Late-stage C-H coupling enables rapid identification of HDAC inhibitors: Synthesis and evaluation of NCH-31 analogues

Article abstract of DOI:10.1021/ml500024s

We previously reported the discovery of NCH-31, a potent histone deacetylase (HDAC) inhibitor. By utilizing our C-H coupling reaction, we rapidly synthesized 16 analogues (IYS-1 through IYS-15 and IYS-Me) of NCH-31 with different aryl groups at the C4-position of 2-aminothiazole core of NCH-31. Subsequent biological testing of these derivatives revealed that 3-fluorophenyl (IYS-10) and 4-fluorophenyl (IYS-15) derivatives act as potent pan-HDAC inhibitor. Additionally, 4-methylphenyl (IYS-1) and 3-fluoro-4-methylphenyl (IYS-14) derivatives acted as HDAC6-insensitive inhibitors. The present work clearly shows the power of the late-stage C-H coupling approach to rapidly identify novel and highly active/selective biofunctional molecules.

Full text of DOI:10.1021/ml500024s

Letter
pubs.acs.org/acsmedchemlett
Late-Stage C−H Coupling Enables Rapid Identification of HDAC
Inhibitors: Synthesis and Evaluation of NCH-31 Analogues
Hiromi Sekizawa,† Kazuma Amaike,† Yukihiro Itoh,‡ Takayoshi Suzuki,*,‡,§ Kenichiro Itami,*,†,∥
and Junichiro Yamaguchi*,†
†Institute of Transformative Bio-Molecules (WPI-ITbM) and Graduate School of Science, Nagoya University, Chikusa, Nagoya
464-8602, Japan
‡Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 13 Taishogun Nishitakatsukasa-Cho, Kita-ku, Kyoto
603-8334, Japan
§JST, PRESTO, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
∥JST, ERATO, Itami Molecular Nanocarbon Project, Nagoya University, Chikusa, Nagoya 464-8602, Japan
S
* Supporting Information
ABSTRACT: We previously reported the discovery of NCH-
31, a potent histone deacetylase (HDAC) inhibitor. By
utilizing our C−H coupling reaction, we rapidly synthesized
16 analogues (IYS-1 through IYS-15 and IYS-Me) of NCH-31
with different aryl groups at the C4-position of 2-amino-
thiazole core of NCH-31. Subsequent biological testing of
these derivatives revealed that 3-fluorophenyl (IYS-10) and 4-fluorophenyl (IYS-15) derivatives act as potent pan-HDAC
inhibitor. Additionally, 4-methylphenyl (IYS-1) and 3-fluoro-4-methylphenyl (IYS-14) derivatives acted as HDAC6-insensitive
inhibitors. The present work clearly shows the power of the late-stage C−H coupling approach to rapidly identify novel and
highly active/selective biofunctional molecules.
KEYWORDS: Histone deacetylase, inhibitor, C−H coupling, HDAC6
istone deacetylases (HDACs) catalyze the removal of
(CTCL).6,7 However, the use of HDAC inhibitors is currently
Hacetyl groups from N-acetylated lysine residues of various limited to CTCL. Therefore, there is a need to develop novel
protein substrates such as histones and α-tubulin.1,2 HDACs
play important roles in various fundamental life processes
potent HDAC inhibitors as anticancer agents.
To date, a number of HDAC inhibitors have been identified,8
including gene expression and cell cycle progression.3 There are
including NCH-31 (Figure 1), which was discovered by one of
us (T.S.).9 Among the HDAC inhibitors reported so far,
currently 18 known HDACs that are organized into several
HDAC6-insensitive inhibitors10 have been reported to show
classes with regard to their DNA sequence similarity: class I
potent and selective cancer cell growth-inhibitory activity by
reactive oxygen species-induced apoptosis.11,12 Following these
findings, we performed further investigation of NCH-31
derivatives, seeking to find potent HDAC inhibitors and
selective HDAC6-insensitive inhibitors. Since the thiol group of
NCH-31 is needed to coordinate the zinc ion in the active site
of class I, II, and IV HDACs,9 we decided to carry out the
structural modification of the arylthiazole amide moiety of
NCH-31 to obtain the desired HDAC inhibitors.
In planning synthesis of NCH-31 derivatives, we realized that
step-economical, late-stage C−H functionalization approach
should be taken rather than following our previous synthesis of
NCH-31 using Hantzsch thiazole synthesis (Figure 2). When
following the classical condensation reaction to make NCH-31
derivatives, one needs to start from acetophenone derivatives to
modify aryl groups; bromination of acetophenones, thiazole
HDACs (HDAC1−3 and HDAC8); class IIa HADCs
(HDAC4, HDAC5, HDAC7, and HDAC10); class IIb
HDACs (HDAC6 and HDAC10); class III HDACs (SIRT1−
7); and class IV HDAC (HDAC11).4 Class I, II, and IV
HDACs (HDAC1−11) are zinc-dependent enzymes, whereas
class III HDACs (SIRT1−7) are NAD+-dependent enzymes.4
Because HDACs are associated with various diseases, especially
cancer, HDAC inhibitors have been developed as therapeutic
agents such as anticancer drugs.5 Indeed, two HDAC inhibitors
vorinostat and romidepsin (Figure 1) have been approved by
the FDA for the treatment of cutaneous T-cell lymphoma
Received: January 20, 2014
Accepted: March 3, 2014
Published: March 3, 2014
Figure 1. HDAC inhibitors: vorinostat, romidepsin, and NCH-31.
© 2014 American Chemical Society
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Figure 2. Synthesis of NCH-31 derivatives through classical and C−H functionalization routes.
Figure 3. Synthesis of NCH-31 analogues (IYS-1−15 and IYS-Me) by C−H coupling. Reaction conditions: (a) EDC·HCl (1.4 equiv), CH2Cl2, 23
°C, 6 h, 80%; (b) AcSK (4.0 equiv), EtOH, 23 °C, 16 h, 98%; (c) ArB(OH)2 (4.0 equiv), Pd(OAc)2 (10 mol %), phen (10 mol %), LiBF4 (1.5
euqiv), TEMPO (1.0 equiv), AcOH (1.0 equiv), DMAc, 100 °C, 10−29%; (d) K2CO3, MeOH, 23 °C; (e) MeI, NaH, DMF, 23 °C; (f) NH2NH2,
CH3CN; then dithiothreitol, NEt3, 23 °C.
formation with thiourea, condensation with carboxylic acids,
and introduction of the thiol unit.13 An ideal, step-economical,
diversity-oriented approach would be to introduce aryl groups
onto a thiazole core of NCH-31 at the late stage of the
synthesis. In doing so, we envisioned that the application of our
recently developed unique Pd catalyst that promotes otherwise-
difficult C4-selective C−H arylation of thiazoles with
arylboronic acids should perfectly match this requirement
(Figure 2).14−16 Considering the preparation of the different
C4-aryl substituents, the present C−H functionalization route
only takes 2n + 2 steps, whereas the classical route takes 5n
steps (n = desired number of thiazole derivatives).
Herein, we demonstrate the synthesis of NCH-31 analogues
by late-stage C−H coupling,17−19 which lead to the rapid
examination of the structure−activity and structure−selectivity
relationships, and identification of new pan-HDAC inhibitors
and HDAC6-insensitive inhibitors that are more potent and
selective than NCH-31.
The synthesis of NCH-31 derivatives commenced with the
condensation of 2-aminothiazole and 7-bromoheptanoic acid,
which are both commercially available compounds, to provide
bromide 1 in 80% yield (Figure 3). Thiolation of 1 by
treatment with potassium thioacetate (AcSK) gave thiazole
amide 2 in excellent yield. Thiazole 2 was then coupled with
various arylboronic acids under our reported conditions for C4-
selective C−H arylation of thiazoles,15 which consists of
Pd(OAc)2 (10 mol %) and 1,10-phenanthroline (phen: 10 mol
%) as a catalyst, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO,
1.0 equiv) as an oxidant, AcOH (1.0 equiv), and LiBF4 (1.5
equiv) in dimethylacetamide (DMAc) at 100 °C, to afford the
corresponding coupling products. These products were then
deacetylated to give IYS-1−15 with virtually complete C4-
selectivity. Unfortunately, arylboronic acids with amino
substituents, heteroaryl substituents, and ortho substituents
did not work under the present conditions. Additionally, 2 was
alkylated at the nitrogen atom of the amide by methyl iodide to
afford 4 and was then C−H arylated at the C4-position and
deacetylated to give IYS-Me. The synthesized NCH-31
analogues (IYS-1−15 and IYS-Me) were tested with an in
vitro assay using human recombinant HDAC1, HDAC6, and
HDAC9, a representative isozyme of Class I, IIb, and IIa
HDACs, respectively (Figure 4). For HDAC1, IYS-1−15
(except IYS-5) showed moderate to excellent inhibition
compared to NCH-31 at 0.1 μM, whereas IYS-Me did not
show HDAC1 inhibition. In the case of HDAC6, a few
compounds displayed moderate to good inhibition; particularly,
IYS-9 and IYS-10 showed more than 70% inhibition at 1 μM,
which is higher than NCH-31. However, IYS-1−5 and 11−14
were totally inactive against HDAC6. IYS-1, IYS-10, IYS-14,
and IYS-15, which bear methyl or fluoro groups on the meta
and/or para positions of the benzene ring, displayed HDAC9
inhibitory activity stronger than NCH-31 at 0.1 μM. These
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NCH-31) with HDAC1 by using Molegro Virtual Docker 5.0.
The simulations were performed based on the reported X-ray
structure of HDAC120 and under the condition that the
catalytic site was set as search space. As a result of these
calculations, the thiolate group of both IYS-15 and NCH-31 is
shown to coordinate to the zinc ion (Figure 5). However, the
Figure 5. (A) View of the conformation of IYS-15 (ball and stick
model) docked in the HDAC1 catalytic core. (B) View of the
conformation of NCH-31 (ball and stick model) docked in the
HDAC1 catalytic core.
Figure 4. HDAC activity in the presence of IYS-1−15 and IYS-Me:
blue bar for HDAC1 (enzyme activity % at 0.1 μM), purple bar for
HDAC6 (enzyme activity % at 1 μM), and brown bar for HDAC9
(enzyme activity % at 0.1 μM).
results indicate that IYS-10 and IYS-15 might be a potent pan-
HDAC inhibitor and that IYS-1 and IYS-14 might be potent
HDAC6-insensitive inhibitors.
The IC50 values of IYS-1, IYS-10, IYS-14, and IYS-15 for
HDAC1, HDAC6, and HDAC9 were also determined (Table
1). In these assays, NCH-31 inhibited HDAC1, HDAC6, and
lowest energy conformation of the aromatic ring portion of
IYS-15 docked in HDAC1 was different from that of NCH-31.
Specifically, the benzene ring of IYS-15 was positioned near His
28 and Phe 150, where the fluorine atom could form an N−H···
F hydrogen bond with His 28 (Figure 5A). However, the
benzene ring of NCH-31 was located adjacent to Tyr 204
where hydrogen bonds were not observed (Figure 5B). These
results indicate that the hydrogen bond formation might
contribute to the higher HDAC1-inhibitory activity of IYS-15.
Next, to understand why introducing a methyl group onto
NCH-31 led to a decrease in HDAC6-inhibitory activity, we
studied the binding mode of the inhibitor (IYS-14 or NCH-31)
with a homology model of HDAC6. Inspection of the simulated
HDAC6/NCH-31 complex showed that the phenyl group of
NCH-31 was positioned in the region delineated by Arg 673,
Phe 679, Phe 680, Leu 749, and Gly 751 (region 1 in Figure
6C), and the amide oxygen of NCH-31 could form a hydrogen
bond with His 651 (Figure 6A). However, the 3-fluoro-4-
methylphenyl group of IYS-14 was not located in region 1
because of a steric repulsion between the methyl group of IYS-
14 and Arg 673, but it was located in region 2 (see Figure 6C)
where it could interact only with Pro 681 (Figure 6B,C). The
conformational change could cause the loss of a hydrogen bond
between the amide oxygen of IYS-14 and His 651 (Figure 6B),
which might lead to a decrease in HDAC6-inhibitory activity of
IYS-14. In addition, the differences in activity between IYS-1
and IYS-8, IYS-5 and IYS-6, and NCH-31 and IYS-Me can be
explained by similar binding model studies (see Figures S1−S6
in the Supporting Information).
Table 1. HDAC1, HDAC6, and HDAC9 Inhibition Data for
NCH-31, IYS-1, IYS-10, IYS-14, and IYS-15
HDAC1 IC50 (μM) HDAC6 IC50 (μM) HDAC9 IC50 (μM)
NCH-31
IYS-1
0.096
0.057
0.049
0.050
0.036
0.23
1.8
0.082
0.042
0.078
0.062
0.057
IYS-10
IYS-14
IYS-15
0.15
6.1
0.55
HDAC9 with IC50 values of 0.096, 0.23, and 0.082 μM,
respectively. As shown in Table 1, IYS-1, IYS-10, IYS-14, and
IYS-15 all showed HDAC1 and HDAC9 inhibitory activity
more potent than NCH-31. As for HDAC6, IYS-10 displayed
slightly more potent activity than NCH-31 (IC50 of IYS-10 =
0.15 μM; IC50 of NCH-31 = 0.23 μM), whereas IYS-1 and IYS-
14 were less potent HDAC6 inhibitors (IC50 of IYS-1 = 1.8
μM; IC50 of IYS-14 = 6.1 μM). In particular, the HDAC6-
inhibitory activity of IYS-14 was 27-fold weaker than that of
NCH-31. Thus, IYS-10 and IYS-15 are potent pan-HDAC
inhibitors and IYS-1 and IYS-14 are potent and selective
HDAC6-insensitive inhibitors.
In summary, we successfully prepared 16 NCH-31 analogues,
which possess various aryl groups at the C4-position of the
thiazole core, by late-stage C−H coupling. Screening of the
NCH-31 analogues identified IYS-10 as a potent pan-HDAC
inhibitor and IYS-14 as a potent and selective HDAC6-
insensitive inhibitor. We believe that this methodology is of
value in future studies for the development of more potent and
selective HDAC inhibitors as well as other biologically active
compounds containing an arylthiazole structure.21−23 The late-
stage C−H coupling approach will find use in many areas to
To explore the origin of the potent HDAC1-inhibitory
activity of IYS-15 as compared to NCH-31, we initially
performed a binding model study of the inhibitor (IYS-15 or
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Figure 6. (A) View of the conformation of NCH-31 (ball and stick
model) docked in the HDAC6 catalytic core. (B) View of the
conformation of IYS-14 (ball and stick model) docked in the HDAC6
catalytic core. (C) Superimposition of IYS-14 (purple) and NCH-31
(green) in the active site of HDAC6.
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ASSOCIATED CONTENT
* Supporting Information
Experimental procedures for biological analysis and chemical
synthesis, and characterization of compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
thiazol-2-carbonsaure. Helv. Chim. Acta 1945, 28, 924.
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Itami, K. Oxidative biaryl coupling of thiophenes and thiazoles with
arylboronic acids through palladium catalysis: otherwise difficult C4-
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AUTHOR INFORMATION
Corresponding Authors
*(T.S.) E-mail: suzukit@koto.kpu-m.ac.jp.
*(K.I.) E-mail: itami@chem.nagoya-u.ac.jp.
*(J.Y.) E-mail: junichiro@chem.nagoya-u.ac.jp.
■
(15) Tani, S.; Uehara, T. N.; Yamaguchi, J.; Itami, K. Programmed
synthesis of arylthiazoles through sequential C−H couplings. Chem.
Sci. 2014, 5, 123−135.
(16) Uehara, T. N.; Yamaguchi, J.; Itami, K. Palladium-catalyzed C−
H and C−N arylation of aminothiazoles with arylboronic Acids. Asian
J. Org. Chem. 2013, 2, 938−942.
Funding
This work was supported by the Funding Program for Next
Generation World-Leading Researchers from JSPS (220GR049
to K.I.), Grants-in-Aid for Scientific Research on Innovative
Areas “Molecular Activation Directed toward Straightforward
Synthesis” (25105720 to J.Y.), KAKENHI (25708005 to J.Y.)
from MEXT, and JST PRESTO program (T.S.). ITbM is
supported by the World Premier Interntional Research Center
(WPI) Initiative, Japan.
(17) Yamaguchi, J.; Yamaguchi, A. D.; Itami, K. C−H bond
functionalization: emerging synthetic tools for natural products and
pharmaceuticals. Angew. Chem., Int. Ed. 2012, 51, 8960−9009.
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rapid construction and late-stage diversification of functional
molecules. Nat. Chem. 2013, 5, 369−375.
(19) Meyer, C.; Schepmann, D.; Yanagisawa, S.; Yamaguchi, J.; Dal
Col, V.; Laurini, E.; Itami, K.; Pricl, S.; Wunsch, B. Pd-catalyzed direct
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C−H bond functionalization of spirocyclic σ1 ligands: generation of a
pharmacophore model and analysis of the reverse binding mode by
docking into a 3D homology model of the σ1 receptor. J. Med. Chem.
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Notes
The authors declare no competing financial interest.
ABBREVIATIONS
HDAC, histone deacetylase; SIRT, sirtuin
■
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