Design, synthesis and biological evaluation of tyrosine-based hydroxamic acid analogs as novel histone deacetylases (HDACs) inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.06.046

Histone deacetylases (HDACs) are a promising target for treating cancer and some other disorders. Herein, based on the structure of our previously reported tetrahydroisoquinoline-based hydroxamic acids, a novel series of tyrosine-based hydroxamic acid derivatives was designed and synthesized as HDACs inhibitors. Compared with tetrahydroisoquinoline-based hydroxamic acids, tyrosine-based hydroxamic acid derivatives exhibited more potent HDAC8 inhibitory activity. However, the antiproliferative activities and HeLa cell nuclear extract inhibition of several selected tyrosine-based hydroxamic acids were moderate.

Full text of DOI:10.1016/j.bmc.2011.06.046

Bioorganic & Medicinal Chemistry 19 (2011) 4437–4444
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of tyrosine-based hydroxamic
acid analogs as novel histone deacetylases (HDACs) inhibitors
Yingjie Zhang a, Jinhong Feng a, Chunxi Liu b, Hao Fang a, Wenfang Xu a,
⇑
a Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji’nan, Shandong 250012, PR China
b Department of Pharmaceutics, School of Pharmacy, Shandong University, Ji’nan, Shandong 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone deacetylases (HDACs) are a promising target for treating cancer and some other disorders.
Herein, based on the structure of our previously reported tetrahydroisoquinoline-based hydroxamic
acids, a novel series of tyrosine-based hydroxamic acid derivatives was designed and synthesized as
HDACs inhibitors. Compared with tetrahydroisoquinoline-based hydroxamic acids, tyrosine-based
hydroxamic acid derivatives exhibited more potent HDAC8 inhibitory activity. However, the antiprolifer-
ative activities and HeLa cell nuclear extract inhibition of several selected tyrosine-based hydroxamic
acids were moderate.
Received 3 May 2011
Revised 14 June 2011
Accepted 15 June 2011
Available online 21 June 2011
Keywords:
Tyrosine derivatives
Histone deacetylases
Inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
Synthesis
1. Introduction
O
H
O
Cancer is a disease characterized by uncontrolled cell growth
and proliferation, which often results from disordered gene expres-
sion, genetic and/or epigenetic. Epigenetic changes refer to herita-
ble changes in gene expression caused by modifications of
chromatin structure other than alteration in DNA sequence.1,2
The fundamental repeating unit of chromatin is the nucleosome
composed of core histone around which DNA coils.3 Some histone
amino acid residues, such as lysines, protrude the nucleosome and
are subject to numerous posttranslational modifications.4 Histone
deacetylases (HDACs) are enzymes responsible for histone lysine
residues deacetylation leading to condensation of chromosomal
DNA and gene transcriptional repression.5–7 Therefore, HDACs are
regarded as promising targets for cancer therapy.
NH
NH
O
O
S
O
H
N
NH
S
HO
N
H
O
O
NH
SAHA
O
FK228
Figure 1. The structures of two approved drugs, SAHA and FK228.
The human HDACs family can be divided into 4 classes. The en-
zymes of classes I (HDACs 1–3 and 8), II (HDACs 4–7, 9 and 10) and
IV (HDACs 11) are all Zn2+-dependent metalloproteases, whereas
the class III HDACs (sirtuins 1–7) are NAD+ dependent.8,9 Indeed,
only Zn2+-dependent HDACs, especially class I and class II isozymes
are involved in promoting tumor cells proliferation, angiogenesis,
migration, resistance to chemotherapy and prohibiting apoptosis
and differentiation.10 Currently, over ten HDACs inhibitors (HDACi)
are in clinical trials as antitumor agents11 and two of them, SAHA
(ZolinzaÒ, Merck, Fig. 1)12 and FK228 (IstodaxÒ, Gloucester Phar-
maceuticals/Celgene, Fig. 1),13 are already on the market.
In our previous study, we designed and synthesized a novel series
of tetrahydroisoquinoline-based hydroxamic acids as potent HDACi
(Fig. 2a), which exhibited excellent in vitro and in vivo antitumor
activities.14,15 In order to investigate the influence of molecular flex-
ibilityon HDACsinhibitory activity, the tetrahydroisoquinoline scaf-
fold (Fig. 2a) was simplified to the tyrosine scaffold (Fig. 2b).
2. Chemistry
Target compounds E, F and H were synthesized according to the
procedures described in Scheme 1. Boc (tert-butoxycarbonyl) group
protection of the starting material L-tyrosine (compound A) led to
compound B, which was condensed with phenylamine in the pres-
ence of DCC and HOBT to afford compound C. Alkylation of the
⇑
Corresponding author. Tel./fax: +86 531 88382264.
E-mail address: wfxu@yahoo.cn (W. Xu).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.046

4438
Y. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4437–4444
O
O
R1
N
R1
N
H
H
N
H
N
H
N
HN
HO
O
R2
HO
O
R2
O
O
a. Tetrahydroisoquinoline-based
b. Tyrosine-based hydroxamic acid
hydroxamic acid
Figure 2. The general structures of tetrahydroisoquinoline-based hydroxamic acids (a) and tyrosine-based hydroxamic acids (b).
O
O
O
OH
OH
N
a
b
c
H
HN
NH2
HN
HO
Boc
HO
HO
Boc
A
B
C
O
O
N
N
d
H
N
H
H
HN
HN
O
HO
O
R2
O
Boc
O
5, R2=
6, R2=
E
O
O
D
O
e
F
.
H HCl
f, g
O
O
d
N
N
H
H
H
N
HN
HN
O
HO
O
O
O
O
O
O
G
H
Scheme 1. Reagents and conditions: (a) (Boc)2O, 1 N NaOH, THF; (b) phenylamine, DCC, HOBT, anhydrous THF; (c) BrCH2COOCH3, K2CO3, anhydrous DMF; (d) NH2OK,
CH3OH; (e) HC1, anhydrous EtOAc; (f) TFA, DCM, Et3N; (g) 3-phenylpropionic acid, TBTU, Et3N, THF.
O
O
O
R1
OH
Cbz
OH
N
a
b
c
H
HN
NH2
HN
HO
HO
HO
HN
Cbz
J01-J03
A
I
O
O
J01-L01, R1=
J02-L02, R1=
J03-L03, R1=
R1
R1
N
N
d
H
O
H
H
N
HN
O
HO
O
Cbz
O
Cbz
K01-K03
O
L01-L03
O
Cl
Scheme 2. Reagents and conditions: (a) Cbz-Cl, 1 M NaOH; (b) R–NH2, DCC, HOBT, anhydrous THF; (c) BrCH2COOCH3, K2C03, anhydrous DMF; (d) NH2OK, CH3OH.
phenol group of compound C with methyl bromoacetate gave com-
pound D, which was treated with NH2OK in methanol to furnish one
target compound E. Boc group of compound E was cleaved in acidic
conditions to give compound F. N-deprotection of compound D fol-
lowed by acylation of the obtained free amine group with 3-phenyl-
propionic acid afforded compound G, which was converted to the
hydroxamic acid derivative H.
Boc protection group of compound N was cleaved by trifluoroacetic
acid in CH2Cl2 giving the free amine intermediate O, which was
subsequently functionalized through either acylation or alkylation
to afford P01–P07. Compounds P01–P07 were then treated with
NH2OK in methanol to give corresponding hydroxamic acids
Q01–Q07.
Cbz (benzoyloxycarbonyl) group protection of L-tyrosine fol-
3. Result and discussion
lowed by condensation with several kinds of amines, alkylation
of the phenol group and final aminolysis of the ester group fur-
nished the desired hydroxamic acids L01–L03 (Scheme 2).
Target compounds Q1–Q7 were synthesized according to the
procedures described in Scheme 3. Compound B was converted
to compound N in two steps as described for compound D. The
Considering that all zinc ion dependent HDACs were highly con-
served in their active sites and HDAC8 could be expressed and
purified in our laboratory, we used HDAC8 as the enzyme source
to screen our target compounds efficiently. Enzyme inhibitory

Y. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4437–4444
4439
O
O
O
O
O
N
OH
N
a
b
c
H
H
HN
O
HN
HN
O
Boc
HO
Boc
HO
Boc
N
O
B
M
O
O
O
O
g
d, P01-P04
or e, P05 or f, P06, P07
N
N
H
H
NH2
O
HN
O
O
O
R2
P01-P07
O
O
O
O
P01-Q01, R2=
P02-Q02, R2=
P05-Q05, R2=
P06-Q06, R2=
O
O
N
H
N
H
O
HN
HO
O
R2
O
P03-Q03, R2=
P04-Q04, R2=
Q01-Q07
P07-Q07, R2=
O
O
Scheme 3. Reagents and conditions: (a) 4-methoxyaniline, DCC, HOBT, anhydrous THF; (b) BrCH2COOCH3, K2CO3, anhydrous DMF; (c) TFA, DCM, saturated Na2CO3; (d)
carboxylic acid, TBTU, Et3N, THF; (e) benzyl bromide, K2CO3, DMF; (f) microwave, RBr, K2CO3, DMF; (g) NH2OK, CH3OH.
Table 1
The structures and HDAC8 inhibitory activity comparison of compounds E, F and H with their parent compounds 7a, 8a,
22c and the positive control SAHA
O
O
N
N
H
H
H
H
N
HN
E, F, H
N
N
HO
O
R
HO
O
R
O
O
7a, 8a, 22c
c
Compounds
SAHAa
IC50 of HDAC8 (
lM)
R
/
1.48 0.20
E
0.227 0.04
1.29 0.15
O
7aa
O
HÁHCl
F
0.467 0.07
8.21 1.68
8aa
H
0.151 0.03
0.759 0.12
22cb
O
a
b
c
Cited from Ref. 14.
Cited from Ref. 15.
Results expressed as the mean standard deviation of at least three separate determinations.
assay (Table 1) revealed that compounds E, F and H exhibited more
potent HDAC8 inhibitory activities relative to SAHA and their cor-
responding tetrahydroisoquinoline analogs 7a, 8a and 22c. Besides,
the results that compounds E and H possessed lower IC50 than
compound F reminded us of the structure–activity relationship
(SAR) obtained from the tetrahydroisoquinoline derivatives,14,15
namely, N-substituents (R2 group, such as the Boc group in com-
pound E and the 3-phenylpropionyl group in compound G) were
beneficial to the inhibitory activity.
To further verify the above SAR and to identify an optimal R1
group, compounds L01–L03 were designed and synthesized
following the methods in Scheme 2. HDAC8 inhibitory activity in
Table 2 showed that compared with compound H (Table 1), com-
pound L01 exhibited superior activity due to the Cbz group located
on the primary amine group. Different R1 groups could also lead to
different inhibitory potency, and compound L02 with 4-methoxy-
phenyl group as R2 group exhibited the most potent activity among
these three compounds. These results promoted us to keep the 4-
methoxyphenyl group fixed and substitute other functional groups
for the Cbz group of L02.
Compounds synthesized in Scheme 3 were designed to prelim-
inarily explore the effect of the N-substituent. The same as the con-
clusion drew from Table 1, tyrosine derivatives Q1–Q7 all showed
preferable HDAC8 inhibitory activities to their corresponding tet-
rahydroisoquinoline analogs (Table 3). Notably, four excellent
inhibitors Q1–Q7 with double-digit nanomolar IC50 were obtained,
and compounds Q1, Q2 and Q3 were almost 1 order of magnitude
more potent than their corresponding secondary amine analogs

4440
Y. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4437–4444
Table 2
The potent anti-HDACs activities of compounds Q1–Q4 pro-
moted us to evaluate their in vitro antiproliferative activities
against human breast tumor cell line (MDA-MB-231), lung tumor
cell line (A549) and colon tumor cell line (HCT-116) since these
three kinds of tumor cell lines were sensitive to our tetrahydroiso-
quinoline-based hydroxamic acid HDACi.14,15 As can be seen in Ta-
ble 4, although the HDAC8 inhibitory activities of these compounds
were all superior to that of SAHA, their antiproliferative activities
were not comparable with that of SAHA. To preliminarily explore
the reason why the antiproliferative activities of the tyrosine deriv-
atives were disappointing, we tested our compounds against HeLa
cell nuclear extract (which contains the panel of HDACs, primarily
HDAC1 and HDAC2) and found that the antiproliferative potency
correlated well with their HeLa cell nuclear extract inhibitory
activity. To be specific, the potent antiproliferative activity of SAHA
was in line with its robust inhibition against HeLa cell nuclear
The structures and HDAC8 inhibitory activity of compounds L01–
L03
O
R1
N
H
H
N
HN
HO
O
Cbz
O
L01-L03
a
Compounds
R1
IC50 of HDAC8 (
lM)
L01
0.197 0.04
O
L02
L03
0.120 0.03
0.213 0.04
Cl
extract (IC50 = 0.107 0.02 lM), and among the four tyrosine
derivatives, compound Q1 with the most similar IC50 value against
HeLa cell nuclear extract to SAHA, exhibited the most effective
antiproliferative activity. However, it should be kept in mind that
linking the enzyme inhibitory activity of HDACi with their cellular
potency is difficult because several other variables should be
considered, such as enzyme isoform selectivity, cellular membrane
permeability, metabolic stability, subcellular localization and
cellular xenobiotics-exporting mechanisms.
The results listed in Table 4 revealed that different with SAHA,
our tyrosine-based hydroxamic acid derivatives exhibited prefera-
ble HDAC8 inhibition. Moreover, compared with the tetrahydroiso-
quinoline-based hydroxamic acid scaffold with little selectivity
against individual HDACs,15 the tyrosine-based hydroxamic acid
scaffold exhibited HDAC8 isoform selectivity to some degree,
a
Results expressed as the mean standard deviation of at
least three separate determinations.
Q5, Q6 and Q7 (the IC50 values of Q1, Q2 and Q3 were
0.087 0.02 M, 0.063 0.01 M and 0.090 0.01 M, whereas
the IC50 values of Q5, Q6 and Q7 were 1.18 0.21 M,
0.660 0.11 M and 0.922 0.13 M, respectively). This tendency
was also observed in the tetrahydroisoquinoline analogs, which
indicated that the oxygen atom of amide group was crucial for en-
zyme inhibition.
l
l
l
l
l
l
Table 3
The structures and HDAC8 inhibitory activity of compounds Q01–Q07 with their parent compounds 25i–25n and 22a
O
O
O
O
N
N
H
H
H
H
N
HN
N
N
HO
O
R2
HO
O
R2
O
O
Q01-Q07
22a, 25i-25n
b
Compounds
IC50 of HDAC8 (
lM)
R2
Q01
25ja
0.087 0.02
0.164 0.028
O
O
Q02
25ia
0.063 0.01
0.141 0.020
Q03
0.090 0.01
0.502 0.17
22aa
O
O
Q04
0.086 0.02
0.114 0.018
25ka
Q05
1.18 0.21
1.92 0.35
25na
Q06
0.660 0.11
1.02 0.23
25ma
Q07
25la
0.922 0.13
1.72 0.36
a
Cited from Ref. 15.
b
Data represent mean values of at least three independent experiments and standard deviations are calculated based on the results
of these experiments.

Y. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4437–4444
4441
Table 4
Antiproliferative and HDACs inhibitory activities of compounds Q01–Q04 with SAHA as positive control
a
Compounds
IC50
HCT-116
(lM)
MDA-MB-231
A549
HeLa cell nuclear extract
HDAC8
Q01
Q02
Q03
Q04
SAHA
2.98 0.24
22.3 3.1
6.59 0.97
14.3 2.2
1.18 0.16
24.0 3.6
>100
54.6 7.9
97.2 17.9
0.69 0.09
1.53 0.23
>100
6.24 1.1
57.3 9.8
0.34 0.07
0.358 0.04
1.23 0.24
0.889 0.12
1.32 0.25
0.107 0.02b
0.087 0.02
0.063 0.01
0.090 0.01
0.086 0.02
1.48 0.20b
a
Results expressed as the mean standard deviation of at least three separate determinations.
Cited from Ref. 15.
b
which could be further derivatized to give rise to HDAC8 selective
inhibitors. Currently, class-selective, even isoform selective HDACs
inhibitors are of great interest, not only as therapeutic agents with
few side effects, but also as molecular probes for exploring the bio-
logical functions of HDAC isoforms.10,16–18
product was used for the following reaction without further puri-
fication. ESI-MS m/z: 282.1 [M+H]+.
5.1.2. (S)-tert-Butyl (3-(4-hydroxyphenyl)-1-oxo-1-(phenyla
mino)propan-2-yl)carbamate (C)
At 0 °C, to a solution of compound B (2.81 g, 10.0 mmol) and
HOBT (1.49 g, 11.0 mmol) in 50 mL of anhydrous THF, was added
a solution of DCC (2.27 g, 11.0 mmol) in 10 mL of anhydrous THF,
and after 20 min, the aniline (1.02 g, 11.0 mmol). After stirring
the mixture at room temperature over night, THF was evaporated
with the residues being taken up in EtOAc (40 mL) and freezed in
refrigerator. Then DCU was filtered off, the filtrate washed with
saturated Na2CO3 (3 Â 10 mL), 1 N HCl (3 Â 10 mL), brine
(3 Â 10 mL) and dried over MgSO4. The solvent was evaporated
to give 3.1 g of crude product compound C as a yellow solid. This
product was used for the following reaction without further puri-
fication. ESI-MS m/z: 356.2 [M+H]+.
4. Conclusions
In summary, on the basis of the structure of our previously re-
ported tetrahydroisoquinoline-based hydroxamic acids, a novel
series of tyrosine-based hydroxamic acid derivatives as HDACi
was designed and synthesized. Interestingly, some structure–activ-
ity relationships (SARs) were generally applicable for both series
compounds, for example, the N-substitutes (R2 group) were bene-
ficial to the inhibitory activity and the derivatives with N-acylation
were more potent than their corresponding analogs with N-alkyl-
ation. Although the tyrosine-based hydroxamic acid derivatives
exhibited more robust HDAC8 inhibitory activity relative to their
parent tetrahydroisoquinoline analogs and the positive control
SAHA, their antiproliferative potency and HeLa cell nuclear extract
inhibition were moderate. Taken together, we speculated that tyro-
sine-based hydroxamic acid scaffold could be used to design
HDAC8 isoform selective inhibitors. Related research is under
way in our laboratory.
Compounds J01–J03, M were synthesized according to the same
procedure as compound C.
5.1.3. (S)-Methyl 2-(4-(2-((tert-butoxycarbonyl)amino)-3-oxo-3-
(phenylamino)propyl)phenoxy)acetate (D)
A mixture of C (2.47 g, 6.93 mmol), K2CO3 (1.91 g, 13.86 mmol)
and methyl bromoacetate (2.12 g, 13.86 mmol) in 40 mL of anhy-
drous DMF was stirred at room temperature for 3 h. The mixture
was poured to 300 mL of H2O and extracted with EtOAc
(3 Â 40 mL). The organic layers were combined, washed with brine
(3 Â 20 mL), dried over MgSO4 and evaporated under vacuum. The
crude product was purified by recrystallization in EtOAc to get 2.8 g
of compound D as a white powder. Yield: 65%, 1H NMR (DMSO-d6) d
1.33 (s, 9H), 2.78 (dd, J = 13.8 Hz, J = 10.2 Hz, 1H), 2.93 (dd,
J = 13.8 Hz, J = 4.8 Hz, 1H), 3.69 (s, 3H), 4.28 (dd, J = 4.8 Hz,
J = 10.2 Hz, 1H), 4.75 (s, 2H), 6.85 (d, J = 8.4 Hz, 2H), 7.06 (t,
J = 7.2 Hz, 1H), 7.24 (d, J = 8.4 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 7.59
(d, J = 8.4 Hz, 2H), 10.03 (s, 1H). ESI-MS m/z: 429.2 [M+H]+.
Compounds K01–K03, N were synthesized according to the
same procedure as compound D.
5. Experimental section
5.1. Chemistry: General procedures
All commercially available starting materials, reagents and sol-
vents were used without further purification unless otherwise sta-
ted. All reactions were monitored by TLC with 0.25 mm silica gel
plates (60GF-254). UV light, iodine stain and ferric chloride were
used to visualize the spots. Silica gel was used for column chroma-
tography purification. Microwave-aided synthesis was performed
using CEM Discover-S microwave synthesis system. Melting points
were determined uncorrected on an electrothermal melting point
apparatus. 1H NMR spectra were recorded on a Bruker DRX spec-
trometer at 600 MHz, d in parts per million and J in hertz, using
TMS as an internal standard. High-resolution mass spectra were
conducted by Shandong Analysis and Test Center in Ji’nan, China.
ESI-MS spectra were recorded on an API 4000 spectrometer.
5.1.4. (S)-tert-Butyl (3-(4-(2-(hydroxyamino)-2-oxoethoxy)-
phenyl)-1-oxo-1-(phenylamino)propan-2-yl)carbamate (E)
To a solution of compound D (0.86 g, 2.0 mmol) in 10 mL of
anhydrous methanol, was added a solution of NH2OK (0.14 g,
6 mmol) in 3.5 mL of anhydrous methanol. The mixture was stirred
for 0.5 h and the solvent was evaporated under vacuum. The resi-
dues were acidified with 2 N HCl until pH 3–4 then extracted with
EtOAc (3 Â 10 mL). The organic layers were combined, washed
with brine (3 Â 10 mL), dried over MgSO4 and evaporated with
the residues being recrystallized with EtOH to give 0.21 g of title
compound E as a white powder. Yield: 24%, mp: 152–154 °C. 1H
5.1.1. (S)-2-((tert-Butoxycarbonyl)amino)-3-(4-hydroxyphenyl)
propanoic acid (B)
To a solution of compound A (3.62 g, 20.0 mmol) in 44 mL of 1 N
NaOH, was added a solution of (Boc)2O (4.80 g, 22.0 mmol) in THF
(10 mL). The solution was kept between pH 9 and 11 by addition of
1 N NaOH. After stirring the mixture at room temperature for 8 h,
THF was evaporated in vacuum with the residues being adjusted to
pH 4–5 with 1 N aqueous citric acid. Then the mixture was ex-
tracted with EtOAc (3 Â 25 mL). The extractions were combined,
washed with brine (3 Â 20 mL), dried over MgSO4 and evaporated
to give 5.30 g of crude product compound B as a white solid. This
NMR (DMSO-d6)
d 1.24+1.33 (s, 9H, cis/trans), 2.77 (dd,
J = 13.8 Hz, 10.8 Hz, 1H), 2.92 (dd, J = 13.8 Hz, 4.8 Hz, 1H), 4.23–
4.27 (m, 1H), 4.41 (s, 2H), 6.87 (d, J = 8.4 Hz, 2H), 7.05 (t,
J = 7.8 Hz, 1H), 7.07 (d, J = 9.0 Hz, 1H), 7.23 (d, J = 8.4 Hz, 2H),
7.31 (t, J = 7.8 Hz, 2H), 7.58 (d, J = 7.8 Hz, 2H), 8.95 (s, 1H), 10.02

4442
Y. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4437–4444
(s, 1H), 10.80 (s, 1H). HRMS (AP-ESI) m/z calcd for C22H27N3NaO6
[M+Na]+ 452.1798. Found: 452.1830.
1H). HRMS (AP-ESI) m/z calcd for C
26H27N3NaO6 [M+Na]+
500.1798. Found: 500.1818.
Compounds H, L01–L03, Q1–Q7 were synthesized according to
the same procedure as compound E.
5.1.4.7.
(S)-3-(4-(2-(Hydroxyamino)-2-oxoethoxy)phenyl)-N-
(4-methoxyphenyl)-2-(3-phenylpropanamido)propanamide
(Q3). Mp: 183–185 °C. 1H NMR (DMSO-d6) d 2.39 (t, J = 7.8 Hz, 2H),
2.73 (t, J = 7.8 Hz, 2H), 2.76 (dd, J = 13.8 Hz, 9.6 Hz, 1H), 2.94 (dd,
J = 13.8 Hz, 4.8 Hz, 1H), 3.72 (s, 3H), 4.40 (s, 2H), 4.58–4.61 (m,
1H), 6.84 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 7.14–7.17 (m,
5H), 7.23 (t, J = 7.8 Hz, 2H), 7.47 (d, J = 9.0 Hz, 2H), 8.23 (d,
J = 8.4 Hz, 1H), 8.96 (s, 1H), 9.93 (s, 1H), 10.81 (s, 1H). HRMS (AP-
ESI) m/z calcd for C27H30N3O6 [M+H]+ 492.2135. Found: 492.2165.
5.1.4.1.
(S)-3-(4-(2-(Hydroxyamino)-2-oxoethoxy)phenyl)-N-
phenyl-2-(3-phenylpropanamido)propanamide (H). Mp: 148–
150 °C. 1H NMR (DMSO-d6) d 2.39 (t, J = 7.8 Hz, 2H), 2.73 (t,
J = 7.8 Hz, 2H), 2.77 (dd, J = 13.8 Hz, 9.6 Hz, 1H), 2.95 (dd,
J = 13.8 Hz, 5.4 Hz, 1H), 4.40 (s, 2H), 4.61–4.65 (m, 1H), 6.85 (d,
J = 8.4 Hz, 2H), 7.05 (t, J = 7.2 Hz, 1H), 7.14–7.15 (m, 4H), 7.17 (t,
J = 7.8 Hz, 1H), 7.22 (t, J = 7.2 Hz, 2H), 7.31 (t, J = 7.8 Hz, 2H), 7.58
(d, J = 8.4 Hz, 2H), 8.27 (d, J = 7.8 Hz, 1H), 8.96 (s, 1H), 10.10 (s,
1H), 10.81 (s, 1H). HRMS (AP-ESI) m/z calcd for C26H28N3O5
[M+H]+ 462.2029. Found: 462.2057.
5.1.4.8. (S)-N-(3-(4-(2-(Hydroxyamino)-2-oxoethoxy)phenyl)-1-
((4-methoxyphenyl)amino)-1-oxopropan-2-yl)-4-phenylbutan-
amide (Q4). Mp: 173–175 °C. 1H NMR (DMSO-d6) d 1.69–1.70 (m,
2H), 2.09 (t, J = 7.8 Hz, 3H), 2.44 (t, J = 7.2 Hz, 3H), 2.77 (dd,
J = 13.8 Hz, 9.6 Hz, 1H), 2.95 (dd, J = 13.8 Hz, 4.8 Hz, 1H), 3.71 (s,
3H), 4.37 (s, 2H), 4.58–4.62 (m, 1H), 6.84 (d, J = 8.4 Hz, 2H), 6.88
(d, J = 9.0 Hz, 2H), 7.12 (d, J = 7.8 Hz, 2H), 7.16 (t, J = 7.8 Hz, 1H),
7.21 (d, J = 7.8 Hz, 2H), 7.26 (t, J = 7.8 Hz, 2H), 7.48 (d, J = 9.0 Hz,
2H), 8.18 (d, J = 7.8 Hz, 1H), 8.96 (s, 1H), 9.95 (s, 1H), 10.80 (s,
1H). HRMS (AP-ESI) m/z calcd for C28H32N3O6 [M+H]+ 506.2291.
Found: 506.2307.
5.1.4.2. (S)-Benzyl (3-(4-(2-(Hydroxyamino)-2-oxoethoxy)phenyl)-
1-oxo-1-(phenylamino) propan-2-yl)carbamate (L01). Mp: 142–
144 °C. 1H NMR (DMSO-d6) d 2.78 (dd, J = 13.8 Hz, 10.8 Hz, 1H),
2.96 (dd, J = 13.8 Hz, 4.8 Hz, 1H), 4.33–4.37 (m, 1H), 4.41 (s, 2H),
4.97–5.03 (m, 2H), 6.87 (d, J = 8.4 Hz, 2H), 7.06 (t, J = 7.2 Hz, 1H),
7.25 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 7.8 Hz, 2H), 7.32–7.36 (m, 5H),
7.60 (d, J = 7.8 Hz, 2H), 7.67 (d, J = 8.4 Hz, 1H), 8.96 (s, 1H), 10.11
(s, 1H), 10.81 (s, 1H). HRMS (AP-ESI) m/z calcd for C25H26N3O6
[M+H]+ 464.1822. Found: 464.1836.
5.1.4.9. (S)-2-(Benzylamino)-3-(4-(2-(hydroxyamino)-2-oxoeth-
oxy)phenyl)-N-(4-methoxyphenyl)propanamide (Q5). Mp: 72–
74 °C. 1H NMR (DMSO-d6) d 2.39 (s, 1H), 2.76–2.79 (m, 1H),
2.86–2.89 (m, 1H), 3.35–3.36 (m, 1H), 3.54–3.56 (m, 1H), 3.71–
3.73 (m, 1H), 3.72 (s, 3H), 4.41 (s, 2H), 6.84–6.88 (m, 4H), 7.14–
7.24 (m, 5H), 7.25–7.27 (m, 2H), 7.47–7.48 (m, 2H), 8.96 (s, 1H),
9.73 (s, 1H), 10.81 (s, 1H). HRMS (AP-ESI) m/z calcd for
5.1.4.3.
(S)-Benzyl
(3-(4-(2-(Hydroxyamino)-2-oxoethoxy)-
phenyl)-1-((4-methoxyphenyl) amino)-1-oxopropan-2-yl)carb-
amate (L02). Mp: 179–181 °C. 1H NMR (DMSO-d6) d 2.77 (dd,
J = 13.2 Hz, 10.8 Hz, 1H), 2.96 (dd, J = 13.2 Hz, 4.2 Hz, 1H), 3.72 (s,
3H), 4.30–4.33 (m, 1H), 4.41 (s, 2H), 4.94–4.99 (m, 2H), 6.86 (d,
J = 8.4 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H),
7.29 (t, J = 7.2 Hz, 2H), 7.30 (t, J = 7.2 Hz, 1H), 7.34 (t, J = 7.2 Hz,
2H), 7.49 (d, J = 9.0 Hz, 2H), 7.63 (d, J = 8.4 Hz, 1H), 8.96 (s, 1H),
9.96 (s, 1H), 10.81 (s, 1H). HRMS (AP-ESI) m/z calcd for
C
25H27N3NaO5 [M+Na]+ 472.1848. Found: 472.1847.
5.1.4.10. (S)-3-(4-(2-(Hydroxyamino)-2-oxoethoxy)phenyl)-
C
26H28N3O7 [M+H]+ 494.1927. Found: 494.1944.
N-(4-methoxyphenyl)-2-(phenethylamino)propanamide (Q6).
Mp: 148–150 °C. 1H NMR (DMSO-d6) d 2.05 (s, 1H), 2.63–2.71 (m,
4H), 2.72–2.75 (m, 1H), 2.83–2.87 (m, 1H), 3.36–3.39 (m, 1H), 3.71
(s, 3H), 4.40 (s, 2H), 6.82–6.86 (m, 4H), 7.11–7.18 (m, 5H), 7.23–
7.26 (m, 2H), 7.39–7.40 (m, 2H), 8.96 (s, 1H), 9.62 (s, 1H), 10.81
(s, 1H). HRMS (AP-ESI) m/z calcd for C26H29N3NaO5 [M+Na]+
486.2005. Found: 486.2049.
5.1.4.4. (S)-Benzyl (1-((2-Chlorophenyl)amino)-3-(4-(2-(hydrox-
yamino)-2-oxoethoxy) phenyl)-1-oxopropan-2-yl)carbamate
(L03). Mp: 160–162 °C. 1H NMR (DMSO-d6) d 2.78 (dd, J = 13.8 Hz,
10.8 Hz, 1H), 2.96 (dd, J = 13.8 Hz, 4.8 Hz, 1H), 4.31–4.33 (m, 1H),
4.41 (s, 2H), 4.93–4.99 (m, 2H), 6.87 (d, J = 8.4 Hz, 2H), 7.12–7.17
(m, 2H), 7.23 (d, J = 8.4 Hz, 2H), 7.25–7.37 (m, 6H), 7.44–7.46 (m,
1H), 7.72 (d, J = 8.4 Hz, 1H), 8.97 (s, 1H), 10.32 (s, 1H), 10.82 (s,
1H). HRMS (AP-ESI) m/z calcd for C25H25ClN3O6 [M+H]+ 498.1432.
Found: 498.1443.
5.1.4.11.
(S)-3-(4-(2-(Hydroxyamino)-2-oxoethoxy)phenyl)-
N-(4-methoxyphenyl)-2-((3-phenylpropyl)amino)propanamide
(Q7). Mp: 87–89 °C. 1H NMR (DMSO-d6) d 1.63–1.68 (m, 2H), 1.95
(s,1H), 2.39–2.55 (m, 4H), 2.70–2.74 (m, 1H), 2.82–2.85 (m, 1H),
3.26–3.31 (m, 1H), 3.71 (s, 3H), 4.39 (s, 2H), 6.84–6.87 (m, 4H),
7.11–7.16 (m, 5H), 7.21–7.24 (m, 2H), 7.45–7.46 (m, 2H), 8.96 (s,
1H), 9.68 (s, 1H), 10.80 (s, 1H). HRMS (AP-ESI) m/z calcd for
5.1.4.5.
(S)-N-(3-(4-(2-(Hydroxyamino)-2-oxoethoxy)phenyl)-
1-((4-methoxyphenyl)amino)-1-oxopropan-2-yl)benzamide
(Q1). Mp: 207–209 °C. 1H NMR (DMSO-d6) d 3.01–3.08 (m, 2H),
3.72 (s, 3H), 4.39 (s, 2H), 4.73–4.79 (m, 1H), 6.87 (d, J = 8.4 Hz,
2H), 6.90 (d, J = 9.0 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.45 (t,
J = 7.8 Hz, 2H), 7.52 (d, J = 9.0 Hz, 2H), 7.53 (t, J = 7.8 Hz, 1H), 7.83
(d, J = 7.8 Hz, 2H), 8.69 (d, J = 8.4 Hz, 1H), 8.95 (s, 1H), 10.08 (s,
1H), 10.78 (s, 1H). HRMS (AP-ESI) m/z calcd for C25H26N3O6
[M+H]+ 464.1822. Found: 464.1841.
C
27H31N3NaO5 [M+Na]+ 500.2161. Found: 500.2192.
5.1.5. (S)-2-Amino-3-(4-(2-(hydroxyamino)-2-oxoethoxy)-
phenyl)-N-phenylpropanamide hydrochloride (F)
To a solution of compound E (0.17 g, 0.40 mmol) in 6 mL of dry
EtOAc, was added a solution of EtOAc (10 mL) saturated by dry HCl
gas. The reaction solution was stirred at room temperature for 7 h
when the precipitation appeared. The suspension was filtered with
the filter being washed with ether to give 0.13 g of desired com-
pound F as a white powder. Yield: 89%, mp: 168–170 °C. 1H NMR
5.1.4.6. (S)-3-(4-(2-(Hydroxyamino)-2-oxoethoxy)phenyl)-N-(4-
methoxyphenyl)-2-(2-phenylacetamido)propanamide (Q2). Mp:
197–199 °C. 1H NMR (DMSO-d6) d 2.80 (dd, J = 13.8 Hz, 9.6 Hz, 1H),
2.97 (dd, J = 13.8 Hz, 4.8 Hz, 1H), 3.39–3.47 (m, 2H), 3.72 (s, 3H),
4.40 (s, 2H), 4.57–4.61 (m, 1H), 6.83 (d, J = 8.4 Hz, 2H), 6.88 (d,
J = 9.0 Hz, 2H), 7.13 (d, J = 7.2 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H),
7.18 (t, J = 7.2 Hz, 1H), 7.24 (t, J = 7.2 Hz, 2H), 7.47 (d, J = 9.0 Hz,
2H), 8.43 (d, J = 8.4 Hz, 1H), 8.98 (s, 1H), 9.97 (s, 1H), 10.83 (s,
(DMSO-d6)
d 3.03 (dd, J = 13.8 Hz, 7.8 Hz, 1H), 3.15 (dd,
J = 13.8 Hz, 6.0 Hz, 1H), 4.18–4.20 (m, 1H), 4.42 (s, 2H), 6.91 (d,
J = 9.0 Hz, 2H), 7.11 (t, J = 7.2 Hz, 1H), 7.22 (d, J = 9.0 Hz, 2H), 7.35
(t, J = 7.2 Hz, 2H), 7.57 (d, J = 7.2 Hz, 2H), 8.33 (br s, 3H), 8.98 (s,
1H), 10.73 (s, 1H), 10.86 (s, 1H). HRMS (AP-ESI) m/z calcd for
C
17H20N3O4 [M+H]+ 330.1454. Found: 330.1471.

Y. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 4437–4444
4443
5.1.6. (S)-Methyl 2-(4-(3-oxo-3-(phenylamino)-2-(3-phenyl-
propanamido)propyl)phenoxy)acetate (G)
under vacuum. The residue was purified by flash column chroma-
tography (petroleum ether/EtOAc 3:1) to give 0.39 g of desired
compound P05 as a white solid. Yield: 81%, 1H NMR (DMSO-d6) d
2.37 (s, 1H), 2.77–2.80 (m, 1H), 2.85–2.89 (m, 1H), 3.33–3.35 (m,
1H), 3.53–3.55 (m, 1H), 3.68 (s, 3H), 3.69–3.70 (m, 1H), 3.72 (s,
3H), 4.75 (s, 2H), 6.82–6.88 (m, 4H), 7.13–7.22 (m, 5H), 7.25–
7.27 (m, 2H), 7.46–7.48 (m, 2H), 9.85 (s, 1H). ESI-MS m/z: 449.2
[M+H]+.
To a solution of compound D (4.28 g, 10.0 mmol) in anhydrous
CH2Cl2 (40 mL), was added 20 mL of trifluoroacetic acid. When the
reaction was finished, saturated Na2CO3 was added to the solution
until the pH reach weak basic. The mixture was separated with
separatory funnel, the organic layer was washed with distilled
water, dried over MgSO4 and evaporated under vacuum to get
2.90 g of white solid. This product was used for the following reac-
tion without further purification. At room temperature, to a solu-
tion of 3-phenylpropionic acid (0.75 g, 5.0 mmol) in anhydrous
THF (20 mL), was added Et3N (0.56 g, 5.5 mmol) followed by 2-
(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoro-
borate (TBTU, 1.78 g, 5.5 mmol). After 15 min, the amine (1.67 g,
5.0 mmol) was added. The stirring was continued for 8 h and then
THF was evaporated. The residues were taken up in EtOAc (40 mL),
washed with 1 N HCl (3 Â 10 mL), saturated Na2CO3 (3 Â 10 mL),
brine (3 Â 10 mL), dried over MgSO4 and evaporated under vac-
uum. The crude product was purified by recrystallization in EtOAc
to get 2.0 g of compound G as a white powder. Yield: 43%, 1H NMR
(DMSO-d6) d 2.38 (t, J = 7.8 Hz, 2H), 2.72 (t, J = 7.8 Hz, 2H), 2.75–
7.79 (m, 1H), 2.92–2.95 (m, 1H), 3.72 (s, 3H), 4.59–4.60 (m, 1H),
4.74 (s, 2H), 6.82 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 7.14
(d, J = 7.8 Hz, 2H), 7.16 (d, J = 9.0 Hz, 2H), 7.17–7.20 (m, 1H), 7.23
(d, J = 7.8 Hz, 2H), 7.47 (d, J = 9.0 Hz, 2H), 8.24 (s, 1H), 9.94 (s,
1H). ESI-MS m/z: 461.2 [M+H]+.
5.1.10. (S)-Methyl 2-(4-(3-((4-methoxyphenyl)amino)-3-oxo-2-
(phenethylamino)propyl)phenoxy)acetate (P06)
A sealed tube containing compound O (0.15 g, 0.41 mmol),
potassium carbonate powder (0.084 g, 0.61 mmol), (2-bromoeth-
yl)benzene (0.11 g, 0.61 mmol) and DMF (4 mL) was subjected to
microwave irradiation at 70 °C for 20 min, at 100 W. After cooling,
the mixture was poured into 20 mL of water and extracted with
EtOAc (3 Â 10 mL). The organic layers were combined, washed
with brine (3 Â 10 mL), dried over MgSO4 and evaporated under
vacuum. The residue was purified by flash column chromatogra-
phy (petroleum ether/EtOAc 3:1) to give 0.13 g of desired com-
pound P06 as a white solid. Yield: 71%, 1H NMR (DMSO-d6) d
2.05 (s, 1H), 2.63–2.71 (m, 4H), 2.72–2.75 (m, 1H), 2.84–2.88 (m,
1H), 3.37–3.39 (m, 1H), 3.69 (s, 3H), 3.71 (s, 3H), 4.78 (s, 2H),
6.80–6.86 (m, 4H), 7.12–7.18 (m, 5H), 7.23–7.25 (m, 2H), 7.40–
7.42 (m, 2H), 8.96 (s, 1H), 9.63 (s, 1H). ESI-MS m/z: 463.2 [M+H]+.
Compounds P07 were synthesized according to the same proce-
dure as compound P06.
Compounds P01–P04 were synthesized according to the same
procedure as compound G.
5.2. Biological materials and methods
5.1.7. (S)-2-(((Benzyloxy)carbonyl)amino)-3-(4-hydroxy-
phenyl)propanoic acid (I)
5.2.1. In vitro HDACs inhibition fluorescence assay
At 0 °C, to a solution of compound A (1.15 g, 6.38 mmol) in 7 mL
of 1 N NaOH, was added Cbz-Cl (1.20 g, 7.01 mmol) dropwise. The
solution was kept between pH 9 and 11 by addition of 1 N NaOH.
After stirring the mixture for 3 h, the solution was adjusted to pH
4–5 with 1 N HCl. Then the mixture was extracted with EtOAc
(3 Â 10 mL). The extractions were combined, washed with brine
(3 Â 10 mL), dried over MgSO4 and evaporated to give 1.85 g of
crude product compound I as light yellow oil. This product was
used for the following reaction without further purification. ESI-
MS m/z: 316.1 [M+H]+.
In vitro HDACs inhibition assays were conducted as previously
described.15 In brief, 10
l
L of enzyme solution (HeLa nuclear ex-
tract or HDAC8) was mixed with various concentrations of tested
compound (50 L). Five minutes later, fluorogenic substrate Boc-
Lys (acetyl)-AMC (40 L) was added, and the mixture was incu-
bated at 37 °C for 30 min and then stopped by addition of 100
l
l
l
L
of developer containing trypsin and TSA. After incubation at
37 °C for 20 min, fluorescence intensity was measured using a
microplate reader at excitation and emission wavelengths of
390 nm and 460 nm, respectively. The inhibition ratios were calcu-
lated from the fluorescence intensity readings of tested wells rela-
tive to those of control wells, and the IC50 values were calculated
using a regression analysis of the concentration/inhibition data.
5.1.8. (S)-Methyl 2-(4-(2-amino-3-((4-methoxyphenyl)amino)-
3-oxopropyl)phenoxy)acetate (O)
To a solution of compound N (4.58 g, 10.0 mmol) in anhydrous
CH2Cl2 (40 mL), was added 20 mL of trifluoroacetic acid. When the
reaction was finished, saturated Na2CO3 was added to the solution
until the pH reach weak basic. The mixture was separated with
separatory funnel, the organic layer was washed with distilled
water, dried over MgSO4 and evaporated under vacuum to get
3.10 g of white solid O. This product was used for the following
reaction without further purification. 1H NMR (DMSO-d6) d 2.66
(dd, J = 13.8 Hz, J = 7.8 Hz, 1H), 2.93 (dd, J = 13.8 Hz, J = 5.4 Hz,
1H), 3.40 (br s, 2H), 3.47 (dd, J = 7.8 Hz, J = 5.4 Hz, 1H), 3.69 (s,
3H), 3.72 (s, 3H), 4.74 (s, 2H), 6.83 (d, J = 8.4 Hz, 2H), 6.88 (d,
J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H),
9.71 (s, 1H). ESI-MS m/z: 359.2 [M+H]+.
5.2.2. MTT assay
In vitro antiproliferative assays were determined as previously
described.15 Briefly, all cell lines were maintained in RPMI1640
medium containing 10% FBS at 37 °C in 5% CO2 humidified incuba-
tor. Cells were passaged the day before dosing into a 96-well cell
plate, allowed to grow for a minimum of 4 h prior to addition of
compounds. After compounds addition, the plates were incubated
for an additional 48 h, and then 0.5% MTT (3-[4,5-dimethyl-2-
thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) solution was
added to each well. After further incubation for 4 h, formazan
formed from MTT was extracted by adding 200 lL of DMSO for
15 min. Absorbance was then determined using an ELISA reader
at 570 nm and the IC50 values were calculated according to the
inhibition ratios.
5.1.9. (S)-Methyl 2-(4-(2-(benzylamino)-3-((4-methoxyphenyl)-
amino)-3-oxopropyl)phenoxy)acetate (P05)
A mixture of O (0.39 g, 1.08 mmol), potassium carbonate pow-
der (0.22 g, 1.62 mmol) and benzyl bromide (0.28 g, 1.62 mmol)
in anhydrous DMF (15 mL) was stirred at room temperature for
4 h. The mixture was poured into 60 mL of water and extracted
with EtOAc (3 Â 20 mL). The organic layers were combined,
washed with brine (3 Â 15 mL), dried over MgSO4 and evaporated
Acknowledgments
This work was supported by National Science and Technology
Major Project (Grant No. 2009ZX09103-118), National Natural
Science Foundation of China (Grant Nos. 90713041, 30772654
and 20272023), Shandong Provincial Natural Science Foundation,

4444
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