Histone deacetylase inhibitors with enhanced enzymatic inhibition effects and potent in vitro and in vivo antitumor activities

Article abstract of DOI:10.1002/cmdc.201300297

In the present work, a series of small molecules were designed and synthesized based on structural optimization. A significant improvement in the enzyme inhibitory activity of these compounds was discovered. Moreover, the tested compounds have moderate preference for classa I HDACs over HDAC6, as demonstrated by enzyme selectivity assays. In vitro antiproliferation assay results show that representative compounds can selectively inhibit the growth of non-solid lymphoma and leukemic cells such as U937, K562, and HL60. In the in vivo antitumor assay, (S)-4-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2- phenylacetamido)-N-hydroxybenzamide (D17) showed better performance than SAHA in blocking U937 tumor growth. Western blot analysis revealed that representative molecules can block the function of both class I HDACs and HDAC6. More importantly, our western blot results reveal that the levels of some oncogenic proteins (p-Akt in the PI3K/AKT/mTOR signal pathway, c-Raf and p-Erk in the MAPK signal pathway) were dramatically down-regulated by our compounds in the U937 cell line rather than MDA-MB-231 cells. This distinction in cellular mechanism might be an important reason why the U937 cell line was found to more sensitive to our HDAC inhibitors than the MDA-MB-231 cell line.

Full text of DOI:10.1002/cmdc.201300297

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DOI: 10.1002/cmdc.201300297
Histone Deacetylase Inhibitors with Enhanced Enzymatic
Inhibition Effects and Potent in vitro and in vivo
Antitumor Activities
Lei Zhang,*[a] Yingjie Zhang,[b] C. James Chou,[c] Elizabeth S. Inks,[c] Xuejian Wang,[d]
Xiaoguang Li,[b] Jinning Hou,[b] and Wenfang Xu*[b]
In the present work, a series of small molecules were designed
and synthesized based on structural optimization. A significant
improvement in the enzyme inhibitory activity of these com-
pounds was discovered. Moreover, the tested compounds
have moderate preference for class I HDACs over HDAC6, as
demonstrated by enzyme selectivity assays. In vitro antiprolif-
eration assay results show that representative compounds can
selectively inhibit the growth of non-solid lymphoma and leu-
kemic cells such as U937, K562, and HL60. In the in vivo antitu-
mor assay, (S)-4-(2-(5-(dimethylamino)naphthalene-1-sulfonami-
do)-2-phenylacetamido)-N-hydroxybenzamide (D17) showed
better performance than SAHA in blocking U937 tumor
growth. Western blot analysis revealed that representative mol-
ecules can block the function of both class I HDACs and
HDAC6. More importantly, our western blot results reveal that
the levels of some oncogenic proteins (p-Akt in the PI3K/AKT/
mTOR signal pathway, c-Raf and p-Erk in the MAPK signal path-
way) were dramatically down-regulated by our compounds in
the U937 cell line rather than MDA-MB-231 cells. This distinc-
tion in cellular mechanism might be an important reason why
the U937 cell line was found to more sensitive to our HDAC in-
hibitors than the MDA-MB-231 cell line.
Introduction
Histone deacetylases (HDACs) are a class of enzymes responsi-
ble for removing acetyl groups from the e-N-acetyl group of
histone lysine residues. To date, 18 HDACs have been discov-
ered, and they are divided into four classes.[1] Class I HDACs in-
clude HDAC 1, 2, 3 and 8; within this class, HDAC 1, 2 and 8
are primarily found in the nucleus, while HDAC 3 is found in
both the nucleus and cytoplasm. Class II HDACs (IIa: HDAC 4,
5, 7 and 9; IIb: HDAC 6 and 10) are able to shuttle in and out
of the nucleus. Members of classes I and II are also called “clas-
sical zinc-dependent” HDACs. Class III HDACs (known as sir-
tuins, Sirt1–7) are a set of NAD-dependent enzymes. The clas-
s IV HDAC (HDAC 11) also exists in both the nucleus and cyto-
plasm but is considered as an atypical isoform and thus placed
in a category of its own based on low sequence similarity to
the others.
Acetylation and deacetylation of histones and non-histone
proteins by histone acetylases (HATs) and HDACs play impor-
tant roles in the regulation of gene expression and transcrip-
tion.[2] The effects include signal transduction, protein phos-
phorylation, cell-cycle regulation, proliferation, apoptosis, car-
diac development, and so on.[3] Overexpression and aberrant
recruitment of HDACs have significant roles in tumorigenesis.[4]
Histone deacetylase inhibitors (HDACIs) have been proven
to alter gene expression by blocking the action of HDACs and
inducing hyperacetylation of histones.[5] A number of structur-
ally diverse HDACIs have shown potent antitumor efficacy in
various stages of clinical trials. Suberoyl anilide hydroxamic
acid (SAHA)[6] and FK228[7] have been approved by the US
Food and Drug Administration (FDA) for the treatment of ad-
vanced cutaneous T-cell lymphoma (CTCL).
[a] Dr. L. Zhang
Department of Pharmacy, School of Medicine, Qingdao University
308 Dengzhou Road, Qingdao, Shandong 266071 (China)
E-mail: lei@qdu.edu.cn
[b] Dr. Y. Zhang, X. Li, J. Hou, Prof. W. Xu
Department of Medicinal Chemistry
In order to develop novel HDACIs as antitumor agents,
a series of small molecules was designed and synthesized
based on virtual screening results.[8] A phenylglycine-contain-
ing structure (4h) was discovered to have good performance
in the enzyme inhibition and tumor cell growth inhibition
assays. Structural modifications were performed in the present
work in discovery of more potent molecules (Scheme 1). To im-
prove the activities of synthesized compounds, aromatic
groups introduced to substitute with the tert-butyloxycarbonyl
(BOC) group of 4h were designed to interact with hydropho-
School of Pharmacy, Shandong University
44 Wenhua Xi Road, Jinan, Shandong 250012 (China)
E-mail: xuwenf@sdu.edu.cn
[c] Prof. C. J. Chou, Dr. E. S. Inks
Department of Drug Discovery & Biomedical Sciences
South Carolina College of Pharmacy, Medical University of South Carolina
280 Calhoun Street, MSC 141, Charleston, SC 29425 (USA)
[d] Dr. X. Wang
School of Pharmacy & Biology Science, Weifang Medicinal University
288 Shengli E Street, Kuiwen, Weifang, Shandong 261042 (China)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300297.
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of the derived molecules were observed, indicating that aro-
matic R groups enhance the inhibitor–receptor interactions by
binding to hydrophobic residues (Phe155, Phe210 and Tyr308;
Table 1). Carbamido-group-containing molecule D18 per-
formed the best in the enzymatic inhibition assay, exhibiting
an IC50 value of 7.5 nm. Molecules with larger aromatic groups
(e.g., naphthalene ring) have potent inhibitory activities: D3
(14.3 nm), D5 (15.6 nm), D16 (23.5 nm) and D17 (9.7 nm). Chlor-
ine substitution of the phenyl ring apparently decreases the
activity as compared with D7, such as D1 (348 nm), D2
(95.5 nm) and D10 (125 nm). However, bromine substitution of
the phenyl group improves the activity (D13: 13.0 nm). Hydro-
phobic groups such as tert-butyl and strongly electrophilic
groups such as trifluoromethyl have a negative effect on the
inhibitory activity (D6: 105 nm, D8: 85.5 nm; D18: 7.5 nm, D19:
47.7 nm). With respect to fluorine and methoxy substituents,
the para position gives rise to more potent derivatives than
the ortho position (D4 vs. D9; D11 vs. D12), although the dif-
ference is not significant. The position of substitution also
plays an important role in the activity of naphthalene-sulfonyl-
containing derivatives; compounds substituted at the 1-posi-
tion (D5) are more potent than those substituted at the 2-posi-
tion (D16). Furthermore, a polar group on the naphthalene
ring improves the enzymatic inhibitory activity of the deriva-
tive (D17). From the above findings, we conclude that larger
aromatic R groups are beneficial to the HDAC inhibitory activi-
ty of the compounds. In terms of the substituents on the aro-
matic ring, polar groups (not strong electrophilic groups) are
favored, and the positions of the substituent groups also affect
the activity.
Scheme 1. Design of new molecules based on the structure of 4h. X group
is CO, SO2 or CONH; R group is substituted aromatic rings.
bic residues (Phe155, Phe210 and Tyr308) in the active site of
HDAC2 (Figure 1).
Results and Discussion
Chemistry
Based on lead structure 4h, a series of phenylglycine-contain-
ing hydroximic acid derivatives were synthesized according to
the procedures described in Scheme 2. The starting material
(S)-2-amino-2-phenylacetic acid was protected by a Boc group;
the other material, 4-aminobenzoic acid, was protected by
a methyl ester. Intermediate (S)-methyl 4-(2-((tert-butoxycarbo-
nyl)amino)-2-phenylacetamido)benzoate (a) was derived by
coupling these two products. Subsequent N-deprotection of
intermediate a and condensation with various acyl chlorides,
sulfonyl chlorides, and aryl amines afforded c1–c19. Target
compounds D1–D19 were derived by treating the correspond-
ing intermediates (c1–c19) with potassium hydroxylamine in
methanol.
To characterize the isoform selectivity of these molecules,
representative compounds D3, D5, D13, D17 and D18 were
tested against HDAC1, HDAC2, HDAC3 and HDAC6 (Table 2).
The result show that these four molecules display preference
for HDAC1, HDAC2 and HDAC3 over HDAC6. As to class I
HDACs, the tested molecules show modest selectivity for
HDAC1 and HDAC3 over HDAC2. D18 exhibits the most potent
activity in the selectivity assay, with IC50 values of 4.8 nm,
Biological evaluation
An enzymatic inhibition assay was performed using HeLa cell
nucleus extract containing a mixture of HDAC isoforms. Com-
pared with 4h, significant improvements in inhibitory activities
Scheme 2. Synthesis of compounds D1–D19. Reagents and conditions: a) (Boc)2O, Et3N, MeOH/H2O (3:1), 8 h, 258C, 86%; b) CH3COCl, CH3OH, 5 h, 758C, 77%;
c) TBTU, Et3N, THF, 8 h, 258C, 75%; d) EtOAc, HCl, 5 h, 258C, 68%; e) RSO2Cl, RCOCl, NaHCO3, THF/H2O, RT, 8 h, 70–82%; 1) RNH2, triphosgene, dioxane, 1108C,
8 h; 2) Et3N, CH2Cl2, RT, 5 h, 43–45% (two steps); f) NH2OK, MeOH, RT, 2 h, 33–54%.
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Table 1. Enzyme inhibitory activities of the derived molecules deter-
mined using HeLa cell nucleus extract containing a mixture of HDAC iso-
forms.
Table 1. (Continued)
Compd
R
IC50 [nm][a]
9.7Æ0.3
Compd
R
IC50 [nm][a]
D17
D1
348.6Æ21.2
D2
D3
D4
95.5Æ4.6
14.3Æ0.9
25.5Æ1.6
D18
D19
7.5Æ0.2
47.7Æ3.3
4h
144.4Æ9.7
145.6Æ7.6
SAHA
D5
15.6Æ1.1
[a] Values represent the mean ÆSD of three independent experiments
performed in triplicate.
D6
D7
105.5Æ9.7
28.7Æ2.2
Table 2. HDAC isoform selectivity of representative compounds.[a]
Compd
IC50 [nm]
HDAC1
HDAC2
HDAC3
HDAC6
D8
85.5Æ6.7
D3
D5
D13
D17
D18
SAHA
28.7Æ1.9
30.7Æ1.3
11.8Æ0.9
24.7Æ1.4
4.8Æ0.3
243.5Æ11.7
170.6Æ9.7
89.2Æ4.7
16.8Æ1.2
23.1Æ1.1
8.5Æ0.3
24.4Æ1.5
5.9Æ0.4
28.1Æ1.8
375.8Æ19.7
294.5Æ11.7
195.8Æ11.9
365.9Æ20.6
294.2Æ22.3
118.2Æ10.8
D9
60.2Æ4.7
106.7Æ8.9
19.5Æ1.4
76.1Æ2.6
256.3Æ11.4
D10
125.5Æ10.6
[a] Values represent the mean ÆSD of three independent experiments
performed in triplicate.
D11
D12
33.6Æ2.3
20.5Æ1.1
19.5 nm, 5.9 nm and 294.2 nm against HDAC1, HDAC2, HDAC3
and HDAC6, respectively.
Modeling
Docking was performed to predict the binding patterns of the
derived molecules in the active site of HDAC2 (Figure 1). There
is a narrow tunnel in the active site of HDAC2, and the zinc ion
is located at the end of the tunnel. An open pocket that is sur-
rounded by hydrophobic residues (Phe155, Phe210 and
Tyr308) lies under the opening of the gap (Figure 1a). D5 (Fig-
ure 1b) and D17 (Figure 1d) are predicted to have similar
binding patterns in the active site of HDAC2. The hydroximic
acid group chelates to the zinc ion giving rise to the tight
binding to the receptor. The linker locating in the tunnel is
predicted to participate in strong hydrophobic interactions
D13
D14
13.7Æ0.7
22.8Æ1.8
D15
D16
34.6Æ2.4
23.5Æ1.7
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these molecules is involved in hydrogen-bonding interaction
with NE2 of His146. In addition, another key residue for hydro-
gen bonding, Asp104, is also predicted to interact through
a hydrogen bond with NH1 of D5 and NH1 of D17. In contrast,
the docking with D16 shows has no hydrogen-bonding inter-
actions with Asp104. This predicted lack of interaction with
the pocket through hydrogen bonding is presumed to be the
reason for the comparatively weak activity of D16 in the
enzyme inhibition assay.
To evaluate the antiproliferation activity of the synthesized
molecules, MTT assays were performed using U937, K562,
HL60, MCF7 PC3 and MDA-MB-231 cell lines. The derived com-
pounds were firstly screened against U937 cells at 2 mm. Mole-
cules exhibiting >50% inhibition were then selected for fur-
ther evaluation in antiproliferation assays against the broader
panel, and their IC50 values were calculated (Table 3). On the
whole, lymphoma and leukemic cell lines (U937, K562, and
HL60) are more sensitive to these molecules than the solid
tumor cell lines (MCF7, PC3, and MDA-MB-231); this is in line
with published preclinical and clinical research results, which
indicate that the therapeutic efficacy of HDACIs is restricted
predominantly to hematological malignancies.[9] Compounds
D3 (1.32 mm) and D17 (1.24 mm) have lower IC50 values than
SAHA in inhibiting the growth of U937 cells. As for K562 cells,
most of the derived compounds (except D11 and D12) show
better performance than SAHA, and D17 is the most active
molecule (IC50 =0.70 mm). Only D11 has a better IC50 value than
SAHA in blocking HL60 cell growth (D11: 0.78; SAHA: 1.44).
Compounds D3 (2.92 mm) and D4 (2.04 mm) outperform SAHA
(5.09 mm) in the MCF7 cell growth inhibition assays. Only three
compounds have larger IC50 values than SAHA in the PC3 cell
assays, of which D18 (IC50 =1.51 mm) is the most active. D3
(IC50 =4.23 mm) demonstrated the best performance in inhibit-
ing the growth of MDA-MB-321 cells.
Figure 1. a) Predicted binding of D5, D16 and D17 to HDAC2 in surface rep-
resentation; the binding pattern of b) D5, c) D16 and d) D17 in the active
site of HDAC2.
with residues around the gap. The benzene ring of the phenyl-
glycine group is predicted to interact with the hydrophobic
area of the opening, further contributing to the binding. Ac-
cording to our modeling results, the external motif binding to
the pocket under the opening has good spatial matching with
the surface of the active site. All these effects in combination
enhance the ligand–receptor interactions. However, according
to our predictions, D16 has a different binding mode (Fig-
ure 1c); in our model, both the phenyl ring and the naphtha-
lene ring in the external motif locate in the opening of the
gap. The hydrogen-bonding interactions predicted to form be-
tween the ligand and the active site of HDAC2 are also likely
to play a significant role in binding. According to the docking
results, the NH group of the hydroximic acid moiety of all
The human leukemic monocyte lymphoma cell line U937
was the most sensitive to the tested molecules (mean IC50
value of 1.51 mm). Therefore, the U937 cell line was selected for
in vivo antitumor assays. The most effective U937 cell growth
inhibitor, D17, which also has the highest water solubility, was
selected for the in vivo antiproliferation assays. As a preliminary
Table 3. Tumor cell growth inhibitory activities of synthesized compounds against a panel of cancer cell lines.[a]
Compd
IC50 [mm]
U937
K562
HL60
MCF7
PC3
3.77Æ0.27
MDA-MB-231
D3
D4
D5
D7
D11
D12
D13
D17
D18
Av.[b]
SAHA
1.32Æ0.07
1.64Æ0.08
1.63Æ0.09
1.77Æ0.06
1.71Æ0.03
1.55Æ0.04
1.39Æ0.03
1.24Æ0.02
1.31Æ0.07
1.51Æ0.05
1.38Æ0.09
1.19Æ0.04
1.59Æ0.06
0.93Æ0.02
1.47Æ0.07
3.16Æ0.11
1.93Æ0.12
1.58Æ0.06
0.70Æ0.01
1.55Æ0.05
1.57Æ0.06
1.86Æ0.07
3.15Æ0.13
4.55Æ0.21
2.88Æ0.16
2.02Æ0.15
0.78Æ0.02
1.80Æ0.09
1.53Æ0.04
3.66Æ0.13
3.83Æ0.29
2.69Æ0.14
1.44Æ0.06
2.92Æ0.15
2.04Æ0.17
8.66Æ0.73
10.8Æ0.93
8.23Æ0.32
6.28Æ0.33
9.71Æ0.44
5.34Æ0.22
5.14Æ0.43
6.57Æ0.41
5.09Æ0.21
4.23Æ0.33
4.78Æ0.44
5.12Æ0.34
6.22Æ0.24
6.67Æ0.25
4.65Æ0.24
4.47Æ0.31
4.46Æ0.28
4.34Æ0.26
4.99Æ0.30
4.70Æ0.35
5.88Æ0.36
10.1Æ0.57
5.67Æ0.35
20.5Æ1.73
3.00Æ0.15
31.6Æ2.33
3.31Æ0.16
1.51Æ0.05
9.48Æ0.66
6.33Æ0.22
[a] Values represent the mean ÆSD of three independent experiments performed in triplicate. [b] The mean (Av.) IC50 value of the test compounds against
each cell line.
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investigation of the in vivo antiproliferative activity of D17,
only one concentration (100 mgkgÀ1 dÀ1) was applied, and
SAHA was used as a positive control. After 16 days of intragas-
tric administration, the mice were euthanized, and tumor
weights were measured. The mean weight of tumors in the
negative control group was found to be 2.66 g (SD: 37%); the
analogous values for SAHA- and D17-treated groups were
2.00 g (SD: 26%) and 1.24 g (SD: 18%), respectively (Fig-
ure 2a). The inhibitory ratio of compound D17 is 53.57%, com-
Figure 3. Dissected tumor tissues taken from mice after intragastric adminis-
tration of 100 mgkgÀ1 dÀ1 of D17 or SAHA as a positive control. All the
tumors were dissected at the 16th day of administration.
results show that these investigated molecules (D3, D5, D13,
D17, and D18) are cell permeable and can inhibit intracellular
and even nuclear HDACs, as determined by monitoring the
acetylation levels of tubulin (target of HDAC6), histones H3
and H4 (targets of HDAC1 and HDAC2), and p21, which is sub-
strate of class I HDACs. All the compounds tested increase the
acetylation levels of tubulin under 6 h treatment, indicating
that the tested molecule can efficiently block the function of
HDAC6 in U937 cells. After 24 h, the effect of D3, D5, and D13
on the increase in acetylated tubulin levels is still significant,
but the action of D17 and D18 are decreased. At both time
points, all the tested compounds show a higher capacity than
SAHA in increasing the levels of the cyclin-dependent kinase
(CDK) inhibitor p21. This indicates that the representative com-
pounds are effective and stable class I HDAC inhibitors. The
tested molecules are more effective at inducing the acetylation
levels of histone H3 than those of histone H4, which correlates
with the enzyme selectivity assay results.
Figure 2. Results of in vivo evaluation of D17. The negative control (vehicle
only) and positive control (SAHA) groups are included for reference.
a) Tumor weight plot; b) inhibitory ratio plot; c) tumor volume plot; d) mice
body weight plot.
HDAC inhibitors are limited by low and ineffective concen-
trations in solid tumors.[9] The FDA-approved drugs SAHA and
FK228 have no clear efficacy in treating relapsed or refractory
breast, colorectal, non-small-cell lung, or metastatic head and
neck cancers, for example, as proven by various clinical
trials.[10] To explore the reason why hematological tumor cell
lines are more sensitive to HDACIs than solid tumor cell lines,
we performed a series of western blot analyses to investigate
the various effects of compounds D3 and D17 on the signaling
cascades between non-solid (U937) and solid (MDA-MB-231)
tumor cell lines (Figure 5). The c-Raf and Erk proteins are two
crucial kinases in activating the MAPK pathway. Phosphorylat-
ed Erk (p-Erk) is the active form of this protein. MDA-MB-231
cells have an overactive MAPK pathway, and the selected mol-
pared with SAHA with a value of 24.97% (Figure 2b). The re-
sults show that D17 exhibits improved antitumor activity over
SAHA. Tumor volumes were measured every two days, and the
results correlate with tumor weights (Figure 2c and Figure 3).
All these results confirm with statistical significance that com-
pound D17 has greater in vivo potency than SAHA. Compound
D17 is safe, as evidenced by the fact that no loss in body
weight was observed (measured every two days; Figure 2d).
Moreover, no clear signs of toxicity in liver and spleen were de-
tected.
Western blot studies were performed to evaluate the intra-
cellular functions of representative compounds (Figure 4). The
Figure 4. Western blot analysis of acetylated histone H3 (Ac-HH3), acetylated histone H4 (Ac-HH4), p21, and acetylated tubulin (Ac-tubulin) in U937 cells after
a) 6 h and b) 24 h of treatment with compounds at 1 mm. Tubulin was used as a loading control.
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compound D17 shows better performance than the positive
control, SAHA. The evidence highlights the value of D17 for
further development. Isoform selectivity studies show that the
representative compounds are generally more prone to bind
to class I HDACs (HDAC1, HDAC2, and HDAC3) than HDAC6.
Western blot assays revealed that the representative molecules
can block the action of both class I HDACs and HDAC6. The
difference in antitumor mechanism between non-solid (U937)
and solid tumor cells (MDA-MB-231) was investigated by west-
ern blot analysis. The MAPK and PI3K/AKT/mTOR signaling
pathways were found to render U937 cells rather than MDA-
MD-231 cells sensitive to the tested molecules.
Experimental Section
General: All commercially available starting materials, reagents,
and solvents were used without further purification. All reactions
were monitored by TLC with 0.25 mm silica gel plates (60GF254);
1
UV light and FeCl3 were used to visualize the spots. H NMR spec-
tra were recorded on a Bruker DRX spectrometer at 600 MHz,
using TMS as an internal standard. High-resolution mass spectra
were performed at the Shandong Analysis and Test Center in
Ji’nan, China. Melting points were determined on an Electrother-
mal melting point apparatus and are uncorrected. Optical rotation
values were measured at room temperature using a modular circu-
lar polarimeter 200 operating at l=589 nm. Maximum absorption
wavelengths of the target compounds were detected for HPLC
analysis. The compounds were dissolved in MeOH at a concentra-
tion of 1 mg per 100 mL and scanned from 500 to 200 nm using
a UV-2102PCS spectrophotometer. All the derived target com-
pounds are >95% pure, as determined by HPLC analysis, per-
formed on an Agilent 1100 HPLC instrument using a Phenomenex
Synergi 4 mm Hydro-RP 80A column (250 mmꢁ4.6 mm). All the
target compounds were eluted with CH3CN and H2O (containing
0.1% formic acid) at various proportions, detected at their maxi-
mum absorption wavelengths.
Figure 5. Western blot analysis of c-Raf, p-Erk, Erk, p-Akt, Akt and histone H3
(HH3) in U937 and MDA-MB-231 cell line after 24 h of treatment with D3
and D17 at 1 mm. Histone H3 was used as a loading control.
ecules cannot lower the signal. However, the tested molecules
can clearly decrease the levels of c-Raf and p-Erk proteins with-
out decreasing the expression of Erk protein in U937 cells. Re-
garding the PI3K/AKT/mTOR cell proliferation pathway, al-
though MDA-MB-231 cells have high levels of Akt protein, p-
Akt levels are very low, indicating that MDA-MB-231 cells do
not depended on the PI3K/AKT/mTOR signaling pathway for
growth and survival. In contrast, although U937 cells have
lower Akt levels, p-Akt levels are very high, which can be sig-
nificantly decreased by compounds D3 and D17. Based on our
results, we can attribute the sensitivity of U937 cells toward
the tested inhibitors to their effects on these two oncogenic
pathways, which are critical for cancer cell survival. To the best
of our knowledge, the work presented herein constitutes the
first report of a mechanistic difference in the antitumor activity
of HDAC inhibitors between non-solid (U937) and solid (MDA-
MB-231) tumor cell lines. Further detailed mechanistic studies
are being carried out to support these findings, and will be re-
ported in due course.
Supporting Information: 1H and 13C NMR spectra, HPLC traces,
and HRMS spectra for final compounds D1–D19 are given in the
Supporting Information.
(S)-Methyl 4-(2-((tert-butoxycarbonyl)amino)-2-phenylacetami-
do)benzoate (a): This compound was synthesized as described in
our previous work.[8b]
(S)-Methyl 4-(2-amino-2-phenylacetamido)benzoate hydrochlo-
ride (b): To a solution of e (3.84 g, 10 mmol) in EtOAc (30 mL), a so-
lution of EtOAc (30 mL) saturated with dry HCl gas was added. The
reaction was stirred at RT for 5 h. The resultant precipitate was iso-
lated by filtration, rinsed with Et2O, and dried to give desired com-
pound b (2.18 g, 68%): MS (ESI): m/z (%): 285.3 [M+H]+; HRMS
(AP-ESI) m/z calcd for C15H16N3O3 [M+H]+ 286.1191, found
286.1186.
Conclusions
A series of HDAC inhibitors was synthesized by structural
modification of a lead compound and evaluated by enzyme in-
hibition assays, in vitro and in vivo antiproliferation assays, iso-
form selectivity tests, and western blot analysis. Significant im-
provements in enzyme inhibitory activity were observed by
changing the Boc group to substituted aromatic groups. The
MTT assays show that the derived molecules are potent anti-
proliferative agents, particularly against non-solid tumor cell
lines such as U937, K562, and HL60. In the in vivo antitumor
assays using athymic nude mice inoculated with U937 cells,
Preparation of c1 and its analogues: Derivatives c2–c17 were pre-
pared as described for c1 (see below).
(S)-Methyl
4-(2-(4-chlorobenzamido)-2-phenylacetamido)ben-
zoate (c1): To a solution of b (0.96 g, 3.0 mmol) in THF (50 mL)/H2O
(1.0 mL), NaHCO3 (1.01 g, 12 mmol) was added. After 10 min, 4-
chlorobenzoyl chloride (0.58 g, 3.3 mmol) was added. The reaction
solution was stirred at RT for 8 h. Then, the solvent was evaporat-
ed, and the residue was taken up in EtOAc (50 mL). The EtOAc so-
lution was washed with saturated aq citric acid (3ꢁ20 mL), saturat-
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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ed aq NaHCO3 (3ꢁ20 mL), and brine (3ꢁ20 mL), dried over MgSO4,
and concentrated in vacuo. Desired compound c1 was obtained
by crystallization from EtOAc as a white powder (0.89 g, 70%):
1H NMR (600 MHz, [D6]DMSO): d=10.76 (s, 1H), 9.20 (d, J=6.6 Hz,
1H), 7.97 (d, J=7.8 Hz, 2H), 7.92 (d, J=8.4 Hz, 2H), 7.76 (d, J=
8.4 Hz, 2H), 7.58 (d, J=7.2 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H), 7.42–
7.39 (m, 2H), 7.36–7.34 (m, 1H), 5.83 (d, J=7.2 Hz, 1H), 3.82 ppm
(s, 3H).
[D6]DMSO): d=10.59 (s, 1H), 8.97 (d, J=9.6 Hz, 1H), 7.88 (d, J=
8.4 Hz, 2H), 7.71 (d, J=8.4 Hz, 2H), 7.52 (d, J=8.4 Hz, 2H), 7.46 (d,
J=8.4 Hz, 2H), 7.37 (d, J=6.6 Hz, 2H), 7.29–7.25 (m, 3H), 5.20 (d,
J=9.6 Hz, 1H), 3.81 ppm (s, 3H).
(S)-Methyl 4-(2-(2-methoxybenzamido)-2-phenylacetamido)ben-
zoate (c11): White powder (0.95 g, 78%): 1H NMR (600 MHz,
[D6]DMSO): d=10.83 (s, 1H), 9.18 (d, J=7.2 Hz, 1H), 7.93 (d, J=
8.4 Hz, 2H), 7.74 (d, J=8.4 Hz, 2H), 7.56 (d, J=7.8 Hz, 2H), 7.54 (d,
J=8.4 Hz, 1H), 7.41 (t, J=7.8 Hz, 2H), 7.33 (t, J=7.2 Hz, 1H), 7.24
(d, J=9.0 Hz, 1H), 7.08 (t, J=7.2 Hz, 1H), 5.87 (d, J=7.2 Hz, 1H),
4.00 (s, 3H), 3.82 ppm (s, 3H).
(S)-Methyl
4-(2-(2-chlorobenzamido)-2-phenylacetamido)ben-
zoate (c2): White powder (0.91 g, 74%): 1H NMR (600 MHz,
[D6]DMSO): d=10.80 (s, 1H), 9.34 (d, J=7.2 Hz, 1H), 7.93 (d, J=
7.8 Hz, 2H), 7.76 (d, J=7.8 Hz, 2H), 7. 58 (d, J=7.8 Hz, 2H), 7.50–
7.44 (m, 3H), 7.40–7.38 (m, 3H), 7.35–7.32 (m, 1H), 5.85 (d, J=
7.2 Hz, 1H), 3.82 ppm (s, 3H).
(S)-Methyl 4-(2-(4-methoxybenzamido)-2-phenylacetamido)ben-
zoate (c12): White powder (0.99 g, 82%): 1H NMR (600 MHz,
[D6]DMSO): d=10.70 (s, 1H), 8.89 (d, J=7.2 Hz, 1H), 7.94 (d, J=
9.0 Hz, 2H), 7.92 (d, J=7.8 Hz, 2H), 7.75 (d, J=7.2 Hz, 2H), 7.57 (d,
J=7.2 Hz, 2H), 7.40 (t, J=7.8 Hz, 2H), 7.34 (t, J=7.8 Hz, 1H), 6.99
(d, J=8.4 Hz, 2H), 5.82 (d, J=7.2 Hz, 1H), 4.02 (s, 3H), 3.81 ppm (s,
3H).
(S)-Methyl
4-(2-(2-naphthamido)-2-phenylacetamido)benzoate
(c3): White powder (0.98 g, 77%): 1H NMR (600 MHz, [D6]DMSO):
d=10.79 (s, 1H), 9.23 (d, J=7.2 Hz, 1H), 8.61 (s, 1H), 8.04 (d, J=
7.8 Hz, 1H), 8.02–7.98 (m, 3H), 7.93 (d, J=9.0 Hz, 2H), 7.78 (d, J=
8.4 Hz, 2H), 7.64–7.59 (m, 4H), 7.44–7.41 (m, 2H), 7.36 (t, J=7.2 Hz,
1H), 5.91 (d, J=7.2 Hz, 1H), 3.82 ppm (s, 3H).
(S)-Methyl
4-(2-(3-bromobenzamido)-2-phenylacetamido)ben-
zoate (c13): White powder (1.03 g, 76%): 1H NMR (600 MHz,
[D6]DMSO): d=10.72 (s, 1H), 9.27 (d, J=6.6 Hz, 1H), 8.16 (s, 1H),
7.93–7.91 (m, 3H), 7.76–7.74 (m, 3H), 7.57 (d, J=7.2 Hz, 2H), 7.45–
7.40 (m, 3H), 7.35 (t, J=7.2 Hz, 1H), 5.81 (d, J=6.6 Hz, 1H),
3.82 ppm (s, 3H).
(S)-Methyl
4-(2-(4-fluorobenzamido)-2-phenylacetamido)ben-
zoate (c4): White powder (0.88 g, 75%): 1H NMR (600 MHz,
[D6]DMSO): d=10.72 (s, 1H), 9.12 (d, J=7.2 Hz, 1H), 8.03–8.01 (m,
2H), 7.92 (d, J=9.0 Hz, 2H), 7.75 (d, J=9.0 Hz, 2H), 7.57 (d, J=
7.8 Hz, 2H), 7.42–7.39 (m, 2H), 7.36–7.33 (m, 1H), 7.31–7.28 (m,
2H), 5.82 (d, J=7.2 Hz, 1H), 3.82 ppm (s, 3H).
(S)-Methyl 4-(2-phenyl-2-(2-propylpentanamido)acetamido)ben-
zoate (c14): White powder (0.91 g, 77%): 1H NMR (600 MHz,
[D6]DMSO): d=10.66 (s, 1H), 8.65 (d, J=7.2 Hz, 1H), 7.91 (d, J=
8.4 Hz, 2H), 7.72 (d, J=8.4 Hz, 2H), 7.48 (d, J=7.8 Hz, 2H), 7.40–
7.37 (m, 2H), 7.33–7.31 (m, 1H), 5.65 (d, J=7.2 Hz, 1H), 3.81 (s,
3H), 1.45–1.40 (m, 2H), 1.28–1.21 (m, 4H), 1.19–1.13 (m, 2H), 0.85
(m, 3H), 0.80 ppm (t, J=7.8 Hz, 3H).
(S)-Methyl 4-(2-(naphthalene-1-sulfonamido)-2-phenylacetami-
1
do)benzoate (c5): White powder (1.09 g, 79%): H NMR (600 MHz,
[D6]DMSO): d=10.47 (s, 1H), 9.15 (d, J=9.6 Hz, 1H), 8.73 (d, J=
8.4 Hz, 1H), 8.13 (d, J=7.2 Hz, 1H), 8.06 (d, J=8.4 Hz, 1H), 7.95 (d,
J=8.4 Hz, 1H), 7.83 (d, J=9.0 Hz, 2H), 7.67–7.65 (m, 1H), 7.61–7.58
(m, 1H), 7.53–7.50 (m, 1H), 7.41 (d, J=8.4 Hz, 2H), 7.29–7.28 (m,
2H), 7.17–7.16 (m, 3H), 5.20 (d, J=9.6 Hz, 1H), 3.81 ppm (s, 3H).
(S)-Methyl
4-(2-phenyl-2-(2-phenylacetamido)acetamido)ben-
zoate (c15): White powder (0.85 g, 73%): 1H NMR (600 MHz,
[D6]DMSO): d=10.77 (s, 1H), 9.02 (d, J=7.8 Hz, 1H), 7.90 (d, J=
9.0 Hz, 2H), 7.72 (d, J=9.0 Hz, 2H), 7.50 (d, J=7.8 Hz, 2H), 7.40–
7.37 (m, 2H), 7.33–7.31 (m, 1H), 7.30–7.28 (m, 4H), 7.22–7.19 (m,
1H), 5.63 (d, J=7.8 Hz, 1H), 3.81 (s, 3H), 3.59 ppm (m, 2H).
(S)-Methyl 4-(2-(4-(tert-butyl)phenylsulfonamido)-2-phenylaceta-
mido)benzoate (c6): White powder (1.03 g, 74%): 1H NMR
(600 MHz, [D6]DMSO): d=10.52 (s, 1H), 8.73 (d, J=9.6 Hz, 1H), 7.84
(d, J=8.4 Hz, 2H), 7.66 (d, J=7.8 Hz, 2H), 7.50 (d, J=8.4 Hz, 2H),
7.37–7.36 (m, 4H), 7.27–7.23 (m, 3H), 5.17 (d, J=9.6 Hz, 1H), 3.80
(s, 3H), 1.17 ppm (s, 9H).
(S)-Methyl 4-(2-(naphthalene-2-sulfonamido)-2-phenylacetami-
1
do)benzoate (c16): White powder (1.08 g, 78%): H NMR (600 MHz,
(S)-Methyl 4-(2-benzamido-2-phenylacetamido)benzoate (c7):
White powder (1.07 g, 72%): 1H NMR (600 MHz, [D6]DMSO): d=
10.72 (s, 1H), 9.07 (d, J=7.2 Hz, 1H), 7.94 (d, J=9.0 Hz, 2H), 7.92
(d, J=9.0 Hz, 2H), 7.75 (d, J=9.0 Hz, 2H), 7.58 (d, J=7.8 Hz, 2H),
7.56–7.54 (m, 1H), 7.47 (t, J=7.8 Hz, 2H), 7.42–7.39 (m, 2H), 7.36–
7.33 (m, 1H), 5.84 (d, J=7.2 Hz, 1H), 3.82 ppm (s, 3H).
[D6]DMSO): d=10.55 (s, 1H), 8.94 (d, J=9.6 Hz, 1H), 8.34 (s, 1H),
7.96–7.94 (m, 2H), 7.90 (d, J=8.4 Hz, 1H), 7.79–7.77 (m, 1H), 7.74
(d, J=9.0 Hz, 2H), 7.61–7.59 (m, 1H), 7.56–7.53 (m, 1H), 7.39–7.36
(m, 4H), 7.23 (t, J=7.2 Hz, 2H), 7.19–7.17 (m, 1H), 5.25 (d, J=
8.4 Hz, 1H), 3.80 ppm (s, 3H).
(S)-Methyl 4-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)-
2-phenylacetamido)benzoate (c17): White powder (1.13 g, 75%):
1H NMR (600 MHz, [D6]DMSO): d=10.40 (s, 1H), 9.06 (d, J=9.6 Hz,
1H), 8.37 (d, J=8.4 Hz, 1H), 8.28 (d, J=7.8 Hz, 1H), 8.12 (d, J=
7.2 Hz, 1H), 7.81 (d, J=8.4 Hz, 2H), 7.55–7.52 (m, 1H), 7.49 (t, J=
7.8 Hz, 1H), 7.40 (d, J=8.4 Hz, 2H), 7.30–7.29 (m, 2H), 7.18–7.16
(m, 4H), 5.17 (d, J=9.6 Hz, 1H), 3.80 (s, 3H), 2.71 ppm (s, 6H).
(S)-Methyl
4-(2-phenyl-2-(4-(trifluoromethyl)phenylsulfonami-
1
do)acetamido)benzoate (c8): White powder (1.08 g, 75%): H NMR
(600 MHz, [D6]DMSO): d=10.61 (s, 1H), 9.14 (d, J=9.6 Hz, 1H), 7.91
(d, J=8.4 Hz, 2H), 7.85 (d, J=9.0 Hz, 2H), 7.75 (d, J=8.4 Hz, 2H),
7.48 (d, J=8.4 Hz, 2H), 7.36 (d, J=7.2 Hz, 2H), 7.26–7.22 (m, 3H),
5.24 (d, J=9.6 Hz, 1H), 3.81 ppm (s, 3H).
(S)-Methyl 4-(2-(3-(2-methoxyphenyl)ureido)-2-phenylacetami-
do)benzoate (c18): To a solution of triphosgene (1.5 g, 5.0 mmol)
in dioxane (40 mL), 2-methoxyaniline (1.23 g, 10 mmol) was added.
The reaction solution was stirred at 1108C for 8 h. The solvent was
evaporated to give a crude residue, which was sufficiently pure for
use without further purification. The residue was redissolved in
CH2Cl2 (50 mL), and b (1.6 g, 5.0 mmol) and Et3N (0.61 g, 6.0 mmol)
were added. The reaction was stirred at RT for 5 h. Then concen-
trated, and the residue was taken up in EtOAc (50 mL). The EtOAc
(S)-Methyl 4-(2-(2,6-difluorophenylsulfonamido)-2-phenylaceta-
mido)benzoate (c9): White powder (1.02 g, 76%): 1H NMR
(600 MHz, [D6]DMSO): d=10.66 (s, 1H), 9.37 (d, J=9.6 Hz, 1H), 7.88
(d, J=9.0 Hz, 2H), 7.57 (d, J=9.0 Hz, 2H), 7.55–7.53 (m, 1H), 7.46
(d, J=9.0 Hz, 2H), 7.31–7.29 (m, 2H), 7.26 (t, J=7.2 Hz, 1H), 7.11 (t,
J=9.0 Hz, 2H), 5.38 (d, J=9.6 Hz, 1H), 3.81 ppm (s, 3H).
(S)-Methyl 4-(2-(4-chlorophenylsulfonamido)-2-phenylacetami-
1
do)benzoate (c10): White powder (1.02 g, 77%): H NMR (600 MHz,
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solution was washed with saturated aq citric acid (3ꢁ20 mL), satu-
rated aq NaHCO3 (3ꢁ20 mL), and brine (3ꢁ20 mL), dried over
MgSO4, and concentrated in vacuo. Desired compound c18 was
obtained by crystallization from EtOAc as a white powder (0.97 g,
128.65, 128.70, 128.94, 129.36, 131.41, 132.50, 134.71, 137.96,
141.91, 164.25, 166.96, 169.69 ppm; HRMS (AP-ESI): m/z [M+H]+
calcd for C26H22N3O4: 440.1610, found: 440.1607; HPLC (l=
272 nm): tR =8.8 min (CH3CN/H2O, 45:55).
1
45%); H NMR (600 MHz, [D6]DMSO): d=10.75 (s, 1H), 8.39 (s, 1H),
(S)-4-Fluoro-N-(2-((4-(hydroxycarbamoyl)phenyl)amino)-2-oxo-1-
phenylethyl)benzamide (D4): Crystallized from EtOAc to give
a white powder (0.22 g, 46%): mp: 240–2428C; [a]2d0 =À5.64 (c=
8.05 (d, J=6.6 Hz, 1H), 7.91 (d, J=8.4 Hz, 2H), 7.89 (d, J=7.8 Hz,
1H), 7.74 (d, J=8.4 Hz, 2H), 7.52 (d, J=7.2 Hz, 2H), 7.42–7.39 (m,
2H), 7.34–7.32 (m, 1H), 6.97 (d, J=7.8 Hz, 1H), 6.89–6.87 (m, 1H),
6.82 (t, J=7.8 Hz, 1H), 5.56 (d, J=7.8 Hz, 1H), 4.02 (s, 3H),
3.81 ppm (s, 3H).
1
1.0, MeOH); H NMR (600 MHz, [D6]DMSO): d=11.12 (s, 1H), 10.65
(s, 1H), 9.10 (d, J=7.2 Hz, 1H), 8.93 (s, 1H), 8.05–8.03 (m, 2H), 7.72
(d, J=8.4, 2H), 7.69 (d, J=9, 2H), 7.59 (d, J=9.0 Hz, 2H), 7.42–7.39
(m, 2H), 7.35 (t, J=7.2 Hz, 1H), 7.30 (t, J=9.0 Hz, 1H), 5.86 ppm (d,
J=7.2 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=58.61, 115.58,
118.98, 127.90, 128.17, 128.43, 128.91, 130.60, 130.62, 137.79,
141.86, 163.66, 164.27, 165.93, 169.61 ppm; HRMS (AP-ESI): m/z
[M+H]+ calcd for C22H19FN3O4: 408.1359, found: 408.1355; HPLC
(l=272 nm): tR =7.7 min (CH3CN/H2O, 40:60).
(S)-Methyl 4-(2-(3-(2,6-diisopropylphenyl)ureido)-2-phenylaceta-
mido)benzoate (c19): Prepared as described for c18 (see above).
White powder (0.61 g, 43%): 1H NMR (600 MHz, [D6]DMSO): d=
10.76 (s, 1H), 7.92 (d, J=8.4 Hz, 2H), 7.74 (s, 2H), 7.52 (d, J=
6.6 Hz, 2H), 7.40 (t, J=6.6 Hz, 2H), 7.33–7.31 (m, 1H), 7.17 (d, J=
7.8 Hz, 2H), 7.10–7.18 (m, 2H), 5.59 (d, J=8.4 Hz, 1H), 3.82 (s, 3H),
3.33 (s, 2H), 1.10 ppm (d, J=6.6 Hz, 12H).
(S)-N-Hydroxy-4-(2-(naphthalene-1-sulfonamido)-2-phenylaceta-
mido)benzamide (D5): Crystallized from EtOAc/hexane (1:1) to
give a white powder (0.30 g, 53%): mp: 152–1548C; [a]2d0 =À7.60
(c=1.0, MeOH); 1H NMR (600 MHz, [D6]DMSO): d=11.07 (s, 1H),
10.33 (s, 1H), 9.09 (d, J=9 Hz, 1H), 8.92 (s, 1H), 8.74 (d, J=8.4 Hz,
1H), 8.14–8.13 (m, 1H), 8.08 (d, J=7.8 Hz, 1H), 7.96 (d, J=8.4 Hz,
1H), 7.68–7.66 (m, 1H), 7.63 (d, J=8.4 Hz, 1H), 7.60 (d, J=7.8 Hz,
2H), 7.52 (t, J=7.8 Hz, 2H), 7.34 (d, J=9 Hz, 2H), 7.29 (d, J=7.8 Hz,
1H), 7.18–7.16 (m, 1H), 5.21 ppm (d, J=8.4 Hz, 1H); HRMS (AP-ESI):
m/z [M+H]+ calcd for C25H22N3O5S: 476.1280, found: 476.1273;
HPLC (l=273 nm): tR =13.0 min (CH3CN/H2O, 40:60).
Preparation of D1–D19: Derivatives D2–D19 were prepared as de-
scribed for D1 (see below).
(S)-4-Chloro-N-(2-((4-(hydroxycarbamoyl)phenyl)amino)-2-oxo-1-
phenylethyl)benzamide (D1): Compound g1 (0.5 g, 1.18 mmol)
was dissolved in a solution of NH2OK (0.56 g, 24 mmol) in metha-
nol (14 mL). After 2 h at RT, the solvent was removed in vacuo. The
residue was acidified to pH 3–4 with saturated aq citric acid solu-
tion, and the solution was extracted with EtOAc (3ꢁ20 mL). The or-
ganic layers were combined, washed with brine (3ꢁ20 mL) and
dried over MgSO4. Desired compound D1 was obtained by crystal-
lization from EtOAc as white powder (0.23 g, 54%): mp: 250–
(S)-4-(2-(4-(tert-Butyl)phenylsulfonamido)-2-phenylacetamido)-N-
hydroxybenzamide (D6): Crystallized from EtOAc/hexane (1:1) to
give a white powder (0.27 g, 48%): mp: 155–1578C; [a]2d0 =À7.77
(c=1.0, MeOH); 1H NMR (600 MHz, [D6]DMSO): d=11.08 (s, 1H),
10.39 (s, 1H), 8.93 (s, 1H), 8.67 (d, J=9.6 Hz, 1H), 7.67 (d, J=
8.4 Hz, 2H), 7.66 (d, J=8.4 Hz, 2H), 7.41 (d, J=9 Hz, 2H), 7.39–
7.27(m, 4H), 7.27 (d, J=8.4 Hz, 2H), 7.25–7.23 (m, 1H), 5.17 (d, J=
10.2, 1H), 1.14 ppm (s, 9H); 13C NMR (75 MHz, [D6]DMSO): d=
60.19, 118.99, 125.88, 126.96, 127.46, 128.01, 128.04, 128.26, 128.76,
137.24, 138.07, 141.19, 155.62, 164.07, 168.17 ppm; HRMS (AP-ESI):
m/z [M+H]+ calcd for C25H28N3O5S: 482.1749, found: 482.1743;
HPLC (l=272 nm): tR =10.6 min (CH3CN/H2O, 45:55).
1
2528C; [a]2d0 =À6.88 (c=1.0, MeOH); H NMR (600 MHz, [D6]DMSO):
d=11.10 (s, 1H), 10.58 (s, 1H), 9.15 (d, J=7.8 Hz, 1H), 8.93 (s, 1H),
7.98 (d, J=8.4 Hz, 2H), 7.72 (d, J=8.4 Hz,), 7.67 (d, J=9.0 Hz,), 7.58
(d J=7.2 Hz, 2H), 7.55 (d, J=7.2 Hz, 1H), 7.42–7.39 (m, 2H), 7.36–
7.34 (m, 1H), 5.84 ppm (d, J=7.8 Hz, 1H); 13C NMR (75 MHz,
[D6]DMSO): d=58.56, 118.96, 127.96, 128.18, 128.34, 128.59, 128.60,
129.03, 132.88, 136.76, 137.60, 141.77, 164.26, 166.04, 169.51 ppm;
HRMS (AP-ESI): m/z [M+H]+ calcd for C22H19ClN3O4: 424.1064,
found: 424.1057; HPLC (l=266 nm): tR =9.6 min (CH3CN/H2O,
45:55).
(S)-2-Chloro-N-(2-((4-(hydroxycarbamoyl)phenyl)amino)-2-oxo-1-
phenylethyl)benzamide (D2): Crystallized from EtOAc to give
a white powder (0.22 g, 45%): mp: 250–2528C; [a]2d0 =À8.20 (c=
(S)-4-(2-Benzamido-2-phenylacetamido)-N-hydroxybenzamide
(D7): Crystallized from EtOAc to give a white powder (0.19 g, 42%):
mp: 229–2308C; [a]2d0 =À6.60 (c=1.0, MeOH); 1H NMR (600 MHz,
[D6]DMSO): d=11.10 (s, 1H), 10.59 (s, 1H), 9.02 (d, J=7.8 Hz, 1H),
8.9 (s, 1H), 7.95 (d, J=7.2 Hz, 2H), 7.72 (d, J=9 Hz, 2H), 7.67 (d, J=
9 Hz, 2H), 7.59 (d, J=7.8 Hz, 1H), 7.56 (t, J=7.2 Hz, 2H), 7.48 (t, J=
7.8 Hz, 2H), 7.46–7.36 (m, 2H), 7.35 (t, J=7.2 Hz, 1H), 5.86 ppm (d,
J=7.2 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=58.49, 118.95,
127.93, 128.17, 128.27, 128.34, 128.60, 128.93, 131.94, 134.13,
137.84, 141.82, 164.27, 166.97, 169.94 ppm; HRMS (AP-ESI): m/z
[M+H]+ calcd for C22H20N3O4: 390.1454, found: 390.1449; HPLC
(l=270 nm): tR =9.9 min (CH3CN/H2O, 35:65).
1
1.0, MeOH); H NMR (600 MHz, [D6]DMSO): d=11.11 (s, 1H), 10.62
(s, 1H), 9.29 (d, J=7.8 Hz, 1H), 8.93 (s, 1H), 7.73 (d, J=9 Hz, 2H),
7.67 (d, J=8.4 Hz, 2H), 7.58 (d, J=7.8 Hz, 2H), 7.48 (d, J=6.6 Hz,
1H), 7.47 (d, J=4.8 Hz, 1H), 7.45 (d, J=8.4 Hz, 1H), 7.41–7.40 (m,
2H), 7.38 (t, J=8.4 Hz, 1H), 7.35–7.32 (m, 1H), 5.86 ppm (d, J=
7.8 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=58.06, 118.97, 127.35,
127.97, 128.25, 128.46, 128.97, 129.76, 129.93, 131.34, 136.55,
137.70, 141.71, 164.24, 166.78, 169.20 ppm; HRMS (AP-ESI): m/z
[M+H]+ calcd for C22H19ClN3O4: 424.1064, found: 424.1057; HPLC
(l=274 nm): tR =7.5 min (CH3CN/H2O, 40:60).
(S)-N-Hydroxy-4-(2-phenyl-2-(4-(trifluoromethyl)phenylsulfona-
mido)acetamido)benzamide (D8): Crystallized from EtOAc/hexane
(S)-N-(2-((4-(Hydroxycarbamoyl)phenyl)amino)-2-oxo-1-phenyl-
ethyl)-2-naphthamide (D3): Crystallized from EtOAc to give
a white powder (0.24 g, 47%): mp: 238–2408C; [a]2d0 =À7.01 (c=
(1:1) to give a white powder (0.23 g, 40%): mp: 200–2028C; [a]d20
=
1
1
À7.20 (c=1.0, MeOH); H NMR (600 MHz, [D6]DMSO): d=11.10 (s,
1H), 10.48 (s, 1H), 9.09 (d, J=9.6 Hz, 1H), 8.94 (s, 1H), 7.92 (d, J=
8.4 Hz, 2H), 7.76 (d, J=8.4 Hz, 2H), 7.67 (d, J=9 Hz, 2H), 7.41 (d,
J=8.4 Hz, 2H), 7.37 (d, J=7.8 Hz, 2H), 7.26 (d, J=8.4 Hz, 2H),
7.23–7.22 (m, 1H), 5.24 ppm (d, J=9 Hz, 1H); 13C NMR (75 MHz,
[D6]DMSO): d=60.18, 119.02, 127.52, 127.54, 127.99, 128.07, 128.79,
128.82, 128.86, 136.72, 140.99, 144.96, 164.08, 167.83 ppm; HRMS
1.0, MeOH); H NMR (600 MHz, [D6]DMSO): d=11.13 (s, 1H), 10.77
(s, 1H), 9.22 (d, J=7.2 Hz, 1H), 8.93 (s, 1H), 8.63 (s, 1H), 8.05 (d, J=
7.8 Hz, 1H), 8.03 (d, J=9.0 Hz, 1H), 8.02 (d, J=8.4 Hz, 1H), 8.00 (d,
J=9.0 Hz, 1H), 7.74–7.71 (m, 2H), 7.65 (d, J=7.8 Hz, 2H), 7.63–7.62
(m, 1H), 7.62–7.60 (m, 1H), 7.44–7.41 (m, 2H), 7.37–7.35 (m, 1H),
5.95 ppm (d, J=7.8 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=
58.66, 119.01, 125.07, 127.13, 127.92, 128.04, 128.16, 128.38, 128.40,
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(AP-ESI): m/z [M+H]+ calcd for C22H19F3N3O5S: 494.0997, found:
494.0993; HPLC (l=269 nm): tR =8.6 min (CH3CN/H2O, 45:55).
found: 468.0554; HPLC (l=270 nm): tR =11.5 min (CH3CN/H2O,
40:60).
(S)-4-(2-(2,6-Difluorophenylsulfonamido)-2-phenylacetamido)-N-
hydroxybenzamide (D9): Crystallized from EtOAc/hexane (1:1) to
give a white powder (0.26 g, 47%): mp: 215–2178C; [a]2d0 =À8.40
(c=1.0, MeOH); 1H NMR (600 MHz, [D6]DMSO): d=11.09 (s, 1H),
10.52 (s, 1H), 9.29 (d, J=9 Hz, 2H), 8.93 (s, 1H), 7.68 (d, J=9 Hz,
2H), 7.56 (m, 1H), 7.49 (d, J=8.4 Hz, 2H), 7.46 (d, J=7.8 Hz, 2H),
7.32–7.9 (m, 2H), 7.27 (d, J=7.2 Hz, 1H), 7.12 (t, J=9 Hz, 2H),
5.37 ppm (d, J=9.6 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=
60.70, 113.45, 113.60, 118.85, 127.60, 128.18, 128.53, 128.87, 135.62,
136.91, 141.25, 158.16, 159.87, 164.18, 167.92 ppm; HRMS (AP-ESI):
m/z [M+H]+ calcd for C21H18F2N3O5S: 462.0935, found: 462.0931;
HPLC (l=267 nm): tR =8.3 min (CH3CN/H2O, 40:60).
(S)-N-Hydroxy-4-(2-phenyl-2-(2-propylpentanamido)acetamido)-
benzamide (D14): Crystallized from EtOAc to give a white powder
(0.23 g, 47%): mp: 267–2698C; [a]2d0 =À7.20 (c=1.0, MeOH);
1H NMR (600 MHz, [D6]DMSO): d=11.09 (s, 1H), 10.52 (s, 1H), 8.92
(s, 1H), 8.61 (d, J=7.2 Hz, 1H), 7.71 (d, J=8.4 Hz, 1H), 7.64 (d, J=
8.4 Hz, 2H), 7.48 (d, J=7.2 Hz, 2H), 7.40–7.37 (m, 2H), 7.33–7.30
(m, 1H), 5.67 (d, J=7.8 Hz, 1H), 1.47–1.43 (m, 1H), 1.28–1.21 (m,
4H), 1.19–1.12 ppm (m, 6H); HRMS (AP-ESI): m/z [M+H]+ calcd for
C23H30N3O4: 412.2236, found: 412.2231; HPLC (l=271 nm): tR =
11.9 min (CH3CN/H2O, 40:60).
(S)-N-Hydroxy-4-(2-phenyl-2-(2-phenylacetamido)acetamido)ben-
zamide (D15): Crystallized from EtOAc to give a white powder
(0.22 g, 46%): mp: 225–2268C; [a]2d0 =À6.70 (c=1.0, MeOH);
1H NMR (600 MHz, [D6]DMSO): d=11.09 (s, 1H), 10.56 (s, 1H), 8.92
(s, 1H), 8.91 (d, J=8.4 Hz, 1H), 7.71 (d, J=9.0 Hz, 2H), 7.63 (d, J=
9.0 Hz, 2H), 7.50 (d, J=7.2 Hz, 2H), 7.40–7.37 (m, 2H), 7.33 (d, J=
7.2 Hz, 1H), 7.29–7.28 (m, 4H), 7.22–7.20 (m, 1H), 5.65 (d, J=
7.8 Hz, 1H), 3.60 ppm (s, 2H); 13C NMR (75 MHz, [D6]DMSO): d=
42.10, 60.20, 118.96, 126.76, 128.22, 128.38, 129.00, 129.52, 136.77,
138.26, 141.68, 163.01, 169.59, 170.61 ppm; HRMS (AP-ESI): m/z
[M+H]+ calcd for C23H22N3O4: 404.1610, found: 404.1607; HPLC
(l=269 nm): tR =9.8 min (CH3CN/H2O, 36:64).
(S)-4-(2-(4-Chlorophenylsulfonamido)-2-phenylacetamido)-N-hy-
droxybenzamide (D10): Crystallized from EtOAc/hexane (1:1) to
give a white powder (0.24 g, 45%): mp: 228–2308C; [a]2d0 =À7.60
(c=1.0, MeOH); 1H NMR (600 MHz, [D6]DMSO): d=11.10 (s, 1H),
10.46 (s, 1H), 8.93 (d, J=7.2 Hz, 1H), 8.92 (s, 1H), 7.72 (d, J=
8.4 Hz, 2H), 7.68 (d, J=9.0 Hz, 2H), 7.47 (d, J=7.2 Hz, 2H), 7.44 (d,
J=8.4 Hz, 2H), 7.38 (d, J=7.8 Hz, 2H), 7.28 (d, J=8.4 Hz, 2H),
7.26–7.25 (m, 1H), 5.20 ppm (d, J=7.2 Hz, 1H); 13C NMR (75 MHz,
[D6]DMSO): d=60.20, 119.05, 128.21, 128.85, 128.98, 129.13, 129.25,
129.30, 136.97, 137.60, 140.03, 141.10, 164.14, 168.04 ppm; HRMS
(AP-ESI): m/z [M+H]+ calcd for C21H19ClN3O5S: 460.0734, found:
460.0725; HPLC (l=269 nm): tR =10.3 min (CH3CN/H2O, 40:60).
(S)-N-Hydroxy-4-(2-(naphthalene-2-sulfonamido)-2-phenylaceta-
mido)benzamide (D16): Crystallized from EtOAc/hexane (1:1) to
give a white powder (0.30 g, 53%): mp: 196–1988C; [a]2d0 =À6.90
(c=1.0, MeOH); 1H NMR (600 MHz, [D6]DMSO): d=11.07 (s, 1H),
10.43 (s, 1H), 8.94 (s, 1H), 8.33 (s, 1H), 7.96 (d, J=8.4 Hz, 1H), 7.94
(d, J=9.0 Hz, 1H), 7.90 (d, J=7.8 Hz, 1H), 7.77 (d, J=8.4 Hz, 1H),
7.60 (t, J=7.2 Hz, 1H), 7.57 (s, 1H), 7.55 (d, J=8.4 Hz, 2H), 7.38 (d,
J=7.2 Hz, 2H), 7.30 (d, J=8.4 Hz, 2H), 7.22 (t, J=7.2 Hz, 2H), 7.17
(d, J=7.2 Hz, 1H), 5.23 ppm (s, 1H); HRMS (AP-ESI): m/z [M+H]+
calcd for C25H22N3O5S: 476.1280, found: 476.1276; HPLC (l=
271 nm): tR =9.6 min (CH3CN/H2O, 43:57).
(S)-N-(2-((4-(Hydroxycarbamoyl)phenyl)amino)-2-oxo-1-phenyl-
ethyl)-2-methoxybenzamide (D11): Crystallized from EtOAc to
give a white powder (0.23 g, 46%): mp: 156–1588C; [a]2d0 =À7.50
(c=1.0, MeOH); 1H NMR (600 MHz, [D6]DMSO): d=11.11 (s, 1H),
10.69 (s, 1H), 9.17 (d, J=7.8 Hz, 1H), 8.93 (s, 1H), 7.91 (d, J=
7.8 Hz, 1H), 7.73 (d, J=9 Hz, 2H), 7.65 (d, J=8.4 Hz, 2H), 7.7–7.54
(m, 3H), 7.42–7.39 (m, 2H), 7.34 (d, J=7.2 Hz, 1H), 7.24 (d, J=
8.4 Hz, 1H), 7.09 (t, J=7.2 Hz, 1H), 5.87 (d, J=7.2 Hz, 1H),
4.01 ppm (s, 3H); 13C NMR (75 MHz, [D6]DMSO): d=56.83, 17.75,
112.89, 119.15, 121.32, 121.37, 127.30, 128.27, 128.43, 129.18,
131.44, 133.67, 138.88, 141.43, 157.94, 164.08, 164.21, 169.28 ppm;
HRMS (AP-ESI): m/z [M+H]+ calcd for C23H22N3O5: 420.1559, found:
420.1553; HPLC (l=270 nm): tR =8.2 min (CH3CN/H2O, 40:60).
(S)-4-(2-(5-(Dimethylamino)naphthalene-1-sulfonamido)-2-phe-
nylacetamido)-N-hydroxybenzamide (D17): Crystallized from
EtOAc/hexane (1:1) to give a white powder (0.30 g, 49%): mp:
152–1548C; [a]2d0 =À6.78 (c=1.0, MeOH); 1H NMR (600 MHz,
[D6]DMSO): d=11.08 (s, 1H), 10.28 (s, 1H), 9.04 (d, J=9.6 Hz, 1H),
8.94 (s, 1H), 8.37 (d, J=8.4 Hz, 1H), 8.29 (d, J=8.4 Hz, 1H), 8.12 (d,
J=7.2 Hz, 1H), 7.60 (d, J=9.0 Hz, 2H), 7.55–7.53 (m, 1H), 7.51–7.48
(m, 1H), 7.31–7.29 (m, 4H), 7.18–7.16 (m, 4H), 5.16 (d, J=9.6 Hz,
1H), 2.71 ppm (s, 6H); HRMS (AP-ESI): m/z [M+H]+ calcd for
C27H27N4O5S: 519.1702, found: 519.1699; HPLC (l=262 nm): tR =
10.2 min (CH3CN/H2O, 40:60).
(S)-N-Hydroxy-4-(2-(4-methoxybenzamido)-2-phenylacetamido)-
benzamide (D12): Crystallized from EtOAc to give a white powder
(0.24 g, 49%): mp: 240–2418C; [a]2d0 =À7.80 (c=1.0, MeOH);
1H NMR (600 MHz, [D6]DMSO): d=11.09 (s, 1H), 10.55 (s, 1H), 8.91
(s, 1H), 8.83 (d, J=7.8 Hz, 1H), 7.95 (d, J=7.2 Hz, 2H), 7.72 (d, J=
9.0 Hz, 2H), 7.67 (d, J=9.0 Hz, 2H), 7.57 (d, J=7.2 Hz, 2H), 7.41–
7.39 (m, 2H), 7.35–7.33 (m, 2H), 7.00 (d, J=8.4 Hz, 2H), 5.84 (d, J=
7.2 Hz, 1H), 3.82 ppm (s, 3H); 13C NMR (75 MHz, [D6]DMSO): d=
55.73, 58.42, 113.81, 118.95, 126.31, 127.94, 128.21, 128.35, 128.92,
130.17, 137.96, 141.82, 162.24, 164.27, 166.42, 169.79 ppm; HRMS
(AP-ESI): m/z [M+H]+ calcd for C23H22N3O5: 420.1559, found:
420.1554; HPLC (l=265 nm): tR =10.1 min (CH3CN/H2O, 40:60).
(S)-N-Hydroxy-4-(2-(3-(2-methoxyphenyl)ureido)-2-phenylaceta-
mido)benzamide (D18): Crystallized from EtOAc to give a white
powder (0.17 g, 33%): mp: 201–2038C; [a]2d0 =À6.40 (c=1.0,
MeOH); 1H NMR (600 MHz, [D6]DMSO): d=11.09 (s, 1H), 10.61 (s,
1H), 8.92 (s, 1H), 8.39 (s, 1H), 8.07 (s, 1H), 8.06 (d, J=7.8 Hz, 1H),
7.88 (d, J=7.8 Hz, 1H), 7.71 (d, J=9.0 Hz, 1H), 7.66 (d, J=8.4 Hz,
2H), 7.52 (d, J=7.8 Hz, 2H), 7.42–7.39 (m, 2H), 7.33 (t, J=7.2 Hz,
1H), 6.97 (d, J=7.8 Hz, 1H), 6.90–6.87 (m, 1H), 6.84–6.82 (m, 1H),
5.57 (d, J=7.8 Hz, 1H), 3.84 ppm (s, 3H); 13C NMR (75 MHz,
[D6]DMSO): d=57.98, 60.19, 111.09, 118.47, 120.88, 121.63, 121.72,
127.11, 127.89, 129.02, 129.06, 129.62, 126.75, 139.06, 141.73,
147.90, 154.98, 164.27, 167.60 ppm; HRMS (AP-ESI): m/z [M+H]+
calcd for C23H23N4O5: 435.1668, found: 435.1662; HPLC (l=
272 nm): tR =8.7 min (CH3CN/H2O, 40:60).
(S)-3-Bromo-N-(2-((4-(hydroxycarbamoyl)phenyl)amino)-2-oxo-1-
phenylethyl)benzamide (D13): Crystallized from EtOAc to give
a white powder (0.26 g, 48%): mp: 234–2368C; [a]2d0 =À6.80 (c=
1
1.0, MeOH); H NMR (600 MHz, [D6]DMSO): d=11.10 (s, 1H), 10.58
(s, 1H), 9.23 (d, J=7.2 Hz, 1H), 8.93 (s, 1H), 8.17 (s, 1H), 7.94 (d, J=
7.8 Hz, 1H), 7.76 (d, J=7.8 Hz, 1H), 7.72 (d, J=9.0 Hz, 2H), 7.67 (d,
J=8.4 Hz, 2H), 7.58 (d, J=7.2 Hz, 2H), 7.45 (d, J=7.8 Hz, 1H), 7.41
(d, J=9.0 Hz, 1H), 7.37–7.34 (m, 1H), 5.84 ppm (d, J=7.8 Hz, 1H);
HRMS (AP-ESI): m/z [M+H]+ calcd for C22H19BrN3O4: 468.0559,
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(S)-4-(2-(3-(2,6-Diisopropylphenyl)ureido)-2-phenylacetamido)-N-
hydroxybenzamide (D19): Crystallized from EtOAc to give a white
powder (0.24 g, 42%): mp: 252–2548C; [a]2d0 =À8.40 (c=1.0,
MeOH); 1H NMR (600 MHz, [D6]DMSO): d=11.12 (s, 1H), 10.67 (s,
1H), 8.95 (s, 1H), 7.77 (s, 1H), 7.71 (d, J=8.4 Hz, 2H), 7.66 (d, J=
7.8 Hz, 2H), 7.52 (d, J=7.8 Hz, 2H), 7.39 (d, J=7.2 Hz, 2H), 7.32 (d,
J=7.2 Hz, 1H), 7.18 (s, 2H), 7.10 (s, 2H), 5.58 (d, J=8.4 Hz, 1H),
3.16–3.13 (m, 2H), 1.11 ppm (d, J=7.2 Hz, 12H); HRMS (AP-ESI): m/
z [M+H]+ calcd for C28H33N4O4: 489.2502, found: 489.2497; HPLC
(l=270 nm): tR =8.8 min (CH3CN/H2O, 48:52).
ment Ethical Review Board of Shandong University (approval No.
201302006).
Western blot analysis: For western blot analysis, total protein ex-
tracts were separated on a polyacrylamide gel, transferred onto
PVDF membranes and blotted as previously described.[12] Briefly,
U937 and MDA-MB-231 cells with different treatment were collect-
ed and lysed with lysis buffer for 30 min then centrifuged for
15 min at 4000 rpm at 48C; the supernatant contained the whole-
cell extracts. Total protein extracts were separated by 12% SDS-
PAGE and transferred onto PVDF membranes (Millipore, cat. no.
IPVH00010). Membranes were blocked with 5% milk in TBS-T for
1 h at room temperature, then incubated with a 1:1000 or 1:2000
dilution of primary antibody overnight at 48C including tubulin
(Sigma), acetylated tubulin (Sigma), histone H3 (Sigma), acetylated
histone H3 (Sigma), acetylated histone H4 (Sigma), p21 (Cell Signal-
ing), c-Raf (Sigma), p-Erk (Sigma), Erk (Sigma), p-Akt (Sigma), and
Akt (Sigma). Membranes were then washed three times and incu-
bated at a 1:2000 dilution of anti-mouse or anti-rabbit HRP-conju-
gated goat secondary antibodies for 2 h at room temperature. Fi-
nally, membranes were washed another three times and developed
by enhanced chemiluminescence (ECL; Millipore, cat. no. WBKL
S0050). Images were obtained using an Image Quant LAS 4000
imager. Complete gel images can be obtained from the Editorial
Office or the authors upon request.
Molecular docking studies
Docking was performed with Sybyl-X 2.0 and Maestro ver. 7.5
(Schrçdinger Inc, supported by Shanghai Institute of Materia
Medica Chinese Academy of Sciences). All the molecules sketched
and minimized by Sybyl-X were ionized and assigned OPLS 2005
force field by LigPrep. The structure of human HDAC2, obtained
from the RCSB Protein Data Bank (PDB entry: 3MAX) was used for
the docking study. The HDAC2 structure was prepared by removal
of waters and ligands, and assignment of OPLS 2005 force field
using Maestro. The active site was defined as a cube of side length
20 ꢂ, centered at the zinc ion by Glide dock. Extra precision was
applied in the docking process; other parameters such as docking
options, charges, core, constraints (zinc ion), and output ligand
poses, were set as default.
Acknowledgements
Biological evaluations
This work was supported by the National Nature Science Founda-
tion of China (grant no. 21172134), the National High Technology
In vitro HDAC inhibition assays: In vitro activity assay against HeLa
cell nuclear extracts has been described in our previous work.[11]
Briefly, HeLa cell nuclear extract solution (10 mL) was mixed with
various concentrations of compound sample (50 mL). Fluorogenic
substrate (40 mL) was added, and after 30 min incubation at 378C,
the reaction was stopped by addition of developer (100 mL) con-
taining trypsin and TSA. Fluorescence intensity was measured after
20 min of reaction using a microplate reader at respective excita-
tion and emission wavelengths of 390 and 460 nm. The IC50 values
were determined based on the fluorescence intensity readings.
The procedures for HDAC1, 2, 3, and 6 assays were similar to that
for HeLa nuclear extracts.
Research
& Development Program of China (grant no.
2011ZX09401-015), the Doctoral Foundation of the Ministry of
Education of China (grant no. 20110131110037), the Independent
Innovation Foundation of Shandong University (IIFSDU), China
(grant no. 2013GN013) and the US National Cancer Institute
(NCI), the US National Institutes of Health (NIH) (grant no.
R01CA163452 to C.J.C.).
Keywords: antitumor agents · cancer · epigenetics · histone
deacetylases · inhibitors · structural optimization
In vitro antiproliferative assays: Tumor cell inhibition was deter-
mined using the MTT method. Briefly, 2000 cells were seeded into
each well of 96-well plates, which were incubated at 378C, 5% CO2
overnight. The cells were then treated with compound sample at
various concentrations for 48 h. Afterward, a 0.5% MTT solution
was added to each well. After 4 h incubation, formazan formed
from MTT was extracted by adding DMSO (200 mL) for 5 min. Opti-
cal density values were then detected at l=570 nm on a micro-
plate reader.
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In vivo antitumor assays against U937 xenografts: For in vivo antitu-
mor efficacy studies, 1.8ꢁ107 U937 cells were inoculated subcuta-
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six weeks old, Slac Laboratory Animals, Shanghai, China). Ten days
after injection, tumors were palpable, and mice were randomized
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PBS solution. During treatment, the body weight of mice was
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euthanized, and tumor weights were measured on an electronic
balance. The experiments were approved by the Animal Experi-
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Received: July 5, 2013
Revised: October 13, 2013
Published online on November 12, 2013
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