Synthesis and biological evaluation of fluoro analogues of antimitotic phenstatin

Article abstract of DOI:10.1016/j.bmc.2013.03.064

With the aim of investigating the influence of fluorine, in particular on the A-ring, a new series of fluoro analogues (7a-l) of phenstatin (3) was synthesized and tested for interactions with tubulin polymerization and evaluated for cytotoxicity on an NCI-60 human cancer cell lines panel. We have shown that the replacement of 3,4,5-trimethoxyphenyl A-ring of phenstatin with 2,4,5-trifluoro-3-methoxyphenyl unit, results in the conservation of both antitubulin and cytotoxic effect. Fluoro isocombretastatin 7k was the most effective anticancer agent in the present study and demonstrated the highest antiproliferative potential on leukemia cell lines SR (GI₅₀ = 15 nM) and HL-60(TB) (GI₅₀ = 23 nM) and on melanoma cell line MDA-MB-435 (GI₅₀ = 19 nM).

Full text of DOI:10.1016/j.bmc.2013.03.064

Bioorganic & Medicinal Chemistry 21 (2013) 2932–2940
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of fluoro analogues of antimitotic
phenstatin
Alina Ghinet a,b,c, Aurélien Tourteau b,d, Benoît Rigo b,c, , Vivien Stocker b,c, Marie Leman b,c
,
⇑
Amaury Farce b,d, Joëlle Dubois e, Philippe Gautret b,c
a Department of Organic Chemistry, ‘Al. I. Cuza’ University of Iasi, Faculty of Chemistry, Bd., Carol I, nr. 11, 700506 Iasi, Romania
b Univ Lille Nord de France, F-59000 Lille, France
c UCLille, EA GRIIOT (4481), Laboratoire de Pharmacochimie, HEI, 13 Rue de Toul, F-59046 Lille, France
d Institut de Chimie Pharmaceutique Albert Lespagnol, EA GRIIOT (4481), IFR114, 3 Rue du Pr Laguesse, B.P. 83, F-59006 Lille, France
e Institut de Chimie des Substances Naturelles, UPR2301 CNRS, Centre de Recherches de Gif, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim of investigating the influence of fluorine, in particular on the A-ring, a new series of fluoro
analogues (7a–l) of phenstatin (3) was synthesized and tested for interactions with tubulin polymeriza-
tion and evaluated for cytotoxicity on an NCI-60 human cancer cell lines panel. We have shown that the
replacement of 3,4,5-trimethoxyphenyl A-ring of phenstatin with 2,4,5-trifluoro-3-methoxyphenyl unit,
results in the conservation of both antitubulin and cytotoxic effect. Fluoro isocombretastatin 7k was the
most effective anticancer agent in the present study and demonstrated the highest antiproliferative
potential on leukemia cell lines SR (GI50 = 15 nM) and HL-60(TB) (GI50 = 23 nM) and on melanoma cell
line MDA-MB-435 (GI50 = 19 nM).
Received 17 January 2013
Revised 24 March 2013
Accepted 26 March 2013
Available online 4 April 2013
Keywords:
Phenstatin
Tubulin
Antimitotic agent
Fluorobenzophenone
Eaton’s reagent
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
with phase III trials under way. CA-4P acts as a vascular disrupting
agent (VDA).7 CA-4P does not induce the common side effects of
The microtubule system, formed of
a
- and b-tubulin heterodi-
existing chemotherapies such as alopecia and bone marrow toxic-
ity. However, cardiovascular toxicity and neurotoxicity are dose
limiting for this compound,8 and also it is prone to double-bond
isomerization,9 leading to the E-isomer which displays dramati-
cally reduced activity.6c Research on combretastatin A-4 in order
to improve in vivo activity has led to the discovery of phenstatin 3
(Fig. 1).10 The water-soluble phenstatin phosphate prodrug 4 and
the parent phenstatin 3 were essentially indistinguishable and
very similar to the combretastatin A-4 phosphate prodrug 2 in
terms of both potency and differential cytotoxicity.10 Nevertheless
this compound has not been advancing in human clinical trials,
and we recently described that phenstatin 3 is subject of many
metabolic transformations as well on the methoxy or hydroxy
groups.11
The replacement of a hydrogen or a hydroxyl group by a
fluorine atom is a strategy widely employed in drug development
to alter the chemical properties, cellular and systemic distribution
and biological activity of drugs,12 and in CA-4 series, replacement
of the ring B hydroxyl group by a fluorine atom led to compounds
with activities in the nanomolar range.13 In phenstatin series, a
fluorine substituent has been placed in all positions of B-ring,
sometimes to obtain synthetic intermediates,14 but activities were
modest15 except for the OH to F replacement (compound 5,
mers, is involved in many essential cell functions1 and is recog-
nized as an attractive pharmacological target for anticancer
drugs.2,3 These cytoskeletal structures are involved in many cellu-
lar functions.1 One of their key roles is in the mitotic spindle, inti-
mately involved in cell division, which bridges chromosomes in the
center and centrosomes at opposite poles of the cell, ultimately
leading to, during the metaphase/anaphase transition, separation
of the sister chromatids and segregation into daughter cells. The
importance of microtubules in cell division makes them an impor-
tant target for anticancer drugs.3 Most of the antimitotic drugs in
clinical use or in development, bind to three major tubulin sites:
the vinca, taxane and colchicine sites.4 Combretastatin A-4 1 binds
to tubulin at the colchicine binding site5 and exhibits very strong
inhibition of a variety of human cancer cell lines.4 The trimethoxy-
phenyl unit and the cisoid disposition of the two aromatic rings are
both essential for activity (Fig. 1).6 Research on combretastatin A-4
to improve water solubility and in vivo activity5 has led to the
water-soluble prodrug combretastatin A-4 phosphate (CA-4P,
Fosbretabulin) 2, currently in phase II human cancer clinical trials7
⇑
Corresponding author. Tel.: + 33 3 28 38 48 58.
E-mail address: benoit.rigo@hei.fr (B. Rigo).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.064

A. Ghinet et al. / Bioorg. Med. Chem. 21 (2013) 2932–2940
2933
O
amine 13 and commercial 3-fluoro-1,4-dimethoxybenzene 14 in
Eaton’s reagent (P2O5/MeSO3H 1:10 w/w). Protected compounds
11–13 were prepared as described previously.11a Surprisingly, the
condensation of 2,4,5-trifluoro-3-methoxybenzoic acid 8 with
chloroacetate 12 was accompanied by the O-demethylation of
the methoxy group at C20 position, followed by the deprotection
of the chloroacetyl group to give catechol 7b as unique product
of the reaction (Scheme 1). Furthermore, a para-demethylation of
the ketone 7e was observed during its formation in Eaton’s condi-
tions to afford compound 7f in 19% yield.
Removal of the protecting groups of benzophenones 7a, 7c, and
7d was easily performed: the chloroacetyl group of ketone 7a
afforded benzophenone 7g in 96% yield under mild basic condi-
tions (sodium acetate, methanol) and the acetamide unit of ke-
tones 7c and 7d was cleaved under acidic conditions (aqueous
10% HCl, methanol) to provide benzophenones 7h and 7i in 76%
and 80%, respectively.
OR
MeO
MeO
MeO
MeO
OR
OMe
B
A
OMe
OMe
OMe
1 R = H, Combretastatin A-4
3 R = H, Phenstatin
4 R = PO3Na2
2 R = PO3Na2, Fosbretabulin (CA-4P)
NH2
O
O
H
N
F
MeO
MeO
NHAc
N
OMe
F
OMe
6 Flubendazole25
516
ITP IC50 = 2.0 µM
(CA-4) IC50 = 1.9 µM
F
F
X
In order to explore the influence of the modification of the con-
nector between the two phenyl rings of phenstatin, we were inter-
ested in the synthesis of fluorinated analogs with an ethylene or a
carbonyl-reduced linker. The direct methylenation of benzophe-
nones 7e and 7g with the Wittig reagent methylenephosphorane,
prepared from methyltriphenylphosphonium bromide and potas-
sium tert-butoxide, afforded isocombretastatins 7j and 7k in mod-
erate 44% and 34% yield, respectively. The reduced form of
phenstatin was previously obtained from Grignard reaction of
the corresponding aldehyde.27 However, the sodium borohydride
reduction of the carbonyl unit of fluorophenstatin 7g, provided a
simpler route to benzhydrol 7l (81%) (Scheme 3).
X: C=O, C=CH2, CH-OH
7
Figure 1. Structure of reference compounds CA-4 1, phenstatin
3
and their
prodrugs 2, 4, structure and inhibitory activity on tubulin polymerization (ITP) of
some biologically active fluorobenzophenones 5,16 and 6,25 and structure of target
fluoro analogues of phenstatin 7.
Fig. 1).16 It is generally considered that the 3,4,5-trimethoxyphenyl
ring A of phenstatin is essential for the biological activity.6a,c,11a,17
However, it has been demonstrated that some modifications of the
A
ring such as 2,3,4-trimethoxyphenyl,18 halogeno-methoxy-
phenyl,19 2,3-methylenedioxy-4-phenyl,20 3,4,5-trimethylphenyl
moieties,21 or even change for a heterocyclic ring22 led to equal
or better activity. In that context, we were interested by the fact
that the old antiprotozoal and anthelmintic compound, flubendaz-
ole 6,23 is an antitubulin polymerization agent,24,25 whose the anti-
tumor properties were recently highlighted,25 partly thanks to its
absence of toxicity and induced neuropathy.26 Taking these facts
in consideration, we decided to study the biological properties of
benzophenones and derivatives, analogues to phenstatin, and
substituted in ring A or ring B by fluorine atoms. It is known that
some substituents placed in the 2-position of ring A are compatible
with good biological activities (OMe,18 NH216). Thus, a fluorine
atom was designed in that position as well as an isoster of these
groups as for blocking a potential metabolic location, leading to
the synthesis of compounds with a 2,4,5-trifluoro-3-methoxy-
phenyl group.
3. Biological evaluation
The synthesized compounds 7a–l were tested for their ability to
interact with tubulin using an assembly assay and compounds 7c,
7e–l were selected and evaluated for cytotoxicity against the 60-
human cancer cell lines panel from the National Cancer Institute
(NCI). The results are summarized in Tables 1–3.
The replacement of the trimethoxyphenyl unit of parent
phenstatin by a 2,4,5-trifluoro-3-methoxyphenyl moiety (com-
pound 7g, Scheme 2) led to the best antitubulin activity in this ser-
ies (7g: IC50 = 28.23 2.79 lM, Table 1). Unexpectedly, the
transformation of the carbonyl bridge of compound 7g in an ole-
finic one (compound 7k) decreased the biological activity (gener-
ally speaking, the isocombretastatins present higher activities
than the corresponding ketones),28 while the reduction of the car-
bonyl linker in compound 7l abolished the antitubulin efficacy in
the same way as for dihydrophenstatin. The modification of the
classical B-ring of compound 7g by a 20,30-dihydroxy-40-methoxy-
phenyl in compound 7b or 30-amino-20,40-dimethoxyphenyl in
compound 7h conserved the antitubulin potential, validating the
structure–activity relationships concerning the C20 position of
2. Chemistry
Fluorophenstatins 7a–f were obtained by reacting commercially
available carboxylic acids 8–10 with protected phenols 11, 12,
R5
O
R5
O
R6
R7
R6
R7
R1
R1
OH
i
+
2
R
2
R
R4
R4
R8
R3
R8
R3
7a: R1=R2=R4=F, R3=R7=OMe, R5=R8=H, R6=OCOCH2Cl 90%
8: R1=R2=R4=F, R3=OMe
11: R5=R8=H, R6=OCOCH2Cl, R7=OMe
12: R5=R7=OMe, R6=OCOCH2Cl, R8=H
13: R5=R7=OMe, R6=NHCOCH3, R8=H
14: R5=R8=OMe, R6=H, R7=F
9: R1=F, R2=OMe, R3=R4=H
10: R1=R2=R3=OMe, R4=H
7b: R1=R2=R4=F, R3=R7=OMe, R5=R6=OH, R8=H 71%
7c: R1=R2=R4=F, R3=R5=R7=OMe, R6=NHCOCH3, R8=H 76%
7d: R1=R4=R8=H, R2=F, R3=R5=R7=OMe, R6=NHCOCH3 85%
7e: R1=R2=R3=R5=R8=OMe, R4=R6=H, R7=F 51%
7f: R1=R3=R5=R8=OMe, R2=OH, R4=R6=H, R7=F 19%
Scheme 1. Reagents and conditions: Eaton’s reagent 4 equiv, 50 °C, 3–14 h.11a

2934
A. Ghinet et al. / Bioorg. Med. Chem. 21 (2013) 2932–2940
phenstatin11a and CA-4 analogs.6 In our previous investigations,11a
the acetylation of 30-amino derivatives and the protection of the
phenol groups by a chloroacetyl function in the benzophenone ser-
ies abolished the potency on microtubule assembly. This behavior
was also observed in the current fluorobenzophenone series, pro-
tected compounds 7a, 7c, and 7d being inactive (Table 1).
In the trimethoxyphenyl series, the modification of ring-B sub-
stitution by a 40-fluoro-20,50-dimethoxyphenyl unit in compound
7e29 resulted in a sevenfold decrease of antitubulin potency com-
pared to parent phenstatin 3, its isocombretastatin analog 7j29
deleting all the activity. Compound 7f (the para-demethylated
derivative of 7e) was also inactive on microtubule assembly.
In vitro cytotoxicity of nine compounds (7c, 7e–l) was assessed
by NCI. They were tested initially at a high single dose (10 lM) in
the full 60-cell panel. Only compounds which satisfied predeter-
mined threshold inhibition criteria have progressed to the 5-dose
screen in order to evaluate their GI50 values (Table 2).
Isocombretastatin derivative 7k exhibited the most important
in vitro cytotoxicity among studied compounds: inhibition of
CCRF-CEM, HL-60(TB), K-562, SR, HCT-15, KM12, SW-620, M14,
MDA-MB-435, MALME-3M, NCI-ADR/RES, and SF-539 cell lines
with GI50 values ranging from 15 to 47 nM (Table 2). The closely
related benzophenone 7g showed decreased cellular activity. The
ketone precursor 7e was more active than the isocombretastatin 7j.
Both showed good cytotoxicity against colon cancer and melanoma
cell lines. 7e also exhibited very potent anti-proliferative activity
against leukemia cell lines K-562 and SR. Amino derivative 7h
was twofold less active than analogue 7g, showing best cytotoxic
activity on MDA-MB-435 melanoma cell line (7g: GI50 = 285 nM,
7h: IC50 = 407 nM, Table 2).
A mean graph midpoints (MG_MID) was calculated for each
derivative selected for GI50 calculation, giving an average activity
parameter over all cell lines for compounds 7e, 7g, 7h, 7j, and
7k. These data indicated that compounds 7e and 7k possessed
the best antiproliferative activity among the tested compounds
(Table 2).
Table 1
Inhibitory activities on tubulin polymerization
b
Compd No.
%TPIa,b
IC50
(lM
SD)
R2
7a
7b
7c
7d
46
93
39
3
N.D.c
29.16 2.04
N.D.
—
0.97112
—
—
N.D.
7e
7f
7g
7h
7i
7j
7k
7l
77
48
88
89
45
44
77
49
99
100
24.04 5.74
N.D.
28.23 2.79
29.20 1.96
N.D.
N.D.
42.18 3.49
N.D.
0.69119
—
0.96761
0.97561
—
—
0.94922
—
0.93782
0.97402
Phenstatin 3
Desoxypodophyllotoxin
3.43 0.70
1.76 0.44
Benzophenones 7c, 7f, and 7i and benzhydrol 7l did not satisfy
predetermined threshold inhibition criteria in the initial single-
dose assay and did not progress to the 5-dose screen. Results from
these studies are reported in Table 3. Fluoro derivatives 7c and 7i
a
b
c
Inhibition of tubulin polymerization at a 100
Values represent mean of two experiments.
Not determined.
lM concentration.
Table 2
Results of the in vitro human cancer cell growth inhibitiona for compounds 7e, 7g, 7h, 7j and 7k
Cell type
Leukemia
Compound
Cell line
Phenstatin 3
7e
7g
7h
7j
7k
b
GI50 (nM)
CCRF-CEM
HL-60(TB)
K-562
—
—
—
—
192
203
46
3310
2020
186
3350
1100
273
340
455
2200
1220
601
666
568
587
547
260
37
23
37
3690
1830
961
30700
3000
752
2980
890
2840
SR
43
969
15
Non-Small
Cell lung
Cancer
Colon
Cancer
NCI-H23
NCI-H460
NCI-H522
HCT-15
KM12
SW-620
M14
MDA-MB-435
MALME-3M
OVCAR-3
NCI/ADR-RES
SK-OV-3
ACHN
RXF 393
SN12C
DU-145
PC-3
SF-268
SF-539
SNB-19
SNB-75
MDA-MB-231/ATCC
HS 578T
BT-549
—
421
115
102
55
50
63
72
25
433
45
68
213
693
144
602
257
207
748
164
419
109
388
194
365
195
3750
2230
1340
1760
1030
703
804
285
9530
1500
789
2440
6840
2470
5160
2080
3720
3850
2470
4390
1740
2090
1600
3130
2880
385
343
25
45
47
45
38
19
44
6.0c
—
—
—
—
—
Melanoma
N.D.e
—
407
11500
2090
1670
8370
7900
4510
5810
3910
10200
6540
3590
5470
1700
8550
3780
34100
4677
—
>10000
521
Ovarian
Cancer
2.0c
—
217
38
94
375
—
—
—
—
1050
6540
2160
498
2440
2240
2670
2160
3300
920
Renal
Cancer
67
176
209
139
140
75
31
67
55
94
Prostate
Cancer
Central
Nervous
System
Cancer
34.0d
—
—
—
—
—
—
—
Breast cancer
945
2190
4120
1820
52
61
295
—
MG-MIDf (nM)
60.1c
a
b
c
d
e
f
Data obtained from NCI’s in vitro 60 cell 5-dose screening.31
GI50 is the molar concentration of synthetic compound causing 50% growth inhibition of tumor cells.
Data obtained from Ref. 10.
Data obtained from Ref. 30.
Not determined.
Average activity parameter over all cell lines for the tested compounds.

A. Ghinet et al. / Bioorg. Med. Chem. 21 (2013) 2932–2940
2935
Table 3
In vitro growth inhibition percentage (GI%) caused by the selected compounds (7c, 7f, 7i, and 7l) against some tumor cell lines in the single-dose assaya
Compd No.
Panel
Most sensitive cell lines
GI%
Positive cytostatic effectb
4/54
Positive cytotoxic effectc
7c
Leukemia
SR
49
83
30
44
31
57
37
21
30
27
81
55
22
21
49
71
61
45
72
53
59
77
100
43
52
81
41
33
53
26
35
0/54
Non-small cell lung cancer
NCI-H522
HOP-62
SNB-75
HCC-2998
KM12
SW-620
UACC-257
UO-31
CNS cancer
Colon cancer
Melanoma
Renal cancer
Prostate cancer
Breast cancer
Ovarian cancer
Leukemia
PC-3
MDA-MB-468
NCI/ADR-RES
SR
MDA-MB-435
HL-60(TB)
K-562
7f
7i
0/58
9/53
0/58
1/53
Melanoma
Leukemia
SR
Non-small cell lung cancer
Colon cancer
NCI-H522
HCC-2998
HCT-15
KM12
SW-620
MDA-MB-435
M14
MCF-7
MDA-MB-468
K-562
Melanoma
Breast cancer
Leukemia
7l
1/59
0/59
SR
Non-small cell lung cancer
Colon cancer
Ovarian cancer
NCI-H522
KM12
NCI/ADR-RES
a
b
c
Data obtained from NCI’s in vitro disease-oriented human tumor cell screen at 10
Ratio between number of cell lines with percentage growth from 0 to 50 and total number of tested cell lines.
Ratio between number of cell lines with percentage growth of <0 and total number of cell lines.
l
M concentration.31
displayed a cytostatic effect on four and nine cell lines, respec-
tively. In addition, 7i demonstrated a minor cytotoxic effect on
MDA-MB-435 cell line (growth percentage: ꢀ2.6% at 10ꢀ5 M con-
centration). Benzophenone 7f and benzhydrol 7l were inactive at
O
F
O
F
OH
OCOCH2Cl
OMe
F
F
F
F
i
OMe
10 lM concentration.
OMe
OMe
7a
7g 96%
OMe
O
OMe
O
4. Molecular modeling
NHCOMe
OMe
NH2
F
F
ii
Docking studies were realized in the colchicine binding site of
tubulin, on new compounds that presented a biological potential
(7b, 7g, 7h and 7k), isocombretastatin A-4 and phenstatin 3
(Fig. 2) in order to gain some insights into their binding mode
and therefore help the design of new pharmacomodulations. Con-
trary to the other compounds, phenstatin 3 occupies an upright
position in the entrance of the pocket (Fig. 2e), most surely because
1
R
1
R
R3
R3
OMe
R2
R2
7c: R1=R3=F, R2=OMe
7d: R1=OMe, R2=R3=H
7h: R1=R3=F, R2=OMe 76%
7i: R1=OMe, R2=R3=H 80%
Scheme 2. Reagents and conditions: (i) AcONaꢁ3H2O 4.5 equiv, MeOH, reflux, 2 h;
(ii) aqueous 10% HCl, MeOH, reflux, 12–16 h.
CH2 OMe
O
OMe
OMe
MeO
MeO
MeO
i
F
F
MeO
OMe
OMe
OMe
OMe
7j 44%
7e
CH2
OH
O
OH
OH
OMe
F
F
OH
F
F
F
F
iii
ii
OMe
F
F
OMe
F
OMe
7l 81%
OMe
7k 34%
7g
Scheme 3. Reagents and conditions: (i) CH3PPh3Br 2 equiv, tBuOK 5 equiv, toluene, 80 °C, 18 h; (ii) CH3PPh3Br 2 equiv, tBuOK 5 equiv, THF, rt, 24 h; (iii) NaBH4 2 equiv, EtOH/
H2O, rt, 3 h.

2936
A. Ghinet et al. / Bioorg. Med. Chem. 21 (2013) 2932–2940
Leu 252
Leu 252
Val 181
Val 181
Leu 255
Leu 255
(a)
(b)
Ala 250
Leu 252
Val 181
Leu 255
Lys 352
(c)
(d)
(e)
(f)
Figure 2. Structure and docking of fluorophenstatins in the tubulin binding site: (a) compound 7b, (b) compound 7g, (c) compound 7h, (d) compound 7k, (e) superimposition
of compounds 7b and 7g with phenstatin 3 (in orange), and (f) superimposition of compound 7k with isocombretastatin A-4 (in purple).
of the somewhat larger volume of its triple methoxy substituted
cycle compared to the less voluminous fluorines. However, the B
cycle is well superposed with the corresponding ring of its newly
synthesized analogues. It nonetheless looses the hydrogen bond
tying the other molecules to Val 181, which is compensated by a
hydrogen bond between its para methoxy and Ala 250. Compound
7b showed preferentially a conformation lying flatly in the bottom
of the binding site, with its halogenated ring forming hydrophobic
contacts with Leu 252 and 255. The meta hydroxyl on the B ring
was involved in a hydrogen bond with the skeleton of Val 181. This
conformation accounted for just below a half of the solutions
(Fig. 2a).
Compound 7g behaved essentially the same way, with half of
the solutions occupying the binding site in the same conformation
as compound 7b and forming the same interactions (Fig. 2b).
Compound 7h, on the contrary, displayed a rather fuzzy collec-
tion of conformations in this region of the binding site. Moreover,
these conformations split equally between two general orienta-
tions, pointing the nitrogen either toward the right or the left of
the pocket. A secondary set of conformations gathered six closely

A. Ghinet et al. / Bioorg. Med. Chem. 21 (2013) 2932–2940
2937
related solutions that were placed upright in the binding site, with
the nitrogen in the bottom. It formed two hydrogen bonds, one be-
tween the nitrogen and the backbone carbonyl of Lys 352, the
other between the methoxy of the A cycle and the backbone of
Ala 250. The difference in placement of this compound when com-
pared with its congeners appears to be made up by a stronger
hydrogen bond net (Fig. 2c).
were recorded on a Varian 640-IR FT-IR Spectrometer. Elemental
analyses (C, H, N) of new compounds were determined by ‘Pôle
Chimie Moléculaire’, Faculté de Sciences Mirande, Université de
Bourgogne, Dijon, France.
6.1.2. General procedure A for Friedel–Crafts reactions in the
presence of Eaton’s reagent
Compound 7k (Fig. 2d) displayed again a horizontal placement
in the binding site. However, it showed two sets of conformations
differing by their orientation. The more numerous had also a better
score and was very similar to compound 7g. It formed the same
hydrogen bond with Val 181. The second cluster was rotated to
point the cycles upward and had no hydrogen bond to stabilise it.
Comparing phenstatin 3 and isocombretastatin A-4 (Fig. 2e and f),
it is interesting to note that the binding mode of isocombrestatin
A-4 is also a flat positioning in the bottom of the pocket, where
the trimethoxy barely fits and pushes the whole molecule toward
Val 181. This notable difference between phenstatin and isocom-
bretastatin A-4 despite their structural closeness may be related
to a possible interaction with Lys 352. Although oriented away
from the pocket, this residue could anchor phenstatin by both it
lateral methoxy and its linker carbonyl, while isocombretastatin
A-4 misses this last and would be therefore less stabilised by a
phenstatin-like conformation displaying much less contact with
the binding site.
Eaton’s reagent was prepared from phosphorus pentoxide
(P2O5) and methanesulfonic acid (CH3SO3H) (weight ratio P2O5/
CH3SO3H 1:10). The mixture was heated at 40 °C under nitrogen
atmosphere until complete homogeneity. Benzoic acid (1.15–
1.5 equiv) and aromatic derivative (1.0 equiv) were then added to
Eaton’s reagent. The mixture was heated at 50 °C under inert atmo-
sphere for 3–14 h. After cooling to room temperature, the reaction
medium was diluted with dichloromethane and carefully poured
into a separatory funnel containing sodium bicarbonate aqueous
solution (50% NaHCO3) (neutralization to pH 7). The aqueous solu-
tion was extracted with dichloromethane, and the combined or-
ganic layers were dried (MgSO4). Solvent was removed under
reduced pressure to produce a brownish oil. The crude product
was purified by Flash chromatography on RediSep packed columns
to provide pure fluorobenzophenones 7a–f.
6.1.2.1. 2-Methoxy-5-(2,4,5-trifluoro-3-methoxybenzoyl)phenyl
chloroacetate (7a).
The general procedure A was followed
using 2,4,5-trifluoro-3-methoxybenzoic acid 8 (12.8 g, 62.0 mmol),
2-methoxyphenyl chloroacetate 11 (10.4 g, 51.6 mmol) and Eaton’s
reagent (4.6 g P2O5 in 31.4 mL CH3SO3H). The mixture was heated
at 50 °C for 14 h. The final brown oil was purified by flash chroma-
tography with EtOAc/n-heptane 3:7 and recrystallized from abso-
lute EtOH to give pure chloroacetate 7a (18.1 g, 90%) as a white
solid; mp (EtOH) 121–123 °C; TLC Rf (EtOAc/n-heptane
5:5) = 0.78; 1H (CDCl3, 400 MHz) d (ppm) 3.97 (s, 3H, OCH3), 4.05
(t, J = 1.3 Hz, 3H, OCH3), 4.24 (s, 2H, OCOCH2Cl), 6.89 (d,
J = 8.2 Hz, 1H, ArH), 7.00 (ddd, J = 9.0, 8.3, 5.6 Hz, 1H, ArH), 7.38
(q, J = 2.2, 1.5 Hz, 1H, ArH), 7.42 (t, J = 1.3 Hz, 1H, ArH). 19F NMR
(CDCl3, 376 MHz) d (ppm) ꢀ147.21 (m, J = 20.7, 16.4, 8.3 Hz, 1F,
ArF), ꢀ139.02 (dddd, J = 23.2, 20.6, 13.5, 9.1 Hz, 1F, ArF), ꢀ131.8
(m, J = 13.6, 6.5 Hz, 1F, ArF). 13C NMR (CDCl3, 100 MHz) d 40.5
(CH2), 56.3 (CH3), 60.9 (t, J = 3.6 Hz, CH3), 107.6 (sym m, CH),
114.6 (CH), 117.1 (CH), 124.5 (d, J = 1.8 Hz, CH), 130.8 (C), 145.5
(sym m, C), 146.3 (C), 146.9 (dd, J = 11.8, 3.5 Hz, C), 147.1 (dd,
J = 14.8, 5.6 Hz, C), 148.6 (C), 150.8 (dd, J = 8.3, 2.1 Hz, C), 151.3
5. Conclusions
The synthesis and the biological evaluation of a new family of
fluorobenzophenones 7a–i are reported. In summary, we have
shown that the replacement of trimethoxyphenyl ring A of phenst-
atin with 2,4,5-trifluoro-3-methoxyphenyl unit results in conser-
vation of an antitubulin and cytotoxic effect, less active than the
parent compound, but which could counterbalance the metabolic
degradation observed with phenstatin,11 especially the O-deme-
thylations of methoxy groups on ring A. The replacement of the
carbonyl bridge of the fluorobenzophenone 7g with an olefinic
group in compound 7k resulted in an increase of the antiprolifera-
tive potential, while reducing the carbonyl linker in compound 7l
abolished the biological efficacy. A docking study of the com-
pounds also hint that the replacement of methoxy groups by fluo-
rine in phenstatin analogs give them a putative binding mode
similar to that of isocombretastatin A-4 due to a decreased bulk
of the corresponding cycle. Further investigation on the metabolic
profile of the best candidates issued from this study will be real-
ized in due course.
(C), 164.7 (C), 189.7 (C). IR
1099. Calcd for C17H12ClF3O5: C, 52.53; H, 3.11. Found: C, 52.40;
H, 3.47.
m
cmꢀ1: 1755, 1655, 1605, 1430,
6. Experimental section
6.1. Chemistry
6.1.2.2.
methoxyphenyl)methanone (7b).
was followed using 2,4,5-trifluoro-3-methoxybenzoic acid
(2,3-Dihydroxy-4-methoxyphenyl)-(2,4,5-trifluoro-3-
The general procedure A
8
(2.5 g, 12.1 mmol), 2,6-dimethoxyphenyl chloroacetate 12 (1.9 g,
8.1 mmol) and Eaton’s reagent (0.9 g P2O5 in 6.1 mL CH3SO3H).
The mixture was heated at 50 °C for 12 h. The final brown oil
was purified by flash chromatography with EtOAc/n-heptane 3:7
to provide the deprotected and mono O-demethylated pure com-
pound 7b (1.9 g, 71%) as a yellow solid; mp (EtOAc/n-heptane)
120–121 °C; TLC Rf (EtOAc/n-heptane 3:7) = 0.17; 1H (CDCl3,
400 MHz) d (ppm) 3.98 (s, 3H, OCH3), 4.09 (s, 3H, OCH3), 5.54
(s, 1H, ArOH), 6.51 (d, J = 9.2 Hz, 1H, ArH), 6.97 (sym m, 1H, ArH),
6.98 (d, J = 9.2 Hz, 1H, ArH), 11.89 (s, 1H, ArOH). 19F
(CDCl3, 376 MHz) d (ppm) ꢀ146.37 (ddd, J = 20.5, 12.4, 9.6 Hz, 1F,
ArF), ꢀ138.92 (dddd, J = 22.4, 20.5, 13.5, 9.5 Hz, 1F, ArF), ꢀ132.42
6.1.1. Materials and methods
Starting materials are commercially available and were used
without further purification. Melting points were measured on a
MPA 100 OptiMeltÒ apparatus and are uncorrected. NMR spectra
were acquired at 400 MHz for 1H NMR, at 100 MHz for 13C NMR,
and at 376 MHz for 19F NMR on a Varian 400 MHz Premium
ShieldedÒ spectrometer. Chemical shifts (d) are given in ppm rela-
tive to CDCl3 (7.26 ppm; 77.1 ppm). Splitting patterns are designed
as: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, mul-
tiplet and sym m, symmetric multiplet. Coupling constants J are re-
ported in hertz (Hz). Thin layer chromatographies were realized on
Macherey Nagel silica gel plates with a fluorescent indicator and
were visualized with UV-lamp at 254 and 366 nm. Column chro-
matographies were performed using a CombiFlash Rf Companion
(Teledyne-Isco System) and RediSep packed columns. IR spectra
(sym m, 1F, ArF). 13C (CDCl3, 100 MHz)
d 56.4 (CH3), 62.2
(t, J = 3.1 Hz, CH3), 103.3 (CH), 109.9 (dd, J = 20.2, 3.1 Hz, CH),
114.4 (C), 121.9 (sym m, C), 125.5 (d, J = 2.4 Hz, CH), 133.5 (C),
138.3 (m, C), 145.9 (ddd, J = 255.0, 15.0, 5.3 Hz, C), 147.4 (ddd,

2938
A. Ghinet et al. / Bioorg. Med. Chem. 21 (2013) 2932–2940
J = 248.5, 11.7, 3.1 Hz, C), 148.5 (dt, J = 249.6, 3.3 Hz, C), 150.8 (C),
153.1 (C), 194.3 (C). IR
cmꢀ1: 3467, 1633, 1597, 1508, 1463,
1429, 1278, 1095, 1066, 777. Calcd for C15H11F3O5: C, 54.89; H,
(CH3), 57.1 (CH3), 60.9 (CH3), 101.6 (CH), 107.4 (2CH), 115.3 (CH),
123.9 (C), 132.8 (C), 141.4 (C), 142.7 (C), 151.9 (C), 152.9 (2C),
m
155.5 (C), 193.9 (C). IR m
cmꢀ1: 2944, 1638, 1213, 1126. Calcd for
19
3.38. Found: C, 54.77; H, 3.17.
C
18
H FO6: C, 61.71; H, 5.47. Found: C, 61.31; H, 5.21.
6.1.2.3.
zoyl)phenyl]acetamide (7c).
followed using 2,4,5-trifluoro-3-methoxybenzoic acid 8 (2.0 g,
9.7 mmol), N-(2,6-dimethoxyphenyl)acetamide 13 (1.3 g,
N-[2,6-Dimethoxy-3-(2,4,5-trifluoro-3-methoxyben-
6.1.2.6. (4-Fluoro-2,5-dimethoxyphenyl)-(4-hydroxy-3,5-dime-
thoxyphenyl)methanone (7f)29
By-product from the syn-
The general procedure A was
.
thesis of benzophenone 7e; white solid (0.4 g, 19%); mp (EtOAc/
n-heptane) 122–124 °C; TLC Rf (EtOAc/n-heptane 3:7) = 0.61; 1H
(CDCl3, 400 MHz) d (ppm) 3.70 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
3.89 (s, 6H, 2OCH3), 5.98 (s, 1H, ArOH), 6.81 (d, J = 12.8 Hz, 1H,
ArH), 7.02 (d, J = 9.5 Hz, 1H, ArH), 7.11 (s, 2H, ArH). 19F (CDCl3,
376 MHz) d (ppm) ꢀ127.7 (dd, J = 12.3, 9.6 Hz, 1F, ArF). 13C (CDCl3,
100 MHz) d 56.3 (2CH3), 57.1 (CH3), 60.9 (CH3), 101.6 (CH), 107.4
(2CH), 115.3 (CH), 124.1 (C), 129.1 (C), 139.9 (C), 146.6 (2C),
151.7 (C), 152.8 (C), 155.3 (C), 193.6 (C). Calcd for C17H17FO6: C,
60.71; H, 5.09. Found: C, 60.76; H, 5.00.
6.5 mmol) and Eaton’s reagent (0.9 g P2O5 in 6.1 mL CH3SO3H). The
mixture was heated at 50 °C for 14 h. The final brown oil was puri-
fied by flash chromatography with EtOAc/n-heptane 5:5 to give pure
acetamide 7c (1.9 g, 76%) as a white solid; mp (EtOAc/n-heptane)
160–162 °C; TLC Rf (EtOAc/n-heptane 75:25) = 0.21; 1H (CDCl3,
400 MHz) d (ppm) 2.15 (br s, 3H, NHCOCH3), 3.58 (s, 3H, OCH3),
3.92 (s, 3H, OCH3), 4.02 (s, 3H, OCH3), 6.60 (br s, 1H, ArNH), 6.79
(d, J = 8.6 Hz, 1H, ArH), 7.18 (sym m, 1H, ArH), 7.56 (d, J = 8.7 Hz, 1H,
ArH). 19F (CDCl3, 376 MHz) d (ppm) ꢀ145.27 (m, 1F, ArF), ꢀ139.81
(m, 1F, ArF), ꢀ131.77 (sym m, 1F, ArF). 13C (CDCl3, 100 MHz) d 23.2
(CH3), 56.3 (CH3), 62.1 (CH3), 62.2 (t, J = 3.1 Hz, CH3), 106.8 (CH),
110.6 (dd, J = 20.2, 3.1 Hz, CH), 119.3 (sym m, C), 124.0 (sym m, C),
130.6 (CH), 137.9 (sym m, C), 147.1 (sym m, C), 149.2 (sym m, C),
151.7 (sym m, C), 156.4 (d, J = 3.1 Hz, C), 156.8 (sym m, C), 159.4
(C), 166.8 (C), 188.6 (C). Due to the presence of the two methoxy
groups next to the acetamide function, the signal corresponding to
6.1.3. (3-Hydroxy-4-methoxyphenyl)-(2,4,5-trifluoro-3-
methoxyphenyl)methanone (7g)
Chloroacetate 7a (18.0 g, 46.3 mmol) and sodium acetate (AcO-
Naꢁ3H2O) (28.4 g, 208.7 mmol) were dissolved in MeOH (50 mL).
The solution was refluxed for 2 h. After cooling at rt, the mixture
was concentrated under reduced pressure. The residue was taken
into distilled water. The resulting precipitate was filtered, washed
with water several times to remove remaining sodium acetate and
recrystallized from absolute EtOH to obtain benzophenone 7g
(13.9 g, 96%) as a white solid; mp (EtOH) 112–115 °C; Rf (EtOAc/n-
heptane 5:5) = 0.32; 1H (CDCl3, 400 MHz) d (ppm) 3.99 (s, 3H,
OCH3), 4.07 (t, J = 1.3 Hz, 3H, OCH3), 5.74 (br s, 1H, ArOH), 6.91
(d, J = 8.2 Hz, 1H, ArH), 7.01 (ddd, J = 9.3, 8.1, 5.6 Hz, 1H, ArH), 7.36
(q, J = 2.2, 1.5 Hz, 1H, ArH), 7.41 (t, J = 1.3 Hz, 1H, ArH). 19F NMR
(CDCl3, 376 MHz) d (ppm) ꢀ146.67 (m, J = 20.4, 16.4, 8.2 Hz, 1F,
ArF), ꢀ139.43 (dddd, J = 23.2, 20.4, 13.5, 9.5 Hz, 1F, ArF), ꢀ131.8
(m, J = 13.6, 6.7 Hz, 1F, ArF). 13C NMR (CDCl3, 100 MHz) d 56.2
(CH3), 62.1 (t, J = 3.9 Hz, CH3), 109.9 (CH), 110.4 (dd, J = 20.2,
3.9 Hz, CH), 115.5 (CH), 123.9 (d, J = 1.6 Hz, CH), 130.2 (C), 144.7
(dd, J = 14.7, 4.7 Hz, C), 145.6 (C), 146.0 (dd, J = 11.8, 3.2 Hz, C),
147.2 (dd, J = 14.8, 5.5 Hz, C), 148.0 (C), 150.5 (dd, J = 7.7, 2.4 Hz,
the methyl group at 23.2 ppm is very weak. IR
m
cmꢀ1: 3210, 1660,
1595, 1510, 1461, 1349, 1089, 995. Calcd for C18H16O5NF3: C,
56.40; H, 4.21; N, 3.65. Found: C, 56.09; H, 4.30; N, 3.67.
6.1.2.4.
phenyl]acetamide (7d).
lowed using 3-fluoro-4-methoxybenzoic acid
11.8 mmol), N-(2,6-dimethoxyphenyl)acetamide
N-[3-(3-Fluoro-4-methoxybenzoyl)-2,6-dimethoxy-
The general procedure A was fol-
9
13
(2.0 g,
(1.5 g,
7.8 mmol) and Eaton’s reagent (0.7 g P2O5 in 4.9 mL CH3SO3H).
The mixture was heated at 60 °C for 5 h. The final product precip-
itates in dichloromethane. After filtration of the obtained precipi-
tate, the pure acetamide 7d (2.3 g, 85%) is obtained as a white
solid; mp (CH2Cl2) 179–180 °C; TLC Rf (EtOAc) = 0.30; 1H (CDCl3,
400 MHz) d (ppm) 2.18 (br s, 3H, NHCOCH3), 3.62 (s, 3H, OCH3),
3.91 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 6.68 (br s, 1H, NHCOCH3),
6.77 (d, J = 8.9 Hz, 1H, ArH), 6.98 (t, J = 8.0 Hz, 1H, ArH), 7.34
(d, J = 8.0 Hz, 1H, ArH), 7.60 (d, J = 8.9 Hz, 1H, ArH), 7.64 (d,
J = 12.0 Hz, 1H, ArH). 19F (CDCl3, 376 MHz) d (ppm) ꢀ134.58
(s, 1F, ArF). 13C (CDCl3, 100 MHz) d 23.4 (CH3), 56.2 (CH3), 56.3
(CH3), 62.1 (CH3), 106.4 (CH), 112.1 (d, J = 1.6 Hz, CH), 117.1 (d,
J = 19.4 Hz, CH), 125.1 (m, C), 127.8 (m, C), 129.4 (CH), 130.8 (d,
J = 5.4 Hz, CH), 150.6 (C), 151.9 (d, J = 11.1 Hz, C), 153.1 (C), 155.4
(d, J = 20.0 Hz, C), 157.6 (C), 167.2 (C), 192.9 (C). Due to the pres-
ence of the two methoxy groups next to the acetamide function,
the signal corresponding to the methyl group at 23.2 ppm is very
C), 151.4 (C), 189.5 (C). IR
m
cmꢀ1: 3421, 1647, 1579, 1436, 1352,
1099, 1020, 788. Calcd. for C15H11F3O4: C, 57.70; H, 3.55. Found: C,
57.40; H, 3.47.
6.1.4. General procedure B for deprotection of acetamide
function
A mixture of acetamide 7c or 7d (1 equiv) and a 10% hydrochlo-
ric acid solution (5–50 mL) in methanol (10–50 mL) was stirred at
reflux for 12–16 h. The final suspension was neutralized to pH 7 by
slow addition of an ammoniacal solution. The aqueous solution
was extracted with dichloromethane. The organic phase was dried
(MgSO4), filtered and concentrated in vacuo. The residue was
recrystallized from Et2O to afford pure aniline 7h or 7i.
weak. IR
m
cmꢀ1: 3257, 1648, 1608, 1515, 1280, 1123, 1094,
1019, 833. Calcd for C18H18O5NF: C, 62.24; H, 5.22; N, 4.03. Found:
C, 62.20; H, 4.94; N, 4.17.
6.1.2.5.
(4-Fluoro-2,5-dimethoxyphenyl)-(3,4,5-trimethoxy-
The general procedure A was fol-
6.1.4.1.
(3-Amino-2,4-dimethoxyphenyl)(2,4,5-trifluoro-3-
The general procedure B
phenyl)methanone (7e)29
.
methoxyphenyl)methanone (7h).
lowed using 2-fluoro-1,4-dimethoxybenzene 14 (1.0 g, 6.4 mmol),
3,4,5-trimethoxybenzoic acid 10 (2.0 g, 9.6 mmol) and Eaton’s re-
agent (0.8 g of P2O5 in 5.4 mL of CH3SO3H) at 60 °C for 3 h. The
crude product was purified by column chromatography on silica
gel with EtOAc/n-heptane 3:7 to give the pure product 7e as a
white solid (1.1 g, 51%); mp (EtOAc/n-heptane) 110–112 °C; TLC
Rf (EtOAc/n-heptane 3:7) = 0.75; 1H (CDCl3, 400 MHz) d (ppm)
3.70 (s, 3H, OCH3), 3.85 (s, 6H, 2OCH3), 3.87 (s, 3H, OCH3), 3.94
(s, 3H, OCH3), 6.81 (d, J = 12.0 Hz, 1H, ArH), 7.03 (d, J = 9.8 Hz, 1H,
ArH), 7.07 (s, 2H, ArH). 19F (CDCl3, 376 MHz) d (ppm) ꢀ127.2 (dd,
J = 12.2, 9.5 Hz, 1F, ArF). 13C (CDCl3, 100 MHz) d 56.3 (2CH3), 56.5
was followed using acetamide 7c (0.1 g, 0.3 mmol), 10% HCl
(5 mL) in methanol (10 mL). The mixture was stirred at reflux for
16 h. The crude product was purified by column chromatography
on silica gel with EtOAc/n-heptane 1:9 and recrystallized from
absolute EtOH to give the pure product 7h as a yellow solid
(0.09 g, 76%); mp (EtOH) 161–162 °C; TLC Rf (EtOAc/n-heptane
75:25) = 0.18; 1H (CDCl3, 400 MHz) d (ppm) 3.63 (s, 3H, OCH3),
3.93 (s, 3H, OCH3), 3.97 (br s, 2H, ArNH2), 4.03 (s, 3H, OCH3), 6.65
(d, J = 8.6 Hz, 1H, ArH), 6.97 (d, J = 8.6 Hz, 1H, ArH), 7.13 (ddd,
J = 12.0, 5.9, 1.6 Hz, 1H, ArH). 19F (CDCl3, 376 MHz) d (ppm)
ꢀ145.93 (ddd, J = 21.1, 17.1, 9.0 Hz, 1F, ArF), ꢀ140.17 (dddd,

A. Ghinet et al. / Bioorg. Med. Chem. 21 (2013) 2932–2940
2939
J = 23.2, 20.5, 13.8, 9.6 Hz, 1F, ArF), ꢀ132.10 (sym m, 1F, ArF). 13C
(CDCl3, 100 MHz) d 55.9 (CH3), 61.1 (CH3), 62.1 (t, J = 4.0 Hz,
CH3), 105.7 (CH), 110.7 (dd, J = 20.4, 4.0 Hz, CH), 120.5 (CH),
124.3 (sym m, C), 125.0 (C), 130.1 (C), 137.82 (sym m, C), 146.4
(ddd, J = 256.3, 15.6, 5.5 Hz, C), 146.6 (C), 147.0 (ddd, J = 247.3,
11.7, 4.0 Hz, C), 150.4 (dt, J = 256.3, 4.0 Hz, C), 151.8 (C), 189.3
(t, J = 1.9 Hz, C). IR
1079, 950, 781. Calcd for C16H14F3O4N: C, 56.31; H, 4.13; N, 4.10.
Found: C, 56.79; H, 3.88; N, 4.17.
n-heptane 1:9. The pure Wittig product 7k was obtained as a beige
oil (0.1 g, 34%); TLC Rf (EtOAc/n-heptane 5:5) = 0.65; 1H (CDCl3,
400 MHz) d (ppm) 3.90 (s, 3H, OCH3), 4.02 (s, 3H, OCH3), 5.30
(s, 1H, CH2), 5.58 (s, 1H, ArOH), 5.68 (s, 1H, CH2), 6.73–6.81 (m, 3H,
ArH), 6.90 (d, J = 2.3 Hz, 1H, ArH). 19F (CDCl3, 376 MHz) d (ppm)
ꢀ152.26 (m, 1F, ArF), ꢀ141.67 (ddd, J = 23.2, 11.6, 2.7 Hz, 1F, ArF),
ꢀ133.63 (q, 1F, ArF). 13C (CDCl3, 100 MHz) d 56.0 (CH3), 62.1 (CH3),
110.3 (2CH), 113.0 (CH), 116.6 (CH2), 118.8 (CH), 133.3 (C), 134.4
(C), 138.7 (C), 141.9 (C), 142.1 (C), 143.0 (C), 145.5 (C), 146.6 (C),
m
cmꢀ1: 3459, 3366, 1642, 1580, 1499, 1355,
150.1 (C). IR
m
cmꢀ1: 3444, 2921, 2851, 1738, 1605, 1505, 1464,
6.1.4.2. (3-Amino-2,4-dimethoxyphenyl)(3-fluoro-4-methoxy-
1431, 1356, 1260, 1091, 1023, 947, 763. Calcd for
4C7H16ꢁC16H13F3O3: C, 63.58; H, 5.11. Found: C, 63.31; H, 5.30.
1
phenyl)methanone (7i).
The general procedure B was fol-
lowed using acetamide 7d (2.0 g, 5.8 mmol), 10% HCl (50 mL) in
methanol (50 mL). The mixture was stirred at reflux for 12 h. The
crude product was purified by column chromatography on silica
gel with EtOAc/n-heptane 4:6 to give the pure product 7i as a yellow
solid (1.4 g, 80%); mp (EtOAc/n-heptane) 80–82 °C; TLC Rf (EtOAc/
n-heptane 4:6) = 0.21; 1H (CDCl3, 400 MHz) d (ppm) 3.65 (s, 3H,
OCH3), 3.92 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.99 (br s, 2H, ArNH2),
6.65 (d, J = 8.5 Hz, 1H, ArH), 6.79 (d, J = 8.5 Hz, 1H, ArH), 6.97 (t,
J = 8.5 Hz, 1H, ArH), 7.61 (ddd, J = 8.5, 2.2, 1.0 Hz, 1H, ArH), 7.64
(dd, J = 12.7, 2.2 Hz, 1H, ArH). 19F (CDCl3, 376 MHz) d (ppm)
ꢀ134.97 (q, J = 10.9, 8.2 Hz, 1F, ArF). 13C (CDCl3, 100 MHz) d 55.9
(CH3), 56.3 (CH3), 61.3 (CH3), 105.5 (CH), 111.9 (d, J = 2.4 Hz, CH),
117.4 (d, J = 19.3 Hz, CH), 118.9 (CH), 125.1 (C), 127.6
(d, J = 3.1 Hz, CH), 129.7 (C), 131.2 (d, J = 4.7 Hz, C), 145.5 (C),
150.2 (C), 151.7 (d, J = 10.9 Hz, C), 153.0 (C), 193.7 (d, J = 2.4 Hz,
6.1.6. 5-[Hydroxy(2,4,5-trifluoro-3-methoxyphenyl)methyl]-2-
methoxyphenol (7l)
An aqueous solution of sodium borohydride (3.7 g, 98.1 mmol
in 140 mL distilled water) was added dropwise to a solution of
benzophenone 7g (13.3 g, 44.6 mmol) in absolute ethanol
(190 mL). After stirring for 3 h at room temperature, the reaction
media is neutralized (pH 7) with a hydrochloric acid solution
(1.5 M) and extracted with ethyl acetate (3 ꢂ 100 mL). Combined
organic layers were dried over magnesium sulfate and concen-
trated under reduced pressure. The final precipitate was washed
with water, collected by filtration to give pure benzhydrol 7l
(10.8 g, 81%) as a white solid; mp (H2O) 128–130 °C; Rf (EtOAc/
n-heptane 5:5) = 0.69; 1H (CDCl3, 400 MHz) d (ppm) 3.88 (s, 3H,
OCH3), 4.00 (t, J = 1.2 Hz, 3H, OCH3), 5.63 (br s, 2H, ArOH and
CHOH), 5.98 (br s, 1H, CHOH), 6.81 (d, J = 8.4 Hz, 1H, ArH), 6.88
(dd, J = 8.4, 2.5 Hz, 1H, ArH), 6.92 (d, J = 2.6 Hz, 1H, ArH), 7.09
(sym m, 1H, ArH). 19F NMR (CDCl3, 376 MHz) d (ppm) ꢀ152.85
(ddd, J = 20.4, 13.6, 5.5 Hz, 1F, ArF), -140.47 (sym m, J = 24.4,
21.7, 13.6, 10.9 Hz, 1F, ArF), ꢀ138.75 (sym m, J = 13.6, 5.5 Hz, 1F,
ArF). 13C NMR (CDCl3, 100 MHz) d 56.0 (CH3), 62.0 (CH3), 69.0
(CH), 107.6 (dd, J = 20.3, 4.7 Hz, CH), 110.6 (CH), 112.6 (CH),
C). IR
m
cmꢀ1: 3457, 3352, 1647, 1606, 1432, 1279, 1256, 1127,
1068, 1023, 817, 762. Calcd for C16H16FO4N: C, 62.95; H, 5.28; N,
4.59. Found: C, 62.79; H, 5.67; N, 4.57.
6.1.5. General procedure C for Wittig reaction
A mixture of potassium tert-butoxide (5 equiv) and methyltri-
phenylphosphonium bromide (2 equiv) in THF or toluene was stir-
red at rt or 80 °C for 1 h. The benzophenone 7e or 7g (1 equiv)
dissolved in THF or toluene was added and the mixture was stirred
at rt or 80 °C for 18–24 h. The solution was poured into water, then
extracted in dichloromethane and dried (MgSO4). The solvent was
removed in vacuo. The crude product was purified by column chro-
matography on silica gel to obtain the Wittig compound 7j or 7k.
118.0 (CH), 135.4 (C), 145.8 (C), 146.4 (C). IR
m
cmꢀ1: 3396, 3264,
1510, 1475, 1087, 1039. Calcd. for C15H13F3O4: C, 57.33; H, 4.17.
Found: C, 56.94; H, 4.48.
6.2. Tubulin assembly studies
Sheep brain tubulin was purified according to the method of She-
lanski31 bytwocyclesassembly–dissamblyandthendissolvedinthe
assembly buffer containing 0.1 M MES, 0.5 mM MgCl2, 1 mM EGTA,
and 1 mM of GTP (pH 6.6) to give a tubulin concentration of about
2–3 mg/mL. Tubulin assembly was monitored by fluorescence
according to reported procedure32 using DAPI as fluorescent mole-
cule. 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 tubulin by 50% compared to
the rate in the absence of compound. The IC50 values for all com-
pounds were compared to the IC50 of phenstatin and desoxypodo-
phyllotoxine andmeasuredthesamedayunderthesameconditions.
6.1.5.1. 5-[1-(4-Fluoro-2,5-dimethoxyphenyl)ethenyl]-1,2,3-tri-
methoxybenzene (7j)29
.
The general procedure C was
followed using benzophenone 7e (0.5 g, 1.4 mmol), potassium
tert-butoxide (0.8 g, 7.1 mmol) and methyltriphenylphosphonium
bromide (1.0 g, 2.8 mmol) in toluene (20 mL) at 80 °C. The crude
product was purified by column chromatography on silica gel with
EtOAc/n-heptane 5:5 to give the pure Wittig product 7j as a white
solid (0.2 g, 44%); mp (EtOAc/n-heptane) 77–79 °C; Rf (EtOAc/
n-heptane 4:6) = 0.54; 1H (CDCl3, 400 MHz) d (ppm) 3.64 (s, 3H,
OCH3), 3.80 (s, 6H, 2OCH3), 3.85 (s, 3H, OCH3), 3.93 (s, 3H, OCH3),
5.32 (d, J = 1.8 Hz, 1H, CH2), 5.59 (d, J = 1.8 Hz, 1H, CH2), 6.54
(s, 2H, ArH), 6.66 (d, J = 8.6 Hz, 1H, ArH), 6.74 (d, J = 8.6 Hz, 1H,
ArH). 19F NMR (CDCl3, 376 MHz) d (ppm) ꢀ132.71 (dd, J = 11.2,
9.5 Hz, 1F, ArF). 13C NMR (CDCl3, 100 MHz) d 55.9 (2CH3), 56.2
(CH3), 60.5 (CH3), 60.9 (CH3), 104.2 (2CH), 106.2 (CH), 115.1
(CH2), 121.1 (CH), 128.1 (C), 137.8 (C), 138.4 (C), 145.0 (C), 146.5
6.3. Cell proliferation assay
The compounds were tested against a panel of 60 human cancer
cell lines at the National Cancer Institute, Bethesda, MD.33a The
cytotoxicity studies were conducted using a 48 h exposure proto-
col using the sulforhodamine B assay.33b
(C), 147.5 (C), 152.8 (C), 154.8 (C). IR
Calcd. for C19H21FO5: C, 65.51; H, 6.08. Found: C, 65.09; H, 6.51.
m
cmꢀ1: 2927, 1574, 1124.
6.1.5.2.
vinyl]phenol (7k).
2-Methoxy-5-[1-(2,4,5-trifluoro-3-methoxyphenyl)
The general procedure C was followed
6.4. Molecular modeling
using benzophenone 7g (0.3 g, 1.0 mmol), methyltriphenylphos-
phonium bromide (0.7 g, 2.0 mmol) and potassium tert-butoxide
(0.5 g, 4.8 mmol) in THF (20 mL) at rt for 24 h. The crude product
was purified by column chromatography on silica gel with EtOAc/
The crystallographic structure of a heterodimer of
a and
b-tubuline was taken from the 1sa034 entry of the RCSB Protein
Data Bank (http://www.pdb.org)35 and the binding site was

2940
A. Ghinet et al. / Bioorg. Med. Chem. 21 (2013) 2932–2940
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Acknowledgments
The authors gratefully acknowledge the ‘Conseil Régional Nord-
Pas-de-Calais’ for the A.G., A.T. and V.S.’s PhD scholarships and the
NCI (National Cancer Institute) for the biological evaluation of
compounds on their 60-cell panel. The authors would like to thank
Dr D. Perry for reviewing this manuscript.
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