Discovery of Selective Histone Deacetylase 6 Inhibitors Using the Quinazoline as the Cap for the Treatment of Cancer

Article abstract of DOI:10.1021/acs.jmedchem.5b01342

Novel selective histone deacetylase 6 (HDAC6) inhibitors using the quinazoline as the cap were designed, synthesized, and evaluated for HDAC enzymatic assays. N-Hydroxy-4-(2-methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)butanamide, 23bb, was the

Full text of DOI:10.1021/acs.jmedchem.5b01342

Article
pubs.acs.org/jmc
Discovery of Selective Histone Deacetylase 6 Inhibitors Using the
Quinazoline as the Cap for the Treatment of Cancer
Zhuang Yang,†,‡,# Taijin Wang,‡,# Fang Wang,‡,# Ting Niu,§ Zhuowei Liu,∥ Xiaoxin Chen,∥
Chaofeng Long,∥ Minghai Tang,‡ Dong Cao,‡ Xiaoyan Wang,‡ Wei Xiang,‡ Yuyao Yi,§ Liang Ma,‡
Jingsong You,*,† and Lijuan Chen*,†,‡
†State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy and Cancer Center, College of Chemistry,
Sichuan University, Chengdu, 610064, China
‡State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy and Cancer Center, West China Hospital of
Sichuan University, Chengdu, 610041, China
§Department of Hematology and Research Laboratory of Hematology, West China Hospital of Sichuan University, Chengdu, 610041,
China
∥Guangdong Zhongsheng Pharmaceutical Co., Ltd., Dongguan, Guangdong 523325, China
S
* Supporting Information
ABSTRACT: Novel selective histone deacetylase 6 (HDAC6)
inhibitors using the quinazoline as the cap were designed,
synthesized, and evaluated for HDAC enzymatic assays. N-
Hydroxy-4-(2-methoxy-5-(methyl(2-methylquinazolin-4-yl)-
amino)phenoxy)butanamide, 23bb, was the most potent
selective inhibitor for HDAC6 with an IC50 of 17 nM and
showed 25-fold and 200-fold selectivity relative to HDAC1 and
HDAC8, respectively. In vitro, 23bb presented low nanomolar
antiproliferative effects against panel of cancer cell lines.
Western blot analysis further confirmed that 23bb increased
acetylation level of α-tubulin in vitro. 23bb has a good pharmacokinetic profile with oral bioavailability of 47.0% in rats. In in vivo
efficacy evaluations of colorectal HCT116, acute myelocytic leukemia MV4-11, and B cell lymphoma Romas xenografts, 23bb
more effectively inhibited the tumor growth than SAHA even at a 4-fold reduced dose or ACY-1215 at the same dose. Our results
indicated that 23bb is a potent oral anticancer candidate for selective HDAC6 inhibitor and deserves further investigation.
icity.17,19−22 In addition, most of the current HDAC inhibitors
INTRODUCTION
■
show a lack of visible efficacy against solid tumors,5,13 which
As one of the epigenetic targets, histone deacetylases
does really limit their application for the treatment of broad
(HDACs), which are responsible for deacetylation of lysine
residues in histone and non-histone substrates,1 have recently
spectrum of cancer. To avoid the side effects and achieve the
potency against solid tumors, an increasing number of
emerged as an important target in the development of
anticancer agents.2−6 The 18 isoforms of HDAC are
investigations are focusing on the development of isotype-
selective HDAC inhibitors, especially those targeting the
categorized into four groups: class I (HDACs 1, 2, 3, and 8),
class II (class IIa (HDACs 4, 5, 7, and 9) and class IIb (HDACs
6 and 10)), and class IV (HDAC11) HDACs are all zinc-
dependent deacetylases that are mechanistically distinct from
NAD+-dependent class III HDACs.7−9 During past inves-
tigations on HDAC inhibition, a number of inhibitors have
been reported, and some of them were licensed or at various
stages of clinical evaluation (Figure 1).10,11 Vorinostat
(SAHA),12−15 romidepsin (FK-228),13 belinostat (PXD-
101),16,17 and panobinostat (LBH-589)18 have gained FDA
approvals for the treatment of cutaneous T-cell lymphoma, T-
cell lymphoma, and multiple myeloma. However, most of them
are class I selective (FK-228, PXD-101) or pan-HDAC
inhibitors (SAHA, LBH-589). These nonselective or partially
selective HDAC inhibitors usually lead to undesirable biological
responses, such as fatigue, nausea/vomiting, and cardiotox-
isoform of HDAC6.9,10,23−37
HDAC6, which is expressed primarily in the cytoplasm,
removes the acetyl group from lysine residues in a number of
non-histone substrates, including α-tubulin, Hsp90.38−40 In
contrast to the lethal effect of HDAC1−3 genetic ablation, it
has been reported that mice with HDAC6-knocked out are
viable.26,41 These results confirmed that HDAC6 selective
inhibitors may have fewer side effects than pan-HDAC
inhibitors or HDAC1−3 isoform selective inhibitors. The
discovery of tubacin, a first reported selective HDAC6 inhibitor,
Special Issue: Epigenetics
Received: June 20, 2015
© XXXX American Chemical Society
A
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Figure 1. Examples of HDAC inhibitors.
Figure 2. Binding site of HDAC6 homology model and design of novel inhibitors selective for HDAC6.
which was identified in 2003 during a high-throughput screen
of 7392 compounds, has prompted numerous investigations
toward the development of HDAC6inhibitors, such as tubastain
A, HPOB,14 and ACY-1215.42,43 Especially, ACY-1215 is
currently in phase II clinical trials to treat multiple myeloma.44
Such promising results drew our attention to develop novel,
selective, and highly effective small molecule inhibitors of
HDAC6.
rim differs greatly between the three isozymes. In order to
identify the subtle difference, HDAC8, HDAC6 homology
model and optimized HDAC1 were aligned by PyMol-1.5.3.
The classic structure of HDAC inhibitors consists of a zinc
binding group (ZBG) that chelates the active site Zn2+ ion, a
linker, and a surface recognition cap group that interacts with
the amino acid residues present at the surface of the HDAC.
From the results of residue alignment, we found that among
three protein structures, the residues of ZBG and linker region
have a high degree of consistency and similar conformation.
However, the forms and conformations of amino acids have a
big difference in the cap region. This is thought to be due to the
flexibility of loops (loop 1, Ser80-Tyr86; loop 2, Glu202-
Phe207 in HDAC6 model) (Supporting Information Table 1).
Through comparing residues of cap region in HDAC1,
HDAC6, and HDAC8 structures, we found that Phe82 (loop
1) and Met198 (loop 2) are critical for forming the surface
groove which is composed of the following residues: Phe136,
Ser84, Phe82, Met198, Phe196. But the residues of similar
function are absent in HDAC1 and HDAC8 structures (Figure
2 and Supporting Information Figure S1). And literature
surveys also indicated that the cap group may contribute an
RESULTS AND DISCUSSION
■
Initial Molecular Docking Studies. The lack of crystal
structure led to more difficulty in the designing of HDAC6
selective inhibitors than the pan-HDAC inhibitors and
HDAC1−3-selective inhibitors. Intrigued by designing highly
selective inhibitors of HDAC6 isoform, we performed the
protein structure alignment of HDAC1 (PDB code 4BKX),
HDAC6 (homology model), and HDAC8 (PDB code 2V5W).
For HDAC6, a homology model was generated by multiple-
thread alignments, as described by Yang Zhang’s research group
through a Web server (I-TASSER).45 Analysis of the Zn ion
binding pockets of HDAC1, HDAC6, and HDAC8 revealed
that, while the active pocket is relatively conserved, the channel
B
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a
Scheme 1. Synthesis of 7a−d
a
Reagents and conditions: (a) NaH, MOMCl, DMF, 0 °C to rt; (b) H2, Pd/C (10%), EtOH, rt; (c) (i) (CH2O)n, MeONa, MeOH, rt; (ii) NaBH4,
reflux; (d) (i) 4-chloro-2-methylquinazoline, cat. conc HCl, (Me)2CHOH, rt; (ii) EtOAc, HCl (g), rt; (e) Br(CH2)nCOOEt, Cs2CO3, CH3CN,
reflux; (g) 50% NH2OH aq, NaOH, CH2Cl2/MeOH (1:2), rt.
a
Scheme 2. Synthesis of 10a−d
a
Reagents and conditions: (a) (i) 4-chloroquinazoline, cat. conc HCl, (Me)2CHOH, rt; (ii) EtOAc, HCl (g), rt; (b) Br(CH2)nCOOEt, Cs2CO3,
CH3CN, reflux; (c) 50% NH2OH aq, NaOH, CH2Cl2/MeOH (1:2), rt.
a
Scheme 3. Synthesis of 16a−d
a
Reagents and conditions: (a) guanidine carbonate, t-BuOH, t-BuOK; (b) POCl3, reflux; (c) (i) 4, cat. conc HCl, (Me)2CHOH, rt; (ii) EtOAc, HCl
(g), rt; (d) Br(CH2)nCOOEt, Cs2CO3, CH3CN, reflux; (e) 50% NH2OH aq, NaOH, CH2Cl2/MeOH (1:2), rt.
important function to the selective activity. Hence, a series of
fragments, which comfortably occupy the surface groove and
form robust interaction, were designed to target this area. By
molecular docking results and the feasibility analysis of
synthesis (Supporting Information Figure S2 and S3), we
chose 2-methylquinazoline as starting point, which is found in
many bioactive compounds,28,46−48 and has been used in the
research of HDAC inhibitor with some exciting results
yielded.28
shown in Scheme 1. Commercially available p-nitrophenol (1)
was the starting material for the synthesis. The phenolic
hydroxyl group was first protected by chloromethyl methyl
ether (MOMCl) to give 2. Then reduction of 2 led to 3, which
was further N-methylated by reacting with paraformaldehyde in
MeONa−MeOH solution and reduction by NaBH4 sequen-
tially. The resulting aniline 4 was coupled with 4-chloro-2-
methylquinazoline, giving the intermediate compound 5. Then
we used the Br(CH2)nCOOEt (n = 1, 3−5) to react with 5 to
obtain compounds 6a−d, which had different length alkyl
chains. Those compounds were directly converted to
hydroxamic acid compounds 7a−d by NH2OH.
Chemistry. At the beginning of our research, we
synthesized a series compounds with 2-methylquinazoline as
the capping groups. The synthetic route to compounds 7a−d is
C
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a
Scheme 4. Synthesis of 23aa−ae and 23ba−be
a
Reagents and conditions: (a) NaH, MOMCl, DMF, 0 °C to rt; (b) H2, Pd/C (10%), EtOH, rt; (c) (i) (CH2O)n, MeONa, MeOH, rt; (ii) NaBH4,
reflux; (d) (i) 4-chloro-2-methylquinazoline, cat. conc HCl, (Me)2CHOH, rt; (ii) EtOAc, HCl (g), rt; (e) Br(CH2)nCOOEt, Cs2CO3, CH3CN,
reflux; (g) 50% NH2OH aq, NaOH, CH2Cl2/MeOH (1:2), rt.
a
Scheme 5. Synthesis of 27
a
Reagents and conditions: (a) Br(CH2)nCOOEt, Cs2CO3, CH3CN, reflux; (b) H2, Pd/C (10%), EtOH, rt; (c) 4-chloro-2-methylquinazoline, cat.
conc HCl, (Me)2CHOH, rt; (d) 50% NH2OH aq, NaOH, CH2Cl2/MeOH (1:2), rt.
To search the structure and relationship analysis (SAR) of
cap, we also synthesized two kinds of HDAC inhibitors which
used different 2-methylquinazoline analogues as the capping
group. The first one was quinazoline without substituent group
at C-2 position (Scheme 2). The synthetic route was similar to
Scheme 1, which using 4-chloroquinazoline replaced 4-chloro-
2-methylquinazoline to couple with 4, giving 10a−d. Scheme 3
shows the preparation of another quinazoline derivatives using
a five fat ring that replaced the phenyl ring of quinazoline. 13,
which was synthesized by 11 cyclizing with guanidine carbonate
under the action of t-BuOK and chlorinated with POCl3,
reacted with 4 and the subsequent reactions were same as
Scheme 1, ging compounds 16a−d.
Then we focused on the linker region. As shown in Scheme
4, the m-nitrophenol (17a) and 2-methoxy-5-nitrophenol
(17b) were used as the starting materials, and the following
reactions were similar as in Scheme 1. Eventually, we got the
compounds 23aa−ad and 23ba−bd.
On the basis of the most potent compound 23bb, we also
synthesized the compound 27 to evaluate the function of N-
methyl group, and the synthesis is shown in Scheme 5. 17b was
reacted with ethyl 4-bromobutyrate, giving 24, which was
further hydrogenated by hydrogen. Then compound 25 was
coupled with 4-chloro-2-methylquinazoline, and the resulting
compound 26 was converted to hydroxamic acid compound 27.
Biological Evaluation. In Vitro HDAC Isoform-Selec-
tivity of the Compounds. The synthesized compounds were
evaluated for the inhibitory activities on the HDAC1, HDAC6,
and HDAC8 isoforms and SAHA, ACY-1215 as positive
controls. The influence of various quinazoline analogues caps
on HDAC6 is summarized in Table 1. Compounds 7a−d,
which used the 2-methylquinazoline’s capping group and
various chain length linkers with different lengths at the C-4
position, had an efficiently selective inhibition activity of
HDAC6. The most potent compound 7a, with IC50 value of 8.6
nM on HDAC6, displayed the best selectivity of HDAC6
versus HDAC1 (20-fold) and HDAC8 (137-fold). The
selective inhibitory activity of HDAC6 further decreased with
an increase in carbon chain length, and the IC50 values of 7b (n
= 3), 7c (n = 4), and 7d (n = 5) on HDAC6 were 196, 57, and
34 nM, respectively. Additionally, the selectivity toward
HDAC6 versus HDAC1 and HDAC8 was also decreased.
The same results were also observed in the classes with
quinazoline (10a−d) and 2-methyl-6,7-dihydro-5H-cyclopenta-
[d]pyrimidine (16a−d) as the capping group. In the two
classes, the short carbon chain length exhibited low nanomolar
inhibition (10a and 16a) on HDAC6 and good selectivity
D
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Table 1. Enzyme Inhibition Data for 7a−e, 10a−e, and 16a−e
a
IC50 (nM)
selective fold
compd
7a
n
HDAC1
HDAC6
8.6
196 32
HDAC8
HDAC1/6
HDAC8/6
1
3
4
5
1
3
4
5
1
3
4
5
172 30
84 14
1
1181 53
1465 60
1698 189
1510 323
1422 79
2457 20
20.0
0.4
3.6
8.0
17.3
2.2
4.1
9.3
15.5
4.9
3.4
6.0
0.7
4.2
137.3
7.5
7b
7c
204 23
272 36
242 27
57
34
14
34
49
19
17
40
7
7
1
4
7
5
3
3
29.8
44.4
101.6
72.3
27.1
55.7
174.9
154.8
31.5
50.2
11.5
28.2
7d
10a
10b
10c
76
9
203
6
1326
2
10d
16a
176 47
264 55
195 35
212 32
120 24
1058 207
2974 627
6190 876
1987 101
1003 176
172 45
16b
16c
63 11
16d
SAHA
ACY-1215
20
15
9
2
3
11
38
1
5
2
254 57
a
Compounds were tested in the 10-dose IC50 mode in duplicate with 3-fold serial dilutions starting at 10 μM. The IC50 values are the mean of at
least two experiments.
Table 2. Enzyme Inhibition Data for 23aa−ad and 23ba−bd
a
IC50 (nM)
selective fold
comp
23aa
n
HDAC1
HDAC6
HDAC8
>1000
HDAC1/6
HDAC8/6
1
3
4
5
1
3
4
5
>1000
>1000
>1000
>1000
>1000
>1000
23ab
23ac
>1000
>1000
306 90
>1000
23ad
23ba
23bb
23bc
23bd
27
73
2
>1000
>1000
422
>1000
1
17
111 30
23
>1000
2
3398 487
5189 648
1531 78
>1000
24.8
2.3
199.9
46.7
66.6
253 23
160
3
5
7.0
>1000
SAHA
ACY-1215
11
38
1
15
9
3
172 45
254 57
0.7
4.2
11.5
28.2
5
2
a
Compounds were tested in the 10-dose IC50 mode in duplicate with 3-fold serial dilutions starting at 10 μM. The IC50 values are the mean of at
least two experiments.
toward to HDAC1 and HDAC8. Interestingly, compounds 7d,
10d, 16d with a carbon chain length of five carbons (n = 5) also
showed low nanomolar inhibition on HDAC6 but decreased
selectivity on HDAC1 and HDAC8. These results suggested
that the length of the hydroxamic acid side chain is important
for the activity and selectivity of HDAC6. When the carbon
chain was at the C-4 position, the optimal length of the carbon
chain linker should be one carbon for the activity and selectivity
of HDAC6. Uniquely, compound 16b, which used a five-fatty
ring to replace the phenyl ring of quinazoline and a three-
carbon linker, also showed 155-fold selectivity of HDAC6
versus HDAC8. All the tested compounds exhibited HDAC6
inhibitory activity and selectivity comparable to or superior to
that of SAHA and ACY-1215, demonstrating that the strategy
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of introducing quinazoline analogues as the cap group is
successful.
Table 4. HDAC Inhibition Activity of Compound 23bb
a
IC50 (nM)
To investigate the position of side chain on the HDAC6
activity, we changed the hydroxamic acid side chain from C-4 to
C-3 position and with (23ba−bd) or without methoxyl group
(23aa−ad) at the C-4 position. As shown in Table 2, the
activities of compounds 23aa−ad on HDAC6 were all
decreased with various carbon chain lengths, and the IC50
values of 23aa−ad on HDAC6 were above 1 μM. In contrast,
23bb−bd with methoxyl group at the C-4 position dramatically
increased the activity and selectivity on HDAC6. 23bb−bd
significantly inhibited the HDAC6, especially compound 23bb,
which had achieved 25-fold selectivity to HDAC1 and 200-fold
selectivity to HDAC8 with an IC50 value of 17 nM on HDAC6.
These results indicated that the substitute on C-4 was necessary
to keep the activity and selectivity on HDAC6 and introduction
of a methoxyl group to the C-4 position was a successful
strategy for the selectivity of HDAC6. Interestingly, 23ba, with
one carbon chain and compound 27, where the methyl was
replaced by hydrogen, completely lost their activity of HDAC1,
-6, and -8 with IC50 values above 1 μM. Compound 23bb, with
three-carbon chain length, was the best one for the selectivity
and activity on HDAC6. In comparison to 7a, 23bb increased
two carbon chain lengths at the C-3 position, and its
maintained activity on HDAC6 may contribute to the change
of the carbon chain from the C-4 to C-3 position.
isoform
23bb
ACY-1215
Class I HDACs
LBH-589
SAHA
HDAC1
HDAC2
HDAC3
HDAC8
422
1
7
9
38
95
5
9
1
3
2
5
0.1
11
1
386
439
0.05
0.1
35 11
135 27
254 57
30
5
3398 487
0.4
172 45
Class IIa HDACs
>10000
HDAC4
HDAC5
HDAC7
HDAC9
>10000
>10000
>10000
>10000
338 17
190 35
4354 267
888 26
>10000
>10000
>10000
>10000
>10000
>10000
>10000
Class IIb HDACs
HDAC6
17
2
9
2
4
4
0.2
1
15
3
HDAC10
1176 168
194 54
Class IV HDAC
>10000
170 10
HDAC11
>10000
4112 84
>10000
a
Compounds were tested in the 10-dose IC50 mode in duplicate with
3-fold serial dilutions starting at 10 μM. The IC50 values are the mean
of at least two experiments.
HDACs (HDAC4, -5, -7, -9, -11), 23bb did not show
appreciable inhibition at 10 μM. The HDAC6 selectivity of
compound 23bb is superior to ACY-1215, LBH-589, and
SAHA. Although the ACY-1215 is known as a selective
HDAC6 inhibitor, the selective indexes of HDAC6 with class I
isoforms were lower than 23bb. The results indicate that 23bb
is a potential candidate for further study as a selective HDAC6
inhibitor.
In Vitro Antiproliferative Activities of 23bb. The
antiproliferative activities against 11 kinds of hematological
tumors (myelomaU266, RPMI8226 cells, human leukemia
MV4-11, K562 cells, and human B cell lymphoma Ramos cells)
or solid tumors (ovarian cancer A2780s, SKOV-3 cells, breast
cancer SKBR3 cells, liver cancer HepG2 cells, lung cancer
H460, A549 cells, cervical cancer HeLa cells and colon cancer
HCT116, HT29 cells) cell lines of the compound 23bb were
evaluated by MTT, and the SAHA and ACY-1215 were as
positive control. Compound 23bb showed significant anti-
proliferative potential with the IC50 values ranging from 14 to
104 nM in these tumor cell lines. In contrast, the selective
inhibitor ACY-1215 was weakly active to all the cell lines, with
IC50 values above 5 μM. SAHA was weakly active to the solid
tumor cell lines, such as A2780s, SKOV-3, SKBR3, HepG2,
H460, A549, HCT116, and HT29, with IC50 values above 1
μM. It turned out that the antiproliferative activities of 23bb
were better than ACY-1215 and SAHA (Table 5), and 23bb
could be used in the therapy of solid tumors.
Selective Upregulation of the Aceylation Level of α-
Tubulin. The effects of 23bb, ACY-1215, LBH-589, and SAHA
on the acetylation level of histone H3 (a known substrate for
HDACs 1, 2, and 3) and α-tubulin (a known substrate for
HDAC6), the biomarkers of HDAC inhibition,50 in HCT116
and MV4-11 cells were measured by Western blot. As shown in
Figure 3, in agreement with the relative potency in HDAC6
inhibition and Tub-Ac assays, 23bb and ACY-1215 induced Ac-
α-tubulin in a concentration-dependent manner and could
upgrade the Ac-α-tubulin level at 10 nM, while increase of
histone acetylation was only observed at high concentration of
1000 nM. LBH-589 and SAHA, the nonselective pan-HDAC
Then 7a, 10a, 16a, and 23bb, which showed over 15-fold
selectivity of HDAC6 versus HDAC1 and 100-fold selectivity of
HDAC6 versus HDAC8, were evaluated in tubulin acetylation
(Tub-Ac)27,39,49 and H3 acetylation (Ac-H3) as a surrogate for
cellular HDAC6 inhibition. As shown in Table 3, compounds
Table 3. Tubulin Acetylation and H3 Acetylation Induction
and Antiproliferative Activities against A375 and HeLa Cells
of Selected Compounds
a
b
EC50 (nM)
IC50 (nM)
compd
Tub-Ac Ac-H3
A375
HeLa
7a
26.2
49.4
50.8
58.9
2
6
5
6
456.5 24
1260 187
1767 302
1741 156
213 11
432 27
2851 54
171 20
379 36
2217 62
49 20
10a
16a
23bb
45
7
a
EC50 value values of tubulin acetylation and Ac-H3 are based on
b
ELISA experiments run in duplicate in HeLa cells. IC50 = compound
concentration required to inhibit tumor cell proliferation by 50%. Data
are expressed as the mean SEM from the dose−response curves of
at least three independent experiments.
showed strong Tub-Ac activities and low Ac-H3 induction in
cellular assays, which were in line with the in vitro HDAC
inhibitory activities. We then evaluated the antiproliferative
activity on human malignant melanoma A375 cells and cervical
cancer HeLa cells, 23bb showed the most potent activities with
IC50 values of 50 and 49 nM on A375 and HeLa cells,
respectively.
Since 23bb showed both potent antiproliferative activity and
selectivity of HDAC6, it was further determined for the activity
on the other HDAC isoforms. As summarized in Table 4,
compound 23bb was potent at 400 nM against HDAC1−3 and
at micromolar level against HDAC8 and HDAC10. 23bb
showed 25-fold and 200-fold selectivity of HDAC6 relative to
the inhibition of HDAC1 and HDAC8 in comparison to 4-fold
and 21-fold of ACY-1215. With regard to class IIa and class IV
F
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Docking Results. The molecular docking with compound
23bb in HDAC6 was performed, and the results are shown
Figure 4. The hydroxamic tail of compound 23bb forms key H-
bonds with Tyr298 and Glu255. These hydrogen bond forces
are critical for stabilizing inhibitors in a specific pose chelating
with Zn ion. More importantly, the quinazoline of compound
23bb occupies the cap region and comfortably locks into the
surface groove. The ring of quinazoline could form π−π
stacking interaction with residues of Phe136 and Phe82,
hydrophobic interaction with residues of Ile85, Met198, and
Gly135. Additionally, the nitrogen atom of quinazoline could
form H-bond with residues of Gly135. And yet in the same
docking procedures in HDAC1 and HDAC8, compound 23bb
could not enough occupy the cap region and form the similar
interaction (Supporting Information Figure S4). And this
shows good agreement with the results of biological activity.
Pharmacokinetic Studies. 23bb (dissolved in ddH2O for
intravenous administration and 0.5% CMC-Na aqueous
solution for oral dosing) was administered to SD rats
intravenously (iv) at 12 mg/kg body weight and orally at 12
mg/kg body weight. Blood samples were taken, and the plasma
was analyzed for concentration of 23bb using an LC−MS/MS
system. As shown in Table 6, low clearance and long terminal
half-life are observed in 23bb. The oral bioavailability of 23bb is
excellent in rats and the bioavailability was up to 47.0%,
Table 5. Activities of 23bb and SAHA against various tumor
cell Lines
a
IC50 (nM)
tumor type
multiple
myeloma
cell line
U266
23bb
ACY-1215
>5000
SAHA
14
6
581 96
RPMI8226
MV4-11
K562
15
4
1468 310
>5000
423 54
85 19
444 43
>1000
leukemia
60 32
63 18
71 31
>5000
B cell
lymphoma
Ramos
>5000
ovarian
A2780s
SKOV-3
SKBR3
HepG2
H460
46 11
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>1000
>1000
>1000
>1000
>1000
>1000
711 245
>1000
>1000
50
22
41
8
8
1
breast
liver
lung
55 13
104 29
49 20
35 21
72 37
A549
cervical cancer
colon
HeLa
HCT116
HT29
a
IC50 = compound concentration required to inhibit tumor cell
proliferation by 50%. Data are expressed as the mean SEM from the
dose−response curves of at least three independent experiments.
inhibitors, increased both Ac-H3 and Ac-α-tubulin levels at low
concentration.
Figure 3. Western blot analysis of acetylated α-tubulin, acetylated histone H3 inHCT116, and MV4-11cell lines after 6 h of treatment with
compound 23bb, ACY-1215, LBH-589, and SAHA at 10, 100, 1000, 10000 nM. GAPDH was used as a loading control.
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Antitumor Activity in Vivo. We first evaluated the in vivo
efficacy of 23bb using a HCT116 xenograft Balb/c nude mouse
model and a MV4-11 xenograft NOD/SCID mouse model,
with SAHA administrated for comparison. The administration,
dosing schedules, and results are presented in Table 7.
As displayed in Table 7, 23bb reduced the tumor growth in
both the hematological tumor MV4-11 xenograft model and
solid tumor HCT116 xenograft model, and the tumor
inhibitory effects were superior to positive drug SAHA. The
significant antitumor activities were observed by intravenous
administration of 23bb at 50 mg/kg on MV4-11 and HCT116
xenograft models. The growth of MV4-11 and HCT116 cancer
cell xenografts was suppressed by 55.0% and 76.3% (percent of
tumor mass change [TGI] values) after iv administration of
23bb at 50 mg/kg. In contrast, SAHA had no inhibitory activity
at the same dose on the MV4-11 xenograft model and only
showed 36.6% tumor growth inhibition on HCT116 xenograft
model. We also established the HCT116 xenograft model to
investigate the antitumor activity of oral administration of
23bb. As displayed in Table 7, the TGI value of oral
administration of 23bb (25 mg/kg) on HCT116 xenograft
model was 60.4%, which was superior to the SAHA group (100
mg/kg, 59.2%). Additionally, the body weight decrease is
acceptable and no other adverse effects were observed upon
treatment with 23bb (Figure 5).
Figure 4. Compound 23bb docked into HDAC6 (homology model).
Table 6. Pharmacokinetic Parameters Tested in Vivo
Since LBH589 is a new pan-inhibitor of HDAC approved by
FDA for the treatment of hematologic cancer, we further
established a Ramos xenograft NOD/SCID female B cell
lymphoma model to compare the in vivo activity of 23bb with
ACY1215 and LBH589. As shown in Table 7, 23bb inhibited
the tumor growth dose-dependently; the TGI values were
22.5% and 58.79% at 40 and 80 mg/kg, respectively, by oral
administration. In contrast, ACY-1215 had no effect at 40 mg/
kg. Although LBH-589 at ip 10 mg/kg administration caused
the tumor reduction with the TGI values of 16.6%, the toxicity
(body weight of mice decreased and three of eight mice died
during experimental period) of LBH 589 was observed. Those
results indicated that 23bb is more effective and safe in contrast
to ACY-1215 and LBH-589, suggesting that 23bb could be
used as a novel potent compound for further research on the
therapy of both hematological tumor and solid tumor.
23bb
route
iv
po
a
N
6
6
dose (mg/kg)
12
12
b
CL (L h−1 kg−1
)
7.008
12.877
154.811
1242.234
238.133
9.62
c
Vss (L/kg)
61.263
2434.117
2213.217
7.658
d
AUC0−inf (μg/L·h)
e
Cmax (μg/L)
f
T1/2 (h)
g
F (%)
47.0
a
b
c
Numbers of rats. Systemic clearance. Volume of distribution
d
following intravenous dosing. Area under the curve following
intravenous dosing, integrated drug concentration with respect to
time and integrated drug concentration with respect to time following
oral dosing. Maximum plasma concentration following intravenous
dosing. plasma half-life. Percent oral bioavailability.
e
f
g
CONCLUSIONS
■
To develop novel HDAC6 selective inhibitors with a
quinazoline cap group, a series of hydroxamic acid analogues
were prepared and evaluated for bioactivity in vitro and in vivo.
suggesting that 23bb is suitable both for iv and oral dosing as
an anticancer agent.
Table 7. Summary of Tumor Growth Inhibition
administration
toxicity
a
tumor model
MV4-11
compd
23bb
dose (mg/kg)
schedule
route
body weight change (%)
death
antitumor activity, tumor mass change (%)
50
50
50
50
25
100
40
80
40
10
Q2D × 12
Q2D × 12
Q2D × 9
Q2D × 9
Q2D × 8
Q2D × 8
Q2D × 6
Q2D × 6
Q2D × 6
Q2D × 6
iv
−3.8
0/6
0/6
0/7
0/7
0/7
0/7
0/8
1/8
0/8
3/8
55.0
0
SAHA
23bb
ip
HCT116
HCT116
Ramos
iv
−5.7
−3.0
−3.8
−6.1
76.3
36.6
60.4
59.2
22.5
58.79
−6.4
16.6
SAHA
23bb
ip
po
po
po
po
po
ip
SAHA
23bb
ACY-1215
LBH-589
−1.9
a
Q2D, every 2 days.
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Figure 5. Inhibitory effect of 23bb on the xenograft models: (A) 23bb inhibited tumor growth on the MV4-11 xenograft model; (B) 23bb inhibited
tumor growth on the HCT116 xenograft model; (C) 23bb inhibited tumor growth on the Ramos xenograft model.
The inhibitory activities on the HDAC1, HDAC6, and HDAC8
isoforms of the synthesized compounds and a SAR analysis
revealed that the strategy successfully achieved the goal to find
a novel selective and highly efficient small molecule inhibitor of
HDAC6. The most potent compound 23bb displayed at least
22.7-fold selectivity for HDAC6 over the other isoforms with
an IC50 value of 17 nM. The antiproliferation assay revealed
that the IC50 values of 23bb ranged from 14 to 104 nM against
11 diverse cancer cell lines, representing both the hematological
and solid models. Subsequently, the Western blot analysis
further confirmed the selectivity profile of compound 23bb.
23bb, with the oral bioavailability of 47.0% in rats, showed
substantial antitumor activity in both solid and hematologic
tumor models. 23bb caused tumor growth inhibition of 55.0%
in human AML MV4-11 xenograft model and 76.3%, 60.4% in
human colorectal HCT116 xenograft model after iv treatment
with 50 mg/kg and po treatment with 25 mg/kg, respectively,
which was more efficient than SAHA groups (0 for MV4-11
xenograft models and 36.6%, 59.2% for HCT116 xenograft
models, 50 mg/kg, ip, and 100 mg/kg, po, respectively). In a
Ramos xenograft NOD/SCID female B cell lymphoma model,
23bb exhibited superior inhibitory activity on tumor growth to
ACY-1215 and LBH-589, suggesting that 23bb is a potential
and promising oral anticancer agent with selective HDAC6
inhibitory properties.
EXPERIMENTAL SECTION
■
Chemistry. All the chemical solvents and reagents, which were
analytically pure without further purification, were commercially
available. TLC was performed on 0.20 mm silica gel 60 F254 plates
(Qingdao Haiyang Chemical, China). 1H NMR and 13C NMR spectra
were on a Bruker Avance 400 spectrometer (Bruker Company,
Germany), using TMS as an internal standard. Chemical shifts were
given in ppm (parts per million). Mass spectra were recorded on Q-
TOF Priemier mass spectrometer (Micromass, Manchester, U.K.).
The purity of each compound (>95%) was determined on an Waters
e2695 series LC system (column, Xtimate C18, 4.6 mm × 150 mm, 5
μm; mobile phase, methanol (90%)/H2O (10%) to methanol (20%)/
H2O (80%); flow rate, 1.0 mL/min; UV wavelength, 254−400 nm;
temperature, 25 °C; injection volume, 10 μL).
General Procedures of Method A for the Synthesis of 2, 18a,
and 18b. Phenol (200 mmol) was dissolved in DMF (500 mL), and
the resulting solution was cooled to 0 °C. 60% NaH (15.36 g, 400
mmol, 2 equiv) was added in portions. After 0.5 h, the MOMCl (30.4
mL, 400 mmol, 2 equiv) was added dropwise. The reaction mixture
was moved to room temperature and monitored by TLC (petroleum
ether/ethyl acetate, 2:1). When the reaction was completed, the
resulting solution was poured into water (5 L), affording a yellow
solid. This solid was collected by filtration and purified by
recrystallization from EtOH to give the title compounds as yellow
acicular crystal.
General Procedures of Method B for the Synthesis of 3, 19a,
19b, and 25. The nitro compound was dissolved in EtOH (500 mL),
and 10% Pd/C (5%) was added. Then the mixture was stirred at room
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temperature under H2. When the reaction was finished, the mixture
was filtered by Celite and washed by ethyl acetate. The filtrate was
concentrated in vacuo to give the title compounds as red solid, used in
the next step without further purification.
2.48 (d, J = 5.7 Hz, 5H), 2.02 (p, J = 6.8 Hz, 2H), 1.18 (t, J = 7.1 Hz,
3H).
General Procedures of Method E for the Synthesis of 6a−e,
9a−e, 15a−e, 22aa−ae, 22ba−be, and 24. The phenol
intermediate (1 mmol, 1 equiv) and Cs2CO3 (652 mg, 2 mmol, 2
equiv) were added into 5 mL of CH3CN, and the resulting mixture
was heated at reflux for 0.5 h. Then the bromo-fatty acid ethyl ester (2
mmol, 2 equiv) was added dropwise. After 2 h, the solvent was filtered
through Celite. The filtrate was concentrated in vacuo to yield a yellow
oily product, without further purification.
General Procedures of Method C for the Synthesis of 4, 20a,
and 20b. An amount of 500 mL of MeOH was added into a 1 L
bottle and cooled to 0 °C. Na (13.8 g, 600 mmol, 5 equiv) was added
in portions. The resulting mixture was moved to room temperature.
When Na was dissolved, aniline (120 mmol, 1 equiv) and
paraformaldehyde (5 g, 168 mmol, 1.4 equiv) were added. The
mixture was stirred overnight. Then NaBH4 (4.54 g, 120 mmol, 1
equiv) was added, and the resulting solution was reflux for 2 h and
concentrated in vacuo. An amount of 1 L of NaOH (1 N) was added
to the residue, affording a white solid. This solid was collected by
filtration and dried to give the title compound.
General Procedures of Method D for the Synthesis of 5, 8,
14, 21a, 21b, and 26. (i) Quinazoline analogues (85 mmol, 1 equiv)
and N-methylaniline (85 mmol, 1 equiv) were added to 500 mL of
(Me)2CHOH. Then 1 mL of concentrated HCl was added to the
mixture and stirred at room temperature. The reaction generated a lot
of yellow solid precipitation, which was collected by filtration and
basified by saturated NaHCO3 aqueous solution. The resulting mixture
was extracted with ethyl acetate (3 × 200 mL). The organic layer was
collected without further treatment. (ii) The previously collected
organic layer was placed into a three-neck bottle. Then HCl gas was
passed into the solvent, which was stirred at room temperature. The
reaction was monitored by TLC (petroleum ether/ethyl acetate, 1:2).
With the reaction going on, a lot of yellow solid was formed. When the
reaction was finished, the mixture was filtered. The filter cake was
added into saturated NaHCO3 aqueous solution. The resulting
mixture was filtered again, giving a white solid which was recrystallized
with EtOH to give the title compound as white solid.
Ethyl 2-(4-(Methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)acetate (6a). 6a was obtained from compound 5 and
1
ethyl bromoacetate as described for method E. H NMR (400 MHz,
CDCl3) δ: 7.74 (d, J = 8.3 Hz, 1H), 7.51 (ddd, J = 8.3, 6.7, 1.6 Hz,
1H), 7.10 (d, J = 8.9 Hz, 2H), 7.03−6.93 (m, 2H), 6.91 (d, J = 8.9 Hz,
2H), 4.63 (s, 2H), 4.28 (q, J = 7.1 Hz, 2H), 3.58 (s, 3H), 2.72 (s, 3H),
1.30 (t, J = 7.1 Hz, 3H).
Ethyl 4-(4-(Methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)butanoate (6b). 6b was obtained from compound 5 and
ethyl 4-bromobutyrate as described for method E. 1H NMR (400
MHz, CDCl3) δ: 7.74 (d, J = 8.3 Hz, 1H), 7.55−7.48 (m, 1H), 7.09 (d,
J = 8.8 Hz, 2H), 7.02 (d, J = 7.7 Hz, 1H), 6.97 (dd, J = 11.1, 4.1 Hz,
1H), 6.88 (d, J = 8.8 Hz, 2H), 4.16 (q, J = 7.2 Hz, 2H), 4.02 (t, J = 6.1
Hz, 2H), 3.57 (s, 3H), 2.72 (s, 3H), 2.53 (t, J = 7.3 Hz, 2H), 2.18−
2.08 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H).
Ethyl 5-(4-(Methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)pentanoate (6c). 6c was obtained from compound 5
1
and ethyl 5-bromovalerate as described for method E. H NMR (400
MHz, CDCl3) δ: 7.73 (d, J = 8.3 Hz, 1H), 7.55−7.47 (m, 1H), 7.09 (d,
J = 8.8 Hz, 2H), 7.02 (d, J = 7.7 Hz, 1H), 6.99−6.93 (m, 1H), 6.88 (d,
J = 8.8 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.98 (s, 2H), 3.57 (s, 3H),
2.71 (s, 3H), 2.40 (t, J = 6.6 Hz, 2H), 1.84 (s, 4H), 1.26 (t, J = 7.1 Hz,
3H).
4-(Methyl(2-methylquinazolin-4-yl)amino)phenol (5). 5 was
obtained from compound 4 and 4-chloro-2-methylquinazoline as
described for method D. H NMR (400 MHz, DMSO-d6) δ: 9.71 (s,
Ethyl 6-(4-(Methyl(2-methylquinazolin-4-yl)amino)-
1
phenoxy)hexanoate (6d). 6d was obtained from compound 5 and
1
ethyl 6-bromohexanoate as described for method E. H NMR (400
1H), 7.68 (d, J = 8.1 Hz, 1H), 7.62 (t, J = 7.1 Hz, 1H), 7.12 (d, J = 8.6
Hz, 2H), 7.08 (t, J = 7.2 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.85 (d, J =
8.6 Hz, 2H), 3.52 (s, 3H), 2.62 (s, 3H).
4-(Methyl(quinazolin-4-yl)amino)phenol (8). 8 was obtained
from compound 4 and 4-chloroquinazoline as described for method D.
1H NMR (400 MHz, DMSO-d6) δ: 10.04 (s, 1H), 8.91 (d, J = 1.8 Hz,
1H), 7.86 (d, J = 7.9 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.28 (t, J = 8.4
Hz, 1H), 7.24 (d, J = 7.3 Hz, 2H), 6.91 (d, J = 8.3 Hz, 3H), 3.64 (d, J
= 1.1 Hz, 3H).
MHz, CDCl3) δ: 7.73 (d, J = 8.3 Hz, 1H), 7.50 (dd, J = 10.8, 4.1 Hz,
1H), 7.08 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.0 Hz, 1H), 6.99−6.92 (m,
1H), 6.87 (d, J = 8.7 Hz, 2H), 4.13 (dd, J = 14.3, 7.1 Hz, 2H), 3.96 (t,
J = 6.2 Hz, 2H), 3.57 (s, 3H), 2.71 (s, 3H), 2.34 (t, J = 7.4 Hz, 2H),
1.88−1.77 (m, 2H), 1.77−1.68 (m, 2H), 1.57−1.47 (m, 2H), 1.26 (t, J
= 7.1 Hz, 3H).
Ethyl 2-(4-(Methyl(quinazolin-4-yl)amino)phenoxy)acetate
(9a). 9a was obtained from compound 8 and ethyl bromoacetate as
described for method E. 1H NMR (400 MHz, CDCl3) δ: 8.80 (s, 1H),
7.84 (d, J = 8.4 Hz, 1H), 7.60−7.55 (m, 1H), 7.15−7.08 (m, 2H), 7.05
(d, J = 3.6 Hz, 2H), 6.94−6.87 (m, 2H), 4.67 (s, 2H), 4.15 (q, J = 7.2
Hz, 2H), 3.60 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H).
Ethyl 4-(4-(Methyl(quinazolin-4-yl)amino)phenoxy)-
butanoate (9b). 9b was obtained from compound 8 and ethyl 4-
bromobutyrate as described for method E. 1H NMR (400 MHz,
CDCl3) δ: 8.80 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.60−7.55 (m, 1H),
7.15−7.08 (m, 2H), 7.05 (d, J = 3.6 Hz, 2H), 6.94−6.87 (m, 2H), 4.16
(q, J = 7.2 Hz, 2H), 4.03 (t, J = 6.1 Hz, 2H), 3.60 (s, 3H), 2.54 (t, J =
7.2 Hz, 2H), 2.18−2.09 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H).
4-(Methyl(2-methyl-6,7-dihydro-5H-cyclopenta[d]pyr-
imidin-4-yl)amino)phenol (14). 14 was obtained from compound 4
1
and compound 13 as described for method D. H NMR (400 MHz,
DMSO-d6) δ: 9.58 (s, 1H), 7.10−7.00 (m, 2H), 6.80−6.72 (m, 2H),
3.32 (s, 3H), 2.58 (t, J = 7.7 Hz, 2H), 2.40 (s, 3H), 1.79 (t, J = 7.2 Hz,
2H), 1.68−1.59 (m, 2H).
3-(Methyl(2-methylquinazolin-4-yl)amino)phenol (21a). 21a
was obtained from compound 20a and 4-chloro-2-methylquinazoline
1
as described for method D. H NMR (400 MHz, DMSO-d6) δ: 9.67
(s, 1H), 8.53 (d, J = 7.8 Hz, 1H), 7.85−7.77 (m, 2H), 7.70 (d, J = 7.4
Hz, 1H), 7.62−7.52 (m, 2H), 7.29 (t, J = 8.2 Hz, 1H), 6.80−6.75 (m,
1H), 3.42 (s, 3H), 2.53 (s, 3H).
2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)-
phenol (21b). 21b was obtained from compound 20b and 4-chloro-
2-methylquinazoline as described for method D. 21 g, 84% yield two
steps. 1H NMR (400 MHz, DMSO-d6) δ: 9.31 (s, 1H), 7.64 (d, J = 8.0
Hz, 1H), 7.59 (ddd, J = 8.3, 6.1, 2.0 Hz, 1H), 7.13−7.03 (m, 2H), 6.94
(dd, J = 7.1, 1.9 Hz, 1H), 6.64 (dd, J = 7.4, 2.2 Hz, 2H), 3.79 (s, 3H),
3.47 (s, 3H), 2.58 (s, 3H).
Ethyl 4-(2-Methoxy-5-((2-methylquinazolin-4-yl)amino)-
phenoxy)butanoate (26). 26 was obtained from compound 25
and 4-chloro-2-methylquinazoline as described for method D step (i):
85.3% yield. 1H NMR (400 MHz, DMSO-d6) δ: 9.56 (s, 1H), 8.47 (d,
J = 8.2 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.53
(t, J = 7.5 Hz, 1H), 7.43 (dd, J = 8.7, 2.2 Hz, 1H), 6.98 (d, J = 8.8 Hz,
1H), 4.07 (q, J = 7.1 Hz, 2H), 4.02 (t, J = 6.4 Hz, 2H), 3.77 (s, 3H),
Ethyl 5-(4-(Methyl(quinazolin-4-yl)amino)phenoxy)-
pentanoate (9c). 9c was obtained from compound 8 and ethyl 5-
bromovalerate as described for method E. 1H NMR (400 MHz,
CDCl3) δ: 8.80 (s, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.62−7.54 (m, 1H),
7.15−7.08 (m, 2H), 7.05 (d, J = 3.9 Hz, 2H), 6.94−6.87 (m, 2H), 4.14
(q, J = 7.1 Hz, 2H), 4.00 (t, J = 5.6 Hz, 2H), 3.60 (s, 3H), 2.40 (t, J =
6.9 Hz, 2H), 1.90−1.80 (m, 4H), 1.27 (t, J = 7.1 Hz, 3H).
Ethyl 6-(4-(Methyl(quinazolin-4-yl)amino)phenoxy)-
hexanoate (9d). 9d was obtained from compound 8 and ethyl 6-
1
bromohexanoate as described for method E. H NMR (400 MHz,
CDCl3) δ: 8.80 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.60−7.55 (m, 1H),
7.15−7.08 (m, 2H), 7.05 (d, J = 3.7 Hz, 2H), 6.93−6.87 (m, 2H), 4.14
(q, J = 7.1 Hz, 2H), 3.98 (t, J = 6.4 Hz, 2H), 3.60 (s, 3H), 2.35 (t, J =
7.4 Hz, 2H), 1.89−1.79 (m, 2H), 1.79−1.69 (m, 2H), 1.60−1.48 (m,
2H), 1.26 (t, J = 7.1 Hz, 3H).
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Ethyl 2-(4-(Methyl(2-methyl-6,7-dihydro-5H-cyclopenta[d]-
pyrimidin-4-yl)amino)phenoxy)acetate (15a). 15a was obtained
from compound 14 and ethyl bromoacetate as described for method E.
1H NMR (400 MHz, DMSO-d6) δ: 7.17 (d, J = 8.8 Hz, 2H), 6.94 (d, J
Ethyl 4-(2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)-
amino)phenoxy)butanoate (22bb). 22bb was obtained from
compound 21b and ethyl 4-bromobutyrate as described for method
1
E. H NMR (400 MHz, DMSO-d6) δ: 7.63 (d, J = 8.1 Hz, 1H), 7.57
(t, J = 7.5 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H),
6.96 (d, J = 3.7 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.72 (dd, J = 8.5, 2.4
Hz, 1H), 4.04 (q, J = 7.1 Hz, 2H), 3.90 (t, J = 6.4 Hz, 2H), 3.78 (s,
3H), 3.50 (s, 3H), 2.58 (s, 3H), 2.39 (t, J = 7.3 Hz, 2H), 1.88 (p, J =
6.8 Hz, 2H), 1.16 (t, J = 7.1 Hz, 3H).
Ethyl 5-(2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)-
amino)phenoxy)pentanoate (22bc). 22bc was obtained from
compound 21b and ethyl 5-bromovalerate as described for method E.
1H NMR (400 MHz, DMSO-d6) δ: 7.64 (d, J = 8.2 Hz, 1H), 7.57 (t, J
= 8.9 Hz, 2H), 4.81 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.35 (s, 3H),
2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 1.77 (t, J = 7.2 Hz, 2H), 1.70−
1.59 (m, 2H), 1.21 (t, J = 7.1 Hz, 3H).
Ethyl 4-(4-(Methyl(2-methyl-6,7-dihydro-5H-cyclopenta[d]-
pyrimidin-4-yl)amino)phenoxy)butanoate (15b). 15b was ob-
tained from compound 14 and ethyl 4-bromobutyrate as described for
1
method E. H NMR (400 MHz, DMSO-d6) δ: 7.15 (d, J = 8.7 Hz,
2H), 6.93 (d, J = 8.8 Hz, 2H), 4.06 (q, J = 7.1 Hz, 2H), 3.97 (t, J = 5.9
Hz, 2H), 3.34 (s, 3H), 2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 2.35 (t, J
= 7.0 Hz, 2H), 1.80−1.55 (m, 6H), 1.18 (t, J = 7.1 Hz, 3H).
Ethyl 5-(4-(Methyl(2-methyl-6,7-dihydro-5H-cyclopenta[d]-
pyrimidin-4-yl)amino)phenoxy)pentanoate (15c). 15c was ob-
tained from compound 14 and ethyl 5-bromovalerate as described for
= 7.5 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H),
6.97−6.90 (m, 2H), 6.70 (dd, J = 8.5, 2.3 Hz, 1H), 4.04 (q, J = 7.1 Hz,
3H), 3.88 (t, J = 5.8 Hz, 2H), 3.77 (s, 3H), 3.50 (s, 3H), 2.58 (s, 3H),
2.31 (t, J = 6.9 Hz, 2H), 1.67−1.59 (m, 4H), 1.17 (t, J = 7.1 Hz, 3H).
Ethyl 6-(2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)-
amino)phenoxy)hexanoate (22bd). 22bd was obtained from
compound 21b and ethyl 6-bromohexanoate as described for method
1
method E. H NMR (400 MHz, DMSO-d6) δ: 7.15 (d, J = 8.7 Hz,
2H), 6.93 (d, J = 8.8 Hz, 2H), 4.06 (q, J = 7.1 Hz, 2H), 3.98 (t, J = 5.9
Hz, 2H), 3.34 (s, 3H), 2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 2.37 (t, J
= 7.0 Hz, 2H), 1.83−1.59 (m, 8H), 1.18 (t, J = 7.1 Hz, 3H).
Ethyl 6-(4-(Methyl(2-methyl-6,7-dihydro-5H-cyclopenta[d]-
pyrimidin-4-yl)amino)phenoxy)hexanoate (15d). 15d was ob-
tained from compound 14 and ethyl 6-bromohexanoate as described
for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.15 (d, J = 8.8 Hz,
2H), 6.93 (d, J = 8.8 Hz, 2H), 4.05 (q, J = 7.1 Hz, 2H), 3.96 (t, J = 6.4
Hz, 2H), 3.34 (s, 3H), 2.59 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 2.31 (t, J
= 7.3 Hz, 2H), 1.78 (t, J = 7.2 Hz, 2H), 1.75−1.69 (m, 2H), 1.69−1.54
(m, 4H), 1.47−1.38 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H).
1
E. H NMR (400 MHz, DMSO-d6) δ: 7.64 (d, J = 8.1 Hz, 1H), 7.57
(t, J = 6.8 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H),
6.94 (d, J = 8.8 Hz, 2H), 6.70 (dd, J = 8.5, 2.4 Hz, 1H), 4.04 (q, J = 7.1
Hz, 3H), 3.86 (t, J = 6.5 Hz, 2H), 3.77 (s, 3H), 3.50 (s, 3H), 2.58 (s,
3H), 2.27 (t, J = 7.3 Hz, 4H), 1.65−1.57 (m, 2H), 1.57−1.47 (m, 2H),
1.40−1.28 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H).
Ethyl 4-(2-Methoxy-5-nitrophenoxy)butanoate (24). 24 was
obtained from compound 17b and ethyl 4-bromobutyrate as described
1
for method E. H NMR (400 MHz, DMSO-d6) δ: 9.55 (s, 1H), 8.47
Ethyl 2-(3-(Methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)acetate (22aa). 22aa was obtained from compound 21a
(d, J = 8.0 Hz, 1H), 7.78 (t, J = 7.1 Hz, 1H), 7.70−7.64 (m, 2H), 7.53
(t, J = 7.5 Hz, 1H), 7.43 (dd, J = 8.7, 2.4 Hz, 1H), 6.98 (d, J = 8.8 Hz,
1H), 4.07 (q, J = 7.1 Hz, 2H), 4.01 (t, J = 6.4 Hz, 2H), 3.77 (s, 3H),
2.50−2.45 (m, 5H), 2.07−1.97 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H).
General Procedures of Method F for the Synthesis of
Hydroxamic Acid Derivatives. The ester intermediate (1 mmol, 1
equiv) was dissolved in CH2Cl2 and methanol (1:2, 9 mL). The
resulting solution was cooled to 0 °C, then hydroxamic (50 wt % in
water, 1 mL, 30 mmol, 30 equiv) and NaOH (0.4 g, 10 mmol, 10
equiv) were added. At the temperature, the reaction was stirred for 1 h.
The solvent was then removed under reduced pressure, and the
obtained solid was dissolved in water, which was adjusted to pH 7−8
by acetic acid and extracted with ethyl acetate (3 × 50 mL). The
organic layer was collected and dried over anhydrous MgSO4. After the
removal of MgSO4 by filtration, the filtrate was concentrated in vacuo
to yield a solid product which recrystallized with EtOH to give the title
compound.
1
and ethyl bromoacetate as described for method E. H NMR (400
MHz, DMSO-d6) δ: 7.71 (d, J = 8.2 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H),
7.34 (t, J = 8.1 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 6.99 (d, J = 8.5 Hz,
1H), 6.93 (dd, J = 14.1, 8.0 Hz, 3H), 4.69 (s, 2H), 4.13 (q, J = 7.1 Hz,
2H), 3.31 (s, 3H), 2.54 (s, 3H), 1.19 (t, J = 7.0 Hz, 3H).
Ethyl 4-(3-(Methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)butanoate (22ab). 22ab was obtained from compound
1
21a and ethyl 4-bromobutyrate as described for method E. H NMR
(400 MHz, DMSO-d6) δ: 7.67 (d, J = 8.1 Hz, 1H), 7.60 (t, J = 7.2 Hz,
1H), 7.31 (t, J = 8.1 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 7.01−6.97 (m,
1H), 6.94 (s, 1H), 6.88 (s, 1H), 6.82 (d, J = 8.0 Hz, 1H), 4.13 (t, J =
7.0 Hz, 2H), 4.01 (q, J = 7.1 Hz, 2H), 3.31 (s, 3H), 2.59 (s, 3H), 2.40
(t, J = 7.1 Hz, 2H), 1.98−1.89 (m, 2H), 1.14 (t, J = 7.1 Hz, 3H).
Ethyl 5-(3-(Methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)pentanoate (22ac). 22ac was obtained from compound
1
21a and ethyl 5-bromovalerate as described for method E. H NMR
N-Hydroxy-2-(4-(methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)acetamide (7a). 7a was obtained from compound 6a as
described for method F: mp 156−158 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.86 (s, 1H), 9.00 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H),
7.59 (t, J = 7.4 Hz, 1H), 7.21 (d, J = 8.8 Hz, 2H), 7.06 (t, J = 7.2 Hz,
1H), 7.00 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.6 Hz, 1H), 4.49 (s, 2H),
3.50 (s, 3H), 2.59 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 164.06,
162.33, 161.03, 156.05, 151.71, 141.57, 131.84, 127.50, 127.09, 125.64,
124.05, 116.02, 114.14, 66.03, 42.30, 26.12. MS (ESI, m/z): 339.16 [M
+ H]+.
N-Hydroxy-4-(4-(methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)butanamide (7b). 7b was obtained from compound 6b as
described for method F: mp 174−176 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.43 (s, 1H), 8.75 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H),
7.58 (t, J = 7.4 Hz, 1H), 7.18 (d, J = 8.6 Hz, 2H), 7.06 (t, J = 7.4 Hz,
1H), 6.97 (d, J = 8.3 Hz, 3H), 3.97 (t, J = 6.1 Hz, 2H), 3.49 (s, 3H),
2.59 (s, 3H), 2.14 (t, J = 7.2 Hz, 2H), 2.02−1.88 (m, 2H). 13C NMR
(101 MHz, DMSO-d6) δ: 168.56, 162.32, 161.00, 156.85, 151.71,
140.97, 131.81, 127.48, 127.16, 125.65, 124.02, 115.78, 114.13, 67.07,
42.32, 28.66, 26.12, 24.75, 18.53. MS (ESI, m/z): 367.18 [M + H]+.
N-Hydroxy-5-(4-(methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)pentanamide (7c). 7c was obtained from compound 6c as
described for method F: mp 155−157 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.38 (s, 1H), 8.70 (s, 1H), 7.65 (d, J = 7.8 Hz, 1H),
(400 MHz, DMSO-d6) δ: 7.66 (d, J = 8.2 Hz, 1H), 7.60 (t, J = 7.3 Hz,
1H), 7.31 (t, J = 8.1 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.96 (t, J = 8.1
Hz, 2H), 6.86 (s, 1H), 6.80 (d, J = 7.8 Hz, 1H), 4.10 (t, J = 7.1 Hz,
2H), 4.04−3.97 (m, 2H), 3.31 (s, 3H), 2.59 (s, 3H), 2.36 (t, J = 7.1
Hz, 2H), 1.68 (dd, J = 14.1, 7.2 Hz, 2H), 1.60 (dd, J = 14.6, 7.4 Hz,
2H), 1.13 (t, J = 7.1 Hz, 3H).
Ethyl 6-(3-(Methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)hexanoate (22ad). 22ad was obtained from compound
21a and ethyl 6-bromohexanoate as described for method E. 1H NMR
(400 MHz, DMSO-d6) δ: 7.66 (d, J = 8.2 Hz, 1H), 7.59 (t, J = 7.3 Hz,
1H), 7.31 (t, J = 8.1 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 6.96 (dd, J =
9.6, 3.9 Hz, 2H), 6.87 (s, 1H), 6.81 (d, J = 8.2 Hz, 1H), 4.04 (dq, J =
14.6, 7.2 Hz, 6H), 3.31 (s, 3H), 2.59 (s, 3H), 2.34−2.22 (m, 4H), 1.67
(dd, J = 14.4, 7.2 Hz, 2H), 1.59 (dd, J = 14.9, 7.5 Hz, 2H), 1.55−1.47
(m, 2H), 1.40 (dd, J = 14.1, 6.7 Hz, 2H), 1.16 (dt, J = 14.3, 7.2 Hz,
6H).
Ethyl 2-(2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)-
amino)phenoxy)acetate (22ba). 22ba was obtained from com-
1
pound 21b and ethyl bromoacetate as described for method E. H
NMR (400 MHz, DMSO-d6) δ: 7.64 (d, J = 8.1 Hz, 1H), 7.58 (t, J =
7.4 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02−6.96 (m, 2H), 6.92 (d, J =
2.2 Hz, 1H), 6.75 (dd, J = 8.5, 2.2 Hz, 1H), 4.74 (s, 2H), 4.01 (q, J =
7.1 Hz, 2H), 3.80 (s, 3H), 3.48 (s, 3H), 2.58 (s, 3H), 1.10 (t, J = 7.1
Hz, 3H).
K
DOI: 10.1021/acs.jmedchem.5b01342
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7.58 (t, J = 7.5 Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 7.06 (t, J = 7.1 Hz,
1H), 6.97 (d, J = 8.8 Hz, 3H), 3.98 (t, J = 5.8 Hz, 2H), 3.49 (s, 3H),
2.59 (s, 3H), 2.03 (t, J = 6.9 Hz, 2H), 1.75−1.60 (m, 4H). 13C NMR
(101 MHz, DMSO-d6) δ: 168.87, 162.32, 160.99, 156.96, 151.70,
140.88, 131.79, 127.47, 127.14, 125.65, 124.01, 115.73, 114.13, 67.33,
42.32, 31.87, 28.13, 26.11, 21.75. MS (ESI, m/z): 381.19 [M + H]+.
N-Hydroxy-6-(4-(methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)hexanamide (7d). 7d was obtained from compound 6d as
described for method F: mp 113−115 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.36 (s, 1H), 8.70 (s, 1H), 7.65 (d, J = 7.6 Hz, 1H),
7.62−7.55 (m, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.06 (t, J = 7.1 Hz, 1H),
6.97 (d, J = 8.8 Hz, 3H), 3.96 (t, J = 6.4 Hz, 2H), 3.49 (s, 3H), 2.58 (s,
3H), 1.98 (t, J = 7.2 Hz, 2H), 1.79−1.65 (m, 2H), 1.62−1.50 (m, 2H),
1.45−1.32 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 169.01,
161.68, 160.83, 157.40, 139.98, 132.55, 127.22, 125.89, 124.57, 115.84,
113.60, 67.67, 42.67, 32.18, 28.35, 25.12, 24.87. MS (ESI, m/z):
395.34 [M + H]+.
J = 5.8 Hz, 2H), 3.34 (s, 3H), 2.59 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H),
1.98 (t, J = 6.9 Hz, 2H), 1.80 (t, J = 7.5 Hz, 2H), 1.70−1.60 (m, 4H).
13C NMR (101 MHz, DMSO-d6) δ: 172.38, 168.84, 164.44, 158.87,
157.10, 138.25, 128.71, 114.65, 113.48, 67.32, 39.41, 33.30, 31.86,
29.72, 28.19, 25.35, 21.03. MS (ESI, m/z): 357.10 [M + H]+.
N-Hydroxy-5-(4-(methyl(2-methyl-6,7-dihydro-5H-
cyclopenta[d]pyrimidin-4-yl)amino)phenoxy)pentanamide
(16c). 16c was obtained from compound 15c as described for method
F: mp 150−152 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.39 (s, 1H),
8.71 (s, 1H), 7.15 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H), 3.97 (t,
J = 5.8 Hz, 2H), 3.34 (s, 3H), 2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H),
2.03 (t, J = 6.9 Hz, 2H), 1.78 (t, J = 7.2 Hz, 2H), 1.70−1.60 (m, 6H).
13C NMR (101 MHz, DMSO-d6) δ: 172.39, 168.86, 164.48, 158.92,
157.06, 138.29, 128.70, 114.67, 113.44, 67.29, 39.44, 33.27, 31.88,
29.71, 28.16, 25.45, 21.76, 21.18. MS (ESI, m/z): 371.12 [M + H]+.
N-Hydroxy-6-(4-(methyl(2-methyl-6,7-dihydro-5H-
cyclopenta[d]pyrimidin-4-yl)amino)phenoxy)hexanamide
(16d). 16d was obtained from compound 15d as described for method
F: mp 157−159 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.36 (s, 1H),
8.68 (s, 1H), 7.15 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 3.96 (t,
J = 6.3 Hz, 2H), 3.34 (s, 3H), 2.59 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H),
1.99−1.94 (m, 2H), 1.78 (t, J = 7.2 Hz, 2H), 1.72 (dd, J = 14.1, 7.1 Hz,
2H), 1.64 (dt, J = 15.0, 7.4 Hz, 2H), 1.56 (dt, J = 14.9, 7.3 Hz, 2H),
1.39 (dt, J = 14.5, 7.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ:
172.37, 168.96, 164.48, 158.92, 157.09, 138.26, 128.70, 114.65, 113.42,
67.56, 39.43, 33.26, 32.19, 29.70, 28.40, 25.45, 25.15, 24.89, 21.17. MS
(ESI, m/z): 385.19 [M + H]+.
N-Hydroxy-2-(3-(methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)acetamide (23aa). 23aawas obtained from compound
22aa as described for method F: mp 97−99 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.38 (s, 1H), 8.70 (s, 1H), 7.67 (d, J = 7.9 Hz, 1H),
7.65−7.58 (m, 1H), 7.33 (t, J = 8.1 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H),
7.05−7.00 (m, 2H), 6.88 (s, 1H), 6.84 (d, J = 7.8 Hz, 1H),4.21(s, 2H),
3.47 (s, 3H), 2.57 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 165.32,
162.74, 161.41, 152.25, 148.80, 148.43, 141.31, 132.33, 127.84, 126.11,
124.87, 119.47, 114.76, 113.92, 112.84, 56.29, 42.69, 26.51. MS (ESI,
m/z): 339.04 [M + H]+.
N-Hydroxy-4-(3-(methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)butanamide (23ab). 23ab was obtained from compound
22ab as described for method F: mp 84−86 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.38 (s, 1H), 8.70 (s, 1H), 7.67 (d, J = 7.9 Hz, 1H),
7.65−7.58 (m, 1H), 7.33 (t, J = 8.1 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H),
7.01−6.93 (m, 2H), 6.88 (s, 1H), 6.84 (d, J = 7.8 Hz, 1H), 4.09 (t,J =
7.2 Hz, 2H), 3.31 (s, 3H), 2.61 (s, 3H), 2.06 (t, J = 7.3 Hz, 2H), 1.94−
1.83 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 169.10, 162.71,
161.13, 158.36, 147.89, 132.74, 131.23, 127.53, 124.75, 119.75, 114.72,
114.59, 114.42, 94.39, 56.11, 53.14, 30.30, 26.41, 23.19. MS (ESI, m/
z): 337.14 [M + H]+.
N-Hydroxy-5-(3-(methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)pentanamide (23ac). 23ac was obtained from compound
22ac as described for method F: mp 82−84 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.37 (s, 1H), 8.67 (s, 1H), 7.66 (d, J = 8.1 Hz, 1H),
7.63−7.57 (m, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.09−7.04 (m, 1H),
6.99−6.92 (m, 2H), 6.85 (s, 1H), 6.80 (d, J = 8.0 Hz, 1H), 4.13−4.04
(m, 2H), 3.34 (s, 3H), 2.59 (s, 3H), 1.99 (t, J = 7.2 Hz, 2H), 1.64 (d, J
= 6.8 Hz, 2H), 1.56 (d, J = 7.5 Hz, 2H). 13C NMR (101 MHz, DMSO-
d6) δ: 162.88, 161.12, 158.33, 152.45, 148.19, 132.50, 131.16, 128.04,
126.09, 124.57, 119.59, 114.85, 114.36, 114.22, 94.39, 56.08, 53.15,
32.54, 26.76, 26.62, 23.27. MS (ESI, m/z): 381.37 [M + H]+.
N-Hydroxy-6-(3-(methyl(2-methylquinazolin-4-yl)amino)-
phenoxy)hexanamide (23ad). 23ad was obtained from compound
22ad as described for method F: mp 71−73 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.29 (s, 1H), 8.73 (s, 1H), 7.66 (d, J = 8.1 Hz, 1H),
7.59 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.06 (t, J = 7.5 Hz,
1H), 6.99−6.92 (m, 2H), 6.86 (s, 1H), 6.81 (d, J = 7.9 Hz, 1H), 4.11−
4.02 (m, 2H), 3.30 (s, 3H), 2.59 (s, 3H), 1.95 (t, J = 7.3 Hz, 2H),
1.70−1.62 (m, 2H), 1.53 (dd, J = 14.8, 7.4 Hz, 2H), 1.31 (dd, J = 14.5,
7.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 169.47, 162.89,
161.06, 158.34, 152.45, 148.18, 132.46, 131.15, 128.02, 126.10, 124.51,
N-Hydroxy-2-(4-(methyl(quinazolin-4-yl)amino)phenoxy)-
acetamide (10a). 10a was obtained from compound 9a as described
1
for method F: mp 172−170 °C. H NMR (400 MHz, DMSO-d6) δ:
10.40 (s, 1H), 8.71 (s, 2H), 7.74 (d, J = 7.9 Hz, 1H), 7.69−7.65 (m,
1H), 7.20 (d, J = 8.8 Hz, 2H), 7.18−7.10 (m, 1H), 7.00 (d, J = 9.0 Hz,
1H), 6.98 (d, J = 8.8 Hz, 2H), 4.45 (s, 2H), 3.51 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ: 131.84, 127.50, 127.09, 125.64, 124.05,
116.02, 66.03, 42.30, 26.12. MS (ESI, m/z): 339.16 [M + H]+.
N-Hydroxy-4-(4-(methyl(quinazolin-4-yl)amino)phenoxy)-
butanamide (10b). 10b was obtained from compound 9b as
described for method F: mp 187−189 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.43 (s, 1H), 8.71 (s, 2H), 7.74 (d, J = 7.9 Hz, 1H),
7.67−7.61 (m, 1H), 7.20 (d, J = 8.8 Hz, 2H), 7.18−7.11 (m, 1H), 7.01
(d, J = 8.9 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 3.98 (t, J = 6.3 Hz, 2H),
3.51 (s, 3H), 2.14 (t, J = 7.4 Hz, 2H), 1.99−1.90 (m, 2H). 13C NMR
(101 MHz, DMSO-d6) δ: 168.78, 160.09, 157.02, 153.95, 151.07,
140.68, 131.91, 128.25, 127.22, 125.75, 124.90, 115.98, 115.87, 67.14,
42.46, 27.17. MS (ESI, m/z): 325.26 [M + H]+.
N-Hydroxy-5-(4-(methyl(quinazolin-4-yl)amino)phenoxy)-
pentanamide (10c). 10c was obtained from compound 9c as
described for method F: mp 176−178 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.38 (s, 1H), 8.71 (s, 2H), 7.74 (d, J = 8.1 Hz, 1H),
7.68−7.60 (m, 1H), 7.20 (d, J = 8.8 Hz, 2H), 7.17−7.11 (m, 1H), 7.02
(d, J = 9.0 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 3.98 (t, J = 5.9 Hz, 2H),
3.50 (s, 3H), 2.03 (t, J = 6.9 Hz, 2H), 1.77−1.58 (m, 4H). 13C NMR
(101 MHz, DMSO-d6) δ: 168.88, 160.89, 157.09, 153.98, 151.07,
140.70, 131.96, 128.05, 127.18, 125.73, 124.91, 115.98, 115.78, 67.35,
42.46, 31.88, 28.13, 21.76. MS (ESI, m/z): 367.19 [M + H]+.
N-Hydroxy-6-(4-(methyl(quinazolin-4-yl)amino)phenoxy)-
hexanamide (10d). 10d was obtained from compound 9d as
described for method F: mp 176−178 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 10.35 (s, 1H), 8.71 (s, 2H), 7.74 (d, J = 7.8 Hz, 1H),
7.67−7.61 (m, 1H), 7.19 (d, J = 8.9 Hz, 2H), 7.17−7.11 (m, 1H), 7.01
(d, J = 8.8 Hz, 1H), 6.98 (d, J = 8.9 Hz, 2H), 3.97 (t, J = 6.4 Hz, 2H),
3.50 (s, 3H), 1.98 (t, J = 7.3 Hz, 2H), 1.78−1.65 (m, 2H), 1.63−1.49
(m, 2H), 1.44−1.35 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ:
168.99, 160.88, 157.12, 153.98, 151.07, 140.67, 131.95, 128.04, 127.17,
125.73, 124.89, 115.98, 115.76, 67.62, 42.45, 32.20, 28.37, 25.14,
24.88. MS (ESI, m/z): 381.18 [M + H]+.
N-Hydroxy-2-(4-(methyl(2-methyl-6,7-dihydro-5H-
cyclopenta[d]pyrimidin-4-yl)amino)phenoxy)acetamide (16a).
16a was obtained from compound 15a as described for method F: mp
1
173−175 °C. H NMR (400 MHz, DMSO-d6) δ: 10.86 (s, 1H), 9.00
(s, 1H), 7.18 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 4.48 (s,
2H), 3.34 (s, 3H), 2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 1.78 (t, J =
7.1 Hz, 2H), 1.71−1.59 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ:
172.48, 164.50, 164.10, 158.89, 156.15, 138.97, 128.62, 114.97, 113.51,
66.03, 39.43, 33.27, 29.76, 25.47, 21.21. MS (ESI, m/z): 329.05 [M +
H]+.
N-Hydroxy-4-(4-(methyl(2-methyl-6,7-dihydro-5H-
cyclopenta[d]pyrimidin-4-yl)amino)phenoxy)butanamide
(16b). 16b was obtained from compound 15b as described for method
F: mp 166−168 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.37 (s, 1H),
8.70 (s, 1H), 7.15 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 3.97 (t,
L
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119.69, 114.82, 114.42, 114.32, 94.40, 56.07, 53.33, 32.68, 26.61,
26.50, 25.34. MS (ESI, m/z): 395.22 [M + H]+.
4BKX), we used the crystal structure reported by Millard et al.53 and
ELM2-SANT fragment was deleted. For the above HDAC1 structure
and HDAC6 model to be exploited for protein structure alignment
and molecular docking, the FG-MD method was employed to improve
the local geometry, the torsion angle, and the hydrogen-binding
networks.54 Molecular docking was carried out on the HDAC8,
HDAC1, and HDAC6 model by Autodock4Zn.55
HDAC Enzymes Inhibition Assays. In vitro HDACs inhibition
assay was conducted utilizing 4-amino-7-methylcoumarin (AMC)
labeled Ac-peptide (Ac-peptide-AMC) substrates, a service provided
by Chempartner Company (Shanghai, China). In detail, upon
deacetylation of the substrate, the release of AMC was promoted in
the existence of trypsin. The compounds, diluted to the indicated
concentrations, were mixed with full-length recombinant HDAC
enzymes (BPS Biosciences), trypsin, as well as Ac-peptide-AMC
substrates and incubated at room temperature for 1 h. The
fluorescence intensity was measured with excitation at 355 nm
wavelength and emission at 460 nm wavelength. The inhibition rates
of the tested groups were calculated via comparison with the DMSO
(vehicle) treated group.
α-Tubulin and Histone H3 Acetylation Cytoblot Assay. The
cytoblot assays were conducted generally as described.27,49 The
antiacetylated tubulin antibody, antiacetylated histone H3 antibody
(Santa Cruz), and horseradish peroxidase (HRP) conjugated
secondary antibodies (Jackson) were used. HeLa cells were seeded
into opaque 96-well plates at a density of 5000 cells per well. After cell
attachment, the compounds were added at various indicated
concentrations and incubated for 6 h. The cells were fixed by 3.7%
paraformaldehyde in ice cold TBS at 4 °C for 1 h. After removal of the
fixing solution, the cells were permeabilized via addition of −20 °C
MeOH (5 min incubation at 4 °C). Subsequently, the wells were
washed with 3% nonfat dry milk in TBS. The antibodies were added,
and the plates were incubated for 4 h at 4 °C. Finally, the wells were
washed thrice by TBS following addition of enhanced chemilumi-
nescence reagent. After a short incubation for 5 min, the luminescence
was recorded by a Spectramax M5 microtiter plate luminometer. The
EC50 values were calculated by curve fitting with the software
GraphPad Prism 5.0.
N-Hydroxy-2-(2-methoxy-5-(methyl(2-methylquinazolin-4-
yl)amino)phenoxy)acetamide (23ba). 23ba was obtained from
1
compound 22ba as described for method F: mp 172−174 °C. H
NMR (400 MHz, DMSO-d6) δ: 7.63 (d, J = 8.1 Hz, 1H), 7.57 (t, J =
6.5 Hz, 1H), 7.05 (d, J = 7.7 Hz, 2H), 7.00 (d, J = 8.1 Hz, 1H), 6.89
(d, J = 8.3 Hz, 1H), 6.63 (d, J = 8.1 Hz, 1H), 4.26 (s, 2H), 3.75 (s,
3H), 3.47 (s, 3H), 2.57 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ:
164.39, 162.81, 161.45, 152.09, 148.83, 148.26, 141.36, 132.33, 127.89,
126.17, 124.57, 119.51, 114.70, 113.22, 112.94, 67.50, 56.17, 42.75,
26.58. MS (ESI, m/z): 369.12 [M + H]+.
N-Hydroxy-4-(2-methoxy-5-(methyl(2-methylquinazolin-4-
yl)amino)phenoxy)butanamide (23bb). 23bb was obtained from
1
compound 22bb as described for method F: mp 161−163 °C. H
NMR (400 MHz, DMSO-d6) δ: 10.41 (s, 1H), 8.71 (s, 1H), 7.64 (d, J
= 8.2 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H) 6.99
(d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 1H), 6.68 (dd, J = 8.4, 2.0 Hz,
1H), 3.88 (t, J = 6.2 Hz, 2H), 3.78 (s, 3H), 3.50 (s, 3H), 2.58 (s, 3H),
2.08 (t, J = 7.4 Hz, 2H), 1.94−1.81 (m, 2H). 13C NMR (101 MHz,
DMSO-d6) δ: 168.57, 162.30, 160.89, 151.62, 148.88, 147.69, 140.99,
131.78, 127.40, 125.65, 123.99, 118.08, 114.21, 112.57, 111.49, 67.82,
55.67, 42.26, 28.64, 26.11, 24.66. MS (ESI, m/z): 397.12 [M + H]+.
N-Hydroxy-5-(2-methoxy-5-(methyl(2-methylquinazolin-4-
yl)amino)phenoxy)pentanamide (23bc). 23bc was obtained from
1
compound 22bc as described for method F: mp 172−174 °C. H
NMR (400 MHz, DMSO-d6) δ: 10.35 (s, 1H), 8.69 (s, 1H), 7.64 (d, J
= 8.2 Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 7.00
(d, J = 8.4 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H),
6.69 (dd, J = 8.5, 2.3 Hz, 1H), 3.88 (t, J = 5.6 Hz, 2H), 3.78 (s, 3H),
3.51 (s, 3H), 2.58 (s, 3H), 1.99−1.94 (m, 2H), 1.70−1.52 (m, 4H).
13C NMR (101 MHz, DMSO-d6) δ: 168.84, 162.30, 160.90, 151.63,
148.99, 147.61, 140.99, 131.78, 127.40, 125.68, 123.97, 117.86, 114.21,
112.51, 111.26, 67.94, 55.66, 42.26, 31.86, 28.04, 26.11, 21.77. MS
(ESI, m/z): 411.16 [M + H]+.
N-Hydroxy-6-(2-methoxy-5-(methyl(2-methylquinazolin-4-
yl)amino)phenoxy)hexanamide (23bd). 23bd was obtained from
1
compound 22bd as described for method F: mp 152−154 °C. H
NMR (400 MHz, DMSO-d6) δ: 10.35 (s, 1H), 8.67 (s, 1H), 7.64 (d, J
= 8.1 Hz, 1H), 7.58 (t, J = 7.1 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.99
(d, J = 8.4 Hz, 1H), 6.94 (dd, J = 5.3, 3.0 Hz, 2H), 6.70 (dd, J = 8.4,
2.1 Hz, 1H), 3.85 (t, J = 6.4 Hz, 2H), 3.77 (s, 3H), 3.50 (s, 3H), 2.58
(s, 3H), 1.94 (t, J = 7.2 Hz, 2H), 1.67−1.56 (m, 2H), 1.56−1.44 (m,
2H), 1.37−1.26 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 168.91,
162.30, 160.89, 151.63, 148.98, 147.62, 140.97, 131.77, 127.38, 125.68,
123.94, 117.83, 114.20, 112.52, 111.29, 68.18, 55.65, 42.24, 32.17,
28.25, 26.11, 25.07, 24.85. MS (ESI, m/z): 425.18 [M + H]+.
N-Hydroxy-4-(2-methoxy-5-((2-methylquinazolin-4-yl)-
amino)phenoxy)butanamide (27). 27 was obtained from com-
pound 26 as described for method F: mp 189−191 °C. 1H NMR (400
MHz, DMSO-d6) δ: 10.45 (s, 1H), 9.55 (s, 1H), 8.72 (s, 1H), 8.48 (d,
J = 8.1 Hz, 1H), 7.82−7.74 (m, 1H), 7.66 (dd, J = 7.9, 5.4 Hz, 2H),
7.57−7.50 (m, 1H), 7.45 (dd, J = 8.7, 2.3 Hz, 1H), 6.98 (d, J = 8.8 Hz,
1H), 3.99 (t, J = 6.3 Hz, 2H), 3.78 (s, 3H), 2.17 (t, J = 7.4 Hz, 2H),
2.05−1.94 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 169.09,
163.39, 157.96, 150.63, 147.92, 145.75, 133.30, 133.15, 127.65, 125.58,
123.13, 114.61, 113.70, 112.51, 108.99, 68.07, 56.30, 40.61, 40.40,
40.20, 39.99, 39.78, 39.57, 39.36, 29.25, 26.73, 25.38. MS (ESI, m/z):
383.27 [M + H]+.
Biological Assay Methods. Homology Modeling and
Molecular Simulation. HDAC6 has two catalytic domains (CDI
and CDII), a ubiquitin binding domain, and a dynein binding
domain.51 For HDAC6, a homology model was generated by multiple-
thread alignments, as described by Yang Zhang’s research group
through a Web server (I-TASSER).45 In the process of building
HDAC6 homology model, only the second catalytic subunit (CDII,
Tyr485-Arg835) was generated, as it plays an essential role in catalytic
function. Then the zinc ion, extracted from the crystal structure of
HDAC8−substrate complex (PDB code 2V5W),52 was added into the
corresponding site of HDAC6 structure. For HDAC1 (PDB code
Antiproliferative Assays. A375, A2780s, SKBR3, HepG2, HeLa,
HCT116, A549, and SKOV-3 cells were cultured in DMEM (Gibco,
Milano, Italy). RPMI8226, K562, H460, HT29, and Ramos cells were
cultured in RPMI-1640 medium (Gibco, Milano, Italy). MV4-11 cells
were cultured in IMDM (Gibco, Milano, Italy). All media contained
10% fetal bovine serum (FBS) (Invitrogen, Milano, Italy), 100 units/
mL penicillin (Gibco, Milano, Italy), and 100 μg/mL streptomycin
(Gibco, Milano, Italy). Cells were incubated at 37 °C in a humidified
atmosphere of 5% CO2. Cells in logarithmic phase were seeded into
96-well culture plates at densities of 3000−5000 cells per well and
subsequently treated with various concentrations of compounds for 72
h in final volumes of 200 μL. Upon end point, 20 μL of MTT (5 mg/
mL) was added to each well, and the cells were incubated for an
additional 1−3 h. After carefully removal of the medium, the
precipitates were dissolved in 150 μL of DMSO via mechanically
shaking, and then absorbance values at a wavelength of 570 nm were
taken on a spectrophotometer (Molecular Devices, Sunnyvale, USA).
IC50 values were calculated using percentage of growth versus
untreated control.
Western Blotting. The cells were treated by the indicated ways
before collection. After washing by PBS 2 times, the cells were
resuspended in RIPA lysis buffer (Beyotime Co.). After 30 min of
incubation on ice, the lysates were collected by centrifuging at 12 000g
for 15 min at 4 °C. The protein concentration was measured.
Equivalent samples (20 μg of protein) were subjected to 15% SDS−
PAGE, and then the proteins were transferred onto activated PVDF
membranes. After blocking by 5% nonfat milk for 1 h at room
temperature, the membranes were incubated with the indicated
primary antibodies and subsequently probed by the appropriate
secondary antibodies conjugated to horseradish peroxidase. Immunor-
eactive bands were visualized using enhanced chemiluminescence
(Millipore, USA).
M
DOI: 10.1021/acs.jmedchem.5b01342
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Journal of Medicinal Chemistry
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In Vivo Pharmacokinetics Evaluation in Mice. A 2 mg/mL
dosing solution was preparing by dissolving the appropriate amount of
the compound in ddH2O for iv administration and 0.5% CMC-Na
aqueous solution for oral dosing. SD rats, weighing 200−250 g each,
were obtained from Beijing HFK Bioscience Co., Ltd.. Each tested
compound was separately administered intravenously to a group of six
rats per time point (12 mg/kg dose) by a bolus injection (12 mg/kg
dose) to the tail vein or periorally. At time points 0 (prior to dosing), 5
min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24
h after dosing, a blood sample was collected from each animal via
cardiac puncture and stored in ice (0−4 °C). Plasma was separated
from the blood by centrifugation (4000g for 15 min at 4 °C) and
stored in a freezer at −80 °C. All samples were analyzed for the test
compound by LC−MS/MS (Waters Acquity UPLC system; Waters
Quattro Premier XE). Data were acquired via monitoring of multiple
reactions. Plasma concentration data were analyzed by a standard
noncompartmental method.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors greatly appreciate the financial support from
National Key Programs of China during the 12th Five-Year
Plan Period (Grant 2012ZX09103101-009) and Guangdong
Innovative Research Team Program (Grant 2011Y073).
ABBREVIATIONS USED
■
HDAC, histone deacetylase; ZBG, zinc-binding group; DMSO,
dimethyl sulfoxide; SAHA, suberoylanilide hydroxamic acid;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; SAR, structure−activity relationship
Animal Tumor Models and Treatment. For the MV4-11 and
Ramos xenograft models, MV4-11 and Ramos cells (107 cells in 100
μL of serum-free IMDM) were injected subcutaneously into the right
flanks of 5- to 6-week-old female NOD/SCID mice. For the HCT116
xenograft, HCT116 cells (107 cells in 100 μL of serum-free DMEM)
were injected subcutaneously into the right flanks of 5- to 6-week-old
female Balb/c nude mice. When the size of the formed xenografts
reached 100−150 mm3, the mice were randomly divided (6 mice per
group in MV4-11 model, 8 mice per group in Ramos model, and 7
mice per group in HCT116 model) into control group and treated
groups. The mice in the experimental groups received intravenous (iv)
injection (50 mg/kg, dissolved in physiological saline containing 5%
ethanol and 5% Cremophor EL) or oral administration (25 mg/kg,
dissolved in physiological saline containing 5% ethanol and 5%
Cremophor EL) of 23bb every 2 days. The mice in the vehicle group
received iv injection or oral administration of equal amount of
physiological saline containing 5% ethanol and 5% Cremophor EL.
Those in the SAHA or LBH-589 or ACY-1215 groups (positive
controls) received ip injection (50 mg/kg for SAHA and 10 mg/kg for
LBH-589, dissolved in physiological saline containing 10% DMSO and
45% PEG400 to a concentration of 10 mg/mL) or oral administration
(100 mg/kg for SAHA and 40 mg/kg for ACY-1215, dissolved in the
same way described above) every 2 days. Tumor burden was measured
every 2 days by a caliper. Tumor volume (TV) was calculated using
the following formula: TV = length × width2 × 0.52. The day that
treatment started was defined as day 0. At the end of the experiment,
mice were sacrificed and tumors were collected and weighed. The
animal studies were conducted in conformity with institutional guide
for the care and use of laboratory animals, and all mouse protocols
were approved by the Animal Care and Use Committee of Sichuan
University (Chengdu, Sichuan, China).
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01342.
Results of HDAC1, HDAC6, and HDAC8 structures
comparisons and the binding energy of the comfortable
fragments (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*J.Y.: e-mail, jsyou@scu.edu.cn.
■
*L.C.: phone, +86-28-85164063; fax, +86-28-85164060. e-mail,
chenlijuan125@163.com.
Author Contributions
#Z.Y., T.W., and F.W. contributed equally and are considered as
co-first authors.
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Journal of Medicinal Chemistry
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