Synthesis and in Vitro and in Vivo Biological Evaluation of Tissue-Specific Bisthiazole Histone Deacetylase (HDAC) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01792

A series of bisthiazole-based hydroxamic acids as novel potent HDAC inhibitors was developed during our previous work. In the present work, a new series of highly potent bisthiazole-based compounds were designed and synthesized. Among the prepared compounds, compound H13, which contains an α-(S)-methyl-substituted benzyl group, displays potent inhibitory activity toward human HDACs and several cancer cells lines. Compound H13 has a favorable PK profile and high tissue distribution specificity in the colon, as well as good efficacy in the AOM-DSS mouse model for colitis-associated colonic tumorigenesis.

Full text of DOI:10.1021/acs.jmedchem.9b01792

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Article
Synthesis and in vitro and in vivo biological evaluation of tissue-
specific bisthiazole Histone deacetylase (HDAC) inhibitors
Shu-Wei Zhang, Chao-Jun Gong, Ming-Bo Su, Fei Chen, Ting He, Yang-
Ming Zhang, Qian-Qian Shen, Yi Su, Jian Ding, Jia Li, Yi Chen, and Fa-Jun Nan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01792 • Publication Date (Web): 19 Dec 2019
Downloaded from pubs.acs.org on December 20, 2019
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Synthesis and in vitro and in vivo biological
evaluation of tissue-specific bisthiazole histone
deacetylase (HDAC) inhibitors
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Shu-Wei Zhang,†,§,∥ Chao-Jun Gong,†,∥ Ming-Bo Su,† Fei Chen,† Ting He,† Yang-Ming
Zhang,†,# Qian-Qian Shen,‡ Yi Su‡ Jian Ding,‡ Jia Li,† * Yi Chen, ‡* Fa-Jun Nan†,#*
† State Key Laboratory of Drug Research, the National Center for Drug Screening, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China;
# Yantai Key Laboratory of Nanomedicine & Advanced Preparations, Yantai Institute of Materia
Medica, Shandong, 264000, China；
‡ Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
§ University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, 100049, P. R.
China
∥ These authors contributed equally
KEYWORDS: Histone deacetylase inhibitors; Bisthiazole; Solid tumor.
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ABSTRACT: A series of bisthiazole-based hydroxamic acids as novel potent HDAC inhibitors
was developed during our previous work. In the present work, a new series of highly potent
bisthiazole-based compounds were designed and synthesized. Among the prepared compounds,
compound H13, which contains an α-(S)-methyl-substituted benzyl group, displays potent
inhibitory activity towards human HDACs and several cancer cells lines. Compound H13 has a
favorable PK profile and high tissue distribution specificity in the colon, as well as good efficacy
in the AOM-DSS mouse model for colitis-associated colonic tumorigenesis.
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Introduction
The acetylation and deacetylation of histones play a key role in the structural modification of
nuclear chromatin and are regulated by the activity of histone acetyltransferase (HAT) and histone
deacetylase (HDAC), respectively.1 Abnormal expression of HDACs has been associated with
various types of cancers. HDAC inhibitors (HDACi) can inhibit the activity of HDAC and display
obvious antitumor activity by changing the histone acetylation state, causing tumor cell growth
arrest and inducing differentiation and apoptosis. Thus, HDAC inhibitors are widely used in cancer
therapy.2 To date, five HDAC inhibitors have been approved by the FDA (Figure 1). SAHA3, FK-
228 (romidepsin)4, belinostat5, and chidamide6 are used for treating cutaneous T-cell lymphoma
(CTCL) or peripheral T-cell lymphoma (PTCL), and LBH589 (panobinostat)7 is the first HDAC
inhibitor approved for multiple myeloma (MM).
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O
H
H
H
O
N
N
OH
N
N
HO
S
H
N
O
O
O
H
6
7
8
9
Vorinostat
Belinostat
HN
O
O
O
N
H
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O
NH
O
H
N
NH2
O
H
H
H
N
N
N
NH
OH
N
H
O
O
O
F
S
S
Panobinostat
Chidamide
Romidepsin
Figure 1. Approved HDAC Inhibitors
It is well known that most HDAC inhibitors are highly effective in hematological malignancy.
However, in solid tumors, the results are not as promised. For example, many clinical trials of
SAHA and FK-228 on breast, colorectal, and prostate cancers and other solid tumors have been
conducted. Unfortunately, the results were not ideal, and various side effects such as prolonged
QT intervals, anorexia, weakness, diarrhea, fatigue, vomiting, anemia, and leukocyte reduction
were reported.8 Therefore, the development of new HDAC inhibitors for solid tumor therapy is an
ongoing challenge.
In our previous work, we identified a series of bisthiazole-based HDAC inhibitors that were
inspired by the natural potent HDAC inhibitor largazole. Of those compounds, the simple but
potent compound H1 (Bisthianostat)9,10 was selected as a candidate for further development for
multiple myeloma(NCT03618602). In this paper, we report further structural modifications of
these bisthiazole HDAC inhibitors through changes to the linker group between the hydroxamate
and the bisthiazole core (Figure 2) and through varying the substituents on the core structure. Thus,
we demonstrated that compounds with an α-(S)-methyl-substituted benzyl group as the linker had
excellent potency in vitro and in vivo.
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R3
O
O
O
S
N
O
N
S
N
LINKER
HO
NH
O
N
N
O
H
N
S
H
N
S
S
H
N
HO
N
O
O
O
S
H
R1
N
n-C7H15
S
N
H
R2
H1(Bisthianostat)
Largazole
Previous work
Current work
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Figure 2. From Largazole to Bisthiazole HDAC Inhibitors
Chemistry
First, we kept the bisthiazole core structure and hydroxamates unchanged, but we replaced the
aliphatic linker with heteroalkyl, benzyl or substituted benzyl groups. The syntheses of compounds
H2-H8 are outlined in Scheme 1. Acids 1a9 and 1b9 were condensed with different amines to
afford amides 7-13; treating these amides with hydroxylamine hydrochloride afforded compounds
H2-8 respectively.
Commercially available intermediates 14 and 15 were condensed to afford imine 16.13 Imine 16
was treated with different Grignard reagents to generate sulfinamides 17a-c.14 The
diastereoisomers 17a and 17b could be isolated by silica gel chromatography. Deprotection of
17a-c afforded chiral amines 18a-c. Amines 18a-c were condensed with various acids to afford
amides 19a-c. The amides were then converted to compounds H6a-b and H9 through a similar
route to what is shown in Scheme 1. To investigate the SAR in detail, the (S)-methyl benzyl linker
was maintained, and a series of analogues with different substituents on the left-hand thiazole ring
was designed and synthesized as outlined in Scheme 3. Various bisthiazole acids with substituents
on the left-hand thiazole ring were coupled with chiral amine 18a to give amides 21a-f, which
were converted to the final hydroxamates H10-15 through a route similar to what is shown in
Scheme 2.
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Scheme 1. Synthesis of Compounds H2-8.
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8
9
O
O
O
HN
a
b
S
N
N
S
N
S
N
O
+
N
HO
N
N
N
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S
H
O
O
S
S
N
HO
R
O
O
1a
2
7
H2
O
O
O
b
R
a
R
S
N
S
S
N
+
N
HO
H
N
H
N
N
H2N
S
S
S
N
N
1a
3 R=COOMe
8 R=COOMe
9 R=(E)-CH=CHCOOMe
H3 R=CONHOH
H4 R=(E)-CH=CHCONHOH
4 R=(E)-CH=CHCOOMe
R
O
a
R
b
R
O
+
O
H
N
O
O
O
S
H2N
HO
S
N
S
N
HO
N
O
H
N
O
H
N
N
S
S
S
5 R=H
10 R=H
N
H5 R=H
N
6 R=Me
7 R=-CH2-CH2-
8 R=(CH3)2
11 R=Me
H6 R=Me
H7 R=-CH2-CH2-
H8 R=(CH3)2
1b
12 R=-CH2-CH2-
13 R=(CH3)2
aReagents and conditions: (a) EDCI, HOBt, i-Pr2NEt, DMF, room temperature, 12h. (b)
NH2OH·HCl, DBU, DCM:MeOH=1:1, room temperature, 12h.
Scheme 2. Synthesis of Compounds H6a, H6b and H9.
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RMgX
O
S
R
O
R
R=Me, X=Br
R=i-Pr ,X=Cl
O
S
OHC
S
a
N
H
N
H2N
c
+
NH2
b
COOMe
COOMe
COOMe
COOMe
18a R=(S)-Me
18b R=(S)-i-Pr
17a R=(S)-Me
17b R=(S)-i-Pr
14
15a
16a
8
9
O
S
O
O
S
OHC
S
H2N
a
MeMgBr
b
N
H
N
c
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+
NH2
COOMe
COOMe
COOMe
COOMe
17c
16b
18c
14
15a
O
R
O
O
R
R
S
N
N
H
S
S
N
d
e
N
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N
+
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N
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O
S
S
COOMe
O
N
NH
19a R=(S)-Me
19b R=(S)-i-Pr
19c R=(R)-Me
HO
H6a R=(S)-Me
H6b R=(R)-Me
H9 R=(S)-i-Pr
1b
18a R=(S)-Me
18b R=(S)-i-Pr
18c R=(R)-Me
O
aReagents and conditions: (a) Cs2CO3, DCM, reflux, 18 h. (b) Et2Zn, THF, -78 °C, 4 h. (c) 2 N
HCl/MeOH, DCM, room temperature, 3 h. (d) EDCI, DMAP, DCM, room temperature, 12 h. (e)
NH2OH·HCl, DBU, DCM:MeOH=1:1, room temperature, 12 h.
Scheme 3. Synthesis of Compounds H10-H15.
O
O
O
S
N
S
N
N
N
S
N
a
b
H
H
N
N
HO
+
H2N
N
S
S
O
O
S
COOMe
R1
R1
O
NH
R1
R2
R2
HO
R2
20a R1=Me, R2=H
20b R1=H, R2=Me
20c R1, R2=cyclohexyl
20d R1=Ph,R2=H
20e R1=4-F-Ph, R2=H
20f R1=4-OMe-Ph, R2=H
18a
21a R1=Me, R2=H
21b R1=H, R2=Me
21c R1, R2=cyclohexyl
21d R1=Ph,R2=H
21e R1=4-F-Ph, R2=H
21f R1=4-OMe-Ph, R2=H
H10 R1=Me, R2=H
H11 R1=H, R2=Me
H12 R1, R2=cyclohexyl
H13 R1=Ph,R2=H
H14 R1=4-F-Ph, R2=H
H15 R1=4-OMe-Ph, R2=H
aReagents and conditions: (a) EDCI, DMAP, DCM, room temperature, 12 h. (b) NH2OH·HCl,
DBU, DCM:MeOH=1:1, room temperature, 12 h.
Results and discussion
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HDAC Inhibition Assay
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The inhibitory activities against human HDACs of compounds H1-8 are shown in Table 1. Using
LBH589 (panobinostat) as the control compound, compared to H1, changing the aliphatic linker
to a heteroalkyl group (compound H2, Table 1) or a phenyl group decreased the inhibitory activity
of the compound, and they were much less potent toward the HDACs (compounds H3 and H4,
Table 1). However, the compounds with a benzyl linker showed higher HDAC inhibitory activities
(compounds H5-8), and compound H5, which has no substituents at the benzyl position, was more
potent against human HDACs 1, 3, and 6 than H1 with IC50 values of 30-40 nM (Table 1). Small
α-substituents on the benzyl group were tolerated (compounds H6-8, Table 1).
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Table 1. Inhibitory activity against human HDACs
LINKER
IC50(nM)
Compound
R
HDAC1
HDAC3
HDAC6
H1
H2
81.46±4.91
73.91±3.83
61.49±4.64
NAa
NA
NA
N
H3
H4
H5
H6
1020±200
660±90
690±90
410±60
27.51±0.16
189±17
300±50
N
H
310±60
N
H
23.74±0.98
80.98±15.0
21.91±2.80
41.65±16.1
H
N
H
N
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H7
H8
22.33±2.97
163.4±8
16.29±2.18
16.58±1.69
148±14
H
N
148±10
H
N
7
8
9
LBH589
7.03±1.18
5.83±1.09
10.63±1.30
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NAa = No inhibitory activity observed at 20 µg/mL.
Considering the benzyl position is easily metabolized position in vivo, a substituent at the benzyl
position was introduced to increase the metabolic stability in the same series. Further explorations
focused on analogues with different configurations of α-substituted benzyl groups (Table 2), and
the chirality of the α-methyl position of the benzyl group was crucial to the inhibitory activity. (S)-
Methyl-substituted compound H6a was much more potent than its enantiomer H6b (IC50 values
of 0.04 µM and 0.28 µM, respectively, against HDAC 1, Table 2). When the (S)-methyl group was
exchanged for a bulkier (S)-isopropyl group, the inhibitory activity of the compound was the same
as that of compound H6a (compound H9, Table 2). Preliminary docking study of H6a and H6b
showed the chiral methyl group determines the orientation of the compound in the pocket, S-
enantiomer preferentially makes the hydroxamic acid coordinates to zinc ions in the pocket than
R-enantiomer. Therefore, it is reasonable to take H6a as a new starting point for further exploration
(Information on computational modeling/docking studies is shown in Figure I, SI file).
Table 2. Inhibitory activity against human HDACs
IC50(nM)
Compound
R
HDAC1
HDAC3
HDAC6
H6
H6a
Me
(S)-Me
80.98±15.0
40.92±0.41
189.4±16.7
42.51±3.04
41.65±16.1
14.02±1.74
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H6b
H9
(R)-Me
(S)-i-Pr
LBH589
279±5
38.25±7.29
7.03±1.18
260±22
38.41±7.13
5.83±1.09
211±36
14.15±2.03
10.63±1.30
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Due to the importance of the (S)-methyl group on the benzyl linker, we then investigated a series
of compounds with an (S)-methyl group on the benzyl linker and different substituents on the
bisthiazole core; the results are outlined in Table 3. When R1 and R2 were alkyl groups, the
compounds showed slightly better inhibitory activity than H6a (IC50 values of 10-20 nM,
compounds H10-12, Table 3). Aromatic substituents such as a phenyl group at the 4’ position on
the left-hand thiazole ring greatly improved the potency against HDACs, and substituents on this
phenyl ring were tolerated (compounds H14 and H15, Table 3). The IC50 value of compound H13
was only 4 nM on HDACs (Table 3), and it was selected for further evaluation on a cellular level
as well as an in vivo efficacy study.
Table 3. Inhibitory activity against human HDACs
IC50(nM)
Compound
R1
R2
HDAC1
HDAC3
HDAC6
H6a
H10
H11
H12
H13
H14
H15
H
Me
H
H
H
Me
40.92±0.41 42.51±3.04 14.02±1.74
19.87±0.47 17.06±0.34 18.51±0.45
16.52±0.50 13.88±0.31 19.08±1.55
14.68±0.67 11.35±0.11 18.31±0.84
3.64±0.26 3.02±0.03 4.96±1.86
2.64±0.02 2.54±0.28 2.02±0.10
3.57±0.40 3.78±0.93 2.38±0.17
7.03±1.18 5.83±1.09 10.63±1.30
Cyclohexyl
Ph
4-F-Ph
4-OMe-Ph
LBH589
H
H
H
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Compound H13 was also evaluated in a panel of 11 HDACs and related 3 SIRTs, as listed in
Table 4. Compound H13 inhibits HDACs 1, 2, 3, 5, 6, 8, 10, and 11 in nanomole potency and
did not inhibit HDACs 4, 7 or 9 or any of the SIRTs.
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Table 4. Inhibitory activity of compound H13 against different HDAC family members and
SIRT family members.
Compound
HDAC1
H13
LBH589
3.59±0.34
13.26±2.04
5.61±1.28
>20000
16740±1520
1.59±0.16
>20000
7.03±1.18
22.83±0.87
5.83±1.09
10390±880
>20000
6.09±0.73
>20000
19.79±4.58
>20000
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
HDAC7
HDAC8
HDAC9
HDAC10
HDAC11
SIRT1
IC50(nM)
16.84±4.23
>20000
1.98±0.57
77.82±29.63
>20000
10.24±3.17
121±33
>20000
SIRT2
>20000
>20000
SIRT3
>20000
>20000
Efficacy of compound H13 on the cellular level
While compound H13 was identified as a potent novel inhibitor for HDACs of class I and II, we
evaluated its antiproliferative activity in a panel of solid tumor cell lines. LBH589 (panobinostat)
was used as the control, and potency was assessed based on the concentration required to inhibit
50% of cell growth at 72 h. As is shown in Table 5, Compound H13 displayed highly potent
antiproliferative activities in all the solid tumor cell lines tested (with IC50 values less than 200 nM,
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Table 5). In particular, compound H13 showed the most potent antiproliferative activity against
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NCI-N87 and T47D cells with IC50 values of 28 and 40 nM, respectively.
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Table 5. Antiproliferation Activity of Compound H13 Against Several Cancer Cell Lines
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IC50 (nM)
Cell line
H13
LBH589
21.28±1.61
17.51±2.23
27.85±1.72
9.00±0.94
29.13±0.42
6.98±0.72
8.42±0.77
37.55±4.81
Human liver cancer cell line BEL-7402
Human lung adenocarcinoma cell line NCI-H1975
Human lung squamous cell carcinoma line EBC-1
Human breast cancer cell line MCF-7
80.79±2.49
66.38±24.55
110±46
58.52±25.72
76.56±31.59
40.81±16.76
28.33±8.67
166±7
Human breast cancer cell line MDA-MB-231
Human breast cancer cell line T47D
Human gastric cancer cell line NCI-N87
Human colon cancer cell line HT-29
Pharmacokinetic and distribution study of compound H13
The pharmacokinetic profiles of compound H13 are summarized in Table 5. After oral
administration of a 5 mg/kg dose of H13 to mice, H13 had a Cmax of 138 ng/mL, T1/2 of 4.23 h,
and oral bioavailability of 34.4% (Table 6).
Table 6. Pharmacokinetic Parameters of Compound H13
Tmax
Cmax
AUC0-t
AUC0-∞ MRT T1/2
(h) (ng/mL) (ng•h/mL) (ng•h/mL) (h)
F
Compound
Dose
(h) (%)
/
/
1075
924
1248
944
10.4 8.67
5.22 4.23 34
i.v. 2 mg/kg
p.o. 5 mg/kg
H13
0.67
138
The metabolic stability of compound H13 in human, monkey, canine, rat and mouse liver cells
was evaluated. Compared to compound H19, H13 had better in vivo stability. The mainly
metabolism is undergone glucuronic acid binding and amide bond hydrolytic(Information on H13
metabolites is shown in Table I, SI file). No metabolites related to the phenyl group which attached
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to bisthiazole were found, consider the ease of synthesis and the minimum of molecule weight, we
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chose compound H13 instead of H14 or H15 for the extended profiling and in vivo experiments.
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To investigate the distribution of compound H13 in mice, the concentrations of compound H13
were determined in tissues and plasma at different time points after oral administration of the mice.
Tissue concentrations of compound H13 were analyzed by LC-MS/MS. The tissue distribution of
H13 in mice showed that the plasma exposure was low in mice after p.o. administration (dose of
50 mg/kg), while high exposure was found in the small intestine and colon. Drug concentrations
in tissues after 1 hour were on the following order: stomach> small intestine> liver> kidney> colon>
lung> ovary> uterus> plasma> brain> testis. After six hours of administration, H13 still
maintained a high concentration in the colon(Figure 3A). The similar results of tissue distribution
were found when H13 was given by intravenous administration. After i.v. administration (dose of
10 mg/kg), high exposure in the small intestine and colon was also found as oral administration
(Figure 3B).
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Figure 3. Drug concentration in tissue (A) tissue distribution for oral administration after 1, 6,
and 24 hours. (B) tissue distribution for intravenous administration after 0.33, 1, and 6 hours.
AUC is an important parameter for pharmacokinetic analyses, it represents the total drug exposure
integrated over time (Table 7). The AUC can be applicable to the concentration of drug in tissues.
It is the best estimate of drug delivery and an indicator of response. AUCs after the administration
of H13 was in the following order: stomach> colon> small intestine> liver> kidney> lung> ovary>
uterus> testis> brain. The compound is distributed in colon in 14.7-fold more in AUC than in
plasma. Compound H13, as a novel bisthiazole HDAC with an α-(S)-methyl-substituted benzyl
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group linker, was therefore identified as a potent and high tissue specificity HDAC inhibitor and
moved forward into in vivo studies.
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Table 7. The AUC of H13 in tissues and plasma
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Tissue
AUC (ng•h/g)
Rate (Tissue/Plasma)
stomach
colon
105642
8795
6079
5185
4692
3438
2622
1964
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698
176.5
14.7
10.2
8.66
7.84
5.74
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1.17
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small intestine
liver
kidney
lung
ovary
uterus
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brain
plasma
599
In Vivo Antitumor Efficacy
Because compound H13 has favorable colon tissue specificity, we chose a human colorectal
carcinoma HT-29 xenograftas the solid tumor model for preliminary evaluation and LBH589 as
the control. The data are outlined in Table 8 and Figure 3. Compound H13 demonstrated
significant antitumor activity with T/C % = 37.56%, 59.26%, and 60.58% at the oral administration
of doses of 100 mg/kg, 50 mg/kg and 25 mg/kg, respectively (Table 8, Figure 3). At the same dose
(50 mg/kg), compound H13 was much more effective than LBH589 (T/C = 59.72% vs 93.85%,
Table 7). Compound H13 also showed an acceptable safety profile as indicated by body weight
changes.
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Table 8 Efficacy of Orally Administered compound H13 in a HT-29 Xenograft Model
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Weight
(g)
d0 d21 d0 d21
TV
n
RTV
T/C
(mm3, mean±SD)
Compound
Dose
(mean±SD) (%)
d0
d21
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Vehicle
LBH589
H13
p.o.
12 12 18.1 19.1 173±38 2854±940 16.68±5.10
50 mg/kg p.o.
100 mg/kg p.o.
50 mg/kg p.o.
25 mg/kg p.o.
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18.2 17.8 173±34 2583±863 15.66±5.97 93.85
18.7 17.5 169±36 1032±326 6.27±1.98** 37.56
17.8 18.6 172±44 1602±913 9.96±6.13* 59.72
17.6 18.5 172±34 1682±398 10.11±3.16* 60.58
H13
H13
t Student’s test vs control, *p<0.01
Figure 4. Efficacy of Orally Administered compound H13 in a HT-29 Xenograft Model
The tumor incidence of compound H13 in a colitis-associated colorectal cancer model
For evaluation whether compound H13 directly decreases the incidence of colitis-associated
cancer, we generated an AOM-DSS mouse model for colitis-associated colonic tumorigenesis15
by injecting mice with procarcinogenic AOM followed by 3 cycles of oral DSS administration
(Figure 5A). The mice were sacrificed on d156 following induction, and the body weight of the
mice was monitored throughout the experiment. The tumor incidence was 12.50% and 11.11% in
H13-treated groups, 100 mg/kg and 50 mg/kg respectively (Figure 5C and Table 9). The tumor
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incidence was 77.78% in the vehicle group as expected, while LBH589 administration showed no
effect in this model. Compound H13 was well tolerated, as the animals showed no signs of toxicity
or weight loss over a period of more than 150 days of drug administration (Figure 5B).
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Table 9. The tumor incidence of compound H13
Group
Incidence (%)
Vehicle
77.78
66.67
LBH589 50 mg/kg
H13 100 mg/kg
H13 50 mg/kg
12.50#
11.11#
u test vs vehicle, #p<0.01
Figure 5. (A) Colitis-associated colorectal cancer model (B) Body weight (C) Tumor diameter
and distribution
Although hydroxamic acid has been reported as a mutagen, compounds such as vorinostat and
panobinostat based on this structure have demonstrated clinical benefits in hematological
malignances. In this study, we also found that after three weeks administration, our compound
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exerted potent efficacy against tumor growth in the xenograft models, while nude mice still were
in good condition. More importantly, in the model of induced colorectal cancer, we also found that
the animals were in good condition after treatment for up to three months. As we know, the AOM-
DSS model is an inducible tumorigenesis model. In this model, the compound H13 did not promote
tumorigenesis, but showed promising efficacy. All these data suggest that this compound may have
good safety profile. However, other HDAC warheads without mutagenic potential would be a
better choice for future modifications.
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Conclusion
In conclusion, a series of new bisthiazole HDAC inhibitors was designed and synthesized by
replacing the linker group and varying the substituents on the left-hand thiazole ring. Among the
prepared compounds, compound H13, with an α-(S)-methyl-substituted benzyl group, exhibited
excellent inhibitory activity against human HDACs1, 2, 3, 6, 8, 10 with IC50 values as low as 4
nM, and it displayed good antiproliferative activity against both hematological malignancy and
solid tumor cell lines. Compound H13 had an acceptable oral bioavailability (F = 34.4%, p.o. 5
mg/kg in mice) and showed high tissue distribution specificity. In efficacy studies, H13 showed
good efficacy in solid tumors such as the HT-29 model. Compound H13 can also directly decrease
the incidence of colitis-associated cancer. All these results indicate that compound H13 has
potential in solid tumor therapy. Further preclinical studies are underway, and the results will be
reported in due course.
Experimental Section
General Methods. Starting materials, reagents and solvents were purchased from commercial
suppliers and used without further purification. Anhydrous toluene and DCE were obtained from
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a distillation over sodium wire or CaH2. All non-aqueous reactions were run under an inert
atmosphere (nitrogen or argon) with rigid exclusion of moisture from reagents and all reaction
vessels were oven-dried. Thin-layer chromatography (TLC) was carried out on pre-coated TLC
13
plates with silica gel HSGF 254. Spots were visualized under UV at 254 nm. 1H-NMR and C-
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NMR spectra were measured on a Varian Mercury-VX 300, Varian MR 400, AVANCE III 500 or
AVANCE III 600 spectrometer using deuterated chloroform (CDCl3), deuterated methanol
(CD3OD), deuterated acetone (acetone-d6) and deuterated dimethyl sulfoxide (DMSO-d6) as the
solvent. Chemical shifts are expressed in δ (ppm.). Abbreviations for signal coupling are as follows:
s, singlet; brs, broad singlet; d, doublet; t, triplet; q, quartet; dd, double doublet; m, multiplet.
Coupling constants (J) are given in Hz. HR-MS were measured on a Micromass Ultra Q-Tof.
Purity of all final compounds was 95% or higher. Purity was evaluated by analytical HPLC
chromatograms using Agilent 1200 series LC system equipped with a degasser, a quaternary pump,
an auto sampler, a column oven and a diode array detector. Analytes were separated on a Zorbax
SB C18 column (4.6 × 150 mm, 5 µm). Solvent Awas 0.1% trifluoroacetic acid in H2O, and solvent
B was 100% methanol. Gradient elution: 20% B for 2 min, then 20%-80% B from 2 to 20 min,
80% B was maintained for 5 min, then 80% -20% B from 25 min to 30 min. All compounds were
monitored at 254 nm at room temperature. Flow rate: 1.0 mL/min. The following abbreviations
for solvents and reagents are used; N, N-dimethlformamide (DMF), dimethylsulfoxide (DMSO),
sodium hydroxide (NaOH), 1,2-dichloroethane (DCE), tetrahydrofuran (THF).
5-Isobutyl-[2,2'-bithiazole]-4-carboxylic acid (1a). Under the protection of N2, a solution of 2-
bromothianzole (5.51g, 0.034mol) was added dropwise to a suspension of activated Zn powder
(1.87g, 0.029mol) in dry THF (30mL) and the resulting mixture was heated to reflux for 2h. After
the organozinc reagents was obtained, the reaction mixture was cooled to room temperature.
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Meanwhile, under the protection of N2, Pd (OAc)2 (272mg, 1.2mmol), PPh3 (630mg, 2.4mmol)
and methyl 2-bromo-5-isobutylthiazole-4-carboxylate (9.42g, 0.034mol) was add to dry
toluene(30mL), and the resulting mixture was stirred under room temperature for 2h. Then the
reaction mixture was transfer to the organiczinc reagents, then the obtained mixture was again
heated to reflux for several hours until most of methyl 2-bromo-5-isobutylthiazole-4-carboxylate
was consumed. The reaction mixture was cooled to room temperature and concentrated under
reduced pressure. The residues were diluted with CH2Cl2 and the obtained suspension washed with
1N HCl and brine, respectively. The CH2Cl2 was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The obtained residue was purified by silica gel column chromatography
(PE:EtOAc = 20:1-4:1) to afford methyl 5-isobutyl-[2,2'-bithiazole]-4-carboxylate as pale-yellow
solids (7.96g, 83%). 1H NMR (300MHz, CDCl3): ꢀ 7.86 (d, J = 3.3Hz, 1H), 7.46 (d, J = 3.3Hz,
1H), 3.96 (s, 3H), 3.15 (d, J = 6.9Hz, 2H), 1.95-1.23 (m, 1H), 0.99 (d, J = 6.6Hz, 6H); MS (ESI+)
m/z: 283.0 (M+H) +.
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To a stirred solution of above obtained coupling product methyl 5-isobutyl-[2,2'-bithiazole]-4-
carboxylate (11.28g, 0.04mmol) in MeOH/H2O (1:4, v/v, 50mL) was added solid NaOH (3.00g,
0.07mmol) at 0℃. The resulting mixture was heated to reflux for 1h and the cooled to room
temperature. After removal of MeOH under reduced pressure, the resulting mixture was extracted
with Et2O. The aqueous phase was then acidified with 1N HCl to pH = 2.0, extracted with EtOAc
150mL (50mL×3). The combination EtOAc extract was washed with brine, dried over anhydrous
Na2SO4 and concentrated under reduced pressure to afford respective acid as a yellow solid 1a.1H
NMR (300MHz, CDCl3) ꢀ 7.86 (d, J = 3.3Hz, 1H), 7.46 (d, J = 3.3Hz, 1H), 3.15 (d, J = 6.9Hz,
2H), 1.95-1.23 (m, 1H), 0.99 (d, J = 6.6Hz, 6H); MS (ESI+) m/z: 269.0(M+H) +.
5-Cyclopropyl-[2,2'-bithiazole]-4-carboxylic acid (1b). 1b was obtained in 75% yield as yellow
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solids according to the preparation procedure for 1a. 1H NMR (300MHz, CDCl3): ꢀ 7.85 (d, J =
3.0Hz, 1H), 7.45 (d, J = 3.0Hz, 1H), 3.10-3.14 (m, 1H), 1.31-1.38 (m, 2H), 0.84-0.89 (m, 2H); MS
(ESI+) m/z: 267.0(M+H) +.
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Methyl 1-(5-isobutyl-[2,2'-bithiazole]-4-carbonyl)piperidine-4-carboxylate (7). To a solution
of the bisthiazole carboxyl acid 1a (70mg, 0.26mmol) in 5mL dry DMF were added EDCI (49mg,
0.26mmol), HOBT (35mg, 0.26mmol), 19 (40mg, 0.28mmol) and i-Pr2NEt (99mg, 0.75mmol),
respectively. The resulting mixture was stirred for 12h at room temperature. Then the mixture was
diluted with H2O and extracted with EtOAc (10mL×3). The combined organic phase was washed
with brine, dried over anhydrous Na2SO4, and then concentrated under reduced pressure The
obtained residue was purified by silica gel column chromatography (PE:EtOAc =10:1) to afford
26 (yellow oil, 81.7%).1H NMR (300MHz, CD3OD) ꢀ 7.89 (d, J = 3.3Hz, 1H), 7.71 (d, J = 3.3Hz,
1H), 4.49 (d, J = 13.2Hz, 1H), 3.82(s, 3H), 3.77 (d, J = 13.8Hz, 1H), 3.07-3.26 (m, 2H), 2.84 (d, J
= 7.2Hz, 2H), 2.63-2.70 (m, 1H), 1.87-1.99 (m, 2H), 1.66-1.80 (m, 2H), 0.99 (d, J=6.6Hz, 6H)
3.15 (d, J = 6.9Hz, 2H), 1.95-1.23 (m, 1H).
8-13 was synthesized following procedures similar to that described for compound 7
Methyl 4-(5-isobutyl-[2,2'-bithiazole]-4-carboxamido)benzoate (8). 1H NMR (300MHz,
CDCl3) : δ 9.47 (s, 1H), 8.03 (d, J = 8.7Hz, 2H), 7.88 (d, J = 3.3Hz, 1H), 7.78 (d, J = 8.4Hz, 2H),
7.48 (d, J = 3.3Hz, 1H), 3.89 (s, 3H), 3.30 (d, J = 7.2Hz, 2H), 2.02-2.07 (m, 1H), 1.02 (d, J =
6.6Hz, 6H). 13C NMR (75MHz, CDCl3): δ 166.8, 160.6, 160.0, 156.9, 149.8, 144.3, 143.3, 142.1,
131.0, 125.7, 121.6, 119.1, 52.2, 36.1, 31.2, 22.5, 22.4.
1
Methyl (E)-3-(4-(5-isobutyl-[2,2'-bithiazole]-4-carboxamido)phenyl)acrylate (9) H NMR
(300MHz, CDCl3) : δ 9.04 (s,1H), 8.03 (d, J = 8.7Hz, 2H), 7.89 (d, J = 3.3Hz, 2H), 7.47 (d, J =
16Hz, 1H), 6.45 (d, J = 16Hz, 1H), 3.89 (s, 3H), 3.23 (d, J = 7.2Hz, 2H), 2.01-1.98 (m, 1H), 1H),
0.96 (d, J = 6.6Hz, 6H);
Methyl 4-(1-(5-cyclopropyl-[2,2'-bithiazole]-4-carboxamido)ethyl)benzoate (11).1H NMR
( 300MHz, CDCl3) ꢀ 8.03 (d, J = 8.1Hz, 2H), 7.72 (d, J = 3.3Hz,1H), 7.65 (d, J = 8.1Hz, 1H), 7.49
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(d, J = 8.1Hz, 1H), 7.44 (d, J = 3.3Hz,1H), 5.40-5.28 (m, 1H), 3.91 (s, 3H), 3.47–3.38 (m, 1H),
1.65 (d, J=7.2Hz, 3H), 1.33-1.28 (m, 2H), 0.82-0.78 (m, 2H); MS (ESI+) m/z: 436.1(M+Na) +.
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Benzyl 4-(1-(5-cyclopropyl-[2,2'-bithiazole]-4-carboxamido)cyclopropyl)benzoate (12). H
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NMR (300MHz, CDCl3) ꢀ 8.01 (d, J = 8.7Hz, 1H), 7.85 (d, J = 3.0Hz, 1H), 7.44 (d, J = 3.0Hz,
1H), 5.34 (s, 2H), 3.89 (s, 3H), 3.49-3.38 (m, 1H), 1.53-1.42 (m, 4H), 1.35-1.28 (m, 2H), 0.83-
0.78 (m, 2H); MS (ESI+) m/z: 524.1(M+Na) +.
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methyl 4-(1-(5-cyclopropyl-[2,2'-bithiazol]-4-yl)-2-methyl-1-oxopropan-2-yl)benzoate (13).
1H NMR (300MHz, CDCl3) ꢀ 8.03 (d, J = 8.7Hz, 2H), 7.86 (d, J = 3.0Hz,1H), 7.80 (s, 1H), 7.54
(d, J = 8.7Hz, 2H), 7.45 (d, J = 3.0Hz,1H), 4.36 (q, J = 7.2Hz, 2H), 3.41–3.32 (m, 1H), 1.83 (s,
6H), 1.37 (d, J = 7.2Hz, 3H), 1.28-1.23 (m, 2H), 0.79-0.74 (m, 2H); MS (ESI+) m/z: 464.1(M+Na)
+
.
methyl (S,E)-4-(((tert-butylsulfinyl)imino)methyl)benzoate (16a). To a solution of 14 (5.00g,
30.5mmoL) in 150mL dry DCM were added 15a (4.43g, 36.6mmoL), CsCO3 (12.9g, 36.6mmoL),
the mixture was stirred for 18h at reflux temperature, and then concentrated under reduced pressure,
then the mixture was diluted with H2O and extracted with EtOAc. The resulting mixture use
diatomite to filter. The combined organic phase was washed with brine, dried over anhydrous
Na2SO4, and then concentrated under reduced pressure The obtained residue was purified by silica
gel column chromatography (PE:Acetone=4:1) to afford 16a.1H NMR (300MHz, CDCl3) ꢀ 8.63
(s, 1H), 8.13 (d, J = 8.4Hz, 2H), 7.91 (d, J = 8.4Hz, 2H), 3.94 (s, 3H), 1.27 (s, 9H); MS (ESI+)
m/z: 290.1(M+Na) +.
Methyl (S,E)-4-(2-(tert-butylsulfinyl)vinyl)benzoate (16b). 16b was synthesized following
procedures similar to that described for compound 16a.
Methyl 4-((S)-1-(((S)-tert-butylsulfinyl)amino)ethyl)benzoate (17a). To the solution of Et2Zn
(17mL, 1M in Toluene, 17mmoL) in 50mL dry THF was added MeMgBr (15mL, 1M in Toluene,
15mmoL) at room temperature. After stirred for 30min, the mixture was cooled to -78oC, the 16a
(2.67g, 10 mmoL) in 15mL dry THF was added. The mixture was stirred for 4h at -78oC. After the
mixture was heat up to room temperature. Then the mixture was diluted with saturated and
ammonium chloride solution and extracted with EtOAc. The combined organic phase was washed
with brine, dried over anhydrous Na2SO4, and then concentrated under reduced pressure The
obtained residue was purified by silica gel column chromatography (PE: Acetone=4:1) to afford
17a. 1H NMR (300MHz, CD3Cl3 ) ꢀ 8.01 (d, J = 8.4Hz, 2H), 7.42 (d, J = 8.4Hz, 2H), 4.59 (dq, J
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= 6.6Hz, 3.3Hz, 3H), 1.23 (s, 9H); MS (ESI+) m/z: 306.1(M+Na) +.
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Methyl 4-((S)-1-(((S)-tert-butylsulfinyl)amino)-2-methylpropyl)benzoate (17b). 17b was
synthesized following procedures similar to that described for compound 36a. 1H NMR (300MHz,
CDCl3) ꢀ 8.01 (d, J = 8.4Hz, 2H), 7.41 (d, J = 8.4Hz, 2H), 4.63 (dq, J = 6.3Hz, 3.3Hz, 3H), 1.20
(s, 9H); MS (ESI+) m/z: 306.1(M+Na) +.
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Methyl 4-((R)-1-(((R)-tert-butylsulfinyl)amino)ethyl)benzoate (17c). 36c was synthesized
following procedures similar to that described for compound 36a 1H NMR (300MHz, CDCl3) ꢀ
7.99 (d, J = 6.9Hz, 2H), 7.33 (d, J = 6.9Hz, 2H), 4.19 (t, J = 5.7Hz,1H), 3.90 (s, 3H), 3.47 (d, J =
5.1Hz, 1H), 2.27-2.14 (m, 1H), 1.23 (s, 9H), 0.92 (dd, J = 6.9Hz, 3H), 0.79 (d, J = 6.9Hz, 2H);
MS (ESI+) m/z: 334.2(M+Na) +. [α]D20 =+33.4 (c 1.81, CHCl3). This sample was measured on an
Autopol VI, serial number 90079, manufactured by Rudolph Research Analytical,Hackettstown,
NJ. The literature reports that R-configuration compound [α]2D0 =-33.4 (c 1.81, CHCl3)12, 36c was
therefore confirmed as S-configuration compound.
Methyl (S)-4-(1-aminoethyl)benzoate (18a). To a stirred solution of 17a (1.18g, 4.16 mmoL) in
5mL DCM, 10mL 2N HCl/MeOH was added at 0℃, the mixture was stirred for 3h at room
temperature and then concentrated under reduced pressure. The obtained residue hen the mixture
was diluted with saturated and sodium carbonate solution and extracted with EtOAc. The
combined organic phase was washed with brine, dried over anhydrous Na2SO4, and then
concentrated under reduced pressure to obtain 18a without further purification (700mg, 94%,
yellow oil).
Methyl (S)-4-(1-amino-2-methylpropyl)benzoate(18b). 18b was synthesized following
procedures similar to that described for compound 18a.
Methyl (R)-4-(1-aminoethyl)benzoate(18c). 37c was synthesized following procedures similar
to that described for compound 18a.
Methyl (S)-4-(1-(5-cyclopropyl-[2,2'-bithiazole]-4-carboxamido)ethyl)benzoate(19a). To a
solution of the bisthiazole carboxyl acid 1b (63mg, 0.25mmoL) in 3mL dry DCM were added 18a
(50mg, 0.28mmoL) and DMAP (45mg, 0.38mmoL), respectively. The resulting mixture was
stirred for 10min at room temperature. Then the EDCI (71mg, 0.38mmoL) was added to the
resulting mixture under N2 atmosphere at 0℃. The resulting mixture was stirred for overnight at
room temperature. The obtained mixture was diluted with 1N HCl and extracted with EtOAc
(10mL×3). The combined organic phase was washed with brine, dried over anhydrous Na2SO4,
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and then concentrated under reduced pressure The obtained residue was purified by silica gel
column chromatography (PE:EtOAc=4:1) to afford 19a.（79mg, 76.5%, white solid）1H NMR
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(300MHz, CDCl3) ꢀ 8.01 (d, J = 8.1Hz, 2H), 7.82 (d, J = 3.0Hz,1H), 7.66 (d, J = 7.8Hz,1H), 7.47
(d, J = 8.1Hz, 2H), 7.41(d, J = 3.0Hz,1H), 5.37-5.27 (m, 1H), 3.88 (s, 3H), 3.46-3.37 (m, 1H), 1.62
(d, J = 7.2Hz, 3H), 1.31-1.26 (m, 2H), 0.81-0.75 (m, 2H); MS (ESI+) m/z: 436.4(M+Na)+.
19b,c was synthesized following procedures similar to that described for compound 19a.
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Methyl
(S)-4-(1-(5-cyclopropyl-[2,2'-bithiazole]-4-carboxamido)-2-
1
methylpropyl)benzoate(19b). H NMR (300MHz, CDCl3 ) ꢀ 8.02(d, J=8.4Hz, 2H), 7.85(d,
J=3.3Hz, 1H), 7.66(d, J=7.8Hz, 1H), 7.45(d, J=3.3Hz, 1H), 7.42(d, J=8.4Hz, 2H), 4.95 (t, J=8.4Hz,
2H), 3.90(s, 3H), 3.45-3.36(m, 1H), 2.26-2.15(m, 1H), 1.32-1.25(m, 2H), 1.05(d, J=6.9Hz, 3H),
0.94(d, J=6.9Hz, 2H), 0.82-0.76(m, 2H), 0.74(d, J=6.0Hz, 6H); MS (ESI) m/z: 464.1(M+Na+)
Methyl (R)-4-(1-(5-cyclopropyl-[2,2'-bithiazole]-4-carboxamido)ethyl)benzoate(19c). 1H
NMR (300MHz, CDCl3) ꢀ 8.03(d, J=8.1Hz, 2H), 7.84(d, J=3.3Hz,1H), 7.66(d, J=7.8Hz,1H),
7.49(d, J=8.1Hz, 2H), 7.43(d, J=3.3Hz,1H), 5.38-5.28(m, 1H), 3.90(s, 3H), 3.47-3.38(m, 1H),
1.64(d, J=6.9Hz, 3H), 1.32-1.28(m, 2H), 0.82-0.77(m, 2H); MS (ESI) m/z: 436.4(M+Na+)
21e-f was synthesized following procedures similar to that described for compound 19a.
Methyl
(S)-4-(1-(5-cyclopropyl-5'-methyl-[2,2'-bithiazole]-4-
carboxamido)ethyl)benzoate(21a). 1H NMR(300MHz, CDCl3) ꢀ 8.03(d, J=8.4Hz, 2H), 7.65(d,
J=7.8Hz,1H), 7.50(s,1H), 7.48(d, J=8.4Hz, 2H), 5.39-5.25(m, 1H), 3.91(s, 3H), 3.45-3.37(m, 1H),
2.53 (s, 3H), 1.63 (d, J=6.9Hz, 3H), 1.31-1.27(m, 2H), 0.82-0.76(m, 2H); MS (ESI) m/z:
450.0(M+Na+)
Methyl
(S)-4-(1-(5-cyclopropyl-4'-methyl-[2,2'-bithiazole]-4-
carboxamido)ethyl)benzoate(21b). 1H NMR (300MHz, CDCl3) ꢀ 8.03 (d, J= 8.4 Hz, 2H), 7.66
(d, J = 7.8 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 6.99 (s, 1H), 5.37–5.28 (m, 1H), 3.90 (s, 3H), 3.47–
3.38 (m, 1H), 2.48 (s, 3H), 1.64 (d, J = 7.2 Hz, 3H), 1.31–1.28 (m, 2H), 0.82–0.76 (m, 2H). MS
(ESI) m/z: 450.1(M+Na+)
Methyl
(S)-4-(1-(5-cyclopropyl-2-(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)thiazole-4-
carboxamido)ethyl)benzoate(21c). 1H NMR (300MHz, CDCl3) ꢀ 8.02 (d, J = 8.1 Hz, 2H), 7.66
(d, J = 7.8 Hz, 1H), 7.48 (d, J = 8.1 Hz, 2H), 5.36–5.27 (m, 1H), 3.90 (s, 3H), 3.45–3.36 (m, 1H),
2.81 (s, 4H), 1.88 (s, 4H), 1.63 (d, J = 6.9 Hz, 3H), 1.32–1.22 (m, 2H), 0.80–0.73 (m, 2H). MS
(ESI) m/z: 468.2 (M+H+)
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Methyl
(S)-4-(1-(5-cyclopropyl-5'-phenyl-[2,2'-bithiazole]-4-carboxamido)ethyl)benzoate
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methyl(21d). H NMR(300MHz, CDCl3) ꢀ 8.04 (d, J = 8.1 Hz, 2H), 8.00 (s, 1H), 7.67 (d, J = 7.8
Hz, 1H), 7.62 (dd, J = 8.1, 1.2 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.47–7.36 (m, 3H), 5.40–5.28
(m, 1H), 3.91 (s, 3H), 3.49–3.38 (m, 1H), 1.66 (d, J = 7.2 Hz, 3H), 1.34–1.28 (m, 2H), 0.83–0.78
(m, 2H). MS (ESI) m/z: 490.1(M+H+)
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(S)-4-(1-(5-Cyclopropyl-5'-(4-fluorophenyl)-[2,2'-bithiazole]-4-
carboxamido)ethyl)benzoate(21e). 1H NMR(300MHz, CDCl3) ꢀ 8.03 (d, J = 8.1 Hz, 2H), 7.73
(s, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.60–7.56 (m, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.13 (t, J = 8.4 Hz,
2H), 5.39–5.29 (m, 1H), 3.91 (s, 3H), 3.47–3.38 (m, 1H), 1.65 (d, J = 6.9 Hz, 3H), 1.33–1.28 (m,
2H), 0.82–0.78 (m, 2H). MS (ESI) m/z: 508.1(M+H+)
Methyl
(S)-4-(1-(5-cyclopropyl-5'-(4-methoxyphenyl)-[2,2'-bithiazole]-4-
1
carboxamido)ethyl)benzoate (21f). H NMR ( 300MHz, CDCl3 ) ꢀ 8.04 (d, J = 8.4 Hz, 2H),
7.89 (s, 1H), 7.68 (d, J = 8.1Hz, 1H), 7.54 (d, J = 9.0 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 6.98–6.92
(d, J = 9.0 Hz, 2H), 5.39–5.29 (m, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 3.47–3.38 (m, 1H), 1.65 (d, J =
6.9 Hz, 3H), 1.34–1.26 (m, 2H), 0.84–0.77 (m, 2H).
N-hydroxy-1-(5-isobutyl-[2,2'-bithiazole]-4-carbonyl)piperidine-4-carboxamide (H2). To a
solution of 7(82mg, 0.21mmol) in 6mL DCM and 3mL MeOH add DBU (63mg, 0.42mmol)，
then add hydroxylamine (50 wt. % in water) 0.3mL. The resulting mixture was stirred for 12 hours
at 30oC. Then the mixture was acidified with 1N HCl to pH = 5.0-6.0, concentrated under reduced
pressure. The obtained residue was dissolved in EtOAc. This EtOAc solution was washed with
brine, dried over anhydrous Na2SO4 and then concentrated under reduced pressure. The obtained
residue was purified by silica gel column chromatography (CH2Cl2:MeOH=10:1) to afford
compound H2 (pale yellow solid, 69.2% ) 1H NMR (400MHz, DMSO-d6) ꢀ 10.49 (s, 1H), 8.73
(s, 1H), 7.99 (d, J = 4Hz, 1H), 7.94 (d, J = 4Hz, 1H), 4.50 (d, J = 12Hz, 1H), 3.66 (d, J = 12Hz,
1H), 3.07(t, J = 12Hz, 1H), 2.81-2.74 (m, 3H), 2.34-2.28 (m, 1H), 1.92-1.86 (m, 1H), 1.76-1.69
(m, 1H), 1.64-1.60 (m, 1H), 1.57-1.51 (m, 1H), 0.92 (d, J=8Hz, 6H); 13C NMR (100MHz, DMSO-
d6) ꢀ 171.2, 162.7, 160.7, 157.9, 147.7, 144.7, 141.5, 123.4, 46.5, 41.4, 35.1, 30.8, 29.3, 28.6, 22.4,
19.9. HRMS (TOF ESI) calcd for C17H22N4O3S2 [M+H]: 395.1206, [M+Na]: 417.1026;
found:395.1202, 417.1025. HPLC analysis: retention time = 7.564 min; peak area, 99.76%; eluent
A, 0.1% TFAin water; eluent B, CH3OH; isocratic (1:1) over 30min with a flow rate of 1 mL min−1.
H3-15 was synthesized following procedures similar to that described for compound H2
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N-(4-(hydroxycarbamoyl)phenyl)-5-isobutyl-[2,2'-bithiazole]-4-carboxamide (H3). 1H NMR
(300MHz, CD3OD+CDCl3): δ 9.45(s, 1H), 7.60(d, J=3.3Hz, 1H), 7.49(s, 4H), 7.35(d, J=3.3Hz,
1H), 2.99(d, J=5.7Hz, 2H), 1.80-1.71(m, 1H), 0.72(d, J=9.0Hz, 6H). 6 (m, 1H), 1.76-1.69 (m, 1H),
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1.64-1.60 (m, 1H), 1.57-1.51 (m, 1H), 0.92 (d, J=8Hz, 6H); C NMR (125MHz, DMSO-d6) ꢀ
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163.3, 160.7, 160.2, 157.1, 147.8, 145.0, 144.6, 141.3, 128.4, 128.0, 124.0, 120.2, 35.4, 31.0, 22.5.
HRMS (TOF ESI) calcd for C18H18N4O3S2 [M+H]: 403.0893, [M+Na]: 425.0713; found:
403.0891, 425.0716. HPLC analysis: retention time = 6.308min; peak area, 97.73%; eluent A, 0.1%
TFA in water; eluent B, CH3OH; isocratic (1:1) over 30min with a flow rate of 1 mL min−1.
(E)-N-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)phenyl)-5-isobutyl-[2,2'-bithiazole]-4-
1
carboxamide (H4). H NMR (400MHz, DMSO-d6) : δ10.74 (s, 1H), 10.30 (s, 1H), 9.04 (s,1H),
8.03 (s,2H), 7.89 (d, J = 8Hz, 2H), 7.47 (d, J = 16Hz, 1H), 6.45 (d, J = 16Hz, 1H), 3.23 (d, J =
13
8Hz, 2H), 2.01-1.98 (m, 1H), 1.76 (d, J=12Hz, 1H), 0.96 (d, J=4Hz, 6H); C NMR (100MHz,
DMSO-d6) δ 163.5, 160.5, 160.2, 157.1, 147.7, 145.0, 144.6, 139.9, 138.4, 130.8, 128.5, 124.0,
121.0, 118.2, 35.4, 31.0, 22.5, 21.8, 15.0; HRMS (TOF ESI) calcd for C20H20N4O3S2 [M+H]:
429.1050, [M+Na]: 451.0869; found: 429.1059, 451.0869. HPLC analysis: retention time = 5.909
min; peak area, 96.28%; eluentA, 0.1% TFAin water; eluent B, CH3OH; isocratic (1:1) over 30min
with a flow rate of 1 mL min−1.
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5-Cyclopropyl-N-(4-(hydroxycarbamoyl)benzyl)-[2,2'-bithiazole]-4-carboxamide (H5). H
NMR (500MHz, DMSO-d6): δ 11.18 (s, 1H), 9.00 (s, 1H), 8.96-8.93 (m, 1H), 7.98-7.96 (m,2H),
7.54 (d, J = 10Hz, 2H), 7.42 (d, J = 5Hz, 2H), 4.54 (d, J = 10Hz, 2H), 3.37-3.34 (m, 1H), 1.31-
13
1.27 (m, 2H), 0.82-0.79 (m, 2H), C NMR (125MHz, DMSO-d6) δ 164.6, 162.1, 160.3, 154.7,
153.8, 144.7, 144.5, 143.4, 131.8, 127.7, 127.4, 123.7, 42.5, 14.3, 10.6. HRMS (TOF ESI) calcd
for C18H16N4O3S2 [M+H]: 401.0737, [M+Na]: 423.0556; found: 401.0739, 423.0558. HPLC
analysis: retention time = 7.078 min; peak area, 98.95%; eluent A, 0.1% TFA in water; eluent B,
CH3OH; isocratic (1:1) over 30min with a flow rate of 1 mL min−1.
5-Cyclopropyl-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-[2,2'-bithiazole]-4-carboxamide
1
(H6). H NMR ( 300MHz, DMSO-d6 ) ꢀ 11.15 (s, 1H), 8.99 (s, 1H), 8.60 (d, J = 7.5Hz, 2H),
8.00-7.97 (m, 2H), 7.72 (d, J = 7.8Hz, 2H), 7.50 (d, J = 7.8Hz, 2H), 5.22-5.17 (m, 1H), 3.25-3.21
(m, 1H), 1.54 (d, J = 6.9Hz, 3H), 1.27-1.24 (m, 2H), 0.79-0.77 (m, 2H); MS (ESI+) m/z: 415.1
(M+H) +.
(S)-5-Cyclopropyl-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-[2,2'-bithiazole]-4-
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carboxamide(H6a). 1H NMR(300MHz, DMSO-d6) ꢀ 11.17(s, 1H), 9.01 (s, 1H), 8.63(d, J=8.4Hz,
1H), 7.98 (s, 2H), 7.71(d, J=8.4Hz, 2H),7.50(d, J=8.4Hz, 2H), 5.24-5.14(m, 1H), 3.27-3.20(m,
1H), 1.55(d, J=6.9Hz, 3H), 1.29-1.20(m, 2H), 0.81-0.75(m, 2H); MS (ESI) m/z: 437.1(M+Na+)
(S)-5-Cyclopropyl-N-(1-(4-(hydroxycarbamoyl)phenyl)-2-methylpropyl)-[2,2'-bithiazole]-4-
carboxamide(H6b). 1H NMR(300MHz, DMSO-d6) ꢀ 11.15(s, 1H), 9.00 (s, 1H), 8.52(d, J=8.7Hz,
1H), 7.97(s, 2H), 7.72(d, J=7.8Hz, 2H), 7.51(d, J=7.8Hz, 2H), 4.72(t, J=9.0Hz, 1H), 3.20-3.14(m,
1H), 2.35-2.21(m, 1H), 1.25-1.22(m, 2H), 1.03-1.01(m,2H), 0.77(d, J=6.0Hz, 6H); 13C NMR
(100MHz, DMSO-d6) ꢀ 161.5, 160.3, 154.8, 153.0, 151.2, 145.2, 144.6, 143.7, 132.5, 127.6, 125.8,
123.6, 59.5, 32.6, 20.4, 19.9, 14.2, 14.1, 10.4; MS (ESI) m/z: 465.1(M+Na+) HPLC analysis:
retention time = 4.445 min; peak area, 98.60%; eluent A, 0.1% TFA in water; eluent B, CH3OH;
isocratic (1:1) over 30min with a flow rate of 1 mL min−1.
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5-Cyclopropyl-N-(1-(4-(hydroxycarbamoyl)phenyl)cyclopropyl)-[2,2'-bithiazole]-4-
1
carboxamide (H7). H NMR (400MHz, DMSO-d6) ꢀ 11.13 (s, 1H), 9.07 (s, 1H), 8.96 (s, 1H),
7.97 (s, 1H) , 7.67 (d, J = 8.0Hz, 2H), 7.29 (d, J = 8.0Hz, 2H), 3.27-3.23 (m, 1H), 1.41-1.38 (m,
4H), 1.33-1.25 (m, 2H), 0.82-0.78 (m, 2H);13C NMR (100MHz, DMSO-d6) ꢀ 162.7, 160.3, 154.6,
153.8, 147.3, 144.9, 144.5, 130.6, 127.2, 125.1, 123.8, 34.8, 19.3, 14.4, 10.5. HRMS (TOF ESI)
calcd for C20H18N4O3S2 [M+H]: 427.0893, [M+Na]: 449.0713; found: 427.0891, 449.0708. HPLC
analysis: retention time = 4.535 min; peak area, 96.49%; eluent A, 0.1% TFA in water; eluent B,
CH3OH; isocratic (1:1) over 30min with a flow rate of 1 mL min−1.
4-(1-(5-Cyclopropyl-[2,2'-bithiazol]-4-yl)-2-methyl-1-oxopropan-2-yl)-N-hydroxybenzamide
(H8).1H NMR (500MHz, DMSO-d6) ꢀ 11.15 (s, 1H), 8.99 (s, 1H), 8.16 (s, 1H), 7.99 (s, 2H), 7.71
(d, J=10Hz, 2H), 7.51 (d, J=10Hz, 2H), 3.16-3.12 (m, 1H), 1.74 (s, 6H), 1.26-1.23 (m, 2H), 0.79-
0.77 (m, 2H); 13C NMR (125MHz, DMSO-d6) ꢀ 164.7, 161.1, 160.2, 154.6, 153.2, 151.0, 145.2,
144.6, 131.2, 127.2, 125.3, 123.6, 55.7, 29.6, 14.3, 10.4; HRMS (TOF ESI) calcd for
C20H20N4O3S2 [M+H]: 429.1050, [M+Na]: 451.0869; found: 429.3191, 451.0868. HPLC analysis:
retention time = 4.090 min; peak area, 98.68%; eluent A, 0.1% TFA in water; eluent B, CH3OH;
isocratic (1:1) over 30min with a flow rate of 1 mL min−1.
(R)-5-Cyclopropyl-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-[2,2'-bithiazole]-4-
carboxamide(H9). 1H NMR(400MHz, DMSO-d6) ꢀ 11.15(s, 1H), 8.99 (s, 1H), 8.61(d, J=8.0Hz,
1H), 7.97 (s, 2H), 7.72(d, J=8.0Hz, 2H), 7.50(d, J=8.1Hz, 2H), 5.24-5.17(m, 1H), 3.33-3.19(m,
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d6) ꢀ 164.6, 161.4, 160.3, 154.7, 153.5, 148.1, 145.0, 144.5, 131.8, 127.4, 126.7, 123.7, 48.4, 22.1,
14.2, 10.5; HRMS (TOF ESI) calcd for C21H22N4O3S2 [M+H]: 415.0893, [M+Na]: 437.0713;
found: 415.0885, 437.0710. HPLC analysis: retention time = 4.303 min; peak area, 98.78%; eluent
A, 0.1% TFAin water; eluent B, CH3OH; isocratic (1:1) over 30min with a flow rate of 1 mL min−1.
(S)-5-Cyclopropyl-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-5'-methyl-[2,2'-bithiazole]-4-
carboxamide(H10). (45mg, 35.4%, pale yellow solid.1H NMR (400MHz, DMSO-d6) ꢀ 9.56 (s,
1H), 8.45 (d, J = 4 Hz 1H), 7.64 (m, 3H), 7.42 (d, J = 4 Hz, 2H), 5.17 (m, 1H), 3.22 (m, 1H), 2.52
(s, 3H),1.54 (d, J = 4 Hz, 3H), 1.26-1.24 (m, 2H), 0.77–0.76 (m, 2H). 13C NMR (125MHz, DMSO-
d6) ꢀ 161.3, 158.4, 155.0, 152.8, 151.2, 145.5, 144.9, 142.5, 137.7, 132.5, 126.5, 125.9, 48.3, 22.2,
14.1, 12.2, 10.4. HRMS (TOF ESI) calcd for C20H20N4O3S2 [M+H]: 429.1050, [M+Na]: 451.0869;
found: 428.9721, 451.0864. HPLC analysis: retention time = 4.563min; peak area, 99.59%; eluent
A, 0.1% TFAin water; eluent B, CH3OH; isocratic (1:1) over 30min with a flow rate of 1 mL min−1.
(S)-5-Cyclopropyl-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-4'-methyl-[2,2'-bithiazole]-4-
carboxamide(H11). 1H NMR (400MHz, DMSO-d6) ꢀ 9.56 (s, 1H), 8.50 (d, J = 4 Hz, 1H), 7.63(d,
J = 4 Hz, 2H), 7.52 (s, 1H), 7.42 (d, J = 4 Hz, 2H), 5.18 (m, 1H), 3.23 (m, 1H), 2.41 (s, 3H), 1.54
(d, J = 4 Hz, 3H), 1.26 – 1.24 (m, 2H), 0.78 – 0.77 (m, 2H). 13C NMR (125MHz, DMSO-d6) ꢀ
161.3, 159.3, 154.8, 154.0, 153.2, 151.2, 145.6, 145.0, 132.4, 126.5, 125.9, 118.0, 48.3, 22.1, 17.1,
14.2, 10.5. HRMS (TOF ESI) calcd for C20H20N4O3S2 [M+H]: 429.1050, [M+Na]: 451.0869;
found: 429.1051, 451.0869. HPLC analysis: retention time = 4.472 min; peak area, 97.58%; eluent
A, 0.1% TFAin water; eluent B, CH3OH; isocratic (1:1) over 30min with a flow rate of 1 mL min−1.
(S)-5-Cyclopropyl-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-2-(4,5,6,7-
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tetrahydrobenzo[d]thiazol-2-yl)thiazole-4-carboxamide(H12). H NMR (400MHz, DMSO-
d6) ꢀ 11.16 (s, 1H), 8.99 (s, 1H), 8.52 (d, J=8.0Hz, 1H), 7.72 (d, J=8.0Hz, 2H), 7.49 (d, J=8.0Hz,
2H), 5.20-5.16 (m, 1H), 3.23-3.18 (m, 1H) , 2.85-2.82 (m, 2H), 2.74-2.71 (m, 2H), 1.82 (s, 3H),
1.54 (d, J=8.0Hz, 3H), 1.25–1.21 (m, 2H), 0.78–0.74 (m, 2H). 13C NMR (100MHz, DMSO-d6) ꢀ
164.6, 161.4, 156.3, 155.1, 152.9, 152.0, 148.1, 144.8, 132.8, 131.8, 127.4, 126.7, 48.4, 26.6, 23.6,
23.0, 22.7, 22.1, 14.1, 10.4; HRMS (TOF ESI) calcd for C23H24N4O3S2 [M+H]: 469.1363, [M+Na]:
491.1182; found: 469.1363, 491.1179. HPLC analysis: retention time = 12.040 min; peak area,
97.37%; eluent A, 0.1% TFA in water; eluent B, CH3OH; isocratic (1:1) over 30min with a flow
rate of 1 mL min−1.
(S)-5-Cyclopropyl-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-5'-phenyl-[2,2'-bithiazole]-4-
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carboxamide(H13). H NMR (300MHz, DMSO-d6) ꢀ 11.16 (s, 1H), 9.00 (s, 1H), 8.62 (d,
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(m, 1H), 1.56 (d, J=7.2Hz, 3H), 1.28–1.23 (m, 2H), 0.81–0.79 (m, 2H). C NMR (125MHz,
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DMSO-d6) ꢀ 164.6, 161.3, 158.8, 154.5, 153.7, 148.1, 145.1, 141.6, 140.7, 131.8, 130.6, 129.9,
129.6, 127.4, 127.1, 126.7, 48.4, 22.1, 14.3, 10.6. HRMS (TOF ESI) calcd for C25H22N4O3S2
[M+H]: 491.1206, [M+Na]: 513.1026; found: 491.1202, 513.1029. [α]2D0 =+54.7 (c 1.92, CHCl3).
This sample was measured on an Autopol VI, serial number 90079, manufactured by Rudolph
Research Analytical, Hackettstown. HPLC analysis: retention time = 12.582 min; peak area,
97.65%; eluent A, 0.1% TFA in water; eluent B, CH3OH; isocratic (1:1) over 30min with a flow
rate of 1 mL min−1.
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(S)-5-Cyclopropyl-5'-(4-fluorophenyl)-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-[2,2'-
bithiazole]-4-carboxamide(H14). 1H NMR (500MHz, DMSO-d6) ꢀ 11.17(s, 1H), 9.00(s, 1H),
8.61(d, J=10 Hz, 1H), 8.37(s, 1H), 7.84(d, J=10 Hz, 2H), 7.75(d, J=5Hz, 2H), 7.52 (d, J=10Hz,
2H), 7.37-7.34(m, 2H), 5.23–5.20 (m, 1H), 3.27–3.23(m, 1H), 1.57 (d, J=10Hz, 3H), 1.30–1.26
(m, 2H), 0.81–0.80 (m, 2H). 13C NMR (125MHz, DMSO-d6) ꢀ 164.6, 161.9, 161.3, 158.8, 154.4,
153.7, 148.1, 145.1, 140.8, 140.5, 131.8, 129.4, 129.3, 127.4, 127.2, 126.7, 117.0, 116.8, 48.4,
22.2, 14.3, 10.5. HRMS (TOF ESI) calcd for C25H21FN4O3S2 [M+H]: 509.1112, [M+Na]:
531.0931; found: 509.1108, 531.0930. HPLC analysis: retention time = 5.230 min; peak area,
95.16%; eluent A, 0.1% TFA in water; eluent B, CH3OH; isocratic (1:1) over 30min with a flow
rate of 1 mL min−1.
(S)-5-Cyclopropyl-N-(1-(4-(hydroxycarbamoyl)phenyl)ethyl)-5'-(4-methoxyphenyl)-[2,2'-
bithiazole]-4-carboxamide(H15). 1H NMR (500MHz, DMSO-d6) ꢀ 11.17(s, 1H), 9.00(s, 1H),
8.59 (d, J =10Hz, 1H), 8.26 (s, 1H), 7.75-7.70(m, 4H), 7.52 (d, J = 10Hz, 2H), 7.06(d, J = 10Hz,
2H), 5.23–5.21(m, 1H), 3.82(s, 3H), 3.27–3.24 (m, 1H), 2.51(d, J = 10Hz, 3H), 1.29–1.25 (m, 2H),
0.82–0.79 (m, 2H). 13C NMR (125MHz, DMSO-d6) ꢀ 164.6, 161.3, 160.4, 157.7, 154.6, 153.4,
148.1, 145.0, 141.8, 139.5, 131.8, 128.6, 127.4, 126.7, 123.1, 115.4, 55.8, 48.4, 22.1, 14.3, 10.5;
HRMS (TOF ESI) calcd for C26H24N4O3S2 [M+H]: 521.1312, [M+Na]: 531.0931; found:
521.1309, 543.1131. HPLC analysis: retention time = 5.129 min; peak area, 95.74%; eluent A, 0.1%
TFA in water; eluent B, CH3OH; isocratic (1:1) over 30min with a flow rate of 1 mL min−1.
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.
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Additional information for the HDAC Inhibition Assay, pharmacokinetic and distribution study of
compound H13 and descriptions of the colitis-associated colorectal cancer model, docking study，
as well as NMR spectrums and HPLC spectrums for compounds H2-15, experimental procedures
and characterization of new chemical entities (PDF)
Molecular string files for all the final target compounds (CSV)
AUTHOR INFORMATION
Corresponding Author
* Email: fjnan@simm.ac.cn(F.N)
* Email: ychen@simm.ac.cn(Y.C)
* Email: jli@simm.ac.cn(J.L)
ACKNOWLEDGMENTS
Funding Sources
This work was supported by grants from The National Natural Science Foundation of China
(30725049), the National Science and Technology Major Project (2018ZX09711002-011-006),
the "Personalized Medicines——Molecular Signature-based Drug Discovery and Development",
Strategic Priority Research Program of the Chinese Academy of Sciences, Grant No.
XDA12020106 , and Shanghai Science and Technology Committee (No. 19431906400).
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