Efficient Synthesis and Bioevaluation of Novel Dual Tubulin/Histone Deacetylase 3 Inhibitors as Potential Anticancer Agents

Article abstract of DOI:10.1021/acs.jmedchem.1c00413

Novel dual HDAC3/tubulin inhibitors were designed and efficiently synthesized by combining the pharmacophores of SMART (tubulin inhibitor) and MS-275 (HDAC inhibitor), among which compound 15cwas found to be the most potent and balanced HDAC3/tubulin dual inhibitor with high HDAC3 activity (IC₅₀= 30 nM) and selectivity (SI > 1000) as well as excellent antiproliferative potency against various cancer cell lines, including an HDAC-resistant gastric cancer cell line (YCC3/7) with IC₅₀values in the range of 30-144 nM. Compound 15cinhibited B16-F10 cancer cell migration and colony formation. In addition, 15cdemonstrated significantin vivoantitumor efficacy in a B16-F10 melanoma tumor model with a better TGI (70.00%, 10 mg/kg) than that of the combination of MS-275 and SMART. Finally, 15cpresented a safe cardiotoxicity profile and did not cause nephro-/hepatotoxicity. Collectively, this work shows that compound 15crepresents a novel tubulin/HDAC3 dual-targeting agent deserving further investigation as a potential anticancer agent.

Full text of DOI:10.1021/acs.jmedchem.1c00413

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Article
Efficient Synthesis and Bioevaluation of Novel Dual Tubulin/Histone
Deacetylase 3 Inhibitors as Potential Anticancer Agents
Xiaopeng Peng,† Jingxuan Chen,† Ling Li, Zhiqiang Sun, Jin Liu, Yichang Ren, Junli Huang,
and Jianjun Chen*
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ABSTRACT: Novel dual HDAC3/tubulin inhibitors were designed and efficiently synthesized by combining the pharmacophores
of SMART (tubulin inhibitor) and MS-275 (HDAC inhibitor), among which compound 15c was found to be the most potent and
balanced HDAC3/tubulin dual inhibitor with high HDAC3 activity (IC50 = 30 nM) and selectivity (SI > 1000) as well as excellent
antiproliferative potency against various cancer cell lines, including an HDAC-resistant gastric cancer cell line (YCC3/7) with IC50
values in the range of 30−144 nM. Compound 15c inhibited B16-F10 cancer cell migration and colony formation. In addition, 15c
demonstrated significant in vivo antitumor efficacy in a B16-F10 melanoma tumor model with a better TGI (70.00%, 10 mg/kg) than
that of the combination of MS-275 and SMART. Finally, 15c presented a safe cardiotoxicity profile and did not cause nephro-/
hepatotoxicity. Collectively, this work shows that compound 15c represents a novel tubulin/HDAC3 dual-targeting agent deserving
further investigation as a potential anticancer agent.
(SAHA, Figure 1) is used clinically in patients with central T
cell lymphoma. In addition, benzamide containing HDAC
inhibitor 2 (MS-275, Figure 2), a highly effective and selective
inhibitor of HDAC1 and HDAC3, is currently under
investigation in clinical trials to treat advanced multiple
myeloma.23 Despite the great success HDAC inhibitors have
enjoyed clinically, and their further use was hindered by the
almost inevitable drug-resistance and dose-limiting adverse
effects (e.g., cardiotoxicity).24−26 Moreover, current HDAC
inhibitors have not shown significant efficacy against solid
tumors.27 Therefore, the search for new strategies that can
potentially overcome these challenges has been intensified in
the past decade, for example, the development of isoform-
selective HDAC inhibitors, which have the potential to reduce
toxicities associated with pan-HDAC inhibitors. In particular,
1. INTRODUCTION
Epigenetic regulation is essential to many biological processes
and is associated with the onset and progression of various
human diseases such as cancer.1,2 Among the numerous
epigenetic enzymes, histone deacetylases (HDACs) have been
extensively explored for decades due to their significance in a
variety of diseases, including cancer,3−6 inflammation,7 neuro-
logical disorders,8,9 and infections.10 HDACs are grouped into
four classes based on their homology: class I (HDACs 1, 2, 3,
and 8), class II (HDACs 4, 5, 6, 7, 9, and 10), and class IV
(HDAC 11), which are enzymatically active via a Zn2+-
dependent mechanism, whereas class III HDACs (SIRT1-7)
are NAD-dependent and are associated with aging.11
Overexpression of HDACs is commonly observed in
different types of cancers such as liver, breast, lung, and gastric
cancers.12,13 Therefore, HDACs are regarded as promising
antitumor drug targets. So far, HDAC inhibitors (HDACi)
have achieved great success as cancer therapeutics for the
treatment of various cancers.14,15 To date, five HDAC
inhibitors (1-SAHA, 2-Belinostat, 3-Panobinostat, 4-Romidep-
sin, and 5-Chidamide, Figure 1) have been approved by FDA
for treating hematologic malignancies.16−22 Among them, 1
Received: March 8, 2021
Published: June 7, 2021
https://doi.org/10.1021/acs.jmedchem.1c00413
© 2021 American Chemical Society
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Figure 1. Pharmacophore model of marketed HDAC inhibitors.
Figure 2. Design of tubulin/HDAC dual inhibitors. (A) Docking analysis of SMART in the colchicine-binding site of tubulin (PDB: 4O2B). (B)
90° clockwise rotation of tubulin in complex with SMART along the x-axis. (C) Design strategy of SMART-based HDAC and Tubulin dual
inhibitors.
HDAC3 is an HDAC isoenzyme that plays an important role
in cancer, inflammation, and neurodegenerative diseases.
Several reports have demonstrated the significant role of
HDAC3 and its downstream signaling pathways in cancer.28,29
Therefore, the development of selective HDAC3 inhibitors
would be of great importance for cancer treatment. In addition
to the development of isoform-selective HDAC inhibitors,
combination therapies using HDACi and other anticancer
agents (e.g., kinase inhibitors, HSP90 inhibitors, tubulin
inhibitors) are also developed to enhance the antitumor
efficacies.30−32 However, the polypharmacological nature of
drug cocktails is often associated with poor patient compliance,
complex dosing, unpredictable pharmacokinetics and pharma-
codynamics, and drug−drug interactions.33,34
An alternative strategy is to develop single molecules with
multitargeting capabilities, which may provide more robust and
durable therapeutic effects as well as benign toxicity
profiles.35−37 In the past decade, numerous HDAC-based
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a
Scheme 1. Novel procedure to SMART analogues
a
Reagents and Conditions: (a) i: NaNO2, HCl, THF, 0 °C; ii: KI, 92%; (b) CuI, PdCl2(PPh3)2, triethylamine, N,N-dimethylformamide (DMF), 90
°C, 74%; (c) RuCl3, PhI(OAc)2, dichloromethane (DCM)/H2O = 4/1, rt, 64%; (d) CuBr2, CHCl3/ethyl acetate = 1/2, 78 °C, 98%; and (e)
EtOH, 87 °C, 89−96%.
a
Scheme 2. Synthesis of Compounds 10a−i and 12a−h
a
Reagents and Conditions: (a) K2CO3, DMF, 80 °C, 73%; (b) LiOH, MeOH/H2O, rt, 100%; (c) o-phenylenediamine, EDCI, HOBt,
triethylamine, DMF, rt, 93%; (d) NH2OTHP, EDCI, HOBt, triethylamine, DMF, rt, 88%; and (e) HCl/1,4-doxane, DCM, rt, 96%
multitargeting antitumor agents have been discovered,38−45
some of them have entered clinical trials; for instance, CUDC-
101, a small molecule EGFR/HDAC dual inhibitor currently
undergoing phase I clinical trials, showed excellent antitumor
efficacy in a variety of preclinical models.46,47
It has been reported that the combination of SAHA and
vincristine (a tubulin inhibitor) could achieve synergistic
effects in leukemia,48 which provides a theoretical basis for
designing dual tubulin/HDAC inhibitors. Several groups have
reported the discovery of novel tubulin/HDAC dual inhibitors
in recent years.30,49−53 For instance, Diana and co-workers
designed dual tubulin/HDAC inhibitors that demonstrated
potent anticancer activities.54 Despite these advancements, the
therapeutic value of dual tubulin/HADC inhibitors (in
particular, dual inhibitors with HDAC-isoform-selectivity)
remains to be further explored.
Microtubules, composed of α/β-tubulin, play a key role in
multiple cellular processes such as mitotic spindle formation
and are regarded as an important target for anticancer
therapeutics.55−58 We have previously reported several types
of tubulin inhibitors targeting the colchicine-binding site with
high anticancer activities, for example, SMART, ABI, and
SAI.59−61 We also explored tubulin-based dual-targeting
molecules recently by designing a series of novel tubulin/
PD-L1 inhibitors, which showed good target engagement and
excellent antitumor activities in vivo.62 In a continuation of our
research interest in tubulin-based dual-targeting strategies, we
designed tubulin/HDAC3 dual inhibitors based on SMART
and MS-275. We confirmed for the first time that these dual-
targeting compounds exhibited more potent anticancer
activities in vitro and in vivo than the drug combination of
single-target compounds, as detailed below.
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a
Scheme 3. Synthesis of Compounds 15a−i and 17a−i
a
Reagents and Conditions: (a) EDCI, HOBt, triethylamine, DMF, rt, 95%; (b) LiOH, MeOH/H2O, rt, 100%; (c) o-phenylenediamine, EDCI,
HOBt, Triethylamine, DMF, rt, 91%; (d) NH2OTHP, EDCI, HOBt, triethylamine, DMF, rt, 85%; and (e) HCl/1,4-dioxane, DCM, rt, 97%
a
Scheme 4. Synthesis of Compounds 20a−c and 22a−c
a
Reagents and Conditions: (a) KI, K2CO3, MeCN, 59%; (b) LiOH, MeOH/H2O, rt, 100%; (c) o-phenylenediamine, EDCI, HOBt, triethylamine,
DMF, rt, 93%; (d) NH2OTHP, EDCI, HOBt, triethylamine, DMF, rt, 87%; and (e) HCl/1,4-dioxane, DCM, rt, 91%
variety of structures and to which the CAP group of HDAC
inhibitors bind. Therefore, it is conceivable that the CAP
moiety in the pharmacophore model of HDAC inhibitors
could be substituted by SMART to generate new classes of
tubulin/HDAC molecules. Based on the above analysis, we
designed two series of target compounds by combining
SMART with the zinc-binding group (ZBG) of SAHA or
MS-275, as shown below in Figure 2C.
2. RESULTS AND DISCUSSION
2.1. Structure-Based Design of Novel Tubulin/HDAC
Dual Inhibitors. The rationale for designing new tubulin/
HDAC dual inhibitors commenced by analyzing the docking
poses of SMART in the colchicine-binding site of tubulin
[PDB ID: 4O2B].63 As shown in Figure 2A,B, the para-
position on the phenyl A-ring of the SMART core is directly
exposed to the solvent, thus could serve as the reaction site for
conjugating via a linker with the HDAC pharmacophore (e.g.,
the hydroxamic acid moiety). Our previous structure−activity
relationship studies of SMART also indicated that different
substituents, including bulky groups at the phenyl A-ring, were
well tolerated.61 In addition, at the binding pocket of HDACs,
there is a large hydrophobic domain that can accommodate a
3. CHEMISTRY
The approach we previously used for the synthesis of SMART
was based on a five-step process, which includes the synthesis
of the Weinreb amides followed by reacting with appropriate
lithium reagents or Grignard reagents under harsh reaction
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a
Scheme 5. Synthesis of Compounds 25a and 27a
a
Reagents and Conditions: (a) CuI, PdCl2(PPh3)2, triethylamine, DMF, 90 °C, 71%; (b) LiOH, MeOH/H2O, rt, 100%; (c) o-phenylenediamine,
EDCI, HOBt, triethylamine, DMF, rt, 96%; (d) NH2OTHP, EDCI, HOBt, triethylamine, DMF, rt, 89%; and (e) HCl/1,4-dioxane, DCM, rt, 94%
a
Scheme 6. Synthesis of Compounds 30a and 32a
a
Reagents and Conditions: (a) Pd(OAc)2, PPh3, triethylamine, DMF, 110 °C, 52%; (b) LiOH, MeOH/H2O, rt, 100%; (c) o-phenylenediamine,
EDCI, HOBt, triethylamine, DMF, rt, 91%; (d) NH2OTHP, EDCI, HOBt, triethylamine, DMF, rt, 83%; and (e) HCl/1,4-dioxane, DCM, rt, 94%
conditions (−78 °C, strictly waterless).61 Although the method
is efficient to prepare various SMART analogues for initial
biological screening, the harsh reaction conditions and the use
of highly water-reactive/flammable/explosive organo-metallic
reagents limited the preparation of SMART and analogues at a
large scale. Therefore, more practical strategies for accessing
structurally diversified SMART are still desirable. Hence, we
developed a new procedure for the efficient synthesis of
SMART and the key SMART analogues (7a/7b/7c) as
illustrated in Scheme 1. First, commercially available 3,4,5-
trimethoxyaniline 1 was converted to 3,4,5-trimethoxy
iodobenzene 2 under Sandmeyer reaction conditions. Second,
a Sonogashira coupling of intermediate 2 with propyne
afforded intermediate 3 in high yields (74%). Subsequently,
the oxidation of the alkyne group of 3 using hyper-iodonium
reagents provided the diketone derivative 4, followed by
treatment with CuBr2 to obtain the brominated intermediate 5,
which was cyclized with various substituted-thiobenzamides 6
to produce the key SMART analogues (7a/7b/7c) with 39−
41% overall yield, higher than that (16−18%) of our previous
procedure to obtain SMART compounds.
The syntheses of target compounds 10a−i and 12a−h were
carried out following the synthetic route outlined in Scheme 2.
The reaction of the phenol group of SMART analogue 7a with
halo-substituted esters yielded the corresponding alkyl aryl
ethers 8 in high yield (73%). Hydrolysis of the alkyl ester of 8
generated the respective carboxylic acids, followed by coupling
with o-phenylenediamine to give target compounds 10a−i or
with NH2OTHP to obtain amides 11. The removal of the
THP-protecting group by hydrochloric acid provided target
compounds 12a−h.
The preparation of target compounds 15a−i and 17a−i is
illustrated in Scheme 3. Amidation of the amine group of
SMART analogue 7b with carboxylate, using EDCI/HOBt as
the coupling reagent, led to the formation of ester
intermediates 13, which were converted to the corresponding
acids 14 in the presence of LiOH solution. Using a similar
protocol shown in Scheme 2, target compounds (15a−i and
17a−i) were prepared according to the procedures outlined in
Scheme 3.
The procedure for synthesizing target compounds 20a−c
and 22a−c is depicted in Scheme 4. First, coupling of SMART
analogue 7b with haloalkane esters afforded the corresponding
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a
Table 1. Antiproliferative Activity of Target Compounds against Four Cancer Cell Lines (IC50, μM)
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Table 1. continued
a
IC50 values are the mean of at least three independent assays, presented as mean standard deviation (SD).
esters 18, which were hydrolyzed to the acid intermediates 19,
followed by a similar reaction shown in Schemes 2 and 3 to
produce target compounds 20a−c and 22a−c.
analogue 7c with methyl acrylate, utilizing the Mizoroki−Heck
cross-coupling reaction. The acrylate compound 28 was
transformed into target compounds 30a and 32a using a
procedure similar to that described for compounds 25a and
27a.
The target compounds 25a, 27a, 30a, and 32a were
synthesized according to the procedures presented in Schemes
5 and 6. In Scheme 5, coupling of the bromo-substituted
SMART analogue 7c with terminal alkyne esters resulted in the
formation of the corresponding esters 23 with good yield
(71%) under standard Sonogashira coupling conditions.
Hydrolysis of 23 gave the carboxylic acid 24, which was
condensed with o-phenylenediamine to afford target com-
pound 25a. In addition, intermediate 24 could also undergo a
coupling reaction with NH2OTHP, followed by deprotection
of the THP-protecting group to produce target compound 27a,
in a manner similar to that described above. As shown in
Scheme 6, the acrylate derivative 28 was obtained in modest
yield (52%) by reacting the corresponding aryl halide SMART
4. BIOLOGICAL EVALUATION
4.1. In Vitro Antiproliferative Assay. The newly
synthesized compounds were evaluated by an MTT assay for
their antiproliferative activities against four cancer cell lines,
including HCT-116 (human colon cancer cell), B16-F10
(mouse melanoma cell), Jurkat (human T lymphocyte cell),
and A549 (human lung cancer cell) cells. The two single-target
compounds, SMART (tubulin inhibitor) and MS-275 (HDAC
inhibitor), were used as reference drugs. As shown in Table 1,
most of the tested compounds exhibited modest to high
cytotoxic activities with IC50 values in the low nanomolar to
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Figure 3. Effect of SMART, MS-275, SMART + MS-275, and 15c on the B16-F10 cells.
low micromolar range. First, we analyzed the impact of linkers
on the activities. Changing the linker from phenolic ether (e.g.,
10f) or phenylamine (e.g., 20a, 20b) to an amide (15a−h) led
to increased activity (e.g., average IC50 values of 1.50 μM 1.56
μM, and 75.5 nM for 10f, 20a, 15c, respectively). Interestingly,
the chain length of the linker had a negative influence on the
antiproliferative activity (15c vs 15d, 15e, 15f), and the best
antiproliferative activity was achieved for compounds with
value of 73 nM. These results suggest that the hybrid
compound 15c may achieve synergetic effects by targeting
HDAC and tubulin simultaneously and, thus, may be
advantageous over the cocktail therapy (SMART and MS-
275) in terms of pharmacokinetics and pharmacodynamics as
aforementioned.
4.3. Effects of HDAC/Tubulin Dual Inhibitors on
HDAC Inhibitor-Resistant Gastric Cancer Cells. Previous
studies indicate that HDAC inhibitors showed low activity
against gastric cancer cell lines due to the overexpression of
multidrug-resistance proteins.64 To experimentally validate
whether the newly synthesized HDAC/tubulin dual inhibitors
possessed the ability to overcome resistance to HDAC
inhibitors, we examined the antiproliferative activity of several
compounds in an HDAC inhibitor-resistant cancer cell line
(YCC3/7) and the corresponding sensitive cell line (YCC11),
with MS-275 as a positive control. As presented in Table 2,
three carbons, for example, compound 15c (average IC50
=
75.5 nM), which was more potent than the two positive
controls (SMART and MS-275 average IC50 = 317 nM, 1.59
μM).
We also synthesized several new analogues bearing an alkene
or alkyne linker (e.g., 15h, 17h, 30a, and 32a), as alkenes are
widely used as the linker in HDAC-based multitargeting
agents. The derivatives with an alkene linker (15h, 17h, 30a,
and 32a) exhibited modest antiproliferative activities (average
IC50 ranging from 0.94 to 5.68 μM). In particular, compounds
30a and 32a with an alkene linker displayed more potent
activity (average IC50 = 0.94, 2.96 μM) than the corresponding
alkyne linker-bearing compound 25a (average IC50 = 5.67
μM). In addition, the impact of rigidity of linkers was studied,
and no improvement of the antiproliferative activity was
observed for compounds (e.g., 15h, 15i, 17h, 17i) with a rigid
linker (e.g., phenyl ring, thiophene).
Next, we investigated the effects of ZBG on the activity. In
general, compounds containing the benzamide N-phenylamine
ZBG showed better activity toward four cancer cell lines, and
the potency decreased slightly when the ZBG was switched
from the benzamide N-phenylamine group (e.g., Series 15,
average IC50 = 1.36 μM) to the hydroxamic acid moiety (e.g.,
Series 17 compounds, average IC50 = 3.28 μM).
Finally, most hybrid compounds (in particular, compound
15c) displayed higher or comparable antiproliferative activity
on all studied cell lines when compared to the single-target
HDACi (MS-275) or a tubulin inhibitor (SMART).
In summary, the combination of the two pharmacophores
(tubulin inhibitors and HDAC inhibitors) is beneficial for
improving antiproliferative potency.
4.2. Comparison of Antiproliferative Effects between
Dual-Acting HDAC/Tubulin Inhibitors and Combination
of Single-Target Agents. To confirm the beneficial effects of
the dual-acting compounds, we evaluated the antiproliferative
activities using four different regimens: SMART, MS-275, 15c,
and the combination of SMART + MS-275, against B16-F10
cells. As shown in Figure 3, MS-275 exhibited low activity with
an IC50 value of 2.38 μM, SMART displayed moderate activity
with an IC50 value of 910 nM, while the combination of
SMART and MS-275 (half the dose) demonstrated nearly
equal potency with an IC50 value of 1.15 μM. To our delight,
the dual-acting compound 15c was significantly more active
than the combination of SMART and MS-275 with an IC50
Table 2. Antiproliferative Activity of Compounds against
HDAC Inhibitor-Resistant Cells
IC50 (μM)
compound
YCC11
YCC3/7
MS-275
SMART
12c
15c
15f
5.56 1.21
1.69 0.61
0.92 0.14
0.30 0.08
3.68 0.23
4.09 1.11
12.91 2.05
1.94 0.52
1.28 0.12
0.56 0.13
5.31 1.06
6.14 2.13
32a
MS-275 was less active against YCC3/7 cells (IC50 = 12.91
μM) compared to YCC11 (IC50 = 5.56 μM). SMART showed
moderate activity against both lines (IC50 = 1.69 and 1.94 μM
for YCC11 and YCC3/7), while compound 15c retained high
activities against both cell lines with IC50 values of 0.30 and
0.56 μM for YCC11 and YCC3/7, respectively, and a
resistance index (RI) of 1.9. Compound 15c was 23-fold and
7-fold more active than MS-275 and SMART in the YCC3/7
cells, suggesting a clear and definite advantage of this dual-
acting molecule. A similar trend was also observed in
compounds 12c, 15f, and 32a. Taken together, these results
indicate that the dual-acting molecules could potentially
overcome resistance to HDAC inhibitors and may offer
therapeutic advantages over MS-275.
4.4. Inhibition of HDACs and HDAC-Isoform Selec-
tivity. Having identified the high antiproliferative activities of
the hybrid inhibitors, we next investigated the inhibitory
activity of the newly synthesized compounds against several
recombinant HDAC enzymes, including HDAC1, HDAC3,
HDAC6, HDAC7, and HDAC8, at a concentration of 1 μM.
As shown in Figure 4, compounds containing an o-phenyl-
enediamine group (series 10, 15, 20, 25, 30) exhibited potent
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Figure 4. Inhibition profiles of the newly synthesized compounds (1 μM) against HDAC isoforms 1, 3, 6, 7, and 8.
Table 3. In Vitro Inhibition of Recombinant HDACs 1, 3, 6, 7, and 8 by the Newly Synthesized Compounds
a
IC50 (μM) SD
b
ID
10f
12c
12g
12h
15a
15c
15d
15e
15f
15g
15h
17b
17c
17d
17e
17h
20a
20b
25a
30a
32a
SMART
MS-275
HDAC1
HDAC3
HDAC6
HDAC7
HDAC8
SI
7.49 1.17
6.27 0.94
7.19 1.03
6.86 0.99
4.07 0.86
9.04 1.49
8.12 1.26
8.53 1.75
9.88 1.91
10.53 2.05
14.39 3.07
7.36 1.41
10.12 2.21
16.61 3.53
19.10 5.42
17.46 4.50
9.80 1.42
10.71 2.24
9.77 1.71
11.04 3.07
8.49 1.19
0.47 0.12
9.56 0.06
12.60 3.11
8.86 1.13
0.36 0.27
0.03 0.01
0.18 0.05
0.39 0.11
1.73 0.50
3.46 0.71
3.96 0.85
12.33 2.11
13.62 2.53
13.07 2.37
12.24 2.03
12.97 2.78
1.76 0.79
0.38 0.10
1.98 0.83
0.17 0.05
19.12 4.79
27.13 7.11
1.15 0.12
1.98 0.39
2.07 0.45
21.06 2.36
31.04 6.44
36.37 7.18
27.25 7.91
29.15 6.90
36.26 10.06
30.22 9.36
1.86 0.23
1.73 0.16
2.55 0.23
1.94 0.22
1.05 0.13
27.07 6.88
23.78 5.39
20.23 4.86
22.53 3.45
1.59 0.10
47.51 8.43
43.22 8.09
40.05 7.99
37.55 6.52
49.78 9.14
42.71 9.52
58.28 7.60
45.26 10.07
44.11 8.70
46.20 9.73
43.01 9.05
46.66 9.04
37.15 6.83
35.70 6.05
32.31 5.89
28.27 5.52
41.60 10.27
38.67 9.56
30.61 8.56
47.46 7.53
63.21 9.80
7.49 1.17
8.84 1.25
7.98 1.31
7.10 1.02
12.02 2.10
11.62 3.23
8.74 2.21
8.50 2.17
10.16 2.97
2.08 0.67
0.80 0.12
5.14 1.02
6.32 1.63
3.28 0.91
4.17 1.28
3.90 1.04
9.11 2.20
8.28 1.79
6.55 1.08
3.11 0.75
2.71 0.25
101
38
20
18
139
1423
323
116
25
22
54
26
21
14
17
27
24
102
16
280
40
c
c
c
c
c
NA
NA
NA
NA
NA
0.19 0.03
0.83 0.17
9.32 1.73
39.56 7.25
17.84 4.58
209
a
b
c
IC50 values are the mean of at least three independent assays, presented as mean SD. SI = maximum value/minimum value. NA = not active.
and selective inhibition against HDAC3 (>70% at 1 μM) over
other four HDAC isoforms, whereas the compound bearing
the hydroxamic acid moiety (series 12, 17, 32a) displayed an
HDAC6 (>50% inhibition at 1 μM) preference over HDAC1/
3/7/8. SMART and MS-275 were used as positive controls in
this assay. As expected, the tubulin inhibitor SMART did not
inhibit any HDAC isoforms at a tested concentration of 1 μM,
while the HDAC inhibitor MS-275 was more potent on
HDAC1/3 isoforms than other isoforms, which is consistent
with previous reports.65,66
Subsequently, we determined the IC50 values of these
compounds against different HDAC isoforms. As depicted in
Table 3, all o-phenylenediamine-bearing compounds demon-
strated potent HDAC3 inhibition with IC50 values in the
nanomolar range (30−3960 nM) and relatively high selectivity
indices (SI > 22). The structure−activity relationship (SAR)
studies indicated that the conjugated compounds bearing a
long linker (6-carbon, 15f) or an aromatic ring (15g) or an
olefin chain (15h) could interfere with the recognition by and
binding of the CAP group to the binding pocket of the
HDAC3 enzyme, finally leading to a weak inhibitory activity
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Figure 5. Inhibition of tubulin polymerization in vitro. (A, B) Tubulin was exposed to 10 μM of selected compounds: vehicle control (5% dimethyl
sulfoxide (DMSO)), colchicine, SMART, 10f, 12c, 15a, 15c,15d, 15e, 15h, 20b, 30a, and 32a. (C) Inhibition of tubulin polymerization by
SMART. (D) Inhibition of tubulin polymerization by compound 15c. (E) SMART exhibited a dose-dependent inhibition of tubulin
polymerization. (F) Compound 15c exhibited a dose-dependent inhibition of tubulin polymerization..
against HDAC3. Compounds bearing rigid linkers, such as an
aromatic ring (15g) or an olefin chain (15h), could increase
the inhibitory activity and selectivity against HDAC8 (anti-
HDAC8 IC50 = 2.08 and 0.80 μM for 15g and 15h). Notably,
compared to the positive control MS-275, compound 15c
showed a dramatically improved potency against HDAC3
(IC50 = 0.03 vs 0.83 μM) and excellent selectivity over the
other HDAC isoforms with a >1000-fold selectivity over
HDAC6/7 and a >300-fold selectivity over HDAC1/8. In
addition, the hydroxamic acid-bearing molecules (series 12, 17,
and 32) showed significantly lowered activity against HDAC3
with a moderate selectivity for HDAC6 (SI: 21−40). Taken
together, the results suggest that these compounds, in
particular compound 15c with high antiproliferative and
selective HDAC3 activity, deserve further investigation.
4.5. In Vitro Tubulin Polymerization Assay. To confirm
whether the newly synthesized dual-acting compounds could
inhibit tubulin polymerization as that of SMART,61 we
evaluated the inhibitory effects of selected compounds on
tubulin polymerization with the two known tubulin inhibitors,
colchicine and SMART, as positive controls. These com-
pounds (10f, 12c, 15a, 15c, 15d, 15e, 15h, 20b, 30a, 32a)
were chosen based on their balanced HDAC-inhibitory
activity, HDAC3 selectivity, and antiproliferative potency. At
a concentration of 10 μM, all of the compounds inhibited
tubulin polymerization to some extent with the inhibition rate
ranging from 18 to 70% (Figure 5A,B, inhibition rate = 18, 39,
65, 70, 28, 24, 67, 19, 30, 47% for 10f, 12c, 15a, 15c, 15d, 15e,
15h, 20b, 30a, 32a, respectively, after 20 min treatment), as
compared to the vehicle control. Among them, compound 15c
showed the highest tubulin-inhibitory activity with an
inhibition rate of 70%, which is comparable to that of
colchicine (59%) and SMART (78%). Next, we further
assessed the tubulin-inhibitory activity of compound 15c and
SMART at various concentrations (0, 0.8, 1.5, 3, 6, 12.5, 25
μM). 15c potently inhibited tubulin polymerization with an
IC50 value of 12.2 μM, similar to that of SMART (IC50 = 7.9
μM, Figure 5C,D). Moreover, compound 15c inhibited tubulin
polymerization in a concentration-dependent manner (Figure
5E,F), which correlated well with its antiproliferative effects.
4.6. Antimicrotubule Effects in B16-F10 Cells. Micro-
tubule dynamics play an important role in cell growth,
especially in the proliferation of cancer cells.67 To further
confirm whether the dual-acting molecules maintain the
mechanisms of action as tubulin polymerization inhibitors,
15c was investigated for its in vitro microtubule polymerization
activity in B16-F10 cells by immunofluorescence staining assay
and SMART was employed as the positive control. As shown
in Figure 6, the microtubule network of cells in the control
group (DMSO, Figure 6A) demonstrated normal organizations
and arrangements with the hair-like microtubule network of
the slim/fibrous microtubules (green) wrapping around the
nucleus (blue) of cells. In contrast, when the cells were treated
with SMART (100 nM, Figure 6B) and 15c (100 nM, Figure
6C) for 6 h, the microtubule network was disrupted effectively.
These results implied that compound 15c could destabilize
microtubules by inhibiting tubulin polymerization and
disturbing microtubule networks, similar to SMART.
4.7. Inhibition of Cancer Cell Migration and Colony
Formation. Cancer cells are able to migrate to distant organs,
leading to the formation of metastasized tumors.68 Our
previous studies and other research groups have shown that
microtubules play an important role in cell migration and
motility and that SMART could inhibit cancer cell migration
efficiently.69 Therefore, we performed a wound-healing assay
to confirm the effects of compound 15c on cell migration in
comparison with SMART. B16-F10 cells were treated with 0,
25, 50, and 100 nM compound 15c and SMART (100 nM) for
24 h. As shown in Figure 7A, the cells in the control group
were able to migrate into the wound channel, recovering 53.27
3.18% of the area. When treated with 25, 50, and 100 nM of
compound 15c and SMART (100 nM), the ratio of the wound
area was 27.32 3.56, 17.63 2.79, 7.18 1.01, and 16.24
2.93%, respectively. The results revealed that compound 15c
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cells.43,75 To investigate whether our compounds could cause
cell death by inducing apoptosis, B16-F10 cells were treated
with DMSO, MS-275, SMART, or various concentrations of
compound 15c for 48 h, stained with Annexin V-FITC and PI,
and analyzed by flow cytometry. As shown in Figure 8C,
compounds 15c caused a dose-dependent induction of
apoptosis in B16-F10 cells. The percentages of apoptotic
cells for compound 15c were 10.9, 39.3, and 67.1% at the
concentrations of 25, 50, and 100 nM, respectively. Notably, at
the same concentration of 100 nM, the apoptosis-inducing
abilities of 15c in B16-F10 (percentage of apoptotic cells:
67.1%, Figure 8D) cells were significantly higher than MS-275
and SMART (38.7 and 29.6%, respectively). These results
suggest that the new HDAC/tubulin dual inhibitors could
efficiently induce apoptosis of B16-F10 cells and achieve
synergistic effects at cellular levels.
4.9. Western Blot Analysis of Acetyl-Histone 3 Levels
in B16-F10 Cells. Western blot analysis was performed to
confirm the intracellular effects of compound 15c on HDAC3.
As shown in Figure 9, similarly to MS-275, compound 15c
increased the intracellular levels of the HDAC3 substrate
acetyl-histone H3 (Ac-H3) in a dose-dependent manner. It is
worth noting that at a low concentration of 200 nM, the effect
of 15c on acetyl-histone H3 was comparable to that of MS-275
at a high concentration (1.0 μM). Furthermore, the results
were in line with the enzyme inhibition results shown in Table
3 that 15c possessed superior HDAC3-inhibitory activity to
MS-275.
Figure 6. Effects of SMART and compound 15c on microtubules.
B16-F10 cells were treated with vehicle control 0.1% DMSO (A),
compound SMART (100 nM) (B), and compound 15c (100 nM)
(C) for 6 h. Microtubules were visualized with an anti-β-tubulin
antibody (green), and the cell nucleus was visualized with DAPI
(blue). Fluorescence images were collected using an LSM 880 laser
confocal microscope (Carl Zeiss, Germany).
4.10. Molecular Docking of 15c in Tubulin and
HDAC3. To investigate the binding interactions of compound
15c with tubulin and HDAC3, molecular docking studies were
performed. As shown in Figure 10A,B, compound 15c binds
well to HDAC3. The benzamide group of compound 15c
chelates Zn2+ in a bidentate manner and forms a hydrogen
bond with Leu-29, the aliphatic chains occupy the hydrophobic
channel, and the TMP (3,4,5-trimethoxyphenyl) group of the
CAP moiety forms additional hydrogen bonds with the Phe-
199 residue at the entrance of the active site. The aliphatic
linker is projected into the hydrophobic cavity of HDAC3.
Moreover, the benzamide group of the ZBG section formed
π−π stacking interaction with Tyr-107 of HDAC3.
The binding mode of compound 15c in the colchicine site of
tubulin is displayed in Figure 10C,D. The benzamide ZBG
moiety interacts with the terminal NH2 group of Val-257 via a
hydrogen bond. In addition, the linker extends out of the
binding site and forms a hydrogen bond with Lys-352. The
TMP (3,4,5-trimethoxyphenyl) group of the scaffold is
engaged in a hydrogen-bonding network with Cys-241 and
Val-238. The well-defined binding modes of 15c in HDAC3
and tubulin may explain the high inhibitory activities against
HDAC3 and tubulin.
4.11. In Vivo Antitumor Efficacy Studies. Having
identified the high activity at both cellular and enzymatic/
biochemical levels, we next evaluated the in vivo antitumor
potency of compound 15c in a B16-F10 tumor. C57BL/6 mice
bearing melanoma tumors were divided into 5 groups and
administered intraperitoneally for 14 consecutive days with the
following regimen: vehicle control, SMART (10 mg/kg per
day), MS-275 (10 mg/kg per day), combination (SMART +
MS-275, 5 mg/kg + 5 mg/kg), and compound 15c (10 mg/
kg). Tumor growth inhibition (TGI) was calculated at the end
of treatment. As shown in Figure 11A, treatment with 15c,
SMART, MS-275, and combination therapy suppressed tumor
displayed a better inhibitory effect on cancer cell migration
than SMART. Furthermore, the wound closure was potently
suppressed by 15c in a dose-dependent manner, as compared
to the control (Figure 7B). In the colony formation assay using
B16-F10 cells, compound 15c was able to suppress the
formation of colonies in a dose-dependent manner, with the
colony-forming ability of B16-F10 cells being totally sup-
pressed after 24 h treatment with 100 nM of compound 15c
(Figure 7D). In addition, the suppressing effects of compound
15c were stronger than that of SMART (Figure 7C).
4.8. Effect of Compound 15c on the Cell Cycle and
Apoptosis. Tubulin polymerization inhibitors and HDAC
inhibitors have been previously reported to disrupt cell mitosis
and induce cell cycle arrest.65,70−74 To probe the effects of
compound 15c on the cell cycle, a flow cytometry analysis was
performed (Figure 8). B16-F10 cells were treated with vehicle,
compound 15c, or reference compounds (MS-275 and
SMART). As compared to the vehicle (ratio of the G2/M
phase is 5.76%), treatment of MS-275 and SMART at 100 nM
for 48 h led to an increase of the G2/M phase of the cell cycle
to 42.17 and 76.58%, respectively (Figure 8A), which is in line
with the previous reports.69 The percentages of the G2/M
phase cell cycle arrested by compound 15c at concentrations of
25, 50, and 100 nM were 15.98, 66.32, and 83.26%,
respectively. At a concentration of 100 nM, compound 15c
significantly induced cell cycle arrest in the G2/M phase with
the effects being more prominent than that of SMART and
MS-275, indicating that hybridization of the pharmacophores
of HDAC inhibitors and tubulin inhibitors to a single molecule
could efficiently increase the percentage of cell cycle arrest at
the G2/M phase (Figure 8B).
Previous studies demonstrated that the inhibition of HDAC
and/or tubulin activity could induce apoptosis of cancer
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Figure 7. Effects on the B16-F10 cells migration and colony formation. (A) Representative images of the wound area in a wound-healing assay.
Images were obtained at 0 h and 24 h after treatment with compound 15c with 0, 25, 50, and 100 nM and SMART (100 nM). (B) Histograms
display the length of the scratches at 0 h and 24 h after treatments with compound 15c with 0, 25, 50, and 100 nM and SMART (100 nM) (n = 3,
**P < 0.05 versus DMSO control). (C) Histograms display the number of colonies at different concentrations after treatments with compound 15c
(0, 25, 50, 100 nM) and SMART (0, 25, 50, 100 nM) (n = 3, **P < 0.05 versus DMSO control). (D) Number of colonies was counted after
treating B16-F10 cells with the indicated concentrations of compound 15c and SMART for 24 h (n = 3, **P < 0.05 versus DMSO control).
growth with TGIs of 70, 25, 47, and 53%, respectively. No
significant body weight loss or adverse effects was observed
(Figure 11B) during treatment. Tumor weights in the
combination therapy group were lower than those in the
monotherapy groups (Figure 11C), indicating that combining
SMART with MS-275 was favorable for antitumor efficacy.
Notably, compound 15c demonstrated the highest in vivo
antitumor efficacy among the 5 groups with a TGI of 70%.
Both the tumor weights (Figure 11C) and tumor volumes
(Figure 11D) were decreased significantly when compared to
SMART or MS-275 monotherapy and combination therapy
(SMART + MS-275). Taken together, these results highlighted
the advantages of dual tubulin/HDAC3 inhibitors for cancer
treatment.
4.12. Effects on Acetyl-Histone 3 (Ac-H3) Expression
Levels in Melanoma Tumor Tissues. After the in vivo study,
immunohistochemistry was conducted to analyze the ex-
pression of the acetyl-histone 3 (Ac-H3) protein. As shown in
Figure 12, treatment with 15c, MS-275, or the combination of
MS-375+SMART increased Ac-H3 expression obviously in
tumor tissues with positive ratios of 36.7, 27.9, and 16.3%,
respectively, whereas no significant change in the expression of
Ac-H3 was observed for the control or SMART treatment
group.
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Figure 8. Cell cycle arrest and apoptosis induced by MS-275, SMART, and 15c. (A) MS-275, SMART, and 15c induced G2/M arrest in B16-F10
cells. B16-F10 cells were incubated with MS-275 (100 nM), SMART (100 nM), or varying concentrations of 15c (25, 50, and 100 nM) for 48 h.
Cells treated with DMSO were used for comparison. (B) Histograms display the percentage of cell cycle distribution after treatment with 15c and
reference compounds. (C) Compound 15c induced apoptosis in B16-F10 cells. B16-F10 cells were exposed to MS-275, SMART, and the
increasing concentrations of compound 15c (25, 50, and 100 nM) for 48 h. Cells treated with DMSO were used for comparison. The percentages
of cells in each stage of cell apoptosis were quantified by flow cytometry: necrosis cells (upper left quadrant); late apoptotic cells (upper right
quadrant); live cells (bottom left quadrant); and early apoptotic cells (bottom right quadrant). (D) Histograms displayed the percentage of cell
cycle distribution after treatment with compound 15c and reference compounds.
Figure 9. (A) B16-F10 cells were treated with dimethyl sulfoxide (DMSO) or compound 15c (20, 50, 100, and 200 nM) and MS-275 (1.0 μM) for
12 h. The levels of Ac-H3 and Histone 3 were determined by immunoblotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as
a loading control. Data are representative of three independent experiments. (B) ImageJ was used to quantify the Western blot data.
4.13. In Vivo Safety Profiling of 15c. To assess the in
vivo safety profiles of 15c, we conducted pathohistological
analysis utilizing H&E staining of major organ tissue sections
(heart, liver, kidney) collected at the end of the in vivo study.
As shown in Figure 13A, no apparent morphological
abnormalities in the kidney and liver of different treatment
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Figure 10. Binding interactions between compound 15c and target proteins (HDAC3 and Tubulin): (A) proposed binding mode of 15c in the
active site of HDAC3 (PDB: 4A69); (B) 90° clockwise rotation of the dimeric HDAC3 in complex with 15c along the x-axis. (C) Proposed
binding mode of 15c in the active site of tubulin (PDB: 4O2B); (D) 90° clockwise rotation of tubulin in complex with 15c along the x-axis. The
figures were generated using PyMol (http://www.pymol.org/).
groups were observed. However, there were morphological
changes in the cardiac tissues from the MS-275 treatment
group, implying the potential risk of heart damage caused by
MS-275.
5. CONCLUSIONS
In this work, we designed, synthesized, and bioevaluated for
the first time a series of dual HDAC3/tubulin inhibitors by
combining the pharmacophores of the tubulin inhibitor
SMART and the HDAC inhibitor MS-275. Among the
newly synthesized molecules, compound 15c was identified
to be the most potent and balanced HDAC3/tubulin dual
inhibitor with high HDAC3 activity (IC50 = 30 nM,
biochemical assay) and selectivity (SI of HDAC3 > 1000) as
well as potent antiproliferative activity against various cancer
cell lines, including colon cancer, melanoma, leukemia, and
nonsmall cell lung cancer (IC50 values in the range of 30−140
nM, MTT assay). Notably, 15c was nearly equally potent
against the HDAC inhibitor-resistant cancer cell line (YCC3/
7) and the corresponding sensitive cell line (YCC11). In
addition, 15c was able to inhibit B16-F10 cancer cell migration
and colony formation. It also induced apoptosis in B16-F10
cells with a significant G2/M cell cycle arrest. Compound 15c
acted by inhibiting tubulin polymerization in vitro and
increasing the level of acetyl-H3 protein in a dose-dependent
manner. Importantly, the therapeutic potential of compound
15c was validated in an in vivo B16-F10 tumor model in which
it exhibited a TGI of 70.0%, which was much more efficacious
than that of MS-275 and SMART and even more effective than
the combination of MS-275 and SMART. Finally, 15c
demonstrated a safe cardiotoxicity profile and did not cause
nephro- and hepatotoxicity. Although several groups have
reported the discovery of novel and potent HDAC/tubulin
dual inhibitors as anticancer agents in recent years,30,49−54 the
low or lack of HDAC-isoform selectivity may have hindered
Cardiotoxicity, such as arrhythmias, is the common side
effect of several major classes of HDAC inhibitors.76−80 In
clinical trials, electrocardiogram (ECG) abnormalities are often
monitored to alert the occurrence of cardiovascular toxicity of
drugs. Therefore, the in vivo cardiotoxicity of tested
compounds in the above animal experiments was determined
by ECG. As shown in Figure 13B, the HDAC inhibitor MS-
275 induced QTc interval shortening and inversion of the S-T
wave, indicating a potential cardiovascular risk such as
myocardial ischemia. On the other hand, there was no
significant difference in the ECG of control, SMART,
combination, and 15c-treated groups. Additionally, a normal
heart rate was observed for 15c. Collectively, these results
suggest that the tubulin/HDAC3 dual-targeting compound
15c has a safe cardiotoxicity profile.
To further investigate the safety profiles of compound 15c,
we conducted serum biochemistry analysis. As displayed in
Figure 13C, compound 15c had no obvious nephrotoxicity and
hepatotoxicity in tested mice compared to the control group,
as assessed by the indicators of liver function (e.g., alanine
aminotransferase-ALT, albumin-ALB, aspartate aminotransfer-
ase-AST, aspartate aminotransferase-AST, alanine amino-
transferase-ALT, total bilirubin-TBIL, albumin/globulin-A/G)
and kidney function (e.g., globulin-GLO, creatinine-CRE,
UREA).
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Figure 11. In vivo antitumor efficacy study. After administering vehicle, SMART (10 mg/kg per day), MS-275 (10 mg/kg per day), SMART + MS-
275 (10 mg/kg per day), and compound 15c (10 mg/kg per day) for 14 days, mice were sacrificed, and the tumors were weighed. (A) Images of
tumor size at 14 days after treatment. (B) Body weight changes of mice during treatment. (C) Tumor weights for each group. **P < 0.05 vs control
group. (D) Tumor volume changes for each group. Note: SM: SMART and MS: MS-275.
Figure 12. Immunohistochemistry of Ac-H3 expressions in melanoma tumor tissues. Formalin-fixed paraffin-embedded sections from melanoma
tumor tissues were stained with H&E stain. **P < 0.05 vs control group. ImageJ was used to quantify the Ac-H3 positive ratio.
their further development. The dual HDAC3/tubulin inhib-
itors described in this work demonstrated not only high
antitumor efficacy but also excellent HDAC3 selectivity (SI >
1000), which can offer advantages over reported HDAC/
tubulin dual inhibitors. Taken together, the above analysis
suggests that the novel dual HDAC3/tubulin inhibitors would
be promising for further development as potential anticancer
agents.
6. EXPERIMENTAL SECTION
6.1. General Methods. Solvents and reagents were purchased
from commercial sources and used without further purification. The
reference compound MS-275 was obtained from InvivoChem
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Figure 13. In vivo safety profiling. (A) Pathological sections of major tissues (heart, liver, kidney) were obtained from mice bearing melanoma
tumors. Organs were stained with hematoxylin and eosin (H&E) and captured the representative images. (B) Evaluation of in vivo cardiotoxicity of
15c by ECG and heart rate measurement in C57BL/6 mice. (C) Serum biochemistry analysis of indicators of kidney and liver function in mice of
the above animal experiments.
(Libertyville, IL 60048). All of the reactions were monitored by thin-
layer chromatography (TLC) using silica gel GF/UV 254. Column
chromatography was carried out using commercially available silica
1
gel (200−300 mesh) under pressure. The H and 13C NMR spectra
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were recorded on Bruker AV-400 spectrometer, with TMS as an
internal standard. High-resolution mass spectrometry (HRMS) was
obtained on a Micromass Q-TOF mass spectrometer. The purity of
the tested compounds was ≥95%, as estimated by HPLC
(SHIMADZU LC-20A, UV detection at 254 nm) analysis on the
C18 column (4.6 mm × 150 mm, 5 μm). HPLC conditions: injection
volume: 10 μL, flow rate, 1 mL/min with a mobile phase of H2O/
MeOH; H2O/MeOH = 55/45 was initially held for 3 min, followed
by a linear gradient from 55/45 to 5/95 = H2O/MeOH over 15 min,
which was then held for 12 min.
6.2. General Procedure for the Preparation of Dual-Target
Compounds. 6.2.1. Synthesis of SMART. 6.2.1.1. General Proce-
dure for Intermediate 2. To a stirred solution of 3,4,5-trimethoxyani-
line 1 (2.05 g, 11.20 mmol) in THF (20 mL) was added 4 M aqueous
HCl (28.00 mL), and the solution was cooled in an ice water bath. A
solution of NaNO2 (926.42 mg, 13.43 mmol) in H2O (10.00 mL) was
added dropwise. After 20 min, a solution of KI (4.64 g, 28.00 mmol)
in H2O (10 mL) was added and stirred for 10 min in the ice water
bath. Then, the solution was slowly warmed up to room temperature
and stirred for 1 h before 1 M aqueous Na2S2O3 was added until the
color of the mixture did not change. The phases were separated, and
the aqueous phase was extracted with EtOAc (25 mL × 3). The
combined organic layers were washed with H2O (5 mL × 2) and
brine (5 mL × 1), dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was purified by column chromatography on silica
gel (PE/EtOAc = 40/1-20/1) to provide intermediate 2 (3.02 g, 92%
yield) as a yellow liquid.
6.2.1.2. General Procedure for Intermediate 3. To a stirred
solution of intermediate 2 (3.02 g, 10.27 mmol) in DMF (20.00 mL),
triethylamine (3.12 g, 30.81 mmol), PdCl2(PPh3)2 (360.39 mg, 0.51
mmol), and CuI (195.57 mg, 1.03 mmol) were added to intermediate
2 solution under a nitrogen atmosphere protect. Finally, a solution of
the propargyne (0.49 g, 12.32 mmol) in DMF (4 mL/mmol) was
added to the mixture and stirred at 90 °C for 16 h. After cooling, ethyl
acetate was added to the crude mixture and filtered with Celite. Then,
the solvent was removed in vacuo, and the residue was diluted with
H2O and extracted with EtOAc (3 × 20 mL). The combined organic
layer was then washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo to provide the crude product, which was
directly subjected to silica gel column chromatography and eluted
with petroleum PE/EtOAc (25/1, v/v) to give intermediate 3 as a
white power (1.56 g, 74%).
6.2.1.3. General Procedure for Intermediate 4. To a stirred
solution of intermediate 3 (1.56 g, 7.56 mmol) in dichloromethane
(12.00 mL) was added oxidant PhI(OAc)2 (7.31 g, 22.69 mmol) and
catalyst RuCl3 (156.89 mg, 0.76 mmol) in an ice water bath. Then,
water (3.00 mL, dichloromethane: H2O = 4:1, v/v) was added. The
reaction will be very violent; pay attention to slow down the stirring
speed. The solution was slowly warmed up to room temperature and
stirred for 2 h. After cooling, dichloromethane was added to the crude
mixture and extracted with dichloromethane (3 × 20 mL). The
combined organic layer was then washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to provide the crude
product, which was directly subjected to silica gel column
chromatography and eluted with petroleum PE/EtOAc (30/1, v/v)
to give intermediate 4 as a yellow solid (1.15 g, 64%).
removed under reduced pressure, and the residue was extracted with
ethyl acetate and washed with brine. The organic layer was dried over
anhydrous Na2SO4 and concentrated. The crude product was purified
by silica gel column chromatography to yield the target key
intermediate SMART 7a/7b/7c (1.57 g, 89%).
6.4. Synthesis of Dual-Target Compounds 10a−i and 12a−
h. 6.4.1. General Procedure for Intermediate 8. To a solution of
intermediate 7a (1.00 g, 2.69 mmol) in 10 mL of DMF was added
K2CO3 (930.28 mg, 6.73 mmol) at 80 °C. The mixture was stirred at
80 °C for 0.5 h, then the halo-substituted esters (2.96 mmol) were
added dropwise to the mixture and stirred for 3 h at 80 °C. After
confirming that the reaction was complete using TLC analysis, the
solution was cooled to room temperature. The solvent was diluted
with H2O, and 1 M hydrochloric acid solution, pH = 7, was added
until the precipitation occurs. Then, the mixture was filtered, and the
filtered residue was obtained and further dried to yield intermediate 8
(73% yield).
6.4.2. General Procedure for Intermediate 9. To a solution of
intermediate 8 (1.0 mmol, 1.0 equiv) in 30 mL of MeOH was added
LiOH solution (2.5 mmol, 2.5 equiv, dissolved in 10 mL of H2O) in
an ice-bath. The mixture was stirred at room temperature for 2 h until
the reaction was complete. The solvent was removed in vacuo, the
residue was diluted with H2O, and 4 M hydrochloric acid solution, pH
= 2, was added. Then, the mixture was filtered, and the filtered residue
was obtained and further dried to yield intermediate 9 (100% yield).
6.4.3. General Procedure for Target Compounds 10a−i. To a
solution of 9 (1.0 mmol, 1.0 equiv) in DMF (15 mL) were added
triethylamine (2.5 mmol, 2.5 equiv), HOBt (1.2 mmol, 1.2 equiv),
and EDCI (1.2 mmol, 1.2 equiv) and stirred for 10 min. Then, the o-
phenylenediamine (1.1 mmol, 1.1 equiv) was added. After stirring for
4 h at room temperature until the reaction was complete, water was
added to the reaction mixture. Then, the mixture was filtered, and the
filtered residue was obtained and further dried to obtain compound
10a−i as a yellow solid (93% yield).
6.4.4. General Procedure for Intermediate 11. To a solution of 9
(1.0 mmol, 1.0 equiv) in DMF (15 mL) were added triethylamine
(2.5 mmol, 2.5 equiv), HOBt (1.2 mmol, 1.2 equiv), and EDCI (1.2
mmol, 1.2 equiv) and stirred for 10 min. Then, O-(tetrahydro-2H-
pyran-2-yl) hydroxylamine (NH2OTHP) (1.1 mmol, 1.1 equiv) was
added. After stirring for 4 h at room temperature until the reaction
was complete, water was added to the reaction mixture. Then, the
mixture was filtered, and the filtered residue was obtained and further
dried to obtain intermediate 11 as a white solid (88% yield).
6.4.5. General Procedure for Target Compounds 12a−h. The
THP-protected intermediate 11 was dissolved in 4 M HCl in 1,4-
dioxane (0.1 mmol/mL dioxane) and left to stir for 4 h. The reaction
was monitored by TLC, which indicated a complete conversion. The
solution was dried in vacuo, and the crude mixture was purified by
chromatography on a silica gel column with dichloromethane/MeOH
(40:1) as the eluent to give a white solid (96% yield).
6.5. Synthesis of Dual-Target Compounds 15a−i and 17a−i.
6.5.1. General Procedure for Intermediate 13. To a solution of
compound 7b (1.0 mmol, 1.0 equiv) in DMF (15 mL) were added
triethylamine (2.5 mmol, 2.5 equiv), HOBt (1.2 mmol, 1.2 equiv),
and EDCI (1.2 mmol, 1.2 equiv) and stirred for 10 min. Then,
carboxylate derivatives were added (1.1 mmol, 1.1 equiv). After
stirring for 4 h at room temperature and monitored by TLC, which
indicated a complete conversion, water was added to the reaction
mixture. Then, the mixture was filtered, and the filtered residue was
obtained and further dried to afford intermediate 13 as a yellow solid
(95% yield).
6.5.2. General Procedure for Intermediate 14. To a solution of
intermediate 13 (1.0 mmol, 1.0 equiv) in 30 mL of MeOH was added
LiOH solution (2.5 mmol, 2.5 equiv, dissolved in 10 mL of H2O) in
an ice-bath. The mixture was stirred at room temperature for 2 h until
the reaction was complete. The solvent was removed in vacuo, the
residue was diluted with H2O, and 4 M hydrochloric acid solution, pH
= 2, was added. Then, the mixture was filtered, and the filtered residue
was obtained and further dried to yield intermediate 14 as a white
solid (100% yield).
6.2.1.4. General Procedure for Intermediate 5. To a suspension of
CuBr2 (3.23 g, 14.48 mmol) in 30 mL of ethyl acetate was added
intermediate 4 (1.15 g, 4.83 mmol) in 15 mL of chloroform and
refluxed overnight. Afterward, the reaction contents were filtered
through a silica plug and the filtrate evaporated in a rotary evaporator
carefully with contents warmed at room temperature, yielding
intermediate 5 as a clear yellow oil (1.51 g, 98%). The product was
sufficiently pure for the following steps.
6.3. General Procedure for SMART. Intermediate 5 (1.51 g,
4.73 mmol) was added to a solution of substituted-thiobenzamide 6
(synthesis from benzamide and Lawesson’s reagent) (5.68 mmol) in
ethanol at ambient temperature. The solution was refluxed for 8 h.
After confirming that the reaction was complete using TLC analysis,
the solution was cooled to room temperature. The solvent was
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6.5.3. General Procedure for Target Compounds 15a−i. To a
solution of 14 (1.0 mmol, 1.0 equiv) in DMF (15 mL) were added
triethylamine (2.5 mmol, 2.5 equiv), HOBt (1.2 mmol, 1.2 equiv),
and EDCI (1.2 mmol, 1.2 equiv) and stirred for 10 min. Then, o-
phenylenediamine (1.1 mmol, 1.1 equiv) was added. After stirring for
4 h at room temperature until the reaction was complete, water was
added to the reaction mixture. Then, the mixture was filtered, and the
filtered residue was obtained and further dried to provide compound
15a−i as a yellow solid (91% yield).
6.5.4. General Procedure for Intermediate 16. To a solution of 14
(1.0 mmol, 1.0 equiv) in DMF (15 mL) were added triethylamine
(2.5 mmol, 2.5 equiv), HOBt (1.2 mmol, 1.2 equiv), and EDCI (1.2
mmol, 1.2 equiv) and stirred for 10 min. Then, O-(tetrahydro-2H-
pyran-2-yl) hydroxylamine (NH2OTHP) (1.1 mmol, 1.1 equiv) was
added. After stirring for 4 h at room temperature until the reaction
was complete, water was added to the reaction mixture. Then, the
mixture was filtered, and the filtered residue was obtained and further
dried to obtain intermediate 16 as a white solid (85% yield).
6.5.5. General Procedure for Target Compounds 17a−i. The
THP-protected intermediate 16 was dissolved in 4 M HCl in 1,4-
dioxane (0.1 mmol/mL dioxane) and left to stir for 4 h. The reaction
was monitored by TLC, which indicated a complete conversion. The
solution was dried in vacuo, and the crude mixture was purified by
chromatography on a silica gel column with dichloromethane/MeOH
(40:1) as the eluent to give a white solid (97% yield).
6.6. Synthesis of Dual-Target Compounds 20a−c and 22a−
c. 6.6.1. General Procedure for Intermediate 18. A suspension of 7b
(1.0 mmol, 1.0 equiv), halo-substituted esters (1.2 mmol, 1.2 equiv),
potassium iodide (1.5 mmol, 1.5 equiv), and anhydrous potassium
carbonate (2.5 mmol, 2.5 equiv) in 20 mL of acetonitrile was refluxed
under nitrogen for 15 h. The mixture was cooled and filtered, and the
filtrate was dissolved in dichloromethane and extracted with aqueous
sodium bicarbonate solution and then with water. The solution was
dried over anhydrous Na2SO4 and filtered. After the concentration in
vacuo, the crude residue was purified by silica gel column
chromatography using the solvent system 15% ethyl acetate in
hexane as an eluent to give intermediate 18 (59% yield) as a light
yellowish crystal.
6.6.2. General Procedure for Intermediate 19. To a solution of
compound 18 (1.0 mmol, 1.0 equiv) in 30 mL of MeOH was added
LiOH solution (2.5 mmol, 2.5 equiv, dissolved in 10 mL of H2O) in
an ice-bath. The mixture was stirred at room temperature for 2 h until
the reaction was complete. The solvent was removed in vacuo, the
residue was diluted with H2O, and 4 M hydrochloric acid solution, pH
= 2, was added. Then, the mixture was filtered, and the filtered residue
was obtained and further dried to yield intermediate 19 as a yellow
solid (100% yield).
6.6.3. General Procedure for Target Compounds 20a−c. To a
solution of 19 (1.0 mmol, 1.0 equiv) in DMF (15 mL) were added
triethylamine (2.5 mmol, 2.5 equiv), HOBt (1.2 mmol, 1.2 equiv),
and EDCI (1.2 mmol, 1.2 equiv) and stirred for 10 min. Then, o-
phenylenediamine (1.1 mmol, 1.1 equiv) was added. After stirring for
4 h at room temperature until the reaction was complete, water was
added to the reaction mixture. Then, the mixture was filtered, and the
filtered residue was obtained and further dried to provide compound
20a−c as a yellow solid (93% yield).
6.6.4. General Procedure for Intermediate 21. To a solution of 19
(1.0 mmol, 1.0 equiv) in DMF (15 mL) were added triethylamine
(2.5 mmol, 2.5 equiv), HOBt (1.2 mmol, 1.2 equiv), and EDCI (1.2
mmol, 1.2 equiv) and stirred for 10 min. Then, O-(tetrahydro-2H-
pyran-2-yl) hydroxylamine (NH2OTHP) (1.1 mmol, 1.1 equiv) was
added. After stirring for 4 h at room temperature until the reaction
was complete, water was added to the reaction mixture. Then, the
mixture was filtered, and the filtered residue was obtained and further
dried to obtain intermediate 21 as a white solid (87% yield).
6.6.5. General Procedure for Target Compounds 22a−c. The
THP-protected intermediate 21 was dissolved in 4 M HCl in 1,4-
dioxane (0.1 mmol/mL dioxane) and left to stir for 4 h. The reaction
was monitored by TLC, which indicated a complete conversion. The
solution was dried in vacuo, and the crude mixture was purified by
chromatography on a silica gel column with dichloromethane/MeOH
(40:1) as the eluent to give a white solid (91% yield).
6.7. Synthesis of Dual-Target Compounds 25a and 27a.
6.7.1. General Procedure for Intermediate 23. To a solution of
bromo-substituted SMART 7c (1.00 g, 2.30 mmol) in DMF (10.00
mL), methyl 4-pentynoate (309.81 mg, 2.76 mmol), PdCl2(PPh3)2
(80.81 mg, 0.12 mmol), and Et3N (582.50 mg, 5.76 mmol) were
added under N2. The reaction mixture was degassed and stirred under
a N2 atmosphere for 20 min before the addition of CuI (43.85 mg,
0.23 mmol). The reaction mixture was then stirred at 90 °C for 12 h
before it was diluted with diethyl ether (10 mL). The content was
poured into a separate funnel and washed with 0.1 M HCl (10 mL),
H2O (10 mL), and brine (10 mL), respectively. The organic phase
was dried over anhydrous Na2SO4 and concentrated in vacuo. The
residue was purified by column chromatography (PE/EtOAc = 20:1)
on silica gel to provide a yellow solid intermediate 23 (762.30 mg,
71% yield).
6.7.2. General Procedure for Intermediate 24. To a solution of
compound 23 (762.30 mg, 1.64 mmol) in 30 mL of MeOH was
added LiOH solution (98.02 mg, 4.09 mmol, dissolved in 3 mL of
H2O) in an ice-bath. The mixture was stirred at room temperature for
2 h until the reaction was complete. The solvent was removed in
vacuo, the residue was diluted with H2O, and 4 M hydrochloric acid
solution, pH = 2, was added. Then, the mixture was filtered, and the
filtered residue was obtained and further dried to yield intermediate
24 as a yellow solid (739.04 mg, 100% yield).
6.7.3. General Procedure for Target Compounds 25a. To a
solution of 24 (739.04 mg, 1.64 mmol) in DMF (10 mL) were added
triethylamine (414.08 mg, 4.09 mmol), HOBt (265.41 mg, 1.96
mmol), and EDCI (376.53 mg, 1.96 mmol) and stirred for 10 min.
Then, o-phenylenediamine (194.71 mg, 1.80 mmol) was added. After
stirring for 4 h at room temperature until the reaction was complete,
water was added to the reaction mixture. Then, the mixture was
filtered, and the filtered residue was obtained and further dried to
provide compound 25a as a yellow solid (853.12 mg, 96% yield).
6.7.4. General Procedure for Intermediate 26. To a solution of 24
(739.04 mg, 1.64 mmol) in DMF (10 mL) were added triethylamine
(414.08 mg, 4.09 mmol), HOBt (265.41 mg, 1.96 mmol), and EDCI
(376.53 mg, 1.96 mmol) and stirred for 10 min. Then, O-(tetrahydro-
2H-pyran-2-yl) hydroxylamine (NH2OTHP) (210.92 mg, 1.80
mmol) was added. After stirring for 4 h at room temperature until
the reaction was complete, water was added to the reaction mixture.
Then, the mixture was filtered, and the filtered residue was obtained
and further dried to provide compound 26 as a yellow solid (806.96
mg, 89% yield).
6.7.5. General Procedure for Target Compounds 27a. The THP-
protected intermediate 26 was dissolved in 4 M HCl in 1,4-dioxane
(0.1 mmol/mL dioxane) and left to stir for 4 h. The reaction was
monitored by TLC, which indicated a complete conversion. The
solution was dried in vacuo, and the crude mixture was purified by
chromatography on a silica gel column with dichloromethane/MeOH
(40:1) as the eluent to give a white solid (643.56 mg, 94% yield).
6.8. Synthesis of Dual-Target Compounds 30a and 32a.
6.8.1. General Procedure for Intermediate 28. Compound 7c (1.00
g, 2.30 mmol), methyl acrylate (237.87 mg, 2.76 mmol), Pd(OAc)2
(25.85 mg, 0.12 mmol), PPh3 (60.93 mg, 0.23 mmol), and Et3N
(582.50 mg, 5.76 mmol) were dissolved in DMF (5 mL). The
reaction mixture was stirred at 110 °C for 12 h. After that, the
reaction mixture was cooled to rt and the reaction mixture was
extracted with EtOAc (3 × 10 mL), the combined organic phase was
washed with H2O (5 × 5 mL) and brine (10 mL), dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography (PE/EtOAc = 20:1) on silica gel
to provide a white oil-liquid 28 (563.27 mg, 52% yield).
6.8.2. General Procedure for Intermediate 29. To a solution of
compound 28 (563.27 mg, 1.28 mmol) in 30 mL of MeOH was
added LiOH solution (76.69 mg, 3.20 mmol, dissolved in 3 mL of
H2O) in an ice-bath. The mixture was stirred at room temperature for
2 h until the reaction was complete. The solvent was removed in
vacuo, the residue was diluted with H2O, and 4 M hydrochloric acid
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solution, pH = 2, was added. Then, the mixture was filtered, and the
filtered residue was obtained and further dried to yield intermediate
29 as a white solid (542.45 mg, 100% yield).
1H NMR (400 MHz, DMSO) δ 9.11 (s, 1H), 8.53 (d, J = 4.8 Hz,
1H), 7.95 (d, J = 3.6 Hz, 2H), 7.63 (s, 2H), 7.14 (d, J = 7.6 Hz, 1H),
7.09 (d, J = 8.4 Hz, 2H), 6.88 (t, J = 7.2 Hz, 1H), 6.71 (d, J = 7.6 Hz,
1H), 6.52 (t, J = 7.6 Hz, 1H), 4.80 (s, 2H), 4.05 (t, J = 5.6 Hz, 2H),
3.86 (s, 6H), 3.79 (s, 3H), 2.33 (t, J = 7.2 Hz, 2H). 13C NMR (101
MHz, DMSO) δ 185.14, 173.60, 171.63, 167.35, 154.28, 152.88,
142.20, 132.20, 127.49, 125.61, 119.74, 116.27, 108.38, 60.63, 56.38,
51.57, 33.63. M.p.: 125.5−127.7 °C. HRMS (ESI-Q-TOF) m/z: [M +
H]+ calculated for C28H28N3O6S: 534.1621, found: 534.1625. HPLC:
tR 17.7 min, purity 98.2%.
6.8.5.6. N-(2-Aminophenyl)-4-(4-(4-(3,4,5-trimethoxybenzoyl)-
thiazol-2-yl)phenoxy)butanamide (10c). Yellow solid, yield: 29%.
1H NMR (400 MHz, DMSO) δ 9.16 (s, 1H), 8.56 (s, 1H), 7.97 (d, J
= 8.4 Hz, 2H), 7.63 (s, 2H), 7.14 (dd, J = 14.8, 8.0 Hz, 3H), 6.89 (s,
1H), 6.71 (d, J = 7.9 Hz, 1H), 6.54 (d, J = 7.2 Hz, 1H), 4.83 (s, 2H),
4.13 (s, 2H), 3.87 (s, 6H), 3.80 (s, 3H), 2.03 (d, J = 33.5 Hz, 4H).
13C NMR (101 MHz, DMSO) δ 184.97, 173.37, 152.84, 142.28,
132.19, 129.46, 128.41, 125.79, 115.61, 108.35, 99.93, 60.59, 56.35,
51.73, 30.25, 24.50. M.p.: 124.8−126.1 °C. HRMS (ESI-Q-TOF) m/
z: [M + H]+ calculated for C29H30N3O6S: 548.1771, found: 548.1771.
HPLC: tR 18.0 min, purity 99.0%.
6.8.5.7. N-(2-Aminophenyl)-5-(4-(4-(3,4,5-trimethoxybenzoyl)-
thiazol-2-yl)phenoxy)pentanamide (10d). Yellow solid, yield: 28%.
1H NMR (400 MHz, DMSO) δ 9.14 (s, 1H), 8.55 (s, 1H), 7.95 (d, J
= 8.0 Hz, 2H), 7.64 (s, 2H), 7.13 (dd, J = 17.4, 8.0 Hz, 3H), 6.89 (t, J
= 7.3 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 6.54 (t, J = 7.3 Hz, 1H), 4.82
(s, 2H), 4.10 (s, 2H), 3.87 (s, 6H), 3.80 (s, 3H), 2.40 (s, 2H), 1.79 (s,
4H). 13C NMR (101 MHz, DMSO) δ 184.62, 170.96, 167.28, 160.77,
153.90, 152.47, 141.90, 131.82, 129.08, 128.04, 125.74, 125.31,
123.51, 116.18, 115.90, 115.26, 107.97, 67.55, 60.21, 55.97, 35.31,
28.15, 21.88. M.p.: 120.1−121.2 °C. HRMS (ESI-Q-TOF) m/z: [M +
H]+ calculated for C30H32N3O6S: 562.1934, found: 562.1935. HPLC:
tR 17.4 min, purity 96.2%.
6.8.3. General Procedure for Target Compound 30a. To a
solution of 29 (542.45 mg, 1.27 mmol) in DMF (10 mL) were added
triethylamine (322.55 mg, 3.19 mmol), HOBt (206.74 mg, 1.53
mmol), and EDCI (293.30 mg, 1.53 mmol) and stirred for 10 min.
Then, o-phenylenediamine (151.67 mg, 1.40 mmol) was added. After
stirring for 4 h at room temperature until the reaction was complete,
water was added to the reaction mixture. Then, the mixture was
filtered, and the filtered residue was obtained and further dried to
provide compound 30a as a yellow solid (598.39 mg, 91% yield).
6.8.4. General Procedure for Intermediate 31. To a solution of 29
(542.45 mg, 1.27 mmol) in DMF (10 mL) were added triethylamine
(322.55 mg, 3.19 mmol), HOBt (206.74 mg, 1.53 mmol), and EDCI
(293.30 mg, 1.53 mmol) and stirred for 10 min. Then, the O-
(tetrahydro-2H-pyran-2-yl)hydroxylamine (NH2OTHP) (164.30 mg,
1.40 mmol) was added. After stirring for 4 h at room temperature
until the reaction was complete, water was added to the reaction
mixture. Then, the mixture was filtered, and the filtered residue was
obtained and further dried to provide compound 31 as a yellow solid
(556.81 mg, 83% yield).
6.8.5. General Procedure for Target Compound 32a. The THP-
protected intermediate 31 (556.81 mg, 1.06 mmol) was dissolved in 4
M HCl in 1,4-dioxane (0.1 mmol/mL dioxane) and left to stir for 4 h.
The reaction was monitored by TLC, which indicated a complete
conversion. The solution was dried in vacuo, and the crude mixture
was purified by chromatography on a silica gel column with
dichloromethane/MeOH (40:1) as the eluent to give a white solid
(439.16 mg, 94% yield).
6.8.5.1. (2-(4-Hydroxyphenyl)thiazol-4-yl)(3,4,5-
trimethoxyphenyl)methanone (7a). Yellow solid, yield: 45%. 1H
NMR (400 MHz, DMSO) δ 9.61 (s, 1H), 8.80 (s, 1H), 8.39 (d, J =
7.2 Hz, 2H), 8.30 (d, J = 9.2 Hz, 2H), 7.61 (s, 2H), 3.87 (s, 6H), 3.80
(s, 3H). 13C NMR (101 MHz, DMSO) δ 185.01, 165.21, 154.93,
152.95, 148.84, 142.49, 138.31, 132.27, 131.95, 128.02, 125.14,
108.39, 60.65, 56.45. M.p.: 123.0−125.4 °C. HRMS (ESI-Q-TOF)
m/z: [M + H]+ calculated for C19H18NO5S: 372.0827, found:
372.0830.
6 . 8 . 5 . 2 . ( 2 - ( 4 - A m i n o p h e n y l ) t h i a z o l - 4 - y l) ( 3 , 4 , 5 -
trimethoxyphenyl)methanone (7b). Yellow solid, yield: 44%. 1H
NMR (400 MHz, DMSO) δ 8.40 (s, 1H), 7.70 (d, J = 8.0 Hz, 3H),
7.65 (s, 2H), 6.66 (d, J = 7.9 Hz, 2H), 5.77 (d, J = 14.3 Hz, 2H), 3.87
(s, 8H), 3.80 (s, 4H). 13C NMR (101 MHz, DMSO) δ 184.25,
164.75, 154.50, 152.51, 148.39, 142.05, 137.87, 131.83, 131.51,
127.57, 124.70, 107.96, 60.20, 56.01. M.p.: 120.7−122.3 °C. HRMS
(ESI-Q-TOF) m/z: [M + H]+ calculated for C19H19N2O4S: 371.0987,
found: 371.0996.
6.8.5.8. N-(2-Aminophenyl)-6-(4-(4-(3,4,5-trimethoxybenzoyl)-
thiazol-2-yl)phenoxy)hexanamide (10e). Yellow solid, yield: 37%.
1H NMR (400 MHz, DMSO) δ 9.12 (s, 1H), 8.54 (s, 1H), 7.94 (d, J
= 7.8 Hz, 2H), 7.64 (s, 2H), 7.16 (d, J = 7.1 Hz, 1H), 7.08 (s, 3H),
6.88 (d, J = 6.8 Hz, 1H), 6.72 (d, J = 7.4 Hz, 1H), 6.54 (d, J = 6.5 Hz,
1H), 4.82 (s, 2H), 4.05 (d, J = 5.1 Hz, 3H), 3.87 (s, 7H), 3.79 (s,
3H), 2.33 (d, J = 17.3 Hz, 3H), 1.72 (dd, J = 25.7, 8.7 Hz, 5H), 1.58
(d, J = 8.7 Hz, 2H), 1.45 (d, J = 24.3 Hz, 4H). 13C NMR (101 MHz,
DMSO) δ 184.67, 173.41, 171.19, 167.38, 160.89, 154.02, 152.57,
141.99, 131.91, 129.64, 129.28, 129.14, 128.12, 125.80, 125.38,
125.20, 123.67, 116.28, 116.00, 115.32, 114.80, 108.09, 60.31, 56.06,
51.28, 35.79, 33.31, 28.48, 25.15. M.p.: 122.7−124.2 °C. HRMS (ESI-
Q-TOF) m/z: [M + H]+ calculated for C31H34N3O6S: 576.2090,
found: 576.2096. HPLC: tR 18.2 min, purity 98.8%.
6.8.5.9. N-(2-Aminophenyl)-7-(4-(4-(3,4,5-trimethoxybenzoyl)-
thiazol-2-yl)phenoxy)heptanamide (10f). Yellow solid, yield: 48%.
1H NMR (400 MHz, DMSO) δ 9.11 (s, 1H), 8.53 (d, J = 4.8 Hz,
6 . 8 . 5 . 3 . ( 2 - ( 4 - B r o m o p h e n y l ) t h i a z o l - 4 - y l) ( 3 , 4 , 5 -
trimethoxyphenyl)methanone (7c). Yellow solid, yield: 47%. 1H
NMR (400 MHz, DMSO) δ 8.67 (s, 1H), 7.98 (d, J = 8.5 Hz, 2H),
7.76 (d, J = 8.5 Hz, 2H), 7.60 (s, 2H), 3.86 (s, 6H), 3.79 (s, 3H) 13C
NMR (101 MHz, DMSO) δ 184.93, 152.52, 143.13, 141.73, 132.47,
131.39, 130.41, 128.34, 123.81, 107.98, 60.25, 56.02. M.p.: 123.7−
125.2 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C19H17BrNO4S: 433.9983, found: 433.9980.
1H), 7.95 (d, J = 3.9 Hz, 2H), 7.63 (s, 2H), 7.14 (d, J = 7.5 Hz, 1H),
7.09 (d, J = 8.4 Hz, 2H), 6.88 (t, J = 7.4 Hz, 1H), 6.71 (d, J = 7.7 Hz,
1H), 6.53 (t, J = 7.4 Hz, 1H), 4.80 (s, 2H), 4.08−4.01 (m, 2H), 3.86
(s, 6H), 3.79 (s, 3H), 2.33 (s, 2H), 1.74 (s, 3H), 1.67−1.57 (m, 2H),
1.47 (s, 3H), 1.38 (d, J = 21.9 Hz, 4H). 13C NMR (101 MHz,
DMSO-d6) δ 184.72, 171.28, 167.37, 162.43, 160.86, 153.94, 152.53,
141.93, 131.88, 129.14, 128.10, 125.78, 125.36, 125.14, 123.62,
116.27, 115.97, 115.31, 108.02, 67.83, 60.28, 56.04, 35.86, 35.74,
30.84, 28.52, 25.29. M.p.: 131.3−133.0 °C. HRMS (ESI-Q-TOF) m/
z: [M + H]+ calculated for C32H36N3O6S: 590.2247, found: 590.2243.
HPLC: tR 18.4 min, purity 99.1%.
6.8.5.4. N-(2-Aminophenyl)-2-(4-(4-(3,4,5-trimethoxybenzoyl)-
thiazol-2-yl)phenoxy)acetamide (10a). Yellow solid, yield: 30%.
1H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.57 (s, 1H), 8.00 (d,
J = 8.5 Hz, 2H), 7.64 (s, 2H), 7.19 (d, J = 8.4 Hz, 2H), 7.15 (d, J =
7.6 Hz, 1H), 6.94 (t, J = 7.3 Hz, 1H), 6.74 (d, J = 7.8 Hz, 1H), 6.56
(t, J = 7.5 Hz, 1H), 4.90 (s, 2H), 4.82 (s, 2H), 3.87 (s, 6H), 3.80 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 184.62, 167.15, 153.92,
152.47, 141.90, 131.81, 129.21, 128.08, 127.99, 127.45, 127.30,
127.15, 126.69, 116.10, 107.97, 68.92, 60.21, 55.97. M.p.: 147.1−
149.0 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C27H26N3O6S: 520.1464, found: 520.1467. HPLC: tR 18.6 min, purity
97.2%.
6.8.5.10. N-(2-Aminophenyl)-4-((4-(4-(3,4,5-trimethoxybenzoyl)-
thiazol-2-yl)phenoxy)methyl)benzamide (10g). Yellow solid, yield:
1
31%. H NMR (400 MHz, DMSO) δ 9.68 (s, 1H), 8.56 (s, 1H),
8.04−7.94 (m, 4H), 7.68−7.56 (m, 4H), 7.48 (dd, J = 36.8, 6.8 Hz,
2H), 7.20 (s, 3H), 6.97 (d, J = 6.5 Hz, 1H), 6.79 (d, J = 7.5 Hz, 1H),
6.60 (s, 1H), 5.27 (d, J = 25.7 Hz, 2H), 4.91 (s, 2H), 3.87 (s, 6H),
3.80 (s, 3H).13C NMR (101 MHz, DMSO-d6) δ 184.62, 169.87,
167.17, 165.06, 160.33, 160.25, 153.95, 152.49, 143.16, 141.92,
140.05, 137.83, 136.09, 134.20, 131.82, 129.38, 129.21, 128.09,
6.8.5.5. N-(2-Aminophenyl)-3-(4-(4-(3,4,5-trimethoxybenzoyl)-
thiazol-2-yl)phenoxy)propanamide (10b). Yellow solid, yield: 31%.
8465
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Article
128.01, 127.56, 127.48, 127.33, 127.17, 126.73, 126.55, 125.60,
123.27, 116.29, 116.15, 115.72, 115.63, 108.00, 69.05, 60.23, 55.98.
M.p.: 135.2−138.9 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C33H30N3O6S: 596.1777, found: 596.1779. HPLC: tR
16.1 min, purity 96.9%.
C24H27N2O7S: 487.1461, found: 486.1469. HPLC: tR 14.0 min, purity
98.4%.
6.8.5.17. N-Hydroxy-6-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
1
yl)phenoxy)hexanamide (12e). White solid, yield: 27%. H NMR
(400 MHz, DMSO) δ 11.91 (s, 1H), 8.47 (s, 1H), 8.24 (s, 1H), 7.82
(d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.7 Hz, 2H), 6.85 (s, 2H), 4.01 (t, J =
6.4 Hz, 2H), 3.74 (d, J = 7.0 Hz, 6H), 3.71 (s, 3H), 2.23 (t, J = 7.2
Hz, 2H), 1.76−1.70 (m, 2H), 1.59−1.52 (m, 2H), 1.46−1.39 (m,
2H). 13C NMR (101 MHz, DMSO) δ 174.45, 165.20, 160.44, 152.40,
149.43, 145.61, 138.23, 131.82, 129.70, 127.81, 125.51, 115.16,
105.54, 67.67, 60.15, 55.90, 33.66, 29.04, 28.36, 24.29. M.p.: 126.3−
127.8 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C25H29N2O7S: 501.1617, found: 501.1611. HPLC: tR 14.1 min, purity
97.8%.
6.8.5.11. (E)-N-(2-Aminophenyl)-3-(4-((4-(4-(3,4,5-
trimethoxybenzoyl)thiazol-2-yl)phenoxy)methyl)phenyl)-
1
acrylamide (10h). Yellow solid, yield: 34%. H NMR (400 MHz,
DMSO) δ 9.41 (s, 1H), 8.56 (s, 1H), 7.98 (dd, J = 8.5, 4.4 Hz, 2H),
7.75 (t, J = 8.7 Hz, 1H), 7.70−7.64 (m, 2H), 7.63 (s, 2H), 7.57−7.51
(m, 2H), 7.34 (d, J = 7.7 Hz, 1H), 7.20 (dd, J = 8.3, 5.9 Hz, 2H),
6.95−6.88 (m, 1H), 6.75 (d, J = 7.9 Hz, 1H), 6.66 (d, J = 16.1 Hz,
1H), 6.59 (d, J = 7.6 Hz, 1H), 5.24 (d, J = 5.6 Hz, 2H), 4.94 (s, 2H),
3.87 (s, 6H), 3.79 (s, 3H). 13C NMR (101 MHz, DMSO) δ 184.64,
167.18, 163.47, 153.93, 152.48, 141.91, 141.60, 139.11, 138.13,
134.58, 131.82, 129.22, 128.25, 128.09, 127.80, 127.55, 125.80,
124.71, 123.49, 122.59, 117.41, 116.27, 116.01, 115.67, 114.68,
107.98, 60.22, 55.98, 48.73. M.p.: 141.0−143.7 °C. HRMS (ESI-Q-
TOF) m/z: [M + H]+ calculated for C35H32N3O6S: 622.1934, found:
622.1930. HPLC: tR 17.4 min, purity 98.6%.
6.8.5.18. N-Hydroxy-7-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
1
yl)phenoxy)heptanamide (12f). White solid, yield: 30%. H NMR
(400 MHz, DMSO) δ 10.34 (s, 1H), 8.66 (s, 1H), 8.55 (s, 1H), 7.94
(d, J = 8.4 Hz, 2H), 7.63 (s, 2H), 7.09 (d, J = 8.4 Hz, 2H), 4.04 (t, J =
5.9 Hz, 2H), 3.87 (s, 6H), 3.81 (d, J = 9.5 Hz, 3H), 1.96 (t, J = 7.1
Hz, 2H), 1.57−1.46 (m, 2H), 1.42 (s, 2H), 1.37−1.26 (m, 4H). 13C
NMR (101 MHz, DMSO) δ 184.62, 169.12, 167.29, 160.80, 153.91,
152.48, 141.91, 131.83, 129.08, 128.04, 125.10, 115.25, 107.98, 67.76,
60.22, 55.98, 32.20, 28.46, 28.31, 25.19, 25.05. M.p.: 121.3−122.9 °C.
HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for C26H31N2O7S:
515.1774, found: 515.1778. HPLC: tR 13.9 min, purity 98.2%.
6.8.5.19. N-Hydroxy-4-((4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
6 . 8 . 5 . 1 2 . ( E ) - N - ( 2 - A m i n o p h en y l) -4 -( 4 - ( 4- ( 3, 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenoxy)but-2-enamide (10i). Yel-
1
low solid, yield: 26%. H NMR (400 MHz, DMSO) δ 9.16 (s, 1H),
8.60 (s, 1H), 8.04 (s, 2H), 7.63 (s, 2H), 7.21 (d, J = 39.0 Hz, 3H),
6.90 (s, 3H), 6.72 (s, 2H), 6.54 (s, 2H), 5.25 (s, 1H), 4.85 (s, 2H),
3.83 (d, J = 28.4 Hz, 9H). 13C NMR (101 MHz, DMSO-d6) δ 185.64,
169.16, 167.18, 152.90, 142.42, 132.19, 128.68, 125.84, 123.66,
117.14, 116.52, 116.19, 108.38, 69.61, 60.63, 56.40. M.p.: 130.1−
131.2 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C29H28N3O6S: 546.1621, found: 546.1623. HPLC: tR 17.7 min, purity
98.0%.
1
yl)phenoxy)methyl)benzamide (12g). White solid, yield: 32%. H
NMR (400 MHz, DMSO) δ 10.62 (s, 1H), 8.82 (s, 1H), 8.61 (s, 1H),
8.34 (d, J = 7.6 Hz, 2H), 8.21 (d, J = 4.4 Hz, 2H), 8.05 (d, J = 8.4 Hz,
2H), 7.94 (d, J = 8.8 Hz, 2H), 7.64 (s, 2H), 5.15 (s, 2H), 3.88 (s,
6H), 3.80 (s, 3H). 13C NMR (101 MHz, DMSO) δ 167.10, 154.00,
152.49, 142.21, 141.10, 131.79, 129.18, 127.85, 127.06, 120.62,
107.97, 71.85, 60.22, 55.98.1. M.p.: 121.9−123.1 °C. HRMS (ESI-Q-
TOF) m/z: [M + H]+ calculated for C27H25N2O7S: 521.1304, found:
521.1301. HPLC: tR 14.0 min, purity 99.1%.
6.8.5.13. N-Hydroxy-2-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
yl)phenoxy)acetamide (12a). White solid, yield: 25%. 1H NMR
(400 MHz, DMSO) δ 10.46 (s, 1H), 8.79 (s, 1H), 8.53 (s, 1H), 7.92
(d, J = 8.3 Hz, 2H), 7.63 (s, 2H), 7.02 (d, J = 8.3 Hz, 2H), 4.54 (s,
2H), 3.86 (s, 6H), 3.79 (s, 4H). 13C NMR (101 MHz, DMSO) δ
184.58, 167.31, 153.91, 152.46, 141.88, 131.81, 129.06, 127.83,
125.07, 115.32, 107.96, 60.20, 55.97, 29.00. M.p.: 123.2−124.6 °C.
HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for C21H21N2O7S:
445.0991, found: 445.0993. HPLC: tR 13.5 min, purity 96.4%.
6.8.5.14. N-Hydroxy-3-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
6.8.5.20. (E)-N-Hydroxy-3-(4-((4-(4-(3,4,5-trimethoxybenzoyl)
thiazol-2-yl) phenoxy) methyl) phenyl) acrylamide (12h). White
solid, yield: 26%. 1H NMR (400 MHz, DMSO) δ 10.48 (s, 1H), 9.06
(s, 1H), 8.56 (s, 1H), 7.98 (d, J = 8.7 Hz, 2H), 7.65−7.55 (m, 4H),
7.51 (d, J = 8.1 Hz, 2H), 7.42−7.34 (m, 1H), 7.19 (d, J = 8.6 Hz,
2H), 6.48 (d, J = 15.9 Hz, 1H), 5.23 (s, 2H), 3.87 (s, 6H), 3.80 (s,
3H). 13C NMR (101 MHz, DMSO) δ 184.65, 167.14, 166.25, 159.93,
153.92, 152.48, 142.64, 141.90,131.81, 128.02, 126.46, 126.08,
125.78, 122.24, 116.12, 115.80, 107.96, 67.00, 60.21, 55.98. M.p.:
134.4−136.1 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C29H27N2O7S: 547.1461, found: 547.1464. HPLC: tR 15.1 min, purity
97.7%.
1
yl)phenoxy)propanamide (12b). White solid, yield: 24%. H NMR
(400 MHz, DMSO) δ 10.18 (s, 1H), 8.64 (s, 1H), 8.57 (s, 1H), 7.97
(d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.63 (s, 3H), 4.19 (s,
2H), 3.87 (s, 6H), 3.80 (s, 3H), 2.35 (d, J = 4.6 Hz, 2H). 13C NMR
(101 MHz, DMSO) δ 184.63, 171.50, 168.92, 167.17, 153.94, 152.46,
141.89, 131.78, 129.29, 127.13, 119.30, 107.95, 64.27, 60.19, 55.95,
32.14. M.p.: 121.3−123.0 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C22H23N2O7S: 459.1148, found: 459.1150. HPLC: tR
13.2 min, purity 96.6%.
6 . 8 . 5 . 2 1 . N 1 - ( 2 - A m i n o p h e n y l ) - N 3 - ( 4 - ( 4 - ( 3 , 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenyl)malonamide (15a). Yellow
1
solid, yield: 40%. H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H),
10.07 (s, 1H), 8.62 (s, 1H), 8.55 (s, 1H), 8.06 (s, 5H), 7.99 (s, 2H),
7.63 (s, 3H), 7.27 (d, J = 7.2 Hz, 1H), 7.00 (s, 1H), 6.79 (d, J = 7.6
Hz, 1H), 6.63 (d, J = 7.7 Hz, 1H), 5.05 (s, 2H), 3.82 (dd, J = 27.1,
12.8 Hz, 11H). 13C NMR (101 MHz, DMSO) δ 185.12, 167.37,
159.43, 158.84, 154.45, 152.91, 152.84, 143.12, 142.35, 142.29,
140.46, 132.20, 132.11, 130.12, 128.92, 127.49, 127.38, 126.11,
122.42, 121.28, 116.79, 116.68, 108.39, 60.63, 60.58, 56.40, 56.30,
18.95. M.p.: 127.3−128.8 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C28H27N4O6S: 547.1573, found: 547.1567. HPLC: tR
13.8 min, purity 99.4%.
6.8.5.15. N-Hydroxy-4-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
1
yl)phenoxy)butanamide (12c). White solid, yield: 28%. H NMR
(400 MHz, DMSO) δ 10.43 (s, 1H), 8.72 (s, 1H), 8.55 (s, 1H), 7.96
(d, J = 7.1 Hz, 2H), 7.63 (s, 2H), 7.09 (d, J = 6.9 Hz, 2H), 4.05 (s,
2H), 3.87 (s, 6H), 3.79 (s, 3H), 2.15 (s, 2H), 1.97 (s, 3H). 13C NMR
(101 MHz, DMSO) δ 1184.63, 168.59, 167.26, 160.66, 153.91,
152.48, 141.91, 131.82, 129.64, 129.11, 128.05, 125.22, 115.28,
107.98, 67.15, 60.22, 55.98, 29.02, 28.64. M.p.: 126.4−127.9 °C.
HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for C23H25N2O7S:
473.1304, found: 473.1300. HPLC: tR 14.0 min, purity 99.7%.
6.8.5.16. N-Hydroxy-5-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
6 . 8 . 5 . 2 2 . N 1 - ( 2 - A m i n o p h e n y l ) - N 4 - ( 4 - ( 4 - ( 3 , 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenyl)succinamide (15b). Yellow
solid, yield: 37%. 1H NMR (400 MHz, DMSO) δ 10.30 (s, 1H), 9.20
(s, 1H), 8.67 (s, 2H), 8.56 (s, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.77 (d, J
= 7.8 Hz, 2H), 7.13 (d, J = 7.5 Hz, 1H), 6.89 (s, 1H), 6.70 (d, J = 7.7
Hz, 1H), 6.53 (d, J = 7.5 Hz, 1H), 4.86 (s, 2H), 3.87 (s, 6H), 3.79 (s,
3H), 2.81 (s, 4H). 13C NMR (101 MHz, DMSO) δ 176.75, 166.46,
154.17, 152.50, 141.97, 134.74, 131.96, 131.71, 130.42, 127.93,
126.84, 107.98, 60.22, 56.00, 28.55. M.p.: 125.3−127.0 °C. HRMS
1
yl)phenoxy)pentanamide (12d). White solid, yield: 26%. H NMR
(400 MHz, DMSO) δ 10.40 (s, 1H), 8.71 (s, 1H), 8.54 (s, 1H), 7.94
(d, J = 8.2 Hz, 2H), 7.64 (s, 2H), 7.08 (d, J = 8.2 Hz, 3H), 4.04 (s,
2H), 3.87 (s, 6H), 3.79 (s, 3H), 2.04 (d, J = 6.6 Hz, 2H), 1.76−1.64
(m, 4H). 13C NMR (101 MHz, DMSO) δ 184.58, 168.95, 167.28,
160.76, 153.94, 152.48, 141.92, 131.82, 129.06, 128.03, 125.15,
115.25, 108.00, 67.44, 60.35, 60.23, 55.98, 31.89, 28.09, 21.74. M.p.:
123.2−125.7 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
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Article
(ESI-Q-TOF) m/z: [M + H]+ calculated for C29H29N4O6S: 561.1730,
found: 561.1736. HPLC: tR 13.5 min, purity 98.7%.
6.94 (s, 1H), 6.76 (s, 1H), 6.59 (d, J = 6.7 Hz, 1H), 5.00 (s, 2H), 3.88
(s, 6H), 3.80 (s, 3H).13C NMR (101 MHz, DMSO) δ 184.67, 167.04,
162.57, 162.00, 154.03, 152.50, 141.93, 141.66, 141.10, 134.68,
133.18, 131.79, 129.53, 127.93, 127.30, 126.24, 124.83, 122.74,
119.81, 116.13, 115.91, 107.98, 60.22, 55.99, 54.89. M.p.: 137.1−
138.2 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C29H27N4O6S: 559.1573, found: 559.1570. HPLC: tR 17.3 min, purity
97.7%.
6 . 8 . 5 . 2 3 . N 1 - ( 2 - A m i n o p h e n y l ) - N 5 - ( 4 - ( 4 - ( 3 , 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenyl)glutaramide (15c). Yellow
solid, yield: 39%. 1H NMR (400 MHz, DMSO) δ 10.23 (s, 1H), 9.11
(s, 1H), 8.57 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz,
2H), 7.63 (s, 2H), 7.18 (d, J = 7.7 Hz, 1H), 6.89 (t, J = 7.4 Hz, 1H),
6.71 (d, J = 7.8 Hz, 1H), 6.54 (d, J = 7.3 Hz, 1H), 4.83 (s, 2H), 3.87
(s, 7H), 3.80 (s, 3H), 2.42 (dt, J = 14.1, 7.1 Hz, 4H), 2.00−1.86 (m,
2H).13C NMR (101 MHz, DMSO) δ 184.67, 171.35, 167.21, 153.96,
152.48, 141.92, 131.80, 129.32, 127.14, 125.73, 125.39, 123.45,
119.37, 116.11, 115.83, 107.97, 60.22, 55.97, 35.74, 34.91, 21.06.
M.p.: 125.3−127.6 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C30H31N4O6S: 575.1886, found: 575.1885. HPLC: tR
14.3 min, purity 98.3%.
6 . 8 . 5 . 2 9 . N 2 - ( 2 - A m i n o p h e n y l ) - N 5 - ( 4 - ( 4 - ( 3 , 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenyl)thiophene-2,5-dicarboxa-
mide (15i). Yellow solid, yield: 23%. 1H NMR (400 MHz, DMSO) δ
10.17 (s, 1H), 9.08 (s, 1H), 8.57 (s, 2H), 8.31 (s, 1H), 7.96 (d, J = 8.8
Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.63 (s, 2H), 7.14 (d, J = 7.6 Hz,
1H), 6.89 (d, J = 6.8 Hz, 1H), 6.70 (d, J = 7.6 Hz, 1H), 6.54 (t, J
=13.2 Hz, 1H), 4.80 (s, 2H), 3.87 (s, 6H), 3.80 (s, 3H). 13C NMR
(101 MHz, DMSO) δ 169.16, 167.37, 154.02, 152.90, 142.42, 141.98,
138.55, 132.19, 128.68, 125.84, 123.66, 117.14, 116.52, 108.38, 60.63,
55.96. M.p.: 135.3−136.8 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C31H27N4O6S2: 615.1294, found: 615.1299. HPLC: tR
17.3 min, purity 98.1%.
6 . 8 . 5 . 2 4 . N 1 - ( 2 - A m i n o p h e n y l ) - N 6 - ( 4 - ( 4 - ( 3 , 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenyl)adipamide (15d). Yellow
1
solid, yield: 30%. H NMR (400 MHz, DMSO) δ 10.20 (s, 1H),
9.10 (s, 1H), 8.57 (s, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.79 (d, J = 5.7
Hz, 2H), 7.63 (s, 2H), 7.15 (s, 1H), 6.89 (s, 1H), 6.72 (s, 1H), 6.53
(s, 1H), 4.82 (s, 2H), 3.87 (s, 7H), 3.80 (s, 3H), 2.41−2.26 (m, 4H),
1.66 (s, 2H), 1.60 (s, 2H).13C NMR (101 MHz, DMSO) δ 184.62,
173.21, 171.56, 170.97, 167.18, 153.95, 152.47, 141.90, 131.78,
129.27, 127.12, 125.70, 125.31, 123.50, 119.32, 116.14, 115.86,
107.96, 60.19, 56.01, 55.96, 51.20, 36.35, 33.02, 25.02, 18.53. M.p.:
132.7−134.3 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C31H33N4O6S: 589.2043, found: 589.2047. HPLC: tR 13.1 min, purity
96.8%.
6.8.5.30. N1-Hydroxy-N3-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-
1
2-yl)phenyl)malonamide (17a). White solid, yield: 20%. H NMR
(400 MHz, DMSO) δ 10.32 (s, 1H), 10.26 (s, 1H), 8.68 (s, 1H), 8.39
(s, 1H), 8.15 (d, J = 8.3 Hz, 2H), 7.64 (d, J = 3.9 Hz, 3H), 7.48 (d, J =
8.3 Hz, 2H), 6.66 (d, J = 8.4 Hz, 2H), 3.87 (d, J = 3.0 Hz, 6H), 3.79
(d, J = 2.7 Hz, 3H), 2.81 (s, 2H). 13C NMR (101 MHz, DMSO) δ
184.74, 173.32, 171.53, 167.26, 154.00, 152.53, 141.97, 141.66,
131.85, 129.70, 129.38, 127.20, 119.40, 108.01, 99.57, 60.28, 56.02,
36.12. M.p.: 145.7−147.0 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C22H22N3O7S: 472.1100, found: 472.1109. HPLC: tR
16.5 min, purity 97.9%.
6 . 8 . 5 . 2 5 . N 1 - ( 2 - A m i n o p h e n y l ) - N 7 - ( 4 - ( 4 - ( 3 , 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenyl)heptanediamide (15e). Yel-
low solid, yield: 37%. 1H NMR (400 MHz, DMSO) δ 10.18 (s, 1H),
9.08 (s, 1H), 8.57 (s, 1H), 7.97 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.4
Hz, 2H), 7.63 (s, 2H), 7.15 (d, J = 7.7 Hz, 1H), 6.88 (s, 1H), 6.71 (d,
J = 7.8 Hz, 1H), 6.52 (s, 1H), 4.80 (s, 2H), 3.87 (s, 6H), 3.80 (s, 3H),
2.41−2.29 (m, 4H), 1.64 (dd, J = 14.5, 7.2 Hz, 4H), 1.38 (d, J = 7.1
Hz, 2H). 13C NMR (101 MHz, DMSO) δ 184.66, 171.65, 171.09,
167.19, 153.94, 152.47, 141.85, 141.68, 131.79, 129.29, 127.13,
125.66, 125.26, 123.54, 119.31, 116.13, 115.85, 107.95, 60.20, 55.96,
36.36, 35.61, 25.07, 24.81, 18.53. M.p.: 125.3−127.8 °C. HRMS (ESI-
Q-TOF) m/z: [M + H]+ calculated for C32H35N4O6S: 603.2199,
found: 603.2207. HPLC: tR 15.7 min, purity 98.3%.
6.8.5.31. N1-Hydroxy-N4-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-
1
2-yl)phenyl)succinamide (17b). White solid, yield: 25%. H NMR
(400 MHz, DMSO) δ 10.46 (s, 1H), 10.25 (s, 1H), 8.65 (s, 1H), 8.57
(s, 1H), 8.15 (d, J = 8.0 Hz, 2H), 7.63 (s, 2H), 7.48 (d, J = 8.0 Hz,
2H), 3.87 (s, 6H), 3.80 (s, 3H), 2.81 (s, 4H). 13C NMR (101 MHz,
DMSO) δ 184.01, 176.66, 166.37, 154.08, 152.41, 141.88, 138.55,
134.36, 131.62, 130.38, 127.85, 126.77, 107.89, 99.45, 60.17, 55.96,
28.51. M.p.: 135.7−136.9 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C23H24N3O7S: 486.1257, found: 486.1251. HPLC: tR
16.7 min, purity 98.7%.
6.8.5.32. N1-Hydroxy-N5-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-
6 . 8 . 5 . 2 6 . N 1 - ( 2 - A m i n o p h e n y l ) - N 8 - ( 4 - ( 4 - ( 3 , 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenyl)octanediamide (15f). Yel-
low solid, yield: 32%. 1H NMR (400 MHz, DMSO) δ 10.41 (s,
1H), 9.28 (s, 1H), 8.60 (s, 1H), 7.99 (d, J = 7.1 Hz, 2H), 7.85 (d, J =
6.7 Hz, 2H), 7.67 (s, 2H), 7.21 (d, J = 6.6 Hz, 1H), 6.91 (s, 1H), 6.74
(d, J = 7.3 Hz, 1H), 6.56 (s, 1H), 4.90 (s, 2H), 3.91 (s, 6H), 3.83 (s,
3H), 2.38 (d, J = 7.0 Hz, 4H), 2.33 (s, 2H), 1.64 (s, 3H), 1.33 (s,
3H). 13C NMR (101 MHz, DMSO) δ 173.37, 171.75, 167.25, 152.48,
141.80, 131.80, 127.09, 125.21, 119.34, 115.87, 107.98, 60.23, 55.98,
51.17, 38.87, 33.23, 28.47, 24.86, 24.31. M.p.: 128.5−130.1 °C.
HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for C33H37N4O6S:
617.2356, found: 617.2359. HPLC: tR 15.4 min, purity 98.5%.
6 . 8 . 5 . 2 7 . N 1 - ( 2 - A m i n o p h e n y l ) - N 4 - ( 4 - ( 4 - ( 3 , 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenyl)terephthalamide (15g). Yel-
low solid, yield: 32%. 1H NMR (400 MHz, DMSO) δ 10.66 (s, 1H),
9.82 (d, J = 9.4 Hz, 1H), 8.61 (s, 1H), 8.11 (d, J = 8.6 Hz, 3H), 8.07−
7.96 (m, 2H), 7.65 (s, 1H), 7.19 (d, J = 7.4 Hz, 1H), 6.99 (t, J = 6.9
Hz, 1H), 6.80 (d, J = 7.9 Hz, 1H), 6.61 (t, J = 7.2 Hz, 1H), 4.94 (s,
2H), 3.89 (s, 6H), 3.81 (s, 3H). 13C NMR (101 MHz, DMSO) δ
186.42, 181.51, 171.29, 165.11, 154.42, 152.91, 143.67, 137.41,
132.21, 128.29, 128.12, 127.48, 127.24, 127.08, 123.44, 121.11,
116.63, 116.52, 108.40, 60.64, 56.39. M.p.: 133.1−134.2 °C. HRMS
(ESI-Q-TOF) m/z: [M + H]+ calculated for C33H29N4O6S: 609.1730,
found: 609.1735. HPLC: tR 18.2 min, purity 96.8%.
1
2-yl)phenyl)glutaramide (17c). Yellow solid, yield: 20%. H NMR
(400 MHz, DMSO) δ 10.39 (s, 1H), 10.19 (s, 1H), 8.57 (s, 1H), 8.39
(s, 1H), 7.97 (d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.6 Hz, 2H), 7.63 (s,
2H), 3.87 (s, 9H), 3.80 (s, 3H), 2.40 (t, J = 7.3 Hz, 2H), 2.29 (t, J =
7.3 Hz, 2H), 1.89−1.75 (m, 2H). 13C NMR (101 MHz, DMSO) δ
184.63, 174.13, 171.17, 167.18, 153.96, 152.47, 152.42, 141.90,
141.61, 131.79, 129.29, 127.81, 127.13, 119.34, 113.69, 107.96, 60.20,
55.96, 55.93, 35.45, 32.93, 20.31. M.p.: 144.1−146.2 °C. HRMS (ESI-
Q-TOF) m/z: [M + H]+ calculated for C24H26N3O7S: 500.1413,
found: 500.1418. HPLC: tR 16.3 min, purity 95.9%.
6.8.5.33. N1-Hydroxy-N6-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-
2-yl)phenyl)adipamide (17d). Yellow solid, yield: 21%. 1H NMR
(400 MHz, DMSO) δ 10.36 (s, 1H), 10.18 (s, 1H), 8.67 (s, 1H), 8.57
(s, 1H), 7.97 (d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.63 (s,
2H), 3.87 (s, 6H), 3.80 (s, 3H), 2.34 (d, J = 6.5 Hz, 2H), 1.99 (d, J =
6.9 Hz, 2H), 1.56 (s, 4H). 13C NMR (101 MHz, DMSO) δ 184.62,
174.33, 171.47, 167.18, 153.95, 152.46, 141.89, 141.64, 131.78,
129.29, 127.13, 119.31, 107.95, 60.19, 55.95, 36.17, 33.40, 24.54,
24.10. M.p.: 144.6−147.2 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C25H28N3O7S: 514.1570, found: 514.1577. HPLC: tR
15.9 min, purity 98.5%.
6.8.5.34. N1-Hydroxy-N7-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-
2-yl)phenyl)heptanediamide (17e). White solid, yield: 29%. 1H
NMR (400 MHz, DMSO) δ 10.33 (s, 1H), 10.16 (s, 1H), 8.65 (s,
1H), 8.57 (s, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.77 (d, J = 8.7 Hz, 2H),
7.63 (s, 2H), 3.87 (s, 6H), 3.80 (s, 3H), 2.34 (t, J = 7.3 Hz, 2H), 1.95
(t, J = 7.3 Hz, 2H), 1.59 (dd, J = 14.9, 7.5 Hz, 2H), 1.55−1.46 (m,
2H), 1.29 (dd, J = 9.3, 5.3 Hz, 2H).13C NMR (101 MHz, DMSO) δ
6 . 8 . 5 . 2 8 . N 1 - ( 2 - A m i n o p h e n y l ) - N 4 - ( 4 - ( 4 - ( 3 , 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenyl)fumaramide (15h). Yellow
solid, yield: 24%. 1H NMR (400 MHz, DMSO) δ 10.79 (s, 1H), 9.70
(s, 1H), 8.59 (s, 1H), 8.03 (d, J = 7.6 Hz, 2H), 7.88 (d, J = 7.8 Hz,
2H), 7.63 (s, 2H), 7.36−7.24 (m, 1H), 7.19 (d, J = 14.8 Hz, 1H),
8467
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Article
184.63, 171.59, 169.01, 167.17, 153.93, 152.46, 141.88, 141.66,
131.78, 129.28, 127.12, 127.04, 119.28, 107.94, 60.19, 55.95, 36.31,
32.12, 28.20, 24.89, 24.72. M.p.: 134.2−136.5 °C. HRMS (ESI-Q-
TOF) m/z: [M + H]+ calculated for C26H30N3O7S: 528.1726, found:
528.1723. HPLC: tR 16.6 min, purity 97.2%.
131.82, 130.42, 127.93, 126.84, 108.28, 60.22, 56.00. M.p.: 134.6−
136.2 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C28H29N4O5S: 533.1780, found: 533.1789. HPLC: tR 17.3 min, purity
97.8%.
6.8.5.41. N-(2-Aminophenyl)-7-((4-(4-(3,4,5-trimethoxybenzoyl)-
6.8.5.35. N1-Hydroxy-N8-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-
2-yl)phenyl)octanediamide (17f). White solid, yield: 28%. 1H
NMR (400 MHz, DMSO) δ 10.32 (s, 1H), 10.16 (s, 1H), 8.64 (s,
1H), 8.57 (s, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.77 (d, J = 8.3 Hz, 2H),
7.63 (s, 2H), 3.87 (s, 6H), 3.80 (s, 3H), 2.34 (t, J = 7.2 Hz, 2H),
2.04−1.90 (m, 2H), 1.58 (d, J = 6.8 Hz, 2H), 1.53−1.41 (m, 4H). 13C
NMR (101 MHz, DMSO) δ 184.64, 171.65, 169.07, 167.18, 153.94,
152.46, 141.89, 141.68, 131.78, 129.62, 129.29, 127.12, 127.04,
119.30, 107.95, 60.19, 55.95, 29.05, 29.00, 28.55, 28.37, 26.53, 24.90,
13.92. M.p.: 133.3−135.0 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C27H32N3O7S: 542.1883, found: 542.1881. HPLC: tR
16.1 min, purity 98.5%.
thiazol-2-yl)phenyl)amino)heptanamide (20c). White solid, yield:
1
30%. H NMR (400 MHz, DMSO) δ 10.17 (s, 1H), 8.57 (s, 1H),
7.97 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 7.63 (s, 2H), 7.14
(d, J = 7.2 Hz, 1H), 6.93−6.85 (m, 1H), 6.71 (d, J = 7.8 Hz, 1H),
6.54 (d, J = 7.2 Hz, 1H), 5.82 (s, 1H), 4.80 (s, 2H), 3.87 (s, 6H), 3.80
(s, 3H), 3.62 (s, 2H), 2.39−2.23 (m, 4H), 1.61 (s, 3H), 1.10−0.96
(m, 3H). 13C NMR (101 MHz, DMSO) δ 184.97, 173.37, 167.25,
152.48, 141.80, 131.80, 127.09, 125.21, 119.34, 115.87, 107.98, 60.23,
55.98, 51.17, 33.23, 28.47, 24.86, 24.31. M.p.: 146.4−148.1 °C.
HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for C32H37N4O5S:
589.2406, found: 589.2409. HPLC: tR 17.4 min, purity 96.8%.
6.8.5.42. N-Hydroxy-2-((4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
yl)phenyl)amino)acetamide (22a). White solid, yield: 24%. 1H NMR
(400 MHz, DMSO) δ 10.18 (s, 1H), 8.73 (s, 1H), 8.57 (s, 1H), 7.97
(d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.64 (s, 2H), 5.86 (s,
1H), 3.88 (s, 6H), 3.80 (s, 3H), 3.72 (s, 2H), 2.39− 2.23 (m, 4H),
1.61 (s, 3H), 1.10−0.96 (m, 3H). 13C NMR (101 MHz, DMSO) δ
177.98, 167.22, 153.12, 152.53, 150.90, 143.97, 134.78, 132.08,
131.82, 130.42, 127.93, 126.84, 108.28, 61.23, 57.07, 53.53. M.p.:
120.1−121.2 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C21H22N3O6S: 444.1151, found: 444.1156. HPLC: tR 18.1 min, purity
98.5%.
6.8.5.43. N-Hydroxy-3-((4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
yl)phenyl)amino)propanamide (22b). White solid, yield: 18%.1H
NMR (400 MHz, DMSO) δ 10.25 (s, 1H), 8.69 (s, 1H), 8.53 (s, 1H),
7.84 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 7.54 (s, 2H), 5.83
(s, 1H), 3.87 (s, 6H), 3.75 (s, 3H), 3.72 (s, 2H), 2.62 (dd, J = 4.4 Hz,
4.0 Hz, 2H), 1.61 (s, 3H), 1.10−0.96 (m, 3H). 13C NMR (101 MHz,
DMSO) δ 181.35, 158.39, 154.45, 152.52, 141.12, 132.24, 127.43,
126.17, 122.47, 121.28, 116.46, 108.39, 61.45, 56.38, 52.38, 16.30.
M.p.: 132.6−133.9 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for: C22H24N3O6S: 458.1308, found: 458.1302. HPLC: tR
18.1 min, purity 95.9%.
6.8.5.36. N1-Hydroxy-N4-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-
2-yl)phenyl)terephthalamide (17g). White solid, yield: 24%. 1H
NMR (400 MHz, DMSO) δ 11.40 (s, 1H), 10.62 (s, 1H), 9.17 (s,
1H), 8.61 (s, 1H), 8.06 (dd, J = 9.7, 7.6 Hz, 4H), 8.00 (d, J = 8.8 Hz,
2H), 7.90 (d, J = 8.3 Hz, 2H), 7.64 (s, 2H), 3.88 (s, 6H), 3.80 (s,
3H). 13C NMR (101 MHz, DMSO) δ 184.68, 167.10, 154.00, 152.49,
147.10, 142.21, 141.10, 131.79, 129.18, 127.85, 127.06, 120.62,
107.97, 60.22, 55.98. M.p.: 131.0−132.2 °C. HRMS (ESI-Q-TOF)
m/z: [M + H]+ calculated for C27H24N3O7S: 534.1257, found:
534.1262. HPLC: tR 16.0 min, purity 99.1%.
6.8.5.37. N1-Hydroxy-N4-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-
1
2-yl)phenyl)fumaramide (17h). White solid, yield: 24%. H NMR
(400 MHz, DMSO) δ 10.77 (s, 1H), 10.72 (s, 1H), 8.81 (s, 1H), 8.60
(s, 1H), 8.02 (d, J = 8.6 Hz, 2H), 7.85 (d, J = 8.6 Hz, 2H), 7.63 (s,
2H), 7.12 (d, J = 15.1 Hz, 1H), 6.86 (d, J = 15.2 Hz, 1H), 3.88 (s,
6H), 3.80 (s, 3H). 13C NMR (101 MHz, DMSO) δ 184.64, 171.65,
169.07, 167.18, 153.94, 152.46, 141.89, 141.68, 131.78, 129.62,
129.29, 127.12, 127.04, 119.30, 107.95, 60.19, 55.95. M.p.: 140.1−
141.7 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C23H22N3O7S: 484.1100, found: 484.1105. HPLC: tR 15.5 min, purity
97.2%.
6.8.5.38. N2-Hydroxy-N5-(4-(4-(3,4,5-trimethoxybenzoyl) thiazol-
2-yl) phenyl)thiophene-2,5-dicarboxamide (17i). White solid, yield:
6.8.5.44. N-Hydroxy-7-((4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
1
yl)phenyl)amino)heptanamide (22c). White solid, yield: 20%. H
1
25%. H NMR (400 MHz, DMSO) δ 11.49 (s, 1H), 10.73 (s, 1H),
NMR (400 MHz, DMSO) δ 10.16 (s, 1H), 8.65 (s, 1H), 8.57 (s, 1H),
7.96 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.63 (s, 2H), 5.82
(s, 1H), 3.87 (s, 6H), 3.79 (s, 3H), 3.56 (s, 2H), 2.33 (t, J = 7.2 Hz,
2H), 2.01−1.92 (m, 4H), 1.57 (t, J = 7.2 Hz, 2H), 1.49 (t, J = 6.8 Hz,
2H).13C NMR (101 MHz, DMSO) δ 184.63, 171.50, 168.92, 167.17,
153.94, 152.46, 141.89, 131.78, 129.29, 127.13, 119.30, 107.95, 60.19,
55.95, 36.26, 32.14, 24.77, 21.25. M.p.: 129.7−131.2 °C. HRMS (ESI-
Q-TOF) m/z: [M + H]+ calculated for C26H32N3O6S: 514.1934,
found: 514.1937. HPLC: tR 18.2 min, purity 97.2%.
9.33 (s, 1H), 8.60 (s, 1H), 8.11 (d, J = 3.8 Hz, 1H), 8.04 (d, J = 8.6
Hz, 2H), 7.97 (d, J = 8.6 Hz, 2H), 7.68 (s, 1H), 7.64 (s, 2H), 3.87 (d,
J = 5.9 Hz, 6H), 3.79 (d, J = 4.3 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 184.65, 167.03, 160.83, 153.98, 152.48, 141.91, 140.92,
131.77, 129.57, 127.94, 127.80, 127.06, 124.91, 120.70, 107.96, 60.21,
55.97. M.p.: 134.1−136.5 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C25H22N3O7S2: 540.0821, found: 540.0822. HPLC: tR
16.1 min, purity 96.6%.
6.8.5.39. N-(2-Aminophenyl)-2-((4-(4-(3,4,5-trimethoxybenzoyl)-
6.8.5.45. N-(2-Aminophenyl)-5-(4-(4-(3,4,5-trimethoxybenzoyl)-
thiazol-2-yl)phenyl)pent-4-ynamide (25a). White solid, yield: 23%.
1H NMR (400 MHz, DMSO) δ 9.28 (s, 1H), 8.65 (s, 1H), 8.00 (d, J
thiazol-2-yl)phenyl)amino)acetamide (20a). White solid, yield:
1
30%. H NMR (400 MHz, DMSO) δ 10.23 (s, 1H), 7.79 (d, J =
8.5 Hz, 2H), 7.65 (s, 2H), 7.15 (d, J = 7.6 Hz, 1H), 6.91 (t, J = 7.0
Hz, 1H), 6.75 (d, J = 8.6 Hz, 2H), 6.72 (s, 1H), 6.71−6.66 (m, 1H),
6.54 (t, J = 7.2 Hz, 1H), 5.82 (s, 1H), 4.84 (s, 2H), 3.99 (d, J = 5.8
Hz, 2H), 3.87 (s, 6H), 3.80 (d, J = 5.0 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 183.32, 168.33, 158.38, 158.88, 154.45, 152.91, 152.84,
143.82, 142.35, 140.46, 132.20, 127.49, 127.38, 126.11, 122.42,
121.28, 116.79, 116.68, 108.39, 60.69, 56.39, 18.30. M.p.: 141.8−
143.1 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C27H27N4O5S: 519.1624, found: 519.1633. HPLC: tR 16.4 min, purity
98.4%.
= 7.3 Hz, 2H), 7.60 (s, 2H), 7.55 (d, J = 7.7 Hz, 2H), 7.14 (d, J = 7.3
Hz, 1H), 6.90 (d, J = 6.8 Hz, 1H), 6.72 (d, J = 7.5 Hz, 1H), 6.55 (d, J
= 6.8 Hz, 1H), 4.84 (s, 2H), 3.86 (s, 6H), 3.79 (s, 3H), 2.77 (s, 2H),
2.64 (s, 2H). 13C NMR (101 MHz, DMSO) δ 184.76, 169.53, 166.61,
154.20, 152.57, 142.27, 142.06, 132.36, 131.78, 130.38, 126.61,
126.16, 125.69, 123.18, 116.25, 115.87, 108.05, 60.32, 56.07, 34.71,
15.55. M.p.: 140.2−141.9 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+
calculated for C30H28N3O5S: 542.1671, found: 542.1674. HPLC: tR
18.2 min, purity 97.9%.
6.8.5.46. N-Hydroxy-5-(4-(4-(3,4,5-trimethoxybenzoyl)thiazol-2-
6.8.5.40. N-(2-Aminophenyl)-3-((4-(4-(3,4,5-trimethoxybenzoyl)-
1
yl)phenyl)pent-4-ynamide (27a). White solid, yield: 33%. H NMR
thiazol-2-yl)phenyl)amino)propanamide (20b). White solid, yield:
1
(400 MHz, DMSO) δ 10.51 (s, 1H), 8.83 (s, 1H), 8.67 (s, 1H), 8.01
(d, J = 6.8 Hz, 2H), 7.61 (s, 2H), 7.54 (d, J = 7.0 Hz, 2H), 3.87 (s,
6H), 3.80 (s, 3H), 2.68 (s, 2H), 2.27 (s, 2H). 13C NMR (101 MHz,
DMSO) δ 184.62, 172.85, 166.48, 154.13, 152.48, 141.97, 132.25,
131.69, 130.29, 126.52, 125.32, 107.98, 92.36, 80.00, 60.21, 55.98,
32.92, 14.86. M.p.: 133.7−135.2 °C. HRMS (ESI-Q-TOF) m/z: [M +
28%. H NMR (400 MHz, DMSO) δ 10.15 (s, 1H), 8.41 (s, 1H),
7.76 (d, J = 8.6 Hz, 2H), 7.65 (s, 2H), 7.20−7.13 (m, 1H), 6.96−6.84
(m, 1H), 6.72 (t, J = 8.0 Hz, 3H), 6.57−6.49 (m, 1H), 6.44 (s, 1H),
5.75 (s, 1H), 4.85 (s, 2H), 3.87 (s, 6H), 3.79 (s, 3H), 3.43 (dd, J =
12.5, 6.5 Hz, 2H), 2.64 (t, J = 6.8 Hz, 2H). 13C NMR (101 MHz,
DMSO) δ 177.89, 167.22, 153.12, 152.53, 143.97, 134.78, 132.08,
8468
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Article
H]+ calculated for C24H23N2O6S: 467.1199, found: 467.1193. HPLC:
tR 17.7 min, purity 98.0%.
6.9.3. In Vitro Tubulin Polymerization Assay. Pig brain micro-
tubule protein was isolated via three cycles of temperature-dependent
assembly/disassembly in 100 mM PIPES (pH 6.5), 1 mM GTP, 1
mM MgSO4, 2 mM EGTA, and 1 mM 2-mercaptoethanol. Glycerol
and phenylmethylsulfonyl fluoride were added to 4 M and 0.2 mM,
respectively, in the first cycle of polymerization. Tubulin was prepared
from microtubule protein by phosphocellulose (P11) chromatography
and stored at −70 °C.
Tubulin was mixed with different concentrations of a compound in
PEM buffer (100 mM PIPES, 1 mM MgCl2, and 1 mM EGTA)
containing 1 mM GTP and 5% glycerol. Microtubule polymerization
was detected using a spectrophotometer at 340 nm at 37 °C. The
plateau absorbance values were used for calculations.
6.9.4. Immunofluorescence Staining. B16-F10 cells were seeded
on glass coverslips in 24-well plates and then treated with vehicle
control 0.1% DMSO, compound SMART (60 nM), compound 15c
(60 nM), and compound 32a (60 nM) for 6 h. The cells were fixed
with 4% paraformaldehyde and then penetrated with PBS containing
0.2% Triton X-100. After blocking for 30 min by adding 100 μL of
goat serum albumin at room temperature, the cells were incubated
with a monoclonal antibody (anti-β-tubulin) at 37 °C for 1 h. Then,
the cells were washed three times with PBS following staining with the
fluorescence secondary antibody. Nucleus was labeled with 4,6-
diamidino-2-phenylindole (DAPI). The cells were finally visualized
using a fluorescence microscope.
6.9.5. Wound-Healing Assay. B16-F10 cells were seeded and
incubated in a six-well plate overnight. Scratches were made in
confluent monolayers by a 200 μL pipette tip. Then, wounds were
washed twice with media to remove debris and uprooted cells. The
cells were treated with SMART (60 nM) and different concentrations
(0, 20, 40, 60 nM) of compound 15c. Images were obtained using
phase-contrast microscopy at 0 and 24 h. The migration distance of
cells in the wound area was measured manually.
6.9.6. Colony Formation Assay. B16-F10 cells (1000 units) were
counted and seeded in 6-well plates. After being cultured for 24 h at
37 °C, the medium was replaced with a medium added with
compound SMART and 15c at the indicated concentrations of 0, 20,
40, and 60 nM. After 24 h treatment, the medium was changed to
normal. Cultured for another 10−14 days, the colonies were fixed
with 4% paraformaldehyde for 30 min and stained with 0.1% Crystal
Violet for 15 min. Following washing with PBS, the colonies were
then photographed and quantified using ImageJ.
6.9.7. Analysis of Cancer Cell Apoptosis. B16-F10 cells were
seeded into 6-well plates and cultured overnight. After treating with
15c for 48 h, the cells were harvested and suspended in a binding
buffer containing Annexin V-FITC and PI and then analyzed with a
flow cytometer (BD FACSCanto II).
6.9.8. Cell Cycle Analysis. B16-F10 cells (1000 units) were seeded
into 6-well plates and cultured overnight. The cells were treated with
15c (25, 50, 100 nM), SMART (100 nM), and MS-275 (100 nM) for
48 h. The collected cells were fixed at 4 °C with 70% ethanol
overnight. The fixed cells were washed and resuspended using PBS
with RNase A and PI. After being incubated for 30 min in the dark,
the samples were analyzed for the DNA content with flow cytometry
(BD FACSCanto II).
6 . 8 . 5 . 4 7 . ( E ) - N - ( 2 - A m i n o p h en y l) -3 -( 4 - (4 - (3 , 4 , 5 -
trimethoxybenzoyl)thiazol-2-yl)phenyl)acrylamide (30a). White
solid, yield: 29%. 1H NMR (400 MHz, DMSO) δ 9.44 (s, 1H),
8.68 (s, 1H), 8.11 (d, J = 7.9 Hz, 2H), 7.80 (d, J = 7.9 Hz, 2H), 7.64
(s, 2H), 7.59 (s, 1H), 7.37 (d, J = 7.6 Hz, 1H), 7.02 (d, J = 15.9 Hz,
1H), 6.93 (t, J = 7.2 Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H), 6.59 (t, J = 7.4
Hz, 1H), 4.96 (s, 2H), 3.88 (s, 6H), 3.81 (s, 3H).13C NMR (101
MHz, DMSO) δ 184.65, 166.65, 163.24, 154.21, 152.52, 142.00,
141.59, 138.40, 137.22, 133.15, 131.72, 130.35, 128.63, 127.00,
125.88, 124.70, 123.75, 116.16, 108.01, 60.24, 56.02. M.p.: 137.3−
138.5 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C28H26N3O5S: 516.1515, found: 516.1518. HPLC: tR 16.8 min, purity
96.0%.
6.8.5.48. (E)-N-Hydroxy-3-(4-(4-(3,4,5-trimethoxybenzoyl)-
thiazol-2-yl)phenyl)acrylamide (32a). White solid, yield: 12%.1H
NMR (400 MHz, DMSO) δ 10.85 (s, 1H), 9.01 (d, J = 92.2 Hz, 1H),
8.66 (s, 1H), 8.06 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H), 7.63
(s, 2H), 7.51 (d, J = 15.8 Hz, 1H), 6.58 (d, J = 15.8 Hz, 1H), 3.87 (s,
6H), 3.80 (s, 3H). 13C NMR (101 MHz, DMSO) δ 184.68, 166.71,
162.51, 154.23, 152.55, 142.04, 137.32, 137.18, 133.06, 131.75,
130.33, 128.54, 126.96, 120.68, 108.04, 60.29, 56.04. M.p.: 141.7−
143.0 °C. HRMS (ESI-Q-TOF) m/z: [M + H]+ calculated for
C22H21N2O6S: 441.1042, found: 441.1044. HPLC: tR 16.8 min, purity
95.3%.
6.9. Biological Assays. 6.9.1. Cell Culture and Cytotoxicity
Assay. The cancer cell lines were purchased from American Type
Culture Collection (ATCC). The cytotoxicity of all compounds was
evaluated against the HCT-116 (human colon cancer cell), B16-F10
(mouse melanoma cell), and A549 (human lung cancer cell) cell lines
using the standard MTT assay in vitro, Jurkat (human T lymphocyte
cell) treating CCK-8 assay. All of the cancer cell lines were cultured in
RPMI-1640 medium supplemented with 10% fetal bovine serum
(FBS). The cells were seeded into 96-well plates at a density of 5000
cells/well. After being incubated overnight, the cells were treated with
100 μL of medium with 0.1% DMSO containing our antitubulin
compounds at different concentrations and cultured at 37 °C for
another 48 h. The MTT or CCK-8 (0.5 mg/mL in PBS) was added
and incubated for another 4 h, and then the absorbance was detected
with a microplate reader at a wavelength of 570 nm. The IC50 values
were calculated by nonlinear regression analysis using GraphPad
Prism. All of the experiments were repeated in at least three
independent experiments.
6.9.2. In Vitro HDAC Inhibition Assay. The HDAC1 (#ab101661),
HDAC6 (#ab42632), and HDAC7 (#ab101660) enzymes were
purchased from Abcam, HDAC3 (#BML-SE515-0050) was pur-
chased from ENZO Inc, and HDAC8 (#H90-30H-05) was purchased
from SignalChem. The reaction mixture contained 25 mM Tris (pH
8.0), 1 mM MgCl2, 0.1 mg/mL BSA, 137mM NaCl, 2.7 mM KCl,
HDAC (HDAC1: 7.2 ng/well; HDAC3: 3.4 ng/well; HDAC6: 15
ng/well; HDAC7: 1.6 ng/well; HDAC8: 22 ng/well) in a total
volume of 40 μL. The test compounds (3-fold dilution, 10
concentrations) were diluted in 10% DMSO, and 5 μL of the
dilution was added and preincubated with purified recombinant
HDAC at room temperature for 5 min before substrate addition.
Finally, the enzyme−substrate (Ac-Leu-Gly-Lys (Ac)-AMC, 10 μM
for HDAC1, 3, and 6; Ac-Leu-Gly-Lys (Tfa)-AMC, 2 μM for
HDAC7, 8) was added and the plate was incubated at 37 °C for 30
min in a final volume of 50 μL. The reaction was quenched with 50
μL of HDAC assay developer (1 mg/mL trypsin and 2 μM TSA in
assay buffer) for 30 min at room temperature. The assay was
performed by quantitating the fluorescent product amount in solution
following an enzyme reaction. Fluorescence was then analyzed with an
excitation of 350−360 nm and an emission wavelength of 450−460
nm at a TECAN microtiter plate reader. The IC50 values were
calculated using nonlinear regression with normalized dose−response
fit using Prism GraphPad software. All experiments were independ-
ently performed at least three times.
6.9.9. Western Blot Analysis. B16-F10 cells were seeded (5 × 105
cells/well) into 6-well transparent plates (Corning), exposed to
compounds 15c at 0.1, 0.5, 1.0, and 2.0 μM for 12 h, and then
harvested and washed with PBS three times. Then, the cells were
lysed with RIPA Cell Lysis Buffer on ice for 30 min. The cell lysates
were centrifuged at 12 000g for 20 min at 4 °C. The supernatant was
collected and the BCA Protein Assay was used to determine the
protein concentration. Equal amounts of protein (30 μg) were
separated by sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE). Then, the proteins were transferred to
poly(vinylidene fluoride) membranes and were blocked with 5%
BSA for 2 h. The membranes were incubated with the primary
antibody overnight at 4 °C and were washed with TBST three times.
Then, the mixture was incubated with the secondary antibody for 1.5
h. The membranes were washed with TBST three times. The
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Article
immunoblots were visualized by Odyssey Infrared Imaging. The
antibodies, including acetylated histone 3 (#ab177177), histone 3
(#9715S), and GAPDH (#sc-47724), were purchased from Abcam.
6.9.10. Molecular Modeling. The molecular modeling studies were
performed with Schrodinger Molecular Modeling Suite 2011
(Schrodinger LLC, New York, NY) running on a Dell window
workstation. The crystal structures of DAMA-colchicine (PDB:
4O2B) and HDAC3 (PDB: 4A69) were retrieved from the RCSB
Protein Data Bank (http://www.rcsb.org/pdb). 15c was prepared
using the Ligprep module, and they were docked into the colchicine/
HDAC3 site by the Glide module in the Schrodinger Suite. The best
docking complexes were subject to restricted molecular dynamics to
release any strains using the Macromodel module with the OPLS-
2005 force field. The ligand and its surrounding residues within 20 Å
could move freely, while the outer atoms are frozen.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00413.
1
Copies of Compounds H NMR, 13C NMR, HRMS,
HPLC spectra (PDF)
Molecular formula strings (CSV)
4A69 (HDAC3 PDB) (PDB)
4O2B (Tubulin PDB) (PDB)
AUTHOR INFORMATION
Corresponding Author
■
Jianjun Chen − School of Pharmaceutical Sciences, Southern
Medical University, Guangzhou 510515, China;
orcid.org/0000-0001-5668-6572; Email: jchen21@
smu.edu.cn
6.9.11. In Vivo Antitumor Evaluation. The animal experiment was
performed with the approval of the National Institutional Animal
Care and Ethical Committee of Southern Medical University. C57
mice, 6−8 weeks old, purchased from SPF (Beijing) Biotechnology
Co., Ltd., were used to study the inhibition effect of 15c on the
subcutaneous transplanted model of melanoma cells. Logarithmic
growth phase B16-F10 cells (4 × 106 per mL) were suspended in PBS
before injecting into mice. Tumors were established by inoculating
subcutaneously with 200 μL containing 3 × 105 cells into each mouse.
Mice were divided into control or treatment groups (n = 8) randomly.
Test compounds were dissolved in a mixed solvent, including ethanol,
polyoxyethylene castor oil, and physiological saline (1.25:1.25:7.5) to
produce the desired concentrations. The vehicle control solution was
formulated with the solvent only. A total of 200 μL of the drug
treatment or vehicle control was administered via i.p. injection once a
day for 14 days. Body weight was monitored during the entire
experiment period to assess acute toxicity. Tumor volume was
measured with a caliper and calculated using the formula a × b2 × 0.5,
where a and b represent the larger and smaller diameters, respectively.
Mice were sacrificed 14 days after the initiation of the experiment, and
the tumors were weighed. Tumor Growth Inhibition value (TGI) was
calculated using the formula: TGI(%) = [1 − Wt/Wv] × 100%, where
Wt and Wv are the mean tumor weight of the treatment group and
vehicle control. The harvested organs (liver, kidney) were fixed in 4%
paraformaldehyde, processed into paraffin routinely, stained with
hematoxylin and eosin (H&E), and captured using a microscope.
6.9.12. Immunohistochemistry (IHC). Formalin-fixed paraffin-
embedded tumor sections were submitted to LandM Co., Ltd. to
conduct the immunohistochemistry assay. In brief, tumor sections
were deparaffinized, rehydrated, and immersed in 3% H2O2 to
inactivate endogenous peroxidase. For antigen retrieval, slides were
incubated in buffer with pH = 9 at 80 °C overnight. After PBS
washing, the slides were blocked with fat-free milk and incubated with
primary antibody (Ac-H3) at 4 °C overnight. The secondary antibody
(IgG) was added, and then Mayer’s hematoxylin solution was used for
counterstaining. For hematoxylin, the tumor sections were incubated
in hematoxylin solution and then counterstained with eosin, followed
by microscope observation and photography.
Authors
Xiaopeng Peng − School of Pharmaceutical Sciences, Southern
Medical University, Guangzhou 510515, China
Jingxuan Chen − School of Pharmaceutical Sciences, Southern
Medical University, Guangzhou 510515, China
Ling Li − School of Pharmaceutical Sciences, Southern Medical
University, Guangzhou 510515, China
Zhiqiang Sun − School of Pharmaceutical Sciences, Southern
Medical University, Guangzhou 510515, China
Jin Liu − School of Pharmaceutical Sciences, Southern Medical
University, Guangzhou 510515, China
Yichang Ren − School of Pharmaceutical Sciences, Southern
Medical University, Guangzhou 510515, China
Junli Huang − School of Pharmaceutical Sciences, Southern
Medical University, Guangzhou 510515, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00413
Author Contributions
†X.P. and J.C. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the scientific research project of
high-level talents (No. C1051008) in Southern Medical
University of China; the Thousand Youth Talents Program
(No. C1080092) from the Organization Department of the
CPC Central Committee, China; and the International Science
and Technology Cooperation Projects of Guangdong Province
(No. G819310411).
6.9.13. In Vivo Cardiotoxicity by ECG and Blood Cell Analysis.
The animal experiment was performed with the approval of the
National Institutional Animal Care and Ethical Committee of
Southern Medical University. C57 mice, 6−8 weeks old, purchased
from SPF(Beijing) Biotechnology Co., Ltd., were used to study the
cardiotoxicity of the tested compounds. The tested compounds were
administered via intragastric gavage once a day for 14 days. The
instrument used: PowerLab (ADI Instruments Castle Hill, NSW,
Australia). Electrodes were inserted into the right forelimb (cathode),
left limb (anode), and right hind limb (ground) subcutaneously. The
right hind limb serves as the reference electrode. The whole blood was
also collected into a heparin sodium anticoagulation tube used for
blood cell analysis.
ABBREVIATIONS USED
■
HDAC, histone deacetylase; HDACi, histone deacetylase
inhibitor; SMART, 4-substituted methoxybenzoyl-aryl-thia-
zoles; SAI, substituted-2-aryl imidazoles; ABP, 6-Aryl-2-
Benzoyl-Pyridine/Phenyl; TMP, trimethoxyphenyl; RI, resist-
ance index; SI, selectivity index; TGI, tumor growth inhibition;
H&E, hematoxylin and eosin; HRMS, high-resolution mass
spectrometry
REFERENCES
■
(1) Nagy, C.; Turecki, G. Transgenerational epigenetic inheritance:
an open discussion. Epigenomics 2015, 7, 781−790.
8470
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J. Med. Chem. 2021, 64, 8447−8473

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(2) Daniel, M.; Tollefsbol, T. O. Epigenetic linkage of aging, cancer
and nutrition. J. Exp. Biol. 2015, 218, 59−70.
(24) Guha, A.; Armanious, M.; Fradley, M. G. Update on cardio-
oncology: Novel cancer therapeutics and associated cardiotoxicities.
Trends Cardiovasc. Med. 2019, 29, 29−39.
(25) Schiattarella, G. G.; Sannino, A.; Toscano, E.; Cattaneo, F.;
Trimarco, B.; Esposito, G.; Perrino, C. Cardiovascular effects of
histone deacetylase inhibitors epigenetic therapies: Systematic review
of 62 studies and new hypotheses for future research. Int. J. Cardiol.
2016, 219, 396−403.
(3) Mohammadi, A.; Sharifi, A.; Pourpaknia, R.; Mohammadian, S.;
Sahebkar, A. Manipulating macrophage polarization and function
using classical HDAC inhibitors: Implications for autoimmunity and
inflammation. Crit. Rev. Oncol. Hematol. 2018, 128, 1−18.
(4) McClure, J. J.; Li, X.; Chou, C. J. Advances and challenges of
HDAC inhibitors in cancer therapeutics. Adv. Cancer Res. 2018, 183−
211.
(26) Yeon, M.; Kim, Y.; Jung, H. S.; Jeoung, D. Histone deacetylase
inhibitors to overcome resistance to targeted and immuno therapy in
metastatic melanoma. Front. Cell Dev. Biol. 2020, 8, 486.
(27) Wang, P.; Wang, Z.; Liu, J. Role of HDACs in normal and
malignant hematopoiesis. Mol. Cancer 2020, 19, No. 5.
(28) Minami, J.; Suzuki, R.; Mazitschek, R.; Gorgun, G.; Ghosh, B.;
Cirstea, D.; Hu, Y.; Mimura, N.; Ohguchi, H.; Cottini, F.; Jakubikova,
J.; Munshi, N. C.; Haggarty, S. J.; Richardson, P. G.; Hideshima, T.;
Anderson, K. C. Histone deacetylase 3 as a novel therapeutic target in
multiple myeloma. Leukemia 2014, 28, 680−689.
(29) Leus, N. G.; van den Bosch, T.; van der Wouden, P. E.; Krist,
K.; Ourailidou, M. E.; Eleftheriadis, N.; Kistemaker, L. E.; Bos, S.;
Gjaltema, R. A.; Mekonnen, S. A.; Bischoff, R.; Gosens, R.; Haisma,
H. J.; Dekker, F. J. HDAC1-3 inhibitor MS-275 enhances IL10
expression in RAW264.7 macrophages and reduces cigarette smoke-
induced airway inflammation in mice. Sci. Rep. 2017, 7, No. 45047.
(30) Zhang, X.; Kong, Y.; Zhang, J.; Su, M.; Zhou, Y.; Zang, Y.; Li,
J.; Chen, Y.; Fang, Y.; Zhang, X.; Lu, W. Design, synthesis and
biological evaluation of colchicine derivatives as novel tubulin and
histone deacetylase dual inhibitors. Eur. J. Med. Chem. 2015, 95, 127−
135.
(5) West, A. C.; Johnstone, R. W. New and emerging HDAC
inhibitors for cancer treatment. J. Clin. Invest. 2014, 124, 30−39.
(6) Witt, O.; Deubzer, H. E.; Milde, T.; Oehme, I. HDAC family:
What are the cancer relevant targets? Cancer Lett. 2009, 277, 8−21.
(7) Glauben, R.; Sonnenberg, E.; Zeitz, M.; Siegmund, B. HDAC
inhibitors in models of inflammation-related tumorigenesis. Cancer
Lett. 2009, 280, 154−159.
(8) Falkenberg, K. J.; Johnstone, R. W. Histone deacetylases and
their inhibitors in cancer, neurological diseases and immune disorders.
Nat. Rev. Drug Discovery 2014, 13, 673−691.
(9) Dhaliwal, M. K.; Giovanella, B. C.; Pathak, S. Cytogenetic
characterization of two human milk-derived cell line (HBL-100)
passages differing in tumorigenicity. Anticancer Res. 1990, 10, 113−
117.
(10) Grabiec, A. M.; Potempa, J. Epigenetic regulation in bacterial
infections: targeting histone deacetylases. Crit. Rev. Microbiol. 2018,
44, 336−350.
(11) Arrowsmith, C. H.; Bountra, C.; Fish, P. V.; Lee, K.; Schapira,
M. Epigenetic protein families: a new frontier for drug discovery. Nat.
Rev. Drug Discovery 2012, 11, 384−400.
(31) Zhang, X.; Zhang, J.; Tong, L.; Luo, Y.; Su, M.; Zang, Y.; Li, J.;
Lu, W.; Chen, Y. The discovery of colchicine-SAHA hybrids as a new
class of antitumor agents. Bioorg. Med. Chem. 2013, 21, 3240−3244.
(32) Nervi, C.; De Marinis, E.; Codacci-Pisanelli, G. Epigenetic
treatment of solid tumours: a review of clinical trials. Clin. Epigenet.
2015, 7, No. 127.
(12) Barneda-Zahonero, B.; Parra, M. Histone deacetylases and
cancer. Mol. Oncol. 2012, 6, 579−589.
(13) Conway, S. J.; Woster, P. M.; Shen, J. K.; Georg, G.; Wang, S.
Epigenetics: novel therapeutics targeting epigenetics. J. Med. Chem.
2015, 58, 523−524.
(14) Peng, X.; Liao, G.; Sun, P.; Yu, Z.; Chen, J. An overview of
HDAC inhibitors and their synthetic routes. Curr. Top. Med. Chem.
2019, 19, 1005−1040.
(33) Morphy, R.; Kay, C.; Rankovic, Z. From magic bullets to
designed multiple ligands. Drug Discovery Today 2004, 9, 641−651.
(34) Anighoro, A.; Bajorath, J.; Rastelli, G. Polypharmacology:
challenges and opportunities in drug discovery. J. Med. Chem. 2014,
57, 7874−7887.
(35) Hesham, H. M.; Lasheen, D. S.; Abouzid, K. A. M. Chimeric
HDAC inhibitors: Comprehensive review on the HDAC-based
strategies developed to combat cancer. Med. Res. Rev. 2018, 38,
2058−2109.
(36) Singh, H.; Kinarivala, N.; Sharma, S. Multi-targeting anticancer
agents: rational approaches, synthetic routes and structure activity
relationship. Anticancer Agents Med. Chem. 2019, 19, 842−874.
(37) Peters, J. U. Polypharmacology - foe or friend? J. Med. Chem.
2013, 56, 8955−8971.
(38) Chen, J.; Luo, B.; Wen, S.; Pi, R. Discovery of a novel rhein-
SAHA hybrid as a multi-targeted anti-glioblastoma drug. Invest. New
Drugs 2020, 38, 755−764.
(39) Peng, X.; Sun, Z.; Kuang, P.; Chen, J. Recent progress on
HDAC inhibitors with dual targeting capabilities for cancer treatment.
Eur. J. Med. Chem. 2020, 208, No. 112831.
(40) He, S.; Dong, G.; Wu, S.; Fang, K.; Miao, Z.; Wang, W.; Sheng,
C. Small molecules simultaneously inhibiting p53-murine double
minute 2 (MDM2) interaction and histone deacetylases (HDACs):
discovery of novel multitargeting antitumor agents. J. Med. Chem.
2018, 61, 7245−7260.
(15) Ho, T. C. S.; Chan, A. H. Y.; Ganesan, A. Thirty years of
HDAC inhibitors: 2020 insight and hindsight. J. Med. Chem. 2020, 63,
12460−12484.
(16) Zagni, C.; Floresta, G.; Monciino, G.; Rescifina, A. The search
for potent, small-molecule HDACIs in cancer treatment: a decade
after vorinostat. Med. Res. Rev. 2017, 37, 1373−1428.
(17) Li, Z.; Zhu, W. G. Targeting histone deacetylases for cancer
therapy: from molecular mechanisms to clinical implications. Int. J.
Biol. Sci. 2014, 10, 757−770.
(18) Manal, M.; Chandrasekar, M. J.; Gomathi Priya, J.; Nanjan, M.
J. Inhibitors of histone deacetylase as antitumor agents: A critical
review. Bioorg. Chem. 2016, 67, 18−42.
(19) Bailey, H.; Stenehjem, D. D.; Sharma, S. Panobinostat for the
treatment of multiple myeloma: the evidence to date. J. Blood Med.
2015, 269−276.
(20) Ververis, K.; Hiong, A.; Karagiannis, T. C.; Licciardi, P. V.
Histone deacetylase inhibitors (HDACIs): multitargeted anticancer
agents. Biol.: Targets Ther. 2013, 7, 47−60.
(21) Grant, C.; Rahman, F.; Piekarz, R.; Peer, C.; Frye, R.; Robey, R.
W.; Gardner, E. R.; Figg, W. D.; Bates, S. E. Romidepsin: a new
therapy for cutaneous T-cell lymphoma and a potential therapy for
solid tumors. Expert Rev. Anticancer Ther. 2010, 10, 997−1008.
(22) Mann, B. S.; Johnson, J. R.; Cohen, M. H.; Justice, R.; Pazdur,
R. FDA approval summary: vorinostat for treatment of advanced
primary cutaneous T-cell lymphoma. Oncologist 2007, 12, 1247−
1252.
(23) Hess-Stumpp, H.; Bracker, T. U.; Henderson, D.; Politz, O.
MS-275, a potent orally available inhibitor of histone deacetylases–the
development of an anticancer agent. Int. J. Biochem. Cell Biol. 2007,
39, 1388−1405.
(41) Dong, G.; Chen, W.; Wang, X.; Yang, X.; Xu, T.; Wang, P.;
Zhang, W.; Rao, Y.; Miao, C.; Sheng, C. Small molecule inhibitors
simultaneously targeting cancer metabolism and epigenetics: discov-
ery of novel nicotinamide phosphoribosyltransferase (NAMPT) and
histone deacetylase (HDAC) dual inhibitors. J. Med. Chem. 2017, 60,
7965−7983.
(42) Liang, X.; Zang, J.; Li, X.; Tang, S.; Huang, M.; Geng, M.;
Chou, C. J.; Li, C.; Cao, Y.; Xu, W.; Liu, H.; Zhang, Y. Discovery of
novel Janus Kinase (JAK) and histone deacetylase (HDAC) dual
8471
https://doi.org/10.1021/acs.jmedchem.1c00413
J. Med. Chem. 2021, 64, 8447−8473

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
inhibitors for the treatment of hematological malignancies. J. Med.
Chem. 2019, 62, 3898−3923.
(43) Huang, Y.; Dong, G.; Li, H.; Liu, N.; Zhang, W.; Sheng, C.
Discovery of Janus Kinase 2 (JAK2) and histone deacetylase (HDAC)
dual inhibitors as a novel strategy for the combinational treatment of
Leukemia and invasive fungal infections. J. Med. Chem. 2018, 61,
6056−6074.
(44) Thakur, A.; Tawa, G. J.; Henderson, M. J.; Danchik, C.; Liu, S.;
Shah, P.; Wang, A. Q.; Dunn, G.; Kabir, M.; Padilha, E. C.; Xu, X.;
Simeonov, A.; Kharbanda, S.; Stone, R.; Grewal, G. Design, synthesis,
and biological evaluation of quinazolin-4-one-based hydroxamic acids
as dual PI3K/HDAC inhibitors. J. Med. Chem. 2020, 63, 4256−4292.
(45) Liu, T.; Wan, Y.; Xiao, Y.; Xia, C.; Duan, G. Dual-target
inhibitors based on HDACs: novel antitumor agents for cancer
therapy. J. Med. Chem. 2020, 63, 8977−9002.
(46) Lai, C. J.; Bao, R.; Tao, X.; Wang, J.; Atoyan, R.; Qu, H.; Wang,
D. G.; Yin, L.; Samson, M.; Forrester, J.; Zifcak, B.; Xu, G. X.;
DellaRocca, S.; Zhai, H. X.; Cai, X.; Munger, W. E.; Keegan, M.;
Pepicelli, C. V.; Qian, C. CUDC-101, a multitargeted inhibitor of
histone deacetylase, epidermal growth factor receptor, and human
epidermal growth factor receptor 2, exerts potent anticancer activity.
Cancer Res. 2010, 70, 3647−3656.
(59) Li, L.; Quan, D.; Chen, J.; Ding, J.; Zhao, J.; Lv, L.; Chen, J.
Design, synthesis, and biological evaluation of 1-substituted -2-aryl
imidazoles targeting tubulin polymerization as potential anticancer
agents. Eur. J. Med. Chem. 2019, 184, No. 111732.
(60) Chen, J.; Wang, Z.; Li, C. M.; Lu, Y.; Vaddady, P. K.; Meibohm,
B.; Dalton, J. T.; Miller, D. D.; Li, W. Discovery of novel 2-aryl-4-
benzoyl-imidazoles targeting the colchicines binding site in tubulin as
potential anticancer agents. J. Med. Chem. 2010, 53, 7414−7427.
(61) Lu, Y.; Li, C. M.; Wang, Z.; Ross, C. R., 2nd; Chen, J.; Dalton,
J. T.; Li, W.; Miller, D. D. Discovery of 4-substituted methoxybenzoyl-
aryl-thiazole as novel anticancer agents: synthesis, biological
evaluation, and structure-activity relationships. J. Med. Chem. 2009,
52, 1701−1711.
(62) Yang, X.; Cheng, B.; Xiao, Y.; Xue, M.; Liu, T.; Cao, H.; Chen,
J. Discovery of novel CA-4 analogs as dual inhibitors of tubulin
polymerization and PD-1/PD-L1 interaction for cancer treatment.
Eur. J. Med. Chem. 2020, 213, No. 113058.
(63) Prota, A. E.; Danel, F.; Bachmann, F.; Bargsten, K.; Buey, R.
M.; Pohlmann, J.; Reinelt, S.; Lane, H.; Steinmetz, M. O. The novel
microtubule-destabilizing drug BAL27862 binds to the colchicine site
of tubulin with distinct effects on microtubule organization. J. Mol.
Biol. 2014, 426, 1848−1860.
(47) Cai, X.; Zhai, H. X.; Wang, J.; Forrester, J.; Qu, H.; Yin, L.; Lai,
C. J.; Bao, R.; Qian, C. Discovery of 7-(4-(3-ethynylphenylamino)-7-
methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide (CUDc-101) as
a potent multi-acting HDAC, EGFR, and HER2 inhibitor for the
treatment of cancer. J. Med. Chem. 2010, 53, 2000−2009.
(48) Chao, M. W.; Lai, M. J.; Liou, J. P.; Chang, Y. L.; Wang, J. C.;
Pan, S. L.; Teng, C. M. The synergic effect of vincristine and
vorinostat in leukemia in vitro and in vivo. J. Hematol. Oncol. 2015, 8,
No. 82.
(49) Wu, Y. W.; Hsu, K. C.; Lee, H. Y.; Huang, T. C.; Lin, T. E.;
Chen, Y. L.; Sung, T. Y.; Liou, J. P.; Hwang-Verslues, W. W.; Pan, S.
L.; HuangFu, W. C. A novel dual HDAC6 and tubulin inhibitor,
MPT0B451, displays anti-tumor ability in human cancer cells in vitro
and in vivo. Front. Pharmacol. 2018, 9, 205.
(50) Wang, B.; Chen, X.; Gao, J.; Su, L.; Zhang, L.; Xu, H.; Luan, Y.
Anti-tumor activity evaluation of novel tubulin and HDAC dual-
targeting inhibitors. Bioorg. Med. Chem. Lett. 2019, 29, 2638−2645.
(51) Schmitt, F.; Gosch, L. C.; Dittmer, A.; Rothemund, M.;
Mueller, T.; Schobert, R.; Biersack, B.; Volkamer, A.; Hopfner, M.
Oxazole-bridged combretastatin A-4 derivatives with tethered
hydroxamic acids: structure(-)activity relations of new inhibitors of
HDAC and/or tubulin function. Int. J. Mol. Sci. 2019, 20, 383.
(52) Mourad, A. A. E.; Mourad, M. A. E.; Jones, P. G. Novel
HDAC/tubulin dual inhibitor: design, synthesis and docking studies
of alpha-phthalimido-chalcone hybrids as potential anticancer agents
with apoptosis-inducing activity. Drug Des., Dev. Ther. 2020, 14,
3111−3130.
(64) Zhu, Y.; Das, K.; Wu, J.; Lee, M. H.; Tan, P. RNH1 regulation
of reactive oxygen species contributes to histone deacetylase inhibitor
resistance in gastric cancer cells. Oncogene 2014, 33, 1527−1537.
(65) Rosato, R. R.; Almenara, J. A.; Grant, S. The histone
deacetylase inhibitor MS-275 promotes differentiation or apoptosis
in human leukemia cells through a process regulated by generation of
reactive oxygen species and induction of p21CIP1/WAF1 1. Cancer
Res. 2003, 63, 3637−3645.
(66) Lauffer, B. E.; Mintzer, R.; Fong, R.; Mukund, S.; Tam, C.;
Zilberleyb, I.; Flicke, B.; Ritscher, A.; Fedorowicz, G.; Vallero, R.;
Ortwine, D. F.; Gunzner, J.; Modrusan, Z.; Neumann, L.; Koth, C.
M.; Lupardus, P. J.; Kaminker, J. S.; Heise, C. E.; Steiner, P. Histone
deacetylase (HDAC) inhibitor kinetic rate constants correlate with
cellular histone acetylation but not transcription and cell viability. J.
Biol. Chem. 2013, 288, 26926−26943.
(67) Ayoub, A. T.; Abou El-Magd, R. M.; Xiao, J.; Lewis, C. W.;
Tilli, T. M.; Arakawa, K.; Nindita, Y.; Chan, G.; Sun, L.; Glover, M.;
Klobukowski, M.; Tuszynski, J. Antitumor activity of lankacidin group
antibiotics is due to microtubule stabilization via a paclitaxel-like
mechanism. J. Med. Chem. 2016, 59, 9532−9540.
(68) Condeelis, J.; Pollard, J. W. Macrophages: obligate partners for
tumor cell migration, invasion, and metastasis. Cell 2006, 124, 263−
266.
(69) Li, G.; Wang, Y.; Li, L.; Ren, Y.; Deng, X.; Liu, J.; Wang, W.;
Luo, M.; Liu, S.; Chen, J. Design, synthesis, and bioevaluation of
pyrazolo[1,5-a]pyrimidine derivatives as tubulin polymerization
inhibitors targeting the colchicine binding site with potent anticancer
activities. Eur. J. Med. Chem. 2020, 202, No. 112519.
(70) Liu, S.; Cheng, H.; Kwan, W.; Lubieniecka, J. M.; Nielsen, T.
O. Histone deacetylase inhibitors induce growth arrest, apoptosis, and
differentiation in clear cell sarcoma models. Mol. Cancer Ther. 2008, 7,
1751−1761.
(71) Chun, P. Histone deacetylase inhibitors in hematological
malignancies and solid tumors. Arch. Pharm. Res. 2015, 38, 933−949.
(72) Li, Y.; Seto, E. HDACs and HDAC inhibitors in cancer
development and therapy. Cold Spring Harbor Perspect. Med. 2016, 6,
No. a026831.
(73) Wang, Q.; Arnst, K. E.; Wang, Y.; Kumar, G.; Ma, D.; White, S.
W.; Miller, D. D.; Li, W.; Li, W. Structure-guided design, synthesis,
and biological evaluation of (2-(1H-indol-3-yl)-1H-imidazol-4-yl)-
(3,4,5-trimethoxyphenyl) methanone (ABI-231) analogues targeting
the colchicine binding site in tubulin. J. Med. Chem. 2019, 62, 6734−
6750.
(53) Aboeldahab, A. M. A.; Beshr, E. A. M.; Shoman, M. E.; Rabea,
S. M.; Aly, O. M. Spirohydantoins and 1,2,4-triazole-3-carboxamide
derivatives as inhibitors of histone deacetylase: Design, synthesis, and
biological evaluation. Eur. J. Med. Chem. 2018, 146, 79−92.
(54) Lamaa, D.; Lin, H. P.; Zig, L.; Bauvais, C.; Bollot, G.; Bignon,
J.; Levaique, H.; Pamlard, O.; Dubois, J.; Ouaissi, M.; Souce, M.;
Kasselouri, A.; Saller, F.; Borgel, D.; Jayat-Vignoles, C.; Al-
Mouhammad, H.; Feuillard, J.; Benihoud, K.; Alami, M.; Hamze, A.
Design and synthesis of tubulin and histone deacetylase inhibitor
based on iso-combretastatin A-4. J. Med. Chem. 2018, 61, 6574−6591.
(55) Mukhtar, E.; Adhami, V. M.; Mukhtar, H. Targeting
microtubules by natural agents for cancer therapy. Mol. Cancer
Ther. 2014, 13, 275−284.
(56) Kuppens, I. E. Current state of the art of new tubulin inhibitors
in the clinic. Curr. Clin. Pharmacol. 2006, 1, 57−70.
(57) Dumontet, C.; Jordan, M. A. Microtubule-binding agents: a
dynamic field of cancer therapeutics. Nat. Rev. Drug Discovery 2010, 9,
790−803.
(74) Chen, J.; Ahn, S.; Wang, J.; Lu, Y.; Dalton, J. T.; Miller, D. D.;
Li, W. Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III)
analogues targeting tubulin polymerization as antiproliferative agents.
J. Med. Chem. 2012, 55, 7285−7289.
(58) Jordan, M. A.; Wilson, L. Microtubules as a target for
anticancer drugs. Nat. Rev. Cancer 2004, 4, 253−265.
8472
https://doi.org/10.1021/acs.jmedchem.1c00413
J. Med. Chem. 2021, 64, 8447−8473

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(75) Liu, S.; Zhang, K.; Zhu, Q.; Shen, Q.; Zhang, Q.; Yu, J.; Chen,
Y.; Lu, W. Synthesis and biological evaluation of paclitaxel and
vorinostat co-prodrugs for overcoming drug resistance in cancer
therapy in vitro. Bioorg. Med. Chem. 2019, 27, 1405−1413.
(76) Alexandre, J.; Moslehi, J. J.; Bersell, K. R.; Funck-Brentano, C.;
Roden, D. M.; Salem, J. E. Anticancer drug-induced cardiac rhythm
disorders: Current knowledge and basic underlying mechanisms.
Pharmacol. Ther. 2018, 189, 89−103.
(77) Zamorano, J. L.; Lancellotti, P.; Rodriguez Munoz, D.;
Aboyans, V.; Asteggiano, R.; Galderisi, M.; Habib, G.; Lenihan, D.
J.; Lip, G. Y. H.; Lyon, A. R.; Lopez Fernandez, T.; Mohty, D.;
Piepoli, M. F.; Tamargo, J.; Torbicki, A.; Suter, T. M. 2016 ESC
position paper on cancer treatments and cardiovascular toxicity
developed under the auspices of the ESC committee for practice
guidelines: the task force for cancer treatments and cardiovascular
toxicity of the European society of cardiology (ESC). Eur. Heart J.
2016, 37, 2768−2801.
(78) Locatelli, M.; Criscitiello, C.; Esposito, A.; Minchella, I.;
Goldhirsch, A.; Cipolla, C.; Curigliano, G. QTc prolongation induced
by targeted biotherapies used in clinical practice and under
investigation: a comprehensive review. Target Oncol. 2015, 10, 27−43.
(79) Sager, P. T.; Balser, B.; Wolfson, J.; Nichols, J.; Pilot, R.; Jones,
S.; Burris, H. A. Electrocardiographic effects of class 1 selective
histone deacetylase inhibitor romidepsin. Cancer Med. 2015, 4, 1178−
1185.
(80) Rathkopf, D. E.; Picus, J.; Hussain, A.; Ellard, S.; Chi, K. N.;
Nydam, T.; Allen-Freda, E.; Mishra, K. K.; Porro, M. G.; Scher, H. I.;
Wilding, G. A phase 2 study of intravenous panobinostat in patients
with castration-resistant prostate cancer. Cancer Chemother. Pharma-
col. 2013, 72, 537−544.
8473
https://doi.org/10.1021/acs.jmedchem.1c00413
J. Med. Chem. 2021, 64, 8447−8473