Quantum chemistry calculation-aided structural optimization of combretastatin A-4-like tubulin polymerization inhibitors: Improved stability and biological activity

Article abstract of DOI:10.1021/acs.jmedchem.5b00118

A potent combretastatin A-4 (CA-4) like tubulin polymerization inhibitor 22b was found with strong antitumor activity previously. However, it easily undergoes cis-trans isomerization under natural light, and the resulting decrease in activity limits its further applications. In this study, we used quantum chemistry calculations to explore the molecular basis of its instability. Aided by the calculations, two rounds of structural optimization of 22b were conducted. Accelerated quantitative light stability testing confirmed that the stability of these designed compounds was significantly improved as predicted. Among them, compounds 1 and 3b displayed more potent inhibitory activity on tumor cell growth than 22b. In addition, the potent in vivo antitumor activity of compound 1 was confirmed. Quantum chemistry calculations were used in the optimization of stilbene-like molecules, providing new insight into stilbenoid optimization and important implications for the future development of novel CA-4-like tubulin polymerization inhibitors.

Full text of DOI:10.1021/acs.jmedchem.5b00118

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Article
Quantum Chemistry Calculation-Aided Structural Optimization
of Combretastatin A-4-like Tubulin Polymerization
Inhibitors: Improved Stability and Biological Activity
Junhang Jiang, Canhui Zheng, Kongkai Zhu, Jia Liu, Nannan Sun,
Chongqing Wang, Hualiang Jiang, Ju Zhu, Cheng Luo, and Youjun Zhou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00118 • Publication Date (Web): 17 Feb 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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Quantum Chemistry Calculation-Aided Structural
Optimization of Combretastatin A-4-like Tubulin
Polymerization Inhibitors: Improved Stability and
Biological Activity
a,†
a,†,*
b,†
a
a
a
Junhang Jiang, Canhui Zheng,
Kongkai Zhu, Jia Liu, Nannan Sun, Chongqing Wang,
b,c
a,*
b,*
a,*
Hualiang Jiang, Ju Zhu, Cheng Luo, and Youjun Zhou
a
School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203.
c
School of Life Science and Technology, Shanghai Tech University, Shanghai 200031, China
KEYWORDS: stilbenoids, combretastatin A-4, tubulin polymerization inhibitors, cis-trans
photoisomerization, quantum chemistry calculation
ABSTRACT: A potent combretastatin A-4 (CA-4) like tubulin polymerization inhibitor 22b was
found with strong anti-tumor activity previously. However, they easily undergo cis-trans
isomerization under natural light, and the resulting decrease in activity limits its further
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applications. In this study, we used quantum chemistry calculations to explore the molecular
basis of its instability. Aided by the calculations, two round of structural optimization of 22b
were conducted. Accelerated quantitative light stability testing confirmed that the stability of
these designed compounds was significantly improved as predicted. Among them, compounds 1
and 3b displayed more potent inhibitory activity on tumor cell growth than 22b. In addition, the
potent in vivo antitumor activity of compound 1 was confirmed. Quantum chemistry calculations
were used in the optimization of stilbene-like molecules, providing new insight into stilbenoid
optimization and important implications for the future development of novel CA-4-like tubulin
polymerization inhibitors.
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Introduction
Stilbenoids are abundant in natural products and are divided into Z-type and E-type based on the
1
-5
configuration of their central double bond. Key examples include combretastatins (Chart 1)
1, 6
and resveratrol. Combretastatins are strong tubulin polymerization inhibitors binding to the
1
, 7-9
colchicine site and can strongly block tumor growth with a vascular-disrupting effect.
Several derivates of combretastatin A-4 (CA-4) have entered clinical trials. It is well-known that,
for the CA-4-like tubulin polymerization inhibitors, two hydrophobic rings (rings A and B) in the
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, 8, 10, 11
Z-configuration of the linking bridge were necessary to bind to the active site.
However,
these compounds have a central double bond that can undergo cis-trans isomerization, greatly
11-13
changing their overall configuration and, as a consequence, reducing their biological activity.
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Chart 1. The structures of combretastatins.
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4
In our early study, we found a new CA-4-like tubulin polymerization inhibitor 22b (Scheme 1).
It also has two hydrophobic rings in the Z-configuration of the linking bridge similar to CA-4,
and showed a similar binding mode to the tubulin protein. The 22b compound has a strong
affinity to tubulin and shows strong anti-tumor activity, which has been confirmed in vivo. Its
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relatively low toxicity also makes it an active compound worthy of further study. However, we
found later that it is prone to cis-trans isomerization under natural light to yield Z-22b (Scheme
1
). The E and Z populations achieved equilibrium in dimethylsulfoxide (DMSO) solution after 7
days, and the Z-isomer showed greatly reduced biological activity. This easy isomerization limits
the utilization and storage of 22b.
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Theoretical calculations have significantly contributed to drug discovery and design. Because
of their high accuracy, quantum mechanical methods have been used in all phases of computer-
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aided drug design and development, especially in facilitating drug lead discovery and lead
optimization. For example, free energy-guided molecular design has been successfully applied in
lead optimization for non-nucleoside inhibitors of human immunodeficiency virus (HIV)-1
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reverse transcriptase. However, no efficient quantum mechanical methods have been reported
for the optimization of CA-4-like tubulin polymerization inhibitors.
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In this study, we used time-dependent density functional theory (TD-DFT) calculations to
explore the mechanism of the easy cis-trans photoisomerization of 22b. Aided by the quantum
chemistry calculation, two round of structural optimization of 22b were conducted. Accelerated
quantitative light stability testing and ultraviolet-visible spectrophotometric analysis confirmed
that the stability of these designed compounds was significantly improved as predicted. Among
them, compounds 1 and 3b displayed more potent inhibitory activity on tumor cell growth than
2
2b. In addition, the potent in vivo antitumor activitiy of compound 1 was confirmed,
encouraging further study of these designed anti-tumor compounds. Quantum chemistry
calculations were used in the optimization of stilbene-like molecules, providing new insight into
stilbenoid optimization and important implications for the future development of novel CA-4-
like tubulin polymerization inhibitors.
Scheme 1. The cis–trans photoisomerization of 22b
Results and discussions
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8-23
Quantum chemical calculations. According to the literature,
the cis-trans isomerization of
stilbenoids is photoinduced. In this isomerization process, the electron might be excited from the
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ground state (S0) to an excited state (Scheme 1). Electron transitions mainly occur between the
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first excited state (S1) and the ground state.
As illustrated in Table 1, TD-DFT at the
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B3LYP/6-31+G(d) and B3LYP/6-311++G(d,p) levels was employed to calculate the vertical
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excitation energy of molecule 22b in Gaussian 09. The vertical transition from S0 to S1 in 22b
was found to be an optically allowed ππ* with an oscillator strength of 0.2983 by TD-
DFT/B3LYP/6-31+G(d), which represents the transition from the highest occupied molecular
orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO) (Figure 1). Analysis of the
frontier orbitals determined that this transition was mainly located on the double bond linking the
phenyl moieties. As indicated by this calculation, the wavelength required for the vertical
excitation of 22b is in the visible spectrum, allowing it to be quickly isomerized in natural light.
The energy gap between the HOMO and LUMO (Table 3) of 22b is also an indicator of its
instability.
Figure 1. The frontier orbitals of 22b involved in the S0S1 transitions
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Table 1. The vertical excitation energies and excitation wavelengths of the four lowest excited
states of 22b
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B3LYP (6-31+G(d))
B3LYP (6-311++G(d,p))
State
S1
E
ex (eV)
os
 (nm)
402.77
358.80
350.88
314.46
E
ex (eV)
os
 (nm)
401.39
356.75
348.62
312.65
3.0783
3.4556
3.5335
3.9428
0.2983
0.0767
0.0736
0.0501
3.0889
3.4754
3.5564
3.9656
0.2921
0.0736
0.0738
0.0481
S2
S3
S4
E
ex is the vertical excitation energy in eV; os is the oscillator strength;  is the excitation
wavelength in nm.
Table 2. Vertical transition excitation energies (S0 to S1) of the eight compounds
S0S1 (6-31+G(d))
S0S1 (6-311++G(d,p))
E
ex (eV)
 (nm)
402.77
334.89
326.80
326.79
332.31
339.02
320.66
324.48
E
ex (eV)
 (nm)
401.39
332.93
326.46
326.45
331.15
337.79
319.45
323.28
22b
CA-4
1
3.0783(0.2983)
3.7023(0.4311)
3.7939(0.2909)
3.7940(0.2909)
3.7310(0.3781)
3.6571(0.3926)
3.8665(0.3242)
3.8210(0.3617)
3.0889(0.2921)
3.7240(0.3884)
3.7979(0.2750)
3.7979(0.2750)
3.7440(0.3594)
3.6704(0.3710)
3.8812(0.3070)
3.8352(0.3449)
2
3a
3b
4a
4b
E
ex is the vertical excitation energy in eV with oscillator strength in parentheses;  is the
wavelength in nm.
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Table 3. The energy gaps between the HOMO and LUMO orbitals of the eight compounds
HOMO
LUMO
Energy Gap
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-
6-
6-
6-
6-
6-
3
1+G(d)
311++G(d,p)
-0.20715
31+G(d)
-0.07735
311++G(d,p)
-0.07801
31+G(d)
0.12828
311++G(d,p)
0.12914
2
2b
-0.205 63
CA
-
0.19894
-0.20122
-0.04790
-0.04838
0.15104
0.15284
-
4
1
-0.19608
-0.19608
-0.20266
-0.20574
-0.20251
-0.20606
-0.19812
-0.19812
-0.20469
-0.20829
-0.20474
-0.20884
-0.03875
-0.03875
-0.04910
-0.05517
-0.04265
-0.04846
-0.04026
-0.04026
-0.05017
-0.05668
-0.04392
-0.05024
0.15733
0.15733
0.15356
0.15057
0.15986
0.1576
0.15786
0.15786
0.15452
0.15161
0.16082
0.1586
2
3
a
3
b
4
a
4
b
The energy unit is hartree where 1 hartree = 27.2114 eV.
As seen from the LUMO, the carbonyl group and the central double bond linking the phenyl
moieties contribute most to the π* orbital. Consequently, if the carbonyl group is changed to a
group with no double bond, the energy of the LUMO could be decreased and the energy gap
between the HOMO and LUMO could be increased, thereby increasing the stability of the
molecule. And with respect to conjugation, if the carbonyl group was changed to a group with no
double bond, the conjugation of the entire molecule could be decreased, which could increase the
excited state energy. Increased excited state energy could also make the molecule more stable.
Guided by this analysis, two optical isomers (S isomer 1 and R isomer 2, Scheme 2) were
designed by changing the carbonyl group in 22b to a hydroxyl group. A similar approach was
used to calculate the vertical excitation energies of optical isomers 1 and 2 (Table 2). The results
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showed that the two excitation energies were almost identical. Both were significantly higher
than 22b and were even slightly higher than CA-4. As expected, the excitation wavelength
needed for isomerization was significantly shortened. The energy gaps between the HOMO and
LUMO (Table 3) of these two molecules also indicated their increased stability. In addition, they
may have similar biological activity to 22b due to the minimal change in their spatial
configuration. Besides, in order to further testify our conjecture, we also calculated the vertical
excitation energy and energy gaps between the HOMO and LUMO of another compound, which
designed by changing the carbonyl group to -C=N-NH2, retaining the double bond. The data (not
shown) indicated that the stability of this compound is not significantly increased compared to
22b.
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After calculation, these two compounds 1 and 2 were synthesized and their stabilities were
confirmed to be improved relative to 22b (see below). Then, another two optical isomers (S
isomer 3a and R isomer 4a, Scheme 2) were designed by changing the connection between the
six-membered ring of 1 and 2 from the B ring to the A ring. These compounds could have
similar improved stability, because their core skeleton (E)-1-benzylidene-1,2,3,4-
tetrahydronaphthalen-2-ol were identical. It was confirmed by the theoretical study, which
showed that the vertical excitation energy of compounds 3a and 4a were equivalent to that of
compounds 1 and 2, and significantly higher than 22b (Table 2 and 3). In addition, they also
have two hydrophobic rings in the Z-configuration of the linking bridge, similar to CA-4, and
may have similar biological activity.
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O
O
O
3,4,5-trimethoxy
O
O
A
Na
-benzylidene
I
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AcOH/piperidine
B
EtOH
O
O
O
CH Cl2
2
O
22b
(
a)
(b)
OH
OH
O
O
(S)
(R)
A
A
O
2
O
O
B
B
O
O
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1
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O
O
substituted
p-anisaldehyde
O
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O
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six-step
route
O
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O
A
II
AcOH/piperidine
OH
O
B
2
CH Cl2
O
R
O
5a-5h
(a)
(b)
OH
OH
R =H
F
a
(S)
(R)
b
c
d
e
O
O
O
CN
NH2
OH
A
A
O
B
B
R
O
O
(c)
R
CONH2 f
NO
O
O
2
g
h
CH3
3
a-3h
Scheme 2. Synthesis of target compounds. Reagents and conditions: (a) BH
methyl-CBS-oxazaborolidine, anhydrous THF, -20 °C; 1 h, 77% - 93%; (b) BH •Me S, (S)-(-)-2-
4a-4h
3
2
•Me S, (R)-(+)-2-
3
2
methyl-CBS-oxazaborolidine, anhydrous THF, -20 °C; 1 h, 78% - 87%; (c) Fe powder, AcOH,
75% EtOH, 70 °C, 0.5 h, 83% and 85% respectively.
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Chemical synthesis. The E isomer 22b was prepared by the Knoevenagel reaction from the key
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intermediate 6-methoxy-3,4-dihydronaphthalen-2(1H)-one according to our prior work.
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Based on the high enantio-selectivity of the Corey-Bakshi-Shibata (CBS) catalysts, the CBS
reduction was selected to produce compounds 1 and 2 (Scheme 2 I). Compound 22b was treated
with borane-methyl sulfide complex catalyzed by (R)-(+)-2-methyl-CBS-oxazaborolidine in
anhydrous THF at -20 °C to yield S isomer 1 (93% yield, ee: 98.53%). Similarly, R isomer 2 was
obtained using (S)-(-)-2-methyl-CBS-oxazaborolidine (87% yield, ee: 95.76%). Single crystal X-
ray analysis data verified their absolute configurations.
The synthetic route to the key intermediate of 22b could not be used to prepare 5,6,7-trimethoxy-
3,4-dihydronaphthalen-2(1H)-one, the key intermediate of compound 5, because the
corresponding starting materials were not commercially available. We used a more indirect route
(
six steps) to afford it, which then was reacted with substituted panisaldehydes to afford
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compound 5 (Scheme 2 II). We prepared enantiomers 3a-3h and 4a-4h using the same methods
as in the synthesis of 1 and 2. The CBS reduction gave good yields (77% to 93%) and high
enantiomeric excesses (87% ee to 99% ee).
Light stability test and ultraviolet-visible spectrophotometric analysis. Stability testing
experiments were conducted to confirm our predictions. More than a week was needed for
compound 22b to reach cis-trans isomerization equilibrium under natural light, and this time
changed with the light intensity. Thus, accelerated quantitative stability testing was performed
with a high-power xenon lamp and a visible light filter. We used this equipment to irradiate
samples 22b, 1, 2, 3a, 4a and CA-4 in DMSO. HPLC was used to analyze the sample changes
2
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and LC-HRMS to determine the sample structures. After irradiation, the Z-configuration
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isomers of these compounds were produced with content changes as a function of irradiation
time as shown in Figure 2.
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50
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53
54
55
56
57
58
59
60
Figure 2. Changes in the seven compounds over time after visible light irradiation. Data are
expressed as the mean ± SD of the percentage of E isomer at each time point.
The results showed that cis-trans isomerization equilibrium of 22b was achieved after 1.5 hours
with 52.8% of 22b remaining. However, the change in 1, 2, 3a and 4a was markedly slower than
22b and was even slower than CA-4. There were 98.9%, 99.2%, 99.4% 99.1% and 99.1% of 1, 2,
3a, 4a and CA-4 remaining after 1.5 hours, and 90.2%, 92.0%, 92.6%, 93.0%, and 84.0%
remaining after 22 hours, respectively. Indeed, there was no obvious change in these compounds
even after 7 days under natural light. The above results were consistent with our expectations.
The cis-trans isomerization stability of compounds 1, 2, 3a and 4a was substantially improved
relative to 22b and CA-4 under natural light.
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1
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2
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4
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8
9
0
Figure 3. The UV-vis absorption spectra of the seven compounds
The UV-vis absorption spectra of the compounds indicate at which wavelengths they can excite
30
their electrons to higher anti-bonding molecular orbitals.
Thus, we used UV-vis
spectrophotometry to analyze samples 22b, 1, 2, 3a, 4a and CA-4 in DMSO. The results (Figure
3) showed that compound 22b had a broad absorption peak at 375.5 nm that covered a large
range of the wavelengths in the visible region. In contrast, the absorption peaks of 1, 2, 3a, and
4
a were shifted to shorter wavelengths than 22b (299.0, 296.5, 296.5, and 294.5 nm,
respectively). These wavelength peaks were even slightly shorter than for CA-4, which is near
08.5 nm. These results were consistent with our theoretical calculations. The UV-vis absorption
3
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spectra of compounds 1, 2, 3a, and 4a did not overlap with any of the wavelengths in the visible
range. Thus, their electron transitions were difficult to excite using visible light. Consequently,
their cis-trans isomerization under natural light was markedly decreased.
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Biological Activity in vitro. We evaluated the biological activities of compounds 1, 2, 3a and 4a,
which had improved stability relative to 22b, on the HT-29 human colon cancer and SKOV3
human ovarian cancer cell line (Table 4). Compound 1 showed high inhibition of tumor cell
growth, even higher than 22b, and equivalent inhibition to CA-4 in the HT-29 cell line. However,
its enantiomer compound 2 exhibited only middling activity. These results suggested that the
chirality of the hydroxyl group had little effect on the compounds’ stability but a significant
effect on their biological activity. The S isomer was favorable with respect to biological activity.
However, compounds 3a and 4a unexpectedly showed only moderate tumor cell growth
inhibitory activity. It suggested that the change in the connection between the six-membered ring
from B ring to A ring reduced the biological activity. The evaluation of the inhibition of tubulin
polymerization showed similar results (Table 5). The docking binding mode of these compounds
(
Figure S28) could mainly explain the observed structure-activity relationships.
Table 4. The tumor cell growth inhibitory activities of the target compounds
Inhibition of tumor cell growth (IC50, μM)
Compd.
HT-29
0.001
SKOV3
CA-4
0.001
0.002
22b
0.13 ± 0.015
0.001
1
0.001
2
0.90 ± 0.083
2.02 ± 0.21
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
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3
a
a
2.46 ± 0.12
2.60 ± 0.28
0.003 ± 0.001
0.005 ± 0.001
0.16 ± 0.026
0.26 ± 0.059
0.55 ± 0.10
1.59 ± 0.21
2.56 ± 0.36
0.22 ± 0.028
0.20 ± 0.013
0.91 ± 0.085
0.79 ± 0.098
0.55 ± 0.081
>10
7.03 ± 0.59
3.78 ± 0.43
0.001
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
b
3
c
0.02 ± 0.003
0.74 ± 0.056
1.06 ± 0.11
1.44 ± 0.37
4.64 ± 0.64
0.73 ± 0.11
0.72 ± 0.12
0.60 ± 0.087
1.87 ± 0.13
1.59 ± 0.08
1.92 ± 0.078
>10
3
d
3
e
3
f
3g
3
4
h
b
4
c
4
d
4
e
4
f
4g
4
h
3.97 ± 0.54
1.34 ± 0.69
IC50 values are the mean of at least three independent determinations.
Table 5. The tubulin assembly inhibitory activities of the target compounds
Compd.
CA-4
1.77±
0.42
22b
3.93±
0.40
1
2
3a
4a
3b
IC50 ± SD
0.41±
0.08
5.7±
>10
>10
>10
(
μM)
0.60
Second round of structural optimization. We then conducted a second round of structural
optimization of compounds 3a and 4a to improve their biological activity while maintaining their
stability. According to the structure-activity relationship of CA-4-like tubulin polymerization
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inhibitors, diverse substituents were introduced into the position 3 of the B ring, the change of
7
, 8, 31, 32
which has an obvious effect on antitumor activity.
Take isomerize compounds 3b and 4b
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with fluorine substituted at position 3 for example, the results of the calculations (Table 2 and
Table 3) showed that the vertical excitation energies and the excitation wavelengths needed to
these compounds were similar to compounds 3a and 4a. They were also predicted to have similar
stability in natural light. The diverse substituted S isomer compounds 3b-3h and R isomer
compounds 4b-4h were prepared using similar synthetic routes (Scheme 2 II). The biological
activity evaluations of these compounds (Table 4) showed that the compounds 3 and 4 with
fluorine, cyan, amino and hydroxyl substituted on position 3 showed higher inhibition activities
of tumor cell growth than 3a and 4a, respectively. Furthermore, compound 3b had higher
activity than 22b and equivalent activity to CA-4, and compound 3c had higher activity than 22b
in the HT-29 cell line. The activities of tubulin polymerization of compound 3b were also
improved relative to that of 3a (Table 5). The docking binding mode of compound 3b (Figure
S28) suggested that the substituent on the position 3 of the B ring could form additional
interaction with α subunit of tubulin. The accelerated quantitative light stability testing and UV-
vis spectrophotometric analysis confirmed that the stability of compound 3b was also improved,
and its excitation wavelengths were substantially reduced relative to 22b and CA-4 (Figures 2
and 3).
Biological Activity in vivo.
Because compound 1 had a slightly higher calculated vertical excitation energy and tubulin
assembly inhibitory activity than 3b, it was selected to further determine the in vivo antitumor
activitiy in the human colon cancer HT-29 xenograft model. CA-4 and cyclophosphamide (CTX)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
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5
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5
5
5
5
5
5
5
6
were chosen as the reference and positive control. The doses of 50, 150 mg/kg for compound 1
and 50 mg/kg for CA-4 were chosen. As shown in Figure 4 and Table 6, obviously and dose-
dependently antitumor effects were observed in mice treated with compound 1 on day 18, which
was administered intraperitoneally (ip) once a day. At the dose of 150 mg/kg, compound 1
achieved a T/C ratio of 37%, lower than 52% of CA-4 at the dose of 50 mg/kg. However, the
loss of the body weight in animals treated with compound 1 was not obvious than that of CA-4 at
this dose. The antitumor potency of compound 1 seemed higher than the lead compound 22b,
which achieved a similar T/C ratio at the dose of 150 mg/kg with that of CA-4 at the dose of 50
0
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0
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2
3
4
5
6
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9
0
1
4
mg/kg in our early study.
Figure 4. The in vivo antitumor activitiy of compound 1. HT-29 tumor-bearing nude mice were
administered 150 mg/kg, 50mg/kg of compound 1 or 50mg/kg of CA-4 or vehicle alone
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intraperitoneally once a day. The figure shows the tumor volume recorded at the indicated days
after treatments. Data are expressed as mean ± SEM of tumor volume at each time point.
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Table 6. Inhibition of human xenograft growth in vivo by compound 1.
Tumor volume
Body weight
Body
3
weight
Dosing
(mean ± SD, cm )
(mean ± SD, g)
Mice (n)
d0/d18
RTV T/C
Vt/Vo (%)
Compd.
schedule
change
(mg/kg)
Vo (d0)
Vt (d18)
d0
d18
(mean,
g)
1
1
150×18d
50×18d
6/6
6/6
0.116±0.012 0.630±0.07**
0.112±0.01 1.0±0.09**
0.116±0.01 0.897±0.15**
5.4
8.9
37
61
52
20
/
23.3±0.95 25.1±1.06
22.3±0.93 24.6±0.63
22.6±0.96 24.3±1.06
22.8±0.98 23.4±1.08
22.8±1.01 25.3±1.04
1.8
2.3
1.7
0.6
2.5
CA-4
CTX
Control
50×18d
6/6
7.7
30×7d
6/6
0.117±0.02
0.116±0.01
0.34±0.04**
1.71±0.15
2.9
vehicle×18d
12/12
14.7
Data are expressed as mean ± SEM *P<0.05, **P<0.01 vs. control.
Conclusion
In conclusion, 22b is a potent tubulin polymerization inhibitor but with poor stability. It easily
undergoes cis-trans isomerization under natural light, which limits its applications. In this study,
we explored the molecular basis of its instability by TD-DFT calculations and conducted two
rounds of structural optimizations aided by quantum chemistry calculations. Its carbonyl was
reduced to a hydroxyl to give the S-configuration compound 1 and R-configuration compound 2.
Then the connection of its six-membered ring was changed from the B ring to the A ring to
produce compounds 3a-3h and 4a-4h.
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1
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2
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2
2
2
2
2
3
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4
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5
6
As predicted, the accelerated quantitative light-stability testing and UV-vis spectrophotometric
analysis results showed that the excitation wavelengths of our purposefully designed compounds
was reduced, and their stability under natural light was improved relative to both 22b and CA-4.
The inhibitory activity evaluation of tumor cell growth and tubulin polymerization revealed
improved results for compounds 1 and 3b relative to 22b. In addition, the potent in vivo
antitumor activitiy of compound 1 was confirmed, encouraging further study of these designed
anti-tumor compounds.
0
1
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0
Stilbenoids are natural compounds with a variety of important biological activities, but the
photoisomerization problem has been a common challenge in optimization work. Most quantum
chemistry calculation research has focused on the mechanisms of photoisomerization but has not
been concerned about optimizing these compounds to improve their stability and maintain their
biological activity. The methods we used in this study offer an important framework for future
modifications of stilbene-like molecules aided by theoretical calculations to improve their
stability.
Materials and methods
Chemistry. Melting points were measured on an electrically heated RK-Z melting point
1
13
apparatus and are uncorrected. The H nuclear magnetic resonance (NMR) and C NMR spectra
were recorded on a Bruker AVANCE III HD 500 MHz (Bruker Biospin, Switzerland) or an
AVANCE II600 spectrometer (Bruker Biospin, Switzerland), using trimethylsilyl (TMS) as an
internal standard and CDCl as the solvent. Chemical shifts are given in ppm (δ), and the spectral
3
data are consistent with the assigned structures. The mass spectra were recorded on a Micromass
Qtof-Micro LC−MS or a Bruker MicroToF II ESI LC-MS. Silica gel column chromatography
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was performed with silica gel 200-300 mesh. All of the solvents and reagents were analytically
pure and, when necessary, were purified and dried using standard protocols. All of the starting
materials were commercially available unless otherwise indicated. Yields of purified products
were not optimized. The purity of each key compound (>95%) was determined on an Agilent
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1100 series liquid chromatography (LC) system (column, ZORBAX Eclipse XDB-C18; mobile
phase, methanol (70%)/H O; UV wavelength, absorbance at 254 nm). The chiral purity of each
2
key compound (>90%) was determined on an Agilent 1100 series LC system (column,
CHIRALPAK AD-H; mobile phase, isopropanol (25%)/n-hexane; UV wavelength, absorbance
at 254 nm). All of the UV-vis spectroscopic work was carried out on a UV-vis
spectrophotometer (SHIMADAZU UV-2600, Japan). The compounds 22b and 5 were
14, 27, 28
synthesized by the routes we reported previously.
General procedure for the synthesis of 1, 3a−3c and 3e−3h. A solution of 22b or 5 (0.71
mmol) in tetrahydrofuran (THF) (1 mL) was added dropwise slowly at -20 °C under a nitrogen
atmosphere to a mixture of 0.14 mL (0.14 mmol) of a 1.0 M toluene solution of (R)-(+)-2-
methyl-CBS-oxazaborolidine and 0.7 mL (0.70 mmol) of a borane-methyl sulfide complex of a
2
1
.0 M toluene solution in anhydrous THF (3 mL). The reaction mixture was stirred at -20 °C for
h and was quenched by the addition of 4 mL of methanol followed by 20 mL of saturated
sodium bicarbonate. The mixture was extracted with ethyl acetate (2×100 mL). The combined
organic phase was washed with brine, dried over Na SO , filtered, and concentrated in
2
4
vacuo. The residue crude was purified by flash column chromatography (gradient elution:
petroleum ether/ethyl acetate, 0−80%) to obtain the pure product.
General procedure for the synthesis of 1, 3a−3c and 3e−3h. A solution of 22b or 5 (0.70
mmol, 1.0 equiv) in tetrahydrofuran (THF) (1 mL) was added dropwise slowly at -20 °C under a
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1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
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4
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5
6
nitrogen atmosphere to a mixture of 0.14 mL (0.14 mmol, 0.2 equiv) of a 1.0 M toluene solution
of (R)-(+)-2-Methyl-CBS-oxazaborolidine and 0.7 mL (0.70 mmol, 1.0 equiv) of a borane-
methyl sulfide complex of a 2.0 M toluene solution in anhydrous THF (3 mL). The reaction
mixture was stirred at -20 °C for 1 h and was quenched by the addition of 4 mL of methanol
followed by 20 mL of saturated sodium bicarbonate. The mixture was extracted with ethyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2 4
acetate (2×100 mL). The combined organic phase was washed with brine, dried over Na SO ,
filtered, and concentrated in vacuo. The residue crude was purified by flash column
chromatography (gradient elution: petroleum ether/ethyl acetate, 0−80%) to obtain the pure
product.
(S,E)-6-Methoxy-1-(3,4,5-trimethoxybenzylidene)-1,2,3,4-tetrahydronaphthalen-2-ol
(1).
White needles from hexane/ethyl acetate, yield 93%, mp 125−126 °C; High-performance LC
1
(
HPLC): 99.05%, ee: 98.53%, tR: 11.4 min (major), 13.3 min (minor); H NMR (500 MHz,
CDCl
3
) δ 7.26 (d, J = 8.7 Hz, 1H), 6.66 (d, J = 2.5 Hz, 1H), 6.53 – 6.52 (m, 3H), 6.50 (dd, J =
8
2
1
5
.8, 2.7 Hz, 1H), 4.52 (dd, J = 6.3, 3.3 Hz, 1H), 3.85 (s, 3H), 3.76 (s, 3H), 3.71 (s, 6H), 3.14 –
13
.82 (m, 2H), 2.23 – 2.05 (m, 2H), 1.74 (b, 1H); C NMR (125 MHz, CDCl
3
) δ 159.23, 153.07,
39.38, 139.06, 137.13, 133.29, 131.45, 124.64, 122.78, 112.83, 111.59, 106.32, 72.73, 60.96,
+
+
25 5
6.06, 55.19, 31.50, 26.26; HRMS (ES+) m/z found 357.1698 (M+H ), while C21H O (M+H )
requires 357.1697.
S,E)-5,6,7-Trimethoxy-1-(4-methoxybenzylidene)-1,2,3,4-tetrahydronaphthalen-2-ol (3a).
(
White solid, yield 83%, mp 66−67°C; HPLC: 99.15%, ee: 90.32%, tR: 9.4 min (major), 12.7 min
1
(
minor); H NMR (600 MHz, CDCl
3
) δ 7.26 (dd, J = 8.9, 0.7 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H),
6
.65 (s, 1H), 6.63 (s, 1H), 4.53 (dd, J = 5.9, 3.0 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 3.81 (s, 3H),
1
3
3
3.37 (s, 3H), 2.98 -2.84 (m, 2H), 2.19 – 2.09 (m, 2H), 1.69 (b, 1H); C NMR (125 MHz, CDCl )
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δ 158.55, 150.86, 150.58, 141.71, 138.41, 130.50, 130.30, 127.52, 123.99, 123.40, 113.73,
+
108.90, 72.45, 60.75, 60.31, 55.29, 31.14, 20.03; HRMS (ES+) m/z found 357.1690 (M+H ),
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
while C21
25 5
H O (M+H ) requires 357.1697.
(
S,E)-1-(3-Fluoro-4-methoxybenzylidene)-5,6,7-trimethoxy-1,2,3,4-tetrahydronaphthalen-2-
ol (3b). White solid, yield 86%, mp 106−107 °C; HPLC: 99.49%, ee: 99.40%, tR: 6.4 min
1
(major), 8.1 min (minor); H NMR (600 MHz, CDCl ) δ 7.06 (dd, J = 12.5, 2.0 Hz, 1H), 7.02 –
3
6
3
.98 (m, 1H), 6.85 (t, J = 8.6 Hz, 1H), 6.57 (s, 2H), 4.49 (dd, J = 6.7, 3.0 Hz, 1H), 3.88 (s, 3H),
.87 (s, 3H), 3.86 (s, 3H), 3.38 (s, 3H), 2.99 – 2.79 (m, 2H), 2.19 – 2.03 (m, 2H), 1.68 (b, 1H);
1
3
3
C NMR (150 MHz, CDCl ) δ 152.95, 151.32, 150.96, 150.75, 146.52, 146.45, 142.04, 139.61,
1
31.09, 131.04, 127.04, 125.44, 124.16, 121.94, 116.87, 116.75, 113.23, 108.83, 72.32, 60.79,
+
6
0.34, 56.35, 55.43, 31.23, 20.13; HRMS (ES+) m/z found 375.1600 (M+H ), while C21
H
24FO
5
+
(M+H ) requires 375.1603.
(
S,E)-5-((2-Hydroxy-5,6,7-trimethoxy-3,4-dihydronaphthalen-1(2H)-ylidene)methyl)-2-
methoxybenzonitrile (3c). White powder, yield 80%, mp 150−151 °C; HPLC: 99.93%, ee:
2
D
0
1
99.14%, [α]
= -23.158 (c=0.38, MeOH), tR: 9.4 min (major), 12.3 min (minor); H NMR (600
MHz, CDCl
3
) δ 7.52 (d, J = 2.1 Hz, 1H), 7.46 (dd, J = 8.7, 2.2 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H),
6
.55 (s, 1H), 6.43 (s, 1H), 4.49 (dd, J = 7.4, 2.8 Hz, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.87 (s, 3H),
3.36 (s, 3H), 2.99 – 2.80 (m, 2H), 2.23 – 2.13 (m, 1H), 2.08 (td, J = 13.4, 6.8 Hz, 1H), 1.79 (b,
1
3
1
1
5
H). C NMR (150 MHz, CDCl
3
) δ 159.89, 151.13, 150.88, 142.27, 140.89, 135.51, 134.34,
30.98, 126.74, 124.39, 120.13, 116.22, 111.13, 108.55, 101.68, 72.00, 60.82, 60.35, 56.17,
+
+
5
5.50, 31.38, 20.34; HRMS (ES+) m/z found 382.1648 (M+H ), while C22H24NO (M+H )
requires 382.1649.
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(
S,E)-1-(3-Hydroxy-4-methoxybenzylidene)-5,6,7-trimethoxy-1,2,3,4-
1
tetrahydronaphthalen-2-ol (3e). White solid, yield 84%, mp 108−109 °C, HPLC: 99.72%; H
NMR (600 MHz, CDCl ) δ 6.91 (d, J = 2.0 Hz, 1H), 6.80 – 6.73 (m, 2H), 6.64 (s, 1H), 6.58 (s,
H), 5.56 (s, 1H), 4.49 (dd, J = 6.2, 3.4 Hz, 1H), 3.87 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H), 3.37 (s,
0
1
2
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4
5
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8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1
3
1
1
3
13
3
H), 2.95 – 2.80 (m, 2H), 2.17 – 2.06 (m, 2H), 1.73 (b, 1H); C NMR (150 MHz, CDCl ) δ
50.80, 150.58, 145.55, 145.37, 141.80, 138.61, 131.26, 127.31, 124.00, 123.47, 121.33, 115.31,
10.51, 109.08, 72.53, 60.78, 60.33, 56.02, 55.39, 31.03, 19.95; HRMS (ES+) m/z found
+
+
73.1645(M+H ), while C H O (M+H ) requires 373.1646.
2
1
25
6
(
S,E)-5-((2-Hydroxy-5,6,7-trimethoxy-3,4-dihydronaphthalen-1(2H)-ylidene)methyl)-2-
methoxybenzamide (3f). White powder, yield 83%, mp 62−63 °C; HPLC: 99.94%, ee: 99.17%,
1
tR: 14.1 min (major), 23.9 min (minor); H NMR (600 MHz, CDCl
3
) δ 8.17 (d, J = 2.2 Hz, 1H),
7
5
.68 (s, 1H), 7.40 (dd, J = 8.6, 2.2 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.63 (s, 1H), 6.52 (s, 1H),
.87 (s, 1H), 4.51 (dd, J = 6.5, 2.8 Hz, 1H), 3.95 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 3.00 – 2.79
13
(m, 2H), 2.19 – 2.07 (m, 2H), 1.85 (b, 1H); C NMR (150 MHz, CDCl
3
) δ 166.82, 156.60,
50.95, 150.63, 141.92, 139.73, 134.29, 133.51, 131.16, 127.25, 124.26, 122.02, 120.64, 111.21,
08.83, 72.22, 60.78, 60.32, 56.13, 55.39, 31.10, 20.15; HRMS (ES+) m/z found 400.1757
1
1
+
+
6
(M+H ), while C22H26NO (M+H ) requires 400.1755.
(S,E)-5,6,7-Trimethoxy-1-(4-methoxy-3-nitrobenzylidene)-1,2,3,4-tetrahydronaphthalen-2-
ol (3g). Light yellow solid, yield 81%, mp 106−107 °C; HPLC: 98.81%, ee: 94.25%, tR: 10.0
1
min (major), 12.2 min (minor); H NMR (600 MHz, CDCl ) δ 7.80 (d, J = 1.5 Hz, 1H), 7.46 (dd,
3
J = 8.6, 1.7 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.57 (s, 1H), 6.49 (s, 1H), 4.50 (dd, J = 7.7, 2.7 Hz,
1
H), 3.94 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H), 3.39 (s, 3H), 2.99 – 2.82 (m, 2H), 2.22 – 2.15 (m,
1
3
1
3
H), 2.08 (td, J = 13.4, 6.8 Hz, 1H); C NMR (150 MHz, CDCl ) δ 151.41, 151.16, 150.96,
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142.36, 141.29, 139.61, 135.06, 130.54, 126.66, 126.13, 124.41, 119.83, 113.18, 108.43, 72.02,
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1
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5
5
5
5
5
6
+
6
0.83, 60.36, 56.62, 55.55, 31.41, 20.38; HRMS (ES+) m/z found 402.1541 (M+H ), while
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
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0
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8
9
0
1
2
3
4
5
6
7
8
9
0
+
C
21
H
24NO
7
(M+H ) requires 402.1548.
(S,E)-5,6,7-Trimethoxy-1-(4-methoxy-3-methylbenzylidene)-1,2,3,4-tetrahydronaphthalen-
2
-ol (3h). White solid, yield 77%, mp 54−55 °C; HPLC: 98.65%, ee: 97.42%, tR: 5.7 min
1
(
major), 6.8 min (minor); H NMR (600 MHz, CDCl
3
) δ 7.12 – 7.09 (m, 2H), 6.73 – 6.69 (m,
1H), 6.67 (s, 1H), 6.60 (s, 1H), 4.50 (t, J = 4.5 Hz, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.81 (s, 3H),
1
3
3
.34 (s, 3H), 2.96 – 2.77 (m, 2H), 2.14 (s, 3H), 2.14 – 2.09 (m, 2H), 1.60 (b, 1H); C NMR (150
MHz, CDCl ) δ 156.79, 150.81, 150.52, 141.67, 137.81, 131.50, 129.69, 127.82, 127.50, 126.29,
3
123.97, 109.73, 108.89, 72.68, 60.78, 60.33, 55.38, 55.30, 30.98, 19.89, 16.08; HRMS (ES+)
+
+
27 5
m/z found 371.1854 (M+H ), while C22H O (M+H ) requires 371.1853.
General procedure for the synthesis of 2, 4a−4c and 4e−4h. The compounds were synthesized
using CBS reduction conditions as previously described in the synthesis of 1, 3a−3c and 3e−3h,
in which a mixture of 22b or 5 (0.70 mmol, 1.0 equiv) in THF (1 mL), 0.14 mL (0.14 mmol, 0.2
equiv) of a 1.0 M toluene solution of (S)-(-)-2-Methyl-CBS-oxazaborolidine and 0.7 mL (0.70
mmol, 1.0 equiv) of a borane-methyl sulfide complex of a 2.0 M toluene solution in anhydrous
THF (3 mL) was used.
(
R,E)-6-Methoxy-1-(3,4,5-trimethoxybenzylidene)-1,2,3,4-tetrahydronaphthalen-2-ol
(2).
White needles from hexane/ethyl acetate, yield 87%, mp 123−124 °C; HPLC: 99.30%, ee:
1
9
5.76%, tR: 13.3 min (major), 11.4 min (minor); H NMR (500 MHz, CDCl ) δ 7.27 (d, J = 8.7
3
Hz, 1H), 6.66 (d, J = 2.5 Hz, 1H), 6.54 – 6.53 (m, 3H), 6.50 (dd, J = 8.8, 2.7 Hz, 1H), 4.53 (dd, J
= 6.3, 3.3 Hz, 1H), 3.85 (s, 3H), 3.77 (s, 3H), 3.71 (s, 6H), 3.15 – 2.85 (m, 2H), 2.23 – 2.06 (m,
1
3
2
H), 1.72 (b, 1H); C NMR (125 MHz, CDCl ) δ 159.23, 153.07, 139.38, 139.06, 137.13,
3
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33.29, 131.46, 124.64, 122.79, 112.82, 111.59, 106.32, 72.73, 60.96, 56.06, 55.18, 31.49, 26.26;
+
+
24 5
HRMS (ES+) m/z found 379.1518 (M+Na ), while C21H O Na (M+Na ) requires 379.1516. X-
0
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ray quality crystals were obtained via slow evaporation of an ethyl acetate solution in air.
Crystallographic data for 2 (Figure S1 and Table S1) have been deposited in the Cambridge
Crystallographic Data Centre as supplementary publication number CCDC 1032584.
(R,E)-5,6,7-Trimethoxy-1-(4-methoxybenzylidene)-1,2,3,4-tetrahydronaphthalen-2-ol (4a).
White solid, yield 80%, mp 61−62°C; HPLC: 99.00%, ee: 98.82%, tR: 12.7 min (major), 9.4 min
1
(
minor); H NMR (600 MHz, CDCl
3
) δ 7.27 (s, 1H), 7.25 (s, 1H), 6.83 (s, 1H), 6.82 (s, 1H), 6.65
(s, 1H), 6.63 (s, 1H), 4.53 (dd, J = 6.0, 3.0 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 3.81 (s, 3H), 3.37
1
3
(
s, 3H), 2.99 – 2.83 (m, 2H), 2.20 – 2.10 (m, 2H), 1.70 (b, 1H); C NMR (125 MHz, CDCl
3
) δ
158.58, 150.87, 150.61, 141.73, 138.37, 130.51, 130.28, 127.47, 123.98, 123.49, 113.74, 108.91,
+
72.53, 60.76, 60.33, 55.30, 31.12, 19.98; HRMS (ES+) m/z found 357.1691 (M+H ), while
+
C
21
H
25
O
5
(M+H ) requires 357.1697.
(
R,E)-1-(3-Fluoro-4-methoxybenzylidene)-5,6,7-trimethoxy-1,2,3,4-tetrahydronaphthalen-
2-ol (4b). White solid, yield 83%, mp 107−108 °C; HPLC: 98.56%, ee: 99.38%, tR: 8.1 min
1
(
major), 6.4 min (minor); H NMR (600 MHz, CDCl
3
) δ7.06 (dd, J = 12.5, 2.1 Hz, 1H), 7.02 –
7
.04 (m, 1H), 6.85 (t, J = 8.6 Hz, 1H), 6.57 (s, 2H), 4.49 (dd, J = 7.0, 2.8 Hz, 1H), 3.88 (s, 3H),
3.87 (s, 3H), 3.86 (s, 3H), 3.38 (s, 3H), 2.98 – 2.78 (m, 2H), 2.20 – 2.03 (m, 2H), 1.69 (b, 1H);
1
3
3
C NMR (150 MHz, CDCl ) δ 152.95, 151.32, 150.96, 150.74, 146.51, 146.44, 142.03, 139.61,
131.09, 131.05, 127.05, 125.44, 124.17, 121.94, 116.87, 116.75, 113.23, 108.83, 72.31, 60.79,
+
60.34, 56.35, 55.43, 31.23, 20.14; HRMS (ES+) m/z found 375.1592 (M+H ), while C21
H
24FO
5
+
(
M+H ) requires 375.1603.
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(
R,E)-5-((2-Hydroxy-5,6,7-trimethoxy-3,4-dihydronaphthalen-1(2H)-ylidene)methyl)-2-
7
8
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1
1
1
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6
methoxybenzonitrile (4c). White powder, yield 79%, mp 151−152 °C; HPLC: 98.85%, ee:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
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8
9
0
2
D
0
1
98.99%, [α]
= 20.583 (c=0.37, MeOH), tR: 12.3 min (major), 9.3 min (minor); H NMR (600
MHz, CDCl
3
) δ7.52 (d, J = 2.1 Hz, 1H), 7.46 (ddd, J = 8.7, 2.2, 0.6 Hz, 1H), 6.87 (d, J = 8.8 Hz,
1H), 6.55 (s, 1H), 6.43 (s, 1H), 4.49 (dd, J = 7.3, 2.5 Hz, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.87 (s,
1
3
3
H), 3.36 (s, 3H), 2.99 – 2.80 (m, 2H), 2.23 – 2.02 (m, 2H), 1.76 (b, 1H); C NMR (150 MHz,
) δ159.88, 151.13, 150.88, 142.27, 140.90, 135.51, 134.34, 130.98, 126.74, 124.39, 120.12,
16.22, 111.13, 108.55, 101.68, 72.00, 60.82, 60.35, 56.17, 55.50, 31.38, 20.35; HRMS (ES+)
CDCl
3
1
+
+
5
m/z found 382.1631 (M+H ), while C22H24NO (M+H ) requires 382.1649.
(R,E)-1-(3-Hydroxy-4-methoxybenzylidene)-5,6,7-trimethoxy-1,2,3,4-
tetrahydronaphthalen-2-ol (4e). White solid, yield 78%, mp 51−52 °C, HPLC: 98.18%; H
NMR (600 MHz, CDCl ) δ 6.94 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.3, 2.0 Hz, 1H), 6.77 (d, J =
.3 Hz, 1H), 6.67 (s, 1H), 6.61 (s, 1H), 5.55 (s, 1H), 4.52 (s, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3.88
1
3
8
1
3
(s, 3H), 3.40 (s, 3H), 2.99 – 2.81 (m, 2H), 2.20 – 2.06 (m, 2H), 1.71 (b, 1H); C NMR (150
MHz, CDCl ) δ 150.80, 150.58, 145.56, 145.38, 141.80, 138.62, 131.27, 127.32, 124.00, 123.47,
21.33, 115.31, 110.51, 109.09, 72.53, 60.77, 60.33, 56.02, 55.39, 31.03, 19.95; HRMS (ES+)
3
1
+
+
m/z found 373.1639(M+H ), while C21
25 6
H O (M+H ) requires 373.1646.
(
R,E)-5-((2-Hydroxy-5,6,7-trimethoxy-3,4-dihydronaphthalen-1(2H)-ylidene)methyl)-2-
methoxybenzamide (4f). White powder, yield 82%, mp 61−62 °C; HPLC: 99.64%, ee: 98.38%,
1
tR: 23.8 min (major), 14.2 min (minor); H NMR (600 MHz, CDCl
3
) δ 8.17 (d, J = 2.3 Hz, 1H),
7
5
2
.68 (s, 1H), 7.40 (dd, J = 8.6, 2.3 Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H), 6.63 (s, 1H), 6.52 (s, 1H),
.84 (s, 1H), 4.51 (dd, J = 6.7, 2.6 Hz, 1H), 3.95 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 3.32 (s, 3H),
1
3
3
.98 – 2.79 (m, 2H), 2.18 – 2.06 (m, 2H), 1.73 (b, 1H); C NMR (150 MHz, CDCl ) δ 166.85,
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56.61, 150.95, 150.63, 141.92, 139.74, 134.29, 133.50, 131.16, 127.26, 124.26, 122.01, 120.64,
11.21, 108.83, 72.21, 60.78, 60.32, 56.13, 55.39, 31.11, 20.16; HRMS (ES+) m/z found
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
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2
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4
5
6
7
8
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0
+
+
6
400.1755 (M+H ), while C22H26NO (M+H ) requires 400.1755.
(R,E)-5,6,7-Trimethoxy-1-(4-methoxy-3-nitrobenzylidene)-1,2,3,4-tetrahydronaphthalen-2-
ol (4g). Light yellow solid, yield 82%, mp 104−105 °C; HPLC: 99.34%, ee: 96.74%, tR: 12.2
1
min (major), 10.0min (minor); H NMR (600 MHz, CDCl
3
) δ 7.80 (d, J = 2.2 Hz, 1H), 7.46 (dd,
J = 8.8, 2.1 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.57 (s, 1H), 6.49 (s, 1H), 4.50 (dd, J = 6.7, 3.4 Hz,
1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H), 3.39 (s, 3H), 2.99 – 2.81 (m, 2H), 2.22 – 2.15 (m,
13
1H), 2.08 (td, J = 13.4, 6.3 Hz, 1H); C NMR (150 MHz, CDCl ) δ 151.40, 151.15, 150.95,
3
142.35, 141.30, 139.59, 135.08, 130.55, 126.68, 126.13, 124.41, 119.81, 113.18, 108.43, 71.99,
+
60.83, 60.36, 56.61, 55.55, 31.40, 20.40; HRMS (ES+) m/z found 402.1541 (M+H ), while
+
C
21
H
24NO
7
(M+H ) requires 402.1548.
(
R,E)-5,6,7-Trimethoxy-1-(4-methoxy-3-methylbenzylidene)-1,2,3,4-tetrahydronaphthalen-
2-ol (4h). White solid, yield 78%, mp 72−73 °C; HPLC: 99.21%, ee: 87.58%, tR: 6.8 min
1
(
major), 5.7 min (minor); H NMR (600 MHz, CDCl
3
) δ 7.12 – 7.09 (m, 2H), 6.72 (d, J = 9.0 Hz,
1H), 6.67 (s, 1H), 6.60 (s, 1H), 4.50 (t, J = 4.7 Hz, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.81 (s, 3H),
1
3
3
.34 (s, 3H), 2.97 – 2.80 (m, 2H), 2.14 (s, 3H), 2.14 – 2.09 (m, 2H), 1.68 (b, 1H); C NMR (150
) δ 156.79, 150.81, 150.52, 141.67, 137.81, 131.50, 129.69, 127.81, 127.50, 126.29,
23.97, 109.73, 108.89, 72.68, 60.78, 60.33, 55.38, 55.30, 30.98, 19.89, 16.08; HRMS (ES+)
MHz, CDCl
3
1
+
+
27 5
m/z found 371.1848 (M+H ), while C22H O (M+H ) requires 371.1853.
General procedure for the synthesis of 3d and 4d. A mixture of Fe powder (1.6 mmol, 10.0
equiv) in ethanol (75%, 10 mL) and acetic acid (2 mL) was heated to reflux for one hour then the
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compound 3g or 4g (0.16 mmol, 1.0 equiv) was added. After 30 min the mixture was filtered
through celite and the filtrate was extracted with ethyl acetate (2×50 mL). The combined organic
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phase was washed with brine, dried over Na SO , filtered, and concentrated in vacuo. The
residue crude was purified by flash column chromatography (petroleum ether/ethyl acetate=2:1)
to obtain the pure product.
(
S,E)-1-(3-Amino-4-methoxybenzylidene)-5,6,7-trimethoxy-1,2,3,4-tetrahydronaphthalen-2-
ol (3d). Light pale solid, yield 83%, mp 61−62 °C; HPLC : 98.48%, ee: 97.02%, tR: 11.8 min
1
(
(
major), 17.8 min (minor); H NMR (600 MHz, CDCl
3
) δ 6.72 (s, 1H), 6.71 (s, 1H), 6.70 – 6.65
m, 2H), 6.57 (s, 1H), 4.49 (t, J = 4.8 Hz, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.38 (s,
1
3
3H), 2.97 – 2.77 (m, 2H), 2.16 – 2.05 (m, 2H); C NMR (150 MHz, CDCl
3
) δ 150.72, 150.49,
1
7
46.48, 141.61, 137.85, 135.69, 130.61, 127.50, 124.22, 123.88, 119.78, 115.66, 110.25, 109.02,
+
2.62, 60.77, 60.32, 55.58, 55.38, 30.93, 19.88; HRMS (ES+) m/z found 372.1804 (M+H ),
+
while C21
H
26NO
5
(M+H ) requires 372.1806.
(R,E)-1-(3-Amino-4-methoxybenzylidene)-5,6,7-trimethoxy-1,2,3,4-tetrahydronaphthalen-
2
-ol (4d). Light pale solid, yield 85%, mp 56−58 °C; HPLC : 98.03%, ee: 92.66%, tR: 17.6 min
1
(
(
major), 11.7 min (minor); H NMR (600 MHz, CDCl
3
) δ 6.72 (s, 1H), 6.70 – 6.66 (m, 3H), 6.57
s, 1H), 4.49 (t, J = 4.6 Hz, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.38 (s, 3H), 2.98 – 2.75
1
3
(m, 2H), 2.15 – 2.04 (m, 2H); C NMR (150 MHz, CDCl
3
) δ 150.73, 150.51, 146.44, 141.63,
1
6
37.82, 135.88, 130.60, 127.47, 124.30, 123.87, 119.66, 115.54, 110.25, 109.04, 72.68, 60.77,
+
0.32, 55.58, 55.38, 30.93, 19.85; HRMS (ES+) m/z found 372.1803 (M+H ), while C21H26NO
5
+
(M+H ) requires 372.1806.
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Quantum chemical calculation. All of the quantum computations were performed using
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Gaussian 09. First, the compounds were optimized at the ab initio B3LYP/6-31+G (d) and
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B3LYP/6-311++G (d,p) computational levels. Frequency calculations were carried out to
confirm that the obtained geometries corresponded to energetic minima. Then, TD-DFT at the
same levels was used to calculate the vertical excitation energies. The frontier orbitals of the
three molecules involved in the S0S1 transitions were visualized using GaussView 5.0.
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2
Stability test. The samples were dissolved in DMSO to a concentration of 1.5×10 M. The
sample solutions (4 mL) were irradiated using a high-power xenon lamp (Perfect Light PLS-
SXE300, China) with a visible light filter under a nitrogen atmosphere. Next, HPLC (LC-20AD,
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Shimadzu, Japan, column ZORBAX Eclipse XDB-C18; mobile phase, methanol (70%)/H O;
UV wavelength, absorbance at 254 nm) was used to measure changes in the sample and the
percentages of the various components in the mixture. For the newly produced substances, LC-
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HRMS were used to determine the samples’ structure.
Ultraviolet-visible spectrophotometric analysis. For spectrophotometric analysis, suitable
-2
dilutions from working standard solutions of the compounds in DMSO (1.0×10 M) were
-5
generated (1.4×10 M). The prepared solutions were incubated in quartz cuvettes in a UV-vis
spectrophotometer (SHIMADAZU UV-2600, Japan) and scanned over the range of 200-800 nm.
The reference used for these measurements was DMSO.
Assay for tumor cell growth inhibition. Cytotoxic effects were examined in the SKOV3 human
ovarian cancer and HT-29 human colon carcinoma cell lines by MTT assay. The absorbance at
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570 nm was recorded on a fully automatic enzyme-labeled meter (Labsystems Inc. Wellscan
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MK-2, Finland). Compounds were tested in triplicate in at least three independent assays, and the
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average median inhibitory concentration (IC50) values were reported.
Assay for Tubulin Polymerization Inhibition. Bovine brain tubulin (Cytoskeleton, Inc., USA)
>99% pure, 3 mg/mL) in 100 μL of general tubulin buffer at 0 °C was placed in a half-area 96-
(
well plate prewarmed at 37 °C in the presence of the tested compounds at varying concentrations.
The reaction was started by warming the samples to 37 °C. The mass of polymer formed was
monitored by turbidimetry at 340 nm every 1 min for 60 min with a multifunction microplate
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reader (Biotek Synergy 4, USA).
Assay for in vivo antitumor activity. BALB/c nude female mice (18−20 g) were obtained from
Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Once the HT-29 xenografts
3
reached a size of around 100 mm , mice were randomly assigned into appropriate groups (six
animals/treatment and twelve animals for the control group). The drugs or vehicle were
administered by ip injection once a day. After completing the treatment schedule and the
evaluation period, tumor bearing mice were euthanized. Tumor volume (TV) was calculated by
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the formula: TV= (ab )/2 where a is the length and b is the width of the tumor nodules
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determined once per 3 days by caliper measurements and recorded along with body weights.
ASSOCIATED CONTENT
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