Design, synthesis and biological evaluation of novel macrocyclic bisbibenzyl analogues as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.06.007

A series of novel macrocyclic bisbibenzyl analogues was designed, synthesized, and evaluated for their antiproliferative activity in vitro. All of the compounds were tested in five anthropic cancer cell lines, including a multidrug-resistant phenotype. Among these novel molecules, compounds 88, 92 and 94 displayed excellent anticancer activity against Hela, k562, HCC1428, HT29, and PC-3/Doc cell lines, with average IC₅₀ values ranging from 2.23 μM to 3.86 μM, and were more potent than the parental compound marchantin C and much more potent than the positive control Adriamycin. In addition, the mechanism of action of compound 88 was investigated by cell cycle analysis and a tubulin polymerization assay in HCC1482 cells. The binding mode of compound 88 to tubulin was also investigated utilizing a molecular docking study. In conclusion, the present study improves our understanding of the action of bisbibenzylbased tubulin polymerization inhibitors and provides a new molecular scaffold for the further development of antitumor agents that target tubulin.

Full text of DOI:10.1016/j.ejmech.2016.06.007

Accepted Manuscript
Design, synthesis and biological evaluation of novel macrocyclic bisbibenzyl
analogues as tubulin polymerization inhibitors
Bin Sun, Lin Li, Qing-wen Hu, Fei Xie, Hong-bo Zheng, Huan-min Niu, Hui-qing Yuan,
Hong-xiang Lou
PII:
S0223-5234(16)30484-6
10.1016/j.ejmech.2016.06.007
EJMECH 8668
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 March 2016
Revised Date: 5 June 2016
Accepted Date: 6 June 2016
Please cite this article as: B. Sun, L. Li, Q.-w. Hu, F. Xie, H.-b. Zheng, H.-m. Niu, H.-q. Yuan, H.-
x. Lou, Design, synthesis and biological evaluation of novel macrocyclic bisbibenzyl analogues as
tubulin polymerization inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.06.007.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel macrocyclic
bisbibenzyl analogues as tubulin polymerization inhibitors
Bin Suna,b,, Lin Li b, Qing-wen Hu b, Fei Xie b, Hong-bo Zheng b, Huan-min Niu c, Hui-qing Yuan c,
and Hong-xiang Lou a,b
*
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 novel macrocyclic bisbibenzyl analogues was designed, synthesized, and evaluated for their
antiproliferative activity in vitro. All of the compounds were tested in five anthropic cancer cell lines,
including a multidrug-resistant phenotype. Among these novel molecules, compounds 88, 92 and 94
displayed excellent anticancer activity against Hela, k562, HCC1428, HT29, and PC-3/Doc cell lines,
with average IC50 values ranging from 2.23 ꢀM to 3.86 ꢀM, and were more potent than the parental
compound marchantin C and much more potent than the positive control Adriamycin. In addition, the
mechanism of action of compound 88 was investigated by cell cycle analysis and a tubulin
polymerization assay in HCC1482 cells. The binding mode of compound 88 to tubulin was also
investigated utilizing a molecular docking study. In conclusion, the present study improves our
understanding of the action of bisbibenzyl-based tubulin polymerization inhibitors and provides a new
molecular scaffold for the further development of antitumor agents that target tubulin.
Keywords: Marchantin C; Bisbibenzyls; Analogues; Tubulin polymerization inhibitors; Anticancer
* Corresponding authors: Hong Xiang 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 play crucial roles in the growth and functions of eukaryotic cells, such as division,
shape maintenance and intracellular transport and are recognized as one of the most important
molecular targets for cancer chemotherapy [1-4]. Microtubules are formed by a dynamic process that
involves the polymerization and depolymerization of α and β-tubulin heterodimers. Drugs interfering
with this dynamic equilibrium could lead to arrested cell division and eventually to apoptosis [5-8].
The inhibitors of tubulin can be mainly divided into two groups: microtubule destabilizers, such as
combretastatin A-4, colchicine and vinca alkaloids, and microtubule stabilizers, such as taxanes [9-11].
Some of them (taxanes and vinca alkaloids) have been widely used in the clinical treatment of diverse
human cancers for decades [12-14]. However, the clinical use of these anti-tubulin drugs is associated
with problems of drug resistance and complex synthesis and isolation procedures [15-18]. Because of
these undesirable limitations, there is an urgent need to develop novel antimitotic agents for cancer
therapy.
Bisbibenzyls are a series of phenolic natural products that are mainly found in bryophytes. These
natural products exhibit versatile and excellent biological activities, including antimicrobial, antifungal,
antioxidative, muscle-relaxing, cytotoxic and multidrug resistance reversal activities [19-26]. The
remarkable biological profile and interesting macrocyclic structure of bisbibenzyls make them
promising resources for new drug discovery. Marchantin C, a natural macrocyclic bisbibenzyl, was first
discovered in the liverwort Marchantia tosana by Asakawa and co-workers, and now has been
discovered from more than 10 liverwort species [19, 27-32]. It was found to be an excellent cytotoxic
agent against KB and P388 leukemia cell lines, with IC50 values 10 and 8.5 ꢀM, respectively. We
previously discovered that marchantin C could arrest the cell cycle in the G2/M phase by inhibiting
cellular microtubule assembly, specifically promoting cellular microtubule depolymerization, and this
antiproliferative mechanism is similar to that of the anti-tubulin agents colchicine and combretastatin
A-4 [33]. Morita et al. has also reported the antimitotic activity of isoplagiochin A and isoplagiochin B,
which have the similar bisbibenzyl skeleton with marchantin C [19]. Recently, we discovered that
marchantin C and its analogues also exhibit multidrug resistance-reversing activity by inhibiting the
P-glycoprotein [34]. From the study mentioned above, marchantin C shows considerable promise as a
lead compound, and its further structural optimization provides the potential for the discovery of more
effective anti-tubulin agents.

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In the present study, a series of novel marchantin C analogues were designed, synthesized, and
evaluated for cytotoxic activity by the MTT assay. Among these analogues, 88, 92 and 94 display
excellent anticancer activity. The mechanism of action of the potent compound 88 was investigated
using cell cycle analysis, a tubulin polymerization assay and molecular modeling. These studies
indicate that the antiproliferative activity of marchantin C analogues was achieved through an
antimitotic effect by inhibiting tubulin polymerization.
2. Results and Discussion
2.1. Design of marchantin C analogues
Our modification strategy of marchantin C was first based on the understanding of the interaction
between marchantin C and tubulin. Dr Iwai et al. have reported the docking research of marchantin
E with influenza PA endonuclease, which provided a very good precedent for the molecular
modeling research of marchantins [20]. In a previous study, the macrocyclic bisbibenzyl compound
was found to be a colchicine site binder on tubulin [35]. We anticipate that marchantin C should also
target the colchicine-binding site in tubulin, due to its bisbibenzyl skeleton and microtubule-
depolymerizing activity. To obtain an exact stereochemical structure of marchantin C for the molecular
modeling study, its crystal structure was determined by X-ray diffraction analysis, and the crystal data
are presented in the supporting information. Marchantin C was then docked into the colchicine-binding
site of tubulin (PDB code 1SA0) using the GOLD (Genetic Optimization for Ligand Docking) program
[36]. In the resulting binding structure (Fig. 1), marchantin C fit well to the binding site of colchicine
on tubulin, and the hydroxyl group on the arene C might form a hydrogen bond with the carbonyl
group of Ser 178. Beside the ligand-protein potential interactions mentioned above, we were also able
to observe that the carbonyl group of Thr 353 was closed to the arene A of marchantin C. We then
predicted that the introduction of a hydrogen bond donor, such as a hydroxyl group, to the
meta-position of the oxygen atom on arene A might facilitate the formation of a strong hydrogen bond
between the ligand and protein. Accordingly, this modification might lead to a cytotoxic potency
enhancement against tumor cell lines.

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Fig. 1. Proposed binding mode of marchantin C as green stick model in the colchicine binding site
(PDB code 1SA0). The native ligand, DAMA-colchicine, is shown in magenta thin wire model.
Hydrogen bonds are shown as dotted red lines, and the distance between ligand and protein is less than
3 Å. Molecular modeling was perform by GLOD software.
[Figure 1 here]
Moreover, to define the structure-activity relationship of bisbibenzyl analogues and to discover
more potent tubulin inhibitors, a series of novel marchantin C analogues were then designed.
According to the strategy shown in Fig. 2, we focused on modifications of the four phenyl rings by
introducing a hydroxyl group or bromine atom to arenes A-D, while maintaining the bisbibenzyl
skeleton, which was regarded as a fundamental structural core for the antitumor activity. We also
changed the ethylene bridge to a double bond and determined the effect of this stereochemical
structural change on the cytotoxic activity.

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Fig. 2. Modification strategy for marchantin C analogues
[Figure 2 here]
2.2. Chemistry
The general synthesis of the marchantin C analogues is outlined in Schemes 1-4. The synthetic
route of analogues 53-57 began with the Ullmann coupling of the appropriately substituted methyl
4-bromobenzoates 1-3 with 3-hydroxy-4-methoxybenzaldehyde (4), resulting in the formation of
diphenyl ethers 5-7 [34]. Compounds 5-7 were then reduced with sodium borohydride to give the
benzyl alcohols 8-10, followed by the reaction with triphenylphosphonium bromide, affording 11-13 in
two steps [34]. Compounds 19-22 were prepared by the Ullmann coupling of the protected 2-hydroxy-
benzaldehyde (14) or 2-hydroxy-3-methoxybenzaldehyde (15) with the appropriate 3-bromo-
benzaldehydes 16-18 in good yield. The building blocks 11-13 and 19-22 were combined by an
intermolecular Wittig reaction in the presence of potassium carbonate and 18-crown-6 and followed by
hydrogenation over Pd/C to give the bibenzyls 23-27. The carboxylic ester group of 23-27 was then
reduced with lithium aluminum hydride and subsequently deprotected with HCl/H2O to give
compounds 33-37. The reaction of 33-37 with triphenylphosphonium bromide afforded compounds
38-42. The cyclization of 38-42 by means of an intramolecular Wittig reaction was achieved with
sodium methoxide, leading to key macrocyclic intermediates 43-47. Finally, 43-47 could be
hydrogenated to give compounds 48-52, followed by demethylation to afford compounds 53-57
(Scheme 1).
However, the Ullmann coupling of the protected 2-hydroxy-4-methoxybenzaldehyde (58) or
2-hydroxy-5-methoxybenzaldehyde (59) with 3-bromo-benzaldehyde (60) failed, and this might be
because the phenolic group of compounds 58 was deactivated by the methoxyl group, and compound

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59 was deactivated by the electronic effect. 2-Bromo-4-methoxybenzaldehyde (63), 2-bromo-5-
methoxybenzaldehyde (64) and the protected 2-hydroxybenzaldehyde (65) were then used as starting
materials for the Ullmann coupling, and diphenyl esters 66 and 67 were obtained, accordingly.
Compounds 66 and 67 were then reduced with sodium borohydride to give the benzyl alcohols 68-69,
followed by reaction with triphenylphosphonium bromide, affording 70-71 in two steps. The diphenyl
ether
75,
as
the
second
building
block,
was
synthesized
from
2-methoxy-5-((tetrahydro-2H-pyran-2-yloxy)methyl)phenol (73) and 4-bromo-benzaldehyde (74) by
the Ullmann reaction. The building blocks 70-71 and 75 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 bibenzyls 76 and 77. Compounds 76 and 77 were deprotected by HCl/H2O to give compounds
78 and 79, and the analogues 86 and 87 were finally obtained by following the protocol described for
analogues 53-57 (Scheme 2). In addition, during the preparation of analogues 54 and 86, two important
macrocyclic intermediates 44 and 82 were prepared in large amounts. The methyl ether cleavage of 44
and 82 was achieved by aluminum iodide, providing analogues 88 and 89, respectively (Scheme 3).
The preparation method for the brominated analogues is shown in Scheme 4. We have previously
synthesized three brominated analogues of marchantin C with N-bromosuccinimide (NBS) and
evaluated their anticancer activity [31]. In this paper, a more convenient and efficient method was
applied for the bromination. Marchantin C was treated with HBr in the presence of DMSO in ethyl
acetate [37], and analogues 90-95 were provided by HPLC separation.

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Scheme 1. Synthesis of compounds 53-57. Reagents and conditions: (a) CuO, K2CO3, Py, reflux, (yields
65%-71%); (b) NaBH4, THF, 0 ꢀ to r.t. (yields 88%-91%); (c) PPh3HBr, MeCN, reflux, (yields
86%-90%); (d) i. K2CO3, 18-crown-6, DCM, reflux; ii. Pd/C (10%), H2, Et3N, EtOAc, r.t. (yields
74%-82%, two steps); (e) LiAlH4, THF, -40 ꢀto rt. (yields 83%-88%); (f) HCl/THF (1:1), r.t. (yields
87%-91%); (g) NaOMe, DCM, r.t. (yields 85%-92%); (h) Pd/C (10%), H2, EtOAc, r.t. (yields
94%-98%); (i) BBr3, DCM, -40 ꢀto r.t. (yields 86%-91%).

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[Scheme 1 here]
Scheme 2. Synthesis of compounds 86-87. Reagents and conditions: (a) CuO, K2CO3, Py, reflux, (yields
70%-74%); (b) NaBH4, THF, 0 ꢀ to r.t. (yields 90%-96%); (c) PPh3HBr, MeCN, reflux, (yields
86%-90%); (d) 2,3-Dihydropyran, p-toluenesulfonic acid, DCM, (yield 89%); (e) i. K2CO3, 18-crown-6,
DCM, reflux; ii. Pd/C (10%), H2, Et3N, EtOAc, r.t. (yields 85%-88%, two steps); (f) HCl/THF (1:1), r.t.;
(g) NaOMe, DCM, r.t. (yields 80%-81%); (h) Pd/C (10%), H2, EtOAc, r.t. (yields 90%-93%); (i) BBr3,
DCM, -40 ꢀto r.t. (yields 71%-76%).
[Scheme 2 here]

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Scheme 3. Synthesis of compounds 88-89. Reagents and conditions: (a) BBr3, DCM, –78 ꢀ to r.t.
(yields 16%-34%).
[Scheme 3 here]
Scheme 4. Synthesis of compounds 90-95. Reagents and conditions: (a) HBr, DMSO, EtOAc, (overall
yield 84%).
[Scheme 4 here]
2.3. Biological evaluation
2.3.1. In vitro antiproliferative activity and structure-activity relationship (SAR) analysis
The cytotoxic activity of the synthesized analogues (53-57 and 86-95) was evaluated in four
human cancer cell lines (Hela, k562, HCC1428, and HT29) and one paclitaxel-resistant cell line
(PC-3/Doc). Adriamycin (ADR) was used as a positive control. 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. All of the synthesized analogues consistently exhibited anticancer activity.
Compounds 88, 92 and 94 displayed excellent cytotoxic activity, with average IC50 values ranging from

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2.23 ꢀM to 3.86 ꢀM, indicating them to be more potent than ADR (average IC50 = 3.94 ꢀM) and
marchantin C (average IC50 = 13.51 ꢀM). Surprisingly, the cytotoxicities of those three compounds
against the human breast cancer HCC1428 cell line (IC50 = 1.26, 1.09 and 1.40 ꢀM for 88, 92 and 94,
respectively) were comparable to or even better than that of the reference drug ADR (IC50 = 1.56 ꢀM).
These compounds also showed obvious anticancer activity against the paclitaxel-resistant cell line, with
IC50 values of 3.56, 5.75 and 4.95 ꢀM, respectively, and were much more potent than the reference
marchantin C (IC50 = 12.00 ꢀM) and ADR (IC50 = 15.17 ꢀM).
The structure-activity relationships of these novel compounds were analyzed as follows:
Effects of hydroxyl substitutions on arene A, B and D: As shown in Table 1, the introduction of a
hydroxyl group at the C-10’ of arene A led to potent compound 54 (average IC50 = 8.45 ꢀM), which
showed improved anticancer activity in four cancer cell lines compared with marchantin C (average
IC50 = 13.51 ꢀM), and this result is consistent with our anticipation mentioned above. However, a shift
of the hydroxyl group from C-10’ to C-11’ caused decreased activity (average IC50 = 17.36 ꢀM for
compound 53). Compounds 55, 86 and 87, with different hydroxyl substitution patterns on arene B,
showed a comparable antitumor activity to marchantin C (average IC50 = 11.37, 11.36 and 12.16 ꢀM for
55, 86 and 87, respectively), indicating that the removal or introduction of a hydroxyl group on arene B
does not provide any obvious effect on the activity. When the hydroxyl group was introduced to the
C-4 or C-6 of arene D (compounds 56 and 57), the activity was slightly diminished. All of these
suggest that no potential hydrogen bond acceptor is present close to arenes B or D, and this finding is
consistent with results of molecular modeling studies of marchantin C to tubulin.
Table 1
In Vitro Cytotoxicity of marchantin C analogues in five cancer cell lines
a
Compd
IC50 (ꢀM
HCC1428
16.85±3.92 11.41±2.15 24.98±4.25 16.23±1.20
)
PC-3/Doc
N/Tb
Hela
k562
HT29
Average
17.36
8.45
53
54
55
56
57
86
87
88
89
90
N/Tb
12.52±4.53 9.86±1.01
3.51±0.44
7.92±1.65
9.50±1.91
N/Tb
N/Tb
N/Tb
N/Tb
12.87±1.02 13.48±1.33 9.64±2.05
11.37
17.30
15.94
11.36
12.16
2.72
17.77±4.56 20.12±2.11 11.31±1.02 20.00±5.44
18.08±1.36 13.69±3.99 16.44±2.96 15.56±1.04
12.54±2.47 13.96±2.54 5.52±1.09 13.46±2.21
14.60±1.02 15.63±1.73 10.04±2.21 8.35±1.56
N/Tb
2.77±0.52
3.36±0.54
1.26±0.16
2.66±0.42
3.56±0.58
N/Tb
16.68±1.98 19.54±2.67 13.29±2.66 17.22±3.33
12.62±1.75 13.32±1.42 14.24±1.25 11.42±2.01
16.68
11.42
5.51±1.93

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91
10.35±1.59 5.78±0.74
5.57±0.73 2.86±0.88
2.56±0.41
1.09±0.39
6.80±1.63
4.01±1.01
6.02±0.68
5.75±1.04
6.30
3.86
92
93
94
16.39±3.89 19.44±3.35 21.88±6.98 13.97±2.20 14.60±2.35
1.64±0.14 1.32±0.09 1.40±0.24 1.86±0.99 4.95±1.11
17.26
2.23
95
15.31±1.02 10.84±1.96 7.02±1.49 13.49±3.39 11.10±2.47
20.2±3.20 11.96±1.77 12.30±4.32 11.28±1.88 12.00±2.86
11.55
13.51
3.94
Marchantin C
Adriamycin
1.06±0.21
0.47±0.09
1.56±0.15
1.46±0.29
15.17±1.21
a
The IC50 values (ꢀM) are the concentrations corresponding to 50% inhibition of each cell line; Mean
values based on three independent experiments; b N/T means not tested.
[Table 1 here]
Effects of the double bond: As shown in Table 1, the double bond impacted the cytotoxic activity
positively (54 vs 88) or negatively (86 vs 89) to some extent. Compound 88, with a double bond at C-7’
(8’), displayed potent anticancer activity, with an average IC50 value of 2.72 ꢀM, being more effective
than 54 and five-fold better than the parent marchantin C. As shown in the molecular modeling studies
below (Fig. 3), the double bond changed the stereochemical structure of the bisbibenzyl skeleton and
made the hydroxyl group on arene A closer to the carbonyl group of Thr 353, which might facilitate the
formation of a hydrogen bond between the ligand and the protein. The double bond at C-7 (8) caused
the diminished activity of compound 89 compared with 86, and the resulting stereochemical skeleton
might exhibit weakened fitness as a tubulin-binding pocket.
Fig. 3. Proposed binding mode of compound 88 as green stick model in the colchicine binding site

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(PDB code 1SA0). Hydrogen bonds are shown as dotted red lines, and the distance between ligand and
protein is less than 3 Å.
[Figure 3 here]
Effects of bromine atom substitutions: A moderate cytotoxicity was observed when arene B was
substituted at the C-4’ position with a bromine atom (average IC50 = 11.42 ꢀM for compound 90). A
two-fold increase in the activity was observed in compound 91 (average IC50 = 6.30 ꢀM) when the
bromine atom on arene B was relocated to the C-6’ position. In addition, compound 92, with two
bromine atoms at C-4’ and C-6’ of arene B, exhibited excellent anticancer activity, with an average IC50
value of 3.86 ꢀM, three-fold better than the that of the parent marchantin C. To our surprise, the
introduction of bromine atoms at the C-6’ position on arene B and the C-13 position on arene C led to
the most potent compound 94 (average IC50 = 2.23 ꢀM), which demonstrated a six-fold improved
cytotoxic activity compared to marchantin C. Interestingly, the substitution position of the bromine
atom on arene B was critical for the cytotoxicity because a shift from 6’-bromo to 4’-bromo resulted in
an obvious decrease in the activity (average IC50 = 17.26 ꢀM for 93 compared with 2.23 ꢀM for 94).
Unexpectedly, the tri-bromination of marchantin C did not significantly enhance the activity, and the
IC50 value of 95 was 11.55 ꢀM, which was comparable to that of marchantin C. All these suggest that
the addition of bromine at the 6’-position of arene B is critical for the enhancement of the antitumor
activity, but the tri-bromination or multi-bromination might decrease this contribution.
The further antiproliferative activity of compound 88 in HCC1428 cells was detected by the
xCELLigence system, and vincristine (VCR) was used as a positive control. As shown in Fig. 4,
compound 88 inhibited HCC1428 cell proliferation within 5 h and decreased the number of cells
markedly within 20 h, showing a similar role to VCR. After treatment for approximately 60 h, the
effect of 88 was close to that of VCR. The cell growth curve showed that compound 88 could inhibit
HCC1428 cell proliferation and subsequently induce cell death.

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Fig. 4. HCC1428 cells were treated with compound 88 as indicated. Cells were then plated 2000
cells/well into E-plate 16 and analyzed using a xCELLigence RTCA DP instrument. Results are
representatives of three independent experiments.
[Figure 4 here]
2.3.2. Cell cycle analysis
After the antiproliferative activity evaluation, we extended our work to the mechanism study.
Flow cytometry was used to analyze the effects of the marchantin C analogues on the cell growth and
division. HCC1428 cells was treated with compound 88 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, 9.17% of the
HCC1428 cells were in the G2/M phase. Compound 88 increased the proportion of cells in the G2/M
phase, and approximately 64.93% were found in the G2/M phase when treated with 3 ꢀM compound 88
for 24 h. However, a decline in the G2/M population (34.67%) occurred after treatment with 6 ꢀM
88, concomitant with a dramatic increase in the sub-G1 population, indicating increased cell death.
These results demonstrate that bisbibenzyl derivative 88 could induce cell cycle arrest at the G2/M
phase, which is consistent with the results obtained for classical tubulin-targeting drugs.
Fig. 5. Compound 88 induced HCC1428 cells cycle arrest at G2/M phase. HCC1428 cells were treated
with 3ꢀM and 6ꢀM of compound 88 for 24 h, and then trypsinized, fixed and stained with PI to

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measure cell cycle profile by flow cytometry. Control cells were treated with DMSO alone. Results are
representatives of three independent experiments.
[Figure 5 here]
2.3.3. Immunofluorescence staining
The significant cell growth inhibitory properties of marchantin C analogues supported by their
obvious G2/M phase arresting properties encouraged us to investigate the further biological mechanism.
Given the microtubule-depolymerization activity of marchantin C in culture cells, we next examined
the effect of compound 88 on the cytoskeleton network by immunofluorescent staining techniques. The
cellular microtubule networks were visualized by confocal microscopy. As shown in Fig. 6, intact
microtubules arrays could be observed in untreated cells. However, during treatment with increasing
dosages of 88, the microtubule networks were decreased and dispersed in the cytoplasm, which was
similar to the result observed with 1 ꢀM VCR treatment. These results indicate that 88 could affect the
cellular microtubule dynamics.
Fig. 6. HCC1428 cells were treated with compound 88 as indicated for 24 h and then fixed and
immunostained with monoclonal anti-α-tubulin antibody (red) and DAPI (blue). One micromolar of

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Vincristine (VCR) and same amount of DMSO were used as controls. Results are representatives of
three independent experiments. Bar = 10 ꢀm.
[Figure 6 here]
2.3.4. Tubulin polymerization inhibition
We next investigated the inhibition of tubulin polymerization by the selected potent compound 88
and compared it with the results using a positive control VCR and negative control taxol. DMSO was
used as a blank control. Bovine brain tubulin was incubated with compound 88 at concentrations of 5
and 10 ꢀM. VCR and taxol were both used at a concentration of 5 ꢀM. As shown in Fig. 7, paclitaxel
increased the absorbance obviously and immediately, indicating that it enhanced tubulin
polymerization. In contrast, VCR and 88 decreased the absorbance, indicating that the tubulin
polymerization was inhibited. Compound 88 showed stronger inhibition than VCR at the two tested
concentrations. These results clearly indicate that compound 88 significantly inhibits the
polymerization of tubulin in vitro.
Fig. 7. Effect of compound 88 on tubulin polymerization in vitro. Purified bovine brain tubulin
was incubated in the presence of Taxol, VCR, DMSO (control), and compound 88 under the
indicated concentrations at 37ꢀ, and absorbance readings were recorded every minute for 1 h.
[Figure 7 here]
2.4. Molecular modeling
The binding mode of active compound 88 was determined at the colchicine binding site on tubulin
using in silico molecular docking protocols. Docking was performed using the GOLD (Genetic
Optimization for Ligand Docking) program. In the resulting hypothetical structure (Fig. 3), the
hydroxyl group on arene C may form a hydrogen bond with the carbonyl group of Ser 178. The

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hydroxyl group on arene A is involved in a potential hydrogen bond with the carbonyl group of Thr 353.
It is also possible that arene B forms a CH-π interaction with the methyl group of Ala 316. The
combination of the CH-π interaction and hydrogen bonds could play a crucial role in the improved
tubulin inhibitory activity of derivative 88. In addition, the introduction of a bromine atom to
marchantin C could change the electron distribution on the phenyl rings, which may create a stronger
hydrogen bond with Ser 178 and enable CH-π interaction with Ala 316, and this might be the reason
for the improved potency of the brominated analogues.
3. Conclusions
In summary, a series of novel marchantin C analogues was designed, synthesized and evaluated
for their antiproliferative activity against five anthropic cancer cell lines. The structure-activity
relationships were investigated by introducing hydroxyl groups and/or bromine atoms into arenes A-D.
Several novel analogues showed excellent antiproliferative activity comparable to that of the positive
control ADR. Those compounds were also effective against the paclitaxel-resistant cancer cell line. The
cell cycle analysis and immunofluorescence microscopy observations revealed that marchantin C
derivative 88 could arrest cancer cells at the G2/M phase by destroying the microtubule networks.
Further mechanism of action studies confirmed that marchantin C analogues maintained their ability by
inhibiting tubulin polymerization at the colchicine-binding site. Molecular modeling provided insights
into the binding mode of marchantin C analogues in tubulin. Marchantin C analogues represent a
potent, new class of tubulin inhibitors, and compound 88 could serve as lead compounds in the further
optimization for the discovery of novel anticancer agents.
4. Experimental section
4.1. Chemistry
Chemicals were commercially available and used as received without further purification.
Solvents (THF, DCM and CH3CN) 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
mesh). The NMR spectra were recorded on a Bruker Spectospin spectrometer at 600 MHz (13C 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 (CDCl3, 7.28 ppm). Semi-prep. HPLC:
Agilent 1100-G1310A isopump equipped with a G1322A degasser, a G1314A VWD detector (210 nm)

ACCEPTED MANUSCRIPT
and a ZORBAX SB-C18 column (9.4 × 250 mm, 5-ꢀm). Abbreviations used 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 a LTQ Orbitrap mass spectrometer.
4.1.1. Methyl 4-(5-formyl-2-methoxyphenoxy)-2-methoxy benzoate (6)
This compound was prepared from methyl 2-methoxyl-4-bromobenzoate 2 (8.55 g, 35.01 mmol)
and 3-hydroxy-4-methoxybenzaldehyde 4 (5.31 g, 35.01 mmol) by means of a procedure similar to that
used for 5. After concentration of the solution, the residue was purified by flash column
chromatography (SiO2), eluting with a 3:2 solution of petroleum ether-DCM to afford 6 (7.85 g, 71%)
as an orange solid; mp 125-126 °C. 1H NMR (CDCl3) δ 9.87 (s, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.76 (dd,
J = 8.4, 1.9 Hz, 1H), 7.60 (d, J = 1.9 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 2.1 Hz, 1H), 6.38
(dd, J = 8.7, 2.2 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.86 (s, 3H); MS (ESI) 317 (M+H)+.
4.1.2. Methyl 4-(5-formyl-2-methoxyphenoxy)-3-methoxy benzoate (7)
This compound was prepared from 3-hydroxy-4-methoxybenzaldehyde (4) and methyl
4-bromo-3-methoxybenzoate (3) in 69% yield by following the procedure described for 6. Yellow solid;
mp 121-122 °C. 1H NMR (CDCl3) 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 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)+.
4.1.3. Methyl 4-(5-(hydroxymethyl)-2-methoxyphenoxy)-2-methoxybenzoate (9)
This compound was prepared from compound 6 by following the procedure described for 8, yield
90%, white solid; mp 85-86 °C.1H NMR (600 MHz, CDCl3) δ 7.79 (d, J = 8.7 Hz, 1H), 7.22 (dd, J =
8.4, 2.1 Hz, 1H), 7.11 (d, J = 2.1 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 2.3 Hz, 1H), 6.38 (dd, J
= 8.7, 2.3 Hz, 1H), 4.65 (s, 2H), 3.87 (s, 3H), 3.87 (s, 3H), 3.83 (s, 3H); MS (ESI) 319 (M+H)+.
4.1.4. Methyl 4-(5-((bromotriphenylphosphoranyl)methyl)-2-methoxyphenoxy)-2-methoxybenzoate (12)
This compound was prepared from compound 9 by following the procedure described for 11, yield
91%, white solid; mp 233-234 °C. 1H NMR (CDCl3) δ 7.77-7.76 (m, 15 H), 7.34-7.32 (m, 2 H), 6.84 (d,
J = 7.2 Hz, 1 H ), 6.50 (s, 1 H), 6.46 (s, 1 H), 6.11 (d, J = 8.4 Hz, 1 H), 5.44 (d, J = 14.4 Hz, 2 H), 3.88
(s, 3 H), 3.83 (s, 3 H), 3.75 (s, 3 H); MS (ESI) 563 (M-Br)+.
4.1.5. (4-Methoxy-3-(2-methoxyl-4-(methoxycarbonyl)phenoxy)benzyl)triphenylphos-phonium Bromide
(13)

ACCEPTED MANUSCRIPT
This compound was prepared from compound 10 by following the procedure described for 11,
1
yield 87%, white solid; mp 215-216 °C. H NMR (CDCl3) δ 7.83-7.70 (m, 9 H), 7.67-7.60 (m, 6 H),
7.57 (d, 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)+.
4.1.6. 3-(2-(1,3-Dioxan-2-yl)phenoxy)benzaldehyde (19)
A mixture of 2-(1,3-dioxan-2-yl)phenol (14, 10 g, 0.05 mol) [38], 3-bromo-benzaldehyde (16, 8.8
g, 0.05 mmol), potassium carbonate (13.5 g, 0.09 mol) and cupric oxide (0.95 g, 5.9 mmol) in pyridine
(50 mL) was stirred under reflux for 12 h. The pyridine was distilled off in vacuo and the residue was
extracted with ethyl acetate (200 mL). The solution was concentrated and the residue was purified by
flash column chromatograph (Al2O3), eluting with a 2:1 solution of petroleum ether-DCM to afford 19
1
(12.6 g, 81%) as a yellow solid; white solid; mp 129-130 °C. H NMR (CDCl3) δ 9.98 (s, 1H), 7.78 (dd,
J = 7.8, 1.7 Hz, 1H), 7.66-7.60 (m, 1H), 7.51-7.49 (m, 2H), 7.37-7.34 (m, 1H), 7.28-7.24 (m, 2H), 6.92
(dd, J = 8.2, 1.0 Hz, 1H), 5.82 (s, 1H), 4.22 (ddd, J = 11.9, 4.9, 1.1 Hz, 2H), 3.93 (ddd, J = 12.4, 3.9,
2.5 Hz, 2H), 2.28-2.19 (m, 1H), 1.44-1.40 (m, 1H) ; MS (ESI) 285 (M+H)+.
4.1.7. 3-(2-(1,3-Dioxan-2-yl)-6-methoxyphenoxy)benzaldehyde (20)
This compound was prepared from 2-(1,3-dioxan-2-yl)-6-methoxyphenol (15) and
3-bromo-benzaldehyde (16) by following the procedure described for 19, yield 86%, white solid; mp
1
127-128 °C. H NMR (CDCl3) δ 9.95 (s, 1 H), 7.55 (d, J = 7.2 Hz, 1 H), 7.43 (t, J = 7.8 Hz, 1 H), 7.37
(d, J = 8.4 Hz, 2 H), 7.30 (t, J = 7.8 Hz, 1 H), 7.15 (d, J = 5.4 Hz, 1 H), 7.02 (d, J = 7.8 Hz, 1 H), 5.69
(s, 1 H), 4.14 (d, J = 7.8 Hz, 2 H), 3.85 (t, J = 12.0 Hz, 2 H), 3.74 (s, 3 H), 2.21-2.16 (m, 1 H), 1.37 (d,
J = 13.2 Hz, 1 H); MS (ESI) 315 (M+H)+.
4.1.8. 5-(2-(1,3-Dioxan-2-yl)-6-methoxyphenoxy)-2-methoxybenzaldehyde (21)
This compound was prepared from 2-(1,3-dioxan-2-yl)-6-methoxyphenol (15) and
5-bromo-2-methoxybenzaldehyde (17) by following the procedure described for 19, yield 80%, white
solid; mp 119-120 °C. 1H NMR (CDCl3) δ 10.42 (s, 1 H), 7.37 (s, 1 H), 7.34 (d, J = 7.5 Hz, 1 H), 7.25
(t, J = 7.9 Hz, 1 H), 7.06 (d, J = 8.7 Hz, 1 H), 6.99 (d, J = 8.0 Hz, 1 H), 6.89 (d, J = 8.9 Hz, 1 H), 5.68
(s, 1 H), 4.16-4.14 (m, 2 H), 3.90 (s, 3 H), 3.85 (t, J = 11.9 Hz, 2 H), 3.72 (s, 3 H), 2.25-2.14 (m, 1H),
1.36 (d, J = 13.6 Hz, 1 H); MS (ESI) 345 (M+H)+.
4.1.9. 3-(2-(1,3-Dioxan-2-yl)-6-methoxyphenoxy)-4-methoxybenzaldehyde (22)

ACCEPTED MANUSCRIPT
This compound was prepared from 2-(1,3-dioxan-2-yl)-6-methoxyphenol (15) and
5-bromo-4-methoxybenzaldehyde (18) by following the procedure described for 19, yield 79%, white
1
solid; mp 127-128 °C. H NMR (CDCl3) δ 9.71 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 7.4 Hz,
1H), 7.28-7.25 (m, 1H), 7.08 (d, J = 7.9 Hz, 1H), 7.01-6.99 (m, 2H), 5.71 (s, 1H), 4.12 (d, J = 10.8 Hz,
2H), 4.06 (s, 3H), 3.82 (t, J = 11.7 Hz, 2H), 3.71 (s, 3H), 2.26-2.09 (m, 1H), 1.34 (d, J = 13.3 Hz, 1H);
MS (ESI) 345 (M+H)+.
4.1.10. Methyl 4-(5-(3-(2-(1,3-dioxan-2-yl)-6-methoxyphenoxy)phenethyl)-2-methoxy-phenoxy)-3-
methoxybenzoate (23)
Potassium carbonate (4.45 g, 32.22 mmol) and a trace of 18-crown-6 were added to a solution of
13 (10.34 g, 16.11 mmol) and 20 (5.06 g, 16.11 mmol) in anhydrous DCM (50 mL), 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 (Al2O3),
eluting with a 3:1 solution of hexane-dichloromethane to afford a mixture of Z/E isomers (8.57 g, 89%)
as a yellow oil. Pd/C 10% (0.7 g) and triethylamine (16 mL) were then added to a solution of isomers
(7.89 g, 13.20 mmol) in ethyl acetate (100 mL). The suspension was stirred under H2 for 24 h at room
temperature. The mixture was filtered, and concentrated to afford 23 (7.54 g, 95%) as a yellow solid;
mp 100-101 °C. 1H NMR (CDCl3) δ 7.66 (s, 1 H), 7.56 (d, J = 8.4 Hz, 1 H ), 7.35 (d, J = 7.8 Hz, 1 H),
7.26 (t, d, J = 7.8 Hz, 1 H), 6.99 (d, J = 8.4 Hz, 2 H), 6.91 (d, J = 8.4 Hz, 1 H), 6.84 (s, 1 H), 6.76-6.74
(m, 2 H), 6.68 (d, J = 7.8 Hz, 1 H), 6.61 (d, J = 8.4 Hz, 1 H), 5.68 (s, 1 H), 4.17-4.14 (m, 2 H), 3.99 (s,
3 H), 3.92 (s, 3 H), 3.86-3.82 (m, 2 H), 3.79 (s, 3 H), 3.71 (s, 3 H), 2.86-2.83 (m, 4 H), 2.21-2.19 (m, 1
H), 1.36 (d, J = 13.8 Hz, 1 H); MS (ESI) 623 (M+Na)+.
4.1.11.
Methyl
4-(5-(3-(2-(1,3-dioxan-2-yl)-6-methoxyphenoxy)phenethyl)-2-methoxyphenoxy)-2-
methoxybenzoate (24)
This compound was prepared from 12 and 20 by following the procedure described for 23, yield
82%, white solid; mp 95-96 °C. 1H NMR (CDCl3) δ 7.77 (d, J = 7.8 Hz, 1H ), 7.35 (d, J = 7.2 Hz, 1H),
7.24 (t, J = 7.8 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.03-6.97 (m, 2H), 6.91 (d, J = 7.8 Hz, 1H), 6.87 (s, 1
H), 6.76-6.74 (m, 2H), 6.68 (d, J = 4.8 Hz, 1H), 6.60 (s, 1H), 6.32 (d, J = 7.8 Hz, 1H), 5.67 (s, 1H),
4.12 (m, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.81-3.80 (m, 2H), 3.76 (s, 3H), 3.68 (s, 3H), 2.84 (s, 4H),
2.18-2.16 (m, 1H), 1.33-1.29 (m, 2H); MS (ESI) 623 (M+Na)+.
4.1.12. Methyl 4-(5-(3-(2-(1,3-dioxan-2-yl)phenoxy)phenethyl)-2-methoxyphenoxy)benzoate (25)

ACCEPTED MANUSCRIPT
This compound was prepared from 11 and 19 by following the procedure described for 23, yield
1
75%, white solid; mp 104-105 °C. H NMR (CDCl3) δ 8.02-7.96 (m, 2H), 7.74 (dd, J = 7.7, 1.7 Hz,
1H), 7.28-7.25 (m, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.17 (td, J = 7.6, 0.9 Hz, 1H), 7.00 (dd, J = 8.3, 2.1 Hz,
1H), 6.94 (d, J = 8.4 Hz, 1H), 6.90-6.87 (m, 3H), 6.87-6.85 (m, 1H), 6.84 (d, J = 2.2 Hz, 1H), 6.81 (t, J
= 1.8 Hz, 1H), 6.79 (dd, J = 8.2, 0.9 Hz, 1H), 5.87 (s, 1H), 4.27-4.20 (m, 2H), 3.95 (td, J = 12.4, 2.5 Hz,
2H), 3.91 (s, 3H), 3.79 (s, 3H), 2.91-2.83 (m, 4H), 2.29-2.21 (m, 1H), 1.45-1.38 (m, 1H); MS (ESI) 563
(M+Na)+.
4.1.13.
Methyl
4-(5-(3-(2-(1,3-dioxan-2-yl)-6-methoxyphenoxy)-4-methoxyphenethyl)-2-methoxy-
phenoxy)benzoate (26)
This compound was prepared from 11 and 22 by following the procedure described for 23, yield
1
80%, white solid; mp 118-119 °C. H NMR (CDCl3) δ 7.97 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 7.8 Hz,
1H), 7.23 (t, J = 8.0 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 6.92 (dd, J = 8.3, 1.8 Hz, 1H), 6.88 (d, J = 8.4
Hz, 1H), 6.84 (t, J = 8.2 Hz, 3H), 6.73 (d, J = 1.7 Hz, 1H), 6.64 (dd, J = 8.1, 1.7 Hz, 1H), 6.38 (d, J =
1.7 Hz, 1H), 5.74 (s, 1H), 4.11 (dd, J = 11.2, 4.6 Hz, 2H), 3.93 (s, 3H), 3.88 (s, 3H), 3.80 (t, J = 11.2
Hz, 2H), 3.73 (s, 3H), 3.66 (s, 3H), 2.68 (s, 4H), 2.22-2.11 (m, 1H), 1.31 (d, J = 13.3 Hz, 1H); MS (ESI)
623 (M+Na)+.
4.1.14.
Methyl
4-(5-(5-(2-(1,3-dioxan-2-yl)-6-methoxyphenoxy)-2-methoxyphenethyl)-2-methoxy-
phenoxy)benzoate (27)
This compound was prepared from 11 and 21 by following the procedure described for 23, yield
74%, white solid; mp 99-100 °C. 1H NMR (CDCl3) δ 7.98 (d, J = 7.0 Hz, 2H), 7.34 (d, J = 7.4 Hz, 1H),
7.23 (t, J = 6.0 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 6.94-6.89 (m, 4H), 6.74 (s,
1H), 6.70 (d, J = 8.6 Hz, 1H), 6.63 (d, J = 5.7 Hz, 1H), 5.69 (s, 1H), 4.25-4.08 (m, 2H), 3.90 (s, 3H),
3.85 (t, J = 12.2 Hz, 2H), 3.78 (s, 3H), 3.75 (s, 3H), 3.70 (s, 3H), 2.85-2.81 (m, 4H), 2.27-2.13 (m, 1H),
1.36 (d, J = 13.1 Hz, 1H) ; MS (ESI) 623 (M+Na)+.
4.1.15.
(4-(5-(3-(2-(1,3-Dioxan-2-yl)-6-methoxyphenoxy)phenethyl)-2-methoxyphenoxy)-2-methoxy-
phenyl)methanol (29)
A solution of 24 (13.11 g, 23.72 mmol) in anhydrous 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
ammonium chloride (10 mL). THF was removed in vacuo and the resulting mixture was diluted with

ACCEPTED MANUSCRIPT
DCM (100 mL), washed with saturated aqueous NaCl (20 mL × 3) and dried over sodium sulfate. The
solvent was removed in vacuo and the residue was purified by flash chromatography (SiO2) eluting
with DCM to yield 29 (12.30g, 88%), colorless oil; 1H NMR (CDCl3) δ 7.36 (d, J = 6.9 Hz, 1H), 7.23 (t,
J = 8.0 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 8.2 Hz, 1H), 6.94 (dd, J
= 8.4, 1.7 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.83 (d, J = 1.7 Hz, 1H), 6.79 – 6.72 (m, 2H), 6.68 (dd, J =
8.1, 1.7 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H), 6.35 (dd, J = 8.2, 2.1 Hz, 1H), 5.68 (s, 1H), 4.61 (s, 2H),
4.11 (dd, J = 11.3, 4.4 Hz, 2H), 3.81-3.79 (m, 2H), 3.78 (s, 3H), 3.76 (s, 3H), 3.67 (s, 3H), 2.83 (s, 4H),
2.17-2.14 (m, 1H), 1.59 -1.30 (m, 1H); MS (ESI) 573 (M+H)+.
4.1.16. (4-(5-(3-(2-(1,3-Dioxan-2-yl)phenoxy)phenethyl)-2-methoxyphenoxy)phenyl)methanol (30)
This compound was prepared from 25 by following the procedure described for 29, yield 83%,
colorless oil; 1H NMR (CDCl3) δ 7.74 (dd, J = 7.7, 1.7 Hz, 1H), 7.30-7.28 (m, 3H), 7.28-7.25 (m, 1H),
7.22 (t, J = 7.8 Hz, 1H), 7.16 (td, J = 7.6, 0.9 Hz, 1H), 6.92 (d, J = 1.1 Hz, 2H), 6.90-6.88 (m, 2H),
6.86-6.82 (m, 1H), 6.81-6.78 (m, 1H), 6.77 (dd, J = 9.7, 1.1 Hz, 2H), 5.87 (s, 1H), 4.65 (s, 2H),
4.26-4.19 (m, 2H), 3.95 (td, J = 12.4, 2.4 Hz, 2H), 3.83 (s, 3H), 2.90-2.76 (m, 4H), 2.31-2.16 (m, 1H),
1.45-1.39 (m, 1H); MS (ESI) 513 (M+H)+.
4.1.17.
(4-(5-(5-(2-(1,3-Dioxan-2-yl)-6-methoxyphenoxy)-2-methoxyphenethyl)-2-methoxyphenoxy)
phenyl)methanol (32)
This compound was prepared from 27 by following the procedure described for 29, yield 79%,
colorless oil; 1H NMR (CDCl3) δ 7.34-7.28 (m, 3H), 7.23 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 5.9 Hz, 2H),
6.92-6.90 (m, 3H), 6.83 (s, 1H), 6.73 (s, 1H), 6.69 (d, J = 8.7 Hz, 1H), 6.60 (d, J = 8.5 Hz, 1H), 5.67 (s,
1H), 4.63 (s, 2H), 4.14 (d, J = 10.1 Hz, 2H), 3.84-3.82 (m, 2H), 3.81 (s, 3H), 3.74 (s, 3H), 3.69 (s, 3H),
2.81-2.79 (m, 4H), 2.25-2.10 (m, 1H), 1.35 (d, J = 12.8 Hz, 1H); MS (ESI) 573 (M+H)+.
4.1.18.
benzaldehyde (34)
Compound 29 (12.33g, 21.5mmol) was dissolved in a solution of ethanol (100 mL) and 10 % aq
2-(3-(3-(4-(Hydroxymethyl)-3-methoxyphenoxy)-4-methoxyphenethyl)phenoxy)-3-methoxy-
HCl (20 mL). The resulting mixture was then stirred at room temperature for 12 h. Sat aq sodium
bicarbonate (150 mL) was added and the ethnol was removed in vacuo. The resulting mixture was
extracted with CH2Cl2 (200 mL) , washed with saturated aqueous NaCl (25 mL × 3) and dried over
sodium sulfate. The solution was concentrated to yield 34 (9.64 g, 87%) as white solid; mp 86-87 °C.
1H NMR (600 MHz, CDCl3) δ 10.26 (d, J = 0.8 Hz, 1H), 7.57 (dd, J = 7.8, 1.5 Hz, 1H), 7.33 (td, J =

ACCEPTED MANUSCRIPT
8.0, 0.7 Hz, 1H), 7.28-7.23 (m, 2H), 7.17 (t, J = 7.9 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.88 (dd, J = 8.3,
2.0 Hz, 1H), 6.84 (d, J = 7.7 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 6.70 (t, J = 2.4 Hz, 1H), 6.68-6.64 (m,
1H), 6.62 (d, J = 2.3 Hz, 1H), 6.39 (dd, J = 8.3, 2.3 Hz, 1H), 4.51 (s, 2H), 3.85 (s, 3H), 3.80 (s, 3H),
3.79 (s, 3H), 2.8-2.79 (m, 4H); MS (ESI) 515 (M+H)+.
4.1.19. 2-(3-(3-(4-(Hydroxymethyl)phenoxy)-4-methoxyphenethyl)phenoxy)benzaldehyde (35)
This compound was prepared from 30 by following the procedure described for 34, yield 89%,
white solid; mp 90-91 °C. 1H NMR (CDCl3) δ 10.50 (s, J = 2.3 Hz, 1H), 7.94 (dd, J = 7.8, 1.8 Hz, 1H),
7.51 (ddd, J = 8.4, 7.3, 1.8 Hz, 1H), 7.30-7.28 (m, 3H), 7.23-7.15 (m, 1H), 6.97 (d, J = 7.5 Hz, 1H),
6.92 (s, 2H), 6.91-6.87 (m, 3H), 6.84-6.79 (m, 2H), 6.75 (s, 1H), 4.66 (s, 2H), 3.83 (s, 3H), 2.91-2.82
(m, 4H); MS (ESI) 455 (M+H)+.
4.1.20. 2-(3-(3-(4-(hydroxymethyl)phenoxy)-4-methoxyphenethyl)-4-methoxyphenoxy)-3-methoxy-
benzaldehyde (37)
This compound was prepared from 32 by following the procedure described for 34, yield 92%,
1
white solid; mp 94-95 °C. H NMR (DMSO-d6) δ 10.23 (s, 1 H), 7.54 (d, J = 7.2 Hz, 1 H), 7.30-7.27
(m, 4 H), 7.23 (d, J = 7.8 Hz, 1 H), 6.91-6.90 (m, 3 H), 6.81 (s, 1 H), 6.67 (d, J = 8.4 Hz, 1 H), 6.65 (s,
1 H), 6.60-6.58 (m, 1 H), 4.65 (s, 2 H), 3.82 (s, 3 H), 3.77 (s, 3 H), 3.78 (s, 3 H), 2.82-2.78 (m, 4 H);
MS (ESI) 515 (M+H)+.
4.1.21.
(4-(5-(3-(2-Formyl-6-methoxyphenoxy)phenethyl)-2-methoxyphenoxy)-3-methoxybenzyl)
triphenylphosphonium bromide (38)
Compound 33 (5.32 g, 10.35 mmol) and triphenylphosphonium bromide (3.72 g, 10.86 mmol)
was dissolved in anhydrous CH3CN (50 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% ethanol in dichloromethane, to yield 38 as white solid (8.12 g, 89%); mp 235-236 °C.
1H NMR (CDCl3) δ 7.77-7.63 (m, 15 H), 7.53 (d, J = 7.8 Hz, 1 H), 7.31 (t, J = 7.8 Hz, 1 H), 7.27 (t, J =
7.8 Hz, 1 H), 7.11 (t, J = 7.8 Hz, 1 H), 7.02 (s, 1 H), 6.83 (d, J = 8.4 Hz, 1 H), 6.80 (d, J = 8.4 Hz, 2 H),
6.65 (s, 1 H), 6.61-6.59 (m, 2 H), 6.51 (s, 2 H), 5.41 (d, J = 7.8 Hz, 2 H), 3.81 (s, 3 H), 3.76 (s, 3 H),
2.77 (s, 4 H); MS (ESI) 759 (M-Br)+.
4.1.22.
2-(3-(3-(4-((Bromotriphenylphosphoranyl)methyl)-3-methoxyphenoxy)-4-methoxyphenethyl)
phenoxy)-3-methoxybenzaldehyde (39)
This compound was prepared from 34 by following the procedure described for 38, yield 90%,

ACCEPTED MANUSCRIPT
1
white solid; mp 221-222 °C. H NMR (CDCl3) δ 10.23 (s, 1H), 7.80-7.71 (m, 9H), 7.67-7.63 (m, 6H),
7.54 (dd, J = 7.7, 1.7 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 7.31-7.28 (m, 2H), 7.14 (t, J = 7.8 Hz, 1H), 6.88
(d, J = 1.1 Hz, 2H), 6.83 (d, J = 7.6 Hz, 1H), 6.74 (s, 1H), 6.66-6.64 (m, 1H), 6.62 (dd, J = 8.5, 2.9 Hz,
1H), 6.36 (d, J = 2.2 Hz, 1H), 6.21 (dd, J = 8.2, 2.0 Hz, 1H), 5.25 (d, J = 13.4 Hz, 2H), 3.81 (s, 3H),
3.79 (s, 3H), 3.17 (s, 3H), 2.87-2.76 (m, 4H); MS (ESI) 759 (M-Br)+.
4.1.23.
benzaldehyde (40)
This compound was prepared from 35 by following the procedure described for 38, yield 85%,
2-(3-(3-(4-((Bromotriphenylphosphoranyl)methyl)phenoxy)-4-methoxyphenethyl)phenoxy)
white solid; mp 230-231 °C. 1H NMR (CDCl3) δ 10.49 (s, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.79-7.74 (m,
9H), 7.67-7.63 (m, 6H), 7.57 (t, J = 7.4 Hz, 1H), 7.52-7.48 (m, 3H), 7.29 (t, J = 7.5 Hz, 1H), 7.18 (t, J
= 7.5 Hz, 1H), 7.04 (dd, J = 8.7, 2.5 Hz, 2H), 6.97 (d, J = 7.6 Hz, 1H), 6.91-6.84 (m, 3H), 6.81-6.76 (m,
2H), 5.40 (d, J = 13.8 Hz, 2H), 3.80 (s, 3H), 2.88-2.80 (m, 4H); MS (ESI) 699 (M-Br)+.
4.1.24. 2-(3-(3-(4-((Bromotriphenylphosphoranyl)methyl)phenoxy)-4-methoxyphenethyl)-4-methoxy-
phenoxy)-3-methoxybenzaldehyde (42)
This compound was prepared from 37 by following the procedure described for 38, yield 92%,
1
white solid; mp 244-245 °C. H NMR (CDCl3) δ 9.95 (s, 1H), 7.75-7.69 (m, 9H), 7.60-7.58 (m, 6H),
7.55 (dt, J = 7.5, 1.2 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.33 (dd, J = 2.5, 1.4 Hz, 1H), 7.22 (ddd, J = 8.1,
2.6, 1.0 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 6.96-6.91 (m, 2H), 6.82 (d, J = 8.4 Hz, 1H), 6.80-6.74 (m,
2H), 6.59 (d, J = 8.5 Hz, 2H), 6.26 (t, J = 2.1 Hz, 1H), 5.29 (d, J = 13.8 Hz, 2H), 3.81 (s, 3H), 3.78 (s, 3
H), 3.79 (s, 3H), 2.87-2.79 (m, 4H); MS (ESI) 759 (M-Br)+.
4.1.25. Macrocycle (stilbene bridge) (43)
A solution of phosphonium salt 38 (2.58 g, 3.07 mmol) in anhydrous dichloromethane (150
mL/mmol) was added dropwise (5 h/mmol) to a suspension of sodium methoxide (1.35 g, 24.6 mmol)
in anhydrous dichloromethane (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 dichloromethane, to provide the compound
43 as white solid (1.19 g, 81%); mp 155-156 °C. 1H NMR (CDCl3) δ 7.23 (t, J = 7.8 Hz, 1 H), 7.11 (d,
J = 7.2 Hz, 1 H), 7.00 (d, J = 7.2 Hz, 1 H), 6.94 (d, J = 8.4 Hz, 2 H), 6.88 (d, J = 7.2 Hz, 2 H),
6.81-6.79 (m, 2 H), 6.75 (d, J = 7.8 Hz, 1 H), 6.65 (d, J = 16.2 Hz, 1 H), 6.57-6.55 (m, 2 H), 6.15 (d, J
= 7.8 Hz, 1 H), 5.72 (s, 1 H), 3.98 (s, 3 H), 3.92 (s, 3 H), 3.74 (s, 3 H), 3.04-2.83 (m, 4 H); MS (ESI)

ACCEPTED MANUSCRIPT
503 (M+Na)+.
4.1.26. Macrocycle (stilbene bridge) (44)
This compound was prepared from 39 by following the procedure described for 43, yield 83%,
o
1
white solid; mp 149-150 C . H NMR (CDCl3) δ 7.25 (d, J = 7.6 Hz, 1 H), 7.22 (t, J = 7.6 Hz, 1 H ),
7.15 (dd, J = 1.6 Hz, J = 7.6 Hz, 1 H), 6.97 (dd, J = 1.6 Hz, J = 7.6 Hz, 1 H), 6.95-6.92 (m, 2 H ), 6.88
(d, J = 8.0 Hz, 1 H), 6.84 (d, J = 8.0 Hz, 1 H), 6.80 (dd, J = 1.6 Hz, J = 7.6 Hz, 1 H), 6.59 (dd, J = 2.0
Hz, J = 8.4 Hz, 1 H), 6.53 (d, J = 7.2 Hz, 1 H), 6.51-6.47 (m, 2H), 6.19 (dd, J = 1.6 Hz, J = 7.6 Hz, 1
H), 5.72 (d, J = 2.0 Hz, 1 H), 3.98 (s, 3H), 3.91 (s, 3H), 3.69 (s, 3H), 3.11-2.81 (m, 4H). 13C NMR
(CDCl3) δ 158.5, 157.9, 156.0, 152.7, 150.3, 146.7, 142.9, 141.5, 135.2, 132.8, 131.2, 128.4, 127.9,
126.6, 126.2, 125.4, 121.9, 119.6, 118.2, 116.9, 115.4, 114.7, 113.0, 111.7, 111.6, 105.4, 56.3, 56.2,
55.5, 34.4, 31.3. MS (ESI) 481 (M+H)+.
4.1.27. Macrocycle (stilbene bridge) (45)
This compound was prepared from 40 by following the procedure described for 43, yield 80%,
1
white solid; mp 180-181 °C. H NMR (CDCl3) δ 7.48 (dd, J = 1.2 Hz, J = 3.2 Hz, 1 H ),7.34 (td, J =
1.2 Hz, J = 7.6 Hz, 1 H), 7.27-7.23 (m, 1 H), 7.20 (dd, J = 1.2 Hz, J = 8.0 Hz, 1 H), 7.15 (d, J = 8.4 Hz,
2 H), 6.91 (d, J = 8.4 Hz, 2 H), 6.87-6.83 (m, 3 H), 6.78 (dd, J = 2.0 Hz, J = 8.4 Hz, 1 H), 6.63 (d, J =
16 Hz, 1 H), 6.51 (d, J = 7.6 Hz, 1 H), 6.42 (d, J = 16 Hz, 1 H), 6.09 (dd, J = 2.4 Hz, J = 8.4 Hz, 1 H),
5.68 (d, J = 2.0 Hz, 1 H), 3.96 (s, 3 H), 2.99-2.88 (m, 4 H); 13C NMR (CDCl3) δ 158.9, 155.2, 152.7,
150.4, 146.8, 143.0, 137.5, 135.1, 131.9, 131.5, 131.2, 130.9, 128.8, 128.6, 126.9, 126.4, 125.3, 122.8,
122.5, 121.9, 119.4, 116.8, 114.6, 113.1, 111.7, 56.2, 34.0, 30.7; MS (ESI) 421 (M+H)+.
4.1.28. Macrocycle (stilbene bridge) (46)
This compound was prepared from 41 by following the procedure described for 43, yield 86%,
white solid; mp 174-175 oC . 1H NMR (CDCl3) δ 7.16 (d, J = 8.5 Hz, 2H), 7.11 (t, J = 7.9 Hz, 1H), 7.02
(dd, J = 7.7, 1.3 Hz, 1H), 6.96-6.91 (m, 3H), 6.88 (d, J = 8.2 Hz, 1H), 6.81 (dd, J = 8.2, 2.0 Hz, 2H),
6.64 (d, J = 15.9 Hz, 1H), 6.56-6.53 (m, 2H), 6.46-6.44 (m, 1H), 5.70 (d, J = 2.0 Hz, 1H), 3.99 (s, 3H),
3.93 (s, 3H), 3.53 (s, 3H), 3.03-2.95 (m, 2H), 2.86-2.79 (m, 2H); 13C NMR (151 MHz, CDCl3) δ 155.1,
151.0, 150.6, 147.3, 147.2, 146.3, 143.7, 137.2, 134.6, 133.9, 131.3, 130.1, 130.0, 127.0, 123.7, 123.0,
121.7, 119.7, 119.2, 117.9, 114.0, 113.0, 111.6, 111.4, 56.6, 56.4, 56.2, 31.3, 29.2; MS (ESI) 481
(M+H)+.
4.1.29. Macrocycle (stilbene bridge) (47)

ACCEPTED MANUSCRIPT
This compound was prepared from 42 by following the procedure described for 43, yield 76%,
white solid; mp 203-204 °C. 1H NMR (CDCl3) δ 7.39 (d, J = 8.4 Hz, 2 H ), 7.22-7.20 (m, 2 H), 7.05 (d,
J = 8.4 Hz, 2 H), 7.01-6.97 (m, 2 H), 6.89-6.86 (m, 2 H), 6.83 (d, J = 7.8 Hz, 1 H), 6.75 (dd, J = 1.8 Hz,
J = 7.8 Hz, 1 H), 6.55 (d, J = 9.0 Hz, 1 H), 6.46 (dd, J = 2.4 Hz, J = 8.4 Hz, 1 H), 5.36 (d, J = 1.8 Hz, 1
H), 3.97 (s, 3 H), 3.93 (s, 3 H), 3.57 (s, 3 H), 2.69-2.58 (m, 4 H); 13C NMR (CDCl3) δ 155.5, 152.6,
125.5, 151.8, 150.4, 146.0, 142.7, 137.7, 136.0, 132.2, 132.1, 130.5, 130.0, 127.0, 125.0, 122.8, 120.6,
118.8, 116.9, 115.6, 113.9, 111.1, 110.9, 110.8, 55.6, 55.4, 52.9, 35.0, 33.1; MS (ESI) 481 (M+H)+.
4.1.30. Macrocyclic derivative (48)
Palladium on activated carbon (10% Pd, 480 mg) were added to a solution of compound 43 (4.80
g, 10 mmol) in ethyl acetate (150 mL). The suspension was stirred under H2 for 24 h at room
temperature. The reaction mixture was filtered, and the solution was concentrated to provide the
compound 48 as a white solid (4.70 g, 95%); mp 166-167 °C. 1H NMR (CDCl3) δ 7.24 (t, J = 7.8 Hz, 1
H), 7.11 (d, J = 7.2 Hz, 1 H), 6.90 (t, J = 7.2 Hz, 1 H), 6.85 (d, J = 7.8 Hz, 2 H), 6.78 (d, J = 7.8 Hz, 1
H), 6.62 (s, 2 H), 6.59 (d, J = 7.8 Hz, 1 H), 6.50 (d, J = 7.2 Hz, 1 H), 6.43 (d, J = 7.8 Hz, 1 H), 6.29 (d,
J = 7.8 Hz, 1 H), 5.47 (s, 1 H), 3.92 (s, 3 H), 3.69 (s, 3 H), 3.61 (s, 3 H), 3.07-3.05 (m, 4 H), 2.86 (s, 2
H), 2.78 (s, 2 H); 13C NMR (CDCl3) δ 158.0, 152.3, 151.4, 147.6, 146.4, 142.0, 141.1, 140.9, 140.1,
136.6, 133.4, 127.9, 125.3, 123.2, 122.4, 122.3, 121.2, 120.8, 115.0, 114.8, 112.8, 111.8, 111.4, 110.1,
56.0, 55.7, 55.6, 36.5, 36.4, 34.6, 30.2; MS (ESI) 505 (M+Na)+.
4.1.31. Macrocyclic derivative (49)
This compound was prepared from 44 by following the procedure described for 48, yield 96%,
white solid; mp 155-156 oC . 1H NMR (CDCl3) δ 7.21 (t, J = 8.0 Hz, 1H), 7.11 (dd, J = 7.9, 1.4 Hz, 1H),
6.92 (t, J = 8.0 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.84 (dd, J = 8.1, 2.1 Hz, 2H), 6.80 (dd, J = 8.2, 2.0
Hz, 1H), 6.63 (t, J = 2.2 Hz, 1H), 6.43 (dd, J = 8.2, 2.0 Hz, 1H), 6.38-6.30 (m, 2H), 6.19 (dd, J = 8.0,
2.2 Hz, 1H), 5.64 (d, J = 2.0 Hz, 1H), 3.93 (s, 3H), 3.69 (s, 3H), 3.64 (s, 3H), 3.02 (s, 4H), 2.91-2.89
(m, 2H), 2.80-2.78 (m, 2H); 13C NMR (151 MHz, CDCl3) δ 158.3, 157.6, 153.9, 152.3, 148.3, 146.7,
141.9, 141.1, 137.5, 133.7, 130.6, 128.1, 127.1, 125.2, 122.6, 122.0, 121.6, 116.0, 115.2, 113.1, 112.0,
111.6, 110.0, 104.6, 56.1, 55.8, 55.2, 36.1, 34.3, 30.3, 28.9; MS (ESI) 505 (M+Na)+.
4.1.32. Macrocyclic derivative (50)
This compound was prepared from 45 by following the procedure described for 48, yield 95%,
1
white solid; mp 178-179 °C. H NMR (CDCl3) δ 7.43-7.38 (m, 1H), 7.12 (t, J = 7.9 Hz, 1H), 7.10-7.07

ACCEPTED MANUSCRIPT
(m, 2H), 7.05-7.02 (m, 2H), 6.89 (d, J = 8.2 Hz, 1H), 6.81-6.78 (m, 1H), 6.78-6.73 (m, 2H), 6.72-6.66
(m, 3H), 6.55-6.51 (m, 1H), 6.06 (d, J = 2.0 Hz, 1H), 5.54 (s, 1H), 3.90 (s, 3H), 3.19-3.13 (m, 2H),
3.06-3.00 (m, 2H), 2.95-2.91 (m, 2H), 2.87-2.79 (m, 2H); 13C NMR (151 MHz, CDCl3) δ 155.9, 154.8,
153.8, 145.4, 144.3, 143.0, 137.9, 133.3, 131.9, 131.4, 130.0, 129.3, 126.7, 123.6, 123.4, 122.7, 120.1,
118.9, 116.6, 116.5, 116.4, 115.0, 37.1, 35.6, 34.5, 30.1; MS (ESI) 445 (M+Na)+.
4.1.33. Macrocyclic derivative (51)
This compound was prepared from 46 by following the procedure described for 48, yield 98%,
white solid; mp 145-146 oC . 1H NMR (CDCl3) δ 7.19 (t, J = 7.9 Hz, 1H), 7.15 (dd, J = 7.8, 1.4 Hz, 1H),
6.94 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.3 Hz, 1H), 6.82 (ddd, J = 10.2, 8.1, 1.7 Hz, 2H), 6.76 (d, J = 8.3
Hz, 1H), 6.53 (d, J = 8.4 Hz, 2H), 6.47 (dd, J = 8.3, 1.9 Hz, 1H), 6.18 (d, J = 2.0 Hz, 1H), 5.87 (d, J =
1.9 Hz, 1H), 3.97 (s, 3H), 3.90 (s, 3H), 3.75 (s, 3H), 3.09-2.90 (m, 4H), 2.89-2.47 (m, 4H); 13C NMR
(151 MHz, CDCl3) δ 153.4, 152.7, 148.0, 146.9, 146.8, 146.7, 142.6, 136.6, 135.1, 133.2, 132.2, 129.4,
125.0, 121.9, 121.5, 120.6, 120.2, 116.1, 115.7, 111.9, 111.5, 110.3, 56.3, 56.1, 55.8, 34.0, 33.0, 31.0,
27.9; MS (ESI) 505 (M+Na)+.
4.1.34. Macrocyclic derivative (52)
This compound was prepared from 47 by following the procedure described for 48, yield 97%,
white solid; mp 156-157 oC . 1H NMR (CDCl3) δ 7.21 (t, J = 8.0 Hz, 1H), 7.08 (dd, J = 7.8, 1.3 Hz, 1H),
7.01 (d, J = 8.4 Hz, 2H), 6.85 (dd, J = 11.7, 4.7 Hz, 2H), 6.81 (dd, J = 8.2, 2.0 Hz, 1H), 6.73 (d, J = 8.4
Hz, 2H), 6.71 (d, J = 3.1 Hz, 1H), 6.45 (d, J = 8.9 Hz, 1H), 6.24 (dd, J = 8.9, 3.1 Hz, 1H), 5.49 (d, J =
1.9 Hz, 1H), 3.93 (s, 3H), 3.67 (s, 3H), 3.26 (s, 3H), 3.07-2.98 (m, 4H), 2.83-2.69 (m, 4H); 13C NMR
(151 MHz, CDCl3) δ 152.7, 152.5, 152.4, 151.9, 148.4, 146.6, 141.5, 139.4, 137.0, 134.6, 131.0, 129.6,
125.1, 122.7, 122.0, 121.6, 117.1, 115.7, 111.6, 111.5, 110.9, 110.4, 56.2, 55.9, 55.8, 36.4, 36.1, 31.0,
30.6; MS (ESI) 505 (M+Na)+.
4.1.35. Macrocyclic derivative (53)
A solution of boron tribromide (6.31 g, 25.22 mmol) in anhydrous dichloromethane (20 mL) was
added dropwise to a stirred solution of compound 48 (1.52 g, 3.15 mmol) in anhydrous
dichloromethane (20 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. The ice-cold water was added, and the reaction
mixture was stirred vigorously for 1 h. The solution was then diluted with dichloromethane (50 mL),
washed with saturated aqueous NaCl (20 mL × 3) and dried over sodium sulfate. The solution was

ACCEPTED MANUSCRIPT
concentrated, and the residue was purified by silica gel column chromatography, eluating with
1
dichloromethane, to provide compound 53 as white solid (1.26 g, 91%); mp 161-162 °C. H NMR
(CDCl3) δ 7.18 (t, J = 7.9 Hz, 1H), 7.03 (d, J = 6.5 Hz, 1H), 6.98 (t, J = 7.9 Hz, 1H), 6.92 (d, J = 8.1
Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.80 (dd, J = 8.1, 1.8 Hz, 1H), 6.71 (s, 1H), 6.66 (s, 1H), 6.55-6.45
(m, 3H), 6.29 (d, J = 7.5 Hz, 1H), 5.50 (d, J = 1.7 Hz, 1H), 5.05 (s, 1H), 4.82 (s, 1H), 3.00 (s, 4H),
2.86-2.78 (m, 4H); 13C NMR (CDCl3) δ 156.8, 148.6, 147.4, 144.8, 143.2, 142.9, 141.2, 139.6, 139.0,
136.2, 133.2, 128.9, 126.1, 123.7, 123.2, 122.1, 121.8, 121.0, 116.7, 115.5, 115.4, 115.0, 114.4, 111.8,
36.3, 35.9, 34.7, 30.4; MS (ESI) 463 (M+Na)+; HRMS calcd for C28H24O5Na 463.1516, found:
463.1510 (M+Na)+.
4.1.36. Macrocyclic derivative (54)
This compound was prepared from 49 by following the procedure described for 53, yield 88%,
white solid; mp 140-141 oC . 1H NMR (CDCl3) δ 7.19 (t, J = 7.9 Hz, 1H), 7.08 (dd, J = 7.8, 1.4 Hz, 1H),
6.97 (t, J = 7.9 Hz, 1H), 6.90 (dd, J = 8.1, 2.8 Hz, 3H), 6.76 (dd, J = 8.1, 1.9 Hz, 1H), 6.72 (s, 1H), 6.53
(dd, J = 8.1, 2.3 Hz, 1H), 6.38 (d, J = 7.6 Hz, 1H), 6.24 (d, J = 8.1 Hz, 1H), 6.22 (d, J = 2.0 Hz, 1H),
5.56 (d, J = 2.1 Hz, 1H), 5.56 (s, 1H), 4.96 (s, 1 H), 4.90 (s, 1 H), 3.12-2.94 (m, 4H), 2.90-2.86 (m, 2H),
2.82-2.69 (m, 2H); 13C NMR (151 MHz, CDCl3) δ 156.8, 153.8, 153.5, 148.7, 145.9, 143.3, 143.1,
139.7, 136.6, 132.8, 131.3, 128.9, 126.3, 125.7, 123.6, 122.5, 121.9, 115.7, 115.5, 115.0, 114.7, 113.9,
111.6, 109.2, 36.1, 34.5, 30.3, 29.4; MS (ESI) 463 (M+Na)+; HRMS calcd for C28H24O5Na 463.1516,
found: 463.1510 (M+Na)+.
4.1.37. Macrocyclic derivative (55)
This compound was prepared from 50 by following the procedure described for 53, yield 86%,
white solid; mp 139-140 oC . 1H NMR (CDCl3) δ 7.44-7.39 (m, 1H), 7.14-7.07 (m, 3H), 7.02 (d, J = 8.3
Hz, 2H), 6.86 (d, J = 8.3 Hz, 1H), 6.84-6.79 (m, 2H), 6.77 (dd, J = 8.1, 1.9 Hz, 1H), 6.70 (d, J = 8.3 Hz,
2H), 6.63 (d, J = 7.6 Hz, 1H), 6.57 (d, J = 1.7 Hz, 1H), 6.02 (d, J = 2.0 Hz, 1H), 3.17-3.11 (m, 2H),
3.05-2.98 (m, 2H), 2.96-2.90 (m, 2H), 2.86-2.79 (m, 2H); 13C NMR (151 MHz, CDCl3) δ 156.2, 154.6,
154.1, 147.8, 147.7, 142.9, 137.3, 133.8, 132.3, 131.4, 129.8, 129.1, 126.7, 123.6, 122.9, 122.5, 120.4,
118.6, 117.4, 116.9, 116.1, 112.0, 56.1, 37.2, 35.8, 34.6, 30.2; MS (ESI) 409 (M+H)+; HRMS calcd for
C28H28O3N1 426.2068 found: 426.2064 (M+NH4)+.
4.1.38. Macrocyclic derivative (56)
This compound was prepared from 51 by following the procedure described for 53, yield 90%,

ACCEPTED MANUSCRIPT
white solid; mp 225-226 oC .1H NMR (CDCl3) δ 7.21 (t, J = 7.9 Hz, 1H), 7.13 (dd, J = 7.8, 1.4 Hz, 1H),
6.95 (d, J = 8.6 Hz, 2H), 6.92 (dd, J = 8.0, 1.8 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.2 Hz,
1H), 6.78 (dd, J = 8.2, 2.0 Hz, 1H), 6.55 (dd, J = 8.5, 2.0 Hz, 3H), 6.25 (d, J = 1.8 Hz, 1H), 5.85 (d, J =
1.9 Hz, 1H), 5.73 (s, 1H), 5.46 (s, 1H), 5.04 (s, 1H), 3.16-3.09 (m, 4H), 2.98-2.96 (m, 2H), 2.76-2.74
(m, 2H); 13C NMR (151 MHz, CDCl3) δ 153.4, 148.9, 145.6, 143.5, 143.4, 143.2, 140.5, 136.6, 133.7,
132.7, 132.3, 129.5, 126.3, 123.0, 122.0, 121.0, 120.3, 115.3, 115.2, 115.1, 114.4, 114.3, 33.2, 32.4,
30.3, 28.6; MS (ESI) 463 (M+Na)+; HRMS calcd for C28H24O5Na 463.1516, found: 463.1510
(M+Na)+.
4.1.39. Macrocyclic derivative (57)
This compound was prepared from 51 by following the procedure described for 53, yield 90%,
1
white solid; mp 124-125 oC . H NMR (CDCl3) δ 7.18 (t, J = 7.9 Hz, 1H), 7.04 (dd, J = 7.8, 1.4 Hz, 1H),
7.01 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 8.0 Hz, 1H), 6.90 (dd, J = 8.0, 1.5 Hz, 1H), 6.82 (dd, J = 8.1, 2.0
Hz, 1H), 6.75 (d, J = 3.1 Hz, 1H), 6.70 (d, J = 8.2 Hz, 2H), 6.50 (d, J = 8.7 Hz, 1H), 6.31 (dd, J = 8.7,
3.1 Hz, 1H), 5.62 (s, 1H), 5.41 (d, J = 1.9 Hz, 1H), 4.96 (s, 1H), 3.54 (s, 1H), 3.08-3.06 (m, 2H),
3.00-2.92 (m, 2H), 2.80-2.69 (m, 4H); 13C NMR (151 MHz, CDCl3) δ 152.2, 150.9, 149.4, 148.8, 146.7,
143.9, 140.1, 139.8, 136.5, 132.4, 129.8, 129.4, 126.1, 122.2, 122.0, 121.4, 117.4, 116.8, 115.5, 115.4,
114.3, 112.3, 36.1, 35.9, 31.6, 30.8; MS (ESI) 463 (M+Na)+; HRMS calcd for C28H24O5Na 463.1516,
found: 463.1510 (M+Na)+.
4.1.40. 2-(3-(1,3-Dioxan-2-yl)phenoxy)-5-methoxybenzaldehyde (66)
This compound was prepared from 2-bromo-5-methoxybenzaldehyde (63) and
3-(1,3-dioxan-2-yl)phenol (65) in 74% yield by following the procedure described for 6, white solid;
mp 123-124 °C. 1H NMR (CDCl3) δ 10.40 (s, 1H), 7.41 (s, 1H), 7.35 (d, J = 7.4 Hz, 1H), 7.18 (s, 1H),
7.13 (d, J = 5.9 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.86 (s, 1H), 5.49 (s, 1H),
4.26 (d, J = 10.6 Hz, 2H), 3.99 (t, J = 11.9 Hz, 2H), 3.86 (s, 3H), 2.26-2.20 (m, 1H), 1.45 (d, J = 13.4
Hz, 1H); MS (ESI) 315 (M+H)+.
4.1.41. 2-(3-(1,3-Dioxan-2-yl)phenoxy)-4-methoxybenzaldehyde (67)
This compound was prepared from 2-bromo-4-methoxybenzaldehyde (64) and 3-(1,3-dioxan-2-yl)
1
phenol (65) in 70% yield by following the procedure described for 6, white solid; mp 111-112 °C. H
NMR (CDCl3) δ 10.36 (s, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.39 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 6.7 Hz,
1H), 7.28 (s, 1H), 7.05 (d, J = 7.1 Hz, 1H), 6.71 (d, J = 8.5 Hz, 1H), 6.34 (s, 1H), 5.51 (s, 1H), 4.28 (d,