Click approach to the discovery of 1,2,3-triazolylsalicylamides as potent Aurora kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.06.047

A series of 1,2,3-triazolylsalicylamide derivatives has been developed from the antiproliferative agent 7 and was evaluated for their Aurora kinase inhibitory activity. The novel 1,2,3-triazolylsalicylamide scaffold could be readily assembled by Cu(I)-cat

Full text of DOI:10.1016/j.bmc.2014.06.047

Bioorganic & Medicinal Chemistry 22 (2014) 4855–4866
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Click approach to the discovery of 1,2,3-triazolylsalicylamides
as potent Aurora kinase inhibitors
Doohee Song a, Yunjeong Park a, Jieun Yoon a, Waqar Aman b, Jung-Mi Hah b, Jae-Sang Ryu a,
⇑
a College of Pharmacy & Graduate School of Pharmaceutical Sciences, Global Top 5 Research Program, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Gu,
Seoul 120-750, Republic of Korea
b Department of Pharmacy, College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu,
Ansan, Kyeonggi-do 426-791, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,2,3-triazolylsalicylamide derivatives has been developed from the antiproliferative agent 7
and was evaluated for their Aurora kinase inhibitory activity. The novel 1,2,3-triazolylsalicylamide scaf-
fold could be readily assembled by Cu(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition, allowing rapid
access to the structurally diverse analogues. The synthesized 1,2,3-triazolylsalicylamide derivatives
revealed a significant Aurora kinase inhibitory activity. In particular, 8g inhibited Aurora A with IC50 val-
Received 23 May 2014
Revised 20 June 2014
Accepted 22 June 2014
Available online 30 June 2014
ues of 0.37
study.
l
M. The critical role of phenolic –OH in the binding was confirmed by a molecular modeling
Keywords:
Aurora kinase
Click chemistry
Library
Ó 2014 Elsevier Ltd. All rights reserved.
1,2,3-Triazole
Anticancer
1. Introduction
centrosomes leading to a monopolar spindle formation and accu-
mulation in mitosis, and the inhibition of Aurora B induces mitotic
Aurora kinases are members of serine/threonine kinases regu-
lating cell division.1 Three human isoforms of Aurora kinase (Aurora
A, B and C)2 have distinct functions and different subcellular local-
izations during cell mitosis. Aurora A localizes to the centrosome
and is involved in centrosome maturation and dipolar spindle for-
mation,3 while Aurora B localizes to the centromeres, the central
spindle and the spindle midbody4 and is involved in centrosome
separation, chromosome segregation and cytokinesis. It is a chro-
mosome passenger protein kinase and regulates the phosphoryla-
tion of histone H3 at serine 10.5 Aurora C localizes to the spindle
poles late in mitosis and is also considered as a chromosome pas-
senger complementing the Aurora B function in mitotic cells.6
An overexpression of Aurora A and Aurora B causes an over-
phosphorylation of normal cell cycle targets and an aberrant phos-
phorylation of cytoplasmic targets, leading to chromosomal insta-
bility, oncogenic transformation, tumor progression and the
development of chemoresistance.7 Indeed, an overexpression of
Aurora kinases is frequently observed in various cancer cells such
as in colon, pancreas, breast, lung, thyroid cancers and in leuke-
mia.8 A number of notable recent researches have validated Aurora
kinases as an attractive anti-cancer drug target. The inhibition of
Aurora A triggers an abnormal formation of spindles and immature
exit resulting in polyploid cell formation.9
Since 1 (VX-680) entered human clinical trials as the first
Aurora kinase inhibitor,10 many Aurora-selective small-molecule
inhibitors have been developed and are currently undergoing pre-
clinical and clinical assessments. The selected examples include 2
(SNS-314),11 3 (MLN8237),12 4 (MK-5108),13 5 (AZD1152)14 and
6 (GSK1070916)15 (Fig. 1). Based on the Aurora subfamily selectiv-
ity, 1 (VX-680) and 2 (SNS-314) are classified as pan-Aurora
inhibitors; 3 (MLN-8237) and 4 (MK-5108) are Aurora A specific
inhibitors; 5 (AZD-1152) and 6 (GSK-1070916) are Aurora B spe-
cific inhibitors. Despite intensive effort to date, no Aurora-selective
drug has been approved by the Food and Drug Administration
(FDA) yet. As part of our ongoing program towards the
development of anti-cancer kinase inhibitors, we have developed
1,2,3-triazolylsalicylamide inhibitors showing Aurora A kinase
selectivity. Herein, we report the design, synthesis and biological
evaluation of 1,2,3-triazolylsalicylamide inhibitors.
2. Results and discussion
2.1. Design
In our previous report, we have identified antiproliferative
agent 7 through a rapid synthesis of natural product lavendustin-
mimetic small molecules using the click fragment assembly.16
⇑
Corresponding author. Tel.: +82 2 3277 3008; fax: +82 2 3277 2851.
E-mail address: ryuj@ewha.ac.kr (J.-S. Ryu).
http://dx.doi.org/10.1016/j.bmc.2014.06.047
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

4856
D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
H
N
Cl
F
S
N
N
N
O
S
NH
N
MeO
N
H
N
N
NH
N
N
O
N
CO2H
OMe
N
S
Cl
N
H
N
H
N
N
H
1 (VX-680)
2 (SNS-314)
3 (MLN8237)
HO
OH
P
F
O
Cl
O
N
N
N
NH
O
CO2H
N
N
HN
N
N
N
H
N
O
NH
S
N
N
H
N
N
N
O
H
F
4 (MK-5108)
5 (AZD1152)
6 (GSK1070916)
Figure 1. Aurora kinase inhibitors.
The identified 1,2,3-triazolylsalicylamide 7 inhibited both, CCRF-
CEM and HCT116 cancer cell lines with GI50 values of 6.41
and 4.37 M, respectively. However, compound 7 showed no EGFR
Accordingly, we decided to develop novel 1,2,3-triazolylsalicyla-
mide scaffold inhibitors targeting Aurora kinases.
lM
l
1,2,3-Triazole is a metabolically stable and versatile connecting
unit. Although 1,2,3-triazole does not occur in nature, a number of
synthetic 1,2,3-triazole compounds show various biological activi-
ties such as anticancer, antifungal, antibacterial, anti-HIV and anti-
tuberculosis activities.19 1,2,3-Triazole acts as a hydrogen bonding
acceptor, thereby improves water solubility and facilitates binding
to biological targets.20 Especially, 1,2,3-triazole scaffold offers
several advantages in the development of kinase inhibitors: (i)
1,2,3-triazole can be a mimic of the purine of ATP binding to the
active site in protein kinases; (ii) 1,2,3-triazole can be a bioisostere
of flat heteroaromatic rings such as imidazole, pyrazole and purine,
inhibitory activity when it was subjected to the in vitro kinase
assay against EGFR kinase. This was an unexpected result because
natural product lavendustin has been known to inhibit EGFR
kinase.17 In order to get an insight of the kinase inhibitory profile
of compound 7, we tested it at a single dose concentration of
10 l
M over a panel of 40 kinases at Reaction Biology Corporation.18
As shown in Fig. 2, compound 7 moderately inhibited kinase activ-
ities of mutant EGFR (L858R), GSK-3b and PIM1 at the test concen-
tration. Particularly, it exhibited a very good selectivity for Aurora
A kinase among 40 kinases with an inhibition percentage of 74.5%.
OCH3
N
O
N
N
F3C
N
H
HO
OCH3
7
100
90
80
70
60
50
40
30
20
10
0
Kinase
Figure 2. % Inhibition of compound 7 at 10 lM concentration over 40 kinases.

D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
4857
Ar
N
N
N
O
O
OCH3
OCH3
CuSO4
Na Ascorbate
Ar
N3
N
O
N
N
F3C
N
H
F3C
N
H
t-BuOH, H2O
rt
F3C
N
H
HO
HO
13h
9a: Ar = 3,5-bis(trifluoromethyl)phenyl (99%)
9b: Ar = 3,5-difluorophenyl (47%)
9c: Ar = p-(dimethylamino)phenyl (57%)
9d: Ar = p-cyanophenyl (31%)
HO
7
9e: Ar = 5-chloro-2,4-dimethoxyphenyl (47%)
9f: Ar = m-chlorophenyl (71%)
9g: Ar = p-(methoxycarbonyl)phenyl (47%)
9h: Ar = m-tolyl (86%)
9i: Ar = p-tolyl (61%)
9j: Ar = p-fluorophenyl (97%)
OCH3
OCH3
N
N
N
O
R2
1N NaOH
CH3OH
N
H
80 oC, 1h
9k: Ar = pyridin-3-yl (78%)
9l: Ar = thiophen-3-yl (83%)
R1
8
9
9m: Ar = m-acetamidophenyl (85%)
9n: Ar = m-(methoxycarbonyl)phenyl (86%)
9o: Ar = pyridin-2-yl (27%)
9p: Ar = 1-methyl-1H-imidazol-5-yl (54%)
9q: Ar = p-carboxyphenyl (96%)
9r: Ar = m-carboxyphenyl (98%)
N
N
N
O
Ar
1N NaOH
CH3OH
F3C
N
H
80 oC, 1h
HO
Scheme 2. Synthesis of 9a–r.
Figure 3. Design of 1,2,3-triazolylsalicylamide derivatives.
(compound 8 in Fig. 3). Next, we replaced the 3,5-dimethoxyphenyl
group with various aryl groups without changing the 3-(trifluoro-
methyl)phenyl group (compound 9 in Fig. 3). A variety of substitu-
ents could be readily installed by Cu(I)-catalyzed azide–alkyne
1,3-dipolar cycloaddition (CuAAC). Due to easy accessibility of var-
ious commercial alkynes, the 3,5-dimethoxyphenyl group could be
intensively modified. In addition, 1,2,3-triazolylbenzamide deriva-
tive was also designed to explore the role of –OH in the 1,2,3-triaz-
olylsalicylamide scaffold.
which are frequently found in kinase inhibitors; (iii) 1,2,3-triazole
can be easily assembled by ‘click chemistry’; (iv) a variety of substit-
uents can be easily introduced by using commercial alkynes or
azides. Therefore, we envisioned a number of 1,2,3-triazolylsalicyla-
mide derivatives modifying compound 7 (Fig. 3). In our design, we
conserved the 1,2,3-triazolylsalicylamide moiety of compound 7
and modified the 3-(trifluoromethyl)phenyl group with substituted
phenyl groups without changing the 3,5-dimethoxyphenyl group
OCH3
O
O
O
a. CDI, CH3CN
R2
OCH3
a. H2SO4, H2O
N3
reflux
NaNO2, 0 o
C
N3
NH2
HO
N
H
HO
CuSO4
b. NaN3, H2O
b. TEA, reflux
R1
R1
Na Ascorbate
t-BuOH, H2O, rt
R1
0
oC
rt
13a: R1= H, R2
= -CF , (91%)
m
3
10a: R1= H
11a: R1= H (100%)
11b: R1= OH (98%)
R2
10b: R1= OH
13b: R1= OH, R2 = H, (42%)
13c: R1= OH, R2 = m-Me, (61%)
13d: R1= OH, R2 = m-OCH3, (52%)
13e: R1= OH, R2 = m-Cl, (5%)
NH2
12a: R2= m-CF3
12b: R2= H
12c: R2= m-Me
12d: R2= -OCH
13f: R1= OH, R2
= -CF , (17%)
o
3
m
3
13g: R1= OH, R2 = m-COOMe, (21%)
13h: R1= OH, R2 = m-CF3, (22%)
12e: R2= m-Cl
12f: R2= o-CF3
12g: R2= m-COOMe
OCH3
OCH3
N
N
N
O
R2
N
H
R1
8a: R1= H, R2= m-CF3, (98%)
8b: R1= OH, R2= H, (73%)
8c: R1= OH, R2= m-Me, (80%)
8d: R1= OH, R2= m-OCH3, (74%)
8e: R1= OH, R2= m-Cl, (51%)
8f: R1= OH, R2= o-CF3, (82%)
8g: R1= OH, R2= -COOMe, (71%)
m
1N NaOH, CH3OH
80 oC, 1 h
8h: R1= OH, R2= m-COOH, (70%)
Scheme 1. Synthesis of 8a–g.

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D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
Table 1
Inhibitory activity of 1,2,3-triazolylsalicylamides 8 and 9 against AurA and AurBa
N
N
N
O
R2
Ar
N
H
R1
8a-h, 9a-r
Compound
R1
R2
Ar
% Inhibitionb
AurA
AurB
8a
8b
8c
8d
8e
8f
8g
8h
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
H
m-CF3
H
m-CH3
m-OCH3
m-Cl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Bis(trifluoromethyl)phenyl
3,5-Difluorophenyl
p-(Dimethylamino)phenyl
p-Cyanophenyl
5-Chloro-2,4-dimethoxyphenyl
m-Chlorophenyl
p-(Methoxycarbonyl)phenyl
m-Tolyl
p-Tolyl
p-Fluorophenyl
30.0
88.8
4.2
4.4
65.9
11.8
50.3
56.3
21.3
71.3
34.8
80.4
39.5
16.2
36.2
76.7
41.7
23.0
30.6
20.0
21.7
53.6
25.3
71.4
57.3
44.9
42.3
27.4
39.6
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
—
83.3
91.8
22.4
90.2
67.9
91.0
88.9
45.7
92.8
94.4
94.7
92.6
92.5
85.7
79.3
75.8
91.3
92.5
90.5
78.0
46.4
63.5
52.5
<0.001
o-CF3
m-CO2CH3
m-CO2H
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
—
Pyridin-3-yl
Thiophen-3-yl
m-Acetamidophenyl
m-(Methoxycarbonyl)phenyl
Pyridin-2-yl
1-Methyl-1H-imidazol-5-yl
p-Carboxyphenyl
9q
9r
m-Carboxyphenyl
—
Staurosporine IC50
(
lM)
0.001
a
Enzymatic assay was conducted by Reaction Biology Corporation (http://www.reactionbiology.com).
b
Compounds were tested at 10
l
M in the presence of 1
l
M ATP. Data shown are the average of duplicated assays. % Inhibition is calculated by subtracting % enzyme
activity from 100.
2.2. Synthesis
8h was synthesized from hydrolysis of the corresponding methyl
benzoate analogue 8g.
Eight different azide building blocks bearing benzamide or
salicylamide were prepared and coupled with 1-ethynyl-3,5-dime-
thoxybenzene via CuAAC as shown in Scheme 1. The synthesis of
azidobenzamide 13a and azidosalicylamides 13b–h commenced
from commercially available 3-aminobenzoic acid (10a) and
5-aminosalicylic acid (10b). The addition of aqueous solution of
NaNO2 to a H2SO4 solution of 10a or 10b formed diazonium salts,
which were then treated with NaN3 to afford azide 11a or 11b in
100% and 98% yield, respectively. The following coupling reactions
with substituted anilines 12a–g in the presence of 1,10-carbonyldi-
imidazole yielded azidobenzamide 13a and azidosalicylamides
13b–h. The coupling reaction of 3-azidobenzoic acid (11a) and
substituted aniline 12a proceeded well and gave azidobenzamide
13a in 91% yield, whereas the coupling reactions of 5-azidosalicylic
acid (11b) and substituted anilines 12a–g depended on the elec-
tronic properties of substituted anilines. The coupling reactions
of 12c and 12d having an electron-donating group on R2 provided
the amides 13c and 13d in 61% and 52% yields, respectively. On the
other hand, the coupling reactions of 12a, 12e–g containing an
electron-withdrawing group on R2 were sluggish and afforded
the corresponding amide 13e–h in low yields (5–22%) due to the
poor nucleophilicity of aniline nitrogen. With azide building blocks
13a–h in hand, we performed click reactions in the presence of
CuSO4 (20 mol %), sodium ascorbate (100 mol %) and alkyne (2.5
equiv) in t-BuOH/H2O as the standard click condition optimized
and used for the synthesis of 7. Then, the benzoic acid analogue
Next, we synthesized the second 1,2,3-triazolylsalicylamide
derivatives (compound 9) bearing the common N-(3-(trifluoro-
methyl)phenyl)salicylamide moiety. The CuAAC reactions were
carried out with azidosalicylamide 13h as an azide coupling part-
ner and various aromatic alkynes as alkyne coupling partners as
shown in Scheme 2. All reactions smoothly produced the corre-
sponding 1,2,3-triazolylsalicylamides 9a–p in 27–99% yields under
standard click conditions. The following saponifications of the
methyl esters 9g and 9n afforded the corresponding benzoic acid
analogues 9q and 9r in 96% and 98% yields, respectively.
2.3. Biological screening
An in vitro kinase assay was performed at a single dose concen-
tration of 10 lM over Aurora A and Aurora B to evaluate the Aurora
kinase inhibitory activity of the synthesized 1,2,3-triazolylsalicyla-
mide compounds. Table 1 shows % inhibition of kinase activity as
an average of duplicate assay compared to the known non-
selective kinase inhibitor, Staurosporine, as a positive control.
Among twenty six 1,2,3-triazolylsalicylamides, eleven analogues
(8e, 8g, 9a, 9d–h and 9l–n) showed high potency over 90% inhibi-
tion against Aurora A, while the mean inhibitions were moderate
in the rest of the compounds. Interestingly, these 11 analogues
were relatively less active against Aurora B (20.0–80.4% inhibition
at 10
lM). Meanwhile, it is worth noting that 1,2,3-triazolylbenza-
mide analogue 8a showed almost a lack of the mean inhibition

D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
4859
Table 2
IC50 of selected 1,2,3-triazolylsalicylamides over AurAa
b
8Compound
Structure
IC50 (lM)
AurA
1.89
AurB
OCH3
N
N
N
O
8e
47.5
3.58
Cl
N
H
OCH3
OCH3
HO
N
N
N
O
H3CO
8g
0.37
N
H
OCH3
CF3
O
HO
N
O
N
N
9a
9d
9e
0.69
1.44
0.72
13.8
NDc
31.9
F3C
F3C
N
H
CF3
HO
N
O
N
N
CN
N
H
HO
H3CO
N
O
N
N
OCH3
F3C
F3C
F3C
F3C
N
H
Cl
Cl
HO
N
O
N
N
NDc
NDc
NDc
NDc
N
H
HO
9f
1.56
1.50
3.83
1.88
N
O
O
N
N
N
H
HO
OCH3
9g
9h
9l
N
N
N
O
N
N
N
H
HO
O
N
N
S
F3C
F3C
N
H
HO
O
N
N
N
H
HO
9m
4.16
21.8
NH
O
N
O
N
N
F3C
N
9n
1.82
63.2
H
OCH3
HO
O
Staurosporine
0.001
0.006
a
b
c
Enzymatic assay was conducted by Reaction Biology Corporation (http://www.reactionbiology.com).
IC50 was determined in the presence of 10
Not determined.
lM ATP.
against both Aurora A and Aurora B, suggesting that the phenolic –
OH of the salicylamide scaffold plays a key role in the binding of
inhibitors to Aurora kinase.
Fig. 3), indicating that 3-(trifluoromethyl)phenyl moiety could be
favorably accommodated into the binding pocket of Aurora kinase.
IC50 values for these selected compounds were determined in a
After a single-dose preliminary screening, eleven 1,2,3-triazol-
ylsalicylamides 8e, 8g, 9a, 9d–h, 9l–n showing over 90% inhibition
against Aurora A were selected for further study (Table 2). Nine out
of eleven selected compounds belonged to compound 9 series and
bore the common 3-(trifluoromethyl)phenyl moiety (blue in
10-dose IC50 mode with 3-fold serial dilution starting at 30 lM
as listed in Table 2. Most of compounds inhibited Aurora A with
IC50 values in the nanomolar to low micromolar range. Especially,
compounds 8g, 9a and 9e revealed nanomolar IC50 values. Com-
pound 8g carrying –CO2CH3 group was the best among a series

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D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
stirring, unless otherwise noted. Air- and moisture-sensitive liq-
uids and solutions were transferred via syringe or stainless-steel
cannula. TLC was performed on 0.25 mm E. Merck silica gel 60
F254 plates and visualized under UV light (254 nm) or by staining
with cerium ammonium molybdenate (CAM), potassium perman-
ganate (KMnO4) or p-anisaldehyde. Flash chromatography was
performed on E. Merck 230ꢀ400 mesh silica gel 60. All reagents
were purchased from commercial suppliers, and used without
further purification unless otherwise noted. Solvents were dis-
tilled from proper drying agents (CaH2 or Na wire) under Ar
atmosphere at 760 mmHg. All moisture- and/or oxygen-sensitive
solids were handled and stored in a glovebox under N2. NMR
spectra were recorded on Varian Unity 400 instruments at
24 °C. Chemical shifts are expressed in ppm relative to TMS (1H,
0 ppm), CDCl3 (1H, 7.26 ppm; 13C, 77.2 ppm); coupling constants
are expressed in Hz. High resolution mass spectra electrospray
ionization (HRMS-ESI) was obtained on an Agilent technologies
6220 TOF LC/MS spectrometer or JEOL JMS-600 W 70 eV (Electron
Ionization).
Glu194
Gln190
Lys175
Figure 4. Docking poses for compound 8g (green) overlaid to SNS-314 (2) (cyan) in
the kinase domain of AurA. The docking structure shows that the salicylamide
group of 8g makes hydrogen-bonding network with Glu194 and Lys175.
of compounds and exhibited IC50 values of 0.37 lM. Notably, most
compounds showed some selectivity toward Aurora A and 5–50
times more strongly inhibited Aurora A than Aurora B. Compound
8g was about 10-fold more active for Aurora A than for Aurora B.
4.1.2. General procedure for the synthesis of azide 11
To a solution of 10 (36.0 mmol) in H2SO4 (27 mL) and H2O
(144 mL) was added a solution of NaNO2 (43.2 mmol) in H2O
(27 mL) at 0 °C. After stirring for 1.5 h at 0 °C, a solution of NaN3
(61.2 mmol) in H2O (22 mL) was added dropwise at 0 °C. The
resulting suspension was stirred for 1.5 h at 0 °C and for 15 h at
rt. After the completion of reaction, the mixture was extracted with
EtOAc (5 ꢁ 100 mL). The combined organic extracts were washed
with brine, dried over anhyd MgSO4, filtered, and evaporated to
afford azide 11.
2.4. Modeling
In order to better understand the observed SAR and the critical
effect of the phenolic –OH of salicylamides, we underwent docking
studies with compound 8g and Aurora A kinase (PDB code:
3d15).11 As shown in Figure 4, compound 8g was docked into the
binding pocket of Aurora A kinase using the crystal structure of
humanized mouse Aurora A co-crystallized with compound 2
(SNS-314) and the most stable docking pose was aligned with com-
pound 2 in the binding site. Compounds 8g and 2 occupy the same
binding pocket in a similar pose. Especially, the 2-aminothiazole
urea moiety of compound 2 forms a hydrogen-bonding network
with Lys175 and Glu194. Likewise, compound 8g interacts with
Lys175, Glu194 and Gln190 through hydrogen bonding. The car-
bonyl group of salicylamide scaffold acts as a hydrogen bonding
acceptor and forms a hydrogen bond with the side chain N–H of
Lys175, whereas the phenolic –OH of salicylamide scaffold acts
as a hydrogen bonding donor and makes another hydrogen bond
with the carboxylate of Glu194. Therefore, the benzamide scaffold
without phenolic –OH would lose the key hydrogen bonding inter-
action with Glu194, resulting in a significant decrease of activity,
which is consistent with the observed SAR data. In addition, meth-
ylester group also forms hydrogen bonding with Gln190.
4.1.2.1. 3-Azidobenzoic acid (11a).
Following the general
procedure for 11, using 3-aminobenzoic acid (10a) (5.00 g,
36.0 mmol), NaNO2 (3.00 g, 43.2 mmol), and NaN3 (4.00 g,
61.2 mmol). The resulting suspension was stirred for 1.5 h at 0 °C
and for 15 h at rt to afford azide 11a (5.87 g, 100%) as a gray solid.
TLC: Rf 0.22 (20:1 CH2Cl2/MeOH). 1H NMR (400 MHz, CDCl3): d 7.90
(ddd, J = 8.0, 2.4, 1.6 Hz, 1H), 7.78 (dd, J = 2.4, 1.6 Hz, 1H), 7.48 (t,
J = 8.0 Hz, 1H) 7.27 (ddd, J = 8.0, 2.4, 1.6 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6): d 166.4, 139.9, 132.6, 130.3, 125.8, 123.5,
119.4. HRMS m/z calcd for C7H5N3O2 163.0382; found 163.0384.
4.1.2.2. 5-Azido-2-hydroxybenzoic acid (11b).
Following the
general procedure for 11, using 5-aminosalicylic acid (10b) (15.4 g,
100 mmol), NaNO2 (8.32 g, 120 mmol), and NaN3 (11.1 g,
170 mmol). The resulting suspension was stirred for 1.5 h at 0 °C
and for 13 h at rt to afford 11b (17.5 g, 98%) as a reddish brown
solid. TLC: Rf 0.43 (5:1 EtOAc/MeOH). 1H NMR (400 MHz, DMSO-
d6): d 11.24 (br s, 1H), 7.42 (d, J = 2.8 Hz, 1H), 7.28 (dd, J = 8.8,
2.8 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H). 13C NMR (100 MHz, DMSO-
d6): d 170.9, 158.3, 130.2, 126.6, 119.7, 118.9, 113.9. HRMS m/z
calcd for C7H5N3O3 179.0331; found 179.0332.
3. Conclusions
In conclusion, we have developed Aurora kinase inhibitors
containing novel 1,2,3-triazolylsalicylamide scaffold, which could
be rapidly assembled by facile ‘click chemistry’: Cu(I)-catalyzed
1,3-dipolar cycloaddition between alkynes and azides. The repre-
sentative compound 8g exhibited inhibitory activity against Aur-
ora A kinase with an IC50 value of 0.37 lM. This compound could
be a good candidate for the further design of potent and selective
antiproliferative agents targeting Aurora kinase. The molecular
modeling study using compound 8g elucidated the phenolic –OH
of salicylamide scaffold is essential for the hydrogen bonding of
8g to Glu194 of Aurora A.
4.1.3. General procedure for the synthesis of 13
A solution of 3-azidobenzoic acid 11 (5.00 mmol) and 1,10-car-
bonyldiimidazole 90% (1.40 g, 7.50 mmol) in anhyd CH3CN (6 mL)
was stirred for 3 h at 80 °C under Ar atmosphere. Then, the corre-
sponding aniline 12 (15.0 mmol) and triethylamine (1.00 mL,
7.50 mmol) were added at 80 °C. The resulting solution was stir-
red at 80 °C for 24 h. After the completion of the reaction, the
reaction mixture was diluted with CH2Cl2 (200 mL), neutralized
with 1 N HCl (200 mL), stirred at rt for 30 min, and extracted with
CH2Cl2 (2 ꢁ 200 mL). The combined organic extracts were dried
over anhyd MgSO4, filtered, and concentrated by rotary evapora-
tion. The residue was purified by column chromatography to
afford 13.
4. Experimental
4.1. Chemistry
4.1.1. General
All reactions were performed in flame-dried glassware fitted
with a glass stopper under positive pressure of Ar with magnetic

D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
4861
4.1.3.1.
(13a).
3-Azido-N-(3-(trifluoromethyl)phenyl)benzamide
Following the general procedure for 13, using 3-azido-
5-azido-2-hydroxybenzoic acid (11b) (896 mg, 5.00 mmol), 1,10-
carbonyldiimidazole 90% (1.40 g, 7.50 mmol), 3-chloroaniline
(12e) (1.60 mL, 15.0 mmol), and triethylamine (1.00 mL,
7.50 mmol) in CH3CN (6 mL). The reaction mixture was stirred
for 16 h at 80 °C. Column chromatography (3:1 hexane/EtOAc)
afforded 13e (78.5 mg, 5%) as a brown solid. TLC: Rf 0.39 (3:1 hex-
ane/EtOAc). Mp: 156.5ꢀ158.5 °C. 1H NMR (400 MHz, DMSO-d6): d
11.55 (br s, 1H), 10.51 (s, 1H), 7.92 (t, J = 2.0 Hz, 1H), 7.65–7.59
(m, 2H), 7.40 (t, J = 8.4 Hz, 1H), 7.21ꢀ7.19 (m, 2H), 7.05 (d,
J = 8.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): d 165.9, 155.7,
140.3, 133.8, 131.2, 131.1, 131.0, 125.1, 124.6, 120.9, 120.1,
119.8, 119.5. HRMS m/z calcd for C13H9ClN4O2 288.0414; found
288.0419.
benzoic acid (11a) (816 mg, 5.00 mmol), 1,10-carbonyldiimidazole
90% (1.40 g, 7.50 mmol), 3-(trifluoromethyl)aniline (12a)
(1.90 mL, 15.0 mmol), and triethylamine (1.00 mL, 7.50 mmol) in
anhyd CH3CN (6 mL). The reaction mixture was stirred at 80 °C
for 24 h. Column chromatography (4:1 hexane/EtOAc) afforded
13a (1.40 g, 91%) as a brown solid. TLC: Rf 0.38 (4:1 hexane/EtOAc).
Mp: 110ꢀ112 °C. 1H NMR (400 MHz, CDCl3): d 7.94 (s, 1H),
7.87ꢀ7.85 (m, 2H), 7.60 (ddd, 1H, J = 7.6, 1.6, 0.8 Hz), 7.56 (t,
J = 2.0 Hz, 1H), 7.53ꢀ7.48 (m, 2H), 7.43 (d, J = 7.6 Hz, 1H), 7.24
(ddd, J = 7.6, 2.0, 0.8 Hz, 1H). 13C NMR (100 MHz, CDCl3): d 164.9,
139.9, 139.7, 136.1, 130.2, 129.9, 129.2 (q, JC–F = 31.5 Hz), 124.4,
124.1 (q, JC–F = 271.0 Hz), 123.9, 122.5, 120.1 (q, JC–F = 3.6 Hz),
118.2, 116.5 (q, JC–F = 3.6 Hz). HRMS m/z calcd for C14H9F3N4O
306.0728; found 306.0730.
4.1.3.6. 5-Azido-2-hydroxy-N-(2-(trifluoromethyl)phenyl)benz-
amide (13f).
Following the general procedure for 13, using
5-azido-2-hydroxybenzoic acid (11b) (896 mg, 5.00 mmol), 1,10-
carbonyldiimidazole 90% (1.40 g, 7.50 mmol), 2-(trifluoro-
methyl)aniline (12f) (1.90 mL, 15.0 mmol), and triethylamine
(1.00 mL, 7.50 mmol) in CH3CN (6 mL). The reaction mixture was
stirred for 24 h at 80 °C. Column chromatography (3:1 hexane/
EtOAc) afforded 13f (280 mg, 17%) as a brown solid. TLC: Rf 0.17
(5:1 hexane/EtOAc). Mp: 129.5ꢀ131.5 °C. 1H NMR (400 MHz,
DMSO-d6): d 12.06 (br s, 1H), 10.82 (br s, 1H), 8.19 (d, J = 8.0 Hz,
1H), 7.83ꢀ7.73 (m, 2H), 7.12 (d, J = 2.8 Hz, 1H), 7.42 (t, J = 8.0 Hz,
1H), 7.25 (dd, J = 8.8, 2.8 Hz, 1H), 7.08 (d, J = 8.8 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6): d 163.7, 154.3, 135.2, 133.3, 130.9, 126.2,
126.1, 125.4, 125.0, 123.8 (q, JC–F = 271.7 Hz), 120.9 (q, JC–F = 29.3 Hz),
120.2, 118.8, 118.6. HRMS m/z calcd for C14H9F3N4O2 322.0678;
found 322.0682.
4.1.3.2. 5-Azido-2-hydroxy-N-phenylbenzamide (13b).
Fol-
lowing the general procedure for 13, using 5-azido-2-hydroxyben-
zoic acid (11b) (896 mg, 5.00 mmol), 1,10-carbonyldiimidazole 90%
(1.40 g, 7.50 mmol), aniline (12b) (1.40 mL, 15.0 mmol), and trieth-
ylamine (1.00 mL, 7.50 mmol) in anhyd CH3CN (6 mL). The reaction
mixture was stirred at 80 °C for 2 h. Column chromatography (4:1
hexane/EtOAc) afforded 13b (537 mg, 42%) as a brown solid. TLC:
Rf 0.37 (3:1 hexane/EtOAc). Mp: 160ꢀ162 °C. 1H NMR (400 MHz,
CDCl3): d 11.76 (s, 1H), 7.81 (br s, 1H), 7.58 (d, J = 7.6 Hz, 2H),
7.42 (t, J = 7.6 Hz, 2H), 7.23 (t, J = 7.6 Hz, 1H), 7.19 (dd, J = 8.8,
2.8 Hz, 1H), 7.10 (d, J = 2.8 Hz, 1H), 7.06 (d, J = 8.8 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6): d 165.2, 155.5, 138.0, 130.3, 128.7,
124.4, 124.3, 120.9, 119.2, 118.9, 118.8. HRMS m/z calcd for
C13H10N4O2 254.0804; found 254.0801.
4.1.3.7.
3-(5-Azido-2-hydroxybenzoylamino)benzoic
acid
4.1.3.3. 5-Azido-2-hydroxy-N-m-tolylbenzamide (13c).
Fol-
methyl ester (13g).
Following the general procedure for
lowing the general procedure for 13, using 5-azido-2-hydroxyben-
zoic acid (11b) (500 mg, 2.80 mmol), 1,10-carbonyldiimidazole 90%
(780 mg, 4.10 mmol), m-tolylamine (12c) (1.00 mL, 8.37 mmol),
13, using 5-azido-2-hydroxybenzoic acid (11b) (896 mg,
5.00 mmol), 1,10-carbonyldiimidazole 97% (1.25 g, 7.50 mmol),
3-aminobenzoic acid methyl ester (12g) (2.27 g, 15.0 mmol),
and triethylamine (1.00 mL, 7.50 mmol) in CH3CN (7.5 mL). The
reaction mixture was stirred for 16 h at 80 °C. Column chroma-
tography (4:1 hexane/EtOAc) afforded 13g (332 mg, 21%) as a
brown solid. TLC: Rf 0.11 (5:1 hexane/EtOAc). Mp: 160ꢀ162 °C.
1H NMR (400 MHz, DMSO-d6): d 11.61 (br s, 1H), 10.76 (br s,
1H), 8.40 (t, J = 1.6 Hz, 1H), 7.94 (dm, J = 8.0 Hz, 1H) 7.73 (dt,
J = 8.0, 1.6 Hz, 1H), 7.65 (d, J = 2.8 Hz, 1H), 7.53 (t, J = 8.0 Hz,
1H), 7.21 (dd, J = 8.8, 2.8 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H) 3.88 (s,
and triethylamine (560 lL, 4.10 mmol) in anhyd CH3CN
(3.50 mL). The reaction mixture was stirred for 2.5 h at 80 °C. Col-
umn chromatography (6:1 hexane/EtOAc) afforded 13c (460 mg,
61%) as a yellow solid. TLC: Rf 0.44 (4:1 hexane/EtOAc). Mp:
141ꢀ143 °C. 1H NMR (400 MHz, DMSO-d6): d 11.81 (br s, 1H),
10.40 (s, 1H), 7.68 (d, J = 2.8 Hz, 1H), 7.52 (s, 1H), 7.49 (d,
J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.21 (dd, J = 8.8, 2.8 Hz,
1H), 7.04 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H), 2.32 (s, 3H).
13C NMR (100 MHz, DMSO-d6): d 165.2, 155.5, 138.0, 137.9,
130.3, 128.6, 125.0, 124.4, 121.4, 119.2, 118.8, 118.7, 118.1, 21.1.
HRMS m/z calcd for C14H12N4O2 268.0960; found 268.0961.
3H). 13C NMR (100 MHz, DMSO-d6):
d 166.0, 165.5, 155.4,
138.5, 130.4, 130.2, 129.3, 125.3, 124.8, 124.5, 121.2, 119.3,
119.0, 118.8, 52.2. HRMS m/z calcd for C15H12N4O4 312.0859;
found 312.0856.
4.1.3.4.
(13d).
5-Azido-2-hydroxy-N-(3-methoxyphenyl)benzamide
Following the general procedure for 13, using
4.1.3.8. 5-Azido-2-hydroxy-N-(3-(trifluoromethyl)phenyl)benz-
5-azido-2-hydroxybenzoic acid (11b) (896 mg, 5.00 mmol), 1,10-
carbonyldiimidazole 90% (1.40 g, 7.50 mmol), 3-methoxyaniline
(12d) (1.70 mL, 15.0 mmol), and triethylamine (1.00 mL,
7.50 mmol) in anhyd CH3CN (6 mL). The reaction mixture was stir-
red for 15 h at 80 °C. Column chromatography (4:1 hexane/EtOAc)
afforded 13d (738 mg, 52%) as a brown solid. TLC: Rf 0.45 (5:1 hex-
ane/EtOAc). Mp: 145ꢀ147 °C. 1H NMR (400 MHz, DMSO-d6): d
11.72 (br s, 1H), 10.59 (br s, 1H), 7.64 (d, J = 2.8 Hz, 1H), 7.40 (t,
J = 2.4 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.25ꢀ7.18 (m, 2H), 7.01 (d,
J = 8.8 Hz, 1H), 6.72 (d, J = 7.6 Hz, 1H), 3.76 (s, 3H). 13C NMR
(100 MHz, DMSO-d6): d 165.2, 159.6, 155.3, 139.2, 130.4, 129.6,
124.4, 119.3, 119.2, 118.9, 113.1, 109.9, 106.6, 55.1. HRMS m/z
calcd for C14H12N4O3 284.0909; found 284.0911.
amide (13h).
Following the general procedure for 13, using
5-azido-2-hydroxybenzoic acid (11b) (1.79 g, 10.0 mmol) 1,10-car-
bonyldiimidazole 97% (2.70 g, 15.0 mmol), and 3-(trifluoro-
methyl)aniline (12a) (3.80 mL, 30.0 mmol) and triethylamine
(2.1 mL, 15.0 mmol) in CH3CN (11 mL). The reaction mixture was
stirred for 13.5 h at 80 °C. Column chromatography (6:1 hexane/
EtOAc) afforded 13h (700 mg, 22%) as a yellow solid. TLC: Rf 0.40
(4:1 hexane/EtOAc). Mp: 149ꢀ151 °C. 1H NMR (400 MHz, DMSO-
d6): d 11.50 (br s, 1H), 11.04 (br s, 1H), 8.22 (s, 1H), 7.97 (d,
J = 7.6 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 2.8 Hz, 1H), 7.48
(d, J = 7.6 Hz, 1H), 7.19 (dd, J = 8.8, 2.8 Hz, 1H), 7.01 (d, J = 8.8 Hz,
1H). 13C NMR (100 MHz, DMSO-d6):
d 165.5, 155.1, 138.9,
130.4, 129.9, 129.5 (q, JC–F = 31.6 Hz), 124.4, 124.3, 124.1 (d,
JC–F = 270.3 Hz), 120.5 (d, JC–F = 3.8 Hz), 119.3, 119.2, 118.8, 116.8
(d, JC–F = 3.7 Hz). HRMS m/z calcd for C14H9F3N4O2 322.0678; found
322.0674.
4.1.3.5.
(13e).
5-Azido-N-(3-chlorophenyl)-2-hydroxybenzamide
Following the general procedure for 13, using

4862
D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
4.1.4. General procedure for the synthesis of 1,2,3-triazole 8 and
9 [click reaction]
m/z (rel int): 431 ([M+H]+, 100), 338 ([MꢀC7H8]+, 6). HRMS m/z
calcd for C24H22N4O4 430.1631; found 430.1634.
Azide 13 (300
lmol), alkyne (750
lmol), CuSO4 (9.6 mg,
60
lmol) and sodium ascorbate (60.0 mg, 300
l
mol) were dis-
4.1.4.4.
5-(4-(3,5-Dimethoxyphenyl)-1H-1,2,3-triazol-1-yl)-2-
solved in 1:1 t-BuOH/H2O (3 mL). The mixture was stirred for 2 h
at rt. After the completion of the reaction, the mixture was filtered
and washed with H2O (70 mL) (solid A). The filtrate was extracted
with EtOAc (3 ꢁ 70 mL). The combined organic extracts were dried
over anhyd MgSO4, filtered, and concentrated by rotary evapora-
tion (residue B). The solid A and residue B were combined and puri-
fied by column chromatography to afford 8 or 9.
hydroxy-N-(3-methoxyphenyl)benzamide (8d).
the general procedure for 8, using 13d (85.3 mg, 300
Following
mol), 1-
mol), CuSO4
l
ethynyl-3,5-dimethoxybenzene (121 mg, 750
l
(9.6 mg, 60 mol) and sodium ascorbate (60.0 mg, 300
l
lmol) in
1:1 t-BuOH/H2O (3 mL). The reaction mixture was stirred for 47 h
at 40 °C. Column chromatography (5:1 hexane/EtOAc ? 20:1 CH2-
Cl2/MeOH) afforded 8d (98.8 mg, 74%) as a brown solid. TLC: Rf
0.50 (20:1 CH2Cl2/MeOH). Mp: 183ꢀ185 °C. 1H NMR (400 MHz,
DMSO-d6): d 11.93 (br s, 1H), 10.67 (br s, 1H), 9.28 (s, 1H), 8.38
(d, J = 2.8 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.45 (t, J = 1.6 Hz, 1H),
7.29–7.28 (m, 2H), 7.19 (d, J = 8.8 Hz, 1H), 7.14 (d, J = 2.4 Hz, 2H),
6.73 (m, 1H), 6.51 (t, J = 2.4 Hz, 1H), 3.82 (s, 6H), 3.77 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): d 164.8, 161.0, 159.6, 157.5, 147.2,
139.3, 132.1, 129.6, 128.8, 125.2, 121.3, 120.1, 119.7, 118.4,
112.9, 109.8, 106.4, 103.2, 100.2, 55.3, 55.1. LRMS m/z (rel int):
447 ([M+H]+, 100), 356 ([MꢀC3H6O3]+, 10), 338 ([MꢀC7H8O]+, 4).
HRMS m/z calcd for C24H22N4O5 446.1590; found 446.1592.
4.1.4.1. 3-(4-(3,5-Dimethoxyphenyl)-1H-1,2,3-triazol-1-yl)-N-(3-
(trifluoromethyl)phenyl)benzamide (8a).
general procedure for 8, using 13a (95.8 mg, 300
Following the
mol), 1-ethy-
l
nyl-3,5-dimethoxybenzene (121 mg, 750
l
mol), CuSO4 (9.6 mg,
60 mol) and sodium ascorbate (60.0 mg, 300
l
lmol) in 1:1 t-
BuOH/H2O (3 mL). The reaction mixture was stirred for 2 h at rt.
Column chromatography (5:1 hexane/EtOAc ? 40:1 CH2Cl2/
MeOH) afforded 8a (138 mg, 98%) as a brown solid. TLC: Rf 0.58
(20:1 CH2Cl2/MeOH). Mp: 160ꢀ162 °C. 1H NMR (400 MHz,
DMSO-d6): d 10.79 (br s, 1H), 9.47 (s, 1H), 8.54 (t, J = 2.0 Hz, 1H),
8.27 (s, 1H), 8.20 (dm, J = 8.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 8.09
(d, J = 8.0 Hz, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H),
7.50 (d, J = 8.0 Hz, 1H), 7.16 (d, J = 2.0 Hz, 2H), 6.54 (t, J = 2.0 Hz,
1H), 3.83 (s, 6H). 13C NMR (100 MHz, DMSO-d6): d 164.8,
161.0, 147.4, 139.7, 136.7, 136.2, 131.9, 130.3, 130.0, 129.4
(d, JC–F = 30.8 Hz), 127.8, 124.2 (d, JC–F = 270.0 Hz), 123.9, 123.1,
120.2, 119.2, 116.5, 116.4, 103.3, 100.3, 55.3. LRMS m/z (rel int):
469 ([M+H]+, 100), 437 ([MꢀCH3O]+, 12). HRMS m/z calcd for
4.1.4.5.
N-(3-Chlorophenyl)-5-(4-(3,5-dimethoxyphenyl)-1H-
1,2,3-triazol-1-yl)-2-hydroxybenzamide (8e).
general procedure for 8, using 13e (29.5 mg, 100
Following the
lmol), 1-ethy-
nyl-3,5-dimethoxybenzene (40.6 mg, 250
l
mol), CuSO4 (3.2 mg,
20 mol) and sodium ascorbate (20.0 mg, 100
l
l
mol) in 1:1 t-
BuOH/H2O (3 mL). The reaction mixture was stirred for 66 h at rt.
Column chromatography (5:1 hexane/EtOAc ? 30:1 CH2Cl2/
MeOH) afforded 8e (23.1 mg, 51%) as a brown solid. TLC: Rf 0.47
(20:1 CH2Cl2/MeOH). Mp: 226ꢀ228 °C. 1H NMR (400 MHz,
DMSO-d6): d 10.60 (br s, 1H), 9.09 (s, 1H), 8.29 (d, J = 2.0 Hz, 1H),
7.91–7.87 (m, 2H), 7.54 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H),
7.17 (d, J = 8.0 Hz, 1H), 7.07 (d, J = 2.4 Hz, 2H), 7.00 (m, 1H), 6.47
(t, J = 2.4 Hz, 1H), 3.90 (s, 6H). 13C NMR (100 MHz, DMSO-d6): d
165.5, 161.5, 157.9, 147.8, 140.0, 133.8, 132.4, 131.2, 129.3,
126.0, 124.7, 122.0, 120.7, 120.6, 120.2, 119.6, 119.0, 103.9,
100.8, 55.9. LRMS m/z (rel int): 451 ([M+H]+, 100), 356 ([MꢀC2H3-
O2Cl]+, 25). HRMS m/z calcd for C23H19ClN4O4 450.1096; found
450.1096.
C24H19F3N4O3 468.1409; found 468.1410.
4.1.4.2. 5-(4-(3,5-Dimethoxyphenyl)-1H-1,2,3-triazol-1-yl)-2-
hydroxy-N-phenylbenzamide (8b).
procedure for 8, using 13b (76.6 mg, 300
Following the general
mol), 1-ethynyl-3,5-
mol)
mol) in 1:1 t-BuOH/H2O
l
dimethoxybenzene (121 mg, 750
lmol), CuSO4 (9.6 mg, 60 l
and sodium ascorbate (60.0 mg, 300
l
(3 mL). The reaction mixture was stirred for 48 h at rt. Column chro-
matography (5:1 hexane/EtOAc ? 20:1 CH2Cl2/MeOH) afforded 8b
(90.6 mg, 73%) as a brown solid. TLC: Rf 0.48 (20:1 CH2Cl2/MeOH).
Mp: 202ꢀ204 °C. 1H NMR (400 MHz, DMSO-d6): d 11.97 (s, 1H),
10.55 (br s, 1H), 9.28 (s, 1H), 8.41 (d, J = 2.8 Hz, 1H), 7.97 (dd,
J = 2.8, 8.8 Hz, 1H), 7.74 (d, J = 7.6 Hz, 2H), 7.40 (t, J = 7.6 Hz, 2H),
7.22 (d, J = 8.8 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.14 (d, J = 2.0 Hz,
2H), 6.52 (t, J = 2.0 Hz, 1H), 3.82 (s, 6H). 13C NMR (100 MHz,
DMSO-d6): d 164.9, 160.9, 157.7, 147.1, 138.1, 132.1, 128.8, 128.7,
125.2, 124.3, 121.2, 120.7, 120.1, 119.5, 118.4, 103.2, 100.2, 55.3.
LRMS m/z (rel int): 417 ([M+H]+, 100), 309 ([MꢀC7H7O]+, 12). HRMS
m/z calcd for C23H20N4O4 416.1485; found 416.1487.
4.1.4.6.
5-(4-(3,5-Dimethoxyphenyl)-1H-1,2,3-triazol-1-yl)-2-
hydroxy-N-(2-(trifluoromethyl)phenyl)benzamide (8f).
lowing the general procedure for 8, using 13f (34.6 mg, 100
1-ethynyl-3,5-dimethoxybenzene (40.1 mg, 250
Fol-
lmol),
l
mol), CuSO4
(3.2 mg, 20 mol) and sodium ascorbate (20.0 mg, 100
l
lmol) in
1:1 t-BuOH/H2O (3 mL). The reaction mixture was stirred for
3.5 h at rt. Column chromatography (5:1 hexane/EtOAc ? 20:1
CH2Cl2/MeOH) afforded 8f (39.7 mg, 82%) as a brown solid. TLC:
Rf 0.47 (20:1 CH2Cl2/MeOH). Mp: 188ꢀ190 °C. 1H NMR (400 MHz,
DMSO-d6): d 12.50 (br s, 1H), 10.87 (br s, 1H), 9.32 (s, 1H), 8.53
(d, J = 2.8 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.03 (dd, J = 8.8, 2.8 Hz,
1H), 7.80 (d, J = 8.0 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.43 (t,
J = 8.0 Hz, 1H), 7.27 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 2.4 Hz, 2H),
6.51 (t, J = 2.4 Hz, 1H), 3.82 (s, 6H). 13C NMR (100 MHz,
DMSO-d6): d 163.3, 160.9, 156.7, 147.2, 135.2, 133.4, 132.1,
129.3, 126.2, 126.0, 125.9, 125.4, 124.1 (q, JC–F = 270 Hz), 122.3,
120.1, 118.6, 118.4, 103.3, 100.2, 99.3, 55.3. LRMS m/z (rel int):
485 ([M+H]+, 100), 338 ([MꢀC7H5F3]+, 4). HRMS m/z calcd for
C24H19F3N4O4 484.1358; found 484.1354.
4.1.4.3.
5-(4-(3,5-Dimethoxyphenyl)-1H-1,2,3-triazol-1-yl)-2-
hydroxy-N-(m-tolyl)benzamide (8c).
procedure for 8, using 13c (80.8 mg, 300
Following the general
mol), 1-ethynyl-3,5-
mol)
mol) in 1:1 t-BuOH/H2O
l
dimethoxybenzene (121 mg, 750
lmol), CuSO4 (9.6 mg, 60 l
and sodium ascorbate (60.0 mg, 300
l
(3 mL). The reaction mixture was stirred for 5.5 h at rt. Column
chromatography (5:1 hexane/EtOAc ? 20:1 CH2Cl2/MeOH) affor-
ded 8c (104 mg, 80%) as a brown solid. TLC: Rf 0.48 (20:1 CH2Cl2/
MeOH). Mp: 204ꢀ206 °C. 1H NMR (400 MHz, DMSO-d6): d 12.00
(br s, 1H), 10.48 (br s, 1H), 9.27 (s, 1H), 8.42 (d, J = 2.8 Hz, 1H),
7.97 (dd, J = 8.8, 2.8 Hz, 1H), 7.56 (s, 1H), 7.54 (d, J = 7.6 Hz, 1H),
7.28 (t, J = 7.6 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.14 (d, J = 2.4 Hz,
2H), 6.98 (d, J = 7.6 Hz, 1H), 6.52 (t, J = 2.4 Hz, 1H), 3.82 (s, 6H),
2.33 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d 164.8, 162.0, 160.9,
157.8, 147.1, 138.1, 138.0, 132.1, 128.7, 128.6, 125.3, 125.0,
121.2, 120.1, 119.4, 118.4, 117.9, 103.2, 100.2, 55.3, 21.1. LRMS
4.1.4.7. Methyl 3-(5-(4-(3,5-dimethoxyphenyl)-1H-1,2,3-triazol-
1-yl)-2-hydroxybenzamido)benzoate (8g).
general procedure for 8, using 13g (31.2 mg, 100
Following the
mol), 1-ethy-
l
nyl-3,5-dimethoxybenzene (40.5 mg, 250
l
mol), CuSO4 (3.2 mg,
20 mol) and sodium ascorbate (20.0 mg, 100
l
l
mol) in 1:1

D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
4863
t-BuOH/H2O (1 mL). The reaction mixture was stirred for 5 h at
60 °C. Column chromatography (5:1 hexane/EtOAc ? 20:1 CH2Cl2/
MeOH) afforded 8g (33.6 mg, 71%) as a brown solid. TLC: Rf 0.12
(30:1 CH2Cl2/MeOH). Mp: 273ꢀ275 °C. 1H NMR (400 MHz, DMSO-
d6): d 11.84 (br s, 1H), 10.70 (br s, 1H), 9.28 (s, 1H), 8.44 (t,
J = 1.6 Hz, 1H), 8.41 (d, J = 2.8 Hz, 1H), 8.00–7.97 (m, 2H), 7.75
(dm, J = 8.0 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.23 (d, J = 8.8 Hz, 1H),
7.14 (d, J = 2.4 Hz, 2H), 6.52 (t, J = 2.4 Hz, 1H), 3.89 (s, 3H), 3.82 (s,
6H). 13C NMR (100 MHz, DMSO-d6): d 166.0, 165.3, 160.9, 157.9,
147.1, 138.6, 132.1, 130.2, 129.3, 128.6, 125.3, 125.2, 124.8, 121.7,
121.1, 120.2, 119.4, 118.4, 103.2, 100.2, 55.3, 52.2. LRMS m/z (rel
int): 475 ([M+H]+, 100), 441 ([MꢀCH5O]+, 20), 338 ([MꢀC8H8O2]+,
6). HRMS m/z calcd for C25H22N4O6 474.1539; found 474.1538.
(s, 1H), 8.37 (d, J = 2.8 Hz, 1H), 8.25 (s, 1H), 7.98–7.95 (m, 2H),
7.75 (d, J = 9.2 Hz, 2H), 7.64 (t, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz,
1H), 7.21 (d, J = 8.8 Hz, 1H), 6.82 (d, J = 9.2 Hz, 2H), 2.95 (s, 6H).
13C NMR (100 MHz, DMSO-d6): d 165.4, 157.2, 150.2, 147.9,
139.1, 130.0, 129.5 (q, JC–F = 31.8 Hz), 129.0, 126.2, 125.2, 124.2,
124.1 (q, JC–F = 270.9 Hz), 121.1, 120.5 (q, JC–F = 3.1 Hz), 119.6,
118.3, 118.0, 117.7, 116.6 (q, JC–F = 3.8 Hz), 112.3, 40.0. LRMS m/z
(rel int): 468 ([M+H]+, 100). HRMS m/z calcd for C24H20F3N5O2
467.1569; found 467.1569.
4.1.4.11. 5-(4-(4-Cyanophenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-
N-(3-(trifluoromethyl)phenyl)benzamide (9d).
the general procedure for 9, using 13h (96.7 mg, 300
Following
mol), 4-eth-
l
ynylbenzonitrile (98.3 mg, 750
l
mol), CuSO4 (9.6 mg, 60
lmol)
4.1.4.8. 5-(4-(3,5-Bis(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-
and sodium ascorbate (59.4 mg, 300
lmol) in 1:1 t-BuOH/H2O
2-hydroxy-N-(3-(trifluoromethyl)phenyl)benzamide (9a).
lowing the general procedure for 9, using 13h (96.7 mg, 300
Fol-
mol),
mol),
(3 mL). The reaction mixture was stirred for 2 h at 80 °C. Column
chromatography (3:1 hexane/EtOAc) afforded 9d (42.0 mg, 31%)
as a yellow solid. TLC: Rf 0.40 (4:1 hexane/EtOAc). Mp:
l
l
1-ethynyl-3,5-bis(trifluoromethyl)benzene (133
CuSO4 (9.6 mg, 60
300 mol) in 1:1 t-BuOH/H2O (3 mL).The reaction mixture was
lL, 750
l
mol) and sodium ascorbate (59.4 mg,
244ꢀ246 °C. 1H NMR (400 MHz, DMSO-d6): d 11.79 (br s, 1H),
10.86 (br s, 1H), 9.45 (s, 1H), 8.38 (d, J = 2.4 Hz, 1H), 8.25 (s, 1H),
8.15–8.13 (m, 2H), 7.99–7.96 (m, 4H), 7.63 (t, J = 8.0 Hz, 1H), 7.50
(d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.8 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6): d 165.1, 157.9, 145.6, 139.2, 134.8, 133.1, 130.1, 129.5
(q, JC–F = 31.7 Hz), 128.3, 125.8, 125.4, 124.1 (q, JC–F = 270.8 Hz),
124.0, 121.6, 121.4, 120.5 (m), 120.0, 118.8, 118.5, 116.6 (q,
JC–F = 3.9 Hz), 110.4. LRMS m/z (rel int): 450 ([M+H]+, 100), 304
([MꢀC7H4F3]+, 52). HRMS m/z calcd for C23H14F3N5O2 449.1100;
found 449.1100.
l
stirred for 2.5 h at rt. Column chromatography (30:1 CH2Cl2/
MeOH) afforded 6h (166 mg, 99%) as an ivory solid. TLC: Rf 0.24
(3:1 hexane/EtOAc). Mp: 194ꢀ196 °C. 1H NMR (400 MHz,
DMSO-d6): d 11.82 (br s, 1H), 10.75 (br s, 1H), 9.63 (s, 1H), 8.57
(s, 2H), 8.38 (d, J = 2.8 Hz, 1H), 8.25 (s, 1H), 8.10 (s, 1H),
7.96–7.93 (m, 2H), 7.62 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H),
7.25 (d, J = 9.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): d 164.9,
157.3, 144.5, 139.1, 132.9, 131.1 (q, JC–F = 32.4 Hz), 130.0, 129.5
(q, JC–F = 31.7 Hz), 128.5, 125.4 (m), 125.1, 124.1 (q, JC–F
=
270.8 Hz), 123.2 (q, JC–F = 270.8 Hz), 124.1, 122.7, 121.6, 121.3
(m), 120.5 (m), 120.1, 118.4, 116.6 (m). LRMS m/z (rel int): 561
([M+H]+, 100), 541 ([MꢀF]+, 5). HRMS m/z calcd for C24H13F9N4O2
560.0895; found 560.0894.
4.1.4.12.
zol-1-yl)-2-hydroxy-N-(3-(trifluoromethyl)phenyl)benzamide
(9e). Following the general procedure for 9, using 13h
(63.7 mg, 200 mol) and 1-chloro-5-ethynyl-2,4-dimethoxyben-
zene (106 mg, 500 mol) CuSO4 (6.4 mg, 40 mol) and sodium
ascorbate (39.6 mg, 200 mol) in 1:1 t-BuOH/H2O (2 mL). The
5-(4-(5-Chloro-2,4-dimethoxyphenyl)-1H-1,2,3-tria-
l
l
l
4.1.4.9.
hydroxy-N-(3-(trifluoromethyl)phenyl)benzamide
(9b). Following the general procedure for 9, using 13h
(96.7 mg, 300 mol), 1-ethynyl-3,5-difluorobenzene (55.0 L,
450 mol), CuSO4 (4.8 mg, 30 mol) and sodium ascorbate
(29.7 mg, 150 mol) in 1:1 t-BuOH/H2O (3 mL). The reaction mix-
ture was stirred for 3.25 h at rt. Then, additional CuSO4 (9.6 mg,
60.0 mol) and 1-ethynyl-3,5-difluorobenzene (18.0 L, 150 mol)
5-(4-(3,5-Difluorophenyl)-1H-1,2,3-triazol-1-yl)-2-
l
reaction mixture was stirred for 3 h at rt and for 16.5 h at 60 °C.
Column chromatography (5:1 ? 1:1 hexane/EtOAc) afforded 9e
(48.6 mg, 47%) as a brown solid. TLC: Rf 0.41 (20:1 CH2Cl2/MeOH).
Mp: 266.5ꢀ268.5 °C. 1H NMR (400 MHz, DMSO-d6): d 11.75 (br s,
1H), 10.79 (br s, 1H), 8.86 (s, 1H), 8.39 (d, J = 2.8 Hz, 1H), 8.25 (s,
1H), 8.13 (s, 1H), 8.01 (dd, J = 8.8, 2.8 Hz, 1H), 7.98 (d, J = 8.0 Hz,
1H), 7.64 (t, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.21 (d,
J = 8.8 Hz, 1H), 6.93 (s, 1H), 4.03 (s, 3H), 3.96 (s, 3H). 13C NMR
(100 MHz, DMSO-d6): d 165.6, 157.6, 155.6, 155.1, 141.8, 139.0,
130.0, 129.5 (q, JC–F = 31.5 Hz), 128.7, 126.9, 125.8, 124.3, 124.1
(q, JC–F = 271.0 Hz), 121.4, 121.1, 120.5, 119.4, 118.2, 116.8
(q, JC–F = 3.6 Hz), 112.5, 111.9, 97.8, 56.4, 56.2. LRMS m/z (rel int):
519 ([M+H]+, 100), 338 ([MꢀC7H4F3Cl]+, 8). HRMS m/z calcd for
l
l
l
l
l
l
l
l
were added and stirred for 45 h at rt. Column chromatography (4:1
hexane/EtOAc) afforded 9b (64.6 mg, 47%) as a light-yellow solid.
TLC: Rf 0.10 (6:1 hexane/EtOAc). Mp: 345ꢀ347 °C. 1H NMR
(400 MHz, DMSO-d6): d 11.76 (s, 1H), 10.80 (s, 1H), 9.38 (s, 1H),
8.33 (d, J = 2.8 Hz, 1H), 8.23 (s, 1H), 7.95–7.92 (m, 2H), 7.63 (dd,
JH–F = 9.6 Hz, JH–H = 2.4 Hz, 2H), 7.61 (t, J = 8.0 Hz, 1H), 7.48 (d, J =
8.0 Hz, 1H), 7.25 (tt, JH–F = 9.6 Hz, JH–H = 2.4 Hz, 1H), 7.22 (d,
J = 8.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): d 165.0, 162.9 (dd,
JC–F = 244.7, 13.9 Hz), 157.5, 145.2, 139.1, 133.8, 130.1, 129.5
(q, JC–F = 31.5 Hz), 128.5, 125.3, 124.1, 124.0 (q, JC–F = 270 Hz), 121.5,
121.2, 120.5 (m), 120.1, 118.4, 116.6 (m), 108.2 (d, JC–F = 26.4 Hz),
103.4 (t, JC–F = 26.4 Hz). LRMS m/z (rel int): 461 ([M+H]+, 100).
HRMS m/z calcd for C22H13F5N4O2 460.0959; found 460.0957.
C24H18ClF3N4O4 518.0969; found 518.0970.
4.1.4.13. 5-(4-(3-Chlorophenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-
N-(3-(trifluoromethyl)phenyl)benzamide (9f). Following
the general procedure for 9, using 13h (48.3 mg, 150 mol),
1-chloro-3-ethynylbenzene (46.2 L, 375 mol), CuSO4 (5.0 mg,
30.0 mol) and sodium ascorbate (30.0 mg, 150 mol) were
l
l
l
l
l
dissolved in 1:1 t-BuOH/H2O (1.5 mL). The reaction mixture was
stirred for 3 h at rt and 1 h at 60 °C. Column chromatography
(6:1 hexane/EtOAc ? 30:1 CH2Cl2/MeOH) afforded 9f (49.0 mg,
71%) as a brown solid. TLC: Rf 0.41 (20:1 CH2Cl2/MeOH). Mp:
260ꢀ262 °C. 1H NMR (400 MHz, DMSO-d6): d 11.76 (br s, 1H),
10.76 (br s, 1H), 9.36 (s, 1H), 8.38 (d, J = 2.8 Hz, 1H), 8.25 (s, 1H),
8.01–7.97 (m, 3H) 7.93 (dt, J = 7.6, 1.2 Hz, 1H), 7.64 (t, J = 7.6 Hz,
1H), 7.55 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.45 (ddd,
J = 7.6, 2.4, 1.2 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6): d 165.2, 157.3, 144.8, 139.1, 133.8, 132.4,
130.1, 130.0, 129.5 (q, JC–F = 31.3 Hz), 128.7, 128.0, 125.3, 124.9,
4.1.4.10. 5-(4-(4-(Dimethylamino)phenyl)-1H-1,2,3-triazol-1-yl)-2-
hydroxy-N-(3-(trifluoromethyl)phenyl)benzamide (9c).
lowing the general procedure for 9, using 13h (96.7 mg, 300
Fol-
mol),
l
4-ethynyl-N,N-dimethylaniline (112
lL, 750
lmol), CuSO4 (9.6 mg,
60 mol) and sodium ascorbate (59.4 mg, 300
l
l
mol) in 1:1
t-BuOH/H2O (3 mL). The reaction mixture was stirred for 20 h at
rt. Column chromatography (2:1 hexane/EtOAc ? 20:1 CH2Cl2/
MeOH) afforded 9c (80.0 mg, 57%) as
a dark brown solid.
TLC: Rf 0.24 (2:1 hexane/EtOAc). Mp: 223.5ꢀ225.5 °C. 1H NMR
(400 MHz, DMSO-d6): d 11.73 (br s, 1H), 10.83 (br s, 1H), 9.02

4864
D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
124.2, 124.1 (q, JC–F = 270.0 Hz), 123.8, 121.4, 120.5, 120.0, 119.5,
118.3, 116.6. LRMS m/z (rel int): (pos) 459 ([M+H]+, 100). HRMS
m/z calcd for C22H14ClF3N4O2 458.0757; found 458.0757.
and sodium ascorbate (30.0 mg, 150 lmol) were dissolved in 1:1
t-BuOH/H2O (1.5 mL). The reaction mixture was stirred for 1.5 h
at 40 °C. Column chromatography (5:1 hexane/EtOAc ? 20:1
CH2Cl2/MeOH) afforded 9j (61.4 mg, 97%) as a brown solid. TLC:
Rf 0.35 (20:1 CH2Cl2/MeOH). Mp: 255ꢀ257 °C. 1H NMR (400 MHz,
DMSO-d6): d 11.76 (br s, 1H), 10.95 (br s, 1H), 9.24 (s, 1H), 8.37
(d, J = 2.8 Hz, 1H), 8.25 (s, 1H), 8.00ꢀ7.95 (m, 4H), 7.63 (t,
J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.35 (t, J = 8.8 Hz, 2H), 7.20
(d, J = 8.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): d 165.2, 162.0
4.1.4.14.
Methyl
4-(1-(4-hydroxy-3-((3-(trifluoro-
methyl)phenyl)carbamoyl)phenyl)-1H-1,2,3-triazol-4-yl)benzo-
ate (9g).
(128 mg, 400
(161 mg, 1.00 mmol), CuSO4 (12.8 mg, 80.2
ascorbate (78.2 mg, 395
Following the general procedure for 9, using 13h
mol) and 4-ethynylbenzoic acid methyl ester
mol) and sodium
mol) were dissolved in 1:1 t-BuOH/H2O
l
l
l
(d, JC–F = 253.2 Hz), 157.4, 146.3, 139.1, 130.0, 129.5 (q, JC–F =
(4 mL). The reaction mixture was stirred for 5 h at rt and for 59 h
at 40 °C. Column chromatography (5:1 hexane/EtOAc ? 20:1
CH2Cl2/MeOH) afforded 9g (90.0 mg, 47%) as a brown solid. TLC:
Rf 0.57 (20:1 CH2Cl2/MeOH). Mp: 258ꢀ260 °C. 1H NMR (400 MHz,
DMSO-d6): d 11.79 (br s, 1H), 11.14 (br s, 1H), 9.41 (d, J = 2.4 Hz,
1H), 8.38 (s, 1H), 8.25 (s, 1H), 8.11 (d, AB system, J = 8.8 Hz, 2H),
8.09 (d, AB system, J = 8.8 Hz, 2H), 7.97ꢀ7.95 (m, 2H), 7.63 (t, J =
7.6 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.18 (m, 1H), 3.89 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): d 166.0, 165.3, 157.6, 146.2, 139.1,
134.8, 130.1, 130.0, 129.6 (q, JC–F = 31.5 Hz), 129.0, 128.6, 125.4,
124.2, 124.1 (q, JC–F = 263.7 Hz), 121.5, 121.0, 120.5 (m), 119.9,
118.4, 117.6, 116.7 (m), 52.2. LRMS m/z (rel int): 483 ([M+H]+,
100). HRMS m/z calcd for C24H17F3N4O4 482.1202; found 482.1206.
31.7 Hz), 128.7, 127.4 (q, JC–F = 8.1 Hz), 126.9, 125.3, 124.2, 124.1
(q, JC–F = 270.0 Hz), 121.4, 120.5, 119.9, 119.7, 118.3, 116.6, 115.9
(d, JC–F = 21.2 Hz). LRMS m/z (rel int): 443 ([M+H]+, 100), 413
([MꢀCHO]+, 6). HRMS m/z calcd for C22H14F4N4O2 442.1053; found
442.1051.
4.1.4.18. 2-Hydroxy-5-(4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)-N-
(3-(trifluoromethyl)phenyl)benzamide (9k).
Following the
general procedure for 9, using 13h (48.3 mg, 150
lmol), 3-ethynyl-
pyridine (15.5 mg, 150
sodium ascorbate (30.0 mg, 150
BuOH/H2O (1.5 mL). The reaction mixture was stirred for 22 h at
rt. Additional CuSO4 (10.0 mg, 60.0 mol) was added and stirred
lmol), CuSO4 (4.8 mg, 30
lmol) and
lmol) were dissolved in 1:1 t-
l
for 22 h at 80 °C. Column chromatography (30:1 CH2Cl2/MeOH)
afforded 9k (50.0 mg, 78%) as a brown solid. TLC: Rf 0.29 (20:1
CH2Cl2/MeOH). Mp: 238.5ꢀ240.5 °C. 1H NMR (400 MHz, DMSO-d6):
d 10.90 (br s, 2H), 9.36 (s, 1H), 9.19 (br s, 1H), 8.61 (br s, 1H),
8.37 (s, 1H), 8.31 (d, J = 8.0 Hz, 1H), 8.25 (s, 1H), 7.97ꢀ7.95 (m,
2H), 7.63 (t, J = 8.0 Hz, 1H), 7.56 (br s, 1H), 7.50 (d, J = 8.0 Hz, 1H),
7.23 (d, J = 8.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): d 165.1,
163.4, 157.5, 149.1, 146.5, 144.5, 139.1, 132.4, 130.0, 129.3 (q,
JC–F = 31.5 Hz), 128.6, 125.4, 125.3, 124.1, 124.0 (q, JC–F = 269.4 Hz),
121.5, 120.5, 120.4, 120.0, 118.4, 116.6 (q, JC–F = 3.7 Hz). LRMS
m/z (rel int): 426 ([M+H]+, 100). HRMS m/z calcd for C21H14F3N5O2
425.1100; found 426.1104.
4.1.4.15. 2-Hydroxy-5-(4-(m-tolyl)-1H-1,2,3-triazol-1-yl)-N-(3-
(trifluoromethyl)phenyl)benzamide (9h).
Following the
mol), m-tolyl-
mol) and
mol) were dissolved in 1:1 t-
general procedure for 9, using 13h (47.7 mg, 150
l
acetylene (48.4
lL, 375 lmol), CuSO4 (4.8 mg, 30 l
sodium ascorbate (30.0 mg, 150
l
BuOH/H2O (1.5 mL). The reaction mixture was stirred for 1.5 h at
40 °C. Column chromatography (5:1 hexane/EtOAc ? 20:1 CH2Cl2/
MeOH) afforded 9h (56.8 mg, 86%) as a brown solid. TLC: Rf 0.35
(20:1 CH2Cl2/MeOH). Mp: 235.5ꢀ237.5 °C. 1H NMR (400 MHz,
DMSO-d6): d 11.75 (br s, 1H), 10.85 (br s, 1H), 9.24 (s, 1H), 8.39
(d, J = 2.8 Hz, 1H), 8.25 (s, 1H), 8.00ꢀ7.97 (m, 2H), 7.79 (s, 1H),
7.74 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz,
1H), 7.38 (t, J = 8.0 Hz, 1H), 7.23ꢀ7.19 (m, 2H), 2.39 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): d 165.3, 157.4, 147.3, 139.1, 138.1,
130.2, 130.0, 129.5 (q, JC–F = 31.5 Hz), 128.9, 128.8, 128.7, 125.8,
125.3, 124.4 (q, JC–F = 270.0 Hz), 124.2, 122.5, 121.2, 120.5, 119.8,
119.6, 118.3, 116.7, 21.1. LRMS m/z (rel int): 439 ([M+H]+, 100).
HRMS m/z calcd for C23H17F3N4O2 438.1304; found 438.1310.
4.1.4.19. 2-Hydroxy-5-(4-(thiophen-3-yl)-1H-1,2,3-triazol-1-yl)-
N-(3-(trifluoromethyl)phenyl)benzamide
the general procedure for 9, using 13h (48.3 mg, 150
ethynylthiophene (37.0 L, 375 mol), CuSO4 (4.8 mg, 30
and sodium ascorbate (30.0 mg, 150 mol) were dissolved in 1:1
(9l).
Following
mol), 3-
mol)
l
l
l
l
l
t-BuOH/H2O (1.5 mL). The reaction mixture was stirred for 22 h
at rt. Column chromatography (30:1 ? 10:1 CH2Cl2/MeOH) affor-
ded 9l (53.7 mg, 83%) as a brown solid. TLC: Rf 0.61 (20:1 CH2Cl2/
MeOH). Mp: 275ꢀ277 °C. 1H NMR (400 MHz, DMSO-d6): d 11.75
(br s, 1H), 10.88 (br s, 1H), 9.11 (s, 1H), 8.36 (d, J = 2.8 Hz, 1H),
8.25 (s, 1H), 7.98ꢀ7.95 (m, 2H), 7.94 (dd, J = 3.2, 1.2 Hz, 1H), 7.70
(dd, J = 4.8, 1.2 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.59 (dd, J = 4.8,
1.2 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.21 (d, J = 8.8 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6): d 165.3, 157.4, 143.8, 139.1, 131.6,
130.1, 129.5 (q, JC–F = 31.3 Hz), 128.7, 127.4, 125.8, 125.3, 124.2,
124.1 (q, JC–F = 274 Hz), 121.4, 121.3, 120.5 (m), 119.8, 119.5,
118.4, 116.7(m). LRMS m/z (rel int): 431 ([M+H]+, 100). HRMS
m/z calcd for C20H13F3N4O2S 430.0711; found 430.0709.
4.1.4.16.
2-Hydroxy-5-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)-N-(3-
(trifluoromethyl)phenyl)benzamide (9i).
Following the gen-
eral procedure for 9, using 13h (47.9 mg, 150
lmol), p-tolylacety-
lene (47.6
lL, 375
l
mol), CuSO4 (4.8 mg, 30
lmol) and sodium
ascorbate (30.0 mg, 150
l
mol) were dissolved in 1:1 t-BuOH/H2O
(1.5 mL). The reaction mixture was stirred for 1.5 h at 40 °C. Col-
umn chromatography (5:1 hexane/EtOAc ? 20:1 CH2Cl2/MeOH)
afforded 9i (39.8 mg, 61%) as a brown solid. TLC: Rf 0.33 (20:1
CH2Cl2/MeOH). Mp: 457ꢀ459 °C. 1H NMR (400 MHz, DMSO-d6): d
11.76 (br s, 1H), 11.01 (br s, 1H), 9.19 (s, 1H), 8.38 (d, J = 2.4 Hz,
1H), 8.25 (s, 1H), 7.98–7.96 (m, 2H), 7.84 (d, J = 8.0 Hz, 2H), 7.63
(t, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H),
7.19 (d, J = 8.8 Hz, 1H), 2.35 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): d 165.4, 157.9, 147.4, 139.2, 137.7, 130.1, 129.6, 129.5
(q, JC–F = 31.5 Hz), 128.7, 127.6, 125.4, 125.3, 124.3, 124.2
(q, JC–F = 270.0 Hz), 121.4, 120.5, 119.7, 119.3, 118.5, 116.7, 20.9.
LRMS m/z (rel int): 439 ([M+H]+, 100). HRMS m/z calcd for
4.1.4.20.
5-(4-(3-Acetamidophenyl)-1H-1,2,3-triazol-1-yl)-2-
hydroxy-N-(3-(trifluoromethyl)phenyl)benzamide (9m).
lowing the general procedure for 9, using 13h (48.3 mg, 150
Fol-
mol),
l
N-(3-ethynylphenyl)acetamide (59.7 mg, 375
(4.8 mg, 30 mol) and sodium ascorbate (30.0 mg, 150
were dissolved in 1:1 t-BuOH/H2O (1.5 mL). The reaction mixture
was stirred for 19 h at rt. Additional CuSO4 (4.8 mg, 30 mol) was
l
mol), CuSO4
l
l
mol)
C23H17F3N4O2 438.1304; found 438.1312.
l
4.1.4.17. 5-(4-(4-Fluorophenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-
added and stirred for 20 h at 80 °C. Column chromatography
(30:1 CH2Cl2/MeOH) afforded 9m (61.7 mg, 85%) as a brown
solid. TLC: Rf 0.16 (30:1 CH2Cl2/MeOH). Mp: 258ꢀ260 °C. 1H
NMR (400 MHz, DMSO-d6): d 11.79 (br s, 1H), 10.89 (br s, 1H),
N-(3-(trifluoromethyl)phenyl)benzamide
the general procedure for 9, using 13h (48.0 mg, 150
orophenylacetylene (49.0 L, 375 lmol), CuSO4 (4.8 mg, 30 lmol)
(9j).
Following
l
mol), 4-flu-
l

D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
4865
10.08 (br s, 1H), 9.19 (s, 1H), 8.40 (d, J = 2.4 Hz, 1H), 8.26ꢀ8.24
(m, 2H), 7.99 (dd, J = 8.8, 2.4 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H),
7.64 (t, J = 8.0 Hz, 1H), 7.59ꢀ7.55 (m, 2H), 7.50 (d, J = 8.0 Hz,
1H), 7.40 (t, J = 8.0 Hz, 1H), 7.21 (d, J = 8.8 Hz, 1H), 2.07 (s, 3H).
13C NMR (100 MHz, DMSO-d6): d 168.5, 165.5, 157.7, 147.2,
139.9, 139.1, 130.8, 130.1, 129.5 (q, JC–F = 31.3 Hz), 129.4,
128.7, 125.5, 124.4 (q, JC–F = 270.0 Hz), 124.3, 121.4, 120.5 (q,
JC–F = 3.7 Hz), 120.4, 119.7, 119.6, 118.9, 118.4, 116.7 (q,
JC–F = 3.7 Hz), 115.9, 24.1. LRMS m/z (rel int): 482 ([M+H]+, 100).
HRMS m/z calcd for C24H18F3N5O3 481.1362; found 481.1359.
(400 MHz, DMSO-d6): d 11.84 (br s, 1H), 10.99 (br s, 1H), 9.04
(s, 1H), 8.37 (d, J = 2.8 Hz, 1H), 8.24 (s, 1H), 7.99–7.96 (m, 2H),
7.78 (s, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.32
(s, 1H), 7.21 (d, J = 8.8 Hz, 1H), 3.88 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): d 165.4, 157.9, 139.2, 138.3, 131.7, 130.1, 129.5 (q,
JC–F = 31.4 Hz), 128.7, 128.4, 125.5, 124.2, 124.1 (q, JC–F = 270.0 Hz),
122.8, 121.6, 120.5, 120.1, 119.9, 118.5, 116.7 (q, JC–F = 3.7 Hz),
33.1. LRMS m/z (rel int): 429 ([M+H]+, 100). HRMS m/z calcd for
C20H15F3N6O2 428.1209; found 428.1215.
4.1.5. General procedure for the synthesis of 8h, 9q, and 9r
[hydrolysis reaction]
4.1.4.21. Methyl 3-(1-(4-hydroxy-3-((3-(trifluoromethyl)phenyl)car-
The suspension of methyl ester (100 lmol) in 1 N NaOH
bamoyl)phenyl)-1H-1,2,3-triazol-4-yl)benzoate (9n).
the general procedure for 9, using 13h (112 mg, 350
Following
mol), 3-ethy-
(1 mL), MeOH (1 mL) was stirred for 1 h at 80 °C. After the com-
pletion of the reaction, MeOH was evaporated. The residue was
acidified with 1 N HCl (pH 5) and extracted with EtOAc
(3 ꢁ 10 mL). The combined organic extracts were dried over
anhyd MgSO4, filtered, and concentrated by rotary evaporation
to afford carboxylic acid.
l
nylbenzoic acid methyl ester (62.0 mg, 380
lmol), CuSO4 (22.3 mg,
140 mol) and sodium ascorbate (69.0 mg, 350
l
l
mol) were dis-
solved in 1:1 t-BuOH/H2O (4 mL). The mixture was stirred for 2 h
at rt and 44 h at 80 °C. Column chromatography (4:1 hexane/
EtOAc ? 30:1 CH2Cl2/MeOH) afforded 9n (145 mg, 86%) as a
brown solid. TLC: Rf 0.10 (5:1 hexane/EtOAc). Mp: 214ꢀ216 °C.
1H NMR (400 MHz, DMSO-d6): d 11.77 (br s, 1H), 10.85 (br s, 1H),
9.41 (s, 1H), 8.54 (t, J = 1.2 Hz, 1H), 8.40 (d, J = 2.8 Hz, 1H), 8.25
(s, 1H), 8.23 (dt, J = 8.0, 1.2 Hz, 1H), 8.00 (dd, J = 8.8, 2.8 Hz, 1H),
7.98ꢀ7.96 (m, 2H), 7.67 (t, J = 7.6 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H),
7.51 (d, J = 7.6 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 3.91 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): d 166.1, 165.3, 157.5, 146.3, 139.1,
130.9, 130.5, 130.1, 129.9, 129.7, 129.4 (q, JC–F = 31.5 Hz), 128.8,
128.7, 125.8, 125.4, 124.3, 124.2 (q, JC–F = 270.0 Hz), 121.4, 120.6
(m), 120.3, 119.9, 118.4, 116.8 (m), 52.4. LRMS m/z (rel int):
483 ([M+H]+, 100), 413 ([MꢀCF3]+, 3). HRMS m/z calcd for
4.1.5.1. 3-(5-(4-(3,5-Dimethoxyphenyl)-1H-1,2,3-triazol-1-yl)-2-
hydroxybenzamido)benzoic acid (8h).
eral procedure above, methyl ester 8g (33.6 mg, 70.0
Following the gen-
mol) in
l
1 N NaOH (0.7 mL) and MeOH (0.7 mL) afforded 8h (22.4 mg,
70%) as a light brown solid. TLC: Rf 0.16 (30:1 CH2Cl2/MeOH).
Mp: 235ꢀ237 °C. 1H NMR (400 MHz, DMSO-d6): d 13.02 (br s,
1H), 11.90 (br s, 1H), 10.91 (br s, 1H), 9.27 (s, 1H), 8.40–8.38 (m,
2H), 7.99–7.96 (m, 1H), 7.95 (dd, J = 8.8, 2.8 Hz, 1H), 7.72 (dt,
J = 8.0, 1.2 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.21 (m, 1H), 7.14 (d,
J = 2.4 Hz, 2H), 6.51 (t, J = 2.4 Hz, 1H), 3.82 (s, 6H). 13C NMR
(100 MHz, DMSO-d6): d 167.1, 165.2, 161.0, 157.9, 147.1, 138.5,
132.1, 131.4, 129.1, 128.6, 125.3, 125.0, 124.8, 121.4, 121.2,
120.1, 119.5, 118.4, 103.2, 100.2, 55.3. LRMS m/z (rel int): 461
([M+H]+, 100), 413 ([MꢀCH3O2]+, 10), 338 ([MꢀC7H6O2]+, 9). HRMS
m/z calcd for C24H20N4O6 460.1383; found 460.1386.
C24H17F3N4O4 482.1202; found 482.1200.
4.1.4.22. 2-Hydroxy-5-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-N-
(3-(trifluoromethyl)phenyl)benzamide (9o).
Following the
general procedure for 9, using 13h (35.4 mg, 110
l
mol), 2-ethynyl-
pyridine (28.0
sodium ascorbate (21.8 mg, 110
l
L, 280
l
mol), CuSO4 (7.0 mg, 44.0
lmol) and
4.1.5.2. 4-(1-(4-Hydroxy-3-((3-(trifluoromethyl)phenyl)carbam-
l
mol) were dissolved in 1:1 t-
oyl)phenyl)-1H-1,2,3-triazol-4-yl)benzoic acid (9q).
ing the general procedure above, methyl ester 9g (48.2 mg,
100 mol) in 1 N NaOH (1 mL) and MeOH (1 mL) afforded 9q
Follow-
BuOH/H2O (1 mL). The mixture was stirred for 1 h at rt and for
42 h at 60 °C. After the completion of the reaction, pyridine
(0.1 mL) was added to the crude mixture to improve poor solubil-
ity. Column chromatography (30:1 CH2Cl2/MeOH) afforded 9o
(12.5 mg, 27%) as a brown solid. TLC: Rf 0.43 (30:1 CH2Cl2/MeOH).
Mp: 274.5ꢀ276.5 °C. 1H NMR (400 MHz, acetone-d6): d 8.93 (s, 1H),
8.65 (d, J = 4.0 Hz, 1H), 8.59 (d, J = 2.8 Hz, 1H), 8.26 (s, 1H), 8.20 (d,
J = 7.6 Hz, 1H), 8.14 (dd, J = 8.8, 2.8 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H),
7.93 (td, J = 7.6, 1.6 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.55 (d,
J = 8.0 Hz, 1H), 7.37 (ddd, J = 7.6, 4.0, 1.2 Hz, 1H), 7.26 (d,
J = 8.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): d 165.8, 158.3,
149.8, 149.6, 148.3, 139.1, 137.6, 130.3, 129.7 (q, JC–F = 31.5 Hz),
128.6, 128.3, 125.8, 124.6, 124.2 (q, JC–F = 270.0 Hz), 123.6, 121.4,
120.8, 120.0, 119.2, 118.6, 117.7 (q, JC–F = 3.7 Hz). LRMS m/z (rel
int): 426 ([M+H]+, 100). HRMS m/z calcd for C21H14F3N5O2
425.1100; found 425.1106.
l
(44.6 mg, 96%) as a light brown solid. TLC: Rf 0.10 (1:1 hexane/
EtOAc). Mp: 309.5ꢀ311.5 °C. 1H NMR (400 MHz, DMSO-d6): d
13.02 (br s, 1H), 11.77 (br s, 1H), 10.77 (br s, 1H), 9.41 (s, 1H),
8.40 (d, J = 2.8 Hz, 1H), 8.25 (s, 1H), 8.07ꢀ8.04 (m, 4H), 8.00 (dd,
J = 8.8, 2.8 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H),
7.51 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6): d 167.0, 165.2, 157.4, 146.3, 139.1, 134.4,
130.2, 130.1, 130.0, 129.5 (d, JC–F = 31.3 Hz), 128.7, 125.4, 125.3,
124.2, 124.1 (d, JC–F = 270.0 Hz), 121.4, 120.8, 120.5, 119.9, 118.3,
116.7. LRMS m/z (rel int): 469 ([M+H]+, 100). HRMS m/z calcd for
C23H15F3N4O4 468.1045; found 468.1042.
4.1.5.3. 3-(1-(4-Hydroxy-3-((3-(trifluoromethyl)phenyl)carbam-
oyl)phenyl)-1H-1,2,3-triazol-4-yl)benzoic acid (9r). Follow-
ing the general procedure above, methyl ester 9n (48.2 mg,
100 mol) in 1 N NaOH (1 mL) and MeOH (1 mL) afforded 9r
4.1.4.23. 2-Hydroxy-5-(4-(1-methyl-1H-imidazol-5-yl)-1H-
1,2,3-triazol-1-yl)-N-(3-(trifluoromethyl)phenyl)benzamide
l
(44.9 mg, 98%) as a brown solid. TLC: Rf 0.37 (20:1 CH2Cl2/MeOH).
Mp: 299ꢀ301 °C. 1H NMR (400 MHz, DMSO-d6): d 11.76 (br s, 1H),
10.78 (br s, 1H), 9.42 (s, 1H), 8.53 (t, J = 1.2 Hz, 1H), 8.42 (d,
J = 2.8 Hz, 1H), 8.25 (s, 1H), 8.20 (dt, J = 8.0, 1.2 Hz, 1H), 8.02 (dd,
J = 8.8, 2.8 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.95 (dt, J = 8.0, 1.2 Hz,
1H), 7.66–7.62 (m, 2H), 7.51 (d, J = 7.6 Hz, 1H), 7.23 (d, J = 8.8 Hz,
1H). 13C NMR (100 MHz, DMSO-d6): d 167.1, 165.3, 157.5, 146.5,
139.1, 131.6, 130.7, 130.1, 129.7, 129.4, 129.3 (q, JC–F = 31.5 Hz),
128.9, 128.8, 126.0, 125.3, 124.2, 124.1 (q, JC–F = 268.5 Hz), 121.3,
120.5, 120.2, 119.9, 118.3, 116.7. LRMS m/z (rel int): 469
(9p).
(48.3 mg, 150
380 mol), CuSO4 (9.6 mg, 60.0
(29.7 mg, 150 mol) in 1:1 t-BuOH/H2O (1.5 mL). The mixture
Following the general procedure for 9, using 13h
mol), 5-ethynyl-1-methyl-1H-imidazole (40.0 L,
mol) and sodium ascorbate
l
l
l
l
l
was stirred for 2.5 h at rt and for 23 h at 60 °C. After the completion
of the reaction, pyridine (0.1 mL) was added to the crude mixture
to
improve
poor
solubility.
Column
chromatography
(40:1 ? 10:1 CH2Cl2/MeOH) afforded 9p (34.8 mg, 54%) as a white
solid. TLC: Rf 0.11 (20:1 CH2Cl2/MeOH). Mp: 259ꢀ261 °C. 1H NMR

4866
D. Song et al. / Bioorg. Med. Chem. 22 (2014) 4855–4866
([M+H]+, 100). HRMS m/z calcd for C23H15F3N4O4 468.1045; found
Supplementary data
468.1043.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.06.047.
4.2. Enzyme screening
Kinase assays were performed at Reaction Biology Corporation
using the ‘HotSpot’ assay platform. Kinase Assay Protocol. Reaction
Buffer: base Reaction buffer; 20 mM Hepes (pH 7.5), 10 mM MgCl2,
1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM
DTT, 1% DMSO. Reaction procedure: to a freshly prepared buffer
solution was added any required cofactor for the enzymatic reac-
tion, followed by the addition of the selected kinase at a concentra-
References and notes
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tion of 20
compound dissolved in DMSO was added to the reaction mixture
in the 10 M concentration. Compounds were tested in a 10-dose
IC50 mode with 3-fold serial dilution starting at 30 M for IC50
determination. 33P-ATP (specific activity 10
Ci/L) was added to
lM. The contents were mixed gently, and then the
l
l
l
the mixture in order to initiate the reaction, and the mixture was
incubated at room temperature for 2 h. Staurosporine was used
as a control compound in a five-dose IC50 mode with 10-fold serial
dilutions starting at 20
lM, and the reaction was carried out at
10 M ATP concentration.
l
4.3. Modeling
The 3D X-ray protein structure of Aurora A as a complex with
compound 2 was obtained from the PDB (code: 3d15)11 and pre-
pared using Protein Preparation Wizard of the Schrodinger Maestro
program. All water molecules were removed from the structure
and it was selected as a template. The structure of 8g was drawn
using Chemdraw, and its 3D conformation was generated using
the Schrodinger LigPrep program with the OPLS 2005 force field.
Molecular docking of compound 8g into the structure of Aurora
A was carried out using Schrodinger Glide (whole software pack-
age; Version 9.2).When making grid file, default condition was
selected except for hinge hydrogen bonding constraint. In order
to acquire high docking score and more plausible docking pose,
the docking result iteratively conducted in the condition of SP
(standard precision) and XP (extra precision) also was followed
by revision of docking pose through substructure energy minimi-
zation using Schrodinger Macro Model.
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Acknowledgments
This research was supported by the Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(NRF-2012R1A2A2A01011831).