A conjugated mTOR/MEK bifunctional inhibitor as potential polypharmacological anticancer agent: the prototype compound discovery

Article abstract of DOI:10.1007/s00044-020-02502-x

mTOR/MEK bifunctional inhibitors have the potential to surmount the drug resistance aroused from cross talk between PI3K/Akt/mTOR (PAM) and Ras/MEK/ERK pathways. Herein, we report the discovery of a conjugated dual-targeted molecule, compound 13, as the p

Full text of DOI:10.1007/s00044-020-02502-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02502-x
ORIGINAL RESEARCH
A conjugated mTOR/MEK bifunctional inhibitor as potential
polypharmacological anticancer agent: the prototype
compound discovery
Qiangqiang Tao1 Fang Fang1,2 Jiaming Li1,2 Yong Wang1 Can Zhao1 Jingtai Liang1 Xiaodong Ma1,2
Hao Wang3
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Received: 17 November 2019 / Accepted: 6 January 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
mTOR/MEK bifunctional inhibitors have the potential to surmount the drug resistance aroused from cross talk between
PI3K/Akt/mTOR (PAM) and Ras/MEK/ERK pathways. Herein, we report the discovery of a conjugated dual-targeted
molecule, compound 13, as the prototype mTOR/MEK bifunctional inhibitor. It exhibited moderately high inhibitory
activity against mTOR and MEK1 with IC50 values of 0.19 μM and 0.98 μM, respectively. In particular, it displayed
attractive antiproliferative activity against both A549 (GI50 = 4.66 μM) and HCT116 (GI50 = 5.47 μM) cell lines. To our
knowledge, it has been the first example of a conjugated mTOR/MEK bifunctional inhibitor. In addition, from this proof-of-
principle study, it has become evident that the single-agent dual inhibition of mTOR and MEK can be fulfilled via covalently
attaching mTOR kinase inhibitor to an allosteric MEK inhibitor.
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Keywords Drug resistance Cross talk mTOR/MEK bifunctional inhibitor mTOR kinase inhibitor Allosteric MEK
inhibitor
Introduction
inhibitors (KIs) suffer from insufficient therapeutic response
attributed to the redundancy in cancer-related survival path-
ways, thereby hindering their further progress. The PI3K/Akt/
mTOR (PAM) pathway inhibitors fit into the scenario, where
monotherapy upregulates compensatory cellular signaling
(Heavey et al. 2016). Ras/MEK/ERK cascade, another pivotal
intracellular survival signaling (Chappell et al. 2011; Tsai and
Nussinov 2013), is involved in molecular reciprocity with
PAM pathway. The cross talk and feedback loop between
them exist due to interaction at various signaling nodes,
exemplified by mTOR (Holt et al. 2012; Zhang et al. 2011;
Chow et al. 2017; Eser et al. 2013; Yang et al. 2012). Besides,
the concurrent deregulation of both pathways contributes to
the etiology of a variety of malignancies (Garcia-Garcia et al.
2015). To conquer the bypass mechanism and achieve
synergism, drug cocktails simultaneously modulating PAM
and Ras/MEK/ERK pathways have been investigated
throughout recent years (Cante-Barrett et al. 2016; Pitts et al.
2014; Mohan et al. 2015; Park et al. 2015). However, owing
to the limitations of drug cocktails, including the combined
off-target effects, complicated pharmacokinetics and ther-
apeutic regimen, as well as potential drug-drug interactions,
the exploration of polypharmacological agents ablating
As a major concern in the research community, drug resis-
tance against single pathway inhibition has posed a con-
siderable obstacle to cancer treatment (Ma et al. 2018).
Particularly, a plethora of clinically investigated kinase
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02502-x) contains supplementary
material, which is available to authorized users.
* Xiaodong Ma
o-omaxiaodong@163.com
* Hao Wang
wanghao986118@163.com
1
School of Pharmacy, Anhui University of Chinese Medicine,
Hefei 230012, China
2
Department of Medicinal Chemistry, Anhui Academy of Chinese
Medicine, Hefei 230012, China
3
Department of Clinical Laboratory, The First Affiliated Hospital of
University of Science and Technology of China, Hefei 230001,
China

Medicinal Chemistry Research
Fig. 1 MXD-42 (1) and its
binding mode with mTOR
Fig. 2 RO-5126766 (2), its
binding mode with MEK1 (Van
Dort et al. 2015), and the
generation of MEK-binding
fragment
multiple disease-related pathways has been given increasing
importance (Ma et al. 2018).
tumor cell lines (Ma et al. 2015). According to the mole-
cular docking analysis, its piperazine moiety oriented
toward the solvent-exposed region. This was consistent with
the structure–activity relationships (SARs) of these quino-
line derivatives, which illustrated appropriate derivatization
at the piperazine did not dramatically alter the enzymatic
activity. In view of these, the piperazine of 1 may serve as a
synthetic handle for attaining conjugated mTOR/MEK
bifunctional inhibitors via being attached to MEK inhibitor
through a chemical linkage. The binding mode of RO-
5126766 (2, Fig. 2), an allosteric MEK inhibitor under
clinical evaluation (phase I, https://clinicaltrials.gov/), with
MEK1 demonstrated its pyridine moiety extended out
towards the solvent-exposed region with the terminal
nitrogen of the sulphonylurea conferring a vector for
structural derivatization (Lito et al. 2014). Furthermore,
SARs study of 2 showed replacement of the pyrimidin-2-
yloxy group with a dimethylaminocarbamate moiety
enhanced MEK affinity (Iikura et al. 2011; Van Dort et al.
2015). Although replacing the 3-fluoropyridine-2-
aminosulfonamide group with a simpler 3-(sulfonamido)
phenyl moiety slightly lowered the activity, it simplified the
As a well-established therapeutic target along PAM
cascade, mTOR has been concomitantly inhibited with
MEK in drug combinations for improving the therapeutic
outcome. For instance, combination of AZD-8055 (ATP-
competitive mTOR inhibitor) and selumetinib (MEK inhi-
bitor) culminated in the reciprocal pathway inhibition and
increased apoptosis in tumor tissue from mouse xenograft
models of non-small cell lung cancer and colorectal carci-
noma (Holt et al. 2012). In addition, the synergism between
them was also observed in treating rhabdomyosarcoma
(Renshaw et al. 2013). Nonetheless, to our knowledge, no
mTOR/MEK bifunctional inhibitors have been disclosed so
far. Bifunctional inhibitors of this kind and the recently
reported PI3K/MEK dual inhibitors (Van Dort et al. 2017)
have the potential to provide novel remedies for cancer
treatment.
During our previous work in search for mTOR KI, a
promising quinoline derivative MXD-42 (1, Fig. 1) was
identified with remarkable inhibitory activity against
mTOR, as well as potent antiproliferative activity against

Medicinal Chemistry Research
covalent linking to the mTOR-binding fragment (Fig. 2).
Based on the considerations stated above, we have recently
conducted a proof-of-principle study to validate the feasi-
bility of achieving mTOR/MEK dual inhibition via cova-
lently linking mTOR KI to an allosteric MEK inhibitor.
Herein, we communicate our discovery of a conjugated
mTOR/MEK1 dual-targeted inhibitor from 1 and 2 (Fig. 3).
of 1-(bromomethyl)-3-nitrobenzene 4 in the presence of
NaH. The resultant intermediate 5 was then condensed with
resorcinol to give the coumarin derivative 6. N, N-dimethyl
carbamylation of its hydroxyl functionality and the fol-
lowing reduction of nitro group provided the aniline deri-
vative 8. Afterwards, it was condensed with 3-
chloropropane-1-sulfonyl chloride to afford the sulfona-
mide 9. Subsequent N-methylation at the sulfonamide
moiety of 9 furnished 10 as the key intermediate. It was
subjected to nucleophilic substitution with 1 to provide the
conjugated compound 11. Condensation of 1 with 4-((tert-
butoxycarbonyl)amino)butanoic acid, and the subsequent
removal of the Boc-protecting group generated the primary
amine, which was used directly for the next step without
further purification. Ultimately, the newly afforded Boc-
deprotected product underwent nucleophilic substitution
with 10 to provide the conjugated compound 13.
Results and discussion
Chemistry
The synthesis of conjugated compounds 11 and 13 was
displayed in Scheme 1. Ethyl acetoacetate 3, as the starting
material, firstly experienced benzylation upon the treatment
Reagents and conditions
(a) NaH (60%), tetrahydrofuran (THF), 0 °C to rt, N2; (b)
resorcinol, H2SO4 (conc.), 0 °C to rt; (c) K2CO3, dimethyl-
carbamyl chloride, N, N-dimethylformamide (DMF), 40 °C;
(d) SnCl2 (dihydrate), EtOAc (EA), 50 °C, N2; (e) 3-
chloropropane-1-sulfonyl chloride, pyridine, rt; (f) MeI,
Cs2CO3, DMF, rt; (g) 1, K2CO3, KI, triethylamine (TEA),
CH3CN, reflux, N2; (h) 4-((tert-butoxycarbonyl)amino)
Fig. 3 The design rationale of target conjugated mTOR/MEK
bifunctional inhibitors
Scheme 1 Synthetic route for
target compounds

Medicinal Chemistry Research
Table 1 The enzymatic and antiproliferative activities of target compounds
mTOR
MEK1
A549
HCT116
Cpd.
Linker
(IC50, μM)
(IC50, μM) (GI50, μM)
(GI50, μM)
2.36
4.97
0.98
a
a
11
13
0.19
4.66
5.47
---
---
0.014
>10
>10
2.80
5.07
5.06
3.23
MXD-42
0.16
RO-5126766
a Unidentified.
butanoic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide hydrochloride (EDCI), 1-hydroxybenzotriazole
(HOBT), TEA, dichloromethane (DCM), rt; (i) (1) tri-
fluoroacetic acid (TFA), DCM, 0 °C to rt; (2) 10, K2CO3, KI,
CH3CN, reflux, N2.
evident that the linker was an important structural factor
affecting potency. Thus, in further SAR study, the impact of
various chemical linkers on activity would be probed.
Molecular docking
The in vitro biological evaluation of target
conjugated compounds
Molecular docking analysis of 13 was then conducted to
elucidate its possible binding modes with mTOR and
MEK1. As illustrated by Fig. 4a, the mTOR-binding frag-
ment of 13 was engaged in H-bond contacts with residues
Val2240 in the hinge region and Tyr2225 in the affinity
pocket. Meanwhile, the linker tethered to the piperazine
moiety, as well as the MEK-binding fragment, was located
in a solvent-exposed region. When docked into the allos-
teric pocket of MEK1 (Fig. 4b), the MEK-binding fragment
of 13 conferred H-bond interactions with residues Val211,
Ser212, and Asp208. Similarly, the linker and the mTOR-
binding fragment extended out of the catalytic cleft. The
capability of 13 to form H-bonds with critical residues in
the mTOR and MEK1 catalytic clefts may account for its
dual inhibitory activity.
The conjugated compounds 11 and 13 were evaluated in the
biochemical assay against mTOR and MEK1, as well as the
antiproliferative assay against A549 and HCT116 cancer cell
lines. Compound 13 exhibited moderately potent inhibitory
activity against both mTOR and MEK1 with IC50 values of
0.19 μM and 0.98 μM, respectively. Besides, it demonstrated
attractive antiproliferative activity against A549 and HCT116
cell lines with GI50 values of 4.66 μM and 5.47 μM, respec-
tively (Table 1).
Importantly, although its mTOR inhibitory activity and
MEK1 inhibitory activity were inferior to MXD-42 and RO-
5126766, respectively, 13 exerted comparable antiproliferative
activity to both reference compounds against the tested two
cell lines. This discrepancy between the enzymatic activity and
cellular activity indicated that a synergism might be fulfilled by
13 via concurrent inhibition of mTOR and MEK1. The sub-
micromolar bifunctional enzymatic activity and single-digit
micromolar cellular potency rendered 13 as a potential lead for
further optimization. When comparing the enzymatic activity
of compounds 11 and 13, which share the same mTOR-
binding fragment and MEK-binding fragment, it became
Conclusions and perspectives
The reciprocal pathway inhibition, along with the enhanced
antitumor efficacy, resulted from mTOR inhibitor/MEK
inhibitor drug cocktail has provided a fundamental basis for
the exploration of polypharmacological agents targeting
both mTOR and MEK. Besides the potential to evade the

Medicinal Chemistry Research
Fig. 4 The molecular docking of
compound 13 into the catalytic
pocket of mTOR (a) and MEK1
(b)
bypass mechanism underlying the resistance to singe path-
way inhibition, the bifunctional inhibitors of mTOR and
MEK are capable of surmounting the drawbacks of drug
cocktails. From this proof-of-principle study, it became
evident that the single-agent dual inhibition of mTOR and
MEK can be accomplished via covalently attaching mTOR
KI to an allosteric MEK inhibitor. Compound 13, emerging
from this strategy, exerted moderately high inhibitory
activity against mTOR and MEK1 with IC50 values of
0.19 μM and 0.98 μM, respectively. Particularly, it exhibited
comparable antiproliferative activity to both parent com-
pounds against A549 and HCT116 cell lines. To our
knowledge, 13 has been the first conjugated mTOR/MEK
bifunctional inhibitor reported up to date. However, this
prototype dual-targeted inhibitor demands further optimi-
zation of the enzyme affinity and the drug-like property.
Hence, in the future SAR investigation, MEK-binding
fragments generated from more potent allosteric MEK
inhibitors will be incorporated, and the chemical diversity
of the covalent linkers will be broadened.
Experimental section
Chemistry
All the reagents and solvents were purchased from common
commercial suppliers. If necessary, purification was carried
out prior to use. NMR spectra were recorded on a Bruker
Avance 400 II (400 MHz) spectrometer in the indicated
solvent. While ESI-MS spectra were obtained by Bruker
Esquire-LC-00075 spectrometer, HRMS spectra were
recorded on Waters Q-TOF Micromass. Melting points
were uncorrected and determined on a WRS-1B melting
point apparatus. Flash column chromatography was per-
formed using silica gel (200–300 mesh).
The preparation of ethyl 2-(3-nitrobenzyl)-3-oxobutanoate
(5)
The intermediate was prepared according to a reported
protocol (Iikura et al. 2011). To a suspension of NaH (60%,

Medicinal Chemistry Research
1
1.01 g, 25.3 mmol) in THF (30 mL) was added ethyl acet-
oacetate (2.9 mL, 23.0 mmol) at 0 °C, and the resultant
mixture was stirred at the same temperature for 0.5 h.
Afterwards, the solution of 1-(bromomethyl)-3-nitro-
benzene (5.00 g, 23.0 mmol) in THF (20 mL) was added
dropwise. The reaction mixture was stirred at room tem-
perature overnight, and quenched with ice water. Following
extraction with EA, the organic layer was washed with
brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The crude product was subjected to flash column
chromatography [EA/petroleum ether (PE) = 1:30] to give
the title compound as a yellow oil. Yield: 53%.
pale solid. Yield: 93%; H NMR (400 MHz, DMSO-d6): δ
7.84 (d, 8.8 Hz, 1H), 7.25 (d, 2.0 Hz, 1H), 7.18 (dd, 2.4 Hz,
8.8 Hz, 1H), 6.95–6.86 (m, 1H), 6.43–6.33 (m, 3H), 4.96 (s,
2H), 3.84 (s, 2H), 3.08 (s, 3H), 2.94 (s, 3H), 2.44 (s, 3H);
ESI-MS: m/z = 353 [M + H]+; m.p. 155–156 °C.
The preparation of 3-(3-((3-chloropropyl)sulfonamido)
benzyl)-4-methyl-2-oxo-2H-chromen-7-yl
dimethylcarbamate (9)
To a solution of 8 (1.5 g, 4.26 mmol) in pyridine (6 mL) was
added 3-chloropropane-1-sulfonyl chloride (0.83 g, 4.69 mmol),
and the resultant reaction mixture was stirred at room tem-
perature for 1 h. Then, the reaction mixture was subjected to
flash column chromatography (DCM/EA = 10:1–8:1) to give
The preparation of 7-hydroxy-4-methyl-3-(3-nitrobenzyl)-
2H-chromen-2-one (6)
1
the title intermediate as a pale solid. Yield: 81%; H NMR
The intermediate was prepared according to a reported
protocol (Iikura et al. 2011). To a mixture of resorcinol
(0.83 g, 7.55 mmol) and 5 (2.00 g, 7.55 mmol) was added
H2SO4 (conc., 1.5 mL) at 0 °C, and the resultant mixture
was stirred at room temperature overnight. After pouring the
reaction mixture to ice water, the formed precipitate was
filtered off, and washed successively with H2O and MeOH
to provide the title intermediate as a slight yellow powder.
Yield: 61%. m.p. 216–217 °C.
(400 MHz, DMSO-d6): δ 9.79 (s, 1H), 7.85 (d, 8.8 Hz, 1H),
7.29–7.22 (m, 2H), 7.18 (dd, 2.4 Hz, 8.8 Hz, 1H), 7.12–7.04
(m, 2H), 6.99 (d, 7.6 Hz, 1H), 3.97 (s, 2H), 3.68 (t, 6.4 Hz, 2H),
3.23–3.14 (m, 2H), 3.07 (s, 3H), 2.94 (s, 3H), 2.47 (s, 3H),
2.12–2.03 (m, 2H); ESI-MS: m/z = 493 [M + H]+; m.p.
162–165 °C.
The preparation of 3-(3-((3-chloropropyl)-N-
methylsulfonamido)benzyl)-4-methyl-2-oxo-2H-chromen-7-
yl dimethylcarbamate (10)
The preparation of 4-methyl-3-(3-nitrobenzyl)-2-oxo-2H-
chromen-7-yl dimethylcarbamate (7)
The intermediate was prepared according to a reported
protocol (Van Dort et al. 2015). The mixture of 9 (1.0 g,
2.03 mmol), MeI (0.94 g, 6.70 mmol), Cs2CO3 (1.32 g,
4.06 mmol), and DMF was stirred at room temperature for
3 h. Afterwards, the reaction mixture was extracted with
EA, and washed successively with H2O and brine. The
organic layer was dried over anhydrous Na2SO4, and con-
centrated in vacuo to provide the crude product. Further
flash column chromatography (DCM/EA = 10:1) gave the
To a solution of 6 (3.11 g, 10.0 mmol) in DMF (20 mL)
were added anhydrous K2CO3 (1.52 g, 11.0 mmol) and
dimethylcarbamyl chloride (1.1 mL, 12.0 mmol). The
resultant reaction mixture was stirred at 40 °C for 4 h, and
ice water was added. After collection of the crude product
by filtration, flash column chromatography (EA/PE = 1:3)
was performed to give the title intermediate as a pale solid.
1
1
Yield: 89%; H NMR (400 MHz, DMSO-d6): δ 8.15–8.11
title intermediate as a white foam. Yield: 94%; H NMR
(m, 1H), 8.10–8.05 (m, 1H), 7.87 (d, 8.8 Hz, 1H), 7.72 (d,
7.6 Hz, 1H), 7.58 (t, 8.0 Hz, 1H), 7.26 (d, 2.4 Hz, 1H), 7.20
(dd, 2.4 Hz, 8.8 Hz, 1H), 4.14 (s, 2H), 3.07 (s, 3H), 2.94 (s,
3H), 2.52 (s, 3H); ESI-MS: m/z = 383 [M + H]+; m.p.
192–194 °C.
(400 MHz, DMSO-d6): 7.84 (d, 8.8 Hz, 1H), 7.37–7.23 (m,
4H), 7.22–7.13 (m, 2H), 4.01 (s, 2H), 3.69 (t, 6.4 Hz, 2H),
3.28–3.19 (m, 5H), 3.07 (s, 3H), 2.94 (s, 3H), 2.48 (s, 3H),
2.12–2.03 (m, 2H); ESI-MS: m/z = 507 [M + H]+.
The preparation of 3-(3-((3-(4-(4-((3′-carbamoyl-[3,6′
-biquinolin]-4′-yl)amino)-2-(trifluoromethyl)phenyl)
piperazin-1-yl)-propyl)-N-methylsulfonamido)benzyl)-4-
methyl-2-oxo-2H-chromen-7-yl dimethylcarbamate (11)
The preparation of 3-(3-aminobenzyl)-4-methyl-2-oxo-2H-
chromen-7-yl dimethylcarbamate (8)
To a solution of 7 (1.00 g, 2.72 mmol) in EA was added
SnCl2 (dihydrate) (3.10 g, 13.6 mmol), and the resultant
mixture was stirred at 50 °C under N2 atmosphere for 4 h.
After adding EA to the mixture, it was quenched with
saturated NaHCO3 solution at 0 °C, and washed with water.
The organic layer was dried over anhydrous Na2SO4, and
concentrated in vacuo to provide the title intermediate as a
The mixture of 1 (145 mg, 0.25 mmol), 10 (106 mg,
0.21 mmol), K2CO3 (58 mg, 0.42 mmol), KI (70 mg,
0.42 mmol), TEA (58 μL, 0.42 mmol), and anhydrous
CH3CN (2 mL) was refluxed under N2 atmosphere for 12 h.
Afterwards, the mixture was concentrated in vacuo, and the
residue was directly subjected to flash column

Medicinal Chemistry Research
chromatography (EA/MeOH/TEA = 50:5:1–100:15:2) to
vacuo, the Boc-deprotected product was afforded as a slight
yellow foam, which was directly used for the next reaction
without further purification. ESI-MS: m/z = 628 [M + H]+.
The mixture of 10 (0.095 g, 0.19 mmol), the Boc-
deprotected product (0.24 g, 0.38 mmol, calculated as the
pure product), K2CO3 (0.052 g, 0.38 mmol), KI (0.063 g,
0.38 mmol), and anhydrous CH3CN (2 mL) was refluxed
under N2 atmosphere for 8 h. Then, the mixture was con-
centrated in vacuo, and DCM/MeOH (1:1, V:V) was added
to the residue. After filtration, the filtrate was concentrated
in vacuo. The residue was subjected to flash column chro-
matography (EA/MeOH/TEA = 50:5:1–100:15:2) to give
13 as a slight yellow hygroscopic solid. Yield: 62% (for two
steps). 1H NMR (400 MHz, DMSO-d6): 10.34 (s, 1H), 9.04
(s, 1H), 8.97 (s, 1H), 8.65–8.52 (m, 1H), 8.35 (s, 1H), 8.28
(d, 8.8 Hz, 1H), 8.19 (s, 1H), 8.12 (d, 8.8 Hz, 1H), 8.05 (d,
8.4 Hz, 1H), 8.00 (d, 8.0 Hz, 1H), 7.87–7.75 (m, 2H),
7.72–7.59 (m, 2H), 7.49 (d, 8.4 Hz, 1H), 7.42 (s, 1H),
7.37–7.22 (m, 4H), 7.21–7.09 (m, 2H), 4.06–3.92 (m, 2H),
3.63–3.50 (m, 4H), 3.28–3.18 (m, 7H), 3.05 (s, 3H), 2.92 (s,
3H), 2.88–2.75 (m, 6H), 2.49–2.42 (m, 5H), 2.03–1.90 (m,
2H), 1.85–1.73 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
170.06, 168.99, 160.82, 153.27, 153.23, 152.11, 150.56,
149.19, 149.09, 148.18, 146.82, 146.78, 146.22, 141.41,
140.77, 140.04, 133.61, 133.10, 131.94, 130.48, 129.80,
129.50, 129.11, 128.73, 128.25, 127.44, 127.22, 126.69,
126.47, 126.43 (q, JC–F = 270.2 Hz), 126.38, 126.23,
125.69, 124.37, 123.93, 122.94, 122.87, 120.75, 118.40,
118.18 (q, JC–F = 5.8 Hz), 117.26, 114.53, 109.57, 53.42,
53.01, 47.03, 46.00, 45.66, 45.39, 41.60, 37.96, 36.33,
36.13, 32.08, 29.55, 22.29, 20.84, 15.31; ESI-HRMS: m/z
calcd for C58H58F3N9O8S [M + H]+ 1098.4159, found
1098.4156; m.p. 147–148 °C.
give the title compound as a slight yellow hygroscopic
1
solid. Yield: 57%. H NMR (400 MHz, DMSO-d6): 10.49
(brs, 1H), 9.56 (brs, 1H), 9.17–8.78 (m, 2H), 8.62 (s, 1H),
8.47–7.95 (m, 5H), 7.92–7.60 (m, 4H), 7.59–6.86 (m, 9H),
4.01 (s, 3H), 3.28–2.84 (m, 23H), 2.14–1.94 (m, 2H); 13C
NMR (100 MHz, DMSO-d6): 168.68, 160.81, 153.25,
153.24, 152.10, 149.11, 148.16, 146.84, 146.81, 141.36,
140.13, 140.10, 133.87, 133.20, 131.77, 129.99 (q, JC–F
=
3.8 Hz), 129.89, 129.86, 129.61, 129.15, 128.71, 128.29,
127.41, 127.27, 126.69, 126.59, 126.57, 126.41, 126.34,
126.29, 126.24, 125.47, 124.86, 123.96, 123.58 (q, JC–F
=
270.0 Hz), 122.93, 122.85, 120.56, 119.45, 118.38, 117.25,
109.54, 52.07, 45.79, 37.99, 36.32, 36.12, 32.12, 20.72,
15.31, 14.04; ESI-HRMS: m/z calcd for C54H51F3N8O7S
[M + H]+ 1013.3632, found 1013.3636; m.p. 134–137 °C.
The preparation of tert-butyl (4-(4-(4-((3′-carbamoyl-[3,6′-
biquinolin]-4′-yl)amino)-2-(trifluoromethyl)phenyl)
piperazin-1-yl)-4-oxobutyl)carbamate (12)
The solution of 4-((tert-butoxycarbonyl)amino)butanoic acid
(212 mg, 1.04 mmol), EDCI (301 mg, 1.57 mmol) and
HOBT (141 mg, 1.04 mmol) in DCM (4 mL) was stirred at
room temperature for 1 h. Then, 1 (301 mg, 0.52 mmol) and
TEA (432 μL, 3.12 mmol) were added successively, and the
resultant mixture was stirred at room temperature for 4 h.
After quenching with saturated NaHCO3 solution at 0 °C, the
organic layer was dried over anhydrous Na2SO4, and con-
centrated in vacuo. The residue was subjected to flash col-
umn chromatography (EA/MeOH/TEA = 150:3:2–150:4:2)
to give the title intermediate as a slight yellow solid. Yield:
1
70%; H NMR (400 MHz, DMSO-d6): 10.31 (s, 1H), 9.05
(d, 2.0 Hz, 1H), 8.96 (s, 1H), 8.58 (d, 1.6 Hz, 1H), 8.34 (d,
1.6 Hz, 1H), 8.29 (dd, 2.0 Hz, 8.8 Hz, 1H), 8.17 (brs, 1H),
8.13 (d, 8.4 Hz, 1H), 8.07 (d, 8.4 Hz, 1H), 8.02 (d, 8.0 Hz,
1H), 7.84–7.77 (m, 1H), 7.72–7.60 (m, 2H), 7.51 (d, 8.8 Hz,
1H), 7.41 (d, 2.4 Hz, 1H), 7.30 (dd, 2.0 Hz, 8.4 Hz, 1H),
6.83 (t, 4.8 Hz, 1H), 3.65–3.46 (m, 4H), 3.02–2.92 (m, 2H),
2.90–2.75 (m, 4H), 2.34 (t, 7.2 Hz, 1H), 1.71–1.57 (m, 2H),
1.38 (s, 9H); ESI-MS: m/z = 728 [M + H]+; m.p.
131–135 °C.
Biology
In vitro enzymatic assay
The Lance Ultra assay and Mobility Shift assay were per-
formed to identify the mTOR inhibitory activity and MEK1
inhibitory activity, respectively.
In the Lance Ultra assay, the compound solutions were
prepared at fourfold of the final concentrations via serial
dilution with DMSO and the subsequent dilution with the
kinase buffer. The kinase solution was prepared with
the kinase buffer at fourfold of the final concentration. The
substrate solution containing ULight-4E-BP1 peptide
(Thr37/46, PE) and ATP was prepared with the kinase buffer
at twofold of the final concentration. The kinase reaction
mixture in each well was composed of 2.5 μL of compound
solution, 2.5 μL of kinase solution, and 5 μL of substrate
solution. After being incubated at room temperature for
0.5 h, 10 μL of kinase detection buffer containing EDTA
The preparation of 3-(3-((3-((4-(4-(4-((3′-carbamoyl-[3,6′-
biquinolin]-4′-yl)amino)-2-(trifluoromethyl)phenyl)
piperazin-1-yl)-4-oxobutyl)amino)-propyl)-N-
methylsulfonamido)benzyl)-4-methyl-2-oxo-2H-chromen-7-
yl dimethylcarbamate (13)
The intermediate 12 was dissolved in DCM (4 mL), and to
the solution was added TFA (1 mL) dropwise at 0 °C.
Subsequently, the resultant mixture was stirred at room
temperature for 4 h. After concentrating the mixture in

Medicinal Chemistry Research
and Eu-anti-phospho-4E-BP1 antibody (Thr37/46, PE) was
added to each well of the assay plate. Before reading on
Envision, the mixture need be mixed briefly with centrifuge
and was allowed to equilibrate for 1 h. After calculating
the percent inhibition from lance signal, the curves were
fitted with four-parameter logistic model in XLFit to give
IC50 values.
mTOR or MEK1, the final binding conformation in mTOR
or MEK was determined according to the calculated energy.
Acknowledgements The authors acknowledge the financial support of
the Natural Science Foundation of Anhui Province (1808085QH261),
the Key Project of Natural Science Research in Universities of Anhui
Province (KJ2019A1004), and University-Enterprise Cooperative
Projects (2018HZ6 and 2019HZ078).
In the Mobility Shift assay, the compound solutions were
prepared at fivefold of the final concentrations via serial
dilution with DMSO and the subsequent dilution with the
kinase buffer. Both the kinase solution and the peptide solu-
tion containing FAM-labeled peptide and ATP were prepared
with the kinase buffer at 2.5-fold of the final concentration.
The kinase reaction mixture in each well was composed of
5 μL of compound solution, 10 μL of kinase solution, and
10 μL of substrate solution. 25 μL of stop buffer [2×, 100 mM
HEPES (pH = 7.5), 0.015% Brij-35, 0.2% coating reagent,
50 mM EDTA] was added to quench the kinase reaction.
After the data collection on Caliper, the percent inhibition
was calculated, and the curves were fitted with four-
parameter logistic model in XLFit to give IC50 values.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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