HDAC-Bax Multiple Ligands Enhance Bax-Dependent Apoptosis in HeLa Cells

Article abstract of DOI:10.1021/acs.jmedchem.0c01454

Inspired by the synergistic effect of BTSA1 (a Bax activator) and SAHA (a histone deacetylase (HDAC) inhibitor) in HeLa cell growth suppression, a series of novel HDAC-Bax multiple ligands were designed rationally. Compound 23, which possesses similar HDAC inhibitory activity relative to SAHA and Bax affinity comparable to BTSA1, exhibits a superior growth suppression against HeLa cells, and its antiproliferative activities are 15-fold and 3-fold higher than BTSA1 and SAHA, respectively. The better antiproliferative activity and lower cytotoxicity of compound 23 indicated that our HDAC-Bax multiple ligand design strategy achieved success. Further studies suggested that compound 23 could enhance Bax-dependent apoptosis by upregulating Bax, followed by inducing the conformational activation of Bax. To our knowledge, we first report HDAC-Bax multiple ligands and demonstrate a new paradigm for the treatment of solid tumors by enhancing Bax-dependent apoptosis.

Full text of DOI:10.1021/acs.jmedchem.0c01454

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HDAC−Bax Multiple Ligands Enhance Bax-Dependent Apoptosis in
HeLa Cells
Tao Liang, Yi Zhou, Reham M. Elhassan, Xuben Hou, Xinying Yang, and Hao Fang*
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ABSTRACT: Inspired by the synergistic effect of BTSA1 (a Bax
activator) and SAHA (a histone deacetylase (HDAC) inhibitor) in
HeLa cell growth suppression, a series of novel HDAC−Bax
multiple ligands were designed rationally. Compound 23, which
possesses similar HDAC inhibitory activity relative to SAHA and
Bax affinity comparable to BTSA1, exhibits a superior growth
suppression against HeLa cells, and its antiproliferative activities
are 15-fold and 3-fold higher than BTSA1 and SAHA, respectively.
The better antiproliferative activity and lower cytotoxicity of
compound 23 indicated that our HDAC−Bax multiple ligand
design strategy achieved success. Further studies suggested that
compound 23 could enhance Bax-dependent apoptosis by
upregulating Bax, followed by inducing the conformational activation of Bax. To our knowledge, we first report HDAC−Bax
multiple ligands and demonstrate a new paradigm for the treatment of solid tumors by enhancing Bax-dependent apoptosis.
have led to the development of a series of potent Bax
activators, such as Bax activator 8, BIF-44 as well as
BTSA119−21 (Figure 2). BTSA1 (IC50 = 250 nM), the most
potent Bax activator, exhibits good antiproliferative activity
against human acute myeloid leukemia (AML) xenografts with
acceptable toxicity.19 Bax activators open up a new world for
the accurate modulation of Bax-dependent apoptosis by
flipping the “on switch” of Bax and demonstrate a new
paradigm for pharmacologic induction of apoptosis.
Efforts of two decades have led to the development of five
approved HDAC inhibitors (Vorinostat (SAHA), Romidepsin
(FK228), Panobinostat (LBH-589), Belinostat (PXD101), and
Chidamide (CS055), Figure 2) for the treatment of cutaneous
T-cell lymphomas (CTCL), multiple myeloma (MM), and
peripheral T-cell lymphomas (PTCL).22−26 Accumulating
evidence suggested that HDAC inhibition could upregulate
Bax,27−29 and we speculated that the combination of Bax
activators and HDAC inhibitors may enhance Bax-dependent
apoptosis and achieve better antiproliferation activities
compared with single agents. To confirm our hypothesis, we
combined BTSA1 and SAHA (1:1) in HeLa cells. Upon
combination of both ligands, a better antiproliferative activity
INTRODUCTION
■
As one of the most common gynecologic cancers, cervical
cancer has a great effect on reproduction and even leads to
causes of mortality in women.1 The treatment of cervical
cancer has changed dynamically over the past 20 years,2 and
more than two decades of efforts have promoted various
effective therapeutic regimens, especially drug combination.3−6
Accumulating evidence revealed that Bax is an essential key
apoptotic mediator in cervical carcinoma cells, and activating
Bax may be a new strategy for the treatment of cervical cancer.7
Bax, a key proapoptotic protein of the Bcl-2 family, is the
cardinal regulator of the mitochondrial pathway.8 Upon Bax
conformational activation, oligomerization at the mitochon-
drial outer membrane (MOM) and mitochondrial outer
membrane permeabilization (MOMP) occur, followed by the
release of cytochrome c, Smac/DIABLO, and other apopto-
genic factors, which turn on the caspase cascade of
apoptosis9−13 (Figure 1).
The activation of Bax could be achieved in an indirect
manner (loss of antiapoptotic function) or a direct manner
(gain of proapoptotic function).14−17 For a long period of
time, inhibiting the function of antiapoptotic proteins with Bcl-
2, Mcl-1, or Bcl-XL inhibitors is the most common indirect
manner of Bax activation. However, identification of the first
Bax activator BAM7 (half-maximal inhibitory concentration
(IC50) = 3.3 μM, Figure 2), which targets the Bax activation
trigger site and induces conformational activation of Bax,
provides blueprints for the development of alternative
apoptosis regulators to induce Bax-dependent apoptosis
directly.18 Over 5 years of structural optimization efforts
Received: August 21, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01454
© XXXX American Chemical Society
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A

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Figure 1. Apoptosis induced by Bax activation.
Figure 2. Bax activators and pharmacophores of representative histone deacetylase (HDAC) inhibitors.
Figure 3. Combination (A) and the combination index (B) of BTSA1 and SAHA.
(IC50 = 0.81 μM) was observed in relation to monotherapy
(BTSA1, IC50 = 11.47 μM; SAHA, IC50 = 2.65 μM, Figure
3A). We further analyzed the results using the Chou−Talalay
method and calculated the combination index (CI) value with
CompuSyn according to the dose−effect relationship. The
combination index value of 0.73 indicated that the
combination of SAHA and BTSA1 exhibits moderate
synergistic effects in the growth suppression of HeLa cells
B
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Figure 4. (A) Binding mode of BTSA1; (B) interactions between BTSA1 and Bax (PDB ID: 2K7W); (C) design of HDAC−Bax multiple ligands.
a
Scheme 1. Synthetic Route of Compounds 2−4
a
Reagents and conditions: (a) (i) NaNO2, HCl, H2O, 0 °C, 30 min; (ii) CH3COONa, ethyl benzoylacetate, EtOH, 0 °C, 1 h, 54%; (b) 2-
bromoacetophenone or 4-(2-bromoacetyl)benzoic acid, thiosemicarbazide, EtOH, 80 °C, 47−48%; (c) isobutyl chlorocarbonate, N-
methylmorpholine, THF, NH2OH·HCl, KOH, MeOH, room temperature (rt), 6 h, 45%.
a
Scheme 2. Synthetic Route of Compounds 20−24
a
Reagents and conditions: (a) HATU, DIPEA, CH2Cl2, rt, overnight, 67−99%; (b) CuBr2, ethyl acetate/CHCl3 (1:1), 80 °C, 3 h, 63−80%; (c)
thiosemicarbazide, MeOH, 80 °C, 3 h, 33−68%; (d) NH2OH·HCl, KOH, MeOH, rt, 6 h, 29−77%.
(Figure 3B). Encouraged by the moderate synergistic effect of
the combination of BTSA1 and SAHA, we set out to explore
more.
application of drug combination.34 However, multitarget
drugs could solve these problems perfectly on the basis of
retaining efficacy.35−37 Therefore, we set out to develop
HDAC−Bax multiple ligands, which could achieve HDAC
inhibition and Bax conformational activation, for the treatment
of cervical cancer.
Actually, it can be challenging to design multitarget agents
because of the completely different structures of target
proteins, and matching potencies at two separate targets with
a single chemical entity is extremely difficult.38 Our group
designed and developed histone deacetylase and proteasome
dual inhibitors using pharmacophore models.39 To merge two
Drug combination is widely used in the treatment of a wide
range of dreadful diseases, such as cancer and acquired
immunodeficiency syndrome (AIDS), because of the syner-
gistic therapeutic effect, dose and toxicity reduction, and to
minimize or delay the induction of drug resistance.30−33
Despite the benefit of drug combination, complex clinical trial
design, complicated drug−drug interactions (DDIs), poor
patient compliance, as well as unpredictable pharmacokinetics
(PK) and pharmacodynamic (PD) relationship limit the
C
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a
Scheme 3. Synthetic Route of Compounds 46−52
a
Reagents and conditions: (a) K2CO3, rt, overnight, 83−94%; (b) CuBr2, ethyl acetate/CHCl3 (1:1), 80 °C, 3 h, 53−84%; (c) thiosemicarbazide,
MeOH, 80 °C, 3 h, 52−78%; (d) NH2OH·HCl, KOH, MeOH, rt, 6 h, 55−83%.
different fragments (HDAC inhibitors and Bax activators),
docking experiments (Figure 4A,B) and molecular dynamics
(MD) simulation (Figure S71) were performed. BTSA1 was
calculated to bind in the hydrophobic region at the
juxtaposition of helices α1 and α6, and the thiazole group of
BTSA1 lies adjacent to a presumed hinge site for loop opening
upon initiation of the conformation activation of Bax.
Moreover, the phenyl group attached to the pyrazolone core
accommodates the Bax surface properly. This fragment is
essential for Bax affinity, and replacement of the phenyl group
with the methyl group resulted in decreased affinity.19
Therefore, the pyrazolone core, the thiazole group, and the
phenyl group attached to the pyrazolone are crucial for BTSA1
to achieve Bax affinity. HDAC inhibitors have a well-accepted
pharmacophore: the zinc binding group (ZBG), the linker, as
well as the cap group40 (Figure 2), and these fragments are
essential for HDAC inhibitory activity. Given the importance
of the functional fragments (pyrazolone core, thiazole group,
and phenyl group attached to the pyrazolone), we set up to use
these functional fragments as the cap group and introduce a
linker and ZBG at the solvent-exposed fragment, the 4-
phenylthiazole group (Figure 4C).
To our knowledge, we first explored the combination of Bax
activator and HDAC inhibitor and designed a series of novel
HDAC−Bax multiple ligands rationally. Potent compound 23,
which possesses similar HDAC inhibitory activity relative to
SAHA and Bax affinity comparable to BTSA1, exhibits better
growth suppression against HeLa cells, and its antiproliferative
activity is 15-fold and 3-fold better than BTSA1 and SAHA,
respectively. Herein, we report the design, synthesis, and
biological evaluation of our HDAC−Bax multiple ligands.
bromoacetyl)benzoic acid in boiling ethanol for 3 h. Moreover,
target compound 4 was synthesized by converting the carboxyl
group of target compound 3 into hydroxamic acid using
isobutyl chlorocarbonate, N-methylmorpholine, and hydroxyl-
amine hydrochloride in tetrahydrofuran (THF). For the
second series of target compounds, 4-carboxybenzaldehyde
was coupled with different amino acid derivatives for the
synthesis of intermediates 5−9 by amide coupling reactions in
the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
(HATU) and N,N-diisopropylethylamine (DIPEA) in
CH2Cl2. Copper(II) bromide was simply used for the
bromination reactions to prepare intermediates 10−14,
which were converted into key intermediates 15−19 in a
manner similar to that described for the preparation of
compound 2. Finally, intermediates 15−19 were converted
into target compounds 20−24 in the presence of hydroxyl-
amine hydrochloride and potassium hydroxide in N,N-
dimethylformamide (DMF) (Scheme 2). As for the third
series of HDAC−Bax multiple ligands, different alkylation
reactions of 4′-hydroxyacetophenone were performed to
obtain intermediates 25−31 in the presence of K2CO3 and
KI in DMF. Then, the syntheses of target compounds 32−52
were similar to that described for the preparation of the second
series of target compounds 15−24 (Scheme 3).
Biological Results. Fluorescence Polarization Binding
Assay. The binding affinities of target compounds for the
recombinant Bax protein were measured in vitro by the
fluorescence polarization assay (FPA) that target compounds
displace a fluorescein-labeled stapled peptide of the BIM BH3
helix, FITC-BIM from the Bax trigger site.16 As shown in Table
1, most target compounds demonstrated similar Bax affinities
because of the same fragments of Bax activators. Moreover, the
most potent compound 23, which displaced FITC-BIM from
the Bax trigger site with an EC50 value of 392.9 nM, exhibited
Bax affinity comparable to that of BTSA1 (target compound 2,
EC50 = 278.1 nM). Docking studies (Figure S70) revealed that
compound 23 and BTSA1 share a similar binding mode within
the Bax rear site. Structure−activity relationship (SAR)
analysis indicated that the linker and the ZBG group of our
RESULTS AND DISCUSSION
■
Chemistry. The synthetic methods for target compounds
are depicted in Scheme 1−3. Thiazol-2-amine was transformed
into diazonium salt, which was used to synthesize ethyl (Z)-3-
oxo-3-phenyl-2-(2-(thiazol-2-yl)hydrazono)propanoate (1) in
the presence of ethyl benzoylacetate. Target compounds 2 and
3 were prepared starting from intermediate 1 and thiosemi-
carbazide in the presence of 2-bromoacetophenone or 4-(2-
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a
Table 1. HDAC Inhibitory Activities and Bax Affinities of Target Compounds
a
All compounds were assayed three times, and the results are expressed as mean standard error of the mean (SEM).
HDAC−Bax multiple ligands display different effects on Bax
affinities. Short linkers exhibited slight effects on Bax affinities,
whereas longer linkers or bulkier linkers impair the affinities for
Bax, except for compound 50. The longer and bulkier linkers of
HDAC−Bax multiple ligands appear to affect the binding
poses of the Bax activator fragments of target compounds
within the Bax rear site.
In Vitro HeLa Cell Extract Inhibitory Assay. We next
evaluated the HDAC inhibitory activities of all target
compounds using the HeLa cell extract (containing primarily
HDAC1 and HDAC2) with SAHA as the reference. The
results in Table 1 suggest that some compounds exhibit good
inhibitory activities against the HeLa cell extract. Target
compound 2 (BTSA1, IC50 > 10 000 nM) without the ZBG
E
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Table 2. Selective Profiles of Compound 23 for Bcl-2 Family Proteins and the HDAC Isoform
a
IC50 (nM)
Bcl-2 family proteins
Mcl-1
HDACs isoform
Bcl-2
Bcl-XL
HDAC1
HDAC2
HDAC8
23
BTSA1
SAHA
>10 000
>10 000
ND
>10 000
>10 000
ND
>10 000
>10 000
ND
66.0 2.3
ND
39.8 2.1
197.9 8.1
ND
128.9 4.5
1086.8 42.1
ND
2736.9 151.0
b
a
b
The results are expressed as mean SEM (n ≥ 2). ND, not detected.
Figure 5. Upregulation and activation of the Bax in HeLa cells. Western blot analysis of samples incubated with compound 23, BTSA1, SAHA, and
BTSA1 + SAHA for 6 h (A) and 12 h (B); immunoprecipitation assay of samples incubated with compound 23, BTSA1, SAHA, and BTSA1 +
SAHA for 6 h (C) and 12 h (D).
group and target compound 3 (IC50 > 10 000 nM) with the
carboxyl group as ZBG are inactive due to the poor chelation
with the zinc ion of HDACs. Moreover, target compound 4
(IC50 = 557.3 nM) with hydroxamic acid as ZBG showed mild
inhibitory activities against the HeLa cell extract because its
linker is too short to accommodate the deep catalytic channel
of HDACs (HDAC1 and HDAC2), and this prevents ZBG
from chelating with the zinc ion well. Therefore, a suitable
linker and ZBG are necessary pharmacophores for HDAC
inhibitor activities. For the second series of HDAC−Bax
multiple ligands, we explored the suitable linkers of target
compounds. Compounds 21 and 23, which possess suitable
induce Bax activation directly instead of inhibiting the function
of antiapoptotic proteins (Bcl-2, Mcl-1, and Bcl-XL), the
affinities of compound 23 for Bcl-2, Mcl-1, and Bcl-XL were
determined using the competitive fluorescence polarization
binding Assay. Compound 23 showed no significant inhibition
against Bcl-2, Mcl-1, and Bcl-XL at the concentration of 10 μM
(Table 2). In other words, compound 23 targets Bax
specifically without binding to antiapoptotic proteins (Bcl-2,
Mcl-1, and Bcl-XL). On the other hand, we further explored
HDAC isoform inhibitory profiles of compound 23, and it
showed good inhibitory activities against HDAC1 (IC50 = 66.0
nM) and HDAC2 (IC50 = 197.9 nM) and mild inhibition over
HDAC8 (IC50 = 1086.8 nM). The result is consistent with the
in vitro HeLa cell extract inhibitory assay.
Upregulation and Activation of Bax in HeLa Cells. Our
HDAC−Bax multiple ligands contain two fragments of the
HDAC inhibitor as well as a Bax activator, and they are
supposed to upregulate and activate Bax. To confirm this,
Western blot and immunoprecipitation assays were performed
in HeLa cells. Following compound 23, BTSA1, and SAHA
treatment for 6 and 12 h, HeLa cells were harvested and lysed.
Lysates were first analyzed to explore the expression of the Bax
protein with the anti-Bax primary antibody (Cell Signaling cat.
2772) using Western blot. As shown in Figure 5A,B,
compound 23 and the positive reference SAHA increase the
acetylation of H3 (a major substrate of class I HDACs),
indicating that they exhibit good HDAC inhibition. Moreover,
compound 23 and SAHA could upregulate Bax compared with
the control (CTL).
linkers, displayed good HDAC inhibitory activities (IC50
87.5 and 62.8 nM, respectively), whereas compound 20 (IC50
= 207.4 nM) with a shorter linker and compound 24 (IC50
=
=
1341.5 nM) with a longer linker showed weaker HDAC
inhibition. As for the third series of target compounds, they
share a similar structure−activity relationship (SAR) with the
second series of compounds. Compounds 48, 49, and 51 could
bury into the HDAC catalytic channel and chelate with zinc
ions properly, which give them good HDAC inhibitory
activities. In addition, potent target compound 23, which
exhibits good Bax affinity and HDAC inhibitory activity, was
chosen for subsequent biological evaluation.
Selectivity Profile of Bcl-2 Family Proteins and HDACs.
Bax activation can be achieved in an indirect manner (loss of
antiapoptotic function with antiapoptotic protein inhibitors) or
a direct manner (gain of proapoptotic function with Bax
activators). To identify that our HDAC−Bax multiple ligands
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Figure 6. (A) Antiproliferative activities of compound 23, BTSA1, and SAHA; (B) apoptotic morphologic features of HeLa cells in response to
compound 23 (1 μM).
Figure 7. (A) TUNEL assay in HeLa cells treated with 1 μM compound 23, BTSA1, and SAHA; (B) apoptosis induction of compound 23,
BTSA1, and SAHA in HeLa cells.
Subsequently, Bax conformational activation was determined
with the conformation-specific primary antibody 6A7 (Santa
Cruz, sc-23959), which recognizes the exposure of the Bax
activation epitope. Lysates were immunoprecipitated with
protein A agarose beads (Cell Signaling cat. 9863) and the
anti-Bax antibody 6A7 overnight at 4 °C and then the beads
were collected and analyzed using Western blot with the other
anti-Bax primary antibody (Cell Signaling cat. 2772). Figure
5C,D suggests that compound 23 could induce the
conformation activation of Bax more effectively in a dose-
dependent manner compared with BTSA1. In addition, the
ability of compound 23 to induce Bax conformation activation
is comparable to that of BTSA1/Vorinostat cotreatment.
These results indicated that compound 23 could achieve two
proapoptotic functions in HeLa cells: (1) upregulate Bax and
(2) induce the conformation activation of Bax.
Cell Viability and Morphologic Features. We evaluated the
antiproliferative activity of compound 23 against HeLa cells. In
the range of concentrations tested, compound 23 exhibits a
better property concerning suppression of HeLa cell growth
(IC50 = 0.86 μM) compared with BTSA1 (IC50 = 11.47 μM)
and SAHA (IC50 = 2.67 μM) (Figure 6A), and its
antiproliferative activities are 3 and 15 times better than
those SAHA and BTSA1, respectively. Compound 23, which
possesses HDAC inhibitory activity and Bax affinity com-
parable to SAHA and BTSA1, respectively, exhibits better
antiproliferative activity against HeLa cells, illuminating the
fact that our HDAC−Bax multiple ligand design strategy
achieves success.
Moreover, we monitored the morphology changes of HeLa
cells in response to compound 23 (1 μM). Upon treatment of
HeLa cells with compound 23, a wide range of apoptotic
morphologic features was observed, such as progressive cellular
shrinkage, membrane blebbing, and the formation of apoptotic
bodies (Figure 6B). Given the apoptotic morphologic features
of HeLa cells, we speculated that our compound 23 suppresses
the growth of HeLa cells by inducing apoptosis.
Terminal Deoxynucleotidyl Transferase (TdT) Deoxyur-
idine Triphosphate (dUTP) Nick-End Labeling (TUNEL) Assay
and Flow Cytometry Analysis. The terminal deoxynucleotidyl
transferase (TdT) dUTP nick-end labeling (TUNEL) assay
was designed and performed to confirm whether compound 23
induces apoptosis in HeLa cells. The strong green fluorescence
of HeLa cells (Figure 7A) treated with compound 23 (1 μM)
indicated that compound 23 could induce apoptosis in HeLa
cells effectively. To further determine the apoptotic rate of
HeLa cells in response to compound 23, flow cytometry
analysis (annexin V/propidium iodide (PI) staining) was
conducted. Compound 23 induces apoptosis of HeLa cells
effectively at the indicated concentration, and over 60% of
apoptotic HeLa cells were detected after incubating with 3 μM
compound 23 for 48 h (Figure 7B). However, the positive
controls SAHA and BTSA1 exhibit weaker apoptosis induction
(apoptotic rates are 47.1 and 19.2%, respectively). In addition,
compound 23 also induces apoptosis more effectively in HeLa
cells at lower concentrations (0.3 and 1 μM) compared to the
positive controls.
Mitochondrial Depolarization Assay (mtΔΨ) and Cas-
pase-3 Activation Assay. Bax conformational activation,
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Figure 8. (A) TMRE mitochondrial potential assay in HeLa cells treated with serial dilution doses of compound 23, BTSA1, and SAHA at
indicated time points; (B) caspase-3 activity in HeLa cells incubated with indicated concentrations of compound 23, BTSA1, and SAHA.
Figure 9. (A) Apoptosis induction in HeLa cells Bax−/− of compound 23, BTSA1, and SAHA; (B) viability assay of HeLa cells Bax−/− treated with
compound 23, BTSA1, and SAHA for 48 h.
translocation, and oligomerization at the mitochondrial outer
membrane (MOM) result in mitochondrial outer membrane
permeabilization (MOMP), followed by the release of
cytochrome c, Smac/Diablo, and other mitochondrial factors,
thereby initiating the caspase cascade of apoptosis.33 There-
fore, we monitored mitochondrial potential changes and
caspase-3 activation of HeLa cells in response to compound
23, BTSA1, and SAHA. Upon compound 23, BTSA1, and
SAHA treatment, the mitochondrial potential changes were
determined with tetramethylrhodamine ethyl ester (TMRE,
Sigma cat. 87917). Mitochondrial potential loss was detected
in HeLa cells incubated with compound 23, BTSA1, and
SAHA, but our compound 23 could induce mitochondrial
depolarization more effectively in a dose-responsive manner
and a time-dependent manner (Figure 8A).
In addition, caspase-3 activation in response to compound
23 was determined with the caspase-3 fluorometric assay kit
(Biovision, K105-200) in HeLa cells. The results (Figure 8B)
revealed that upon treatment of HeLa cells with compound 23
for different times (6, 12, 18, and 24 h), the amount of
activated caspase-3 is significantly increased compared with the
control (CTL). All tested compounds induced the activation of
caspase-3 in a dose-dependent manner and a time-dependent
manner, and compound 23 exhibits the best property
concerning caspase-3 activation.
protein was knocked down using siRNA (RIBOBIO, cat.
stB0003578A). Subsequently, the apoptotic rates of HeLa cells
Bax−/− in response to compound 23, BTSA1, and SAHA were
determined using flow cytometry analysis (annexin V/PI
staining). Figure 9A suggests that Bax knockdown greatly
impaired the ability of compound 23 to induce apoptosis in
HeLa cells Bax−/− (14.8% apoptotic rate at 3 μM) and even
abolished the ability of BTSA1 to induce apoptosis, whereas
Bax knockdown shows less impact on SAHA.
We further evaluated the antiproliferative activities of
compound 23, BTSA1, and SAHA against HeLa cells
Bax−/−. Compound 23 and BTSA1 exhibited weaker
antiproliferative activities against HeLa cells Bax−/− (Figure
9B, compound 23, IC50 = 7.55 μM; BTSA1, IC50 > 20 μM)
compared with wild-type HeLa cells (compound 23, IC50
=
0.86 μM; BTSA1, IC50 = 11.47 μM). Bax knockdown greatly
impaired the antiproliferative activity of compound 23 and
even abolished the antiproliferative activity of BTSA1.
However, the antiproliferative activity of SAHA against HeLa
cells Bax−/− (IC50 = 4.66 μM) is comparable to that of wild-
type HeLa cells (IC50 = 2.65 μM).
These results indicated that compound 23 and BTSA1
suppress the growth of HeLa cells by inducing Bax-dependent
apoptosis. However, Bax knockdown, which abolishes Bax-
dependent apoptosis, greatly impaired the apoptosis induction
of compound 23 and BTSA1 in HeLa cells Bax−/−, followed by
weaker antiproliferative activities.
Bax Knockdown in HeLa Cells. To prove that compound
23 can induce Bax-dependent apoptosis in HeLa cells, the Bax
H
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Figure 10. (A) Cytotoxicity of compound 23, BTSA1, and SAHA on normal HL-7702 cells; (B) stability of compound 23 in rat plasma.
In Vitro Cytotoxicity Studies. Toxicity study is an inevitable
issue in drug development, and the cytotoxicities of compound
23, BTSA1, and SAHA were evaluated using healthy HL-7702
cells (human normal liver cells). All tested compounds
exhibited acceptable cytotoxicity (Figure 10A), whereas
BTSA1 (IC50 = 54.2 μM) and compound 23 (IC50 = 65.2
μM) exhibited lower cytotoxicity compared with SAHA (IC50
= 27.4 μM). Given the good antiproliferative activity against
HeLa cells and acceptable cytotoxicity over normal HL-7702
cells, we conclude that compound 23 kills HeLa cells
effectively and spares healthy cells.
Stability of Compound 23 in Rabbit Plasma. A
preliminary investigation of the stability of compound 23
was performed. After incubating compound 23 with rabbit
plasma at 37 °C for indicated times (0, 0.5, 1, 3, 6, 12, and 24
h), samples were analyzed using high-performance liquid
chromatography (HPLC). The result (Figure 10B) shows that
compound 23 is stable in rabbit plasma.
EXPERIMENTAL SECTION
■
Biology. Reagents. Primary antibodies were purchased from Cell
Signaling Technology (CST), Santa Cruz, Abcam, and Beyotime
Biotechnology. HeLa cells (ATCC, CCL-2) were purchased from the
American-type culture collection (ATCC). Salt, organic solvents, and
other reagents were from Sigma-Aldrich, Cell Signaling Technology
(CST), Biovision, Beyotime Biotechnology, and Ribobio Technology.
Production of Recombinant Protein. Human, recombinant Bax
was expressed in transformed Escherichia coli BL21 (DE3), and
protein expression was induced with 0.5 mM isopropyl β-D-1-
thiogalactopyranoside (IPTG) in kanamycin-containing Luria Broth.
For specific methods and more details, please refer to the Supporting
Information.
Calculation of the Combination Index. The combination of
BTSA1 and SAHA was performed, and the results were analyzed
using the Chou−Talalay method.30 The combination index (CI)
value was calculated with CompuSyn according to the dose-inhibition
relationship of the combination of SAHA and BTSA1 (1:1) or single
drugs. Final CI results were the average of detailed CI values at
inhibition rates of 0.5, 0.75, and 0.9 and expressed as mean SEM.
Moreover, synergism is subdivided into (near) additive (CI = 1.0),
slight synergism (CI value is within 0.85−1.0), moderate synergism
(CI value is within 0.7−0.85), synergism (CI value is within 0.3−0.7),
strong synergism (CI value is within 0.1−0.3), and very strong
synergism (CI < 0.1).
Fluorescence Polarization Binding Assay. Bax Binding Assay.
Direct binding curves were generated by incubating FITC-BIM (50
nM) with indicated dilutions of full-length His-Bax, and fluorescence
polarization was measured at 30 min on an Envision station
(PerkinElmer) under the condition of an excitation wavelength
(480 nm) and an emission wavelength (535 nm). For competition
assays, a serial dilution of test compounds or acetylated BIM (Ac-
BIM) was incubated with full-length His-Bax (500 nM) for 30 min,
followed by the addition of FITC-BIM (50 nM). After 20 min of
incubation, fluorescence polarization was measured on an Envision
station (PerkinElmer). EC50 or IC50 values were calculated by
nonlinear regression analysis of dose−response curves with Graphpad
Prism software.
Bcl-2, Mcl-1, and Bcl-XL Binding Assays. 5-FAM Bid-BH3 direct
binding curves to His-Bcl-2, His-Mcl-1, and His-Bcl-XL were
generated by incubating the fluorescent peptide (50 nM) with the
indicated dilution of full-length recombinant purified proteins, and
fluorescence polarization was measured at 30 min on an Envision
station (PerkinElmer) under the condition of an excitation wave-
length (485 nm) and an emission wavelength (535 nm). As for
competition assays, a serial dilution of the tested compounds or ABT-
199 was incubated with full-length His-Bcl-2 (500 nM), His-Mcl-1
(250 nM), or His-Bcl-XL (200 nM) for 30 min, and 5-FAM Bid-BH3
(10 nM) was added. After 20 min, fluorescence polarization was
measured on an Envision station (PerkinElmer). EC50 or IC50 values
were calculated by nonlinear regression analysis of dose−response
curves with GraphPad Prism software.
CONCLUSIONS
■
Encouraged by the better antiproliferative activity of the
combination of BTSA1 and SAHA compared with a single
drug, a series of novel HDAC−Bax multiple ligands were
designed rationally. Preliminary evaluation revealed that all of
the target compounds exhibit good profiles in a preliminary
screening concerning HDAC inhibitory activities, Bax affinities,
and Bax selectivity. Compound 23 possesses similar HDAC
inhibitory activity relative to SAHA and Bax affinity
comparable to BTSA1, whereas its antiproliferative activities
are 15- and 3-fold better than BTSA1 and SAHA, respectively.
The good antiproliferative activity and acceptable cytotoxicity
of compound 23 indicated that the design strategy of HDAC−
Bax multiple ligands is successful in HeLa cell growth
suppression. In other words, the two fragments (HDAC
inhibitor and Bax activator) of compound 23 achieve
synergistic effects in suppression of HeLa cell growth.
Moreover, a series of experiments confirmed that our
compound 23 could upregulate Bax and induce the
conformation activation of Bax, followed by initiating the
intrinsic apoptosis pathway, thereby enhancing Bax-dependent
apoptosis. In addition, the ability of compound 23 to induce
Bax conformation activation is comparable to that of BTSA1/
Vorinostat cotreatment. In summary, we first report the design,
synthesis, and biological evaluations of HDAC−Bax multiple
ligands, and our studies display a new paradigm for the
treatment of solid tumor by enhancing Bax-dependent
apoptosis.
HDAC Inhibitory Assay. A fluorescence assay was used to evaluate
HDAC inhibitory activities of target compounds, with SAHA as the
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reference. Briefly, an enzyme solution (HeLa nuclear extract, HDAC1,
HDAC2, and HDAC8) was treated with a serial dilution of the tested
compounds, followed by 5 min of incubation at 37 °C, and then the
fluorogenic substrate Boc-Lys (acetyl)-AMC or Boc-Lys (triflour-
oacetyl)-AMC was added. After 30 min of incubation at 37 °C, the
reaction was quenched by the addition of a developer containing
trypsin and trichostatin A (TSA). The suspension was incubated at 37
°C for 20 min, and the fluorescence intensity was monitored using a
microplate reader at the excitation and emission wavelengths of 390
and 460 nm. The fluorescence intensity readings of the tested wells
relative to those of the control wells were used to calculate the
inhibition ratios and the IC50 values.
Western Blot. After treating with compound 23, BTSA1, SAHA, or
vehicle (0.2% dimethyl sulfoxide (DMSO)) for 6 and 12 h, HeLa cells
were harvested and lysed with a radioimmunoprecipitation assay
(RIPA) buffer containing 1 mM phenylmethylsulfonyl fluoride
(PMSF) as well as 1% phosphatase inhibitor cocktail solution on
ice for 30 min. Lysates were centrifugated at 4 °C, 14 000 rpm, and
the supernatants were collected and boiled in loading buffer. Protein
samples were electrophoretically separated on 12.5% sodium dodecyl
sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gel
(EpiZyme, PAGE gel fast preparation kit), transferred to immobi-
lon-FL poly(vinylidene fluoride) (PVDF) membranes (Millipore),
and subjected to immunoblotting. For visualization of proteins,
membranes were blocked in TBST containing 5% nonfat dry milk.
After washing three times with TBST, the membranes were incubated
with primary antibodies overnight at 4 °C in a 1:1000 dilution. Then,
membranes were washed with TBST and incubated with a secondary
antibody in a 1:2000 dilution. Finally, the ECL reaction was
performed using the Boster ECL kit, and membranes were detected
on Amersham Imager 680. Proteins were detected following primary
antibodies: Bax (Cell Signaling cat. 2772), Ac-H3 (Cell Signaling cat.
13998), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(Beyotime, AF1186).
Immunoprecipitation Assay. Incubated with compound 23,
BTSA1, SAHA, or vehicle (0.2% DMSO) for 6 and 12 h, HeLa
cells were harvested and lysed with NP-40 buffer containing 1 mM
PMSF and 1% phosphatase inhibitor cocktail solution on ice for 30
min. Lysates were immunoprecipitated with protein A agarose beads
(Cell Signaling cat. 9863) and the conformation-specific primary
antibody 6A7 (Santa Cruz, sc-23959) that recognizes the exposure of
the Bax activation epitope overnight at 4 °C. The beads were
collected by centrifugation and then washed three times with NP-40
buffer, followed by boiling in a loading buffer. Protein samples were
analyzed using Western blot in the presence of the anti-Bax primary
antibody (Cell Signaling cat. 2772) and GAPDH (Beyotime,
AF1186).
Cell Viability and Cytotoxicity Studies. HeLa cells and HeLa cells
Bax−/− were cultured with the Roswell Park Memorial Institute
(RPMI) 1640 medium (10% fetal bovine serum (FBS)) at 37 °C in a
5% CO2 humidified incubator. In brief, the cells were seeded at
3500−4000 cells per well (100 μL/well) in 96-well plates for 8 h,
followed by incubating with the indicated dilution of the tested
compound (100 μL/well) for 48 h. The solution of 0.5% 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (10 μL/
well) was added, and the mixture was incubated for another 4 h. A
total of 150 μL of DMSO was added and shaken for dissolution for 15
min at 37 °C. Absorbance was determined with a microtiter-plate
reader at 570 nm to calculate the inhibition ratios and the IC50 values.
Moreover, cytotoxicity studies of compound 23, TBSA1, and
SAHA were evaluated in healthy HL-7702 cells (human normal liver
cells) in a manner that was described for cell viability assays.
Cell Morphologic Features. HeLa cells were seeded at 1 × 105
cells per well (2 mL/well) in six-well clear-bottom plates. After 8 h of
incubation at 37 °C in a 5% CO2 humidified incubator, the solution of
compound 23 (1 μM) was added, followed by incubating for 6, 12,
24, and 36 h. Then, cell morphology changes were monitored using a
Zeiss Axio Observer A1 fluorescence microscope.
HeLa cells. HeLa cells (1 × 105) were incubated with 1 μM
compound 23, BTSA1, SAHA, or vehicle (0.2% DMSO) in six-well
clear-bottom plates for 48 h and then washed with prechilled
phosphate-buffered saline (PBS) two times. Then, these cells were
fixed with paraformaldehyde for 30 min at rt. After treating PBS
containing 0.5% Triton X-100 for 5 min, cells were washed with PBS
three times, followed by the addition of a mixture of terminal
deoxynucleotidyl transferase and FITC-12-dUTP. Finally, cells were
washed two times carefully and green fluorescence was observed using
laser confocal microscopy.
Flow Cytometry Analysis. Flow cytometry analysis was performed
in HeLa cells and HeLa cells Bax−/−. Cells (1 × 105 cells/well) were
incubated with different concentrations (0.3, 1, and 3 μM) of
compound 23, BTSA1, SAHA, or vehicle (0.2% DMSO) in six-well
clear-bottom plates for 48 h. Subsequently, cells were harvested by
centrifugation and washed with cold PBS buffer. Cells were
resuspended in 195 μL of annexin V−fluorescein isothiocyanate
(FITC) binding buffer, and 2.5 μL of annexin V−FITC and 5 μL of
propidium iodide (PI) were added. The samples were incubated for
15 min at room temperature and analyzed using flow cytometry.
Mitochondrial Depolarization Assay (mtΔΨ). Cells (1 × 105
cells/well) were seeded in six-well clear-bottom plates and incubated
with the indicated dilution of compound 23, BTSA1, SAHA, or
vehicle (0.2% DMSO). Cells were stained with 100 nM
tetramethylrhodamine ethyl ester (TMRE, Sigma cat. 87917) for 30
min at 37 °C. Subsequently, cells were collected by centrifugation and
washed with PBS to eliminate background fluorescence. They were
transferred into a black 96-well plate, and the fluorescence intensity
was detected by a Thermo Varioskan microplate reader (Ex: 540 nm;
Em: 579 nm).
Caspase-3 Activation Assay. HeLa cells were incubated with
various concentrations (0.3, 1, and 3 μM) of potent compound 23,
BTSA1, SAHA, or vehicle (0.2% DMSO) for different times (6, 12,
18, and 24 h); then, 1 × 106 cells were collected and washed with
PBS. Cells were resuspended with 50 μL of chilled cell lysis buffer on
ice for 10 min, and the lyases were transferred into a 96-well plate.
Then, 50 μL of 2× reaction buffer (containing 10 mM dithiothreitol
(DTT)) and 5 μL of caspase-3 substrates (1 mM DEVE-AFC) were
added to each sample, and the mixture was incubated at 37 °C for 1.5
h. The fluorescence intensity was tested in a microplate reader at the
excitation and emission wavelengths of 400 and 505 nm. The results
are expressed as fold increase in the caspase activity of apoptotic cells
over that of noninduced cells.
Cellular Transfections. HeLa cells were transfected with siRNA of
Bax to silence the Bax protein with the riboFECT CP transfection kit
(RIBOBIO, Cat. C10511-05) according to the manufacturer’s
protocol. Generally, HeLa cells were incubated with a mixture of
1× riboFECT CP buffer, 50 nM siRNA of Bax as well as the
riboFECT CP reagent and cultured with an RPMI 1640 medium
(10% FBS) at 37 °C in a 5% CO2 humidified incubator. After 24 and
72 h, cells were harvested and lysed with a RIPA buffer containing 1
mM PMSF and a 1% phosphatase inhibitor cocktail solution. Lysates
were used to monitor the transfection efficiency with Western blot.
Stability of Compound 23 in Rat Plasma. First, 50 μL of
compound solution (2 mg/mL in DMSO) and 200 μL of rabbit
plasma were incubated at 37 °C for indicated times (0, 0.5, 1, 3, 6, 12,
and 24 h). Then, 600 μL of acetonitrile was added into each sample
aliquot, followed by centrifugation (14 000 rpm, 10 min) and
filtration. Samples were analyzed using HPLC with a C18 column
(150 mm × 4.6 mm, 5 μm, Diamonsil) at a flow rate of 1 mL/min.
The mixture of 85% methanol and 15% H2O containing 0.1% formic
acid was used as the mobile phase.
Molecular Docking and Molecular Dynamics Simulation. The
NMR structure of Bax (PDB ID: 2K7W) was selected for molecular
docking experiments using GLIDE (Glide, version 11.5, Schrodinger).
After protein preparation and grid generation, BTSA1 was docked
into the Bax trigger site and top-scoring binding poses were chosen as
the proposed binding modes. Then, the lowest-energy structure pose
from docking was subjected to molecular dynamics (MD) simulation
using DESMOND (DESMOND, version 3, Schrodinger); please refer
TUNEL Assay. Terminal deoxynucleotidyl transferase (TdT) dUTP
nick-end labeling (TUNEL) assay was designed to detect apoptotic
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to a previous report.19 Clustering and analysis of the trajectory were
performed with MAESTRO tools (Maestro, version 10.0, Schro-
dinger).
Moreover, compound 23 was docked into Bax in a manner similar
to those described for the docking studies of BTSA1, and the results
are displayed in the Supporting Information.
8.14−8.11 (m, 2H), 8.06−8.01 (m, 3H), 7.73 (d, J = 4.1 Hz, 1H),
7.60−7.51 (m, 3H), 7.38 (d, J = 4.0 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 179.37, 167.55, 155.33, 153.46, 149.60, 149.30, 138.42,
132.15, 130.88, 130.47, 130.41, 130.36, 130.33, 128.99, 128.50,
126.37, 114.52, 111.81. HRMS (AP-ESI) m/z, calcd for
C22H14N6O3S2, ([M + H]+): 475.0642, found: 475.0634. HPLC tR
= 6.454 min (97.78% purity).
Chemistry. In our work, all start materials, reagents, and solvents
are analytical grade and used without further purification unless
otherwise stated. Reactions were monitored by thin-layer chromatog-
raphy (TLC) on 0.25 mm silica gel plates (60GF-254), and the spots
were visualized with UV light, chloride ferric, and iodine vapor.
Melting points were determined by the RY-1 electrothermal melting
(Z)-N-Hydroxy-4-(2-(5-oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
benzamide (4). To a solution of compound 3 (1.59 g, 4 mmol) in
DMF (30 mL) at 0 °C, isobutyl chloroformate (0.61 mL, 4.8 mmol)
and N-methylmorpholine (0.58 mL, 5.2 mmol) were added and the
mixture was stirred for 30 min. It was filtered, and the filtrate was
added to freshly prepared hydroxylamine (0.53 g, 16 mmol) in
methanol. After stirring the mixture for another 6 h at room
temperature, the reaction mixture was poured into 120 mL of H2O.
Filter and the crude product was purified by silica gel column
chromatography to obtain the title compound 0.65 g, yield: 45%, mp
1
point apparatus. H NMR and 13C NMR spectra were obtained on a
Brucker DRX spectrometer at 400 MHz. Chemical shifts are shown in
parts per million (ppm) relative to tetramethylsilane (TMS) as the
internal standard and are significant. 1H NMR data are reported in the
following order: multiplicity (m, multiplet; q, quartet; t, triplet; d,
doublet; s, singlet) number of protons. High-resolution mass spectra
(HRMS) were conducted on an Agilent 6510 quadrupole time-of-
flight liquid chromatography/mass spectrometer (LC/MS) delivered
with electrospray ionization (ESI). The purities of all of the target
compounds were determined by reversed-phase (RP)-HPLC with a
Shimadzu LC-20AT system and a C18-silica column (Thermo, 4.6 ×
150 mm2, 5 μm). Elution was methanol/water (80:20−90:10), and
the flow rate was 1 mL/min. All of the final compounds achieved a
minimum of 95% purity.
1
194−195 °C. H NMR (400 MHz, DMSO-d6) δ 11.29 (s, 1H), 9.05
(s, 1H), 8.23−7.93 (m, 5H), 7.86 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 3.9
Hz, 1H), 7.55 (q, J = 7.2 Hz, 3H), 7.35 (d, J = 3.9 Hz, 1H). 13C NMR
(101 MHz, DMSO-d6) δ 179.76, 164.39, 155.40, 149.41, 137.04,
133.69, 132.33, 131.20, 130.35, 130.26, 129.25, 128.91, 128.51,
127.90, 126.23, 114.76, 110.95. HRMS (AP-ESI) m/z, calcd for
C22H15N7O3S2, ([M + H]+): 490.0751, found: 490.0746. HPLC tR =
16.209 min (98.64% purity).
Methyl 5-(4-Acetylbenzamido)pentanoate (5). To a solution of 4-
carboxybenzaldehyde (1.64 g, 10 mmol), DIPEA (1.65 mL, 10 mmol)
and HATU (4.56 g, 12 mmol) were added, and the mixture was
stirred for 30 min at room temperature. After adding methyl 5-
aminopentanoate hydrochloride (2.01 g, 11 mmol), the mixture was
stirred at room temperature overnight. The reaction solution was
washed with 1 M HCl, saturated NaHCO3, and saturated brine. The
organic phase was dried and concentrated in vacuo to give the crude
product. The crude product was purified by silica gel column
chromatography to obtain a white solid 2.35 g, yield: 85%, mp 89−90
°C. 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 8.1 Hz, 2H), 7.87 (d,
J = 8.0 Hz, 2H), 6.48 (s, 1H), 3.69 (s, 3H), 3.49 (q, J = 6.2 Hz, 2H),
2.64 (s, 3H), 2.40 (t, J = 6.7 Hz, 2H), 1.72 (qd, J = 14.1, 7.0 Hz, 4H).
Methyl 6-(4-Acetylbenzamido)hexanoate (6). The title com-
pound was synthesized from 4-carboxybenzaldehyde and methyl 6-
aminohexanoate hydrochloride in a manner similar to that described
Ethyl (Z)-3-Oxo-3-phenyl-2-(2-(thiazol-2-yl)hydrazono)-
propanoate (1). To a solution of 2-aminothiazole (8 g, 80 mmol)
in 6 M HCl (112 mL) at 0 °C, a solution of sodium nitrite (6.62 g, 96
mmol) in water (64 mL) was added dropwise while the temperature
of the reaction mixture was maintained below 5 °C. When the
addition was completed, the resulting diazonium salt was stirred for
30 min at 0 °C. A solution of sodium acetate (38 g, 460.8 mmol) in
water (80 mL) was added to a solution of active ethyl benzoylacetate
(12.30 g, 64 mmol) in ethanol (240 mL). The mixture was cooled to
0 °C, and the diazonium salt solution was added over a period of
several mines. Then, the mixture was stirred for 1 h at 0 °C and for
another 1 h at room temperature. Ethanol was reduced in vacuo and
then extracted with ethyl acetate (200 mL × 3). The organic phase
was combined and dried with anhydrous magnesium sulfate. Then,
the organic phase was concentrated in vacuo to give the crude
product, followed by purification using silica gel column chromatog-
raphy with petroleum ether/ethyl acetate to obtain an orange-red oil
10.5 g, yield: 54%. 1H NMR (400 MHz, CDCl3) δ 12.80 (s, 1H), 7.96
(d, J = 7.7 Hz, 2H), 7.61 (t, J = 7.4 Hz, 2H), 7.48 (t, J = 7.7 Hz, 2H),
7.39 (d, J = 3.6 Hz, 1H), 6.84 (d, J = 3.5 Hz, 1H), 4.37 (q, J = 7.2 Hz,
2H), 1.32 (t, J = 7.1 Hz, 3H).
1
for the preparation of compound 5, yield: 67%, mp 96−98°C. H
NMR (400 MHz, CDCl3) δ 8.00 (d, J = 7.9 Hz, 2H), 7.86 (d, J = 7.9
Hz, 2H), 6.38 (s, 1H), 3.67 (s, 3H), 3.49 (q, J = 6.6 Hz, 2H), 2.64 (s,
3H), 2.34 (t, J = 7.3 Hz, 2H), 1.76−1.60 (m, 4H), 1.50−1.34 (m,
2H).
Methyl 7-(4-Acetylbenzamido)heptanoate (7). The title com-
pound was synthesized from 4-carboxybenzaldehyde and methyl 7-
aminoheptanoate hydrochloride in a manner similar to that described
(Z)-5-Phenyl-2-(4-phenylthiazol-2-yl)-4-(2-(thiazol-2-yl)-
hydrazono)-2,4-dihydro-3H-pyrazol-3-one (2). To a solution of 2-
bromoacetophenone (0.33 g, 1.66 mmol) in ethanol (20 mL),
thiosemicarbazide (0.15 g, 1.66 mmol) was added, and the mixture
was stirred for 1 h at room temperature. When the addition of the
solution of intermediate 1 (0.5 g, 1.66 mmol) in ethanol was
completed, it was refluxed for 3 h. The reaction mixture was cooled
down, and the product was isolated by filtration. It was washed with
cool ethanol as well as diethyl ether, and the title compound was
1
for the preparation of compound 5, yield: 96%, mp 101−103 °C. H
NMR (400 MHz, DMSO-d6) δ 8.62 (t, J = 5.1 Hz, 1H), 8.02 (d, J =
7.6 Hz, 2H), 7.94 (d, J = 8.0 Hz, 2H), 3.57 (s, 3H), 3.26 (dd, J = 12.9,
6.5 Hz, 2H), 2.62 (s, 3H), 2.30 (t, J = 7.3 Hz, 2H), 1.53 (s, 4H), 1.30
(s, 4H).
Methyl 4-((4-Acetylbenzamido)methyl)benzoate (8). The title
compound was synthesized from 4-carboxybenzaldehyde and methyl
4-(aminomethyl)benzoate hydrochloride in a manner similar to that
described for the preparation of compound 5, yield: 99%, mp 132−
134°C. 1H NMR (600 MHz, CDCl3) δ 8.03−7.99 (m, 4H), 7.90 (d, J
= 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 6.78 (t, J = 6.0 Hz, 1H), 4.72
(d, J = 5.9 Hz, 2H), 3.91 (s, 3H), 2.63 (s, 3H).
1
obtained as a red solid 0.4 g, yield: 47%, mp 230−231 °C. H NMR
(400 MHz, DMSO-d6) δ 8.17 (d, J = 7.2 Hz, 2H), 8.01 (d, J = 7.6 Hz,
2H), 7.85 (s, 1H), 7.73 (d, J = 4.1 Hz, 1H), 7.61−7.53 (m, 3H), 7.48
(t, J = 7.6 Hz, 2H), 7.42−7.33 (m, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 155.17, 153.36, 150.36, 149.45, 134.58, 132.48, 130.97,
130.44, 130.37, 129.22, 129.00, 128.47, 126.41, 114.50, 109.48.
HRMS (AP-ESI) m/z, calcd for C21H14N6OS2, ([M + H]+):
431.0743, found: 431.0739. HPLC tR = 7.605 min (99.01% purity).
(Z)-4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)hydrazineylidene)-
4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)benzoic Acid (3). The title
compound was synthesized from 4-(2-bromoacetyl)benzoic acid,
thiosemicarbazide, and intermediate 1 in a manner similar to that
described for the preparation of compound 2, yield: 48%, mp 268−
269 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 7.4 Hz, 2H),
Methyl (E)-3-(4-((4-Acetylbenzamido)methyl)phenyl)acrylate (9).
The title compound was synthesized from 4-carboxybenzaldehyde
and methyl (E)-3-(4-(aminomethyl)phenyl)acrylate hydrochloride in
a manner similar to that described for the preparation of compound 5,
1
yield: 71%, mp 175−176°C. H NMR (400 MHz, DMSO-d6) δ 9.28
(t, J = 5.7 Hz, 1H), 8.04 (q, J = 8.1 Hz, 4H), 7.70 (d, J = 7.8 Hz, 2H),
7.66 (d, J = 16.0 Hz, 1H), 7.38 (d, J = 7.8 Hz, 2H), 6.63 (d, J = 16.0
Hz, 1H), 4.53 (d, J = 5.8 Hz, 2H), 3.73 (s, 3H), 2.63 (s, 3H).
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Methyl 5-(4-(2-Bromoacetyl)benzamido)pentanoate (10). To a
solution of methyl 5-(4-acetylbenzamido)pentanoate (2.3 g, 8.3
mmol) in chloroform (40 mL) and ethyl acetate (40 mL), copper(II)
bromide (3.71 g, 16.6 mmol) was added. Then, the reaction mixture
was refluxed for 3 h. It was filtered, and the filtrate was concentrated
under reduced pressure. The residues were dissolved with ethyl
acetate and washed with water. They were dried over, and the solvent
was removed. The crude product was purified by silica gel column
chromatography to obtain a white solid 2.24 g, yield: 72%, mp 82−84
°C. 1H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 8.1 Hz, 2H), 7.90 (d,
J = 8.1 Hz, 2H), 6.48 (s, 1H), 4.46 (s, 2H), 3.69 (s, 3H), 3.49 (q, J =
6.2 Hz, 2H), 2.40 (t, J = 6.7 Hz, 2H), 1.80−1.65 (m, 4H).
Methyl 6-(4-(2-Bromoacetyl)benzamido)hexanoate (11). The
title compound was synthesized from intermediate 6 in a manner
similar to that described for the preparation of compound 10, yield:
70%, mp 97−99 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.66 (s, 1H),
8.07 (d, J = 8.0 Hz, 2H), 7.96 (d, J = 7.7 Hz, 2H), 4.98 (s, 2H), 3.57
(s, 3H), 3.26 (dd, J = 12.4, 6.2 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H),
1.64−1.43 (m, 4H), 1.38−1.26 (m, 2H).
Methyl 7-(4-(2-Bromoacetyl)benzamido)heptanoate (12). The
title compound was synthesized from intermediate 7 in a manner
similar to that described for the preparation of compound 10, yield:
80%, mp 99−101 °C. 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 7.9
Hz, 2H), 7.87 (d, J = 7.9 Hz, 2H), 6.23 (s, 1H), 4.46 (s, 2H), 3.67 (s,
3H), 3.47 (dd, J = 13.3, 6.6 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.64
(dd, J = 14.1, 7.1 Hz, 4H), 1.50−1.33 (m, 4H).
Methyl 4-((4-(2-Bromoacetyl)benzamido)methyl)benzoate (13).
The title compound was synthesized from intermediate 8 in a manner
similar to that described for the preparation of compound 10, yield:
76%, mp 126−129 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.36 (dd, J
= 13.5, 6.7 Hz, 1H), 8.14−8.01 (m, 4H), 7.94 (d, J = 7.7 Hz, 2H),
7.47 (d, J = 7.9 Hz, 2H), 5.00 (s, 2H), 4.58 (d, J = 5.7 Hz, 2H), 3.84
(s, 3H).
benzamido)heptanoate (17). The title compound was synthesized
from intermediate 12, ethyl (Z)-3-oxo-3-phenyl-2-(2-(thiazol-2-yl)-
hydrazono)propanoate, and thiosemicarbazide in a manner similar to
that described for the preparation of compound 15, yield: 60%, mp
1
219−220 °C. H NMR (400 MHz, DMSO-d6) δ 8.50 (t, J = 5.2 Hz,
1H), 8.17 (d, J = 6.8 Hz, 2H), 8.07 (d, J = 7.8 Hz, 2H), 7.98 (s, 1H),
7.93 (d, J = 7.8 Hz, 2H), 7.73 (d, J = 3.7 Hz, 1H), 7.63−7.49 (m,
3H), 7.37 (d, J = 3.9 Hz, 1H), 3.58 (s, 3H), 3.27 (q, J = 6.1 Hz, 2H),
2.31 (t, J = 7.3 Hz, 2H), 1.54 (m, 4H), 1.32 (m, 4H).
Methyl (Z)-4-((4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
benzamido)methyl)benzoate (18). The title compound was
synthesized from intermediate 13, ethyl (Z)-3-oxo-3-phenyl-2-(2-
(thiazol-2-yl)hydrazono)propanoate, and thiosemicarbazide in a
manner similar to that described for the preparation of compound
1
15, yield: 33%, mp 182−183 °C. H NMR (600 MHz, DMSO-d6) δ
9.20 (t, J = 6.0 Hz, 1H), 8.17 (d, J = 7.4 Hz, 2H), 8.12 (dd, J = 9.5,
7.6 Hz, 2H), 8.04 (d, J = 6.5 Hz, 1H), 8.01 (d, J = 9.1 Hz, 2H), 7.97−
7.93 (m, 2H), 7.73 (d, J = 4.1 Hz, 1H), 7.60−7.53 (m, 4H), 7.51−
7.47 (m, 2H), 7.37 (d, J = 4.0 Hz, 1H), 4.59 (d, J = 5.9 Hz, 2H), 3.85
(s, 3H).
Methyl (E)-3-(4-((4-(2-((Z)-5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
benzamido)methyl)phenyl)acrylate (19). The title compound was
synthesized with intermediate 14, ethyl (Z)-3-oxo-3-phenyl-2-(2-
(thiazol-2-yl)hydrazono)propanoate, and thiosemicarbazide in a
manner similar to that described for the preparation of compound
1
15, yield: 68%, mp 250−251 °C. H NMR (400 MHz, DMSO-d6) δ
9.16 (t, J = 5.6 Hz, 1H), 8.16 (d, J = 6.7 Hz, 2H), 8.11 (d, J = 7.8 Hz,
2H), 8.00 (d, J = 7.0 Hz, 3H), 7.74 (d, J = 3.8 Hz, 1H), 7.70 (d, J =
7.6 Hz, 2H), 7.66 (d, J = 16.3 Hz, 1H), 7.56 (d, J = 6.5 Hz, 2H), 7.38
(t, J = 6.0 Hz, 3H), 6.62 (d, J = 16.0 Hz, 1H), 4.53 (d, J = 5.4 Hz,
2H), 3.72 (s, 3H).
(Z)-N-(5-(Hydroxyamino)-5-oxopentyl)-4-(2-(5-oxo-3-phenyl-4-
(2-(thiazol-2-yl)hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)-
thiazol-4-yl)benzamide (20). Hydroxylamine hydrochloride (2.92 g,
42 mmol) and potassium hydroxide (3.53 g, 63 mmol) were dissolved
in 21 and 15 mL of methyl alcohol, respectively. The solution of
potassium hydroxide was added into the solution of hydroxylamine
hydrochloride at 0 °C. After 10 min, the reaction mixture was filtered.
To the solution of compound 15 (1.76 g, 3 mmol) in DMF (8 mL),
freshly prepared hydroxylamine solution was added. The reaction
mixture was stirred for 3 h, and the solvent was removed in vacuo.
Then, 10 mL of water was added, and the solution was acidified to pH
= 4 and then filtered. The crude product was purified by silica gel
column chromatography as a red solid 0.97 g, yield: 55%, mp 219−
220 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.54 (t, J =
5.2 Hz, 1H), 8.17 (d, J = 6.9 Hz, 2H), 8.08 (d, J = 7.9 Hz, 2H), 7.98
(s, 1H), 7.94 (d, J = 7.8 Hz, 2H), 7.73 (d, J = 3.7 Hz, 1H), 7.61−7.50
(m, 3H), 7.37 (d, J = 3.6 Hz, 1H), 3.27 (t, J = 5.1 Hz, 2H), 1.99 (t, J
= 6.2 Hz, 2H), 1.62−1.45 (m, 4H). 13C NMR (101 MHz, DMSO-d6)
δ 179.49, 174.94, 169.56, 166.22, 155.34, 153.47, 149.51, 136.87,
134.23, 133.06, 131.03, 130.34, 129.76, 128.93, 128.51, 128.15,
126.10, 114.64, 110.95, 39.48, 32.54, 29.31, 23.29. HRMS (AP-ESI)
m/z, calcd for C27H24N8O4S2, ([M + H]+): 589.1435, found:
589.1422. HPLC tR = 8.293 min (98.95% purity).
(Z)-N-(6-(Hydroxyamino)-6-oxohexyl)-4-(2-(5-oxo-3-phenyl-4-(2-
(thiazol-2-yl)hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)-
thiazol-4-yl)benzamide (21). The title compound was synthesized
with intermediate 16 in a manner similar to that described for the
preparation of compound 20, yield: 46%, mp >280 °C. 1H NMR (400
MHz, DMSO-d6) δ 10.37 (s, 1H), 8.67 (s, 1H), 8.54 (s, 1H), 8.19 (d,
J = 6.3 Hz, 2H), 8.07 (d, J = 8.0 Hz, 2H), 7.93 (d, J = 7.9 Hz, 2H),
7.89 (s, 1H), 7.68 (d, J = 2.7 Hz, 1H), 7.50 (dd, J = 14.1, 7.2 Hz, 3H),
7.30 (d, J = 3.5 Hz, 1H), 3.26 (d, J = 6.4 Hz, 2H), 1.96 (t, J = 7.1 Hz,
2H), 1.53 (s, 4H), 1.30 (d, J = 6.5 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 182.94, 169.59, 166.24, 155.91, 153.45, 150.30, 149.03,
141.82, 137.26, 134.00, 133.22, 129.14, 128.59, 128.14, 126.03,
122.52, 116.12, 110.20, 49.07, 32.72, 29.42, 26.63, 25.43. HRMS (AP-
ESI) m/z, calcd for C28H26N8O4S2, ([M + H]+): 603.1591, found:
603.1589. HPLC tR = 13.034 min (97.31% purity).
Methyl (E)-3-(4-((4-(2-Bromoacetyl)benzamido)methyl)phenyl)-
acrylate (14). The title compound was synthesized from intermediate
9 in a manner similar to that described for the preparation of
compound 10, yield: 63%, mp 149−151 °C. H NMR (400 MHz,
CDCl3) δ 8.05 (d, J = 8.1 Hz, 2H), 7.91 (d, J = 8.0 Hz, 2H), 7.68 (d, J
= 16.0 Hz, 1H), 7.52 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 7.9 Hz, 2H),
6.54 (s, 1H), 6.43 (d, J = 16.0 Hz, 1H), 4.69 (d, J = 5.7 Hz, 2H), 4.45
(s, 2H), 3.81 (s, 3H).
1
Methyl (Z)-5-(4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
benzamido)pentanoate (15). To the solution of methyl 5-(4-(2-
bromoacetyl)benzamido)pentanoate (0.42 g, 1 mmol) in methanol
(20 mL), thiosemicarbazide (0.09 g, 1 mmol) was added, and the
mixture was stirred for 1 h at room temperature. When the addition of
the solution of ethyl (Z)-3-oxo-3-phenyl-2-(2-(thiazol-2-yl)-
hydrazono)propanoate (0.36 g, 1 mmol) in methanol (10 mL) was
completed, it was refluxed for another 3 h. The reaction mixture was
cooled down, and the product was isolated by filtration. It was washed
with cold methanol and diethyl ether, and compound 15 was obtained
1
as a red solid 0.28 g, yield: 64%, mp 196−197 °C. H NMR (400
MHz, DMSO-d6) δ 8.53 (t, J = 5.4 Hz, 1H), 8.16 (d, J = 6.7 Hz, 2H),
8.08 (d, J = 7.9 Hz, 2H), 7.98 (s, 1H), 7.93 (d, J = 8.1 Hz, 2H), 7.73
(d, J = 4.0 Hz, 1H), 7.60−7.51 (m, 3H), 7.37 (d, J = 3.9 Hz, 1H),
3.59 (s, 3H), 3.28 (d, J = 5.6 Hz, 2H), 2.36 (t, J = 6.7 Hz, 2H), 1.66−
1.48 (m, 4H).
Methyl (Z)-6-(4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
benzamido)hexanoate (16). The title compound was synthesized
with intermediate 11, ethyl (Z)-3-oxo-3-phenyl-2-(2-(thiazol-2-yl)-
hydrazono)propanoate, and thiosemicarbazide in a manner similar to
that described for the preparation of compound 15, yield: 59%, mp
1
199−200 °C. H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H), 8.16
(s, 2H), 8.07 (s, 2H), 8.01−7.84 (m, 3H), 7.73 (s, 1H), 7.55 (s, 3H),
7.37 (s, 1H), 3.58 (s, 3H), 3.26 (s, 2H), 2.37−2.24 (m, 2H), 1.61−
1.45 (m, 4H), 1.39−1.29 (m, 2H).
Methyl (Z)-7-(4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
L
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Article
(Z)-N-(7-(Hydroxyamino)-7-oxoheptyl)-4-(2-(5-oxo-3-phenyl-4-
(2-(thiazol-2-yl)hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)-
thiazol-4-yl)benzamide (22). The title compound was synthesized
with intermediate 17 in a manner similar to that described for the
preparation of compound 20, yield: 77%, mp 187−188 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.35 (s, 1H), 8.51 (t, J = 5.2 Hz, 1H), 8.17
(d, J = 6.8 Hz, 2H), 8.07 (d, J = 7.8 Hz, 2H), 7.98 (s, 1H), 7.93 (d, J =
7.9 Hz, 2H), 7.73 (d, J = 3.7 Hz, 1H), 7.62−7.50 (m, 3H), 7.37 (d, J
= 3.7 Hz, 1H), 3.27 (d, J = 6.2 Hz, 2H), 1.95 (t, J = 7.2 Hz, 2H),
1.63−1.44 (m, 4H), 1.38−1.22 (m, 4H). 13C NMR (101 MHz,
DMSO-d6) δ 179.77, 169.61, 166.18, 155.35, 153.40, 149.65, 149.53,
136.83, 134.31, 132.81, 131.07, 130.37, 129.92, 128.96, 128.52,
128.17, 126.09, 114.61, 110.96, 39.70, 32.73, 29.55, 28.85, 26.75,
25.59. HRMS (AP-ESI) m/z, calcd for C29H28N8O4S2, ([M + H]+):
617.1748, found: 617.1735. HPLC tR = 11.52 min (97.88% purity).
(Z)-N-Hydroxy-4-((4-(2-(5-oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
benzamido)methyl)benzamide (23). The title compound was
synthesized with intermediate 18 in a manner similar to that
described for the preparation of compound 20, yield: 29%, mp 217−
(400 MHz, CDCl3) δ 7.92 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.4 Hz,
2H), 4.02 (t, J = 6.3 Hz, 2H), 3.68 (s, 3H), 2.55 (s, 3H), 2.44−2.31
(m, 2H), 1.94−1.79 (m, 2H), 1.72 (dt, J = 15.2, 7.4 Hz, 2H), 1.52
(dt, J = 15.1, 7.7 Hz, 2H).
Methyl 7-(4-Acetylphenoxy)heptanoate (28). The title compound
was synthesized with 4′-hydroxyacetophenone and methyl 7-
bromoheptanoate in a manner similar to that described for the
1
preparation of compound 25, yield: 90%, mp 63−65 °C. H NMR
(400 MHz, CDCl3) δ 7.92 (d, J = 8.1 Hz, 2H), 6.91 (d, J = 8.2 Hz,
2H), 4.02 (t, J = 6.4 Hz, 2H), 3.67 (s, 3H), 2.55 (s, 3H), 2.33 (t, J =
7.4 Hz, 2H), 1.89−1.76 (m, 2H), 1.74−1.62 (m, 2H), 1.50 (dt, J =
14.8, 7.3 Hz, 2H), 1.40 (dt, J = 14.7, 7.4 Hz, 2H).
Methyl 8-(4-Acetylphenoxy)octanoate (29). The title compound
was synthesized with 4′-hydroxyacetophenone and methyl 8-
bromooctanoate in a manner similar to that described for the
1
preparation of compound 25, yield: 92%, mp 52−54 °C. H NMR
(400 MHz, CDCl3) δ 7.92 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.4 Hz,
2H), 4.01 (t, J = 6.4 Hz, 2H), 3.67 (s, 3H), 2.55 (s, 3H), 2.32 (t, J =
7.4 Hz, 2H), 1.79 (dq, J = 13.4, 6.6 Hz, 2H), 1.64 (dt, J = 12.1, 6.2
Hz, 2H), 1.56−1.44 (m, 2H), 1.43−1.26 (m, 4H).
1
219 °C. H NMR (400 MHz, DMSO-d6) δ 12.86 (s, 1H), 11.18 (s,
Methyl 4-((4-Acetylphenoxy)methyl)benzoate (30). The title
compound was synthesized with 4′-hydroxyacetophenone and methyl
4-(bromomethyl)benzoate in a manner similar to that described for
1H), 9.15 (s, 1H), 8.99 (s, 1H), 8.19 (d, J = 6.3 Hz, 1H), 8.11 (d, J =
7.9 Hz, 1H), 8.05−7.84 (m, 6H), 7.72 (d, J = 7.7 Hz, 2H), 7.66 (d, J
= 2.9 Hz, 1H), 7.56−7.37 (m, 4H), 7.26 (d, J = 3.1 Hz, 1H), 4.54 (s,
2H). 13C NMR (101 MHz, DMSO-d6) δ 179.58, 166.45, 164.64,
155.43, 153.53, 149.67, 149.44, 143.48, 137.21, 133.89, 133.73,
131.79, 131.20, 130.25, 129.15, 128.90, 128.53, 128.31, 127.63,
127.46, 126.21, 114.79, 111.07, 42.99. HRMS (AP-ESI) m/z, calcd
for C30H22N8O4S2, ([M + H]+): 623.1278, found: 623.1270. HPLC tR
= 14.884 min (97.12% purity).
N-(4-((E)-3-(Hydroxyamino)-3-oxoprop-1-en-1-yl)benzyl)-4-(2-
((Z)-5-oxo-3-phenyl-4-(2-(thiazol-2-yl)hydrazineylidene)-4,5-dihy-
dro-1H-pyrazol-1-yl)thiazol-4-yl)benzamide (24). The title com-
pound was synthesized with intermediate 19 in a manner similar to
that described for the preparation of compound 20, yield: 36%, mp
251−253°C. 1H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H), 10.09
(s, 1H), 9.15 (t, J = 5.9 Hz, 1H), 8.18 (d, J = 6.7 Hz, 2H), 8.10 (d, J =
7.8 Hz, 2H), 8.00 (d, J = 7.9 Hz, 2H), 7.94 (s, 1H), 7.77−7.64 (m,
3H), 7.60 (d, J = 15.9 Hz, 1H), 7.53 (m, 3H), 7.38 (d, J = 7.7 Hz,
2H), 7.32 (d, J = 3.3 Hz, 1H), 6.50 (d, J = 15.9 Hz, 1H), 4.53 (d, J =
5.6 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 181.07, 168.08,
166.42, 155.60, 153.45, 149.26, 144.21, 142.59, 137.31, 133.70,
133.29, 131.97, 129.87, 128.90, 128.81, 128.73, 128.58, 128.29,
128.21, 127.94, 127.20, 126.17, 119.20, 115.14, 110.80, 42.99. HRMS
(AP-ESI) m/z, calcd for C32H24N8O4S2, ([M + H]+): 649.1435,
found: 649.1445. HPLC tR = 9.004 min (98.36% purity).
Methyl 4-(4-Acetylphenoxy)butanoate (25). A solution of 4′-
hydroxyacetophenone (2.7 g, 20 mmol) in DMF was mixed with
K2CO3 (8.3 g, 60 mmol); then, methyl 4-bromobutanoate (3.77 mL,
30 mmol) and KI (0.1 g) were added to the solution. The mixture was
stirred at room temperature overnight, and the mixture was poured
into cool water. It was extracted with ethyl acetate three times, and
the organic phases were combined. It was dried over with anhydrous
magnesium sulfate for 0.5 h, and then it was filtered. The solvent was
removed, and the crude product was purified using silica gel column
chromatography to obtain a white solid 3.94 g, yield: 83%, mp 64−66
°C. 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 8.2 Hz, 2H), 6.91 (d,
J = 8.3 Hz, 2H), 4.08 (t, J = 6.0 Hz, 2H), 3.70 (s, 3H), 2.55 (s, 3H),
2.53 (d, J = 7.4 Hz, 2H), 2.14 (p, J = 6.6 Hz, 2H).
1
the preparation of compound 25, yield: 88%, mp 119−121 °C. H
NMR (400 MHz, CDCl3) δ 8.07 (d, J = 7.8 Hz, 2H), 7.94 (d, J = 8.3
Hz, 2H), 7.50 (d, J = 7.9 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H), 5.19 (s,
2H), 3.93 (s, 3H), 2.56 (s, 3H).
Methyl (E)-3-(4-((4-Acetylphenoxy)methyl)phenyl)acrylate (31).
The title compound was synthesized with 4′-hydroxyacetophenone
and methyl (E)-3-(4-(bromomethyl)phenyl)acrylate in a manner
similar to that described for the preparation of compound 25, yield:
1
84%, mp 150−151 °C. H NMR (400 MHz, CDCl3) δ 7.94 (d, J =
8.3 Hz, 2H), 7.68 (t, J = 15.4 Hz, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.45
(d, J = 7.9 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 6.45 (d, J = 16.0 Hz,
1H), 5.15 (s, 2H), 3.81 (s, 3H), 2.55 (s, 3H).
Methyl 4-(4-(2-Bromoacetyl)phenoxy)butanoate (32). The title
compound was synthesized with intermediate 25 and copper(II)
bromide in a manner similar to that described for the preparation of
compound 10, yield: 70%, mp 90−92 °C. 1H NMR (400 MHz,
CDCl3) δ 7.96 (d, J = 7.9 Hz, 2H), 6.94 (d, J = 8.2 Hz, 2H), 4.40 (s,
2H), 4.10 (dd, J = 12.4, 6.3 Hz, 2H), 3.70 (s, 3H), 2.54 (t, J = 7.1 Hz,
2H), 2.22−2.09 (m, 2H).
Methyl 5-(4-(2-Bromoacetyl)phenoxy)pentanoate (33). The title
compound was synthesized with intermediate 26 and copper(II)
bromide in a manner similar to that described for the preparation of
compound 10, yield: 83%, mp 61−62 °C. 1H NMR (400 MHz,
CDCl3) δ 7.95 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 4.40 (s,
2H), 4.05 (d, J = 5.4 Hz, 2H), 3.68 (s, 3H), 2.41 (t, J = 6.3 Hz, 2H),
1.92−1.77 (m, 4H).
Methyl 6-(4-(2-Bromoacetyl)phenoxy)hexanoate (34). The title
compound was synthesized with intermediate 27 and copper(II)
bromide in a manner similar to that described for the preparation of
compound 10, yield: 65%, mp 61−63 °C. 1H NMR (400 MHz,
CDCl3) δ 7.96 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 8.3 Hz, 2H), 4.40 (s,
2H), 4.04 (t, J = 6.1 Hz, 2H), 3.68 (s, 3H), 2.36 (t, J = 7.4 Hz, 2H),
1.90−1.79 (m, 2H), 1.78−1.67 (m, 2H), 1.57−1.47 (m, 2H).
Methyl 7-(4-(2-Bromoacetyl)phenoxy)heptanoate (35). The title
compound was synthesized with intermediate 28 and copper(II)
bromide in a manner similar to that described for the preparation of
compound 10, yield: 57%, mp 60−61 °C. 1H NMR (400 MHz,
CDCl3) δ 7.96 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 8.3 Hz, 2H), 4.40 (s,
2H), 4.08−4.00 (m, 2H), 3.67 (s, 3H), 2.33 (t, J = 7.3 Hz, 2H),
1.89−1.77 (m, 2H), 1.68 (dd, J = 14.9, 7.4 Hz, 2H), 1.50 (dt, J = 14.2,
6.9 Hz, 2H), 1.40 (dt, J = 14.6, 7.4 Hz, 2H).
Methyl 8-(4-(2-Bromoacetyl)phenoxy)octanoate (36). The title
compound was synthesized with intermediate 29 and copper(II)
bromide in a manner similar to that described for the preparation of
compound 10, yield: 53%, mp 62−64 °C. 1H NMR (400 MHz,
CDCl3) δ 7.96 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 4.40 (s,
2H), 4.03 (t, J = 6.3 Hz, 2H), 3.67 (s, 3H), 2.32 (t, J = 7.4 Hz, 2H),
Methyl 5-(4-Acetylphenoxy)pentanoate (26). The title compound
was synthesized with 4′-hydroxyacetophenone and methyl 5-
bromopentanoate in a manner similar to that described for the
1
preparation of compound 25, yield: 90%, mp 56−57 °C. H NMR
(400 MHz, CDCl3) δ 7.92 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz,
2H), 4.03 (d, J = 5.2 Hz, 2H), 3.68 (s, 3H), 2.55 (s, 3H), 2.41 (t, J =
6.2 Hz, 2H), 1.92−1.76 (m, 4H).
Methyl 6-(4-Acetylphenoxy)hexanoate (27). The title compound
was synthesized with 4′-hydroxyacetophenone and methyl 6-
bromohexanoate in a manner similar to that described for the
1
preparation of compound 25, yield: 94%, mp 46−48 °C. H NMR
M
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1.89−1.75 (m, 2H), 1.64 (dt, J = 14.5, 7.4 Hz, 2H), 1.55−1.44 (m,
2H), 1.44−1.31 (m, 4H).
7.63−7.49 (m, 3H), 7.37 (d, J = 3.7 Hz, 1H), 7.00 (d, J = 8.0 Hz,
2H), 4.01 (t, J = 6.2 Hz, 2H), 3.58 (s, 3H), 2.30 (t, J = 7.6 Hz, 2H),
1.72 (dd, J = 13.4, 6.6 Hz, 2H), 1.60−1.50 (m, 2H), 1.42 (d, J = 6.7
Hz, 2H), 1.32 (m, 4H).
Methyl 4-((4-(2-Bromoacetyl)phenoxy)methyl)benzoate (37).
The title compound was synthesized with intermediate 30 and
copper(II) bromide in a manner similar to that described for the
preparation of compound 10, yield: 84%, mp 121−123 °C. 1H NMR
(400 MHz, CDCl3) δ 8.07 (d, J = 7.7 Hz, 2H), 7.98 (d, J = 8.2 Hz,
2H), 7.50 (d, J = 7.9 Hz, 2H), 7.03 (d, J = 8.2 Hz, 2H), 5.21 (s, 2H),
4.39 (s, 2H), 3.93 (s, 3H).
Methyl (Z)-4-((4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)methyl)benzoate (44). The title compound was synthesized
from intermediate 37, ethyl (Z)-3-oxo-3-phenyl-2-(2-(thiazol-2-yl)-
hydrazono)propanoate, and thiosemicarbazide in a manner similar to
that described for the preparation of compound 15, yield: 61%, mp
215−216 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 6.9 Hz,
2H), 8.00 (d, J = 7.5 Hz, 2H), 7.94 (d, J = 8.1 Hz, 2H), 7.72 (d, J =
3.7 Hz, 1H), 7.69 (s, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.60−7.50 (m,
3H), 7.37 (d, J = 3.7 Hz, 1H), 7.12 (d, J = 8.2 Hz, 2H), 5.27 (s, 2H),
3.86 (s, 3H).
Methyl (E)-3-(4-((4-(2-Bromoacetyl)phenoxy)methyl)phenyl)-
acrylate (38). The title compound was synthesized with intermediate
31 and copper(II) bromide in a manner similar to that described for
1
the preparation of compound 10, yield: 75%, mp 140−141 °C. H
NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 16.0
Hz, 1H), 7.56 (d, J = 7.8 Hz, 2H), 7.45 (d, J = 7.8 Hz, 2H), 7.03 (d, J
= 8.4 Hz, 2H), 6.46 (d, J = 16.0 Hz, 1H), 5.16 (s, 2H), 4.39 (s, 2H),
3.81 (s, 3H).
Methyl (E)-3-(4-((4-(2-((Z)-5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)methyl)phenyl)acrylate (45). The title compound was
synthesized from intermediate 38, ethyl (Z)-3-oxo-3-phenyl-2-(2-
(thiazol-2-yl)hydrazono)propanoate, and thiosemicarbazide in a
manner similar to that described for the preparation of compound
Methyl (Z)-4-(4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)butanoate (39). The title compound was synthesized from
intermediate 32, ethyl (Z)-3-oxo-3-phenyl-2-(2-(thiazol-2-yl)-
hydrazono)propanoate, and thiosemicarbazide in a manner similar
to that described for the preparation of compound 15, yield: 52%, mp
196−198 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 7.0 Hz,
2H), 7.91 (d, J = 8.0 Hz, 2H), 7.72 (d, J = 3.7 Hz, 1H), 7.66 (s, 1H),
7.61−7.51 (m, 3H), 7.36 (d, J = 3.7 Hz, 1H), 7.01 (d, J = 8.1 Hz,
2H), 4.04 (t, J = 6.1 Hz, 2H), 3.62 (s, 3H), 2.50−2.46 (m, 2H),
2.10−1.94 (m, 2H).
Methyl (Z)-5-(4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)pentanoate (40). The title compound was synthesized from
intermediate 33, ethyl (Z)-3-oxo-3-phenyl-2-(2-(thiazol-2-yl)-
hydrazono)propanoate, and thiosemicarbazide in a manner similar
to that described for the preparation of compound 15, yield: 54%, mp
173−174 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 6.9 Hz,
2H), 7.91 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 3.8 Hz, 1H), 7.66 (s, 1H),
7.63−7.48 (m, 3H), 7.37 (d, J = 3.8 Hz, 1H), 7.01 (d, J = 8.1 Hz,
2H), 4.13−3.94 (m, 2H), 3.60 (s, 3H), 2.40 (t, J = 6.8 Hz, 2H), 1.73
(dd, J = 12.3, 6.5 Hz, 4H).
Methyl (Z)-6-(4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)hexanoate (41). The title compound was synthesized from
intermediate 34, ethyl (Z)-3-oxo-3-phenyl-2-(2-(thiazol-2-yl)-
hydrazono)propanoate, and thiosemicarbazide in a manner similar
to that described for the preparation of compound 15, yield: 71%, mp
198−200 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 6.9 Hz,
2H), 7.91 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 3.7 Hz, 1H), 7.66 (s, 1H),
7.63−7.50 (m, 3H), 7.36 (d, J = 3.7 Hz, 1H), 7.00 (d, J = 8.1 Hz,
2H), 4.00 (t, J = 6.2 Hz, 2H), 3.59 (s, 3H), 2.34 (t, J = 7.3 Hz, 2H),
1.73 (dd, J = 13.7, 6.6 Hz, 2H), 1.61 (dt, J = 14.7, 7.2 Hz, 2H), 1.54−
1.38 (m, 2H).
Methyl (Z)-7-(4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)heptanoate (42). The title compound was synthesized from
intermediate 35, ethyl (Z)-3-oxo-3-phenyl-2-(2-(thiazol-2-yl)-
hydrazono)propanoate, and thiosemicarbazide in a manner similar
to that described for the preparation of compound 15, yield: 68%, mp
146−147 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 7.0 Hz,
2H), 7.90 (d, J = 8.0 Hz, 2H), 7.72 (d, J = 3.7 Hz, 1H), 7.66 (s, 1H),
7.54 (t, J = 9.9 Hz, 3H), 7.36 (d, J = 3.8 Hz, 1H), 7.00 (d, J = 8.2 Hz,
2H), 4.00 (t, J = 6.2 Hz, 2H), 3.59 (s, 3H), 2.32 (t, J = 7.3 Hz, 2H),
1.72 (dd, J = 13.6, 6.7 Hz, 2H), 1.63−1.51 (m, 2H), 1.48−1.39 (m,
2H), 1.39−1.30 (m, 2H).
1
15, yield: 78%, mp 222−223 °C. H NMR (400 MHz, DMSO-d6) δ
8.16 (d, J = 6.8 Hz, 2H), 7.93 (d, J = 8.2 Hz, 2H), 7.76 (d, J = 7.8 Hz,
2H), 7.72 (d, J = 4.0 Hz, 1H), 7.71−7.64 (m, 2H), 7.53 (m, 5H), 7.37
(d, J = 3.7 Hz, 1H), 7.11 (d, J = 8.3 Hz, 2H), 6.66 (d, J = 16.0 Hz,
1H), 5.21 (s, 2H), 3.73 (s, 3H).
(Z)-N-Hydroxy-4-(4-(2-(5-oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)butanamide (46). The title compound was synthesized
with intermediate 39 in a manner similar to that described for the
preparation of compound 20, yield: 67%, mp 242−244 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.72 (s, 1H), 8.21 (d, J = 6.5
Hz, 2H), 7.91 (d, J = 8.1 Hz, 2H), 7.64 (s, 1H), 7.57−7.38 (m, 4H),
7.25 (d, J = 2.7 Hz, 1H), 7.00 (d, J = 8.2 Hz, 2H), 4.01 (t, J = 6.1 Hz,
2H), 2.16 (t, J = 7.2 Hz, 2H), 2.02−1.90 (m, 2H). 13C NMR (101
MHz, DMSO) δ 182.87, 169.13, 158.71, 155.61, 153.40, 149.95,
149.71, 141.20, 133.19, 129.11, 128.85, 128.58, 127.88, 127.70,
123.08, 115.82, 115.00, 106.49, 67.32, 29.24, 25.34. HRMS (AP-ESI)
m/z, calcd for C25H21N7O4S2, ([M + H]+): 548.1169, found:
548.1170. HPLC tR = 13.493 min (99.75% purity).
(Z)-N-Hydroxy-5-(4-(2-(5-oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)pentanamide (47). The title compound was synthesized
with intermediate 40 in a manner similar to that described for the
preparation of compound 20, yield: 83%, mp 153−155 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.69 (s, 1H), 8.19 (d, J = 6.6
Hz, 2H), 7.91 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 3.2 Hz, 1H), 7.57 (s,
1H), 7.49 (dt, J = 21.4, 7.3 Hz, 3H), 7.29 (d, J = 3.2 Hz, 1H), 7.01 (d,
J = 8.3 Hz, 2H), 4.01 (d, J = 5.8 Hz, 2H), 2.04 (t, J = 6.6 Hz, 2H),
1.80−1.61 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 181.73,
169.44, 158.86, 155.43, 153.42, 149.97, 149.74, 138.27, 132.38,
129.60, 128.75, 128.51, 127.72, 127.61, 125.59, 115.48, 115.00,
106.82, 67.56, 32.40, 28.68, 22.28. HRMS (AP-ESI) m/z, calcd for
C26H23N7O4S2, ([M + H]+): 562.1326, found: 562.1320. HPLC tR =
6.208 min (96.77% purity).
(Z)-N-Hydroxy-6-(4-(2-(5-oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)hexanamide (48). The title compound was synthesized
with intermediate 41 in a manner similar to that described for the
preparation of compound 20, yield: 55%, mp 132−134 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.35 (s, 1H), 8.16 (d, J = 6.9 Hz, 1H), 7.91
(d, J = 8.2 Hz, 1H), 7.72 (d, J = 3.5 Hz, 1H), 7.66 (s, 1H), 7.60−7.49
(m, 1H), 7.36 (d, J = 3.7 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 4.00 (t, J
= 6.0 Hz, 1H), 1.99 (t, J = 7.1 Hz, 1H), 1.78−1.69 (m, 1H), 1.56 (dd,
J = 14.4, 7.1 Hz, 1H), 1.48−1.37 (m, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 179.00, 172.51, 169.52, 159.01, 154.98, 153.43, 150.29,
149.32, 132.75, 130.92, 130.42, 130.31, 128.99, 128.44, 127.75,
127.29, 114.99, 114.52, 107.31, 67.86, 32.72, 28.94, 25.67, 21.53.
HRMS (AP-ESI) m/z, calcd for C27H25N7O4S2, ([M + H]+):
576.1482, found: 576.1481. HPLC tR = 6.721 min (99.98% purity).
Methyl (Z)-8-(4-(2-(5-Oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)octanoate (43). The title compound was synthesized from
intermediate 36, ethyl (Z)-3-oxo-3-phenyl-2-(2-(thiazol-2-yl)-
hydrazono)propanoate, and thiosemicarbazide in a manner similar
to that described for the preparation of compound 15, yield: 76%, mp
166−167 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 6.8 Hz,
2H), 7.90 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 3.4 Hz, 1H), 7.66 (s, 1H),
N
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(Z)-N-Hydroxy-7-(4-(2-(5-oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)heptanamide (49). The title compound was synthesized
with intermediate 42 in a manner similar to that described for the
preparation of compound 20, yield: 64%, mp 127−128 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.16 (d, J = 6.9 Hz, 2H), 7.91
(d, J = 8.0 Hz, 2H), 7.72 (d, J = 3.5 Hz, 1H), 7.65 (s, 1H), 7.61−7.49
(m, 3H), 7.36 (d, J = 3.5 Hz, 1H), 7.01 (d, J = 8.1 Hz, 2H), 4.01 (t, J
= 6.2 Hz, 2H), 1.96 (t, J = 7.2 Hz, 2H), 1.78−1.68 (m, 2H), 1.52 (dd,
J = 14.6, 7.2 Hz, 2H), 1.47−1.39 (m, 2H), 1.36−1.28 (m, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 179.45, 169.57, 159.03, 155.01,
153.40, 150.28, 149.37, 132.71, 131.00, 130.40, 130.21, 129.00,
128.46, 127.76, 127.30, 115.02, 114.53, 107.30, 67.91, 32.69, 29.09,
28.84, 25.75, 25.56. HRMS (AP-ESI) m/z, calcd for C28H27N7O4S2,
([M + H]+): 590.1639, found: 590.1628. HPLC tR = 12.577 min
(97.32% purity).
(Z)-N-Hydroxy-8-(4-(2-(5-oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)octanamide (50). The title compound was synthesized with
intermediate 43 in a manner similar to that described for the
preparation of compound 20, yield: 60%, mp 144−145 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.32 (s, 1H), 8.16 (d, J = 7.1 Hz, 2H), 7.91
(d, J = 8.2 Hz, 2H), 7.72 (d, J = 3.6 Hz, 1H), 7.65 (s, 1H), 7.61−7.49
(m, 3H), 7.36 (d, J = 3.7 Hz, 1H), 7.01 (d, J = 8.1 Hz, 2H), 4.01 (t, J
= 6.2 Hz, 2H), 1.95 (t, J = 7.2 Hz, 2H), 1.80−1.67 (m, 2H), 1.57−
1.47 (m, 2H), 1.45−1.38 (m, 2H), 1.37−1.21 (m, 4H). 13C NMR
(101 MHz, DMSO-d6) δ 179.08, 169.62, 159.02, 154.98, 153.42,
150.29, 149.32, 132.76, 130.94, 130.40, 130.27, 128.98, 128.44,
127.75, 127.26, 114.98, 114.53, 107.28, 67.93, 32.73, 29.16, 29.01,
29.00, 25.92, 25.56. HRMS (AP-ESI) m/z, calcd for C29H29N7O4S2,
([M + H]+): 604.1795, found: 604.1787. HPLC tR = 5.994 min
(99.01% purity).
of final products; flow cytometry analysis in HeLa cells
and HeLa cells Bax−/−; cellular transfections; docking
results of compound 23 with Bax; and molecular
dynamics simulation of BTSA1 with Bax (PDF)
Molecular formula strings (CSV)
Docking results of BTSA1 with Bax (PDB)
Docking results of compound 23 with Bax (PDB)
AUTHOR INFORMATION
Corresponding Author
■
Hao Fang − Department of Medicinal Chemistry, Key
Laboratory of Chemical Biology (Ministry of Education), School
of Pharmaceutical Sciences, Cheeloo College of Medicine,
Shandong University, Jinan, Shandong 250012, P. R. China;
orcid.org/0000-0002-2879-5146; Phone: 86-531-
88382019; Email: haofangcn@ sdu.edu.cn
Authors
Tao Liang − Department of Medicinal Chemistry, Key
Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Cheeloo College of Medicine,
Shandong University, Jinan, Shandong 250012, P. R. China
Yi Zhou − Department of Medicinal Chemistry, Key Laboratory
of Chemical Biology (Ministry of Education), School of
Pharmaceutical Sciences, Cheeloo College of Medicine,
Shandong University, Jinan, Shandong 250012, P. R. China;
orcid.org/0000-0002-1282-2461
Reham M. Elhassan − Department of Medicinal Chemistry, Key
Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Cheeloo College of Medicine,
Shandong University, Jinan, Shandong 250012, P. R. China
Xuben Hou − Department of Medicinal Chemistry, Key
Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Cheeloo College of Medicine,
Shandong University, Jinan, Shandong 250012, P. R. China;
orcid.org/0000-0002-8346-9001
(Z)-N-Hydroxy-4-((4-(2-(5-oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)methyl)benzamide (51). The title compound was synthe-
sized with intermediate 44 in a manner similar to that described for
1
the preparation of compound 20, yield: 60%, mp 216−217 °C. H
NMR (400 MHz, DMSO-d6) δ 11.22 (s, 1H), 9.03 (s, 1H), 8.18 (d, J
= 7.0 Hz, 2H), 7.93 (d, J = 8.1 Hz, 2H), 7.78 (d, J = 7.6 Hz, 2H), 7.69
(d, J = 3.3 Hz, 1H), 7.62 (s, 1H), 7.59−7.46 (m, 5H), 7.31 (d, J = 3.2
Hz, 1H), 7.11 (d, J = 8.1 Hz, 2H), 5.22 (s, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 180.80, 172.51, 164.43, 158.43, 155.27, 153.40, 149.94,
140.67, 135.92, 132.73, 131.87, 129.99, 129.96, 129.90, 128.83,
128.51, 127.93, 127.77, 127.52, 115.44, 115.00, 107.22, 69.18. HRMS
(AP-ESI) m/z, calcd for C29H21N7O4S2, ([M + H]+): 596.1169,
found: 596.1156. HPLC tR = 15.510 min (99.59% purity).
Xinying Yang − Department of Medicinal Chemistry, Key
Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Cheeloo College of Medicine,
Shandong University, Jinan, Shandong 250012, P. R. China
(E)-N-Hydroxy-3-(4-((4-(2-((Z)-5-oxo-3-phenyl-4-(2-(thiazol-2-yl)-
hydrazineylidene)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4-yl)-
phenoxy)methyl)phenyl)acrylamide (52). The title compound was
synthesized with intermediate 45 in a manner similar to that described
for the preparation of compound 20, yield: 70%, mp 186−188 °C. 1H
NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 9.13 (s, 1H), 8.16 (d, J
= 6.9 Hz, 2H), 7.93 (d, J = 8.2 Hz, 2H), 7.71 (d, J = 3.6 Hz, 1H), 7.66
(s, 1H), 7.53 (ddd, J = 31.3, 21.8, 11.8 Hz, 8H), 7.35 (d, J = 3.6 Hz,
1H), 7.11 (d, J = 8.2 Hz, 2H), 6.48 (d, J = 15.8 Hz, 1H), 5.19 (s, 2H).
13C NMR (101 MHz, DMSO-d6) δ 179.19, 163.15, 158.56, 155.07,
153.46, 150.17, 149.38, 138.83, 138.42, 134.86, 133.34, 131.07,
130.32, 129.79, 128.95, 128.64, 128.46, 128.07, 127.79, 127.75,
119.66, 115.42, 114.63, 107.48, 69.35. HRMS (AP-ESI) m/z, calcd
for C31H23N7O4S2, ([M + H]+): 622.1326, found: 622.1320. HPLC tR
= 5.343 min (97.91% purity).
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01454
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors genuinely thank the Advanced Medical Research
Institute, Shandong University, for their help in the experi-
ments. They thank Renxiao Wang from Shanghai institute of
Organic Chemistry, Chinese Academy of Sciences, for his
donation of E. coli produced recombinant N-terminal His-
tagged human Bcl-2, Mcl-1, and Bcl-XL proteins. This work
was supported by the National Nature Science Foundation of
China (21672127 and 81874288), the Major Project of
Science and Technology of Shandong Province
(2017CXGC1401), the Shandong Provincial Natural Science
Foundation (ZR2019LZL004), the Fundamental Research
Funds of Shandong University (2019GN045), and the Joint
Research Funds for Shandong University and Karolinska
Institute (SDU-KI-2019-06).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01454.
Production of recombinant Bax proteins; synthesis of
stapled peptides; 1H NMR, 13C NMR, and HRMS
spectra of all target compounds; HPLC chromatogram
O
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ABBREVIATIONS
■
HDAC, histone deacetylase; MOM, mitochondrial outer
membrane; MOMP, mitochondrial outer membrane perme-
abilization; BAM7, Bax activator; BTSA1, Bax activator; AML,
acute myeloid leukemia; SAHA, Vorinostat, HDAC inhibitor;
CTCL, cutaneous T-cell lymphoma; PTCL, peripheral T-cell
lymphoma; MM, multiple myeloma; CI, combination index;
AIDS, acquired immunodeficiency syndrome; DDI, drug−drug
interaction; PK, pharmacokinetics; PD, pharmacodynamic;
ZBG, zinc binding group; SAR, structure−activity relationship;
THF, tetrahydrofuran; HATU, 1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-
fluorophosphate; DIPEA, N,N-diisopropylethylamine; DMF,
N,N-dimethylformamide; FPA, fluorescence polarization assay;
FITC-BIM, fluorescein isothiocyanate-labeled stapled peptide
of the BIM BH3 helix; IC50, half-maximal inhibitory
concentration; TUNEL, terminal deoxynucleotidyl transferase
(TdT) dUTP nick-end labeling; PI, propidium iodide; TMRE,
tetramethylrhodamine ethyl ester; SEC, size exclusion
chromatography; Ac-BIM, acetylated stapled peptide of the
BIM BH3 helix; His-Bax, histidine-labeled Bax protein; EC50,
concentration for 50% of maximal effect; 5-FAM Bid-BH3, 5-
carboxyfluorescein-labeled Bid-BH3 helix; His-Bcl-2, histidine-
labeled Bcl-2 protein; His-Bcl-XL, histidine-labeled Bcl-XL
protein; ABT-199, Bcl-2 inhibitor; AMC, 7-amino-4-methyl-
coumarin; TSA, trichostatin A, HDAC inhibitor; DMSO,
dimethyl sulfoxide; PMSF, phenylmethanesulfonyl fluoride,
protease inhibitors; SDS, sodium dodecylsulfate; PAGE,
polyacrylamide gel electrophoresis; PVDF, poly(vinylidene
fluoride); GAPDH, glyceraldehyde-3-phosphate dehydrogen-
ase; FBS, fetal bovine serum; MTT, 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; HL-7702,
human normal liver cells; PBS, phosphate-buffered saline;
DTT, dithiothreitol; DEVE-AFC, caspase-3 substrates; TLC,
thin-layer chromatography
(16) Gavathiotis, E.; Reyna, D. E.; Davis, M. L.; Bird, G. H.;
Walensky, L. D. BH3-triggered structural reorganization drives the
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