Synthesis and antiplatelet activity of ethyl 4-(1-benzyl-1H-indazol-3-yl)benzoate (YD-3) derivatives

Article abstract of DOI:10.1016/j.bmc.2007.10.070

Previously, ethyl 4-(1-benzyl-1H-indazol-3-yl)benzoate (YD-3) was identified by us as the first non-peptide protease-activated receptor 4 (PAR4) antagonist. To continue on our development of novel anti-PAR4 agents, YD-3 was used as a lead compound and a series of its derivatives were synthesized and evaluated for their selective anti-PAR4 activity. Through structure-activity relationship (SAR) study, we identified the important functional groups contributing to anti-PAR4 activity, and these functional groups were kept intact during subsequent structural modification. Several new compounds with anti-PAR4 activity comparable to YD-3 were identified. Among them, ethyl 4-[1-(3-chlorobenzyl)-1H-indazol-3-yl]benzoate (33) showed the most potent inhibitory effect on PAR4-mediated platelet aggregation, ATP release, and P-selectin expression. On the other hand, ethyl 4-(1-phenyl-1H-indazol-3-yl)benzoate (83) exhibited dual inhibitory effects on PAR4 and thromboxane formation from arachidonic acid. The above findings can be used as guidelines for development of novel antiplatelet drug candidates.

Full text of DOI:10.1016/j.bmc.2007.10.070

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1262–1278
Synthesis and antiplatelet activity of ethyl
4-(1-benzyl-1H-indazol-3-yl)benzoate (YD-3) derivatives
Hua-Sin Chen,a Sheng-Chu Kuo,a Che-Ming Teng,b Fang-Yu Lee,c Jih-Pyang Wang,d
Yu-Chun Lee,a Chiung-Wen Kuo,a Ching-Che Huang,a
Chin-Chung Wue,* and Li-Jiau Huanga,*
aGraduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan
bPharmacological Institute, College of Medicine, National Taiwan University, Taiwan
cYung-Shin Pharmaceutical Industry Co., Ltd, Tachia, Taichung, Taiwan
dDepartment of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan
eGraduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan
Received 30 August 2007; revised 19 October 2007; accepted 20 October 2007
Available online 25 October 2007
Abstract—Previously, ethyl 4-(1-benzyl-1H-indazol-3-yl)benzoate (YD-3) was identified by us as the first non-peptide protease-acti-
vated receptor 4 (PAR4) antagonist. To continue on our development of novel anti-PAR4 agents, YD-3 was used as a lead
compound and a series of its derivatives were synthesized and evaluated for their selective anti-PAR4 activity. Through struc-
ture–activity relationship (SAR) study, we identified the important functional groups contributing to anti-PAR4 activity, and these
functional groups were kept intact during subsequent structural modification. Several new compounds with anti-PAR4 activity com-
parable to YD-3 were identified. Among them, ethyl 4-[1-(3-chlorobenzyl)-1H-indazol-3-yl]benzoate (33) showed the most potent
inhibitory effect on PAR4-mediated platelet aggregation, ATP release, and P-selectin expression. On the other hand, ethyl 4-(1-phe-
nyl-1H-indazol-3-yl)benzoate (83) exhibited dual inhibitory effects on PAR4 and thromboxane formation from arachidonic acid.
The above findings can be used as guidelines for development of novel antiplatelet drug candidates.
ꢀ 2008 Published by Elsevier Ltd.
1. Introduction
In the previous study, we showed that ethyl 4-(1-
benzyl-1H-indazol-3-yl)benzoate (YD-3) represents
the first selective non-peptide PAR4 antagonist.7
YD-3 selectively inhibited platelet aggregation caused
by PAR4 agonist peptides at submicromolar concen-
trations (IC50 * 0.13 lM). Although YD-3 alone
hardly affected platelet aggregation induced by
thrombin, it was able to act synergistically with
PAR1 antagonists to inhibit thrombin-induced plate-
let aggregation and P-selectin expression.8 In order
to identify the key function groups for antiplatelet
activity in YD-3, and to develop potent PAR4
antagonist, YD-3 was selected as lead compound.
A series of its derivatives were synthesized and eval-
uated for selective anti-PAR4 activity. Their struc-
ture–activity relationships (SAR) were established
and utilized in subsequent structural modification.
Compounds exhibiting superior and unique activities
were further examined for mechanism of action to
establish guidelines for further development of drug
candidates.
Thrombin-mediated platelet activation and aggregation
are critical for arterial thrombosis in unstable angina
and myocardial infarction.1 Human platelet expresses
two thrombin receptors PAR1, the principal receptor,
and PAR4, which serves as an auxiliary factor to signal
platelet activation.2–4 It has been reported that pharma-
cological or genetic blockade of PAR1 or PAR4 inhibits
arterial thrombosis in animal models.5,6 Moreover, be-
cause PAR antagonists do not affect the ability of
thrombin to cleave fibrinogen, they probably cause few-
er bleeding complications than thrombin inhibitors.
Therefore, PAR1 and PAR4 represent promising targets
for development of new antithrombotic drugs.
Keywords: Protease-activated receptor 4 (PAR4) antagonist; Struc-
ture–activity relationships (SAR); YD-3 derivatives.
*
Corresponding authors. Tel.: +886 7 3121101x2669; fax: +886 7
3114773 (C.-C.W); tel./fax: +886 22030760 (L.-J.H); e-mail
4
addresses: ccwu@kmu.edu.tw (C.-C.W); ljhuang@mail.cmu.edu.tw
(L.-J.H)
0968-0896/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.10.070

H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
1263
2. Results and discussion
followed by esterification with ethyl alcohol, to give the
corresponding key intermediates (17, 19) and target
compound 18.
2.1. Chemistry
The key intermediates (17, 19) for synthesis of target
compounds were prepared according to Scheme 1.9
First, 1-(N-morpholino)cyclohexene (1) was treated sep-
arately with two methoxy carbonylbenzoyl chlorides (2,
4) in the presence of Et3N, followed by acid treatment
(20% HCl) under reflux, to yield the corresponding ke-
tones (7, 8). Condensation of compounds 7 and 8 with
NH2NH2 Æ H2O afforded the corresponding tetrahydro-
indazoles (9, 10) that were then dehydrogenated over
Pd/C, under elevated temperature, to produce the corre-
sponding indazoles (11, 13). Subsequent methylation of
compound 11 with methyl iodide gave methyl 4-(1-
methyl-1H-indazol-3-yl)benzoate (12). The above-syn-
thesized compounds 11–13 were subjected to hydrolysis,
Scheme 2 illustrates the synthetic method for com-
pounds 30–41, 45–47, 50, 51, and 56–59. First, the solu-
tion of compound 17 in EtOH was benzylated with
benzyl chloride in the presence of EtONa to yield a mix-
ture of compounds 30 (mp 79–81 ꢁC, major product)
and 31 (mp 112–113 ꢁC, minor product). Mass spectra
[m/z, 356 (M+)] and elemental analysis data indicated
that they are N1- and N2-benzyl regioisomers with the
same molecular formula C23H20N2O2. Having the same
characteristic IR, NMR, and Mass spectra as previously
reported for YD-3,10 compound 30 was identified as the
N1-benzyl isomer. On the other hand, the structure of
compound 31 was confirmed by HMBC NMR spectra.
As indicated by the HMBC correlation in Figure 1,
O
O
O
O
N+
O
O
a
b
N
R4"
R3"
+
Cl
R4"
R4"
R3"
R3"
7 8
,
5 6
,
2 4
,
1
R4"
R4"
R3"
R3"
c
d
N
N
N
H
N
H
11 13
,
9 10
,
R4"
R3"
e
11
N
N
CH3
2 5 7 9
,
,
,
R3" = H, R4" = COOMe
4 6 8 10
,
,
,
R3" = COOMe, R4" = H
12
11
12
13
14
15
16
17
18
19
R3" = H, R4" = COOMe
R3" = H, R4" = COOMe
R4"
R4"
R3" = COOMe, R4" = H
R3"
R3"
R1 = H, R3" = H, R4" = COOH
R1 = CH3, R3" = H, R4" = COOH
R1 = H, R3" = COOH, R4" = H
R1 = H, R3" = H, R4" = COOEt
R1 = CH3, R3" = H, R4" = COOEt
R1 = H, R3" = COOEt, R4" = H
(1) f
h
11 12 13
,
,
(2) g
N
N
N
N
R1
R1
17 18 19
,
,
14 15 16
,
,
Scheme 1. Reagents and conditions: (a) Et3N, CHCl3, reflux, 3 h; (b) 20% HCl, reflux, 5 h; (c) 98% NH2NH2 Æ H2O, MeOH, reflux, 30 min; (d)
trans-decalin, 10% Pd/C, reflux, 24 h; (e) methyl iodide, MeONa, MeOH, rt, 12 h; (f) MeOH, 10% NaOH, reflux, 1 h; (g) 10% HCl; (h) 99.5% EtOH,
p-TSA, reflux, 2 h.

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H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
O
OEt
O
O
N
OEt
OEt
a
CH2Cl
+
+
N
N
N
N
N
H
30 YD-3
(
)
31
20
17
R4"
21 32
,
R = 2-Cl, R3" = H, R4" = COOEt
R = 3-Cl, R3" = H, R4" = COOEt
R = 4-Cl, R3" = H, R4" = COOEt
R = 2-F, R3" = H, R4" = COOEt
R = 3-F, R3" = H, R4" = COOEt
R = 4-F, R3" = H, R4" = COOEt
R = 2-OCH3, R3" = H, R4" = COOEt
R = 3-OCH3, R3" = H, R4" = COOEt
R = 4-OCH3, R3" = H, R4" = COOEt
R = H, R3" = COOEt, R4" = H
R4"
R3"
22 33
,
R3"
23 34
,
a
24 35
,
CH2Cl
+
N
25 36
,
N
N
R
26 37
,
N
H
27 38
,
28 39
,
R
20 29
32 41
17 19
,
29 40
,
20 41
,
17 R3" = H, R4" = COOEt
19
R3" = COOEt, R4" = H
O
OEt
42 45
,
Ar =
N
N
43 46
,
Ar =
Ar CH2Cl
N
N
CH2Ar
42 44
46 47
,
Ar =
N
45 47
a
O
N
O
N
OEt
OEt
Ar CH2OH
48 49
,
b
48 50
,
Ar =
Ar =
O
N
49 51
,
N
H
CH2Ar
50 51
,
17
O
R1I
52 55
a
O
OEt
52 56
,
R1 = C2H5
R1 = C3H7
53 57
,
54 58
,
R1 = CH(CH3)2
R1 = C4H9
N
N
R1
55 59
,
56 59
Scheme 2. Reagents and conditions: (a) EtONa, 99.5% EtOH, rt, 12 h; (b) 1,10-(azodicarbonyl)dipiperidine, tri-n-butylphosphine, toluene, reflux,
16 h.
the 3J-correlation from C3-carbon to N-CH2-, H-20, and
H-60 observed in compound 31 differed from the 3J-cor-
relation seen in compound 30.
the antiplatelet activity of the minor product 31 was
found to be extremely weak, the minor byproducts
obtained during synthesis of compounds 32–40 were nei-
ther purified nor tested for activities. Following the same
synthetic method, the intermediate 19 was benzylated
with benzyl chloride to afford the corresponding major
product 41. Similarly, intermediate 17 was alkylated
Next, the key intermediate 17 was subjected to alkyl-
ation with various substituted benzyl chlorides (21–29)
to yield corresponding major products (32–40). Since

H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
1265
COOCH2CH3
4'
5'
COOCH2CH3
3'
2'
6'
4'
5'
6'
1'
4
7
3'
2'
3a
3
N
1
5
1'
4
2
6
3a
3
N
5
6
N
7a
6"
CH2
CH2
1"
2"
2"
3"
2
N
1
7a
1"
6"
7
5"
4"
3"
5" 4"
31
30 YD-3
(
)
Compound 30: HMBC correlations
13C
Compound 31: HMBC correlations
1H 13C
1H
H-4
C-6, C-7a
H-4
C-6, C-7a
H-5
C-3a, C-7
H-5
C-3a, C-7
H-6
C-4, C-7a
H-6
C-4, C-7a
H-7
C-3a, C-5
H-7
C-3a, C-5
H-2", 6"
H-3", 5"
H-2', 6'
H-3', 5'
N-CH2-
-CH2-, C-6", 2", C-4"
C-1", C-5", 3", C-4"*
C-3, C-4', C-6', 2'
4'-COOCH2CH3, C-1', C-5', 3'
C-2", 6", C-7a
H-2", 6"
H-3", 5"
H-2', 6'
H-3', 5'
N-CH2-
-CH2-, C-6", 2", C-4"
C-1", C-5", 3", C-4"*
C-3, C-4', C-6', 2'
4'-COOCH2CH3, C-1', C-5', 3'
C-3, C-2", 6"
*: 2JCH correlation; others are 3JCH correlation
Figure 1. HMBC correlations of compounds 30 and 31.
with various picolyl chlorides (42–44) to obtain corre-
sponding picolyl derivatives (45–47). Alternatively, the
intermediate 17 was reacted with 2- or 3-hydroxymethyl
furan (48, 49) using Mitsunobu reaction to provide the
corresponding furyl derivatives (50, 51). Next, com-
pound 17 was treated with various alkyl halides
(52–55) to afford the corresponding major N1-alkyl
derivatives (56–59).
converted into the target compound 83 by oxidative
cyclization.
2.2. Antiplatelet activity
In washed human platelets, U46619 (2 lM, a stable
thromboxane A2 mimetic), collagen (10 lg/mL), throm-
bin (0.05 U/mL), SFLLRN (10 lM, a PAR1 agonist
peptide), and GYPGKF (1.5 mM, a PAR4 agonist pep-
tide) all elicited about 80–90% aggregation. The effect of
test compounds on platelet aggregation was assessed
and the results are summarized in Table 1. As indicated
in Table 1, the lead compound YD-3 demonstrated
selective and potent inhibitory activity against GYP-
GKF-induced platelet aggregation with an IC50 value
of 0.13 lM. However, it showed only weak inhibitory
effect (IC50 > 10 lM) toward platelet aggregation in-
duced by U46619, collagen, thrombin, and SFLLRN.
Such results agree with the data in the literature,7 and
reconfirmed that YD-3 is a selective and potent anti-
PAR4 agent.
Starting from a different synthetic approach, shown in
Scheme 3, compound 30 (YD-3) was first hydrolyzed,
with 10% NaOH, into a carboxylic acid (60) which
was converted into an acid chloride (61) by treatment
with SOCl2. The so-formed compound 61 was then
either esterified with various alkyl alcohols to produce
corresponding esters (62–64) or treated with various
amines to give amides (65–69). Moreover, compound
30 was reduced with Ca(BH4)2 into a carbinol derivative
(70) which was subsequently oxidized with MnO2 into
an aldehyde derivative (71). The so-formed aldehyde
(71) was either converted into a Schiff base (72) by treat-
ment with methylhydroxyamine, or was subjected to
condensation with malonic acid to yield an acrylic acid
derivative (73) which was subsequently esterified into
compound 74.
The replacement of the 400-ethoxycarbonyl group of YD-
3 with a methoxycarbonyl group (compound 62) re-
tained its potency. Its IC50 toward GYPGKF-induced
platelet aggregation raised from 0.13 to 0.56 lM, equiv-
alent to the decrease of potency to about 1/4 that of YD-
3. The attempt to replace the 400-ethoxycarbonyl moiety
of lead compound with different ester groups (63, 64)
raised the IC50 to greater than 10 lM toward platelet
aggregation caused by all the tested inducers. This
seemed to suggest that 400-ethoxycarbonyl group is the
optimized ester moiety for YD-3. The reposition of the
The 1-phenyl analog of YD-3, compound 83, was pre-
pared according to our previously reported method.10
As described in Scheme 4, a 4-ethoxycarbonylphenyl
phenyl ketone (79) was first prepared, which was then
transformed into a phenylhydrazone (82) by reaction
with phenylhydrazine. Following consecutive treatment
with Pb(OAc)4 and BF3 Æ Et2O, compound 82 was

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H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
O
OR
N
N
62
63
64
R = CH3
R = C3H7
R = CH(CH3)2
62 64
O
N
O
N
O
N
OCH2CH3
OH
Cl
d
O
N
NHR
(1) a
(2) b
c
65
66
67
68
69
R = H
N
N
N
R = CH3
e
N
R = C2H5
R = C3H7
R = CH(CH3)2
61
60
30 YD-3
(
)
65 69
f
NOCH3
N
O
N
OH
N
H
h
72
g
N
COOEt
COOH
N
i
N
j
N
N
71
70
N
N
74
73
Scheme 3. Reagents and conditions: (a) MeOH, 10% NaOH, reflux, 1 h; (b) 10% HCl; (c) SOCl2, CH2Cl2, reflux, 16 h; (d) substituted alcohol, reflux,
2 h; (e) substituted amine, rt, 30 min; (f) Ca(BH4)2, THF, reflux, 6 h; (g) MnO2, acetone, rt, 20 h; (h) O-methylhydroxylamine, 99.5% EtOH, reflux,
3 h; (i) malonic acid, pyridine, 80 ꢁC, 3 h; (j) 99.5% EtOH, p-TSA, reflux, 2 h.
ethoxycarbonyl group of YD-3 from 400- to 300-position
(41) drastically impaired its antiplatelet activity. Hydro-
lysis, or reduction, of the 400-ethoxycarbonyl group of
YD-3 into carboxylic acid (60) and 400-carbinol (70),
respectively, also lowered the antiplatelet activity. Fur-
thermore, the substitution of the 400-ethoxycarbonyl
group of YD-3 with amide groups (65–69) also remark-
ably decreased the antiplatelet activity. Among them,
compound 67 was the only one that retained selective
but relatively weak anti-PAR 4 activity. Next, the sub-
stitution of the 400-ethoxycarbonyl group of YD-3 with
400-CH@NOCH3 (72) or –CH@CH–COOCH2CH3 (74)
also decreased the antiplatelet activity.
The above SAR results indicated that the 400-ethoxycar-
bonyl group on YD-3 is the major contributing func-
tional group for its antiplatelet activity. Any
modification of this moiety of YD-3 lowered its anti-
platelet activity. Therefore, during subsequent structural
modification of the 1-benzyl group of YD-3, the 400-eth-
oxycarbonyl group was kept intact. Next, the removal of
the 1-benzyl group, or the aromatic moiety on the 1-ben-
zyl group, from YD-3 yielded compounds 17 and 18,
respectively, with selective but weaker anti-PAR4 activ-
ity. On the other hand, replacing the N1-methyl group of
compound 18 with N1-ethyl (56), N1-propyl (57), N1-iso-
propyl (58), and N1-butyl (59) groups all results in

H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
1267
O
O
O
a
b
CH3
+
O
Cl
CH3
OH
78
75
76
77
O
O
OEt
O
OEt
c
d
N
O
N
NH
HN
OEt
79
81
(
E
-form)
81
(Z-form)
O
N
OEt
O
Ac
OEt
O
e
f
N
N
N
82
83
Scheme 4. Reagents and conditions: (a) AlCl3, rt, 12 h; (b) HOAc, H2SO4, CrO3, 100 ꢁC, 4 h; (c) 99.5% EtOH, p-TSA, reflux, 2 h; (d)
phenylhydrazine, HOAc, 99.5% EtOH, reflux, 12 h; (e) Pb(OAc)4, CH2Cl2, 0–5 ꢁC, 30 min; (f) BF3 Æ Et2O, reflux, 1 h.
reduced activity. The removal of the –CH2– moiety from
1-benzyl group of YD-3 led to a 1-phenyl derivative (83)
with a shift in antiplatelet pattern from PAR4 specific
inhibition to dual inhibition against both collagen- and
GYPGKF-induced platelet aggregation. As compari-
son, the replacement of the 1-benzyl group with 1-picol-
yl group (45–47) or 1-furfuryl group (50, 51) maintained
the selective anti-PAR4 activity. The relocation of the
1-benzyl group to N2-position resulted in 2-benzyl deriv-
ative (31) with considerable loss of antiplatelet activity.
The above findings seem to suggest that the presence
of benzyl group at the N1-position of indazole ring is
important for optimum activity. Consequently, during
our subsequent structural modification, the benzyl
group was fixed at the N1-position while various substit-
uents were introduced into it.
comparable with YD-3. Finally, the introduction of
methoxy group onto the 1-benzyl group of YD-3 led
to 1-(methoxybenzyl) derivatives (38–40) with slightly
attenuated anti-PAR4 activity.
From the above SAR study, we have obtained the fol-
lowing important information: (1) Both the 400-ethoxy-
carbonyl and 1-benzyl groups on YD-3 were identified
as the important functional groups contributing to the
selective anti-PAR4 activity of YD-3 derivatives studied.
The relocation of either one of them led to significant
loss in activity. (2) The substitution of the 400-ethoxycar-
bonyl group on YD-3 with other esters, amides, carbox-
ylic acid, or carbinol moieties decreased its activity
significantly. (3) The introduction of chloro, fluoro
atom, or methoxy group onto the 1-benzyl group of
YD-3 maintained its selective and potent anti-PAR 4
activity, but led to considerable fluctuation in potency.
(4) The replacement of the 1-benzyl group of YD-3 with
1-alkyl, 1-picoyl, or 1-furfuryl group maintained the
selective anti-PAR4 activity. (5) The removal of the
1-benzyl group, or the phenyl moiety on the 1-benzyl
group, from YD-3 led to selective but weaker anti-
PAR4 activity. (6) The removal of the -CH2- moiety
from the 1-benzyl group of YD-3 afforded a 1-phenyl
derivative with shift in antiplatelet pattern from PAR4
specific inhibition to dual inhibition against collagen-
and GYPGKF-induced platelet aggregation.
First, the substitution of chloro atom onto different sites
of the 1-benzyl group led to 1-chlorobenzyl derivatives
(32–34) with selective and potent anti-PAR4 activity.
Among them, 1-(4-chlorobenzyl) derivative (34) was
found to be equally potent as YD-3, whereas the 1-(3-
chlorobenzyl) derivative (33), with an IC50 of 0.08 lM,
is more potent than the leading YD-3. Similar substitu-
tion of fluoro atom onto the 1-benzyl group afforded
1-(fluorobenzyl) derivatives (35–37) with selective and
potent anti-PAR4 activity. In particular, the activity of
1-(2-fluorobenzyl) derivative (35) was found to be

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H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
Table 1. The inhibitory effects of YD-3 and its derivatives on human platelet aggregation induced by U46619, collagen, thrombin, SFLLRN, and
GYPGKF
O
OEt
R4"
R3"
N
N
N
N
R1
17 18 30 32 41 45 47
,
,
,
,
,
,
31
50 51 56 60 62 70 72
,
,
,
,
74 83
,
Compound
R1
R030
R040
IC50 (lM)
U46619
Collagen
Thrombin
SFLLRN
GYPGKF
YD-3
62
63
64
41
60
65
66
67
68
69
70
72
74
17
18
56
57
58
59
83
45
46
47
50
51
31
32
33
34
35
36
37
38
39
40
Benzyl
Benzyl
H
H
H
H
COOC2H5
COOCH3
COOC3H7
COOCH(CH3)2
H
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.13 0.02
0.56 0.03
>10
Benzyl
Benzyl
>10
>10
>10
>10
Benzyl
Benzyl
COOC2H5
>10
>10
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
—
H
H
H
H
H
H
H
H
H
COOH
CONH2
>10
>10
Benzyl
Benzyl
>10
>10
CONHCH3
CONHC2H5
CONHC3H7
>10
Benzyl
Benzyl
>10
5.64 0.40
>10
>10
9.56 0.51
>10
>10
Benzyl
Benzyl
CONHCH(CH3)2
CH2OH
>10
>10
Benzyl
Benzyl
CH@NOCH3
CH@CHCOOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
—
>10
>10
>10
H
CH3
>10
>10
5.65 0.30
0.27 0.02
0.36 0.02
0.49 0.02
0.65 0.04
0.67 0.01
2.64 0.29
0.29 0.03
5.55 0.31
5.63 0.17
0.34 0.03
0.58 0.03
>10
C2H5
C3H7
>10
>10
CH(CH3)2
C4H9
>10
>10
Phenyl
4.81 0.14
>10
>10
20-Picoyl
30-Picoyl
40-Picoyl
20-Furyl
>10
>10
30-Furyl
>10
>10
—
20-Chlorobenzyl
30-Chlorobenzyl
40-Chlorobenzyl
20-Fluorobenzyl
30-Fluorobenzyl
40-Fluorobenzyl
20-Methoxybenzyl
30-Methoxybenzyl
40-Methoxybenzyl
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
>10
>10
0.23 0.05
0.08 0.01
0.12 0.01
0.11 0.00
0.27 0.03
0.19 0.01
0.47 0.01
0.26 0.01
0.62 0.03
>10
>10
>10
>10
>10
>10
>10
Human platelets were incubated with tested sample or 0.3% DMSO at 37 ꢁC for 3 min, then U46619 (2 lM), collagen (10 lg/mL), thrombin (0.05 U/
mL), SFLLRN (10 lM), or GYPGKF (1.5 mM) was added to trigger the aggregation. Values are presented as means SE.
YD-3: positive control.
In addition to platelet aggregation, activated platelets
also release the contents of their dense- (platelet agonists
such as ADP and serotonin) and a-granules (P-selectin,
fibrinogen, and growth factors), which are important for
thrombosis, inflammation, and atherogenesis.11 In the
present study, the secretion of dense- and a-granule con-
tents in platelets was evaluated by ATP release (which is
co-released with ADP from dense granules) and P-selec-
tin expression, respectively. YD-3 and three representa-
tive derivatives (compounds 33, 45, and 83) were

H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
1269
Table 4. Effects of YD-3, 33, 45, and 83 on the thromboxane B2 (TxB2)
formation in human platelets stimulated with arachidonic acid
tested for their effect on platelet granule secretion. Com-
pound 33 was chosen for its highest potency against
PAR4, while compound 45 was selected for its ease of
converting into an HCl salt which may expedite the dos-
age preparation process. Finally, compound 83 was se-
lected due to its exhibition of different antiplatelet
pattern. The inhibitory effect of YD-3, and compounds
33, 45, and 85 against PAR4-mediated ATP release is
summarized in Table 2. As shown, the inhibitory effect
of compound 33 at 0.1 and 0.2 lM concentration was
slightly better than that of YD-3, meanwhile, complete
inhibition of ATP release was observed in compound
45 at the concentration of 1 lM, and in compound 85
at 5 lM. In comparison, the inhibitory effect of the
above compounds against PAR4-mediated P-selectin
expression is shown in Table 3. As shown, the inhibitory
effect of compound 33 at 0.05 and 0.1 lM concentration
was also slightly better than that of YD-3. Similar to
their potency in their inhibition of ATP release, com-
plete inhibition of P-selectin expression was observed
in compound 45 at the concentration of 1 lM, and in
compound 83 at 5 lM.
TxB2
(ng/3 · 108 platelets)
Resting
Control
YD-3
33
0.52 0.04
1589.4 266.1
1408.3 266.2
1368.4 219.1
1013.7 301.4
24.4 4.4***
5 lM
5 lM
5 lM
5 lM
45
83
Washed human platelets were preincubated with DMSO or test com-
pounds at 37 ꢁC for 3 min and then arachidonic acid (150 lM) was
added. Reactions were terminated by EDTA (2 mM) and indometh-
acin (50 lM) 5 min after the addition of arachidonic acid. Values are
presented as means SEM (n = 3). ***P < 0.001 as compared with
control.
to anti-PAR4 effect, there is other effect of compound 83
in human platelets. It is known that platelet stimulation
by collagen is largely dependent on the release of arachi-
donic acid (AA) and its subsequent conversion via
cyclooxygenase to thromboxane A2, and therefore can
be prevented by cyclooxygenase inhibitors.12 To exam-
ine the effect of compound 83 on thromboxane forma-
tion in human platelets, we used exogenous AA as a
precursor, which circumvents receptor stimulation. As
shown in Table 4, compound 83 (5 lM) inhibited AA-
induced thromboxane formation by 98.5%. In parallel,
platelet aggregation caused by AA was also abolished
in the presence of compound 83 (data not shown). These
results suggest that compound 83 is able to inhibit the
metabolism of AA at the cyclooxygenase level. In con-
trast, YD-3, 33, and 45, even at the highest concentra-
tions used, had no effect on AA-induced thromboxane
formation and platelet aggregation, which indicated that
they are all pure PAR4 antagonists. The dual inhibition
of PAR4 and thromboxane formation by compound 83
may provide broader spectrum of antiplatelet activity
and represents a lead for the development of new anti-
platelet drugs.
Compound 83 inhibited both GYPGKF- and collagen-
induced platelet aggregation, suggesting that in addition
Table 2. Effects of YD-3, 33, 45, and 83 on ATP release from activated
platelets
Released ATP
(ng/3 · 108 platelets)
Resting
Control
YD-3
1.3 0.1
100.2 18.9
12.7 2.4***
74.4 14.7
8.9 3.6***
58.9 15.4
3.7 0.3***
1.8 0.4***
0.2 lM
0.1 lM
0.2 lM
0.1 lM
1 lM
33
45
83
5 lM
Washed human platelets were incubated with DMSO (vehicle control)
or test compounds at 37 ꢁC for 3 min, then GYPGKF (1.5 mM) was
added to trigger ATP release. Released ATP was measured by a
luciferase/luciferin kit as described in Section 4. Results are presented
as means SEM (n = 3). ***P < 0.001 as compared with the control.
3. Conclusion
Table 3. Effect of YD-3, 33, 45, and 83 on P-selectin expression
Through the SAR study of YD-3 derivatives, we have
identified the important functional groups contributing
to the selective and potent anti-PAR4 activity. We also
identified compound 33 as a more potent anti-PAR4
agent than YD-3. Furthermore, compound 83 was
found to exhibit dual inhibitory effect on PAR4 and
thromboxane formation. The above finding will provide
guidelines for development of novel antiplatelet drug
candidates.
% Positive P-selectin
expression
Resting
Control
YD-3
1.7 0.2
88.7 2.4
0.1 lM
0.05 lM
0.1 lM
0.05 lM
1 lM
7.7 0.8***
67.2 5.1**
5.2 2.0***
39.3 11.0**
1.2 0.6***
1.6 0.8***
33
45
83
5 lM
Washed human platelets were pre-incubated with DMSO or test
compounds for 3 min and thentreated with or without GYPGKF in
the presence of FITC-conjugated anti-CD62P for 15 min at room
temperature. The samples were then fixed and analyzed by flow
cytometry. The levels of P-selectin expression were expressed as the
percentages of cells positive for anti-CD62P. Results are presented as
means SEM. (n = 3). ***P < 0.001, **P < 0.01 as compared with the
control.
4. Experimental
All of the solvents and reagents were obtained commer-
cially and used without further purification. Reactions
were monitored by thin-layer chromatography, using
Merck plates with fluorescent indicator. Column chro-
matography was performed on silica gel.

1270
H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
Melting points were determined with a Yanaco MP-
500D melting point apparatus and are uncorrected. IR
spectra were recorded on Shimadzu IR-440 or Nicolet
Impact 400 FT-IR spectrophotometers as KBr pellets.
NMR spectra were obtained on a Bruker Avance
DPX-200 FT-NMR spectrometer in DMSO-d6 or
CDCl3. The following abbreviations are used: s, singlet;
d, doublet; dd, double doublet; t, triplet; q, quartet; m,
multiplet; and br, broad. MS spectra were measured
with an HP 5995 GC-MS instrument. The UV spectra
were recorded on a Shimadzu UV-160A UV–vis record-
ing spectrophotometer as methanolic solution. Elemen-
tal analyses (C, H, and N) were performed on a
Perkin-Elmer 2400 Series II CHNS/O analyzer or Ele-
mentar vario EL III Heraeus CHNOS Rapid F002
and the results were within 0.4% of the calculated
values.
4.2. Methyl 4-(1-methyl-1H-indazol-3-yl)benzoate (12)
Into the solution of compound 11 (1.0 g, 0.004 mol) in
absolute EtOH (50 mL) was added EtONa (0.9 g,
0.014 mol). The mixture was stirred at 25 5 ꢁC for
30 min. Methyl iodide (1.14 g, 0.008 mol) was added
dropwise, and the mixture was stirred at 25 5 ꢁC for
12 h. The solid precipitate so formed was directly ex-
tracted several times with CHCl3. The combined extract
was concentrated in vacuo for solvent removal. The res-
idue was chromatographed (silica gel-CHCl3) to afford
compound 12, yield 0.52 g, 48.9%; mp 114–116 ꢁC; MS
(EI, 70 eV): m/z 266 (M+); found: C, 72.18; H, 5.30;
N, 10.53. C15H12N2O2 requires: C, 72.16; H, 5.32; N,
10.52; UV kmax (loge): 211 (4.3), 318 (4.2); IR (KBr):
1
1609 (C@N), 1709 (C@O) cmꢀ1; H NMR (200 MHz,
DMSO-d6): d 3.88 (s, 3H), 4.13 (s, 3H), 7.24–7.31 (m,
1H), 7.44–7.51 (m, 1H), 7.72 (d, 1H, J = 8.5 Hz), 8.05–
8.09 (m, 2H), 8.11 (d, 1H, J = 5.7 Hz), 8.12–8.16 (m,
2H); 13C NMR (50 MHz, DMSO-d6): d 35.91, 52.37,
110.65, 120.96 (C · 2), 121.97, 126.62, 126.78 (C · 2),
128.57, 130.08 (C · 2), 138.10, 140.84, 141.53, 166.26.
4.1. Methyl 4-(1H-indazol-3-yl)benzoate (11)
(A) Into a solution of 1-(N-morpholino)cyclohexene
(1) (33.4 g, 0.2 mol) and triethylamine (28 mL) in
CHCl3 (100 mL) was added dropwise the solution of
terephthalic acid monomethyl ester chloride (2)
(19.9 g, 0.2 mol) in CHCl3 (40 mL) at 45 ꢁC. The reac-
tion mixture was allowed to react for 3 h. Then 20%
HCl (20 mL) was added, and the mixture was heated
under reflux for 5 h and then allowed to stand at
25 5 ꢁC. The CHCl3 layer was collected and washed
with H2O and dried over MgSO4 and then evapo-
rated. The residue was chromatographed (silica
gel-CHCl3/n-hexane = 1/2) to afford methyl 4-[(2-oxo-
4.3. Methyl 3-(1H-indazol-3-yl)benzoate (13)
(A) Into a solution of mono-methyl isophthalate (3)
(10.0 g, 0.053 mol), thionyl chloride (6 mL, 0.082 mol),
and dimethylformamide (DMF) (0.5 mL) in CH2Cl2
(100 mL) at reflux temperature to afford methyl 3-(chol-
rocarbonyl)benzoate (4).
(B) Into a solution of 1-(N-morpholino)cyclohexene (1)
(10.0 g, 0.06 mol), triethylamine (60 mL), compound 4
(19.9 g, 0.2 mol), and CHCl3 (150 mL) were allowed to
react as in the preparation of compound 7 to afford
methyl 3-[(2-oxocyclohexyl)carbonyl]benzoate (8).
cyclohexyl)carbonyl]benzoate
86.5%; mp 91–92 ꢁC.
(7),
yield
45.19 g,
(B) Compound 7 (15.8 g, 0.06 mol) was dissolved in
MeOH (100 mL), then 3.4 mL of 98% NH2NH2 Æ H2O
was added dropwise at reflux. The mixture was allowed
to react for 30 min and was then concentrated in vacuo
until there is about 35 mL solution left. The residue was
allowed to precipitate during cooling. The solid precipi-
tate was washed with EtOH and dried to yield methyl 4-
(4,5,6,7-tetrahydro-1H-indazol-3-yl)benzoate (9), yield
13.84 g, 90.1%; mp 184–187 ꢁC.
(C) Into a solution of compound 8 (5.0 g, 0.0192 mol),
98% NH2NH2 Æ H2O (3.4 mL), and MeOH (100 mL)
were allowed to react as in the preparation of compound
9 to afford 3-(4,5,6,7-tetrahydro-1H-indazol-3-yl)benzo-
ate (10), yield 4.5 g, 90.0%; mp 133–136 ꢁC.
(D) Into a solution of compound 10 (4.0 g, 0.058 mol),
trans-decalin (100 mL), and 10% Pd/C (5.0 g) were
allowed to react as in the preparation of compound 11
to afford methyl 3-(1H-indazol-3-yl)benzoate (13), yield
9.85 g, 82.0%; mp 103–104 ꢁC.
(C) Into the solution of compound 9 (13.0 g, 0.05 mol)
in trans-decahydronaphthalene (trans-decalin) was
added 10% Pd/C (2.7 g), and the mixture was heated un-
der reflux for 24 h and then concentrated to dry in vacuo
in oil bath. The residue was chromatographed (silica gel-
ethyl acetate/n-hexane = 1/3) to afford compound 11,
yield 10.42 g, 82.7%; mp 175–178 ꢁC; MS (EI, 70 eV):
m/z 252 (M+); found: C, 71.40; H, 4.77; N,
11.11. C15H12N2O2 requires: C, 71.42; H, 4.79; N,
11.10; UV kmax (loge): 211 (4.3), 316 (4.1); IR (KBr):
4.4. 4-(1H-Indazol-3-yl)benzoic acid (14)
Compound 11 (1.0 g, 0.004 mol) in MeOH (5 mL) was
added 10% NaOH (20 mL). The mixture was stirred at
25 5 ꢁC for 30 min. The mixture was heated under re-
flux for 1 h and then cooled to 5 1 ꢁC. Then 10% HCl
was added dropwise until white solid was no longer pre-
cipitated in ice-bath. The solid was washed several times
with H2O to afford compound 14, yield 0.83 g, 87.2%;
mp 231–234 ꢁC; MS (EI, 70 eV): m/z 238 (M+); found:
C, 70.59; H, 4.24; N, 11.78. C15H12N2O2 requires: C,
70.58; H, 4.23; N, 11.76; UV kmax (loge): 211 (4.2),
317 (4.1); IR (KBr): 1616 (C@N), 1684 (C@O), 3014
1609 (C@N), 1697 (C@O), 3306 (–NH) cmꢀ1 1H
;
NMR (200 MHz, DMSO-d6): d3.88 (s, 3H), 7.20–7.28
(m, 1H), 7.34–7.46 (m, 1H), 7.62 (d, 1H, J = 8.2 Hz),
8.08 (d, 2H, J = 8.6 Hz), 8.13 (d, 1H, J = 9.6 Hz), 8.17
(d, 2H, J = 8.6 Hz), 13.48 (br, 1H); 13C NMR
(50 MHz, DMSO-d6): d 52.36, 111.06, 120.35, 120.79,
121.78, 126.55, 126.86 (C · 2), 128.566, 130.05 (C · 2),
138.59, 141.89, 142.15, 166.30.
(–OH), 3231 (–NH) cmꢀ1
;
1H NMR (200 MHz,

H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
1271
DMSO-d6): d 7.22–7.26 (m, 1H), 7.40–7.44 (m, 1H), 7.62
(d, 1H, J = 8.4 Hz), 8.07–8.16 (m, 5H), 13.42 (br, 1H);
13C NMR (50 MHz, DMSO-d6): d 111.30, 120.63,
121.08, 121.98, 126.78, 127.03 (C · 2), 130.06, 130.46
(C · 2), 138.44, 142.17, 142.58, 167.65.
18, yield 0.73 g, 65.2%; mp 63–64 ꢁC; MS (EI, 70 eV): m/z
280 (M+); found: C, 72.87; H, 5.73; N,
9.98. C15H12N2O2 requires: C, 72.84; H, 5.75; N, 9.99;
UV kmax (loge): 210 (4.3), 318 (4.2); IR (KBr): 1607
(C@N), 1707 (C@O) cmꢀ1
;
1H NMR (200 MHz,
DMSO-d6): d 1.34 (t, 3H, J = 7.0 Hz), 4.13 (s, 3H),
4.33 (q, 2H, J = 7.0 Hz), 7.24–7.31 (m, 1H), 7.44–7.52
(m, 1H), 7.73 (d, 1H, J = 8.0 Hz), 8.07 (d, 2H,
J = 8.3 Hz), 8.15 (d, 3H, J = 8.3 Hz); 13C NMR
(50 MHz, DMSO-d6): d 14.44, 60.98, 110.67, 120.97
(C · 2), 121.98, 126.63, 126.77 (C · 3), 128.83, 130.05
(C · 2), 138.05, 140.86, 141.52, 165.76.
4.5. 4-(1-Methyl-1H-indazol-3-yl)benzoic acid (15)
Compound 12 (1.0 g, 0.0038 mol) was allowed to react as
in the preparation of compound 14 to afford compound
15, yield 0.82 g, 85.6%; mp 234–237 ꢁC; MS (EI, 70 eV):
m/z 252 (M+); found: C, 71.43; H, 4.78; N, 10.99.
C15H12N2O2 requires: C, 71.42; H, 4.80; N, 11.03; UV
k
max (loge): 211 (4.3), 319 (4.2); IR (KBr): 1609 (C@N),
4.9. Ethyl 3-(1H-indazol-3-yl)benzoate (19)
1
1684 (C@O), 2983 (–OH) cmꢀ1; H NMR (200 MHz,
DMSO-d6): d 4.12 (s, 3H), 7.23–7.30 (m, 1H), 7.43–7.51
(m, 1H), 7.71 (d, 1H, J = 8.5 Hz), 8.04–8.15 (m, 5H),
12.97 (br, 1H); 13C NMR (50 MHz, DMSO-d6): d
35.88, 110.61, 120.99, 121.90, 126.54, 126.67 (C · 3),
129.79, 130.23 (C · 2), 137.71, 141.04, 141.51, 167.35.
Compound 16 (1.5 g, 0.007 mol) was allowed to react as
in the preparation of compound 17 to afford compound
19, yield 0.61 g, 36.0%; mp 112–113 ꢁC; MS (EI, 70 eV):
m/z 266 (M+); found: C, 72.20; H, 5.31; N, 10.54.
C15H12N2O2 requires: C, 72.16; H, 5.30; N, 10.52; UV
kmax (loge): 215 (4.5), 306 (4.7); IR (KBr): 1719
1
4.6. 3-(1H-Indazol-3-yl)benzoic acid (16)
(C@O) cmꢀ1; H NMR (200 MHz, DMSO-d6): d 1.34
(t, 3H, J = 7.0 Hz), 4.34 (q, 2H, J = 7.0 Hz), 7.24 (t,
1H, J = 7.5 Hz), 7.42 (t, 1H, J = 7.6 Hz), 7.60–7.71 (m,
2H), 7.95–8.08 (m, 2H), 8.28 (d, 1H, J = 7.8 Hz), 8.57
(s, 1H), 13.39 (br, 1H); 13C NMR (50 MHz, DMSO-
d6): d 14.41, 61.15, 111.06, 120.15, 120.52, 121.64,
126.52, 127.42 (C · 2), 128.36, 129.71, 130.76, 131.38,
134.46, 141.88, 142.29, 165.88.
Compound 13 (1.0 g, 0.004 mol) was allowed to react as
in the preparation of compound 14 to afford compound
16, yield 0.8 g, 93.0%; mp 187–190 ꢁC; MS (EI, 70 eV):
m/z 238 (M+); found: C, 70.60; H, 4.20; N, 11.79.
C15H12N2O2 requires: C, 70.58; H, 4.23; N, 11.76; UV
kmax (loge): 215 (4.5), 306 (4.7); IR (KBr): 1684
(C@O), 3449 (–OH) cmꢀ1
;
1H NMR (200 MHz,
DMSO-d6): d 7.23 (t, 1H, J = 7.5 Hz), 7.42 (t, 1H,
J = 7.5 Hz), 7.60–7.68 (m, 2H), 7.95–8.07 (m, 2H), 8.23
(d, 1H, J = 7.7 Hz), 8.57 (s, 1H), 13.42 (br, 1H); 13C
NMR (50 MHz, DMSO-d6): d 111.05, 120.16, 120.53,
121.57, 126.46, 127.51, 128.59, 129.49, 130.89, 132.09,
134.26, 141.88, 142.47, 167.61.
4.10. Ethyl 4-(1-benzyl-1H-indazol-3-yl)benzoate (30;
YD-3) and ethyl 4-(2-benzyl-2H-indazol-3-yl)benzoate
(31)
Into the solution of compound 17 (4.0 g, 0.015 mol) in
absolute EtOH (150 mL) was added EtONa (3.5 g,
0.052 mol). The mixture was stirred at 25 5 ꢁC for
30 min. Then benzyl chloride (20) (10.1 g, 0.08 mol)
was added dropwise, and the mixture was stirred at
25 5 ꢁC for 12 h. The solid precipitate so formed was
directly extracted several times with CHCl3. The com-
bined extract was concentrated in vacuo for solvent
removal. The residue was chromatographed (silica gel-
CHCl3) to afford compounds 30 (YD-3) and 31. YD-3,
yield 2.90 g, 54.3%; mp 79–81 ꢁC; MS (EI, 70 eV): m/z
356 (M+); found: C, 77.48; H, 5.68; N, 7.88.
C23H20N2O2 requires: C, 77.51; H, 5.66; N, 7.86; UV
kmax (loge): 211 (4.7), 320 (4.6); IR (KBr): 1611
4.7. Ethyl 4-(1H-indazol-3-yl)benzoate (17)
Compound 14 (1.0 g, 0.0042 mol) and toluene-4-sulfonic
acid monohydrate (p-TSA) (0.5 g, 0.0026 mol) were dis-
solved in absolute EtOH (100 mL). The solution was
heated under reflux for 2 h and then allowed to stand
at 25 5 ꢁC. The solution was chromatographed (silica
gel-ethyl acetate/n-hexane = 2/3) to afford compound
17, yield 0.94 g, 84.1%; mp 133–135 ꢁC; MS (EI,
70 eV): m/z 266 (M+); found: C, 72.18; H, 5.29; N,
10.52. C15H12N2O2 requires: C, 72.16; H, 5.30; N,
10.52; UV kmax (loge): 211 (4.2), 317 (4.0); IR (KBr):
(C@N), 1721 (C@O) cmꢀ1
;
1H NMR (200 MHz,
1609 (C@N), 1697 (C@O), 3306 (–NH) cmꢀ1
;
NMR (200 MHz, DMSO-d6): 1.33 (t, 3H,
1H
DMSO-d6): d 1.32 (t, 3H, J = 7.1 Hz), 4.33 (q, 2H,
J = 7.1 Hz), 5.75 (s, 2H), 7.22–7.30 (m, 6H), 7.44 (t,
1H, J = 7.0 Hz), 7.78 (d, 1H, J = 8.5 Hz,), 8.06–8.10
(m, 2H), 8.12 (d, 1H, J = 7.5 Hz), 8.15–8.18 (m, 2H);
13C NMR (50 MHz, DMSO-d6): d 14.40, 52.33, 60.97,
110.77, 121.14, 121.30, 122.14, 126.85, 126.95 (C · 2),
127.60 (C · 2), 127.83, 128.82 (C · 2), 129.03, 130.03
(C · 2), 137.41, 137.91, 141.23, 141.71, 165.75. Com-
pound 31, yield 0.47 g, 8.8%; mp 112–113 ꢁC; MS (EI,
70 eV): m/z 356 (M+); found: C, 77.49; H, 5.65; N,
7.87. C23H20N2O2 requires: C, 77.51; H, 5.66; N, 7.86;
UV kmax (loge): 211 (4.7), 321 (4.3); IR (KBr): 1607
d
J = 7.0 Hz), 4.32 (q, 2H, J = 7.0 Hz), 7.20–7.27
(m,1H), 7.38–7.45 (m, 1H), 7.62 (d, 1H, J = 8.4 Hz),
8.08 (d, 2H, J = 8.4 Hz), 8.12 (d, 1H, J = 9.2 Hz), 8.16
(d, 2H, J = 8.4 Hz), 13.47 (br, 1H); 13C NMR
(50 MHz, DMSO-d6): d 14.41, 60.94, 111.05, 120.37,
120.78, 121.77, 126.53, 126.84 (C · 2), 128.86, 130.00
(C · 2), 138.53, 141.91, 142.21, 165.80.
4.8. Ethyl 4-(1-methyl-1H-indazol-3-yl)benzoate (18)
Compound 15 (1.0 g, 0.004 mol) was allowed to react as
in the preparation of compound 17 to afford compound
(C@N), 1708 (C@O) cmꢀ1
;
1H NMR (200 MHz,
DMSO-d6): d 1.31 (t, 3H, J = 7.1 Hz), 4.33 (q, 2H,

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H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
J = 7.1 Hz), 5.70 (s, 2H), 6.98–7.04 (m, 2H), 7.09–7.14
(m, 1H), 7.19–7.24 (m, 3H), 7.27–7.34 (m,1H), 7.56 (d,
1H, J = 8.4 Hz), 7.69 (d, 3H, J = 8.3 Hz), 8.09 (d, 2H,
J = 8.3 Hz); 13C NMR (50 MHz, DMSO-d6): d 14.35,
54.39, 61.20, 117.51, 120.12, 121.19, 122.62, 126.52,
127.15 (C · 2), 127.84, 128.78 (C · 2), 129.84 (C · 2),
130.07 (C · 3), 133.75, 134.62, 137.04, 147.80, 165.48.
(C · 2), 129.08, 129.49 (C · 2), 130.04 (C · 2), 132.53,
136.43, 137.80, 141.22, 141.92, 165.74.
4.11.4. Ethyl 4-[1-(2-fluorobenzyl)-1H-indazol-3-yl]ben-
zoate (35). Compound 17 (4.0 g, 0.015 mol), EtONa
(3.5 g, 0.052 mol), and 2-fluorobenzyl chloride (24)
(21.7 mL, 0.15 mol) were allowed to react as in the prep-
aration of compound 30 (YD-3) to afford compound 35,
yield 3.19 g, 56.9%; mp 94–96 ꢁC; MS (EI, 70 eV): m/z
374 (M+); C23H19FN2O2; UV kmax (loge): 211 (4.7),
4.11. Preparation of ethyl 4-[1-(substituted benzyl)-1H-
indazol-3-yl]benzoate (32–40)
320 (4.5); IR (KBr): 1612 (C@N), 1719 (C@O) cmꢀ1
;
4.11.1. Ethyl 4-[1-(2-chlorobenzyl)-1H-indazol-3-yl]ben-
zoate (32). Compound 17 (4.0 g, 0.015 mol), EtONa
(3.5 g, 0.052 mol), and 2-chlorobenzyl chloride (21)
(24.3 mL, 0.15 mol) were allowed to react as in the prep-
aration of compound 30 (YD-3) to afford compound 32,
yield 3.20 g, 54.6%; mp 93–95 ꢁC; MS (EI, 70 eV): m/z
390.5 (M+); found: C, 70.70; H, 4.89; N, 7.15.
C23H19ClN2O2 requires: C, 70.68; H, 4.90; N, 7.17;
UV kmax (loge): 211 (4.7), 321 (4.6); IR (KBr): 1609
1H NMR (200 MHz, DMSO-d6): d 1.33 (t, 3H,
J = 7.1 Hz), 4.33 (q, 2H, J = 7.1 Hz), 5.80 (s, 2H),
7.11–7.32 (m, 5H), 7.44–7.52 (m, 1H), 7.80 (d, 1H,
J = 8.5 Hz), 8.05–8.16 (m, 5H); 13C NMR (50 MHz,
DMSO-d6): d 14.41, 46.27, 60.99, 110.64, 115.74,
121.16 (C · 2), 122.23, 124.17, 124.93, 126.99 (C · 3),
129.10, 130.03, 130.18 (C · 2), 130.27, 137.77, 141.34,
141.99, 160.21, 165.74.
(C@N), 1711 (C@O) cmꢀ1
;
1H NMR (200 MHz,
4.11.5. Ethyl 4-[1-(3-fluorobenzyl)-1H-indazol-3-yl]ben-
zoate (36). Compound 17 (4.0 g, 0.015 mol), EtONa
(3.5 g, 0.052 mol), and 3-fluorobenzyl chloride (25)
(21.7 mL, 0.15 mol) were allowed to react as in the prep-
aration of compound 30 (YD-3) to afford compound 36,
yield 2.97 g, 53.0%; mp 86–87 ꢁC; MS (EI, 70 eV): m/z
374 (M+); C23H19FN2O2; UV kmax (loge): 211 (4.7),
DMSO-d6): d 1.32 (t, 3H, J = 7.1 Hz), 4.32 (q, 2H,
J = 7.1 Hz), 5.82 (s, 2H), 6.87 (dd, 1H, J = 1.7,
7.6 Hz), 7.18–7.34 (m, 3H), 7.42–7.51 (m, 2H), 7.74 (d,
1H, J = 8.5 Hz), 8.04–8.18 (m, 5H); 13C NMR
(50 MHz, DMSO-d6): d 14.41, 49.96, 60.98, 110.65,
121.16 (C · 2), 122.27, 127.00 (C · 3), 127.74, 129.11,
129.38, 129.72 (C · 2), 130.02 (C · 2), 132.23, 134.74,
137.73, 141.62, 142.16, 165.72.
320 (4.5); IR (KBr): 1611 (C@N), 1707 (C@O) cmꢀ1
;
1H NMR (200 MHz, DMSO-d6): d 1.33 (t, 3H,
J = 7.1 Hz), 4.33 (q, 2H, J = 7.1 Hz), 5.78 (s, 2H),
7.07–7.14 (m, 3H), 7.24–7.36 (m, 2H), 7.43–7.51 (m,
1H), 7.82 (d, 1H, J = 8.5 Hz), 8.06–8.18 (m, 5H); 13C
NMR (50 MHz, DMSO-d6): d 14.41, 51.64, 60.99,
110.71, 114.33, 114.76, 121.21, 121.27, 122.27, 123.64,
127.02 (C · 3), 129.11, 130.05 (C · 2), 130.93, 137.78,
140.23, 141.29, 141.99, 162.37, 165.74.
4.11.2. Ethyl 4-[1-(3-chlorobenzyl)-1H-indazol-3-yl]ben-
zoate (33). Compound 17 (4.0 g, 0.015 mol), EtONa
(3.5 g, 0.052 mol), and 3-chlorobenzyl chloride (22)
(24.3 mL, 0.15 mol) were allowed to react as in the prep-
aration of compound 30 (YD-3) to afford compound 33,
yield 2.88 g, 49.2%; mp 76–77 ꢁC; MS (EI, 70 eV): m/z
390.5 (M+); found: C, 70.71; H, 4.89; N, 7.16.
C23H19ClN2O2 requires: C, 70.68; H, 4.90; N, 7.17;
UV kmax (loge): 211 (4.6), 320 (4.5); IR (KBr): 1609
4.11.6. Ethyl 4-[1-(4-fluorobenzyl)-1H-indazol-3-yl]ben-
zoate (37). Compound 17 (4.0 g, 0.015 mol), EtONa
(3.5 g, 0.052 mol), and 4-fluorobenzyl chloride (26)
(21.7 mL, 0.15 mol) were allowed to react as in the prep-
aration of compound 30 (YD-3) to afford compound 37,
yield 3.13 g, 55.8%; mp 79–80 ꢁC; MS (EI, 70 eV): m/z
374 (M+); C23H19FN2O2; UV kmax (loge): 211 (4.6),
(C@N), 1713 (C@O) cmꢀ1
;
1H NMR (200 MHz,
DMSO-d6): d 1.33 (t, 3H, J = 7.0 Hz), 4.33 (q, 2H,
J = 7.0 Hz), 5.78 (s, 2H), 7.20–7.37 (m, 5H), 7.44–7.52
(m, 1H), 7.83 (d, 1H, J = 8.4 Hz), 8.06–8.17 (m, 5H);
13C NMR (50 MHz, DMSO-d6): d 14.42, 51.52, 61.01,
110.71, 121.25 (C · 2), 122.32, 126.27, 127.03 (C · 3),
127.45, 127.86, 129.12, 130.07 (C · 2), 130.83, 133.40,
137.75, 139.91, 141.28, 142.02, 165.74.
320 (4.4); IR (KBr): 1609 (C@N), 1711 (C@O) cmꢀ1
;
1H NMR (200 MHz, DMSO-d6): d 1.32 (t, 3H,
J = 7.1 Hz), 4.32 (q, 2H, J = 7.1 Hz), 5.74 (s, 2H),
7.08–7.22 (m, 2H), 7.26–7.39 (m, 3H), 7.41–7.49 (m,
1H), 7.80 (d, 1H, J = 8.5 Hz), 8.06–8.17 (m, 5H); 13C
NMR (50 MHz, DMSO-d6): d 14.39, 51.52, 60.97,
110.72, 115.63 (C · 2), 121.16, 121.30, 122.18, 126.96
(C · 3), 129.06, 129.76 (C · 2), 130.02 (C · 2), 133.62,
137.85, 141.14, 141.84, 161.83, 165.74.
4.11.3. Ethyl 4-[1-(4-chlorobenzyl)-1H-indazol-3-yl]ben-
zoate (34). Compound 17 (4.0 g, 0.015 mol), EtONa
(3.5 g, 0.052 mol), and 4-chlorobenzyl chloride (23)
(24.3 mL, 0.15 mol) were allowed to react as in the prep-
aration of compound 30 (YD-3) to afford compound 34,
yield 3.39 g, 57.9%; mp 89–91 ꢁC; MS (EI, 70 eV): m/z
390.5 (M+); found: C, 70.70; H, 4.88; N, 7.18.
C23H19ClN2O2 requires: C, 70.68; H, 4.90; N, 7.17;
UV kmax (loge): 211 (4.7), 320 (4.5); IR (KBr): 1611
4.11.7.
Ethyl
4-[1-(2-methoxybenzyl)-1H-indazol-3-
yl]benzoate (38). Compound 17 (4.0 g, 0.015 mol),
EtONa (3.5 g, 0.052 mol), and 2-methoxybenzyl chlo-
ride (27) (21 mL, 0.15 mol) were allowed to react as in
the preparation of compound 30 (YD-3) to afford com-
pound 38, yield 3.42 g, 56.9%; mp 80–81 ꢁC; MS (EI,
70 eV): m/z 386 (M+); found: C, 74.67; H, 5.72; N,
7.22. C24H22N2O3 requires: C, 74.59; H, 5.74; N, 7.25;
UV kmax (loge): 211 (4.7), 320 (4.5); IR (KBr): 1611
(C@N), 1719 (C@O) cmꢀ1
;
1H NMR (200 MHz,
DMSO-d6): d 1.33 (t, 3H, J = 7.1 Hz), 4.33 (q, 2H,
J = 7.1 Hz), 5.76 (s, 2H), 7.24–7.39 (m, 5H), 7.43–7.50
(m, 1H), 7.80 (d, 1H, J = 8.4 Hz), 8.06–8.17 (m, 5H);
13C NMR (50 MHz, DMSO-d6): d 14.42, 51.51, 61.00,
110.72, 121.20, 121.28, 122.25, 126.99 (C · 3), 128.84

H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
1273
(C@N), 1713 (C@O) cmꢀ1
;
1H NMR (200 MHz,
1H, J = 8.4 Hz), 7.98 (d, 1H, J = 7.8 Hz), 8.07 (d, 1H,
J = 8.02 Hz), 8.25 (d, 1H, 7.8 Hz), 8.55 (s, 1H); 13C
NMR (50 MHz, DMSO-d6): d 14.41, 52.23, 61.16,
110.76, 120.89, 121.08, 122.02, 126.86, 127.30, 127.55
(C · 2), 127.80, 128.56, 128.83 (C · 2), 129.76, 130.83,
131.50, 133.89, 137.53, 141.22, 141.84, 165.83.
DMSO-d6): d 1.28 (t, 3H, J = 7.0 Hz), 3.77 (s, 3H),
4.28 (q, 2H, J = 7.0 Hz), 5.62 (s, 2H), 6.76–6.78 (m,
2H), 6.96 (d, 1H, J = 8.2 Hz), 7.18–7.25 (m, 2H), 7.36–
7.40 (m, 1H), 7.65 (d, 1H, J = 8.3 Hz), 8.00–8.11 (m,
5H); 13C NMR (50 MHz, DMSO-d6): d 14.61, 47.59,
55.88, 61.19, 111.04, 111.37, 120.83, 121.28 (C · 2),
122.25, 125.32, 126.96, 127.11 (C · 2), 128.75, 129.16,
129.52, 130.23 (C · 2), 138.17, 141.70, 141.82, 156.96,
165.98.
4.13. Ethyl 4-[1-(pyridin-2-ylmethyl)-1H-indazol-3-
yl]benzoate (45)
Compound 17 (4.0 g, 0.015 mol), EtONa (4.2 g,
0.062 mol), and 2-picolyl chloride hydrochloride (42)
(4.92 g, 0.03 mol) were allowed to react as in the prepa-
ration of compound 30 (YD-3) to afford compound 45,
yield 1.4 g, 26.3%; mp 80–82 ꢁC; MS (EI, 70 eV): m/z
357 (M+); found: C, 73.99; H, 5.34; N, 11.73.
C22H19N3O2 requires: C, 73.93; H, 5.36; N, 11.76; UV
kmax (loge): 211 (4.6), 320 (4.5); IR (KBr): 1611
4.11.8.
Ethyl
4-[1-(3-methoxybenzyl)-1H-indazol-3-
yl]benzoate (39). Compound 17 (4.0 g, 0.015 mol),
EtONa (3.5 g, 0.052 mol), and 3-methoxybenzyl chlo-
ride (28) (21 mL, 0.15 mol) were allowed to react as in
the preparation of compound 30 (YD-3) to afford com-
pound 39, yield 2.87 g, 49.5%; mp 93–94 ꢁC; MS (EI,
70 eV): m/z 386 (M+); found: C, 74.54; H, 5.71; N,
7.23. C24H22N2O3 requires: C, 74.59; H, 5.74; N, 7.25;
UV kmax (loge): 211 (4.7), 320 (4.4); IR (KBr): 1609
(C@N), 1709 (C@O) cmꢀ1
;
1H NMR (200 MHz,
DMSO-d6): d 1.32 (t, 3H, J = 7.1 Hz), 4.32 (q, 2H,
J = 7.1 Hz), 5.84 (s, 2H), 7.10 (d, 1H, J = 7.8 Hz),
7.24–7.31 (m, 2H), 7.41–7.45 (m, 1H), 7.67–7.76 (m,
2H), 8.07 (d, 2H, J = 8.5 Hz), 8.14 (d, 3H, J = 8.5 Hz),
8.49 (d, 1H, J = 4.7 Hz); 13C NMR (50 MHz, DMSO-
d6): d 14.41, 54.21, 61.00, 110.92, 121.09, 121.23,
121.82, 122.19, 123.09, 126.96 (C · 3), 129.05, 130.03
(C · 2), 137.39, 137.85, 141.71, 141.90, 149.53, 156.67,
165.77.
(C@N), 1713 (C@O) cmꢀ1
;
1H NMR (200 MHz,
DMSO-d6): d 1.29 (t, 3H, J = 7.1 Hz), 3.64 (s, 3H),
4.29 (q, 2H, J = 7.1 Hz), 5.68 (s, 2H), 6.74–6.85 (m,
3H), 7.12–7.23 (m, 2H), 7.40–7.43 (m, 1H), 7.75 (d,
1H, J = 8.4 Hz), 8.02–8.14 (m, 5H); 13C NMR
(50 MHz, DMSO-d6): d 14.63, 52.44, 55.42, 61.21,
111.02, 113.07, 113.83, 119.86, 121.37, 121.48, 122.39,
127.17 (C · 3), 129.24, 130.27 (C · 3), 138.11, 139.16,
141.47, 141.91, 159.79, 165.98.
4.14. Ethyl 4-[1-(pyridin-3-ylmethyl)-1H-indazol-3-
yl]benzoate (46)
4.11.9.
Ethyl
4-[1-(4-methoxybenzyl)-1H-indazol-3-
yl]benzoate (40). Compound 17 (4.0 g, 0.015 mol),
EtONa (3.5 g, 0.052 mol), and 4-methoxybenzyl chlo-
ride (29) (21 mL, 0.15 mol) were allowed to react as in
the preparation of compound 30 (YD-3) to afford com-
pound 40, yield 2.90 g, 50.1%; mp 89–90 ꢁC; MS (EI,
70 eV): m/z 386 (M+); found: C, 74.65; H, 5.72; N,
7.22. C24H22N2O3 requires: C, 74.59; H, 5.74; N, 7.25;
UV kmax (loge): 211 (4.7), 320 (4.5); IR (KBr): 1609
Compound 17 (4.0 g, 0.015 mol), EtONa (4.2 g,
0.062 mol), and 3-picolyl chloride hydrochloride (43)
(4.92 g, 0.03 mol) were allowed to react as in the prepa-
ration of compound 30 (YD-3) to afford compound 46,
yield 0.24 g, 4.4%; MS (EI, 70 eV): m/z 357 (M+); found:
C, 73.88; H, 5.34; N, 11.80. C22H19N3O2 requires: C,
73.93; H, 5.36; N, 11.76; UV kmax (loge): 211 (4.6),
(C@N), 1697 (C@O) cmꢀ1
;
1H NMR (200 MHz,
320 (4.4); IR (KBr): 1610 (C@N), 1709 (C@O) cmꢀ1
;
DMSO-d6): d 1.27 (t, 3H, J = 7.1 Hz), 3.61 (s, 3H),
4.27 (q, 2H, J = 7.1 Hz), 5.60 (s, 2H), 6.79 (d, 2H,
J = 8.6 Hz), 7.16–7.21 (m, 1H), 7.23 (d, 2H,
J = 8.6 Hz), 7.34–7.42 (m, 1H), 7.72 (d, 1H,
J = 8.4 Hz), 8.02–8.13 (m, 5H); 13C NMR (50 MHz,
DMSO-d6): d 14.58, 52.12, 55.39, 61.17, 110.98, 114.37
(C · 2), 121.31, 121.57, 122.26, 126.93, 127.10 (C · 2),
129.20, 129.33 (C · 2), 129.46, 130.23 (C · 2), 138.19,
141.21, 141.77, 159.21, 165.99.
1H NMR (200 MHz, DMSO-d6): d 1.29 (t, 3H,
J = 7.0 Hz), 4.29 (q, 2H, J = 7.1 Hz), 5.78 (s, 2H),
7.25–7.32 (m, 2H), 7.40–7.45 (m, 1H), 7.59–7.61 (m,
1H), 7.85 (d, 1H, J = 8.2 Hz), 8.02–8.13 (m, 5H), 8.43
(d, 1H, J = 4.7 Hz), 8.59 (d, 1H, J = 2.2 Hz); 13C
NMR (50 MHz, DMSO-d6): d 14.64, 50.01, 61.23,
110.92, 121.36, 121.50, 122.54, 124.21, 127.26 (C · 3),
129.33, 130.27 (C · 2), 133.23, 135.74, 137.96, 141.46,
142.33, 149.26, 149.44, 165.96.
4.12. Ethyl 3-(1-benyl-1H-indazol-3-yl)benzoate (41)
4.15. Ethyl 4-[1-(pyridin-4-ylmethyl)-1H-indazol-3-
yl]benzoate (47)
Compound 19 (4.0 g, 0.015 mol), EtONa (3.5 g,
0.052 mol), and benzyl chloride (20) (10.1 g, 0.08 mol)
were allowed to react as in the preparation of compound
30 (YD-3) to afford compound 41, yield 3.01 g, 52.0%;
mp 54–55 ꢁC; MS (EI, 70 eV): m/z 356 (M+); found: C,
77.55; H, 5.68; N, 7.89. C23H20N2O2 requires: C,
77.51; H, 5.66; N, 7.86; UV kmax (loge): 217 (4.6), 311
Compound 17 (4.0 g, 0.015 mol), EtONa (4.2 g,
0.062 mol), and 4-picolyl chloride hydrochloride (44)
(9.84 g, 0.06 mol) were allowed to react as in the prepa-
ration of compound 30 (YD-3) to afford compound 47,
yield 0.89 g, 16.6%; mp 90–92 ꢁC; MS (EI, 70 eV): m/z
357 (M+); found: C, 73.98; H, 5.33; N, 11.74.
C22H19N3O2 requires: C, 73.93; H, 5.36; N, 11.76; UV
kmax (loge): 211 (4.7), 320 (4.4); IR (KBr): 1611
1
(4.7); IR (KBr): 1603 (C@N), 1718 (C@O) cmꢀ1; H
NMR (200 MHz, DMSO-d6):
J = 7.1 Hz), 4.36 (q, 2H, J = 7.1 Hz), 5.75 (s, 2H),
d
1.34 (t, 3H,
(C@N), 1708 (C@O) cmꢀ1 1H NMR (200 MHz,
;
7.23–7.35 (m, 6H), 7.45 (m, 1H), 7.66 (m, 1H), 7.78 (d,
DMSO-d6): d 1.31 (t, 3H, J = 7.1 Hz), 4.31 (q, 2H,

1274
H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
J = 7.1 Hz), 5.78 (s, 2H), 7.27–7.33 (m, 2H), 7.42–7.46
(m, 1H), 7.64 (d, 1H, J = 7.9 Hz), 7.75 (d, 1H,
J = 8.5 Hz), 8.05 (d, 2H, J = 8.3 Hz), 8.12 (d, 3H,
J = 8.3 Hz), 8.44 (dd, 1H, J = 1.6, 4.8 Hz), 8.58 (d, 1H,
J = 2.2 Hz); 13C NMR (50 MHz, DMSO-d6): d 14.63,
50.01, 61.22, 110.90, 121.51, 121.63, 122.52, 124.20,
127.25 (C · 3), 129.34, 130.26 (C · 2), 133.22, 135.73,
137.97, 141.46, 142.34, 149.26, 149.34, 165.96.
MS (EI, 70 eV): m/z 294 (M+); found: C, 73.49; H,
6.14; N, 9.49. C18H18N2O2 requires: C, 73.45; H, 6.16;
N, 9.52; UV kmax (loge): 211 (4.4), 318 (4.2); IR
(KBr): 1611 (C@N), 1699 (C@O) cmꢀ1 1H NMR
;
(200 MHz, DMSO-d6): d 1.34 (t, 3H, J = 7.2 Hz), 1.45
(t, 3H, J = 7.2 Hz), 4.32 (q, 2H, J = 7.2 Hz), 4.54 (q,
2H, J = 7.2 Hz), 7.25–7.29 (m, 1H), 7.45–7.48 (m, 1H),
7.75 (d, 1H, J = 8.4 Hz), 8.08 (d, 2H, J = 8.4 Hz),
8.12–8.15 (m, 3H); 13C NMR (50 MHz, DMSO-d6): d
14.69, 15.35, 43.89, 61.24, 110.79, 121.32 (C · 2),
122.25, 126.83, 127.08 (C · 2), 129.11, 130.29 (C · 2),
138.39, 140.90, 141.30, 166.06.
4.16. Ethyl 4-[1-(2-furylmethyl)-1H-indazol-3-yl]benzoate
(50)
Compound 17 (4.0 g, 0.015 mol), tri-n-butylphosphine
(7.74 mL, 0.03 mol), 1,10-(azodicarbonyl)dipiperidine
(7.5 g, 0.03 mol), and 2-furfuryl alcohol (48) (2.63 g,
0.03 mol) were dissolved in anhydrous toluene
(50 mL). The mixture was heated under reflux for 16 h
and then allowed to stand at 25 5 ꢁC. The solid precip-
itate so formed was directly extracted several times with
diethyl ether. The combined extract was concentrated in
vacuo for solvent removal. The residue was chromato-
graphed (silica gel-CHCl3) to afford compound 50, yield
2.83 g, 54.6%; mp 80–82 ꢁC; MS (EI, 70 eV): m/z 346
(M+); found: C, 72.84; H, 5.25; N, 8.11. C21H18N2O3 re-
quires: C, 72.82; H, 5.24 N, 8.09; UV kmax (loge): 210
(4.3), 320 (4.2); IR (KBr): 1612 (C@N), 1717 (C@O)
4.19. Ethyl 4-(1-propyl-1H-indazol-3-yl)benzoate (57)
Compound 17 (4.0 g, 0.015 mol), EtONa (3.5 g,
0.052 mol), and propyl iodide (53) (14.5 mL, 0.15 mol)
were allowed to react as in the preparation of compound
30 (YD-3) to afford compound 57, yield 2.69 g, 58.3%;
MS (EI, 70 eV): m/z 308 (M+); found: C, 74.06; H,
6.57; N, 9.05. C19H20N2O2 requires: C, 74.00; H, 6.54;
N, 9.08; UV kmax (loge): 210 (4.3), 320 (4.3); IR
(KBr): 1609 (C@N), 1702 (C@O) cmꢀ1 1H NMR
;
(200 MHz, DMSO-d6): d 0.86 (t, 3H, J = 7.4 Hz), 1.34
(t, 3H, J = 7.1 Hz), 1.87–1.92 (m, 2H), 4.36 (q, 2H,
J = 7.1 Hz), 4.45 (t, 2H, J = 6.9 Hz), 7.25–7.29 (m,
1H), 7.44–7.48 (m, 1H), 7.76 (d, 1H, J = 8.5 Hz), 8.08
(d, 2H, J = 8.4 Hz), 8.12–8.15 (m, 3H); 13C NMR
(50 MHz, DMSO-d6): d 11.80, 14.85, 23.51, 50.55,
61.39, 111.05, 121.34, 121.45, 122.36, 126.99, 127.26
(C · 2), 129.28, 130.45 (C · 2), 138.56, 141.47, 141.70,
166.21.
1
cmꢀ1; H NMR (200 MHz, DMSO-d6): d 1.33 (t, 3H,
J = 7.1 Hz), 4.33 (q, 2H, J = 7.1 Hz), 5.76 (s, 2H), 6.40
(dd, 1H, J = 1.9, 3.2 Hz), 6.55 (d, 1H, J = 3.0 Hz),
7.24–7.31 (m, 1H), 7.44–7.51 (m, 1H), 7.56–7.57 (m,
1H), 7.84 (d, 1H, J = 8.5 Hz), 8.05–8.16 (m, 5H); 13C
NMR (50 MHz, DMSO-d6): d 14.40, 45.67, 60.98,
109.26, 110.85 (C · 2), 121.07, 121.29, 122.21, 126.85,
126.96 (C · 2), 129.07, 130.03 (C · 2), 137.80, 141.09,
141.70, 143.38, 150.07, 165.75.
4.20. Ethyl 4-(1-isopropyl-1H-indazol-3-yl)benzoate (58)
Compound 17 (4.0 g, 0.015 mol), EtONa (3.5 g,
0.052 mol), and isopropyl iodide (54) (14.5 mL,
0.15 mol) were allowed to react as in the preparation
of compound 30 (YD-3) to afford compound 58, yield
1.65 g, 35.7%; MS (EI, 70 eV): m/z 308 (M+); found:
C, 74.05; H, 6.52; N, 9.01. C19H20N2O2 requires: C,
74.00; H, 6.54; N, 9.08; UV kmax (loge): 210 (4.3), 319
4.17. Ethyl 4-[1-(3-furylmethyl)-1H-indazol-3-yl]benzoate
(51)
Compound 17 (4.0 g, 0.015 mol) and 3-furfuryl alcohol
(49) (2.63 g, 0.03 mol) were allowed to react as in the
preparation of compound 50 to afford compound 51,
yield 2.49 g, 48.0%; mp 56–57 ꢁC; MS (EI, 70 eV): m/z
346 (M+); found: C, 72.87; H, 5.22; N, 8.06.
C21H18N2O3 requires: C, 72.82; H, 5.24 N, 8.09; UV
kmax (loge): 210 (4.6), 320 (4.3); IR (KBr): 1611
1
(4.2); IR (KBr): 1609 (C@N), 1704 (C@O) cmꢀ1; H
NMR (200 MHz, DMSO-d6): d1.34 (t, 3H,
J = 7.1 Hz), 1.54 (d, 6H, J = 6.6 Hz), 4.34 (q, 2H,
J = 7.1 Hz), 5.06–5.09 (m, 1H), 7.25–7.28 (m, 1H),
7.43–7.47 (m, 1H), 7.79 (d, 1H, J = 8.5 Hz), 8.08 (d,
2H, J = 8.3 Hz), 8.11–8.15 (m, 3H); 13C NMR
(50 MHz, DMSO-d6): d 14.87, 22.67 (C · 2), 50.38,
61.39, 110.97, 121.45, 121.51, 122.43, 126.82, 127.28
(C · 2), 129.24, 130.44 (C · 2), 138.72, 140.70, 141.32,
166.24.
(C@N), 1717 (C@O) cmꢀ1
;
1H NMR (200 MHz,
DMSO-d6): d 1.30 (t, 3H, J = 7.0 Hz), 4.34 (q, 2H,
J = 7.0 Hz), 5.55 (s, 2H), 6.37 (d, 1H, J = 0.8 Hz),
7.23–7.27 (m, 1H), 7.43–7.44 (m, 1H), 7.53–7.54 (m,
1H), 7.75–7.76 (m, 1H), 7.81 (d, 1H, J = 8.5 Hz), 8.05
(d, 2H, J = 8.3 Hz), 8.09 (d, 1H, J = 8.1 Hz), 8.12 (d,
2H, J = 8.3 Hz); 13C NMR (50 MHz, DMSO-d6): d
14.65, 43.93, 61.21, 110.89, 111.00, 121.33, 121.48,
121.56, 122.35, 126.98, 127.19 (C · 2), 129.21, 130.25
(C · 2), 138.16, 141.10, 141.34, 141.73, 144.24, 165.98
4.21. Ethyl 4-(1-butyl-1H-indazol-3-yl)benzoate (59)
Compound 17 (4.0 g, 0.015 mol), EtONa (3.5 g,
0.052 mol), and butyl iodide (55) (17.0 mL, 0.15 mol)
were allowed to react as in the preparation of compound
30 (YD-3) to afford compound 59, yield 2.42 g, 50.2%;
MS (EI, 70 eV): m/z 322 (M+); found: C, 74.55; H,
6.90; N, 8.65. C20H22N2O2 requires: C, 74.51; H, 6.88;
N, 8.69; UV kmax (loge): 210 (4.4), 320 (4.2); IR
4.18. Ethyl 4-(1-ethyl-1H-indazol-3-yl)benzoate (56)
Compound 17 (4.0 g, 0.015 mol), EtONa (3.5 g,
0.052 mol), and ethyl iodide (52) (12.1 mL, 0.15 mol)
were allowed to react as in the preparation of compound
30 (YD-3) to afford compound 56, yield 3.02 g, 68.4%;
(KBr): 1607 (C@N), 1701 (C@O) cmꢀ1 1H NMR
;

H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
1275
(200 MHz, DMSO-d6): d 0.89 (t, 3H, J = 7.3 Hz), 1.26–
1.31 (m, 2H), 1.34 (t, 3H, J = 7.0 Hz), 1.82–1.89 (m,
2H), 4.35 (q, 2H, J = 7.0 Hz), 4.48 (t, 2H, J = 6.9 Hz),
7.25–7.29 (m, 1H), 7.44–7.48 (m, 1H), 7.76 (d, 1H,
J = 8.5 Hz), 8.09 (d, 2H, J = 8.2 Hz), 8.12–8.15 (m,
3H); 13C NMR (50 MHz, DMSO-d6): d 14.17, 14.86,
20.12, 32.17, 48.75, 61.39, 111.01, 121.37, 121.46,
122.36, 126.99, 127.25 (C · 2), 129.28, 130.45 (C · 2),
138.56, 141.45, 141.58, 166.21.
2H), 7.23–7.40 (m, 8H), 8.03–8.08 (m, 2H), 8.14 (d,
1H, J = 10.9 Hz), 8.18–8.21 (m, 2 H); 13C NMR
(50 MHz, CDCl3): d 10.31, 21.93, 53.02, 66.34, 109.63,
120.98, 121.34, 121.92, 126.33, 126.92 (C · 4), 127.59,
128.53 (C · 2), 129.33, 129.83 (C · 2), 136.36, 137.84,
140.92, 142.74, 166.36.
4.25. Isopropyl 4-(1-benzyl-1H-indazol-3-yl)benzoate (64)
Compound 60 (1.0 g, 0.003 mol) and isopropanol
(15 mL) were allowed to react as in the preparation of
compound 62 to afford compound 64, yield 0.51 g,
46.7%; mp 75–77 ꢁC; MS (EI, 70 eV): m/z 370 (M+);
found: C, 77.84; H, 6.01; N, 7.55. C24H22N2O2 requires:
C, 77.81; H, 5.99; N, 7.56; UV kmax (loge): 211 (4.7), 320
4.22. 4-(1-Benzyl-1H-indazol-3-yl)benzoic acid (60)
Compound 30 (YD-3) (1.0 g, 0.0028 mol) was allowed to
react as in the preparation of compound 14 to afford
compound 60, yield 0.83 g, 89.8%; mp 203–206 ꢁC; MS
(EI, 70 eV): m/z 328 (M+); found: C, 76.84; H, 4.90;
N, 8.50. C21H16N2O2 requires: C, 76.81; H, 4.91; N,
8.53; UV kmax (loge): 211 (4.6), 320 (4.5); IR (KBr):
1
(4.6); IR (KBr): 1605 (C@N), 1713 (C@O) cmꢀ1; H
NMR (200 MHz, CDCl3): d 1.42 (d, 6H, J = 6.2 Hz),
5.24–5.37 (m, 1H), 5.69 (s, 2H), 7.20–7.39 (m, 8H),
8.03–8.07 (m, 2H), 8.12 (d, 1H, J = 10.5 Hz), 8.17–8.20
(m, 2H); 13C NMR (50 MHz, CDCl3): d 21.81 (C · 2),
53.00, 68.19, 109.66, 121.00, 121.42, 121.95, 126.35,
126.87 (C · 2), 126.97 (C · 2), 127.61, 128.55 (C · 2),
129.77, 129.83 (C · 2), 136.41, 137.78, 140.95, 142.76,
165.81.
1572 (C@N), 1624 (C@O), 3441 (–OH) cmꢀ1 1H
;
NMR (200 MHz, DMSO-d6): d 5.74 (s, 2H), 7.23–7.30
(m, 6H), 7.45 (t, 1H, J = 6.9 Hz), 7.79 (d, 1H,
J = 8.4 Hz), 8.05–8.08 (m, 2H), 8.10 (d, 1H,
J = 3.8 Hz), 8.11–8.16 (m, 2H); 13C NMR (50 MHz,
DMSO-d6): d 52.28, 110.76, 121.26 (C · 2), 122.11,
126.86 (C · 3), 127.62 (C · 2), 127.84, 128.84 (C · 3),
130.24 (C · 2), 137.44 (C · 2), 141.20, 141.86, 167.39.
4.26. 4-(1-Benzyl-1H-indazol-3-yl)benzamide (65)
4.23. Methyl 4-(1-benzyl-1H-indazol-3-yl)benzoate (62)
Compound 60 (1.0 g, 0.003 mol) and NH3(g) in toluene
(30 mL) were reacted for 30 min at 25 5 ꢁC. The mix-
ture was directly extracted several times with ethyl ace-
tate. The combined extract was concentrated in vacuo
for solvent removal. The residue was chromatographed
(silica gel-ethyl acetate/CHCl3 = 2/1) to afford com-
pound 65, yield 0.37 g, 36.7%; mp 182–185 ꢁC; MS
(EI, 70 eV): m/z 327 (M+); found: C, 77.07; H, 5.24;
N, 12.87. C21H17N3O requires: C, 77.04; H, 5.23; N,
12.84; UV kmax (log e): 211 (4.3), 317 (4.2); IR (KBr):
Into a solution of compound 60 (1.0 g, 0.003 mol) in
CH2Cl2 (50 mL) was added thionyl chloride (5 mL,
0.068 mol). The mixture was reacted under reflux for
16 h and then allowed to stand at 25 5 ꢁC. The mix-
ture was concentrated to dry in vacuo in water bath to
afford 4-(1-benzyl-1H-indazol-3-yl)benzoyl chloride
(61), then MeOH (15 mL) was added into the residue.
The solution was reacted under reflux for 2 h and then
allowed to stand at 25 5 ꢁC. The solution was concen-
trated in vacuo for solvent removal. The residue was
chromatographed (silica gel-CHCl3) to afford com-
pound 62, yield 0.56 g, 53.3%; mp 95–97 ꢁC; MS (EI,
70 eV): m/z 328 (M+); found: C, 77.20; H, 5.32; N,
8.19. C22H18N2O2 requires: C, 77.17; H, 5.30; N, 8.18;
UV kmax (loge): 211 (4.6), 320 (4.5); IR (KBr): 1609
1558 (C@N), 1636 (C@O), 3099 (-NH) cmꢀ1
;
1H
NMR (200 MHz, DMSO-d6): d 5.74 (s, 2H), 7.26–7.31
(m, 5H), 7.41–7.45 (m, 2H), 7.79 (d, 1H, J = 8.5 Hz),
7.99–8.05 (m, 2H), 8.07 (d, 1H, J = 7.7 Hz), 8.09–8.16
(m, 2H); 13C NMR (50 MHz, DMSO-d6): d 52.23,
110.69, 121.24 (C · 2), 121.98, 126.62 (C · 2), 126.82,
127.61 (C · 2), 127.82, 128.42 (C · 2), 128.84 (C · 2),
133.54, 136.05, 137.51, 141.17, 142.08, 167.78.
(C@N), 1705 (C@O) cmꢀ1
;
1H NMR (200 MHz,
CDCl3-d1): d 3.97 (s, 3H), 5.69 (s, 2H), 7.22–7.41 (m,
8H), 8.04–8.12 (m, 2H), 8.15 (d, 1H, J = 9.9 Hz), 8.18–
8.22 (m, 2H); 13C NMR (50 MHz, CDCl3-d1): d 51.91,
53.02, 109.64, 120.98, 121.42, 121.90, 126.36, 126.92
(C · 4), 127.61, 128.54 (C · 2), 128.94, 129.88 (C · 2),
136.36, 137.96, 140.92, 142.68, 166.79.
4.27. 4-(1-Benzyl-1H-indazol-3-yl)-N-methylbenzamide
(66)
Compound 60 (1.0 g, 0.003 mol) and methylamine(g) in
toluene (30 mL) were allowed to react as in the prepara-
tion of compound 65 to afford compound 66, yield
0.42 g, 40.0%; mp 187–189 ꢁC; MS (EI, 70 eV): m/z
341 (M+); found: C, 77.44; H, 5.62; N, 12.33.
C22H19N3O requires: C, 77.40; H, 5.61; N, 12.31; UV
kmax (log e): 211 (4.7), 317 (4.5); IR (KBr): 1552
4.24. Propyl 4-(1-benzyl-1H-indazol-3-yl)benzoate (63)
Compound 60 (1.0 g, 0.003 mol) and n-propanol
(15 mL) were allowed to react as in the preparation of
compound 62 to afford compound 63, yield 0.47 g,
43.3%; mp 57–58 ꢁC; MS (EI, 70 eV): m/z 370 (M+);
found: C, 77.84; H, 5.98; N, 7.58. C24H22N2O2 requires:
C, 77.81; H, 5.99; N, 7.56; UV kmax (loge): 211 (4.3), 321
(C@N), 1636 (C@O), 3018 (-NH) cmꢀ1 1H NMR
;
(200 MHz, DMSO-d6): d 2.80 (d, 3H, J = 4.3 Hz), 5.74
(s, 2H), 7.22–7.29 (m, 6H), 7.45 (t, 1H, J = 7.0 Hz),
7.78 (d, 1H, J = 8.3 Hz), 7.95–7.98 (m, 2H), 8.02 (d,
1H, J = 12.7 Hz), 8.11–8.15 (m, 2H), 8.52 (q, 1H,
J = 4.4 Hz); 13C NMR (50 MHz, DMSO-d6): d 26.52,
52.23, 110.69, 121.23 (C · 2), 121.97, 126.68 (C · 2),
1
(4.3); IR (KBr): 1611 (C@N), 1709 (C@O) cmꢀ1; H
NMR (200 MHz, CDCl3): d 1.07 (t, 3H,J = 7.5 Hz),
1.79–1.89 (m, 2H), 4.33 (t, 2H,J = 6.6 Hz), 5.69 (s,

1276
H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
126.81, 127.60 (C · 2), 127.82, 127.98 (C · 2), 128.84
(C · 2), 133.76, 135.86, 137.51, 141.17, 142.06, 166.49.
109.60, 120.96, 121.32, 121.87, 126.33, 126.92 (C · 3),
127.12 (C · 4), 127.58, 128.52 (C · 2), 133.72, 136.41,
140.91, 142.78, 166.17.
4.28. 4-(1-Benzyl-1H-indazol-3-yl)-N-ethylbenzamide
(67)
4.31. [4-(1-Benzyl-1H-indazol-3-yl)phenyl]methanol (70)
Compound 60 (1.0 g, 0.003 mol) and ethylamine(g) in
toluene (30 mL) were allowed to react as in the prepara-
tion of compound 65 to afford compound 67, yield
0.47 g, 43.3%; mp 161–164 ꢁC; MS (EI, 70 eV): m/z
355 (M+); found: C, 77.69; H, 5.98; N, 11.79.
C23H21N3O requires: C, 77.72; H, 5.96; N, 11.82; UV
kmax (log e): 211 (4.3), 317 (4.1); IR (KBr): 1547
Anhydrous calcium chloride (CaCl2, granular, 2.0 g,
0.018 mol) was added into the solution of sodium
borohydride (NaBH4, 2.0 g, 0.052 mol) in anhydrous
THF (100 mL). The mixture was stirred at 25 5 ꢁC
for 4 h. Then compound 30 (YD-3) (1.0 g, 0.0028 mol)
was added. The above mixture was heated under reflux
for 6 h and then allowed to stand at 25 5 ꢁC. The
reaction mixture was then poured into the ice-water
(300 mL). The mixture was extracted several times with
CH2Cl2. The combined extract was concentrated in va-
cuo for solvent removal. The residue was chromato-
graphed (silica gel-ethyl acetate/n-hexane = 2/3) to
afford compound 70, yield 0.7 g, 79.7%; mp 102–
104 ꢁC; MS (EI, 70 eV): m/z 314 (M+); found: C,
80.27; H, 5.79; N, 8.89. C21H18N2O requires: C,
80.23; H, 5.77; N, 8.91; UV kmax (loge): 211 (4.7),
(C@N), 1631 (C@O), 3125 (-NH) cmꢀ1 1H NMR
;
(200 MHz, DMSO-d6): d 1.14 (t, 3H, J = 7.2 Hz),
3.23–3.43 (m, 2H), 5.74 (s, 2H), 7.19–7.32 (m, 6H),
7.45 (t, 1H, J = 7.3 Hz), 7.78 (d, 1H, J = 8.5 Hz), 7.96–
8.00 (m, 2H), 8.03 (d, 1H, J = 10.1 Hz), 8.09–8.15 (m,
2H), 8.55 (t, 1H, J = 5.4 Hz); 13C NMR (50 MHz,
DMSO-d6): d 15.05, 34.29, 52.22, 110.68, 121.23
(C · 2), 121.95, 126.63 (C · 2), 126.80, 127.58 (C · 2),
127.80, 128.03 (C · 2), 128.83 (C · 2), 133.94, 135.83,
137.51, 141.18, 142.09, 165.76.
320 (4.5); IR (KBr): 1614 (C@N), 3418 (–OH) cmꢀ1
;
1H NMR (200 MHz, DMSO-d6): d 4.57 (d, 2H,
J = 5.7 Hz), 5.27 (t, 1H, J = 5.7 Hz), 5.71 (s, 2H),
7.18–7.30 (m, 6H), 7.38–7.45 (m, 1H), 7.47 (d, 2H,
J = 8.2 Hz), 7.75 (d, 1H, J = 8.5 Hz), 7.80 (d, 2H,
J = 8.2 Hz), 8.07 (d, 1H, J = 8.2 Hz); 13C NMR
(50 MHz, DMSO-d6): d 52.11, 62.94, 110.50, 121.24
(C · 2), 121.55, 126.64, 126.84 (C · 2), 127.18 (C · 2),
127.59 (C · 2), 127.76, 128.80 (C · 2), 131.86, 137.68,
141.12, 142.51, 142.99.
4.29. 4-(1-Benzyl-1H-indazol-3-yl)-N-propylbenzamide
(68)
Compound 60 (1.0 g, 0.003 mol) and propylamine
(15 mL) in toluene (30 mL) were allowed to react as in
the preparation of compound 65 to afford compound
68, yield 0.45 g, 40.1%; mp 135–137 ꢁC; MS (EI,
70 eV): m/z 369 (M+); found: C, 78.04; H, 6.28; N,
11.35. C24H23N3O requires: C, 78.02; H, 6.27; N,
11.37; UV kmax (loge): 211 (4.5), 317 (4.4); IR (KBr):
4.32. 4-(1-Benzyl-1H-indazol-3-yl)benzaldehyde (71)
1543 (C@N), 1628 (C@O), 3152 (–NH) cmꢀ1
;
1H
NMR (200 MHz, DMSO-d6):
d
0.90 (t, 3H,
Into a solution of compound 70 (1.0 g, 0.0032 mol) in
acetone (30 mL), manganese(IV) oxide (0.55 g,
0.0064 mol) was added, and the mixture was stirred
for 20 h at 25 5 ꢁC. The reaction mixture was concen-
trated in vacuo for solvent removal. The residue was
chromatographed (silica gel-ethyl acetate/n-hexane =
1/3) to afford compound 71, yield 0.8 g, 80.1%; mp
91–92 ꢁC; MS (EI, 70 eV): m/z 312 (M+); found: C,
80.83; H, 5.13; N, 8.93. C21H16N2O requires: C, 80.75;
H, 5.16; N, 8.97; UV kmax (loge): 211 (4.7), 320 (4.5);
J = 7.3 Hz), 1.49–1.60 (m, 2H), 3.19–3.29 (m, 2H), 5.74
(s, 2H), 7.21–7.32 (m, 6H), 7.45 (t, 1H, J = 8.0 Hz),
7.78 (d, 1H, J = 8.4 Hz), 7.96–8.00 (m, 2H), 8.02 (d,
1H, J = 9.9 Hz), 8.09–8.15 (m, 2H), 8.55 (t, 1H,
J = 5.5 Hz); 13C NMR (50 MHz, DMSO-d6): d 11.72,
22.65, 41.24, 52.22, 110.70, 121.23 (C · 2), 121.97,
126.64 (C · 2), 126.82, 127.59 (C · 2), 127.82, 128.07
(C · 2), 128.84 (C · 2), 133.98, 135.82, 137.52, 141.18,
142.10, 165.97.
1
IR (KBr): 1603 (C@N), 1692 (C@O) cmꢀ1; H NMR
4.30. 4-(1-Benzyl-1H-indazol-3-yl)-N-isopropylbenzamide
(69)
(200 MHz, DMSO-d6): d 5.71 (s, 2H), 7.19–7.26 (m,
6H), 7.37–7.41 (m, 1H), 7.74 (d, 1H, J = 8.5 Hz), 7.98
(d, 2H, J = 8.3 Hz), 8.10 (d, 1H, J = 8.2 Hz), 8.18 (d,
2H, J = 8.3 Hz), 10.00 (s, 1H, H-23); 13C NMR
(50 MHz, DMSO-d6): d 52.60, 111.02, 121.40, 121.58,
122.52, 127.15, 127.52 (C · 2), 127.85 (C · 2), 128.09,
129.06 (C · 2), 130.65 (C · 2), 135.60, 137.54, 139.32,
141.45, 141.83, 193.05.
Compound 60 (1.0 g, 0.003 mol) and isopropylamine
(15 mL) in toluene (30 mL) were allowed to react as in
the preparation of compound 65 to afford compound
69, yield 0.56 g, 49.9%; mp 169–171 ꢁC; MS (EI,
70 eV): m/z 369 (M+); found: C, 78.06; H, 6.26; N,
11.35. C24H23N3O requires: C, 78.02; H, 6.27; N,
11.37; UV kmax (log e): 211 (4.4), 317 (4.3); IR (KBr):
4.33. 4-(1-Benzyl-1H-indazol-3-yl)benzaldehyde O-meth-
yloxime (72)
1628 (C@N), 1723 (C@O), 3157 (-NH) cmꢀ1
;
1H
NMR (200 MHz, DMSO-d6):
d
1.30 (d, 6H,
J = 6.5 Hz), 4.29–4.39 (m, 1H), 5.67 (s, 2H), 6.07 (d,
1H, J = 7.5 Hz), 7.19–7.35 (m, 6H), 7.37 (d, 2H,
J = 3.7 Hz), 7.90 (d, 2H, J = 8.3 Hz), 8.02 (d, 1H,
J = 8.5 Hz), 8.08 (d, 2H, J = 8.3 Hz); 13C NMR
(50 MHz, DMSO-d6): d 22.67 (C · 2), 41.74, 52.96,
Into a solution of compound 71 (1.0 g, 0.0032 mol) in
(50 mL),
(1.2 mL, 0.014 mol) was added. The mixture was
heated at reflux for 3 h, and then allowed to stand
at 25 5 ꢁC. The reaction mixture was extracted
99.5%
EtOH
O-methylhydroxylamine

H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
1277
several times with CH2Cl2. The combined extract was
4.36. Ethyl 4-(1-phenyl-1H-indazol-3-yl)benzoate (83)
concentrated in vacuo for solvent removal. The resi-
due was chromatographed (silica gel-ethyl acetate/n-
hexane = 1/8) to afford compound 72, yield 0.78 g,
71.5%; mp 65–67 ꢁC; MS (EI, 70 eV): m/z 341 (M+);
found: C, 77.44; H, 5.59; N, 12.34. C22H19N3O re-
quires: C, 77.40; H, 5.61; N, 12.31; UV kmax (log e):
210 (4.7), 320 (4.4); IR (KBr): 1611 (C@N), 2940
(A) Into a solution of aluminum chloride (85.0 g,
0.64 mol) in toluene (75) (90 mL) was added dropwise
the benzoyl chloride (76) (50 mL, 0.43 mol) in ice-
bath. The mixture was heated at 100 ꢁC for 2 h and
then allowed to stand at 25 5 ꢁC. The mixture was
concentrated to dry in vacuo in water bath to afford
4-methylbenzophenone (77), yield 60.21 g, 70.8%; mp
54–56 ꢁC.
1
(–CH) cmꢀ1; H NMR (200 MHz, DMSO-d6): d 3.92
(s, 3H), 5.73 (s, 2H), 7.21–7.30 (m, 6H), 7.35–7.43
(m, 1H), 7.75 (d, 2H, J = 8.3 Hz), 7.76 (d, 1H, J =
8.4 Hz), 8.05 (d, 2H, J = 8.3 Hz), 8.11 (d, 1H,
J = 8.2 Hz), 8.29 (s, 1H); 13C NMR (50 MHz,
DMSO-d6): d 52.24, 61.90, 110.66, 121.22 (C · 2),
121.91, 126.80, 127.24 (C · 2), 127.59 (C · 3), 127.67
(C · 2), 128.83 (C · 2), 131.41, 134.82, 137.53,
141.19, 142.20, 148.67.
(B) Into
a
solution of compound 77 (25.0 g,
0.127 mol) in glacial acetic acid (130 mL), chromium
oxide (40.0 g, 0.4 mol) and H2SO4 (25 mL) were
added. The mixture was heated at 100 ꢁC for 2 h
and then allowed to stand at 25 5 ꢁC to afford
4-benzoylbenzoic acid (78), yield 21.74 g, 75.6%; mp
197–200 ꢁC.
4.34. (2E)-3-[4-(1-Benzyl-1H-indazol-3-yl)phenyl]acrylic
acid (73)
(C) Compound 78 (20.0 g, 0.0884 mol) was allowed to
react as in the preparation of compound 17 to afford
ethyl 4-benzoylbenzoate (79), yield 15.96 g, 71.0%; mp
47–49 ꢁC.
Into a solution of compound 71 (1.0 g, 0.0032 mol) in
toluene (30 mL), malonic acid (2.5 g, 0.024) and piperi-
dine (3.5 g, 0.041 mol) were added. The mixture was
heated at 80 ꢁC for 3 h and then allowed to stand at
25 5 ꢁC. The reaction mixture was concentrated in va-
cuo for solvent removal. The residue was chromato-
graphed (silica gel-CH2Cl2/EtOH = 16/1) to afford
compound 73, yield 0.98 g, 78.7%; mp 186–189 ꢁC; MS
(EI, 70 eV): m/z 354 (M+); found: C, 78.02; H, 5.11;
N, 7.87. C23H18N2O2 requires: C, 77.95; H, 5.12; N,
7.90; UV kmax (loge): 211 (4.6), 321 (4.4); IR (KBr):
1605 (C@C), 1630 (C@N), 1674 (C@O), 3030 (–OH)
(D) Into a solution of compound 79 (10.0 g, 0.039 mol)
in absolute EtOH (100 mL), phenylhydrazine (80) (8.4 g,
0.078 mol) and glacial acetic acid (1 mL) were added.
The mixture was heated under reflux for 24 h and then
allowed to stand at 25 5 ꢁC to afford ethyl 4-[(phen-
ylhydrazono)(phenyl)methyl]benzoate (81), yield 8.8 g,
65.6%; mp 135–137 ꢁC.
(E) Into a solution of lead tetraacetate (20.0 g) in
CH2Cl2 (100 mL) was added the solution of com-
pound 81 (10.0 g, 0.029 mol) in CH2Cl2 (100 mL).
The mixture was reacted for 30 min in the ice-bath.
Then boron trifluoride etherate (200 mL) was added
dropwise and the mixture was heated under reflux
for 30 min. The reaction mixture was poured into
the ice-bath to afford compound 83, yield 5.34 g,
53.8%; mp 85–87 ꢁC; MS (EI, 70 eV): m/z 342 (M+);
found: C, 77.22; H, 5.32; N, 8.15. C22H18N2O2 re-
quires: C, 77.17; H, 5.30 N, 8.18; UV kmax (loge):
242 (4.6), 303 (4.2); IR (KBr): 1598 (C@N), 1723
1
cmꢀ1; H NMR (200 MHz, DMSO-d6): d 5.69 (s, 2H),
6.54 (d, 1H, J = 16.0 Hz), 7.18–7.27 (m, 6H), 7.37–7.41
(m, 1H), 7.61 (d, 1H, J = 16.0 Hz), 7.74 (d, 1H,
J = 9.3 Hz), 7.78 (d, 2H, J = 8.4 Hz), 8.00 (d, 2H,
J = 8.3 Hz), 8.08 (d, 1H, J = 8.2 Hz); 13C NMR
(50 MHz, DMSO-d6): d 52.46, 110.91, 119.67, 121.46
(C · 2), 122.21, 127.05, 127.50 (C · 2), 127.85 (C · 2),
128.06, 129.06 (C · 2), 129.31 (C · 2), 134.05, 135.30,
137.71, 141.38, 142.37, 143.89, 168.08.
4.35. Ethyl (2E)-3-[4-(1-benzyl-1H-indazol-3-
yl)phenyl]acrylate (74)
(C@O) cmꢀ1 1H NMR (200 MHz, CDCl3): d 1.45
;
(t, 3H, J = 7.1 Hz), 4.44 (q, 2H, J = 7.1 Hz), 7.26–
7.51 (m, 3H), 7.57 (d, 2H, J = 8.1 Hz), 7.76–7.83 (m,
3H), 8.10 (d, 1H, J = 8.10 Hz), 8.17 (d, 2H, J =
8.72 Hz), 8.21 (d, 2H, J = 8.16 Hz); 13C NMR
(50 MHz, CDCl3): d 14.15, 60.91, 110.66, 121.13,
122.16, 122.93 (C · 3), 126.80, 127.05, 127.16 (C · 2),
129.30 (C · 2), 129.70, 129.85 (C · 2), 137.41, 137.68,
140.25, 144.63, 166.26.
Compound 73 (1.0 g, 0.0028 mol) was allowed to react
as in the preparation of compound 17 to afford com-
pound 74, yield 0.8 g, 75.0%; mp 87–89 ꢁC; MS (EI,
70 eV): m/z 382 (M+); found: C, 78.57; H, 5.77; N,
7.30. C25H22N2O2 requires: C, 78.51; H, 5.80 N, 7.32;
UV kmax (loge): 211 (4.7), 321 (4.4); IR (KBr): 1605
(C@C), 1630 (C@N), 1705 (C@O) cmꢀ1 1H NMR
;
(200 MHz, DMSO-d6): d 1.22 (t, 3H, J = 7.0 Hz), 4.15
(q, 2H, J = 6.9 Hz), 5.69 (s, 2H), 6.63 (d, 1H,
J = 16.1 Hz), 7.17–7.27 (m, 6H), 7.36–7.44 (m, 1H),
7.67 (d, 1H, J = 16.2 Hz), 7.73 (d, 1H, J = 9.0 Hz),
7.81 (d, 2H, J = 8.3 Hz), 8.00 (d, 2H, J = 8.1 Hz), 8.07
(d, 1H, J = 7.7 Hz); 13C NMR (50 MHz, DMSO-d6): d
14.65, 52.46, 60.53, 110.90, 118.52, 121.45 (C · 2),
122.20, 127.04, 127.48 (C · 2), 127.85 (C · 2), 128.06,
129.05 (C · 2), 129.45 (C · 2), 133.82, 135.52, 137.68,
141.38, 142.32, 144.35, 166.71.
4.37. Preparation of washed human platelets and mea-
surement of platelet aggregation
Human blood anticoagulated with acid citrate dextrose
was obtained from healthy human volunteers and the
platelet suspension was then prepared according to the
procedures described previously.13 Platelets were finally
suspended in Tyrode’s solution containing Ca2+ (2 mM)
at a concentration of 3 · 108 platelets/mL. Platelet

1278
H.-S. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1262–1278
aggregation was measured turbidimetrically with a light-
transmission aggregometer. The extent of platelet aggre-
gation was measured as the maximal increase of light
transmission within 5 min after the addition of
stimulators.
say kits according to the procedure described by the
manufacturer.
Acknowledgments
4.38. ATP release assay
The investigation was supported by research grants to
S.C. Kuo and L.J. Huang from the National Science
Council of the Republic of China (NSC 93-2320-B-
039-003) and China Medical University (CMU 93-PC-
06) in Taiwan.
ATP release from platelet dense granule was measured
as described previously.13 Washed human platelets
were pre-incubated with DMSO or test compounds
for 3 min and thentreated with GYPGKF for addi-
tional 5 min. The reactions were stopped with EDTA
(5 mM), and the samples were immediatelycentrifuged
at 4 ꢁC. ATP was measured in the supernatantsby the
addition of a luciferase/luciferin reagent in a micro-
plate luminometer.
References and notes
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4.39. Measurement of P-selectin expression
P-selectin expression in platelets was measured as de-
scribed previously.14 Washed human platelets were
pre-incubated with DMSO or test compounds for
3 min and then treated with or without GYPGKF in
the presence of FITC-conjugated anti-CD62P for
15 min at room temperature. The samples were then
fixed at 4 ꢁC with 1% paraformaldehyde. Flow cytomet-
ric analysis was performed on a flow cytometer. Platelets
were identified by logarithmic signal amplification for
forward and side scatter. The levels of P-selectin expres-
sion were expressed as the percentages of cells positive
for anti-CD62P.
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4.40. Measurement of thromboxane B2 formation
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Because thromboxane A2 is very unstable and rapidly
converted to more stable metabolite thromboxane B2,
we thus measured the latter instead of thromboxane
A2. After challenge of platelets with arachidonic acid
for 5 min, EDTA (2 mM) and indomethacin (50 lM)
were added. The platelet suspensions were centrifuged
for 3 min at 13,000 rpm, and the thromboxane B2 in
the supernatants was assayed using enzyme immunoas-