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

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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ACCEPTED MANUSCRIPT

Dual potent ALK and ROS1 inhibitors combating drug-resistant mutants:

synthesis

and

biological

evaluation

of

aminopyridine-containing

diarylaminopyrimidine derivatives

Ming Guo^{a, 1}, Daiying Zuo^{c, 1}, Junlong Zhang^a, Lingyun Xing^a, Wenfeng Gou^c, Feng Jiang^a, Nan Jiang^a, Dajun

*

Zhang^b,*, Xin Zhai^a,

^aKey Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical

University, Shenyang 110016, China.

^bKey Lab. of Behavioral and Cognitive Neuroscience of Liaoning Province, Shenyang Medical College, Shenyang

110034, China.

^cDepartment 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 ALK^WT, ROS1^WT, ALK^L1196Mand ALK^G1202Rkinases with IC₅₀of 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 IC₅₀values

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.

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 IC₅₀values in double-digit nanomolars. Interestingly, the

most prominent compound F-1 displayed highlighted potency on H2228, Karpas299, HCC78 and Ba/F3^G1202Rcells with

IC₅₀values 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/F3^G1202Rcell (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 IC₅₀values

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 IC₅₀values 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 IC₅₀values 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 IC₅₀values below 3.9 nM. Especially, F-1 showed an outstanding inhibition on ALK^L1196M( IC₅₀= 1.3 nM)

and ALK^G1202R( 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 IC₅₀value of 9.4 nM against ALK^G1202R, 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 ALK^L1196Mand ALK^G1202Rwas 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 IC₅₀value of double-digit nanomolars. Therein, F-1 exhibited the

most prominent cytotoxicity with IC₅₀value of 30 nM against Ba/F3^G1202RALK-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 IC₅₀values 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 IC₅₀of 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 Na₂SO₄and 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.

ACCEPTED MANUSCRIPT

4.2.1. MTT assay

Cells (H2228, Karpas299, HCC78, A549 and EA^G1202Rcancer cell lines) were seeded into 96-well plates at a density of

approximate 5×10⁴per 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 IC₅₀was 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. IC₅₀was 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).

ACCEPTED MANUSCRIPT

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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/F3^G1202RA549

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

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

F-8

0.09±0.01

0.11±0.02

0.23±0.03

0.30±0.05

>1

F-9

>1

F-10

F-11

F-12

F-13

F-14

F-15

F-16

F-17

F-18

>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

0.12±0.03

>1

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OMe

OPr-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

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

0.78±0.09

0.74±0.08

0.85±0.01

>1

H

0. 09±0.01

0.17±0.02

0.44±0.03

>1

0.087±0.01 0.24±0.04

0.026±0.01 0.041±0.01

>1

crizotinib

ceritinib

0.058±0.01 >1

^aValues are the means of at least three independent experiments.

^bSD: 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

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 IC₅₀value in double-digit nanomolars.

F-1 possessed remarkable potency against ALK and ROS1 kinases with IC₅₀values below 3.9 nM.

F-1 could down-regulate the phosphorylation of ALK and its downstream proteins in a dose-dependent

manner.

Article Doi

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

DOI: 10.1016/j.ejmech.2018.09.012

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

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

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