Design, synthesis and preliminary biological evaluation of indoline-2,3-dione derivatives as novel HDAC inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.05.048

Histone deacetylases (HDACs) are zinc-dependent or NAD⁺ dependent enzymes and play a critical role in the process of tumor development. Herein a series of indoline-2,3-dione derivatives have been designed and synthesized as potential HDACs inhibitors. The preliminary biological evaluation showed that most compounds synthesized have exhibited moderate Hela cell nuclear extract inhibitory activities, among which compound 25a (IC₅₀ = 10.13 nM) has shown the best efficacy. The anti-proliferative activities of some of these compounds were also discussed.

Full text of DOI:10.1016/j.bmc.2015.05.048

Accepted Manuscript
Design, synthesis and preliminary biological evaluation of indoline-2,3-dione
derivatives as novel HDAC inhibitors
Kang Jin, Shanshan Li, Xiaoguang Li, Jian Zhang, Wenfang Xu, Xuechen Li
PII:
S0968-0896(15)00470-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.05.048
BMC 12344
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
3 March 2015
26 May 2015
27 May 2015
Please cite this article as: Jin, K., Li, S., Li, X., Zhang, J., Xu, W., Li, X., Design, synthesis and preliminary biological
evaluation of indoline-2,3-dione derivatives as novel HDAC inhibitors, Bioorganic & Medicinal Chemistry (2015),
doi: http://dx.doi.org/10.1016/j.bmc.2015.05.048
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Design, synthesis and preliminary biological evaluation of
indoline-2,3-dione derivatives as novel HDAC inhibitors
a
a
a
a
a*,†
Kang Jin , Shanshan Li , Xiaoguang Li , Jian Zhang ,Wenfang Xu , and Xuechen
b,*,†
Li
a
Department of Medicinal Chemistry, School of Pharmacy, Shandong University,
Jinan, Shandong 250012, China
b
Department of Chemistry, The University of Hong Kong Pokfulam Road, Hong
Kong (P.R. China)
*
†
Corresponding author.
These authors contributed equally.
Abstract
+
Histone deacetylases (HDACs) are Zinc-dependent or NAD dependent enzymes and
play a critical role in the process of tumor development. Herein a series of
indoline-2,3-dione derivatives have been designed and synthesized as potential
HDACs inhibitors. The preliminary biological evaluation showed that most
compounds synthesized have exhibited moderate Hela cell nuclear extract inhibitory
activities, among which compound 25a (IC50= 10.13 nM) has shown the best efficacy.
The anti-proliferative activities of some of these compounds were also discussed.
Keywords: Indoline-2,3-dione derivatives; HDAC inhibitors; Synthesis; Biological
evaluation; QSAR
*Corresponding Author: Wenfang Xu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong
University 44, West Culture Road, Jinan 250012, P. R. China
TEL +86-531-88382264; fax: +86 531 88382264; E-mail: wfxu@yahoo.cn(W. Xu).
*
Corresponding Author: Xuechen Li
Department of Chemistry, The University of Hong Kong Pokfulam Road, Hong Kong
P.R. China)
TEL: (852) 2219 4992; E-mail: xuechenl@hku.hk.
(

1. Introduction
Cancer has long been known as the most tortuous and life-threatening disease
in the world. Every year, tens of thousands of people lose their lives due to the
lack of effective treatments, especially the effective anti-tumor drugs. In recent
years, a great many of relevant anti-cancer targets, such as the histone
deacetylases (HDACs), have been reported.
+
Histone deacetylases (HDACs), as Zinc-dependent or NAD dependent
enzymes, are responsible for the deacetylation of lysine residues located in various
[1]
protein substrates, such as nucleosomal histones . As a result of the deacetylation,
the increased positive charge density on the N-termini of nucleosomal histones
can strengthen the binding to the negatively charged chromosomal DNA, resulting
[2-4]
in the repression of the genetic transcription . In this way, HDACs play a
critical role in remodeling chromatin and down-regulating many genes expression.
In addition, many other non-histone proteins have also been reported as HDACs
substrates recently, such as molecular chaperones, cytoskeletal proteins,
transcription factors and nuclear import factors, which are related to the survival
[5]
pathways and cell growth . The hypo-acetylation of many histones and
non-histone proteins has been known to be associated with many pathogenic
[
6]
mechanisms, especially carcinogenesis .
HDACs in the human can be categorized into four classes. ClassⅠ(HDACs
1
-3 and 8), Ⅱ(HDACs 4-7,9 and 10) and Ⅳ(HDACs 11) are Zinc-dependent
+
[7]
enzymes, while the class Ⅲ HDACs (sirtuins 1–7) are NAD dependent . It has
been shown that the dysregulation of class I and Ⅱ HDACs, especially class I
isozymes, has been linked to the process of tumor cells proliferation,
[
8-15]
angiogenesis, invasion and metastasis
. Over the past few years, numerous
novel and efficient HDAC inhibitors (HDACi) have been reported as potential
[
16]
antitumor agents, among which, SAHA (Zolinza®, Merck, Fig. 1 ) and FK228
[17]
(
Istodax®, Gloucester Phar-maceuticals/Celgene, Fig. 1) , have already been on
the market.
As we can see in the Figure 1, a powerful HDACi generally contains three

domains: a zinc binding group (ZBG), a hydrophobic cap group and a linker in a
[
1]
certain length connecting the ZBG and a cap group . According to the previous
work in our group, compound 10c is excellent APN inhibitor, which also
possesses similar structural characteristic as the HDAC inhibitors. Hence, we
wished to design and synthesize a series of novel indoline-2,3-dione derivatives
(
Fig. 2) as potential HDACs inhibitors in order to search for more valuable
candidates for the tumor therapy. In this report, the synthesis, biological
evaluation and docking studies of these compounds were discussed.

2. Chemistry
The target compounds were all synthesized by taking advantage of some
commonly used chemical reagents in the laboratory. As shown in the scheme 1,
the crucial intermediate 3 was synthesized through the Sandmeyer reaction using
4
-bromoaniline. Compound 2, prepared from the reaction of 1, hydroxylamine
hydrochloride and chloral hydrate, could be converted to 3 with concentrated
sulfuric acid at 90℃. Compound 4 could be obtained by the reaction between
methyl 3 and ethane-1,2-diol, which was then coupled with methyl
2
-bromoacetate to produce compound 5. After the hydrolysis of methyl ester, the
condensation between amino acid methyl esters of different lengths with the
carboxylic acid group of compound 6 proceeded to afford 7a-7d. The target
compounds 8a-8d were obtained by the treatment of 7a-7d with NH
2
OK in
methanol.
The target compounds 16a-16e were synthesized follwoing the same
sequence as 7a-7d by using different substituted anilines as the starting material,
as shown in scheme 2. Compounds 11b-11c were coupled with propane-1,3-diol
or butane-1,4-diol under acidic environment to give rise to 17a-17d. Following
the procedure noted before, target compounds 21a-21d were thus prepared
(
scheme 3). In addition, the intermediate 17c could react with methyl
-bromopropanoate or methyl 4-bromobutanoate to give 22a-22b, which then
3
could be used to synthesize other two target compounds 25a-25b just as the steps
provided before (scheme 4).
Finally, compound 27 was obtained by the condensation of
benzene-1,2-diamine and 26 which was derived from hydrolysis of 20c (scheme
5
).

3. Results and discussion
All the target compounds were tested using HeLa cell nuclear extract (which
mainly contains HDAC1 and HDAC2) as the enzyme source and the results were
listed in Table 1 to Table 5. As the data shown in Table 1 and Table 2, different
lengths of the side chain would lead to the different HDACs inhibitory activities.
Compared with compounds 8a-d, compound 16c displayed a better potency,
which indicated that the length of this side chain was much more suitable for
using in HDACs inhibitors.
In order to explore the effect of different substituents in R position,
1
compounds 16a-f were synthesized and assayed for their potential HDACs
inhibitory activities. From the data of Table 2, compound 16c with a bromide
group on the benzene ring exhibited the most powerful inhibitory activity. This
may be due to the fact that the negative inductive effect of bromide is weaker
than fluorine and chlorine, so that the ꢀ-electron density on the benzene ring is a
little denser. Thus, the ꢀ-ꢀ interaction between benzene and the residues in the
rim of the HDACs active site is slightly stronger. This also explained why
compound 16f with a nitrogroup in R position had an extremely poorer activity
1
compared to the other compounds in Table 2. In addition, the activity of
compound 16c is better than compound 16d and 16e, which may be related to the
size of group R
1
and the steric hindrance effect between R and the active site of
1
HDACs, and bromine atom is much more applicable to be used in the further
development of HDACs inhibitors in this experiment.
The compounds in Table 3 were specifically designed to investigate the
effect of ketal spiro rings at the 3-postion of indolin-2-one skeleton. The data in
Table 3 showed that compounds 21a-b and 21c-d exhibited better activities than
1
6b and 16c respectively. It is likely that the enlargement of heterocycle had
influenced the steric hindrance and enhanced the ability to form the hydrophobic
effect. Otherwise, 6-membered heterocyclic derivatives 21a and 21c displayed a
higher potency for the inhibition of HDACs than 7-membered heterocyclic
counterparts 21b and 21d respectively. This may be explained by that the

6
-membered heterocyclic ring is more stable and suitable for the hydrophobic
effect than 7-membered ones.
After the effect of the substituent in R
been determined, two compounds with longer side chains have been synthesized
Table 4). Compared with 21c, compounds 25a exhibited a better inhibitory
1
and the size of heterocyclic ring have
(
activity but the inhibitory potency of 25b was a little lower. This showed that the
length of the side chain of compound 25a was fitted better to occupy the active
site of HDACs.
Compounds in Table 5 were synthesized to study the effect of hydroximic
acid. From the data, we could see that compounds 20c and 26 showed very weak
inhibitory potency. Although 27 was more potent, it was still not as good as 21c
and contrast SAHA. This means that hydroximic acid had stronger zinc-chelating
activity than o-phenylenediamine, hydroxyl and methoxyl groups. And it could
bind to the active site of HDACs much more solidly.
To further assay the anti-tumor inhibitory activity of these derivatives,
several potent compounds were selected to test their antiproliferative effects on
twelve common tumor cell lines with SAHA as the positive control. The result
showed in Table 6 suggested that compound 25a showed stronger inhibitory
ability than other inhibitors against most tumor lines, especially several
suspension cell lines such as K562 cell (chronic myeloid leukemia, CML), U937
cell (histiocytic lymphoma), HEL cell (leukemia cell line) and KG1 cell
(
leukemia cell line). In addition, other solid tumor cells were also very sensitive
to our compounds except for MDA-MB-231 cell (breast cancer). Based on this
information, a subcutaneous H7402 xenograft model was established. Once the
3
volume of tumors became about 140mm , mice were dosed with compound 25a
or SAHA for 16 days. The gavage administration of compound 25a and SAHA
were at different dosages, 60mg/kg/d and 100mg/kg/d separately. During the
treatment, no significant body weight variation and toxicity were detected. The
relative increment ratio (T/C) of 25a was 62.50%, meanwhile SAHA was 75.53%.
Both the SAHA-treated group and the 25a-treated group were significantly

different from the control group (p < 0.05) by two-tailed t test. As the result
shown in Figure 3, the in vivo antitumor efficacy of compound 25a (60mg/kg/d)
was a little better than that of SAHA (100mg/kg/d). Since the dosage of 25a was
far less than that of SAHA, the potency of 25a was much higher compared to that
of SAHA.
Aiming to investigate the HDAC isoform selectivity of the
indoline-2,3-dione derivatives, we selected compound 25a to assay enzyme
inhibition against HDAC1, HDAC3 and HDAC6. The results (Table 7)
confirmed that our indoline-2,3-dione inhibitors exhibited well selectivity against
individual HDACs.
In order to investigate the interaction between target compounds and
HDACs, we selected compound 25a to dock into the active site of HDAC8 (PDB
entry: 1T64) using a sybyl/Sketch module and optimized via Powell’s method by
the Tripos force field with convergence criterion set at 0.05 kcal/(Å mol), and
assigned with the Gasteiger-Hückel method. The docking study of 25a and the
active site of HDAC8 was performed using Sybyl/FlexX module. The active site
was defined as 8.0 Å radius circles around TSA in the co-crystal structure (PDB
entry: 1T64). Other docking parameters utilized in the program were kept default.
As the result shown in Figure 4, hydroxamic acid group can form a chelated
structure with the zinc ion in the centre of active site and the side chain can
occupy the long and narrow passage which links the zinc and the outside world.
To obtain the detailed binding mode, a 2D pattern was created as well (Figure 5),
which suggested that the isatin structure of compound 25a can form hydrophobic
2
06
207
interaction with Gly and Phe around the opening of the passage.

4. Conclusion
Based on the previous work in our laboratory, a series of indoline-2,3-dione
derivates were designed and synthesized as HDAC inhibitors. Among them, most
compounds exhibited potent activities in the in vitro HDAC inhibition assay and
antiproliferative assay. Especially, the compounds 21c, 25a and 25b showed
low-nanomolar IC50 values. Compound 25a was also evaluated for the in vivo
antitumor assay in a H7402 xenograft mice model, and exhibited more potent
efficacy than that of approved drug SAHA. Further structure transformation of
2
5a for more effective HDAC inhibitors is underway in our laboratory.

5
.
Experiment
.1. Chemistry: general procedures
All the starting reactants, solvents, and catalysts were commercially
5
available unless otherwise stated. All solvents, except for those aqueous
media, were purified by standard techniques in order to exclude the moisture.
All reactions were monitored by thin-layer chromatography with 0.25 mm
silica gel plates (60GF-254). The spots were visualized with UV light, iodine
1
vapor or ferric chloride. H NMR spectra were determined on a Brucker
DRX spectrometer at 600 MHz for 1H, ꢁin parts per million (ppm) and J in
Hertz. ESI-MS were recorded on an API 4000 spectrometer, and HRMS were
conducted by Shandong Analysis and Test Center in Ji’nan, China. Melting
points were recorded using an uncorrected electrothermal melting point
apparatus. Parts of targeted products were analysed by HPLC on an Agilent
1100 HPLC instrument using a Phenomenex Synergi 4ꢂ Hydro-RP 80A
column (250 mm ×4.6 mm) through one of the following methods. Method A:
compounds 21a, 21b, 25a were eluted with 50% methanol/50% water
(
containing 0.1% formic acid) over 15 min, with a flow rate of 2.0 mL/min
and detection at 267 nm. Method B: compounds 25b were eluted with 55%
methanol/45% water (containing 0.1% formic acid) over 15 min, with a flow
rate of 2.0 mL/min and detection at 267 nm. Method C: compounds 27 were
eluted with 65% methanol/35% water (containing 0.1% formic acid) over 10
min, with a flow rate of 2.0 mL/min and detection at 267 nm.
N-(4-bromophenyl)-2-(hydroxyimino)
-bromoindoline-2,3-dione
acetamide
(2),
(3),
(4),
5
5
’-bromospiro[[1,3]dioxolane-2,3’-indolin]-2’-one
Methyl-2-(5'-bromo-2'- oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acetate
(
[
5),
2-(5'-bromo-2'-oxospiro
[1,3]dioxolane-2,3'-indoline]-1'-yl)-N-(2-(hydroxyamino)-2-oxoethyl)aceta

mide (7a), 2-(5'-fluoro-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acetic
acid
(14a)
and
its
analogues14b-f,
methyl-2-(5'-chloro-2'-oxospiro[[1,3]dioxane-2,3'-indoline]
-1'-yl)acetate
(
18a) and its analogues 18c-d, were prepared based on the procedures
[18]
described previously
.
General procedure for the preparation of 8a and its Analogue 8b-d.
.1.1. 2-(5'-bromo-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)-N-(2-(hydrox
yamino)-2-oxoethyl)acetamide (8a).
5
To a solution of 1.5 N NH
0.5 g, 1.25 mmol) added. The mixture was stirred at room temperature for
about 1.5 h and then concentrated in vacuum. The residue was dissolved in
0mL EtOAc. 1N HCl was added to the solution while shaking the container
until the PH of aqueous layer became weakly basic. The organic layer was
separated using a separatory funnel, dried with Na SO , and then evaporated
2
-OK in methanol (10mL) was compound 7a
(
5
2
4
under low pressure to obtain the crude product. Finally, the crude product
was purified by column chromatography (P/E=1:1) to get compound 8a as
1
white solid (0.26 g, 52.0% yield). mp=176-177℃. . H NMR (600 MHz,
DMSO) ꢁ 10.57 (s, 1H), 8.86 (d, J = 1.3 Hz, 1H), 8.57 (t, J = 5.8 Hz, 1H),
7
.61 – 7.56 (m, 2H), 6.92 – 6.90 (m, 1H), 4.35 – 4.32 (m, 4H), 4.32 – 4.30
m, 2H), 3.65 (d, J = 5.8 Hz, 2H). HRMS (AP-ESI) m/z Calcd for
[M+H]+ 400.0066, found 400.0141.
.1.2. 4-(2-(5'-bromo-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acetamido)
(
14 3 6
C H14BrN O
5
-
N-hydroxybutanamide (8b).
1
White solid, yield 41.3%. mp=159-160℃. H NMR (600 MHz, DMSO)
10.36 (s, 1H), 8.70 (s, 1H), 8.27 (t, J = 5.5 Hz, 1H), 7.60 (dd, J = 6.8, 2.0
ꢁ
Hz, 2H), 6.90 – 6.87 (m, 1H), 4.33 (dd, J = 4.1, 2.7 Hz, 2H), 4.32 – 4.30 (m,
2
1
4
H), 4.25 (s, 2H), 3.05 (dd, J = 12.9, 6.7 Hz, 2H), 1.96 (t, J = 7.5 Hz, 2H),
.66 – 1.60 (m, 2H). HRMS (AP-ESI) m/z Calcd for C16
28.0379, found 428.0447.
3 6
H18BrN O [M+H]+

5
5
5
.1.3. 6-(2-(5'-bromo-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acetamido)
-
N-hydroxyhexanamide (8c).
1
White solid, yield 57.4%. mp=158-159℃. H NMR (600 MHz, DMSO)
10.34 (s, 1H), 8.67 (s, 1H), 8.23 (t, J = 5.5 Hz, 1H), 7.62 – 7.58 (m, 2H),
.87 (d, J = 8.3 Hz, 1H), 4.34 – 4.32 (m, 2H), 4.32 – 4.30 (m, 2H), 4.24 (s,
H), 3.05 (dd, J = 12.7, 6.7 Hz, 2H), 1.94 (t, J = 7.4 Hz, 2H), 1.51 – 1.44 (m,
H), 1.40 (dt, J = 14.6, 7.1 Hz, 2H), 1.27 – 1.20 (m, 2H). HRMS (AP-ESI)
ꢁ
6
2
2
3 6
m/z Calcd for C18H22BrN O [M+H]+ 456.0692, found 456.0766.
.1.4. (E)-3-(4-(2-(5'-bromo-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acet
amido)phenyl)-N-hydroxyacrylamide (8d).
1
White solid, yield 53.2%. mp=223-224℃. H NMR (600 MHz, DMSO)
ꢁ
10.71 (s, 1H), 10.19 (s, 1H), 9.00 (s, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.52 (d,
J = 8.3 Hz, 2H), 7.45 (d, J = 2.4 Hz, 1H), 7.40 (d, J = 15.7 Hz, 1H), 7.36 (dd,
J = 8.7, 2.4 Hz, 1H), 6.49 (d, J = 8.8 Hz, 1H), 6.37 (d, J = 15.8 Hz, 1H), 4.17
(
t, J = 6.8 Hz, 2H), 4.07 (t, J = 6.9 Hz, 2H), 3.94 (s, 2H). HRMS (AP-ESI)
m/z Calcd for C21 [M+H]+ 488.0379, found 488.0423.
3 6
H18BrN O
.1.5. Methyl4-(2-(5'-fluoro-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acet
amido)benzoate(15a )
A solution of compound 14a (0.50 g, 1.87 mmol) in 30ml anhydrous
THF was prepared, then N-methylmorpholine (0.25 mL, 2.24 mmol) and
isobutyl chloroformate (0.29 mL, 2.24 mmol) were added successively at
-
15℃. The reaction was kept at the same temperature for 30 min. And a
solution of compound 4-Amino-benzoic acid methyl ester (0.31 g, 2.06 mmol)
in 20mL THF was added dropwise to the mixture. 1 h later, the cooling bath
was moved away, and the mixture was stirred at room temperature for
anothre 20 h. THF was evaporated in vacuum with the residue being
dissolved in 50mL EtOAc. The EtOAc solution was washed with saturated
citric acid, saturated NaHCO
3 2
and brine in turn, dried over Na SO4, and
concentrated with a rotary evaporator to obtain crude product. Finally, the

crude product was purified by column chromatography (P/E=5:1) to get
compound 15a as white solid (0.60 g, 80.2% yield).
Compounds 15b-f were synthesized by the same method.
5
5
5
5
5
.1.6. Methyl-4-(2-(5'-chloro-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)ace
tamido)benzoate (15b).
White solid, 82.1% yield.
.1.7. Methyl-4-(2-(5'-bromo-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)ace
tamido)benzoate (15c).
White solid, 79.6% yield.
.1.8. Methyl-4-(2-(2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acetamido)be
nzoate (15d).
White solid, 81.4% yield.
.1.9. Methyl-4-(2-(5'-methyl-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)ac
etamido)benzoate (15e).
White solid, 82.5% yield.
.1.10. Methyl-4-(2-(5'-nitro-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acet
amido)benzoate (15f)
White solid, 76.3% yield.
Compounds 16a-f were synthesized by the same method as compound
8a.
General procedure for the preparation of 16a and its Analogue 16b-f.
.1.11. 4-(2-(5'-fluoro-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acetamido)
5
-
N-hydroxybenzamide (16a).
1
White solid, yield 46.2%. mp=248-249℃. H NMR (600 MHz, DMSO)
11.12 (s, 1H), 10.72 (s, 1H), 10.63 (s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.70
ꢁ
(
dd, J = 22.7, 8.8 Hz, 2H), 7.63 (d, J = 8.8 Hz, 1H), 7.36 (dd, J = 7.6, 2.8 Hz,
1
2
H), 7.27 (td, J = 9.1, 2.7 Hz, 1H), 7.09 – 7.05 (m, 1H), 4.55 (d, J = 8.7 Hz,
H), 4.36 (dd, J = 4.6, 2.7 Hz, 2H), 4.32 (dd, J = 4.7, 2.6 Hz, 2H). HRMS
(
3 6
AP-ESI) m/z Calcd for C19H16FN O [M+H]+ 402.1023, found 402.1091.

5.1.12. 4-(2-(5'-chloro-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acetamido)
-
N-hydroxybenzamide (16b).
1
White solid, yield 43.9%. mp=201-202℃. H NMR (600 MHz, DMSO)
11.12 (s, 1H), 10.70 (s, 1H), 10.61 (s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.72 (d,
ꢁ
J = 8.8 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.52 (dd,
J = 4.3, 2.2 Hz, 1H), 7.49 (dd, J = 8.4, 1.9 Hz, 1H), 7.10 (dd, J = 8.4, 5.6 Hz,
1
H), 4.56 (d, J = 8.6 Hz, 2H), 4.35 (dd, J = 4.2, 2.8 Hz, 2H), 4.33 (d, J = 3.7
Hz, 2H). HRMS (AP-ESI) m/z Calcd for C19
found 418.0796.
3 6
H16ClN O [M+H]+ 418.0728,
5.1.13. 4-(2-(5'-bromo-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acetamido)
-
N-hydroxybenzamide (16c).
1
White solid, yield 54.3%. mp=263-264℃. H NMR (600 MHz, DMSO)
11.12 (s, 1H), 10.62 (s, 1H), 8.95 (s, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.63 (dd,
ꢁ
J = 20.6, 8.2 Hz, 4H), 7.04 (d, J = 8.1 Hz, 1H), 4.54 (s, 2H), 4.34 (d, J = 5.3
Hz, 4H). HRMS (AP-ESI) m/z Calcd for C19
found 462.0268.
3 6
H16BrN O [M+H]+ 462.0222,
5.1.14. N-hydroxy-4-(2-(2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)acetamid
o)benzamide (16d).
1
White solid, yield 54.3%. mp=184-185℃. H NMR (600 MHz, DMSO)
ꢁ
11.12 (s, 1H), 10.72 (s, 1H), 10.62 (s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.72 (d,
J = 8.8 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.43 –
.38 (m, 2H), 7.10 (t, J = 7.5 Hz, 1H), 7.03 (dd, J = 8.4, 4.7 Hz, 1H), 4.54 (d,
J = 8.9 Hz, 2H), 4.39 – 4.36 (m, 2H), 4.31 (dd, J = 4.5, 2.3 Hz, 2H). HRMS
AP-ESI) m/z Calcd for C19 [M+H]+ 384.1117, found 384.1187.
7
(
17 3 6
H N O
5.1.15. N-hydroxy-4-(2-(5'-methyl-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl
)
acetamido)benzamide (16e).
1
White solid, yield 48.7%. mp=194-195℃. H NMR (600 MHz, DMSO)
11.12 (s, 1H), 10.69 (s, 1H), 10.60 (s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.72 (d,
ꢁ
J = 8.8 Hz, 1H), 7.70 – 7.67 (m, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.24 – 7.18 (m,
H), 6.91 (dd, J = 8.0, 5.1 Hz, 1H), 4.51 (d, J = 8.9 Hz, 2H), 4.36 (dd, J = 4.0,
2

2.4 Hz, 2H), 4.30 – 4.28 (m, 2H), 2.28 (s, 3H). HRMS (AP-ESI) m/z Calcd
19 3 6
for C20H N O
[M+H]+ 398.1274, found 398.1333.
5.1.16. N-hydroxy-4-(2-(5'-nitro-2'-oxospiro[[1,3]dioxolane-2,3'-indoline]-1'-yl)a
cetamido)benzamide (16f).
1
White solid, yield 41.5%. mp=136-137℃. H NMR (600 MHz, DMSO)
ꢁ
11.21 (s, 1H), 10.81 (s, 1H), 10.13 (s, 1H), 8.24 (d, J = 2.8 Hz, 1H), 8.10
(
dd, J = 9.2, 2.8 Hz, 1H), 7.96 (dd, J = 8.7, 3.6 Hz, 1H), 7.93 (d, J = 8.8 Hz,
1H), 7.82 (d, J = 8.8 Hz, 1H), 7.79 (dd, J = 8.8, 3.9 Hz, 1H), 7.76 – 7.73 (m,
1H), 4.24 (d, J = 5.4 Hz, 1H), 4.12 (dd, J = 4.6, 2.6 Hz, 1H), 4.06 (dd, J = 4.6,
2.4 Hz, 1H), 4.03 (dd, J = 14.2, 7.1 Hz, 1H), 3.82 (s, 2H). HRMS (AP-ESI)
m/z Calcd for C19
H
16
N
4
8
O [M+H]+ 429.0968, found 429.1059.
General procedure for the preparation of 21a and its Analogue
1b-d.
2
Compounds 21a-d were synthesized by the same method as
compound 16a-f.
5.1.17. 4-(2-(5'-chloro-2'-oxospiro[[1,3]dioxane-2,3'-indoline]-1'-yl)acetamido)-
N-hydroxybenzamide (21a).
1
White solid, yield 34.2%. mp=133-134℃. H NMR (600 MHz, DMSO)
ꢁ
11.13 (s, 1H), 10.71 (s, 1H), 10.22 (s, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.73 (d,
J = 8.7 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.47 –
.42 (m, 2H), 7.07 – 7.05 (m, 1H), 4.72 (dt, J = 19.5, 9.8 Hz, 2H), 4.58 (d, J
8.7 Hz, 2H), 3.96 (dd, J = 9.7, 5.2 Hz, 2H), 2.24 – 2.14 (m, 1H), 1.73 –
.67 (m, 1H). HRMS (AP-ESI) m/z Calcd for C20 [M+H]+
32.0884, found 432.0953.
7
=
1
4
3 6
H18ClN O
5.1.18. 4-(2-(5'-chloro-2'-oxospiro[[1,3]dioxepane-2,3'-indoline]-1'-yl)acetamido
)
-N-hydroxybenzamide (21b).
1
White solid, yield 33.7%. mp=134-135℃. H NMR (600 MHz, DMSO)
11.13 (s, 1H), 10.69 (s, 1H), 10.23 (s, 1H), 7.73 (s, 1H), 7.71 (dd, J = 4.7,
.6 Hz, 2H), 7.70 – 7.68 (m, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.44 (dd, J = 8.4,
ꢁ
2

1
.4 Hz, 1H), 7.07 (dd, J = 6.1, 2.3 Hz, 1H), 4.56 (s, 2H), 4.27 (d, J = 3.0 Hz,
H), 4.03 (d, J = 4.3 Hz, 2H), 1.73 (d, J = 3.9 Hz, 4H). HRMS (AP-ESI) m/z
2
3 6
Calcd for C21H20ClN O [M+3H]+ 448.1041, found 448.1106.
5.1.19. 4-(2-(5'-bromo-2'-oxospiro[[1,3]dioxane-2,3'-indoline]-1'-yl)acetamido)-
N-hydroxybenzamide (21c).
1
White solid, yield 40.3%. mp=142-143℃. H NMR (600 MHz, DMSO)
ꢁ
11.12 (s, 1H), 10.75 (s, 1H), 10.66 (s, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.72 (d,
J = 8.7 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.52 (d, J
2.2 Hz, 1H), 7.50 – 7.48 (m, 1H), 7.11 – 7.09 (m, 1H), 4.56 (d, J = 8.6 Hz,
H), 4.34 (d, J = 6.5 Hz, 4H), 1.19 (dd, J = 28.9, 14.1 Hz, 2H).
=
2
5.1.20. 4-(2-(5'-bromo-2'-oxospiro[[1,3]dioxepane-2,3'-indoline]-1'-yl)acetamido
)
-N-hydroxybenzamide (21d).
1
White solid, yield 35.1%. mp=130-131℃. H NMR (600 MHz, DMSO)
10.74 (s, 1H), 10.65 (s, 1H), 10.18 (s, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.81 (d,
ꢁ
J = 2.0 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.69 – 7.67 (m, 1H), 7.63 (d, J =
8
.8 Hz, 1H), 7.57 (dd, J = 8.4, 2.0 Hz, 1H), 7.05 – 7.01 (m, 1H), 4.56 (d, J =
.7 Hz, 2H), 4.27 (d, J = 12.2 Hz, 2H), 4.04 – 4.01 (m, 2H), 1.73 (s, 4H).
8
HRMS (AP-ESI) m/z Calcd for C21
92.0614.
H20ClN
3
6
O [M+H]+ 492.0515, found
4
General procedure for the preparation of 25a and its Analogue 25b.
Compounds 23a-b were synthesized by the same method as compound
14a, and Compounds 25a-b were synthesized by the same method as
compound 16a.
5.1.21. 4-(3-(5'-bromo-2'-oxospiro[[1,3]dioxane-2,3'-indoline]-1'-yl)propanamid
o)-N-hydroxybenzamide (25a).
1
White solid, yield 63.7%. mp=159-160℃. H NMR (600 MHz, DMSO)
ꢁ
11.11 (s, 1H), 10.39 (s, 1H), 10.23 (s, 1H), 7.88 – 7.86 (d, J = 8.8 Hz, 1H),

7
.71 – 7.66 (dd, J = 8.4, 20.4 Hz, 2H), 7.61 – 7.60 (m, 2H), 7.51 (s, 1H),
.16– 7.14 (d, J = 7.8 Hz, 1H), 4.72– 4.68 (t, J = 9.6 Hz, 2H), 3.85– 3.82 (t, J
7
=
7.2 Hz, 4H), 2.69– 2.67 (t, J = 7.2 Hz, 2H), 2.17– 2.15 (t, J = 7.2 Hz, 1H),
.69– 1.66 (d, J =13.2 Hz, 1H). HRMS (AP-ESI) m/z Calcd for
[M+H]+ 490.0535, found 490.0611.
1
C
21
H20BrN
3
O
6
5.1.22. 4-(4-(5'-bromo-2'-oxospiro[[1,3]dioxane-2,3'-indoline]-1'-yl)butanamido)
-
N-hydroxybenzamide (25b).
1
White solid, yield 58.4%. mp=160-161℃. H NMR (600 MHz, DMSO)
11.09 (s, 1H), 10.15 (s, 1H), 10.11 (s, 1H), 7.70 – 7.68 (d, J = 8.4 Hz, 2H),
ꢁ
7
.64 – 7.62 (d, J = 8.4 Hz, 3H), 7.53 (s, 1H), 7.13 – 7.12 (d, J = 7.8 Hz, 1H),
.73– 4.70 (t, J = 10.2 Hz, 2H), 3.92 – 3.91 (d, J = 8.4 Hz, 2H), 3.69– 3.67 (t,
4
J = 6.6 Hz, 2H), 2.43 – 2.39 (dd, J = 10.2, 15.6 Hz, 2H), 2.17– 2.15 (t, J =
.6 Hz, 1H), 1.88– 1.85 (t, J = 13.8 Hz, 2H), 1.69 – 1.67 (d, J = 12.0 Hz, 1H).
HRMS (AP-ESI) m/z Calcd for C22 [M+H]+ 504.0692, found
04.0759.
6
3 6
H22BrN O
5
General procedure for the preparation of20c,26, and 27.
5.1.23. Methyl-4-(2-(5'-bromo-2'-oxospiro[[1,3]dioxane-2,3'-indoline]-1'-yl)aceta
mido)benzoate (20c).
1
White solid, yield 80.1%. mp=234-235℃. H NMR (600 MHz, DMSO)
ꢁ
10.75 (s, 1H), 7.93 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.58 (dd, J
8.4, 2.1 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 4.72
=
(
dd, J = 11.8, 9.3 Hz, 2H), 4.57 (s, 2H), 3.98 – 3.93 (m, 2H), 3.82 (s, 3H),
.23 – 2.15 (m, 1H), 1.69 (d, J = 13.5 Hz, 1H). HRMS (AP-ESI) m/z Calcd
[M+H]+ 475.0426, found 475.0500.
2
for C21
H
19BrN
2
O
6
5.1.24. 4-(2-(5'-bromo-2'-oxospiro[[1,3]dioxane-2,3'-indoline]-1'-yl)acetamido)b
enzoic acid (26).
To a solution of KOH (0.3 g, 5.36 mmol) in 50 mL alcohol and 25 mL
H
2
O was added compound 20c (1.43 g, 3.00 mmol). Keep the reaction for 1h
at room temperature. The alcohol was removed in vacuum. Then 1N HCl

solution was added to the remaining mixture gradually until the solution
became acidic and a white precipitate was generated. Concentrated the
mixture and compound 26 was obtained as white solid (1.32 g, 95.5% yield),
1
mp=202-203℃. H NMR (600 MHz, DMSO) ꢁ 12.69 (d, J = 69.0 Hz, 1H),
10.71 (s, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.68 (d, J = 8.6 Hz, 2H), 7.58 (dd, J =
8.4, 1.4 Hz, 1H), 7.54 (s, 1H), 7.02 (t, J = 8.4 Hz, 1H), 4.72 (t, J = 10.8 Hz,
2H), 4.57 (s, 2H), 3.98 – 3.92 (m, 2H), 2.23 – 2.16 (m, 1H), 1.70 (d, J = 13.4
Hz, 1H). HRMS (AP-ESI) m/z Calcd for C20
found 461.0337.
2 6
H17BrN O [M+H]+ 461.0270,
5.1.25. N-(2-aminophenyl)-4-(2-(5'-bromo-2'-oxospiro[[1,3]dioxane-2,3'-indoline
]
-1'-yl)acetamido)benzamide (27).
Compound 26 ( 0.69 g, 1.5 mmol) was dissolved in 30 mL anhydrous
THF. Then N-methylmorpholine (0.36mL, 3.3mmol) and isobutyl
chloroformate (0.23mL, 1.8mmol) was added to this solution at -15℃ and
stirred the mixture for 1h at the some temperature. Then o-phenylenediamine
(
0.18g, 1.65mmol) was added to the mixture and stirred for 1h at -15℃ and
for another 12h at room temperature. Concentrated and removed the solvent
with a rotary evaporator. The residue was dissolved in 50mL EtOAc and
washed with brine (10mL×2). Then the organic phase was dried over
2 4
anhydrous Na SO and concentrated under low pressure to get the crude
product. Finally, compound 27 was separated by column chromatography
1
(
P/E=2:1) as reddish brown solid (0.70 g, 84.6% yield), mp=228-229℃. H
NMR (600 MHz, DMSO) ꢁ 10.67 (s, 1H), 9.59 (s, 1H), 7.97 (d, J = 8.2 Hz,
2H), 7.69 (d, J = 8.4 Hz, 2H), 7.62 – 7.57 (m, 1H), 7.55 (s, 1H), 7.15 (d, J =
7.6 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.96 (t, J = 7.5 Hz, 1H), 6.78 (d, J =
7.9 Hz, 1H), 6.59 (t, J = 7.4 Hz, 1H), 4.88 (s, 2H), 4.73 (t, J = 10.8 Hz, 2H),
4.58 (s, 2H), 3.96 (d, J = 7.5 Hz, 2H), 2.24 – 2.15 (m, 1H), 1.70 (d, J = 13.3
Hz, 1H). HRMS (AP-ESI) m/z Calcd for C20
found 551.0924.
2 6
H17BrN O [M+H]+ 551.0852,

5.2. Biological evaluation
5.2.1. In vitro HDACs inhibition fluorescence assay.
The HDACs enzyme extracted from Hela nuclear was used in this
testing procedure. First, 50ꢂL of tested compound solution in a variety of
concentrations was mixed with 10ꢂL of HDACs enzyme solution. Five
minutes later, 40ꢂL Boc-Lys (acetyl)-AMC was added as fluorogenic
substrate, and then the mixture was incubated for 30 min at 37 ℃ .
Finally,100ꢂL Trypsin solution was added in order to stop the hydrolytic
reaction. After incubation for another 20 min at 37℃ , the hydrolysate was
monitored using a UV-vis spectrophotometer through the change in the
absorbance measured at 390 nm and 460 nm.
.2.2. In vitro antiproliferative assay.
5
All cell lines were grown in RPMI1640 medium with 10% FBS at 37℃
in 5% CO
2
humidified incubator. First, cells were pated in 96-well plates
(
5000/well), and allowed to grow for at least 4 hours. Then different
concentrations of compounds were added. The plates were put in the
incubator to keep on cultivating for another 48 hours. Then 1% MTT
(
3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) solution
was added to each well. Four hours later, DMSO was added to dissolve
formazan and mixed for 15 min. Finally, the optical density values were
measured using a UV-vis spectrophotometer at 570nm.
5.2.3. In vivo antitumor assay against H7402 xenograft.
In order to investigate the in vivo antitumor capability, H7402 human
7
hepatoma cells (6.88×10 ) were inoculated subcutaneously in the right axilla
of each male athymic nude mouse (4-6 weeks old, Beijing HFK Bioscience
Co. Ltd.). Eight days later, the average volume of all the tumors had grown
3
to 140mm , and then nude mice were grouped randomly into treatment and
control groups (seven mice per group). Treatment groups received 60mg/kg/d
compound 25a or 100mg/kg/d compound SAHA respectively, and blank
control group received PBS solution in an equal volume. Since the day of

gavage administration, tumor volumes and body weight was measured every
three days. Tumor volumes were measured using a vernier caliper through
2
the equation (V=ab /2, where a and b represent the longest and shortest
diameter, respectively). Relative increment ratio(T/C) was calculated based
on the following formula:
T/C (%) = the treatment group (T) RTV/the blank control group (C) RTV
where RTV, named relative tumor volume =V
measured at the end of treatment; V : the tumor volume measured at the
beginning of grouping).
t 0 t
/V (V : the tumor volume
0

Acknowledgments
This work was supported by National Science and Technology Specific Projects
of China (No. 2009ZX09103-118) and National Nature Science Foundation of China
(
(
Nos. 90713041 and 30772654) and National Natural Science Foundation of China
Grant No. 21172134).
References and notes
1. Zhang, Y.; Feng, J.; Jia, Y.; Wang, X.; Zhang, L.; Liu, C.; Fang, H.; Xu,
W. J Med Chem 2011, 54, 2823.
2
3
. Hassig, CA.; Schreiber, SL. Curr Opin Chem Biol 1997, 1, 300.
. Tony, Kouzarides. Current Opinion in Genetics & Development 1999, 9,
4
0.
4
5
6
. Wolffe, AP. Science 1996, 272, 371.
. Glozak, MA.; Sengupta, N.; Zhang, X.; Seto, E. Gene 2005, 363, 15.
. Choi, E.; Lee, C.; Park, JE.; Seo, JJ.; Cho, M.; Kang, JS.; Kim, HM.; Park,
SK.; Lee, K.; Han, G. Bioorganic & Medicinal Chemistry Letters 2011, 21,
1
218.
7
8
9
1
. de Ruijter, AJ.; van Gennip, AH.; Caron, HN.; Kemp, S.; van Kuilenburg,
AB. Biochem J 2003, 370, 737.
. Halkidou, K.; Gaughan, L.; Cook, S.; Leung, HY.; Neal, DE.; Robson, CN.
Prostate 2004, 59, 177.
. Choi, JH.; Kwon, HJ.; Yoon, BI.; Kim, JH.; Han, SU.; Joo, HJ.; Kim, DY.
Jpn J Cancer Res 2001, 92, 1300.
0. Wilson, AJ.; Byun, DS.; Popova, N.; Murray, LB.; L'Italien, K.; Sowa, Y.;
Arango, D.; Velcich, A.; Augenlicht, LH.; Mariadason, JM. J Biol Chem
2
006, 281, 13548.
1
1. Zhang, Z.; Yamashita, H.; Toyama, T.; Sugiura, H.; Ando, Y.; Mita, K.;
Hamaguchi, M.; Hara, Y.; Kobayashi, S.; Iwase, H. Breast Cancer Res
Treat 2005, 94, 11.
1
1
1
1
2. Zhu, P.; Martin, E.; Mengwasser, J.; Schlag, P.; Janssen, KP.; Göttlicher,
M. Cancer Cell 2004, 5, 455.
3. Huang, BH.; Laban, M.; Leung, CH.; Lee, L.; Lee, CK.; Salto-Tellez, M.;
Raju, GC.; Hooi, SC. Cell Death Differ 2005, 12, 395.
4. Song, J.; Noh, JH.; Lee, JH.; Eun, JW.; Ahn, YM.; Kim, SY.; Lee, SH.;
Park, WS.; Yoo, NJ.; Lee, JY.; Nam, SW. APMIS 2005, 113, 264.
5. Zhang, Z.; Yamashita, H.; Toyama, T.; Sugiura, H.; Omoto, Y.; Ando, Y.;
Mita, K.; Hamaguchi, M.; Hayashi, S.;, Iwase, H. Clin Cancer Res 2004,

1
0 ,6962.
1
1
1
6. Grant, S.; Easley, C.; Kirkpatrick P. Nat Rev Drug Discov 2007, 6, 21.
7. Campas-Moya C. Drugs Today (Barc) 2009, 45, 787.
8. Kang, J.; Xiaopan, Z.; Chunhua, M.; Yingying, X.; Yumei, Yuan.; Wenfang,
X. Bioorg. Med. Chem 2013, 21, 2663.
O
H
NH NH
O
O
S
O
H
N
NH
S
N
OH
H
O
O
NH
O
O
Cap group
ZBG
Linker
SAHA
FK228
Figure 1. The structures of SAHA and FK228.

n
R1= F,Cl, Br, H, CH3, NO2
R2= -(CH )-, -(CH ) -,
O
R1
O
O
Br
O
O
2
2 3
O
N
-(CH2)5-
O
N
O
m
H
N
HN
R2
R3= -NH-OH, -OH, -OCH3,
HN
H2N
OH
R3
O
m= 1, 2, 3
n= 0, 1, 2
1
0c
Figure 2. The new HDAC inhibitors derived from compound 10c.

1
1
1
1
600
400
200
000
Blank control
8
6
4
2
00
00
00
00
0
SAHA (100mg/kg/d)
2
5a (60mg/kg/d)
1
4
7
10
13
16
Figure 3. Antitumor activity of 25a and SAHA against H7402 human
tumor xenografts implanted in nude mice. The values are mean tumor volume in at
ɑ
least three experiments. Values are the mean of three experiments. The standard
derivations are <20% of the mean.

Figure 4. The FlexX docking result of 25a with HDAC8 (PDB entry: 1T64).

Figure 5. The docking result of 25a shown by LIGPLOT.

O
O
Br
Br
Br
Br
O
a
O
b
c
d
O
O
N
NH2
N
H
N
H
N
H
OH
1
2
3
4
O
O
O
O
Br
O
Br
O
Br
O
g
Br
O
e
f
O
O
O
O
N
N
N
N
O
O
O
O
O
OH
HN
HN
R2
R
2
O
NH
OH
5
6
7a-7d
8a-8d
O
O
Scheme 1. (1)R
Cl CH(OH) , NH
toluene, 130℃; (d) BrCH
KOH, HCl; (f) amino acid methyl esters with different lengths, isobutyl
chloroformate, NMM, THF, -20℃; (g) NH OK, CH OH.
2
= CH
2
, (CH
SO
COOCH
2
)
3
, (CH
, 85℃; (b) H
, TBAB, K
2
)
5
,
. (2)Reagents and conditions: (a)
3
2
2
OH, Na
2
4
2
SO , 90℃; (c) ethane-1,2-diol, TsOH,
CO , KI, acetone, 60℃; (e) EtOH,
3
4
2
3
2
2
3

O
O
R1
R1
R1
R1
O
d
a
O
b
c
O
O
N
NH
2
N
H
N
H
N
H
OH
9a-9f
10a-10f
11a-11f
12a-12f
O
O
O
O
O
O
R
1
O
R
1
O
R
1
g
R
1
e
f
O
O
O
O
N
N
N
N
O
O
O
O
O
OH
HN
15a-15f
O
HN
16a-16f
O
1
3a-13f
14a-14f
O
HN
OH
Scheme 2. (1)R
Cl CH(OH) , NH
toluene, 130℃; (d) BrCH
KOH, HCl; (f) methyl-4-aminobenzoate, isobutyl chloroformate, NMM, THF,
20℃; (g) NH OK, CH OH.
1
= F, Cl, Br, H, CH
OH, Na SO , 85℃; (b) H
COOCH , TBAB, K
3
, NO
SO
CO
2
. (2)Reagents and conditions: (a)
, 90℃; (c) ethane-1,2-diol, TsOH,
, KI, acetone, 60℃; (e) EtOH,
3
2
2
2
4
2
4
2
3
2
3
-
2
3

n
n
n
O
O
O
O
R1
R1
O
R1
O
e
R
1
O
f
h
d
O
O
O
O
N
H
N
H
N
N
O
O
1
1b-11c
17a-17d
18a-18d
19a-19d
OH
O
n
n
O
O
R1
O
R
1
O
g
O
O
N
N
O
O
HN
HN
O
O
O
HN
OH
2
0a-20d
21a-21d
Scheme 3. (1) R
propane-1,3-diol or butane-1,4-diol, TsOH, toluene, 130℃; (d) BrCH
TBAB, CO KI, acetone, 60 ℃ ; (e) EtOH, KOH, HCl; (f)
methyl-4-aminobenzoate, isobutyl chloroformate, NMM, THF, -20℃; (g) NH OK,
CH OH.
1
= Cl or Br; n= 1 or 2. (2)Reagents and conditions: (h)
2
COOCH
3
,
K
2
3
,
2
3

O
O
O
O
Br
O
i
Br
O
e
Br
O
f
Br
O
g
O
O
O
O
N
H
N
N
N
O
O
O
m
m
m
O
OH
HN
O
17c
22a-22b
23a-23b
24a-24b
O
O
Br
O
O
N
O
m
HN
O
HN
OH
2
5a-25b
Scheme 4. (1)m= 2 or 3. (2)Reagents and conditions: (i) BrCH
Br(CH COOCH , TBAB, K CO , KI, acetone, 60℃; (e) EtOH, KOH, HCl; (f)
methyl-4-aminobenzoate, isobutyl chloroformate, NMM, THF, -20℃; (g) NH OK,
CH OH.
2 2 3
CH COOCH or
2
)
3
3
2
3
2
3