Targeting Cysteine Located Outside the Active Site: An Effective Strategy for Covalent ALKi Design

Article abstract of DOI:10.1021/acs.jmedchem.0c01707

Potent inhibitors of ALK are highly desired because of the occurrence of drug resistance. We herein firstly report the development of a rationally designed inhibitor, Con B-1, which can covalently bind to Cys1259, a cysteine located outside the ALK active site by linking a warhead with Ceritinib through a 2,2′-Oxybis(ethylamine) linker. The in vitro and in vivo assays showed ConB-1 is a potent selective ALKi with low toxicity to normal cells. In addition, the molecule showed significant improvement of anticancer activities and potential antidrug resistant activity compared with Ceritinib, demonstrating the covalent inhibitor of ALK can be a promising drug candidate for the treatment of NSCLC. This work may provide a novel perspective on the design of covalent inhibitors.

Full text of DOI:10.1021/acs.jmedchem.0c01707

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Article
Targeting Cysteine Located Outside the Active Site: An Effective
Strategy for Covalent ALKi Design
Guoyi Yan,¶ Xinxin Zhong,¶ Chunlan Pu,¶ Lin Yue, Huifang Shan, Suke Lan, Meng Zhou, Xueyan Hou,
Jie Yang, Deyu Li, Shilong Fan,* and Rui Li*
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ABSTRACT: Potent inhibitors of ALK are highly desired because of the occurrence of drug resistance. We herein firstly report the
development of a rationally designed inhibitor, Con B-1, which can covalently bind to Cys1259, a cysteine located outside the ALK
active site by linking a warhead with Ceritinib through a 2,2′-Oxybis(ethylamine) linker. The in vitro and in vivo assays showed
ConB-1 is a potent selective ALKi with low toxicity to normal cells. In addition, the molecule showed significant improvement of
anticancer activities and potential antidrug resistant activity compared with Ceritinib, demonstrating the covalent inhibitor of ALK
can be a promising drug candidate for the treatment of NSCLC. This work may provide a novel perspective on the design of
covalent inhibitors.
residue in the active pocket.17 However, more than half of
INTRODUCTION
■
kinase targets bear no such cysteine residue.11a,18 In this work,
ALK is a member of the family of insulin-like tyrosine kinase
we describe the design of covalent inhibitors targeting the
Cys1259 located outside the active pocket of ALK by
incorporating a warhead to Ceritinib utilizing a linker. Through
exploring the linker spacing and component, ConB-1 was
identified, which showed potent, highly selective ALK
inhibition activity and superior anticancer activity. This work
showcases a strategy of covalent inhibitor design by targeting
the cysteine outside the active site, which might be applicable
receptors.1 It was proved that ALK is a strong carcinogenic
gene that enhances the development of a variety of cancers.2
The incidence of ALK gene rearrangement in NSCLC is 3−
7%.3 Patients diagnosed with ALK-rearranged lung cancer can
benefit from treatment with ALK tyrosine kinase inhibitors.4
FDA-approved drugs that target the abnormal ALK protein are
Crizotinib,5 Ceritinib,6 Alectinib,7 Brigatinib,8 and Lorlatinib.9
Nevertheless, acquired resistance to these drugs usually
emerges after one year of treatment, and it is still an urgent
need to develop novel and potent ALKis.10
to other therapeutic targets (Figure 1).
RESULTS AND DISCUSSION
■
Covalent inhibitors have attracted more and more attention
in dealing with the insufficient activity and drug resistance in
drug research because of the improved potency and prolonged
duration of action.11 Meanwhile, covalent inhibitors especially
with acrylamide as the warhead, such as Osimertinib,12
Ibrutinib,13 Neratinib14 and Afatinib,15 which have all been
approved by the FDA in recent years, can also be safe.16
With the development of structural biology and computer-
aided drug-design technology, the rational design of covalent
inhibitors has made great progress by targeting the cysteine
Structure-Guided Design of ALK Covalent Inhibitors.
To develop a covalent inhibitor for ALK, Ceritinib, a potent
Received: September 29, 2020
Published: January 20, 2021
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condensation reaction was in progress between intermediate
d1 and c1 by stirring with HATU and DIPEA in DMF to
afford target molecule Con B-1.
In Vitro Bioassays and SAR. The biochemical potency of
the compounds to ALK were examined at two concentrations
(200 nM and 20 nM). Among all the molecules, four
compounds in Con A series (Con A-1, 2, 3, 4) and three in
Con B series (Con B-1, 2, 3) showed potent inhibition of ALK
(Figure 3a, Table S1). It is worth noticing that the molecues
with n2 = 1 showed low inhibitory activity against ALK
compared with other molecules, which might be ascribed to
the lower flexibility of the methylene group that could not
allow the warhead to adopt the suitable direction to form a
covalent bond with the terminal Cys1259.
To investigate the functional consequence of inhibiting ALK
in cells, five cancer cell lines (H3122, H2228, H1299, A549,
and Hela) were treated with the compounds at different
concentrations, and the growth was monitored through the
MTT assay (Table S2). Among the cell lines, H3122 and
H2228 were reported as ALK-positive human NSCLC cell
lines.19,21 As despicted in Figure 3b, five of the compounds
displayed potent antiproliferative activities (Con A-1, Con A-
3, Con A-4, Con B-1, and Con B-3), and four of them showed
advantaged bioactivity compared with Ceritinib (Figure 3c).
On the other hand, the toxicity of covalent inhibitors should
not be underestimated. The biologically active molecules were
tested against normal human liver cell line LO2 to learn their
toxicities. As a result, at a concentration of 5 μM, all the
compounds showed a similar inhibition rate to the cells
(Figure 3d, Table S3), indicating that compared with Ceritinib,
the covalent inhibition of ALK do not increase the toxicity to
normal cells. Because of the highest potency in in vitro
bioactivity and low toxicity to normal cells, Con B-1 was
selected for further biological and mechanism investigation.
High selectivity may be the most important feature in the
design of a safe and effective covalent inhibitor.11a,22 In this
study, to learn the selectivity of Con B-1, several targets
(IGF1R, INSR, FLT3, and FGFR2) were selected because they
were the main targets of Ceritinib.6 As a result, Con B-1
Figure 1. (a) Schematic illustration of molecular design in this work;
(b) Molecular structure components of Con B-1.
ALKi approved by FDA for treatment of NSCLC, was used as
the template (Figure 2a). Through observing the cocrystal
structure of ALK and Ceritinib (pdb: 4MKC),19 we noticed
that Cys1259, although located outside of the Ceritinib
binding site, can still be a promising residue for covalent
targeting by linking a warhead with Ceritinib through a linker
(Figure 2b). As for the warhead, acrylamide was selected as the
electrophile because of its convenient synthesis and inertness
toward glutathione.20 The length and composition of the linker
are crucial for the activity, because without a proper linker, the
warhead with linker may not only fail to form a covalent bond
with Cys1259 but also impede the noncovalent binding of
Ceritinib part with ALK. So, in this study, two kind of linkers
with different lengths were examined (the structures were
depicted in Figure 2c)
Chemistry. The synthetic approach for the target
compounds were outlined in Schemes 1 and 2. The
compounds in the two series shared similar synthetic methods.
For Con B-1 as an example, firstly, installation of the
acrylamide group on a1 under basic conditions afforded b1
in overall moderate yield; secondly, tert butoxycarbonyl moiety
of intermediate b1 was removed by stirring in trifluoroacetic
acid for 30 min to obtain compound c1; finally, amide
showed high selectivity, with IC value of 0.5 nM to ALK
50
comparing with 7.1, 12.6, 875, and >1000 nM to IGF1R,
INSR, FLT3, and FGFR2, respectively (Figure 4a, Table S4).
Figure 2. (a) Chemical structure of Ceritinib; (b) Binding mode of Ceritinib in ALK (PDB ID:4MKC); (c) Chemical structures of designed
molecules.
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a
Scheme 1. Synthesis of Compounds in Con B Series
a
Reagents and conditions: (i) acryloyl chloride, DIPEA, THF, ice bath; (ii) TFA, 50 °C; (iii) for n2 = 1: Ceritinib, tert-Butyl bromoacetate, K2CO3,
Acetonitrile, reflux, TFA; for n2 = 2: Ceritinib, Methyl acrylate, Et3N, Methanol, rt, NaOH, Methanol/H2O, 60 °C; (iv) HATU, DIPEA, DMF, rt.
a
Scheme 2. Synthesis of Compounds in Con A Series
a
Reagents and conditions: (i) acryloyl chloride, DIPEA, THF, ice bath; (ii) TFA, 50 °C; (iii) for n2 = 1: Ceritinib, tert-Butyl bromoacetate, K2CO3,
Acetonitrile, reflux, TFA; for n2 = 2: Ceritinib, Methyl acrylate, Et3N, Methanol, rt, NaOH, Methanol/H2O, 60 °C; (iv) HATU, DIPEA, DMF, rt.
Determination of Covalent Inhibition of ConB-1. First,
to prove the ability of acrylamide in binding with the Cys1259,
a new molecule (Re-ConB-1) with the double bond of
acrylamide group changed to a single bond was synthesized,
and it showed a dramatic decrease by 25-fold (0.5 nM vs 13.2
nM) in binding activity against ALK when compared with
ConB-1 (Figure 4b,c), demonstrating the great contribution of
covalent binding to the bioactivity. Next, to further investigate
the covalent binding ability, ConB-1 was incubated with L-
cysteine (the molar ratio of L-cysteine and Con B-1 = 1:5), and
LC-MS analysis revealed a peak in the LC trace with a mass of
891.3649 Da, corresponding to addition of one molecule of L-
cysteine to ConB-1 (Figure.S1). Furthermore, to confirm that
ConB-1 can covalently bind to ALK, an excess amount of
ConB-1 was preincubated with ALK for 4 h, and a peak at m/z
20276.81 corresponding to the complex of ALK with ConB-1
was observed, demonstrating that Con B-1 could efficiently
form a single covalent bond with ALK (Figure 5).
Long Time Inhibitory Effect and ATP Competitive
Experiment of Con B-1 on ALK. To monitor the influence
of the remote covalent bond to the inhibitory activity with
time, ALK and Con B-1 were incubated for 30 min at room
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Figure 3. (a) Inhibition rate (%) of compounds against ALK at two concentrations; (b) Antiproliferative activities of molecules targeting human
cancer cell line H3122; (c) Chemical structures of molecules with advantaged anticancer bioactivities compared with Ceritinib; (d)
Antiproliferative activities of molecules targeting human normal cell line LO2.
Figure 4. (a) Selectivity of Con B-1 against ALK, IGF1R, INSR, FLT3, and FGFR2; (b) Chemical structure of Con B-1 and Re-Con B-1; (c)
Inhibition rates of the molecules (Con B-1, Re-Con B-1, and Ceritinib) against ALK at different concentrations.
temperature. The free compounds were removed through the
column, and the substrate was added to start the reaction. The
reaction rate was monitored for 2 h. In this experiment, normal
enzyme activity group was set as the positive control group,
and the nonenzyme group was the negative control group
(Figure 6a). As a result, during the 2 h incubation period, the
conversion rate was low compared with positive control,
demonstrating the continuous potent inhibitory effect of Con
B-1 to ALK. It should be pointed out that the conversion rate
was slowly growing during the incubation period, suggesting
that Ceritinib as kinase-recognition scaffold in this molecule
can leave the binding pocket through thermodynamic motion.
Because of the restriction of the terminal covalent bond, the
free Ceritinib group might enter the binding pocket again,
which might indicate the phenomenon that the kinase
inhibitory activity of Con B-1 is higher than Ceritinib.
Apart from that, to identify whether Con B-1 is competitive
for ATP, the inhibitory effect of Con B-1 to ALK at a high
concentration of ATP (1 mM) was conducted. After a 10 min
incubation, as depicted in Figure 6b, Con B-1 still holds an
advantage compared with Ceritinib at a high ATP concen-
tration; meanwhile, the IC50 value (1.4 nM) of Con B-1 to
ALK at a high concentration of ATP was upregulated (0.5 nM
at ATP Km). The above experiments indicated that the
binding of kinase-recognition scaffold of Con B-1 to ALK is
reversible, and the covalent bond outside the binding pocket
can improve the binding affinity to the target protein.
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in Figure 9, the expression of p-ALK, p-AKT, and STAT3 were
decreased in response to Con B-1 treatment. Meanwhile, the
expression of AKT and p-STAT3 was slightly downregulated
after the treatment of increasing concentration of Con B-1.
Collectively, these data indicated that ALK signal pathway can
be effectively inhibited by treatment with Con B-1 in H3122
cell line.
Pharmacokinetic Studies of Con B-1. The pharmacoki-
netic properties of Con B-1 were evaluated in Sprague−
Dawley (SD) rats. As shown in Table 1, Con B-1 showed a
plasma clearance of 16.98 mL·kg−1·min−1 administered
intravenously in rats, whereas oral administration in rats gave
half-time (T1/2) of 2.89 h. Oral bioavailability (F) of 12.1% was
achieved. Apparently, the oral bioavailability of the molecule
was not ideal compared with Ceritinib,6 which can be ascribed
to the large molecular weight and the amide bonds in the
molecule. Further optimizations could be conducted in the
future, and we will report it on due course.
In Vivo Antiproliferative Activity of Compound Con
B-1. The in vivo antitumor activity of Con B-1 was conducted
based on the novel covalent binding mode and promising
cellular activity. As depicted in Figure 10, the growth of
xenograft tumors was inhibited by Con B-1 in a dose-
dependent manner. TGIs of 62%, 49.7% and 39.7% were
observed in the H3122 xenograft model at dose of 50, 20, and
10 mg/kg, respectively. Ceritinib as positive control in this
assay was less active than Con B-1 with TGI of 34% at dose of
20 mg/kg. In addition, Con B-1 did not cause significant
weight loss during the treatment period demonstrating its low
toxicity.
Figure 5. ConB-1 covalently modified ALK in MS assay. Mass spectra
of of (a) ALK alone, (b) ALK preincubated with ConB-1
(MW:769.3).
Bioactivity of Con B-1 to Mutant ALK and Cell Lines.
ALK-L1196M, ALK-G1202R and the corresponding cell lines
BaF3-ALK-L1196M, BaF3-ALK-G1202R were employed to
identify the possible antidrug resistant activity of ConB-1. As
depicted in Figure 7a, Con B-1 was more active than Ceritinib
in ALK-L1196M but less active in ALK-G1202R. In BaF3
models of ALK mutant cell lines, Con B-1 showed similar
activity (Figure 7b). It was assumed that in ALK-G1202R, the
binding mode of Con B-1 was changed and unable to form an
effective covalent bond with Cys1259, which led to the
decrease of the binding affinity of ConB-1.
Binding Mode Analysis of Con B-1. To further
investigate the binding mode of Con B-1 with ALK, molecular
modeling studies were conducted. It was shown that in the
ATP binding pocket, Con B-1 adopts a similar binding mode
with Ceritinib. Apart from that, with the optimized linker, the
acrylamide group succeeded in binding with the Cys1259
residue, which endowed con B-1 with higher bioactivity
compared with Ceritinib (Figure 8a,b).
CONCLUSIONS
■
In this work, by fine-tuning of the type and length of linker
attached to Ceritinib, a first-in-class ALK covalent inhibitor
targeted the Cys1259, which located outside the ATP binding
pocket was discovered.
Compared with Ceritinib, as a covalent molecule, Con B-1
has several advantages, including the improvement of ALK
inhibition activity, the enhancement of antiproliferative
activity, and higher inhibitory activity on drug-resistant cell
lines.
It should also be pointed out that, different with the
traditional covalent compound, due to the restriction of the
covalent bond located outside of ALK active pocket, the
Ceritinib moiety might leave the ATP pocket through the
Western Blot Analysis of Con B-1. To examine the
effects of Con B-1 on ALK signaling, Western blot analysis was
performed. After 24 h treatment of H3122 cells with Con B-1
at different concentrations, the expression levels of p-ALK,
AKT, p-AKT, STAT3, and p-STAT3 were evaluated. As shown
Figure 6. Inhibitory effect of Con B-1 on (a) ALK in 2 h incubation and (b) higher ATP concentration.
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Figure 7. Bioactivity of Con B-1 to mutant ALK and cell lines. (a) Bioactivity of Con B-1 to ALK mutations; (b) Bioactivity of Con B-1 to BaF3
models of ALK mutations.
Figure 8. (a) Binding mode of Con B-1 in ALK; (b) Overlap of the binding modes of Ceritinib (green carbon atoms) and Con B-1 (gray carbon
atoms). In molecular modeling study, the X-ray crystal structure of ALK (PDB ID: 4MKC) was obtained as starting point.19
molecular dynamics, which is consistent with the ATP
competitive experiments and long time inhibitory effect.
It has been estimated that only 39% of kinases (200 of 518)
contain a cysteine in the ATP-binding pocket, which restricted
the design of covalent inhibitors.11a,18 The strategy of targeting
cysteine outside the active site in this work may expand the
range of proteins that can be targeted for covalent inhibition.
EXPERIMENTAL SECTION
■
Human Cell Lines. H3122 (Human nonsmall cell lung cancer),
H2228 (Human nonsmall cell lung cancer), H1299 (Human
nonsmall cell lung cancer), A549 (Human lung cancer cells), HeLa
(Human cervical cancer), and LO2 (human normal hepatocyte line)
were obtained from the American Type Culture Collection
(Manassas, VA, U.S.A.). BaF3-ALK-G1202R and BaF3-ALK-L1196
M were from Sundia MediTech Company, Ltd. All cell lines were
maintained at 37 °C under a humidified 5% CO2 atmosphere
according to supplier recommendations.
Chemical Synthesis. All solvents and reagents obtained from
commercial sources were used without further purification. Flash
Figure 9. Western blot analysis of key ALK signaling proteins.
column chromatography was performed using silica gel from Qingdao
1
Haiyang. H NMR and 13C NMR spectra were recorded on a Bruker
AMX 400 spectrometer and were calibrated using TMS or residual
deuterated solvent as an internal reference (CDCl3: 1H, δ = 7.26 ppm;
Table 1. Pharmacokinetic Parameters for Con B-1 in Rats
a
b
IV (1 mg/kg)
PO (10 mg/kg)
CL (mL/min/kg)
16.98
Vss (ml/kg)
2806.62
Tmax (h)
2.67
Cmax (ng/mL)
157
AUCinf (ng·h/ml)
859
T1/2 (h)
2.89
F(%)
12.1
a
b
Vehicle: 5% DMA/10% Solutol HS 15/85% saline. Vehicle: 5% DMA/10% Solutol HS 15/85% saline.
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Figure 10. Pharmacodynamic profile of Con B-1 in vivo. (a) Growth inhibitory effect of Con B-1 on established H3122 xenografts in female
BALB/c nude mice (N = 6 per group); (b) Body weight of the mices during the dosage period.
13C, δ = 77.16 ppm, DMSO-d6 2.50 ppm, CD3OD 3.31 ppm). All of
the target compounds were examined by HPLC, and the purity of the
biologically tested compounds was ≥95%
= 18.3, 2H), 3.35 (s, 4H), 3.13 (t, J = 11.7, 1H), 2.18 (d, J = 12.2,
3H), 2.13−1.92 (m, 4H), 1.37−1.16 (m, 13H). HRMS (DART-
TOF) calculated for C30H39ClN5O5S [M + H]+ m/z 616.2360, found
616.2347.
Synthesis of tert-Butyl (2-(2-acrylamidoethoxy)ethyl)carbamate
(b1). To a solution of acryloyl chloride (181 mg, 2 mmol) in THF (15
mL) at 0 °C, was added a solution of tert-butyl (2-(2-aminoethoxy)-
ethyl)carbamate (a1, 408 mg, 2 mmol) and DIPEA (417 mg, 3
mmol) in THF. The reaction mixture was stirred for 1 h under the ice
bath. Upon completion, the reaction was quenched with water and
extracted with EtOAc. The organic layer was washed twice with water,
dried over anhydrous sodium sulfate, filtered, and concentrated under
reduced pressure. The crude material was purified by column
chromatography to afford target molecule as a colorless oil (392
mg, yield 76%). 1H NMR (400 MHz, CDCl3) δ = 6.22 (dd, J = 17.0,
2.0, 1H), 6.13 (dd, J = 17.0, 9.9, 1H), 5.55 (dd, J = 9.9, 2.0, 1H),
3.53−3.47 (m, 2H), 3.46 (s, 4H), 3.27−3.18 (m, 2H), 1.37 (s, 9H).
HRMS (DART-TOF) calculated for C12H23N2O4 [M + H]+ m/z
259.1658, found 259.1645.
Synthesis of tert-Butyl (2-(2-(2-acrylamidoethoxy)ethoxy)ethyl)-
carbamate (b2). The title compound was obtained from tert-butyl
(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate following a similar
synthesis procedure to that of b1 (colorless oil, yield 69%). 1H
NMR (400 MHz, CDCl3) δ = 6.30 (dd, J = 17.0, 1.3, 1H), 6.23−6.06
(m, 1H), 5.64 (d, J = 10.1, 1H), 3.70−3.51 (m, 9H), 3.40−3.24 (m,
2H), 1.46 (d, J = 6.6, 9H). HRMS (DART-TOF) calculated for
C14H27N2O5 [M + H]+ m/z 303.1920, found 303.1893.
Synthesis of 3-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)-
amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-methylphenyl)-
piperidin-1-yl)propanoic Acid (d2). To a solution of Ceritinib (330
mg, 0.6 mmol) in methanol (50 mL) was added methyl acrylate (78
mg, 0.9 mmol) and Et3N (240 mg, 2.4 mmol). The solution was
stirred for 8 h. After that, the methanol was removed through reduced
pressure, and then the mixture was dissolved with EtOAc and washed
with water for 3 times. The organic layer was dried over anhydrous
sodium sulfate, concentrated, and purified by flash chromatography
on a silica gel column to afford the intermediate as a white solid (348
mg, yield 90%). The intermediate was dissolved in 10 mL of
methanol/H2O (1:1), and to this solution NaOH (200 mg, 5 mmol)
was added and stirred at 60 °C for 10 h. After that, the methanol was
removed through reduced pressure, and the pH of water layer was
adjusted to 2 with dilute hydrochloric acid. Then the mixture was
dissolved with EtOAc and washed with water for 3 times. The organic
layer was dried over anhydrous sodium sulfate, concentrated, and
purified by flash chromatography on silica gel column to afford the
1
intermediate as a white solid (323 mg, yield 95%). H NMR (400
MHz, CDCl3) δ = 9.51 (s, 1H), 8.58 (d, J = 8.4, 1H), 8.16 (d, J = 1.2,
1H), 8.03 (d, J = 3.2, 1H), 7.93 (d, J = 7.9, 1H), 7.62 (dd, J = 11.3,
4.4, 1H), 7.58−7.51 (m, 1H), 7.27 (d, J = 6.5, 1H), 6.75 (d, J = 37.6,
1H), 4.56 (dt, J = 11.1, 5.6, 1H), 3.51−3.14 (m, 3H), 2.81 (ddd, J =
47.2, 24.3, 12.3, 3H), 2.17 (d, J = 8.1, 3H), 1.34 (dd, J = 14.8, 6.5,
13H). HRMS (DART-TOF) calculated for C31H41ClN5O5S [M +
H]+ m/z 630.2517, found 630.2508.
Synthesis of tert-Butyl (13-oxo-3,6,9-trioxa-12-azapentadec-14-
en-1-yl)carbamate (b3). The title compound was obtained from tert-
butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate fol-
lowing a similar synthesis procedure to that of b1 (colorless oil,
1
yield 62%). H NMR (400 MHz, CDCl3) δ = 6.35−6.25 (m, 1H),
Synthesis of N-(2-(2-(3-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)propanamido)ethoxy)ethyl)-
acrylamide (Con B-1). b1 (46 mg, 0.2 mmol) was dissolved in 5 mL
of trifluoroacetic acid and stirred at 50 °C for 1 h. Subsequently,
DCM (10 mL) was added into the reaction solution, and the mixture
was concentrated under reduced pressure to obtain brown oil (c1).
The resulting crude intermediate was used in the next step without
further purification. To a solution of the above intermediate, d2 (126
mg, 0.2 mmol), and HATU (45.6 mg, 0.12 mmol) in DMF (10 mL)
was added DIPEA (38.7 mg, 0.3 mmol). After being stirred for 15 h,
the reaction was quenched with water, separated, dried over
anhydrous sodium sulfate, concentrated, and purified by flash
chromatography on silica gel column to afford the title compound
as a white solid (65 mg, yield 42%). 1H NMR (400 MHz, CDCl3) δ =
9.51 (s, 1H), 8.58 (d, J = 8.4, 1H), 8.16 (s, 1H), 8.03 (s, 1H), 7.98−
7.90 (m, 1H), 7.62 (t, J = 7.3, 1H), 7.54 (d, J = 10.5, 1H), 7.25 (d, J =
8.8, 2H), 6.76 (s, 1H), 6.28 (dd, J = 17.0, 1.3, 1H), 6.15 (dd, J = 17.0,
10.1, 1H), 5.62 (dd, J = 10.2, 1.3, 1H), 4.62−4.47 (m, 1H), 3.68−
3.41 (m, 8H), 3.24 (ddd, J = 37.5, 22.1, 8.9, 3H), 2.86−2.66 (m, 3H),
2.49 (t, J = 6.2, 2H), 2.26 (t, J = 11.3, 3H), 2.16 (s, 3H), 1.76 (dt, J =
21.8, 11.9, 5H), 1.34 (dd, J = 19.4, 6.5, 13H). 13C NMR (101 MHz,
CDCl3) δ = 172.78, 165.78, 157.47, 155.35, 144.69, 138.50, 134.63,
6.24−6.08 (m, 1H), 5.62 (d, J = 10.1, 1H), 3.70−3.59 (m, 10H),
3.58−3.50 (m, 4H), 3.33 (t, J = 13.0, 2H), 1.45 (d, J = 8.4, 9H). 13C
NMR (101 MHz, CDCl3) δ = 165.69, 156.06, 130.94, 126.22, 79.23,
70.40, 70.37, 70.21, 70.14, 70.13, 69.77, 40.31, 39.25, 28.39.
Synthesis of 2-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)-
amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-methylphenyl)-
piperidin-1-yl)acetic Acid (d1). To a solution of Ceritinib (560 mg, 1
mmol) in acetonitrile (15 mL) was added tert-butyl bromoacetate
(195 mg, 1 mmol) and K2CO3 (414 mg, 3 mmol), and the resulting
solution was stirred under reflux conditions for 5 h. Subsequently, the
reaction was quenched with water and extracted with EtOAc, and the
organic layer was dried over anhydrous sodium sulfate, concentrated,
and purified by flash chromatography on the silica gel column to
afford the intermediate as a yellow solid (511 mg, yield 76%). The
intermediate was dissolved in 20 mL of TFA and stirred for 5 h. After
that, 10 mL of DCM was added into the reaction solution, and the
mixture was concentrated under reduced pressure to obtain a brown
oil. The mixture was purified by flash chromatography on a silica gel
column to afford the title compound as a white solid (421 mg, yield
90%). 1H NMR (400 MHz, MeOD) δ = 8.29 (d, J = 7.9, 1H), 8.23 (s,
1H), 7.99 (d, J = 7.9, 1H), 7.70 (t, J = 7.7, 1H), 7.51 (t, J = 7.6, 1H),
7.40 (s, 1H), 6.92 (s, 1H), 4.72−4.59 (m, 1H), 4.13 (s, 2H), 3.82 (t, J
1564
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131.29, 130.80, 128.02, 127.38, 126.45, 124.93, 123.63, 123.14,
120.82, 111.15, 105.90, 72.02, 69.99, 69.48, 55.48, 54.26, 53.94, 39.35,
38.79, 37.79, 32.58, 22.28, 18.93, 15.37. HRMS (DART-TOF)
calculated for C38H53ClN7O6S [M + H]+ m/z 770.3467, found
770.3418.
17.0, 10.2, 1H), 5.61 (td, J = 9.8, 3.4, 1H), 4.67−4.52 (m, 1H), 3.66−
3.48 (m, 12H), 3.32−3.21 (m, 1H), 3.06 (s, 2H), 2.98 (d, J = 11.4,
2H), 2.74−2.61 (m, 1H), 2.31 (dd, J = 11.5, 9.2, 2H), 2.16 (s, 3H),
1.82−1.67 (m, 4H), 1.41−1.30 (m, 12H). 13C NMR (101 MHz,
CDCl3) δ = 170.89, 165.58, 157.48, 155.36, 144.69, 138.50, 137.32,
134.63, 131.29, 130.85, 127.83, 127.29, 126.41, 124.92, 123.63,
123.13, 120.79, 111.10, 105.85, 71.82, 70.43, 70.13, 70.09, 69.84,
62.10, 55.48, 54.99, 39.26, 38.80, 37.60, 32.94, 22.30, 18.94, 15.37.
HRMS (DART-TOF) calculated for C37H51ClN7O6S [M + H]+ m/z
756.3310, found 756.3319.
Synthesis of N-(2-(2-(2-(3-(4-(4-((5-Chloro-4-((2-
(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-5-isopro-
poxy-2-methylphenyl)piperidin-1-yl)propanamido)ethoxy)ethoxy)-
ethyl)acrylamide (Con B-2). The title compound was obtained from
b2 and d2 following a similar synthesis procedure to that of Con B-1
1
(white solid, yield 39%). H NMR (400 MHz, CDCl3) δ = 9.51 (s,
Synthesis of N-(1-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-2-oxo-6,9,12-trioxa-3-azatetradecan-
14-yl)acrylamide (Con B-6). The title compound was obtained from
b3 and d1 following a similar synthesis procedure to that of Con B-1
1H), 8.58 (d, J = 8.3, 1H), 8.16 (s, 1H), 8.02 (s, 1H), 7.93 (dd, J =
8.0, 1.5, 1H), 7.67−7.59 (m, 1H), 7.53 (s, 1H), 7.31−7.26 (m, 1H),
6.78 (s, 1H), 6.28 (dd, J = 17.0, 1.6, 2H), 6.12 (dd, J = 17.0, 10.2,
1H), 5.68−5.56 (m, 1H), 4.54 (dt, J = 12.1, 6.1, 1H), 3.69−3.41 (m,
11H), 3.25 (dq, J = 13.0, 6.5, 1H), 3.14 (d, J = 10.9, 2H), 2.69 (dd, J
= 26.2, 14.6, 3H), 2.46 (t, J = 6.2, 2H), 2.27−2.12 (m, 5H), 1.78 (dd,
J = 33.0, 11.0, 5H), 1.44−1.29 (m, 12H). 13C NMR (101 MHz,
CDCl3) δ = 172.45, 165.61, 157.48, 155.36, 144.69, 138.51, 134.62,
131.29, 130.86, 127.90, 127.28, 126.38, 124.94, 123.64, 123.12,
120.77, 111.20, 71.87, 70.43, 70.12, 69.86, 55.48, 54.25, 53.97, 39.27,
38.99, 32.62, 29.70, 22.27, 18.94, 15.37. HRMS (DART-TOF)
calculated for C40H57ClN7O7S [M + H]+ m/z 814.3729, found
814.3760.
1
(white solid, yield 21%). H NMR (400 MHz, CDCl3) δ = 9.52 (s,
1H), 8.58 (t, J = 7.2, 1H), 8.16 (d, J = 2.9, 1H), 8.02 (s, 1H), 7.93
(dd, J = 8.0, 1.5, 1H), 7.67−7.60 (m, 1H), 7.57−7.47 (m, 2H), 7.27−
7.23 (m, 1H), 6.79 (s, 1H), 6.28 (dt, J = 7.7, 2.7, 1H), 6.19−6.08 (m,
1H), 5.67−5.58 (m, 1H), 4.69−4.52 (m, 1H), 3.68−3.49 (m, 16H),
3.32−3.21 (m, 1H), 3.08 (d, J = 17.3, 2H), 2.98 (d, J = 11.4, 2H),
2.30 (dt, J = 11.3, 5.7, 2H), 2.21−2.12 (m, 3H), 1.82−1.65 (m, 4H),
1.42−1.29 (m, 12H). 13C NMR (101 MHz, CDCl3) δ = 170.82,
165.57, 157.48, 155.35, 144.70, 138.50, 137.36, 134.64, 131.29,
130.93, 127.78, 127.23, 126.28, 124.91, 123.63, 123.13, 120.78,
111.07, 105.84, 71.74, 70.56, 70.50, 70.24, 70.19, 69.83, 62.09, 55.48,
54.99, 39.29, 38.82, 37.62, 32.93, 29.69, 22.30, 18.94, 15.37. HRMS
(DART-TOF) calculated for C41H59ClN7O8S [M + H]+ m/z
844.3834, found 844.3829.
Synthesis of tert-Butyl (3-Acrylamidopropyl)carbamate (f1). The
title compound was obtained from tert-butyl (3-aminopropyl)-
carbamate following a similar synthesis procedure to that of b1
(white solid, yield 71%). 1H NMR (400 MHz, CDCl3) δ = 6.25−6.15
(m, 1H), 6.09 (dd, J = 17.0, 9.9, 1H), 5.54 (dd, J = 9.9, 1.8, 1H), 3.28
(td, J = 12.0, 4.9, 2H), 3.10 (dd, J = 11.8, 5.8, 2H), 1.65−1.52 (m,
2H), 1.36 (s, 9H). HRMS (DART-TOF) calculated for C11H21N2O3
[M + H]+ m/z 229.1552, found 229.1545.
Synthesis of tert-Butyl (4-Acrylamidobutyl)carbamate (f2). The
title compound was obtained from tert-butyl (4-aminobutyl)-
carbamate following similar a synthesis procedure to that of b1
(white solid, yield 76%). 1H NMR (400 MHz, CDCl3) δ = 6.27 (dd, J
= 17.0, 1.6, 2H), 6.13 (dd, J = 17.0, 10.2, 1H), 5.62 (dd, J = 10.2, 1.6,
1H), 4.72 (s, 1H), 3.35 (q, J = 6.4, 2H), 3.13 (s, 2H), 1.62−1.49 (m,
4H), 1.44 (s, 9H). 13C NMR (101 MHz, CDCl3) δ = 165.74, 156.23,
130.99, 126.13, 79.27, 39.20, 28.41, 27.71, 26.50. HRMS (DART-
TOF) calculated for C12H23N2O3 [M + H]+ m/z 243.1709, found
243.1701.
Synthesis of tert-Butyl (6-Acrylamidohexyl)carbamate (f3). The
title compound was obtained from tert-butyl (6-aminohexyl)-
carbamate following a similar synthesis procedure to that of b1
(colorless oil, yield 68%). 1H NMR (400 MHz, CDCl3) δ = 6.28 (dd,
J = 17.0, 1.4, 1H), 6.12 (dd, J = 17.0, 10.2, 1H), 5.62 (dd, J = 10.2,
1.3, 1H), 3.32 (dd, J = 13.1, 6.7, 2H), 3.11 (dd, J = 12.6, 6.2, 2H),
1.60−1.41 (m, 13H), 1.34 (dd, J = 8.8, 5.6, 4H). HRMS (DART-
TOF) calculated for C14H26N2O3Na [M + Na]+ m/z 293.1841, found
293.1856.
Synthesis of N-(15-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl) amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)-13-oxo-3,6,9-trioxa-12-
azapentadecyl)acrylamide (Con B-3). The title compound was
obtained from b3 and d2 following similar synthesis procedure to that
of Con B-1 (white solid, yield 42%). 1H NMR (400 MHz, CDCl3) δ
= 9.51 (s, 1H), 8.59 (d, J = 8.3, 1H), 8.20−8.14 (m, 1H), 8.06−7.97
(m, 2H), 7.93 (dd, J = 8.0, 1.5, 1H), 7.68−7.59 (m, 1H), 7.53 (s,
1H), 7.28−7.22 (m, 1H), 6.78 (s, 1H), 6.28 (dd, J = 17.0, 1.6, 1H),
6.14 (dd, J = 17.0, 10.2, 1H), 5.62 (dd, J = 10.1, 1.6, 1H), 4.63−4.48
(m, 1H), 3.72−3.56 (m, 12H), 3.55−3.43 (m, 4H), 3.27 (dq, J =
13.7, 6.9, 1H), 3.12 (d, J = 11.4, 2H), 2.78−2.64 (m, 3H), 2.44 (t, J =
6.4, 2H), 2.16 (s, 4H), 1.86−1.65 (m, 5H), 1.42−1.28 (m, 14H). 13C
NMR (101 MHz, CDCl3) δ = 172.69, 165.72, 157.49, 155.35, 144.69,
138.51, 137.29, 134.64, 131.28, 130.92, 127.80, 127.23, 126.33,
124.90, 123.62, 123.12, 120.75, 111.11, 105.84, 71.77, 70.43, 70.38,
70.24, 70.10, 69.96, 55.48, 54.31, 54.00, 39.30, 39.01, 38.03, 32.73,
29.69, 22.28, 18.94, 15.37. HRMS (DART-TOF) calculated for
C30H38Cl2N5O4S [M + H]+ m/z 634.2022, found 634.2013.
Synthesis of N-(2-(2-(2-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)acetamido)ethoxy)ethyl)acrylamide
(Con B-4). The title compound was obtained from b1 and d1
following a similar synthesis procedure to that of Con B-1 (white
solid, yield 33%). 1H NMR (400 MHz, CDCl3) δ = 9.51 (s, 1H), 8.58
(t, J = 6.3, 1H), 8.16 (d, J = 2.6, 1H), 8.03 (d, J = 6.3, 1H), 7.93 (dd, J
= 8.0, 1.4, 1H), 7.66−7.59 (m, 1H), 7.54 (s, 1H), 7.46 (t, J = 5.9,
1H), 7.25 (d, J = 8.9, 2H), 6.77 (s, 1H), 6.34−6.26 (m, 1H), 6.23−
6.11 (m, 1H), 5.66−5.60 (m, 1H), 4.65−4.49 (m, 1H), 3.69−3.45
(m, 9H), 3.26 (dt, J = 13.7, 6.8, 1H), 3.07 (s, 2H), 2.98 (d, J = 11.4,
2H), 2.32 (dd, J = 11.7, 9.6, 2H), 2.17 (d, J = 13.0, 3H), 1.84−1.62
(m, 4H), 1.43−1.28 (m, 12H). 13C NMR (101 MHz, CDCl3) δ =
171.16, 165.63, 157.48, 155.35, 144.69, 138.51, 137.18, 134.64,
131.29, 130.81, 127.86, 127.28, 126.42, 124.92, 123.64, 123.13,
120.80, 111.01, 105.84, 71.81, 70.16, 69.43, 62.11, 55.48, 55.01, 39.36,
38.52, 37.64, 32.94, 22.29, 18.93, 15.37. HRMS (DART-TOF)
calculated for C37H51ClN7O6S [M + H]+ m/z 756.3310, found
756.3319.
Synthesis of tert-Butyl (8-Acrylamidooctyl)carbamate (f4). The
title compound was obtained from tert-butyl (8-aminooctyl)-
carbamate following a similar synthesis procedure to that of b1
(colorless oil, yield 67%). 1H NMR (400 MHz, CDCl3) δ = 6.27 (dd,
J = 17.0, 1.2, 1H), 6.11 (dd, J = 17.0, 10.2, 1H), 5.62 (dd, J = 10.2,
1.1, 1H), 4.56 (s, 1H), 3.09 (dd, J = 12.6, 6.2, 2H), 1.59−1.39 (m,
13H), 1.28 (d, J = 17.7, 9H). HRMS (DART-TOF) calculated for
C16H30N2O3Na [M + Na]+ m/z 321.2154, found 321.2149.
Synthesis of N-(3-(3-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)propanamido)propyl)acrylamide
(Con A-1). The title compound was obtained from f1 and d2
following a similar synthesis procedure to that of Con B-1 (white
solid, yield 52%). 1H NMR (400 MHz, CDCl3) δ = 9.50 (s, 1H), 8.58
Synthesis of N-(2-(2-(2-(2-(4-(4-((5-Chloro-4-((2-
(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-5-isopro-
poxy-2-methylphenyl)piperidin-1-yl)acetamido)ethoxy)ethoxy)-
ethyl)acrylamide (Con B-5). The title compound was obtained from
b2 and d1 following a similar synthesis procedure to that of Con B-1
1
(white solid, yield 37%). H NMR (400 MHz, CDCl3) δ = 9.51 (s,
1H), 8.58 (t, J = 6.1, 1H), 8.16 (d, J = 3.0, 1H), 8.02 (s, 1H), 7.93
(dd, J = 8.0, 1.5, 1H), 7.67−7.59 (m, 1H), 7.54 (d, J = 6.3, 1H),
7.30−7.26 (m, 1H), 6.79 (s, 1H), 6.35−6.23 (m, 2H), 6.13 (dd, J =
1565
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(d, J = 8.4, 1H), 8.14 (d, J = 8.9, 2H), 7.93 (d, J = 7.9, 1H), 7.62 (t, J
= 7.8, 1H), 7.54 (s, 1H), 7.27−7.22 (m, 1H), 6.77 (s, 1H), 6.35−6.22
(m, 1H), 6.14 (dd, J = 17.0, 10.2, 1H), 5.63 (d, J = 10.2, 1H), 4.61−
4.48 (m, 1H), 3.47−3.21 (m, 6H), 3.12 (d, J = 11.3, 2H), 2.78−2.64
(m, 3H), 2.45 (t, J = 6.2, 2H), 2.23−2.08 (m, 5H), 1.83 (d, J = 13.3,
2H), 1.77−1.60 (m, 4H), 1.34 (dd, J = 19.1, 6.5, 13H). 13C NMR
(101 MHz, CDCl3) δ = 173.40, 165.87, 157.49, 155.38, 155.34,
144.69, 138.52, 137.19, 134.64, 131.25, 127.79, 127.17, 126.01,
124.90, 123.66, 123.11, 120.74, 111.00, 105.82, 71.74, 55.47, 54.38,
54.01, 38.10, 35.77, 35.60, 32.85, 32.69, 29.89, 22.26, 18.93, 15.37.
HRMS (DART-TOF) calculated for C37H51ClN7O5S [M + H]+ m/z
740.3361, found 740.3400.
Synthesis of N-(4-(3-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)propanamido)butyl)acrylamide (Con
A-2). The title compound was obtained from f2 and d2 following a
similar synthesis procedure to that of Con B-1 (white solid, yield
67%). 1H NMR (400 MHz, CDCl3) δ = 9.51 (s, 1H), 8.58 (d, J = 8.3,
1H), 8.16 (s, 1H), 8.03 (s, 1H), 7.93 (dd, J = 8.0, 1.4, 1H), 7.62 (dd, J
= 11.4, 4.3, 1H), 7.54 (s, 1H), 7.31−7.23 (m, 2H), 6.75 (s, 1H), 6.75
(s, 1H), 6.33−6.22 (m, 1H), 6.17−6.06 (m, 1H), 4.55 (dt, J = 12.1,
6.1, 1H), 3.46−3.11 (m, 7H), 2.90−2.68 (m, 3H), 2.49 (t, J = 5.9,
2H), 2.31 (t, J = 11.4, 2H), 2.16 (s, 3H), 1.88 (d, J = 12.7, 2H), 1.76−
1.67 (m, 2H), 1.65−1.53 (m, 4H), 1.35 (dd, J = 20.1, 6.5, 12H). 13C
NMR (101 MHz, CDCl3) δ = 172.69, 165.83, 157.46, 155.36, 155.33,
144.70, 138.49, 136.78, 134.66, 131.27, 130.98, 127.93, 127.27,
126.19, 124.88, 123.63, 123.15, 120.80, 110.97, 105.86, 71.87, 55.49,
54.31, 53.88, 39.13, 38.53, 37.77, 32.57, 32.00, 27.18, 26.48, 22.26,
18.93, 15.37. HRMS (DART-TOF) calculated for C38H53ClN7O5S
[M + H]+ m/z 754.3517, found 754.3531.
Synthesis of N-(6-(3-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)propanamido)hexyl)acrylamide (Con
A-3). The title compound was obtained from f3 and d2 following a
similar synthesis procedure to that of Con B-1 (white solid, yield
62%). 1H NMR (400 MHz, CDCl3) δ = 9.52 (s, 1H), 8.58 (d, J = 8.4,
1H), 8.16 (s, 1H), 8.04 (d, J = 10.3, 2H), 7.93 (dd, J = 7.9, 1.3, 1H),
7.67−7.58 (m, 1H), 7.52 (s, 1H), 7.25 (d, J = 9.7, 1H), 6.78 (d, J =
16.4, 1H), 6.31−6.20 (m, 1H), 6.09 (dd, J = 17.0, 10.2, 1H), 5.90 (s,
1H), 5.64−5.54 (m, 1H), 4.58−4.46 (m, 1H), 3.27 (tq, J = 13.9, 6.8,
5H), 3.13 (d, J = 11.3, 2H), 2.72 (dd, J = 12.2, 6.9, 3H), 2.43 (t, J =
6.1, 2H), 2.27−2.13 (m, 5H), 1.85 (d, J = 12.6, 2H), 1.67 (dd, J =
22.0, 12.1, 2H), 1.60−1.47 (m, 4H), 1.42−1.29 (m, 16H). 13C NMR
(101 MHz, CDCl3) δ = 172.49, 165.61, 157.47, 155.37, 144.65,
138.50, 137.10, 134.63, 131.29, 131.01, 127.95, 127.44, 126.09,
124.91, 123.62, 123.13, 120.84, 111.20, 105.90, 72.02, 55.49, 54.36,
53.87, 39.11, 38.58, 37.94, 32.84, 32.37, 29.43, 29.39, 26.22, 26.10,
22.29, 18.93, 15.37. HRMS (DART-TOF) calculated for
C40H57ClN7O5S [M + H]+ m/z 782.3830, found 782.3863.
Synthesis of N-(8-(3-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)propanamido)octyl)acrylamide (Con
A-4). The title compound was obtained from f4 and d2 following a
similar synthesis procedure to that of Con B-1 (white solid, yield
57%). 1H NMR (400 MHz, CDCl3) δ = 9.52 (s, 1H), 8.58 (d, J = 8.3,
1H), 8.16 (s, 1H), 8.02 (s, 2H), 7.93 (dd, J = 8.0, 1.4, 1H), 7.67−7.58
(m, 1H), 7.53 (s, 1H), 7.27 (d, J = 6.0, 1H), 6.75 (s, 1H), 6.26 (ddd, J
= 17.0, 6.5, 1.4, 1H), 6.10 (td, J = 17.4, 9.1, 1H), 5.66−5.53 (m, 1H),
4.61−4.45 (m, 1H), 3.39−3.09 (m, 8H), 2.73 (dd, J = 14.2, 5.1, 3H),
2.46 (t, J = 6.0, 2H), 2.24 (t, J = 11.1, 2H), 2.17 (s, 3H), 1.86 (d, J =
12.1, 2H), 1.71 (dd, J = 22.6, 11.9, 2H), 1.60−1.43 (m, 5H), 1.41−
1.28 (m, 21H). 13C NMR (101 MHz, CDCl3) δ = 172.20, 165.51,
157.47, 155.36, 144.70, 138.50, 134.64, 131.29, 131.00, 127.90,
127.29, 126.09, 124.91, 123.61, 123.14, 120.82, 110.98, 105.92, 71.84,
55.49, 54.33, 53.85, 39.51, 39.03, 37.80, 32.67, 29.47, 29.40, 29.09,
29.05, 26.85, 26.75, 22.28, 18.93, 15.37. HRMS (DART-TOF)
calculated for C42H61ClN7O5S [M + H]+ m/z 810.4143, found
810.4150.
5). The title compound was obtained from f1 and d1 following a
similar synthesis procedure to that of Con B-1 (white solid, yield
35%). 1H NMR (400 MHz, CDCl3) δ = 9.51 (s, 1H), 8.58 (d, J = 8.2,
1H), 8.15 (s, 1H), 8.01 (s, 1H), 7.93 (dd, J = 8.0, 1.5, 1H), 7.66−7.60
(m, 2H), 7.57 (s, 1H), 7.27−7.21 (m, 1H), 6.81 (s, 1H), 6.72 (t, J =
6.0, 1H), 6.28 (dd, J = 17.0, 1.5, 1H), 6.15 (dd, J = 17.0, 10.2, 1H),
5.64 (dd, J = 10.2, 1.5, 1H), 4.66−4.54 (m, 1H), 3.38 (td, J = 12.4,
6.4, 4H), 3.27 (dq, J = 13.7, 6.8, 1H), 3.10 (s, 2H), 2.99 (t, J = 10.9,
2H), 2.39 (d, J = 14.2, 2H), 2.16 (s, 3H), 1.85−1.69 (m, 6H), 1.35
(dd, J = 23.4, 6.5, 12H). 13C NMR (101 MHz, CDCl3) δ = 165.90,
157.48, 155.36, 155.32, 144.74, 138.50, 137.20, 134.64, 131.28,
131.22, 127.78, 127.13, 126.01, 124.92, 123.66, 123.13, 120.78,
111.05, 105.80, 71.69, 61.86, 55.48, 55.08, 37.53, 35.88, 35.70, 32.80,
29.80, 29.69, 22.28, 18.94, 15.37. HRMS (DART-TOF) calculated for
C36H49ClN7O5S [M + H]+ m/z 726.3204, found 726.3223.
Synthesis of N-(4-(2-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)acetamido)butyl)acrylamide (Con A-
6). The title compound was obtained from f2 and d1 following a
similar synthesis procedure to that of Con B-1 (white solid, yield
33%). 1H NMR (400 MHz, CDCl3) δ = 9.51 (s, 1H), 8.58 (d, J = 8.2,
1H), 8.15 (s, 1H), 8.06 (d, J = 5.2, 1H), 8.02 (s, 1H), 7.93 (dd, J =
8.0, 1.5, 1H), 7.68−7.59 (m, 1H), 7.54 (s, 1H), 7.30−7.22 (m, 1H),
6.75 (s, 1H), 6.33 (t, J = 5.5, 1H), 6.25 (dd, J = 17.0, 1.6, 1H), 6.12
(dd, J = 17.0, 10.1, 1H), 5.60 (dd, J = 10.1, 1.6, 1H), 4.64−4.48 (m,
1H), 3.44−3.15 (m, 7H), 2.86−2.68 (m, 3H), 2.47 (t, J = 6.2, 2H),
2.28 (t, J = 11.3, 2H), 2.16 (s, 3H), 1.87 (d, J = 12.5, 2H), 1.78−1.55
(m, 7H), 1.34 (dd, J = 20.3, 6.5, 12H). 13C NMR (101 MHz, CDCl3)
δ = 165.75, 157.48, 155.35, 144.71, 138.50, 134.64, 131.28, 130.98,
127.85, 127.21, 126.20, 124.91, 123.65, 123.13, 120.76, 111.04,
105.83, 71.78, 61.99, 55.48, 55.06, 39.22, 38.52, 37.57, 32.79, 29.69,
27.60, 26.45, 22.29, 18.94, 15.37. HRMS (DART-TOF) calculated for
C37H51ClN7O5S [M + H]+ m/z 740.3361, found 740.3348.
Synthesis of N-(6-(2-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)acetamido)hexyl)acrylamide (Con A-
7). The title compound was obtained from f3 and d1 following a
similar synthesis procedure to that of Con B-1 (white solid, yield
36%). 1H NMR (400 MHz, CDCl3) δ = 9.51 (s, 1H), 8.57 (t, J = 7.1,
1H), 8.16 (d, J = 3.0, 1H), 8.02 (s, 1H), 7.93 (d, J = 7.8, 1H), 7.62 (t,
J = 7.6, 1H), 7.56 (d, J = 8.2, 1H), 7.25 (d, J = 8.7, 1H), 6.79 (s, 1H),
6.27 (d, J = 17.0, 1H), 6.11 (dd, J = 17.0, 10.3, 1H), 5.82 (s, 1H), 5.62
(d, J = 10.2, 1H), 4.58 (dt, J = 12.3, 6.1, 1H), 3.29 (dq, J = 20.8, 6.7,
5H), 3.01 (dd, J = 30.0, 19.2, 3H), 2.67 (dd, J = 23.3, 12.7, 1H), 2.35
(d, J = 9.6, 2H), 2.22−2.11 (m, 3H), 2.05 (d, J = 8.8, 1H), 1.77 (dd, J
= 25.4, 11.2, 4H), 1.65−1.48 (m, 5H), 1.46−1.29 (m, 15H). 13C
NMR (101 MHz, CDCl3) δ = 165.60, 155.36, 144.72, 138.50, 134.63,
131.29, 131.00, 127.27, 126.17, 124.94, 123.64, 123.13, 120.81,
111.09, 105.85, 71.83, 55.48, 55.00, 39.12, 38.54, 37.55, 29.70, 29.33,
26.11, 26.07, 22.29, 18.94, 15.37. HRMS (DART-TOF) calculated for
C39H55ClN7O5S [M + H]+ m/z 768.3674, found 768.3659.
Synthesis of N-(8-(2-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)acetamido)octyl)acrylamide (Con A-
8). The title compound was obtained from f4 and d1 following a
similar synthesis procedure to that of Con B-1 (white solid, yield
29%). 1H NMR (400 MHz, CDCl3) δ = 9.51 (s, 1H), 8.58 (d, J = 8.2,
1H), 8.16 (d, J = 3.2, 1H), 8.04 (d, J = 18.4, 1H), 7.93 (dd, J = 8.0,
1.6, 1H), 7.66−7.59 (m, 1H), 7.56 (d, J = 11.3, 1H), 7.24 (s, 1H),
6.80 (d, J = 13.0, 1H), 6.30−6.23 (m, 1H), 6.07 (dd, J = 17.0, 10.3,
1H), 5.68−5.58 (m, 2H), 4.64−4.52 (m, 1H), 3.39−3.24 (m, 5H),
3.00 (dd, J = 26.5, 15.3, 3H), 2.34 (t, J = 10.7, 2H), 1.75 (dd, J = 32.2,
10.2, 4H), 1.53 (d, J = 6.6, 5H), 1.40−1.30 (m, 19H). 13C NMR (101
MHz, CDCl3) δ = 165.52, 157.48, 155.36, 144.71, 138.50, 134.63,
131.29, 130.96, 127.86, 127.32, 126.18, 124.93, 123.64, 123.14,
120.83, 111.12, 100.00, 71.85, 62.05, 55.49, 55.00, 39.54, 38.90, 37.60,
32.88, 29.70, 29.49, 29.02, 26.75, 22.30, 18.93, 15.37. HRMS (DART-
TOF) calculated for C41H59ClN7O5S [M + H]+ m/z 796.3987, found
796.3981.
Synthesis of N-(3-(2-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-
methylphenyl)piperidin-1-yl)acetamido)propyl)acrylamide (Con A-
ALK Inhibition Assays. The ALK activity assay was conducted
through Mobility shift assay. Briefly, the compound, wild-type ALK
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(Carna, 08-518), ALK-L1196 M (Carna, 08-529), ALK-G1202R
(Carna, 08-544), Kinase substrate22 and ATP (Km, Sigma) were
diluted in kinase buffer to the indicated concentrations. The assay
plate was covered and incubated at room temperature for 10 min. To
learn the inhibitory activity of Con B-1 at higher ATP concentration,
a concentration of 1 mM was applied in this study. The data were
collected on Caliper EZ Reader and presented in Excel. The curves
were fitted by Graphpad Prism 5.0.
Cell Viability Assay. Cells were cultured at 37 °C with 5% CO2 in
RPMI 1640 or DMEM, supplemented with 10% (v/v) fetal bovine
serum (Gibco), and 1% (v/v) penicillin− streptomycin (HyClone).
Cells in logarithmic phase were seeded in a 96-well plate at 2−5 × 103
cells per well for 24 h (37 °C, 5% CO2). Then, an equal volume of
medium containing various concentrations of test compounds was
added to each well. After 72 h, MTT was added, and the cells were
incubated for an additional 1−4 h. The absorbance values (OD) of
the 96-well plate was measured at 450 nm using a Spectra MAX M5
microplate spectrophotometer (Molecular Devices). The IC50 values
were the means of at least three independent experiments and
calculated by GraphPad Prism 5 software.
Molecular Modeling. To predict the possible binding mode of
Con B-1 with ALK, Molecular modeling was performed by using
autodock 4.0. The X-ray crystal structure of ALK (PDB ID: 4MKC)
was obtained as starting point.
Mass Spectrometry Analysis for Identifying Covalent
Binding Nature of ConB-1. L-Cysteine was incubated with
DMSO or a 5-fold molar excess of ConB-1 for 8 h at 4 °C in
water. Reactions were then analyzed by LC−MS using a Shimadzu
LC and autosampler system (Shimadzu) interfaced to an LTQ ion
trap mass spectrometer.
ALK samples were incubated with 20 molar excess of the ConB-1
for 4 h at rt before being quenched by adding 0.1% trifluoroacetic acid
(aq.). The resulting samples were separated over an HPLC column
with 5−95% acetonitrile in water as an eluent. The MS data were
analyzed using an Agilent LC/MSD Time-of-Flight Mass Spectrom-
eter equipped with an electrospray ionization source.
Inhibitory Effect of Con B-1 on ALK in 2 h Incubation. In this
experiment, 70 fold the final concentration of ALK (87.5 nM) and
200-fold the IC50 (100 nM) of Con B-1 were incubated for 30 min at
room temperature. The free compounds were removed through the
column, and the substrate was added to start the reaction. Then the
reaction rate was monitored for 2 h to determine whether the
combination of the compound and the enzyme is reversible or
irreversible. The normal enzyme activity group was set as the positive
control group, and the nonenzyme group was the negative control
group.
Western Blotting. H3122 cells were treated with Con B-1 at the
concentrations of 0, 10, 50, 200, and 500 nM for 24 h at 37 °C, then
the cells were harvested, washed in ice-cold PBS, and lysed with RIPA
buffer, protease inhibitors, phosphatase cocktails A and B, and PMSF
(1 mM). Protein concentration was determined by the BCA Protein
Assay Kit (Beyotime#p0012s). The samples were subjected to SDS−
PAGE and then transferred onto PVDF membranes (Millpore). The
membranes were incubated overnight at 4 °C with the primary
antibody in 5% BSA/TBST buffer with gentle shaking, then washed
with 1× TBS/T 3 times, followed by incubation for 1 h with a 1/5000
dilution of secondary HRP antibody in 5% nonfat milk/TBST.
Antibody information: p-ALK (Cell Signaling Technology; catalogue
no. 3341s), AKT (wanleibio; catalogue no. WL0003b), p-AKT
(wanleibio; catalogue no. WL02908), STAT3 (abcam; catalogue no.
ab68153), p-STAT3 (Cell Signaling Technology; catalogue no.
9145T), GAPDH (Cell Signaling Technology; catalogue no. 2118).
The target blots were detected with chemiluminescence system.
Pharmacokinetic Studies. Male SD rats (200−220 g, N = 3 per
group) were dosed intravenously with 1 mg/kg of Con B-1 prepared
in 5% DMA/10% Solutol HS 15/85% saline, and orally with 10 mg/
kg in 0.5% CMC-Na. Blood samples were taken at 0 (prior to dosing),
0.25, 0.5, 1, 2, 4, 6, 12, and 24 h following oral dosing and at 0 (prior
to dosing), 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h following intravenous
dosing. The samples were collected from the jugular vein and stored
in ice (0−4 °C). Plasma was obtained from the blood samples by
centrifugation (8000 rpm for 6 min at 2−8 °C) and stored at −80 °C.
All samples of the compound were determined by LC−MS/MS
(Shimadzu; API 4000). The assay lower limit of quantitation (LLOQ)
was 1 ng/mL for Con B-1 in plasma. The pharmacokinetic
parameters were analyzed by noncompartmental methods using
WinNonlin 5.2 (accomplished by Sichuan XPiscoric Inc.).
In Vivo Xenograft Studies. The female BALB/c nude mice were
purchased (Beijing HFK Bioscience Co. ltd., Beijing, China). H3122
cells were harvested during the exponential-growth phase and washed
twice with serum-free medium. Mice (6−7 weeks old and weighed
18−22 g) were injected subcutaneously with 5 × 106 H3122 cells,
which were suspended in 0.1 mL of serum and antibiotic-free growth
medium. The tumors were allowed to grow to 150−200 mm3, at
which point the mice were divided randomly (6 mice for each group).
The mice were dosed orally with Con B-1 (10, 20, 50 mg/kg/d,
dissolved in 10% NMP/90% PEG300, vehicle (10% NMP/90%
PEG300), and Ceritinib (positive control, 20 mg/kg/d, dissolved in
10% NMP/90% PEG300). The body weight and tumor volume were
measured every 3 days. The tumor volume was determined with
Vernier calipers and calculated as follows: tumor volume = a × b2/2
(a, long diameter; b, short diameter). Percentage of tumor growth
inhibition (TGI) was determined after 15 days of treatment as the
reduction in size of tumors in treated groups vs vehicle controls. All
experimental protocols and animal handling procedures were
approved by the Institutional Animal Care and Use Committee of
Sichuan University.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01707.
■
sı
Inhibition rate (%) of compounds against ALK;
antiproliferative activities of various cancer cell lines;
antiproliferative activities of active compounds against
human normal cell LO2; selectivity of Con B-1; covalent
adduct nature of ConB-1; HPLC traces for Con A-3,
Con A-4, Con B-1, and Con B-3 (PDF)
Molecular formula strings and some data (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Rui Li − State Key Laboratory of Biotherapy, Collaborative
Innovation Center of Biotherapy and Cancer Center, West
China Hospital of Sichuan University, Chengdu 610041,
China; Email: lirui@scu.edu.cn
Shilong Fan − The Ministry of Education Key Laboratory of
Protein Science, Beijing Advanced Innovation Center for
Structural Biology, Beijing Frontier Research Center for
Biological Structure, Collaborative Innovation Center for
Biotherapy, School of Life Sciences, Tsinghua University,
Beijing 100000, China; Email: fanshilong@
mail.tsinghua.edu.cn
Authors
Guoyi Yan − State Key Laboratory of Biotherapy,
Collaborative Innovation Center of Biotherapy and Cancer
Center, West China Hospital of Sichuan University, Chengdu
610041, China; Department of Hepatobiliary Pancreatic
Surgery, Henan Provincial People’s Hospital, Zhengzhou
450000, China; orcid.org/0000-0002-3953-6190
Xinxin Zhong − State Key Laboratory of Biotherapy,
Collaborative Innovation Center of Biotherapy and Cancer
Center, West China Hospital of Sichuan University, Chengdu
610041, China
1567
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Article
Chunlan Pu − State Key Laboratory of Biotherapy,
Collaborative Innovation Center of Biotherapy and Cancer
Center, West China Hospital of Sichuan University, Chengdu
610041, China; orcid.org/0000-0003-4892-1979
Lin Yue − State Key Laboratory of Biotherapy, Collaborative
Innovation Center of Biotherapy and Cancer Center, West
China Hospital of Sichuan University, Chengdu 610041,
China
Huifang Shan − State Key Laboratory of Biotherapy,
Collaborative Innovation Center of Biotherapy and Cancer
Center, West China Hospital of Sichuan University, Chengdu
610041, China
Suke Lan − State Key Laboratory of Biotherapy, Collaborative
Innovation Center of Biotherapy and Cancer Center, West
China Hospital of Sichuan University, Chengdu 610041,
China
Meng Zhou − Engineering Research Center for the
Development and Application of Ethnic Medicine and TCM,
Ministry of Education & State Key Laboratory of Functions
and Applications of Medicinal Plants, Guizhou Medical
University, Guiyang 550000, China
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01707
Author Contributions
¶G.Y., X.Z., and C.P. contributed equally to this work. G.Y.Y.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We greatly appreciate the financial support from National
Natural Science Foundation of China (grants 81773195 and
81472780) and Sichuan Science and Technology Program
2021YJ0220
ABBREVIATIONS USED
■
ALK, anaplastic lymphoma kinase; ALKi, anaplastic lymphoma
kinase inhibitors; NSCLC, nonsmall cell lung cancer; HATU,
hexafluorophosphate azabenzotriazole tetramethyl uranium;
DIPEA, N,N-diisopropylethylamine; DMF, dimethylforma-
mide; MTT, methyl thiazolyl tetrazolium; SAR, structure
activity relationship; LC-MS, liquid chromatography−mass
spectroscopy; SD rats, Sprague−Dawley rats; TGIs, tumor
growth inhibitions; TFA, Trifluoroacetic acid; DCM, dichloro-
methane
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