Discovery of novel quinoline-based mTOR inhibitors via introducing intra-molecular hydrogen bonding scaffold (iMHBS): The design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2015.11.003

A series of quinoline derivatives featuring the novelty of introducing intra-molecular hydrogen bonding scaffold (iMHBS) were designed, synthesized and biologically evaluated for their mTOR inhibitory activity, as well as anti-proliferative efficacies aga

Full text of DOI:10.1016/j.bmc.2015.11.003

Bioorganic & Medicinal Chemistry 23 (2015) 7585–7596
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel quinoline-based mTOR inhibitors via introducing
intra-molecular hydrogen bonding scaffold (iMHBS): The design,
synthesis and biological evaluation
⇑
Xiaodong Ma, Xiaoqing Lv, Ni Qiu, Bo Yang, Qiaojun He, Yongzhou Hu
Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of quinoline derivatives featuring the novelty of introducing intra-molecular hydrogen bonding
scaffold (iMHBS) were designed, synthesized and biologically evaluated for their mTOR inhibitory activ-
ity, as well as anti-proliferative efficacies against HCT-116, PC-3 and MCF-7 cell lines. As a result, six com-
pounds exhibited significant inhibition against mTOR with IC50 values below 35 nM. Compound 15a, the
most potent mTOR inhibitor reported herein (IC50 = 14 nM), also displayed the most favorable cellular
Received 11 September 2015
Revised 26 October 2015
Accepted 4 November 2015
Available online 4 November 2015
activities, with the IC50 values of 0.46, 0.61 and 0.24 lM against HCT-116, PC-3 and MCF-7, respectively.
Keywords:
Besides, several compounds in this series were identified to be selective over class I PI3Ks. Further west-
ern blot analysis of 16b, a representative compound in this series, highlighted their advantage in sur-
mounting the S6K/IRS1/PI3K negative feedback loop upon dual inhibition of mTORC1 and mTORC2. In
addition to the remarkable activity, 15a demonstrated acceptable stability in simulated gastric fluid
(SGF), simulated intestinal fluid (SIF) and liver microsome, thereby being valuable for extensive in vivo
investigation.
Quinoline derivatives
mTOR inhibitor
Anti-proliferative efficacy
Selectivity
Negative feedback loop
Stability
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
ison, ATP-competitive mTOR modulators (mTOR kinase inhibitors)
simultaneously inhibiting mTORC1 and mTORC2 may conquer the
Nowadays, the medicinal potential of the mammalian target of
rapamycin (mTOR), a key node of PI3K/Akt/mTOR cascade, has
been validated by several rapalogues (rapamycin and its semi-syn-
thetic analogs) prescribed for cancer treatment in clinic.1 In cells,
mTOR assembles into two functionally distinct multi-protein com-
plexes, known as mTORC1 and mTORC2.2 While mTORC1 locates
downstream of Akt, serving as a crucial regulator of translation
and cell proliferation, mTORC2 resides upstream of Akt,3 activating
it upon phosphorylation on Ser473. Rapalogues, such as rapamy-
cin, everolimus and temsirolimus, selectively suppress the activity
of mTORC1 through an allosteric mechanism. However, the inhibi-
tion is partial due to the failure in attenuating the kinase activity of
mTOR.4 In addition, rapalogues rarely interfere with the activity of
mTORC2, except in a small subset of cell lines.5 More importantly,
the rapalogue treatment releases the S6K/IRS1/PI3K negative feed-
back loop, which consequently upregulates Akt and undermines
the anti-proliferative efficacy of mTORC1 inhibition.6 With these
shortcomings, the application of allosteric mTOR inhibitors has
been confined to a limited number of tumor types.7–9 In compar-
hyperactivation of Akt induced by the negative feedback loop,
hence broadening anti-tumor spectrum and improving therapeutic
efficacy.7,10
Throughout the past decade, numerous ATP-competitive mTOR
inhibitors have been advanced into clinical trial, including mTOR
selective (AZD-2014,11,12 MLN-012813) and dual mTOR/PI3K inhi-
bitors (BEZ-235,14–16 GSK-2126458,17 XL-765,18,19 PF-04691502,20
etc.). During the pursuit of ATP-competitive mTOR inhibitors, Torin
2 (Fig. 1) inspired our interest by virtue of its remarkable mTOR
inhibitory activity and structural differentiation from other mole-
cules belonging to this class. Despite its favorable bioavailability
and overall exposure, Torin 2 displayed a short elimination half-life
of 0.72 h following oral administration.10 Besides, a majority of
analogs sharing the same benzonaphthridinone core as Torin 2 also
suffered from poor PK profiles due to the instability in liver micro-
some and limited solubility.21
According to our insight into the binding mode of Torin 2 with
mTOR,22 the N1 of the quinoline conferred a critical H-bond con-
tact to residue Val2240 in the hinge region. Meanwhile, the amino
pyridine moiety of Torin 2 was projected into the inner hydropho-
bic pocket of mTOR catalytic cleft. The
a,b-unsaturated lactam
⇑
fused to the C-3 and C-4 positions of quinoline was important for
constraining the pendant phenyl ring to the bioactive conformation,
Corresponding author. Tel./fax: +86 571 88208460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
http://dx.doi.org/10.1016/j.bmc.2015.11.003
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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X. Ma et al. / Bioorg. Med. Chem. 23 (2015) 7585–7596
Figure 1. The design rationale for the quinoline derivatives with iMHBS.
in which the trifluoromethyl group packed into an N-lobe pocket.
However, as Michael-acceptor, the ,b-unsaturated lactam was
liable to be captured in vivo, thereby accounting for the short elim-
ination half-life of Torin 2. Considering this, our optimization prior-
itized obviating the metabolically instable a,b-unsaturated lactam
and introducing an intra-molecular H-bond to mimic it. Accord-
ingly, a series of quinoline derivatives were designed via a de-con-
struction approach to break the lactam of Torin 2 (Fig. 1). These
quinolines were characterized by a C-3 amide functionality and a
C-4 substituted aniline, upon which a pseudo-ring was desired to
form between the amide oxygen and the secondary amine hydro-
gen through a six-membered intra-molecular H-bond.
di-methylation at the benzyl position and catalytic hydrogenation
to afford the substituted aniline 2. According to the procedure
reported in the literature,23 condensation of 4-bromoaniline 3 with
diethyl ethoxymethylenemalonate 4 (EMME), followed by the
intra-molecular cyclization, provided the quinoline derivative 5.
Afterwards, treatment of 5 with POCl3 gave the C-4 chlorine quino-
line derivative 6, which experienced SNAr reaction with corre-
sponding substituted aniline to furnish intermediates 7a and 7b.
Subsequent aminolysis converted the C-3 ester moiety of 7a or
7b into corresponding amide functionality. The newly formed
8a–c finally underwent Suzuki coupling with corresponding borate
to generate target compounds 9a–h.
a
We envisioned this type of intra-molecular hydrogen bonding
scaffold (iMHBS) not only restricted the basic conformation of
Torin 2, but also eliminated the metabolically susceptible site.
Besides, another advantage of the iMHBS designed herein lay in
the reduced ring members compared to Torin 2, thereby being ben-
eficial for improving the solubility. This research was focused on
the preparation, in vitro evaluation, as well as structure–activity
relationship (SAR) study of these quinolines to validate the
iMHBS strategy and screen promising compounds for further
development.
The preparation of compounds bearing C-4 elongated substruc-
ture was displayed in Scheme 2. SNAr reaction of N-Boc-piperazine
with 10 and the following catalytic hydrogenation provided 11 as
an important fragment. It was then subjected to SNAr reaction
with 6 to afford the intermediate 12. After a sequence of aminoly-
sis, Suzuki coupling and removal of the Boc-protecting group, tar-
get compounds 15a–d and intermediate 15e were obtained as the
hydrochlorides. Further acylation or sulfonylation at the piperazine
furnished target compounds 16a–c.
As shown in Scheme 3, compound 19 was obtained via removal
of the Boc-protecting group, propionylation and subsequent Suzuki
coupling upon utilization of 13a as the starting material. After
removing the Boc-protecting group of 13a, the newly formed pro-
duct was treated with chloroacetyl chloride to give 18 as an inter-
mediate. It was then subjected to nucleophilic attack by N-methyl
piperazine and Suzuki coupling with corresponding borate to
furnish 20 as the target compound.
2. Results and discussion
2.1. Chemistry
The synthetic route for compounds 9a–h was outlined in
Scheme 1. Firstly, 4-nitrophenylacetonitrile 1 was subjected to
Scheme 1. Reagents and conditions: (a) (1) MeI, TBAB, DCM/H2O, rt; (2) H2, Pd/C, MeOH, rt; (b) (1) 80 °C; (2) Ph2O, 250 °C; (c) DMF, POCl3, reflux; (d) 2 or 3-
trifluoromethylaniline, AcONa, HAc, rt; (e) NH3 (EtOH solution, concentrated) or MeNH2 (EtOH solution, concentrated), 80 °C; (f) corresponding borate, K2CO3, Pd(PPh3)4, H2O/
1,4-dioxane, 100 °C.

X. Ma et al. / Bioorg. Med. Chem. 23 (2015) 7585–7596
7587
Scheme 2. Reagents and conditions: (a) (1) N-Boc-piperazine, K2CO3, DMF, 40 °C; (2) H2, Pd/C, MeOH, rt; (b) 6, AcONa, HAc, rt; (c) NH3 (EtOH solution, concentrated), MeNH2
(EtOH solution, concentrated) or cyclopropylamine, 80 °C; (d) corresponding borate, K2CO3, Pd(PPh3)4, H2O/1,4-dioxane, 100 °C; (e) HCl (EtOAc solution, concentrated), 0 °C to
rt; (f) Et3N, AcCl or MsCl, THF, 0 °C.
Scheme 3. Reagents and conditions: (a) (1) TFA, DCM, 0 °C to rt; (2) Et3N, propionyl chloride or chloroacetyl chloride, THF, 0 °C to rt; (b) corresponding borate, K2CO3, Pd
(PPh3)4, H2O/1,4-dioxane, 100 °C; (c) (1) N-methyl piperazine, K2CO3, CH3CN, 80 °C; (2) corresponding borate, K2CO3, Pd(PPh3)4, H2O/1,4-dioxane, 100 °C.
2.2. mTOR inhibitory activity
Replacement of the methyl group with cyclopropyl resulted in a
further loss in activity, as illustrated by the activity of 15c versus
15b. Additionally, C-6 heterocycle served as another critical factor
to affect activity, with aminopyridine or quinoline more beneficial
than methoxypyridine or N-methyl pyrazole. Compounds 9f and
15d, both featuring a C-6 methoxypyridine moiety, were approxi-
mately 20-fold less potent than corresponding aminopyridine
derivative 9d and quinoline derivative 15a. Similarly, neither the
C-6 N-methyl pyrazole derivative 9a nor 16a exhibited favorable
activity as respective aminopyridine counterpart 9d and quinoline
counterpart 16c did. In comparison, alteration in C-4 aniline only
had a slight impact on activity of these compounds, as exemplified
by the C-6 aminopyridine derivatives 9d, 9g and 19. Despite varied
substitution on the aniline, they displayed comparable mTOR inhi-
bitory activity with IC50 values being 30, 51 and 21 nM, respec-
tively. Besides, further derivatization at the piperazine failed to
enhance potency, according to the data of compounds 15a, 16b
and 20.
All the target compounds were evaluated for their inhibitory
activity against mTOR via lance ultra assay with PI-103, an
mTOR/PI3K dual inhibitor, as the positive control. According to
the results summarized in Table 1, nearly half of them (9b, 9d,
9g, 15a, 16b, 16c, 19 and 20) exhibited remarkable inhibition
against mTOR with IC50 values at two-digit nanomolar level. In par-
ticular, six quinoline derivatives, including 9b, 9d, 15a, 16b, 19 and
20, displayed comparable mTOR inhibitory activity to PI-103 with
IC50 values lower than 35 nM.
From the data for mTOR inhibition, some valuable SARs can be
deduced. As for the C-3 amide functionality, introduction of N-sub-
stituent was detrimental for activity. For example, compounds 9c,
9e and 15b bearing a methyl substituent at the nitrogen demon-
strated significantly compromised activity compared to corre-
sponding N-unsubstituted counterparts 9b, 9d and 15a. With the
N-substituent being bulkier, the activity decreased sharply.

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Table 1
In vitro biological activities of the target quinoline derivatives
Compd
Substituted phenyl
C-6 heterocycle
Amide N-substituent
mTOR (IC50, nM)
1105
HCT-116 (IC50
17.26
,
lM)
PC-3 (IC50
,
lM)
MCF-7 (IC50
, lM)
9a
H
>50
1.25
9b
9c
9d
9e
9f
H
33
514
30
4.56
8.73
0.67
4.66
2.57
1.83
2.82
7.12
0.83
1.74
1.66
1.08
0.95
1.30
0.52
0.49
1.58
0.41
Me
H
Me
H
280
674
51
9g
H
9h
H
H
361
14
3.17
0.46
6.39
0.61
0.60
0.24
15a
15b
15c
15d
Me
806
>3000
284
a
a
a
a
a
a
H
2.05
0.99
0.78
16a
16b
16c
H
H
H
1225
31
14.71
1.29
1.00
7.08
3.90
3.73
2.69
0.44
0.36
73
19
20
H
H
21
24
2.40
1.46
0.57
0.97
0.20
0.99
0.21
0.44
0.05
PI-103
BEZ-235
16
21b
a
Not identified.
IC50 value reported in the literature.
b
2.3. Anti-proliferative activity
under phase II clinical trial, was employed as the positive control.
On the whole, compounds with two-digit nanomolar mTOR inhibi-
tory activity (9b, 9d, 9g, 15a, 16b, 16c, 19 and 20) also demon-
strated attractive potency against all the tested cell lines. As
shown in Table 1, the IC50 values were at low micromolar or sub-
micromolar level without exception. Among them, 15a, the most
Encouraged by the favorable enzymatic activity of the prepared
quinoline derivatives, we subsequently evaluated their anti-prolif-
erative efficacies against several human tumor cell lines, including
HCT-116, PC-3 and MCF-7. BEZ-235, an mTOR/PI3K dual inhibitor

X. Ma et al. / Bioorg. Med. Chem. 23 (2015) 7585–7596
7589
remarkable mTOR inhibitor in this series, exhibited the most favor-
able cellular activities as well with IC50 values of 0.46, 0.61 and
0.24 lM against HCT-116, PC-3 and MCF-7, respectively. Besides,
9d also displayed distinguished efficacy against all the three cell
lines with IC50 values at submicromolar level. Throughout the
tested cell lines, MCF-7 was regarded to be the most susceptible
to these quinoline derivatives. Against the cell line, six compounds
of mTORC1 and mTORC2 therefore provided a powerful evidence
for the capability to prevent the feedback loop upon 16b treat-
ment. On the contrary, rapamycin failed to downregulate pAKT
(Ser473) despite the decrease in the level of pS6(Ser235) at
0.5 lM. Instead, rapamycin treatment culminated in the upregula-
tion of pAKT(Ser473), which indicated the release of the negative
feedback loop.
exhibited IC50 values below 0.5
IC50 values lower than 3.0 M.
lM, and the other ones showed
l
2.6. In vitro stability
2.4. Selectivity over class I PI3Ks
To direct the in vivo development of these quinoline deriva-
tives, 15a was then evaluated for its stability in simulated gastric
fluid (SGF), simulated intestinal fluid (SIF) and liver microsome.
As a result, almost no degradation was observed upon incubation
of this compound in both SGF and SIF after 45 min. Besides, it
demonstrated acceptable stability in rat liver microsome (RLM)
with nearly 50% left at 30 min. Meanwhile, favorable stability
was exhibited by 15a in human liver microsome (HLM) with 80%
and 73% remaining at 30 min and 60 min, respectively.
Compounds 9b, 15a and 16b in this series were further evalu-
ated for their inhibitory activities against PI3K , PI3Kb, PI3K
and PI3Kd with PI-103 as the positive control. As summarized in
Table 2, none of them displayed significant inhibition against the
four class I PI3Ks at the concentration of 1.0 lM. Thereby, 9b,
a
c
15a and 16b, as potent mTOR inhibitors, simultaneously exhibited
acceptable specificity over class I PI3Ks.
2.5. Western blot analysis
2.7. Molecular docking
To highlight the merits of mTOR kinase inhibitors, 16b, as a rep-
resentative of this series, was further investigated for its potential
to obviate the S6K/IRS1/PI3K negative feedback loop via western
blot analysis in MCF-7 cell line (Fig. 2). Herein, pAKT(Ser473)
served as the biomarker for inhibition of mTORC2, while pS6
(Ser235) was an indicator for suppression of mTORC1. As a conse-
quence, BEZ-235 and 16b induced a remarkable downregulation of
For probing the possible binding mode of these quinoline
derivatives, compound 15a was docked into the mTOR catalytic
cleft based on the cocrystal structure of Torin 2 complexed with
mTOR. According to Figure 3a and b, the nitrogen of quinoline tem-
plate and C-3 amide hydrogen engaged in two H-bonds with the
critical residue Val2240 in hinge region. Meanwhile, the hydroxyl
of residue Tyr2225 that locating at the inner hydrophobic pocket
served as an H-bond donor for the quinoline at the C-6 position
of quinoline core. In addition to these H-bond interactions, the
both pAKT(Ser473) and pS6(Ser235) at 0.5
lM. Particularly, a com-
plete downregulation of pAKT(Ser473) was accomplished by 16b
at the concentration as low as 0.1 lM. The simultaneous inhibition
quinoline template made a
p–p contact to the indole moiety of
Trp2239, which was also observed in the cocrystal structure of
Torin 2 complexed with mTOR. As formerly hypothesized, the C-
3 amide oxygen and C-4 secondary amine hydrogen were within
H-bond distance and the intra-molecular H-bond formed between
them restricted the orientation of the C-4 substituted aniline. In
this conformation, the trifluoromethyl group packed into the N-
lobe pocket (leu2185, Pro 2169, etc.). As for compound 15b, how-
ever, the N-methyl amide moiety at the C-3 position had to rotate
away from the indole of Trp2239 for obviating the unfavorable
contact (Fig. 3c). Consequently, the H-bond interaction with
Val2240 was weakened and the H-bond contact to Tyr2225
decreased due to the concerted movement of the quinoline core,
which accounted for the compromised activity of 15b.
Table 2
The inhibitory activities of compounds 9b, 15a and 16b against class I PI3Ks
Compd
PI3K
a
PI3Kb
PI3K
c
PI3Kd
9b
15a
16b
PI-103
26.9%a
32.3%a
2427b
12b
10.3%a
1.0%a
>5000b
13b
ꢀ2.4%a
6.7%a
939b
48b
ꢀ12.8%a
16.7%a
>5000b
10b
a
Inhibition rate at 1.0
IC50 value (nM).
lM.
b
3. Conclusion
Built upon our insight into the cocrystal structure of Torin 2
complexed with mTOR, a novel structural series of quinoline
derivatives were obtained via introducing iMHBS to mimic the
bioactive conformation of Torin 2. All the target quinolines were
evaluated for their mTOR inhibitory activity and the majority were
tested for the anti-proliferative efficacies against HCT-116, PC-3, as
well as MCF-7 cell lines. In general, the quinolines with two-digit
nanomolar mTOR inhibitory activity, namely 9b, 9d, 9g, 15a, 16b,
16c, 19 and 20, also demonstrated attractive potency against all
the tested cell lines. In particular, compound 15a, the most potent
mTOR inhibitor in this series (IC50 = 14 nM), simultaneously dis-
played the most favorable cellular activities, with respective IC50
value of 0.46, 0.61 and 0.24 lM against HCT-116, PC-3 and MCF-
7. Besides, several compounds in this series, including 9b, 15a
and 16b, were identified to be selective over class I PI3Ks. The
advantage of these mTOR kinase inhibitors over rapalogues was
further validated by western blot analysis in MCF-7 cell line, which
Figure 2. Western blot analysis of the quinoline derivative 16b in MCF-7 cell line.

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X. Ma et al. / Bioorg. Med. Chem. 23 (2015) 7585–7596
Figure 3. Molecular docking of quinoline derivatives 15a (a and b) and 15b (c) into the catalytic cleft of mTOR.
highlighted the capability of 16b, a representative in this series,
to conquer the S6K/IRS1/PI3K negative feedback loop upon the
inhibition of both mTOR complexes. Moreover, 15a showed accept-
able gastrointestinal and liver microsomal stability in vitro,
thereby meriting extensive in vivo investigation.
Subsequently, ethanol formed during the reaction was removed
under reduced pressure and diphenyl ether (50 mL) was added to
the resultant white solid. After being heated at 250 °C for 6 h, the
mixture was cooled and petroleum ether (PE, 50 mL) was added.
The precipitated product was collected, washed successively with
PE and EtOAc, and dried in vacuo to give the intermediate as a
white solid. Yield: 61%; 1H NMR (500 MHz, DMSO-d6): d 12.46 (s,
1H, OH), 8.60 (s, 1H, Ar-H), 8.23 (d, 2.5 Hz, 1H, Ar-H), 7.88 (dd,
8.5 Hz, 2.5 Hz, 1H, Ar-H), 7.61 (d, 8.5 Hz, 1H, Ar-H), 4.23 (q,
7.0 Hz, 2H, CH2), 1.29 (t, 7.0 Hz, 3H, CH3). ESI-MS: m/z = 296 [M
+H]+; m/z = 294 [MꢀH]ꢀ.
4. Experimental
4.1. Chemistry
The reagents and solvents for reaction were purchased from
common commercial suppliers. If necessary, purification was car-
ried out prior to use. Melting points were uncorrected and deter-
mined on a Büchi B-540 apparatus (Büchi Labortechnik, Flawil,
Switzerland). 1H NMR and 13C NMR spectra were recorded on
500 MHz and 125 MHz instruments (Bruker Bioscience, Billerica,
MA, USA), respectively, with tetramethylsilane (TMS) as internal
standard. ESI mass spectral data were obtained by Esquire-LC-
00075 spectrometer (Bruker Bioscience). Flash column chromatog-
raphy was performed using silica gel (200–300 mesh). HPLC was
performed using an Agilent 1200 system with UV detection at
254 nm, eluting with a binary solvent system A and B or C and B
[A: H2O; B: CH3OH; C: H2O with 0.12% ammonium acetate (W/
V)]. Analytical purity of all compounds was >95% unless stated
otherwise.
4.1.3. Ethyl 6-bromo-4-chloroquinoline-3-carboxylate (6)
To a suspension of 5 (4.00 g, 13.6 mmol) in POCl3 (15.0 mL) was
added catalytic amount of N,N-dimethylformamide (DMF) and the
mixture was heated to reflux for 4 h. It was then cooled to room
temperature and carefully poured onto ice. After vigorous agita-
tion, the precipitate was filtered, washed with water and dissolved
in DCM. The solution was subsequently washed with saturated
NaHCO3 solution, brine, dried with anhydrous Na2SO4 and concen-
trated in vacuo to provide the title intermediate as a light yellow
solid. Yield: 93%; 1H NMR (500 MHz, CDCl3): d 9.21 (s, 1H, Ar-H),
8.47 (d, 1.5 Hz, 1H, Ar-H), 8.02 (d, 9.0 Hz, 1H, Ar-H), 7.95 (dd,
1.5 Hz, 9.0 Hz, 1H, Ar-H), 4.40 (q, 7.0 Hz, 2H, CH2), 1.60 (t, 7.0 Hz,
3H, CH3). ESI-MS: m/z = 314 [M+H]+.
4.1.1. 2-(4-Aminophenyl)-2-methylpropanenitrile (2)
4.1.4. General procedure for the preparation of intermediates 7a
and 7b (A)
To
a suspension of 4-nitrophenylacetonitrile 1 (1.0 equiv,
4.50 g, 27.8 mmol), tetrabutyl ammonium bromide (TBAB,
0.448 g, 1.39 mmol, 0.05 equiv) and iodomethane (11.8 g,
83.4 mmol, 3.0 equiv) in dichloromethane (DCM, 40.0 mL) was
added sodium hydroxide (2.78 g, 69.5 mmol, 2.5 equiv) in water
(40.0 mL) dropwise. After stirring at room temperature for 8 h,
the organic layer was separated, washed with brine, dried with
anhydrous Na2SO4 and concentrated in vacuo. Then the residue
was subjected to flash column chromatography using EA/PE
(1:14–1:7) as eluent to furnish the di-methylated product as a
white crystal. Yield: 83%; ESI-MS: m/z = 191 [M+H]+. The di-methy-
lated product (4.38 g, 23.1 mmol) and 10% Pd/C (10% of the sub-
strate, w/w) were shacked in methanol (60.0 mL) and the
mixture was stirred at room temperature under H2 (balloon) atmo-
sphere overnight. After this time, Pd/C was filtered and the filtrate
was concentrated in vacuo to provide 2 as a colorless oil, which
slowly solidified and was used for the next step without purifica-
tion. Yield: 95%. ESI-MS: m/z = 161 [M+H]+.
To a solution of 6 (1.0 equiv) in acetic acid (2 mL/1 mmol sub-
strate) AcONa (1.4 equiv) and substituted aniline (1.0 equiv), such
as 2 or 3-trifluoromethylaniline, were added. The resultant mixture
was stirred for 0.5 h at room temperature and quenched with
water. After the rude product was totally precipitated, it was fil-
tered, washed with water and dissolved in DCM. The solution
was then washed with saturated NaHCO3 solution, brine and dried
over anhydrous Na2SO4. Following removal of solvent in vacuo, the
residue was purified via flash column chromatography using EA/PE
(1:6) as eluent to afford corresponding ethyl 6-bromo-4-anilino-3-
carboxylate quinoline derivative 7a or 7b as light yellow solid.
4.1.4.1.
Ethyl
6-bromo-4-((4-(2-cyanopropan-2-yl)phenyl)
amino)quinoline-3-carboxylate (7a).
Light yellow solid; yield:
83%; ESI-MS: m/z = 438 [M+H]+.
4.1.4.2. Ethyl 6-bromo-4-((3-(trifluoromethyl)phenyl)amino)
4.1.2. Ethyl 6-bromo-4-hydroxyquinoline-3-carboxylate (5)
quinoline-3-carboxylate (7b).
Light yellow solid; yield:
91%; 1H NMR (500 MHz, DMSO-d6): d 9.70 (s, 1H, NH), 8.78 (s,
1H, Ar-H), 8.52 (d, 1.5 Hz, 1H, Ar-H), 7.95 (dd, 1.5 Hz, 9.0 Hz, 1H,
Ar-H), 7.92 (d, 9.0 Hz, 1H, Ar-H), 7.49 (t, 8.5 Hz, 1H, Ar-H), 7.33
A mixture of 4-bromoaniline 3 (10.0 g, 58.48 mmol, 1.0 equiv)
with diethyl ethoxymethylenemalonate
4
(EMME, 12.63 g,
58.48 mmol, 1.0 equiv) was warmed in oil bath at 80 °C for 2 h.

X. Ma et al. / Bioorg. Med. Chem. 23 (2015) 7585–7596
7591
(s, 1H, Ar-H), 7.32 (d, 2.0 Hz, 1H, Ar-H), 7.27 (dd, 2.0 Hz, 8.5 Hz, 1H,
Ar-H), 3.81 (q, 7.0 Hz, 2H, CH2), 1.01 (t, 7.0 Hz, 3H, CH3). ESI-MS:
m/z = 439 [M+H]+.
Ar-H), 7.85 (dd, 2.0 Hz, 9.0 Hz, 1H, Ar-H), 7.79 (d, 2.0 Hz, 1H, Ar-
H), 7.76 (br s, 1H, NH), 7.43 (d, 8.5 Hz, 2H, Ar-H), 7.40 (d, 1.5 Hz,
1H, Ar-H), 7.08 (d, 8.5 Hz, 2H, Ar-H), 6.22 (d, 1.5 Hz, 1H, Ar-H),
3.48 (s, 3H, Pyrazole N-CH3), 1.66 (s, 6H, CH3ꢁ2); ESI-MS: m/z =
411 [M+H]+; mp 258–260 °C; HPLC: tR = 12.29 min, flow rate
0.8 mL/min, COSMOSIL 5C18-MS-II column (4.6ID ꢁ 250 mm), rt,
eluent A-80%, eluent B-20%.
4.1.5. General procedure for the preparation of intermediates
8a–c (B)
A mixture containing 7a or 7b and the concentrated solution of
NH3 in EtOH (4 mL/1 mmol substrate) in a sealed tube was heated
at 80 °C for 8 h. It was then cooled to ꢀ20 °C and filtered to collect
the filtrate. After removal of the solvent, the residue was purified
via flash column chromatography using EA/PE/TEA (30:10:1) as
the eluent to afford corresponding 6-bromo-4-(phenylamino)
quinoline-3-carboxamide derivative 8a or 8c as light yellow solid.
The intermediate 8b was prepared by treatment of 7a with the
concentrated solution of MeNH2 in EtOH (2 mL/1 mmol substrate)
in a sealed tube at 80 °C for 2 h. The mixture was then cooled and
the solvent was removed in vacuo. The resultant residue was
purified via flash column chromatography using EA/PE/TEA
(60:40:1–60:20:1) as eluent to afford the desired intermediate as
light yellow solid.
4.1.6.2.
biquinoline]-30-carboxamide (9b).
40-((4-(2-Cyanopropan-2-yl)phenyl)amino)-[3,60-
Light yellow solid; yield:
65%; 1H NMR (500 MHz, DMSO-d6): 10.64 (br s, 1H, NH), 9.00 (s,
1H, Ar-H), 8.83 (d, 2.5 Hz, 1H, NH), 8.47 (d, 2.5 Hz, 1H, NH), 8.23
(dd, 1.5 Hz, 8.5 Hz, 2H, Ar-H), 8.19 (d, 1.5 Hz, 1H, Ar-H), 8.08 (d,
8.5 Hz, 1H, Ar-H), 8.01 (d, 7.5 Hz, 1H, Ar-H), 7.97 (d, 7.5 Hz, 1H,
Ar-H), 7.78–7.75 (m, 1H, Ar-H), 7.69 (s, 1H, Ar-H), 7.65–7.62 (m,
1H, Ar-H), 7.49 (d, 8.5 Hz, 2H, Ar-H), 7.15 (d, 8.5 Hz, 2H, Ar-H),
1.70 (s, 6H, CH3ꢁ2); 13C NMR (125 MHz, DMSO-d6): d 170.03,
150.64, 149.56, 149.50, 149.01, 147.27, 143.16, 136.96, 133.63,
133.51, 132.37, 130.76, 130.28, 130.01, 129.19, 128.75, 127.86,
127.67, 126.56, 125.20, 124.06, 121.73, 120.79, 113.48, 36.77,
28.95; ESI-MS: m/z = 458 [M+H]+; mp 200–203 °C; HPLC:
4.1.5.1.
6-Bromo-4-((4-(2-cyanopropan-2-yl)phenyl)amino)
Light yellow solid; yield:
tR = 7.28 min, flow rate 1.0 mL/min, DiamonsilTM C18
4.6 ꢁ 200 mm, rt, eluent A-50%, eluent B-50%.
5l
quinoline-3-carboxamide (8a).
46%; 1H NMR (500 MHz, DMSO-d6): d 10.21 (s, 1H, NH), 8.94 (s,
1H, Ar-H), 8.16 (br s, 1H, NH), 8.00 (d, 2.0 Hz, 1H, Ar-H), 7.88 (d,
9.0 Hz, 1H, Ar-H), 7.82 (dd, 2.0 Hz, 9.0 Hz, 1H, Ar-H), 7.65 (br s,
1H, NH), 7.39 (d, 9.0 Hz, 2H, Ar-H), 7.01 (d, 9.0 Hz, 2H, Ar-H),
1.65 (s, 6H, CH3ꢁ2). ESI-MS: m/z = 409 [M+H]+.
4.1.6.3. 40-((4-(2-Cyanopropan-2-yl)phenyl)amino)-N-methyl-
[3,60-biquinoline]-30-carboxamide (9c).
Light yellow solid;
yield: 82%; 1H NMR (500 MHz, DMSO-d6): 9.94 (s, 1H, NH), 9.10
(d, 2.0 Hz, 1H, Ar-H), 8.78 (s, 1H, Ar-H), 8.61 (d, 2.0 Hz, 1H, Ar-H),
8.49 (d, 2.0 Hz, 1H, Ar-H), 8.44 (q, 4.5 Hz, 1H, NH), 8.26 (dd,
2.0 Hz, 9.0 Hz, 1H, Ar-H), 8.08 (d, 9.0 Hz, 1H, Ar-H), 8.05–8.00 (m,
2H, Ar-H), 7.79–7.75 (m, 1H, Ar-H), 7.67–7.62 (m, 1H, Ar-H), 7.44
(d, 9.0 Hz, 2H, Ar-H), 7.10 (d, 9.0 Hz, 2H, Ar-H), 2.52 (d, 4.5 Hz,
3H, N-CH3), 1.68 (s, 6H, CH3ꢁ2); 13C NMR (125 MHz, DMSO-d6):
d 167.78, 150.73, 149.84, 149.34, 147.34, 147.12, 142.83, 136.51,
134.03, 133.56, 132.52, 130.84, 130.23, 129.73, 129.21, 128.80,
127.96, 127.65, 126.18, 125.22, 123.22, 121.35, 121.32, 114.98,
36.73, 28.95, 26.31; ESI-MS: m/z = 472 [M+H]+; mp 248–250 °C;
4.1.5.2. 6-Bromo-4-((4-(2-cyanopropan-2-yl)phenyl)amino)-N-
methylquinoline-3-carboxamide (8b).
Light yellow solid;
yield: 89%; 1H NMR (500 MHz, DMSO-d6): d 9.63 (s, 1H, NH),
8.72 (s, 1H, Ar-H), 8.36 (q, 4.5 Hz, 1H, NH), 8.31 (d, 2.0 Hz, 1H,
Ar-H), 7.87 (d, 9.0 Hz, 1H, Ar-H), 7.84 (dd, 2.0 Hz, 9.0 Hz, 1H, Ar-
H), 7.36 (d, 9.0 Hz, 2H, Ar-H), 7.00 (d, 9.0 Hz, 2H, Ar-H), 2.42 (d,
4.5 Hz, 3H, Ar-H), 1.65 (s, 6H, CH3ꢁ2). ESI-MS: m/z = 423 [M+H]+.
4.1.5.3. 6-Bromo-4-((3-(trifluoromethyl)phenyl)amino)quino-
HPLC: tR = 7.63 min, flow rate 1.0 mL/min, DiamonsilTM C18 5
4.6 ꢁ 200 mm, rt, eluent A-50%, eluent B-50%.
l
line-3-carboxamide (8c).
Light yellow solid; yield: 52%; 1H
NMR (500 MHz, CDCl3): d 10.94 (s, 1H, NH), 9.53 (br s, 1H, NH),
9.01 (s, 1H, Ar-H), 7.93 (d, 9.0 Hz, 1H, Ar-H), 7.79–7.71 (m, 2H,
Ar-H), 7.45–7.38 (m, 2H, Ar-H), 7.27 (s, 1H, Ar-H), 7.16 (d, 7.5 Hz,
1H, Ar-H). ESI-MS: m/z = 410 [M+H]+.
4.1.6.4.
6-(6-Aminopyridin-3-yl)-4-((4-(2-cyanopropan-2-yl)
phenyl)amino)quinoline-3-carboxamide (9d).
Light yellow
solid; yield: 79%; 1H NMR (500 MHz, DMSO-d6): d 10.68 (s, 1H,
NH), 8.97 (s, 1H, Ar-H), 8.31 (s, 1H, Ar-H), 7.99–7.90 (m, 3H, Ar-
H), 7.76 (s, 2H, NHꢁ2), 7.48 (d, 8.5 Hz, 2H, Ar-H), 7.31 (dd,
2.0 Hz, 8.5 Hz, 1H, Ar-H), 7.11 (d, 8.5 Hz, 2H, Ar-H), 6.43 (d,
8.5 Hz, 1H, Ar-H), 6.18 (s, 2H, NHꢁ2), 1.72 (s, 6H, CH3ꢁ2); 13C
NMR (125 MHz, DMSO-d6): d 170.31, 159.83, 149.66, 148.94,
148.79, 146.59, 143.33, 136.73, 135.62, 134.78, 130.52, 129.05,
126.49, 125.20, 123.33, 121.72, 121.38, 120.65, 113.09, 108.35,
36.75, 28.95; ESI-MS: m/z = 423 [M+H]+; mp 266–267 °C; HPLC:
tR = 9.17 min, flow rate 0.8 mL/min, COSMOSIL 5C18-MS-II column
(4.6ID ꢁ 250 mm), rt, eluent A-80%, eluent B-20%.
4.1.6. General procedure for the preparation of target
compounds 9a–h (C)
To a solution of the intermediate 8a (or 8b, 8c) (1.0 equiv) in
H2O/1,4-dioxane (1:3, V/V) were added anhydrous potassium
carbonate (1.1 equiv), corresponding borate (1.1 equiv), such as
1-methyl-1H-pyrazole-5-boronic acid pinacol ester, quinoline-3-
boronic acid pinacol ester, 2-aminopyridine-5-boronic acid pinacol
ester or 2-methoxypyridine-5-boronic acid pinacol ester, and Pd
(Ph3P)4 (0.08 equiv). The resultant suspension was stirred at
100 °C for 8 h under N2 atmosphere. After this time, the reaction
mixture was cooled and concentrated in vacuo. The residue was
treated with EtOAc, and following filtration, the filtrate was
washed with water and brine. Afterwards, the organic layer was
dried over anhydrous Na2SO4 and evaporated in vacuo. The residue
was subjected to flash column chromatography using EA/MeOH/
TEA (160:3:2) as eluent, which afforded compounds 9a–h as light
yellow solid.
4.1.6.5.
phenyl)amino)-N-methylquinoline-3-carboxamide
(9e).
Light yellow solid; yield: 70%; 1H NMR (500 MHz,
6-(6-Aminopyridin-3-yl)-4-((4-(2-cyanopropan-2-yl)
DMSO-d6): 9.98 (s, 1H, NH), 8.74 (s, 1H, Ar-H), 8.50 (q, 4.5 Hz,
1H, NH), 8.10 (d, 2.0 Hz, 1H, Ar-H), 8.02–7.88 (m, 3H, Ar-H), 7.48
(dd, 2.0 Hz, 8.5 Hz, 1H, Ar-H), 7.42 (d, 9.0 Hz, 2H, Ar-H), 7.04 (d,
9.0 Hz, 2H, Ar-H), 6.46 (d, 8.5 Hz, 1H, Ar-H), 6.12 (s, 2H, NH),
2.56 (d, 4.5 Hz, 3H, N-CH3), 1.68 (s, 6H, CH3ꢁ2); 13C NMR
(125 MHz, DMSO-d6): d 167.52, 159.36, 149.01, 148.09, 146.50,
146.19, 142.54, 135.80, 135.26, 134.67, 129.93, 128.34, 125.68,
124.72, 122.94, 120.73, 120.69, 120.01, 114.11, 107.87, 36.20,
28.44, 25.88; ESI-MS: m/z = 437 [M+H]+; mp 215–218 °C; HPLC:
4.1.6.1. 4-((4-(2-Cyanopropan-2-yl)phenyl)amino)-6-(1-methyl-
1H-pyrazol-5-yl)quinoline-3-carboxamide (9a).
Light yel-
low solid; yield: 73%; 1H NMR (500 MHz, DMSO-d6): 10.63 (s, 1H,
NH), 9.04 (s, 1H, Ar-H), 8.33 (br s, 1H, NH), 8.01 (d, 9.0 Hz, 1H,

7592
X. Ma et al. / Bioorg. Med. Chem. 23 (2015) 7585–7596
tR = 7.23 min, flow rate 0.9 mL/min, COSMOSIL 5C18-MS-II column
(4.6ID ꢁ 250 mm), rt, eluent A-75%, eluent B-25%.
the filtrate was concentrated in vacuo to provide the 11 as a white
solid after recrystallization from EtOH. Yield: 87%. ESI-MS:
m/z = 346 [M+H]+; mp 140–141 °C.
4.1.6.6. 4-((4-(2-Cyanopropan-2-yl)phenyl)amino)-6-(6-meth-
oxypyridin-3-yl)quinoline-3-carboxamide (9f).
Light yel-
4.1.8. Ethyl 6-bromo-4-((4-(4-(tert-butoxycarbonyl)piperazin-1-
yl)-3-(trifluoromethyl)phenyl)amino)quinoline-3-carboxylate
(12)
low solid; yield: 74%; 1H NMR (500 MHz, DMSO-d6): 10.73 (s, 1H,
NH), 8.99 (s, 1H, Ar-H), 8.30 (br s, 1H, NH), 8.11 (d, 2.5 Hz, 1H,
Ar-H), 8.03 (dd, 1.5 Hz, 8.5 Hz, 1H, Ar-H), 8.00 (d, 8.5 Hz, 1H, Ar-
H), 7.86 (d, 1.5 Hz, 1H, Ar-H), 7.73 (br s, 1H, NH), 7.71 (dd,
2.5 Hz, 8.5 Hz, 1H, Ar-H), 7.50 (d, 8.5 Hz, 2H, Ar-H), 7.14 (d,
8.5 Hz, 2H, Ar-H), 6.84 (d, 8.5 Hz, 1H, Ar-H), 3.86 (s, 3H, OCH3),
1.73 (s, 6H, CH3ꢁ2); 13C NMR (125 MHz, DMSO-d6): d 169.75,
163.12, 149.80, 148.88, 148.55, 144.85, 142.68, 137.24, 136.58,
132.91, 130.18, 128.92, 128.40, 126.12, 124.73, 122.48, 121.57,
119.95, 112.34, 110.57, 53.21, 36.23, 28.34; ESI-MS: m/z = 438 [M
+H]+; mp 250–252 °C; HPLC: tR = 8.15 min, flow rate 1.0 mL/min,
The preparation of 12 is according to general procedure A via
the SNAr reaction between 6 and 11 in the presence of AcONa.
Light yellow solid; yield: 89%; 1H NMR (500 MHz, DMSO-d6):
9.68 (s, 1H, NH), 8.84 (s, 1H, Ar-H), 8.54 (d, 2.0 Hz, 1H, Ar-H),
7.95 (dd, 2.0 Hz, 9.0 Hz, 1H, Ar-H), 7.92 (d, 9.0 Hz, 1H, Ar-H), 7.53
(d, 8.5 Hz, 1H, Ar-H), 7.35 (d, 2.5 Hz, 1H, Ar-H), 7.27 (dd, 2.5 Hz,
8.5 Hz, 1H, Ar-H), 3.82 (q, 2.0 Hz, 2H, CH2), 3.51–3.37 (m, 4H, Piper-
azine-CH2ꢁ2), 2.77 (t, 4.5 Hz, 4H, Piperazine-CH2ꢁ2), 1.43 (s, 9H,
Boc-CH3ꢁ3), 1.01 (t, 2.0 Hz, 3H, CH3); ESI-MS: m/z = 623 [M+H]+.
DiamonsilTM C18 5
l
4.6 ꢁ 200 mm, rt, eluent A-60%, eluent B-40%.
4.1.9. General procedure for the preparation of intermediates
13a–c
4.1.6.7. 6-(6-Aminopyridin-3-yl)-4-((3-(trifluoromethyl)phenyl)
amino)quinoline-3-carboxamide (9g). Light yellow solid;
The synthetic procedure of intermediates 13a–c is similar to
general procedure B, except for the intermediate 13c. It was pre-
pared by treatment of 12 with cyclopropylamine (1 mL/100 mg
substrate) in the sealed tube at 80 °C for 24 h. Afterwards, the
excessive cyclopropylamine was removed in vacuo and the residue
was purified via flash column chromatography using EA/PE/TEA
(60:40:1–60:20:1) as eluent to afford 13c as light yellow solid.
yield: 67%; 1H NMR (500 MHz, DMSO-d6): 10.12 (s, 1H, NH), 8.89
(s, 1H, Ar-H), 8.18–8.10 (m, 2H, Ar-H), 8.09–7.96 (m, 3H, Ar-H,
NH), 7.64 (br s, 1H, NH), 7.63 (s, 1H, Ar-H), 7.48 (t, 8.0 Hz, 1H,
Ar-H), 7.37–7.30 (m, 2H), 7.25 (d, 8.0 Hz, 1H, Ar-H), 6.50 (d,
8.0 Hz, 1H, Ar-H), 6.20 (s, 2H, NH); 13C NMR (125 MHz, DMSO-
d6): d 169.45, 159.91, 149.97, 148.69, 148.64, 146.58, 146.21,
144.80, 135.83, 135.67, 130.55, 130.27 (JC–F 31.25 Hz), 129.20,
124.57 (JC–F 271.25 Hz), 123.39, 123.17, 121.69, 120.22, 118.68
(JC–F 2.50 Hz), 116.03, 115.91 (JC–F 2.50 Hz), 108.42; ESI-MS: m/
z = 424 [M+H]+; mp 275–277 °C; HPLC: tR = 7.32 min, flow rate
0.9 mL/min, COSMOSIL 5C18-MS-II column (4.6ID ꢁ 250 mm), rt,
eluent A-75%, eluent B-25%.
4.1.9.1. tert-Butyl 4-(4-((6-bromo-3-carbamoylquinolin-4-yl)
amino)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate
(13a).
Light yellow solid; yield: 53%; 1H NMR (500 MHz,
DMSO-d6): 9.91 (s, 1H, NH), 8.90 (s, 1H, Ar-H), 8.21 (d, 2.0 Hz,
1H, Ar-H), 8.08 (br s, 1H, NH), 7.92 (d, 9.0 Hz, 1H, Ar-H), 7.89 (dd,
2.0 Hz, 9.0 Hz, 1H, Ar-H), 7.61 (br s, 1H, NH), 7.46 (d, 8.5 Hz, 1H,
Ar-H), 7.29 (d, 3.0 Hz, 1H, Ar-H), 7.18 (dd, 3.0 Hz, 8.5 Hz, 1H, Ar-
H), 3.49–3.36 (m, 4H, Piperazine-CH2ꢁ2), 2.77 (t, 4.5 Hz, 4H, Piper-
azine-CH2ꢁ2), 1.43 (s, 9H, Boc-CH3ꢁ3); ESI-MS: m/z = 594 [M+H]+.
4.1.6.8.
line]-30-carboxamide (9h).
40-((3-(Trifluoromethyl)phenyl)amino)-[3,60-biquino-
Light yellow solid; yield: 76%;
1H NMR (500 MHz, CDCl3): 12.62 (br s, 1H, NH), 9.75 (s, 1H, Ar-
H), 8.74 (s, 1H, Ar-H), 8.51 (d, 8.5 Hz, 1H, Ar-H), 8.15 (d, 9.0 Hz,
1H, Ar-H), 8.12 (d, 9.0 Hz, 1H, Ar-H), 7.86 (s, 1H, Ar-H), 7.84 (s,
1H, Ar-H), 7.76–7.72 (m, 2H, Ar-H), 7.71 (d, 8.5 Hz, 1H, Ar-H),
7.66–7.62 (m, 1H, Ar-H), 7.60–7.58 (m, 1H, Ar-H), 7.56 (s, 1H, Ar-
H), 7.47 (d, 8.5 Hz, 1H, Ar-H); 13C NMR (125 MHz, DMSO-d6): d
168.73, 150.68, 149.29, 149.01, 148.97, 146.85, 145.87, 144.11,
133.94, 133.21, 131.97, 130.48, 130.12, 129.85, 129.79 (JC–F
32.50 Hz), 129.60, 128.69, 128.28, 127.47, 127.27, 123.82 (JC–F
275.00 Hz), 122.85, 122.62, 121.19, 118.46 (JC–F 3.75 Hz), 115.62;
ESI-MS: m/z = 459 [M+H]+; mp 212–215 °C; HPLC: tR = 10.36 min,
4.1.9.2. tert-Butyl 4-(4-((6-bromo-3-(methylcarbamoyl)quino-
lin-4-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-car-
boxylate (13b).
Light yellow solid; yield: 86%; 1H NMR
(500 MHz, DMSO-d6): 9.48 (s, 1H, NH), 8.66 (s, 1H, Ar-H), 8.52 (s,
1H, Ar-H), 8.38 (q, 4.5 Hz, NH), 7.90 (d, 1.0 Hz, 2H, Ar-H), 7.46 (d,
8.5 Hz, 1H, Ar-H), 7.25 (d, 2.5 Hz, 1H, Ar-H), 7.18 (dd, 2.5 Hz,
8.5 Hz, 1H, Ar-H), 3.49–3.39 (m, 4H, Piperazine-CH2ꢁ2), 2.77 (t,
4.5 Hz, 4H, Piperazine-CH2ꢁ2), 2.35 (d, 4.5 Hz, 3H, N-CH3), 1.43
(s, 9H, Boc-CH3ꢁ3); ESI-MS: m/z = 608 [M+H]+.
flow rate 1.0 mL/min, DiamonsilTM C18 5
A-60%, eluent B-40%.
l
4.6 ꢁ 200 mm, rt, eluent
4.1.9.3. tert-Butyl 4-(4-((6-bromo-3-(cyclopropylcarbamoyl)
quinolin-4-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-
4.1.7. tert-Butyl 4-(4-amino-2-(trifluoromethyl)phenyl)
carboxylate (13c).
Light yellow solid; yield: 57%; 1H NMR
piperazine-1-carboxylate (11)
(500 MHz, DMSO-d6): 9.33 (s, 1H, NH), 8.66 (s, 1H, Ar-H), 8.59 (t,
1.0 Hz, 1H, Ar-H), 8.43 (d, 3.5 Hz, 1H, NH), 7.91 (d, 1.0 Hz, 2H, Ar-
H), 7.46 (d, 8.5 Hz, 1H, Ar-H), 7.23 (d, 2.5 Hz, 1H, Ar-H), 7.13 (dd,
2.5 Hz, 8.5 Hz, 1H, Ar-H), 3.49–3.38 (m, 4H, Piperazine-CH2ꢁ2),
2.77 (t, 4.5 Hz, 4H, Piperazine-CH2ꢁ2), 2.36–2.27 (m, 1H, N-CH),
1.43 (s, 9H, Boc-CH3ꢁ3), 0.60–0.54 (m, 2H, CHꢁ2), 0.48–0.42 (m,
2H, CHꢁ2); ESI-MS: m/z = 634 [M+H]+.
To a suspension of anhydrous potassium carbonate (7.30 g,
53.0 mmol, 1.2 equiv) and 1-chloro-4-nitro-2-(trifluoromethyl)
benzene 10 (10.0 g, 44.3 mmol, 1.0 equiv) in DMF (25 mL) was
added N-Boc-piperazine (8.23 g, 44.3 mmol, 1.0 equiv) at room
temperature. The resultant mixture was stirred at 40 °C for 12 h
and then evaporated in vacuo to remove the solvent. Afterwards,
the residue was treated with EtOAc and water. The organic layer
was washed with brine, and dried over anhydrous Na2SO4. Follow-
ing removal of EtOAc in vacuo, the residue slowly solidified to give
tert-butyl 4-(4-nitro-2-(trifluoromethyl)phenyl)piperazine-1-car-
boxylate as light yellow solid. Yield: 92%, ESI-MS: m/z = 376 [M
+H]+. The resultant product (6.11 g, 16.3 mmol) and 10% Pd/C
(10% of the substrate, w/w) were shacked in methanol (100 mL)
and the suspension was stirred at room temperature under H2 (bal-
loon) atmosphere overnight. After this time, Pd/C was filtered and
4.1.10. General procedure for the preparation of intermediates
14a–e
The synthetic procedure of intermediates 14a–e is according to
general procedure C.
4.1.10.1. tert-Butyl 4-(4-((30-carbamoyl-[3,60-biquinolin]-40-yl)
amino)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate
(14a).
Light yellow solid; yield: 72%; 1H NMR (500 MHz,

X. Ma et al. / Bioorg. Med. Chem. 23 (2015) 7585–7596
7593
DMSO-d6): 10.28 (s, 1H, NH), 9.07 (d, 2.5 Hz, 1H, Ar-H), 8.95 (s, 1H,
Ar-H), 8.59 (d, 1.5 Hz, 1H, Ar-H), 8.35 (d, 1.0 Hz, 1H, Ar-H), 8.29 (dd,
1.5 Hz, 8.5 Hz, 1H, Ar-H), 8.17 (br s, 1H, NH), 8.13 (d, 8.5 Hz, 1H,
Ar-H), 8.06 (d, 8.0 Hz, 1H, Ar-H), 8.02 (d, 8.5 Hz, 1H, Ar-H), 7.83–
7.77 (m, 1H, Ar-H), 7.70–7.66 (m, 1H, Ar-H), 7.65 (br s, 1H, NH),
7.52 (d, 8.5 Hz, 1H, Ar-H), 7.41 (d, 2.5 Hz, 1H, Ar-H), 7.29 (dd,
2.5 Hz, 8.5 Hz, 1H, Ar-H), 3.48–3.38 (m, 4H, Piperazine-CH2ꢁ2),
2.79 (t, 4.5 Hz, 4H, Piperazine-CH2ꢁ2), 1.43 (s, 9H, Boc-CH3ꢁ3);
ESI-MS: m/z = 643 [M+H]+.
this process, a little hydrochloride of the Boc-deprotected product
was precipitated and the suspension was stirred at the room tem-
perature for 3 h. After removal of solvent, the resultant solid was
washed with diethyl ether and dried in vacuo to afford target com-
pound 15a (or 15b–d) or the intermediate 15e.
4.1.11.1. Hydrochloride of 40-((4-(piperazin-1-yl)-3-(trifluo-
romethyl)phenyl)amino)-[3,60-biquinoline]-30-carboxamide
(15a).
Yellow solid; yield: 97%; 1H NMR (500 MHz, DMSO-d6):
12.11 (s, 1H, HCl), 9.45 (s, 1H, Piperazine-NH), 9.31 (s, 2H, NH, Ar-
H), 9.15 (s, 1H, Ar-H), 9.10 (s, 1H, Ar-H), 8.98 (s, 1H, Ar-H), 8.64 (d,
9.0 Hz, 1H, Ar-H), 8.30 (d, 9.0 Hz, 1H, Ar-H), 8.28 (br s, 1H, NH),
8.18 (d, 8.0 Hz, 1H, Ar-H), 8.15 (d, 8.0 Hz, 1H, Ar-H), 7.94–7.91
(m, 1H, Ar-H), 7.81–7.78 (m, 1H, Ar-H), 7.74 (s, 1H, Ar-H), 7.72–
7.68 (m, 2H, Ar-H, NH), 7.60 (d, 8.5 Hz, 1H, Ar-H), 3.25–3.17 (m,
4H, Piperazine-CH2ꢁ2), 3.16–3.08 (m, 4H, Piperazine-CH2ꢁ2);
ESI-MS: m/z = 543 [M+H]+; mp 238–240 °C; HPLC: tR = 10.96 min,
4.1.10.2. tert-Butyl 4-(4-((30-(methylcarbamoyl)-[3,60-biquino-
lin]-40-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-car-
boxylate (14b).
Light yellow solid; yield: 79%; 1H NMR
(500 MHz, DMSO-d6): 9.70 (s, 1H, NH), 9.34 (d, 2.0 Hz, 1H, Ar-H),
8.76 (d, 2.0 Hz, 1H, Ar-H), 8.72 (s, 1H, Ar-H), 8.70 (s, 1H, Ar-H),
8.43 (q, 4.5 Hz, 1H, Ar-H), 8.32 (dd, 1.5 Hz, 8.5 Hz, 1H, Ar-H), 8.12
(d, 8.5 Hz, 1H, Ar-H), 8.10–8.04 (m, 2H, Ar-H), 7.84–7.77 (m, 1H,
Ar-H), 7.72–7.66 (m, 1H, Ar-H), 7.51 (d, 8.5 Hz, 1H, Ar-H), 7.34 (d,
2.5 Hz, 1H, Ar-H), 7.27 (dd, 2.5 Hz, 8.5 Hz, 1H, Ar-H), 3.51–3.38
(m, 4H, Piperazine-CH2ꢁ2), 2.79 (t, 4.5 Hz, 4H, Piperazine-
CH2ꢁ2), 2.40 (d, 4.5 Hz, 3H, CH3), 1.43 (s, 9H, Boc-CH3ꢁ3); ESI-
MS: m/z = 657 [M+H]+.
flow
rate
0.8 mL/min,
COSMOSIL
5C18-MS-II
column
(4.6ID ꢁ 250 mm), rt, eluent C-60%, eluent B-40%.
4.1.11.2. Hydrochloride of N-methyl-40-((4-(piperazin-1-yl)-3-
(trifluoromethyl)phenyl)amino)-[3,60-biquinoline]-30-carbox-
amide (15b).
Light yellow solid; yield: 93%; 1H NMR
4.1.10.3.
biquinolin]-40-yl)amino)-2-(trifluoromethyl)phenyl)piper-
azine-1-carboxylate (14c).
Light yellow solid; yield: 65%; 1H
tert-Butyl
4-(4-((30-(cyclopropylcarbamoyl)-[3,60-
(500 MHz, DMSO-d6): 12.10 (s, 1H, HCl), 10.01 (s, 1H, Piperazine-
NH), 9.85 (s, 1H, NH), 9.77 (s, 1H, Ar-H), 9.60 (s, 2H, Ar-H), 8.78
(s, 1H, Ar-H), 8.69–8.64 (m, 2H, NH), 8.27 (d, 7.5 Hz, 2H, Ar-H),
8.22 (d, 7.5 Hz, 1H, Ar-H), 8.06–7.95 (m, 1H, Ar-H), 7.92–7.80 (m,
1H, Ar-H), 7.75–7.62 (m, 2H, Ar-H), 7.54 (d, 7.5 Hz, 1H, Ar-H),
3.27–2.97 (m, 8H, Piperazine-H), 2.25 (s, 3H); 13C NMR
(125 MHz, DMSO-d6): d 164.02, 152.32, 148.93, 146.23, 144.39,
140.72, 140.55, 138.21, 137.17, 134.06, 133.76, 132.89, 131.62,
129.71, 129.64, 129.06, 128.44, 126.25 (JC–F 27.50 Hz), 125.28,
124.14, 123.62, 123.96 (JC–F 272.50 Hz), 122.54, 121.59, 120.17,
113.06, 50.28, 43.81, 25.84; ESI-MS: m/z = 557 [M+H]+; mp 267–
270 °C; HPLC: tR = 11.50 min, flow rate 0.8 mL/min, COSMOSIL
5C18-MS-II column (4.6ID ꢁ 250 mm), rt, eluent C-60%, eluent
B-40%.
NMR (500 MHz, DMSO-d6): 9.51 (s, 1H, NH), 9.39 (d, 2.0 Hz, 1H, Ar-
H), 8.79 (d, 2.0 Hz, 1H, Ar-H), 8.78 (d, 2.5 Hz, 1H, Ar-H), 8.71 (s, 1H,
Ar-H), 8.46 (d, 3.5 Hz, 1H, NH), 8.33 (dd, 2.0 Hz, 8.5 Hz, 1H, Ar-H),
8.13 (d, 8.5 Hz, 1H, Ar-H), 8.10–8.07 (m, 2H, Ar-H), 7.84–7.78 (m,
1H, Ar-H), 7.72–7.66 (m, 1H, Ar-H), 7.50 (d, 8.5 Hz, 1H, Ar-H),
7.32 (d, 2.5 Hz, 1H, Ar-H), 7.22 (dd, 2.5 Hz, 8.5 Hz, 1H, Ar-H),
3.49–3.39 (m, 4H, Piperazine-CH2ꢁ2), 2.79 (t, 4.5 Hz, 4H, Piper-
azine-CH2ꢁ2), 2.42–2.35 (m, 1H, N-CH), 1.44 (s, 9H, Boc-CH3ꢁ3),
0.52–0.43 (m, 2H, CHꢁ2), 0.12–0.05 (m, 2H, CHꢁ2); ESI-MS: m/
z = 683 [M+H]+.
4.1.10.4. tert-Butyl 4-(4-((3-carbamoyl-6-(6-methoxypyridin-3-
yl)quinolin-4-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-
1-carboxylate (14d).
4.1.11.3. Hydrochloride of N-cyclopropyl-40-((4-(piperazin-1-yl)-
Light yellow solid; yield: 81%; 1H NMR
3-(trifluoromethyl)phenyl)amino)-[3,60-biquinoline]-30-carbox-
(500 MHz, DMSO-d6): 10.35 (s, 1H, NH), 8.94 (s, 1H, Ar-H), 8.23
(d, 2.0 Hz, 1H, Ar-H), 8.19 (br s, 1H, NH), 8.06 (dd, 2.0 Hz, 8.5 Hz,
1H, Ar-H), 8.04 (s, 1H, Ar-H), 8.02–8.00 (m, 1H, Ar-H), 7.89 (dd,
2.5 Hz, 8.5 Hz, 1H, Ar-H), 7.67 (br s, 1H, NH), 7.54 (d, 8.5 Hz, 1H,
Ar-H), 7.36 (d, 2.5 Hz, 1H, Ar-H), 7.28 (dd, 2.5 Hz, 8.5 Hz, 1H, Ar-
H), 6.90 (d, 8.5 Hz, 1H, Ar-H), 3.89 (s, 3H, OCH3), 3.49–3.39 (m,
4H, Piperazine-CH2ꢁ2), 2.82 (t, 4.5 Hz, 4H, Piperazine-CH2ꢁ2),
1.43 (s, 9H, Boc-CH3ꢁ3); ESI-MS: m/z = 623 [M+H]+.
amide (15c).
Yellow solid; yield: 96%; 1H NMR (500 MHz,
DMSO-d6): 11.79 (s, 1H, HCl), 9.77 (s, 1H, Piperazine-NH), 9.65 (s,
1H, NH), 9.34 (br s, 1H, NH), 9.29 (s, 2H, Ar-H), 8.81 (s, 1H, Ar-H),
8.75 (d, 3.5 Hz, 1H, Ar-H), 8.69 (d, 8.5 Hz, 1H, Ar-H), 8.30 (d,
8.5 Hz, 1H, Ar-H), 8.23–8.19 (m, 2H, Ar-H), 7.97–7.94 (m, 1H, Ar-
H), 7.84–7.80 (m, 1H, Ar-H), 7.68–7.53 (m, 3H, Ar-H), 3.24–3.16
(m, 4H, Piperazine-CH2ꢁ2), 3.15–3.04 (m, 4H, Piperazine-CH2ꢁ2),
2.24–2.15 (m, 1H, CH), 0.53–0.46 (m, 2H, CH2), 0.23–0.14 (m, 2H,
CH2); 13C NMR (125 MHz, DMSO-d6): d 164.54, 152.33, 148.85,
147.49, 144.78, 138.76 (JC–F 6.25 Hz), 138.31, 137.60, 134.98,
133.15, 132.81, 131.62, 129.47, 129.16, 128.56, 128.41, 126.59
(JC–F 27.50 Hz), 125.62, 125.52, 123.97 (JC–F 271.25 Hz), 123.96,
122.40 (JC–F 6.25 Hz), 121.76, 121.37, 120.43, 113.12, 50.28,
43.85, 22.89, 5.59; ESI-MS: m/z = 583 [M+H]+; mp 257–259 °C;
HPLC: tR = 10.00 min, flow rate 0.8 mL/min, COSMOSIL 5C18-MS-
II column (4.6ID ꢁ 250 mm), rt, eluent C-50%, eluent B-50%.
4.1.10.5. tert-Butyl 4-(4-((3-carbamoyl-6-(1-methyl-1H-pyrazol-
5-yl)quinolin-4-yl)amino)-2-(trifluoromethyl)phenyl)piper-
azine-1-carboxylate (14e).
Light yellow solid; yield: 74%; 1H
NMR (500 MHz, DMSO-d6): 10.33 (s, 1H, NH), 9.02 (s, 1H, Ar-H),
8.27 (br s, 1H, NH), 8.07 (d, 8.5 Hz, 1H, Ar-H), 7.93 (d, 1.5 Hz, 1H,
Ar-H), 7.90 (dd, 1.5 Hz, 8.5 Hz, 1H, Ar-H), 7.73 (br s, 1H, NH),
7.49 (d, 8.5 Hz, 1H, Ar-H), 7.47 (d, 2.0 Hz, 1H, Ar-H), 7.34 (d,
2.5 Hz, 1H, Ar-H), 7.22 (dd, 2.5 Hz, 8.5 Hz, 1H, Ar-H), 6.31 (d,
2.0 Hz, 1H, Ar-H), 3.60 (s, 3H, Pyrazole N-CH3), 3.50–3.38 (m, 4H,
Piperazine-CH2ꢁ2), 2.77 (t, 4.5 Hz, 4H, Piperazine-CH2ꢁ2), 1.43
(s, 9H, Boc-CH3ꢁ3); ESI-MS: m/z = 596 [M+H]+.
4.1.11.4. Hydrochloride of 6-(6-methoxypyridin-3-yl)-4-((4-
(piperazin-1-yl)-3-(trifluoromethyl)phenyl)amino)quinoline-3-
carboxamide (15d).
Yellow solid; yield: 95%; 1H NMR
(500 MHz, DMSO-d6): 12.27 (s, 1H, HCl), 9.64 (s, 2H, Piperazine-
NH, NH (C-4 position of quinoline)), 8.99 (s, 1H, Ar-H), 8.70 (br s,
1H, NH), 8.43–8.35 (m, 3H, NH, Ar-H), 8.24 (d, 8.5 Hz, 1H, Ar-H),
8.15 (dd, 1.5 Hz, 8.5 Hz, 1H, Ar-H), 7.76–7.67 (m, 3H, Ar-H), 7.59
(d, 8.5 Hz, 1H, Ar-H), 6.94 (d, 8.5 Hz, 1H, Ar-H), 3.89 (s, 3H,
4.1.11. General procedure for the preparation of target
compounds 15a–d or the intermediate 15e (D)
To
a solution of intermediate 14a (or 14b–e) in EtOAc
(20 mL/1 mmol substrate) was added EtOAc, saturated with hydro-
gen chloride (10 mL/1 mmol substrate), dropwise at 0 °C. During

7594
X. Ma et al. / Bioorg. Med. Chem. 23 (2015) 7585–7596
OCH3), 3.27–3.09 (m, 8H, Piperazine-H); 13C NMR (125 MHz,
DMSO-d6): d 166.95, 164.06, 153.89, 149.24, 145.65, 143.48,
138.30, 138.03, 137.84, 135.76, 132.92, 129.32, 127.68, 126.64
(JC–F 28.75 Hz), 125.83, 123.91 (JC–F 271.25 Hz), 123.03, 122.67,
121.59, 119.52, 111.26, 110.77, 53.95, 50.33, 43.75; ESI-MS: m/
z = 523 [M+H]+; mp 266–269 °C; HPLC: tR = 8.25 min, flow rate
0.8 mL/min, COSMOSIL 5C18-MS-II column (4.6ID ꢁ 250 mm), rt,
eluent C-80%, eluent B-20%.
8.5 Hz, 1H, Ar-H), 3.60–3.49 (m, 4H, Piperazine-CH2ꢁ2), 2.84 (t,
4.5 Hz, 2H, Piperazine-CH2), 2.79 (t, 4.5 Hz, 2H, Piperazine-CH2),
2.05 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6): d 169.52,
168.80, 151.05, 149.69, 149.56, 147.31, 147.26, 146.80, 141.20,
134.10, 133.56, 132.44, 130.96, 130.29, 129.97, 129.23, 128.73,
127.92, 127.71, 127.06 (JC–F 27.50 Hz), 126.21, 124.88, 124.21
(JC–F 271.25 Hz), 123.39, 121.21, 118.70 (JC–F 5.00 Hz), 114.96,
53.93, 53.48, 46.75, 41.85,21.71; ESI-MS: m/z = 585 [M+H]+; mp
261–262 °C; HPLC: tR = 7.06 min, flow rate 1.0 mL/min, DiamonsilTM
4.1.11.5. Hydrochloride of 6-(1-methyl-1H-pyrazol-5-yl)-4-((4-
C18 5
l
4.6 ꢁ 200 mm, rt, eluent A-50%, eluent B-50%.
(piperazin-1-yl)-3-(trifluoromethyl)phenyl)amino)quinoline-3-
carboxamide (15e).
Light yellow solid; yield: 92%; 1H NMR
4.1.12.3.
40-((4-(4-(Methylsulfonyl)piperazin-1-yl)-3-(trifluo-
(500 MHz, DMSO-d6): 12.17 (s, 1H, HCl), 9.42 (s, 2H, NH, Piper-
azine-NH), 9.08 (s, 1H, Ar-H), 8.48 (s, 1H, NH), 8.42 (s, 1H, NH),
8.27 (d, 8.5 Hz, 1H, Ar-H), 8.21 (dd, 1.5 Hz, 8.5 Hz, 1H, Ar-H), 7.78
(s, 1H, Ar-H), 7.72 (d, 2.0 Hz, 1H, Ar-H), 7.67 (dd, 2.0 Hz, 8.5 Hz,
1H, Ar-H), 7.58 (d, 8.5 Hz, 1H, Ar-H), 7.53 (d, 1.5 Hz, 1H, Ar-H),
6.40 (d, 1.5 Hz, 1H, Ar-H), 3.81 (s, 3H, Pyrazole N-CH3), 3.22–3.17
(m, 4H, Piperazine-CH2ꢁ2), 3.12–3.07 (m, 4H, Piperazine-
CH2ꢁ2); ESI-MS: m/z = 496 [M+H]+.
romethyl)phenyl)amino)-[3,60-biquinoline]-30-carboxamide
(16c).
Light yellow solid; yield: 77%; 1H NMR (500 MHz,
DMSO-d6): 10.28 (s, 1H, NH), 9.06 (d, 2.5 Hz, 1H, Ar-H), 8.96 (s,
1H, Ar-H), 8.61 (d, 2.5 Hz, 1H, Ar-H), 8.36 (d, 2.0 Hz, 1H, Ar-H),
8.29 (dd, 2.0 Hz, 8.5 Hz, 1H, Ar-H), 8.16 (br s, 1H, NH), 8.13 (d,
8.5 Hz, 1H, Ar-H), 8.07 (d, 7.5 Hz, 1H, Ar-H), 8.02 (d, 7.5 Hz, 1H,
Ar-H), 7.82–7.78 (m, 1H, Ar-H), 7.70–7.66 (m, 1H, Ar-H), 7.64 (br
s, 1H, NH), 7.59 (d, 8.5 Hz, 1H, Ar-H), 7.41 (d, 2.5 Hz, 1H, Ar-H),
7.32 (dd, 2.5 Hz, 8.5 Hz, 1H, Ar-H), 3.28–3.17 (m, 4H, Piperazine-
CH2ꢁ2), 3.01–2.89 (m, 7H, Piperazine-CH2ꢁ2, Methylsulfonyl-
CH3); 13C NMR (125 MHz, DMSO-d6): d 169.44, 151.05, 149.72,
149.51, 147.33, 147.14, 146.48, 141.39, 134.17, 133.60, 132.45,
130.93, 130.31, 130.02, 129.24, 128.76, 127.94, 127.74, 127.08
(JC–F 27.50 Hz), 126.45, 124.82, 124.18 (JC–F 271.25 Hz), 123.36,
121.26, 118.59 (JC–F 6.25 Hz), 115.10, 53.11, 46.50, 34.40; ESI-MS:
m/z = 621 [M+H]+; mp 260–261 °C; HPLC: tR = 8.80 min, flow rate
4.1.12. General procedure for the preparation of target
compounds 16a–c (E)
Compounds 16a–c were prepared from 15a or 15e. To the
hydrochloride (1.0 equiv), anhydrous THF and Et3N (3.0 equiv)
were added. Subsequently, to the resultant suspension, acetyl chlo-
ride or mesyl chloride (1.2 equiv) in anhydrous THF was added
dropwise at 0 °C. The reaction mixture was stirred at the same
temperature for 1.5 h and concentrated in vacuo. The residue
was treated with DCM and saturated NaHCO3 solution, and the
organic layer was washed with brine. Following removal of DCM
in vacuo, the residue was subjected to flash column chromatogra-
phy with EA/MeOH/TEA (150:3:2, for the acylated product) or EA/
MeOH/TEA (300:3:3, for the mesylated product) as the eluent to
give target compounds 16a–c.
1.0 mL/min, DiamonsilTM C18 5
eluent B-40%.
l
4.6 ꢁ 200 mm, rt, eluent A-60%,
4.1.13. Synthetic procedure for the intermediates 17 and 18
To a solution of 13a (1.0 equiv) in DCM (10 mL/1 mmol sub-
strate) was added TFA (TFA/DCM = 1:4, V/V) dropwise at 0 °C.
The reaction mixture was then stirred at the room temperature
for 6 h and concentrated in vacuo. Afterwards, the resultant oil
and anhydrous Et3N (3.0 equiv) were dissolved in anhydrous THF
(10 mL/1 mmol substrate). To the solution, propionyl chloride
(1.2 equiv) in anhydrous THF (5 mL/1 mmol substrate) was added
dropwise at 0 °C. The reaction mixture was stirred at the same
temperature for 1.5 h and concentrated in vacuo. The residue
was dissolved in DCM, washed with saturated NaHCO3 solution
and brine. Following removal of the solvent in vacuo, the residue
was subjected to flash column chromatography with EA as the elu-
ent to afford 17 as a slight yellow solid. The preparation of 18 was
similar to that of 17, only with chloroacetyl chloride in place of
propionyl chloride. The eluent for the flash column chromatogra-
phy of 18 was EA/PE/TEA (200:100:3).
4.1.12.1.
fonyl)piperazin-1-yl)-3-(trifluoromethyl)phenyl)amino)quino-
line-3-carboxamide (16a).
Light yellow solid; yield: 72%; 1H
6-(1-Methyl-1H-pyrazol-5-yl)-4-((4-(4-(methylsul-
NMR (500 MHz, DMSO-d6): 10.32 (s, 1H, NH), 9.02 (s, 1H, Ar-H),
8.26 (br s, 1H, NH), 8.07 (d, 8.5 Hz, 1H, Ar-H), 7.93 (d, 1.5 Hz, 1H,
Ar-H), 7.90 (dd, 1.5 Hz, 8.5 Hz, 1H, Ar-H), 7.73 (br s, 1H, NH),
7.55 (d, 8.5 Hz, 1H, Ar-H), 7.47 (d, 1.5 Hz, 1H, Ar-H), 7.35 (d,
2.5 Hz, 1H, Ar-H), 7.24 (dd, 2.5 Hz, 8.5 Hz, 1H, Ar-H), 6.31 (d,
1.5 Hz, 1H, Ar-H), 3.61 (s, 3H, Pyrazole N-CH3), 3.29–3.18 (m, 4H,
Piperazine-CH2ꢁ2), 2.96 (s, 3H, Methylsulfonyl), 2.91 (t, 4.5 Hz,
4H, Piperazine-CH2ꢁ2); 13C NMR (125 MHz, DMSO-d6): d 168.93,
150.45, 148.83, 147.21, 145.90, 141.76, 141.15, 137.92, 130.60,
130.08, 126.77, 126.45 (JC–F 27.50 Hz), 126.08, 124.28, 123.31
(JC–F 283.75 Hz), 120.37, 117.97 (JC–F 5.00 Hz), 114.87, 114.73,
106.31, 52.44, 45.97, 37.11, 33.76; ESI-MS: m/z = 574 [M+H]+; mp
246–248 °C; HPLC: tR = 10.15 min, flow rate 0.9 mL/min, COSMOSIL
5C18-MS-II column (4.6ID ꢁ 250 mm), rt, eluent A-75%, eluent
B-25%.
4.1.13.1. 6-Bromo-4-((4-(4-propionylpiperazin-1-yl)-3-(trifluo-
romethyl)phenyl)amino)quinoline-3-carboxamide
(17).
Light yellow solid; yield: 74% for two steps; ESI-MS:
m/z = 550 [M+H]+.
4.1.13.2.
6-Bromo-4-((4-(4-(2-chloroacetyl)piperazin-1-yl)-3-
4.1.12.2. 40-((4-(4-Acetylpiperazin-1-yl)-3-(trifluoromethyl)phe-
(trifluoromethyl)phenyl)amino)quinoline-3-carboxamide
(18).
nyl)amino)-[3,60-biquinoline]-30-carboxamide (16b).
Light
Light yellow solid; yield: 68% for two steps; 1H NMR
yellow solid; yield: 84%; 1H NMR (500 MHz, DMSO-d6): 10.29 (br
s, 1H, NH), 9.06 (d, 2.0 Hz, 1H, Ar-H), 8.96 (s, 1H, Ar-H), 8.59 (d,
2.0 Hz, 1H, Ar-H), 8.36 (d, 2.0 Hz, 1H, Ar-H), 8.29 (dd, 2.0 Hz,
8.5 Hz, 1H, Ar-H), 8.17 (br s, 1H, NH), 8.13 (d, 8.5 Hz, 1H, Ar-H),
8.07 (d, 7.5 Hz, 1H, Ar-H), 8.02 (d, 7.5 Hz, 1H, Ar-H), 7.82–7.78
(m, 1H, Ar-H), 7.70–7.66 (m, 1H, Ar-H), 7.65 (br s, 1H, NH), 7.51
(d, 8.5 Hz, 1H, Ar-H), 7.41 (d, 2.5 Hz, 1H, Ar-H), 7.30 (dd, 2.5 Hz,
(500 MHz, DMSO-d6): 9.94 (br s, 1H, NH), 8.90 (s, 1H, Ar-H), 8.23
(d, 2.0 Hz, 1H, Ar-H), 8.08 (br s, 1H, NH), 7.92 (d, 9.0 Hz, 1H, Ar-
H), 7.89 (dd, 2.0 Hz, 9.0 Hz, 1H, Ar-H), 7.61 (br s, 1H, NH), 7.45
(d, 8.5 Hz, 1H, Ar-H), 7.31 (d, 2.5 Hz, 1H, Ar-H), 7.19 (dd, 2.5 Hz,
8.5 Hz, 1H, Ar-H), 4.44 (s, 2H, Ar-H), 3.65–3.48 (m, 4H, Piper-
azine-CH2ꢁ2), 2.85 (t, 4.0 Hz, 2H, Piperazine-CH2), 2.81 (t, 4.0 Hz,
2H, Piperazine-CH2); ESI-MS: m/z = 570 [M+H]+.

X. Ma et al. / Bioorg. Med. Chem. 23 (2015) 7585–7596
7595
4.1.14. 6-(6-Aminopyridin-3-yl)-4-((4-(4-propionylpiperazin-1-
yl)-3-(trifluoromethyl)phenyl)amino)quinoline-3-carboxamide
(19)
The synthetic procedure for compound 19 is according to that of
9a–h. The eluent for the flash column chromatography of 19 was
EA/MeOH/TEA (160:3:2).
well of the assay plate. The reaction mixture was incubated at
room temperature for 1 h, and then stopped by kinase quench buf-
fer containing EDTA and Eu-anti-phospho-4E-BP1 antibody. Before
reading on a plate reader, the mixture need be mixed briefly with
centrifuge and was allowed to equilibrate for 60 min. The inhibi-
tion rate was calculated as (max ꢀ Lance signal)/(max ꢀ min) ꢁ
100%. Herein, ‘max’ stands for DMSO control, while ‘min’ stands
for Lance signal of no enzyme control. Finally, data was presented
in MS Excel and the curves fitted by Graphpad 5.0. Equation used
was: Y = Bottom + (Top ꢀ Bottom)/(1 + (IC50/X)^HillSlope).
Light yellow solid; yield: 59%; 1H NMR (500 MHz, DMSO-d6):
10.34 (br s, 1H, NH), 8.91 (s, 1H, Ar-H), 8.20 (br s, 1H, NH), 8.06
(d, 2.5 Hz, 1H, Ar-H), 8.01–7.95 (m, 2H, Ar-H), 7.88 (s, 1H, Ar-H),
7.68 (br s, 1H, NH), 7.51 (d, 8.5 Hz, 1H, Ar-H), 7.47 (dd, 2.5 Hz,
8.5 Hz, 1H, Ar-H), 7.36 (d, 2.5 Hz, 1H, Ar-H), 7.24 (dd, 2.5 Hz,
8.5 Hz, 1H, Ar-H), 6.46 (d, 8.5 Hz, 1H, Ar-H), 6.19 (s, 2H, NH),
3.65–3.48 (m, 4H, Piperazine-CH2ꢁ2), 2.84 (t, 4.5 Hz, 2H, Piper-
azine-CH2), 2.80 (t, 4.5 Hz, 2H, Piperazine-CH2), 2.37 (q, 7.5 Hz,
2H, CH2), 1.02 (t, 7.5 Hz, 3H, CH3); ESI-MS: m/z = 564 [M+H]+; mp
224–226 °C; HPLC: tR = 7.11 min, flow rate 0.9 mL/min, COSMOSIL
5C18-MS-II column (4.6ID ꢁ 250 mm), rt, eluent A-70%, eluent
B-30%.
4.3. Cell proliferation assay
Cell proliferation was evaluated by sulforhodamine B (SRB)
assay. In detail, HCT116, PC3 and MCF-7 cells were seeded into
96-well plates, cultured overnight and then exposed to serial con-
centrations of compounds for 72 h. Subsequently, cells were
washed with PBS and fixed with 10% (w/v) trichloroacetic acid at
4 °C for 1 h. Afterwards, the cells were stained for 30 min with
0.4% SRB dissolved in 1% acetic acid. Then the cells were washed
by 1% acetic acid for 5 times, and protein-bound dye was extracted
with 10 mmol unbuffered Tris base. The absorbance was measured
at 515 nm using a multiscan spectrum (Thermo Electron Co., Van-
taa, Finland). The inhibition rate on proliferation of each well was
calculated as (A515 control cells ꢀ A515 treated cells)/A515 con-
trol cells ꢁ 100%. The average IC50 values were determined by Logit
method from at least two independent tests.
4.1.15. 40-((4-(4-(2-(4-Methylpiperazin-1-yl)acetyl)piperazin-1-yl)-
3-(trifluoromethyl)phenyl)amino)-[3,60-biquinoline]-30-carbox-
amide (20)
To
a solution of 18 (1.0 equiv) in anhydrous acetonitrile
(10 mL/1 mmol substrate) was added N-Methyl piperazine
(1.1 equiv) and K2CO3 (1.5 equiv). The reaction mixture was
allowed to reflux under N2 atmosphere for 4 h. Afterwards, the sol-
vent was removed in vacuo and the residue was dissolved in DCM,
washed with water and brine. Following removal of DCM, the resi-
due was used for the next step without purification. Subsequent
Suzuki coupling of the rude product with quinoline-3-boronic acid
pinacol ester was carried out according to the general procedure C.
The eluent for the flash column chromatography of 20 was
EA/MeOH/TEA (20:2:1).
4.4. Class I PI3Ks selectivity assay
PI3K
cent assay. PI3K
a
inhibitory activity was assessed by Kinase-Glo Lumines-
was purchased from Invitrogen and ATP, DMSO,
a
as well as EDTA were purchased from Sigma. The kinase buffer
contained 50 mM HEPES (pH 7.5), 3 mM MgCl2, 1 mM EDTA,
100 mM NaCl, 0.03% CHAPS and 2 mM DTT. The kinase solution
was prepared by dissolving the kinase in the kinase buffer and
the substrate solution was prepared by dissolving PIP2 and ATP
in the kinase reaction buffer. Compound solution, kinase solution
and substrate solution were added successively to the well of the
assay plate. The reaction mixture was incubated at room tempera-
ture for 1 h, and then stopped by Kinase-Glo reagent. Before read-
ing on a plate reader for luminescence, the mixture need be mixed
briefly with centrifuge and was allowed to equilibrate for 15 min.
The percent inhibition was calculated as 100 ꢀ (max ꢀ sample
RLU)/(max ꢀ min) ꢁ 100. Herein, ‘max’ stands for the RLU of no
enzyme control, while ‘min’ means the RLU of DMSO control.
Finally, data was presented in MS Excel and the curves fitted by
Graphpad 5.0. Equation used was: Y = Bottom + (Top ꢀ Bottom)/
(1 + (IC50/X)^HillSlope).
PI3Kb inhibitory activity was assessed by ADP-Glo Luminescent
assay. PI3Kb was purchased from Millipore and ATP, DMSO, as well
as EDTA were purchased from Sigma. The kinase reaction mixture
was incubated at room temperature for 1 h. Afterwards, a little
amount of reaction mixture was transferred to a new 384 plate
and equal amount of ADP-Glo reagent was added to each well of
the new assay plate to stop the reaction. After being mixed briefly
with centrifuge, the resultant mixture was shaken slowly on the
shaker and equilibrated for 40 min. Kinase detection reagent was
then added to each wells. The mixture was shaken 1 min, equili-
brated for 60 min before reading on a plate reader for lumines-
cence. The percent inhibition was calculated as (max ꢀ sample
RLU)/(max ꢀ min) ꢁ 100. Herein, ‘max’ stands for the RLU of DMSO
Light yellow solid; yield: 48% for two steps; 1H NMR (500 MHz,
DMSO-d6): 10.30 (s, 1H, NH), 9.06 (s, 1H, Ar-H), 8.96 (s, 1H, Ar-H),
8.61 (s, 1H, Ar-H), 8.37 (s, 1H, Ar-H), 8.30 (d, 8.5 Hz, 1H, Ar-H), 8.19
(br s, 1H, NH), 8.13 (d, 8.0 Hz, 1H, Ar-H), 8.06 (d, 8.0 Hz, 1H, Ar-H),
8.02 (d, 8.0 Hz, 1H, Ar-H), 7.81–7.78 (m, 1H, Ar-H), 7.73–7.58 (m,
2H, Ar-H, NH), 7.49 (d, 8.5 Hz, 1H, Ar-H), 7.43 (s, 1H, Ar-H), 7.30
(d, 8.5 Hz, 1H, Ar-H), 3.69–3.50 (m, 4H, Piperazine-CH2ꢁ2), 3.29
(s, 2H, CH2), 3.13–2.96 (m, 2H, Piperazine-CH2), 2.88–2.77 (m,
4H, Piperazine-CH2ꢁ2), 2.73–2.56 (m, 6H, Piperazine-CH2ꢁ3),
2.36 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6): d 169.53,
167.72, 151.04, 149.67, 149.62, 147.40, 147.35, 146.81, 141.26,
134.10, 133.57, 132.46, 130.99, 130.28, 129.98, 129.24, 128.74,
127.95, 127.71, 127.07 (JC–F 27.50 Hz), 126.14, 124.97, 124.23
(JC–F 272.50 Hz), 123.49, 121.23, 118.81, 114.98, 60.21, 54.23,
53.53, 51.46, 46.16, 44.67; ESI-MS: m/z = 683 [M+H]+; mp 211–
213 °C; HPLC: tR = 11.99 min, flow rate 0.8 mL/min, COSMOSIL
5C18-MS-II column (4.6ID ꢁ 250 mm), rt, eluent C-80%, eluent B-20%.
4.2. mTOR inhibition assay
mTOR inhibitory activity was assessed by lance ultra assay.
mTOR was purchased from Millipore and ATP, DMSO, as well as
EDTA were purchased from Sigma. The kinase buffer contained
50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EDTA, 3 mM MnCl2,
0.01% Tween-20 and 2 mM DTT. The kinase solution was prepared
by dissolving the kinase in the kinase buffer and the substrate solu-
tion was prepared by dissolving ULight-4E-BP1 peptide substrate
and ATP in the kinase reaction buffer. Compound solution, kinase
solution and substrate solution were added successively to the

7596
X. Ma et al. / Bioorg. Med. Chem. 23 (2015) 7585–7596
control, while ‘min’ means the RLU of no enzyme control. Finally,
data was presented in MS Excel and the curves fitted by Graphpad
5.0. Equation used was: Y = Bottom + (Top ꢀ Bottom)/(1 + (IC50/X)
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The co-crystal structure of mTOR in complex with Torin 2 (PDB
code 4JSX) was used for the docking calculation in C-DOCKER mod-
ule (Discovery Studio, version 2.5; Accelrys, San Diego, CA, USA,
2008). After removal of Torin 2 and solvent molecules, the
CHARMm-force field was applied to the protein. The active site
was determined according to the location of Torin 2 in mTOR
enzyme and each ligand was docked into the defined site. The final
binding conformations of them were determined based on the
calculated CDOCKING ENERGE.
Acknowledgments
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N.; Jiang, J.; Kirk, C. J.; Laidig, G. J.; Lewis, E. R.; Lu, Y.; Muchamuel, T.; Parlati, F.;
Ring, E.; Shenk, K. D.; Shields, J.; Shwonek, P. J.; Stanton, T.; Sun, C. M.; Sylvain,
C.; Woo, T. M.; Yang, J. J. Med. Chem. 2009, 52, 3028.
The authors thank Jianyang Pan (Institute of Pharmaceutical
Informatics, Zhejiang University, China) for performing NMR spec-
trometry and mass spectrometry for structural characterization.