Dual potent ALK and ROS1 inhibitors combating drug-resistant mutants: Synthesis and biological evaluation of aminopyridine-containing diarylaminopyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2018.09.012

To identify ALK and ROS1 dual inhibitors conferring resistance to ALK secondary mutations, especially ‘gatekeeper’ L1196 M and the most predominant ceritinib-resistant G1202R mutations, a series of novel 2,4-diarylaminopyrimidine analogues were designed and synthesized by incorporating 2-alkoxy-6-alicyclic aminopyridinyl motifs. The biological evaluations on cellular and enzymatic assays led to identification of compound F-1, which turned out to be effective against ALK^WT, ROS1^WT, ALK^L1196M and ALK^G1202R kinases with IC₅₀ of 2.1 nM, 2.3 nM, 1.3 nM and 3.9 nM, respectively, superior to crizotinib and ceritinib. Moreover, F-1 exhibited significant cytotoxicity on ALK-addicted Karpas299, H2228, and Ba/F3 cell expressing G1202R mutant, as well as ROS1-positive HCC78 cell with IC₅₀ values ranging from 10 nM to 43 nM. Notably, F-1 was capable of suppressing phospho-ALK and its relative downstream signaling pathways, and eventually, inducing cell apoptosis in a dose-dependent manner in Karpas-299 cell. Together, F-1 is validated as a promising ALK/ROS1 dual inhibitor great potential for G1202R ALK mutation cancers.

Full text of DOI:10.1016/j.ejmech.2018.09.012

Accepted Manuscript
Dual potent ALK and ROS1 inhibitors combating drug-resistant mutants: Synthesis
and biological evaluation of aminopyridine-containing diarylaminopyrimidine
derivatives
Ming Guo, Daiying Zuo, Junlong Zhang, Lingyun Xing, Wenfeng Gou, Feng Jiang,
Nan Jiang, Dajun Zhang, Xin Zhai
PII:
S0223-5234(18)30784-0
10.1016/j.ejmech.2018.09.012
EJMECH 10723
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 June 2018
Revised Date: 4 September 2018
Accepted Date: 4 September 2018
Please cite this article as: M. Guo, D. Zuo, J. Zhang, L. Xing, W. Gou, F. Jiang, N. Jiang, D. Zhang, X.
Zhai, Dual potent ALK and ROS1 inhibitors combating drug-resistant mutants: Synthesis and biological
evaluation of aminopyridine-containing diarylaminopyrimidine derivatives, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.09.012.
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Dual potent ALK and ROS1 inhibitors combating drug-resistant mutants:
synthesis
and
biological
evaluation
of
aminopyridine-containing
diarylaminopyrimidine derivatives
Ming Guoa, 1, Daiying Zuoc, 1, Junlong Zhanga, Lingyun Xinga, Wenfeng Gouc, Feng Jianga, Nan Jianga, Dajun
*
Zhangb,*, Xin Zhaia,
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China.
b Key Lab. of Behavioral and Cognitive Neuroscience of Liaoning Province, Shenyang Medical College, Shenyang
110034, China.
c Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China.
Abstract
To identify ALK and ROS1 dual inhibitors conferring resistance to ALK secondary mutations, especially ‘gatekeeper’
L1196M and the most predominant ceritinib-resistant G1202R mutations, a series of novel 2,4-diarylaminopyrimidine
analogues were designed and synthesized by incorporating 2-alkoxy-6-alicyclic aminopyridinyl motifs. The biological
evaluations on cellular and enzymatic assays led to identification of compound F-1, which turned out to be effective
against ALKWT, ROS1WT, ALKL1196M and ALKG1202R kinases with IC50 of 2.1 nM, 2.3 nM, 1.3 nM and 3.9 nM,
respectively, superior to crizotinib and ceritinib. Moreover, F-1 exhibited significant cytotoxicity on ALK-addicted
Karpas299, H2228, and Ba/F3 cell expressing G1202R mutant, as well as ROS1-positive HCC78 cell with IC50 values
ranging from 10 nM to 43 nM. Notably, F-1 was capable of suppressing phospho-ALK and its relative downstream
signaling pathways, and eventually, inducing cell apoptosis in a dose-dependent manner in Karpas-299 cell. Together,
F-1 is validated as a promising ALK/ROS1 dual inhibitor great potential for G1202R ALK mutation cancers.
Keywords: ALK/ROS1; Dual inhibitors; 2,4-Diarylaminopyrimidines; Synthesis; G1202R mutants
1. Introduction
Oncogenic gene fusions caused by chromosomal rearrangements have been recognized as important drivers across
many cancers [1]. Wherein, anaplastic lymphoma kinase (ALK) constitutes a part of the oncogenic fusion proteins
involving echinoderm microtubule-associated protein like 4 (EML4)-ALK and nucleophosmin (NPM)-ALK [2,3].
Identified as the most frequent oncogenic driver, EML4-ALK and NPM-ALK are aberrantly expressed due to
translocations or point mutations in a subset of cancers including non-small cell lung cancer (NSCLC), anaplastic large
cell lymphoma (ALCL) and neuroblastoma tumors [4,5]. Therefore, ALK fusions have been regarded as the most
promising oncogenic drug target which enabled the rapid discovery of potent ALK inhibitors [6].
ROS1, a ‘druggable’ orphan receptor tyrosine kinase, was firstly identified in glioblastoma in 1987, and subsequently
reported in NSCLC in 2007 [7]. ROS1 was vulnerable to chromosomal rearrangements resulting in transforming gene
fusions including CD74-ROS1, SDC4-ROS1 etc., which defined a molecular subtype of NSCLC [8,9]. ROS1 is
Corresponding author.
Corresponding author.
E-mail address: zhangdajun2008@126.com (D.J. Zhang); zhaixin_syphu@126.com (X. Zhai).
1
These authors contributed equally to this work.

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evolutionarily related to ALK for that both of them share substantial sequence homology in their kinase domains. As a
result, patients with ROS1-rearranged NSCLC shared many clinical characteristics in common with ALK-rearrangement
persons [10]. Hence, the therapeutic efficacy of targeted ALK inhibitors could be effectively evaluated in preclinical
ROS1-rearranged NSCLC cases, providing a rationale for its assessment in patients with advanced ROS1-rearranged
NSCLC [11].
Crizotinib, the first approved multi-targeted ALK inhibitor in 2011 which was also available for ROS1 rearrangement,
has become prescription medicine to treat patients with metastatic NSCLC harboring ALK and ROS1 rearrangements
[2,12,13]. However, despite a high response rate of 60% in ALK-positive NSCLC patients, unfortunately, tumor relapse
was invariably progressed after one year of treatment due to the emergence of resistant mutation [14]. A dozen of
targeted point mutations have been observed in ALK kinase domain including G1269A, L1152R, C1156Y, S1206Y,
G1202R and insertion 1151Tins, especially the prominent L1196M (the ‘gatekeeper’ mutation) (22% ~ 36%) [15]. To
combat various ALK-resistant mutants, many efforts have been done to guarantee more effective ALK inhibitors,
motivating the development of structurally diverse ‘second-generation’ ALK inhibitors. Ceritinib is the second approved
ALK inhibitor effective for treatment of crizotinib-resistant mutations such as L1196M and G1269A, but it was invalid
on other mutations presented by the obstacle G1202R which accounts for approximate 75% of all identified ALK
secondary mutations [16, 17]. Successively, alectinib [18] and brigatinib [19] was approved in 2015 and in 2017
respectively which were significantly efficient against the majority of crizotinib-resistant mutants except for G1202R
[20]. Similarly, some other ALK inhibitors are under clinical assessment currently such as ALK/ROS1 dual inhibitor
lorlatinib (phase III, Pfizer) [21] as well as ALK candidates ensartinib (phase II, Ignyta) [22] and enterctinib (phase II,
Xcovery) [23]. However, to the best of our knowledge, none of the ALK candidates effective in combating the tough
G1202R mutation has been successfully achieved (Fig. 1) [18, 24]. Therefore, discovery of novel ALK inhibitors capable
of conferring G1202R potency is urgently needed.
(Fig. 1 should be listed here)
In this perspective, molecular design was performed based on the cocrystal structure of ceritinib with ALK to develop
ALK inhibitors resistant to G1202R and L1196M mutants. In view of the pivotal role of 2,4-diarylaminopyrimidines
(DAAPs) skeleton [25, 26], DAAPs nucleus were retained to exert the key hydrogen bond interactions with ALK
residues. Recently, the ‘head’ isopropylsulfonyl group and the ‘tail’ piperidinyl motif in ceritinib have been investigated
extensively, indicating that modifications on both ‘head’ and ‘tail’ moieties were tolerant [27-29]. Hence, versatile
substituted acylamines instead of the isopropylsulfonyl fragment were incorporated in ceritinib and various amino groups
were embedded based on the bioisosterism principle.
The G1202R mutant is located at the hydrophobic binding pocket adjacent to the solvent front of the ALK domain.
The large basic residue Arg1202 was near to the isopropoxy group of ceritinib, and the unfavorable steric hindrance is
likely responsible for the loss on potency in the G1202R mutation [30]. Based on this analysis, we shifted the bulky
isopropoxy moiety to a tiny methoxy or hydrogen atom to improve activity against G1202R mutant by diminishing the
incompatible steric obstacle. Meanwhile, a larger isopropylthio group was introduced as matched moiety. Pyridinyl
skeleton as ‘privileged medicinal scaffold’ had been applied in drug design widely. The ‘necessary nitrogen atom’ is
prone to be a versatile high-impact design element to achieve pharmacological profiles [31]. In this perspective, the
2-alkoxypyridin-3-yl motif was attached to 2-amino group on DAAPs framework expecting to exert extra interaction
between pyridinyl motif with ALK catalytic domain. Furthermore, versatile alicyclic amines as solubilizing ‘tails’ were
integrated into the pyridinyl group and, herein, a series of 2,4-diarylaminopyrimidine analogues bearing alicyclic
aminopyridinyl fragments were designed successfully.
In this study, the target compounds were subjected to anti-proliferative evaluation and enzymatic assays. The intensive

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exploration on the outstanding compound F-1 was propelled by morphology analysis and western blot assay, and results
manifested that F-1 possessed excellent anti-cancer activity through down-regulating the expression of ALK and its
downsteam pathway, and progressing into apoptosis eventually.
2. Results and discussion
2.1. Chemistry
All compounds (F-1~F-28) were synthesized as depicted in Scheme 1. Commercially available benzene-1,2-diamine
were condensed regioselectively with 4-Cl group of 2,4,5-trichloropyrimidine in the presence of
N,N-diisopropylethylamine (DIPEA) in isopropanol to provide intermediate A. Then intermediate A was acylated by
substituted acyl chloride or sulfonyl chloride in the company of pyridine in dichloromethane to afford intermediates
B-1~B-4 in overall 78~85% yields [32]. Chloro group on 2,6-dichloro-3-nitropyridine was conveniently converted to
methoxy (isopropoxy or isopropylthio) group under the conditions of sodium hydride in corresponding methanol
(isopropanol or isopropyl mercaptan) solution to generate intermediates C-1~C-3. 2-chloro-5-nitropyridine and C-1~C-3
were subjected to amination with alicyclic amines to furnish intermediates D-1~D-16 [33]. In the subsequent step, the
nitro group of D-1~D-16 was reduced using palladium on activated carbon in methanol to give the ammoniated
derivatives E-1~E-16, which eventually underwent a nucleophilic substitution catalyzed by 4-methyl-benzenesulfonic
acid to furnish the target compounds F-1~F-28 in satisfactory yields.
(Scheme 1 should be listed here)
1
The structures of all target compounds were identified by MS, H NMR and 13C NMR spectra. Interestingly, the
existence of C=C bonds on N-phenylbut-2-enamide in compounds F-10~F-13 could lead to E- or Z-isomeric forming.
According to the reported data of N-phenylbut-2-enamide motif, the coupling constants of olefinic protons on C-3
position were observed for the E-configuration (3J = 12.0 ~ 20.0 Hz) and Z-isomer (3J = 10.0 ~ 12.0 Hz) [34, 35]. At
same time, the chemical shifts of 3H were altered from 6.00 ~ 7.00 ppm to 5.00 ~ 6.35 ppm as the configurations varied
from E- to Z-isomer. Therefore, the configuration of carbon-carbon double bond could be identified according to
1
3
coupling constants and chemical shifts of H protons. For an instance, HNMR (400 MHz) analysis of F-11, the
observed chemical shift (
exclusively.
δ
= 6.80 ppm) and the resultant coupling constant ( 3J =13.72 Hz) confirmed E-isomer
2.2.1. In vitro antiproliferative activity and SARs study.
All compounds (F-1~F-28) were investigated for in vitro antiproliferative activities against a panel of cancer cell lines,
including ALK-addicted Karpas299 (ALCL cell) expressing NPM-ALK, H2228 (NSCL cell) harboring EML4-ALK and
ROS1-positive HCC78 (human lung adenocarcinoma cell) harboring SLC34A2-ROS1. Importantly, ALK-expressive
Ba/F3 cell engineered to express G1202R mutation was examined to identify the potency on ALK secondary mutations.
Meanwhile, EGFR-positive A549 (NSCL cell) independent on ALK and ROS1 were adopted to evaluate the potential
off-target effects. Crizotinib and ceritinib served as positive controls, and the pharmacological data indicated as
half-maximal inhibitory concentration (IC50) were outlined on Table 1.
As expected, the results showed that most of the compounds exerted promising or significant anti-proliferative
activities in a set of ALK- and ROS1-addicted cancer cells with IC50 values in double-digit nanomolars. Interestingly, the
most prominent compound F-1 displayed highlighted potency on H2228, Karpas299, HCC78 and Ba/F3G1202R cells with
IC50 values of 12 nM, 10 nM, 43 nM and 30 nM, respectively, comparable or superior to ceritinib and crizotinib. It
noteworthy that F-1 showed amazing potency on Ba/F3G1202R cell (IC50 = 30 nM) overmatched the positive controls
dramatically. Likewise, the similar inhibition on G1202R-expressed Ba/F3 cell were observed for the majority of title
compounds, addressing the potency on ALK G1202R mutant and rationalizing the molecular design by incorporating the
2-alkoxy-6-alicyclic aminopyridinyl motif. As a general trend, most compounds showed significantly enhanced potency

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against Karpas299 cell relative to others. By contrast, they were less sensitive on A549 cell with the IC50 values
exceeding 1 µM, demonstrating the on-target effect of these derivatives.
We began by replacing the isopropylsulfonyl group (‘head’) in ceritinib with acylamino motif including methyl
sulfonamide (F-1~F-9, F-22~F-27), cyclopropyl sulfonamide (F-14~F-20, F-28), acetylamine (F-21) and butenamide
(F-10~F-13). The structure-activity relationships (SARs) based on IC50 values manifested that variations of ‘head’ would
led to vital alterations on cytotoxicities. Obviously, embedment of methyl sulfonamide (e.g. F-1, F-7) gave rise to a 2.8-
to 6.3-fold increase in cytotoxicity against all tested cells relative to cyclopropyl sulfonamide analogues (e.g. F-15, F-19).
In contrast, the bulky butenamide fragment weakened the biological activity evidently (F-2 v.s. F-11), suggesting groups
at this region were somewhat sensitive to substituents’ sizes.
Owning to the multiple pharmacological profiles of ‘privileged’ pyridinyl scaffold, we inserted 2-alkoxypyridin-3-yl
motif to position-2 on DAAPs skeleton. Aiming to diminish the steric obstacle between isopropoxy fragments in ceritinib
with back pocket on Arg1202 ALK mutant region, further optimization on pyridinyl moiety was performed by
embedding tiny methoxy or hydrogen atom. As a result, replacing the isopropoxy with methoxy led to an obvious
increase in activity. By comparison, shifting isopropoxy (F-22) to bulky isopropoxythio motif (F-24) resulted in declined
potency by more than10-fold. The same trend was discovered in another matched molecular pairs (F-3, F-23 v.s. F-25).
Turn to analogs F-26~F-28 bearing unsubstituted pyridinyl skeleton, interestingly, removal of methoxy group generated a
2.3- to 6.6-fold reduction in potency (F-1 v.s. F-26; F-15 v.s. F-28), in good agreement with the low expanding lipophilic
pocket at G1202R site.
To determine substitution effects of the terminal amino groups, versatile secondary amines as solubilizing ‘tails’ were
introduced (Table 1). Pharmacological data indicated that variation of the amino ‘tails’ did change the cytotoxicity
correspondingly. Piperidine or pyrrolidine derivatives, represented by F-3, F-4, F-16 and F-25, were not active against at
least 2-3 tested cell lines, most likely due to the absence of the cationic nitrogen or oxygen atoms. Likewise, once the
pyrrolidine group was ‘opened’ to diethylamine, an even decreased activity was observed, such as F-3 v.s. F-4; F-17 v.s.
F-18, highlighting the importance of hydrophilicity of ‘tails’ exposing in the solvent region. Based on this perspective,
introduction of hydrophilic groups, such as 3-hydroxyazetidinyl in F-7 and 3-hydroxyethylpiperazinyl in F-9, were
envisioned for the enhanced activity. Interestingly, compounds F-1 and F-15 bearing 4-methylpiperazinyl ‘tail’ turned
out to be the most active according to the best IC50 values among all compounds, while replacement of the
4-methylpiperazinyl group by morphino (F-14) or thiomorphino (F-5) led to a decreased liveness by about 2-fold. The
main SARs would provide valuable results to guide further molecular design.
(Table 1 should be listed here)
2.2.2. In vitro enzymatic assay.
To ascertain the potent target and evaluate antitumor potential, the highly active compounds F-1, F-5, F-7, F-9, F-15
and F-21 were selected for further in vitro enzymatic assay based on the cellular results. The potency was examined
against wild-type ALK, ALK secondary mutations (the "gatekeeper" L1196M and ceritinib-resistant G1202R), ROS1 and
non-relative EGFR kinase, and the inhibitory results were summarized in Table 2.
As depicted in Table 2, among the six tested hits, F-1 showed the most excellent potency on the majority of tested
kinase with IC50 values below 3.9 nM. Especially, F-1 showed an outstanding inhibition on ALKL1196M ( IC50 = 1.3 nM)
and ALKG1202R ( IC50= 3.9 nM). Analog F-1 was almost 11 to 23-fold more active relative to ceritinib (IC50 = 35 nM,
91nM), and was found superior to crizotinib dramatically (IC50 =380, >1000 nM). On contrast, the activity of F-22
bearing the bulk isopropoxypyridinyl moiety was declined with IC50 value of 9.4 nM against ALKG1202R, which was found
to be 2 to 3-fold decreased relative to the corresponding methoxypyridinyl analogues. It indicates that the steric crash
with G1202R is an unfavorable framework to ALK inhibition. Importantly, F-7 bearing 3-hydroxyazetidinyl group

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revealed outstanding potency against wild-type ROS1( IC50 = 1.6 nM ) and ALK ( IC50 = 1.4 nM ) which was superior to
all other analogs and reference drugs. However, its inhibitions on ALKL1196M and ALKG1202R was comparable to other
molecules.
Differ from the clear SARs in cellular assay, the enzymatic results were indiscrimination in ROS1 and ALK tested
groups attributed to the overall high potency under the experimental conditions, as was in accordance with current
structurally analogical ALK inhibitors such as ceritinib. As respected, all tested compounds abolished enzymatic
activities once examining to EGFR, reflecting the desired selectivity over EGFR kinases. In consideration of results from
both enzymatic and cellular assays, compound F-1 was taken forward for further in-depth investigation.
(Table 2 should be listed here)
2.2.3. Inhibitory effects of F-1 against ALK and downstream signaling.
It is reported that the inhibition of ALK would result in significant down-regulation of downstream signaling covering
p-Akt and p-STAT3 expressions [35].
To further illuminate the cellular targeting activity of F-1 on ALK and its
downstream signaling pathways, western blot assay was investigated following F-1 treatment at the indicated
concentrations for 24 h in Karpas299 cells, and the results were exhibited in Fig. 2.
According to the results, F-1 inhibited the phosphorylation of ALK obviously and substantially suppress the
phosphorylation of Akt and STAT3 in a dose-dependent manner. A complete abolishment on ALK phosphorylation can
be clearly appreciated at a low dose of 100 nM, and inhibition could be observed even at 30 nM. Meanwhile, clear
inhibition of p-Akt and p-STAT3 were also detected at 100 nM, indicating the promising activity of F-1 in suppressing
relevant expression of ALK downstream signaling. The reduction of cellular p-ALK, p-AKT as well as p-STAT3
confirmed that F-1 was a promising ALK inhibitor.
(Fig. 2 should be listed here)
2.2.4. Effects of F-1 in inducing cell apoptosis.
It is known that apoptosis is one of the important standards to assess the effect of anti-cancer agents, and several
current ALK inhibitors exert antiproliferative potency via inducing cell apoptosis [36]. Therefore, the effect of F-1 on
apoptotic morphological change was further investigated by means of Acridineorange (AO)/ethidium bromide (EB)
double fluorescence staining [37] and Hoechst 33258 nucleic acid staining assays [38]. AO is capable of penetrating
through intact membranes of normal cells and coloring DNA as green fluorescence, while EB is only taken up by
apoptotic cells coloring DNA as orange fluorescence. Therefore, the transition of fluorescence from green to orange
indicated that the membranes of normal cells were broken and nuclear DNAs were fragmented on account of apoptosis.
Hoechst 33258 nuclear counterstain is part of fluorescent dyes that emits blue fluorescence once coupled with DNA, as
to investigate the apoptotic morphological changes of nuclear chromation.
F-1 (25 nM and 50 nM) or ceritinib (50 nM) were treated with AO/EB and Hoechst 33258 stains in Karpas299 cells,
respectively, and the resultant apoptosis photograph were shown in Fig. 3. In upper part, uniformly stained green nuclei
in control group suggested the intact cell membranes and nuclei in AO/EB staining. While, the increase of light green
fluorescence was detected followed by F-1 (25 nM) treatment. Importantly, the irregular orange-red fluorescence in
Karpas299 cells was examined in F-1 (50 nM) and ceritinib (50 nM) treated groups, indicating the late apoptosis cells
were markedly emerged.
Consistently, the similar F-1-induced cell apoptosis could be observed in Hoechst 33258 stains in Karpas299 cells. As
depicted in Fig.4 (the lower part), cells in control group displayed homogeneous blue fluorescence. Surprisingly, F-1 (50
nM) or ceritinib (50 nM) treated cells showed unclear shrinkage and fragmentation obviously. In contrast, the early

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apoptotic cells were noticed in F-1 (25 nM) treatment group inferior to F-1 (50 nM) section, revealing that F-1 promoted
Karpas299 cell apoptosis in a dose-dependent manner.
(Fig. 3 should be listed here)
3. Conclusion
In conclusion, in an attempt to develop promising ALK and ROS1 dual inhibitors capable of overcoming L1196M and
the most predominant ceritinib-resistant G1202R mutants, we designed a series of new 2,4-diarylaminopyrimidine
analogues by embedding the 2-alkoxy-6-alicyclic aminopyridinyl frameworks adjusted to fit for the back pocket on
G1202R mutation domain, leading to potent inhibitors against both wild-type and resistant mutants of ALK. All
compounds were subjected to anti-proliferative evaluation against a panel of ALK-addicted cells including Karpas299,
H2228 and ROS1-positive HCC78 cell, especially the Ba/F3 cell expression the challenging G1202R mutant, which
resulted in discovery of several potent compounds with IC50 value of double-digit nanomolars. Therein, F-1 exhibited the
most prominent cytotoxicity with IC50 value of 30 nM against Ba/F3G1202R ALK-expressive cell, indication the efficiency
on ceritinib-resistant G1202R mutations. The primary SARs studies based on cellular assay regulated the optimal
structural moieties which were benefit for new molecular design.
Further enzymatic examination in vitro on ALK and ROS1 led to the identification of F-1, which possessed remarkable
activity with IC50 values of 2.1 nM and 2.3 nM, respectively. Especially, F-1 was effective in suppressing the ALK
resistant mutations including the gatekeeper L1196M and the challenging G1202R mutants with IC50 of 1.3 nM and 3.9
nM, superior to crizotinib and ceritinib, regarded as a prominent ALK/ROS1dual inhibitor. The intensive exploration on
F-1 was propelled by western blot assay and morphology analysis. Notably, F-1 could suppress the phosphorylation of
ALK and inhibit its downstream signaling pathways in a dose-dependent manner, and manifested excellent
apoptosis-induced activity in the AO/EB and Hoechst33258 stains eventually. All of these findings supported F-1 as an
attractive ALK/ROS1 dual candidate to combat both resistant mutants of ALK including G1202R.
4. Experimental Procedures
4.1. Chemistry
Unless noted, all materials were gained from commercially available sources and were put in use without further
purification. Melting points of all compounds were obtained on a Büchi Melting Point B-540 apparatus
(BüchiLabortechnik, Switzerland) which were uncorrected. Mass spectra (MS) were performed in ESI mode on Agilent
1100 LC-MS (Agilent, palo Alto, USA). 1H-NMR and 13C-NMR spectra were taken using Bruker spectrometers (Bruker
Bioscience, Billerica, USA) with TMS as an internal standard. Column chromatography was run on silica gel (200-300
mesh) from Qingdao Ocean Chemicals (Qingdao, China).
4.1.1. N1-(2,5-dichloropyrimidin-4-yl)benzene-1,2-diamine (A)
Benzene-1,2-diamine (17.7 g, 0.16 mol) together with 2,4,5-trichloropyrimidine (30.0 g, 0.16 mol) and DIPEA (54.0
mL, 0.33 mol) was added in isopropanol (0.3 L). The reaction mixture was heated and stirred for 2.0 h at 80 °C. The
mixture was filtered when TLC showed the reaction was completed. The cake was resuspended in water and adjusted the
pH to 7 with hydrochloric acid. The mixture was filtered after stirring for 30 minutes. The white solid was collected to
give A in a 96.5% yield after trituration in dichloromethane and filtration. MS (ESI) m/z: 255.0 [M+H]+, 253.1 [M-H]-。
4.1.2. General procedure for preparation of compounds (B-1~B-4)
To a solution of A (20.0 g, 0.079 mol) in dichloromethane (DCM, 0.1 L) was added pyridine (19.0 g, 0.24 mol). Then
substituted acyl chloride or sulfonyl chloride (0.09 mol) was added slowly under the circumstance of ice-salt baths. After
reacting at room temperature for 2.5 h, water (30 mL) was poured into the mixture after reacting at room temperature for
2.5 h and the solvent was evaporated under reduced pressure. The crude product was resuspended in water and adjusted
the pH to 7 with hydrochloric acid, then filtered and washed with water to give B-1~B-4.

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4.1.2.1. N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)methanesulfonamide (B-1).
Yield: 85.4%; MS (ESI) m/z: 333.2 [M+H]+, 331.0 [M-H]-.
4.1.2.2. N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)cyclopropanesulfonamide (B-2).
Yield: 81.7%; MS (ESI) m/z: 359.4 [M+H]+.
4.1.2.3. (E)-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)but-2-enamide (B-3).
Yield: 91.6%; MS (ESI) m/z: 322.9 [M+H]+, 345.2 [M+Na]+.
4.1.2.4. N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)acetamide (B-4).
Yield: 89.4%; MS (ESI) m/z: 297.0 [M+H]+, 319.1 [M+Na]+.
4.1.3. General procedure for preparation of compounds (C-1~C-3)
2,6-dichloro-3-nitropyridine (20.0 g, 0.10 mol) was added in the component solvent of RXH ( 0.1mol) and
tetrahydrofuran (THF, 0.1 L) , then sodium hydride (4.6 g, 0.11 mol) was mixed in batches in the context of ice-salt
baths. After reacting at room temperature for 6 h, the mixture was poured into water (1 L) and stirred for 30 minutes.
Then the yellow solid was collected by filtered and purified by column chromatography through petroleum ether/ethyl
acetate as an eluent to gain white solid C-1~C-3.
4.1.3.1. 6-chloro-2-methoxy-3-nitropyridine (C-1).
Yield: 71.8%; MS (ESI) m/z: 188.9 [M+H]+.
4.1.3.2. 6-chloro-2-isopropoxy-3-nitropyridine (C-2).
Yield: 75.3%; MS (ESI) m/z: 217.5 [M+H]+.
4.1.3.3. 6-chloro-2-(isopropylthio)-3-nitropyridine (C-3).
Yield: 73.7%; MS (ESI) m/z: 233.3 [M+H]+.
4.1.4. General procedure for preparation of compounds (D-1~D-16)
To a solution of C-1~C-3 (5.3 mmol) or 2-chloro-5-nitropyridine (5.3 mmol) in THF (5 mL) was added K2CO3 (10.6
mmol) and different alicyclic amines (0.011 mol). The reaction mixture was heated and stirred for 3.0 h at 50 °C. The
solvent was evaporated under reduced pressure whereafter water was poured into the mixture and stirring for 1h.
Production D-1~D-16 was gained after filtration.
4.1.4.1. 1-(6-methoxy-5-nitropyridin-2-yl)-4-methylpiperazine (D-1)
Yield: 82.4%; MS (ESI) m/z: 275.3 [M+H]+.
4.1.4.2. 2-methoxy-3-nitro-6-(piperidin-1-yl)pyridine (D-2)
Yield: 87.6%; MS (ESI) m/z: 238.1 [M+H]+.
4.1.4.3. 2-methoxy-3-nitro-6-(pyrrolidin-1-yl)pyridine (D-3)
Yield: 94.8%; MS (ESI) m/z: 224.0 [M+H]+.
4.1.4.4. N,N-diethyl-6-methoxy-5-nitropyridin-2-amine (D-4)
Yield: 78.4%; MS (ESI) m/z: 226.0 [M+H]+.
4.1.4.5. 4-(6-methoxy-5-nitropyridin-2-yl)thiomorpholine (D-5)
Yield: 85.5%; MS (ESI) m/z: 256.1 [M+H]+.
4.1.4.6. 1-ethyl-4-(6-methoxy-5-nitropyridin-2-yl)piperazine (D-6)
Yield: 75.4%; MS (ESI) m/z: 267.3 [M+H]+.
4.1.4.7. 1-(6-methoxy-5-nitropyridin-2-yl)azetidin-3-ol (D-7)
Yield:84.4%; MS (ESI) m/z: 226.4 [M+H]+.
4.1.4.8. 1-(6-methoxy-5-nitropyridin-2-yl)-3-methylpiperazine (D-8)
Yield: 85.7%; MS (ESI) m/z: 253.3 [M+H]+.
4.1.4.9. 2-(4-(6-methoxy-5-nitropyridin-2-yl)piperazin-1-yl)ethan-1-ol (D-9)
Yield: 79 %; MS (ESI) m/z: 283.3 [M+H]+.
4.1.4.10. 4-(6-methoxy-5-nitropyridin-2-yl)morpholine (D-10)

ACCEPTED MANUSCRIPT
Yield: 71.1%; MS (ESI) m/z: 240.2 [M+H]+.
4.1.4.11. 1-(6-isopropoxy-5-nitropyridin-2-yl)-4-methylpiperazine (D-11)
Yield: 81.2%; MS (ESI) m/z: 281.5 [M+H]+.
4.1.4.12. 2-isopropoxy-3-nitro-6-(pyrrolidin-1-yl)pyridine (D-12)
Yield: 86.7%; MS (ESI) m/z: 252.3 [M+H]+.
4.1.4.13. 1-(6-(isopropylthio)-5-nitropyridin-2-yl)-4-methylpiperazine (D-13)
Yield: 84.2%; MS (ESI) m/z: 297.2 [M+H]+.
4.1.4.14. 2-(isopropylthio)-3-nitro-6-(pyrrolidin-1-yl)pyridine (D-14)
Yield: 75.6%; MS (ESI) m/z: 268.4 [M+H]+.
4.1.4.15. 1-methyl-4-(5-nitropyridin-2-yl)piperazine (D-15)
Yield: 76.5%; MS (ESI) m/z: 223.1 [M+H]+.
4.1.4.16. 5-nitro-2-(pyrrolidin-1-yl)pyridine (D-16)
Yield: 72.4%; MS (ESI) m/z: 194.5 [M+H]+.
4.1.5. General procedure for preparation of compounds (E-1~E-16)
To a mixture of D-1~D-16 (0.14 mol ) in THF ( 15 mL) was added Pd/C (0.2N) while maintaining the temperature
about 35 °C and stirring about 6 h under the condition of hydrogen gas. After the mixture reacted for about 6 h, filtered
the suspension and the resultant solution was evaporated to give the oily residue, which was solidified by ether to give
the compounds E-1~E-16.
4.1.5.1. 2-methoxy-6-(4-methylpiperazin-1-yl)pyridin-3-amine (E-1)
Yield: 71.1%; MS (ESI) m/z: 223.3 [M+H]+.
4.1.5.2. 2-methoxy-6-(piperidin-1-yl)pyridin-3-amine (E-2)
Yield: 76.3%; MS (ESI) m/z: 208.0 [M+H]+.
4.1.5.3. 2-methoxy-6-(pyrrolidin-1-yl)pyridin-3-amine (E-3)
Yield: 81.8%; MS (ESI) m/z: 194.3 [M+H]+.
4.1.5.4. N2,N2-diethyl-6-methoxypyridine-2,5-diamine (E-4)
Yield: 80.2%; MS (ESI) m/z: 196.3 [M+H]+.
4.1.5.5. 2-methoxy-6-thiomorpholinopyridin-3-amine (E-5)
Yield: 75.6%; MS (ESI) m/z: 226.5 [M+H]+.
4.1.5.6. 6-(4-ethylpiperazin-1-yl)-2-methoxypyridin-3-amine (E-6)
Yield: 78.8%; MS (ESI) m/z: 237.2 [M+H]+.
4.1.5.7. 1-(5-amino-6-methoxypyridin-2-yl)azetidin-3-ol (E-7)
Yield: 71.2%; MS (ESI) m/z: 196.4 [M+H]+.
4.1.5.8. 2-methoxy-6-(3-methylpiperazin-1-yl)pyridin-3-amine (E-8)
Yield: 77.0%; MS (ESI) m/z: 223.3 [M+H]+.
4.1.5.9. 2-(4-(5-amino-6-methoxypyridin-2-yl)piperazin-1-yl)ethan-1-ol (E-9)
Yield: 79.2%; MS (ESI) m/z: 253.6 [M+H]+.
4.1.5.10. 2-methoxy-6-morpholinopyridin-3-amine (E-10)
Yield: 75.5%; MS (ESI) m/z: 210.3 [M+H]+.
4.1.5.11. 2-isopropoxy-6-(4-methylpiperazin-1-yl)pyridin-3-amine (E-11)
Yield: 82.1%; MS (ESI) m/z: 251.4 [M+H]+.
4.1.5.12. 2-isopropoxy-6-(pyrrolidin-1-yl)pyridin-3-amine (E-12)
Yield: 84.6%; MS (ESI) m/z: 223.4 [M+H]+.
4.1.5.13. 2-(isopropylthio)-6-(4-methylpiperazin-1-yl)pyridin-3-amine (E-13)
Yield: 78.6%; MS (ESI) m/z: 267.3 [M+H]+.

ACCEPTED MANUSCRIPT
4.1.5.14. 2-(isopropylthio)-6-(pyrrolidin-1-yl)pyridin-3-amine (E-14)
Yield: 72.3%; MS (ESI) m/z: 238.3 [M+H]+.
4.1.5.15. 6-(4-methylpiperazin-1-yl)pyridin-3-amine (E-15)
Yield: 71.3%; MS (ESI) m/z:193.5 [M+H]+.
4.1.5.16. 6-(pyrrolidin-1-yl)pyridin-3-amine (E-16)
Yield: 76.5%; MS (ESI) m/z:164.2 [M+H]+.
4.1.6. General procedure for preparation of compounds (F-1~F-28)
To a solvent of E-1~E-16 (1.5 mmol) in isopropanol was added p-methylbenzenesulfonic acid (1.5 mmol) and
B-1~B-4 (1.5 mmol) under stirring. Afterwards the mixture was heated to reflux and stirred for 10 h. The mixture was
filtered after cooling to room temperature to obtain the solid. The crude product was resuspended in water and adjusted
the pH to 8 with sodium bicarbonate, then extracted with ethyl acetate (20 mL) for 3 times and the combined organic
layers were washed with water, brine, dried over Na2SO4 and the solvent was evaporated under reduced pressure. After
purifying through column chromatography using dichloromethane/ methanol as an eluent, the target products F-1~F-28
were obtained in satisfactory yields.
4.1.6.1.
N-(2-((5-chloro-2-((2-methoxy-6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfo
namide (F-1)
Yield: 80.6%; m.p.:209.6-213.4 °C; MS (ESI) m/z:519.3 [M+H]+; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.32 (s,
1H), 8.47 (s, 1H), 8.10 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.33 (dd, J = 7.3, 1.8 Hz, 1H), 7.14
(ddd, J = 9.0, 5.8, 1.4 Hz, 2H), 6.25 (d, J = 8.5 Hz, 1H), 3.77 (s, 3H), 3.46-3.41 (m, 4H), 2.95 (s, 3H), 2.46-2.40 (m, 4H),
2.24 (s, 3H). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 159.5, 155.8, 155.6, 155.0, 154.8, 136.1, 135.3, 129.0, 127.4,
127.1, 124.7, 124.3, 112.7, 104.0, 98.3, 54.8(2C), 53.1, 46.2, 45.4 (2C), 29.5.
4.1.6.2.
N-(2-((5-chloro-2-((2-methoxy-6-(piperidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(F-2)
Yield: 79.7%; m.p.:156.7-160.4 °C; MS (ESI) m/z:504.4 [M+H]+; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.30 (s,
1H), 8.41 (d, J = 11.8 Hz, 1H), 8.12-8.05 (m, 2H), 8.01 (s, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.33-7.29 (m, 1H), 7.16 (dd, J =
15.9, 8.3 Hz, 2H), 6.23 (d, J = 8.5 Hz, 1H), 3.76 (s, 3H), 3.46 (d, J = 4.9 Hz, 4H), 2.97 (s, 3H), 1.58 (s, 6H).
4.1.6.3.
N-(2-((5-chloro-2-((2-methoxy-6-(pyrrolidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(F-3)
Yield: 82.3%; m.p.:183.6-184.2 °C;MS (ESI) m/z: 490.4 [M+H]+; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.30 (s,
1H), 8.40 (d, J = 5.2 Hz, 1H), 8.09 (d, J = 4.0 Hz, 1H), 8.05 (s, 2H), 7.46 (d, J = 8.5 Hz, 1H), 7.33-7.31 (m, 1H), 7.14 (dd,
J = 14.8, 7.7 Hz, 2H), 5.89 (d, J = 8.3 Hz, 1H), 3.78 (s, 3H), 3.38 (t, J = 6.5 Hz, 4H), 2.97 (s, 3H), 1.95 (t, J = 6.5 Hz,
4H).
4.1.6.4.
N-(2-((5-chloro-2-((6-(diethylamino)-2-methoxypyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(F-4)
Yield: 82.0%; m.p.:153.2-156.7 °C; MS (ESI) m/z: 492.4 [M+H]+; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.30 (s,
1H), 8.40 (s, 1H), 8.07 (d, J = 15.8 Hz, 3H), 7.42 (d, J = 6.3 Hz, 1H), 7.32 (s, 1H), 7.13 (d, J = 3.7 Hz, 2H), 6.03 (dd, J =
8.2, 1.7 Hz, 1H), 3.76 (d, J = 1.9 Hz, 3H), 3.47 (d, J = 5.2 Hz, 4H), 2.97 (s, 3H), 1.18-1.11 (m, 6H). 13C-NMR (100 MHz,
DMSO-d6) δ (ppm): 160.0, 156.5, 155.6, 155.1, 153.4, 137.5, 135.7, 128.4, 127.7, 127.4, 124.4, 124.1, 110.0, 103.6, 96.4,
52.8, 42.7 (2C), 29.5, 13.5 (2C).
4.1.6.5.

ACCEPTED MANUSCRIPT
N-(2-((5-chloro-2-((2-methoxy-6-thiomorpholinopyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(F-5)
Yield: 84.7%; m.p.:160.3-162.4 °C; MS (ESI) m/z: 520.4 [M-H]-; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.30 (s,
1H), 8.44 (d, J = 44.7 Hz, 1H), 8.08 (s, 1H), 8.06 (s, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.36 – 7.30 (m, 1H), 7.12 (dd, J = 9.9,
4.7 Hz, 2H), 6.27 (d, J = 8.5 Hz, 1H), 5.75 (s, 1H), 3.85 (dd, J = 10.6, 5.7 Hz, 4H), 3.77 (s, 3H), 2.94 (s, 3H), 2.62 (dd, J
= 6.0, 3.7 Hz, 4H). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 159.6, 156.0, 155.7, 155.0, 153.7, 136.7, 135.2, 129.3,
127.1, 126.8, 124.6, 124.2, 112.2, 104.0, 98.6, 55.4, 53.1, 48.1 (2C) , 25.4(2C).
4.1.6.6.
N-(2-((5-chloro-2-((6-(4-ethylpiperazin-1-yl)-2-methoxypyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfona
mide (F-6)
Yield: 86.3%; m.p.:133.2-137.3 °C; MS (ESI) m/z: 533.4 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.46 (s,
1H), 8.07 (d, J = 2.6 Hz, 2H), 7.99 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.38 – 7.28 (m, 1H), 7.20 – 7.09 (m, 2H), 6.24 (d, J
= 8.5 Hz, 1H), 3.78 (s, 3H), 3.47 – 3.39 (m, 4H), 2.95 (s, 4H), 2.40 (dd, J = 14.3, 7.1 Hz, 3H), 1.23 (s, 2H), 1.08 – 1.01
(m, 3H).
4.1.6.7.
N-(2-((5-chloro-2-((6-(3-hydroxyazetidin-1-yl)-2-methoxypyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfo
namide (F-7)
Yield: 78.6%; m.p.:186.8-187.3 °C; MS (ESI) m/z: 490.4 [M-H]-; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.34 (s,
1H), 8.49 (s, 1H), 8.04 (d, J = 3.6 Hz, 2H), 7.36 (d, J = 8.3 Hz, 1H), 7.34-7.27 (m, 1H), 7.18-6.98 (m, 2H), 6.45 (t, J =
5.7 Hz, 1H), 6.03 (d, J = 8.3 Hz, 1H), 5.36 (d, J = 5.2 Hz, 1H), 3.91 (dd, J = 11.2, 6.0 Hz, 1H), 3.76 (s, 3H), 3.68 (dd, J =
11.0, 4.5 Hz, 1H), 3.58 (dd, J = 11.1, 5.8 Hz, 1H), 3.47-3.38 (m, 1H), 3.31-3.22 (m, 1H), 2.93 (s, 3H). 13C-NMR (150
MHz, DMSO-d6) δ (ppm): 159.0, 155.1, 155.1, 154.4, 154.3, 135.7, 134.7, 128.6, 126.6, 126.3, 124.0, 123.6, 112.1,
103.4, 97.6, 58.4, 52.8(2C), 52.5, 28.9.
4.1.6.8.
N-(2-((5-chloro-2-((2-methoxy-6-(3-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfo
namide (F-8)
Yield: 77.2%; m.p.:240.3-244.1 °C; MS (ESI) m/z: 519.4 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.67 (s,
1H), 8.07 (s, 1H), 8.04 (s, 1H), 7.99 (s, 1H), 7.61 (d, J = 8.3 Hz, 1H), 7.30 (d, J = 7.4 Hz, 1H), 7.04 (dd, J = 16.5, 8.3 Hz,
2H), 6.29 (d, J = 8.4 Hz, 1H), 4.09 (d, J = 10.8 Hz, 2H), 3.79 (s, 3H), 3.41 – 3.31 (m, 1H), 3.12 (d, J = 7.7 Hz, 1H), 2.88
(s, 3H), 2.85 (d, J = 9.1 Hz, 2H), 2.56 (d, J = 11.9 Hz, 1H), 1.23 (s, 1H), 1.14 (d, J = 6.2 Hz, 3H). 13C-NMR (100 MHz,
DMSO-d6) δ (ppm): 159.6, 155.7, 155.6, 154.7, 154.4, 136.3, 134.4, 131.2, 125.5, 125.0, 124.3, 123.1, 112.9, 104.1, 98.4,
65.4, 53.1, 51.5, 50.4, 44.5, 18.5, 15.6.
4.1.6.9.
N-(2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methoxypyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)met
hanesulfonamide (F-9)
Yield: 82.3%; m.p.: 188.6-192.0°C; MS (ESI) m/z: 549.5 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 1H), 8.49
(s, 1H), 8.07 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.41 – 7.27 (m, 1H), 7.13 (dd, J = 9.2, 5.2 Hz,
2H), 6.24 (d, J = 8.5 Hz, 1H), 4.46 (s, 1H), 3.77 (s, 3H), 3.55 (t, J = 6.2 Hz, 2H), 3.43 (s, 4H), 2.94 (s, 3H), 2.57 – 2.52
(m,3H), 2.46 (t, J = 6.2 Hz, 2H), 1.23 (s, 1H). 13C-NMR (150 MHz, DMSO-d6) δ (ppm): 159.5, 155.6, 155.6, 154.9,
154.8, 136.2, 135.2, 129.1, 127.1, 126.8, 124.5, 124.1, 112.6, 103.9, 98.1, 60.7, 58.8, 53.3(2C), 53.0, 45.5 (2C), 29.4.
4.1.6.10.
(E)-N-(2-((5-chloro-2-((2-methoxy-6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)but-2-ena
mide (F-10)
Yield: 85.6%; m.p.:173.2-176.9 °C; MS (ESI) m/z: 507.5 [M-H]-; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 10.01 (s,
1H), 8.43 (s, 1H), 8.02 (s, 1H), 7.80 (s, 1H), 7.64 (dd, J = 11.9, 6.4 Hz, 2H), 7.34-7.31 (m, 1H), 7.20-7.16 (m, 3H), 6.18

ACCEPTED MANUSCRIPT
(dd, J = 18.1, 10.2 Hz, 2H), 3.78 (s, 3H), 3.40 (s, 4H), 2.44-2.37 (m, 4H), 2.22 (s, 3H), 1.13 (d, J = 6.1 Hz, 3H).
4.1.6.11.
(E)-N-(2-((5-chloro-2-((2-methoxy-6-(piperidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)but-2-enamide
(F-11)
Yield: 83.9%; m.p.:168.2-171.3 °C; MS (ESI) m/z: 494.5 [M+H]+; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 10.06 (s,
1H), 8.50 (s, 1H), 8.00 (s, 1H), 7.80 (s, 1H), 7.72 (dd, J = 7.4, 1.8 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.31 (dd, J = 7.3, 2.1
Hz, 1H), 7.22-7.12 (m, 2H), 6.86 (dq, J = 13.7, 6.8 Hz, 1H), 6.18 (m, 2H), 3.77 (s, 3H), 3.43 (d, J = 5.2 Hz, 4H), 1.57 (s,
6H) , 1.23 (s, 3H).
4.1.6.12.
(E)-N-(2-((5-chloro-2-((2-methoxy-6-(pyrrolidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)but-2-enamide
(F-12)
Yield: 80.2%; m.p.:180.0-184.7 °C; MS (ESI) m/z: 480.5 [M+H]+; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.35 (s,
1H), 8.46 (s, 1H), 8.14 (s, 1H), 8.08 (s, 1H), 8.02 (s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 7.1 Hz, 1H), 7.25-7.08 (m,
2H), 6.27 (d, J = 8.4 Hz, 1H), 3.78 (s, 3H), 3.72 (s, 4H), 3.39 (s, 4H), 2.54 (s, 1H), 0.92 (d, J = 6.3 Hz, 2H), 0.84 (d, J =
1.2 Hz, 2H).
4.1.6.13.
(E)-N-(2-((5-chloro-2-((2-methoxy-6-(3-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)but-2-ena
mide (F-13)
Yield: 79.7%; m.p.:165.4-163.2 °C; MS (ESI) m/z:509.5 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ (ppm)13.31 (s,
1H), 10.02 (s, 1H), 8.50 (s, 1H), 8.01 (s, 1H), 7.81 (s, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.36 – 7.28
(m, 1H), 7.23 – 7.09 (m, 2H), 6.86 (dt, J = 13.8, 6.9 Hz, 1H), 6.28 – 6.12 (m, 2H), 4.00 (d, J = 12.5 Hz, 2H), 3.78 (s, 3H),
2.99 (d, J = 11.0 Hz, 1H), 2.71 (ddd, J = 22.4, 10.4, 8.2 Hz, 3H), 2.40 – 2.25 (m, 1H), 1.87 (d, J = 6.8 Hz, 3H), 1.06 (d, J
= 6.3 Hz, 3H). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 167.3, 165.1, 159.2, 156.1, 154.9, 154.4, 141.3, 135.0, 132.4,
131.2, 126.6, 125.8, 125.5, 125.2, 125.1, 112.8, 104.0, 98.1, 53.1, 52.6, 50.4, 45.5, 45.2, 19.5, 18.0.
4.1.6.14.
N-(2-((5-chloro-2-((2-methoxy-6-morpholinopyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)cyclopropanesulfonamide
(F-14)
Yield: 81.3%; m.p.:151.2-154.3 °C; MS (ESI) m/z:530.5 [M-H]-; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.35 (s, 1H),
8.46 (s, 1H), 8.14 (s, 1H), 8.08 (s, 1H), 8.02 (s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 7.1 Hz, 1H), 7.25-7.08 (m, 2H),
6.27 (d, J = 8.4 Hz, 1H), 3.78 (s, 3H), 3.72 (s, 4H), 3.39 (s, 4H), 2.54 (s, 1H), 0.92 (d, J = 6.3 Hz, 2H), 0.84 (d, J = 1.2
Hz, 2H).
4.1.6.15.
N-(2-((5-chloro-2-((2-methoxy-6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)cyclopropane
sulfonamide (F-15)
Yield:81.9%; m.p.:180.9-195.4 °C; MS (ESI) m/z:543.5 [M-H]-; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.36 (s, 1H),
8.46 (s, 1H), 8.13 (s, 1H), 8.07 (s, 1H), 8.02 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.14 (dt, J = 14.7,
6.2 Hz, 2H), 6.26 (d, J = 8.5 Hz, 1H), 3.77 (s, 3H), 3.48-3.41 (m, 4H), 2.55 (td, J = 7.8, 4.0 Hz, 1H), 2.47-2.40 (m, 4H),
2.24 (s, 3H), 0.95-0.81 (m, 4H). 13C-NMR (150 MHz, DMSO-d6) δ (ppm): 159.5, 155.7, 155.5, 155.0, 154.8, 136.3,
135.5, 128.4, 128.1, 127.3, 124.2, 123.9, 112.6, 103.8, 98.2, 54.7(2C), 53.0, 46.2, 45.4 (2C), 29.4, 5.6(2C).
4.1.6.16.
N-(2-((5-chloro-2-((2-methoxy-6-(piperidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)cyclopropanesulfonam
ide (F-16)
Yield: 82.6%; m.p.:96.3-100.6 °C; MS (ESI) m/z:530.5 [M+H]+; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.34 (s, 1H),
8.43 (s, 1H), 8.10 (s, 1H), 8.07 (s, 2H), 7.53 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.25-7.06 (m, 2H), 6.24 (d, J =
8.5 Hz, 1H), 3.77 (s, 3H), 3.47 (d, J = 5.1 Hz, 3H), 2.55 (td, J = 7.9, 4.1 Hz, 1H), 1.59 (s, 6H), 0.97-0.89 (m, 2H), 0.83 (t,

ACCEPTED MANUSCRIPT
J = 5.9 Hz, 2H).
4.1.6.17.
N-(2-((5-chloro-2-((2-methoxy-6-(pyrrolidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)cyclopropanesulfona
mide (F-17)
Yield: 79.9%; m.p.:169.0-171.2 °C; MS (ESI) m/z:514.4 [M-H]-; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.35 (s, 1H),
8.41 (d, J = 4.9 Hz, 1H), 8.14-7.93 (m, 3H), 7.53-7.44 (m, 1H), 7.37-7.32 (m, 1H), 7.12 (dt, J = 14.2, 7.9 Hz, 2H), 5.90
(d, J = 8.4 Hz, 1H), 3.78 (s, 3H), 3.38 (t, J = 6.5 Hz, 4H), 2.59-2.53 (m, 1H), 1.95 (t, J = 6.6 Hz, 4H), 0.95-0.82 (m, 4H).
13C-NMR (100 MHz, DMSO-d6) δ (ppm): 160.0, 156.5, 155.5, 155.0, 153.4, 136.9, 135.7, 128.3, 127.1, 126.5, 124.0,
123.5, 110.4, 103.6, 97.1, 52.8, 46.9(2C), 29.6, 25.5(2C), 5.6(2C).
4.1.6.18.
N-(2-((5-chloro-2-((6-(diethylamino)-2-methoxypyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)cyclopropanesulfonami
de (F-18)
Yield: 75.3%; m.p.:132.1-134.0 °C; MS (ESI) m/z:516.4 [M-H]-; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.35 (s, 1H),
8.42 (s, 1H), 8.11 (s, 1H), 8.05 (s, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.37-7.26 (m, 2H), 7.11 (dt, J = 13.9, 7.0 Hz, 2H), 6.04
(d, J = 8.5 Hz, 1H), 3.76 (s, 3H), 3.47 (q, J = 6.9 Hz, 4H), 2.60-2.54 (m, 1H), 1.18-1.11 (m, 6H), 0.95-0.91 (m, 2H), 0.86
(dd, J = 8.2, 4.0 Hz, 2H).
4.1.6.19.
N-(2-((5-chloro-2-((6-(3-hydroxyazetidin-1-yl)-2-methoxypyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)cyclopropane
sulfonamide (F-19)
Yield: 79.7%; m.p.:113.4-119.8 °C; MS (ESI) m/z:516.4 [M-H]-; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.31 (s, 1H),
8.43 (s, 1H), 8.04 (s, 1H), 8.03 (s, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.09 (d, J = 7.3 Hz, 1H), 6.41 (t,
J = 5.8 Hz, 1H), 6.04 (d, J = 8.3 Hz, 1H), 5.33 (d, J = 4.4 Hz, 1H), 3.92 (d, J = 4.3 Hz, 1H), 3.76 (s, 3H), 3.68 (dd, J =
11.0, 4.4 Hz, 1H), 3.58 (dd, J = 11.1, 5.8 Hz, 1H), 3.44 (dd, J = 13.4, 6.3 Hz, 1H), 3.38 (dd, J = 12.8, 5.7 Hz, 1H), 3.27
(dd, J = 13.4, 6.0 Hz, 1H), 2.59 – 2.51 (m, 1H), 0.91 (d, J = 7.7 Hz, 2H), 0.84 (d, J = 3.6 Hz, 2H). 13C-NMR (100 MHz,
DMSO-d6) δ (ppm): 160.0, 156.8, 155.5, 155.1, 137.2, 135.7, 128.4, 127.2, 124.0, 123.6, 110.6, 103.6, 98.8, 70.0, 65.4,
53.0, 48.6(2C), 45.4, 29.5, 5.1(2C).
4.1.6.20.
N-(2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methoxypyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)cycl
opropanesulfonamide (F-20)
Yield: 82.7%; m.p.:178.9-184.2 °C; MS (ESI) m/z:575.4 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ (ppm): (s, 1H),
8.46 (s, 1H), 8.08 (s, 1H), 8.07 (s, 1H), 8.02 (d, J = 6.1 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.17
(s, 1H), 7.15 – 7.04 (m, 1H), 6.25 (d, J = 8.5 Hz, 1H), 4.45 (s, 1H), 3.78 (s, 3H), 3.55 (t, J = 6.1 Hz, 2H), 3.47 – 3.40 (m,
4H), 2.58 – 2.52 (m, 4H), 2.46 (t, J = 6.2 Hz, 2H), 1.23 (s, 1H), 0.91 (d, J = 7.9 Hz, 2H), 0.84 (d, J = 4.1 Hz, 2H).
4.1.6.21.
N-(2-((5-chloro-2-((2-methoxy-6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)acetamide
(F-21)
Yield:89.2%; m.p.:112.3-113.6 °C; MS (ESI) m/z:483.4 [M+H]+; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 10.01 (s,
1H), 8.37 (s, 1H), 8.02 (s, 1H), 7.83 (s, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.26 (dd, J = 7.2, 2.0 Hz,
1H), 7.21-7.11 (m, 2H), 6.19 (d, J = 8.5 Hz, 1H), 3.78 (s, 3H), 3.44-3.37 (m, 4H), 2.44-2.37 (m, 4H), 2.22 (s, 3H), 2.09 (s,
3H).
4.1.6.22.
N-(2-((5-chloro-2-((2-isopropoxy-6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesu
lfonamide (F-22)
Yield: 88.3%; m.p.:140.8-144.9 °C; MS (ESI) m/z:547.4 [M+H]+; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.48 (s,

ACCEPTED MANUSCRIPT
1H), 8.48 (s, 1H), 8.08 (s, 1H), 7.94 (d, J = 6.4 Hz, 1H), 7.87 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H),
7.24-7.15 (m, 2H), 6.21 (d, J = 8.4 Hz, 1H), 5.14 (dt, J = 12.2, 6.1 Hz, 1H), 3.47 (s, 4H), 2.96 (s, 3H), 2.63 (s, 4H), 2.37
(s, 3H), 1.22 (d, J = 6.2 Hz, 6H). 13C-NMR (150 MHz, DMSO-d6) δ (ppm): 159.1, 155.9, 155.0, 154.1, 153.8, 135.1,
134.8, 129.0, 127.5, 127.3, 125.0, 124.9, 113.5, 103.9, 98.1, 68.1, 53.8(2C), 45.9, 44.6 (2C), 22.3(2C), 8.9.
4.1.6.23.
N-(2-((5-chloro-2-((2-isopropoxy-6-(pyrrolidin-1-yl)pyridin-3-yl)amino)pyrimidin4-yl)amino)phenyl)methanesulfonamid
e (F-23)
Yield: 85.6%; m.p.:151.3-154.6 °C; MS (ESI) m/z:516.4 [M-H]-; 1H-NMR (400 MHz, DMSO-d6) δ (ppm-d6): 9.31 (s,
1H), 8.44 (s, 1H), 8.05 (s, 1H), 8.00 (s, 1H), 7.83 (s, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.40-7.30 (m, 1H), 7.22-7.07 (m, 2H),
5.83 (d, J = 8.4 Hz, 1H), 5.25-5.10 (m, 1H), 3.36 (d, J = 6.2 Hz, 4H), 2.95 (s, 3H), 1.94 (t, J = 6.4 Hz, 4H), 1.21 (d, J =
6.2 Hz, 6H).
4.1.6.24.
N-(2-((5-chloro-2-((2-(isopropylthio)-6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methan
esulfonamide (F-24)
1
Yield: 87.5%; m.p.:167.9-169.2 °C; MS (ESI) m/z:563.4 [M+H]+; H-NMR (400 MHz, DMSO-d6) δ (ppm)：9.30 (s,
1H), 8.80 (s, 1H), 8.58 (s, 1H), 8.15 (d, J = 3.2 Hz, 1H), 7.87 (s, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.41 (dd, J = 7.6, 1.3 Hz,
1H), 7.30-7.24 (m, 1H), 7.20-7.17 (m, 1H), 6.72 (d, J = 8.2 Hz, 1H), 3.84 (dt, J = 13.2, 6.5 Hz, 1H), 3.13 (s, 4H), 2.94 (s,
3H), 2.40 (s, 4H), 2.21 (s, 3H), 1.23 (s, 6H).
4.1.6.25.
N-(2-((5-chloro-2-((2-(isopropylthio)-6-(pyrrolidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfon
amide (F-25)
1
Yield: 85.0%; m.p.:121.8-124.9 °C; MS (ESI) m/z:532.4 [M-H]-; H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.29 (s,
1H), 8.34 (d, J = 20.8 Hz, 2H), 8.06 (d, J = 18.9 Hz, 2H), 7.30 (d, J = 6.7 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 7.09 (s, 2H),
6.13 (d, J = 8.5 Hz, 1H), 3.87 (dt, J = 13.6, 6.8 Hz, 1H), 3.42 (s, 4H), 2.98 (s, 3H), 1.97 (t, J = 6.3 Hz, 4H), 1.29 (d, J =
6.8 Hz, 6H).
4.1.6.26.
N-(2-((5-chloro-2-((6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(F-26)
1
Yield: 88.3%; m.p.:134.6-138.8 °C; MS (ESI) m/z:489.4 [M+H]+; H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.18 (s,
1H), 8.56 (s, 1H), 8.25 (s, 1H), 8.14 (s, 1H), 7.99 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.54-7.10 (m, 4H), 6.74 (d, J = 8.9 Hz,
1H), 3.51 (s, 4H), 2.96 (s, 3H), 2.74 (s, 4H), 2.44 (s, 3H).
4.1.6.27.
N-(2-((5-chloro-2-((6-(pyrrolidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(F-27)
Yield: 89.2%; m.p.:169.6-174.3 °C; MS (ESI) m/z:460.4 [M+H]+; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.39 (s,
1H), 8.98 (s, 1H), 8.55 (s, 1H), 8.20-7.98 (m, 3H), 7.66 (d, J = 7.3 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.21 (d, J = 7.0 Hz,
1H), 7.17 (d, J = 7.5 Hz, 1H), 6.32 (d, J = 8.9 Hz, 1H), 3.34-3.27 (m, 4H), 2.93 (s, 3H), 1.98-1.88 (m, 4H).
4.1.6.28.
N-(2-((5-chloro-2-((6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)cyclopropanesulfonamid
e (F-28)
Yield: 87.8%; m.p.:150.1-153.9 °C; MS (ESI) m/z:513.4 [M-H]-; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.21 (s, 1H),
8.54 (s, 1H), 8.26 (s, 1H), 8.15 (s, 1H), 8.03 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 7.7 Hz,
1H), 7.21 (t, J = 7.6 Hz, 1H), 7.12 (d, J = 7.9 Hz, 1H), 6.77 (d, J = 8.9 Hz, 1H), 3.56 (s, 4H), 2.89 (s, 4H), 2.73 (s, 1H),
2.29 (s, 3H), 0.92-0.87 (m, 2H), 0.84 (d, J = 3.8 Hz, 2H).
4.2. Biological section.

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4.2.1. MTT assay
Cells (H2228, Karpas299, HCC78, A549 and EAG1202R cancer cell lines) were seeded into 96-well plates at a density of
approximate 5×104 per well (depends on the cell growth rate). After 24 h, triplicate wells were treated with media and
various concentrations of tested compounds. 72 h later, the drug containing medium was replaced by fresh medium 100
mL with MTT solution (5 mg/mL). After 4 h of incubation, the medium bearing MTT was removed, and 100 mL of
DMSO solution was added to each well. The plates were gently agitated until the purple formazan crystals were
dissolved, and the OD490 was determined by a microplate reader (MK3, Thermo, Germany). The data were calculated
and plotted as the percent viability compared to the control. The IC50 was defined as the concentration that reduced the
absorbance of the untreated wells by 50% of the vehicle in the MTT assay.
4.2.2. In vitro enzyme assay.
The in vitro enzymatic assays were performed by Mobility shift assay. The solution of peptide substrates, ATP,
appropriate kinase (Carna), and various concentrations of tested compounds were mixed with the kinase reaction buffer
(50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT), with blank DMSO as the negative control. The
kinase reaction was initiated by the addition of tyrosine kinase proteins diluted in 39 µL of kinase reaction buffer solution
and incubated at 28 °C for 1 h. Then, 25 µL of stop buffer (100 mM HEPES, pH = 7.5, 0.015% Brij-35, 0.2% Coating
Reagent #3, 50 mM EDTA) was added to stop reaction. The data were cellected on Caliper at 320 nm and 615 nm and
converted to inhibition values. IC50 was presented in MS Excel and the curves fitted by XLfit excel add-in version 4.3.1.
4.2.3. Western blotting.
Western blotting analysis was performed as described Karpas299 cells were treated with F-1 in 0, 30, 100 and 200 nM,
respectively. After treatment with F-1 for 24 h, cells were collected. The protein in the cytoplasm was extracted,
quantified with protein assay reagent and electrophoresed on SDS-polyacrylamide gel until the proteins of different
molecular weights were separated. Then, the different proteins contained in the SDS-polyacrylamide gel were transferred
to polyvinylidene difluoride (PVDF) membranes. Subsequently, non-specific binding was blocked with 5% (w/v)
skimmed milk for 3h. Then the membrane was incubated with respective primary antibodies at 4 °C overnight. After
being washed with PDS trice for 10min, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated
secondary antibodies (1: 3000) for 2h. The enhanced chemiluminescence reagent was used to identify the specific bands.
Densitometry analysis on immune blots was performed using ImageJ2 software.
4.2.4. Morphology analysis of apoptotic cells and nuclear morphology assays.
Apoptotic morphological changes of Karpas299 cells were detected by AO/EB staining. Briefly, cells were seeded in
six-well plates for 24 h, and then treated with ceritinib (50 nM) or different concentrations (25 nM and 50 nM) of F-1 for 48
h. The cells were washed with phosphate buffer saline (PBS) and then stained with AO/EB mixed solution (AO:EB = 1:1)
for a quarter. The stained cells were washed twice with PBS and observed by fluorescence microscope (Olympus, Tokyo,
Japan).
To investigate the apoptotic morphological changes of nuclear chromatin in Karpas 299 cells, Hoechst33258 staining
was performed. In brief, cells were seeded in six-well plates overnight, and then treated with ceritinib (50 nM) or different
concentrations (25 nM and 50 nM) of F-1. After 48 h, the medium was discarded and the cells were washed with PBS.
After that, the cells were stained with Hoechst33258 solution for 15 min. The stained cells were washed twice with PBS
and observed by fluorescence microscope (Olympus, Tokyo, Japan).
Acknowledgments
This work was supported by National Natural Science Foundation of China (No. 81673308), Key
Research
Plan
Project of Department of Science & Technology of Liaoning Province (No. 2017225058), Natural Science Foundation
of Liaoning Province (No. 201602703), and Development Project of Ministry of Education Innovation Team (No.
IRT1073).

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Legends
Figure 1. Structures of approved ALK inhibitors and target compounds
Scheme 1. Reagents and conditions: (a) DIPEA, i-PrOH, 80°C, 2h; (b) Substituted acyl chloride or sulfonyl chloride,pyridine, DCM,
r.t., 2.5h. (c) R2XH, NaH, THF, r.t., 6h; (d)alicyclic amine, K2CO3, THF, 50°C, 3h; (e)H2, Pd/C, MeOH, 30°C, 6h; (f) B-1~B-4,
p-TsOH, i-PrOH, 80°C, 5h.
Table 1. Structures and antiproliferative activities of compounds F-1~F-28.
Table 2. Inhibitory effects of selected compounds against ALK and ALK secondary mutations (L1196M and G1202R), ROS1 and
EGFR.
Figure 2. F-1 Suppresses ALK and its downstream signaling. Inhibition of cellular phosphorylation of ALK.Akt, and STAT3 by F-1
(30, 100 and 200 nM) in Karpas299 cells investigated by western blot analysis.
Figure 3. F-1 induces apoptosis in Karpas299 cells. Karpas299 cells were treated with F-1 (25 and 50 nM) or ceritinib (50 nM) for
24 h, then stained with AO/EB (the upper part) or Hoechst 33258 (the lower part), and photographed by Olympus inverted
fluorescence microscope (scale bar = 50 µM).

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Table 1. Structures and antiproliferative activities of compounds F-1~F-28.
IC50 (µM)± SD a
Compd.
F-1
-R1
- R2
-R3
H2228
Karpas299
HCC78
Ba/F3G1202R A549
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
0.012±0.01 0.01±0.01
0.043±0.02 0.03±0.01
>1
0.13±0.03
0.17±0.01
0.36±0.05
0.04±0.01
0.08±0.01
0.06±0.01
0.19±0.02
0.24±0.02
0.31±0.04
0.08±0.02
0.74±0.01
0.33±0.04
0.13±0.02
0.18±0.02
0.05±0.01
>1
>1
F-2
0.71±0.06
F-3
>1
F-4
0.024±0.01 0.027±0.01
0.85±0.07
>1
F-5
0.95±0.08
0.88±0.09
F-6
0.042±0.01 0.06±0.01
0.035±0.01 0.032±0.01
>1
F-7
0.49±0.05
0.07±0.01
0.27±0.02
0.36±0.04
0.38±0.05
>1
0.39±0.04
0.038±0.01
0.072±0.01
0.08±0.01
0.22±0.04
0.58±0.08
0.055±0.05
0.028±0.10
0.48±0.05
0.12±0.01
0.46±0.05
0.44±0.06
0.11±0.01
0.41±0.02
0.21±0.03
0.16±0.02
0.92±0.14
0.48±0.05
0.27±0.03
0.39±0.06
0.24±0.03
0.26±0.03
>1
>1
F-8
0.09±0.01
0.11±0.02
0.23±0.03
0.30±0.05
>1
>1
F-9
>1
F-10
F-11
F-12
F-13
F-14
F-15
F-16
F-17
F-18
>1
>1
>1
0.09±0.01
0.06±0.01
0.27±0.04
0.41±0.05
0.64±0.05
0.17±0.02
0.09±0.01
0.63±0.08
>1
>1
>1
>1
>1
0.12±0.03
>1

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OMe
OMe
OMe
OPr-i
OPr-i
SPr-i
SPr-i
H
0.05±0.01
0.11±0.01
0.09±0.01
0.13±0.02
0.14±0.02
0.26±0.06
0.09±0.01
0.13±0.02
0.32±0.02
0.07±0.02
0.08±0.01
0.07±0.01
0.04±0.01
0.09±0.06
0.88±0.01
0.85±0.04
>1
>1
F-19
F-20
F-21
F-22
F-23
F-24
F-25
F-26
F-27
F-28
0.018±0.01 0.035±0.01
>1
0.16±0.02
0.027±0.01
0.70±0.06
0.88±0.09
0.82±0.07
>1
0.046±0.01 0.018±0.01
0.41±0.05
0.42±0.05
0.98±0.09
0.08±0.01
0.48±0.05
0.14±0.03
0.31±0.02
0.34±0.05
0.75±0.08
0.04±0.01
0.08±0.01
0.06±0.01
>1
>1
0.78±0.09
0.74±0.08
0.85±0.01
>1
H
H
0. 09±0.01
0.17±0.02
0.44±0.03
>1
>1
0.087±0.01 0.24±0.04
0.026±0.01 0.041±0.01
>1
>1
crizotinib
ceritinib
0.058±0.01 >1
a Values are the means of at least three independent experiments.
b SD: standard deviation.

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Table 2. Inhibitory effects of selected compounds against ALK and ALK secondary mutations (L1196M and
G1202R), ROS1 and EGFR.
IC50 (µM)
Compd.
ALK
2.1
L1196M ALK
G1202R ALK
ROS1
2.3
EGFR
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
1.3
1.6
2.0
2.2
3.9
4.8
380
35
F-1
3.9
7.9
2.8
6.1
F-5
1.4
5.2
1.6
F-7
3.2
7.8
3.4
F-9
3.9
6.0
4.6
F-15
4.2
9.4
2.0
F-22
32.2
2.9
>1000
6.7
crizotinib
ceritinib
2.0
91
aValues are the means of at least two independent experiments.

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Twenty-eight novel 2,4-diarylaminopyrimidine derivatives were designed and synthesized.
F-1 inhibited a panel of ALK-addicted cancer cells with IC50 value in double-digit nanomolars.
F-1 possessed remarkable potency against ALK and ROS1 kinases with IC50 values below 3.9 nM.
F-1 could down-regulate the phosphorylation of ALK and its downstream proteins in a dose-dependent
manner.