Synthesis, biological evaluation and modeling studies of terphenyl topoisomerase II α inhibitors as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2015.03.010

We report the synthesis and evaluation of a series of novel terphenyls. Compound 17 had the most potent anticancer activity, indicating that the phenolic hydroxyl was a key group. A DNA relaxation test showed that compound 17 had a strong inhibitory effec

Full text of DOI:10.1016/j.ejmech.2015.03.010

Accepted Manuscript
Synthesis, Biological Evaluation and Modeling Studies of Terphenyl Topoisomerase
IIα Inhibitors as Anticancer Agents
Jin Qiu, Baobing Zhao, Wanxia Zhong, Yuemao Shen, Houwen Lin
PII:
S0223-5234(15)00169-5
10.1016/j.ejmech.2015.03.010
EJMECH 7755
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 December 2014
Revised Date: 4 March 2015
Accepted Date: 4 March 2015
Please cite this article as: J. Qiu, B. Zhao, W. Zhong, Y. Shen, H. Lin, Synthesis, Biological Evaluation
and Modeling Studies of Terphenyl Topoisomerase IIα Inhibitors as Anticancer Agents, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.03.010.
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ACCEPTED MANUSCRIPT
B
A
CoMFA contour map
CoMSIA contour map
CoMFA and CoMSIA contour maps with compound 17
provide useful insight into designing novel TOP inhibitory.

ACCEPTED MANUSCRIPT
Synthesis, Biological Evaluation and Modeling Studies of
Terphenyl Topoisomerase IIα Inhibitors as Anticancer Agents
a, §
b,§
a,§
b,*
a,* *
Jin Qiu, Baobing Zhao, Wanxia Zhong, Yuemao Shen, Houwen Lin
a
Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Pujian Road 160, Shanghai 200127, People’s
Republic of China
b
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong
University, No. 44 West Wenhua Road, Jinan, Shandong 250012, People’s Republic of China
§
These authors contributed equally to this work.
*
Corresponding author. Tel.: +86-531-88382108
* Corresponding author. Tel.: +86-021-68383346
*
E-mail addresses: yshen@sdu.edu.cn. (Yuemao Shen), franklin67@126.com (Houwen Lin
)
1

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ABSTRACT
We report the synthesis and evaluation of a series of novel terphenyls. Compound 17 had the most
potent anticancer activity, indicating that the phenolic hydroxyl was a key group. A DNA relaxation
test showed that compound 17 had a strong inhibitory effect on TOP2α, but not on TOP1, which was
consistent with the docking analysis results. We performed a 3D-QSAR study using CoMFA and
CoMSIA to determine, for the first time, the chemical-biological relationship in the inhibition of TOP
2
by terphenyls. The CoMFA and CoMSIA model had good modeling statistics: leave-one-out q of
2
2
0
.605 and 0.622, r of 0.998 and 0.994, and r
(test set) of 0.742 and 0.660. These results suggest
pred
that the ortho-phenolic hydroxyl on ring A is important for producing terphenyls with more efficacious
activity.
Keywords: terphenyls; topoisomerase; 3D-QSAR
2

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1
. Introduction
Topoisomerases (TOP) are essential for managing the topological state of DNA [1, 2]. Several
compounds may increase TOP-mediated DNA duplex breakage and kill cancer cells [3]. Different
types of compounds inhibited TOP were reported recently, such as 2-substituted amidoanthraquinones
and benzoxazole-containing derivatives inhibited TOP1 [4], fluorinated purine analogues [5] and
dihydroxylated 2,4-diphenyl-6-thiophen-2-yl pyridine derivatives inhibited TOP2 [6], hydroxylated
2
,4-diphenyl indenopyridine derivatives inhibited TOP2α [7]. Type II topoisomerases (TOP2s) are
notable antitumor targets because they can transiently cleave both strands of a DNA duplex to enable
the transport of another DNA segment [8]. TOP2α, one of the TOP2 isoforms, is highly expressed in
rapidly growing cancer cells [9, 10]. Therefore, TOP2α is considered to be an important therapeutic
target for developing TOP2α-targeting antitumor drugs [11, 12].
Recently, we discovered 8 p-terphenyl derivatives that have potent cytotoxicity against the human
breast carcinoma MDA-MB-435 cell line, mediated through ROS generation, cell cycle arrest and
apoptosis [13]. Compound [1,1’:4’,1”-Terphenyl]-2’,3,3’,4,4”-pentaol (X1, Figure 1) was found to
only inhibit TOP2α activity, but not TOP1 [13]. Based on the structure of compound X1, we
synthesized a series of novel terphenyls (Figure 1) and evaluated their cytotoxic activity against the
MDA-MB-435 cell line (Table 1). Among these compounds, compound 17 was slightly more potent
than X1 ( IC50 = 4.1 ± 0.39). As shown in Figure 2, the inhibitory activity of compound 17 against
TOP2α was slightly stronger than that of VP-16 and its inhibitory activity against TOP2α and TOP1
was consistent with DOCK analysis results (Figure 3). To obtain a detailed and quantitative view of
ligand binding, we used the comparative molecular field analysis (CoMFA) [14] and comparative
molecular similarity indices analysis (CoMSIA) [15] methods. In this paper, for the first time, we
report CoMFA and CoMSIA studies using a database of terphenyls that inhibit TOP. The QSAR
models showed high quantitative correlations with good predictive abilities.
Figure 1.
Scheme 1-4..
2
. Results
3

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2
.1. Chemistry
A series of terphenyls was synthesized as depicted in Schemes 1-4.
Scheme 1. The synthesis was started from commercially available 4-bromo-2-hydroxybenzoic
acid and 5-bromosalicylic acid. These compounds were subjected to esterification and were coupled
with 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi (1,3,2-dioxaborolane). This process resulted in compounds 21
and 22. Then, these compounds were coupled with 23 [13] to obtain compounds 1 and 2 respectively.
Using KOH to hydrolyze compounds 1 and 2 resulted in compounds 3 and 4, respectively. Compounds
5
and 6 were obtained by the demethylation of 3 and 4 using boron tribromide.
Scheme 2. 1,4-dibromo-2,3-dimethoxybenzene (compound 24) [13] was coupled with
,4-dimethoxyphenylboronic acid to produce compound 25 and compound 16, and the latter was
3
demethylated to obtain compound 17. Compound 25 was coupled with 4,4,4', 4', 5,5,5',
'-octamethyl-2,2'-bi (1,3,2-dioxaborolane), without separation, and then was directly coupled with
5
methyl 4-bromobenzoate (compound 26) to obtain compounds 7, 18 and 19. Compound 7 was
demethylated to produce compound 8.
Scheme 3. 4-bromo-2-nitrophenol and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi (1,3,2-dioxaborolane)
were coupled through a Suzuki reaction; after the reaction was finished and without further treatment, a
second reaction coupling the compound with 1,4-dibromo-2,3-dimethoxybenzene (compound 24) was
performed to obtain compounds 9 and 20.
Scheme 4. Three equivalents of commercially available boric acid compounds (compounds 27, 28
and 29) were coupled with 1 equivalent of the corresponding brominated compounds (compound 24,
3
0 and 31) to obtain compounds 10, 11, 12, 13 and 14, respectively. Compound 15 was obtained by the
demethylation of compound 12.
2
.2. Cytotoxicity evaluation of compounds 1-20 against MDA-MB-435 cells
Our previous studies had shown that the terphenyl compounds had more potent active on
MDA-MB-435 cells line than on other cell lines, so we only use this cell line in this study. The
4

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cytotoxicity of the compounds was expressed as IC , which is defined as the concentration of
50
compound that results in 50% growth inhibition. Each value represents the mean ± SD of three
independent experiments. The data are summarized in Table 1. These results demonstrate that the
phenolic hydroxyl is a key group for activity and that the activities of compounds 3, 4, 5 and 8, which
had a substituted carboxyl group, were very low.
Table 1.
2
.3. Inhibition of TOP2α
Compounds 2, 12 and 17, which had IC50 values lower than 20 µM, were evaluated for their
ability to inhibit TOP1 and TOP2α. The results are shown in Figure 2.
Figure 2.
Compound 12 had inhibitory activity against TOP2α, and compound 17 slightly inhibited TOP1
and intensely inhibited TOP2α. Additionally, the data presented in Figure 2B showed that compound
1
7 and VP-16 are almost equal active in inhibiting TOP2α activity.
2
.4. Compound 17 mechanism of action
To study the effect of compound 17 on TOP2α-mediated DNA cleavage, circular supercoiled
plasmid (SC) DNA was used as the substrate in an inhibition model assay to investigate the mode of
inhibition; this assay was used in our previous work [13]. The results indicated that compound 17 was a
TOP suppressor but not a poison (data not shown).
Figure 3.
2
.5. DOCKING
In our previous studies we found that compound X3 inhibited TOP1 and TOP2α, and we found
compound 17 only inhibited TOP2α in this study. So, in order to find the binding site of these
compounds to TOP, molecular docking was used to explore the binding of compounds X3 and 17 to
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TOP1 and TOP2α (Figure 3). The main difference was that X3 could form hydrogen bonds with the
TOP1 residues GLU-356 and ASN-722, whereas 17 could form hydrogen bonds with TOP1 residue
ASP-533 (Figure 3A). For TOP2α, compound X3 could form hydrogen bonds with residue ASP-541,
whereas 17 could form hydrogen bonds with residues ASP-541 and DG1-10 (Figure 3B). These results
were consistent with the TOP inhibition findings.
2
.6. 3D-QSAR models and statistics
3
D-QSAR is used to correlate compound activity with the interaction fields surrounding the
compounds. All compounds, based on minimum energy conformers, were aligned using a terphenyl
nucleus as a template. In CoMFA and CoMSIA, partial least-squares (PLS) regression is used to derive
and statistically validate models [16].
2
The 3D-QSAR models were evaluated based on the cross-validated correlation coefficient (q ),
2
non-cross-validated correlation coefficient (r ), standard error of estimate (SEE), F test value, and
2
evaluation of the model with a test set (r pred). The statistical results listed in Table 2 indicate that the
CoMFA and CoMSIA models built upon this set of compounds were statistically reliable.
Table 2.
2
CoMFA and CoMSIA analysis of the statistical parameters indicated that q = 0.605 and 0.622 for
2
the best main fraction of 4 and 5; typically, a representation model with q > 0.5 has good prediction
2
ability. Additionally, r = 0.998 and 0.994, SEE was 0.030 and 0.050, and F = 960.9 and 242.5.
The training set consisted of 13 compounds, and their pIC , predicted values and residual values
5
0
are presented in Table 3.
Table 3.
2
.7. External validation of CoMFA and CoMSIA models
The predictive ability of the CoMFA and CoMSIA models was assessed using a test set of 5
internal molecules (compounds 1, 2, 7, 9, and 10) and 5 external molecules (compounds C1-5, Figure
), which are shown in Table 4.
4
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Figure 4.
Table 4.
2
The predictive correlation coefficient r
(test set) was 0.742 and 0.660 for the CoMFA and
pred
CoMSIA models, respectively (Table 2). The correlation between the predicted activities and the
experimental activities is depicted in Figure 5.
Figure 5.
2
.8. Analysis of 3D-QSAR contour maps
The coefficient × standard deviation (coeff*stddev) contour maps of the 3D-QSAR models for the
CoMFA and CoMSIA models had good consistency in the electrostatic fields (Figure 6A and B). We
observed that electrostatic groups were acceptable and steric substitutions were unacceptable. These
results were similar to the DOCK results.
The coeff*stddev maps suggest regions in the model space where adding steric bulk or changing
the electrostatic charge at specific loci in the core molecule should increase (or decrease) target
activity.[14, 15] Briefly, the green and yellow contours represent favorable and unfavorable steric
substitutions, respectively, whereas the red and blue contours represent a favorable effect from more
electronegative and more electropositive substitutions, respectively.
Figure 6.
3
. Discussion
In conclusion, we present a simple and rapid protocol for terphenyl synthesis. All of the
synthesized compounds were evaluated in MDA-MB-435 cells, and compound 17 displayed the
highest cytotoxicity. The TOP2α inhibitory test and docking study of compound 17 suggested that the
phenolic hydroxyl group was important for potency. We used CoMFA and CoMSIA to describe the
quantitative chemical-biological relationship of terphenyl TOP2α inhibition for the first time. High
2
2
quantitative correlations were obtained with good results, as evidenced by the high q and r values.
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2
Additionally, the high r
value indicated high predictive power for the activities of untested
pred
compounds.
Overall, the current study investigated the anticancer activities and TOP2α inhibition activities of
novel variations of p-terphenyl derivatives. The antitumor and TOP2α inhibition activities of
compound 17, combined with the results of the DOCK and 3D-QSAR analyses, demonstrated that the
ortho-phenolic hydroxyl of ring A was the key group for compound activity. These results provide
insight into the key contributions for functional groups of these molecules, which could be used to
develop novel synthetic candidates that inhibit TOP2α.
4
. Experimental section
4
.1. Chemistry
4
.1.1. Chemical Syntheses: General Methods
All melting points were determined on a micro melting point apparatus and were uncorrected.
1
13
H-NMR and C-NMR spectra were obtained on a Brucker Avance-600 NMR spectrometer or an
Inova-600 NMR spectrometer in the indicated solvents. Chemical shifts are expressed in ppm (δ units)
relative to the TMS signal as an internal reference. TLC was performed on Silica Gel GF254 and spots
were visualized by iodine vapor or by UV light irradiation (254 nm). Flash column chromatography
was performed on a column packed with Silica Gel 60 (200-300 mesh). Solvents were reagent grade
and, when necessary, they were purified and dried using standard methods. The concentration of the
reaction solutions was performed using a rotary evaporator at reduced pressure. The purity of the
product (> 95%) was assessed by reversed-phase HPLC using an Agilent 1200 series and an analytical
C18 column using 50% MeOH as solvent system and a flow rate of 1 mL/min with detection
wavelength at 254 nm.
4
.1.2. General Parallel Procedure a (Scheme 1-4.)
The appropriate bromo derivatives (1 eq.), appropriate phenyl boronic acids (1.5 eq.) and
KFꢀ2H O (3.0 eq.) were dissolved in dioxane and the three resulting mixtures were deoxygenated with
2
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a stream of N . After 10 min, PdCl (dppf) (0.05 eq.) was added, and each mixture was brought to reflux
2
2
and stirred under N for 5–22 h until the reaction was complete, followed by detection using TLC.
2
Then, each solution was cooled to room temperature. Next, each solution was poured into a mixture of
H O and ethyl acetate, and the two phases were separated. The aqueous layer was washed with ethyl
2
acetate, and the organic phases were combined and washed with brine. The ethyl acetate layer was
dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. Each crude
product was purified via chromatography or Sephadex LH-20.
4
.1.3. General Parallel Procedure b (Scheme 1-4.)
The corresponding compounds (1 eq.) were added to CH Cl . Then, BBr (3 eq.) was added to
2
2
3
each solution and the resulting reaction mixtures were allowed to warm to room temperature for 20 h
and treated as follows: Each solution was poured into ice water, followed by warming to ambient
temperature, after which each solution was washed twice with ethyl acetate. The combined organic
layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure.[17]
The purification of each crude product yielded the corresponding derivatives.
4
.1.4. General Parallel Procedure c (Scheme 1-4.)
The corresponding compounds (1 eq.) were added to 50% MeOH. Then, KOH (1.1 eq.) was added
to each solution and refluxed for 1 h. Then, each mixture was cooled to room temperature and treated
using standard methods.
4
.1.5. General Parallel Procedure d (Scheme 1-4.)
The appropriate bromo derivatives (1 eq.) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi
(
1,3,2-dioxaborolane) (1.1 eq.) and KAc (3.0 eq.) were dissolved in dioxane, and the three resulting
mixtures were deoxygenated under a stream of N . After 10 min, PdCl (dppf) (0.05 eq.) was added, and
2
2
each mixture was brought to 60-80°C and stirred under N for 2–4 h until the reaction was complete,
2
followed by detection using TLC. After the reaction was finished, without further treatment the crude
product was subjected to a second coupling reaction.
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4
.1.6. General Parallel Procedure e (Scheme 1-4.)
The appropriate bromo derivatives (1 eq.), appropriate phenyl boronic acids (3 eq.) and KF (3.0
eq.) were respectively dissolved in dioxane, and the three resulting mixtures were deoxygenated under
a stream of N . After 10 min, PdCl (dppf) (0.05 eq.) was added, and each mixture was brought to reflux
2
2
and was stirred under N for 5–22 h until the reaction was complete, followed by detection using TLC.
2
Then, each solution was cooled to room temperature. Next, each solution was poured into a mixture of
H O and ethyl acetate and the two phases were separated. The aqueous layer was washed with ethyl
2
acetate, and the organic phases were combined and then washed with brine. The ethyl acetate layer was
dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. Each crude
product was purified by chromatography using petroleum ether/ethyl acetate, which yielded the
corresponding derivatives.
4
.1.7. 4,4’’-dihydroxy-2’,3’-dimethoxy [1,1’:4’,1’’-terphenyl]-3-carboxylic acid
methyl ester (1)
The resulting residue was purified by silica gel chromatography (10/1 PET/EtOAc) to afford the
1
desired product as a white solid (55.6 %): mp 178-180 °C; H NMR (600 MHz, (CD ) CO, rt) δ 10.80
3
2
(
s, 1H, OH), 8.43 (s, 1H, OH), 8.08 (s, 1H, H2) 7.79 (d, J = 8.4 Hz, 1H, H6) 7.46 (d, J = 7.2 Hz, 2H,
H2”, H6”), 7.16(s, 2H, H5’, H6’), 7.07 (d, J = 8.4 Hz, 1H, H5) 6.94 (d, J = 7.8 Hz, 2H, H3”, H5”), 4.01
13
(
s, 3H, COOCH ), 3.70 (s, 3H, OCH ), 3.67 (s, 3H, OCH ) C NMR (150 MHz, (CD ) CO, rt) δ
3 3 3 3 2
1
1
1
70.5 (CO), 160.7 (C4), 156.9 (C4”), 151.07 (C2’), 151.06 (C3’),136.7 (C6), 135.5 (C2), 133.3 (C1”),
30.2 (C2”), 130.2 (C6”), 130.1 (C1), 129.3 (C1’), 129.1 (C4’), 125.3 (C6’), 124.9 (C5’), 117.2 (C5),
15.1 (C3”), 115.1 (C5”), 112.1 (C3), 60.0 (COOCH ), 59.8 (OCH ), 52.1 (OCH ); HRMS (ESI) m/z
3
3
3
+
[
M+H] Calcd. for C H O : 381.1338, Found: 381.1359.
22 21 6
4
.1.8. 3,4’’-dihydroxy-2’,3’-dimethoxy [1,1’:4’,1’’-terphenyl]-4- carboxylic acid
methyl ester (2)
The resulting residue was purified by silica gel chromatography (10/1 PET/EtOAc) to afford the
1
desired product as a white solid (62.1 %): mp 140-142 °C; H NMR (600 MHz, (CD ) CO, rt) δ 10.79
3
2
(
s, 1H, OH), 8.45 (s, 1H, OH), 7.91 (d, J = 8.4 Hz, 1H, H2) 7.47 (d, J = 8.4 Hz, 2H, H2”, H6”),
1
0

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7
.18~7.21 (brs, 4H, H5, H5’, H6, H6’), 6.95 (d, J = 8.4 Hz, 2H, H3”, H5”) 4.01 (s, 3H, COOCH ), 3.73
3
1
3
(
s, 3H, OCH ), 3.67 (s, 3H, OCH ). C NMR (150 MHz, (CD ) CO, rt) δ 170.4 (CO), 161.3 (C3),
3 3 3 2
1
1
1
57.0 (C4”), 151.3 (C2’), 151.1 (C3’), 146.0 (C1), 136.5 (C1”), 133.2 (C1’), 130.2 (C2”), 130.2 (C6”),
29.5 (C5), 128.9 (C4’), 125.3 (C6’), 125.1 (C5’), 120.4 (C6), 117.6 (C2), 115.1 (C3”), 115.1 (C5”),
+
10.9 (C4), 60.3 (COOCH ), 59.8 (OCH ), 52.0 (OCH ) ; HRMS (ESI) m/z [M+H] Calcd. for
3
3
3
C H O : 381.1338, Found: 381.1334.
2
2
21
6
4
.1.9. 4,4’’-dihydroxy-2’,3’-dimethoxy [1,1’:4’,1’’-terphenyl]- 3-carboxylic acid (3)
The resulting residue was purified by sephadex LH-20 chromatography (1/1 MeOH/Acetone) to
1
afford the desired product as a grey white solid(58.4 %): mp 250-253 °C; H NMR (600 MHz,
(
CD ) CO, rt) δ 11.18 (s, 1H, OH), 8.50 (s, 1H, OH), 8.15 (s, 1H, H2), 7.80 (d, J = 9 Hz, 1H, H6),
3 2
7
.47(d, J = 7.8 Hz, 2H, H2”, H6”), 7.19 (d, J = 9.6 Hz, 1H, H5’), 7.17 (d, J = 9.6 Hz, 1H, H6’), 7.07(d,
13
J = 9 Hz, 1H, H5), 6.95 (d, J = 7.2 Hz, 2H, H3”, H5”), 3.72 (s, 3H, OCH), 3.68 (s, 3H, OCH);
C
NMR (150 MHz, (CD ) CO, rt) δ 171.8 (CO), 161.3 (C4), 156.8 (C4”), 151.1 (C2’), 151.1 (C3’), 136.8
3
2
(
(
C6), 135.4 (C1”), 133.3 (C1), 130.7 (C2), 130.2 (C2”), 130.2 (C6”), 129.2 (C4’), 129.1 (C1’), 125.3
C5’), 124.9 (C6’), 117.1 (C5), 115.0 (C3”), 115.0 (C5”), 112.0 (C3), 60.0 (OCH ), 59.8 (OCH );
3
3
+
HRMS (ESI) m/z [M+H] Calcd. for C H O : 367.1182, Found: 367.1180.
2
1
19
6
4
.1.10. 3,4’’-dihydroxy-2’,3’-dimethoxy [1,1’:4’,1’’-terphenyl]- 4-carboxylic acid (4)
The resulting residue was purified by sephadex LH-20 chromatography (1/1 MeOH/Acetone) to
1
afford the desired product as a grey white solid (76.3 %): mp 244-247 °C; H NMR (600 MHz,
(
CD ) CO, rt) δ 7.97 (d, J = 7.8 Hz, 1H, H6), 7.48 (d, J = 7.8 Hz, 2H, H2”, H6”), 7.19~7.22 (m, 4H,
3 2
13
H2, H3’, H5, H5’), 6.95 (d, J = 7.2 Hz, 2H, H3”, H5”), 3.74 (s, 3H, OCH), 3.68 (s, 3H, OCH);
C
NMR (150 MHz, (CD ) CO, rt) δ 171.6 (CO), 161.7 (C3), 156.9 (C4”), 151.3 (C3’), 151.1 (C2’), 146.0
3
2
(
(
C1), 136.4 (C1”), 133.2 (C4’), 130.2 (C2”), 130.2 (C6”), 130.0 (C5), 128.9 (C1’), 125.3 (C5’), 125.1
-
C6’), 120.3 (C6), 117.5 (C2), 115.0 (C3”), 115.0 (C5”), 110.8 (C4); HRMS (ESI) m/z [M-H] Calcd.
for C H O : 365.1025, Found: 365.1029.
2
1
17
6
4
.1.11. 2’,3’,4,4’’-tetrahydroxy [1,1’:4’,1’’-terphenyl]-3-carboxylic acid (5)
1
1

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The reaction maxture was poured into ice water, and the resulting precipitate was filtered to afford
1
the desired product as a grey white solid (63.1%): mp > 260 °C; H NMR (600 MHz, (CD ) CO, rt) δ
3
2
8
1
.20 (s, 1H, H2), 7.83 (d, J = 8.4 Hz, 1H, H6), 7.46 (d, J = 7.8 Hz, 2H, H2”, H6”), 7.05 (d, J = 8.4 Hz,
13
H, H5), 6.91~6.93 (3H, H3”, H5”, H6’) 6.88 (d, J = 7.8 Hz, 1H, H5’); C NMR (150 MHz,
(
CD ) CO, rt) δ 171.7 (CO), 160.9 (C4), 156.5 (C4”), 142.7 (C2’), 142.2 (C3’), 136.9 (C6), 130.7 (C2),
3 2
1
1
3
30.3 (C2”), 130.3 (C6”), 129.6 (C1”), 129.3 (C1), 128.2 (C4’), 126.1 (C1’), 121.2 (C5’), 120.9 (C6’),
-
17.0 (C5), 115.1 (C3”), 115.1 (C5”), 111.9 (C3); HRMS (ESI) m/z [M-H] Calcd. for C H O :
19
13
6
37.0712, Found: 337.0704.
4
.1.12. 2’,3,3’,4’’-tetrahydroxy [1,1’:4’,1’’-terphenyl]-4-carboxylic acid (6)
The resulting residue was purified by sephadex LH-20 chromatography (1/1 MeOH/Acetone) to
1
afford the desired product as a grey white solid (85.7 %): mp > 260 °C; H NMR (600 MHz,
(
CD ) CO, rt) δ 7.95 (d, J = 8.4 Hz, 1H, H6), 7.46 (d, J = 7.8 Hz, 2H, H2”, H6”), 7.28 (s, 1H, H2), 7.25
3 2
(
d, J = 8.4 Hz, 1H, H5), 6.98 (d, J = 8.4 Hz, 1H, H5’), 6.93 (d, J = 7.8 Hz, 2H, H3”, H5”), 6.89 (d, J =
1
3
8
.4 Hz, 1H, H6’); C NMR (150 MHz, (CD ) CO, rt) δ 171.7 (CO), 161.9 (C3), 156.8 (C4”), 146.5
3 2
(
(
(
C1), 143.2 (C3’), 142.4 (C2’), 130.3 (C2”), 130.3 (C6”), 130.1 (C5), 129.3 (C4’), 129.1 (C1’), 125.9
C1”), 121.2 (C5’), 121.0 (C6’), 120.2 (C6), 117.5 (C2), 115.2 (C3”), 115.2 (C5”), 110.5 (C4); HRMS
-
ESI) m/z [M-H] Calcd. for C H O : 337.0712, Found: 337.0713.
1
9
13
6
4
.1.13. 2’,3,3’,4-tetramethoxy[1,1’:4’,1’’-terphenyl] -4’’-carboxylic acid (7)
The resulting residue was purified by silica gel chromatography (10/1 PET/EtOAc) to afford the
1
desired product as a white solid (21.1 %): mp 241-243 °C; H NMR (600 MHz, DMSO-d6, rt) δ 13.00
(
s, 1H, COOH), 8.04 (d, J = 8.4 Hz, 2H, H2”, H6”), 7.68 (d, J = 8.4 Hz, 2H, H3”, H5”) 7.24 (d, J = 8.4
Hz, 1H, H6’), 7.21 (d, J = 7.8 Hz, 1H, H5’), 7.16 (s, 1H, H2), 7.12 (d, J = 8.4 Hz, 1H, H6), 7.06 (d, J =
13
8
.4 Hz, 1H, H5), 3.82 (s, 6H, OCH ), 3.66 (s, 3H, OCH ), 3.65 (s, 3H, OCH ); C NMR (150 MHz,
3 3 3
DMSO-d6, rt) δ 167.7 (CO), 151.2 (C3), 151.1 (C4), 148.8 (C2’), 148.75 (C6’), 142.5 (C1”), 136.0
(
(
C1), 133.9 (C4”), 130.2 (C1’), 129.9 (C4’), 129.8 (C2”), 129.8 (C6”), 129.6 (C3”), 129.6 (C5”), 125.9
C6), 125.7 (C6’), 121.6 (C5’), 113.1 (C2), 112.1 (C5), 61.1 (OCH ), 60.9 (OCH ), 56.0 (OCH ), 56.0
3
3
3
1
2

ACCEPTED MANUSCRIPT
+
(
OCH ); HRMS (ESI) m/z [M+H] Calcd. for C H O : Calcd. for C H O : 395.1495, Found:
3
23 23
6
23 23
6
3
95.1485.
4
.1.14. 2’,3,3’,4-tetrahydroxy [1,1’:4’,1’’-terphenyl] -4’’-carboxylic acid (8)
The resulting residue was purified by sephadex LH-20 chromatography (1/1 MeOH/Acetone) to
1
afford the desired product as a white solid (73.3 %): mp > 260 °C; H NMR (600 MHz, CD OD, rt) δ
3
8
7
.08 (d, J = 7.2 Hz, 2H, H2”, H6”), 7.75 (d, J = 7.8 Hz, 2H, H3”, H5”), 7.09 (s, 1H, H2), 6.95 (d, J =
.8 Hz, 1H, H6), 6.90 (d, J = 7.8 Hz, 1H, H5’), 6.86 (d, J = 7.2 Hz, 1H, H6’), 6.85 (d, J = 6.6 Hz, 1H,
13
H5); C NMR (150 MHz, CD OD, rt) δ 168.6 (CO), 144.7 (C4), 144.4 (C3), 143.8 (C3’), 143.2
3
(
(
[
C2’), 142.3 (C1”), 130.0 (C1), 129.4 (C4”), 129.2 (C2”), 129.2 (C6”), 128.8 (C3”), 128.8 (C5”), 128.4
C4’), 126.2 (C1’), 121.0 (C5’), 120.9 (C6’), 120.4 (C6), 116.0 (C2), 114.9 (C5); HRMS (ESI) m/z
-
M-H] Calcd. for C H O : 337.0712, Found: 337.0718.
1
9
13
6
4
.1.15. 3,3’’-dinitro-2’,3’-dimethoxy -4,4’’-dihydroxy [1,1’:4’,1’’-terphenyl] (9)
The resulting residue was purified by silica gel chromatography (10/1 PET/EtOAc) to afford the
1
desired product as a grey yellow solid (43.6 %): mp 175-177 °C; H NMR (600 MHz, CDCl , rt) δ
3
1
0.67 (s, 2H, OH), 8.36 (s, 2H, H2, H2”), 7.88 (d, J = 8.4 Hz, 2H, H6, H6”), 7.26 (d, J = 9 Hz, 2H, H5,
1
3
H5”), 7.19 (s, 2H, H5’, H6’); C NMR (150 MHz, CDCl , rt) δ 154.4 (C4), 154.4 (C4”), 151.1 (C2’),
3
1
1
51.1 (C3’), 138.7 (C6), 138.7 (C6”), 133.5 (C3), 133.5 (C3”), 133.4 (C1), 133.4 (C1”), 130.1 (C1’),
30.1 (C4’), 125.2 (C2), 125.2 (C2”), 125.1 (C5’), 125.1 (C6’), 119.8 (C5), 119.8 (C5”); HRMS (ESI)
-
m/z [M-H] Calcd. for C H N O : 411.0828, Found: 411.0832.
2
0
15
2
8
4
.1.16. 4,4’’-dihydroxy-2’,3’-diethoxy [1,1’:4’,1’’-terphenyl] (10)
The resulting residue was purified by silica gel chromatography (5/1 PET/EtOAc) to afford the
product as a white solid then was purified by sephadex LH-20 chromatography (MeOH) to afford the
1
desired product as a white solid (64.2 %): mp 105-107 °C; H NMR (600 MHz, CD3OD, rt) δ7.43(d, J
=
8.4 Hz, 4H, H2, H2”, H6, H6”), 7.09(s, 2H, H5’, H6’), 6.86(d, J = 8.4 Hz, 4H, H3, H3”, H5, H5”),
13
3
.84(q, J = 7.2 Hz, 4H, OCH ), 1.18(t, J = 7.2 Hz, 6H, CH ). C NMR (150 MHz, CD3OD, rt) δ 156.4
2 3
(
C4), 156.4 (C4”), 150.0 (C2’), 150.0 (C3’), 135.0 (C1), 135.0 (C1”), 130.0 (C2), 130.0 (C2”), 130.0
1
3

ACCEPTED MANUSCRIPT
(
(
C6), 130.0 (C6”), 129.5 (C1’), 129.5 (C4’), 124.9 (C5’), 124.9 (C6’), 114.5 (C3), 114.5 (C3”), 114.5
-
C5), 114.5 (C5”), 68.4 (OCH ), 68.4 (OCH ), 14.6 (CH ), 14.6 (CH ); HRMS (ESI) m/z [M-H]
2
2
3
3
Calcd. for C H O : 349.1440, Found: 349.1455.
2
2
21
4
4
.1.17. 3,3’’,4,4’’-tetramethoxy-2’,3’-diethoxy [1,1’:4’,1’’-terphenyl] (11)
The resulting residue was purified by silica gel chromatography (10/1 PET/EtOAc) to afford the
1
desired product as a withe solid (31.2 %): mp 101-103 °C. H NMR (600 MHz, CDCl3, rt) δ 7.26 (d, J
=
1.8 Hz, 2H, H2, H2”), 7.16 (s, 2H, H5’, H6’), 7.15 (dd, J = 8.4, 1.8 Hz, 2H, H6, H6”), 6.96 (d, J =
8
.4 Hz, 2H, H5, H5”) , 3.96 (s, 6H, OCH ), 3.95 (s, 6H, OCH ), 3.85 (q, J = 7.2 Hz,4H, OCH ), 1.23
3
3
2
1
3
(
t,6H, CH CH ). C NMR (150 MHz, CDCl3, rt) δ 150.3 (C3), 150.3 (C3”), 148.3 (C4), 148.3 (C4”),
2 3
1
1
48.2 (C2’), 148.2 (C3’), 135.1 (C1), 135.1 (C1”), 131.1 (C1’), 131.1 (C4’), 125.3 (C6), 125.3 (C6”),
21.4 (C5’), 121.4 (C6’), 112.6 (C2), 112.6 (C2”), 110.8 (C5), 110.8 (C5”), 69.0 (OCH CH ), 69.0
2
3
(
(
OCH CH ), 55.9 (OCH ), 55.9 (OCH ), 55.9 (OCH ), 55.9 (OCH ), 15.9 (OCH CH ), 15.9
2 3 3 3 3 3 2 3
+
OCH CH ); HRMS (ESI) m/z [M+H] Calcd. for C H O : 439.2120, Found: 439.2119.
2
3
26 31
6
4
.1.18. 4,4’’-dihydroxy-2’,3’-dimethoxy [1,1’:4’,1’’-terphenyl] (12)
The resulting residue was purified by silica gel chromatography (10/1 PET/EtOAc) to afford the
product as a white solid then was purified by sephadex LH-20 chromatography (MeOH) to afford the
1
desired product as a white solid (41.6 %): mp 178-180 °C; H NMR (600 MHz, CD3OD, rt) δ 7.42(d, J
=
8.4 Hz, 4H, H2, H2”, H6, H6”), 7.10(s, 2H, H5’ H6’), 6.86(d, J = 8.4 Hz, 4H, H3, H3”, H5, H5”),
1
3
3
.65(s, 6H, OCH3). C NMR (150 MHz, CD3OD, rt) δ 156.5 (C4), 156.5 (C4”), 150.8 (C2’), 150.8
(
(
(
C3’), 134.7 (C1), 134.7 (C1”), 129.9 (C2), 129.9 (C2”), 129.9 (C6), 129.9 (C6”), 129.3 (C1’), 129.3
C4’), 125.0 (C5’), 125.0 (C6’), 114.6 (C3), 114.6 (C3”), 114.6 (C5), 114.6 (C5”), 59.6 (OCH ), 59.6
3
-
OCH ); HRMS (ESI) m/z [M-H] Calcd. for C H O : 321.1127, Found: 321.1145.
3
20 17
4
4
.1.19. 4,4’’-dimethoxy-2’,3’-diacetoxy[1,1’:4’,1’’-terphenyl] (13)
The resulting residue was purified by silica gel chromatography (5/1 PET/EtOAc) to afford the
1
desired product as a withe solid (28.9 %): mp 178-180 °C; H NMR (600 MHz, CDCl3, rt) δ 7.43(d, J
=
7.8 Hz, 4H, H2, H2”, H6, H6”), 7.35(s, 2H, H5’ H6’), 6.98(d, J = 7.8 Hz, 4H, H3, H3”, H5, H5”),
1
4

ACCEPTED MANUSCRIPT
1
3
3
1
1
1
.87(s, 4H, OCH ) , 2.14(S, 6H OCOCH ). C NMR (150 MHz, CDCl3, rt) δ 168.3 (CO), 168.3 (CO),
3 3
59.2 (C4), 159.2 (C4”), 140.4 (C2’), 140.4 (C3’), 134.9 (C1’), 134.9 (C4’), 129.9 (C2), 129.9 (C2”),
29.9 (C6), 129.9 (C6”), 129.2 (C1), 129.2 (C1”), 127.8 (C5’), 127.8 (C6’), 113.9 (C3), 113.9 (C3”),
13.9 (C5), 113.9 (C5”), 55.3 (OCH ), 55.3 (OCH ), 20.5 (COCH ), 20.5 (COCH ) HRMS (ESI) m/z
3
3
3
3
+
[
M+Na] Calcd. for C H O Na: 429.1314, Found: 429.1302.
24 22 6
4
.1.20. 3,3’’,4,4’’-tetramethoxy-2’,3’-diacetoxy[1,1’:4’,1’’-terphenyl] (14)
The resulting residue was purified by silica gel chromatography (3/1 PET/EtOAc) to afford the
1
desired product as a withe solid (19.4 %): mp 196-197 °C; H NMR (600 MHz, CDCl3, rt) δ 7.38(s,
2
H, H5’, H6’), 7.05(d, J = 8.4 Hz, 2H, H6, H6”), 7.02(s, 2H, H2, H2”), 6.95(d, J = 7.8 Hz, 2H, H5,
H5”) , 3.95(s, 6H, OCH ), 3.92(s, 6H, OCH ), 2.15(s, 6H, COCH ). 13C NMR (150 MHz, CDCl3, rt)
3
3
3
δ 168.3 (CO), 168.3 (CO), 148.7 (C3), 148.7 (C3”), 148.6 (C4), 148.6 (C4”), 140.4 (C2’), 140.4 (C3’),
1
1
35.2 (C1’), 135.2 (C4’), 129.5 (C1), 129.5 (C1”), 127.9 (C5’), 127.9 (C6’), 121.2 (C6), 121.2 (C6”),
11.9 (C2), 111.9 (C2”), 111.1 (C5), 111.1 (C5”), 55.9 (OCH ), 55.9 (OCH ), 55.9 (OCH ), 55.9
3
3
3
+
(
OCH ), 20.6 (COCH ), 20.6 (COCH ) HRMS (ESI) m/z [M+Na] Calcd. for C H O Na: 489.1526,
3 3 3 26 26 8
Found: 489.1511.
4
.1.21. [1,1’:4’,1”-Terphenyl]- 2’,3’,4,4’’-tetraol (15)
The resulting residue was purified by recrystallization in acetone to afford the desired product as a
1
withe solid (52.1%): mp > 260 °C; H NMR (600 MHz, (CD ) CO, rt) δ 7.46 (d, J = 7.8 Hz, 4H, H2,
3
2
13
H2”, H6, H6”), 6.91 (d, J = 7.8 Hz, 4H, H3, H3”, H5, H5”), 6.84 (s, 2H, H5’, H6’); C NMR (150
MHz, (CD ) CO, rt) δ 156.4 (C4), 156.4 (C4”), 142.3 (C2’), 142.3 (C3’), 130.2 (C2), 130.2 (C2”),
3
2
1
1
2
30.2 (C6), 130.2 (C6”), 129.6 (C1), 129.6 (C1”), 127.4 (C1’), 127.4 (C4’), 121.0 (C5’), 121.0 (C6’),
-
15.0 (C3), 115.0 (C3”), 115.0 (C5), 115.0 (C5”); HRMS (ESI) m/z [M-H] Calcd. for C H O :
18
13
4
93.0814, Found: 293.0809.
4
.1.22. 2’,3,3’,3’’,4,4’’-hexamethoxy-[1,1’:4’,1’’-terphenyl] (16)
The resulting residue was purified by silica gel chromatography (15/1 PET/EtOAc) to afford the
1
desired product as a withe solid (12.3 %): mp 130-132 °C; H NMR (600 MHz, (CD ) CO, rt) δ 7.22 (s,
3
2
1
5

ACCEPTED MANUSCRIPT
2
H, H5’, H6’), 7.17 (s, 2H, H2, H2”), 7.14 (d, J = 8.4 Hz, 2H, H6, H6”) 7.03 (d, J = 8.4 Hz, 2H, H5,
13
H5”), 3.88 (s, 6H, OCH ), 3.87 (s, 6H, OCH ), 3.70 (s, 6H, OCH ) C NMR (150 MHz, (CD ) CO,
3
3
3
3 2
rt) δ 151.1 (C3), 151.1 (C3”),149.1 (C2’), 149.1 (C3’), 148.9 (C4), 148.9 (C4”), 134.8 (C1), 134.8
(
C1”), 130.8 (C1’), 130.8 (C4’), 125.2 (C6), 125.2 (C6”), 121.4 (C5’), 121.4 (C6’), 113.2 (C2), 113.2
(
C2”), 111.6 (C5), 111.6 (C5”), 59.9 (OCH ), 59.9 (OCH ), 55.3 (OCH ), 55.3 (OCH ), 55.2 (OCH ),
3
3
3
3
3
+
5
5.2 (OCH ) ; HRMS (ESI) m/z [M+H] Calcd. for C H O : 411.1808, Found: 411.1814.
3 24 27 6
4
.1.23. [1,1’:4’,1”-Terphenyl]-2’,3,3’,3’’,4,4’’-hexaol (17)
The resulting residue was purified by sephadex LH-20 chromatography (1/1 MeOH/CH Cl ) to
2
2
1
afford the desired product as a grey white solid (80.0 %): mp > 260 °C; H NMR (600 MHz, (CD ) CO,
3
2
rt) δ 7.98 (s, 2H, OH), 7.91 (s, 2H, OH), 7.43 (s, 2H, OH), 7.13 (s, 2H, H5’,H6’), 6.95 (d, J = 8.4 Hz,
13
2
H, H5, H5”), 6.88 (d, J = 8.4 Hz, 2H, H6, H6”), 6.81 (s, 2H, H3, H3”); C NMR (150 MHz,
(
CD ) CO, rt) δ 144.7 (C4), 144.7 (C4”), 144.3 (C3), 144.3 (C3”), 142.3 (C2’), 142.3 (C3’), 130.2
3
2
(
C1), 130.2 (C1”), 127.4 (C1’), 127.4 (C4’), 120.8 (C6), 120.8 (C6”), 120.7 (C5’), 120.7 (C6’), 116.3
-
(
C2), 116.3 (C2”), 115.2 (C6), 115.2 (C6”); HRMS (ESI) m/z [M-H] Calcd. for C H O : 325.0712,
18
13
6
Found: 325.0708.
4
.1.24. dimethyl biphenyl-4,4'-dicarboxylate (18)
The resulting residue was purified by silica gel chromatography (5/1 PET/EtOAc) to afford the
1
desired product as a withe solid (29.7 %): mp 108-110 °C; H NMR (600 MHz, CDCl , rt) δ 8.15 (d, J
3
13
=
8.4 Hz, 4H, H3, H3’, H5, H5’), 7.71 (d, J = 8.4 Hz, 4H, H2, H2’, H6, H6’), 3.97 (s, 6H, OCH3).
C
NMR (150 MHz, CDCl , rt) δ 166.8 (CO), 166.8 (CO), 144.3 (C1), 144.3 (C1’), 130.2 (C2), 130.2
3
(
C2’), 130.2 (C6), 130.2 (C6’), 129.7 (C4), 129.7 (C4’), 127.2 (C3), 127.2 (C3’), 127.2 (C5), 127.2
+
(
C5’), 52.2 (OCH ), 52.2 (OCH ) ; HRMS (ESI) m/z [M+H] Calcd. for C H O : 271.0970, Found:
3
3
16 15
4
2
71.0977.
4
.1.25. 2,3,3',4'-tetramethoxybiphenyl (19)
The resulting residue was purified by silica gel chromatography (15/1 PET/EtOAc) to afford the
1
desired product as a yellow oil (10.4 %): H NMR (600 MHz, (CD ) CO, rt) δ 7.15 (s, 1H, H2),
3
2
1
6

ACCEPTED MANUSCRIPT
7
3
1
1
.06~7.11 (m, 2H, H6, H6’), 6.99~7.02 (t, 2H, H5, H5’) 6.94 (d, J = 7.8 Hz, 1H, H4’), 3.89 (s, 3H),
13
.86 (s, 6H), 3.61 (s, 3H) C NMR (150 MHz, (CD ) CO, rt) δ 153.4 (C2’), 148.9 (C3’), 148.8 (C3),
3
2
46.6 (C4), 135.5 (C1), 131.1 (C1’), 123.9 (C6’), 122.2 (C5’), 121.4 (C6), 113.4 (C5), 111.6 (C2),
11.6 (C4’), 59.6 (OCH ), 55.3 (OCH ), 55.2 (OCH ), 55.19 (OCH ).
3
3
3
3
4
.1.26. 3,3’-dinitro -4,4’’-dihydroxy diphenyl (20)
The resulting residue was purified by silica gel chromatography (15/1 PET/EtOAc) to afford the
1
desired product as a yellow solid (25.5 %): mp > 260 °C; H NMR (600 MHz, DMSO-d6, rt) δ11.16 (s,
2
H, OH), 8.15 (s, 2H, H2, H2’), 7.87 (d, J = 8.4 Hz, 2H, H6, H6’), 7.20 (d, J = 9 Hz, 2H, H5, H5’);
C NMR (150 MHz, DMSO-d6,, rt) δ 151.7 (C4), 151.7 (C4’), 137.9 (C3), 137.9 (C3’), 133.2 (C6),
33.2 (C6’), 129.5 (C1), 129.5 (C1’), 122.9 (C2), 122.9 (C2’), 120.1 (C5), 120.1 (C5’).
1
3
1
4
.1.27. methyl 2-hydroxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate
(21)
The resulting residue was purified by silica gel chromatography (15/1 PET/EtOAc) to afford the
1
desired product as a withe solid (53.3 %): H NMR (600 MHz, CDCl ) δ 11.01 (s, 1H), 8.31 (s, 1H),
3
13
7
.88 (dd, J = 7.4, 1.2 Hz, 1H), 6.97 (d, J = 7.4 Hz, 1H), 3.95 (s, 3H), 1.34 (s, 12H); C NMR (150
MHz, CDCl ) δ 170.7, 164.0, 141.96, 137.3, 117.1, 112.1, 83.8, 77.2, 77.0, 76.8, 52.2, 24.9.
3
4
.1.28. methyl 2-hydroxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate
(22)
The crude product was purified by chromatography using petroleum ether/ethyl acetate 30:1 to
1
yield the corresponding derivatives (41.1%). H NMR (600 MHz, CDCl , rt) δ 10.62 (s, 1H), 7.83 (d, J
3
=
6.8 Hz, 1H), 7.44 (s, 1H), 7.30 (d, J = 6.8 Hz, 1H), 3.97 (s, 3H), 1.37 (s, 12H).
4
.1.29. 4-bromo-2,3,3',4'-tetramethoxybiphenyl (25)
The resulting residue was purified by silica gel chromatography (15/1 PET/EtOAc) to afford the
1
desired product as a withe solid (73.3 %): H NMR (600 MHz, Acetone) δ 7.35 (d, J = 8.4 Hz, 1H,
H6), 7.17 (s, 1H, H2), 7.06~7.09 (2H, H5’, H6’), 7.00 (d, J = 8.4 Hz, 1H, H5), 3.92 (s, 3H, OCH3),
1
7

ACCEPTED MANUSCRIPT
1
3
3
1
1
.86 (s, 6H, OCH ), 3.66 (s, 3H, OCH ); C NMR (150 MHz, Acetone) δ 151.8 (C3’), 151.1 (C2’),
3 3
49.2 (C3), 149.1 (C4), 135.9 (C1), 129.8 (C1’), 127.7 (C5’), 126.4 (C6’), 121.4 (C6), 115.7 (C4’),
13.0 (C2), 111.7 (C5), 60.2 (OCH ), 60.17 (OCH ), 55.4 (OCH ), 55.3 (OCH ).
3
3
3
3
4
.1.30. 1,4-dibromo-2,3-diethoxybenzene (30)
0
0
C, 1,2-Benzenediol,3,6-dibromo (360mg), anhydrous potassium carbonate (747mg), anhydrous
acetone (3ml), diethyl sulfate (0.71ml). After being stirred overnight, the mixture was quenched with
sufficient quantum aqueous ammonia and extracted with ethyl acetate. The aqueous layer was washed
with ethyl acetate. The organic phases were combined, washed with brine, dried over anhydrous
sodium sulfate and evaporated to dryness under reduced pressure. The crude product was purified by
chromatography using petroleum ether/ethyl acetate 20:1 to yield the corresponding derivatives
1
13
(
71.1%). H NMR (600 MHz, CDCl , rt) δ 7.19(s, 2H), 4.14(q, 4H), 1.44(t, 6H); C NMR (150 MHz,
3
CDCl , rt) δ 151.0, 151.0, 128.5, 128.5, 117.5, 117.5, 69.7, 69.7, 15.7, 15.7.
3
4
.1.31. 3,6-dibromo-1,2-phenylene diacetate (31)
3
,6-dibromobenzene-1,2-diol [13] was acetylated by acetic anhydride gained 31.
4
4
.2. Biological Assay
.2.1. In vitro cytotoxicity assays
The cytotoxicity was measured by the MTT assay as described in the literature [18, 19]. The cells
plated in the wells of 96-well plates (Falcon, USA) were treated in triplicate with various
concentrations of compounds for 72 h in 5% CO incubator at 37°C. After fresh medium being
2
changed, a 20 ꢁL aliquot of MTT solution (5 mg/mL) was added and incubated for 4 h at 37°C. Then,
1
00 ꢁL of triplex solution (10% SDS, 5% isobutanol, 12 mM HCl) was added to each well and
incubated overnight at 37°C. The absorbance of each well was determined by a microplate reader
(
M-3350, Bio-Rad) with a 590 nm wavelength. Growth inhibition rates were calculated with the
OD control well −OD treated well
Inhibition rate =
×100%
OD control well −OD blank well
following equation:
1
8

ACCEPTED MANUSCRIPT
4
.2.2. DNA gel electrophoresis assay of topoisomerases
Plasmid pBR322 DNA and purified calf thymus DNA TOP1 were purchased from TakaRa
Biotechnology (Dalian) Co., Ltd., recombinant human TOP2α was obtained from TopoGENINC
USA). All experiments were done at least in duplicate to confirm the results.
(
4
.2.3. DNA relaxation assay
DNA TOP1 inhibition assay was performed as described previously [20] with minor
modifications. The test compounds were dissolved in DMSO at 20 mM as stock solution. The activity
of DNA TOP1 was determined by assessing the relaxation of supercoiled DNA pBR322. The mixture
of 0.5 ꢁg of plasmid pBR322 DNA and 1 units of TOP1 was incubated without and with the prepared
compounds at 37 °C for 30 min in the relaxation buffer (10 mM Tris-HCl (pH 7.9), 150 mM NaCl,
0
.1% bovine serum albumin, 1 mM spermidine, 5% glycerol). The reaction in the final volume of 20
L was terminated by adding 2.5 ꢁL of the stop solution containing 5% sarcosyl, 0.0025%
bromophenol blue, and 25% glycerol. DNA samples were then electrophoresed on a 1% agarose gel at
V /cm for 2 h with a running buffer of TAE. Gels were stained for 30 min in an aqueous solution of
ꢁ
5
ethidium bromide (0.5 ꢁg/mL) and photographed under UV light.
DNA TOP2α inhibitory activity of compounds was measured as follows [21]. Briefly, the mixture
of 0.5 ꢁg of supercoiled pBR322 plasmid DNA and 1 units of TOP2α was incubated without and with
the prepared compounds in the assay buffer (10 mM Tris-HCl (pH 7.9) containing 50 mM NaCl, 50
mM KCl, 5 mM MgCl , 1 mM EDTA, 1 mM ATP, and 15 lg/mL bovine serum albumin) for 30 min at
2
3
0 °C. The reaction in a final volume of 20 ꢁL was terminated by the addition of 3 ꢁL of 7 mM EDTA.
Reaction products were analyzed on a 1% agarose gel at 5 V /cm for 2 h with a running buffer of TAE.
Gels were stained for 30 min in an aqueous solution of ethidium bromide (0.5 ꢁg/mL) and
photographed under UV light.
4
.3. DOCK
The molecular docking was conducted using AutoDock Vina 1.1.2 [22] an open-source program
for doing molecular docking. Compounds 17 and X3 were ultimately converted to the PDBQT format
1
9

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using AutoDock Tools 1.5.6 [http:/mgltools.scripps.edu], which is required for AutoDock Vina. The
3
-dimensional (3D) structure of TOP1 and TOP2α were downloaded from the Protein Data Bank
PDBID: 1T8I and 4FM9, respectively). Using AutoDock Tools, the PDB (1T8I) structure was
converted from a pdb file to a pdbqt file and the search grid was identified as center_x: 21.95, center_y:
4.434, and center_z: 27.801 with dimensions size_x: 16, size_y: 16, and size_z: 16. Then the
(
-
compound 17 and X3 were docked into the binding site of topoisomerase IIa as described for
topoisomerase I. The grid site and dimensions are center_x: 26.529, center_y: 103.004, and center_z:
3
6.787 and size_x: 16, size_y: 16, and size_z: 16 respectively. For Vina docking, the default
parameters were used if it was not mentioned [23, 24]. The best-scoring pose as judged by the Vina
docking score was chosen and visually analyzed using PyMOL.
4
.4. 3D-QSAR Modeling and Validation
Sybyl X 1.1 was used to perform the 3D-QSAR analyses. Two complementary methods were
used: the Comparative Molecular Field Analysis (CoMFA) and the Comparative Molecular Similarity
Indices Analysis (CoMSIA) [25]. For CoMFA field generation, we used the standard steric and
electrostatic fields. The steric, electrostatic, hydrophobic, hydrogen bond donor and hydrogen bond
acceptor fields were used for CoMSIA. Gasteiger-Hückel charges were assigned to all compounds.
Other field settings were default. Partial least-squares (PLS) regression methods were used to derive
models in this study. Leave-one-out (LOO) cross-validation was used to identify the optimum number
of components. LOO approaches evaluate the predictability and over fitting of a regression model with
2
the cross-validated correlation coefficient q . The non-cross-validated models were built with the
optimum number of components over the entire training set and evaluated with the correlation
2
coefficient (r ), standard error of estimate (SEE), and F-test value. The resulting models were further
2
2
validated using a test set of 10 compounds. The predictive r (r pred) indicates the correlation between
the predicted and experimental activities of this test set.
Acknowledgments
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This work was supported by a grant from the 973 Program (2010CB833802), NSFC Projects
81273384, 90913024).
(
2
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Figure 1. Compounds 1-20 and X1-3.
Figure 2. TOP1 (A) and TOP2α (B) inhibitory effects of compounds 2, 12 and 17 in agarose relaxation
assay. Negatively supercoiled pBR322 (SC) and relaxed DNA (RLX) are shown. 100 µM OPT
(
10-hydroxy camptothecin) and VP16 (etoposide) were employed as positive controls.
Figure 3. Compounds 17 and X3 were docked into the TOP1 (A) and TOP2α (B)binding pockets.
Compound 17 is shown in gray and compound X3 is displayed in cyan. The docking results are
illustrated by PyMoL [http://www.pymol.org/].
Figure 4. Structure of compounds C1-5
Figure 5. Scatter plots of predicted pIC values vs. experimental pIC values. The plots were obtained
5
0
50
from the CoMFA (A) and CoMSIA (B) models. The training set contains 13 compounds (black
squares), and the test set contains 10 compounds (red triangles).
Figure 6. Contour maps of the CoMFA (A) and CoMSIA (B) models with compound 17. The green
and yellow contours represent favorable and unfavorable steric substitutions, respectively, whereas the
red and blue contours indicate a favorable effect from more electronegative and more electropositive
substitutions, respectively.
a
Scheme 1-4. Synthesis route of compounds 1-20 .
a
Table 1. Effect of compounds 1-20 on MDA-MB-435 cell proliferation
Table 2. Summary of 3D-QSAR model statistics.
Table 3. Experimental pIC50 and predicted pIC50 values of the training set from the CoMFA and
a
CoMSIA models.
2
4

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Table 4. Experimental pIC and predicted pIC values of the test set from the CoMFA and CoMSIA
50
50
a
models.
2
5

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Table 1. The effect of compounds 1-20 on the proliferations of MDA-MB-435 cells
Compound IC (µM) Compound IC (µM) Compound IC (µM)
a
5
0
50
50
1
2
3
4
5
6
7
44.80±1.48
20.90±1.36
>100
8
>100
15
16
>100
9
61.80±2.83
41.80±1.39
>100
90.80±1.57
3.96±1.07
>100
10
11
12
13
14
17
>100
18
99.80±2.91
36.80±1.79
>100
14.70±1.53
96.80±2.97
>100
19
23.10±1.28
>100
20
VP-16
2.81±1.65
a
The cytotoxicity of compounds was expressed as IC . The IC is defined as the concentration of compound that
50
50
resulted in 50% inhibition of growth rate. Values represent means±SD of three individual experiments. VP-16
etoposide) was used as positive compound.
(

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Table 2. Summary of 3D-QSAR Model Statistics
Statistical parameters CoMFA(IC ) CoMSIA(IC )
5
0
50
no. of components
4
5
2
q2 (cross-validated r )
0.605
0.998
0.03
0.622
0.994
0.05
2
r
std error of estimate
F value
960.9
0.74
242.5
0.66
2
r
(for test set)
pred
a
Cross-validated correlation coefficient from LOO.
b
Non-cross-validated correlation coefficient.
Correlation coefficient for test set predictions.
c

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Table 3. Experimental pIC50 and predicted pIC50 values of the training set from the CoMFA and
a
CoMSIA models.
CoMFA
CoMSIA
Training set Experimental
Predicted Residual Predicted Residual
3
4
5
6
8
4
3.99
4.05
0.01
3.941
4.089
4.018
4.387
4.005
4.032
4.871
3.99
0.059
-0.089
-0.0171
0.0472
-0.005
-0.032
-0.0383
0.0241
0.003
4
-0.05
4.0009
4.4342
4
4.018
4.404
3.981
3.985
4.866
3.997
3.982
4.062
5.406
4.696
5.363
-0.0171
0.0302
0.019
1
1
2
3
5
6
7
4
0.015
1
1
1
1
1
4.8327
4.0141
4
-0.0333
0.0171
0.018
3.997
4.057
5.387
4.646
5.401
4.0419
5.4023
4.6876
5.3872
-0.0201
-0.0037
-0.0084
0.0242
-0.0151
0.0153
0.0416
-0.0138
X2
X1
a
pIC50 = −log (IC ), if the IC > 100, we determined the pIC = 4.
50
50
50
b
The IC50 values of compound X1 and X2 were 0.39 ± 0.03 and 32.67 ± 6.03.