Synthesis of novel 7-oxo and 7-hydroxy trifluoroallocolchicinoids with cytotoxic effect

Article abstract of DOI:10.1016/j.bmc.2012.02.043

The synthesis of 7-oxo and 7-hydroxy trifluoroallocolchicinoids was achieved through the intramolecular cyclization of o-phenyl-β- phenylalanines. The resulting compounds were evaluated for their cytotoxic activity against KB cells and their inhibitory ef

Full text of DOI:10.1016/j.bmc.2012.02.043

Bioorganic & Medicinal Chemistry 20 (2012) 2614–2623
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel 7-oxo and 7-hydroxy trifluoroallocolchicinoids
with cytotoxic effect
Elizabeth Chosson a,b, Francesca Santoro a,c, Christophe Rochais a, Jana Sopkova-de Oliveira Santos a,
Rémi Legay a, Sylviane Thoret d, Thierry Cresteil d, Maria Stefania Sinicropi c, Thierry Besson b,
Patrick Dallemagne a,
⇑
a Université de Caen Basse Normandie, Centre d’Etudes et de Recherche sur le Médicament de Normandie UPRES EA 4258 – FR CNRS 3038 INC3M,
UFR des Sciences Pharmaceutiques, Bd Becquerel, 14032 Caen cedex, France
b Université de Rouen, CNRS UMR 6014, C.O.B.R.A. & FR3038, I.R.C.O.F., Rue Tesnière, 76130 Mont Saint-Aignan, France
c Department of Pharmaceutical Sciences, University of Calabria, 87036 Arcavacata di Rende, Italy
d Institut de Chimie des Substances Naturelles, CNRS UPR 230, 1 Avenue de la terrasse, 91198 Gif sur Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of 7-oxo and 7-hydroxy trifluoroallocolchicinoids was achieved through the intramolecular
cyclization of o-phenyl-b-phenylalanines. The resulting compounds were evaluated for their cytotoxic
activity against KB cells and their inhibitory effect towards the polymerization of tubulin. The results
yielded some potent cytotoxic compounds with correlated partial antitubulin effect.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 19 December 2011
Revised 10 February 2012
Accepted 16 February 2012
Available online 3 March 2012
Keywords:
Colchicin
Allocolchicinoid
Betaphenylalanine
Cytotoxicity
Tubulin
Allocolchicine 1, as well as colchicine 2, its supposed biogenetic
precursor, is an alkaloid isolated from Colchicum autumnale
L. (Fig. 1).1–3 The tubulin polymerization inhibitory activity these
two natural compounds exert confers on them a disturbing effect
towards the microtubule-dependent cell functions and an antimi-
totic potentiality. These activities show also that the tropolone ring
of colchicine is not absolutely necessary to the antitubulin effect
and can be replaced by a benzene ring.4 This discovery has led to
numerous works aiming at synthesizing various allocolchicinoids,
among which N-acetylcolchinol (NAC, 3) and its methylether
(NCME or NSC51046, 4) which bind to the colchicine binding site
of tubulin. These inhibitors have gained considerable attention
by virtue of having been found to be active against a number of
cancer cell lines and more interestingly against some multi-drug
resistant lines expressing P-glycoprotein.5–8 NAC further exhibits
a potent antivascular effect resulting in disruption of the endothe-
lial cells of tumor blood vessels, tumor ischemia and necrosis.9 For
this reason, the dihydrogenphosphate and prodrug of NAC
(ZD6126, 5) has reached the Phase II clinical trials in USA for the
treatment of metastatic renal cell carcinoma, before these trails
have been arrested due to a cardiac toxicity.
For a long time, allocolchicinoids were obtained by transforma-
tion of colchicine itself,10–13 thus limiting the range of structural
variations. Recently however, syntheses of novel allocolchicinoids
were reported and some SAR studies have been realized.5,14–19
The latter have noticeably established that, contrarily to the two
benzene rings, the seven-membered central one does not seem
to take part productively in the binding to tubulin but lies in an
entropic contribution by suppressing free rotation around the pivot
bond between the least square planes of the benzene rings.20
The antitubulin effect of the allocolchicinoids appearing thus
correlated with the torsional angle between these rings, structural
modifications affecting the cycloheptene ring of novel allocolchici-
noids are requested to enhance the SAR knowledge and to poten-
tially select novel antitubulin agents with therapeutic interest.
Within this context, we choose to synthesize 7-oxo 6 and
7-hydroxy 7 trifluoroallocolchicinoids in order to evaluate for the
first time the incidence on the biological activity of a sp2 configu-
ration conferred to the carbon C7 of the dibenzocycloheptene sys-
tem relatively to a sp3 configuration. The synthesis of these
compounds is herein reported through the intramolecular cycliza-
tion of o-phenyl-b-phenylalanines 8 together with an evaluation of
⇑
Corresponding author.
E-mail address: patrick.dallemagne@unicaen.fr (P. Dallemagne).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.043

E. Chosson et al. / Bioorg. Med. Chem. 20 (2012) 2614–2623
2615
O
O
O
O
O
O
O
H
O
H
O
H
O
MeO2C
RO
O
N
N
N
O
O
O
(-)-allocolchicine (1)
(-)-colchicine (2)
NAC (R = H), (3)
NSC51046 or NCME (R = Me),(4)
ZD6126 (R = P(O)(OH)2), (5)
Figure 1. Structure of (ꢀ)-allocolchicine (1), (ꢀ)-colchicine (2) and some allocolchicinoids (3–5).
their abilities to inhibit assembly of tubulin and the growth of KB
cell lines (Fig. 2).
tion (Scheme 1, Method B). According to this novel sequence, the
overall yield with which the hydrochloric salt of 8a was obtained
starting from 10 was of 17%, the cinnamic acid 11 being concom-
itantly synthesized. This pathway was then applied to the cou-
pling of 10 with 2,3,4-trimethoxyphenylboronic acid and to the
coupling of its 3-methoxy 13 and 3-hydroxy derivatives 14 with
3,4-dimethoxy- and 2,3,4-trimethoxyphenyl-boronic acids under
microwave irradiation (Scheme 2). According to this sequence,
six biphenylcarboxaldehydes 12a–f and the corresponding b-ami-
noacids 8a–f were synthesized with yields ranging from 15% to
25%.
A mixture of TFA and TFA2O was then used to achieve the one-
pot intramolecular cyclization of 8a–f, according to the method we
have previously described for the cyclization of b-aminoacids in
activated aromatic series into various indanones, thia-, oxa- and
azaindanones (Scheme 3).27–29 Applied to 8a–f, the reaction led
to the expected N-(7-oxo-6,7-dihydro-5H-dibenzo [a,c][7]cyclo-
hepten-5-yl)-2,2,2-trifluoroacetamides 6a–f with yields ranging
from 50% to 60%. Only 8d led concomitantly to the formation of
the indanone derivative 15. Fortunately, the cyclization of 8a, 8c
and 8e never took place on the 20 position of the phenyl ring. This
one-pot method of cyclization, already used by Boyé starting from
biphenylpropionic acids,30 further prevents the O-demethylation
which usually occurs during Friedel–Crafts cyclisation of methoxy
derivatives using Lewis acids. It also led to compounds whose tri-
fluoracetylamino groups can exert some metabolic poisonous
properties and thus can enhance their cytotoxic effect.
1. Results and discussion
1.1. Chemistry
Despite their potential interest as useful building blocks in
medicinal chemistry,21 o-phenyl-b-phenylalanines 8 were at the
beginning of our work almost unknown. In fact, only a 20-carboxy-
phenyl derivative was described in literature and synthesized
through the ring opening of an oxodibenzaze-pinylacetic acid.22
We undertook the synthesis of these scaffolds firstly starting from
racemic 2-bromo-3-amino-3-phenylpropionic acid 9 which we
previously synthesized according to a Rodionov–Johnson proce-
dure23,24 applied to 2-bromobenzal-dehyde 10 (Scheme 1, Method
A).25 A subsequent Suzuki–Miyaura cross coupling was then
achieved between 9, previously N-protected as a trifluoracetamide,
and 3,4-dimethoxyphenyl-boronic acid, using Pd tetrakistriphenyl-
phosphine as catalyst in DME and an aqueous solution of NaHCO3
as a base. The sequence led to the N-deprotected o-(30,40-dime-
thoxyphenyl)-b-phenylalanine 8a isolated under its hydrochloric
salt which was precipitated at the end of the reaction by acidificat-
ion and saturation of the aqueous solution. The overall yield to ob-
tain 8a starting from 10 was of 6%. This poor yield was the
consequence of side products formation: 2-bromocinnamic acid
during the Rodionov–Johnson step and 2-(30,40-dimethoxy-
phenyl)cinnamic acid 11 during the Suzuki–Miyaura reaction,
respectively.
Finally, the reduction of the carbonyl group of 6a–f was
achieved using sodium borohydride in methanol (Scheme 3). The
reaction led to the alcohols (7a–f) with yields ranging from 50%
to 85%. The latter were formed as a mixture of their cis and trans
diastereoisomers among which only the trans dimethoxy deriva-
tive (7a) has been separated by fractional crystallizations. Its con-
figuration was established on the basis of 2D NMR data, completed
In order to enhance this yield, the sequence has been reversed,
starting with the cross coupling of 10 with 3,4-dimetho-
xyphenylboronic acid according to the method we very recently
described,26 followed by the involvement of the resulting un-
known biphenylcarboxaldehyde 12a in a Rodionov–Johnson reac-
R
R
R
OH
R
O
R
R
N H2
N
N
OH
H
H
CF3
CF3
O
O
O
6
7
8
Figure 2. Retrosynthetic access to 7-oxo (6) and 7-hydroxy (7) allocolchicinoids through intramolecular cyclization of o-phenyl-b-phenylalanines (8).

2616
E. Chosson et al. / Bioorg. Med. Chem. 20 (2012) 2614–2623
Br
Br
O
i
NH2
9
10
OH
ii
O
iii
Method A
iii
iv
O
O
O
O
O
O
O
Method B
vi
v
+
NH2
, HCl
OH
O
8a
11
12
OH
O
Scheme 1. Synthetic pathways for the synthesis of compounds 9, 11, 12a and 8a. Reagents: Method A, (i) CH2(CO2H)2, AcONH4, EtOH; (ii) (CF3CO)2O, Et2O; (iii) 3,4-
(MeO)2C6H3B(OH)2, Pd(PPh3)4, NaHCO3, DME, H2O; (iv) HCl, NaCl, H2O; Method B, (v) CH2(CO2H)2, AcONH4, EtOH; (vi) HCl, H2O.
O
O
R2
O
R2
O
Br
O
i
ii
R1
R1
R1
NH
iii
2 , HCl
O
OH
8a-f
10,13,14
12a-f
O
a
b
c
d
e
f
10 13
14
R1
R2
H
H
H
OMe OMe OH OH
OMe OMe
R1
H OMe OH
OMe
H
H
Scheme 2. Synthetic pathway for the synthesis of compounds 12a–f and 8a–f (Method B). Reagents: (i) 3,4-substituted C6H3B(OH)2, Pd(PPh3)4, K3PO4ꢁH2O, DME under
microwave irradiation (6 bars, 80–150 watts); (ii) CH2(CO2H)2, AcONH4, EtOH; (iii) HCl, H2O.
O
O
O
O
O
2
2
O
R
2
R
R
i
iii
O
O H
N H
ii
1
1
2 , HCl
1
R
R
R
O H
8a-f
8d
N
N
H
CF
H
3
CF
3
O
O
7a-f
O
6a-f
i
ii
O
O
O
O
O
a
b
c
d
e
f
H
1
H
H
OMe OMe OH
OMe
R
R
O H
N
O
OH
H
CF
3
2
OMe
H
H
OMe
N
O
O
H
CF
3
15
O
( Trans)-7a
Scheme 3. Synthetic pathway for the synthesis of compounds 6a–f, 7a–f and 15. Reagents: (i) TFA; (ii) TFA2O; (iii) NaBH4, MeOH.
by 1D NOESY experiments and was confirmed by X-ray crystallo-
graphic analysis (Fig. 3). The latter also showed a dihedral angle
of 49.2° between the two aromatic rings of 7a, in a similar manner
as for colchicine itself.
1.2. Biology
Compounds synthesized in this work were tested for
their in vitro cytotoxicity against KB cells at 10ꢀ5 and 10ꢀ6
M

E. Chosson et al. / Bioorg. Med. Chem. 20 (2012) 2614–2623
2617
interesting since 6d was prepared under its racemic mixture,
whereas NCME is an optically active compound.
Inhibition of the polymerization of tubulin appears partially
correlated with the cytotoxicity exerted towards KB and 6d, the
most cytotoxic derivative showed an IC50 against tubulin of
12
l
M. This inhibitory effect remains however lower compared
to NCME (IC50 = 0.7
l
M).8 On the other hand, the alcohol (7b), to-
tally devoid of cytotoxic activity, surprisingly inhibits tubulin in
a same order of magnitude than 6d.
Concerning the SAR, 7-keto compounds appear always more
cytototoxic comparatively to the corresponding 7-hydroxy com-
pounds. In a similar manner, keto compounds inhibit more
strongly tubulin than their hydrogenated analogs, except for the
alcohol 7b. On the other hand, the absence of a methoxy group
in C1 position (R2) appears detrimental for both cytotoxicity and
tubulin inhibition since 6a, 7a, 7c, 6e and 7e were devoid of activ-
Figure 3. View of crystal structure of ( Trans)-7a. Displacement ellipsoids are
drawn at the 50% probability level. H-atoms are shown as small spheres of arbitrary
radii.
ity (only 6c weakly inhibits tubulin with IC50 = 60 lM). This result
is consistent with the SAR established for colchicine and colchici-
noids, this methoxy group being important in setting the correct
conformation of the keto compounds.30 Finally, for compounds
with R2 = OMe, an additional OMe group in C9 position (R1) seems
favorable to the activities (compound 6d vs. 6b). It can be replaced
by a hydroxyl group but with a decrease of activity (compound 6f).
Interestingly, the indanone (15) bearing four methoxy groups
showed also a moderate cytotoxic effect against KB cells.
concentrations in triplicate, using docetaxel at 2.5 10ꢀ10 M as
reference. The results are expressed in term of inhibition percent-
age at each concentrations and IC50 were measured for percentage
greater than 80% at 10ꢀ5 M (Table 1). The inhibition of the poly-
merization of tubulin was also evaluated at 6.7 10ꢀ5 M in triplicate
using deoxypodophyllotoxin (Dppt) as reference. The results are
expressed in term of inhibition percentage and IC50 were measured
for percentage greater than 45% (Table 1).
2. Conclusion
The results show that among the thirteen tested compounds,
three exerted a cytotoxic activity towards KB cells with micromo-
lar IC50 (6b, 7d and 15) and two with submicromolar values (6d
and 6f). The most potent compound was the tetramethoxy one
(6d) with an IC50 of 45 nM in the same order of magnitude than
those of NCME (IC50 = 19 nM).8 This result appears particularly
We have during this work developed a synthesis of novel 7-keto
and 7-hydroxy trifluoroallocolchicinoids, some of which (com-
pounds 6d and 6f) showing a good cytotoxic activity against KB
cells, correlated with a partial tubulin inhibitory effect. This prob-
ably accounts for a multiple mechanism of action implying other
Table 1
Percentage of inhibition of KB cells growing at various concentrations and IC50 for deoxypodophyllotoxin (Dppt) and compounds 6a–f, 7a–f and 15
O
O
10
O
O
O
11
2
9
2
R
O
O
R
O
OH
H
1
7
1
R
R
N
O
3
CF
3
N
N
H
CF
3
H
CF
3
O
O
O
O
6a-f
7a-f
15
R1
R2
Compd
KB cells inhibition
Tubulin inhibition
Inh% at 10ꢀ5
M
Inh% at 10ꢀ6
M
IC50 SD (lM)
Inh% at 6.7 10ꢀ5
M
IC50 SD (lM)
H
H
6a
7a
6b
7b
6c
7c
6d
7d
6e
7e
6f
7f
15
Dppt
NCME
92
9
91
8
38
9
96
94
40
2
96
60
97
100
—
0
0
18
0
0
0
98
0
0
0
37
0
0
100
—
—
—
12
23
48
89
63
2
89
18
24
13
62
29
33
100
—
—
80
13
60
—
12
—
—
H
OMe
H
2.950 0.650
—
—
—
0.045 0.005
1.595 0.410
—
7
4
5
OMe
OMe
OH
OH
OMe
H
3
—
—
37
—
OMe
0.330 0.120
—
1.250 0.340
0.003 0.001
0.019 0.001a
2
—
—
—
—
—
—
—
3.2 0.2
0.7 0.1a
values for N-acetylcolchinol methylether (NCME) from.8
a

2618
E. Chosson et al. / Bioorg. Med. Chem. 20 (2012) 2614–2623
targets. Another compound (7b) exhibited also such an antitubulin
effect but without cytotoxicity. If this lack of activity is not due to
the incapacity of 7b to reach into intracellular sites, this compound
could also constitute an interesting hit to develop novel agents
able to inhibit more specifically this target. The extension of this
synthetic procedure to enantiomeric starting materials and also
to heterocyclic series is currently under investigation. It could lead
to novel original derivatives with enhanced properties.
3.2.2. 20,30,40-Trimethoxybiphenyl-2-carbaldehyde (12b)
Starting from 2-bromobenzaldehyde (1.7 mmol, 315 mg,
199 lL), 2,3,4-trimethoxyphenylboronic acid (2.5 mmol, 540 mg),
K3PO4ꢁH2O (5.1 mmol, 1.17 g) and Pd(PPh3)4 (0.085 mmol,
982 mg), 12b was obtained as a pale yellow powder (323 mg,
70%); LC–MS tR = 10.82 min, [ESI+] m/z [M+H]+ 273. Other spectral
data are in agreement with those found in the literature.18
3.2.3. 30,4,40-Trimethoxybiphenyl-2-carbaldehyde (12c)
Starting from 2-bromo-5-methoxybenzaldehyde (3 mmol,
645 mg), 3,4-dimethoxyphenylboronic acid (4.5 mmol, 819 mg),
K3PO4ꢁH2O (9 mmol, 1.91 g) and Pd(PPh3)4 (0.15 mmol, 1.73 g),
12c was obtained as pale yellow solid (635 mg, 77%); mp 104–
105c °C; 13C NMR (100 MHz, CDCl3) d 192.5 (CHO), 158.9, 148.9,
148.8, 138.9, 134.5, 132.0, 130.1, 122.9, 121.4, 113.1, 110.9,
109.7, 55.9 (2C, OCH3), 55.6 (OCH3); LC–MS tR = 6.72 min, [ESI+]
m/z [M+H]+ 273. Other spectral data are in agreement with those
found in the literature.31
3. Experimental section
3.1. Chemistry
Materials and methods: All commercial solvents and reagents
were used as-received. The microwave reactions were performed
using a Biotage Initiator Microwave oven; temperatures were mea-
sured with an IR-sensor and reaction times given as hold times.
Flash chromatography was realized on a spot two apparatus; col-
umn: SiO2; eluent: cyclohexane/ethyl acetate. Melting points were
determined on a Kofler melting point apparatus and were uncor-
rected. IR spectra were recorded on KBr discs; only selected absor-
bances were quoted. TLC were carried out on 5 ꢂ 10 pre-coated
plates with silica gel GF254 type 60 using an ethyl acetate cyclo-
hexane mixture (1:1) as solvent; LC/MS (ESI) analyses were real-
ized with a separating module using the following gradient: A
(95%)/B (5%) to A (5%)/B (95%) in 5 min; this ratio was hold during
2 min before return to initial conditions in 1 min. Initial conditions
were then maintained for 2 min (A: H2O, B: CH3CN; each contain-
ing HCOOH: 0.1%; Column: C18, flow: 0.4 mL min-1). MS detection
was performed by positive or negative ESI. High resolution mass
spectra were performed at 40 eV by electronic impact (HREIMS)
or positive or negative electrospray (HRESIMS). 1H and 13C NMR
spectra were recorded, respectively, at 400 and 100 MHz using
CDCl3, DMSO-d6, CD3OD or (CD3)2CO as solvents. 2D NMR spectra
and 1D NOESY experiments were recorded at 500 MHz. The appar-
ent multiplicity is described as s (singlet), d (doublet), dd (doublet
of doublets), t (triplet), m (multiplet); chemical shifts d are re-
ported in parts per million with the solvent resonance as the inter-
nal standard; coupling constants J are given in Hertz.
3.2.4. 20,30,4,40-Tetramethoxybiphenyl-2-carbaldehyde (12d)
Starting from 2-bromo-5-methoxybenzaldehyde (1.7 mmol,
365 mg), 2,3,4-trimethoxyphenylboronic acid (2.5 mmol, 540
mg), K3PO4ꢁH2O (5.1 mmol, 1.17 g) and Pd(PPh3)4 (0.085 mmol,
982 mg), 12d was obtained as a pale yellow solid (443 mg, 86%);
LC–MS tR = 6.93 min, [ESI+] m/z [M+H]+ 303. Other spectral data
are in agreement with those found in the literature.18
3.2.5. 4-Hydroxy-30,40-dimethoxybiphenyl-2-carbaldehyde (12e)
Starting from 2-bromo-5-hydroxybenzaldehyde (3 mmol,
603 mg), 3,4-dimethoxyphenylboronic acid (4.5 mmol, 819 mg),
K3PO4ꢁH2O (9 mmol, 1.91 g) and Pd(PPh3)4 (0.15 mmol, 1.73 g),
12e was obtained as a pale yellow solid (561 mg, 72%); mp 185–
186 °C; IR (KBr) k (cmꢀ1) 3424 (OH), 1679 (CO), 1605, 1498,
1253; 1H NMR (400 MHz, CDCl3) d 9.94 (s, 1H, CHO), 7.44 (d, 1H,
Jmeta = 2.9 Hz), 7.36 (d, 1H, Jortho = 8.8 Hz), 7.15 (dd, 1H, Jortho
=
8.8 Hz, Jmeta = 2.9 Hz), 6.95 (d, 1H, Jortho = 8.8 Hz), 6.87 (m, 2H),
5.21 (s, 1H, OH), 3.95 (s, 3H, OCH3), 3.92 (s, 3H, OCH3); 13C NMR
(100 MHz, CDCl3) d 192.5 (CHO), 155.1, 148.9, 148.7, 138.9,
134.7, 132.3, 130.0, 122.9, 121.2, 113.1, 112.8, 110.9, 56.0 (2C,
OCH3); LC–MS tR = 5.92 min, [ESIꢀ] m/z [MꢀH]ꢀ 257; HREIMS
[M+] m/z 258.08921 (calcd for C15H14O4 258.08919).
3.2. General procedure for the synthesis of biphenylcarbalde-
hydes (12a–f)
3.2.6. 4-Hydroxy-20,30,40-trimethoxybiphenyl-2-carbaldehyde
(12f)
To degassed solutions of 1 equiv of substituted 2-bromo-benz-
aldehydes 10, 13 or 14 in DME were added 1.5 equiv of boronic
species, 3 equiv of K3PO4ꢁH2O and 0.05 equiv of Pd(PPh3)4; the sus-
pensions were heated under microwave irradiation (6 bars, 80–
150 watts) for 1 h 30 min at 150 °C. The resulting mixtures were
filtered (washed with Et2O or EtOAc) and concentrated under re-
duced pressure. The crude products were then purified by flash
chromatography.
Starting from 2-bromo-5-hydroxy-benzaldehyde (1.5 mmol,
301.5 mg), 2,3,4-trimethoxyphenylboronic acid (2.25 mmol,
477 mg), K3PO4ꢁH2O (4.5 mmol, 1.04 g) and Pd(PPh3)4
(0.075 mmol, 866 mg), 12f was obtained as pale yellow solid
(296 mg, 68%); mp 130–131 °C; IR (KBr) k (cmꢀ1) 3454 (OH),
1680 (CO), 1602, 1486, 1286; 1H NMR (400 MHz, CDCl3) d 9.78
(s, 1H, CHO), 7.56 (d, 1H, Jmeta = 2.9 Hz, H3), 7.28 (d, 1H,
Jortho = 8.8 Hz, H6), 7.18 (dd, 1H, Jortho = 8.8Hz, Jmeta = 2.9 Hz, H5),
6.97 (d, 1H, Jortho = 7.8 Hz), 6.78 (d, 1H, Jortho = 7.8 Hz), 3.93 (s, 6H,
OCH3), 3.53 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3) d 193.2
(CHO), 155.8, 153.7, 151.1, 141.9, 134.4, 133.8, 132.7, 125.7,
124.3, 121.6, 112.7, 107.5, 61.1 (OCH3), 60.6 (OCH3), 56.0 (OCH3);
LC–MS tR = 6.04 min, [ESIꢀ] m/z [MꢀH]ꢀ 287; HREIMS [M+] m/z
288.09936 (calcd for C16H16O5 288.09975).
3.2.1. 30,40-Dimethoxybiphenyl-2-carbaldehyde (12a)
Starting from 2-bromobenzaldehyde (1 mmol, 185 mg, 117 lL),
3,4-dimethoxyphenyl boronic acid (1.5 mmol, 273 mg), K3PO4ꢁH2O
(3 mmol, 637 mg) and Pd(PPh3)4 (0.05 mmol, 577 mg), 12a was ob-
tained as a colorless oil (173 mg, 71%); IR (KBr) k (cmꢀ1) 1688 (CO),
1598, 1517, 1247, 1025; 1H NMR (400 MHz, CDCl3) d 10.00 (s, 1H,
CHO), 8.01 (d, 1H Jortho = 7.8 Hz), 7.63 (dd, 1H, Jortho = 7.8, Jmeta
=
3.2.7. 3-Amino-3-(2-bromophenyl)propanoic acid (9)
1.9 Hz), 7.47 (m, 2H), 6.94 (d, 1H, Jortho = 8.8 Hz), 6.91 (m, 2H),
3.95 (s, 3H, OCH3), 3.91 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3)
d 192.7 (CHO), 149.3, 148.8, 146.2, 145.7, 133.9, 133.5, 130.7,
127.7, 127.6, 122.9, 113.2, 110.9, 55.9 (2C, OCH3); LC–MS
tR = 10.63 min, [ESI+] m/z [M+H]+ 243, [M+HꢀH2O]+ 225; HREIMS
[M+] m/z 242.093876 (calcd for C15H14O3 242.094294).
2-Bromo-benzaldehyde 10 (50 mmol, 9.25 g) was refluxed in
EtOH (150 mL) with 1 equiv of malonic acid (50 mmol, 5.20 g)
and 2 equiv of ammonium acetate (100 mmol, 7.70 g) for 6 h; the
resulting precipitate was triturated in distilled water to separate
the expected amino acid 9 from the corresponding diammonium
2-bromobenzylidene-propanedioate that was concomitantly

E. Chosson et al. / Bioorg. Med. Chem. 20 (2012) 2614–2623
2619
formed and which remained in water. Compound 9 was obtained
as a colorless solid (3.5 g, 29%) which was used without further
purification; mp 200–201 °C; IR (KBr) k (cmꢀ1) 2924, 2360, 1686
(CO), 1621, 1465, 1284, 1223, 1023. Other spectral data are in
agreement with those found previously by us.25
tion, the aqueous layer was concentrated under reduced pressure;
addition of acetone to the residue gave 8a as a colorless precipitate
(165 mg, 24%) which was used without further purification.
3.2.9. 3-Amino-3-(20,30,40-trimethoxybiphenyl-2-yl)propanoic
acid hydrochloride (8b)
The latter was obtained according to the method B used for 8a
starting from 12b (2.28 mmol, 620 mg). Although the same work
up was applied, 8b could not be isolated from the starting material
and was engaged under this form in the cyclisation step.
3.2.8. 3-Amino-3-(30,40-dimethoxybiphenyl-2-yl)propanoic acid
hydrochloride (8a) and 3-(30,40-di-methoxybiphenyl-2-yl)prop-
2-enoic acid (11)
Method A; TFA2O (100 mmol, 14 mL) was added dropwise to a
suspension of 2-bromo-3-amino-3-phenylpropanoic acid
9
(10 mmol, 2.44 g) in Et2O (30 mL). The reaction mixture was stirred
for 1 h and then the solvent was concentrated under reduced pres-
sure. The residue was triturated with distilled water to afford after
filtration the expected 3-(2-bromophenyl)-3-trifluoro-acetylami-
nopropanoic acid which was obtained as a colorless solid (3.3 g,
3.2.10. 3-Amino-3-(30,4,40-trimethoxybiphenyl-2-yl)propanoic
acid hydrochloride (8c)
The latter was obtained according to the method B used for 8a,
starting from 12c (2.3 mmol, 630 mg). At the end of the run time,
distilled water was added to the reaction mixture and EtOH was
removed under reduced pressure; the aqueous layer was then
acidified with HCl 12 M and stirred until a bright yellow precipitate
was obtained (cinnamic acid); after filtration, the aqueous layer
was concentrated under reduced pressure; addition of acetone to
the residue gave 8c as a colorless precipitate (196 mg, 26%) which
was used without further purification; mp 232–233 °C; IR (KBr) k
(cmꢀ1) 3033 (OH), 1716 (CO), 1615, 1497,1408, 1245, 1174,
1016; 1H NMR (400 MHz, CD3OD) d 7.26 (d, 1H, Jortho = 8.8 Hz),
7.18 (d, 1H, Jmeta = 1.9 Hz), 7.02 (m, 2H), 6.93 (d, 1H, Jmeta = 1.9 Hz),
6.86 (dd, 1H, Jortho = 7.8 Hz, Jmeta = 1.9 Hz), 4.79 (m, 1H, H3), 3.87 (s,
3H, OCH3), 3.86 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 2.99 (m, 2H,
H2a,b); 13C NMR (100 MHz, CD3OD) d 172.7 (CO2H), 161.2, 150.3,
150.1, 136.3, 135.5, 133.6, 133.4, 123.2, 115.6, 114.5, 113.0,
112.3, 56.5 (2C, OCH3), 56.1 (OCH3), 49.0 (under solvent residual
peaks), 39.4; LC–MS tR = 4.36 min, [ESIꢀ] m/z [MꢀH]ꢀ 330; HREIMS
[M+] m/z 331.14131(calcd for C18H21NO5 331.14194).
quant.) and used without further purification; IR (KBr) k (cmꢀ1
)
3300, 2360, 1706 (CO), 1559, 1261-1185-1156 (CF3), 1023. Other
spectral data are in agreement with those found in the literature.25
To a degassed solution of the latest N-protected amino acid
(9.4 mmol, 3.2 g) in DME (100 mL) were added 1.1 equiv of (3,4-
dimethoxyphenyl)boronic acid (10 mmol, 1.88 g), 4 equiv of NaH-
CO3 (37.6 mmol, 3.16 g) in H2O (50 mL) and 0.05 equiv of Pd(PPh3)4
(0.47 mmol, 0.54 g); the suspension was refluxed for 48 h under
nitrogen, filtered after cooling and evaporated to dryness in vac-
uum. The crude residue was then dissolved in water and washed
with CHCl3; the resulting aqueous layer was acidified with a solu-
tion of HCl 6 M until pH 1 and extracted with CHCl3; after drying
over MgSO4 and filtration, the solvent was removed in vacuum
and 2-(30,40-dimethoxybiphenyl) cinnamic acid (11) crystallized
in the round flask as a colorless solid (10 mg <1%). Mp 166–
167 °C; IR (KBr) k (cmꢀ1) 3430, 1693 (CO), 1622, 1521, 1325,
1252, 1027; 1H NMR (400 MHz, CDCl3) d 7.87 (d, 1H, 3J = 16.6 Hz,
Hethylen.), 7.70 (d, 1H, Jortho = 7.8 Hz), 7.42 (m, 3H), 6.94 (d, 1H,
Jortho = 8.8 Hz), 6.86 (m, 2H), 6.40 (d, 1H, 3J = 16.6 Hz, Hethylen),
3.94 (s, 3H, OCH3), 3.88 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3)
d 172.0 (CO2H), 148.6 (2C), 146.2, 142.9, 132.3, 132.2, 130.4,
130.2, 127.4, 127.0, 122.3, 118.1, 113.1, 110.9, 55.9 (2C); EIMS m/
z 284 [M+], 239 [MꢀCO2H]+., HREIMS [M+] m/z 284.10542 (calcd
for C17H16O4 284.10484).
The remaining aqueous layer was saturated with NaCl and stir-
red for 3 h at room temperature to give crystals of the expected
deprotected biphenyl amino acid 8a. The latter was obtained as
colorless crystals which was used without further purification
(600 mg, 19%); mp 242–243 °C; IR (KBr) k (cmꢀ1) 3140, 2911,
1730 (CO), 1522, 1498, 1247, 1221, 1141, 1109, 1020; 1H NMR
(400 MHz, DMSO-d6)⁄ d 8.75 (s, 2H, NH2), 7.88 (d, 1H, Jortho = 7.5 Hz,
H3), 7.47 (m, 1H, H4), 7.42 (m, 1H, H5), 7.27 (dd, 1H, Jortho = 8.0 Hz,
Jmeta = 1.0 Hz, H6), 7.04 (d, 1H, Jortho = 8.5 Hz, H50), 7.02 (d, 1H,
Jmeta = 1.5 Hz, H20), 6.91 (dd, 1H, Jortho = 8.5 Hz, Jmeta = 1.5 Hz, H60),
4.60 (m, 1H, H3), 3.81 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.08 (dd,
1H, 2J = 16 Hz, 3J = 7.5 Hz, H2a), 2.94 (dd, 1H, 2J = 16 Hz,
3J = 7.5 Hz, H2b); 13C NMR (100 MHz, DMSO-d6)⁄ d 171.0 (C1),
148.7 (C30), 148.5 (C40), 142.3 (C1), 135.0 (C2), 132.7 (C10), 130.9
(C6), 128.9 (C5), 128.3 (C4), 127.3 (C3), 121.9 (C60), 113.6 (C20),
112.0 (C50), 56.0 (2C, OCH3), 47.6 (C3), 39.2 (C2); LC–MS
tR = 6.17 min, [ESI+] m/z [M+H]+ 302; HRESIMS [MꢀH]ꢀ m/z
300.1234 (calcd for C17H18NO4 300.1236).
3.2.11. 3-Amino-3-(20,30,4,40-tetramethoxybiphenyl-2-
yl)propanoic acid hydrochloride (8d)
The latter was obtained according to the method B used for 8a,
starting from 12d (2.07 mmol, 625 mg). At the end of the run time,
EtOH was evaporated until 2 mL left; distilled water was added until
a yellow precipitate was obtained (cinnamic acid); after filtration,
EtOH was removed in vacuum and the aqueous layer was acidified
until pH 1; the resulting layer was washed three times with Et2O
to remove the corresponding benzylidene malonic acid; the aqueous
layer was concentrated under reduced pressure; addition of acetone
to the residue gave 8d as a colorless precipitate (191 mg, 25%) which
was used without further purification; mp 191–192 °C; IR (KBr) k
(cmꢀ1) 3154, 1724 (CO), 1608, 1489,1402, 1294, 1240, 1079; 1H
NMR (400 MHz, CD3OD)⁄ d 7.26 (d, 1H, Jortho = 8.8 Hz, H6), 7.20 (d,
1H, Jmeta = 1.9 Hz, H3), 7.05 (dd, 1H, Jortho = 8.8 Hz, Jmeta = 1.9 Hz,
H5), 6.98 (d, 1H, Jortho = 8.8 Hz, H60), 6.91 (m, 1H, H50), 4.50 (dd,
1H, 3J = 8.8 Hz, 3J = 5.8 Hz, H3), 3.89 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 3.83 (s, 3H, OCH3), 3.59 (s, 3H, OCH3), 2.91 (dd, 1H,
2J = 17.5 Hz, 3J = 8.8 Hz, H2a), 2.75 (dd, 1H, 2J = 17.6 Hz, 3J = 5.9 Hz,
H2b); 13C NMR (100 MHz, CD3OD)⁄ d 173.7 (C1), 161.4 (C4), 155.2
(C40), 152.0 (C30), 143.5 (C20), 137.7 (C2), 133.5 (C6), 131.2 (C1),
127.4 (C10), 127.1 (C60), 115.6 (C5), 112.3 (C3), 109.8 (C50), 61.8
(OCH3), 61.5 (OCH3), 56.6 (OCH3), 56.1 (OCH3), 50.4 (C3), 39.2
(C2); LC–MS tR = 4.57 min, [ESIꢀ] m/z 360 [MꢀH]ꢀ; HRESIMS
[M+H]+ m/z 362.1596 (calcd for C19H24NO6 362.1604).
⁄d assignments according to 2D experiments (COSY, HMBC).
Method B; a solution of 30,40-dimethoxybiphenyl-2-carbalde-
hyde (12a) (2 mmol, 483 mg), 1 equiv of malonic acid (2 mmol,
208 mg) and 2 equiv of ammonium acetate (4 mmol, 308 mg)
was refluxed in EtOH (20 mL) for 20 h. Water (10 mL) was then
added to the reaction mixture and EtOH was removed under re-
duced pressure; the aqueous layer was then acidified with HCl
12 M and stirred until 11 was precipitated (10 mg <1%); after filtra-
⁄d assignments according to 2D experiments (COSY, HMBC).
3.2.12. 3-Amino-3-(4-hydroxy-30,40-dimethoxybiphenyl-2-yl)
propanoic acid hydrochloride (8e)
The latter was obtained according to the method B used for 8a,
starting from 12e (2.13 mmol, 550 mg). At the end of the run time,
EtOH was evaporated until 2 mL left; distilled water was added
until a yellow precipitate was obtained (cinnamic acid); after

2620
E. Chosson et al. / Bioorg. Med. Chem. 20 (2012) 2614–2623
filtration, EtOH was removed in vacuum and the aqueous layer was
acidified until pH 1; the resulting phase was washed three times
with Et2O to remove the corresponding benzylidene malonic acid;
the aqueous layer was evaporated to dryness under reduced pres-
sure; addition of acetone to the residue gave 8e as a colorless solid
(75 mg, 10%) which was used without further purification; mp
240–241 °C; IR (KBr) k (cmꢀ1) 3044 (OH), 1734 (CO), 1496, 1403,
1244; 1H NMR (400 MHz, CD3OD) d 7.15 (d, 1H, Jortho = 8.8 Hz),
7.06 (d, 1H, Jmeta = 2.9 Hz), 7.02 (d, 1H, Jortho = 7.8 Hz), 6.93 (d, 1H,
Jmeta = 1.9 Hz), 6.87 (m, 2H), 4.75 (dd, 1H, J = 5.8 Hz, H3), 3.87 (s,
3H, OCH3), 3.84 (s, 3H, OCH3), 3,02 (dd, 1H, 2J = 17.6 Hz,
3J = 7.8 Hz, H2a), 2.94 (dd, 1H, 2J = 17.6 Hz 3J = 5.8 Hz, H2b); 13C
NMR (100 MHz, CD3OD) d 172.5 (CO2H), 157.0, 148.3, 147.7,
139.1, 132.8, 131.4, 131.2, 121.3, 114.7, 113.2, 112.8, 111.6, 55.5
(2C, OCH3), 48.7, 41.1; LC–MS tR = 4.10 min, [ESIꢀ] m/z [MꢀH]ꢀ
316; HREIMS [M+] m/z 317.12591 (calcd for C17H19NO5
317.12629).
(cmꢀ1) 2928, 1713–1682 (CO), 1586, 1336,1163, 1113; 1H NMR
(400 MHz, (CD3)2CO) d 9.26 (br s, 1H, NH), 7.47 (m, 4H, H1-4),
6.90 (s, 1H, H8), 5.56 (m, 1H, H5), 3.94 (s, 6H, OCH3), 3.58 (s, 3H,
OCH3), 3.31 (dd, 1H, 2J = 17.6 Hz, 3J = 3.9 Hz, H6a), 3.04 (dd, 1H,
2J = 17.6 Hz, 3J = 12.8 Hz, H6b); 13C NMR (100 MHz, (CD3)2CO) d
201.4 (C7), 159.6 (q, 2J = 36 Hz, NHCOCF3), 154.5, 151.9, 146.7,
139.1, 136.1, 132.7, 132.6, 128.9, 128.8, 128.1, 122.8, 116.2 (q,
1J = 286 Hz, CF3), 107.4, 61.6 (OCH3), 61.2 (OCH3), 56.4 (OCH3),
53.2 (C6), 48.6 (C5); LC–MS tR = 6.68 min, [ESIꢀ] m/z [MꢀH]ꢀ
408; HREIMS [M+] m/z 409.11491 (calcd for C20H18F3NO5
409.11367).
3.3.3. N-(3,9,10-Trimethoxy-7-oxo-6,7-dihydro-5H-
dibenzo[a,c][7] cyclohepten-5-yl)-2,2,2-trifluoroacetamide (6c)
Starting from 8c (0.46 mmol, 169 mg), 6c was obtained as a pale
yellow solid (100 mg, 53%); mp 178–179 °C; IR (KBr) k (cmꢀ1
)
3315, 2928, 2850, 1722 (CO), 1667 (CO), 1598, 1498, 1273, 1245-
1210-1166 (CF3), 1041; 1H NMR (400 MHz, (CD3)2CO) d 9.22 (br
d, 1H, 3J = 6.8 Hz, NH), 7.48 (d, 1H, Jortho = 8.8 Hz, H1), 7.17 (s,
1H), 7.04 (s, 1H), 6.99 (dd, 1H, Jortho = 8.8 Hz, Jmeta = 2.9 Hz, H2),
6.94 (d, 1H, Jmeta = 2.9 Hz, H4), 5.55 (m, 1H, H5), 3.98 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.25 (dd, 1H,
2J = 18.5 Hz, 3J = 2.9 Hz, H6a), 3.09 (dd, 1H, 2J = 18.5 Hz,
3J = 11.9 Hz, H6b); 13C NMR (100 MHz, (CD3)2CO) d 200.3 (C7),
160 (C3), 153.9, 153.7 (q, 2J = 36 Hz, NHCOCF3), 149.7, 140.5,
132.8, 132.5, 131.2, 129.6, 115.6 (q, 1J = 288 Hz, CF3), 113.7,
113.1, 112.1, 109.4, 56.2 (OCH3), 56.1 (OCH3), 55.6 (OCH3), 52.6
(C6), 48.7 (C5); LC–MS tR = 6.49 min, [ESIꢀ] m/z [MꢀH]ꢀ 408; HRE-
IMS [M+] m/z 409.11502 (calcd for C20H18F3NO5 409.11367).
3.2.13. 3-Amino-3-(4-hydroxy-20,30,40-trimethoxybiphenyl-2-
yl)pro-panoic acid (8f)
The latter was obtained according to the method B used for 8a
starting from 12f, (1.96 mmol, 567 mg). At the end of the run time,
8f has precipitated in the medium and was obtained after filtration
as a colorless powder (160 mg, 23%) which was used without fur-
ther purification; mp >260 °C; IR (KBr) k (cmꢀ1) 3237 (OH), 1615
(CO), 1595, 1487,1410, 1292, 1094; 1H NMR (400 MHz, DMSO-d6,
90 °C) d 6.99 (d, 1H, Jmeta = 2.9 Hz, H3), 6.92 (d, 1H, Jortho = 7.8 Hz),
6.80 (m, 2H), 6.70 (dd, 1H, Jortho = 7.8 Hz, Jmeta = 2.9 Hz, H5), 3.99
(m, 1H, CH), 3.83 (s, 6H, OCH3), 3.78 (s, 3H, OCH3), 2.29 (m, CH2,
H2); 13C NMR (100 MHz, DMSO-d6) d 172.9 (CO2H), 157.0, 152.8,
151.1, 141.7, 140.2, 131.4, 127.4, 126.5, 125.4, 114.5, 112.7,
108.0, 60.6 (OCH3), 60.4 (OCH3), 55.8 (OCH3), 49.2, 40.1; LC–MS
tR = 4.24 min, [ESIꢀ] m/z [MꢀH]ꢀ 346; HRESIMS [M+H]+ m/z
348.1435 (calcd for C18H22NO6 348.1447).
3.3.4. N-(3,9,10,11-Tetramethoxy-7-oxo-6,7-dihydro-5H-
dibenzo [a,c][7]cyclohepten-5-yl)-2,2,2-trifluoroacetamide (6d)
Starting from 8d (0.25 mmol, 90 mg), 6d was obtained as a col-
orless solid (41 mg, 37%) which was separated from 15 using flash
chromatography; mp 90–91 °C; IR (KBr) k (cmꢀ1) 3430, 2925, 2849,
1727 (CO), 1679 (CO), 1613, 1579, 1485, 1336, 1210-1163-1111
3.3. General procedure for the synthesis of dibenzocyclohepten-
ones (6a–f) and phenylindanone (15)
(CF3), 1088; 1H NMR (400 MHz, (CD3)2CO)
d
9.25 (d,
1H,3J = 6.8 Hz, NH), 7.41 (d, 1H, Jortho = 8.8 Hz, H1), 6.95 (m, 2H,
H2&H4), 6.88 (s, 1H, H8), 5.56 (m, 1H, H5), 3.91 (s, 6H, OCH3),
3.82 (s, 3H, OCH3), 3.56 (s, 3H, OCH3), 3.28 (dd, 1H, 2J = 17.5 Hz,
3J = 3.9 Hz, H6a), 3.02 (dd, 1H, 2J = 17.5 Hz, 3J = 13.6 Hz, H6b); 13C
NMR (100 MHz, (CD3)2CO) d 201.5 (C7), 160.4 (C3), 157.1 (q,
2J = 36 Hz, HNCOCF3), 154.1, 151.8, 146.8, 140.7, 135.9, 134.1,
124.7, 124.5, 117 (q, 1J = 285 Hz, CF3), 113.2, 108.7, 107.6, 61.4
(OCH3), 61.2 (OCH3), 56.4 (OCH3), 55.5 (OCH3), 53.2 (C6), 48.5
(C5); LC–MS tR = 6.40 min, [ESIꢀ] m/z [MꢀH]ꢀ 438; HREIMS [M+]
m/z 439.12413 (calcd for C21H20F3NO6 439.12423).
A solution of b-amino acid 8a–f in TFA was stirred at room tem-
perature for 1 h; then, 25 equiv of TFA2O were added and the mix-
ture was refluxed for 3 h. The reaction mixture was evaporated to
dryness under reduced pressure and water was added; the result-
ing precipitate was filtered and purified by flash chromatography.
3.3.1. N-(9,10-Dimethoxy-7-oxo-6,7-dihydro-5H-dibenzo[a,c-[7]
cyclohepten-5-yl)-2,2,2-trifluoroacetamide (6a)
Starting from 8a (1.8 mmol, 600 mg), 6a was obtained as a col-
orless powder (410 mg, 61%); mp 205–206 °C; IR (KBr) k (cmꢀ1
)
3335, 2962, 1725 (CO),1702 (CO), 1518, 1359, 1273, 1215-1183-
1167 (CF3), 1026; 1H NMR (400 MHz, CDCl3)⁄ d 7.45 (m, 2H, H1,
H2), 7.40 (m, 1H, H3), 7.30 (d, 1H, Jortho = 7.8 Hz, H4), 7.28 (s, 1H,
H8), 6.95 (s, 1H, H11), 6.73 (d, 1H, 3J = 4.4 Hz, NH), 5.46 (m, 1H,
H5), 4.01 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 3.22 (m, 2H, H6a,b);
13C NMR (100 MHz, CDCl3)⁄ d 199.1 (C7), 156.4 (q, 2J = 38 Hz,
NHCOCF3), 153.1 (C10), 149.2 (C9), 137.0 (C11b), 136.5 (C4),
132.0 (C11a), 131.1 (C1), 130.4 (C7a), 129.2 (C2&C4a), 128.6 (C3),
115.4 (q, 1J = 288 Hz, CF3), 112.6 (C11), 111.4 (C8), 56.3 (OCH3),
56.2 (OCH3), 51.9 (C6), 49.1 (C5); LC–MS tR = 9.18 min, [ESIꢀ] m/z
[MꢀH]ꢀ 378; HRESIMS [MꢀH]ꢀ m/z 378.0938 (calcd for
C19H15F3NO4 378.0953).
3.3.5. N-[4-Methoxy-3-oxo-7-(2,3,4-trimethoxyphenyl)-2,3-
dihydro-1H-inden-1-yl]-2,2,2-trifluoroacetamide (15)
Starting from 8d (90 mg, 0.25 mmol), 15 was obtained as a col-
orless solid (27 mg, 24%) which was separated from 6d using flash
chroma-tography; mp 210–211 °C; IR (KBr) k (cmꢀ1) 3295, 2940,
1724 (CO), 1702 (CO), 1486, 1462, 1204-1178-1100 (CF3), 1090;
1H NMR (400 MHz, CDCl3) d 7.48 (d, 1H, Jortho = 8.3 Hz), 7.00 (d,
1H, Jortho = 8.3 Hz), 6.83 (d, 1H, Jortho = 8.3 Hz), 6.72 (d, 1H,
Jortho = 8.3 Hz), 5.52 (br s, 1H, NH), 4.05 (m, 1H, H1), 3.99 (s, 3H,
OCH3), 3.89 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.66 (s, 3H, OCH3),
3.19 (dd, 1H, 2J = 18.5 Hz, 3J = 7.8 Hz, H2a), 2.52 (dd, 1H,
2J = 18.5 Hz, 3J = 3.5 Hz, H2b);13C NMR (100 MHz, CDCl3) d 200.4
⁄d assignments according to 2D experiments (COSY, HMBC).
2
(C3), 156.4 (q, JCF = 37 Hz,NHCOCF3), 157.2, 154.1, 151.4, 150.4,
1
142.4, 138.7, 128.9, 124.8, 124.3, 124.2, 115.3 (q, JCF = 286 Hz,
3.3.2. N-(9,10,11-Trimethoxy-7-oxo-6,7-dihydro-5H-
CF3), 111.6, 107.9, 61.2 (OCH3), 60.8 (OCH3), 56.1 (OCH3), 56.0
(OCH3), 47.0, 44.7; LC–MS tR = 6.07 min, [ESIꢀ] m/z [MꢀH]ꢀ 438;
HREIMS m/z [M+] 439.12638 (calcd for C21H20F3NO6 439.12423).
dibenzo[a,c][7] cyclohepten-5-yl)-2,2,2-trifluoroacetamide (6b)
Starting from crude 8b, 6b was obtained as a colorless solid
(103 mg, 11% overall yield from 12b); mp 82–83 °C; IR (KBr) k

E. Chosson et al. / Bioorg. Med. Chem. 20 (2012) 2614–2623
2621
3.3.6. N-(3-Hydroxy-9,10-dimethoxy-7-oxo-6,7-dihydro-5H-
dibenzo[a,c][7]cyclohepten-5-yl)-2,2,2-trifluoroacetamide (6e)
Starting from 8e (0.25 mmol, 80 mg), 6e was obtained as a col-
orless solid (50 mg, 50%); mp 226–227 °C; IR (KBr) k (cmꢀ1) 2924,
1727–1706 (CO), 1641, 1618, 1496, 1360, 1275, 1210, 1169; 1H
NMR (400 MHz, (CD3)2CO)⁄ d 9.22 (d, 1H, 3J = 7.5 Hz, NH), 8.83 (s,
1H, OH), 7.39 (d, 1H, Jortho = 8.8 Hz, H1), 7.18 (s, 1H, H8), 7.02 (s,
1H, H11), 6.90 (m, 2H, H2&H4), 5.50 (m, 1H, H5), 3.99 (s, 3H,
OCH3), 3.89 (s, 3H, OCH3), 3.25 (dd, 1H, 2J = 18.6 Hz, 3J = 11.7 Hz,
H6a), 3.10 (dd, 1H, 2J = 18.6 Hz, 3J = 2.9 Hz, H6b); 13C NMR
with a shaped pulse to observe dipolar coupling due to NOE with
the irradiated peaks.
3.4.2. N-(7-Hydroxy-9,10,11-trimethoxy-6,7-dihydro-5H-
dibenzo [a,c][7]-cyclohepten-5-yl)-2,2,2-trifluoroacetamide
(7b)
Starting from 6b (0.067 mmol, 28 mg), 7b was obtained as a col-
orless solid (16 mg, 58%); mp 98–99 °C; IR (KBr) k (cmꢀ1) 2923,
2849, 1712 (CO), 1599, 1485, 1404, 1329, 1215-1191-1145 (CF3),
1077, 1004; 1H NMR (400 MHz, (CD3)2CO) d⁄ 9.04 (brs, 1H, NH),
7.41 (m, 4H, H1-4), 7.18 (s, 1H, H8), 4.79 (m, 1H, H5), 4.57 (d,
1H, 3J = 4.9 Hz, OH), 4.32 (m, 1H, H7), 3.90 (s, 3H, OCH3), 3.86 (s,
3H, OCH3), 3.53 (s, 3H, OCH3), 2.64 (td, 1H, 2J = 11.7 Hz,
3J = 6.8 Hz, H6a), 2.22 (td,1H, 2J = 11.7 Hz, 3J = 6.8 Hz, H6b); 13C
NMR (100 MHz, (CD3)2CO)⁄ d 157.0 (q, 2J = 36 Hz, HNCOCF3),
154.3, 151.4, 142.3, 139.5, 139.1, 134.1, 131.0, 128.5, 128.3,
127.5, 122.8, 117.0 (q, 1J = 285 Hz, CF3), 103.9, 68.3 (C7), 61.1
(OCH3), 61.0 (OCH3), 56.2 (OCH3), 49.7 (C6), 47.5 (C5); LC–MS
tR = 6.25 min, [ESIꢀ] m/z 410 [MꢀH]ꢀ; HREIMS [M+] m/z
411.13031 (calcd for C20H20F3NO5 411.12932).
(100 M Hz, (CD3)2CO)⁄
d 200.4 (C7), 158.4 (C3), 156.1 (q,
2J = 38 Hz, NHCOCF3), 153.8 (C10), 149.5 (C9), 140.6 (C4a), 132.9
(C11a), 132.8 (C1), 131.0 (C7a), 128.5 (C11b), 116.2 (q,
1J = 285 Hz, CF3), 115.9 (C4), 113.1 (C11), 112.0 (C8), 110.1 (C2),
56.2 (OCH3), 56.1 (OCH3), 52.6 (C6), 48.7 (C5); LC–MS tR = 5.84 min,
[ESIꢀ] m/z 394 [MꢀH]ꢀ; HREIMS [M+] m/z 395.09889 (calcd for
C19H16F3NO5 395.09802).
⁄d assignments according to 2D experiments (COSY, HMBC).
3.3.7. N-(3-Hydroxy-9,10,11-trimethoxy-7-oxo-6,7-dihydro-5H-
dibenzo[a,c][7]cyclohepten-5-yl)-2,2,2-trifluoroacetamide (6f)
Starting from 8f (0.29 mmol, 100 mg) 6f was obtained as a color-
less solid (57 mg, 46%); mp 122–123 °C; IR (KBr) k (cmꢀ1) 2938, 1713
(CO), 1678 (CO), 1585, 1487, 1336, 1212-1163-1110 (CF3), 999; 1H
NMR (400 MHz, (CD3)2CO) d 9.21 (br s 1H, NH), 8.70 (s, 1H, OH),
7.30 (d, 1H, Jortho = 7.8 Hz, H1), 6.86 (m, 3H, H2, H4&H8), 5.52 (m,
1H, H5), 3.90 (s, 6H, OCH3), 3.54 (s, 3H, OCH3), 3.24 (dd, 1H,
2J = 18.5 Hz, 3J = 3.9 Hz, H6a), 3.01 (dd, 1H, 2J = 18.5 Hz,
3J = 13.6 Hz, H6b); 13C NMR (100 MHz, (CD3)2CO) d 201.6 (C7),
158.3, 157.1 (q, 2J = 36 Hz, HNCOCF3), 153.9, 151.9, 146.7, 140.7,
135.9, 134.1, 125.1, 123.3, 116.7 (q, 1J = 286 Hz, CF3), 115.2, 109.6,
107.5 (C8), 61.3 (OCH3), 61.2 (OCH3), 56.4 (OCH3), 53.1 (C6), 48.6
(C5); LC–MS tR = 6.08 min, [ESIꢀ] m/z 424 [MꢀH]ꢀ; HRESIMS
[M+H]+ m/z 426.1153 (calcd for C20H19F3NO6 426.1164).
⁄7b is a mixture of 2 diastereoisomers (85% trans, 15% cis); only
trans isomer NMR signals are described here.
3.4.3. N-(7-Hydroxy-3,9,10-trimethoxy-6,7-dihydro-5H-dibenzo
[a,c][7]-cyclohepten-5-yl)-2,2,2-trifluoroacetamide (7c)
Starting from 6c (0.06 mmol, 25 mg), 7c was obtained as a col-
orless powder (17 mg, 68%); mp 246–247 °C; IR (KBr) k (cmꢀ1
)
3255, 3078, 2931, 1729 (CO), 1611, 1493, 1242, 1211-1178-1143
(CF3), 1042; 1H NMR (400 MHz, (CD3)2CO) d⁄ 9.03 (d, 1H,
3J = 7.8 Hz, NH), 7.35 (d, 1H, Jortho = 8.8 Hz, H1), 6.98 (m, 4H), 4.81
(m, 1H, H5), 4.56 (d, 1H, 3J = 4.9 Hz, OH), 4.45 (m, 1H, H7), 3.89
(s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 2.73 (td, 1H,
2J = 11.7 Hz, 3J = 6.8 Hz, H6a), 2.33 (td, 1H, 2J = 11.7 Hz, 3J = 6.8 Hz,
H6b); 13C NMR (100 MHz, (CD3)2CO)⁄ d 160.1 (C3), 159.2 (q,
2J = 36 Hz, HNCOCF3) 149.9, 149.3, 140.4, 135.1, 131.1, 130.3 (C1),
128.8, 116.2 (q, 1J = 286 Hz, CF3),112.9, 112.8, 109.9, 108.4, 68.2
(C7), 56.3 (2C, OCH3), 55.5 (OCH3), 49.8 (C6), 48.3 (C5); LC–MS
tR = 6.09 min, [ESIꢀ] m/z 410 [MꢀH]ꢀ; HRESIMS [MꢀH]ꢀ m/z
410.1201 (calcd for C20H19F3NO5 410.1215).
3.4. General procedure for the synthesis of
dibenzocycloheptenols (7a–f)
NaBH4 (2 equiv) was added portion-wise to a solution of the
dibenzocycloheptenones (6a–f) in MeOH (15 mL). The reaction
mixture was then stirred for 2 h at room temperature before the
solvent was removed under vacuum; addition of water led to the
expected alcohols 7a–f which were isolated by filtration and puri-
fied by recrystallization.
⁄7c is a mixture of 2 diastereoisomers (65% trans, 35% cis); only
trans isomer NMR signals are described here.
3.4.4. N-(7-Hydroxy-3,9,10,11-tetramethoxy-6,7-dihydro-5H-
dibenzo[a,c][7]cyclohepten-5-yl)-2,2,2-trifluoroacetamide (7d)
Starting from 6d (0.045 mmol, 20 mg), 7d was obtained as a col-
orless solid (13 mg, 65%); mp 190–191 °C; IR (KBr) k (cmꢀ1) 2928,
2852, 1706, 1610, 1485, 1458, 1236-1189-1152 (CF3), 1073; 1H
NMR (400 MHz, (CD3)2CO) d⁄ 8.91 (d, 1H, 3J = 7.8 Hz NH), 7.24 (d,
1H, Jortho = 7.8 Hz, H1), 7.04 (s, 1H, H8), 6.80 (m, 2H, H2&H4),
4.64 (td, 1H, 2J = 11.7, 3J = 7.8, H5), 4.44 (d, 1H, 3J = 3.9 Hz, OH),
4.21 (m, 1H, H7), 3.77 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.69 (s,
3H, OCH3), 2.51 (td, 1H, 2J = 11.7 Hz, 3J = 6.8 Hz, H6a), 2.08 (td,
1H, 2J = 11.7 Hz, 3J = 6.8 Hz, H6b); 13C NMR (100 MHz, (CD3)2CO)⁄
d 160.1 (C3), 157.2 (q, 2J = 38 Hz, NHCOCF3), 154.1 (C10), 151.5
(C9), 142.3, 141.1, 139.0, 132.2 (C1), 126.2, 122.2, 115.8 (q,
1J = 288 Hz, CF3), 112.3 (C2), 109.2 (C4), 103.9 (C8), 68.4 (C7),
61.0 (2C, OCH3), 56.2 (OCH3), 55.4 (OCH3), 49.8 (C6), 47.4 (C5);
LC–MS tR = 6.34 min, [ESIꢀ] m/z 440 [MꢀH]ꢀ; HREIMS [M+] m/z
441.13934 (calcd for C21H22F3NO6 441.13988).
3.4.1. N-(7-Hydroxy-9,10-dimethoxy-6,7-dihydro-5H-
dibenzo[a,c] [7]-cyclohepten-5-yl)-2,2,2-trifluoroacetamide (7a)
Starting from 6a (0.37 mmol, 140 mg), 7a (cis + trans diastereo-
isomers) was obtained as a colorless solid (120 mg, 85%); recrystal-
lization from Et2O gave the (+/ꢀ) trans diastereoisomers; mp
240–241 °C; IR (KBr) k (cmꢀ1) 3547, 3297, 1707 (CO), 1566, 1506,
1217-1187-1142 (CF3), 1023; 1H NMR (400 MHz, DMSO-d6)⁄
d
10.02 (d, 1H, 3J = 7.8 Hz, NH), 7.37 (m, 3H, H1, H2&H3), 7.23 (m,
2H, H4,H8), 6.98 (s, 1H, H11), 5.47 (d, 1H, 3J = 3.9 Hz, OH), 4.57
(m, 1H, H5), 4.20 (m, 1H, H7), 3.82 (s, 6H, OCH3), 2.65 (td, 1H,
2J = 12.1 Hz, 3J = 7.3 Hz, H6a), 2.08 (td, 1H, 2J = 11.7 Hz, 3J = 7.3 Hz,
H6b); 13C NMR (100 MHz, DMSO-d6)⁄ d 155.6 (q, 2J = 36 Hz,
HNCOCF3), 148.6 (C9), 147.7 (C10), 137.9 (C4a), 137.4 (C11b),
134.4 (C7a), 128.2 (C1), 127.5 (C11a,C2), 127.4 (C3), 122.4 (C4),
115.9 (q, 1J = 288 Hz, CF3), 111.8 (C11), 107.3 (C8), 66.5 (C7), 55.7
(OCH3), 55.5 (OCH3), 48.4 (C5), 47.0 (C6); LC–MS tR = 8.53 min,
[ESIꢀ] m/z 380 [MꢀH]ꢀ; HREIMS [M+] m/z 381.11794 (calcd for
⁄7d is a mixture of 2 diastereoisomers (90% trans, 10% cis); only
trans isomer NMR signals are described here.
C
19H18F3NO4 381.11876).
3.4.5. N-(3,7-Dihydroxy-9,10-dimethoxy-6,7-dihydro-5H-
dibenzo[a,c][7]-cyclohepten-5-yl)-2,2,2-trifluoroacetamide (7e)
Starting from 6e (20 mg, 0.05 mmol) 7e was obtained as a col-
orless powder (13 mg, 65%); mp >260 °C; IR (KBr) k (cmꢀ1) 3291,
⁄
The trans configuration was established on the basis of 2D
NMR homonuclear (COSY) and heteronuclear (HSQC, HMBC) data,
completed by 1D NOESY experiments using selective refocusing

2622
E. Chosson et al. / Bioorg. Med. Chem. 20 (2012) 2614–2623
2933, 1727, 1618, 1588, 1493, 1217-1191-1141 (CF3), 1052, 1025;
1H NMR (400 MHz, (CD3)2CO) d⁄ 9.00 (d, 1H, 3J = 6.8 Hz, NH), 8.63
(s, 1H, C3-OH), 7.31 (s, 1H, H8), 7.23 (d, 1H, Jortho = 7.8 Hz, H1),
6.94 (s, 1H, H11), 6.88 (d, 1H, Jmeta = 2.9 Hz, H4), 6.84 (dd, 1H,
Jortho = 7.8 Hz, Jmeta = 2.9 Hz, H2), 4.76 (m, 1H, H5), 4.56 (d, 1H,
3J = 4.9 Hz, OH), 4.46 (m, 1H, H7), 3.87 (s, 6H, OCH3), 2.72 (td,
1H,2J = 11.7 Hz, 3J = 6.8 Hz, H6a), 2.29 (td, 1H, 2J = 11.7 Hz,
3J = 6.8 Hz, H6b); 13C NMR (100 M Hz, (CD3)2CO) d 158.0 (C3),
150.0 (C10), 149.7 (q, 2J = 36 Hz, NHCOCF3), 149.6 (C9), 140.6,
135.4, 130.4 (C1), 130.1, 129.5, 115.6 (q, 1J = 286 Hz, CF3), 115.1,
113.3, 110.9, 108.9 (C8), 68.3 (C7), 56.5 (OCH3), 56.4 (OCH3), 49.9
(C6), 48.5 (C5); LC–MS tR = 5.38 min, [ESIꢀ] m/z 396 [MꢀH]ꢀ; HRE-
IMS [M+] m/z 397.11267 (calcd for C19H18F3NO5 397.11367).
⁄7e is a mixture of 2 diastereoisomers (85% trans, 15% cis); only
trans isomer NMR signals are described here.
After 72 h exposure MTS reagent (Promega) was added and incu-
bated for 3 h at 37 °C: the absorbance was monitored at 490 nm
and results expressed as the inhibition of cell proliferation calcu-
lated as the ratio [(OD490 treated/OD490 control)ꢂ100]. For IC50
determinations (50% inhibition of cell proliferation) experiments
were performed in separate duplicate.
6. Tubulin polymerization inhibition
Sheep brain microtubule proteins were purified by two cycles of
assembly /disassembly at 37 °C/0 °C in MES buffer: 100 mM MES
(2-[N-morpholino]ethane sulfonic acid, pH 6.6), 1 mM EGTA (eth-
yleneglycolbis[ß-aminoethyl
ether]-N,N,N0,N0-tetracetic
acid),
0.5 mM MgCl2. All samples were dissolved in DMSO. Basically,
inhibition of tubulin polymerization was conducted as initially de-
scribed in35 the evaluated compound (1
l
L) was added to microtu-
3.4.6. N-(3,7-Dihydroxy-9,10,11-trimethoxy-6,7-dihydro-5H-di-
benzo[a,c][7]cyclohepten-5-yl)-2,2,2-trifluoroacetamide (7f)
Starting from 6f (0.047 mmol, 20 mg) 7f was obtained as a col-
orless solid (10 mg, 50%); mp >260 °C; IR (KBr) k (cmꢀ1) 2931, 1725
(CO), 1586, 1485, 1401, 1337, 1220-1184-1143 (CF3), 1067; 1H
NMR (400 MHz, (CD3)2CO) d⁄ 8.96 (br s, 1H, NH), 8.56 (s, 1H,
OH), 7.26 (d, 1H, Jortho = 8.8 Hz, H1), 7.15 (s, 1H, H8), 6.84 (m, 2H,
H2,H4), 4.72 (m, 1H, H5), 4.53 (d, 1H, 3J = 3.9 Hz, OH), 4.35 (m,
1H, H7), 3.88 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.51 (s, 3H,
OCH3), 2.63 (m, 1H, H6a), 2.18 (m, 1H, H6b); 13C NMR (100 MHz,
(CD3)2CO) d 157.8 (C3), 153.9 (q, 2J = 38 Hz, NHCOCF3), 153.8,
151.4, 141.0, 138.9, 133.3, 132.2 (C1), 124.9, 122.3, 114.7 (q,
1J = 282 Hz, CF3), 114.4 (C2), 110.1 (C4), 103.9 (C8), 68.4 (C7),
61.0 (OCH3), 60.9 (OCH3), 56.2 (OCH3), 49.8 (C6), 47.4 (C5); LC–
MS tR = 5.63 min, [ESIꢀ] m/z 426 [MꢀH]ꢀ; HRESIMS [M+H]+ m/z
428.1309 (calcd for C20H21F3NO6 428.1321).
bule solution (150
lL) and incubated for 10 min at 37 °C followed
by 5 min at 0 °C. The tubulin polymerization rate was measured by
turbidimetry at 350 nm using deoxypodophyllotoxin (DPPT) as ref-
erence compound. Compounds were first tested at 6.7 10ꢀ5 M and
results were given as the percentage of inhibition for the less active
compounds. Compounds showing higher activities were then
tested at concentrations ranged 33–2 lM to determine their IC50.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.02.043.
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Crystallographic data of 7a: Crystal size: 0.35 ꢂ 0.21 ꢂ 0.18 mm.
Formula C19H18F3NO4, formula weight 381.34, crystal system
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c = 17.706 (7) Å,
(11) Å3, Z = 4, calculated density = 1.355 g/cm3,
Rint = 0.0631, R[F2 > 2 (F2)] = 0.0631, wR(F2) = 0.1385.
a
= 90°, b = 102.325 (14)°,
c
= 90°, V = 1869.7
l
= 0.114 mmꢀ1
,
r
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The human epidermoid carcinoma KB cell lines were obtained
from ECACC (Salisbury, UK) and grown in D-MEM medium supple-
mented with 10% fetal calf serum (Invitrogen), in the presence of
penicilline, streptomycine and fungizone in 75 cm3 flask under
5% CO2. Cells were plated in 96-well tissue culture microplates at
a density of 650 cells/well in 200
with compounds dissolved in DMSO with compound concentra-
tions ranged 0.5–10 M using a Biomek 3000 automate (Beckman).
Controls received the same volume of DMSO (1% final volume).
ll medium and treated 24 h later
l

E. Chosson et al. / Bioorg. Med. Chem. 20 (2012) 2614–2623
2623
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