Design, synthesis, biological evaluation and molecular modeling study of novel macrocyclic bisbibenzyl analogues as antitubulin agents

Article abstract of DOI:10.1016/j.ejmech.2017.02.021

A series of macrocyclic bisbibenzyls with novel skeletons was designed, synthesized, and evaluated for antiproliferative activity against five anthropic cancer cell lines. Among these novel molecules, compound 47 displayed excellent anticancer activity ag

Full text of DOI:10.1016/j.ejmech.2017.02.021

Accepted Manuscript
Design, synthesis, biological evaluation and molecular modeling study of novel
macrocyclic bisbibenzyl analogues as antitubulin agents
Bin Sun, Lin Li, Qing-wen Hu, Hong-bo Zheng, Hui Tang, Huan-min Niu, Hui-qing
Yuan, Hong-xiang Lou
PII:
S0223-5234(17)30086-7
10.1016/j.ejmech.2017.02.021
EJMECH 9217
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 October 2016
Revised Date: 7 February 2017
Accepted Date: 8 February 2017
Please cite this article as: B. Sun, L. Li, Q.-w. Hu, H.-b. Zheng, H. Tang, H.-m. Niu, H.-q. Yuan, H.-
x. Lou, Design, synthesis, biological evaluation and molecular modeling study of novel macrocyclic
bisbibenzyl analogues as antitubulin agents, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.02.021.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, biological evaluation and molecular modeling
study of novel macrocyclic bisbibenzyl analogues as antitubulin
agents
a,b,
b
b
b
b
c
Bin Sun , Lin Li , Qing-wen Hu , Hong-bo Zheng , Hui Tang , Huan-min Niu , Hui-qing Yuan
c
a,b
,
and Hong-xiang Lou
*
a
National Glycoengineering Research Center, Shandong University, Jinan 250012, P. R. China
b
Key Laboratory of Natural Products & Chemical Biology, Ministry of Education, Shandong
University, Jinan, 250012, P.R. China
c
School of Medicine, Shandong University, Jinan 250012, P.R. China
ABSTRACT
A series of macrocyclic bisbibenzyls with novel skeletons was designed, synthesized, and evaluated for
antiproliferative activity against five anthropic cancer cell lines. Among these novel molecules,
compound 47 displayed excellent anticancer activity against HeLa, k562, HCC1428, HT29 and
PC-3/Doc cell lines, with IC50 values ranging from of 1.51 µM to 5.51 µM, which were more potent
than the parent compound, marchantin C. Compounds 44 and 55 with novel bisbibenzyl skeletons also
exhibited significantly improved antiproliferative potency. Structure-activity relationship (SAR)
analyses of these synthesized compounds were also performed. In addition, compound 47 effectively
inhibited tubulin polymerization in HCC1482 cells and induced HCC1482 cell cycle arrest at the G2/M
phase in a concentration-dependent manner. The binding mode of compound 47 to tubulin was also
investigated utilizing a molecular docking study. In conclusion, the present study discovered several
potent antitubulin compounds with novel bisbibenzyl skeletons, and our systematic studies revealed
new scaffolds that target tubulin and mitosis and provide progress towards the discovery of novel
antitumor drugs discovery.
Keywords: Bisbibenzyls; Tubulin polymerization inhibitors; Anticancer; Molecular modeling
*
Corresponding authors: Hongxiang Lou, Ph.D.
Tel: +86-531-88382051; Fax: +86-531-88382019.
E-mail address: louhongxiang@sdu.edu.cn (H. Lou)

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1
. Introduction
Microtubules are recognized as one of the most important molecular targets for cancer therapies
due to their role as one of the major cytoskeletal components in eukaryotic cells and their crucial
function in cell shape maintenance, division, protein trafficking and intracellular transport[1-4].
Tubulin–interacting drugs are used widely and successfully in the treatment of a variety of human
cancers by interfering with microtubule dynamics, a process that controls the balance between
microtubule assembly and microtubule disassembly [5-10]. Three major binding sites for tubulin
inhibitors have been identified as the vinca alkaloid-, taxanes-, and colchicine-binding sites. Although
antitubulin agents that interact with the vinca alkaloid- or taxanes-binding sites are widely used in
clinical cancer therapy, none of the colchicine-binding site drugs are even approved in clinical
oncology [11-17]. In fact, most of the colchicine-binding agents have high potency, selective toxicity
toward tumor vasculature, and relatively simple structures for optimization, and they show a promising
ability to overcome P-glycoprotein-mediated multidrug resistance [18-25]. Therefore, agents that
interact with the colchicine-binding site show promise for the development of novel and potent
anticancer drugs and have attracted great interest from medicinal chemists in recent years.
Macrocyclic bisbibenzyls are a series of phenolic natural products that are mainly found in
bryophytes. Each bisbibenzyl skeleton comprises four aromatic rings (arene A-D) and two ethano
bridges, with lunularin as the biosynthetic origin (Fig. 1) [26]. All of these compounds can be divided
into several sub-classes that are distinguished by the connectivity between the two lunularin sub-units,
which is produced by oxidative coupling to form either biaryl or biaryl ether linkages [27, 28].
Marchantins, isomarchantins, neomarchantins and riccardin II are four main sub-classes of
macrocyclic bisbibenzyls that have two diaryl ether sub-units as a common structural feature (Fig. 1)
and occupy approximately 25% of all the natural macrocyclic bisbibenzyls discovered to date. These
compounds exhibit versatile and excellent biological activities, including antimicrobial, antioxidative,
cytotoxic, and multidrug resistance reversal activities [29-36]. The remarkable biological profiles of
these natural products make them promising resources for new drug discovery. Among these
compounds, marchantin C, a natural macrocyclic bisbibenzyl, was discovered to be a potent tubulin
inhibitor that targets the colchicine-binding site and the compound was also found to exhibit multidrug
resistance-reversing activity by inhibiting the P-glycoprotein [37-39]. In addition, its bisbibenzyl
scaffold is similar to the cis-stilbenoid anti-tubulin agents, such as combretastatins. We have recently

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reported a series of marchantin C analogues as potent tubulin polymerization inhibitors in which the
substitutive groups on the aromatic rings were varied [40]. Continuing our search strategy for novel
tubulin inhibitors, we extended our studies to modification of the marchantin C skeleton. In this paper,
a panel of novel marchantin C analogues was designed, synthesized and examined as potential tubulin
targeting agents. The result of this effort, described below, indicated that these novel marchantin C
analogues represent a new class of potent antitubulin agents.
Fig. 1. Modification strategy for macrocyclic bisbibenzyl analogues
[
Figure 1 here]
2
. Results and Discussion
2
.1. Design of marchantin C analogues
In the previous study, marchantin C and its derivatives were found to be potent antimitotic agents
that target tubulin, and their macrocyclic bisbibenzyl skeletons were found to fit sterically in the
colchicine-binding site. To define the structure-activity relationships (SARs) of bisbibenzyl analogues

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and to develop potential new drug candidates, a series of novel marchantin C analogues were designed.
According to the modification strategy shown in Fig. 1, we first systematically varied the substitutive
position of the ether bond on the arenes B, C and D of marchantin C, which gave five novel bisbibenzyl
skeletons, BBM-B-m class, BBM-C-o class, BBM-C-p class, BBM-D-o class and BBM-D-p class, as
well as two natural bisbibenzyl skeletons, the riccardin II and neomarchantin classes. This modification
could change the stereochemical structure of the macrocyclic bisbibenzyls, and we speculated that
some of these novel skeletons might be more complementary to the colchicine-binding site (Fig. S1).
Second, we changed the ethylene bridge to a double bond and determined the effect of this further
stereochemical structural change on the cytotoxic activity. Finally, we focused on modification of the
phenyl rings via introduction of hydroxyl groups or methoxyl groups on arenes A-D to assess the
substituent effects on the antiproliferative activity, since those two substituents were mostly founded in
the natural bisbibenzyl compounds.
2
.2. Chemistry
All of the marchantin C analogues were prepared by eight independent synthetic routes, and the
general procedure is outlined in Schemes 1-8. The synthetic route of analogues 53-57 began with
Ullmann coupling of the appropriately substituted methyl 4-bromobenzoate 1-2 with
3
-hydroxy-4-methoxybenzaldehyde (3), resulting in the formation of diphenyl ethers 4-5. Compounds
-5 were then reduced with sodium borohydride to give the benzyl alcohols, followed by reaction with
4
triphenylphosphonium bromide to afford 6-7 in two steps. Compounds 13-16 were prepared by
Ullmann coupling of the protected 2-hydroxy-3-methoxybenzaldehyde (8) with the appropriate
2
-bromo-benzaldehydes 9-12 in good yield. The building blocks 6-7 and 13-16 were combined by an
intermolecular Wittig reaction in the presence of potassium carbonate and 18-crown-6 followed by
hydrogenation over Pd/C to give the bibenzyls 17-21. The carboxylic ester group of 17-21 was then
reduced with lithium aluminum hydride and subsequently deprotected with HCl/H O to give
2
compounds 22-26. The reaction of 22-26 with triphenylphosphonium bromide afforded compounds
2
7-31. The cyclization of 27-31 by means of an intramolecular Wittig reaction was achieved with
sodium methoxide, leading to key macrocyclic intermediates 32-36. 32-36 were then hydrogenated to
give compounds 37-41. Finally, 32, 39 and 37-41 were demethylated by boron tribromide to afford
compounds 42-47 (Scheme 1). The position of the methoxyl group in compound 47 was determined
from a NOE spectrum, as shown in Fig. 2. Correlations of H -15’ with H-4’ were determined in
3

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compound 47.
Compounds 55, 65, 77, 78, 88, 96, 109, 110, 120 and 121 were prepared by a protocol similar to
that used for compounds 42-47, and the synthetic routes are depicted in Schemes 2-8.
Scheme 1. Synthesis of macrocyclic compounds 32-47. Reagents and conditions: (a) CuO, K CO , Py,
2
3
reflux, (yields 63%-76%); (b) i. NaBH , THF, 0 ℃ to r.t.; ii. PPh HBr, MeCN, reflux, (yields 78%-89%,
4
3

ACCEPTED MANUSCRIPT
two steps); (c) i. K CO , 18-crown-6, DCM, reflux; ii. Pd/C (10%), H , Et N, EtOAc, r.t. (yields
2
3
2
3
7
1%-80%, two steps); (d) i. LiAlH , THF, -40 ℃ to r.t.; ii. HCl/EtOH (1:10), r.t. (yields 80%-86%, two
4
steps); (e) PPh HBr, MeCN, reflux, (yields 86%-93%); (f) NaOMe, DCM, r.t. (yields 80%-88%); (g)
3
Pd/C (10%), H , EtOAc, r.t. (yields 95%-99%); (h) BBr , DCM, -40 ℃ to r.t. (yields 51%-86%).
2
3
[
Scheme 1 here]
Fig. 2. Key NOESY correlations (dashed blue arrows) for compound 47.
Figure 2 here]
[
Scheme 2. Synthesis of macrocyclic compounds 53-55. Reagents and conditions: (a) CuO, K CO , Py,
2
3
reflux, (yield 65%); (b) i. K CO , 18-crown-6, DCM, reflux; ii. Pd/C (10%), H , Et N, EtOAc, r.t.
2
3
2
3

ACCEPTED MANUSCRIPT
(
yield 83%, two steps); (c) i. LiAlH , THF, -40 ℃ to r.t.; ii. HCl/EtOH (1:10), r.t. (yield 85%, twp
4
steps); (d) PPh HBr, MeCN, reflux, (yield 92%); (e) NaOMe, DCM, r.t. (yield 80%); (f) Pd/C (10%),
3
H , EtOAc, r.t. (yields 96%); (g) BBr , DCM, -40 ℃ to r.t. (yield 86%).
2
3
[
Scheme 2 here]
Scheme 3. Synthesis of macrocyclic compounds 63-65. Reagents and conditions: (a) CuO, K CO , Py,
2
3
reflux, (yield 61%); (b) LiAlH , THF, -40 ℃ to r.t. (yield 88%); (c) PPh HBr, MeCN, reflux, (yield
4
3
9
0%); (d) i. K CO , 18-crown-6, DCM, reflux; ii. Pd/C (10%), H , Et N, EtOAc, r.t. (yield 80%, two
2 3 2 3
steps); (e) i. LiAlH , THF, -40 ℃ to r.t.; ii. HCl/EtOH (1:10), r.t. (yield 87%, twp steps); (f) NaOMe,
4
DCM, r.t. (yield 73%); (g) Pd/C (10%), H , EtOAc, r.t. (yields 92%); (h) BBr , DCM, -40 ℃ to r.t.
2
3
(
yield 82%).
[
Scheme 3 here]

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Scheme 4. Synthesis of macrocyclic compounds 73-78. Reagents and conditions: (a) CuO, K CO , Py,
2
3
reflux, (yield 70%); (b) i. K CO , 18-crown-6, DCM, reflux; ii. Pd/C (10%), H , Et N, EtOAc, r.t.
2
3
2
3
(
(
(
yield 80-86%); (c) i. LiAlH , THF, -40 ℃ to r.t.; ii. HCl/EtOH (1:10), r.t.; iii. PPh HBr, MeCN, reflux,
4 3
yield 70-74%, three steps); (d) NaOMe, DCM, r.t. (yields 71-77%); (e) Pd/C (10%), H , EtOAc, r.t.
2
yields 92-96%); (f) BBr , DCM, -40 ℃ to r.t. (yields 79-82%).
3
[
Scheme 4 here]

ACCEPTED MANUSCRIPT
Scheme 5. Synthesis of macrocyclic compounds 87-88. Reagents and conditions: (a) CuO, K CO , Py,
2
3
reflux, (yield 75%); (b) HCl/EtOH (1:10), r.t. (yield 79%); (c) NaBH , THF, 0 ℃ to r.t. (yield 86%); (d)
4
PPh HBr, MeCN, reflux, (yield 88%); (e) i. K CO , 18-crown-6, DCM, reflux; ii. Pd/C (10%), H ,
3
2
3
2
Et N, EtOAc, r.t. (yield 83%, two steps); (f) i. LiAlH , THF, -40 ℃ to r.t.; ii. HCl/EtOH (1:10), r.t.,
3
4
(
yield 73%, two steps); (g) i. NaOMe, DCM, r.t.; ii. Pd/C (10%), H , EtOAc, r.t. (yields 75%); (h) BBr ,
2 3
DCM, -40 ℃ to r.t. (yields 73%).
[
Scheme 5 here]

ACCEPTED MANUSCRIPT
Scheme 6. Synthesis of macrocyclic compounds 95-96. Reagents and conditions: (a) CuO, K CO , Py,
2
3
reflux, (yield 76%); (b) i. HCl/EtOH (1:10), r.t.; ii. NaBH , THF, 0 ℃ to r.t. (yield 61%, two steps); (c)
4
PPh HBr, MeCN, reflux, (yield 82%); (d) i. K CO , 18-crown-6, DCM, reflux; ii. Pd/C (10%), H ,
3
2
3
2
Et N, EtOAc, r.t. (yield 88%); (e) i. LiAlH , THF, -40 ℃ to r.t.; ii. HCl/EtOH (1:10), r.t., (yield 72%,
3
4
two steps); (f) i. NaOMe, DCM, r.t.; ii. Pd/C (10%), H , EtOAc, r.t. (yields 77%); (g) BBr , DCM,
2
3
-40 ℃ to r.t. (yields 79%).
[
Scheme 6 here]

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Scheme 7. Synthesis of macrocyclic compounds 105-110. Reagents and conditions: (a) CuO, K CO , Py,
2
3
reflux, (yields 54-73%); (b) i. K CO , 18-crown-6, DCM, reflux; ii. Pd/C (10%), H , Et N, EtOAc, r.t.;
2
3
2
3
iii. LiAlH , THF, -40 ℃ to r.t. (yields 64-76%, three steps); (c); i. HCl/EtOH (1:10), r.t.; ii. PPh HBr,
4
3
MeCN, reflux, (yields 74-81%, two steps); (d) NaOMe, DCM, r.t. (yields 81-86%); (e) Pd/C (10%), H2,
EtOAc, r.t. (yields 93-96%); (f) BBr , DCM, -40 ℃ to r.t. (yields 73-80%).
3
[
Scheme 7 here]
Scheme 8. Synthesis of macrocyclic compounds 116-121. Reagents and conditions: (a) CuO, K CO , Py,
2
3
reflux, (yields 77%); (b) i. K CO , 18-crown-6, DCM, reflux; ii. Pd/C (10%), H , Et N, EtOAc, r.t.
2
3
2
3
(
yields 66-75%, two steps); (c) i. LiAlH , THF, -40 ℃ to r.t.; ii. HCl/EtOH (1:10), r.t.; iii. PPh HBr,
4 3
MeCN, reflux, (yields 66-70%, three steps); (d) NaOMe, DCM, r.t. (yields 83-87%); (e) Pd/C (10%),
H , EtOAc, r.t. (yields 95-96%); (f) BBr , DCM, -40 ℃ to r.t. (yields 74-80%).
2
3
[
Scheme 8 here]
2
2
.3. Biological evaluation
.3.1. In vitro antiproliferative activity and structure-activity relationship (SAR) analysis
The cytotoxic activity of the synthesized analogues (42-47, 55-56, 77-78, 88, 96, 109-110, and
20-121) was evaluated in four human cancer cell lines (HeLa, k562, HCC1428, and HT29) and one
1
paclitaxel-resistant cell line (PC-3/Doc) in parallel with adriamycin (ADR) and marchantin C as
positive references. The cells were treated with each compound for 48 h, and the cell viability was
assessed using the MTT method. The results are summarized in Table 1.

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Among these newly synthesized analogues, compounds 44, 47 and 55 exhibited excellent
cytotoxic activity, with average IC50 values ranging from 1.96 µM to 8.95 µM, and they were more
potent than the parent marchantin C (average IC = 15.13 µM). In addition, compound 47 was the most
50
potent cytotoxic agent with an average IC value of 2.67 µM, which was more potent than that of the
50
reference drug ADR (average IC = 4.44 µM). Moreover, the antiproliferative activity of compound 47
5
0
against the human breast cancer cell line and the paclitaxel-resistant cell line (IC = 1.51 µM for
50
HCC1428 and IC = 2.67 µM for PC-3/Doc, respectively) was obviously superior to that of reference
5
0
ADR (IC = 1.77 µM for HCC1428 and IC = 16.89 µM for PC-3/Doc).
50
50
Fig. 3. Three-dimensional structures of compounds 42, 55, 77, 88, 96, 109, 120 and marchantin C; The
IC50 represents the average IC50 value of each compound against four human cancer cell lines (HeLa,
k562, HCC1428, and HT29); The aromatic carbons that are included in the macrocyclic ring are
indicated by a blue cross symbol.
[
Figure 3 here]
The structure-activity relationships of these novel marchantin C analogues were analyzed as
follows:
Effects of the bisbibenzyl skeletons: As shown in Fig. 3, for the first step, we systematically
modified the marchantin C structure by varying the position of the ether bond on arenes B, C and D
while keeping the hydroxyl groups positioned ortho to the ether bond, which was similar to the

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substitutive patterns of marchantin C. This gave us seven novel macrocyclic analogues with completely
different bisbibenzyl skeletons: 42 (BBM-D-o skeleton), 55 (BBM-D-p skeleton), 77 (BBM-B-m
skeleton), 88 (BBM-C-p skeleton), 96 (BBM-C-o skeleton), 109 (neomarchantin skeleton) and 120
(
riccardin II skeleton). The biological activities of these seven compounds are shown in Table 1. The
results of the antiproliferative evaluation indicated that all seven analogues consistently exhibited
anticancer activity. Interestingly, when the 18-membered macrocyclic ring of marchantin C was
converted to a 17-membered bisbibenzyl ring, the resulting analogues 42 and 96 showed decreased
anticancer potency, with average IC50 values of 18.65 and 18.29 µM, respectively. To our surprise,
when the bisbibenzyl ring of marchantin C was expanded to a 19-membered ring, compounds 55, 77
and 88 displayed enhanced cytotoxicity, with average IC50 values of 9.34, 10.16 and 10.27 µM,
respectively. Unexpectedly, further expansion of the bisbibenzyl to a 20-membered ring led to
compounds 109 and 120 (average IC = 17.42 and 14.93 µM for 109 and 120, respectively), which
5
0
showed a significant decrease in potency compared to compounds 55, 77 and 88. In addition, the
anticancer activities of compounds 109 and 120 were comparable to or even less potent than that of
marchantin C. Based on these data, we find that the trend in anticancer activity for bisbibenzyl
skeletons is 19-membered rings﹥18-membered rings﹥20-membered rings﹥17-membered rings.
These results suggest that the macrocyclic ring strain and conformational changes may affect molecular
antitumor activity to some extent, which is consistent with our prediction mentioned above.
Furthermore, compound 46, which contains a double bond at C-7’ (8’), was also prepared, and this
compound displayed moderate anticancer activity with an average IC50 value of 16.94 µM, which is
comparable to that of compound 42. This indicated that the introduction of a double bond to the
bisbibenzyl skeleton does not significantly impact the anticancer potency.
Effects of the substituents: After confirming the effects of the bisbibenzyl skeleton on the
anticancer activity, we then focused on modifying the bisbibenzyls through introduction of a hydroxyl
or methoxyl group to the arene rings in order to evaluate the effect of the substituent. As shown in Fig.
1
, we started our substituent modification work from compound 55 as a result of its excellent
anticancer potency. However, the introduction of a hydroxyl group at the C-5 position on arene D
decreased the cytotoxicity (average IC = 22.20 µM for compound 65). When the hydroxyl group was
5
0
introduced to another potent compound, 77, at position C-13’ on arene A, the anticancer activity was
also diminished (compound 78, average IC = 15.57 µM). In addition, the introduction of a hydroxyl
50

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group to compounds 109 and 120 also resulted in decreased activity (average IC50 = 25.86 µM and
2
0.00 µM for compounds 110 and 121, respectively).
Surprisingly, introduction of a hydroxyl group to the less potent compound 42 at the C-5 position
of arene D resulted in compound 43 (average IC50 value of 12.18 µM), which exhibited improved
anticancer activity compared to compound 42. In addition, compound 44 (average IC =8.13 µM) with
50
two hydroxyl groups at positions C-4 and C-5 of arene D, displayed a further increase in potency over
compound 43. When the substituent was changed from a hydroxyl group to a methoxyl group on arene
B, the most potent compound 47 was obtained, with an average IC value of 1.96 µM. However, when
50
a hydroxyl group was introduced to the C-13’ position on arene A to give compound 45, the anticancer
activity was distinctly diminished compared to 43. This is consistent with the observations made for
compounds 78 and 121. All of these observations suggest that hydrogen bond donors or acceptors are
present close to arene D, while both arene A and arene B are located near to the hydrophobic pocket.
These findings are consistent with the results of molecular modeling studies discussed below (Fig. 8).
Finally, it is noteworthy that the introduction of a hydroxyl group ortho to the ether bond led to a
decrease in activity (42 vs 45, 55 vs 65, 77 vs 78, 109 vs 110 and 120 vs 121) and that the anticancer
potencies within this series of analogues vary significantly with the substituents on the arene rings.
Table 1. In Vitro cytotoxicity of marchantin C derivatives in four cancer cell lines
a
IC (µM)
5
0
Compound
HeLa
K562
Hcc1428
Ht29
PC-3/Doc
Average
18.65
12.18
8.04
b
42
43
44
45
46
47
55
65
77
78
88
96
21.52±4.10 21.63±3.97 19.04±4.05 12.39±1.87
N/T
b
15.91±3.77 14.05±3.08 10.78±2.07
10.39±2.98 6.16±1.15 8.46±3.69
7.98±1.16
7.51±4.17
N/T
7.70±1.59
b
26.81±4.65 25.29±4.44 22.02±3.57 15.94±2.43
N/T
22.52
16.94
2.67
b
21.86±2.01 19.23±3.08 17.06±2.95
9.60±1.10
1.98±0.23
7.57±1.52
N/T
2.61±0.67
1.74±0.55
8.52±1.00
1.51±0.31
9.25±1.23
5.51±1.41
10.45±2.01
10.92±1.25
9.34
b
27.44±5.89 24.91±5.02 19.49±3.53 16.96±3.70
11.71±2.22 7.02±1.05 10.65±1.97 7.98±1.96
19.52±4.19 10.89±2.30 19.52±2.28 12.36±2.41
15.75±2.58 6.06±1.46 7.69±1.11 11.59±2.96
N/T
22.20
10.16
15.57
10.27
18.29
17.42
25.86
14.93
20.00
14.99
13.43±2.61
b
N/T
b
N/T
b
23.56±4.01 11.92±2.55 21.81±3.02 15.86±3.45
18.89±3.61 15.73±3.90 22.40±2.05 12.65±2.59
28.30±4.65 29.88±4.97 25.15±3.76 20.11±4.24
22.70±3.44 10.25±2.28 15.28±3.28 11.49±2.00
29.45±5.21 15.56±1.99 20.12±4.41 14.85±2.99
N/T
b
1
09
N/T
b
110
N/T
b
1
1
20
21
N/T
b
N/T
Marchantin C 21.22±3.55 12.95±2.03 13.36±2.10 12.99±1.95 14.45±2.88

ACCEPTED MANUSCRIPT
Adriamycin
1.23±0.15
0.57±0.10
1.77±0.23
1.76±0.14
16.89±2.29
4.44
a
The IC values (µM) are the concentrations corresponding to 50% inhibition of each cell line growth;
5
0
b
Mean values based on three independent experiments; N/T means not tested.
Table 1 here]
[
The antiproliferative activity of the most potent compound 47 in HCC1428 cells was further
monitored using the xCELLigence system, and vincristine (VCR) was used as a positive control. As
shown in Fig. 4, compound 47 inhibited HCC1428 cell proliferation within 5 h and decreased the
number of cells markedly within 20 h, which shows a role similar to VCR. After treatment for
approximately 60 h, the effect of 47 was close to that of VCR. The cell growth curve shows that
compound 47 inhibited HCC1428 cell proliferation and subsequently induced cell death.
Fig. 4. HCC1428 cells were treated with compound 47 as indicated. Cells were then plated 2000
cells/well into E-plate 16 and analyzed using a xCELLigence RTCA DP instrument. The
results are representative of three independent experiments.
[
Figure 4 here]
2
.3.2. Cell cycle analysis
After the antiproliferative activity evaluation, we extended our work to a mechanistic study. Flow
cytometry was used to analyze the effects of the marchantin C analogues on cell growth and division.
HCC1428 cells were treated with compound 47 for 24 h. DMSO was used as a vehicle control, and
VCR was used as a positive control. As shown in Fig. 5, in the vehicle group, 12.93% of the HCC1428
cells were in the G /M phase. Compound 47 increased the proportion of cells in the G /M phase to
2
2
approximately 37.70% and 85.34% when treated for 24 h at concentrations of 3 µM and 6 µM,
respectively. These results demonstrate that compound 47 induces cell cycle arrest at the G /M phase in
2

ACCEPTED MANUSCRIPT
a dose-dependent manner, which is consistent with the results obtained for classical tubulin-targeting
drugs.
Fig. 5. Compound 47 induced HCC1428 cell cycle arrest at the G /M phase. HCC1428 cells were
2
treated with 3 µM and 6 µM concentrations of compound 47 for 24 h, and then trypsinized, fixed and
stained with PI to measure the cell cycle profile by flow cytometry. Control cells were treated with
DMSO alone. The results are representative of three independent experiments.
[
Figure 5 here]
2
.3.3. Immunofluorescence staining
The significant cell growth inhibitory properties of the marchantin C analogues and their obvious
G /M phase arresting properties encouraged us to further investigate the biological mechanism. Given
2
the microtubule-depolymerization activity of marchantin C in culture cells, we next examined the
effect of compound 47 on the cytoskeleton network using immunofluorescent staining techniques. The
cellular microtubule networks were visualized by confocal microscopy. As shown in Fig. 6, intact
microtubule arrays could be observed in untreated cells. However, during treatment with increasing
dosages of 47, the microtubule networks decreased and dispersed in the cytoplasm, which was similar
to the result observed for a 1 µM VCR treatment. These results indicate that 47 affects the cellular
microtubule dynamics. In addition, effect of the less potent compound 77 on the cytoskeleton network
was also tested. As shown in Fig. 6, compound 77 displayed very weak cytoskeleton network
disrupting activity at the concentration of 3 µM compared with compound 47, and as increasing the
dosages of 77 to 6 µM and 10 µM, the microtubule networks decreased and dispersed, obviously,
which indicated that the antiproliferative activity of these compounds was closely related to the
inhibition of tubulin polymerization.

ACCEPTED MANUSCRIPT
Fig. 6. HCC1428 cells were treated with compound 47 and 77 as indicated for 24 h and then fixed and
immunostained with monoclonal anti-α-tubulin antibody (red) and DAPI (blue). One micromolar
concentrations of VCR and DMSO were used as controls. The results are representative of three
independent experiments. Bar = 10 µm.
[
Figure 6 here]
2
.3.4. Tubulin polymerization inhibition
We next investigated the inhibition of tubulin polymerization by the selected potent compound 47

ACCEPTED MANUSCRIPT
and compared it to the results from a positive control (VCR) and a negative control (taxol). DMSO was
used as a blank control. Bovine brain tubulin was incubated with compound 47 at concentrations of 5
and 10 µM. VCR and taxol were both used at a concentration of 5 µM. As shown in Fig. 7, paclitaxel
caused an immediate and significant increase in the absorbance, indicating an enhancement of tubulin
polymerization. In contrast, VCR and 47 caused a decrease in the absorbance, indicating that the
tubulin polymerization was inhibited. Compound 47 showed stronger inhibition than VCR at the two
tested concentrations. These results clearly indicate that compound 47 significantly inhibits the
polymerization of tubulin in vitro.
Fig. 7. Effect of compound 47 on tubulin polymerization in vitro. Purified bovine brain tubulin
was incubated in the presence of taxol, VCR, DMSO (control), and compound 47 under the
indicated concentrations at 37℃, and absorbance readings were recorded every minute for 1 h.
[
Figure 7 here]
2
.4. Molecular modeling
The excellent bioactivity of bisbibenzyl derivatives encouraged us to investigate the possible
mechanism of action at the molecular level, and more specifically, the binding mode of active
compound 47 to tubulin. In a previous study, a macrocyclic bisbibenzyl compound was found to be a
colchicine site binder on tubulin. We anticipated that 47 would also target the colchicine-binding site in
tubulin due to its bisbibenzyl skeleton and microtubule-depolymerizing activity. Compound 47 was
then docked into the colchicine-binding site on tubulin using the GOLD (Genetic Optimization for
Ligand Docking) program. The binding mode of compound 47 was studied further by energy
minimization. In the resulting hypothetical structure (Fig. 8A), the hydroxyl group on C-4 of arene D
may form hydrogen bonds with the residues Lys 352 and Asn 349. In addition, the hydroxyl group on
C-5 of arene D is involved in a potential hydrogen bond with the carbonyl group of Val 315. It is also
possible that arene B forms a CH-π interaction with the methyl group of Leu 248. We believe that the

ACCEPTED MANUSCRIPT
combination of the CH-π interaction and the hydrogen bonds could play a crucial role in the excellent
antitubulin activity of derivative 47. Furthermore, as shown in Fig. 8B, the methoxyl group on arene B
could fit well into the hydrophobic pocket formed by residues Gln 247, Leu 248, Lys 352 and Ala 354,
which might contribute to its enhanced potency compared to compound 44.
Fig. 8. Proposed binding mode of compound 47 in the colchicine-binding site (PDB code 1SA0). (A)
The binding mode of 47 (cyan) in the binding site with hydrogen bonds shown as dotted red where the
distance between the ligand and the protein is less than 3 Å. (B) Docking of 47 (grey) into the binding
site overlaid with a model of DAMA-colchicine (magenta stick). The methoxyl group could fit well
into the hydrophobic pocket formed by residues Gln 247, Leu 248, Lys 352 and Ala 354.
[
Figure 8 here]
3
. Conclusions
In this report, a series of novel marchantin C analogues was designed, synthesized and evaluated
for their antiproliferative activity against five anthropic cancer cell lines, resulting in the discovery of
several novel antitumor agents as tubulin polymerization inhibitors. Some of those analogues were also
effective against the paclitaxel-resistant cancer cell line. Compound 47 was identified as the most
potent compound, with IC50 values of 1.51-5.51 µM, which were comparable or superior to the
reference drug ADR and much more potent than the parent marchantin C. As a probe in mechanistic
studies, compound 47 arrested cancer cells in the G2/M phase in a concentration-dependent manner
and disrupted cellular microtubules. Further mechanistic studies confirmed that compound 47 gained
its activity by inhibiting tubulin polymerization at the colchicine-binding site. Molecular modeling
provided insight into the binding mode of the marchantin C analogue in tubulin. Structure-activity
relationships concluded that (1) the macrocyclic bisbibenzyl skeleton is the basic structure for the

ACCEPTED MANUSCRIPT
antitubulin activity, (2) strain within the macrocyclic ring affects the molecular antitumor activity to
some extent, with a 19-membered ring being most active, and (3) the nature and location of the
substituents on the arene rings play a critical role in the antiproliferative activity. These marchantin C
analogues with novel bisbibenzyl skeletons represent a new class of tubulin inhibitors, and compound
4
4
4
7 could serve as a lead compound for further optimization in the discovery of novel antimitotic agents.
. Experimental section
.1. Chemistry
Chemicals were commercially available and used as received without further purification.
Solvents (THF, MeCN and DCM) were dried and freshly distilled before use according to procedures
reported in the literature. Reactions were monitored by thin-layer chromatography using Merck plates
with fluorescent indicator. Column chromatography was carried out on silica gel or alumina (200-300
13
mesh). The NMR spectra were recorded on a Bruker Spectrospin spectrometer at 600 MHz ( C NMR
at 150 MHz) using TMS as an internal standard. The chemical shifts are reported in parts per million
1
(
ppm δ) referenced to the residual H resonance of the solvent (CDCl , 7.28 ppm). Abbreviations used
3
in the splitting pattern were as follows: s=singlet, d=doublet, t=triplet, quin=quintet, m=multiplet, and
br=broad. Mass spectral analyses were performed at the Shandong University-Chemical Analysis
Center. All HRMS spectra (ESI) were obtained on an LTQ Orbitrap mass spectrometer.
General procedure 1 (GP 1) for the Ullmann reaction
A mixture of phenol (0.05 mol) (compounds 3, 8, 67, and 97-98), bromo-benzaldehyde (1.05
equiv.) (compounds 1-2, 9-12, 48, 56, and 66), potassium carbonate (2 equiv.) and cupric oxide (0.3
equiv.) in pyridine (50 mL) was stirred under reflux for 12 h. The pyridine was distilled off in vacuo
and the residue was extracted with EtOAc (200 mL). The solution was concentrated and the residue
was purified by flash column chromatography (SiO or Al O ), eluting with a hexane-DCM mixture, to
2
2
3
yield the pure product as a solid in moderate yield.
General procedure 2 (GP 2) for the preparation of the phosphonium salts
Benzyl alcohol (25 mmol) (compounds 4-5, 22-26, 51, 58, 61, 69-70, 81, 85, 90, 93, 103-104, and
1
14-115) and triphenylphosphonium bromide (1.05 quiv.) were dissolved in anhydrous CH CN (50
3
mL), the resulting mixture was then refluxed for 3 h, the solvent was removed in vacuo and the residue
was purified by silica gel column chromatography, eluting with 5% methanol in DCM, to yield the
pure product as a white solid in satisfactory yield.

ACCEPTED MANUSCRIPT
General procedure 3 (GP 3) for the intramolecular Wittig reaction
A solution of phosphonium salt (compounds 27-31, 52, 62, 71-72, 86, 94, 103-104, and 114-115)
in anhydrous DCM (150 mL/mmol) was added dropwise (5 h/mmol) to a suspension of MeONa (8
equiv.) in anhydrous DCM (50 mL/mmol). The reaction mixture was stirred for 15 h at room
temperature. Insoluble material was filtered off, the solvent was removed in vacuo and the residue was
purified by silica gel column chromatography, eluting with DCM, to provide the desired compound in
satisfactory yield.
General procedure 4 (GP 4) for the catalytic hydrogenation of stilbenes and benzyl ethers
Palladium on activated carbon (10% Pd, 100 mg/mmol) was added to a solution of stilbene or
benzyl ether (compounds 37-41, 54, 64, 75-76, 87, 95, 107-108, and 118-119) in EtOAc (100-150 mL).
The suspension was stirred under H for 24 h at room temperature. The reaction mixture was filtered,
2
and the solution was concentrated to provide the product as a white solid in satisfactory yield.
General procedure 5 (GP 5) for the cleavage of aryl methyl ethers
A solution of boron tribromide (8 equiv.) in anhydrous DCM (10 mL) was added dropwise to a
stirred solution of aryl methyl ether (compounds 42-45, 46-47, 55, 65, 77-78, 88, 96, 109-110, and
1
20-121) (1 mmol) in anhydrous DCM (10 mL) at -78 ˚C. The reaction mixture was stirred at -78 ˚C
for 3 h and then was allowed to warm up to room temperature within 12 h. Ice-cold water was then
added, and the reaction mixture was stirred vigorously for 1 h. The solution was then diluted with
DCM (50 mL), washed with sat aq NaCl and dried over sodium sulfate. The solution was concentrated,
and the residue was purified by silica gel column chromatography, eluting with DCM, to provide a
white solid in satisfactory yield.
Methyl 4-(5-formyl-2-methoxyphenoxy) benzoate (4).
This compound was prepared from methyl 4-bromobenzoate (1) and 3-hydroxy-4-methoxy-
1
benzaldehyde (3) in 76% yield by following GP 1. Orange solid; mp 118-120 ˚C. H NMR (CDCl ) δ
3
9
8
.87 (s, 1 H), 8.02-7.99 (m, 2 H), 7.76 (dd, J = 8.2, 2.1 Hz, 1 H), 7.59 (d, J = 2.1 Hz, 1 H), 7.14 (d, J =
+
.3 Hz, 1 H), 6.94 (d, J = 8.7 Hz, 2 H), 3.91 (s, 3 H), 3.90 (s, 3 H); MS (ESI) 287 (M+H) .
Methyl 4-(5-formyl-2-methoxyphenoxy)-3-methoxybenzoate (5).
This compound was prepared from 3-hydroxy-4-methoxybenzaldehyde (3) and methyl
1
4
-bromo-3-methoxybenzoate (2) in 63% yield by following GP 1. Yellow solid; mp 109-110 ˚C. H
NMR (CDCl ) δ 9.83 (s, 1 H), 7.70 (d, J = 1.8 Hz, 1 H), 7.69 (s, 1 H), 7.61 (dd, J = 1.8 Hz, 8.4 Hz, 1
3

ACCEPTED MANUSCRIPT
H), 7.42 (d, J = 1.8 Hz, 1 H), 7.13 (d, J = 8.4 Hz, 1 H), 6.81 (d, J = 8.4 Hz, 1 H), 3.95 (s, 3 H), 3.94 (s,
+
3
H), 3.93 (s, 3 H); MS (ESI) 317 (M+H) .
(4-Methoxy-3-(4-(methoxycarbonyl)phenoxy)benzyl)triphenylphosphonium Bromide (6).
Sodium borohydride (0.34 g, 9.16 mmol) was added to a solution of 4 (7.21 g, 22.81 mmol) in
THF (25 mL) over 15 min at 0 ˚C. The reaction mixture was then stirred at room temperature for 3 h.
Water (10 mL) and 1M HCl (10 mL) were added and the THF was evaporated in vacuo. The resulting
mixture was extracted with DCM (15 mL), washed with sat aq NaCl, and dried over sodium sulfate.
The solution was concentrated to obtain a crude oil. This crude oil and triphenylphosphonium bromide
(
23 mmol) were then dissolved in anhydrous CH CN (50 mL), the resulting mixture was then refluxed
3
for 3 h, the solvent was removed in vacuo and the residue was purified by silica gel column
chromatography, eluting with 5% methanol in DCM, to yield the pure product as a white solid, mp
1
2
17-219 ˚C. H NMR (CDCl ) δ 7.90 (d, J = 9.0 Hz, 2 H), 7.80-7.73 (m, 9 H), 7.65-7.58 (m, 6 H), 7.27
3
(
(
s, 1 H), 6.84 (d, J = 8.4 Hz, 1 H), 6.69 (d, J = 8.7 Hz, 2 H), 6.51 (s, 1 H), 5.46 (d, J = 14 Hz, 2 H), 3.90
+
s, 3 H), 3.73 (s, 3 H); MS (ESI) 533 (M-Br) .
(4-Methoxy-3-(2-methoxyl-4-(methoxycarbonyl)phenoxy)benzyl)triphenylphosphonium Bromide
(7).
This compound was prepared from compound 5 by following the procedure described for 6, yield
1
7
8%, white solid; mp 230-231 ˚C. H NMR (CDCl ) δ 7.83-7.70 (m, 9 H), 7.67-7.60 (m, 6 H), 7.57 (d,
3
J = 1.8 Hz, 1 H), 7.36 (s, 1 H), 7.03 (d, J = 1.8 Hz, 1 H), 6.91 (dd, J = 1.8 Hz, 8.4 Hz, 1 H), 6.87 (d, J =
8
.4 Hz, 1 H), 6.79 (d, J = 8.4 Hz, 1 H), 5.40 (d, J = 14 Hz, 2 H), 3.92 (s, 3 H), 3.89 (s, 3 H), 3.83 (s, 3
+
H); MS (ESI) 563 (M-Br) .
2
-(2-(1,3-Dioxan-2-yl)-6-methoxyphenoxy)benzaldehyde (13).
1
This compound was prepared from 8 and 9 in 69% yield by following GP 1, yellow oil. H NMR
(
CDCl ) δ 10.78 (s, 1H), 7.92 (dd, J = 7.7, 1.6 Hz, 1H), 7.41-7.38 (m, 1H), 7.36 (d, J = 7.9 Hz, 1H),
3
7
5
1
.29 (t, J = 10.9 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 6.62 (d, J = 8.4 Hz, 1H),
.69 (s, 1H), 4.14 (d, J = 7.4 Hz, 2H), 3.83 (t, J = 11.5 Hz, 2H), 3.72 (s, 3H), 2.18 (qd, J = 12.6, 6.3 Hz,
+
H), 1.37 (d, J = 13.5 Hz, 1H); MS (ESI) 315 (M+H) .
2
-(2-(1,3-Dioxan-2-yl)-6-methoxyphenoxy)-5-methoxybenzaldehyde (14).
This compound was prepared from 8 and 10 in 67% yield by following GP 1, colorless oil. H
NMR (CDCl ) δ 10.76 (s, 1 H), 7.39 (d, J = 3.0 Hz, 1 H), 7.36 (d, J = 7.8 Hz, 1 H), 7.28 (dd, J = 3.0 Hz,
1
3

ACCEPTED MANUSCRIPT
7
1
1
.8 Hz, 1 H), 7.00 (d, J = 7.2 Hz, 2 H), 6.61 (d, J = 9.0 Hz, 1 H), 5.73 (s, 1 H), 4.18 (dd, J = 4.8 Hz,
0.8 Hz, 2 H), 3.88 (t, J = 10.8 Hz, 2 H), 3.84 (s, 3 H), 3.72 (s, 3 H), 2.25-2.16 (m, 1 H), 1.39 (d, J =
+
3.2 Hz, 1 H); MS (ESI) 345 (M+H) .
2
-(2-(1,3-Dioxan-2-yl)-6-methoxyphenoxy)-4,5-dimethoxybenzaldehyde (15).
This compound was prepared from 8 and 11 in 76% yield by following GP 1, colorless oil. H
NMR (CDCl ) δ 10.62 (s, 1H), 7.37 (s, 1H), 7.35 (dd, J = 8.2, 1.1 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H),
1
3
6
3
.99 (dd, J = 8.1, 1.1 Hz, 1H), 6.24 (s, 1H), 5.76 (s, 1H), 4.18 (dd, J = 10.7, 3.5 Hz, 2H), 3.91 (s, 3H),
.87 (t, J = 11.5 Hz, 2H), 3.72 (s, 3H), 3.68 (s, 3H), 2.36-2.11 (m, 1H), 1.40 (d, J = 13.5 Hz, 1H) ; MS
+
(
ESI) 375 (M+H) .
6
-(2-(1,3-Dioxan-2-yl)-6-methoxyphenoxy)benzo[d][1,3]dioxole-5-carbaldehyde (16).
1
This compound was prepared from 8 and 12 in 76% yield by following GP 1, colorless oil. H
NMR (CDCl ) δ 10.58 (s, 1H), 7.35 (d, J = 7.0 Hz, 1H), 7.32 (s, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.00 (d,
3
J = 7.3 Hz, 1H), 6.16 (s, 1H), 5.98 (s, 2H), 5.69 (s, 1H), 4.18 (d, J = 7.2 Hz, 2H), 3.87 (t, J = 11.4 Hz,
+
2
H), 3.75 (s, 3H), 2.26-2.13 (m, 1H), 1.39 (d, J = 13.5 Hz, 1H) ; MS (ESI) 359 (M+H) .
Methyl 4-(5-(2-(2-(1,3-dioxan-2-yl)-6-methoxyphenoxy)phenethyl)-2-methoxyphenoxy)-benzoate
(17).
Potassium carbonate (4.45 g, 32.22 mmol) and a trace of 18-crown-6 were added to a solution of 6
10.34 g, 16.11 mmol) and 13 (5.06 g, 16.06 mmol) in anhydrous DCM, the resulting mixture was
(
stirred under reflux for 24 h. The insoluble material was then filtered off and the filtrate was
concentrated to provide the orange oil that was purified by flash column chromatography (Al O ),
2
3
eluting with a 3:1 solution of hexane-dichloromethane to afford a mixture of Z/E isomers as a yellow
oil. Pd/C 10% (0.7 g) and triethylamine (16 mL) were then added to the solution of isomers in ethyl
acetate (100 mL). The suspension was stirred under H for 24 h at room temperature. The mixture was
2
1
filtered, and concentrated to afford 17 (6.87 g, 75%) as colorless oil. H NMR (CDCl ) δ 8.02-7.92 (m,
3
2
H), 7.37 (dd, J = 7.9, 1.4 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 7.12 (dd, J = 8.3, 2.1 Hz, 1H), 7.06 (dd, J
7.4, 1.6 Hz, 1H), 7.04-7.00 (m, 1H), 6.98 (dd, J = 8.2, 1.4 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.95 (d,
J = 2.1 Hz, 1H), 6.91-6.86 (m, 3H), 6.43 (d, J = 8.2 Hz, 1H), 5.67 (s, 1H), 4.15 (dd, J = 10.7, 3.5 Hz,
=
2
H), 3.91 (s, 3H), 3.79 (s, 3H), 3.66 (s, 3H), 2.26-2.15 (m, 1H), 1.37-1.31 (m, 1H); MS (ESI) 571
+
(
M+H) .
Methyl 4-(5-(2-(2-(1,3-dioxan-2-yl)-6-methoxyphenoxy)-5-methoxyphenethyl)-2-methoxyphenoxy)

ACCEPTED MANUSCRIPT
benzoate (18).
This compound was prepared from 6 and 14 by following the procedure described for 17, yield
1
7
7
6
3
1
1%, colorless oil. H NMR (CDCl ) δ 7.97 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 7.4 Hz, 1H), 7.24 (t, J =
3
.7 Hz, 1H), 7.12 (d, J = 7.5 Hz, 1H), 6.97 (d, J = 7.6 Hz, 3H), 6.89 (d, J = 8.2 Hz, 2H), 6.64 (s, 1H),
.54 (d, J = 8.3 Hz, 1H), 6.36 (d, J = 8.7 Hz, 1H), 5.68 (s, 1H), 4.16 (d, J = 9.1 Hz, 2H), 3.91 (s, 3H),
.84 (t, J = 10.8 Hz, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.66 (s, 3H), 3.09-2.96 (m, 4H), 2.27-2.13 (m, 1H),
+
.35 (d, J = 13.7 Hz, 1H); MS (ESI) 601 (M+H) .
Methyl
4-(5-(2-(2-(1,3-dioxan-2-yl)-6-methoxyphenoxy)-4,5-dimethoxyphenethyl)-2-methoxy-
phenoxy) benzoate (19).
This compound was prepared from 6 and 15 by following the procedure described for 17, yield
1
7
8
1
3
9%, colorless oil. H NMR (CDCl ) δ 8.01-7.91 (m, 2H), 7.36 (dd, J = 7.9, 1.4 Hz, 1H), 7.25 (t, J =
3
.0 Hz, 1H), 7.09 (dd, J = 8.4, 2.1 Hz, 1H), 6.99-6.94 (m, 3H), 6.91-6.84 (m, 2H), 6.56 (s, 1H), 6.15 (s,
H), 5.71 (s, 1H), 4.17 (dd, J = 11.4, 4.6 Hz, 2H), 3.90 (s, 3H), 3.89-3.82 (m, 2H), 3.79 (s, 3H), 3.79 (s,
H), 3.66 (s, 3H), 3.60 (s, 3H), 3.10-2.96 (m, 4H), 2.24-2.20 (m, 1H), 1.40-1.33 (m, 1H); MS (ESI) 631
+
(
M+H) .
Methyl 4-(5-(2-(2-(1,3-dioxan-2-yl)-6-methoxyphenoxy)-5-methoxyphenethyl)-2-methoxy- phenoxy)
-
3-methoxybenzoate (20)
This compound was prepared from 7 and 14 by following the procedure described for 17, yield
1
8
7
1
3
2
0%, colorless oil. H NMR (CDCl ) δ 7.64 (s, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 7.0 Hz, 1H),
3
.24 (t, J = 7.0 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H), 7.02-6.90 (m, 3H), 6.64 (s, 1H), 6.61 (d, J = 8.1 Hz,
H), 6.53 (d, J = 8.3 Hz, 1H), 6.36 (d, J = 8.6 Hz, 1H), 5.68 (s, 1H), 4.16 (d, J = 8.8 Hz, 2H), 3.98 (s,
H), 3.91 (s, 3H), 3.86-3.84 (m, 2H), 3.78 (s, 3H), 3.72 (s, 3H), 3.67 (s, 3H), 3.17-2.96 (m, 4H),
+
.29-2.14 (m, 1H), 1.36-1.34 (m, 1H); MS (ESI) 631 (M+H) .
Methyl 4-(5-(2-(5-(2-(1,3-dioxan-2-yl)-6-methoxyphenoxy)benzo[d][1,3]dioxol-6-yl) ethyl)-2-
methoxyphenoxy)benzoate (21)
This compound was prepared from 6 and 16 by following by following the procedure described
1
for 17, yield 74%, colorless oil. H NMR (CDCl ) δ 7.32 (dd, J = 7.6, 1.4 Hz, 1H), 7.20 (t, J = 8.0 Hz,
3
1
H), 7.15 (s, 1H), 7.10 (d, J = 8.1 Hz, 2H), 6.88 (dd, J = 8.0, 2.1 Hz, 1H), 6.80-6.76 (m, 2H), 6.70 (d, J
8.4 Hz, 2H), 6.62 (d, J = 1.9 Hz, 1H), 6.43 (s, 1H), 5.98 (s, 1H), 5.79 (s, 2H), 5.65 (s, 1H), 4.16 (d, J
=

ACCEPTED MANUSCRIPT
=
7.3 Hz, 2H), 3.83-3.76 (m, 2H), 3.70 (s, 3H), 3.66 (s, 3H), 3.59 (s, 3H), 2.97-2.91 (m, 2H), 2.86-2.80
+
(
m, 2H), 2.20-2.14 (m, 1H), 1.34 (d, J = 13.6 Hz, 1H); MS (ESI) 615 (M+H) .
2
-(2-(3-(4-(Hydroxymethyl)phenoxy)-4-methoxyphenethyl)phenoxy)-3-methoxybenzaldehyde (22).
A solution of 17 (13.11 g, 23.72 mmol) in THF (25 mL) was added dropwise to a stirred
suspension of lithium aluminum hydride (1.75 g, 46.07 mmol) in anhydrous THF (30 mL). The
resulting mixture was stirred at room temperature for 2.5 h and carefully hydrolysed with sat aq NH Cl
4
(
10 mL). THF was removed in vacuo and the resulting mixture was diluted with DCM (70 mL),
washed with saturated aqueous NaCl and dried over sodium sulfate. The solvent was removed in vacuo
to yield crude oil. The crude oil was then dissolved in a solution of ethanol (100 mL) and 10 % aq HCl
(
20 mL). The resulting mixture was then stirred at room temperature for 12 h. Sat aq sodium
bicarbonate was added and the ethanol was removed in vacuo. The resulting mixture was extracted
with DCM, washed with saturated aqueous NaCl and dried over sodium sulfate. The solution was
1
concentrated to yield 22 (9.20 g, 80%) as colorless oil. H NMR (CDCl ) δ 10.19 (s, 1H), 7.57 (dd, J =
3
7
.9, 1.5 Hz, 1H), 7.32 (td, J = 8.0, 0.7 Hz, 1H), 7.28-7.26 (m, 2H), 7.25 (dd, J = 8.2, 1.5 Hz, 1H), 7.10
(
dd, J = 7.5, 1.6 Hz, 1H), 7.06-7.01 (m, 2H), 6.95 (d, J = 8.4 Hz, 1H), 6.93 (td, J = 7.6, 1.6 Hz, 1H),
6
3
.90-6.86 (m, 2H), 6.84 (d, J = 2.0 Hz, 1H), 6.40 (dd, J = 8.2, 0.7 Hz, 1H), 4.64 (s, 2H), 3.83 (s, 3H),
+
.71 (s, 3H), 3.13-2.99 (m, 4H); MS (ESI) 485 (M+H) .
2
-(2-(3-(4-(Hydroxymethyl)phenoxy)-4-methoxyphenethyl)-4-methoxyphenoxy)-3-methoxybenzald
ehyde (23).
This compound was prepared from compound 18 by following the procedure described for 22,
1
yield 78%, colorless oil. H NMR (CDCl ) δ 10.20 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.0 Hz,
3
1
H), 7.21 (t, J = 8.0 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 6.97-6.90 (m, 3H), 6.86 (d, J = 8.0 Hz, 2H), 6.60
(
(
s, 1H), 6.56 (d, J = 8.0 Hz, 1H), 6.30 (d, J = 8.6 Hz, 1H), 4.59 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.66
+
s, 3H), 3.07-2.98 (m, 4H); MS (ESI) 515 (M+H) .
2
-(2-(3-(4-(Hydroxymethyl)phenoxy)-4-methoxyphenethyl)-4,5-dimethoxyphenoxy)-3-methoxybenz
aldehyde (24).
This compound was prepared from compound 19 by following the procedure described for 22,
1
yield 71%, colorless oil. H NMR (CDCl ) δ 10.19 (s, 1H), 7.56 (dd, J = 7.9, 1.5 Hz, 1H), 7.30 (td, J =
3
8
.0, 0.8 Hz, 1H), 7.28-7.24 (m, 2H), 7.23 (dd, J = 8.2, 1.5 Hz, 1H), 6.98 (dd, J = 8.3, 2.1 Hz, 1H), 6.93
(
d, J = 8.4 Hz, 1H), 6.89-6.85 (m, 2H), 6.78 (d, J = 2.1 Hz, 1H), 6.57 (s, 1H), 6.00 (s, 1H), 4.62 (s, 2H),

ACCEPTED MANUSCRIPT
+
3
.83 (s, 3H), 3.77 (s, 3H), 3.70 (s, 3H), 3.60 (s, 3H), 3.05-2.93 (m, 4H); MS (ESI) 545 (M+H) .
-(2-(3-(4-(Hydroxymethyl)-2-methoxyphenoxy)-4-methoxyphenethyl)-4-methoxyphenoxy)-3-meth
2
oxybenzaldehyde (25).
This compound was prepared from compound 20 by following by following the procedure
1
described for 22, yield 71%, colorless oil. H NMR (CDCl ) δ 10.19 (s, 1H), 7.69 (s, 1H), 7.56 (d, J =
3
8
3
2
.0 Hz, 1H), 7.30 (d, J = 7.4 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H), 7.00-6.85 (m,
H), 6.61 (s, 1H), 6.60 (d, J = 8.0 Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H), 6.37 (d, J = 8.0 Hz, 1H), 4.68 (s,
+
H), 3.88 (s, 3H), 3.74 (s, 3H), 3.70 (s, 3H), 3.66 (s, 3H), 3.17-2.96 (m, 4H); MS (ESI) 545 (M+H) .
2
-(5-(3-(4-(Hydroxymethyl)phenoxy)-4-methoxyphenethyl)benzo[d][1,3]dioxol-6-yloxy)-3-methox
ybenzaldehyde (26).
This compound was prepared from compound 21 by following by following the procedure
1
described for 22, yield 76%, colorless oil. H NMR (CDCl ) δ 10.06 (s, 1H), 7.46 (dd, J = 7.8, 1.3
3
Hz, 1H), 7.21 (t, J = 8.1 Hz, 1H), 7.17 (s, 1H), 7.14 (d, J = 8.1 Hz, 2H), 6.90 (dd, J = 8.3, 1.9 Hz,
1
1
H), 6.87-6.82 (m, 2H), 6.80 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 1.9 Hz, 1H), 6.47 (s, 1H), 5.91 (s,
H), 5.77 (s, 2H), 4.55 (s, 2H), 3.75 (s, 3H), 3.64 (s, 3H), 2.95-2.89 (m, 2H), 2.87-2.82 (m, 2H);
+
MS (ESI) 529 (M+H) .
2
-(2-(3-(4-((Bromotriphenylphosphoranyl)methyl)phenoxy)-4-methoxyphenethyl)phenoxy)-3-meth
oxybenzaldehyde (27).
This compound was prepared from 22 in 88% yield by following GP 2, white solid; mp 167-168
1
˚
C. H NMR (CDCl ) δ 10.19 (s, 1H), 7.80-7.72 (m, 9H), 7.68-7.65 (m, 6H), 7.49 (td, J = 7.6, 2.9 Hz,
3
2
6
3
H), 7.33 (t, J = 7.9 Hz, 1H), 7.09 (d, J = 6.9 Hz, 1H), 6.99 (dd, J = 8.2, 1.7 Hz, 4H), 6.92-6.88 (m, 2H),
.78 (d, J = 2.0 Hz, 1H), 6.67 (d, J = 7.8 Hz, 2H), 6.37 (d, J = 8.0 Hz, 1H), 5.34 (d, J = 13.6 Hz, 2H),
+
.80 (s, 3H), 3.70 (s, 3H), 3.10-2.97 (m, 4H); MS (ESI) 729 (M-Br) .
2
-(2-(3-(4-((Bromotriphenylphosphoranyl)methyl)phenoxy)-4-methoxyphenethyl)-4-methoxypheno
xy)-3-methoxybenzaldehyde (28).
This compound was prepared from 23 in 90% yield by following GP 2, white solid; mp 155-156
1
˚
C. H NMR (CDCl ) δ 10.20 (s, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.82-7.76 (m, 9H), 7.67-7.66 (m, 6H),
3
7
.36 (d, J = 8.0 Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 6.99-6.93 (m, 3H), 6.87 (d, J
=
8.6 Hz, 2H), 6.62 (s, 1H), 6.59 (d, J = 8.0 Hz, 1H), 6.33 (d, J = 8.0 Hz, 1H), 5.35 (d, J = 13.4 Hz, 2H),
+
3
.81 (s, 3H), 3.74 (s, 3H), 3.68 (s, 3H), 3.04-2.95 (m, 4H); MS (ESI) 759 (M-Br) .

ACCEPTED MANUSCRIPT
2
-(2-(3-(4-((Bromotriphenylphosphoranyl)methyl)phenoxy)-4-methoxyphenethyl)-4,5-dimethoxyp
henoxy)-3-methoxybenzaldehyde (29).
This compound was prepared from 24 in 90% yield by following GP 2, white solid; mp 174-175
1
˚
C. H NMR (CDCl ) δ 10.24 (s, 1H), 7.80-7.72 (m, 9H), 7.69-7.65 (m, 6H), 7.31 (d, J = 7.8 Hz, 1H),
3
7
1
2
.30 (d, J = 1.5 Hz, 1H), 7.02 (d, J = 8.4 Hz, 2H), 6.96 (dd, J = 8.3, 2.0 Hz, 2H), 6.90 (d, J = 7.8 Hz,
H), 6.78 (d, J = 2.0 Hz, 1H), 6.70 (d, J = 8.1 Hz, 2H), 6.60 (s, 1H), 6.01 (s, 1H), 5.38 (d, J = 13.6 Hz,
H), 3.81 (s, 3H), 3.78 (s, 3H), 3.71 (s, 3H), 3.59 (s, 3H), 3.02-2.99 (m, 2H), 2.94-2.90 (m, 2H); MS
+
(
ESI) 789 (M-Br) .
2
-(2-(3-(4-((Bromotriphenylphosphoranyl)methyl)-2-methoxyphenoxy)-4-methoxyphenethyl)-4-me
thoxyphenoxy)-3-methoxybenzaldehyde (30).
This compound was prepared from 25 in 87% yield by following GP 2, white solid; mp 156-157
1
˚
C. H NMR (CDCl ) δ 10.20 (s, 1H), 7.86-7.75 (m, 9H), 7.72-7.65 (m, 6H), 7.67 (s, 1H), 7.55 (d, J =
3
8
3
.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 8.4 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 7.05-6.92 (m,
H), 6.64 (s, 1H), 6.61 (d, J = 8.0 Hz, 1H), 6.57 (d, J = 8.0 Hz, 1H), 6.32 (d, J = 8.0 Hz, 1H), 5.36 (d, J
=
13.6 Hz, 2H), 3.87 (s, 3H), 3.73 (s, 3H), 3.68 (s, 3H), 3.65 (s, 3H), 3.12-2.90 (m, 4H); MS (ESI) 789
+
(
M-Br) .
Macrocycle (stilbene bridge) (32).
This compound was prepared from 27 in 83% yield by following GP 3, white solid; mp 162-163
1
˚
C. H NMR (CDCl ) δ 7.27 (d, J = 6.0 Hz, 1H ), 7.23 (d, J = 8.0 Hz, 1H), 7.13 (dd, J = 1.2 Hz, J = 7.6
3
Hz, 1H), 7.01 (dd, J = 1.2 Hz, J = 8.4 Hz, 1H), 6.97-6.96 (m, 1H ), 6.92 (d, J = 8.0 Hz, 1H), 6.90-6.87
(
m, 2H), 6.77-6.72 (m, 3H), 6.26 (d, J = 8.0 Hz, 1H), 6.12 (d, J = 16.0 Hz, 1H), 5.21 (d, J = 2.0 Hz,
1
3
1
1
H), 4.00 (s, 3H), 3.88 (s, 3H), 3.01-2.71 (m, 4H); C NMR (CDCl ) δ 165.5, 165.4, 153.3,151.7,
3
46.2, 142.1, 140.2, 134.3, 133.2, 131.6, 128.1, 127.7, 125.8, 123.4, 123.3, 121.7, 121.2, 118.4, 113.8,
+
1
12.9, 112.0, 119.9, 56.3, 56.2, 29.0, 24.0. MS (ESI) 451 (M+H) .
Macrocycle (stilbene bridge) (33).
This compound was prepared from 28 in 83% yield by following GP 3, white solid; mp 186-187
1
˚
C. H NMR (CDCl ) δ 7.31 (s, 1 H), 7.24 (t, J = 7.8 Hz, 1 H), 7.13 (d, J = 7.8 Hz, 2 H), 7.00 (d, J =
3
8
6
.4 Hz, 2 H), 6.93-6.86 (m, 3 H), 6.88 (d, J = 15.6 Hz, 1 H), 6.49 (d, J = 8.4 Hz, 1 H), 6.38 (s, 1 H),
.20 (d, J = 8.4 Hz, 1 H), 6.11 (d, J = 15.6 Hz, 1 H), 5.24 (s, 1 H), 4.00 (s, 3 H), 3.89 (s, 3 H), 3.58 (s, 3
13
H), 3.06 (s, 4 H); C NMR (CDCl ) δ 156.6, 154.0, 153.3, 151.7, 150.8, 146.1, 142.4, 140.3, 134.4,
3

ACCEPTED MANUSCRIPT
1
2
32.9, 132.4, 131.4, 129.4, 125.6, 121.8, 118.4, 114.5, 113.8, 113.4, 111.9, 111.8, 110.2, 56.2, 55.6,
+
9.1, 24.2; MS (ESI) 503 (M+Na) .
Macrocycle (stilbene bridge) (34).
This compound was prepared from 29 in 80% yield by following GP 3, white solid; mp 173-174
1
˚
C. H NMR (CDCl ) δ 7.28-7.27 (m, 2 H), 7.23 (t, J = 8.4 Hz, 1 H ), 7.12 (d, J = 7.8 Hz, 2 H), 7.01 (d,
3
J = 7.8 Hz, 2 H), 6.91 (d, J = 2.4 Hz, 1 H), 6.89 (d, J = 4.8 Hz, 1 H), 6.86 (dd, J = 1.2 Hz, J = 8.4 Hz, 1
H), 6.33 (s, 1 H), 6.08 (d, J = 9.6 Hz, 1 H), 5.91 (s, 1 H), 5.24 (d, J = 1.2 Hz, 1 H), 3.99 (s, 3 H), 3.89 (s,
1
3
3
1
H), 3.62 (s, 3 H), 3.52 (s, 3 H), 2.95-2.57 (m, 4 H); C NMR (CDCl ) δ 156.0, 152.5, 151.1, 149.8,
3
47.7, 146.7, 145.6, 142.7, 141.7, 139.6, 133.8, 131.9, 130.9, 125.1, 121.1, 119.1, 117.9, 113.5, 111.9,
+
1
11.4, 111.3, 98.8, 55.9, 55.7, 55.6, 55.5, 28.8, 23.0; MS (ESI) 511 (M+H) .
Macrocycle (stilbene bridge) (35).
This compound was prepared from 30 in 87% yield by following GP 3, white solid; mp 183-184
1
˚
C. H NMR (CDCl ) δ 7.22 (d, J = 8.4 Hz, 1 H), 6.97 (d, J = 7.2 Hz, 1 H), 6.94 (d, J = 7.2 Hz, 1 H),
3
6
.91 (s, 1 H), 6.87 (d, J = 16.2 Hz, 1 H), 6.85 (d, J = 7.8 Hz, 1 H), 6.83 (d, J = 16.2 Hz, 1 H), 6.80 (d, J
8.4 Hz, 1 H), 6.79-6.65 (m, 4 H), 6.55-6.51 (m, 1 H), 5.54 (s, 1 H), 3.98 (s, 3 H), 3.91 (s, 3 H), 3.86
=
13
(
s, 3 H), 3.71 (s, 3 H), 2.85-2.69 (m, 4 H); C NMR (CDCl ) δ 157.3, 152.8, 152.1, 149.4, 146.7, 144.0,
3
1
41.4, 137.3, 135.0, 134.5, 131.6, 130.4, 128.8, 128.7, 125.4, 123.7, 122.0, 119.4, 117.4, 115.6, 113.7,
+
1
11.9, 111.4, 108.9, 55.9, 55.6, 55.2, 54.3, 34.6, 33.0; MS (ESI) 511 (M+H) .
Macrocycle (stilbene bridge) (36).
This compound was prepared from 31 in 88% yield by following GP 3, white solid; mp 195-196
1
˚
C. H NMR (CDCl ) δ 7.16 (s, 1 H), 7.12 (d, J = 8.4 Hz, 1 H), 7.03 (d, J = 8.4 Hz, 2 H), 6.89 (d, J =
3
8
.4 Hz, 1 H), 6.86 (d, J = 15.6 Hz, 1 H), 6.80 (d, J = 8.4 Hz, 2 H), 6.75 (dd, J = 1.8 Hz, J = 8.4 Hz, 2
H), 6.20 (s, 1 H), 6.00 (d, J = 15.6 Hz, 1 H), 5.85 (s, 1 H), 5.71 (s, 2 H), 5.11 (d, J = 1.8 Hz, 1 H), 3.90
13
(
s, 3 H), 3.78 (s, 3 H), 2.96-2.43 (m, 4 H); C NMR (CDCl ) δ 156.7, 153.2, 151.8, 151.1, 146.2, 145.4,
3
1
1
42.1, 141.4, 140.3, 134.3, 132.8, 132.4, 131.5, 125.9, 121.8, 120.6, 118.4, 113.7, 112.0, 111.8, 107.7,
+
00.8, 96.3, 56.2, 56.1, 29.3, 23.8. MS (ESI) 495 (M+H) .
Macrocyclic derivative (37).
This compound was prepared from 32 in 96% yield by following GP 4, white solid; mp 200-201
1
˚
C. H NMR (CDCl ) δ 7.27 (dd, J = 2.0 Hz, J = 8.4 Hz, 1 H), 7.22 (t, J = 8.0 Hz, 1 H), 7.13-7.10 (m, 2
3
H), 6.99 (dd, J = 2.4 Hz, J = 8.0 Hz, 1 H), 6.93 (td, J = 1.6 Hz, J = 7.6 Hz, 1 H), 6.82 (t, J = 8.8 Hz, 1