Structure-activity relationship study of thiazolyl-hydroxamate derivatives as selective histone deacetylase 6 inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.06.036

Several human diseases are associated with aberrant epigenetic pathways mediated by histone deacetylases (HDACs), especially HDAC6, a class IIb HDACs, which has emerged as an attractive target for neurodegenerative and autoimmune disease therapeutics. In a previous study, we developed the novel HDAC6-selective inhibitor 9a ((E)-N-hydroxy-4-(2-styrylthiazol-4-yl)butanamide) and showed that it has anti-sepsis activity in vivo. In this study, we conducted structure-activity relationship (SAR) studies to optimize the activity and selectivity of HDAC6, synthesizing its derivatives with various aliphatic linker sizes and cap structures. We identified 6u ((E)-N-hydroxy-3-(2-(4-fluorostyryl)thiazol-4-yl)propanamide), which has nanomolar inhibition activity and a 126-fold selectivity for HDAC6 over HDAC1. Through the docking analyses of 6u against HDAC subtypes, we revealed the importance of the optimal aliphatic linker size, as well as the electronic substituent effect and rigidity of the aryl cap group. Thus, we suggest a new rationale for the design of HDAC6-selective inhibitors.

Full text of DOI:10.1016/j.bmc.2019.06.036

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-activity relationship study of thiazolyl-hydroxamate derivatives as
selective histone deacetylase 6 inhibitors
Gibeom Nama, Jun Min Junga, Hyun-Ju Park⁎, Seung Yeop Baek, Ki Seon Baek, Hui yeon Mok,
Da Eun Kim, Young Hoon Jung⁎
School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do 16419, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Several human diseases are associated with aberrant epigenetic pathways mediated by histone deacetylases
(HDACs), especially HDAC6, a class IIb HDACs, which has emerged as an attractive target for neurodegenerative
and autoimmune disease therapeutics. In a previous study, we developed the novel HDAC6-selective inhibitor 9a
((E)-N-hydroxy-4-(2-styrylthiazol-4-yl)butanamide) and showed that it has anti-sepsis activity in vivo. In this
study, we conducted structure-activity relationship (SAR) studies to optimize the activity and selectivity of
HDAC6, synthesizing its derivatives with various aliphatic linker sizes and cap structures. We identified 6u ((E)-
N-hydroxy-3-(2-(4-fluorostyryl)thiazol-4-yl)propanamide), which has nanomolar inhibition activity and a 126-
fold selectivity for HDAC6 over HDAC1. Through the docking analyses of 6u against HDAC subtypes, we re-
vealed the importance of the optimal aliphatic linker size, as well as the electronic substituent effect and rigidity
of the aryl cap group. Thus, we suggest a new rationale for the design of HDAC6-selective inhibitors.
Computer-aided drug design
SAR of thiazolyl-hydroxamates
Selective HDAC6 inhibitors
1. Introduction
genetic ablation of HDAC1-3 is lethal, but the knockout of HDCA6 does
not affect mice. Therefore, HDAC6 inhibitor-induced side effects may
Post-translational modification (PTM) of histones dramatically ef-
fects gene expression. Since the emergence of epigenetics and PTMs,
numerous genes and diseases have been found to be associated with
histone PTMs. The control of the modification processes has been an
attractive approach in drug development,1 and, to date, histone acet-
ylation and methylation modulators have mainly been developed as
cancer therapeutics.2 Histone acetylation is modulated by histone
acetyltransferase (HAT) and deacetylase (HDAC). HDAC subtypes are
grouped into 4 classes and 18 subtypes. Class I, II, and IV are zinc-
dependent enzymes: class I (HDAC 1, 2, 3, and 8), class IIa (HDAC 4, 5,
7, and 9), class IIb (HDAC 6, and 10), and class IV (HDAC11). Class III
members are NAD+-dependent enzymes known as sirtuins. The char-
acteristics of each subtype and their selective inhibitors have been well
investigated.3,4 A majority of the FDA-approved anticancer HDAC in-
hibitors are nonselective class I HDAC inhibitors.
be few, permitting their application in various diseases, including
neurodegenerative and autoimmune diseases. In neurodegenerative
diseases like Huntington’s and Charcot-Marie-Tooth neuropathy, tu-
bulin and microtubule acetylation increases intracellular axonal trans-
port.8,9 Increasing ubiquitination by Hsp90 acetylation is another sug-
gested therapeutic strategy.10 The mechanism of the anti-inflammatory
effect of HDAC6 inhibitors is also intensely being studied. HDAC6 in-
hibitors block MAPK signaling by MKP-1 acetylation,11,12 enhance the
suppressive capacity of regulatory T cells,13,14 and inhibit immune cells
by altering microtubule dynamics and ubiquitination,15–17 Our interest
also lies in the development of selective HDAC6 inhibitors with less
cytotoxicity, applicable to the disease models described above.
In our previous efforts, we identified the novel selective HDAC6
inhibitor 9a (Fig. 1), with a thiazole linker and phenyl cap group, by
computational database screening and molecular design.18
HDAC6 is a unique subtype of HDACs. It is a class IIb HDAC, with
two catalytic domains mainly expressed in the cytoplasm.5 Unlike other
HDACs, HDAC6 is a non-specific histone deacetylase and modifies cy-
toplasmic proteins such as α-tubulin,6 Hsp90, and cortactin. The acet-
ylation of α-tubulin regulates the structure of the cytoskeleton.7 The
The IC50 values of 9a was found to be 199 nM for HDAC6 and
13.8 μM for HDAC1.18 Most zinc-dependent HDAC inhibitors consist of
three pharmacophoric elements: the surface capping group, the ali-
phatic linker unit for channel binding, and the catalytic zinc-binding
group. In this study, we optimized the scaffold of 9a by altering not
⁎ Corresponding authors.
E-mail addresses: hyunju85@skku.edu (H.-J. Park), yhjung@skku.edu (Y.H. Jung).
a These authors equally contributed to this work.
https://doi.org/10.1016/j.bmc.2019.06.036
Received 23 May 2019; Received in revised form 18 June 2019; Accepted 20 June 2019
0968-0896/©2019PublishedbyElsevierLtd.
Pleasecitethisarticleas:GibeomNam,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.06.036

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 1. Known HDAC6 selective inhibitors
and our HDAC6 inhibitor lead 9a.
selectivity, we focused on modifying the cap group (m and R).
In this study, the para-fluorophenethyl cap group dramatically in-
creased the inhibition potency of HDAC6 (6e and 6u). In comparing the
HDAC6 IC50 of analogues with para-substituted phenethyl moieties
(Table 1), we found that electronegative hydrogen bond acceptor sub-
stituents potentiate or sustain HDAC6 activity (6f, 6g and 6l), while
donor groups decrease it (6i and 6o′). The meta-fluorophenethyl-con-
taining analogue (6m) showed somewhat decreased activity and se-
lectivity for HDAC6. Analogues having di-/tri-substituted fluoro, hy-
droxyl, and methoxy phenyl groups did not show improved inhibition
activity. Electronic features of substituents in the phenyl ring seem a
more critical factor for activity than steric bulkiness. However, sub-
stitution of the phenethyl cap group had no significantly effect on
HDAC6 selectivity. Derivatives with a heteroaryl ethyl cap group (6q,
6r, and 6s) also showed low HDAC6 selectivity.
Fig. 2. HDAC6 inhibitor scaffold.
only the length of aliphatic linker, but also the structure and rigidity of
the cap group (Fig. 2).
The designed derivatives were evaluated by preliminary docking
analyses against HDAC6 (PDB: 5EDU)19 and HDAC1 (PDB: 4BKX),20
synthesized and in vitro HDAC1 and HDAC6 enzymatic assays were
performed. As a result, we identified the more potent and selective
HDAC6 inhibitor 6u, with margin of safety and HDAC6 selectivity at
the cellular level.
2. Results and discussion
Cap group rigidity was used to evaluate HDAC6 selectivity. The IC50
and selectivity ratio of each pair of derivatives with an ethyl or ethenyl
group in their m position were compared (Fig. 3). Although compounds
with a phenethyl cap (6a, 6e, 6f, and 6g) have higher HDAC inhibitory
activity, all compounds with a phenylethene cap (6t, 6u, 6v and 6w)
show increased HDAC6 selectivity, revealing that cap group rigidifi-
cation improves HDAC subtype selectivity.
2.1. Synthesis of derivatives of 9a
We designed 27 HDAC6 selective inhibitor candidates, using 9a as a
lead structure (Table 1), to evaluate the effects of the aliphatic chain
length (m and n, Fig. 2), presence of a single or double bond in the m
position, and modification of the cap group (R), on the selectivity and
inhibition activity for HDAC6.
The HDAC6 selectivity of derivatives with flexible phenethyl caps is
unaffected by para-halogen substituents, which affect both the HDAC6
inhibition activity and selectivity of derivatives with rigid phenylethene
caps; the activity and selectivity decrease in the order
F > Br ≥ Cl > H. The electronic and steric effects of the halogen
substituent may play a more crucial role in the interaction of the rigid
cap group with the binding site of HDAC6, compared with that of the
flexible cap group.
We previously confirmed that the cis-double bond in the m position,
which is part of the cap group, is detrimental to HDAC6 activity (Data
not shown). Our previous docking model of 9a also revealed that if the
linker m contains a cis-double bond, the drug will not fit in the active
site pocket, due to huge steric crash.18 Therefore, all compounds with a
double bond in the m position are in the trans form.
As depicted in Scheme 1, the synthesis of desired thiazole hydro-
xamate 6 was initiated from readily available amides 1 and 4-keto-
pentanoic acid (3). Treatment of amide 1 with Laswsson’s reagent
yielded thioamide 2.18 Bromination and esterification from 3 gave
bromo ester 4. The thiazole esters 5a-5x were obtained by cyclization
of 2 with 4 in MeOH solvent.21 Finally, the thiazole esters 5a-5x were
reacted with hydroxyl amine to give thiazole hydroxamates 6a-6x.22
We therefore performed extensive docking analysis of 6u to eluci-
date its HDAC6 selectivity, by examining possible binding poses of 6u
in HDAC6.
2.3. Docking simulation to elucidate the HDAC6 selectivity of 6u
The correlation between the cap group rigidity and HDAC6 se-
lectivity is demonstrated by comparing the flexible docking results of
6u with those of 6e. The flexible docking of 6e with a p-fluorophenethyl
cap generated an ensemble of diverse conformations spread out at the
active site pocket entrance (Fig. 4). In contrast, the poses of 6u with a p-
fluorophenylethene cap consisted of limited conformations aligned in
the restricted area. Therefore, we examined in detail whether this cap
group interacts with unconserved residues between HDAC1 and
HDAC6.
2.2. Structure-activity relationship of HDAC6 inhibitors
To measure inhibition activity, in vitro HDAC1 and HDAC6 enzy-
matic assays were performed, and their activities were compared using
their IC50 and selectivity indexes (IC50, HDAC1/IC50, HDAC6) (Table 1). In
compounds with a phenyl cap group (6a-6d), C chain linker length
changes (m = 1 or 2; n = 2 or 3) dramatically affected inhibition ac-
tivity, but not HDAC6 selectivity. Therefore, the total length of the
aliphatic chain (m + n) was optimized to 4, permitting the derivative
(6a) with m = n = 2 to show the highest activity. After optimizing the
aliphatic chain length to m = n = 2, we evaluated the derivatives with
different bond states of m and aromatic cap groups.
The residues composing the rim of the catalytic channel, where
most HDACs, including HDAC1 and HDAC6, possess two hydrophobic
grooves, are also generally conserved with minor differences (Fig. 5A
and B). A large groove is composed of His, Pro, Leu residues, and highly
conserved in all HDACs. A small groove is located right next to the Leu
residue of a large groove and is composed of a single Tyr (HDAC1 and
2) or Phe (HDAC3, 4, 6, 7, and 8) residue. In the X-ray crystal structures
of complexes of HDAC inhibitors and class I HDACs, the cap moiety of
inhibitors is mostly positioned at a large hydrophobic groove.19,25
There are two distinctive residues present at the pocket entrance of
HDAC6: Ser568 and Met682. Ser568 is located between two parallel
The binding pocket of HDAC can be divided into three parts: zinc-
containing catalytic site, hydrophobic channel, and solvent-accessible
entrance. As illustrated in Supplementary Fig. S1, as opposed to dif-
ferent pocket size, the amino acid residues inside the binding pocket of
all published human HDACs are highly conserved,23 whereas those in
the entrance region are diverse. Therefore, to improve HDAC6
2

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 1
Structures and the HDAC1 and HDAC6 inhibition activity of compounds.
Name
Structure
HDAC1 IC50 (nM)
HDAC6 IC50 (nM)
Selectivity index
(HDAC1/HDAC6)
6a
118.4
14.20
30.89
8.705
3.83
6b
19101
7567
1455
212.5
13.13
6c
6d
6e
15915
1158
7329
283.2
42.30
839.1
95.04
21.23
391.9
60.98
8.275
18.97
12.18
13.95
296.2
6f
90.05
79.81
43.92
20.42
15.33
15.89
6.200
6.330
5.87
5.02
6g
6h
83.71
470.1
51.90
150.2
50.68
56.89
15.93
17.22
1.65
6.86
6h′
6i
6j
1060
179.3
30.04
85.35
110.4
37.70
35.36
12.42
1.08
119.7
6k
6l
164.6
98.02
259.6
63.05
51.69
85.75
39.07
43.6
9.235
12.14
10.33
4.21
2.25
6.84
6m
37.95
(continued on next page)
3

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 1 (continued)
Name
Structure
HDAC1 IC50 (nM)
HDAC6 IC50 (nM)
Selectivity index
(HDAC1/HDAC6)
6n
278.3
130.4
20.22
5.075
13.76
6o
103
38.37
61.42
561.8
17.69
210.9
1.68
2.01
6o′
1132
435.6
6p
291.6
118.1
97.07
26.95
3.00
0.80
6p′
15707
13980
19531
11130
6q
6r
558.1
141.4
382.5
152.4
101
42.13
6.53
7.27
33.77
9.375
6s
6t
335.6
91.35
54.2
13.19
785.8
6.19
25486
19010
1386
18.39
6u
6v
6w
5432
695.0
7316
8612
42.98
592.1
309.4
7.870
192.4
145.6
126.38
33.06
53.68
19573
16610
6x
394.5
3.511
174.6
1.456
116.6
8.922
36.92
4.422
3.38
0.39
TSA
SAHA
131.5
21.40
70.83
13.93
1.86
(continued on next page)
4

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 1 (continued)
Name
Structure
HDAC1 IC50 (nM)
HDAC6 IC50 (nM)
Selectivity index
(HDAC1/HDAC6)
Tubastatin A [24]
2977
880.0
11.14
2.614
267.24
To investigate the binding pose in the catalytic channel (Fig. 5D),
the hydroxamate group forms a bidentate chelation with Zn ion, and
the thiazole linker stacks in between Phe620 and Phe680. The or-
ientation of thiazole was fixed to the sulfur atom of Ser568, which is a
unique residue of HDAC6, and orients the cap group toward the small
hydrophobic groove. In derivatives with longer linkers (n = 3C) such as
6c and 6d, the thiazole ring deviates from the space between two Phe
residues, and the π-π interaction cannot be formed (results not shown).
The docking results therefore confirm that the optimal aliphatic chain
length of m = n = 2 is important for the HDAC6 inhibitor to fit in the
zinc-binding domain and cap group of the appropriate region. It espe-
cially confirms that m = trans-ethenyl is important for cap moiety lo-
calization to the small groove, which permits HDAC6 selectivity.
Scheme 1. Reagents and conditions: (a) Lawesson’s reagent, THF, rt, 2 h,
100%; (b) Br2, MeOH, 0 °C, 2 h, then 80 °C, 2 h, 55%; (c) MeOH, overnight
reflux, 2 h, 40 ∼ 60% (d) NH2OH · HCl, 8 N NaOH, MeOH, 10 ∼ 20%
side-chain phenyl rings of Phe620 and Phe680, conserved as Asp in
other HDACs. Met682, a unique residue of HDAC6, is distinctively or-
ientated in the next loop structure position, to provide additional sur-
face in the small hydrophobic groove. These unique structural features
of HDAC6 may enable ligand selectivity. The results of docking mod-
eling suggest the active conformation of 6u poses in the small hydro-
phobic groove at the pocket entrance.
2.4. Cellular assays of compound 6u
Western blot analysis was conducted to confirm HDAC6 inhibition
of 6u at cellular level, using cell extracts of HeLa and RAW 264.7 cells,
respectively. We compared the effect of 6u with that of the well-known
HDAC6 inhibitor tubastatin A,24 on tublin acetylation in HeLa cells
(Fig. 6). Treatment with both compounds increased tubulin acetylation
and equivalently increased histone acetylation, due to mild class I
HDAC inhibition. These results suggest that the HDAC6 inhibitor ability
of 6u is equivalent to that of tubastatin A in cells. Previous study results
showed that the HDAC6 inhibitor 9a (see Fig. 1) suppresses the ex-
pression of LPS treatment-induced pro-inflammatory cytokines in RAW
264.7 cells, and has anti-sepsis effects in vivo.12,18 To compare the
cellular HDAC6 inhibition ability of 6u with that of the pan-HDAC in-
hibitor SAHA, we examined their effects on α-tubulin acetylation in
RAW 264.7 cells.6 As shown in Fig. 7, 6u increases tubulin acetylation
in a concentration-dependent manner. It also increased histone H3
acetylation, but at higher concentrations than SAHA.
In examining the molecular interactions of 6u with the small groove
residues of HDAC6 (Fig. 5C), we found that the p-fluorophenyl ring in
the cap group of 6u interacts with Phe679 forms (π-π interaction)
(angle = 31.1°, distance = 4.2 Å). With respect to electrostatic inter-
actions, we found that an electropositive guanidinium side chain of
Arg673 is located above Phe679, where the para-substituted halogen
atoms can form favorable attractive interactions. The fluorine atom
aligns with Arg673, and they work as counterparts. The corresponding
residue in HDAC1 is Cys273, which is unfavorable to electrostatic in-
teractions. The observed dramatic decrease in the potency of the p-
chloro (6v) and p-bromo (6w) derivatives may be owed to the de-
creased electronegativity and effect of atom size.
Fig. 3. Compounds with alkane-alkene linker pairs in their m position, and their IC50 values.
5

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 4. Binding poses of 6e and 6u in the CDII of HDAC6 (PDB code 5EDU) generated using Schrödinger XP-Glide flexible docking. Rigidification of the cap group
localized the cap moiety to a smaller hydrophobic groove. The Connolly surface of binding site was generated and rendered in opaque solid gray.
2.5. Microsomal stability assay
purification, unless otherwise stated. All reactions were performed
under an inert atmosphere (nitrogen). All melting points were obtained
on an Electrothermal IA9000 series digital melting point apparatus and
were uncorrected. Mass spectra (MS) were obtained with ultra-high
performance liquid chromatography-quadrupole-time-of-flight mass
spectrometry, using electrospray ionization (ESI). The instrument con-
sists of an Acquity UPLC system (Waters Co., Milford, MA, USA) cou-
pled to a Waters Acquity Xevo G2 Q-TOF system (Waters Corp.,
Manchester, UK). Nuclear magnetic resonance spectra (1H and 13C
NMR) were obtained for CDCl3, CD3OD, and DMSO-d6 on a Bruker
Unity 300 MHz and Varian Unit 400 MHz spectrometer. Chemical shifts
are reported in parts per million (ppm) relative to, respectively, residual
CDCl3 δH (7.26 ppm), CDCl3 δC (77.0 ppm), CD3OD δH (3.31 ppm),
CD3OD δC (49.00 ppm), DMSO-d6 δH (2.50 ppm), and DMSO-d6 δC
(39.5 ppm) as internal standards. Resonance patterns are reported with
the notations s (singlet), d (doublet), t (triplet), q (quartet), and m
(multiplet). Coupling constants (J) are reported in hertz (Hz). For thin
layer chromatography, Kieselgel 60 F254 (Merck) was coated on the
plates, and E. Merck Kieselgel 60 (230–400 mesh) was used for flash
column chromatography.
To evaluate the in vivo stability of 6g and 6u, we performed mi-
crosomal stability assays in both mice and human microsomes. The
residual concentration of 6g in mice and human microsomes sig-
nificantly decreased after 30 min, regardless of the presence of NADPH.
In contrast, 6u concentrations in human microsomes decreased negli-
gibly, and it showed low intrinsic clearance (Table 2). Overall, 6u has
acceptable submicromolar cytotoxicity (result not shown) and human
hepatic tolerance in vitro.
3. Conclusion
Based on the structure of our lead HDAC6 inhibitor 9a, we designed
27 compounds for further optimization of its activity and selectivity.
We aimed at optimizing the linker length, and the substituents and
bond state of the cap group. HDAC1 and HDAC6 enzymatic assays were
performed to evaluate the potency of their HDAC6 selectivity. The
linker length optimized to m = n = 2 and cap moiety rigidification
were critical for improving HDAC6 selectivity. In this study, only the
para-fluoro phenyl cap in conjugation with the trans-ethenyl group (6u)
was found to sustain nanomolar HDAC6 potency and increase HDAC6
selectivity over 126-fold. To establish the molecular basis for the
HDAC6 selectivity of 6u, we performed intensive molecular docking
analyses, using the structures of all available HDAC subtypes. Docking
results suggested that cap rigidification permitted HDAC6 selectivity by
localizing the cap group to the small hydrophobic groove, and inter-
acting with unconserved residues in HDAC6. Although our scaffold is a
classical linear hydroxamate pan-HDAC inhibitor like SAHA, with the
manipulation of the aliphatic linker length and the substituent and
simple rigidification of the aryl cap group, we constructed the novel
HDAC6-selective scaffold 6u. Because compound 6u showed sub-
micromolar cytotoxicity in HeLA cells (Data now shown) and negligibly
metabolization in human microsomes, we are currently conducting in
vivo therapeutic application of 6u in a mouse model of neurodegen-
erative disease, the results of which will be published in due course.
4.1.1.1. Methyl 5-bromo-4-oxopentanoate. To a solution of levulinic acid
(10 g, 86.12 mmol) in methanol (80 mL), bromine (4.41 mL,
86.12 mmol) was added dropwise at 0 °C. The reaction mixture was
stirred for 2 h at 0 °C, and then for 2 h at 80 °C. The methanol was
evaporated, and the solution was neutralized to pH 7 using saturated
sodium bicarbonate. The aqueous layer was extracted using methylene
chloride, and the organic layer was washed with water and brine, dried
over magnesium sulfate and concentrated. The residue was purified
using column chromatography (Hex:EtOAc = 5:1) to afford a pale-
yellow liquid compound (8.8 g, 48% yield). 1H NMR (400 MHz, CDCl3)
δ 3.96 (s, 2H), 3.69 (s, 3H), 2.98–2.95 (t, J = 15 Hz, 2H), 2.68–2.65 (t,
J = 15 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ 200.51, 172.78, 51.79,
34.39, 34.12, 27.99.
4.1.1.2. Methyl 6-bromo-5-oxohexanoate. To
a
solution of 4-
acetylbutyric acid (5 g, 38.42 mmol) in methanol (48 mL), bromine
(1.98 mL, 38.42 mmol) was added dropwise at 0 °C. The reaction
mixture was stirred for 2 h at 0 °C, and then for 2 h at 80 °C. The
solution was evaporated and neutralized to pH 7 using saturated
sodium bicarbonate. The aqueous layer was extracted with methylene
chloride, and the organic layer was washed with water and brine, dried
over magnesium sulfate, and concentrated. The residue was purified
4. Material and methods
4.1. Chemistry
4.1.1. General methods
Commercially available reagents were used without additional
6

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 5. Superposition of the cocrystalized TSA (yellow carbon) in HDAC6 (PDB id 5EDU) with the QPLD docking model 6u (green carbon). (A) Overlay of the X-ray
structures of HDAC subtypes. The two unique residues, Ser568 and Met 682 in HDAC6 CD2 are indicated. Ser568 locates between two Phe residues. In other HDACs,
the Ser568 residue is conserved as Asp, and Met682 as Gly. (B) Top view showing two hydrophobic grooves with lipophilic potential at the binding pocket entrance.
(C) Electrostatic potential surfaces around the pocket entrance and composing residues. The distance and angle plane between the cap group of 6u and Phe679 are
shown (figures are drawn in Schrödinger release 2018-2). (D) H-bond and π-π interaction are represented as yellow and blue dashed lines, respectively. Zn ion, gray
sphere.
using column chromatography (Hex:EtOAc = 5:1) to afford
a
(0.16 mmol mL−1), bromo ester (1–1.5 equiv.) was added and stirred at
70 °C under reflux overnight. After the reaction was completed, me-
thanol was removed under reduced pressure. The residue was extracted
with ethyl acetate and washed with water and brine. The organic layer
was dried with MgSO4 and concentrated. The crude product was pur-
ified by flash chromatography on a silica gel (hexane–ethyl acetate) to
obtain a thiazole compound.
compound as pale-yellow liquid (4.63 g, 54% yield). 1H NMR
(400 MHz, CDCl3) δ 3.87 (s, 2H), 3.67 (s, 3H), 2.77–2.66 (m, 2H),
2.40–2.35 (t, J = 15 Hz, 2H), 2.00–1.90 (m, 2H); 13C NMR (100 MHz,
CDCl3): δ 215.45, 201.31, 51.72, 38.66, 34.12, 32.79, 18.92.
4.1.2. General procedure for the preparation of thiazole esters21
To a solution of the appropriate thioamide (1 equiv.) in methanol
Fig. 6. Western blotting results of the HDAC6 inhibition effect of 6u and tubastatin A in HeLa cells.
7

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 7. Western blotting results of the HDAC6 inhibition effect of 6u and SAHA in RAW 264.7 cells.
4.1.2.1. Methyl 3-(2-phenethylthiazol-4-yl)propanoate (5a). Colorless oil
in 52% yield. 1H NMR (300 MHz, CDCl3) δ: 7.30–7.16 (m, 5H), 6.82 (s,
1H), 3.80 (s, 3H), 3.30–2.25 (t, J = 15 Hz, 2H), 3.11–3.06 (m, 4H),
2.76–2.71 (t, J = 15 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ: 196.54,
196.37, 176.48, 160.52, 158.94, 152.36, 152.30, 140.78, 70.52, 51.79,
34.12, 32.63, 27.19.
4.1.2.7. Methyl
3-(2-(4-bromophenethyl)thiazol-4-yl)propanoate
(5g). Colorless oil in 96% yield. 1H NMR (400 MHz, CDCl3) δ:
7.41–7.39 (d, J = 8 Hz, 2H), 7.08–7.06 (d, J = 8 Hz, 2H), 6.78 (s,
1H), 3.68 (s, 3H), 3.26–3.22 (t, J = 15 Hz, 2H), 3.09–3.03 (m, 4H),
2.75–2.71 (t, J = 15 Hz, 2H).
4.1.2.8. Methyl
3-(2-(4-methoxyphenethyl)thiazol-4-yl)propanoate
(5h). Colorless oil in 95% yield. 1H NMR (400 MHz, CDCl3) δ:
7.13–7.11 (d, J = 8 Hz, 2H), 6.84–6.82 (d, J = 8 Hz, 2H), 6.78 (s,
1H), 3.79 (s, 3H), 3.69 (s, 3H), 3.25–3.21 (t, J = 15 Hz, 2H), 3.09–3.00
(m, 4H), 2.76–2.72 (t, J = 15 Hz, 2H).
4.1.2.2. Methyl-3-(2-benzylthiazol-4-yl)propanoate (5b). Colorless oil in
50% yield. 1H NMR (300 MHz, CDCl3) δ: 7.36–7.25 (m, 5H), 6.89 (s,
1H), 4.24 (s, 3H), 3.63 (s, 3H), 3.22–3.17 (t, J = 15 Hz, 2H), 3.00–2.95
(t, J = 15 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ: 200.51, 190.47,
170.97, 159.43, 156.32, 153.27, 152.64, 150.22, 140.53, 140.53,
51.79, 34.12, 27.19.
4.1.2.9. Methyl 3-(2-(4-((tert-butoxycarbonyl)amino)phenethyl)thiazol-4-
yl)propanoate (5i). Colorless oil in 41% yield. 1H NMR (400 MHz,
CDCl3) δ: 7.31–7.29 (d, J = 8 Hz, 2H), 7.11–7.08 (d, J = 8 Hz, 2H),
7.01 (s, 1H), 3.68 (s, 3H), 3.28–3.24 (t, J = 15 Hz, 2H), 3.05–3.00 (m,
4H), 2.75–2.71 (t, J = 15 Hz, 2H), 1.53 (s, 9H).
4.1.2.3. Methyl 4-(2-benzylthiazol-4-yl)butanoate (5c). Colorless oil in
51% yield. 1H NMR (300 MHz, CDCl3) δ: 7.34–7.23 (m, 5H), 6.83 (s,
1H), 4.36 (s, 2H), 3.69 (s, 3H), 3.24–3.19 (t, J = 15 Hz, 2H), 3.05–3.00
(t, J = 15 Hz, 2H), 2.86–2.76 (m,2H); 13C NMR (100 MHz, CDCl3) δ:
200.51, 190.47, 170.97, 159.43, 156.32, 153.27, 152.64, 150.22,
140.53, 51.79, 34.47, 34.12, 27.19.
4.1.2.10. Methyl
3-(2-(4-cyanophenethyl)thiazol-4-yl)propanoate
(5j). Colorless oil in 44% yield. 1H NMR (400 MHz, CDCl3) δ:
7.58–7.56 (d, J = 8 Hz, 2H), 7.30–7.28 (d, J = 8 Hz, 2H), 6.79 (s,
1H), 3.68 (s, 3H), 3.30–3.26 (t, J = 15 Hz, 2H), 3.18–3.14 (t,
J = 15 Hz, 2H), 3.08–3.04 (t, J = 15 Hz, 2H), 2.75–2.71 (t,
J = 15 Hz, 2H).
4.1.2.4. Methyl 4-(2-phenethylthiazol-4-yl)butanoate (5d). Colorless oil
in 51% yield. 1H NMR (300 MHz, CDCl3) δ: 7.34–7.20 (m, 5H), 6.79 (s,
1H), 3.72 (s, 3H), 3.43–3.38 (t, J = 15 Hz, 2H), 3.22–3.17 (t, J = 15 Hz,
2H), 3.04–2.99 (t, J = 15 Hz, 2H), 2.54–2.49 (t, J = 15 Hz, 2H),
2.2–2.10 (m, 2H); 13C NMR (100 MHz, CDCl3) δ: 196.54, 196.37,
176.48, 160.52, 158.94, 152.36, 152.30, 140.78, 70.52, 51.79, 36.47,
34.12, 32.63, 27.19.
4.1.2.11. Methyl 3-(2-(4-(trifluoromethyl)phenethyl)thiazol-4-yl)propanoate
(5k). Colorless oil in 74% yield. 1H NMR (400 MHz, CDCl3) δ: 7.55–7.53
(d, J = 8 Hz, 2H), 7.32–7.30 (d, J = 8 Hz, 2H), 6.80 (s, 1H), 3.68 (s, 3H),
3.30–3.26 (t, J = 15 Hz, 2H), 3.18–3.14 (t, J = 15 Hz, 2H), 3.09–3.05 (t,
J = 15 Hz, 2H), 2.75–2.71 (t, J = 15 Hz, 2H).
4.1.2.5. Methyl 3-(2-(4-fluorophenethyl)thiazol-4-yl)propanoate (5e). Yellow
oil in 46% yield. 1H NMR (300 MHz, CDCl3) δ: 7.29–7.16 (m, 4H), 6.82 (s,
1H), 3.67 (s, 3H), 3.30–2.25 (t, J = 15 Hz, 2H), 3.11–3.06 (m, 4H),
2.76–2.71 (t, J = 15 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ: 196.52,
176.48, 160.52, 158.94, 152.36, 152.30, 140.78, 70.52, 51.79, 34.12,
32.63, 27.19.
4.1.2.12. Methyl
3-(2-(4-nitrophenethyl)thiazol-4-yl)propanoate
(5l). Colorless oil in 91% yield. 1H NMR (400 MHz, CDCl3) δ:
8.15–8.13 (d, J = 8 Hz, 2H), 7.35–7.33 (d, J = 8 Hz, 2H), 6.80 (s,
1H), 3.68 (s, 3H), 3.33–3.29 (t, J = 15 Hz, 2H), 3.23–3.20 (t,
J = 15 Hz, 2H), 3.08–3.05 (t, J = 15 Hz, 2H), 2.75–2.71 (t,
J = 15 Hz, 2H).
4.1.2.6. Methyl
3-(2-(4-chlorophenethyl)thiazol-4-yl)propanoate
(5f). Colorless oil in 65% yield. 1H NMR (400 MHz, CDCl3) δ:
7.26–7.24 (d, J = 8 Hz, 2H), 7.13–7.11 (d, J = 8 Hz, 2H), 6.78 (s,
1H), 3.68 (s, 3H), 3.26–3.22 (t, J = 15 Hz, 2H), 3.08–3.04 (m, 4H),
2.76–2.72 (t, J = 15 Hz, 2H).
4.1.2.13. Methyl
3-(2-(3-fluorophenethyl)thiazol-4-yl)propanoate
(5m). Colorless oil in 65% yield. 1H NMR (400 MHz, CDCl3) δ:
7.27–7.21 (m, 1H), 6.98–6.96 (d, J = 12 Hz, 1H), 6.92–6.88 (m,
Table 2
Microsomal stability assay results. The intrinsic clearance and residual concentrations of verapamil, 6u, and 6g after 30 min of incubation.
Mouse
Human
Compound
NADPH(−)
0 min
NADPH(+)
0 min
CLh,int
NADPH(−)
0 min
NADPH(+)
0 min
CLh,int
(μL/min/mg)
(μL/min/mg)
30 min
5 min
15 min
30 min
30 min
5 min
15 min
30 min
Veraparmil
100.0%
100.0%
100.0%
99.3%
95.8%
79.5%
92.6%
106.9%
96.9%
52.1%
99.8%
83.0%
18.1%
68.6%
61.2%
N.D.
216.8
98.5
70.1
100.0%
100.0%
100.0%
96.2%
112.4%
66.4%
90.6%
61.6%
105.1%
81.8%
40.6%
103.4%
70.9%
13.0%
94.6%
38.3%
125.9
7.7
6u
6g
25.2%
33.8%
106.3%
105.0%
63.8
8

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
2H), 6.79 (s, 1H), 3.68 (s, 3H), 3.28–3.24 (t, J = 15 Hz, 2H),
3.11–3.05 (m, 4H), 2.76–2.72 (t, J = 15 Hz, 2H).
3.11 (t, J = 7.4 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H).
4.1.2.24. (E)-Methyl 3-(2-(2-(1H-indol-3-yl)vinyl)thiazol-4-yl)propanoate
(5x). Red oil in 44% yield. 1H NMR (300 MHz, CDCl3) δ: 8.31 (bs, 1H),
7.67–7.65 (d, J = 6 Hz, 2H), 7.62–7.59 (d, J = 9 Hz, 1H), 7.36–7.33 (d,
J = 9 Hz, 1H), 7.21–7.08 (m, 2H), 6.89 (s, 1H), 6.79 (s, 1H), 3.68 (s,
3H), 2.82–2.77 (t, J = 15 Hz, 2H), 2.39–2.34 (t, J = 15 Hz, 2H); 13C
NMR (100 MHz, CDCl3) δ: 200.98, 195.46, 175.81, 160.42, 154.52,
150.99, 149.34, 142.65, 140.23, 138.48, 136.57, 120.48, 114.73,
113.28, 50.64, 35.29, 31.52.
4.1.2.14. Methyl
3-(2-(3,4-difluorophenethyl)thiazol-4-yl)propanoate
(5n). Colorless oil in 67% yield. 1H NMR (400 MHz, CDCl3) δ:
7.09–6.96 (m, 2H), 6.90–6.87 (m, 1H), 6.79 (s, 1H), 3.68 (s, 3H),
3.26–3.22 (t, J = 15 Hz, 2H), 3.09–3.03 (m, 4H), 2.75–2.71 (t,
J = 15 Hz, 2H).
4.1.2.15. Methyl 3-(2-(3,4-dimethoxyphenethyl)thiazol-4-yl)propanoate
(5o). Colorless oil in 94% yield. 1H NMR (400 MHz, CDCl3) δ:
6.81–6.72 (m, 3H), 6.79 (s, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.68 (s,
3H), 3.27–3.23 (t, J = 15 Hz, 2H), 3.10–3.01 (m, 4H), 2.76–2.72 (t,
J = 15 Hz, 2H).
4.1.3. General procedure for the preparation of hydroxamate22
To a solution of the appropriate thiazole ester (1 equiv.) in methanol
(0.1 mmol mL−1), hydroxylamine hydrochloride (2 equiv.) and 8 N-
sodium hydroxide (0.5 mmol mL−1) were added at 0 °C. The reaction
mixture was stirred for 1.5 h at room temperature. After the reaction
was completed, methanol was removed under reduced pressure. The
residue was extracted with ethyl acetate and washed with water and
brine. The organic layer was dried with MgSO4 and concentrated. The
crude product was recrystallized with ethyl acetate or methylene
chloride.
4.1.2.16. Methyl 3-(2-(3,4,5-trimethoxyphenethyl)thiazol-4-yl)propanoate
(5p). Colorless oil in 83% yield. 1H NMR (400 MHz, CDCl3) δ: 6.80 (s,
1H), 6.42 (s, 2H), 3.83 (s, 6H), 3.82 (s, 3H), 3.68 (s, 3H), 3.28–3.24 (t,
J = 15 Hz, 2H), 3.10–3.01 (m, 4H), 2.76–2.72 (t, J = 15 Hz, 2H).
4.1.2.17. Methyl
3-(2-(2-(furan-2-yl)ethyl)thiazol-4-yl)propanoate
(5q). Colorless oil in 42% yield. 1H NMR (400 MHz, CDCl3) δ:
7.33–7.32 (dd, J = 2, 0.8 Hz, 1H), 6.79 (s, 1H), 6.28–6.27 (dd,
J = 3.2, 2 Hz, 1H), 6.02–6.01 (dd, J = 3.2, 2 Hz, 1H), 3.68 (s, 3H),
3.33–3.29 (t, J = 15 Hz, 2H), 3.13–3.05 (m, 4H), 2.76–2.72 (t,
J = 15 Hz, 2H).
4.1.3.1. N-Hydroxy-3-(2-phenethylthiazol-4-yl)propanamide (6a). White
solid in 19% yield. mp 124.4–125.3 °C. HRMS: calcd for C14H16N2O2S
[M−H]+ 275.0862, found 275.0854. 1H NMR (300 MHz, DMSO-d6) δ:
7.32–7.18 (m, J = 15 , 9 Hz, 5H), 6.78 (s, 1H), 3.30–2.25 (t, J = 15 Hz,
2H), 3.11–3.06 (m, 4H), 2.76–2.71 (t, J = 15 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) δ: 196.54, 196.37, 176.48, 160.52, 158.94,
152.36, 152.30, 140.78, 70.52, 34.12, 32.63, 27.19.
4.1.2.18. Methyl
3-(2-(2-(thiophen-2-yl)ethyl)thiazol-4-yl)propanoate
(5r). Colorless oil in 75% yield. 1H NMR (400 MHz, CDCl3) δ:
7.15–7.13 (dd, J = 5.2, 1.2 Hz, 1H), 6.93–6.91 (dd, J = 5.2, 3.6 Hz,
1H), 6.83–6.82 (dd, J = 3.6, 1.2 Hz, 1H), 6.82 (s, 1H), 3.68 (s, 3H),
3.36–3.33 (m, 4H), 3.11–3.07 (t, J = 15 Hz, 2H), 2.78–2.74 (t,
J = 15 Hz, 2H).
4.1.3.2. N-Hydroxy-3-(2-benzylthiazol-4-yl)propanamide
(6b). White
solid in 17% yield. mp 135.1–135.3 °C. HRMS: calcd for C13H14N2O2S
[M−H]+ 261.0705, found 261.0698. 1H NMR (300 MHz, DMSO-d6) δ:
7.32–7.21 (m, J = 15, 6 Hz, 5H), 6.76 (s, 1H), 4.24 (s, 2H), 2.83–2.78
(t, J = 15 Hz, 2H), 2.39–2.34 (t, J = 15 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6) δ: 200.51, 190.47, 170.97, 159.43, 156.32, 153.27, 152.64,
150.22, 140.53, 140.53, 34.12, 31.14.
4.1.2.19. Methyl
3-(2-(2-(1H-indol-3-yl)ethyl)thiazol-4-yl)propanoate
(5s). Red oil in 46% yield. 1H NMR (300 MHz, CDCl3) δ: 8.17 (s,
1H), 7.62–7.59 (d, J = 9 Hz, 1H), 7.37–7.34 (d, J = 9 Hz, 1H),
7.22–7.08 (m, 2H), 7.00 (s, 1H), 6.81 (s, 1H), 3.68 (s, 3H), 3.39–3.34
(t, J = 15 Hz, 2H), 3.24–3.19 (t, J = 15 Hz, 2H), 2.71–2.66 (t,
J = 15 Hz, 2H), 2.24–2.19 (t, J = 15 Hz, 2H); 13C NMR (100 MHz,
CDCl3) δ: 210.78, 190.72, 162.48, 155.28, 154.32, 152.47, 150.35,
150.20, 138.79, 135.44, 129.87, 125.44, 50.62, 46.42, 35.29, 31.52,
26.49.
4.1.3.3. N-Hydroxy-4-(2-benzylthiazol-4-yl)butanamide
(6c). White
solid in 19% yield. mp 137.3–137.6 °C. HRMS: calcd for C14H16N2O2S
[M−H]+ 275.0834, found 275.0647. 1H NMR (300 MHz, DMSO-d6) δ:
7.31–7.20 (m, J = 15, 6 Hz, 5H), 6.75 (s, 1H), 4.25 (s, 2H), 2.82–2.77
(t, J = 15 Hz, 2H), 2.39–2.34 (t, J = 15 Hz, 2H), 2.10–2.00 (m, 2H); 13
C
NMR (100 MHz, DMSO-d6) δ: 200.51, 190.47, 170.97, 159.43, 156.32,
4.1.2.20. (E)-Methyl 3-(2-strylthiazol-4-yl)propanoate (5t). Colorless oil
in 60% yield. 1H NMR (300 MHz, CDCl3) δ: 7.56–7.53 (d, J = 9 Hz, 2H),
7.43–7.24 (m, 5H), 6.90 (s, 1H), 3.65 (s, 3H), 3.14–3.09 (t, J = 15 Hz,
2H), 2.81–2.76 (t, J = 9 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ: 172.64,
168.93, 158.27, 137.22, 136.11, 130.29, 128.35, 121.95, 114.92,
51.27, 33.25, 31.52, 26.49.
153.27, 152.64, 150.22, 140.53, 140.53, 34.47, 34.12, 27.19.
4.1.3.4. N-Hydroxy-4-(2-phenethylthiazol-4-yl)butanamide (6d). White
solid in 22% yield. mp 154.7–155.3 °C. HRMS: calcd for C15H18N2O2S
[M−H]+ 289.1146, found 289.1137. 1H NMR (300 MHz, DMSO-d6) δ:
7.31–7.20 (m, J = 15, 9 Hz, 5H), 6.75 (s, 1H), 3.31–2.26 (t, J = 15 Hz,
2H), 3.12–3.07 (t, J = 15 Hz, 9 Hz, 5H), 2.82–2.77 (t, J = 15 Hz, 2H),
2.39–2.34 (t, J = 15 Hz, 2H), 2.09–1.99 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) δ: 196.54, 196.37, 176.48, 160.52, 158.94, 152.36, 152.30,
140.78, 70.52, 51.79, 36.47, 34.12, 32.63, 27.19.
4.1.2.21. (E)-Methyl
3-(2-(4-fluorostyryl)thiazol-4-yl)propanoate
(5u). Yellow oil in 44% yield. 1H NMR (300 MHz, CDCl3) δ:
7.54–7.51 (d, J = 9 Hz, 2H), 7.32–7.18 (m, 4H), 6.90 (s, 1H), 3.65
(s, 3H), 2.81–2.76 (t, J = 15 Hz, 2H), 2.63–2.58 (t, J = 15 Hz, 2H);
13C NMR (100 MHz, CDCl3) δ: 172.64, 158.27, 137.22, 136.11,
130.29, 128.35, 121.95, 114.92, 51.27, 33.25, 31.52, 26.49.
4.1.3.5. N-Hydroxy-3-(2-(4-fluorophenethyl)thiazol-4-yl)propanamide
(6e). Light yellow solid in 32% yield. mp 118.4–118.9 °C. HRMS: calcd
for C14H16FN2O2S [M+H]+ 295.0918, found 295.0917. 1H NMR
(300 MHz, DMSO-d6) δ: 7.32–7.19 (m, 4H), 6.78 (s, 1H), 3.30–3.25
4.1.2.22. Methyl 3-(2-(4-chlorostyryl)thiazol-4-yl)propanoate (5v). Colorless
oil in 46% yield. 1H NMR (400 MHz, CDCl3) δ: 7.34–7.29 (d, J = 8 Hz, 2H),
7.13–7.11 (d, J = 8 Hz, 2H), 6.92 (s, 1H), 3.68 (s, 3H), 3.26–3.22 (t,
J = 15 Hz, 2H), 3.08–3.04 (m, 4H), 2.76–2.72 (t, J = 15 Hz, 2H).
(t, J = 15 Hz, 2H), 3.11–3.06 (m, 4H), 2.76–2.71 (t, J = 15 Hz, 2H); 13
C
NMR (100 MHz, DMSO-d6) δ: 196.54, 176.48, 160.52, 158.94, 152.36,
152.30, 140.78, 70.52, 34.12, 32.63, 27.19.
4.1.2.23. (E)-Methyl
3-(2-(4-bromostyryl)thiazol-4-yl)propanoate
4.1.3.6. N-Hydroxy-3-(2-(4-chlorophenethyl)thiazol-4-yl)propanamide
(5w). Colorless oil in 32% yield. 1H NMR (400 MHz, CDCl3) δ:
(6f). White solid in 38% yield. mp 160.6–162.5 °C. HRMS: calcd for
7.54–7.47 (m, 2H), 7.43–7.18 (m, 5H), 6.90 (s, 1H), 3.69 (s, 3H),
C
14H14ClN2O2S [M−H]+ 309.0475, found 309.0465. 1H NMR
9

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
(400 MHz, DMSO-d6) δ: 7.28–7.26 (d, J = 8 Hz, 2H), 7.19–7.17 (d,
J = 8 Hz, 2H), 7.02 (s, 1H), 3.29–3.26 (t, J = 15 Hz, 2H), 3.09–3.05 (t,
J = 15 Hz, 2H), 3.06–3.02 (t, J = 15 Hz, 2H), 2.50–2.46 (t, J = 15 Hz,
2H); 13C NMR (400 MHz, DMSO-d6) δ: 170.41, 170.25, 154.53, 138.93,
131.76, 129.80, 128.13, 113.41, 34.92, 34.06, 31.87, 26.52.
3.06–3.02 (t, J = 15 Hz, 2H), 2.50–2.46 (t, J = 15 Hz, 2H); 13C NMR
(400 MHz, DMSO-d6) δ: 170.39, 170.26, 164.15, 161.73, 154.54,
143.03, 142.95, 129.82, 129.74, 124.07, 124.04, 114.93, 114.72,
113.39, 112.77, 112.55, 35.28, 33.96, 31.88, 26.54.
4.1.3.14. N-Hydroxy-3-(2-(3,4-difluorophenethyl)thiazol-4-yl)
4.1.3.7. N-Hydroxy-3-(2-(4-bromophenethyl)thiazol-4-yl)propanamide
propanamide (6n). White solid in 36% yield. mp 143.9–145.6 °C.
(6g). White solid in 55% yield. mp 159.3–161.3 °C. HRMS: calcd for
HRMS: calcd for
C
14H13F2N2O2S [M−H]+ 311.0670, found
C
14H14BrN2O2S [M−H]+ 352.9960, found 352.9959. 1H NMR
311.0666. 1H NMR (400 MHz, DMSO-d6) δ: 7.18–7.11 (m, 2H), 7.03
(s, 1H), 7.03–6.98 (m, 1H), 3.30–3.26 (t, J = 15 Hz, 2H), 3.09–3.05 (t,
J = 15 Hz, 2H), 3.06–3.02 (t, J = 15 Hz, 2H), 2.50–2.46 (t, J = 15 Hz,
2H); 13C NMR (400 MHz, DMSO-d6) δ: 170.24, 170.14, 154.60, 124.64,
124.61, 124.58, 124.55, 117.04, 116.87, 116.81, 116.63, 113.42,
34.66, 33.95, 31.86, 26.53.
(400 MHz, DMSO-d6) δ: 7.47–7.45 (d, J = 8 Hz, 2H), 7.23–7.21 (d,
J = 8 Hz, 2H), 7.08 (s, 1H), 3.24–3.20 (t, J = 15 Hz, 2H), 3.02–2.98 (t,
J = 15 Hz, 2H), 2.90–2.86 (t, J = 15 Hz, 2H), 2.35–2.31 (t, J = 15 Hz,
2H); 13C NMR (400 MHz, DMSO-d6) δ: 168.70, 155.41, 140.33, 131.59,
131.25, 119.65, 113.60, 34.75, 34.34, 32.21, 27.28.
4.1.3.8. N-Hydroxy-3-(2-(4-methoxyphenethyl)thiazol-4-yl)propanamide
4.1.3.15. N-Hydroxy-3-(2-(3,4-dimethoxyphenethyl)thiazol-4-yl)
(6h). White solid in 64% yield. mp 140.4–142.5 °C. HRMS: calcd for
propanamide (6o). White solid in 66% yield. mp 135.6–136.8 °C.
HRMS: calcd for C16H19N2O4S [M−H]+ 335.1066, found 335.1066.
1H NMR (400 MHz, DMSO-d6) δ: 7.02 (s, 1H), 6.87–6.85 (d, J = 8 Hz,
1H), 6.80–6.79 (d, J = 4 Hz, 1H), 6.75–6.73 (dd, J = 8, 1.6 Hz, 1H),
3.80 (s, 3H), 3.80 (s, 3H), 3.28–3.25 (t, J = 15 Hz, 2H), 3.06–3.00 (m,
4H), 2.50–2.46 (t, J = 15 Hz, 2H); 13C NMR (400 MHz, DMSO-d6) δ:
170.97, 170.24, 154.41, 148.99, 147.66, 133.07, 120.48, 113.31,
112.16, 111.74, 55.12, 55.03, 35.32, 34.52, 31.87, 26.52.
C
15H17N2O3S [M−H]+ 305.0965, found 305.0960. 1H NMR (400 MHz,
DMSO-d6) δ: 7.16–7.14 (d, J = 8 Hz, 2H), 7.05 (s, 1H), 6.88–6.86 (d,
J = 8 Hz, 2H), 3.80 (s, 3H), 3.30–3.27 (t, J = 15 Hz, 2H), 3.10, 3.03 (m,
4H), 2.53–2.50 (t, J = 15 Hz, 2H); 13C NMR (400 MHz, DMSO-d6) δ:
174.95, 174.18, 162.26, 158.31, 136.01, 133.02, 117.43, 117.24,
58.17, 38.83, 38.55, 35.81, 30.45.
4.1.3.9. N-Hydroxy-3-(2-(4-aminophenethyl)thiazol-4-yl)propanamide
(6i). White solid in 28% yield. mp 154.2–155.1 °C. HRMS: calcd for
4.1.3.16. N-Hydroxy-3-(2-(3,4,5-trimethoxyphenethyl)thiazol-4-yl)
propanamide (6p). White solid in 44% yield. mp 150.3–150.6 °C.
HRMS: calcd for C17H21N2O5S [M−H]+ 365.1173, found 365.1171.
1H NMR (400 MHz, DMSO-d6) δ: 7.10 (s, 1H), 6.51 (s, 2H), 3.74 (s, 6H),
3.61(s, 3H), 3.25–3.21 (t, J = 15 Hz, 2H), 2.97–2.93 (t, J = 15 Hz, 2H),
2.91–2.88 (t, J = 15 Hz, 2H), 2.36–2.32 (t, J = 15 Hz, 2H); 13C NMR
(400 MHz, DMSO-d6) δ: 169.46, 168.73, 155.40, 153.17, 136.59,
136.24, 113.52, 106.14, 60.42, 56.24, 35.99, 34.77, 32.22, 27.31.
C
14H16N3O2S [M−H]+ 290.0971, found 290.0963. 1H NMR (400 MHz,
DMSO-d6) δ: 6.88 (s, 1H), 6.84–6.82 (d, J = 8 Hz, 2H), 6.56–6.54 (d,
J = 8 Hz, 2H), 3.11–3.07 (t, J = 15 Hz, 2H), 2.93–2.89 (t, J = 15 Hz,
2H), 2.84–2.80 (t, J = 15 Hz, 2H), 2.37–2.33 (t, J = 15 Hz, 2H); 13C
NMR (400 MHz, DMSO-d6) δ: 171.27, 170.25, 154.29, 145.40, 129.82,
128.74, 115.55, 113.26, 35.09, 34.78, 31.89, 26.50.
4.1.3.10. N-Hydroxy-3-(2-(4-cyanophenethyl)thiazol-4-yl)propanamide
(6j). White solid in 29% yield. mp 149.5–152.4 °C. HRMS: calcd for
4.1.3.17. N-Hydroxy-3-(2-(2-(furan-2-yl)ethyl)thiazol-4-yl)propanamide
C
15H14N3O2S [M−H]+ 300.0805, found 300.0807. 1H NMR (400 MHz,
(6q). White solid in 33% yield. mp 127.0–128.1 °C. HRMS: calcd for
DMSO-d6) δ: 7.76–7.74 (d, J = 8 Hz, 2H), 7.48–7.46 (d, J = 8 Hz, 2H),
7.09 (s, 1H), 3.29–3.25 (t, J = 15 Hz, 2H), 3.14–3.10 (t, J = 15 Hz, 2H),
2.90–2.86 (t, J = 15 Hz, 2H), 2.35–2.31 (t, J = 15 Hz, 2H); 13C NMR
(400 MHz, DMSO-d6) δ: 170.23, 169.86, 154.66, 146.24, 131.98,
129.36, 118.41, 113.50, 109.79, 35.50, 33.50, 31.84, 26.50.
C
12H13N2O3S [M−H]+ 265.0656, found 265.0647. 1H NMR (400 MHz,
DMSO-d6) δ: 7.53–7.52 (dd, J = 2, 0.8 Hz, 1H), 7.11 (s, 1H), 6.35–6.34
(dd, J = 3.2, 2 Hz 1H), 6.14–6.13 (dd, J = 3.2, 0.8 Hz, 1H), 3.26–3.23
(t, J = 15 Hz, 2H), 3.06–3.03 (t, J = 15 Hz, 2H), 2.90–2.86 (t,
J = 15 Hz, 2H), 2.35–2.31 (t, J = 15 Hz, 2H); 13C NMR (400 MHz,
DMSO-d6) δ: 168.70, 155.42, 154.19, 142.02, 113.73, 110.88, 106.39,
32.18, 31.59, 27.91, 27.18.
4.1.3.11. N-Hydroxy-3-(2-(4-(trifluoromethyl)phenethyl)thiazol-4-yl)
propanamide (6k). White solid in 63% yield. mp 140.4–142.5 °C.
HRMS: calcd for
C
15H14F3N2O2S [M−H]+ 343.0735, found
4.1.3.18. N-Hydroxy-3-(2-(2-(thiophen-2-yl)ethyl)thiazol-4-yl)
propanamide (6r). White solid in 56% yield. mp 122.9–123.2 °C. HRMS:
calcd for C12H13N2O2S2 [M−H]+ 281.0417, found 281.0418. 1H NMR
(400 MHz, DMSO-d6) δ: 7.32–7.31 (dd, J = 4.8, 1.2 Hz, 1H), 7.11 (s,
1H), 6.94–6.92 (dd, J = 4.8, 3.2 Hz 1H), 6.90–6.89 (dd, J = 3.2, 1.2 Hz,
1H), 3.27–3.25 (m, 4H), 2.90–2.86 (t, J = 15 Hz, 2H), 2.35–2.31 (t,
J = 15 Hz, 2H); 13C NMR (400 MHz, DMSO-d6) δ: 168.69, 168.65,
155.44, 143.18, 127.36, 125.64, 124.45, 113.74, 35.04, 32.22, 29.63,
27.31.
343.0728. 1H NMR (400 MHz, DMSO-d6) δ: 7.59–7.57 (d, J = 8 Hz,
2H), 7.42–7.40 (d, J = 8 Hz, 2H), 7.03 (s, 1H), 3.35–3.31 (t, J = 15 Hz,
2H), 3.20–3.16 (t, J = 15 Hz, 2H), 3.07–3.03 (t, J = 15 Hz, 2H),
2.50–2.46 (t, J = 15 Hz, 2H); 13C NMR (400 MHz, DMSO-d6) δ:
170.23, 170.13, 154.61, 144.84, 144.83, 128.86, 124.96, 113.44,
35.30, 33.78, 31.85, 26.53.
4.1.3.12. N-Hydroxy-3-(2-(4-nitrophenethyl)thiazol-4-yl)propanamide
(6l). White solid in 44% yield. mp 163.2–164.1 °C. HRMS: calcd for
C
14H14N3O4S [M−H]+ 320.0715, found 320.0705. 1H NMR (400 MHz,
4.1.3.19. N-Hydroxy-3-(2-(2-(1H-indol-3-yl)ethyl)thiazol-4-yl)
propanamide (6s). Light red solid in 21% yield. mp 129.3–131.2 °C.
HRMS: calcd for C16H16N3O2S [M−H]+ 312.0814, found 312.0807. 1H
NMR (300 MHz, DMSO-d6) δ: 8.17 (s, 1H), 7.62–7.59 (d, J = 9 Hz, 1H),
7.37–7.34 (d, J = 9 Hz, 1H), 7.22–7.08 (m, 2H), 6.98 (s, 1H), 6.73 (s,
1H), 3.41–3.36 (t, J = 15 Hz, 2H), 3.27–3.22 (t, J = 15 Hz, 2H)
2.82–2.77 (t, J = 15 Hz, 2H), 2.39–2.34 (t, J = 15 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) δ: 210.78, 190.72, 162.48, 155.28, 154.32,
152.47, 150.35, 150.20, 138.79, 135.44, 129.87, 125.44, 50.64,
46.42, 35.29, 31.52, 26.49.
DMSO-d6) δ: 8.16–8.14 (d, J = 8 Hz, 2H), 7.56–7.54 (d, J = 8 Hz, 2H),
7.10 (s, 1H), 3.32–3.28 (t, J = 15 Hz, 2H), 3.20–3.16 (t, J = 15 Hz, 2H),
2.90–2.86 (t, J = 15 Hz, 2H), 2.35–2.31 (t, J = 15 Hz, 2H); 13C NMR
(400 MHz, DMSO-d6) δ: 168.62, 155.49, 149.31, 146.53, 130.32,
123.86, 113.73, 35.06, 33.86, 32.20, 27.28.
4.1.3.13. N-Hydroxy-3-(2-(3-fluorophenethyl)thiazol-4-yl)propanamide
(6m). White solid in 62% yield. mp 144.9–145.7 °C. HRMS: calcd for
C
14H14FN2O2S [M−H]+ 293.0764, found 293.0760. 1H NMR
(400 MHz, DMSO-d6) δ: 7.31–7.26 (m, 1H), 7.03 (s, 1H), 7.01–6.91
(m, 3H), 3.31–3.27 (t, J = 15 Hz, 2H), 3.12–3.08 (t, J = 15 Hz, 2H),
4.1.3.20. N-Hydroxy-(E)-3-(2-styrylthiazol-4-yl)propanamide
10

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
(6t). White solid in 20% yield. mp 166.9–167.8 °C. HRMS: calcd for
J = 15 Hz, 2H), 2.88–2.84 (t, J = 15 Hz, 2H), 2.40–2.36 (t, J = 15 Hz,
2H); 13C NMR (400 MHz, DMSO-d6) δ: 172.08, 168.32, 151.97, 145.58,
144.18, 130.91, 119.54, 116.29, 115.99, 115.56, 34.69, 33.86, 31.69,
25.82.
C
14H15N2O2S [M+H]+ 275.0661, found 275.0643. 1H NMR (300 MHz,
DMSO-d6) δ: 7.54–7.51 (d, J = 9 Hz, 2H), 7.41–7.22 (m, 5H), 6.88 (s,
1H), 3.14–3.09 (t, J = 15 Hz, 2H), 2.81–2.76 (t, J = 9 Hz, 2H); 13C
NMR (100 MHz, DMSO-d6) δ: 172.64, 168.93, 158.27, 137.22, 136.11,
130.29, 128.35, 121.95, 114.92, 33.25, 31.52, 26.49.
4.1.4.3. N-Hydroxy-3-(2-(3,4,5-trihydroxyphenethyl)thiazol-4-yl)
propanamide (6p′). White solid in 33% yield. mp 165.2–166.3 °C.
HRMS: calcd for C14H15N2O5S [M−H]+ 323.0813, found 323.0854.
1H NMR (400 MHz, DMSO-d6) δ: 7.36 (s, 1H), 6.14 (s, 2H), 3.27–3.23
(t, J = 15 Hz, 2H), 2.96–2.93 (t, J = 15 Hz, 2H), 2.80–2.76 (t,
J = 15 Hz, 2H), 2.66–2.63 (t, J = 15 Hz, 2H); 13C NMR (400 MHz,
DMSO-d6) δ: 173.84, 172.33, 151.50, 146.50, 131.86, 130.15, 115.76,
107.59, 34.89, 33.70, 33.04, 25.
4.1.3.21. N-Hydroxy-(E)-3-(2-(4-fluorostyryl)thiazol-4-yl)propanamide
(6u). Light yellow solid in 28% yield. mp 154.7–155.3 °C. HRMS: calcd
for C14H14FN2O2S [M−H]+ 291.0615, found 291.0604. 1H NMR
(300 MHz, DMSO-d6) δ: 7.54–7.51 (d, J = 9 Hz, 2H), 7.32–7.18 (m,
4H), 6.90 (s, 1H), 2.75–2.70 (t, J = 15 Hz, 2H), 2.52–2.47 (t, J = 15 Hz,
2H); 13C NMR (100 MHz, DMSO-d6) δ: 172.64, 158.27, 137.22, 136.11,
130.29, 128.35, 121.95, 114.92, 33.25, 31.52, 26.49.
4.2. Biology and computational studies
4.1.3.22. N-Hydroxy-(E)-3-(2-(4-chlorostyryl)thiazol-4-yl)propanamide
(6v). Yellow solid in 27% yield. mp 176.2–176.8 °C. HRMS: calcd for
4.2.1. Compounds and reagents
C
14H12ClN2O2S [M−H]+ 307.0311, found 307.0308. 1H NMR
SAHA (SML0061), tubastatin A (SML0044), and anti-β-actin anti-
body (a mouse monoclonal antibody) (A1978) were purchased from
Sigma-Aldrich. Antibodies for acetyl K40 α-tubulin (ab179484), α-tu-
bulin (ab52866), and histone H3 (ab1791) were purchased from
Abcam. Rabbit polyclonal anti-acetyl-histone H3 antibody (06-599) was
purchased from Millipore. Secondary anti-rabbit and anti-mouse anti-
bodies labeled with horseradish peroxide (sc-2030 and sc-2005) were
purchased from Santa Cruz Biotechnology. DMEM high glucose media
(SH30243.01), FBS (SH30084.03), and penicillin-streptomycin solution
(SV30010) were purchased from Hyclone. Other chemicals and re-
agents were obtained from Sigma-Aldrich, unless otherwise mentioned.
(400 MHz, DMSO-d6) δ: 10.05–10.35 (m, 1H), 8.85–8.61 (m, 1H),
7.73 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 2.8 Hz, 2H), 7.28 (s, 3H),
3.00–2.88 (m, 2H), 2.40–2.31(m, 2H); 13C NMR (400 MHz, DMSO-d6)
δ: 174.08, 165.69, 156.97, 135.11, 133.61, 132.38, 129.35, 129.30,
122.64, 114.75, 33.48, 26.86.
4.1.3.23. N-Hydroxy-(E)-3-(2-(4-bromostyryl)thiazol-4-yl)propanamide
(6w). Yellow solid in 21% yield. mp 172.4–172.6 °C. HRMS: calcd for
C
14H12BrN2O2S [M−H]+ 352.9791, found 352.9769. 1H NMR
(400 MHz, DMSO-d6) δ: 10.07–10.36 (m, 1H), 8.87–8.67 (m, 1H),
7.73–7.22 (m, 7H), 2.94 (t, J = 7.6 Hz, 2H), 2.37 (t, J = 7.7 Hz, 2H);
13C NMR (400 MHz, DMSO-d6) δ: 168.00, 165.53, 157.13, 135.44,
132.40, 132.25, 129.62, 122.66, 114.84, 32.18, 27.34
4.2.2. Ligand preparation for docking
Docking study were carried out by Glide27 in Schrödinger maestro
1128 (operated under CentOS release
7 and windows 10) and
4.1.3.24. N-Hydroxy-(E)-3-(2-(2-(1H-indol-3-yl)vinyl)thiazol-4-yl)
propanamide (6x). Light red solid in 20% yield. mp 169.2–171.4 °C.
HRMS: calcd for C16H14N3O2S [M−H]+ 312.0815, found 312.0807. 1H
NMR (300 MHz, DMSO-d6) δ: 8.31 (bs, 1H), 7.67–7.65 (d, J = 6 Hz,
2H), 7.62–7.59 (d, J = 9 Hz, 1H), 7.36–7.33 (d, J = 9 Hz, 1H),
7.21–7.08 (m, 2H), 6.89 (s, 1H), 6.79 (s, 1H), 2.82–2.77 (t,
J = 15 Hz, 2H), 2.46–2.41 (t, J = 15 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6) δ: 200.98, 195.46, 175.81, 160.42, 154.52, 150.99, 149.34,
142.65, 140.23, 138.48, 136.57, 120.48, 114.73, 113.28, 35.29, 31.52.
Sybyl X 2.1.129 (operated under windows 7) software. In Schrödinger,
sketched ligands were prepared using the LigPrep module. Ligands
were charged and minimized using the OPLS3 force field. We set out to
generate possible ionized states at pH 5–9, check metal binding state,
and include original state options, to generate neutral and deprotonated
states. All generated states were used for docking. In Sybyl, ligands
were sketched in neutral states and energy minimized using the con-
jugated gradient method in the Tripos force field with the Gasteiger-
Hückel charge method, until
a convergence value of 0.001 kcal
Å−1 mol−1
.
4.1.4. General procedure for 6h′, 6o′, and 6p′ synthesis26
To a solution of the appropriate methoxyphenethyl thiazole (6h, 6o,
and 6p) (1 equiv.) in methylene chloride (0.1 mmol mL−1), boron tri-
bromide (1.5 equiv) was added dropwise at 0 °C, and the reaction
mixture was stirred for 10–30 min at room temperature. After the re-
action was completed, the reaction mixture was cooled to 0 °C and
quenched with methanol. Methanol was then removed under reduced
pressure, and the residue was recrystallized with acetonitrile or me-
thylene chloride.
4.2.3. Preparation of protein structures
The crystal structures of human HDAC1 (PDB: 4BKX) and HDAC6
(PDB: 5EDU) were downloaded from RCSB Protein Data Bank (https://
www.rcsb.org/). Downloaded structures were refined as follows: in
Schrödinger, ‘Protein Preparation’ module was used. Missing side
chains and loops were filled in using Prime, and metal ions included in
protein were set to create zero-order bonds. For final refinements, H-
bond assessment and restrained minimization were conducted in de-
fault option. In Sybyl, human HDAC6 (PDB ID: 5EDU) was prepared
using ‘Protein Preparation’ module. All water molecules and un-
necessary molecules were removed. Ligands were extracted and
charged with Gasteiger-Hückel charge, and Kollman-all charge was
used for protein.
4.1.4.1. N-Hydroxy-3-(2-(4-hydroxyphenethyl)thiazol-4-yl)propanamide
(6h′). White solid in 32% yield. mp 156.3–157.1 °C. HRMS: calcd for
C
14H15N2O3S [M−H]+ 291.0872, found 291.0837. 1H NMR (400 MHz,
DMSO-d6) δ: 7.30 (s, 1H), 7.04–7.02 (d, J = 8 Hz, 2H), 6.69–6.67 (d,
J = 8 Hz, 2H), 3.31–3.27 (t, J = 15 Hz, 2H), 2.95–2.90 (m, 4H),
2.39–2.35 (t, J = 15 Hz, 2H); 13C NMR (400 MHz, DMSO-d6) δ:
171.97, 168.35, 156.24, 152.11, 130.24, 129.82, 115.65, 115.61,
34.29, 33.91, 31.70, 25.85.
4.2.4. Glide docking
Grids were generated using receptor grid generation module from
prepared protein structures. Center of grids were calculated from co-
crystalized ligands or nearby residues. Grid box sizes were auto-
matically defined or defined as 15 Å if the size was too small. Generated
grids were used for both XP glide30 and QPLD docking31. XP docking
was proceeded in default setting and gerated 10 poses per ligand for
comparison. For QPLD, precision setting of initial docking, and XP was
used for redocking.
4.1.4.2. N-Hydroxy-3-(2-(3,4-dihydroxyphenethyl)thiazol-4-yl)
propanamide (6o′). White solid in 28% yield. mp 162.3–162.6 °C.
HRMS: calcd for C14H14N2O4S [M−H]+ 307.0757, found 307.0753.
1H NMR (400 MHz, DMSO-d6) δ: 7.32 (s, 1H), 6.65–6.62 (m, 2H),
6.49–6.47 (d, J = 8 Hz, 1H), 3.29–3.25 (t, J = 15 Hz, 2H), 2.96–2.93 (t,
11

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
4.2.5. Surflex-dock docking
compound. ( )-Verapamil hydrochloride, 1 nM NADPH, 0.5 mg/ml
total protein of microsomes (Human liver microsomes ultra pooled
donor: Corning Gentest™, 452117; mouse liver microsome and CD-1
mice: Corning Gentest™ 452701), and 0.1 M phosphate buffer (pH 7.4).
The incubation plates were then incubated at 37 °C for 0, 5, 15, and
30 min respectively. Additionally, assays without NADPH were used to
evaluate metabolic stability in the absence of NADPH at 37 °C for 0 and
30 min. The reaction was terminated by adding acetonitrile. The re-
acted solution was transferred onto a filter plate and analyzed using
UPLC-MS. The assay was performed in duplicate.
The docking was conducted using prepared HDAC6 crystal struc-
ture, using Docking suite module. Protomol was generated with co-
crystalized TSA structure as the template, with the threshold to 0.50
and bloat to 5. The Cscore calculation option was checked, and max-
imum output poses was set to 50. Only poses with Cscore > 3 were
considered as docking hits.
4.2.6. HDAC enzymatic assay
The inhibition activity of all the synthesized 9a derivatives and the
reference compounds against HDAC1 and HDAC6 were evaluated using
BPS bioscience assay kits (Fluorogenic HDAC1 assay kit [50061] and
Fluorogenic HDAC6 [50076]). Enzymatic assays were conducted in
accordance with the manufacturer’s protocols. After a reaction, half of
the reactant volume (50 μl) was loaded into white 96-well plates
(Costar 4693). Fluorescence was detected using a Glomax-Multi plus
detection system from Promega, at an excitation wavelength range of
350–380 nm, and emission range of 440–470 nm. We designed the final
concentration of DMSO to be 0.1 vol% in reaction volume to minimize
the effects of DMSO.24 For the reactions, all mixtures were incubated in
a 37 °C water bath for 30 min, and at room temperature for 15 min after
adding 50 μl of HDAC developer. All assays were repeated at least three
times. Relative enzyme activity was calculated using Microsoft Excel.
Dose-response curves and IC50 values were generated using the
GraphPad Prism v5 software.
Acknowledgments
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by
the Ministry of Education (NRF-2012R1A5A2A28671860).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.06.036.
References
1. Arrowsmith CH, Bountra C, Fish PV, Lee K, Schapira M. Epigenetic protein families: a
new frontier for drug discovery. Nat Rev Drug Discov. 2012;11:384–400.
2. Morera L, Lubbert M, Jung M. Targeting histone methyltransferases and demethy-
lases in clinical trials for cancer therapy. Clin Epigenet. 2016;8:57.
3. Thaler F, Mercurio C. Towards selective inhibition of histone deacetylase isoforms:
what has been achieved, where we are and what will be next. ChemMedChem.
2014;9:523–526.
4.2.7. Cell culture
Raw 264.7 and HeLa cells were purchased from the Korea Cell Line
Bank and cultured in DMEM supplemented with 10% fetal bovine
serum and 1% penicillin-streptomycin solution. All cell lines were
grown at 37 °C in a humidified incubator with an atmosphere of 95% air
and 5% CO2.
4. Di Micco S, Chini MG, Terracciano S, Bruno I, Riccio R, Bifulco G. Structural basis for
the design and synthesis of selective HDAC inhibitors. Bioorg Med Chem.
2013;21:3795–3807.
5. Wagner FF, Olson DE, Gale JP, et al. Potent and selective inhibition of histone dea-
cetylase 6 (HDAC6) does not require a surface-binding motif. J Med Chem.
2013;56:1772–1776.
4.2.8. Immunoblotting
6. Hubbert C, Guardiola A, Shao R, et al. HDAC6 is a microtubule-associated deacety-
lase. Nature. 2002;417:455–458.
Raw 264.7 and HeLa cells were seeded in 6-well plates (30006, SPL
life science) at a density of 2.5 × 105/mL and incubated for 24 h. After
washing with PBS, the drug-diluted media were replaced, and they
were again incubated for 24 h. 6u, SAHA, and tubastatin A at con-
centrations of 0.3–3 μM and 0.1–10 μM were used to treat Raw 264.7
and HeLa cells, respectively. The final DMSO concentration was 0.1 vol
% of reaction volume, in all control and drug-treated wells. Proteins in
the Raw 264.7 cells were harvested using Ripa buffer (R0278, Sigma-
7. Janke C, Montagnac G. Causes and consequences of microtubule acetylation. Curr
Biol. 2017;27:R1287–R1292.
8. Dompierre JP, Godin JD, Charrin BC, et al. Histone deacetylase 6 inhibition com-
pensates for the transport deficit in Huntington's disease by increasing tubulin
acetylation. J Neurosci. 2007;27:3571–3583.
9. Mo Z, Zhao X, Liu H, et al. Aberrant GlyRS-HDAC6 interaction linked to axonal
transport deficits in Charcot-Marie-Tooth neuropathy. Nat Commun. 2018;9:1007.
10. Li G, Jiang H, Chang M, Xie H, Hu L. HDAC6 alpha-tubulin deacetylase: a potential
therapeutic target in neurodegenerative diseases. J Neurol Sci. 2011;304:1–8.
11. Cao W, Bao C, Padalko E, Lowenstein CJ. Acetylation of mitogen-activated protein
kinase phosphatase-1 inhibits Toll-like receptor signaling. J Exp Med.
2008;205:1491–1503.
Aldrich) and the EpiQuikTM nuclear extraction kit
I (OP-0002,
Epigentek), and the Nuclear extract kit (Active motif 40010) was used
to extract proteins in the HeLa cells, following the manufacturer’s
protocols. These proteins were quantified using the Bradford assay (Bio-
rad 500-0006). Protein samples were electrophoresed on 10 and 15%
SDS-polyacrylamide gels, transferred onto PVDF membranes, and
blocked with 8% skim milk in TBST (10 mM Tris-HCl, pH 7.5, 150 nM
NaCl and 0.1% Tween 20) overnight. They were then probed with
various concentrations of primary antibodies diluted in TBST for 2 h at
room temperature, per the manufacturer’s instructions. Various con-
centrations of secondary antibodies labeled with horseradish perox-
idase and diluted with TBST (dilution ration, 1:4000) for 2 h at room
temperature, were used. The immune-reactive bands were obtained
using the enhanced chemiluminescence (ECL) (ECL-PS250, Dongin LS)
system detection kit with a ChemiDOC imaging system (Dacinch-
chemi™). Acetyl α-tubulin, α-tubulin, and β-actin were detected in
single membranes by repeating antibody detach using EzReprobe (WSE-
7240, ATTO), and blocking. Acetyl histone H3 and histone H3 were also
detected in single membranes using the same protocol.
12. Kim SJ, Baek KS, Park HJ, Jung YH, Lee SM. Compound 9a, a novel synthetic histone
deacetylase inhibitor, protects against septic injury in mice by suppressing MAPK
signalling. Br J Pharmacol. 2016;173:1045–1057.
13. de Zoeten EF, Wang L, Butler K, et al. Histone deacetylase 6 and heat shock protein
90 control the functions of Foxp3(+) T-regulatory cells. Mol Cell Biol.
2011;31:2066–2078.
14. Kalin JH, Butler KV, Akimova T, Hancock WW, Kozikowski AP. Second-generation
histone deacetylase 6 inhibitors enhance the immunosuppressive effects of Foxp3+
T-regulatory cells. J Med Chem. 2012;55:639–651.
15. Yan B, Xie SB, Liu Z, et al. HDAC6 deacetylase activity is critical for lipopoly-
saccharide-induced activation of macrophages. PLoS ONE. 2014;9.
16. Into T, Inomata M, Niida S, Murakami Y, Shibata K. Regulation of MyD88 ag-
gregation and the MyD88-dependent signaling pathway by sequestosome 1 and
histone deacetylase 6. J Biol Chem. 2010;285:35759–35769.
17. Li D, Liu Y, Yang Y, et al. Looped host defense peptide CLP-19 binds to microtubules
and inhibits surface expression of TLR4 on mouse macrophages. J Immunol.
2013;190:6083–6092.
18. Yoo J, Kim SJ, Son D, et al. Computer-aided identification of new histone deacetylase
6 selective inhibitor with anti-sepsis activity. Eur J Med Chem. 2016;116:126–135.
19. Hai Y, Christianson DW. Histone deacetylase 6 structure and molecular basis of
catalysis and inhibition. Nat Chem Biol. 2016;12:741–747.
20. Millard CJ, Watson PJ, Celardo I, et al. Class I HDACs share a common mechanism of
regulation by inositol phosphates. Mol Cell. 2013;51:57–67.
4.2.9. Microsomal stability assay
21. Balasubramanian G, Narayanan S, Andiappan L, et al. In vivo effective dibenzo[b, d]
furan-1-yl-thiazoles as novel PDE-4 inhibitors. Bioorgan Med Chem.
2016;24:5702–5716.
The microsomal assay was conducted as previously reported.32
Various concentrations of the reaction solutions were prepared. Each
reaction volume contained 1 μM of the test (6u and 6g) or reference
22. Ugwu DI, Ezema BE, Eze FU, Ayogu JI, Ezema CG, Ugwuja DI. Synthesis and
12

G. Nam, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
biological applications of hydroxamates. Am J Org Chem. 2014;4:26–51.
docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem.
23. Miyake Y, Keusch JJ, Wang LL, et al. Structural insights into HDAC6 tubulin dea-
cetylation and its selective inhibition. Nat Chem Biol. 2016;12:748–754.
24. Butler KV, Kalin J, Brochier C, Vistoli G, Langley B, Kozikowski AP. Rational design
and simple chemistry yield a superior, neuroprotective HDAC6 inhibitor, tubastatin
A. J Am Chem Soc. 2010;132:10842–10846.
2004;47:1739–1749.
28. Schrödinger Release 2017-3: Schrödinger, LLC, New York, NY, 2017; 2017.
29. Certara, L. P. SX. 2.1.1 ed. SYBYL molecular modeling software, St. Louis, MO; 2013.
30. Friesner RA, Murphy RB, Repasky MP, et al. Extra precision glide: docking and
scoring incorporating a model of hydrophobic enclosure for protein− ligand com-
plexes. J Med Chem. 2006;49:6177–6196.
25. Porter NJ, Mahendran A, Breslow R, Christianson DW. Unusual zinc-binding mode of
HDAC6-selective hydroxamate inhibitors. Proc Natl Acad Sci USA.
2017;114:13459–13464.
31. Cho AE, Rinaldo D. Extension of QM/MM docking and its applications to metallo-
proteins. J Comput Chem. 2009;30:2609–2616.
26. Patent Reference: WO2012112946, page 128.
32. Yoon DO, Zhao X, Son D, et al. SAR studies of indole-5-propanoic acid derivatives to
develop novel GPR40 agonists. ACS Med Chem Lett. 2017;8:1336–1340.
27. Friesner RA, Banks JL, Murphy RB, et al. Glide: a new approach for rapid, accurate
13