Identification of a terphenyl derivative that blocks the cell cycle in the G0-G1 phase and induces differentiation in leukemia cells

Article abstract of DOI:10.1021/jm060253o

To further explore the SAR of resveratrol-related trans-stilbene derivatives, here we describe the synthesis of (a) a series of 3,5-dimethoxy analogues in which a variety of substituents were introduced at positions 2′, 3′, 4′, and 5′ of the stilbene scaf

Full text of DOI:10.1021/jm060253o

3012
J. Med. Chem. 2006, 49, 3012-3018
Identification of a Terphenyl Derivative that Blocks the Cell Cycle in the G0-G1 Phase and
Induces Differentiation in Leukemia Cells
Marinella Roberti,*,† Daniela Pizzirani,† Maurizio Recanatini,† Daniele Simoni,‡ Stefania Grimaudo,§ Antonietta Di Cristina,§
Vincenzo Abbadessa,§ Nicola Gebbia,|| and Manlio Tolomeo§
Dipartimento di Scienze Farmaceutiche, UniVersita` di Bologna, Via Belmeloro 6, 40126 Bologna, Italy,
Dipartimento di Scienze Farmaceutiche, UniVersita` di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy, and
DiVisione di Ematologia e SerVizio AIDS and Dipartimento di Oncologia, Policlinico ‘P. Giaccone’,
UniVersita` di Palermo, Via del Vespro 129, 90127 Palermo, Italy
ReceiVed March 6, 2006
To further explore the SAR of resveratrol-related trans-stilbene derivatives, here we describe the synthesis
of (a) a series of 3,5-dimethoxy analogues in which a variety of substituents were introduced at positions
2′, 3′, 4′, and 5′ of the stilbene scaffold and (b) a second group of derivatives (2-phenylnaphthalenes and
terphenyls) that incorporate a phenyl ring as a bioisosteric replacement of the stilbene alkenyl bridge. We
thoroughly characterized all of the new compounds with respect to their apoptosis-inducing activity and
their effects on the cell cycle. One of the new derivatives, 13g, behaved differently from the others, as it
was able to block the cell cycle in the G0-G1 phase and also to induce differentiation in acute myelogenous
leukemia HL60 cells. Compared to resveratrol, the synthetic terphenyl 13g showed a more potent apoptotic
and differentiating activity. Moreover, it was active on both multidrug resistance and Bcr-Abl-expressing
cells that were resistant to resveratrol.
Chart 1a
Introduction
The stilbene scaffold is a basic element for a number of
biologically active natural and synthetic compounds, and in
accordance with Evans’ definition, it can be considered as a
privileged structure.1 One of the most relevant and studied
stilbenes is resveratrol (1a, 3,4′,5-trihydroxy-trans-stilbene;
Chart 1), a phytoalexin present in grapes and other food
products,2,3 endowed with several different biological properties
and able to act and bind at different cellular targets.4,5
The interesting chemopreventive6,7 and chemotherapeutic7-11
properties associated with resveratrol offer promises for the
design of new chemoterapeutic agents, and in this context,
several studies have been reported describing the synthesis and
antitumor evaluation of these types of stilbene derivatives.12-16
Resveratrol has also been shown to induce apoptosis in
different cancer cell lines.7,17-19 Since reduced apoptosis has
been implicated in the development and progression of malig-
nant tumors20,21 and in the occurrence of chemoresistant
phenotypes,22-25 resveratrol-induced apoptosis might therefore
contribute to its antitumor activity. However, resveratrol is not
a potent cytotoxic compound when compared with other
chemotherapeutic drugs. Recently, we observed in different
cancer cell lines that, to obtain the induction of apoptosis in
50% of cells, a resveratrol concentration about 10 times higher
than its IC50 was necessary.26
Because of our ongoing interest in finding new apoptosis-
inducing agents, we are particularly intrigued by molecular
scaffolds that allow the parallel synthesis of substituted deriva-
tives. Actually, the parallel procedure is suitable for rapidly
obtaining variously substituted analogues, particularly when
structure-based design strategy is not applicable due to the lack
of knowledge of a specific target. Thus, we recently synthesized
a Overall yield.
a small library of resveratrol analogues bearing the 3,5-
dimethoxy motif at the A phenyl ring and amino, methoxy, and
hydroxy functions at the 3′- and/or 4′-positions.26 Many deriva-
tives turned out to be more active than resveratrol as apoptosis-
inducing agents in HL60 leukemia cells. Besides, some of them
were active toward resistant HL60R cells, and their activity was
higher than that of several classic chemotherapeutic agents. Also,
considering the importance of the 3,5-dimethoxy motif in
conferring proapoptotic activity to stilbene-based compounds,
* Corresponding author. Tel. +39-051-2099738. Fax: +39-051-2099734.
E-mail: marinella.roberti@unibo.it.
† Universita` di Bologna.
‡ Universita` di Ferrara.
§ Divisione di Ematologia e Servizio AIDS, Universita` di Palermo.
|| Dipartimento di Oncologia, Universita` di Palermo.
10.1021/jm060253o CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/18/2006

A Terphenyl DeriVatiVe that Blocks the Cell Cycle
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 3013
Scheme 1a
a Reagents and conditions: (a) Pd(Ph3P)4, aq Na2CO3, toluene/EtOH 3:1, reflux, 5 h; Bu4NF, THF, rt, 3 h.
we compared the cellular effects of pterostilbene (2a) and 3′-
hydroxypterostilbene (2b) with those of their corresponding
natural 3,5-hydroxy analogues, resveratrol (1a) and piceatannol
(1b), respectively.27 The 3,5-dimethoxylated compounds differed
with respect to 1a and 1b not only in their pattern of activity
on sensitive and resistant cells but also regarding the effects on
the cell cycle and apoptotic pathways. Moreover, both 2a and
2b were less toxic to normal hemopoietic stem cells than to the
leukemia and lymphoma cells.
substitution of the alkenyl bridge. Thus, we obtained both
2-phenylnaphthalenes 7a,c,d (Schemes 1 and 2), and terphenyls
13a-i (Schemes 1 and 2), all of which present the resveratrol-
like pattern of oxygenated phenyl rings, i.e., one para-substituted
and one di-meta-substituted phenyl connected by an unsaturated/
aromatic system. One of these compounds, 13g, behaved
differently than the others, as it was able to block the cell cycle
in the G0-G1 phase and also to induce differentiation in HL60
cells.
To further explore the SAR of this class of derivatives, here
we describe the synthesis of a series of resveratrol analogues
(3a-h, Chart 1) in which a variety of substituents were
introduced at positions 2′, 3′, 4′, and 5′ of the stilbene scaffold
while the 3,5-dimethoxy motif was maintained. We thoroughly
characterized all of the new derivatives with respect to both
their apoptosis-inducing activity and their effects on cell cycle
by carrying out experiments on leukemia cell lines HL60,
HL60R (MDR expressing), and K562 (apoptosis resistant).
Some of the compounds turned out to be active as apoptosis-
inducing agents; however, they did not reveal significant profile
variations in terms of cytotoxicity and effects on cell cycle with
respect to resveratrol and our previous stilbene derivatives.26
Given these results, we aimed at increasing the structural
diversity of the new molecules by synthesizing a second series
of derivatives, which incorporate a phenyl ring as a bioisosteric
Chemistry
The compounds were obtained following different parallel
synthetic pathways (Schemes 1 and 2) carried out in a Carousel
reaction station.
To prepare the trans-stilbene derivatives 3a-h (Chart 1), we
took advantage of a standard Wittig reaction between the
appropriate aromatic aldehydes and the aromatic ylide26,28 (see
the Supporting Information).
The 2-phenylnaphthalene derivatives and the terphenyls were
obtained as shown in Scheme 1. A cross-coupling Suzuki
reaction between 3,5-dimethoxyphenylboronic acid 429a,b and
the appropriate 2-bromonaphthalene derivatives 5, 6, or bro-
moiodobenzene (ortho, meta, or para isomers) in the presence
of a catalytic amount of tetrakis(triphenylphosphine)palladium

3014 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Roberti et al.
Scheme 2a
a Reagents and conditions: (a) BBr3, CH2Cl2, -78 °C, 18 h.
Table 1. IC50 (µM ( SE) and AC50 (µM ( SE) Values of Resveratrol (1a), Pterostilbene (2a), Stilbene Derivatives 3a-h, 2-Phenylnaphthalenes
7a,c,d, and Terphenyls 13a-i in Sensitive HL60, MDR HL60R, and Bcr-Abl-Expressing K562 Cells
HL60
HL60R
K562
a
b
a
b
a
b
compounds
IC50
AC50
IC50
AC50
IC50
AC50
resveratrol (1a)
pterostilbene (2a)
3a
3b
3c
3d
3e
3f
3g
3h
7a
7c
5.0 ( 0.8
35 ( 4
50 ( 6
75 ( 5
35 ( 4
58 ( 5
4.0 ( 0.3
15 ( 2
18 ( 2
29 ( 5
79 ( 6
>100
91 ( 18
19 ( 1
48 ( 7
30 ( 3
>100
23 ( 2
>100
60 ( 7
42 ( 3
68 ( 7
>100
8.0 ( 0.9
34 ( 5
42 ( 6
73 ( 8
>100
>100
>100
16 ( 2
53 ( 3
23 ( 3
>100
20 ( 2
80 ( 12
28 ( 2
16 ( 2
23 ( 4
58 ( 9
12 ( 3
>100
28 ( 2
10 ( 3
24 ( 2
26 ( 1
10 ( 1
8.0 ( 0.8
18 ( 3
16 ( 1
23 ( 4
>100
>100
85 ( 11
>100
45 ( 7
72 ( 8
70 ( 13
25 ( 4
28 ( 2
60 ( 9
55 ( 7
75 ( 5
>100
9.0 ( 0.6
23 ( 5
>100
2.5 ( 0.3
3.5 ( 0.4
11 ( 2
50 ( 2
82 ( 10
98 ( 10
>100
14 ( 2
20 ( 2
>100
84 ( 6
>100
76 ( 3
>100
>100
>100
10 ( 1
33 ( 6
92 ( 15
32 ( 4
>100
42 ( 5
20 ( 4
35 ( 5
>100
14 ( 2
10 ( 2
14 ( 2
6.0 ( 0.8
20 ( 2
20 ( 2
8.0 ( 0.6
55 ( 4
84 ( 13
42 ( 5
>100
7d
28 ( 4
13a
13b
13c
13d
13e
13f
13g
13h
13i
2.5 ( 0.2
75 ( 7
4.8 ( 0.7
50 ( 7
48 ( 2
>100
27 ( 3
50 ( 5
18 ( 3
8.0 ( 0.7
75 ( 12
75 ( 9
38 ( 4
5.0 ( 0.3
7.0 ( 0.4
7.0 ( 1.2
27 ( 3
10 ( 3
15 ( 2
25 ( 3
63 ( 5
9.0 ( 0.7
24 ( 4
48 ( 5
60 ( 9
>100
3.5 ( 0.3
40 ( 3
a Concentration able to inhibit 50% of cell growth. b Concentration able to induce apoptosis in 50% of cells.
gave the 2-phenylnaphthalene derivatives 7a,b and the biphenyl
dimethoxy derivatives 8-10, respectively. A second Suzuki
cross-coupling between compounds 8-10 and the boronic acids
11 and 12 provided the required terphenyls 13a-f. Removal
of the tert-butyldimethylsilyl (TBDMS) group from derivative
7b using tetrabutylammonium fluoride afforded compound 7c.
Finally, the parallel demethylation of trimethoxy derivatives 7a
and 13b,d,f with BBr3 afforded 7d and 13g-i (Scheme 2).
Compounds 7a,c,d were recently described by Minutolo et al.30
within a series of resveratrol analogues tested against human
breast cancer cells.
effects of each compound on cell cycle were studied by flow
cytometry after staining cells with propidium iodide. Moreover,
for compounds able to block cells in the G0-G1 phase, we
investigated the ability to induce cell differentiation by evaluat-
ing the expression of CD11b, CD11c, and CD14 on HL60 cells.
Results
The antiproliferative and apoptosis-inducing activities of the
new compounds are summarized in Table 1, and these data are
compared to those obtained with natural stilbenes resveratrol
(1a) and its 3,5-dimethoxy analogue pterostilbene (2a). Anti-
proliferative and apoptosis-inducing activities were expressed
as IC50 (concentration able to inhibit 50% of cell growth) and
AC50 (concentration able to induce apoptosis in 50% of cells),
respectively.
Within the 3,5-dimethoxystilbene series 3a-h, the best results
were obtained with compound 3c bearing a 4′-methoxy function.
The activity of 2′-hydroxy derivative 3a was higher than that
of pterostilbene (2a) on HL60 cells, but it became lower on
HL60R and K562 cells. When the hydroxy function was moved
Biology
All compounds considered in this study were tested for their
antiproliferative and proapoptotic activities on sensitive acute
myelogenous leukemia HL60 cells, multidrug resistance (MDR)-
expressing HL60R cells, and Bcr-Abl-expressing K562 cells.
The antiproliferative activity of each compound was evaluated
by counting cells with an automatic cell counter; apoptosis was
evaluated by morphological assay and annexine V test. The

A Terphenyl DeriVatiVe that Blocks the Cell Cycle
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 3015
to the 3′ position, as in compound 3b, the cytotoxic effects
decreased on both HL60 and HL60R cells. In the subset of the
B-ring disubstituted compounds, the introduction of the 2′-
hydroxy and 3′-methoxy functions, as in compound 3d,
increased the proapoptotic activity on all cell lines tested in this
study. The potency dropped by removing the 3′-methoxy group
and introducing a 5′-Cl as in 3f. By appending an ethoxy
function in 3′-position of pterostilbene, we obtained compound
3e, which showed a better activity on HL60 cells with respect
to the parent compound 2a. The analogue 3g bearing 3′,5′-
dimethoxy substitution was slightly less active than pterostilbene
(2a), and the activity decreased further with the 3′,5′-di-tert-
butyl analogue 3h, which was the least active one of this series.
In summary, these 3,5-dimethoxy-trans-stilbenes showed a
cytotoxic profile comparable to that of other stilbene derivatives
earlier synthesized by us,26,27 even though some compounds of
the previous series were more potent on HL60 cells.
Regarding the naphthalene derivatives 7a,c,d, compound 7c,
which may be considered a pterostilbene analogue, showed an
interesting activity on MDR-expressing HL60R cells, while the
trimethoxy derivative 7a was poorly active in all cell lines. The
cytotoxic activity of 7d was similar to that of the parent
compound resveratrol (1a).
Among the terphenyl derivatives, compounds 13a,c,e,g were
effective antiproliferative and proapoptotic agents; in contrast,
compounds 13b,d,h showed a lower cytotoxic activity. Com-
pounds 13f and 13i were the most potent compounds of the
series.
Figure 1. Cell cycle distribution of HL60 cells exposed for 24 h to
some representative stilbene, naphthalene, and terphenyl compounds
presented in this study: (a) control, (b) 3d (30 µM), (c) 3c (10 µM),
(d) 7c (20 µM), (e) 13f (30 µM), (f) 13i (20 µM).
Despite some evident effects of the structural changes on both
the antiproliferative and proapoptotic actions of compounds, no
clear SAR pattern could be identified throughout the series.
The effects of compounds on cell cycle were evaluated in
HL60 cells by flow cytometry after staining cells with propidium
iodide. Analysis of sub-G0-G1 (apoptotic peak), G0-G1, S, and
G2-M peaks revealed that all compounds presented in this study
caused a recruitment of cells in S or G2-M phases, except for
the terphenyl 13g, which blocked cells in G0-G1. In Figure
1b-f, the flow cytometric analyses of the most representative
derivatives 3c, 3d, 7c, 13f, and 13i are reported, compared to
untreated HL60 cells (Figure 1a). Compound 3d (Figure 1b)
caused a clear recruitment of cells in S phase and a decrease of
cells in the G2-M phase; compounds 7c and 13f (Figure 1d,e)
induced a recruitment of cells in S and G2-M phases and a
decrease of the G0-G1 peak, and compounds 3c and 13i (Figure
1c,f) caused a marked block of cells in the G2-M phase. All
compounds induced the appearance of a sub-G0-G1 apoptotic
peak. Similar results were previously described for resveratrol,
piceatannol, and other natural stilbenes.27,31a-e Figure 2b-d
shows the effects of compound 13g on cell cycle after 8 h
(Figure 2b), 24 h (Figure 2c), and 96 h (Figure 2d). The block
of cell cycle in G0-G1 is evident in Figure 2b-d when
compared to the control (Figure 2a). An apoptotic sub-G0-G1
peak was observed in HL60 cells exposed to 13g for 24 and 96
h (Figure 2c,d). In contrast, the sub-G0-G1 peak was very low
in the sample exposed to 13g for 8 h (Figure 2b).
Figure 2. Cell cycle distribution of HL60 cells exposed for 8 h (b)
and 24 h (c) to 50 µM of 13g; (d) cell cycle distribution of HL60 cells
exposed for 96 h to 10 µM of 13g; (a) control.
in HL60 cells. Moreover, we compared the differentiating effect
of 13g with that of resveratrol (1a), because previous evidence
indicated that resveratrol was able to induce cell differentiation
in some promyelocytic leukemia cell lines.32 Cells were exposed
to 10 µM 13g or 10 µM resveratrol. After 72 h, the expression
of CD14 (monocytic marker), CD11b, and CD11c (granulocytic
markers) was evaluated by flow cytometry. About 60% of HL60
cells exposed to 13g became CD14, CD11b, and CD11c
positive, while less than 25% of HL60 cells exposed to
resveratrol became positive to these markers (Table 2). As
shown in Figure 3a-f, HL60 cells differentiated in metamy-
elocytes, myelocytes, monocytes, and granulocytes after 4 days
exposure to 10 µM 13g. Of interest, some monocytes derived
from HL60 cells exposed for 4 days to 10 µM 13g became
specialized macrophages able to phagocyte fragments of apo-
ptotic cells (Figure 3f).
Given the above results, although 13g was not the most potent
compound as regards the cytotoxic activity, we considered it
worth further study, because it was the only one able to arrest
the cell cycle in the G0-G1 phase. The G0-G1 recruitment
appeared after 8 h when HL60 cells were exposed to 50 µM of
13g; using a lower concentration (10 µM), we observed a G0-
G1 block after 96 h (Figure 2d).
Considering the ability of 13g to induce a stable block in the
G0-G1 phase, we investigated its effect on cell differentiation

3016 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Roberti et al.
Figure 3. Morphologic changes observed in HL60 cells (promyelocytic acute leukemia) after 72-96 h exposure to 13g (10 µM): (a) control
(promyelocytes); (b) myelocytes and a metamyelocyte (72 h after exposure to 13g); (c) myelocyte-, metamyelocyte-, and monoblast-derived cells
(96 h after exposure); (d) metamyelocyte- and monoblast-derived cells (96 h after exposure); (e) band neutrophil (96 h after exposure); (f) phagocytosis
of apoptotic bodies by a macrophage (96 h after exposure).
we showed the high potency of 3′-hydroxypterostilbene (2b).27
Table 2. Expression of Monocytic (CD14) and Granulocytic (CD11b,
CD11c) Markers in HL60 Cells before (control) or 4 days after
Treatment with 10 µM 13g or 10 µM Resveratrol (1a)
None of the variations presently introduced into the B ring of
the compounds allowed us to improve the antiproliferative and
compounds
CD14 (%)
CD11b (%)
CD11c (%)
proapoptotic profile of our lead 2b. We studied not only sensitive
HL60 cells but also resistant HL60R and Bcr-Abl-expressing
K562 cells: despite some good potency showed by stilbene
derivatives 3c and 3d on sensitive cells, the antiproliferative
and eventually the proapoptotic actions were not maintained
against resistant and Bcr-Abl-expressing cells. Among the
naphthalene-based compounds 7a,c,d, none gave better results
with respect to the parent stilbene derivatives 3c, pterostilbene
(2a), and resveratrol (1a) in terms of antiproliferative and
proapoptotic activities. More interesting were the results ob-
tained with the terphenyl derivatives 13a-i. Among the
compounds belonging to this class, the best results regarding
the proapoptotic activity were obtained with the differently
substituted derivatives 13e, 13f, and 13i, suggesting that the
ortho-substitution motif in the terphenyl architecture plays a
prevalent role in determining this type of activity.
control
13g
resveratrol (1a)
0.2
58
23
0
62
24
0
60
22
In contrast, other trihydroxylated resveratrol-like compounds
(7d, 13h, 13i) did not induce differentiation.
Discussion
As an extension of our previous studies26,27 on the SAR of
trans-stilbene derivatives, we synthesized a series of 3,5-
dimethoxy analogues in which a variety of substituents were
introduced at positions 2′, 3′, 4′, and 5′ of the stilbene scaffold.
Moreover, in an attempt to increase the diversity of the
compounds while the basic structural motif of resveratrol was
retained, we obtained both some naphthalene and some terphenyl
derivatives. Notably, the latter two structures do not bear the
ethylene double bond that is the main reason for the chemical
and metabolic instability of stilbene compounds.33a-c By the
use of this approach, we earlier disclosed a terphenyl analogue
of stilbene-based arotinoid that surprisingly supported cell
growth and inhibited apoptosis.34 The same strategy was also
recently exploited by Minutolo et al., who carried out a study
on the ceramide-mediated proapoptotic activity of some vari-
ously hydroxylated and methoxylated naphthalenes.30
All of the compounds presented in this paper were prepared
through parallel synthesis, taking advantage of the standard
Wittig reaction for stilbenes and of cross-coupling Suzuki
protocol for naphthalenes and terphenyls. This synthetic ap-
proach proved to be efficient to rapidly obtain small sets of
compounds and appropriate when random substitutions have
to be explored in the lack of either a specific target or a clearly
defined SAR pattern.
The lack of clearly identifiable SARs within the whole series
as well as within each of the subsets (stilbenes, naphthalenes,
terphenyls) points to a complex mechanism of action for all of
these molecules. In principle, however, this is not surprising
given their at least qualitative stereoelectronic similarity to
resveratrol, whose mechanism of apoptosis induction is still
controversial. We have already discussed the discrepancies of
opinion about this aspect in a previous paper.27
Although the trihydroxylated terphenyl 13g was not the most
potent compound presently synthesized, it showed the most
peculiar biological profile. In fact, 13g caused a block of cell
cycle in the G0-G1 phase, while resveratrol,31a-e as well as the
other stilbenes or stilbene mimetics studied in this and previous
works,26,27 induced a recruitment of cells in S or G2-M phases.
Moreover, whereas at higher concentration 13g was able to
induce apoptosis both in sensitive and resistant cell lines, when
used at low concentration (10 µM) for 72-96 h, it was able to
induce a differentiation of HL60 cells. This is indeed a
remarkable feature, if one considers that currently there is only
one drug (all-trans-retinoic acid, ATRA) available in dif-
ferentiation therapy to treat patient with acute promyelocytic
leukemia (APL).35a-b Remarkably, however, the differentiating
Initially, the stilbene compounds 3a-h were studied in vitro
for both cell growth inhibition and the ability to induce apoptosis
in leukemia and lymphoma cells. In our previous work,26 we
pointed out the importance of a hydroxy function at the C-3′
position of the stilbene scaffold, and in a further development,

A Terphenyl DeriVatiVe that Blocks the Cell Cycle
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 3017
two phases were separated. The aqueous layer was washed with
Et2O, and the organic phases were combined and washed with 1
M NaOH followed by brine. The ethereal solution was dried over
Na2SO4 and evaporated. Purification of each crude product by flash
chromathography using petroleum ether/ethyl acetate yielded the
corresponding terphenyl derivatives 13a-f.
effects of 13g are, in part, different from those of ATRA, as
the latter induces in vitro a prevalent granulocytic differentiation
of APL blasts, while 13g induces a granulocytic and monocytic
differentiation.36a-b Moreover, no morphological aspects of
macrophages able to phagocyte apoptotic cells have been
described for ATRA.
General Parallel Procedure for Demethylation to Trihydroxy
Derivatives 7d, 13 g-i. In four distinct reactors, the trimethoxy
derivatives 7a and 13b,d,f (1 equiv) were dissolved in anhydrous
CH2Cl2 (10 mL) at -78 °C. Then, BBr3 (1 M in CH2Cl2, 3 equiv)
was added to each solution, and the resulting reaction mixture was
allowed to warm to room temperature for 20 h, cooled at 0 °C,
and treated as follows. Each solution was poured into H2O, and
the two phases were separated. The aqueous layer was washed twice
with CH2Cl2, and the organic phases were combined and washed
with a 1 M solution of sodium thiosulfate followed by H2O. The
organic layer was dried over Na2SO4 and evaporated to dryness.
Purification of each crude product by flash chromathography using
petroleum ether/ethyl acetate yielded the corresponding trihydroxy
derivatives 7d, 13g-i.
Recently, Asou et al. first described the differentiating
properties of resveratrol in a series of myeloblastic leukemia
cells.32 The differentiating effects of resveratrol were higher in
NB4 and U935 cell lines but were poor in HL60 cells. The
mechanism by which resveratrol induces differentiation is not
known, although it has been suggested that a decrease in activity
of NF-κB could be associated with the terminal differentiation
induced by the compound. In the present work, we observed
that resveratrol was able to induce the expression of differentiat-
ing markers in less than 25% of HL60 cells, while 13g induced
differentiation in about 60% of cells. Of interest, 13g induced
a myeloid differentiation of HL60 cells with the appearance of
cells in different phases of differentiation; after 96 h of treatment,
metamyelocytes and band neutrophils were the most representa-
tive cells. The presence of macrophages able to phagocyte
apoptotic bodies suggests that differentiation induced by 13g
is not only morphological but also functional. Finally, in a
previous work, some of us37 observed that ATRA was unable
to induce apoptosis in HL60 cells after 48 h of treatment; in
contrast, 13g was able to induce apoptosis after 24 h.
In conclusion, compared to resveratrol, the synthetic terphenyl
13g showed a more potent apoptotic and differentiating activity.
Moreover, 13g was active on both MDR and Bcr-Abl-expressing
cells that were resistant to resveratrol. For these characteristics,
13g could be considered a promising lead to obtain proapoptotic
and differentiating compounds for the treatment of acute
leukemias. As a final observation from a chemical perspective,
we want to remark that the terphenyl scaffold proved to be a
versatile architecture for the identification of small molecules
useful as chemical probes to study cell functions and mecha-
nisms.
Acknowledgment. This work was supported by a PRIN
Project Grant from MIUR, Italy.
Supporting Information Available: General chemical methods,
detailed biological protocols, general methods for the synthesis of
stilbene derivatives 3a-h, synthesis of 3,5-dimethoxyphenylboronic
acid 4, physical and spectroscopic data for compounds 3a-h, 7a-
d, and 13a-i, and elemental analysis results. This material is
available free of charge via the Internet at http://pubs.acs.org.
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30, 91-113.
Experimental Section
General Parallel Procedure for the Synthesis of Derivatives
7a, b, 8-10. In five distinct reactors, 2-Br-naphthalenes 5 and 6
and o-, m-, and p-iodobromobenzene (1.0 equiv) were dissolved in
toluene (10 mL). Then, 3,5-dimethoxyphenylboronic acid 4 (2
equiv) in EtOH (3 mL) and aqueous Na2CO3 (2 M, 3.0 equiv) were
added to each reactor, and the resulting mixtures were deoxygenated
with a stream of N2. After 10 min, Pd(PPh3)4 (0.05 equiv) was
added, and each mixture was brought to reflux, allowed to stir under
N2 for 5 h, cooled to room temperature, and treated as follows.
Each solution was poured into a mixture of H2O and Et2O, and the
two phases were separated. The aqueous layer was washed with
Et2O, and the organic phases were combined and washed with 1
M NaOH followed by brine. The ethereal solution was dried over
Na2SO4 and evaporated. Purification of each crude product by flash
chromathography using petroleum ether/ethyl acetate 9.5:0.5 or 9.9:
0.1 yielded the corresponding (3,5-dimethoxyphenyl)naphthalenes
7a,b and the biphenyldimethoxy derivatives 8-10.
General Parallel Procedure for the Synthesis of Terphenyl
Derivatives 13a-f. Each of the three bromobiphenyldimethoxy
derivatives 8-10 (2.0 equiv) was dissolved in toluene (20 mL) and
each was partitioned in two distinct reactors. 4-Substituted-
phenylboronic acid 11 or 12 (2 equiv), in EtOH (3 mL), and aqueous
Na2CO3 (2M, 3.0 equiv) were added to each set of three reactors
containing 8-10, and the resulting six mixtures were deoxygenated
with a stream of N2. After 10 min, Pd(PPh3)4 (0.05 equiv) was
added, and each mixture was brought to reflux, allowed to stir under
N2 for 3-12 h, cooled to room temperature, and treated as follows.
Each solution was poured into a mixture of H2O and Et2O, and the
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