Design, synthesis and biological evaluation of novel dithiocarbamate-substituted diphenylaminopyrimidine derivatives as BTK inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.07.043

Bruton's tyrosine kinase (BTK) has emerged as an attractive target related to B-lymphocytes dysfunctions, especially hematologic malignancies and autoimmune diseases. In our study, a series of diphenylaminopyrimidine derivatives bearing dithiocarbamate moieties were designed and synthesized as novel BTK inhibitors for treatment of B-cell lymphoma. Among all these compounds, 30ab (IC₅₀ = 1.15 ± 0.19 nM) displays similar or more potent inhibitory activity against BTK than spebrutinib (IC₅₀ = 2.12 ± 0.32 nM) and FDA approved drug ibrutinib (IC₅₀ = 3.89 ± 0.57 nM), which is attributed to close binding of 30ab with BTK predicted by molecular docking. In particular, 30ab exhibits enhanced anti-proliferative activity against B-lymphoma cell lines at the IC₅₀ concentration of 0.357 ± 0.02 μM (Ramos) and 0.706 ± 0.05 μM (Raji), respectively, almost 10-fold better than ibrutinib and spebrutinib. In addition, 30ab displays stronger selectivity on B-cell lymphoma over other cancer cell lines than spebrutinib. Furthermore, 30ab efficiently blocks BTK downstream pathways and results in apoptosis of cancer cells. In vivo xenograft model evaluation demonstrates the significant efficacy and broad safety margin of 30ab in treatment of B-cell lymphoma. We propose that compound 30ab is a candidate for further study and development based on our current findings.

Full text of DOI:10.1016/j.bmc.2019.07.043

Journal Pre-Proof
Design, synthesis and biological evaluation of novel dithiocarbamate-substitu-
ted diphenylaminopyrimidine derivatives as BTK inhibitors
Zheng Zhai, Ridong Li, Xinyu Bai, Xianling Ning, Zhiqiang Lin, Xuyang Zhao,
Yan Jin, Yuxin Yin
PII:
S0968-0896(19)30696-0
https://doi.org/10.1016/j.bmc.2019.07.043
BMC 15027
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Bioorganic & Medicinal Chemistry
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26 July 2019
Please cite this article as: Zhai, Z., Li, R., Bai, X., Ning, X., Lin, Z., Zhao, X., Jin, Y., Yin, Y., Design, synthesis
and biological evaluation of novel dithiocarbamate-substituted diphenylaminopyrimidine derivatives as BTK
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Design, synthesis and biological evaluation of novel dithiocarbamate-
substituted diphenylaminopyrimidine derivatives as BTK inhibitors.
Zheng Zhai 1,2, Ridong Li1, Xinyu Bai1, Xianling Ning1, Zhiqiang Lin1, Xuyang Zhao1,
Yan Jin1 and Yuxin Yin1,3,4
2
1Institute of Systems Biomedicine, Department of Physiology, School of Basic
Medical Sciences, Peking University Health Science Center, Beijing, China.
3Peking-Tsinghua Center for Life Sciences, Peking University Health Science Center,
Beijing, China.
4 Correspondence
Yuxin Yin, M.D., Ph.D.
Professor and Director
Institute of Systems Biomedicine
Peking University Health Science Center
Beijing 100191, P.R. China
Phone: (86) 10-8280-1237
Fax: (86) 10-8280-1380
E-mail: yinyuxin@hsc.pku.edu.cn
Abstract
Bruton’s tyrosine kinase (BTK) had emerged as an attractive target related to B-
lymphocytes dysfunctions, especially hematologic malignancies and autoimmune
diseases. In our study, a series of diphenylaminopyrimidine derivatives bearing
dithiocarbamate moieties were designed and synthesized as novel BTK inhibitors for
treatment of B-cell lymphoma. Among all these compounds, 30ab (IC50 = 1.15 ± 0.19
nM) displayed similar or more potent inhibitory activity against BTK than spebrutinib
(IC50 = 2.12 ± 0.32 nM) and FDA approved drug ibrutinib (IC50 = 3.89 ± 0.57 nM),
which was attributed to close binding of 30ab with BTK predicted by molecular
docking. In particular, 30ab exhibited enhanced anti-proliferative activity against B-
lymphoma cell lines at the IC50 concentration of 0.357 ± 0.02 μM (Ramos) and 0.706
± 0.05 μM (Raji) respectively, almost 10-fold better than ibrutinib and spebrutinib. In
addition, 30ab displayed stronger selectivity on B-cell lymphoma over other cancer
cell lines than spebrutinib. Furthermore, 30ab efficiently blocked BTK downstream
pathways and resulted in apoptosis of cancer cells. In vivo xenograft model evaluation
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demonstrated the significant efficacy and broad safety margin of 30ab in treatment of
B-cell lymphoma. We proposed that compound 30ab was a candidate for further
study and development based on our preliminary findings.
Keywords:
Bruton's tyrosine kinase (BTK); Diphenylaminopyrimidines; Dithiocarbamate;
Antitumor activity
1. Introduction
Bruton’s tyrosine kinase (BTK) belongs to Tec family non-receptor tyrosine kinase
that predominantly expressed in B cells and myeloid cells [1]. Its absence was initially
anticipated to be associated with human genetic disorder in X-linked
agammaglobulinemia (XLA) patients who manifested the symptoms of primary
immunodeficiency disorders and susceptible to infection, due to lack of mature B-
lymphocytes and antibodies [2]. Not until the identification of BTK functions did
researchers realized its significance in determining the differentiation, maturation and
activation of B cells [3], where upon recognizing specific antigen, the engagement of
BCR recruits intricate downstream scaffold proteins and kinases to achieve the
phosphorylation of BTK tyrosine residues. Phosphorylated BTK further triggers
multiple signal pathways, such as PI3K/Akt, NFAT and STAT5 pathway, especially
the canonical PLCγ mediated activation of PKC/NF-κB and MAPK pathways [4]
fulfill the pivotal role of BTK in regulating the expression of crucial genes for the
proliferation of B cells. Although BTK serves as an indispensable dominator in
maintaining fundamental B cell functions, constitutively activation exacerbates B
cells hyper-function and abnormal proliferation, resulting in autoimmune diseases and
tumorigenesis, respectively. It has been elucidated that numerous B-cell-derived
malignancies imputes to dysregulated BTK functions [5], in the case of non-
Hodgkin’s lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic
lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Waldenstroms
macroglobunemia (WM) and multiple myeloma (MM) . Given the integral functions
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and restricted expression pattern, BTK has received great attention as a therapeutic
target for the treatment of B-cell malignancies [6].
The first BTK inhibitor ibrutinib (1) approved by FDA for the treatment of a panel
of B cell malignancies, firstly, approved for MCL in 2013, CLL in 2014 and
subsequently the treatment of WM in 2015. Ibrutinib showed remarkable improved
response rate and elongated survival rate over traditional chemotherapy in clinical
trial, which monumentally proved that BTK targeting therapeutic regimen effectively
inhibits B-cell malignancies [7]. Acalabrutinib (2) arises as the first FDA approved
second-generation BTK inhibitor for the treatment of MCL in 2017, demonstrates
enhanced potency for BTK and selectivity over other kinases than ibrutinib, which
attenuates untoward effects and toxicities [8]. Spebrutinib (3) is a represented BTK
inhibitor in advanced clinical trial for the treatment of CLL with generally safety,
long-term circulation and notable inhibitory effect on B-cell malignancies. (Fig. 1).
An array of structural related compounds have also progressed to preclinical or
clinical trial studies, such as GS-4059 [9], HM-71224 [10, 11], QL-47 [12, 13]. Apart
from these above covalent BTK inhibitors, some reversible agents [14] including
GDC-0834 [15, 16], GDC-0853 [17], G-744 [18], RN-486 [19] were exquisitely
designed with better selectivity and safety for immunological indications [20] and
ibrutinib-resistant BTK C481S mutation [21, 22]. Nevertheless, drug resistant and
severe side effects are still inevitable during the treatment [23], which signifies the
urgent need of developing novel BTK inhibitors with improved efficacy and reduced
toxicity.
Fig. 1. Structures of known BTK inhibitors approved by FDA or in advanced clinical trial.
Encouraged by the present success in clinical trials, spebrutinib has attracted quite a
number of researches to further modify it as lead compound, aiming to improve the
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pharmacokinetic properties and efficacy. Spebrutinib shares
a
common
diphenylaminopyrimidine pharmacophore with several tyrosine kinase inhibitors,
such as ALK (4) [24], FAK (5) [25, 26], EGFR (6) [27-29] inhibitors, prompting that
diphenylaminopyrimidines are versatile privileged scaffolds in developing a panel of
kinase inhibitors. In addition, spebrutinib as well as ibrutinib and acalabrutinib are
irreversible BTK inhibitor [30] with acrylamide group covalently binding to Cys481
of BTK, which deprives the kinase activity of BTK thoroughly. Since both of the
diphenylaminopyrimidines and acrylamide moieties are essential for activity, the
optimization of spebrutinib focus on its side chain. It was reported that the
introduction of sulfonamide (7) [31] to spebrutinib enhanced the binding affinity to
BTK, phosphamide (8) [32] and amino acid (9) [33] substituted side chain increased
the formation of hydrogen bonds with BTK, other functional group modifications (10)
[34] also promoted the interaction between compounds and BTK protein. These
studies exemplified recent achievements in improving the potency of spebrutinib to a
limited extent by functional groups modification. Alternatively, conjugating the
diphenylaminopyrimidine pharmacophore with novel anti-cancer scaffolds holds
promising prospect. (Fig. 2).
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Fig. 2. Known kinase inhibitors with diphenylaminopyrimidines pharmacophores.
Dithiocarbamate is a well-known structure with synthetic applications and
biological activities, such as antimicrobial, antiviral, anti-inflammation and treatment
of chronic alcoholism, being extensively studied for a long time. Dithiocarbamates
widely exist in natural products with tumor-preventing effects, such as Brassinin (11).
Disulfiram (12), a simple dithiocarbamate containing drug, highlights profound effect
in discouraging alcohol intake since its approval for nearly 50 years and recently
shown to be effective against diverse cancer types [35]. Growing number of studies
have reported novel hybrid molecules generated by combining available
pharmacophores with dithiocarbamate entities [36]. For example, dithiocarbamate
linked β-carboline derivatives (13) [37] displayed profound apoptosis inducing ability
by DNA binding and DNA topoisomerase II inhibition. Emetine-dithiocarbamate
derivatives (14) [38] showed appreciable potency in both androgen receptor positive
and negative prostate cancer cell lines. Our group has incorporated dithiocarbamate
moieties into multiple kinase inhibitors and successfully discovered compounds with
significant antitumor activities and novel mechanisms of action. Dithiocarbamate-
anilinoquinazoline analogs as EGFR inhibitors (15) showed improved activity than
lapatinib, part of which were identified as potential dual or multiple kinases inhibitors.
Dithiocarbamate-diarylaminopyrimidine derivatives (16) [39] exhibited significant
antiproliferative activity on FAK overexpressed cancer cell lines, meanwhile
prevented metastasis through inhibiting migration and anti-angiogenesis. Furthermore,
we conducted extensive research on pyruvate kinase M2 (PKM2) modulators,
including pyridin-3-ylmethyl carbamodithioicesters PKM2 activators (17) [40] and
dithiocarbamate-naphthoquinone PKM2 inhibitors (18) [41, 42], both of them
performed satisfactory enzymatic potent and anti-proliferative activity toward a series
of cancer cell lines. In addition, our synthetic dual dithiocarbamates compounds (19)
[43] displayed notable anti-proliferative effect in phenotypic screening and following
mechanism evaluation revealed that they induced apoptosis and inhibited proliferation
through Aurora-A kinase inhibition [44]. Above studies document that
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dithiocarbamate is an excellent scaffold in anti-cancer drug development (Fig. 3).
Fig. 3. Dithiocarbamate derivatives with antitumor activity.
Harnessing the strategy of conjugating-pharmacophore in rational ligand design [45,
46], herein we reported a serial of compounds with the combination of
dipenylaminopyrimidine and dithiocarbamate structures, which was the pioneer work
of dithiocarbamate-containing BTK inhibitors as far as we concerned (Fig. 4). On
purpose of systematic structure-activity relationship study, we successfully obtained
compounds with different linker and various amine moieties, ultimately came up with
potential drug candidates 30ab through in vitro, in silico and in vivo evaluation.
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Fig.4. Designed strategy of dipenylaminopyrimidine-dithiocarbamate derivatives as BTK inhibitor.
2. Results and discussion
2.1 Chemistry
The general synthetic route was outlined in scheme1. Commercially available 2,4-
dichloro-5-fluoropyrimidine 20 was substituted by 3-nitroaniline 21 under basic
condition of DIEA, which region-selectively replaced the C-4 chlorine to afford the
intermediate 22. In the meantime, 4-nitrophenol 23 reacted with various bromides to
afford different ether intermediates 24a-c, which were reduced to the corresponding
amine intermediates 25a-c by Fe-NH4Cl. Then, the second nucleophilic substitution
of pyrimidine in intermediate 22 took place at C-2 position by amine intermediates
25a-c in the present of TFA. The coupling reaction afforded intermediates 26a-c,
which were further reduced by Fe-NH4Cl and acylated with acryloyl chlorine to form
intermediates 28a-c. To avoid Michael-addition reaction on acryl amide and improve
chemo-selectivity, we replaced the terminal chlorine with more active iodine by
halogen exchange to achieve the conversion of intermediates 28a-c to 29a-c. Scheme
2 depicted the preparation of dipenylaminopyrimidine compounds 31a-b with amide
substituted side chain, intermediate 22 was converted to intermediate 36 by several
steps, including reduction, acylation and coupling reactions under similar conditions
in Scheme 1. In order to reveal the function of acryl amide moiety, we synthesized
compound 32 without α, β-unsaturated group and the routine was described in scheme
3. It simply took one step to furnish intermediate 27a with propionyl amide group and
bypassed the halogen exchange step due to the absence of Michael receptor group.
Finally, a series of amines reacted with CS2 in the present of TEA in acetone to afford
carbamodithioic salts, which substituted all the halogen-substituted intermediates and
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produced dithiocarbamate deriveratives as final compounds 30-32.
Scheme 1. Synthesis of compounds 30. Reagents and conditions: (a) DIEA, i-PrOH, 80℃, 8 h; (b) n = 2, 1-bromo-
2-chloroethane; n = 3, 1-bromo-3-chloropropane; n = 4, 1-bromo-3-chlorobutane; K2CO3, CH3CN, 80℃, 12-24h;
(c) Fe-NH4Cl, EtOH-H2O, 80℃, 1h; (d) TFA, i-PrOH, 90℃, 12h; (e) Fe-NH4Cl, EtOH-H2O, 80℃, 2h; (f) acryloyl
chloride, Na2CO3, DMF, 0.5h; (g) NaI, acetone, 60℃, 48h; (h) (i) CS2, substituted amines, TEA, acetone, rt - 60℃,
1.5h.
Scheme 2.Synthesis of compounds 31a-b. Reagents and conditions: (a) Fe-NH4Cl, EtOH-H2O, 80℃, 2h; (b)
acryloyl chloride, Na2CO3, DMF, 0.5h; (c)TFA, i-PrOH, 90℃, 12h; (d) Fe-NH4Cl, EtOH-H2O, 80℃, 2h; (e) n = 1,
chloroacetyl chloride; n = 2, 3-chloropropionyl chloride; Na2CO3, DMF, 0.5h; (f) CS2, 3-(aminomethyl)pyridine,
TEA, DMF, rt - 60℃, 4h.
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Scheme 3. Synthesis of compound 32. Reagents and conditions:(a) propionyl chloride, Na2CO3, DMF, 0.5h; (b)
CS2, 3-(aminomethyl)pyridine, TEA, DMF, 60℃, 4h.
2.2 Biological evaluation
Table 1.
Biological evaluation of compounds with different amine substitutions.
Compd.
R
BTK inhibitory
activity
Antiproliferative activity
(IC50 ± SD, μM)
(IC50 ± SD, nM) Ramos
Raji
30a
30b
30c
30d
30e
30f
30g
8.21 ± 2.15
15.0 ± 1.9
98.2 ± 8.6
91.7 ± 7.5
1.44 ± 0.48
23.3 ± 3.8
240.8 ± 30.5
5.94 ± 1.59
7.17 ± 1.64
3.44 ± 0.35
9.31 ± 1.78
4.32 ± 0.89
9.53 ± 1.83
3.54 ± 0.68
>40
8.64 ± 1.73
9.58 ± 1.06
13.3 ± 2.54
13.8 ± 2.08
13.7 ± 0.99
>40
30h
125.2 ± 10.1
9.21 ± 1.89
20.9 ± 2.8
30i
30j
2.50 ± 0.95
5.09 ± 1.48
9.46 ± 0.78
6.29 ± 0.83
9.75 ± 1.25
115.2 ± 22.6
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30k
30l
154.5 ± 23.4
83.6 ± 18.4
29.9 ± 2.8
27.1 ± 3.0
2.97 ± 0.53
7.65 ± 1.85
2.87 ± 0.31
2.29 ± 0.63
15.3 ± 2.4
13.4 ± 2.0
4.24 ± 0.12
6.13 ± 0.74
30m
30n
30o
30p
35.6 ± 6.3
56.9 ± 9.5
1.90 ± 0.15
1.02 ± 0.07
3.90 ± 0.48
3.93 ± 0.35
30q
30r
68.3 ± 12.9
80.3 ± 13.3
3.74 ± 0.59
2.76 ± 0.28
7.76 ± 0.83
3.53 ± 0.41
30s
30t
263.7 ± 50.7
385.9 ± 48.1
14.5 ± 1.4
20.6 ± 2.9
>40
15.0 ± 2.5
30u
30v
30w
30x
30y
30z
283.5 ± 20.5
10.8 ± 2.4
2.07 ± 0.47
1.38 ± 0.28
3.22 ± 0.15
1.24 ± 0.15
1.03 ± 0.10
2.25 ± 0.38
>40
2.22 ± 0.30
1.28 ± 0.04
1.15 ± 0.26
1.22 ± 0.21
0.561 ± 0.08
5.68 ± 1.90
6.90 ± 1.89
8.58 ± 2.48
48.6 ± 7.3
30aa
30ab
30ac
>500
>40
>40
1.15 ± 0.19
18.5 ± 2.5
0.357 ± 0.02
2.67 ± 0.15
0.706 ± 0.05
10.3 ± 2.0
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30ad
30ae
30af
7.36 ± 1.88
30.7 ± 6.4
8.49 ± 2.56
2.12 ± 0.32
3.89 ± 0.57
0.574 ± 0.18
2.73 ± 0.74
4.42 ± 1.11
5.44 ± 0.83
1.88 ± 0.52
0.501 ± 0.10
4.84 ± 1.02
6.57 ± 1.89
21.6 ± 3.5
17.7 ± 2.6
Spebruti
nib
/
/
Ibrutinib
Table 2
Biological evaluation of compounds with various side chains.
Compd.
R’
X
n
R
BTK
Antiproliferative activity
inhibitory
activity
(IC50 ± SD, μM)
Ramos
Raji
(IC50 ± SD,
nM)
30ag
30ah
O
O
3
3
13.9 ± 2.5
4.36 ± 0.75
1.36 ± 0.24
4.98 ± 0.83
8.25 ± 1.23
63.2 ± 4.7
30ai
30aj
30ak
O
O
O
3
4
4
2.39 ± 0.45
35.4 ± 4.8
1.42 ± 0.11
15.4 ± 2.4
1.02 ± 0.12
10.2 ± 1.9
4.45 ± 0.88
130.6 ± 20.5 2.92 ± 0.25
30al
31a
31b
32
O
4
1
2
2
16.5 ± 5.2
18.4 ± 4.9
23.7 ± 3.5
>500
1.49 ± 0.36
1.13 ± 0.21
1.22 ± 0.20
>40
1.05 ± 0.15
0.31 ± 0.03
0.34 ± 0.08
>40
NHCO
NHCO
O
2.2.1 In vitro BTK enzymatic inhibition assay.
In order to screen the inhibitory effect of compounds on BTK protein, all
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TM
compounds have been tested through ADP-Glo
kinase assay (Table. 1& 2).
Compound 30e (IC50 = 1.44 ± 0.48 nM) with dimethyl amine and 30a (IC50 = 8.21 ±
2.15 nM) with methyl amine showed comparative BTK inhibition activity to that of
ibrutinib (IC50 = 3.89 ± 0.57 nM) and spebrutinib (IC50 = 2.12 ± 0.32 nM). However,
compounds with longer chain amines led to decrease activity against BTK, for
example, compound 30b (IC50 = 15.0 ± 1.9 nM), 30d (IC50 = 91.7 ± 7.5 nM) and 30f
(IC50 = 23.3 ± 3.8 nM) showed impaired activity. Compound 30g, 30h and 30c with
even longer or branched chain displayed sharply loss of enzymatic activities.
Cyclopropyl amine substituted 30i (IC50 = 2.50 ± 0.95 nM) had significantly better
activity than cyclohexamine counterpart 30j (IC50 = 115.2 ± 22.6 nM), which may
also due to steric effect. Saturated heterocyclic amines are widely acknowledged as a
common class of scaffolds in medicinal chemistry, but all compounds with saturated
heterocyclic amines (30k-30u, 30ah, 30ak) failed to show improved activity against
BTK. The morpholinyl 30m (IC50 = 29.9 ± 2.8 nM), 1-(2,6-dimethyl)morpholinyl 30n
(IC50 = 27.1 ± 3.0 nM) and 4-methylpiperazynyl 30o (IC50 = 35.6 ± 6.3 nM)
substitution compounds demonstrated moderate inhibitory activities compared to
spebrutinib. However, increasing steric hindrance of the substitutions on piperazinyl
ring (30p-30u) caused the loss of activity gradually. Subsequently, the attempt of
incorporating aromatic containing amines brought about the significant activity
enhancement. Benzylamine derivative 30v (IC50 = 10.8 ± 2.4 nM) showed potent
activity, which was further improved by substitutions with either electron-withdraw-
groups (EWG) or electron-donating-groups (EDG) on various position of benzene
ring, such as the p-methoxyl 30w (IC50 = 5.68 ± 1.90 nM), m-fluorine 30x (IC50 =
6.90 ± 1.89 nM) and p-cyanide 30y (IC50 = 8.58 ± 2.48 nM) substitution derivatives
showed stronger potency regardless of electronic effects and position, but introduction
of multi-substituents on the benzene ring (30z, IC50 = 48.6 ± 7.3 nM) reversed the
trend, partly because of steric hindrance. We further investigated the effect of
aromatic heterocycles containing nitrogen atom, in which 3-aminomethyl pyridine
30ab (IC50 = 1.15 ± 0.19 nM) demonstrated the best activity among 2-aminomethyl
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pyridine 30ac (IC50 = 18.5 ± 2.5 nM), 4-aminomethyl pyridine 30ad (IC50 = 7.36 ±
1.88 nM), 2-aminomethyl pyrazine 30af (IC50 = 8.49 ± 2.56 nM). It was also found
that secondary amines showed decreased activity (30aa, 30ae), suggested steric effect
and restriction of spatial conformation were crucial for compounds activity. Having
the best amine substitution in hand, we studied the influence of linker between
dipenylaminopyrimidine and dithiocarbamate moieties. Although increasing length of
carbon chain slightly impaired activity, it was rather negligible comparing with the
influence of amine moieties (30e vs 30ag, 30aj; 30p vs 30ah, 30ak; 30ab vs 30ai,
30al). At the same time, various length amide linker failed to surmount the activity of
30ab (30ab vs 31a, 31b). Finally, replacement of the acrylamide moiety with
proponylamide 32 resulted in completely loss of activity, indicated that the
acrylamide unit was crucial to BTK inhibitory activity as a covalent inhibitor. In
summary, we obtained a number of target compounds with moderate to excellent
potency against BTK through preliminary enzymatic screening.
2.2.2 SAR investigation on anti-proliferative activity against B lymphoma cell lines
In order to determine in vitro anti-proliferation activity of the target compounds, we
selected two typical cell lines derived from human B-cell lymphoblastic leukemia,
namely, Ramos and Raji, in which BTK was overexpressed, for the evaluations of cell
proliferation. These cell lines showed consistent tendency towards different
compounds as expected, so the SAR was summarized based on the both results. All
the compounds listed in Table. 1 and Table. 2 as well as two known BTK inhibitors,
ibrutinib and spebrutinib, which served as positive control, were tested with MTS cell
proliferation assay. Initially, we examined the role of different amine moieties on
compounds’ cytotoxicity. The anti-proliferation activity of compounds bearing simple
linear (30a, 30b, 30e, 30f) and branched (30c) aliphatic amines with carbon chain
shorter than two atoms remained basically consistent with spebrutinib, which was also
compliant to allylamine 30d and cyclic amine (30i, 30j). However, the activities of
compounds with dipropylamine 30g and di-n-butylamine 30h lost dramatically,
indicated the chain length and steric effect were critical for cytotoxicity. As for
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aliphatic amines substitutions, cellular based antiproliferation assay demonstrated
correlation trend with BTK inhibition. Next, we tested some representative secondary
cyclic amine moieties, in which compound with isopropyl piperazinyl 30p showed
slightly improved cytotoxic activity than spebrutinib and was the best among others,
such as, pyrrolidinyl 30k, piperidinyl 30l, morpholinyl (30m, 30n) and piperazinyl
(30o, 30q-30u). The trend that bulky substitutes on piperazinyl moiety (30s-30u)
diminished the activity was somewhat similar to the steric effect of long chain
aliphatic amines. In contrast, despite some of the above secondary cyclic amines (30s-
30u) failed to show desirable enzymatic inhibition, they still exhibited moderate anti-
proliferative activity in different cell lines. The discrepancy between enzymatic and
cellular based assay may be due to off-target effect caused by bulky hydrophobic
group substituted piperazine-dithiocarbamte moieties, whose antitumor activity was
previously reported [47], therefore, these compounds were excluded from further
evaluation. Whereas, benzyl moiety (30v) exerted impressive improvement in
cytotoxicity that provided consistency result with the enzymatic assay, although the
activity changes were not pronounced with further substitutes on the aromatic ring,
including p-methoxy 30w, m-fluorine 30x, p-cyanide30y and 3,4,5-trimethoxy 30z.
Nevertheless, the introduction of hetero-atom containing aromatic groups culminated
compounds with the most potent against B lymphoma cell lines along with the best
enzymatic activity. The number and position of N-atom had been optimized, in which
3-aminomethyl pyridine 30ab elicited the best activity, compared to 2-aminomethyl
pyridine 30ac, 4-aminomethyl pyridine 30ad, 2-aminomethyl pyrazine and other
secondary aromatic amino substitutes (30aa, 30ae). It showed that the anti-
proliferation IC50 of compound 30ab reached nanomolar level, which was almost 10-
fold better than spebrutinib. In accordance with the BTK inhibitory results, diverse
side chains length and substitutions had negligible impacts on compounds’ activities.
The most potent compound 30ab in both enzymatic and cellular screening assay was
prioritized to perform further evaluation.
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2.2.3. Prediction of binding mode
Fig. 5. Predicted binding mode of compounds with BTK. (A) Alignment of the co-crystallized ligand
with molecular docking results. The co-crystallized ligand was shown in orange sticks, CNX-774, in
pink sticks, 30ab, in blue sticks, Cys-481 residue, in green sticks. (B), (C) and (D) depicted 2D
interaction of compound 30e, 30p and 30ab with BTK respectively. Pink arrows indicated hydrogen
bonds and black dashes showed covalent bonds.
Molecular docking, as a reliable method in simulating the interaction of ligand and
protein, assisted the interpretation of SAR result and rational drug design.
Accordingly three typical compound 30e, 30p and 30ab were selected to reveal the
impact of different amine groups on binding mode. By way of model validation, 30ab
was docked into the ATP binding pocket of available BTK co-crystal structure (PDB
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code: 5P9K) [48] in parallel with its original ligand (CNX-774) (Fig. 5A), in which
typical three-dimensional binding configuration of the both were illustrated. Hereafter,
the plausible intermolecular binding patterns of the three compounds were predicted,
where similar binding mode was found in their shared functional groups, that is, the
acrylamide warhead covalently bind to the Cys481 of BTK, the
dipenylaminopyrimidine pharmacophore compactly occupied the hydrophobic pocket
with tight hydrogen bond formed by N-3 of the pyrimidine ring and Met477. Beyond
the retained structures, distinctive amine groups in their dithiocarbamate moieties
exhibited different interactions with BTK. The dimethyl amine group in 30e formed
polar interaction with Asn484 that contributed to its BTK binding affinity (Fig. 5B).
The protonated piperazine moiety in 30p formed salt bond with Glu488, which may
lead to tighter binding with BTK (Fig. 5C). Compound 30ab poised the 3-
aminomethyl pyridine moiety in a suitable position to created extra hydrogen bond
interaction with Glu407, thus strengthened the exterior solvent accessible region
binding (Fig. 5D). Overall, molecular docking prediction offered reasonable
explanation for SAR between various amine moieties.
2.2.4. Cell cytotoxicity against a panel of cancerous and normal cell lines
Table 3.
In vitro cytotoxicity of compound 30ab against cancerous and normal cell lines.
Cell line
Original
IC50 ± SD (μM)
30ab
Spebrutinib
5.44 ± 0.83
21.6 ± 3.5
12.6 ± 1.5
20.3 ± 2.1
6.70 ± 1.25
>40
Ramos
Burkitt's lymphoma
0.357 ± 0.02
0.706 ± 0.05
3.27 ± 0.79
>40
Raji
Burkitt's lymphoma
K-562
Chronic myelogenous leukemia
Anaplastic large cell lymphoma
Acute T cell leukemia
Karpass299
Jurkat
2.49 ± 0.37
35.4 ± 3.5
17.4 ± 2.4
16.2 ± 1.9
MDA-MB-231
MCF7
Breast adenocarcinoma
Breast adenocarcinoma
Hepatocellular carcinoma
29.0 ± 1.5
33.5 ± 3.8
Hep G2
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786-O
HeLa
PC-3
Renal cell adenocarcinoma
4.96 ± 1.04
8.74 ± 1.20
8.25 ± 0.99
10.2 ± 1.5
3.20 ± 0.34
17.9 ± 1.3
33.4 ± 2.5
28.4 ± 2.2
19.3 ± 2.0
12.8 ± 2.7
8.62 ± 2.32
14.9 ± 2.1
17.4 ± 1.8
22.2 ± 1.7
18.9 ± 1.6
1.98 ± 0.18
38.5 ± 2.4
32.5 ± 3.0
22.6 ± 1.7
16.0 ± 1.3
27.6 ± 2.5
4.36 ± 1.07
Cervical adenocarcinoma
Prostate adenocarcinoma
HT-29
A549
H1299
H3122
HEF
Colorectal adenocarcinoma
Non-small cell lung cancer
Non-small cell lung cancer
Non-small cell lung cancer
Embryo fibroblast (normal cell)
Lung fibroblast (normal cell)
Skin fibroblast (normal cell)
Breast epithelium (normal cell)
HELF
BJ
MCF 10A
Since dipenylaminopyrimidine structure exists in numerous kinase inhibitors with
antitumor activities, whether our compounds posed the feature of selectivity over cell
lines or potent as broad antitumor agents warrant further evaluation. We next tested
the best in class compound 30ab against a panel of established cell lines using
compounds spebrutinib as reference. Compared to spebrutinib, compound 30ab
displayed enhanced antiproliferative activity against BTK overexpression cell lines
(Fig.6) with similar cytotoxicity to unrelated cancerous and normal cell lines (Table.
3), which implied that 30ab had considerable selectivity and safety profile.
Fig. 6. Compound 30ab inhibited B-lymphoma cell proliferation in a dose depend manner.
2.2.5. Effects of compound on cell signaling pathways and apoptosis.
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Fig.7. Western blot analysis for compounds’ effect in Ramos cell. A. Compound 30ab treatment
inhibited BTK mediated downstream signal pathways. B. Compound 30ab induced cell apoptosis.
To gain better insight of the compounds’ influence on cellular mechanism, the
signaling pathways of Ramos were examined through western blot analysis, taking
compound 30ab as a representative compound in comparison to spebrutinib and
ibrutinib. Ramos cell were at first treated with gradient concentrations of compounds,
upon anti-IgM stimulation the phosphorylation of BTK itself as well as downstream
targets PLCγ2, Erk and Akt were detected. As showed in Fig.7A, all of the four
inhibitors robustly blocked BTK autophosphorylation at site Y223, confirming their
binding capability with BTK in mechanism. Furthermore, 30ab inhibited BTK-
mediated phosphorylation of PLCγ2 (Y1217) in a dose-depend manner with greater
efficacy than spebrutinib and ibrutinib at the concentration of 10μM. What’s more,
similar effect on Akt phosphorylation were observed for all compounds as expected,
suggesting their inhibition on growth-dependent Akt/mTOR pathway. The inactive
analogue compound 32, whose electrophilic acrylamide was replaced by a non-
reactive propionamide, exhibited diminished inhibition on PLCγ2 phosphorylation,
which demonstrated the stringent requirement for irreversible binding group.
Intriguingly, in contrast to the little impact of spebrutinib and ibrutinib on Erk
pathway, compound 30ab and 32 triggered an abnormal increasing of Erk
phosphorylation at higher concentration, underlined the unique function of
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compound’s activity on antiproliferation. It was speculated that our compounds were
granted with polyphamacological properties [49] owe to dithiocarbamates
substitutions, which implemented their potential to manipulate several pathways
synergistically to eliminate cancer cells. Thereby, apoptosis-inducing abilities of
compounds were studied taking cleaved PARP1 as an intrinsic apoptosis marker to
reflect caspase-3 activation. Impressively, the treatment of compound 30ab intensely
induced cleavage of PARP1 to a greater extent than spebrutinib and ibrutinib at 10μM
level (Fig.7B). Taken together, compound 30ab efficiently inhibited the activation of
BCR pathway and downstream proliferation signal through BTK inhibition, and
induced apoptosis through caspase-3 activation.
2.2.6. In vivo antitumor activity.
The antitumor efficacy of compound 30ab was also evaluated by Ramos cells
xenograft mouse model in vivo (Fig. 8). Animals were assigned into four groups,
model control group, spebrutinib positive control group, small dose group and large
dose group, respectively. The administration of drug was repeated for 2 weeks by
intraperitoneal injection daily. The tumor growth inhibition (TGI) values of
compound 30ab at 30mg/kg/day was 21.5% while at the same dose the TGI value of
spebrutinib was 28.9%. At the higher dose, 60mg/kg/day, compound 30ab reached
the TGI value of 57.8%, exhibited promising antitumor activity in vivo. In addition,
body weight change of the animals was not significant among groups and there were
no severe morphological lesions in histologic tissue sections, which indicated that
compound 30ab had no major systemic toxicity during the treatment of B cell
lymphoma. Based on the above in vivo pharmacodynamics results, we believed that
the capacity of 30ab in effectively suppressing tumors growth in vivo made it
worthwhile for further development as drug candidate.
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Fig. 8. In vivo effects of compounds 30ab on a xenograft mouse model. (A) Tumor volume growth
during treatment; (B) body weight changes in 14 days; (C) Average tumor weight for each group; (D)
Images of tumor tissues collected from mice; (E) H&E staining of tissues (40×) from control and
higher doses of compound 30ab treated group. * p<0.05, ** p<0.01.
3. Conclusion
In summary, we have designed and synthesized a series of dipenylaminopyrimidine
bearing dithiocarbamate moieties as novel BTK inhibitors. A comprehensive study on
SAR has been carried out on kinase inhibition and anti-proliferation based on lead
compound spebrutinib. Most of the compounds with dithiocarbamate substitution
manifested elevated anti-proliferation activity, with IC50 value lower than spebrutinib,
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despite improvement was various in different cases. Among them, some compounds
(30e, 30ab) showed stronger potency to block the BTK enzyme than spebrutinib.
Particularly, 30ab was identified as the most promising compound against BTK (IC50
= 1.15nM), which suppressed proliferation of two typical B-cell leukemia cell lines
with IC50 of 0.357μM and 0.706μM, respectively. The interaction of 30ab and BTK
protein was visually explicated by molecular docking prediction as well. Moreover,
the mechanism study revealed that 30ab potently inhibited phosphorylation of the
hierarchical BTK downstream mediator and induced apoptosis in Ramos cells.
Combining favorable kinase inhibition and anti-proliferation in vitro, 30ab also
showed anti-tumor efficacy similar to spebrutinib in a xenograft mouse model and
well-tolerated at treatment doses. Significantly, our novel molecules propelled the
treatment of lymphoma and potent as drug candidates. Further optimization and
mechanism will be reported in due course.
4. Experimental section
4.1. General methods for chemistry
Solvents and reagents were purchased from commercial resources and were used in synthesis
without further purification unless otherwise stated. 1H NMR and 13C NMR spectra were detected on a
Bruker AVANCEIII-400 spectrometer in deuterium solvent, such as DMSO-d6 or CDCl3. Chemical
shifts(δ) were reported in ppm unit relative to TMS as internal standard, coupling constants J expressed
in Hertz. High resolution mass spectrum (HRMS) was performed on Thermo Scientific Orbitrap Elite
MS. All the reactions were monitored by thin layer chromatography (TLC) plates and detected with
UV light or iodine vapor staining. Column chromatography separations were carried out on Silica Gel
(200-300 mesh) using petroleum ether, ethyl acetate and methanol as eluents.
4.2.1. Procedure for the preparation of intermediate compound 22.
In
a flask containing i-PrOH (20mL), 4-nitroaniline (10mmol, 1.0 eq), 2,4-dicholo-5-
fluoropyrimiding (12mmol, 1.2eq) and DIEA (10mmol, 1.0 eq) were charged. The reaction mixture
was heated to reflux at 90℃ for 8h. The reaction was cooled to room temperature and yellow solid
precipitate was filtrated and wash with cold ethyl ether to give the product 22, which were used without
further purification. Yellow solid, Yield: 76%.1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.72 (t,
J= 2.1 Hz, 1H), 8.41 (d, J = 3.3 Hz, 1H), 8.14 (dd, J = 8.2, 1.4 Hz, 1H), 7.98 – 7.89 (m, 1H), 7.65 (t, J
= 8.2 Hz, 1H).13C NMR (101 MHz, DMSO-d6) δ 153.11 (d, J = 3.4 Hz), 151.17 (d, J = 11.8 Hz),
148.30, 145.70 (d, J = 259.7 Hz), 142.79 (d, J = 21.1 Hz), 139.70, 130.46, 127.30, 118.67, 115.57.
4.2.2. Procedure for the preparation of intermediate compounds 24a-c.
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To a solution of 4-nitrophenol (10mmol, 1.0 eq) in 25mL acetonitrile was added K2CO3 (10mmol,
1eq) at room temperature and stirred for 30min, afterwards, different haloalkanes (15mmol, 1.5 eq)
were added dropwise. 1-Bromo-2-chloroethane (for 24a,n = 2); 1-bromo-3-chloropropane (for 24b, n =
3); 1-bromo-3-chlorobutane (for 24c, n = 4). The mixtures were stirred at 80℃ for 12-24h. The
reactions were monitoring with TLC (petroleum ether: EtOAc = 4:1). Upon completion, water was
added, the aqueous layer was extracted with EtOAc (25mL×3) and the combined organic layers were
washed with brine. Evaporated the solvent to give the crude products 24a-c.
4.2.3. Procedure for the preparation of intermediate compounds 25a-c.
The intermediate products 24a-c (10mmol, 1eq) were dissolved in 20mL ethanol, iron powder
(50mmol, 5eq), NH4Cl (15mmol, 1.5eq) and 10mL H2O were added to the solution under stirred, and
the mixture was heated at 80℃ for 1h. The reaction was filtrated and the residue was washed with
EtOAc. The combined filtration was added water, extracted with EtOAC (30mL×3), washed with brine
and dried over Na2SO4. After evaporation, the residue was purified by column chromatography
(petroleum ether: EtOAc = 2:1) to obtain analogue 25a-c.
4.2.3.1. 4-(2-chloroethoxy)aniline (25a).
White solid, Yield: 73%. 1H NMR (400 MHz, CDCl3) δ 6.79 (d, J = 8.7 Hz, 1H), 6.66 (d, J = 8.6 Hz,
1H), 4.17 (t, J = 5.9 Hz, 1H), 3.79 (t, J = 5.9 Hz, 1H), 3.45 (s, 1H).13C NMR (101 MHz, CDCl3) δ
151.23, 140.82, 116.35, 116.31, 69.06, 42.20.
4.2.4. Procedure for the preparation of intermediate compounds 26a-c.
To a mixture of intermediate 22 (5mmol, 1eq) and intermediates 25a-c(5.5mmol, 1.1eq)in 30mL i-
PrOH, TFA (10mmol, 2eq) was added dropwise. The resulting solution was heated at 90℃ for 12h.
The corresponding products 26a-c were filtrated after cooled to room temperature and washed with
cold ether.
4.2.4.1. N2-(4-(2-chloroethoxy)phenyl)-5-fluoro-N4-(3-nitrophenyl)pyrimidine-2,4-
diamine (26a).
White solid, Yield: 79%. 1H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 10.25 (s, 1H), 8.51 (t, J =
2.0 Hz, 1H), 8.36 (d, J = 4.7 Hz, 1H), 8.20 (d, J = 7.5 Hz, 1H), 7.98 (dd, J = 8.2, 1.7 Hz, 1H), 7.62 (t, J
= 8.2 Hz, 1H), 7.39 (d, J = 8.9 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 4.24 – 4.18 (m, 2H), 3.97 – 3.91 (m,
2H).13C NMR (101 MHz, DMSO-d6) δ 155.02, 152.63, 152.03 (d, J = 11.9 Hz), 148.22, 140.91, 139.02,
138.45, 131.69, 130.37, 128.90, 123.39, 119.51, 117.33, 115.15, 68.61, 43.54.
4.2.5. Procedure for the preparation of intermediate compounds 27a-c.
The intermediate products 26a-c (5mmol, 1eq) were dissolved in 30mL ethanol, iron powder
(25mmol, 5eq), NH4Cl (7.5mmol, 1.5eq) and 5mL H2O were added to the solution under stirred, and
the mixture was heated at 80℃ for 2h. The reaction was filtrated and the residue was washed with
EtOAc. The combined filtration was added water, extracted with EtOAC (30mL×3), washed with brine
and evaporated the solvent to obtain the analogue 27a-c.
4.2.6. Procedure for the preparation of intermediate compounds 28a-c.
A mixture of intermediate products 27a-c (2mmol, 1eq), EtOAc (20 mL) and Na2CO3 (2mmol, 1eq)
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was stirred under ice bath, then acryloyl chloride (2.1mol, 1.05eq) was added dropwise and the
resulting mixture was allowed to room temperature and stirred for 30 min. The reaction was quenched
with water and extracted with EtOAc (10mL×3), washed with brine, dried over Na2SO4. The solvent
was removed under reduced pressure, and the residue was purified with column chromatography to
give the products 28a-c (petroleum ether: EtOAc = 1:1)
4.2.6.1.
N-(3-((2-((4-(2-chloroethoxy)phenyl)amino)-5-fluoropyrimidin-4-
yl)amino)phenyl)acrylamide (28a).
White solid, Yield: 87%.1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.39 (s, 1H), 9.01 (s, 1H),
8.08 (d, J = 3.6 Hz, 1H), 7.96 (s, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 7.9 Hz, 1H), 7.43 (d, J =
7.9 Hz, 1H), 7.30 (t, J = 8.1 Hz, 1H), 6.80 (d, J = 8.8 Hz, 2H), 6.48 (dd, J = 16.9, 10.1 Hz, 1H), 6.28 (d,
J = 16.9 Hz, 1H), 5.77 (d, J = 10.2 Hz, 1H), 4.21 – 4.13 (m, 2H), 3.95 – 3.88 (m, 2H).13C NMR (101
MHz, DMSO-d6) δ 163.58,156.12 (d, J = 2.8 Hz), 152.95, 150.25 (d, J = 10.9 Hz), 142.07,141.25 (d, J
= 19.3 Hz), 139.62, 139.52 (d, J = 4.6 Hz), 135.02, 132.38, 129.11, 127.34, 120.72, 117.86, 115.31,
114.89, 113.95, 68.62, 43.62.
4.2.7. Procedure for the preparation of intermediate compounds 29a-c.
The intermediate products 28a-c (1mmol, 1eq) and NaI (5mmol, 5eq) were dissolved in acetone
(10mL) and heated to reflux for 48h. The NaCl solid precipitated as the reaction proceeded. After
completion of the reaction monitored with TLC (petroleum ether: EtOAc = 4:1), the mixture was
diluted with EtOAc and water, extracted with EtOAc (10mL×3), washed with brine and finally
evaporated the solvent to give intermediate compound 29a-c for the next step without purification.
4.3. General procedure for the synthesis of compounds 30.
To a solution of amines (1.2mmol, 1.2eq) in acetone (15mL) was added TEA (3mmol, 3eq) and CS2
(2mmol, 2eq), the resulting solution was stirred for 30min under room temperature. Then the
intermediates 29a-c (1mmol, 1eq) were added by one-portion and heated the mixture to reflux for 1h.
After the completion of the reaction, the solvent was removed under reduced pressure. Purification the
residue using column chromatography (petroleum ether/EtOAc/MeOH) to give the target compounds
30.
4.3.1.2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl methylcarbamodithioate(30a).
White solid, Yield: 39%,Mp: 173.6-174.8℃.1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 10.02
(d, J = 3.5 Hz, 1H), 9.36 (s, 1H), 8.97 (s, 1H), 8.07 (d, J = 3.3 Hz, 1H), 7.96 (s, 1H), 7.55 (d, J = 8.7 Hz,
2H), 7.50 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 6.78 (d, J = 8.7 Hz,
2H), 6.47 (dd, J = 16.9, 10.1 Hz, 1H), 6.27 (d, J = 16.8 Hz, 1H), 5.75 (d, J = 11.2 Hz, 1H), 4.07 (t, J =
6.3 Hz, 2H), 3.57 (t, J = 6.2 Hz, 2H), 3.06 (d, J = 4.2 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ
196.37, 163.59, 156.16 (d, J = 2.5 Hz), 153.13, 150.25 (d, J = 10.7 Hz), 142.07, 141.25 (d, J = 19.0 Hz),
139.62, 139.53 (d, J = 6.4 Hz), 134.85, 132.38, 129.10, 127.31, 120.73, 117.84, 115.32, 114.78, 113.94,
66.97, 34.29, 33.85.HRMS (ESI) m/z calcd for C23H24FN6O2S2[M + H]+ 499.13807. Found: 499.13693.
4.3.2.2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl ethylcarbamodithioate(30b).
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1
Yellow solid, Yield: 29%, Mp: 162.2-162.8 ℃. H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H),
10.04 (t, J = 4.9 Hz, 1H), 9.37 (s, 1H), 8.97 (s, 1H), 8.06 (d, J = 3.7 Hz, 1H), 7.94 (s, 1H), 7.54 (d, J =
9.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.28 (t, J = 8.1 Hz, 1H), 6.78 (d, J = 9.0
Hz, 2H), 6.46 (dd, J = 17.0, 10.1 Hz, 1H), 6.26 (dd, J = 17.0, 1.9 Hz, 1H), 5.75 (dd, J = 10.1, 1.9 Hz,
1H), 4.06 (t, J = 6.5 Hz, 2H), 3.62-3.55 (m, 4H), 1.14 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO-
d6) δ 195.42, 163.57,156.12 (d, J = 2.6 Hz), 153.13, 150.25 (d, J = 10.7 Hz), 142.05, 141.19 (d, J =
19.0 Hz), 139.60,139.52 (d, J = 4.6 Hz), 134.81, 132.38, 129.11, 127.32, 120.73, 117.84, 115.31,
114.76, 113.92, 66.98, 42.28, 33.65, 13.54.HRMS (ESI) m/z calcd for C24H26FN6O2S2 [M + H]+
513.15372. Found: 513.15381.
4.3.3.2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl isopropylcarbamodithioate (30c).
White solid, Yield: 27%, Mp: 156.1-157.2 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.95
(d, J = 7.4 Hz, 1H), 9.38 (s, 1H), 8.98 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.95 (s, 1H), 7.55 (d, J = 9.0 Hz,
2H), 7.51 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.0 Hz,
2H), 6.47 (dd, J = 16.9, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 1.9 Hz, 1H), 5.76 (dd, J = 10.1, 1.9 Hz, 1H),
4.58 (dq, J = 13.4, 6.6 Hz, 1H), 4.07 (t, J = 6.5 Hz, 2H), 3.57 (t, J = 6.5 Hz, 2H), 1.18 (d, J = 6.6 Hz,
6H).13C NMR (101 MHz, DMSO-d6) δ 194.36, 163.57, 156.14 (d, J = 2.5 Hz), 153.13, 150.24 (d, J =
11.0 Hz), 142.06, 141.24 (d, J = 18.4 Hz), 139.61, 139.52 (d, J = 5.7 Hz), 134.82, 132.38, 129.10,
127.32, 120.71, 117.83, 115.30, 114.75, 113.92, 67.00, 49.22, 33.53, 21.46.HRMS (ESI) m/z calcd for
C25H28FN6O2S2[M + H]+ 527.16937. Found: 527.16919.
4.3.4. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl allylcarbamodithioate (30d).
White solid, Yield: 37%, Mp: 160.1-161.4 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.24 (t, J = 5.2 Hz,
1H), 10.12 (s, 1H), 9.38 (s, 1H), 8.98 (s, 1H), 8.08 (d, J = 3.7 Hz, 1H), 7.96 (s, 1H), 7.55 (d, J = 9.0 Hz,
2H), 7.51 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.0 Hz,
2H), 6.48 (dd, J = 17.0, 10.1 Hz, 1H), 6.28 (dd, J = 17.0, 1.9 Hz, 1H), 5.88 (ddd, J = 22.7, 10.6, 5.6 Hz,
1H), 5.76 (dd, J = 10.1, 1.9 Hz, 1H), 5.18 (ddd, J = 13.7, 11.7, 1.5 Hz, 2H), 4.26 (t, J = 5.4 Hz, 2H),
4.08 (t, J = 6.5 Hz, 2H), 3.59 (t, J = 6.4 Hz, 2H).13C NMR (101 MHz, DMSO-d6) δ 196.43, 163.57,
156.13 (d, J = 2.6 Hz), 153.11, 150.24 (d, J = 10.7 Hz), 142.06, 141.23 (d, J = 20.6 Hz), 139.61, 139.52
(d, J = 5.4 Hz), 134.84, 133.29, 132.38, 129.11, 127.33, 120.71, 117.84, 117.47, 115.31, 114.76,
113.93, 66.94, 49.42, 33.83.HRMS (ESI) m/z calcd for C25H26FN6O2S2[M + H]+ 525.15372. Found:
525.15430.
4.3.5. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl dimethylcarbamodithioate (30e).
White solid, Yield: 42%, Mp: 221.3-222.1 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.38 (s,
1H), 8.98 (s, 1H), 8.07 (d, J = 3.6 Hz, 1H), 7.95 (s, 1H), 7.54 (d, J = 8.9 Hz, 2H), 7.50 (d, J = 8.3 Hz,
1H), 7.42 (d, J = 8.0 Hz, 1H), 7.28 (t, J = 8.1 Hz, 1H), 6.78 (d, J = 8.9 Hz, 2H), 6.47 (dd, J = 16.9, 10.1
Hz, 1H), 6.27 (dd, J = 16.9, 1.5 Hz, 1H), 5.78 – 5.72 (m, 1H), 4.10 (t, J = 6.3 Hz, 2H), 3.62 (t, J = 6.3
Hz, 2H), 3.48 (s, 3H), 3.37 (s, 3H).13C NMR (101 MHz, DMSO-d6) δ 195.17, 163.57, 156.12 (d, J =
2.7 Hz), 153.10, 150.24 (d, J = 11.0 Hz), 142.05, 141.21 (d, J = 20.4 Hz), 139.60, 139.51 (d, J = 5.0
Hz), 134.83, 132.37, 129.12, 127.33, 120.72, 117.83, 115.29, 114.76, 113.90, 66.48, 45.63, 41.79,
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36.34.HRMS (ESI) m/z calcd for C24H26FN6O2S2[M + H]+ 513.15372. Found: 513.15308.
4.3.6. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl diethylcarbamodithioate (30f).
White solid, Yield: 35%, Mp: 188.6-189.7 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.37 (s,
1H), 8.98 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.96 (s, 1H), 7.55 (d, J = 9.0 Hz, 2H), 7.51 (d, J = 8.0 Hz,
1H), 7.42 (d, J = 8.1 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.80 (d, J = 9.0 Hz, 2H), 6.47 (dd, J = 17.0, 10.1
Hz, 1H), 6.27 (dd, J = 17.0, 1.9 Hz, 1H), 5.75 (dd, J = 10.1, 1.9 Hz, 1H), 4.11 (t, J = 6.4 Hz, 2H), 3.98
(q, J = 7.0 Hz, 2H), 3.77 (q, J = 7.0 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 1.27 – 1.16 (m, 6H).13C NMR
(101 MHz, DMSO-d6) δ 193.88, 163.56, 156.15 (d, J = 2.8 Hz), 153.11, 150.23 (d, J = 10.6 Hz),
142.06, 141.26 (d, J = 19.6 Hz), 139.61, 139.52 (d, J = 6.2 Hz), 134.86, 132.39, 129.10, 127.27, 120.70,
117.81, 115.28, 114.79, 113.90, 66.55, 49.75, 47.07, 35.87, 12.84, 11.82.HRMS (ESI) m/z calcd for
C26H30FN6O2S2 [M + H]+ 541.18502. Found: 541.18500.
4.3.7. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl dipropylcarbamodithioate (30g).
White solid, Yield: 40%, Mp: 163.8-164.6 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.38
(s, 1H), 8.98 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.95 (s, 1H), 7.55 (d, J = 9.1 Hz, 2H), 7.51 (d, J = 8.2 Hz,
1H), 7.42 (d, J = 8.1 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.1 Hz, 2H), 6.47 (dd, J = 17.0, 10.1
Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.75 (dd, J = 10.1, 2.0 Hz, 1H), 4.11 (t, J = 6.4 Hz, 2H), 3.93
– 3.85 (m, 2H), 3.71 – 3.65 (m, 2H), 3.63 (t, J = 6.5 Hz, 2H), 1.77 – 1.62 (m, 4H), 0.89 (dt, J = 14.9,
7.4 Hz, 6H).13C NMR (101 MHz, DMSO-d6) δ 194.62, 163.55, 156.14 (d, J = 2.8 Hz), 153.12, 150.23
(d, J = 10.7 Hz), 142.06, 141.24 (d, J = 20.0 Hz), 139.61, 139.52 (d, J = 6.1 Hz), 134.85, 132.40,
129.09, 127.26, 120.70, 117.81, 115.28, 114.80, 113.89, 66.55, 56.84, 54.32, 35.96, 20.81, 19.59, 11.45,
11.40.HRMS (ESI) m/z calcd for C28H34FN6O2S2 [M + H]+ 569.21632. Found: 569.21490.
4.3.8. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl dibutylcarbamodithioate (30h).
White solid,Yield: 31%, Mp: 150.5-151.3 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.37 (s,
1H), 8.98 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.95 (s, 1H), 7.55 (d, J = 9.0 Hz, 2H), 7.51 (d, J = 8.4 Hz,
1H), 7.42 (d, J = 8.4 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.1 Hz, 2H), 6.47 (dd, J = 17.0, 10.1
Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.75 (dd, J = 10.1, 2.0 Hz, 1H), 4.11 (t, J = 6.4 Hz, 2H), 3.97
– 3.89 (m, 2H), 3.75 – 3.67 (m, 2H), 3.63 (t, J = 6.4 Hz, 2H), 1.71 – 1.60 (m, 4H), 1.38 – 1.24 (m, 4H),
0.91 (td, J = 7.3, 5.3 Hz, 6H).13C NMR (101 MHz, DMSO-d6) δ 194.37, 163.54,156.15 (d, J = 2.7 Hz),
153.12, 150.22 (d, J = 10.9 Hz), 142.06, 141.25 (d, J = 19.6 Hz), 139.61,139.53 (d, J = 6.5 Hz), 134.86,
132.41, 129.08, 127.24, 120.68, 117.80, 115.27, 114.80, 113.88, 66.55, 55.07, 52.54, 35.95, 29.45,
28.33, 20.01, 19.96, 14.16, 14.04.HRMS (ESI) m/z calcd for C30H38FN6O2S2 [M + H]+ 597.24762.
Found: 597.24750.
4.3.9. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl cyclopropylcarbamodithioate (30i).
Yellow solid, Yield: 37%, Mp: 184.4-184.9℃. 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H),
10.07 (s, 1H), 9.38 (s, 1H), 8.99 (s, 1H), 8.08 (s, 1H), 7.95 (s, 1H), 7.55 (d, J = 6.1 Hz, 2H), 7.50
(d, J=6.4 Hz, 1H), 7.42 (d, J = 6.3 Hz, 1H), 7.30 (d, J = 7.2 Hz, 1H), 6.79 (s, 2H), 6.47 (dd, J =
25

JOURNAL PRE-PROOF
15.9, 10.4 Hz, 1H), 6.27 (d, J = 16.6 Hz, 1H), 5.76 (d, J = 5.3 Hz, 1H), 4.09 (t, J = 6.3 Hz, 2H),
3.60 (t, J = 6.3 Hz, 2H), 3.43-3.31 (m, 1H), 0.73 (dd, J = 40.8, 13.5 Hz, 4H).13C NMR (101 MHz,
DMSO-d6) δ 196.78, 163.56, 156.12, 153.09, 150.22 (d, J = 9.7 Hz), 142.04, 141.26 (d, J = 19.5
Hz), 139.58, 139.51 (d, J = 5.5 Hz), 134.83, 132.37, 129.11, 127.34, 120.68, 117.81, 115.28,
114.74, 113.89, 66.90, 33.76, 30.58, 7.87, 6.60.HRMS (ESI) m/z calcd for C25H26FN6O2S2[M +
H]+ 525.15372. Found: 525.15295.
4.3.10. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl cyclohexylcarbamodithioate (30j).
White solid, Yield: 34%, Mp: 134.4-135.6 ℃. 1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.94
(d, J = 7.6 Hz, 1H), 9.37 (s, 1H), 8.98 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.95 (s, 1H), 7.55 (d, J = 9.1 Hz,
2H), 7.51 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.1 Hz,
2H), 6.47 (dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H),
4.34 – 4.22 (m, 1H), 4.07 (t, J = 6.5 Hz, 2H), 3.58 (t, J = 6.5 Hz, 2H), 1.97-1.70 (m, 4H), 1.66 – 1.03
(m, 6H).
13C NMR (101 MHz, DMSO-d6) δ 194.42, 163.56, 156.14 (d, J = 2.4 Hz),153.13,150.23 (d, J = 10.8
Hz), 142.05, 141.24 (d, J = 20.8 Hz), 139.60, 139.52 (d, J = 6.0 Hz), 134.82, 132.38, 129.10, 127.31,
120.70, 117.82, 115.29, 114.75, 113.91, 67.00, 56.47, 33.59, 31.45, 25.52, 25.10.13C NMR (101 MHz,
DMSO-D6) δ 156.14 (d, J = 2.4 Hz), 150.23 (d, J = 10.8 Hz), 141.24 (d, J = 20.8 Hz), 139.52 (d, J =
6.0 Hz).HRMS (ESI) m/z calcd for C28H32FN6O2S2[M + H]+ 567.20067. Found: 567.20062.
4.3.11. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl pyrrolidine-1-carbodithioate (30k).
White solid, Yield: 38%, Mp: 199.6-200.3 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.38
(s, 1H), 8.99 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.95 (s, 1H), 7.55 (d, J = 9.1 Hz, 2H), 7.51 (d, J = 8.3 Hz,
1H), 7.42 (d, J = 8.6 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.1 Hz, 2H), 6.47 (dd, J = 17.0, 10.1
Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 4.11 (t, J = 6.4 Hz, 2H), 3.80
(t, J = 6.9 Hz, 2H), 3.64 (t, J = 6.5 Hz, 4H), 2.08 – 1.98 (m, 2H), 1.91 (p, J = 6.5 Hz, 2H).13C NMR
(101 MHz, DMSO-d6) δ 190.80, 163.56, 156.13 (d, J = 2.8 Hz), 153.09, 150.23 (d, J = 10.7 Hz),
142.05, 141.24 (d, J = 19.0 Hz), 139.60, 139.52 (d, J = 5.3 Hz), 134.84, 132.38, 129.11, 127.31, 120.70,
117.82, 115.27, 114.75, 113.88, 66.61, 55.65, 51.07, 35.25, 26.05, 24.21.HRMS (ESI) m/z calcd for
C26H28FN6O2S2[M + H]+ 539.16937. Found: 539.16986.
4.3.12. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl piperidine-1-carbodithioate (30l).
White solid, Yield: 26%, Mp: 197.3-198.4 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H),
9.38 (s, 1H), 8.99 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.96 (s, 1H), 7.55 (d, J = 9.0 Hz, 2H), 7.51 (d,
J = 8.0 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.0 Hz, 2H), 6.47
(dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 1.9 Hz, 1H), 5.75 (dd, J = 10.1, 2.0 Hz, 1H), 4.23
(s, 2H), 4.11 (t, J = 6.4 Hz, 2H), 3.91 (d, J = 4.9 Hz, 2H), 3.65 (t, J = 6.4 Hz, 2H), 1.61 (dd, J =
25.1, 4.5 Hz, 6H).13C NMR (101 MHz, DMSO-d6) δ 193.69, 163.57, 156.12 (d, J = 2.8 Hz),
153.13, 150.25 (d, J = 10.8 Hz), 142.06, 141.19 (d, J = 18.9 Hz), 139.61, 139.52 (d, J = 4.7 Hz),
134.83, 132.39, 129.10, 127.29, 120.73, 117.83, 115.29, 114.78, 113.91, 66.61, 53.07, 51.44,
35.93, 26.26, 25.67, 24.01.HRMS (ESI) m/z calcd for C27H30FN6O2S2[M + H]+ 553.18502. Found:
26

JOURNAL PRE-PROOF
553.18414.
4.3.13.
2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl morpholine-4-carbodithioate (30m).
White solid, Yield: 80%, Mp:217.8-218.5℃.1H NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H),
9.32 (s, 1H), 8.93 (s, 1H), 8.01 (d, J = 3.6 Hz, 1H), 7.90 (s, 1H), 7.49 (d, J = 8.9 Hz, 2H), 7.45 (d,
J = 8.1 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.23 (t, J = 8.1 Hz, 1H), 6.73 (d, J = 8.9 Hz, 2H), 6.42
(dd, J = 16.9, 10.1 Hz, 1H), 6.22 (d, J = 16.9 Hz, 1H), 5.70 (d, J = 10.1 Hz, 1H), 4.19 (s, 2H), 4.07
(t, J = 6.3 Hz, 2H), 3.90 (s, 2H), 3.62 (s, 6H).13C NMR (101 MHz, DMSO-d6) δ 195.58,
163.52,156.07 (d, J = 2.6 Hz),153.02,150.18 (d, J = 10.9 Hz), 142.00, 141.17 (d, J = 20.2 Hz),
139.55,139.47 (d, J = 5.0 Hz), 134.81, 132.32, 129.06, 127.28, 120.66, 117.77, 115.23, 114.71,
113.83, 66.39, 66.01, 65.98, 51.78, 50.68, 35.74. HRMS (ESI) m/z calcd for C26H28FN6O3S2[M +
H]+555.16428. Found: 555.16376.
4.3.14.2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl 2,6-dimethylmorpholine-4-carbodithioate(30n).
White solid,Yield: 43%, Mp: 196.3-197.5 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.38 (s,
1H), 8.98 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.95 (t, J = 1.7 Hz, 1H), 7.55 (d, J = 9.1 Hz, 2H), 7.51 (d, J
= 8.2 Hz, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.1 Hz, 2H), 6.47 (dd, J =
17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 5.28 (s, 1H), 4.43
(s, 1H), 4.14 (t, J = 6.3 Hz, 2H), 3.68 (t, J = 6.3 Hz, 2H), 3.63-3.55 (m, 2H), 3.17 – 2.69 (m, 2H), 1.15
(d, J = 6.2 Hz, 6H).13C NMR (101 MHz, DMSO-d6) δ 195.41, 163.56, 156.14 (d, J = 2.8 Hz),
153.06,150.23 (d, J = 10.8 Hz), 142.06, 141.25 (d, J = 20.2 Hz), 139.62,139.53 (d, J = 5.9 Hz), 134.89,
132.39, 129.10, 127.30, 120.70, 117.82, 115.28, 114.77, 113.89, 71.24, 66.45, 56.38, 55.21, 35.89,
18.81.HRMS (ESI) m/z calcd for C28H32FN6O3S2[M + H]+ 583.19558. Found: 583.19489.
4.3.15.2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl 4-methylpiperazine-1-carbodithioate (30o).
Yellow solid,Yield: 52%, Mp: 185.2-186.5℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H),
9.37 (s, 1H), 8.98 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.96 (s, 1H), 7.55 (d, J = 9.0 Hz, 2H), 7.51 (d,
J = 8.0 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.28 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.0 Hz, 2H), 6.47
(dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 1.9 Hz, 1H), 5.75 (dd, J = 10.1, 1.9 Hz, 1H), 4.24
(s, 2H), 4.12 (t, J = 6.3 Hz, 2H), 3.92 (s, 2H), 3.66 (t, J = 6.3 Hz, 2H), 2.44 – 2.33 (m, 4H), 2.20 (s,
3H).13C NMR (101 MHz, DMSO-d6) δ 195.11, 163.57, 156.14 (d, J = 2.6 Hz), 153.08,150.23 (d, J
= 10.7 Hz), 142.07,141.26 (d, J = 19.4 Hz), 139.62, 139.53 (d, J = 5.8 Hz), 134.88, 132.40, 129.11,
127.31, 120.69, 117.81, 115.27, 114.77, 113.88, 66.50, 54.42, 51.52, 50.00, 45.51, 35.94.HRMS
(ESI) m/z calcd for C27H31FN7O2S2[M + H]+ 568.19592. Found:568.19537.
4.3.16. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl 4-isopropylpiperazine-1-carbodithioate (30p).
White solid,Yield: 63%, Mp: 194.4-196.3 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.37 (s,
1H), 8.98 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.95 (s, 1H), 7.55 (d, J = 9.1 Hz, 2H), 7.52 (d, J = 8.3 Hz,
1H), 7.42 (d, J = 8.7 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.1 Hz, 2H), 6.47 (dd, J = 17.0, 10.1
Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 4.23 (s, 2H), 4.12 (t, J = 6.4
27

JOURNAL PRE-PROOF
Hz, 2H), 3.91 (s, 2H), 3.66 (t, J = 6.3 Hz, 2H), 2.69 (hept, J = 6.4 Hz, 1H), 2.51 (dd, J = 5.4, 3.6 Hz,
4H), 0.97 (d, J = 6.5 Hz, 6H).13C NMR (101 MHz, DMSO-d6) δ 194.72, 163.56, 156.14 (d, J = 2.8 Hz),
153.09, 150.23 (d, J = 10.8 Hz),142.06, 141.25 (d, J = 19.2 Hz), 139.61, 139.53 (d, J = 6.1 Hz), 134.87,
132.39, 129.10, 127.29, 120.69, 117.81, 115.27, 114.78, 113.88, 66.53, 53.94, 52.07, 50.62, 48.21,
35.87, 18.57.HRMS (ESI) m/z calcd for C29H35FN7O2S2[M + H]+ 596.22722. Found: 596.22754.
4.3.17. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl 4-(2-hydroxyethyl)piperazine-1-carbodithioate (30q).
White solid,Yield: 32%, Mp: 177.2-179.4 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.38 (s,
1H), 8.99 (s, 1H), 8.07 (d, J = 3.3 Hz, 1H), 7.96 (s, 1H), 7.55 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 7.8 Hz,
1H), 7.42 (d, J = 7.8 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 6.79 (d, J = 8.7 Hz, 2H), 6.47 (dd, J = 16.9, 10.1
Hz, 1H), 6.27 (d, J = 16.9 Hz, 1H), 5.76 (d, J = 10.1 Hz, 1H), 4.49 (s, 1H), 4.25 (s, 2H), 4.12 (t, J = 6.1
Hz, 2H), 3.93 (s, 2H), 3.66 (t, J = 6.0 Hz, 2H), 3.53 (s, 2H), 2.51 (t, J = 7.9 Hz, 6H), 2.44 (t, J = 5.9 Hz,
2H).13C NMR (101 MHz, DMSO-d6) δ 194.88, 163.56, 156.13 (d, J = 2.6 Hz), 153.07, 150.22 (d, J =
10.8 Hz), 142.05, 141.25 (d, J = 18.4 Hz), 139.61,139.52 (d, J = 5.7 Hz), 134.86, 132.38, 129.11,
127.32, 120.68, 117.80, 115.27, 114.76, 113.88, 66.50, 59.93, 58.94, 52.98, 51.64, 50.29, 35.90.HRMS
(ESI) m/z calcd for C28H33FN7O3S2[M + H]+ 598.20648. Found: 598.20544.
4.3.18. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl 4-acetylpiperazine-1-carbodithioate (30r).
Yellow solid, Yield: 57%, Mp: 165.3-166.5 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.39
(s, 1H), 9.00 (s, 1H), 8.08 (d, J = 3.7 Hz, 1H), 7.95 (s, 1H), 7.55 (d, J = 9.1 Hz, 2H), 7.51 (d, J = 8.1 Hz,
1H), 7.42 (d, J = 8.6 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.1 Hz, 2H), 6.47 (dd, J = 17.0, 10.1
Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 4.44-4.18 (m, 2H), 4.14 (t, J =
6.3 Hz, 2H), 4.09 – 3.91 (m, 2H), 3.68 (t, J = 6.3 Hz, 2H), 3.60 (dd, J = 10.8, 7.2 Hz, 4H), 2.03 (s,
3H).13C NMR (101 MHz, DMSO-d6) δ 195.56, 169.14, 163.56, 156.11 (d, J = 2.8 Hz), 153.07, 150.24
(d, J = 10.8 Hz), 142.05, 141.20 (d, J = 20.5 Hz), 139.60, 139.52 (d, J = 4.4 Hz), 134.86, 132.38,
129.11, 127.34, 120.71, 117.83, 115.28, 114.76, 113.88, 66.42, 51.07, 49.72, 44.90, 35.93,
21.69.HRMS (ESI) m/z calcd for C28H31FN7O3S2[M + H]+ 596.19083. Found: 596.19122.
4.3.19. tert-butyl 4-(((2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl)thio)carbonothioyl)piperazine-1-carboxylate (30s).
White solid, Yield: 39%, Mp: 162.8-163.4 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.39
(s, 1H), 8.99 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.95 (s, 1H), 7.55 (d, J = 9.0 Hz, 2H), 7.51 (d, J = 8.1 Hz,
1H), 7.42 (d, J = 8.1 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.0 Hz, 2H), 6.47 (dd, J = 17.0, 10.1
Hz, 1H), 6.27 (dd, J = 17.0, 1.9 Hz, 1H), 5.76 (dd, J = 10.1, 1.9 Hz, 1H), 4.26 (s, 2H), 4.13 (t, J = 6.3
Hz, 2H), 3.97 (s, 2H), 3.68 (t, J = 6.2 Hz, 2H), 3.52 – 3.44 (m, 4H), 1.42 (s, 9H).13C NMR (101 MHz,
DMSO-d6) δ 195.58, 163.56,156.10 (d, J = 2.5 Hz), 154.16, 153.07, 150.25 (d, J = 10.7 Hz), 142.05,
141.17 (d, J = 19.9 Hz), 139.60,139.51 (d, J = 3.4 Hz), 134.85, 132.38, 129.10, 127.32, 120.72, 117.83,
115.28, 114.76, 113.89, 79.86, 66.42, 51.25, 49.71, 35.94, 28.49.HRMS (ESI) m/z calcd for
C31H37FN7O4S2[M + H]+ 654.23270. Found: 654.23297.
4.3.20. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl 4-phenylpiperazine-1-carbodithioate (30t).
28

JOURNAL PRE-PROOF
White solid, Yield: 25%, Mp: 205.8-206.6 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.39
(s, 1H), 9.00 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.95 (s, 1H), 7.56 (d, J = 9.0 Hz, 2H), 7.52 (d, J = 8.1 Hz,
1H), 7.43 (d, J = 8.0 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 7.24 (dd, J = 8.4, 7.5 Hz, 2H), 6.94 (d, J = 8.0
Hz, 2H), 6.81 (t, J = 8.3 Hz, 3H), 6.48 (dd, J = 17.0, 10.1 Hz, 1H), 6.28 (dd, J = 17.0, 1.9 Hz, 1H), 5.76
(dd, J = 10.1, 1.9 Hz, 1H), 4.40 (s, 2H), 4.15 (t, J = 6.3 Hz, 2H), 4.10 (s, 2H), 3.70 (t, J = 6.3 Hz, 2H),
3.36 – 3.24 (m, 4H).13C NMR (101 MHz, DMSO-d6) δ 195.22, 163.56, 156.12 (d, J = 2.8 Hz), 153.09,
150.49, 150.23 (d, J = 10.8 Hz), 142.05, 141.21 (d, J = 19.0 Hz), 139.61, 139.53 (d, J = 5.1 Hz). 134.86,
132.38, 129.51, 129.11, 127.33, 120.71, 119.73, 117.82, 115.92, 115.28, 114.78, 113.87, 66.48, 51.26,
49.82, 48.04, 35.92.HRMS (ESI) m/z calcd for C32H33FN7O2S2[M + H]+ 630.21157. Found: 630.21112.
4.3.21. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl 4-benzylpiperazine-1-carbodithioate (30u).
White solid, Yield: 57%, Mp: 167.8-168.4 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.39
(s, 1H), 9.00 (s, 1H), 8.08 (d, J = 3.7 Hz, 1H), 7.96 (s, 1H), 7.55 (d, J = 9.0 Hz, 2H), 7.52 (d, J = 8.2 Hz,
1H), 7.42 (d, J = 8.2 Hz, 1H), 7.37 – 7.24 (m, 6H), 6.79 (d, J = 9.0 Hz, 2H), 6.47 (dd, J = 17.0, 10.1 Hz,
1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz, 1H), 4.27 (s, 2H), 4.12 (t, J = 6.3 Hz,
2H), 3.94 (s, 2H), 3.66 (t, J = 6.3 Hz, 2H), 3.52 (s, 2H), 2.46 (s, 4H).13C NMR (101 MHz, DMSO-d6) δ
194.95, 163.55, 156.14 (d, J = 2.7 Hz), 153.07, 150.22 (d, J = 10.8 Hz), 142.05, 141.26 (d, J = 19.7 Hz),
139.61, 139.53 (d, J = 5.8 Hz), 138.02, 134.87, 132.38, 129.41, 129.11, 128.71, 127.59, 127.33, 120.67,
117.82, 115.27, 114.76, 113.87, 66.48, 61.75, 52.42, 51.60, 50.21, 35.94.HRMS (ESI) m/z calcd for
C33H35FN7O2S2[M + H]+ 644.22722. Found: 644.22760.
4.3.22. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl benzylcarbamodithioate (30v).
Yellow solid, Yield: 49%, Mp: 140.2-140.8 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.56 (t, J = 5.6 Hz,
1H), 10.13 (s, 1H), 9.38 (s, 1H), 8.99 (s, 1H), 8.08 (d, J = 3.7 Hz, 1H), 7.96 (s, 1H), 7.56 (d, J = 9.0 Hz,
2H), 7.51 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.38 – 7.25 (m, 6H), 6.79 (d, J = 9.0 Hz, 2H),
6.48 (dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 1.9 Hz, 1H), 5.76 (dd, J = 10.1, 1.9 Hz, 1H), 4.88
(d, J = 5.6 Hz, 2H), 4.10 (t, J = 6.4 Hz, 2H), 3.61 (t, J = 6.4 Hz, 2H).13C NMR (101 MHz, DMSO-d6) δ
196.89, 163.57, 156.14 (d, J = 2.8 Hz), 153.11, 150.24 (d, J = 10.8 Hz), 142.06, 141.25 (d, J = 19.1 Hz),
139.61, 139.52 (d, J = 5.6 Hz), 137.73, 134.86, 132.38, 129.11, 128.85, 128.16, 127.69, 127.33, 120.70,
117.83, 115.30, 114.77, 113.93, 66.93, 50.20, 33.92.HRMS (ESI) m/z calcd for C29H28FN6O2S2[M +
H]+ 575.16937. Found: 575.16913.
4.3.23. 2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-
yl)amino)phenoxy)ethyl 4-methoxybenzylcarbamodithioate (30w).
White solid, Yield: 71%, Mp: 132.3-133.7 ℃.1H NMR (400 MHz, DMSO-d6) δ 10.49 (t, J = 5.5 Hz,
1H), 10.13 (s, 1H), 9.39 (s, 1H), 9.00 (s, 1H), 8.08 (d, J = 3.7 Hz, 1H), 7.96 (s, 1H), 7.55 (d, J = 9.0 Hz,
2H), 7.51 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.31 – 7.23 (m, 3H), 6.90 (d, J = 8.6 Hz, 2H),
6.78 (d, J = 9.0 Hz, 2H), 6.47 (dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 1.9 Hz, 1H), 5.78 – 5.71
(m, 1H), 4.79 (d, J = 5.5 Hz, 2H), 4.08 (t, J = 6.4 Hz, 2H), 3.73 (s, 3H), 3.60 (t, J = 6.4 Hz, 2H).13C
NMR (101 MHz, DMSO-d6) δ 196.38, 163.57, 159.01, 156.13 (d, J = 2.6 Hz), 153.09, 150.23 (d, J =
10.7 Hz), 142.05, 141.25 (d, J = 18.9 Hz), 139.60, 139.52 (d, J = 5.6 Hz), 134.84, 132.36, 129.69,
129.62, 129.11, 127.35, 120.67, 117.81, 115.28, 114.74, 114.24, 113.89, 66.92, 55.54, 49.77,
29