Design, synthesis and anti-tumor activity study of novel histone deacetylase inhibitors containing isatin-based caps and o-phenylenediamine-based zinc binding groups

Article abstract of DOI:10.1016/j.bmc.2017.03.036

As a hot topic of epigenetic studies, histone deacetylases (HDACs) are related to lots of diseases, especially cancer. Further researches indicated that different HDAC isoforms played various roles in a wide range of tumor types. Herein a novel series of HDAC inhibitors with isatin-based caps and o-phenylenediamine-based zinc binding groups have been designed and synthesized through scaffold hopping strategy. Among these compounds, the most potent compound 9n exhibited similar if not better HDAC inhibition and antiproliferative activities against multiple tumor cell lines compared with the positive control entinostat (MS-275). Additionally, compared with MS-275 (IC₅₀ values for HDAC1, 2 and 3 were 0.163, 0.396 and 0.605?μM, respectively), compound 9n with IC₅₀ values of 0.032, 0.256 and 0.311?μM for HDAC1, 2 and 3 respectively, showed a moderate HDAC1 selectivity.

Full text of DOI:10.1016/j.bmc.2017.03.036

Accepted Manuscript
Design, Synthesis and Anti-tumor Activity Study of Novel Histone Deacetylase
Inhibitors Containing Isatin-based Caps and O-phenylenediamine-based Zinc
Binding Groups
Shuai Gao, Jie Zang, Qianwen Gao, Xuewu Liang, Qinge Ding, Xiaoyang Li,
Wenfang Xu, C. James Chou, Yingjie Zhang
PII:
S0968-0896(17)30137-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.036
BMC 13634
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 January 2017
12 March 2017
14 March 2017
Please cite this article as: Gao, S., Zang, J., Gao, Q., Liang, X., Ding, Q., Li, X., Xu, W., Chou, C.J., Zhang, Y.,
Design, Synthesis and Anti-tumor Activity Study of Novel Histone Deacetylase Inhibitors Containing Isatin-based
Caps and O-phenylenediamine-based Zinc Binding Groups, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.03.036
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Design, Synthesis and Anti-tumor Activity Study of Novel
Histone Deacetylase Inhibitors Containing Isatin-based Caps
and O-phenylenediamine-based Zinc Binding Groups
Shuai Gao a, Jie Zang a, Qianwen Gao a, Xuewu Liang a, Qinge Ding a, Xiaoyang Li b,
Wenfang Xu a, C. James Chou b and Yingjie Zhang a*
aDepartment of Medicinal Chemistry, School of Pharmacy, Shandong University, 44
West Culture Road, 250012 Ji’nan, Shandong, P.R. China
bDepartment of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy,
Medical University of South Carolina, Charleston, South Carolina 29425, United States
Abstract: As a hot topic of epigenetic studies, histone deacetylases (HDACs) are
related to lots of diseases, especially cancer. Further researches indicated that different
HDAC isoforms played various roles in a wide range of tumor types. Herein a novel
series of HDAC inhibitors with isatin-based caps and o-phenylenediamine-based zinc
binding groups have been designed and synthesized through scaffold hopping strategy.
Among these compounds, the most potent compound 9n exhibited similar if not better
HDAC inhibition and antiproliferative activities against multiple tumor cell lines
compared with the positive control entinostat (MS-275). Additionally, compared with
MS-275 (IC50 values for HDAC1, 2 and 3 were 0.163, 0.396 and 0.605 µM,
respectively), compound 9n with IC50 values of 0.032, 0.256 and 0.311 µM for
HDAC1, 2 and 3 respectively, showed a moderate HDAC1 selectivity.
Keywords: anticancer； epigenetics； HDAC inhibitors； isatin
Corresponding Authors:
*(Y.Z.) Phone: 86-531-88382009.E-mail: zhangyingjie@sdu.edu.cn
1. Introduction
The onset and progression of cancer, traditionally seen as a genetic disease, are
revealed to be associated with epigenetic changes along with genetic abnormalities in
recent years.1 Histone deacetylases (HDACs) as novel targets for the discovery of
anticancer agents are a series of enzymes that regulate histone deacetylation, which
may be the best understood type of epigenetic modifications.2 HDACs can enrich the
histone tails with positive charges via removing an acetyl group from the ɛ-amino
group of lysine residue, forcing the compaction of the chromatin containing
electronegative DNA. The packaged chromatin prevents transcription factors from
DNA promoters, thereby repressing the expression of genes, including various

tumor-suppressor genes.3 In addition, a mass of non-histone proteins with diverse
biological functions are also substrates of HDACs, such as transcription factors (p53),
nuclear factors (NF-κB), α-tubulin, heat-shock protein 90 (Hsp90), estrogen receptor
(ERα), DNA repair protein Ku70 and so on.4 18 mammalian HDACs have been
identified and grouped into classes I–IV based on their homology to the yeast
(saccharomyces cerevisiae) HDACs.5 Class I HDACs comprising HDAC1, 2, 3 and 8
are homologous to the yeast transcriptional regulator reduced potassium
dependency-3 (RPD3). Class II HDACs which are structurally close to yeast histone
deacetylase1 (Had1) are divided into two subfamilies. HDAC4, 5, 7 and 9 compose
the class IIa HDACs, while HDAC6 and 10 constitute the class IIb HDACs. Class III
HDACs consisting of seven isoforms (SIRT1-7) are most related to the yeast silent
information regulator (sir 2) family. HDAC11, the lastly discovered isoform, is the
only member of class IV HDAC which shares the homology with class I and II
HDACs. The classical HDACs (class I, II, and IV) are Zn2+ dependent enzymes,
whereas class III HDACs needs the co-factor NAD+ for their activity.3
Higher expression levels of class I HDACs were observed in tumor cells than in
normal cells. HDAC1 has been implicated in the initiation and progression of various
types of tumor. For instance, high level of HDAC1 is associated with rapid tumor cell
proliferation and genomic instability in prostate cancer.6 Moreover, the knockdown of
HDAC1 in leukemia cells K562, HL-60, and U937 significantly inhibited cell cycle
progression and cell proliferation.7 HDAC2 overexpression is an independent marker
of poor prognosis in patients with oral, prostate, ovarian, endometrial or gastric
cancer.8 HDAC3 always forms a stable complex with the nuclear receptor
co-repressor (N-CoR) and the silencing mediator of retinoic and thyroid receptors
(SMRT), which are relevant to a wide range of biological processes, such as cell
differentiation, proliferation and apoptosis.9-11 Therefore, the discovery of novel class
I-selective HDAC inhibitors (HDACIs) as antineoplastic agents are of interest for
clinical use.
In recent decades, more than 20 chemical entities have been studied as HDACIs in
different clinical trials. Among them, five compounds were successfully accepted by
U.S.FDA or Chinese FDA (CFDA) as novel anti-cancer agents (Fig. 1). Vorinostat
(SAHA) and romidepsin (FK228) were approved by U.S.FDA for
the chemotherapeutics against refractory cutaneous T-cell lymphoma (CTCL).
Belinostat (PXD-101) and panobinostat (LBH-589) were permitted by U.S.FDA to
remedy for relapsed or refractory peripheral T-cell lymphoma (PTCL) and multiple
myeloma, respectively. Unlike the above four pan-HDACIs, chidamide
(CS055/HBI-8000) which obtained the approval from CFDA as a monotherapy for
relapsed and refractory peripheral T-cell lymphoma (PTCL) is an isoform-selective
HDACI.12 Chidamide, as well as its benzamide derivatives entinostat (MS-275) and
CI-994 (Fig. 2), shows more potent inhibition against class I HDACs (HDAC1, 2, and
3) than other classes. The pharmacophore of most HDACIs comprises three units:
zinc binding group (ZBG) binding the Zn2+ at the bottom of the active site; cap group
(CAP) specifically interacting with the amino acid residues around the entrance of the
active site; a linker that connects the ZBG and CAP occupying the 11 Å channel of

the active site (Fig. 1).13, 14
Figure 1. Pharmacophore model and chemical structures of representative HDACIs
As shown in Fig. 2, indoline-2, 3-dione also called isatin is an endogenous
molecule with a wide range of biological activities, including antiviral activity,
antitumor and antiangiogenic activities, antibacterial, antitubercular, antifungal,
anticonvulsant and anxyolytic activities.15 For example, indirubin as a derivative of
isatin is the active ingredient of Danggui Longhui Wan used to treat chronic
myelocytic leukemia (CML).16 In our pioneering work, a series of hydroxamic acid
derivatives containing isatin-based caps have been synthesized and evaluated as
potential HDACIs. Meanwhile, a benzamide-based molecule 9a has been obtained by
splicing isatin to the structure of the clinical HDACI, CI-994.17Although compound
9a and the positive control MS-275 exhibited similar HDACs inhibition, 9a exhibited
inferior in vitro antiproliferative activity. Herein, structural modification of the lead
compound 9a was carried out through scaffold hopping strategy (Fig. 2) to find more
potent HDACIs.
Figure 2. The design of HDACIs through scaffold splicing and hopping strategy.
2. Results and discussion
2.1 Chemistry

Scheme 1. (1) X = Br, Cl or F; n = 0 or 1. (2) Reagents and conditions: (a) Cl3CH(OH) 2, NH2OH,
Na2SO4, 60-85 °C; (b) concentrated H2SO4, 80 °C; (c) ethane-1, 2-diol or propane-1, 3-diol, TsOH,
toluene, 140°C; (d) BrCH2COOCH3 or BrCH2CH2COOCH3, TBAB, K2CO3, KI, acetone, 60 °C; (e)
KOH, H2O, THF/CH3OH, r.t., then acidified to pH 1 with HCl; (f) methyl-4-aminobenzoate or methyl
4-(aminomethyl) benzoate, TBTU, Et3N, THF/CH2Cl2, r.t.; (g) NaOH, H2O, THF/CH3OH, reflux; then
acidified to pH 1 with HCl; (h) o-phenylenediamine, TBTU, Et3N, THF/CH2Cl2, r.t.
The synthesis of compounds 9a-9o was shown in Scheme 1. Reaction of starting
materials 1a-1c, hydroxylamide hydrochloride and chloral hydrate led to compounds
2a-2c, respectively. Sandmeyer reaction was used for the synthesis of compounds
3a-3c under the condition of concentrated sulfuric acid at 80 °C. Compounds 4a-4f
obtained from 3a-3c by ketalization reaction in the presence of ethane-1, 2-diol or
propane-1, 3-diol were then reacted with methyl 2-bromoacetate or methyl
3-bromopropanoate to get compounds 5a-5h. The hydrolysis of 5a-5h was
implemented to produce intermediates 6a-6h, which further underwent condensation
reaction with methyl-4-aminobenzoate or methyl 4-(aminomethyl) benzoate to afford
7a-7o. Compounds 8a-8o were hydrolytic products of 7a-7o, respectively. Finally,
target compounds 9a-9o were obtained by condensation of o-phenylenediamine and
compounds 8a-8o in the presence of 2-(1H-benzotriazole-1-yl)-1, 1, 3,
3-tetramethyluronium tetrafluoroborate (TBTU).
Condensation of 6a-6f with methyl 6-aminohexanoate hydrochloride, followed by
hydrolysis and condensation with o-phenylenediamine led to target compounds
12a-12f (Scheme 2).
Scheme 2. (1) X = Br, Cl or F; n = 0 or 1. (2) Reagents and conditions: (a) Methyl 6-aminohexanoate
hydrochloride, TBTU, Et3N, THF/CH2Cl2, r.t.; (b) NaOH, H2O, THF/CH3OH, reflux; then acidified to
pH 1 with HCl; (c) o-phenylenediamine , TBTU, Et3N, THF/CH2Cl2, r.t.
Unlike Scheme 1 and 2, the synthetic route affording desired compounds with an
aromatic R1 was illustrated in Scheme 3. Firstly, the amino group of
4-bromo-2-nitroaniline (compound 13) was protected by di-tert-butyl dicarbonate to
get compound 14, and then thienyl or phenyl group was introduced through Suzuki

coupling to get 15a or 15b. Compounds 16a and 16b derived from the reduction of
15a and 15b were reacted with 19 to get 20a and 20b, respectively. At last, the
deprotected compounds 21a and 21b were coupled with intermediates 6b and 6e to
give 22a-22d, which were treated with hydrogen chloride in ethyl acetate to remove
the Boc group to yield the desired compounds 23a-23d.
Scheme 3. (1)R1 = thienyl or phenyl. (2) Reagents and conditions: (a) (Boc)2O, Et3N, DMAP, CH2Cl2,
r.t.; (b) thiophene-2-boronic acid or phenylboronic acid, Pd[P(Ph3)4], Na2CO3, 1, 4-dioxane, H2O,
90 °C; (c) Pd/C, H2, CH3OH/EtOAc, r.t.; (d) (CF3CO)2O, 0 °C; (e) SOCl2, THF; reflux; (f) pyridine, r.t.;
(g) K2CO3, H2O, THF/CH3OH, 70 °C; (h) o-phenylenediamine, TBTU, Et3N, THF/CH2Cl2, r.t.; (i)
EtOAc/HCl, r.t.
The preparation of the compound 30 with fluoro-substituent was illustrated in
Scheme 4. Boc protection of the starting material 24 followed by reduction led to 26.
Reaction of 26 with 19 led to 27, which was transformed to the target compound 30
using similar methods described in Scheme 3.

Scheme 4. Reagents and conditions: (a) (Boc)2O, Et3N, DMAP, CH2Cl2, r.t.; (b) Pd/C, H2,
CH3OH/EtOAc, r.t.; (c) pyridine, r.t.; (d) K2CO3, H2O, THF/CH3OH, 70 °C; (e) o-phenylenediamine,
TBTU, Et3N, THF/CH2Cl2, r.t.; (f) EtOAc/HCl, r.t.
2.2 HeLa nuclear extract inhibitory assay
HeLa nuclear extract was used as the enzyme source to evaluate the HDACs
inhibitory activities of our target compounds with MS-275 as the positive control.
Firstly, the X group of the isatin-based cap in 9a was replaced by different halogens to
get analogs 9b and 9c. Then compounds 9e-9f were synthesized with different sizes of
ketal spiro ring at the 3-postion of the isatin-based caps. In the following work, the
length of the linker was extended by replacing the methyl-4-aminobenzoate with
methyl 4-(aminomethyl) benzoate according to the structure of MS-275 so as to gain
compounds 9g-9l. Meanwhile, compounds 9m, 9n and 9o with different linkers
relative to compounds 9d, 9e and 9h respectively were synthesized to evaluate the
effect of linker length on HDAC inhibition.
Results in Table 1 showed that compounds 9a-9c with six-membered spirocycle
had better HDAC inhibitory activities than their five-membered spirocycle analogs
9d-9f, respectively. The same structure-activity relationship could also be observed
between 9g-9i (n=1) and 9j-9l (n=0), though not that robust. Among compounds
9a-9c (m=0), bromo- or chloro-substituted compounds (9a, 9b) exhibited better
HDAC inhibition than fluoro-substituted compound 9c. The same structure-activity
relationship was also observed among 9d-9f (m=0), but not among 9g-9i (m=1) or
9j-9l (m=1). Compounds 9m and 9n with the ethidine linker in the y position
displayed more potent activities than their counterparts 9d and 9e with the methylene
linker, respectively. However, compound 9o with the ethidine linker in the y position
exhibited similar potency to its analog 9h with the methylene linker. Above
structure-activity relationships indicated that cap part and the linker part could
simultaneously affect the HDAC inhibitory potency of these benzamide-based
HDACIs.
Table 1. HDAC InhibitoryActivity of Compounds 9a-9o, and MS-275
Compd
X
n
m
y
IC50 of HeLa
nuclear
extract (µM)a
-Br
-Cl
-F
-Br
-Cl
1
1
1
0
0
0
0
0
0
0
1
1
1
1
1
1.44 0.42
1.08 0.08
3.75 0.65
2.66 0.45
2.17 0.18
9a
9b
9c
9d
9e

-F
0
1
1
1
0
0
0
1
1
1
0
1
1
1
1
1
1
0
0
1
1
1
1
1
1
1
1
2
2
2
>6.00
9f
-Br
-Cl
-F
-Br
-Cl
-F
Br
Cl
Cl
2.00 0.13
1.25 0.08
1.56 0.72
2.14 0.73
1.93 0.34
1.84 0.62
0.51 0.03
0.43 0.19
1.03 0.25
1.12 0.21
9g
9h
9i
9j
9k
9l
9m
9n
9o
MS-275
a Results expressed as the mean standard deviation of at least three separate
determinations.
Then, compounds 12a-12f with the similar aliphatic linker as RG2833 were
synthesized.18 Results in Table 2 showed that the aliphatic linker, however, may
decrease the HDAC inhibitory activities of the target compounds. This should be
associated with the loss of the π-π stacking interaction between aliphatic linker of
12a-12f and the two parallel phenylalanine residues composing the hydrophobic
channel of HDACs active site.19, 20
Table 2. HDAC InhibitoryActivity of Compounds 12a-12f, and MS-275
Compd
X
n
IC50 of HeLa
nuclear extract
(µM)a
-Br
-Cl
-F
-Br
-Cl
-F
1
1
1
0
0
0
8.42 1.59
8.30 0.87
6.50 0.09
7.53 1.32
9.70 2.83
27.18 0.28
1.12 0.21
12a
12b
12c
12d
12e
12f
MS-275
a Results expressed as the mean standard deviation of at least three separate
determinations.
Besides a hydrophobic 11 Å channel, class I HDACs active site also contains a 14
Å internal cavity at the bottom of the active site.21, 22 The 14 Å cavity as an escaping
route for acetate following deacetylation could be used for the design of HDAC1/2
selective inhibitors.23, 24 Thereby, we designed and synthesized several
5-substituted-o-phenylenediamines 23a-23d, which should be HDAC1/2 selective
over HDAC3. Moreover, one 4-fluorine-o-phenylenediamine analog 30 was also

synthesized. As shown in Table 3, compounds 23a-23d and 30 exhibited significantly
decreased inhibitory activities toward HeLa nuclear extract.
Table 3. HDAC InhibitoryActivity of Compounds 23a-23d, 30, and MS-275
Compd
X
n
R1
R2
IC50 of HeLa
nuclear
extract
(µM)a
Cl
Cl
Cl
1
0
1
H
H
H
>100.0
23a
23b
23c
>100.0
>100.0
Cl
Br
0
1
H
F
>100.0
23d
H
>100.0
30
1.12 0.21
MS-275
a Results expressed as the mean standard deviation of at least three separate
determinations.
2.3 In vitro antiproliferative assay
Based on the results of HDACs inhibitory assay, several compounds were selected
to test their antitumor activities against five cancer cell lines, including K562 (chronic
myelogenous leukemia cell), Molt-4 (human T lymphoblast; acute lymphoblastic
leukemia cell), HEL (human erythrocyte leukemia cell), PC-3 (human prostate cancer
cell) and SK-N-BE(2) (human neuroblastoma) (Table 4). Generally, the
antiproliferative activities of these compounds were consistent with their HDACs
inhibitory activities. Compounds 9m and 9n with the most potent HDACs inhibitory
potency also exhibited the most potent antiproliferative activities, which were
comparable to that of MS-275.
Table 4.In vitro antiproliferative activity for selected HDAC inhibitors
Compd
IC50 (µM)a
K562
Molt-4
HEL
PC-3
SK-N-BE(2)

NDb
1.75
2.83
1.54
1.48
5.28
1.32
ND
2.27
0.69
1.15
0.53
0.58
2.75
0.48
ND
ND
9a
9d
1.24
2.46
0.75
0.70
5.13
0.65
1.27
11.35
4.82
3.23
10.19
4.89
0.93
1.92
0.96
1.01
8.98
0.33
9h
9m
9n
23a
MS-275
a Assays were performed in replicate (n≥2), the SD values are <20% of the mean.
b Not determined.
2.4. HDAC class I and class IIa Whole-cell assay
Whole-cell HDAC inhibition assay in HEL cells was used to evaluate the HDACs
class selectivity of five representative compounds. Results in Table 5 showed that
except 9a and 23a, which was not active for both class I and class IIa HDACs, all
tested o-phenylenediamine-based HDACIs exhibited selectivity for class I HDACs
over class IIa.
Table 5.HDAC class I and IIa cellular activity for selected HDAC inhibitorsa
Compd
HDAC class I cellular IC50 HDAC class IIa cellular
(µM) b
>2.00
0.61
IC50 (µM) c
>2.00
9a
>2.00
9d
9h
0.93
>2.00
0.37
>2.00
9m
0.51
>2.00
9n
>2.00
0.43
>2.00
>2.00
23a
MS-275
a Assays were performed in replicate (n≥2), the SD values are <20% of the mean.
b Boc-Lys (acetyl)-AMC substrate.
c Boc-Lys (triflouroacetyl)-AMC substrate.
2.5. In Vitro HDACs Isoform Selectivity Fluorescence Assay
The compounds were tested for their inhibition against HDAC1, 2 and 3 to further
explore their HDAC isoform selective profiles (Table 6). MS-275 was used as the
positive control. As expected, 5-substituted-o-phenylenediamine 23a exhibited
dramatic HDAC1/2 selectivity over HDAC3. It is worth noting that compared with
MS-275, compound 9n exhibited moderate HDAC1 selectivity over HDAC2 and
HDAC3.
Table 6. HDACs inhibitory activity and isoform selectivity for selected HDAC
inhibitors

Compd
IC50 of
HDAC1
(µM) a
0.046
IC50 of
HDAC2
(µM) a
0.128
IC50 of
HDAC3
(µM) a
0.667
HDAC2/HDAC1 HDAC3/HDAC1
isoform
selectivity
2.8
isoform
selectivity
14.5
9d
0.137
0.012
0.032
0.023
0.163
0.223
0.026
0.256
0.179
0.396
0.096
0.019
0.311
151.8
0.605
1.6
2.2
8.0
7.8
2.3
0.7
9h
1.6
9m
9.7
9n
6600
3.7
23a
MS-275
a Assays were performed in replicate (n≥2), the SD values are <20% of the mean.
2.6. Docking study
To explore the binding mode of these compounds in HDACs, compounds 9h and
9n were docked into the active site of HDAC1 (PDB code 5ICN). Results in Fig. 3
showed that 9h and 9n shared a similar binding mode. For both compounds, the
isatin-based caps were solvent-exposed (Fig. 3a) and the o-phenylenediamine-based
zinc binding groups chelated the Zn2+ (Fig. 3b). However, compared with 9h, the
phenyl group in the linker part of 9n could form more favorable π-π stacking
interaction with the two parallel phenyl groups of Phe 150 and Phe 205 in HDAC1
(Fig. 3b). This could rationalize the better HDAC1 inhibitory activity of 9n.
Figure 3. Proposed binding modes of compounds 9h (pink) and 9n (blue) in the
active site of HDAC1 (derived by modification of PDB code 5ICN using Tripos
SYBYL-X 1.3). The zinc ion is shown as a green sphere.

3. Conclusions
A novel series of compounds with isatin-based caps and o-phenylenediamine-based
zinc binding groups as HDACIs were designed and synthesized based on the previous
work in our laboratory.17All synthesized target compounds were evaluated for their
HDAC inhibition using HeLa nuclear extract. Given their better HDACs inhibitory
potency, compounds 9d, 9h, 9m, 9n and 23a were chosen for the antiproliferative
assays, and compounds 9m and 9n showed promising antiproliferative effects which
were consistent with their potent HDAC inhibitory potency. More importantly,
compared with the MS-275, compound 9n presented moderate HDAC1 selectivity
over HDAC2 and HDAC3, which could be used as a lead compound to find more
selective HDAC1 inhibitors.
4. Experimental section
4.1 Chemistry
4.1.1. Materials and Methods.
All materials, reagents and solvents used without further purification unless
otherwise stated were purchased from commercial sources. All reactions were
monitored by TLC with 0.25 mm silica gel plates (60 GF-254). UV light, iodine stain
and ninhydrin were used to visualize the spots. 1H and 13C were recorded on a Bruker
DRX spectrometer at 300/400 MHz with tetramethylsilane (TMS) as an internal
reference, δ in parts per million (ppm) and J in Hertz. High-resolution mass spectra
were obtained from Shandong Analysis and Test Centre in Ji’nan, China. ESI-MS
spectra were recorded on an API 4000 spectrometer. Melting points were determined
on an electrothermal melting point apparatus without correcting. Silica gel was used
for column chromatography purification. The purity of the tested compounds was
assessed by an Agilent 1200 HPLC instrument. An ODS HYPERSIL column (4.6 mm
× 250 mm, 5µm) was used with 30% water / 70% methanol (method A for compounds
9a-9d, 9n-9o, 12a-12f, 23a-23d and 30) or 35% water / 65% methanol (method B for
compounds 9e-9m) over 18 min at a flow rate of 1.0 mL/min with detection at 254
nm. All final compounds were determined to be at least 95% pure by HPLC analysis.
(E)-N-(4-bromophenyl)-2-(hydroxyimino) acetamide (2a), 5-bromoindoline-2,
3-dione (3a), 5-bromospiro [indoline-3, 2'-[1, 3] dioxan]-2-one (4a), methyl
2-(5-bromo-2-oxospiro [indoline-3, 2'-[1,3]dioxan]-1-yl) acetate (5a),
2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetic acid (6a), methyl
4-(2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)benzoate (7a),
4-(2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)benzoic acid
(8a),
N-(2-aminophenyl)-4-(2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetam
ido)benzamide (9a), methyl
6-(2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)hexanoate (10a)

and their analogues 2b-2c, 3b-3c,4b-4f, 5b-5h, 6b-6h, 7b-7o and 10b-10f in relevant
steps were synthesized as described previously.17
4.1.2. General procedure for the preparation of compounds 8b and its analogue
8c-8o.
4.1.2.1.
4-(2-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)benzoic acid
(8b) To a solution of compound 7b (0.29 g, 0.67 mmol) in 6 mL THF and 3 mL
CH3OH was added 3 M aqueous NaOH (0.7 mL). Keep the reaction for 2 h at room
temperature (r.t.). Then 1 M aqueous HCl was added to the mixture gradually until the
solution became acidic. After filtration, compound 8b was obtained as white solid
(0.20 g, 72% yield). Mp: 226-228 °C, ESI-MS m/z: 417.8 [M + H]+
4.1.2.2.
4-(2-(5-Fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)benzoic acid
(8c)
White solid, 91% yield. Mp: >250 °C, ESI-MS m/z: 401.1 [M + H]+
4.1.2.3.
4-(2-(5-Bromo-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)benzoic
acid (8d)
White solid, 92% yield. Mp: >250 °C, ESI-MS m/z: 447.0 [M + H]+
4.1.2.4.
4-(2-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)benzoic
acid (8e)
White solid, 78% yield. Mp: >250 °C, 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s,
1H), 10.70 (s, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 2.1
Hz, 1H), 7.48 (dd, J = 8.4, 2.2 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 4.56 (s, 2H), 4.37 –
4.32 (m, 4H).
4.1.2.5.
4-(2-(5-Fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)benzoic
acid (8f)
White solid, 86% yield. Mp: >250 °C, 1H NMR (400 MHz, DMSO-d6) δ 12.76 (s,
1H), 10.71 (s, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.69 (d, J = 8.7 Hz, 2H), 7.37 (dd, J = 7.6,
2.6 Hz, 1H), 7.28 (td, J = 9.2, 2.7 Hz, 1H), 7.08 (dd, J = 8.6, 4.0 Hz, 1H), 4.56 (s, 2H),
4.38 – 4.31 (m, 4H).
4.1.2.6.
4-((2-(5-Bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)methyl)ben
zoic acid (8g)
White solid, 88% yield. Mp: >250 °C, ESI-MS m/z: 475.1 [M + H]+
4.1.2.7.
4-((2-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)methyl)ben
zoic acid (8h)
White solid, 91% yield. Mp: 228-230 °C, 1H NMR (400 MHz, DMSO-d6) δ 12.88 (s,
1H), 8.85 (t, J = 5.9 Hz, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.45 (dd, J = 8.4, 2.2 Hz, 1H),
7.42 – 7.39 (m, 2H), 7.38 (s, 1H), 6.96 (d, J = 8.4 Hz, 1H), 4.72 (td, J = 11.5, 2.4 Hz,

2H), 4.39 (s, 2H), 4.37 (s, 2H), 3.98 – 3.91 (m, 2H), 2.26 – 2.12 (m, 1H), 1.74 – 1.66
(m, 1H).
4.1.2.8.
4-((2-(5-Fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)methyl)benz
oic acid (8i)
White solid, 90% yield. Mp: >250 °C, ESI-MS m/z: 415.1 [M + H]+
4.1.2.9.
4-((2-(5-Bromo-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)methyl)be
nzoic acid (8j)
White solid, 71% yield. Mp: >250 °C, ESI-MS m/z: 461.0 [M + H]+
4.1.2.10.
4-((2-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)methyl)be
nzoic acid (8k)
White solid, 93% yield. Mp: >250 °C, 1H NMR (400 MHz, DMSO-d6) δ 12.88 (s, 1H),
8.85 (t, J = 5.9 Hz, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.52 – 7.45 (m, 2H), 7.38 (d, J = 8.3
Hz, 2H), 6.99 (dd, J = 7.6, 1.3 Hz, 1H), 4.38 (s, 2H), 4.37 (s, 2H), 4.36 – 4.28 (m,
4H).
4.1.2.11.
4-((2-(5-Fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)methyl)be
nzoic acid (8l)
White solid, 59% yield. Mp: 230-232 °C, ESI-MS m/z: 401.1 [M + H]+
4.1.2.12.
4-(3-(5-Bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)propanamido)benzoic
acid (8m)
White solid, 86% yield. Mp: >250 °C, 1H NMHR (400 MHz, DMSO-d6) δ 12.52 (s,
1H), 10.32 (s, 1H), 7.92 – 7.85 (m, 2H), 7.65 (d, J = 8.7 Hz, 2H), 7.61 (dd, J = 8.4,
2.1 Hz, 1H), 7.51 (d, J = 2.0 Hz, 1H), 7.14 (t, J = 5.7 Hz, 1H), 4.70 (td, J = 11.4, 2.2
Hz, 2H), 3.97 – 3.88 (m, 4H), 2.69 (t, J = 7.0 Hz, 2H), 2.23 – 2.10 (m, 1H), 1.72 –
1.63 (m, 1H).
4.1.2.13.
4-(3-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)propanamido)benzoic
acid (8n)
White solid, 95% yield. Mp: >250 °C, ESI-MS m/z: 431.1 [M + H]+
4.1.2.14.
4-((3-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)propanamido)methyl)
benzoic acid (8o)
White solid, 98% yield. Mp: >250 °C, ESI-MS m/z: 445.1 [M + H]+
4.1.3. General procedure for the preparation of compounds 9b and its analogue
9c-9o.
4.1.3.1.
N-(2-Aminophenyl)-4-(2-(5-chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acet
amido)benzamide (9b)
To a solution of 8b (0.15 g, 0.36 mmol) in dry THF and CH2Cl2 was added TBTU
(0.14 g, 0.43 mmol), followed by Et3N (0.2 mL, 1.44 mmol). After 40 min in room

temperature, o-phenylenediamine (0.05 g, 0.43 mmol) was added. The mixture was
stirred at the same temperature overnight. Then the solvent was evaporated under
vacuum. The residue was dissolved in EtOAc and washed with brine (3 × 20 mL).
Organic phase was combined and dried by MgSO4 overnight. The EtOAc was
removed under low pressure to give a crude material that was purified via silica-gel
column chromatography (CH2Cl2/CH3OH = 100:1), yielding the desired compound
9b as a reddish white solid (0.25 g, 66% yield). Mp: >250 °C, 1H NMR (400 MHz,
DMSO-d6) δ 10.67 (s, 1H), 9.60 (s, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 8.7 Hz,
2H), 7.52 – 7.39 (m, 2H), 7.16 (d, J = 7.5 Hz, 1H), 7.09 (d, J = 8.2 Hz, 1H), 7.02 –
6.92 (m, 1H), 6.78 (dd, J = 7.9, 0.9 Hz, 1H), 6.65 – 6.55 (m, 1H), 4.89 (s, 2H), 4.74 (t,
J = 10.5 Hz, 2H), 4.59 (s, 2H), 3.99-3.95 (m, 2H), 2.27 – 2.13 (m, 1H), 1.73-1.69 (m,
1H). 13C NMR (100 MHz, DMSO-d6) δ 171.34, 165.66, 165.10, 143.61, 141.80,
141.77, 131.15, 129.82, 129.30, 128.72, 127.41, 127.15, 126.87, 124.45, 123.91,
118.70, 116.74, 116.62, 111.78, 93.44, 61.17, 42.79, 25.21. HRMS (AP-ESI) m/z
calcd for C26H23ClN4O5 [M + H]+ 507.1430, found 507.1563. HPLC tR = 8.37 min,
97.5%
4.1.3.2.
N-(2-Aminophenyl)-4-(2-(5-fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acet
amido)benzamide (9c)
White solid, 51% yield. Mp: >250 °C, 1H NMR (400 MHz, DMSO-d6) δ 10.66 (s,
1H), 9.59 (s, 1H), 7.97 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H), 7.30 (dd, J = 7.7,
2.6 Hz, 1H), 7.25 (td, J = 9.2, 2.7 Hz, 1H), 7.16 (d, J = 7.5 Hz, 1H), 7.07 (dd, J = 8.6,
4.1 Hz, 1H), 6.96 (t, J = 7.6 Hz, 1H), 6.78 (d, J = 8.7 Hz, 1H), 6.59 (t, J = 7.9 Hz, 1H),
4.88 (s, 2H), 4.75 (dd, J = 11.5, 9.3 Hz, 2H), 4.58 (s, 2H), 3.97 (dd, J = 11.4, 2.9 Hz,
2H), 2.27 – 2.13 (m, 1H), 1.72-1.69 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ
171.57, 165.77, 165.02, 160.19, 157.81, 143.62, 141.82, 139.05, 129.80, 129.30,
128.47, 128.39, 127.15, 126.87, 123.90, 118.69, 117.73, 117.50, 116.73, 116.61,
112.35, 112.10, 111.27, 111.20, 93.54, 61.09, 42.80, 25.21. HRMS (AP-ESI) m/z
calcd for C26H23FN4O5 [M + H]+ 491.1725, found 491.1851. HPLC tR = 5.64 min,
95.4%
4.1.3.3.
N-(2-Aminophenyl)-4-(2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)ac
etamido)benzamide (9d)
White solid, 60% yield. Mp: >250 °C,1H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H),
9.59 (s, 1H), 7.95 (t, J = 12.3 Hz, 2H), 7.69 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 6.8 Hz,
2H), 7.15 (d, J = 7.6 Hz, 1H), 7.07 (d, J = 9.0 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 6.78
(d, J = 7.8 Hz, 1H), 6.59 (t, J = 7.4 Hz, 1H), 4.89 (s, 2H), 4.57 (s, 2H), 4.39 – 4.30 (m,
4H). 13C NMR (100 MHz, DMSO-d6) δ 172.84, 165.60, 165.11, 143.70, 143.60,
141.77, 134.68, 129.86, 129.28, 127.88, 127.13, 126.85, 126.81, 123.93, 118.73,
116.74, 116.62, 115.27, 112.57, 101.52, 66.39, 46.18, 43.09. HRMS (AP-ESI) m/z
calcd for C25H21BrN4O5 [M + H]+ 537.0768, found 537.0769. HPLC tR = 6.50 min,
95.3%
4.1.3.4.

N-(2-Aminophenyl)-4-(2-(5-chloro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)ac
etamido)benzamide (9e)
White solid, 55% yield. Mp: >250 °C,1H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H),
9.58 (s, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.69 (d, J = 8.7 Hz, 2H), 7.53 (d, J = 2.1 Hz,
1H), 7.50 (dd, J = 8.4, 2.2 Hz, 1H), 7.16 (d, J = 7.0 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H),
7.00 – 6.93 (m, 1H), 6.78 (dd, J = 8.0, 1.2 Hz, 1H), 6.59 (td, J = 7.8, 1.2 Hz, 1H), 4.90
(s, 2H), 4.58 (s, 2H), 4.39 – 4.30 (m, 4H).13C NMR (100 MHz, DMSO-d6) δ 172.96,
165.63, 165.10, 143.61, 143.26, 141.77, 131.87, 129.85, 129.29, 127.70, 127.14,
126.86, 126.47, 125.18, 123.91, 118.72, 116.74, 116.61, 112.10, 101.56, 66.38, 43.11.
HRMS (AP-ESI) m/z calcd for C25H21ClN4O5 [M + H]+ 493.1273, found 493.1410.
HPLC tR = 8.36 min, 97.7%
4.1.3.5.
N-(2-Aminophenyl)-4-(2-(5-fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)ac
etamido)benzamide (9f)
White solid, 66% yield. Mp: >250 °C,1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H),
8.25 (t, J = 5.4 Hz, 1H), 7.26 (dd, J = 7.7, 2.7 Hz, 1H), 7.23 – 7.14 (m, 2H), 6.93 –
6.83 (m, 2H), 6.71 (dd, J = 8.0, 1.2 Hz, 1H), 6.53 (td, J = 7.7, 1.4 Hz, 1H), 4.81 (s,
2H), 4.73 (td, J = 11.5, 2.4 Hz, 2H), 4.26 (s, 2H), 3.99 – 3.88 (m, 2H), 3.09 (q, 6.6 Hz,
2H), 2.32 (t, J = 7.4 Hz, 2H), 2.25 – 2.10 (m, 1H), 1.72 – 1.65 (m, 1H), 1.65 – 1.55 (m,
2H), 1.52 – 1.41 (m, 2H), 1.39 – 1.29 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ
172.95, 171.55, 166.13, 142.28, 140.55, 126.19, 125.76, 124.09, 118.35, 118.11,
116.69, 116.40, 112.88, 112.64, 111.41, 111.33, 101.67, 66.23, 42.39, 39.12, 36.16,
29.26, 26.57, 25.47. HRMS (AP-ESI) m/z calcd for C25H21FN4O5 [M + H]+ 477.1569,
found 477.1717. HPLC tR = 11.55 min, 95.2%
4.1.3.6.
N-(2-Aminophenyl)-4-((2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)ace
tamido)methyl)benzamide (9g)
White solid, 34% yield. Mp: 219-221 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.65 (s,
1H), 8.88 (t, J = 5.7 Hz, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.59 (dd, J = 8.4, 1.9 Hz, 1H),
7.52 (d, J = 1.9 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 7.7 Hz, 1H), 6.97 (t, J =
7.6 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 7.8 Hz, 1H), 6.60 (t, J = 7.4 Hz,
1H), 4.86 (s, 2H), 4.72 (t, J = 10.6 Hz, 2H), 4.39 (d, J = 7.0 Hz, 4H), 3.94 (dd, J =
11.1, 2.9 Hz, 2H), 2.27 – 2.09 (m, 1H), 1.69 (d, J = 13.4 Hz, 1H).13C NMR (100 MHz,
DMSO-d6) δ 171.09, 166.58, 165.54, 143.61, 143.00, 142.23, 133.82, 129.08, 128.33,
127.45, 127.17, 127.11, 126.66, 123.80, 116.66, 114.88, 112.13, 93.36, 61.13, 42.52,
42.13, 25.21. HRMS (AP-ESI) m/z calcd for C27H25BrN4O5 [M + H]+ 565.1081, found
565.1086. HPLC tR = 11.94 min, 98.3%
4.1.3.7.
N-(2-Aminophenyl)-4-((2-(5-chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)ace
tamido)methyl)benzamide (9h)
White solid, 31% yield. Mp: 203-205 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.65 (s,
1H), 8.87 (t, J = 5.8 Hz, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.46 (dd, J = 8.4, 2.1 Hz, 1H),
7.44 – 7.41 (m, 2H), 7.40 (s, 1H), 7.18 (d, J = 7.6 Hz, 1H), 6.98 (t, J = 7.2 Hz, 2H),
6.79 (d, J = 7.3 Hz, 1H), 6.61 (t, J = 7.4 Hz, 1H), 4.90 (s, 2H), 4.73 (t, J = 10.5 Hz,

2H), 4.41 (s, 2H), 4.39 (s, 2H), 3.97-3.94 (m, 2H), 2.27 – 2.12 (m, 1H), 1.72-1.69 (m,
1H). 13C NMR (100 MHz, DMSO-d6) δ 171.19, 166.61, 165.55, 143.62, 143.00,
141.79, 133.72, 131.05, 128.75, 128.33, 127.45, 127.33, 127.18, 126.96, 124.41,
123.78, 116.72, 116.59, 111.64, 93.40, 61.11, 42.52, 42.15, 25.20. HRMS (AP-ESI)
m/z calcd for C27H25ClN4O5 [M + H]+ 521.1586, found 521.1702. HPLC tR = 10.54
min, 98.1%
4.1.3.8.
N-(2-Aminophenyl)-4-((2-(5-fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)ace
tamido)methyl)benzamide (9i)
White solid, 39% yield. Mp: 198-200 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.64 (s,
1H), 8.87 (t, J = 5.7 Hz, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.28
(dd, J = 7.6, 2.3 Hz, 1H), 7.25 – 7.20 (m, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.01 – 6.91
(m, 2H), 6.79 (d, J = 7.7 Hz, 1H), 6.60 (t, J = 7.4 Hz, 1H), 4.90 (s, 2H), 4.74 (dd, J =
11.2, 9.7 Hz, 2H), 4.43 – 4.36 (m, 4H), 3.95 (dd, J = 10.8, 3.0 Hz, 2H), 2.25 – 2.11 (m,
1H), 1.71-1.68 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 171.41, 166.71, 165.54,
160.15, 157.77, 143.63, 143.03, 139.05, 133.72, 128.32, 127.45, 127.18, 126.95,
123.78, 117.63, 117.40, 116.72, 116.59, 112.32, 112.07, 111.12, 111.04, 93.52, 61.03,
42.51, 42.18, 25.21. HRMS (AP-ESI) m/z calcd for C27H25FN4O5 [M + H]+ 505.1882,
found 505.1986. HPLC tR = 6.89 min, 97.2%
4.1.3.9.
N-(2-Aminophenyl)-4-((2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)a
cetamido)methyl)benzamide (9j)
White solid, 18% yield. Mp: 231-233 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.63 (s,
1H), 8.86 (t, J = 5.8 Hz, 1H), 7.95 (d, J = 8.1 Hz, 2H), 7.65 – 7.58 (m, 2H), 7.39 (d, J
= 8.2 Hz, 2H), 7.17 (d, J = 7.5 Hz, 1H), 7.01 – 6.92 (m, 2H), 6.78 (d, J = 9.1 Hz, 1H),
6.60 (t, J = 7.5 Hz, 1H), 4.91 (s, 2H), 4.39 (s, 2H), 4.37 (s, 2H), 4.39-4.31 (m, 4H).13C
NMR (100 MHz, DMSO-d6) δ 172.68, 166.53, 165.57, 143.72, 143.57, 142.98,
134.56, 133.77, 128.32, 127.82, 127.46, 127.13, 126.90, 123.85, 116.75, 116.62,
115.18, 112.41, 101.51, 66.35, 42.55, 42.47. HRMS (AP-ESI) m/z calcd for
C26H23BrN4O5 [M + H]+ 551.0928, found 551.0926. HPLC tR = 7.93 min, 95.8%
4.1.3.10.
N-(2-Aminophenyl)-4-((2-(5-chloro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)a
cetamido)methyl)benzamide (9k)
1
White solid, 41 % yield. Mp: >250 °C, H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H),
8.85 (t, J = 5.9 Hz, 1H), 7.95 (d, J = 8.1 Hz, 2H), 7.53 – 7.46 (m, 2H), 7.39 (d, J = 8.2
Hz, 2H), 7.17 (d, J = 7.3 Hz, 1H), 7.03 – 6.93 (m, 2H), 6.79 (dd, J = 8.0, 1.3 Hz, 1H),
6.60 (td, J = 7.7, 1.2 Hz, 1H), 4.89 (s, 2H), 4.39 (s, 2H), 4.37 (s, 2H), 4.36 – 4.28 (m,
4H). 13C NMR (100 MHz, DMSO-d6) δ 172.80, 166.56, 165.56, 143.60, 143.27,
142.99, 133.75, 131.70, 128.32, 127.62, 127.46, 127.16, 126.94, 126.56, 125.12,
123.81, 116.73, 116.61, 111.94, 101.55, 66.34, 42.53, 42.48. HRMS (AP-ESI) m/z
calcd for C26H23ClN4O5 [M + H]+ 507.1430, found 507.1538. HPLC tR = 7.17 min,
98.3%
4.1.3.11.

N-(2-Aminophenyl)-4-((2-(5-fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)a
cetamido)methyl)benzamide (9l)
White solid, 50% yield. Mp: 230-232 °C, 1H NMR (300 MHz, DMSO-d6) δ 9.62 (s,
1H), 8.84 (t, J = 5.9 Hz, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 7.34
(dd, J = 7.7, 2.7 Hz, 1H), 7.31 – 7.23 (m, 1H), 7.20 – 7.14 (m, 1H), 6.97 (ddd, J = 9.5,
6.1, 1.7 Hz, 2H), 6.78 (dd, J = 8.0, 1.3 Hz, 1H), 6.60 (td, J = 7.7, 1.3 Hz, 1H), 4.88 (s,
2H), 4.42 – 4.36(m, 4H), 4.35-4.30 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ
173.04, 166.66, 165.55, 160.34, 157.96, 143.61, 143.02, 140.55, 133.74, 128.32,
128.06, 127.76, 127.46, 127.17, 126.94, 123.80, 118.36, 118.12, 116.72, 116.60,
112.94, 112.69, 111.46, 111.39, 66.26, 42.49. HRMS (AP-ESI) m/z calcd for
C26H23FN4O5 [M + H]+ 491.1725, found 491.1836. HPLC tR = 9.09 min, 95.2%
4.1.3.12.
N-(2-Aminophenyl)-4-(3-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)pro
panamido)benzamide (9m)
White solid, 44% yield. Mp: 218-220 °C, 1H NMR (400 MHz, DMSO-d6) δ 10.28 (s,
1H), 9.57 (s, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.67 (d, J = 8.7 Hz, 2H), 7.62 (dd, J = 8.4,
2.0 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.19 – 7.13 (m, 2H), 7.00 – 6.94 (m, 1H), 6.79
(dd, J = 7.9, 0.9 Hz, 1H), 6.63 – 6.57 (m, 1H), 4.89 (s, 2H), 4.72 (dd, J = 11.5, 9.4 Hz,
2H), 3.98 – 3.89 (m, 4H), 2.70 (t, J = 7.0 Hz, 2H), 2.25 – 2.11 (m, 1H), 1.69 (d, J =
13.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 170.98, 169.46, 165.17, 143.58,
142.14, 141.58, 134.07, 129.54, 129.32, 129.14, 127.25, 127.12, 126.85, 123.96,
118.78, 116.76, 116.62, 114.98, 112.18, 93.20, 61.07, 36.16, 34.72, 25.19. HRMS
(AP-ESI) m/z calcd for C27H25BrN4O5 [M + H]+ 565.1081, found 565.1078. HPLC tR
= 18.11 min, 97.5%
4.1.3.13.
N-(2-Aminophenyl)-4-(3-(5-chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)pro
panamido)benzamide (9n)
White solid, 25% yield. Mp: 220-222 °C, 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s,
1H), 9.55 (s, 1H), 7.94 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 7.49 (dd, J = 8.4,
2.2 Hz, 1H), 7.41 (d, J = 2.2 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.16 (d, J = 6.9 Hz,
1H), 7.02 – 6.92 (m, 1H), 6.78 (dd, J = 8.0, 1.2 Hz, 1H), 6.60 (td, J = 7.7, 1.3 Hz, 1H),
4.89 (s, 2H), 4.72 (td, J = 11.5, 2.4 Hz, 2H), 4.03 – 3.87 (m, 4H), 3.32 (s, 2H), 2.70 (t,
J = 7.0 Hz, 2H), 2.24 – 2.10 (m, 1H), 1.74 – 1.63 (m, 1H). 13C NMR (100 MHz,
DMSO-d6) δ 171.08, 169.46, 165.18, 143.52, 142.14, 141.16, 131.20, 129.55, 129.13,
129.02, 127.39, 127.10, 126.84, 124.55, 124.00, 118.79, 116.80, 116.65, 111.68,
93.26, 61.06, 36.18, 34.73, 25.19. HRMS (AP-ESI) m/z calcd for C27 H25ClN4O5 [M +
H]+ 521.1586, found 521.1579. HPLC tR = 10.44 min, 95.8%
4.1.3.14.
N-(2-Aminophenyl)-4-((3-(5'-chloro-2'-oxospiro[[1,3]dioxane-2,3'-indolin]-1'-yl)p
ropanamido)methyl)benzamide (9o)
White solid, 37% yield. Mp: 171-173 °C,1H NMR (400 MHz, DMSO-d6) δ 9.61 (s,
1H), 8.56 (t, J = 5.9 Hz, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.44 (dd, J = 8.3, 2.2 Hz, 1H),
7.41 (d, J = 2.0 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.16 (d, J = 7.3 Hz, 1H), 7.11 (d, J
= 8.3 Hz, 1H), 7.01 – 6.93 (m, 1H), 6.78 (dd, J = 8.0, 1.3 Hz, 1H), 6.65 – 6.56 (m,

1H), 4.89 (s, 2H), 4.74 (td, J = 11.5, 2.5 Hz, 2H), 4.32 (d, J = 5.8 Hz, 2H), 3.98 – 3.91
(m, 2H), 3.88 (t, J = 7.0 Hz, 2H), 2.52 (t, J = 8.7 Hz, 2H), 2.25 – 2.11 (m, 1H), 1.75 –
1.66 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 171.04, 170.16, 165.54, 143.55,
143.21, 141.17, 133.58, 131.15, 128.98, 128.23, 127.37, 127.12, 126.91, 124.50,
123.87, 116.76, 116.63, 111.72, 93.27, 61.08, 42.35, 36.49, 33.66, 25.22. HRMS
(AP-ESI) m/z calcd for C28 H27ClN4O5 [M + H]+ 535.1743, found 535.1742. HPLC tR
= 8.14 min, 97.3%
4.1.4. General procedure for the preparation of compounds 11a and its analogue
11a-11f.
4.1.4.1.
6-(2-(5-Bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)hexanoic
acid (11a) To a solution of compound 10a (0.20 g, 0.43 mmol) in 6 mL THF and 3
mL CH3OH was added 3 M aqueous NaOH (0.42 mL). Keep the reaction for 2 h at
room temperature. Then 1 M aqueous HCl was added to the mixture gradually until
the solution became acidic. After filtration, the compound 11a was obtained as white
solid (0.18 g, 93% yield). ESI-MS m/z: 455.1 [M + H]+
4.1.4.2.
6-(2-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)hexanoic
acid (11b)
White solid, 89% yield. Mp: 143-145 °C, ESI-MS m/z: 411.1 [M + H]+
4.1.4.3.
6-(2-(5-Fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)hexanoic
acid (11c)
White solid, 93% yield. Mp: 144-146 °C, ESI-MS m/z: 395.2 [M + H]+
4.1.4.4.
6-(2-(5-Bromo-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)hexanoic
acid (11d)
White solid, 74% yield. Mp: 165-167 °C, ESI-MS m/z: 441.1 [M + H]+
4.1.4.5.
6-(2-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)hexanoic
acid (11e)
White solid, 98% yield. Mp: 170-172 °C, 1H NMR (400 MHz, DMSO-d6) δ 11.98 (s,
1H), 8.22 (t, J = 5.5 Hz, 1H), 7.50 – 7.44 (m, 2H), 6.92 (dd, J = 8.0, 0.6 Hz, 1H), 4.38
– 4.28 (m, 4H), 4.25 (s, 2H), 3.05 (q, J = 6.7 Hz, 2H), 2.19 (t, J = 7.4 Hz, 2H), 1.54 –
1.45 (m, 2H), 1.45 – 1.37 (m, 2H), 1.32 – 1.22 (m, 2H).
4.1.4.6.
6-(2-(5-Fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)hexanoic
acid (11f)
White solid, 79% yield. Mp: 133-135 °C, 1H NMR (300 MHz, DMSO-d6) δ 11.98 (s,
1H), 8.24 (t, J = 5.4 Hz, 1H), 7.32 (dd, J = 7.7, 2.7 Hz, 1H), 7.30 – 7.20 (m, 1H), 6.90
(dd, J = 8.6, 4.1 Hz, 1H), 4.41 – 4.26 (m, 4H), 4.24 (s, 2H), 3.06 (q, 6.6 Hz, 2H), 2.20
(t, J = 7.3 Hz, 2H), 1.59 – 1.46 (m, 2H), 1.45 – 1.36 (m, 2H), 1.33 – 1.19 (m, 2H).
4.1.5. General procedure for the preparation of compounds 12a and its analogue
12b-12f.

4.1.5.1.
N-(2-Aminophenyl)-6-(2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acet
amido)hexanamide (12a)
To a solution of 11a (0.20 g, 0.44 mmol) in dry THF and CH2Cl2 was added TBTU
(0.17 g, 0.53 mmol), followed by Et3N (0.25 mL, 1.76 mmol). After 40 min in room
temperature, o-phenylenediamine (0.06 g, 0.53 mmol) was added. The mixture was
stirred at the same temperature overnight. Then the solvent was evaporated under
vacuum. The residue was dissolved in EtOAc and washed with brine (3 × 20 mL).
Organic phase was combined and dried by MgSO4 overnight. The EtOAc was
removed under vacuum to give a crude material that was purified via silica-gel
column chromatography (CH2Cl2/CH3OH = 100:1), yielding the desired compound
12a as white solid (0.25 g, 54% yield). Mp: 139-140 °C, 1H NMR (400 MHz,
DMSO-d6) δ 9.08 (s, 1H), 8.24 (t, J = 5.0 Hz, 1H), 7.58 – 7.48 (m, 2H), 7.16 (d, J =
7.7 Hz, 1H), 6.93 – 6.80 (m, 2H), 6.71 (d, J = 7.9 Hz, 1H), 6.54 (t, J = 7.5 Hz, 1H),
4.80 (s, 2H), 4.71 (t, J = 10.6 Hz, 2H), 4.26 (s, 2H), 3.92 (d, J = 2.8 Hz, 2H), 3.09 (dd,
J = 12.2, 6.3 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H), 2.25 – 2.10 (m, 1H), 1.68 (d, J = 13.4
Hz, 1H), 1.60 (dt, J = 14.7, 7.4 Hz, 2H), 1.51 – 1.41 (m, 2H), 1.38 – 1.28 (m, 2H). 13C
NMR (100 MHz, DMSO-d6) δ 171.54, 171.02, 166.04, 142.34, 142.25, 133.90,
129.06, 127.13, 126.15, 125.75, 124.08, 116.66, 116.38, 114.80, 112.06, 93.35, 61.11,
60.21, 42.02, 39.15, 36.17, 29.25, 26.58, 25.47, 25.21. HRMS (AP-ESI) m/z calcd for
C25H29BrN4O5 [M + H]+ 545.1394, found 545.1395. HPLC tR = 8.54 min, 96.6%
4.1.5.2.
N-(2-Aminophenyl)-6-(2-(5-chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acet
amido)hexanamide (12b)
White solid, 53% yield. Mp: 123-125 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.10 (s,
1H), 8.26 (t, J = 5.4 Hz, 1H), 7.45 – 7.39 (m, 2H), 7.16 (d, J = 7.2 Hz, 1H), 6.89 (dd,
J = 7.8, 3.7 Hz, 2H), 6.71 (d, J = 7.2 Hz, 1H), 6.54 (t, J = 7.5 Hz, 1H), 4.72 (t, J =
10.5 Hz, 2H), 4.27 (s, 2H), 3.95-3.92 (m, 2H), 3.09 (q, J = 6.4 Hz, 2H), 2.32 (t, J =
7.4 Hz, 2H), 2.25 – 2.11 (m, 1H), 1.70-1.67 (m, 1H), 1.65 – 1.55 (m, 2H), 1.50-1.43
( m, 2H), 1.37-1.29 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 171.54, 171.11,
166.07, 142.35, 141.81, 131.04, 128.70, 127.25, 126.16, 125.77, 124.41, 124.04,
116.65, 116.37, 111.58, 93.39, 61.08, 42.02, 39.13, 36.16, 29.26, 26.57, 25.48, 25.20.
HRMS (AP-ESI) m/z calcd for C25H29ClN4O5 [M + H]+ 501.1899, found 501.2034.
HPLC tR = 7.71 min, 98.5%
4.1.5.3.
N-(2-Aminophenyl)-6-(2-(5-fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acet
amido)hexanamide (12c)
White solid, 42% yield. Mp: 129-131 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s,
1H), 8.25 (t, J = 5.4 Hz, 1H), 7.26 (dd, J = 7.7, 2.7 Hz, 1H), 7.23 – 7.14 (m, 2H), 6.93
– 6.83 (m, 2H), 6.71 (dd, J = 8.0, 1.2 Hz, 1H), 6.53 (td, J = 7.7, 1.4 Hz, 1H), 4.81 (s,
2H), 4.73 (td, J = 11.5, 2.4 Hz, 2H), 4.26 (s, 2H), 3.99 – 3.88 (m, 2H), 3.09 (q, 6.6 Hz,
2H), 2.32 (t, J = 7.4 Hz, 2H), 2.25 – 2.10 (m, 1H), 1.72 – 1.65 (m, 1H), 1.65 – 1.55 (m,
2H), 1.52 – 1.41 (m, 2H), 1.39 – 1.29 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ
171.54, 171.33, 166.17, 142.36, 139.08, 128.46, 128.39, 126.16, 125.77, 124.05,

117.61, 117.37, 116.65, 116.37, 112.25, 112.00, 111.05, 110.97, 61.01, 42.06, 39.12,
36.16, 29.27, 26.58, 25.48, 25.21. HRMS (AP-ESI) m/z calcd for C25H29FN4O5 [M +
H]+ 485.2195, found 485.2249. HPLC tR = 5.46 min, 96.6%
4.1.5.4.
N-(2-Aminophenyl)-6-(2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)ac
etamido)hexanamide (12d)
White solid, 31% yield. Mp: 158-160 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s,
1H), 8.25 (s, 1H), 7.58 (d, J = 7.8 Hz, 2H), 7.16 (d, J = 7.6 Hz, 1H), 6.88 (t, J = 9.2
Hz, 2H), 6.71 (d, J = 7.7 Hz, 1H), 6.53 (t, J = 7.3 Hz, 1H), 4.81 (s, 2H), 4.37 – 4.28
(m, 4H), 4.25 (s, 2H), 3.13 – 3.02 (m, 2H), 2.31 (t, J = 7.2 Hz, 2H), 1.63-1.56 (m, 2H),
1.47-1.42 (m, 2H), 1.37 – 1.28 (m, 2H).13C NMR (100 MHz, DMSO-d6) δ 172.59,
171.55, 166.00, 143.73, 142.34, 134.56, 127.76, 126.83, 126.15, 125.76, 124.08,
116.67, 116.38, 115.10, 112.36, 101.50, 66.32, 42.36, 39.15, 36.17, 29.24, 26.57,
25.47. HRMS (AP-ESI) m/z calcd for C24H27BrN4O5 [M + H]+ 531.1238, found
531.1656. HPLC tR = 6.00 min, 95.9%
4.1.5.5.
N-(2-Aminophenyl)-6-(2-(5-chloro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)ac
etamido)hexanamide (12e)
White solid, 69% yield. Mp: 170-172 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.10 (s,
1H), 8.25 (t, J = 5.2 Hz, 1H), 7.48 (s, 1H), 7.47 – 7.42 (m, 1H), 7.16 (d, J = 7.8 Hz,
1H), 6.93-6.87 (m, 2H), 6.71 (d, J = 7.9 Hz, 1H), 6.55 (t, 7.4Hz, 1H), 4.84 (s, 2H),
4.38 – 4.27 (m, 4H), 4.25 (s, 2H), 3.08 (dd, J = 12.5, 6.4 Hz, 2H), 2.31 (t, J = 7.3 Hz,
2H), 1.65 – 1.54 (m, 2H), 1.49-1.42 (m, 2H), 1.36-1.28 (m, 2H).13C NMR (100 MHz,
DMSO-d6) δ 172.71, 171.54, 166.02, 143.28, 142.33, 131.71, 127.54, 126.48, 126.16,
125.77, 125.06, 124.05, 116.67, 116.38, 111.89, 101.54, 66.31, 42.36, 39.19, 36.15,
29.25, 26.57, 25.54. HRMS (AP-ESI) m/z calcd for C24H27ClN4O5 [M + H]+ 487.1743,
found 487.1842. HPLC tR = 5.53 min, 95.3%
4.1.5.6.
N-(2-Aminophenyl)-6-(2-(5-fluoro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)ac
etamido)hexanamide (12f)
White solid, 46% yield. Mp: 183-185 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.73 (s,
1H), 8.87 (t, J = 5.4 Hz, 1H), 7.98 (d, J = 7.8 Hz, 2H), 7.63 – 7.50 (m, 3H), 7.45 –
7.37 (m, 4H), 7.37 – 7.32 (m, 2H), 7.31 – 7.22 (m, 2H), 6.98 (dd, J = 8.4, 3.8 Hz, 1H),
6.88 (d, J = 8.3 Hz, 1H), 5.13 (s, 2H), 4.45 – 4.37 (m, 4H), 4.37 – 4.25 (m, 4H).
13C NMR (100 MHz, DMSO-d6) δ 173.05, 166.67, 165.72, 160.34, 157.96, 143.20,
143.09, 140.65, 140.53, 133.70, 129.27, 128.64, 128.38, 127.47, 126.50, 125.98,
125.27, 125.17, 124.09, 118.36, 118.13, 117.02, 112.96, 112.71, 111.47, 111.39,
101.68, 66.31, 42.52. HRMS (AP-ESI) m/z calcd for C24H27FN4O5 [M + H]+
471.2083, found 471.2183. HPLC tR = 4.26 min, 97.6%
4.1.6. tert- Butyl (4-Bromo-2-nitrophenyl) carbamate (14)
4-Bromo-2-nitroaniline (13) (1.0 g, 4.61 mmol) was dissolved in dry CH2Cl2 (15 mL),
and then Et3N (1.92 mL, 13.82 mmol) was added followed by (Boc)2O (1.51 g, 6.91
mmol) and DMAP (0.06 g, 0.50 mmol) solubilized in dry CH2Cl2. After 1 h in room
temperature, the solution was washed with water, dried by Na2SO4 and concentrated

under low pressure. The residue was washed with petroleum ether (PE) and EtOAc to
afford the product 14 as a yellow solid (1.80 g, 55% yield).1H NMR (400 MHz,
DMSO-d6) δ 8.43 (d, J = 2.2 Hz, 1H), 8.11 (dd, J = 8.8, 2.5 Hz, 1H), 7.64 (d, J = 8.3
Hz, 1H), 1.35 (s, 9H).
4.1.7. tert-Butyl (2-Nitro-4-(thiophen-2-yl) phenyl) carbamate (15a) and
tert-butyl (3-nitro-[1, 1'-biphenyl]-4-yl) carbamate (15b)
4.1.7.1. tert-Butyl (2-Nitro-4-(thiophen-2-yl) phenyl) carbamate (15a)
Compound 14 (0.40 g, 1.26 mmol) and 2-thienylboronic acid were dissolved in
degassed 1, 4-dioxane, and then Pd[P(Ph3)4] was added quickly. Degassed 3M
aqueous Na2CO3 (1.25 mL) was added into the solution, which was degassed for 10
min by sparging with argon gas. The reaction mixture was heated at 90 °C for 5h
under argon atmosphere. The solvent was removed with rotary evaporator. EtOAc
was added. The organic layer was washed with brine, dried over anhydrous MgSO4
and purified by chromatography on a silica gel column (PE/EtOAc = 90:1) to obtain
compound 15a as a pure yellow powder (0.35 g, 87% yield). 1H NMR (400 MHz,
DMSO-d6) δ 9.63 (s, 1H), 8.17 (d, J = 2.4 Hz, 1H), 7.94 (dd, J = 8.5, 2.3 Hz, 1H),
7.69 (d, J = 8.5 Hz, 1H), 7.63-7.58 (m, 2H), 7.16 (dd, J = 5.2, 3.5 Hz, 1H), 1.45 (s,
9H).
4.1.7.2. tert-Butyl (3-Nitro-[1, 1'-biphenyl]-4-yl) carbamate (15b)
Yellow pure solid, 25% yield. ESI-MS m/z: 337.6 [M + Na]+.
4.1.8. tert-Butyl (2-Amino-4-(thiophen-2-yl)phenyl)carbamate (16a) and
tert-butyl (3-amino-[1,1'-biphenyl]-4-yl)carbamate (16b)
4.1.8.1. tert-Butyl (2-Amino-4-(thiophen-2-yl)phenyl)carbamate (16a)
Compound 15a (0.53 g, 1.66 mmol) was solubilized in methanol and EtOAc (6 + 16
mL) and stirred at room temperature for 20 h with Pd/C (0.05 g, 10% w/w) under
hydrogen atmosphere. The catalyst was filtered off, and the solvent was removed and
washed with PE to give compound 16a as a white solid (0.47 g, 98% yield). 1H NMR
(400 MHz, DMSO-d6) δ 8.34 (s, 1H), 7.44-7.42 (m, 1H), 7.29 (dd, J = 3.5, 1.2 Hz,
1H), 7.26 (d, J = 8.5 Hz, 1H), 7.11 (dd, J = 5.3, 3.5 Hz, 1H), 6.97 (d, J = 2.0 Hz, 1H),
6.78 (dd, J = 8.5, 2.2 Hz, 1H), 5.04 (s, 2H), 1.45 (s, 9H).
4.1.8.2. tert-Butyl (3-Amino-[1,1'-biphenyl]-4-yl)carbamate (16b)
White solid, 90% yield. Mp: 171-173 °C, ESI-MS m/z: 307.4 [M + Na]+
4.1.9. 4-((2,2,2-Trifluoroacetamido)methyl)benzoic acid (18)
Trifluoroacetic anhydride (4 mL) was dropwise added to compound 17 (1.00 g, 6.62
mmol) under the ice baths. The suspension was stirred at room temperature for 2 h,
and quenched by adding ice water. Compound 18 was collected via filtration and
dried under vacuum to get a white solid (1.50 g, 93% yield). 1H NMR (400 MHz,
DMSO-d6) δ 12.96 (s, 1H), 10.09 (t, J = 5.9 Hz, 1H), 7.93 (d, J = 8.3 Hz, 2H), 7.39 (d,
J = 8.4 Hz, 2H), 4.47 (d, J = 6.0 Hz, 2H).
4.1.10. tert-Butyl (4-(Thiophen-2-yl)-2-(4-((2,2,2-trifluoroacetamido) methyl)
benzamido) phenyl) carbamate (20a) and tert-butyl
(3-(4-((2,2,2-trifluoroacetamido)methyl) benzamido)-[1,1'-biphenyl]-4-yl)
carbamate (20b)
4.1.10.1. tert-Butyl (4-(Thiophen-2-yl)-2-(4-((2,2,2-trifluoroacetamido) methyl)

benzamido) phenyl) carbamate (20a) To a solution of compound 18 (0.5 g, 2.02
mmol) in anhydrous THF (7 mL) was added SOCl2 (0.74 mL, 10.11 mmol) slowly
under ice baths. The mixture was heated at reflux for 3 h, and concentrated under
vacuum to get crude material 19 as yellow oil. The residue solubilized in pyridine (3
mL) was added to the solution of the compound 16a (0.43 g, 1.50 mmol) in pyridine
(3 mL) slowly under ice baths. The mixture was stirred overnight at room temperature.
Pyridine was removed. The residue was extracted with CH2Cl2 (3×20 mL) and washed
with 1 M HCl (3×20 mL), saturated NaHCO3 (3× 20 mL) and brine (3× 20 mL). The
solvent was evaporated under vacuum to give the crude product which was
recrystallized by EtOAc. Finally, the desired compound 20a was achieved as a white
solid (0.55 g, 71% yield).
4.1.10.2. tert-Butyl (3-(4-((2,2,2-Trifluoroacetamido)methyl)
benzamido)-[1,1'-biphenyl]-4-yl) carbamate (20b)
White solid, 52% yield. 1H NMR (300 MHz, DMSO-d6) δ 10.12 (t, J = 6.0 Hz, 1H),
9.96 (s, 1H), 8.80 (s, 1H), 8.00 (d, J = 8.3 Hz, 2H), 7.88 (d, J = 2.1 Hz, 1H), 7.71 –
7.62 (m, 3H), 7.53 (dd, J = 8.5, 2.2 Hz, 1H), 7.50 – 7.43 (m, 4H), 7.36 (t, J = 7.3 Hz,
1H), 4.50 (d, J = 5.9 Hz, 2H), 1.47 (s, 9H).
4.1.11. tert-Butyl
(2-(4-(Aminomethyl)benzamido)-4-(thiophen-2-yl)phenyl)carbamate (21a) and
tert-butyl (3-(4-(aminomethyl)benzamido)-[1,1'-biphenyl]-4-yl)carbamate (21b)
4.1.11.1. tert-Butyl
(2-(4-(Aminomethyl)benzamido)-4-(thiophen-2-yl)phenyl)carbamate (21a)
To a solution of compound 20a (0.37 g, 0.72 mmol) in THF (4 mL), methanol (3 mL)
and water (2 mL) was added K2CO3 (0.40 g, 2.88 mmol). The mixture was kept at
reflux for 6 h, and then cooled to room temperature. The solution was concentrated
and dissolved in CH2Cl2 (30 mL) again. After washing with water, the product 21a
precipitated from the solvent and filtered to obtain a white solid (0.30 g, 100% yield).
4.1.11.2. tert-Butyl
(3-(4-(Aminomethyl)benzamido)-[1,1'-biphenyl]-4-yl)carbamate (21b)
White solid, 88% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.13 (d, J = 8.1 Hz, 1H),
7.93 (d, J = 8.1 Hz, 2H), 7.58 – 7.48 (m, 3H), 7.45 (d, J = 8.0 Hz, 2H), 7.37 (d, J =
8.1 Hz, 1H), 6.94 – 6.86 (m, 1H), 4.24 (d, J = 6.3 Hz, 1H), 3.79 (s, 2H), 1.45 (s, 9H).
4.1.12. General procedure of preparation of 22a and its analogues 22b-22d
4.1.12.1. tert-Butyl
(2-(4-((2-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)methyl)
benzamido)-4-(thiophen-2-yl)phenyl)carbamate (22a)
Compound 6b (0.19 g, 0.65 mmol) was dissolved in anhydrous THF and CH2Cl2, then
TBTU (0.25 g, 0.77 mmol) was added followed by Et3N (0.33 mL, 2.36 mmol). After
40 min, another reactant, 22a (0.25 g, 0.59 mmol) was added. The mixture solution
was stirred at room temperature overnight. Solvent was concentrated and dissolved in
EtOAc. The organic layer was washed with saturated NaHCO3 (3× 20 mL) and brine
(3× 20 mL), dried by MgSO4, and the solution was evaporated in vacuum. The crude
product was purified by silica chromatography column (CH2Cl2/ CH3OH = 100:1) to
get compound 22a as a white solid (0.41 g, 89% yield). Mp: 176-178 °C, 1H NMR

(300 MHz, DMSO-d6) δ 9.95 (s, 1H), 8.93 (t, J = 5.9 Hz, 1H), 8.77 (s, 1H), 7.97 (d, J
= 8.3 Hz, 2H), 7.84 (d, J = 2.1 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.56 – 7.51 (m, 2H),
7.48 – 7.41 (m, 5H), 7.14 (dd, J = 5.1, 3.6 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 4.73 (td,
J = 11.3, 2.1 Hz, 2H), 4.42 (s, 2H), 4.41 (s, 2H), 3.99 – 3.90 (m, 2H), 2.28 – 2.10 (m,
1H), 1.74 – 1.65 (m, 1H), 1.46 (s, 9H).
4.1.12.2. tert-Butyl
(2-(4-((2-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)methy
l)benzamido)-4-(thiophen-2-yl)phenyl)carbamate (22b)
White solid, 89% yield. Mp: 174-176 °C, 1H NMR (300 MHz, DMSO-d6) δ 10.01 (s,
1H), 8.99 (t, J = 5.9 Hz, 1H), 8.80 (s, 1H), 7.98 (d, J = 8.3 Hz, 2H), 7.83 (d, J = 2.1
Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.54 (dd, J = 3.6, 1.5 Hz, 2H), 7.51 – 7.45 (m, 4H),
7.43 (s, 1H), 7.13 (dd, J = 5.1, 3.6 Hz, 1H), 7.02 (dd, J = 7.7, 1.1 Hz, 1H), 4.41 (s,
2H), 4.39 (s, 2H), 4.37 – 4.30 (m, 4H), 1.46 (s, 9H).
4.1.12.3. tert-Butyl
(3-(4-((2-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)methyl)
benzamido)-[1,1'-biphenyl]-4-yl)carbamate (22c)
White solid, 60% yield. Mp: 141-143 °C, 1H NMR (300 MHz, DMSO-d6) δ 9.91 (s,
1H), 8.88 (t, J = 5.9 Hz, 1H), 8.78 (s, 1H), 7.96 (d, J = 8.3 Hz, 2H), 7.87 (d, J = 2.1
Hz, 1H), 7.65 (d, J = 8.3 Hz, 3H), 7.55 – 7.41 (m, 7H), 7.40 – 7.33 (m, 1H), 6.98 (d, J
= 8.3 Hz, 1H), 4.79 – 4.67 (m, 2H), 4.41 (s, 4H), 4.00 – 3.90 (m, 2H), 2.26 – 2.10 (m,
1H), 1.75 – 1.65 (m, 1H), 1.47 (s, 9H).
4.1.12.4. tert-Butyl
(3-(4-((2-(5-Chloro-2-oxospiro[indoline-3,2'-[1,3]dioxolan]-1-yl)acetamido)methy
l)benzamido)-[1,1'-biphenyl]-4-yl)carbamate (22d)
White solid, 62% yield. Mp: 183-185 °C, 1H NMR (400 MHz, DMSO-d6) δ 10.08 (s,
1H), 9.04 (s, 1H), 8.84 (s, 1H), 8.00 (dd, J = 8.2, 3.9 Hz, 2H), 7.88 (d, J = 1.3 Hz, 1H),
7.66 (dd, J = 8.2, 3.2 Hz, 3H), 7.52 (dd, J = 8.5, 2.2 Hz, 1H), 7.51 – 7.43 (m, 6H),
7.36 (t, J = 7.3 Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H), 4.44 – 4.38 (m, 4H), 4.38 – 4.30 (m,
4H), 1.47 (s, 9H).
4.1.13. General procedure for the preparation of 23a and its analogues 23b-23d
4.1.13.1.
N-(2-Amino-5-(thiophen-2-yl)phenyl)-4-((2-(5-chloro-2-oxospiro[indoline-3,2'-[1,
3]dioxan]-1-yl)acetamido)methyl)benzamide (23a)
Compound 22a (0.40 g, 0.57 mmol) was solubilized in the solution of EtOAc (4 mL)
saturated by anhydrous HCl gas. After 5 h, the precipitate was collected by filter as a
yellow solid. The crude product was washed with saturated NaHCO3, extracted with
EtOAc, and purified by chromatography on a silica gel column (CH2Cl2/CH3OH =
80:1) to afford compound 23a as a white solid (0.11 g, 32% yield). Mp: 208-210 °C,
1H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 8.88 (t, J = 5.8 Hz, 1H), 7.99 (d, J =
8.1 Hz, 2H), 7.49 (d, J = 1.6 Hz, 1H), 7.46 (dd, J = 8.4, 2.2 Hz, 1H), 7.44-7.43 (m,
2H), 7.41 (s, 1H), 7.36 (dd, J = 5.0, 0.7 Hz, 1H), 7.31 (dd, J = 8.3, 2.1 Hz, 1H), 7.25
(d, J = 2.7 Hz, 1H), 7.06 (dd, J = 5.0, 3.7 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.83 (d, J
= 8.4 Hz, 1H), 5.16 (s, 2H), 4.73 (dd, J = 11.5, 9.4 Hz, 2H), 4.41 (s, 2H), 4.40 (s,2H),

3.96 (dd, J = 11.4, 2.9 Hz, 2H), 2.27 – 2.13 (m, 1H), 1.72-1.69 (m, 1H). 13C NMR
(100 MHz, DMSO-d6) δ 171.19, 166.62, 165.72, 144.71, 143.51, 143.12, 141.80,
133.59, 131.05, 128.75, 128.69, 128.40, 127.45, 127.33, 124.42, 123.85, 123.70,
122.72, 121.42, 116.79, 111.64, 93.41, 61.11, 42.53, 42.16, 25.21. HRMS (AP-ESI)
m/z calcd for C31H27ClN4O5S [M + H]+ 603.1463, found 603.1598. HPLC tR = 16.93
min, 95.5%
4.1.13.2.
N-(2-Amino-5-(thiophen-2-yl)phenyl)-4-((2-(5-chloro-2-oxospiro[indoline-3,2'-[1,
3]dioxolan]-1-yl)acetamido)methyl)benzamide (23b)
White solid, 47% yield. Mp: 198-200 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.84 (s,
1H), 8.90 (t, J = 5.9 Hz, 1H), 7.99 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 1.7 Hz, 1H), 7.52 –
7.47 (m, 2H), 7.43 (s, 1H), 7.39 (dd, J = 6.5, 1.1 Hz, 2H), 7.36 (dd, J = 8.3, 2.0 Hz,
1H), 7.29 (d, J = 3.3 Hz, 1H), 7.07 (dd, J = 5.0, 3.7 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H),
6.91 (d, J = 8.3 Hz, 1H), 4.40 (s, 2H), 4.39(s, 2H), 4.37 – 4.27 (m, 4H).13C NMR (100
MHz, DMSO-d6) δ 172.80, 166.59, 165.81, 144.36, 143.27, 143.23, 133.44, 131.71,
128.76, 128.44, 127.62, 127.47, 126.55, 125.13, 124.36, 124.14, 121.96, 117.92,
111.95, 101.55, 66.34, 42.53, 42.48. HRMS (AP-ESI) m/z calcd for C30H25ClN4O5S
[M + H]+ 589.1307, found 589.1436. HPLC tR = 11.31 min, 95.4%
4.1.13.3
N-(4-Amino-[1,1'-biphenyl]-3-yl)-4-((2-(5-chloro-2-oxospiro[indoline-3,2'-[1,3]dio
xan]-1-yl)acetamido)methyl)benzamide (23c)
White solid, 65% yield. Mp: 218-220 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.73 (s,
1H), 8.88 (t, J = 5.9 Hz, 1H), 7.99 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 7.5 Hz, 2H), 7.54
(d, J = 1.5 Hz, 1H), 7.46 (dd, J = 8.4, 2.2 Hz, 1H), 7.44 – 7.36 (m, 5H), 7.34 (dd, J =
8.3, 2.1 Hz, 1H), 7.24 (t, J = 7.3 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 8.3 Hz,
1H), 5.11 (s, 2H), 4.73 (t, J = 10.5 Hz, 2H), 4.41 (s, 2H), 4.39(s, 2H), 3.97-3.93 (m,
2H), 2.19 (m, 1H), 1.71-1.68 (m, 1H).13C NMR (100 MHz, DMSO-d6) δ 171.20,
166.62, 165.71, 143.27, 143.07, 141.80, 140.66, 133.70, 131.05, 129.27, 128.75,
128.60, 128.38, 127.47, 127.33, 126.50, 125.97, 125.28, 125.17, 124.42, 124.05,
116.99, 111.64, 93.41, 61.11, 42.53, 42.16, 25.21. HRMS (AP-ESI) m/z calcd for
C33H29ClN4O5 [M + H]+ 597.1899, found 597.1966. HPLC tR = 19.26 min, 95.1%
4.1.13.4.
N-(4-Amino-[1,1'-biphenyl]-3-yl)-4-((2-(5-chloro-2-oxospiro[indoline-3,2'-[1,3]dio
xolan]-1-yl)acetamido)methyl)benzamide (23d)
White solid, 98% yield. Mp: 191-192 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.74 (s,
1H), 8.89 (t, J = 5.9 Hz, 1H), 7.99 (d, J = 8.2 Hz, 2H), 7.55 (m, 3H), 7.51 – 7.47 (m,
2H), 7.43 – 7.37 (m, 4H), 7.34 (dd, J = 8.3, 2.2 Hz, 1H), 7.24 (t, J = 7.3 Hz, 1H), 7.00
(d, J = 8.6 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 5.11 (s, 2H), 4.40 (s, 2H), 4.38 (s, 2H),
4.37 – 4.28 (m, 4H).13C NMR (100 MHz, DMSO-d6) δ 172.80, 166.58, 165.73,
143.27, 143.07, 140.67, 133.72, 131.71, 129.27, 128.60, 128.38, 127.62, 127.47,
126.56, 126.49, 125.98, 125.27, 125.13, 124.07, 116.99, 111.94, 101.55, 66.34, 42.54,
42.48. HRMS (AP-ESI) m/z calcd for C32H27ClN4O5 [M + H]+ 583.1743, found
583.1787. HPLC tR = 12.89 min, 97.3%
4.1.14. tert-Butyl

(5-Fluoro-2-(4-((2,2,2-trifluoroacetamido)methyl)benzamido)phenyl)carbamate
(27)
Using the synthetic method for compounds 20a, compounds 19 and 26 gave the
compound 27 as a white solid, 57% yield. 1H NMR (400 MHz, DMSO-d6) δ 10.12 (s,
1H), 9.81 (s, 1H), 8.80 (s, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.53 (dd, J = 11.1, 2.9 Hz,
1H), 7.45 (dd, J = 12.4, 7.3 Hz, 3H), 6.97 (td, J = 8.5, 3.0 Hz, 1H), 4.48 (s, 2H), 1.45
(s, 9H).
4.1.15. tert-Butyl (2-(4-(Aminomethyl)benzamido)-5-fluorophenyl)carbamate
(28)
Using the synthetic method for compounds 21a, compound 27 gave the compound 28
as a white solid, 98% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.13 (d, J = 8.1 Hz,
1H), 7.93 (d, J = 8.1 Hz, 2H), 7.58 – 7.48 (m, 3H), 7.45 (d, J = 8.0 Hz, 2H), 7.37 (d, J
= 8.1 Hz, 1H), 6.94 – 6.86 (m, 1H), 4.24 (d, J = 6.3 Hz, 1H), 3.79 (s, 2H), 1.45 (s,
9H).
4.1.16. tert-Butyl
(2-(4-((2-(5-Bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan]-1-yl)acetamido)methyl)
benzamido)-5-fluorophenyl)carbamate (29)
Using the synthetic method for compounds 22a, compounds 6a and 28 gave the
compound 29 as a white solid, 46% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.78 (s,
1H), 8.88 (t, J = 5.9 Hz, 1H), 8.77 (s, 1H), 7.94 (d, J = 8.1 Hz, 2H), 7.58 (dd, J = 8.4,
2.0 Hz, 1H), 7.52 (td, J = 6.0, 3.0 Hz, 2H), 7.49 – 7.41 (m, 3H), 7.00-6.91 (m, 2H),
4.72 (dd, J = 11.6, 9.3 Hz, 2H), 4.39 (d, J = 4.1 Hz, 4H), 3.95 (dd, J = 8.1, 6.4 Hz,
2H), 2.25-2.15 (m, 1H), 1.71-1.67 (m, 1H), 1.45 (s, 9H).
4.1.17.
N-(2-Amino-4-fluorophenyl)-4-((2-(5-bromo-2-oxospiro[indoline-3,2'-[1,3]dioxan
]-1-yl)acetamido)methyl)benzamide (30)
Using the synthetic method for compounds 23a, compound 29 gave the compound 30
as a white solid, 98% yield. Mp: 237-239 °C, 1H NMR (400 MHz, DMSO-d6) δ 9.58
(s, 1H), 8.88 (t, J = 5.7 Hz, 1H), 7.95 (d, J = 8.1 Hz, 2H), 7.59 (dd, J = 8.4, 1.9 Hz,
1H), 7.53 (d, J = 1.9 Hz, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.11 (dd, J = 8.3, 6.6 Hz, 1H),
6.92 (d, J = 8.4 Hz, 1H), 6.54 (dd, J = 11.2, 2.8 Hz, 1H), 6.36 (td, J = 8.5, 2.7 Hz, 1H),
5.24 (s, 2H), 4.72 (dd, J = 11.5, 9.6 Hz, 2H), 4.39 (d, J = 8.5 Hz, 4H), 3.94 (dd, J =
11.3, 2.9 Hz, 2H), 2.25 – 2.11 (m, 1H), 1.69 (d, J = 13.4 Hz, 1H). 13C NMR (100
MHz, DMSO-d6) δ 171.08, 166.59, 165.82, 143.03, 142.22, 133.91, 133.60, 129.07,
128.35, 127.41, 127.11, 119.74, 114.88, 112.13, 102.60, 102.38, 102.04, 101.78,
93.34, 61.21, 42.52, 42.11, 25.20. HRMS (AP-ESI) m/z calcd for C27H24BrFN4O5
[M + H]+ 583.0987, found 583.0983. HPLC tR = 8.81 min, 97.2%
4.2. Biological materials and methods
4.2.1. In vitro HDAC Inhibition Fluorescence Assay
In vitro HDAC inhibition assays were conducted as previously described.25 In brief,
10 µL of enzyme solution (HeLa nuclear extract, HDAC1, 2 or 3) was mixed with
various concentrations of 50 µL tested compounds (0.039, 0.39, 1.56, 6.25, 25, 100
µM). The mixture was incubated at 37 °C for 1 h, then fluorogenic substrate Boc-Lys
(acetyl)-AMC (40µL) was added, and the suspension was incubated at 37 °C for

another 1 h. The reaction was ended by addition of 100 µL of developer containing
trypsin and TSA. After incubation under the same situation for 20 min, fluorescence
intensity was monitored through a microplate reader at excitation and emission
wavelengths of 390 and 460 nm, respectively. The inhibition ratios were calculated
from the fluorescence intensity readings of tested wells relative to those of control
wells, and the IC50 values were calculated using a regression analysis of the
concentration/inhibition data.
4.2.2. In vitro antiproliferative assay
Cell antiproliferative assay was determined by MTT [(3-[4,
5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide)] method as previously
described.25 All cell lines were cultured in RPMI 1640 medium containing 10% FBS
at 37 °C in a 5% CO2 humidified incubator. Cells were plated into 96-well plates
(5000/well), and permitted to grow for at least 4 h. After addition of different
concentrations of compounds, cells were maintained for 48 h. A solution of 0.5%
MTT was added to each well. After incubation for another 4 h, the formazan formed
from MTT was extracted by adding 200 µL of DMSO for 15 min. Then the optical
density values were measured using a UV-vis spectrophotometer at 570 nm.
4.2.3. Whole-cell HDAC inhibition assay
The whole-cell HDAC inhibition assay was based on assays published by Tessier P.
et al. 26, Hildmann C. et al. 27 and Marek L. et al. 28 with minor modifications.
Different subtypes of HDACs have intrinsic differences in substrate recognition. The
lysine analogs Boc-Lys (acetyl)-AMC and Boc-Lys (triflouroacetyl)-AMC could
permeate into cells and function as cellular class I and class IIa HDAC substrates,
respectively. After deacetylation of the lysine residue, the product could be
hydrolyzed by trypsin to release 7-amino-4-methylcoumarin (AMC), a well-studied
fluorophore detectable by standard fluorescence measurements.
HEL cells were seeded into 96-well black tissue culture plates at a density of 8× 103
cells/well in a total volume of 81 µL of culture medium and cultivated for 24 h under
standard cell culture conditions. The volume of 9 µL of tested compounds with
various concentrations was added to each well. After 3 h, 10 µL of 3 mM Boc-Lys
(Ac)-AMC or Boc-Lys-(ε-trifluormethylacetyl)-AMC was added to reach a final
concentration of 300 µM. Cells were placed with the substrates for 3 h in 37 °C
incubator with 5% CO2. The reaction was stopped by adding 100 µL/well
lysis/developer buffer mix (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1
mM MgCl2, 1% Nonidet-P40, 2.0 mg/mL trypsin, 10 µM TSA). After final incubation
for 3 h, fluorescence was measured at excitation of 360 nm and emission of 470 nm.
4.2.4. Molecular Docking studies
Compounds were docked into the active site of HDAC1 (PDB entry: 5ICN) using
Tripos SYBYL-X 1.3. Before docking process, the protein structure retrieved from
PDB site was treated by deleting water molecules, FF99 charges. A 100-step
minimization process was performed to further optimize the protein structure. The
molecular structures were generated with Sybyl/Sketch module and optimized using
Powell’s method with the Tripos force field with convergence criterion setting at
0.005 kcal/ (Åmol) and assigned charges with the Gasteiger-Hückel method.

Molecular docking was carried out via the Sybyl/Surflex-Dock module. Other
docking parameters were kept to the default values.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of interest.
Acknowledgments
This work was supported by National Natural Science Foundation of China (Grant
Nos. 21302111, 81373282), National High-Tech R&D Program of China (863
Program) (Grant No. 2014AA020523), Young Scholars Program of Shandong
University (YSPSDU, Grant NO. 2016WLJH33), Major Project of Science and
Technology of Shandong Province (2015ZDJS04001).
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