Synthesis of 4-(2-phenylhydrazono)-1-(4-phenylthiazol-2-yl)-1H-pyrazol- 5(4H)-one compounds and characterization of their affinities to anti-apoptotic Bcl-2 family proteins

Article abstract of DOI:10.1002/cjoc.201300426

Organic compounds containing the thiazol-2-yl-1H-pyrazol-5(4H)-one moiety are known to be associated with versatile pharmacological applications. In this study, we describe the methods for preparing 4-(2-phenylhydrazono)-1-(4- phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one compounds. A set of 26 compounds were synthesized with overall yields ranging between 37% -92%. They were tested in a fluorescence polarization-based binding assay against three anti-apoptotic Bcl-2 family proteins, including Bcl-x_L, Bcl-2, and Mcl-1. Our results indicate that this class of compounds are not effective inhibitors of these anti-apoptotic Bcl-2 family proteins. Their apoptosis-inducing effects are possibly due to BAX activation as suggested by Gavathiotis et al. in their recent study. However, other possibilities should not be ignored. In addition, a crystal structure obtained by us reveals that the exocyclic double bond in the molecular structure of this class of compounds is in the (Z)-configuration This class of compounds are not effective binders of anti-apoptotic Bcl-2 family proteins. Their apoptosis-inducing effects are possibly due to BAX activation rather than Bcl-2 inhibition as suggested by a recent study. Copyright

Full text of DOI:10.1002/cjoc.201300426

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DOI: 10.1002/cjoc.201300426
Synthesis of 4-(2-Phenylhydrazono)-1-(4-phenylthiazol-2-yl)-
1H-pyrazol-5(4H)-one Compounds and Characterization of
Their Affinities to Anti-apoptotic Bcl-2 Family Proteins
Shicheng Shi, Li Han, Mi Zhou, Yangfeng Li, Zhen Liu, Biao Yu, and Renxiao Wang*
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 200032, China
Organic compounds containing the thiazol-2-yl-1H-pyrazol-5(4H)-one moiety are known to be associated with
versatile pharmacological applications. In this study, we describe the methods for preparing 4-(2-phenylhydrazono)-
1-(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one compounds. A set of 26 compounds were synthesized with overall
yields ranging between 37%－92%. They were tested in a fluorescence polarization-based binding assay against
three anti-apoptotic Bcl-2 family proteins, including Bcl-xL, Bcl-2, and Mcl-1. Our results indicate that this class of
compounds are not effective inhibitors of these anti-apoptotic Bcl-2 family proteins. Their apoptosis-inducing ef-
fects are possibly due to BAX activation as suggested by Gavathiotis et al. in their recent study. However, other
possibilities should not be ignored. In addition, a crystal structure obtained by us reveals that the exocyclic double
bond in the molecular structure of this class of compounds is in the (Z)-configuration.
Keywords apoptosis inducer, BAX activation, Bcl-2 inhibition, protein-protein interaction, small-molecule in-
hibitor
tions.[1-6] In our previous studies, we synthesized this
class of compounds and screened them using MTT as-
says on several tumor cell lines, including A549, HeLa
and MDA-MB-231 cells. Indeed, some of them exhib-
ited obvious cytotoxicity (CG50＝1－20 μmol•L−1) on
Introduction
Organic compounds containing the thiazol-2-yl-1H-
pyrazol-5(4H)-one moiety (Figure 1), obtained either
from natural resources or chemical synthesis, are known
to be associated with versatile pharmacological applica-
OEt
SO3H
SO2NH2
HN
N
O
HN
N
O
HN
N
O
S
S
S
N
N
N
N
N
N
N
N
N
(c)
(b)
(a)
EtO
R1
HN
N
O
HN
N
O
S
S
N
N
N
N
N
N
R2
(e)
(d) BAM7
Figure 1 Some known compounds containing the thiazol-2-yl-1H-pyrazol-5(4H)-one moiety, which were reported as (a) causing revert
cholesterol accumulation in Niemann-Pick C cells,[4] (b) treating neurological and neurodegenerative diseases,[5] (c) shp-2 inhibitor,[6] (d)
apoptosis inducer by BAX activation[8] and (e) apoptosis inducer described in our previous study.[7]
*
E-mail: wangrx@sioc.ac.cn; Tel.: 0086-021-54925128
Received May 28, 2013; accepted June 24, 2013; published online XXXX, 2013.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201300426 or from the author.
Chin. J. Chem. 2013, XX, 1—6
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Shi et al.
COMMUNICATION
these tumor cells, and our preliminary studies indicated
that this class of compounds induced apoptosis in tumor
cell lines.[7] However, the exact molecular mechanism
of the apoptosis-inducing effects of this class of com-
pounds remains unknown.
fication, however, was not systematically performed by
Gavathiotis et al. in their study.
In this study, we describe a set of efficient synthetic
methods for preparing this class of compounds. All of
the 26 obtained compounds, including BAM7, were
tested in a fluorescence polarization-based in vitro
binding assay to measure their binding affinities to three
anti-apoptotic Bcl-2 family proteins, i.e. Bcl-xL, Bcl-2,
and Mcl-1. Our results indicate that this class of com-
pounds do not bind effectively with any of these three
Bcl-2 family proteins. Our study thus provides an addi-
tional support to Gavathiotis’ conclusion that this class
of compounds induce apoptosis through BAX activation
rather than Bcl-2 inhibition.
Recently, Gavathiotis et al.[8] reported an interesting
study regarding the regulation of Bcl-2 family proteins
with small-molecule compounds. Bcl-2 family proteins
are key regulators of the apoptotic pathway. Anti-
apoptotic members, such as Bcl-2, Bcl-xL, and Mcl-1,
sequester the Bcl-2 homology 3 (BH3) death domains of
pro-apoptotic members, such as BAX, to maintain cell
survival.[9-11] The BH3-binding groove on anti-apoptotic
Bcl-2 family proteins has been considered as target for
developing small-molecule apoptosis inducers as poten-
tial anti-cancer therapies.[12] A number of inhibitors of
Bcl-2 family proteins have been successfully developed
during the past decade or so.[13-30] But Gavathiotis et al.
attempted to find compounds that can mediate direct
BAX activation to induce apoptosis. A total of 750000
molecules were examined by means of computational
screening. Finally, 100 of them were selected and tested.
Based on the results of NMR and biochemical analyses,
they demonstrated that one compound engaged the
BAX trigger site and promoted the functional oligo-
merization of BAX. The molecule was observed to in-
duce cell death in a BAX-dependent manner.
Experimental
Organic synthesis
The target compounds 3a－3t and 3u－3z were
prepared with the synthetic methods illustrated in
Schemes 1 and 2, respectively. In brief, target com-
pounds were synthesized by three steps: Firstly, substi-
tuted ethyl acetoacetate underwent coupling reaction
with aromatic diazonium salts in aqueous solution to
yield 1. Then, 1 was cyclized with thiosemicarbazide
under reflux in HOAc to give 2. Finally, heated in di-
oxane, 2 and phenacyl bromide underwent the Hantzsch
thiazole reaction to yield compounds 3a－3t (Scheme
1).[31,32] When R1 was CH2OH, hydroxylmethyl group
would esterify with HOAc under the reaction condition.
Therefore, the synthetic route of compounds 3u－3z
was altered as: Ethyl acetoacetate was treated with thio-
semicarbazide in 5% HCl solution, and was then heated
with NaOAc in EtOH to obtain 5, which existed in its
enamine form. Then, 5 was coupled with diazonium
salts to produce 6. The final step was also the cycliza-
tion reaction with phenacyl bromide to obtain the target
compounds 3u－3z (Scheme 2).[33]
Gavathiotis’ study drew our attention since the most
active compound described by them, i.e. BAM7 in Fig-
ure 1, belongs to the same class of thiazol-2-yl-1H-
pyrazol-5(4H)-one compounds tested in our previous
studies. Their results provide hints to understanding the
molecular mechanism of this class of compounds. Since
both activation of BAX and inhibition of anti-apoptotic
Bcl-2 family proteins will induce apoptosis through the
mitochondrial pathway,[9-11] it is also necessary to verify
if this class of compounds actually interact with
anti-apoptotic Bcl-2 family proteins. This type of veri-
Scheme 1 General synthetic methods for obtaining hydrazono-thiazolyl-pyrazolone compounds
R3
O
OEt
S
NH2
R1
R1
O
S
H
O
N
O
c
HN
a, b
H2N
HN
d
N
N
N
N
N
N
R1
N
R2
N
R1
R2
2a － 2h
R2
1a － 1w
3a － 3t
Reagents and conditions: (a) NaNO2, HCl, 0－5 ℃; (b) substituted ethyl acetoacetate, NaOAc, EtOH, r.t., 1 h; (c) thiosemicarbazide, HOAc,
reflux, 4 h; (d) α-bromoacetophenone, dioxane, reflux, 0.5 h.
Scheme 2 Revised synthetic methods for obtaining hydrazono-thiazolyl-pyrazolone derivatives 6a－6e
R1
S
S
O
O
R3
S
O
O
O
HN
d
a, b
c
H2N
HN
H2N
N
N
N
N
R1
OEt
N
N
N
N
HN
5
6a － 6e
3u － 3z
Reagents and conditions: (a) thiosemicarbazide, 5% HCl, H2O, r.t., 1 h; (b) EtOAc, EtOH, reflux, 4 h; (c) substituted aniline, NaNO2, HCl,
0－5 ℃; (d) α-bromoacetophenone, dioxane, reflux, 0.5 h.
2
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Synthesis of 4-(2-Phenylhydrazono)-1-(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one Compounds
The synthetic methods for preparing the necessary
starting material or intermediates as well as spectral data
of compounds 3a－3z are given in the Supporting In-
formation.
In this study, each compound was tested against all
three target proteins (Bcl-xL, Bcl-2 and Mcl-1) at three
different concentrations, i.e. 1, 10 and 50 μmol•L−1. At
each concentration, the average FP value of three paral-
lel measurements was used to compute inhibition ratio
using the following equation.
Crystal structure determination
Growth of single crystals was attempted for 3y. Sin-
gle crystals in good quality were obtained using the fol-
lowing condition. The compound sample (10 mg) was
dissolved in DCM (0.5 mL) and n-hexane (2.0 mL) in a
5 mL sample bottle. The mixture was then transferred
into a 60 mL reagent bottle containing 20 mL n-hexane.
The solution was allowed to vaporize for several days to
obtain the crystals.
X-ray crystal diffraction data were collected on a
Bruker Apex II CCD diffractometer operating at 50 kV
and 30 mA using Mo Kα radiation (λ＝0.71073 Å) at
293 K. Main parameters of this crystal structure are:
C21H19N5O2S, Mr＝405.48 g/mol, crystal size 0.31 mm
×0.12 mm×0.09 mm. Triclinic, space group P-1, a＝
5.0025(6) Å, b＝12.4830(15) Å, c＝16.0296(19) Å, α＝
75.651(3)°, β＝85.409(3)°, γ＝81.677(3)°, V＝958.5(2)
Å3, Z＝2, Dc＝1.045 g•cm−3, λ＝0.71073 Å, μ(Mo Kα)
＝0.198 mm−1. This crystal structure has been deposited
in the Cambridge Crystal Data Center (access number:
927666).
mP －mP
max
Inhibition＝
test ×100%
mP －mP
max
NC
Here, mPmax refers to the FP signal detected when the
protein was incubated with the Bid-BH3 peptide, mPNC
refers to the FP signal detected from the negative con-
trol, i.e. the Bid-BH3 peptide alone, and mPtest refers to
the FP signal detected when the compound under test
was added at a certain concentration. Because most
compounds tested in this study did not exhibit signifi-
cant binding affinities to three target proteins, no addi-
tional attempt was made to determine accurate IC50
values or inhibition constants for these compounds.
Inhibition rates measured at 50 μmol•L−1 for all
compounds are summarized in Table 1. The full results
can be found in the Supporting Information. Detailed
descriptions of the methods used for expression and
purification of three proteins, the FP-based assay, and
data processing can be found in one of our recent publi-
cations.[26]
In vitro binding assay
Fluorescence polarization (FP)-based assay was em-
ployed in our study to measure the binding affinities of
small-molecule compounds to three human anti-apop-
totic Bcl-2 family proteins, i.e. Bcl-xL, Bcl-2, and Mcl-1.
In this binding assay, a 26-residue peptide derived from
the BH3 domain on the Bid protein with 5-carboxy-
fluorescein (5-FAM) on the N-terminus, i.e. 5-FAM-
QEDIIRNIARHLAQVGDSM-DRSIPPG, was used as
the fluorescence tracer. Our dose-dependent saturation
experiments determined that this Bid-BH3 peptide
bound to Bcl-xL with Kd＝41 nmol•L−1, to Bcl-2 with
Kd＝89 nmol•L−1, and to Mcl-1 with Kd＝29 nmol•L−1.
In each measurement, the Bid-BH3 peptide at a total
concentration of 10 nmol•L−1 was incubated with the
protein first. Total concentration of the protein was set
to five times of the Kd value of the fluorescence tracer,
i.e. 205 nmol•L−1 in the case of Bcl-xL, 445 nmol•L−1 in
the case of Bcl-2, and 145 nmol•L−1 in the case of Mcl-1.
Competitive binding of a given compound was charac-
terized quantitatively by monitoring the changes in FP
signals upon the addition of the compound at a series of
doses. As a validation, ABT-263, a well-known Bcl-2
inhibitor was tested in our binding assay as a positive
control. Our measured inhibition constants (Ki) of
ABT-263 against Bcl-xL and Bcl-2 were lower than 1
nmol•L−1, which was consistent with the data reported
in literature.[13,14] Our measured Ki value of ABT-263
against Mcl-1 was 1.6 μmol•L−1, which was also basi-
cally consistent with the reported data of 0.55
μmol•L−1.[14]
Results and Discussion
In this study, a total of 26 compounds were obtained
(Table 1). These compounds were synthesized by the
methods illustrated in Scheme 1 and Scheme 2. Differ-
ent substituent groups were installed at three sites on the
framework to explore a preliminary structure-activity
relationship of this class of compounds. As for R1, some
small polar groups were considered to improve solubil-
ity since the framework of this class of compounds is
apparently not very soluble. As for R2, three hydropho-
bic groups of different sizes, i.e. methyl, isopropyl, and
phenyl groups were chosen by considering synthetic
feasibility. R3 was basically a substituted phenyl or
naphthyl group, which resembles the chemical struc-
tures of the same class of compounds investigated by
other researchers (Figure 1).
In this study, we obtained the crystal structure of 3y,
i.e. compound BAM7 described in Gavathiotis’ study.[8]
The crystal structure is shown in Figure 2. One can see
that this molecule has a rather flat shape, in which the
four 5-member and 6-member rings are aligned basi-
cally in one plane. The torsion angle between atoms
C(5)-N(4)-N(5)-C(8) is −179.8° indicating that this
“linker” part also locates on the same plane. Since the
other compounds synthesized by us (Table 1) are dif-
ferent from 3y only in terminal substituent groups, it is
reasonable to assume that their three-dimensional struc-
tures are similar to that of 3y.
Chin. J. Chem. 2013, XX, 1—6
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3

Shi et al.
COMMUNICATION
Table 1 Inhibitory activities of compounds 3a－3z on three anti-apoptotic Bcl-2 family proteins
R1
HN
N
O
S
N
N
N
R3
R2
Inhibition rate at 50 μmol•L−1/%
Compd. ID
Symbol
R1
R2
R3
Yield%
Ref.a
Bcl-xL
Bcl-2
14
43
39
14
−6
−6
31
4
Mcl-1
BCL-SSC-01
BCL-SSC-02
BCL-SSC-03
BCL-SSC-04
BCL-SSC-05
BCL-SSC-06
BCL-SSC-07
BCL-SSC-08
BCL-SSC-09
BCL-SSC-10
BCL-SSC-11
BCL-SSC-12
BCL-SSC-13
BCL-SSC-14
BCL-SSC-15
BCL-SSC-16
BCL-SSC-17
BCL-SSC-18
BCL-SSC-19
BCL-SSC-20
BCL-SSC-21
BCL-SSC-22
BCL-SSC-23
BCL-SSC-24
BCL-SSC-25
BCL-SSC-26
3a
3b
3c
3d
3e
3f
3-OCH3
2-CH2OH
3-CH2OH
2-OCH3
Me
Me
Me
i-Pr
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
3-OMe-C6H4
2-Naphthyl
74
92
75
72
83
64
89
70
72
85
55
59
37
68
79
81
64
85
43
41
63
67
58
54
62
56
17
35
30
−19
−3
6
7
23
−12
−32
25
30
39
35
30
30
34
46
47
9
[7]
[7]
3-CO2CH3 i-Pr
3-OCH3
3-NO2
i-Pr
i-Pr
Ph
3g
3h
3i
−2
16
9
3-CO2CH3
2-CO2CH3
2-NO2
Ph
4
3j
Ph
35
9
37
17
41
39
−9
20
14
0
3k
3l
2-OCH3
3-OCH3
3-NO2
Ph
Ph
5
3m
3n
3o
3p
3q
3r
3s
Ph
19
−13
7
2-OCH3
3-CH2OH
2-CO2CH3
3-OCH3
Me
Me
Me
i-Pr
2-Naphthyl
28
43
13
34
19
18
56
53
16
38
38
44
2-Naphthyl
12
−3
7
2-Naphthyl
3-CO2CH3 i-Pr
3-CH2OH
3-CH2OAc Me 3,4-(OMe)2-C6H3
2-Naphthyl
2
Me 3,4-(OMe)2-C6H3
29
−9
23
21
0
9
3t
−9
49
43
25
31
31
34
3u
3v
3w
3x
3y
3z
4-SO2NH2
3-COOH
3-Br
Me
Me
Me
4-OMe-C6H4
4-OMe-C6H4
4-OMe-C6H4
[7]
[7]
3-Br
Me 3,4-(OMe)2-C6H3
25
25
14
2-OEt
Me
Me
Ph
[8]
2-OEt
4-OMe-C6H4
a
A few compounds were reported in our previous study (ref. [7]) or Gavathiotis’ study (ref. [8]). All of the other compounds are new
compounds that have not been reported in literature before.
(E)-3y. Computations were performed using the
A notable feature in the three-dimensional structure
of 3y is the intramolecular hydrogen bonds formed be-
tween N(5)－H…O(1) and N(5)－H…O(2) (Figure 2).
This bifurcate hydrogen bond network further immobi-
lizes the 1H-pyrazol-5(4H)-one ring, the linker part, and
the substituent phenyl ring in one common plane. Due
to the existence of this hydrogen bond, the C(5)＝N(4)
double bond is of the (Z)-configuration. Note that
Gavathiotis et al. derived a binding mode of BAM-7 (3y)
in complex with BAX through molecular modeling, in
which the C(5) ＝N(4) double bond was of the
(E)-configuration.[8]
It is reasonable to expect that the (Z)-configuration
of 3y is energetically favored. In order to prove this, we
employed density functional theory computations to
estimate the energy difference between (Z)-3y and
Figure 2 Crystal structure of 3y (BAM-7). Formula C21H19N5-
O2S, molecular weight 405.4729. Cambridge Crystallographic
Data Center (CDCC) deposition number: 927666.
4
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Synthesis of 4-(2-Phenylhydrazono)-1-(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one Compounds
Gaussian 09 package. Geometry optimization of both
structures was performed at the B3LYP/6-311＋＋
G(d,p) level. Both optimized structures were proved to
be minima on the potential energy surface by vibrational
frequency analysis (Figure 3). Single-point energies
were then computed for each structure in gas phase as
well as in water solvent using a PCM solvation model.
The computed energy difference between (Z)-3y and
(E)-3y is 8.79 kcal/mol in gas phase and 5.03 kcal/mol
in water, respectively. This energy gap is significant
even in water. Accordingly, it is reasonable to expect
that BAM-7 binds to BAX in its low-energy
(Z)-configuration rather than the (E)-configuration. In
other words, binding of BAM-7 to BAX is not likely to
compensate the energy penalty needed for breaking the
intramolecular hydrogen bond in the (Z)-configuration
and converting this molecule into the (E)-configuration.
Thus, we believe that the binding mode of BAM-7 to
BAX shown in Gavathiotis’ study is not correct.
But other possible apoptosis-inducing mechanisms
for this class of compounds should not be completely
ruled out. As implied by their versatile biological effects
reported by different researchers (Figure 1), we specu-
late that this class of compounds actually has multiple
molecular targets in cell. In fact, apoptosis is controlled
by such a complex biological network. Many factors
have contributions to this process. For example, some
Bcl-2 inhibitors were originally identified as mecha-
[15]
nism-based apoptosis inducers, such as HA14-1
and
gossypol[16,17]. But it was revealed later in more sophis-
ticated studies that these compounds did not function
solely as BH3 mimetics since their cytotoxic activity
was not dependent on Bax or Bak.[34] Gavathiotis et al.
demonstrated nicely with Bax and/or Bak knockout
mouse embryo fibroblasts (MEFs) that BAM7 triggers
in vitro BAX oligomerization, BAX-mediated pore
formation and BAX-dependent cell death.[8] In fact, they
also tested 16 other analogs of BAM7 and found that
those analogs did not function in the same manner. Thus,
the role of this class of compounds as apoptosis induc-
ers still needs to be interpreted with extreme care.
Conclusions
In this study, we have synthesized a set of 26 4-(2-
phenylhydrazono)-1-(4-phenylthiazol-2-yl)-1H-pyrazol-
5(4H)-one compounds. The crystal structure of one se-
lected compound clearly reveals that the exocyclic dou-
ble bond on the core moiety is in the (Z)-configuration
due to the formation of intramolecular hydrogen bonds.
All obtained compounds were tested against three
anti-apoptotic Bcl-2 family proteins in binding assay.
Our results indicate that generally speaking, they are not
effective Bcl-2 inhibitors. Thus, their apoptosis-induc-
ing effects are possibly due to BAX activation as sug-
gested by Gavathiotis et al. rather than Bcl-2 inhibition.
However, as implied by the versatile biological effects
of this class of compounds, the molecular mechanism of
their apoptosis-inducing effects is perhaps more sophis-
ticated than solely BAX activation.
Figure 3 Difference in the structures and energies of (Z)-3y
(top) and (E)-3y (bottom). Energies were computed at the
B3LYP/6-311＋＋G(d,p) level.
Acknowledgements
Binding affinities of this class of compounds to three
anti-apoptotic Bcl-2 family proteins measured in our
study are also summarized in Table 1. One can see that
about half of these compounds have weak interactions
with three Bcl-2 proteins. For example, compounds 3u
and 3v exhibited inhibition rates around 50% at the
highest concentration tested in our study (50 μmol•L−1).
Other compounds basically do not interact with three
Bcl-2 proteins. We thus conclude that this class of
compounds, if the molecular framework is not changed
significantly, are not effective inhibitors of anti-apop-
totic Bcl-2 family proteins. Their apoptosis-inducing
effects should be attributed to other molecular mecha-
nisms, possibly through BAX activation as reported by
Gavathiotis et al. in their recent study.[8]
The authors are grateful to the financial supports
from the Chinese National Natural Science Foundation
(Nos. 81172984, 21072213, 21002117, 21102168,
21102165 and 20921091), the Chinese Ministry of Sci-
ence and Technology (863 High-Tech Grant, No.
2012AA020308) and the Chinese Academy of Sciences.
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Shi et al.
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