Synthesis and structure-activity relationships of new antiproliferative and proapoptotic retinoid-related biphenyl-4-yl-acrylic acids

Article abstract of DOI:10.1016/j.bmc.2007.04.057

Atypical retinoids, or retinoid-related molecules (RRMs), represent a class of proapoptotic agents with a promising potential in the treatment of neoplastic diseases. In the present work, the synthesis and structure-activity relationship of a series of 3′-adamantan-1-yl-biphenyl-4-yl-acrylic acids substituted in ring A were studied. The synthesized compounds were evaluated for their antiproliferative activity in a human promyelocitic leukemia cell line (NB4), and in an ovarian carcinoma cell system including IGROV-1, carrying a functional wild-type p53, and a cisplatin-resistant subline, IGROV-1/Pt-1. The presence of at least one oxygenated substituent in positions 4′ or 5′ appears determinant for the antiproliferative activity. With two substituents of this kind the activity increases, particularly in the case of alkylenedioxy compounds. The activation of DNA damage response as indicated by phosphorylation of H2AX histone, RPA-2 protein, and p53 at serine 15 by the most apoptotic compounds provides additional support to the hypothesis that the genotoxic stress is a critical event mediating apoptosis induction by compounds of this group.

Full text of DOI:10.1016/j.bmc.2007.04.057

Bioorganic & Medicinal Chemistry 15 (2007) 4863–4875
Synthesis and structure–activity relationships of new
antiproliferative and proapoptotic retinoid-related
biphenyl-4-yl-acrylic acids
Raffaella Cincinelli,a Sabrina Dallavalle,a,* Raffaella Nannei,a Lucio Merlini,a
Sergio Penco,b Giuseppe Giannini,b Claudio Pisano,b Loredana Vesci,b
Fabiana Fosca Ferrara,b Valentina Zuco,c Chiara Zanchic and Franco Zuninoc
a
`
Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, Via Celoria 2, 20133 Milano, Italy
bSigma-tau, R&D, Pomezia, Italy
Unita di Farmacologia Antitumorale Preclinica, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy
c
`
Received 18 October 2006; revised 18 April 2007; accepted 27 April 2007
Available online 3 May 2007
Abstract—Atypical retinoids, or retinoid-related molecules (RRMs), represent a class of proapoptotic agents with a promising
potential in the treatment of neoplastic diseases. In the present work, the synthesis and structure–activity relationship of a series
of 30-adamantan-1-yl-biphenyl-4-yl-acrylic acids substituted in ring A were studied. The synthesized compounds were evaluated
for their antiproliferative activity in a human promyelocitic leukemia cell line (NB4), and in an ovarian carcinoma cell system includ-
ing IGROV-1, carrying a functional wild-type p53, and a cisplatin-resistant subline, IGROV-1/Pt-1. The presence of at least one
oxygenated substituent in positions 40 or 50 appears determinant for the antiproliferative activity. With two substituents of this kind
the activity increases, particularly in the case of alkylenedioxy compounds. The activation of DNA damage response as indicated by
phosphorylation of H2AX histone, RPA-2 protein, and p53 at serine 15 by the most apoptotic compounds provides additional sup-
port to the hypothesis that the genotoxic stress is a critical event mediating apoptosis induction by compounds of this group.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
We have recently reported that a novel adamantyl reti-
noid, ST1926 (1), is a potent inducer of apoptosis in a
variety of tumor cell lines and exhibits efficacy against
solid tumor models, with an improved pharmacological
profile.7 The evidence that ST1926 enhances the proa-
poptotic and antitumor activity of other clinically effec-
tive agents supports the potential interest of novel
combination approaches with the use of agents which
induce apoptosis through different mechanisms. The
pattern of cellular response to ST1926 indicated the acti-
vation of DNA damage response, thus supporting that
the induction of genotoxic stress is implicated in mediat-
ing drug-induced apoptosis.8–10
Retinoids may have clinical efficacy in cancer chemopre-
vention. The chemopreventive properties of agents of
this class include growth inhibitory, prodifferentiating,
proapoptotic, and antiangiogenic activities.1,2 Synthetic
retinoids containing the adamantyl moiety represent a
promising group of novel agents of this class, because
they are characterized by a potent proapoptotic activity
in a large variety of tumor cells. They exhibit antitumor
activity in in vivo models with acceptable side effects.3
The detailed molecular mechanism involved in apopto-
sis induction by adamantyl retinoids is not clearly de-
fined. These retinoids have been shown to induce
apoptosis by a mechanism which is independent of reti-
noid receptor signaling pathway.4–6
In our previous work we explored the putative key
portions of the molecule, that is, the carboxylic group,
the double bond, and the adamantan-1-yl moiety, by
introducing substituents or structural modifications to
these moieties.7 We then evaluated the effects of the
changes on the activity of the derivatives, using appro-
priate cellular and molecular models. The structure–
activity relationship study suggested that the structural
Keywords: Retinoids; Adamantyl; Antiproliferative activity; Apopto-
sis; Synthesis.
*
Corresponding author. Tel.: +39 0250316816; fax: +39 0250316801;
e-mail: Sabrina.Dallavalle@unimi.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.057

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R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
compound 2, whose synthesis has been described in
COOH
our previous paper.7 We applied an efficient procedure
that relies on the use of triethylsilane as a reducing agent
and a catalytic system consisting of palladium(II)acetate
and the bidentate phosphine ligand 1,3-bis(diph-
enylphosphino)propane (dppp). The reaction was highly
chemoselective and the bromo group was completely
unaffected under these conditions.11
B
A
HO
1
ST 1926
Chart 1.
requirements for optimal activity of this class of com-
pounds are rather strict, meaning that the carboxylic
group, the E unsubstituted double bond, and a bulky
lipophilic group in position 30 are necessary to maintain
The synthesis of compound 6, in which the OH group
was moved from its original position, has already been
described (Scheme 1).7
high antiproliferative activity. Thus compound
1
remained the most potent compound of the series
(Chart 1).
Compounds 9–15 were obtained as outlined in Scheme 2.
First, 2-(1-adamantyl)-4-bromophenol7 7 was alkylated
with methyl iodide and then cross-coupled with p-form-
ylbenzeneboronic acid to give the aldehyde 8. Wittig
condensation with methoxycarbonylmethylenetriphenyl-
phosphorane and final basic hydrolysis led to compound
9. Condensation of aldehyde 8 with carbethoxybromom-
ethylenetriphenylphosphorane led to a mixture of the
two diastereomeric esters 10a and 10b, that were sepa-
rated by chromatography. Basic hydrolysis of 10b
led to the corresponding acid 10d, while hydrolysis of
Z-10a afforded the elimination product 11 together with
the acid 10c.
The present work was designed to gain further insight
into the critical requirements linking structural features
and biological activity of this class of biphenyl-4-yl-ac-
rylic acids. Thus we planned to explore the effect of sub-
stituents on the ring A of the biphenyl system. In
particular we wanted to evaluate the role of the oxygen
atom in this portion of the ligand, first removing and
then replacing the phenolic OH by other polar functions
while maintaining the adamantyl group at the same
position.
Interestingly, we noticed that removal of the phenolic
OH group led to a strong decrease of cytotoxic activity,
meaning that an oxygenated function is a critical struc-
tural feature. However, differently from other key por-
tions of the molecule, where small changes in structure
had a pronounced influence on biological activity,
replacement of the phenolic OH or addition of other
oxygenated groups in the adjacent position led to com-
pounds endowed with potent antitumor activity, there-
fore worth of further investigation.
The introduction of an aminomethyl moiety was accom-
plished by alkylation of compound 77 followed by
palladium-catalyzed cross-coupling with methyl p-bromo-
cinnamate and hydrolysis with HCl 37% in dioxane to
give the acid 15. The corresponding acetyl derivative
13 was obtained by Friedel–Crafts (FC) alkylation of
7 with N-(hydroxymethyl)-acetamide, Suzuki coupling,
and basic hydrolysis.
Compounds substituted with two oxygenated moieties
were prepared as depicted in Schemes 3–5. A FC alkyl-
ation of 40-bromobiphenyl-4-ol with adamantan-1-ol led
12
2. Chemistry
to compound 16. Formylation of this latter by (CH2O)n
led mainly to compound 17, that was converted into the
acid 22 by a palladium-catalyzed cross-coupling Heck
reaction followed by hydrolysis. Compound 18, obtained
in small amount from the formylation reaction, was used
as a starting material for the synthesis of 21, with the usual
sequence of reactions.
First, we planned to deoxygenate the phenol moiety of 1
to test the role of the hydroxy group. Attempts to obtain
the desired compound from 1, via the corresponding tri-
flate, were unsuccessful. So we performed the reduction
reaction starting from the triflate of the less hindered
Br
Br
Br
a
b
2
3
4
OTf
OH
e,f
c,d
COOH
COOH
5
6
OH
Scheme 1. Reagents and conditions: (a) TfO2, Py, 3 h, rt, 87%; (b) Et3 SiH/dppp, Pd(OAc)2, DMF, 24 h, 60 ꢁC, 42%; (c) TOTP, Pd(OAc)2, TEA,
methyl acrylate, 32 h, reflux, 67%; (d) LiOHÆH2O, THF/H2O, rt, overnight, 63%; (e) TOTP, Pd(OAc)2, TEA, methyl acrylate, 2 days, reflux, 50%; (f)
LiOHÆH2O, THF/H2O, rt, overnight, 80%.

R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
4865
COOH
COOH
11
9
H CO
3
H CO
3
c,d
a,b
h
10a R=COOEt (Z)
10b R=COOEt (E)
10c R=COOH (Z)
10d R=COOH (E)
R
CHO
f
e
Br
g
H CO
3
H CO
3
8
COOH
Br
l,m
Br
i
HO
HO
HO
HO
HO
+
-
CH NH Cl
2
3
15
CH NHCOCH Cl
2
2
14
7
n
COOH
o,p
Br
CH NHCOCH
CH NHCOCH
2
3
2
3
13
12
Scheme 2. Reagents and conditions: (a) NaH, MeI, DMF, 2 h, 20 ꢁC 78%; (b) Pd tetrakis, 4-formylbenzeneboronic acid, 2 M Na2CO3, toluene, 2 h,
reflux, 72%; (c) Ph3 PCH = COOMe, chloroform, 3 h, reflux, 86%; (d) NaOH, methanol, 7 h, reflux, 70%; (e) Ph3PCBr = COOEt, chloroform, 14 h,
reflux, 87%; (f) KOH, methanol, 1 h, reflux, 7%; (g) LiOH, THF/H2O, rt, overnight, 89%; (h) KOH, methanol, 1 h, reflux, 68%; (i)
ClCH2CONHCH2OH, CH3COOH/H2SO4 9:1, 20 h, rt, 87%; (l) bis(pinacolato)diboron, KOAc, PdCl2(dppf), then methyl p-Br-cinnamate,
PdCl2(dppf), Na2CO3 2 M, 3 h, 100 ꢁC, 27% ; (m) HCl 37%, dioxane, 23 h, reflux, 92%; (n) CH3CONHCH2OH, CH3COOH:H2SO4 9:1, 5 days, rt,
37%; (o) bis(pinacolato)diboron, KOAc, PdCl2(dppf), then methyl p-Br-cinnamate, PdCl2(dppf), Na2CO3 2 M, 100 ꢁC, 3 h, 43% ; (p) LiOH THF/
H2O, rt, overnight, 72%.
COOH
COOH
22
HO
HO
21
CHO
b,c
OH
d,e
Br
Br
Br
a
+
HO
HO
HO
17
16
18
CHO
OH
f, g, h
m
Br
Br
H CO
3
23
HO
19
OH
i, l
OH
n, o, p
COOH
COOH
24
20
H CO
3
H CO
3
OCH
OH
3
Scheme 3. Reagents and conditions: (a) (CH2O)n, SnCl4, 2,6-lutidine, toluene, 7 h, 95 ꢁC, 60% (17), 34% (18); (b) methyl acrylate, TOTP, Et3N,
Pd(OAc)2, 110 ꢁC, 66%; (c) LiOH THF/H2O, rt, overnight, 83%; (d) methyl acrylate, TOTP, Et3N, Pd(OAc)2, 110 ꢁC, 33%; (e) LiOH THF/H2O, rt,
overnight, 91%; (f) CH3I, K2CO3, acetone, 6 h, reflux, 90%; (g) MCPBA, CH2Cl2, 1 h, reflux, 47%; (h) KOH, MeOH, 1 h, rt, then HCl, 100%; (i)
methyl acrylate, TOTP, Et3N, Pd(OAc)2, 67%; (l) NaOH, methanol, 2 h, reflux, 20% (m) MCPBA, CH2Cl2, 20 h, reflux, 53%; (n) methyl acrylate,
TOTP, Et3N, Pd(OAc)2, 84%; (o) CH3 I, NaH, DMF, rt, 2 h, 19%; (p) LiOH THF/H2O, rt, 4 days, 87%.

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R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
ron and methyl p-bromocinnamate14 followed by the
usual saponification with LiOH furnished the acid 27.
Br
Br
a
HO
HO
Aldehyde 30 was obtained by FC alkylation and palla-
dium-catalyzed cross-coupling of the bromobenzene
29a. Wittig condensation with methoxycarbonylmethy-
lenetriphenylphosphorane followed by standard base
hydrolysis conditions led to acid 31a. The acid 31b
was prepared via palladium-catalyzed Suzuki one-pot
condensation with methyl 4-bromocinnamate followed
by hydrolysis.
OCH
OCH
26
25
3
3
COOH
b,c
27
HO
OCH
3
Scheme 4. Reagents and conditions: (a) 1-adamantanol, CH2Cl2,
concd H2SO4, rt, 1 h, 60%; (b) bis(pinacolato)diboron, PdCl2(dppf),
KOAc, methyl p-bromocinnamate, Na2CO3 2 M and PdCl2(dppf),
70%; (c) LiOH THF/H2O, rt, overnight, 98%.
The two diastereomeric esters 32 and 33 were obtained
by Wittig condensation of 30a with ethoxycarbonyl-
bromomethyltriphenylphosphorane. Final hydrolysis,
accompanied by elimination in the case of 32, led to
acids 34 and 35.
Methylation of 17 with CH3I in acetone, followed by
Baeyer–Villiger oxidation13 and hydrolysis with KOH
in methanol, afforded compound 23. The acid 24 was
then obtained by Heck coupling and hydrolysis. Bae-
yer–Villiger oxidation on the aldehyde 17 gave the cate-
chol 19 that was immediately subjected to Heck
coupling with methyl acrylate. The high instability of
the obtained catechol required methylation of both the
phenolic groups with methyl iodide and NaH in
DMF. Hydrolysis of the ester gave the desired carbox-
ylic acid 20.
3. Antiproliferative activity
The effects of the selected compounds on cell growth
were determined in a panel of human tumor cells,
including an ovarian carcinoma cell line (IGROV-1), a
subline selected for resistance to cisplatin and character-
ized by p53 mutation (IGROV-1/Pt1), and a leukemia
cell line (NB4). The results are reported in Table 1.
4. Cytodifferentiation test
The synthesis of compounds 27–35 is illustrated in
Schemes 4,5. Friedel–Crafts alkylation of 2-methoxy-4-
bromophenol led to compound 26. Palladium-catalyzed
Suzuki one-pot cross-coupling with bis(pinacolato)dibo-
The obtained compounds were also tested in vitro for
their cytodifferentiating activity on the leukemia cell line
NB4.
h, i
CHO
COOH
Br
Br
a
b
c,d
O
O
( )nO
O
( )nO
O
( )nO
O
30
31a n=1
31b n=2
29a n=1
29b n=2
28a n=1
28b n=2
e
COOEt
Br
Br
32
COOH
+
COOEt
O
O
O
33
O
g
O
f
Br
COOH
O
34
35
O
O
Scheme 5. Reagents and conditions: (a) 1-adamantanol, CH2Cl2, concd H2SO4, 4 h, rt; (b) Pd(PPh3)4, 4-formylbenzeneboronic acid, Na2CO3 2 M,
EtOH, toluene, 7 h, reflux, 70%; (c) Ph3P = CHCOOMe, CHCl3; (d) LiOH, THF/H2O; (e) Ph3P = CBrCOOEt, CHCl3, 8 h, reflux; (f) LiOH, THF/
H2O 89%; (g) KOH, MeOH, 1 h, reflux, 40%; (h) bis(pinacolato)diboron, KOAc, PdCl2(dppf), methyl p-Br-cinnamate, Na2CO3, 50%; (i) LiOH,
THF/H2O, rt, overnight, 91%.

R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
4867
Table 1. Antiproliferative activity of selected compounds on IGROV-
1, IGROV-1/Pt1, and NB4 cellular lines
Compound
IC50 (lM)
IGROV-1
IGROV-1/Pt1
NB4
1
0.23 0.08
18.8
0.40 0.08
21.9
n.d.
0.082 0.005
n.d.
5
6
1.31 0.02
1.19 0.5
30.6
1.2 0.09
1.1 0.07
41 2.4
9
1.43 0.58
>10
10d
10c
11
13
15
20
21
22
24
27
31a
31b
34
35
ꢀ20
ꢀ20
17.9 2.2
n.d.
1.70 0.60
1.24 0.07
0.45 0.3
0.71 0.10
0.76 0.57
1.64 0.03
0.57 0.14
0.52
4.8 1.9
2.89 0.02
0.39 0.18
2.4 0.57
1.04 0.57
2.72 0.57
0.97 0.33
n.d.
0.99 0.03
0.18 0.008
1.89 0.07
0.19 0.01
1.61 0.1
0.55 0.03
0.27 0.02
0.26 0.009
0.11 3.4
2.0 0.03
72.3 4.4
0.39 0.17
0.23 0.07
3.6
0.46 0.09
0.48 0.25
4.80 2.4
34.10
36.6
IC50 (lM) is the concentration required for 50% inhibition of cell
growth ( SD).
Figure 1. Time course of cell cycle perturbations in ovarian carcinoma
IGROV-1 cells, following treatment with selected retinoids. Cells were
treated with the drug at IC80 and the cell cycle was analyzed by
FACScan analysis of PI-stained cells at 24, 48, and 72 h of treatment.
One experiment representative of three is reported.
5. Results and discussion
As a first step we checked the role of the phenolic OH
group in 40 position, by preparing the deoxy compound
5. This modification induced a dramatic decrease of the
antiproliferative activity, thus indicating the importance
of such a group for the activity. Shifting the OH to the 50
position (compound 6) or methylation of the 40-OH
(compound 9) restored the activity, so that the minimum
requirement is the presence of an oxygen atom in at least
one of the two positions. The introduction of two oxy-
genated functions (compounds 20, 24, 27, 31a, 31b) sub-
stantially maintained the activity, the two compounds
with a ring (31a, 31b) being the most active. Probably
in this case the oxygen atoms are more exposed to the
interaction with the receptor with respect to the less
accessible oxygens in the methoxy derivatives. Also the
introduction of a carbon atom linked to a heteroatom
in the position adjacent to the OH (compounds 13, 15,
21, 22) maintained the activity.
a marker of cell death. This behavior was less marked
for compound 24.
All compounds effective in cell growth inhibition were
also active as proapoptotic agents (Table 2). Compound
24 exhibited an appreciably reduced ability to induce
apoptosis. This observation was consistent with a lower
sub-G1 peak (Fig. 1). All tested compounds were able to
activate DNA damage response as indicated by phos-
phorylation of H2AX histone, RPA-2 protein, and p53
at serine 15 (Fig. 2). Compound 9 was the most effective
in inducing phosphorylation of H2AX histone, which is
known to be the most sensitive marker of double-strand
DNA breaks.15 Again compound 24, which is a weaker
apoptosis inducer, was also less effective in activation of
a genotoxic stress response. This observation provides
additional support to the hypothesis that the genotoxic
stress is a critical event mediating apoptosis induction
by compounds of this groups.
For two compounds (9 and 31a) some modifications in
the distal part of the molecule were made, in order to
check whether the structural requirements for activity
held also in this series.7 In fact the compounds with a
bromoacrylic chain 10c–d and 35 appeared inactive,
whereas the propynoic acid derivatives (11 and 34)
maintained the activity, as expected.7
Table 2. Apoptosis induction in IGROV-1 cells by selected retinoids
IGROV-1
% of apoptosis
1
51
55
50
47
45
30
20
3
15
31a
9
5
5
In this new series compound 31b was the most potent
agent with activity comparable with 1 in all tested cell
lines. Cell cycle analysis of drug-treated cells revealed
21
31b
24
4
4
a
marked perturbation of cell cycle progression
Apoptosis was determined at 72 h following exposure to IC80 by
morphological analysis of propidium iodide-stained cells. The results
are expressed as percentage of apoptotic cells versus total cell number.
Values are the mean standard deviation of three independent
experiments.
(Fig. 1). The most active compounds of this series
(e.g., 9, 24, 31a, 31b, 15) caused accumulation of cells
in the G1/S phases with the appearance in a time-depen-
dent manner of a sub-G1 peak, which is considered to be

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R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
In conclusion, we have explored which structural fea-
control 1 31a
9
24 21 31b 15
tures confer strong antiproliferative activity and proa-
poptotic properties in a class of atypical retinoids.
These results can be useful for the modulation of the
pharmacological potential of these compounds.
γ-H2AX
RPA
phospho-p53 (Ser15)
β-tubulin
6. Experimental
Figure 2. Induction of DNA damage response in IGROV-1 cells
exposed to equitoxic drug concentration of selected retinoids (IC80) for
6 h. Cells were lysed and processed for Western blotting. Control
loading is shown by b-tubulin.
6.1. General chemical methods
All reagents and solvents were of reagent grade or were
purified by standard methods before use. Melting points
were determined in open capillaries on a Buchi melting
¨
As already reported for compound 1,8 atypical adaman-
tyl retinoids caused modulation of MAP kinases impli-
cated in stress response (e.g., p38 and JNK). We
investigated the ability of 31a to activate these kinases
as detected by their enhanced phosphorylation
(Fig. 3). In IGROV-1 cells, activation of p38 and JNK
was more marked following exposure to 31a, in spite
of a comparable proapoptotic activity. Activation of
stress kinases by compound 1 was more evident in
IGROV-1/Pt1 cells, characterized by p53 mutation and
by reduced sensitivity to these agents, thus indicating
no correlation between stress response and apoptosis.
These observations suggest that the activation of MAP
kinase pathways plays a protective role in the cellular
stress response to these agents.
point apparatus and are uncorrected. Column chroma-
tography was carried out on flash silica gel (Merck
230–400 mesh). TLC analysis was conducted on silica
gel plates (Merck 60F254). NMR spectra were recorded
at 300 MHz with a Bruker instrument. Chemical shifts
(d values) and coupling constants (J values) are given
in ppm and Hz, respectively. Mass spectra were
recorded at an ionizing voltage of 70 eV on a Finnigan
TQ70 spectrometer. The relative intensities of mass
spectrum peaks are listed in parentheses. HPLC analysis
of the mixture of diastereoisomers was performed on an
HP 1050 quaternary pump fitted with a Rheodyne injec-
tor (20 lL loop) and a HP-1050 Diode-Array detector.
Chromatograms were recorded at 360 and 400 nm.
The column was a Rainin C18, 25 · 0.4 cm, flow
1 mL/min, with a gradient from CH3CN/H2O 30:70 to
100:0, in 20 min.
A preliminary evaluation of the ability of compounds of
this series to induce differentiation of NBA leukemia
cells indicated that some compounds had cytodifferenti-
ating activity comparable to (10c, 11) or better (9, 31b)
than the standard ATRA (all-trans retinoic acid) in a
dose-dependent manner, thus supporting an additional
therapeutic interest for these compounds.
6.2. General procedure for Heck condensation (A)
A round flask containing a mixture of 0.73 mmol of the
appropriate aryl bromide, 1.17 mmol of methyl acrylate,
5 mg of Pd (OAc)2, and 27 mg of tri-o-tolylphosphine in
0.3 mL of Et3N was immersed in an oil bath preheated
at 50 ꢁC and kept at 100–110 ꢁC for 2–12 h. Iced water
was then added, followed by 2 N HCl. The aqueous
phase was extracted repeatedly with ethyl acetate, then
the organic phases were dried over Na2SO4, filtered,
and evaporated. The crude product was purified by flash
chromatography and, when necessary, crystallized from
diisopropyl ether.
6.3. General procedure for ester hydrolysis (B)
The appropriate ester (0.16 mmol) was suspended in a
solution of 34 mg (0.815 mmol) of LiOHÆH2O in
8.3 mL of THF/H2O 3:2 and stirred at room tempera-
ture in the dark overnight. After evaporation of THF
the remaining aqueous phase was washed with hexane,
diethyl ether and then acidified with HCl 2 N (0.4
mL). The precipitated white solid was filtered and dried.
No further purification was necessary.
6.4. General procedure for one-pot Suzuki condensation
(C)
Figure 3. Effect of compounds 1 and 31a on JNK and p38 activation in
IGROV-1 and IGROV-1/Pt1 cells exposed to the drugs at their
respective IC80. Western blot analysis was performed with phospho-
specific antibodies after 18 h of treatment. Anti-p38 and JNK blots are
shown as control of protein expression and anti-actin as control of
protein loading.
A small amount of the appropriate aryl bromide
(0.6 mmol), 166 mg (0.65 mmol) of bis(pinacolato)dibo-
ron, 174 mg (1.78 mmol) of KOAc, and 13 mg

R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
4869
(0.018 mmol) of PdCl2(dppf) were dissolved in 36 mL of
dioxane and the mixture was stirred at 100 ꢁC for 1–2 h
under nitrogen. After cooling the solution at room tem-
J = 16.0 Hz), 7.30–7.80 (9H, m, 8Ar + CH@). Anal.
calcd for C25H26O2: C, 83.76; H, 7.31. Found: C,
83.84; H, 7.22.
perature,
methyl
4-bromocinnamate
(287 mg,
1.19 mmol), PdCl2(dppf) (13 mg, 0.018 mmol) and 2 M
Na2CO3 (1.48 mmol, 0.74 mL) were added and the mix-
ture was heated at 80–100 ꢁC for 1–3 h. The solution
was cooled at room temperature, diluted with water,
acidified with 2 N HCl (2.4 mL), and extracted with
ethyl acetate. The organic phase was washed with brine,
dried over Na2SO4, filtered, and evaporated. The crude
product was purified by flash chromatography.
6.4.4. 40-Methoxy-30-(adamantan-1-yl)biphenyl-4-carbal-
dehyde (8). To a suspension of sodium hydride (60% in
mineral oil, 0.402 g, 10.1 mmol) in 11 mL of DMF, 2-
(1-adamantyl)-4-bromophenol (7) (2.75 g, 8.38 mmol)
was slowly added, while maintaining the temperature
at 20 ꢁC. The mixture was stirred at room temperature
for 1 h, then methyl iodide (0.73 mL, 11.7 mmol) was
added. After having stirred for additional 2 h at 20 ꢁC,
the solution was poured into water and extracted with
diethyl ether. The organic phase was dried and evapo-
rated. The residue was purified by flash chromatography
(hexane) to give 2.1 g (78%) of 4-bromo-1-methoxy-2-
(adamantan-1-yl)-benzene, mp 136 ꢁC. 1H NMR
(300 MHz, CDCl3) d: 1.75 (6H, s, 6Ad), 2.05 (9H, s,
9Ad), 3.80 (3H, s, OMe), 6.72 (1H, d, 1Ar,
J = 8.0 Hz), 7.24 (1H, dd, 1Ar, J = 8.0, 2.2 Hz), 7.28
(1H, d, 1Ar, J = 2.2 Hz).
6.4.1. Trifluoromethanesulfonic acid 40-bromo-5-(ada-
mantan-1-yl)-biphenyl-3-yl ester (3). To a solution of
5-(1-adamantanyl)-40-bromobiphenyl-3-ol (2)7 (840 mg,
2.19 mmol) in 10 mL of pyridine at 0 ꢁC trifluorome-
thanesulfonic anhydride (0.41 mL, 2.41 mmol) was
slowly added. The resulting mixture was stirred at 0 ꢁC
for 30 min and then allowed to warm at room tempera-
ture and stirred for 3 h. The mixture was then poured
into water and extracted with diethyl ether. The organic
extracts were washed sequentially with water, 2 N HCl,
water, brine, dried, and concentrated to give a yellow
oil. Purification by flash chromatography (hexane/ethyl
acetate 95:5) afforded 980 mg (87%) of the title com-
pound. 1H NMR (300 MHz, CDCl3) d: 1.73 (6H, s,
6Ad), 1.95 (3H, s, 3Ad), 2.10 (6H, s, 6Ad), 7.20 (2H,
br s, 2Ar), 7.35 (2H, m, 2Ar), 7.50 (1H, s, 1Ar), 7.58
(2H, m, 2Ar). Anal. calcd for C23H22BrF3O3S: C,
53.60; H, 4.30. Found: C, 53.75; H, 4.18.
To a solution of 1.95 g (6.06 mmol) of 4-bromo-1-meth-
oxy-2-(adamantan-1-yl)-benzene in 12.2 mL of toluene,
6.06 mL (12.1 mmol) of 2 M aqueous Na2CO3, 0.210 g
(0.182 mmol) of tetrakis-triphenylphosphine-palladium
and a solution of 1 g (6.67 mmol) of 4-formylbenzene-
boronic acid in 2.83 mL of ethanol were added. The
mixture was refluxed for 2 h under a stream of nitrogen,
then it was cooled at room temperature, diluted with
dichloromethane, and washed with brine. The organic
phase was dried, filtered, and evaporated. The residue
was purified by flash chromatography (hexane/ethyl ace-
tate 85:15) to give 1.5 g (72%) of the desired aldehyde,
6.4.2. 40-Bromo-3-(adamantan-1-yl)-biphenyl (4). To a
mixture of the above triflate (68 mg, 0.13 mmol),
Pd(OAc)2 (0.6 mg, 2.6.10–3 mmol), and dppp (1.1 mg
2.6.10–3 mmol) in 1 mL of DMF at 60 ꢁC was added
Et3SiH (0.325 mmol, 52.5 lL). The stirring was contin-
ued for 1.5 h and during this time the solution changed
color from red to yellow. After dilution with diethyl
ether the mixture was successively washed with water,
saturated aqueous sodium bicarbonate, and brine, then
it was dried and evaporated. The crude was purified
by flash chromatography (hexane/dichloromethane
1
mp 196 ꢁC. H NMR (300 MHz, CDCl3) d: 1.80 (6H,
s, 6Ad), 2.10 (9H, s, 9Ad), 3.90 (3H, s, OMe), 6.98
(1H, d, 1Ar, J = 8.0 Hz), 7.47 (1H, dd, 1Ar, J = 8.0,
2.2 Hz), 7.48 (1H, d, J = 2.2 Hz), 7.70 (2H, d, 2Ar,
J = 8.3 Hz), 7.90 (2H, d, 2Ar, J = 8.3 Hz), 10.0 (1H, s).
Anal. calcd for C24H26O2: C, 83.20; H, 7.56. Found:
C, 83.08; H, 7.70.
6.4.5. 3-(40-Methoxy-30-(adamantan-1-yl)biphenyl-4-yl)-
acrylic acid (9). Compound 8 (658 mg, 1.90 mmol) and
635 mg (1.90 mmol) of methoxycarbonylmethylenetri-
phenylphosphorane were dissolved in 10 mL of chloro-
form and the solution was refluxed for 3 h in a current
of nitrogen. Chloroform was evaporated and the crude
product was purified by flash chromatography (dichlo-
romethane/hexane 40:60) to give 655 mg (86%) of 3-
(40-methoxy-30-(adamantan-1-yl)biphenyl-4-yl)-acrylic
acid methyl ester, mp 212 ꢁC. 1H NMR (300 MHz,
CDCl3) d: 1.73 (6H, s, 6Ad), 2.04 (3H, s, 3Ad), 2.10
(6H, s, 6Ad), 3.76 (3H, s, OMe), 3.82 (3H, s, OMe),
6.40(1H, d, CH@, J = 17,0 Hz), 6.88 (1H, d, 1Ar,
J = 8.6 Hz), 7.35 (1H, dd, 1Ar, J = 8.6, 2.2 Hz), 7.41
(1H, d, 1Ar, J = 2.2 Hz), 7.45–7.60 (4H, m, 4Ar), 7.67
(1H, d, CH@, J = 17,0 Hz). The above ester (120 mg,
0.298 mmol) was suspended in 17 mL of a solution of
0.7 N NaOH in methanol and the mixture was refluxed
for 7 h. After evaporation of methanol, the residue was
treated with water, acidified with 6 N HCl, and the pre-
cipitate was filtered to afford 80 mg (70%) of the pure
1
99:1) to obtain 20 mg (42%) of the title compound. H
NMR (300 MHz, CDCl3) d: 1.85 (6H, s, 6Ad), 2.01
(3H, s, 3Ad), 2.15 (6H, s, 6Ad), 7.30–7.60 (8H, m,
8Ar). Anal. calcd for C22H23Br: C, 71.94; H, 6.31.
Found: C, 71.86; H, 6.35.
6.4.3. 3-(30-(Adamantan-1-yl)-biphenyl-4-yl)-acrylic acid
(5). Compound 4 (100 mg, 0.27 mmol) was reacted with
methyl acrylate according to the general procedure A, to
obtain 67 mg (67%) of 3-(30-(1-adamantyl)-biphenyl-4-
1
yl)-acrylic acid methyl ester, mp 122–123 ꢁC. H NMR
(300 MHz, CDCl3) d: 1.73 (6H, s, 6Ad), 1.95 (3H, s,
3Ad), 2.10 (6H, s, 6Ad), 3.78 (3H, s, OMe), 6.48 (1H,
d, CH@, J = 16.0 Hz), 7.35–7.40 (3H, m, 3Ar), 7.50–
7.65 (5H, m, 5Ar), 7.75 (1H, d, CH@, J = 16.0 Hz).
The above ester (67 mg, 0.18 mmol) was hydrolyzed
according to the general procedure B, to afford 40 mg
(63%) of the title compound, mp 268–269 ꢁC. 1H
NMR (300 MHz, DMSO-d6) d: 1.67 (6H, s, 6Ad), 1.98
(3H, s, 3Ad), 2.03 (6H, s, 6Ad), 6.55 (1H, d, CH@,

4870
R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
1
product, mp >240 ꢁC. H NMR (300 MHz, DMSO-d6)
d: 1.67 (6H, s, 6Ad), 1.98 (3H, s, 3Ad), 2.03 (6H, s,
6Ad), 3.74 (3H, s, OMe), 6.45 (1H, d, CH@,
J = 16.2 Hz), 6.97 (1H, d, 1Ar, J = 8.5 Hz), 7.34 (1H,
d, 1Ar, J = 2.3 Hz), 7.43 (1H, dd, 1Ar, J = 8.5,
2.3 Hz), 7.53 (1H, d, CH@, J = 16.2 Hz), 7.57 (2H, d,
2Ar, J = 8.4 Hz), 7.65 (2H, d, 2Ar, J = 8.4 Hz). Anal.
calcd for C26H28O3: C, 80.38; H, 7.26. Found: C,
80.34; H, 7.31.
6Ad), 2.09 (9H, s, 9Ad), 3.85 (3H, s, OMe), 7.08 (1H, d,
1Ar, J = 8.0 Hz), 7.44 (1H, d, 1Ar, J = 2.0 Hz), 7.55
(1H, dd, 1Ar, J = 8.0, 2.0 Hz), 7.65 (2H, d, 2Ar,
J = 8.0 Hz), 7.72 (2H, d, 2Ar, J = 8.0 Hz). MS (EI) m/z
342 (MꢁCO2). A small amount (7%) of acid 10c was ob-
tained as a side product of this reaction. 1H NMR
(300 MHz, DMSO-d6) d: 1.70 (6H, s, 6Ad), 2.05 (3H, s,
3Ad), 2.10 (6H, s, 6Ad), 3.85 (3H, s, OMe), 7.05 (1H, d,
1Ar, J = 8.3 Hz), 7.43 (1H, d, 1Ar, J = 1.2 Hz), 7.53
(1H, dd, 1Ar, J = 8.3, 1.2 Hz), 7.70 (2H, d, 2Ar,
J = 8.3 Hz), 7.96 (2H, d, 2Ar, J = 8.3 Hz), 8.25 (1H, s,
CH@). Anal. calcd for C26H27BrO3: C, 66.81; H, 5.82.
Found: C, 66.76; H, 5.92.
6.4.6. Z-2-Bromo-3-[40-methoxy-30-(adamantan-1-yl)biphe-
nyl-4-yl]-acrylic acid ethyl ester (10a) and E-2-bromo-3-
[40-methoxy-30-(adamantan-1-yl)biphenyl-4-yl]-acrylic acid
ethyl ester (10b). A solution of 2.78 g (8.02 mmol) of
aldehyde 8 and 3.43 g (8.02 mmol) of carbethoxybro-
momethylenetriphenylphosphorane in 43 mL of chloro-
form was refluxed for 14 h under nitrogen. The solvent
was evaporated and the residue was purified by flash
chromatography (hexane/dichloromethane 45:55) to ob-
tain 2.89 g of the Z diastereoisomer (10a), and 700 mg of
the E diastereoisomer (10b). Yield 87%, mp (Z) 129 ꢁC,
mp (E) 135 ꢁC. 1H NMR Z isomer (300 MHz, CDCl3) d:
1.42 (3H, t, Et, J = 6.0 Hz), 1.83 (6H, s, 6Ad), 2.14 (9H,
s, 9Ad), 3.90 (3H, s, OMe), 4.37 (2H, q, Et, J = 6.0 Hz),
6.97 (1H, d, 1Ar, J = 8.0 Hz), 7.45 (1H, dd, 1Ar, J = 8.0,
2.2 Hz), 7.54 (1H, d, 1Ar, J = 2.2 Hz), 7.65 (2H, d, 2Ar,
J = 8.4 Hz), 7.95 (2H, d, 2Ar, J = 8.4 Hz), 8.25 (1H, s,
CH@). Anal. calcd for C28H31BrO3: C, 67.88; H, 6.31.
Found: C, 67.73; H 6.39.
6.4.9.
N-[5-Bromo-2-hydroxy-3-(adamantan-1-yl)-ben-
zyl]acetamide (12). A finely pulverized mixture of 2-(1-
adamantanyl)-4-bromophenol (7) (500 mg, 163 mmol)
and N-(hydroxymethyl)acetamide (145 mg, 1.63 mmol)
was added portionwise, at 10 ꢁC while stirring, to
1.63 mL of a solution of CH3COOH/H2SO4 concd 9:1.
After the mixture was stirred at room temperature for
5 days, ice was added and the yellow solid was filtered
and washed with water. Purification by flash chromato-
graphy (hexane/ethyl acetate 60:40) gave 225 mg (37%)
1
of the pure product, mp 216 ꢁC H NMR (300 MHz,
CDCl3) d: 1.77 (6H, s, 6Ad), 2.04 (3H, s, 3Ad), 2.05
(3H, s, CH3), 2.10 (6H, s, 6Ad), 4.25 (2H, d, CH2N,
J = 6.8 Hz), 6.28 (1H, t, NH, J = 6.8 Hz), 7.05 (1H, d,
1Ar, J = 2.6 Hz), 7.23 (1H, d, 1Ar, J = 2.6 Hz). Anal.
calcd for C19H24BrNO2: C, 60.32; H. 6.39; N. 3.70.
Found: C, 60.45; H, 6.32; N, 3.75.
1H NMR E isomer (300 MHz, CDCl3) d: 1.42 (3H, t, Et,
J = 5.0 Hz), 1.80 (6H, s, 6Ad), 2.10 (3H, s, 3Ad), 2.15
(6H, s, 6Ad), 3.87 (3H, s, OMe), 4.25 (2H, q, Et,
J = 5.0 Hz), 6.93 (1H, d, 1Ar, J = 8.3 Hz), 7.33 (2H, d,
2Ar, J = 8.5 Hz), 7.37 (1H, s, CH@), 7.40 (1H, dd,
1Ar, J = 8.3, 2.2 Hz), 7.48 (1H, d, 1Ar, J = 2.2 Hz),
7.52 (2H, d, 2Ar, J = 8.5 Hz). Anal. calcd for
C28H31BrO3: C, 67.88; H. 6.31. Found: 67.71; H, 6.34.
6.4.10. E-3-[30-(Acetylaminomethyl)-40-hydroxy-50-(ada-
mantan-1-yl)biphenyl-4-yl]-acrylic acid (13). An amount
of 195 mg (0.516 mmol) of 12 was reacted with methyl
p-bromocinnamate according to the general procedure
A. Purification by flash chromatography (ethyl acetate/
dichloromethane 9:1) afforded 103 mg (43%) of 3-[30-
(acetylaminomethyl)-40-hydroxy-50-(adamantan-1-yl)bi-
phenyl-4-yl]-acrylic acid methyl ester, mp 163 ꢁC. 1H
NMR (300 MHz, CDCl3) d: 1.80 (6H, s, 6Ad), 2.05
(3H, s, OAc), 2.08 (3H, s, 3Ad), 2.20 (6H, s, 6Ad),
3.80 (3H, s, OMe), 4.40 (2H, d, CH2N, J = 6.0 Hz),
6.32 (1H, t, NH, J = 6.0 Hz), 6.69 (1H, d, CH@,
J = 15.8 Hz), 7.19 (1H, d, 1Ar, J = 2.6 Hz), 7.44 (1H,
d, 1Ar, J = 2.6 Hz), 7.54–7.58 (4H, m, 4Ar), 7.72 (1H,
d, CH@, J = 15.8 Hz).
6.4.7.
E-3-(30-Adamantan-1-yl-40-methoxy-biphenyl-4-
yl)-2-bromo-acrylic acid (10d). The ester 10b (150 mg,
0.303 mmol) was hydrolyzed according to the general
procedure B for one night in the dark. The precipitated
solid was filtered to give 126 mg (89%) of the pure
1
product, mp 234 ꢁC, H NMR (300 MHz, DMSO/d6)
d: 1.70 (6H, s, 6Ad), 2.00 (3H, s, 3Ad), 2.08 (6H, s,
6Ad), 3.80 (3H, s, OMe), 7.05 (1H, d, 1Ar,
J = 8.5 Hz), 7.35 (1H, s, CH@), 7.40 (1H, d, 1Ar,
J = 2.4 Hz), 7.42 (2H, d, 2Ar, J = 8.4 Hz), 7.50 (1H,
dd, 1Ar, J = 8.5, 2.4 Hz), 7.65 (2H, d, 2Ar, J =
8.4 Hz). Anal. calcd for C26H27BrO3: C, 66.81; H,
5.82. Found: C, 66.90; H, 5.87.
The above ester (100 mg, 0.218 mmol) was hydrolyzed
as described in the general procedure B, to obtain
70 mg (72%) of the pure product, mp 264 ꢁC. 1H
NMR (300 MHz, acetone-d6) d: 1.83 (6H, s, 6Ad),
2.07 (3H, s, 3Ad), 2.25 (6H, s, 6Ad), 4.36 (2H, d,
CH2N, J = 6.0 Hz), 6.53 (1H, d, CH@, J = 15.8 Hz),
7.40 (1H, d, 1Ar, J = 2.3 Hz), 7.48 (1H, d, 1Ar,
J = 2.3 Hz), 7.64 (2H, d, 2Ar, J = 8.3 Hz), 7.69 (1H, d,
CH@, J = 15.8 Hz), 7.73 (2H, d, 2Ar, J = 8.3 Hz), 8.37
(1H, t, NH, J = 6.0 Hz), 10.41 (1H, s). MS (EI) m/z
445 (55, M+), 386 (100).
6.4.8. [40-Methoxy-30-(adamantan-1-yl)biphenyl-4-yl]pro-
pynoic acid (11) and Z-3-(30-(Adamantan-1-yl)-40-meth-
oxy-biphenyl-4-yl)-2-bromo-acrylic acid (10c). A suspension
of 2.89 g (5.85 mmol) of the ester 10a in 16 mL of a 25%
solution of potassium hydroxide in methanol was refluxed
for 1 h. The solvent was evaporated and water was added
to the residue, then HCl 12 N was slowly added. The
formed solid was filtered and dried. Crystallization from
diisopropylether gave 1.5 g (68%) of compound 11, mp
6.4.11. N-[5-Bromo-2-hydroxy-3-(adamantan-1-yl)-ben-
zyl]-2-chloroacetamide (14). A finely pulverized mixture
of 2-(adamantan-1-yl)-4-bromophenol (7) (500 mg,
1
246 ꢁC. H NMR (300 MHz, DMSO-d6) d: 1.75 (6H, s,

R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
4871
163 mmol) and 2-chloro-N-(hydroxymethyl)acetamide
(201 mg, 1.63 mmol) was added portionwise, at 10 ꢁC
while stirring, to 1.63 mL of a solution of CH3COOH/
H2SO4 concd 9:1. After the mixture was stirred at room
temperature for 20 h, ice was added and the white solid
was filtered and washed with water, to obtain 586 mg
(87%) of the pure product, mp 154 ꢁC. 1H NMR
(300 MHz, CDCl3) d: 1.75 (6H, s, 6Ad), 2.10 (9H, s,
9Ad), 4.10 (2H, s, CH2Cl), 4.35 (2H, d, CH2N,
J = 6.0 Hz), 7.13 (1H, d, 1Ar, J = 2.3 Hz), 7.25 (1H, d,
1Ar, J = 2.3 Hz), 7.35 (1H, t, NH, J = 6.0 Hz), 9.0
(1H, br s, OH). Anal. calcd for C19H23BrClNO2: C,
55.29; H, 5.62; N, 3.39. Found: C, 55.40; H, 5.49; N,
3.49.
phy (hexane/ethyl acetate 95:5) to give 1.3 g (60%) of
the title compound, mp 189 ꢁC. H NMR (300 MHz,
1
CDCl3) d: 1.8 (6H, s, Ad), 1.65 (3H, s, Ad), 2.19 (6H,
s, Ad), 7.40 (2H, d, 2Ar, J = 8.3 Hz), 7.54 (2H, d, 2Ar,
J = 8.3 Hz), 7.56 (1H, d, 1Ar, J = 2.3 Hz), 7.64 (1H, d,
1Ar, J = 2.3 Hz), 9.94 (1H, s, OH), 11.8 (1H, s, CHO).
Anal. calcd for C23H23BrO2: C, 67.16; H, 5.64. Found:
C, 67.22; H, 5.73.
6.4.14. 40-Bromo-3-hydroxymethyl-5-(adamantan-1-yl)-
biphenyl-4-ol (18). To a solution of 16 (300 mg,
0.782 mmol) and 2,6-lutidine (0.799 mg, 0.872ll)
in 1.72 mL of freshly distilled toluene 0.219 mL
(1.87 mmol) were added with a syringe. The reaction
mixture turned yellow with a yellow precipitate. The
mixture was stirred at room temperature for 20 min,
then solid paraformaldehyde (93.8 mg, 3.182 mmol)
was added and the reaction mixture was stirred for
additional 20 min, then heated at 95 ꢁC for 5 h. After
cooling at room temperature, water and 1 N HCl were
added. The aqueous phase was extracted with ethyl
acetate, the extract washed with brine, dried over
Na2SO4, and evaporated. The crude product was puri-
fied by flash chromatography (hexane/ethyl acetate
85:15) to give 110 mg (34%) of the title compound,
mp 178 ꢁC. 1H NMR (300 MHz, CDCl3) d: 1.83
(6H, s, 6Ad), 2.16 (3H, s, 3Ad), 2.24 (6H, s, 6Ad),
4.93 (2H, s, CH2OH), 7.08 (1H, d, 1Ar, J = 1.6 Hz),
7.38 (1H, d, 1Ar, J = 1.6 Hz), 7.40 (2H, d, 2Ar,
J = 8.2 Hz), 7.53 (2H, d, 2Ar, J = 8.2 Hz). Anal. calcd
for C23H25BrO2: C, 66.83; H, 6.10. Found: 66.96; H,
6.09.
6.4.12. E-3-{30-[(2-Chloroacetylamino)-methyl]-40-hydro-
xy-50-(adamantan-1-yl)biphenyl-4-yl}-acrylic acid (15).
An amount of 3.25 g (7.87 mmol) of 14 was reacted with
methyl 4-bromocinnamate according to the general pro-
cedure C. Purification by flash chromatography (dichlo-
romethane) gave 866 mg (27%) of 3-{30-[(2-
chloroacetylamino)-methyl]-40-hydroxy-50-(adamantan-
1-yl)biphenyl-4-yl}-acrylic acid methyl ester, mp 127 ꢁC.
1H NMR (300 MHz, CDCl3) d: 1.78 (6H, s, 6Ad), 2.08
(3H, s, 3Ad), 2.19 (6H, s, 6Ad), 3.82 (3H, s, OMe),
4.13 (2H, s, CH2Cl), 4.47 (2H, d, CH2N, J = 6.0 Hz),
6.44 (1H, d, CH@, J = 15.0 Hz), 7.25 (1H, d, 1Ar,
J = 2.3 Hz), 7.40 (1H, t, NH, J = 6.0 Hz), 7.45 (1H, d,
1Ar, J = 2.3 Hz), 7.54–7.60 (4H, m, 4Ar), 7.72 (1H, d,
CH@, J = 15.0 Hz), 9.07 (1H, s, OH).
To a solution of 850 mg (1.72 mmol) of the above ester
in 34 mL of dioxane, 10.2 mL of concentrated HCl was
added. The solution was refluxed for 23 h. The solvent
was evaporated, the residue taken up with water, and
the solid formed was filtered and dried, to obtain
700 mg (92%) of the pure product, mp >300 ꢁC. 1H
NMR (300 MHz, DMSO-d6) d: 1.72 (6H, s, 6Ad), 2.03
(3H, s, 3Ad), 2.14 (6H, s, 6Ad), 4.09 (2H, q, CH2N,
J = 5.3 Hz), 6.54 (1H, d, CH@ J = 15.8 Hz), 7.43 (1H,
d, 1Ar, J = 2.3 Hz), 7.56 (1H, d, 1Ar, J = 2.3 Hz), 7.58
(1H, d, CH@, J = 15.8 Hz), 7.64 (2H, d, 2Ar,
J = 7.1 Hz), 7.73 (2H, d, 2Ar, J = 7.1 Hz), 8.15 (3H, br
s), 8.92 (1H, s). Anal. calcd for C26H30ClNO3 : C,
70.98; H, 6.87; N, 3.18. Found: C, 71.09; H, 6.80; N,
3.10.
6.4.15. 40-Bromo-5-(adamantan-1-yl)biphenyl-3,4-diol (19).
An amount of 300 mg (0.73 mmol) of compound 17 was
dissolved in 3 mL of dry dichloromethane and then
180 mg (0.73 mmol) of 3-chloroperoxybenzoic acid was
added. The mixture was refluxed for 20 h. The solvent
was evaporated, the residue was dissolved in ethyl ace-
tate, washed twice with NaHCO3 (20%), brine, and
dried over Na2SO4. Purification by flash chromatogra-
phy (hexane/ethyl acetate 90:10) afforded 155 mg
(53%) of the pure product, mp 178 ꢁC. 1H NMR
(300 MHz, CDCl3) d: 1.78 (6H, s, 6Ad), 2.10 (3H,s,
3Ad), 2.15 (6H, s, 6Ad), 6.90 (1H, d, 1Ar, J = 2.3 Hz),
7.00 (1H, d, 1Ar, J = 2.3 Hz), 7.35 (2H, d, 2Ar,
J = 8.3 Hz), 7.48(2H, d, 2Ar, J = 8.3 Hz). Anal. calcd
for C22H23BrO2: C, 66.17; H, 5.81. Found: C, 66.30;
H, 5.97.
6.4.13. 40-Bromo-4-hydroxy-5-(adamantan-1-yl)biphenyl-
3-carbaldehyde (17). To a solution of 2 g (5.22 mmol) of
4-(40-bromophenyl)-2-(adamantan-1-yl)-phenol (16) and
1.12 g (1.22 mL, 10.44 mmol) of 2,6-lutidine in 11 mL of
freshly distilled toluene, 0.68 g (2.61 mmol, 0.3 mL) of
SnCl4 was added over 10 min under a nitrogen atmo-
sphere. A pale yellow precipitate was formed. The mix-
ture was allowed to stir at room temperature for 20 min,
then solid paraformaldehyde (0.626 g, 20.88 mmol) was
added in one portion and the reaction mixture was stir-
red for additional 10 min, then heated at 90–95 ꢁC for
7 h. After having cooled at room temperature, 30 mL
of water and 6 mL of HCl 1 M were added. The aqueous
phase was extracted with ethyl acetate, the extract
washed with brine, dried over Na2SO4, and evaporated.
The crude product was purified by flash chromatogra-
6.4.16. E-3-[30,40-Dimethoxy-50-(adamantan-1-yl)biphe-
nyl-4-yl]-acrylic acid (20). An amount of 150 mg
(0.376 mmol) of compound 19 was reacted with methyl
acrylate (51.7 mg, 0.602 mmol) according to the general
procedure A. The crude product was purified by flash
chromatography (dichloromethane/methanol 97:3) to
give 127 mg (84%) of the highly unstable 3-[30,40-dihy-
droxy-50-(1-adamantyl)-biphenyl-4-yl]-acrylic acid methyl
1
ester, mp 208 ꢁC. H NMR (300 MHz, CDCl3) d: 1.79
(6H, s, 6Ad), 2.10 (3H,s, 3Ad), 2.20 (6H, s, 6Ad), 3.81
(3H, s, OMe), 6.42 (1H, d, CH@, J = 16.0 Hz), 6.98
(1H, d, 1Ar, J = 2.3 Hz), 7.05 (1H, d, 1Ar, J = 2.3 Hz),
7.48 (4H, m, 4Ar), 7.70(1H, d, CH@, J = 16.0 Hz).

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R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
To a suspension of sodium hydride (60% in mineral oil,
14 mg, 0.360 mmol) in 0.4 mL of DMF, 50 mg
(0.124 mmol) of the above ester was slowly added, while
maintaining the temperature at 20 ꢁC. The mixture was
then stirred at room temperature for 1 h, then methyl io-
dide (100 lL, 0.322 mmol) was added. After stirring for
an additional hour, the mixture was poured into water
and extracted with ethyl acetate. The organic phase
was dried over Na2SO4, filtered, and evaporated. The
residue was purified by flash chromatography (hexane:
ethyl acetate 85:15) to give 10 mg (19%) of 3-[30,40-dime-
thoxy-50-(adamantan-1-yl)biphenyl-4-yl]-acrylic acid methyl
obtained after purification by flash chromatography
(hexane/ethyl acetate 3:7) and crystallization from diiso-
propylether. Yield 66%, mp 245 ꢁC. 1H NMR
(300 MHz, CDCl3) d: 1.56 (6H, s, Ad), 2.07 (3H, s,
Ad), 2.20 (6H, s, Ad), 3.58 (3H, s, OMe), 6.46 (1H, d,
CH@, J = 15.0 Hz), 7.59 (m, 4H, 4Ar), 7.60 (1H, d,
1Ar, J = 2.3 Hz), 7.70 (1H, d, 1Ar, J = 2.3 Hz), 7.73
(1H, d, CH@, J = 15.0 Hz), 9.95 (1H, s, OH), 11.9
(1H, s, CHO).
The above ester was hydrolyzed as described in general
procedure B to obtain the corresponding acid, yield
1
1
ester, mp 117 ꢁC. H NMR (300 MHz, CDCl3) d: 1.79
83%, mp > 300 ꢁC. H NMR (300 MHz, DMSO-d6) d:
(6H, s, 6Ad), 2.10 (3H,s, 3Ad), 2.14 (6H, s, 6Ad), 3.81
(3H, s, OMe), 3.91 (6H, s, 2 OMe), 6.45 (1H, d, CH@,
J = 15.7 Hz), 7.00 (1H, d, 1Ar, J = 2.2 Hz), 7.08 (1H,
d, 1Ar, J = 2.2 Hz), 7.57 (4H, m, 4Ar), 7.70 (1H, d,
CH@, J = 16.0 Hz), MS (EI) m/z 432 (M+).
1.54 (6H, s, Ad), 2.08 (3H, s, Ad), 2.16 (6H, s, Ad),
6.55 (1H, d, CH@, J = 16.0 Hz), 7.59 (1H, d, CH@,
J = 16.0 Hz), 7.66–8.18 (5H, m, 5Ar), 8.03 (1H, d,
1Ar, J = 2.3 Hz), 10.05 (1H, s, CHO), 12.05 (1H, s,
OH). MS (EI) m/z 402 (M+).
The above ester (24 mg, 0.055 mmol) was hydrolyzed
according to the general procedure B for 4 days in the
dark. The precipitated solid was filtered to give 20 mg
(87%) of the pure product, mp 215 ꢁC, 1H NMR
(300 MHz, acetone-d6) d: 1.83 (6H, s, 6Ad), 2.08
(3H,s, 3Ad), 2.16 (6H, s, 6Ad), 3.90 (3H, s, OMe),
3.96 (3H, s, OMe), 6.55 (1H, d, CH@, J = 15.8 Hz),
7.17 (1H, d, 1Ar, J = 2.2 Hz), 7.25 (1H, d, 1Ar,
J = 2.2 Hz), 7.70–7.98 (4H, m, 4Ar), 7.93 (1H, d,
CH@, J = 15.8 Hz). MS (EI) m/z 418 (M+).
6.4.19. 40-Bromo-4-methoxy-5-(adamantan-1-yl)biphenyl-
3-ol (23). A mixture of 17 (345 mg, 0.839 mmol), MeI
(502 mg, 3.54 mmol), and K2CO3 (167 mg, 1.21 mmol)
in dry acetone (15 mL) was refluxed for 6 h. The solvent
was evaporated, the residue was suspended in water and
extracted with ethyl acetate. The organic phase was
dried over Na2SO4, filtered, and evaporated to give
322 mg (90%) of 40-bromo-4-methoxy-5-(adamantan-1-
yl)biphenyl-3-carbaldehyde, mp. 145 ꢁC. 1H NMR
(300 MHz, CDCl3) d: 1.75 (6H, s, Ad), 2.14 (9H, s,
Ad), 3.72 (3H, s, OMe), 7.19 (2H, d, 2Ar, J = 8.3 Hz),
7.54 (2H, d, 2Ar, J = 8.3 Hz), 7.69 (1H, d, 1Ar,
J = 1.9 Hz), 7.87 (1H, d, 1Ar, J = 1.9 Hz), 10.2 (1H, s,
CHO).
6.4.17. E-3-[40-Hydroxy-30-hydroxymethyl-50-(adaman-
tan-1-yl)biphenyl-4-yl]-acrylic acid (21). An amount of
100 mg (0.242 mmol) of 18 was reacted with methyl
acrylate (33.3 mg, 0.387 mmol) as described previously
(B). The residue was purified by flash chromatography
(hexane/ethyl acetate 70: 30) to give 33 mg (33%) of
the pure 3-[40-hydroxy-30-hydroxymethyl-50-(adaman-
tan-1-yl)biphenyl-4-yl]-acrylic acid methyl ester, mp
205 ꢁC, 1H NMR (300 MHz, CDCl3) d: 1.92 (6H, s,
6Ad), 2.13 (3H,s, 3Ad), 2.20 (6H, s, 6Ad), 3.95 (3H, s,
OMe), 4.90(2H, s, CH2OH), 6.43 (1H, d, CH@,
J = 16.0 Hz), 7.10 (1H, d, 1Ar, J = 1.6 Hz), 7.42 (1H,
d, 1Ar, J = 1.6 Hz), 7.53–7.60 (4H, m, 4Ar), 7.70 (1H,
d, CH@, J = 16.0 Hz), 7.94 (1H, s).
To 220 mg (0.517 mmol) of the above aldehyde dis-
solved in 2 mL of dry dichloromethane, 354 mg
(1.034 mmol) of 3-chloroperoxybenzoic acid (70%) was
added portionwise. The solution was refluxed for 1 h.
A yellow solid formed during the reaction. Dichloro-
methane was removed and the residue was dissolved in
ethyl acetate, washed twice with NaHCO3 (20%), with
brine, and then dried over Na2SO4. Purification by flash
chromatography (hexane/ethyl acetate 97:3) gave 66 mg
(21%) of the phenol 23 and 85 mg (26%) of formic acid
5-adamantan-1-yl-40-bromo-4-methoxy-biphenyl-3-yl es-
1
The above ester (33 mg, 0.0789 mmol) was hydrolyzed
as described before (B) to obtain 29 mg (91%) of the
pure acid, mp > 300 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d: 1.73 (6H, s, 6Ad), 2.03 (3H,s, 3Ad),
2.14 (6H, s, 6Ad), 5.88 (1H, t, –OH, J = 5.3 Hz),
4.66 (2H, d, CH2OH, J = 5.3 Hz), 6.50 (1H, d,
CH@, J = 15.7 Hz), 7.30 (1H, d, 1Ar, J = 1.6 Hz),
7.35(1H, d, 1Ar, J = 1.6 Hz), 7.58 (1H, d, CH@,
J = 15.7 Hz), 7.60 (2H, d, 2Ar, J = 8.3 Hz), 7.68 (2H,
d, 2Ar, J = 8.3 Hz), 8.85 (1H, s, OH) 12.3 (1H, br s,
COOH). MS (EI) m/z 404 (30), 386 (100), 305 (70),
240 (57), 165 (20).
ter, mp 103 ꢁC. H NMR (300 MHz, CDCl3) d: 1.55
(6H, s, Ad), 2.12 (9H, s, Ad), 3.87 (3H, s, OMe), 7.15
(1H, d, 1Ar, J = 2.3 Hz), 7.33 (1H, d, 1Ar, J = 2.3 Hz),
7.38 (2H, d, 2Ar, J = 8.3 Hz), 7.55 (2H, d, 2Ar,
J = 8.3 Hz), 8.30 (1H, s).
A solution of 80 mg (0.181 mmol) of the above com-
pound in 4 mL of methanol was treated with 100 lL
of 10% KOH. After stirring for 1 h, the solvent was re-
moved, the residue was suspended in water and acidified
with 10% HCl. The formed yellow solid was filtered and
dried. The title compound was obtained quantitatively
1
(75 mg). H NMR (300 MHz, CDCl3) d: 1.78 (6H, s,
6.4.18. E-3-[30-Formyl-40-hydroxy-50-(adamantan-1-yl)-
biphenyl-4-yl]-acrylic acid (22). Compound 17 was
subjected to Heck condensation as described in general
procedure A. The pure 3-(30-formyl-40-hydroxy-50-(ada-
mantan-1-yl)biphenyl-4-yl)-acrylic acid methyl ester was
Ad), 2.12 (9H, s, Ad), 3.85 (3H, s, OMe), 7.00 (2H, d,
2Ar, J = 2.2 Hz), 7.40 (2H, d, 2Ar, J = 8.3 Hz), 7.55
(2H, d, 2Ar, J = 8.3 Hz), 8.30 (1H, s). Anal. calcd for
C23H25BrO2: C, 66.83; H, 6.10. Found: C, 67.00; H,
6.01.

R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
4873
6.4.20.
E-3-[30-Hydroxy-40-methoxy-50-(adamantan-1-
d: 1.73 (6H, s, 6Ad), 2.00 (3H,s, 3Ad), 2.12 (6H, s,
6Ad), 3.86 (3H, s, OMe), 6.50 (1H, d, CH@,
J = 16.0 Hz), 7.00 (1H, d, 1Ar, J = 1.9 Hz), 7.58 (1H,
d, CH@, J = 16.0 Hz), 7.63 (2H, d, J = 7.8 Hz), 7.70
(2H, d, J = 7.8 Hz), 8.54 (1H, s), 12.3 (1H, br s); MS
(EI) m/z 404 (M+).
yl)biphenyl-4-yl]-acrylic acid (24). Compound 23
(125 mg, 0.302 mmol) was reacted with methyl acrylate
(41.5 mg, 0.483 mmol) according to the general proce-
dure A. Purification by flash chromatography (hexane/
ethyl acetate 80:20) afforded 84 mg (67%) of 3-(30-hydro-
xy-40-methoxy-50-[(adamantan-1-yl)-biphenyl-4-yl]-ac-
rylic acid methyl ester, mp 197 ꢁC. 1H NMR (300 MHz,
CDCl3) d: 1.80 (6H, s, Ad), 2.11 (9H, s, Ad), 3.82 (3H, s,
OMe), 6.45 (1H, d, CH@, J = 15.8 Hz), 7.08 (2H, d,
2Ar, J = 2.2 Hz), 7.55 (4H, m, 4 Ar), 7.70 (1H, d,
CH@, J = 15.8 Hz).
6.4.23.
6-Bromo-4-(adamantan-1-yl)benzo[1,3]dioxole
(29a). To a solution of 4-bromo-1,2-methylenedioxyben-
zene (28a) (1.5 g, 7.31 mmol) and 1-adamantanol
(1.11 g, 7.31 mmol) in 3.7 mL of dichloromethane,
0.4 mL of concentrated sulfuric acid was added. The
mixture was stirred for 4 h at room temperature, poured
into water, neutralized with sodium bicarbonate, and ex-
tracted with dichloromethane. The solvent was dried
and evaporated. Crystallization from diisopropylether
gave 1.06 g (47%) of the desired compound, mp
133 ꢁC. 1H NMR (300 MHz, CDCl3) d: 1.80 (6H, s,
6Ad), 2.01 (6H, s, 6Ad), 2.12 (3H, s, 3Ad), 5.95 (2H,
s, OCH2), 6.87 (2H, d, 2Ar, J = 2.8 Hz). Anal. calcd
for C17H19BrO2: C, 60.91; H, 5.71. Found: 61.02; H,
5.80.
The above ester (57 mg, 0.136 mmol) was treated with
7.8 mL of a 0.7 N NaOH solution in methanol under re-
flux for 2 h. After evaporation of methanol and addition
of water, the mixture was acidified with 6 N HCl and the
solid was filtered. Purification by flash chromatography
(ethyl acetate/hexane 95:5) gave 10 mg (20%) of the pure
1
product, mp >300 ꢁC, H NMR (300 MHz, acetone-d6)
d: 1.83 (6H, s, Ad), 2.08 (3H, s, 3Ad), 2.28 (6H, s, 6Ad),
3.92 (3H, s, OMe), 6.55 (1H, d, CH@, J = 15.0 Hz), 7.08
(1H, d, 1Ar, J = 2.3 Hz), 7.14 (1H, d, 1Ar, J = 2.3 Hz),
7.67 (2H, d, 2Ar, J = 8.3 Hz), 7.70 (1H, d, CH@,
J = 15.0 Hz), 7.72 (2H, d, 2Ar, J = 8.3 Hz). Anal. calcd
for C26H28O4: C, 77.20; H, 6.98. Found: C, 77.26; H,
7.07.
6.4.24. 4-[7-(Adamantan-1-yl)benzo[1,3]dioxol-5-yl]benz-
aldehyde (30). To a solution of 875 mg (2.61 mmol) of
29a in 5.3 mL of toluene, 2.61 mL (5.22 mmol) of a
2 N aqueous solution of Na2CO3, 90.5 mg (0.08 mmol)
of Pd(PPh3)4, and 430 mg (2.87 mmol) of 4-formylben-
zeneboronic acid (previously suspended in 1.2 mL of
ethanol) were added. The mixture was refluxed under
nitrogen for 7 h, then extracted with ethyl acetate. The
organic phase was washed with brine, dried, and evapo-
rated. The crude product was purified by flash chroma-
tography (hexane/ethyl acetate 9:1) and crystallized
from diethyl ether to give 658 mg (70%) of the desired
6.4.21.
4-Bromo-2-methoxy-6-(adamantan-1-yl)phenol
(26). To a solution of 408 mg (1.97 mmol) of 4-bro-
moguaiacol (25) and 300 mg (1.97 mmol) of 1-adamant-
anol in 1 mL of dichloromethane, 108 mL (1.97 mmol)
of concd sulfuric acid (98%) was added. The mixture
was stirred for 1 h at room temperature, poured into
water, neutralized with sodium bicarbonate, and ex-
tracted with dichloromethane.The organic phase was
then dried over Na2SO4, filtered, and evaporated. The
residue was purified by flash chromatography (hexane/
ethyl acetate 95:5) to give 394 mg (60%) of the pure
1
aldehyde, mp 205 ꢁC. H NMR (300 MHz, CDCl3) d:
1.80 (6H, s, 6Ad), 2.09 (9H, s, 9Ad), 6.02 (2H, s,
OCH2), 7.01 (1H, d, 1Ar, J = 1.9 Hz), 7.04 (1H, d,
1Ar, J = 1.9 Hz), 7.68 (2H, d, 2Ar, J = 8.2 Hz), 7.92
(2H, d, 2Ar, J = 8.2 Hz), 10.07 (1H, s). Anal. calcd for
C24H24O3: C, 79.97; H, 6.71. Found: C, 80.12; H, 6.67.
1
product, mp 159–160 ꢁC, H NMR (300 MHz, CDCl3)
d: 1.78 (6H, s, 6Ad), 2.08 (9H, s, 9Ad), 3.85 (3H, s,
OMe), 5.90 (1H, s), 6.85 (1H, d, 1Ar, J = 2.2 Hz), 6.93
(1H, d, 1Ar, J = 2.2 Hz). Anal. calcd for C17H21BrO2:
C, 60.54; H, 6.28. Found: C, 60.63; H, 6.19.
6.4.25.
E-3-{4-[7-(Adamantan-1-yl)benzo[1,3]dioxol-5-
yl]-phenyl}-acrylic acid (31a). A solution of 300 mg
(0.832 mmol) of 30 and 278 mg (0.832 mmol) of meth-
oxycarbonylmethylenetriphenylphosphorane in 4.5 mL
of chloroform was refluxed under nitrogen for 5 h.
The solvent was evaporated and the residue was purified
by flash chromatography (hexane/dichloromethane
45:55) to give 298 mg (86%) of 3-{4-[7-(adamantan-1-
yl)benzo[1,3]dioxol-5-yl]-phenyl}-acrylic acid methyl es-
6.4.22. E-3-[40-hydroxy-30-methoxy-50-(adamantan-1-yl]-
biphenyl-4-yl]-acrylic acid (27). An amount of 200 mg
(0.593 mmol) of compound 26 was reacted with methyl
4-bromocinnamate (287 mg, 1.13 mmol) as described
in the general procedure C. Purification by flash chro-
matography (dichloromethane/hexane 50:50) gave
172 mg (70%) of 3-[40-hydroxy-30-methoxy-50-(adaman-
tan-1-yl)biphenyl-4-yl]acrylic acid methyl ester, mp
190 ꢁC. 1H NMR (300 MHz, CDCl3) d: 1.80 (6H, s,
6Ad), 2.08 (3H,s, 3Ad), 2.20 (6H, s, 6Ad), 3.81 (3H, s,
OMe), 3.95 (3H, s, OMe), 6.08 (1H, s), 6.45 (1H, d,
CH@, J = 16.0 Hz), 6.98 (1H, d, 1Ar, J = 1.9 Hz), 7.08
(1H, d, 1Ar, J = 1.9 Hz), 7.48–7.63 (4H, m, 4Ar), 7.73
(1H, d, CH@, J = 16.0 Hz), MS (EI) m/z 432 (M+).
1
ter, mp 205 ꢁC. H NMR (300 MHz, CDCl3) d: 1.72
(6 H, s, 6Ad), 2.06 (9H, s, 9Ad), 3.80 (3H, s, OMe),
5.97 (2H, s, OCH2O), 6.44 (1H, d, CH@, J = 16.0 Hz),
6.95 (1H, d, 1Ar, J = 1.9 Hz), 6.98 (1H, d, 1Ar,
J = 1.86 Hz), 7.52–7.60 (4H, m, 4Ar), 7.71 (1H, d,
CH@, J = 16.0 Hz).
The above ester (200 mg, 0.48 mmol) was hydrolyzed as
usual (B) to afford 150 mg (78%) of the pure product,
1
The above ester (90 mg, 0.215 mmol) was hydrolyzed as
described above (B) to obtain 85 mg (98%) of the pure
product, mp 218 ꢁC. H NMR (300 MHz, DMSO-d6)
mp > 300 ꢁC, H NMR (300 MHz, DMSO-d6) d: 1.72
(6H, s, 6Ad), 2.01 (9H, s, 9Ad), 6.01 (2H, s, OCH2O),
1
6.52 (1H, d, CH@, J = 16.2 Hz), 6.99 (1H, d, 1Ar,

4874
R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
J = 1.9 Hz), 7.14 (1H, d, 1Ar, J = 1.9 Hz), 7.58 (1H, d,
CH@, J = 16.2 Hz), 7.60 (2H, d, 2Ar, J = 8.5 Hz), 7.70
(2H, d, 2Ar, J = 8.5 Hz), 12.35 (1H, br s, COOH). Anal.
calcd for C26H26O4: C, 77.59; H, 6.51. Found: C, 77.64;
H, 6.42.
CH2O), 6.97 (1H, d, 1Ar, J = 1.9 Hz), 7.00 (1H, d,
1Ar, J = 1.9 Hz), 7.56 (2H, d, 2Ar, J = 8.3 Hz), 7.90
(2H, d, 2Ar, J = 8.3 Hz), 8.22 (1H, s, CH@). Anal. calcd
for C28H29BrO4: C, 66.01; H, 5.74. Found: C, 66.18; H,
5.54.
6.4.26. 7-Bromo-5-(adamantan-1-yl)-2,3-dihydro-benzo-
[1,4]dioxine (29b). To a solution of 4-bromo-1,2-ethylen-
edioxybenzene (500 mg, 2.28 mmol) (28b) and 1-ada-
mantanol (347 mg, 2.28 mmol) in 1.15 mL of
dichloromethane, 0.125 mL (2.28 mmol) of concen-
trated sulfuric acid was added. The mixture was stirred
for 2 h at room temperature, poured into water, neutral-
ized with sodium bicarbonate, and extracted with
dichloromethane. The organic phase was evaporated
to obtain a crude that was purified by flash chromatog-
raphy (hexane/ethyl acetate 95:5) to give 280 mg (35%)
of the pure product, mp 166–168 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d: 1.70 (6H, s, 6Ad), 2.00 (9H,
s, 9Ad), 4.23 (4H, s, OCH2), 6.74 (1H, d, 1Ar,
J = 2.6 Hz), 6.88 (1H, d, 1Ar, J = 2.6 Hz).Anal. calcd
for C18H21BrO2: C, 61.90; H, 6.06. Found: C, 61.85;
H, 6.01.
1H NMR E isomer (300 MHz, CDCl3) d: 1.20 (3H, t,
Et, J = 7.5 Hz), 1.75 (6H, s, 6Ad), 2.02 (9H, s, 9Ad),
4.24 (2H, q, Et, J = 7.5 Hz), 5.97 (2H, s, CH2O), 6.92
(1H, d, 1Ar, J = 1.8 Hz), 6.98 (1H, d, 1Ar,
J = 1.8 Hz), 7.31 (2H, d, 2Ar, J = 8.3 Hz), 7.38 (1H,
s, CH@), 7.49 (2H, d, 2Ar, J = 8.3 Hz). Anal. calcd
for C28H29BrO4: C, 66.01; H, 5.74. Found: C, 66.12;
H, 5.66.
6.4.29. {4-[7-(Adamantan-1-yl)benzo[1,3]dioxol-5-yl]-phe-
nyl}-propynoic acid (34). A suspension of 350 mg
(0.687 mmol) of the ester 32 in 2.4 mL of a 25% solution
of potassium hydroxide in methanol was refluxed for
1 h. The solvent was evaporated and water was added
to the residue, then HCl 12 N was slowly added. The
precipitated solid was filtered and dried. Crystallization
from diisopropylether gave 109 mg (40%) of the desired
compound, mp 204 ꢁC. 1H NMR (300 MHz, DMSO-d6)
d: 1.76 (6H, s, 6Ad), 2.03(9H, s, 9Ad), 6.02 (2H, s,
CH2O), 6.98 (1H, d, 1Ar, J = 1.9 Hz), 7.14 (1H, d,
1Ar, J = 1.9 Hz), 7.54 (2H, d, 2Ar, J = 8.3 Hz), 7.64
(2H, d, 2Ar, J = 8.3 Hz). MS (EI) m/z 356 (MꢁCO2).
6.4.27. 3-{4-[8-(Adamantan-1-yl)-2,3-dihydro-benzo[1,4]-
dioxin-6-yl]-phenyl}-acrylic acid (31b). Suzuki one-pot
condensation between 200 mg (0.573 mmol) of com-
pound 28b with methyl 4-bromocinnamate was carried
out as described in the general part (C). The residue
was purified by flash chromatography (dichlorometh-
ane/hexane 1.1) to give 124 mg (50%) of 3-{4-[8-(ada-
mantan-1-yl)-2,3-dihydro-benzo[1,4]dioxin-6-yl]-phe-
nyl}-acrylic acid methyl ester, mp 209 ꢁC. 1H NMR
(300 MHz, CDCl3) d: 1.78 (6H, s, 6Ad), 2.08 (3H, s,
3Ad), 2.13 (6H, s, 6Ad), 3.83 (3H, s, OMe), 4.33 (4H,
s, OCH2), 6.45 (1H, d, CH@, J = 16.0 Hz), 7.00 (1H,
d, 1Ar, J = 2.3 Hz), 7.05 (1H, d, 1Ar, J = 2.3 Hz),
7.50–7.63 (4H, m, 4Ar), 7.70 (1H, d, CH@, J = 16.0 Hz).
6.4.30. E-2-Bromo-3-[4-(7-adamantan-1-yl)benzo[1,3]diox-
ol-5-yl)-phenyl]-acrylic acid (35). The ester 33 (73 mg,
0.143 mmol) was hydrolyzed as usual (B) to afford
57 mg (89%) of the pure product, mp 239 ꢁC, 1H
NMR (300 MHz, DMSO-d6) d: 1.72 (6H, s, 6Ad), 2.00
(9H, s, 9Ad), 5.98 (2H, s, OCH2), 6.95 (1H, d, 1Ar,
J = 1.2 Hz), 7.15 (1H, d, 1Ar, J = 1.2 Hz), 7.31 (1H, s,
CH@), 7.39 (2H, d, 2Ar, J = 8.5 Hz), 7.56 (2H, d, 2Ar,
J = 8.5 Hz), 13.4 (1H, br s). Anal. calcd for C26H25BrO4:
C, 64.87; H, 5.23. Found: C, 64.98; H, 5.12.
The above ester (70 mg, 0.163 mmol) was hydrolyzed as
usual (B) to afford 62 mg (91%) of the pure product, mp
288 ꢁC. H NMR (300 MHz, DMSO-d6) d: 1.71 (6H, s,
6.5. Cellular sensitivity to drugs
1
6Ad), 2.02 (3H, s, 3Ad), 2.08 (6H, s, 6Ad), 4.25 (4H, s,
OCH2), 6.50 (1H, d, CH@, J = 15.6 Hz), 6.97 (1H, d,
1Ar, J = 1.9 Hz), 7.02 (1H, d, 1Ar, J = 1.9 Hz), 7.56
(1H, d, CH@, J = 15.6 Hz), 7.58 (2H, d, 2Ar,
J = 8.25 Hz), 7.69 (2H, d, 2Ar, J = 8.3 Hz). MS (EI)
m/z 416 (M+).
In ovarian carcinoma cells, cellular sensitivity to drugs
was evaluated by growth-inhibition assay after 72-h
drug exposure. Cells in the logarithmic phase of growth
were seeded in duplicate into 6-well plates. Twenty-four
hours after seeding, the drug was added to the medium.
Cells were harvested 72 h after drug exposure and
counted with a cell counter. In leukemia cells, drug
exposure was 24 h and the growth inhibition was as-
sessed after 72 h. IC50 is defined as the drug concentra-
tion causing a 50% reduction of cell number compared
with that of untreated control.
6.4.28. Z-2-Bromo-3-[4-(7-adamantan-1-yl)benzo[1,3]diox-
ol-5-yl)-phenyl]-acrylic acid ethyl ester (32) and E-2-
bromo-3-[4-(7-adamantan-1-yl)benzo[1,3]dioxol-5-yl)-phe-
nyl]-acrylic acid ethyl ester (33). A solution of 360 mg
(0.999 mmol) of 30 and 472 mg (0.999 mmol) of
ethoxycarbonylbromomethylenetriphenylphosphorane in
5.3 mL of chloroform was refluxed under nitrogen for
8 h. The solvent was evaporated and the crude product
was purified by flash chromatography (hexane/dichloro-
methane 45:55) to give 369 mg (72%) of the Z isomer
(32), mp 150 ꢁC, and 82 mg (16%) of the E isomer
(33), mp 188 ꢁC. 1H NMR Z isomer (300 MHz, CDCl3)
d: 1.39 (3H, t, Et, J = 7.5 Hz), 1.80 (6H, s, 6Ad), 2.09
(9H, s, 9Ad), 4.37 (2H, q, Et, J = 7.5 Hz), 5.98 (2H, s,
6.6. Determination of apoptosis
Apoptosis was determined in ovarian carcinoma
IGROV-1 cells by TUNEL assay following 72 h-expo-
sure to the drug.8 Treated cells were fixed in 4% parafor-
maldehyde, for 60 min, at room temperature, washed
and resuspended in ice-cold PBS. The in situ cell death
detection kit fluorescein (Roche, Mannheim, Germany)
was used according to the manufacturers’ instructions

R. Cincinelli et al. / Bioorg. Med. Chem. 15 (2007) 4863–4875
4875
3. Dallavalle, S.; Zunino, F. Exp. Opin. Ther. Pat. 2005, 15,
1625–1635.
`
4. Garattini, E.; Giannı, M.; Terao, M. Curr. Pharm. Des.
2004, 10, 433–448.
5. Kagechika, H.; Shudo, K. J. Med. Chem. 2005, 48, 5875–
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6. Lotan, R. J. Biol. Regul. Homeost. Agents 2003, 17,
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7. (a) Cincinelli, R.; Dallavalle, S.; Merlini, L.; Penco, S.;
Pisano, C.; Carminati, P.; Giannini, G.; Vesci, L.; Gaet-
ano, C.; Illy, B.; Zuco, V.; Supino, R.; Zunino, F. J. Med.
Chem. 2003, 46, 909–912; (b) Cincinelli, R.; Dallavalle, S.;
Nannei, R.; Carella, S.; De Zani, D.; Merlini, L.; Penco,
S.; Garattini, E.; Giannini, G.; Pisano, C.; Vesci, L.;
Carminati, P.; Zuco, V.; Zanchi, C.; Zunino, F. J. Med.
Chem. 2005, 48, 4931–4946.
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Pisano, C.; Zanier, R.; Giordano, V.; Garattini, E.;
Zunino, F. Cell Death Diff. 2004, 11, 280–289.
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Pisano, C.; Barbarino, M.; Zanier, R.; Bucci, F.; Aulicino,
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and the samples were analyzed by flow cytometry (Bec-
ton Dickinson).
6.7. Cell cycle analysis
The cell cycle distribution was analyzed in propidium io-
dide-stained cells by FACScan flow cytometry, as
described.8
6.8. Western blot analysis
Cells were treated for the indicated times with seven se-
lected compounds (Fig. 2) at a cytotoxic concentration
corresponding to IC80. Analysis of each MAP protein
and their activated (i.e., phosphorylated) form was per-
formed as previously reported.8
6.9. Cytodifferentiation
The nitro blue tetrazolium (NBT) reduction assay16 was
performed on extracts of phorbol myristate acetate-
stimulated cells and the percentage of NBT+ cells was
11. Kotsuki, H.; Datta, P. K.; Hayakawa, H.; Suenaga, H.
Synthesis 1995, 1348–1350.
12. Casiraghi, G.; Casnati, G.; Puglia, G.; Sartori, G.;
Terenghi, G. J. Chem. Soc., Perkin Trans. I 1980, 1862–
1865.
determined with
a
spectrophotometric assay at
540 nm. NB4 tumor cells were exposed at sub-toxic con-
centrations of the novel retinoids for 3 days.
13. Godfrey, J. M.; Sargent, M. V. J. Chem. Soc., Perkin
Trans. 1 1974, 1353–1354.
References and notes
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`
16. Giannı, M.; Kalac¸, Y.; Ponzanelli, I.; Rambaldi, A.;
Terao, M.; Garattini, E. Blood 2001, 97, 3234–3243.