1-Phenyl-4-benzoyl-1H-1,2,3-triazoles as orally bioavailable transcriptional function suppressors of Estrogen-related receptor α

Article abstract of DOI:10.1021/jm4003928

Estrogen-related receptor α is a potential candidate target for therapeutic treatment of breast cancer. We describe the discovery and structure-activity relationship study of a series of 1-phenyl-4-benzoyl-1H-1,2, 3-triazoles as novel suppressors of ERRα

Full text of DOI:10.1021/jm4003928

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Article
1-Phenyl-4-benzoyl-1H-1,2,3-triazoles as Orally Bioavailable
Transcriptional Function Suppressors of Estrogen-Related Receptor #
Shilin xu, Xiaoxi Zhuang, Xiaofen Pan, zhang zhang, Lei
Duan, Yingxue Liu, Lianwen zhang, Xiaomei Ren, and Ke Ding
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4003928 • Publication Date (Web): 08 May 2013
Downloaded from http://pubs.acs.org on May 17, 2013
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1-Phenyl-4-benzoyl-1H-1,2,3-triazoles as Orally
Bioavailable Transcriptional Function Suppressors
of Estrogen-Related Receptor α
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Shilin Xu,†,§ Xiaoxi Zhuang,† Xiaofen Pan,† Zhang Zhang,† Lei Duan,† Yingxue Liu,† Lianwen
Zhang,† Xiaomei Ren,*,† and Ke Ding*,†
†Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese
Academy of Sciences, #190 Kaiyuan Avenue, Guangzhou 510530, China
§University of Chinese Academy of Sciences, # 19 Yuquan Road, Beijing 100049, China
*Corresponding author: Tel: +86-20-32015276. Fax: +86-20-32015299.
E-Mail: ding_ke@gibh.ac.cn
KEYWORDS Estrogen-related receptor, Transcriptional function, Suppressors, 1, 2, 3-
Triazoles, Breast cancer
ABSTRACT Estrogen-related receptor α is a potential candidate target for therapeutic treatment
of breast cancer. We describe the discovery and structure-activity relationship study of a series of
1-phenyl-4-benzoyl-1H-1,2,3-triazoles as novel suppressors of ERRα transcriptional functions.
The most promising compound, 2-aminophenyl-(1-(3-isopropylphenyl)-1H-1,2,3-triazol-4-
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yl)methan-one (14n) potently suppressed the transcriptional functions of ERRα with IC50 = 0.021
ꢀM in a cell-based reporter gene assay and also decreased both the mRNA levels and the protein
levels of ERRα and the downstream targets. This compound inhibited the proliferation and
migration of breast cancer cells with high level of ERRα. Preliminary pharmacokinetic studies
suggested that it possessed a good pharmacokinetic profile with an oral bioavailability of 71.8%.
The compounds may serve as novel small molecule probes for further validation of ERRα as a
molecular target for anticancer drug development.
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INTRODUCTION
Estrogen-related receptors (ERRs) are the orphan members of a nuclear receptor (NR) super-
family which contains three isoforms (ERRα, ERRβ and ERRγ).1 These isoforms share
significant homology with the classical estrogen receptors (ERs), but they do not bind with
endogenous estrogens or any known natural ligands. ERRα and ERRβ are the first two orphan
nuclear receptors identified by a low stringency hybridization screen in 1988,2 and ERRγ was
discovered 10 years later.3 ERRα is expressed predominately in metabolically active tissues such
as intestine, brown adipose tissue and skeletal muscles, and functions as a key regulator of energy
metabolism. ERRβ is important in early development, but its expression is highly restricted in
adult stages and it can only be detected at low levels in adult eyes, inner ear, heart and kidneys.
The exact function of ERRγ remains elusive, but it is mainly expressed in the spinal cord and
central nervous system.3c, 4
Collectively, studies have demonstrated that ERRα plays a critical role in the regulation of the
energy of metabolism by controlling the transcriptional functions and/or protein expression of
key components involved in tricarboxylic acid (TCA) metabolism, oxidative phosphorylation
(OXPHOS) and glycolysis. These key components include peroxisome proliferator-activated
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receptor γ coactivator-1α (PGC-1α),5 medium-chain acyl-coenzyme A dehydrogenase (MCAD)6
and pyruvate dehydrogenase kinase 4 (PDK4).7 In addition, genetic and functional analyses have
revealed the functional cross-talk between ERRα and ERα at multiple steps of the transcriptional
process by which, through competition or cooperation they interact with similar co-regulators.8
Modulation of ERRα activity through small molecules may be seen as a novel strategy for
clinical management of estrogen-linked diseases such as breast cancer.1b
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Breast cancer is one of the most common cancers diagnosed in the United States and after lung
cancer, is the leading cause of cancer deaths in women. Hormonal therapy medications, which
include aromatase inhibitors, selective estrogen receptor modulators, and estrogen receptor
downregulators, have achieved a significant advantage in the clinical treatment of most breast
cancer patients but about 10-20% of breast cancers are diagnosed as triple-negative [TNBC,
negative for estrogen receptors (ER-), progesterone receptors (PR-), and HER2 (HER2-)] or
basal-like (BLBC), for which the traditional hormone therapies are ineffective.9 There is
therefore an urgent need for alternative therapies for effective and comprehensive management of
breast cancer with different mechanism of action.
There are several reports implying that ERRα might be a valid candidate target for therapeutic
treatment of breast cancer, including ER-negative breast cancer.8, 10, 11 For instance, Ariazi et al.
reported that ERRα is over-expressed in ERα-negative breast cancer and could serve as an
unfavorable biomarker in human breast cancer11b and Suzuki et al. also found that expression of
ERRα is associated with an increased risk of recurrence and adverse clinical outcome.12
Furthermore, small interfering RNA (siRNA) targeting ERRα in vitro was shown to substantially
reduce the migration of the ERα-negative MDA-MB-231 cells, a BLBC cell line, and suppress
tumor growth in vivo in an xenograft model.13
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Several classes of ERRα inverse agonists or agonists, which could suppress or improve
ERRα’s transcriptional functions, have been discovered (Figure 1).14 Some of the synthetic
ERRα inverse agonists have been reported to inhibit tumor development and progression by
disrupting the interaction of ERRs with their co-activators or their constitutive transcriptional
activities, but, despite these advances, no ERRα modulators have demonstrated efficacy in
humans. As a part of our continuing program on ERR modulators,14f we report the discovery and
structural optimization of a series of 1-phenyl-4-benzoyl-1H-1,2,3-triazoles derivatives as new
suppressors of ERRα transcriptional functions, which may produce lead compounds for further
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development of novel anti-breast cancer drugs targeting ERRα.
N
N
O
OH
F3C
OMe
O
CH3
S
N
H
CF3
N
N
N
H3C
H3C
CF3
2 (XCT790)14b
114a
O
OMe
HN
S
N
S
O
S
O
N
CF3
N
314c
CN
414d
OH
O
N
HO
O
O
N
OH
OH
614f
5 (Kaempferol)14e
Figure 1. Structures of reported ERRα modulators (1-6)
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CHEMISTRY
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The synthesis of new ERRα suppressors 14a-ab is shown in Scheme 1. The substituted
benzaldehydes (7) used as starting materials were either first protected with 3,4-dihydro-2H-
pyran (DHP) to give the derived benzaldehydes (8a-c) which were reacted with Grignard reagent
ethynylmagnesium bromide to produce 9a-c or directly converted into intermediates 9d-h and 9j-
k through a Grignard reaction. Compounds 9 underwent a copper-catalyzed cycloaddition with
the azido-substituted benzene (11) to provide the triazoles (12). Compounds 12a-c were oxidized
with MnO2 to afford 13a-c, which were deprotected with PPTS in ethanol to furnish 14a-c.
Alternatively, compound 14l was prepared by deprotection followed by dehydroxylation using
triethylsilane and trifluoroacetic acid. 12d-h was directly oxidized with MnO2 to give products
14d-h. In addition, 12d, 12j-k and 12m-aa were hydrogenated to the corresponding aromatic
amines 12i, 16j-k and 16m-aa which were subsequently oxidized to the final products 14i-k and
14m-aa, and direct hydrolysis of the methyl ester 14w yielded the carboxylic acid 14ab.
Scheme 1. Syntheses of compounds 14a-ab
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iPr
iPr
iPr
4
5
6
7
8
9
O
CHO
HO
O
CHO
N
N
N
N
N
N
a
e
f
N
N
N
HO
THPO
THPO
THPO
THPO
HO
7a:
7b:
7c:
4-OH
3-OH
2-OH
8a:
8b:
8c:
4-OTHP
3-OTHP
2-OTHP
12a:
12b:
14a:
14b:
14c:
4-OTHP
3-OTHP
4-OH
3-OH
2-OH
13a:
13b:
13c:
4-OTHP
3-OTHP
2-OTHP
e
b
iPr
iPr
iPr
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12
13
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HO
HO
CHO
HO
N
N
HO
N
N
N
N
HO
g
f
b
N
N
N
R1
15
14l
12c
R1
9a:
9b:
9c:
9d:
9e:
9f:
9g:
9h:
9j:
7d:
7e:
7f:
7g:
7h:
R1 = 2-NO2
R1 = 2-Me
R1 = 2-F
R1 = 2-CF3
R1 = 2-OMe
R1 = 3-NO2
R1 = 4-NO2
R1 = 4-OTHP
R1 = 3-OTHP
R1 = 2-OTHP
R1 = 2-NO2
R1 = 2-Me
iPr
iPr
HO
O
e
N
N
N
N
7j:
7k:
d
R1 = 2-F
N
N
R1 = 2-CF3
R1 = 2-OMe
R1 = 3-NO2
R1 = 4-NO2
R1
R1
9k:
12d:
14d:
14e:
14f:
14g:
14h:
R1 = 2-NO2
R1 = 2-Me
R1 = 2-F
R1 = 2-CF3
R1 = 2-OMe
R1 = 2-NO2
R1 = 2-Me
R1 = 2-F
R1 = 2-CF3
R1 = 2-OMe
12e:
12f:
12g:
12h:
h
R2
R2
c
12i: R1 = 2-NH2
14i: R1 = 2-NH2
H2N
N3
11
10a:
10m:
10n:
10o:
10p:
R2 = 4-iPr
R2
R2
R2
HO
HO
O
R2 = 2-iPr
R2 = 3-iPr
R2 = 3-Et
R2 = 3-Me
N
N
N
N
e
h
N
N
N
N
N
H2N
R1
R1
10q: R2 = 3-CF3
12j:
12k:
10r:
10s:
10t:
10u:
10v:
10w:
10x:
10y:
R1 = 3-NO2, R2 = 4-iPr
R1 = 4-NO2, R2 = 4-iPr
R2 = 3-H
14j:
14k:
R1 = 3-NH2, R2 = 4-iPr
R1 = 4-NH2, R2 = 4-iPr
16
R2 = 3-tBu
R2 = 3-Ph
R2 = 3-OPh
R2 = 3-OBn
R2 = 3-COOMe
R2 = 3-COOEt
R2 = 3-COOPr
12m:
12n:
12o:
12p:
12q:
12r:
12s:
12t:
12u:
12v:
R1 = 2-NO2, R2 = 2-iPr
R1 = 2-NO2, R2 = 3-iPr
R1 = 2-NO2, R2 = 3-Et
R1 = 2-NO2, R2 = 3-Me
R1 = 2-NO2, R2 = 3-CF3
R1 = 2-NO2, R2 = 3-H
R1 = 2-NO2, R2 = 3-tBu
R1 = 2-NO2, R2 = 3-Ph
R1 = 2-NO2, R2 = 3-OPh
R1 = 2-NO2, R2 = 3-OBn
14m:
14n:
14o:
14p:
14q:
14r:
14s:
14t:
14u:
14v:
14w:
14x:
14y:
14z:
14aa:
14ab:
R1 = 2-NH2, R2 = 2-iPr
R1 = 2-NH2, R2 = 3-iPr
R1 = 2-NH2, R2 = 3-Et
R1 = 2-NH2, R2 = 3-Me
R1 = 2-NH2, R2 = 3-CF3
R1 = 2-NH2, R2 = 3-H
R1 = 2-NH2, R2 = 3-tBu
R1 = 2-NH2, R2 = 3-Ph
R1 = 2-NH2, R2 = 3-OPh
R1 = 2-NH2, R2 = 3-OBn
R1 = 2-NH2, R2 = 3-COOMe
R1 = 2-NH2, R2 = 3-COOEt
R1 = 2-NH2, R2 = 3-COOPr
R1 = 2-NH2, R2 = 3-CN
R1 = 2-NH2, R2 = 3-SO2NH2
R1 = 2-NH2, R2 = 3-COOH
10z: R2 = 3-CN
10aa:
R2 = 3-SO2NH2
12w:
12x:
12y:
12z:
R1 = 2-NO2, R2 = 3-COOMe
R1 = 2-NO2, R2 = 3-COOEt
R1 = 2-NO2, R2 = 3-COOPr
R1 = 2-NO2, R2 = 3-CN
i
12aa:
R1 = 2-NO2, R2 = 3-SO2NH2
Reactions and conditions: (a) 3, 4-Dihydro-2H-pyran (1.5 equiv), PPTS (0.05 equiv),
dichloromethane, rt, 80%-98%. (b) Ethynyl magnesium bromide (1.2 equiv), THF, 0 ℃ to rt,
90%-96%. (c) Concentrated hydrochloride (5 equiv), NaNO2 (1.2 equiv), NaN3 (1.2 equiv), H2O,
EtOAc, 0 ℃ to rt, 98%. (d) Sodium ascorbate (0.3 equiv), CuSO4 (0.1 equiv), tBuOH/H2O (1:1,
v/v), rt, 67%-75%. (e) MnO2 (4 equiv), dichloromethane, rt, 78%-92%. (f) PPTS (0.05 equiv),
ethanol, 50 ℃ , 80%-95%. (g) Triethylsilane (3 equiv), trifluoroacetic acid (3 equiv),
dichloromethane, rt, 81%. (h) Pd/C (10%), H2, methanol, rt. (i) LiOH (5 equiv), THF/H2O, rt,
90%.
RESULTS AND DISCUSSION
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To identify new molecules capable of modulating the transcriptional functions of ERRα, we
conducted the screen on a carbonyl-focused chemical library using a well-established mammalian
two-hybrid reporter gene assay, where 293FT cells were transiently transfected with expression
plasmids encoding fusion protein containing GAL4 DNA binding domain and human ERRα
ligand binding domain, and PGC-1α, respectively, together with luciferase reporter constructs.14f
Several compounds were identified which could potently modulate the transcriptional activity of
ERRα, including 8-ethyl-2-phenyl-4H-pyrido[1,2-α]pyrimidin-4-one (6) which improved
ERRα’s functions,14f while (4-hydroxyphenyl)(1-(4-isopropylphenyl)-1H-1,2,3-triazol-4-
yl)methanone (14a) strongly suppressed its activity at a concentration of 10 ꢀM. Further
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evaluation showed the IC50 value of compound 14a to be 1.59 ꢀM, about 6 times more potent
than the standard reference compound 5,14e but approximate 4 folds less active than 2 (Table
1).14b
Extensive structural optimization of the lead compound (14a) was conducted to improve its
suppressive activity on the transcriptional functions of ERRα, as measured in this reporter assay,
and the results are summarized in Table 1. We first investigated the potential impact of the
hydroxyl group in ring A on the bioactivity by altering its position. The position of the hydroxyl
group was found to strongly affect the inhibitory activity on ERRα’s transcriptional functions.
Although the meta-hydroxy derivative (14b) displayed similar potency to that of 14a, the
suppressive effect on ERRα’s transcriptional functions was improved about 4 folds by moving
the hydroxyl group from the para- (14a) to the ortho-position (14c); 14c inhibited ERRα’s
transcriptional functions with an IC50 value of 0.39 ꢀM. Further investigation suggested the
ortho-hydroxyl group in 14c could be replaced by an NO2 (14d) or a Me (14e) group with no
significant effect on the bioactivity, but when the ortho-hydroxyl group in 14c was replaced by F
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(14f), CF3 (14g) or OMe (14h), the resulting compounds were obviously less potent. For instance,
the ortho-CF3 substituted compound 14g failed totally to inhibit ERRα at concentrations as high
as 50 ꢀM. The potency was improved about 10-fold when the ortho-hydroxyl group in 14c was
replaced with an amino group (14i), which strongly suppressed ERRα’s transcriptional functions
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with IC50 value of 0.041 ꢀM, while the meta- (14j) or para-NH2 (14k) analogues were clearly
less potent. The investigation also suggested that the carbonyl group was necessary for the
compounds to display strong suppression of ERRα. For instance, compound 14l, in which the
carbonyl group was replaced by a methylene, was 4-times less potent than the corresponding
carbonyl compound 14c.
We further investigated the impact of the substituent in ring B on the ERRα suppressive
activity by adopting compound 14i as a new starting point and found that the position of the
substituent in this ring significantly affected the biological activity of the resulting compounds.
For instance, when the para-isopropyl in 14i was moved to the ortho- position (14m), the
potency was decreased about 100-fold. However, the compound bearing a meta-isopropyl group
(14n) was about twice as potent as the original compound 14i. The structure-activity relationship
study also clearly revealed that an isopropyl at the meta position might be optimal for the
compounds to suppress the transcriptional functions of ERRα. A smaller or larger hydrophobic
group at this position was found to be detrimental to the ERRα suppressing activity. For instance,
the compounds with H (14r), ethyl (14o), methyl (14p), or CF3 (14q) at the meta position in ring
B displayed IC50 values of 0.43, 0.20, 2.17, 15.89 ꢀM, respectively, numbers that are about 20,
10, 100, and 800-fold greater than that of compound 14n. The compounds bearing larger
hydrophobic groups such as t-butyl (14s), phenyl (14t), or phenoxyl (14u) were also less potent
than 14n, displaying IC50 values of 0.094, 0.65, 1.74 ꢀM, respectively. When ring B was
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substituted with a meta-benzoxyl group (14v), the ERRα suppressing potency was totally
abolished. Further investigation showed that the meta-isopropyl group in 14n could be replaced
by ester substituents such as COOMe (14w), COOEt (14x), COOPr (14y) and still retained
ERRα suppressing activity, but the potency was obviously decreased. However, when the meta-
isopropyl group in 14n was replaced with a hydrophilic group such as nitrile (14z), sulfonamide
(14aa) or carboxyl (14ab), the resulting compounds totally failed to modulate ERRα’s
transcriptional functions.
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Table 1. Substituent effects on suppression of ERRα transcriptional functions in a mammalian
two-hybrid reporter gene assay.
Compds
R1
R2
R3
R4
R5
R6
IC50 (ꢀM)a
40-50%
inhibition@ 1.0ꢀM
(0.37ꢀMb)
2
9.64 0.50
1.59 0.48
1.44 0.35
0.39 0.01
0.9 0.25
5
H
H
H
OH
H
OH
H
H
H
H
H
H
H
H
H
i-Pr
i-Pr
i-Pr
i-Pr
14a
14b
14c
14d
OH
NO2
H
H
H
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Me
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
0.89 0.27
9.53 0.20
>50
14e
14f
14g
14h
14i
10
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CF3
OMe
NH2
H
H
H
H
H
2.07 0.67
0.041 0.014
0.31 0.08
0.16 0.02
H
H
NH2
H
H
14j
14k
H
NH2
1.54 0.42
14l
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
i-Pr
H
H
i-Pr
Et
H
H
H
H
H
H
H
H
3.95 1.29
0.021 0.009
0.20 0.07
2.17 0.03
15.89 6.87
0.43 0.04
0.094 0.012
0.65 0.04
14m
14n
14o
14p
14q
14r
14s
14t
H
H
Me
CF3
H
H
H
H
t-Bu
Ph
H
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NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OPh
OBn
H
H
H
H
H
H
H
H
1.74 0.26
>50
14u
14v
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CO2Me
CO2Et
CO2Pr
CN
0.072 0.02
0.10 0.08
1.04 0.09
>50
14w
14x
14y
14z
SO2NH2
>50
14aa
CO2H
>50
14ab
a The biological activities were determined by using a well-established mammalian two-hybrid
reporter gene assay. 293FT cells were transiently transfected with expression plasmids encoding
fusion protein containing GAL4 DNA binding domain and human ERRα ligand binding domain,
and PGC-1α, respectively, together with luciferase reporter constructs. The reported results are
means of three independent experiments performed in duplicate. b Reported data. 14b
Given its strong potency in the cell-based reporter gene assay, the ERRα suppressing activity of
14n was further validated by investigating its effect on the expression of ERRα and downstream
target genes by using real-time PCR and Western blot analysis. It is known that ERRα is
important in regulation of the transcriptional functions and/or protein expression of key
compounds in tricarboxylic acid (TCA) metabolism, oxidative phosphorylation (OXPHOS) and
glycolysis, such as MCAD6 and PDK47. Thus, the impact of compound 14n on these functions
was investigated first at both the mRNA and protein levels. As shown in Figure 2, 14n dose-
dependently and potently reduced the mRNA levels of PDK4 in a quantitative real-time PCR
assay, whereas the mRNA levels of MCAD were not reduced significantly (Figure 2A).
Interestingly, the protein levels of both PDK4 and MCAD decreased dose-dependently after a 48-
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h treatment by a Western blot analysis (Figure 2B). It was also observed that both the mRNA and
protein levels of ERRα were also obviously decreased after the treatment with 14n, which might
be caused by an autoregulatory loop of ERRα.15
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Figure 2. Compound 14n down-regulates the transcriptional functions of ERRα. (A) 14n
reduced the mRNA levels of ERRα, PDK4 and MCAD in a quantitative real-time PCR assay.
MCF-7 breast cancer cells were treated with vehicle (DMSO) or 0-10 ꢀM of 14n for 48 hrs
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followed by RT-PCR analysis of mRNA expression. (B) 14n decreased the protein levels of
ERRα, PDK4 and MCAD in dose-dependent manners, determined by a Western blot analysis.
MCF-7 breast cancer cells were treated with vehicle (DMSO) or 0-20 ꢀM of 14n for 24 hrs.
Lysates from treated cells were loaded. Multiple proteins were detected, ERRα at 52kD, PDK4 at
46kD, and MCAD at 43kD in size. Results are the means or representatives of three independent
experiments (*p < 0.05).
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The anti-proliferative effects of compounds were also investigated using ER-positive MCF-7
and ER-negative MDA-MB-231 breast cancer cells, both of which positively express ERRα.16
The results demonstrated that the anti-proliferative potencies of compounds translated well from
their activities in the heterologous reporter gene assay (Table 2 and Supporting Information).
Compounds 14i, 14n and 14s displayed the most potent ERRα suppressing activities in the
reporter assay and also inhibited the growth of MCF-7 breast cancer cells with IC50 values of 6.7,
3.7 and 17.2 ꢀM, respectively. The compounds also displayed comparable potencies in inhibiting
the proliferation of ER-negative MDA-MB-231 breast cells, indicating they might be used as
lead compounds for development of new anticancer agents with which to treat triple-negative
breast cancer.
Table 2. In Vitro antiproliferative activities of the representative analogues against MCF-7 and
MDA-MB-231 breast cancer cells
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Cell Assay, IC50 (ꢀM)a
6
7
Compd
MCF-7b
6.7
MDA-MB-231c
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8.5
6.9
14i
14n
14s
3.7
17.2
14.9
a MCF-7 and MDA-MB-231 cells were treated with 6 concentration ranging from 0.016-50 ꢀM
of the compounds for 72 hrs. The antiproliferative activities of the compounds were evaluated
using the MTT assay. All results are mean values of three independent experiments performed in
duplicate. MCF-7 is an ER-positive human breast cancer cell line. MDA-MB-231 is an ER-
negative human breast cancer cell line.
b
c
Studies have demonstrated that ERRα plays an important role in modulating the motility of
cancer cells. Selective suppression of ERRα with siRNA has been shown to substantially reduce
the migration of the ERRα-positive MDA-MB-231 and MCF-7 breast cancer cells.13, 16 Therefore,
we first investigated the effect of compound 14n on MCF-7 and MDA-MB-231 cell motility by
using a Boyden Chamber assay. It was shown that 24-h treatment with compound 14n does-
dependently reduced the numbers of both MCF-7 (Figure 3A, 3B) and MDA-MB-231 (Figure
3C, 3D) cells that migrated toward serum in the lower chamber, indicating its strong inhibitory
effect on the motility of breast cancer cells. The migration inhibitory effect of 14n on MCF-7
cells was further validated by using a well-established wound healing assay. It was shown that
treatment with compound 14n moderately inhibited the migrating process in MCF-7 cells,
suppressing the wound closure by ~32% and ~40% at concentrations of 5.0 and 10.0 ꢀM (p <
0.01), respectively, compared with the untreated control (Figure 3E and 3F).
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Figure 3. Compound 14n potently inhibits migration of MCF-7 and MDA-MB-231 breast cancer
cells. (A, B) Compound 14n dose-dependently decreased the number of migrated MCF-7 cells
toward serum in the lower transwell chamber. (C, D) Compound 14n decreased the number of
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migrated MDA-MB-231 cells toward serum in the lower transwell chamber. (E, F) 14n reduced
MCF-7 cell migration into a denuded area in a wound-healing assay. Results are the means of at
least three independent experiments (*p < 0.05, **p <0.01).
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Given its promising in vitro biological activity, a preliminary pharmacokinetic (PK) study of
14n was conducted in Sprague-Dawley male rats. As shown in Table 3, compound 14n displayed
good pharmacokinetic properties, with an oral bioavailability of 71.8% and an in vivo half-life of
1.5 h.
Table 3. Pharmacokinetic Profile of 14n in Sprague-Dawley Rats.a
Dose
(mg/kg)
Cmax
(ꢀg/L)
tmax
(h)
t1/2
(h)
AUC(0-∞)
(ꢀg*h/L)
Oral bioavailability
F(%)
Route
iv
5
9594
0.033
1.25
2.1
1.5
4217
71.8
po
25
5115
15132
aEstimated from mean concentration time data (n = 4 rats per time point).
In summary, a series of 1-phenyl-4-benzoyl-1H-1,2,3-triazole analogues were identified as
functional suppressors of the transcriptional functions of ERRα. ERRα suppressing functions of
compound 14n were investigated by a cell-based reporter gene assay, real-time PCR and a
Western blot assay. Furthermore, the representative compound 14n demonstrated inhibitory
potency against the growth of both ER-positive MCF-7 and ER-negative MDA-MB-231 breast
cancer cells with positive ERRα, and a dose-dependent reduction in the migration of breast
cancer cells in vitro. The results are further evidence that ERRα may be a useful target for
treatment of breast cancer and also serve to identify compounds that could be used as leads in
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drug discovery efforts to discover compounds that could be used to treat breast cancer including
ER-negative breast cancer. Further structural optimization to improve pharmacokinetic properties
and pharmacodynamic evaluation in vivo of this class of compounds are in progress and will be
reported in due course.
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EXPERIMENTAL SECTION
General Methods for Chemistry. All reagents and solvents were used as purchased from
commercial sources. Flash chromatography was performed using silica gel (300 mesh). All
reactions were monitored by TLC, using silica gel plates with fluorescence F254 and UV light
1
13
visualization. H NMR and C NMR spectra were recorded on a Brucker AV-400 spectrometer
at 400MHz and Brucker AV-500 spectrometer at 125 MHz. Coupling constants (J) are expressed
in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million (ppm) units relative to
an internal control (TMS). Low resolution ESI-MS were recorded on an Agilent 1200 HPLC-
MSD mass spectrometer and high resolution ESI-MS on an Applied Biosystems Q-STAR Elite
ESI-LC-MS/MS mass spectrometer. Infrared (IR) spectra were recorded on a Brucker
TENSOR27 spectrometer. The purity of compounds was determined by reverse-phase high
performance liquid chromatography (HPLC) analysis to be >95%. HPLC instrument: Dionex
Summit HPLC (Column: Diamonsil C18, 5.0 ꢀm, 4.6 × 250mm (Dikma Technologies); detector:
PDA-100 photodiode array; injector: ASI-100 autoinjector; pump: p-680A). A flow rate of 1.0
mL/min was used with mobile phase of MeOH in H2O with 0.1% modifier (ammonia or
trifluoroacetate, v/v).
4-(Tetrahydro-2H-pyran-2-yloxy)benzaldehyde (8a).17 General procedure for syntheses of
8a-c. 3,4-Dihydro-2H-pyran (5.6 mL, 61.4 mmol) was added to a solution 7a (5 g, 40.9 mmol) in
anhydrous CH2Cl2 (150 mL) under argon, followed by pyridinium p-toluenesulfonate (0.5 g, 2
mmol). The reaction mixture was stirred at room temperature overnight. The crude reaction
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mixture was washed 3X with saturated aqueous NaHCO3 and brine, dried with Na2SO4, filtered,
5
6
and concentrated in vacuo. The resultant crude material was purified by column chromatography
7
8
9
1
(SiO2, PE/EtOAc stepwise elution, 10:1 to 6:1) to give 8a (8.3 g, 98% yield, yellow oil). H
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NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 7.85-7.81 (m, 2H), 7.17-7.15 (m, 2H), 5.55-5.53 (m,
1H), 3.88-3.82 (m, 1H), 3.66-3.61 (m, 1H), 2.05-1.96 (m, 1H), 1.91-1.87 (m, 2H), 1.74-1.67 (m,
2H), 1.64-1.59 (m, 1H).
1-(4-(Tetrahydro-2H-pyran-2-yloxy)phenyl)prop-2-yn-1-ol (9a).18 General procedure for
syntheses of 9a-h and 9j-k. A solution of 8a (6g, 29 mmol) in anhydrous THF (30 mL) was
cooled to 0 ℃ in an ice bath. Ethynyl magnesium bromide (0.5 M in THF, 69.6 mL, 34.8 mmol)
was then added dropwise. The reaction was allowed to warm to room temperature and
maintained for 2 hrs, at which time TLC analysis indicated complete consumption of the starting
aldehyde. The reaction was quenched by addition of saturated aqueous NH4Cl solution and the
reaction mixture was extracted several times with Et2O. The combined ether extracts were
washed with brine and dried over anhydrous Na2SO4. Filtration and removal of the solvent gave
an oil that was subjected to flash column chromatography (SiO2, PE/EtOAc stepwise elution,
1
10:1 to 5:1) to give the title compound. (6.5 g, 96%, yellow oil). H NMR (400 MHz, CDCl3) δ
7.46 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 5.43 (t, J = 3.2 Hz, 1H), 5.41 (s, 1H), 3.91-
3.85 (m, 1H), 3.61-3.57 (m, 1H), 2.65 (d, J = 1.6 Hz, 1H), 2.34 (s, 1H), 2.04-1.96 (m, 1H), 1.88-
1.84 (m, 2H), 1.70-1.58 (m, 3H).
1-Azido-4-isopropylbenzene (11a).19 General procedure for syntheses of 11a and 11m-aa. To
a 0 ℃ solution of 4-isopropylaniline (10a) (5.4 g, 40 mmol) in EtOAc (80 mL) was added
concentrated hydrochloride (16 mL), followed by a solution of NaNO2 (3.31 g, 48 mmol) in 50
mL H2O by consecutive dropwise addition over a period of 10 min with stirring. After stirring for
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a further 1 hr, a solution of NaN3 (3.12g, 48 mmol) in H2O (50 mL) was added to the above
reaction mixture with stirring. The reaction mixture was warm to room temperature and stirred
for 4 h. Then the solution was poured into H2O, extracted with EtOAc, dried over Na2SO4,
filtered and concentrated in vacuo to give 11a (6.3 g, 98%, brown oil), which was used for the
next step without further purification.
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(1-(4-Isopropylphenyl)-1H-1,2,3-triazol-4-yl)(4-(tetrahydro-2H-pyran-2-yloxy)phenyl)-
methanol (12a). General procedure for syntheses of 12a-h, 12j-k and 12m-aa. Sodium ascorbate
(89 mg, 0.45 mmol), and CuSO4 (24 mg, 0.15 mmol) were added in single portions to a solution
of 9a (348 mg, 1.5 mmol) and 1-azido-4-isopropylbenzene (11a) (266 mg, 1.65 mmol) in 1:1
tBuOH/H2O (20 mL). The reaction mixture was stirred at room temperature under an argon
atmosphere overnight and then was concentrated in vacuo, diluted with H2O and extracted with
EtOAc. The combined organic phases were dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by chromatography (SiO2, PE/CH2Cl2 stepwise
1
elution, 1:3 to CH2Cl2) to give the title compound (395 mg, 67%, white solid). H NMR (400
MHz, DMSO-d6) δ 8.58 (s. 1H), 7.80 (d, J = 8.4, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.8
Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 6.01 (d, J = 4.4 Hz, 1H), 5.84 (d, J = 4.4 Hz, 1H), 5.45-5.43
(m, 1H), 3.77-3.72 (m, 1H), 3.54-3.49 (m, 1H), 2.99-2.93 (m, 1H), 1.88-1.69 (m, 3H), 1.63-1.50
(m, 3H), 1.23 (d, J = 6.8 Hz, 6H).
(1-(4-Isopropylphenyl)-1H-1,2,3-triazol-4-yl)(4-(tetrahydro-2H-pyran-2-
yloxy)phenyl)methanone (13a). General procedure for syntheses of 13a-c, 14d-k and 14m-
14aa. MnO2 (265 mg, 3 mmol) was added to a solution of 12a (300 mg, 0.76 mmol) in CH2Cl2
(20 mL). After stirring at room temperature overnight, excess MnO2 was removed by filtration of
the reaction mixture through a pad of Celite. The filtrate was washed sequentially with H2O and
brine, and dried over anhydrous Na2SO4. Filtration and removal of the solvent afforded a residue
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that was purified by flash column chromatography (SiO2, PE/EtOAc stepwise elution, 10:1 to
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1
5:1) to give the title compound. (274 mg, 92%, white solid). H NMR (400 MHz, DMSO-d6) δ
7
8
9
9.49 (s, 1H), 8.34-8.32 (m, 2H), 7.94 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H), 7.23-7.20 (m,
2H), 5.68 (t, J = 3.2 Hz, 1H), 3.78-3.72 (m, 1H), 3.63-3.58 (m, 1H), 3.32 (s, 1H), 3.06-2.95 (m,
1H), 1.93-1.75 (m, 3H), 1.68-1.54 (m, 3H), 1.26 (d, J = 7.2 Hz, 6H).
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(4-Hydroxyphenyl)(1-(4-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone (14a). General
procedure for syntheses of 14a-c and 15. Pyridinium p-toluenesulfonate (5mg, 0.02 mmol) was
added to a solution of 13a (154 mg, 0.39 mmol) in EtOH (20 mL). The reaction mixture was then
warmed to 50 ℃ and stirred for 2 hrs. After the complete consumption of the starting material,
the solution was concentrated in vacuo and subjected to flash column chromatography (SiO2,
1
PE/EtOAc, 5:1 to CH2Cl2/EtOAc, 20:1) to give 14a (107mg, 89%, white solid). H NMR (400
MHz, DMSO-d6) δ 10.51 (s, 1H), 9.44 (s, 1H), 8.28 (d, J = 8.8 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H),
7.50 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 3.05-2.95 (m, 1H), 1.25 (d, J = 7.2 Hz, 6H).
13C NMR (125 MHz, DMSO-d6) δ 182.97, 162.49, 149.75, 147.61, 134.09, 132.78, 127.73,
127.66, 127.33, 120.69, 115.28, 33.07, 23.64. HRMS (ESI) for C18H17N3O2 [M+H]+, Calcd:
308.1394, Found: 308.1388. HPLC purity: 96.8%. Melting point: 199.0-201.6 ℃.
(3-Hydroxyphenyl)(1-(4-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone (14b). 14b
(white solid) was synthesized by a method similar to that described for 14a. 1H NMR (400 MHz,
DMSO-d6) δ 9.86 (s, 1H), 9.50 (s, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.6 Hz, 1H), 7.68-
7.67 (m, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.41 (t, J = 8.0 Hz, 1H), 7.12-7.09 (m, 1H), 3.06-2.95 (m,
1H), 1.25 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 185.34, 157.83, 150.31, 147.60,
138.15, 134.48, 130.10, 128.25, 128.13, 121.37, 121.22, 120.96, 116.75, 33.54, 24.11. HRMS
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(ESI) for C18H17N3O2 [M+H]+, Calcd: 308.1394, Found: 308.1388. HPLC purity: 99.26%.
Melting point: 182.9-183.6 ℃.
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(2-Hydroxyphenyl)(1-(4-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone (14c). 14c
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(yellow solid) was synthesized by a method similar to that described for 14a. H NMR (400
MHz, DMSO-d6) δ 11.40 (s, 1H), 9.53 (s, 1H), 8.39 (dd, J = 1.6, 8.0 Hz, 1H), 7.93 (d, J = 8.4
Hz, 2H), 7.59-7.55 (m, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.05-7.02 (m, 2H), 3.06-2.95 (m, 1H), 1.25
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(d, J = 7.2 Hz, 6H). C NMR (125 MHz, DMSO-d6) δ 188.26, 160.29, 149.90, 147.26, 135.47,
133.96, 131.94, 127.85, 127.69, 121.59, 120.75, 119.13, 117.42, 33.08, 23.64. HRMS (ESI) for
C18H17N3O2 [M+H]+, Calcd: 308.1394, Found: 308.1393. HPLC purity: 98.19%. Melting point:
116.7-117.3 ℃.
(1-(4-Isopropylphenyl)-1H-1,2,3-triazol-4-yl)(2-nitrophenyl)methanone (14d). 14d (yellow
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solid) was synthesized from 12d by a method similar to that described for 13a. H NMR (400
MHz, DMSO-d6) δ 9.65 (s, 1H), 8.29 (d, J = 7.6 Hz, 1H), 7.99-7.96 (m, 1H), 7.90-7.86 (m, 3H),
7.80 (dd, J = 1.2, 7.6 Hz, 1H), 7.50 (d, J = 8.8 Hz, 2H), 3.05-2.95 (m, 1H), 1.24 (d, J = 6.8 Hz,
6H). 13C NMR (125 MHz, DMSO-d6) δ 185.32, 150.08, 147.01, 146.53, 134.83, 134.33, 133.83,
131.95, 129.53, 127.73, 126.51, 124.29, 120.77, 33.08, 23.63. HRMS (ESI) for C18H16N4O3
[M+H]+, Calcd: 337.1295, Found: 337.1294. HPLC purity: 97.79%. Melting point: 123.1-123.9
℃.
(1-(4-Isopropylphenyl)-1H-1,2,3-triazol-4-yl)(o-tolyl)methanone (14e). 14e (white solid)
was synthesized by a method similar to that described for 14d. 1H NMR (400 MHz, DMSO-d6) δ
9.46 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 7.6 Hz, 1H), 7.52-7.48 (m, 3H), 7.39-7.34 (m,
2H), 3.05-2.95 (m, 1H), 2.38 (s, 3H), 1.25 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ
188.82, 149.84, 147.58, 137.47, 136.72, 134.01, 131.01, 129.57, 127.65, 127.46, 125.40,
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120.75,33.07, 23.64, 19.72. HRMS (ESI) for C19H19N3O [M+H]+, Calcd: 306.1601, Found:
306.1600. HPLC purity: 97.96%. Melting point: 88.2-89.4 ℃.
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(2-Fluorophenyl)(1-(4-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone (14f). 14f (white
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solid) was synthesized by a method similar to that described for 14d. H NMR (400 MHz,
DMSO-d6) δ 9.57 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.83-7.79 (m, 1H), 7.73-7.68 (m, 1H), 7.50
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(d, J = 8.8 Hz, 2H), 7.43-7.38 (m, 2H), 3.05-2.95 (m, 1H), 1.25 (d, J = 6.8 Hz, 6H). C NMR
(125 MHz, DMSO-d6) δ 184.15, 160.73, 158.72, 149.94, 147.07, 134.08, 134.01, 133.93, 130.86,
127.69, 127.30, 126.39, 126.28, 124.51, 124.48, 120.76, 116.42, 116.25, 33.08, 23.63. HRMS
(ESI) for C18H16FN3O [M+H]+, Calcd: 310.1350, Found: 310.1351. HPLC purity: 99.71%.
Melting point: 118.7-119.4 ℃.
(1-(4-Isopropylphenyl)-1H-1,2,3-triazol-4-yl)(2-(trifluoromethyl)phenyl)methanone (14g).
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14g (white solid) was synthesized by a method similar to that described for 14d. H NMR (400
MHz, DMSO-d6) δ 9.61 (s, 1H), 7.94 (d, J = 6.8 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.87-7.78 (m,
3H), 7.50 (d, J = 8.4 Hz, 2H), 3.05-2.95 (m, 1H), 1.25 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz,
DMSO-d6) δ 187.07, 150.03, 146.88, 137.24, 133.84, 132.27, 130.91, 129.04, 127.69, 127.53,
126.64, 126.60, 126.42, 126.16, 124.78, 122.60, 120.80, 33.07, 23.61. HRMS (ESI) for
C19H16F3N3O [M+H]+, Calcd: 360.1318, Found: 360.1314. HPLC purity: 98.46%. Melting point:
111.0-111.4 ℃.
(1-(4-Isopropylphenyl)-1H-1,2,3-triazol-4-yl)(2-methoxyphenyl)methanone (14h). 14h
(white solid) was synthesized by a method similar to that described for 14d. 1H NMR (400 MHz,
CDCl3) δ 8.53 (s, 1H), 7.69-7.65 (m, 3H), 7.52 (t, J = 7.2 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.10-
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7.04 (m, 2H), 3.83 (s, 3H), 3.05-2.95 (m, 1H), 1.30 (d, J = 7.2 Hz, 6H). C NMR (125 MHz,
CDCl3) δ 187.86, 158.15, 150.56, 148.72, 134.35, 132.98, 130.43, 128.01, 127.87, 124.98,
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120.81, 120.46, 111.92, 55.89, 33.87, 23.84. HRMS (ESI) for C19H19N3O2 [M+H]+, Calcd:
322.1550, Found: 322.1550. HPLC purity: 99.88%. Melting point: 105.3-106.8 ℃.
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2-(Hydroxy(1-(4-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methyl)phenol (15). 15 (white
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solid) was synthesized from 12c by a method similar to that described for 14a. H NMR (400
MHz, DMSO-d6) δ 9.42 (s, 1H). 8.46 (s, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.46-7.41 (m, 3H), 7.10-
7.06 (m, 1H), 6.83-6.77 (m, 2H), 6.16 (d, J = 4.8 Hz, 1H), 5.83 (d, J = 4.8 Hz, 1H), 3.01-2.91 (m,
1H), 1.23 (d, J = 7.2 Hz, 6H).
2-((1-(4-Isopropylphenyl)-1H-1,2,3-triazol-4-yl)methyl)phenol (14l). Triethylsilane (152
ꢀL, 1 mmol) and trifluoroacetic acid (73 ꢀL, 1 mmol) were added to a solution of 15 (100 mg,
0.32 mmol) in CH2Cl2 (5 mL) under argon with stirring at 0 ℃. The solution was stirred at room
temperature overnight, then quenched by the addition of saturated aqueous NaHCO3. The
mixture was extracted with CH2Cl2 and the combined organic layers were dried with Na2SO4,
filtered and evaporated in vacuo. The residue was purified by flash column chromatography
(SiO2, PE/EtOAc stepwise elution, 5:1 to 4:1) to give the title compound (77 mg, 81% yield,
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white solid). H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.40 (s, 1H), 7.77 (d, J = 8.8 Hz,
2H), 7.42 (d, J = 8.8 Hz, 2H), 7.09-7.02 (m, 2H), 6.82 (d, J = 7.6 Hz, 1H), 6.73 (t, J = 7.6 Hz,
1H), 3.98 (s, 2H), 3.00-2.93 (m, 1H), 1.23 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ
154.82, 148.68, 146.78, 134.74, 129.86, 127.51, 127.34, 125.34, 120.57, 119.89, 118.90, 114.96,
32.97, 25.49, 23.66. HRMS (ESI) for C18H19N3O [M+H]+, Calcd: 294.1601, Found: 294.1600.
HPLC purity: 99.89%. Melting point: 147.5-149.3 ℃.
(2-Aminophenyl)(1-(4-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone (14i). General
procedure for syntheses of 14i-k and 14m-aa. Pd/C (10%, 23 mg) was added to a solution of 12d
(223 mg, 0.66 mmol) in MeOH (20 mL). After two vacuum/H2 cycles to replace air inside the
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reaction flask with H2, the reaction mixture was stirred at room temperature under a hydrogen
balloon for 4 h. The reaction mixture was filtered through a pad of celite and concentrated in
vacuo to obtain (2-aminophenyl)(1-(4-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanol (12i),
which was used for the next step without further purification. The intermediate 12i was dissolved
in CH2Cl2 (20 mL) and MnO2 (230 mg, 2.64 mmol) was added to this solution. After stirring at
room temperature overnight, The reaction mixture was filtered through a pad of celite,
concentrated in vacuo, and purified with flash chromatography (SiO2, PE/CH2Cl2 stepwise
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elution, 1:1 to 1:2) to give 14i (140 mg, 63% for two steps, yellow solid). H NMR (400 MHz,
DMSO-d6) δ 9.34 (s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.4 Hz,
2H), 7.35-7.31 (m, 3H), 6.86 (d, J = 8.4 Hz, 1H), 6.61 (t, J = 7.6 Hz, 1H), 3.05-2.95 (m, 1H),
1.25 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 186.14, 152.33, 149.67, 148.43,
134.58, 134.14, 133.65, 127.65, 126.90, 120.66, 116.73, 116.26, 114.41, 33.07, 23.66. HRMS
(ESI) for C18H18N4O [M+H]+, Calcd: 307.1553, Found: 307.1553. HPLC purity: 96.22%.
Melting point: 114.0-114.8 ℃.
(3-Aminophenyl)(1-(4-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone
(14j).
14j
(yellow solid) was synthesized by a method similar to that described for 14i. 1H NMR (400 MHz,
DMSO-d6) δ 9.44 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.45-7.42 (m, 2H),
7.23 (t, J = 7.8 Hz, 1H), 6.89-6.87 (m, 1H), 5.41 (s, 2H), 3.06-2.95 (m, 1H), 1.25 (d, J = 6.8 Hz,
6H). 13C NMR (100 MHz, DMSO-d6) δ 185.39, 149.63, 148.64, 147.04, 137.19, 133.93, 128.68,
127.43, 127.22, 120.60, 118.56, 117.52, 114.48, 32.86, 23.39. HRMS (ESI) for C18H18N4O
[M+H]+, Calcd: 307.1553, Found: 307.1554. HPLC purity: 98.43%. Melting point: 175.9-177.4
℃.
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(4-Aminophenyl)(1-(4-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone
(14k).
14k
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(white solid) was synthesized by a method similar to that described for 14i. 1H NMR (400 MHz,
DMSO-d6) δ 9.34 (s, 1H), 8.15 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.4 Hz,
2H), 6.65 (d, J = 8.8 Hz, 2H), 6.26 (s, 2H), 3.05-2.95 (m, 1H), 1.25 (d, J = 6.8 Hz, 6H). 13C
NMR (125 MHz, DMSO-d6) δ 181.74, 154.22, 149.60, 148.19, 134.18, 132.77, 127.64, 126.72,
123.68, 120.61, 112.54, 33.07, 23.66. HRMS (ESI) for C18H18N4O [M+H]+, Calcd: 307.1553,
Found: 307.1553. HPLC purity: 99.84%. Melting point: 200.6-203.4 ℃.
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(2-Aminophenyl)(1-(2-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone (14m). 14m
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(yellow oil) was synthesized by a method similar to that described for 14i. H NMR (400 MHz,
DMSO-d6) δ 9.07 (s, 1H), 8.46 (dd, J = 1.2, 8.4 Hz, 1H), 7.65-7.62 (m, 2H), 7.49-7.41 (m, 2H),
7.35-7.31 (m, 3H), 6.86 (d, J = 8.4 Hz, 1H), 6.62 (t, J = 7.9 Hz, 1H), 2.64-2.57 (m, 1H), 1.16 (d,
J = 6.8 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 186.17, 152.33, 147.75, 144.25, 134.57,
134.47, 133.62, 130.96, 130.81, 126.84, 126.78, 126.73, 116.75, 116.28, 114.40, 27.64, 23.40.
HRMS (ESI) for C18H18N4O [M+H]+, Calcd: 307.1553, Found: 307.1553. HPLC purity: 95.59%.
(2-Aminophenyl)(1-(3-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone
(14n).
14n
(yellow solid) was synthesized by a method similar to that described for 14i. 1H NMR (400 MHz,
DMSO-d6) δ 9.41 (s, 1H), 8.40-8.38 (m, 1H), 7.90 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.54 (t, J =
8.0 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.35-7.31 (m, 3H), 6.86 (d, J = 8.0 Hz, 1H), 6.61 (t, J = 7.6
Hz, 1H), 3.08-2.98 (m, 1H), 1.28 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 186.18,
152.32, 150.63, 148.43, 136.24, 134.60, 133.65, 129.80, 127.22, 127.00, 118.57, 118.11, 116.74,
116.29, 114.43, 33.42, 23.60. HRMS (ESI) for C18H18N4O [M+H]+, Calcd: 307.1553, Found:
307.1552. HPLC purity: 99.15%. Melting point: 104.4-105.6 ℃.
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(2-Aminophenyl)(1-(3-ethylphenyl)-1H-1,2,3-triazol-4-yl)methanone (14o). 14o (yellow
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solid) was synthesized by a method similar to that described for 14i. H NMR (400 MHz,
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DMSO-d6) δ 9.38 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.53 (t,
J = 7.8 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.35-7.32 (m, 3H), 6.86 (d, J = 8.4 Hz, 1H), 6.61 (t, J =
7.4 Hz, 1H), 2.74 (q, J = 7.6 Hz, 2H), 1.26 (t, J = 7.6 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ
186.14, 152.33, 148.43, 145.94, 136.21, 134.60, 133.64, 129.76, 128.61, 126.94, 119.91, 117.92,
116.73, 116.27, 114.42, 27.98, 15.30. HRMS (ESI) for C17H16N4O [M+H]+, Calcd: 293.1397,
Found: 293.1397. HPLC purity: 97.53%. Melting point: 107.8-109.5 ℃.
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(2-Aminophenyl)(1-m-tolyl-1H-1,2,3-triazol-4-yl)methanone (14p). 14p (yellow solid) was
synthesized by a method similar to that described for 14i. 1H NMR (400 MHz, DMSO-d6) δ 9.36
(s, 1H), 8.39 (dd, J = 1.2, 8.0 Hz, 1H), 7.87 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 7.8 Hz,
1H), 7.37-7.31 (m, 4H), 6.86 (d, J = 8.4 Hz, 1H), 6.63-6.60 (m, 1H), 2.44 (s, 3H). 13C NMR (125
MHz, DMSO-d6) δ 186.11, 152.32, 148.42, 139.67, 136.11, 134,59, 133.62, 129.74, 129.65,
126.87, 120.97, 117.64, 116.73, 116.25, 114.41, 20.85. HRMS (ESI) for C16H14N4O [M+H]+,
Calcd: 279.1240, Found: 279.1234. HPLC purity: 98.56%. Melting point: 140.4-142.2 ℃.
(2-Aminophenyl)(1-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methanone
(14q).
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14q (yellow solid) was synthesized by a method similar to that described for 14i. H NMR (400
MHz, DMSO-d6) δ 9.57 (s, 1H), 8.43 (s, 1H), 8.39-8.35 (m, 2H), 7.94-7.87 (m, 2H), 7.36-7.32
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(m, 3H), 6.87 (d, J = 8.4 Hz, 1H), 6.62 (t, J = 7.6 Hz, 1H). C NMR (125 MHz, DMSO-d6) δ
185.95, 152.35, 148.48, 136.70, 134.68, 133.56, 131.27, 130.65, 130.39, 127.45, 125.69, 124.59,
122.44, 117.47, 117.44, 116.76, 116.21, 114.45. HRMS (ESI) for C16H11F3N4O [M+H]+, Calcd:
333.0958, Found: 333.0956. HPLC purity: 97.9%. Melting point: 162.4-163.3 ℃.
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(2-Aminophenyl)(1-phenyl-1H-1,2,3-triazol-4-yl)methanone (14r). 14r (yellow solid) was
synthesized by a method similar to that described for 14i. 1H NMR (400 MHz, DMSO-d6) δ 9.40
(s, 1H), 8.40 (dd, J = 1.2, 8.4 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.66-7.62 (m, 2H), 7.57-7.53 (m,
1H), 7.35-7.31 (m, 3H), 6.86 (d, J = 8.4 Hz, 1H), 6.63-6.59 (m, 1H). 13C NMR (125 MHz,
DMSO-d6) δ 168.11, 152.34, 148.50, 136.17, 134.61, 133.65, 129.87, 129.17, 126.95, 120.58,
116.73, 116.24, 114.42. HRMS (ESI) for C15H12N4O [M+H]+, Calcd: 265.1084, Found:
265.1083. HPLC purity: 99.32%. Melting point: 95.3-96.1 ℃.
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(2-Aminophenyl)(1-(3-tert-butylphenyl)-1H-1,2,3-triazol-4-yl)methanone
(14s).
14s
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(yellow oil) was synthesized by a method similar to that described for 14i. H NMR (400 MHz,
DMSO-d6) δ 9.46 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.98 (s, 1H), 7.84-7.81 (m, 1H), 7.59-7.53
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(m, 2H), 7.35-7.32 (m, 3H), 6.86 (d, J = 8.0 Hz, 1H), 6.61 (t, J = 7.6 Hz, 1H), 1.36 (s, 9H). C
NMR (125 MHz, DMSO-d6) δ 186.21, 152.94, 152.32, 148.44, 136.10, 134.58, 133.67, 129.55,
127.12, 126.04, 117.93, 117.66, 116.73, 116.30, 114.41, 34.82, 30.90. HRMS (ESI) for
C19H20N4O [M+H]+, Calcd: 321.1710, Found: 321.1708. HPLC purity: 99.12%.
(2-Aminophenyl)(1-(biphenyl-3-yl)-1H-1,2,3-triazol-4-yl)methanone (14t). 14t (yellow
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solid) was synthesized by a method similar to that described for 14i. H NMR (400 MHz,
DMSO-d6) δ 9.57 (s, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.30 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.87-
7.84 (m, 3H), 7.73 (t, J = 7.8 Hz, 1H), 7.53 (t, J = 7.4 Hz, 2H), 7.44 (t, J = 7.2 Hz, 1H), 7.36-7.32
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(m, 3H), 6.87 (d, J = 8.4 Hz, 1H), 6.62 (t, J = 7.4 Hz, 1H). C NMR (125 MHz, DMSO-d6) δ
186.14, 152.33, 148.48, 141.87, 138.67, 136.79, 134.61, 133.64, 130.49, 129.00, 128.18, 127.25,
127.00, 119.48, 118.68, 116.74, 116.29, 114.44. HRMS (ESI) for C21H16N4O [M+H]+, Calcd:
341.1397, Found: 341.1396. HPLC purity: 99.11%. Melting point: 174.6-177.0 ℃.
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(2-Aminophenyl)(1-(3-phenoxyphenyl)-1H-1,2,3-triazol-4-yl)methanone
(14u).
14u
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(yellow solid) was synthesized by a method similar to that described for 14i. 1H NMR (400 MHz,
DMSO-d6) δ 9.43 (s, 1H), 8.35 (dd, J = 1.4, 8.2 Hz, 1H), 7.81 (dd, J = 1.6, 8.0 Hz, 1H), 7.71 (t, J
= 2.2 Hz, 1H), 7.63 (t, J = 8.2 Hz, 1H), 7.48-7.43 (m, 2H), 7.34-7.30 (m, 3H), 7.24-7.21 (m, 1H),
7.16-7.12 (m, 3H), 6.85 (d, J = 8.0 Hz, 1H), 6.62-6.58 (m, 1H). 13C NMR (125 MHz, DMSO-d6)
δ 186.03, 157.85, 155.77, 152.33, 148.45, 137.40, 134.63, 133.62, 131.44, 130.24, 127.05,
124.24, 119.21, 118.59, 116.73, 116.20, 115.12, 114.41, 110.50. HRMS (ESI) for C21H16N4O2
[M+H]+, Calcd: 357.1346, Found: 357.1345. HPLC purity: 99.34%. Melting point: 92.6-94.2 ℃.
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(2-Aminophenyl)(1-(3-(benzyloxy)phenyl)-1H-1,2,3-triazol-4-yl)methanone (14v). 14v
(yellow solid) was synthesized by a method similar to that described for 14i. 1H NMR (400 MHz,
DMSO-d6) δ 9.43 (s, 1H), 8.39 (dd, J = 1.2, 8.0 Hz, 1H), 7.72 (t, J = 2.0 Hz, 1H), 7.64-7.62 (m,
1H), 7.54 (t, J = 7.8 1H), 7.50 (d, J = 7.2 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.37-7.31 (m, 4H),
7.18 (dd, J = 1.8, 8.2 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.61 (t, J = 7.6 Hz, 1H), 5.24 (s, 2H). 13C
NMR (125 MHz, DMSO-d6) δ 186.08, 159.25, 152.34, 148.45, 137.16, 136.54, 134.62, 133.64,
130.87, 128.46, 127.98, 127.80, 127.01, 116.74, 116.24, 115.74, 114.43, 112.70, 106.98, 69.73.
HRMS (ESI) for C22H18N4O2 [M+H]+, Calcd: 371.1503, Found: 371.1499. HPLC purity:
96.33%. Melting point: 144.3-146.6 ℃.
Methyl 3-(4-(2-aminobenzoyl)-1H-1,2,3-triazol-1-yl)benzoate (14w). 14w (yellow solid)
was synthesized by a method similar to that described for 14i. 1H NMR (400 MHz, DMSO-d6) δ
9.52 (s, 1H), 8.56 (s, 1H), 8.38-8.31 (m, 2H), 8.10 (d, J = 8.0 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H),
7.36-7.32 (m, 3H), 6.87 (d, J = 8.4 Hz, 1H), 6.62 (t, J = 7.8 Hz, 1H), 3.93 (s, 3H). 13C NMR (125
MHz, DMSO-d6) δ 186.02, 165.21, 152.35, 148.51, 136.48, 134.66, 133.62, 131.27, 130.56,
129.53, 127.26, 125.16, 120.94, 116.75, 116.23, 114.45, 52.56. HRMS (ESI) for C17H14N4O3
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Journal of Medicinal Chemistry
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[M+H]+, Calcd: 323.1139, Found: 323.1139. HPLC purity: 99.80%. Melting point: 175.6-176.5
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Ethyl 3-(4-(2-aminobenzoyl)-1H-1,2,3-triazol-1-yl)benzoate (14x). 14x (yellow solid) was
synthesized by a method similar to that described for 14i. 1H NMR (400 MHz, CDCl3) δ 8.78 (d,
J = 1.2 Hz, 1H), 8.76 (s, 1H), 8.76-8.43 (m, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.09-8.07 (m, 1H),
7.67 (t, J = 8.0 Hz, 1H), 7.38-7.34 (m, 1H), 6.79-6.73 (m, 2H), 6.32 (s, 2H), 4.45 (q, J = 7.2 Hz,
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2H), 1.44 (t, J = 7.2 Hz, 3H). C NMR (125 MHz, CDCl3) δ 186.49, 165.09, 151.64, 149.97,
136.64, 135.05, 134.38, 132.51, 130.25, 130.15, 125.91, 124.85, 121.44, 117.59, 116.93, 116.22,
61.73, 14.30. HRMS (ESI) for C18H16N4O3 [M+H]+, Calcd: 337.1295, Found: 337.1296. HPLC
purity: 99.92%. Melting point: 132.5-132.9 ℃.
Propyl 3-(4-(2-aminobenzoyl)-1H-1,2,3-triazol-1-yl)benzoate (14y). 14y (yellow solid) was
synthesized by a method similar to that described for 14i. 1H NMR (400 MHz, CDCl3) δ 8.77 (d,
J = 8.0 Hz, 1H), 8.66 (s, 1H), 8.44-8.43 (m, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.09-8.06 (m, 1H),
7.67 (t, J = 8.0 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 6.79-6.72 (m, 2H), 6.32 (s, 2H), 4.35 (t, J = 6.8
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Hz, 2H), 1.86-1.81 (m, 2H), 1.06 (t, J = 7.2 Hz, 3H). C NMR (125 MHz, CDCl3) δ 186.49,
165.16, 151.64, 149.96, 136.66, 135.05, 134.38, 132.52, 130.24, 130.16, 125.91, 124.85, 121.43,
117.59, 116.93, 116.21, 67.28, 22.06, 10.49. HRMS (ESI) for C19H18N4O3 [M+H]+, Calcd:
351.1452, Found: 351.1451. HPLC purity: 99.45%. Melting point: 118.6-119.7 ℃.
3-(4-(2-Aminobenzoyl)-1H-1,2,3-triazol-1-yl)benzonitrile (14z). 14z (yellow solid) was
synthesized by a method similar to that described for 14i. 1H NMR (400 MHz, DMSO-d6) δ 9.49
(s, 1H), 8.58 (s, 1H), 8.41-8.35 (m, 2H), 8.03 (d, J = 7.6 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 7.36-
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7.32 (m, 3H), 6.87 (d, J = 8.4 Hz, 1H), 6.64-6.60 (m, 1H). C NMR (125 MHz, DMSO-d6) δ
185.84, 152.38, 148.49, 136.63, 134.71, 133.53, 132.74, 131.25, 127.35, 125.25, 124.11, 117.72,
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