Discovery and extensive in vitro evaluations of NK-HDAC-1: A chiral histone deacetylase inhibitor as a promising lead

Article abstract of DOI:10.1021/jm201496g

Herein, further SAR studies of lead compound NSC746457 (Shen, J.; Woodward, R.; Kedenburg, J. P.; Liu, X. W.; Chen, M.; Fang, L. Y.; Sun; D. X.; Wang. P. G.J. Med. Chem. 2008, 51, 7417-7427) were performed, including the replacement of the trans-styryl moiety with a 2-substituted benzo-hetero aromatic ring and the introduction of a substituent onto the central methylene carbon. A promising chiral lead, S-(E)-3-(1-(1-(benzo[d]oxazol-2-yl)-2-methylpropyl)-1H-1,2,3- triazol-4-yl)-N-hydroxyacrylamide (12, NK-HDAC-1), was discovered and showed about 1 order of magnitude more potency than SAHA in both enzymatic and cellular assays. For the in vitro safety tests, NK-HDAC-1 was far less toxic to nontransformed cells than tumor cells and showed no significant inhibition activity against CYP-3A4. The pharmaceutical properties (LogD, solubility, liver micrsomal stability (t1/2), plasma stability (t1/2), and apparent permeability) strongly suggested that NK-HDAC-1 might be superior to SAHA in bioavailability and in vivo half-life.

Full text of DOI:10.1021/jm201496g

Article
pubs.acs.org/jmc
Discovery and Extensive in Vitro Evaluations of NK-HDAC-1: A Chiral
Histone Deacetylase Inhibitor as a Promising Lead
Jingli Hou,‡,∥ Zhonghua Li,‡,∥ Qinghong Fang,‡ Congran Feng,‡ Hanwen Zhang,‡ Weikang Guo,‡
Huihui Wang,‡ Guoxian Gu,‡ Yinping Tian,‡ Pi Liu,‡ Ruihua Liu,‡ Jianping Lin,‡ Yi-kang Shi,§
Zheng Yin,†,‡ Jie Shen,*,†,‡ and Peng George Wang*,†,‡
†State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China
‡College of Pharmacy, Nankai University, Tianjin,China
§National Glycoengineering Research Center, Shandong University, 44 West Wenhua Road, Jinan, China
S
* Supporting Information
ABSTRACT: Herein, further SAR studies of lead compound
NSC746457 (Shen, J.; Woodward, R.; Kedenburg, J. P.; Liu, X.
W.; Chen, M.; Fang, L. Y.; Sun; D. X.; Wang. P. G. J. Med.
Chem. 2008, 51, 7417−7427) were performed, including the
replacement of the trans-styryl moiety with a 2-substituted
benzo-hetero aromatic ring and the introduction of a
substituent onto the central methylene carbon. A promising
chiral lead, S-(E)-3-(1-(1-(benzo[d]oxazol-2-yl)-2-methyl-
propyl)-1H-1,2,3-triazol-4-yl)-N-hydroxyacrylamide (12, NK-
HDAC-1), was discovered and showed about 1 order of magnitude more potency than SAHA in both enzymatic and cellular
assays. For the in vitro safety tests, NK-HDAC-1 was far less toxic to nontransformed cells than tumor cells and showed no
significant inhibition activity against CYP-3A4. The pharmaceutical properties (LogD, solubility, liver micrsomal stability (t1/2),
plasma stability (t1/2), and apparent permeability) strongly suggested that NK-HDAC-1 might be superior to SAHA in
bioavailability and in vivo half-life.
dependent HDACi: a Zn2+ binding group (ZBG) positioned at
the bottom of a highly conserved “tube-like” active site, a “cap”
INTRODUCTION
The reversible acetylation and deacetylation modifications of
■
region which can interact with the rim of the HDAC active
lysine residues of histones can regulate gene expression via
remodeling chromatin architechture.2−8 Histone acetyltrans-
ferases (HATs) can relax chromatin and epigenetically promote
gene transcription. Conversely, histone deacetylases (HDACs)
pocket, and a linker which fits in the hydrophobic “tube”
(Figures 1 and 2).
In our recent works,1 click chemistry was employed to afford
a highly efficient combinatorial approach to constructing a
can condense chromatin and epigenetically prevent gene
library of new HDACi candidates, and a hit compound
transcription. Besides histones, a large number of nonhistone
(NSC746457) was discovered which showed comparable
client proteins of HDACs and HATs were identified, such as
activity to SAHA. The triazole ring was of suitable size to fit
transcription factors, hormone receptors, chaperone protein,
cytoskeletal protein, etc.9,10 Accordingly, multiple antitumor
into the narrow active pocket of HDAC protein. Moreover, the
triazole ring was correctly positioned to form a π−π interaction
with two reserved phenylalanine residues in the docking
structure of NSC746457 with HDAC2 (Figure 2). This
indicated that, besides the role of linker moiety, the triazole
ring in our design also contributed to the binding affinity.
In this article, lead optimizations of NSC746457 were
performed (Scheme 1), focusing on two aspects: (1) the
introduction of a side chain at the methylene carbon atom and
(2) the replacement of trans-styryl moiety with a 2-substituted
benzo-hetero ring. The methylene carbon atom is at the joint
position between the cap region and the linker moiety of
NSC746457, and in the computational complex structure, this
mechanisms of HDACi were suggested, including alteration of
gene expression, degradation of Hsp90 client proteins,
increased production of reactive oxygen species, induction of
cell cycle arrest, etc. To date, there are more than 10 HDACi
molecules entering into clinical trials (Figure 1). SAHA and
Romidepsin, the first two marketed HDACi drugs, were
approved by the FDA in 2006 and 2009, respectively, for the
treatment of cutaneous T-cell lymphoma (CTCL).
There are 18 HDAC isozymes encoded in the human
genome, and they are divided into four classes. Class III
isozymes, including Sirt1 to Sirt7, utilize NAD+ as a cofactor.
The other three classes (Class I, HDAC1, 2, 3, and 8; Class II,
HDAC 4, 5, 6, and 9; and Class IV, HDAC 11) are Zn-
dependent isozymes. Co-crystals of HDAC bound with
HDACi11 highlight a common molecular scaffold for Zn-
Received: November 7, 2011
Published: March 21, 2012
© 2012 American Chemical Society
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Figure 1. Structures of TSA and some HDACi molecules in clinical trials or clinical use.
intrinsic restriction might direct the cap region of the HADCi
molecule to properly be in contact with some surface groove of
HDAC. There are four reasons for us to attempt the
replacement of the trans-styryl moiety with a 2-substituted
benzo-hetero ring: (1) trans-styryl can be considered as a
bioisoster of the 2-substituted-hetero aromatic ring, and they
share a similar structural scaffold; therefore, such a replacement
would maintain the HDAC inhibition potency of the hit
compound. (2) The possible epoxide metabolite of the styryl
moiety could be a potential carcinogenic alkylation reagent16
and such a replacement would avoid this toxicity concern for
drug development. (3) The newly introduced side chain at the
methylene carbon generates a chiral center which can be
conveniently derived from chiral amino acid reactants if the
trans-styryl moiety was replaced with a 2-substituted benzo-
hetero ring. (4) Some benzo-hetero rings, such as indole and
benzofuran, were successfully employed in the cap regions of
some HDACi molecules in clinical trials (Figure 1). Taken
together, the lead optimization studies herein were planned to
proceed in four stages (Scheme 1), which are thoroughly
discussed in the following sections.
Figure 2. Docking of NSC746457 (our hit compound) into the active
pocket of human HDAC2 protein.
methylene carbon atom is positioned at the open rim of the
HDAC active pocket, surrounding which are several shallow
grooves (Figure 2). Inspired by previous HDACi lead
optimization study results,12−15 we envisioned that introduc-
tion of a side chain at the methylene carbon would enhance the
HDACi/HDAC binding affinity: (a) This side chain might be
in contact with some shallow groove on the surface of the rim
and therefore contribute to the binding affinity. (b) This side
chain would restrict the free rotation of the two single bonds
which flanked the newly generated chiral center. Such an
Scheme 1. Flow Chart of Work Plan in This Article
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a
Scheme 2. Syntheses of Target Compounds in Stage 1: 1−3
a
Reagents and conditions: (a) NaN3, DMSO, room temperature; (b) CuSO4, sodium ascorbate, rt; (c) TFA, triisopropylsilane, DCM, rt.
a
Table 1. In Vitro Efficacy Assays of Compounds in Stage 1.
a
GI50 (μM)
a
b
b
c
c
c
c
c
IC50 (nM) HDACs
A549
HepG2
K562
SW-620
Colo-205
OVCR-3
PC-3
SAHA
67
3.1
1.9
2.1
4.7
3.5
3.2
8.8
6.1
>20
0.50
0.55
2.6
0.63
0.48
1.5
4.0
1.1
1.5
1.1
2.0
1.1
3.6
1.9
1.6
1.3
2.9
1.6
NSC746457
104
41
1
2
3
105
178
0.72
1.2
d
d
d
d
d
>20
ND
ND
ND
ND
ND
a
b
c
Mean of three independent triplicate experiments. MTT data from our lab. Selected MTS data from the program of NCI-60-human-tumor-cell-
d
line-anticancer-drug-screen. ND, not determined.
inhibition assay data. Compound 2 was therefore taken as the
optimal parent lead to enter into stage 2 studies.
RESULT AND DISCUSSION
■
Stage 1: Replacement of trans-Styryl with a 2-
Substituted Benzo-Hetero Aromatic Ring. In this stage,
the trans-styryl moiety was replaced with a 2-substituted
benzoxazole ring, benzothiazole ring, or benzimidazole ring,
resulting in three newly designed HDACi molecules 1−3.
These three compounds were prepared from 2-chloromethyl
substituted benzo-hetero compounds 1c−3c, which were
obtained from the known procedures.17−19 Compounds 1c−
3c were then converted to the azido derivatives 1b−3b via
nucleophilic substitution by using sodium azide solution in
DMSO. Cu(I)-mediated Huisgen cycloaddition reactions of the
known alkyne compound 141 were carried out with each of the
azido derivatives 1b−3b to afford the click products 1a−3a.
Finally, the PMB protecting group was removed by TFA
(Scheme 2) to provide the target compounds 1−3. In HDAC
Stage 2: Introduction of a Side Chain onto the Middle
Methylene Carbon Atom. Initially in this stage, β-substituted
racemic amino acid derivatives were employed as the reactants
to prepare racemic target compounds 4−11, which were the
branched derivatives of compound benzoxazole 2. As shown in
Scheme 3, amidation reaction of N-Boc protected amino acid
reactants with 2-aminophenol, followed by the intramolecular
Mitsunobu reactions, afforded intermediates 5c−11c. For 5c−
9c, the N-Boc protecting group was removed by the treatment
with TFA in DCM; for 10c and 11c, a harsher acidic condition
using 4 M hydrochloric acid in dioxane was employed to
simultaneously remove both the N-Boc group and the O-Boc
group. After routine workup manipulations, the obtained
amino-group containing intermediates were directly converted
to the azido compounds 5b−11b via the Cu(II)-mediated diazo
transfer reaction. Huisgen cycloaddition reactions of the known
alkyne compound 14 were carried out with 5b−11b using the
catalyst of CuI·(POEt)3 to afford the click products 5a−11a.
The intermediate 4a was prepared via the amidation reaction of
11a. Finally, the PMB protecting group was removed by TFA
to provide the target compounds 4−11. In vitro efficacy (Table
2) showed that target compounds in this stage with a middle-
size and hydrophobic substitution group (6, iPr; 8, sec-Bu; 9,
iso-Bu) showed dramatically improved potency. Large sub-
stitution (7: Bn) or a negatively charged substitution (e.g., 10
or 11 with a carboxylic acid group) led to the decrease in
potency. The target compound with a small substituent (5:
Me) slightly enhanced the potency. For compound 4, which
had a primary amide substituent, it was more potent than
parent compound 2 in the HDAC inhibition assay, whereas the
opposite trend was observed in MTT tests. Such inconsistency
might originate from its poor permeability due to the high polar
surface area of compound 4. To further explore the SAR, the
benzothiazole derivative 6′, which was the counterpart of
benzoxazole derivative 6, was prepared. The synthesis of 6′
followed exactly the same synthetic procedure of 6 (Scheme3)
using 2-aminothiopenol as the starting material, except that the
initial amidation reaction product, after routine workup and
without column purification, was directly employed in the next
inhibition assays (Table 1), benzothiazole derivative 1 (IC50
=
41 nM) was approximately two times more potent than
NSC746457 (IC50 = 104 nM); benzoxazole derivative 2 (IC50 =
105 nM) was comparable to NSC746457; benzimidazole
derivative 3, (IC50 = 178 nM) was approximately two times less
potent than NSC746457. DFT calculations using Schrodinger
Jaguar 7.820 revealed that compound 3 could form an
intramolecular hydrogen bond to partially freeze the dominant
conformation, which, however, was not like the preferred
conformation of the hit compound in the docking structure
(Figures 2 and 3). Such a conformation restriction was not
observed in the DFT calculation for compound 2. In cell
viability assays, the weak potency of compound 3 was also
observed; however, compound 2 was slightly more potent than
compound 1, showing results inconsistent to the HDAC
Figure 3. DFT calculations of favorable conformations for compounds
2 and 3.
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a
Scheme 3. Syntheses of Recemic Target Compounds in Stage 2: 4−11 and 6′
a
Reagents and conditions: (a) EDC, HOBT, DCM; (b) DIAD,PPh3, TEA; (c) (i) TFA, DCM, (ii) TfN3, NaHCO3, CuSO4; (d) (i) 4 M HCl in
dioxane, (ii) TfN3, NaHCO3, CuSO4; (e) CuI·(POEt)3, DIPEA, THF, rt; (f) N-methyl piperidine, isobutyl chloroformate, NH3, −30 °C to −20 °C;
(g) TFA, triisopropylsilane, DCM, rt.
a
Table 2. In Vitro Efficacy Assays of Compounds in Stage 2
a
b
IC50 (nM)
HDACs
GI50 (μM)
compds
A549
HepG2
MDA-MB-231
SAHA
67
105
49
86
23
5
3.1
3.5
6.1
14.0
2.9
1.0
1.9
5.9
1.7
2.2
>20
>20
1.0
2
4.7
1.4
4
21.8
2.4
9.5
5
1.2
6
0.4
0.17
0.93
5.5
Figure 4. Effects of SAHA, compounds 1, 2, 6, and 6′ on histone H3
acetylation expression at 5 μM after 24 h.
6′
7
1.9
219
4
4.4
Stage 3: Optically Pure Enantiomers of Compound 6.
The S-enatiomer 12 and the R-enatiomer 13 were prepared and
evaluated in this stage. As shown in Scheme 4, the synthetic
procedures of these two enantiomers were exactly the same as
that of the racemic compound 6, except that the N-Boc
protecting group was replaced with the N-Cbz protecting
group, which could be removed in neutral conditions to avoid
the racemization risks. Four pairs of enantiomers, including 12c
and 13c, 12b and 13b, 12a and 13a, and 12 and 13 were all
well characterized by chiral chromatography. The S-enatiomer
12 (IC50 = 7 nM) was more potent than R-enatiomer 13 (IC50
= 31 nM) in HDAC inhibition assays. Additionally, the
inhibition capacities of compound 12 and SAHA against
HDACs in living cells were compared. After the incubation
with compound 12 or SAHA, AcH3 levels in MDA-MB-231
breast cancer cells were tested by Western blot analysis. As
shown in Figure 5A, compound 12 and SAHA dose-
dependently increased the acetylation of histone H3 after the
cells were treated for 24 h. However, 5.0 μM compound 12
more significantly elevated the acetylation of histone H3
8
0.52
0.66
>20
>20
0.19
0.28
>20
>20
9
19
140
333
10
11
a
b
Mean of three independent triplicate experiments. MTT data from
our lab.
Mitsunobu reaction. Similar to the trend for compounds 1 and
2, the benzothiazole derivative 6′ was more potent in the
enzyme inhibition assay and less potent in MTT tests than
benzoxazole derivative 6. It presumably was due to the
metabolism viability of the sulfur-containing scaffold. To verify
the hypothesis, we assessed the differential effects of the four
compounds on the status of histone acetylation in MDA-MB-
231 breast cancer cells. As shown in Figure 4, Western blot
analysis showed that 5.0 μM compounds 1 and 6′ induced
significantly less acetylation of histone H3 compared to that of
5.0 μM compounds 2 and 6, respectively. Compound 6 was
therefore taken as the optimal parent lead to enter into stage 3
studies.
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a
Scheme 4. Syntheses of Chiral Target Compounds in Stage 3: 12 and 13
a
Reagents and conditions: (a) EDC, HOBT, DCM; (b) DIAD,PPh3, TEA; (c) (i) Pd/C, H2, (ii) TfN3, NaHCO3, CuSO4; (d) CuI·(POEt)3, DIPEA,
THF, rt; (e) TFA, triisopropylsilane, DCM, rt.
each isomer, two hydrophobic interactions with the rim region
of HDAC2 protein were observed: (1) the benzoxazole ring
with a shallow groove on the surface and (2) the isopropyl
group with the Phe210 residue. For both of these two
hydrophobic interactions, docking structures indicated that S-
isomer 12 had a tighter interaction than R-isomer 13.
When a ligand molecule forms a complex with a protein, the
molecular mobility is restricted to cause a loss of binding
affinity due to the entropic penalty. Such a loss will be
minimized if (1) the ligand’s mobility is restricted by some
intrinsic structural features; and (2) the dominant conforma-
tion of the ligand itself is close to its preferred conformation in
the complex structure. Under such a guideline, our DFT
calculations of single bond rotation barrier energies revealed an
Figure 5. (A) Dose-dependent effect of compound 12 and SAHA on
histone H3 acetylation.(B) Time-dependent effect of compound 12 on
histone H3 acetylation at the concentration of 0.5 μM.
entropic contribution of the isopropyl substituent. The rotation
energy barrier of the single bond of compound 12 was 3.9 kcal/
mol higher than that of compound 2 (Figure 7). Additionally,
as for the preferred conformations of compound 12 in the
compared to that of 5.0 μM SAHA. Even 0.1 μM compound 12
can dramatically induce the acetylation of histone H3. The
acetylation of histone H3 after compound 12 treatment at 0.5
μM for 3 h was detected and it increased in a time-dependent
manner for 24 h (Figure 5B).
docking complex and by itself in gas phase, the CiPro−Cchiral
−
Ntriazole−Ctriazole dihedral angles were 300° and 270°,
respectively. In another word, the calculated low energy
conformation of compound 12 by itself happened to be close
to its dominant conformation in the docking structure, and the
binding affinity was therefore enhanced. Compound 12 was
named as NK-HDAC-1 to enter into the next stage.
Both 12 and 13 were docked into the active site of the
reported human HDAC2 crystal structure21 (Figure 6). For
Figure 6. Docking of 12 (in yellow) and 13 (in cyan) into the active pocket of human HDAC2.
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Figure 7. Relaxed coordinate scan using the QM method DFT (b3lyp) with the QM basic set 6-31G**. The software is Jaguar, version 7.8
(Schrodinger, Inc., New York, NY, 2010). Dihedral angle (H −Cchiral−Ntriazole−Ctriazole or Ci‑pro−Cchiral−Ntriazole−Ctriazole) from 0° to 360°, increment
̈
1
5°, total 73 point.
Stage 4: Extensive in Vitro Assays to Evaluate the
Potential of NK-HDAC-1 as a New Drug Candidate. After
the completion of HDAC inhibition assays, NK-HDAC-1 and
its enantiomer 13 were both submitted to the National Cancer
Institute (NCI) for acceptance into the human cancer cell line
screening program (Table 3). For the 60 cell lines, the GI50
values of NK-HDAC1 mainly ranged between 100 to 500 nM.
In detail, the GI50 values against leukemia cell lines ranged from
0.072 to 0.19 μM; the GI50 values against nonsmall cell lung
tumor cell lines ranged from 0.010 to 0.54 μM; the GI50 values
against colon cancer cell lines ranged from 0.038 to 0.47 μM;
the GI50 values against CNS cancer cell lines ranged from 0.12
to 0.44 μM; the GI50 values against melanoma cell lines ranged
from 0.044 to 0.22 μM; the GI50 values against ovarian cancer
cell lines ranged from 0.027 to 0.70 μM; the GI50 values against
renal cancer cell lines ranged from 0.044 to 0.362 μM; the GI50
values against prostate cancer cell lines ranged from 0.12 to
0.16 μM; and the GI50 values against breast cancer cell lines
ranged from 0.051 to 0.52 μM. In general, compound 12 was
approximately 5-fold more potent than the R-enantiomer 13
and approximately 1 order of magnitude more potent than
SAHA. Similar to SAHA, no significant specificities were
observed among the tumor cells from different tissues. Besides
the GI50 data of tumor cell lines from NCI, the GI50 values of
NK-HDAC-1 and SAHA against nontransformed human breast
cells MCF-10A were determined in our lab. GI50 values of NK-
HDAC-1 against tumor breast cells were approximately 3 to 30
times lower than that of nontransformed breast cancer cells.
Correspondingly, the difference for SAHA was 2 to 20 times. In
summary, cell viability assay data indicated that NK-HDAC-1
should be more potent while having fewer in vivo side effects
than SAHA.
Extensive in vitro pharmacokinetic (PK) related properties of
NK-HDAC-1 were determined (Table 4). Its molecular weight
is 327.1; its solubility in pH 7.4 PBS buffer is 20 ug/mL; and its
LogD7.4 value is 2.0. All these physco-chemical properties are
well acceptable for an oral drug candidate. The half-lives of NK-
HDAC-1 in artificial gastric fluid, artificial intestinal fluid,
mouse microsome, and mouse plasma are 9.4 h, > 24 h, 32 h,
and 195 min respectively; comparatively, the corresponding
data for SAHA are 22 h, > 24 h, 3.3 h, and 82 min. The
apparent permeability (Papp A to B) of NK-HDAC-1 at 10 μM is
6.61 × 10−6 cm/s, which is nearly four times higher than that
reported for SAHA (1.70 × 10−6).22 Additionally, the efflux
ratio of NK-HDAC-1 in Caco-2 permeability experiments is
1.15, indicating that NK-HDAC-1 should not be a substrate for
P glycoprotein transporters. Since coadministration with other
antitumor drugs is currently a main clinical research area for
HDACi drug candidates, inhibition assays of human CYP
enzyme by NK-HDAC-1 were performed. The IC50 values of
NK-HDAC-1 and SAHA against human CYP3A4, a major CYP
isozyme involved in the metabolism of approximately half of
the marketed drugs, were 31.9 μM and 24.8 μM respectively,
indicating that the possible drug−drug interaction side effects
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a,b
Table 3. GI50 Values of NK-HDAC-1 (Compound 12) in MTS and MTT Tests (μM)
12
13
SAHA
12
13
SAHA
Leukemia
CNS Cancer
0.33
CCRF-CEM
HL-60(TB)
K-562
0.11
0.35
0.28
0.28
0.30
0.35
0.80
1.3
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
0.63
0.31
0.75
0.85
0.41
0.34
1.6
0.19
0.12
1.6
0.088
0.072
0.090
0.50
0.40
0.40
0.24
2.0
MOLT-4
SR
0.43
0.63
0.79
1.6
0.25
0.18
Non-Small Cell Lung Cancer
Melanoma
0.044
0.016
0.18
A549/ATCC
EKVX
0.16
0.24
0.010
0.54
0.14
0.14
0.15
0.013
0.30
1.6
LOX IMVI
MALME-3M
M14
0.16
0.063
0.41
0.24
0.35
0.52
0.25
0.23
0.40
1.0
0.68
0.070
1.3
1.3
0.32
1.3
HOP-92
4.0
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
4.0
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
0.091
0.087
0.22
0.50
1.3
0.35
0.25
0.39
0.13
1.0
0.79
0.79
0.50
1.0
0.10
0.63
0.50
0.40
0.088
0.14
Colon Cancer
0.14
Renal Cancer
0.17
COLO 205
HCT-116
HCT-15
HT29
0.25
0.18
0.96
0.22
0.41
0.29
0.79
0.40
2.5
786−0
A498
0.34
0.26
0.47
0.35
0.79
0.16
0.23
3.2
0.038
0.18
1.6
0.47
ACHN
RXF 393
SN 12C
TK-10
UO-31
0.21
1.3
0.079
0.79
2.0
0.18
1.3
KM12
0.23
0.36
2.0
SW-620
0.13
0.63
0.044
0.060
0.63
0.50
Ovarian Cancer
0.056
Nontransformed Human Breast Cell Line
c
c
d
c
IGROV1
0.19
0.47
2.8
1.3
MCF-10A
1.6
ND
9.6
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
0.22
1.3
Breast Cancer
0.15
0.70
4.0
MDA-MB-468
HS 578T
BT-549
0.25
1.3
0.63
5.0
0.14
0.40
0.24
0.095
0.26
0.79
0.50
0.16
1.0
0.58
0.084
0.052
0.28
0.18
0.91
0.45
1.3
0.027
T-47D
0.051
0.50
2.5
0.14
MDA-MB-231
MCF7
0.52
0.18
2.0
Prostate Cancer
PC-3
0.12
0.16
0.28
0.35
2.0
1.3
DU-145
a
b
c
d
Mean of three independent triplicate experiments. All MTS data from NCI. MTT data from our lab. ND = not determined.
due to CYP3A4 inhibition by NK-HDAC-1 should be
negligible.
provided strong evidence that the in vivo PK properties of NK-
HDAC-1 might be greatly superior to those of SAHA. In fact,
our preliminary in vivo PK experiments using Balb/c mice
showed that the bioavailability of NK-HDAC-1 was greater
than 80%.23 To the best of our knowledge, both of these two
data were the best in class. In summary, our current data
provided strong evidence to warrant future in vivo preclinical
studies for NK-HDAC-1.
CONCLUSIONS
■
Conformationally constrained HDAC inhibitors bearing
benzoxazole or benzothiazole cap moieties were synthesized
via click chemistry. A promising chiral lead compound, NK-
HDAC-1, with an isopropyl substituent showed approximately
10-fold greater potency than SAHA in vitro. The SAR data of
efficacy were well rationalized by computational studies.
Besides the hydrophobic interactions between NK-HDAC-1
and HDAC2 protein, this article specially addressed the
significance of the rotation barrier of a single bond to the
binding affinity, which would provide inspiring clues for future
drug design programs. Additionally, the molecular weight of
NK-HDAC-1 is only 327.1, which is lower than most of the
HDACi drugs and candidates in clinical trials. It leaves great
space for future lead optimization. Poor PK properties,
including short in vivo half-life and low bioavailability, were
the major problems for most of hydroxamate-containing
HDACi in clinical trials. The in vitro PK data in this article
EXPERIMENTAL SECTION
■
HDAC Enzymes Assay. The compounds were screened using
purified HeLa cell nuclear extracts. The HDAC enzyme assay was
based on the homogeneous fluorescence release assay in which
deacetylation of the subtract, Ac-Arg-Gly-Lys(Ac)-AMC, generates a
fluorophore that can be detected by a fluorometer.
Inhibition of Cell Growth in Vitro. Cells were seeded in 96-well
plates and treated for 72 h with six serial compound dilutions. Cell
viability was measured using an MTT assay which was performed
following the manufacturer’s protocol. The GI50 value was obtained
using Calcusyn software (Biosoft,Cambridge, UK). The results were
derived from three independent experiments performed in triplicate.
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raphy was performed on silica gel (200−300 mesh normal phase from
Table 4. PK Related in Vitro Properties of SAHA and NK-
HDAC-1
1
Qingdao, or 200−400 mesh reverse phase from MED). H and 13C
a
NMR spectra were obtained on a Bruker Avance 300 or 400
spectrometer, using tetramethylsilane as an internal standard. High
resolution mass spectra (HRMS) were obtained on a QFT−ESI mass
spectrometer.
SAHA
264.2
NK-HDAC-1
327.1
molecular weight
c
solubility in pH 7.4 PBS
buffer
>170 μg/
20 μg/mL
b
Compound purity was determined by HPLC analysis on a
Shimadzu LD-20A system with an ODS-C18 column (4.6 × 150
mm, 5 μm) 1.2 mL/min over 15 min by UV at 220 nm. Enantiomeric
excess (ee) determinations were carried out by chiral HPLC on a
Chiral AD-H column, eluting with hexane/isopropanol/MeOH
(containing 0.1% TFA) = 40:40:20 and detecting by UV at 254 nm.
General Procedure for the Preparation of Click HDACi 1−13
and 6′. Triisopropylsilane (3 mmol, 0.6 mL, 3 equiv), PMB protected
substrate (1a−13a and 6′a, 1 mmol, 1.0 equiv), and TFA (2 mL) were
added in sequence to a reaction vessel containing DCM (40 mL). The
mixture was stirred at room temperature and monitored by TLC.
Upon completion, the reaction mixture was diluted with acetonitrile
(100 mL) and then neutralized with DOWEX Marathon WBA anion
exchange resin (from Aldrich). The resin was washed with acetonitrile
(50 mL × 2). The combined organic solution was evaporated to
provide a solid, which was purified to afford the final product.
(E)-3-(1-((Benzo[d]thiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)-
N-hydroxyacrylamide (1). Compound 1 was synthesized from
compound 1a following the general procedure The crude product was
purified via C-18 reverse phase chromatography (water/acetonitrile
from 6:1 to 2.5:1) to give the product as a light yellow solid in 65%
yield. 1H NMR (500 MHz, DMSO-d6) δ 10.82 (br, s, 1H), 9.05 (br, s,
1H), 8.54 (s, 1H), 8.09 (apparent d, J = 7.9 Hz, 1H), 7.98 (apparent d,
J = 8.1 Hz, 1H), 7.50−7.53 (m, 1H), 7.40−7.46 (m, 2H), 6.63 (d, J =
15.8 Hz, 1H), 6.18 (s, 2H); 13C NMR (125 MHz, DMSO-d6) δ 165.1,
162.3, 152.2, 143.3, 134.8, 126.8, 126.5, 125.7, 125.6, 122.8, 122.5,
120.0, 50.7. HRMS (−ESI) calcd for C13H10N5O2S [M − H]−
300.0561; found, 300.0554. Anal. RP-HPLC tR = 8.12 min
(acetonitrile/water (containing 0.1% TFA) = 25:75; purity = 100%).
(E)-3-(1-((Benzo[d]oxazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)-
N-hydroxyacrylamide (2). Compound 2 was synthesized from
compound 2a following the general procedure. The crude product was
purified via C-18 reverse phase chromatography (water/acetonitrile
mL
e
c
LogD in pH 7.4 PBS
buffer
1.0
2.0
c
c
in vitro stability (t1/2
)
artificial gastric
fluid
22 h
9.4 h
c
c
artificial
intestinal fluid
>24 h
>24 h
c
c
plasma (mouse) 3.3 h
32 h
c
c
micrisome
(mouse)
82 min
195 min
c
c
human CYP 3A4
inhibition
24.8 μM
31.9 μM
Caco-2 permeability assay Papp B to A
at 10 μM
1.70 × 10−6
7.61 × 10−6
e
c
cm/s
cm/s
f
Papp A to B
ND
6.61 × 10−6
c
cm/s
d
f
c
efflux ratio
ND
1.15
a
b
Mean of three independent triplicate experiments. See ref 27,
c
d
determined in neutral water. Data from our lab. Defined by the ratio
of Papp B to A over Papp A to B; see ref 22; ND = not deterimined;
e
f
Histone H3 Acetylation Assay. MDA-MB-231 cells plated in a
35-mm dish were treated with compound 12 or SAHA at different
concentrations for 24 h before the cells were trypsinized and washed
with PBS. The treated cells were then lysed with RIPA lysis buffer
containing 50 mM Tris−HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA,
2 mM EGTA, 1 mM dithiothreitol, 1% Nonidet P-40, 0.1% SDS,
protease inhibitors (1 mM PMSF, 5 mg/mL aprotinin, 5 mg/mL
leupeptin, and 5 mg/mL pepstatin), and phosphatase inhibitors (20
mM β-glycerophosphate, 50 mM NaF, and 1 mM Na3VO4) for 30
min at 4 °C, and the supernatant was obtained after centrifuged at
12,000g, 4 °C for 20 min. The protein concentration was quantified by
a BCA protein assay reagent kit (Beyotime Institute of Biotechnol-
ogy). Identical amounts of protein (60 μg) from each sample were
applied for SDS−PAGE and then transferred to the polyvinylidene
difluoride membrane (Millipore, USA). The acetylation level was
detected by primary antibody Antiacetyl-Histon H3 (Millipore, USA)
and HRP-linked goat antirabbit secondary antibody (Santa Cruz
Biotechnology, USA). The membrane was developed using enhanced
chemiluminescence Western blot detection reagents (Millipore, USA).
In Vitro ADME Studies. Solubility and logD were measured using
the published methods.24 Microsomal stability studies, coca-2
permeability assay, and CYP3A4 inhibition were performed accroding
to published methods by Centaurus Biopharma, Ltd.
1
from 6:1 to 2.5:1) to give the product as white solid in 50% yield. H
NMR (500 MHz, DMF-d7) δ 10.99 (br, s,1H), 9.38 (br, s, 1H), 8.70
(s, 1H), 7.74−7.77 (m, 2H), 7.57 (d, J = 15.7 Hz, 1H), 7.42−7.49 (m,
2H), 6.85 (d, J = 15.7 Hz, 1H), 6.23 (s, 2H); 13C NMR (125 MHz,
DMF-d7) δ 160.7, 151.0, 144.1, 140.9, 127.2, 126.0, 125.7, 125.0,
120.31, 120.17, 111.0, 46.9 (one peak less due to overlap with solvent
DMF-d7). HRMS(−ESI) calcd for C13H10N5O3[M − H]− 284.0789;
found, 284.0783. Anal. RP-HPLC tR = 6.01 min (acetonitrile/water
(containing 0.1% TFA) = 25:75; purity = 100%).
(E)-3-(1-((1H-Benzo[d]imidazol-2-yl)methyl)-1H-1,2,3-triazol-
4-yl)-N-hydroxyacrylamide (3). Compound 3 was synthesized from
compound 3a following the general procedure. The crude product was
purified via C-18 reverse phase chromatography (water/acetonitrile
Molecular Docking. AutoDock 4.225 was used for the docking
calculations. The AutodockTools26 package was employed to generate
the docking input files and to analyze the docking results. The charge
of Znic and charge of O* in R-CONHO*H was set to +2 and −1,
respectively. The torsion angle of O−C−N−O* is set to 32.8°. A grid
box size of 18.75 × 18.75 × 18.75 Å3 with a spacing of 0.375 Å was
implemented and covered almost the entire HDAC221 protein surface.
Lamarckian genetic algorithm runs were set at 200 runs, with a
population size of 200, a quaternion step of 30.0°, and a torsion step of
30.0°. All other parameter values were default values.
1
from 7:1 to 4:1) to give the product as a white solid in 50% yield. H
NMR (500 MHz, DMSO-d6 + CF3COOD) δ 8.52 (s, 1H), 7.76 (br, s,
2H), 7.51 (br, s, 2H), 7.48 (d, J = 16 Hz, 1H), 6.67 (d, J = 15.7 Hz,
1H), 6.25 (s, 2H) (two active protons less); 13C NMR (125 MHz,
DMSO-d6 + CF3COOD) δ 162.8, 147.7, 143.9, 131.5, 127.3, 126.5,
120.5, 118.3, 116.3, 114.6, 114.4, 112.5, 45.2. HRMS(−ESI) calcd for
C13H11N6O2 [M − H]− 283.0949; found, 283.0945. Anal. RP-HPLC
tR = 9.82 min (acetonitrile/water (containing 0.1% TFA) = 10:90;
purity = 100%)
4-(4-((E)-2-(Hydroxycarbamoyl)vinyl)-1H-1,2,3-triazol-1-yl)-
4-(benzo[d]oxazol-2-yl)butanamide (4). To a stirred solution of
compound 11a (0.1 mmol, 47 mg, 1.0 equiv.) in dry THF (1 mL) at
−30 °C was added N-methyl piperidine (12 μL, 1.1 equiv.), followed
by isobutyl chloroformate (15 μL, 1.1 equiv). The reaction mixture
was allowed to warm to −20 °C over 30 min, and then gaseous
ammonia was then bubbled through the reaction mixture for 15 min.
After warming to rt over 1 h, the reaction mixture was diluted with
water, and the organic layer was separated. The aqueous layer was
DFT Calculation. The geometries of compounds 2 and 3 were
optimized using the DFT (B3LYP) method with the 6-31G** basis
set. The relaxed coordinate scan of compounds 12 and 2 was
performed using the DFT (B3LYP) method with the 6-31G** basis
set. The dihedral angle (H1−Cchiral−Ntriazole−Ctriazole or Ci‑pro−Cchiral
−
Ntriazole−Ctriazole) was increased from 0° to 360° with a step of 5°. All
the DFT calculations were performed using Jaguar 7.8.20
Chemistry. All reagents were purchased from Alfa Aesar and used
without further purification. Thin-layer chromatography (TLC) was
carried out on silica GF254 plates (Qingdao). Column chromatog-
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extracted with DCM, and the combined organic extracts were dried
over Na2SO4 and concentrated under reduced pressure to give the
desired compound 4a as a light yellow solid. The PMB group was
removed following the general procedure to afford the crude product.
The crude product was purified via C-18 reverse phase chromatog-
raphy (water/acetonitrile from 7:1 to 4:1) to give the product as a
[M − H]− 374.1259; found, 374.1250. Anal. RP-HPLC tR = 6.78 min
(acetonitrile/water (containing 0.1% TFA) = 40:60; purity = 94.0%).
(E)-3-(1-(1-(Benzo[d]oxazol-2-yl)-2-methylbutyl)-1H-1,2,3-
triazol-4-yl)-N-hydroxyacrylamide (8). Compound 8 was synthe-
sized from compound 8a following the general procedure. The crude
product was purified via C-18 reverse phase chromatography (water/
acetonitrile from 5:1 to2.5:1) to give the product as white solid in 45%
yield. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (br, s, 1H), 9.12 (br, s,
1H), 8.69 (s, 1H), 7.82 (d, J = 7.3 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H),
7.39−7.48 (m, 3H), 6.64 (d, J = 15.7 Hz, 1H), 6.16 (d, J = 9.4 Hz,
1H), 2.68−2.75 (m, 1H), 1.19−1.26 (m, 1H), 1.06−1.13 (m, 1H),
0.99 (d, J = 6.7 Hz, 3H), 0.86 (t, J = 7.3 Hz, 3H); 13C NMR (100
MHz, DMSO-d6) δ 162.2, 161.6, 150.0, 143.3, 139.9, 126.6, 126.1,
125.1, 124.7, 120.2, 120.1, 111.2, 62.3, 37.4, 24.4, 15.3, 10.5.
HRMS(+ESI) calcd for C17H20N5O3 [M + H]+ 347.1561; found,
342.1566. Anal. RP-HPLC tR = 9.00 min (acetonitrile/water
(containing 0.1% TFA) = 35:65; purity = 99.4%).
1
white solid (32%, 2 steps). H NMR (400 MHz, DMSO-d6) δ 10.87
(s, 1H), 9.11 (s, 1H), 8.67 (s, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.74 (d, J
= 7.5 Hz, 1H), 7.40−7.47 (m, 3H),7.34 (s, 1H), 6.88(s, 1H), 6.65 (d, J
= 15.7 Hz, 1H), 6.37 (apparent t, J = 7.4 Hz, 1H), 2.75−2.81 (m, 1H),
2.56−2.67 (m, 1H), 2.06−2.20 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 172.5, 162.1, 150.3, 143.4, 140.0, 126.0, 125.0, 124.5,
120.18, 120.12, 111.2, 57.4, 30.2, 27.8 (two peaks less due to overlap).
HRMS(−ESI) calcd for C16H15N6O4 [M − H]− 356.1160; found,
356.1161. Anal. RP-HPLC tR = 6.83 min (acetonitrile/water
(contaning 0.1% TFA) = 20:75; purity = 98.5%).
(E)-3-(1-(1-(Benzo[d]oxazol-2-yl)ethyl)-1H-1,2,3-triazol-4-yl)-
N-hydroxyacrylamide (5). Compound 5 was synthesized from
compound 5a following the general procedure. The crude product was
purified via C-18 reverse phase chromatography (water/acetonitrile
(E)-3-(1-(1-(Benzo[d]oxazol-2-yl)-3-methylbutyl)-1H-1,2,3-
triazol-4-yl)-N-hydroxyacrylamide (9). Compound 9 was synthe-
sized from compound 9a following the general procedure. The crude
product was purified via C-18 reverse phase chromatography (water/
acetonitrile from 5:1 to 2.5:1) to give the product as a white solid in
1
from 5:1 to 3:1) to give the product as a white solid in 45% yield. H
NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H), 7.78−7.81 (m, 1H),
7.72−7.75 (m, 1H), 7.39−7.46 (m, 3H), 6.64 (d, J = 15.8 Hz, 1H),
6.48 (q, J = 7.0 Hz, 1H), 2.05 (d, J = 7.0 Hz, 3H) (two active proton
less); 13C NMR (100 MHz, DMSO-d6) δ 163.0, 162.3, 150.3, 143.3,
140.1, 126.7, 125.9, 124.9, 124.2, 120.13, 120.05, 111.1, 53.6, 18.3.
HRMS(−ESI) calcd for C14H12N5O3 [M − H]− 298.0946; found,
298.0948. Anal. RP-HPLC tR = 4.89 min (acetonitrile/water
(containing 0.1% TFA) = 30:70, purity = 97.9%).
1
48% yield. H NMR (400 MHz, DMSO-d6) δ 10.86 (br, s, 1H), 9.14
(br, s, 1H), 8.71 (s, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.74 (d, J = 7.5 Hz,
1H), 7.40−7.46 (m, 3H), 6.65 (d, J = 15.7 Hz, 1H), 6.41 (dd, J1 = 5.6
Hz, J2 = 9.8 Hz, 1H), 2.33−2.40 (m, 1H), 2.42−2.46 (m, 1H), 1.31−
1.37 (m, 1H), 0.98 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H); 13C
NMR (100 MHz, DMSO-d6) δ 162.5, 162.2, 150.2, 143.2, 139.9,
126.6, 125.9, 124.9, 124.4, 120.1, 111.1, 56.3, 40.3, 24.2, 22.3, 21.2.
HRMS(+ESI) calcd for C17H20N5O3 [M + H]+ 342.1561; found,
342.1567. Anal. RP-HPLC tR = 9.96 min (acetonitrile/water
(containing 0.1% TFA) = 35:65; purity = 99.3%)
(E)-3-(1-(1-(Benzo[d]oxazol-2-yl)-2-methylpropyl)-1H-1,2,3-
triazol-4-yl)-N-hydroxyacrylamide (6). Compound 6 was synthe-
sized from compound 6a following the general procedure. The crude
product was purified via C-18 reverse phase chromatography (water/
acetonitrile from 5:1 to 3:1) to give the product as a white solid in 44%
yield. 1H NMR (400 MHz, DMSO-d6) δ: 8.66 (s, 1H), 7.82 (d, J = 7.3
Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.40−7.48 (m, 3H), 6.65 (d, J =
15.7 Hz, 1H), 6.11 (d, J = 9.1 Hz, 1H), 2.89−2.97 (m, 1H), 1.04 (d, J
= 6.4 Hz, 3H), 0.87 (d, J = 6.4 Hz, 3H) (two active proton less). 13C
NMR(100 MHz, DMSO-d6) δ: 162.2, 161.6, 150.0, 143.3, 140.0,
126.5, 126.1, 125.0, 124.6, 120.2, 111.2, 63.3, 31.4, 19.0, 18.3 (one
peak less due to overlap). HRMS(−ESI) calcd for C16H16N5O3 [M −
H]− 326.1259; found, 326.1266. Anal. RP-HPLC tR = 4.88 min
(acetonitrile/water (containing 0.1% TFA) = 40:60; purity = 99.1%).
(E)-3-(1-(1-(Benzo[d]thiazol-2-yl)-2-methylpropyl)-1H-1,2,3-
triazol-4-yl)-N-hydroxyacrylamide (6′). Compound 6′ was synthe-
sized from compound 6′a following the general procedure. The crude
product was purified via C-18 reverse phase chromatography (water/
acetonitrile from 5:1 to2:1) to give the product as a white solid in 48%
3-(4-((E)-2-(Hydroxycarbamoyl)vinyl)-1H-1,2,3-triazol-1-yl)-
3-(benzo[d]oxazol-2-yl)propanoic Acid (10). Compound 10 was
synthesized from compound 10a following the general procedure. The
crude product was purified via C-18 reverse phase chromatography
(water/acetonitrile from 7:1 to 4:1) to give the product as a white
1
solid in 59% yield. H NMR (400 MHz, DMSO-d6) δ 12.85 (br, s,
1H), 10.84 (s, 1H), 9.07 (s, 1H), 8.70 (s, 1H), 7.72−7.81 (m, 2H),
7.39−7.46 (m, 3H), 6.59−6.65 (m, 2H), 3.70 (dd, J1 = 17.2 Hz, J2 =
6.7 Hz, 1H), 3.56 (dd, J1 = 17.2 Hz, J2 = 8.1 Hz, 1H); 13C NMR (100
MHz, DMSO-d6) δ 170.2, 162.2, 161.6, 150.2, 143.0, 139.8, 126.6,
126.0, 125.0, 124.9, 120.13, 120.06, 111.1, 54.2, 36.4. HRMS(−ESI)
calcd for C15H12N5O5 [M − H]− 342.0844; found, 342.0852. Anal.
RP-HPLC tR = 5.88 min (acetonitrile/water (containing 0.1% TFA) =
25:75; purity = 96.2%).
4-(4-((E)-2-(Hydroxycarbamoyl)vinyl)-1H-1,2,3-triazol-1-yl)-
4-(benzo[d]oxazol-2-yl)butanoic Acid (11). Compound 11 was
synthesized from compound 11a following the general procedure. The
crude product was purified via C-18 reverse phase chromatography
(water/acetonitrile from 7:1 to 4:1) to give the product as a white
1
yield. H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.10 (s, 1H),
8.67 (s, 1H), 8.06−8.15 (m, 2H), 7.47−7.58 (m, 3H), 7.43 (d, J = 15.7
Hz, 1H), 6.66 (d, J = 15.7 Hz, 1H), 6.15 (d, J = 9.7 Hz, 1H), 2.87−
2.96 (m, 1H), 0.99 (d, J = 6.4 Hz, 3H), 0.87 (d, J = 6.3 Hz, 3H); 13C
NMR (100 MHz, DMSO-d6) δ 167.5, 162.3, 151.9, 143.1, 134.7,126.7,
126.6, 124.7, 123.1, 122.5, 120.1, 67.5, 33.2, 19.2, 18.7. HRMS(−ESI)
calcd for C16H16N5O2S [M − H]− 342.1030; found, 342.1031. Anal.
RP-HPLC tR = 5.32 min (acetonitrile/water (containing 0.1% TFA) =
40:60, purity = 96.1%).
(E)-3-(1-(1-(Benzo[d]oxazol-2-yl)-2-phenylethyl)-1H-1,2,3-tri-
azol-4-yl)-N-hydroxyacrylamide (7). Compound 7 was synthesized
from compound 7a following the general procedure The crude
product was purified via C-18 reverse phase chromatography (water/
acetonitrile from 5:1 to3:1) to give the product as a white solid in 61%
yield. 1H NMR (500 MHz, DMSO-d6) δ 1.80 (br, s, 1H), 9.04 (br, s,
1H), 8.59 (s, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H),
7.39−7.45 (m, 2H), 7.34 (d, J = 15.7 Hz, 1H), 7.21−7.22 (m, 4H),
7.16−7.19 (m, 1H), 6.63−6.66 (m, 1H), 6.58 (d, J = 15.7 Hz, 1H),
3.93 (dd, J1 = 5.5 Hz, J2 = 14.2 Hz,1H), 3.79 (dd, J1 = 10.5 Hz, J2 =
13.8 Hz, 1H); 13C NMR(125 MHz, DMF-d7) δ 162.8, 162.5, 150.7,
143.6, 140.5, 136.0, 129.5, 128.9, 127.5, 127.2, 126.6, 125.5, 125.2,
120.7, 120.6, 111.7, 59.4, 38.1. HRMS(−ESI) calcd for C20H16N5O3
1
solid in 55% yield. H NMR (400 MHz, DMSO-d6) δ 10.85 (br, s,
1H), 9.10 (br, s, 1H), 8.68 (s, 1H), 7.82 (d, J = 6.9 Hz, 1H), 7.74 (d, J
= 8.2 Hz, 1H), 7.40−7.47 (m, 2H), 6.65 (d, J = 15.7 Hz, 1H), 6.40
(dd, J1 = 6.1 Hz, J2 = 8.8 Hz, 1H), 2.75−2.84 (m, 1H), 2.58−2.69 (m,
1H), 2.23−2.39 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 173.1,
162.3, 161.9, 150.3, 143.3, 140.0, 126.7, 126.0, 125.0, 124.6, 120.20,
120.16, 111.2, 57.1, 29.5, 27.5. HRMS(−ESI) calcd for C16H14N5O5
[M − H]− 356.1000; found, 356.1008. Anal. RP-HPLC tR = 6.17 min
(acetonitrile/water (containing 0.1% TFA) = 25:75; purity = 95.3%).
(E)-3-(1-((S)-1-(Benzo[d]oxazol-2-yl)-2-methylpropyl)-1H-
1,2,3-triazol-4-yl)-N-hydroxyacrylamide (12). Compound 12 was
synthesized from compound 12a following the general procedure. The
crude product was purified via C-18 reverse phase chromatography
(water/acetonitrile from 5:1 to 3:1) to give the product as a white
1
solid in 42% yield, ee = 91.3%. H NMR (400 MHz, DMSO-d6) δ
10.80 (br, s, 1H), 9.10 (br, s, 1H), 8.66 (s, 1H), 7.82 (d, J = 7.9 Hz,
1H), 7.76 (d, J = 7.4 Hz), 7.39−7.48 (m, 3H), 6.65 (d, J = 15.7 Hz),
6.11 (d, J = 9.2 Hz, 1H), 2.88−2.97 (m, 1H), 1.03 (d, J = 6.6 Hz, 3H),
0.86(d, J = 6.6 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 162.3,
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161.6, 150.1, 143.3, 139.9, 126.6, 126.1, 125.0, 124.7, 120.2, 120.1,
111.2, 63.4, 31.4, 19.0, 18.3. HRMS(−ESI) calcd for C16H16N5O3 [M
− H]− 326.1259; found, 326.1265.
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(E)-3-(1-((R)-1-(Benzo[d]oxazol-2-yl)-2-methylpropyl)-1H-
1,2,3-triazol-4-yl)-N-hydroxyacrylamide (13). Compound 13 was
synthesized from compound 13a following the general procedure. The
crude product was purified via C-18 reverse phase chromatography
(water/acetonitrile from 5:1 to 3:1) to give the product as white solid
in 44% yield, ee = 92.1%. 1H NMR (400 MHz, DMSO-d6) δ 10.83 (br,
s, 1H), 9.09 (br, s, 1H), 8.67 (s, 1H), 7.83 (d, J = 7.2 Hz, 1H), 7.76 (d,
J = 7.3 Hz, 1H), 7.40−7.48 (m, 3H), 6.65 (d, J = 15.8 Hz, 1H), 6.11
(d, J = 9.2 Hz, 1H), 2.88−2.97 (m, 1H), 1.03 (d, J = 6.7 Hz, 3H), 0.86
(d, J = 6.6 Hz, 3H); 13C NMR(100 MHz, DMSO-d6) δ 162.3, 161.6,
150.1, 143.2, 139.9, 126.7, 126.1, 125.0, 124.7, 120.2, 120.1, 111.2,
63.3, 31.4, 19.0, 18.3. HRMS(−ESI) calcd for C16H16N5O3 [M − H]−
326.1259; found, 326.1266.
ASSOCIATED CONTENT
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S
* Supporting Information
Full experimental details, NMR spectra, and chiral HPLC. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (86) 022-23500781. Fax: (86) 022-23505369. E-mail:
pwang@nankai.edu.cn.
Author Contributions
∥These authors contributed equally to this work.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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This study was supported by the National 973 Basic Research
Program of China (No. 2010CB529100) and National Natural
Science Foundation of China (No. 21072105). The HDAC
inhibition assay was assisted by Dr. Wenfang Xu from Institute
of Medicinal Chemistry School of Pharmaceutical Sciences,
Shandong University. The cancer cell line MTS studies were
performed by the NIH with the assistance from Dr. Robert
Woodward.
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