A novel atypical retinoid endowed with proapoptotic and antitumor activity

Article abstract of DOI:10.1021/jm025593y

The novel atypical retinoid E-3-(4′-hydroxy-3′-adamantylbiphenyl-4-yl)acrylic acid (ST1926, 4) exhibited a potent antiproliferative activity on a large panel of human tumor cells. Despite almost complete loss of ability to activate RARs, the compound was an effective apoptosis inducer and surprisingly produced DNA damage, that likely contributes to the proapoptotic activity. Following oral administration, 4 was well tolerated and caused tumor growth inhibition in the ovarian carcinoma, A2780/DX, and in the human melanoma, MeWo, growing in nude mice, thus supporting the therapeutic interest of the novel agent.

Full text of DOI:10.1021/jm025593y

J . Med. Chem. 2003, 46, 909-912
909
Letters
A Novel Atyp ica l Retin oid En d ow ed w ith
P r oa p op totic a n d An titu m or Activity
†
†
Raffaella Cincinelli, Sabrina Dallavalle,
†
‡
‡
F igu r e 1. Chemical structure of 1.
Sch em e 1. Synthesis of 4a
Lucio Merlini, Sergio Penco, Claudio Pisano,
Paolo Carminati, Giuseppe Giannini,
Loredana Vesci, Carlo Gaetano, Barbara Illy,
Valentina Zuco, Rosanna Supino, and
‡
‡
‡
§
|
⊥
⊥
Franco Zunino*,⊥
Dipartimento di Scienze Molecolari Agroalimentari,
Universit a` di Milano, Milano, Italy,
R & D, Sigma-tau, Pomezia, Italy,
Unit a` Operativa Farmacologia Preclinica,
Istituto Nazionale dei Tumori, Via Venezian 1,
0133 Milano, Italy, Laboratorio Patologia Vascolare,
2
Istituto Dermopatico dell’Immacolata, Rome, Italy, and
Laboratorio Terapia Genica e Biologia Vascolare,
Istituto Cardiologico “Fondazione I. Monzino”, Milano, Italy
Received October 10, 2002
Abst r a ct : The novel atypical retinoid E-3-(4′-hydroxy-3′-
adamantylbiphenyl-4-yl)acrylic acid (ST1926, 4) exhibited a
potent antiproliferative activity on a large panel of human
tumor cells. Despite almost complete loss of ability to activate
RARs, the compound was an effective apoptosis inducer and
surprisingly produced DNA damage, that likely contributes
to the proapoptotic activity. Following oral administration, 4
was well tolerated and caused tumor growth inhibition in the
ovarian carcinoma, A2780/DX, and in the human melanoma,
MeWo, growing in nude mice, thus supporting the therapeutic
interest of the novel agent.
a
Reagents and conditions: (i) 1-adamantanol, H2SO4/AcOH 1:9,
2
5 °C, 3 days (quantitative); (ii) methyl acrylate, tri-(o-tolyl)phos-
phine, Pd(OAc)2, Et3N, 100 °C, 4 h (98%); (iii) LiOH‚H2O, THF/
H2O 1:1, 25 °C overnight (94%).
involved in several biological processes, but have a
marginal role in the treatment of neoplastic diseases.
Retinoids exert their biological effects through ability
to bind to and activate specific receptors which function
as transcriptional factors, but it is likely that the broad
biological effects associated with retinoids are mediated
The efficacy of chemotherapeutic treatment of tumors
is limited by the development of drug resistance. Among
the multiple mechanisms involved, it is likely that
defects in apoptotic pathways are responsible for re-
duced cell ability to undergo drug-induced apoptosis.
Indeed, tumor progression has been associated with
2
by distinct pathways.
1
multiple changes including resistance to apoptosis.
“Atypical retinoids” do not fit into the classical concept
of ligand-receptor interaction: they activate certain
RARs (retinoic acid receptors) as well as exert a growth-
regulatory or apoptogenic activity that is not receptor
mediated. 4-Hydroxyphenylretinamide (4HPR) and 6-[3-
(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxy-
lic acid (AHPN/CD437) belonging to this class are
considered retinoids because they bind to and transac-
This evidence emphasizes the interest for identifica-
tion of novel strategies to facilitate the cells to undergo
apoptosis. Among novel apoptosis-based approaches,
“
atypical” retinoids display significant potential as
2
therapeutic agents in tumor treatment. The cellular
basis of their proapoptotic activity remains unclear.
However, it is evident that some novel retinoid-related
compounds possess unique mechanism(s) of action. In
particular, a relevant feature of these agents is the
ability to activate p53-independent apoptosis, because
p53 mutation, a frequent alteration of tumor cells, is
implicated in resistance to DNA-damaging agents. Clas-
sical retinoids, structural analogues of vitamin A, are
3
tivate RARs. In particular, CD437 (1, Figure 1) is
known as RARγ-selective retinoid. However, the in-
volvement of RARs in biological activity of 1 remains
uncertain. Compound 1 is active in retinoid-resistant
cells and retinoid antagonists cannot block this activity.4
Preclinical and clinical data have already supported the
potential of atypical retinoids as cancer therapeutic and
5
,6
*
To whom correspondence should be addressed. Phone:
chemopreventive agents.
+
39 0223902267. Fax: +39 0223902692. e-mail: Franco.zunino@
Focusing our attention on the RAR-independent
induction of apoptosis, we synthesized new atypical
retinoids. Here we report the discovery of a new lead
compound endowed with potent antiproliferative and
pro-apoptotic activities.
istitutotumori.mi.it.
†
Universit a` di Milano.
Sigma-tau.
Istituto Dermopatico dell’Immacolata
Istituto Nazionale dei Tumori.
‡
§
⊥
|
Istituto Cardiologico “Fondazione I. Monzino”.
1
0.1021/jm025593y CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/19/2003

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10 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Letters
F igu r e 2. Transactivation and luciferase assay in HeLa cells. RAREluc reporter construct (2 µg) was cotransfected with 0.5 µg
of pcDNA3 RARR, pcDNA3 RARâ, and pcDNA3 RARγ expression vectors together with 0.5 µg of CMV âgal expression vector for
evaluation of transfection efficiency. 24 h after transfection, cells were incubated respectively with 0.1 µM all-trans-retinoic acid
(
2
(
ATRA) or ST1926 (4) or CD437 (1) or equivalent amount of control solvent. Luciferase assays were performed after additional
4 h using the Luciferase Assay System (Promega). Luciferase activity was measured using an A9904V TopCount luminometer
Packard). Counts per second were corrected for protein concentration and transfection efficiency. Data are mean values of three
independent experiments ((SD) performed in duplicate.
Ta ble 1. Antiproliferative Effect of 4 and 1a
IC50 (µM)
Ta ble 2. Time Course of Apoptosis in Response to 4 or 1a
IGROV-1 DU145
cell line
tumor type
p53 status
4
1
4
1
4
1
IGROV-1
A2780/DX
DU145
LNCaP
PC3
H460
HCT116
LoVo
ovarian carcinoma wild-type 0.23 ( 0.09 0.35 ( 0.04
0.1 ( 0.05 n.d.
prostate carcinoma mutant
wild-type 0.12 ( 0.09 0.32 ( 0.05
null 0.21 ( 0.06 0.43 ( 0.01
24 h
42 ( 9
46 (11
44 ( 5
22 ( 6
15 ( 5
38 ( 2
24 ( 3
44 ( 4
50 ( 2
16 ( 1
33 ( 8
30 ( 5
4
7
8 h
2 h
0.1 ( 0.02 0.28 ( 0.05
a
Apoptosis was determined by morphological analysis of pro-
NSCLC
colon carcinoma
wild-type 0.19 ( 0.06 0.43 ( 0.05
wild-type 0.32 ( 0.05 0.68 ( 0.02
wild-type 0.15 ( 0.03 0.49 ( 0.04
pidium iodide-stained cells following exposure to IC80 (0.5 and 1
µM for 4 and 1, respectively). The results are expressed as
percentage of apoptotic cells versus total cell number. Values are
the mean ( standard deviation of three independent experiments.
A431
GBM
cervix carcinoma
glioblastoma
mutant
mutant
mutant
null
0.25 ( 0.07 0.51 ( 0.1
0.18 ( 0.02 0.55 ( 0.03
0.25 ( 0.02 0.42 ( 0.12
0.25 ( 0.03 0.67 ( 0.07
Me665/2/21 melanoma
SAOS
U2OS
osteosarcoma
linkers. These spacers were designed in order to obtain
conformationally minimized structures in which the
distance between the two groups was maintained in a
range of values as close as possible to 11.4 Å. Among
the numerous compounds synthesized, we identified a
very promising derivative in which the naphthalene ring
was replaced by a styrene moiety.
The lead compound of this class of new retinoids, ST
1926 (4), was prepared in a high-yielding three-step
sequence on a multigram scale (Scheme 1). Friedel-
Crafts reaction of 1-adamantanol with commercially
available 4-hydroxy-4′-bromobiphenyl gave quantita-
tively compound 2 that was subjected to Heck reaction
with methyl acrylate to afford the ester 3 (98%). E-3-
(4′-Hydroxy-3′-adamantylbiphenyl-4-yl)acrylic acid (4)
was obtained (94%) by hydrolysis of 3 with LiOH in
THF/H2O.
wild-type 0.26 ( 0.09 0.44 ( 0.03
a
Cells were exposed to 4 or 1 for 72 h. Antiproliferative activity
was measured by cell counting and expressed as concentration
required for 50% inhibition of cell growth (IC50). IC50 values were
determined by dose-response curves.
The strategy for the design was devised using as a
reference compound 1. We assumed that functional
groups critical for activity were the hydroxy group, the
lipophilic adamantyl moiety, and the carboxylic func-
tion. On the basis of molecular mechanics calculations
we obtained a minimal energy conformation for 1, in
which the distance between the C1 of the adamantyl
group and the carboxylic carbon was calculated to be
ca. 11.4 Å. Thus we planned to prepare a series of
analogues keeping the key structural and pharmaco-
logically important groups of 1 connected by different

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 911
F igu r e 3. Cell cycle perturbation. Cell cycle distribution of
U2-OS cells at different times of treatment with the IC80 of
the two drugs. FACS analysis was performed on propidium
iodide stained cells.
F igu r e 5. (A) Effect of ST1926 4 delivered orally (qdx2/wx3w)
starting from day 1, against the A2780/DX human ovarian
carcinoma. (B) Effect of 4, delivered orally (qdx5/wx2w) start-
ing from day 6, against the MeWo human melanoma.
0
.1 to 0.3 µM, which are pharmacologically relevant. The
growth-inhibitory activity of 4 was apparently p53-
independent, because the cellular effects were compa-
rable in cells with functional or defective p53. Among
the tested cell lines, prostate carcinoma cells exhibited
an exquisite sensitivity to 4. The cellular effects of 4
and 1 were studied in more detail in an ovarian
carcinoma cell line, IGROV-1, carrying a functional
F igu r e 4. ST1926 4-induced DNA damage in IGROV-1 and
H460 cells as determined by alkaline elution. Cells were
treated for 6 h with the drug, lysed, and processed as reported
9
by Supino et al. For IGROV-1 cells, the data are mean values
of three independent experiments. In the inset: percentage
of DNA retention on the filter in function of the elution time
in IGROV-1 cells. 9, untreated cells; b, 1000 rad; 2, 4, 1 µM;
7
wild-type p53 and a p53-mutant prostate carcinoma cell
1
, 4, 0.5 µM; O, 4, 0.25 µM.
8
line, DU145. In both cell systems 4 was an effective
The capacity of 4 and 1 to transcriptionally activate
a retinoic acid response element reporter construct
RAREluc) in the presence or absence of specific retinoic
apoptosis inducer, since an induction of apoptosis
(already evident at 24 h) was found at concentrations
causing antiproliferative effects (IC80) (Table 2). Again
4 was more potent and effective than 1 in both cell
systems. 4 caused cell accumulation in G1/S or S phase
of cell cycle depending on tumor cells. In osteosarcoma
U2-OS cells, the 4-induced arrest in S phase was more
pronounced than that caused by 1 (Figure 3).
The impressive potency of 4 as an antiproliferative
agent, and the accumulation of treated cells in S phase
of cell cycle suggested a drug interference with DNA
replication. Indeed, an appreciable extent of DNA
breaks was detected in IGROV-1 ovarian carcinoma and
H460 lung carcinoma cells following 6 h exposure to 4
(Figure 4). It is unlikely that these lesions reflect an
early manifestation of apoptosis since apoptosis could
be detected only at 24 h. Alkaline elution experiments
in different conditions should provide additional infor-
mation on the mechanism and nature of these DNA
lesions. Despite the compound ability to induce poten-
(
acid receptor isoforms were studied, in comparison to
ATRA, in transient transfection experiments performed
in HeLa cells. While ATRA strongly activated all recep-
tor isoforms (about 100 fold), 4 failed to significantly
stimulate transcription in presence of RARR and RARâ
receptors compared to RAREluc alone (Figure 2). A
marginal increase in luciferase activity, however, was
observed in the presence of RARγ overexpression. Thus,
although structurally related to retinoids, 4 was char-
acterized by an almost complete loss of typical retinoid
features.
Compound 4 was studied in a large panel of human
tumor cell lines including p53-defective cells (Table 1).
Treatment for 72 h with 4 caused a dose-dependent
growth inhibition in all tumor cell lines. 4 was at least
-fold more potent than 1. The concentrations of 4
required for 50% growth inhibition (IC50) ranged from
2

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12 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Letters
tially lethal lesions and its potency, 4 was well tolerated
in vivo following oral administration. Using a daily
treatment schedule (qdx5/wx4w), oral administration of
Fondazione Hoepli. We thank Drs. Maria Luisa Cervoni
and Vincenzo Giordano for their support in animal
models.
4
caused a significant tumor growth inhibition in a
human ovarian carcinoma, A2780/DX, and in a human
melanoma, MeWo, growing in nude mice (Figure 5).
Under similar conditions, 1 did not exhibit activity
against the melanoma model.
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(
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(
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Con clu sion s
In comparison with 1, the related compound 4 shows
an even lesser ability to activate RARs. Notwithstanding
its nonretinoid profile, 4 shows a potent antiproliferative
activity on a large panel of human tumor cells. The large
spectrum of growth-inhibitory activity and the evidence
of antitumor activity in human tumor xenografts sup-
port the therapeutic potential of the novel agent for
cancer treatment. The therapeutic interest of the novel
compound is also emphasized by the compound ability
to induce genotoxic lesions which likely contribute to
the proapoptotic activity. The mechanism of the induc-
tion of DNA lesions is unclear, because a direct drug-
DNA interaction is unexpected. Taking into account the
unique pharmacological profile, characterized by good
tolerability and broad spectrum of activity, 4 represents
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agents with potential applicability in the treatment of
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Ack n ow led gm en t. This work was partially sup-
ported by the Associazione Italiana Ricerca sul Cancro,
Milano, by the Consiglio Nazionale delle Ricerche,
Roma, by the Ministero della Salute, Roma, and by the
J M025593Y