Discovery of tofacitinib derivatives as orally active antitumor agents based on the scaffold hybridization strategy

Article abstract of DOI:10.1016/j.ejmech.2020.112601

In this work, a novel series of tofacitinib analogs were designed and synthesized based on the scaffold hybridization strategy and then evaluated for their antiproliferative activity toward three gastric cancer cell lines, leading to the identification of compound C18 which exhibited potent inhibitory activity against MGC-803 cell lines with an IC₅₀ value of 2.68 μM. Compound C18 could effectively inhibit the colony formation, suppress the cell migration and induce apoptosis of MGC-803 cells through activating the p38 and JNK signaling pathways, while C18 showed no obvious effect on the cell cycle distribution in MGC-803 cells. In addition, C18 could initiate mitochondrial dysfunction of MGC-803 cells. Besides, in vivo antitumor studies indicated that C18 could inhibit gastric cancer tumor growth in vivo without obvious global toxicity.

Full text of DOI:10.1016/j.ejmech.2020.112601

Journal Pre-proof
Discovery of Tofacitinib Derivatives as Orally Active Antitumor Agents Based on the
Scaffold Hybridization Strategy
Xiao-Jing Shi, Shuai Wang, Xiao-Jing Li, Xiao-Han Yuan, Li-Juan Cao, Bin Yu, Hong-
Min Liu
PII:
S0223-5234(20)30573-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112601
EJMECH 112601
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 March 2020
Revised Date: 31 May 2020
Accepted Date: 15 June 2020
Please cite this article as: X.-J. Shi, S. Wang, X.-J. Li, X.-H. Yuan, L.-J. Cao, B. Yu, H.-M. Liu, Discovery
of Tofacitinib Derivatives as Orally Active Antitumor Agents Based on the Scaffold Hybridization
Strategy, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112601.
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Discovery of Tofacitinib Derivatives as Orally Active Antitumor Agents Based on
the Scaffold Hybridization Strategy
Xiao-Jing Shi a,#, Shuai Wang a,b,#, Xiao-Jing Li a, #, Xiao-Han Yuan a, Li-Juan Cao a,
Bin Yu a,c,* and Hong-Min Liu a,*
a
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001,
China;
b
Gordon Center for Medical Imaging, Massachusetts General Hospital and Harvard
Medical School, Boston, MA 02129, USA;
c State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing,
210023, China;
* Corresponding authors:
Dr. Bin Yu (zzuyubin@hotmail.com)
Prof. Hong-Min Liu (liuhm@zzu.edu.cn)
# Xiao-Jing Shi, # Shuai Wang, and #Xiao-Jing Li contributed equally to this work.
1 / 40

Abstract:
In this work, a novel series of tofacitinib analogs were designed and synthesized
based on the scaffold hybridization strategy and then evaluated for their
antiproliferative activity toward three gastric cancer cell lines, leading to the
identification of compound C18 which exhibited potent inhibitory activity against
MGC-803 cell lines with an IC50 value of 2.68 μM. Compound C18 could effectively
inhibit the colony formation, suppress the cell migration and induce apoptosis of
MGC-803 cells through activating the p38 and JNK signaling pathways, while C18
showed no obvious effect on the cell cycle distribution in MGC-803 cells. In addition,
C18 could initiate mitochondrial dysfunction of MGC-803 cells. Besides, in vivo
antitumor studies indicated that C18 could inhibit gastric cancer tumor growth in vivo
without obvious global toxicity.
Keywords: Tofacitinib; Scaffold hybridization; [1,2,4]triazolo[1,5-a]pyrimidines;
Antitumor activity;
1. Introduction
Tofacitinib, an FDA-approved JAK inhibitor for the treatment of myeloproliferative
disorders, has been found to induce G1 cell-cycle arrest and inhibit tumor growth in
Epstein-Barr virus-associated T and natural killer cell lymphoma cells [1]. Besides,
the Wiita AP group reported that tofacitinib could be used as an anti-myeloma
therapeutic to reverse growth-promoting effects of the bone marrow
microenvironment
[2].
As
a
subtype
of
purine
analogs,
[1,2,4]triazolo[1,5-a]pyrimidine has been reported to possess diverse pharmacological
properties [3-7]. In our previously published work, we also found that the
[1,2,4]triazolo[1,5-a]pyrimidine derivatives showed promising inhibitory activity
against human cancer cells [7-10]. The 3-oxopropanenitrile moiety in tofacitinib is
crucial to its cysteine covalent binding [11]. The binding site locates at the protein
active site and is buried inside the hydrophobic pocket. Thus, removal of this moiety
and substituting with a non-Michael system structure could give new compounds with
interesting or unexpected bioactivity. In order to obtain new promising antitumor
2 / 40

agents, we herein rationally designed and synthesized novel tofacitinib analogues by
replacing the 3-oxopropanenitrile moiety in tofacitinib with the privileged
[1,2,4]triazolo[1,5-a]pyrimidine scaffold and also evaluated their antitumor activity
against gastric cancers in vitro and in vivo. The scaffold hybridization strategy would
be a feasible option to design new bioactive compounds.
Figure 1. Design of tofacitinib analog C18 based the scaffold hybridization strategy.
2. Results and Discussion
2.1. Chemistry
Compounds B1-B27 were synthesized following the previously reported method [8].
As shown in Scheme 1, to a solution of 4H-1,2,4-triazole-3-amine in acetic acid was
added the corresponding β-ketoester, affording compounds A1-A4. The cyclized
compounds A1-A4 was then dissolved in POCl3, and the solution was kept under
o
reflux at 90 C, producing the corresponding compounds B1-B4. Compounds B1-B4
then
reacted
with
N-methyl-N-((3S,4R)-4-methylpiperidine-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
in ethanol, yielding compounds C1-C4. Besides, we obtained the intermediates
Q1-Q22 from 5-amino-4H-1,2,4-triazole-3-thiol by reacting with different alkyl
halides. Compounds Q1-Q22 were then subjected to the cyclization through reacting
with different β-ketoesters to give compounds A5-A25. Compounds A5-A25 were
then chlorinated by POCl3 to give compounds B5-B27, which then reacted with
3 / 40

N-methyl-N-((3S,4R)-4-methylpiperidine-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
to produce compounds C5-C27. The compounds synthesized are shown in Fig. 2.
Scheme 1. Synthesis of compounds C1-C27. Reagents and conditions: (a) Acetic acid,
o
120 oC, 2-15 h, reflux. (b) POCl3, 90 C, 3-5 h, reflux; (c) Et3N, EtOH, rt, 5 h; (d)
Na2CO3, acetone, 60 oC, 3-5 h, reflux.
4 / 40

Figure 2. Modifications based on the score scaffold for SARs studies
5 / 40

2.2. Biological evaluation of tofacitinib derivatives.
Tofacitinib, an FDA-approved JAK inhibitor for the treatment of myeloproliferative
disorders, has been reported to induce G1 cell-cycle arrest and inhibit tumor growth in
Epstein-Barr virus-associated T and natural killer cell lymphoma cells [1]. Initially,
the tofacitinib analogue C1 with the 2-cyano acetyl replaced with the
[1,2,4]triazolo[1,5-a]pyrimidine scaffold was found to possess good inhibitory
activity toward four different cancer cell lines (Table 1), particularly for MGC-803
cells. Tofacitinib and 5-FU were used as controls. Compound C1 showed better
potency against the tested cancel cells than 5-FU. The data suggest that modifications
at the NH of the piperidine ring could improve the antiproliferative activity.
Table 1. In vitro antiproliferative activity of C1, Tofacitinib and 5-FU against selected
human cancer cell lines
IC50 (μM)
Compound
MGC-803
4.35±0.64
>100
H1975
22.2±1.35
>100
PC-9
20.85±1.32
>100
MCF-7
33.78±0.73
>100
C1
Tofacitinib
5-FU
3.79 ± 0.49
2.67±0.43
3.25±0.51
4.76±1.01
Inspired by the interesting results, we next performed extensive structural
modifications around the [1,2,4]triazolo[1,5-a]pyrimidine scaffold, generating
compounds C1-C27. These compounds were evaluated for their antiproliferative
activity against three gastric cancer cell lines (MGC-803, HGC-27, and BGC-823) by
the MTT assay. 5-FU was used as a positive control. As shown in Table 2, we firstly
introduced an ethyl group and a phenyl group to replace the methyl group at position
R1, affording compounds C2 and C3, respectively, but both of them showed
significantly decreased activity toward the selected cancer cells. Introduction of a
cyclopropyl ring in C1 yielded compound C4, which failed to improve its
antiproliferative activity. The data may suggest that the methyl group at position R1
6 / 40

was preferred over other large groups, and the activity of C1-C4 decreased
correspondingly with the increase of the steric hindrance of R1.
Table 2. In vitro antiproliferative activity of C1-C4 against selected cell lines.
IC50 (μM)
Compound
MGC-803
4.35±0.64
80.17±1.31
84.87±1.93
55.88±1.75
3.79 ± 0.49
HGC-27
11.32±1.05
＞100
BGC-823
67.36±1.83
82.23±1.92
＞100
C1
C2
C3
＞100
50.54±1.70
5.75 ± 1.15
＞100
C4
5-Fu
5.89±0.78
With compound C1 in hand, we next explored further SARs by installing different R2
to the triazolopyrimidine ring. As shown in Table 3, most of these compounds
displayed moderate to good antiproliferative activity against the selected gastric
cancer cell lines. Introduction of different alkyl groups to R2 led to compounds C5-C9,
which showed decreased but acceptable antiproliferative activity toward MGC-803
and HGC-27. While for BGC-823 cells, these compounds had better antiproliferative
activity than C1. Intriguingly, Compound C10 with the benzyl mercapto group at R2
position showed almost the same cytotoxic activity with compound C1 against
MGC-803, HGC-27 and about 5-fold increase of potency toward BGC-823 relative to
compound C1. We then explored the effect of the length of the carbon chain between
sulfydryl and phenyl on the antiproliferative activity. Compared to C10, C24 bearing
the –(CH2)2Ph group showed no obvious improvement of the antiproliferative activity,
while C25 bearing –(CH2)3Ph group displayed no antitumor activity, indicating the
length of the side chain was important for the activity. Molecular fine-turning of
substituents on the phenyl ring of C10 was further performed to improve its
antiproliferative activity, generating compounds C11-C20. As shown in Table 2,
installation of different halogen atom into the phenyl ring exhibited moderate to
strong inhibition against the selected gastric cancer cells. Compared to compound C1,
7 / 40

compounds C11-C12, C15-C17 and C19-C20 possessed decreased but acceptable
antiproliferative activity, while compound C13 (4-Br) showed almost the same
activity toward MGC-803. To our delight, compound C18 bearing strong
electron-withdrawing 4-CF3 group exhibited better antiproliferative activity toward all
the selected cancer cells. Compound C14 bearing a bromine atom at the 2-position of
the phenyl ring had reduced activity toward MGC-803 and BGC-823 cells compared
to C13 with a bromine atom at the 4-position of the phenyl ring. Besides, we also
replaced the benzyl ring with three different aryl heterocyclic groups, generating
compounds C21-C23, none of which had increased antiproliferative activity
compared to C18. In order to further investigate the SARs, we then incorporated
additional methyl and n-pentyl groups to the R3 position in compound C12,
respectively, resulting in compounds C26 and C27, which had significantly reduced
antiproliferative activity compared to C12.
Table 3. In vitro antiproliferative activity of C1-C27 against selected cell lines.
IC50 (μM)
Compound
MGC-803
HGC-27
BGC-823
15.54±1.19
20.94±1.32
51.16±1.71
C5
32.55±1.51
18.07±1.26
9.54±0.98
9.17±0.96
5.87±0.77
11.87±0.67
16.04±1.21
4.02±0.6
35.21±1.55
35.12±1.55
21.36±1.33
11.5±1.06
40.88±1.95
62.05±1.79
36.54±1.56
16.29±1.21
14.35±1.16
44.31±1.65
26.33±1.42
28.03±1.45
81.13±1.91
59.58±1.68
69.86±1.84
C6
C7
C8
C9
13.91±1.14
25.99±1.42
18.35±1.26
19.47±1.29
21.32±1.33
29.36±1.88
18.22±1.26
C10
C11
C12
C13
C14
C15
C16
26.98±1.43
26.95±1.43
15.22±1.18
8 / 40

5.86±0.77
2.68±0.43
52.02±1.72
9.85±0.99
＞100
15.15±1.18
9.54±1.87
＞100
69.53±1.84
23.31±0.91
98.88±1.99
18.94±1.28
＞100
C17
C18
C19
C20
C21
19.18±1.28
＞100
21.21±1.33
＞100
＞100
C22
C23
C24
C25
C26
C27
5-Fu
＞100
6.19±0.79
＞100
＞100
11.84±1.07
＞100
＞100
28.03±1.45
＞100
90.49±0.98
＞100
＞100
＞100
＞100
32.28±1.51
5.89±0.78
3.79 ± 0.49
5.75 ± 1.15
Encouraged by the above interesting results, we also investigated the possible toxicity
of these tofacitinib analogues toward GES-1 (normal human gastric epithelial cell
line). As shown in Table 4, compounds C1-C27 showed moderate to weak inhibition
toward GES-1, among which the most potent compound C18 (IC50 = 2.68 μM against
MGC-803) inhibited GES-1 with an IC50 value of 17.32 μM, exhibiting 6.5-fold
selectivity.
Table 4. In vitro inhibitory activity of C1-C27 against GES-1
IC50(μM)
IC50 (μM)
Compound
Compound
GES-1
GES-1
36.56±1.76
＞100
57.85±1.76
＞100
C1
C2
C3
C4
C5
C6
C15
C16
C17
C18
C19
C20
82.45±1.76
17.32±1.76
＞100
＞100
＞100
67.08±1.76
＞100
87.46±1.76
9 / 40

＞100
＞100
40.18±1.7
＞100
C7
C8
C21
C22
C23
C24
C25
C26
C27
5-Fu
27.92±1.76
98.16±1.76
67.18±1.76
12.33±1.76
＞100
C9
66.21±1.76
＞100
C10
C11
C12
C13
C14
99.16±1.46
＞100
55.59±1.46
＞100
2.37±0.93
2.3. The effect of C18 on colony formation, migration and cell apoptotic morphology
of MGC-803 cells.
In view of the potent antiproliferative activity of compound C18 against MGC-803,
we then explored the effect of C18 on colony formation, migration and cell apoptotic
morphologies. As shown in Figs. 3A and 3B, when MGC-803 cells were treated with
indicated concentrations (1, 2 and 3 μM) of C18 for 9 days, the colony number of
C18 treated groups significantly decreased and the inhibition rate of colony formation
was about 31%, 54%, 73%, respectively, compared with that of control group.
Besides, as depicted in Figs. 3C and 3D, compound C18 could
concentration-dependently inhibit the cell migratory ability of MGC-803 cells. In
addition, as shown in Figs. 3E and 3F, MGC-803 cells treated with C18 exhibited
apoptosis-related morphologies, such as cell rounding up, cell debris and nuclear
fragmentation and condensation. These results indicated that compound C18 could
effectively inhibit the proliferation and survival of MGC-803 cells.
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Figure 3. The effect of C18 on cell proliferation, migration and cell apoptotic
morphologies of MGC-803 cells. (A) and (B) Clonogenicity of the MGC-803 cells
treated with indicated concentrations (0, 1, 2 and 3 μM) of C18 for 9 days. (C) and (D)
Cell migration in MGC-803 cells treated with indicated concentrations (0, 5, 7.5 and
10 μM) of C18 for 24 h was determined by the wound healing assay. (E) Morphology
and (F) the nuclear morphology changes of MGC-803 cells treated with C18 at the
indicated concentrations (0, 5, 7.5 and 10 μM). All results performed above are
presented as mean ± SD from three independent experiments. ** P < 0.01, compared
with the control group.
2.4. The effect of C18 on cell apoptosis and cell cycle distribution of MGC-803 cells
In order to further investigate the mechanism of the inhibitory effect caused by C18
on gastric cancer, we next performed flow cytometric analysis to quantitatively
analyze the apoptosis-related change in MGC-803 cells. As shown in Figs. 4A and 4C,
11 / 40

treatment of MGC-803 cells with different concentrations (5, 7.5 and 10 μM) of C18
for 24 h resulted in a significant corresponding increase of FITC-Annexin V/PI
positive population, the apoptotic percentage was up to 40.7% for the group treated
with 10 μM of C18, significantly higher than that of the control group (7.7%).
Similarly, as shown in Figs. 4B and 4D, MGC-803 cells treated with the same
concentration of C18 for 48 h also significantly increased the percentage of the
apoptotic population, and the apoptotic percentage for the group treated with 10 μM
of C18 was 82.1%. In addition, the cell cycle distribution treated with C18 at the
indicated concentrations (0, 5, 7.5, and 10 μM) for 24 h were also investigated by
flow cytometry. However, C18 treatment did not cause any significant alteration of
cell cycle distribution in MGC-803 cells (Fig. 4E). These above results indicated that
C18 could concentration- and time-dependently induce both early and late apoptosis
in MGC-803 cells while it showed no obvious effect on cell cycle distribution.
12 / 40

Figure 4. The effect of C18 on the cell apoptosis and cell cycle distribution in
MGC-803 cells. (A) and (B) MGC-803 cells were treated by C18 for 24 or 48 h;
apoptosis cells were detected by using the Annexin V-FITC/PI double staining and
analyzed by flow cytometry. (C) and (D) are the quantitation of those apoptotic cells
in (A) and (B). (E) MGC-803 cells were treated with C18 at the indicated
concentrations (0, 5, 7.5, and 10 μM). After treatment for 24 h, the cell cycle
distributions were then analyzed by flow cytometry. All results performed above are
presented as mean ± SD from three independent experiments. ** P < 0.01, compared
with the control group.
2.5. C18 induced mitochondrial dysfunction in MGC-803 cells
In order to further explore the underlying mechanism of apoptosis induced by C18,
the effect on the mitochondrial signal pathway, one of the most important
apoptosis-related pathway [12, 13], was then investigated in MGC-803 cells after
13 / 40

treatment with C18. Changes in mitochondrial membrane potential (MMP) could be
used to reflect the mitochondrial function, and JC-1 dye was used to detect MMP.
With the decrease of MMP, JC-1 dye exhibits potential-dependent released from
mitochondria to the cytoplasm, accomplished by a fluorescence emission shift from
red to green and an increase green/ red fluorescence ratio [14]. After treatment of
MGC-803 cells with C18 (5, 7.5 and 10 μM) for 24 h, C18 increased the green
fluorescence intensity in a dose-dependent manner (Fig. 5A). As shown in Figs. 5B
and 5C, compared with the control group, the ratio of the green/red fluorescence
intensity increased in a concentration-dependent manner, the ratio was up to 8.73-fold
for the group treated with 10 μM of C18.
The bcl-2 family proteins, consisting of anti-apoptotic and pro-apoptotic members,
play key roles in mitochondrial apoptotic pathway. Once apoptosis occurs, the
expression of anti-apoptotic protein decreases, while the expression of pro-apoptotic
protein increases, further inducing the release of cytochrome c into the cytoplasm
from mitochondrial and subsequently activating caspases that cleave a set of cellular
proteins to cause apoptotic changes [14, 15]. As shown in Figs. 5D-5I, C18 markedly
decreased the expression of anti-apoptotic proteins Bcl-2 and Mcl-1, and increased
expression of pro-apoptotic protein Bax, which further activated the release of
cytochrome c from mitochondrial to cytoplasm in a concentration-dependent manner.
14 / 40

Figure 5. C18 induced the mitochondrial dysfunction of MGC-803 cells and changed
the expression of mitochondria-related apoptotic proteins. MGC-803 cells were
treated with C18 for 24 h, then the cells were stained with JC-1 and imaged by
fluorescent microscopy (A) or quantitatively analyzed by flow cytometry (B) and (C).
(D) C18 changed the expressions of Bcl-2 family (Bcl-2, Mcl-1, Bax) proteins and
induced release of cytochrome c; (E-I) Statistical analysis of these proteins expression
change induced by C18. All results performed above are presented as mean ± SD
from three independent experiments. * P < 0.05 and ** P < 0.01, compared with the
control group.
Moreover, we also performed western blot assay to examine the effect of C18 on the
activation of mitochondrial dysfunction related caspases including caspase 9, caspase
15 / 40

3, PARP, and a DNA damage marker γH2A.X. As shown in Fig. 6, C18 treatment
up-regulated expression levels of cleavage caspase 9, cleavage caspase 3, and
cleavage caspase 7, cleavage PARP, γH2A.X and down-regulated expression levels of
pro caspase 9, pro caspase 3 and pro caspase 7. These above results showed that C18
could increase active caspase level, which might mediate the apoptosis in MGC-803
cells.
Figure 6. The expressions of caspase 9, caspase 3, caspase 7, PARP, and γH2A.X
after C18 treatment in MGC-803 cells were detected by Western blot analysis.
MGC-803 cells were treated with C18 (5, 7.5, and 10 μM) for 24 h, and the
expression levels and quantization of caspase 9, caspase 3, caspase 7, PARP, and
γH2A were detected by western blot. All results performed above are presented as
mean ± SD from three independent experiments. * P < 0.05 and ** P < 0.01,
compared with the control group.
16 / 40

2.6. C18 induces apoptosis of MGC-803 cells partly through activating the p38 and
JNK signaling pathways
The key mitogen-activated protein kinase (MAPK) pathway, containing the
extracellular signal-regulated kinase 1/2 (Erk1/2), p38, c-jun NH2-terminal kinase
(JNK), and Erk5, is involved in regulating many stress-induced physiological
processes, including cell growth, and apoptosis [16]. Among these MAPKs, Erk 1/2
were identified as growth factor-stimulated proteins which mediate cell motility,
proliferation, differentiation and survival [17]. JNK and p38 kinases are activated in
response to various stimuli, such as oxidant stress and DNA damage and previous
studies showed that p38 and JNK pathway activation can promote apoptosis [18, 19].
Erk5 could be also activated by a variety of extracellular stimuli, such as cellular
stresses and growth factors, to regulate processes such as proliferation and other
critical functions [20]. In this study, western blot results showed that the total
expressions of Erk1/2, p38, JNK, Erk5 as well as phosphorylated Erk1/2 and ERK5
were found to be unchanged in MGC-803 cells treated with different concentrations
of C18, while the expressions of phosphorylated p38 and JNK increased significantly
(Figs. 7A-7I). To verify whether p38 and JNK sub-MAPK pathway could play a
major important role in C18-induced cell cytotoxicity, MGC-803 cells were
pre-incubated with 4 μM of p38 inhibitor (SB203580) or 20 μM of JNK inhibitor
(SP600125) for 4 h before C18 treatment. As depicted in Fig. 7J, both the p38
inhibitor and the JNK inhibitor could increase the cell viability, indicating that
C18-induced apoptosis in MGC-803 cells was partly mediated by the p38 and JNK
MAPK pathways.
17 / 40

Figure 7. C18 activated p38 and JNK MAPK family proteins in MGC-803 cells.
(A)-(I) Western blot and quantitated analysis of MAPK proteins (Phospho- and total
forms of Erk1/2, p38, JNK, and Erk5) in MGC-803 cells treated with C18. (J)
MGC-803 cells were pretreated with p38 and JNK inhibitors: SB203580, and
SP600125, respectively, followed by treatment with 10 μM C18 for 24 h; The cell
viability was determined by the MTT assay and expressed as a percentage of the
control. All results performed above are presented as mean ± SD from three
independent experiments. *P < 0.05，** P < 0.01，and ## P < 0.01, compared with the
control group.
2.7 In vivo antitumor studies of C18 on xenograft model bearing MGC-803 cells.
Encouraged by the potent antiproliferative activity of C18 toward gastric cancer
MGC-803 cells in vitro, we next determined the in vivo antitumor effect of C18 on
xenograft model bearing MGC-803 cells by subcutaneous implantation, and 5-FU was
18 / 40

used as a positive control. The mouse body weight and tumor size were measured
every 3 days. As shown in Fig. 8A, C18 did not cause obvious changes in body
weight and general health, compared with the controls. The average tumor volume of
C18 treatment groups significantly decreased in comparison with the controls (Fig.
8B). After treatment with 12.5 mg/Kg and 25 mg/Kg of C18, the corresponding
average tumor weight decreased to 56.8 % and 53.0% of that treated with 0.5%
CMC-Na (Figs. 8C and 8D). Histopathological examination of the heart, liver, spleen,
lung, and kidney revealed no signs of toxicity after C18 treatment (Fig. 8E). All of
these data indicated that C18 was orally active toward xenograft model bearing
MGC-803 cells without obvious global toxicity.
19 / 40

Figure 8. The anti-tumor growth effect of C18 on xenograft model bearing MGC-803
cells. MGC-803 cells were transplanted subcutaneously to the right sides of SCID
mice and subjected to C18 (12.5 and 25 mg/kg), 5-FU (10 mg/kg) and 0.5% CMC-Na
for 21 days. (A) The bodyweight and (B) tumor size were measured every 2 days
from MGC-803 mice after C18 treatment. (C) Photographs of tumors and (D) the final
tumor weight in each group. (E) Histopathological examination of major organs in
C18-treated mice. Data are presented as means ± SD. * P < 0.05 was considered
statistically significant compared with the control.
20 / 40

3. Conclusions
In this work, we designed and synthesized a novel series of tofacitinib analogs based
the scaffold hybridization strategy and then evaluated their preliminary inhibitory
activity toward human cancer cell lines, leading to the discovery of C18. C18 showed
potent inhibitory activity against MGC-803 cell. Mechanistic studies showed that
compound C18 could effectively inhibit the colony formation, limit the cell migratory
ability of MGC-803 cells and induce apoptosis of MGC-803 cells, while C18 showed
no obvious effect on the cell cycle distribution. In addition, C18 could induce the
mitochondrial dysfunction and apoptosis through activation of the p38 and JNK
signaling pathways in MGC-803 cells. Besides, in vivo antitumor studies indicated
that C18 could inhibit gastric cancer tumor growth in vivo without obvious global
toxicity.
4. Experimental section
4.1. General Information.
Reagents and solvents were purchased from commercial sources and used without
further purification. Thin-layer chromatography (TLC) was carried out on glass plates
coated with silica gel (Qingdao Haiyang Chemical Co., G60F-254) and visualized by
UV light (254 nm). The products were purified by column chromatography over silica
gel (Qingdao Haiyang Chemical Co., 200–300 mesh). Melting points were
determined on an X-5 micromelting apparatus and are uncorrected. All the NMR
spectra were recorded with a Bruker DPX 400 MHz spectrometer with TMS as the
internal standard in CDCl3 or DMSO-d6. Chemical shifts are given as δ ppm values
relative to TMS. High-resolution mass spectra (HRMS) were recorded on a Waters
Micromass Q-T of Micromass spectrometer by electrospray ionization (ESI).
4.2. The general method for the synthesis of Q1-Q22.
Compounds Q1-Q22 were synthesized following the previously reported method as
described for the synthesis of Q1 [8]. 3-Amino-5-mercapto-1,2,4-triazole (1.0 g, 8.61
mmol) reacted with different n-bromopropane (9.47 mmol) in the presence of sodium
carbonate (1.37g, 12.92 mmol) and sodium iodide (129.06 mg, 0.861 mmol) in
21 / 40

acetone (30 mL). Upon completion of the reaction monitored by thin-layer
chromatography, Na2CO3 and NaI were removed via filtration. The filtrate was
concentrated under vacuum and then dissolved in EtOAc. The organic layer was
washed with water (2 ×10 mL) and brine (2 × 20 mL), which was then dried over
anhydrous MgSO4. After filtration, the solvent was removed under vacuum. The
resulting residue was then purified by column chromatography, affording product Q1.
4.3. The general method for the synthesis of A1-A27.
Compounds A1-A27 were prepared following the previously reported method as
described for A1 [8]. Q1 (1 g, 6.32 mmol) reacted with ethyl acetoacetate (1.23 g,
o
9.48 mmol) in AcOH (10 mL) at 120 C for 3-6 h. Upon completion, the reaction
mixture was cooled to room temperature and the white precipitate formed was filtered
and washed with water to afford the desired compound A1.
4.4. The general method for the synthesis of B1-B27.
Compounds B1-B27 were prepared following the previously reported method [8].
A1 was stirred in POCl3 (excess) at 90 oC for 3-5 h. The resultant oil was added
dropwise to ice cold bath. The resulting aqueous mixture was extracted with CH2Cl2
(4 × 30 mL) and the organic layers were washed by saturated aqueous NaHCO3 (3×10
mL). The organic layers were dried over MgSO4 and concentrated under reduced
pressure to give the yellow solid B1, which was used directly without further
purification.
4.5. The general method for the synthesis of C1-C27.
The general method was described for the synthesis of C1. Compound B1 (150 mg,
0.618 mmol) was dissolved in ethanol (2 mL), followed by addition of compound
N-methyl-N-((3S,4R)-4-methyl piperidine-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
(151.61mg, 0.618 mmol) and triethylamine (93.80 mg, 926.97 mmol), the mixture
was stirred at room temperature for 5 h. Upon completion monitored by thin-layer
chromatography, the resultant mixture was concentrated under reduced pressure, the
residue was then purified by column chromatography to generate compound C1.
22 / 40

o
1
Compound C1, white solid, yield: 45 %. m.p.: 152-156 C. H NMR (400 MHz,
CDCl3) δ 8.26 (dd, J = 7.5, 6.5 Hz, 2H), 7.08 (d, J = 3.6 Hz, 1H), 6.55 (s, 1H), 6.20 (s,
1H), 5.48 (s, 1H), 4.19 (dd, J = 12.1, 6.3 Hz, 1H), 4.02 (dd, J = 12.6, 4.1 Hz, 2H),
3.75 – 3.65 (m, 1H), 3.53 (d, J = 1.5 Hz, 3H), 2.58 – 2.45 (m, 4H), 2.00 (d, J = 26.0
13
Hz, 2H), 1.11 (d, J = 5.9 Hz, 3H). C NMR (100 MHz, CDCl3) δ 164.90, 158.07,
157.23, 154.15, 151.87, 150.31, 120.51, 103.15, 102.26, 95.00, 52.07, 49.83, 46.39,
35.69, 32.41, 30.72, 25.19, 15.30. HRMS (ESI): m/z calcd for C19H24N9 (M+H)-,
378.2155; found, 378.2155.
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1
Compound C2, white solid, yield: 62 %. m.p.: 174-179 C. H NMR (400 MHz,
CDCl3) δ 8.32 (d, J = 15.6 Hz, 2H), 8.10 (dd, J = 6.2, 2.6 Hz, 2H), 7.54 – 7.44 (m,
3H), 7.09 (d, J = 3.5 Hz, 1H), 6.78 (s, 1H), 6.60 (d, J = 3.5 Hz, 1H), 5.60 (d, J = 4.5
Hz, 1H), 4.33 (dd, J = 12.7, 6.1 Hz, 1H), 4.24 – 4.15 (m, 1H), 4.09 (dd, J = 12.6, 3.8
Hz, 1H), 3.75 (dd, J = 10.3, 6.3 Hz, 1H), 3.60 (d, J = 10.1 Hz, 3H), 2.63 (s, 1H), 2.53
(m, 1H), 2.16 – 1.98 (m, 2H), 1.15 (d, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ 162.01, 158.14, 157.63, 154.75, 152.03, 150.99, 150.64, 137.40, 130.69, 128.80,
127.66, 120.21, 103.11, 102.46, 92.01, 65.84, 51.92, 50.15, 46.67, 35.81, 32.57, 30.81,
15.47, 15.26. HRMS (ESI): m/z calcd for C24H25N9 (M+H)-, 440.2311; found,
440.2310.
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1
Compound C3, white solid, yield: 81 %. m.p.: 161-165 C. H NMR (400 MHz,
CDCl3) δ 8.29 (d, J = 7.7 Hz, 2H), 7.11 (d, J = 3.6 Hz, 1H), 6.58 (d, J = 3.5 Hz, 1H),
6.23 (s, 1H), 5.52 (d, J = 4.5 Hz, 1H), 4.22 (dd, J = 12.6, 6.5 Hz, 1H), 4.07 (m, 2H),
3.76 – 3.67 (m, 1H), 3.56 (s, 3H), 2.83 (q, J = 7.6 Hz, 2H), 2.51 (m, 1H), 2.11 – 1.94
23 / 40

(m, 2H), 1.34 (t, J = 7.6 Hz, 3H), 1.14 (d, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 169.83, 158.08, 157.33, 154.15, 151.92, 150.59, 150.41, 120.41, 103.11,
102.29, 94.02, 51.98, 49.90, 46.42, 35.70, 32.45, 31.89, 30.71, 15.34, 13.13. HRMS
(ESI): m/z calcd for C20H26N9 (M+H)-, 392.2311; found, 392.2310.
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1
Compound C4, white solid, yield: 61 %. m.p.: 151-157 C. H NMR (400 MHz,
CDCl3) δ 8.25 (d, J = 20.1 Hz, 2H), 7.07 (d, J = 3.5 Hz, 1H), 6.56 (d, J = 3.5 Hz, 1H),
5.45 (d, J = 4.6 Hz, 1H), 4.03 (d, J = 4.4 Hz, 3H), 3.54 (s, 4H), 3.19 – 2.94 (m, 4H),
13
2.56 – 2.40 (m, 1H), 2.26 – 1.97 (m, 4H), 1.13 (d, J = 7.0 Hz, 3H). C NMR (100
MHz, CDCl3) δ 174.85, 158.09, 157.31, 154.05, 151.95, 150.55, 146.44, 120.22,
110.11, 103.04, 102.35, 52.70, 51.14, 47.05, 35.56, 34.74, 32.35, 31.26, 29.46, 24.01,
15.57. HRMS (ESI): m/z calcd for C21H26N9 (M+H)-, 404.2311; found, 404.2312.
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Compound C5, white solid, yield: 75 %. m.p.: 163-167 C. H NMR (400 MHz,
CDCl3) δ 8.30 (s, 1H), 7.09 (d, J = 3.6 Hz, 1H), 6.58 (d, J = 3.5 Hz, 1H), 6.12 (s, 1H),
5.50 (d, J = 4.7 Hz, 1H), 4.26 (dd, J = 12.7, 6.3 Hz, 1H), 4.07 – 3.90 (m, 2H), 3.72 –
3.63 (m, 1H), 3.56 (s, 3H), 3.07 (td, J = 7.0, 1.3 Hz, 2H), 2.52 (s, 4H), 2.09 – 1.91 (m,
3H), 1.74 – 1.61 (m, 2H), 1.12 (d, J = 7.0 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 166.42, 164.23, 158.08, 157.68, 152.02, 150.58, 149.18, 120.26,
103.06, 102.36, 94.38, 51.90, 49.85, 46.36, 35.82, 33.12, 32.49, 30.74, 25.05, 22.98,
15.45, 13.19. HRMS (ESI): m/z calcd for C22H30N9S (M+H)-, 452.2345; found,
452.2343.
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Compound C6, white solid, yield: 66 %. m.p.: 136-142 C. H NMR (400 MHz,
CDCl3) δ 8.29 (s, 1H), 7.09 (d, J = 3.6 Hz, 1H), 6.58 (d, J = 3.5 Hz, 1H), 6.13 (s, 1H),
24 / 40

5.97 – 5.78 (m, 1H), 5.50 (d, J = 4.1 Hz, 1H), 5.17 (d, J = 16.9 Hz, 1H), 4.98 (d, J =
10.0 Hz, 1H), 4.27 (dd, J = 12.7, 6.0 Hz, 1H), 4.13 – 3.87 (m, 2H), 3.73 (d, J = 6.8 Hz,
2H), 3.64 (dd, J = 10.1, 6.0 Hz, 1H), 3.57 (s, 3H), 2.51 (d, J = 9.1 Hz, 3H), 2.47 (d, J
13
= 7.0 Hz, 1H), 2.13 – 1.90 (m, 2H), 1.12 (d, J = 7.0 Hz, 3H). C NMR (100 MHz,
CDCl3) δ 165.60, 164.35, 158.08, 157.69, 152.02, 150.59, 149.24, 133.38, 120.24,
117.76, 103.07, 102.36, 94.47, 51.92, 49.90, 46.38, 35.84, 33.84, 32.46, 30.77, 25.07,
15.47. HRMS (ESI): m/z calcd for C22H28N9S (M+H)-, 450.2188; found, 450.2189.
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1
Compound C7, white solid, yield: 76 %. m.p.: 126-130 C. H NMR (400 MHz,
CDCl3) δ 8.29 (s, 1H), 7.09 (d, J = 3.5 Hz, 1H), 6.57 (d, J = 3.4 Hz, 1H), 6.15 (s, 1H),
5.49 (d, J = 3.9 Hz, 1H), 4.24 (dd, J = 12.7, 6.4 Hz, 1H), 4.00 (dd, J = 12.6, 3.7 Hz,
2H), 3.84 (s, 2H), 3.69 (dd, J = 10.3, 5.9 Hz, 1H), 3.55 (s, 3H), 2.52 (s, 4H), 2.34 (s,
13
2H), 2.11 – 1.95 (m, 4H), 1.14 (d, J = 7.0 Hz, 3H). C NMR (100 MHz, CDCl3) δ
164.56, 164.39, 158.06, 157.76, 152.06, 150.56, 149.26, 120.35, 103.07, 102.29,
94.60, 79.30, 70.86, 65.84, 52.02, 49.86, 46.42, 35.78, 32.42, 30.77, 25.09, 22.71,
19.38, 15.44. HRMS (ESI): m/z calcd for C22H26N9S (M+H)-, 448.2032; found,
448.2031.
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Compound C8, white solid, yield: 53 %. m.p.: 135-139 C. H NMR (400 MHz,
CDCl3) δ 8.29 (s, 1H), 7.09 (d, J = 3.6 Hz, 1H), 6.55 (d, J = 3.5 Hz, 1H), 6.10 (s, 1H),
s3.60 (m, 1H), 3.54 (s, 3H), 3.11 – 2.92 (m, 2H), 2.52 – 2.40 (m, 4H), 2.01 – 1.87 (m,
13
2H), 1.09 (d, J = 7.0 Hz, 4H), 0.48 – 0.37 (m, 2H), 0.18 – 0.07 (m, 2H). C NMR
(100 MHz, CDCl3) δ 166.40, 164.25, 158.06, 157.66, 152.05, 150.50, 149.18, 120.35,
103.08, 102.31, 94.40, 51.83, 49.84, 46.38, 36.97, 35.87, 32.49, 30.75, 25.05, 15.46,
25 / 40

10.87, 5.71, 5.70. HRMS (ESI): m/z calcd for C23H30N9S (M+H)-, 464.2345; found,
464.2343.
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Compound C9, white solid, yield: 49 %. m.p.: 145-148 C. H NMR (400 MHz,
CDCl3) δ 8.30 (s, 1H), 7.10 (d, J = 3.6 Hz, 1H), 6.58 (d, J = 3.6 Hz, 1H), 6.12 (s, 1H),
5.52 (d, J = 4.5 Hz, 1H), 4.26 (dd, J = 12.7, 6.2 Hz, 1H), 4.08 – 3.90 (m, 2H), 3.72 –
3.62 (m, 1H), 3.57 (s, 3H), 3.03 (m, 2H), 2.51 (d, J = 11.2 Hz, 4H), 2.03 – 1.88 (m,
2H), 1.76 (t, J = 14.2 Hz, 2H), 1.68 – 1.49 (m, 4H), 1.19 – 1.03 (m, 6H), 0.84 (m, 2H).
13C NMR (100 MHz, CDCl3) δ 166.75, 164.20, 158.08, 157.67, 152.01, 150.53,
149.19, 120.23, 103.08, 102.42, 94.39, 51.80, 49.86, 46.37, 38.11, 37.82, 35.86, 32.54,
32.41, 30.74, 26.22, 25.99, 25.06, 15.48. HRMS (ESI): m/z calcd for C26H36N9S
(M+H)-, 506.2814; found, 506.2812.
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Compound C10, white solid, yield: 65 %. m.p.: 182-185 C. H NMR (400 MHz,
CDCl3) δ 8.32 (s, 1H), 7.34 (t, J = 7.3 Hz, 2H), 7.22 (m, 2H), 7.10 (d, J = 3.5 Hz, 1H),
6.60 (d, J = 3.4 Hz, 1H), 6.17 (s, 1H), 5.55 (d, J = 4.4 Hz, 1H), 4.38 (s, 2H), 4.31 (dd,
J = 12.8, 6.1 Hz, 1H), 4.11 – 4.02 (m, 1H), 3.93 (dd, J = 12.7, 3.9 Hz, 1H), 3.68 (dd, J
= 10.3, 6.1 Hz, 1H), 3.57 (d, J = 12.7 Hz, 3H), 2.57 (s, 3H), 2.54 – 2.47 (m, 1H), 2.38
(s, 2H), 2.08 – 1.91 (m, 2H), 1.17 (d, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
165.77, 164.38, 158.07, 157.68, 151.99, 150.49, 149.27, 137.21, 128.83, 128.34,
127.17, 120.31, 103.06, 102.28, 94.54, 51.80, 50.01, 46.46, 35.92, 35.34, 32.50, 30.77,
25.09, 15.57. HRMS (ESI): m/z calcd for C26H30N9S (M+H)-, 500.2345; found,
500.2342.
26 / 40

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Compound C11, white solid, yield: 78 %. m.p.: 173-175 C. H NMR (400 MHz,
CDCl3) δ 8.27 (s, 1H), 7.30 – 7.21 (m, 2H), 7.05 (d, J = 3.4 Hz, 1H), 6.85 (t, J = 8.6
Hz, 2H), 6.55 (d, J = 3.4 Hz, 1H), 6.14 (s, 1H), 5.50 (d, J = 4.0 Hz, 1H), 4.38 – 4.22
(m, 3H), 4.04 – 3.85 (m, 2H), 3.63 (dd, J = 10.3, 6.1 Hz, 1H), 3.54 (s, 3H), 2.58 –
13
2.45 (m, 4H), 2.00 – 1.93 (m, 2H), 1.12 (d, J = 7.0 Hz, 3H). C NMR (100 MHz,
CDCl3) δ 165.52, 164.45, 163.16, 160.71, 158.08, 157.66, 151.93, 150.44, 149.29,
133.06, 130.45, 130.37, 120.34, 115.14 (d, J = 21.5 Hz), 103.07, 102.24, 94.59, 51.85,
49.96, 46.46, 35.95, 34.51, 32.46, 30.78, 25.07, 15.55. HRMS (ESI): m/z calcd for
C26H29FN9S (M+H)-, 518.2251; found, 518.2253.
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Compound C12, white solid, yield: 47 %. m.p.: 168-172 C. H NMR (400 MHz,
CDCl3) δ 8.44 (d, J = 8.6 Hz, 1H), 8.16 (s, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.66 – 7.53
(m, 4H), 6.97 (d, J = 3.5 Hz, 1H), 6.59 (s, 1H), 6.51 (d, J = 3.5 Hz, 1H), 6.18 (s, 1H),
5.48 (d, J = 4.0 Hz, 1H), 4.51 (q, J = 14.7 Hz, 2H), 4.29 (dd, J = 12.7, 5.8 Hz, 1H),
4.07 – 3.97 (m, 1H), 3.90 (dd, J = 12.8, 3.6 Hz, 1H), 3.53 (s, 3H), 2.54 (s, 3H), 2.43 (s,
13
2H), 2.00 – 1.81 (m, 2H), 1.05 (d, J = 7.0 Hz, 3H). C NMR (100 MHz, CDCl3) δ
165.33, 164.50, 158.07, 157.71, 151.99, 150.59, 149.28, 139.74, 131.69, 130.29,
129.86, 127.48, 122.24, 120.23, 103.01, 102.34, 94.61, 51.73, 50.13, 46.54, 36.01,
34.62, 32.51, 30.79, 25.11, 15.64. HRMS (ESI): m/z calcd for C26H29ClN9S (M+H)-,
534.1955; found, 534.1956.
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Compound C13, white solid, yield: 69 %. m.p.: 152-155 C. H NMR (400 MHz,
CDCl3) δ 8.20 (s, 1H), 7.21 (dd, J = 8.4, 1.7 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 6.98 (d,
J = 3.6 Hz, 1H), 6.47 (d, J = 3.5 Hz, 1H), 6.06 (s, 1H), 5.42 (d, J = 4.4 Hz, 1H), 4.27
27 / 40

– 4.12 (m, 3H), 3.97 – 3.87 (m, 1H), 3.80 (dd, J = 12.7, 3.9 Hz, 1H), 3.61 – 3.49 (m,
1H), 3.46 (s, 3H), 2.45 (s, 3H), 2.41 – 2.28 (m, 2H), 1.98 – 1.78 (m, 2H), 1.04 (d, J =
13
7.0 Hz, 3H). C NMR (100 MHz, CDCl3) δ 165.34, 164.47, 158.08, 157.65, 151.96,
150.50, 149.29, 136.50, 131.38, 130.50, 121.04, 120.33, 103.07, 102.29, 94.62, 60.40,
51.80, 49.99, 46.47, 35.96, 34.58, 32.46, 30.78, 25.09, 15.57, 14.20. HRMS (ESI):
m/z calcd for C26H29BrN9S (M+H)-, 578.1450; found, 578.1448.
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1
Compound C14, white solid, yield: 63 %. m.p.: 166-171 C. H NMR (400 MHz,
CDCl3) δ 8.20 (s, 1H), 7.38 (s, 1H), 7.18 (dd, J = 17.1, 7.2 Hz, 3H), 6.98 (dd, J = 8.7,
6.0 Hz, 2H), 6.50 (d, J = 3.4 Hz, 1H), 6.07 (s, 1H), 5.45 (s, 1H), 4.31 – 4.10 (m, 3H),
4.01 – 3.88 (m, 1H), 3.78 (dd, J = 12.7, 3.4 Hz, 1H), 3.50 (s, 4H), 2.57 – 2.32 (m, 6H),
1.99 – 1.85 (m, 2H), 1.05 (d, J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 165.61,
164.37, 158.05, 157.65, 151.91, 150.46, 149.27, 136.80, 132.70, 130.99, 128.86,
127.38, 124.66, 120.29, 103.05, 102.26, 94.57, 65.85, 51.90, 50.03, 46.44, 35.88,
35.75, 32.44, 30.72, 29.69, 25.10, 15.53. HRMS (ESI): m/z calcd for C26H29BrN9S
(M+H)-, 578.1450; found, 578.1451.
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Compound C15, white solid, yield: 84 %. m.p.: 182-186 C. H NMR (400 MHz,
CDCl3) δ 8.25 (s, 1H), 7.98 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 7.06 (s, 1H),
6.55 (s, 1H), 6.17 (s, 1H), 5.49 (s, 1H), 4.37 – 4.22 (m, 3H), 3.99 – 3.82 (m, 2H), 3.57
(d, J = 20.3 Hz, 4H), 2.63 (d, J = 21.3 Hz, 1H), 2.54 – 2.40 (m, 4H), 2.06 – 1.85 (m,
2H), 1.12 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 164.78, 164.67, 158.06,
157.67, 151.84, 150.41, 149.32, 146.92, 145.26, 129.58, 123.45, 120.34, 103.07,
102.25, 94.77, 60.40, 51.85, 49.87, 46.45, 36.00, 34.36, 32.36, 30.82, 29.68, 25.09,
28 / 40