Design, synthesis and biological evaluation of novel hydroxamic acid based histone deacetylase 6 selective inhibitors bearing phenylpyrazol scaffold as surface recognition motif

Article abstract of DOI:10.1016/j.bmc.2017.08.029

In recent years, inhibition of HDAC6 became a promising therapeutic strategy for the treatment of cancer and HDAC6 inhibitors were considered to be potent anti-cancer agents. In this work, celecoxib showed moderate degree of HDAC6 inhibition activity and selectivity in preliminary enzyme inhibition activity assay. A series of hydroxamic acid derivatives bearing phenylpyrazol moiety were designed and synthesized as HDAC6 inhibitors. Most compounds showed potent HDAC6 inhibition activity. 11i was the most selective compound against HDAC6 with IC₅₀ values of 0.020 μM and selective factor of 101.1. Structure-activity relationship analysis indicated that locating the linker group at 1′ of pyrazol gave the most selectivity. The most compounds 11i (GI₅₀ = 3.63 μM) exhibited 6-fold more potent than vorinostat in HepG2 cells. Considering of the high selectivity against HDAC6 and anti-proliferation activity, such compounds have potential to be developed as anti-cancer agents.

Full text of DOI:10.1016/j.bmc.2017.08.029

Accepted Manuscript
Design, synthesis and biological evaluation of novel hydroxamic acid based
histone deacetylase 6 selective inhibitors bearing phenylpyrazol scaffold as sur-
face recognition motif
Jinyu Yang, Gaoliang Cheng, Qihao Xu, Shenglin Luan, Shuxiang Wang, Dan
Liu, Linxiang Zhao
PII:
S0968-0896(17)31490-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.029
BMC 13932
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
20 July 2017
15 August 2017
17 August 2017
Please cite this article as: Yang, J., Cheng, G., Xu, Q., Luan, S., Wang, S., Liu, D., Zhao, L., Design, synthesis and
biological evaluation of novel hydroxamic acid based histone deacetylase 6 selective inhibitors bearing
phenylpyrazol scaffold as surface recognition motif, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.08.029
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Design, synthesis and biological evaluation of novel hydroxamic acid based histone deacetylase 6 selective
inhibitors bearing phenylpyrazol scaffold as surface recognition motif
Jinyu Yang, Gaoliang Cheng, Qihao Xu, Shenglin Luan, Shuxiang Wang, Dan Liu*, Linxiang Zhao*
Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China
*Corresponding authors.
Tel./Fax: +86 024 4352 0221.
E-mail address: sammyld@163.com (D. Liu);
linxiang.zhao@vip.sina.com (L. Zhao)

Abstract
In recent years, inhibition of HDAC6 became a promising therapeutic strategy for the treatment of cancer and
HDAC6 inhibitors were considered to be potent anti-cancer agents. In this work, celecoxib showed moderate degree of
HDAC6 inhibition activity and selectivity in preliminary enzyme inhibition activity assay. A series of hydroxamic acid
derivatives bearing phenylpyrazol moiety were designed and synthesized as HDAC6 inhibitors. Most compounds
showed potent HDAC6 inhibition activity. 11i was the most selective compound against HDAC6 with IC50 values of
0.020 µM and selective factor of 101.1. Structure-activity relationship analysis indicated that locating the linker group
at 1’ of pyrazol gave the most selectivity. The most compounds 11i (GI50 = 3.63 μM) exhibited 6-fold more potent than
vorinostat in HepG2 cells. Considering of the high selectivity against HDAC6 and anti-proliferation activity, such
compounds have potential to be developed as anti-cancer agents.
Keywords: Phenylpyrazole derivatives; HDAC6 selective inhibitor; Anti-proliferative activity; Structure-activity
relatonship.

1. Introduction
The acetylation level of histone regulated by histone acetyl transferases (HATs) and histone deacetylases (HDACs)
plays an important role in the development of many diseases such as HIV, HCV, cancer etc.[1, 2]. The 18 isoforms of
HDACs are grouped into four classes: class I (HDACs 1, 2, 3 and 8), class II (HDACs 4, 5, 6, 7, 9 and 10), class
III(SIRT1-7) and class IV (HDAC 11). The HDACs can be classified into two categories based on their mechanisms:
zinc-dependent deacetylases (class I, II and IV) and class III NAD+-dependent (class III) deacetylases [3]. Inhibition of
HDACs has been proved to be an effective therapeutic strategy for cancers[4]. There are five agents have been approved
by FDA or CFDA (Vorinostat[5], Romidepsin[6], Panobinostat[7], Belinostat[8] and Chidamide[9]) for the treatment of
lymphoma and multiple myeloma. However, all the approved drugs are class I selective or pan-HDAC inhibitors which
have multiple side-effects as reported[10]
.
HDAC6, which is primarily located at cytoplasm, deacetylated lysine residues of many non-histone substrates,
including Hsp90, α-tubulin and Ku70[11, 12]. Recently, The dysregulated expression of HDAC6 was proved to be related
to many diseases exemplified by HIV[13], Alzheimer's disease[14], inflammation[15] and cancer[16]. Development of
HDAC6 selective inhibitors as novel anticancer agents seemed to be preferable than the pan-inhibitors, considering the
fact that HDAC6 selective inhibitors showed fewer side-effects[17]. To date, Tubacin[18], ACY-1215[19], tubastatin A[20]
and other HDAC6 selective inhibitors have been reported by many groups. Among them, ACY-1215 is the most
promising molecule of which phase II clinical trial for treating multiple myeloma has been completed. All these
achievements prompted us to develop potent HDAC6 inhibitors with high selectivity. Considering all the reported
structures, a HDAC6 inhibitor can be divided into three pharmacophores: (1) zinc-binding group (ZBG), interacting
with the Zn2+ at the bottom of the active site; (2) linker group, matching the hydrophobic tunnel of HDAC6; (3) surface
recognition motif (SRM), covering the entrance of the active pocket.

Figure 1. Structures of some reported HDAC6 inhibitors
In our previous study, a small library of approved drugs was screened for inhibitory activity of HDAC6 using
fluorescence assay. Celecoxib, an anti-inflammatory drug, which is also accounted as a carbonic anhydrase II inhibitor
for its zinc binding capacity[21], showed moderate degree of HDAC6 inhibition and selectivity (IC50 = 0.643 µM and SF
= 1.8). In addition, celecoxib was well tolerated and had no clinically significant adverse effects with the total daily
dosage of 400 mg in a six weeks trial[22]. Considering the activity, selectivity and security, we chose celecoxib as an
outstanding lead compound for the development of HDAC6 selective inhibitors in this study. We designed and
synthesized a series of phenylpyrazole derivatives based on celecoxib. Several modifications of celecoxib were
performed to improve the potency and selectivity: (1) to increase HDAC6 binding affinity, the sulfanilamide group was
replaced by hydroxamic acid in order to chelate Zn2+ by more coordination bond; (2) based on the conception that SRM
structures strongly influence the selectivity of compounds against HDAC isotype[23], a SAR study for the SRM by
changing the location of linker group on pyrazole was proceed to optimize HDAC6 selectivity of the lead compound; (3)
to deeply discuss the effect of linker group on the activity and selectivity against HDAC6, the phenyl was replaced by
several linear alkyls of different lengths.
Figure 2. Rational design of Celecoxib derivatives
2. Results and discussion
2.1. Chemistry

The synthetic route to compound 8 was illustrated as Scheme 1. A condensation reaction of p-methylacetophenone
5a with trifluoroacetic acid ethyl ester in THF at room temperature was performed to obtain dione 6a, which was
coupled with sulfonamidophenylhydrazine hydrochloride in the next step. Intermediate 7 was reacted with 50%
hydroxylamine to afford the designed hydroxamic acid 8.
Scheme 1. Synthesis of target compound 8. Reagents and conditions: (a) Ethyl trifluoroacetate, NaOMe, THF, reflux,
5h; (b) Methyl 4-hydrazinylbenzoate, EtOH, reflux, 4h; (c) 50% NH2OH, NaOH, MeOH, r.t., 3h.
Compounds 11a-11p were synthesized from substituted acetophenones 5a-5c by the route displayed in Scheme 2.
Compounds 6a-6g were treated with hydrazine hydrate in acetic acid at 120oC to give intermediates 9a-9g.
Intermediates 9a-9g were reacted with methyl 4-(bromomethyl)benzoate or methyl 4-(2-bromoethyl)benzoate, then the
ester groups of products were converted into the corresponding hydroxamic acids of 11a-11p.
Scheme 2. Synthesis of target compounds 11a-11p. Reagents and conditions: (a) Substituted carboxylic ethyl ether,
NaH, anhydrous THF, N2, r.t., overnight; (b) hydrazine hydrate, acetic acid, reflux, 2h; (c) methyl 4-bromomethyl
benzoate or methyl 4-(2-bromoethyl)benzoate, K2CO3, MeCN, reflux, 4h; (d) 50% NH2OH, NaOH, MeOH, r.t., 3h.
The synthetic route to compounds 16a-16f was depicted in Scheme 3. Intermediate 14 was synthesized from
4-fluoroacetophenone through condensation, cyclization and hydrolysis successively. Intermediate 14 was coupled with
appropriate amino acid methyl esters to afford the required amides 15a-15f, which were treated with 50%

hydroxylamine to produce 16a-16f.
Scheme 3. Synthesis of target compounds 16a-16f. Reagents and conditions: (a) Dimethyl oxalate, NaOMe, diethyl
ether, r.t., overnight; (b) hydrazine hydrate, acetic acid, reflux, 2h; (c) NaOH, H2O, reflux, 2h; (d) appropriate amino
acid methyl esters, HOBt, EDCI, TEA, DMF, r.t., overnight; (e) 50% NH2OH, NaOH, MeOH, r.t., 3h.
2.2 In vitro HDAC inhibition and selectivity
The HDAC inhibition activities of novel compounds against HeLa nuclear extracts and recombinant human
HDAC1, 2, 3, 6, 8 enzymes were investigated, using vorinostat and Rolinostat as positive controls. As shown in Table 1
and Table 2, the ZBG motif had a significant influence on HDAC inhibition activity. Compound 8 (IC50= 0.359 µM)
with a hydroxamic acid group showed more potent activity against HDAC6 than celecoxib (IC50= 0.643 µM). The
substituent groups on benzene ring of SRM also have influence on the selectivity. When the linker is on 1’ of the
pyrazol, the activity against HDAC6 follows the trends of 11e (R1 = Me, IC50 = 0.029 µM), 11g (R1 = 3, 4-OCH2O, IC50
= 0.036 µM) and 11i (R1 = F, IC50 = 0.020 µM), suggesting a bulk substituent is unfavorable for HDAC6 inhibition
activity. For the R2 group, the selectivity and activities decreased when the substituent getting larger (exemplified by 11i,
11k and 11m). For the distance between pyridine and the phenyl in linker, carbon length with n = 1 (11a and 11b) gave
the superior activities with IC50s of 0.027 µM and 0.016 µM against HDAC6, respectively. Systematic evaluation
reflected that the position of linker on pyrazol largely influenced HDAC6 selectivity (1’ substituted > 2’- substituted >
3’- substituted). For example, compound 11i is the most selective HDAC6 inhibitor with a SF of 101.1 which higher
than 11j (SF = 60.0) and 16a (SF = 38.0). For the linker group, benzene ring is favorable for HDAC6 selectivity and the
length with 5 or 6 carbon is favorable for HDAC inhibition activity (exemplified by 16a, 16e and 16f).
Table 1. The HDAC inhibitory activities of compounds 8 and 11a-11p.

IC50 (µM)
Selective
factor (SF)
HDAC6/1
1.8
Linker
Compd.
R1
R2
n
position
HDACs HDAC1 HDAC2 HDAC3 HDAC6 HDAC8
1
1
1
2
1
2
1
2
Me
Me
Me
Me
Me
Me
Me
CF3
CF3
CF3
CF3
CF3
CF3
Me
0
0
1
1
2
2
1
1
1.637
1.139
0.231
0.033
1.460
0.121
0.022
0.013
1.185
0.836
0.925
0.110
1.071
0.352
0.694
0.133
ND
ND
0.643
0.359
0.027
0.016
0.540
0.078
0.029
0.007
ND
>5
>5
>5
>5
>5
>5
>5
Celecoxib
8
0.919
1.216
0.047
0.635
0.549
0.076
0.041
0.736
0.128
0.021
0.816
0.080
0.053
0.026
2.3
34.3
11a
6.9
11b
11c
2.0
4.5
11d
11e
23.9
Me
Me
19.0
11f
3, 4-O
1
2
Me
Me
1
1
0.016
0.008
0.430
0.024
0.135
0.040
0.092
0.036
0.036
0.005
>5
>5
11.9
4.8
11g
11h
CH2O
3, 4-O
CH2O
1
2
1
2
1
2
1
2
F
F
F
F
F
F
F
F
Me
Me
1
1
1
1
1
1
1
1
1.439
0.040
1.792
0.165
3.827
0.476
>5
2.021
0.480
3.611
0.487
>5
1.974
0.114
1.503
0.391
2.883
0.362
>5
1.205
0.069
1.844
0.228
1.942
0.290
>5
0.020
0.008
0.069
0.035
0.167
0.107
0.420
0.326
0.031
0.009
>5
>5
101.1
60.0
52.3
13.9
11i
11j
Et
>5
11k
Et
>5
11l
n-propyl
n-propyl
benzyl
benzyl
>5
11m
0.541
>5
>5
5.1
11n
>5
11o
1.028
0.055
ND
1.883
0.085
0.100
0.763
0.031
0.066
0.619
0.010
0.037
>5
5.8
2.7
11p
2.139
>5
Vorinostat
ACY-1215
11.1
Table 2. The HDAC inhibitory activities of compounds 16a-16f.
IC50 (µM)
HDAC1 HDAC2 HDAC3 HDAC6
Seletivity
Compd.
16a
X
factor
HDACs
HDAC8
HDAC6/1
0.022
0.038
0.017
0.009
0.001
>5
38.0
3.5
-(CH2)2-
-(CH2)3-
>5
>5
>5
>5
>5
>5
>5
16b
16c
2.603
3.741
1.835
1.426
1.057

-(CH2)4-
-(CH2)5-
-(CH2)6-
0.644
0.058
0.043
0.055
ND
1.039
0.066
0.079
0.085
0.100
0.883
0.042
0.040
0.031
0.066
0.671
0.036
0.035
0.010
0.037
0.275
0.029
0.022
0.031
0.009
>5
3.8
2.3
16d
16e
3.710
2.951
2.139
>5
3.6
16f
2.7
Vorinostat
Rolinostat
11.1
2.3 Cell growth inhibition assay
The antiproliferative effects of novel compounds were tested against A549 (human lung cancer) and HpeG2
(human liver cancer) cell lines with Vorinostat as a positive control.
The results of the anti-proliferation assay of synthesized compounds are summarized in Table 3. Most
hydroxamate analogues manifested significant anti-proliferative activities against two tumor cell lines. Most compounds
showed more potent anti-proliferation activities than vorinostat against HepG2 cells. Replacement of the methyl of 11e
(GI50 = 13.34 μM) by 3, 4-OCH2O- led to decline of anti-proliferative activity (11g, GI50 = 16.48 μM). However,
replacement of the methyl of 11e by F led to increase of anti-proliferative activity (11i, GI50 = 3.63 μM). Comparison of
the activities of compound 11a (GI50 = 7.5 μM) and compound 11b (GI50 = 8.58 μM) suggested that compounds with
the linker group on 1’ of the pyrazol were more potent than the corresponding 2’ substituent compounds against HepG2
cell. This phenomenon could be further confirmed by 11e, 11g, 11i vs. 11f, 11h, 11j. These results suggesting that a
small R1 group is favorable for the inhibition activity against HepG2 cell, which is identical with the HDAC6 selectivity.
For 16a-16f, 16f displayed the optimal activity (GI50 = 1.98 μM against A549 cells) suggesting the length of linker
group with 6 carbon is favorable for inhibition activity. Comparison of the activities of 16a (GI50 = 20.22 μM) and 16f
(GI50 = 1.98 μM) indicated that flexible linker group is beneficial for anti-proliferation activity.
Table 3. The anti-proliferative activities of indicated compounds and SAHA against two representative tumor cell lines
GI50 (µM)
GI50 (µM)
Compd.
Compd.
A549
5.02
HepG2
21.96
>100
7.5
A549
11.38
16.72
19.17
16.03
15.21
20.22
HepG2
30.02
7.76
Vorinostat
11j
11k
11l
>100
10.63
15.52
29.33
>100
8
16.68
6.66
11a
11b
11c
11d
8.58
11m
11n
16a
23.96
27.58
16.75
38.13

12.38
11.16
23.18
28.06
32.99
13.34
21.18
16.48
20.76
3.63
46.88
>100
>100
7.09
76.03
88.01
>100
47.58
23.17
11e
11f
11g
11h
11i
16b
16c
16d
16e
16f
1.98
2.4 Docking study
Compound 11i was found to be an excellent HDAC6 selective inhibitor (IC50 = 0.020 µM and SF = 101.1) and
anticancer agent (GI50 = 3.63 μM). The possible binding modes of 11i on HDAC1 (PDB ID: 4BKX), HDAC 2 (PDB ID:
4LXZ), HDAC 6 (PDB ID: 5EDU) and 11g on HDAC6 were explored using the Discovery Studio 3.0/CDOCKER
protocol (Figure 3).
Docking studies revealed that HDAC6 contains a deeper, wider and more hydrophobic pocket formed by Phe679,
Phe680, Met682, Leu749 and Gly750 than other HDAC isoforms. Analysis of the docked complexes suggested that
compound 11i could only occupy the hydrophobic pocket of HDAC6 suitably with a π–π stack between benzene ring
and Phe679 for the high capacity of hydrophobic pocket (Figure 3C). However, 11i showed very different
conformations when binding to HDAC1 and HDAC3 (Figure 3A, 3B). This clear difference probably made the
selectivity of 11i against HDAC6. Notably, the hydrophobic pocket could only accommodate a benzene ring without a
bulky substituent group, which explained the low selectivity of 11g (Figure 3D). By analyzing the events mentioned
above, we drew the conclusion that the hydrophobic pocket is a characteristic of HDAC6 and a benzene ring with a
small substituent at SAM moiety is significative for HDAC6 selectivity.

Figure 3. Possible binding modes of 11i to HDAC1 (A), HDAC2 (B), HDAC6 (C) and 11g to HDAC6 (D).
3. Conclusion
In our study, a series of phenylpyrazole hydroxamate analogues have been designed and synthesized. The
synthesized compounds were investigated for their enzyme inhibitory activities against HDAC1, 2, 3, 6 and 8 as
well as their in vitro antiproliferative activities against three human cancer cell lines including A549 and HepG2.
Compound 11i demonstrated the supreme HDAC6 selectivity, which was much higher than Vorinostat and
Rolinostat. Compound 11i also showed the supreme antiproliferative activity against HepG2, which is 6-fold more
potent than Vorinostat. Molecular docking analysis showed that the phenyl group with a small substituent group is
crucial for HDAC6 selectivity. This study provides further possibility of developing HDAC6 selectivity inhibitors
for the treatment of cancer.
4. Experimental section
4.1 Chemistry
The melting points were determined on an electrically heated X-4 digital visual melting point apparatus and were
1
uncorrected. Mass spectra (MS) were determined on a Finnigan MAT/USA spectrometer (LC-MS). H NMR and 13C
NMR spectrum were recorded on Bruker AV-400 or ARX 600 spectrometers with tetramethylsilane (TMS) used as the
internal standard. Chemical shifts were reported in ppm (δ). High–resolution mass spectra were obtained on Bruker

microTOF–Q in the ESI mode (HR–ESI–MS). All reactions were performed with commercially available reagents and
they were used without further purification. All reactions were monitored by thin-layer chromatography (TLC) carried
on fluorescent precoated plates GF254 (Qindao Haiyang Chemical, China) and detection of the components was made
by short UV light. Column chromatography was performed with silica gel 60 (200–300 mesh).
4.1.1 The synthesis of N-hydroxy-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzamide (8)
A solution of p-methylacetophenone (0.67 g, 5 mmol) in dry THF (20 mL) was added dropwise to NaH (0.24 g, 10
mmol) in dry THF (50 mL) under nitrogen atomosphere and the mixture was stirred for 1h at 0oC. Then a solution of
ethyl trifluoroacetate (0.9mL, 7.5 mmol) in dry THF (10 mL) was added dropwise. The mixture was warmed to room
temperature for 12h. The reaction was quenched with saturation NaHCO3 and extracted with ethyl acetate (50 mL × 3).
The combined organic extracts were washed with brine (50 mL), dried with Na2SO4 and evaporated. Finally, the
resulting residue was purified by column chromatography on silica gel as indicated to give 6a as canary yellow solid.
To a solution of 6a (0.46 g, 2 mmol) in ethyl alcohol (50 mL) was added 4-hydrazinyl-N-hydroxybenzamide
hydrochloride (0.53 g, 2.6 mmol). Then the temperature was raised to 70oC for 5h. The mixture solution was
concentrated to dryness, added water (50 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic
extracts were washed with brine (30 mL), dried with Na2SO4 and concentrated. Finally, the resulting residue was
purified by column chromatography on silica gel as indicated to give 7 as white solid.
To a solution of 7 (0.36 g, 1 mmol) in methyl alcohol (30 mL) were added 1 M aqueous solution of NaOH (3 mL)
and NH2OH (50 wt % in water, 3 mL) dropwise successively at 0oC. The reaction was warmed to room temperature for
3h. The mixture solution was concentrated to dryness, and the obtained solid was dissolved in water. The pH was
adjusted to 7 with 1M aqueous solution of HCl. The resulting precipitate was filtered to give 8 as white solid. Yield:
63.2%. Mp: 104.5-105.9°C. ESI-MS: m/z, 360.3 [M-H]-. 1H-NMR (600 MHz, DMSO-d6) δ: 11.33 (s, 1H), 9.13 (s, 1H),
7.80 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 7.21 – 7.18 (m, 4H), 7.17 (s, 1H), 2.30 (s, 3H). 13C-NMR (151 MHz,
DMSO) δ: 145.19, 140.87, 139.03, 132.90, 129.56, 129.43, 128.77, 128.00, 126.86, 125.60, 125.54, 105.91, 20.86. .

HRMS (ESI+) m/z calcd for C18H14F3N3O2 [M-H]- 360.1038, found: 360.1047.
4.1.2 General procedure for the synthesis of 11a-11p
Compounds 6b-6g were synthesized from 5a-5c with appropriate carboxylic ethyl ether by the synthetic method of
6a.
To a solution of 6a-6g (10 mmol) in acetic acid (50 mL) was added hydrazine hydrate (0.76 g, 11 mmol). The
temperature was raised to 80°C for 4 h. When the mixture was cooled to room temperature, the resulting solution was
poured into water (100 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic extracts were washed
with brine (50 mL), dried with Na2SO4 and concentrated. Finally, the resulting residue was purified by column
chromatography on silica gel as indicated to give 9a-9g.
To a solution of 9a-9g (5 mmol) in acetonitrile (100 mL) was added methyl 4-(bromomethyl)benzoate (1.37 g, 6
mmol) or methyl 4-(2-bromoethyl)benzoate (1.46 g, 6mmol). Then Cs2CO3 (3.26 g, 10 mmol) was added and the
temperature was raised to 70oC for 5h. The mixture solution was concentrated to dryness, added water (100 mL) and
extracted with ethyl acetate (80 mL × 3). Finally, the organic layers were combined, washed with brine, dried with
Na2SO4 and evaporated. Finally, the resulting residue was purified by column chromatography on silica gel as indicated
to give 10a-10p as white solid.
To a solution of 10a-10p (1 mmol) in methyl alcohol (30 mL) were added 1 M aqueous solution of NaOH (3 mL)
and NH2OH (50 wt % in water, 3 mL) dropwise successively at 0oC. The reaction was warmed to room temperature for
3h. The mixture solution was concentrated to dryness, and the obtained solid was dissolved in water (10 mL). The pH
was adjusted to 7 with a 1M aqueous solution of HCl. The resulting precipitate was filtered to give 11a-1p as white
solid.
4.1.2.1 N-hydroxy-4-((5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)benzamide (11a)
1
Yield: 27.1%. Mp: 171.7-173.8°C. ESI-MS: m/z, 374.2 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 11.17 (s, 1H),
9.02 (s, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 7.9 Hz, 2H), 7.05 (d, J = 8.3 Hz, 2H), 6.94

(s, 1H), 5.50 (s, 2H), 2.34 (s, 3H). 13C-NMR (101 MHz, DMSO) δ: 164.26, 146.07, 140.06, 139.62, 132.65, 130.03,
129.14, 127.76, 127.10, 126.04, 105.25, 53.47, 21.29. HRMS (ESI+) m/z calcd for C19H16F3N3O2 [M-H]- 374.1195,
found: 374.1187.
4.1.2.2 N-hydroxy-4-((3-(p-tolyl)-5-(trifluoromethyl)-1H-pyrazol-1-yl)methyl)benzamide (11b)
1
Yield: 41.5%. Mp: 152.6-153.8°C. ESI-MS: m/z, 374.1 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 10.99 (s, 1H),
9.05 (s, 1H), 7.77 (m, 1H), 7.76 (m, 1H), 7.73 (m, 1H), 7.71 (m, 1H), 7.26 – 7.24 (m, 4H), 7.24 (s, 1H), 5.57 (s, 2H),
2.33 (s, 3H). 13C-NMR (151 MHz, DMSO) δ: 150.51, 139.41, 138.04, 132.46, 129.65, 129.48, 128.77, 127.33, 126.88,
126.72, 125.43, 105.55, 53.91, 20.92. HRMS (ESI+) m/z calcd for C19H16F3N3O2 [M-H]- 374.1195, found: 374.1195.
4.1.2.3 N-hydroxy-4-(2-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)benzamide (11c)
1
Yield: 25.4%. Mp: 98.7-99.8°C. ESI-MS: m/z, 388.3 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 11.14 (s, 1H),
8.98 (s, 1H), 7.59 (d, J = 8.2 Hz, 2H), 7.26 (s, 1H), 7.25 (s, 1H), 7.15 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.2 Hz, 2H), 6.75
(s, 1H), 4.37 (t, J = 7.2 Hz, 2H), 3.10 (t, J = 7.2 Hz, 2H), 2.35 (s, 3H). 13C-NMR (101 MHz, DMSO-d6) δ: 150.42,
141.46, 138.25, 132.50, 131.56, 129.84, 129.66, 129.40, 129.19, 128.89, 127.42, 125.77, 105.14, 52.09, 35.86, 21.31.
HRMS (ESI+) m/z calcd for C20H18F3N3O2 [M-H]- 388.1357, found: 388.1387.
4.1.2.4 N-hydroxy-4-(2-(3-(p-tolyl)-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)benzamide (11d)
1
Yield: 40.1%. Mp: 78.6-79.8°C. ESI-MS: m/z, 388.2 [M-H]-. H-NMR (600 MHz, DMSO) δ: 11.15 (s, 1H), 8.99
(s, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 7.31 (s, 1H), 7.26 (s, 1H), 7.24 (d, J = 3.0 Hz, 2H), 7.23 (s,
1H), 4.47 (t, J = 7.2 Hz, 2H), 3.24 (t, J = 7.1 Hz, 2H), 2.33 (s, 3H). 13C-NMR (101 MHz, DMSO-d6) δ: 145.77, 141.48,
139.35, 131.58, 129.83, 129.79, 129.37, 129.21, 129.12, 129.02, 127.38, 126.16, 104.68, 51.11, 35.55, 21.29. HRMS
(ESI+) m/z calcd for C20H18F3N3O2 [M-H]- 388.1357, found:388.1275.
4.1.2.5 N-hydroxy-4-(3-methyl-5-(p-tolyl)-1H-pyrazol-1-yl)benzamide (11e)
1
Yield: 29.8%. Mp: 110.5-111.9°C. ESI-MS: m/z, 320.1 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 11.15 (s, 1H),
9.00 (s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.25 (q, J = 8.2 Hz, 4H), 7.03 (d, J = 8.3 Hz, 2H), 6.21 (s, 1H), 5.30 (s, 2H), 2.32

(s, 3H), 2.20 (s, 3H). 13C-NMR (151 MHz, DMSO-d6) δ: 164.31, 147.58, 144.59, 141.65, 138.36, 132.12, 129.77,
128.55, 127.72, 127.48, 126.77, 106.16, 52.24, 21.15, 13.74. HRMS (ESI+) m/z calcd for C19H19N3O2 [M-H]- 320.1477,
found: 320.1476.
4.1.2.6 N-hydroxy-4-(5-methyl-3-(p-tolyl)-1H-pyrazol-1-yl)benzamide (11f)
1
Yield: 44.0%. Mp: 125.8-127.6°C. ESI-MS: m/z, 320.3 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 11.17 (s, 1H),
9.01 (s, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 7.19 (t, J = 7.9 Hz, 4H), 6.51 (s, 1H), 5.37 (s, 2H), 2.30
(s, 3H), 2.24 (s, 3H). 13C-NMR (151 MHz, DMSO-d6) δ: 163.95, 149.33, 140.81, 140.06, 136.59, 131.99, 130.79,
129.23, 127.31, 126.85, 124.98, 102.72, 51.80, 20.89, 10.89. HRMS (ESI+) m/z calcd for C19H19N3O2 [M-H]- 320.1477,
found: 320.1477.
4.1.2.7 4-(5-(benzo[d][1,3]dioxol-5-yl)-3-methyl-1H-pyrazol-1-yl)-N-hydroxybenzamide (11g)
1
Yield: 25.7%. Mp: 175.3-176.7°C. ESI-MS: m/z, 350.1 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 11.12 (s, 1H),
9.01 (s, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.03 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 8.0 Hz, 1H), 6.92 (d,
J = 1.6 Hz, 1H), 6.83 (d, J = 1.7 Hz, 1H), 6.82 (d, J = 1.7 Hz, 1H), 6.19 (s, 1H), 6.05 (s, 2H), 5.29 (s, 2H), 2.18 (s, 3H)..
13C-NMR (101 MHz, DMSO-d6) δ: 164.21, 148.00, 147.55, 144.37, 141.70, 132.22, 129.93, 127.56, 126.88, 126.76,
124.34, 122.68, 109.16, 109.06, 106.36, 101.86, 52.32, 13.83. HRMS (ESI+) m/z calcd for C19H17N3O4 [M-H]-
350.1219, found: 350.1311.
4.1.2.8 4-(3-(benzo[d][1,3]dioxol-5-yl)-5-methyl-1H-pyrazol-1-yl)-N-hydroxybenzamide (11h)
1
Yield: 43.5%. Mp: 151.8-153.1°C. ESI-MS: m/z, 350.3 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 11.15 (s, 1H),
9.03 (s, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.27 (dd, J = 16.2, 5.6 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.0 Hz, 1H),
6.48 (s, 1H), 6.02 (s, 2H), 5.36 (s, 2H), 2.22 (s, 3H). 13C-NMR (151 MHz, DMSO-d6) δ: 163.96, 149.10, 147.67, 146.66,
140.74, 140.06, 132.02, 127.89, 127.28, 126.84, 118.63, 108.51, 105.44, 102.68, 101.00, 51.76, 10.88. HRMS (ESI+)
m/z calcd for C19H17N3O4 [M-H]- 350.1219, found: 350.1217.
4.1.2.9 4-(5-(4-fluorophenyl)-3-methyl-1H-pyrazol-1-yl)-N-hydroxybenzamide (11i)

Yield: 22.4%. Mp: 142.5-144.1°C. ESI-MS: m/z, 324.1 [M-H]-. 1H-NMR (400 MHz, DMSO-d6) δ: 11.16 (s, 1H),
9.04 (s, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.41 (dd, J = 8.5, 5.5 Hz, 2H), 7.27 (t, J = 8.8 Hz, 2H), 7.02 (d, J = 8.1 Hz,
2H), 6.25 (s, 1H), 5.30 (s, 2H), 2.20 (s, 3H). 13C-NMR (101 MHz, DMSO-d6) δ: 163.86, 162.90, 160.47, 148.41,
140.53, 140.34, 132.22, 130.12, 130.10, 127.27, 127.00, 126.92, 126.84, 115.63, 115.42, 102.91, 51.85, 10.90. HRMS
(ESI+) m/z calcd for C18H16FN3O2 [M-H]- 324.1227, found: 324.1154.
4.1.2.10 4-(3-(4-fluorophenyl)-5-methyl-1H-pyrazol-1-yl)-N-hydroxybenzamide (11j)
1
Yield: 40.1%. Mp: 178.3-178.9°C. ESI-MS: m/z, 324.2 [M-H]-. H-NMR (400 MHz, DMSO-d6) δ: 11.19 (s, 1H),
9.03 (s, 1H), 7.79 (dd, J = 8.7, 5.6 Hz, 2H), 7.71 (d, J = 8.2 Hz, 2H), 7.21 (t, J = 8.3 Hz, 4H), 6.55 (s, 1H), 5.39 (s, 2H),
2.24 (s, 3H). 13C-NMR (101 MHz, DMSO-d6) δ: 163.97, 163.38, 160.93, 147.31, 143.20, 141.14, 131.86, 130.67,
130.59, 127.20, 126.80, 126.77, 126.53, 116.01, 115.80, 106.30, 52.01, 13.42.. HRMS (ESI+) m/z calcd for
C18H16FN3O2 [M-H]- 324.1227, found: 324.1206.
4.1.2.11 4-(3-ethyl-5-(4-fluorophenyl)-1H-pyrazol-1-yl)-N-hydroxybenzamide (11k)
Yield: 23.3%. Mp: 169.2-171.1°C. ESI-MS: m/z, 338.1 [M-H]-. 1H NMR (600 MHz, DMSO) δ: 11.12 (s, 1H), 9.02
(s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.44 – 7.40 (m, 2H), 7.29 – 7.25 (m, 2H), 7.01 (d, J = 8.3 Hz, 2H), 6.29 (s, 1H), 5.32 (s,
2H), 2.58 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H).. 13C-NMR (101 MHz, DMSO-d6) δ: 163.87, 163.36, 160.91,
144.73, 143.07, 141.16, 131.87, 130.67, 130.59, 127.18, 126.89, 126.86, 126.49, 116.00, 115.79, 104.89, 52.08, 21.03,
13.82.. HRMS (ESI+) m/z calcd for C19H18FN3O2 [M-H]- 338.1383, found: 338.1308.
4.1.2.12 4-(5-ethyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)-N-hydroxybenzamide (11l)
1
Yield: 39.5%. Mp: 166.3-167.5°C. ESI-MS: m/z, 338.2 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 11.17 (s, 1H),
9.01 (s, 1H), 7.81 (dd, J = 8.5, 5.7 Hz, 2H), 7.70 (d, J = 8.2 Hz, 2H), 7.23 – 7.17 (m, 4H), 6.59 (s, 1H), 5.39 (s, 2H),
2.58 (q, J = 7.5 Hz, 2H), 1.17 (t, J = 7.5 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6) δ: 164.02, 162.89, 160.47, 148.46,
146.42, 140.82, 132.00, 130.17, 130.14, 127.30, 127.01, 126.93, 126.82, 115.62, 115.41, 101.22, 51.77, 18.31, 12.78.
HRMS (ESI+) m/z calcd for C19H18FN3O2 [M-H]- 338.1383, found: 338.1384.

4.1.2.13 4-(5-(4-fluorophenyl)-3-propyl-1H-pyrazol-1-yl)-N-hydroxybenzamide (11m)
Yield: 26.4%. Mp: 150.1-151.5°C. ESI-MS: m/z, 352.1 [M-H]-. 1H NMR (600 MHz, DMSO) δ: 11.14 (s, 1H), 8.99
(s, 1H), 7.64 (d, J = 8.2 Hz, 2H), 7.43 – 7.40 (m, 2H), 7.28 – 7.24 (m, 2H), 7.00 (d, J = 8.2 Hz, 2H), 6.28 (s, 1H), 5.32 (s,
2H), 2.53 (t, J = 7.5 Hz, 2H), 1.67 – 1.60 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6) δ: 163.95,
163.34, 160.90, 147.16, 143.05, 141.25, 131.82, 130.65, 129.63, 127.18, 126.83, 126.56, 126.41, 115.99, 115.77, 105.35,
52.06, 29.80, 22.38, 13.92. HRMS (ESI+) m/z calcd for C20H20FN3O2 [M-H]- 352.1540, found: 352.1445.
4.1.2.14 4-(3-(4-fluorophenyl)-5-propyl-1H-pyrazol-1-yl)-N-hydroxybenzamide (11n)
1
Yield: 44.3%. Mp: 170.2-170.8°C. ESI-MS: m/z, 352.2 [M-H]-. H-NMR (400 MHz, DMSO-d6) δ: 11.17 (s, 1H),
9.02 (s, 1H), 7.81 (dd, J = 8.8, 5.6 Hz, 2H), 7.70 (d, J = 8.2 Hz, 2H), 7.24 – 7.16 (m, 4H), 6.59 (s, 1H), 5.40 (s, 2H),
2.55 (t, J = 7.6 Hz, 2H), 1.63 – 1.51 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6) δ: 164.02,
162.89, 160.47, 148.47, 144.89, 141.00, 131.97, 130.14, 129.74, 127.28, 127.02, 126.94, 126.77, 115.62, 115.41, 101.78,
51.75, 26.83, 21.48, 13.73. HRMS (ESI+) m/z calcd for C20H20FN3O2 [M-H]- 352.1540, found: 352.1547.
4.1.2.15 4-(3-benzyl-5-(4-fluorophenyl)-1H-pyrazol-1-yl)-N-hydroxybenzamide (11o)
Yield: 21.6%. Mp: 115.4-117.6°C. ESI-MS: m/z, 400.1 [M-H]-. 1H NMR (600 MHz, DMSO) δ 11.15 (s, 1H), 9.02
(s, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.42 – 7.39 (m, 2H), 7.30 (d, J = 1.4 Hz, 2H), 7.29 (s, 2H),
7.27 – 7.24 (m, 2H), 7.02 (d, J = 8.2 Hz, 2H), 6.25 (s, 1H), 5.35 (s, 2H), 3.92 (s, 2H).. 13C-NMR (101 MHz, DMSO-d6)
δ: 163.85, 163.39, 160.94, 151.02, 143.35, 141.07, 140.09, 131.89, 130.69, 130.61, 128.68, 128.44, 127.19, 126.64,
126.47, 126.30, 126.11, 116.01, 115.79, 105.92, 52.17, 34.06. HRMS (ESI+) m/z calcd for C24H20FN3O2 [M-H]-
400.1540, found: 400.1450.
4.1.2.16 4-(5-benzyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)-N-hydroxybenzamide (11p)
1
Yield: 38.3%. Mp: 179.5-181.3°C. ESI-MS: m/z, 400.3 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 11.17 (s, 1H),
9.01 (s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.78 (dd, J = 6.1, 2.8 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.29 – 7.26 (m, 2H), 7.21
– 7.19 (m, 2H), 7.19 (t, J = 1.8 Hz, 2H), 7.13 (d, J = 8.3 Hz, 2H), 6.50 (s, 1H), 5.37 (s, 2H), 4.02 (s, 2H). 13C-NMR (101

MHz, DMSO-d6) δ: 163.96, 162.94, 160.51, 148.64, 143.85, 140.54, 137.94, 131.95, 129.88, 129.46, 128.68, 128.65,
127.18, 127.07, 126.99, 126.61, 115.64, 115.43, 103.16, 51.93, 30.97. HRMS (ESI+) m/z calcd for C24H20FN3O2 [M-H]-
400.1540, found: 400.1565.
4.1.3 General procedure for the synthesis of 16a-16f
To a solution of 1-(4-fluorophenyl)ethanone (1.38 g, 10mmol) and dimethyl oxalate (1.18 g, 10mmol) in diethyl
ether (150 mL) was added dropwise NaOMe (0.65 g, 12mmol) in MeOH (20 mL). The mixture was stirred for 12h at
room temperature and then filtrated under reduced pressure to give 12.
To a solution of 12 (2.24 g, 10 mmol) in acetic acid (50 mL) was added hydrazine hydrate (0.76 g, 11 mmol). The
temperature was raised to 80°C for 4 h. When the mixture was cooled to room temperature, the resulting solution was
poured into water (100 mL). The resulting precipitate was then hydrolyzed into carboxylic acids 14 with sodium
hydroxide (12 mmol) in 50°C for 4 h. Acidify the mixture to pH 2 with 2M HCl, the precipitate was filtered under
reduced pressure.
To a solution of 14 (1.03 g, 5 mmol) in THF (20 mL) were added 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide
(EDCI, 1.15 g, 6 mmol), 1-hydroxy-1H-benzotriazole (HOBt, 0.81 g, 6mmol) and appropriate amino acid methyl ester
hydrochloride (6 mmol). The mixture was stirred at room temperature for 24 h and then poured into water (100 mL),
extracted with ethyl acetate (50 mL × 3). Finally, the organic layers were combined, washed with brine, dried with
Na2SO4 and evaporated. Finally, the resulting residue was purified by column chromatography on silica gel as indicated
to give 15a-15f as white solid.
To a solution of 15a-15f (1 mmol) in methyl alcohol (30 mL) were added 1 M aqueous solution of NaOH (3 mL)
and NH2OH (50 wt % in water, 3 mL) dropwise successively at 0oC. The reaction was warmed to room temperature for
3h. The mixture solution was concentrated to dryness, and the obtained solid was dissolved in water (10 mL). The pH
was adjusted to 7 with a 1M aqueous solution of HCl. The resulting precipitate was filtered under reduced pressure to
give 16a-16f as white solid.

4.1.3.1 5-(4-fluorophenyl)-N-(4-(hydroxycarbamoyl)benzyl)-1H-pyrazole-3-carboxamide (16a)
1
Yield: 54.8%. Mp: 245.2-247.5°C. ESI-MS: m/z, 353.1 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 13.70 (s, 1H),
11.17 (s, 1H), 8.99 (s, 1H), 8.83 (s, 1H), 7.84 (s, 2H), 7.70 (s, 2H), 7.38 (d, J = 6.6 Hz, 2H), 7.31 (d, J = 4.9 Hz, 2H),
7.07 (s, 1H), 4.49 (s, 2H). 13C-NMR (101 MHz, DMSO-d6) δ: 164.51, 163.62, 161.19, 143.28, 131.76, 129.75, 128.86,
127.82, 127.56, 127.37, 127.32, 116.47, 116.25, 114.14, 103.02, 42.24. HRMS (ESI+) m/z calcd for 353.1040.
4.1.3.2 5-(4-fluorophenyl)-N-(3-(hydroxyamino)-3-oxopropyl)-1H-pyrazole-3-carboxamide (16b)
1
Yield: 57.9%. Mp: 192.7-193.5°C. ESI-MS: m/z, 291.0 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 13.62 (s, 1H),
10.47 (s, 1H), 8.76 (s, 1H), 8.13 (t, J = 4.7 Hz, 1H), 7.85 (d, J = 6.5 Hz, 1H), 7.79 (d, J = 4.4 Hz, 1H), 7.33 (d, J = 8.6
Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.05 (s, 1H), 3.45 (dd, J = 12.4, 6.2 Hz, 2H), 2.26 (t, J = 6.2 Hz, 2H). 13C-NMR NMR
(101 MHz, DMSO) δ: 167.59, 161.59, 160.89, 147.87, 142.66, 127.64, 127.57, 127.07, 116.22, 116.01, 102.71, 35.31,
32.45. HRMS (ESI+) m/z calcd for C13H13FN4O3 [M-H]- 291.0972, found: 291.0899.
4.1.3.3 5-(4-fluorophenyl)-N-(4-(hydroxyamino)-4-oxobutyl)-1H-pyrazole-3-carboxamide (16c)
1
Yield: 55.3%. Mp: 142.5-143.1°C. ESI-MS: m/z, 305.1 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 13.63 (s, 1H),
10.40 (s, 1H), 8.72 (s, 1H), 8.23 (s, 1H), 7.83 (s, 2H), 7.30 (s, 2H), 7.05 (s, 1H), 3.24 (dd, J = 12.8, 6.5 Hz, 2H), 2.07 –
1.94 (m, 2H), 1.78 – 1.70 (m, 2H). 13C-NMR (101 MHz, DMSO) δ: 168.94, 163.20, 160.78, 147.71, 142.44, 127.47,
127.43, 127.14, 116.08, 115.88, 102.62, 38.30, 30.08, 25.53.. HRMS (ESI+) m/z calcd for C14H15FN4O3 [M-H]-
305.1128, found: 305.1097.
4.1.3.4 5-(4-fluorophenyl)-N-(5-(hydroxyamino)-5-oxopentyl)-1H-pyrazole-3-carboxamide (16d)
1
Yield: 50.6%. Mp: 107.9-108.6°C. ESI-MS: m/z, 319.1 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 13.59 (s, 1H),
10.34 (s, 1H), 8.66 (s, 1H), 8.16 (s, 1H), 7.82 (d, J = 27.0 Hz, 3H), 7.29 (d, J = 35.7 Hz, 3H), 7.03 (s, 1H), 3.23 (d, J =
5.4 Hz, 2H), 2.04 – 1.93 (s, 2H), 1.58 – 1.44 (s, 5H). 13C-NMR (101 MHz, DMSO-d6) δ: 169.10, 161.57, 158.63,
148.10, 142.57, 127.61, 127.57, 127.08, 116.21, 116.00, 102.70, 38.17, 32.11, 29.08, 22.78. HRMS (ESI+) m/z calcd for
C15H17FN4O3 [M-H]- 319.1285, found: 319.1260.

4.1.3.5 5-(4-fluorophenyl)-N-(6-(hydroxyamino)-6-oxohexyl)-1H-pyrazole-3-carboxamide (16e)
1
Yield: 49.7%. Mp: 114.2-115.0°C. ESI-MS: m/z, 333.2 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 13.59 (s, 1H),
10.33 (s, 1H), 8.65 (s, 1H), 8.13 (s, 1H), 7.83 (s, 2H), 7.30 (s, 2H), 7.03 (s, 1H), 3.23 (s, 2H), 1.95 (t, J = 7.3 Hz, 2H),
1.54 – 1.48 (m, 4H), 1.27 (s, 2H). 13C-NMR (101 MHz, DMSO-d6) δ: 169.17, 162.83, 161.23, 148.15, 142.56, 127.59,
127.33, 127.07, 116.10, 116.01, 102.72, 38.48, 32.34, 29.17, 26.17, 25.02. HRMS (ESI+) m/z calcd for C16H19FN4O3
[M-H]- 333.1441, found: 333.1368.
4.1.3.6 5-(4-fluorophenyl)-N-(7-(hydroxyamino)-7-oxoheptyl)-1H-pyrazole-3-carboxamide (16f)
1
Yield: 53.3%. Mp: 92.5-93.7°C. ESI-MS: m/z, 347.1 [M-H]-. H-NMR (600 MHz, DMSO-d6) δ: 13.59 (s, 1H),
10.33 (s, 1H), 8.65 (s, 1H), 8.13 (s, 1H), 7.83 (s, 2H), 7.30 (s, 2H), 7.03 (s, 1H), 3.23 (s, 2H), 1.95 (t, J = 7.3 Hz, 2H),
1.54 – 1.48 (m, 4H), 1.27 (s, 2H). 13C-NMR (101 MHz, DMSO-d6) δ: 169.18, 163.17, 160.75, 147.57, 142.55, 127.46,
127.37, 127.08, 116.06, 115.85, 102.65, 38.55, 32.32, 29.21, 28.43, 26.27, 25.19.. HRMS (ESI+) m/z calcd for
C17H21FN4O3 [M-H]- 347.1598, found: 347.1518.
4.2. Biological assays
4.2.1 HDAC inhibition fluorescence assay
In this HDACs assay, HeLa nuclear extracts (Enzo Life Sciences, USA) were used as a source of histone
deacetylase. The recombinant human HDAC1, 2, 3, 6 and 8 were purchased from BPS Bioscience (USA). All reactions
were performed in the black half area 96–well microplates. A serial dilution of the inhibitors (5 µL/well) and enzymes
(5 µL/well) were pre-incubated in HDAC buffer (10 µL/well) at 25°C for 15 min, and then fluorogenic substrate (5
µL/well) Boc–Lys(Ac)–AMC (for HeLa NuEx, HDAC1, 2, 3 and 6 isoforms) or Boc–Lys(TFA)–AMC (for HDAC 8
isoform) was added. After incubation at 37°C for 60 min, the mixture was stopped by the addition of developer (25
µL/well) for 10 min. Fluorescence intensity was measured using the Thermo Scientific Varioskan Flash Station at
excitation and emission wavelengths of 355 (or 360 for the HeLa NuEx) and 460 nm, respectively. The IC50 values were
extracted by curve fitting the dose/response slopes.

4.2.2 Anti-proliferative assays
Cells were provided by the Shanghai Cell Bank, Chinese Academy of Sciences. The cells were cultured in media,
DMEM (High Glucose) for A549, MEM for HepG2 with 10% FBS and antibiotics (100 units/mL penicillin G sodium
and 100 ng/mL streptomycin). All cells were incubated in a Thermo/Forma Scientific CO2 water jacketed incubator with
5% CO2 in air at 37°C. The anti-proliferative activities of target compounds were evaluated by the methyl thiazolyl
tetrazolium bromide (MTT) assay. Cells (1.5-3.5×104 cells/well in 100 µL medium) were incubated for 24 h, then
various concentrations of each compound mixed in 100 μL medium were added to each well. After another 96 h of
incubation at 37°C, MTT solution (50 μL of 2 mg/mL) was added per well and the cultures were continued for an
additional 4 h. The medium was removed by aspiration and the cells were dissolved in 200 μL DMSO. The absorbance
at 570 nm was measured in the 96-well plate reader. Growth inhibition was calculated and expressed as the ratio of the
cell number in the treated group to that of the untreated group. The GI50 values were calculated according to inhibition
ratios.

Acknowledgment
This work was supported by the National Nature Science Foundation of China (No. 81473086), Nature Science
Foundation of Liaoning Province (No. 2015020728-301).

References and notes
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