Pyridoxine as a template for the design of antiplatelet agents

Article abstract of DOI:10.1016/j.bmcl.2004.06.079

We describe the synthesis and biological activity of novel pyridoxine derivatives as potential antiplatelet agents. The B₆ vitamers have been shown to display beneficial therapeutic effects in cardiovascular related disorders. The design of novel antiplatelet agents using pyridoxine as a template has led to the discovery of a class of novel cardio- and cerebro-protective agents. The present study describes the synthesis of several of these derivatives along with the antiplatelet and antiischemic activity of derivative 16.

Full text of DOI:10.1016/j.bmcl.2004.06.079

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4747–4750
Pyridoxine as a template for the design of antiplatelet agents
Wenlian Zhang,a John Yao,a Vinh Pham,a Tara Whitney,a Doug Froese,a
Albert D. Friesen,b Linda Stang,c Chen Xu,d Ashfaq Shuaib,d James M. Diakura and
Wasim Haquea,
*
aCanam Bioresearch Inc., 6-1200 Waverley Street, Winnipeg, MB, Canada R3T 0P4
bMedicure Inc., 6-1200 Waverley Street, Winnipeg, MB, Canada R3T 0P4
cSpecial Coagulation Laboratory, University of Alberta Hospital, Canada T6G 2B7
dStroke Research Laboratory, Department of Medicine, University of Alberta, Canada T6G 2B7
Received 4 December 2003; revised 22 June 2004; accepted 23 June 2004
Available online 19 July 2004
Abstract—The B6 vitamers have been shown to display beneficial therapeutic effects in cardiovascular related disorders. The design
of novel antiplatelet agents using pyridoxine as a template has led to the discovery of a class of novel cardio- and cerebro-protective
agents. The present study describes the synthesis of several of these derivatives along with the antiplatelet and antiischemic activity
of derivative 16.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The activation and aggregation of platelets are key
events in the pathophysiology of thrombotic diseases.1
Interestingly, these two processes also play roles in
hemostasis.2 In the context of a pathological role, plate-
let adhesion and aggregation at the site of atheroscle-
rotic lesions in coronary arteries is a primary cause of
cerebral ischemia, unstable angina, and myocardial
infarction. These diseases continue to be major causes
of morbidity and mortality, thus, there remains a need
for antiplatelet drugs that are both efficacious and act
rapidly. Thrombus formation proceeds by initial platelet
binding to the site of vascular injury followed by plate-
let–platelet cohesion. Platelets lack a nucleus and com-
pensate by storing an array of pre-synthesized
molecules inside their granules. In addition, these anu-
cleate cells express a number of receptor types that dis-
tinguish platelets from other types of cells. These
receptors govern platelet reactivity, which can be initi-
ated by a wide range of agonists and adhesive proteins.
Some of the key receptor-mediated platelet aggregation
events include activation by collagen, ADP, and throm-
bin receptor-activating peptide (TRAP) agonists.3 Anti-
platelet drugs have found clinical application in the
secondary prevention of vascular events including acute
myocardial infarction, stroke, and cardiovascular
death.4 Aspirin is the most widely used antiplatelet
agent, and its mechanism of action involves irreversible
inactivation of cyclooxygenase 1 for the lifespan of the
platelet. The newer antiplatelet alternative clopidogrel,
is an irreversible ADP receptor antagonist, and there
is a recent trend toward combining two mechanistically
different antiplatelet agents in an effort to reduce coro-
nary events.5 The co-enzyme form of vitamin B6,
namely pyridoxal 50-phosphate (PLP), has been
reported to be a mild (mM) inhibitor of ADP-, epineph-
rine-, collagen-, arachidonic acid-, and thrombin-
induced platelet aggregation in in vitro experiments.6
In addition, benzamidine, pentamidine, and PLP were
found to block platelet adhesion onto artificial surfaces.7
However, results from in vivo studies on the antiplatelet
effects of vitamin B6 and its vitamers in humans are still
controversial.8 For example, in human platelet rich plas-
ma the inhibition of ADP- and collagen-induced platelet
aggregation were dramatically attenuated in the pres-
ence of albumin, presumably as a result of Schiff base
formation between PLP 1 (Fig. 1) and the active lysines
of the serum protein. More recently, it has been shown
that pyridoxal 2 and pyridoxine 3 can suppress GPIIb
promoter activity.9 These two vitamers were more effi-
cient at reducing GPIIb expression than 1, presumably
due to their ability to more efficiently cross the cell
Keywords: Pyridoxine; Antiplatelet; Antiischemic.
*
Corresponding author. Tel.: +1-204-487-2328; e-mail: whaque@
canambioresearch.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.079

4748
W. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4747–4750
Thus, reaction of amino compound 811 with either 4-
HO
O
H
fluorophenylisocyanate 9a or 4-fluorophenylisothiocya-
nate 9b under reflux in toluene gave the corresponding
urea/thiourea derivative in reasonable yield. Subsequent
removal of the silyl protecting groups was accomplished
by treatment with tetrabutylammonium fluoride,
yielding 10 and 11, respectively. We also carried out
the functionalization of 3 with benzamidine and biph-
enylamidine moieties as shown in Scheme 3. Starting
from aldehyde 12,12 formation of the Schiff base by reac-
tion with 4-aminobenzonitrile in the presence of a cata-
lytic amount of 4-toluenesulfonic acid followed by
reduction of the imine with sodium borohydride gave
compound 13. Subjection of this compound to the
classical Pinner reaction13 provided benzamidine 14.14
Repetition of this reaction sequence from 12 with 4-
amino-40-cyanobiphenyl proceeded via 15 to give the
corresponding biphenylamidine 16.14 Compound 16
was treated with 1equiv HCl to give the hydrochlo-
ride salt, which was then used for the subsequent in vitro
biological and ex vivo studies.
HO
HO
OH
OR
N
N
1 R = PO3H2
2 R = H
3
Figure 1.
membrane. We postulated that since the B6 vitamer
structures possess mild antiplatelet activity, this scaffold
could serve as a basic template to design more potent
platelet aggregation inhibitors. We selected the pyridox-
ine vitamer 3 as the template for further elaboration.
Modification of scaffold 3 was initiated by preparation
of bromide 4, which was obtained by following pub-
lished protocol.10 This intermediate was unstable, there-
fore, it was used without delay in the subsequent
reaction. Addition of 4 to a stirred suspension of tetraz-
ole and powdered potassium carbonate in acetonitrile at
0ꢁCgave the isopropylidene 5-terazole derivative in low
yield,11 as outlined in Scheme 1. Subsequent treatment
with aqueous acetic acid at 80ꢁCgave the 5-tetrazole-
pyridoxine 5. This process was repeated with aminotet-
razole and 2-methylimidazole to give compounds 6
and 7, respectively.
For determination of the inhibition of platelet aggrega-
tion, platelet rich plasma (PRP) was obtained by draw-
ing whole blood into sodium citrate tubes (3.2%) and
centrifuging. Platelet poor plasma (PPP) was obtained
by centrifuging the remainder of the sample until the
platelets were removed. The PRP was adjusted to a
count of 280·109/L with PPP. Incubations were done
with 200lL of platelets and 25lL of the compound
stock solution (4.5mM), giving an approximate final
platelet count of 250·109/L and compound concentra-
tion of 500lM. This mixture was incubated for 30min
at rt, and the baseline transmittance measured with an
aggregometer. Agonist (25lL) was then added to
achieve a final concentration of collagen (1 and 5lg/
mL), ADP (4lM), and TRAP (12lM). The percent
reduction in platelet aggregation induced by the pyr-
idoxine derivatives is shown in Table 1. Compound 16
appears to attenuate platelet aggregation by inhibiting
ADP-induced events.
The preparation of arylurea 10 and arylthiourea 11 was
done according to the procedures shown in Scheme 2.
N
N
O
5 R =
HO
HO
N
N
N
N
O
Br
a,b
R
NH2
N
6 R =
7 R =
N
N
N
N
4
5-7
N
Scheme 1. Synthesis of 5-substituted pyridoxine derivatives. Reagents
and conditions: (a) K2CO3, C HCN, 0ꢁC, tetrazole, 5-aminotetrazole
3
or 2-methylimidazole, 30min, (5: 26%, 6: 19%, 7: 44%); (b) HOAc/H2O
4/1, 80ꢁC, 1h.
Ischemia-reperfusion injury is a root cause of a many of
the important cardiovascular diseases including myocar-
dial infarction and thrombotic stroke. Myocardial ische-
mia is a condition that occurs when the uptake of
oxygen in the heart is below the level needed to maintain
the rate of cellular oxidation.15 Prolonged myocardial
ischemia leads to significant tissue injury and ultimately
myocyte necrosis. Myocardial infarct size is well estab-
lished as an important predictor of cardiac function,
and thus prognosis.16
TBDPSO
N=C=R
TBDPSO
NH2
+
N
F
8
9a R = O
9b R = S
a,b
HO
HO
F
Vitamer 1 has previously been reported to reduce dam-
age induced by ischemia-reperfusion injury.17 More re-
cently, the poly(ADP-ribose) polymerase inhibitor
HO-3089 has been reported to impede ADP-induced
platelet aggregation as well as to exert protection in ani-
mal models of cardiac and brain ischemia-reperfusion
injury.18 We therefore investigated the ability of the ami-
dine derivative 16 to reduce infarct size in the male
Wistar rat model of left anterior descending coronary
artery occlusion (25min) and reperfusion (2h) according
R
NH NH
N
10 R = O
11 R = S
Scheme 2. Synthesis of arylurea and arylthiourea substituted pyridox-
ine analogs. Reagents and conditions: (a) Tol, reflux, 9a or 9b, 16h,
(with 9a: 79%, with 9b: 65%); (b) TBAF, THF, rt, 16h, (10: 89%, 11:
60%).

W. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4747–4750
4749
O
O
HO
HO
H
NH
NH
O
O
d,e
a,b
NH
CN
HO
O
NH
N
N
N
13
12
14
c,b
O
NH
NH2
O
HO
d,e
NH
CN
NH
N
N
15
16
Scheme 3. Synthesis of amidine functionalized pyridoxines. Reagents and conditions: (a) 4-aminobenzonitrile, C6H6/cat p-TsOH, reflux, 18h, (61%);
(b) NaBH4, HOAc, 4ꢁC–rt, 15min, (93%); (c) 4-amino-40-cyanobiphenyl, C6H6/cat p-TsOH, reflux, 18h; (d) HCl/EtOH, 4ꢁC–rt, 16h; (e) 2M NH3–
MeOH, 80ꢁC, sealed tube, 2h, (14: 82%; 16: 63%).
Table 1. Percent reduction of platelet aggregation induced by agonists
collagen, ADP, and TRAP
Table 3. Infarct volume reduction after treatment with compound 16
Concentration of
16 (mg/kg)
Infarct volume
n (Number of animals)
Compound Concn
(mM)
Collagen
ADP
TRAP
(4lM) (12lM)
5 (lg/mL) 1 (lg/mL)
0
40.0
30.5
26.5a
2.9a
10
10
10
10
10
20
40
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.25
0.1
0
4
0
4
0
0
0
0
3
5
0
6
4
7
a P < 0.05.
6
0
1
0
2
7
1
0
1
3
10
11
14
16
1
0
0
0
4
23
10
93
41
NDa
18
79
82
63
66
6
24
30
1
NDa
88
96
93
5
by embolizing a preformed clot into the middle cerebral
artery.21 1h After embolization, doses of 10, 20, or
40mg/kg of 16 were intravenously infused over 60min.
Cerebral infarct volume20 was significantly reduced
when evaluated 48h after embolization, compared to
control as shown in Table 3.
a ND=not done.
to previously published protocol.19 A comparison of
compounds 1 (positive control) and 16 (bolus injection
of 0.1 and 0.07mmol/kg, respectively) for their ability
to reduce infarct size relative to saline control is shown
in Table 2. Treatment of rats with the test compound for
5min prior to ischemia led to a reduction in infarct size
of 31% for compound 1 and 30% for compound 16.
Compound 16 was also evaluated in an ex vivo model of
thrombogenesis22 for its ability to inhibit platelet adhe-
sion. This compound was administered to pigs at three
doses (1, 10, and 30mg/kg), and platelet adhesion was
monitored in an ex vivo Plexiglas chamber by measuring
the deposition of 111In-labeled platelets as per estab-
lished protocol.23 Compound 16 reduced platelet adhe-
sion by 66% (n=8, mean and SEM, P<0.05 vs
baseline) at high shear rate (3397/s) only at the highest
dose.24
Currently, thrombolysis is the only approved treatment
for stroke. Unfortunately, this approach can only be ap-
plied in less than 5% of patients presenting with an acute
ischemic stroke. Thus, new drugs are urgently needed to
complement thrombolysis in acute ischemic stroke.20
We tested compound 16 in an embolic model of focal
cerebral ischemia in rats. Focal ischemia was induced
ADP is a primary platelet agonist, and plays an impor-
tant role in platelet aggregation. Platelet aggregation
was measured in whole blood from pigs treated with 1,
10, and 30mg/kg of 16 using ADP (40lM) as the agon-
ist. Impedance aggregometry measurements25 revealed
that even at the lowest administered dose of 16, ADP-in-
duced whole blood aggregation was completely abol-
ished (n=4, mean and SEM, P<0.05 vs baseline).24
Table 2. Ischemia-reperfusion study, %reduction in infarct size after
bolus injection of 1 (0.1mmol/kg) and 16 (0.07mmol/kg)
Compound
AARa
ISb
n
%Reduction in IS
Our initial goal was to enhance the mild antiplatelet
activity of the vitamin B6 scaffold by installing a phar-
macologically active auxillary such as a phenyl- or bi-
phenyl-amidine at the 5-position of this scaffold. The
metabolite of this scaffold, P5P, is also known to possess
beneficial antiischemic properties.17 Modification of the
Saline
52 ( 3.1)
49 ( 5.8)
46 ( 2.7)
48 ( 3.1)
34 ( 2.8)c
32 ( 3.3)c
10
10
8
1
16
31
30
a Area at risk (AAR), standard error (SEM) is given in parentheses.
b Infarct size (IS) as %AAR.
c P < 0.05.

4750
W. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4747–4750
13. Pinner, A. Die Imidoaether und ihre Derivate; Oppenheim:
Berlin, 1892; p 1.
natural vitamer 3 with a biphenylamidine moiety has led
to compound 16. This modification has improved the
vitamer scaffold in terms of its antiplatelet properties,
while maintaining the scaffolds ability to attenuate ische-
mia-reperfusion induced damage. We were intrigued by
observation that this compound retained two seemingly
unrelated activities. Additional modification of this scaf-
fold is currently underway in an effort to further
improve its antiplatelet activity.
14. Compound 14: 1H NMR (300MHz, CD3OD) d 7.86 (1H,
br s), 7.65 (2H, d, J=8.9Hz), 6.79 (2H, d, J=8.9Hz), 4.98
(2H, s), 4.47 (2H, s), 2.45 (3H, s). ES MS (C15H19N4O2),
calcd for MH+: 287.34; found: 287.29. Compound purity
was determined by RP-HPLC (C18, Zorbax SB-C8,
Agilent Technologies) using gradients of acetonitrile and
0.1% (v/v) TFA in water. The retention time for 14 was
10.5min; purity: 95.3%. Compound 16: 1H NMR
(300MHz, CD3OD) d 7.90 (1H, br s), 7.84 (2H, d,
J=9.0Hz), 7.79 (2H, d, J=9.0Hz), 7.56 (2H, d,
J=8.7Hz), 6.77 (2H, d, J=8.7Hz), 4.99 (2H, s), 4.40
(2H, s), 2.44 (3H, s). ES MS (C21H23N4O2), calcd for
MH+: 363.43; found: 363.36. Compound purity was
determined by RP-HPLC (C18, Zorbax SB-C8, Agilent
Technologies) using gradients of methanol and 16.7mM
aqueous Na2HPO4, pH3.0. The retention time for 16 was
14.3min; purity: 99.3%.
References and notes
1. Clutton, P.; Folts, J. D.; Freedman, J. E. Pharmacol. Res.
2001, 44, 255.
2. Ni, H.; Freedman, J. Transfus. Apheresis Sci. 2003, 28,
257.
15. Ferrari, R.; Pepi, P.; Ferrari, F.; Nesta, F.; Benigno, M.;
Visioli, O. Am. J. Cardiol. 1998, 82(5A), 2K.
16. Dellborg, M.; Herlitz, J.; Risenfors, M.; Swedberg, K. Int.
J. Cardiol. 1993, 40, 167.
17. Dhalla, N. S.; Sethi, R.; Dakshinamurti, K. U.S. Patent
6,043,259.
18. Alexy, T.; Toth, A.; Marton, Z.; Horvath, B.; Koltai, K.;
Feher, G.; Kesmarky, G.; Kalai, T.; Hideg, K.; Sumegi,
B.; Toth, K. J. Cardiovasc. Pharmacol. 2004, 43, 423.
19. Zacharowski, K.; Blackburn, B.; Thiemermann, C. Eur. J.
Pharmacol. 2001, 418, 105.
20. (a) Wang, C.; Yang, T.; Shuaib, A. J. Neurosci. Methods
2001, 109, 147; (b) Experimental details of the cerebral
ischemia study will be presented elsewhere.
21. Yang, Y.; Shuaib, A.; Li, Q. J. Neurosci. Methods 1998,
84, 9.
22. Mehri, Y.; Provost, P.; Chauvet, P.; Theoret, J.-F.;
Phillips, M. L.; Latour, J.-G. Arterioscler. Thromb. Vasc.
Biol. 1999, 19, 372.
3. Clemetson, K. J. In Platelets; Michelson, A., Ed.;
Academic Press: San Diego, 2002; pp 65–84.
4. Ault, K. A.; Cannon, C. P.; Mitchell, J.; McCahan, J.;
Tracy, R. P.; Novotny, W. F.; Reimann, J. D.; Braunwald,
E. 1999, 33, 634.
5. Helft, G.; Osende, J. I.; Worthley, S. G.; Zaman, A. G.;
Rodriguez, O. J.; Lev, E. I.; Farkouh, M. E.; Fuster, V.;
Badimon, J. J.; Chesebro, J. H. Arterioscler. Thromb.
Vasc. Biol. 2000, 20, 2316.
6. (a) Subbarao, K.; Kuchibholta, J.; Kakkar, V. V.
Biochem. Pharmacol. 1979, 28, 531; (b) Krishnamurthi,
S.; Westwick, J.; Kakkar, V. V. Thromb. Haemostas. 1982,
48, 136.
7. Gammell, C. H. J. Lab. Clin. Med. 1998, 131, 84.
8. (a) Schoene, N. W.; Chanmugam, P.; Reynolds, R. D. Am.
J. Clin. Nutr. 1986, 43, 825; (b) van Wyk, V.; Luus, H. G.;
Heyns, A. du P. Thromb. Res. 1992, 66, 657.
9. Chang, S.-J.; Chuang, H.-J.; Chen, H.-H. J. Nutr. Sci.
Vitaminol. 1999, 45, 471.
23. Badimon, L.; Turitto, V.; Rosemark, J. A.; Badimon, J. J.;
Fuster, V. J. Lab. Clin. Med. 1988, 110, 706.
24. The full details of this biological study will be reported
elsewhere.
25. Rand, M. L.; Leung, R.; Packham, M. A. Transfus.
Apheresis Sci. 2003, 28, 307.
10. Imperiali, B.; Roy, R. S. J. Org. Chem. 1995, 60, 1891.
11. Haque, W. U.S. Patent 6,417,204 B1, 2002.
12. (a) Korytnyk, W.; Ikawa, M. Methods Enzymol. 1970,
18A, 524; (b) Pham, V.; Zhang, W.; Chen, V.; Whitney, T.;
Yao, J.; Froese, D.; Friesen, A. D.; Diakur, J. M.; Haque,
W. J. Med. Chem. 2003, 46, 3680.