Design, synthesis and evaluation of novel metalloproteinase inhibitors based on l-tyrosine scaffold

Article abstract of DOI:10.1016/j.bmc.2012.08.014

A series of novel l-tyrosine derivatives were designed, synthesized and assayed for their inhibitory activities on matrix metalloproteinase 2 (MMP-2) and histone deacetylase 8 (HDAC-8). The results showed that these l-tyrosine derivatives exhibited inhibitory profiles against MMP-2 and HDAC-8. The compounds 6h (IC₅₀ = 0.013 ± 0.001 μM) and 6j (IC ₅₀ = 0.017 ± 0.001 μM) were equal potent MMP-2 inhibitors to the positive control NNGH (IC₅₀ = 0.014 ± 0.001 μM). As for HDAC-8 inhibition, some of the hydroxamate compounds, such as 6d (IC ₅₀ = 3.6 ± 0.2 μM) and 6c (IC₅₀ = 5.8 ± 0.5 μM), were equal potent to the positive control SAHA (IC₅₀ = 1.6 ± 0.1 μM). Structure-activity relationships were also briefly discussed.

Full text of DOI:10.1016/j.bmc.2012.08.014

Bioorganic & Medicinal Chemistry 20 (2012) 5738–5744
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of novel metalloproteinase inhibitors based
on L-tyrosine scaffold
⇑
Xian-Chao Cheng, Run-Ling Wang , Zhen-Ke Dong, Jing Li, Yao-Yuan Li, Rong-Rong Li
Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin
300070, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel
ities on matrix metalloproteinase 2 (MMP-2) and histone deacetylase 8 (HDAC-8). The results showed
that these -tyrosine derivatives exhibited inhibitory profiles against MMP-2 and HDAC-8. The com-
pounds 6h (IC50 = 0.013 0.001 M) and 6j (IC50 = 0.017 0.001 M) were equal potent MMP-2 inhibi-
tors to the positive control NNGH (IC50 = 0.014 0.001 M). As for HDAC-8 inhibition, some of the
hydroxamate compounds, such as 6d (IC50 = 3.6 0.2 M) and 6c (IC50 = 5.8 0.5 M), were equal potent
L-tyrosine derivatives were designed, synthesized and assayed for their inhibitory activ-
Received 11 July 2012
Revised 8 August 2012
Accepted 9 August 2012
Available online 22 August 2012
L
l
l
l
l
l
Keywords:
to the positive control SAHA (IC50 = 1.6 0.1
discussed.
lM). Structure–activity relationships were also briefly
L-tyrosine derivatives
Synthesis
Ó 2012 Elsevier Ltd. All rights reserved.
MMP-2 inhibitors
HDAC-8 inhibitors
IC50
1. Introduction
identify more potent MMP-2 inhibitors, using scaffold hopping
strategy, we replaced -hydroxyproline with -tyrosine scaffold to
L
L
The matrix metalloproteinases (MMPs) are a family of structur-
ally related zinc-dependent endoproteinases that degrade and re-
model structural proteins in the extracellular matrix.1 They
include more than 20 subtypes, among which MMP-2 is highly in-
volved in the process of tumor invasion and metastasis and has
been considered as a promising target for cancer therapy.2,3
It has been reported that besides the catalytic activity center
zinc (II) ion of MMP-2, there are two hydrophobic domains, which
are called S10 pocket and S1 pocket, respectively. S10 pocket, the
key domain of MMP-2, is deeper and narrower than that of most
other MMP subtypes, and S1 pocket is solvent exposed.4,5 Cur-
rently identified MMP-2 inhibitors shared the following structural
character and binding mode: (1) a zinc binding group (ZBG, such as
hydroxamate and carboxylate) capable of chelating the active site
zinc ion; (2) one at least functional group which provides a hydro-
gen bond interaction with the enzyme backbone; (3) one or more
side chains which undergo effective interactions with the enzyme
subsites, such as S10 pocket and S1 pocket.6,7
form the new integrated structural pattern (see Fig. 1). R1 group
can be various alkyl groups, such as benzyl and phenylethyl group.
R2 group can be various acyl groups, such as benzoyl, acetyl or
methylsulfonyl group. COR3 group can be hydroxamate, carboxyl-
ate or carboxyl group. R1 group and R2 group might occupy the
S10 and S1 pockets, respectively, while the COR3 group might che-
late the active site zinc ion. The tyrosine scaffold might bond to the
enzyme backbone.
2. Results and discussion
The target compounds were synthesized via the route as shown
in Scheme 1. Starting from N-Boc-L-tyrosine methyl ester (1) as a
chiral template, the important intermediate (S)-methyl 2-amino-
3-(4-alkyloxyphenyl)propanoate (3a–b) was prepared via
etherization11 and deprotection of Boc by CF3COOH.12 Acylation
of 3a–b with various acyl chlorides produced compounds 4a–j.13
Further hydrolysis of 4a–j provided the carboxyl acid compounds
5a–j,10 and ammonolysis of 4a–j with NH2OK provided the final
hydroxamate compounds 6a–j.9 These target compounds have
not been reported in literature and their chemical structures were
confirmed by IR, 1H NMR, 13C NMR and ESI-MS.
In our previous work, the
L-hydroxyproline scaffold-based
MMP-2 inhibitors were studied, and some active compounds were
reported in the literature.8–10 Some of these compounds (see Fig. 1)
showed low nanomolar inhibitory activity for MMP-2. In order to
The newly synthesized L-tyrosine derivatives were assayed for
the inhibitory activities on MMP-2 and histone deacetylase 8
(HDAC-8). Similarly as MMP-2, HDAC-8 is also a zinc-dependent
metalloproteinase involved in the process of tumor invasion and
⇑
Corresponding author. Tel.: +86 022 23542805; fax: +86 022 23542014.
E-mail addresses: chengxianchao2000@yahoo.com.cn (X.-C. Cheng), wangrun-
ling@tijmu.edu.cn (R.-L. Wang).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.014

X.-C. Cheng et al. / Bioorg. Med. Chem. 20 (2012) 5738–5744
5739
O
O
R2
scaff old
hopping
virtual
R2 NH
R3
screening
R3
N
R group
R1
optimization
O
O
R1
Figure 1. Construction of
L-tyrosine based compounds database.
O
O
Boc NH
O
O
Boc NH
H2N
O
O
a
b
R1
O
R1
O
HO
1
2a-b
3a-b
c
O
R2 NH
OH
O
O
R2 NH
R2 NH
O
NHOH
d
e
R1
O
R1
O
R1
O
6a-j
4a-j
5a-j
Scheme 1. Reagents: (a) R1Cl, K2CO3, NaI; (b) CF3COOH; (c) R2Cl, Na2CO3; (d) NaOH, HCl; (e) NH2OK.
metastasis.14 Thus the assay was performed on both of MMP-2 and
HDAC-8 so as to identify the compound selectivity. NNGH
(N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid),
a potent inhibitor of MMP-2, was used as the positive control of
MMP-2 assay. SAHA (suberoylanilide hydroxamic acid), a potent
HDACs inhibitor that has been registered as antitumor drug, was
used as the positive control of HDAC-8 assay.
the hydroxamate group was a more potent ZBG than carboxyl group
as shown in the activity order of 6a-j and their predecessors.
Among compounds 6a–j, while fixing R2 group, the R1 group
was altered as benzyl and phenylethyl group. So the differences
in the inhibitory activities of these compounds were caused by
the R1 group length. Introduction of longer group (6f–j) displayed
higher activity. The same rule could also be seen among com-
pounds 5a–j and 4a–j.
The results showed that these L-tyrosine derivatives exhibited
inhibitory activities against MMP-2 and HDAC-8 (Table 1). Most
of these compounds, especially the hydroxamate compounds,
could inhibit MMP-2 at nanomolar concentrations and HDAC-8 at
micromolar concentrations, thus exhibiting selective inhibition of
MMP-2 against HDAC-8.
As for MMP-2 inhibition, the compounds 6h and 6j were equal
potent to the positive control NNGH. The Flexible docking of the
compounds 6h and 6j with MMP-2 was done using Discovery Stu-
dio 3.0 software and the results were shown in Figures 2 and 3. The
phenylethyloxyl group occupied the deep S10 pocket of MMP-2,
and the hydroxamate group chelated the active site zinc ion 166.
Compounds 6h and 6j interacted well with MMP-2 active site,
especially the deep S10 pocket and zinc ion 166, consistent with
the binding mode of the original ligand I52, thus showing high
inhibitory activities against MMP-2.
As for HDAC-8 inhibition, some of the hydroxamate com-
pounds, such as 6d and 6c, were equal potent to the positive con-
trol SAHA. Figure 4 showed the docking result of compound 6d
with HDAC-8 active site (the broken line showing SAHA). The
molecule inserted into the pocket and the hydroxamate group
was found to be close to zinc ion 237 in the active site and che-
lated well with the distance of 1.82 and 2.15 Å, respectively. The
binding mode of 6d with HDAC-8 active site was similar to that of
SAHA, which could explain the high potency of 6d. Among com-
pounds 6a–j, shorter R1 group (benzyloxyl) and R2 group (acetyl
and methanesulfonyl) exhibited higher activity, and this principle
could also be seen among compounds 5a–j (except compounds 5b
and 5g).
The different inhibitory profile might be explained by the active
site structural differences. MMP-2 was a zinc-dependent endopep-
tidase that could cut the peptide to parts from the specific amino
acid residue of peptide. However, HDAC-8 was a class of enzyme
Compounds 6a–j were more potent than their predecessors 5a–j.
This activity difference was caused by the ZBG (COR3), which was
the only structural difference between 6a–j and their predecessors.
The ZBG is hydroxamate (CONHOH) for 6a–j and carboxyl group for
their predecessors, respectively. Both of these two groups could
chelate zinc ion at catalytic activity center of the enzyme. However,
that removed acetyl groups (O@C–CH3) from an e-N-acetyl lysine
amino acid on a histone. Due to the structural differences between
MMP-2 and HDAC-8, there were different structural requirements
for their respective inhibitors.

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X.-C. Cheng et al. / Bioorg. Med. Chem. 20 (2012) 5738–5744
Table 1
The structures and IC50 values of L-tyrosine derivatives
O
R2 NH
R3
R1
O
a
No.
Structure
IC50 (lM)
R1
R2
R3
MMP-2
HDAC-8
4a
4b
4c
4d
4e
4f
4g
4h
4i
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CO
p-CH3C6H4CO
CH3CO
CH3SO2
p-CH3C6H4SO2
C6H5CO
p-CH3C6H4CO
CH3CO
CH3SO2
p-CH3C6H4SO2
C6H4CO
p-CH3C6H4CO
CH3CO
CH3SO2
p-CH3C6H4SO2
C6H5CO
p-CH3C6H4CO
CH3CO
CH3SO2
p-CH3C6H4SO2
C6H5CO
p-CH3C6H4CO
CH3CO
CH3SO2
p-CH3C6H4SO2
C6H5CO
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
67.5 6.9
83.7 9.5
27.6 2.5
17.8 1.6
10.7 1.1
74.6 7.4
85.5 7.8
12.5 1.3
2.4 0.2
6.3 0.5
9.8 0.9
8.1 0.8
3.0 0.3
1.4 0.1
2.5 0.3
7.6 0.7
4.5 0.5
0.75 0.07
0.12 0.01
0.27 0.02
1.7 0.1
0.46 0.04
0.037 0.002
0.067 0.006
0.025 0.002
0.9 0.1
>1000
>1000
871.5 73.2
753.7 72.6
>1000
C6H5CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
>1000
>1000
825.1 82.9
>1000
961.8 85.4
110.5 10.9
>1000
36.9 5.2
47.2 5.1
504.7 48.9
253.4 33.6
385.6 40.5
120.9 14.6
102.3 12.7
>1000
4j
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
OH
6a
6b
6c
6d
6e
6f
6g
6h
6i
NHOH
NHOH
NHOH
NHOH
NHOH
NHOH
NHOH
NHOH
NHOH
NHOH
9.3 1.0
30.7 4.1
5.8 0.5
3.6 0.2
42.4 4.3
27.8 3.2
52.5 4.9
14.4 1.1
9.5 1.0
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2CH2
p-CH3C6H4CO
CH3CO
CH3SO2
3.9 0.3
0.013 0.001
0.029 0.002
0.017 0.001
6j
p-CH3C6H4SO2
82.5 9.3
H
N
N
S
OH
NNGH
SAHA
0.014 0.001
/
O O
O
O
O
H
N
/
1.6 0.1
N
H
OH
O
a
IC50 values are mean of three experiments, standard deviation is given.
The above structure–activity relationship studies encouraged us
4. Experimental
to further design novel-scaffold-based MMP-2 and HDAC-8 inhib-
itors, which would be reported later.
4.1. Synthetic methods and spectroscopic details
Melting points were determined using X-6 digital display binoc-
ular microscope (uncorrected). Infrared spectra were measured on a
nicolet nexus 470 FT-IR spectrometer using smear KBr crystal or KBr
plate. 1H NMR spectra were recorded on a Bruker Avance (400 MHz)
spectrometer; J values are in Hz. 13C NMR spectra were recorded on a
BrukerAvance(400 MHz)spectrometer.Massspectrawererecorded
on an electro-spray ionization mass spectrometer as the value m/z.
Flash column chromatography was performed using 300 mesh silica
gel. The yields were calculated by the last step reaction.
3. Conclusions
In conclusion, a series of novel L-tyrosine derivatives was de-
signed and synthesized. These compounds exhibited inhibitory
activities against MMP-2 and HDAC-8. The compounds 6h and
6j were equal potent to the positive control NNGH. As for
HDAC-8 inhibition, some of the hydroxamate compounds, such
as 6d and 6c, were equal potent to the positive control SAHA.
SAR studies indicated that introduction of longer R1 group favored
the inhibitory activity against MMP-2, while shorter R1 group and
R2 group exhibited higher HDAC-8 inhibitory activity. The flexible
docking was consistent with the above SAR results. Further assays
of these compounds on cell culture and animal models were
underway.
4.1.1. (S)-Methyl 3-(4-(substituted oxy)phenyl)-2-(tert-
butoxycarbonylamino)propanoate (2)
N-Boc-L-tyrosine methyl ester (1) (29.5 g, 100 mmol) and ben-
zyl chloride (or phenylethyl chloride) (200 mmol) were dissolved

X.-C. Cheng et al. / Bioorg. Med. Chem. 20 (2012) 5738–5744
5741
Figure 2. Flexible docking result of compound 6h with MMP-2 (the broken line
showing the original ligand I52).
Figure 4. Flexible docking result of compound 6d with HDAC-8 (the broken line
showing SAHA).
(s, 3H, OCH3), 3.12–2.98 (m, 2H, CH2), 1.47 (s, 9H, C(CH3)3). ESI-
MS: 401.6 (M+NH4).
4.1.1.2. (S)-Methyl 2-(tert-butoxycarbonylamino)-3-(4-pheneth-
oxyphenyl)propanoate (2b).
White solid, 84.2%, mp 78.3–
80.4 °C. 1H NMR (CDCl3, d): 7.290 (d, 1H, N–H, J = 6.8 Hz), 7.248–
7.223 (m, 5H, Ar-H), 7.052 (d, 2H, Ar-H, J = 8.0 Hz), 6.846 (d, 2H,
Ar-H, J = 8.4 Hz), 5.024 (s, 1H, CH), 3.962 (t, 2H, CH2, J = 6.0 Hz),
3.737 (s, 3H, CH3O), 3.056 (q, 2H, CH2, J = 4.8 Hz), 2.833 (t, 2H,
CH2O, J = 7.6 Hz), 1.455 (s, 9H, (CH3)3C). ESI-MS: 438.2 (M+K).
4.1.2. (S)-Methyl 2-amino-3-(4-(substituted oxy)phenyl)
propanoate (3)
On an ice bath, 2a or 2b (10 mmol) was added to a solution of
trifluoroacetic acid and dichloromethane (1:1, 10 ml). The mixture
was reacted for 5 h and TLC was used to monitor completion. The
solvent was evaporated to give yellow oil, which was dissolved in
saturated NaHCO3 solution to PH7. After extraction with dichloro-
methane, the organic layer was evaporated to give oil, which was
purified by flash column chromatography (ethyl acetate:n-hex-
ane = 1:1) and 3a or 3b was obtained as white crystal.
Figure 3. Flexible docking result of compound 6j with MMP-2 (the broken line
showing the original ligand I52).
4.1.2.1. (S)-Methyl 2-amino-3-(4-(benzyloxy)phenyl)propanoate
(3a).
84%, mp 158–160 °C, 1H NMR (DMSO-d6, d): 8.52 (s, 2H,
in acetone (1000 ml), then potassium carbonate (27.6 g, 200 mmol)
and sodium iodide (catalytic amount) were added to the solution.
The mixture was rufluxed for 10 h, and TLC was used to monitor
the reaction. The resulting mixture was filtrated and the filtrate
was evaporated to give yellow oil. The oil was further purified by
flash column chromatography (ethyl acetate/n-hexane = 1/5) and
2 was obtained.
NH2), 7.46–7.33 (m, 5H, Ar-H), 7.14 (d, 2H, Ar-H, J = 8.80 Hz), 6.98
(d, 2H, Ar-H, J = 8.80 Hz), 5.09 (s, 2H, OCH2), 4.21 (t, 1H, CH,
J = 7.00 Hz), 3.68 (s, 3H, OCH3), 3.20–3.00 (m, 2H, CH2). ESI-MS:
286.1 (M+1).
4.1.2.2. (S)-Methyl 2-amino-3-(4-phenethoxyphenyl)propano-
ate (3b).
93.6%, mp: 109.5–112.1 °C. 1H NMR (DMSO-d6, d):
4.1.1.1. (S)-Methyl 3-(4-(benzyloxy)phenyl)-2-(tert-butoxycar-
7.275 (d, 2H, Ar-H, J = 7.6 Hz), 7.222 (d, 3H, Ar-H, J = 6.8 Hz),
7.080 (d, 2H, Ar-H, J = 8.8 Hz), 6.835 (d, 2H, Ar-H, J = 8.8 Hz),
3.920 (t, 2H, NH2, J = 6.4 Hz), 3.642 (s, 1H, CH), 3.588 (s, 1H, CH3),
3.175 (s, 2H, CH2), 2.832–2.710 (m, 4H, CH2CH2). ESI-MS: 300.2
(M+1).
bonylamino)propanoate (2a).
Yellow solid, 96%, mp 52.0–
54.2 °C. 1H NMR (CDCl3, d): 7.45–7.31 (m, 5H, Ar-H), 7.08 (d, 2H,
Ar-H, J = 8.00 Hz), 6.93 (d, 2H, Ar-H, J = 8.00 Hz), 5.21 (d, 1H, NH,
J = 8.00 Hz), 5.03 (s, 2H, CH2O), 4.59 (d, 1H, CH, J = 7.20 Hz), 3.70

5742
X.-C. Cheng et al. / Bioorg. Med. Chem. 20 (2012) 5738–5744
4.1.3. (S)-Methyl 2-substituted amido-3-(4-(substituted oxy)
phenyl)propanoate (4)
Ar-H), 6.827 (d, 2H, Ar-H, J = 7.6 Hz), 4.591 (q, 1H, CH, J = 8.0 Hz),
3.907 (t, 2H, CH2, J = 6.4 Hz), 3.634 (s, 3H, CH3), 3.061 (m, 2H,
CH2), 2.712 (t, 2H, CH2, J = 7.6 Hz), 2.352 (s, 3H, CH3). ESI-MS:
418.2(M+1).
Compound 3a or 3b (10 mmol) was dissolved in dichlorometh-
ane (100 ml) and Na2CO3 (2.12 g, 20 mmol) was added. Acyl chlo-
ride (10 mmol) in dichloromethane (100 ml) was added to the
solution. The mixture was reacted for 18 h and TLC was used to
monitor the completion. The filtrate was evaporated to give oil,
which was purified by flash column chromatography (ethyl ace-
tate:n-hexane = 1:1) and 4 was obtained.
4.1.3.8. (S)-Methyl 2-acetamido-3-(4-(phenylethyloxy)phenyl)
propanoate (4h).
White crystal, 56.9%, mp: 117.7–119.1 °C.
1H NMR (DMSO-d6, d): 8.277–8.296 (d, 1H, NH, J = 7.6 Hz), 7.101–
7.307 (m, 7H, Ar-H), 6.829–6.848 (d, J = 7.6 Hz, 2H, Ar-H), 4.381–
4.399 (d, 1H, CH, J = 7.2 Hz), 3.914–3.945 (m, 2H, CH2), 3.591 (s,
3H, CH3), 2.508–2.954 (m, 4H, CH2CH2), 1.796 (s, 3H, CH3). ESI-
MS: 342.2(M+1).
4.1.3.1. (S)-Methyl 2-benzamido-3-(4-(benzyloxy)phenyl)pro-
panoate (4a).
Yellow crystal, 79%, mp 128.3–130.5 °C, 1H
NMR (DMSO-d6, d): 8.83 (d, 1H, NH, J = 8.00 Hz), 7.80 (d, 2H, Ar-
H, J = 8.00 Hz), 7.54 (t, 1H, Ar-H, J = 7.20 Hz), 7.48–7.29 (m, 7H,
Ar-H), 7.22 (d, 2H, Ar-H, J = 8.00 Hz), 6.92 (d, 2H, Ar-H,
J = 8.00 Hz), 5.04 (s, 2H, OCH2), 4.60 (m, 1H, CH), 3.64 (s, 3H,
OCH3), 3.12–2.99 (m, 2H, CH2). ESI-MS: 390.3 (M+1).
4.1.3.9. (S)-Methyl 2-methanesulfonyl amido-3-(4-(phenyleth-
yloxy)phenyl)propanoate (4i).
Yellow crystal, 81.2%, mp:
64.8–65.2 °C. 1H NMR (DMSO-d6, d): 7.829 (s, 1H, NH), 7.299–
7.161 (m, 7H, Ar-H), 6.857 (d, 2H, Ar-H, J = 8.4 Hz), 4.100 (t, 1H,
CH, J = 6.0 Hz), 3.924 (t, 2H, CH2, J = 6.4 Hz), 3.629 (s, 3H, CH3O),
2.971–2.499 (m, 4H, CH2CH2), 2.765 (s, 3H, SO2CH3). ESI-MS:
376.2(M-1).
4.1.3.2. (S)-Methyl 2-(p-methyl)benzamido-3-(4-(benzyloxy)
phenyl)propanoate (4b).
White crystal, 66%, mp: 157.4–
159.3 °C. 1H NMR (DMSO-d6, d): 8.727 (d, 1H, N–H, J = 8.0 Hz),
7.723 (d, 2H, Ar-H, J = 8.4 Hz), 7.429–7.203 (m, 9H, Ar-H), 6.913
(d, 2H, Ar-H, J = 8.8 Hz), 5.040 (s, 2H, CH2O), 4.619–4.562 (m, 1H,
CH), 3.630 (s, 3H, CH3O), 3.112–2.994 (m, 2H, CH2), 2.352 (s, 3H,
CH3). ESI-MS: 402.2 (M-1).
4.1.3.10. (S)-Methyl 2-(4-methylbenzene-1-sulfonyl amido)-3-
(4-(phenylethyloxy)phenyl)propanoate (4j).
White crystal,
61%, mp: 89.0–91.4 °C. 1H NMR (CDCl3, d): 7.622 (d, 2H, Ar-H,
J = 8.4 Hz), 7.265 (d, 2H, Ar-H, J = 7.6 Hz), 7.203 (q, 6H, Ar-H and
N–H, J = 4.4 Hz), 6.951 (d, 2H, Ar-H, J = 8.4 Hz), 6.736 (d, 2H, Ar-
H, J = 8.4 Hz), 4.125 (m, 1H, C–H), 3.896 (t, 2H, CH2, J = 6.4 Hz),
3.467 (s, 3H, CH3O), 2.945 (m, 2H, CH2), 2.788 (t, 2H, CH2,
J = 7.6 Hz), 2.366 (s, 3H, Ar-CH3). ESI-MS: 452.2(M-1).
4.1.3.3. (S)-Methyl 2-acetamido-3-(4-(benzyloxy)phenyl)pro-
panoate (4c).
Yellow crystal, 55.7%, mp: 133.6–134.7 °C. 1H
NMR (DMSO-d6, d): 8.275 (d, 1H, NH, J = 8.0 Hz), 7.363–7.442 (m,
2H, Ar-H), 7.300–7.336 (t, 3H, Ar-H, J = 7.2 Hz), 7.130–7.109 (d,
2H, Ar-H, J = 8.4 Hz), 6.927–6.906 (d, 2H, Ar-H, J = 8.4 Hz), 5.060
(s, 2H, OCH2), 4.358–4.414 (m, 1H, CH), 3.580 (s, 3H, OCH3),
2.768–2.951 (m, 2H, CH2), 1.787 (s, 3H, COCH3). ESI-MS:
328.2(M+1).
4.1.4. (S)-3-(4-(Substituted oxy)phenyl)-2-(substituted amido)
propanoic acid (5)
Compound 4 (2.75 mmol) was dissolved in 14 ml methanol and
28 ml NaOH (1 mol/L) was added. The solution was stirred at room
temperature for 4 h and TLC was used to monotor the completion.
The solvent was evaporated to give yellow solid. The yellow solid
was dissolved in 50 ml water and 1 mol/L HCl was added to pH
2-3. The yellow solid was precipitated, which was washed by water
to give 5.
4.1.3.4. (S)-Methyl 2-methanesulfonyl amido-3-(4-(benzyloxy)
phenyl)propanoate (4d).
Yellow crystal, 80.7%, mp: 122.3–
124.9 °C. 1H NMR (DMSO-d6, d): 7.811 (d, 1H, NH, J = 8.8 Hz),
7.441–7.302 (m, 5H, Ar-H), 7.185 (d, 2H, Ar-H, J = 8.1 Hz), 6.943
(d, 2H, J = 7.9 Hz, Ar-H), 5.083 (s, 2H, CH2), 4.096 (q, 1H,
J = 8.7 Hz, CH), 3.628 (s, 3H, CH3O), 2.975–2.748 (m, 2H, CH2),
2.564 (s, 3H, CH3). ESI-MS: 749.4 (2 M+Na).
4.1.4.1.
acid (5a).
(S)-3-(4-(Benzyloxy)phenyl)-2-(benzamido)propanoic
Yellow crystal, 90.7%, mp: 163.9–165.4 °C. 1H
4.1.3.5. (S)-Methyl 2-(4-methylbenzene-1-sulfonyl amido)-3-(4-
NMR (DMSO-d6, d): 12.712 (s, 1H, OH), 8.649–8.669 (d, 1H, NH,
J = 8.0 Hz), 7.797–7.815 (d, 2H, Ar-H, J = 7.2 Hz), 7.228–7.529 (m,
10H, Ar-H), 6.903–6.922 (d, 2H, Ar-H, J = 7.6 Hz), 5.034 (s, 2H,
OCH2), 4.574–4.631 (m, 1H, CH), 2.979–3.145 (m, 2H, CH2). ESI-
MS: 750.5 (2M-1).
(benzyloxy)phenyl)propanoate (4e).
White crystal, 55%, mp:
143.5–146.6 °C. 1H NMR (DMSO-d6, d): 8.350 (s, 1H, N–H), 7.449–
7.380 (m, 6H, Ar-H), 7.354–7.332 (m, 1H, Ar-H), 7.260 (d, 2H, Ar-
H, J = 8.4 Hz), 7.011 (d, 2H, Ar-H, J = 8.4 Hz), 6.840 (d, 2H, Ar-H,
J = 8.4 Hz), 5.058 (s, 2H, CH2O), 3.898–3.838 (m, 1H, CH), 3.333
(s, 3H, CH3O), 2.831 (q, 1H, CH2, J = 6.8 Hz), 2.680 (q, 1H, CH2,
J = 6.8 Hz), 2.348 (s, 3H, ArCH3). ESI-MS: 438.2 (M-1).
4.1.4.2.
propanoic acid (5b).
(S)-3-(4-(Benzyloxy)phenyl)-2-(p-methylbenzamido)
Yellow crystal, 90.6%, mp: 141.2–
4.1.3.6. (S)-Methyl 2-benzamido-3-(4-(phenylethyloxy)phenyl)
143.1 °C. 1H NMR (DMSO-d6, d): 8.562 (d, 1H, N–H, J = 8.0 Hz),
7.718 (d, 2H, Ar-H, J = J = 8.0 Hz), 7.428–7.215 (m, 9H, Ar-H),
6.905 (d, 2H, Ar-H, J = 8.4 Hz), 5.031 (s, 2H, CH2O), 4.561–4.515
(m, 1H, CH), 3.098–3.004 (m, 2H, CH2), 2.350 (s, 3H, CH3). ESI-
MS: 388.1 (M-1).
propanoate (4f).
White crystal, 60.3%, mp: 130.1–131.7 °C.
1H NMR (DMSO-d6, d): 8.810–8.830 (d, 1H, NH, J = 8.0 Hz),
7.944–7.963 (d, 2H, Ar-H, J = 7.8 Hz), 7.800–7.963 (d, 2H, Ar-H),
7.799–7.817 (d, 2H, Ar-H, J = 7.6 Hz), 7.438–7.542 (m, 4H, Ar-H),
7.171–7.268 (m, 4H, Ar-H), 6.823–6.845 (d, 2H, Ar-H, J = 8.8 Hz),
5.047–5.103 (m, 1H, CH), 3.889–3.920 (t, 2H, OCH2, J = 6.4 Hz),
3.642 (s, 3H, CH3), 3.030–3.179 (m, 2H, CH2), 2.691–2.730 (t, 2H,
CH2, J = 8.0 Hz). ESI-MS: 402.3(M-1).
4.1.4.3.
acid (5c).
(S)-3-(4-(Benzyloxy)phenyl)-2-(acetamido)propanoic
White crystal, 81.2%, mp: 183.4–185.2 °C. 1H NMR
(DMSO-d6, d): 12.712 (s, 1H, OH), 8.124–8.143 (d, 1H, NH,
J = 7.6 Hz), 7.328–7.452 (m, 5H, Ar-H), 7.136–7.154 (d, 2H, Ar-H,
J = 7.2 Hz), 6.912–6.931 (d, 2H, Ar-H, J = 7.6 Hz), 5.063 (s, 2H,
OCH2), 4.350-4.362 (m, 1H, CH), 2.745–2.993 (m, 2H, CH2), 1.789
(s, 3H, CH3). ESI-MS: 625.4 (2M-1).
4.1.3.7. (S)-Methyl 2-(p-methylbenzamido)-3-(4-(phenylethyl-
oxy)phenyl)propanoate (4g).
White crystal, 83.4%, mp:
111.5–113.8 °C. 1H NMR (DMSO-d6, d): 8.701 (d, 1H, NH,
J = 8.0 Hz), 7.721 (d, 2H, Ar-H, J = 7.6 Hz), 7.273–7.184 (m, 9H,

X.-C. Cheng et al. / Bioorg. Med. Chem. 20 (2012) 5738–5744
5743
4.1.4.4. (S)-3-(4-(Benzyloxy)phenyl)-2-(methanesulfonyl amido)
4.1.5.1.
propanamide (6a).
(S)-N-Hydroxy-3-(4-benzyloxyphenyl)-2-(benzamido)
Yellow crystal, 30%, mp:167.2–168.8 °C.
propanoic acid (5d).
White crystal, 94.4%, mp: 146.3–
147.0 °C. 1H NMR (DMSO-d6, d): 12.890 (s, 1H, OH), 7.628 (d, 1H,
NH, J = 8.8 Hz), 7.448–7.321 (m, 5H, Ar-H), 7.200 (d, 2H, Ar-H,
J = 8.8 Hz), 6.943 (d, 2H, Ar-H, J = 8.8 Hz), 5.080 (s, 2H, CH2),
3.969 (m, 1H, CH), 3.000–2.706 (m, 2H, CH2), 2.539 (s, 3H, CH3).
ESI-MS: 697.5 (2 M-1), 721.3 (2 M+Na).
IR (KBr, cmꢀ1): 3294.50 (OH and NH), 2919.85 (CH), 1658.49
(C@O), 1176.39 (C–O). 1H NMR (DMSO-d6, d): 10.80 (s, 1H, OH),
8.91 (s, 1H, ONH), 8.63 (d, 1H, NH, J = 8.40 Hz), 7.81 (d, 2H, Ar-H,
J = 7.20 Hz), 7.50 (d, 1H, Ar-H, J = 7.20 Hz), 7.46–7.31 (m, 7H, Ar-
H), 7.24 (d, 2H, Ar-H, J = 8.40 Hz), 6.90 (d, 2H, Ar-H, J = 8.40 Hz),
5.02 (s, 2H, OCH2), 4.54 (m, 1H, CH), 3.00–2.80 (m, 2H, CH2). 13C
NMR (DMSO-d6, d): 170.2 (C@O), 168.7 (C@O), 154.5 (C aro),
137.8 (C aro), 135.4 (C aro), 131.3 (C aro), 129.5 (4 ꢁ C aro),
128.3 (4 ꢁ C aro), 126.4 (4 ꢁ C aro), 112.7 (2 ꢁ C aro), 72.6 (CH2),
55.6 (CH), 35.2 (CH2). ESI-MS: 391.1 (M+1), 413.0 (M+Na), 389.3
(M-1).
4.1.4.5. (S)-3-(4-(Benzyloxy)phenyl)-2-(4-methylbenzene-1-sul-
fonyl amido)propanoic acid (5e).
White crystal, 71.4%, mp:
96.0–98.3 °C. 1H NMR (DMSO-d6, d): 7.650 (s, 1H, N–H), 7.460–
7.333 (m, 7H, Ar-H), 7.241 (d, 2H, Ar-H, J = 8.0 Hz), 7.023 (d, 2H,
Ar-H, J = 8.4 Hz), 6.813 (d, 2H, Ar-H, J = 8.8 Hz), 5.043 (s, 2H,
CH2O), 3.593 (t, 1H, CH, J = 5.6 Hz), 3.342 (s, 1H, OH), 2.853-2.699
(m, 2H, CH2), 2.334 (s, 3H, CH3). ESI-MS: 424.1 (M-1).
4.1.5.2. (S)-N-Hydroxy-3-(4-benzyloxyphenyl)-2-(p-methylben-
zamido)propanamide (6b).
Yellow crystal, 22.5%, mp:
4.1.4.6. (S)-3-(4-(Phenylethyloxy)phenyl)-2-(benzamido)propa-
noic acid (5f).
126.5–128.0 °C. IR (KBr, cmꢀ1): 3422.46 (OH and NH), 2921.71
(CH), 1632.60 (C@O), 1177.08 (C–O). 1H NMR (DMSO-d6, d):
10.769 (s, 1H, NHOH), 8.889 (s, 1H, NHOH), 8.509 (d, 1H, CONH,
J = 8.0 Hz), 7.730 (d, 2H, Ar-H, J = 7.2 Hz), 7.428–7.231 (m, 9H, Ar-
H), 6.900 (d, 2H, Ar-H, J = 8.4 Hz), 5.042 (s, 2H, CH2O), 4.530 (d,
1H, CH, J = 5.6 Hz), 2.944 (s, 2H, CH2), 2.344 (s, 3H, CH3). 13C
NMR (DMSO-d6, d): 168.9 (C@O), 167.2 (C@O), 155.8 (C aro),
142.1 (C aro), 135.6 (C aro), 132.1 (C aro), 129.5 (4 ꢁ C aro),
128.3 (3 ꢁ C aro), 126.7 (5 ꢁ C aro), 114.8 (2 ꢁ C aro), 71.2 (CH2),
55.1 (CH), 37.5 (CH2), 22.3 (CH3). ESI-MS: 403.2 (M-1).
Yellow crystal, 84.2%, mp: 181.5–183.3 °C. 1H
NMR (DMSO-d6, d): 8.315–8.333 (d, 1H, NH, J = 7.2 Hz), 7.755 (s,
2H, Ar-H), 6.872–7.755 (m, 12H, Ar-H), 7.170–7.440 (m, 10H, Ar-
H), 6.767–6.894 (m, 2H, Ar-H), 4.419–4.429 (s, 1H, CH), 3.886 (s,
2H, OCH2), 3.003–3.128 (m, 2H, CH2), 2.706 (m, 2H, CH2). ESI-MS:
388.2 (M-1).
4.1.4.7. (S)-3-(4-(Phenylethyloxy)phenyl)-2-(p-methylbenzami-
do)propanoic acid (5g).
White crystal, 91.7%, mp: 96.2–
98.9 °C. 1H NMR (DMSO-d6, d): 8.116 (s, 1H, NH), 7.646 (d, 2H,
Ar-H, J = 7.6 Hz), 7.289–7.104 (m, 9H, Ar-H), 6.756 (d, 2H, Ar-H,
J = 8.0 Hz), 4.321 (d, 1H, CH, J = 4.8 Hz), 3.886 (t, 2H, CH2,
J = 6.4 Hz), 3.140–2.986 (m, 2H, CH2), 2.708 (t, 2H, CH2,
J = 7.6 Hz), 2.340 (s, 3H, CH3). ESI-MS: 404.2 (M+1).
4.1.5.3.
propanamide (6c).
(S)-N-Hydroxy-3-(4-benzyloxyphenyl)-2-(acetamido)
Yellow crystal, 19.8%, mp: 169.2–
170.9 °C. IR (KBr, cmꢀ1): 3314.27 (OH and NH), 3046.70 (CH),
1636.15 (C@O), 1181.77 (C–O). 1H NMR (DMSO-d6, d): 10.630 (s,
1H, OH), 8.816 (s, 1H, NH–O), 8.123-8.144 (d, 1H, NHCO,
J = 8.4 Hz), 7329–7.452 (m, 5H, Ar-H), 7.124–7.143 (d, 2H, Ar-H,
J = 7.6 Hz), 6.898–6.917 (d, 2H, Ar-H, J = 7.6 Hz), 5.056 (s, 2H,
OCH2), 4.305–4.325 (m, 1H, CH), 2.658–2.863 (m, 2H, CH2), 1.757
(s, 3H, COCH3). 13C NMR (DMSO-d6, d): 170.4 (C@O), 168.7
(C@O), 156.9 (C aro), 136.1 (C aro), 129.1 (2 ꢁ C aro), 128.6
(3 ꢁ C aro), 126.9 (3 ꢁ C aro), 114.5 (2 ꢁ C aro), 71.1 (CH2), 54.5
(CH), 37.2 (CH2), 22.6 (CH3). ESI-MS: 679.6 (2 M+Na).
4.1.4.8. (S)-3-(4-(Phenylethyloxy)phenyl)-2-(acetamido)propa-
noic acid (5h).
White crystal, 78.3%, mp: 172.8–174.3 °C. 1H
NMR (DMSO-d6, d): 12.585–12.598 (d, 1H, OH, J = 7.2 Hz), 8.093–
8.111 (d, 1H, NH, J = 7.6 Hz), 7.107–7.282 (m, 7H, Ar-H), 6.817–
6.835 (d, 2H, Ar-H, J = 7.2 Hz), 4.328 (s, 1H, CH), 3.923 (s, 2H,
CH2), 2.711–2.784 (m, 3H, CH3), 1.994–2.010 (m, 2H, CH2), 1.780
(m, 4H, CH2). ESI-MS: 326.5 (M-1).
4.1.4.9. (S)-3-(4-(Phenylethyloxy)phenyl)-2-(methanesulfonyl
4.1.5.4. (S)-N-Hydroxy-3-(4-benzyloxyphenyl)-2-(methanesulfo-
amido)propanoic acid (5i).
White crystal, 93.9%, mp:113.4–
nyl amido)propanamide (6d).
White crystal, 23.2%, mp:
115.3 °C. 1H NMR (DMSO-d6, d): 12.867 (s, 1H, OH), 7.615 (d, 1H,
NH, J = 8.8 Hz), 7.287–7.176 (m, 7H, Ar-H), 6.859 (d, 2H, Ar-H,
J = 8.4 Hz), 4.004–3.918 (m, 3H, CH+CH2), 2.996–2.713 (m, 4H,
CH2CH2), 2.573 (s, 3H, CH3). ESI-MS: 362.1 (M-1).
153.3–156.0 °C. IR (KBr, cmꢀ1): 3423.89 (OH and NH), 2929.82
(CH), 1627.91 (C@O), 1150.38 (C–O). 1H NMR (DMSO-d6, d):
7.427–7.322 (m, 6H, Ar-H+NH), 7.174 (d, 2H, Ar-H, J = 8.4 Hz),
6.939 (d, 2H, Ar-H, J = 8.4 Hz), 5.076 (s, 2H, CH2), 3.800 (q, 1H,
CH, J = 8.4 Hz), 3.340 (s, 1H, CH3), 2.843–2.676 (m, 2H, CH2). 13C
NMR (DMSO-d6, d): 168.9 (C@O), 156.4 (C aro), 136.5 (C aro),
129.4 (2 ꢁ C aro), 128.4 (3 ꢁ C aro), 127.2 (3 ꢁ C aro), 114.1
(2 ꢁ C aro), 71.5 (CH2), 54.8 (CH), 44.1 (CH3), 35.2 (CH2). ESI-MS:
363.1 (M-1).
4.1.4.10.
(S)-3-(4-(phenylethyloxy)phenyl)-2-(4-methylben-
Yellow crystal,
zene-1-sulfonyl amido)propanoic acid (5j).
49.5%, mp: 126.5–128.5 °C. 1H NMR (DMSO-d6, d): 12.780 (s, 1H,
O–H), 8.361 (d, 1H, N–H, J = 8.8 Hz), 8.000 (s, 1H, Ar-H), 7.459 (t,
2H, Ar-H, J = 8.0 Hz), 7.440–7.173 (m, 6H, Ar-H), 6.999 (t, 2H, Ar-
H, J = 8.4 Hz), 6.740 (t, 2H, Ar-H, J = 8.0 Hz), 3.917 (m, 1H, C–H),
3.321 (m, 2H, CH2), 2.853–2.650 (m, 4H, CH2CH2), 2.324 (s, 3H,
CH3). ESI-MS: 440.1 (M+1).
4.1.5.5. (S)-N-Hydroxy-3-(4-benzyloxyphenyl)-2-(4-methylben-
zene-1-sulfonyl amido)propanamide (6e).
White crystal,
21.6%, mp: 170.7–173.4 °C. IR (KBr, cmꢀ1): 3356.87 (OH),
3261.25 (NH), 2922.13 (CH), 1609.44 (C@O), 1156.73 (C–O). 1H
NMR (DMSO-d6, d): 7.770 (d, 1H, SO2NH, J = 8.0 Hz), 7.500–7.317
(m, 7H, Ar-H), 7.201 (d, 2H, Ar-H, J = 8.4 Hz), 6.943 (d, 2H, Ar-H,
J = 8.4 Hz), 6.794 (t, 2H, Ar-H, J = 8.0 Hz), 5.041 (s, 2H, CH2O),
3.700 (m, 1H, OH), 3.175–2.600 (m, 2H, CH2), 2.357 (t, 3H, CH3,
J = 8.0 Hz). 13C NMR (DMSO-d6, d): 168.5 (C@O), 156.1 (C aro),
141.9 (C aro), 137.2 (C aro), 136.3 (C aro), 129.1 (4 ꢁ C aro),
128.3 (5 ꢁ C aro), 127.8 (3 ꢁ C aro), 114.4 (2 ꢁ C aro), 71.1 (CH2),
54.4 (CH), 35.9 (CH2), 21.8 (CH3). MS m/z: 439.2 (M-1).
4.1.5. (S)-N-hydroxy-3-(4-substituted oxyphenyl)-2-(substituted
amino)propanamide (6)
To a solution of compound 5 (2 mmol) in methanol (7 ml) at
room temperature, was added dropwise a solution of NH2OK
(6 mmol) in methanol (3.4 ml). The mixture was stirred at room
temperature for 24 h and the solvent was evaporated in vacuo.
The residue was purified by flash column chromatography (ethyl
acetate:n-hexane = 1:1) to give 6.

5744
X.-C. Cheng et al. / Bioorg. Med. Chem. 20 (2012) 5738–5744
4.1.5.6.
(S)-N-Hydroxy-3-(4-phenylethyloxyphenyl)-2-(benz-
4.2. Biological evaluation
amido)propanamide (6f).
Yellow crystal, 17.9%, mp: 168.5–
170.2 °C. IR (KBr, cmꢀ1): 3271.53 (OH and NH), 2922.55 (CH),
1633.33 (C@O), 1177.90 (C–O). 1H NMR (DMSO-d6, d): 10.783 (s,
1H, OH), 8.892 (s, 1H, CONH), 8.590–8.611 (d, 1H, Ar-CONH,
J = 8.4 Hz), 7.802–7.821 (d, 2H, Ar-H, J = 7.6 Hz), 7.170–7.513 (m,
10H, Ar-H), 6.809–6.829 (d, 2H, Ar-H, J = 8.0 Hz), 4.531–4.546 (t,
1H, CH, J = 6.0 Hz), 3.883–3.914 (t, 2H, OCH2), 2.943 (t, 2H, CH2),
2.689–2.727 (m, 2H, CH2), 1.970 (t, 2H, CH2). 13C NMR (DMSO-d6,
d): 169.5 (C@O), 167.9 (C@O), 156.2 (C aro), 138.5 (C aro), 134.3
(C aro), 132.3 (C aro), 129.5 (2 ꢁ C aro), 128.6 (5 ꢁ C aro), 127.8
(4 ꢁ C aro), 125.5 (C aro), 114.2 (2 ꢁ C aro), 67.1 (CH2), 54.2 (CH),
37.9 (CH2), 35.3 (CH2). ESI-MS: 403.3 (M-1).
4.2.1. MMP-2 inhibition assay
The L-tyrosine derivatives were assayed for the inhibitory activ-
ities against MMP-2 using MMP-2 Colorimetric Drug Discovery Kit.
The assay buffer was pipetted into each desired well of the micro-
plate and equilibrated to 37 °C. 20
trol, inhibitor NNGH, and test inhibitor wells. Then 20
inhibitor was added to the inhibitor NNGH wells only, and desired
volume of test inhibitor was added to appropriate wells. The plate
was incubated for 30 min at 37 °C to allow inhibitor/enzyme inter-
l
l MMP-2 was added to the con-
ll NNGH
action. Then 10 ll BML-P125-9090 substrate was added to start
reaction. Continuously read plates at A412 nm in the microplate
reader and record data at 1 min time intervals for 10 min. Data
analysis was performed using OriginPro 7.5 software and the IC50
values were obtained.
4.1.5.7. (S)-N-Hydroxy-3-(4-phenylethyloxyphenyl)-2-(p-meth-
ylbenzamido)propanamide (6g).
White crystal, 51.2%, mp:
149.9–152.2 °C. IR (KBr, cmꢀ1): 3291.14 (OH and NH), 2921.45
(CH), 1629.84 (C@O), 1177.63 (C–O). 1H NMR (DMSO-d6, d):
10.739 (s, 1H, OH), 8.863 (s, 1H, NHOH), 8.479 (d, 1H, CONH,
J = 8.4 Hz), 7.725 (d, 2H, Ar-H, J = 8.0 Hz), 7.286–7.152 (m, 9H, Ar-
H), 6.810 (d, 2H, Ar-H, J = 8.0 Hz), 4.528 (d, 1H, CH, J = 6.0 Hz),
3.898 (t, 2H, CH2, J = 6.4 Hz), 2.947 (d, 2H, CH2, J = 10.0 Hz), 2.708
(t, 2H, CH2, J = 7.2 Hz), 2.342 (s, 3H, CH3). 13C NMR (DMSO-d6, d):
169.4 (C@O), 167.6 (C@O), 156.6 (C aro), 141.3 (C aro), 138.3 (C
aro), 131.3 (C aro), 129.3 (4 ꢁ C aro), 128.5 (3 ꢁ C aro), 127.4
(4 ꢁ C aro), 125.3 (C aro), 114.3 (2 ꢁ C aro), 67.6 (CH2), 54.1 (CH),
37.3 (CH2), 35.2 (CH2), 21.2 (CH2). ESI-MS: 417.3 (M-1).
4.2.2. HDAC-8 inhibition assay
The L-tyrosine derivatives were further assayed for the inhibitory
activities against HDACs using HDAC-8 Fluorimetric Drug Discovery
Kit. Firstly, HDAC-8 assay buffer, diluted SAHA or test inhibitors
were added to appropriate wells of the microplate. Then substrate
solution and the HDAC-8 assay buffer were warmed for diluting
the enzyme to assay temperature. The chilled and undiluted
HDAC-8 was added to the warmed buffer, then the diluted HDAC-
8 was added to all wells except those that are to be ‘No Enzyme Con-
trols’. By adding diluted substrate (25 ll) to each well and mixing
thoroughly, HDAC-8 reactions were initiated and proceeded for de-
4.1.5.8. (S)-N-Hydroxy-3-(4-phenylethyloxyphenyl)-2-(acetam-
sired length of time, which were then stopped by addition of Fluor
ido)propanamide (6h).
Yellow crystal, 20.5%, mp: 144.6–
de LysÒ Developer II (50
ll). The plate was incubated at room tem-
146.1 °C. IR (KBr, cmꢀ1): 3294.83 (OH and NH), 2936.75 (CH),
1635.42 (C@O), 1162.83 (C–O). 1H NMR (DMSO-d6, d): 10.660 (s,
1H, OH), 8.852 (s, 1H, CONH), 8.148–8.168 (d, 1H, CONH,
J = 8.0 Hz), 7.112–7.285 (m, 7H, Ar-H), 6.806–6.825 (d, 2H, Ar-H,
J = 7.6 Hz), 4.311-4.347 (m, 1H, CH), 3.903–3.918 (m, 2H, OCH2),
3.177 (m, 2H, CH2), 2.658–2.747 (m, 2H, CH2). 13C NMR (DMSO-
d6, d): 170.2 (C@O), 169.2 (C@O), 156.2 (C aro), 138.3 (C aro),
129.4 (2 ꢁ C aro), 128.4 (3 ꢁ C aro), 127.2 (2 ꢁ C aro), 125.5 (C
aro), 114.4 (2 ꢁ C aro), 67.4 (CH2), 54.0 (CH), 37.7 (CH2), 35.3
(CH2), 22.6 (CH3). ESI-MS: 341.2 (M-1).
perature (30 °C) for at least 45 min. Signal was stable for at least
60 min. Finally, the samples were read in a microplate reading fluo-
rimeter capable of excitation at a wavelength in the range 350–
380 nm and detection of emitted light in the range 440–460 nm.
The IC50 values were obtained using OriginPro 7.5 software.
Acknowledgments
This work was supported by National Natural Science Founda-
tion of China (21202120), The Funded Project for Science & Tech-
nology Development of Tianjin Higher Education (20100101),
China Postdoctoral Science Foundation funded project
(20100480655), Tianjin Medical University New Century Talent
Foundation, and Tianjin Medical University Undergraduate Re-
search Project (ZR05).
4.1.5.9.
(S)-N-Hydroxy-3-(4-phenylethyloxyphenyl)-2-(meth-
Yellow crystal,
anesulfonyl amido)propanamide (6i).
26.4%, mp: 137.1–139.9 °C. IR (KBr, cmꢀ1): 3440.47 (OH and NH),
2930.63 (CH), 1583.57 (C@O), 1149.06 (C–O). 1H NMR (DMSO-d6,
d): 7.287–7.132 (m, 8H, Ar-H and SO2N–H), 6.798 (d, 2H, Ar-H,
J = 8.0 Hz), 3.911 (t, 2H, CH2, J = 6.4 Hz), 3.707 (s, 1H, CH), 2.916
(s, 1H, CH2), 2.749–2.687 (m, 6H, CH2+CH3). 13C NMR (DMSO-d6,
d): 170.0 (C@O), 156.5 (C aro), 138.1 (C aro), 129.4 (2 ꢁ C aro),
128.3 (3 ꢁ C aro), 127.1 (2 ꢁ C aro), 125.6 (C aro), 114.7 (2 ꢁ C
aro), 67.6 (CH2), 55.1 (CH), 43.8 (CH3), 35.9 (CH2), 35.1 (CH2).
ESI-MS: 377.2 (M-1).
References and notes
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