Synthesis and antiproliferative activity of a new compound containing an α-methylene-γ-lactone group

Article abstract of DOI:10.1021/jm025518n

The biological activity of compound 9 obtained by introducing an α-methylene-γ-butyrolactone group into 3-(4-hydroxyphenyl)propionic acid, 1, was studied for possible effects on HL-60 cells, murine splenocytes, and human peripheral mononuclear cells (PBMC). 9 induced apoptosis in the HL-60 cell line and has a clear capacity to inhibit proliferation induced in murine splenocytes and PBMC by different mitogenic agents with no apparent toxic side effects. 9 was synthesized from 1, and its structure and stereochemistry were elucidated by spectroscopic methods.

Full text of DOI:10.1021/jm025518n

2358
J . Med. Chem. 2002, 45, 2358-2361
Sch em e 1a
Syn th esis a n d An tip r olifer a tive Activity
of a New Com p ou n d Con ta in in g a n
r-Meth ylen e-γ-La cton e Gr ou p
Antonio G. Gonza´lez,† Margarita Herna´ndez Silva,‡
J uan I. Padro´n,† Francisco Leo´n,† Eduardo Reyes,§
Melchor AÄ lvarez-Mon,§ J uan P. Pivel,
J ose´ Quintana,| Francisco Este´vez,| and
J aime Bermejo*,†
Instituto Universitario de Bio-Orga´nica
“Antonio Gonza´lez”sInstituto de Productos Naturales y
Agrobiologı´a, CSIC, Avenida Astrofı´sico F. Sa´nchez 3,
38206 La Laguna, Tenerife, Canary Islands, Spain,
Facultad de Ciencias, Departamento de Ciencias Ba´sicas,
Universidad del Bio-Bio, Casilla 447, Chillan, Chile,
Departamento de Medicina, Universidad de Alcala´,
Alcala´ de Henares, Madrid, Spain, CSIC Associate Unit,
Industrial Farmace´utica Cantabria, c/ Azcona 46,
28028 Madrid, Spain, and Departamento de Bioqu´ımica,
Universidad de Las Palmas de Gran Canaria, Spain
Received March 6, 2002
Abstr a ct: The biological activity of compound 9 obtained by
introducing an R-methylene-γ-butyrolactone group into 3-(4-
hydroxyphenyl)propionic acid, 1, was studied for possible
effects on HL-60 cells, murine splenocytes, and human pe-
ripheral mononuclear cells (PBMC). 9 induced apoptosis in the
HL-60 cell line and has a clear capacity to inhibit proliferation
induced in murine splenocytes and PBMC by different mito-
genic agents with no apparent toxic side effects. 9 was
synthesized from 1, and its structure and stereochemistry were
elucidated by spectroscopic methods.
In tr od u ction . Propionic acid derivatives1 are very
important substances, both commercially and medically
speaking, within the group of nonsteroidal antiinflam-
matory therapeutic agents. Ibuprofen1 was the first of
a series of products within this subgroup, which now
includes naproxen,2 ketoprofen,3 and fenbufen.4 The
a
Reagents: (a) LiAlH4, THF, reflux 6 h, 50%; (b) CH3I, acetone,
K2CO3, 95%; (c) PCC, CH2Cl2, 1.5 h, room temp, 52%; (d) DABCO,
7 days, room temp, 90%; (e) NBS, (CH3)2S, 0 °C to room temp, 24
h, 92%; (f) Sn, (CH3CH2)2O, HOAc, p-(TsOH), C6H6, reflux 9 h,
70%.
are nonselective inhibitors of cyclooxygenases.5 The
structural similarity of ibuprofen and 3-(4-hydroxyphen-
yl)propionic acid, 1, originally obtained as the major
product from the fern Asplenium onopteris6 prompted
a pharmacological study of this molecule once it had
been modified as shown in Scheme 1.
The antitumoral activity of sesquiterpene lactones
was known well before their allergenic function and is
related to the presence of the R-methylene-γ-lactone
group and its propensity for Michael-type additions with
-SH or -NH nucleophiles in compounds that may play
an important role in the mechanisms through which
lactones carry out their biological activity.7
pharmacodynamic characteristics of these products are
similar, with different degrees of antiinflammatory,
antipyretic, analgesic, and antiplatelet activity, and all
According to Hanson et al.,8 the R-methylene-γ-
lactone group in vernolepin and elephantopin with
biological -NH either reacts very slowly (as in the case
of lysine) or not at all (the case of guanine) while with
the -SH (as occurs with cysteine) the reaction rate is
similar to that of the iodoacetate, a common reagent for
* To whom correspondence should be addressed. Phone: (34) 922-
250766. Fax: (34) 922-318571. E-mail: jbermejo@ull.es.
† Instituto Bio-Orga´nicasInstituto de Productos Naturales.
‡ Universidad Bio-Bio.
§ Universidad de Alcala´.
Industrial Farmace´utica Cantabria.
| Universidad de las Palmas de Gran Canaria.
10.1021/jm025518n CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/11/2002

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2359
F igu r e 2. Photomicrographs of representative fields of HL-
60 cells stained with bisbenzimide trihydrochloride to evaluate
chromatin condensation after treatment for 24 h with no drug
(a) or 20 µM of 9 (b).
F igu r e 1. Representative experiment to demonstrate the
effects of 9 on the growth of HL-60 cells. Cells were seeded at
densities of 1 × 104 per well, and after 24 h exposure, MTT
assays were performed.17
the sulfhydryl group.9 It therefore seems clear that the
antitumoral activity of sesquiterpene lactones is related
to the ability of the R-methylene-γ-lactone group to bind
to biological nucleophiles.
Apoptotic cell death plays a critical role in normal cell
development, tissue homeostasis, and the regulation of
the immune system.10 Many anticancer drugs have been
shown to cause the death of sensitive cells through the
induction of apoptosis.
In this work we report the synthesis and biological
activity of a compound containing an R-methylene-γ-
butyrolactone group.
F igu r e 3. HL-60 cells were counted and scored for apoptotic
chromatin condensation by quantitative fluorescent micros-
copy. Columns represent the mean number of cells counted
that undergo apoptosis as a percentage of 500 total cells
randomly counted in triplicate samples; bars represent the
standard deviation SD.
Ch em ist r y. The 5-[2-(4-methoxyphenyl)ethyl]-3-
methylen-4-methyldihydrofuran-2-one (9) derivative11
was synthesized as follows. 3-(4-Hydroxyphenyl)propi-
onic acid, 1, isolated for the first time as a natural
product from the fern Asplenium onopteris,6 was used
as starting material. Reduction of 1 with LiAlH4 gave
compound 2, which, when methylated, afforded methyl
ether 3. This was then oxidized with pyridinium chlo-
rochromate to give aldehyde 4.12
Reaction of an acetaldehyde, 5, with methyl acrylate,
6, in the presence of 1,4-diazabicyclo[2,2,2]octane (DAB-
CO) yielded 7,13 and treatment of 7 with N-bromosuc-
cinimide (NBS) afforded 8 regio- and stereoselectively.14
2-Carbomethoxyallylation of 4 with 2-carbomethoxyallyl
bromide 8 in the presence of metallic tin and catalytic
amounts of acetic and p-toluenesulfonic acids yielded
915 (Scheme 1). The stereochemistry of the lactone ring
was determined in accordance with Lo¨ffer et al.16
In d u ction of Ap op tosis by Com p ou n d 9. Com-
pound 9 was found to inhibit the growth of HL-60 cells
in culture. The inhibitory concentration IC50 of com-
pound 9 was 4 µM at 24 h (Figure 1). To determine
whether the extensive cell death observed in HL-60 cells
after treatment with 9 was due to apoptosis or necrosis,
we performed two independent assays. The first of these
involved QFM (quantitative fluorescent microscopy).18
Treatment of HL-60 cells with 9 induced apoptosis
identified by characteristic morphological changes in-
cluding chromatin condensation, compaction along the
periphery of the nucleus, and nuclear segmentation into
three or more chromatin fragments (Figure 2). When
quantitative fluorescent microscopy was used, apoptosis
was observed to be dose-dependent (Figure 3).
F igu r e 4. HL-60 cells were treated with 5 and 20 µM
compound 9 and harvested at 6 and 24 h, and DNA fragmen-
tation was monitored by electrophoresis in 2% agarose gels.
results in endonucleolytic DNA cleavage (Figure 4) and
would increase with drug concentration. This phenom-
enon was associated with a decrease in p32 CPP32
proenzyme level (Figure 5).
In Vitr o An tip r olifer a tive Effects. The p-hydroxy-
phenylpropionic acid derivative 9 was tested in vitro for
its immunomodulator effect and its impact on the
proliferative response of peripheral blood mononuclear
cells (PBMC) to T lymphocyte mitogens. PBMC were
purified from healthy human donors and cultured in the
presence of optimal doses of the indicated mitogens in
the presence or absence of different concentrations of
Second, DNA fragmentation was analyzed. HL-60
cells were treated with 9 for 6 and 24 h, and DNA
samples were subjected to gel electrophoresis. The
results showed that exposure to 5 µM of compound 9

2360 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Letters
F igu r e 5. HL-60 cells were treated with 9 and harvested at
12 and 24 h. Total cell lysates were analyzed by immunoblot-
ting with anti-CPP32 antibody.
F igu r e 7. Interleukin 2 (IL-2) postpones the inhibitory effect
of 9 on PBMC proliferation.
F igu r e 6. Compound 9 inhibits the proliferative response of
PBMC to mitogens.
9, as previously described.19 The presence of 9 in the
culture medium induced a marked dose-dependent
inhibitory effect on the [3H]-thymidine intake of mito-
genic stimulated PBMC (Figure 6).
F igu r e 8. Compound 9 inhibits the proliferative response of
splenocytes to mitogens.
We found that 9 inhibits the growth of HL-60 myeloid
leukemia cells in culture (IC50 ) 4 µM) and that the
mechanism by which it achieves this cytotoxic effect is
by activating apoptosis. Treatment of HL-60 cells with
9 induces morphological changes and internucleosomal
DNA fragmentation characteristic of apoptotic cell death
and also is associated with activation of caspase-3 by
inducing cleavage of the proenzyme procaspase-3. How-
ever, 9 does not induce toxicity in T lymphocytes or in
murine splenocytes. These findings are very similar to
those found for the thiol alkylating agent imexon, a
cyanoaziridine derivative that induces apoptosis in
multiple myeloma,21 whereas normal lymphocytes are
less sensitive.22
The proliferative response of PBMC to PHA, Con A,
immobilized anti-CD3, immobilized anti-CD3 plus PHA,
and immobilized anti-CD3 plus Con A, and stimulation
was significantly decreased by 9 at 0.04, 0.04, 4, 0.04,
and 4 µM, respectively. The presence of these concen-
trations of 9 in the culture medium did not induce
significant variations in the percentage of viable cells
in the PBMC cultures measured by trypan blue exclu-
sion and flow cytometry.20 Taking into account the
relevant role played by recombinant human interleukin
2 (IL-2) in the regulation of T lymphocyte proliferation,
we investigated the effects of this cytokine on the
inhibitory activity shown by 9 on the proliferative
response of PBMC to T lymphocyte mitogens. Saturated
amounts of IL-2 (100 UI/mL) were added to PBMC
cultures in the presence of PHA and 9. IL-2 clearly
postponed the suppressor effect of 9 upon the prolifera-
tive response of PBMC to PHA stimulation (Figure 7).
Similar results were obtained in the presence of the
other mitogenic signals analyzed (data not shown).
The lactone showed its ability to inhibit proliferation
induced in murine splenocytes by different mitogenic
agents (Con A, anti-CD3, and LPS) but not that induced
by PHA (Figure 8).
In summary, we found that compound 9, which differs
in its primary cellular targeting from classical chemo-
therapeutic drugs, possesses extremely interesting prop-
erties with regard to its apoptosis-inducing ability in
HL-60 cells.
Ack n ow led gm en t. This research was supported in
part by FEDER Grant 1FD1997-1831 (J .B.B), in part
by the Fondo de Investigaciones Sanitarias (FIS) Grant
00/0806 (M.A and E.R), and in part by Direccio´n General

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J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2361
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