Discovery of 2,4-pyrimidinediamine derivatives as potent dual inhibitors of ALK and HDAC

Article abstract of DOI:10.1016/j.ejmech.2021.113672

Combination of anaplastic lymphoma kinase (ALK) inhibitor with histone deacetylases (HDAC) inhibitor could exert synergistically anti-proliferative effects on ALK positive non-small cell lung cancer (NSCLC) na?ve or resistant cells. In this work, we designed and synthesized a series of 2,4-pyrimidinediamine derivatives as dual ALK and HDAC inhibitors based on pharmacophore merged strategy. Among which, compound 10f displayed the most potent and balanced inhibitory activity against ALK (IC₅₀ = 2.1 nM) and HDAC1 (IC₅₀ = 7.9 nM), respectively. In particular, 10f was also potent against the frequently observed Crizotinib-resistant ALK^L1196M (IC₅₀ = 1.7 nM) as well as the Ceritinib-resistant ALK^G1202R (IC₅₀ = 0.4 nM) mutants. In antiproliferative activity assay, 10f exhibited impressive activity on ALK-addicted cancer cell lines at low micromole concentrations, which was comparable to that of Crizotinib and Ceritinib. Further flow cytometric analysis indicated that 10f could effectively induce cell death via cell apoptosis and cell cycle arrest. Taken together, these results suggested 10f would be a promising lead compound for the ALK-positive NSCLC treatment, especially the Ceritinib- or Crizotinib-resistant NSCLC.

Full text of DOI:10.1016/j.ejmech.2021.113672

European Journal of Medicinal Chemistry 224 (2021) 113672
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of 2,4-pyrimidinediamine derivatives as potent dual
inhibitors of ALK and HDAC
,
,
,
,
,
Tao Pan a 1, Yanrong Dan a 1, Dafeng Guo a 1, Junhao Jiang a b, Dongzhi Ran a b, Lin Zhang a,
Binghua Tian a, Jianyong Yuan a b, Yu Yu a *, Zongjie Gan a
,
,
b,
, b, **
a Department of Medicinal Chemistry, College of Pharmacy, Chongqing Medical University, Chongqing 400016, PR China
b Chongqing Research Center for Pharmaceutical Engineering, Chongqing Medical University, Chongqing 400016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Combination of anaplastic lymphoma kinase (ALK) inhibitor with histone deacetylases (HDAC) inhibitor
could exert synergistically anti-proliferative effects on ALK positive non-small cell lung cancer (NSCLC)
naïve or resistant cells. In this work, we designed and synthesized a series of 2,4-pyrimidinediamine
derivatives as dual ALK and HDAC inhibitors based on pharmacophore merged strategy. Among which,
compound 10f displayed the most potent and balanced inhibitory activity against ALK (IC50 ¼ 2.1 nM)
and HDAC1 (IC50 ¼ 7.9 nM), respectively. In particular, 10f was also potent against the frequently
observed Crizotinib-resistant ALKL1196M (IC50 ¼ 1.7 nM) as well as the Ceritinib-resistant ALKG1202R
(IC50 ¼ 0.4 nM) mutants. In antiproliferative activity assay, 10f exhibited impressive activity on ALK-
addicted cancer cell lines at low micromole concentrations, which was comparable to that of Crizoti-
nib and Ceritinib. Further flow cytometric analysis indicated that 10f could effectively induce cell death
via cell apoptosis and cell cycle arrest. Taken together, these results suggested 10f would be a promising
lead compound for the ALK-positive NSCLC treatment, especially the Ceritinib- or Crizotinib-resistant
NSCLC.
Received 28 May 2021
Received in revised form
22 June 2021
Accepted 24 June 2021
Available online 29 June 2021
Keywords:
ALK/HDAC dual Inhibitors
Pharmacophore merged strategy
2,4-Pyrimidinediamine
NSCLC
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
player in NSCLC development, and the development of inhibitors
for blocking ALK signaling pathways represents an effective ther-
Chromosomal rearrangement of the anaplastic lymphoma ki-
nase (ALK) gene has been identified as one of the most frequent
oncogenic drivers that are associated with many malignancies [1],
which include diffuse large B-cell lymphoma (DLBCL) [2], inflam-
matory myofibroblastic tumors (IMTs) [3], and non-small cell lung
cancer (NSCLC) [4]. For instance, echinoderm microtubule-
associated protein-like 4 (EML4)-ALK gene fusion, the predomi-
nant ALK rearrangement that is associated with NSCLC, can pro-
mote dimerization and constitutive phosphorylation of ALK
proteins, thereby leading to aberrant proliferation and survival of
cancer cells via continuous activation of ALK and its downstream
signaling pathways [5]. Hence, ALK was proven to be a critical
apeutic strategy for the treatment of ALK-driven NSCLC [6e8].
Crizotinib (Fig. 1), which became the first small molecular ALK
inhibitor to be approved by the FDA in 2011, initially demonstrated
potent efficacy against ALK-rearranged NSCLC [9]. Unfortunately,
patients who are treated with Crizotinib eventually relapse within
1e2 years due to acquired drug resistance [10,11]. To overcome the
acquired resistance to Crizotinib, second-generation and third-
generation ALK inhibitors with improved potency and selectivity,
such as Ceritinib [12], Alectinib [13], Brigatinib [14] and Lorlatinib
[15] (Fig. 1), are subsequently administered. These newly approved
inhibitors exhibit remarkable antitumor activity in Crizotinib-
sensitive and Crizotinib-resistant NSCLC models and provide clin-
ical benefit to patients who have progressed following Crizotinib
treatment. However, similar to Crizotinib, drug resistance to next-
generation ALK-TKIs inevitably arises due to multiple resistance
mechanisms, which include the emergence of new secondary
mutations in the kinase domain, bypass pathway activation, and
epithelial-to-mesenchymal transition (EMT), among others
[16e19].
* Corresponding author. Department of Medicinal Chemistry, College of Phar-
macy, Chongqing Medical University, Chongqing 400016, PR China.
** Corresponding author. Chongqing Research Center for Pharmaceutical Engi-
neering, Chongqing Medical University, Chongqing 400016, PR China.
E-mail addresses: yuyu3519@163.com (Y. Yu), gzj@cqmu.edu.cn (Z. Gan).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113672
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Fig. 1. Chemical structures of ALK inhibitors.
Amino residue mutations have been identified as the most
common reason for drug resistance [20]. For example, in contrast
with the gatekeeper mutation L1196 M, which predominantly
confers resistance to Crizotinib [21], the solvent front G1202R
mutation, has been regarded as one of the most frequent causes of
resistance to second-generation inhibitors [16,22,23]. Therefore,
the discovery and development of effective ALK inhibitors that
could counteract G1202R-induced resistance have attracted sub-
stantial attention [24,25]. Recently, it was found that an unfavor-
able steric obstruction caused by the bulky group in ALK inhibitors
with 1202R residue is likely responsible for their loss of potency
against the G1202R mutant [23]. Replacing the piperidine group in
Ceritinib with aliphatic amines or glycine, could attenuate and
diminish the steric hindrance, thereby resulting in novel com-
pounds with enhanced activities against this mutant [26,27].
Consequently, these findings would be beneficial for the develop-
ment of novel and potent ALK inhibitors that can overcome the
resistance that is caused by the G1202R mutation.
hydrophobic linker and a zinc-binding group (ZBG) [32]. The cap
group is critical for HDAC selectivity and can accommodate variable
structures. Hence, the N4-(2-(isopropylsulfonyl)phenyl)-N2-
phenylpyrimidine motif in Ceritinib, which shows satisfactory
binding affinity with ALKwt according to the cocrystal structure
(PDB: 4MKC), was selected as the cap group [12]. Additionally, a
flexible long-chain aliphatic group was chosen as the linker to
replace the piperidine in Ceritinib to avoid steric hindrance.
Meanwhile, a zinc-binding group (ZBG), such as hydroxamic acid,
was incorporated into the tail of the sidechain to chelate the cat-
alytic zinc ion in HDACs, with the objective of dual inhibition.
Finally, a series of diarylaminopyrimidine derivatives with potent
and balanced inhibitory activities against ALK, including ALKG1202R
and HDACs were designed and synthesized in this study (Fig. 2).
,
2. Results and discussion
2.1. Chemistry
Histone deacetylases (HDACs) are a class of enzymes that
remove acetyl groups from lysine residues in histones and play
important roles in the proliferation, differentiation and apoptosis of
tumor cells [28]. In addition to the G1202R mutation that is iden-
tified as a common resistance mechanism for Ceritinib, accumu-
lating evidence has shown that EMT mediated by HDACs is also
associated with the sensitivity to the second-generation ALK in-
hibitors [16], while combination with HDAC inhibitors could
improve the efficacy of ALK inhibitors in combating drug resistance
[29]. For example, Cho and coworkers demonstrated that
combining Ceritinib with an HDAC inhibitor, namely, Panobinostat,
could have synergistic antiproliferative effects on NSCLC naïve or
resistant cells and xenografts [30]. Another study similarly showed
that co-treatment with HDAC inhibitor quisinostat could restore
the sensitivity of lung adenocarcinoma cells to Critzotinib [31].
Therefore, the success of the synergy between ALK inhibitors and
HDAC inhibitors in increasing anti-ALK activity to circumvent drug
resistance provided a rational combinational approach in NSCLC
therapeutics.
The synthetic route to target compounds 6a-t is illustrated in
Scheme 1. The key intermediate, namely, 2,5-dichloro-N-(2-(iso-
propylsulfonyl)phenyl)pyrimidin-4-amine (1), was prepared ac-
cording to procedures from the literature [33]. Briefly, compound 1
readily reacted with substituted methyl 4-aminobenzoate or 3-
aminobenzoate 2a-h to produce compounds 3a-h. Hydrolysis of
compounds 3a-h under basic conditions provided corresponding
carboxylic intermediates 4a-h, which subsequently condensed
with methyl aminoalkanoates in CH2Cl2 in the presence of 1-ethyl-
3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDCI)
and 1-hydroxybenzotriazole (HOBT) to produce important ester
intermediates 5b-t. Finally, the transformation of compounds 5b-t
into the target hydroxamic acid compounds 6a-t was realized with
a freshly prepared NH2OH/KOH methanol solution at 0 ꢀC with
moderate yields.
Another series of target compounds 10a-j and 16a-m were
prepared via a similar synthetic procedure, as illustrated in Scheme
2 and Scheme 3, respectively. In Scheme 2, intermediates 8a-c were
obtained with moderate to satisfactory yields by treating 1 with
commercially available starting materials m-phenylenediamine 7a,
p-phenylenediamine 7b and 2-methyl-m-phenylenediamine 7c
under hydrochloride-promoted conditions. The coupling of re-
sultants 8a-c with monomethyl dicarboxylates of various chain
lengths generated corresponding compounds 9a-j, which were
Based on these studies, we hypothesized that developing a
single molecule that can concurrently inhibit both ALK and HDACs
would be a potential strategy to overcome resistance. To design
dual inhibitors, a pharmacophore merging approach was utilized.
As illustrated in Fig. 2, almost all approved HDAC inhibitors share a
common pharmacophore: a surface recognition “CAP” group, a
2

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Fig. 2. Design strategy for a dual inhibitor that targets ALK, including ALKG1202R, and HDACs.
Scheme 1. Reagents and conditions: (a) HCl, i-PrOH, 90 ꢀC, 6 h, 57.1%e72.4%; (b) 35% NaOH aqueous solution, 50 ꢀC, 3 h, 62.5%e97.7%; (c) corresponding methyl aminoalkanoates,
EDCI, HOBT, DIPEA, CH2Cl2, r.t., 12 h, 37.9%e71.2%; and (d) NH2OH, KOH, CH3OH, 0 ꢀC, 2 h, 20.6%e66.6%.
directly converted into hydroxamic acid target compounds 10a-j by
NH2OH/KOH in dry methanol. In Scheme 3, intermediates 13a-m
were afforded in a similar way employing aromatic amines 11a-j
and substituted-2,4-dichloropyrimidines 12a-e as starting mate-
rials. Next, condensation of 13a-m with monomethyl suberate
catalyzed by EDCI/HOBT produced compounds 14a-m, which were
finally transferred into hydroxamic acid target compounds 16a-m
under the same reaction conditions.
The synthetic route to desired compound 20 is illustrated in
Scheme 4. Reaction of compound 1 with 4-bromoaniline in the
catalysis of hydrochloride in isopropanol produced intermediate 18
which subsequently underwent a standard Heck coupling reaction
with methyl acrylate in the presence of Pd(OAC)2 to yield inter-
mediate 19. Finally, treatment of compound 19 with NH2OH/KOH in
methanol yielded target compound 20.
employed as positive controls to validate our hypothesis. Fortu-
nately, as presented in Table 1, all designed compounds retained
significant potency toward ALKwt at the 10 nM concentration and
remained as potent as Crizotinib, thereby demonstrating that
modifications to the piperidine part would be permissible. More
interestingly, a subset of these analogs demonstrated promising
potency against HDAC1 kinase simultaneously, especially com-
pound 6f, displayed HDAC inhibitory activity that was comparable
to that of SAHA, with an IC50 value of 12 nM. This encouraging
outcome demonstrated the rationality of our design strategy and
prompted us to further investigate preliminary SAR. Unsurprisingly,
it was found that shortening the linker length (compounds 6a-d)
dramatical decreased the HDAC1 inhibitory activities, which was
likely due to the attenuated chelating interaction between the
hydroxamic acid group and the zinc cation [34]. For example,
compound 6a (IC50 ¼ 495 nM) was over 10-fold less potent than
compound 6f. A similar trend was also observed for aniline de-
rivatives 10a-f, among which compound 10f, which has an optimal
length of six carbons, elicited the most potent inhibitory activity,
with an IC50 value of 7.9 nM as well. However, in contrast to the
linker length, which plays a key role in HDAC inhibition, the carbon
number had no readily observable influence on ALK inhibition
2.2. Biological evaluation
2.2.1. In vitro ALK and HDAC kinase inhibition assays of target
compounds
All synthetic compounds were subjected to ALKwt and HDAC1
enzymatic inhibition assays in which SAHA and Crizotinib were
3

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Scheme 2. Reagents and conditions: (a) HCl, i-PrOH, 90 ꢀC, 6 h, 42.6%e59.9%; (b) corresponding mono-methyl dicarboxylates, EDCI, HOBT, DIPEA, CH2Cl2, r.t., 12 h, 31.5%e65.4%; and
(c) NH2OH, KOH, CH3OH, 0 ꢀC, 2 h, 40.0%e81.2%.
Scheme 3. Reagents and conditions: (a) DIPEA, i-PrOH, 85 ꢀC, 6 h or NaH, DMF, 0 ꢀC, 2 h, 30.2%e85.7%; (b) TsOH, i-PrOH, 90 ꢀC, 6 h, 55.4%e76.6%; (c) monomethyl suberate, EDCI,
HOBT, DIPEA, DMF, r.t., 12 h, 30.5%e77.2%; and (d) NH2OH, KOH, CH3OH, 0 ꢀC, 2 h, 68.5%e89.3%.
based on the results for compounds 6a-f and 10a-i.
with the corresponding Cl- and OCH3-substituted compounds (6h
vs 6j and 6k, and 6m vs 6l), hence, a small electron-donating group
in the N2-phenyl ring may be favorable for HDAC1 inhibition. In
addition, shifting the linker from the para-to meta-position resul-
ted in compounds 6n-t, which exhibited reduced potency
To explore the substituent effects on the binding efficiency, CH3,
Cl and OCH3 groups were introduced at the C2-position or C3-
position of the N2-phenyl ring. Target compounds that containing
CH3 groups were slightly more active against HDAC1 compared
4

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Scheme 4. Reagents and conditions: (a) HCl, i-PrOH, 90 ꢀC, 6 h; (b) methyl acrylate, Pd(OAc)2, tri(o-tolyl)phosphine, CH3CN, DMF, 90 ꢀC, 6 h; and (c) NH2OH, KOH, CH3OH, 0 ꢀC, 2 h.
compared to compounds 6a-d with the same carbon chain in
enzyme assays. This SAR result suggested that the para-substituted
side chain might be more accessible for occupation of the tunnel in
HDAC proteins. Optimization was further conducted to identify the
contributions of various linkers to the potency. Unfortunately,
replacement of the alkyl with a cinnamic linkage as in Panobinostat
led to compound 20, which suffered an HDAC1 inhibition potency
loss.
cancer cells, compounds 6f and 10f demonstrated almost 3- to 20-
fold higher anticancer activities than the reference compounds
Ceritinib and Crizotinib. Especially in H2228 cells, compounds 6f
and 10f displayed potent activities with IC50 values of 0.18 and
0.13 mM, respectively. These results demonstrate that the dual in-
hibitors may exhibit enhanced antiproliferative capacities.
2.2.3. Effect of compound 10f on the proliferative ability of cancer
cells
Next, we replaced 2-isopropylsulphonyl with different groups
and continued to further investigate the SARs based on 10f. As
shown in Table 2, when 2-isopropylsulphonyl was substituted by
CH3, OCH3, Cl, and Br at different positions, a dramatic loss of ALK
potency was observed, which demonstrated that 2-
isopropylsulphonyl group was important and showed a decisive
effect on ALK inhibitory (Table 2, 16a-l). On the other hand, the
target compounds with different substituents such as F, OCH3, and
CH3 groups at R5-position in pyrimidine ring were well tolerated in
both ALK and HDAC1 as compound 10f dose (Table 2, 16k-m).
In view of the encouraging potencies that were demonstrated by
compounds 6f and 10f on ALKwt and HDAC1, the activity and
selectivity profiles of these two compounds were further evaluated
using two common ALK mutant enzymes and three HDAC kinase
subtypes. Overall, as presented in Table 3, both selected compounds
displayed almost equivalent inhibition toward HDAC1, HDAC2 and
HDAC6, which is in line with the positive control SAHA. Moreover,
compounds 6f (IC50 ¼ 2.4 nM) and 10f (IC50 ¼ 1.7 nM) showed
satisfactory inhibitory activities against the ALKL1196M mutant,
which was regarded as being responsible for Crizotinib
(IC50 ¼ 16 nM) resistance. More strikingly, in the case of the
ALKG1202R mutant, to which the drug resistance of Ceritinib is
attributed, compound 10f (IC50 ¼ 0.4 nM) showed almost 10-fold
Since compound 10f showed desirable activity according to
enzyme and cell viability assays, it was selected for further explo-
ration. Colony formation was conducted to determine the effect of
compound 10f on the proliferative ability of cancer cells. In this
assay, MDA-MB-231 and SH-SY5Y cells were selected and exposed
to various concentrations of compound 10f (0.1
mM, 0.2 mM, and
0.4 M) for 72 h. Then, the cells were cultured in a fresh medium for
m
another two weeks. The results in Fig. 3 demonstrate that the
numbers of clones in the 10f treatment group were significantly
reduced compared with those in the control group. The results
demonstrate that the proliferation activities of MDA-MB-231 and
SH-SY5Y cells were remarkably decreased by compound 10f, which
is in accordance with the CCK-8 assay results.
2.2.4. Effects of compound 10f on cell migration and repair
capability
In addition, a cell scratch assay was conducted to examine the
effects of compound 10f on cell migration and repair capability.
MDA-MB-231 and SH-SY5Y cells were cultured with compound 10f
at three concentrations (0.1 mM, 0.2 mM, and 0.4 mM) for 48 h. The
migration of both tested cell lines was apparently suppressed by
compound 10f (Fig. 4). The wound of MDA-MB-231 in the control
group showed 30.3% closure after 48 h, while the wound was
superior activity to Ceritinib (IC50
¼
3.3 nM) and fourth-
generation ALK inhibitor Repotrectinib (IC50 ¼ 4.5 nM), thereby
suggesting that compound 10f might be a promising ALK inhibitor
for overcoming the G1202R mutant.
decreased to 3.7% with 10f treatment (0.4 mM). A similar result was
observed in SH-SY5Y cells, as the wound was 2.8% in the 10f
treatment group versus 15.1% in the control group. These results
demonstrate that compound 10f can suppress cell migration and
repair capability.
2.2.2. Anti-proliferative activities of compounds 6f and 10f against
cancer cell lines
A CCK-8 assay was conducted to evaluate the antiproliferative
activities of compounds 6f and 10f on the HepG2 (liver cancer),
MDA-MB-231 (breast cancer), A549, H2228 (NSCLC), SK-N-BE2, and
SH-SY5Y (neuroblastoma) cancer cell lines. As shown in Table 4,
both compounds 6f and 10f displayed similar activities to positive
2.2.5. Effects of compound 10f on cell apoptosis and cell cycle
To preliminarily investigate whether compound 10f induces
apoptosis of MDA-MB-231 cells, AO/EB and Hoechst 33258 staining
were conducted after compound 10f treatment for 24 h. According
to the results of AO/EB staining, the orange-red fluorescence was
emerged in the cells after treated with different concentrations of
controls SAHA and Ceritinib, with approximately 1 mM IC50 values
for the MDA-MB-231, A549 and HepG2 cell lines. Moreover, for the
SK-N-BE2, SH-SY5Y (F1174L) and H2228 (EML4-ALK) ALK-addicted
10f (1.0
mM, 2.0 mM, and 4.0 mM) or Ceritinib (4.0 mM) (Fig. 5A).
Moreover, the results of Hoechst 33258 staining further supported
5

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Table 1
Inhibitory activities of target compounds against the ALKwt and HDAC1 kinases.
Cops.
R1
H
R2
R3
H
HDAC1 inhibitiona (%)
ALKwt inhibition (%)
@10 nM
@10 nM
0.1
@100 nM
NDb
@100 nM
ND
6a
81.1
6b
6c
H
H
H
H
ꢁ1
ꢁ2
3
4
86.7
91.2
97.3
97.9
6d
6e
6f
H
H
H
H
H
H
H
H
8
31
85.2
89.4
84.5
97.8
98.1
87.7
70.3
98.8
H
55
49
48
35
45
24
101
100
93
98.1
H
102.7
101.5
101.1
97.8
6g
6h
6i
CH3
CH3
Cl
78
93
6j
Cl
67
100.4
6

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Table 1 (continued)
Cops.
R1
R2
R3
H
HDAC1 inhibitiona (%)
ALKwt inhibition (%)
@10 nM
@10 nM
26
@100 nM
74
@100 nM
100.9
6k
OCH3
99.0
6l
H
H
OCH3
CH3
H
45
58
ꢁ3
86
96
ND
97.3
93.1
ND
98.5
97.3
ND
6m
6n
H
6o
6p
H
H
H
H
ꢁ1
4
81.0
85.7
99.1
98.0
4
44
6q
6r
H
H
H
H
23
35
65
84
81.0
83.2
97.7
97.3
6s
6t
H
H
H
41
7
84
49
82.6
86.8
96.6
98.5
CH3
10a
10b
H
H
H
H
ꢁ10
ꢁ2
ꢁ7
92.1
ND
98.2
ND
ND
(continued on next page)
7

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Table 1 (continued)
Cops.
R1
H
R2
R3
H
HDAC1 inhibitiona (%)
ALKwt inhibition (%)
@10 nM
@10 nM
4
@100 nM
17
@100 nM
98.5
10c
87.6
10d
10e
H
H
H
H
ꢁ8
20
90
84.7
86.5
99.0
98.8
52
10f
H
H
H
54
38
87
83
80.8
87.0
96.6
98.1
10g
H
10h
H
H
10
55
8
41
87.2
98.0
10i
10j
20
H
H
H
H
86
39
3
89.4
76.9
71.6
98.0
98.5
101.1
CH3
H
0
Crizotinib
SAHA
2.6 nM
12 nM
a
The values indicate the kinase inhibition percentages at 10 nM and 100 nM, which are the average values from two independent experiments.
Not determined.
b
the results of the AO/EB staining. As shown in Fig. 5B, the uneven
apoptosis. As shown in Fig. 6A, both cell lines displayed an
increasing dose-dependent apoptosis rate after 24 h of incubation
with compound 10f. In MDA-MB-231 cells, the apoptosis rate was
blue fluorescence was gradually enhanced accompanied with the
escalation of the compound concentration, and the apoptosis pro-
moting effect of compound 10f was stronger than that of Ceritinib
increased from 5.92% (control) to 19.27% (10f, 4.0
apoptosis rate in SH-SY5Y cells that were treated with compound
10f was 22.26% at a concentration of 1.0 M, which was comparable
to that of Ceritinib at 1.0 M (Fig. 6C).
mM). Likewise, the
at 4.0 mM.
Flow cytometry was conducted to further determine whether
the anti-proliferation effect that was induced by compound 10f was
related to apoptosis in MDA-MB-231 and SH-SY5Y cells. Both cell
lines were incubated with compound 10f at three concentrations
m
m
Next, Annexin V-FITC/PI was conducted to determine compound
10f induced cell cycle arrest in MDA-MB-231 and SH-SY5Y cells.
Then, the DNA content was analyzed to identify the effect of
compound 10f on the cell cycle (Fig. 6B). In MDA-MB-231 cells, the
percentage of cells in the G2 phase when treated with compound
10f increased significantly from 13.23% to 48.70%, 75.94% and
(1.0
(1.0
m
M, 2.0
mM, and 4.0 mM) and with positive control Ceritinib
mM) for 24 h. Annexin V-FITC and PI staining methods were
used for apoptosis analysis, and the percentage of apoptotic cells
was defined as the sum of the percentages for early and late
8

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Table 2
Inhibitory activities of target compounds 16a-m against the ALKwt and HDAC1 kinases.
Cops.
R1
R2
R3
R4
R5
HDAC1 inhibitiona (%)
ALKwt inhibition (%)
@10 nM
@100 nM
@10 nM
@100 nM
16a
16b
16c
16d
16e
16f
16g
16h
16i
H
CH3
H
H
Cl
Br
H
CH3
H
H
H
CH3
H
H
H
OCH3
H
OCH3
OCH3
H
H
H
H
CH3
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
OCH3
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CF3
F
84
99
99
ND
97
90
88
97
ND
94
89
94
4.9
1.2
0.2
7.2
3.1
0.2
1.2
ꢁ0.7
3.1
4.9
66.3
36.9
27.8
4.9
30.1
21.2
25.0
20.0
4.9
91
ND b
86
67
67
83
ND
65
17.6
7.6
96.0
16j
16k
H
H
H
56
70
H
16l
16m
a
H
H
H
H
H
H
CH3
63
68
93
91
83.5
60.4
97.9
94.9
OCH3
The values indicate the kinase inhibition percentages at 10 nM and 100 nM, which are the average values from two independent experiments.
Not determined.
b
Table 3
88.07% in a dose-dependent manner. This trend was also observed
in SH-SY5Y cells. Compared with the untreated group, the per-
centage of cells in the G2 phase increased from 23.89% to 50.99%,
which was accompanied by a dramatic loss of G1-and S-phase
populations (Fig. 6D). According to these results, compound 10f
could induce cell death via cell apoptosis and G2 phase arrest.
Inhibitory activities of compounds 6f and 10f against ALK and HDAC kinasesa.
Compd.
IC50 (nM)
HDAC1 HDAC2 HDAC6 ALKwt ALKL1196M ALKG1202R
6f
10f
SAHA
Ceritinib
Crizotinib
12
7.9
12
ND
ND
15
9.3
12
ND
ND
ND
6.7
4.4
10
ND
ND
ND
4.3
2.4
1.7
ND
0.25
16
1.0
0.4
ND
3.3
12
2.1
NDb
<1.0
2.6
2.2.6. Cellular effects of compound 10f on HDAC- and ALK-mediated
signaling pathways
A Western blot analysis was conducted to further evaluate the
intracellular effects of compound 10f on the HDAC- and ALK-
Repotrectinib ND
ND
ND
4.5
a
The reported data are the mean values from two independent experiments.
Not determined.
b
Table 4
Anti-proliferative activities of compounds 6f and 10f against cancer cell lines a
.
Compd.
Cell lines/IC50
HepG2
(mM)
MDA-MB-231
A549
SK-N-BE2 (ALK wt)
SH-SY5Y(F1174L)
H2228 (EML4-ALK)
6f
10f
SAHA
Ceritinib
Crizotinib
0.91 0.19
0.64 0.07
1.43 0.17
4.08 0.27
0.32 0.06
0.39 0.02
0.46 0.06
0.61 0.09
1.82 0.63
0.79 0.06
3.42 0.21
0.46 0.08
0.59 0.02
2.16 0.10
0.41 0.12
0.22 0.01
0.05 0.01
0.77 0.11
2.66 0.20
0.78 0.03
0.34 0.01
0.11 0.02
0.73 0.04
0.37 0.10
0.32 0.01
0.18 0.11
0.13 0.05
NDb
1.98 0.13
2.62 0.21
a
The reported data are the mean values from three independent experiments.
Not determined.
b
9

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Fig. 3. Effects of compound 10f on the antiproliferative activities of MDA-MB-231 and SH-SY5Y cells according to colony formation experiments. SH-SY5Y and MDA-MB-231 cells
(400/well) were cultured with concentrations of compound 10f of 0.1, 0.2 and 0.4 M for 3 days. Then, the cells were imaged after an additional 12 days of drug-free medium
m
incubation. A bar chart representation of the colonies that formed after treatment with compound 10f is presented. The data are presented as the mean SD of three independent
experiments. Colony counts are presented in the bar chart. The data are presented as the mean SD (n ¼ 3), with **p＜0.01 and ***p＜0.001 compared with the control.
Fig. 4. Representative images and quantitative analysis of a cell scratch assay for the MDA-MB-231 and SH-SY5Y cell lines. The healing rates are presented as the mean SD (n ¼ 3),
where **p＜0.01 and ***p＜0.001, compared with the control.
Fig. 5. MDA-MB-231 cells were treated with compound 10f (1.0, 2.0, 4.0
mM) or Ceritinib (4.0
mM) for 24 h, then stained with AO/EB (A) or Hoechst 33258 (B) and photographed.
10

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Fig. 6. (A) Flow cytometry was used to identify the impact of compound 10f on cell apoptosis in the MDA-MB-231 and SH-SY5Y cell lines. Both cell lines were treated with
compound 10f and Ceritinib for 24 h and stained with an FITC Annexin V/PI kit. Cells in the upper-right quadrant correspond to PI-positive/Annexin V-positive, late apoptotic or
necrotic cells, and cells in the lower-right quadrant correspond to early apoptotic cells. (B) Both cell lines were treated with compound 10f at concentrations of 1.0, 2.0 and 4.0 mM
11

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
mediated signaling pathways. After exposure to compound 10f (at
1.0 mM, 2.0 mM, and 4.0 mM) for 24 h, increasing levels of intracel-
increased G1202R mutant activity of compound 10f over that of
Ceritinib (Fig. 10D).
lular acetylated histone 3 (Ac-H3), which is regarded as a marker of
HDAC 1/2/3 inhibition, were observed in the MDA-MB-231 and SH-
SY5Y cell lines as expected, although the efficacy was inferior to
that of SAHA (Fig. 7). Meanwhile, the selected compound was also
found to suppress the ALK-mediated signaling pathway in a dose-
dependent manner (Fig. 8). Accordingly, it was demonstrated that
the levels of phosphorylation of ALK and its key downstream pro-
teins, namely, p-AKT and p-ERK, were significantly blocked by
As depicted in Fig. 11A, the proposed binding mode of com-
pound 10f to HDAC2 was also similar to that of SAHA. For instance,
two coordination bonds were formed between the oxygen atoms
on the hydroxamic acid group and the zinc atom in the active site of
HDAC2, which may be the key contributors to the comparable po-
tency of compound 10f against HDAC2 with that of SAHA. It was
also found that the amide group in compound 10f participated in a
new hydrogen bond interaction with amino reside D104 (Fig. 11B).
compound 10f at a 4.0 mM concentration in both tested cell lines
with respect to the control group. Taken together, these findings
show that compound 10f acted as a dual inhibitor that could inhibit
both the HDAC 1/2/3- and ALK-mediated signaling pathways.
3. Conclusion
In summary, a series of 2,4-pyrimidinediamine derivatives that
target HDAC and ALK were designed and synthesized in this study.
Enzyme assays demonstrated that most of these new compounds
could simultaneously inhibit ALK and HDAC proteins, and repre-
sentative compound 10f exhibited potent inhibitory activity with
IC50 values of 2.1 nM and 7.9 nM against ALK and HDAC1, respec-
tively. Moreover, compound 10f also showed impressive enzyme
activity against the challenging Crizotinib-resistant ALKL1196M
(IC50 ¼ 1.7 nM) and Ceritinib-resistant ALKG1202R mutations
(IC50 ¼ 0.4 nM) and was approximately 10-fold more potent than
Crizotinib and Ceritinib. The following cytotoxicity experiments
showed that compound 10f possessed comparable antiproliferative
activity to Crizotinib and Ceritinib against ALK-addicted cell lines.
Preliminary mechanistic studies showed that the levels of phos-
phorylated ALK and Ac-H3 were significantly inhibited or increased
in both tested cell lines after treatment with compound 10f, as was
found for Ceritinib and SAHA. These results suggested that com-
pound 10f might be a promising lead compound for the treatment
of NSCLC, especially Ceritinib- or Crizotinib-resistant NSCLC.
2.2.7. Protein kinase selectivity and pharmacokinetic (PK) profiles
of compound 10f
Since the isopropoxy moiety in Ceritinib was responsible for the
kinase selectivity and metabolic liability [35], compound 10f was
further evaluated against a panel of 123 kinases to characterize its
protein kinase selectivity profile at 1
mM (Fig. 9 and Table S1).
Similar to Ceritinib, compound 10f exhibited satisfactory inhibition
of the ALK, IGF-1R, InsR and FLT kinases. However, compound 10f
also led to over 95% inhibition of the Aurora, CDK and FGFR kinases.
The kinase profiling indicated a relatively low kinase selectivity of
compound 10f and demonstrated the necessity of the isopropoxy
group.
Next, compound 10f was selected to investigate in vivo phar-
macokinetic characteristics. Compound 10f was administered
intravenously (iv) at 1 mg/kg or orally (po) at 10 mg/kg to Sprague-
Dawley (SD) rats. As presented in Table 5, the clearance rate (CL) of
compound 10f was 88.6 mL/min/kg, hence, the compound was
rapidly cleared with a short terminal half-life of 0.42 h. The oral
bioavailability was also measurable, however, no results were
detected, which was likely due to the low permeability or aqueous
solubility of 10f [36]. These results suggested that 10f has poor PK
properties, and further optimization of 10f is necessary prior to
in vivo studies.
4. Experimental
4.1. Chemistry
All reagents were obtained from Adamas, tansoole and Macklin
and were used without further purification unless otherwise noted.
The melting point of the sample was determined by X-4 micro-
scopic melting point instrument. Routine 1H NMR and 13C HNMR
spectra were noted from solution in deuterated chloroform (CDCl3)
or dimethyl sulfoxide (DMSO‑d6) with tetramethylsilane (TMS) as
internal standard using a Bruker-600 MHz spectrometer. Chemical
shifts are reported in ppm and coupling constants (J) in Hz. HRMS
spectra were acquired with an Aglient 6470/AB Sciex 4600 Triple
Quad LC-MS apparatus. Column chromatography was performed
on silica gel (200e300 mesh).
2.3. Molecular docking studies
To better explain the interaction between compound 10f and
ALK or HDAC, molecular docking studies of compound 10f in ALKwt
(PDB ID: 4MKC) and HDAC2 (PDB ID: 4LXZ) were conducted using
Softwire Discovery Studio 2018. As shown in Fig. 10A, compound
10f could occupy the ATP binding site of ALK similarly to Ceritinib.
The 2-aminopyrimidine fragment could interact with M1199 via a
hydrogen bond in the hinge region, which are critical for binding to
ALK. In addition, the oxygen of the sulfonyl group was also found to
form two hydrogen bonds with the amino group of the K1150
amino residue. Moreover, the “tail” of compound 10f, which was
oriented toward the solvent-exposed area, formed an additional
carbon hydrogen bond with R1120, which might enhance the af-
finity of 10f with ALKwt (Fig. 10B).
4.1.1. General procedure for the synthesis of intermediates 3a-h
2,5-Dichloro-N-(2-(isopropylsulfonyl)phenyl)pyrimidin-4-
amine (1.02 g, 2.95 mmol, 1) and methyl 4-aminobenzoate (0.48 g,
3.17 mmol, 2a) was dissolved in 20 mL isopropanol and 0.1 mL HCl
solution (37%) was added. The resulting mixture was then stirred at
90 ꢀC for 6 h. After reaction completion, the reaction mixture was
extracted with 100 mL EtOAc, then the organic layer was washed
with saturated NaCl aqueous solution twice, dried overnight with
anhydrous Na2SO4 and filtered. The resulting filtrate was concen-
trated under vacuum and can be directly used without further
purification.
In contrast to the ALKwt model, detrimental bump interactions
were induced between the piperidine ring of Ceritinib and the
R1202 side chain in the G1202R mutant model, as illustrated in
Fig. 10C. In comparison, the aliphatic chain of compound 10f un-
dergoes less steric collision with R1202, as we expected. Interest-
ingly, the hydroxamic acid group were engaged in three additional
hydrogen bonds with R1202 and G1121, which explains the
for 24 h. Then, the cells were fixed in ice-cold 70% ethanol at 4 ꢀC and stained with propidium iodide dye, and the cell cycle was evaluated via flow cytometry. The bar graph shows
the percentages of cells in apoptosis (C) or the G1, S and G2 phases (D).
12

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Fig. 7. Inhibitory effects of compound 10f on Ac-H3 in the MDA-MB-231 (A) and SH-SY5Y (B) cell lines according to Western blot analysis.
Fig. 8. Western blot analysis of ALK and its downstream pathway after treatment with compound 10f in the MDA-MB-231 (A) and SH-SY5Y (B) cell lines.
Fig. 9. (A). Distribution of kinases inhibited by compound 10f within the human kinome. The color for inhibition is indicated. Enzymatic activities were determined in single-dose
duplicates at concentrations of 1
mM. Staurosporine served as a positive control. The experiment was performed by ICE bioscience (Beijing, China). (B). Inhibition rate of compound
10f against some representative kinases.
13

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
Table 5
PK properties of compound 10f in SD rats (N ¼ 3).
Comp.
Dose (mg/kg)
1
Route
iv.
AUC0-∞ (h$ng/mL)
Cmax (ng/mL)
1480.0
CL (mL/min/kg)
88.6
MRT0-∞ (h)
0.13
Vss (L/kg)
3.3
t1/2 (h)
0.42
10f
205.1
Fig. 10. (A). The overlap of the proposed binding modes of compound 10f (Grey) and Ceritinib (Purple) in ALKwt (PDB ID: 4MKC). (B). The predicted binding mode for compound 10f
in the binding site cavity of ALKwt (C). The predicted binding modes of both Ceritinib and compound 10f in the binding site cavity of ALKG1202R. (D). The predicted binding modes of
compound 10f in the binding site cavity of ALKG1202R. Green dashed lines indicate hydrogen bonds.
Fig. 11. (A). The overlap of the proposed binding modes of compound 10f (Grey) and SAHA (Purple) in HDAC2 (PDB ID: 4LXZ). (B). The predicted binding mode of compound 10f in
the binding site cavity of HDAC2. Green dashed lines indicate hydrogen bonds, and grey dashed lines indicate metal interactions.
4.1.2. General procedure for the synthesis of intermediates 4a-h
235e237 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 12.87 (s, 1H), 9.74 (s,
1H), 9.52 (s, 1H), 8.61 (s, 1H), 8.34 (s, 1H), 8.19 (s, 1H), 7.89 (dd,
J ¼ 4.8, 3.0 Hz, 1H), 7.85 (dd, J ¼ 8.0, 1.5 Hz, 1H), 7.72 (t, J ¼ 7.4 Hz,
1H), 7.54 (d, J ¼ 7.7 Hz, 1H), 7.37 (q, J ¼ 7.5 Hz, 2H), 3.47e3.43 (m,
1H), 1.17 (d, J ¼ 6.7 Hz, 6H). MS (ESI) m/z: [MþH]þ: 447.1.
4.1.2.1. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)benzoic acid (4a). 3a (1.80 g, 3.61 mmol) was
added into 15 mL 35% NaOH aqueous solution and 5 mL EtOH. The
mixture was then stirred at 50 ꢀC for 3 h and the solution finally
became clear. The pH of the reaction mixture was adjusted to 5e6
with 2 N HCl, the precipitate was collected by filtration, washed
with water and dried under vacuum to afford 1.60 g of intermediate
4a as a white solid. Yield: 91.6%. M.p. 272e274 ꢀC. 1H NMR
4.1.2.3. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-2-methylbenzoic acid (4c). White solid. Yield:
93.5%. M.p. > 250 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 12.39 (s, 1H),
(600 MHz, DMSO‑d6)
d
12.55 (s, 1H), 9.94 (s, 1H), 9.48 (s, 1H), 8.52
9.82 (s,1H), 9.45 (s,1H), 8.52 (d, J ¼ 7.2 Hz,1H), 8.35 (s,1H), 7.88 (dd,
J ¼ 8.0, 1.4 Hz, 1H), 7.81e7.73 (m, 2H), 7.59e7.49 (m, 2H), 7.47e7.40
(m, 1H), 3.51e3.41 (m, 1H), 2.42 (s, 3H), 1.16 (d, J ¼ 6.8 Hz, 6H). MS
(ESI) m/z: [MþH]þ: 461.1.
(d, J ¼ 7.5 Hz,1H), 8.37 (s,1H), 7.89 (dd, J ¼ 7.9,1.4 Hz,1H), 7.85e7.81
(m, 1H), 7.79 (d, J ¼ 8.8 Hz, 2H), 7.74 (d, J ¼ 8.7 Hz, 2H), 7.50e7.43
(m, 1H), 3.51e3.40 (m, 1H), 1.16 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z:
[MþH]þ: 447.1.
4.1.2.4. 2-Chloro-4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
4.1.2.2. 3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)benzoic acid (4b). White solid. Yield: 62.5%. M.p.
amino)pyrimidin-2-yl)amino)benzoic acid (4d). White solid. Yield:
97.7%. M.p. 98e100 ꢀC.1H NMR (600 MHz, DMSO‑d6)
d 12.89 (s,1H),
14

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
10.03 (s, 1H), 9.47 (s, 1H), 8.47 (d, J ¼ 7.5 Hz,1H), 8.39 (s, 1H), 7.94 (d,
J ¼ 1.4 Hz, 1H), 7.88 (dd, J ¼ 8.0, 1.5 Hz, 1H), 7.84e7.80 (m, 1H), 7.75
(d, J ¼ 8.7 Hz, 1H), 7.57 (dd, J ¼ 8.6, 1.5 Hz, 1H), 7.47e7.42 (m, 1H),
3.50e3.42 (m, 1H), 1.16 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ:
481.0.
(d, J ¼ 8.8 Hz, 2H), 7.69 (d, J ¼ 8.8 Hz, 2H), 7.44 (t, J ¼ 7.6 Hz,1H), 3.58
(s, 3H), 3.51e3.41 (m,1H), 3.28e3.23 (m, 2H), 2.36 (t, J ¼ 7.4 Hz, 2H),
1.77 (p, J ¼ 7.2 Hz, 2H), 1.16 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z:
[MþH]þ: 546.2.
4.1.3.3. Methyl
amino)pyrimidin-2-yl)amino)benzamido)pentanoate
White solid. Yield: 68.4%. M.p. 161e164 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
1H), 8.28 (t, J ¼ 5.6 Hz, 1H), 7.88 (dd, J ¼ 7.9, 1.2 Hz, 1H), 7.85e7.79
(m, 1H), 7.73 (d, J ¼ 8.8 Hz, 2H), 7.70 (d, J ¼ 8.7 Hz, 2H), 7.44 (t,
J ¼ 7.6 Hz, 1H), 3.58 (s, 3H), 3.50e3.41 (m, 1H), 3.27e3.22 (m, 2H),
2.34 (t, J ¼ 7.2 Hz, 2H), 1.60e1.48 (m, 4H), 1.17 (d, J ¼ 6.8 Hz, 6H). MS
(ESI) m/z: [MþH]þ: 560.2.
5-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
4.1.2.5. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
(5d).
imidin-2-yl)amino)-2-methoxybenzoic acid (4e). White solid. Yield:
97.7%. M.p. 214e215 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 12.11 (s,
d
9.81 (s, 1H), 9.49 (s, 1H), 8.57 (d, J ¼ 6.7 Hz, 1H), 8.35 (s,
1H), 9.86 (s, 1H), 9.48 (s, 1H), 8.54 (d, J ¼ 7.3 Hz, 1H), 8.39 (s, 1H),
7.88 (dd, J ¼ 8.0, 1.5 Hz, 1H), 7.82e7.76 (m, 1H), 7.62 (d, J ¼ 8.6 Hz,
1H), 7.46e7.40 (m, 2H), 7.35 (d, J ¼ 8.4 Hz, 1H), 3.63 (s, 3H),
3.52e3.45 (m, 1H), 1.17 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ:
477.1.
4.1.2.6. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
4.1.3.4. Methyl
amino)pyrimidin-2-yl)amino)benzamido)hexanoate
solid. Yield: 56.2%. M.p. 144e147 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
6-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
imidin-2-yl)amino)-3-methoxybenzoic acid (4f). White solid. Yield:
(5e). White
95.3%. M.p. 88e90 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 10.05 (s, 1H),
9.17 (s, 1H), 8.50 (s, 1H), 8.19 (d, J ¼ 7.9 Hz, 1H), 7.93 (dd, J ¼ 7.9,
1.4 Hz, 1H), 7.85e7.75 (m, 2H), 7.57 (t, J ¼ 7.6 Hz, 1H), 7.49 (d,
J ¼ 1.7 Hz, 1H), 7.32 (dd, J ¼ 8.4, 1.3 Hz, 1H), 3.88 (s, 3H), 3.52e3.39
(m, 1H), 1.12 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ: 477.1.
d
9.71 (s, 1H), 9.57 (s, 1H), 8.66 (s, 1H), 8.42 (t, J ¼ 5.6 Hz, 1H), 8.33 (s,
1H), 8.03 (s, 1H), 7.84 (dd, J ¼ 7.9, 1.3 Hz, 1H), 7.76 (d, J ¼ 6.7 Hz, 1H),
7.70e7.64 (m,1H), 7.42 (d, J ¼ 7.7 Hz,1H), 7.39e7.29 (m, 2H), 3.58 (s,
3H), 3.50e3.41 (m, 1H), 3.27e3.21 (m, 2H), 2.36 (t, J ¼ 7.4 Hz, 2H),
1.75 (q, J ¼ 7.2 Hz, 2H), 1.38e1.30 (m, 2H), 1.28e1.23 (m, 2H), 1.17 (d,
J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ: 574.2.
4.1.2.7. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-3-methylbenzoic acid (4g). White solid. Yield:
95.3%. M.p. 234e237 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 12.70 (s,
4.1.3.5. Methyl
amino)pyrimidin-2-yl)amino)benzamido)heptanoate
White solid. Yield: 70.0%. M.p. 152e154 ꢀC. 1H NMR (600 MHz,
CDCl3)
7-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
1H), 9.59 (s, 1H), 9.02 (s, 1H), 8.53 (d, J ¼ 8.4 Hz, 1H), 8.28 (s, 1H),
7.83e7.78 (m, 2H), 7.75 (dd, J ¼ 8.3, 1.9 Hz, 1H), 7.68 (d, J ¼ 8.4 Hz,
1H), 7.57e7.52 (m, 1H), 7.36e7.30 (m, 1H), 3.51e3.40 (m, 1H), 2.28
(s, 3H), 1.17 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ: 461.1.
(5f).
d
9.66 (s, 1H), 8.51 (d, J ¼ 8.3 Hz, 1H), 8.17 (d, J ¼ 2.5 Hz, 1H),
7.93 (dd, J ¼ 9.0, 4.0 Hz, 1H), 7.73e7.71 (m, 2H), 7.66 (t, J ¼ 7.1 Hz,
1H), 7.62e7.60 (m, 2H), 7.5e7.45 (m, 1H), 7.30 (t, J ¼ 7.6 Hz, 1H),
6.21e6.12 (m, 1H), 3.68 (s, 3H), 3.50e3.36 (m, 2H), 3.24e3.22 (m,
1H), 2.33e2.31 (m, 2H), 1.68e1.58 (m, 4H), 1.43e1.39 (m, 4H), 1.31
(d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ: 588.2.
4.1.2.8. 3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-2-methylbenzoic acid (4h). White solid. Yield:
95.3%. M.p. 230e233 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 12.91 (s,
1H), 9.59 (s, 1H), 9.12 (s, 1H), 8.45 (s, 1H), 8.22 (s, 1H), 7.77 (d,
J ¼ 7.8 Hz, 1H), 7.62 (d, J ¼ 7.6 Hz, 1H), 7.51 (d, J ¼ 7.7 Hz, 1H), 7.46 (s,
1H), 7.30 (t, J ¼ 7.7 Hz,1H), 7.25 (t, J ¼ 7.5 Hz,1H), 3.47e3.40 (m, 1H),
2.34 (s, 3H), 1.16 (d, J ¼ 6.7 Hz, 6H). MS (ESI) m/z: [MþH]þ: 461.1.
4.1.3.6. Methyl
6-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)-2-methylbenzamido)hexanoate (5g).
White solid. Yield: 64.9%. M.p. 130e132 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
9.72 (s, 1H), 8.53 (d, J ¼ 8.3 Hz, 1H), 8.15 (s, 1H), 7.94 (dd,
4.1.3. General procedure for the synthesis of intermediates 5b-t
J ¼ 7.9,1.3 Hz,1H), 7.71e7.64 (m,1H), 7.58 (s, 1H), 7.40e7.38 (m, 2H),
7.34e7.29 (m, 2H), 5.88 (s, 1H), 3.68 (s, 3H), 3.48e3.41 (m, 2H),
3.29e3.20 (m, 1H), 2.42 (s, 3H), 2.36 (t, J ¼ 7.4 Hz, 2H),1.70e1.62 (m,
4H), 1.49e1.40 (m, 2H), 1.33 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z:
[MþH]þ: 588.2.
4.1.3.1. Methyl
3-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)benzamido)propanoate (5b). To
a
100 mL round bottle was charged with 4a (0.80 g,1.80 mmol), HOBT
(0.27 g, 2.04 mmol), EDCI (0.40 g, 2.02 mmol), DIPEA (0.50 g,
3.83 mmol) and 20 mL DCM. The resulting solution was warmed to
30 ꢀC, stirred at this temperature for 0.5 h, and methyl 3-
aminopropionate hydrochloride (0.29 g, 2.07 mmol) was added
and stirred for another 12 h. Then the reaction mixture was added
into 40 mL water and extracted by DCM. The combined organic
layer was successively washed with saturated NaCl twice, dried
overnight with anhydrous Na2SO4, and filtered under reduced
pressure. The resulting filtrate was concentrated under vacuum and
purified by column chromatography on silica gel with DCM and
methanol (30:1) to afford 0.60g of 5b as a white crystal. Yield: 57.1%.
4.1.3.7. Methyl
7-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)-2-methylbenzamide) heptanoate (5h).
White solid. Yield: 47.0%. M.p. 78e80 ꢀC. 1H NMR (600 MHz, CDCl3)
d
9.68 (s, 1H), 8.51 (d, J ¼ 8.2 Hz, 1H), 8.15 (s, 1H), 7.93 (dd, J ¼ 8.0,
1.5 Hz,1H), 7.69e7.61 (m,1H), 7.39e7.37 (m, 3H), 7.33e7.27 (m, 2H),
5.76 (t, J ¼ 5.6 Hz,1H), 3.67 (s, 3H), 3.45e3.40 (m, 2H), 3.31e3.19 (m,
1H), 2.41 (s, 3H), 2.32 (t, J ¼ 7.5 Hz, 2H), 1.69e1.58 (m, 4H),
1.44e1.36 (m, 4H), 1.32 (d, J ¼ 6.9 Hz, 6H). MS (ESI) m/z: [MþH]þ:
602.2.
M.p. 148e150 ꢀC. 1H NMR (600 MHz, CDCl3)
d 9.63 (s, 1H), 8.50 (d,
J ¼ 8.3 Hz,1H), 8.18 (s, 1H), 7.93 (d, J ¼ 7.9 Hz,1H), 7.72 (d, J ¼ 8.2 Hz,
2H), 7.67 (t, J ¼ 7.7 Hz, 1H), 7.61 (d, J ¼ 8.1 Hz, 2H), 7.35e7.28 (m,
2H), 6.81 (s, 1H), 3.77e3.66 (m, 5H), 3.28e3.19 (m, 1H), 2.67 (t,
J ¼ 5.6 Hz, 2H), 1.32 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ: 532.1.
4.1.3.8. Methyl
phenyl)amino)pyrimidin-2-yl)amino)benzamido)hexanoate
White solid. Yield: 42.6%. M.p. 135e136 ꢀC. 1H NMR (600 MHz,
CDCl3)
J ¼ 8.0, 1.5 Hz, 1H), 7.84 (d, J ¼ 2.1 Hz, 1H), 7.75e7.67 (m, 2H), 7.50 (s,
1H), 7.35e7.29 (m, 2H), 6.42 (t, J ¼ 5.6 Hz, 1H), 3.67 (s, 3H),
3.49e3.45 (m, 2H), 3.27e3.19 (m, 1H), 2.34 (t, J ¼ 7.4 Hz, 2H),
1.74e1.62 (m, 4H), 1.48e1.40 (m, 2H), 1.31 (d, J ¼ 6.9 Hz, 6H). MS
(ESI) m/z: [MþH]þ: 608.1.
6-(2-chloro-4-((5-chloro-4-((2-(isopropylsulfonyl)
(5i).
d
9.68 (s, 1H), 8.45 (d, J ¼ 8.2 Hz, 1H), 8.18 (s, 1H), 7.94 (dd,
4.1.3.2. Methyl
amino)pyrimidin-2-yl)amino)benzamido)butanoate
solid. Yield: 71.2%. M.p. 190e192 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
9.81 (s,1H), 9.48 (s,1H), 8.56 (d, J ¼ 7.3 Hz,1H), 8.35 (s,1H), 8.30 (t,
J ¼ 5.6 Hz, 1H), 7.88 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.84e7.78 (m, 1H), 7.72
4-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
(5c). White
d
15

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
4.1.3.9. Methyl
7-(2-chloro-4-((5-chloro-4-((2-(isopropylsulfonyl)
1H), 7.36 (t, J ¼ 7.8 Hz, 1H), 7.24 (d, J ¼ 7.5 Hz, 1H), 7.19 (s, 1H), 6.31
(s, 1H), 3.68 (s, 3H), 3.44 (q, J ¼ 6.4 Hz, 2H), 3.28e3.20 (m, 1H), 2.38
(t, J ¼ 7.0 Hz, 2H), 1.71 (d, J ¼ 6.1 Hz, 3H), 1.66e1.61 (m, 1H), 1.32 (d,
J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ: 560.2.
phenyl)amino)pyrimidin-2-yl)amino)benzamido)heptanoate
(5j).
White solid. Yield: 37.9%. M.p. 130e132 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) d 9.86 (s, 1H), 9.47 (s, 1H), 8.50 (s, 1H), 8.41e8.32 (m, 1H),
8.23 (t, J ¼ 5.6 Hz, 1H), 7.88 (dd, J ¼ 7.8, 1.4 Hz, 2H), 7.85e7.79 (m,
1H), 7.51 (d, J ¼ 7.9 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz, 1H), 7.28 (d, J ¼ 8.4 Hz,
1H), 3.58 (s, 3H), 3.50e3.42 (m, 1H), 3.21e3.14 (m, 2H), 2.31 (t,
J ¼ 7.4 Hz, 2H), 1.58e1.51 (m, 2H), 1.51e1.44 (m, 2H), 1.36e1.24 (m,
4H), 1.17 (t, J ¼ 8.3 Hz, 6H). MS (ESI) m/z: [MþH]þ: 622.2.
4.1.3.16. Methyl
amino)pyrimidin-2-yl)amino)benzamido)hexanoate
solid. Yield: 52.2%. M.p. 108e110 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
6-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
(5r). White
d
9.67 (s, 1H), 9.56 (s, 1H), 8.64 (s, 1H), 8.32 (d, J ¼ 7.4 Hz, 2H), 8.01
(s, 1H), 7.83 (d, J ¼ 7.6 Hz, 1H), 7.75 (d, J ¼ 6.9 Hz, 1H), 7.67 (d,
J ¼ 7.9 Hz, 1H), 7.41 (d, J ¼ 7.4 Hz, 1H), 7.36e7.29 (m, 2H), 3.57 (s,
3H), 3.44 (dd, J ¼ 7.5, 5.0 Hz, 1H), 3.22e3.18 (m, 2H), 2.29 (t,
J ¼ 7.4 Hz, 2H), 1.59e1.42 (m, 4H), 1.34e1.23 (m, 2H), 1.16 (d,
J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ: 574.2.
4.1.3.10. Methyl
6-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)-2-methoxybenzamido)hexanoate (5k).
White solid. Yield: 39.6%. M.p. 150e152.0 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
9.67 (s, 1H), 8.51 (d, J ¼ 8.3 Hz, 1H), 8.19 (d, J ¼ 2.3 Hz, 1H),
8.13 (d, J ¼ 8.6, 2.8 Hz, 1H), 7.93 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.80 (t,
J ¼ 5.5 Hz, 1H), 7.69e7.63 (m, 1H), 7.49e7.40 (m, 1H), 7.37 (s, 1H),
7.32e7.27 (m, 1H), 7.18 (d, J ¼ 8.6, 1.4 Hz, 1H), 3.85 (s, 3H), 3.67 (s,
3H), 3.51e3.42 (m, 2H), 3.29e3.22 (m, 1H), 2.39e2.28 (m, 2H),
1.74e1.59 (m, 4H), 1.50e1.42 (m, 2H), 1.31 (d, J ¼ 6.8 Hz, 6H). MS
(ESI) m/z: [MþH]þ: 604.2.
4.1.3.17. Methyl
amino)pyrimidin-2-yl)amino)benzamido)heptanoate
White solid. Yield: 50.0%. M.p. 128e129 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 9.70 (s, 1H), 9.56 (s, 1H), 8.65 (s, 1H), 8.39e8.29 (m, 2H),
8.01 (s, 1H), 7.83 (dd, J ¼ 8.0, 1.3 Hz, 1H), 7.74 (d, J ¼ 6.8 Hz, 1H), 7.67
(t, J ¼ 7.5 Hz, 1H), 7.41 (d, J ¼ 7.7 Hz, 1H), 7.36e7.30 (m, 2H), 3.57 (s,
3H), 3.48e3.41 (m, 1H), 3.22e3.17 (m, 2H), 2.28 (t, J ¼ 7.4 Hz, 2H),
1.56e1.42 (m, 4H), 1.30e1.20 (m, 4H), 1.16 (d, J ¼ 6.8 Hz, 6H). MS
(ESI) m/z: [MþH]þ: 588.2.
7-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
(5s).
d
4.1.3.11. Methyl
6-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)-3-methoxybenzamido)hexanoate (5l).
White solid. Yield: 38.6%. M.p. 118e120 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
9.60 (s, 1H), 8.51 (t, J ¼ 8.6 Hz, 1H), 8.38e8.30 (m, 1H), 8.18
(d, J ¼ 7.0 Hz, 1H), 7.94 (dd, J ¼ 8.1, 1.9 Hz, 1H), 7.89e7.78 (m, 1H),
7.73e7.66 (m, 1H), 7.44 (s, 1H), 7.36e7.27 (m, 1H), 7.21 (dd, J ¼ 8.4,
1.7 Hz, 1H), 6.18 (s, 1H), 3.93 (s, 3H), 3.69 (s, 3H), 3.52e3.41 (m, 2H),
3.30e3.21 (m, 1H), 2.43e2.31 (m, 2H), 1.71e1.63 (m, 4H), 1.50e1.39
(m, 2H), 1.32 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ: 604.2.
4.1.3.18. Methyl
6-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)-2-methylbenzamido)hexanoate (5t).
White solid. Yield: 45.2%. M.p. 123e125 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
9.64 (s, 1H), 8.44 (d, J ¼ 8.4 Hz, 1H), 8.05 (s, 1H), 7.80 (d,
J ¼ 7.9 Hz, 1H), 7.73 (d, J ¼ 8.0 Hz, 1H), 7.45 (t, J ¼ 7.8 Hz, 1H),
7.20e7.11 (m, 2H), 7.09 (d, J ¼ 7.5 Hz, 1H), 6.73 (s, 1H), 5.76 (s, 1H),
3.59 (s, 3H), 3.45e3.32 (m, 2H), 3.20e3.12 (m, 1H), 2.32e2.23 (m,
5H), 1.63e1.53 (m, 4H), 1.38e1.33 (m, 2H), 1.24 (d, J ¼ 6.7 Hz, 6H).
MS (ESI) m/z: [MþH]þ: 588.2.
4.1.3.12. Methyl
6-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)-3-methylbenzamido)hexanoate (5m).
White solid. Yield: 45.2%. M.p. 99e102 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
9.73 (s, 1H), 8.49 (d, J ¼ 8.4 Hz, 1H), 8.15 (s, 1H), 8.06 (d,
J ¼ 8.4 Hz, 1H), 7.91 (d, J ¼ 7.9 Hz, 1H), 7.64 (s, 1H), 7.63e7.42 (m,
2H), 7.28 (s, 1H), 7.07 (s, 1H), 6.19 (s, 1H), 3.67 (s, 3H), 3.53e3.41 (m,
2H), 3.29e3.19 (m, 1H), 2.44e2.30 (m, 5H), 1.74e1.61 (m, 4H), 1.43
(t, J ¼ 7.7 Hz, 2H), 1.32 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ:
588.2.
4.1.4. General procedure for the synthesis of 6a-t
4.1.4.1. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-hydroxybenzamide (6a). A solution of potas-
sium hydroxide (5.6 g, 100 mmol) in methanol (14 mL) was added
to a stirred solution of hydroxylamine hydrochloride (4.67 g,
67.2 mmol) in methanol (24 mL) at 0 ꢀC. The reaction mixture was
stirred at this temperature for 1 h. The precipitate was removed by
filtration and the filtrate was collected to provide fresh hydroxyl-
amine solution which was stored in a refrigerator before use. 3a
(0.20g, 0.43 mmol) was weighed into 20 mL freshly prepared hy-
droxylamine methanol solution and stirred at room temperature
for 2 h. The pH value of reaction mixture was adjusted to 6e7 with
saturated NH4Cl solution. The white solid was precipitated, filtered
and washed with water and 0.10 g of 6a was obtained as white solid
by recrystallization of methanol. Yield: 52.1%. M.p. 175e176 ꢀC. 1H
4.1.3.13. Methyl
amino)pyrimidin-2-yl)amino)benzamido)propanoate
White solid. Yield: 63.7%. M.p. 142e144 ꢀC. 1H NMR (600 MHz,
CDCl3)
3-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
(5o).
d
9.75 (s, 1H), 8.54 (d, J ¼ 8.3 Hz, 1H), 8.16 (s, 1H), 7.91 (dd,
J ¼ 8.0, 1.5 Hz, 2H), 7.77e7.71 (m, 1H), 7.63e7.58 (m, 1H), 7.42 (d,
J ¼ 7.7 Hz, 2H), 7.37 (t, J ¼ 7.8 Hz, 1H), 7.28 (s, 1H), 6.82 (s, 1H), 3.71
(s, 3H), 3.27e3.20 (m,1H), 2.65 (t, J ¼ 5.9 Hz, 2H), 2.05e1.93 (m, 2H),
1.32 (d, J ¼ 6.9 Hz, 6H). MS (ESI) m/z: [MþH]þ:532.1.
4.1.3.14. Methyl
amino)pyrimidin-2-yl)amino)benzamido)butanoate
solid. Yield: 59.0%. M.p. 138e140 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
4-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
NMR (600 MHz, DMSO‑d6) d 9.84 (s, 1H), 8.94 (s, 1H), 8.54 (s, 1H),
(5p). White
8.36 (s, 1H), 7.96e7.78 (m, 2H), 7.69e7.65 (m, 4H), 7.45 (s, 1H), 3.47
(s, 1H), 1.16 (d, J ¼ 5.6 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d
9.68 (s, 1H), 9.57 (s, 1H), 8.66 (d, J ¼ 6.2 Hz, 1H), 8.39 (t, J ¼ 5.6 Hz,
d 164.47, 157.71, 155.60, 143.24, 138.31, 135.40, 131.46, 127.85,
1H), 8.32 (s, 1H), 8.03 (s, 1H), 7.83 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.76 (d,
J ¼ 7.4 Hz, 1H), 7.67 (t, J ¼ 7.7 Hz, 1H), 7.43 (d, J ¼ 7.7 Hz, 1H),
7.36e7.30 (m, 2H), 3.58 (s, 3H), 3.49e3.40 (m, 1H), 3.27e3.23 (m,
2H), 2.35 (t, J ¼ 7.4 Hz, 2H), 1.76 (p, J ¼ 7.2 Hz, 2H), 1.17 (d, J ¼ 6.8 Hz,
6H). MS (ESI) m/z: [MþH]þ: 546.2.
125.86, 125.72, 125.15, 124.60, 118.61, 105.93, 55.25, 15.32. HRMS m/
z calcd for C20H21ClN5O4S [MþH]þ 462.0997, found: 462.1001.
4.1.4.2. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(3-(hydroxyamino)-3-oxopropyl)benzamide
(6b). White solid. Yield: 40.4%. M.p. 156e158 ꢀC. 1H NMR
4.1.3.15. Methyl
amino)pyrimidin-2-yl)amino)benzamido)pentanoate
White solid. Yield: 63.4%. M.p. 58e60 ꢀC. 1H NMR (600 MHz, CDCl3)
9.67 (s, 1H), 8.56 (d, J ¼ 8.3 Hz, 1H), 8.17 (s, 1H), 7.91 (d, J ¼ 9.3 Hz,
2H), 7.74 (d, J ¼ 7.8 Hz, 1H), 7.60 (t, J ¼ 7.7 Hz, 1H), 7.42 (d, J ¼ 7.6 Hz,
5-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
(600 MHz, DMSO‑d6) d 10.43 (s,1H), 9.83 (s,1H), 8.83 (s,1H), 8.54 (s,
(5q).
1H), 8.39 (s, 1H), 8.35 (s, 1H), 7.89 (d, J ¼ 7.8 Hz, 1H), 7.82 (d,
J ¼ 7.3 Hz, 1H), 7.80e7.71 (m, 4H), 7.65 (d, J ¼ 8.3 Hz, 1H), 7.45 (t,
J ¼ 7.2 Hz, 1H), 3.49 (s, 1H), 3.47e3.42 (m, 2H), 2.26 (t, J ¼ 7.0 Hz,
d
2H), 1.16 (d, J ¼ 6.6 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 167.81,
16

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
166.24, 157.72, 155.60, 143.28, 135.36, 131.42, 128.21, 127.84, 127.59,
125.67, 125.02, 124.49, 118.61, 118.40, 106.02, 55.23, 36.45, 33.04,
15.32. HRMS m/z calcd for C23H26ClN6O5S [MþH]þ 533.1368, found:
533.1368.
(dd, J ¼ 7.9, 1.4 Hz, 1H), 7.82e7.75 (m, 1H), 7.50 (s, 1H), 7.47e7.38 (m,
2H), 7.21 (d, J ¼ 8.3 Hz, 1H), 3.49e3.41 (m, 1H), 3.22e3.15 (m, 2H),
2.25 (s, 3H), 1.96 (t, J ¼ 7.4 Hz, 2H), 1.60e1.44 (m, 4H), 1.32e1.26 (m,
2H), 1.17 (d, J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 169.52,
169.15, 157.89, 155.71, 155.45, 141.29, 138.38, 136.44, 135.49, 131.44,
130.98, 128.13, 125.36, 124.67, 124.41, 120.97, 116.32, 105.56, 55.28,
39.22, 32.73, 29.39, 26.59, 25.38, 20.52, 15.33. HRMS m/z calcd for
4.1.4.3. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(4-(hydroxyamino)-4-oxobutyl)benzamide
(6c). White solid. Yield: 40.0%. M.p. 177e178 ꢀC. 1H NMR (600 MHz,
C
27H34ClN6O5S [MþH]þ 589.1994, found: 589.1991.
DMSO‑d6)
d 10.39 (s, 1H), 9.81 (s, 1H), 9.46 (s, 1H), 8.70 (s, 1H), 8.56
(d, J ¼ 7.0 Hz,1H), 8.36 (d, J ¼ 6.7 Hz,1H), 8.32 (t, J ¼ 5.4 Hz,1H), 7.88
(dd, J ¼ 7.9, 1.3 Hz, 1H), 7.84e7.80 (m, 1H), 7.73 (d, J ¼ 8.7 Hz, 2H),
7.69 (d, J ¼ 8.7 Hz, 2H), 7.44 (t, J ¼ 7.5 Hz, 1H), 3.50e3.42 (m, 1H),
3.27e3.18 (m, 2H), 2.01 (t, J ¼ 7.5 Hz, 2H), 1.78e1.70 (m, 2H), 1.16 (d,
4.1.4.8. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)-2-
methylbenzamide (6h). White solid. Yield: 66.6%. M.p. 146e147 ꢀC.
1H NMR (600 MHz, DMSO‑d6)
d 10.36 (s, 1H), 9.66 (s, 1H), 9.47 (s,
J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d
168.94, 165.75,
1H), 8.68 (s,1H), 8.56 (s,1H), 8.33 (s,1H), 8.08 (t, J ¼ 5.5 Hz,1H), 7.88
(dd, J ¼ 7.9, 1.2 Hz, 1H), 7.81e7.77 (m, 1H), 7.50 (s, 1H), 7.47e7.39 (m,
2H), 7.21 (d, J ¼ 8.3 Hz, 1H), 3.52e3.44 (m, 1H), 3.21e3.15 (m, 2H),
2.25 (s, 3H), 1.95 (t, J ¼ 7.4 Hz, 2H), 1.59e1.43 (m, 4H), 1.36e1.24 (m,
157.25, 155.20, 155.10, 142.72, 137.84, 134.94, 130.99, 130.09, 127.33,
125.12, 124.52, 124.10, 117.96, 105.47, 54.81, 38.86, 30.06, 25.42,
14.85. HRMS m/z calcd for C24H28ClN6O5S [MþH]þ 547.1530, found:
547.1539.
4H), 1.17 (d, J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 169.59,
169.16, 157.89, 155.71, 155.45, 141.28, 138.38, 136.39, 135.48, 131.44,
131.02, 128.11, 125.37, 124.68, 124.40, 120.98, 116.34, 105.56, 55.29,
39.27, 32.73, 29.52, 28.82, 26.69, 25.62, 20.49, 15.33. HRMS m/z
calcd for C28H36ClN6O5S [MþH]þ 603.2156, found: 603.2169.
4.1.4.4. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(5-(hydroxyamino)-5-oxopentyl)benzamide
(6d). White solid. Yield: 60.6%. M.p.135e137 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
d 9.84 (s, 1H), 8.57 (s, 1H), 8.41e8.32 (m, 2H), 7.88 (d,
J ¼ 7.8 Hz, 1H), 7.84 (t, J ¼ 7.3 Hz, 2H), 7.75 (d, J ¼ 8.6 Hz, 2H),
7.73e7.55 (m, 4H), 7.45 (t, J ¼ 7.4 Hz,1H), 3.52e3.45 (m,1H), 3.22 (d,
J ¼ 5.8 Hz, 2H), 1.99 (t, J ¼ 6.5 Hz, 2H), 1.61e1.42 (m, 4H), 1.17 (d,
4.1.4.9. 2-Chloro-4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)-N-(6-(hydroxyamino)-6-oxohexyl)
benzamide (6i). White solid. Yield: 31.5%. M.p. 168e169 ꢀC. 1H NMR
J ¼ 6.7 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d169.47, 166.10,
(600 MHz, DMSO‑d6) d 10.36 (s, 1H), 9.88 (s,1H), 9.47 (s,1H), 8.68 (s,
157.72, 155.65, 155.53, 143.15, 138.30, 135.47, 131.43, 128.71, 128.21,
127.85, 124.55, 118.62, 118.42, 105.90, 55.36, 55.28, 32.49, 29.36,
23.27, 15.32. HRMS m/z calcd for C25H30ClN6O5S [MþH]þ 561.1681,
found: 561.1680.
1H), 8.50 (s, 1H), 8.38 (s, 1H), 8.25 (t, J ¼ 5.6 Hz, 1H), 7.97e7.86 (m,
2H), 7.86e7.78 (m,1H), 7.51 (d, J ¼ 8.1 Hz,1H), 7.44 (t, J ¼ 7.6 Hz,1H),
7.28 (d, J ¼ 8.4 Hz,1H), 3.52e3.44 (m,1H), 3.20e3.15 (m, 2H),1.95 (t,
J ¼ 7.4 Hz, 2H), 1.45e1.55 (m, 4H), 1.32e1.20 (m, 2H), 1.16 (d,
J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 169.53, 166.50,
4.1.4.5. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide
(6e). White solid. Yield: 65.3%. M.p.167e169 ꢀC. 1H NMR (600 MHz,
157.56, 155.63, 142.41, 138.48, 138.19, 135.69, 131.48, 130.63, 130.23,
129.56, 125.71, 124.84, 124.69, 119.18, 117.35, 106.27, 55.24, 39.35,
32.72, 29.18, 26.52, 25.34, 15.32. HRMS m/z calcd for
DMSO‑d6)
d
10.35 (s, 1H), 9.83 (s, 1H), 9.49 (s, 1H), 8.68 (s, 1H), 8.56
C
26H31Cl2N6O5S [MþH]þ 609.1454, found: 609.1463.
(s, 1H), 8.36 (s, 1H), 8.28 (t, J ¼ 5.3 Hz, 1H), 7.89 (d, J ¼ 7.9 Hz, 1H),
7.83 (t, J ¼ 7.7 Hz,1H), 7.72 (d, J ¼ 8.6 Hz, 2H), 7.69 (d, J ¼ 8.5 Hz, 2H),
7.45 (t, J ¼ 7.6 Hz, 1H), 3.52e3.41 (m, 1H), 3.35e3.15 (m, 2H), 1.96 (t,
J ¼ 7.3 Hz, 2H), 1.55e1.47 (m, 4H), 1.33e1.24 (m, 2H), 1.17 (d,
4.1.4.10. 2-Chloro-4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)
benzamide (6j). White solid. Yield: 61.5%. M.p. 194e196 ꢀC. 1H NMR
J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d
169.54, 166.10,
(600 MHz, DMSO‑d6) d 10.34 (s, 1H), 9.87 (s,1H), 9.47 (s,1H), 8.66 (s,
157.72, 155.67, 155.56, 143.13, 138.31, 135.40, 131.46, 128.18, 127.91,
125.57, 124.96, 124.56, 118.43, 105.93, 55.27, 34.10, 32.72, 29.52,
26.63, 25.42, 15.32. HRMS m/z calcd for C26H32ClN6O5S [MþH]þ
575.1838, found: 575.1825.
1H), 8.50 (s, 1H), 8.37 (s, 1H), 8.23 (t, J ¼ 5.5 Hz, 1H), 7.93e7.86 (m,
2H), 7.83 (t, J ¼ 7.8 Hz, 1H), 7.52 (d, J ¼ 8.1 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz,
1H), 7.28 (d, J ¼ 8.4 Hz, 1H), 3.52e3.44 (m, 1H), 3.23e3.10 (m, 2H),
1.95 (t, J ¼ 7.4 Hz, 2H), 1.55e1.43 (m, 4H), 1.37e1.21 (m, 4H), 1.17 (d,
J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 169.59, 166.51,
4.1.4.6. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide
(6f). White solid. Yield: 49.2%. M.p. 184e186 ꢀC. 1H NMR (600 MHz,
157.56, 155.63, 142.41, 138.19, 135.68, 131.48, 130.61, 130.26, 129.55,
125.71, 124.85, 124.68, 119.19, 117.37, 106.26, 95.46, 55.24, 39.40,
32.74, 29.32, 28.79, 26.60, 25.61, 15.33. HRMS m/z calcd for
DMSO‑d6)
d
10.37 (s, 1H), 9.83 (s, 1H), 9.49 (d, J ¼ 0.9 Hz,1H), 8.71 (s,
C
27H33Cl2N6O5S [MþH]þ 623.1610, found: 623.1618.
1H), 8.57 (d, J ¼ 5.4 Hz, 1H), 8.36 (s, 1H), 8.28 (t, J ¼ 5.4 Hz, 1H), 7.89
(d, J ¼ 7.9 Hz, 1H), 7.83 (t, J ¼ 7.8 Hz, 1H), 7.73 (d, J ¼ 8.7 Hz, 2H), 7.70
(d, J ¼ 8.6 Hz, 2H), 7.45 (t, J ¼ 7.6 Hz, 1H), 3.51e3.46 (m, 1H),
3.25e3.20 (m, 2H), 1.95 (t, J ¼ 7.4 Hz, 2H), 1.51 (d, J ¼ 6.0 Hz, 4H),
1.35e1.22 (m, 4H), 1.17 (d, J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz,
4.1.4.11. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(6-(hydroxyamino)-6-oxohexyl)-2-
methoxybenzamide (6k). White solid. Yield: 55.2%. M.p.
167e169 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 10.35 (s, 1H), 9.81 (s,
DMSO‑d6)
d
169.59, 166.10, 157.73, 155.67, 155.54, 143.12, 138.31,
1H), 9.47 (s, 1H), 8.68 (s, 1H), 8.54 (s, 1H), 8.37 (s, 1H), 7.99 (t,
J ¼ 5.3 Hz, 1H), 7.87 (d, J ¼ 7.7 Hz, 1H), 7.78 (t, J ¼ 7.7 Hz, 1H), 7.70 (d,
J ¼ 8.5 Hz, 1H), 7.49e7.40 (m, 2H), 7.38 (d, J ¼ 8.2 Hz, 1H), 3.72 (s,
3H), 3.52e3.44 (m, 1H), 3.27e3.22 (m, 2H), 1.95 (t, J ¼ 7.2 Hz, 2H),
1.55e1.48 (m, 4H), 1.34e1.25 (m, 2H), 1.17 (d, J ¼ 6.7 Hz, 6H). 13C
135.45, 131.45, 128.20, 127.93, 125.53, 124.97, 124.54, 118.43, 105.91,
55.28, 32.70, 29.64, 28.84, 26.72, 25.58, 15.32. HRMS m/z calcd for
C
27H34ClN6O5S [MþH]þ 589.1994, found: 589.1998.
4.1.4.7. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(6-(hydroxyamino)-6-oxohexyl)-2-
methylbenzamide (6g). White solid. Yield: 50.5%. M.p. 161e162 ꢀC.
NMR (151 MHz, DMSO‑d6) d 169.53, 164.74, 158.03, 157.68, 155.63,
155.44, 144.48, 138.31, 135.44, 131.70, 131.47, 125.37, 124.63, 124.46,
115.91, 111.17, 106.25, 102.42, 56.05, 55.27, 39.34, 32.71, 29.47, 26.60,
25.37, 15.32. HRMS m/z calcd for C27H34ClN6O6S [MþH]þ 605.1949,
found: 605.1963.
1H NMR (600 MHz, DMSO‑d6)
d 10.35 (s, 1H), 9.64 (s, 1H), 9.46 (s,
1H), 8.66 (s,1H), 8.56 (s,1H), 8.33 (s,1H), 8.06 (t, J ¼ 5.6 Hz,1H), 7.87
17

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
4.1.4.12. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(6-(hydroxyamino)-6-oxohexyl)-3-
methoxybenzamide (6l). White solid. Yield: 61.5%. M.p. 93e95 ꢀC.
4.1.4.17. 3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(5-(hydroxyamino)-5-oxopentyl)benzamide
(6q). White solid. Yield: 46.3%. M.p. 196e198 ꢀC. 1H NMR
1H NMR (600 MHz, DMSO‑d6)
d
10.35 (s, 1H), 9.51 (s, 1H), 8.66 (s,
(600 MHz, DMSO‑d6) d 10.36 (s,1H), 9.68 (s,1H), 9.56 (s,1H), 8.67 (s,
1H), 8.50 (s, 1H), 8.44e8.21 (m, 3H), 7.96 (s, 1H), 7.85 (d, J ¼ 7.7 Hz,
1H), 7.72 (s, 1H), 7.50 (s, 1H), 7.40 (d, J ¼ 7.7 Hz, 2H), 3.89 (s, 3H),
3.46 (s, 1H), 3.25 (d, J ¼ 5.9 Hz, 2H), 1.96 (t, J ¼ 7.1 Hz, 2H), 1.53 (s,
4H), 1.29 (d, J ¼ 6.8 Hz, 2H), 1.16 (d, J ¼ 6.7 Hz, 6H). 13C NMR
2H), 8.37 (t, J ¼ 5.1 Hz, 1H), 8.33 (s, 1H), 8.02 (s, 1H), 7.84 (d,
J ¼ 7.8 Hz, 1H), 7.77 (d, J ¼ 7.0 Hz, 1H), 7.68 (t, J ¼ 7.5 Hz, 1H), 7.42 (d,
J ¼ 7.6 Hz, 1H), 7.39e7.28 (m, 2H), 3.53e3.41 (m, 1H), 3.30e3.22 (m,
2H),1.98 (t, J ¼ 7.0 Hz, 2H), 1.57e1.49 (m, 4H),1.17 (d, J ¼ 6.8 Hz, 6H).
(151 MHz, DMSO‑d6)
d
169.54, 165.96, 157.76, 156.18, 155.57, 149.37,
13C NMR (151 MHz, DMSO‑d6)
d 169.41, 166.77, 159.51, 158.10,
138.25, 135.37, 131.43, 131.23, 129.64, 125.33, 124.68, 124.44, 120.36,
119.97, 110.12, 105.95, 56.36, 55.30, 32.72, 29.72, 29.51, 26.64, 25.42,
15.31. HRMS m/z calcd for C27H34ClN6O6S [MþH]þ 605.1944, found:
605.1944.
155.71, 155.30, 140.50, 138.43, 135.84, 135.41, 131.40, 128.73, 124.56,
124.03, 122.69, 120.90, 119.63, 105.60, 55.35, 39.42, 32.46, 29.22,
23.21, 15.33. HRMS m/z calcd for C25H30ClN6O5S [MþH]þ 561.1681,
found: 561.1683.
4.1.4.18. 3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide
(6r). White solid. Yield: 44.8%. M.p. 90e92 ꢀC. 1H NMR (600 MHz,
4.1.4.13. 4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(6-(hydroxyamino)-6-oxohexyl)-3-
methylbenzamide (6m). White solid. Yield: 20.6%. M.p. 178e180 ꢀC.
DMSO‑d6)
d 10.36 (s, 1H), 9.70 (s, 1H), 9.57 (s, 1H), 8.68 (s, 2H), 8.37
1H NMR (600 MHz, DMSO‑d6)
d 10.35 (s, 1H), 9.60 (s, 1H), 9.00 (s,
(s, 1H), 8.33 (s, 1H), 8.03 (s, 1H), 7.84 (d, J ¼ 7.7 Hz, 1H), 7.77 (d,
J ¼ 5.9 Hz, 1H), 7.68 (d, J ¼ 6.8 Hz, 1H), 7.43 (d, J ¼ 7.5 Hz, 1H),
7.39e7.24 (m, 2H), 3.52e3.44 (m, 1H), 3.22 (t, J ¼ 5.9 Hz, 2H),
2.03e1.95 (m, 2H), 1.59e1.45 (m, 4H), 1.36e1.22 (m, 2H), 1.17 (d,
1H), 8.67 (s,1H), 8.54 (d, J ¼ 7.1 Hz,1H), 8.35 (s,1H), 8.25 (s,1H), 7.80
(d, J ¼ 7.5 Hz, 1H), 7.73 (s, 1H), 7.68 (d, J ¼ 7.7 Hz, 1H), 7.59e7.52 (m,
2H), 7.31 (t, J ¼ 7.1 Hz,1H), 3.51e3.40 (m,1H), 3.26 (t, J ¼ 5.1 Hz, 2H),
2.26 (s, 3H), 1.96 (d, J ¼ 6.4 Hz, 2H), 1.53 (s, 4H), 1.30 (s, 2H), 1.17 (d,
J ¼ 6.7 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 169.52, 166.75,
J ¼ 6.3 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 169.53, 166.20,
158.10, 155.72, 155.29, 148.65, 140.48, 138.43, 135.86, 135.41, 131.40,
128.74, 124.53, 123.97, 122.70, 120.93, 119.65, 105.60, 55.36, 32.71,
29.35, 26.60, 25.40, 15.33, 14.56. HRMS m/z calcd for C26H32ClN6O5S
[MþH]þ 575.1838, found: 575.1840.
159.03, 155.92, 155.24, 140.81, 138.60, 135.21, 132.00, 131.41, 130.93,
129.79, 125.37, 124.99, 123.98, 123.63, 123.25, 105.19, 55.38, 40.53,
32.73, 29.45, 26.61, 25.42, 18.60, 15.32. HRMS m/z calcd for
C
27H34ClN6O5S [MþH]þ 589.2000, found: 589.2007.
4.1.4.19. 3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide
(6s). White solid. Yield: 49.2%. M.p.182e184 ꢀC. 1H NMR (600 MHz,
4.1.4.14. 3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-hydroxybenzamide (6n). White solid. Yield:
36.8%. M.p. 95e96 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d
9.69 (s, 1H),
DMSO‑d6) d 10.35 (s, 1H), 9.70 (s, 1H), 9.57 (s, 1H), 8.68 (s, 2H), 8.36
8.66 (d, J ¼ 6.0 Hz, 3H), 8.33 (s, 1H), 7.96 (s, 1H), 7.88e7.67 (m, 4H),
(s, 1H), 8.33 (s, 1H), 8.03 (s, 1H), 7.84 (d, J ¼ 7.8 Hz, 1H), 7.77 (d,
J ¼ 6.2 Hz, 1H), 7.68 (t, J ¼ 7.0 Hz, 1H), 7.43 (d, J ¼ 7.5 Hz, 1H),
7.39e7.28 (m, 2H), 3.50e3.42 (m, 1H), 3.22 (d, J ¼ 6.0 Hz, 2H), 1.94
7.37 (t, J ¼ 7.6 Hz, 1H), 7.34e7.29 (m, 2H), 3.48e3.42 (m, 1H), 1.17 (d,
J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 164.79, 158.05,
155.65,155.36,140.62,138.43,135.53, 133.99, 131.39,128.85, 124.68,
124.17,124.08, 122.58,120.49,119.17,105.70, 55.34,15.33. HRMS m/z
calcd for C20H21ClN5O4S [MþH]þ 462.0997, found: 462.1003.
(t, J ¼ 7.1 Hz, 2H), 1.49 (s, 4H), 1.27 (s, 4H), 1.17 (d, J ¼ 6.5 Hz, 6H). 13
C
NMR (151 MHz, DMSO‑d6)
d 169.56, 166.77, 158.10, 155.72, 155.28,
140.47, 138.43, 135.89, 135.40, 131.41, 128.74, 124.52, 124.00, 123.95,
122.69, 120.94, 119.66, 105.60, 55.36, 32.71, 31.30, 29.48, 28.83,
26.72, 25.57, 15.33. HRMS m/z calcd for C27H34ClN6O5S [MþH]þ
589.1994, found: 589.1997.
4.1.4.15. 3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(3-(hydroxyamino)-3-oxopropyl)benzamide
(6o). White solid. Yield: 52.6%. M.p.119e121 ꢀC. 1H NMR (600 MHz,
4.1.4.20. 3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(6-(hydroxyamino)-6-oxohexyl)-2-
methylbenzamide (6t). White solid. Yield: 33.8%. M.p. 56e58 ꢀC. 1H
DMSO‑d6)
d 9.68 (s, 1H), 8.65 (s, 2H), 8.43 (s, 1H), 8.32 (s, 1H), 8.03
(s, 1H), 7.84 (d, J ¼ 7.6 Hz, 1H), 7.77 (d, J ¼ 6.9 Hz, 1H), 7.69 (t,
J ¼ 7.2 Hz, 1H), 7.42 (d, J ¼ 7.4 Hz, 1H), 7.38e7.29 (m, 2H), 3.51e3.40
(m, 3H), 2.25 (t, J ¼ 7.1 Hz, 2H), 1.17 (d, J ¼ 6.7 Hz, 6H). 13C NMR
NMR (600 MHz, DMSO‑d6)
d 10.34 (s, 1H), 9.56 (s, 1H), 9.05 (s, 1H),
8.68 (s, 1H), 8.55 (s, 1H), 8.28 (s, 1H), 8.21 (s, 1H), 7.78 (d, J ¼ 7.7 Hz,
1H), 7.61 (t, J ¼ 7.5 Hz,1H), 7.44 (d, J ¼ 7.8 Hz,1H), 7.32e7.19 (m, 2H),
7.12 (d, J ¼ 7.4 Hz, 1H), 3.50e3.41 (m, 1H), 3.20 (t, J ¼ 6.1 Hz, 2H),
2.19 (s, 3H), 2.01e1.94 (m, 2H), 1.55e1.46 (m, 4H), 1.32e1.26 (m,
(151 MHz, DMSO‑d6) d 167.74, 166.81, 158.08, 155.70, 155.30, 140.54,
138.41, 135.53, 135.43, 131.39, 128.76, 124.59, 124.09, 124.02, 122.79,
120.88, 119.58, 105.63, 55.36, 36.56, 32.86, 15.34. HRMS m/z calcd
for C23H26ClN6O5S [MþH]þ 533.1368, found: 533.1378.
2H),1.16 (d, J ¼ 6.7 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 169.55,
159.57, 155.96, 155.17, 139.57, 138.66, 135.53, 131.35, 130.95, 127.65,
125.85, 124.81, 124.08, 123.58, 123.38, 122.96, 104.70, 55.36, 49.07,
39.20, 32.71, 29.29, 26.55, 25.35, 15.33, 15.24. HRMS m/z calcd for
4.1.4.16. 3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)-N-(4-(hydroxyamino)-4-oxobutyl)benzamide
(6p). White solid. Yield: 46.3%. M.p.176e178 ꢀC. 1H NMR (600 MHz,
C
27H34ClN6O5S [MþH]þ 589.1994, found: 589.1993.
DMSO‑d6)
d 10.40 (s, 1H), 9.70 (s, 1H), 9.57 (s, 1H), 8.73 (s, 1H), 8.66
(s, 1H), 8.42 (t, J ¼ 5.5 Hz, 1H), 8.33 (s, 1H), 8.04 (s, 1H), 7.84 (d,
J ¼ 7.9, 1.1 Hz, 1H), 7.77 (d, J ¼ 7.2 Hz, 1H), 7.68 (t, J ¼ 7.6 Hz,1H), 7.44
(d, J ¼ 7.7 Hz, 1H), 7.39e7.30 (m, 2H), 3.52e3.44 (m, 1H), 3.29e3.17
(m, 2H), 2.01 (t, J ¼ 7.5 Hz, 2H), 1.78e1.67 (m, 2H), 1.17 (d, J ¼ 6.8 Hz,
4.1.5. General procedure for the synthesis of intermediates 8a-c
4.1.5.1. N2-(4-aminophenyl)-5-chloro-N4-(2-(isopropylsulfonyl)
phenyl)pyrimidine-2,4-diamine (8a). Synthesized using the prepa-
ration method of 3a using 5.00 g (14.50 mmol) of 1 and 1.94 g
(18.00 mmol) of p-phenylenediamine (7a) in 20 mL IPA and 0.3 mL
HCl solution (37%) was added, and 3.20 g of 8a was obtained as
white solid; yield: 52.9%. M.p. 218e219 ꢀC. 1H NMR (600 MHz,
6H). 13C NMR (151 MHz, DMSO‑d6)
d 169.33, 166.85, 158.09, 155.73,
155.29, 140.51, 138.41, 135.75, 135.41, 133.07, 131.40, 128.75, 124.57,
124.00, 122.73, 120.92, 119.64, 105.61, 55.36, 40.89, 30.50, 25.76,
15.33. HRMS m/z calcd for C24H28ClN6O5S [MþH]þ 547.1525, found:
547.1523.
DMSO‑d6)
d 9.47 (s, 1H), 9.08 (s, 1H), 8.65 (s, 1H), 8.19 (s, 1H),
7.84e7.81 (m, 1H), 7.67 (s, 1H), 7.42e7.26 (m, 1H), 7.18 (d, J ¼ 7.4 Hz,
18

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
2H), 6.52 (d, J ¼ 8.7 Hz, 2H), 4.83 (s, 2H), 3.51e3.36 (m, 1H),
1.26e1.01 (m, 6H). MS (ESI) m/z: [MþH]þ: 418.1.
J ¼ 7.9 Hz, 1H), 7.63 (t, J ¼ 7.8 Hz, 1H), 7.47 (s, 4H), 7.28 (s, 1H), 7.25
(d, J ¼ 7.7 Hz, 1H), 7.13 (s, 1H), 4.13 (q, J ¼ 7.1 Hz, 2H), 3.26e3.21 (m,
1H), 2.37 (t, J ¼ 7.5 Hz, 2H), 2.33 (t, J ¼ 7.4 Hz, 2H),1.83e1.71 (m, 4H),
1.71e1.63 (m, 3H), 1.32 (t, J ¼ 5.6 Hz, 6H). MS (ESI) m/z: [MþH]þ:
574.2.
4.1.5.2. N2-(3-aminophenyl)-5-chloro-N4-(2-(isopropylsulfonyl)
phenyl)pyrimidine-2,4-diamine (8b). White solid. Yield: 59.9%. M.p.
141e144 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 9.52 (s, 1H), 9.26 (s,
1H), 8.74 (d, J ¼ 8.3 Hz, 1H), 8.26 (d, J ¼ 8.4 Hz, 1H), 7.83 (dd, J ¼ 7.9,
1.5 Hz, 1H), 7.77e7.33 (m, 1H), 7.39e7.33 (m, 1H), 6.89 (t, J ¼ 7.9 Hz,
1H), 6.83 (s, 1H), 6.77 (d, J ¼ 7.9 Hz, 1H), 6.23 (dd, J ¼ 7.9, 1.3 Hz, 1H),
4.94 (s, 2H), 3.48e3.41 (m, 1H), 1.17 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z:
[MþH]þ: 418.1.
4.1.6.6. Methyl
8-((4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)phenyl)amino)-8- oxooctanoate (9f).
White solid. Yield: 55.7%. M.p. 174e176 ꢀC. 1H NMR (600 MHz,
CDCl3)
d
9.62 (s, 1H), 8.55 (d, J ¼ 7.9 Hz, 1H), 8.12 (s, 1H), 7.90 (dd,
J ¼ 7.9, 1.5 Hz, 1H), 7.65e7.58 (m, 1H), 7.46 (s, 4H), 7.25 (d, J ¼ 5.0 Hz,
2H), 7.18 (s, 1H), 3.67 (s, 3H), 3.26e3.18 (m, 1H), 2.38e2.66 (m, 4H),
1.77e1.71 (m, 2H), 1.69e1.57 (m, 2H), 1.47e1.35 (m, 4H), 1.31 (d,
J ¼ 6.9 Hz, 6H). MS (ESI) m/z: [MþH]þ: 588.2.
4.1.5.3. N2-(3-amino-4-methylphenyl)-5-chloro-N4-(2-(iso-
propylsulfonyl)phenyl)pyrimidine-2,4-diamine (8c). White solid.
Yield: 42.6%. M.p. 232e233 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 9.52
(s, 1H), 9.21 (s, 1H), 8.75 (d, J ¼ 7.8 Hz, 1H), 8.23 (s, 1H), 7.83 (dd,
J ¼ 7.9,1.5 Hz,1H), 7.79e7.71 (m,1H), 7.37e7.32 (m,1H), 6.84 (s,1H),
6.80 (d, J ¼ 8.1 Hz, 1H), 6.73 (dd, J ¼ 8.0, 1.7 Hz, 1H), 4.72 (s, 2H),
3.48e3.40 (m, 1H), 2.01 (s, 3H), 1.18 (t, J ¼ 6.3 Hz, 6H). MS (ESI) m/z:
[MþH]þ: 432.0.
4.1.6.7. Methyl
5-((3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)phenyl)amino)-5-oxopentanoate (9g).
White solid. Yield: 62.6%. M.p. > 250 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
d
9.84 (s, 1H), 9.56 (s, 2H), 8.71 (d, J ¼ 6.4 Hz, 1H), 8.28 (s,
1H), 7.93e7.78 (m, 2H), 7.68 (t, J ¼ 7.6 Hz,1H), 7.35 (t, J ¼ 7.5 Hz, 2H),
7.24e7.09 (m, 2H), 3.59 (s, 3H), 3.48e3.40 (m, 1H), 2.39e2.30 (m,
4H), 1.86e1.77 (m, 2H), 1.18 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z:
[MþH]þ: 546.2.
4.1.6. General procedure for the synthesis of intermediates 9a-j
4.1.6.1. Methyl
3-((4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)phenyl)amino)-3-oxopropanoate (9a).
Synthesized using the preparation method of 5b using 8a (0.80 g,
1.91 mmol), HOBT (0.27 g, 2.04 mmol), EDCI (0.40 g, 2.02 mmol),
DIPEA (0.50 g, 3.83 mmol) and monomethyl malonate hydrochlo-
ride (0.27 g, 2.02 mmol) in 20 mL DCM, and 0.57 g of 9a was ob-
tained as white solid. Yield: 56.1%. M.p. 180e181 ꢀC. 1H NMR
4.1.6.8. Methyl
6-((3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)phenyl)amino)-6-oxohexanoate (9h).
White solid. Yield: 64.7%. M.p. 110e112 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
d
9.81 (s, 1H), 9.55 (s, 2H), 8.70 (d, J ¼ 7.4 Hz, 1H), 8.29 (s,
1H), 7.90e7.79 (m, 2H), 7.71e7.65 (m, 1H), 7.37e7.31 (m, 2H),
7.20e7.13 (m, 2H), 3.58 (d, J ¼ 4.0 Hz, 3H), 3.47e3.41 (m, 1H),
2.36e2.24 (m, 5H), 1.60e1.48 (m, 3H), 1.17 (d, J ¼ 6.8 Hz, 6H). MS
(ESI) m/z: [MþH]þ:560.0.
(600 MHz, CDCl3)
d
9.61 (s, 1H), 9.17 (s, 1H), 8.55 (d, J ¼ 8.4 Hz, 1H),
8.14 (s, 1H), 7.94e7.88 (m, 1H), 7.63 (s, 1H), 7.49 (s, 4H), 7.27 (s, 1H),
7.15 (s, 1H), 3.82 (s, 3H), 3.51 (s, 2H), 3.29e3.18 (m, 1H), 1.31 (d,
J ¼ 6.9 Hz, 6H). MS (ESI) m/z: [MþH]þ: 518.1.
4.1.6.9. Ethyl
7-((3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
4.1.6.2. Methyl
4-((4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)phenyl)amino)-7-oxoheptanoate (9i).
amino)pyrimidin-2-yl)amino)phenyl)amino)-4-oxobutanoate (9b).
White solid. Yield: 55.7%. M.p. > 250 ꢀC. 1H NMR (600 MHz,
White solid. Yield: 61.4%. M.p. 206e209 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
d
9.80 (s, 1H), 9.55 (d, J ¼ 7.1 Hz, 2H), 8.71 (d, J ¼ 6.2 Hz,
CDCl3)
d
9.61 (s, 1H), 8.55 (d, J ¼ 8.4 Hz, 1H), 8.13 (s, 1H), 7.90 (d,
1H), 8.28 (s, 1H), 7.88e7.80 (m, 2H), 7.68 (t, J ¼ 7.5 Hz, 1H),
7.38e7.29 (m, 2H), 7.22e7.12 (m, 2H), 4.03 (q, J ¼ 7.0 Hz, 2H),
3.47e3.41 (m, 1H), 2.28 (t, J ¼ 5.8 Hz, 5H), 1.47e1.62 (m, 5H),
1.34e1.25 (m, 3H), 1.17 (d, J ¼ 6.6 Hz, 6H). MS (ESI) m/z: [MþH]þ:
574.2.
J ¼ 7.9 Hz, 1H), 7.65e7.57 (m, 2H), 7.52e7.44 (m, 4H), 7.25 (d,
J ¼ 7.6 Hz, 1H), 7.09 (s, 1H), 3.72 (s, 3H), 3.24 (s, 1H), 2.78 (t,
J ¼ 6.5 Hz, 2H), 2.68 (t, J ¼ 6.5 Hz, 2H), 1.31 (d, J ¼ 6.9 Hz, 6H). MS
(ESI) m/z: [MþH]þ: 532.1.
4.1.6.3. Methyl
5-((4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
4.1.6.10. Methyl
6-((5-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)phenyl)amino)-5-oxopentanoate (9c).
amino)pyrimidin-2-yl)amino)-2-methylphenyl)amino)-6-
White solid. Yield: 65.4%. M.p. 180e183 ꢀC. 1H NMR (600 MHz,
oxohexanoate (9j). White solid. Yield: 31.5%. M.p. 134e137 ꢀC. 1H
CDCl3)
d
9.61 (s, 1H), 8.55 (d, J ¼ 8.4 Hz, 1H), 8.13 (s, 1H), 7.90 (d,
NMR (600 MHz, CDCl3)
d
9.59 (s, 1H), 8.57 (d, J ¼ 8.3 Hz, 1H), 8.14 (d,
J ¼ 7.9 Hz, 1H), 7.63 (t, J ¼ 7.8 Hz, 1H), 7.46 (s, 5H), 7.25 (d, J ¼ 7.6 Hz,
1H), 7.16 (s, 1H), 3.70 (s, 3H), 3.27e3.19 (m, 1H), 2.50e2.39 (m, 4H),
2.10e2.01 (m, 2H), 1.31 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z: [MþH]þ:
546.1.
J ¼ 9.1 Hz, 1H), 7.94 (s, 1H), 7.90 (d, J ¼ 7.0 Hz, 1H), 7.60 (t, J ¼ 7.6 Hz,
1H), 7.48e7.41 (m, 1H), 7.24 (t, J ¼ 7.7 Hz, 1H), 7.16e7.07 (m, 2H),
7.05 (s, 1H), 3.68 (s, 3H), 3.29e3.20 (m, 1H), 2.45e2.34 (m, 4H), 2.24
(s, 3H), 1.78e1.70 (m, 4H), 1.31 (d, J ¼ 6.8 Hz, 6H). MS (ESI) m/z:
[MþH]þ: 574.2.
4.1.6.4. Methyl
6-((4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)phenyl)amino)-6-oxohexanoate (9d).
4.1.7. General procedure for the synthesis of 10a-j
White solid. Yield: 55.6%. M.p. 180e181 ꢀC. 1H NMR (600 MHz,
4.1.7.1. N1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
CDCl3)
d
9.62 (s, 1H), 8.56 (d, J ¼ 8.4 Hz, 1H), 8.13 (s, 1H), 7.90 (d,
imidin-2-yl)amino)phenyl)-N3-hydroxymalonamide
(10a).
J ¼ 7.9 Hz,1H), 7.63 (t, J ¼ 7.8 Hz, 1H), 7.52e7.45 (m, 4H), 7.41 (s, 1H),
7.25 (d, J ¼ 7.6 Hz, 1H), 7.10 (s, 1H), 3.69 (s, 3H), 3.28e3.18 (m, 1H),
2.40 (t, J ¼ 6.4 Hz, 4H), 1.78e1.71 (m, 4H), 1.31 (d, J ¼ 6.8 Hz, 6H). MS
(ESI) m/z: [MþH]þ: 560.2.
Synthesized using the preparation method of 6a using 9a (0.20 g,
0.38 mmol) in 20 mL hydroxylamine methanol solution, and 0.12 g
of 10a was obtained as white solid. Yield: 60.3%. M.p. 148e149 ꢀC.
1H NMR (600 MHz, DMSO‑d6)
d 10.62 (s, 1H), 10.04 (s, 1H), 9.52 (s,
1H), 9.47 (s, 1H), 8.99 (s, 1H), 8.60 (s, 1H), 8.28 (s, 1H), 7.86 (d,
J ¼ 7.9 Hz, 1H), 7.74 (t, J ¼ 7.6 Hz, 1H), 7.52 (d, J ¼ 7.0 Hz, 2H), 7.45 (d,
J ¼ 8.2 Hz, 2H), 7.40 (t, J ¼ 7.5 Hz, 1H), 3.52e3.42 (m, 1H), 3.10 (s,
4.1.6.5. Methyl
7-((4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)
amino)pyrimidin-2-yl)amino)phenyl)amino)-7-oxoheptanoate (9e).
White solid. Yield: 55.7%. M.p. 175e176 ꢀC. 1H NMR (600 MHz,
2H), 1.17 (d, J ¼ 6.6 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 165.30,
CDCl3)
d
9.62 (s, 1H), 8.56 (d, J ¼ 8.4 Hz, 1H), 8.13 (s, 1H), 7.90 (d,
164.04, 158.14, 155.73, 155.35, 138.50, 136.05, 135.25, 133.88, 131.41,
19

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
125.08, 124.63, 124.15, 120.53, 119.86, 104.94, 60.23, 55.32, 42.42,
15.33, 14.56. HRMS m/z calcd for C22H24ClN6O5S [MþH]þ: 519.1212,
found: 519.1210.
28.89, 25.56, 25.52, 15.33. HRMS m/z calcd for C27H34ClN6O5S
[MþH]þ: 589.1998, found: 589.1998.
4.1.7.7. N1-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
4.1.7.2. N1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)phenyl)-N5-hydroxyglutaramide
(10g).
White solid. Yield: 81.2%. M.p. 217e218 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
imidin-2-yl)amino)phenyl)-N4-hydroxysuccinamide
(10b).
White solid. Yield: 40.0%. M.p. 151e153 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 10.44 (s, 1H), 9.88 (s, 1H), 9.50e9.44 (m, 2H), 8.73 (s,
d
10.41 (s, 1H), 9.86 (s, 1H), 9.57 (d, J ¼ 8.6 Hz, 2H), 8.72 (s,
d
2H), 8.30 (s, 1H), 7.88e7.82 (m, 2H), 7.70 (t, J ¼ 7.7 Hz, 1H),
7.38e7.32 (m, 2H), 7.23e7.16 (m, 2H), 3.52e3.48 (m, 1H), 2.31 (t,
J ¼ 7.4 Hz, 2H), 2.01 (t, J ¼ 7.4 Hz, 2H), 1.81e1.76 (m, 2H), 1.18 (d,
1H), 8.60 (s, 1H), 8.27 (s, 1H), 7.86 (d, J ¼ 7.9 Hz, 1H), 7.74 (t,
J ¼ 7.6 Hz,1H), 7.50 (d, J ¼ 7.4 Hz, 2H), 7.45 (d, J ¼ 8.3 Hz, 2H), 7.40 (t,
J ¼ 7.5 Hz, 1H), 3.48e3.42 (m, 1H), 2.54 (t, J ¼ 7.1 Hz, 2H), 2.28 (t,
J ¼ 7.1 Hz, 2H),1.16 (d, J ¼ 6.6 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 171.08, 169.19,
158.17, 155.61, 155.19, 140.65, 139.87, 138.49, 135.35, 131.38, 128.89,
124.39, 124.02, 123.89, 115.51, 113.84, 111.65, 105.36, 55.40, 36.07,
32.13, 21.64, 15.34. HRMS m/z calcd for C24H28ClN6O5S [MþH]þ:
547.1525, found: 547.1532.
d
170.22, 168.87, 158.17, 155.74, 155.34, 138.51, 135.66, 135.25,
134.30, 131.41, 125.05, 124.56, 124.15, 120.52, 119.68, 104.86, 55.32,
31.87, 27.98, 15.32. HRMS m/z calcd for C23H26ClN6O5S [MþH]þ:
533.1368, found: 533.1368.
4.1.7.8. N1-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
4.1.7.3. N1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)phenyl)-N6-hydroxyadipamide
(10h). White
imidin-2-yl)amino)phenyl)-N5-hydroxyglutaramide
(10c).
White solid. Yield: 66.3%. M.p. 159e160 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 10.42 (s, 1H), 9.83 (s, 1H), 9.54e9.41 (m, 2H), 8.72 (s,
solid. Yield: 71.0%. M.p. 215e216 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d
10.41 (s, 1H), 9.89 (s, 1H), 9.57 (d, J ¼ 6.6 Hz, 2H), 8.71 (s, 2H), 8.30
d
(s, 1H), 7.90e7.80 (m, 2H), 7.69 (t, J ¼ 7.7 Hz, 1H), 7.37e7.33 (m, 2H),
7.21 (d, J ¼ 8.1 Hz, 1H), 7.16 (t, J ¼ 8.0 Hz, 1H), 3.49e3.43 (m, 1H),
2.29 (d, J ¼ 6.8 Hz, 2H),1.98 (t, J ¼ 6.7 Hz, 2H),1.54 (d, J ¼ 3.0 Hz, 4H),
1H), 8.61 (s, 1H), 8.28 (s, 1H), 7.86 (d, J ¼ 7.8 Hz, 1H), 7.74 (t,
J ¼ 7.5 Hz, 1H), 7.53e7.46 (m, 4H), 7.40 (t, J ¼ 7.5 Hz, 1H), 3.49e3.41
(m, 1H), 2.35e2.27 (m, 2H), 2.02 (t, J ¼ 7.1 Hz, 2H), 1.86e1.71 (m,
1.18 (d, J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 171.43,
2H), 1.17 (d, J ¼ 6.5 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d
170.83,
169.43, 158.17, 155.60, 155.19, 140.63, 139.89, 138.49, 135.37, 131.37,
128.89, 124.34, 124.00, 123.87, 115.52, 113.87, 111.68, 105.34, 55.40,
36.61, 32.63, 25.35, 25.31, 15.34. HRMS m/z calcd for C25H30ClN6O5S
[MþH]þ: 561.1681, found: 561.1680.
169.23, 158.17, 155.72, 155.31, 138.51, 135.66, 135.29, 134.35, 131.39,
124.93, 124.61, 124.11, 120.50, 119.84, 104.81, 55.33, 36.01, 32.15,
21.74, 15.32. HRMS m/z calcd for C24H28ClN6O5S [MþH]þ: 547.1525,
found: 547.1530.
4.1.7.9. N1-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
4.1.7.4. N1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)phenyl)-N7-hydroxyheptanediamide
(10i).
White solid. Yield: 65.3%. M.p. 202e203 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
imidin-2-yl)amino)phenyl)-N6-hydroxyadipamide
(10d). White
solid. Yield: 68.4%. M.p. 199e200 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
10.37 (s,1H), 9.77 (s,1H), 9.47 (s, 2H), 8.68 (s,1H), 8.60 (s, 1H), 8.27
d
10.35 (s, 1H), 9.83 (s, 1H), 9.57 (d, J ¼ 3.3 Hz, 2H),
d
8.75e8.68 (m, 2H), 8.30 (s, 1H), 7.89e7.80 (m, 2H), 7.70 (t, J ¼ 7.7 Hz,
1H), 7.40e7.34 (m, 2H), 7.21e7.14 (m, 2H), 3.52e3.45 (m, 1H), 2.28
(t, J ¼ 7.3 Hz, 2H), 1.95 (t, J ¼ 7.4 Hz, 2H), 1.59e1.48 (m, 4H),
1.31e1.25 (m, 2H), 1.18 (d, J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz,
(s, 1H), 7.85 (d, J ¼ 7.8 Hz, 1H), 7.74 (t, J ¼ 7.3 Hz, 1H), 7.54e7.48 (m,
4H), 7.40 (t, J ¼ 7.4 Hz, 1H), 3.50e3.39 (m, 1H), 2.28 (d, J ¼ 5.8 Hz,
2H), 1.99 (d, J ¼ 6.4 Hz, 2H), 1.54 (s, 4H), 1.17 (d, J ¼ 6.5 Hz, 6H). 13
C
NMR (151 MHz, DMSO‑d6)
d
171.10, 169.42, 158.18, 155.74, 155.33,
DMSO‑d6) d 171.51, 169.51, 158.17, 155.60, 155.19, 140.64, 139.91,
138.51, 135.69, 135.25, 134.31, 131.40, 125.14, 124.69, 124.12, 120.54,
119.82, 104.86, 55.32, 36.59, 32.66, 25.38, 25.36, 15.33. HRMS m/z
calcd for C25H30ClN6O5S [MþH]þ: 561.1681, found: 561.1686.
138.50, 135.36, 131.38, 128.90, 124.36, 123.99, 123.86, 115.49, 113.83,
111.64, 105.36, 55.39, 36.68, 32.64, 28.75, 25.43, 25.33, 15.34. HRMS
m/z calcd for C26H32ClN6O5S [MþH]þ: 575.1838, found: 575.1850.
4.1.7.5. N1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
4.1.7.10. N1-(5-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)
pyrimidin-2-yl)amino)-2-methylphenyl)-N6-hydroxyadipamide (10j).
White solid. Yield: 65.3%. M.p. 194e196 ꢀC. 1H NMR (600 MHz,
imidin-2-yl)amino)phenyl)-N7-hydroxyheptanediamide
(10e).
White solid. Yield: 51.0%. M.p. 195e196 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 10.35 (s, 1H), 9.77 (s, 1H), 9.52e9.42 (m, 2H), 8.68 (s,
d
DMSO‑d6) d 10.36 (s, 1H), 9.55e9.44 (m, 2H), 9.21 (s, 1H), 8.67 (s,
1H), 8.60 (s, 1H), 8.27 (s, 1H), 7.86 (d, J ¼ 6.1 Hz, 1H), 7.74 (s, 1H),
7.54e7.46 (m, 4H), 7.40 (s, 1H), 3.45 (s, 1H), 2.27 (s, 2H), 1.95 (s, 2H),
1.59e1.52 (m, 4H), 1.27 (s, 2H), 1.17 (s, 6H). 13C NMR (151 MHz,
2H), 8.28 (s, 1H), 7.89e7.78 (m, 1H), 7.73 (t, J ¼ 7.5 Hz, 1H), 7.61 (s,
1H), 7.42e7.31 (m, 2H), 7.07 (d, J ¼ 8.3 Hz, 1H), 3.48e3.42 (m, 1H),
2.29 (s, 2H), 2.12 (s, 3H), 1.98 (s, 2H), 1.54 (s, 4H), 1.17 (d, J ¼ 6.8 Hz,
DMSO‑d6)
d
171.20, 169.52, 158.17, 155.76, 155.34, 138.51, 135.67,
6H). 13C NMR (151 MHz, DMSO‑d6)
d 171.25, 169.43, 158.12, 155.69,
135.26, 134.34, 131.41, 125.08, 124.59, 124.13, 120.52, 119.81, 104.87,
155.18, 138.50, 138.32, 136.79, 135.43, 131.38, 130.31, 126.04, 124.45,
123.95, 123.88, 117.29, 105.08, 55.38, 36.04, 32.64, 25.49, 25.36,
17.79, 15.34. HRMS m/z calcd for C26H32ClN6O5S [MþH]þ: 575.1838,
found: 575.1837.
55.32, 36.64, 32.64, 28.75, 25.42, 25.38, 15.33. HRMS m/z calcd for
C
26H32ClN6O5S [MþH]þ: 575.1838, found: 575.1834.
4.1.7.6. N1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyr-
imidin-2-yl)amino)phenyl)-N8-hydroxyoctanediamide
(10f).
White solid. Yield: 81.2%. M.p. 172e174 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 10.33 (s, 1H), 9.75 (s, 1H), 9.46 (s, 2H), 8.65 (s, 1H), 8.60
4.1.8. General procedure for the synthesis of intermediates 13a-m
Aniline (1.02 g, 10.96 mmol, 11a) and 2,4,5-trichloropyrimidine
(2.04 g, 11.22 mmol, 12a) was dissolved in 20 mL isopropanol and
DIPEA (1.46 g, 11.32 mmol) was added. The resulting mixture was
then stirred at 90 ꢀC for 6 h. After reaction completion, the reaction
mixture was extracted with 100 mL EtOAc, then the organic layer
was washed with saturated NaCl aqueous solution twice, dried
overnight with anhydrous Na2SO4 and filtered. The resulting filtrate
(13a) was concentrated under vacuum and can be directly used
without further purification. 13b-j was synthesized using the
d
(s, 1H), 8.26 (s, 1H), 7.85 (dd, J ¼ 7.9, 1.3 Hz, 1H), 7.73 (t, J ¼ 7.6 Hz,
1H), 7.49 (d, J ¼ 8.3 Hz, 2H), 7.45 (d, J ¼ 8.9 Hz, 2H), 7.39 (t, J ¼ 7.6 Hz,
1H), 3.53e3.40 (m, 1H), 2.26 (t, J ¼ 7.4 Hz, 2H), 1.94 (t, J ¼ 7.4 Hz,
2H), 1.65e1.52 (m, 2H), 1.53e1.44 (m, 2H), 1.27 (s, 4H), 1.16 (d,
J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 171.26, 169.57,
158.18, 155.73, 155.33, 138.51, 135.66, 135.24, 134.35, 131.41, 124.99,
124.58, 124.11, 120.54, 119.83, 104.87, 55.33, 36.75, 32.72, 28.92,
20

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
analog method that used to synthesize 13a.
4.1.10.6. Methyl 8-((4-((4-((2-bromophenyl)amino)-5-
To a solution of 2-(isopropylsulfonyl)aniline (2.04 g, 21.92 mmol,
11j) in DMF (20 mL) was added sodium hydride (1.04 g,
24.96 mmol, 60% in mineral oil) at 0 ꢀC. After stirring for 0.5 h, 2,4-
dichloro-5-fluoropyrimidine (3.66 g, 22.05 mmol, 12b) was added
to the reaction mixture followed by warming the mixture to room
temperature. After stirring for 2 h, the reaction mixture was
quenched with ice and diluted with excess water. The precipitate
was filtered and the solid was dried to obtain 13k as a brown solid
which can be directly used without further purification. 13l-m was
synthesized using the analog method that used to synthesize 13k.
chloropyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoate
White solid. Yield: 62.4%. M.p. 205e207 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 9.63 (s,1H), 9.23 (s, 1H), 8.76 (s,1H), 8.12 (s,1H), 7.75 (d,
J ¼ 8.0 Hz, 1H), 7.70 (d, J ¼ 6.9 Hz, 1H), 7.45 (t, J ¼ 7.6 Hz, 1H), 7.31 (d,
J ¼ 7.8 Hz, 2H), 7.26 (t, J ¼ 7.1 Hz, 3H), 3.58 (s, 3H), 2.29 (t, J ¼ 7.2 Hz,
2H), 2.23 (t, J ¼ 7.4 Hz, 2H), 1.58e1.51 (m, 4H), 1.28 (s, 4H).
(15f).
d
4.1.10.7. Methyl 8-((4-((5-chloro-4-((3-methoxyphenyl)amino)pyr-
imidin-2-yl)amino)phenyl)amino)-8-oxooctanoate
(15g). White
solid. Yield: 70.3%. M.p. 128e130 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d
9.69 (s, 1H), 9.26 (s, 1H), 8.75 (s, 1H), 8.12 (s, 1H), 7.51 (d, J ¼ 8.9 Hz,
2H), 7.38 (d, J ¼ 8.9 Hz, 2H), 7.29 (d, J ¼ 7.8 Hz, 1H), 7.26e7.21 (m,
2H), 6.71 (dd, J ¼ 8.1, 1.9 Hz, 1H), 3.73 (s, 3H), 3.57 (s, 3H), 2.29 (t,
J ¼ 7.4 Hz, 2H), 2.25 (t, J ¼ 7.4 Hz, 2H), 1.61e1.54 (m, 2H), 1.54e1.49
(m, 2H), 1.29 (dd, J ¼ 8.0, 4.7 Hz, 4H).
4.1.9. General procedure for the synthesis of intermediates 14a-m
Synthesized using the preparation method of 3a using 1.00 g
(4.18 mmol) of 13a and 0.45 g (4.17 mmol) of p-phenylenediamine
(7a) in 20 mL IPA and 0.80g (4.17 mmol) TsOH was added, and 0.6 g
of 14a was obtained as purple solid which can be directly used
without further purification. 14b-m was synthesized using the
analog method that used to synthesize 14a.
4.1.10.8. Methyl
8-((4-((5-chloro-4-((5-methoxy-2-methylphenyl)
amino)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoate (15h).
Brown solid. Yield: 75.2%. M.p. 149e152 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) d 9.62 (s,1H), 9.16 (s, 1H), 8.62 (s, 1H), 8.06 (s,1H), 7.35 (d,
4.1.10. General procedure for the synthesis of intermediates 15a-m
J ¼ 8.4 Hz, 2H), 7.24 (d, J ¼ 8.6 Hz, 2H), 7.21 (d, J ¼ 8.4 Hz, 1H), 6.94
(d, J ¼ 1.9 Hz, 1H), 6.83 (dd, J ¼ 8.4, 2.1 Hz, 1H), 3.74 (s, 3H), 3.58 (s,
3H), 2.29 (t, J ¼ 7.3 Hz, 2H), 2.23 (t, J ¼ 7.4 Hz, 2H), 2.09 (s, 3H),
1.56e1.51 (m, 4H), 1.30e1.27 (m, 4H).
4.1.10.1. Methyl
8-((4-((5-chloro-4-(phenylamino)pyrimidin-2-yl)
amino)phenyl)amino)-8-oxooctanoate (15a). Synthesized using the
preparation method of 5b using 14a (0.60 g, 1.93 mmol), HOBT
(0.52 g, 3.86 mmol), EDCI (0.74 g, 3.86 mmol), DIPEA (1.00 g,
7.75 mmol) and monomethyl suberate hydrochloride (0.44g,
1.96 mmol) in 20 mL DMF, and 0.40 g of 15a was obtained as brown
solid. Yield: 66.6%. M.p. 198e200 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
4.1.10.9. Methyl 8-((4-((5-chloro-4-((3,4-dimethoxyphenyl)amino)
pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoate
Pink solid. Yield: 77.2%. M.p. 144e146 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 9.69 (s,1H), 9.19 (s, 1H), 8.70 (s,1H), 8.07 (s,1H), 7.50 (d,
(15i).
d
9.70 (s, 1H), 9.24 (s, 1H), 8.80 (s, 1H), 8.12 (s, 1H), 7.66 (d, J ¼ 7.6 Hz,
d
2H), 7.50 (d, J ¼ 8.4 Hz, 2H), 7.42e7.33 (m, 4H), 7.14 (t, J ¼ 7.3 Hz,
1H), 3.58 (s, 3H), 2.29 (t, J ¼ 7.3 Hz, 2H), 2.25 (t, J ¼ 7.3 Hz, 2H), 1.56
(d, J ¼ 6.9 Hz, 2H), 1.53 (d, J ¼ 7.0 Hz, 2H), 1.29 (s, 4H).
J ¼ 8.7 Hz, 2H), 7.35 (d, J ¼ 8.7 Hz, 2H), 7.18 (s, 1H), 7.14 (d, J ¼ 8.1 Hz,
1H), 6.92 (d, J ¼ 8.6 Hz, 1H), 3.78 (s, 3H), 3.69 (s, 3H), 3.58 (s, 3H),
2.29 (t, J ¼ 7.2 Hz, 2H), 2.24 (t, J ¼ 7.4 Hz, 2H), 1.57e1.51 (m, 4H),1.29
(d, J ¼ 6.9 Hz, 4H).
4.1.10.2. Methyl
amino)phenyl)amino)-8-oxooctanoate (15b). White solid. Yield:
70.2%. M.p. 168e170 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
9.61 (s, 1H),
8-((4-((5-chloro-4-(o-tolylamino)pyrimidin-2-yl)
4.1.10.10. Methyl
8-((4-((4-((3-methoxyphenyl)amino)-5-(tri-
d
fluoromethyl)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoate
9.13 (s, 1H), 8.67 (s, 1H), 8.05 (s, 1H), 7.37e7.28 (m, 4H), 7.28e7.23
(m, 2H), 7.21 (d, J ¼ 8.8 Hz, 2H), 3.58 (s, 3H), 2.29 (t, J ¼ 7.4 Hz, 2H),
2.22 (t, J ¼ 7.4 Hz, 2H), 2.17 (s, 3H), 1.59e1.49 (m, 4H), 1.30e1.26 (m,
4H).
(15j). Grey solid. Yield: 67.6%. M.p. 143e146 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
d 9.90 (s, 1H), 9.58 (s, 1H), 8.63 (s, 1H), 8.34 (s, 1H), 7.60
(d, J ¼ 8.5 Hz, 2H), 7.37 (s, 2H), 7.17 (d, J ¼ 5.6 Hz, 2H), 7.00 (s, 1H),
6.48 (d, J ¼ 7.9 Hz, 1H), 3.58 (s, 6H), 2.33e2.29 (m, 4H), 1.63e1.59
(m, 2H), 1.56e1.53 (m, 2H), 1.32 (s, 4H).
4.1.10.3. Methyl 8-((4-((5-chloro-4-(m-tolylamino)pyrimidin-2-yl)
amino)phenyl)amino)-8-oxooctanoate (15c). White solid. Yield:
4.1.10.11. Methyl
8-((4-((5-fluoro-4-((2-(isopropylsulfonyl)phenyl)
50.4%. M.p. 178e180 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 9.69 (s, 1H),
amino)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoate (15k).
Brown solid. Yield: 43.2%. M.p. 178e179 ꢀC. 1H NMR (600 MHz,
9.24 (s, 1H), 8.72 (s, 1H), 8.10 (s, 1H), 7.53e7.47 (m, 3H), 7.39 (d,
J ¼ 8.6 Hz, 3H), 7.24e7.21 (m, 1H), 6.96 (d, J ¼ 7.4 Hz, 1H), 3.58 (s,
3H), 2.30 (d, J ¼ 10.8 Hz, 4H), 2.25 (t, J ¼ 7.3 Hz, 2H), 1.59e1.46 (m,
5H), 1.29 (s, 4H).
DMSO‑d6)
d
9.72 (s, 1H), 9.45 (d, J ¼ 2.1 Hz, 1H), 9.32 (s, 1H), 8.64 (d,
J ¼ 8.1 Hz, 1H), 8.23 (d, J ¼ 3.0 Hz, 1H), 7.85 (dd, J ¼ 8.0, 1.4 Hz, 1H),
7.76e7.73 (m, 1H), 7.51 (d, J ¼ 8.8 Hz, 2H), 7.44 (d, J ¼ 8.9 Hz, 2H),
7.38 (t, J ¼ 7.6 Hz, 1H), 3.58 (s, 3H), 3.49 (m, 1H), 2.28 (m, 4H),
1.60e1.55 (m, 2H), 1.58e1.50 (m, 2H), 1.32e1.28 (m, 4H), 1.16 (d,
J ¼ 6.8 Hz, 6H).
4.1.10.4. Methyl
amino)phenyl)amino)-8-oxooctanoate (15d). White solid. Yield:
69.8%. M.p. 200e201 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
9.68 (s,
8-((4-((5-chloro-4-(p-tolylamino)pyrimidin-2-yl)
d
1H), 9.19 (s, 1H), 8.73 (s, 1H), 8.08 (s, 1H), 7.55e7.48 (m, 4H), 7.37 (d,
J ¼ 8.2 Hz, 2H), 7.16 (d, J ¼ 7.7 Hz, 2H), 3.58 (s, 3H), 2.33 (s, 2H), 2.29
(d, J ¼ 5.9 Hz, 3H), 2.25 (d, J ¼ 6.9 Hz, 2H), 1.60e1.51 (m, 4H), 1.29 (s,
4H).
4.1.10.12. Methyl 8-((4-((4-((2-(isopropylsulfonyl)phenyl)amino)-5-
methylpyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoate (15l).
Brown solid. Yield: 30.5%. M.p. 195e196 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
d
9.70 (s, 1H), 9.13 (s, 1H), 9.01 (s, 1H), 8.71 (d, J ¼ 7.8 Hz,
1H), 8.03 (s, 1H), 7.85e7.81 (m, 1H), 7.71 (t, J ¼ 7.4 Hz, 1H), 7.54 (d,
J ¼ 8.7 Hz, 2H), 7.43 (d, J ¼ 8.8 Hz, 2H), 7.32 (t, J ¼ 7.6 Hz, 1H),
3.58e3.57 (m, 3H), 3.47e3.41 (m, 1H), 2.31e2.29 (m, 2H), 2.26 (t,
J ¼ 7.4 Hz, 2H), 2.11 (s, 3H), 1.59e1.56 (m, 2H), 1.55e1.52 (m, 2H),
1.30 (s, 4H), 1.16 (d, J ¼ 6.8 Hz, 6H).
4.1.10.5. Methyl
imidin-2-yl)amino)phenyl)amino)-8-oxooctanoate
solid. Yield: 72.1%. M.p. 201e202 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
9.64 (s, 1H), 9.24 (s, 1H), 8.78 (s, 1H), 8.12 (s, 1H), 7.69 (s, 1H), 7.60
8-((4-((5-chloro-4-((2-chlorophenyl)amino)pyr-
(15e). White
d
(d, J ¼ 7.6 Hz, 1H), 7.41 (d, J ¼ 7.6 Hz, 1H), 7.38e7.29 (m, 3H), 7.26 (d,
J ¼ 7.0 Hz, 2H), 3.58 (s, 3H), 2.29 (d, J ¼ 6.5 Hz, 2H), 2.23 (s, 2H), 1.53
(d, J ¼ 6.2 Hz, 4H), 1.28 (s, 4H).
4.1.10.13. Methyl 8-((4-((4-((2-(isopropylsulfonyl)phenyl)amino)-5-
methoxypyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoate
21

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
(15 m). Brown solid. Yield: 39.2%. M.p. 100e101 ꢀC. 1H NMR
J ¼ 7.4 Hz, 2H), 1.61e1.52 (m, 2H), 1.52e1.43 (m, 2H), 1.32e1.23 (m,
(600 MHz, DMSO‑d6)
d
9.70 (s, 1H), 9.55 (s, 1H), 9.05 (s, 1H), 8.85 (d,
4H). 13C NMR (151 MHz, DMSO‑d6)
d 173.89, 171.10, 158.12, 157.62,
J ¼ 8.4 Hz, 1H), 7.99 (s, 1H), 7.81 (dd, J ¼ 6.4, 5.4 Hz, 1H), 7.71 (t,
J ¼ 7.4 Hz,1H), 7.57 (d, J ¼ 8.8 Hz, 2H), 7.45 (d, J ¼ 8.8 Hz, 2H), 7.30 (t,
J ¼ 7.7 Hz, 1H), 3.89 (s, 3H), 3.58 (s, 3H), 3.44e3.40 (m, 1H), 2.30 (t,
J ¼ 7.3 Hz, 2H), 2.26 (t, J ¼ 7.4 Hz, 2H), 1.61e1.55 (m, 2H), 1.55e1.51
(m, 2H), 1.30 (s, 4H), 1.17 (d, J ¼ 6.7 Hz, 6H).
157.05, 155.09, 136.33, 136.22, 136.19, 133.57, 133.53, 129.98, 128.06,
127.69, 119.64, 119.17, 36.70, 32.70, 32.66, 28.92, 28.87, 25.54. HRMS
m/z calcd for C24H27Cl2N6O3 [MþH]þ 517.1522, found: 517.1522.
4.1.11.6. N1-(4-((4-((2-bromophenyl)amino)-5-chloropyrimidin-2-yl)
amino)phenyl)-N8-hydroxyoctanediamide (16f). Brown solid. Yield:
4.1.11. General procedure for the synthesis of 16a-m
85.0%. M.p. 250e252 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 10.22 (s,
4.1.11.1. N1-(4-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)
phenyl)-N8-hydroxyoctanediamide (16a). Synthesized using the
preparation method of 6a using 15a (0.40 g, 0.83 mmol) in 20 mL
hydroxylamine methanol solution, and 0.28 g of 16a was obtained
as Brown solid. Yield: 70.3%. M.p. 223e225 ꢀC. 1H NMR (600 MHz,
1H), 9.66 (d, J ¼ 9.1 Hz,1H), 9.24 (s,1H), 8.69 (s,1H), 8.12 (s,1H), 7.75
(d, J ¼ 7.7 Hz, 1H), 7.68 (s, 1H), 7.45 (t, J ¼ 7.3 Hz, 1H), 7.31 (s, 2H),
7.26 (s, 4H), 2.23 (t, J ¼ 6.9 Hz, 2H), 1.94 (s, 2H), 1.55 (s, 2H), 1.48 (s,
2H), 1.25 (d, J ¼ 14.9 Hz, 4H). 13C NMR (151 MHz, DMSO‑d6)
d 171.11,
171.10, 158.10, 157.03, 155.07, 155.03, 137.85, 136.22, 133.51, 133.11,
128.67, 127.95, 121.67, 119.63, 119.16, 96.47, 51.65, 36.69, 33.70,
28.92, 25.53, 24.79. HRMS m/z calcd for C24H27BrClN6O3 [MþH]þ
561.1017, found: 561.1020.
DMSO‑d6)
d 10.33 (s, 1H), 9.72 (s, 1H), 9.24 (s, 1H), 8.80 (s, 1H), 8.64
(s, 1H), 8.12 (s, 1H), 7.66 (d, J ¼ 7.6 Hz, 2H), 7.50 (d, J ¼ 8.6 Hz, 2H),
7.41e7.30 (m, 4H), 7.14 (t, J ¼ 7.3 Hz, 1H), 2.25 (t, J ¼ 7.3 Hz, 2H), 1.94
(t, J ¼ 7.3 Hz, 2H), 1.61e1.53 (m, 2H), 1.53e1.45 (m, 2H), 1.32e1.26
(m, 4H). 13C NMR (151 MHz, DMSO‑d6)
d
171.17, 169.53, 158.19,
4.1.11.7. N1-(4-((5-chloro-4-((3-methoxyphenyl)amino)pyrimidin-2-
156.36, 155.11, 139.16, 136.11, 133.82, 128.75, 124.29, 123.71, 119.83,
119.73, 103.98, 36.73, 32.71, 28.92, 28.89, 25.57, 25.53. HRMS m/z
calcd for C24H28ClN6O3 [MþH]þ 483.1911, found: 483.1902.
yl)amino)phenyl)-N8-hydroxyoctanediamide (16g). White solid.
Yield: 89.3%. M.p. 200e201 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 9.80
(s, 1H), 9.26 (s, 1H), 8.76 (s, 1H), 8.12 (s, 1H), 7.52 (d, J ¼ 8.8 Hz, 2H),
7.41 (d, J ¼ 8.7 Hz, 2H), 7.30 (d, J ¼ 7.5 Hz, 1H), 7.28e7.14 (m, 3H),
6.71 (d, J ¼ 7.2 Hz, 1H), 3.74 (s, 3H), 2.26 (t, J ¼ 7.4 Hz, 2H), 1.95 (t,
J ¼ 7.1 Hz, 2H), 1.59e1.55 (m, 2H), 1.49 (s, 2H), 1.27 (s, 4H). 13C NMR
4.1.11.2. N1-(4-((5-chloro-4-(o-tolylamino)pyrimidin-2-yl)amino)
phenyl)-N8-hydroxyoctanediamide (16b). White solid. Yield: 85.3%.
M.p. 193e195 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d
10.33 (s, 1H), 9.61
(151 MHz, DMSO‑d6) d 171.19, 159.83, 158.18, 156.30, 155.16, 140.29,
(s, 1H), 9.13 (s, 1H), 8.66 (d, J ¼ 9.5 Hz, 2H), 8.06 (s, 1H), 7.36e7.29
(m, 4H), 7.26 (dt, J ¼ 7.4, 6.1 Hz, 2H), 7.22 (d, J ¼ 8.8 Hz, 2H), 2.22 (t,
J ¼ 7.4 Hz, 2H), 2.18 (s, 3H), 2.02e1.80 (m, 2H), 1.58e1.52 (m, 2H),
1.51e1.46 (m, 2H), 1.29e1.23 (m, 4H). 13C NMR (151 MHz, DMSO‑d6)
136.06, 133.89, 129.44, 119.80, 119.74, 115.75, 109.92, 109.23, 104.06,
100.00, 55.47, 36.73, 32.59, 28.92, 28.89, 25.58. HRMS m/z calcd for
C
25H30ClN6O4 [MþH]þ 513.2017, found: 513.2013.
d
171.04, 169.55, 158.20, 157.47, 154.65, 137.66, 136.47, 135.36,
4.1.11.8. N1-(4-((5-chloro-4-((5-methoxy-2-methylphenyl)amino)
pyrimidin-2-yl)amino)phenyl)-N8-hydroxyoctanediamide
(16h).
Brown solid. Yield: 87.6%. M.p. 219e221 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 10.31 (s, 1H), 9.64 (s, 1H), 9.15 (s, 1H), 8.62 (s, 1H), 8.04
133.25, 130.73, 128.40, 126.67, 126.61, 119.63, 118.77, 103.38, 36.69,
32.72, 28.92, 28.88, 25.55, 25.52, 18.43. HRMS m/z calcd for
C
25H30ClN6O3 [MþH]þ 497.2068, found: 497.2059.
d
(d, J ¼ 12.6 Hz, 1H), 7.35 (d, J ¼ 8.3 Hz, 2H), 7.25 (d, J ¼ 8.4 Hz, 2H),
7.21 (d, J ¼ 8.2 Hz, 1H), 6.94 (s, 1H), 6.82 (d, J ¼ 7.4 Hz, 1H), 3.70 (s,
3H), 2.23 (t, J ¼ 7.2 Hz, 2H), 2.08 (s, 3H), 1.94 (t, J ¼ 6.8 Hz, 2H), 1.55
(s, 2H), 1.48 (s, 2H), 1.29e1.20 (m, 4H). 13C NMR (151 MHz,
4.1.11.3. N1-(4-((5-chloro-4-(m-tolylamino)pyrimidin-2-yl)amino)
phenyl)-N8-hydroxyoctanediamide (16c). Brown solid. Yield: 80.2%.
M.p. 208e210 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 10.20 (s, 1H), 9.73
(s, 1H), 9.24 (s, 1H), 8.72 (s, 1H), 8.10 (s, 1H), 7.61e7.44 (m, 4H), 7.40
(d, J ¼ 8.7 Hz, 3H), 7.23 (t, J ¼ 7.7 Hz,1H), 6.97 (d, J ¼ 7.4 Hz,1H), 2.32
(s, 3H), 2.25 (t, J ¼ 7.3 Hz, 2H), 2.02e1.86 (m, 2H), 1.59e1.54 (m, 2H),
1.51e1.46 (m, 2H), 1.27 (d, J ¼ 10.9 Hz, 4H). 13C NMR (151 MHz,
DMSO‑d6) d 194.95, 182.88, 171.06, 158.20, 158.14, 157.36, 154.68,
138.33, 136.45, 133.32, 131.18, 126.86, 119.64, 118.81, 113.65, 112.35,
55.63, 36.76, 36.70, 28.93, 28.90, 25.62, 25.57, 17.58. HRMS m/z
calcd for C26H32ClN6O4 [MþH]þ 527.2174, found: 527.2170.
DMSO‑d6)
d 171.17, 169.51, 158.20, 156.37, 155.11, 138.99, 138.05,
136.09, 133.83, 128.62, 125.05, 124.27, 120.82, 119.74, 119.68, 103.88,
4.1.11.9. N1-(4-((5-chloro-4-((3,4-dimethoxyphenyl)amino)pyr-
36.75, 32.70, 28.91, 28.88, 25.57, 25.54, 21.64. HRMS m/z calcd for
imidin-2-yl)amino)phenyl)-N8-hydroxyoctanediamide
(16i).
White solid. Yield: 80.3%. M.p. 162e164 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 10.37 (s, 1H), 9.77 (s, 1H), 9.23 (s, 1H), 8.74 (s, 1H), 8.08
C
25H30ClN6O3 [MþH]þ 497.2068, found: 497.2065.
d
4.1.11.4. N1-(4-((5-chloro-4-(p-tolylamino)pyrimidin-2-yl)amino)
(s, 1H), 7.49 (d, J ¼ 8.5 Hz, 2H), 7.38 (s, 2H), 7.22 (s, 1H), 7.19 (s, 1H),
7.15 (d, J ¼ 8.0 Hz, 1H), 6.93 (d, J ¼ 8.6 Hz, 1H), 3.79 (s, 3H), 3.70 (s,
3H), 2.26 (t, J ¼ 7.3 Hz, 2H),1.95 (t, J ¼ 7.3 Hz, 2H), 1.59e1.53 (m, 2H),
1.52e1.46 (m, 2H), 1.26 (d, J ¼ 6.6 Hz, 4H). 13C NMR (151 MHz,
phenyl)-N8-hydroxyoctanediamide (16d). White solid. Yield: 88.7%.
M.p. 233e235 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 10.19 (s, 1H), 9.76
(s, 1H), 9.20 (s, 1H), 8.73 (s, 1H), 8.08 (s, 1H), 7.55e7.44 (m, 5H), 7.39
(d, J ¼ 8.7 Hz, 2H), 7.16 (d, J ¼ 8.1 Hz, 2H), 2.33 (s, 3H), 2.26 (t,
J ¼ 7.3 Hz, 2H), 1.95 (t, J ¼ 7.1 Hz, 2H), 1.59e1.54 (m, 2H), 1.49 (s, 2H),
DMSO‑d6)
d 171.17, 169.58, 158.05, 156.66, 154.48, 148.89, 146.20,
136.12, 133.79, 132.16, 119.72, 119.66, 116.43, 112.02, 109.28, 103.74,
56.26, 55.85, 36.72, 32.72, 28.92, 28.89, 25.59, 25.52. HRMS m/z
calcd for C26H31ClN6O5Na [MþNa]þ 565.1942, found: 565.1941.
1.27 (d, J ¼ 11.8 Hz, 4H). 13C NMR (151 MHz, DMSO‑d6)
d 171.17,
158.20, 156.49, 154.88, 136.54, 136.17, 133.77, 133.45, 129.23, 123.91,
119.73, 119.71, 103.90, 36.75, 31.62, 30.30, 28.93, 28.89, 25.59, 21.06.
HRMS m/z calcd for C25H30ClN6O3 [MþH]þ 497.2068, found:
497.2063.
4.1.11.10. N1-hydroxy-N8-(4-((4-((3-methoxyphenyl)amino)-5-(tri-
fluoromethyl)pyrimidin-2-yl)amino)phenyl)octanediamide
Brown solid. Yield: 82.7%. M.p. 180e182 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 10.35 (s, 1H), 9.93 (s, 1H), 9.58 (s, 1H), 8.65 (d,
(16j).
4.1.11.5. N1-(4-((5-chloro-4-((2-chlorophenyl)amino)pyrimidin-2-yl)
d
amino)phenyl)-N8-hydroxyoctanediamide (16e). White solid. Yield:
J ¼ 15.6 Hz, 2H), 8.34 (s, 1H), 7.60 (d, J ¼ 8.4 Hz, 2H), 7.36 (d,
J ¼ 5.6 Hz, 2H), 7.17 (s, 2H), 7.00 (s, 1H), 6.48 (d, J ¼ 7.7 Hz, 1H), 3.58
(s, 3H), 2.32 (t, J ¼ 7.2 Hz, 2H), 1.95 (t, J ¼ 7.2 Hz, 2H), 1.64e1.57 (m,
2H), 1.53e1.47 (m, 2H), 1.30 (s, 4H). 13C NMR (151 MHz, DMSO‑d6)
80.1%. M.p. 231e233 ꢀC. 1H NMR (600 MHz, DMSO‑d6)
d 10.32 (s,
1H), 9.65 (s, 1H), 9.23 (s, 1H), 8.78 (s, 1H), 8.65 (s, 1H), 8.12 (s, 1H),
7.69 (d, J ¼ 6.8 Hz, 1H), 7.59 (d, J ¼ 8.0 Hz, 1H), 7.41 (t, J ¼ 7.1 Hz, 1H),
7.32 (m, 3H), 7.26 (d, J ¼ 8.6 Hz, 2H), 2.23 (t, J ¼ 7.4 Hz, 2H), 1.94 (t,
d
171.57, 169.56, 161.07, 159.85, 155.92, 141.33, 137.07, 133.40, 129.41,
22

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
126.43, 124.34, 123.23, 119.49, 112.45, 107.67, 105.90, 55.21, 36.85,
the product 19. 19 (0.19 g, 0.4 mmol) was weighed into 20 mL
freshly prepared hydroxylamine methanol solution and stirred at
room temperature for 2 h. The pH value of reaction mixture was
adjusted to 6e7 with saturated NH4Cl solution. The white solid was
precipitated, filtered and washed with water and 0.08 g of 20 was
obtained. Yield: 42.2%. M.p. 162e164 ꢀC. 1H NMR (600 MHz,
32.73, 31.91, 30.47, 28.91, 25.59, 25.54. HRMS m/z calcd for
C
26H30F3N6O4 [MþH]þ 547.2281, found: 547.2277.
4.1.11.11. N1-(4-((5-fluoro-4-((2-(isopropylsulfonyl)phenyl)amino)
pyrimidin-2-yl)amino)phenyl)-N8-hydroxyoctanediamide
(16k).
White solid. Yield: 78.5%. M.p. 188e190 ꢀC. 1H NMR (600 MHz,
DMSO‑d6) 10.33 (s, 1H), 9.73 (s, 1H), 9.45 (s, 1H), 9.32 (s, 1H), 8.64
DMSO‑d6)
d 10.67 (s, 1H), 9.79 (s, 1H), 9.49 (s, 1H), 8.97 (s, 1H), 8.57
d
(s, 1H), 8.34 (s, 1H), 7.88 (d, J ¼ 7.7 Hz, 1H), 7.82 (t, J ¼ 7.5 Hz, 1H),
7.67 (d, J ¼ 7.6 Hz, 2H), 7.51e7.34 (m, 4H), 6.39e6.28 (m, 1H),
3.49e3.42 (m, 1H), 1.16 (d, J ¼ 6.6 Hz, 6H). 13C NMR (151 MHz,
(d, J ¼ 7.4 Hz, 2H), 8.23 (d, J ¼ 2.4 Hz,1H), 7.85 (d, J ¼ 7.7 Hz,1H), 7.74
(t, J ¼ 7.6 Hz,1H), 7.51 (d, J ¼ 8.4 Hz, 2H), 7.45 (d, J ¼ 8.7 Hz, 2H), 7.39
(t, J ¼ 7.5 Hz, 1H), 3.53e3.47 (m, 1H), 2.26 (t, J ¼ 7.2 Hz, 2H), 1.94 (t,
J ¼ 7.2 Hz, 2H), 1.60e1.54 (m, 2H), 1.53e1.47 (m, 2H), 1.28 (s, 4H),
DMSO‑d6)
d 163.71, 157.74, 155.66, 155.54, 141.83, 138.69, 138.35,
135.42, 131.46, 128.58, 128.46, 125.52, 124.93, 124.50, 119.58, 116.95,
105.79, 55.28, 15.32. HRMS m/z calcd for C22H23ClN5O4S [MþH]þ
488.1154, found: 488.1158.
1.17 (d, J ¼ 6.7 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 171.20,
169.57, 155.95, 149.34, 149.28, 141.97, 140.34, 138.59, 136.21, 135.46,
133.94, 131.55, 123.98, 119.91, 119.86, 100.00, 55.45, 36.74, 32.73,
28.92, 28.89, 25.57, 25.52, 15.30. HRMS m/z calcd for C27H34FN6O5S
[MþH]þ 573.2295, found: 573.2294.
4.2．. Cell viability assay
All cancer cell lines (A549, HepG-2, MDA-MB-231, SH-SY5Y,
H2228 and SK-N-BE2) were purchased from Cell Bank of China
Science Academy (Shanghai, China). Cells were maintained in
DMEM/F12 medium (Gibco, US) supplemented with 10% fetal
bovine serum (Gibco, US) at 37 ꢀC with 5% CO2. Cell viability was
determined with cell counting kit-8 (CCK-8) assay. Briefly, six
cancer cell lines were seeded into 96-well plates at a density of
5 ꢂ 103 cells/well. After 24 h, the cells were treated with different
4.1.11.12. N1-hydroxy-N8-(4-((4-((2-(isopropylsulfonyl)phenyl)
amino)-5-methylpyrimidin-2-yl)amino)phenyl)octanediamide (16l).
White solid. Yield: 69.5%. M.p. 209e211 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
d 10.33 (s, 1H), 9.71 (s, 1H), 9.12 (s, 1H), 9.00 (s, 1H), 8.72
(d, J ¼ 7.9 Hz, 1H), 8.65 (s, 1H), 8.03 (s, 1H), 7.82 (d, J ¼ 7.9 Hz, 1H),
7.71 (t, J ¼ 7.7 Hz,1H), 7.54 (d, J ¼ 8.5 Hz, 2H), 7.43 (d, J ¼ 8.6 Hz, 2H),
7.32 (t, J ¼ 7.6 Hz, 1H), 3.44 (dt, J ¼ 13.4, 6.7 Hz, 1H), 2.26 (t,
J ¼ 7.1 Hz, 2H), 2.10 (s, 3H),1.94 (t, J ¼ 6.9 Hz, 2H), 1.59e1.55 (m, 2H),
1.51e1.47 (m, 2H), 1.27 (s, 4H), 1.16 (d, J ¼ 6.7 Hz, 6H). 13C NMR
concentrations of compounds (0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8
After 72 h, the drug containing medium in each well was replaced
by 100 L fresh medium and 10 L CCK-8 (Yeasen, China) and
mM).
(151 MHz, DMSO‑d6)
d
171.16, 169.57, 158.68, 158.57, 157.07, 139.74,
m
m
136.58, 135.26, 133.56, 131.36, 124.00, 123.07, 119.89, 119.86, 119.78,
99.99, 55.31, 36.74, 32.72, 28.93, 28.89,25.58, 25.52, 15.32, 13.14.
incubated for extra 90 min. The OD450nm value was measured using
a microplate reader (BioTek, US). The data were calculated as the
percent inhibition compared with control. The IC50 was the con-
centration at 50% inhibition ratio after fitting the curve in SPSS 17.0
software.
HRMS m/z calcd for
C
28H37N6O5S [MþH]þ 569.2546, found:
569.2541.
4.1.11.13. N1-hydroxy-N8-(4-((4-((2-(isopropylsulfonyl)phenyl)
amino)-5-methoxypyrimidin-2-yl)amino)phenyl)octanediamide
(16 m). White solid. Yield: 68.5%. M.p. 167e169 ꢀC. 1H NMR
4.3．. In Vitro ALK/HDAC1 inhibition assay
(600 MHz, DMSO‑d6)
d
10.33 (s,1H), 9.70 (s,1H), 9.55 (s,1H), 9.05 (s,
HDAC1 Enzyme Assay. The assays were carried out by Sundia
MediTech Company, Ltd. Briefly, different concentrations of com-
pounds were incubated with recombinant HDAC1 (BPS Biosciences,
San Diego, CA, USA) at room temperature for 15 min, which was
followed by adding mixture of Ac-peptide-AMC substrate and
trypsin to initiate the coupled reaction in Tris-based assay buffer.
Fluorescent AMC released from substrate was kinetically measured
at RT in Synergy2 (BioTek, US) using filter sets as
excitation ¼ 355 nm and emission ¼ 460 nm. IC50 values were
calculated by GraphPad Prism software (California, US).
1H), 8.84 (s, 1H), 8.65 (s, 1H), 7.99 (s, 1H), 7.81 (dd, J ¼ 8.0, 1.4 Hz,
1H), 7.71 (s, 1H), 7.57 (d, J ¼ 8.9 Hz, 2H), 7.45 (d, J ¼ 8.9 Hz, 2H), 7.30
(s, 1H), 3.89 (s, 3H), 3.41 (d, J ¼ 6.7 Hz, 1H), 2.26 (t, J ¼ 7.4 Hz, 2H),
1.94 (t, J ¼ 7.4 Hz, 2H), 1.57 (s, 2H), 1.49 (s, 2H), 1.30e1.26 (m, 4H),
1.17 (d, J ¼ 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 171.13,
169.57, 154.10, 153.29, 151.06, 139.26, 136.94, 135.38, 135.25, 133.31,
131.43, 123.42, 122.83, 122.73, 119.97, 119.27, 58.04, 55.37, 36.74,
32.73, 28.93, 28.89, 25.59, 25.52, 15.36. HRMS m/z calcd for
C
28H37N6O6S [MþH]þ 585.2495, found: 585.2493.
ALK Enzyme Assay. The assays were carried out by Sundia
4.1.12. General procedure for the synthesis of 20
MediTech Company, Ltd. To make 100x solution from 10
fold serial dilution for total of 8 concentrations in DMSO. Transfer
250 nL samples to 384-well plate. Add 10 L enzyme mix to assay
plate with final concentration of 1.25 nМ ALK.10 L 1x Kinase buffer
was used as the negative control. Pre-incubate enzyme and sam-
ples at RT for 10 min. Add 15 L substrate mix containing final
concentration of 30 М ATP and 3 М kinase substrate to assay
plate and incubate at RT for 25 min. 30 L stop buffer containing
EDTA will be added to stop the reaction. Read conversion rate with
Caliper EZ Reader. The dose-response curve is fitted with GraphPad
Prism 5 and IC50 calculated by log(inhibitor) vs. response -Variable
slope.
mM with 3-
To a solution of 1 in 20 mL isopropanol and 0.1 mL HCl solution
(37%) was added 1 (1.02 g, 2.95 mmol) and 4-bromoaniline (0.54 g,
3.17 mmol, 2a). The resulting mixture was then stirred at 90 ꢀC for
6 h. After reaction completion, the reaction mixture was extracted
with 100 mL EtOAc, then the organic layer was washed with
saturated NaCl aqueous solution twice, dried overnight with
anhydrous Na2SO4 and filtered. The resulting filtrate was concen-
trated under vacuum afford 18 as white solid. To a 50 mL three-
necked round bottle was charged with 18 (2.4g, 5 mmol), methyl
acrylate (0.86 g, 10 mmol), Pd(OAc)2 (0.12 g), Tri(o-tolyl)phosphine
(0.31 g, 1 mmol), 5 mL CH3CN and 10 mL DMF. The resulting solu-
tion was warmed to 90 ꢀC and stirred at this temperature for 6 h
under N2 atmosphere. When the reaction was completed, 100 mL
water was added and the resulting mixture was extracted with
ethyl acetate twice. The combined organic layer was successively
washed with H2O and then brine, then dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure to give
m
m
m
m
m
m
4.4．. Cloning formation assay
MDA-MB-231 or SH-SY5Y cells were seeded into 24-well plates
at a density of 200 cells/well, and exposed in different concentra-
tions of compound 10f (0.1, 0.2, 0.4 mM) for three days. After that,
23

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
the drug containing medium was replaced by fresh drug-free me-
dium and cells were cultured for two weeks until the colonies
emerged. Then, cells were coped by crystal violet formalin solution
for 30 min. Finally, the plates were washed and dried for images
recording. ImageJ was used for colonies counting.
4 ꢀC overnight and afterward, 1/3000 horseradish peroxidase (HRP)
labeled secondary antibody at room temperature for 30 min.
Finally, the bands were detected by a chemiluminescence imaging
system (CLiNX, Shanghai, China) after incubation in ECL reagent.
Antibody against b-actin, as well as secondary antibody was pur-
chased from Beyotime institute of biotechnology (Shanghai, China).
ALK was purchased from Proteintech (Wuhan, China). Phosphor-
ALK (Y1278), AKT, phosphor-AKT (Ser473), ERK, phosphor-ERK
(Thr202/Tyr204), Histone H3, acetyl- Histone H3 (Lys9/14/18/23/
27) and ECL were purchased from Affinity Biosciences (Changzhou,
China). All other kits were purchased from Beyotime institute of
biotechnology (Shanghai, China).
4.5. Cell scratch assay
MDA-MB-231 or SH-SY5Y cells were seeded into 6-well plates at
a density of 5 ꢂ 105 cells/well. 24 h later, a straight line was
scratched at the bottom of each well by a pipette. After washing
with PBS for three times, cells were treated with different con-
centrations of 10f (0.1, 0.2, 0.4 mM) for 48 h. Inverted microscope
( ꢂ 200, Nikon, Japan) was used for images recording and width
4.9. Pharmacokinetic study
(W) of wounds were quantified by ImageJ software.
The pharmacokinetics analysis of 10f was conducted in male
SpragueꢁDawley rats (Beijing Vital River Laboratory Animal Tech-
nology Co., Ltd). Compound 10f was dissolved in a mixture of 20%
DMSO þ40% PEG þ20% 5% glucose solution and administrated via
the oral route at 10 mg/kg or administrated via the intravenous
route at 1 mg/kg. Blood samples were collected from each animal
via posterior orbital vein at the specific time points (2min、5min、
15min、30min、1 h、2 h、4 h、6 h、8 h、12 h) and plasma was
separated from the blood by centrifugation and stored in a freezer
at ꢁ80 ꢀC. The sample analysis was performed on a LCꢁMS/MS
system. (mass spectrometer API 4000, using an Hedera ODS-2
W0h ꢁ W48h
Healing rate ð%Þ ¼
ꢂ 100%
W0h
4.6. AO/EB and Hoechst 33258 staining analysis
Apoptosis were detected by AO/EB or Hoechst 33258 staining in
MDA-MB-231 cells. Briefly, cells were seeded into 6-well plates,
then treated with compound 10f (1.0, 2.0, 4.0
mM) or Ceritinib
(40 nM) for 24 h. In terms of AO/EB staining method, cells were
collected and washed with phosphate-buffered saline (PBS). The
cells resuspended with an appropriate amount of PBS were counted
and adjusted to a density of (0.2e5) ꢂ 106/mL. Next, the suspension
was stained with AO/EB mixed solution (AO: EB: dilution buffer ¼ 1
: 1: 8) for 15 min and evaluated with a fluorescence microscope. In
Hoechst 33258 staining, the cells were fixed with 0.5 mL fixative for
10 min. Then cells were washed with PBS for twice and stained with
Hoechst 33258. After washing with PBS, anti-fluorescence
quenching was added and each well was detected with a fluores-
cence microscope (Olympus, Tokyo, Japan).
column (100 ꢂ 2.1 mm, 5
mm), Shimadzu Corporation).
4.10. Molecular docking
The cocrystal structure of wild-type ALK complexed with Cer-
itinib (PDB ID: 4MKC), and HDAC2 complexed with SAHA (PDB ID:
4LXZ) were obtained from the Protein Data Bank (http://www.rcsb.
org). The G1202R mutant ALK model was built using 4MKC as a
template. The Prepare Protein protocol in Discovery Studio 2017R2
(DS) (www.3dsbiovia.com) was used for preparing the protein
structures. The ligands were optimized using the Gaussian 09
(www.gaussian.com) by the density functional theory method at
the B3LYP/6-311G** level. The binding models were visualized
using CDOCKER protocol in DS. To verify the reliability of the
docking results, Ceritinib and SAHA were re-docked to the active
sites, respectively. The docking results with a root mean square
deviation (RMSD) of heavy atoms less than 2.0 Å indicates that the
docking method is feasible and the docking results are credible
[37].
4.7. Flow cytometric analysis
Apoptosis and cell cycle were detected by flow cytometric.
MDA-MB-231 or SH-SY5Y cells were seeded into 6-well plates.
After the plates were incubated overnight, cells in each well were
treated with different concentrations of 10f (1.0, 2.0, 4.0
Ceritinib (1.0 M) for 24 h. In terms of apoptosis analysis, cells were
gathered and washed with phosphate-buffered saline (PBS). Cells
were then resuspended in cold PBS and incubated with 5
Annexin V-FITC and 5 L propidium iodine (PI) for 30 min at room
mM) or
m
mL
m
Declaration of competing interest
temperature. Finally, the samples were analyzed using flow cy-
tometer. In terms of cell cycle analysis, cells were resuspended in
cold ethanol (70%). Subsequently, cells were treated with RNase A
and PI. Finally, the samples were detected with a flow cytometer
(CytoFLEX, Beckman Coulter, US).
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
4.8. Western blot analysis
We appreciate the financial support from National Natural Sci-
ence Foundation of China (No. 21907013, and No. 81172097) and
the Scientific and Technological Research Program of Chongqing
Municipal Education Commission (KJQN201900431).
The MDA-MB-231 or SH-SY5Y cells were seeded into 6-well
plates and treated with different concentrations of 10f (1.0, 2.0,
4.0 mM) or Ceritinib (1.0 mM) or SAHA (1.0 mM). After 24 h, cells in
each well were gathered and decomposed with lysis buffer. Sub-
sequently, the lysates were mixed with loading buffer and boiled
for 15 min. The samples were separated with SDS-PAGE and then
transferred to polyvinylidene fluoride (PVDF) membranes, followed
by incubated with 5% (w/v) bovine serum albumin (BSA) for 90 min.
The membranes were incubated with 1/1000 primary antibody at
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113672.
24

T. Pan, Y. Dan, D. Guo et al.
European Journal of Medicinal Chemistry 224 (2021) 113672
D.R. Camidge, Mechanisms of resistance to crizotinib in patients with ALK
gene rearranged non-small cell lung cancer, Clin. Canc. Res. 18 (2012)
1472e1482.
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