The synthesis and evaluation of new benzophenone derivatives as tubulin polymerization inhibitors

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The synthesis and evaluation of new benzophenone derivatives as

tubulin polymerization inhibitors

Received 00th January 20xx,

Accepted 00th January 20xx

Shun Zhang, Baijiao An, Jun Yan, Ling Huang, Xingshu Li*

DOI: 10.1039/x0xx00000x

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Inspired by the potent inhibition activity of phenstatin and millepachine against cancer cell growth, a series of new

benzophenone derivatives were synthesized and evaluated as tubulin polymerization inhibitors. Among them, compound,

10a exhibited 0.029 – 0.062μΜ of the IC₅₀for five human cancer cell lines, which is much better than that of the two

leading compounds. Flow cytometric analysis showed that 10a induces G2/M phase arrest and apoptosis in A549 cells.

Cellular studies revealed that the induction of apoptosis by 10a was associated with a collapse of mitochondrial membrane

potential (MMP). Overall, the current study demonstrates that the benzophenone derivatives are promising anticancer

agents by targeting tubulin.

stabilizing agents.³⁰Millepachine, with a chalcone structure

(Figure. 1), which was first isolated from the millettia

pachycarpa, exhibited potent cytotoxic against a variety of

Introduction

human cancer cells.³¹In our study, to search for new structural

compounds as anticancer agents, we have developed a new

series of ortho-(3,4,5-trimethoxybenzoyl)-acetanilides as novel

anti-cancer agent.³²In this work, inspired by the structure of

millepachine and phenstatin, we disclose the synthesis,

evaluation and the mechanism study of a series of new

benzophenone derivatives as tubulin polymerization inhibitors.

Discovering highly effective agents with low toxicity for the

treatment of cancer is an important goal for the

pharmaceutical chemists. In the past decades, to develop

anticancer agents which targeting microtubule stabilizing or

microtubule destabilizing have attracted much attention.^1-5

Tubulin play a critical role in a variety of essential cellular

processes such as the movement of organelles, intracellular

transportation, and the formation and maintenance of cell

shape and so on.^6-8Some tubulin inhibitors, such as Colchicin,⁹

Vinblastine^10-11and Noscapinoids, ¹²have been used in clinical

or widely studded. Especially, Colchicine, are known as typical

agents targeting colchicine binding site. Recently, many

compounds targeting the colchicine binding site have been

reported. Combretastatin A-4 (CA-4), a prominent tubulin

polymerization inhibitor,¹³which strongly inhibits tubulin

polymerization by binding to the colchicine binding site, its

disodium phosphate form (CA-4P) has been approved for

clinical trials.^14-15CA-4 analogues including benzophenone

derivatives¹⁶such as phenstatin and diphenylamine derivatives

such as MPC6827¹⁷and its analogue^18-19also have been

developed. And recently tubulin inhibitor including chalcone

analogue²⁰and others^21-22also have been reported. For

developing novel and more effective anticancer agents, many

studies of the modification of CA-4 have appeared in recent

years.^23-27Among them, phenstatin, (Figure. 1) which the cis

double bond structure of CA-4 was replaced by carbonyl

group, exhibited potent inhibition of cancer cell growth.^28-29

Nature products are another source of new microtubule-

OCH₃

O

H₃CO

OH

N

H₃CO

OH

N

OCH₃

CA-4

Phenstatin

MPC-6827

OCH₃

O

OH

N

O

N

Cl

H

O

OCH₃

H₃CO

OCH₃

O

Millepachine

MPC-6827 analogue

10a

Figure 1 The structures of CA-4, phenstatin, MCP-6827, MCP-6827analogue,

Millepachine and 10a

Results and discussion

Chemistry

The general synthesis routes of the iso-MIL analogues are

listed in Schemes 1-2. First, the key intermediate was

obtained with vanillina as the starting material which reacted

.

3

1

with 3-chloro-3-methyl-1-butyne and then heated in pyridine

at 120 °C overnight with an overall yield of 70-75% for the two-

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006,

China. E-mail: lixsh@mail.sysu.edu.cn; Fax: +86-20-3994-3050; Tel: +86-20-3994-

3050

step. Then, the reaction of

3 with a series of aryl lithium

reagents generated in situ form bromobenzene or its

† Electronic supplementary information (ESI) available: NMR spectrums, HPLC and

HR-MS of target compounds. See DOI: 10.1039/x0xx00000x

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analogues and n-BuLi gave compound 4a-4h at the -78°C. The

oxidation of 4a-4h with pyridinium chlorochromate (PCC)

provides target compound 5a-5h (scheme 1). On the other

hand, the protection of bromobenzene analogues containing

hydroxyl bromobenzene (6a-6b) with MOM was performed by

treating them with chloromethyl methyl ether and sodium

hydride in dichloromethane.

Biological evaluation

In vitro human cancer cell lines growth inhibition. MTT ((3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide))

assay was first carried out for evaluation the antiproliferative

activities of the new iso-MIL analogues toward five human

cancer cell lines A549 (non-small cell lung carcinoma), HeLa

(human epithelial cervical cancer cell line), HCT-116 (human

colon cancer cell line), A2780 (human ovarian cancer cell line)

and MGC-803 (human gastric cancer cell line), and the results

were listed in Table 1. It could be seen from table 1 that most

of the synthetic compounds gave good anti-proliferative

activity with IC₅₀values in sub-micromol level. It is interesting

that compound 10a

,

the replacement of 3,4,5-

trimethoxyphenyl moiety of phenstatin by 8-methoxy-2,2-

dimethyl-2H-chromen-6-yl, provided the best results with 35

nM of IC₅₀value for A549 cell line, 29 nM for HeLa, 38 nM for

HCT-116, 62 nM for A2780 and 49 nM for MGC-803,

respectively, which were much better than the reference

compound phenstatin (IC₅₀value for A549: 179 nM, HeLa: 42

nM, HCT116: 123 nM, A2780: 190 nM, Mgc803: 904 nM,

respectively). Preliminary structure−acꢀvity relaꢀonship

analysis indicated that the 3-hydroxyl and 4-methoxy group in

B ring of the target compounds is necessary for inhibitory

activity. Compounds 5a, bearing 4-methoxy group but no 3-

hydroxyl group in B ring, exhibited 0.196 to 1.02μΜ of the IC₅₀

values, while its analogues, 5b and 5c, with 3- or 2-methoxy

Scheme 1 The synthesis of target compounds

Chloro-3-methylbut-1-yne, CuCl₂·2H₂O, DBU, CH₃CN, 0°C, 5 h, 81.0%. (b) Pyridine,

120 °C, 12 h, 85.0%. (c) substituted bromobenzene, n-BuLi, dry THF, -78°C, 12h,

42.9-65.3%. (d) PCC CH₂Cl₂3-5h, 43.1-76.5%.

5. Reagents and conditions: (a) 3-

，

group in B ring, provided only good to moderated results (5b

:

0.545 to 4.95μΜ for five cancer cell lines, 5c: more than 10μΜ

Using the same method as the synthesis of 5, compounds 9a-

of the IC₅₀values for four cancer cell lines besides 0.985μΜ for

9b were obtained, which were de-protected by in the presence

of HCl in mathanol to give the target compound 10a-10b.

Finally, compound 11 was straightforwardly based on wittig

reaction following a simple known method, and compound

A549. Compounds 5d

instead of hydroxyl group, all gave sub-micromol level of the

IC₅₀values for four cancer cell lines but not as potent as 10a

, 5e and 5f, with fluorine atom or methyl

.

To evaluate the effect of sulphur atom in synthesized

compound to the antiproliferative activities, we prepared

compound 5g, which possesses a methylmercapto group in 4-

position of the B ring. 5g gave nearly the same activity level

comparing with 5a in the most cases, but provided better

result in MGC-803 line (5a: 1.02μΜ of the IC₅₀, 5g: 0.50μΜ).

The results also indicated that with two or three methoxyl

groups were unfavourable for the activities; 12 exhibited 0.717

to 3.056 of the IC₅₀values and 5h gave more than 10μΜ of the

IC₅₀values in all of the five cancer lines. Inspired by the

excellent antiproliferative activity of iso-CA-4, we synthesised

11. Compound 11 showed the same level activities in all the

cell lines compared with 10a, but slightly lower than that of

iso-CA-4. Considering the excellent antiproliferative activity

and the briefness of the preparation, 10a was selected to

further study.

10a was treated with CH₃I to give target compound 12

.

Scheme 2 The synthesis of target compounds 10a-10b

,

11

, 12. (e) Chloromethyl

methyl ether, DIPEA, 0°C, 5h, 95.1-96.1%. (f)

3

, n-BuLi, dry THF, -78°C, 12h, 44.3-

55.4%. (g) PCC, CH₂Cl₂, 3-5h, 37.0-49.1%. (h) HCl, CH₃OH, 10h, 32.1-41.5%. (i)

Ethyl-triphenylphosphonium chloride, n-BuLi, dry THF, -78°C, 12h, HCl, CH₃OH,

10h, 95.1%. (j) NaH, CH₃I, CH₂Cl₂, 48h, 81.5%.

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Table 1 Antiproliferative activity of compounds against five human cancer cell lines^a

IC₅₀^b(μΜ)

HCT116

Compounds

A549

Hela

A2780

0.476±0.13

4.627±0.13

>10

Mgc803

1.020±0.25

4.95±0.34

>10

5a

0.196±0.10

0.545±0.011

0.985±0.03

0.226±0.11

0.327±0.19

0.106±0.01

0.25±0.01

>10

0.458±0.02

1.23±0.23

>10

0.549±0.12

1.318±0.05

>10

5b

5c

5d

0.279±0.06

1.390±0.05

0.125±0.03

0.325±0.04

>10

0.334±0.08

1.607±0.12

0.107±0.03

0.306±0.04

>10

0.242±0.08

0.358±0.07

0.449±0.02

0.636±0.10

>10

0.766±0.13

8.378±0.13

0.285±0.08

0.503±0.06

>10

5e

5f

5g

5h

10a

0.035±0.01

1.490±0.03

0.059±0.02

0.729±0.23

0.179±0.03

3.750±0.12

0.020±0.04

0.0295±0.01

1.890±0.09

0.043±0.01

3.056±0.07

0.042±0.02

5.070±0.17

0.046±0.06

0.038±0.01

1.430±0.23

0.050±0.09

0.758±0.06

0.123±0.05

5.613±0.09

0.032±0.08

0.0624±0.03

2.680±0.56

0.039±0.06

0.717±0.10

0.190±0.02

3.600±0.14

0.038±0.012

0.049±0.01

1.390±0.01

0.079±0.02

0.998±0.12

0.904±0.06

8.900±0.23

0.067±0.04

10b

11

12

phenstatin

Millepachine

Colchicin

^aCell lines were treated with different concentrations of the compounds for 48 h. Cell viability was measured by MTT assay as described in the Experimental Section. ^b

IC₅₀values are indicated as the mean±SD (standard error) of three independent experiments.

Selectivity of 10a towards normal cells and cancer cells.

a potent tubulin polymerization inhibitor with IC₅₀value of

To examine the cytotoxicity of 10a towards human normal 4.011±0.14 μΜ, which exhibited slightly weaker than Colchicin

cells (Table 2), we use BJ cell (human dermal fibroblasts) and (IC₅₀: 3.01±0.12 μΜ, Fig. 2B).

HLF cell (human embryonic lung fibroblast) for the MTT assay

comparing with A549 cell (non-small cell lung carcinoma). The

results in Table 2 showed that compound 10a has far more

potent antiproliferative activity than millepachine and

phenstatin. Furthermore, 10a gave 158-fold and 571-fold

selectivity ratios to BJ cell and HLF cell, respectively, which

demonstrate the hypotoxicity of 10a towards the two kinds of

human normal cells.

Table 2. Antiproliferative activity of compound 10a, millepachine, phenstatin, against

human normal cells.

IC₅₀^a(μΜ)

HLF

Selectivity ratio^b

(HLF/BJ)

Figure 2 Effect of compound 10a and colchicin on mircotubule dynamics. Purified

tubulin protein at 10 mM in a reaction buffer was incubated at 37 °C in the

absence (control) or presence of 10a (A) (10 μΜ, 5 μΜ, 3.75 μΜ, 2.5 μΜ, or 1.25

μΜ) and colchicine (B) (7.5 μΜ, 5 μΜ, 1 μΜ). Polymerizations are followed by an

increase in fluorescence emission at 410 nM over a 60 min period at 37 °C

(excitation wavelength is 340 nM).

Compounds

A549

BJ

10a

0.035±0.01

5.539 ±0.09

>20

158/>571

>5/>5

Millepachine 3.750±0.12

phenstatin 0.179±0.03

2.421±0.11

14/>112

^aData are presented as the mean ± SE from the dose-response cures of at least

three independent experiments. ^bSelectivity ratio = (IC₅₀human normal cells) /

(IC₅₀A549).

Disruption of Microtubule Dynamics.

To investigate the morphological alterations of cancer cells

caused by compound 10a, A549 cells were exposed to

different concentrations of 10a (25, 50 and 75 nM) for 12 h.

From Fig. 3 we can see the morphological alterations such as

microtubule shrinkage, membrane blebbing and chromatin

condensation indeed occurred under the laser scanning

confocal microscope that are normally associated with the

occurrence of apoptosis.

In vitro Inhibition of Tubulin Polymerization.

To determine whether 10a to be

a potent tubulin

polymerization inhibitor, the tubulin polymerisation assay

which was described by Bonne, D. et al. with moderate

modification was performed and Colchicin was used as the

reference compound. Fig. 2A showed that compound 10a was

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Apoptosis assay

To evaluate the ability of compound 10a to induce cell death,

we used flow cytometry with propidium iodide (PI), which only

stains DNA and enters dead cells, and fluorescent

immunolabeling of the protein annexin-V (V-FITC), which

selectively binds to the early-apoptotic cells that expose on

their surface the phospholipid phosphatidylserine. The results

in Fig. 4A indicated that when A549 cells were incubated with

10a at different concentrations (25, 50, and 75 nM) or DMSO

(0.01%) for 48 h, the percentage of early and late apoptosis

cells were 5.94%, 38.16%, and 69.85.7%, respectively (Figure

3A). When the incubation time was extended to 72h, 21.13%,

69.18%, and 84.58% of the early and late apoptosis cells were

obtained (Fig. 4C). These data indicated that 10a induce cell

apoptosis in a concentration- and time-dependent manner.

Analysis of cell cycle arrest

To evaluate the arrest effect of compound 10a on the cell

cycle, we study the effect on cell cycle progression using

propidium iodide (PI) staining by flow cytometry analysis in

A549 cells. After treated with DMSO (0.01%) or compound 10a

at varies concentrations (25, 50, and 75 nM) for 24 h, the cells

were harvested and analysed by flow cytometry (Fig. 5) which

indicated compound 10a resulted in significant cell-cycle arrest

at G2/M phase, and showed a dose-dependent manner

(38.78%, 52.11%, 68.16% G2/M cell cycle arrest were

observed, respectively, comparing with 18.29% of that of the

control). The percentage of cells at G2/M phase increased to

45.6%, 59.14% and 68.22% for 48h, respectively, which

showed a time-dependent manner. These results revealed that

compound 10a might has the same fashion as CA-4, which

arrest cell-cycle progression at G2/M phase due to

microtubule depolymerization and cytoskeleton disruption

(Fig. 5).

Figure 3 Effects of compound 10a on mircrotubule dynamics in vitro. A549 cells

were treated with compound 10a at different concentration (25, 50, and 75 nM)

or DMSO (0.01%) for 12 h. Nucleuses were stained with Hochest 33342 (blue),

and microtubules were stained with mouse anti-α-tubulin conjugated with Alex

flour 488 (green). Images were taken using LSM 570 laser confocal microscope.

Figure 4 10a induced the apoptosis of A549 cells. A549 cells were treated with 10a at

the indicated concentrations for 48 h (A) and 72h (C). Then, the cells were trypsinized,

harvested and stained with Annexin V-FITC and PI solution for flow cytometry. The

percentages of cells in each stage of apoptosis were quantitated by flow cytometry.

(B1, upper left quadrant) necrotic cells; (B2, upper right quadrant) late apoptotic cells;

(B3, bottom left quadrant) live cells; and (B4, bottom right quadrant) early apoptotic

cells. Representative images from five independent experiments were shown.

Representative images from three independent experiments are shown 48h(B)

and 72h(D).

Figure

5 Cell cycle arrest effect of 10a. Effects of compounds 10a on DNA

content/cell following the treatment of A549 cells at different concentration (25,

50, and 75 nM) or DMSO (0.01%) for 24 h(A) and 48 h(B). The experiments were

performed at least three times, and the results of the representative experiments

are shown. Representative images from three independent experiments are

shown 24h(C) and 48h(D).

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10a induced Mitochondrial Dysfunction.

The X-ray crystal structure of tubulin complex with colchicine

As mitochondria play a vital role in the progression of (PDB entry 1SA0) was downloaded from RCSB PDB. The crystal

apoptosis, decreased mitochondrial membrane potential structure consists of four similar chains of domains and one

(MMP, ΔΨm) has been known as an early event in apoptotic linear chain of helix. The colchicine binding site is between two

cells. To detect the MMP, we use flow cytometry analysis and of the domains (Fig. 7A), the domains are named chain A and B

fluorescence microscope for this study. Figure 6A indicated to distinguish. The molecular docking procedure was

that after incubation of the cells with 10a from 25 to 75 nM, performed with the glide protocol of Schrodinger Maestro

the green fluorescence intensity (JC-1 monomers), increased 10.2. Compound 10a was docked into the colchicine site on

from 1.33% to 88.23%, while the red fluorescence intensity the basis of binding location of colchicine in the crystal

(JC-1 aggregates) decreased from 98.55% to 10.94%. Figure 6B, structure. The docking included all atoms as receptor and

the confocal microscopy assay showed that the cells converted using extra precision with flexible ligand sampling. The ligand-

from the aggregate to the monomers form, which was protein complex with the optimum score (Fig. 7B) was selected

consisted with results of flow cytometry analysis. These results as the initial structure for MD simulations and subsequent

implied that compound 10a induced MMP collapse and MM-PB(GB)SA binding free energy calculation.

mitochondrial dysfunction and eventually triggered apoptotic The MD simulations were carried out using the AMBER12³³

cell death.

molecular simulation package for both colchicine binding

crystal structure and compound 10a docking complex. The

AMBER99SB³⁴force field was employed for the protein and

the TIP3P model³⁵was used for solvent water molecules. The

force field parameters of ligands (colchicine and 10a) were

generated from AMBER GAFF force field. After three steps of

minimizations, heating and equilibrium, 10ns MD simulations

under NVT ensemble were carried out for both systems. ³⁶The

representative snapshots of binding mode are shown in Fig.7C

and D. The trajectory of last 2ns were chosen for MM-

PB(GB)SA binding free energy calculations. The Generalized

Born(GB) method performs better in this study with -

45.5878 kcal/mol for colchicine and -39.5153 kcal/mol for

compound 10a. The binding free energy for 10a is similar with

colchicine indicating 10a is a good tubulin polymerization

inhibitor in colchicine binding site.

Colchicine

Compound 10a

A chain

B chain

A chain

B chain

D

C

Leu242

Cys241

Ser178

Cys241

Leu248

Colchicine

Compound 10a

Thr202

Met259

Figure 6 Compound 10a decreased the mitochondrial membrane potential of

A549 cells. The A549 cells were treated with 10a at the different concentration

(25, 50, and 75 nM) or DMSO (0.01%) for 48 h, followed by incubation with the

fluorescence probe JC-1 for 30 min. Then, the cells were analysed by flow

cytometry (A) or fluorescence microscope (B). The experiments were performed

at least three times, and the results of the representative experiments are

shown.

Val181

Thr199

Figure 7. (A) Colchicine binding site in crystal structure. (B) Compound 10a

optimum docking pose in colchicine binding site. (C) and (D) Representative

snapshots for colchicine and compound 10a binding modes after MD simulations.

The key residues interacted with the compound 10a and colchicine are shown as

stick models with the corresponding colour of their ribbon. Hydrogen bonds

between the key residues and compound 10a are shown as dash lines.

Docking

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To a solution of

2

(2.18g, 10mmol) in pyridine (50ml) was

Conclusions

stirred at 120°C, and then the mixture was stirred for 10h.

After the solvent was removed in vacuum, the residue was

subjected to column chromatography (Petroleum ether/ethyl

It is well known that sometimes the minute structural

modification might result in great changes of the bioactivity.

Inspired by the structure of phenstatin and millepachine, we

have developed a series of new benzophenone derivatives as

acetate = 5:1) to to get the target compound 3.

8-methoxy-2,2-dimethyl-2H-chromene-6-carbaldehyde (3)

tubulin polymerization inhibitors. The optimal compound 10a

,

1

White solid, yield: 85.0%. H NMR (400 MHz, CDCl₃) δ 9.72 (s,

bearing 8-methoxy-2,2-dimethyl-2H-chromen-6-yl moiety

instead of the 3,4,5-tri-methoxyphenyl moiety of phenstatin,

exhibited much better antiproliferative activity in five tested

cancer cell lines (e.g. IC50 (μΜ) for A549 cell, 10a: 0.035;

Phenstatin: 0.179). Mechanism study indicated that 10a is a

good tubulin polymerization inhibitor with 4.01μΜ of the IC₅₀

value targeting the colchicines-binding site. Others including

10a induce cell apoptosis, arrest cell-cycle progression, induce

mitochondrial dysfunction and the morphological alterations

of A549 cell were also studied. The further research of 10a

such as in vivo anti-tumor evaluation leading to anticancer

drugs is in progress.

1H), 7.23 (d, J = 1.6 Hz, 1H), 7.08 (d, J = 1.7 Hz, 1H), 6.28 (d, J =

9.9 Hz, 1H), 5.61 (d, J = 9.9 Hz, 1H), 3.84 (s, 3H), 1.44 (s, 6H).

General procedure for the preparation of 4, 8a-8b

n-Butyllithium (0.8mL, 2.5M) was added dropwise to a solution

of substituted bromobenzene (1.0mmol) in anhydrous

THF(15mL). The mixture was stirred vigorously at -78°C under

a nitrogen atmosphere for 1h, and then, 8-methoxy-2,2-

dimethyl-2H-chromene-6-carbaldehyde (3) (218mg, 1.0mmol)

was added. After the mixture was stirred for another 12h, a

solution of saturated NH₄Cl was added to quench the reaction.

Ethyl acetate was added to the solution and the organic layers

were separated, dried over anhydrous Na₂SO₄, and evaporated

in vacuum to give the crude product which was purified by

Experimental

column chromatography to obtain the product

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-

methoxyphenyl)methanol (4a)

4 or 8a-8b.

Chemistry. All reagents were analytically pure without further

purification, and all chemistry solvents (THF, CH₂Cl₂) were

reagent-grade and were dried and freshly distilled before the

necessary step. ¹H NMR and ¹³C NMR spectra were recorded in

CDCl₃on a Bruker AvanceIII spectrometer, and chemical shifts

are reported as parts per million (ppm) with tetramethylsilane

as the internal standard. Melting points (MP) were measured

with SRS-OptiMelt automated melting point instrument. High-

resolution mass spectra (HR-MS) were recorded using an

Agilent LC–MS 6120 instrument. The high-performance

liquidchromatography (HPLC) were run on TC-C18 column (4.6

White oil, yield: 61.7%. IR (KBr, cm^-1) 3507, 2972, 2934, 2837,

1640, 1584, 1482, 1253, 1130, 710.¹H NMR (400 MHz, CDCl₃) δ

7.27 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 6.79 (d, J = 1.7

Hz, 1H), 6.58 (d, J = 1.7 Hz, 1H), 6.25 (d, J = 9.8 Hz, 1H), 5.67 (s,

1H), 5.59 (d, J = 9.8 Hz, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 2.39 (s,

1H), 1.46 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 159.09 (s),

148.33 (s), 141.36 (s), 136.27 (s), 131.10 (s), 127.93 (s), 122.48

(s), 121.76 (s), 117.02 (s), 113.92 (s), 110.79 (s), 76.61 (s),

75.65 (s), 56.37 (s), 55.39 (s), 27.99 (d, J = 2.4 Hz). HRMS (ESI)

(m/z) [M+Na] ⁺calcd for C₂₀H₂₂O₄, 349.1410; found, 349.1419.

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(3-

×

250 mm, 5μm) with two different solvent gradients

(methanol/ water=85:15), and a flow rate of 0.50mL/min. All

synthesized compounds were determined at least 95% of

purity by HPLC.

methoxyphenyl)methanol (4b)

White oil, yield: 65.3%. IR (KBr, cm^-1) 3506, 2976, 2934, 2828,

1

1640, 1585, 1506, 1246, 1133, 724. H NMR (400 MHz, CDCl₃)

General procedure for the preparation of 2

δ 7.27 – 7.20 (m, 1H), 6.98 – 6.90 (m, 2H), 6.83 – 6.75 (m, 2H),

3-chloro-3-methyl-1-butyne (1.53g, 15mmol) was added slowly 6.59 (d, J = 1.7 Hz, 1H), 6.24 (d, J = 9.8 Hz, 1H), 5.67 (s, 1H),

to a stirred of Vanillina ( ) (1.52g, 10mmol), CuCl₂·2H₂O (5mg, 5.59 (d, J = 9.8 Hz, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 2.50 (s, 1H),

1

0.3mol %), DBU (1.5ml, 15mmol) in CH₃CN (100mL) at 0°C, and 1.45 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 159.78 (s), 148.34 (s),

the reaction was monitored by TCL. After stirred for 5 hours, 145.61 (s), 141.51 (s), 135.93 (s), 131.10 (s), 129.55 (s), 122.44

the solvent was removed under reduced pressure. Water was (s), 121.79 (s), 118.95 (s), 117.17 (s), 112.92 (s), 112.18 (s),

added and the mixture was extracted by ethyl acetate. The 110.89 (s), 76.63 (s), 75.98 (s), 56.37 (s), 55.33 (s), 27.99 (d, J =

+

organic layer was dried over sodium sulfate and evaporated in 3.0 Hz). HRMS (ESI) (m/z) [M+Na] calcd for C₂₀H₂₂O₄,

vacuum to give the crude product which was purified by flash 349.1410; found, 349.1409.

column chromatography (Petroleum ether/ethyl acetate = 5:1) (8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(2-

to get white solid (

2).

methoxyphenyl)methanol (4c)

White oil, yield: 58.4%. IR (KBr, cm^-1) 3524, 2966, 2934, 2836,

3-methoxy-4-((2-methylbut-3-yn-2-yl)oxy)benzaldehyde (2)

1

White solid, yield: 81.0%. H NMR (400 MHz, CDCl₃) δ 9.88 (s, 1646, 1585, 1472, 1263, 1134, 759. H NMR (400 MHz, CDCl₃)

1

1H), 7.64 (d, J = 8.7 Hz, 1H), 7.43 (s, 1H), 7.42 (d, J = 6.7 Hz, 1H), δ 7.29 – 7.24 (m, 1H), 7.21 (dd, J = 7.5, 1.5 Hz, 1H), 6.94 (t, J =

3.89 (s, 3H), 2.64 (s, 1H), 1.74 (s, 6H).

7.2 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 1.8 Hz, 1H), 6.60

(d, J = 1.8 Hz, 1H), 6.25 (d, J = 9.8 Hz, 1H), 5.96 (s, 1H), 5.59 (d, J

= 9.8 Hz, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 3.05 (s, 1H), 1.46 (s,

General procedure for the preparation of 3

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6H). ¹³C NMR (101 MHz, CDCl₃) δ 156.91 (s), 148.16 (s), 141.19 12.8 Hz, 3H), 1.46 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 148.31

(s), 135.27 (s), 132.13 (s), 130.91 (s), 128.83 (s), 127.96 (s), (s), 141.44 (s), 140.88 (s), 137.53 (s), 135.95 (s), 131.11 (s),

122.67 (s), 121.63 (s), 120.92 (s), 117.13 (s), 111.14 (s), 110.84 127.09 (s), 126.65 (s), 122.37 (s), 121.75 (s), 117.08 (s), 110.80

(s), 76.54 (s), 72.05 (s), 56.37 (s), 55.57 (s), 28.01 (s). HRMS (s), 76.61 (s), 75.60 (s), 56.32 (s), 27.95 (d, J = 3.4 Hz), 15.92 (s).

+

(ESI) (m/z) [M+Na] calcd for C₂₀H₂₂O₄, 349.1410; found, HRMS (ESI) (m/z) [M+Na] ⁺calcd for C₂₀H₂₂O₃S, 365.1182;

349.1417.

found, 365.1186.

(3-fluoro-4-methoxyphenyl)(8-methoxy-2,2-dimethyl-2H-

chromen-6-yl)methanol (4d)

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(3,4,5-

trimethoxyphenyl)methanol (4h)

White oil, yield: 44.4%. IR (KBr, cm^-1) 3506, 2970, 2935, 2842, White oil, yield: 55.2%. IR (KBr, cm^-1) 3498, 2971, 2948, 2834,

1

1628, 1585, 1499, 1272, 1124, 725. H NMR (400 MHz, CDCl₃) 1638, 1585, 1455, 1229, 1124, 725. H NMR (400 MHz, CDCl₃)

δ 7.10 (dd, J = 12.2, 2.0 Hz, 1H), 7.05 (d, J = 9.2 Hz, 1H), 6.90 (t, δ 6.80 (d, J = 1.8 Hz, 1H), 6.63 – 6.57 (m, 3H), 6.26 (d, J = 9.8

J = 8.5 Hz, 1H), 6.75 (d, J = 1.8 Hz, 1H), 6.56 (d, J = 1.8 Hz, 1H), Hz, 1H), 5.64 (s, 1H), 5.61 (d, J = 9.8 Hz, 1H), 3.82 (s, 3H), 3.82

6.25 (d, J = 9.8 Hz, 1H), 5.63 (s, 1H), 5.61 (d, J = 9.8 Hz, 1H), (S, 9H), 2.63 (s, 1H), 1.46 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ

3.86 (s, 3H), 3.80 (s, 3H), 2.52 (s, 1H), 1.46 (s, 6H). ¹³C NMR 153.04 (s), 148.11 (s), 141.29 (s), 139.67 (s), 136.96 (s), 135.88

(101 MHz, CDCl₃) δ 152.28 (d, J = 245.8 Hz), 148.30 (s), 146.84 (s), 130.98 (s), 122.31 (s), 121.61 (s), 117.09 (s), 110.87 (s),

(d, J = 10.9 Hz), 141.47 (s), 137.17 (d, J = 5.5 Hz), 135.80 (s), 103.50 (s), 76.51 (s), 75.77 (s), 60.76 (s), 56.22 (s), 56.03 (s),

131.16 (s), 122.31 (s), 122.22 (d, J = 3.4 Hz), 121.77 (s), 117.00 27.80 (d, J = 2.0 Hz). HRMS (ESI) (m/z) [M+Na] ⁺calcd for C₂₂

(s), 114.42 (d, J = 19.0 Hz), 113.21 (d, J = 1.8 Hz), 110.68 (s), H₂₆O₆, 409.1622; found, 409.1622.

76.65 (s), 75.06 (s), 56.33 (s), 56.29 (s), 27.92 (d, J = 3.7 Hz). (8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-3-

HRMS (ESI) (m/z) [M+Na] ⁺calcd for C₂₀H₂₁O₄F, 367.1316; (methoxymethoxy)phenyl)methanol (8a)

White oil, yield: 55.4%. ¹H NMR (400 MHz, CDCl₃) δ 7.21 (d, J =

found, 367.1324.

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-3-

1.9 Hz, 1H), 6.97 (dd, J = 8.3, 1.9 Hz, 1H), 6.86 (d, J = 8.4 Hz,

methylphenyl)methanol (4e)

1H), 6.82 (d, J = 1.8 Hz, 1H), 6.61 (d, J = 1.8 Hz, 1H), 6.26 (d, J =

White oil, yield: 52.3%. IR (KBr, cm^-1) 3504, 2968, 2928, 2853, 9.8 Hz, 1H), 5.69 (s, 1H), 5.61 (d, J = 9.8 Hz, 1H), 5.22 (s, 2H),

1

1739, 1585, 1508, 1255, 1124, 725. H NMR (400 MHz, CDCl₃) 3.87 (s, 3H), 3.83 (s, 3H), 3.50 (s, 3H), 2.02 (s, 1H), 1.46 (s, 6H).

δ 7.13 (d, J = 9.4 Hz, 2H), 6.80 (d, J = 1.7 Hz, 1H), 6.76 (d, J = 8.1 (8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(3-methoxy-4-

Hz, 1H), 6.59 (d, J = 1.6 Hz, 1H), 6.25 (d, J = 9.8 Hz, 1H), 5.64 (s, (methoxymethoxy)phenyl)methanol (8b)

White oil, yield: 44.3%. ¹H NMR (400 MHz, CDCl₃) δ 7.09 (d, J =

1H), 5.59 (d, J = 9.8 Hz, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 2.36 (s,

1H), 2.19 (s, 3H), 1.45 (d, J = 1.3 Hz, 6H). ¹³C NMR (101 MHz,

8.3 Hz, 1H), 6.95 (d, J = 1.9 Hz, 1H), 6.86 (dd, J = 8.2, 1.9 Hz,

CDCl₃) δ 157.26 (s), 148.25 (s), 141.25 (s), 136.38 (s), 135.80 (s),

1H), 6.81 (d, J = 1.8 Hz, 1H), 6.60 (d, J = 1.8 Hz, 1H), 6.26 (d, J =

131.01 (s), 129.12 (s), 126.73 (s), 125.09 (s), 122.52 (s), 121.71

9.8 Hz, 1H), 5.68 (s, 1H), 5.61 (d, J = 9.8 Hz, 1H), 5.21 (s, 2H),

(s), 116.96 (s), 110.77 (s), 109.78 (s), 76.56 (s), 75.70 (s), 56.35

3.85 (s, 3H), 3.82 (s, 3H), 3.50 (s, 3H), 2.34 (s, 1H), 1.46 (s, 7H).

(s), 55.44 (s), 27.97 (d, J = 2.4 Hz), 16.41 (s). HRMS (ESI) (m/z)

[M+Na] ⁺calcd for C₂₁H₂₄O₄, 363.1567; found, 367.1566.

General procedure for the preparation of 5, 9a-9b

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-2-

To a solution of

4 or 8a-8b (1.0mmol) in 15ml of CH₂Cl₂, PCC

methylphenyl)methanol (4f)

(258mg 1.2mmol) and silica gel (258mg) were added and the

reaction was monitored by TCL. After stirred for 5 h, the

mixture was filtered and the filtrates were concentrated under

vacuum to give the residue which was purified by silica gel

column chromatography to obtain the target compound as a

white solid.

White oil, yield: 42.9%. IR (KBr, cm^-1) 3402, 2974, 2915, 2840,

1

1611, 1577, 1464, 1247, 1080, 725. H NMR (400 MHz, CDCl₃)

δ 7.36 (d, J = 8.5 Hz, 1H), 6.75 (dd, J = 9.3, 2.0 Hz, 2H), 6.69 (d, J

= 2.5 Hz, 1H), 6.52 (d, J = 1.6 Hz, 1H), 6.23 (d, J = 9.8 Hz, 1H),

5.82 (s, 1H), 5.59 (d, J = 9.8 Hz, 1H), 3.79 (s, 3H), 3.78 (s, 3H),

2.27 (s, 1H), 2.23 (s, 3H), 1.45 (d, J = 1.7 Hz, 6H). ¹³C NMR (101

MHz, CDCl₃) δ 158.74 (s), 148.19 (s), 141.24 (s), 137.05 (s),

135.49 (s), 134.03 (s), 130.95 (s), 127.65 (s), 122.47 (s), 121.65

(s), 117.39 (s), 116.21 (s), 111.33 (s), 110.91 (s), 76.52 (s),

72.77 (s), 56.27 (s), 55.20 (s), 27.90 (d, J = 2.9 Hz), 19.62 (s).

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-

methoxyphenyl)methanone (5a)

white solid, yield 75.7%. mp: 89.5°C -91.8°C. ¹H NMR (400 MHz,

CDCl₃) δ 7.81 (s, 1H), 7.79 (s, 1H), 7.32 (d, J = 1.7 Hz, 1H), 7.08

(d, J = 1.7 Hz, 1H), 6.98 (s, 1H), 6.96 (s, 1H), 6.31 (d, J = 9.9 Hz,

1H), 5.67 (d, J = 9.8 Hz, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 1.53 (s,

6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.57 (s), 162.89 (s), 148.22

(s), 146.03 (s), 132.29 (s), 131.20 (s), 130.78 (s), 130.26 (s),

122.26 (s), 121.95 (s), 120.77 (s), 113.54 (s), 113.44 (s), 77.82

(s), 56.37 (s), 55.58 (s), 28.31 (s). HRMS (ESI) (m/z) [M+H] ⁺

calcd for C₂₀H₂₀O₄, 325.1434; found, 325.1441. Purity: 98.6%

(by HPLC).

+

HRMS (ESI) (m/z) [M+Na] calcd for C₂₁H₂₄O₄, 363.1567;

found, 367.1572.

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-

(methylthio)phenyl)methanol (4g)

White oil, yield: 60.1%. IR (KBr, cm^-1) 3445, 2972, 2929, 2849,

1

1638, 1577, 1481, 1263, 1142, 742. H NMR (400 MHz, CDCl₃)

δ 7.30 (d, J = 8.3 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 1.7

Hz, 1H), 6.59 (d, J = 1.7 Hz, 1H), 6.26 (d, J = 9.8 Hz, 1H), 5.71 (d,

J = 2.4 Hz, 1H), 5.61 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H), 2.49 (d, J =

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(3-

methoxyphenyl)methanone (5b)

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white solid, yield 63.7%. mp:83.8°C -85.4°

C. ¹H NMR (400 (s), 131.10 (d, J = 11.7 Hz), 130.82 (s), 122.88 (s), 121.88 (s),

MHz, CDCl3) δ 7.43 – 7.34 (m, 2H), 7.33 – 7.28 (m, 2H), 7.15 – 120.74 (s), 116.62 (s), 112.85 (s), 110.17 (s), 77.96 (s), 56.33 (s),

+

7.05 (m, 2H), 6.30 (d, J = 9.9 Hz, 1H), 5.67 (d, J = 9.9 Hz, 1H), 55.39 (s), 28.36 (s), 20.60 (s). HRMS (ESI) (m/z) [M+H] calcd

3.90 (s, 3H), 3.86 (s, 3H), 1.58 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) for C₂₁H₂₂O₄, 339.1591; found, 339.1596 Purity: 96.0% (by

δ 195.36 (s), 159.54 (s), 148.30 (s), 146.55 (s), 139.69 (s), HPLC).

131.16 (s), 129.61 (s), 129.22 (s), 122.79 (s), 122.42 (s), 121.88 (8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-

(s), 120.76 (s), 118.26 (s), 114.22 (s), 113.38 (s), 77.97 (s), (methylthio)phenyl)methanone (5g)

56.38 (s), 55.56 (s), 28.36 (s). HRMS (ESI) (m/z) [M+H] ⁺calcd

white solid, yield 44.5%. mp:117.1°C -118.2°

C. ¹H NMR (400

for C₂₀H₂₀O₄, 325.1434; found, 325.1442. Purity: 96.8% (by

HPLC).

MHz, CDCl₃) δ 7.72 (d, J = 7.3 Hz, 2H), 7.34 (s, 1H), 7.30 (d, J =

7.5 Hz, 2H), 7.08 (s, 1H), 6.30 (d, J = 9.5 Hz, 1H), 5.67 (d, J = 9.5

Hz, 1H), 3.91 (s, 3H), 2.54 (s, 3H), 1.53 (s, 6H). ¹³C NMR (101

MHz, CDCl₃) δ 194.72 (s), 148.30 (s), 146.31 (s), 144.52 (s),

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(2-

methoxyphenyl)methanone (5c)

white solid, yield 63.7%. mp:131.7°C -133.1°

C. ¹H NMR (400 134.39 (s), 131.22 (s), 130.46 (s), 129.86 (s), 124.88 (s), 122.42

MHz, CDCl₃) δ 7.50 – 7.39 (m, 2H), 7.30 (d, J = 8.6 Hz, 1H), 7.04 (s), 121.88 (s), 120.78 (s), 113.36 (s), 77.90 (s), 56.37 (s), 28.33

+

(d, J = 7.4 Hz, 1H), 7.00 (d, J = 6.9 Hz, 2H), 6.25 (d, J = 9.9 Hz, (s), 14.98 (s). HRMS (ESI) (m/z) [M+H] calcd for C₂₀H₂₀O₃S,

1H), 5.63 (d, J = 9.9 Hz, 1H), 3.89 (s, 3H), 3.77 (s, 3H), 1.51 (s, 341.1206; found, 341.1211 Purity: 97.6% (by HPLC).

6H). ¹³C NMR (101 MHz, CDCl₃) δ 195.08 (s), 157.08 (s), 148.32 (8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(3,4,5-

(s), 146.98 (s), 131.40 (s), 130.86 (s), 130.09 (s), 129.18 (s), trimethoxyphenyl)methanone (5h)

123.06 (s), 121.90 (s), 120.74 (s), 120.39 (s), 112.48 (s), 111.48

white solid, yield 70.5%. mp:151.8°C -152.9°

C. ¹H NMR (400

(s), 78.00 (s), 56.32 (s), 55.77 (s), 28.39 (s).HRMS (ESI) (m/z)

[M+H] ⁺calcd for C₂₀H₂₀O₄, 325.1434; found, 325.1445. Purity:

98.4% (by HPLC).

MHz, CDCl₃) δ 7.35 (s, 1H), 7.14 (s, 1H), 7.03 (s, 2H), 6.33 (d, J =

9.9 Hz, 1H), 5.69 (d, J = 9.8 Hz, 1H), 3.94 (s, 3H), 3.92 (s, 3H),

3.89 (s, 6H), 1.54 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.71

(s), 152.89 (s), 148.19 (s), 146.41 (s), 141.52 (s), 133.44 (s),

131.29 (s), 129.73 (s), 122.42 (s), 121.80 (s), 120.88 (s), 113.55

(3-fluoro-4-methoxyphenyl)(8-methoxy-2,2-dimethyl-2H-

chromen-6-yl)methanone (5d)

white solid, yield 43.1%. mp:121.2

°

C

-122.1

°

C. ¹H NMR (400 (s), 107.39 (s), 78.00 (s), 61.09 (s), 56.42 (s), 28.42 (s). HRMS

+

MHz, CDCl₃) δ 7.63 – 7.55 (m, 2H), 7.31 (s, 1H), 7.06 (s, 1H), (ESI) (m/z) [M+H] calcd for C₂₂H₂₄O₆, 385.1646 found,

7.03 (t, J = 8.2 Hz, 1H), 6.31 (d, J = 9.9 Hz, 1H), 5.68 (d, J = 9.8 385.1638 Purity: 100.0% (by HPLC).

Hz, 1H), 3.98 (s, 3H), 3.91 (s, 3H), 1.53 (s, 6H). ¹³C NMR (101 (8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-3-

MHz, CDCl₃) δ 193.45 (s), 153.03 (s), 151.21 (d, J = 10.8 Hz), (methoxymethoxy)phenyl)methanone (9a)

1

White oil, yield 49.1%. H NMR (400 MHz, CDCl₃) δ 7.65 (d, J =

149.47 (d, J = 219.5 Hz), 146.38 (s), 131.33 (s), 131.07 (d, J =

5.1 Hz), 129.67 (s), 127.30 (d, J = 3.2 Hz), 122.29 (s), 121.87 (s),

1.7 Hz, 1H), 7.49 (dd, J = 8.4, 1.7 Hz, 1H), 7.32 (d, J = 1.4 Hz,

120.84 (s), 117.80 (d, J = 19.2 Hz), 113.37 (s), 112.24 (d, J = 1.0

1H), 7.11 (d, J = 1.5 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.31 (d, J =

+

Hz), 77.97 (s), 56.43 (s), 28.37 (s). HRMS (ESI) (m/z) [M+H]

9.9 Hz, 1H), 5.67 (d, J = 9.8 Hz, 1H), 5.27 (s, 2H), 3.97 (s, 3H),

calcd for C₂₀H₁₉O₄F, 343.1340; found, 343.1338 Purity: 95.0%

3.90 (s, 3H), 3.52 (s, 3H), 1.53 (s, 6H).

(by HPLC).

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(3-methoxy-4-

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-3-

(methoxymethoxy)phenyl)methanone (9b)

methylphenyl)methanone (5e)

White oil, yield 37.0%.¹H NMR (400 MHz, CDCl₃) δ 7.44 (d, J =

white solid, yield 76.5%. mp:145.6°C -147.1°

C. ¹H NMR (400

1.9 Hz, 1H), 7.34 (dt, J = 3.8, 1.9 Hz, 2H), 7.19 (d, J = 8.4 Hz,

1H), 7.11 (d, J = 1.8 Hz, 1H), 6.32 (d, J = 9.9 Hz, 1H), 5.68 (d, J =

9.8 Hz, 1H), 5.33 (s, 2H), 3.95 (s, 3H), 3.91 (s, 3H), 3.55 (s, 3H),

1.53 (s, 6H).

MHz, CDCl₃) δ 7.65 (d, J = 6.5 Hz, 2H), 7.32 (s, 1H), 7.08 (s, 1H),

6.87 (d, J = 9.0 Hz, 1H), 6.31 (d, J = 9.9 Hz, 1H), 5.67 (d, J = 9.8

Hz, 1H), 3.91 (s, 3H), 3.91 (s, 3H), 2.27 (s, 3H), 1.53 (s, 6H). ¹³C

NMR (101 MHz, CDCl₃) δ 194.87 (s), 161.19 (s), 148.18 (s),

145.93 (s), 132.58 (s), 131.16 (s), 130.48 (s), 130.30 (s), 130.22

(s), 126.75 (s), 122.24 (s), 122.02 (s), 120.76 (s), 113.50 (s),

108.96 (s), 77.79 (s), 56.40 (s), 55.64 (s), 28.34 (s), 16.41 (s).

HRMS (ESI) (m/z) [M+H] ⁺calcd for C₂₁H₂₂O₄, 339.1591; found,

339.1592 Purity: 98.4% (by HPLC).

General procedure for the preparation of 7a-7b

DIPEA (258mg 2mmol) and Chloromethyl methyl ether

(121mg, 1.5mmol) were added dropwise to the 6a-6b (201mg,

1mmol) solution in anhydrous CH₂Cl₂(20ml) over 5 minutes at

0°C. After stirred for 3 hours, the mixture was quenched with

saturated NaHCO₃solution, extracted by EtOAc. The combined

organic extracts was dried over MgSO₄and evaporated in

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-2-

methylphenyl)methanone (5f)

white solid, yield 76.5%. mp:116.2°C -117.9°

C. ¹H NMR (400

vacuum to give a 7a-7b

.

MHz, CDCl₃) δ 7.38 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H), 6.98 (s, 1H),

6.81 (s, 1H), 6.75 (d, J = 8.4 Hz, 1H), 6.26 (d, J = 9.9 Hz, 1H),

5.65 (d, J = 9.9 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 2.37 (s, 3H),

1.51 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 196.64 (s), 160.94 (s),

148.35 (s), 146.61 (s), 139.87 (s), 133.31 – 133.24 (m), 131.31

4-bromo-1-methoxy-2-(methoxymethoxy)benzene (7a)

1

Yellow oil, yield 95.1%. H NMR (400 MHz, CDCl₃) δ 7.29 (d, J =

2.3 Hz, 1H), 7.09 (dd, J = 8.6, 2.3 Hz, 1H), 6.75 (d, J = 8.7 Hz,

1H), 5.20 (s, 2H), 3.85 (s, 3H), 3.51 (s, 3H).

4-bromo-2-methoxy-1-(methoxymethoxy)benzene (7b)

8 | J. Name., 2012, 00, 1-3

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Yellow oil, yield 96.1%. ¹H NMR (400 MHz, CDCl₃) δ 6.99 (s, white solid, yield 95.1%. mp:137.3

ARTICLE

°

C -139.0°

C. ¹H NMR (400

3H), 5.16 (s, 2H), 3.83 (s, 3H), 3.48 (s, 3H).

MHz, CDCl₃) δ 6.98 (s, 1H), 6.85 (d, J = 8.3 Hz, 1H), 6.81 (d, J =

8.3 Hz, 1H), 6.77 (s, 1H), 6.62 (s, 1H), 6.26 (d, J = 9.8 Hz, 1H),

5.62 (d, J = 9.4 Hz, 2H), 5.29 (d, J = 1.8 Hz, 2H), 3.91 (s, 3H),

3.82 (s, 3H), 1.49 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 149.31

(s), 147.85 (s), 146.42 (s), 145.21 (s), 141.79 (s), 135.13 (s),

133.97 (s), 131.00 (s), 122.48 (s), 121.42 (s), 120.36 (s), 119.08

(s), 114.67 (s), 112.53 (s), 112.15 (s), 110.21 (s), 76.75 (s),

General procedure for the preparation of 10a-10b

Compound 9a-9b (384mg, 1mmol) was dissolved in methanol

(10mL) and then HCl (12N, 1mL) was added. After stirred for

3h, the reaction mixture was neutralized with NaHCO₃,

extracted with ethyl acetate and purified by flash column

chromatography (Petroleum ether /ethyl acetate = 5:1) to

+

56.38 (s), 56.07 (s), 28.07 (s). HRMS (ESI) (m/z) [M+H] calcd

for C₂₁H₂₂O₄, 339.1591 found, 339.1587 Purity: 99.8% (by

HPLC).

provided the target compound 10a-10b

.

(3-hydroxy-4-methoxyphenyl)(8-methoxy-2,2-dimethyl-2H-

chromen-6-yl)methanone (10a)

General procedure for the preparation of 12

white solid, yield 41.5%. mp:116.9°C -118.4°

C. ¹H NMR (400

To a solution of 10a (340mg, 1mmol) in anhydrous THF (15ml),

MHz, CDCl₃) δ 7.41 (s, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.33 (s, 1H),

7.09 (d, J = 1.6 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.31 (d, J = 9.9

Hz, 1H), 5.71 (s, 1H), 5.67 (d, J = 9.9 Hz, 1H), 3.98 (s, 3H), 3.91

(s, 3H), 1.52 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.56 (s),

150.03 (s), 148.22 (s), 146.11 (s), 145.26 (s), 131.55 (s), 131.16

(s), 130.08 (s), 123.49 (s), 122.40 (s), 121.98 (s), 120.73 (s),

116.26 (s), 113.45 (s), 109.80 (s), 77.83 (s), 56.38 (s), 56.18 (s),

NaH (60%, 60mg, 1.5mmol) and CH₃I (213mg, 1.5mmol) was

added at 0°C. After stirred for 48h at room temperature, the

reaction mixture was extracted with ethyl acetate, removed

the solvents in reduced pressure, and purified by flash column

chromatography (Petroleum ether /ethyl acetate = 5:1) to give

(3,4-dimethoxyphenyl)(8-methoxy-2,2-dimethyl-2H-chromen-

6-yl)methanone (12).

+

28.30 (s). HRMS (ESI) (m/z) [M+H] calcd for C₂₀H₂₀O₅,

(3,4-dimethoxyphenyl)(8-methoxy-2,2-dimethyl-2H-chromen-6-

yl)methanone (12)

341.1384 found, 341.1381 Purity: 98.2% (by HPLC).

(4-hydroxy-3-methoxyphenyl)(8-methoxy-2,2-dimethyl-2H-

chromen-6-yl)methanone (10b)

yellow oil, yield 81.5%.

¹H NMR (400 MHz, CDCl₃) δ 7.44 (s,

white solid, yield 32.1%. mp:168.7°C -170.5°

C. ¹H NMR (400

1H), 7.39 (d, J = 8.3 Hz, 1H), 7.32 (s, 1H), 7.10 (d, J = 1.4 Hz,

1H), 6.91 (d, J = 8.3 Hz, 1H), 6.32 (d, J = 9.9 Hz, 1H), 5.68 (d, J =

9.8 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.91 (s, 3H), 1.53 (s, 6H).

¹³C NMR (101 MHz, CDCl₃) δ 194.50 (s), 152.57 (s), 148.91 (s),

148.13 (s), 146.00 (s), 131.20 (s), 130.80 (s), 130.20 (s), 124.80

(s), 122.17 (s), 121.88 (s), 120.76 (s), 113.50 (s), 112.21 (s),

109.73 (s), 77.80 (s), 56.35 (s), 56.12 (s), 28.30 (s). HRMS (ESI)

MHz, CDCl₃) δ 7.44 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.31 (s, 1H),

7.10 (s, 1H), 6.96 (d, J = 8.2 Hz, 1H), 6.31 (d, J = 7.2 Hz, 2H),

5.68 (d, J = 9.8 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 3H), 1.53 (s, 6H).

¹³C NMR (101 MHz, CDCl₃) δ 194.56 (s), 149.81 (s), 148.14 (s),

146.69 (s), 146.00 (s), 131.21 (s), 130.43 (s), 130.23 (s), 125.59

(s), 122.18 (s), 121.91 (s), 120.78 (s), 113.57 (s), 111.99 (s),

77.81 (s), 56.36 (s), 56.20 (s), 28.30 (s). HRMS (ESI) (m/z) [M+H]

+

(m/z) [M+H] calcd for C₂₁H₂₂O₅, 355.1540 found, 355.1536

+

Purity: 99.8% (by HPLC).

calcd for C₂₀H₂₀O₅, 341.1384 found, 341.1380 Purity: 99.0%

(by HPLC).

Biological. The cell lines (A549, HeLa, A2780, HCT116, Mgc803,

BJ, HLF) were purchased from the Laboratory Animal Service

Center at Sun Yat-sen University (Guangzhou, China). We

General procedure for the preparation of 11

To a solution of ethyl-triphenylphosphonium chloride (392mg

1.2mmol) in anhydrous THF (15mL) at -78°C under a nitrogen

atmosphere, n-Butyllithium (0.8mL, 2.5M) was added in

dropwise. The resulting yellow solution was stirred for 1 h and

then warmed to room temperature stirred for 1h. After a

solution of 9a (384mg 1mmol) in dry THF was added at -78°C

and warmed to room temperature. Once completed, the

reaction mixtures were stopped with saturated NH₄Cl solution

and extracted with ethyl acetate, The organic layer was

washed with brine, dried and filtered. Removal of the solvent

performed the tubulin polymerisation assay using

a

commercial kit (cytoskeleton, cat. #B011P) which was

purchased from Cytoskeleton (Danvers, MA, USA). FITC-

conjugated mouse anti-tubulin antibody was purchased from

Cell Signaling Technology (Danvers, MA, USA). Goat anti-

mouse IgG/Alexa-Fluor 488 antibody was obtained from

Invitrogen (Camarillo, California, USA). A purified brain tubulin

polymerisa- tion kit was purchased from Cytoskeleton

(Danvers, MA, USA). Annexin-V/FITC and Cell cycle was

purchased from Keygen Biotech, China. MTT was purchased

gave

a residue that was purified by flash column

from Sigma, USA. A lipophilic cationic dye, 5,5

′,6,6′-

chromatography (Petroleum ether/ethyl acetate = 8:1) to

furnish yellow oil, which was dissolved in methanol (10mL).

HCl (12N, 1ml) was added, remove the MOM and the product

2-methoxy-5-(1-(8-methoxy-2,2-dimethyl-2H-chromen-6-

yl)vinyl)phenol (11) was getted.

tetrachloro-1,1 ′,3,3 ′-tetraethyl-benzimidazolcarbocyanine

(JC-1) was obtained from Beyotime, China.

Biological assay such as cell culture, MTT assay, in vitro tubulin

polymerisation assay can be seen Supporting Information.

2-methoxy-5-(1-(8-methoxy-2,2-dimethyl-2H-chromen-6-

yl)vinyl)phenol (11)

Acknowledgements

J. Name., 2013, 00, 1-3 | 9

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We thank the National Natural Science Foundation of China

(No. 21302235, 20972198), Guangdong Natural Science

Foundation (2014A030313124) and Ph.D. Programs

Foundation of Ministry of Education of China

(20120171120045) for financial support of this study.

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The synthesis and evaluation of new benzophenone derivatives as tubulin polymerization inhibitors

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