Parallel synthesis and cytotoxicity evaluation of a polyamine-quinone conjugates library

Article abstract of DOI:10.1021/jm800637b

A library of 24 derivatives designed by combining two natural products-derived fragments was prepared and tested to determine their anticancer potential in HT29 colon cancer cells. All library members inhibit cell proliferation as measured by MTT mitochondrial functional assay, with IC ₅₀ values in the 1-100 μM range. Entry 1b caused apoptotic EGFR-mediated intracellular signaling. Thus, polyamino-quinones emerged as readily accessible and easily diversified scaffolds for anticancer lead discovery.

Full text of DOI:10.1021/jm800637b

J. Med. Chem. 2008, 51, 5463–5467
5463
Parallel Synthesis and Cytotoxicity Evaluation of a Polyamine-Quinone Conjugates Library
Maria Laura Bolognesi,*,† Natalia Calonghi,‡ Chiara Mangano,‡ Lanfranco Masotti,‡ and Carlo Melchiorre*,†
Department of Pharmaceutical Sciences, Alma Mater Studiorum, UniVersity of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Department of
Biochemistry “G. Moruzzi”, Alma Mater Studiorum, UniVersity of Bologna, Via Irnerio 48, 40126 Bologna, Italy
ReceiVed May 28, 2008
A library of 24 derivatives designed by combining two natural products-derived fragments was prepared
and tested to determine their anticancer potential in HT29 colon cancer cells. All library members inhibit
cell proliferation as measured by MTT mitochondrial functional assay, with IC50 values in the 1-100 µM
range. Entry 1b caused apoptotic EGFR-mediated intracellular signaling. Thus, polyamino-quinones emerged
as readily accessible and easily diversified scaffolds for anticancer lead discovery.
Introduction
Proliferative disorders are expected to be the major cause of
death in the 21st century. To identify new chemical entities for a
more effective treatment of cancer, drug designers can follow many
strategies, but the crucial decision is always the selection of a
suitable starting point from the vast chemical space.1 In this respect,
natural products can be viewed as evolved privileged structures2
and biologically prevalidated leads, in other words, as molecules
that have probably evolved evolutionarily to exert highly specialized
functions. Therefore, they represent the biologically relevant and
prevalidated fractions of chemical space explored by nature so
far.3,4 Indeed, natural products continue to provide an important
source of new anticancer leads, with about 74% of anticancer
compounds being either natural or natural product-derived prod-
ucts.5 Natural molecules have potent antitumor properties and have
provided multiple active compounds in the past.6 The chemical
diversity, the structural complexity, and the inherent biologic
activity of natural products make them ideal candidates for new
therapeutics.7-10 Natural products not only disrupt aberrant signal-
ing pathways leading to cancer (i.e., proliferation, deregulation of
apoptosis, angiogenesis, invasion, and metastasis) but also synergize
with chemotherapy and radiotherapy.
Figure 1. Early hit 1b and general formula of the designed library
compounds.
several small-molecule RTK inhibitors recently approved for
clinical use worldwide.18
Polyamines have long been associated with cell growth and
cancer: their important roles in angiogenesis and invasion have
recently been identified, and now polyamine analogues are being
developed as anticancer drugs to target polyamine metabolic
enzymes and inhibit polyamine biosynthesis.19,20 Besides modulat-
ing cell growth, some analogues also play an important role in
cell apoptosis by regulating gene expression.21
Therefore, following the concept that a library built from selected
natural products should yield better hits at a higher rate than
classical compound libraries, we planned to design a small library
combining aromatic quinone scaffolds with polyamines, varying
from diamines to triamines and tetramines, as potential new
chemical entities against cancer (Figure 1).
We recently reported preliminary biological results showing that
a polyaminoquinone derivative designed following this rationale,
FR18 (1b) (Figure 1), shows cytotoxic activity in a human colon
carcinoma cell line by activating apoptosis.22 This encouraged us
to perform SAR studies, synthesizing a focused polyaminoquinone
library. Herein, to our knowledge, we disclose the first parallel
synthesiswithapolymer-assistedpurificationofapolyamine-quinone
conjugates library together with their antiproliferative properties.
All of these considerations prompted us to design a combi-
natorial library of potential anticancer agents by using as
molecular building blocks two natural scaffolds with great
anticancer potential such as quinones and polyamines.
Naphthoquinones11 and anthraquinones12,13 have a wide spec-
trum of anticancer activity: they covalently bind to and intercalate
into DNA, inhibit DNA replication and RNA transcription, act as
DNA topoisomerase II poisons, produce oxidative stress, and
induce DNA breakage and chromosomal aberrations.14,15 More-
over, quinones, such as emodin16 and shikonin,17 are able to
modulate receptor tyrosine kinase (RTK)a activity, an innovative
molecular target for successful mechanism-based cancer therapies.
RTKs are mediators of cellular proliferation, and their mutations
are often associated with hyperplasia and tumor development.
Targeted inhibition of RTKs has therefore become an attractive
therapeutic strategy in the treatment of cancer and has resulted in
* To whom correspondence should be addressed. Phone: +39 0512099700.
Fax: +39 0512099734. E-mail: for M.L.B., marialaura.bolognesi@unibo.it;
for C.M., carlo.melchiorre@unibo.it.
Results and Discussion
In designing the library, we decided to combine three quinone
building blocks with eight polyamines, generating a library of 24
compounds (Chart 1). To introduce diversity into the quinone
subunit, we selected the naphthoquinone (1) of 1b, and two higher
quinone homologues, anthraquinone (2) and naphtacenequinone
†
Department of Pharmaceutical Sciences, University of Bologna.
Department of Biochemistry “G. Moruzzi”, University of Bologna.
‡
a Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth
factor receptor; PAT, polyamine transporter; RTK, receptor tyrosine kinase;
SS, serum starved.
10.1021/jm800637b CCC: $40.75
2008 American Chemical Society
Published on Web 08/13/2008

5464 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Chart 1. Building Blocks for the Designed Library
Brief Articles
Scheme 1a
a Reaction conditions: (a) CH2Cl2, air, rt, 6 h; (b) CH2Cl2, rt, overnight; (c) CF3COOH, CH2Cl2, rt, 4 h.
Table 1. IC50 Values for the Cellular Proliferation Inhibition (MTT Assay) of Library Compounds in HT29 Colon Cancer Cells
(3). In contrast to the first two quinones, the latter has been less
investigated as a molecular scaffold endowed with anticancer
properties.
Natural polyamines spermidine (f) and spermine (h) and six
synthetic variants (a-e, g) were chosen to explore the role of the
polyamine backbone in relation to the quinone component. In fact,
the selected polyamines differ in chain length and number of
nitrogen atoms within the carbon chains, thus allowing study of
the length and number of cationic sites required for optimal activity.
For the synthesis of the library, we initially focused on a solid-
phase approach. Attachment of the polyamine to a solid support
via one terminal amine function, allowing subsequent reaction of
the other with the appropriate quinone, appeared to be advantageous
because of a sort of selective asymmetric protection. Unfortunately,

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5465
approach (see Scheme 1 in Supporting Information).29 Michael
addition of N-BOC-diaminopentane (5) to acrylonitrile provided
in 91% yield derivative 6, which was subsequently transformed
into the BOC-protected nitrile 7. Raney-nickel reduction of 7 gave
the desired product 4g. Regarding diamines 4a-d, we adapted a
protocol developed by Krapcho.30 The success of this method relies
on the use of a large excess of the diamine with respect to the
BOC2O (8:1). Amine excess was easily removed by washing the
crude reaction repeatedly with warm water, affording the desired
monoprotected diamines with yields ranging from 70% to 84%
(Scheme 2 in Supporting Information).
Figure 2. Confocal laser scanning micrographs of EGF and EGFR in
control and 1b treated cells. EGF fluorescence is shown in green, EGFR
staining in red. White pixels represent the colocalization of the two proteins.
To achieve an initial picture of the anticancer properties of the
library members, 1a-h, 2a-h, and 3a-h were evaluated for their
antiproliferative activity in HT29 cells, a human colon adenocar-
cinoma cell line. The cells were treated with the test compounds
at concentrations ranging from 0.1 to 400 µM for 24 h, and cell
viability was assessed through measurements of mitochondrial
dehydrogenase activities using the MTT method. To compare the
antiproliferative potencies in terms of concentrations required for
50% of the effect, all analogues were tested in dose-dependent
manner, and the obtained IC50 values are listed in Table 1. The
results revealed that all compounds were capable of inducing a
decrease of cell growth. No major differences were observed within
the library, all members possessing similar IC50 values in the
micromolar range. Decanediamine derivatives (series d) were less
uniform in their behavior, with derivative 3d being the less
cytotoxic of the series and anthracene analogue 2d standing out as
the most efficacious compound (IC50 ) 4.1 µM). Conversely,
natural polyamine analogues belonging to the spermidine (f) and
spermine (h) series showed only slight differences in the IC50
values. This might suggest that in these compounds the polyamine
moiety is more relevant than the quinone one for biological activity.
This finding might be rationalized by the fact that both are natural
polyamines, in principle more capable of recognizing a biological
counterpart. Of the remaining compounds, five of them possessed
an IC50 between 1 and 10 µM, the others between 10 and 100 µM.
As mentioned above, we have previously shown that in HT29
1b is active at concentrations as low as 10 nM and inhibits EGF
binding to its receptor (EGFR). Furthermore, flow cytometric
analysis showed that 1b induces an arrest in the S phase of the
cell cycle and apoptotic death.22 From this peculiar pharmacological
profile, 1b seems to have possible therapeutic applications in colon
cancer. Because of the structural proximity, it was assumed that
the synthesized compounds would similarly interfere with the
binding mechanisms of EGF to its receptor. Fluorescence confocal
microscopy was used to detect specific binding of 1b to EGFR,
and nine representative compounds were properly selected for
further investigation using this technique. We designated 2b and
3b as close analogues of 1b, and series e and h as prototypical
examples of a triamine and a tetramine.
the bond between the amino group and the quinone is very labile,
as observed in the synthesis of natural products having this
functionality.23 Thus, we decided to construct a library via solution-
phase synthesis and examined the possible routes to incorporating
a properly protected polyamine into the 2-position of the quinone.
In accordance with the widely reported reactivity of the quinone
system in the 1,4-Michael addition, amino-quinones are usually
synthesized from primary amines and quinone itself. The unsatur-
ated nature of the quinone is maintained, using either excess
quinone or other oxidizing agents such as atmospheric oxygen.
The same transformation can also be achieved through nucleophilic
substitution chemistry, starting from quinones bearing good leaving
groups such as methoxy groups and halogen atoms.24
We explored both approaches and found the first more practical
in terms of lower number of formed byproduct and greater
commercial accessibility of starting materials. Moreover, the latter
was reported to require very drastic conditions.25 However, to
obtain a quantitative conversion, the Michael addition should be
carried out using an excess of starting amine. We circumvented
this drawback using scavenger resins to facilitate the purification
process. Amine excess was then scavenged through the use of
polymer-supported sulfonyl chloride and trisamine to yield the
library compounds, which, after deprotection, possessed adequate
purity for direct biological screening. The developed synthetic route
is shown in Scheme 1. More specifically, quinones 1-3 reacted
with protected polyamines 4a-h in a ratio 1:2 for 6 h. Upon
completion of the reaction, resins were added to sequester the
remaining reactants. Simple filtration and evaporation of the
solvents afforded highly purified products. Deprotection of the BOC
groups was accomplished by treatment with trifluoroacetic acid in
CH2Cl2 to obtain the target compounds as trifluoroacetate salts.
The overall yields ranged from 47% to 96% based on mass
recovery, with an average of 90% and 75% for compounds of series
1 and 2, respectively. Notably, the yield dropped (55%) for
compounds of series 3 due to the very low solubility of naphtacene
derivatives. The HPLC purities for 1a-h and 2a-h ranged from
80% to 99% with an average purity level of 94%. Because of their
low solubility, it was impracticable to obtain HPLC analysis of
compound of series 3. Gratifyingly, the biological screening data
obtained on some library compounds were highly reproducible and
consistent with those obtained for the same compounds made by
traditional chemistry.
For confocal microscopy, HT29 were grown on glass coverslips
for 24 h and serum starved (SS) for an additional 24 h before being
exposed to a 1 µM concentration of each compound for 30 min.
Untreated as well as series 1 compounds treated cells were then
stimulated with fluorescent EGF, while samples treated with series
2 and 3 compounds were stimulated with nonfluorescent EGF
because 2 and 3 compounds show intrinsic fluorescence. The
analysis reveals that in EGF stimulated cells, EGF and EGFR form
a granular complex localized in the plasma membrane, whose
presence greatly diminishes in 1b treated, EGF stimulated HT29.
This indicates that concomitant treatment with 1b and EGF prevents
the formation of the complex (Figure 2). We have proposed that
1b binds competitively to the extracellular domain of the receptor,
and this interaction does not prevent the RTK activation and causes
A prerequisite for the chosen strategy was an easy access to
large amounts of partially protected polyamines (4a-h), having
only one free primary amine unmasked. The ready removal in
mildly acidic conditions, compatible with the stability of the
amino-quinone bond, made BOC a superior protecting group with
respect to the others for the purpose of this library. Thus,
norspermine (e),26 spermidine (f),27 and spermine (h)28 derivatives
were synthesized by following reported procedures, whereas
protected homospermidine (g) was prepared using the acrylonitrile

5466 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Brief Articles
Figure 3. Confocal laser scanning micrographs (a) and cell cycle analysis (b) of control and 1e and 1h treated HT29 cells. (a) Cells were SS,
treated with 1e or 1h and stimulated with fluorescent EGF (green). (b) Flow cytometric analysis of cells treated with 1e or 1h for 24 h.
apoptotic, EGFR-mediated intracellular signaling.22 Surprisingly
enough, confocal microscopy analysis showed that EGF binding
to EGFR is not perturbed by 1e and 1h administration, as indicated
by the equal intensity of EGF fluorescent signal in untreated and
treated samples (Figure 3a). In addition, 24 h treatment with 1e
and 1h does not affect HT29 proliferation, as shown by flow
cytometric cell cycle analysis (Figure 3b). For class 2 and 3
compounds, fluorescence confocal analysis has been performed by
taking advantage of their fluorescent properties and staining EGFR
in indirect immunofluorescence (see Figure 1 in Supporting
Information). Again, 2b, 2e, and 2h and 3b, 3e, and 3h do not
colocalize with EGFR, suggesting that 1 µM administration of each
of them does not interfere with the binding of EGF to EGFR.
Accordingly, no effect on cell proliferation has been observed for
these compounds. In conclusion, these studies indicate that the
series 1 has no further experimental use, but for 1b, given its proven
ability to interfere with EGF/EGFR interactions. Series 2 and 3,
on the other hand, promise a successful use in cellular internaliza-
tion and transport studies, given their fluorescent properties.
In this respect, cancer cells frequently demonstrate an elevated
polyamine uptake activity, importing high level of exogenous
polyamines through the polyamine transporter system (PAT).31
Thus, it is a promising strategy for anticancer therapies to exploit
toxic compounds that selectively can gain entry into the cell via
PAT.32 Notably, the conjugates of this study appear particularly
intriguing, because they may utilize PAT to enter cells where they
may exert cytotoxic effects through a pleiotropic mechanism of
action. This suggests a potential for specific targeting of tumors
that have enhanced PAT activity over their normal cell counterparts.
In this regard, notwithstanding the enormous financial investment
and many efforts, effective drugs are not available yet for anticancer
therapy, which is an urgent need and unmet goal. A possible reason
might be that cancer, like other diseases such as neurodegenerative
syndromes, diabetes, and cardiovascular diseases, is a disorder that
involves multiple pathogenic factors.33,34 Thus, a drug able to hit
a single target, albeit with outstanding affinity and selectivity, may
not be clinically effective for the treatment of a multifactorial
disease.35 Nowadays, besides therapies based on cocktails or
combinations of drugs with different therapeutic mechanisms, to
overcome this problem, a new design strategy is emerging based
on the assumption that a single molecule is able to simultaneously
modulate multiple targets relevant for a given complex pathology.36,37
All these considerations clearly establish the polyamine-
quinone conjugates as promising structures for the development
of new antitumor agents that, besides the modulation of EGFR,
might have potential multiple anticancer activities (DNA intercala-
tion, formation of covalent DNA adducts, topoisomerase poisoning,
and free radical effects) in addition to a successful drug delivery
via PAT. Therefore, compounds incorporating these natural product
fragments are being synthesized and will be reported in due course.
Experimental Section
General Information. Chemical reagents and scavenger resins
were purchased from Sigma Aldrich, Fluka, and Lancaster (Italy).
Dichloromethane was distilled from calcium chloride. The library
was synthesized using a Syncore Reactor manual synthesizer with
evaporation system (BUCHI Italia) using a 24-well liquid-phase
reaction block. Filtration was performed using 4 mL disposable
polypropylene syringe Extract-Clean filter columns (Alltech). TLC
were performed on 0.20 mm silica gel 60 F254 plates (Merck,
Germany). Nuclear magnetic resonance spectra (NMR) were
recorded at 200 and 300 MHz on Varian VXR 200 and 300
spectrometers and reported in parts per million. Low-resolution mass
spectra EI-MS and ESI-MS were recorded on a VG707EH-F and
Waters ZQ 4000 apparatus, respectively.
General Procedure for the Synthesis of Polyamine-Quinone
Conjugate Library Members. Quinones 1-3 (0.3 mmol), dry
CH2Cl2 (10 mL), and protected polyamines 4a-h (0.6 mmol) were
added in parallel in 24 vials (30 mL). The mixtures were shaken at
200 rpm in open vials at room temperature for 6 h. After the reaction
was completed, polymer-supported sulfonyl chloride (100-200
mesh, loading 1.5-2.0 mmol/g) (440 mg) and polymer-supported
trisamine (PS-trisNH, 200-400 mesh, loading 1.1 mmol/g) (270
mg) were added to each reactor to scavenge the excess of unreacted
polyamine. After shaking overnight, the suspensions were filtered
to remove the resins and the solutions recovered were evaporated.
The crude products were dissolved in CH2Cl2 (4 mL), and
CF3COOH (4 mL) was added. After shaking for 4 h, the solvent
was evaporated under reduced pressure, and the solids obtained
were triturated with ether to afford the trifluoroacetate salts in
acceptable yields (see Supporting Information).
Acknowledgment. This research was supported by grants
from MIUR (FIRB RBNE03FH5Y) and the University of
Bologna (C.M.) and by a grant from MIUR 60% 2007 (L.M.).
We thank C.I.R.B. for the use of the confocal microscope.
Supporting Information Available: Characterization data, a
table of HPLC analysis data for target compounds, synthetic
procedures for 4a-d and 4g, biological methods, and confocal laser
scanning micrographs and cell cycle analysis for 2b, 2e, 2h, 3b,
3e, and 3h. This material is available free of charge via the Internet
at http://pubs.acs.org.
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