Discovery and biological evaluation of novel androgen receptor antagonist for castration-resistant prostate cancer

Article abstract of DOI:10.1016/j.ejmech.2019.03.041

Prostate cancer (PC) is the second most common malignancy in men worldwide. Among current therapies, two antiandrogens, Abiraterone Acetate and Enzalutamide (Enza) have become the standard of care for patients with metastatic castration-resistant prostate

Full text of DOI:10.1016/j.ejmech.2019.03.041

European Journal of Medicinal Chemistry 171 (2019) 265e281
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery and biological evaluation of novel androgen receptor
antagonist for castration-resistant prostate cancer
a
a
b
a
a
a
Jiang Yu , Lanxi Zhang , Guoyi Yan , Peiting Zhou , Chaoguo Cao , Fei Zhou ,
c
a, c, *
Xinghai Li , Yuanwei Chen
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy,
Chengdu, 610041, China
Department of Hepatobiliary Pancreatic Surgery, Henan Province People's Hospital, Zhengzhou, 450003, China
Hinova Pharmaceuticals Inc, 4th Floor, Building RongYao A, No. 5, Keyuan South Road, Chengdu, 610041, China
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 December 2018
Received in revised form
Prostate cancer (PC) is the second most common malignancy in men worldwide. Among current ther-
apies, two antiandrogens, Abiraterone Acetate and Enzalutamide (Enza) have become the standard of
care for patients with metastatic castration-resistant prostate cancer (mCRPC). Here, we designed and
synthesized a new series of nonsteroidal compounds deriving from the hybridization of Abiraterone (Abi)
and Enzalutamide, among which compound 4a featuring the diphenylamine scaffold was identified as a
potent and cell selective androgen receptor (AR) antagonist. In cell proliferation assays, compound 4a
exhibited better antiproliferative activities than Enzalutamide against AR-overexpressing VCaP cells and
14 March 2019
Accepted 16 March 2019
Available online 22 March 2019
Keywords:
Androgen receptor antagonist
F876L mutation
Enzalutamide resistance
Castration-resistant prostate cancer
22Rv1 cells bearing H874Y-mutated AR. In addition, 4a suppressed the activity of AR-F876L mutant that
confers resistance to Enzalutamide and efficiently blocked R1881-induced PSA and FKBP5 gene
expression. In competitive binding assay, compound 4a displayed higher binding affinity to AR than
Enzalutamide. These results suggest compound 4a as a potential candidate to treat Enza-resistant CRPC.
©
2019 Published by Elsevier Masson SAS.
1
. Introduction
most recently Apalutamide (formerly known as ARN509) for non-
metastatic CRPC [11e13]. Abiraterone, a steroidal antiandrogen,
blocked dihydrotestosterone (DHT) biosynthesis through sup-
pressing the 17-hydroxylase/17,20-lyase (CYP17A1), which repre-
Prostate cancer (PC) is the second most commonly diagnosed
male malignancy and the fifth leading cause of cancer mortality in
men worldwide [1]. The androgen receptor (AR), a ligand-
dependent transcription factor, is highly expressed throughout
various stages of prostate cancer [2,3]. The dysregulation of AR
signaling is crucial for the occurrence and progression of prostate
cancer. Multiple mechanisms have been identified to confirm
maintained AR signaling in CRPC, including AR amplifications,
increased AR expression and the presence of point mutations in
ligand binding domain (LBD) [4e6].
sents
a breakthrough for the treatment of CRPC [14e16].
Enzalutamide and Apalutamide are second-generation AR antago-
nists with superior binding affinity to the ligand binding domain of
AR (AR-LBD) than the first-generation inhibitor, Bicalutamide
[17,18]. Other second-generation AR antagonists (Fig. 1A) like Dar-
olutamide (formerly known as ODM-201), Galeterone (Gal) and
HC-1119, have entered clinical trials [19e21]. As a multi-target oral
small molecule inhibitor, Galeterone blocked AR signaling through
AR-LBD antagonism, AR degradation and inhibition of CYP17A1
[22]. Unfortunately, Galeterone failed in the phase III clinical trial
(ARMOR3-SV) and the company announced the discontinuation of
their phase II expansion (ARMOR2) as well on August 22, 2016 [23].
Despite the success achieved in CRPC with second generation
androgen deprivation therapies (ADT), such as Abiraterone [24,25]
and Enzalutamide [12,26], resistance to both therapies have been
observed. The proposed mechanisms of acquired and primary
resistance include up-regulation of systemic and intratumoral
Until 2010, the docetaxel-based chemotherapy was the only
strategy known to extend survival in patients with mCRPC [7e10].
Since 2011, three AR-axis inhibitors (Fig. 1A) have been approved by
FDA including Abiraterone Acetate, Enzalutamide for mCRPC and
*
Corresponding author. State Key Laboratory of Biotherapy and Cancer Center,
West China Hospital, Sichuan University and Collaborative Innovation Center for
Biotherapy, Chengdu, 610041, China.
E-mail address: ywchen@scu.edu.cn (Y. Chen).
https://doi.org/10.1016/j.ejmech.2019.03.041
0223-5234/© 2019 Published by Elsevier Masson SAS.

266
J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
Abbreviations
IC50
50% inhibitory concentration;
CCK-8cell counting kit-8PSA cell counting kit-8PSAprostate
AR
androgen receptor
specific antigen
AR-LBD
PC
ligand binding domain of AR
prostate cancer
castration-resistant prostate cancer
metastatic castration-resistant prostate cancer
androgen deprivation therapies
Enzalutamide
FKBP5
wt-AR
EGFP
TGI
DCM
DMF
DMA
PEG
FK506 Binding Protein 5
wild-type AR
enhanced green fluorescence protein
tumor growth inhibition
dichloromethane
N,N-Dimethyl formamide
N,N-dimethylacetamide
poly(ethylene glycol)
CRPC
mCRPC
ADT
Enza
Abi
Abiraterone
Galeterone
Gal
CYP17A1 Cytochrome P450 17A1
DHT dihydrotestosterone
7-OHP
17a-hydroxy progesterone.
androgen biosynthesis, AR gene amplification or overexpression
and AR mutations in the ligand-binding domain [27]. Recently, a
F876L missense mutation in AR-LBD has been identified, which
confers an antagonist-to-agonist switch driving the resistance to
Enzalutamide and ARN-509 [28,29]. Other recurrent point muta-
tions in AR-LBD including L701H, W741C (Bicalutamide resistance),
H874Y and T877A (Flutamide resistance) are present in approxi-
mately 15e20% of CRPC cases, a frequency that grows to over 60%
when AR gene amplification is included [30,31].
study, based on the structures of FDA-approved Enzalutamide and
Abiraterone, a new class of compounds were designed, synthesized
and evaluated.
2. Chemistry
Recently, the utilization of molecular hybridization strategy has
led to the discovery of many active molecules [32e34]. Although
Enzalutamide and Abiraterone feature different scaffolds, the
structural similarity between these two antiandrogens attracted
our attention. As described in Fig. 1B, both of them bear similar
Therefore, there is an unmet and urgent need to develop novel
and potent antiandrogens to block the AR signaling in CRPC. In this
Fig. 1. (A) Examples of antiandrogen. (B) Design of novel compounds.

J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
267
three-ring structure and the ring 1 and ring 3 of Enzalutamide
match well with those of Abiraterone. Thus, the resulting hybrid
molecules were expected to maintain activity on AR or CYP17
enzyme. Encouragingly, several compounds deriving from Preg-
nenolone or Galeterone with similar three-ring scaffold have been
reported to potently inhibit the activities of AR or CYP17 enzyme
prostate cancer cells [17] via a CCK-8 cell proliferation assay (SAB,
CP002). Compounds with IC50 value lower than 1 M were next
m
tested in AR-overexpressing VCaP cells [38] or 22Rv1 cells bearing
H874Y-mutated AR [39,40]. To rule out the non-AR-mediated
toxicity, AR-negative human prostate cancer cells (PC-3) and non-
cancer originated human hepatocyte cells (L-02) were also used.
To develop novel inhibitors, we initially designed compounds
with tetrahydroisoquinoline, indoline and tetrahydroquinoline
fragments (1a-k). As presented in Table 1, compounds bearing
tetrahydroquinoline scaffold (1h-j) showed superior activity
against LNCaP/AR cells, although poor inhibitory activities toward
VCaP and 22Rv1 cells were observed.
[
35,36]. Therefore, to develop novel type of compounds we started
our research from hybrid molecule bearing a bicyclic linker at the
position of ring 2.
To obtain desired compounds 1a-k, intermediate 3 was syn-
thesized following literature procedure [37] as shown in Scheme 1.
The subsequent nucleophilic substitution reaction of 4-Fluoro-2-
(
trifluoromethyl) benzonitrile with corresponding amine (2e4)
Then we simplified the tetrahydroquinoline scaffold by opening
its piperidine ring. The resulting diphenylamine analogues were
synthesized and evaluated. As presented in Table 2, replacing the
followed by Suzuki coupling yielded compounds 1a-k. In addition,
compound 4b was successfully prepared by a two-step procedure.
Nucleophilic substitution reaction using 4-Fluoro-2-(tri-
fluoromethyl) benzonitrile (8) and 3-Iodophenol provided corre-
sponding diphenyl ether (9), which underwent Suzuki coupling to
achieve compound 4b.
CF
activities against LNCaP/AR. Comparing compounds 2b, 2d, 2f with
2a, 2c, 2e, the introduction of methyl group at R position had little
3
group (2a, 2b) with Cl (2c, 2d) caused a moderate increase in
2
effect on the inhibitory activity toward LNCaP/AR cells. In addition,
Scheme 2 describes the general procedure for the synthesis of
diphenylamine analogues (2a-h, 3a-n, 4a, 4c-e). Diazotization of
arylamine (10) by nitrous acid generated in situ and the substitution
of N2 group by iodide resulted in product 12. Next, intermediates
the phenyl substituted analogues 2g and 2h exhibited significantly
decreased activity against LNCaP/AR with IC50 values of 1.29
and 2.78 M, respectively. This result indicates the heteroaromatic
substituent on ring B is of great importance. Noteworthily, com-
pound 2f bearing a methyl group at R position exhibited improved
potency toward VCaP cells with an IC50 value of 3.92 М, suggesting
mM
m
17e20 were obtained from 12 or commercially available 11 through
2
palladium-catalyzed cross coupling and N-alkylation reactions.
Finally, various ring C were introduced by Suzuki coupling or Cu-
catalyzed C-N coupling reactions (2a-h, 3a-n). The desired com-
pound 4a was synthesized using 4-((5-bromo-2-methylphenyl)
amino)-2-chlorobenzonitrile (15) and 3,5-dimethylisoxazole-4-
boronic acid pinacol ester via Suzuki coupling reaction. To ach-
ieve compounds 4c-e, intermediate 22 was prepared from
commercially available 5-Bromo-2-methylaniline (21) through
Suzuki coupling reaction. The subsequent palladium-catalyzed C-N
coupling afforded desired compounds 4e and intermediate 23,
which underwent N-alkylation reaction to yield compounds 4d and
m
that the N-ethyl substituted diphenylamine scaffold was worthy of
further optimization.
1 2
Next, we kept Cl and methyl group as substituents at R and R
positions and optimized ring C to achieve compounds 3a-n
(Table 3). Among them, five compounds (3c, 3d, 3e, 3i, 3j) exhibited
favorable antiproliferative activity with IC50 values ranging from
1.03 to 5.38 mM toward VCaP and 22Rv1 cells. And 3,5-
dimethylisoxazole (3e) was identified as the preferred ring C,
which was chosen as the terminal heteroaromatic ring for further
modification.
4
c, respectively.
As presented in Table 4, replacing N atom (4a) with O atom (4b)
3
at R position led to obvious decrease in LNCaP/AR inhibitory ac-
tivity. Compound 4c bearing 2-pyridyl group for ring A was found
to be more potent than compound with 4-pyridyl group (4d).
3
. Results and discussion
Importantly, compound 4a featuring NH group as R
exhibited more desirable anticancer activity toward VCaP cells
IC50 ¼ 0.70 M) than N-ethyl substituted compound 3e
3
substituent
3
.1. Discovery of compound 4a
(
m
All compounds were firstly evaluated in LNCaP/AR human
ꢀ
ꢀ
Scheme 1. Synthesis of compounds 1a-k and 4b. Reagents and conditions: (a) H
2
NOH-HCl, MeOH, reflux, 1 h; AlH(Bu-i)
2
, DCM, 0 C-rt, overnight; (b) NaH, DMF, 0 C-rt, 2 h; (c)
ꢀ
ꢀ
Suzuki coupling, boronic acid/ester, Pd(PPh
3
)
, K
4 2
3 2
CO , H O, 1,4-Dioxane, 90 C, 6 h; (d) K
2 3
CO , DMSO, 110 C, 2 h.

268
J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
ꢀ
Scheme 2. Synthesis of compounds 2a-h, 3a-n, 4a and 4c-e. Reagents and conditions: (a) H
2
SO
4
, NaNO
, K
2
, H
CO
2
O, KI, MeCN, 0 C-rt, overnight; (b) Pd
2
(dba)
3
, DavePhos, Cs
2
CO
3
, MeCN,
3
CO ,
ꢀ
ꢀ
reflux, 5 h; (c) Bromoethane, NaH, DMF, 0 C-rt, 2 h; (d) Suzuki coupling, boronic acid/ester, Pd(PPh
3
)
4
2
3
, H
2
O, 1,4-Dioxane, 90 C, 6 h; (e) Benzimidazole/imidazole, CuI, Cs
2
ꢀ
ꢀ
DMF, 120 C, 40 h; (f) Pyrazole, CuI, NaH, DMSO, 120 C, overnight.
(
IC50 ¼ 1.20
m
M) and better cell selectivity than 2-pyridyl
on AR-F876L mutant was next measured via dual luciferase re-
porter assay using AR luciferase reporter system (CCS-1019L; QIA-
GEN). As shown in Fig. S1, R1881 significantly induced the
expression of luciferase even at a concentration of 0.3 nM. Satisfy-
ingly, compound 4a was found to suppress 1 nM R1881-induced
AR-F876L transactivation in a dose-dependent manner (Fig. 3A).
substituted compound 4c.
3
.2. Compound 4a antagonizes wild-type AR and the AR-F876L
mutant
As expected, treatment with high concentration (10
of Enzalutamide caused obvious increase in AR-F876L transcription
compared to those of low concentration (0.1 M and 1 M), which
mM and 20 mM)
To investigate whether our compounds suppress AR-regulated
gene transcription in prostate cancer cells, qRT-PCR analysis was
performed on VCaP cells. As illustrated in Fig. 2A, compounds 2f,
m
m
was in accord with Moilanen's report [41]. On the other hand, both
of Enzalutamide and compound 4a potently suppressed the tran-
scriptional activity of wild-type AR (wt-AR) (Fig. 3B). Furthermore,
to measure the effect of compound 4a on AR nuclear translocation,
WPMY-1 cells were transfected with enhanced green fluorescence
protein (EGFP) tagged ARs. As shown in Fig. 3CeD, compound 4a
prevented wt-AR and AR-F876L mutant from translocating into the
nucleus in the presence of 0.5 nM R1881. In comparison to 4a,
treatment with Enzalutamide increased the nuclear translocation
of AR-F876L mutant but not wt-AR. These results confirm that
compound 4a effectively suppress the activity of wt-AR and the AR-
F876L mutant in vitro, indicating that compound 4a has the po-
tential to overcome Enzalutamide resistance.
3
d, 3i, 4a and 4e caused significant inhibition of R1881-induced
PSA and FKBP5 mRNA expression at a concentration of 5 M. And
m
the most potent compound 4a reduced PSA and FKBP5 mRNA
expression in a concentration-dependent manner (Fig. 2B). Thus,
given its favorable cell selectivity and potent anti-cancer activities
against different PC cells (Fig. 2C), compound 4a was selected for
further studies. Next, the direct interaction between 4a and AR-LBD
was demonstrated via a competitive AR binding assay. Compound
3
4
a was measured in competition with radio ligand [ H] R1881 in
cytosolic lysates from LNCaP cells using endogenous hormone
progesterone and synthetic androgen R1881 as standards. As
shown in Fig. 2D, R1881 and progesterone displayed strong binding
potency to AR-LBD with IC50 values of 4.6 nM and 13.7 nM
respectively, which indicated the feasibility of this assay system.
Satisfyingly, compound 4a (IC50 ¼ 641.3 nM) was found ~8-fold
more effective than Enzalutamide (IC50 ¼ 5336.0 nM), suggesting
that compound 4a antagonizes AR by targeting the ligand binding
domain. However, the binding affinity of Enzalutamide was lower
than that in other papers [17,18,33], possibly because of the
different source of AR protein and different radioactive ligand used.
The emergence of mutant ARs have been reported to be linked
to resistance to first- and second-generation AR antagonists. A
missense mutation (F876L) in AR-LBD was identified to mediate
Enzalutamide resistance [28]. Therefore, the effect of compound 4a
3.3. Molecular modeling
To investigate the possible binding mode of compound 4a,
molecular docking simulation was performed using Autodock 4.0
[42]. Compounds were docked into the active site of AR protein
crystal structures (PDB ID: 3V49) [43]. As shown in Fig. 4B, the
cyano group of compound 4a formed a key hydrogen bond inter-
action with Arg752, which was similar to that of Enzalutamide. The
methyl group on benzene ring of 4a formed hydrophobic in-
teractions with the side chains of Phe764, Leu704, Met780. And the

J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
269
Table 1
In vitro antiproliferative activities of 1a-k. .
Table 2
a
a
In vitro antiproliferative activities of 2a-h. .
Compd
Enza
B
C
IC50 (
mM)
Compd
Enza
R
_
1
R
_
2
C
IC50
(mM)
LNCaP/AR VCaP
22Rv1 PC-3 L-02
LNCaP/AR VCaP
22Rv1 PC-3 L-02
_
_
0.19
0.76
50%@30 >30
>30
NT
17.10
NT
1
1
1
1
a
b
c
2.53
3.50
NT
4.79
6.94
NT
_
0.19
0.58
50%@30 >30
>30
>30
17.10
NT
2
2
2
2
2
a
b
c
CF
CF
Cl
Cl
Cl
3
3
H
>30
>30
>30
>30
>30
19.00
16.30
>30
0.98
1.67
0.98
NT
NT
NT
NT
Me
H
0.56
0.33
0.32
0.36
>30
>30
>30
>30
NT
23.20
15.00
NT
d
>30
25.90 25.10 NT
d
e
Me
H
>30
1
1
1
1
e
f
1.24
NT
NT
NT
NT
NT
NT
NT
NT
>30
21.00
11.50
0.12
NT
NT
2f
Cl
Me
0.40
3.92
>30
>30
NT
g
h
NT
NT
2
2
g
Cl
Cl
H
1.29
2.78
NT
NT
NT
NT
NT
NT
13.50
12.30 7.97
10.50
h
Me
>30
>30
1
1
i
j
0.07
0.24
>30
>30
15.00 28.70 6.38
a
IC50, 50% inhibitory concentration. IC50 values are the mean value of at least two
experiments with duplicate measurements. The deviations were less than 40%. 50%
30, 50% inhibition at 30 M. NT, not tested.
@
m
10.39 9.80
NT
NT
(
P ¼ 0.0672).
1
k
1.42
>30
NT
NT
3.5. Antitumor effect of compound 4a in vivo
a
IC50, 50% inhibitory concentration. IC50 values are the mean value of at least two
To assess the potency of compound 4a in vivo, the pharmaco-
experiments with duplicate measurements. The deviations were less than 40%. 50%
30, 50% inhibition at 30 M. NT, not tested.
kinetic study was conducted in Sprague-Dawley rats. The plasma
level of 4a was measured after a single oral dose of 30 mg/kg. Based
on the concentration-time curve (Fig. 6A), PK parameters of com-
pound 4a were calculated. As summarized in Fig. 6B, the maximum
plasma concentration (Cmax ¼ 2320.0 ng/ml) was achieved at 0.5 h
after administration. Acceptable half-life (T ¼ 4.4 h) and the area
@
m
3
-methyl substituent on isoxazole moiety of compound 4a had
contact with the hydrophobic pocket formed by the residues of
Leu873, His874 and Met742. Differently, Enzalutamide formed
additional hydrogen bond interactions through its nitrogen and
fluorine atoms with the side chains of Gln711 and His874 (Fig. 4A).
And the sigma-pi interaction between benzene ring of Enzaluta-
mide and Met742 was also observed. Therefore, the distinctive
binding modes of compound 4a and Enzalutamide with AR might
contribute to their different inhibitory abilities toward the AR-
F876L mutant and 22Rv1 cells bearing H874Y-mutated AR in vitro.
1
/2
under the curve (AUC0-t ¼ 4041.9 ng*h/ml) were observed. Thus, we
next evaluated the antitumor efficacy of compound 4a in vivo. The
BALB/c nude male mice were subcutaneously injected with VCaP
cells. When the average tumor volume reached approximately
3
150 mm , the mice were castrated or sham-operated. When tumors
3
regrew to 200e350 mm , oral treatments with compound 4a,
Enzalutamide or vehicle were initiated once a day for 40 days.
Given its faster tumor growth than castrated groups, the sham-
operated mice were administered with vehicle for only 15 days
and the tumors were collected solely. As shown in Fig. 6C, the tu-
mor growth was inhibited by compound 4a with 44.7% tumor
growth inhibition (TGI), which was equal to that of Enzalutamide
43.0%). The tumor weights of each group were respectively
recorded at the end of treatment (Fig. 6D). And no significant
weight loss was observed during the treatment (Fig. S2).
3.4. Compound 4a has little effect on CYP17A1 hydroxylase
Since compound 4a is the hybrid of Abiraterone and Enzaluta-
mide, it is expected to still inhibit the catalytic activity of CYP17A1
enzyme. Thus, we measured the effect of 4a on the production of
(
1
7
a
-hydroxyprogesterone (17-OHP) from progesterone, which was
catalyzed by CYP17A1. As shown in Fig. 5, Abiraterone completely
suppressed the formation of 17-OHP at a concentration of 10 M.
m
And nearly 50% inhibition of CYP17A1 hydroxylase was also
observed by Galeterone at the same concentration. Interestingly,
out of our expectation, compound 4a slightly blocked the produc-
tion of 17-OHP, which didn't reach statistical significance
4. Conclusions
In conclusion, a new series of nonsteroidal AR antagonists
deriving from the hybridization of Enzalutamide and Abiraterone

270
J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
Table 3
In vitro antiproliferative activities of 2d, 3a-n. .
Table 4
a
a
In vitro antiproliferative activities of 3e, 4a-e. .
Compd
A
R
3
IC50
(mM)
Compd
Enza
C
IC50 (mM)
LNCaP/AR VCaP
22Rv1 PC-3
L-02
LNCaP/AR
VCaP
22Rv1
PC-3
L-02
Enza
3e
_
_
0.19
0.19
50%@30 >30
>30
17.10
8.67
_
0.19
0.32
50%@30
>30
>30
>30
>30
>30
17.10
15.00
1.20
0.70
NT
2.99
3.50
NT
4.84
2d
4
4
4
a
0.43
1.67
0.63
23.30 11.30
3a
0.29
0.70
0.32
2.34
NT
>30
>30
5.38
15.70
15.30
9.03
NT
bb
c
NT
NT
3
3
b
c
NT
0.87
5.43
4.80
4.42
3.19
5.78
4
4
d
e
2.88
3.70
2.01
1.97
NT
NT
NT
NT
NT
NT
3
3
d
e
0.17
0.19
1.67
1.20
4.33
2.99
7.29
4.84
6.23
8.67
a
IC50, 50% inhibitory concentration. IC50 values are the mean value of at least two
experiments with duplicate measurements. The deviations were less than 40%. 50%
30, 50% inhibition at 30 M. NT, not tested.
Compound without methyl group on ring B.
@
m
3
3
3
f
0.48
0.49
0.38
4.01
>30
NT
>30
>30
>30
>30
NT
NT
NT
b
g
h
>30
H874Y-mutated AR. The direct interaction between compound 4a
and AR-LBD suggested that 4a antagonized AR by targeting the
ligand binding domain. Importantly, compound 4a efficiently
suppressed the activities of wt-AR and the AR-F876L mutant that
confers resistance to Enzalutamide. The antitumor activity of 4a
was also proved in castration-resistant VCaP xenograft model
in vivo. Therefore, these results make compound 4a promising lead
compound for the treatment of Enza-resistant CRPC. Further efforts
have focused on the structural optimization of compound 4a to
improve the pharmacokinetic profile and antitumor activity in vivo.
22.00
3
i
0.33
1.27
2.00
11.00
12.70
3
2
3
j
0.48
0.40
0.41
2.90
3.92
>30
1.03
NT
3.58
NT
1.73
NT
b
k
5. Experimental section
16.50
>30
NT
5
.1. Cell culture
3
3
l
1.98
0.39
25.30
8.56
NT
3.57
8.16
NT
NT
LNCaP/AR cells were gift from Kang Cheng Biotech (KC BIO,
Sichuan, China). Other cells used were purchased from the Cell
Bank of Chinese Academy of Sciences (Shanghai, China). LNCaP/AR,
m
3.04
22Rv1 (TCHu100) and L-02 (GNHu6) cells were cultured in RPMI-
1
640, VCaP (TCHu220) and WPMY-1 (GNHu36) cells in DMEM,
2
h
n
2.78
2.55
>30
>30
NT
NT
>30
PC-3 (SCSP-532) in a mixture of DMEM and F12 (1:1) nutrient
medium supplemented with 10% (v/v) fetal bovine serum (Gibco)
and 1% (v/v) penicillin/streptomycin (HyClone, SV30010). Cells
3
20.00
NT
NT
ꢀ
were grown in an incubator with 5% CO
2
at 37 C.
a
IC50, 50% inhibitory concentration. IC50 values are the mean value of at least two
5.2. CCK-8 cell proliferation assay
experiments with duplicate measurements. The deviations were less than 40%. 50%
30, 50% inhibition at 30 M. NT, not tested.
@
m
The cells were seeded in 96-well plate at a density of
4
3
2
ꢁ 10 cells for VCaP and 1e3 ꢁ 10 cells per well for other cells.
were designed and synthesized. Among them, compound 4a
bearing a diphenylamine scaffold was identified as the most potent
compound with better inhibitory activities than Enzalutamide
against AR-overexpressing VCaP cells and 22Rv1 cells bearing
After 24 h, an equal volume of medium containing various con-
centrations of compounds was added to each well (9-point and 3-
fold serial dilution starting at 30 mM). Cells were treated for 6 days
and then CCK-8 reagent (SAB, CP002) was added. After an addi-
tional 1e4 h incubation, the absorbance value (OD) was measured

J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
271
Figure 2. (A, B) qRT-PCR analysis for 0.2 nM R1881-induced PSA and FKBP5 mRNA expression in VCaP cells treated with (A) compounds at a concentration of 5
m
M; (B) compound
4
a at different concentrations for 24 h. The relative mRNA expression was normalized to GAPDH. Data were expressed as the mean ± SD (n ¼ 3). * indicates significance as p < 0.05,
** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to samples treated with DMSO by two-tailed t-test. NS, no significant difference. (C) Growth inhibitory curves of compound
3
4
a in different cell lines. (D) Binding affinity of 4a to AR-LBD. IC50, 50% inhibitory concentration. 1 nM radioligand [ H] R1881 and LNCaP cytosol were used.
at 450 nm via spectrophotometry (Thermo Multiskan MK3). IC50
was the mean value of at least two independent experiments with
duplicate measurements and calculated by GraphPad Prism 5
software.
5.4. Competitive AR binding assay
3
The assay was performed using [ H] R1881 (PerkinElmer, Cat:
NET590250UC, Lot: 2133648) and LNCaP cytosol containing wild-
type AR and T877A mutant. Transfer 1
ence compounds and testing compounds to assay plate (8-point
and 4-fold serial dilution starting at 10 M for 4a and Enza or
starting at 1 M for Progesterone and R1881.1 l of 2 mM Proges-
terone was transferred to assay plate for nonspecific binding and
DMSO was used for total binding. Then 100 l of cell cytosol stocks
and 100 l of radio ligand (final concentration was 1 nM) were
added into the plate. The plate was sealed and incubated at 4 C for
ml of serial dilution refer-
m
5
.3. QPCR
m
m
Cells were maintained in 6-well plates in DMEM supplemented
m
with 5% charcoal stripped FBS and 1% penicillin-streptomycin for
m
4
0
9
8 h and then treated with combination of antiandrogen and
.2 nM R1881 (Nanjing Chemlin Chemical Industrial Co., Ltd., CAS:
65-93-5). RNA was extracted using TRIzol reagent (Invitrogen,
ꢀ
2
4 h. When binding completed, 100
m
l of radioligand adsorption
ꢀ
buffer was added, which was shook at 4 C for 15 min. Then, the
plate was centrifuged for 20 min at 4600 rpm, 4 C. 100
15596026) and cDNA was subsequently synthesized using reverse
ꢀ
ml of su-
conversion kit (abm, G492). Diluted cDNA and gene-specific
primers were mixed with EvaGreen qPCR MasterMix (abm,
MasterMix-S) and the transcripts were measured by CFX96™ Real-
Time PCR Detection Systems (BIO-RAD). Primers were purchased
from GeneCopoeia Inc (PSA: HQP009633, FKBP5: HQP057374). The
relative expression of PSA or FKBP5 was quantified and normalized
to GAPDH (GeneCopoeia, HQP006940). The positive control,
Enzalutamide, was a gift from Hinova Pharmaceuticals Inc.
pernatant was transferred into scint-tube (PerkinElmer, Cat:
000192) followed by addition of 2 ml Ultima Gold cocktail (Per-
6
kinElmer, Cat: 6013329, Lot: 77e16371). TriCap was used for scin-
tillation counting. Calculate the “Inhibition [% Control]" using the
equation: %Inh ¼ (1-Background subtracted Assay value/Back-
ground subtracted HC value) *100 (Accomplished by WuXi Apptec
Co., Ltd., China)
(
Sichuan, China).

272
J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
Fig. 3. (A) Compound 4a inhibited R1881-induced transactivation of AR-F876L. PC-3 cells maintained in medium supplemented with charcoal stripped FBS were treated with
compound and 1 nM R1881 for 24 h. The Firefly luciferase signal was normalized to Renilla luciferase signal. Data were expressed as the mean ± SEM (n ¼ 3). (B) Compound 4a and
Enzalutamide inhibited the transcriptional activity of wt-AR. HEK293 cells maintained in medium supplemented with dialyzed FBS were treated with compound in the presence of
2
nM testosterone for 24 h. (C, D) Nuclear localization of transfected (C) AR-F876L mutant and (D) wt-AR in WPMY-1 cells treated with vehicle or 10 mM compound in the presence
or absence of 0.5 nM R1881 for 24 h. The nuclei were labeled with Hoechst 33342 (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to
the Web version of this article.)
5
.5. Dual luciferase reporter assay
transfection, cells were treated with combination of compound and
nM testosterone for another 24 h. Luciferase activity was
measured by the addition of 100 l Steady-Glo reagent per well
2
AR-negative PC-3 cells were grown in 96-well plates in a
m
mixture of DMEM and F12 (1:1) nutrient medium supplemented
with 5% charcoal stripped FBS. Within 24 h, cells were transiently
cotransfected with 50 ng F876L AR plasmid (established by Sangon
Biotech Co., Ltd., Shanghai, China.) and 50 ng reporter plasmid
(Promega, E2550) and the signal was read on Envision (Accom-
plished by WuXi Apptec Co., Ltd., China)
5.7. AR nuclear translocation
(
QIAGEN, CCS-1019L) per well using EndoFectinTM-Max trans-
fection reagent (GeneCopoeia, EF003) according to the manufac-
turer's instructions. 24 h after transfection, cells were treated with
combination of compound and 1 nM R1881 for another 24 h. Firefly
and Renilla luciferase activities were measured by the Dual Lucif-
erase Kit (Promega, E1910) according to the manufacturer's in-
structions. The luminescence was recorded on a configurable multi-
mode microplate reader (BioTek, Synergy H1) and the Firefly
luciferase signal was normalized to that of the Renilla as relative
luciferase units.
WPMY-1 cells were grown in 48-well plate in DMEM nutrient
medium supplemented with 5% charcoal stripped FBS. Within 6 h,
cells were transiently cotransfected with 200 ng AR-F876L EGFP
plasmid (established by Sangon Biotech Co., Ltd., Shanghai, China.)
TM
per well using EndoFectin -Max transfection reagent (GeneCo-
poeia, EF003) according to the manufacturer's instructions. The
second day, cells were treated with combination of compound and
.5 nM R1881 for another 24 h. Hoechst 33342 (Sigma, B-2261), the
fluorescent stain for DNA, was added into each well according to
the manufacturer's instructions. After an additional 30 min incu-
bation at room temperature, nutrient medium was removed and
the cells were washed with PBS buffer solution twice. Visualization
performed on Zeiss Observer D1 fluorescence microscope (AX10
cam HRC).
0
5.6. Wt-AR reporter assay
HEK293 cells were seeded into 96-well plate in 89% DMEM
nutrient medium (Gibco, 31053028) supplemented with 10% Dia-
lyzed FBS (Biological Industries, 04-011-1A) and 1% GlutaMax. At
the same time, cells were transiently cotransfected with 5 ng AR
plasmid (Origene, RC235415) and 100 ng reporter plasmid (Prom-
ega, E1360) per well using transfection reagent (Promega, E2311)
according to the manufacturer's instructions. 24 h after
5.8. Molecular modeling
The possible binding modes of compound 4a and Enzalutamide
with androgen receptor were analyzed by Autodock 4.0 [42]. The X-

J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
273
Fig. 4. The predicted binding modes and 2D diagram of (A) Enzalutamide and (B) compound 4a in complex with AR (PDB ID: 3V49).
ray crystal structure of AR (PDB ID:3V49) [43] was obtained from
5.9. CYP17A1 hydroxylase assay
Protein Database online (www.rcsb.org). The binding site of the
ligand was chosen as active site and the grid dimensions were set to
The catalytic activity of CYP17 enzyme was evaluated by
measuring the production of 17 -hydroxyprogesterone. The reac-
tion was carried out in a 50 l final volume containing 1 pmol lysate
of CYP17A1 (Origene, LY400030), 10 M compound or 0.5% DMSO,
M Progesterone (MedChem Express, MCE) and 1 mM NADPH
3
2
0 ꢁ 20 ꢁ 20 Å . A total of 20 conformations of each compound
a
were generated for molecular docking. The other parameters were
as default.
m
m
50 m
(
Roche) in buffer (50 mM Tris, pH 7.4 and 5 mM MgCl
2
). The
ꢀ
mixture was incubated at 37 C for 30 min, which was quenched
with 30
hydroxyprogesterone was analyzed by LC-MS/MS (Shimadzu LC-
0AD; API 4000). Abiraterone (E1203570010) and Galeterone
ml 20% trichloroacetic acid. The production of 17a-
2
(
(
AK323649) were purchased from Sun Chemical Technology
Shanghai) Co., Ltd. (Accomplished by Sichuan XPiscoric Inc.)
5.10. Pharmacokinetic studies
Compound was formulated in PEG200/DMA (9:1, v/v) for a
single dose of 30 mg/kg in male SD rats (N ¼ 3). Blood samples were
withdrawn at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h from retrobulbar
ꢀ
vein and stored on ice (0e4 C). Heparinized plasma was obtained
ꢀ
by centrifugation (8000 rpm for 5 min at 4 C) and then stored
ꢀ
at ꢂ20 C. Compound was separated from plasma samples via
protein precipitation with methol. The concentration of plasma
drug was determined by LC-MS/MS (Shimadzu LC-20AD; API
Fig. 5. Inhibition of CYP17A1 hydroxylase. 10 mM compounds were used. Data were
4000). The pharmacokinetic parameters were analyzed by non-
expressed as the mean ± SD (n ¼ 2). * indicates the significance as p < 0.05 and **
compartmental method using WinNonlin 5.2 (Accomplished by
Sichuan XPiscoric Inc.).
p < 0.01 by two-tailed t-test compared to samples treated with DMSO. NS, no signifi-
cant difference. 17-OHP, 17a-hydroxy progesterone.

274
J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
Fig. 6. (A) The concentration-time curve of compound 4a in rats. (B) Pharmacokinetic parameters of compound 4a. Compound was formulated in 10% DMA and 90% PEG200 for a
single oral dose of 30 mg/kg. (C) Growth inhibitory effects of 4a and Enzalutamide on CRPC xenografts. (D) Individual VCaP tumor weight of each group. The tumor volume and
weight were expressed as the mean ± sem (N ¼ 8e10).
5.11. In vivo xenograft study
5.12. Synthetic procedures and characterization
BALB/c nude male mice (5 weeks old from HFK Bioscience Co.
5.12.1. General chemistry
7
Ltd., Beijing, China) were subcutaneously injected with 1 ꢁ 10
VCaP cells in 150
54234) (1:1). When the average tumor volume reached
Reagents and solvents were obtained from commercial sources
and used as received. Flash column chromatography was carried
out using Biotage Isolera One apparatus with Agela flash column
ml mixture of DMEM medium and Matrigel (BD,
3
1
3
50e200 mm , the mice were castrated or sham-operated under
silica-CS. NMR spectra were recorded on Bruker AMX 400 spec-
1
Nembutal anesthesia and then randomized into groups (n ¼ 8e10).
trometer
(Bruker
Company,
Germany,
H ¼ 400 MHz,
3
13
When castrated tumor regrew to 200e350 mm , oral treatments
C ¼ 101 MHz). Chemical shifts were reported in parts per million
with 4a (30 mg/kg, qd), Enzalutamide (30 mg/kg, qd) or vehicle
were initiated and continued for 40 days. For all in vivo studies,
PEG200/DMA (9:1, v/v) was used as a vehicle. The body weight and
tumor volume were monitored twice weekly and the tumor volume
(ppm) and referenced to an internal standard of tetramethylsilane
1
(TMS) or residual deuterated solvent (DMSO‑d
6
, 2.50 ppm for
H
1
3
1
3
NMR and 39.52 ppm for C NMR; CDCl , 7.26 ppm for H NMR and
13
77.16 ppm for C NMR). Purity of prepared compounds was
determined by high-performance liquid chromatography (HPLC)
analysis, performed on Dionex ultimate 3000 HPLC instrument
using Waters e2695 Series chromatographs and Waters xBridge
2
3
was calculated according to the formula W ꢁ L/2 (mm ), wherein
W was the short diameter and L was the long diameter. Percentage
of tumor growth inhibition (TGI) was calculated as [1 ꢂ (T ꢂ T
C ꢂ C )] ꢁ 100, where T and T were the mean tumor volumes on
the final day and first day of treatment for the test groups; and
likewise C and C were the mean tumor volume of vehicle group.
0
)/
(
0
0
column (5
m
m, 4.6 mm ꢁ 150 mm). All key compounds were at least
95% pure. HRMS spectra were recorded on Q-TOF Premier mass
spectrometer (Micromass, Manchester, UK).
0
All animal experiments have been approved by Institutional Animal
Care and Treatment Committee of Sichuan University in China
(
IACUC number: 20100318).
5.12.2. General procedure a for the synthesis of 1a-k
To a solution of 4-Bromo-1-indanone (1.05 g, 5.0 mmol) in

J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
275
MeOH (14.0 ml) hydroxylamine hydrochloride (1.4 g, 20.0 mmol)
was added. The resulting mixture was heated to reflux for 1 h and
then cooled to room temperature. The mixture was concentrated
136.05, 135.60, 134.37, 134.05, 133.80, 127.37, 126.78, 125.44, 124.13,
116.98, 114.38, 110.09, 95.74, 48.55, 44.53, 29.04. HRMS (ESI) m/z for
þ
C22H17F
ysis: MeOH-H
3
N3 [MþH] , calcd 380.1296, found 380.1366. HPLC anal-
under vacuum. DCM (15.0 ml) and 50% NaHCO
added. The mixture was vigorously stirred for another 20min and
the organic phase was separated, dried over anhydrous Na SO
3
(15.0 ml) were
2
O (70:30), 7.41 min, 94.01% purity.
2
4
,
5.12.2.3. 4-(6-(pyrimidin-5-yl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-
filtered and concentrated under reduced pressure. The obtained
white solid (5.0 mmol) in DCM (30.0 ml) at 0 C was treated with
(trifluoromethyl)benzonitrile, 1c. The title compound was obtained
ꢀ
1
following general procedure A. H NMR (400 MHz, CDCl
3
)
d
9.22 (s,
AlH(Bu-i)
2
(20.0 ml, 30.0 mmol, 1.5 M in toluene) dropwise and
1H), 8.96 (s, 2H),7.67 (d, J ¼ 8.8 Hz, 1H), 7.57e7.32 (m, 3H), 7.17 (d,
stirred for 30 min. The reaction mixture was warmed to room
temperature, stirred for another 12 h and quenched with cold water
and extracted with DCM. The organic phase was dried over anhy-
J ¼ 1.7 Hz, 1H), 7.01 (dd, J ¼ 8.7, 2.0 Hz, 1H), 4.62 (s, 2H), 3.74 (t,
13
J ¼ 5.8 Hz, 2H), 3.13 (t, J ¼ 5.7 Hz, 2H). C NMR (101 MHz, CDCl
3
)
d
157.63, 154.85, 151.53, 136.07, 134.40, 134.08, 133.80, 133.38,
drous Na
bromo-tetrahydroquinoline (3) was obtained as a yellow solid
600.0 mg, yield 56.6%) after flash column chromatography using a
2
SO
4
, filtered and concentrated under vacuum. Pure 5-
127.69, 126.75, 125.42, 124.02, 121.38, 116.93, 114.43, 110.19, 95.90,
þ
48.51, 44.48, 29.02. HRMS (ESI) m/z for C21H16F
3
N4 [MþH] , calcd
(
381.1249, found 381.1243. HPLC analysis: MeOH-H
2
O (70:30),
solvent gradient of 5e25% ethyl acetate in hexanes.
4.70 min, 97.98% purity.
The mixture of 60% dispersion of NaH (80.0 mg, 2.0 mmol) in
mineral oil and 5-bromo-tetrahydroquinoline (3) (212.0 mg,
5.12.2.4. 4-(6-(quinolin-3-yl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-
ꢀ
1.0 mmol) in dry DMF (4.0 ml) was stirred at 0 C for 30 min. 4-
(trifluoromethyl)benzonitrile, 1d. The title compound was obtained
1
Fluoro-2-(trifluoromethyl) benzonitrile (378.2 mg, 2.0 mmol) was
following general procedure A. H NMR (400 MHz, CDCl
3
)
d
9.18 (d,
added and the mixture was warmed to room temperature. After
J ¼ 1.5 Hz, 1H), 8.32 (s, 1H), 8.15 (d, J ¼ 8.4 Hz, 1H), 7.90 (d, J ¼ 8.1 Hz,
1H), 7.74 (t, J ¼ 7.6 Hz,1H), 7.67 (d, J ¼ 8.8 Hz,1H), 7.66e7.47 (m, 3H),
7.38 (d, J ¼ 7.9 Hz, 1H), 7.17 (s, 1H), 7.01 (dd, J ¼ 8.8, 1.9 Hz, 1H), 4.63
2
h, the reaction mixture was quenched with cold water and
extracted with ethyl acetate. The organic phase was washed with
water twice and then dried over anhydrous Na SO , filtered, and
concentrated under vacuum. Pure 4-(5-bromo-3,4-
13
2
4
(s, 2H), 3.75 (t, J ¼ 5.8 Hz, 2H), 3.15 (t, J ¼ 5.7 Hz, 2H). C NMR
3
(101 MHz, CDCl ) d 151.58, 149.71, 147.45, 137.04, 136.04, 135.66,
dihydroquinolin ꢂ1(2H)-yl)-2-(trifluoromethyl)benzonitrile (6)
was obtained as a yellow solid (100.0 mg, yield 26.2%) after flash
column chromatography using a solvent of 10% ethyl acetate in
hexanes. For the synthesis of intermediate 5 and 7, commercially
available 2 and 4 were used respectively by the procedure
described for intermediate 6.
134.36, 134.05, 133.22, 132.76, 129.58, 129.28, 128.00, 127.41, 127.18,
127.11, 125.88, 124.15, 121.45, 117.03, 114.35, 110.12, 95.69, 48.54,
þ
44.57, 29.10. HRMS (ESI) m/z for C26H19F
430.1453, found 430.1491. HPLC analysis: MeOH-H
3
N
3
[MþH] , calcd
2
O (70:30),
18.66 min, 94.30% purity.
Under N
Pd(pph (30.0 mg, 0.026 mmol), 2.0 M aq Na
.78 mmol) and 1-Methyl-1H-pyrazole-5-boronic acid pinacol
2
atmosphere, a mixture of 6 (100.0 mg, 0.26 mmol),
5.12.2.5. 4-(5-(pyridin-3-yl)indolin-1-yl)-2-(trifluoromethyl)benzo-
3
)
4
2
CO (0.29 ml,
3
nitrile, 1e. The title compound was obtained following general
1
0
procedure A. H NMR (400 MHz, CDCl
3
)
d
8.83 (d, J ¼ 1.7 Hz, 1H),
ester (108.2 mg, 0.52 mmol) in 1,4-Dioxane (0.65 ml) was heated to
8.63e8.49 (m, 1H), 7.92e7.80 (m, 1H), 7.73 (t, J ¼ 13.4 Hz, 1H), 7.54
ꢀ
9
0 C and stirred for 6 h. The reaction mixture was cooled, diluted
(d, J ¼ 2.2 Hz, 1H), 7.50 (s, 1H), 7.46e7.30 (m, 4H), 4.13 (t, J ¼ 8.3 Hz,
13
with ethyl acetate, washed with water, dried over anhydrous
Na SO , filtered and concentrated under vacuum. Purification on
2H), 3.30 (t, J ¼ 8.3 Hz, 2H). C NMR (101 MHz, CDCl
3
) d 148.13,
2
4
147.90, 146.78, 143.95, 136.03, 134.43, 134.11, 133.78, 133.50, 131.70,
silica using a solvent gradient of 10e30% ethyl acetate in hexanes
yielded the desired compound 1j (77.0 mg, 77.5%). Compounds 1a-
k were prepared according to general procedure as described for
compound 1j using corresponding aryl bromide 2e4 and the
appropriate boronic acid or boronic acid pinacol ester. The char-
acterization data for compounds 1a-k were provided below.
126.49, 124.39, 123.61, 121.14, 117.75, 116.42, 113.42, 110.61, 98.89,
þ
52.08, 28.04. HRMS (ESI) m/z for C21H15F
366.1140, found 366.1187. HPLC analysis: MeOH-H
3
N
3
[MþH] , calcd
2
O (70:30),
7.69 min, 99.47% purity.
5.12.2.6. 4-(5-(pyrimidin-5-yl)indolin-1-yl)-2-(trifluoromethyl)ben-
zonitrile, 1f. The title compound was obtained following general
1
5.12.2.1. 4-(6-(pyridin-3-yl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-(tri-
procedure A. H NMR (400 MHz, CDCl
3
)
d
9.17 (s, 1H), 8.94 (s, 2H),
fluoromethyl)benzonitrile, 1a. The title compound was obtained
7.77 (d, J ¼ 8.6 Hz, 1H), 7.55 (d, J ¼ 2.0 Hz, 1H), 7.51 (s, 1H), 7.47e7.32
1
13
following general procedure A. H NMR (400 MHz, CDCl
3
)
d
8.86 (s,
(m, 3H),4.15 (t, J ¼ 8.4 Hz, 2H), 3.32 (t, J ¼ 8.3 Hz, 2H). C NMR
1
H), 8.62 (s, 1H), 7.92e7.82 (m, 1H), 7.67 (d, J ¼ 8.8 Hz, 1H), 7.48 (dd,
3
(101 MHz, CDCl ) d 157.06, 154.36, 146.62, 144.80, 136.06, 134.47,
134.15, 133.91, 127.80, 126.48, 124.14, 123.80, 121.07, 118.03, 116.29,
J ¼ 7.9, 1.7 Hz, 1H), 7.45 (s, 1H), 7.39 (dd, J ¼ 7.7, 4.8 Hz, 1H), 7.33 (d,
J ¼ 7.9 Hz, 1H), 7.16 (d, J ¼ 2.5 Hz, 1H), 7.00 (dd, J ¼ 8.8, 2.6 Hz, 1H),
113.64, 110.72, 99.38, 52.22, 27.86. HRMS (ESI) m/z for
13
þ
4
.60 (s, 2H), 3.73 (t, J ¼ 5.9 Hz, 2H), 3.11 (t, J ¼ 5.8 Hz, 2H). C NMR
C21H18F
HPLC analysis: MeOH-H
3
N
4
O [M þ CH
3
OH þ H] , calcd 399.1433, found 399.1431.
(
101 MHz, CDCl3)
d
151.58, 148.66, 148.21, 137.00, 136.04, 135.55,
2
O (70:30), 4.82 min, 99.84% purity.
134.30, 134.00, 132.75, 127.31, 126.87, 125.61, 124.06, 123.59, 117.03,
1
14.33, 110.05,100.03, 95.62, 48.51, 44.55, 29.05. HRMS (ESI) m/z for
5.12.2.7. 4-(5-(quinolin-3-yl)indolin-1-yl)-2-(trifluoromethyl)benzo-
þ
C22H17F
ysis: MeOH-H
3
N3 [MþH] , calcd 380.1296, found 380.1366. HPLC anal-
nitrile, 1g. The title compound was obtained following general
1
2
O (70:30), 7.25 min, 96.10% purity.
procedure A. H NMR (400 MHz, CDCl
8
3
)
d
9.17 (d, J ¼ 2.2 Hz, 1H),
.27 (d, J ¼ 2.0 Hz, 1H), 8.14 (d, J ¼ 8.4 Hz, 1H), 7.88 (d, J ¼ 8.0 Hz,
5
.12.2.2. 4-(6-(pyridin-4-yl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-
1H), 7.82e7.67 (m, 2H), 7.64 (s, 1H), 7.62e7.52 (m, 3H), 7.42 (s, 1H),
13
(
trifluoromethyl)benzonitrile, 1b. The title compound was obtained
7.40 (s, 1H), 4.15 (t, J ¼ 8.3 Hz, 2H), 3.34 (t, J ¼ 8.3 Hz, 2H). C NMR
(101 MHz, CDCl 149.62, 147.14, 146.76, 143.96, 136.04, 134.44,
1
following general procedure A. H NMR (400 MHz, CDCl
3
)
d
8.67 (d,
3
) d
J ¼ 5.3 Hz, 2H), 7.67 (d, J ¼ 8.8 Hz, 1H), 7.52 (dd, J ¼ 11.2, 6.9 Hz, 4H),
134.12, 133.63, 133.25, 132.36, 131.71, 129.22, 127.89, 127.11, 126.78,
7
.34 (d, J ¼ 7.9 Hz, 1H), 7.16 (d, J ¼ 2.2 Hz, 1H), 7.00 (dd, J ¼ 8.8,
124.61, 123.85, 121.13, 117.78, 116.43, 113.44, 110.71, 98.91, 52.20,
þ
2
2
.4 Hz, 1H), 4.61 (s, 2H), 3.73 (t, J ¼ 5.9 Hz, 2H), 3.12 (t, J ¼ 5.8 Hz,
27.95. HRMS (ESI) m/z for C25H17F
found 416.1356. HPLC analysis: MeOH-H
3
N
3
[MþH] , calcd 416.1296,
13
H). C NMR (101 MHz, CDCl
3
)
d
151.50, 150.28, 147.80, 137.26,
2
O (70:30), 19.93 min,

276
J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
9
6.98% purity.
under vacuum. Purification on silica using a solvent of 10% ethyl
acetate in hexanes yielded the intermediate 12 as a white solid
5
.12.2.8. 4-(5-(pyridin-3-yl)-3,4-dihydroquinolin-1(2H)-yl)-2-(tri-
(1.71 g, yield 62.5%).
fluoromethyl)benzonitrile, 1h. The title compound was obtained
Under N
1.0 mmol), 3-bromoaniline (172.0 mg, 1.0 mmol), Cs
1.0 mmol), Pd (dba) (45.8 mg, 0.05 mmol) and Davephos (31.5 mg,
2
atmosphere, a mixture of intermediate 12 (263.5 mg,
1
following general procedure A. H NMR (400 MHz, CDCl
3
)
d
8.63
2
CO (325.8 mg,
3
(
7
7
2
dd, J ¼ 4.8, 1.6 Hz, 2H), 7.67 (m, 2H), 7.51 (d, J ¼ 2.3 Hz, 1H),
2
3
.43e7.37 (m, 1H), 7.34 (dd, J ¼ 8.7, 2.4 Hz, 1H), 7.30e7.26 (m, 1H),
0.08 mmol) in MeCN (6.0 ml) was heated to reflux for 5 h. The re-
action mixture was cooled to room temperature and diluted with
ethyl acetate. The resulting mixture was filtered off and the solution
was concentrated under vacuum to give a crude product 16 that
was purified by column chromatography on silica using a solvent of
10% ethyl acetate in hexanes. The desired compound 16 was ob-
tained as a yellow solid (220.0 mg, yield 72%). For the synthesis of
intermediate 13e15, compound 12 or commercially available 11
and corresponding aromatic amine were used respectively
following the procedure described for 16.
.26e7.19 (m, 1H), 7.00 (dd, J ¼ 7.2, 1.5 Hz, 1H), 3.68 (t, J ¼ 6.6 Hz,
13
H), 2.70e2.55 (m, 2H), 2.03e1.91 (m, 2H). C NMR (101 MHz,
151.16, 149.93, 148.54, 140.74, 138.48, 136.59, 136.26,
35.76, 134.10, 133.72, 130.25, 126.57, 125.01, 123.10, 120.67, 119.62,
3
CDCl ) d
1
1
C
16.62, 115.33, 98.89, 48.25, 25.10, 24.58. HRMS (ESI) m/z for
þ
H
22 16
F
3
N
3
Na [MþNa] , calcd 402.1194, found 402.1166. HPLC
2
analysis: MeOH-H O (70:30), 8.01 min, 99.47% purity.
5
.12.2.9. 4-(5-(pyridin-4-yl)-3,4-dihydroquinolin-1(2H)-yl)-2-(tri-
fluoromethyl)benzonitrile, 1i. The title compound was obtained
following general procedure A. H NMR (400 MHz, DMSO‑d ) d 8.66
6
60% dispersion of NaH (35.0 mg, 0.875 mmol) in mineral oil was
added portion-wise to a solvent of 16 (110.0 mg, 0.35 mmol) in dry
DMF (4.0 ml) at 0 C. The reaction mixture was stirred for 30 min
and then warmed to room temperature. Bromoethane (76.3 mg,
0.7 mmol) was added. After an additional 2 h, the mixture was
quenched with water and extracted with ethyl acetate. The organic
1
ꢀ
(
d, J ¼ 3.7 Hz, 2H), 7.94 (d, J ¼ 8.5 Hz, 1H), 7.57 (s, 1H), 7.50 (d,
J ¼ 8.3 Hz, 1H),7.42 (d, J ¼ 4.2 Hz, 2H), 7.36 (d, J ¼ 7.8 Hz, 1H),7.28 (t,
J ¼ 7.7 Hz, 1H), 7.04 (d, J ¼ 7.2 Hz, 1H), 3.72 (t, J ¼ 6.0 Hz, 2H), 2.59 (t,
13
J ¼ 5.6 Hz, 2H), 1.88 (m, J ¼ 5.5 Hz, 2H). C NMR (101 MHz, CDCl
3
)
d
151.23, 149.83, 148.41, 140.77, 139.61, 135.78, 134.20, 133.70,
2 4
phase was washed twice with water, dried over anhydrous Na SO ,
1
4
3
8
29.61, 126.71, 124.25, 123.86, 121.01, 119.69, 116.48, 115.35, 99.12,
filtered and concentrated under vacuum. Pure product 20 was
obtained as a yellowish green solid (90.0 mg, yield 77.0%) after flash
column chromatography using a solvent of 10% ethyl acetate in
hexanes. For the synthesis of 17e19, intermediate 13e16 were used
by the procedure described for intermediate 20, respectively.
A suspension of 20 (133.5 mg, 0.4 mmol), imidazole (36.8 mg,
þ
8.27, 25.02, 24.52. HRMS (ESI) m/z for C22H17F
80.1296, found 380.1364. HPLC analysis: MeOH-H
3
N
3
[MþH] , calcd
2
O (70:30),
.27 min, 99.56% purity.
5
1
.12.2.10. 4-(5-(1-methyl-1H-pyrazol-5-yl)-3,4-dihydroquinolin-
(2H)-yl)-2-(trifluoromethyl)benzonitrile, 1j. The title compound
2 3
0.54 mmol), Cs CO (260.6 mg, 0.8 mmol) and CuI (17.5 mg,
1
was obtained following general procedure A. H NMR (400 MHz,
CDCl
0.09 mmol) in DMF (2.0 ml) was degassed under a stream of ni-
ꢀ
3
)
d
7.66 (d, J ¼ 8.7 Hz, 1H), 7.56 (d, J ¼ 1.7 Hz, 1H), 7.51 (d,
trogen over 10 min and then heated to 120 C and stirred for 40 h.
J ¼ 1.9 Hz, 1H), 7.35 (dd, J ¼ 8.6, 2.2 Hz, 1H), 7.29 (d, J ¼ 8.1 Hz, 1H),
The resulting mixture was cooled to room temperature, diluted
with ethyl acetate, washed twice with water, dried over anhydrous
Na SO , filtered and concentrated under vacuum. Purification on
2 4
silica using a solvent gradient of 10e50% ethyl acetate in hexanes
yielded the desired compound 2c (40.0 mg, yield 31.2%) as a white
solid. Compounds 2a, 2b, 2d and 3m were prepared according to
general procedure B as described for compound 2c using corre-
sponding aryl bromide (17e19).
7
3
2
.21 (t, J ¼ 7.8 Hz, 1H), 6.98 (d, J ¼ 7.4 Hz, 1H), 6.24 (d, J ¼ 1.7 Hz, 1H),
.73 (s, 3H), 3.69 (t, J ¼ 6.5 Hz, 2H), 2.58e2.43 (t, 2H), 2.07e1.91 (m,
13
H). C NMR (101 MHz, CDCl
3
)
d
151.15, 141.32, 140.75, 138.59,
135.72, 134.22, 133.79, 131.38, 130.99, 126.43, 125.29, 121.16, 120.04,
116.37, 115.64, 106.78, 99.52, 48.38, 36.78, 24.76, 24.15. HRMS (ESI)
þ
m/z for C21
H F
17 3
N
4
Na [MþNa] , calcd 405.1303, found 405.1315.
2
HPLC analysis: MeOH-H O (70:30), 7.83 min, 98.50% purity.
Compounds 2e-h, 3b-l and 3n were obtained from 19 or 20 and
appropriate boronic acid via Suzuki coupling reaction as described
in general procedure A for compound 1a-k. The characterization
data for compounds 2a-h and 3b-n are provided below.
5
1
.12.2.11. 4-(5-(3,5-dimethylisoxazol-4-yl)-3,4-dihydroquinolin-
(2H)-yl)-2-(trifluoromethyl)benzonitrile, 1k. The title compound
1
was obtained following general procedure A. H NMR (400 MHz,
CDCl
7.65 (d, J ¼ 8.7 Hz, 1H), 7.51 (d, J ¼ 2.3 Hz, 1H), 7.34 (dd,
3
) d
J ¼ 8.7, 2.4 Hz,1H),7.26 (dd, J ¼ 8.0, 0.8 Hz, 2H),7.19 (t, J ¼ 7.8 Hz,1H),
5.12.3.1. 4-((3-(1H-imidazol-1-yl)phenyl)(ethyl)amino)-2-(tri-
6
2
CDCl
1
4
.86 (dd, J ¼ 7.4, 1.1 Hz, 1H), 3.79e3.53 (m, 2H), 2.56e2.37 (t, 2H),
fluoromethyl)benzonitrile, 2a. The title compound was obtained
13
1
.29 (s, 3H), 2.15 (s, 3H), 2.04e1.91 (m, 2H). C NMR (101 MHz,
165.70,159.29,151.19,140.80,135.77,134.08,133.73,131.78,
29.73, 126.66, 125.81, 120.65, 119.72, 116.48, 115.32, 115.22, 98.96,
following general procedure B. H NMR (400 MHz, CDCl
3
)
d
7.88 (s,
3
)
d
1H), 7.64e7.53 (m, 2H), 7.38 (dd, J ¼ 8.1, 1.2 Hz, 1H), 7.29 (s, 1H),
7.25e7.16 (m, 3H), 7.02 (d, J ¼ 2.3 Hz, 1H), 6.85 (dd, J ¼ 8.8, 2.4 Hz,
13
8.17, 24.71, 24.54, 11.51, 10.61. HRMS (ESI) m/z for C22
H
18
3
F N
3
ONa
1H), 3.87 (q, J ¼ 7.1 Hz, 2H), 1.31 (t, J ¼ 7.1 Hz, 3H). C NMR
þ
[
MþNa] , calcd 420.1300, found 420.1286. HPLC analysis: MeOH-
(101 MHz, CDCl
3
) d 150.44, 146.22, 139.32, 135.93, 134.49, 134.14,
2
H O (70:30), 9.317 min, 99.32% purity.
132.05, 130.90, 126.14, 123.85, 121.13, 120.00, 119.83, 116.59, 116.00,
1
11.64, 96.86, 47.23, 12.28. HRMS (ESI) m/z for C19H16F3N4
þ
5
.12.3. General procedure B for the synthesis of 2a-h, 3b-l and 3n
solution of 4-amino-2-chlorobenzonitrile (10) (1.6 g,
0.5 mmol) in MeCN (60.0 ml) and water (18.0 ml) was cooled to
[MþH] , calcd 357.1247, found 357.1299. HPLC analysis: MeOH-H
2
O
A
(70:30), 4.05 min, 99.56% purity.
1
0
ꢀ
C. Sulfuric acid (1.8 ml) and 2.0 M aqueous sodium nitrite
5.12.3.2. 4-((5-(1H-imidazol-1-yl)-2-methylphenyl)(ethyl)amino)-2-
(
840.0 mg, 6.0 ml) was slowly added, respectively. Thereafter po-
(trifluoromethyl)-benzonitrile, 2b. The title compound was obtained
1
tassium iodide (3.3 g, 20.0 mmol) dissolved in 5.0 ml of water was
added dropwise. The reaction mixture was allowed to warm up to
room temperature and stirred overnight. The organic phase was
evaporated, the resulting suspension was dissolved in ethyl acetate
following general procedure B. H NMR (400 MHz, DMSO‑d
6
)
d
8.33
(s, 1H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.70 (dd, J ¼ 8.3, 2.2 Hz, 1H), 7.62 (t,
J ¼ 5.5 Hz, 1H), 7.58 (d, J ¼ 8.3 Hz, 1H), 7.11 (s, 1H), 6.80 (d,
1
3
J ¼ 26.2 Hz, 2H), 3.83 (s, 2H), 2.07 (s, 3H), 1.20 (t, J ¼ 7.1 Hz, 3H).
C
and washed three times with 10% aqueous NaHSO
3
. The organic
NMR (101 MHz, CDCl
3
) d 150.19, 143.34, 137.12, 136.13, 135.41,
phase was dried over anhydrous Na SO , filtered and concentrated
2
4
134.50, 134.32, 133.51, 130.75, 123.78, 121.79, 120.98, 118.06, 116.85,

J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
277
1
14.31, 109.50, 95.91, 46.45, 17.50, 12.37. HRMS (ESI) m/z for
J ¼ 8.2 Hz, 1H), 3.71 (s, 2H), 2.14 (s, 3H), 1.29 (t, J ¼ 7.1 Hz, 3H). 13C
þ
C20H18F3N4 [MþH] , calcd 371.1405, found 371.1462. HPLC anal-
3
NMR (101 MHz, CDCl ) d 151.75, 142.83, 141.28, 139.78, 138.17,
ysis: MeOH-H
2
O (70:30), 4.76 min, 98.28% purity.
135.46, 134.64, 132.44, 128.94, 127.70, 127.66, 126.88, 126.69, 117.73,
1
12.28, 110.66, 99.00, 46.27, 17.50, 12.27. HRMS (ESI) m/z for
þ
5
.12.3.3. 4-((3-(1H-imidazol-1-yl)phenyl)(ethyl)amino)-2-
chlorobenzonitrile, 2c. The title compound was obtained following
general procedure B. 1H NMR (400 MHz, CDCl 7.88 (s, 1H), 7.57
C22H19ClN2Na [MþNa] , calcd 369.1134, found 369.1119. HPLC
2
analysis: MeOH-H O (70:30), 5.25 min, 96.10% purity.
3
) d
(
7
t, J ¼ 8.0 Hz,1H), 7.41 (d, J ¼ 8.8 Hz,1H), 7.35 (dd, J ¼ 8.0, 1.2 Hz,1H),
5.12.3.9. 2-Chloro-4-(ethyl(2-methyl-5-(1H-pyrazol-4-yl)phenyl)
.29 (s, 1H), 7.25e7.12 (m, 3H), 6.77 (d, J ¼ 2.4 Hz, 1H), 6.62 (dd,
amino)benzonitrile, 3b. The title compound was obtained following
1
J ¼ 8.8, 2.4 Hz, 1H), 3.82 (q, J ¼ 7.1 Hz, 2H), 1.29 (t, J ¼ 7.1 Hz, 3H). 13C
general procedure B. H NMR (400 MHz, CDCl
3
)
d
7.85 (s, 2H), 7.45
NMR (101 MHz, CDCl3)
d
151.41, 146.36, 139.31, 137.95, 135.55,
(dd, 1H), 7.36 (d, J ¼ 8.4 Hz, 2H), 7.22 (d, 1H), 6.54 (s, 1H), 6.36 (d,
1
1
[
H
34.60, 131.80, 130.87, 126.07, 119.92, 119.51, 117.98, 117.13, 114.56,
12.72, 100.98, 47.06, 12.47. HRMS (ESI) m/z for C18H16ClN4
MþH]þ, calcd 323.0985, found 323.1052. HPLC analysis: MeOH-
O (70:30), 4.51 min, 97.31% purity.
J ¼ 7.6 Hz, 1H), 3.70 (s, 2H), 2.10 (s, 3H), 1.28 (t, J ¼ 11.6, 4.5 Hz, 3H).
13
3
C NMR (101 MHz, CDCl ) d 151.70, 142.84, 138.18, 134.70, 132.53,
131.18, 127.25, 126.26, 125.44, 117.68, 116.73, 112.28, 110.63, 108.71,
2
106.05, 99.04, 46.33, 17.39, 12.41. HRMS (ESI) m/z for C19H18ClN4
þ
[
MþH] , calcd 337.1142, found 337.1210. HPLC analysis: MeOH-H
2
O
5
.12.3.4. 4-((5-(1H-imidazol-1-yl)-2-methylphenyl)(ethyl)amino)-2-
(70:30), 6.48 min, 99.82% purity.
chlorobenzonitrile, 2d. The title compound was obtained following
1
general procedure B. H NMR (400 MHz, DMSO‑d
6
)
d
8.32 (s, 1H),
5.12.3.10. 2-Chloro-4-(ethyl(2-methyl-5-(3-methyl-1H-pyrazol-4-yl)
7
.80 (s, 1H), 7.67 (dd, J ¼ 8.3, 2.3 Hz, 1H), 7.64e7.46 (m, 3H), 7.10 (s,
phenyl)amino)benzonitrile, 3c. The title compound was obtained
following general procedure B. 1H NMR (400 MHz, CDCl ) d 7.68 (s,
3
1
H), 6.68 (s,1H), 6.46 (d, J ¼ 7.5 Hz,1H), 3.77 (s,1H), 2.07 (s, 2H),1.18
13
(
t, J ¼ 7.1 Hz, 3H). C NMR (101 MHz, CDCl3)
d
151.26, 143.54,
1H), 7.45e7.31 (m, 3H), 7.12 (d, J ¼ 1.1 Hz, 1H), 6.54 (s, 1H), 6.37 (d,
138.31, 137.20, 136.18, 135.35, 134.72, 133.40, 130.63, 122.06, 120.93,
J ¼ 8.1 Hz, 1H), 3.68 (s, 2H), 2.45 (s, 3H), 2.12 (s, 3H), 1.28 (t,
118.09, 117.37, 112.55, 110.72, 99.88, 46.40, 17.44, 12.61. HRMS (ESI)
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 151.73, 142.58, 138.17,
þ
m/z for C19H18ClN4 [MþH] , calcd 337.1142, found 337.1145. HPLC
134.70, 134.26, 133.52, 132.32, 130.76, 129.95, 127.89, 127.08, 119.05,
117.70, 112.29, 110.62, 98.98, 46.22, 17.39, 12.35, 11.72. HRMS (ESI)
m/z for C20H19ClN4 [MþH]þ, calcd 351.1298, found 351.1365. HPLC
analysis: MeOH-H2O (70:30), 8.35 min, 98.10% purity.
2
analysis: MeOH-H O (70:30), 4.70 min, 96.49% purity.
5
.12.3.5. 2-Chloro-4-(ethyl(3-(pyrimidin-5-yl)phenyl)amino)benzo-
nitrile, 2e. The title compound was obtained following general
1
procedure B. H NMR (400 MHz, CDCl
3
)
d
9.24 (s, 1H), 8.95 (s, 2H),
5.12.3.11. 2-Chloro-4-(ethyl(2-methyl-5-(1-methyl-1H-pyrazol-5-yl)
7
2
2
.63 (t, J ¼ 7.8 Hz, 1H), 7.58e7.47 (m, 1H), 7.40 (dd, J ¼ 5.3, 3.5 Hz,
phenyl)amino)benzonitrile, 3d. The title compound was obtained
1
H), 7.35e7.27 (m, 1H), 6.74 (d, J ¼ 2.4 Hz, 1H), 6.60 (dd, J ¼ 8.9,
following general procedure B. H NMR (400 MHz, DMSO‑d
6
)
d
7.60
13
.5 Hz, 1H), 3.83 (q, J ¼ 7.1 Hz, 2H), 1.30 (t, J ¼ 7.1 Hz, 3H). C NMR
157.99, 154.89, 151.67, 145.92, 138.03, 136.70,
34.48, 133.51, 131.38, 127.93, 125.95, 125.37, 117.27, 114.08, 112.18,
(d, J ¼ 8.9 Hz, 1H), 7.55 (d, J ¼ 8.0 Hz, 1H), 7.51 (dd, J ¼ 7.9, 1.7 Hz,
(
101 MHz, CDCl3) d
1H), 7.46 (d, J ¼ 1.9 Hz, 1H), 7.38 (d, J ¼ 1.5 Hz, 1H), 6.69 (s, 1H), 6.45
1
(d, J ¼ 1.9 Hz, 2H), 3.86 (s, 3H), 3.78 (s, 2H), 2.11 (s, 3H), 1.17 (t,
þ
13
1
3
00.46, 47.13, 12.44. HRMS (ESI) m/z for C19H16ClN4 [MþH] , calcd
J ¼ 7.1 Hz, 3H). C NMR (101 MHz, DMSO‑d
6
) d 152.17, 142.76,
35.0985, found 335.1060. HPLC analysis: MeOH-H
.44 min, 99.88% purity.
2
O (70:30),
142.12, 138.36, 137.35, 136.73, 135.57, 132.78, 130.48, 129.30, 128.34,
4
117.84, 112.26, 111.73, 106.35, 97.80, 46.27, 38.04, 17.53, 12.53. HRMS
þ
(
ESI) m/z for C20H20ClN4 [MþH] , calcd 351.1298, found 351.1375.
5
.12.3.6. 2-Chloro-4-(ethyl(2-methyl-5-(pyrimidin-5-yl)phenyl)
2
HPLC analysis: MeOH-H O (70:30), 18.75 min, 99.25% purity.
amino)benzonitrile, 2f. The title compound was obtained following
1
general procedure B. H NMR (400 MHz, CDCl
3
)
d
9.22 (s, 1H), 8.94
5.12.3.12. 2-Chloro-4-((5-(3,5-dimethylisoxazol-4-yl)-2-
(
6
3
1
1
s, 2H), 7.54 (s, 2H), 7.39 (d, J ¼ 8.7 Hz, 1H), 7.34 (s, 1H), 6.54 (s, 1H),
methylphenyl)(ethyl)amino)benzoin-trile, 3e. The title compound
was obtained following general procedure B. H NMR (400 MHz,
1
.38 (d, J ¼ 6.5 Hz, 1H), 3.73 (s, 2H), 2.18 (s, 3H), 1.31 (t, J ¼ 6.7 Hz,
13
H). C NMR (101 MHz, CDCl
3
)
d
157.77, 154.72, 151.45, 143.52,
CDCl
3
)
d
7.43 (d, J ¼ 7.9 Hz, 1H), 7.38 (d, J ¼ 8.9 Hz, 1H), 7.21 (dd,
38.29, 137.84, 134.81, 134.42, 133.21, 133.12, 127.66, 126.52, 117.42,
J ¼ 7.8, 1.7 Hz, 1H), 6.98 (d, J ¼ 1.6 Hz, 1H), 6.53 (s, 1H), 6.36 (d,
12.46, 110.69, 99.65, 46.31, 17.62, 12.40. HRMS (ESI) m/z for
J ¼ 8.1 Hz,1H), 3.70 (s, 2H), 2.42 (s, 3H), 2.28 (s, 3H), 2.15 (s, 3H),1.28
þ
13
C20H18ClN4 [MþH] , calcd 349.1142, found 349.1220. HPLC anal-
(t, J ¼ 7.1 Hz, 3H). C NMR (101 MHz, CDCl
3
) d 165.27, 158.31, 151.60,
ysis: MeOH-H
2
O (70:30), 5.25 min, 99.88% purity.
142.74, 138.22, 135.91, 134.76, 132.57, 130.33, 129.65, 128.64, 117.57,
1
15.61, 112.38, 110.59, 99.30, 46.15, 17.48, 12.24, 11.60, 10.80. HRMS
0
þ
5
2
.12.3.7. 4-([1,1 -biphenyl]-3-yl(ethyl)amino)-2-chlorobenzonitrile,
(ESI) m/z for C21H21ClN3O [MþH] , calcd 366.1295, found
g. The title compound was obtained following general procedure
B. H NMR (400 MHz, CDCl
366.1331. HPLC analysis: MeOH-H
2
O (70:30), 9.81 min, 95.97%
1
3
) d 7.62e7.34 (m, 9H), 7.21e7.10 (m, 1H),
purity.
6
.72 (t, J ¼ 3.1 Hz, 1H), 6.56 (dt, J ¼ 6.8, 3.4 Hz, 1H), 3.81 (dq, J ¼ 14.2,
13
7
d
1
.1 Hz, 2H), 1.28 (t, J ¼ 7.1 Hz, 3H). C NMR (101 MHz, CDCl
151.98, 144.99, 143.71, 139.99, 137.97, 134.59, 130.64, 128.96,
27.83, 127.07, 126.45, 126.42, 125.99, 117.47, 113.66, 111.91, 99.65,
3
)
5.12.3.13. 2-Chloro-4-(ethyl(5-(furan-3-yl)-2-methylphenyl)amino)
benzonitrile, 3f. The title compound was obtained following gen-
1
eral procedure B. H NMR (400 MHz, CDCl
3
)
d
7.72 (s, 1H), 7.55e7.46
þ
4
7.04, 12.25. HRMS (ESI) m/z for C21H17ClN2Na [MþNa] , calcd
(m, 1H), 7.46e7.39 (m, 1H), 7.36 (s, 1H), 7.34 (s, 1H), 7.19 (d,
J ¼ 1.5 Hz, 1H), 6.72e6.63 (m, 1H), 6.53 (s, 1H), 6.35 (d, J ¼ 8.0 Hz,
3
55.0978, found 355.0979. HPLC analysis: MeOH-H
2
O (70:30),
13
4
.83 min, 95.97% purity.
1H), 3.70 (s, 2H), 2.10 (s, 3H), 1.28 (t, J ¼ 7.1 Hz, 3H). C NMR
3
(101 MHz, CDCl ) d 151.69, 143.92, 142.76, 138.57, 138.17, 135.15,
0
5
.12.3.8. 2-Chloro-4-(ethyl(4-methyl-[1,1 -biphenyl]-3-yl)amino)
134.64, 132.56, 132.44, 126.40, 125.53, 125.45, 117.67, 112.28, 110.65,
benzonitrile, 2h. The title compound was obtained following gen-
108.53, 98.98, 46.27, 17.35, 12.39. HRMS (ESI) m/z for
1
þ
eral procedure B. H NMR (400 MHz, CDCl
3
)
d
7.56 (td, J ¼ 8.4,1.6 Hz,
C
20
H17ClN
2
ONa [MþNa] , calcd 359.0927, found 359.0927. HPLC
3
H), 7.50e7.38 (m, 3H), 7.39e7.29 (m, 3H), 6.55 (s, 1H), 6.37 (d,
analysis: MeOH-H2O (70:30), 18.74 min, 99.94% purity.

278
J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
5
.12.3.14. 2-Chloro-4-(ethyl(5-(furan-2-yl)-2-methylphenyl)amino)
benzonitrile, 3g. The title compound was obtained following gen-
eral procedure B. 1H NMR (400 MHz, CDCl
1H), 7.74 (ddd, J ¼ 8.4, 6.9, 1.4 Hz, 1H), 7.68 (dd, J ¼ 7.9, 1.9 Hz, 1H),
7.59 (dt, J ¼ 16.4, 4.7 Hz, 1H), 7.52 (d, J ¼ 8.0 Hz, 1H), 7.46 (d,
J ¼ 1.8 Hz,1H), 7.39 (d, J ¼ 8.9 Hz, 1H), 6.58 (s, 1H), 6.41 (d, J ¼ 8.5 Hz,
3
)
d
7.59 (dd, J ¼ 8.0,
13
1
1
.7 Hz, 1H), 7.46 (d, J ¼ 1.4 Hz, 1H), 7.40 (d, J ¼ 1.6 Hz, 1H), 7.37 (s,
1H), 3.77 (d, J ¼ 11.8 Hz, 2H), 2.18 (s, 3H), 1.32 (t, J ¼ 7.1 Hz, 3H).
C
H), 7.35 (d, J ¼ 2.0 Hz, 1H), 6.64 (d, J ¼ 3.3 Hz, 1H), 6.53 (s, 1H), 6.48
3
NMR (101 MHz, CDCl ) d 151.63, 149.47, 147.51, 143.24, 138.26,
(
3
dd, J ¼ 3.3, 1.8 Hz, 1H), 6.36 (d, J ¼ 7.5 Hz, 1H), 3.65 (s, 2H), 2.10 (s,
138.04, 136.65, 134.78, 133.08, 132.92, 132.49, 129.65, 129.31, 128.01,
127.98, 127.93, 127.24, 126.95, 117.59, 112.40, 110.71, 99.36, 46.40,
13
H), 1.28 (t, J ¼ 7.1 Hz, 3H). C NMR (101 MHz)
d 152.89, 151.67,
þ
142.67, 142.31, 138.23, 135.61, 134.77, 132.47, 131.11, 124.53, 123.50,
17.56, 12.48. (ESI) m/z for C25H20ClN3H [MþH] , calcd 398.1346,
117.83, 112.39, 111.88, 110.68, 105.40, 99.10, 46.30, 17.63, 12.43.
2
found 398.1413. HPLC analysis: MeOH-H O (70:30), 20.52 min,
þ
HRMS (ESI) m/z for C20H17ClN2ONa [MþNa] , calcd 359.0927,
97.63% purity.
2
found 359.0920. HPLC analysis: MeOH-H O (70:30), 25.03 min,
9
9.44% purity.
5.12.3.20. 4-((5-(1H-benzo[d]imidazol-1-yl)-2-methylphenyl)(ethyl)
amino)-2-chloro benzonitrile, 3m. The title compound was obtained
5
.12.3.15. 2-Chloro-4-(ethyl(2-methyl-5-(thiophen-2-yl)phenyl)
form 19 and benzimidazole following general procedure B as
1
amino)benzonitrile, 3h. The title compound was obtained following
general procedure B. H NMR (400 MHz, CDCl
described for compound 2c. H NMR (400 MHz, CDCl
3
)
d
8.12 (s,
1
3
)
d
7.55 (dd, J ¼ 7.9,
1H), 7.88 (dd, J ¼ 6.2, 2.8 Hz, 1H), 7.58 (d, J ¼ 8.2 Hz, 1H), 7.50 (d,
J ¼ 2.0 Hz, 1H), 7.48 (d, J ¼ 2.0 Hz, 1H), 7.42 (d, J ¼ 8.8 Hz, 1H),
7.39e7.32 (m, 2H), 7.30 (d, J ¼ 2.1 Hz, 1H), 6.59 (d, J ¼ 1.7 Hz, 1H),
6.50e6.34 (m, 1H), 3.75 (d, J ¼ 6.7 Hz, 2H), 2.21 (s, 3H), 1.31 (t,
1
1
.8 Hz, 1H), 7.42e7.27 (m, 5H), 7.08 (dd, J ¼ 4.8, 3.9 Hz, 1H), 6.54 (s,
H), 6.36 (d, J ¼ 7.7 Hz, 1H), 3.68 (s, 2H), 2.10 (s, 3H), 1.29 (t,
13
J ¼ 7.1 Hz, 3H). C NMR (101 MHz, CDCl
3
) d 151.63, 143.03, 142.71,
1
3
138.14, 135.72, 134.71, 134.52, 132.53, 128.15, 126.42, 125.43, 125.15,
J ¼ 7.1 Hz, 3H). C NMR (101 MHz, CDCl
3
) d 151.28, 143.82, 142.00,
123.37, 117.69, 112.33, 110.70, 99.18, 46.29, 17.49, 12.41. HRMS (ESI)
138.38, 136.81, 136.05,134.89, 133.61, 124.60,123.98, 123.50,123.06,
120.88, 117.35, 112.63, 110.82, 110.15, 100.05, 100.00, 46.40, 17.59,
þ
m/z for C20H18ClN2S [MþH] , calcd 353.0801, found 353.0880.
þ
HPLC analysis: MeOH-H
2
O (70:30), 12.35 min, 99.22% purity.
12.48. (ESI) m/z for C23H20ClN4 [MþH] , calcd 387.1298, found
3
2
87.1392. HPLC analysis: MeOH-H O (70:30), 10.73 min, 99.27%
5
.12.3.16. 2-Chloro-4-(ethyl(2-methyl-5-(pyridin-3-yl)phenyl)
purity.
amino)benzonitrile, 3i. The title compound was obtained following
1
0
0
0
0
0
general procedure B. H NMR (400 MHz, CDCl
H), 8.61 (d, J ¼ 3.7 Hz, 1H), 7.93e7.78 (m, 1H), 7.54 (dd, J ¼ 7.9,
.8 Hz, 1H), 7.47 (d, J ¼ 7.9 Hz, 1H), 7.37 (dd, J ¼ 6.2, 2.7 Hz, 2H), 7.32
3
)
d
8.83 (d, J ¼ 1.6 Hz,
5.12.3.21. 2-Chloro-4-(ethyl(4-methyl-2 ,3 ,4 ,5 -tetrahydro-[1,1 -
1
1
biphenyl]-3-yl)amino) benzonitrile, 3n. The title compound was
1
obtained following general procedure B. H NMR (400 MHz, CDCl
3
)
(
2
d
d, J ¼ 1.7 Hz, 1H), 6.55 (s, 1H), 6.37 (d, J ¼ 8.5 Hz, 1H), 3.72 (s, 2H),
d
7.43e7.30 (m, 2H), 7.28 (s, 1H), 7.09 (d, J ¼ 1.7 Hz, 1H), 6.50 (s, 1H),
13
.16 (s, 3H), 1.30 (t, J ¼ 7.1 Hz, 3H). C NMR (101 MHz, CDCl
3
)
6.32 (d, J ¼ 7.5 Hz, 1H), 6.21e6.08 (m, 1H), 3.67 (d, J ¼ 62.9 Hz, 2H),
151.60, 148.85, 148.08, 143.12, 138.23, 137.95, 136.63, 135.33,
2.53e2.28 (m, 2H), 2.29e2.14 (m, 2H), 2.06 (s, 3H), 1.85e1.73 (m,
1
3
1
9
34.76, 134.14, 132.84, 127.77, 126.70, 123.65, 117.60, 112.36, 110.67,
2H), 1.65 (dtd, J ¼ 12.5, 6.3, 2.8 Hz, 2H), 1.26 (t, J ¼ 7.1 Hz, 4H).
C
þ
9.39, 46.33, 17.52, 12.38. (ESI) m/z for C21H18ClN3Na [MþNa] ,
NMR (101 MHz, CDCl 151.83, 142.66, 142.15, 138.08, 135.35,
3
) d
calcd 370.1087, found 370.1086. HPLC analysis: MeOH-H
2
O (70:30),
134.62, 134.38, 131.69, 125.36, 125.19, 124.49, 117.80, 112.17, 110.59,
8
.13 min, 98.99% purity.
98.68, 46.21, 27.25, 25.85, 22.94, 22.06, 17.32, 12.34. (ESI) m/z for
þ
C22H23ClN2Na [MþNa] , calcd 373.1447, found 373.1454. HPLC
5
.12.3.17. 2-Chloro-4-(ethyl(2-methyl-5-(pyridin-4-yl)phenyl)
2
analysis: MeOH-H O (70:30), 10.13 min, 96.89% purity.
amino)benzonitrile, 3j. The title compound was obtained following
1
general procedure B. H NMR (400 MHz, CDCl
H), 7.60 (dd, J ¼ 7.9, 1.9 Hz, 1H), 7.50 (s, 2H), 7.48 (s, 1H), 7.37 (d,
J ¼ 8.7 Hz, 2H), 6.54 (s, 1H), 6.37 (d, J ¼ 8.1 Hz, 1H), 3.72 (s, 2H), 2.17
3
)
d
8.67 (d, J ¼ 4.8 Hz,
5.12.3.22. 2-Chloro-4-(ethyl(2-methyl-5-(1H-pyrazol-1-yl)phenyl)
amino)benzonitrile, 3a. The intermediate 19 was prepared
following general procedure B. To a solution of 1H-pyrazole
(54.4 mg, 0.8 mmol) in dry DMSO (1.0 ml) was added 60% NaH
2
13
(
s, 3H), 1.30 (t, J ¼ 7.2 Hz, 3H). C NMR (101 MHz, CDCl
50.42, 146.90, 143.18, 138.25, 138.21, 137.84, 134.77, 132.90, 127.72,
26.54, 121.32, 117.55, 112.38, 110.67, 99.43, 46.32, 29.71, 17.61,
2.44. (ESI) m/z for C21H19ClN3 [MþH] , calcd 348.1189, found
48.1259. HPLC analysis: MeOH-H O (70:30), 8.07 min, 99.07%
purity.
3
) d 151.56,
ꢀ
1
(80.0 mg, 2.0 mmol) portion-wise at 0 C. The mixture was warmed
1
1
to room temperature and stirred for 30 min. Thereafter CuI (7.6 mg,
0.04 mmol) and 19 (140.0 mg, 0.4 mmol) were added. The mixture
was degassed under a stream of nitrogen over 10 min and was
þ
3
2
ꢀ
stirred overnight at 120 C. The resulting mixture was cooled to
room temperature, diluted with ethyl acetate and washed twice
5
.12.3.18. 2-Chloro-4-(ethyl(5-(6-fluoropyridin-3-yl)-2-
2 4
with water. The organic layer was dried over anhydrous Na SO ,
methylphenyl)amino)benzonitrile, 3k. The title compound was ob-
tained following general procedure B. H NMR (400 MHz, CDCl )
3
filtered and concentrated under vacuum. The crude product was
purified by column chromatography on silica gel using a solvent
1
d
8.41 (s, 1H), 7.96 (t, J ¼ 6.8 Hz, 1H), 7.46 (dd, J ¼ 21.1, 13.1 Hz, 2H),
gradient of 0e30% ethyl acetate in hexanes to give 3a as a yellow oil
1
7
6
.38 (d, J ¼ 8.7 Hz, 1H), 7.27 (s, 1H), 7.12e6.89 (m, 1H), 6.54 (s, 1H),
(80.0 mg, yield 59%). H NMR (400 MHz, CDCl
3
)
d
8.12 (d, J ¼ 2.5 Hz,
13
.38 (s, 1H), 3.72 (s, 2H), 2.18 (s, 3H), 1.30 (t, J ¼ 7.1 Hz, 3H). C NMR
151.69, 145.89, 145.74, 143.32, 139.69, 138.37,
36.93, 134.90, 133.75, 133.04, 127.80, 126.73, 117.66, 112.52, 110.78,
09.95, 109.57, 99.56, 46.47, 17.64, 12.58. (ESI) m/z for
1H), 7.75 (d, J ¼ 1.6 Hz, 1H), 7.47e7.40 (m, 2H), 7.29 (d, J ¼ 2.0 Hz,
(
3
101 MHz, CDCl ) d
1H), 7.23 (d, J ¼ 8.2 Hz, 1H), 6.91 (s, 1H), 6.55e6.44 (m, 1H), 6.30 (d,
1
3
1
1
J ¼ 7.5 Hz, 1H), 3.72 (s, 2H), 2.07 (s, 3H), 1.27 (t, J ¼ 6.6 Hz, 3H).
C
3
NMR (101 MHz, CDCl ) d 151.45, 143.81, 143.77, 141.74, 135.87,
þ
C21H17ClFN3Na [MþNa] , calcd 388.0993, found 388.0975. HPLC
135.27, 133.24, 132.22, 131.19, 129.78, 120.27, 118.44, 111.41, 107.94,
analysis: MeOH-H O (70:30), 10.36 min, 99.87% purity.
2
106.87, 91.89, 46.36, 17.43, 12.42. HRMS (ESI) m/z for C19H18ClN4
þ
[
MþH] , calcd 337.1142, found 337.1134. HPLC analysis: MeOH-H
2
O
5
.12.3.19. 2-Chloro-4-(ethyl(2-methyl-5-(quinolin-3-yl)phenyl)
(70:30), 9.72 min, 94.71% purity.
amino)benzonitrile, 3l. The title compound was obtained following
1
general procedure B. H NMR (400 MHz, CDCl
1
3
)
d
9.16 (d, J ¼ 2.3 Hz,
5.12.3.23. 2-Chloro-4-((5-(3,5-dimethylisoxazol-4-yl)-2-
methylphenyl)amino)benzonitrile, 4a. The title compound was
H), 8.29 (d, J ¼ 2.1 Hz,1H), 8.14 (d, J ¼ 8.4 Hz,1H), 7.88 (d, J ¼ 7.8 Hz,

J. Yu et al. / European Journal of Medicinal Chemistry 171 (2019) 265e281
279
obtained from 15 to 3,5-dimethylisoxazole-4-boronic acid pinacol
ester via Suzuki coupling reaction as described in general proced-
99.93% purity.
1
ure A for compound 1a-k. H NMR (400 MHz, DMSO)
.63 (d, J ¼ 8.6 Hz, 1H), 7.41 (d, J ¼ 7.6 Hz, 1H), 7.22 (s, 1H), 7.17 (d,
J ¼ 7.5 Hz, 1H), 6.89 (s, 1H), 6.77 (d, J ¼ 8.3 Hz, 1H), 2.40 (s, 3H), 2.23
s, 6H). 13C NMR (101 MHz, DMSO)
165.54, 158.56, 151.65, 138.57,
37.05, 135.85, 132.62, 132.28, 129.05, 126.46, 125.25, 117.76, 115.81,
13.79,112.97, 98.89, 17.88, 11.83, 10.97. (ESI) m/z for C19 16ClN ONa
MþNa] , calcd 360.0880, found 360.0854. HPLC analysis: MeOH-
O (70:30), 6.92 min, 99.58% purity.
d
8.73 (s, 1H),
5.12.4.2. N-(5-(3,5-dimethylisoxazol-4-yl)-2-methylphenyl)-N-ethyl-
pyridin-4-amine, 4d. The title compound was obtained following
general procedure C. H NMR (400 MHz, CDCl )
7
1
3
d
8.18 (d, J ¼ 6.4 Hz,
(
d
2H), 7.43 (d, J ¼ 7.9 Hz, 1H), 7.20 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.01 (d,
J ¼ 1.8 Hz, 1H), 6.36 (d, J ¼ 5.4 Hz, 2H), 3.71 (s, 2H), 2.42 (s, 3H), 2.28
1
1
3
1
[
H
3
(s, 3H), 2.16 (s, 3H), 1.28 (t, J ¼ 7.2 Hz, 3H). C NMR (101 MHz,
þ
3
CDCl ) d 165.24, 158.43, 153.11, 149.07, 142.69, 136.13, 132.36,
H
2
130.22, 129.82, 128.48, 115.70, 107.15, 45.50, 17.48, 12.36, 11.68,
þ
10.86. (ESI) m/z for C19H22N
3
O [MþH] , calcd 308.1685, found
5
.12.3.24. 4-(3-(3,5-dimethylisoxazol-4-yl)phenoxy)-2-(tri-
2 3
308.1775. HPLC analysis: MeOH-H O (70:30) with 0.1% Et N,
fluoromethyl)benzonitrile, 4b. A mixture of 4-fluoro-2-(tri-
fluoromethyl)benzonitrile (8) (378.0 mg, 2.0 mmol), 3-iodophenol
4.01 min, 94.25% purity.
(
440.0 mg, 2.0 mmol) and K
2
CO
3
(414.6 mg, 3.0 mmol) in DMSO
5.12.4.3. N-(5-(3,5-dimethylisoxazol-4-yl)-2-methylphenyl)pyridin-
ꢀ
(
4.0 ml) was heated to 110 C and stirred for 2 h. The reaction
4-amine, 4e. The title compound was obtained following general
1
mixture was cooled to room temperature, diluted with ethyl ace-
tate and washed with water. The organic layer was dried over
anhydrous Na SO , filtered and concentrated under vacuum. Puri-
2 4
fication on silica using a solvent of 10% ethyl acetate in hexanes
yielded the product 9 (670.0 mg, yield 86.1%) as a white solid. The
title compound was obtained from 9 to 3,5-dimethylisoxazole-4-
boronic acid pinacol ester through Suzuki coupling reaction as
procedure C. H NMR (400 MHz, CDCl )
d
8.27 (dd, J ¼ 4.9, 1.5 Hz,
3
2H), 7.35 (d, J ¼ 7.8 Hz, 1H), 7.17 (d, J ¼ 1.7 Hz, 1H), 7.09e6.97 (m,
1H), 6.67 (dd, J ¼ 4.9, 1.5 Hz, 2H), 6.00 (s, 1H), 2.41 (s, 3H), 2.29 (s,
13
3
3H), 2.28 (s, 3H). C NMR (101 MHz, CDCl ) d 165.22, 158.54, 151.48,
149.96, 138.01, 132.00, 131.83, 129.38, 126.22, 124.62, 115.97, 109.28,
þ
17.70, 11.65, 10.87. (ESI) m/z for C H N O [MþH] , calcd 280.1372,
17 18 3
found 280.1444. HPLC analysis: MeOH-H O (70:30) with 0.1% Et N,
2
3
1
described in general procedure A for compound 1a-k. H NMR
4.93 min, 97.75 purity.
(
7
1
400 MHz, CDCl
3
)
d
7.79 (d, J ¼ 8.5 Hz, 1H), 7.53 (t, J ¼ 7.8 Hz, 1H),
.38 (s, 1H), 7.19 (d, J ¼ 7.1 Hz, 2H), 7.08 (d, J ¼ 7.6 Hz, 1H), 6.99 (s,
Author contributions
1
3
H), 2.42 (s, 3H), 2.28 (s, 3H). C NMR (101 MHz, CDCl
3
) d 165.62,
1
61.24, 158.29, 154.40, 136.81, 135.30, 134.97, 133.31, 130.98, 126.51,
J.Y and L.Z contributed equally to this work. The manuscript was
written by J.Y. The design of compounds was performed by J.Y. The
synthesis of compounds was performed by J.Y., L.Z., C.C. Biological
evaluation was accomplished by J.Y., P.Z. and F.Z. The molecular
modeling was performed by G.Y. And X. L provided important
suggestions to this work. All authors have given approval to the
final version of the manuscript.
120.91, 119.99, 119.32, 115.91, 115.60, 115.32, 103.53, 11.65, 10.81.
þ
(
ESI) m/z for C19
H
13
F
3
N
2
O
2
Na [MþNa] , calcd 381.0827, found
3
81.0818. HPLC analysis: MeOH-H
2
O (70:30), 7.33 min, 98.78%
purity.
5.12.4. General procedure C for the synthesis of 4c-e
5
-bromo-2-methylaniline (21) and 3,5-dimethylisoxazole-4-
boronic acid pinacol ester underwent Suzuki coupling reaction as
described in general procedure A for compound 1a-k to give in-
Conflicts of interest
termediate 22. Under N
123.0 mg, 0.6 mmol), 22 (101.0 mg, 0.5 mmol), Cs
.25 mmol), Pd (dba) (23.0 mg, 0.025 mmol) and Davephos
15.7 mg, 0.04 mmol) in MeCN (3.0 ml) was heated to reflux for 5 h.
2
atmosphere, a mixture of 4-iodopyridine
The authors declare no competing financial interest.
Acknowledgment
(
1
(
2
CO (292.5 mg,
3
2
3
The reaction mixture was cooled to room temperature and diluted
with ethyl acetate. The mixture was filtered off and the solution was
concentrated under vacuum to give a crude product 4e that was
purified by column chromatography on silica using a solvent of 10%
MeOH in DCM. The compound 4e was obtained as a yellow oil
We gratefully acknowledge the financial support from National
Natural Science Foundation of China (81472418 and 81672951). We
thank Drs. Wu Du and Lei Fan in Hinova Pharmaceuticals Inc. for
helpful discussions and suggestions to this research. Authors also
wish to thank Dr. Wu Du for his advices to this manuscript.
(
100.0 mg, 71.7%). For the synthesis of intermediate 23, commer-
cially available 2-iodopyridine were used following the procedure
described for 4e.
Appendix A. Supplementary data
Compound 4c and 4d were successfully prepared using 23 or 4e
through N-alkylation reaction as described in general procedure B
for compound 17e19. The characterization data for compounds 4c-
e are provided below.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.03.041.
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5
.12.4.1. N-(5-(3,5-dimethylisoxazol-4-yl)-2-methylphenyl)-N-ethyl-
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1
3
)
d
8.21 (ddd, J ¼ 5.0,
1
.9, 0.8 Hz, 1H), 7.40 (d, J ¼ 7.8 Hz, 1H), 7.32e7.26 (m, 1H), 7.15 (dd,
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3
d
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4] T. Visakorpi, E. Hyytinen, P. Koivisto, M. Tanner, R. Keinanen, C. Palmberg,
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13
H), 2.18 (s, 3H), 1.24 (t, J ¼ 7.1 Hz, 3H). C NMR (101 MHz, CDCl
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)
165.12, 158.56, 157.96, 147.97, 143.47, 136.92, 136.68, 132.06,
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þ
11.66, 10.88. (ESI) m/z for C19H21N
3
ONa [MþNa] , calcd 330.1582,
2
found 330.1618. HPLC analysis: MeOH-H O (55:45), 8.38 min,

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3
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