Synthesis of 1-benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole analogues as novel antiplatelet agents

Article abstract of DOI:10.1021/jm010001h

1-Benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole (28, YC-1) was selected as the lead compound for systemic structural modification. After screening for antiplatelet activity, SARs of YC-1 analogues were established. Among these potent active derivatives, co

Full text of DOI:10.1021/jm010001h

3746
J . Med. Chem. 2001, 44, 3746-3749
Brief Articles
Syn th esis of 1-Ben zyl-3-(5′-h yd r oxym eth yl-2′-fu r yl)in d a zole An a logu es a s Novel
An tip la telet Agen ts
Fang-Yu Lee,†,‡ J in-Cherng Lien,† Li-J iau Huang,† Tsang-Miao Huang,‡ Sheng-Chung Tsai,† Che-Ming Teng,§
Chin-Chung Wu,| Fong-Chi Cheng, and Sheng-Chu Kuo*,†
Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan, Yung-Shin Pharmaceutical
Industry Co., Ltd., Tachia, Taichung, Taiwan, Pharmacological Institute, College of Medicine, National Taiwan University,
Taiwan, Department of Pharmacy, Tajen Institute of Technology, Pingtung, Taiwan, and MSD Pharma Services Taiwan LTD,
Taipei, Taiwan
Received J anuary 3, 2001
1-Benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole (28, YC-1) was selected as the lead compound
for systemic structural modification. After screening for antiplatelet activity, SARs of YC-1
analogues were established. Among these potent active derivatives, compounds 29, 30, 31, 44,
and 45 functioned as potent activators of sGC and inhibitors of PDE5 with potency comparable
to that of YC-1. In addition, compound 58 was found to be a selective and potent inhibitor of
protease-activated receptor type 4 (PAR4)-dependent platelet activation.
In tr od u ction
YC-1, we feel obligated to report our extensive synthesis
effort of YC-1 analogues to further the discovery of
potential antiplatelet agents.
To examine whether the 5′-CH2OH and aromatic ring
of YC-1 may interact with the binding site of sGC, we
prepared a series of YC-1 analogues with a fixed
pyrazole (B) ring and with modifications made on
peripheral 1-benzyl, 5′-CH2OH, and 5,6-positions. In
addition, the C ring was also replaced by benzene ring.
In our previous work on the development of novel
antiplatelet agents, we reported that 1-benzyl-3-(5′-
hydroxymethyl-2′-furyl)indazole (28, YC-1) exhibited
significant inhibitory effects against thrombin-, AA-,
collagen-, and PAF-induced platelet aggregation.1 Due
to its antiplatelet activity of broad spectrum, we as-
sumed that YC-1 interfered with platelet aggregation
through a common pathway. Mechanism investigation
revealed that the in vitro antiplatelet activity of YC-1
was associated with NO-independent activation of soluble
guanylate cyclase (sGC),1,2 although YC-1 and NO
activated sGC in a synergistic manner.3 In addition,
YC-1 was similar to NO to enhance the response of sGC
to CO.3 Unlike NO, YC-1 activated sGC without involv-
ing heme but via binding to allosteric site of sGC which
in turn decreased the dissociation rate of NO and CO.3,4
Accordingly, YC-1 has been recognized as a new anti-
platelet agent with unique mechanism of action.
Herein we report a SAR based on the antiplatelet
activities of these analogues.
Besides, YC-1 demonstrated NO-enhancing capability
via sGC activation and PDE inhibition5 and therefore
may greatly elevate the cGMP level. These unique
activities make YC-1 an ideal lead compound for new
drug development. Furthermore, the ability of YC-1 to
potentiate NO action may be of therapeutic potential,
since it could reduce the dosage of nitrovasodilators and
thus decrease the associated toxicity. Consequently, the
pharmacological activities of YC-1 pertinent to its
clinical efficacy were widely studied in recent years.
The structure-activity relationship (SAR) of YC-1
analogues has not been reported. As the inventors of
Resu lts a n d Discu ssion
Ch em istr y. All key intermediates (23-27, 41, 58, 59)
were synthesized according to Scheme 1. The starting
diaryl ketones (7-11, 52, 53) were treated with ap-
propriate hydrazines (12, 38) to yield the corresponding
hydrazones (13-17, 39, 54, 55) as mixtures of E- and
Z-isomers6 which were converted to indazoles (23-27,
41, 58, 59) via the formation of azo intermediates (18-
22, 40, 56, 57).
The above hydrazones (13-17, 39, 54, 55) were then
treated with Pb(OAc)4 in CH2Cl2 at low temperature.
Subsequently, BF3‚Et2O was added, and the mixture
was heated to yield the desired indazoles (23-27, 41,
58, 59) via cyclization.
* To whom correspondence should be addressed. Fax: 011-886-4-
22030760, 011-886-4-22055105 (update). E-mail: jclien@mail.cmc.edu.tw.
† China Medical College.
‡ Yung-Shin Pharmaceutical Industry Co., Ltd.
§ National Taiwan University.
These indazoles (23-27, 41, 58, 59) were either
reduced with Ca(BH4)2 to afford the corresponding
carbinol derivatives (28-32, 42, 60, 61) or hydrolyzed
| Tajen Institute of Technology.
MSD Pharma Services Taiwan LTD.
10.1021/jm010001h CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/28/2001

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3747
Sch em e 1
An tip la telet Activity. The antiplatelet activities of
1,5,6-substituted 3-(5′-substituted-2′-furyl)indazoles (23-
51) are summarized in Table 1. The lead compound (YC-
1) was found to exhibit significant inhibitory effect
against thrombin-, AA-, collagen-, and PAF-induced
platelet aggregation. Replacing the 1-benzyl group with
phenyl (42) improved antiaggregation potency toward
thrombin, AA, and collagen inducers, whereas impaired
the activity toward PAF-induced aggregation.
Removal of 1-benzyl group of YC-1 to yield compound
I7 dramatically reduced antiplatelet activity, suggesting
that the presence of an aromatic ring at 1-position is
essential for potent antiplatelet activities of broad
spectrum.
We then explored the effects of altering R5 and R6
substituents. Placing an F atom at the 6-position yielded
29 which significantly enhanced the inhibition toward
AA-, collagen-, and PAF-induced aggregation, but re-
duced the inhibition against thrombin-induced aggrega-
tion. Substitution of 6-position with CH3 (30) and OCH3
(31) did not alter the activity. Finally, bridging the 5,6-
positions with -OCH2O- (32) significantly diminished
the activity.
Then, 5′-CH2OH was converted to -CH2OCH3 (44) and
resulted in greatly increased activity. Likewise, the
ether 45 significantly enhanced the inhibition toward
thrombin-induced aggregation, whereas it reduced the
inhibition against AA-, collagen-, and PAF-induced
aggregation. Similar conversion of 30 to 46 resulted in
only a minor change in activity, while conversion of 31
and 32 into 47 and 48 led to reduced activity.
In another approach, amination of the 5′-CH2OH
group provided 51 and considerably improved the
inhibition activity toward PAF- and thrombin-induced
aggregation but was inactive toward AA- and collagen-
induced aggregation. Meanwhile, elimination of the OH
group provided 50, showing markedly increased inhibi-
tion, especially toward thrombin- and PAF-induced
aggregation.
Sch em e 2
Subsequently, the 5′-CH2OH group was converted
into -COOCH3 to give 23, which demonstrated weak
inhibition against AA- and collagen-induced aggregation
and no inhibition toward thrombin- and PAF-induced
aggregation at a concentration as high as 300 µM. The
pattern of preferential inhibition of 23 toward platelet
aggregation differed widely from that of YC-1. Like 23,
esters 24-27 were less potent than their corresponding
5′-CH2OH counterparts 29-32 and had a different
pattern of preferential inhibition from YC-1.
A marked decrease in inhibition potency toward
thrombin- and PAF-induced aggregation for N-phenyl
compounds was observed when 5′-CH2OH (42) was
converted into -COOCH3 (41).
The 5′-COOH derivative 33 was found less potent
than YC-1. Again, as expected, the 5′-COOH derivatives
34-37 were also less potent than their 5′-CH2OH
counterparts (29-32).
to give the corresponding carboxylic acid derivatives
(33-37, 43, 62, 63).
The -CH2OH groups of compounds 28-32 were modi-
fied following Scheme 2 to give their corresponding
methoxy methyl derivatives (44-48). Meanwhile, in-
termediate 49, obtained by treating 28 with BCl3, was
either hydrogenated to yield 5′-methyl derivative (50)
or treated with diethylamine to give its N,N-diethy-
laminomethyl derivative (51).
Replacing the 3-furan ring of YC-1, 23 and 33 with a
benzene ring yielded 58-63, which were generally
weaker than their 3-furyl counterparts (Table 1). How-
ever, compound 58 displayed unexpectedly great inhibi-
tion against thrombin-induced aggregation (IC50 ) 29.3
µM) but very little inhibition toward AA-, collagen-, and
PAF-induced aggregation. Such highly selective anti-

3748 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22
Brief Articles
Ta ble 1. Inhibitory Effect of Compounds 23-37, 41-48, 50-51, 58-63, and I on Platelet Aggregation Induced by Thrombin, AA,
Collagen, and PAFa
IC50 (µM)b
compd
R
R′
R′′
thrombin
AA
collagen
PAF
87.3
145.4
28 (YC-1)
42
-H
-
-
-H
-H
-H
-H
-
-
-CH2OH
-CH2OH
-CH2OH
-CH2OH
-CH2OH
-CH2OH
-CH2OH
-CH3
-CH2OCH3
-CH2OCH3
-CH2OCH3
-CH2OCH3
-CH2OCH3
-CH2NEt2
-COOCH3
-COOCH3
-COOCH3
-COOCH3
-COOCH3
-COOCH3
-COOH
-COOH
-COOH
-COOH
-COOH
-COOH
-
173.0
158.2
>270
217.4
146.9
168.3
238.8
39.4
143.4
115.8
125.9
255.7
>270
101.9
>270
>270
>270
>270
>270
259.8
>270
>270
>270
>270
>270
>270
29.3
54.3
53.8
13.7
17.9
100.9
21.7
49.4
49.7
96.6
47.3
8.2
45.9
32.4
108.2
101.6
55.7
81.1
10.6
94.0
64.2
92.3
136.2
83.7
I
>270
>270
29
30
31
32
50
44
45
46
47
48
51
23
41
24
25
26
27
33
43
34
35
36
37
58
59
60
61
62
63
aspirin
-F
25.2
45.9
53.4
72.0
89.2
-CH3
-OCH3
46.7
104.6
27.5
6.6
64.1
-OCH2O-
163.8
39.6
25.5
108.7
76.3
176.6
195.6
55.9
-H
-H
-H
-H
-H
-H
-H
-F
-CH3
-OCH3
59.3
114.8
113.0
50.4
161.5
18.2
101.1
78.3
156.9
168.4
75.5
>270
179.5
182.5
139.7
150.3
212.4
143.8
122.6
98.1
-OCH2O-
-H
-H
-
-H
-H
-H
-H
-H
-
-F
>270
>270
148.6
-CH3
-OCH3
>270
>270
-OCH2O-
253.5
-H
-
-H
-H
-H
-H
-
-F
>270
>270
>270
>270
>270
>270
164.0
>270
142.9
>270
119.1
150.3
150.0
148.1
184.8
124.7
100.9
51.3
-CH3
-OCH3
-OCH2O-
-H
-COOC2H5
-H
-CH2OH
-H
-COOH
-H
-COOC2H5
-H
-CH2OH
-H
-
-
-
-
>270
192.7
>270
>270
>270
>270
173.6
178.7
235.4
129.6
155.5
20.0
156.1
148.2
>270
-COOH
-
>270
a
Platelets were incubated with test compound at 37 °C for 1 min, then thrombin (0.1 unit/mL), AA (100 µM), collagen (10 µg/mL), or
PAF (2 ng/mL) was added to trigger the aggregation. Values are expressed as mean ( SE from three to six separations. Aspirin acts as
a positive control. The accuracy of IC50 values are within (10%.
b
platelet activity prompted us to further investigate its
mechanism of action.
activity of broad spectrum as YC-1. Therefore, these
nine promising compounds were further tested for the
activation of sGC and inhibition of PDE5.
Thus far, from the antiplatelet activity of these YC-1
analogues, the following SARs can be established.
YC-1 analogues with a benzyl or phenyl group at the
1-position all exhibited potent antiplatelet activity of
broad spectrum. Replacing 5′-CH2OH with -CH2OCH3,
-CH2N(CH2CH3)2, or -CH3 maintained the potent activ-
ity. In contrast, replacing the 5′- CH2OH with -COOCH3
or -COOH resulted in a markedly decreased activity of
narrower spectrum.
Placing an F atom at the 6-position strengthened the
inhibition toward AA-, collagen-, and PAF-induced
aggregation. Conversely, the introduction of a 6-CH3 or
6-OCH3 caused no noticeable effect, while 5,6-OCH2O-
profoundly reduced the activity.
Replacing the furan ring of YC-1, 23, and 33 with a
benzene ring was generally deleterious to the antiplate-
let activity.
On the basis of the results of our antiplatelet screen-
ing, nine indazole derivatives (29-31, 42, 44-46, 50,
and 51) were identified with significant antiplatelet
Activa tion of sGC a n d In h ibition of P DE5. The
sGC activation data of the above nine compounds are
summarized in Table 2. Compounds 29, 30, 31, 44, and
45 all exhibited potent activities comparable to YC-1.
Furthermore, their inhibition activity toward PDE5 was
stronger than that of YC-1. Among them, compounds
31 and 45 demonstrated 10-fold more potency than
YC-1. However, the potency for sGC activation and
PDE5 inhibition did not parallel their antiplatelet
activities. The reason for such discrepancy might be
explainable by that a cGMP-independent mechanism is
involved in the antiplatelet activity.
Con clu sion
Starting from the lead compound, we retained the
pyrazole (B) ring as a nucleus and systematically
modified the peripheral 1-benzyl group, 5,6-substitu-
ents, and 5′-CH2OH, as well as varied the A and C rings,
to obtain several series of YC-1 analogues. After screen-

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 22 3749
Ta ble 2. Activation of sGC and Inhibition of PDE5 of 28-31,
42, 44-46, 50-51a
Therefore, compound 58 can serve as a lead compound
for antiplatelet agents. Details about the action mech-
anism of 58 will be published elsewhere.
Exp er im en ta l Section
Eva lu a tion of An tip la telet Aggr ega tion Activity. An-
tiplatelet aggregation activity was determined as previously
described.13,14
Mea su r em en t of Gu a n yla te Cycla se Activity. Guany-
late cyclase activity was determined as previously described.15
Mea su r em en t of P h osp h od iester a se Activity. Phos-
phodiesterase activity was determined as previously de-
scribed.16
sGC
PDE5
(IC50
(pmol cGMP/
,
compd
control
28 (YC-1) -H -H
29
30
31
44
45
46
42
50
51
R
R′
R′′
-CH2OH
-CH2OH
-CH2OH
min/mg protein) µM)
8.0 ( 3.4
44.6 ( 6.3c
37.2 ( 11.7a
35.1 ( 9.9a
39.9 ( 7.1b
32.0 ( 12.8b
41.8 ( 11.2c
22.1 ( 8.7
27.8 ( 7.0
30.9 ( 3.8
17.9 ( 1.1
ND
31.9
14.0
8.4
3.5
13.8
3.9
Su p p or tin g In for m a tion Ava ila ble: Synthetic methods,
yields, and complete physical and spectral data for compounds
7-65. This material is available free of charge via the Internet
at http://pubs.acs.org.
-H -F
-H -CH3
-H -OCH3 -CH2OH
-H -H
-H -F
-CH2OCH3
-CH2OCH3
-CH2OCH3
-CH2OH
-CH3
-CH2N(CH2CH3)2
Refer en ces
-H -CH3
9.3
36.3
22.3
63.0
63.0
(1) Ko, F. N.; Wu, C. C.; Kuo, S. C.; Lee, F. Y.; Teng, C. M. YC-1, a
novel activator of platelet guanylate cyclase. Blood 1994, 84,
4226-4233.
(2) Wu, C. C.; Ko, F. N.; Kuo, S. C.; Lee, F. Y.; Teng, C. M. YC-1
inhibited human platelet aggregation through NO-independent
activation of soluble guanylate cyclase. Br. J . Pharmacol. 1995,
116, 1973-1978.
(3) Friebe, A.; Koesling, D. Mechanism of YC-1-induced activation
of soluble guanylyl cyclase. Mol. Pharmacol. 1998, 53, 123-127.
(4) Sharma, V. S.; Magde, D.; Kharitonov, V. G.; Koesling, D. Soluble
guanylate cyclase: effect of YC-1 on ligation kinetics with carbon
monoxide. Biochem. Biophys. Res. Commun. 1999, 254, 188-
191.
(5) Galle, J .; Zabel, U.; Hubner, U.; Hatzelmann, A.; Wagner, B.;
Wanner, C.; Schmidt, H. H. Effects of the soluble guanylyl
cyclase activator, YC-1, on vascular tone, cyclic GMP levels and
phosphodiesterase activity. Br. J . Pharmacol. 1999, 127, 195-
203.
(6) Tsai, C. C.; Wang, L. Y.; Huang, L. J .; Teng, C. M.; Lin, T. P.;
Li, H. T.; Kuo, S. C. Synthesis and antiplatelet Activity of 1,3-
disubstituted 5-methylfuro[3,2-c]pyrazoles and related com-
pounds. Chin. Pharm. J . 1995, 47, 407-419.
(7) Kuo, S. C.; Lee, F. Y.; Teng, C. M. 1-(Substituted benzyl)-3-
(Substituted aryl) Condensed Pyrazole Derivatives and Processes
of Making the Same. U.S. Patent 5574168, 1996.
(8) Vu, T. K. H.; Hung, D. T.; Wheaton, V. I.; Coughlin, S. R.
Molecular cloning of a functional thrombin receptor reveals a
novel proteolytic mechanism of receptor activation. Cell 1991,
64, 1057-1068.
(9) Ishihara, H.; Zeng, D.; Connolly, A. J .; Tam, C.; Coughlin, S. R.
Antibodies to protease-activated receptor 3 inhibit activation of
mouse platelets by thrombin. Blood 1998, 91, 4152-4157.
(10) Kahn, M. L.; Zheng, Y. W.; Huang, W.; Bigornia, V.; Zeng, D.;
Moff, S.; Farese, R. V. J .; Tam, C.; Coughlin, S. R. A dual
thrombin receptor system for platelet activation. Nature 1998,
394, 690-694.
(11) Wu, C. C.; Huang, S. W.; Hwang, T. L.; Kuo, S. C.; Lee, F. Y.;
Teng, C. M. YD-3, a novel inhibitor of protease-induced platelet
activation. Br. J . Pharmacol. 2000, 130, 1289-1296.
(12) Xu, W. F.; Andersen, H.; Whitmore, T. E.; Presnell, S. R.; Yee,
D. P.; Ching, A.; Gilbert, T.; Davie, E. W.; Forster, D. C. Cloning
and characterization of human protease-activated receptor 4.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6642-6646.
(13) Born, G. V. R.; Cross, M. J . The aggregation of blood platelet. J .
Physiol. 1963, 168, 178-195.
(14) Teng, C. M.; Chen, W. Y.; Ko, W. C.; Ouyang, C. Antiplatelet
effect of butylidenephthalide. Biochem. Biophys. Acta 1987, 924,
375-382.
(15) Gerzer, R.; Hamet, P.; Ross, A. H.; Lawson, J . A.; Hardman, J .
G. Calcium-induced release from platelet membranes of fatty
acid that modulate soluble guanylate cyclase. J . Pharmacol. Exp.
Ther. 1983, 226, 180.
-H -H
-H -H
IBMX
a
sGC activity in the supernatant fraction of platelet homoge-
nate was determined in the presence of [R-32P]GTP and 82 µM of
test compound at 37 °C for 10 min. Values are presented as pmol
cGMP/min/mg protein. PDE activity fraction was determined in
the presence of [3H]cGMP and various concentrations of test
compound at 37 °C for 10 min. Values are presented as the
concentration (µM) by 50% inhibition of PDE5 (IC50). IBMX (3-
isobutyl-1-methylxanthine) acts as a positive control. ND: not
determined. *P < 0.05. **P < 0.01. ***P < 0.001.
ing for antiplatelet activity, SARs of these YC-1 ana-
logues were established in this study.
Five indazole compounds (29, 30, 31, 45, and 46) were
identified as promising antiplatelet candidates. To-
gether with YC-1, these compounds are currently under
pharmacological investigations.
Another important contribution of our work is the
discovery of compound 58, which demonstrated highly
selective and potent inhibition toward thrombin-induced
aggregation. Mechanism study revealed that compound
58 selectively inhibited platelet aggregation and phos-
phoinositide breakdown induced by thrombin without
affecting the proteolytic activity of thrombin. On the
other hand, compound 58 inhibited thrombin-induced
platelet activation only in the impairment of protease-
activated receptor type 1 (PAR1)8-10 in human platelets.
Previously,11 we suggested that 58 could interfere with
platelet activation elicited by thrombin through the
blockade of a non-PAR1 thrombin receptor. Recently,
after detailed study, we learned that compound 58
inhibited the aggregation of washed human platelets
stimulated by the protease-activated receptor type 4
(PAR4)12 agonist peptide GYPGKF (IC50 0.10 ( 0.01
µM). These recent results indicate that compound 58 is
a selective and potent inhibitor of PAR4-dependent
platelet activation. To our knowledge, compound 58 is
currently the only PAR4 antagonist available, and it
could act as a probe to investigate the functional role of
PAR4-mediated signaling in platelet and other cells.
(16) Hidaka, H.; Asano, T. Human blood platelet 3′,5′-cyclic nucleotid
phosphodiesterase. Isolation of low Km and high Km phosphodi-
esterase. Biochim. Biophys. Acta 1976, 429, 485-497.
J M010001H