Studies on the apoptotic activity of natural and synthetic retinoids: Discovery of a new class of synthetic terphenyls that potently support cell growth and inhibit apoptosis in neuronal and HL-60 cells

Article abstract of DOI:10.1021/jm049080y

New terphenyl derivatives have been synthesized and tested for their effect on cell survival in serum-free cultures. These compounds protected HL60 cells from death and supported their growth with an activity higher than that of the natural 14-hydroxy-retro-retinol. Terphenyls 26 and 28 also possess antiapoptotic activity on neuronal cells, proving them as possible candidates for the treatment of neurodegenerative and ischemic diseases.

Full text of DOI:10.1021/jm049080y

J. Med. Chem. 2005, 48, 4293-4299
4293
Studies on the Apoptotic Activity of Natural and Synthetic Retinoids:
Discovery of a New Class of Synthetic Terphenyls That Potently Support Cell
Growth and Inhibit Apoptosis in Neuronal and HL-60 Cells
Daniele Simoni,*,# Giuseppe Giannini,† Marinella Roberti,| Riccardo Rondanin,# Riccardo Baruchello,#
Marcello Rossi,# Giuseppina Grisolia,# Francesco Paolo Invidiata,‡ Stefania Aiello,‡ Silvia Marino,§
Sabrina Cavallini,§ Anna Siniscalchi,§ Nicola Gebbia, Lucia Crosta, Stefania Grimaudo,b Vincenzo Abbadessa,b
Antonietta Di Cristina,b and Manlio Tolomeob
Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy,
R & D, Sigma-tau, Pomezia, Italy, Dipartimento di Scienze Farmaceutiche, Universita` di Bologna, Bologna, Italy,
Dipartimento Farmacochimico Tossicologico e Biologico, Universita` di Palermo, Palermo, Italy, Dipartimento di Medicina
Clinica e Sperimentale, Sezione di Farmacologia, Universita` di Ferrara, Ferrara, Italy, Consorzio di Ricerca sul Rischio
Biologico in Agricoltura (Co.Ri.Bi.A.), Palermo, Italy, and Divisione di Ematologia e Servizio A.I.D.S., Policlinico,
Universita` di Palermo, Palermo, Italy
Received November 15, 2004
New terphenyl derivatives have been synthesized and tested for their effect on cell survival in
serum-free cultures. These compounds protected HL60 cells from death and supported their
growth with an activity higher than that of the natural 14-hydroxy-retro-retinol. Terphenyls
26 and 28 also possess antiapoptotic activity on neuronal cells, proving them as possible
candidates for the treatment of neurodegenerative and ischemic diseases.
Introduction
Apoptotic processes are widespread biochemical events,
being involved in, for example, development, differen-
tiation, cell proliferation/homoeostasis, regulation and
function of the immune system, and the removal of
defective and therefore harmful cells.1 Thus, dysfunction
or deregulation of the apoptotic program is implicated
in a variety of pathological conditions. Defects in apo-
ptosis can result in cancer,2,3 autoimmune diseases, and
spreading of viral infections, while neurodegenerative
disorders such as Alzheimer and Parkinson diseases,
AIDS, and ischemic diseases are caused or enhanced
by excessive apoptosis.4,5 Increasing evidence suggests
that inhibition of apoptosis in these severe pathologies
may be beneficial as a therapeutic approach to slow
disease progression and to improve a patient’s pros-
pects.4 To this end, the discovery of new compounds able
Figure 1. Natural retinoids and retro-retinoids.
to modulate the apoptotic process, both as activators or
inhibitors, represents an important challenge in me-
dicinal chemistry.
Retinoids, a class of natural and synthetic compounds
structurally related to vitamin A (1, Figure 1), are
known to modulate cell proliferation, apoptosis, and
retinoic acid (3), are known6 to be involved in cell
differentiation, morphogenesis, proliferation, and anti-
neoplastic processes.
Two other bioactive metabolites of vitamin A, namely,
14-hydroxy-retro-retinol (14-HRR) (4) and anhydroret-
inol (AR) (5), are involved in cell proliferation, with
growth-supporting properties for normal lymphocyte
proliferation being ascribed to 4, while 5 was discovered
to act as a natural antagonist triggering growth arrest
and death by apoptosis.7,8 A mutually reversible rela-
tionship exists between these two retro-retinoids be-
cause one can reverse the effects of the other when given
in pharmacological doses.9-11 Another reported metabo-
lite of vitamin A sharing similar activity as 4 is the 13,-
14-dihydroxy retinol (DHR, 6).12 Exploitation of this
relationship holds much promise for therapeutic control
of diseases such as cancer.
differentiation, with different effects depending on the
cellular context. Thus, all-trans-retinoic acid (ATRA, 2)
and its naturally occurring retinoid analogue 9-cis-
* To whom correspondence should be addressed. Phone: +39-0532-
291291. Fax: +39-0532-291296. E-mail: smd@dns.unife.it.
# Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
† Sigma-tau.
| Universita` di Bologna.
‡ Dipartimento Farmacochimico Tossicologico e Biologico, Universita`
di Palermo.
§ Dipartimento di Medicina Clinica e Sperimentale, Universita` di
Ferrara.
Consorzio di Ricerca sul Rischio Biologico in Agricoltura
(Co.Ri.Bi.A.).
b Divisione di Ematologia e Servizio A.I.D.S., Policlinico, Universita`
di Palermo.
10.1021/jm049080y CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/03/2005

4294 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Simoni et al.
Scheme 1a
a (a) (DHQ)2PHAL, K3Fe(CN)6, K2CO3, K2OsO4, CH3SO2NH2,
t-BuOH-H2O; (b) Ac2O, Et3N, DMAP, CH2Cl2; (c) SOCl2, DiPEA,
CH2Cl2 , -90 °C; (d) K2CO3, BHT, MeOH.
Suzuki reaction between 18 and bromoiodobenzene
(ortho, meta, or para isomers) in a mixture of toluene-
ethanol in the presence of a catalytic amount of tetrakis-
triphenylphosphine palladium with aqueous sodium
carbonate solution produced the desired biphenyl alde-
hydes (19-21) in good yields. The subsequent oxidation
led to the corresponding carboxylic acids (22-24), which
underwent Suzuki cross-coupling with the boronic acid
25 in acetonitrile to give the required 26-28 (Scheme
2).
Figure 2. Synthesized retinoids and retro-retinoids.
Our continuous interest13-15 in the field led us to
discover that the apoptotic activity of TTNPB (7)16 and
cis-TTNPB (8)17 (Figure 2) was strictly connected with
the properties of the alkenyl portion.13
In this research context, we have ascertained that
arotinoids such as 918 and the cis derivative 1019 are
not able to activate apoptosis in HL60 cells. Moreover,
9 is surprisingly able to induce cell growth by preventing
apoptosis at 1 × 10-6 M, reproducing to some extent
the growth-supporting properties hitherto ascribed only
to the natural 14-HRR (4) and DHR (6).11,12 We found
a similar unexpected activity for 11,20 another derivative
that we synthesized taking into account the presence
of the trimethoxyphenyl moiety in many bioactive
molecules but not for its cis stereisomer 12.20 To validate
our findings, availability of 14-HRR (4) was required
in order to compare its activity with compounds 9-12.
The literature has reported that configuration of natural
4 could not be clearly assigned, but very interestingly,
both synthetic enantiomers behaved similarly in biologi-
cal assays.8 Thus, we embarked on the synthesis of
compound (+)-(4) and its hypothetical precursor 13 with
(S)-configuration at C-14, testing also the biological
activity of the intermediates 16 and 17 obtained along
the synthetic pathway. Furthermore, we prepared the
terphenyl-based derivatives of 11 and 12, namely,
compounds 26-28, considering the phenyl bridge ring
as a known bioisosteric substitution of the alkenyl
portion in retinoids17,21 to give conformationally re-
stricted stilbene mimetics.
Biological Results and Discussion
In Figure 3a the curves are reporting HL-60 cell
growth cultured in complete medium (containing serum)
or serum-free medium. When cultured in nonsupple-
mented serum-free medium, the cell number decreased
in 72 h from 200 × 103 to 110 × 103 mL-1 and the
percentage of apoptotic cells increased from 0% to 43%
(Figure 3a and Table 1). Flow cytometry analysis
showed a recruitment in the G0-G1 phase of the cell
cycle (Figure 3b-d), and a sub-G0-G1 peak correspond-
ing to apoptotic cells was evident after 48 h of culture
(Figure 3c). Morphologic analysis by fluorescence mi-
croscopy after staining with acridine orange and ethid-
ium bromide of cells cultured for 72 h in serum-free
medium has shown a very low percentage of necrotic
cells (less than 10%). The effects of the compounds
tested in this study on cell growth and apoptosis are
shown in table. Analyses were carried out on the HL60
cell line. In the table are reported the effects of the
compounds used at 1 µM. All compounds induced a dose-
dependent increase of cell growth and an inhibition of
apoptotic cells with a plateau ranging from 0.8 to 1.2
µM; higher concentrations were less effective in sup-
porting cell growth or were cytotoxic (data not shown).
The decrease of living cells (from 200 × 103 to 110 ×
103 mL-1) was partially blocked by (+)-4 at 1 µM to yield
181 × 103 mL-1. (+)-4 also prevented apoptosis in 9%
of cells. At the same concentration, compound 13 was
less effective than (+)-4 in protecting cells from the
effects of the serum deprivation, while the diacetate 16
proved to be the most active, being able to partially
support cell growth and to inhibit the programmed cell
death induced by serum deprivation more efficiently
than 14-HRR (t < 0.01).
Chemistry
The natural retro-retinoid (+)-(4) and its synthetic
precursors 16 and 17 have been obtained through the
original methodology developed by Corey et al.22 Com-
pound 13 has been derived from intermediate 15 as
outlined in Scheme 1. Standard Wittig reactions be-
tween appropriate partners have been used to prepare
the benzoic acid derivatives 9-12. A cross-coupling

Activity of Retinoids
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4295
Scheme 2a
a (a) Pd(Ph3P)4, aqueous Na2CO3, toluene/EtOH; (b) aqueous KMnO4; (c) Pd(Ph3P)4, aqueous Na2CO3, CH3CN.
Table 1. Effects of Different Compounds on Cell Growth and
Apoptosis in HL-60 Cells Cultured for 72 h in
Nonsupplemented Serum-Free Mediuma
of the prevalent role of the terphenyl scaffold rather
than the relative positions of the ortho, meta, or para
junctions in determining the activity of these compounds
came from the data listed in the table.
no. of living cells/mL
compd
(×103)
apoptosis (%)
43 ( 1.56
These very promising results led us to test the most
active of these compounds as antiapoptotic agents on
neuronal cells. It is apparent that excitoxicity is impli-
cated as a mechanism of cell death in acute neurologic
diseases, such as stroke and trauma. The cascade of
events initiated by interaction of excessively released
glutamate with its receptors may result in either
necrosis or apoptosis, depending on the intensity of the
original insult.25,26 Thus, a slow excitotoxic process is
likely to induce a delayed neuronal death with apoptotic
features in chronic neurodegenerative diseases, such as
Alzheimer, Parkinson, and other age-related disorders.27
Neuronal cell death can be experimentally induced by
a variety of neurotoxic agents;28,29 thus, cultured neu-
rons may undergo apoptosis when treated with 0.15 µM
staurosporine for 24 h, with a mechanism involving
cytochrome c release30 or excitotoxic necrosis when
treated with 30 µM glutamate for 10 min.31-33 Interest-
ingly, the terphenyl compounds 26 and 28 used at 1 µM
were able to prevent staurosporine-induced cell death
(Figure 3e), but their protective action was totally
ineffective during the glutamate neurotoxic insult. A
quantification of the neuroprotective effects cannot be
surely made by these methods. However, the increase
in cell survival induced by 26 and 28 in different
experimentally induced cell death could be attributed
to their antiapoptotic action, presumably taking place
upstream from mitochondria. In fact, the method used
here to quantify cell death is based on the mitochondrial
reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) to a dark-blue formazan
product; so it is not suitable for detecting effects
occurring downstream of mitochondria, but it is useful
for determining the point in the death pathway where
agents have their effects.34 The absorbance detected
after staining with MTT is proportional to the number
of viable cells, and results obtained using the MTT assay
were similar to results obtained with cell count. These
results suggest for compounds 26 and 28 a potential
neuroprotective action in chronic neurodegenerative
diseases. Additionally, neither (+)-4 nor 16 proved to
be active on neuronal cells, thus suggesting a possible
untreated control 110 ( 4
14-HRR
9
181 (6.7 (p ) 0.0003)
155 ( 4.47 (p ) 0.0021) 36 ( 1.25 (p ) 0.0004)
34 ( 1.34 (p ) 0.0002)
10
100 ( 4.33 (p ) 0.07)
166 ( 5.3 (p ) 0.0004)
100 ( 4.38 (p ) 0.09)
149 ( 6 (p ) 0.0035)
129 ( 5.5 (p ) 0.062)
207 ( 3.57 (p < 0.0001) 30 ( 1.16 (p ) 0.0021)
129 ( 5.45 (p ) 0.067) 40 ( 1.7 (p ) 0.18)
300 ( 15.4 (p ) 0.0002) 18 ( 6.7 (p < 0.0001)
43 ( 1.8 (p ) 0.29)
36 ( 0.9 (p ) 0.028)
42 ( 1.65 (p ) 0.44)
37 ( 1.38 (p ) 0.024)
41 ( 1.8 (p ) 0.35)
11
12
13
15
16
17
26
27
350 ( 8.3 (p < 0.0001)
220 ( 8.2 (p < 0.0001)
17 ( 0.7 (p < 0.0001)
22 ( 0.9 (p < 0.0001)
28
a Data represent the mean ( SEM of five independent experi-
ments. The difference in the value of the number of living cells
per milliliter and the % apoptosis between each compound and
control was significant (p < 0.05) for compounds 14-HRR, 9, 11,
13, 16, and 26-28. Apoptosis was detected by morphological
analysis as described in Materials and Methods for Biological
Assays. Compounds were used at 1 µM.
The known antiproliferative23 and cytodifferentiat-
ing24 properties of 7 in a variety of cancer cell lines and
the ability of cis-TTNPB (8) to induce apoptosis in HL60
cells13 were in contrast to activities that we found for
their desmethyl derivatives. Removal of the methyl
group led in some cases, as for the cis-stilbenes 10 and
12, to loss of activity, while trans compounds 9 and 11
were able to slow the rate of cell death and, in part, to
support cell growth. This result was quite surprising,
since we were unaware of previously reported data for
synthetic derivatives of retinoids featuring cell-growth-
supporting activity. Taken together, these results fur-
ther support the idea that both stereochemistry and
substitutions at the double bond of stilbene-arotinoids
as TTNPB are playing a crucial role in determining the
apoptotic or antiapoptotic activity of compounds belong-
ing to this family. Interestingly, potent antiapoptotic
activity and cell-growth-supporting activity has been
found for the terphenyl-based compounds 26-28, being
more active than (+)-4 and intermediate 16 (t < 0.01
both). In particular, compound 27 was the most active,
3-fold more active than the natural retro-retinoid (+)-
4, being able to significantly support cell growth (from
200 × 103 to 350 × 103 cells/mL in 72 h) and to reduce
apoptotic HL60 cells from 43% to 17%. Clear evidence

4296 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Simoni et al.
are able to support growth and to inhibit apoptosis in
different cell lines at 1 × 10-6 M. Interestingly, the
activity of these compounds is likely related to the trans
stereochemistry of the double bond. To the best of our
knowledge, synthetic retinoids possessing this biological
profile have not been hitherto reported. Moreover, the
cell-growth-supporting activity of 16, together with its
relatively greater chemical stability, makes this com-
pound an interesting candidate to substitute the natural
14-HRR (4). A noteworthy finding was the disclosure
of a new “lead” featuring a terphenyl architecture with
a very intriguing biological profile and the right re-
quirements for being considered as a potential drug for
treating degenerative diseases. The methoxyphenyl
appendages are not comparable to phenolic moieties,
characteristic of some known neuroprotective com-
pounds active as ROS (reactive oxygen species) scav-
engers.35 Researchers have only recently focused their
interest on compounds endowed with antiapoptotic
activity as potential drugs useful in degenerative and
ischemic diseases. We first describe in this paper that
a new class of synthetic terphenyls are able to support
cell growth and inhibit apoptosis. Although their effects
on survival of neuronal cells are modest, the data
reported in this work should be considered as the first
step for future studies of structure-activity relation-
ships (SARs) to find other compounds belonging to this
class and endowed with more potent antiapoptosis
activity. Moreover, considering that these compounds
are different from a chemical point of view from other
compounds currently studied as antiapoptotic agents
(e.g., anti-caspases) it should be interesting to investi-
gate the effects of combination therapies that include
our terphenyl compounds. Further studies of this class
of natural and synthetic compounds should also provide
a better understanding of the mechanism of action of
natural bioactive retro-retinoids.
Figure 3. (a) Cell growth of HL-60 cells in complete or serum-
free medium, in cells/mL × 103. (b-d) Flow cytometry deter-
mination of apoptosis. HL60 cells were grown in complete (b)
or serum-free medium (c and d): c ) evaluation after 48 h of
culture; d ) evaluation after 72 h of culture. Apoptosis was
determined by FACscan flow cytometry assay using the
CellQuest software. The hypodiploid sub-GoG1 peak, designed
as AP, represents cells undergoing apoptosis. Data reported
in parts b-d are representative of four different experiments
that have produced similar results. (e) Effects of (+)-4, 16, 26-
28 (each 1 µM) on cell viability (% survival) of rat primary
cortical neurons treated with 0.15 µM staurosporine (STS). Cell
viability was evaluated by MTT assay as described in Materi-
als and Methods for Biological Assays. Data are the mean (
SEM of 5-11 experiments in triplicate: (//) P < 0.01 signifi-
cantly different from staurosporine alone, ANOVA followed by
Dunnet’s test. Effects on % survival without staurosporine
were the following (in parentheses is the number of experi-
ments in triplicate): 26, 91 ( 3% (8); 27, 101 ( 4% (8); 28,
106 ( 4 (4).
Experimental Section
Chemistry. Melting points were obtained with a Kofler
apparatus and are uncorrected. Reaction courses and product
mixtures were routinely monitored by thin-layer chromatog-
raphy (TLC) on silica gel precoated F254 Merck plates. Nuclear
magnetic resonance (1H and 13C NMR) spectra were deter-
mined in CDCl3 solution, unless otherwise indicated, with a
Bruker AC-200 spectrometer, and peak positions are given in
part per million downfield from tetramethylsilane as internal
standard. All drying operations were performed over anhy-
drous sodium sulfate. Column chromatography (medium pres-
sure) was carried out with 60-200 mesh silica gel, using the
flash technique. Microanalysis of all new synthesized com-
pounds agreed with calculated values within (0.4% of the
theoretical.
General Procedure for the Synthesis of Stilbene
Compounds. To the phosphonium bromide salt (3,4,5-tri-
methoxybenzyl- or 5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphth-
2-ylmethyl-, 2 mmol) in anhydrous tetrahydrofuran (15 mL)
is added sodium hydride (50% in paraffin, 1.2 equiv), and the
mixture is stirred under argon for 30 min. A solution of
aldehyde in tetrahydrofuran is added dropwise during 30 min,
and stirring is continued for 6 h at room temperature. The
suspension is then poured into water and extracted with
dichloromethane. The organic phase is washed with brine,
dried, and evaporated to give after chromatographic purifica-
tion a mixture of cis/trans-stilbenes that is hydrolyzed without
separation. To a solution of stilbene esters mixture in methanol
(50 mg in 5 mL) is added 10 % LiOH until a slight cloudiness
is attained. The mixture is heated to 60-70 °C until completion
different mechanism of action or difficulties due to
inherent instability or low entrance rate into cells.
Conclusion
Our search for new compounds as apoptosis inducers
led us to discover a novel class of potent antiapoptotic
agents with even higher cell-growth-supporting activity
than the natural retro-retinoids. Thus, simple removal
of the methyl group located on the double bond of
TTNPB (7) provided compounds such as 9 and 11 that

Activity of Retinoids
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4297
3,4,5-Trimethoxy[1,1′;2′,1′′]terphenyl-4′′-carboxylic Acid
of hydrolysis, then cooled, washed once with diethyl ether,
acidified, and extracted with diethyl ether. The organic phase
is evaporated, and the acids are purified and separated by a
chromatographic column.
1
(28). Yield 65%. Mp 166-170 °C. H NMR (CD3OD): δ 3.59
(s, 6H), 3.72 (s, 3H), 6.37 (s, 2H), 7.26 (d, J ) 8.3 Hz, 2H),
7.45 (m, 4H), 7.91 (d, J ) 8.2 Hz, 2H). 13C NMR: δ 56.0, 61.0,
107.3, 127.7, 128.3, 129.9, 130.4, 130.5, 136.2, 137.0, 139.4,
140.6, 147.6, 152.8, 171.7. Anal. (C22H20O5) C, H.
4-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethylnaphthalen-
2-yl)vinyl]benzoic acid. trans-9. White solid. Mp 272-275
1
°C. Yield 38%. H NMR (CDCl3) δ 1.33 (s, 6H), 1.36 (s, 6H),
3,4,5-Trimethoxy[1,1′;3′,1′′]terphenyl-4′′-carboxylic Acid
(27). Yield 76%. Mp 198-201 °C. 1H NMR: δ 3.92 (s, 3H),
3.95 (s, 6H), 6.83 (s, 2H), 7.54-7.65 (m, 3H), 7.74-7.78 (m,
3H), 8.23 (d, J ) 8.4 Hz, 2H). 13C NMR: δ 56.4, 61.1, 104.7,
126.2, 126.4, 127.2, 127.4, 128.2, 129.4, 130.9, 136.9, 137.9,
140.5, 142.3, 146.5, 153.6, 171.7. Anal. (C22H20O5) C, H.
3,4,5-Trimethoxy[1,1′;4′,1′′]terphenyl-4′′-carboxylic Acid
(26). Yield 72%. Mp 243-247 °C. 1H NMR: δ 3.92 (s, 3H),
3.96 (s, 6H), 6.84 (s, 2H), 7.71 (m, 4H), 7.76 (d, J ) 8.4 Hz,
2H), 8.22 (d, J ) 8.2 Hz, 2H). 13C NMR: δ 56.3, 61.1, 104.5,
127.1, 127.6, 127.7, 128.0, 130.4, 130.9, 136.4, 138.8, 141.4,
145.9, 153.6, 170.7. Anal. (C22H20O5) C, H.
Materials and Methods for Biological Assays. Cell
Culture and Culture Conditions. HL60 cells were obtained
from the American Type Culture Collection (Rockville, MD).
Cells were grown in RPMI 1640 (Gibco Grand Island, NY)
containing 10% FCS (Gibco), 100 U/mL penicillin (Gibco), 100
mg/mL streptomycin (Gibco), and 2 mM L-glutamine (Sigma
Chemical Co., St. Louis, MO) in a 5% CO2 atmosphere at 37
°C. The employed serum-free medium is constituted by RPMI
1640 and ITS+1 liquid media supplement (Sigma). ITS+1
liquid media supplement contains 0.5% delipidated bovine
serum albumin, 10 µg/mL insulin, 5.5 µg/mL transferrin, 4.7
µg/mL linoleic acid, and 2 mM L-glutamine (Sigma).
1.73 (s, 4H), 7.10 (d, J ) 16 Hz, 1H), 7.26 (d, J ) 16 Hz, 1H),
7.37 (s, 2H), 7.46 (s, 1H), 7.62 (d, J ) 8.4 Hz, 2H), 8.11 (d, J
) 8.4 Hz, 2H). Anal. (C23H26O2) C, H.
cis-10. Solid. Mp 145-148 °C. Yield 41%. 1H NMR (CDCl3)
δ 1.09 (s, 6H), 1.64 (s, 4H), 6.55 (d, J ) 11.5 Hz, 1H), 6.66 (d,
J ) 11.5 Hz, 1H), 7.00 (d, J ) 8 Hz, 1H), 7.16 (s, 1H), 7.23 (d,
J ) 8 Hz, 1H), 7.39 (d, J ) 8 Hz, 2H), 7.98 (d, J ) 8 Hz, 2H).
Anal.(C23H26O2) C, H.
4-[2-(3,4,5-Trimethoxyphenyl)vinyl]benzoic Acid. trans-
11. Solid. Mp 218-221 °C. Yield 35%. 1H NMR (CDCl3) δ 3.90
(s, 3H), 3.94 (s, 6H), 6.79 (s, 2H), 7.05 (d, 1H, J ) 16.3 Hz),
7.20 (d, 1H, J ) 15.9 Hz), 7.61 (d, 2H, J ) 8.2 Hz), 8.11 (d,
2H, J ) 8.2 Hz). Anal. (C18H18O5) C, H.
cis-12. Solid. Mp 140-143 °C. Yield 33%. 1H NMR (CDCl3)
δ 3.67 (s, 6H), 3.85 (s, 3H), 6.46 (s, 2H), 6.59 (d, 1H, J ) 12.4
Hz), 6.67 (d, 1H, J ) 12.4 Hz), 7.41 (d, 2H, J ) 8.1 Hz), 8.01
(d, 2H, J ) 8.1 Hz). Anal. (C18H18O5) C, H.
General Procedure for Biphenylcarbaldehyde Syn-
thesis (19-21). To a solution of o-, m-, or p-iodobromobenzene
(120 mg, 0.42 mmol) in 1 mL of toluene is added a catalytic
amount (3-5% mol) of tetrakis-triphenylphosphine palladium
and 0.3 mL of aqueous 2 M Na2CO3. A solution of 4-formyl-
benzeneboronic acid (100 mg, 0.66 mmol) in 1 mL of ethanol
is then added, and the mixture is heated to reflux for 3 h in
an argon atmosphere. After cooling, the mixture is extracted
three times with dichloromethane and the joined organic
phases are washed with water and brine, dried, and evapo-
rated under vacuum. The residue is purified by chromatogra-
phy, giving the bromodiphenylaldehyde derivative.
2′-Bromobiphenyl-4-carbaldehyde (19).36 Oil. Yield 30%.
1H NMR: δ 7.26-7.41 (m, 3H), 7.60 (d, J ) 8.1 Hz, 2H), 7.71
(d, J ) 8 Hz, 1H), 7.97 (d, J ) 8.3 Hz, 2H), 10.09 (s, 1H).
3′-Bromobiphenyl-4-carbaldehyde (20).37 Oil. Yield 50%.
1H NMR: δ 7.37, J ) 7.7 Hz, 1H), 7.57 (m, 2H), 7.72 (d, J )
9.2 Hz, 2H), 7.77 (m, 1H), 7.96 (d, J ) 9 Hz, 2H), 10.07 (s,
1H).
Cytotoxicity Assays. To evaluate the number of live and
dead cells, cells were stained with trypan blue and counted
on a hemocytometer. Cells that showed trypan blue uptake
were interpreted as nonviable.
Morphological Evaluation of Apoptosis and Necrosis.
Drugs effects on apoptosis and necrosis were determined
morphologically by fluorescence microscopy after labeling with
acridine orange and ethidium bromide. Cells (2 × 105) were
centrifuged (300 g), and the pellet was resuspended in 25 µL
of the dye mixture. An amount of 10 µL of the mixture was
placed on a microscope slide, covered with a 22 mm2 coverslip,
and examined in oil immersion with a 100× objective using a
fluorescence microscope. Live cells were determined by the
uptake of acridine orange (green fluorescence) and exclusion
of ethidium bromide (red fluorescence) stain. Live and dead
apoptotic cells were identified by perinuclear condensation of
chromatin stained by acridine orange or ethidium bromide,
respectively, and by the formation of apoptotic bodies. Necrotic
cells were identified by uniform labeling with ethidium
bromide.
Determination of Apoptosis by Annexine V. Cells (1 ×
106) were washed with PBS and centrifugated at 200g for 5
min. The cell pellet was suspended in 100 µL of staining
solution containing annexine-V-fluorescein labeling reagent
(Annexine-V-Fluos Staining Kit, Roche Molecular Biochemi-
cals, Mannheim, Germany) and incubated for 15 min at 20
°C. Annexine V positive cells were evaluated by fluorescence
microscopy and flow cytometry.
Neuronal Cell Cultures. Cortical neuronal cultures were
prepared from 1-day old Sprague Dawley rats, as described
by Alho et al.41 The neurons were plated in 24-well culture
vessels (Falcon) coated with 20 µg/mL poly-L-lysine and
cultured in modified neurobasal medium (1 mL) by adding 50
mg/mL gentamycin sulfate, 2 % B27, and 500 µM glutamax
500. After 24-48 h, 5 µM cytosin arabinoside (ARA-C) was
also added to prevent glial cell replication. The neurotoxic
insult was induced on the seventh to ninth day in vitro (7-9
DIV) by adding 0.15 µM staurosporine for 24 h. In another
set of experiments, an amount of 500 µL of medium was
collected and set aside. The cells were intoxicated by adding
30 µM glutamate for 10 min, and then the medium was
removed and replaced with the original one. Drugs to be tested,
dissolved in 50 µL of DMSO, were added to the medium 30
min before the intoxication and maintained until the end of
4′-Bromobiphenyl-4-carbaldehyde (21).38 Solid. Mp 135-
1
140°C. Yield 62%. H NMR: δ 7.50 (d, J ) 8.8 Hz, 2H), 7.62
(d, J ) 8.8 Hz, 2H), 7.72 (d, J ) 8 Hz, 2H), 7.96 (d, J ) 8.6 Hz,
2H), 10.06 (s, 1H).
General Procedure for Oxidation to Biphenylcar-
boxylic Acid (22-24). To a gently boiling solution of bromo-
biphenylcarbaldehyde (19-21, 100 mg) in sodium carbonate
(5 mL) is added potassium permanganate (70 mg in 1 mL of
water), and reflux is maintained for 3 h. After cooling, the
solution is acidified with concentrated hydrochloric acid and
extracted three or more times with diethyl ether. The residue
is used in the next reaction with no further purification.
2′-Bromobiphenyl-4-carboxylic Acid (22).39 Yield 99%.
1
Mp 237-241 °C. H NMR (DMSO-d6): δ 7.40-7.70 (m, 5H),
8.00-8.10 (m, 3H).
3′-Bromobiphenyl-4-carboxylic Acid (23).39 Yield 99%.
1
Mp 250-253 °C. H NMR (DMSO-d6): δ 7.40-7.85 (m, 6H),
8.11 (d, J ) 8.4 Hz, 2H).
4′-Bromobiphenyl-4-carboxylic Acid (24).39,40 Yield 98%.
1
Mp >300 °C. H NMR: δ 7.50 (d, J ) 8.8 Hz, 2H), 7.59-7.68
(m, 4H), 8.16 (d, J ) 8.6 Hz, 2H).
General Procedure for Terphenyl Synthesis. To a
solution of bromobiphenyl acid (22-24, 100 mg) and 3,4,5-
trimethoxybenzene boronic acid (25, 77 mg) in 0.4 M aqueous
sodium carbonate (5 mL) and acetonitrile (5 mL) is added a
catalytic amount (5% mole) of tetrakis-triphenylphosphine
palladium, and the mixture is heated to reflux under argon
atmosphere for 3 h. The suspension is cooled, filtered, and
washed with dichloromethane. The water phase is then
acidified and extracted with diethyl ether. Purification of the
residue of ether extracts gives the terphenyl product.

4298 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
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the experiment. An equal amount of DMSO was added to the
medium in control wells. The primary effects of the drugs were
tested in cultured cells submitted to the same protocol, in the
absence of neurotoxins.
MTT Assay. Cell survival was determined either 24 h after
staurosporine addition or 30 min after glutamate intoxication.
Yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide) is converted to the blue formazan product only
by metabolically active mitochondria, and the absorbance is
proportional to the number of viable cells. MTT (0.5 mg/mL)
was added to the cultures, and the blue color was allowed to
develop for 2 h. After aspiration of the medium, 100 µL of 9:1
isopropyl alcohol/HCl was used to solubilize the blue crystals.
Samples were read at a test wavelength of 570 nm. The
absorbance data are expressed as percentages of control
groups.28
Statistics. Survival data are given as the mean ( SEM of
percent values with respect to control cells. The statistical
significance of the differences has been assessed with the
analysis of the variance (ANOVA), followed by Dunnet’s test
(GraphPad software).
Acknowledgment. This work was supported in part
by the Ministero dell’Universita` e della Ricerca Scien-
tifica e Tecnologica, Rome, Italy. We are grateful to
Professor Gian Piero Pollini for remarkable help during
the writing up of the manuscript revision.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
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