Discovery of novel cyclin-dependent kinase (CDK) and histone deacetylase (HDAC) dual inhibitors with potent in vitro and in vivo anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2020.112073

In the current study, we reported a series of novel 1-H-pyrazole-3-carboxamide-based inhibitors targeting histone deacetylase (HDAC) and cyclin-dependent kinase (CDK). The representative compounds N-(4-((2-aminophenyl)carbamoyl)benzyl)-4-(2,6-dichlorobenzamido)-1H-pyrazole-3-carboxamide (7c) and N-(4-(2-((2-aminophenyl)amino)-2-oxoethyl)phenyl)-4-(2,6-dichlorobenzamido)-1H-pyrazole-3-carboxamide (14a) with potent antiproliferative activities towards five solid cancer cell lines, showed excellent inhibitory activities against HDAC2 (IC₅₀ = 0.25 and 0.24 nM respectively) and CDK2 (IC₅₀ = 0.30 and 0.56 nM respectively). In addition, compounds 7c and 14a significantly inhibited the migration of A375 and H460 cells. Further studies revealed that compounds 7c and 14a could arrest cell cycle in G2/M phase and promote apoptosis in A375, HCT116, H460 and Hela cells, which was associated with increasing the intracellular reactive oxygen species (ROS) levels. More importantly, compound 7c possessed favorable pharmacokinetic properties with the intraperitoneal bioavailability of 63.6% in ICR mice, and potent in vivo antitumor efficacy in the HCT116 xenograft model. Our study demonstrated that compound 7c provides a promising strategy for the treatment of malignant tumors.

Full text of DOI:10.1016/j.ejmech.2020.112073

Journal Pre-proof
Discovery of novel cyclin-dependent kinase (CDK) and histone deacetylase (HDAC)
dual inhibitors with potent in vitro and in vivo anticancer activity
Chunhui Cheng, Fan Yun, Sadeeq Ullah, Qipeng Yuan
PII:
S0223-5234(20)30040-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112073
EJMECH 112073
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 December 2019
Revised Date: 3 January 2020
Accepted Date: 13 January 2020
Please cite this article as: C. Cheng, F. Yun, S. Ullah, Q. Yuan, Discovery of novel cyclin-dependent
kinase (CDK) and histone deacetylase (HDAC) dual inhibitors with potent in vitro and in vivo
anticancer activity, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112073.
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Graphic abstracts

Discovery of Novel Cyclin-dependent Kinase (CDK) and
Histone Deacetylase (HDAC) Dual Inhibitors with Potent In
Vitro and In Vivo Anticancer Activity
Chunhui Cheng†, Fan Yun†, Sadeeq Ullah, Qipeng Yuan*
Key Laboratory of Biomedical Materials of Natural Macromolecules (Beijing University of
Chemical Technology), Ministry of Education. College of Life Science and Technology, Beijing
University of Chemical Technology, 15 Beisanhuan East Road, Beijing 100029, China
† These two authors contributed equally
*E-mail addresses of the corresponding authors: yuanqp@mail.buct.edu.cn
A B S T R A C T
In
1-H-pyrazole-3-carboxamide-based inhibitors targeting histone deacetylase (HDAC)
and cyclin-dependent kinase (CDK). The representative compounds
N-(4-((2-aminophenyl)carbamoyl)benzyl)-4-(2,6-dichlorobenzamido)-1H-pyrazole-3-
carboxamide (7c) and
the
current
study,
we
reported
a
series
of
novel
N-(4-(2-((2-aminophenyl)amino)-2-oxoethyl)phenyl)-4-(2,6-dichlorobenzamido)-1H-
pyrazole-3-carboxamide (14a) with potent antiproliferative activities towards five
solid cancer cell lines, showed excellent inhibitory activities against HDAC2 (IC50
=0.25 and 0.24nM respectively) and CDK2 (IC50 =0.30 and 0.56nM respectively). In
addition, compounds 7c and 14a significantly inhibited the migration of A375 and
H460 cells. Further studies revealed that compounds 7c and 14a could arrest cell
cycle in G2/M phase and promote apoptosis in A375, HCT116, H460 and Hela cells,
which was associated with increasing the intracellular reactive oxygen species (ROS)
levels. More importantly, compound 7c possessed favorable pharmacokinetic
properties with the intraperitoneal bioavailability of 63.6% in ICR mice, and potent in
vivo antitumor efficacy in the HCT116 xenograft model. Our study demonstrated that
compound 7c provides a promising strategy for the treatment of malignant tumors.
Key words: Histone deacetylase, Cyclin-dependent kinase, Antiproliferative
activity, Pharmacokinetic properties, In vivo antitumor activity

1 Introduction
Tumorigenesis is closely associated with gene mutations and abnormal
chromosome modifications which are the category of epigenetic aberrations[1].
Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are epigenetic
enzymes, which jointly governed the acetylation of histone [2, 3]. Overexpressed
HDACs could remove the acetyl group from the terminal lysine residues of histones,
thus silencing the expression of tumor suppressor genes and resulting in tumor
carcinoma[4-9]. HDAC inhibitors have received considerable success for cutaneous
cell lymphoma therapy. Recently, four HDAC inhibitors, vorinostat (SAHA),
romidepsin (FK228), belinostat (PXD-101), and panobinostat (LBH589) have been
approved by U.S. Food and Drug Administration (FDA) for the therapy of
hematological malignancies[10-13]. Chidamide (CS055) has been approved by China
Food and Drug Administration for the treatment of peripheral T-cell lymphoma[14]
(Figure 1A). Single HDAC inhibitors have been proved to be effective for the
treatment of hematological malignancies, but they show low inhibitory activity
against solid tumors[15, 16]. Moreover, many clinical researches have demonstrated
that HDAC inhibitors exploit synergistic effects in combination with other anticancer
agents[17-19]. However, multicomponent drugs raised the risks involved in complex
pharmacokinetic properties, unpredictable drug-drug interactions and different drug
solubilities[20, 21]. On the contrary, developing HDAC-based multitargeting drugs
could provide an effective and practical strategy to overcome the limitations of single
and multicomponent agents.
The dysfunction of cell cycle regulation is a remarkable future of the occurrence
and development of malignant tumors[22]. Cyclin-dependent kinases (CDKs),
belonging to a family of serine/threonine protein kinases, regulate cell cycle and
promote cell growth, proliferation and apoptosis[23, 24]. To date, the CDK family
have consisted of 20 members (CDK1-20), which form a CDK/ cyclin complex with
associated cyclin to exert their function[25]. CDK1, 2, 4 and 6 play a crucial role in
cell cycle, while CDK7, 8 and 9 are involved in transcriptional regulation[26, 27].
Abnormal expression of CDK2/cyclin A or CDK2/cyclin E leads to cell carcinoma,
and overexpression of CDK2 induced by gene mutation has been identified in various
human tumors, containing breast cancer, ovarian carcinoma, bladder cancer,
endometrial carcinoma and gastric carcinoma[28-30]. As a consequence, CDK
inhibitors have been considered as promising antineoplastic agents. So far, two CDK
inhibitors have been approved by FDA: palbociclib[31] and ribociclib[32] for the
treatment of breast cancer and HER2-negative advanced breast cancer respectively
(Figure 1B). Several CDK inhibitors have been at different stages of clinical trials,
for instance, (R)-roscovitine[33, 34], AT-7519[35] and flavopiridol[36, 37].

Fig.1. Chemical structures of approved HDAC (A) and CDK (B) inhibitors
It’s reported that vorinostat and flavopiridol, a pan-CDK inhibitor, have
synergized in leukemia, breast, lung and esophageal cancer[38, 39]. In addition, Fan
et al. reported a series of novel CDK4/9 and HDAC1 dual inhibitors against malignant
cancer[40]. These evidence indicated that designing rationally a multitargeting
molecule inhibiting CDK and HDAC could be a promising strategy for cancer therapy.
Compound
1,
4-(2,6-difluorobenzamido)-N-(4-fluorophenyl)-1H-pyrazole-3-carboxamide,
was
proven to reveal high inhibitory activity against CDK1 and CDK2 enzymes, and good
pharmacokinetic (PK) properties, indicating it to be a lead compound (Figure 2) [33,
35]. Additionally, pharmacophore models of most HDAC inhibitors comprise a cap
group, a linker and a zinc-binding group (ZBG). The cap structure containing a
hydrophobic ring interacts with amino acid residues at the protein surface; the linker
occupies the tubular channel to connect the cap group and the zinc site; the
zinc-binding group, such as hydroxamic acid and 2-aminobenzamide, chelates with
zinc ion and forms hydrogen bonds in the catalytic site (Figure 1)[41, 42].
Considering that there is a large hydrophobic area on the surface of HDAC, we
envisaged that the essential pharmacophore of compound 1 was merged with
hydroxamic acid or o-aminobenzamide moiety to acquire a single molecule that could
inhibit both HDAC as well as CDK enzymes. Furthermore, on basis of the strategy as

shown in Figure 2 , we synthesized a series of dual CDK/ HDAC compounds, and
evaluated their antitumor activity in vitro and in vivo.
Fig.2. Design of multi-target inhibitors against HDAC and CDK.
2. Results and discussion
2.1 Chemistry
The synthetic routes of all target compounds were listed in Scheme 1-3. The
preparation procedures of compounds 7a-7g and 8a-8g were illustrated in Scheme 1.
Commercially available 4-nitro-1H-pyrazole-3-carboxylic acid (2) reacted with
methyl 4-(aminomethyl)benzoate hydrochloride (3) to synthesize 4, which was
reduced by palladium on activated carbon to yield compound 5. Then compound 5
was treated with different substituted benzoyl chlorides to afford the key intermediate
6. The intermediate 6 was hydrolyzed, and then treated with benzene-1,2-diamine to
give the target compounds 7a-7g. In addition, the hydroxamic acids 8a-8g were
directly generated by compound 6 with fresh hydroxylamine solution.
As shown in Scheme 2, firstly, compound 10 was prepared by the reduction of
methyl 4-nitro-1H-pyrazole-3-carboxylate (9) in the presence of palladium on
activated carbon. 10 reacted with various benzoyl chlorides to afford compound 11,
which was subjected to hydrolysis in 1,4-dioxane to give the corresponding acid 12.
After that, the key intermediate 13 was obtained by 12 with ethyl
2-(4-aminophenyl)acetate. The synthesis of hydroxamic acids 15a-15g was similar to
that of 8a-8g. Then target compounds 14a-14g were prepared via the hydrolysis and
condensation reaction from 13. In Scheme 3, compound 12 was treated with available
aminoalkylester hydrochlorides to provide the intermediate 16, which was
consequently converted to final products 17a-17g.

Scheme 1. Synthesis of 7a-7g and 8a-8g a
a Reagents and conditions: (a) EDCI, HOBt, DIEA, DMF, r.t; (b) Pd/C, HCOONH4, THF: IPA=1:1,
reflux; (c) corresponding benzoyl chlorides, triethylamine, 1,4-dioxane, 0 ; (d) NaOH (4 equiv),
1,4-dioxane, 85 ; (e) benzene-1,2-diamine, HBTU, TEA/DMF, r.t; (f) NH2OH·HCl, KOH,
MeOH, r.t.
Scheme 2 Synthesis of 14a-14g and 15a-15g a

a Reagents and conditions: (a) Pd/C, HCOONH4, THF: IPA=1:1, reflux; (b) corresponding benzoyl
chlorides, triethylamine, 1,4-dioxane, 0 ; (c) NaOH (4 equiv), 1,4-dioxane, 85 ; (d) EDCI,
HOBt, DIEA, DMF, r.t; (e) NaOH(4 equiv), 1,4-dioxane, 85 ; (f) benzene-1,2-diamine, HBTU,
TEA/DMF, r.t; (g) NH2OH·HCl, KOH, MeOH, r.t.
Scheme 3 Synthesis of 17a-17g a
a Reagents and conditions: (a) EDCI, HOBt, DIEA, DMF, r.t; (b) NH2OH·HCl, KOH, MeOH, r.t.
2.2 Biological evaluation

2.2.1 Assays of antiproliferative activity and enzyme inhibition
Table 1
Antiproliferative activity and enzyme inhibition data (inhib%) of the target compounds
% cell growth inhibition a
% enzyme inhibition a
HCT116
8µM
H460
HDAC1
10µM
CDK2
10µM
Compd
7a
2µM
8µM
2µM
79.09%
83.75%
73.81%
88.61%
84.38%
81.19%
75.43%
64.49%
59.28%
81.91%
61.67%
71.61%
76.95%
68.78%
78.05%
74.29%
81.63%
69.20%
51.98%
20.42%
30.54%
75.81%
44.86%
25.56%
14.23%
4.52%
73.12%
78.65%
73.77%
62.88%
63.73%
39.79%
23.52%
25.01%
46.24%
68.65%
29.41%
53.84%
51.01%
43.75%
72.88%
60.42%
76.80%
60.01%
29.17%
16.48%
13.63%
62.15%
21.70%
29.94%
9.56%
80.55%
75.01%
78.35%
61.71%
17.40%
57.57%
45.47%
7.33%
13.92%
50.12%
32.83%
11.78%
5.54%
7.25%
5.73%
0.46%
10.37%
45.38%
11.99%
26.58%
23.54%
15.97%
57.33%
16.44%
3.52%
0.37%
3.08%
6.06%
3.12%
10.50%
12.46%
0.04%
11.82%
9.04%
9.50%
1.58%
16.96%
15.05%
3.57%
22.94%
0.40%
13.04%
0.88%
1.73%
26.05%
98.32%
90.41%
97.27%
96.71%
97.73%
97.50%
98.02%
72.58%
67.41%
80.73%
69.52%
98.69%
98.00%
79.18%
89.89%
85.12%
90.61%
78.15%
63.47%
41.29%
57.04%
94.22%
56.35%
44.97%
33.17%
24.51%
34.06%
46.14%
42.14%
24.32%
33.48%
70.13%
35.74%
26.82%
24.37%
96.39%
97.90%
66.84%
90.93%
82.99%
59.09%
53.61%
36.98%
97.18%
65.67%
53.21%
76.82%
51.26%
64.47%
76.14%
53.52%
95.83%
82.39%
91.88%
60.52%
41.71%
33.64%
11.34%
96.60%
44.35%
58.91%
7.65%
7b
7c
7d
7e
7f
7g
8a
8b
27.70%
57.40%
31.62%
29.84%
57.06%
45.64%
67.39%
75.06%
73.77%
8.93%
8c
8d
8e
8f
8g
14a
14b
14c
14d
14e
14f
14g
15a
15b
15c
15d
15e
15f
15g
17a
17b
17c
17d
17e
17f
17g
CS055
SAHA
27.75%
23.37%
26.82%
75.06%
36.99%
30.18%
23.11%
26.56%
21.39%
24.25%
17.90%
21.31%
6.84%
0.84%
21.35%
22.42%
6.52%
14.31%
24.51%
20.09%
4.66%
1.04%
9.38%
1.07%
35.83%
19.37%
27.52%
41.30%
45.79%
32.43%
12.28%
-
3.02%
13.76%
59.41%
14.86%
6.54%
12.08%
4.87%
28.77%
15.77%
25.32%
28.71%
38.24%
68.66%
5.76%
0.91%
15.72%
72.20%
86.06%
10.01%
64.43%
77.79%
-

1
73.97%
57.57%
59.13%
21.32%
-
66.61%
a The values represent the means of at least two separate experiments with SD less than 10%.
The results of antiproliferative activity and enzyme inhibition assays were
displayed in Table 1 with CS055, SAHA and compound 1 as the reference drugs. The
growth inhibition of all compounds was examined at the concentrations of 8µM and
2µM towards human colorectal cancer cell line (HCT116) and human lung cancer cell
line (H460). In addition, we also evaluated the enzyme inhibition for HDAC1 and
CDK2 at 10µM. Compounds 17a-17g with the long alkyl linker at the R3 position
significantly decreased the antiproliferative activity relative to those with the aromatic
ring. The introduction of electron-donating methoxyl group (7g, 8g, 14g and 15g)
gave the weaker activity than that of electron-withdrawing groups, such as 7e, 8e 14e
and 15e. The o-aminobenzamide-based compounds 7b-7d caused a remarkable
increase of activity compared to 8b-8d with hydroxamic acid as ZBG. Compounds
14a-14c, simultaneously substituted with fluorine or chlorine moieties at the R1 and
R2 positions exhibited better activity than those with R2=H, such as 14d-14g.
Moreover, the antiproliferative activities were substantially consistent with enzyme
inhibitory activities. For example, compounds 7a-8a with higher cell growth
inhibitory effects were also more effective against HDAC1 and CDK2 than
compounds 15b-15f.
HDACs or CDKs were overexpressed in many malignant tumors[43-48].
Particularly, it was reported that Class I HDACs showed aberrant upregulation in
more than 75% tumor tissues[49, 50]. Studies have indicated that several novel CDK
or HDAC inhibitors showed potent antiproliferative activity against human colorectal,
lung, malignant, cervical and hepatoma cancer cell lines[51-60]. Consequently, IC50
values of compounds that revealed superior or similar antiproliferative activity to the
positive controls were further investigated towards human cancer cell lines: HCT116,
H460, A375, Hela and SMMC7721. As depicted in Table 2, compounds 7c and 14a,
in which two chlorine atoms were linked at the R1 and R2 positions, exhibited
excellent antiproliferative activities with IC50 values ranging from 0.71 to 7.76µM
against five cell lines. Moreover, these two compounds were more potent for HCT116
cells (IC50 = 0.71 and 1.45µM, respectively) than CS055 (IC50 =2.77µM) and 1 (IC50
=1.96µM). The activity was weakened when R1 was a fluorine atom and the R2
position included fluorine or chlorine (7b and 7c, IC50 from 0.70 to 16.26µM; 14b and
14c, IC50 from 2.57 to 16.46µM). These data clearly proved that the chlorine atoms at
the R1 and R2 positions were the most optimal replacement, which illustrated that
2,6-dichlorobenzamide group was crucial to enhance the potency. However, most
compounds showed poor cell inhibition towards SMMC7721 cells. In addition,
compound 7c was tested for the cell morphology at concentrations of 4, 1 and 0.25µM
respectively against all cancer cells (Figure 3). The image analysis suggested that
compounds could great change cell morphology while inhibiting cell growth.
Next, compounds 7c and 14a were tested in the mouse embryonic fibroblast cells
NIH 3T3 to determine their effects on normal cells. As shown in Table 3, compounds

7c and 14a exhibited low cytotoxicity with IC50 values of 4.47 and 4.64µM
respectively compared with CS055 (IC50= 2.46µM). Considering the antiproliferative
activity and cytotoxicity, compounds 7c and 14a were evaluated for further biological
activity.
Table 2
IC50 Values in 5 cancer cells of selected compounds
Compd
IC50(µM) a
Hela
4.48
HCT116
1.13
0.70
0.71
1.66
0.90
4.80
3.68
0.50
2.40
2.20
1.45
2.57
3.46
2.40
2.77
1.26
1.96
A375
1.70
1.20
1.20
1.59
2.11
2.27
2.66
5.05
6.42
3.01
1.60
2.89
2.97
4.61
4.11
2.68
13.38
H460
7.79
3.63
4.19
6.32
6.36
9.99
7.35
10.57
6.17
23.73
2.63
5.34
4.99
6.88
>32
SMMC7721
15.21
16.26
7.76
7a
7b
3.27
7c
1.83
7d
4.06
6.62
7e
10.47
3.44
12.93
6.85
7f
7g
8.13
8.32
8c
4.84
14.61
21.78
16.69
5.22
8e
16.20
6.61
8f
14a
14b
14c
15a
CS055
SAHA
1
3.15
4.30
14.38
16.46
12.31
25.59
5.274
23.21
8.89
7.15
14.94
2.41
11.92
9.46
8.23
a Values are the means of at least two separate experiments.

Figure 3. The human cancer cell lines were treated with 7c at the indicated concentration or
DMSO (0.1%) for 48 h, and the cells morphology was detected with microscope.
Table 3
Cytotoxicity against the NIH 3T3 normal cell line
Compd
NIN 3T3 IC50 (µM) a
7c
14a
1
CS055
2.46
4.47
4.64
7.85
a Values are the mean of three separate experiments
2.2.2 In vitro HDAC and CDK inhibitory activity assay
Compounds 7c and 14a were selected for in vitro inhibitory assays against
HDACs and CDKs since these compounds showed high potency. It’s evident from
Table 3 that compounds 7c and 14a revealed significant isoform selectivity for
HDAC2 (IC50= 0.25 and 0.24nM respectively) over other HDACs. Compound 7c was
above 40-fold and 4-fold more potent for HDAC1 and HDAC3 compared with CS055.
Compound 14a exhibited comparable HDAC1 inhibition to CS055, whereas for
HDAC3, 14a was less active with IC50 >1000nM. These two compounds have little
inhibition effects on HDAC6 and HDAC8. In addition, among CDKs, compound 7c
and 14a displayed higher inhibitory activity against CDK2 with IC50 values of 0.30
and 0.56nM respectively. It was noteworthy that these two compounds also had
inhibitory activity against CDK1 (7c, IC50 = 8.63nM; 14a, IC50 =12.58nM). The IC50
values of 7c and 14a towards other CDKs were >1000nM. From these results, we can
confirm that compounds 7c and 14a possessed dual HDAC and CDK inhibitory
activity.
Table 4

Inhibitory profile of compounds 7c and 14a against HDACs and CDKs
Compd
IC50 (nM) a
HDAC6,8
>1000
HDAC1
260
-
HDAC2
23.00
-
HDAC3
180
CDK1
-
CDK2
-
CDK4,6,7
-
CS055
1
-
-
29.47
8.63
12.58
9.52
0.30
0.56
>1000
>1000
>1000
7c
6.4
0.25
45
>1000
14a
240
0.24
>1000
>1000
a Values are the means of at least two separate determinations.
2.2.3 Molecular docking study
We successfully performed molecular docking studies to disclose the binding
interactions of the representative compounds (7c and 14a) with HDAC2 and CDK2.
As we speculated, compounds 7c and 14a were approached to the narrow binding-site
channel, and the 2- aminobenzamide moiety chelated with zinc ion at the bottom
catalytic site of HDAC2 (Figure S1Aand S1C in Supporting Information). It’s
observed that both compounds 7c and 14a could form four important hydrogen bonds
with HIS145, HIS146, GLY154 and TYR308 in the HDAC2 active site (Figure 4A
and 4C). In addition, the compound 7c formed two other hydrogen bonding
interactions with ASP181 and PHE210, and the cap group of 14a also formed one
additional hydrogen bond with LEU276. It might explain the reason that the HDAC1
inhibitory activity of compound 7c was superior to that of 14a. Furthermore, the
pyrazole-3-carboxamide group of the compounds 7c and 14a could form hydrogen
bonds to the backbone residues GLU81 and LEU83 in the hinge area of CDK2, which
was essential for CDK2 inhibitory effects of compounds. Besides, 7c formed extra
hydrogen bonding interaction with HIS84 (Figure 4B), while 14a was able to form
similar hydrogen bond with LYS89 (Figure 4D). The molecular docking results
provided a valid explanation for the interactions of compounds (7c and 14a) with
HDAC2 and CDK2, and also unveiled the appropriate dual inhibitor design.

Figure 4. Predicted binding modes of compounds 7c and 14a with HDAC2 (PDB code: 4LXZ)
and CDK2 (PDB code: 1PYE). Interactions between the protein and the ligand are shown as
yellow dotted lines. (A) Predicted binding mode of compound 7c in the active site of HDAC2. (B)
Proposed binding mode of compound 7c with CDK2. (C) Compound 14a interacted with the
active site of HDAC2. (D) Proposed binding mode of compound 14a with CDK2. Protein surface
of HDAC2 and CDK2 docking with compounds 7c and 14a was shown in the Supporting
Information.
2.2.4 Cell scratch assay
Cell scratch assay is considered as an effective method to measure the cell
migration and repair ability. To evaluate the inhibitory effect of the compounds on
cells migration, A375 cells and H460 cells were cultured, and then treated with 7c,
14a, 1, and CS055 for 48h. The images were mentioned in Figure 5. It’s observed that
A375 and H460 cells migration were apparently suppressed by 7c and 14a as
compared to the positive controls 1 and CS055. The inhibitory ability of 14a toward
A375 cells migration was stronger than that of 7c.

Figure 5. The effects of CS055, 1, 7c and 14a on the migration of A375 cells (A) at 0.25µM and
H460 cells (B) at 0.5µM. One representative of three independent experiments is shown.
2.2.5 Cell cycle analysis
To further explore the intracellular mechanisms of the representative compounds,
7c and 14a were carefully conducted for their cell cycle effects on A375 cell line,
HCT116 cell line, H460 cell line and Hela cell line. Cells were treated with 7c and
14a at concentrations of 2, 1 and 0.5µM, as well as control (DMSO) for 24h, which
were measured by the flow cytometry with propidium iodide (PI) staining. As
presented in Figure 6, after treatment with 7c and 14a, cancer cells could show a loss
in the proportion of cells in G0/G1 phase and an obvious increase in G2/M phase
compared to control (DMSO). It’s evidently observed that these compounds
significantly arrested A375 and HCT116 cells in G2/M phase. For instance, the
percentages of A375 cells for 7c and 14a at dose of 2µM were increased from 1.13%
to 57.74% and 66.48% respectively (the data from Figure S2 in Supporting
Information). Taken together, the patterns of 7c and 14a blocking cell cycle were

consistent with that of compound 1, which could be relevant to CDK2 inhibitory
activity (Figure S2 in Supporting Information).
Figure 6. The effects of compounds 7c and 14a on the cell cycle assay of cancer cell lines. (A)
A375 cell line; (B) HCT116 cell line; (C) H460 cell line; (D) Hela cell line.
2.2.6 Cell apoptosis assay
We next wanted to determine that the capacity of compounds 7c and 14a to
induce cell death via apoptosis in A375 cell line, HCT116 cell line, H460 cell line and
Hela cell line. Cells were treated with 7c and 14a, CS055 and compound 1 as the
reference drugs for 48h, which were stained with FITC-Annexin V and propidium
iodide (PI), and then analyzed by flow cytometry. Figure 7 indicated that the
compounds 7c and 14a could expedite cell apoptosis in a concentration-dependent
manner. Compounds 7c and 14a could more effectively induce apoptosis than control
(DMSO) towards all four cell lines. The apoptosis rates of 7c and 14a towards A375
cells were 97.22% and 73.08% respectively, which were higher than that of
compound 1 (66.71%) and CS055 (62.60%) at the concentration of 2µM. The
apoptosis rates of HCT116 cells induced by 7c and 14a were 60.6% and 77.1%
respectively, however, compound 1 and CS055 induced 48.00% and 44.30%
respectively. They showed stronger effects on cell apoptosis against A375 and
HCT116 cells. For H460 and Hela cells, the apoptosis induced by 7c and 14a was
better than that of CS055. Hence, it can be concluded that 7c and 14a could cause cell
death through apoptosis.

Figure 7. Effects of 7c and 14a on the apoptosis of cells. Cells were treated with compounds 7c,
14a, 1 and CS055 for 48h, and measured by flow cytometry with FITC-Annexin V/PI staining.
Data are expressed as means ± SD of the percentages of apoptotic cells from three independent
experiments.
2.2.7. Determination of immunofluorescence and ROS generation

Since the docking results revealed a potent affinity between CDK2 and HDAC1
with the representative compounds, we assessed the effects of 7c and 14a on the
inhibition of CDK2 and the deacetylation of histone H3. Compounds showed
favorable antiproliferative activity for A375 cells which possessed a good cell
adhesion property, so A375 cell line was selected as the model. The
immunofluorometric analysis was illustrated in Figure 8A and 8B. It’s demonstrated
that 7c and 14a could lead to an apparent inhibition of CDK2 compared to control
(DMSO) (Fig. 8A). They also induced an increase of acetylation of H3K9(Fig. 8B),
which suggested that 7c and 14a might alter the acetylation of H3, inhibiting HDAC
activity. These findings elucidated that compounds 7c and 14a acted as efficient
CDK2 and HDAC inhibitors.
Reactive oxygen species (ROS) play a critical role in cellular processes
involving cell growth, proliferation, development, differentiation, senescence and
apoptosis[61, 62]. High levels of intracellular ROS might cause DNA damage, and
ultimately resulted in cell death. The 2,7-dichlorofluorescein diacetate (DCFH-DA) is
frequently applied for intracellular ROS detection. As mentioned in Figure 8C, the
green fluorescence signal was hardly observed in the control group. In contrast, after
treatment with the compounds 7c and 14a, cells displayed strong green fluorescence,
indicating a significant increase in intracellular ROS levels. Accordingly, compounds
7c and 14a might accelerate intracellular ROS accumulation, leading to cancer cell
death.

Figure 8. A375 cells were treated with 7c and 14a at 1µM or DMSO for 12h.
Immunofluorescence images of (A) CDK2, DAPI and (B) AcH3K9, DAPI staining and merge in
untreated or treated cells. (C) A375 cells were treated with 7c and 14a at 1µM or DMSO for
24h.Fluorescence images of intracellular ROS generation.
2.2.8 Pharmacokinetic parameters
Considering that compounds 7c and 14a exhibited excellent in vitro antitumor
activity, the pharmacokinetic properties (PK) were investigated in ICR male mice.
Compounds 7c and 14a were administered intraperitoneally (IP) at a dose of 20mg/kg
respectively. The PK parameters were presented Table 4. The half-life (t1/2) of
compound 7c was 2.61h, the maximum plasma concentration (Cmax) was 7570 ng/mL,
and the area under concentration-time curve (AUC0-∞) was 30700 ng h/mL. However,
compound 14a showed a short half-life of 1.63h, a Cmax of 2170 ng/mL and an
AUC0-∞ of 7200 ng h/mL. In addition, compared with 14a (F=27.8%), compound 7c
showed higher bioavailability with F= 63.6%. The data indicated that compound 7c
possessed better pharmacokinetic properties than 14a.

Table 5
Pharmacokinetic parameters of 7c and 14a in ICR mice
Compd
Dose (mg/kg)
administration
7c
20
i.p.
14a
20
i.p.
t (h)
Tmax (h)
Cmax (ng/mL)
AUC0-∞ (ng h/mL)
MRT0-∞ (ng h/mL)
F (%)
2.61
2.00
7570
30700
3.31
63.6
1.63
1.00
2170
7200
2.36
27.8
½
2.2.9 In vivo antitumor activity of compound 7c
As compound 7c exhibited excellent antiproliferative activity towards HCT116
cells (IC50= 0.71µM) over other cell lines, we decided to evaluate the in vivo
antitumor efficacy of compound 7c in the HCT116 xenograft nude mice models.
When tumors grew to a volume of 100-300 mm3, the BALB/c female mice were
randomly divided into treatment and control groups (6 mice per group). Compound 7c
was intraperitoneally administered at 25 and 12.5 mg/kg once daily (QD) for 21 days.
No significant body weight changes and toxicity signs were observed in the treatment
groups (Fig. 9A). Compound 7c effectively inhibited the growth of HCT116
xenograft tumors compared to control. The tumor growth inhibitions (TGI) of 7c at
12.5 and 25 mg/kg were 37.0% and 51.0% respectively (Fig.9B-D). These results
demonstrated that compound 7c possessed remarkable in vivo antitumor efficacy.

Figure 9. In vivo antitumor potency of compound 7c against HCT116 xenografts models with
intraperitoneal administration. (A) Body weight and (B) tumor volume measurements for 7c
treated mice groups after 21 days. (C) Comparison of the final tumor weights in each group after
the 21 days treatment period of 7c. (D) The picture of dissected HCT116 tumor tissues.
3. Conclusions
In summary, we have successfully designed and synthesized novel dual
HDAC/CDK inhibitors. The representative compounds (7c and 14a) displayed potent
antiproliferative activities against five selected human cancer cell lines with IC50
values ranging from 0.71 to 7.76µM. The compounds 7c and 14a also showed
excellent HDAC and CDK inhibition with IC50 values at the nanomolar level.
Moreover, compounds 7c and 14a significantly arrested cell cycle at G2/M phase and
promoted apoptosis towards A375 cell line, HCT116 cell line, H460 cell line and Hela
cell line. In addition, the immunofluorometric analysis indicated that 7c and 14a
displayed significant inhibition of CDK2 and an increase of the acetylation of H3K9.
They could also enhance ROS levels, and then cause cell death. Furthermore, the in
vivo studies indicated that compound 7c showed good pharmacokinetic profile, and
also exhibited potent antitumor efficacy in the HCT116 xenograft model (TGI=
51.0%).

4. Experimental section
4.1 Chemistry
1H NMR and 13C NMR spectra were recorded on a Bruker AV III 400
spectrometer (400MHz) using DMSO-d6 as solvent. The chemical shifts were
measured in parts per million (ppm) relative to Me4Si as internal standard.
High-resolution mass spectra (HRMS) were obtained on an Agilent Q-TOF 6540
mass spectrometer. All reagents and solvents were purchased from commercial
sources and were used without further purification. The progress of all reactions was
monitored by thin layer chromatography (TLC), and precoated plates with silica gel
F254 were purchased from Qingdao Haiyang Chemical Co. Ltd. All the final
compounds were obtained ≥ 90% purity, as tested by high-performance liquid
TM
TM
chromatography (HPLC) on a Thermo Scientific
UltiMate
3000 systerm
(column, C18, 5mm, 4.6mm × 250mm; mobile phase, gradient elution of methanol/
H2O (0.1% H3PO4); flow rate, 1.0mL/min; temperature, 35°C). Analyses indicated by
the symbols of the elements or functions were within 0.4% of the theoretical values.
4.1.1. methyl 4-((4-nitro-1H-pyrazole-3-carboxamido)methyl)benzoate (4) Yield:
65%; 4-nitro-1H-pyrazole-3-carboxylic acid (3.18mmol, 1.1equiv), methyl
4-(aminomethyl)benzoate hydrochloride (2.89mmol, 1equiv), EDCI (3.47mmol,
1.2equiv), HOBt (3.47mmol, 1.2equiv), DIEA (6.94mmol, 2.4equiv) were mixed in
DMF (20mL). The reaction was stirred for 12h at room temperature. TLC analysis,
using petroleum ether: ethyl acetate (1:2) as the eluent, indicated the complete
reaction. The mixture was diluted with a large amount of water and then extracted
with ethyl acetate. The organic layer was collected and dried by anhydrous sodium
1
sulfate. The solvent was concentrated to obtain 4 as a light purple solid. H NMR
(400MHz, DMSO-d6): δ=7.91(3H, m), 7.50(2H, m), 4.50(2H, s), 3.84(3H, s); HRMS
(ESI) (M+H)+ Calc’d for C13H12N4O5: 305.0887 Found:305.0884.
4.1.2. methyl 4-((4-amino-1H-pyrazole-3-carboxamido)methyl)benzoate (5) Yield:
93%; To a 100mL, one-necked flask, sequentially charged with compound 4
(1.64mmol, 1equiv), HCOONH4 (8.20mmol, 5equiv) in 16mL of the mixed solvent of
tetrahydrofuran and isopropanol (1:1). While stirring vigorously under nitrogen,
palladium 5% on activated carbon (182mg, wetted with ca. 55% Water) was added
into the mixture. The reaction was heated at reflux for 1h. When TLC analysis showed
the complete reaction, the mixture was cooled to ambient temperature, and isolated by
1
filtration. Then the filtrate was concentrated in vacuo to afford 5 as a solid. H NMR
(400MHz, DMSO-d6): δ=7.91(2H, d, J=8.12Hz), 7.44(2H, m), 7.10(2H, s), 4.60(2H,
s), 4.47(2H, m), 3.84(3H, s); HRMS (ESI) (M+H)+ Calc’d for C13H14N4O3: 275.1145
Found:275.1141.
4.1.3.
methyl
4-((4-(2,6-dichlorobenzamido)-1H-pyrazole-3-carboxamido)methyl)benzoate
(6c)

Yield: 87%; A mixture of compound 5 (1.82mmol, 1equiv), triethylamine (2.18mmol,
1.2equiv) was stirred in 20mL of 1,4-dioxane. Then 2,6-dichlorobenzoyl chloride
(2.00mmol, 1.1equiv) was added dropwise into the solution in an ice bath. The
reaction was stirred for 12h at 0 . TLC analysis (petroleum ether: ethyl acetate=1:1)
indicated the complete reaction. The mixture was filtered, and then the filtrate was
evaporated under reduced pressure. The crude product was further purified by flash
column chromatography with an appropriate ethyl acetate/petroleum ether mixture to
provide the product 6c. 1H NMR (400MHz, DMSO-d6): δ=13.48(1H, s), 10.09(1H, s),
9.15(1H, s), 8.37(1H, s), 7.91 (2H, d, J=8.34Hz), 7.36 (5H, m),4.48 (2H, d, J=6.66Hz),
3.83(3H, s); HRMS (ESI) (M+H)+ Calc’d for C20H16Cl2N4O4: 447.0628
Found:447.0619.
Compounds 6a-6b and 6e-6g were prepared according to a similar procedure
described for 6c.
General Procedure for the synthesis of compounds 7a-7g. Compound 6
(1.32mmol, 1equiv) was dissolved in 20mL of 1,4-dioxane. Then aqueous sodium
hydroxide solution (2mol/L, 5equiv) was added into this mixture. The reaction was
heated for 1h at 85 . When TLC analysis indicated the complete reaction, the
reaction mixture was cooled to ambient temperature, concentrated in vacuo, and
acidified with 2N HCl (pH at 5-6) to form precipitation. The precipitate was isolated
by filtration and dried to afford the corresponding acid as a white solid. Subsequently,
2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU,
1.77mmol, 1.5equiv), the aforementioned acid (1.18mmol, 1equiv) and
trimethylamine (1.77mmol, 1.5equiv) were dissolved in DMF (15mL). The mixture
was stirred at room temperature for 10min. Afterwards benzene-1,2-diamine
(1.18mmol, 1equiv) was added into the solution for another 3h. When TLC analysis
(petroleum ether: ethyl acetate=1:3) indicated the complete reaction, the reaction
mixture was diluted with water, and extracted with ethyl acetate. Then the organic
layer was dried by anhydrous Na2SO4 and concentrated in vacuo. The crude product
was further purified by flash column chromatography with an appropriate ethyl
acetate/petroleum ether mixture to provide the target compounds 7a-7g.
4.1.4.
N-(4-((2-aminophenyl)carbamoyl)benzyl)-4-(2,6-difluorobenzamido)-1H-pyrazole-3-
carboxamide (7a) Yield: 75%; HPLC Rt= 3.002min; 1H NMR (400MHz, DMSO-d6):
δ= 9.65(1H, s), 9.21(1H, s), 8.37(1H, s), 7.93(2H, m), 7.61(1H, m), 7.42(2H, m),
7.25(2H, m), 7.17(1H, d, J=7.16Hz), 6.97(1H, m), 6.80(1H, m), 6.60(1H, t,
J=7.59Hz), 4.90(2H, s), 4.51(2H, s); 13C NMR (400MHz, DMSO-d6): δ= 163.86,
160.94, 158.45, 156.70, 143.56, 140.86, 133.70, 133.36, 128.39, 128.30, 128.02,
127.81, 127.70, 127.47, 124.91, 122.07, 121.33, 116.58, 112.98, 112.75, 42.13ppm.
HRMS (ESI) (M+H)+ Calc’d for C25H20F2N6O3: 491.1644 Found:491.1637.
4.1.5.

N-(4-((2-aminophenyl)carbamoyl)benzyl)-4-(2-chloro-6-fluorobenzamido)-1H-pyraz
1
ole-3-carboxamide (7b) Yield: 63%; HPLC Rt= 2.992min; H NMR (400MHz,
DMSO-d6): δ= 13.47(1H, s), 10.23(1H, s), 9.62(1H, s), 8.40(1H, s), 7.92(3H, m),
7.43(5H, m), 7.16(1H, d, J=7.04Hz), 6.97(1H, m), 6.79(1H, m), 6.60(1H, t,
13
J=7.73Hz), 4.93(2H, s), 4.49(2H, d, J=6.00Hz); C NMR (400MHz, DMSO-d6): δ=
163.32, 160.12, 158.33, 157.65, 143.07, 133.12, 132.30, 132.21, 131.13, 131.08,
127.82, 127.77, 126.99, 126.63, 126.42, 125.75, 123.28, 121.44, 120.91, 116.21,
116.07, 115.07, 114.86, 41.57ppm. HRMS (ESI) (M+H)+ Calc’d for C25H20ClFN6O3:
507.1348 Found:507.1340.
4.1.6.
N-(4-((2-aminophenyl)carbamoyl)benzyl)-4-(2,6-dichlorobenzamido)-1H-pyrazole-3-
1
carboxamide (7c) Yield: 45%; HPLC Rt= 2.975min; H NMR (400MHz, DMSO-d6):
δ= 13.45(1H, s), 10.11(1H, s), 9.59(1H, s), 9.15(1H, m), 8.38(1H, s), 7.92(2H, m),
7.56(3H, m), 7.42(2H, m), 7.15(1H, d, J=8.10Hz), 6.96(1H, m), 6.78(1H, m), 6.59(1H,
t, J=7.70Hz), 4.87(2H, s), 4.48(2H, d, J=5.71Hz); 13C NMR (400MHz, DMSO-d6): δ=
163.83, 160.88, 143.56, 135.85, 133.62, 133.30, 132.31, 131.72, 128.86, 128.26,
127.50, 127.11, 126.90, 123.78, 121.92, 121.35, 116.70, 116.56, 42.07ppm. HRMS
(ESI) (M+H)+ Calc’d for C25H20Cl2N6O3: 523.1053 Found: 523.1049.
4.1.7.
N-(4-((2-aminophenyl)carbamoyl)benzyl)-4-benzamido-1H-pyrazole-3-carboxamide
(7d) Yield: 68%; HPLC Rt= 2.962min; 1H NMR (400MHz, DMSO-d6): δ= 13.41(1H,
s), 10.70(1H, s), 9.70(1H, s), 9.23(1H, s), 8.37(1H, s), 7.89(4H, m), 7.60(4H, m),
7.49(2H, m), 7.19(1H, d, J=7.84Hz), 7.02(1H, m), 7.83(1H, d, J=7.56Hz), 6.66(1H,
13
m), 4.58(2H, m), 2.69(2H, m); C NMR (400MHz, DMSO-d6): δ= 165.67, 164.49,
163.13, 143.54, 142.52, 133.73, 133.54, 133.06, 132.49, 129.50, 128.32, 127.45,
127.17, 126.96, 124.33, 123.23, 120.61, 117.53, 117.11, 42.05, 38.70ppm. HRMS
(ESI) (M+H)+ Calc’d for C25H22N6O3: 455.1832 Found: 455.1825.
4.1.8.
N-(4-((2-aminophenyl)carbamoyl)benzyl)-4-(2-chlorobenzamido)-1H-pyrazole-3-car
boxamide (7e) Yield: 60%; HPLC Rt= 2.948min; 1H NMR (400MHz, DMSO-d6): δ=
10.25(1H, s), 9.46(1H, s), 9.19(1H, s), 8.37(1H, s), 7.94(2H, m), 7.72(1H, m),
7.44(6H, m), 7.17(1H, m), 6.97(1H, t, J=7.59Hz), 6.78(1H, d, J=7.68Hz), 6.60(1H, t,
J=8.24Hz), 4.89(2H, s), 4.50(2H, s); 13C NMR (400MHz, DMSO-d6): δ= 165.61,
164.00, 162.92, 143.57, 135.31, 133.61, 132.48, 130.70, 130.38, 130.05, 128.27,
128.14, 127.48, 127.13, 126.91, 123.79, 122.59, 116.72, 116.58, 42.03ppm. HRMS
(ESI) (M+H)+ Calc’d for C25H21ClN6O3: 489.1443 Found: 489.1445.
4.1.9.
N-(4-((2-aminophenyl)carbamoyl)benzyl)-4-(2-fluorobenzamido)-1H-pyrazole-3-carb
1
oxamide (7f) Yield: 67%; HPLC Rt= 2.945min; H NMR (400MHz, DMSO-d6): δ=
13.40(1H, s), 10.88(1H, m), 9.71(1H, s), 9.15(1H, m), 8.41(1H, s), 8.03(1H, m),

7.97(2H, m), 7.66(1H, m), 7.48(2H, m), 7.41(2H, m), 7.20(1H, d, J=7.52Hz), 7.01(1H,
m), 6.85(1H, d, J=7.88Hz), 6.69(1H, m), 4.56(2H, d, J=6.07Hz); 13C NMR (400MHz,
DMSO-d6): δ= 165.21, 163.59, 161.02, 159.00, 158.55, 134.18, 132.98, 131.28,
127.82, 126.99, 126.65, 126.47, 125.22, 124.12, 122.24, 120.70, 120.48, 120.36,
117.45, 116.89, 116.63, 116.40, 41.52, 38.21ppm. HRMS (ESI) (M+H)+ Calc’d for
C25H21FN6O3: 473.1738 Found: 473.1740.
4.1.10.
N-(4-((2-aminophenyl)carbamoyl)benzyl)-4-(2-methoxybenzamido)-1H-pyrazole-3-c
1
arboxamide (7g) Yield: 80%; HPLC Rt= 2.967min; H NMR (400MHz, DMSO-d6):
δ= 11.76(1H, s), 9.66(1H, s), 9.06(1H, s), 8.41(1H, s), 8.12(1H, m), 7.96(2H, m),
7.57(1H, m), 7.48(2H, m), 7.24(2H, m), 7.12(2H, m), 7.02(1H, m), 6.80(1H, m),
6.60(1H, m), 4.90(2H, s), 4.59(2H, d, J=5.98Hz), 4.07(3H, s); 13C NMR (400MHz,
DMSO-d6): δ= 163.29, 160.93, 157.44, 143.25, 143.06, 141.17, 133.53, 133.10,
131.35, 127.77, 127.14, 126.62, 126.36, 124.59, 123.33, 122.51, 120.84, 120.16,
117.60, 116.22, 116.09, 114.42, 112.28, 55.91, 41.54ppm. HRMS (ESI) (M+H)+
Calc’d for C26H24N6O4: 485.1938 Found:485.1929.
General Procedure for the synthesis of 8a-8g. A solution of KOH (2.84g,
50.6mmol) in MeOH (7mL) was added dropwise into a solution of hydroxylamine
hydrochloride (2.34g, 33.7mmol) in MeOH (12mL) in an ice bath. The mixture was
stirred at 0 for 1h, and then was filtered to obtain a fresh solution of NH2OH in
MeOH. Then compound 6 (0.22mmol, 1equiv) was dissolved in 3mL of the
aforementioned fresh solution of NH2OH. The mixture was stirred for 1h at room
temperature. TLC analysis (ethyl acetate: methanol=5:1) indicated the complete
reaction. The reaction mixture was evaporated under reduced pressure, and was
acidified with 2N HCl (pH at 5-6) to form precipitation. The precipitate was isolated
by filtration and dried to afford the corresponding compounds 8a-8g as a white solid.
4.1.11.
4-(2,6-difluorobenzamido)-N-(4-(hydroxycarbamoyl)benzyl)-1H-pyrazole-3-carboxa
1
mide (8a) Yield: 89%; HPLC Rt= 2.993min; H NMR (400MHz, DMSO-d6): δ=
13.46(1H, s), 11.14(1H, s), 10.26(2H, s), 9.08(1H, s), 8.29(1H, s), 7.65(3H, m),
13
7.30(2H, m), 7.18(2H, m), 4.40(2H, m); C NMR (400MHz, DMSO-d6): δ= 164.09,
163.31, 160.44, 157.95, 156.22, 142.68, 132.86, 131.23, 129.34, 127.04, 126.87,
121.59, 113.62, 112.49, 112.24, 41.48ppm. HRMS (ESI) (M+H)+ Calc’d for
C19H15F2N5O4: 416.1171 Found: 416.1168.
4.1.12.
4-(2-chloro-6-fluorobenzamido)-N-(4-(hydroxycarbamoyl)benzyl)-1H-pyrazole-3-car
boxamide (8b) Yield: 81%; HPLC Rt= 2.975min; 1H NMR (400MHz, DMSO-d6): δ=
13.45(1H, s), 11.16(1H, s), 10.20(1H, s), 9.12(1H, s), 9.00(1H, s), 8.38(1H, s),
13
7.69(2H, d, J=8.45Hz), 7.55(1H, m), 7.37(4H, m), 4.46(2H, m); C NMR (400MHz,
DMSO-d6): δ= 164.57, 163.78, 160.61, 158.84, 158.14, 143.16, 132.79, 132.70,
131.76, 129.83, 127.55, 127.35, 126.24, 121.92, 121.40, 115.56, 115.34, 42.00ppm.

HRMS (ESI) (M+H)+ Calc’d for C19H15ClFN5O4: 432.0876 Found: 432.0872.
4.1.13.
4-(2,6-dichlorobenzamido)-N-(4-(hydroxycarbamoyl)benzyl)-1H-pyrazole-3-carboxa
1
mide (8c) Yield: 73%; HPLC Rt= 2.970min; H NMR (400MHz, DMSO-d6): δ=
10.13(1H, s), 9.17(1H, s), 8.39(1H, s), 7.71(2H, d, J=8.40Hz), 7.56(4H, m), 7.38(2H,
m), 4.47(2H, m); 13C NMR (400MHz, DMSO-d6): δ= 164.04, 163.20, 160.45, 142.64,
135.33, 131.80, 131.23, 129.30, 128.34, 127.08, 126.88, 121.43, 41.51ppm. HRMS
(ESI) (M+H)+ Calc’d for C19H15Cl2N5O4: 448.0580 Found: 448.0576.
4.1.14. 4-benzamido-N-(4-(hydroxycarbamoyl)benzyl)-1H-pyrazole-3-carboxamide
(8d) Yield: 82%; HPLC Rt= 2.945min; 1H NMR (400MHz, DMSO-d6): δ= 13.50(1H,
s), 11.24(1H, s), 10.69(1H, s), 9.22(1H, s), 8.36(1H, s), 7.88(2H, d, J=7.48Hz),
7.74(2H, d, J=8.04Hz), 7.58(4H, m), 7.42(2H, d, J=7.94Hz), 4.55(2H, d, J=5.78Hz);
13C NMR (400MHz, DMSO-d6): δ= 164.09, 162.65, 142.68, 133.23, 131.98, 131.27,
129.38, 128.99, 127.16, 127.02, 126.91, 126.68, 122.75, 41.48ppm. HRMS (ESI)
(M+H)+ Calc’d for C19H17N5O4: 380.1360 Found: 380.1358.
4.1.15.
4-(2-chlorobenzamido)-N-(4-(hydroxycarbamoyl)benzyl)-1H-pyrazole-3-carboxamid
1
e (8e) Yield: 76%; HPLC Rt= 2.948min; H NMR (400MHz, DMSO-d6): δ=
13.44(1H, s), 11.17(1H, s), 10.22(1H, s), 9.12(1H, m), 8.99(1H, s), 8.36(1H, s),
7.71(3H, m), 7.58(2H, m), 7.48(1H, m), 7.36(2H, d, J=7.80Hz), 4.47(2H, d,
J=6.39Hz); 13C NMR (400MHz, DMSO-d6): δ= 164.56, 164.04, 162.89, 143.17,
135.27, 133.13, 132.49, 131.76, 130.70, 130.37, 130.04, 129.84, 128.13, 127.55,
127.37, 122.52, 120.94, 41.94ppm. HRMS (ESI) (M+H)+ Calc’d for C19H16ClN5O4:
414.0970 Found:414.0958.
4.1.16.
4-(2-fluorobenzamido)-N-(4-(hydroxycarbamoyl)benzyl)-1H-pyrazole-3-carboxamide
(8f) Yield: 82%; HPLC Rt= 2.972min; 1H NMR (400MHz, DMSO-d6): δ= 13.50(1H,
s), 11.24(1H, s), 10.87(1H, d, J=10.59Hz), 9.16(1H, s), 8.41(1H, s), 8.04(1H, t,
13
J=7.65Hz), 7.74(4H, m), 7.42(4H, m), 4.53(2H, d, J=6.22Hz); C NMR (400MHz,
DMSO-d6): δ= 161.01, 159.01, 158.54, 142.76, 134.25, 134.16, 131.26, 129.36,
127.17, 127.05, 126.90, 125.20, 125.17, 122.27, 120.49, 120.37, 116.62, 116.38,
41.44ppm. HRMS (ESI) (M+H)+ Calc’d for C19H16FN5O4: 398.1265 Found:398.1256.
4.1.17.
N-(4-(hydroxycarbamoyl)benzyl)-4-(2-methoxybenzamido)-1H-pyrazole-3-carboxam
1
ide (8g) Yield: 88%; HPLC Rt= 2.978min; H NMR (400MHz, DMSO-d6): δ=
11.74(1H, s), 11.21(1H, s), 9.00(1H, m), 8.39(1H, s), 8.10(1H, m), 7.72(2H, m),
7.55(1H, m), 7.43(2H, m), 7.21(1H, d, J=8.59Hz), 7.11(1H, t, J=7.62Hz), 4.54(2H, d,
13
J=6.16Hz), 4.05(3H, s); C NMR (400MHz, DMSO-d6): δ= 164.57, 163.76, 161.45,

157.92, 143.45, 134.04, 131.84, 131.72, 127.71, 127.38, 122.98, 121.33, 120.61,
112.77, 56.39, 41.95ppm. HRMS (ESI) (M+H)+ Calc’d for C20H19N5O5: 410.1465
Found:410.1463.
4.1.18. methyl 4-amino-1H-pyrazole-3-carboxylate (10) Yield: 88%; Methyl
4-nitro-1H-pyrazole-3-carboxylate (2.92mmol, 1equiv), HCOONH4 (14.6mmol,
5equiv) was dissolved in 16mL of the mixed solvent of tetrahydrofuran and
isopropanol (1:1). While stirring vigorously under nitrogen, palladium 5% on
activated carbon (182mg, wetted with ca. 55% Water) was added into the mixture.
The reaction was heated at reflux for 1h. Upon cooling to ambient temperature, the
mixture was filtered. The filtrate was evaporated under reduced pressure to afford
compound 10 as a solid. 1H NMR (400MHz, DMSO-d6): δ=12.82(1H, s), 7.09(1H, s),
4.86(2H, s), 3.77(3H, s); HRMS (ESI) (M+H)+ Calc’d for C5H7N3O2: 142.0617
Found:142.0612.
4.1.19. 4-(2,6-dichlorobenzamido)-1H-pyrazole-3-carboxylic acid (12a) Yield: 73%;
A mixture of compound 10 (3.54mmol, 1equiv), triethylamine (4.25mmol, 1.2equiv)
was stirred in 20mL of 1,4-dioxane. Then 2,6-dichlorobenzoyl chloride (3.89mmol,
1.1equiv) was added dropwise into the solution in an ice bath. The reaction was
stirred for 12h at 0 . TLC analysis (petroleum ether: ethyl acetate=1:1) indicated the
complete reaction. The mixture was filtered to obtain the crude product 11a in
1,4-dioxane. Then the filtrate was used in the next step without further isolation. An
aqueous sodium hydroxide solution (2mol/L, 5equiv) was added into the above filtrate.
The reaction was heated at 85 for 1h. When TLC analysis indicated the complete
reaction, the reaction mixture was cooled to ambient temperature, concentrated in
vacuo, and acidified with 2N HCl (pH at 5-6) to form precipitation. The precipitate
was isolated by filtration and dried to afford the corresponding acid 12a as a white
1
solid. H NMR (400MHz, DMSO-d6): δ=13.44(1H, s), 9.87(1H, s), 8.29(1H, s), 7.55
(3H, m); HRMS (ESI) (M+H)+ Calc’d for C11H7Cl2N3O3: 299.9943 Found:299.9929.
Compounds 12b-12g were prepared according to a similar procedure described
for 12a.
4.1.20.
ethyl
2-(4-(4-(2,6-dichlorobenzamido)-1H-pyrazole-3-carboxamido)phenyl)acetate (13a)
Yield: 69%; 12a (2.16mmol, 1equiv), ethyl 2-(4-aminophenyl)acetate (2.16mmol,
1equiv), EDCI (2.59mmol, 1.2equiv), HOBt (2.59mmol, 1.2equiv) and DIEA
(2.59mmol, 1.2equiv) were mixed in DMF (20mL). TLC analysis, using petroleum
ether: ethyl acetate (1:2) as the eluent, indicated the complete reaction. The mixture
was diluted with water and then extracted with ethyl acetate. The organic layer was
collected and dried by anhydrous sodium sulfate. The solvent was concentrated to
1
provide compound 13a as a solid. H NMR (400MHz, DMSO-d6): δ=10.31(1H, s),
10.15(1H, s), 8.44(1H, s), 7.68 (2H, m), 7.58 (4H, m), 7.21 (2H, d, J=8.46Hz), 4.09
(2H, m), 3.61(2H, s), 1.24(3H, m); HRMS (ESI) (M+H)+ Calc’d for C21H18Cl2N4O4:

461.0784 Found:461.0784.
Compounds 13b-13g were prepared according to a similar procedure described
for 13a.
General Procedure for the synthesis of 14a-14g. Compound 13 (1.32mmol,
1equiv) was dissolved in 20mL of 1,4-dioxane. Then aqueous sodium hydroxide
solution (2mol/L, 5equiv) was added into this mixture. The reaction was heated at 85
for 1h. When TLC analysis indicated the complete reaction, the reaction mixture was
cooled to ambient temperature, concentrated in vacuo, and acidified with 2N HCl (pH
at 5-6) to form precipitation. The precipitate was isolated by filtration and dried to
afford the corresponding acid as a white solid. Subsequently, HBTU (1.77mmol,
1.5equiv), the aforementioned acid (1.18mmol, 1equiv) and trimethylamine
(1.77mmol, 1.5equiv) was dissolved in DMF (15 mL). The mixture was stirred at
room temperature for 10min. Afterwards benzene-1,2-diamine (1.18mmol, 1equiv)
was added into the solution for another 5h. When TLC analysis (petroleum ether:
ethyl acetate=1:3) indicated the complete reaction, the mixture was diluted with water,
and extracted with ethyl acetate. Then the organic layer was dried by anhydrous
Na2SO4 and was concentrated in vacuo. The crude product was further purified by
flash column chromatography with an appropriate ethyl acetate/petroleum ether
mixture to provide the target compounds 14a-14g.
4.1.21.
N-(4-(2-((2-aminophenyl)amino)-2-oxoethyl)phenyl)-4-(2,6-dichlorobenzamido)-1H-
pyrazole-3-carboxamide (14a) Yield: 43%; HPLC Rt= 2.983min; 1H NMR (400MHz,
DMSO-d6): δ= 13.60(1H, s), 10.33(1H, s), 10.17(1H, s), 9.37(1H, s), 8.46(1H, s),
7.75(2H, d, J=8.18Hz), 7.57(2H, m), 7.52(1H, m), 7.30(2H, d, J=8.46Hz), 7.16(1H,
m), 6.90(1H, m), 6.73(1H, m), 6.54(1H, m), 3.62(2H, s), 2.69(2H, s); 13C NMR
(400MHz, DMSO-d6): δ= 169.18, 161.74, 160.65, 141.78, 136.58, 135.47, 133.31,
131.73, 131.30, 129.13, 128.31, 125.88, 125.29, 123.34, 121.68, 121.39, 120.57,
116.25, 115.90, 42.12, 38.21ppm. HRMS (ESI) (M+H)+ Calc’d for C25H20Cl2N6O3:
523.1053 Found: 523.1051.
4.1.22.
N-(4-(2-((2-aminophenyl)amino)-2-oxoethyl)phenyl)-4-(2-chloro-6-fluorobenzamido)
-1H-pyrazole-3-carboxamide (14b) Yield: 53%; HPLC Rt= 2.962min;
1H NMR (400MHz, DMSO-d6): δ= 13.59(1H, s), 10.73(1H, d, J=6.67Hz), 10.32(1H,
s), 10.25(1H, s), 9.35(1H, m), 8.45(1H, s), 7.74(2H, t, J=8.92Hz), 7.56(1H, m),
7.46(1H, m), 7.38(2H, m), 7.31(1H, m), 7.16(1H, m), 6.90(1H, m), 6.73(1H, m),
13
6.53(1H, m), 3.61(2H, m), 3.03(2H, s); C NMR (400MHz, DMSO-d6): δ= 169.17,
161.71, 160.19, 158.58, 157.72, 141.74, 136.59, 133.32, 132.23, 131.74, 131.18,
129.13, 125.87, 125.70, 125.29, 123.36, 121.66, 120.55, 116.28, 115.91, 115.03,
114.81, 42.12, 38.21ppm. HRMS (ESI) (M+H)+ Calc’d for C25H20ClFN6O3: 507.1348
Found: 507.1342.

4.1.23.
N-(4-(2-((2-aminophenyl)amino)-2-oxoethyl)phenyl)-4-(2,6-difluorobenzamido)-1H-
pyrazole-3-carboxamide (14c) Yield: 61%; HPLC Rt= 2.972min; 1H NMR (400MHz,
DMSO-d6): δ= 10.34(1H, s), 9.38(1H, s), 8.43(1H, s), 7.76 (2H, d, J=8.36Hz),
7.63(1H, m), 7.27(5H, m), 7.17(1H, m), 6.90(1H, m), 6.73(1H, m), 6.54(1H, m),
4.84(2H, s), 3.62(2H, s); 13C NMR (400MHz, DMSO-d6): δ= 169.17, 161.71, 160.51,
160.44, 158.02,, 157.95, 156.48, 141.92, 136.61, 131.76, 129.15, 125.86, 125.29,
123.28, 120.54, 116.14, 115.82, 112.46, 112.22, 42.13ppm. HRMS (ESI) (M+H)+
Calc’d for C25H20F2N6O3: 491.1644 Found: 491.1645.
4.1.24.
N-(4-(2-((2-aminophenyl)amino)-2-oxoethyl)phenyl)-4-benzamido-1H-pyrazole-3-car
1
boxamide (14d) Yield: 66%; HPLC Rt= 2.963min; H NMR (400MHz, DMSO-d6):
δ= 13.52(1H, s), 10.59(1H, s), 10.37(1H, m), 8.42(1H, s), 7.91(6H, m), 7.62(5H, m),
7.35(2H, m), 3.64(4H, m); 13C NMR (400MHz, DMSO-d6): δ= 163.27, 162.98,
133.76, 133.40, 132.53, 129.67, 129.52, 127.24, 123.57, 121.37, 121.04, 116.69,
116.36ppm. HRMS (ESI) (M+H)+ Calc’d for C25H22N6O3: 455.1832 Found:
455.1826.
4.1.25.
N-(4-(2-((2-aminophenyl)amino)-2-oxoethyl)phenyl)-4-(2-chlorobenzamido)-1H-pyra
1
zole-3-carboxamide (14e) Yield: 67%; HPLC Rt= 2.965min; H NMR (400MHz,
DMSO-d6): δ= 9.38(1H, s), 8.39(1H, s), 7.74(3H, m), 7.59(4H, m), 7.30(2H, d,
J=8.11Hz), 7.16(1H, m), 6.89(1H, m), 6.73(1H, m), 6.53(1H, m), 4.83(2H, s),
3.61(2H, s); 13C NMR (400MHz, DMSO-d6): δ= 169.18, 162.63, 161.97, 141.91,
136.65, 131.87, 131.69, 130.18, 129.92, 129.63, 129.15, 127.60, 125.84, 125.27,
123.29, 120.52, 116.13, 115.81, 42.13ppm. HRMS (ESI) (M+H)+ Calc’d for
C25H21ClN6O3: 489.1443 Found: 489.1444.
4.1.27.
N-(4-(2-((2-aminophenyl)amino)-2-oxoethyl)phenyl)-4-(2-fluorobenzamido)-1H-pyra
1
zole-3-carboxamide (14f) Yield: 70%; HPLC Rt= 2.953min; H NMR (400MHz,
DMSO-d6): δ= 10.82(1H, m), 10.36(1H, s), 9.53(1H, m), 8.46(1H, s), 8.05(1H, m),
7.80(2H, m), 7.68(1H, m), 7.42(4H, m), 7.20(1H, m), 6.95(1H, m), 6.70(1H, m),
13
6.34(1H, m), 4.86(1H, s), 3.65(2H, m); C NMR (400MHz, DMSO-d6): δ= 169.62,
169.20, 162.02, 161.09, 159.10, 158.62, 141.91, 140.38, 134.32, 131.81, 131.31,
129.18, 129.11, 125.22, 123.33, 122.71, 120.63, 117.45, 116.67, 116.44, 116.12,
115.82, 113.99, 42.33ppm. HRMS (ESI) (M+H)+ Calc’d for C25H21FN6O3: 473.1738
Found: 473.1736.
4.1.27.
N-(4-(2-((2-aminophenyl)amino)-2-oxoethyl)phenyl)-4-(2-methoxybenzamido)-1H-p
1
yrazole-3-carboxamide (14g) Yield: 80%; HPLC Rt= 2.977min; H NMR (400MHz,

DMSO-d6): δ= 11.80(1H, s), 9.43(1H, s), 8.46(1H, s), 8.14(1H, m), 7.84(2H, d,
J=8.42Hz), 7.59(1H, m), 7.36(2H, m), 7.26(1H, d, J=8.22Hz), 7.14(3H, m), 6.91(1H,
m), 6.74(1H, m), 6.55(1H, m), 4.87(2H, s), 4.14(3H, s), 3.65(2H, s); 13C NMR
(400MHz, DMSO-d6): δ= 169.74, 162.35, 161.47, 157.98, 142.40, 137.42, 134.11,
131.88, 129.72, 126.33, 125.77, 123.83, 123.50, 121.39, 120.90, 120.58, 116.63,
116.33, 112.85, 56.53, 42.64ppm. HRMS (ESI) (M+H)+ Calc’d for C26H24N6O4:
485.1938 Found:485.1931.
General Procedure for the synthesis of 15a-15g. A solution of KOH (2.84g,
50.6mmol) in MeOH (7mL) was added dropwise into a solution of hydroxylamine
hydrochloride (2.34g, 33.7mmol) in MeOH (12mL) in an ice bath. The mixture was
stirred at 0 for 1h, and then was filtered to obtain a fresh solution of NH2OH in
MeOH. Then compound 13 (0.22mmol, 1equiv) was dissolved in 3mL of the
aforementioned fresh solution of NH2OH. The mixture was stirred for 1h at room
temperature. TLC analysis (ethyl acetate: methanol=5:1) indicated the complete
reaction. The reaction mixture was evaporated under reduced pressure, and was
acidified with 2N HCl (pH at 5-6) to form precipitation. The precipitate was isolated
by filtration and dried to afford the corresponding compounds 15a-15g as a white
solid.
4.1.28.
4-(2,6-dichlorobenzamido)-N-(4-(2-(hydroxyamino)-2-oxoethyl)phenyl)-1H-pyrazole
1
-3-carboxamide (15a) Yield: 71%; HPLC Rt= 2.973min; H NMR (400MHz,
DMSO-d6): δ= 10.66(1H, s), 10.30(1H, s), 10.16(1H, s), 8.83(1H, s), 8.44(1H, s),
13
7.71(2H, d, J=8.67Hz), 7.57(4H, m), 7.20(2H, d, J=8.18Hz), 3.24(2H, s); C NMR
(400MHz, DMSO-d6): δ= 167.05, 160.68, 136.57, 135.47, 131.74, 131.48, 131.29,
128.96, 128.31, 121.66, 120.43ppm. HRMS (ESI) (M+H)+ Calc’d for C19H15Cl2N5O4:
448.0580 Found: 448.0573.
4.1.29.
4-(2-chloro-6-fluorobenzamido)-N-(4-(2-(hydroxyamino)-2-oxoethyl)phenyl)-1H-pyr
1
azole-3-carboxamide (15b) Yield: 76%; HPLC Rt= 2.985min; H NMR (400MHz,
DMSO-d6): δ= 13.64(1H, s), 10.69(1H, s), 10.31(2H, m), 8.86(1H, s), 8.46(1H, s),
7.74(2H, m), 7.58(1H, m), 7.45(2H, m), 7.23(2H, m), 3.27(2H, s); 13C NMR
(400MHz, DMSO-d6): δ= 167.60, 162.19, 160.68, 159.09, 158.21, 137.09, 133.80,
132.73, 132.63, 131.97, 131.68, 129.47, 126.19, 122.17, 121.93, 120.95, 115.53,
115.31ppm. HRMS (ESI) (M+H)+ Calc’d for C19H15ClFN5O4: 432.0876 Found:
432.0876.
4.1.30.
4-(2,6-difluorobenzamido)-N-(4-(2-(hydroxyamino)-2-oxoethyl)phenyl)-1H-pyrazole
1
-3-carboxamide (15c) Yield: 78%; HPLC Rt= 2.977min; H NMR (400MHz,
DMSO-d6): δ= 13.67(1H, s), 10.72(1H, s), 10.34(2H, m), 8.85(1H, s), 8.43(1H, s),