Characterization of the stereochemical structures of 2H-thiazolo[3,2-a]pyrimidine compounds and their binding affinities for anti-apoptotic Bcl-2 family proteins

Article abstract of DOI:10.1002/cmdc.201300159

In a previous study we reported a class of compounds with a 2H-thiazolo[3,2-a]pyrimidine core structure as general inhibitors of anti-apoptotic Bcl-2 family proteins. However, the absolute stereochemical configuration of one carbon atom on the core struct

Full text of DOI:10.1002/cmdc.201300159

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Ambidexterity! The compounds report-
ed herein were characterized as inhibi-
tors of anti-apoptotic Bcl-2 family pro-
teins. Structures in this compound class
contain a chiral center (C4 atom) on the
pyrimidine ring. Interestingly, our study
revealed that the R and S enantiomers
of this compound class have similar
binding affinities for Bcl-xL, Bcl-2, and
Mcl-1.
Y. Xu, M. Zhou, Y. Li, C. Li, Z. Zhang,
B. Yu,* R. Wang*
&& – &&
Characterization of the Stereochemical
Structures of 2H-Thiazolo[3,2-
a]pyrimidine Compounds and Their
Binding Affinities for Anti-apoptotic
Bcl-2 Family Proteins
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DOI: 10.1002/cmdc.201300159
Characterization of the Stereochemical Structures of 2H-
Thiazolo[3,2-a]pyrimidine Compounds and Their Binding
Affinities for Anti-apoptotic Bcl-2 Family Proteins
Yaochun Xu, Mi Zhou, Yan Li, Chengke Li, Zhengxi Zhang, Biao Yu,* and Renxiao Wang*[a]
In a previous study we reported a class of compounds with
a 2H-thiazolo[3,2-a]pyrimidine core structure as general inhibi-
tors of anti-apoptotic Bcl-2 family proteins. However, the abso-
lute stereochemical configuration of one carbon atom on the
core structure remained unsolved, and its potential impact on
the binding affinities of compounds in this class was unknown.
In this study, we obtained pure R and S enantiomers of four se-
lected compounds by HPLC separation and chiral synthesis.
The absolute configurations of these enantiomers were deter-
mined by comparing their circular dichroism spectra to that of
an appropriate reference compound. In addition, a crystal
structure of one selected compound revealed the exocyclic
double bond in these compounds to be in the Z configuration.
The binding affinities of all four pairs of enantiomers to Bcl-xL,
Bcl-2, and Mcl-1 proteins were measured in a fluorescence-po-
larization-based binding assay, yielding inhibition constants (Ki
values) ranging from 0.24 to 2.20 mm. Interestingly, our results
indicate that most R and S enantiomers exhibit similar binding
affinities for the three tested proteins. A binding mode for this
compound class was derived by molecular docking and molec-
ular dynamics simulations to provide a reasonable interpreta-
tion of this observation.
Introduction
As key regulators of apoptosis (programmed cell death), Bcl-2
family proteins have drawn much attention since the late
1990s.[1–3] Anti-apoptotic members of this family such as Bcl-2,
Bcl-xL, and Mcl-1 bind and sequester pro-apoptotic members
such as Bim, Puma, Noxa, Bad, and Bax, thereby preventing mi-
tochondria-mediated cell death. Various tumor cell types are
found to overexpress at least one of these proteins, which pro-
vides a mechanism for those cells to resist apoptosis induced
by chemotherapy or radiation. Recent studies have revealed
that Bcl-2 family proteins also play important roles in other cel-
lular events such as autophagy[4] and cell proliferation.[5] Thus,
the development of small-molecule inhibitors targeting anti-
apoptotic Bcl-2 family proteins has become a promising ap-
proach in the discovery of new anticancer therapies.[6]
effort toward the discovery of new Bcl-2 inhibitors to eventual-
ly reach the goal of an effective anticancer agent.
In our previous study,[23] we reported a class of compounds
with a 2H-thiazolo[3,2-a]pyrimidine core structure as general
inhibitors of anti-apoptotic Bcl-2 family proteins (Figure 1). The
lead compound was discovered by chance. A series of deriva-
Bcl-2 family proteins execute their biological functions
through protein–protein interactions. This type of target is nor-
mally more difficult to address in drug discovery. Despite the
challenge, a range of small-molecule inhibitors of anti-apoptot-
ic Bcl-2 family proteins have been reported thus far.[7–22] Some
of them, such as ABT-263,[7,8] gossypol,[9,10] and GX15-070,[11]
have already been tested in clinical trials. However, no Bcl-2 in-
hibitor has been approved yet for entry into the market as an
anticancer drug. This situation therefore demands continuous
Figure 1. Structures of the compounds previously reported by us (left)[23]
and Feng et al. (right).[26]
tive compounds were then obtained under the guidance of
structure-based drug design to improve potency. Indeed, some
new compounds exhibited promising binding affinities (Ki <
100 nm). Their specific binding to the target proteins was also
confirmed by 15N-HSQC NMR experiments. An advantage of
this compound class is that some members are effective bind-
ers of Mcl-1 as well as Bcl-xL or Bcl-2. Such compounds may be
[a] Y. Xu, M. Zhou, Y. Li, C. Li, Z. Zhang, Prof. B. Yu, Prof. R. Wang
State Key Laboratory of Bioorganic and Natural Products Chemistry
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences
345 Lingling Road, Shanghai 200032 (P.R. China)
E-mail: byu@mail.sioc.ac.cn
wangrx@mail.sioc.ac.cn
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300159.
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used for treating cancer cells that resist Bcl-2/Bcl-xL inhibitors
or other chemotherapies through overexpression of Mcl-1.[24,25]
The structure–activity relationships of this class of com-
pounds were described in our previous study.[23] However, the
absolute stereochemical configuration of the C4 atom on the
core structure remained unsolved. Whether the corresponding
R and S enantiomers elicit different biological effects, as is the
case with some other chiral drugs, was also unknown. Feng
et al. reported a set of compounds with a core structure similar
to ours (Figure 1), which were also characterized as Bcl-2 inhib-
itors (IC50: 2–70 mm).[26] In their work, enantiomers of one se-
lected compound were separated by chiral HPLC. Subsequent
binding assay results indicated that the R enantiomer had
decent binding affinity (IC50 =1.9 mm), whereas the S enantio-
mer was essentially inactive. However, we were skeptical of
their observation, as a predicted binding mode of this com-
pound class derived in our previous study did not quite sup-
port it.
The specific aim of this study was to address this issue. We
synthesized four compounds of this class and obtained their
pure R and S enantiomers by HPLC separation and chiral syn-
thesis. Their absolute configuration was determined by com-
paring their circular dichroism (CD) spectra to that of an appro-
priate reference compound. The crystal structure of one com-
pound was also obtained. All four pairs of enantio-
mers were tested for their binding affinities to three
anti-apoptotic Bcl-2 family proteins in a fluorescence
polarization (FP)-based binding assay. Our results in-
dicate that the binding affinities of most pairs of R
and S enantiomers were similar. A binding mode of
this compound class was proposed by molecular
modeling to interpret this observation.
Scheme 1. Synthesis of thiazolo[3,2-a]pyrimidinone compounds. Reagents
and conditions: a) p-TsOH, H2O, EtOH, reflux, 24 h; b) 2-chloroacetic acid,
AcOH, Ac2O, AcONa, 808C, 24 h.
Results and Discussion
At the initial attempt, we selected and synthesized
two compounds described in our previous study:[23]
2a (BCL-LZH-02) and 2b (BCL-LZH-18). The former
was the lead compound chosen for optimization in
our previous study, and the latter was one of the
best compounds obtained by us so far. These two
compounds were synthesized by procedures outlined
in Scheme 1. The products were then separated by
preparative chiral HPLC to obtain pure enantiomers.
We then attempted to resolve the stereochemical
structures of these two compounds through crystal
structure determination. Growth of single crystals
was attempted for two pairs of enantiomers as well
Figure 2. Crystal structure of deacetylated (S)-2b (CCDC entry 942185).
The resolved crystal structure of deacetylated 2b is shown
as the original racemic mixture. A number of conditions were
tested, but unfortunately all trials failed to obtain single crys-
tals of sufficient quality for structure determination. As an addi-
tional attempt, 2b was deacetylated with saturated K2CO3 in
CH2Cl2/CH3OH (v/v=1:1) to obtain a derivative compound with
a free hydroxy group on the phenyl ring. Good crystals were
obtained from the racemic mixture of this compound and
were used in structure determination.
in Figure 2. It is evident that the chiral C4 atom is in the S con-
figuration in this particular structure. Although this crystal
structure did not really help us to determine the absolute con-
figuration of the obtained enantiomers of 2a or 2b, it resolved
another uncertainty in the stereochemical structure of this
class of compounds: The exocyclic double bond connecting
the five-membered thiazole ring and the rest of the molecule,
that is, C2=C16, is in the Z configuration. Note that the config-
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uration of this double bond
largely determines the overall
molecular shape of this com-
pound class. Accuracy is there-
fore important if one wants to
model the binding mode of this
class of compounds to their
target proteins correctly.
Because we did not succeed
in determining the absolute con-
figuration of this class of com-
pounds through crystal structure
determination, we turned to
chiral synthesis to solve this
problem. Monastrol is a cell-per-
meable, small-molecule inhibitor
of the kinesin Eg5.[27] This mole-
cule is a direct derivative of 4-
aryl-3,4-dihydro-2(1H)-pyrimi-
Figure 3. CD spectra of the first and the second eluted enantiomers of 1c (monastrol). The first eluted enantiomer
((S)-1c, blue line) exhibited a positive Cotton effect around 303 nm and a negative Cotton effect around 235 and
335 nm, whereas the second enantiomer ((R)-1c, green line) exhibited a mirror-image pattern.
done (DHPMs). The absolute
configuration of DHPMs has
been well characterized.[28] We realized that monastrol could
be used as the starting material for the synthesis of this class
of compounds. Thus, monastrol (1c) was synthesized through
the Biginelli reaction (Scheme 1).[29] The racemic product was
then separated by chiral HPLC. Both enantiomers of 1c
(99% ee) were dissolved in methanol to obtain their CD spec-
tra and optical rotation values. CD spectra of the first and the
second eluted enantiomer are shown in Figure 3. By compari-
son with the known CD spectra of DHPMs as reference, the
enantiomer showing a positive Cotton effect at 303 nm was
determined to be the S enantiomer, whereas the other was the
R enantiomer. Measured optical rotation values of (S)- and (R)-
1c were +98 and À98 (c=1.0, methanol), respectively.
After optically pure monastrol was obtained, it was then
used as the starting material to synthesize two new com-
pounds: 2c and 2d (Scheme 1). All obtained enantiomers of
2c–d as well as 2a–b were dissolved in CHCl3 to obtain CD
spectra and optical rotation values. The absolute configuration
of each enantiomer was determined by examining the pattern
observed on its CD spectrum: a positive Cotton effect around
440 nm and a negative effect around 245 nm indicated the S
enantiomer, whereas the mirror-image pattern indicated the R
enantiomer. Optical rotation values were also examined as ad-
ditional proof (see figures S2–S6 in the Supporting Informa-
tion).
We then selected the two enantiomers of 2a to examine
their binding to Bcl-xL by CD titration experiments. (S)-2a and
(R)-2a were tested at six different concentrations up to 50 mm,
and the results are shown in Figure 4. In both cases, CD signals
were essentially proportional to the concentrations of 2a, indi-
cating their dose-dependent binding to Bcl-xL. Although we
were unable to derive quantitative association constants
within the concentration range tested in this experiment, it is
clear that both (S)-2a and (R)-2a bind Bcl-xL with approximate-
ly equal binding affinities.
Figure 4. CD spectra of a) (S)-2a and b) (R)-2a mixed with Bcl-xL, which were
measured in 5 mm Tris buffer with 5% DMSO; 2a was tested at six different
concentrations up to 50 mm.
To obtain quantitative binding affinities for 2a–d to the
three anti-apoptotic Bcl-2 family proteins (Bcl-xL, Bcl-2, and
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Table 1. Binding affinities of the compounds reported in this study for
three anti-apoptotic Bcl-2 family proteins.
Compd[a]
Yield [%]
Ki [mm][b]
Bcl-xL
Bcl-2
Mcl-1
(S)-2a
(R)-2a
(S)-2b
(R)-2b
(S)-2c
(R)-2c
(S)-2d
(R)-2d
62
62
78
78
85
96
68
74
1.10Æ0.66
0.83Æ0.14
0.28Æ0.04
0.55Æ0.06
0.29Æ0.04
0.24Æ0.06
0.34Æ0.06
0.29Æ0.06
0.81Æ0.30
1.10Æ0.37
0.94Æ0.24
1.00Æ0.11
0.56Æ0.17
0.70Æ0.15
0.59Æ0.25
0.54Æ0.06
2.20Æ0.29
0.36Æ0.24
0.32Æ0.05
0.42Æ0.06
0.50Æ0.12
1.20Æ0.36
0.74Æ0.28
0.29Æ0.10
[a] See Scheme 1 for compound structures. [b] Values are the average Æ
standard deviation (SD) of three parallel measurements.
Mcl-1), all enantiomers of these compounds were tested in an
FP-based in vitro binding assay; the results are summarized in
Table 1. Binding affinities of these compounds to three target
proteins range by one order of magnitude, that is, 0.24–
2.20 mm. Interestingly, in most cases the binding affinity of the
S enantiomer is similar to that of the corresponding R enantio-
mer on all three proteins, with a maximum difference of two-
fold. This level of difference is actually close to the intrinsic
limit of this type of binding assay. The only exception is ob-
served for 2a at Mcl-1, for which the binding affinity of the R
enantiomer (Ki =0.36 mm) is sixfold higher than that of the S
enantiomer (Ki =2.20 mm). However, this discrepancy can be
reasonably explained by a predicted binding mode of this
compound class, as discussed below.
Our results disagree with those of Feng et al. obtained with
a similar set of compounds. They reported that at least in one
case, the R enantiomer of their compound was a good binder,
while the S enantiomer was basically inactive.[26] We did not
test their compounds in this study because the samples of
their compounds were not available to us. The exact reason
for the discrepancy between our results and their results is un-
known. One possible explanation is that although our com-
pounds and theirs share a common core structure, these two
sets of compounds are still somewhat different in other re-
gions (Figure 1). For example, the major hydrophobic region
on our compounds and that on the compounds of Feng and
colleagues are at opposite ends of the molecular framework.
Thus, these two sets of compounds may behave differently in
their binding to anti-apoptotic Bcl-2 family proteins.
To seek a reasonable interpretation of our binding assay re-
sults, we selected 2b to examine the binding modes of its S
and R enantiomers with Bcl-xL and Mcl-1 through molecular
modeling. The most stable binding modes of (S)-2b and (R)-2b
with Mcl-1 predicted by us are shown in Figure 5. The binding
modes of these two compounds to Bcl-xL are given in the Sup-
porting Information.
First of all, a reasonable binding mode was obtained for
both (S)-2b and (R)-2b, suggesting that both enantiomers are
effective binders of Mcl-1. A common feature in these two
binding modes is the location of the benzyl indole moiety on
2b in a hydrophobic cavity formed by residues M231, V249,
Figure 5. Binding modes of a) (S)-2b and b) (R)-2b in complex with Mcl-
1 predicted by molecular modeling. Compound 2b is rendered in stick
model, while Mcl-1 is shown as green ribbons. Residues in direct contact
with 2b are shown and labeled explicitly. c) Superimposition of these two
complex structures.
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V253, L267, and F270. This hydrophobic cavity is common
among Bcl-xL, Bcl-2, and Mcl-1. Such hydrophobic interaction is
perhaps the critical factor for binding of 2b to Bcl-xL. Indeed,
the structure–activity relationships of this compound class, ob-
tained in our previous study, indicate that replacement of this
moiety with a phenyl moiety leads to a total loss of binding af-
finity.[23] This interaction pattern is also observed with other
small-molecule Bcl-2 inhibitors such as those in PDB entries
2YXJ,[7] 4EHR,[21] and 4HW2.[22] Moreover, there is a possible hy-
drogen bond formed between the carbonyl group on the thia-
zole ring on 2b and the hydroxy group on the side chain of
T266 in both cases. T266 locates approximately at the center
of the binding groove on Mcl-1, and this hydrogen bond acts
as an anchor for immobilizing (S)-2b or (R)-2b during their
binding.
reasonable, it is not clear from this model why the R enantio-
mer of this compound is a good binder, whereas the S enan-
tiomer is basically inactive. Furthermore, the location of the
chiral C4 atom inside the binding groove is quite different be-
tween our model and Feng’s model. It should be emphasized
that the purpose of our study is not to overturn the results of
Feng et al., but to raise awareness of the disputes on the struc-
ture–activity relationships of this class of compounds as Bcl-2
inhibitors. The ultimate answer will be provided by crystal
complex structures as well as more careful measurements.
Conclusions
In this study we resolved the stereochemical structures of four
2H-thiazolo[3,2-a]pyrimidine compounds and investigated their
binding affinities toward three anti-apoptotic Bcl-2 family pro-
teins. Interestingly, the R and S enantiomers of most tested
compounds exhibited similar binding affinities for all three pro-
teins. According to our predicted binding mode, the chiral part
in the molecular structure of this compound class resides at
a relatively open region in the binding site, and the binding
site is also sufficiently flexible to accommodate both enantio-
mers. Therefore, the difference in chirality does not lead to
a significant difference in binding affinity for this class of com-
pounds. Notably, our results disagree with those reported by
Feng et al. on a similar set of compounds.[26] Readers should
be aware of this dispute. The exact binding mode of this class
of compounds remains to be resolved by experimental meth-
ods. Nevertheless, it is clear, based on the outcomes of this
study, that the benzyl indole moiety plays a key role in main-
taining the binding affinities of this compound class. On the
other hand, the pyrimidine ring along with its substituent
groups require further optimization in order to develop this
class of compounds into even more promising Bcl-2 inhibitors.
Nevertheless, the most apparent difference between the
binding modes of (S)-2b and (R)-2b lies in the orientation of
the substituted phenyl group on the C4 atom, which is a natu-
ral consequence of the different configuration of this atom. In
the case of (S)-2b, this substituted phenyl ring points toward
N260 and R263 at one side of the binding groove. The acetyl
group on it, that is, R1 in Scheme 1, may form a hydrogen
bond with R263. In the case of (R)-2b, the substituted phenyl
ring points toward the other side. Instead, the ester group on
C5, that is, R2 in Scheme 1, may form a hydrogen bond with
N260. This part of the binding groove is relatively open, as it is
close to the C terminus of Mcl-1, providing enough space for
accommodating these two different binding modes. Also, one
can see clearly in the superimposed binding modes of (S)-2b
and (R)-2b that the core structure of 2b must also adjust its
position and orientation in these two cases to allow the substi-
tuted phenyl group to stretch into different directions. In par-
ticular, as mentioned above, the binding affinity of (R)-2a to
Mcl-1 is sixfold higher than that of (S)-2a. Note that this com-
pound does not have any substituent on the phenyl ring
(Scheme 1). If assuming the binding modes of (S)-2a and (R)-
2a are similar to the counterparts of 2b, (S)-2a cannot form
polar interactions with either N260 or R263, whereas (R)-2a
can with the ester group on the C5 atom. Therefore, (R)-2a is
preferred over (S)-2a in binding to Mcl-1.
To summarize, our predicted binding mode for 2b illustrates
the key role of the benzyl indole moiety as well as the thiazole
core moiety in the binding of this class of compounds to Bcl-2
family proteins. However, the substituent groups on C4 and
C5, either in the S or R enantiomer, are not optimal for fitting
this part of the binding groove. The hydrogen bonds formed
by 2b with either N260 or R263 were observed to be unstable
during molecular dynamics (MD) simulations. This explains the
fact that the structure–activity relationships for this class of
compounds are less clear when structural modifications are
made at these two positions.[23]
Experimental Section
Compound synthesis: Four compounds (2a–d) were synthesized
as outlined in Scheme 1. Dihydropyrimidine derivatives 1a–c were
prepared through the classic Biginelli reaction[29] of commercially
available b-keto esters or amides, substituted phenyl aldehydes
and thiourea in ethanol at reflux. The reaction was catalyzed by
para-toluenesulfonic acid monohydrate. Products were then al-
lowed to react with N-substituted 1H-indole-3-carbaldehyde and 2-
chloroacetic acid in the presence of acetic acid, acetic anhydride,
and sodium acetate at 808C to obtain 2a–d with yields in the
range of 62–96% (Table 1).
All NMR spectra were recorded at room temperature on a Bruker
AV-400 400 MHz spectrometer. They were calibrated using residual
undeuterated chloroform (dH =7.26 ppm) and CDCl3 (dC =
77.16 ppm) and undeuterated methanol (dH =3.31 ppm), CD3OD
(dC =49.00 ppm) as internal references. The following abbreviations
are used to designate multiplicities: s=singlet, d=doublet, t=
triplet, q=quartet, m=multiplet, quint=quintet, br=broad. High-
resolution mass spectra (HRMS) were recorded on a Bruker APEXIII
7.0 Tesla ESI-FT mass spectrometer at an emitter voltage of 4000 V
. Spectral data for compounds 1a–1c and 2a–2d are given below.
It is also interesting to compare the binding mode of our
compounds with those reported by Feng et al. For this pur-
pose, we also reproduced the binding mode of Feng’s com-
pound 1 to Bcl-xL as shown in Feng’s study.[26] Detailed descrip-
tions can be found in the Supporting Information. Although
the predicted binding mode of Feng’s compound is actually
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6-Methyl-N,4-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxamide (1a): H NMR (300 MHz, [D6]DMSO): d=10.00 (s, 1H),
9.74 (s, 1H), 9.45 (s, 1H), 7.54 (d, J=8.1 Hz, 2H), 7.38–7.33 (m, 2H),
7.29–7.23 (m, 5H), 7.02 (d, J=7.5 Hz, 1H), 5.40 (d, J=2.4 Hz, 1H),
2.07 ppm (s, 3H).
1.21–1.17 ppm (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=
169.1, 165.4, 165.1, 156.6, 153.2, 150.7, 141.9, 136.5, 135.5, 130.3,
129.5, 129.1, 128.3, 128.0, 127.2, 125.4, 123.8, 121.9, 121.2, 119.0,
113.6, 111.5, 110.5, 108.0, 60.55, 54.8, 50.9, 22.9, 21.2, 14.0 ppm; MS
(ESI): m/z 592 [M+H]+; HRMS (ESI): m/z calcd for C34H30N3O5S+
[M+H]+: 592.1900, obsd: 592.1891; (S)-2c, [a]D25: +1033 (c=0.3,
CHCl3), 99.5% ee, tR =17.8 min; (R)-2c, [a]D25: À1000 (c=0.3, CHCl3),
98.9% ee, tR =23.5 min.
1
Methyl-4-(4-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahy-
dropyrimidine-5-carboxylate (1b): 1H NMR (300 MHz, [D6]DMSO):
d=10.03 (s, 1H), 9.59 (s, 1H), 9.44 (s, 1H), 7.00 (d, J=8.4 Hz, 2H),
6.71 (d, J=8.4 Hz, 2H), 5.06 (d, J=3.6 Hz, 1H), 3.54 (s, 3H),
2.28 ppm (s, 3H).
(Z)-Ethyl-2-((1-([1,1’-biphenyl]-4-ylmethyl)-1H-indol-3-yl)methy-
lene)-5-(3-acetoxyphenyl)-7-methyl-3-oxo-3,5-dihydro-2H-thia-
zolo[3,2-a]pyrimidine-6-carboxylate (2d, BCL-LZH-42A): 1H NMR
(400 MHz, CDCl3): d=8.08 (s, 1H), 7.80–7.79 (m, 1H), 7.56–7.54 (m,
4H), 7.45–7.41 (m, 3H), 7.37–7.28 (m, 6H), 7.26–7.21 (m, 2H), 7.16
(s, 1H), 7.03–7.02 (m, 1H), 6.21 (s, 1H), 5.42 (s, 2H), 4.12–4.10 (q,
J=1.2 Hz, J=5.6 Hz, 2H), 2.52 (s, 3H), 2.26 (s, 3H), 1.21–1.17 ppm
(t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=169.1, 165.4,
165.1, 156.6, 153.2, 150.7, 141.9, 136.5, 135.5, 130.3, 129.4, 129.1,
128.8, 128.3, 1282.0, 127.2, 125.5, 125.4, 123.8, 121.9, 121.2, 118.9,
113.6, 111.4, 110.5, 108.0, 60.5, 54.8, 50.9, 22.9, 21.2, 14.0 ppm; MS
(ESI): m/z 668 [M+H]+; HRMS (ESI): m/z calcd for C40H34N3O5S+
[M+H]+: 668.2213, obsd: 668.2210; (S)-2d, [a]D25: +691 (c=0.3,
CHCl3), 99.7% ee, tR =54.7 min; (R)-2d, [a]D25: À703 (c=0.3, CHCl3),
98.7% ee, tR =86.3 min.
Ethyl-4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (1c, monastrol): 1H NMR (400 MHz,
CD3OD): d=7.14–7.10 (t, J=8 Hz, 1H), 6.77–6.73 (m, 2H), 6.69–6.67
(m, 1H), 5.25 (s, 1H), 4.10–4.08 (q, J=1.2 Hz, J=5.6 Hz, 2H), 2.34 (s,
3H), 1.20–1.16 ppm (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD):
d=175.9, 167.3, 158.7, 146.0, 145.6, 130.6, 118.9, 115.8, 114.5,
103.1, 61.3, 56.3, 17.6, 14.4 ppm; MS (ESI): m/z 293 [M+H]+; HRMS
(ESI): m/z calcd for C14H16N2O3SNa+ [M+Na]+: 315.0773, obsd:
25
315.0779; (S)-1c, [a]D
: +98 (c=1.0, MeOH), 99.6% ee, tR =
5.9 min; (R)-1c, [a]D25: À98 (c=1.0, MeOH), 97.6% ee, tR =7.2 min.
(Z)-2-((1-Benzyl-1H-indol-3-yl)methylene)-7-methyl-3-oxo-N,5-di-
phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-6-carboxamide
1
(2a, BCL-LZH-02): H NMR (300 MHz, [D6]DMSO): d=10.03 (s, 1H),
HPLC and CD spectra: Chiral HPLC separation of 2a and 2b was
performed at 358C using a chromatography system consisting of
a CHIRALPAK IA column (46 mmꢁ250 mm, 10 mm) and a Shimadzu
LC 20 instrument equipped with an SPD-20A UV detector; detec-
tion was at l 254 nm and the flow rate was 1.0 mLminÀ1. MeOH/
CH2Cl2 95:5 and CH2Cl2/CH3CN 95:5 mixtures (v/v) were used as the
eluting solvent for 2a and 2b, respectively. Retention times for (S)-
2a and (R)-2a were 5.6 and 6.7 min, respectively. Retention times
for (S)-2b and (R)-2b were 4.2 and 4.6 min, respectively. The race-
mic mixture of 1c was separated by using a Chiralcel OJ-H column
(i.d.=0.46 cm ꢁ l=25 cm; mobile phase: hexane/EtOH=70:30 v/v;
T=358C). Retention times for (S)-1c and (R)-1c were 5.9 and
7.2 min, respectively.
8.11 (s, 1H), 8.03 (s, 1H), 7.91 (d, J=7.2 Hz, 1H), 7.56 (dd, J=7.8 Hz,
1.5 Hz, 3H), 7.38–7.17 (m, 14H), 7.05 (t, J=7.5 Hz, 1H), 6.24 (s, 1H),
5.61 (s, 2H), 2.13 ppm (d, J=0.6 Hz, 3H); 13C NMR (100 MHz,
CDCl3): d=165.3, 154.8, 143.5, 139.7, 137.5, 136.7, 135.6, 130.3,
129.4, 129.3, 129.2, 129.0, 128.5, 128.1, 127.6, 127.3, 126.8, 124.9,
124.8, 123.9, 122.0, 120.1, 119.1, 113.9, 111.7, 110.6, 56.4, 51.1, 29.8,
21.9 ppm; MS (ESI): m/z 581 [M+H]+; HRMS (ESI): m/z calcd for
C36H29N4O2S+ [M+H]+: 581.2006, obsd: 581.2015; (S)-2a, [a]D
:
25
+851 (c=0.3, CHCl3), 96.2% ee, tR =5.6 min; (R)-2a, [a]D25: À730
(c=0.3, CHCl3), 96.9% ee, tR =6.7 min.
(Z)-Methyl-5-(4-acetoxyphenyl)-2-((1-benzyl-1H-indol-3-yl)methy-
lene)-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-
6-carboxylate (2b, BCL-LZH-18): 1H NMR (300 MHz, CDCl3): d=
8.09 (s, 1H), 7.79–7.76 (m, 1H), 7.46–7.15 (m, 12H), 7.03 (d, J=
8.4 Hz, 2H), 6.22 (s, 1H), 5.37 (s, 2H), 3.67 (s, 3H), 2.52 (s, 3H),
2.26 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=169.2, 166.0, 165.1,
156.7, 153.4, 150.6, 137.8, 136.5, 135.4, 130.3, 129.0, 128.9, 128.3,
127.9, 127.1, 126.6, 125.4, 123.8, 121.9, 121.6, 118.9, 113.7, 111.4,
110.4, 107.9, 54.4, 51.5, 50.9, 22.9, 21.1 ppm; MS (ESI): m/z 578 [M+
H]+, 600 [M+Na]+; HRMS (ESI): m/z calcd for C33H28N3O5S+ [M+
All CD spectra were obtained with a Jasco J-810 spectropolarime-
ter using standard conditions. Sample solutions in methanol were
placed in quartz cells of 10 mm path length, and their concentra-
tions were adjusted to 0.05 mgmLÀ1. Sample solutions were pre-
pared at room temperature. CD spectra were taken with the fol-
lowing settings: scan rate=500 nmminÀ1; bond width=0.1 nm;
response time=1.0 s; accumulations=2 scan. Optical rotations
were measured in a 10 mm quartz cell on a Jasco P-1030 polarime-
ter. HPLC and CD spectra and optical rotation values of com-
pounds 1c, 2a–d are given in the Supporting Information.
H]+: 578.1744, obsd: 578.1751; (S)-2b, [a]D25: +706 (c=0.3, CHCl3),
25
98.0% ee, tR =4.2 min; (R)-2b, [a]D
98.0% ee, tR =4.6 min.
:
À982 (c=0.3, CHCl3),
CD titration was performed for (R)-2a and (S)-2a binding to Bcl-xL.
The concentration of Bcl-xL used in this experiment was 50 mm. Six
different concentrations of 2a (5, 10, 20, 30, 40, and 50 mm) were
tested in 5 mm Tris buffer (pH 7.5) with 5% DMSO. CD signals were
recorded between 300 and 600 nm on a Jasco J-715 spectropo-
larimeter at room temperature in a cuvette of 1 cm path length.
All spectra were accumulated twice at a bandwidth of 1.0 nm and
a resolution of 0.5 nm at a scan speed of 100 nmminÀ1. This ex-
periment was not performed for other compounds reported in this
study, because it consumed too much protein sample on the spec-
tropolarimeter accessible to us and yet was difficult to derive
quantitative binding data.
(Z)-Methyl-2-((1-benzyl-1H-indol-3-yl)methylene)-5-(4-hydroxy-
phenyl)-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimi-
dine-6-carboxylate (deacetylated 2b, BCL-LZH-18A): 1H NMR
(400 MHz, CDCl3): d=8.11 (s, 1H), 7.77–7.75 (m, 1H), 7.42 (s, 1H),
7.32–7.24 (m, 8H), 7.15–7.13 (d, 2H), 6.74–6.72 (d, 2H), 6.17 (s, 1H),
5.36 (s, 2H), 3.66 (s, 3H), 2.52 ppm (s, 3H); MS (ESI): m/z 536 [M+
H]+; HRMS (ESI): m/z calcd for C31H26N3O4S+ [M+H]+: 536.1649,
obsd: 536.1638.
(Z)-Ethyl-5-(3-acetoxyphenyl)-2-((1-benzyl-1H-indol-3-yl)methy-
lene)-7-methyl-3-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidine-
6-carboxylate (2c, BCL-LZH-11A): 1H NMR (400 MHz, CDCl3): d=
8.10 (s, 1H), 7.80–7.78 (m, 1H), 7.39 (s, 1H), 7.34–7.17 (m, 8H),
7.17–7.15 (m, 3H), 7.03–7.02 (m, 1H), 6.21 (s, 1H), 5.37 (s, 2H),
4.12–4.10 (q, J=1.2 Hz, J=5.6 Hz, 2H), 2.52 (s, 3H), 2.27 (s, 3H),
Crystal structure determination: Growth of single crystals was at-
tempted for the R and S enantiomers of 2a and 2b as well as their
racemic mixture. These attempts, however, failed to obtain single
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crystals good enough for structure determination. Nevertheless,
good crystals were obtained from the racemic mixture of deacety-
lated 2b with the following methods: The compound sample
(15 mg) and THF (2.0 mL) were added to a 5 mL sample bottle. The
sample bottle was then transferred into a 60 mL reagent bottle
containing 20 mL petroleum ether. The solution was allowed to
stand for several days to obtain the final crystals.
(Tecan Group Ltd.). In each experiment, a positive control contain-
ing the protein (Bcl-xL, Bcl-2, or Mcl-1), the Bid-BH3 peptide, and
1% (v/v) [D6]DMSO, as well as a negative control containing only
Bid-BH3 peptide, were included on each plate.
Each compound was tested against all three proteins (Bcl-xL, Bcl-2,
and Mcl-1) at seven different concentrations: 1, 10 and 100 nm,
and 1, 10, 50, and 100 mm. At each concentration, the average FP
value of three parallel measurements was used to perform nonlin-
ear fitting to obtain the final binding curve. The nonlinear fitting
was conducted with GraphPad Prism software (version 5). Concen-
tration of the given compound at which 50% of the bound pep-
tide was displaced (IC50) was derived from the binding curve. The
competitive inhibition constant (Ki) of each tested compound was
then calculated with an equation developed by Wang et al.[32] as-
suming formation of a binary complex with the target protein.
X-ray crystal diffraction data were collected on a Bruker Apex II
CCD diffractometer operating at 50 kV and 30 mA using MoKa radi-
ation (l=0.71073 ꢂ) at 133 K. Main features of this crystal structure
are: C39H41N3O6S, Mr =679.81 gmolÀ1
, crystal size 0.35ꢁ0.10ꢁ
0.05 mm, triclinic, space group=P1, a=9.4103(13) ꢂ, b=
11.5966(16) ꢂ, c=15.993(2) ꢂ, a=79.623(2)8, b=85.822(2)8, g=
78.788(2)8, V=1682.6(4) ꢂ3, T=133 K, Z=2, Dcalc =1.342 gcmÀ3, l=
0.71073 ꢂ, m(MoKa)=8.396 mmÀ1. Other details are given in the
Supporting Information. In addition, CCDC 942185 contains the
supplementary crystallographic data for this paper. These data can
be obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Molecular modeling: The binding modes of (S)-2b and (R)-2b
were predicted through molecular docking and MD simulations.
Bcl-xL and Mcl-1 were considered for this purpose. As the first step,
molecular docking was employed to derive a rough binding mode
for 2b with Bcl-xL and Mcl-1. The complex structure between
human Bcl-xL and the Bak-BH3 peptide (PDB entry: 1BXL) was used
in molecular docking of 2b. For the Mcl-1 protein, the structure
from the complex between human Mcl-1 and the Bim-BH3 peptide
(PDB entry: 2PQK) was selected for molecular docking. The molec-
ular structures of (R)-2b and (S)-2b were sketched with SYBYL soft-
ware (version 8.1) and optimized with the MMFF94 force field. Au-
tomatic docking of each molecule to Bcl-xL or Mcl-1 was performed
by using GOLD software (version 5.1, Cambridge Crystallographic
Data Centre). Detailed parameters and settings used in this task
are given in the Supporting Information. A total of 30 final binding
poses were generated for each ligand molecule. These binding
poses were clustered by RMSD values with a cutoff of 2.0 ꢂ. The
binding pose with the highest binding score from the largest clus-
ter was selected as the most reasonable one after visual examina-
tion.
In vitro binding assays: A fluorescence polarization (FP)-based
assay was employed in this study to measure the binding affinities
of small-molecule compounds to three anti-apoptotic Bcl-2 family
proteins: Bcl-xL, Bcl-2, and Mcl-1. Competitive binding of a given
compound was characterized quantitatively by monitoring the
changes in FP signal upon addition of test compound at multiple
concentrations.
The Bcl-xL protein used in our study is a special truncated construc-
tion of the full-length protein, with deletion of residues 45–84 on
a large loop region and residues 210–233 at the C-terminal hydro-
phobic region. The Bcl-2 protein used in our study has the same
construction as that (Bcl-2/Bcl-xL, isoform 2) reported by Fesik
et al.,[30] which is composed of residues 1–34 of human Bcl-2, resi-
dues 29–44 of human Bcl-xL, and residues 92–207 of human Bcl-2.
The Mcl-1 protein used in our study has the same construction as
that (hMcl-1BLR) used in the work of Colman et al.,[31] which is
composed of residues 152–189 of mouse Mcl-1 and residues 209–
327 of human Mcl-1. Methods used for preparing the samples of
these three proteins are given in the Supporting Information.
The selected binding pose of the given compound was then re-
fined through MD simulation using the AMBER program (ver-
sion 12, University of California San Francisco). Each MD simulation
was performed in an explicit water box for 5 ns. Detailed parame-
ters and settings used in our MD simulations are given in the Sup-
porting Information. On each MD trajectory, 5000 snapshots were
retrieved at 1 ps intervals. These snapshots were grouped into clus-
ters based on mass-weighted RMSD values with a cutoff value of
1.5 ꢂ. The snapshot closest to the cluster center in the largest clus-
ter was selected as the representative binding mode. The final se-
lected binding modes of (R)-2b and (S)-2b to Bcl-xL and Mcl-1 are
shown in figures S10 and S11 in the Supporting Information.
A 26-residue peptide derived from the BH3 domain of the Bid pro-
tein with 5-carboxyfluorescein (5-FAM) on the N terminus, that is,
5-(FAM)-QEDIIRNIARHLAQVGDSMDRSIPPG, was used as the fluores-
cence tracer (HD Biosciences). The desired sequence was verified
by amino acid component analysis and MS. The purity of this pep-
tide was >95%, as verified by HPLC. Our dose-dependent satura-
tion experiments determined that this Bid-BH3 peptide binds to
Bcl-xL with Kd =41 nm, to Bcl-2 with Kd =89 nm, and to Mcl-1 with
Kd =29 nm (see figure S7 in the Supporting Information).
In competitive binding measurements, each protein (Bcl-xL, Bcl-2,
or Mcl-1) and the compound under test (in 1% [D6]DMSO solution)
were pre-incubated in the assay buffer (PBS, pH 7.3, 140 mm NaCl,
2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4) at 378C for 30 min. A
solution in PBS of the FAM-Bid peptide (20 mL) was then added to
the solution to produce a final volume of 200 mL and incubated at
378C for another 20 min. The total concentration of the Bid-BH3
peptide used in assay was 10 nm, while the total concentration of
protein was set to fivefold the Kd value of the fluorescence tracer.
Finally, the solutions were transferred into black flat-bottomed
384-well plates (Corning Inc.) with 60 mL per well and three wells
per sample. Polarization signals (in milipolarization units, mP) were
measured at an excitation wavelength of 485 nm and an emission
wavelength of 535 nm on a Tecan Genios Pro Injector Reader
Acknowledgements
The authors are grateful for financial support from the Chinese
National Natural Science Foundation (Grant Nos. 81172984,
21072213, 21002117, 21102168, 21102165, and 20921091) and
the 863 High-Tech program (Grant No. 2012AA020308) from the
Chinese Ministry of Science and Technology.
Keywords: Bcl-2 · binding affinity · chirality · inhibitors ·
protein–protein interactions · small molecules
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