The first examples of ilexgenin A hybrids as a new class of multi-potent, anti-platelet agents

Article abstract of DOI:10.1016/j.cclet.2013.05.021

Seventeen novel ilexgenin A hybrids (IA-aspirin) and (IA-NO), as donor hybrids (IA-NO will release NO in vivo and function as NO donor), were designed and synthesized in order to develop new multi-targeting agents for the treatment of platelet disorders. Their in vitro activities against ADP, AA and thrombin were evaluated. As a result, IA hybrids achieved substantial increases in the three tested pathways compared with IA. Encouragingly, the most potent hybrid compounds 6d and 14d displayed about 8-fold higher potency than aspirin, and 3-fold higher potency than the simultaneous administration of aspirin and IA in inhibiting ADP-induced aggregation with IC₅₀ values of 0.15 mmol/L and 0.14 mmol/L, respectively. The results suggest these IA hybrids are good candidates for multi-target therapies, and especially, may be considered as promising ADP agonists.

Full text of DOI:10.1016/j.cclet.2013.05.021

Chinese Chemical Letters 24 (2013) 723–726
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Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
The first examples of ilexgenin A hybrids as a new class of multi-potent,
anti-platelet agents
a,
Li-Ping Lin a,b, Fei-Hua Wu a, , Jing-Yu Liang
*
*
a School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, China
b Jiangsu Center for Research & Development of Medicinal Plants, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Seventeen novel ilexgenin A hybrids (IA-aspirin) and (IA-NO), as donor hybrids (IA-NO will release NO in
vivo and function as NO donor), were designed and synthesized in order to develop new multi-targeting
agents for the treatment of platelet disorders. Their in vitro activities against ADP, AA and thrombin were
evaluated. As a result, IA hybrids achieved substantial increases in the three tested pathways compared
with IA. Encouragingly, the most potent hybrid compounds 6d and 14d displayed about 8-fold higher
potency than aspirin, and 3-fold higher potency than the simultaneous administration of aspirin and IA
in inhibiting ADP-induced aggregation with IC50 values of 0.15 mmol/L and 0.14 mmol/L, respectively.
The results suggest these IA hybrids are good candidates for multi-target therapies, and especially, may
be considered as promising ADP agonists.
Received 13 April 2013
Received in revised form 22 April 2013
Accepted 6 May 2013
Available online 12 June 2013
Keywords:
Ilexgenin A
Hybrids
Multi-potent anti-platelet activity
ADP agonists
ß 2013 Fei-Hua Wu and Jing-Yu Liang. Published by Elsevier B.V. on behalf of Chinese Chemical
Society. All rights reserved.
1. Introduction
(IC50 = 0.62 mmol/L) platelet aggregation. However, the relatively
weak potency of IA may compromise its application in pharmacy
Cardiovascular disease caused by platelet-mediated thrombus
formation is now a growing public health problem, which leads to
an urgent need to develop more efficacious and less toxic agents for
clinical use [1–4]. However, the difficulty in developing satisfac-
tory therapies for this disorder lies in the complex pathophysiology
of the disease, which includes activation of thromboxane A2 (TXA2)
[arachidonic acid (AA) pathway], adenosine diphosphate (ADP),
thrombin and even involves the inflammatory process. Thus,
multi-target therapy seems to be an attractive preventive
approach for platelet disorders [5,6]. Due to most of the natural
compounds possessing multi-potent characteristics, many scien-
tists have turned their interest to explore a wide variety of
traditional herbal plants. As a consequence, we also have an
interest in studying the plant, Ilex pubescens Hook. et Arn.
(Aquifoliaceae), which is one of the well-known herbs widely
used in China as a traditional Chinese medicine for the treatment
of cardiovascular diseases. Ilexgenin A (IA) (1) is among the
most abundant triterpenes in I. pubescens and has been determined
to be one of its main antithrombotic ingredients (IC50 = 0.71 mmol/
L against thrombin) [7]. We also assayed in this study for the
potency of IA against ADP (IC50 = 1.02 mmol/L) and AA-induced
industry. Therefore, we believe that our attempt in making IA
hybrids to increase its effectiveness is meaningful.
On the other hand, numerous investigations in previous years,
including preparation of aspirin hybrids [8] and nitric oxide-
releasing derivatives [9], have been reported with some encour-
aging results in the inhibition of platelet aggregation. It is well-
known that aspirin has been a first-line agent against cardiovas-
cular death by preventing thrombotic events for a long time [10].
Aspirin inhibits platelet aggregation by irreversibly binding to
cyclooxygenase and blocking the synthesis of thromboxane A2 (AA
pathway). Furthermore, NO also plays an important role in
regulating the inhibition of platelet aggregation and thrombus
formation in cerebrovascular and cardiovascular systems [11].
Herein, a series of novel IA hybrids were designed and synthesized
by coupling aspirin, or NO, to the 24- and (or) the 28-position of IA
through dibromoalkanes intermediates with differing carbon
chain lengths. By combination of IA and aspirin, or NO, together
to exert synergic action in antiplatelet activity, we hope to find new
multi-potent, anti-platelet agents.
2. Experimental
Owing to the difficulty in designing aspirin ester prodrugs, the
synthetic strategy was first to introduce the linker which was then
used to connect the aspirin moiety. Thus, different dibromoalkanes
(1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane
* Corresponding authors.
E-mail addresses: fhwu2000@sina.com (F.-H. Wu), jyliang08@126.com
(J.-Y. Liang).
1001-8417/$ – see front matter ß 2013 Fei-Hua Wu and Jing-Yu Liang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.05.021

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L.-P. Lin et al. / Chinese Chemical Letters 24 (2013) 723–726
and 1,6-dibromohexane, respectively) were initially reacted with 1
to selectively generate major products 24,28-dibromoalkyl ilex-
genin A 2a–d (45%–81%), together with 28-bromoalkyl ilexgenin A
3a–d as minor products (7%–12%). We synthesized compounds 2
and 3 as follows: To a stirred solution of compound 1 (502 mg,
1 mmol) in DMF (5 mL) at room temperature was added K2CO3
(1.105 g, 8 mmol) and stirred for a period of half an hour. Different
dibromoalkanes (8 mmol) in DMF (2 mL) were then added dropwise
over 10 min and stirring continued for a further 2 h. Subsequently,
theclickchemistrymethodwasemployed(Schemes1and2).Firstly,
azide-functionalized derivatives 4, 5 and 11 were prepared through
diazotization of 2, 3 and 10 (0.58 mmol) with sodium azide
(1.2 mmol) using DMF as solvent at 80–90 8C for 4 h. Then two
alkyne-functionalized derivatives 9 and 12 were obtained by
etherification of 1a and 8 (5 mmol) with propargyl bromide
(5 mmol) in the presence of K2CO3 (10 mmol) in DMF. Finally,
compound 9 was converted to IA-aspirin hybrid 9b (65%) through a
reaction with azide 11, whereas compound 12 was converted into
6a–d(80%–85%), and7a–d(68%–72%)byreactingwithazide4and5,
respectively. The yield of 6e was only 10%, however, as the by-
product. In the process, a sample of azide (4, 5 or 11, 0.47 mmol) was
dissolved inCH2Cl2 (5 mL)atroom temperature. Thenthe mixtureof
CuSO4Á5H2O (0.61 mmol) and sodium ascorbate (1.22 mmol) in H2O
(2 mL) was added dropwise under an atmosphere of nitrogen. The
reaction mixture was allowed to heat at reflux for 3 h.
(dd, 2H, J = 1.0, 8.0 Hz), 5.38 (s, 4H), 5.30 (br s, 1H), 4.27–4.34 (m,
4H), 3.86–4.10 (m, 4H), 3.03 (dd, 1H, J = 4.5, 12 Hz), 2.54 (s, 1H),
2.22 (s, 6H), 2.11 (m, 4H), 1.35 (s, 3H), 1.22 (s, 3H), 1.16 (s, 3H), 0.90
(d, 3H, J = 7.0 Hz), 0.72 (s, 3H), 0.66 (s, 3H). 13C NMR (125 MHz,
CDCl3):
d
177.9, 177.2, 169.6 Â 2, 164.4 Â 2, 150.6 Â 2, 142.8 Â 2,
138.7, 134.0 Â 2, 131.8 Â 2, 128.3, 125.9 Â 2, 124.0, 123.7 Â 4,
123.0, 78.2, 73.9, 60.8 Â 2, 60.4 Â 2, 56.4, 54.2, 49.9, 47.8, 47.4,
47.3, 47.2, 42.2, 42.1, 39.7, 38.0, 37.6, 36.7, 32.7, 28.2, 27.3,
27.1 Â 3, 26.9, 26.1, 24.4, 24.0, 20.9 Â 2, 20.2, 17.2, 16.1 Â 2, 13.2.
Compound 6b: ESI-MS (m/z): 1155.7 [M+Na]+. 1H NMR
(500 MHz, CDCl3):
d 8.01, 8.00 (dd, 2H, J = 1.5, 8.0 Hz), 7.68 (s,
2H), 7.56, 7.55 (dd, 2H, J = 1.7, 8.0 Hz), 7.29 (dd, 2H, J = 1.1, 5.5 Hz),
7.09, 7.08 (d, 2H, J = 8.0 Hz), 5.31 (br s, 1H), 5.29 (s, 4H), 4.35 (m,
4H), 3.99–4.17 (m, 4H), 3.03 (dd, 1H, J = 4.5, 12 Hz), 2.26 (s, 6H),
1.55–1.98 (m, 8H), 1.37 (s, 3H), 1.26 (s, 3H), 1.20 (s, 3H), 0.94(d, 3H,
J = 7.0 Hz), 0.76 (s, 3H), 0.69 (s, 3H).
Compound 6c: ESI-MS (m/z): 1183.7 [M+Na]+. 1H NMR
(500 MHz, CDCl3):
d 8.00 (d, 2H, J = 8.0 Hz), 7.64 (s, 2H), 7.56 (d,
2H, J = 1.5, 7.5 Hz), 7.29 (tt, 2H, J = 1.5, 7.5 Hz), 7.08 (d, 2H,
J = 8.0 Hz), 5.32 (br s, 1H), 5.29 (s, 4H), 4.36–4.40 (m, 4H), 3.92–
4.13 (m, 4H), 3.01 (dd, 1H, J = 4.5, 12 Hz), 2.26 (s, 6H), 1.56–2.03 (m,
12H), 1.38 (s, 3H), 1.24 (s, 3H), 1.20 (s, 3H), 0.93 (d, 3H, J = 6.7 Hz),
0.74 (s, 3H), 0.67 (s, 3H).
Compound 6d: ESI-MS (m/z): 1211.7 [M+Na]+; HR-MS
([M+H]+): Calcd: 1189.6434, Found: 1189.6431. 1H NMR
Eight nitroxy-alkyl esters (14, 15), on the other hand, were
prepared by stirring the solution with corresponding Br-displace-
ments 2 and 3 in the presence of AgNO3. To a stirred solution of 2
(0.5 mmol) or 3 (0.5 mmol) in THF:CH3CN (1:1, 5 mL) was added
AgNO3 (1.2 mmol) dropwise over 10 min. The mixture was heated
at reflux for 3 h in dark flasks (Scheme 3).
(500 MHz, CDCl3):
d
7.97 (dd, 2H, J = 1.5, 8.0 Hz), 7.61 Â 2 (s,
2H), 7.52 (ddd, 2H, J = 2.0, 8.0 Hz), 7.25 (ddd, 2H, J = 1.0, 7.5 Hz),
7.04 (dd, 2H, J = 1.0, 8.0 Hz), 5.38 (s, 4H), 5.30 (br s, 1H), 4.27–4.33
(m, 4H), 3.86–4.10 (m, 4H), 3.03 (dd, 1H, J = 4.5, 12 Hz), 2.22 (s, 3H),
2.11 (s, 3H), 1.52–1.74 (m, 16H), 1.35 (s, 3H), 1.22 (s, 3H), 1.16 (s,
3H), 0.90 (d, 3H, J = 7.0 Hz), 0.72 (s, 3H), 0.66 (s, 3H). 13C NMR
The resulting compounds were characterized by 1H NMR, 13C
NMR and mass spectroscopy. The experimental data of selected
compounds were listed as follows.
(125 MHz, CDCl3):
d
177.8, 177.6, 169.5 Â 2, 164.3 Â 2, 150.5 Â 2,
142.4 Â 2, 138.0, 134.0 Â 2, 131.8 Â 2, 128.8, 125.9 Â 2, 123.7 Â 2,
123.6 Â 2, 123.0 Â 2, 78.2, 73.0, 63.9, 63.8, 58.1 Â 2, 56.4, 53.2,
50.2, 50.1, 48.9, 47.8, 46.4, 41.2, 41.1, 39.7, 39.0, 37.3, 37.2, 30.7,
30.0 Â 3, 28.2, 28.1, 28.0, 27.3, 26.0 Â 2, 25.9, 25.4 Â 3, 24.0, 23.7,
23.6, 20.8 Â 2, 20.2, 16.6, 16.0, 13.2.
Compound 6a: ESI-MS (m/z): 1127.5 [M+Na]+. 1H NMR
(500 MHz, CDCl3):
d 7.97 (dd, 2H, J = 1.5, 8.0 Hz), 7.61 (s, 2H),
7[(Schme_1)TD$FIG] .51 (ddd, 2H, J = 2.0, 8.0 Hz), 7.25 (ddd, 2H, J = 1.5, 7.5 Hz), 7.04
Scheme 1. The synthesis of 24- and (or) 28-substituted IA-aspirin hybrids. Reagents and conditions: (a) 1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane and
1,6-dibromohexane, dry K2CO3, DMF, r.t.; (b) NaN3, DMF, 80–90 8C; (c) propargyl bromide, dry K2CO3, DMF, r.t.; (d) CuSO4Á5H2O, sodium ascorbate, CH2Cl2–H2O, reflux.

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L.-P. Lin et al. / Chinese Chemical Letters 24 (2013) 723–726
725
Scheme 2. The synthesis of 3-substituted IA-aspirin hybrids. Reagents and conditions: (a) 1,3-dibromopropane, dry K2CO3, DMF, r.t.; (b) NaN3, DMF, 80–90 8C; (c) propargyl
[(chme_3)TD$FIG]
bS romide, dry K CO , DMF, r.t.; (d) CuSO Á5H O, sodium ascorbate, CH Cl –H O, reflux; (e) BnCl, dry K CO , DMF, r.t.; (f) NaH, THF, 0 8C to reflux, 65% (total yield of 29%).
2
3
4
2
2
2
2
2
3
Scheme 3. The synthesis of 24- and (or) 28-substituted IA-NO hybrids. Reagents and conditions: (a) AgNO3, avoid light, reflux, THF/CH3CN, yield in 56%–68%.
Table 1
Antiplatelet aggregation activity of target compounds and intermediates in vitro.a,c
Compound
Inhibition rate (%) of AA (40
mmol/L)
Inhibition rate (%) of ADP (10
m
mol/L)
Inhibition rate (%) of thrombin
(5 U/mL)
0.1 mmol/L
0.25 mmol/L
0.1 mmol/L
0.25 mmol/L
0.1 mmol/L
0.25 mmol/L
2a
NAb
6.2 Æ 2.0
9.1 Æ 2.4
4.8 Æ 2.3
1.2 Æ 2.3
19.7 Æ 3.2**
20.0 Æ 0.8**
NA
26.2 Æ 2.5**
32.1 Æ 4.4**
37.7 Æ 8.5**
46.1 Æ 4.4**
6.1 Æ 2.0
NA
NA
2b
2.2 Æ 0.3
4.7 Æ 2.2
4.0 Æ 0.8
NA
NA
0.9 Æ 4.5
2c
15.5 Æ 4.3*
17.1 Æ 3.2*
NA
3.1 Æ 3.2
8.5 Æ 3.2
NA
10.3 Æ 3.5
11.4 Æ 4.4
NA
2d
3a
3b
0.3 Æ 0.1
1.7 Æ 3.1
2.0 Æ 0.3
2.1 Æ 1.8
1.1 Æ 0.8
3.9 Æ 4.1
4.6 Æ 2.3
13.9 Æ 2.9**
16.1 Æ 2.7**
16.1 Æ 3.1**
18.0 Æ 2.3**
NA
2.1 Æ 1.5
0.2 Æ 1.1
9.7 Æ 3.2
13.0 Æ 0.4*
8.1 Æ 2.8
1.1 Æ 0.8
13.9 Æ 4.3*
21.7 Æ 2.3**
13.9 Æ 2.9*
19.1 Æ 4.7**
18.3 Æ 3.5**
29.0 Æ 5.3**
NA
12.1 Æ 4.4
15.7 Æ 5.0
24.1 Æ 5.2**
14.4 Æ 4.0**
8.0 Æ 2.7
NA
NA
3c
9.5 Æ 2.1
1.1 Æ 2.4
4.5 Æ 2.6
NA
6.3 Æ 2.8
8.4 Æ 3.0
NA
3d
14.1 Æ 2.6*
4.4 Æ 4.0
4a
4b
8.0 Æ 2.7
NA
3.7 Æ 3.7
11.5 Æ 2.9
6.0 Æ 0.4
20.1 Æ 3.0**
23.1 Æ 2.7**
31.7 Æ 3.7**
37.0 Æ 2.1**
NA
4c
10.9 Æ 1.5
11.0 Æ 1.7
21.3 Æ 3.8**
29.1 Æ 2.2**
30.9 Æ 4.0**
37.2 Æ 2.1**
NA
35.9 Æ 7.5**
41.0 Æ 12.7**
41.3 Æ 3.8**
59.9 Æ 2.2**
69.9 Æ 6.0**
77.2 Æ 12.5**
NA
7.4 Æ 2.5
4.2 Æ 2.6
15.2 Æ 3.1**
11.3 Æ 4.7**
10.5 Æ 4.5*
15.5 Æ 2.5**
NA
4d
6a
6b
6c
6d
6e
7a
8.9 Æ 2.4
10.1 Æ 1.5*
12.1 Æ 4.1**
14.0 Æ 2.3**
NA
16.3 Æ 2.4**
21.1 Æ 3.4**
25.9 Æ 4.0**
27.2 Æ 2.1**
NA
10.9 Æ 4.3*
12.1 Æ 4.5**
13.3 Æ 2.3**
21.0 Æ 2.3**
NA
34.3 Æ 3.7**
48.9 Æ 4.2**
56.9 Æ 4.0**
65.2 Æ 6.5**
NA
11.3 Æ 2.5*
9.3 Æ 2.5*
9.5 Æ 1.5*
12.5 Æ 2.5*
NA
18.1 Æ 1.0*
19.1 Æ 1.4**
26.7 Æ 6.7**
31.0 Æ 2.1**
NA
7b
7c
7d
9b
14a
14b
14c
14d
1
11.3 Æ 0.8*
15.2 Æ 1.2**
16.7 Æ 2.3**
16.9 Æ 3.0**
2.1 Æ 0.2
25.1 Æ 3.2**
27.3 Æ 0.6*
21.3 Æ 7.0**
24.6 Æ 4.2**
30.2 Æ 5.1**
35.1 Æ 2.3**
15.1 Æ 2.3
44.1 Æ 0.2**
49.3.1 Æ 5.0**
16.3 Æ 0.6**
21.2 Æ 3.2**
26.7 Æ 1.3**
28.3 Æ 2.0**
NA
74.3 Æ 10.2**
54.8 Æ 6.2**
60.2 Æ 6.6**
78.7 Æ 10.3**
5.2 Æ 1.8
9.6 Æ 3.2*
8.1 Æ 3.9*
6.2 Æ 3.3
7.7 Æ 0.6
6.7 Æ 0.2
NA
18.6 Æ 3.8*
20.0 Æ 2.5**
20.8 Æ 4.3**
22.3 Æ 5.6*
12.8 Æ 2.9
17.8 Æ 0.9**
19.2 Æ 1.8*
Aspirin
1 + aspirin
NA
10.1 Æ 0.1**
9.3 Æ 2.4
24.3 Æ 0.2**
9.6 Æ 3.0*
a
Data are expressed as mean Æ SD of three experiments. Aggregation rate of ADP was 54.7% Æ 2.9%, AA was 56.2% Æ 1.3% and thrombin was 58.6% Æ 5.4%.
b
c
NA: not active.
Ref. [13].
*
p < 0.05 vs vehicle.
p < 0.01 vs vehicle.
**

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L.-P. Lin et al. / Chinese Chemical Letters 24 (2013) 723–726
Compound 14a: ESI-MS (m/z): 709.4 [M+H]+. 1H NMR (500 MHz,
CDCl3): 5.35(brs,1H), 4.54(m, 4H), 4.08–4.25(m, 4H), 3.10(dd,1H,
J = 4.1, 11.5 Hz), 2.51 (s, 1H), 1.40 (s, 3H), 1.25 (s, 3H), 1.20 (s, 3H),
0.97 (d, 3H, J = 5.6 Hz), 0.77 (s, 3H), 0.76 (s, 3H). 13C NMR (125 MHz,
be thrombin and ADP [14,15]. Therefore, the design and synthesis
d
of IA hybrids to increase their potency against the two platelet
aggregation inductors was a necessary and meaningful attempt for
developing multi-target antiplatelet agents. Furthermore, to the
best of our knowledge, this letter is among the first to report the
synthesis and all-round biological evaluation of IA derivatives in
AA, ADP and thrombin. Thus, the synthesis and evaluation of the
anti-platelet effect of IA hybrids could be an innovative way to
develop effective new anti-platelet agents. Finally, due to the key
role ADP plays in hemostasis and thrombosis and the development
of antiplatelet drugs targeting the P2Y12 receptor, further
investigation aimed at evaluating the activities of IA hybrids as
a new class of P2Y12 receptor antagonists is warranted.
CDCl3):
d
177.8, 177.2, 139.0, 128.3, 78.0, 74.1, 61.9 Â 2, 61.4 Â 2,
56.5, 54.2, 49.9, 48.2, 47.4, 42.7, 41.8, 39.9, 38.2, 37.2, 36.7, 32.5, 28.8,
27.3, 27.1, 26.9, 26.6 Â 2, 26.4, 24.4, 23.5, 21.4, 17.3, 16.2, 15.2, 13.6.
Compound 14b: ESI-MS (m/z): 737.4 [M+H]+. 1H NMR (500 MHz,
CDCl3):d5.34(brs,1H), 4.48(m, 4H), 4.00–4.17(m, 4H), 3.13(dd,1H,
J = 4.2, 12.2 Hz), 2.52 (s, 1H), 1.56–1.87 (m, 8H), 1.40 (s, 3H), 1.27 (s,
3H), 1.21 (s, 3H), 0.94 (d, 3H, J = 6.8 Hz), 0.78 (s, 3H), 0.70 (s, 3H).
Compound 14c: ESI-MS (m/z): 765.5 [M+H]+. 1H NMR
(500 MHz, CDCl3):
d 5.24 (br s, 1H), 4.46 (t, 4H, J = 6.2, 12.5 Hz),
4.10–4.19 (m, 4H), 3.10 (dd, 1H, J = 3.5, 11.2 Hz), 2.50 (s, 1H), 1.66–
1.82 (m, 12H), 1.40 (s, 3H), 1.26 (s, 3H), 1.21 (s, 3H), 0.93 (d, 3H,
J = 6.6 Hz), 0.77 (s, 3H), 0.71 (s, 3 H).
Acknowledgments
Compound 14d: ESI-MS (m/z): 793.2 [M+H]+. 1H NMR
The study was financially supported by a grant from the
Postgraduate Innovation Project of Jiangsu Province, China (No.
CX09B_284Z) and the Natural Science Foundation of Jiangsu
Province, China (No. BK2012378).
(500 MHz, CDCl3):
d 5.35 (br s, 1H), 4.48 (m, 4H), 4.00–4.17 (m,
4H), 3.09 (dd, 1H, J = 4.2, 12.0 Hz), 2.58 (s, 1H), 1.41 (3H, s), 1.27
(3H, s), 1.21 (3H, s), 0.94 (d, 3H, J = 6.6 Hz), 0.77 (s, 3H), 0.70 (s, 3H).
3. Results and discussion
References
Seventeen novel IA-aspirin and IA-NO donor hybrids were
designed and synthesized. Both target hybrids and their inter-
mediates were evaluated for the anti-platelet potency by Born’s
turbidimetric method [12] employing AA, ADP and thrombin as
platelet aggregation inductors, respectively. Aspirin and IA were
used as positive controls. The assay results are summarized in
Table 1. Although the anti-AA activities were less than aspirin, the
disubstituted target hybrid compounds (Inhibition rate for 6a–d in
0.25 mmol/L ranged from 20.1% to 37.0% and for 14a–d ranged
from 18.6% to 22.3%), however, were more potent against thrombin
than aspirin (17.8%). Furthermore, the inhibitory activities against
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ADP (10
mmol/L) of the target hybrids (inhibition rate for 6a–d in
0.25 mmol/L ranged from 41.3% to 77.2% and for 14a–d ranged
from 54.8% to 78.7%) were significantly increased compared with
IA (5.2%) and aspirin (9.3%), either alone or in combination.
Encouragingly, the most potent compounds 6d (77.2%) and 14d
(78.7%) displayed about an 8-fold higher potency than aspirin
(9.3%), and 3-fold higher than the simultaneous administration
(24.3%) of aspirin and IA with IC50 values of 0.15 mmol/L and
0.14 mmol/L, respectively. In addition, the potency of intermedi-
ates was far lower than the corresponding target hybrid
compounds, which was consistent with our expectations that
hybrids would exert synergy for anti-platelet activities.
The above results on anti-platelet activity of IA hybrids also,
from the structure–activity relationship (SAR), prompted us to
suspect that the anti-platelet effect was related to the carbon chain
length and number of the substituents from 24 or (and) 28-COOH.
The disubstituted hybrids were, in vitro, more potent antithrom-
botics than mono-substituted ones (e.g., 2 vs. 3, 6 vs. 7). In addition,
with a longer carbon-chain linker, IA disubstituted derivatives
seem to show stronger inhibitory activities (e.g., 6d vs. 6a–c, 14d vs.
14b–c). Comparison of IA-aspirin hybrids 9b and 6a–d, on the
other hand, showed that the free 3-hydroxy seemed critical for the
anti-platelet activity. It is also interesting to note that replacement
of aspirin with salicylic acid, the by-product hybrid 6e exhibited no
inhibitory effect on platelet aggregation.
(b) D.K. Lee, H.S. Lee, M.D. Huh, et al., Antiplatelet action of Ilexoside D, a
triterpenoid saponin from Ilex pubescens, Arch. Pharm. Res. 14 (1991) 352–356;
(c) Y.N. Han, J.I. Song, I.K. Rhee, Anticoagulant activity of Ilexoside D, a triterpe-
noid saponin from Ilex pubescens, Arch. Pharm. Res. 16 (1993) 209–212.
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activities of ferulic acid esters, J. China Pharm. Univ. 40 (2009) 486–490.
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(b) Z.L. Min, Y.H. Zhang, P. Zhuang, et al., Synthesis and anti-platelet activities of
nitric-oxide releasing derivatives of 3-butylphthalide, J. China Pharm. Univ. 39
(2008) 392–397.
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dose, effectiveness, and side effects, Chest 119 (Suppl. 1) (2001) 39S-63S.
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[12] G.V.R. Born, M.J. Cross, The aggregation of blood platelets, J. Physiol. 168 (1963)
178–195.
[13] Antiplatelet aggregation assays: Blood samples were withdrawn from rat ab-
dominal aorta and mixed with 3.8% trisodium citrate (9:1, v/v), followed by
centrifuging at 1000 rpm for 10 min. The supernatants were collected and used as
platelet rich plasma (PRP). Additional sample were centrifuged at 3000 rpm for
10 min and the supernatants were collected as platelet poor plasma (PPP). The
effect of individual compounds on the AA, ADP and thrombin-induced platelet
aggregation was measured by the Born’s turbidimetric method using a Platelet-
Aggregometer (LBY-NJ Platelet-Aggregometer, Beijing). Briefly, PRP (280 mL) was
pre-treated in duplicated with vehicle (0.5% DMSO), different concentrations of
individual compounds or the reference drugs and exposed to 10 mL of AA (final
concentration 40 mmol/L), ADP (final concentration 10 mmol/L) or thrombin
(final concentration 5 U/mL) incubated at 37 8C for 5 min. Changes in the light
transmittance of the reaction mixture were continuously recorded for 5 min and
the maximal aggregation was also recorded. The anti-platelet aggregation activity
of tested compound was evaluated as inhibition rate (%) which was determined
using the following formula: Inhibition rate (%) = (1 À the maximal aggregation of
compound/the maximal aggregation of control) Â 100.
4. Conclusion
[14] P. Theroux, Antiplatelet therapy: do the new platelet inhibitors add significantly
to the clinical benefits of aspirin? Am. Heart J. 134 (1997) s62–s70.
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detecting antiplatelet effect in patients with coronary artery disease, Biomed.
Pharm. 63 (2009) 608–612.
In summary, the IA hybrids demonstrated significant increases
in anti-platelet activity both against thrombin and ADP compared
with aspirin and IA. It is well-known that platelets can be activated
by several physiological agonists, but the most important seems to