Optimization of P2Y12 Antagonist Ethyl 6-(4-((Benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283) Led to the Discovery of an Oral Antiplatelet Agent with Improved Druglike Properties

Article abstract of DOI:10.1021/acs.jmedchem.8b01971

P2Y₁₂ antagonists are widely used as antiplatelet agents for the prevention and treatment of arterial thrombosis. Based on the scaffold of a known P2Y₁₂ antagonist AZD1283, a series of novel bicyclic pyridine derivatives were designed and synthesized. The cyclization of the ester substituent on the pyridine ring to the ortho-methyl group led to lactone analogues of AZD1283 that showed significantly enhanced metabolic stability in subsequent structure-pharmacokinetic relationship studies. The metabolic stability was further enhanced by adding a 4-methyl substituent to the piperidinyl moiety. Compound 58l displayed potent inhibition of platelet aggregation in vitro as well as antithrombotic efficacy in a rat ferric chloride model. Moreover, 58l showed a safety profile that was superior to what was observed for clopidogrel in a rat tail-bleeding model. These results support the further evaluation of compound 58l as a promising drug candidate.

Full text of DOI:10.1021/acs.jmedchem.8b01971

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Article
Optimization of P2Y12 Antagonist Ethyl 6-(4-
(Benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-
(
methylnicotinate (AZD1283) Led to the Discovery of an
Oral Antiplatelet Agent with Improved Drug-Like Properties
Deyu Kong, Tao Xue, bin guo, Jianjun Cheng, Shunying Liu, Jianhai Wei,
Zhengyu Lu, Haoran Liu, Guoqing Gong, Tian Lan, Wenhao Hu, and Yushe Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01971 • Publication Date (Web): 07 Mar 2019
Downloaded from http://pubs.acs.org on March 8, 2019
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Optimization
of
P2Y12
Antagonist
Ethyl
6-(4-
((Benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-
methylnicotinate (AZD1283) Led to the Discovery of an Oral
Antiplatelet Agent with Improved Drug-Like Properties
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Deyu Kong,†,‡ Tao Xue, Bin Guo, Jianjun Cheng, Shunyin Liu, Jianhai Wei,
‡
‡
▽
†
†
‡
‡
||
||
,†,§
Zhengyu Lu, Haoran Liu, Guoqing Gong, Tian Lan, Wenhao Hu,* and Yushe
Yang*,‡
†Shanghai Engineering Research Center of Molecular Therapeutics and New Drug
Development, School of Chemistry and Molecular Engineering, East China Normal
University, Shanghai, 200062, PR China.
‡State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, PR China.
▽
iHuman Institute, ShanghaiTech University, Shanghai 201210, China
§School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006,
China.
llDepartment of Pharmacology, School of Pharmacy, China Pharmaceutical University,
Nanjing, Jiangsu Province 210009, China.
ABSTRACT: P2Y12 antagonists are widely used as antiplatelet agents for the
prevention and treatment of arterial thrombosis. Based on the scaffold of a known P2Y12
antagonist AZD1283, a series of novel bicyclic pyridine derivatives were designed and
synthesized. The cyclization of the ester substituent on the pyridine ring to the ortho
methyl group led to lactone analogs of AZD1283 that showed significantly enhanced
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metabolic stability in subsequent structure-pharmacokinetic relationship studies. The
metabolic stability was further enhanced by adding a 4-methyl substituent to the
piperidinyl moiety. Compound 58l displayed potent inhibition of platelet aggregation
in vitro as well as antithrombotic efficacy in a rat ferric chloride model. Moreover, 58l
showed a safety profile that was superior to what was observed for clopidogrel in a rat
tail-bleeding model. These results support the further evaluation of compound 58l as a
promising drug candidate.
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INTRODUCTION
Thromboembolic disorders, such as acute myocardial infarction and stroke, are still
1
the most common causes of mortality and morbidity in the developed world. Platelet
activation plays a critical role in thrombotic complications.2, 3 The activation process
involves the production of several platelet activation agonists, including thrombin,
2
thromboxane A
2
and adenosine diphosphate (ADP). ADP is a key factor in platelet
4
activation and aggregation, and its mechanism involves the binding and activation of
two G-protein coupled receptors (GPCRs), P2Y
1
and P2Y12.5 P2Y
is ubiquitously
1
expressed and plays a role in initial activation, while the P2Y12 receptor is primarily
expressed in platelets and plays a central role in the amplification of platelet activation,
aggregation and stable thrombus formation. The P2Y12 receptor has been validated as
an important drug target for the development of antithrombotic drugs.6
The therapeutic success of P2Y12 receptor irreversible antagonist clopidogrel (1,
Figure 1) in preventing atherothrombotic events in patients that have experienced acute
7
coronary syndrome (ACS), confirmed that P2Y12 antagonism is a powerful therapeutic
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strategy in the management and prevention of arterial thrombosis.8, 9 However,
clopidogrel is a thienopyridine prodrug that requires metabolic conversion by the
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cytochrome P450 system to generate the active metabolite that irreversibly binds to
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the P2Y12 receptor, resulting in a slow onset and offset of its pharmacological action.
Therefore, clopidogrel displayed obvious drawbacks, including delayed onset of
1
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action, significant response variability, and insufficient antiplatelet activity in some
patients.14 These drawbacks led to the development of more potent and reliable
reversible P2Y12 receptor antagonists, such as ticagrelor (2)15 and cangrelor16 (3).
However, both of these drugs have their own contraindications and numerous issues
that must be considered in clinical practice. For example, several adverse clinical effects,
such as dyspnea, ventricular pauses and hyperuricemia were observed with ticagrelor,
which could likely be attributed to the inhibition of equilibrative nucleoside transporter
1
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15
1
by ATP analogs. And cangrelor must be administered by intravenous injection.
Figure 1. Structures of clopidogrel (1), ticagrelor (2), cangrelor (3) and AZD1283 (4).
Existing evidence suggests that reversible binding could not only lead to a rapid
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recovery of platelet function but also increase the separation between the antithrombotic
effect and bleeding risk.18, 19 This hypothesis has inspired extensive efforts toward the
discovery of novel, reversible and orally available P2Y12 receptor antagonists with
improved safety profiles.20, 21 Among these antagonists, AZD1283 (4, Figure 1), an
oral, reversible P2Y12 receptor antagonist reported by AstraZeneca, showed potent
antithrombotic efficacy and reduced bleeding effects in animal models and was
advanced into human clinical trials. However, the development of this compound was
terminated prior to phase II studies due to poor absorption and low metabolic stability
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of the ester. Moreover, AZD1283 inhibited three CYP450-metabolizing enzymes,
CYP2C9, CYP3A4 and CYP2C19.22
The X-ray crystal structure of AZD1283 bound to the human P2Y12 receptor showed
that a hydrogen bond between the ester carbonyl and Asn159 is crucial to its high
affinity.24, 25 Knowing that the ester group is susceptible to metabolic degradation, we
envisioned that cyclization of this ester group to the ortho methyl would generate novel
analogs of AZD1283 that retain its P2Y12 activity while having much improved
metabolic stability (Figure 2). Herein, we describe the structure-activity and structure-
pharmacokinetic relationships of these bicyclic pyridine compounds and the
identification of compound 58l as a potential drug candidate for the development of
novel antithrombotic agents.
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Figure 2. Design of novel, fused bicyclic pyridine antiplatelet agents.
RESULTS AND DISCUSSION
CHEMISTRY
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The syntheses of five-membered lactone compounds 21a–g and 22a–e are shown in
Scheme 1. For the synthesis of the right-hand amine moieties, carboxylic acids 5a–d
were used as starting materials, and they were activated with TBTU as a coupling
reagent and then treated with sulfonamides 6 and 9 to produce acyl sulfonamides 7a–
d22 and 10a–c; Subsequent Boc deprotection provided amines 8a–d and 11a–c. To
construct the left-hand fused pyridines, substituted acetoacetates 12a and 12b were used
as starting materials, and they were treated with benzyl alcohol and then debenzylated
to introduce the hydroxyl group (14a–b). The hydroxyl groups were subsequently
protected as silyl ethers to afford 15a–b, which were then subjected to an Aldol
condensation to give intermediates 16a-b. Cyclization of 16a-b with cyanoacetamide
gave pyridones 17a–b. Cleavage of the silyl ether protecting groups and hydrolysis of
the esters gave intermediates 19a–b, which were refluxed with POCl to give lactone-
3
fused pyridines 20a–b. Compounds 20a–b were coupled with amines 8a–d and 11a–c
to afford target compounds 21a–g and 22a–e.
Scheme 1. Synthesis of Compounds 21a–g and 22a–ea
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aReagents and conditions: (a) TBTU, triethylamine (TEA), THF, LiCl, rt, 18 h, 26–90%;
(
b) HCOOH, rt, 20 h, 80–96%; (c) BnOH, NaH, THF, 0 °C to rt, 91–92%; (d) H , Pd/C,
2
EtOH, rt, 86–98%; (e) TBSCl, imidazole, DMAP, THF, rt, 49–70%; (f) DMF/DMA,
80 °C , 72–83%; (g) 2-Cyanoacetamide, NaOEt, EtOH, rt, 81–81%; (h) TBAF, THF, rt,
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85–91%; (i) NaOH, MeOH, rt, 86–96%; (j) POCl , 100 °C , 56–58%;(k) TEA, EtOH,
3
reflux, overnight, 69–85% for 21a–g, 57–82% for 22a–e
Six-membered lactone compounds 33a–c and 34a–b were prepared using a similar
method, as shown in Scheme 2. Ethyl 5-hydroxy-3-oxopentanoate (26) was prepared
and used in place of 14a and 14b in Scheme 1 for the construction of pyridine-fused
six-membered lactone 32. Intermediate 32 were treated with amines 8a, 8c, 8d, 13a and
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3c to provide compounds 33a–c and 34a–b in good yields.
Scheme 2. Synthesis of Compounds 33a–c and 34a–ba
aReagents and conditions: (a) NaH, n-BuLi, THF, rt, 43%; (b) H
, Pd/C, EtOH, rt, 98%;
2
(
c) TBSCl, imidazole, DMAP, THF, rt, 81%; (d) DMF/DMA, 80 °C , 96%; (e) 2-
cyanoacetamide, NaOEt, EtOH, rt, 81%; (f) TBAF, THF, rt, 98%; (g) NaOH, MeOH,
rt, 94%; (h) POCl
3
, 100 °C, 35%; (i) TEA, EtOH, reflux, overnight, 73–78% for 33a–
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c, 79–82% for 34a–b
The synthesis of cyclic ketone compounds 40a–c is depicted in Scheme 3.
Commercially available dimedone 35 was first converted to enaminone 36 and then to
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pyridone 37 via a two-step Hantzsch-type reaction. The α position of ketone 37 was
substituted with a F to give 38a and a Cl to give 38b. These pyridones (37, 38a and
38b) were converted to the corresponding 2-chloropyridines (39a–c) in good yields
under Vilsmeier conditions using SOCl
2
/DMF. The coupling of 39a–c with amine 8a
provided compounds 40a–c.
Scheme 3. Synthesis of Compounds 40a–ca
aReagents and reaction conditions: (a) DMF/DMA, reflux, 99%; (b) malononitrile,
NH
MeCN, PTSA, Ar, 80 °C , 65% for 38c; (d) SOCl
EtOH, reflux, 18 h, 61–78%
4
OAc, CH
3
CN, reflux, 53%; (c) Selectfluor, H
2
SO , MeOH, 70% for 38b, NCS,
4
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, DMF, DCM, 51–62%; (e) DIPEA,
The preparations of compounds 45a–b are outlined in Scheme 4. Ethyl 4-chloro-3-
oxobutanoate (41) was used as the starting material, and it was converted to pyridine
4
2 via a Hantzsch-type reaction. Subsequent chlorination of 42 gave 43, which was
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treated with amine 8a to afford intermediate 44. 44 was reacted with ammonia and
methylamine to afford compounds 45a and 45b, respectively.
Scheme 4. Synthesis of Compounds 45a–ba
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aReagents and reaction conditions: (a) Ac
cyanoacetamide, NaOEt, EtOH, rt, 12 h, 62% for two steps; (c) Oxalyl chloride, DMF,
0 °C, 54%; (d) TEA, EtOH, reflux, 84%; (e) amine, MeOH, rt, 76% for 45a,
O, triethoxymethane, 120 °C, 62%; (b) 2-
2
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methylamine, MeOH, rt, 69% for 45b
Compounds 51a–c were synthesized according to Scheme 5. Bis(2,4-
dimethoxybenzyl)amine 46 was treated with benzylsulfonyl chloride to afford
sulfonamide 47. The α position of the sulfone was deprotonated with NaHMDS and
27
then treated with N-fluorobenzenesulfonimide, methyl iodide or 1,3,2-dioxathiolan-
2
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2,2-oxide to give derivatives 48a–c, respectively. Subsequent deprotection with TFA
gave intermediates 49a–c. For the left-hand fragment, chloropyridine 20a was coupled
to piperidine carboxylic acid to give intermediate 50, which was then coupled to
sulfonamides 49a–c to produce compounds 51a–c in good yields.
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Scheme 5. Synthesis of Compounds 51a–ca
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aReagents and conditions: (a) Benzylsulfonyl chloride, DMAP, THF, rt, 60%; (b)
NaHMDS, THF, –78 °C , Ar, N-fluorobenzenesulfonimide for 48a, CH I for 48b, 1,3,2-
3
dioxathiolan-2,2-oxide (DTD) for 48c, 66–78%; (c) TFA, DCM, rt, 70–82%; (d)
DIPEA, EtOH, reflux, 70%; (e) EDCI, HOBt, DIPEA, 58–73%
Compounds 58a–m were synthesized using an alternative route, as illustrated in
Scheme 6. Derivatives of 55a–i were prepared using various benzyl chlorides (52a–i)
as starting materials. Intermediates 53a–i were readily prepared by refluxing the benzyl
chlorides with thiourea in ethanol, and these compounds were then subjected to
NCS/HCl oxidative chlorination conditions to generate benzylsulfonyl chlorides 54a–
2
8
i. The following four steps in the synthesis of target compounds 58a–m were similar
to those depicted in Scheme 1.
Scheme 6. Synthesis of Compounds 58a–ma
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4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aReagents and conditions: (a) EtOH, reflux, 2 h; (b) NCS, HCl, MeCN, rt; (c) NH
OH,
4
THF, rt, 25–68%; (d) TBTU, TEA, LiCl, rt, 48–84%; (e) HCOOH, rt, 74–91%; (f)
DIPEA, EtOH, reflux, 12 h, 41–74%
Compounds 61 and 62a–d were synthesized according to Scheme 7. Compound 20a
was treated with 4-fluoropiperidine-4-carboxylic acid to give acid 59, which was
converted to acid chloride 60 and then coupled to various sulfonamides to afford
compounds 61 and 62a–d.
Scheme 7. Synthesis of Compounds 61 and 62a–da
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aReagents and conditions: (a) DIPEA, EtOH, reflux, 80%; (b) SOCl2, DMF, DCM; (c)
TEA, DCM, 56–79%
In Vitro Platelet Aggregation. All compounds were evaluated for their ability to
inhibit ADP-induced human platelet-rich plasma (PRP) aggregation.29-31 The platelet-
rich plasma was prepared following a standard protocol, and the aggregation was
measured by a double-channel aggregometer for platelet aggregation (see the
Experimental Section for details).
As an initial exploration, five- or six-membered lactones were designed to replace
the ethyl ester of compound AZD1283 because the lactone group could maintain the
hydrogen bond with the receptor but also potentially improve the metabolic stability of
the compound. As shown in Table 1, this hypothesis was confirmed with five-
membered lactone compound 21a, which exhibited slightly enhanced activity
compared to AZD1283, but six-membered lactone analog 33a showed no activity in the
PRP aggregation assay. Additionally, when a methyl group was introduced to the α
position of the five-membered lactone, a loss of activity was observed (21e versus 21a).
We then converted the lactone to a cyclic ketone or a lactam, both of which are lactone
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bioisosteres, as in compounds 40a–c and 45a–b. Compared to the six-membered
lactone, the simple six-membered cyclic ketone showed improved inhibitory activity
(
40a versus 33a). Again, the introduction of α substituents decreased the activity to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
varying degrees (40b–c versus 40a). Neither of the two lactam analogs 45a–b inhibited
platelet aggregation. These results indicate a very strict SAR at this moiety.
Table 1. In Vitro Antiplatelet Aggregation Potency of Various A-Ring Derivatives
a
a
compd
A-ring
IC50 (μM)
compd
A-ring
IC50 (μM)
AZD1283
3.60
2.35
40b
254.81
>300
>300
>300
2
1a
40c
45a
45b
21e
33a
>300
>300
116.51
4
0a
aADP-induced platelet aggregation ([ADP] =10 μM, n = 2), human platelet-rich plasma
(
hPRP).
Based on the encouraging results of compound 21a, a comprehensive SAR
investigation was implemented around the B-ring and the aryl tail (R) (Table 2).
Changing the piperidine to either an azetidine or a pyrrolidine led to a loss of potency
(
21c–d versus 21a), so the piperidine ring was kept and substituents were introduced to
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1
1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
its 4 position, as this may block the metabolically labile α position of the carbonyl and
therefore improve compound stability. As demonstrated by compound 21b, the
introduction of a methyl group led to a 2-fold decrease in potency compared to
unsubstituted compound 21a, while introducing a fluorine significantly increased the
inhibitory activity (IC50 = 1.21 μM for 62a versus 2.35 μM for 21a). Another potential
site for metabolic degradation is the benzyl position of the sulfonamide, so this position
was structurally modified, and fluoride, cyclopropyl32 or methyl groups were
introduced at this position. However, we noticed that di-fluoro compound 51a was
chemically unstable upon storage under ambient conditions. Methyl- and cyclopropyl-
substituted compounds 51b, 51c and 62b exhibited 2- to 4-fold decreased potencies
compared to compound 24a. These findings showed that substituents at the benzyl
position did not improve the inhibitory activity. Replacement of the benzyl substituent
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
31
with a 5-Cl-thienyl moiety, which is a structural motif present in elinogrel, slightly
decreased the in vitro activity (22a vs 24a). Interestingly, keeping this 5-Cl-thienyl
moiety and adding a methyl substituent at the 4 position of the piperidinyl group
increased the potency by a factor of 3 (22b vs 24a), while fluoro-substituted compound
6
1 showed reduced potency. The structure-activity relationship was not consistent with
that observed in the above benzyl series. Based on these results, the α-substituted five-
membered lactone or six-membered lactone scaffolds were reevaluated. Unfortunately,
neither changes in the B-ring nor the aryl tail group improved the inhibitory activities
(21f–g, 22d–e, 33b–c and 34–b) (IC50 >300 μM).
Table 2. In Vitro Antiplatelet Aggregation Potency of Various A-Ring, B-Ring and
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Aryl Group (R) Analogs.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A-ring
B-ringa
3-aze
R
IC50 (μM)
b
compd
2
1c
>300
>300
6.68
2
1d
3-pyr
4-methyl-pip
4-fluoro-pip
4-pip
21b
62a
1.21
n.d.c
4.16
5
1a
1b
5
4-pip
51c
4-pip
4.10
62b
22a
22b
4-fluoro-pip
4-pip
9.95
4.47
4-methyl-pip
4-fluoro-pip
3-aze
1.64
6
1
37.06
193.12
2
2c
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
1f
3-aze
3-pyr
4-pip
3-aze
3-aze
3-pyr
4-pip
3-aze
>300
>300
>300
>300
>300
>300
>300
>300
2
1g
2d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2e
3
3b
3
3c
3
4a
34b
a3-aze=3-azetidinyl, 3-pyr=3-pyrrolyl, 4-pip=4-piperidinyl, 4-fluoro-pip=4- fluoro-
b
piperidinyl, 4-methyl-pip=4-methyl-piperidinyl. ADP-induced platelet aggregation
c
(
[ADP] =10 μM, n = 2), human platelet-rich plasma (hPRP). n.d. = not determined.
Based on the reported cocrystal structure of AZD1283 bound to P2Y12, the phenyl
tail of AZD1283 occupies a hydrophobic pocket formed by the side chains of F252,
R256, Y259, L276 and K2807.24, 25 The reported SAR of the aromatic substituents at
this position also showed that lipophilic substituents are favored on this phenyl ring.33
Therefore, various small lipophilic substituents were introduced onto the phenyl group
to improve the binding affinity of the compound. As shown in Table 3, the para-fluoro
(
58a) and para-chloro (58h) analogs displayed inhibitory activities comparable to that
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of compound 24a. However, shifting the fluoro substituent from the para- (58a) to the
ortho- (58b) or meta- (58c) positions significantly reduced the activity. In addition, di-
fluoro (58d) or di-chloro substitution (58i) also decreased the potency to varying
degrees. Small alkyl groups, such as methyl (58g), provided comparable activity, but a
trifluoromethyl (58e) or cyano (58f) substituent led to a 2- to 4-fold decrease in activity.
Table 3. In Vitro Antiplatelet Aggregation Potency of Various Phenyl Derivatives
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
a
compd
8a
R
IC50 (μM)
compd
R
IC50 (μM)
5
3.50
58f
8.90
4.28
5
8b
23.72
58g
58h
58i
5
8c
70.49
4.42
5
8d
5.20
30.51
58e
15.90
aADP-induced platelet aggregation ([ADP] =10 μM, n = 2), human platelet-rich plasma
(hPRP).
From the above SAR studies, the A-ring, B-ring and aryl tail groups were each
optimized individually. Finally, we combined these fragments for the last round of
modifications. As shown in Table 4, compounds 58l and 58m exhibited excellent
activities in the hPRP assay, and they were both over 10-fold more potent than their
corresponding 4-fluoro-substituted piperidinyl counterparts (62c and 62d). However,
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the 4-F/CH
3
-substitutedpiperidinyl derivatives (58j–k and 62c–d) did not display more
potent antiplatelet activities than their unsubstituted piperidine counterparts.
Table 4. In Vitro Antiplatelet Aggregation Potency of B-Ring and Aryl Group (R)
Derivatives.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
compd
R1
R2
IC50 (μM)
58j
8.76
58k
35.44
2.94
5
8l
58m
3.01
6
2c
32.76
37.22
62d
aADP-induced platelet aggregation ([ADP] = 10 μM, n = 2), human platelet-rich plasma
(hPRP).
On the basis of their in vitro parameters, a selection of compounds 21a, 22a, 22b and
5
8l were progressed for evaluation of metabolic stabilities in a liver microsome assay,
and the data are listed in Table 5. The liver microsomal stability34, 35 of AZD1283 in rat
liver microsomes was very poor (T1/2 = 6.08 min) but was better in dog microsomes
(T1/2 = 207 min) and human microsomes (T1/2 = 65.0 min). The four new compounds
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were much more stable in rat microsomes (T1/2 = 76.0 – 152 min) and were comparable
(
81.3 and 79.1 min for 21a and 22a) or much more stable (203.9 and 208.3 min for 22b
and 58l) in human microsomes. Compound 22b showed more stable in dog microsomes
than AZD1283. Compound 58l showed the best stability in both rat and human liver
microsomes with the lowest clearance values and longest half-life among these
compounds. This further proved that replacing the ethyl ester of AZD1283 with the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
five-membered lactone could significantly improve the metabolic stability.
Table 5. Microsomal Stability of Selected Compoundsa
b
Clintc
T
1/2
compd
species
MF%d
(
min)
(mL/min/g protein)
rat
dog
6.08
201
345
11.2
32.3
14.5
18.2
25.8
13.8
12.3
26.6
27.6
10.5
10.3
10.6
116.3
10.1
11.0
66.0
38.0
75.0
54.0
43.0
76.0
64.0
42.0
61.7
67.2
65.6
65.3
60.0
66.0
AZD
1
283
human
rat
65.0
145
21a
dog
116
human
rat
81.3
152
2
2a
2b
dog
171
human
rat
79.1
76.0
200.3
203.9
89.1
97.5
208.3
2
dog
human
rat
5
8l
dog
human
a0.33 mg/mL microsomal protein, NADP -regenerating system, [inhibitor], 0.1 μM;
+
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
incubation at 37 °C; samples taken at 0, 7, 17, 30, and 60 min; determination of parent
compound by MS. bT1/2: elimination half-life. Clint: intrinsic body clearance.
dCalculated of metabolic bioavailability.
c
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Pharmacokinetic Analysis. Based on their excellent in vitro activities in the hPRP
assay, compounds 21a, 22a, 22b, 58l, 58m and 62a were selected for evaluation of their
PK properties in Sprague−Dawley (SD) rats along, and AZD1283 was evaluated for
comparison. All compounds were administered orally at a dose of 5 mg/kg. As
summarized in Tables 6, AZD1283 showed poor PK properties, such as an extremely
low plasma concentration (Cmax = 25.9 ± 11 ng/mL) and overall drug exposure (AUC0−∞
=
34 ± 3.34 ng·h/mL). To our delight, all our new five-membered lactone compounds
displayed much better PK properties, with AUCs 8- (62a) to 121-fold (58l) higher than
that of AZD1283. Among these compounds, the overall PK profile of 58l in rats was
the most desirable, as it showed the highest maximal plasma concentration (Cmax = 1661
± 642 ng/mL), highest plasma exposure (AUC0−∞ = 4120 ± 2127 ng·h/mL), and longest
elimination half-life (T1/2 = 2.91 ± 1.09). 5-Chloro-thienyl analogs 22a and 22b
displayed less favorable overall PK properties compared with that of 58l, including
moderate Cmax values and lower plasma exposures. Notably, 22b showed higher plasma
exposure than 22a, which supports the hypothesis that the introduction of a methyl
group at the 4 position of the piperidine ring could improve the PK properties of the
compound. However, the introduction of a fluoro substituent at the same position
(compound 62a) led to decreased plasma exposure and an overall worse PK profile
compared to that of compound 21a, which indicated that although it enhances in vitro
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potency, fluoro substitution negatively impacted the PK properties. Although generally
considered a metabolically liable group, a methyl substituent at the para-position of the
benzene tail not only improved the in vitro antiplatelet activity of 58l but also led to
much better pharmacokinetic properties compared to those of chloro-substituted
derivative 58m (an interesting example of the “magic methyl” effect). The IV PK of
58l in rats also was outstanding, with relatively low clearance (CL = 10.2 ±0.9
mL/min/kg), higher plasma exposure (AUC0-∞ = 8187 ± 673 ng·h/mL), and good oral
bioavailability (F = 51%) at a dose of 5 mg/kg, which displayed much better than that
of AZD1283 (data not shown). Taken together, compound 58l has excellent
pharmacokinetic parameters in rats.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. PK Properties of Selected Compounds in Male Rats after Oral
Administrationa
compd
dose
C
max
T
1/2
T
max
AUC0-∞
MRT
(h)
(mg/kg)
(ng/mL)
(h)
(h)
(ng·h/mL)
AZD
21a
5
5
5
5
5
5
5
25.9 ± 11
1.68 ± 0.37
0.25
0.25
0.25
0.25
0.25
0.25
0.25
34.0 ± 3.34
384 ± 170
417 ± 217
1057 ± 632
4120 ± 2127
1352 ± 754
291 ± 137
2.67 ± 0.77
1.91 ± 0.41
2.31 ± 0.55
1.98 ± 0.70
3.64 ± 0.18
2.58 ± 1.56
3.83 ± 4.38
271 ± 89
225 ± 144
479 ± 67
1.70 ± 0.85
2.40 ± 0.27
1.26 ± 0.45
2.91 ± 1.09
1.90 ± 1.29
2.52 ± 3.0
2
2a
22b
8l
8m
62a
5
1661 ± 642
877 ± 798
149 ± 85
5
aSprague−Dawley rats (male). All values are represented as the mean ± SD.
Abbreviations: Cmax, peak plasma concentration of a drug after administration; Tmax
,
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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time to reach Cmax; T1/2, elimination half-life; AUC, area under the concentration−time
curve; MRT, mean residence time; AZD, AZD1283.
CYP450 Inhibition Assay. Another drawback of AZD-1283 is its high CYP450
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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6
7
8
9
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
inhibition, especially for the CYP2C19 subtype. Therefore, we tested compounds 21a,
2a, 22b, and 58l along with AZD1283 for their abilities to inhibit five major CYP450
2
enzymes, and the results are summarized in Table 7. Indeed, as reported previously,
AZD1283 showed strong inhibition of CYP2C19 (IC50 = 0.399 μM) and moderate
inhibition of CYP2C9 and CYP3A4. The inhibitory activities of the four new
compounds toward CYP2C19 and CYP2C9 were dramatically lower, and 5-chloro-
thienyl substituted compounds 22a and 22b exhibited no CYP3A4 inhibition.
Compounds 21a and 58l moderately inhibited CYP3A4 and were slightly more potent
than AZD1283. However, in conjunction with its very weak inhibition of the other main
subtypes, the overall profile of 58l is comparable to that of AZD1283 in terms of drug-
drug interaction potential. Additionally, by comparing the results of compounds 21a
and 58l with 22a and 22b, it could be speculated that the benzyl group is associated
with CYP3A4 inhibition, which suggested the benzyl tail as a possible site for further
modifications.
Table 7. CYP450 Inhibition of Selected Compounds.
IC50 (μM)a
compd
CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4(M)b CYP3A4(T)c
AZD
1a
>25
>25
6.62
>25
0.399
>25
>25
>25
4.28
8.01
3.64
3.53
2
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2
2a
2b
8l
50
50
50
50
50
25
28.5
7.75
50
50
50
50
50
50
50
50
2
5
1.64
1.11
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
3
3
3
3
3
4
4
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4
4
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4
4
5
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
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9
0
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8
9
0
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9
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9
0
aIC50 refers to the concentration of compound that causes 50% inhibition of the enzyme
b
c
activity. Midazolam was used as the substrate. Testosterone was used as the substrate.
AZD, AZD1283.
+
hERG K Channel Inhibition. To further evaluate the safely profiles of these
compounds, we evaluated compounds 21a, 22a, 22b, and 58l in a human ether-a-go-
go-related gene (hERG) assay, and none of the compounds inhibited hERG at
concentrations as high as 40 μM. This excludes the QT interval prolongation-related
cardiac toxicity of these compounds.
Solubility Assay. Good solubility is the key to druggability. Therefore, we tested
compound 58l and along with AZD1283 for their solubility in three different matrices
(Table 8). To our delight, compound 58l displayed significantly better solubility, with
maximal soluble concentration 6 to 18-fold higher than that of AZD1283.
Table 8. Solubility of AZD1283 and 58l in Different Matrices.
maximal soluble concentration (µM)
compd
intestinal juice
15.61±6.40
gastric juice
0.51±0.20
water
AZD1283
58l
2.99±1.80
90.90±2.69
4.00±2.53
54.2±15.31
In Vivo Antithrombotic Efficacy. Based on its in vitro potency in the hPRP assay
and its excellent overall pharmacokinetic properties, the in vivo antithrombotic efficacy
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of compounds 21a, 22a, 22b, and 58l were tested, and AZD1283 was evaluated for
comparison. The ferric chloride model in Wistar rats was used to determine the efficacy
of those antiplatelet agents. All tested compounds were administered orally at a dose of
10 mg/kg (Figure 3). Among these new five-membered lactone compounds, 21a, 22b,
and 58l resulted in a significant antithrombotic effect, whereas administration of
AZD1283 caused no significant difference relative to the control group. Compound 58l
showed the best antithrombotic effect among these compounds, which was in
accordance with its in vivo PK properties and the liver microsomal stability. To further
determine the antithrombotic effect of compound 58l, we tested it together with
clopidogrel (the most commonly used P2Y12 antagonist) in the same experimental
model (Figure 4). Compound 58l decreased thrombus weight in dose-dependent
manner with an ED50 of 27 mg/kg, compared to that of 7 mg/kg for clopidogrel.
0
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0
Fecl -induced Arterial Thrombosis
3
8
vehicle
AZD1283
6
4
2
0
*
21a
22a
22b
58l
*
**
Dose (10 mg/kg p.o.)
Figure 3. Dose-effects of AZD1283, 21a, 22a, 22b, and 58l on thrombus weight after
FeCl
3
-induced arterial injury in anesthetized Wistar rats at 1.5 h postdosing following
*
oral administration at 10 mg/kg. Data are reported as the mean ± SEM (n = 10). ( p <
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*
*
0.05, p < 0.01).
Bleeding Risk Assessment. Achieving efficient levels of platelet inhibition with
P2Y12 antagonists causes a concomitant increase in the risk of bleeding events.
Therefore, we evaluated the effects of 58l on both bleeding time and bleeding weight
in rats after oral administration. The effects of 58l were studied 1.5 h postdosing in a
tail transection model. The effects of compound 58l were compared to those of
clopidogrel under the same conditions. The ED100 (ED100: 2-fold prolongation of
bleeding time of the control group) was used as the measure of bleeding time, and blood
loss was defined as the amount of blood oozing out of the rat tail after placing a
standardized surgical wound. As shown in Figure 5, 58l and clopidogrel prolonged the
bleeding time in a dose-dependent manner, but the calculated ED100 value of 58l was
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.58 mg/kg, while that of clopidogrel was 2.36 mg/kg. In terms of bleeding weight, in
the absence of any antithrombotic treatment (vehicle group), the surgical blood loss was
quantified at 0.22 g of blood (Table 8). At different doses of clopidogrel (2.5−20 mg/kg),
the surgical blood loss weight increased from 0.44 to 1.99 g of blood. At the tested
doses of compound 58l (2.5−40 mg/kg), the surgical blood loss weight increased from
0
.24 to only 0.66 g.
Recent studies have suggested the application of a “therapeutic window” for the use
of clopidogrel to prevent bleeding and thrombotic complications.36, 37 For the new
generation of P2Y12 antagonists, such as prasugrel and ticagrelor, the concept of a
therapeutic window is becoming more important because of their high potency and the
associated increase in bleeding risk. In our studies in rats (Table 9), we observed that
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at a dose of 40 mg/kg, compound 58l inhibited thrombosis at a rate of 56%, which is
equal to that of clopidogrel at 10 mg/kg (57% inhibition of thrombosis), and these rates
are comparable to the high average rate of inhibition achieved with a daily dose of 75
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mg of clopidogrel. At these therapeutically equivalent doses, compound 58l showed
significantly lower bleeding weight and time compared to clopidogrel (Table 9),
indicating it has a wider therapeutic window than clopidogrel and may therefore have
more potential for use across a broader spectrum of ACS patients with fewer
contraindications.
Figure 4. Dose-effects of clopidogrel and 58l on thrombus weight after FeCl -induced
3
arterial injury in anesthetized Wistar rats at 1.5 h postdosing following oral
administration. 58l (2.5−80 mg/kg), clopidogrel (2.5−20 mg/kg). Data are reported as
*
**
***
the mean ± SEM (n = 10). ( p < 0.05, p < 0.01, p < 0.001).
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Figure 5. Dose-effects of 58l and clopidogrel on tail bleeding time at 1.5 h postdosing
following oral administration in rats. Data are reported as the mean ± SEM (n = 10) (*p
**
***
<
0.05, p < 0.01, p < 0.001).
Table 8. Effects of 58l and Clopidogrel on Blood Loss in Rats
compd
dose (mg/kg)
bleeding weight (g)
Vehicle
-
0.22 ± 0.06
0.24 ± 0.06
0.31 ± 0.07
0.39 ± 0.08
0.43 ± 0.10
0.66 ± 0.11
0.44 ± 0.11
0.70 ± 0.16
1.20 ± 0.24
1.99 ± 0.17
58l
2.5
5
10
2
4
0
0
clopidogrel
2.5
5
10
2
0
Dose-effects of 58l and clopidogrel on tail blood loss at 1.5 h postdosing following oral
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administration in rats. Data are reported as the mean ± SEM (n = 10).
Table 9. Effects of 58l and Clopidogrel on Bleeding Risk after Oral Administration
in Rats.
0
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8
9
0
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dose
mg/kg)
inhibition of
thrombosis (%)
bleeding weight
(g) a
bleeding time
(min) a
compd
(
40
10
56
57
0.66 ± 0.11***
1.20 ± 0.24***
33.09 ± 7.88***
40.50 ±11.81***
5
8l
clopidogrel
a
***
Data are reported as the mean ± SEM (n = 10) ( p < 0.001).
CONCLUSION
In summary, to improve the metabolic stability of the reversible P2Y12 antagonist
AZD1283, a series of bicyclic pyridine analogs were designed and synthesized. Among
these lactone, lactam and cyclic ketone derivatives, a pyridine-fused lactone was the
most effective replacement for the open ester substituent of AZD1283. Further
structural optimization focusing on the B-ring and aryl tail led to the discovery of
several novel analogs, such as 21a, 22a, 22b and 58l, with high in vitro inhibitory
activities in the platelet aggregation assay. Importantly, the pharmacokinetic properties
of these compounds were much better than those of AZD1283 in rats, and they showed
significantly enhanced metabolic stabilities in rat and human microsomes. Furthermore,
among these new five-membered lactone compounds, 21a, 22b, and 58l resulted in a
significant antithrombotic effect in vivo, whereas administration of AZD1283 caused
no significant difference relative to the control group. The best PK properties and
antithrombotic effect in vivo were observed with compound 58l, which was further
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tested in an in vivo model of FeCl -induced carotid artery thrombosis injury to evaluate
3
its antithrombotic efficacy as well as in a rat tail-bleeding model to assess its bleeding
risk. Compound 58l displayed potent and dose-dependent inhibition of platelet
aggregation in vivo and resulted in decreased bleeding time and weight compared to
clopidogrel.
1
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Blood platelets are highly activated within the first one or two weeks in patients after
experiencing ACS, and highly active platelet agents (clopidogrel or ticagrelor) may
only be required for the initial treatment of ACS patients, which should potentially be
switched a maintenance therapy using antiplatelet agents with lower activities but
3
8
superior safety profiles. Our results demonstrate that compound 58l could be ideal for
such maintenance treatment based on its reduced bleeding risk and wider therapeutic
window. Further preclinical evaluation of compound 58l is currently underway.
EXPERIMENTAL SECTION
Chemistry. All solvents and chemicals were used as purchased without further
purification. Room temperature refers to 20−25 °C. Intermediates not described below
were purchased from commercial vendors and were used as supplied unless stated
otherwise. All reactions were monitored using thin-layer chromatography (TLC) on
silica gel F-254 TLC plates. Column chromatography was carried out using silica gel
1
13
(200−300 mesh). H and C NMR spectra were recorded on a Bruker 400, a Bruker
00 or a Bruker 600 NMR spectrometer using solvent residual as an internal standard.
5
Chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (J)
are reported in Hertz (Hz). EI-MS spectra were obtained on a Finnigan MAT95
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