Programmed cell death (PCD) associated with the stilbene motif of arotinoids: Discovery of novel apoptosis inducer agents possessing activity on multidrug resistant tumor cells

Article abstract of DOI:10.1016/S0960-894X(00)00547-3

Considering that the stereochemistry of the C9-C10 alkenyl portion of natural 9-cis-RA, as the one of the olefinic moiety of the previously described isoxazole retinoid 4, seems of particular importance for their apoptotic activity, we prepared a novel class of TTNPB analogues bearing both the cis or trans configuration of the alkenyl portion. The compounds were evaluated in vitro for their cytotoxic and apoptotic activities. We discovered that the cis-TTNPB 9c possesses apoptotic activity comparable with that of the retinoid 4. Moreover, the amino arotinoid 16c showed potent apoptotic activity in HL60 promyelocytic leukemia cells. Interestingly, 16c proved to be a particularly potent apoptosis-inducing agent active in multidrug resistant (MDR) cell lines. Therefore, to the best of our knowledge, 16c may represent the first known aminoarotinoid endowed with potent apoptotic activity in MDR cells. Taken together, these results seem to point out that the cis-stilbene motif of arotinoids may be at least an important feature in conferring cytotoxic and apoptotic activity to this class of compounds. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00547-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2669±2673
Programmed Cell Death (PCD) Associated with the Stilbene
Motif of Arotinoids: Discovery of Novel Apoptosis
Inducer Agents Possessing Activity on Multidrug
Resistant Tumor Cells
Daniele Simoni,a,* Marinella Roberti,b Francesco Paolo Invidiata,c
Riccardo Rondanin,a Riccardo Baruchello,a Cinzia Malagutti,a Angelica Mazzali,a
Marcello Rossi,a Stefania Grimaudo,d Luisa Dusonchet,d Maria Meli,d
Maria Valeria Raimondi,d Natale D'Alessandrod and Manlio Tolomeod
aDipartimento di Scienze Farmaceutiche, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy
b
Á
Dipartimento di Scienze Farmaceutiche, Universita di Bologna, Italy
Á
Á
Dipartimento di Scienze Farmacologiche, Policlinico P. Giaccone, Universita di Palermo, Italy
c
Dipartimento Farmacochimico Tossicologico e Biologico, Universita di Palermo, Italy
d
Received 17 July 2000; revised 21 August 2000; accepted 18 September 2000
AbstractÐConsidering that the stereochemistry of the C9±C10 alkenyl portion of natural 9-cis-RA, as the one of the ole®nic moiety
of the previously described isoxazole retinoid 4, seems of particular importance for their apoptotic activity, we prepared a novel
class of TTNPB analogues bearing both the cis or trans con®guration of the alkenyl portion. The compounds were evaluated in
vitro for their cytotoxic and apoptotic activities. We discovered that the cis-TTNPB 9c possesses apoptotic activity comparable with
that of the retinoid 4. Moreover, the amino arotinoid 16c showed potent apoptotic activity in HL60 promyelocytic leukemia cells.
Interestingly, 16c proved to be a particularly potent apoptosis-inducing agent active in multidrug resistant (MDR) cell lines.
Therefore, to the best of our knowledge, 16c may represent the ®rst known aminoarotinoid endowed with potent apoptotic activity
in MDR cells. Taken together, these results seem to point out that the cis-stilbene motif of arotinoids may be at least an important
feature in conferring cytotoxic and apoptotic activity to this class of compounds. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Retinoids exert most of their e€ects by binding to spe-
ci®c receptors and modulating gene expression. The
diversity of retinoid-induced signalling pathways is
mediated by at least six retinoid receptors which fall
into two subfamilies: retinoic acid receptors (RARs), a,
b and g, and the retinoid X receptors (RXRs), a, b and
g.1 In common with other members of the steroid hor-
mone receptor superfamily, these two subfamilies of
receptors contain a DNA-binding domain and a ligand-
binding domain united by a short hinge region that may
also serve as a nuclear transactivation signal. At the
cellular level, activation of the retinoid receptors can
inhibit cell proliferation, induce di€erentiation and
apoptosis; the induction of apoptosis is related to cell
growth and di€erentiation in various ways, depending
on the cell type. The relative implication of RAR and
RXR receptors in retinoid-induced apoptosis is still
unclear; some observations suggest that cell di€erentia-
tion mediated by retinoic acid is induced by the activation
Retinoids are a class of natural and synthetic com-
pounds structurally related to all-trans-retinoic acid
(ATRA).1,2 These biological agents are known to play a
major role in regulating growth and di€erentiation of
normal, premalignant, and malignant cell types. There-
fore, they are currently being evaluated as preventative
or therapeutic agents in a variety of human cancers.3 It
has been shown that retinoids can augment the activity
of other biological and chemotherapeutic agents, o€er-
ing potentially e€ective combination regimens.3 More-
over, by inducing apoptosis, these compounds may
enhance sensitivity of tumors to cytotoxic agents and
overcome drug resistance.3 5
*Corresponding author. Tel.: +39-532-291-291; fax: +39-532-291-
296; e-mail: smd@dns.unife.it
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00547-3

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D. Simoni et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2669±2673
of RARs, whereas activation of RXRs seems to be
essential for driving cells into apoptosis;6 on the con-
trary, other authors have demonstrated the involvement
of the retinoic acid receptor in apoptosis induced by
retinoids, as shown by the inhibition of apoptosis after
treatment of HL60 cells, with a RARa antagonist.7,8
However, although the mechanisms of retinoid-induced
apoptosis still remain unclear, the clinical ecacy of
most of the novel potent retinoids, i.e. 9-cis-retinoic acid
(9-cis-RA),9 CD437,10 LDG1069 and N-(4-hydro-
xyphenyl)-retinamide (4-HPR)11 seems to be related
to both their di€erentiation- and apoptosis-inducing
activity.12
Since 9-cis-RA is well known to possess both apoptosis-
and di€erentiation-inducing activities, whereas ATRA
induces di€erentiation but not apoptosis, we considered
that the 9-cis con®guration contained in the polyene
side chain of 9-cis-RA, as well as the cis con®guration of
the ole®n contained in 4, may be related to the apopto-
tic activity of these compounds. Consequently, we
planned the synthesis of novel TTNPB-retinoid ana-
logues taking into consideration both the con®guration
E and Z of the stilbene motif. Thus, we discovered that
the cis-TTNPB 9c, which still retains some structural
features of 9-cis-RA, was endowed with a potent apop-
totic activity, being more active than the previously
On the other hand, considering that apoptosis is prob-
ably the major modality by which leukemic cells are
killed by treatment with pharmacological agents, it is
likely that the identi®cation of new retinoids able to
induce apoptosis in di€erent types of tumor cells, inde-
pendently/or not of their di€erentiating activity, could
be an important goal in the cancer chemotherapy. Tak-
ing these considerations into account, we started a study
aimed to design retinoid compounds endowed with
potent apoptotic activity.13 15 We focused our attention
on the identi®cation of the structural features associated
with the apoptotic activity of compounds related to
arotinoids. We took advantage of our previous work on
a new class of isoxazole-containing arotinoids, which
were found to possess interesting apoptotic activity.15 In
particular, we have reported recently on the e€ects of
the substitution of the phenyl ring of the stilbene skele-
ton of TTNPB with an isoxazole heterocycle: while the
E isomer 5 was found to be endowed with an appreci-
able di€erentiating activity, the cis-stilbene derivative 4
was essentially inactive at the level of the retinoid
receptors, but was identi®ed as a potent inducer of
apoptosis. These facts led us to hypothesize the exis-
tence of a non-RAR-mediated mechanism associated
with the cis-stilbene-like motif of 4 and responsible for
the apoptotic activity of the compound. On the other
hand, although 4-HPR and CD437 activate nuclear
receptors, their pro-apoptotic activity appears to be
mediated, in part, through retinoid receptor-independent
pathways.10,11 Interestingly, 4-HPR and CD437 proved
very active in multidrug resistant tumor cells.16 18
described isoxazole retinoid 4. On the other hand,
TTNPB 3, a known potent di€erentiating arotinoid, is
only a poor inducer of apoptosis. We found also that
the amino derivative 16c proved to be a particularly
potent apoptosis-inducing agent active in multidrug
resistant (MDR) cell lines, in particular resistant to
the apoptotic e€ects of several chemotherapeutic
drugs, such as daunorubicin, methotrexate, citarabine,
5-¯uorouracil and others.
In our opinion these ®ndings may provide an important
basis to facilitate the design of novel potent apoptosis-
inducing agents structurally related to the cis-stilbene
motif of arotinoids. The newly synthesized retinoids will
also be important in studies of receptor selectivity aimed
to better clarify the mechanism of action of vitamin A
and of its analogues (retinoids).
Chemistry
For the procurement of the benzoic acid derivatives 3,
9a±c, 10a,b we took advantage (Scheme 1), to some
extent, of known procedures:19 the Wittig reaction
between the carbomethoxybenzaldehydes 7a±c and the
phosphonium salt 6 in the presence of n-butyllithium
produced the esters 8a±c in signi®cant yields. The reac-
tion produced a mixture of the E and Z isomers (7:3)
which was in turn easily hydrolyzed with lithium
hydroxide and the desired carboxylic acid derivatives
puri®ed and separated by ¯ash chromatography.

D. Simoni et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2669±2673
2671
Spectral data of known and new compounds are con-
Results and Discussion
sistent with those of arotinoids previously described in
the literature.19 21 Finally, the Wittig reaction between
the phosphonium salts 6 and 11 and the aldehydes 12a±
d in THF solution and in the presence of NaH as the
base produced in good yields the compounds 13a±e and
14a±e (Scheme 2). The E and Z isomers were separated
and characterized; bond geometry of the compounds
Table 1 shows the cytotoxic activity expressed as IC50
(the drug concentration able to inhibit by 50% cell
growth) and apoptotic activity expressed as AC50 (the
drug concentration able to induce apoptosis in 50% of
cells) of 16 di€erent new retinoid derivatives in the
HL60 promyelocytic leukemia cell line. On the basis of
their e€ects on HL60 cells these compounds could be
classi®ed into four groups: (1) compounds with a mod-
est cytotoxic activity in terms of cell growth inhibition
and unable to activate programmed cell death (3, 13e
and 14e); (2) compounds moderately e€ective in indu-
cing cytotoxic and apoptotic e€ects (9a,b); (3) highly
cytotoxic compounds endowed with a moderate apop-
totic activity (9c, 15a, 15b, 15c, 15d, 16b and 16d); and
(4) compounds endowed with both a high cytotoxic and
1
was established by comparison of the H NMR of the
isomeric pairs. Z isomers showed the ole®nic protons
0.3±0.45 ppm higher ®eld than those of the E isomers.
The coupling constants of the vinylic protons of the E
isomers 13a±e were about 16 Hz whereas the Z isomers
14a±e showed the coupling constants of 12 Hz. The
compounds 13a±d and 14a±d were reduced with Zn/
AcOH at room temperature to give the amine derivatives
15a±d and 16a±d in about 80% yields.
Scheme 1.
Scheme 2.

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D. Simoni et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2669±2673
apoptotic activity (16c). Derivatives 10a,b were devoid
of any activity.
Most of the retinoids tested in this study, including
ATRA, 9-cis-RA and 13-cis-RA, were inactive (data not
shown) in multidrug resistant HL60R cells, K562 leu-
kemia and HUT78 T-cell lymphoma cells. HL60R cells
overexpress P-glycoprotein (P-gp) and are resistant to
apoptosis induced by drugs expelled out of cells by this
multidrug transporter, such as daunorubicin, etoposide,
vincristine, mitoxantrone and others. However, they are
also resistant to apoptosis from drugs which are not
substrates of P-gp, such as methotrexate, cytarabine,
and cisplatin. K562 is a myeloid leukemia cell line
expressing BCR-ABL, an oncogene that confers resis-
tance to apoptosis from di€erent cytotoxic drugs or
radiations.
Previously, we observed that isoxazole arotinoids with
the cis stereochemistry were more ecient inducers of
apoptosis than their correspondent trans stereoisomers.
Here we observed that the cis isomer of the potent dif-
ferentiating agent TTNPB is able to induce apoptosis in
HL60 cells, while the trans isomer has only moderate
e€ects in inhibiting cell proliferation. This was not
observed in the case of the trans isomer of 15a, which is
twice as active in inducing programmed cell death than
the respective cis isomer 16a. However, at di€erence, in
the case of the most active compound 16c, the cis isomer
is clearly the most apoptosis-inducing agent. Therefore,
although we hypothesized that the di€erent stereo-
chemistry of the double bond may be associated with a
di€erent apoptotic activity, this can not be considered
an absolute requisite. However, it appears evident from
the data shown in Table 1 that a relation exists between
apoptosis and the cis con®guration of the alkenyl por-
tion. Therefore, the cis-stilbene motif of arotinoids
seems to be at least an important feature in conferring
cytotoxic and apoptotic activity to this class of reti-
noids. Moreover, it also appears evident that the sub-
stituent's position at the phenyl portion of the TTNPB
analogues is a crucial factor for their activity.
Moreover, K562 cells do not express Fas/APO1 a cell
membrane receptor implicated in the activation of pro-
grammed cell death. In this study, we identi®ed three
new retinoids (16b±d) able to act in these cell lines with
pleiotropic resistance. In HL60R, K562 and HUT78
cells, the compound 16c showed a cytotoxic and apop-
totic activity similar to that observed in the sensitive
HL60 cell line. Also, 16b was active in these cell lines,
but at a concentration about 3±4 times higher than 16c.
On the contrary, 16d was able to inhibit the growth, but
not to induce programmed cell death (Table 2), in the
resistant cells. These data indicate that 16c may be con-
sidered an interesting drug for the therapy of di€erent
types of leukemia, especially those resistant to the con-
ventional treatments and expressing factors like P-gp or
the BCR-ABL oncogene.
Table 1. Cytotoxic and apoptotic activity of di€erent retinoid deri-
vatives in HL60 cells
Compound no.
IC50 (mM)
AC50 (mM)a
Acknowledgement
3
9a
9b
9c
46 (Æ6.2)
52 (Æ7.3)
50 (Æ10)
10 (Æ2.8)
>100
>100
90 (Æ7.8)
80 (Æ9.8)
40 (Æ5.1)
>100
>100
>100
>100
40 (Æ9.7)
85 (Æ6.6)
40 (Æ4.7)
38 (Æ3.8)
90 (Æ6.7)
50 (Æ5.9)
10 (Æ2.0)
38 (Æ4.6)
47 (Æ5.6)
>100
This work was in part supported by the Ministero
dell'Universita e della Ricerca Scienti®ca e Tecnologica
(Rome)
10a
10b
13e
14e
15a
15b
15c
15d
16a
16b
16c
16d
4
>100
50 (Æ6.5)
50 (Æ8.3)
20 (Æ7.0)
61 (Æ6.4)
20 (Æ4.2)
18 (Æ5.1)
42 (Æ8.1)
25 (Æ3.0)
10 (Æ3.0)
18 (Æ4.6)
26 (Æ3.2)
11 (Æ3)
References and Notes
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ATRA
aResults were obtained after 48 h of treatment. Cell cultures and eval-
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Table 2. Cytotoxic and apoptotic activity of retinoid derivatives 16b±d in multidrug resistant HL60R cells and in 9-cis-retinoic acid resistant K562
and HUT78 cellsa
HL60R
K562
HUT78
Compound No
IC50
AC50
IC50
AC50
IC50
AC50
16b
16c
16d
48 (Æ9.0)
15 (Æ4.6)
25 (Æ2.1)
87 (Æ8.9)
25 (Æ5.5)
>100
42 (Æ5.8)
13 (Æ3.9)
50 (Æ8.8)
50 (Æ6.1
27 (Æ6.8)
>100
22 (Æ7.2)
5 (Æ1.4)
43 (Æ7.9)
46 (Æ7.8)
10 (Æ3.8)
>100
aIC50 and AC50 were expressed as mM. Results were obtained after 48 h of treatment. Cell cultures and evaluation of cytotoxicity and apoptosis were
carried out as previously described.15

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2673
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