,
L.I. Pilkington, D. Barker, S. Jones, N.C. Dempsey-Hibbert, Thieno[2,3-b]pyridine derivatives are potent
anti-platelet drugs, inhibiting platelet activation, aggregation and showing synergy with aspirin, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.014.
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ACCEPTED MANUSCRIPT
Novel thienopyridine derivatives are potent anti-platelet drugs, inhibiting platelet
activation, aggregation and showing synergy with aspirin
Naif K. Binsaleha, Catherine A. Wigleya, Kathryn A. Whiteheada, Michelle van Rensburgb, Johannes
Reynissonb, Lisa I. Pilkingtonb, David Barkerb, Sarah Jonesa, and Nina C. Dempsey-Hibberta,
a School of Healthcare Science, Manchester Metropolitan University, Manchester, UK, M1 5GD
b School of Chemical Sciences, The University of Auckland, New Zealand.

ACCEPTED MANUSCRIPT
Thieno[2,3-b]pyridine derivatives are potent anti-platelet drugs,
inhibiting platelet activation, aggregation and showing synergy with
aspirin
Naif K. Binsaleha, Catherine A. Wigleya, Kathryn A. Whiteheada, Michelle van Rensburgb,
Johannes Reynissonb, Lisa I. Pilkingtonb, David Barkerb, Sarah Jonesa and Nina C.
Dempsey-Hibberta,
a School of Healthcare Science, Manchester Metropolitan University, Manchester, UK, M1 5GD
b School of Chemical Sciences, The University of Auckland, New Zealand.
To whom correspondence should be addressed: School of Healthcare Science, Manchester Metropolitan University, Manchester, UK, M1
5GD (N.C.D-H.); School of Chemical Sciences, The University of Auckland, New Zealand (D.B.).
E-mail addresses: n.dempsey-hibbert@mmu.ac.uk (N.C. Dempsey-Hibbert), d.barker@auckland.ac.nz (D. Barker).
Keywords: thienopyridine, platelets, thrombosis, clopidogrel, aspirin
Abstract
Drugs which inhibit platelet function are commonly used to prevent blood clot formation in patients
with Acute Coronary Syndromes (ACS) or those at risk of stroke. The thieno[3,2-c]pyridine class of
therapeutic agents, of which clopidogrel is the most commonly used, target the P2Y12 receptor, and
are often used in combination with acetylsalicylic acid (ASA). Six thieno[2,3-b]pyridine were
assessed for in vitro anti-platelet activity; all derivatives showed effects on both platelet activation and
aggregation, and showed synergy with ASA. Some compounds demonstrated greater activity when
compared to clopidogrel. These compounds, therefore, represent potential novel P2Y12 inhibitors for
improved treatment for patients.
1. Introduction
Acute coronary syndromes (ACS) are life-threatening heart conditions ranging from chest pain
(unstable angina) to myocardial infarction (MI). Typically in ACS, inappropriate platelet-rich
thrombus formation occurs, eventually occluding blood flow, resulting in a lack of oxygen, heart
damage and potential death [1,2]. Platelet hyperactivity is often a major contributory factor in the
development of disorders such as ACS and also stroke due to acute arterial thrombosis [3]. MI and
stroke are currently the two most common causes of morbidity in the developed world [4]. Thus, the
ability to control platelet activity and reduce adverse arterial thrombus formation is a critical tool in
modern clinical practice.
Platelets circulate within the blood in a resting state, but upon contact with a platelet agonist, will
undergo various biochemical and physiological changes to become activated and begin to aggregate
[5]. Adenosine diphosphate (ADP) acts to induce activation via binding with the P2Y1 and P2Y12
receptors, leading to platelet shape change, the release of α- and dense- storage granules, intracellular
mobilization and an influx of Ca2+. There are significantly less numbers of P2Y1 present on the
platelet membrane and these are involved in producing a platelet shape change but only generate a
weak and transient aggregation [5]. In contrast, the P2Y12 receptor is found in very large numbers on

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the platelet cell surface membrane. Although it was initially thought to be platelet specific, P2Y12 has
recently been shown to be present in low numbers on some other cell types such as leukocyte
subtypes, microglia, vascular smooth muscle cells and in some cancer cell lines [6,7]. ADP is also
contained in platelet dense granules whereby initial activation of the platelet results in release of the
dense granules and hence stored ADP which acts to amplify the activation response, an elegant
example of a positive feedback mechanism [4]. Platelet activation by alternative agonists such as
collagen or thrombin also promote dense granule section and therefore ADP is again involved in
amplification of platelet activation. Downstream of the initial P2Y12 receptor activation, GPIIb/IIIa
receptor activation is induced, this results in further degranulation, thromboxane (TXA2) production
and prolonged platelet aggregation [8].
Due to the importance of P2Y12 in platelet activation, P2Y12 inhibitors have been developed which
bind to the receptor, thereby blocking the binding of ADP. Indeed, a combination treatment of a
P2Y12 inhibitor and a COX-1 inhibitor, most commonly clopidogrel and aspirin (acetylsalicylic acid,
(ASA)) results in reduced cardiac events in patients with ACS and patients having undergone
percutaneous coronary intervention (stents) [9]. Prior to FDA (USA Food and Drug Administration)
approval, the thieno[3,2-c]pyridine clopidogrel was shown to reduce the risk of death/MI and stroke
in the CURE (Clopidogrel in Unstable angina to prevent Recurrent Events) and CREDO (Clopidogrel
for the Reduction of Events During Observation) trials, and it was concluded that clopidogrel and
ASA treatment had long-term benefits [9-12]. However, ASA and clopidogrel combination treatment
is usually only recommended for a maximum of 12 months due to the potential for gut damage and
bleeding. Furthermore, not all patients are appropriate for this treatment, as adequate liver function is
needed to metabolise these drugs into their active forms [4,10]. Of those patients that are suitable for
treatment, approximately 4%-30% will be classed as ‘non-responders’ [13]. Currently there are
several proposed reasons for clopidogrel poor/no response which include genetics (CYP2C19*2 loss
of function allele, P2Y12 receptor gene polymorphism), drug interactions, (e.g. Paclitaxel, Statins,
Calcium channel blockers), patient body mass index and co-morbidities such as diabetes, intestinal
conditions and impaired renal function [8,14-20].
More recently a non-thienopyridine-based P2Y12 inhibitor, ticagrelor has been approved for ACS
patients. This is following results from the PLATO (Platelet Inhibition and Patient Outcomes) trial
demonstrating that treatment of ACS patients with ticagrelor significantly reduced the rate of death
from vascular causes, MI and stroke when compared with clopidogrel treatment [21]. However,
paradoxically, this beneficial effect of ticagrelor was not observed within patients from the USA and
Canada that had been enrolled in this trial [22]. It is proposed that this is due to differences in ASA
maintenance doses used in this part of the world. Despite no increase in the rate of major bleeding
overall in the ticagrelor arm of the PLATO trial, haemorrhagic side effects in non-CABG (coronary
artery bypass graft) patients were greater. A further drawback of ticagrelor is the need for increased
administration frequency due to its reversible nature, which in-turn is associated with increased cost.
Prasugrel is a newer thieno[3,2-c]pyridine with an improved efficacy but at the cost of an increased
bleed risk, with the JUMBO-TIMI (Joint Utilization of Medications to Block Platelets Optimally –
Thrombolysis in Myocardial Infarction) study finding significantly more bleeding events in patients
taking prasugrel compared to patients taking clopidogrel [8,23]. However, in a randomised trial, it
was found that prasugrel, was able to overcome the poor outcome for the CYP2C19*2 loss-of-
function allele seen in some clopidogrel non-responders [18]. This suggests that refinement of this
family of compound may be useful in these patients.
More recently, the synthesis of structurally related thieno[2,3-b]pyridines have been reported and
found to have potent phospholipase C (PLC) inhibitory activity[24]. As PLC activity has been linked

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to blood coagulation[25,26], a representative group of these new thienopyridines were assessed for
their anti-platelet activity (Fig 1).
Figure 1. Thieno[2,3-b]pyridines assessed for anti-platelet activity and clinically used clopidogrel, prasugrel (both
thieno[3,2-c]pyridines) and ticagrelor
2. Results
2.1 Chemistry
A representative group of six thieno[2,3-b]pyridines (1a-b, 2, 3a-c) were chosen, displaying
various sized and functionalized cycloalkyl rings as well as various substitutions on the phenyl ring
(Fig 1). The preparation of the three cyclooctyl derivatives (3a-c) is shown below (Scheme 1).
Cyclooctyl carbonitrile 4 was synthesized from cyclooctanone through a two-step procedure -
formation of the corresponding hydroxymethylene salt which was then immediately heated at reflux
with cyanothioacetamide and piperidinium acetate, followed by acidification with acetic acid to
provide the bicyclic thiocarbonitrile 4. Carbonitrile 4 was then condensed with substituted 2-halo-N-
phenylacetamides 5a-c to give the desired thieno[2,3-b]pyridines 3a-c.

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Scheme 1. Synthesis of cyclooctyl thieno[2,3-b]pyridine derivatives.
The preparation of thienopyridines 1a, 1b and 2 was achieved by repeating the same procedure
starting from the appropriate cycloketone (1,3-cyclohexadione for 1a and 1b; cycloheptanone for 2) to
give the corresponding bicyclic thiocarbonitrile which was then reacted with the required 2-halo-N-
phenylacetamides [27-29].
2.2 Biology
2.2.1 Thieno[2,3-b]pyridines inhibit ADP-stimulated platelet activation

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The expression of CD62P (P-selectin) along with PAC1 binding as markers of platelet α-granule
secretion and fibrinogen receptor activation, respectively, were analysed in PRP samples following
treatment with the thienopyridine compounds. The platelet-rich plasma (PRP) was stimulated with
ADP in order to induce platelet activation and hence expression of the two markers. Samples were
treated with clopidogrel (active metabolite), thienopyridine 1-3 or vehicle control for 30 min prior to
ADP stimulation. All thienopyridines 1-3 resulted in a significant decrease in CD62P expression
when compared to ADP-stimulated controls (Fig 1). When PAC1 binding was analysed, all six
thienopyridines 1-3 resulted in inhibition, while clopidogrel was unable to produce the same effect.
More interestingly, three of the six thienopyridines (1a, 1b and 3a) were able to inhibit the expression
of CD62P and PAC1 binding to a greater degree than clopidogrel (Figs 2 & 3). In the case of PAC1
binding, this was also true of 3c, 3b and 2. Thienopyridine 3a appeared to be the most superior
compound at causing this effect. Taken together, these data show that the tested thieno[2,3-
b]pyridines inhibited platelet activation in the presence of ADP and, under these conditions, were
more effective than clopidogrel.
Figure 2. Change in CD62P expression in ADP-stimulated PRP following thienopyridine treatment was assessed by flow
cytometry. Change in expression (i.e. inhibition) relative to control was determined. Data are presented as Mean ± SEM of
four independent blood donors, where n = 2 for each donor. Statistical analysis was performed using the Student’s t-test for
paired data to determine differences from control and from clopidogrel (active metabolite, CLP)-treated samples. Significant
differences from control (*** represents p<0.001) and clopidogrel (ψψψ represents p<0.001) are indicated.

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Figure 3. Change in PAC1 binding in ADP-stimulated PRP following thienopyridine treatment was assessed by flow
cytometry. Change in binding (i.e. inhibition) relative to control was determined. Data are presented as Mean ± SEM of four
independent blood donors, where n = 2 for each donor. Statistical analysis was performed using the Student’s t-test for
paired data to determine differences from control and from clopidogrel (active metabolite, CLP)-treated samples. Significant
differences from control (*** represents p<0.001) and clopidogrel (ψ represents p<0.05 and ψψ represents p<0.01 ) are
indicated.
2.2.2 Thieno[2,3-b]pyridines inhibit ADP-stimulated platelet aggregation in PRP
After assessing platelet activation, light transmission aggregometry (LTA) was used to assess ADP-
stimulated platelet aggregation in PRP following 30 min treatment with 10 µM of clopidogrel or the
novel thienopyridines. This allowed the assessment of platelet function. Treatment with all six
thienopyridines 1-3 resulted in a significant reduction in maximum aggregation when compared to
vehicle-control treated PRP (Fig 4). A paired t-test, to compare Maximum Aggregation (MaxA)
values after thienopyridine treatment with that following clopidogrel treatment, revealed that 3b, 1a,
and 3a caused a significantly greater inhibition of aggregation (p = 0.0009, p = 0.0124 and p = 0.0016
respectively) (Fig 4).
Figure 4. Change in maximum aggregation (MaxA) in ADP-stimulated PRP following thienopyridine treatment was
assessed by light-transmission aggregometry. Change in aggregation (i.e. inhibition) relative to control was determined. Data
are presented as Mean ± SEM of seven independent blood donors, where n = 3 for each donor. Statistical analysis was
performed using the Student’s t-test for paired data to compare each drug-treated sample with the control and with
clopidogrel (active metabolite, CLP)-treated samples. Significant differences from control (*** represents p<0.001) and
clopidogrel (ψ represents p<0.05, ψψ represents p<0.01 and ψψψ represents p<0.001) are indicated.
2.2.3 Thieno[2,3-b]pyridines inhibit collagen-stimulated platelet aggregation in whole blood
Although thienopyridines block ADP-induced aggregation, an effect on collagen-induced
activation should also be observed as the secondary wave of platelet aggregation caused by dense-
granule-derived ADP is inhibited. Indeed, the tested compounds inhibited collagen-induced
aggregation of PRP, but to a lesser degree than ADP-induced aggregation (Fig 5). Interestingly,
platelet aggregation in clopidogrel-treated samples was not significantly different from aggregation in
the untreated samples, whilst the novel thienopyridines appeared more effective at inhibiting collagen-
induced aggregation than clopidogrel, with the exceptions of 3c.

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Figure 5. Change in maximum aggregation (MaxA) in ADP-stimulated PRP following thienopyridine treatment was
assessed by light-transmission aggregometry. Change in aggregation (i.e. inhibition) relative to control was determined. Data
are presented as Mean ± SEM of seven independent blood donors, where n = 3 for each donor. Statistical analysis was
performed using the Student’s t-test for paired data to compare each drug-treated sample with the control and with
clopidogrel (active metabolite, CLP)-treated samples. Significant differences from control (*** represents p<0.001) and
clopidogrel (ψ represents p<0.05, ψψ represents p<0.01 and ψψψ represents p<0.001) are indicated.
2.2.4 Thieno[2,3-b]pyridines inhibit ADP-induced platelet-leukocyte aggregate formation
Following activation, platelets may adhere to local leukocytes (monocytes and neutrophils) via
platelet CD62P binding with P-selectin glycoprotein ligand-1 (PSGL1) on the leukocyte surface.
Platelet-leukocyte aggregates are considered a reliable marker of pro-thrombotic state [30]. Whole
blood samples were pre-treated with clopidogrel, thienopyridine 1-3 or vehicle control for 30 min
prior to ADP stimulation. Samples were double stained with the platelet marker CD42b and the
leukocyte marker CD45 and analysed using flow cytometry. Platelets were identified in the whole
blood sample (Fig 6A) by expression of CD42b and gated (Fig 6B). CD45-positive events within the
platelet gate were identified (Fig 6C). All novel thienopyridines 1-3, resulted in a significant decrease
in the percentage of platelet-leukocyte aggregates when compared to ADP-stimulated controls (Fig 7).
Statistical analyses to compare percentage of aggregates in the thienopyridine-treated samples with
the clopidogrel-treated samples revealed a significant difference following treatment with 1b.
Figure 6. (A) Whole blood was analysed by forward scatter (FSC) and side scatter (SSC). (B) The platelet population was
identified by expression of CD42b and gated (P1). (C) CD45 positive events within the platelet gate (P1) were identified
(P2).

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Figure 7. Change in the percentage of platelet-leukocyte aggregates within the ADP-stimulated whole blood samples
following thienopyridine treatment was assessed by flow cytometry. Change in CD42b+/CD45+ events (i.e. inhibition)
relative to control was determined. Data are presented as Mean ± SEM of four independent blood donors, where n = 2 for
each donor. Statistical analysis was performed using the Student’s t-test for paired data to determine differences from control
(* represents p<0.05, ** represents p<0.01, *** represents p<0.001) and from clopidogrel (active metabolite, CLP)-treated
samples (ψ represents p<0.05).
2.2.5 Thieno[2,3-b]pyridines show synergy with Aspirin
Management of ACS or stroke patients often involves the use of clopidogrel in combination with
ASA based on a duel-hit hypothesis, whereby platelet function is inhibited via simultaneous inhibition
of the P2Y12 receptor and the COX-1 enzyme. We investigated the synergistic action of the novel
thienopyridines 1-3 and ASA, using LTA to assess platelet function.
At the concentrations used in this study, all thienopyridines caused greater inhibition of platelet
aggregation when compared with the inhibition caused by ASA alone (Fig 8) (represented by *). This
was also true of clopidogrel. When thienopyridine-combination treatments were compared with the
respective thienopyridine only treatments, all showed synergy with ASA, with the exception of 3c and
3b (represented by ψ). However, these thienopyridines were shown to have a significant inhibitory
effect when used in isolation. An important facet of this work was to determine whether any of these
novel thienopyridines 1-3 were more potent when used in combination with ASA, than the
combination of clopidogrel and ASA. It was demonstrated that all compounds with the exception of
3c showed significantly greater activity than clopidogrel when used in combination with ASA
(represented by ɸ).

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Figure 8. Change in maximum aggregation (MaxA) in ADP-stimulated PRP following thienopyridine treatment in the
presence or absence of ASA was assessed by LTA. Change in aggregation (i.e. inhibition) relative to control was
determined. Data are presented as Mean ± SEM of seven independent blood donors, where n=3 for each donor. Statistical
analysis was performed using the Student’s t-test for paired data to compare each drug-treated sample with the control and
with clopidogrel (active metabolite, CLP)-treated. * denotes differences from ASA-only treated samples (* represents
p<0.05, ** represents p<0.01 and *** represents p<0.001) . ψ denotes differences from thienopyridine-only treated samples
(ψ represents p<0.05, ψψ represents p<0.01, ψψψ represents p<0.001). ɸ denotes differences between combination treated
samples and clopidogrel+ASA treated samples (ɸ represents p<0.05, ɸɸ represents p<0.01 and ɸɸɸ represents p<0.001).
3. Discussion
Although platelet inhibitors such as clopidogrel and, more recently prasugrel and ticagrelor are
currently used in clinical practice, the continued platelet hyperactivity in some patients taking these
drugs highlights a need for continued refinement of this class of drugs [31]. The present study
provides a significant contribution to the literature on P2Y12 inhibitor therapy by reporting on the use
of novel thieno[2,3-b]pyridine derivatives and their greater activity when compared to clopidogrel.
All six novel compounds significantly inhibited expression of both CD62P and PAC1 when
compared to ADP-stimulated controls. When looking at the inhibition of PAC1, all the compounds
had increased activity when compared with clopidogrel, while only 1a, 1b and 3a showed greater
activity in the inhibition of CD62P. Although both CD62P and PAC1 reflect platelet activation,
CD62P is expressed upon alpha-granule release [32], while PAC1 binds to activated GP IIb/IIIa
(fibrinogen receptor) [33]. CD62P release and fibrinogen receptor activation are not necessarily
simultaneous events during the process of platelet activation [34]. It is therefore important to analyse
more than one marker of platelet activation when examining the effects of anti-platelet drugs. In the
present study, clopidogrel had a very small effect on PAC1 binding, with all six novel thienopyridines
1-3 showing greater efficacy. This difference is likely to be highly important in vivo.
Aggregometry was used in this study to assess platelet function. All the thienopyridines were found
to inhibit ADP-induced platelet aggregation, with 3b, 1a and 3a showing greater efficacy than
clopidogrel. Furthermore, collagen-induced aggregation was also hindered to some degree following
derivative treatment, highlighting the importance of P2Y12 in secondary activation from dense-
granule-derived ADP. LTA is a gold standard measure of platelet function and is included in the

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majority of studies focusing on anti-platelet drugs [14,35-37]. However, strong platelet activation can
occur without significant end-point aggregation and, activated platelets even with poor aggregation
will still exert a systemic effect, increasing inflammation and stimulating further platelet activation.
Therefore, it is critical to monitor both activation status and aggregation to assess the global effects of
anti-platelet drugs.
Platelet-leukocyte aggregate formation has been shown to be increased in patients with ACS [38-
40] and has been suggested as a monitoring tool for risk of MI in these patients [41]. Platelet-
leukocyte aggregates have also been suggested to be a superior marker of platelet activation when
compared with CD62P expression [42]. Our data shows that all tested thienopyridine derivatives 1-3,
as well as clopidogrel, were able to inhibit ADP-induced platelet-leukocyte aggregate formation. This
is in agreement with similar studies that report decreases in platelet-leukocyte aggregate formation
following clopidogrel, prasugrel or cangrelor treatment, both in vitro and in vivo [3, 43-45].
Exploring synergy of the novel thienopyridine derivatives with ASA revealed that platelet
aggregation was inhibited to a greater extent when any of the novel compounds were used in
combination with ASA, when compared with ASA or derivative alone. The exception to this was 3b
which although appeared to show greater efficacy when used as part of the combination treatment,
statistical analysis proved this to be insignificant. More interestingly, when combined with ASA, all
thienopyridines, with the exception of 3c, showed a greater inhibitory effect than clopidogrel+ASA.
These data support results which demonstrate a superior platelet-inhibitory effect of clopidogrel+ASA
compared to either treatment alone [46,47]. Armstrong et al. studied the combination of prasugrel and
ASA on platelet activation in vitro and reported that ASA did not significantly increase the inhibitory
effect of prasugrel [35]. It was proposed that in the presence of strong P2Y12 receptor blockade, ASA
does not provide any benefit. However, our novel derivatives were extremely effective at P2Y12
receptor blockade when used in isolation, and yet were enhanced by ASA. There is a lack of literature
documenting the effects of prasugrel/cangrelor and ASA in vivo, but it appears that ASA-
thienopyridine synergy is P2Y12 inhibitor-specific.
It is clear that these thieno[2,3-b]pyridines have greater activity than the clinically used thieno[3,2-
c]pyridines. Clinically used prasugrel, clopidogrel and cangrelor all affect platelet function to varying
degrees in different patient groups [48-51]. Our derivatives follow this pattern and although this study
has shown that all of them have the ability to inhibit activation and aggregation to some degree, some
demonstrated greater activity than these clinical agents. Taken together, our data show that 1a and 3a
are very effective platelet inhibitors. They are both more effective than clopidogrel at inhibiting the
expression of CD62P and PAC1 in response to ADP stimulation. They are more effective at inhibiting
platelet aggregation both alone and in combination with ASA. This global reduction in both platelet
aggregation and activation highlights these molecules as worthy of further investigation to determine
their potential as P2Y12 inhibitors in the clinical setting.
When comparing the molecular structures of the tested thienopyridines the most consistently active
compound, 1a, has a strong similarity to clopidogrel with both containing a 2-chlorophenyl moiety
linked to a larger heterocyclic group. This similar motif of a 2-substituted phenyl group is also found
in the commonly used drug prasugrel. The active compound 3a interestingly does not contain a 2-
substituent on its phenyl ring. This suggests that alternative substitution patterns in these series of
compounds can still lead to viably active compounds.
Of course, when considering any drug aimed at inhibiting platelet function, it is important to
consider over-effectiveness, with risk of bleeding becoming an issue. Indeed, the superior activity of
prasugrel over clopidogrel has also been associated with increased risk of bleeding in some studies

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[8,23,52]. It will be important to determine whether our derivatives are also associated with increased
bleed risk in vivo.
4. Conclusion
In conclusion, the six thieno[2,3-b]pyridines derivatives tested all possessed anti-platelet activity,
showing inhibitory effects on ADP-induced CD62P expression and PAC1 binding, platelet-leukocyte
aggregate formation and aggregation. The study has involved in vitro testing on platelets obtained
from healthy individuals. Testing the in vitro inhibitory effects of these derivatives on platelets
obtained from patients who are candidates for clopidogrel/prasugrel treatment is necessary not only to
assess activity in these patients, but also to assess whether these derivatives would be useful as
alternative treatments for clopidogrel non-responders.
5. Materials and methods
5.1 Synthesis of compounds
5.1.1 General details
All reactions were carried out under a nitrogen atmosphere in dry, freshly distilled solvents unless
otherwise noted. All NMR spectra were recorded on a Bruker Avance DRX 400 MHz spectrometer at
ambient temperature. Chemical shifts are reported relative to the solvent peak of DMSO (δ 2.50 for
1
1H and δ 39.5 for 13C). H NMR data is reported as position (δ), relative integral, multiplicity (s,
singlet; d, doublet; t, triplet; m, multiplet; br, broad peak), coupling constant (J, Hz), and the
assignment of the atom. 13C NMR data are reported as position (δ) and assignment of the atom. All
NMR assignments were performed using HSQC and HMBC experiments. High-resolution mass
spectroscopy (HRMS) was carried out by electrospray ionisation (ESI) on a Brüker MicroTOF-Q
mass spectrometer. Unless noted, chemical reagents were used as purchased. Acetamides 5a-c were
obtained using literature methods [29,53-55].
5.1.2 2-Oxo-1,2,5,6,7,8,9,10-octahydrocycloocta[b]pyridine-3-carbonitrile 4. To sodium metal
(1.15 g, 0.05 mol) in ether (250 mL) under an atmosphere of nitrogen at room temperature was added
a solution of cyclooctanone (6.3g, 0.05 mol) in ether (50 mL), dropwise. Ethanol (0.25 mL) was then
added and the mixture stirred for 2 d. The mixture was then filtered under nitrogen and the solid
washed with ether and then collected to provide the desired product (6.43g, 73%) as a pale yellow
solid. Salt (1.76 g, 10.0 mmol) was dissolved in H2O (50 mL), followed by addition of
cyanoacetamide (0.84 g, 10.0 mmol), and freshly prepared piperidinium acetate solution (9.5 mL).
(The piperidinium acetate solution was prepared by mixing acetic acid (4.20 mL), water (10 mL), and
piperidine (7.20 mL)). The mixture was heated at reflux overnight before being acidified with acetic
acid (15 mL). The reaction mixture was allowed to cool to r.t. and stirred for a further 12 h before the
residue was filtered off, washed with ice water and collected to give the title compound 4 (1.86 g,
85%) as a white solid which was used in the next reaction without further purification. m.p. > 230 °C.
δH (400 MHz, (CD3)2SO) 1.35 (4H, br s, H-7 and H-8), 1.58 (2H, br s, H-6), 1.67 (2H, br s, H-9),
1
2.57-2.60 (2H, m, H-5), 2.82-2.85 (2H, m, H-10), 7.96 (1H, s, H-4), 13.97 (1H, br s, NH). The H
NMR data was consistent with that previously reported [53].
5.1.3 General procedure A for synthesis of thieno[2,3-b]pyridine-2-carboxamides 3a-c. A mixture of
2-bromo- or 2-chloroacetamides 5a-c (1 equiv), carbonitrile 4 (1 equiv) and anhydrous sodium
carbonate (1.06 equiv) in absolute ethanol was stirred at reflux for 24-48 h. The mixture was cooled to

ACCEPTED MANUSCRIPT
room temperature and the solvent removed in vacuo to give the crude product which was
recrystallized from methanol and washed with small amounts of water to give the thieno[3,2-
e]pyridine-2-carboxamides 3a-c.
5.1.3.1 3-Amino-N-phenyl-5,6,7,8,9,10-hexahydrocycloocta[b]thieno[3,2-e]pyridine-2-carboxamide
3a. The reaction was carried out according to general procedure A using 2-bromo-N-phenylacetamide
5a (0.49 g, 2.3 mmol), carbonitrile 4 (0.5 g, 2.3 mmol) and anhydrous sodium carbonate (0.26 g, 2.4
mmol) in absolute ethanol (10 mL) for 20 h to give the title product 3a (514 mg, 64 %) as a pale
1
1
green-yellow solid. m.p. >230 °C. H NMR (400 MHz; d6-DMSO) H NMR (400 MHz; d6-DMSO)
1.34 (4H, br s, H-7 and H-8), 1.71-1.74 (4H, br m, H-6 and H-9), 2.87 (2H, t, J = 6.0 Hz, H-5), 3.01
(2H, t, J = 6.0 Hz, H-10), 7.04-7.08 (1H, m, H-4'), 7.29-7.33 (4H, br s, NH2 and H-3′), 7.69 (2H, d, J =
8.0 Hz, H-2′), 8.22 (1H, s, H-4), 9.34 (1H, br s, NH); 13C NMR (100 MHz; d6-DMSO) 25.4 and 25.5
(C-7 and C-8), 30.5 (C-9), 31.2 (C-5), 32.1 (C-6), 34.3 (C-10), 96.0 (C-2), 121.1 (C-2'), 123.3 (C-4′),
124.8 (C-3a), 128.3 (C-3′), 130.7 (C-4), 132.2 (C-4a), 139.0 (C-1′), 146.9 (C-3), 156.4 (C-11a), 162.7
(C-10a), 164.0 (C=O); IR: vmax(ATR)/cm-1: 3387, 3293, 2920, 2853, 1648, 1595, 1529, 1435, 1315,
1240, 1057, 748; m/z (ESI+): 352 (MH+, 100 %); HRMS (ESI+) found (MH+): 352.1467 C20H22N3OS
requires 352.1478.
5.1.3.2 3-Amino-N-(4'-methoxyphenyl)-5,6,7,8,9,10-hexahydrocycloocta[b]thieno[3,2-e]pyridine-2-
carboxamide 3b. The reaction was carried out according to general procedure A using 2-chloro-N-(4-
methoxyphenyl)acetamide 5b (0.23 g, 1.2 mmol), carbonitrile 4 (0.25 g, 1.2 mmol) and anhydrous
sodium carbonate (0.15 g, 1.4 mmol) in absolute ethanol (5.0 mL) to give the title product 3b (90 mg,
21%) as a beige solid. m.p. 215-217 °C. 1H NMR (400 MHz; d6-DMSO) 1.34 (4H, br s, H-7 and H-8),
1.66-1.79 (4H, br m, H-6 and H-9), 2.87 (2H, t, J = 6.0 Hz, H-5), 3.01 (2H, t, J = 6.0 Hz, H-10), 3.74
(3H, s, CH3), 6.89 (2H, d, J = 8.9 Hz, H-3'), 7.22 (2H, br s, NH2), 7.56 (2H, d, J = 8.9 Hz, H-2), 8.19
(1H, s, H-4), 9.24 (1H, br s, NH); 13C NMR (100 MHz; d6-DMSO) 25.4 and 25.5 (C-7 and C-8), 30.5
(C-9), 31.2 (C-5), 32.2 (C-6), 34.3 (C-10), 55.2 (CH3), 96.2 (C-2), 113.6 (C-3'), 122.9 (C-2'), 124.9
(C-3a), 125.5 (C-4a), 130.7 (C-4), 132.2 (C-1'), 146.5 (C-3), 155.5 (C-4'), 156.3 (C-10a), 162.6 (C-
11a), 163.9 (C=O); IR: vmax(ATR)/cm-1: 3426, 3326, 2932, 1593, 1509, 1497, 1267, 1247, 1032, 826;
m/z (ESI+): 404 (MNa+, 100%), 382 (MH+, 35%); HRMS (ESI+) found (MNa+): 404.1412
C21H23N3NaO2S requires 404.1403.
5.1.3.3
3-Amino-N-(3'-bromo-2'-methylphenyl)-5,6,7,8,9,10-hexahydrocycloocta[b]thieno[3,2-
e]pyridine-2-carboxamide 3c. The reaction was carried out according to general procedure A using 2-
bromo-N-(3-bromo-2-methylphenyl)acetamide 5c (0.16 g, 0.53 mmol), carbonitrile 4 (0.115 g, 0.53
mmol) and anhydrous sodium carbonate (0.060 g, 0.56 mmol) in absolute ethanol (2 mL) for 48 h to
give the title product 3c (159 mg, 68 %) as an off white solid. m.p. 230-232 °C. 1H NMR (400 MHz;
d6-DMSO) 1.34 (4H, br s, H-7 and H-8), 1.71-1.73 (4H, br m, H-6 and H-9), 2.26 (3H, s, CH3), 2.87
(2H, t, J = 6.0 Hz, H-5), 3.02 (2H, t, J = 6.0 Hz, H-10), 7.15-7.17 (3H, m, H-5' and NH2), 7.31 (1H, d,
J = 8.0 Hz, H-6′), 7.49 (1H, d, J = 8.0 Hz, H-4′), 8.20 (1H, s, H-4), 9.33 (1H, br s, NH); 13C NMR
(100 MHz; d6-DMSO) 25.4 and 25.5 (C-7 and C-8), 30.5 (C-9), 31.2 (C-5), 32.2 (C-6), 34.3 (C-10),
96.1 (C-2), 124.5 (C-1′), 124.9 (C-3a), 126.8 (C-6′), 127.1 (C-5′), 129.7 (C-4′), 130.7 (C-4), 132.2 (C-
4a), 134.1 (C-2′), 138.2 (C-3′), 146.5 (C-3), 156.4 (C-11a), 162.6 (C-10a), 164.2 (C=O); IR:
vmax(ATR)/cm-1: 3415, 3321, 2929, 2854, 1468, 1574, 1517, 1428, 1304, 1258, 1058, 773; m/z (ESI+):
446 (81BrMH+, 100 %), 444 (79BrMH+, 95 %), 360 (20), 227 (50); HRMS (ESI+) found (81BrMH+):
446.0713 C21H2381BrN3OS requires 446.0721. Found (79BrMH+): 444.0730 C21H2379BrN3OS requires
444.0740.

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5.2 Biological methods
5.2.1 Ethics
The Manchester Metropolitan University ethics board granted approval for the study. Healthy
volunteers were recruited for blood collection and gave written informed consent before donating a
blood sample. The study was performed conforming to the Declaration of Helsinki.
Participants who had taken anti-platelet medication, anti-inflammatory medications, herbal
medicines that may interfere with platelet function (Ginko Biolba, St John’s Wort) or Selective
Serotonin Reuptake Inhibitors in the past fortnight were excluded from the study. The study involved
a total of eleven participants (n=3 male, n=8 female, age range 20-38). Samples from some
participants (chosen at random) were used for analysis of multiple markers of platelet activation and
function.
5.2.2 Sample collection and platelet-rich plasma purification
Blood was collected by venipuncture from the participants’ antecubital vein into sodium citrated
vacutainers. For experiments involving platelet-rich plasma (PRP), whole blood was centrifuged at
180 g for 15 min at 20 °C. Following centrifugation, the PRP was aspirated from the top of the
vacutainers and placed into a fresh tube. The PRP was then diluted with an equal volume of Tyrode’s
buffer (NaCl (134 mM), KCl (2.9 mM), Na2HPO4 (0.34 mM), NaHCO3 (12 mM), MgCl2 (1 mM),
HEPES (20 mM), Glucose (5 mM), adjusted to pH 7.4). For light-transmission aggregometry (LTA)
experiments, platelet-poor plasma was also required and was isolated by centrifuging a 500 µL aliquot
of diluted PRP at 5000 g for 5 min, and aspirating the supernatant from the platelet pellet.
5.2.3 Thienopyridine treatment
For experiments involving PRP, a 400 µL volume of diluted PRP was treated with 50 µL
clopidogrel active metabolite (CLP in figures), thienopyridine (final concentrations 10 µM), or
vehicle control (DMSO) for 30 min at 37 °C. In experiments involving whole blood, 500 µL whole
blood was treated with 500 µL clopidogrel active metabolite, thienopyridine (final concentrations 10
µM) or vehicle control for 30 min at 37 °C. In ASA synergy experiments, 400 µL samples of diluted
PRP were treated with 25 µL clopidogrel active metabolite or thienopyridine and 25 µL ASA (final
concentration 30 µM) and incubated for 30 min at 37 °C. Following treatment diluted PRP samples
were analysed using LTA. Thienopyridines were tested separately for their inherent cytotoxicity and it
was discovered that they only exhibited toxicity at the higher 100 µM concentration, after treatment
for 48-72 h.
5.2.4 Platelet activation analysis
A 90 µL volume of drug-treated PRP was activated using 10 µL ADP (final concentration 10 µM)
(Labmedics, UK). After 5 min incubation at room temperature (RT), 10 µL PE-conjugated anti-
human CD62P (BD Biosciences, UK) and 10 µL FITC-conjugated anti-human PAC1 (BD
Biosciences, UK) was added and the samples were incubated for 10 min at 20 °C in the dark at RT. A
100 µL volume of 4% paraformaldehyde (Sigma, UK) was added to fix the samples before addition of
300 µL DPBS (Lonza, UK). Samples were analysed using a FACSVerse Flow cytometer (Becton
Dickinson, UK) using FACSSuite software for analysis. Platelets were gated using FSC/SSC for
size/granularity, and FITC and PE Mean Fluorescence Intensity (MFI) were recorded. Unstimulated
controls (not treated with ADP) were also analysed to ensure a stimulation of CD62P and PAC1.
5.2.5 Light transmission aggregometry

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The drug-treated PRP samples (450 µL volume) were analysed on a Chronolog 700 aggregometer
using ADP (final concentration 10 µM) or collagen (final concentration 1 µg/ml) (Labmedics, UK) as
the agonist. Maximum Aggregation (MaxA) was recorded over 5 mins.
5.2.6 Platelet-leukocyte aggregate analysis
A 5 µL volume of the drug-treated whole blood was added to 55 µL Dulbecco’s Phosphate
Buffered Saline (DPBS) before activation with 10 µL ADP (final concentration 10 µM). After 5 min
incubation at room temperature (RT), 10 µL PE-conjugated anti-human CD45 (BD Biosciences, UK)
and 10 µL FITC-conjugated anti-human CD42b (BD Biosciences, UK) was added and the samples
were incubated for 20 min at 20 °C in the dark. Finally, samples were diluted by addition of 2 ml
DPBS before immediate analysis by flow cytometry. Platelets (CD42b positive events) were gated
and further analysed for CD45 expression. Percentage of CD42b positive events expressing CD45
was recorded. Unstimulated controls (not treated with ADP) were also analysed to ensure low
numbers of platelet-leukocyte aggregates under resting conditions.
5.2.7 Statistical analysis
Student t-tests for paired data were used to compare CD62P, PAC1 and MaxA values in drug-
treated samples with that in control samples. * represents p < 0.05, ** represents p < 0.01, ***
represents p < 0.001 when comparing with control.
ψ represents p < 0.05, ψψ represents p < 0.01, ψψψ represents p < 0.001 when comparing drug-
treated samples with clopidogrel treated samples by paired t-test.
In ASA synergy experiments, t-tests for paired data were used to compare thienopyridine-only
treated samples or ASA-treated samples with thienopyridine-ASA combination treated samples, and
also each thienopyridine-ASA combination treated sample with clopidogrel-ASA treated samples. ɸ
represents differences between combination treated samples and clopidogrel+ASA treated samples.
Acknowledgments
The authors would like to thank the volunteers who kindly donated blood for this study. This work
was supported by the Saudi Arabian Cultural Bureau on behalf of University of Hail, in the form of a
PhD studentship, the Manchester Metropolitan University Healthcare Science Research Centre, and
also by the Auckland Medical Research Foundation.
References
(1)
(2)
(3)
(4)
(5)
(6)
(7)
Clark, M. G.; Beavers, C.; Osborne, J. Heart Lung J. Crit. Care 2015, 44 (2), 141–149.
Morrow, D. Myocardial Infarction: A Companion to Braunwald’s Heart Disease, 1st ed.; Elsevier, 2016.
Gremmel, T.; Eslam, R. B.; Koppensteiner, R.; Lang, I. M.; Panzer, S. Cardiovasc. Ther. 2013, 31 (5), e40–e45.
Zetterberg, F.; Svensson, P. Bioorg. Med. Chem. Lett. 2016, 26 (12), 2739–2754.
E Liverani; LE Kilpatrick; AY Tsygankov; SP Kunapuli. Curr. Drug Targets 2014, 15 (7), 720–728.
Gachet, C. Purinergic Signal. 2012, 8 (3), 609–619.
Sarangi, S.; Pandey, A.; Papa, A.-L.; Sengupta, P.; Kopparam, J.; Dadwal, U.; Basu, S.; Sengupta, S. Med. Oncol.
Northwood Lond. Engl. 2013, 30 (2), 567.
(8)
(9)
Damman, P.; Woudstra, P.; Kuijt, W. J.; de Winter, R. J.; James, S. K. J. Thromb. Thrombolysis 2012, 33 (2), 143–
153.
Oh, P. C.; Ahn, T.; Kim, D. W.; Hong, B.-K.; Kim, D.-S.; Kwan, J.; Choi, C. U.; Yang, Y.-M.; Bae, J. H.; Jung, K.
T.; Choi, W. G.; Jeon, D. W.; Cho, D. K.; Pyun, W. B.; Cha, K. S.; Cha, T.-J.; Chun, K. J.; Kim, Y. D.; Kim, B. S.;
Kim, D.-I.; Kim, T. I. Int. J. Cardiol. 2016, 202, 331–335.

ACCEPTED MANUSCRIPT
(10)
(11)
Emmons, K. L.; Taylor, N. R. Pharmacotherapy 2007, 27 (4), 553–563.
Jain, N.; Li, X.; Adams-Huet, B.; Sarode, R.; Toto, R. D.; Banerjee, S.; Hedayati, S. S. Am. J. Cardiol. 2016, 117
(4), 656–663.
(12)
Li, Z.; Wang, Y.; Zhao, X.; Liu, L.; Wang, D.; Wang, C.; Meng, X.; Li, H.; Pan, Y.; Wang, X.; Wang, C.; Yang, X.;
Zhang, C.; Jing, J.; Xian, Y.; Johnston, S. C.; Wang, Y.; CHANCE Investigators. J. Am. Heart Assoc. 2016, 5 (3),
e003038.
(13)
(14)
Saab, Y. B.; Zeenny, R.; Ramadan, W. H. Ther. Clin. Risk Manag. 2015, 11, 1421–1427.
Bouman, H. J.; van Werkum, J. W.; Rudez, G.; Leebeek, F. W. G.; Kruit, A.; Hackeng, C. M.; Ten Berg, J. M.; de
Maat, M. P. M.; Ruven, H. J. T. Thromb. Haemost. 2010, 103 (2), 379–386.
Fontana, P.; Reny, J.-L. Rev. Med. Suisse 2008, 4 (143), 360–363.
Gremmel, T.; Panzer, S. Thromb. Haemost. 2011, 106 (2), 211–218.
Hulot, J.-S.; Bura, A.; Villard, E.; Azizi, M.; Remones, V.; Goyenvalle, C.; Aiach, M.; Lechat, P.; Gaussem, P.
Blood 2006, 108 (7), 2244–2247.
(15)
(16)
(17)
(18)
(19)
Kim, M. H.; Guo, L. Z.; Shin, E.-S.; Ann, S. H.; Choi, S. Y.; Lee, Y. S.; Kim, T. H. J. Am. Coll. Cardiol. 2016, 67
(13), 208.
Angiolillo, D. J.; Capranzano, P.; Desai, B.; Shoemaker, S. B.; Charlton, R.; Zenni, M. M.; Guzman, L. A.; Bass, T.
A. Thromb. Res. 2009, 124 (3), 318–322.
(20)
(21)
Shinoda, Y.; Kimura, M.; Usami, E.; Asano, H.; Yoshimura, T. Biomed. Rep. 2016, 5 (1), 141–145.
Wallentin, L.; Becker, R. C.; Budaj, A.; Cannon, C. P.; Emanuelsson, H.; Held, C.; Horrow, J.; Husted, S.; James,
S.; Katus, H.; Mahaffey, K. W.; Scirica, B. M.; Skene, A.; Steg, P. G.; Storey, R. F.; Harrington, R. A. N. Engl. J.
Med. 2009, 361 (11), 1045–1057.
(22)
Mahaffey, K. W.; Wojdyla, D. M.; Carroll, K.; Becker, R. C.; Storey, R. F.; Angiolillo, D. J.; Held, C.; Cannon, C.
P.; James, S.; Pieper, K. S.; Horrow, J.; Harrington, R. A.; Wallentin, L.; PLATO Investigators. Circulation 2011,
124 (5), 544–554.
(23)
(24)
Sarafoff, N.; Byrne, R. A.; Sibbing, D. Curr. Pharm. Des. 2012, 18 (33), 5224–5239.
Reynisson, J.; Jaiswal, J. K.; Barker, D.; D’mello, S. A. N.; Denny, W. A.; Baguley, B. C.; Leung, E. Y. Cancer
Cell Int, 2016, 16, 18.
(25)
Munnix, I. C. A.; Strehl, A.; Kuijpers, M. J. E.; Auger, J. M.; van der Meijden, P. E. J.; van Zandvoort, M. A. M.;
oude Egbrink, M. G. A.; Nieswandt, B.; Heemskerk, J. W. M. Arterioscler. Thromb. Vasc. Biol. 2005, 25 (12),
2673–2678.
(26)
(27)
(28)
Putney, J. W.; Tomita, T. Adv. Biol. Regul. 2012, 52 (1), 152–164.
Leung, E.; Hung, J. M.; Barker, D.; Reynisson, J. Med Chem Commun 2014, 5 (1), 99–106.
Arabshahi, H. J.; van Rensburg, M.; Pilkington, L. I.; Jeon, C. Y.; Song, M.; Gridel, L.-M.; Leung, E.; Barker, D.;
Vuica-Ross, M.; Volcho, K. P.; Zakharenko, A. L.; Lavrik, O. I.; Reynisson, J. MedChemComm 2015, 6, 1987–
1997.
(29)
Leung, E.; Pilkington, L. I.; van Rensburg, M.; Jeon, C. Y.; Song, M.; Arabshahi, H. J.; De Zoysa, G. H.; Sarojini,
V.; Denny, W. A.; Reynisson, J.; Barker, D. Bioorg. Med. Chem. 2016, 24 (5), 1142–1154.
Cerletti, C.; Tamburrelli, C.; Izzi, B.; Gianfagna, F.; de Gaetano, G. Thromb. Res. 2012, 129, 263-266.
Franchi, F.; Angiolillo, D. J. Nat. Rev. Cardiol. 2015, 12 (1), 30–47.
(30)
(31)
(32)
(33)
(34)
(35)
Blair, P.; Flaumenhaft, R. Blood Rev. 2009, 23 (4), 177–189.
Shattil, S. J.; Hoxie, J. A.; Cunningham, M.; Brass, L. F. J. Biol. Chem. 1985, 260 (20), 11107–11114.
Naimushin, Y. A.; Mazurov, A. V. Biochem. Biokhimiia 2003, 68 (2), 209–216.
Armstrong, P. C. J.; Leadbeater, P. D.; Chan, M. V.; Kirkby, N. S.; Jakubowski, J. A.; Mitchell, J. A.; Warner, T. D.
J. Thromb. Haemost. JTH 2011, 9 (3), 552–561.
(36)
(37)
(38)
Valenti, R.; Marcucci, R.; Comito, V.; Marrani, M.; Cantini, G.; Migliorini, A.; Parodi, G.; Gensini, G. F.; Abbate,
R.; Antoniucci, D. JACC Cardiovasc. Interv. 2015, 8 (12), 1563–1570.
Vistoli, G.; Brizzolari, A.; Faioni, E.; Razzari, C.; Santaniello, E. Bioorg. Med. Chem. Lett. 2014, 24 (24), 5652–
5655.
Furman, M. I.; Benoit, S. E.; Barnard, M. R.; Valeri, C. R.; Borbone, M. L.; Becker, R. C.; Hechtman, H. B.;
Michelson, A. D. J. Am. Coll. Cardiol. 1998, 31 (2), 352–358.
(39)
(40)
(41)
Ott, I.; Neumann, F.-J.; Gawaz, M.; Schmitt, M.; Scho¨mig, A. Circulation 1996, 94 (6), 1239–1246.
Sarma, J.; Laan, C. A.; Alam, S.; Jha, A.; Fox, K. A. A.; Dransfield, I. Circulation 2002, 105 (18), 2166–2171.
Furman, M. I.; Barnard, M. R.; Krueger, L. A.; Fox, M. L.; Shilale, E. A.; Lessard, D. M.; Marchese, P.; Frelinger,
A. L.; Goldberg, R. J.; Michelson, A. D. J. Am. Coll. Cardiol. 2001, 38 (4), 1002–1006.
Michelson, A. D.; Barnard, M. R.; Krueger, L. A.; Valeri, C. R.; Furman, M. I. Circulation 2001, 104 (13), 1533–
1537.
Harding, S. A.; Sarma, J.; Din, J. N.; Maciocia, P. M.; Newby, D. E.; Fox, K. a. A. Heart Br. Card. Soc. 2006, 92
(9), 1335–1337.
Klinkhardt, U.; Bauersachs, R.; Adams, J.; Graff, J.; Lindhoff-Last, E.; Harder, S. Clin. Pharmacol. Ther. 2003, 73
(3), 232–241.
(42)
(43)
(44)
(45)
(46)
Klinkhardt, U.; Harder, S. Semin. Thromb. Hemost. 2005, 31 (4), 400–403.
Moshfegh, K.; Redondo, M.; Julmy, F.; Wuillemin, W. A.; Gebauer, M. U.; Haeberli, A.; Meyer, B. J. J. Am. Coll.
Cardiol. 2000, 36 (3), 699–705.
(47)
(48)
Serebruany, V. L.; Malinin, A. I.; Jerome, S. D.; Lowry, D. R.; Morgan, A. W.; Sane, D. C.; Tanguay, J.-F.;
Steinhubl, S. R.; O’connor, C. M. Am. Heart J. 2003, 146 (4), 713–720.
Harrington, R. A.; Stone, G. W.; McNulty, S.; White, H. D.; Lincoff, A. M.; Gibson, C. M.; Pollack, C. V. J.;
Montalescot, G.; Mahaffey, K. W.; Kleiman, N. S.; Goodman, S. G.; Amine, M.; Angiolillo, D. J.; Becker, R. C.;

ACCEPTED MANUSCRIPT
Chew, D. P.; French, W. J.; Leisch, F.; Parikh, K. H.; Skerjanec, S.; Bhatt, D. L. N. Engl. J. Med. 2009, 361 (24),
2318–2329.
(49)
(50)
Jia, M.; Li, Z.; Chu, H.; Li, L.; Chen, K. Med. Sci. Monit. Int. Med. J. Exp. Clin. Res. 2015, 21, 1131–1137.
Roe, M. T.; Armstrong, P. W.; Fox, K. A. A.; White, H. D.; Prabhakaran, D.; Goodman, S. G.; Cornel, J. H.; Bhatt,
D. L.; Clemmensen, P.; Martinez, F.; Ardissino, D.; Nicolau, J. C.; Boden, W. E.; Gurbel, P. A.; Ruzyllo, W.;
Dalby, A. J.; McGuire, D. K.; Leiva-Pons, J. L.; Parkhomenko, A.; Gottlieb, S.; Topacio, G. O.; Hamm, C.;
Pavlides, G.; Goudev, A. R.; Oto, A.; Tseng, C.-D.; Merkely, B.; Gasparovic, V.; Corbalan, R.; Cinteză, M.;
McLendon, R. C.; Winters, K. J.; Brown, E. B.; Lokhnygina, Y.; Aylward, P. E.; Huber, K.; Hochman, J. S.;
Ohman, E. M. N. Engl. J. Med. 2012, 367 (14), 1297–1309.
(51)
(52)
Wiviott, S. D.; Antman, E. M.; Gibson, C. M.; Montalescot, G.; Riesmeyer, J.; Weerakkody, G.; Winters, K. J.;
Warmke, J. W.; McCabe, C. H.; Braunwald, E.; TRITON-TIMI 38 Investigators. Am. Heart J. 2006, 152 (4), 627–
635.
Wiviott, S. D.; Braunwald, E.; McCabe, C. H.; Montalescot, G.; Ruzyllo, W.; Gottlieb, S.; Neumann, F.-J.;
Ardissino, D.; De Servi, S.; Murphy, S. A.; Riesmeyer, J.; Weerakkody, G.; Gibson, C. M.; Antman, E. M. N. Engl.
J. Med. 2007, 357 (20), 2001–2015.
(53)
(54)
Pilkington, L. I.; Haverkate, N. A.; van Rensburg, M.; Reynisson, J.; Leung, E.; Barker, D. Synlett 2016.
van Rensburg, M.; Leung, E.; Haverkate, N. A.; Eurtivong, C.; Pilkington, L. I.; Reynisson, J.; Barker, D. Bioorg.
Med. Chem. Lett. 2017, 27 (2), 135–138.
(55)
Hung, J. M.; Arabshahi, H. J.; Leung, E.; Reynisson, J.; Barker, D. Eur. J. Med. Chem. 2014, 86, 420–437.

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Novel thienopyridine derivatives are potent anti-platelet drugs, inhibiting platelet
activation, aggregation and showing synergy with aspirin
Naif K. Binsaleha, Catherine A. Wigleya, Kathryn A. Whiteheada, Michelle van Rensburgb, Johannes
Reynissonb, Lisa I. Pilkingtonb, David Barkerb, Sarah Jonesa, and Nina C. Dempsey-Hibberta,
a School of Healthcare Science, Manchester Metropolitan University, Manchester, UK, M1 5GD
b School of Chemical Sciences, The University of Auckland, New Zealand.
Highlights
•
•
•
•
Thieno[2,3-b]pyridines inhibited platelet activation and aggregation
Thieno[2,3-b]pyridines showed synergy with aspirin
Tested compounds had greater activity to clinically used thieno[3,2-c]pyridines
Thieno[2,3-b]pyridines have been found to be a novel class of P2Y12 inhibitors