Novel quinoline-derived mTOR inhibitors with remarkable enzymatic and cellular activities: Design, synthesis and biological evaluation

Article abstract of DOI:10.1039/c5md00401b

Herein, we reported the preparation and in vitro development of a novel series of quinoline-based mTOR inhibitors, some of which were obtained via introducing a ring-opening strategy. As for enzymatic activity, more than half of these quinoline derivatives exhibited moderate to potent inhibition against mTOR. Among them, six compounds showed IC₅₀ values below 50 nM. In particular, several quinolines exhibited remarkably enhanced anti-proliferative activities against all the three tested tumor cell lines in contrast to the initial lead 9. As a representative in this series, compound 24 demonstrated IC₅₀ values of 0.11, 0.17 and 0.04 μM against HCT-116, PC-3 and MCF-7 cell lines, respectively. Besides, compounds 17 and 24 were identified to be selective over class I PI3Ks. Further Western blot analysis validated the dual inhibition of mTORC1 and mTORC2 as a result of compound 24 treatment in the MCF-7 cell line, which was beneficial for conquering the S6K/IRS1/PI3K negative feedback loop. Moreover, acceptable stability was displayed by compound 17, another representative of this series, in simulated intestinal fluid (SIF), simulated gastric fluid (SGF), as well as rat liver microsome (RLM). By virtue of the favorable biological profiles, several quinolines merit further in vivo investigation.

Full text of DOI:10.1039/c5md00401b

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RESEARCH ARTICLE
Novel quinoline-derived mTOR inhibitors with
remarkable enzymatic and cellular activities:
design, synthesis and biological evaluation
Cite this: DOI: 10.1039/c5md00401b
*
Xiao-Dong Ma, Ni Qiu, Bo Yang, Qiao-Jun He and Yong-Zhou Hu
Herein, we reported the preparation and in vitro development of a novel series of quinoline-based mTOR
inhibitors, some of which were obtained via introducing a ring-opening strategy. As for enzymatic activity,
more than half of these quinoline derivatives exhibited moderate to potent inhibition against mTOR.
Among them, six compounds showed IC50 values below 50 nM. In particular, several quinolines exhibited
remarkably enhanced anti-proliferative activities against all the three tested tumor cell lines in contrast to
the initial lead 9. As a representative in this series, compound 24 demonstrated IC50 values of 0.11, 0.17 and
0.04 μM against HCT-116, PC-3 and MCF-7 cell lines, respectively. Besides, compounds 17 and 24 were
identified to be selective over class I PI3Ks. Further Western blot analysis validated the dual inhibition of
mTORC1 and mTORC2 as a result of compound 24 treatment in the MCF-7 cell line, which was beneficial
for conquering the S6K/IRS1/PI3K negative feedback loop. Moreover, acceptable stability was displayed by
compound 17, another representative of this series, in simulated intestinal fluid (SIF), simulated gastric fluid
(SGF), as well as rat liver microsome (RLM). By virtue of the favorable biological profiles, several quinolines
merit further in vivo investigation.
Received 12th September 2015,
Accepted 25th November 2015
DOI: 10.1039/c5md00401b
www.rsc.org/medchemcomm
only resulted in a partial suppression of mTOR in an allo-
steric manner. Another merit of ATP-competitive inhibitors
1. Introduction
The mammalian target of rapamycin (mTOR) is a critical inte-
grator of extra- and intra-cellular signals that regulates diverse
growth-related processes, exemplified by translation, metabo-
lism, ribosome biogenesis and proliferation.1–5 In cells,
mTOR assembles into two distinct multi-protein complexes:
mTORC1 and mTORC2.6 Functionally, mTORC1 serves as the
downstream regulator of Akt, while mTORC2 emerges
upstream of Akt, activating it upon phosphorylation at
Ser473.7 As a drug discovery target, mTOR has inspired con-
siderable scientific interest, due to its frequent deregulation
in oncology.8–10 Despite intensified development efforts,
mTORC1 selective inhibitors, including rapamycin and its
semi-synthetic analogs (rapalogues),11,12 merely benefit
patients with advanced and metastatic renal cell carcinoma
(RCC).
stems from the dual inhibition of mTORC1 and mTORC2.
In a myriad of cancers, the sole inhibition of mTORC1 by
rapalogues releases the S6K/IRS1/PI3K negative feedback
loop, consequently hyper-activating the upstream regulator
Akt and neutralizing the therapeutic efficacy. Considering
this, the simultaneous inhibition of mTORC2 may conquer
the upregulation of Akt induced by the negative feedback
loop, thereby improving clinical response and broadening
the anti-tumor spectrum. In recent years,
a medicinal
chemistry campaign has been initiated in the exploration
of mTOR kinase inhibitors to address the limitation asso-
ciated with rapalogues.14 This facilitates the clinical pro-
gression of numerous inhibitors belonging to this family,
including BEZ-235 (mTOR/PI3K dual inhibitor, 1),15,16
GSK-2126458 (mTOR/PI3K dual inhibitor, 2),17 SAR-245409
(mTOR/PI3K dual inhibitor, 3),18 PF-04691502 (mTOR/PI3K
dual inhibitor, 4),19 PF-05212384 (mTOR/PI3K dual inhibi-
tor, 5),20 AZD-2014 (mTOR inhibitor, 6),21 MLN-0128
(mTOR inhibitor, 7)22 and CC-223 (mTOR inhibitor, 8)23,24
(Fig. 1).
In respect of cancer therapy, the advantages of mTOR
kinase inhibitors (ATP-competitive mTOR inhibitors) over
rapalogues have been widely accepted.13 Firstly, they lead to a
more complete inhibition of mTOR upon competitive interac-
tion with the ATP-binding domain. In contrast, rapalogues
Our latest exploration of ATP-competitive mTOR inhibitors
has culminated in a novel series of 3,4,6-trisubstituted quino-
line derivatives with favorable enzymatic potency. During the
further optimization to enhance their anti-proliferative activi-
ties, compound 9 in this series attracted our attention due to
Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Phar-
maceutical Sciences, Zhejiang University, Hangzhou 310058, China.
E-mail: huyz@zju.edu.cn; Fax: +86 571 88208460; Tel: +86 571 88208460
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Fig. 1 Some representative mTOR kinase inhibitors under clinical trial.
its structural similarity to BEZ-235 (1), an mTOR/PI3K dual 2. Results and discussion
inhibitor bearing the imidazoij4,5-c]quinolinone template. As
illustrated in the biological data summarized in Fig. 2, 9
2.1. Structural optimization of 8
approximately maintained the potent mTOR inhibitory activ-
ity after de-constructing the five-membered imidazolone
fused to the C-3 and C-4 positions of 1. Thereby, the
imidazolone was regarded to be dispensable for mTOR
inhibition. With this information in hand, our optimization
prioritized breaking imidazolone of the imidazoij4,5-
c]quinolinone derivatives and investigating substituents
distinct from those of 9 at the C-3 and C-4 positions of the
quinoline core. Herein, we described this optimization
approach that leads to the identification of several promising
quinoline-derived mTOR inhibitors, which not only maintained
the potent enzymatic activity, but also exhibited superior anti-
proliferative efficacies to 9.
The potent mTOR inhibitory activity of 9 encouraged our
extensive structural alteration at the C-3 position of the
quinoline core. After reversal of the C-3 amide functionality,
the enzymatic activity decreased 3-fold while the cellu-
lar activity was maintained according to the biological
data of 10 (Table 1). This informed us that the C-3
acetylamide moiety improved the physicochemical proper-
ties of 10 and offset the slight loss in mTOR inhibitory
activity, thereby accounting for the comparable cellular
activities of 9 and 10. Considering the convenience in syn-
thesis, we initiated our investigation on different replace-
ments of the C-3 acetylamide moiety. Unfortunately, none of
these counterparts, including the C-3 methylsulfonamide
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Fig. 2 Structure and in vitro biological activities of compound 9 in contrast to those of BEZ-235.
Table 1 The in vitro biological data of compounds 10–14
Compd
R1
mTOR (IC50, nM)
HCT-116 (IC50, μM)
PC-3 (IC50, μM)
MCF-7 (IC50, μM)
10
11
12
13
14
CH3CO
MeSO2
CF3CO
t-BuCO
(MeSO2)2
114
3.95
>10
>10
>10
>10
2.56
>10
>10
>10
>10
0.44
b
b
a
b
b
1.36
b
b
92% inhibition at 3000 nM. b Not identified.
a
(11), trifluoroacetamide (12), pivaloylamino (13) and
dimethylsulfonamide (14), exhibited improvement in anti-
proliferative activities.
Bearing in mind the importance of C-6 quinoline for activ-
ity, our efforts were then directed at decorating the
substituted aniline at the C-4 position. BGT-226 (15) was an
Table 2 The in vitro biological data of compounds 16–20
Compd
R2
mTOR (IC50, nM)
HCT-116 (IC50, μM)
PC-3 (IC50, μM)
MCF-7 (IC50, μM)
16
17
18
19
20
H
46
22
340
310
17
1.61
0.36
1.45
1.24
0.64
3.44
0.50
5.42
3.82
1.05
0.47
0.11
1.11
0.32
0.30
CH3CO
MeSO2
CF3CO
t-BuCO
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additional dual mTOR/PI3K modulator discovered by Novartis
with the same imidazoij4,5-c]quinolinone platform as 1.25
Upon introducing the ring-opening strategy, compound 16
was obtained after breaking the five-membered imidazolone
and replacing the C-6 methoxypyridine with quinoline that
was proven to be optimum in the former study. To our
delight, this C-3 amino derivative (16) bearing the elongated
C-4 substructure displayed an IC50 value of 46 nM against
mTOR (Table 2). In addition, acceptable anti-proliferative effi-
cacy was demonstrated with IC50 values of 1.61, 3.44 and 0.47
μM against the HCT-116, PC-3 and MCF-7 cell lines, respec-
tively. From our point of view, compound 16 still needed di-
versification to modify the physicochemical profiles, espe-
cially to lower the hydrophilicity. Acetylation of the C-3 amino
moiety of 16 conferred an enhanced enzymatic activity with
an IC50 of 22 nM against mTOR. Besides, the resultant
compound 17 exhibited a 4-fold, 7-fold and 4-fold increase
accessible to a variety of substituents for further modulating
the physicochemical properties. Simple diversification, such
as acylation and sulfonylation, retained the enzymatic activity
as illustrated by the comparable IC50 values of 24 (IC50
=
30 nM) and 25 (IC50 = 25 nM) to 17 (Table 4). However, the
slight improvement in hydrophobicity did improve the anti-
proliferative activities. The N-acetyl piperazine derivative 24
exhibited a 3-fold enhanced anti-proliferative efficacy com-
pared to 17 against all the tested cell lines with IC50 values of
0.11 (HCT-116), 0.17 (PC-3) and 0.04 μM (MCF-7), respec-
tively. The cellular activity of N-sulfonylated piperazine deriva-
tive 25 was also remarkable with IC50 values of 0.43, 0.19 and
0.06 μM against HCT-116, PC-3 and MCF-7, respectively. Sim-
ilar to that observed in compound 20, further increase in
hydrophobicity via replacing the C-3 acetamide of 24 with
pivaloylamino significantly weakened the cellular potency
against MCF-7 by 20-fold, although the enzymatic activity of the
resultant compound 26 varied slightly (IC50 = 36 nM). Up to
this point, the SAR investigation described above enabled the
identification of several potent mTOR inhibitors, including
17, 24 and 25, that displayed considerably enhanced anti-
proliferative efficacies in comparison with the initial lead 9.
in cellular activity against HCT-116 (IC50
= 0.36 μM),
PC-3 (IC50 = 0.50 μM) and MCF-7 (IC50 = 0.11 μM), respec-
tively. Subsequent introduction of methylsulfonamide or
trifluoroacetamide as a C-3 substituent sharply decreased the
mTOR inhibitory activity and the corresponding counterparts
(18 and 19) only showed moderate mTOR inhibition with
IC50 values of 340 nM and 310 nM, respectively. In response
to the compromised enzymatic activity, both compounds
displayed weakened anti-proliferative activities against all the
tested cell lines, especially PC-3. The clear structure–activity
relationships (SARs) deduced from 17–19 may give an expla-
nation for the compromised cellular activities observed for 12
and 13. Although compound 20 maintained the mTOR inhib-
itory activity of 17, the excessive increase in hydrophobicity
caused a slight loss in cellular activity after replacing the C-3
acetylamide with pivaloylamino.
Beyond that of 17, several other substitution patterns at
the phenyl ring of the C-4 aniline, including the 4-cyano,
4-trifluoromethyl, as well as 3,5-dimethyloxy replacements,
were also investigated. However, all of these turned out to be
detrimental to the enzymatic activity according to the biologi-
cal data summarized in Table 3. Consistent with the loss in
mTOR inhibitory activity, the resultant compounds 21–23
demonstrated a significant decrease in anti-proliferative effi-
cacies against the three tumor cell lines.
2.2. Molecular docking
Before diversification at the piperazine moiety, compound 17
was docked into the catalytic cleft for probing its possible
binding mode with mTOR. As illustrated in Fig. 3a, the quin-
oline template conferred a critical H-bond interaction with
the residue Val2240 in the hinge region. Meanwhile, the car-
bonyl of the C-3 amide moiety served as an H-bond receptor
for residue Cys2243. The quinoline replacement at the C-6
position reached into the inner hydrophobic pocket, engag-
ing in an H-bond with residue Tyr2225. In addition to these
H-bond interactions, the trifluoromethyl group packed par-
tially into the N-lobe pocket (Leu2185, Pro2169, etc.). As
described above, the piperazine was directed into a largely
solvent-exposed region, thereby tolerable for further diversifi-
cation. The binding mode of compound 20 was similar to
that of 17 (Fig. 3b). The quinoline core, the C-3 amide and
the C-6 quinoline made three contacts to residues Val2240,
Cys2243 and Tyr2225 via H-bonds. The bulky t-butyl group of
20 successfully avoided the unfavorable interactions with the
As demonstrated in the molecular docking (Fig. 3), the
piperazine of 17 was projected into a vast space, thereby
Table 3 The in vitro biological data of compounds 21–23
Compd
R3
mTOR (IC50, nM)
HCT-116 (IC50, μM)
PC-3 (IC50, μM)
21
22
23
4-CN
4-CF3
3,5-(OCH3)2
>3000
>3000
>3000
>10
>10
>10
>10
>10
>10
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Fig. 3 Molecular docking of quinolines 17 (a) and 20 (b) into the catalytic cleft of mTOR.
Table 4 The in vitro biological data of compounds 24–26
Compd
R2
R4
mTOR (IC50, nM)
HCT-116 (IC50, μM)
PC-3 (IC50, μM)
MCF-7 (IC50, μM)
24
25
26
PI-103
BEZ-235
CH3CO
CH3CO
t-BuCO
CH3CO
MeSO2
CH3CO
30
25
36
14
21
0.11
0.43
>10
0.17
0.19
>10
0.04
0.06
0.79
0.14
0.19
0.05
indole moiety of residue Trp2239, thereby accounting for its
comparable mTOR inhibitory activity to that of 17.
compounds 17–20 as the hydrochlorides. Ultimately, the pi-
perazines of compounds 17 or 20 were acetylated or sulfo-
nylated to generate compounds 24–26.
2.3. Chemistry
2.4 Selectivity over class I PI3Ks
Scheme 1 displays the synthetic route for compounds 10–14
and 21–23. 2-Aminobenzoic acid (27) was employed as the
starting material and was firstly brominated to furnish 28.
Meanwhile, 30 was prepared from nitromethane via the
reported method with minor modification.26 Following con-
densation of 30 with 28, the newly formed 31 was subjected
to an intra-molecular cyclization to afford intermediate 32.
Afterwards, treatment of 32 with phosphorus oxychloride led
to the formation of the key intermediate 33. SNAr reaction of
33 with the corresponding substituted anilines then provided
intermediates 34 or 36a–c. Their subsequent Suzuki coupling
with quinoline-3-boronic acid pinacol ester correspondingly
gave 35 or 37a–c as intermediates. Finally, they underwent
catalytic hydrogenation and acylation or sulfonylation to gen-
erate target compounds 10–14 and 21–23.
As shown in Scheme 2, the intermediate 33 was converted
to 40 after a sequence of SNAr reaction, Suzuki coupling and
catalytic hydrogenation. Subsequent removal of the Boc-
protecting group of 40 resulted in the formation of com-
pound 16 as the hydrochloride. Alternatively, the C-3 amino
functionality of 40 was further acylated or sulfonylated to pro-
vide intermediates 41a–d. Afterwards, their Boc-protecting
groups were removed to furnish the corresponding
Compounds 17 and 24 in this series were further evaluated
for their inhibitory activities against PI3Kα, PI3Kβ, PI3Kγ and
PI3Kδ with PI-103 as the positive control. As summarized in
Table 5, compound 17 exhibited significant inhibition to
none of the four class I PI3Ks at the concentration of 1.0 μM.
Besides, compound 24 also displayed acceptable specificity,
which was 50-fold, >167-fold, 17-fold and 70-fold selective
over PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ, respectively.
2.5 Western blot assay in the MCF-7 cell line
Compound 24, as the representative of this series, was
selected for further Western blot analysis in the MCF-7 cell
line, in which pS6 (Ser235) and pAkt (Ser473) served as the
biomarker for the inhibition of mTORC1 and mTORC2,
respectively (Fig. 4). As we anticipated, the sole inhibition of
mTORC1 by rapamycin led to an obvious increase in the level
of pAkt at the concentration of 0.5 μM, indicating the release
of the S6K/IRS1/PI3K negative feedback loop. In contrast,
compound
24
treatment
induced
simultaneous
downregulation of pS6 (Ser235) and pAkt (Ser473) at the
same concentration, implying the dual inhibition of mTORC1
and mTORC2. Moreover, compound 24 culminated in a
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Scheme 1 Reagents and conditions: (a) NH4Br, H2O2 (30%), HAc, 10 °C to rt; (b) NaOH, H2O, 0 °C; concentrated HCl, ice; (c) 28, EtOH, rt; (d)
AcOK, Ac2O, 120 °C; (e) DMF, POCl3, reflux; (f) corresponding substituted aniline, AcONa, HAc, rt; (g) corresponding borate, K2CO3, PdIJPPh3)4,
H2O/1,4-dioxane, 100 °C; (h) (1) H2, Pd/C, MeOH, rt; (2) AcCl, pyridine, THF, 0 °C (for 10, 21–23); MeSO2Cl, pyridine, 40 °C (for 11); (CF3CO)2O,
pyridine, DCM, rt (for 12); t-BuCOCl, pyridine, THF, 0 °C (for 13); MeSO2Cl, Et3N, THF, rt (for 14).
partial downregulation of pS6 (Ser235) and a complete
downregulation of pAkt (Ser473) at the concentration as low
as 0.1 μM. These experimental results testified the advantage
of 24 over mTORC1 modulator rapamycin in conquering the
S6K/IRS1/PI3K negative feedback loop.
(RLM). As a consequence, no obvious degradation was
observed after 45 min-exposure to both SIF and SGF. In addi-
tion, it demonstrated an acceptable metabolic stability in
RLM, with more than half remaining after 30 min.
3. Experimental
2.6. In vitro stability
3.1. Chemistry
To guide the further in vivo development of these quinolines,
compound 17 was subsequently chosen as their representa-
tive to investigate their stability in simulated intestinal fluid
(SIF), simulated gastric fluid (SGF) and rat liver microsome
The reagents and solvents for the reactions were purchased
from common commercial suppliers. If necessary, purifica-
tion was carried out prior to use. Melting points were
uncorrected and determined on a Büchi B-540 apparatus
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Scheme 2 Reagents and conditions: (a) corresponding substituted aniline, AcONa, HAc, rt; (b) corresponding borate, K2CO3, PdIJPPh3)4, H2O/1,4-
dioxane, 100 °C; (c) H2, Pd/C, MeOH, rt; (d) AcCl, pyridine, THF, 0 °C (for 41a); MeSO2Cl, pyridine, 40 °C (for 41b); (CF3CO)2O, pyridine, DCM, rt
(for 41c); t-BuCOCl, pyridine, THF, 0 °C (41d); (e) HCl (EtOAc solution, concentrated), 0 °C to rt; (f) Et3N, AcCl, THF, 0 °C (for 24 and 26); Et3N,
MeSO2Cl, THF, 0 °C (for 25).
(Büchi Labortechnik, Flawil, Switzerland). 1H NMR and 13C
NMR spectra were recorded on 500 MHz (or 400 MHz) and
125 MHz (or 100 MHz) instruments, respectively, (Bruker Bio-
Table 5 The inhibitory activities of compounds 17 and 24 against class I
PI3Ks
science, Billerica, MA, USA) with tetramethylsilane (TMS) as
Compd
PI3Kα
PI3Kβ
PI3Kγ
PI3Kδ
internal standard. The ESI mass spectral data were obtained
by using an Esquire-LC-00075 spectrometer (Bruker Biosci-
ence). Flash column chromatography was performed using
silica gel (200–300 mesh). HPLC was performed using an
Agilent 1200 system with UV detection at 254 nm, eluting
17
24
PI-103
42.1%a
1500b
12b
1.0%a
>5000b
13b
47.3%a
502b
34.9%a
2075b
10b
48b
a
Inhibition rate at 1.0 μM. b IC50 value (nM).
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(10 mL), a catalytic amount of DMF was added, and the mix-
ture was stirred under reflux for 4 h. Then, it was cooled
and slowly poured onto ice. Afterwards, the precipitate
was filtered, washed with water and dissolved in
dichloromethane (DCM). The solution was subsequently
washed with a saturated NaHCO3 solution, brine, dried with
anhydrous Na2SO4 and concentrated in vacuo. The residue
was purified via flash column chromatography using ethyl
acetate (EA)/petroleum ether (PE) (1 : 10) as the eluent to
afford 33 as a white solid. Yield: 92%; 1H NMR (500 MHz,
CDCl3): 9.21 (s, 1H, Ar–H), 8.47 (s, 1H, Ar–H), 8.02 (d, 9.0
Hz, 1H, Ar–H), 7.95 (d, 9.0 Hz, 1H, Ar–H); ESI-MS: m/z = 287
[M + H]+.
3.1.5. General procedure for the preparation of intermedi-
ates 34, 36a–c and 38 (A). To a solution of 33 (1.0 eq.) in
acetic acid (2 mL per 1 mmol of substrate), AcONa (1.4 eq.)
and 2-(4-aminophenyl)-2-methylpropanenitrile (1.0 eq.) were
added at room temperature. The resultant mixture was
stirred for 0.5 h at the same temperature and quenched with
water. After the crude product was totally precipitated, it was
filtered, washed with water and dissolved in DCM. The resul-
tant solution was subsequently washed with a saturated
NaHCO3 solution, brine, and dried over anhydrous Na2SO4.
Following removal of DCM in vacuo, the residue was purified
via flash column chromatography using EA/PE (1 : 6) as the
eluent to afford 2-(4-((6-bromo-3-nitroquinolin-4-yl)amino)-
phenyl)-2-methylpropanenitrile (34) as a light yellow solid.
The intermediates 36a–c and 38 were prepared according to
the procedure for 34, only with 2-(4-aminophenyl)-2-
methylpropanenitrile replaced by the corresponding
substituted aniline, including 4-aminobenzonitrile (for 36a),
4-(trifluoromethyl)aniline (for 36b), 3,5-dimethoxyaniline (for
36c) and tert-butyl 4-(4-amino-3-(trifluoromethyl)phenyl)-
piperazine-1-carboxylate (for 38).
3.1.5.1. 2-(4-((6-Bromo-3-nitroquinolin-4-yl)amino)phenyl)-2-
methylpropanenitrile (34). Light yellow solid; yield: 86%; 1H
NMR (400 MHz, DMSO-d6): 10.12 (s, 1H, NH), 9.09 (s, 1H, Ar–
H), 8.71 (s, 1H, Ar–H), 8.02 (d, 8.8 Hz, 1H, Ar–H), 7.94 (d, 8.8
Hz, 1H, Ar–H), 7.47 (d, 8.4 Hz, 2H, Ar–H), 7.14 (d, 8.4 Hz, 2H,
Ar–H), 1.68 (s, 6H, CH3 × 2); ESI-MS: m/z = 411 [M + H]+.
3.1.5.2. 4-((6-Bromo-3-nitroquinolin-4-yl)amino)benzonitrile
(36a). Light yellow crystal; yield: 83%; 1H NMR (400 MHz,
DMSO-d6): 10.09 (s, 1H, NH), 9.06 (s, 1H, Ar–H), 8.79 (s, 1H,
Ar–H), 8.02 (d, 8.4 Hz, 1H, Ar–H), 7.95 (d, 8.4 Hz, 1H, Ar–H),
7.30 (d, 8.4 Hz, 2H, Ar–H), 7.11 (d, 8.4 Hz, 2H, Ar–H); ESI-MS:
m/z = 369 [M + H]+.
Fig. 4 Western blot analysis of the quinoline derivative 24 in the MCF-
7 cell line.
with a binary solvent system A and B [A: CH3OH; B: H2O with
0.12% ammonium acetate (w/v)]. Analytical purity of all com-
pounds was >95% unless stated otherwise.
3.1.1. 2-Amino-5-bromobenzoic acid (28). To a suspension
of 2-aminobenzoic acid (27, 6.86 g, 50.0 mmol, 1.0 eq.) and
ammonium bromide (5.39 g, 55.0 mmol, 1.1 eq.) in HAc (50
mL), hydrogen dioxide (6.00 mL, 30%, 55.0 mmol, 1.1 eq.)
was added dropwise at 10 °C. After stirring at room tempera-
ture for 16 h, the reaction mixture was slowly poured into
water and the crude product was precipitated. Following filtra-
tion, it was washed with water and compound 28 was
obtained as a pale solid after recrystallization from water.
Yield: 83%; mp 217–219 °C.
3.1.2. (E)-5-Bromo-2-(2-nitrovinylamino)benzoic acid (31).
To a solution of sodium hydroxide (30.2 g, 756 mmol, 3.0
eq.) in water (70 mL), nitromethane (13 mL, 252 mmol, 1.0
eq.) was dropwise at 0 °C. The reaction mixture was stirred at
the same temperature for 1 h and at room temperature for
another hour. Then, it was slowly poured to a mixture of ice
(60.0 g) and concentrated HCl (90.0 mL). The resultant solu-
tion was immediately combined with a solution of 28 (6.03 g,
27.9 mmol, 0.11 eq.) in EtOH (450 mL) and the mixture was
allowed to be stirred at room temperature for 18 h. Following
filtration, the solid product was washed repeatedly with
water. The cake was sliced into flakes and dried in vacuo at
35 °C to provide the title compound as a yellow solid, which
was used directly for the next step. Yield: 78%.
3.1.5.3. 6-Bromo-3-nitro-N-(4-(trifluoromethyl)phenyl)-
quinolin-4-amine (36b). Light yellow solid; yield: 91%; 1H
NMR (500 MHz, CDCl3): 10.31 (s, 1H, NH), 9.52 (s, 1H, Ar–H),
7.98 (d, 9.0 Hz, 1H, Ar–H), 7.85 (dd, 2.0 Hz, 9.0 Hz, 1H, Ar–
H), 7.78 (d, 2.0 Hz, 1H, Ar–H), 7.68 (d, 8.0 Hz, 2H, Ar–H),
7.22 (d, 8.0 Hz, 2H, Ar–H); ESI-MS: m/z = 412 [M + H]+.
3.1.5.4. 6-Bromo-N-(3,5-dimethoxyphenyl)-3-nitroquinolin-4-
amine (36c). Light yellow solid; yield: 86%; 1H NMR (400
MHz, DMSO-d6): 10.00 (s, 1H, NH), 9.05 (s, 1H, Ar–H), 8.78
(s, 1H, Ar–H), 8.02 (d, 8.8 Hz, 1H, Ar–H), 7.94 (d, 8.8 Hz, 1H,
3.1.3. 6-Bromo-3-nitroquinolin-4-ol (32). A mixture of 31
(5.74 g, 20.0 mmol, 1.0 eq.), potassium acetate (2.35 g, 24.0
mmol, 1.2 eq.) and acetic anhydride (30 mL) was stirred at
120 °C for 2 h. After cooling, the precipitate was filtered and
washed repeatedly with acetic acid, and then with water.
Afterwards, it was dried to give the title product. Yield: 38%;
ESI-MS: m/z = 267 [M + H]−.
3.1.4. 6-Bromo-4-chloro-3-nitroquinoline (33). To a suspen-
sion of 32 (1.61 g, 6.00 mmol) in phosphorus oxychloride
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Ar–H), 6.26 (s, 3H, Ar–H), 3.69 (s, 6H, OCH3 × 2); ESI-MS: m/z
= 404 [M + H]+.
9.0 Hz, 1H, Ar–H), 7.49 (s, 1H, Ar–H), 7.37 (d, 9.0 Hz, 1H, Ar–
H), 3.44–3.37 (m, 4H, piperazine–CH2 × 2), 2.77 (t, 4.5 Hz,
4H, piperazine–CH2 × 2), 1.41 (s, 9H, CH3 × 3); ESI-MS: m/z =
645 [M + H]+.
3.1.7. Synthetic procedures for compounds 10–14, 21–23
and intermediates 41a–d. To a suspension of the intermedi-
ate 35 in methanol, 10% Pd/C (10% of the weight of the sub-
strate) was added. The resultant mixture was stirred under
H2 (balloon) overnight at room temperature. After this time,
Pd/C was filtered and the filtrate was concentrated in vacuo
to provide the C-3 amino derivative as a colorless oil, which
was used for the next step without purification.
3.1.5.5 tert-Butyl 4-(4-((6-bromo-3-nitroquinolin-4-yl)amino)-
2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (38). Light
1
yellow solid; yield: 88%; H NMR (400 MHz, DMSO-d6): 10.16
(s, 1H, NH), 9.09 (s, 1H, Ar–H), 8.71 (s, 1H, Ar–H), 8.03 (d, 9.2
Hz, 1H, Ar–H), 7.95 (d, 9.2 Hz, 1H, Ar–H), 7.52 (d, 8.4 Hz, 1H,
Ar–H), 7.41 (s, 1H, Ar–H), 7.31 (d, 8.4 Hz, 1H, Ar–H), 3.46–
3.40 (m, 4H, piperazine–CH2
× 2), 2.82–2.75 (m, 4H,
piperazine–CH2 × 2), 1.43 (s, 9H, CH3 × 3); ESI-MS: m/z = 596
[M + H]+.
3.1.6. General procedure for the preparation of intermedi-
ates 35, 37a–c and 39 (B). To a solution of 34 (1.0 eq.) in
H2O/1,4-dioxane (1 : 3, v/v, 12 mL per 1 mmol of substrate),
anhydrous potassium carbonate (1.1 eq.), quinoline-3-boronic
acid pinacol ester (1.1 eq.) and PdIJPh3P)4 (0.08 eq.) were
added. The resultant suspension was stirred at 100 °C for 8 h
under N2 atmosphere. After this time, the reaction mixture
was removed from the oil bath, cooled and concentrated in
vacuo. The residue was treated with EtOAc, and following fil-
tration, the filtrate was washed with water and brine. After-
wards, the organic layer was dried over anhydrous Na2SO4
and concentrated in vacuo. The residue was subjected to flash
column chromatography using EA/DCM (1 : 3–1 : 1) as the elu-
ent, which afforded 35 as a yellow solid. The intermediates
37a–c and 39 were prepared according to the procedure for
35, only with 34 replaced by 36a–c or 38 as the substrate.
3.1.6.1. 2-Methyl-2-(4-((3′-nitro-[3,6′-biquinolin]-4′-yl)amino)-
phenyl)propanenitrile (35). Yellow solid; yield: 82%; 1H NMR
(500 MHz, CDCl3): 10.66 (s, 1H, NH), 9.54 (s, 1H, Ar–H), 8.61
(d, 1.5 Hz, 1H, Ar–H), 8.19 (d, 9.0 Hz, 1H, Ar–H), 8.11 (d, 8.5
Hz, 1H, Ar–H), 8.08 (dd, 2.0 Hz, 8.5 Hz, 1H, Ar–H), 7.99 (d,
1.0 Hz, 1H, Ar–H), 7.97 (d, 2.0 Hz, 1H, Ar–H), 7.82 (d, 8.0 Hz,
1H, Ar–H), 7.76–7.72 (m, 1H, Ar–H), 7.62–7.59 (m, 3H, Ar–H),
7.30 (d, 8.5 Hz, 2H, Ar–H), 1.78 (s, 6H, CH3 × 2); ESI-MS: m/z
= 460 [M + H]+.
3.1.6.2. 4-((3′-Nitro-[3,6′-biquinolin]-4′-yl)amino)benzonitrile
(37a). Yellow solid; yield: 77%; ESI-MS: m/z = 418 [M + H]+.
3.1.6.3. 3′-Nitro-N-(4-(trifluoromethyl)phenyl)-[3,6′-
biquinolin]-4′-amine (37b). Yellow solid; yield: 68%; ESI-MS:
m/z = 461 [M + H]+.
3.1.6.4. N-(3,5-Dimethoxyphenyl)-3′-nitro-[3,6′-biquinolin]-4′-
amine (37c). Yellow solid; yield: 79%; 1H NMR (500 MHz,
CDCl3): 10.65 (s, 1H, NH), 9.51 (s, 1H, Ar–H), 8.86 (d, 2.0 Hz,
1H, Ar–H), 8.17–8.15 (m, 3H, Ar–H), 8.08 (dd, 2.0 Hz, 8.5 Hz,
1H, Ar–H), 7.94 (s, 1H, Ar–H), 7.80 (d, 8.0 Hz, 1H, Ar–H),
7.78–7.75 (m, 1H, Ar–H), 7.64–7.61 (m, 1H, Ar–H), 6.58 (t, 2.0
Hz, 1H, Ar–H), 6.42 (d, 2.0 Hz, 2H, Ar–H), 3.77 (s, 6H, OCH3
× 2); ESI-MS: m/z = 453 [M + H]+.
Subsequently, the newly formed C-3 amino derivative (1.0
eq.) and pyridine (1.1 eq.) were dissolved in anhydrous THF
(5 mL per 1 mmol of substrate). Afterwards, a solution of
acetyl chloride (1.1 eq.) in anhydrous tetrahydrofuran (THF,
5 mL per 1 mmol of substrate) was added dropwise to the
mixture at 0 °C. The resultant mixture was stirred at the
same temperature for 30 min and then concentrated in
vacuo. The residue was dissolved in DCM, and the solution
was washed with a saturated NaHCO3 solution and brine.
The organic layer was subsequently dried over anhydrous
Na2SO4 and concentrated in vacuo. Afterwards, the residue
was subjected to flash column chromatography with EA/
MeOH (50 : 1) as the eluent to give the target compound 10
as a light yellow solid.
The solution of the newly formed C-3 amino derivative
(1.0 eq.) and methylsulfonyl chloride (1.0 eq.) in anhydrous
pyridine (4 mL per 1 mmol of substrate) was warmed at 40
°C under N2 atmosphere. After 8 h, the mixture was directly
subjected to flash column chromatography with EA/MeOH
(50 : 1) as the eluent to give the target compound 11 as a light
yellow solid.
The solution of the newly formed C-3 amino derivative
(1.0 eq.), pyridine (1.2 eq.) and trifluoroacetic anhydride (1.2
eq.) in anhydrous DCM (10 mL per 1 mmol of substrate) was
stirred at room temperature under N2 atmosphere for 2 h. Af-
terwards, the reaction mixture was washed with a saturated
NaHCO3 solution and brine. The organic layer was subse-
quently dried over anhydrous Na2SO4 and concentrated in
vacuo. Afterwards, the residue was subjected to flash column
chromatography with EA as the eluent to give the target com-
pound 12.
The newly formed C-3 amino derivative (1.0 eq.) and pyri-
dine (1.1 eq.) were dissolved in anhydrous THF (5 mL per 1
mmol of substrate). Afterwards, a solution of pivaloyl chlo-
ride (1.1 eq.) in anhydrous THF (5 mL per 1 mmol of sub-
strate) was added dropwise to the mixture at 0 °C. The resul-
tant mixture was stirred at the same temperature for 30 min
and then concentrated in vacuo. The residue was dissolved in
DCM, and the solution was washed with a saturated NaHCO3
solution and brine. The organic layer was subsequently dried
over anhydrous Na2SO4 and concentrated in vacuo. After-
wards, the residue was subjected to flash column chromatog-
raphy with EA/MeOH (50 : 1) as the eluent to give the target
compound 13 as a light yellow solid.
3.1.6.5. tert-Butyl 4-(4-((3′-nitro-[3,6′-biquinolin]-4′-yl)amino)-
2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (39). Yellow
solid; yield: 70%; 1H NMR (500 MHz, CDCl3): 10.28 (s, 1H,
NH), 9.28 (d, 2.5 Hz, 1H, Ar–H), 9.12 (s, 1H, Ar–H), 8.82 (d,
2.0 Hz, 1H, Ar–H), 8.74 (d, 2.0 Hz, 1H, Ar–H), 8.42 (d, 8.5 Hz,
1H, Ar–H), 8.16 (d, 8.5 Hz, 1H, Ar–H), 8.08–8.05 (m, 2H, Ar–
H), 7.81–7.78 (m, 1H, Ar–H), 7.69–7.66 (m, 1H, Ar–H), 7.55 (d,
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To a solution of the newly formed C-3 amino derivative
(1.0 eq.) and Et3N (TEA, 4.0 eq.) in anhydrous THF (10 mL
per 1 mmol of substrate), methanesulfonyl chloride (4.0 eq.)
was added at room temperature. The resultant mixture was
stirred at the same temperature for 1 h and then concen-
trated in vacuo. The residue was dissolved in DCM, and the
solution was washed with a saturated NaHCO3 solution and
brine. The organic layer was subsequently dried over anhy-
drous Na2SO4 and concentrated in vacuo. Afterwards, flash
column chromatography of the residue with EA/PE (1: 2–3 : 4)
as the eluent gave the target compound 14.
Compounds 21–23 and the intermediate 41a were pre-
pared according to the procedure for 10, only with 35
replaced by 37a–c or 39 as the starting material. The interme-
diate 41b was prepared according to the procedure for 11,
only with 35 replaced by 39 as the starting material. The
intermediate 41c was prepared according to the procedure
for 12, only with 35 replaced by 39 as the starting material.
The intermediate 41d was prepared according to the proce-
dure for 13, only with 35 replaced by 39 as the starting
material.
(JC–F 34.00 Hz), 149.33, 147.81, 146.87, 145.65, 145.33,
138.88, 138.18, 135.87, 134.04, 131.88, 131.14, 130.31, 128.57,
128.53, 127.48, 125.80, 125.32, 124.44, 123.61, 123.41, 122.00,
120.64, 116.00 (JC–F 230.00 Hz), 112.76, 36.49, 28.33; ESI-MS:
m/z = 526 [M + H]+; mp 205–207 °C; HPLC: tR = 13.90 min,
flow rate 1.0 mL min−1, COSMOSIL 5C18-MS-II column (4.6
ID × 250 mm), rt, eluent A – 70%, eluent B – 30%.
3.1.7.4. N-(4′-((4-(2-Cyanopropan-2-yl)phenyl)amino)-[3,6′-
biquinolin]-3′-yl)pivalamide (13). Light yellow solid; yield: 71%
(for two steps); 1H NMR (500 MHz, DMSO-d6): 9.43 (d, 2.0
Hz, 1H, Ar–H), 8.89 (s, 1H, NH), 8.79 (d, 2.0 Hz, 1H, Ar–H),
8.74 (d, 2.0 Hz, 1H, Ar–H), 8.66 (s, 1H, NH), 8.62 (s, 1H, Ar–
H), 8.24 (dd, 2.0 Hz, 8.5 Hz, 1H, Ar–H), 8.12 (d, 8.5 Hz, 1H,
Ar–H), 8.10–8.05 (m, 2H, Ar–H), 7.80–7.76 (m, 1H, Ar–H),
7.67–7.63 (m, 1H, Ar–H), 7.32 (d, 8.5 Hz, 2H, Ar–H), 6.83 (d,
8.5 Hz, 2H, Ar–H), 1.62 (s, 6H, CH3 × 2), 0.85 (s, 9H, CH3
3); ESI-MS: m/z = 514 [M + H]+; mp 113–115 °C; HPLC: tR
×
=
9.01 min, flow rate 1.0 mL min−1, COSMOSIL 5C18-MS-II col-
umn (4.6 ID × 250 mm), rt, eluent A – 70%, eluent B – 30%.
3.1.7.5. N-(4′-((4-(2-Cyanopropan-2-yl)phenyl)amino)-[3,6′-
biquinolin]-3′-yl)-N-(methylsulfonyl)methanesulfonamide
(14).
3.1.7.1. N-(4′-((4-(2-Cyanopropan-2-yl)phenyl)amino)-[3,6′-
biquinolin]-3′-yl)acetamide (10). Light yellow solid; yield: 63%
(for two steps); 1H NMR (500 MHz, CDCl3): 9.03 (s, 1H, Ar–
H), 9.01 (s, 1H, Ar–H), 8.23–8.18 (m, 2H, Ar–H), 8.14 (s, 1H,
Ar–H), 8.09 (d, 8.5 Hz, 1H, Ar–H), 8.00 (d, 8.0 Hz, 1H, Ar–H),
7.82 (d, 8.0 Hz, 1H, Ar–H), 7.72 (t, 7.5 Hz, 1H, Ar–H), 7.57
(t, 7.5 Hz, 1H, Ar–H), 7.35 (d, 7.5 Hz, 2H, Ar–H), 6.83 (d, 7.5 Hz,
2H, Ar–H), 2.19 (s, 3H, acetyl), 1.70 (s, 6H, CH3 × 2); 13C NMR
Light yellow solid; yield: 39% (for two steps); 1H NMR (500
MHz, DMSO-d6): 8.81 (s, 1H, NH), 8.77 (s, 1H, Ar–H), 8.67 (d,
2.0 Hz, 1H, Ar–H), 8.42 (d, 2.0 Hz, 1H, Ar–H), 8.26 (dd, 2.0
Hz, 8.5 Hz, 1H, Ar–H), 8.14 (d, 8.5 Hz, 1H, Ar–H), 8.01 (d, 8.5
Hz, 1H, Ar–H), 7.99 (d, 2.0 Hz, 1H, Ar–H), 7.96 (d, 8.5 Hz, 1H,
Ar–H), 7.78–7.75 (m, 1H, Ar–H), 7.65–7.61 (m, 1H, Ar–H), 7.50
(d, 8.5 Hz, 2H, Ar–H), 7.07 (d, 8.5 Hz, 2H, Ar–H), 3.55 (s, 6H,
methylsulfonyl × 2), 1.69 (s, 6H, CH3 × 2) ; ESI-MS: m/z = 586
[M + H]+; mp 172–175 °C; HPLC: tR = 7.21 min, flow rate 1.0
mL min−1, COSMOSIL 5C18-MS-II column (4.6 ID × 250 mm),
rt, eluent A – 70%, eluent B – 30%.
3.1.7.6. N-(4′-((4-Cyanophenyl)amino)-[3,6′-biquinolin]-3′-yl)-
acetamide (21). Light yellow solid; yield: 65% (for two steps);
1H NMR (500 MHz, DMSO-d6): 9.67 (s, 1H, NH), 9.30 (d,
2.0 Hz, 1H, Ar–H), 9.25 (s, 1H, NH), 8.93 (s, 1H, Ar–H), 8.73
(d, 2.0 Hz, 1H, Ar–H), 8.46 (d, 2.0 Hz, 1H, Ar–H), 8.24 (dd,
2.0 Hz, 9.0 Hz, 1H, Ar–H), 8.16 (d, 9.0 Hz, 1H, Ar–H), 8.06
(d, 8.5 Hz, 2H, Ar–H), 7.80–7.77 (m, 1H, Ar–H), 7.68–7.64
(m, 1H, Ar–H), 7.60 (d, 8.5 Hz, 2H, Ar–H), 6.78 (d, 9.0 Hz, 2H,
Ar–H), 1.83 (s, 3H, acetyl); ESI-MS: m/z = 430 [M + H]+; mp
165–167 °C.
(125 MHz, DMSO-d6):
δ 168.90, 151.29, 150.05, 147.34,
146.49, 142.58, 138.31, 134.55, 133.97, 133.69, 132.79, 130.63,
130.21, 129.19, 128.86, 128.14, 128.05, 127.65, 125.62, 125.40,
124.09, 122.41, 122.18, 118.41, 36.49, 28.94, 23.11; ESI-MS:
m/z = 472 [M + H]+; mp 141–142 °C; HPLC: tR = 9.42 min,
flow rate 1.0 mL min−1, COSMOSIL 5C18-MS-II column (4.6
ID × 250 mm), rt, eluent A – 70%, eluent B – 30%.
3.1.7.2. N-(4′-((4-(2-Cyanopropan-2-yl)phenyl)amino)-[3,6′-
biquinolin]-3′-yl)methanesulfonamide (11). Light yellow solid;
yield: 46% (for two steps); 1H NMR (500 MHz, DMSO-d6):
9.25 (s, 1H, NH), 8.96 (d, 2.0 Hz, 1H, Ar–H), 8.76 (s, 1H, Ar–
H), 8.64 (s, 1H, NH), 8.53 (d, 2.0 Hz, 1H, Ar–H), 8.20 (dd, 2.0
Hz, 8.5 Hz, 1H, Ar–H), 8.18 (d, 2.0 Hz, 1H, Ar–H), 8.13 (d, 8.5
Hz, 1H, Ar–H), 8.02 (d, 8.5 Hz, 1H, Ar–H), 7.98 (d, 8.5 Hz, 1H,
Ar–H), 7.77–7.74 (m, 1H, Ar–H), 7.66–7.62 (m, 1H, Ar–H), 7.40
(d, 8.5 Hz, 2H, Ar–H), 6.92 (d, 8.5 Hz, 2H, Ar–H), 2.93 (s, 3H,
methylsulfonyl), 1.67 (s, 6H, CH3 × 2); ESI-MS: m/z = 508 [M +
H]+; mp 132–134 °C.
3.1.7.7. N-(4′-((4-(Trifluoromethyl)phenyl)amino)-[3,6′-biquin-
olin]-3′-yl)acetamide (22). Light yellow solid; yield: 52% (for
two steps); 1H NMR (500 MHz, CDCl3): 8.90 (s, 1H, Ar–H),
8.82 (s, 1H, Ar–H), 8.30 (d, 1.5 Hz, 1H, Ar–H), 8.16 (d, 8.5 Hz,
1H, Ar–H), 8.10 (d, 8.0 Hz, 1H, Ar–H), 8.00 (s, 1H, Ar–H), 7.96
(dd, 1.5 Hz, 8.0 Hz, 1H, Ar–H), 7.87 (d, 8.0 Hz, 1H, Ar–H),
7.77–7.73 (m, 1H, Ar–H), 7.62–7.57 (m, 1H, Ar–H), 7.22 (d, 8.0
Hz, 2H, Ar–H), 6.65 (d, 8.0 Hz, 2H, Ar–H), 2.20 (s, 3H, acetyl);
ESI-MS: m/z = 473 [M + H]+; mp 187–188 °C; HPLC: tR = 12.78
min, flow rate 1.0 mL min−1, COSMOSIL 5C18-MS-II column
(4.6 ID × 250 mm), rt, eluent A – 85%, eluent B – 15%.
3.1.7.8. N-(4′-((3,5-Dimethoxyphenyl)amino)-[3,6′-biquinolin]-
3′-yl)acetamide (23). Light yellow solid; yield: 74% (for two
3.1.7.3. N-(4′-((4-(2-Cyanopropan-2-yl)phenyl)amino)-[3,6′-
biquinolin]-3′-yl)-2,2,2-trifluoroacetamide (12). Light yellow
1
solid; yield: 54% (for two steps); H NMR (400 MHz, DMSO-
d6): 11.03 (brs, 1H, NH), 10.62 (brs, 1H, NH), 9.46 (s, 1H, Ar–
H), 9.10 (s, 1H, Ar–H), 8.90 (s, 1H, Ar–H), 8.87 (s, 1H, Ar–H),
8.58 (d, 8.8 Hz, 1H, Ar–H), 8.22 (d, 8.8 Hz, 1H, Ar–H), 8.12 (d,
8.8 Hz, 2H, Ar–H), 7.87–7.81 (m, 1H, Ar–H), 7.74–7.70 (m, 1H,
Ar–H), 7.54 (d, 8.0 Hz, 2H, Ar–H), 7.22 (d, 8.0 Hz, 2H, Ar–H),
1.70 (s, 6H, CH3 × 2); 13C NMR (100 MHz, DMSO-d6): δ 158.20
1
steps); H NMR (500 MHz, DMSO-d6): 9.43 (s, 1H, NH), 9.32
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(d, 2.0 Hz, 1H, Ar–H), 8.80 (s, 1H, NH), 8.71 (d, 2.0 Hz, 1H,
Ar–H), 8.64 (s, 1H, Ar–H), 8.54 (d, 2.0 Hz, 1H, Ar–H), 8.20
(dd, 2.0 Hz, 8.5 Hz, 1H, Ar–H), 8.11 (d, 8.5 Hz, 1H, Ar–H),
8.07 (s, 1H, Ar–H), 8.05 (s, 1H, Ar–H), 7.80–7.76 (m, 1H, Ar–
H), 7.68–7.64 (m, 1H, Ar–H), 6.06 (t, 2.0 Hz, 1H, Ar–H), 5.96
(d, 2.0 Hz, 2H, Ar–H), 3.63 (s, 6H, OCH3 × 2), 1.83 (s, 3H,
acetyl); ESI-MS: m/z = 465 [M + H]+; mp 170–172 °C.
Compounds 17–20 were prepared according to the procedure
for 16, only with 40 replaced by 41a–d as the starting
material.
3.1.8.1.
Hydrochloride
of
N-(4′-((4-(piperazin-1-yl)-3-
(trifluoromethyl)phenyl)amino)-[3,6′-biquinolin]-3′-yl)amino
(16). Light yellow solid; yield: 94%; 1H NMR (500 MHz,
DMSO-d6): 10.69 (s, 1H, HCl), 9.85 (s, 1H, piperazine–NH),
9.61 (s, 1H, NH), 9.54 (s, 2H, Ar–H), 9.06 (s, 1H, Ar–H), 8.73
(s, 1H, Ar–H), 8.40–8.31 (m, 2H, Ar–H), 8.26 (d, 8.5 Hz, 2H,
Ar–H), 8.08–7.98 (m, 1H, Ar–H), 7.93–7.86 (m, 1H, Ar–H), 7.46
(d, 8.5 Hz, 1H, Ar–H), 7.43 (s, 1H, Ar–H), 7.12 (d, 8.5 Hz, 1H,
Ar–H), 3.23–3.14 (m, 4H, piperazine–CH2 × 2), 3.07–2.98 (m,
4H, piperazine–CH2 × 2); 13C NMR (125 MHz, DMSO-d6): δ
145.95, 144.81, 141.07, 139.90, 137.90, 134.64, 134.52, 133.86,
133.80, 133.68, 132.83, 132.52, 132.40, 129.78, 129.60, 128.55,
128.10, 126.45 (JC–F 27.50 Hz), 125.22, 124.25 (JC–F 271.25 Hz),
123.61, 123.17, 123.07, 121.78, 117.71 (JC–F 5.00 Hz), 50.39,
43.86; ESI-MS: m/z = 515 [M + H]+; mp 198–200 °C; HPLC:
tR = 12.76 min, flow rate 1.2 mL min−1, COSMOSIL 5C18-
MS-II column (4.6 ID × 250 mm), rt, eluent A – 60%, eluent
B – 40%.
3.1.7.9. tert-Butyl 4-(4-((3′-acetamido-[3,6′-biquinolin]-4′-
yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate
1
(41a). Light yellow solid; yield: 66% (for two steps); H NMR
(500 MHz, DMSO-d6): 9.37 (s, 1H, NH), 9.35 (d, 2.5 Hz, 1H,
Ar–H), 9.01 (s, 1H, NH), 8.77–8.75 (m, 2H, Ar–H), 8.61 (d, 2.0
Hz, 1H, Ar–H), 8.22 (dd, 2.0 Hz, 9.0 Hz, 1H, Ar–H), 8.11 (d,
9.0 Hz, 1H, Ar–H), 8.08–8.05 (m, 2H, Ar–H), 7.80–7.76 (m, 1H,
Ar–H), 7.68–7.64 (m, 1H, Ar–H), 7.43 (d, 8.5 Hz, 1H, Ar–H),
7.09 (d, 2.5 Hz, 1H, Ar–H), 6.91 (dd, 2.5 Hz, 1H, 8.5 Hz, Ar–
H), 3.45–3.37 (m, 4H, piperazine–CH2 × 2), 2.73 (t, 5.0 Hz,
4H, piperazine–CH2 × 2), 1.68 (s, 3H, acetyl), 1.40 (s, 9H, CH3
× 3); ESI-MS: m/z = 657 [M + H]+.
3.1.7.10. tert-Butyl 4-(4-((3′-(methylsulfonamido)-[3,6′-biquin-
olin]-4′-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-carbox-
ylate (41b). Light yellow solid; yield: 41% (for two steps); 1H
NMR (500 MHz, DMSO-d6): 9.28 (s, 1H, NH), 9.08 (d, 2.5 Hz,
1H, Ar–H), 8.82 (s, 1H, NH), 8.78 (s, 1H, Ar–H), 8.60 (d, 1.5
Hz, 1H, Ar–H), 8.25 (d, 1.5 Hz, 1H, Ar–H), 8.23 (dd, 1.5 Hz,
8.5 Hz, 1H, Ar–H), 8.14 (d, 8.5 Hz, 1H, Ar–H), 8.04 (d, 8.5 Hz,
1H, Ar–H), 8.00 (d, 8.0 Hz, 1H, Ar–H), 7.81–7.75 (m, 1H, Ar–
H), 7.68–7.62 (m, 1H, Ar–H), 7.45 (d, 8.5 Hz, 1H, Ar–H), 7.20
(d, 2.5 Hz, 1H, Ar–H), 7.00 (dd, 2.5 Hz, 8.5 Hz, 1H, Ar–H),
3.1.8.2.
Hydrochloride
of
N-(4′-((4-(piperazin-1-yl)-3-
(trifluoromethyl)phenyl)amino)-[3,6′-biquinolin]-3′-yl)acetamide
(17). Light yellow solid; yield: 97%; 1H NMR (500 MHz,
DMSO-d6): 11.50 (s, 1H, HCl), 9.80 (s, 1H, piperazine–NH),
9.61 (s, 1H, Ar–H), 9.55 (s, 1H, NH), 9.44 (s, 3H, NH, Ar–H ×
2), 8.77 (s, 1H, Ar–H), 8.64 (s, 1H, Ar–H), 8.33 (s, 1H, Ar–H),
8.20 (s, 2H, Ar–H), 7.94 (s, 1H, Ar–H), 7.80 (s, 1H, Ar–H), 7.52
(s, 2H, Ar–H), 7.46 (s, 1H, Ar–H), 3.15 (s, 4H, piperazine–CH2
× 2), 3.08 (s, 4H, piperazine–CH2 × 2), 1.46 (s, 3H, acetyl);
ESI-MS: m/z = 557 [M + H]+; mp 228–230 °C; HPLC: tR = 10.51
min, flow rate 1.2 mL min−1, COSMOSIL 5C18-MS-II column
(4.6 ID × 250 mm), rt, eluent A – 60%, eluent B – 40%.
3.46–3.33 (m, 4H, piperazine–CH2
× 2), 2.96 (s, 3H,
methylsulfonyl), 2.74 (t, 5.0 Hz, 4H, piperazine–CH2 × 2), 1.40
(s, 9H, CH3 × 3); ESI-MS: m/z = 693 [M + H]+.
3.1.7.11. tert-Butyl 4-(4-((3′-(2,2,2-trifluoroacetamido)-[3,6′-
biquinolin]-4′-yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-
carboxylate (41c). Light yellow solid; yield: 69% (for two
steps); ESI-MS: m/z = 711 [M + H]+.
3.1.8.3.
Hydrochloride
of
N-(4′-((4-(piperazin-1-yl)-3-
(trifluoromethyl)phenyl)amino)-[3,6′-biquinolin]-3′-yl)-
methanesulfonamide (18). Light yellow solid; yield: 98%; 1H
NMR (500 MHz, DMSO-d6): 11.21 (s, 1H, HCl), 9.52 (s, 1H,
sulfonamide–NH), 9.32 (s, 3H, piperazine–NH, NH, Ar–H),
9.03 (s, 1H, Ar–H), 8.86 (s, 1H, Ar–H), 8.74 (s, 1H, Ar–H), 8.57
(dd, 1.5 Hz, 8.5 Hz, 1H, Ar–H), 8.35 (d, 8.5 Hz, 1H, Ar–H),
8.16 (d, 8.5 Hz, 1H, Ar–H), 8.11 (d, 8.5 Hz, 1H, Ar–H), 7.93–
7.87 (m, 1H, Ar–H), 7.80–7.74 (m, 1H, Ar–H), 7.66 (s, 1H, Ar–
H), 7.57 (s, 2H, Ar–H), 3.22–3.14 (m, 4H, piperazine–CH2 × 2),
3.13–3.04 (m, 4H, piperazine–CH2 × 2), 2.97 (s, 3H, CH3); ESI-
MS: m/z = 593 [M + H]+; mp 245–246 °C; HPLC: tR = 10.41
min, flow rate 1.2 mL min−1, COSMOSIL 5C18-MS-II column
(4.6 ID × 250 mm), rt, eluent A – 60%, eluent B – 40%.
3.1.7.12. tert-Butyl 4-(4-((3′-pivalamido-[3,6′-biquinolin]-4′-
yl)amino)-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate
1
(41d). Light yellow solid; yield: 77% (for two steps); H NMR
(500 MHz, DMSO-d6): 9.44 (d, 2.5 Hz, 1H, Ar–H), 9.03 (s, 1H,
NH), 8.83–8.80 (m, 2H, Ar–H, NH), 8.73 (d, 2.0 Hz, 1H, Ar–H),
8.64 (s, 1H, Ar–H), 8.25 (dd, 2.0 Hz, 8.5 Hz, 1H, Ar–H), 8.12
(d, 8.5 Hz, 1H, Ar–H), 8.10–8.06 (m, 2H, Ar–H), 7.80–7.76 (m,
1H, Ar–H), 7.68–7.64 (m, 1H, Ar–H), 7.41 (d, 8.5 Hz, 1H, Ar–
H), 7.07 (d, 2.5 Hz, 1H, Ar–H), 6.94 (dd, 2.5 Hz, 8.5 Hz, 1H,
Ar–H), 3.42–3.35 (m, 4H, piperazine–CH2 × 2), 2.70 (t, 5.0 Hz,
4H, piperazine–CH2 × 2), 1.40 (s, 9H, CH3 × 3), 0.86 (s, 9H,
CH3 × 3); ESI-MS: m/z = 699 [M + H]+.
3.1.8. General procedure for the preparation of com-
pounds 16–20. To a solution of intermediate 40 (0.200 mmol)
in EA (2.00 mL), EA saturated with hydrogen chloride (2.00
mL) was added dropwise at 0 °C. During this process, a little
of the hydrochloride of the Boc-deprotected product precipi-
tated and the suspension was stirred at the room tempera-
ture for 3 h. After removal of EA in vacuo, the resultant solid
was washed with diethylether to give the title compound 16.
3.1.8.4. Hydrochloride of 2,2,2-trifluoro-N-(4′-((4-(piperazin-
1-yl)-3-(trifluoromethyl)phenyl)amino)-[3,6′-biquinolin]-3′-
1
yl)acetamide (19). Light yellow solid; yield: 93%; H NMR (500
MHz, DMSO-d6): 11.94 (s, 1H, HCl), 11.19 (s, 1H, piperazine–
NH), 10.02 (s, 1H, NH), 9.81 (s, 1H, Ar–H), 9.73 (brs, 1H,
NH), 9.53 (s, 2H, Ar–H), 8.86 (s, 1H, Ar–H), 8.68 (d, 7.5 Hz,
1H, Ar–H), 8.31 (d, 8.0 Hz, 1H, Ar–H), 8.28 (d, 8.0 Hz, 1H, Ar–
H), 8.24 (d, 7.0 Hz, 1H, Ar–H), 8.05–7.99 (m, 1H, Ar–H), 7.91–
7.79 (m, 1H, Ar–H), 7.65–7.46 (m, 3H, Ar–H), 3.25–2.99 (m,
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7H, piperazine–H), 2.91 (s, 1H, piperazine–H); 13C NMR (125
MHz, DMSO-d6): δ 155.03 (JC–F 37.50 Hz), 149.99, 148.93,
146.47, 146.66, 146.63, 140.68, 137.56, 136.31, 134.46, 133.75,
132.52, 131.69, 129.69, 129.64, 129.38, 128.54, 126.29 (JC–F
28.75 Hz), 125.09, 124.12, 123.90, 122.84, 123.90 (JC–F 271.25
Hz), 121.95, 121.07, 115.67 (JC–F 287.50 Hz), 112.66, 50.16,
43.79; ESI-MS: m/z = 611 [M + H]+; mp 230–231 °C; HPLC: tR
= 14.78 min, flow rate 1.2 mL min−1, COSMOSIL 5C18-MS-II
column (4.6 ID × 250 mm), rt, eluent A – 60%, eluent B –
40%.
150.10, 147.36, 146.30, 144.54, 140.65, 137.48, 134.71, 133.79,
132.74, 130.59, 130.21, 129.19, 128.88, 128.17, 128.09, 127.63,
126.55 (JC–F 28.75 Hz), 125.46, 124.16, 124.41 (JC–F 271.25 Hz),
122.45, 122.19, 122.03, 115.75 (JC–F 6.25 Hz), 54.06, 53.49,
46.81, 41.92, 23.02, 21.71; ESI-MS: m/z = 599 [M + H]+; mp
195–196 °C; HPLC: tR = 10.66 min, flow rate 1.2 mL min−1,
COSMOSIL 5C18-MS-II column (4.6 ID × 250 mm), rt, eluent
A – 70%, eluent B – 30%.
3.1.9.2. N-(4′-((4-(4-(Methylsulfonyl)piperazin-1-yl)-3-
(trifluoromethyl)phenyl)amino)-[3,6′-biquinolin]-3′-yl)acetamide
(25). Light yellow solid; yield: 84%; 1H NMR (500 MHz,
DMSO-d6): 9.43 (s, 1H, NH), 9.39 (s, 1H, Ar–H), 9.09 (brs, 1H,
NH), 8.78 (d, 8.5 Hz, 2H, Ar–H), 8.65 (s, 1H, Ar–H), 8.26 (d,
8.5 Hz, 1H, Ar–H), 8.14 (d, 8.5 Hz, 1H, Ar–H), 8.09 (d, 8.5 Hz,
2H, Ar–H), 7.86–7.76 (m, 1H, Ar–H), 7.73–7.62 (m, 1H, Ar–H),
7.53 (d, 8.5 Hz, 1H, Ar–H), 7.09 (s, 1H, Ar–H), 6.98 (d, 8.5 Hz,
1H, Ar–H), 3.30–3.12 (m, 4H, piperazine–CH2 × 2), 3.02–2.83
(m, 7H, piperazine–CH2 × 2, methylsulfonyl), 1.70 (s, 3H,
CH3, acetyl); ESI-MS: m/z = 635 [M + H]+; mp 165–166 °C;
HPLC: tR = 9.18 min, flow rate 1.2 mL min−1, COSMOSIL
5C18-MS-II column (4.6 ID × 250 mm), rt, eluent A – 70%, el-
uent B – 30%.
3.1.8.5.
Hydrochloride
of
N-(4′-((4-(piperazin-1-yl)-3-
(trifluoromethyl)phenyl)amino)-[3,6′-biquinolin]-3′-yl)pivalamide
(20). Light yellow solid; yield: 98%; 1H NMR (400 MHz,
DMSO-d6): 11.01 (s, 1H, HCl), 9.67 (s, 1H, piperazine–NH),
9.40 (s, 1H, Ar–H), 9.20–9.10 (m, 4H, Ar–H × 2, NH × 2), 8.79
(s, 1H, Ar–H), 8.62 (d, 8.8 Hz, 1H, Ar–H), 8.33 (d, 8.8 Hz, 1H,
Ar–H), 8.14 (d, 8.0 Hz, 2H, Ar–H), 7.90–7.85 (m, 1H, Ar–H),
7.77–7.72 (m, 1H, Ar–H), 7.54 (d, 8.8 Hz, 1H, Ar–H), 7.47 (s,
1H, Ar–H), 7.42 (d, 8.8 Hz, 1H, Ar–H), 3.22–3.15 (m, 4H, pi-
perazine–CH2 × 2), 3.09–3.02 (m, 4H, piperazine–CH2 × 2),
0.83 (s, 9H, CH3 × 3); ESI-MS: m/z = 599 [M + H]+; mp 217–
219 °C; HPLC: tR = 16.66 min, flow rate 1.2 mL min−1,
COSMOSIL 5C18-MS-II column (4.6 ID × 250 mm), rt, eluent
A – 60%, eluent B – 40%.
3.1.9. General procedure for the preparation of com-
pounds 24–26. To compound 17 (1.0 eq.), anhydrous THF
(5 mL per 1 mmol of substrate) and TEA (3.0 eq.) were added.
Subsequently, acetyl chloride (1.2 eq.) in anhydrous THF
(5 mL per 1 mmol of substrate) was added dropwise to the
resultant suspension 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 a sat-
urated NaHCO3 solution and brine. Following removal of
DCM in vacuo, the residue was subjected to flash column
chromatography with EA/MeOH/TEA (50 : 3 : 1) as the eluent
to give the target compound 24.
3.1.9.3. N-(4′-((4-(4-Acetylpiperazin-1-yl)-3-(trifluoromethyl)-
phenyl)amino)-[3,6′-biquinolin]-3′-yl)pivalamide (26). Light
1
yellow solid; yield: 71%; H NMR (500 MHz, DMSO-d6): 9.44
(d, 2.0 Hz, 1H, Ar–H), 9.03 (s, 1H, NH), 8.81 (s, 2H, Ar–H),
8.74 (d, 2.0 Hz, 1H, Ar–H), 8.65 (s, 1H, NH), 8.25 (dd, 2.0 Hz,
8.5 Hz, 1H, Ar–H), 8.12 (d, 8.5 Hz, 1H, Ar–H), 8.10–8.05 (m,
2H, Ar–H), 7.80–7.75 (m, 1H, Ar–H), 7.68–7.64 (m, 1H, Ar–H),
7.40 (d, 8.5 Hz, 1H, Ar–H), 7.08 (d, 2.5 Hz, 1H, Ar–H), 6.95
(dd, 2.5 Hz, 8.5 Hz, 1H, Ar–H), 3.55–3.45 (m, 4H, piperazine–
CH2 × 2), 2.77–2.73 (m, 2H, piperazine–CH2), 2.71–2.67 (m,
2H, piperazine–CH2), 2.01 (s, 3H, acetyl), 0.85 (s, 9H, CH3
3); ESI-MS: m/z = 641 [M + H]+; mp 157–159 °C; HPLC: tR
×
=
16.23 min, flow rate 1.2 mL min−1, COSMOSIL 5C18-MS-II
column (4.6 ID × 250 mm), rt, eluent A – 70%, eluent B –
30%.
Compound 25 was prepared according to the procedure
for 24, only with acetyl chloride replaced by methylsulfonyl
chloride. Besides, the eluent for flash column chromatogra-
phy was EA/MeOH/TEA (200 : 4 : 1).
3.2. mTOR inhibition assay
Compound 26 was prepared according to the procedure
for 24, only with 17 replaced by 20 as the starting material.
Besides, the eluent for flash column chromatography was EA/
MeOH/TEA (200 : 4 : 1).
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 MnCl, 0.01% Tween-20 and 2 mM DTT. The
kinase solution was prepared by dissolving the kinase in the
kinase buffer and the substrate solution was prepared by dis-
solving the ULight-4E-BP1 peptide substrate and ATP in the
kinase reaction buffer. The compound solution, kinase solu-
tion and substrate solution were added successively to the
wells of the assay plate. The reaction mixture was incubated
at room temperature for 1 h, and then stopped by a kinase
quench buffer containing EDTA and Eu-anti-phospho-4E-BP1
antibody. Before reading on a plate reader, the mixture
needed to be mixed briefly using a centrifuge and was
allowed to equilibrate for 60 min. The inhibition rate was
3.1.9.1. N-(4′-((4-(4-Acetylpiperazin-1-yl)-3-(trifluoromethyl)-
phenyl)amino)-[3,6′-biquinolin]-3′-yl)acetamide (24). White
1
solid; yield: 79%; H NMR (500 MHz, DMSO-d6): 9.40 (s, 1H,
NH), 9.37 (d, 3.0 Hz, 1H, Ar–H), 9.07 (brs, 1H, NH), 8.77 (s,
2H, Ar–H), 8.64 (d, 2.5 Hz, 1H, Ar–H), 8.26 (dd, 2.5 Hz, 11.0
Hz, 1H, Ar–H), 8.14 (d, 11.0 Hz, 1H, Ar–H), 8.10–8.06 (m, 2H,
Ar–H), 7.82–7.77 (m, 1H, Ar–H), 7.70–7.65 (m, 1H, Ar–H), 7.44
(d, 11.0 Hz, 1H, Ar–H), 7.12 (d, 3.0 Hz, 1H, Ar–H), 6.96 (dd,
3.0 Hz, 11.0 Hz, 1H, Ar–H), 3.56–3.51 (m, 4H, piperazine–CH2
× 2), 2.82–2.78 (m, 2H, piperazine–CH2), 2.76–2.72 (m, 2H,
piperazine–CH2), 2.03 (s, 3H, acetyl), 1.70 (s, 3H, CH3, acetyl);
13C NMR (125 MHz, DMSO-d6): δ 168.75, 168.63, 151.30,
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calculated as (max − Lance signal)/(max − min) × 100%.
Herein, “max” stands for the DMSO control, while “min”
stands for the Lance signal of the no enzyme control. Finally,
data were presented in MS Excel and the curves fitted by
GraphPad 5.0. The equation used was: Y = Bottom + (Top −
Bottom)/(1 + (IC50/X)^HillSlope).
while “min” means the RLU of the DMSO control. Finally,
data were presented in MS Excel and the curves fitted by
GraphPad 5.0. The equation used was: Y = Bottom + (Top −
Bottom)/(1 + (IC50/X)^HillSlope).
PI3Kβ inhibitory activity was assessed by ADP-Glo Lumi-
nescent assay. PI3Kβ 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 the reaction mixture
was transferred to a new 384-well plate and an equal amount
of the ADP-Glo reagent was added to each well of the new
assay plate to stop the reaction. After being mixed briefly
using a centrifuge, the resultant mixture was shaken slowly
on the shaker and equilibrated for 40 min. The kinase detec-
tion reagent was then added to each well. The mixture was
shaken for 1 min, equilibrated for 60 min before reading on
a plate reader for luminescence. The percent inhibition was
calculated as (max − sample RLU)/(max − min) × 100. Herein,
“max” stands for the RLU of the DMSO control, while “min”
means the RLU of the no enzyme control. Finally, data were
presented in MS Excel and the curves fitted by GraphPad 5.0.
The equation used was: Y = Bottom + (Top − Bottom)/(1 +
(IC50/X)^HillSlope). PI3Kγ and δ inhibitory activities were
evaluated according to the PI3Kβ inhibition assay.
3.3. 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 concentrations of compounds for 72 h. Subsequently,
cells were washed with PBS and fixed with 10% (w/v) trichlo-
roacetic 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 with 1% acetic acid 5 times,
and the protein-bound dye was extracted with 10 mmol of
unbuffered Tris base. The absorbance was measured at 515
nm using a multiscan spectrophotometer (Thermo Electron
Co., Vantaa, Finland). The inhibition rate on proliferation of
each well was calculated as (A515 control cells − A515 treated
cells)/A515 control cells ×100%. The average IC50 values were
determined by the Logit method from at least two indepen-
dent tests.
3.6. Western blot analysis
3.4. Molecular docking
Cells were treated with final concentrations of 0.1 μM or 0.5
μM of BEZ-235, rapamycin, compound 24, and DMSO and in-
cubated at 37 °C for 2 h. Afterwards, the cells were washed
twice with ice-cold PBS and cell lysis buffer was added.
Then the cellular debris was pelleted by centrifugation at
13 000 rpm for 30 min at 4 °C and the supernatant was
collected. For Western blot analysis, the proteins were sepa-
rated on SDS-PAGE and then transferred onto PVDF mem-
branes. The membranes were incubated with antibodies
against pS6 (Ser235) (Cell Signaling Technology), pAkt
(Ser473) (Cell Signaling Technology) and GAPDH (Santa
Cruz), washed with PBST and then incubated with secondary
antibodies. Finally, the proteins were visualized using enhanced
chemiluminescence.
The co-crystal structure of mTOR in complex with Torin2
(PDB code 4JSX) was used for the docking calculation in
C-DOCKER module (Discovery Studio, version 2.5; Accelrys,
San Diego, CA, USA, 2008). After removal of Torin2 and sol-
vent molecules, the CHARMm-force field was applied to the
protein. The active site was determined according to the loca-
tion of Torin2 in the mTOR enzyme. After removal of Torin2
from the receptor, each ligand was docked into the defined
site. Their final binding conformations were determined
based on the calculated CDOCKING ENERGY.
3.5. Class I PI3Ks selectivity assay
PI3Kα inhibitory activity was assessed by Kinase-Glo Lumi-
nescent Kinase assay. PI3Kα was purchased from Invitrogen
and ATP, DMSO, 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 reac-
tion buffer. The compound solution, kinase solution and
substrate solution were added successively to the wells of the
assay plate. The reaction mixture was incubated at room tem-
perature for 1 h, and then stopped by the Kinase-Glo reagent.
Before reading on a plate reader for luminescence, the mix-
ture needed to be mixed briefly using a 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 the no enzyme control,
3.7. In vitro stability assay
The in vitro stability in SIF, SGF and RLM was evaluated
according to the reported assay with some modification.27
4. Conclusions
With the attempt to modify cellular activity of the initial lead
9, a novel series of 3,4,6-trisubstituted quinoline derivatives
were prepared and biologically evaluated in vitro. After suc-
cessive structural optimizations, we discovered several prom-
ising quinolines, including 17, 24 and 25, which exhibited
not only potent mTOR inhibitory activities, but also remark-
ably enhanced anti-proliferative efficacies compared to 9.
These three quinolines were obtained via introducing a ring-
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opening strategy to BGT-226, an imidazoij4,5-c]quinolinone
derivative. Compound 24, as the most attractive one through-
out this series, displayed an IC50 value of 30 nM against mTOR,
along with IC50 values of 0.11, 0.17 and 0.04 μM against the
HCT-116, PC-3 and MCF-7 cell lines, respectively. Besides, com-
pounds 17 and 24 were identified to be selective over class
I PI3Ks. As illustrated by the further Western blot analysis, com-
pound 24 treatment culminated in a simultaneous inhibition of
mTORC1 and mTORC2, thereby beneficial for surmounting the
hyper-activation of Akt induced by the S6K/IRS1/PI3K negative
feedback loop. Moreover, the representative compound in this
series demonstrated acceptable stability in artificial gastric juice,
intestinal juice as well as rat liver microsome. Due to these advan-
tages, compounds 17, 24 and 25 merit further development
to investigate their in vivo biological profiles.
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15 S. M. Maira, F. Stauffer, J. Brueggen, P. Furet, C. Schnell, C.
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Acknowledgements
The authors thank Jianyang Pan (Institute of Pharmaceutical
Informatics, Zhejiang University, China) for performing NMR
spectroscopy and mass spectrometry for structure elucidation.
19 H. M. Cheng, S. Bagrodia, S. Bailey, M. Edwards, J.
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