Bioorganic Chemistry 81 (2018) 689–699

Contents lists available at ScienceDirect

Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

Design, synthesis and biological evaluation N2-(2-alkyoxy-6-aliphatic T

aminopyridin-3-yl)-2,4-diaminepyrimidine derivatives bearing acylamino or
DBTD ‘head’ as potential ALK inhibitors
Lingyun Xing, Tongfei Jing, Junlong Zhang, Ming Guo, Xiuqi Miao, Feng Jiang, Xin Zhai
⁎

Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China

ARTICLE INFO ABSTRACT

Keywords: Aiming to develop promising ALK inhibitors, two series of N2-(2-alkyoxy-6-aliphatic aminopyridin-3-yl)-2,4-
ALK inhibitor diaminepyrimidine derivatives (22a-x and 23a-d) were designed according to scaffold hopping and bioisos-
2,4-diarylaminopyrimidine terism principles. All compounds were efficiently synthesized by concise reactions and anti-proliferative activ-
Pyridinyl group ities on ALK-addicted H2228, Karpas299 cells and EGFR-expressive A549 cell were evaluated by MTT assay.
DBTD
Several compounds exhibited potential cytotoxic activities with IC50 values below 0.10 μM. Five compounds
(22g, 22h, 22l, 22s and 23a) were selected for further enzymatic determination, resulting in the discovery of 22l
against ALK and ALKL1196M with IC50 values of 2.1 nM and 3.8 nM. Particularly, western blot and cell apoptosis
assays identified 22l as a promising ALK inhibitor, which was capable of obviously inhibiting cellular ALK
activity and inducing cell apoptosis. Eventually, molecular docking modes of 22l with ALK confirmed structural
basis in accordance with the SARs analysis.

1. Introduction
Anaplastic lymphoma kinase (ALK) was a single stranded trans-
membrane protein belonging to the insulin receptor tyrosine kinase
family [1]. The oncogenic nucleophosmin (NPM)-ALK fusion gene was
discovered as an ALK translocation product occurring in 80% of ana-
plastic large-cell lymphoma [2]. Echinoderm microtubule-associated
protein-like-4 (EML4)-ALK was the first targetable fusion kinase to be
identified in 4–6% of lung adenocarcinomas [3,4]. ALK fusion genes
had generated substantial interest for the development of potent and
specific ALK inhibitors for treatment of EML4-ALK rearrangement [5].
Recently, a few ALK inhibitors have been successfully approved or in
clinical study (Fig. 1). The first-generation ALK inhibitor crizotinib was
approved in 2011 which had demonstrated clear clinical benefits to
treat ALK-positive NSCLC patients [6]. However, a majority of patients
developed resistance to crizotinib treatment successively [7,8]. There-
fore, several more potent second- and third-generation inhibitors have
been identified to combat disease resistance, including ceritinib [9] and
alectinib [10], brigatinib [11], the pre-registered lorlatinib [12] and the
phase III investigational agent ensartinib [13].
The co-crystal structure of ceritinib with ALK revealed interactions
between ligand and molecular (Fig. 2). The 4-arylaminopyrimidine and
Cl atom on pyrimidine ring were indispensable for activity which
occupied the kinase domain and formed interactions with amino acid
residues [14]. Thus, this structural domain should be remained for
potent activity and selectivity [15]. While hydrophilic piperidinyl
group (‘tail’) extended to solvent and was responsible for a salt-bridge
interaction with Glu1210, tolerating to further modification with var-
ious aliphatic amines [16,17]. The isopropoxy group on ceritinib ske-
leton exhibited higher kinase selectivity on account of combination
with protein pockets. To our disappointed, it had been reported that
ceritinib was ineffective for ALKG1202 mutation duing to large steric
hindrance of isopropoxy moiety [18]. Therefore, isopropoxy group was
replaced with fragments of methoxyl, isopropylthio and hydrogen atom
aiming to investigate the effect of steric hindrance. Additionally, the
replacement of benzene group with pyridinyl group in aromatic ring
system has shown many important effects on molecular which could
properly tune the lipophilicity and improve the metabolism [19–21].
Hence, pyridinyl group as a high-impact moiety has been applied in
drug design [22,23].
Optimization towards different R1 substituted (‘head’) was an ef-
fective approach to enrich structural diversity. Beyond intramolecular
hydrogen bond on isopropyl sulfonyl group, acylamino group was re-
garded as a favorable design strategy which could form hydrogen bond
with receptors either [24]. Meanwhile, the acylamino ‘head’ could
enhance the affinity between molecules and enzymes to increase

⁎
Corresponding author.
E-mail address: zhaixin_syphu@126.com (X. Zhai).

https://doi.org/10.1016/j.bioorg.2018.09.019
Received 22 June 2018; Received in revised form 29 August 2018; Accepted 11 September 2018
Available online 12 September 2018
0045-2068/ © 2018 Elsevier Inc. All rights reserved.
L. Xing et al. Bioorganic Chemistry 81 (2018) 689–699

NH O
Cl N Cl N
N N
O NH H
F N N
N N N
Cl H H
H2N N S O O
O N
O

crizotinib (PF-02341066) ceritinib (LDK378) alectinib (CH5424802)

launched 2011 launched 2014 launched 2014

N O
F Cl
N N O
Cl N N F O N
N
O N Cl O NH
N
N N N H
H H N
P O O H2N N H2 N N
N

brigatinib (AP26113) lorlatinib (PF-06463922) ensartinib (X396)

launched 2017 pre-registered phaseIII

Fig. 1. Structures of ALK inhibitors.

Fig. 2. Design strategies for target compounds (22a-x and 23a-d) based on co-crystal structure of ceritinib with ALK (PDB ID code: 4MKC). (A) The co-crystal
structure of ceritinib with ALK. (B) Structure design strategies of 22a-x and 23a-d.

inhibitory activity. In addition, the rigid 3,4-dihydro-2H-benzo[1,4-b]
thiazine-1,1-dioxide (DBTD) fragment was built as a new structural
motif by scaffold hopping [25], which could enhance the rigidity. Thus,
compounds 23a-d with DBTD ‘head’ were designed successfully to
discuss the binding ability to protein and evaluate its effectivity.
Herein, two series of N2-(2-alkyoxy-6-aliphatic aminopyridin-3-yl)-
2,4-diaminepyrimidine derivatives (22a-x and 23a-d) bearing acyla-
mino or DBTD ‘head’ were designed and synthesized. Compounds were
assayed for the anti-proliferative activity in vitro against three cancer
cell lines H2228, Karpas299 and A549. Subsequently, five compounds
were selected to test in vitro enzymatic evaluation on ALK and
ALKL1196M. To further study, the promising compound 22l was eval-
uated for western blot assay and cell apoptosis. Finally, molecular
docking studies were carried out to identify SARs.
2. Results and discussion
2.1. Chemistry
The synthetic routes to compounds 22a-x and 23a-d were outlined
in Scheme 1. The commercially available 2,4,5-trichloropyrimidine (1)
was reacted with benzene-1,2-diamine (2) to provide intermediate 3 in
96.5% yield. 3 was acylated by different acyl chloride in the presence of
triethylamine to afford the key intermediates 4a-c [26]. Meanwhile,
substitution reaction of compound 2-fluoronitrobenzene (5) with mer-
captoethanol gave 6, which was chlorinated by thionyl chloride to
generate 2-chloroethyl-2-nitrophenyl thioether (7) as a yellow solid. 7
was oxidized by hydrogen peroxide to yeild 8, which was subjected to
reduction by iron powder in the presence of hydrochloric acid to gain 9
in good yield. Intramolecular N-alkylation of 9 in dimethyl formamide
gave desired DBTD analog 10, which was concentrated with 2,4,5-tri-
chloropyrimidine to yield the key intermediate 11
Compound 12 reacted with methanol (isopropanol or isopropyl
mercaptan) in the company of sodium hydride to obtain 13b-d without
further purification. Subsequently, amination of 13a-d by corre-
sponding aliphatic amines with potassium carbonate furnished target
intermediates 14a-c, 14e-g, 15a-g, 16a-c or 17a-c in satisfied yields.
Treatment 14a-c, 14e-g, 15a-15g, 16a-16c or 17a-17c with hydro-
chloric acid as catalysis under activated iron powder in ethanol/water
(9:1) gave rise to 18a-c, 18e-g, 19a-g, 20a-c or 21a-c, which were
reacted with 4a-c and purified on silica gel column to get target pro-
ducts 22a-x. Similarly, the desired compounds 23a-d were accom-
plished using the same method as preparation of 22a-x.

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Scheme 1. Reagents and conditions: (i) DIPEA, ethanol, 80 °C; (ii) the acyl chloride of R1, triethylamine, DCM, rt; (iii) 2-mercaptoethanol, K2CO3, DMF, 110 °C; (iv)
thionyl chloride, toluene, 80 °C; (v) hydrogen peroxide, glacial acetic acid, 100 °C; (vi) iron powder, hydrochloric acid, ethanol/water(9:1), 80 °C; (vii) K2CO3, DMF,
100 °C; (viii) NaH (60%), DMF, 0 ∼ 5 °C; (ix) NaH (60%), methanol(/IPA/isopropyl mercaptan), THF, rt; (x) aliphatic amines, K2CO3, THF, 50 °C; (xi) iron powder,
hydrochloric acid, ethanol/water(9:1), 80 °C; (xii) 4a-d, p-toluenesulfonic acid, IPA, 80 °C.

2.2. Biological evaluation
2.2.1. In vitro anti-proliferative activity and SARs study
MTT assay was employed to evaluate the anti-proliferative char-
acteristics of target compounds to screen the active compounds pre-
liminarily. Compounds 22a-x and 23a-d were investigated on cyto-
toxicity against ALK-addicted H2228 (Human NSCLC cells), Karpas299
(Human transsexual cell lymphoma cell lines) cells and EGFR-ex-
pressive A549 (Human EGFR-positive NSCL cell lines) cell. Using cer-
itinib as the positive control, the results expressed as IC50 values were
shown in Tables 1 and 2.
The pharmacological data indicated that most compounds exhibited
moderate to excellent cytotoxic activities against ALK-addicted H2228
and Karpas299 cells. Compound 22l displayed significant activity
against H2228 and Karpas299 with IC50 values of 0.030 μM and
0.036 μM. Meanwhile, 22l showed poor inhibitory activity against
A549 with IC50 values of 0.65 μM. Compared with 22a-x, compounds
23a-d exhibited obviously reduced activities against H2228 and
Karpas299 cells, indicating the negative effect of DBTD group on cy-
totoxicity. As expected, all compounds showed weak potency against
A549 cells, suggesting the selectivity on ALK cell lines.
As shown in Table 1, anti-proliferative activity of target compounds
would preliminarily define the structure–activity relationships. A small
set of compounds 22a-g with different R3-moiety ‘tail’ were evaluated
for cytotoxic activities, wherein compound 22g bearing morpholinyl
moiety showed the best potency with IC50 values of 0.09 μM, 0.068 μM
and 0.95 μM against H2228, Karpas299 and A549, which identified
polarity ‘tails’ with good hydrophilia property being beneficial for in-
creased potency. Compounds containing azetidin-3-ol (22d) and hy-
droxyethyl piperazinyl (22e) were 2.4- and 6.3-fold more potent
against H2228 than those inserting methyl group in piperazinyl
fragment (22f). Meanwhile, significant potency loss was detected on
compounds 22a-c with diethylamino, piperidyl and pyrrolidinyl groups
due to the lack of hydrophilicity.
As a general trend, among diverse chain R1-moiety, compounds
bearing sulfonyl (22l) and acetyl (22g) showed better activities against
H2228 with IC50 values less than 0.1 μM than compound bearing butyl-
2-acrylamide substitution (22h). Generally, introduction of butyl-2-
acrylamide moiety would result in unsatisfactory activities as indicated
in 22h-k, which illustrated the effect of steric on cellular potency.
Notably, it was found that compound 22l bearing sulfonyl ‘head’ in-
creased ALK potency significantly.
The IC50 values of 22l-x showed that variations of R2 group had a
marked impact on activity. The R2 moiety was introduced with lipo-
philic substituents which occupied the ALK protein lipophilic pocket to
improve the activity. Derivatives 22m-o with isopropyl provided
moderate potency, however, shifting isopropyl to bulk isopropylthio led
to poor activity deducing to the steric hindrance within the hydro-
phobic pocket. For example, the inhibitory activities of 22q and 22r
were decreased extremely with IC50 values more than 1 μM against
tested cell lines. Replacement of isopropyl with hydrogen atom resulted
in compounds 22s-x, which decreased activity, indicating R2-hydro-
phobic pocket should be inserted the motif with appropriate volume.
Thus, methoxyl on R2 moiety (22l) was regarded as the optimal sub-
stituent.
To integrate above factors and process further explore, the effect of
rigid sulfonyl ‘head’ group was studied by scaffold hopping principle.
As illustrated in Table 2, compounds with rigid DBTD ‘head’ were in-
ferior to corresponding compounds bearing acylamino ‘head’. Ex-
tremely, 23d demonstrated dramatic poor effect on H2228 and
Karpas299 cells with IC50 values more than 5 μM. Further analysis on
SARs would be detailed description on molecular docking studies.

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Table 1 Table 2
Cytotoxicity of 22a-x against H2228, Karpas299 and A549 cell lines in vitro Cytotoxicity of 23a-d against H2228, Karpas299 and A549 cell lines in vitro

. .

Compd. R1 R2 R3 IC50 (µM) Compd. R2 R3 IC50 (µM)

H2228 Karpas299 A549 H2228 Karpas299 A549

22a 0.92 0.16 2.16 23a 2.05 0.82 1.62

22b 0.51 0.094 1.06 23b 2.33 1.28 2.84

23c 3.41 2.68 0.44

22c 0.80 0.19 1.23
23d >5 >5 2.26
22d 0.62 0.06 0.54
ceritinib 0.032 0.048 0.79

22e 0.24 0.052 0.18

22f 1.51 1.28 3.11 Table 3

Enzymatic inhibition of five compounds against ALK and ALKL1196M in vitro.
22g 0.09 0.068 0.95 Compd. IC50 (nM)

22h 0.19 0.09 1.12 ALK ALKL1196M

22i 0.98 0.23 1.88 22g 3.6 5.4

22h 5.8 12.8
22j 0.78 0.12 0.97
22l 2.1 3.8
22k 0.37 0.08 0.66 22s 8.3 20.3
23a > 3000 > 3000
22l 0.030 0.036 0.65 ceritinib 2.3 8.9

22m 0.054 0.04 0.32

compound 22l turned out to be superior to ceritinib (IC50 = 2.3 nM)
22n 0.65 0.87 0.62 with IC50 value of 2.1 nM. Likewise, compounds 22g and 22l showed
significant inhibitory activity to ceritinib (IC50 = 8.9 nM) against
22o 0.74 1.66 2.51
ALKL1196M with IC50 values 5.4 nM and 3.8 nM, respectively. Paralleling
22p 0.66 0.84 3.23 with cellular results, 22l was found to be 1.4- and 2.3-fold more potent
than 22h with butyl-2-acrylamide ‘head’ and 22s with hydrogen atom
22q 1.12 1.19 5.22 substitution on pyridinyl group against ALKL1196M, which revealed that
appropriate steric hindrance was favor to improve the activities.
22r 1.47 1.08 4.69

22s H 0.20 0.038 0.287 2.2.3. In western blot assay

Western blot assay was an important method to elucidate the
22t H 0.68 0.37 1.21 modulation of target phosphorylation effect of 22l in the in vitro assay.
Using ceritinib as the positive control, compound 22l and ceritinib were
22u H 0.94 0.61 2.81
examined to determine the abilities to inhibit Karpas299 cell lysates
22v H 0.92 0.11 0.58 (Fig. 3).
As demonstrated in Fig. 3, 22l suppressed ALK expression and re-
22w H 0.67 0.096 0.66 duced phosphorylation of ALK in a dose-dependent suppression manner
from 25 nM to 100 nM, which was comparable to ceritinib. Slight in-
22x H 1.26 1.79 4.23
hibition of ALK expression and phosphorylation could be observed at a
dose of 50 nM, meanwhile, obvious inhibitions were discovered a
ceritinib 0.032 0.048 0.79
concentration of 100 nM. These results addressed 22l did potently in-
hibit protein activity.

2.2.4. Apoptosis analysis

2.2.2. In vitro enzymatic assays
Based on the cellular assays, further enzymatic assays in vitro were
of particular importance to evaluate antitumor potential. Five re-
presentative compounds (22g, 22h, 22l, 22s and 23a) were selected
against ALK and ALKL1196M inhibitory studies. The results were sum-
marized in Table 3, which were comparable to the positive control
ceritinib.
As shown in Table 3, compared with 22l, compound 23a was ob-
served a loss in activity against ALK and ALKL1196M, which indicated
that DBTD group was unfavorable to inhibitory activity. However, op-
timal compounds with chain R1-moiety displayed good inhibitory po-
tency against ALK with IC50 values ranging from 2.1 to 8.3 nM. Notably,
Apoptosis assay elucidated the process for the observed induction of
cellular death, so it played a necessary role to assess the potency of
compound 22l. The cytotoxicity of 22l was further confirmed by an
acridine orange (AO)/ethidium bromide (EB) fluorescent staining assay
against H2228 and Karpas299 cell lines (Fig. 4). This staining solution
was a mixture of AO and EB, in which AO was used for live cell iden-
tification and EB for identification of necrotic cells. The AO permeated
into the intact cell membrane and thus stains live cells to appear green,
whereas, EB stained necrotic cells to appear orange when visualized by
fluorescent microscopy due to disruption of membrane integrity.
Therefore, early apoptotic cells contained bright green condensed
bodies and later apoptotic cells presented colored orange nuclei

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Fig. 3. Mechanism of action of compound 22l and ceritinib in Karpas299 cell lines. Cells were treated with 22l or ceritinib at the indicated concentrations for 6 h and
the levels of protein were evaluated by western blot analysis of cell lysates using specific antibodies.
indicating.
As shown in Fig. 4, H2228 and Karpas299 cells were observed cell
apoptosis in 22l treated groups. It was observed that control cells dis-
played normal cell morphological features with uniformly stained green
nuclei, while compound 22l at the concentration of 160 nM started to
induce morphological changes. At the concentration of 320 nM, the
number of green cells was reduced and orange cells were increased
indicating cells underwent apoptotic stage. Simultaneously, apoptotic
cells were clearly observed at 640 nM. These results explored 22l in-
duced dose-dependent apoptosis of H2228 and Karpas299 cells as evi-
dent by observed hall marks of apoptosis.
2.2.5. Molecular docking studies
Computer aided molecular docking was used to simulate interac-
tions and evaluate active sites. In order to detail the binding mode,
molecular dockings of 22l and 23a were performed based on co-crystal
structures of ALK (PDB ID code: 4MKC) by Discovery Studio 2017
(Fig. 5).
As shown in Fig. 5A, 22l occupied the same kinase domain with
ceritinib. Both cerinitib and 22l were found to form two hydrogen
bonds via the amino and pyrimidine nitrogen atoms onto the backbone
oxygen and nitrogen of Met1199 (Fig. 5B and C), which were essential
for binding to ALK. The Cl atom on pyrimidine ring of 22l formed
hydrophobic interactions with Leu1196 and Leu1256. Regardless of the
size of the aromatic ring, cerinitib and 22l formed alkyl interactions
with Leu1122. Meanwhile, the acylamino group of compound 22l not
only formed hydrogen bond interactions with Lys1150, but also carbon
hydrogen bond interactions with Gly1125 (Fig. 5D), as illustrated the
reasonability of the chain ‘head’. The water-soluble ‘tail’ of 22l was
Fig. 4. AO/EB stained apoptosis of H2228 and Karpas299 cell lines. H2228 and Karpas299 cell were treated with different concentrations of 22l for 24 h, and then
examined by fluorescence microscope.
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Bioorganic Chemistry 81 (2018) 689–699
engaged in carbon hydrogen bond interactions with Asp1203 and
Leu1122, which was similar to ceritinib. These simulation models
proved that 22l had familiar contact to cerinitib with ALK.
It was available from Fig. 5E and F that 23a vanished interactions
with Lys1150 and Gly1125 deducing to the rigid DBTD ‘head’, which
explained the reason of poor activities and the important of the chain
‘head’. Hence, the results from the docking study were in accordance
with the SARs analysis.
3. Conclusions
In this paper, two series of N2-(2-alkyoxy-6-aliphatic aminopyridin-
3-yl)-2,4-diaminepyrimidine derivatives (22a-x and 23a-d) bearing
acylamino or DBTD ‘head’ were designed, synthesized and evaluated for
biological activities. Compared with compounds 22a-x bearing acyla-
mino group, compounds with rigid DBTD ‘head’ (23a-d) decreased the
potency relative. Importantly, 22l possessed remarkable antitumor
potency against H2228 and Karpas299 cells with IC50 values of
0.030 μM and 0.036 μM as well as excellent activity on ALK and
ALKL1196M with IC50 of 2.1 nM and 3.8 nM, respectively. Consistently,
western blot and cell apoptosis tests illustrated 22l could inhibite the
cellular ALK activity and induce cell apoptosis in a dose-dependent
manner. Moreover, molecular docking modes detailed that 22l could
not only form critical hydrogen bonding interactions and hydrophobic
interactions but also overlap well with ceritinib in 3D model, which was
consistent with observed SARs for this class of compounds. In conclu-
sion, the target compound 22l was identified as a promising ALK in-
hibitor for cancer treatments.
L. Xing et al.
Fig. 5. The binding poses of cerinitib, 22l and 23a with ALK (PDB ID code: 4MKC). (A) Composite model of cerinitib (red sticks) and 22l (gray sticks) with ALK. (B)
Docking model of cerinitib with ALK. (C) Docking model of compound 22l with ALK. (D) 2D diagram of the interaction between 22l and ALK. (E) Docking model of
23a (purple sticks) with ALK. (F) 2D diagram of the interaction between 23a and ALK.
4. Experimental section
4.1. Chemistry
All melting points were obtained on a Büchi Melting Point B-540
apparatus (Büchi Labortechnik, Flawil, Switzerland) and were un-
corrected. Mass spectra (MS) were taken in ESI mode on Agilent 1100
LC-MS (Agilent, palo Alto, CA, USA). 1H NMR and 13C NMR spectra
were performed using Bruker spectrometers (Bruker Bioscience, re-
spectively, Billerica, MA, USA) with TMS as an internal standard.
Column chromatography was run on silica gel (200–300 mesh) from
Qingdao Ocean Chemicals (Qingdao, Shandong, China). Unless other-
wise noted, all materials were obtained from commercially available
sources and were used without further purification.
4.1.1. Preparation of N1-(2,5-dichloropyrimidin-4-yl)benzene-1,2-diamine
(3)
2,4,5-trichloropyrimidine 1 (30.0 g, 0.16 mol), benzene-1,2-diamine
2 (17.7 g, 0.16 mol) and N,N-diisopropylethylamine (54.0 mL,
0.33 mol) was added to ethanol (300 mL). The resulting solution was
stirred at 80 °C for 2 h. The reaction mixture was evaporated and the
residue was stirred with aqueous HCl (0.1 M, 10 mL) for 30 min. The
solid was collected by filtration and dried under vacuum. The crude
solid was triturated with dichloromethane to give a solid which was
collected by filtration and dried under vacuum to afford a white solid 3
in 96.5% yield. MS (ESI) m/z: [M+H]+ 255.0.
4.1.2. General procedure for preparation of compounds (4a-c)
Acetylchloride, methylsulfonyl chloride or (E)-but-2-enoyl chloride
(0.094 mol) was added to a mixture of 3 (20.0 g, 0.079 mol) and trie-
thylamine (33.0 mL, 0.24 mol) in DCM (100 mL) at 0 °C for 30 min, then
stirred at room temperature for 2 h. The reaction mixture was evapo-
rated and the residue was stirred with water (200.0 mL). The solid was
collected by filtration and dried under vacuum to give 4a-c.
4.1.2.1. N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)acetamide
(4a). Yield: 89.2%; MS (ESI) m/z: 297.0 [M+H]+.
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Bioorganic Chemistry 81 (2018) 689–699
4.1.2.2. N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)
methanesulfonamide (4b). Yield: 85.8%; MS (ESI) m/z: 333.2 [M+H]+.
4.1.2.3. (E)-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)but-2-
enamide (4c). Yield: 91.1%; MS (ESI) m/z: 322.9 [M+H]+.
4.1.3. Preparation of 2-((2-nitrophenyl)thio)ethan-1-ol (6)
To the solution of mercaptoethanol (16.6 g, 0.21 mol) in dry DMF (1
l) was added 2-fluoronitrobenzene 5 (30.0 g, 0.21 mol) and K2CO3
(58.7 g, 0.42 mol). The reaction mixture was heated with stirring at
110 °C for 6 h. After reacting at room temperature, the reaction mixture
was poured into water and extracted with AcOEt (3 × 100 mL). The
combined organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated under reduced pressure to yield a yellow
solid 6 in 94.2% yield. MS (ESI) m/z(%): 200.1 [M+H]+.
4.1.4. Preparation of 2-chloroethyl-2-nitrophenyl thioether (7)
Thionyl chloride (17.85 g, 0.15 mol) was added dropwise to a so-
lution of 6 (10.0 g, 0.05 mol) in toluene (100 mL) at 50 °C for 1 h, then
refluxed for 10 h. After cooling, the reaction mixture was concentrated
under reduced pressure. The residue was stirred with water. The solid
was collected by filtration and dried under vacuum to gain 7 as a yellow
solid in 93.4% yield. MS (ESI) m/z(%): 218.1 [M+H]+.
4.1.5. Preparation of 1-((2-chloroethyl)sulfonyl)-2-nitrobenzene (8)
To the solution of 7 (10.9 g, 0.05 mol) in CH3COOH (100 mL), 30%
H2O2 (30.5 mL, 0.16 mol) was added dropwise while maintaining the
temperature below 50 °C. After the addition was completed, the mixture
was heated to 100 °C for another 13 h. After cooling, the reaction
mixture was poured into water to give a light yellow precipitate. This
was collected by filtration and air-dried to give 8 as a light yellow solid
in 89.0% yield. MS (ESI) m/z(%): 250.0 [M+H]+.
4.1.6. Preparation of 2-((2-chloroethyl)sulfonyl)aniline (9)
To the solution of 8 (12.9 g, 0.05 mol) in ethanol/water (9:1, 60 mL)
was added hydrochloric acid (7.0 mL) and iron powder (11.6 g,
0.2 mol). The resulting mixture was heated at 80 °C for 3 h, filtered
L. Xing et al.
through celite and concentrated to remove ethanol. The aqueous solu-
tion was extracted with ethyl acetate (3 × 100 mL) and the combined
organic layers were washed with water, brine, dried over Na2SO4 and
concentrated to gain 9 in 67.3% yield. MS (ESI) m/z(%): 220.6 [M
+H]+.
4.1.7. Preparation of 3,4-dihydro-2H-benzo[1,4-b]thiazine-1,1-dioxide
(10)
To the solution of 9 (10.0 g, 0.05 mol) in DMF (100 mL), K2CO3
(18.9 g, 0.14 mol) was added. The mixture was stirred at 100 °C for
10 h. After cooling, the reaction mixture was poured into water to give
a light grey precipitate. This was collected by filtration and air-dried to
give 10 as a light grey solid in 55.4% yield. MS (ESI) m/z(%): 184.0 [M
+H]+.
4.1.8. Preparation of 4-(2,5-dichlorophenyl)-3,4-dihydro-2H-benzo[1,4-b]
thiazine-1,1-dioxide (11)
To the suspension of 60% NaH (0.66 g, 0.016 mol) in DMF (20 mL)
was added 10 (1.0 g, 0.005 mol) at 0 °C. The mixture was stirred for
30 min, and then 1 (1.2 g, 0.007 mol) diluted in DMF (6 mL) was added
slowly. The mixture was warmed to room temperature and stirred
overnight. The reaction mixture was poured into water to give a yellow
precipitate which was collected by filtration. The crude product pur-
ified by column chromatography using dichloromethane/methanol as
an eluent to afford 11 a white solid in 41.8% yield. MS (ESI) m/z(%):
329.8 [M+H]+; 1H NMR (400 MHz, DMSO‑d6) δH: 9.61 (s, 1H), 8.53
(d, J = 8.4 Hz, 1H), 8.30 (s, 1H), 8.01 (dd, J = 8.0, 1.4 Hz, 1H),
7.77–7.69 (m, 1H), 7.33 (dd, J = 17.7, 9.8 Hz, 1H), 6.60 (dd, J = 16.5,
9.4 Hz, 1H), 6.50 (d, J = 16.5 Hz, 1H), 6.04 (d, J = 9.4 Hz, 1H).
4.1.9. General procedure for preparation of compounds (13b-d)
To the suspension of 60% NaH (4.6 g, 0.11 mol) in THF (20 mL) was
added 12 (20.0 g, 0.1 mol) at 0 °C. The mixture was stirred at 0 °C for
30 min, and then methanol, IPA or isopropyl mercaptan (0.1 mol) was
added slowly. The mixture was warmed to room temperature and
stirred for 6 h. The reaction mixture was poured into water to give a
yellow precipitate which was collected by filtration to afford 13b-d.
4.1.9.1. 6-chloro-2-methoxy-3-nitropyridine (13b). Yield: 72.7%; MS
(ESI) m/z: 188.9 [M+H]+.
4.1.9.2. 6-chloro-2-isopropoxy-3-nitropyridine (13c). Yield: 69.1%; MS
(ESI) m/z: 217.1 [M+H]+.
4.1.9.3. 6-chloro-2-(isopropylthio)-3-nitropyridine (13d). Yield: 78.2%;
MS (ESI) m/z: 233.0 [M+H]+.
4.1.10. General procedure for preparation of compounds (14a-c, 14e-g,
15a-g, 16a-c and 17a-c)
A solution of 13a-d (0.005 mol) in THF (20 mL) was added K2CO3
(0.011 mol) and various secondary amines (0.011 mol), which was
stirred at 50 °C. After cooling to room temperature, the reaction mixture
was poured into water. The aqueous solution was extracted with ethyl
acetate (3 × 100 mL) and the combined organic layers were washed
with water, brine, dried over Na2SO4 and concentrated to gain 14a-c,
14e-g, 15a-g, 16a-c or 17a-c.
4.1.10.1. 4-(5-nitropyridin-2-yl)morpholine (14a). Yield: 89.3%; MS
(ESI) m/z: 210.1 [M+H]+.
4.1.10.2. 5-nitro-2-(piperidin-1-yl)pyridine (14b). Yield: 91.0%; MS
(ESI) m/z: 208.1 [M+H]+.
4.1.10.3. N,N-diethyl-5-nitropyridin-2-amine (14c). Yield: 87.6%; MS
(ESI) m/z: 196.3 [M+H]+.
695
Bioorganic Chemistry 81 (2018) 689–699
4.1.10.4. 1-(5-nitropyridin-2-yl)azetidin-3-ol (14e). Yield: 89.0%; MS
(ESI) m/z: 196.1 [M+H]+.
4.1.10.5. 2-(4-(5-nitropyridin-2-yl)piperazin-1-yl)ethan-1-ol
(14f). Yield: 83.4%; MS (ESI) m/z: 253.2 [M+H]+.
4.1.10.6. 3-methyl-1-(5-nitropyridin-2-yl)piperazine (14g). Yield: 86.7%;
MS (ESI) m/z: 222.2 [M+H]+.
4.1.10.7. 4-(6-methoxy-5-nitropyridin-2-yl)morpholine (15a). Yield: 90.1%;
MS (ESI) m/z: 240.0 [M+H]+.
4.1.10.8. 2-methoxy-3-nitro-6-(piperidin-1-yl)pyridine (15b). Yield: 89.4%;
MS (ESI) m/z: 238.1 [M+H]+.
4.1.10.9. N,N-diethyl-6-methoxy-5-nitropyridin-2-amine (15c). Yield:
78.6%; MS (ESI) m/z: 226.0 [M+H]+.
4.1.10.10. 2-methoxy-3-nitro-6-(pyrrolidin-1-yl)pyridine (15d). Yield:
84.2%; MS (ESI) m/z: 224.2 [M+H]+.
4.1.10.11. 1-(6-methoxy-5-nitropyridin-2-yl)azetidin-3-ol (15e). Yield:
80.5%; MS (ESI) m/z: 226.0 [M+H]+.
4.1.10.12. 2-(4-(6-methoxy-5-nitropyridin-2-yl)piperazin-1-yl)ethan-1-ol
(15f). Yield: 79.3%; MS (ESI) m/z: 283.3 [M+H]+.
4.1.10.13. 1-(6-methoxy-5-nitropyridin-2-yl)-3-methylpiperazine
(15g). Yield: 71.6%; MS (ESI) m/z: 253.1 [M+H]+.
4.1.10.14. 4-(6-isopropoxy-5-nitropyridin-2-yl)morpholine (16a). Yield:
89.5%; MS (ESI) m/z: 268.1 [M+H]+.
4.1.10.15. 2-isopropoxy-3-nitro-6-(piperidin-1-yl)pyridine (16b). Yield:
88.4%; MS (ESI) m/z: 266.0 [M+H]+.
4.1.10.16. N2,N2-diethyl-6-isopropoxy-5-nitropyridin-2-amine
(16c). Yield: 85.1%; MS (ESI) m/z: 254.1 [M+H]+.
4.1.10.17. 4-(6-(isopropylthio)-5-nitropyridin-2-yl)morpholine
(17a). Yield: 82.9%; MS (ESI) m/z: 284.1 [M+H]+.
4.1.10.18. 2-(isopropylthio)-3-nitro-6-(piperidin-1-yl)pyridine
(17b). Yield: 86.2%; MS (ESI) m/z: 282.1 [M+H]+.
4.1.10.19. N2,N2-diethyl-6-(isopropylthio)-5-nitropyridin-2-amine
(17c). Yield: 86.8%; MS (ESI) m/z: 270.1 [M+H]+.
4.1.11. General procedure for preparation of compounds (18a-c, 18d-g,
19a-g, 20a-c and 21a-c)
To the solution of 14a-c, 14e-g, 15a-g, 16a-c or 17a-c (0.005 mol)
in ethanol/water (9:1, 30 mL) was added hydrochloric acid (1.0 mL)
and iron powder (1.12 g, 0.02 mol). The resulting mixture was heated at
80 °C for 3 h, filtered through celite and concentrated to remove
ethanol. The aqueous solution was extracted with ethyl acetate
(3 × 100 mL) and the combined organic layers were washed with
water, brine, dried over Na2SO4 and concentrated to gain 18a-c, 18e-g,
19a-g, 20a-c or 21a-c.
4.1.11.1. 6-morpholinopyridin-3-amine (18a). Yield: 79.3%; MS (ESI)
m/z: 180.1 [M+H]+.
4.1.11.2. 6-(piperidin-1-yl)pyridin-3-amine (18b). Yield: 92.6%; MS
(ESI) m/z: 178.1 [M+H]+.
4.1.11.3. N2,N2-diethylpyridine-2,5-diamine (18c). Yield: 87.4%; MS
L. Xing et al.
(ESI) m/z: 166.1 [M+H]+.
4.1.11.4. 1-(5-aminopyridin-2-yl)azetidin-3-ol (18e). Yield: 87.3%; MS
(ESI) m/z: 166.1 [M+H]+.
4.1.11.5. 2-(4-(5-aminopyridin-2-yl)piperazin-1-yl)ethan-1-ol (18f). Yield:
80.7%; MS (ESI) m/z: 223.2 [M+H]+.
4.1.11.6. 6-(3-methylpiperazin-1-yl)pyridin-3-amine (18g). Yield: 82.4%;
MS (ESI) m/z: 192.2 [M+H]+.
4.1.11.7. 2-methoxy-6-morpholinopyridin-3-amine (19a). Yield: 90.1%;
MS (ESI) m/z: 210.1 [M+H]+.
4.1.11.8. 2-methoxy-6-(piperidin-1-yl)pyridin-3-amine (19b). Yield:
89.5%; MS (ESI) m/z: 208.1 [M+H]+.
4.1.11.9. N2,N2-diethyl-6-methoxypyridine-2,5-diamine (19c). Yield:
87.3%; MS (ESI) m/z: 196.1 [M+H]+.
4.1.11.10. 2-methoxy-6-(pyrrolidin-1-yl)pyridin-3-amine (19d). Yield:
86.1%; MS (ESI) m/z: 194.1 [M+H]+.
4.1.11.11. 1-(5-amino-6-methoxypyridin-2-yl)azetidin-3-ol (19e). Yield:
80.6%; MS (ESI) m/z: 196.0 [M+H]+.
4.1.11.12. 2-(4-(5-amino-6-methoxypyridin-2-yl)piperazin-1-yl)ethan-1-
ol (19f). Yield: 79.4%; MS (ESI) m/z: 253.1 [M+H]+.
4.1.11.13. 2-methoxy-6-(3-methylpiperazin-1-yl)pyridin-3-amine
(19g). Yield: 82.1%; MS (ESI) m/z: 223.2 [M+H]+.
4.1.11.14. 2-isopropoxy-6-morpholinopyridin-3-amine (20a). Yield:
86.4%; MS (ESI) m/z: 238.2 [M+H]+.
4.1.11.15. 2-isopropoxy-6-(piperidin-1-yl)pyridin-3-amine (20b). Yield:
84.2%; MS (ESI) m/z: 236.2 [M+H]+.
4.1.11.16. N2,N2-diethyl-6-isopropoxypyridine-2,5-diamine (20c). Yield:
85.7%; MS (ESI) m/z: 224.0 [M+H]+.
4.1.11.17. 2-(isopropylthio)-6-morpholinopyridin-3-amine (21a). Yield:
86.1%; MS (ESI) m/z: 254.1 [M+H]+.
4.1.11.18. 2-(isopropylthio)-6-(piperidin-1-yl)pyridin-3-amine
(21b). Yield: 82.2%; MS (ESI) m/z: 252.2 [M+H]+.
4.1.11.19. N2,N2-diethyl-6-(isopropylthio)pyridine-2,5-diamine
(21c). Yield: 86.5%; MS (ESI) m/z: 240.2 [M+H]+.
4.1.12. General procedure for preparation of compounds (22a-x and 23a-
d)
To the solution of 18a-c, 18e-g, 19a-g, 20a-c or 21a-c (0.005 mol)
in isopropanol was added 4a-c or 11(0.005 mol) and p-toluenesulfonic
(0.005 mol). The resulting mixture was heated at 80 °C for 24 h. After
completion of the reaction as indicated by TLC, the mixture was filtered
immediately. Then the residue was stirred with aqueous NaHCO3. The
aqueous solution was extracted with DCM (3 × 50 mL) and combined
organic layers. The mixture was evaporated and purified on silica gel
(200–300 mesh) using dichloromethane/methanol (2500:1). Then
evaporate the mixture. The solid was dried under vacuum and gained
pure compounds 22a-x and 23a-d.
4.1.12.1. N-(2-((5-chloro-2-((4-(diethylamino)-2-methoxyphenyl)amino)
pyrimidin-4-yl)amino)phenyl)acetamide (22a). Yield: 82.0%; m.p.:
172.3–178.6 °C; MS (ESI) m/z: 454.4 [M−H]−; 1H NMR(400 MHz,
696
Bioorganic Chemistry 81 (2018) 689–699
DMSO‑d6) δH: 10.06 (s, 1H), 8.23 (s, 1H), 7.99 (s, 1H), 7.78 (s, 1H),
7.63 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.24 (dd, J = 8.0,
2.1 Hz, 1H), 7.30–7.14 (m, 2H), 6.20 (d, J = 8.4 Hz, 1H), 3.84 (d,
J = 2.1 Hz, 4H), 3.77 (s, 3H), 2.06 (s, 3H), 1.27 (s, 6H).
4.1.12.2. N-(2-((5-chloro-2-((2-methoxy-4-(piperidin-1-yl)phenyl)
amino)pyrimidin-4-yl)amino)phenyl)acetamide (22b). Yield: 84.5%;
m.p.: 178.3–179.6 °C; MS (ESI) m/z: 468.4 [M+H]+; 1H NMR
(400 MHz, DMSO‑d6) δH: 10.03 (s, 1H), 8.36 (s, 1H), 8.02 (s, 1H),
7.81 (s, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.25
(dd, J = 7.2, 2.0 Hz, 1H), 7.22–7.08 (m, 2H), 6.18 (d, J = 8.5 Hz, 1H),
3.77 (s, 3H), 3.44 (d, J = 5.1 Hz, 4H), 2.09 (s, 3H), 1.57 (s, 6H).
4.1.12.3. N-(2-((5-chloro-2-((2-methoxy-4-(pyrrolidin-1-yl)phenyl)
amino)pyrimidin-4-yl)amino)phenyl)acetamide (22c). Yield: 80.4%;
m.p.: 183.8–185.6 °C; MS (ESI) m/z: 454.4 [M+H]+; 1H NMR
(400 MHz, DMSO‑d6) δH: 10.08 (s, 1H), 8.32 (s, 1H), 8.00 (s, 1H),
7.80 (s, 1H), 7.77–7.72 (m, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.29–7.21
(m, 1H), 7.17–7.10 (m, 2H), 5.84 (d, J = 8.2 Hz, 1H), 3.79 (s, 3H), 3.36
(s, 4H), 2.09 (s, 3H), 1.94 (s, 4H).
4.1.12.4. N-(2-((5-chloro-2-((4-(3-hydroxyazetidin-1-yl)-2-
methoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)acetamide
(22d). Yield: 85.2%; m.p.: 183.8–190.6 °C; MS (ESI) m/z: 454.4
[M−H]−; 1H NMR (400 MHz, DMSO‑d6) δH: 10.01 (s, 1H), 8.31 (s,
1H), 7.99 (s, 1H), 7.79 (s, 1H), 7.73 (s, 1H), 7.41 (d, J = 8.3 Hz, 1H),
7.24 (d, J = 9.3 Hz, 1H), 7.18–7.06 (m, 2H), 6.36 (t, J = 6.0 Hz, 1H),
5.33 (s, 1H), 3.89 (s, 1H), 3.77 (s, 3H), 3.66 (dd, J = 11.1, 4.5 Hz, 1H),
3.56 (dd, J = 11.0, 5.7 Hz, 1H), 3.41 (dd, J = 13.0, 6.5 Hz, 1H), 3.24
(dd, J = 13.3, 5.9 Hz, 1H), 2.09 (s, 3H).
4.1.12.5. N-(2-((5-chloro-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)-2-
methoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)acetamide
(22e). Yield: 79.8%; m.p.:150.1–155.5 °C; MS (ESI) m/z: 513.4 [M
+H]+; 1H NMR (400 MHz, DMSO‑d6) δH: 10.01 (s, 1H), 8.37 (s, 1H),
8.01 (s, 1H), 7.83 (s, 1H), 7.73 (d, J = 6.2 Hz, 1H), 7.61 (d, J = 8.4 Hz,
1H), 7.26 (d, J = 7.8 Hz, 1H), 7.16 (s, 2H), 6.19 (d, J = 8.3 Hz, 1H),
4.46 (s, 1H), 3.78 (s, 3H), 3.54 (t, J = 5.8 Hz, 2H), 3.40 (s, 4H), 2.53 (s,
4H), 2.45 (t, J = 6.0 Hz, 2H), 2.09 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6) δC: 170.07, 159.21, 156.06, 154.95, 154.42, 135.09,
132.32, 131.14, 126.42, 125.74, 125.15 (2C), 112.98, 104.01, 98.18,
60.77, 58.95, 53.34 (2C), 53.09, 45.63 (2C), 29.46, 23.46.
4.1.12.6. N-(2-((5-chloro-2-((2-methoxy-4-(3-methylpiperazin-1-yl)
phenyl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (22f). Yield:
84.5%; m.p.:164.1–164.8 °C; MS (ESI) m/z: 483.4 [M+H]+; 1H NMR
(400 MHz, DMSO‑d6) δH: 10.03 (s, 1H), 8.35 (s, 1H), 8.12 (s, 1H), 7.87
(s, 1H), 7.72 (d, J = 6.2 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.22 (d,
J = 6.9 Hz, 1H), 7.18 (s, 2H), 6.23 (d, J = 8.4 Hz, 1H), 3.78 (s, 3H),
3.40 (s, 4H), 2.53 (s, 4H), 2.09 (s, 3H) 1.39 (s, 3H).
4.1.12.7. N-(2-((5-chloro-2-((2-methoxy-4-morpholinophenyl)amino)
pyrimidin-4-yl)amino)phenyl)acetamide (22g). Yield: 81.3%;
m.p.:154.1–159.5 °C; MS (ESI) m/z: 468.4 [M−H]−; 1H NMR (400 MHz,
DMSO‑d6) δH: 10.04 (s, 1H), 8.39 (s, 1H), 8.03 (s, 1H), 7.85 (s, 1H), 7.72
(d, J = 8.2 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 7.2 Hz, 2.1HZ,
1H), 7.23–7.09 (m, 2H), 6.20 (d, J = 8.5 Hz, 1H), 3.79 (s, 3H), 3.72–3.69
(m, 4H), 3.37–3.34 (m, 4H), 2.09 (s, 3H).
4.1.12.8. (E)-N-(2-((5-chloro-2-((2-methoxy-4-morpholinophenyl)amino)
pyrimidin-4-yl)amino)phenyl)but-2-enamide (22h). Yield: 78.3%;
m.p.:172.3–178.6 °C; MS (ESI) m/z: 496.6 [M+H]+; 1H NMR
(400 MHz, DMSO‑d6) δH: 10.07 (s, 1H), 8.51 (s, 1H), 8.01 (s, 1H),
7.83 (s, 1H), 7.75–7.69 (m, J = 13.9, 1H), 7.65 (d, 1H), 7.38–7.27 (m,
J = 9.0 Hz, 1H), 7.24–7.14 (m, 2H), 6.87 (dq, J = 13.9, 6.9 Hz, 1H),
6.19 (d, J = 8.8 Hz, 2H), 3.79 (s, 3H), 3.76–3.67 (m, 4H), 3.39–3.33
L. Xing et al.
(m, 4H), 1.23 (t, J = 7.2 Hz, 3H).
4.1.12.9. (E)-N-(2-((5-chloro-2-((6-(diethylamino)-2-methoxypyridin-3-yl)
amino)pyrimidin-4-yl)amino)phenyl)but-2-enamide (22i). Yield: 84.8%;
m.p.:193.8–194.6 °C; MS (ESI) m/z: 480.5 [M−H]−; 1H NMR (400 MHz,
DMSO‑d6) δH: 10.07 (s, 1H), 8.33 (s, 1H), 7.90 (s, 1H), 7.83 (s, 1H),
7.75–7.67 (m, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.38–7.27 (m, J = 8.7 Hz,
1H), 7.24–7.14 (m, 2H), 6.87 (dq, J = 12.9, 6.9 Hz, 1H), 6.19 (d,
J = 8.4 Hz, 2H), 3.79 (s, 3H), 3.67–3.56 (m, 4H), 1.26 (t, 6H), 1.43 (t,
J = 6.9 Hz, 3H).
4.1.12.10. (E)-N-(2-((5-chloro-2-((6-(3-hydroxyazetidin-1-yl)-2-
methoxypyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)but-2-enamide
(22j). Yield: 82.5%; m.p.:161.0–162.1 °C; MS (ESI) m/z: 482.4 [M
+H]+; 1H NMR (400 MHz, DMSO‑d6) δH: 10.01 (s, 1H), 8.31 (s, 1H),
7.99 (s, 1H), 7.73 (s, 1H), 7.75–7.67 (m, 1H), 7.56 (d, J = 8.4 Hz, 1H),
7.38–7.27 (m, J = 8.4 Hz, 1H), 7.24–7.14 (m, 2H), 6.87 (dq, J = 13.3,
6.3 Hz, 1H), 6.19 (d, J = 8.5 Hz, 2H), 5.33 (s, 1H), 4.33 (m, 1H), 3.98
(dd, J = 11.1, 4.5 Hz, 2H), 3.79 (s, 3H), 3.68 (dd, J = 11.1, 4.5 Hz, 2H),
1.52 (t, J = 6.8 Hz, 3H).
4.1.12.11. (E)-N-(2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-
2-methoxypyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)but-2-enamide
(22k). Yield: 81.6%; m.p.:161.0–162.1 °C; MS (ESI) m/z: 539.4 [M
+H]+; 1H NMR (400 MHz, DMSO‑d6) δH: 10.02 (s, 1H), 8.33 (s, 1H),
7.83 (s, 1H), 7.77 (s, 1H), 7.75–7.67 (m, J = 13.8, 1H), 7.56 (d,
J = 8.4 Hz, 1H), 7.38–7.27 (m, 1H), 7.24–7.18 (m, 2H), 6.86 (dq,
J = 13.8, 6.9 Hz, 1H), 6.19 (d, J = 8.4 Hz, 2H), 4.46 (s, 1H), 3.79 (s,
3H), 3.55 (t, J = 5.8 Hz, 2H), 3.40 (s, 4H), 2.62 (s, 4H), 2.43 (t,
J = 5.8 Hz, 2H), 1.52 (t, J = 6.9 Hz, 3H).
4.1.12.12. N-(2-((5-chloro-2-((2-methoxy-4-morpholinophenyl)amino)
pyrimidin-4-yl)amino)phenyl)methanesulfonamide (22l). Yield: 85.3%;
m.p.: 181.0–182.1 °C; MS (ESI) m/z: 506.4 [M+H]+; 1H NMR
(400 MHz, DMSO‑d6) δH: 9.32 (s, 1H), 8.46 (s, 1H), 8.12 (s, 1H), 8.08
(s, 1H), 7.98 (s, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H),
7.18 (t, J = 7.8 Hz, 2H), 6.25 (d, J = 8.4 Hz, 1H), 3.78 (s, 3H),
3.75–3.69 (m, 4H), 3.53 (s, 4H), 2.96 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6) δC: 159.44, 155.80, 155.58, 155.05, 154.85, 135.97, 128.89,
127.52, 127.31, 124.80, 124.53, 113.25, 104.06, 98.16, 66.41 (2C),
53.13, 46.05 (2C), 29.16.
4.1.12.13. N-(2-((5-chloro-2-((2-isopropoxy-4-morpholinophenyl)amino)
pyrimidin-4-yl)amino)phenyl)methanesulfonamide (22m). Yield: 78.5%;
m.p.: 176.7–178.2 °C; MS (ESI) m/z: 532.5 [M−H]−; 1H NMR
(400 MHz, DMSO‑d6) δH: 9.34 (s, 1H), 8.48 (s, 1H), 8.08 (s, 1H), 7.93
(d, J = 6.6 Hz, 1H), 7.87 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.35 (dd,
J = 7.4, 1.9 Hz, 1H), 7.21–7.15 (m, 2H), 6.19 (d, J = 8.5 Hz, 1H), 5.14
(dt, J = 12.3, 6.2 Hz, 1H), 3. 37–3.70 (m, 4H), 3.36 (s, 4H), 2.96 (s,
3H), 1.22 (d, J = 6.2 Hz, 6H).
4.1.12.14. N-(2-((5-chloro-2-((2-isopropoxy-4-(piperidin-1-yl)phenyl)
amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (22n). Yield:
84.9%; m.p.: 211.0–212.1 °C; MS (ESI) m/z: 530.5 [M−H]−; 1H NMR
(400 MHz, DMSO‑d6) δH: 9.33 (s, 1H), 8.59 (s, 1H), 8.08–8.03 (m, 1H),
7.99 (d, J = 5.8 Hz, 1H), 7.82 (s, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.31
(dd, J = 7.3, 2.0 Hz, 1H), 7.10–7.05 (m, 2H), 6.19 (d, J = 8.5 Hz, 1H),
5.13 (dt, J = 12.2, 6.2 Hz, 1H), 3.44 (d, J = 5.1 Hz, 4H), 2.91 (s, 3H),
1.58 (s, 6H), 1.23 (d, J = 4.0 Hz, 6H).
4.1.12.15. N-(2-((5-chloro-2-((4-(diethylamino)-2-isopropoxyphenyl)
amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (22o). Yield:
82.2%; m.p.: 196.3–202.8 °C; MS (ESI) m/z: 518.5 [M−H]−; 1H NMR
(400 MHz, DMSO‑d6) δH: 9.29 (s, 1H), 8.42 (s, 1H), 8.05 (s, 1H), 8.00 (s,
1H), 7.86 (s, 1H), 7.45 (d, J = 8.5 Hz, 1H), 7.33 (m, 1H), 7.13 (m,
J = 6.8 Hz, 2H), 5.97 (d, J = 8.5 Hz, 1H), 5.17–5.07 (m, 1H), 3.44 (q,
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Bioorganic Chemistry 81 (2018) 689–699
J = 6.9 Hz, 4H), 2.96 (s, 3H), 1.20 (d, J = 6.2 Hz, 6H), 1.13 (t,
J = 6.9 Hz, 6H).
4.1.12.16. N-(2-((5-chloro-2-((2-(isopropylthio)-4-morpholinophenyl)
amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (22p). Yield:
80.7%; m.p.: 166.9–168.3 °C; MS (ESI) m/z: 548.5 [M−H]−; 1H NMR
(400 MHz, DMSO‑d6) δH: 9.32 (s, 1H), 8.41 (s, 2H), 8.05 (s, 2H), 7.32 (t,
J = 8.5 Hz, 2H), 7.11 (s, 2H), 6.52 (d, J = 8.7 Hz, 1H), 3.83–3.75 (dt,
J = 13.6, 6.8 Hz, 1H), 3.73 (m, J = 13.6, 4H), 3.44 (m, 4H), 2.98 (s,
3H), 1.28 (d, J = 6.8 Hz, 6H).
4.1.12.17. N-(2-((5-chloro-2-((2-(isopropylthio)-4-(piperidin-1-yl)
phenyl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(22q). Yield: 86.4%; m.p.: 186.4–192.1 °C; MS (ESI) m/z: 548.4 [M
+H]+; 1H NMR (400 MHz, DMSO‑d6) δH: 9.79 (s, 1H), 8.69 (s, 1H),
8.29 (s, 1H), 8.08 (s, 1H), 8.00 (s, 1H), 7.26 (t, J = 9.5 Hz, 2H), 6.96 (t,
J = 7.1 Hz, 1H), 6.85 (s, 1H), 6.51 (d, J = 8.7 Hz, 1H), 3.82 (dt,
J = 13.6, 6.9 Hz, 1H), 3.55 (d, J = 5.0 Hz, 4H), 2.86 (s, 3H),
1.71–1.50 (m, 6H), 1.29 (d, J = 6.8 Hz, 6H).
4.1.12.18. N-(2-((5-chloro-2-((4-(diethylamino)-2-(isopropylthio)
phenyl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(22r). Yield: 82.3%; m.p.: 173.0–176.2 °C; MS (ESI) m/z: 536.2 [M
+H]+; 1H NMR (400 MHz, DMSO‑d6) δH: 9.29 (s, 1H), 9.08 (s, 1H),
8.49 (s, 1H), 8.18 (s, 1H), 8.12 (s, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.70
(d, J = 9.4 Hz, 1H), 7.39 (dd, J = 7.8, 1.4 Hz, 1H), 7.31 (td, J = 7.8,
1.4 Hz, 1H), 7.22 (td, J = 7.7, 1.4 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H),
3.43–3.40 (m, 4H), 2.96 (s, 3H), 1.64–1.49 (m, 6H), 1.23 (s, 6H).
4.1.12.19. N-(2-((5-chloro-2-((4-morpholinophenyl)amino)pyrimidin-4-
yl)amino)phenyl)methanesulfonamide (22s). Yield: 83.4%; m.p.:
201.2–202.8 °C; MS (ESI) m/z: 476.3 [M+H]+; 1H NMR (400 MHz,
DMSO‑d6) δH: 9.29 (s, 1H), 9.15 (s, 1H), 8.51 (s, 1H), 8.24 (s, 1H), 8.13
(s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 9.9 Hz, 1H), 7.39 (dd,
J = 7.9, 1.4 Hz, 1H), 7.32 (t, J = 7.1 Hz, 1H), 7.23 (td, J = 7.7, 1H),
6.69 (d, J = 9.1 Hz, 1H), 3.70 (m, 4H), 3.32 (s, 4H), 2.95 (s, 3H).
4.1.12.20. N-(2-((5-chloro-2-((4-(piperidin-1-yl)phenyl)amino)
pyrimidin-4-yl)amino)phenyl)methanesulfonamide (22t). Yield: 82.6%;
m.p.: 164.3–167.3 °C; MS (ESI) m/z: 474.4 [M+H]+; 1H NMR
(400 MHz, DMSO‑d6) δH: 9.30 (s, 1H), 9.07 (s, 1H), 8.50 (s, 1H), 8.18
(d, 1H), 8.11 (s, 1H), 8.00 (s, 1H), 7.68 (dd, J = 9.3, 1.5 Hz, 1H), 7.37
(dd, J = 7.8, 1.3 Hz, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.19 (t, J = 7.8 Hz,
1H), 6.67 (d, J = 8.9 Hz, 1H), 3.41 (d, J = 5.4 Hz, 4H), 2.95 (s, 3H),
1.55 (m, 6H).
4.1.12.21. N-(2-((5-chloro-2-((4-(diethylamino)phenyl)amino)pyrimidin-
4-yl)amino)phenyl)methanesulfonamide (22u). Yield: 81.4%; m.p.:
183.6–186.1 °C; MS (ESI) m/z: 462.4 [M+H]+; 1H NMR (400 MHz,
DMSO‑d6) δH: 8.95 (s, 1H), 8.56 (s, 1H), 8.05 (m, 3H), 7.60 (d,
J = 8.5 Hz, 1H), 7.36 (dd, J = 7.7, 1.5 Hz, 1H), 7.16 (m, 2H), 7.12
(d, 1H), 6.46 (d, J = 9.1 Hz, 1H), 3.45 (q, J = 7.0 Hz, 4H), 2.93 (s, 3H),
1.09 (t, J = 7.0 Hz, 6H).
4.1.12.22. N-(2-((5-chloro-2-((6-(3-hydroxyazetidin-1-yl)pyridin-3-yl)
amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (22v). Yield:
84.3%; m.p.: 169.5–172.2 °C; MS (ESI) m/z: 462.0 [M+H]+; 1H NMR
(400 MHz, DMSO‑d6) δH: 8.92 (s, 1H), 8.59 (s, 1H), 8.34–7.84 (m, 3H),
7.61 (d, J = 9.1 Hz, 1H), 7.35 (dd, J = 9.5 Hz, 4.5 Hz, 1H), 7.27–7.05
(m, 2H), 7.15 (d, 1H), 6.46 (d, J = 3.1 Hz, 1H), 5.38 (s, 1H), 4.43 (m,
1H), 3.98 (dd, J = 8.3, 4.5 Hz, 2H), 3.68 (dd, J = 6.8, 2H), 2.93 (s, 3H).
4.1.12.23. N-(2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)
pyridin-3-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(22w). Yield: 83.9%; m.p.: 181.0–185.1 °C; MS (ESI) m/z: 519.4 [M
+H]+; 1H NMR (400 MHz, DMSO‑d6) δH: 9.25 (s, 1H), 8.86 (s, 1H),
L. Xing et al.
8.34–7.84 (m, 3H), 7.60 (d, J = 9.1 Hz, 1H), 7.35 (dd, J = 7.0, 1.5 Hz,
1H), 7.27–7.05 (m, 2H), 7.17 (d, 1H), 6.46 (d, J = 2.8 Hz, 1H), 4.46 (s,
1H), 3.45 (t, J = 6.2 Hz, 2H), 3.36 (s, 4H), 2.62 (s, 4H), 2.93 (s, 3H),
2.42 (t, J = 5.6 Hz, 2H).
4.1.12.24. N-(2-((5-chloro-2-((6-(3-methylpiperazin-1-yl)pyridin-3-yl)
amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (22x). Yield:
81.8%; m.p.: 193.2–199.2 °C; MS (ESI) m/z: 489.4 [M+H]+; 1H NMR
(400 MHz, DMSO‑d6) δH: 8.95 (s, 1H), 8.56 (s, 1H), 8.34–7.84 (m, 3H),
7.61 (d, J = 10.0 Hz, 1H), 7.35 (dd, J = 8.3, 2.6 Hz, 1H), 7.27–7.05 (m,
2H), 7.17 (d, 1H), 6.46 (d, J = 1.8 Hz, 1H), 3.25 (s, 1H), 3.10 (s, 1H),
2.93 (s, 3H), 2.88 (s, 1H), 2.79 (s, 1H), 2.73 (s, 1H), 2.72 (s, 1H), 2.68
(s, 1H), 1.31 (s, 3H), 1.07(s, 1H).
4.1.12.25. 4-(5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-
methoxypyridin-3-yl)amino)pyrimidin-4-yl)-3,4-dihydro-2H-benzo[1,4- b]
thiazine-1,1-dioxide (23a). Yield: 78.4%; m.p.:190.8–196.0 °C; MS
(ESI) m/z: 546.4 [M+H]+; 1H NMR (400 MHz, CDCl3) δH: 9.13 (s,
1H), 8.47 (d, J = 8.3 Hz, 1H), 8.17 (d, J = 7.5 Hz, 1H), 8.11 (s, 1H),
8.00 (d, J = 8.3 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 6.1 Hz,
1H), 7.03 (s, 1H), 6.51 (m, 2H), 6.08 (dd, J = 6.1 Hz, 4.9 Hz, 2H), 4.02
(s, 1H), 3.94 (s, 3H), 3.74 (s, 2H), 3.55 (s, 4H), 2.76 (s, 4H), 2.71 (d,
J = 4.9 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6) δC: 159.66, 156.37,
155.90, 155.35, 154.90, 137.78, 137.38, 135.20, 129.98, 129.84,
126.46, 123.92, 123.48, 112.09, 98.28, 70.26, 60.77, 58.96, 53.38
(2C), 53.06, 45.51 (2C), 29.47.
4.1.12.26. 4-(5-chloro-2-((2-methoxy-6-(piperidin-1-yl)pyridin-3-yl)
amino)pyrimidin-4-yl)-3,4-dihydro-2H-benzo[1,4-b]thiazine-1,1-dioxide
(23b). Yield: 77.8%; m.p.:158.8–166.8 °C; MS (ESI) m/z: 501.3 [M
+H]+; 1H NMR (400 MHz, CDCl3) δH: 9.17 (s, 1H), 8.46 (s, 1H), 8.00
(m, 4H), 7.60 (s, 1H), 7.15 (s, 1H), 6.55 (s, 2H), 6.27 (s, 1H), 6.03 (s,
1H), 3.94 (s, 3H), 3.46 (s, 4H), 1.71 (s, 4H), 1.26 (s, 2H).
4.1.12.27. 4-(5-chloro-2-((2-methoxy-6-(pyrrolidin-1-yl)pyridin-3-yl)
amino)pyrimidin-4-yl)-3,4-dihydro-2H-benzo[1,4-b]thiazine-1,1-dioxide
(23c). Yield: 80.5%; m.p.:178.5–181.2 °C; MS (ESI) m/z: 487.4 [M
+H]+; 1H NMR (400 MHz, CDCl3) δH: 9.57 (s, 1H), 8.40 (d, J = 8.1 Hz,
1H), 8.01(m, 2H), 7.72 (s, 1H), 7.51 (s, 1H), 7.42 (m, 1H), 7.30 (m,
2H), 6.58–6.50 (m, 2H), 6.06 (d, J = 9.0 Hz, 1H), 5.87 (s, 1H), 3.89 (s,
3H), 3.45 (s, 4H), 2.02 (s, 4H).
4.1.12.28. 4-(5-chloro-2-((2-isopropoxy-6-(piperidin-1-yl)pyridin-3-yl)
amino)pyrimidin-4-yl)-3,4-dihydro-2H-benzo[1,4- b]thiazine-1,1-dioxide
(23d). Yield: 75.3%; m.p.:162.3.5–169.2 °C; MS (ESI) m/z: 529.4 [M
+H]+; 1H NMR (400 MHz, CDCl3) δH: 9.81 (s, 1H), 8.30 (s, 1H), 8.17
(s, 1H), 8.08 (s, 2H), 7.80 (s, 1H), 7.54 (m, 2H), 6.55–6.52 (m, 2H),
6.05 (s, 2H), 3.56–3.49 (m, 7H), 2.23 (s, 4H), 1.90 (s, 2H), 1.72 (s, 4H).
13
C NMR (100 MHz, DMSO‑d6) δC: 159.62, 158.74, 155.88, 155.21,
154.89, 137.77, 136.87, 135.10, 129.98, 129.81, 126.56, 123.95,
123.64, 111.64, 97.85, 67.88, 51.84, 46.43 (2C), 44.64, 25.39 (2C),
24.75, 22.43 (2C).
4.2. MTT assay in vitro
The cytotoxic activities of compounds 22a-x and 23a-d were eval-
uated against H2228, Karpas299 and A549 by the standard MTT assay
in vitro, with ceritinib as the positive control. The cancer cell lines were
cultured in minimum essential medium (MEM) supplement with 10%
fetal bovine serum (FBS). Approximate 4 × 103 cells, suspended in
MEM medium, were plated into each well of a 96-well plate and in-
cubated in 5% CO2 at 37 °C for 24 h. The tested compounds at the in-
dicated final concentrations were added to the culture medium and
incubated for 72 h. Fresh MTT was added to each well at the terminal
concentration of 5 μg/mL, and incubated with cells at 37 °C for 4 h. The
formazan crystals in each well were dissolved in 100 μL DMSO, and the
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Bioorganic Chemistry 81 (2018) 689–699
absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm
(for the reference wavelength) was measured with an ELISA reader. All
of the compounds were tested three times in each of the cell lines. The
results, expressed as IC50 (inhibitory concentration 50%), were the
averages of three determinations and calculated relative to the vehicle
(DMSO) control by the Bacus Laboratories Incorporated Slide Scanner
(Bliss) software.
4.3. In vitro enzymatic assays
The in vitro enzymatic assays versus ALK and ALKL1196M were
evaluated by homogeneous time-resolved fluorescence (HTRF) assay. In
enzymatic assay, the solution of peptide substrates, ATP, appropriate
kinase, and diluted compound was 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 solution 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
60 min. And then add 25 μL of stop buffer (100 mM HEPES, pH 7.5,
0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) to stop re-
action. The plate was read by Caliper at 320 nm and 615 nm. IC50 va-
lues were calculated from the inhibition curves.
4.4. Western blotting
Western blotting analysis was performed as described Karpas299
cells were treated with 22l or ceritinib. After treatment 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. After
incubated overnight at 4 °C with the respective primary antibodies,
horseradish peroxidase (HRP)-conjugated secondary antibodies (1:
3000) was added. The Enhanced chemiluminescence reagent was used
to identify the specific bands. Densitometry analysis on immunoblots
were performed using ImageJ2 software.
4.5. Morphology assays of apoptotic cells
Apoptotic morphological changes of H2228 and Karpas299 cells
were detected by AO/EB staining. Briefly, cells were seeded in six-well
plates for 24 h, then treated with ceritinib or different concentrations of
22l for 48 h. The cells were washed with phosphate buffer saline (PBS)
and then stained with AO/EB mixed solution (AO: EB ¼ 1: 1) for
15 min. The stained cells were washed twice with PBS and observed by
fluorescence microscope.
4.6. Molecular docking
The molecular modeling studies were performed with Discovery
Studio 2017. The protein coordinates (PDB ID code: 4MKC) were
downloaded from the Protein Data Bank (http://www.rcsb.org/pdb/).
In the docking process, the protein protocol was prepared by several
operations, including standardization of atom names and insertion of
missing atoms in residues. Then, the receptor model was typed with the
CHARMm force field and a binding sphere with radius of 12 Å was
defined through the original ligand (ceritinib) as the binding site. The
ceritinib, 22l and 23a were drawn with ChemSketch and fully mini-
mized using the CHARMm force field. Finally, they were docked into
the binding site using the CDOCKER protocol with the default settings.
5. Conflict of interest
The authors have declared no conflict of interest.
L. Xing et al.
Acknowledgements
This work was supported by National Natural Science Foundation of
China, China (No. 81673308), Natural Science Foundation of Liaoning
Province, China (No. 201602703) and Development Project of Ministry
of Education Innovation Team, China (No. IRT1073).
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