A fluorine scan of a tubulin polymerization inhibitor isocombretastatin A-4: Design, synthesis, molecular modelling, and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2017.11.055

A novel series of tubulin polymerization inhibitors, based on fluorinated derivatives of isocombretastatin A-4 was synthesized with the goal of evaluating the effect of these compounds on the proliferative activity. The introduction of fluorine atom was performed on the phenyl ring or at the linker between the two aromatic rings. The modification of isoCA-4 by introduction of difluoromethoxy group at the para-position (3i) and substitution of the two protons of the linker by two fluorine atoms (3m), produced the most active compounds in the series, with IC₅₀ values of 0.15–2.2 nM (3i) and 0.1–2 nM (3m) respectively, against a panel of six cancer cell lines. Compounds 3i and 3m had greater antiproliferative activity in comparison with references CA-4 or isoCA-4, the presence of fluorine group leads to a significant enhancement of the antiproliferative activity. Molecular docking studies indicated that compounds 3i and 3m occupy the colchicine binding site of tubulin. Evaluation of cytotoxicity in Human noncancer cells indicated that the compounds 3i and 3m were practically ineffective in quiescent peripheral blood lymphocytes, and may have a selective antiproliferative activity against cancer cells. Analyses of cell cycle distribution, and morphological microtubules organization showed that compound 3m induced G₂/M phase arrest and, dramatically disrupted the microtubule network.

Full text of DOI:10.1016/j.ejmech.2017.11.055

Accepted Manuscript
A fluorine scan of a tubulin polymerization inhibitor isocombretastatin A-4: Design,
synthesis, molecular modelling, and biological evaluation
Timothée Naret, Jérôme Bignon, Guillaume Bernadat, Mohamed Benchekroun,
Helene Levaique, Christine Lenoir, Joelle Dubois, Alain Pruvost, François Saller,
Delphine Borgel, Boris Manoury, Veronique Leblais, Romain Darrigrand, Sébastien
Apcher, Jean-Daniel Brion, Etienne Schmitt, Frédéric R. Leroux, Mouad Alami,
Abdallah Hamze
PII:
S0223-5234(17)30958-3
10.1016/j.ejmech.2017.11.055
EJMECH 9930
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 September 2017
Revised Date: 12 October 2017
Accepted Date: 20 November 2017
Please cite this article as: Timothé. Naret, Jéô. Bignon, G. Bernadat, M. Benchekroun, H. Levaique,
C. Lenoir, J. Dubois, A. Pruvost, Franç. Saller, D. Borgel, B. Manoury, V. Leblais, R. Darrigrand, Sé.
Apcher, J.-D. Brion, E. Schmitt, Fréé.R. Leroux, M. Alami, A. Hamze, A fluorine scan of a tubulin
polymerization inhibitor isocombretastatin A-4: Design, synthesis, molecular modelling, and biological
evaluation, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.055.
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ACCEPTED MANUSCRIPT
A Fluorine Scan of a Tubulin Polymerization Inhibitor
isoCombretastatin A-4: Design, Synthesis, Molecular Modelling,
and Biological Evaluation
Timothée Naret,a Jérôme Bignon,b Guillaume Bernadat,a Mohamed Benchekroun,a
Helene Levaique,b Christine Lenoir,b Joelle Dubois,b Alain Pruvost,c François Saller,d
Delphine Borgel,d Boris Manoury,e Veronique Leblais,e Romain Darrigrand,f
Sébastien Apcher,f Jean-Daniel Brion,a Etienne Schmitt,g Frédéric R. Leroux,g Mouad
Alami,*,a Abdallah Hamze*,a

ACCEPTED MANUSCRIPT
A Fluorine Scan of a Tubulin Polymerization Inhibitor
isoCombretastatin A-4: Design, Synthesis, Molecular Modelling, and
Biological Evaluation
Timothée Naret,a Jérôme Bignon,b Guillaume Bernadat,a Mohamed Benchekroun,a Helene
Levaique,b Christine Lenoir,b Joelle Dubois,b Alain Pruvost,c François Saller,d Delphine
Borgel,d Boris Manoury,e Veronique Leblais,e Romain Darrigrand,f Sébastien Apcher,f Jean-
Daniel Brion,a Etienne Schmitt,g Frédéric R. Leroux,g Mouad Alami,*,a Abdallah Hamze*,a
aBioCIS, Equipe Labellisée Ligue Contre le Cancer, Univ. Paris-Sud, CNRS, University Paris-Saclay, F-92290, Châtenay Malabry,
France
bInstitut de Chimie des Substances Naturelles, UPR 2301, CNRS, F-91198 Gif sur Yvette, France
cService de Pharmacologie et d’Immunoanalyse (SPI), CEA, INRA, University Paris-Saclay, F-91191 Gif sur Yvette, France.
dINSERM, UMR-S1176, University Paris-Saclay, F-94276 Le Kremlin-Bicêtre, France
eINSERM, UMR-S 1180, Faculté de Pharmacie University Paris-Saclay, F-92290, Châtenay-Malabry, France
fINSERM U1015, University Paris-Saclay, Département d’Immunologie, F-94800, Villejuif, France
gCNRS-Univ. Strasbourg (ECPM), UMR 7509, COHA, 25 Rue Becquerel, F-67087 Strasbourg, France
Corresponding authors. Tel.: +33 1 46 83 54 98. Fax: +33 1 46 83 58 28. Email: abdallah.hamze@u-psud.fr (A. Hamze)
Abstract
A novel series of tubulin polymerization inhibitors, based on fluorinated derivatives of isocombretastatin A-4 was
synthesized with the goal of evaluating the effect of these compounds on the proliferative activity. The introduction of
fluorine atom was performed on the phenyl ring or at the linker between the two aromatic rings. The modification of
isoCA-4 by introduction of difluoromethoxy group at the para-position (3i) and substitution of the two protons of the
linker by two fluorine atoms (3m), produced the most active compounds in the series, with IC50 values of 0.15–2.2 nM
(3i) and 0.1-2 nM (3m) respectively, against a panel of six cancer cell lines. Compounds 3i and 3m had greater
antiproliferative activity in comparison with references CA-4 or isoCA-4, the presence of fluorine group leads to a
significant enhancement of the antiproliferative activity. Molecular docking studies indicated that compounds 3i and 3m
occupy the colchicine binding site of tubulin. Evaluation of cytotoxicity in Human noncancer cells indicated that the
compounds 3i and 3m were practically ineffective in quiescent peripheral blood lymphocytes, and may have a selective
antiproliferative activity against cancer cells. Analyses of cell cycle distribution, and morphological microtubules
organization showed that compound 3m induced G2/M phase arrest and, dramatically disrupted the microtubule
network.
Keywords: isoCA-4, fluorinated analogs, cytotoxic, cancer, tubulin inhibitors.
1

1. Introduction
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The introduction of fluorine atoms into a molecule can completely change its physical and chemical properties, with
important consequences on the biological activities.[1] Even a single fluorine atom can completely change biological
properties of a natural product.[2] In medicinal chemistry programs, fluorine substitutions of a lead compound represent
one of the most utilized modifications during the optimization phase.[3] Since 1957, over 150 fluorinated drugs have
come to the market, this represents about 20% of all pharmaceuticals.[3c, 4] The judicious introduction of fluorine into
a molecule can productively influence interaction processes with the biological target, intrinsic potency, and
pharmacokinetic properties.[5] Usually, the effect of fluorine on the bioactivity of organic compounds is rather subtle
and difficult to predict. Therefore, intense structure−activity relationship studies are usually necessary to pinpoint the
correct position of fluorine in the target molecule.
Microtubules (MTs) are involved in numerous essential cellular functions in several eukaryotic cells, such as cell
growth, division, motility, intracellular trafficking, and the ability to adapt various shapes to interact with the
environment.[6] There is an increasing evidence showing that interfering with MT dynamic equilibrium can engage the
spindle checkpoint and the arrest of cell-cycle progression leading to cell death.[7] Combretastatin A-4 (CA-4, Fig. 1), a
natural tubulin binding agent, is a cis-stilbene isolated from the South African tree Combretum caffrum
(Combretacae)[8], which exhibits in vitro cytotoxic properties in cancer cells.[9] Structure-activity studies showed that
the cis configuration of the stilbene moiety is crucial for its activity.[10] On the other hand, the cis-olefin structure of
CA-4 is associated with thermal and light instability due to its cis-trans isomerization,[11] which compromise the
development of combretastatins as drug candidates.[12] In recent years, our group has designed and synthesized an
extensive 1,1-diarylethylene series of isocombretastatin A-4 analogs (isoCA-4, Fig. 1) in order to overcome double
bond isomerization. [13] This new scaffold allows a similar spatial arrangement between the two aromatic rings as
observed in the non-planar cis-conformation of CA-4 while preventing the undesired conversion to the inactive trans-
configuration.
Fig. 1. Chemical structures of CA-4, isoCA-4, and fluoro-1,1diarylethylenes derivatives.
We had previously described the introduction of fluorine atom on the ring B of isoCA-4 (compound 3j, Table 1)
which showed marked anti-tubulin activity and cytotoxicity.[13b] This favorable biological profile encouraged us
toward the synthesis and evaluation of fluoro-isoCA-4 analogs. To explore the influence of fluorine substitution in
isocombretastatin series, we studied and synthesized a small library of compounds in which, the methoxy group of ring
2

B was substituted by an OCF3 or OCHF2 group, the two hydrogens of the olefinic linker were replaced with fluorine. In
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addition, the same modification was done with different ring A, such as 3,4,5-trimethoxyphenyl, 2,4,6-
trimethoxyphenyl, 3,5-dimethoxyphenyl, or 3,4,5-trifluorophenyl groups.
The fluorinated compounds were then evaluated in vitro for their antiproliferative activity on colorectal carcinoma
cells (HCT116) as well as for the tubulin polymerization inhibition; some of them were further tested for their
antiproliferative activity on other tumor cell lines and their effect on cell cycle distribution.
2. Results and discussion
2.1. Chemistry
N-tosylhydrazones have emerged as a new type of versatile coupling partners for transition metal-catalyzed cross-
coupling reactions and have attracted increasing attention.[13c, 14] Consequently, we realized that this methodology
was perfectly suited for the preparation of derivatives of isoCA-4, as it results in the formation of the requisite double
bond in a one-step procedure, in comparison to classical Wittig reaction which requires at least, a three-step process for
synthesis.
As depicted in Table 1, the key intermediate N-tosylhydrazones derived from the corresponding ketones were coupled
with various aryl halides under our previously reported conditions, using PdCl2(MeCN)2/dppf[15] as the catalytic
system and LiO-t-Bu as the base in dioxane (method A) or a combination of Pd(OAc)2/SPhos and LiO-t-Bu in refluxing
CPME (method B).[16] Accordingly, the desired olefins 3a-l were obtained in good yields (45-95%). Most compounds
were prepared by using method A, however, in the case of steric compound (3c), trifluorophenyl derivative (3k) or
quinoline derivative (3l), method B was more efficient. It should be noted that this coupling also works in the case of
free aniline, and the compound 3f was obtained in a moderate yield.
Recently, we demonstrate that the reduction of the double bond of isoCA-4 leads to isoerianin derivatives with good
antiproliferative activity at nanomolar concentrations against a panel of human cancer cell lines.[17] In this context, we
found this conversion (CH2=C/CH3-C) is appropriate in this series of new molecules. Thus, the 1,1-ethane moiety
derivatives 4 were prepared efficiently by reduction of the corresponding 1,1-diarylethylenes analogs by hydrogenation
in the presence of H2 and Pd/C.
3

Table 1. Synthesis of fluoro-1,1diarylethylenes 3 by the Pd-catalyzed coupling of N-tosylhydrazones 1 with aryl
ACCEPTED MANUSCRIPT
halides, and their conversion to reduced derivatives 4.
Entry
1
ArBr
Isolated yields of 3
Isolated yields of 4
3a, 76%a
2a
4a, 95%
3b, 94%a
3c, 81%b
2
3
2a
2a
3d, 95%a
4b, 93%
4
2b
3e, 75%a
3f, 45%a
5
6
2b
2c
3g, 95%a
3h, 60%a
3i, 65%b,c
3j, 78%a
4c, 88%
4d, 51%
4e, 85%
4f, 97%
7
8
2d
2e
2f
9
MeO
MeO
F
MeO
MeO
F
10
2g
OMe
OMe
OMe
OMe
3k, 50%b,c
3l, 65%b, c
11
12
2h
2f
c
a Prepared according to the method A. b Prepared according to the method B. Overall yield obtained after two
steps, coupling and reduction of NO2 to NH2 using Fe/HCl in EtOH.
4

For the preparation of compounds 3m and 3n (Scheme 1), the benzophenone intermediates were prepared by
ACCEPTED MANUSCRIPT
condensation of (3,4,5-trimethoxyphenyl)magnesium bromide and corresponding benzaldehyde followed by oxidation
of the resulting alcohols with pyridinium chlorochromate (PCC). Then, the previously obtained benzophenones were
treated by diethyl (difluoromethyl)phosphonate in the presence of LiHMDS in THF to furnish the corresponding gem-
difluoro-1,1-diarylethylene derivatives. The nitro intermediate was reduced by iron with catalytic HCl, in a mixture of
EtOH/H2O to give the compound 3m with 50% yield. On the other hand, deprotection of silyl ether with K2CO3 in
MeOH gave the compound 3n, with 60% yield. Finally, the reduced derivative 4g was prepared from 3n by a simple
reduction in the presence of H2 and Pd/C.
Scheme 1. Synthetic procedure for the preparation of compounds 3m, 3n and 4g. a) 3,4,5-trimethoxyphenylmagnesium
bromide, THF, -78 °C; b) PCC, CH2Cl2, -20 °C; c) Diethyl (difluoromethyl)phosphonate, LiHMDS, THF, -78 °C to
reflux; d) Fe, conc. HCl, EtOH:H2O 8:2, reflux; e) K2CO3, MeOH, rt, f) H2/Pd/C, AcOEt, rt.
For the preparation of compounds 3o (scheme 2), the benzophenone intermediate was prepared by condensation of
(3,4,5-trimethoxyphenyl)magnesium bromide and 4-(difluoromethoxy)-3-nitrobenzaldehyde followed by oxidation of
the resulting alcohols with pyridinium chlorochromate (PCC). Then, the previously obtained benzophenone was treated
by diethyl (difluoromethyl)phosphonate in the presence of LiHMDS in THF to furnish the corresponding gem-difluoro-
1,1-diarylethylene derivatives. After treatment with TfOCHF2 in presence of aqueous KOH (6M) in acetonitrile, the
nitro intermediate was reduced by iron with catalytic HCl, in a mixture of EtOH/H2O to give the compound 3o.
Scheme 2. Synthetic procedure for the preparation of compounds 3o. a) 3,4,5-trimethoxyphenylmagnesium bromide,
THF, -78 °C; b) PCC, CH2Cl2, -20 °C; c) Diethyl (difluoromethyl)phosphonate, LiHMDS, THF, -78 °C to reflux; d)
TfOCHF2, KOHaq (6M), ACN, RT; e) Fe, conc. HCl, EtOH:H2O 8:2, reflux.
2.2. Biological results
In vitro antiproliferative activity
In Table 2 and 3 are summarized the in vitro antiproliferative activity of fluoro-isocombretastatines 3a-o, fluoro-
isoerianines derivatives 4a-g, and the reference compounds (isoerianin, isoCA-4, isoNH2CA-4, and CA-4[18]) against
human colon carcinoma cells (HCT116). The results presented in Table 2 indicate that inhibition of cell growth was
5

highly dependent upon the presence and the position of the methoxy substituents on the benzene ring-A (3a-c and 3d-e).
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The absence of the methoxy group in position 4 or switching from 3,4,5-trimethoxyphenyl to 2,4,6-trimethoxyphenyl
lead to a complete loss of activity. The nature and the position of the B-ring substituents markedly influenced the
biological effects. The presence in the meta-position of the ring B, a hydrogen 3a, nitro 3d, fluoride 3g or
trifluoromethoxy group 3h leads to a low cytotoxic activity. In the series of compounds bearing an OCF3 substituent
(3a-g), the most active compound was the 3-amino derivative 3f which inhibited the growth of HCT116 cells with an
IC50 of 200 nM and was 20-fold more active than the corresponding nitro derivative 3d. These results are in accordance
with our previous reports dealing with the significance of 3-NH2 group on the phenyl B-ring. Four of the fluorinated
synthesized compounds, corresponding to the 3’NH2-4’-OCHF2 (3i), 3’-F-4’OMe (3j) phenyl analogs and to difluoro-
isoCA-4 derivatives (3l and 3m) were significantly more active than the rest of the derivatives (0.9 nM ≤ IC50 ≤ 7 nM).
To study the influence of fluorine on activity, we prepared compound 3k, having the 3,4,5-trifluorophenyl motif (ring
A). In comparison to the reference compound, this analog was 50 fold less active than isoNH2CA-4. This observation
confirms again the importance of the methoxy group on ring A. Recently, we showed that trimethoxyphenyl ring can be
replaced by quinoline nucleus without loss of activity.[19] Accordingly, we combined this heterocycle (A) with the best
activity obtained with B-ring containing 3’NH2-4’-OCHF2 groups (3l). A moderate activity was observed with this
compound (3l) to HCT116 cells (IC50 = 20 nM). Finally, to explore further the structure-activity relationship, we studied
the reduction of the double bond of the spacer located between the ring A and B (Table 3).
Fluorinated-isoerianin derivatives (4a-g, Table 3) obtained by the reduction of the double bond of fluro-isoCA-4
derivatives were significantly less active than their olefin congeners. It is worth mentioning that the reduction of the
double bond of compound 3i led to compound 4e that displayed higher cytotoxic activity in comparison to the reference
compound isoerianin (IC50 = 18 nM vs 28 nM).
6

Table 2. Cytotoxic activity of fluoro-isoCA-4 derivatives against HCT116 cells.a
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Linker: Ar(Ar’)C=CH2 or Ar(Ar’)C=CF2
IC50 HCT 116
IC50 HCT 116
(nM)b
Compounds
Compounds
(nM)b
5000 ± 400
7 ± 1
3a
3j
F
NH2
>10000
100 ± 1
3b
3c
3k
3l
F
OMe
F
20.2 ± 4.19
>10000
4000 ± 300
>10000
3d
3e
3m
3n
0.9 ± 0.05
2 ± 1
200 ± 10
9.14 ± 0.034
3f
3o
2000 ± 80
1500 ± 100
1 ± 0.05
3g
3h
3i
isoCA4
isoNH2CA4
CA4
2 ± 0.2
2 ± 0.1
2.3 ± 0.1
aHCT116 human colon carcinoma cells. bCompound concentration required to decrease cell growth by 50%; values
represent the average ± sd of three experiments. cThe IC50 values for CA-4, isoCA-4, and isoNH2CA-4 were determined
in this study.
7

Table 3. Cytotoxic activity of fluoro-isoCA-4 derivatives against HCT116 cells.a
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Linker: Ar(Ar’)CH−CH3
IC50 HCT 116
IC50 HCT 116
(nM)b
Compounds
Compounds
(nM)b
>10000
4a
4e
18 ± 2
3000 ± 150
5000 ± 300
4b
4c
4f
85 ± 6
4g
30 ± 2
28 ± 2c
>10000
4d
Isoerianin
aHCT116 human colon carcinoma cells. bCompound concentration required to decrease cell growth by 50%; values
represent the average ± sd of three experiments. cThe IC50 value isoerianin were determined in this study.
These preliminary in vitro cytotoxic results prompted us to evaluate the most promising molecules in these series
against other human cancer cell lines and for inhibitory effect on tubulin polymerization (Table 4). Fluorinated
compounds 3i, 3j, 3m and 3n were assayed as growth inhibitors of a panel of cancer cell lines, including A549 (lung
carcinoma), K562 (chronic myelogenous leukaemia), doxorubicin-resistant K562R, U87-MG (human primary
glioblastoma), and MCF7 (breast cancer), using isoCA-4, isoNH2CA-4 and CA-4 as reference compounds (Table 4).
Three of the synthesized compounds (3i, 3m, and 3n) were significantly more active than the rest of the derivatives with
IC50 values of 0.15-4 nM, 0.1-2 nM and 0.2-2.2 nM, respectively, in the six cell lines, as compared with 2-20 nM, 2-9
nM and 2.3-180 nM for the reference compounds isoCA-4, isoNH2CA-4, and CA-4. Their antiproliferative activities
were more pronounced against A549 cells in comparison to reference compounds isoCA-4 and natural CA-4.
Compound 3m displayed a subnanomolar activity against four cancer cells line including HCT116, K562, K562R, and
U87-MG. Compound 3m was ~90 fold more active than the reference natural CA-4 against A549 cells line, and it
exhibited the greatest antiproliferative activity among the tested compounds, especially against doxorubicin-resistant
K562R cell line (IC50 = 0.1 nM).
8

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Table 4. Cytotoxic activity of selected fluoro-isoCA-4 derivatives against human tumor cell lines, and tubulin
polymerization inhibition.a
IC50
IC50 (nM)a
(µM)b
TPI
Compounds
HCT116
1 ± 0.05
A549
K562
K562R
U87
MCF7
0.15 ±
0.01
2.2 ± 0.2
4 ± 0.05
0.6 ± 0.01
2 ± 0.05
0.4 ± 0.06
3i
3j
7 ± 1
8 ± 0.3
6 ± 0.4
2 ± 0.1
8 ± 0.5
1 ± 0.05
1.5 ± 0.2
4 ± 0.5
0.1 ±
0.001
3m
0.9 ± 0.05
2 ± 0.05
0.4 ± 0.05
0.2 ± 0.01
2.0 ± 0.1
3n
2 ± 1
2 ± 0.1
2.2 ± 0.3
5 ± 0.3
0.2 ± 0.05
2.2 ± 0.8
0.3 ± 0.01
8 ± 0.3
0.6 ± 0.05
8 ± 0.4
2.0 ± 0.3
2.0 ± 0.3
isoCA-4
2 ± 0.2
20 ± 0.1
isoNH2CA-4
2 ± 0.1
7 ± 0.6
5 ± 0.2
6 ± 0.2
2.0 ± 0.4
23 ± 0,06
9 ± 0.5
2.9 ± 0.8
8 ± 0.5
2.2 ± 0.2
2.1 ± 0.3
CA-4
2.3 ± 0.1
180 ± 30
3 ± 0.25
aIC50 = Compound concentration required to inhibit tumor cell proliferation by 50%; Data are expressed as the mean ±
sd from the dose-response curves of at least three independent experiments. b TPI: tubulin polymerization inhibition,
IC50 is the concentration of compound required to inhibit 50% of the rate of microtubule assembly (average of three
experiments).
Tubulin polymerization inhibition
To investigate whether the antiproliferative activities of compounds 3i, 3j, 3m and 3n, derived from an interaction
with tubulin, these compounds were evaluated for their tubulin polymerization inhibition (TPI, Table 4). For
comparison, isoCA-4, isoNH2CA-4, and CA-4 were used as references. Compounds 3m and 3n inhibited tubulin
assembly and presented significant activity with IC50 in the low micromolar concentrations, comparable with the
reference compounds (CA-4 and isoCA-4, Table 4), these derivatives may act as a highly potent cytotoxic agent
through tubulin polymerization inhibition. Compound 3i strongly inhibited tubulin assembly, with submicromolar
activity (IC50 = 0.42 µM), in comparison to the reference compounds, which was 5 fold more potent than CA-4 as an
inhibitor of tubulin assembly. This latter compound, as described above, was among the more effective as an inhibitor
of cell proliferation. In comparison to compound 3f, the substitution of the 4-OCF3 group at phenyl group by a CHF2
group (compound 3i) considerably enhanced biological activity. The acidity of the hydrogen of the difluoromethyl
group (CF2H) allows stabilizing interactions with electron-withdrawing groups such as a carbonyl,[1a] and the better
9

activity of compound 3i can be explained by the possibility of the formation of a hydrogen bond (See infra, modelling
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section).
Molecular modelling
Compounds 3i, 3m, and 3n displayed the best antiproliferative activity and effective tubulin polymerization
inhibition, we then selected them as the optimized compounds for the following studies. To investigate the potential
binding site of these compounds with the tubulin-microtubule system, molecular modeling studies were achieved. We
performed docking experiments with its structure against the colchicine binding site of tubulin (Fig. 2).[20]
The search for hydrogen bonds in the structure of the complex identified the classic interaction between Cysβ241 and
the trimethoxyphenyl motif for this kind of compounds, as well as the previously described hydrogen bond between a
polar hydrogen of the amino group and backbone carbonyl oxygen of Thrα179.[21] We also wondered whether the
CHF2 group could play a role in the strong protein-ligand interaction that could be measured, and found the
corresponding hydrogen atom was located at a distance of 2.44 Å from the sulfur atom of Metβ259. This finding led us
to hypothesize, that a hydrogen bond could be established between the Metβ259 of tubulin and compound 3i, on
condition that, the C−H bond is polarized enough, a similar phenomenon with CHF2 groups have already been
described.[22] In comparison with isoCA-4 or isoNH2CA-4, which establish only two hydrogen bonds with the tubulin
(Cysβ241 and Val181) the increase in the tubulin polymerization inhibition of 3i might be due to the increase of
hydrogen bond with Thrα179 and Metβ259 residues. In order to confirm and characterize the hydrogen-bond donating
capability of the CF2H group in derivative 3i, we performed DFT pKa calculations. A structure in which the CF2H
group is replaced by a proper methyl group was used as reference. Results showed a 5.6 unit decrease when calculations
were conducted using a water solvation model, and 9.8 unit decrease with a DMSO solvation model (for results, see
experimental section; Molecular modelling part).
10

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Fig. 2. (a), (b), (c) Calculated binding mode for compounds 3i, 3m and 3n in the colchicine binding site of tubulin. The
solvent-accessible surface is colored according to its local polarity (maroon: hydrophobic, cyan: polar). (d) Overlay of
poses from (a), (b) and (c) superimposed with the reference binding mode of isoCA-4 drawn in magenta colour.
Evaluation of cytotoxicity in human non-cancer cells
To obtain a preliminary indication of the cytotoxic potential of these derivatives for normal human cells, the
cytotoxicity of the most active compounds (3i and 3m) was evaluated in proliferating peripheral blood lymphocytes
(PBLs) isolated from two different healthy donors, and compared to that of the reference compound (CA-4). In our
hands, dose-response curves yielded an IC50 value of ~ 0.1-0.5 µM for CA-4, as described previously [23], and the IC50
values were estimated to be at least one order higher for 3i and 3m (Table 5). Indeed, the compounds could only be
tested at a maximal final concentration of 25 µM, which corresponds to a final concentration of DMSO of 0.25%, and
therefore the compounds could not be investigated at higher concentrations. Nevertheless, these results indicated that
our compounds were less cytotoxic than the reference compound (CA-4), with a difference of at least one order of
magnitude.
11

CA-4 was found to be less cytotoxic in quiescent (IC5 > 10 µM) than in proliferating PBLs. Dose-response curves for
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0
3i and 3m were indistinguishable from that of CA-4, and yielded IC50 values > 10 µM for 3i and 3m (Table 5). These
results thus suggested that the compounds 3i and 3m were not more cytotoxic than CA-4 in quiescent PBLs.
Table 5. Cytotoxicity of compounds 3i, 3m and CA-4 for human PBL.a
Donor
CA-4 (IC50)b
3i (IC50)b
Proliferating PBLs
3m (IC50)b
1
2
0.5 µM
0.1 µM
> 10 µM
> 10 µM
> 10 µM
> 10 µM
Quiescent PBLs
1
2
> 10 µM
> 10 µM
> 10 µM
> 10 µM
> 10 µM
> 10 µM
a PBL : Proliferating peripheral blood lymphocytes. b Compound concentration
required to reduce cell growth by 50%
Effect of 3i and 3m on the contractile tone of isolated rat aorta rings
CA-4 related compounds were reported to enhance vasoconstriction of isolated arteries[24] that may cause
hypertensive side effects.[25] In order to assess the possible impact of the novel compounds on vascular tone, we
studied the vascular reactivity of isolated rat aorta rings in the presence of 3i or 3m and compared them to vehicle, CA-
4 or isoNH2CA-4 (10 µM) (Table 6). Addition of CA-4 or isoNH2CA-4 on resting rat aorta rings had no significant
effect compared to vehicle (DMSO 0.1%). Compounds 3i or 3m induced a slight (≈ 2%) contraction that was
significantly higher than control, but which was not statistically different from the effect obtained with CA-4 or
isoNH2CA-4. Besides, although the test compounds induced a slight leftward shift of concentration-contractile response
relationships (CCRR) for PGF2α, a classic vasoconstrictor agonist (see Supporting Information, Fig. A), the related
pharmacological parameters, namely pD2 and Emax, were not significantly altered (Table 6). Overall, novel compounds
3i and 3m slightly increased resting tone of isolated rat aorta rings, without impacting the overall contractile response to
a physiological vasoconstrictor.
Table 6. Evaluation of the effect of compounds 3i, 3m, isoNH2CA-4 and CA-4 on the contractile tone of isolated
rat aorta rings.
Compd.
3i
Effect on basal tone (%)
2.2 ± 0.3**
2.1 ± 0.5*
pD2
Emax (%)
5.87 ± 0.06
5.94 ± 0.08
5.79 ± 0.12
5.73 ± 0.09
5.65 ± 0.06
115.5 ± 2.9
124.8 ± 4.4
117.3 ± 3.4
120.8 ± 3.5
115.4 ± 5.1
3m
1.6 ± 0.4
isoNH2CA-4
CA-4
0.7 ± 0.2
0.2 ± 0.2
Vehicle
The amplitude of the contractile effect induced by the compounds (Compd.) on resting aorta rings is
expressed as percentage of the contractile response obtained with high K+ solution (K60, See
methods). Potency (pD2) and efficacy (Emax, expressed as % of response to K60) of the
vasoconstrictor agonist PGF2α obtained in the presence of different test compounds or vehicle
(DMSO, 0.1%) are also presented. All compounds were used at 10 µM. Data are mean ± SEM. N =
6. *: P < 0.05 and **: P < 0.01 vs Vehicle.
Metabolic stability of 3i, 3m and 3n
Assessing plasma stability early is crucial as compounds that are rapidly degraded in plasma may not have sufficient
concentration to produce activity at the intended target(s). For it, the stability of compounds 3i, 3m, 3n, and isoNH2CA-
4 was studied in human plasma (Table 7). No degradation was observed for 3i, 3m, and 3n when were tested at 5µM for
12

2 h at 37°C in plasma. This result, suggests that these compounds are not sensitive to enzymatic hydrolysis. Procaine,
ACCEPTED MANUSCRIPT
used as a positive control to ensure the integrity of the experiment, was degraded in human plasma at rates consistent
with our previous values. As microsomal enzymes are different than plasma enzymes, the stability of these compounds
was investigated in human microsomes. Human microsomes were incubated for 45 minutes with 5 µM of each
compound with or without NADPH, and the drug was quantified by LC-MS/MS. Verapamil was used as a reference
compound. Accordingly, compound 3m and isoNH2CA-4 seems to be more stable than compound 3i and 3n. The
introduction of fluorine atom on the linker leads to an enhancement of the antiproliferative activity without a significant
modification of metabolic stability.
Table 7. Human plasma and microsomal stabilities and intrinsic clearance values for compounds 3i, 3m, 3n, and
isoNH2CA-4.
Compd.
Human plasma
In vitro intrinsic
Human microsomal
Metabolism not
stability % remaining
clearance Clint
stability % remaining at NADPH dependent
at 120 min
(µl/min/mg protein)
120 min
3.3
(%)
9
100
100
100
94
73
43
64
46
-
3i
13.6
5.0
<5
20
6
3m
3n
11.6
-
isoNH2CA-4
Procaine
Verapamil
52
-
-
181
3.9
5.6
Effect of 3m on the cell cycle
Compound 3m displayed the best antiproliferative activity and metabolic stability, and we then selected it as the
optimized compound for the following studies. To explore whether the cytotoxicity of 3m was due to the cell arrest, we
used a flow cytometer to examine the effect of this compound on cell cycle progression. As shown in Figure 3, after 24h
of treatment, 3m (20 nM) had a dramatic effect on the cell cycle progression, inducing cell cycle arrest at the G2/M
phase in both HCT116 and U87-MG cells.
13

Fig. 3. Effects of compound 3m (blue) on cell cycle distribution in a) HCT116 and b) U87-MG cells at 20 nM, as
ACCEPTED MANUSCRIPT
determined by flow cytometry analysis (DMSO control in red). DNA content was assessed via propidium iodide
staining.
Immunofluoresence studies
As tubulin-microtubule system plays a vital role in the maintenance of cell shape and basic cellular functions, we next
investigated the capacity of the most potent compound, 3m, to alter the microtubule network in U87-MG cells by using
cell-based immunofluorescence staining assays. As shown in Figure 4, in the control group (DMSO), the microtubule
network in the U87-MG cells exhibited normal arrangement and organization (slim and fibrous), and the microtubules
are wrapped around the uncondensed cell nucleus. In contrast, after exposure to 5 nM of compound 3m for 24h, the
staining of tubulin was more diffuse and the microtubule network was disorganized (Fig. 4). These changes were
observed more clearly by increasing the concentration to 10 nM, in this case, the microtubules spindles significantly
shrunk, accompanied by both disorganization of actin network and multinucleation phenomena. These results confirmed
that 3m dramatically disrupted the microtubule and actin in U87-MG cells at lower concentrations.
14

ACCEPTED MANUSCRIPT
Fig. 4. Effect of 3m at the concentration of 5 nM and 10 nM on microtubule network organization. U87-MG cells were
treated with 3m (5 nM and 10 nM) for 24 h. Control was realized by treatment with 0.1% of DMSO. After incubation,
the cells were fixed and stained with monoclonal β-tubulin antibody (red), cell nuclei were stained with DAPI (blue)
and Alexa Fluor® 488 Phalloidin was used to visualize actin cytoskeleton (green).
3. Conclusions
To understand the structure-activity relationship within isocombretastatin series, and to develop more efficacious
antiproliferative derivatives than the natural product (CA-4), we synthesized and evaluated a panel of fluorinated
isocombretastatin A-4 analogs. The position of the fluorine atom on the structure plays a crucial role in the
antiproliferative activity and the tubulin polymerization inhibition. Among all synthesized compounds, 2-
(difluoromethoxy)-5-(1-(3,4,5-trimethoxyphenyl)vinyl)aniline
(3i),
and
5-(2,2-difluoro-1-(3,4,5-
trimethoxyphenyl)vinyl)-2-methoxyaniline (3m) potently inhibit the growth of human colon carcinoma HCT116, lung
carcinoma A549, chronic myelogenous leukaemia K562, doxorubicin-resistant K562R, brain human glioblastoma U87-
MG, and breast cancer MCF7 cells with IC50 values in the nanomolar to subnanomolar range. The most active
15

compound (3m) leads cancer cells to accumulate in the G2/M phase of the cell cycle, in addition, it strongly affected
ACCEPTED MANUSCRIPT
cell shape and microtubule network at low concentration. In comparison to CA-4, compounds 3i and 3m showed very
low cytotoxicity in proliferating lymphocytes obtained from healthy volunteers. When used at 10 µM, 3i and 3m
induced a slight contraction of resting rat aorta rings but did not significantly alter the overall contractile response to
PGF2α, a classic vasoconstrictor agonist. Finally, we propose a binding mode for compounds 3i, 3m, and 3n based on
molecular dynamics simulations that is consistent with the observed structure−activity relationships for this class of
compounds.
4. Experimental
4.1. Chemistry
4.1.1. General considerations
1
The compounds were all identified by usual physical methods, i.e. H-NMR, 13C-NMR, IR, MS. Melting points (mp)
were recorded on a Büchi B-450 apparatus and were uncorrected. NMR spectra were performed on a Bruker AMX 200
(1H, 200 MHz; 13C, 50 MHz; 19F, 88 MHz), Bruker AVANCE 300 or Bruker AVANCE 400 (1H, 300 MHz or 400
1
MHz; 13C, 75 MHz or 100 MHz). Solvent peaks were used as reference values, with CDCl3 at 7.26 ppm for H NMR
and 77.16 ppm for 13C NMR, with (CD3)2SO at 2.50 ppm for 1H NMR and 39.52 ppm for 13C NMR. Chemical shifts δ
are given in ppm, and the following abbreviations are used: singlet (s), doublet (d), doublet of doublet (dd), triplet (t),
quadruplet (q) and multiplet (m). Infrared spectra (IR) were measured on a Bruker Vector 22 spectrophotometer and
were recorded in film (film, cm-1). High resolution mass spectra (HRMS) were recorded on a MicrotofQ Bruker
Daltonics. The purity of the final compounds was determined to be ≥95% by means of analytical high pressure liquid
chromatography - mass spectroscopy (HPLC-MS) on a Waters Alliance 2695 (HPLC) and LCT Premier, ESI-TOF
(MS) system with an XBridge C18 column (2.1 × 150 mm, 3.5 µm) eluted at 0.25 mL/min with Milli-Q water + 0.1 %
formic acid/CH3CN, gradient 95/5 to 0/100 in 20 min. Reaction courses and product mixtures were routinely monitored
by TLC on silica gel (precoated F254 Merck plates), and compounds were visualized under a UVP Mineralight UVGL-
58 lamp (254 nm) and with phosphomolybdic acid/∆, or vanillin/∆.
4.1.2. General procedure for the preparation of N-tosylhydrazone derivatives[26]
To a rapidly stirred suspension of p-toluenesulphonohydrazide (5 mmol) in dry methanol (10 mL) at 60 °C, the ketone
(5 mmol) was added dropwise. Within 5-60 min the N-tosylhydrazone began to precipitate. The mixture was cooled to 0
°C and the product was collected on a Büchner funnel, washed with petroleum ether then was dried in vacuo to afford
the pure product.
4-Methyl-N'-(1-(3,4,5-trimethoxyphenyl)ethylidene)benzenesulfonohydrazide (1a). Compound 1a was prepared
according to the general method. (1.89 g, 79% yield). White solid, mp: 167-168 °C. TLC: Rf = 0.49
(Cyclohexane/AcOEt, 7/3, SiO2). IR (solid): 3356, 3192, 2981, 1577, 1509, 1449, 1408, 1350, 1324, 1230, 1163, 1124,
1055, 1003 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 7.92 (d, J = 8.2 Hz, 2H), 7.74 (s, 1H), 7.31 (d, J = 8.1 Hz, 2H),
6.86 (s, 2H), 3.86 (s, 6H), 3.85 (s, 3H), 2.41 (s, 3H), 2.14 (s, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 152.9 (2C),
152.5 (C), 144.2 (C), 139.6 (C), 135.4 (C), 132.8 (C), 129.5 (2CH), 128.2 (2CH), 103.8 (2CH), 60.9 (CH3), 56.1
(2CH3), 21.6 (CH3), 13.6 (CH3). HRMS (ESI) (M + H)+ m/z calcd for C18H23N2O5S 379.1322 found 379.1325.
4-Methyl-N'-(1-(3,5-dimethoxyphenyl)ethylidene)benzenesulfonohydrazide (1b). Compound 1b was prepared according
to the general method. (1.74 g, 76% yield). White solid, mp: 149-150 °C. TLC: Rf = 0.22 (Cyclohexane/AcOEt, 7/3,
SiO2). IR (solid): 3155, 1585, 1548, 1421, 1363, 1325, 1306, 1198, 1150, 1059, 1035 cm-1. 1H NMR (300 MHz, CDCl3)
16

δ (ppm) 7.92 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 6.78 (dd, J = 2.2, 1.1 Hz, 2H), 6.45 (t, J = 2.1 Hz, 1H), 3.79
ACCEPTED MANUSCRIPT
(s, 6H), 2.41 (s, 3H), 2.11 (s, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 160.6 (2C), 152.3 (C), 144.2 (C), 139.2 (C),
135.4 (C), 129.5 (2CH), 128.2 (2CH), 104.6 (2CH), 101.6 (CH), 55.4 (2CH3), 21.6 (CH3), 13.5 (CH3). HRMS (ESI) (M
+ H)+ m/z calcd for C17H21N2O4S 349.1217 found 349.1215.
4-Methyl-N'-(1-(2,4,6-trimethoxyphenyl)ethylidene)benzenesulfonohydrazide (1c). Compound 1c was prepared
according to the general method. (1.68 g, 89% yield). White solid, mp: 197-199 °C. TLC: Rf = 0.26
(Cyclohexane/AcOEt, 7/3, SiO2). IR (solid): 3190, 1606, 1583, 1496, 1456, 1415, 1382, 1337, 1186, 1156, 1126, 1049
cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 7.82 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 6.11 (s, 2H), 3.84 (s, 3H),
3.63 (s, 6H), 2.46 (s, 3H), 2.10 (s, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 163.1 (C), 157.4 (2C), 151.4 (C), 143.5
(C), 136.4 (C), 129.4 (2CH), 128.0 (2CH), 103.2 (C), 91.0 (2CH3), 55.8 (2CH3), 55.7 (CH3), 23.9 (CH3), 21.7 (CH3).
HRMS (ESI) (M + Na)+ m/z calcd for C18H22N2NaO5S 401.1142 found 401.1137.
4-Methyl-N'-(1-(3,4,5-trifluorophenyl)ethylidene)benzenesulfonohydrazide (1d). Compound 1d was prepared according
to the general method. (1.26 g, 74% yield). White solid, mp: 201-202 °C. TLC: Rf = 0.57 (Cyclohexane/AcOEt, 7/3,
SiO2). IR (solid): 3193, 1597, 1532, 1438, 1401, 1367, 1290, 1259, 1224, 1188, 1121, 1071, 1038, 1018 cm-1. 1H NMR
(300 MHz, DMSO-d6) δ (ppm) 10.78 (s, 1H), 7.49 (dd, J = 9.3, 7.0 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 2.36 (s, 3H), 2.15
(s, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm) 150.2 (ddd, J = 245.6, 9.8, 3.3 Hz, 2CF), 149.8 (d, J = 1.4 Hz, C),
143.6 (C), 139.4 (dt, J = 249.8, 15.0 Hz, CF), 136.0 (C), 134.1 (d, J = 4.0 Hz, C), 129.6 (2CH), 127.6 (2CH), 110.5 (d, J
= 21.6 Hz, 2CH), 21.0 (CH3), 14.0 (CH3). 19F NMR (188 MHz, DMSO-d6) δ (ppm) -132.8 (d, J = 21.6 Hz), -158.3 (t, J
= 21.6 Hz). HRMS (ESI) (M + H)+ m/z calcd for C15H14F3N2O2S 343.0728 found 343.0729.
4.1.3. Aryl bromides derivatives
Aryl bromides 2a-e were purchased from commercial sources and used as such. Aryl bromide 2f was prepared from 4-
bromo-2-nitrophenol and difluoromethyl triflate.
Preparation of difluoromethyl triflate
Triflic acid (1.2 eq., 8.45 g, 5 mL, 56.3 mmol) was introduced into a Schlenk’s vessel using a gas-tight syringe and was
then carefully treated with TiCl4 (1.17 %, 0.104 g, 0.06 mL, 0.547 mmol) under an inert atmosphere and vigorous
stirring. After 5min, the homogeneous solution was evacuated at 13mbar over 10min until gas evolution ceased. The
mixture was cooled to -20 °C and Ruppert's reagent (6.65 g, 7 mL, 46.8 mmol) was very carefully added via syringe,
the mixture was stirred for 5min at -20 °C, and then very slowly rose to room temperature by placing a water bath. The
mixture was stirred 1h more. Volatile materials were distilled off in vacuo (130mbar) and trapped in N2 trap.
The volatile material was recovered from the N2 trap after slow temperature rise under argon, to afford 9.82g of
colourless liquid difluoromethyl trifluoromethanesulfonate (6.97 g, 34.8 mmol, 74 %). 1H NMR (CDCl3, 400 MHz): δ =
6.64 (t, JH-F = 60 Hz, 1H, -CHF2) ppm.
Preparation of 4-bromo-2-nitropheno.[27]
A mixture of 4-bromophenol (1 eq., 5 g, 28.9 mmol), NaNO2 (1 eq., 2 g, 29 mmol), citric acid (1 eq., 5.6 g, 28.9 mmol)
and SiO2 50%, w/w (5.8 g, 96.5 mmol) in hexane (150 mL) was vigorously stirred into a 500mL round-bottom flask
over 1h at room temperature. The solution was filtered, dried with Na2SO4 and filtered again. Hexane was removed by
evaporation. The crude product was purified by silica gel chromatography (cyclohexane/ethyl acetate 98:2) and dried
17

1
3
under vacuum to give a bright yellow solid (3.59 g, 16.47 mmol, 57%). H NMR (CDCl3, 400MHz): δ = 7.11 (d, J =
ACCEPTED MANUSCRIPT
9.2 Hz, 1H), 7.69 (d, 3J = 8.8 Hz, 1H), 8.28 (s, 1H, -OH), 10.52 (s, 1H) ppm.
Preparation of 4-bromo-1-(difluoromethoxy)-2-nitrobenzene (2f). A solution of 4-bromo-2-nitrophenol (1 eq., 2.42 g,
11.1 mmol) and KOH 6M aq. (12.1 eq., 6 M, 22.3 mL, 133 mmol) in MeCN (24 mL) was prepared under inert
atmosphere in
a Schlenk vessel. A water-bath was placed to control exothermicity. Difluoromethyl
trifluoromethanesulfonate (3 eq., 9.8 g, 33.3 mmol) was very carefully added, the mixture was stirred 30 min at room
temperature. The mixture was quenched with water (24 mL) and extracted by diethyl ether (3 x 50 mL). The organic
layers were washed with brine, dried with Na2SO4, filtered and evaporated in vacuo. The crude product was purified by
1
silica gel chromatography (pentane/ether 100:0 to 98:2) to give light brown oil (1.98 g, 7.39 mmol, 66%). H NMR
(CDCl3, 400 MHz): δ = 6.53 (t, 2J = 72.4 Hz, -CHF2), 7.21 (d, 3J = 8.8 Hz, 1H), 7.66 (d, 3J = 6.4 Hz, 1H), 7.97 (s, 1H)
ppm. 19F NMR (CDCl3, 376Mz): δ = - 82.3 (d, 2J = 72 Hz, CHF2) ppm. 13C NMR (CDCl3, 100 MHz): δ = 115.5 (t, 1J
= 264 Hz, -OCHF2), 119.04 (s, C-Br), 125.2 (s), 128.6 (s), 137.2 (s, C(3)), 142.1 (s, C-O), 143.3 (s, C-NO2) ppm. Anal.
calcd for C7H4BrF2NO3: C, 31.37; H, 1.50; Br, 29.81; F, 14.18; N, 5.23; O, 17.91. Found: C, 30.90; H, 1.62; N, 5.06.
4.1.4. General procedure for coupling between N-Tosylhydrazones and arylbromide (method A)
To
a
solution of N-tosylhydrazone (0.6 mmol, 1.1 eq) in distilled dioxane (8 mL) was added
bis(acetonitrile)palladium(II) chloride (0.05 mmol, 5 mol %), and dppp (0.1 mmol, 10 mol %). The resulting suspension
was stirred à RT for 2 min, and cesium carbonate (1.5 mmol, 3.0 eq) was added. Then, the reaction mixture was stirred
for additional 2 min, and aryl bromide (0.5 mmol, 1.0 eq) was added in one portion. The mixture was then heated at
100°C for 3h. The crude reaction mixture was allowed to cool to rt. EtOAc was added, and the mixture was filtrated
through Celite®. The solvent was evaporated under reduced pressure, and the crude product was purified by flash
chromatography on silica gel.
4.1.5. General procedure for coupling between N-Tosylhydrazones and arylbromide (method B)
In this method, the same protocol was used, as mentioned for method A, but Pd(OAc)2 and Sphos were used instead of
PdCl2(MeCN)2 and dppf.
1,2,3-Trimethoxy-5-(1-(4-(trifluoromethoxy)phenyl)vinyl)benzene (3a). Compound 3a was prepared according to
method A. Flash chromatography on silica gel (Cyclohexane/AcOEt, 10/0 to 7/3) afforded 135 mg of 3a (0.38 mmol,
yield 76%). Brown oil. TLC: Rf = 0.29 (Cyclohexane/AcOEt, 9/1, SiO2). 1H NMR (300 MHz, CDCl3) δ (ppm) 7.39 (d,
J = 8.5 Hz, 2H), 7.18 (d, J = 8.7 Hz, 2H), 6.52 (s, 2H), 5.44 (d, J = 3.5 Hz, 2H), 3.88 (s, 3H), 3.82 (s, 6H). 13C NMR (75
MHz, CDCl3) δ (ppm) 153.1 (2C), 149.0 (C), 140.0 (C), 138.1 (C), 136.7 (C), 129.7 (2CH), 120.7 (2CH), 120.6 (q, J =
255.0 Hz, CF3), 114.8 (CH2), 105.7 (2CH), 61.0 (CH3), 56.3 (2CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -55.9 (s).
HRMS (ESI) (M + H)+ m/z calcd for C18H18F3O4 355.1157 found 355.1150.
1,3-Dimethoxy-5-(1-(4-(trifluoromethoxy)phenyl)vinyl)benzene (3b). Compound 3b was prepared according to method
A. Flash chromatography on silica gel (Cyclohexane/Et2O, 10/0 to 7/3) afforded 189 mg of 3b (0.47 mmol, yield 94%).
Yellow oil. TLC: Rf = 0.55 (Cyclohexane/AcOEt, 9/1, SiO2). IR (film): 2939, 1590, 1506, 1456, 1423, 1349, 1253,
1204, 1153, 1050, 1018 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 7.38 (dt, J = 15.0, 3.0 Hz, 2H), 7.17 (dd, J = 8.8, 0.9
Hz, 2H), 6.46 (s, 3H), 5.47 (dd, J = 7.5, 1.0 Hz, 2H), 3.78 (s, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 160.8 (2C),
148.9 (C), 143.3 (C), 140.1 (C), 129.7 (2CH), 120.7 (2CH), 120.6 (q, J = 255.8 Hz, C), 115.2 (CH2), 106.8 (2CH),
18

100.0 (CH), 55.5 (2CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -58.2 (s). HRMS (ESI) (M + H)+ m/z calcd for
ACCEPTED MANUSCRIPT
C17H16F3O3 325.1046 found 325.1048.
1,3,5-Trimethoxy-2-(1-(4-(trifluoromethoxy)phenyl)vinyl)benzene (3c).[28] Compound 3c was prepared according to
method B. Flash chromatography on silica gel (Cyclohexane/AcOEt, 95/5) afforded 143 mg of 3c (0.405 mmol, yield
81%). Colorless oil. TLC: Rf = 0.61 (Cyclohexane/AcOEt, 8/2, SiO2). IR (film): 2840, 1605, 1584, 1507, 1468, 1455,
1
1414, 1337, 1254, 1224, 1203, 1160, 1125, 1052, 1038, 1016 cm-1. H NMR (300 MHz, CDCl3) δ (ppm) 7.35 (d, J =
8.9 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 6.22 (s, 2H), 5.92 (d, J = 1.2 Hz, 1H), 5.25 (d, J = 1.2 Hz, 1H), 3.86 (s, 3H), 3.71
(s, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 161.0 (C), 158.8 (2C), 148.5 (C), 140.1 (C), 139.9 (C), 127.3 (2CH), 120.6
(2CH), 120.5 (q, J = 257 Hz, CF3), 117.3 (CH2), 91.1 (2CH), 56.1 (2CH3), 55.5 (CH3). 19F NMR (188 MHz, CDCl3) δ
(ppm) -58.2 (s). HRMS (ESI) (M + H)+ m/z calcd for C18H18F3O4 355.1152 found 355.1146.
1,2,3-Trimethoxy-5-(1-(3-nitro-4-(trifluoromethoxy)phenyl)vinyl)benzene (3d). Compound 3d was prepared according
to method A. Flash chromatography on silica gel (Cyclohexane/AcOEt, 10/0 to 7/3) afforded 190 mg of 3d (0.475
mmol, yield 95%). White solid, mp: 126-128 °C. TLC: Rf = 0.18 (Cyclohexane/AcOEt, 9/1, SiO2). IR (film): 2941,
1
1998, 1580, 1540, 1505, 1464, 1412, 1344, 1209, 1127, 1007 cm-1. H NMR (300 MHz, CDCl3) δ (ppm) 7.97 (s, 1H),
7.62 (dd, J = 8.6, 1.9 Hz, 1H), 7.41 (dd, J = 8.6, 1.3 Hz, 1H), 6.48 (s, 2H), 5.56 (d, J = 8.2 Hz, 2H), 3.89 (s, 3H), 3.83
(s, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.3 (2C), 147.0 (C), 142.5 (C), 141.3 (C), 140.5 (C), 138.5 (C), 135.2
(C), 133.4 (CH), 125.1 (CH), 122.1 (CH), 120.9 (q, J = 258 Hz, CF3), 116.9 (CH2), 105.5 (2CH), 60.9 (CH3), 56.2
(2CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -57.9 (s). HRMS (ESI) (M + H)+ m/z calcd for C18H17F3NO6 400.1002
found 400.1008.
4-(1-(3,5-dimethoxyphenyl)vinyl)-2-nitro-1-(trifluoromethoxy)benzene (3e). Compound 3e was prepared according to
method A. Flash chromatography on silica gel (Cyclohexane/AcOEt, 95/5) afforded 138 mg of 3e (0.375 mmol, yield
75%). Yellow oil. TLC: Rf = 0.33 (Cyclohexane/AcOEt, 9/1, SiO2). IR (film): 1591, 1542, 1506, 1456, 1424, 1349,
1
1258, 1205, 1157, 1065 cm-1. H NMR (300 MHz, CDCl3) δ (ppm) 7.95 (d, J = 2.2 Hz, 1H), 7.60 (dd, J = 8.6, 2.2 Hz,
1H), 7.39 (d, J = 8.6 Hz, 1H), 6.48 (t, J = 2.2 Hz, 1H), 6.41 (d, J = 2.2 Hz, 2H), 5.58 (d, J = 16.7 Hz, 2H), 3.79 (s, 6H).
13C NMR (75 MHz, CDCl3) δ (ppm) 160.9, 160.4 (2), 146.9, 133.4, 125.1 (2), 122.9, 120.9 (q, J = 258 Hz), 117.3, 13
C
NMR (75 MHz, CDCl3) δ 161.1 (2C), 147.0 (C), 142.5 (C), 141.8 (C), 141.4 (C), 140.4 (C), 133.6 (CH), 125.3 (CH),
123.0 (CH), 120.9 (q, J = 258 Hz, CF3), 117.5 (CH2), 106.7 (2CH), 100.4 (CH), 55.6 (2CH3). 19F NMR (188 MHz,
CDCl3) δ (ppm) -57.9 (s). HRMS (ESI) (M + H)+ m/z calcd for C17H15F3NO5 370.0897 found 370.0896.
2-(Trifluoromethoxy)-5-(1-(3,4,5-trimethoxyphenyl)vinyl)aniline (3f). Compound 3f was prepared according to method
A. Flash chromatography on silica gel (Cyclohexane/AcOEt, 10/0 to 6/4) afforded 83 mg of 3f (0.225 mmol, yield
1
45%). Yellow oil. TLC: Rf = 0.09 (Cyclohexane/AcOEt, 9/1, SiO2. H NMR (300 MHz, CDCl3) δ (ppm) 7.08 (d, J =
9.1 Hz, 1H), 6.77 (d, J = 1.8 Hz, 1H), 6.71 (dd, J = 8.4, 1.6 Hz, 1H), 6.53 (s, 2H), 5.38 (s, 2H), 3.86 (s, 3H), 3.80 (s,
6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.0 (2C), 149.3 (C), 140.8 (C), 138.9 (C), 137.9 (C), 137.0 (C), 136.0 (C),
121.2 (CH), 120.9 (q, J = 258 Hz, CF3), 118.4 (CH), 116.7 (CH), 114.3 (CH2), 105.7 (2CH), 60.9 (CH3), 56.2 (2CH3).
19F NMR (188 MHz, CDCl3) δ (ppm) -55.9 (s). HRMS (ESI) (M + H)+ m/z calcd for C18H19F3NO4 370.1261 found
370.1258.
5-(1-(3-Fluoro-4-methoxyphenyl)vinyl)-1,2,3-trimethoxybenzene (3g). Compound 3g was prepared according to method
A. Flash chromatography on silica gel (Cyclohexane/AcOEt, 100/0 to 97/3) afforded 177 mg of 3g (0.475 mmol, yield
95%). Yellow oil. TLC: Rf = 0.65 (Cyclohexane/AcOEt, 7/3, SiO2). IR (film): 1938, 1651, 1613, 1579, 1516, 1503,
1
1465, 1452, 1411, 1333, 1283, 1270, 1233, 1172, 1124, 1026, 1004 cm-1. H NMR (300 MHz, CDCl3) δ (ppm) 7.33 –
7.13 (m, 3H), 6.52 (s, 2H), 5.49 (d, J = 2.7 Hz, 2H), 3.90 (s, 3H), 3.84 (s, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm)
19

154.3 (d, J = 247.5 Hz, CF), 153.2 (3C), 148.2 (C), 142.1 (d, J = 6.5 Hz, C), 138.3 (C), 136.2 (C), 124.3 (d, J = 3.2 Hz,
ACCEPTED MANUSCRIPT
CH), 123.4 (CH), 120.6 (q, J = 257 Hz, CF3), 117.0 (d, J = 19.2 Hz, CH), 115.7 (CH2), 105.7 (2CH), 61.0 (CH3), 56.3
(2CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -59.1 (s), -179.27 (s). HRMS (ESI) (M + H)+ m/z calcd for C18H17F4O4
373.1063 found 373.1069.
1,2,3-Trimethoxy-5-(1-(4-methoxy-3-(trifluoromethoxy)phenyl)vinyl)benzene (3h). Compound 3h was prepared
according to method A. Flash chromatography on silica gel (Cyclohexane/AcOEt, 100/0 to 96/4) afforded 115 mg of 3h
1
(0.30 mmol, yield 60%). Brown oil. TLC: Rf = 0.33 (Cyclohexane/AcOEt, 9/1, SiO2). H NMR (300 MHz, CDCl3) δ
(ppm) 7.31 – 7.24 (m, 2H), 6.96 (d, J = 8.4 Hz, 1H), 6.54 (s, 2H), 5.39 (d, J = 1.7 Hz, 2H), 3.90 (s, 3H), 3.88 (s, 3H),
3.82 (s, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 152.93 (d, J = 29.6 Hz, C), 151.89 (2C), 148.5 (C), 138.2 (C), 137.7
(C), 136.7 (C), 134.2 (C), 127.8 (CH), 123.0 (CH), 120.8 (q, J = 255 Hz, CF3), 113.7 (CH2), 112.62 (CH), 105.8 (2CH),
61.0 (CH3), 56.3 (3CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -58.6 (s). HRMS (ESI) (M + H)+ m/z calcd for
C19H20F3O5 385.1263 found 385.1262.
2-(Difluoromethoxy)-5-(1-(3,4,5-trimethoxyphenyl)vinyl)aniline (3i). Nitro intermediate (2-(difluoromethoxy)-5-(1-
(3,4,5-trimethoxyphenyl)vinyl)aniline) was synthesized according to method B. Flash chromatography on silica gel
(Cyclohexane/AcOEt, 8/2) afforded 144 mg of nitro intermediate (0.38 mmol, yield 76%). Yellow solid, mp: 95-97 °C.
TLC: Rf = 0.34 (Cyclohexane/AcOEt, 7/3, SiO2). IR (film): 1758, 1622, 1580, 1507, 1465, 1432, 1412, 1347, 1307,
1256, 1236, 1202, 1126, 1037, 1004 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 7.92 (d, J = 2.1 Hz, 1H), 7.58 (dd, J =
8.6, 2.1 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 6.64 (t, J = 56 Hz, 1H), 6.48 (s, 2H), 5.53 (d, J = 11.2 Hz, 2H), 3.89 (s, 3H),
3.83 (s, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.3 (2C), 147.2 (C), 142.5 (d, J = 22.3 Hz, C), 140.1 (C), 138.5
(C), 135.5 (C), 133.5 (CH), 125.0 (CH), 123.2 (CH), 116.5 (CH2), 115.7 (t, J = 264 Hz, CHF2), 105.6 (2CH), 61.0
(CH3), 56.3 (2CH3).19F NMR (188 MHz, CDCl3) δ (ppm) -80.2 (s). HRMS (ESI) (M + H)+ m/z calcd for
C18H17F2NO6Na 404.0922 found 404.0915. After the cross-coupling reaction, amine derivative 3i was obtained by
reduction of nitro group according to the following procedure: Iron powder (212 mg, 3.8 mmol, 10 eq) and concentrated
hydrochloric acid (ca., 1 drop) were added to a solution of nitroaryle (0.38 mmol, 1.0 eq) in EtOH (4.0 mL) and water
(1.0 mL), and the mixture was heated to reflux for 90 min. EtOAc (10 mL) was added to the mixture, and it was dried
with MgSO4. After filtration and evaporation of the solvent, the residue was purified by flash chromatography on silica
gel (Cyclohexane/AcOEt, 8/2) to afforded 114 mg of 3i (0.32 mmol, yield 85%, yield 65% over 2 steps). Yellow oil.
TLC: Rf = 0.41 (Cyclohexane/AcOEt, 7/3, SiO2). IR (film): 3378, 2939, 1757, 1581, 1537, 1504, 1466, 1451, 1412,
1344, 1267, 1237, 1184, 1126, 1099, 1052, 1004 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 7.26 (s, 2H, NH2), 6.97 (d,
J = 8.3 Hz, 1H), 6.76 (d, J = 1.6 Hz, 1H), 6.70 (dd, J = 8.4, 1.7 Hz, 1H), 6.53 (s, 2H), 6.49 (t, J = 56.0 Hz, 1H), 5.36 (d,
J = 4.1 Hz, 2H), 3.86 (s, 3H), 3.81 (s, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.0 (2C), 149.5 (C), 139.7 (C), 138.4
(C), 138.3 (C), 138.0 (C), 137.2 (C), 119.7 (CH), 118.6 (CH), 116.7 (t, J = 193 Hz, CHF2), 116.5 (CH), 114.0 (CH2),
105.8 (2CH), 61.0 (CH3), 56.3 (2CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -77.8 (s). HRMS (ESI) (M + H)+ m/z
calcd for C18H20F2NO4 352.1360 found 352.1354. HPLC: C18 tr: 17.67 min.
5-(1-(3-Fluoro-4-methoxyphenyl)vinyl)-1,2,3-trimethoxybenzene (3j). Compound 3j was prepared according to method
A. Flash chromatography on silica gel (Cyclohexane/AcOEt, 10/0 to 6/4) afforded 124 mg of 3j (0.39 mmol, yield
78%). Yellow oil. TLC: Rf = 0.61 (Cyclohexane/AcOEt, 7/3, SiO2). IR (film): 2938, 1613, 1579, 1516, 1465, 1411,
1333, 1238, 1233, 1172, 1124, 1026, 1004 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 7.12 – 7.05 (m, 2H), 6.90 (t, J =
8.7 Hz, 1H), 6.52 (s, 2H), 5.35 (d, J = 9.8 Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 3.80 (s, 6H). 13C NMR (75 MHz, CDCl3)
δ (ppm) 153.0 (3C), 152.0 (d, J = 243.8 Hz, CF), 148.7 (C), 147.4 (d, J = 10.8 Hz, C), 138.0 (C), 137.0 (C), 134.4 (d, J
= 6.2 Hz, C), 124.1 (d, J = 2.9 Hz, CH), 115.9 (d, J = 18.8 Hz, CH), 113.5 (CH2), 112.9 (C), 105.7 (2CH), 61.0 (CH3),
20

56.3 (CH3), 56.2 (2CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -133.8 (s). HRMS (ESI) (M + H)+ m/z calcd for
ACCEPTED MANUSCRIPT
C18H20FO4 319.1346 found 319.1337. HPLC: C18 tr: 18.90 min.
2-Methoxy-5-(1-(3,4,5-trifluorophenyl)vinyl)aniline (3k). Nitro intermediate (1,2,3-trifluoro-5-(1-(4-methoxy-3-
nitrophenyl)vinyl)benzene) was synthesized according to general procedure B. Flash chromatography on silica gel
(Cyclohexane/AcOEt, 10/0 to 7/3) afforded 87 mg nitro intermediate (0.28 mmol, yield 56%). Yellow oil. TLC: Rf =
0.50 (Cyclohexane/AcOEt, 7/3, SiO2). IR (film): 1619, 1500, 1434, 1359, 1282, 1185, 1128, 1087, 1042, 1016 cm-1 1
H
NMR (300 MHz, CDCl3) δ (ppm) 7.80 (d, J = 2.2 Hz, 1H), 7.45 (dd, J = 8.7, 2.2 Hz, 1H), 7.08 (d, J = 8.7 Hz, 1H), 6.93
(dd, J = 8.1, 6.8 Hz, 2H), 5.49 (d, J = 7.9 Hz, 2H), 3.99 (s, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.0 (C), 151.3
(ddd, J = 249.4, 10.1, 4.5 Hz, 2CF), 145.3 (C), 139.8 (dt, J = 252.0, 15.4 Hz, CF), 139.7 (C), 136.6 (d, J = 4.8 Hz, CH),
133.7 (CH), 132.6 (C), 125.3 (CH), 116.9 (CH2), 113.6 (CH), 112.4 (d, J = 21.8 Hz, 2CH), 56.8 (CH3). 19F NMR (188
MHz, CDCl3) δ (ppm) -132.0 (d, J = 20.5 Hz), -158.6 (t, J = 20.5 Hz). HRMS (ESI) (M + H)+ m/z calcd for
C15H11F3NO3 310.0686 found 310.0691. After the cross-coupling reaction, amine derivative 3k was obtained by
reduction of nitro group according to the procedure described for 3i. Flash chromatography on silica gel
(Cyclohexane/AcOEt, 8/2 to 6/4) afforded 125 mg of 3k (0.45 mmol, yield 90%, yield 50% over 2 steps). Brown oil.
TLC: Rf = 0.80 (Cyclohexane/AcOEt, 5/5, SiO2). IR (film): 3389, 2838, 1620, 1527, 1441, 1361, 1289, 1255, 1218,
1182, 1153, 1042 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 7.03 – 6.91 (m, 2H), 6.76 (d, J = 8.8 Hz, 1H), 6.64 (m,
2H), 5.35 (d, J = 25.1 Hz, 2H), 3.88 (s, 3H), 3.83 (s, 2H). 13C NMR (75 MHz, CDCl3) δ (ppm) 150.95 (ddd, J = 249.2,
6.0, 3.8 Hz, 2CF), 147.6 (C), 139.4 (dt, J = 247.5, 15.4 Hz, CF), 138.1 (d, J = 3.8 Hz, C), 136.1 (C), 133.0 (C), 118.7
(CH), 114.8 (CH), 114.3 (CH2), 112.4 (dd, J = 14.8, 6.3 Hz, 2CH), 110.2 (CH), 55.7 (CH3). 19F NMR (188 MHz,
CDCl3) δ (ppm) -133.3 (d, J = 20.6 Hz), -160.1 (t, J = 20.6 Hz). HRMS (ESI) (M + H)+ m/z calcd for C15H13F3NO
280.0949 found 280.0943. HPLC: C18 tr: 18.57 min.
2-(Difluoromethoxy)-5-(1-(2-methylquinolin-4-yl)vinyl)aniline (3l). ). Compound 3l was prepared according to method
B. After the cross-coupling reaction, amine derivative 3l was obtained by reduction of nitro group according to the
procedure described for 3i. Flash chromatography on silica gel (Cyclohexane/AcOEt, 7/3) afforded 106 mg of 3j (0.325
mmol, yield 65% over 2 steps). Yellow oil. TLC: Rf = 0.42 (Cyclohexane/AcOEt, 7/3, SiO2). IR (film): 3473, 3385,
3324, 3189, 2924, 2853, 1624, 1592, 1562, 1512, 1437, 1412, 1379, 1353, 1307, 1247, 1218, 1198, 1116, 1045 1033
cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 8.05 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.69 – 7.60 (m, 1H), 7.37
(t, J = 7.0 Hz, 1H), 7.21 (s, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.71 (d, J = 1.8 Hz, 1H), 6.66 (d, J = 2.2 Hz, 1H), 6.47 (t, J =
75.0 Hz, 1H), 5.94 (s, 1H), 5.38 (s, 1H), 3.85 (s, 2H), 2.77 (s, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 158.9 (C),
148.4 (C), 148.3 (C), 145.6 (C), 138.7 (C), 138.4 (C), 138.3 (C), 129.5 (CH), 129.0 (CH), 126.0 (CH), 125.8 (CH),
125.4 (C), 122.6 (CH), 121.1 (t, J = 185.3 Hz, CHF2), 120.1 (CH), 117.1 (=CH2), 116.9 (CH), 114.8 (CH), 25.4 (CH3).
19F NMR (376 MHz, CDCl3) δ (ppm) -79.8 (s). HRMS (ESI) (M + H)+ m/z calcd for C19H17F2N2O 327.1309 found
327.1311. HPLC: C18 tr: 10.80 min.
5-(2,2-difluoro-1-(3,4,5-trimethoxyphenyl)vinyl)-2-methoxyaniline (3m).
Synthesis of nitro intermediate: 5-(2,2-difluoro-1-(4-methoxy-3-nitrophenyl)vinyl)-1,2,3-trimethoxybenzene. A solution
of diethyl (difluoromethyl)phosphonate (1.56 g, 8.33 mmol, 5.0 eq) in THF (13 mL) was cooled to -78°C, and then a
1.0 M solution of LiHMDS (8.33 mL, 8.33 mmol, 5 eq) was added dropwise over the course of 10 min. The mixture
was stirred at -78 °C under nitrogen for 30 min. Then a solution of (4-methoxy-3-nitrophenyl)-(3,4,5-
trimethoxyphenyl)methanone (579 mg, 1.67 mmol, 1.0 eq) in THF (13 mL) was added. After 15 min, the reaction flask
was removed from the cooling bath and was heated to reflux for 5 h, and was then cooled to RT. The reaction was
quenched by the addition of H2O (15 mL), and the solvent was removed under vacuum. The aqueous phase was
21

extracted with AcOEt (3 x 12 mL) and the organic phase was washed with brine (25 mL), dried over MgSO4 and
ACCEPTED MANUSCRIPT
concentrated to give a residue which was purified by silica gel chromatography (Cyclohexane/AcOEt, 8/2 to 5/5) to
give 17% of nitro intermediate (108.1 mg, 0.28 mmol) as a yellow oil. TLC: Rf = 0.57 (Cyclohexane/AcOEt, 5/5, SiO2).
IR (film): 2941, 1707, 1621, 1584, 1534, 1507, 1466, 1414, 1349, 1305, 1265, 1236, 1186, 1128, 1091, 1024 cm-1. 1H
NMR (300 MHz, CDCl3) δ (ppm) 7.83 – 7.77 (m, 1H), 7.50 – 7.41 (m, 1H), 7.09 (d, J = 8.8 Hz, 1H), 6.45 (s, 2H), 4.00
(s, 3H), 3.90 (s, 3H), 3.83 (s, 6H). 13C NMR (100 MHz, CDCl3) δ (ppm) 153.9 (t, J = 219.0 Hz, =CF2), 153.5 (2C),
152.2 (C), 139.7 (C), 138.1 (C), 134.9 (t, J = 3.1 Hz, CH), 128.5 (t, J = 3.1 Hz, C), 126.7 (t, J = 3.6 Hz, C), 126.3 (t, J =
3.4 Hz, CH), 113.6 (CH), 107.1 (2CH), 94.8 (t, J = 19.1 Hz, C=CF2), 60.9 (CH3), 56.7 (CH3), 56.3 (2CH3). 19F NMR
(188 MHz, CDCl3) δ (ppm) -83.7 (d, J = 30.7 Hz), -85.4 (d, J = 30.7 Hz). HRMS (ESI) (M + H)+ m/z calcd for
C18H18F2NO6 382.1102 found 382.1104. After the cross-coupling reaction, amine derivative 3m was obtained by
reduction of nitro group according to the procedure described for 3i. Flash chromatography on silica gel
(Cyclohexane/AcOEt, 7/3 to 5/5) afforded 87 mg of 3m (0.25 mmol, yield 50%). Yellow oil. TLC: Rf = 0.74
(Cyclohexane/AcOEt, 5/5, SiO2). IR (film): 3371, 2943, 1714, 1620, 1584, 1512, 1466, 1414, 1348, 1263, 1212, 1178,
1158, 1128, 1059, 1033 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 6.75 (d, J = 8.0 Hz, 1H), 6.68 – 6.60 (m, 2H), 6.47
(s, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.79 (s, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.5 (t, J = 290.3 Hz, CF2), 153.1
(2C), 146.9 (C), 137.4 (C), 135.9 (C), 130.2 (C), 126.7 (C), 120.0 (CH), 116.1 (CH), 110.2 (CH), 107.1 (2CH), 96.2 (t,
J = 18.3 Hz, C=CF2), 61.0 (CH3), 56.2 (2 CH3), 55.6 (CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -86.3 (d, J = 35.9
Hz), -86.7 (d, J = 35.9 Hz). HRMS (ESI) (M + H)+ m/z calcd for C18H20F2NO4 352.1360found 352.1372. HPLC: C18 tr:
15.84 min.
5-(2,2-Difluoro-1-(3,4,5-trimethoxyphenyl)vinyl)-2-methoxyphenol (3n). A solution of difluoromethylphosphonate (1.0
g, 5.32 mmol, 2.0 eq) in THF (20 mL) was cooled to -78°C, and then a 1.06 M solution of LiHMDS in THF (5.02 mL,
5.32 mmol, 2.0 eq) was added dropwise over the course of 5 min. The mixture was stirred at -78 °C under nitrogen for
30 min. Then a solution of (3-((tert-butyldimethylsilyl)oxy)-4-methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone
(1.52 g, 2.66 mmol, 1.0 eq) in THF (20 mL) was added. After 15 min, the reaction flask was removed from the cooling
bath and was heated to reflux for 1 h, and was then cooled to RT. The reaction was quenched by the addition of H2O
(20 mL), and the solvent was removed under vacuum. The aqueous phase was extracted with Et2O (3 x 10 mL) and the
organic phase was washed with brine (15 mL), dried over MgSO4 and concentrated to give a residue which was
dissolved in MeOH (20 mL), treated with K2CO3 (0.734 g, 5.32 mmol, 2.0 eq) and stirring was continued for 2 h. Water
(20 mL) was added and the aqueous phase was extracted with Et2O (3 x 20 mL). The organic phase was washed with
brine (20 mL), dried over MgSO4 and concentrated to give a residue which was purified by silica gel chromatography to
give 560 mg of 3n (1.59 mmol, yield 60%). White solid, mp: 118-119 °C. TLC: Rf 0.30 (Cyclohexane/EtOAc: 7/3). IR
(neat): 3435, 2939, 2840, 1703, 1582, 1509, 1466, 1413, 1352, 1305, 1259, 1175, 1124, 1101, 1059, 1029, 1004 cm-1.
1H NMR (300 MHz, CDCl3) δ (ppm) 6.86 (s, 1H), 6.81 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 8.5 Hz, 1H), 6.47 (s, 2H), 5.79
(s, 1H), 3.86 (s, 6H), 3.78 (s, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.6 (t, J = 290.5 Hz, CF2), 153.09 (2C), 146.1
(C), 145.5 (C), 137.6 (C), 129.9 (C), 127.2 (C), 121.4 (CH), 115.8 (CH), 110.5 (CH), 107.1 (2CH), 96.0 (t, J = 18.5 Hz,
C=CF2), 60.9 (CH3), 56.2 (2CH3), 55.9 (CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -85.80 (d, J = 34.8 Hz), -86.57 (d,
J = 34.8 Hz). HRMS (ESI) (M + H)+ m/z calcd for C18H19F2O5 353.1201 found 353.1204. HPLC: C18 tr: 16.70 min.
5-(2,2-Difluoro-1-(3,4,5-trimethoxyphenyl)vinyl)-2-(difluoromethoxy)aniline (3o).
Synthesis of benzophenone intermediate: (4-(Difluoromethoxy)-3-nitrophenyl)(3,4,5-trimethoxyphenyl)methanone. To a
solution of 4-(difluoromethoxy)-3-nitrobenzaldehyde (746 mg, 3.4 mmol, 1.0 eq) in THF (20 mL) was added slowly by
22

cannule at -10 °C a solution of (3,4,5-trimethoxyphenyl)magnesium bromide lithium chloride (15 mL, 0.35M, 5.25
ACCEPTED MANUSCRIPT
mmol, 1.54 eq) and stirred for 15 min at -10 °C then 1 h at RT. The reaction was then hydrolyzed at 0 °C with saturated
aqueous solution of NH4Cl (50 mL) and extracted with AcOEt (3x40 mL). The combined organic layers were dried
over MgSO4, filtered and concentrated. The crude alcohol was redissolved in DCM (30 mL) and pyridinium
chlorochromate (2.1 g; 9.7 mmol, 2.8 eq) was added in 3 portions over 2 h. The solution was stirred overnight at RT,
then filtered over silica and concentrated under vacum. The residue was purified by flash chromatography
(Cyclohexane/AcOEt, 8/2 to 5/5) to furnish the corresponding benzophenone (950 mg, 72%). White solid, mp: 125-126
°C. TLC: Rf = 0.76 (Cyclohexane/AcOEt: 5/5). IR (neat): 2924, 2853, 1721, 1659, 1613, 1582, 1539, 1503, 1466,
15454, 1414, 1358, 1332, 1260, 1187, 1126, 1095, 1042 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 8.37 (d, J = 1.8 Hz,
1H), 8.08 (dd, J = 8.6, 1.9 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.04 (s, 2H), 6.74 (t, J = 72.0 Hz, 1H), 3.98 (s, 3H), 3.91
(s, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 192.0 (C=O), 153.4 (2C), 145.7 (C), 143.2 (C), 142.2 (C), 135.7 (C),
135.1 (CH), 131.0 (C), 127.6 (CH), 122.6 (CH), 115.4 (t, J = 264.8 Hz, CHF2), 107.9 (2CH), 61.2 (CH3), 56.6 (2CH3).
19F NMR (376 MHz, CDCl3) δ (ppm) -82.3 (s). HRMS (ESI) (M + H)+ m/z calcd for C17H16 F2NO7 384.0895 found
384.0884.
Synthesis of free-hydroxy difluoroethyelene intermediate: 4-(2,2-Difluoro-1-(3,4,5-trimethoxyphenyl)vinyl)-2-
nitrophenol. A solution of diethyl (difluoromethyl)phosphonate (2.33 g, 12.39 mmol, 5.0 eq) in THF (30 mL) was
cooled to -78°C, and then a 1.0 M solution of LiHMDS (12.39 mL, 12.39 mmol, 5.0 eq) was added dropwise over the
course of 10 min. The mixture was stirred at -78 °C under nitrogen for 30 min. Then a solution of (4-(difluoromethoxy)-
3-nitrophenyl)(3,4,5-trimethoxyphenyl)methanone (950 mg, 2.48 mmol, 1.0 eq) in THF (30 mL) was added. After 15
min, the reaction flask was removed from the cooling bath and was heated to reflux for 5 h, and was then cooled to RT.
The reaction was quenched by the addition of H2O (40 mL), and the solvent was removed under vacuum. The aqueous
phase was extracted with AcOEt (3 x 30 mL) and the organic phase was washed with brine (30 mL), dried over MgSO4
and concentrated to give a residue which was purified by silica gel chromatography (Cyclohexane/AcOEt, 8/2 to 5/5) to
give 39% of free-hydroxy nitro intermediate (355 mg, 0.968 mmol) as a yellow oil. TLC: Rf =
0.51(Cyclohexane/AcOEt, 5/5, SiO2). IR (film): 3250, 2940, 2820, 1704, 1630, 1583, 1538, 1509, 1490, 1466, 1452,
1414, 1354, 1320, 1280, 1236, 1184, 1164, 1126, 1081, 1060, 1002 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 10.61 (s,
1H), 8.08 (d, J = 1.4 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 6.45 (s, 2H), 3.89 (s, 3H), 3.82 (s, 6H).
13C NMR (75 MHz, CDCl3) δ (ppm) 154.5 (C), 154.0 (t, J = 292.5 Hz, =CF2), 153.5 (2C), 138.5 (CH), 138.2 (C), 133.6
(C), 128.6 (C), 126.8 (C), 125.6 (CH), 120.3 (CH), 107.1 (2CH), 94.8 (t, J = 17.3 Hz, C=CF2), 61.0 (CH3), 56.3 (2CH3).
19F NMR (188 MHz, CDCl3) δ (ppm) -86.3 (d, J = 35.9 Hz), -86.7 (d, J = 35.9 Hz). HRMS (ESI) (M - H)- m/z calcd for
C17H14 F2NO6 366.0789 found 366.0791.
Synthesis of nitro intermediate: 5-(1-(4-(Difluoromethoxy)-3-nitrophenyl)-2,2-difluorovinyl)-1,2,3-trimethoxybenzene.
A solution of 4-(2,2-Difluoro-1-(3,4,5-trimethoxyphenyl)vinyl)-2-nitrophenol (355 mg, 0.968 mmol, 1.0 eq) and KOH
6M aq. (2 mL, 6 M, 11.71 mmol, 12.1 eq.) in MeCN (3 mL) was prepared under inert atmosphere in a Schlenk vessel.
A water-bath was placed to control exothermicity. Difluoromethyl trifluoromethanesulfonate (581 mg, 2.90 mmol, 3.0
eq.) was very carefully added, the mixture was stirred 30 min at room temperature. The mixture was quenched with
water (25 mL) and extracted by diethyl ether (3 x 20 mL). The organic layers were washed with brine, dried with
MgSO4, filtered and evaporated in vacuo. The crude product was purified by silica gel chromatography
(Cyclohexane/AcOEt, 7/3 to 5/5) to give 90% of OCHF2 nitro intermediate (364 mg, 0.872 mmol) as a yellow oil. TLC:
Rf = 0.64(Cyclohexane/AcOEt, 5/5, SiO2). IR (film): 2943, 1705, 1585;1539, 1508, 1467, 1415, 1350, 1302, 1256,
1
1238, 1187, 1165, 1127, 1100, 1059, 1003 cm-1. H NMR (300 MHz, CDCl3) δ (ppm) 7.85 (d, J = 2.0 Hz, 1H), 7.52
23

(dd, J = 8.6, 2.1 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 6.65 (t, J = 72.7 Hz, 1H), 6.45 (s, 2H), 3.88 (s, 3H), 3.82 (s, 6H). 13C
ACCEPTED MANUSCRIPT
NMR (75 MHz, CDCl3) δ (ppm) 154.18 (t, J = 294.8 Hz, =CF2), 153.6 (2C), 142.65 (C), 142.00 (C), 138.30 (C), 134.6
(CH), 133.1 (t, J = 4.0 Hz, C), 127.7 (CH), 126.1 (CH), 123.2 (CH), 115.6 (t, J = 264.0 Hz, CHF2), 107.1 (2CH), 94.7
(dd, J = 21.5, 16.8 Hz, C=CF2), 60.9 (CH3), 56.3 (2CH3). 19F NMR (376 MHz, CDCl3) δ (ppm) -82.2 (s), -83.4 (d, J =
26.8 Hz), -85.9 (d, J = 26.8 Hz). HRMS (ESI) (M + H)+ m/z calcd for C18H16 F4NO6 418.0908 found 418.0905. To
prepare the final compound 3o, amine group was reduced according to the procedure described for 3i. Flash
chromatography on silica gel (Cyclohexane/AcOEt, 6/4) to afforded 57 mg of 3o (0.148 mmol, yield 17%). Yellow oil.
TLC: Rf = 0.69 (Cyclohexane/AcOEt, 5/5, SiO2). IR (film): 2943, 1705, 1585, 1539, 1508, 1467, 143, 1415, 1350,
1
1302, 1256, 1238, 1187, 1165, 1127, 1100, 1059, 1003 cm-1. H NMR (300 MHz, CDCl3) δ 7.02 (d, J = 8.3 Hz, 1H),
6.70 (s, 1H), 6.66 (d, J = 8.4 Hz, 1H), 6.51 (t, J = 74.2 Hz, 1H), 6.47 (s, 2H), 3.89 (s, 3H), 3.82 (s, 6H). 19F NMR (376
MHz, CDCl3) δ (ppm) -79.8 (s), -86.6 (d, J = 32.7 Hz), -87.4 (d, J = 32.7 Hz). 13C NMR (75 MHz, CDCl3) δ (ppm)
153.7 (t, J = 291.0 Hz, =CF2), 153.3 (2C), 138.60 (C), 137.86 (C), 137.72 (C), 132.38 (C), 129.53 (C), 119. 9 (CH),
119.8 (CH), 117.5 (CH), 116.6 (t, J = 258.8 Hz, CHF2), 107.1 (2CH), 95.9 (t, J = 18.8 Hz, C=CF2), 61.0 (CH3), 56.3
(2CH3). HRMS (ESI) (M + H)+ m/z calcd for C18H18F4NO4 388.1172 found 388.1178. HPLC: C18 tr: 18.05 min.
4.1.6. General procedure for reduction of diarylethylene bond.
To a round-bottom flask provided with a two-way cock, diarylethylene (0.5 mmol) was dissolved in AcOEt (10 mL).
Under argon, Pd/C (10 mol %) was added and the flask was purged with hydrogen three times. Then, the black mixture
was vigorously stirred under hydrogen pressure at 25°C overnight. The flask was then purged with argon three times,
and the mixture was filtered through Celite®. The solvent was evaporated under reduced pressure and the crude residue
was purified by flash chromatography on silica gel.
1,2,3-Trimethoxy-5-(1-(4-(trifluoromethoxy)phenyl)ethyl)benzene (4a). Flash chromatography on silica gel
(Cyclohexane/AcOEt, 10/0 to 7/3) afforded 169 mg of 4a (0.475 mmol, yield 95%). Colorless oil. TLC: Rf = 0.33
1
(Cyclohexane/AcOEt, 9/1, SiO2). IR (film): 2936, 1589, 1506, 1460, 1419, 1331, 1257, 1159, 1127, 1010 cm-1. H
NMR (300 MHz, CDCl3) δ (ppm) 7.23 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 6.40 (s, 2H), 4.09 (q, J = 7.2 Hz,
1H), 3.82 (s, 3H), 3.82 (s, 6H), 1.61 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.3 (2C), 147.6 (C),
145.0 (C), 141.5 (C), 136.6 (C), 128.9 (2CH), 121.0 (2CH), 120.6 (q, J = 255.0 Hz, CF3), 104.8 (2CH), 60.8 (CH3), 56.2
(2CH3), 44.5 (CH), 22.2 (CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -58.3 (s). HRMS (ESI) (M + H)+ m/z calcd for
C18H20F3O4 357.1308 found 357.1311.
2-(Trifluoromethoxy)-5-(1-(3,4,5-trimethoxyphenyl)ethyl)aniline (4b). Flash chromatography on silica gel
(Cyclohexane/AcOEt, 10/0 to 6/4) afforded 173 mg of 4b (0.465 mmol, yield 93%). Yellow oil. TLC: Rf = 0.12
(Cyclohexane/AcOEt, 9/1, SiO2). IR (film): 2936, 1590, 1508, 1461, 1419, 1250, 1167, 1127, 1008 cm-1. 1H NMR (300
MHz, CDCl3) δ (ppm) 7.07 (d, J = 8.5 Hz, 1H), 6.72 (s, 1H), 6.67 (d, J = 8.8 Hz, 1H), 6.41 (s, 2H), 3.99 (q, J = 7.1 Hz,
1H), 3.82 (s, 9H), 1.58 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.3 (2C), 146.1 (C), 141.5 (C), 137.5
(C), 135.4 (C), 121.5 (CH), 120.9 (q, J = 258 Hz, CF3), 118.8 (CH), 116.9 (CH), 104.8 (2CH), 61.0 (CH3), 56.3 (2CH3),
44.6 (CH), 22.0 (CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -58.2 (s). HRMS (ESI) (M + H)+ m/z calcd for
C18H21F3NO4 372.1417 found 372.1416.
5-(1-(3-Fluoro-4-(trifluoromethoxy)phenyl)ethyl)-1,2,3-trimethoxybenzene (4c). Flash chromatography on silica gel
(Cyclohexane/AcOEt, 100/0 to 97/3) afforded 165 mg of 4c (0.44 mmol, yield 88%). Yellow oil. TLC: Rf = 0.69
1
(Cyclohexane/AcOEt, 7/3, SiO2). H NMR (300 MHz, CDCl3) δ (ppm) 7.23 (td, J = 8.3, 1.1 Hz, 1H), 7.10 – 7.00 (m,
2H), 6.41 (s, 2H), 4.09 (q, J = 7.2 Hz, 1H), 3.85 (s, 3H), 3.85 (s, 6H), 1.63 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz,
24

CDCl3) δ (ppm) 154.5 (d, J = 250.5 Hz, CF), 153.4 (2C), 147.5 (d, J = 5.7 Hz, C), 140.7 (C), 136.9 (C), 134.6 (d, J =
ACCEPTED MANUSCRIPT
13.2 Hz, C), 123.6 (CH), 123.5 (CH), 120.6 (q, J = 257.3 Hz, CF3), 116.3 (d, J = 18.7 Hz, CH), 104.8 (2CH), 61.0
(CH3), 56.3 (2CH3), 44.6 (CH), 21.9 (CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -59.3 (s), -129.0 (s). HRMS (ESI) (M
+ H)+ m/z calcd for C18H19F4O4 375.1214 found 375.1216.
1,2,3-Trimethoxy-5-(1-(4-methoxy-3-(trifluoromethoxy)phenyl)ethyl)benzene (4d). Flash chromatography on silica gel
(Cyclohexane/AcOEt, 100/0 to 96/4) afforded 98 mg of 4d (0.255 mmol, yield 51%). Yellow oil. TLC: Rf = 0.45
1
(Cyclohexane/AcOEt, 9/1, SiO2). H NMR (300 MHz, CDCl3) δ (ppm 7.18 – 7.02 (m, 2H), 6.93 (d, J = 8.4 Hz, 1H),
6.41 (s, 2H), 4.05 (q, J = 7.2 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.83 (s, 6H), 1.61 (d, J = 7.2 Hz, 3H). 13C NMR (75
MHz, CDCl3) δ (ppm) 153.3 (2C), 150.4 (C), 141.7 (C), 139.0 (C), 138.0 (C), 136.6 (C), 126.9 (CH), 122.2 (CH), 120.8
(q, J = 258 Hz, CF3), 113.0 (CH), 104.8 (2CH), 61.0 (CH3), 56.2 (3CH3), 44.2 (CH), 22.2 (CH3). 19F NMR (188 MHz,
CDCl3) δ (ppm) -58.5 (s). HRMS (ESI) (M + H)+ m/z calcd for C19H22F3O5 387.1414 found 387.1413.
2-(Difluoromethoxy)-5-(1-(3,4,5-trimethoxyphenyl)ethyl)aniline (4e). Flash chromatography on silica gel
(Cyclohexane/AcOEt, 10/0 to 6/4) afforded 150 mg of 4e (0.425 mmol, yield 85%). Yellow oil. TLC: Rf = 0.36
(Cyclohexane/AcOEt, 7/3, SiO2). IR (film): 3375, 1967, 1623, 1590, 1509, 1462, 1419, 1378, 1330, 1236, 1201, 1126,
1034, 1006 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 6.94 (d, J = 8.4 Hz, 1H), 6.59 (m, 2H), 6.43 (t, J = 73 Hz, 1H),
6.42 (s, 2H), 3.97 (q, J = 7.0 Hz, 1H), 3.82 (s, 9H), 1.57 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.2
(2C), 144.8 (C), 141.8 (C), 138.6 (C), 136.9 (C), 136.4 (C), 120.1 (CH), 117.6 (CH), 116.9 (t, J = 258 Hz, CHF2), 115.8
(CH), 104.8 (2CH), 61.0 (CH3), 56.2 (2 CH3), 44.6 (CH), 22.0 (CH3). 19F NMR (188 MHz, CDCl3) δ (ppm) -77.6 (s).
HRMS (ESI) (M + H)+ m/z calcd for C18H22F2NO4 354.1517 found 354.1512.
5-(1-(3-Fluoro-4-methoxyphenyl)ethyl)-1,2,3-trimethoxybenzene (4f). Flash chromatography on silica gel
(Cyclohexane/AcOEt, 10/0 to 6/4) afforded 155 mg of 4f (0.485 mmol, yield 97%). Yellow oil. TLC: Rf = 0.61
(Cyclohexane/AcOEt, 7/3, SiO2). IR (film): 2965, 2839, 1735, 1588, 1507, 1465, 1418, 1331, 1274, 1235, 1183, 1126,
1069, 1008 cm-1. 1H NMR (300 MHz, CDCl3) δ (ppm) 7.05 – 6.73 (m, 3H), 6.40 (s, 2H), 4.01 (q, J = 7.1 Hz, 1H), 3.86
(s, 3H), 3.82 (s, 9H), 1.58 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.3 (2C), 152.4 (d, J = 240 Hz,
CF), 145.9 (d, J = 10.9 Hz, C), 141.9 (C), 139.6 (d, J = 5.4 Hz, C), 136.5 (C), 123.0 (d, J = 3.1 Hz, CH), 115.3 (d, J =
18.4 Hz, CH), 113.4 (CH), 104.7 (2CH), 61.0 (CH3), 56.5 (CH3), 56.2 (2CH3), 44.2 (CH), 22.1 (CH3). 19F NMR (188
MHz, CDCl3) δ (ppm) -133.4 (s). HRMS (ESI) (M + H)+ m/z calcd for C18H22FO4 321.1502 found 321.1496.
5-(2,2-Difluoro-1-(3,4,5-trimethoxyphenyl)ethyl)-2-methoxyphenol (4g). Flash chromatography on silica gel
(Cyclohexane/AcOEt, 10/0 to 6/4) afforded 160 mg of 4g (0.452 mmol, yield 90%). Colorless oil. TLC: Rf = 0.61
1
(Cyclohexane/AcOEt, 5/5, SiO2). H NMR (300 MHz, CDCl3) δ (ppm) 7.26 (d, J = 0.6 Hz, 1H), 6.89 (d, J = 1.1 Hz,
1H), 6.81 (d, J = 2.2 Hz, 1H), 6.50 (s, 2H), 6.23 (td, J = 55.9, 4.2 Hz, 1H), 5.62 (s, 1H), 4.23 (td, J = 15.9, 4.2 Hz, 1H),
3.88 (s, 3H), 3.83 (s, 9H). 13C NMR (75 MHz, CDCl3) δ (ppm) 153.4 (2C), 146.09 (C), 145.8 (C), 137.5 (C), 132.9 (C),
130.2 (C), 120.7 (CH), 117.0 (t, J = 243.0 Hz, CHF2), 115.3 (CH), 110.8 (CH), 106.3 (2CH), 61.0 (CH3), 56.3 (2 CH3),
56.1 (CH3), 54.6 (t, J = 20.7 Hz, CH-CHF2). 19F NMR (188 MHz, CDCl3) δ (ppm) -116.11 (d, J = 5.5 Hz). HRMS (ESI)
(M + H)+ m/z calcd for C18H21F2O5 355.1352 found 355.1354.
4.2. Biology
4.2.1. Cell culture and proliferation assay
Cancer cell lines were obtained from the American type Culture Collection (Rockville, USA) and were cultured
according to the supplier’s instructions. U87-MG cells were grown in Dulbecco minimal essential medium (DMEM)
25

containing 4.5 g/L glucose supplemented with 10% FCS and 1% glutamine. Human K562 and K562R (Doxo-resistant),
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HCT116, A549 and MCF7 cells were grown in RPMI 1640 containing 10% FCS and 1% glutamine. All cell lines were
maintained at 37°C in a humidified atmosphere containing 5% CO2. Cell viability was assessed using Promega
CellTiter-Blue reagent according to the manufacturer’s instructions. Cells were seeded in 96-well plates (5 X 103 cells
per well) containing 50 µL growth medium. After 24 h of culture, the cells were supplemented with 50 µL of the test
compound dissolved in DMSO (less than 0.1% in each preparation). After 72 h of incubation, 20 µL of resazurin was
added for 2 h before recording fluorescence (λex = 560 nm, λem = 590 nm) using a Victor microtiter plate fluorimeter
(Perkin–Elmer, USA). The IC50 value corresponds to the concentration of test compound that caused a decrease of 50%
in fluorescence of drug-treated cells compared with untreated cells. Experiments were performed in triplicate.
PBLs from healthy donors were obtained by separation on Lymphoprep (Fresenius KABI Norge AS) gradient. After
extensive washing, cells were resuspended (1.0 × 106 cells/mL) in RPMI-1640 with 10% fetal bovine serum and
incubated overnight. For cytotoxicity evaluations in proliferating PBL cultures, nonadherent cells were resuspended at 5
× 105 cells/mL in growth medium containing 2.5 µg/mL PHA (Irvine Scientific). Different concentrations of the test
compounds were added, and viability was determined 72 h later by the MTT test. For cytotoxicity evaluations in resting
PBL cultures, nonadherent cells were resuspended (5 × 105 cells/mL) and treated for 72 h with the test compounds, as
described above. J. Med. Chem. 2015, 58, 3209−3222
4.2.2. Tubulin binding assay
Sheep brain tubulin was purified according to the method of Shelanski[29] by two cycles of assembly-disassembly
and then diluted in the assembly buffer containing 0.1m MES, 0.5 mm MgCl2, 1mm EGTA and 1 mm GTP (pH 6.6) to
give a tubulin concentration of about 10 µM. Tubulin assembly was monitored and recorded continuously by
fluorescence according to reported procedure[30] using DAPI as the fluorescent molecule. Assays were realized on 96-
well plates prepared with Biomek NKMC and Biomek 3000 from Beckman Coulter and read at 37 °C on Wallac Victor
fluorimeter from Perkin Elmer. The IC50 value of each compound was determined as the concentration required to
decrease the maximum assembly rate of tubulin by 50% compared to the rate in the absence of the compound. The IC50
values for all compounds were compared to the IC50 of isoCA-4 and CA-4 measured the same day under the same
conditions.
4.2.3. Cell Cycle Analysis
Exponentially growing cancer HCT116 and U87-MG cells were incubated with compound 3m at 20 nM in DMSO for
24 h. Cell-cycle profiles were determined by flow cytometry on a FC500 flow cytometer (Beckman-Coulter, France) as
described previously.[31]
4.2.4. Immunofluorescence Staining Assays
U87-MG cells were seeded on Lab-Tek™ Chamber Slides (Thermo Fisher Scientific) and incubated for 24 h in the
presence or absence of 3m at the concentrations of 5 and 10 nM. Control was realized by adding 0.1 % of DMSO
during incubation. Cells were then fixed for 30 min. with 2% paraformaldehyde at room temperature and washed with
Dulbecco's phosphate-buffered saline (DPBS). After permeabilization with 0.25% Triton X-100 for 5 min., U87-MG
cells were washed in DPBS and incubated for 1 h in DPBS containing 1% Bovine Serum Albumin (BSA) and 10%
normal goat serum (DAKO cytomation) for blocking. Cells were then incubated overnight at 4 °C with the mouse anti-β
tubulin primary antibody (Thermo Fisher Scientific). Thereafter, the cells were washed with 0.2% Tween in DPBS and
26

incubated simultaneously for 2 h at room temperature with Alexa-Fluor 647 goat anti-mouse IgG secondary antibody
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and Hoechst 33258 (Thermo Fisher Scientific). Cells were then washed with DPBS and incubated for 30 min at room
temperature with Alexa-Fluor 488 Phalloidin (Thermo Fisher Scientific) to perform F-actin labeling. Fluorescence
images were collected by Nikon TE2000 inverted microscope with Nikon 1.4 NA DIC optics, 60X oil immersion
objective (Nikon).
4.2.5.Cytotoxicity evaluations in peripheral blood lymphocytes (PBL)
The cytotoxicity of the synthesized compounds was evaluated in quiescent and proliferating peripheral blood
lymphocytes (PBLs), essentially as described previously.[23] Briefly, stock solutions of the different compounds were
prepared by dissolving them in DMSO at a concentration of 10 mM. Peripheral blood mononuclear cells were obtained
from three different healthy donors by density gradient centrifugation using Ficoll-Paque PREMIUM (GE Healthcare),
plated at a density of 106/mL in RPMI-1640 medium containing 10% fetal calf serum (complete RPMI-1640 medium),
and cultured overnight at 37°C. Non-adherent cells were harvested and diluted at 5.105/mL in complete RPMI-1640
medium containing (for proliferating PBLs) or not (for quiescent PBLs) phytohemagglutinin (PHA, Irvine Scientific,
final concentration 2.5 µg/mL). Cells (90 µL) were then added to 10 µL of either DMSO or serial dilutions of the
compounds (0.001-25 µM, final concentrations) in 96-well culture plates, and cultured for 72 hours at 37°C. Viability
was determined by a MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega).
4.2.6. Molecular modelling
X-ray structures of five different tubulin cocrystals were retrieved from the PDB[32] (accession codes 1SA0, 1SA1,
3HKC, 3HKD, 3HKE) and prepared using the Protein Preparation Wizard from the Schrödinger suite,[33] including
optimization of the hydrogen bond network and a short minimization with position restraints on heavy atoms using the
OPLS_2005 force field .[34] Coordinates for compounds 3i, 3l, 3m, and isoCA-4 were generated using MarvinSktech
module from the JChem suite v15.12.7.[35] Ligands were the freely docked in the colchicine binding site located
between chains C and D using the ensemble docking procedure available in GOLD v5.2.2 [36] over the five aligned
tubulin structures. CHEMPLP with default parameters was used as an objective function.[37] Structures of complexes
were exported and loaded in Chimera v1.10.2 for examination (including hydrogen bond detection, close contact
analysis and calculation of solvent-accessible surface) and depiction.[38]
4.2.7. DFT pKa calculations
Predictions of pKa values were conducted using Jaguar pKa prediction module Jaguar, version 8.9, Schrödinger, LLC,
New York, NY, 2015 with default parameters.
Results of pKa calculations:
Goup
CH3
CH3
CF2H
CF2H
Solvent
Water
DMSO
Water
pKa
29.8
46.1
24.2
36.3
DMSO
4.2.8. Metabolic Stability Study of Compound 3i, 3m, and 3l
The UPLC−MS/MS system consisted of a Waters ACQUITY UPLC® System with an Acquity UPLC BEH RP18 1.7
µm, 2.1x50 mm column and a reversed phase gradient over a run time of 5.5 minutes. Mobile phase A was 0.2% formic
27

acid in water and mobile phase B 0.2% formic acid in methanol coupled to a Waters XEVO™ TQ-Mass Spectrometer
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operating in positive ion electrospray MRM mode. All solvents and chemicals were of LC/MS grade and were
purchased from VWR International.
Human plasma was obtained from Etablissement Français du Sang (EFS, Les Ulis, France). Compounds at 5 µM were
incubated in plasma at 37°C from 0 to 120 minutes. The human liver microsomes (HLM) were obtained from Biopredic
(Rennes, France) and NADPH was from Sigma-Aldrich (Lyon, France). Incubations were performed from 0 to 45
minutes. For each time points, a preparation containing potassium phosphate buffer (pH 7.4), HLM and the tested
compound was pre-incubated at 37°C for 5 minutes. The addition of NADPH was starting the incubation. The final
concentration of the incubation was 100 mM, 1 mM and 5 µM for phosphate buffer, NADPH, and the tested compound,
respectively. At last, microsome concentration was 1 mg/mL. At each time points, for plasma stability as for
microsomal stability, the incubation was stopped by addition of acetonitrile. After vortexing and centrifugation, the
supernatant was directly injected in the UPLC-MS/MS system in the case of microsomal incubation samples. For
plasma stability, supernatant was evaporated under a nitrogen stream and the dry extract was reconstituted with water
containing 0.2% of formic acid/methanol; 75/25; v/v. The concentrations of 3i, 3m, and 3l were determined by
UPLC−MS/MS.
4.2.9. Pharmacological studies on vascular reactivity of isolated rat aorta rings
All procedures on animals complied with the European directive 2010/63/EU of the European Parliament on the
protection of animals used for scientific purposes. Adult Wistar rats (300 - 400 g) were purchased from Janvier Labs
(Le Genest-Saint-Isle, France) and housed at the animal facility ANIMEX (Faculty of Pharmacy, Université Paris-Sud;
authorizations obtained from the French Ministry of Agriculture, Fisheries and Food (no. D- 92-283, December 13th,
2012). Rats were anesthetized with pentobarbital sodium (200 mg.kg-1, i.p.) and aorta was quickly isolated and placed
in ice-cold physiological saline solution (PSS, in mM : NaCl 119, KCl 4.7, CaCl2(H2O)2 2.5, MgSO4(H2O)7 1.2,
KH2PO4 1.2, NaHCO3, 25, D-glucose 11, aerated with a mixture of O2-CO2 at 95% and 5%, respectively). Aorta was
cleaned from fat and adventitial tissue and sliced into 2-3 mm aortic rings. Rings were mounted in standard individual
organ bath chambers filled with PSS maintained at 37°C and aerated with 95% O2 and 5% CO2 (pH 7.4) and used for
isometric tension recording.[39] Vessels were passively stretched to their optimal resting force (2 g) and equilibrated for
1 h. Then, proper vessel function was controlled by evaluating its contractile response to a 60 mM KCl depolarizing
solution (PSS containing 60 mM KCl by equimolar substitution with NaCl, K60). Rings developing a contraction below
1.5 g were discarded. Secondly, relaxant response to acetylcholine (10 µM), reflecting intact endothelial function, was
assessed. Rings relaxing less than 15% of the amplitude of the contractile response to K60 were discarded. Following
four washes with PSS, test compounds (10 µM) or vehicle (DMSO 0.1%) were added to the bath and allowed to
incubate for 10 min. The amplitude of the contractile effect induced by the addition of the compounds was recorded.
Then increasing concentrations of the vasoconstrictor agent prostaglandin F2α (PGF2α; 1 nM to 30 µM) were
sequentially added, to obtain concentration-contractile response relationships (CCRR). Emax was defined as the
maximal contractile response, obtained with 30 µM PGF2a. Tension values were normalized to Emax and CCRR for
PGF2a were fitted with the Hill equation using GraphPad Prism software (GraphPad software, Inc., La Jolla, CA,
USA), allowing to determine EC50 (M) and pD2, defined as: -log(EC50), for each experiment. All data were expressed as
mean ± SEM, where “N” represents the number of rats. Contractile responses were expressed as percentage of
contractile response to K60 solution. Statistical analysis were performed using GraphPad Prism software and Kruskal-
28