Design, synthesis, and evaluation of novel boronic-chalcone derivatives as antitumor agents

Article abstract of DOI:10.1021/jm030213+

A series of boronic-chalcone derivatives were synthesized and tested for antitumor activity against human breast cancer cell lines. The results show the boronic-chalcones are more toxic to breast cancer cells compared to normal breast cells than other known chalcones.

Full text of DOI:10.1021/jm030213+

J . Med. Chem. 2003, 46, 2813-2815
2813
Letters
in edible plants. Chemically they consist of open-chain
flavonoids in which the two aromatic rings are joined
by a three-carbon R,â-unsaturated carbonyl system.
Licochalcone-A, a chalcone derivative found in the
licorice root, has been associated with a wide variety of
anticancer effects.16 Chalcones inhibit the proliferation
of both established and primary ovarian cancer cells.17
In vivo, chalcones have been demonstrated to be effec-
tive as antitumor agents in skin carcinogenesis18,19
and chemopreventive agents in several experimental
models.20-22 Recent studies have shown that these
chalcones induce apoptosis in variety of cell types,
including breast cancers.23-26 Biochemical experiments
have shown that these compounds could disrupt the
MDM2/p53 protein complex, releasing p53 from both the
p53/MDM2 and DNA-bound p53/MDM2 complexes.27
However, carboxylic acid analogues of chalcone 3g and
9 reported in the literature27 are equally toxic to both
normal and malignant breast epithelial cells. The toxic-
ity to normal breast cells may be due to MDM2/p53-
independent mechanisms. Therefore, a chalcone deriva-
tive that could bind strongly and irreversibly to disrupt
MDM2 protein complexes may be selectively toxic to
MDM2 overexpressing breast cancer cells. A novel
boronic-chalcone strategy outlined in this paper should
overcome the limiting lack of specificity of carboxylic
acid analogues of chalcones.
Design , Syn th esis, a n d Eva lu a tion of
Novel Bor on ic-Ch a lcon e Der iva tives a s
An titu m or Agen ts
Srinivas K. Kumar, Erin Hager, Catherine Pettit,
Hallur Gurulingappa, Nancy E. Davidson, and
Saeed R. Khan*
Division of Experimental Therapeutics, Sidney Kimmel
Comprehensive Cancer Center at J ohns Hopkins,
Baltimore, Maryland 21231
Received March 18, 2003
Abstr a ct: A series of boronic-chalcone derivatives were syn-
thesized and tested for antitumor activity against human
breast cancer cell lines. The results show the boronic-chalcones
are more toxic to breast cancer cells compared to normal breast
cells than other known chalcones.
In tr od u ction . Breast cancer is expected to account
for 203 500 new cancer cases and 39 600 deaths in
2002.1 Although major advances have been made in
early detection, prevention, and treatment, the need for
more effective therapy in the fight against late-stage
breast cancer continues. Currently there is no curative
treatment for women with metastatic breast cancer once
they have failed adjuvant therapies. New, effective
cytotoxic agents with novel mechanisms of action are
therefore urgently needed for the treatment of women
with metastatic breast cancer.
The mouse double minute 2 (MDM2) oncogene has
been suggested as a target for breast cancer therapy.2,3
It is amplified or overexpressed in human breast cancer.
The oncoprotein MDM2 inhibits the tumor suppressor
protein p53 by binding to the p53 transactivation
domain. The p53 gene is inactivated in human cancer
either by mutations or by binding to oncogenic proteins
such as MDM2.4-7 In breast tumors, overexpression of
MDM2 inactivates an otherwise intact p53, disabling
the genome integrity checkpoint and allowing cell cycle
progression of defective cells.8 Studies comparing MDM2
overexpression and p53 mutation concluded that these
are mutually exclusive events, supporting the notion
that the primary impact of MDM2 amplification in
cancer cells is the inactivation of the endogenous wild-
type p53.7 It has been shown recently that a peptide
homologue of p53 is sufficient to induce p53-dependent
death of cells overexpressing MDM2.9 This result pro-
vides clear evidence that disruption of the p53/MDM2
complex might be effective in cancer therapy. It has
been shown that MDM2 additionally has a role in tumor
growth p53-independent mechanisms.10-15
Boronic chalcone analogues have been previously
described. These compounds have been used as fluores-
cent probes that may be useful for detection of fluo-
rides28 and saccharides such as glucose that may be
applicable to the design of biosensors for diabetes.28
Previous studies27,29 on the binding modes of carboxylic
acid analogues of chalcones 3g and 9 with MDM2
revealed that the carboxylic acid group could be placed
near the base of lysine51 (K51), which is found in a salt
bridge interaction with glutamic acid25 (E25). It was
presumed that the acid group of the chalcone forms a
salt bridge with K51 and simultaneously breaks the salt
bridge with E25 of the MDM2. We envisioned that the
boronic acid analogue might form a stronger salt bridge
with K51 of MDM2 than the corresponding carboxylic
acid analogue of chalcones.
Here we investigate the potential value of MDM2 as
a drug target for breast cancer therapy by inhibiting
MDM2 expression with specific boronic-chalcone ana-
logues. We hypothesize that anti-MDM2 boronic-chal-
cone analogues will selectively inhibit growth of breast
cancer cells. To test our hypothesis, we systematically
designed a set of boronic acid-chalcone derivatives and
tested their ability to selectively kill breast cancer vs
normal breast epithelial cells. To our knowledge, there
are no reports investigating the anticancer activity of
boronic-chalcones on different cancer cell lines. Thus the
identification of new boronic-chalcone analogues will be
important in the continued development of this class of
agents as anticancer drugs.
Chalcones are a class of anticancer agents that have
shown promising therapeutic efficacy for the manage-
ment of human cancers. Chalcones, considered as the
precursor of flavonoids and isoflavonoids, are abundant
* To whom correspondence should be addressed. Tel: (410) 614 0200.
Fax: (410) 614 8397. E-mail: khansa@jhmi.edu.
10.1021/jm030213+ CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/30/2003

2814 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Letters
Ta ble 1. Chalcones Inhibit Growth of Human Breast Cell
Ch a r t 1. Structures of Boronic Chalcones Used in This
Work
Linesa
compd MDA-MB-435 MDA-MB-231 Wt-MCF7 MCF-10A MCF-12A
3a
3b
3c
3d
3e
3f
3g
6
7
8
9
10
11
10
3.5
16
8.8
8.8
18
9
4.5
18
4
13
15
15
8.8
9.5
8.5
8.8
9.5
44
9
8
11
8
18
15
23
7.0
5.0
6.0
7.8
8.5
9
13
7
9.5
5.5
15
75
18
25
18
17
44
13
15
38
18
12
63
38
63
11
22
39
38
38
15
30
100
15
28
38
60
9
19
a
Sch em e 1a
IC50 values expressed in µM; see biology section for details of
the MTT assay.
Normal breast epithelial cell lines, MCF-10A and MCF-
12A, were maintained in 5% and 10% horse serum in
DMEM:Ham’s F12 media, respectively, supplemented
with 2 mM glutamine, 100 units/mL penicillin/strepto-
mycin, 0.02 µg/mL EGF, 0.01 mg/mL insulin, and 0.1
µg/mL cholera toxin.
a
Reagents: (a) KOH, MeOH, reflux.
Sch em e 2b
Cells were incubated at 37 °C in a 5% CO2 atmo-
sphere. The MTT colorimetric assay was used to deter-
mine growth inhibition.34 Cells were plated in 96-well
plates and allowed to attach for 24 h. Chalcone deriva-
tives were dissolved in DMSO at 10 mM concentrations.
Cells were expose in quadruplicate well to chalcone
concentrations of 0.5-100 µM for 96 h. After 96 h the
media was aspirated, and 100 µL of 1 mg/mL MTT
solution (Sigma Chemical Co.) diluted in serum free
media was added to each well. After 4 h of incubation,
the MTT solution was removed and 200 µL of 1:1 (v/v)
solution of DMSO:ethanol was added to each well to
b
Reagents: (a) KOH, MeOH, reflux. (b) NaH, pinacol (bromo-
methyl)boronate, THF. (c) NaOH, H2O.
Boronic acids are Lewis acids and isosteres of car-
boxylic acid. The pKa’s of boronic acids are ∼9-10, and
therefore at physiological pH, boronic acids remain un-
ionized.30 Thus, a coordinate covalent bond (boron-
nitrogen) can be formed between a electron-deficient
boronic acid moiety and electron-donating amino group,
which may strongly enhance binding of boronic-chal-
cones with the lysine51 of MDM2 at neutral pH when
compared to the corresponding carboxylic acid analogue
of chalcones. Here we report the design and synthesis
of a series of potent antitumor boronic-chalcone deriva-
tives. Compound 7 displays the highest activity for the
breast cancer cell lines that we tested. This compound
has been shown to be 6-9-fold less toxic to normal MCF-
12A cell lines.
Ch em istr y. Chart 1 shows the compound series used
in this study. Compounds 3a -g, 6, 8, 9, 10, and 11 were
synthesized according to standard Claisen-Schmidt
aldol condensation protocols as previously published31,32
(Scheme 1). Compound 7 was prepared by treating
compound 6 with pinacol (bromomethyl)boronate in the
presence of sodium hydride in DMSO and further
deprotection in alkaline condition33 (Scheme 2).
dissolve formazan crystals. The absorbance at A540
nm
was determined on a plate reader. IC50 values were
determined from log plots of percent of control vs
concentration. Each compound was assayed twice in
quadruplicate.
Resu lts a n d Discu ssion . IC50 values were used to
determine growth inhibition in the presence of chalcone
derivatives. Of particular interest are compounds able
to differentially inhibit growth such that human breast
cancer cell lines are inhibited, but normal breast epi-
thelial cells are significantly less inhibited. Compound
3a , 3d , 3e, 7, 8, 10, and 11 are 5-10-folds more toxic
to human breast cancer cell lines compared to normal
breast epithelial cell lines (Table 1). In the presence of
these compounds, cell growth in the human breast
cancer cell lines MDA-MB-435, MDA-MB-231, and wt-
MCF7 is inhibited, indicated by the range of low IC50
values from 3.5 to 23. Cell growth in the normal breast
epithelial cell lines MCF-10A and MCF-12A is less
inhibited, shown by higher IC50 values ranging from 11
to 75.
Con clu sion s. Here we describe a series of boronic-
chalcones that inhibit growth of human breast cell lines
at micromolar concentrations. Several are particularly
interesting as they preferentially inhibit growth of
human breast cancer cell lines. The results show the
boronic-chalcones are more toxic to breast cancer cells
compared to normal breast cells than other known
chalcones. These antitumor compounds will now be
tested in various other cancer cell lines. Structure-
Biology. Human breast cancer MDA-MB-231 (estro-
gen receptor negative) and wtMCF7 (estrogen receptor
positive) cells were maintained in DMEM medium
supplemented with 5% fetal bovine serum, 2 mM
glutamine, and 100 units/mL penicillin/streptomycin.
MDA-MB-435 (estrogen receptor negative human breast
cancer cells) was maintained in IMEM medium supple-
mented with 5% fetal bovine serum and 2 mM glutamine.

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