Novel capsaicin analogues as potential anticancer agents: Synthesis, biological evaluation, and in silico approach

Article abstract of DOI:10.1002/ardp.201400233

A novel class of benzo[d][1,3]dioxol-5-ylmethyl alkyl/aryl amide and ester analogues of capsaicin were designed, synthesized, and evaluated for their cytotoxic activity against human and murine cancer cell lines (B16F10, SK-MEL-28, NCI-H1299, NCI-H460, SK-BR-3, and MDA-MB-231) and human lung fibroblasts (MRC-5). Three compounds (5f, 6c, and 6e ) selectively inhibited the growth of aggressive cancer cells in the micromolar (mM) range. Furthermore, an exploratory data analysis pointed at the topological and electronic molecular properties as responsible for the discrimination process regarding the set of investigated compounds. The findings suggest that the applied designing strategy, besides providing more potent analogues, indicates the aryl amides and esters as well as the alkyl esters as interesting scaffolds to design and develop novel anticancer agents. 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Full text of DOI:10.1002/ardp.201400233

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
1
Full Paper
Novel Capsaicin Analogues as Potential Anticancer Agents:
Synthesis, Biological Evaluation, and In Silico Approach
Mariana C. F. C. B. Damião1, Kerly F. M. Pasqualoto2, Adilson K. Ferreira3, Sarah F. Teixeira3,
Ricardo A. Azevedo3, José A. M. Barbuto3,4, Fanny Palace-Berl5, Gilberto C. Franchi-Junior6,
Alexandre E. Nowill6, Maurício T. Tavares1, and Roberto Parise-Filho1
1
Department of Pharmacy, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil
Biochemistry and Biophysical Laboratory, Butantan Institute, São Paulo, SP, Brazil
Laboratory of Tumor Immunology, Department of Immunology, Institute of Biomedical Sciences,
2
3
University of São Paulo, São Paulo, SP, Brazil
Cell and Molecular Therapy Center (NUCEL-NETCEM), Faculty of Medicine, University of São Paulo,
4
São Paulo, SP, Brazil
5
Department of Biochemical-Pharmaceutical Technology, School of Pharmaceutical Sciences,
University of São Paulo, São Paulo, SP, Brazil
Onco-Hematological Child Research Center, Faculty of Medical Sciences, State University of Campinas,
6
Campinas, SP, Brazil
A novel class of benzo[d][1,3]dioxol-5-ylmethyl alkyl/aryl amide and ester analogues of capsaicin were
designed, synthesized, and evaluated for their cytotoxic activity against human and murine cancer cell
lines (B16F10, SK-MEL-28, NCI-H1299, NCI-H460, SK-BR-3, and MDA-MB-231) and human lung fibroblasts
(MRC-5). Three compounds (5f, 6c, and 6e) selectively inhibited the growth of aggressive cancer cells in
the micromolar (mM) range. Furthermore, an exploratory data analysis pointed at the topological and
electronic molecular properties as responsible for the discrimination process regarding the set of
investigated compounds. The findings suggest that the applied designing strategy, besides providing
more potent analogues, indicates the aryl amides and esters as well as the alkyl esters as interesting
scaffolds to design and develop novel anticancer agents.
Keywords: Bioisosterism / Capsaicin analogues / Cytotoxic activity / Exploratory data analysis / Molecular modification
Received: June 9, 2014; Revised: July 28, 2014; Accepted: August 15, 2014
DOI 10.1002/ardp.201400233
Additional supporting information may be found in the online version of this article at the publisher's web-site.
:
Introduction
discover novel potent and selective antineoplastic drugs can be
considered as a big challenge for medicinal chemists [2–5].
Programmed cell death is a natural process to remove
unwanted cells, such as those with potentially harmful
mutations, aberrant substratum attachment, or alterations in
cell-cycle control. Deregulation of apoptosis can disrupt the
delicate balance between cell proliferation and cell death, and
can lead to diseases, as cancer, for instance [6, 7]. In this
regard, it is important to modulate the apoptotic signaling
pathways to constitute a key event in the antitumor activities.
In the past few years, the potential of capsaicin for inducing
apoptosis in a wide variety of tumors has been demonstrat-
ed [8–13], but its strong pungency and nociceptive activity [14]
limit its application in food or medicinal fields. Capsaicin
Cancer is the second leading cause of human death in
developed and developing countries, accounting for nearly
one in five deaths [1]. Although there are many therapeutic
strategies, high systemic toxicity, low selectivity, and drug
resistance are the main problems in the treatment of cancer.
Therefore, to find new therapeutic targets for cancer and
Correspondence: Dr. Roberto Parise-Filho, Department of Pharmacy,
School of Pharmaceutical Sciences, University of Sao Paulo, Prof. Lineu
Prestes Avenue, 580, 05508-000 Sao Paulo, Brazil.
E-mail: roberto.parise@usp.br
Fax: þ55 11 3091-3815
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Bioisosterism strategy is commonly used to improve thera-
peutic properties, such as potency, selectivity and pharmaco-
kinetics of key compounds, or generate incremental
innovation [16]. The replacement of amide bonds, particular-
ly, by suitable bioisoster groups, which maintain similar
geometric, electronic, or hydrogen bonding properties,
has been well succeeded and extensively applied [17].
Additionally, the methylcatechol group was modified in
order to achieve the 1,3-benzodioxole bicyclic system, which
is a privileged structural feature present in anticancer agents,
such as podophylotoxin and derivatives [18, 19]. Furthermore,
the lipophilic chain was replaced by various alkyl/aryl groups
to maintain the hydrophobic character on this specific region
of the molecule.
The novel sets were synthesized and evaluated in vitro for
their cytotoxicity in the following cell lines: Murine melano-
ma (B16F10), human melanoma (SK-MEL-28), human lung
cancer (NCI-H1299 and NCI-H460), breast adenocarcinoma
(SK-BR-3 and MDA-MB-231), and human lung fibroblast
(MRC-5), through the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide) assay.
Figure 1. Chemical structure of RPF101.
might be considered as a potential lead, for instance, to the
rational design of a novel class of antineoplastic drugs with
improved pharmacokinetics and pharmacodynamics profiles.
Recently, our group reported the synthesis and antitumoral
activity of RPF101 (Fig. 1), a structural analogue of capsaicin
designed by applying the ligand-based strategy [15].
RPF101 was more potent than its prototype by inducing
apoptosis and cell cycle arrest at the G2/M phase through
disruption of the microtubule network in the MCF-7 breast
cancer cells. In our efforts to develop more potent and
selective anticancer agents, combined with the relevant and
promising profile of RPF101, a novel series of benzo[1,3]dioxol-
5-ylmethyl alkyl/aryl amides and esters were also designed
through the ligand-based drug design (LBDD) strategy.
Thereby, the amidic bond present in capsaicin was preserved
(5a–f) or replaced by an isosteric ester group (6a–e) (Fig. 2).
Herein, in silico studies, comprising molecular modeling
and chemometric methods, were also carried out to (i) select
the energetically favorable conformation of each compound
and, then, (ii) calculate its molecular properties (electronic,
steric, hydrophobic, topological, geometric), which were used
Figure 2. Chemical structure of capsaicin and general structure of capsaicin analogues.
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Novel Capsaicin Analogues as Potential Anticancer Agents
3
to (iii) perform the exploratory data analysis (hierarchical
cluster analysis, HCA, and principal components analysis,
PCA) [20–22]. The findings were helpful to establish some
qualitative structure–property relationships, since the calcu-
lated molecular properties are directly related to the
compounds’ chemical structures, which are responsible for
the molecular recognition process.
capsaicin was used as the standard compound. The biological
endpoint was determined as the concentration, which causes
fifty percent of cell growth inhibition (IC50). The cytotoxicity
did not differ greatly from the chemical nature and size of the
substituent groups. Indeed, all compounds showed similar
values of cytotoxicity. Over all, three compounds in particular
[5f (R ¼ 4-OMe-Ph), 6c (n-hexanoyl), and 6e (R ¼ 3,4-Cl-Ph)] have
shown promising cytotoxic profile when compared to
capsaicin (see Table 1).
Results and discussion
Regarding Table 1, the amide analogue 5f (methoxyl group
attached to the aromatic ring) induced cytotoxic effects on
B16F10 and H1299 cells (IC50 ¼ 87 and 187 mM, respectively).
These results reveal the methoxyl amide as the most
promising compound of the set of capsaicin analogues.
Additionally, compound 5f is more potent against B16F10
(IC50 ¼ 87 mM) than the alkyl ester, 6c (n-hexanoyl analogue)
(IC50 ¼ 130 mM). The ester analogue (6c) contains a more
flexible substituent group at the R position and was more
effective against SK-MEL-28 (IC50 ¼ 85 mM) cell line, differently
from the other analogues (5f and 6e) and the prototype,
capsaisin. These results have suggested that the alkyl ester
compound presents an interesting biological profile, mainly,
against melanoma cell lines.
Chemistry
An efficient and simple two-step methodology was employed
to obtain capsaicin analogues. Amide (5a–f) and ester (6a–e)
products were synthesized by treatment of 3a–3j with oxalyl
chloride in CH2Cl2, at room temperature for 2 h, followed
by acylation of benzo[d][1,3]dioxol-5-ylmethanamine 1 and
benzo[d][1,3]dioxol-5-ylmethanol 2. Formation of acyl chlor-
ides from carboxylic acids, for further acylation was applied
due to its wide applicability and common use. In this regard,
both synthetic procedures were suitable for amide and
ester formation and provided satisfactory yields (values
between 60% and 74%). The synthetic steps are depicted in
Scheme 1 [23, 24].
The ester analogue, 6e (3,4-dichloro attached to the
aromatic ring) induced preferentially cytotoxic effects in
the H1299 cells (IC50 ¼ 172 mM) in comparison to the
prototype. However, 5f, 6c, and 6e compounds were inactive
in human non-small cell lung cancer (NCI-H460) and breast
adenocarcinoma (SK-BR-3 and MDA-MB-231) cells.
Interestingly, compounds 5f, 6c, and 6e did not present
cytotoxicity against human fetal lung fibroblasts (MRC-5),
indicating that these molecules exhibit a certain selective
profile in comparison to capsaicin (IC50 ¼ 10 mM). Then, our
Biological activity
Cell viability assay
The synthesized derivatives 5a–f, 6a–e were evaluated for
their in vitro cytotoxic activity against a panel of tumor and
non-tumor cells [murine melanoma (B16F10), human mela-
noma (SK-MEL-28), non-small cell lung cancer (NCI-H1299 and
NCI-H460), breast adenocarcinoma (SK-BR-3 and MDA-MB-231),
and human fetal lung fibroblasts (MRC-5)] in a range from 50.0
to 200.0 mM. The assays were carried out in triplicate, and
Scheme 1. Synthetic route of benzo[d][1,3]dioxol-5-ylmethyl amides (5a–f) and esters (6a–e). Reagents and conditions: (a) oxalyl chloride,
CH2Cl2, cat. DMF, r.t., 2 h; (b) Et3N, cat. DMAP, CH2Cl2, 0°C, 24 h.
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Table 1. Cytotoxic activity (IC50mM) of compounds 5f, 6c, and 6e.
Cell lines
B16F10
SK-MEL-28
NCI-H1299
NCI-H460
SK-BR-3
MDA-MB-231
MRC-5
Compounds
R
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
Capsaicin
5f
117
87
ꢀ200
ꢀ200
ꢀ200
120
ꢀ200
75
ꢀ200
48
ꢀ200
10
ꢀ200
187
6c
6e
130
85
ꢀ200
ꢀ200
ꢀ200
ꢀ200
ꢀ200
ꢀ200
ꢀ200
ꢀ200
ꢀ200
ꢀ200
ꢀ200
172
findings have indicated that the designing strategy applied
here has produced more selective and active analogues than
the prototype, capsaicin.
Additionally, the benzo[d][1,3]dioxol-5-ylmethyl aryl/alkyl
capsaicin bioisosters, such as 5f and 6c, might be interesting
scaffolds for optimization against lung cancer.
RMSD value found for capsaicin was already expected since
the molecular model was built up using a crystallographic
structure that shares little resemblance with the prototype.
The intramolecular energy contributions (i.e., thermo-
dynamic properties) found for the selected lowest-energy
conformations from MD simulation at 310 K were the
following: Stretching (ESTRETCH), bending (EBEND), torsional
(ETORS), Lennard–Jones or 1–4 interactions (E1,4), van der Waals
(EvdW), electrostatic (ECHARGE), hydrogen bonding (EHb), and
solvation (ESOLV). The summation of all energy contributions,
which correspond to the total potential energy (ETOT), was
also considered.
To the set of designed molecules, ETOT values varied from
2.70 to 36.33 kcal/mol, of which capsaicin and 5b presented
higher (more positive) values (31.81 and 36.33 kcal/mol,
respectively) than the other compounds. This fact reflects
more degrees of freedom, once both molecules present a
carbonic side chain of nine atoms (see Supplementary
Information, Table S2).
Then, 48 descriptors or molecular properties of different
nature were calculated (thermodynamic, hydrophobic, elec-
tronic, topological, geometric, and steric) for each compound.
A table composed of 12 rows (samples plus capsaicin) and
48 columns (independent variables or descriptors or molecular
properties) was generated and used as input for the exploratory
data analysis (PCA and HCA). All calculated descriptors and
their methods are shown in the Supplementary Information
(Table S1).
Molecular modeling approach
The purpose of the theoretical approach was to find energeti-
cally more favorable conformations for capsaicin and its
analogues (5a–f and 6a–e), calculate the molecular properties
and, finally, provide some insights regarding structure–activity
relationships by exploratory data analysis. Molecular dynamics
(MD) simulations (1 ns at 310K) of all compounds reached the
thermodynamic equilibrium between 400,000 and 600,000
simulation steps. The lowest-energy conformer of each molecule
was selected from the equilibrium region of the conformational
ensemble profile (CEP). Additionally, root mean square
deviation (RMSD) values between the atomic positions of the
selected conformers and the atomic positions of the energy-
minimized structures were computed as a criterion to verify
whether the structural integrity was maintained after simu-
lations. Higher RMSD values indicate loss of structural integrity
during MD simulations if there is considered a crystallographic
structure as template or reference [25].
The RMSD value found between the lowest-energy conform-
er and the initial energy-minimized structure of capsaicin was
0.53 Å and lower than 0.09 Å for its analogues. The higher
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Novel Capsaicin Analogues as Potential Anticancer Agents
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Exploratory analysis
15.28%, 9.59%, and 6.92%, respectively, of total variance from
the original data, meaning they did not influence significant-
ly the samples separation process, and, then, were not
considered to further discussion.
Exploratory data analysis allows the investigation of inter-
samples relationships through either similarity indices, or
linear combinations from the original data set. The findings
can be used to establish structure–molecular property
relationships, since the calculated descriptors are directly
related to the compounds’ chemical structure.
The unsupervised PCA aimed to correlate the descriptors to
find other uncorrelated variables, the principal components
(PCs), which can describe the information from the original
data set [20, 21].
The PCA findings from exploratory data analysis are shown
in Fig. 3. According to the factors selection (Fig. 3a), the two
first factors or PCs discriminated more than sixty percent
(61.93%) of total variance from the original data. PC1, or factor
1, explained 34.90% of total variance whereas PC2, or factor 2,
described 27.03%. Also PC3, PC4, and PC5 discriminated
PC1, or factor 1, seems to be responsible for splitting the
samples according to their isosteric functional groups.
Amides, including capsaicin, were positioned at the positive
side of PC1 axis whereas the ester analogues remained at the
negative side. Compound 5a, amide analogue with short side
chain, is an exception to the pattern observed in factor 1.
Interestingly, PC2 or factor 2 has considered mostly the
kind of substitution in the amide or ester bond position
(R group), whether aryl or alkyl substituent group. Then,
regarding PC2, the compounds were separated into three
smaller groups: A, which presents amides with long side
chains (capsaicin and 5b); B that comprises ester analogues
with aliphatic side chains (6a, 6b, and 6c); and C, which
Figure 3. PCA findings: (a) Factors selection, factor 1 and 2 discriminated 61.93% of total variance from original data; (b) scores plot for PC1
(factor 1) versus PC2 (factor 2), the samples were classified mainly into three groups according to functionality and/or substitution [A (amides
with long side chain: capsaicin and 5b), B (alkyl ester analogues: 6a, 6b, and 6c), and C (aryl amides and esters: 5d, 5f, 5c, 6e, and 6d)]; (c)
loading values for the calculated descriptors regarding factor (or PC) 1 and 2; (d) outliers diagnosis through the sample residuals versus
Mahalanobis distance. The sample residual threshold (green line) is based upon 95% probability limit (set internally in Pirouette program).
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
contains the compounds with aromatic substituent groups
(5d, 5f, 5c, 6e, and 6d). The amide analogues 5e (nitro-
substituted) and 5a (short side chain) remained alone and
more distant from the rest due to their structural features at
the R position.
It is noteworthy that compounds 5f, 6c, and 6e, experimen-
tally evaluated, were in B and C groups, and positioned closely
to the PC1 axis. This information will be useful for the
designing of new analogues, since molecules that are
positioned around PC1 axis would be likely more promising
against tumor cell lines since they share molecular properties
with the three tested compounds.
The loadings’ table, presented in Fig. 3c, provides informa-
tion about which descriptors or molecular properties mostly
influenced the samples discrimination. In PC1, or factor 1
(34.9%), topological (Randic index, Platt index, number of
routable bonds) and steric/hydrophobic (molar refractivity,
MR) molecular properties presented higher loading values
(absolute values; >0.3). In PC2, or factor 2 (27.03%), electronic
(the lowest unoccupied molecular orbital energy, Elumo),
topological (Hacceptor, Harary index) and steric/hydrophobic
(MR) molecular properties influenced more the samples
separation. Also, their loading values varied from 0.39 to 0.51
(absolute values).
Moreover, the plot of sample residual versus Mahalanobis
distances [26] (Fig. 3d) indicated there were no outliers. The
sample residual threshold (light green line) is based upon a
95% of confidence level interval set internally in Pirouette
3.11 [27]. The compounds did not exceed a threshold value,
meaning the calculated properties were sufficient to describe
the structural features of the entire data set.
Complementary findings were obtained for both methods,
PCA and HCA, reinforcing particularly the discrimination
pattern given by PC1and PC2, as can be seen in the
dendrogram of samples (Fig. 4). HCA analysis presented
higher distinction between samples and classified them into
four sub-clusters (A, B, C, and E) when a similarity cursor of
0.6 (dashed line) was considered as reference.
The compounds were grouped, in general, accordingly to
their functional groups (ester or amide) and nature of side
chain (alkyl or aryl). Considering ester analogues, compounds
were mainly grouped into two clusters: A (68.8% similarity)
and C (66.3% similarity). Cluster A was composed of alkyl
esters and cluster C of aryl esters. Regarding amide analogues,
compounds were grouped in cluster B (69.8% similarity)
and E (64.1% similarity). Cluster B was composed by aryl
amides substituted by electron donating groups (4-OCH3,
4-CH3), while cluster E comprises amides with long alkyl
Figure 4. Dendrogram found for HCA of samples. Considering a similarity cursor of 0.6 (dotted line), capsaicin derivatives were grouped into
four sub-clusters: A (68.8% similarity), B (69.8% similarity), C (66.3% similarity), and E (64.1% similarity). The clusters were basically formed
regarding the isosteric group (amide or ester) and nature of side chain (alkyl or aryl).
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Novel Capsaicin Analogues as Potential Anticancer Agents
7
Figure 5. LUMO map found for compounds 5f (A) and 6a (B). Color ranges from ꢁ0.02 (intense red) to 0.02 (intense blue) (GaussView
3.0) [28]. The compounds are represented as ball and stick model. The carbon atoms are in gray, nitrogen in blue, hydrogen in white, and
oxygen atoms are in red.
chains (capsaicin and 5b). Compound 5a, a short side chain
amide, was not grouped into any clusters when a similarity
cursor of 60% was considered. The same behavior was
observed for compounds 5e and 5d (cluster D; 21.5%
similarity), which contain electron withdrawing substituents
(3-NO2, 3,4-Cl).
range against B16F10, SK-MEL-28 and NCI-H1299 cell lines.
The chemometric study (PCA and HCA) has revealed
interesting points regarding structure–property relation-
ships. PCA findings indicated that topological (Randic index)
and electronic properties (Elumo) might be important for
improving the biological activity. The investigated set of
bioisoster molecules can be considered as promising scaffolds
to be explored. Indeed, new substituted aryl/alkyl amides and
esters are going to be synthesized in order to obtain potential,
selective and non-toxic anticancer agents, particularly against
melanoma and lung cancer.
It is well known that molecular properties are directly
dependent upon the chemical structure. Steric properties
(including geometric and topological properties), for in-
stance, are related to the molecular shape. Electronic and
stereo chemical properties can be considered as the most
important requirements in the molecular recognition
process. Regarding PCA findings, among the highest loading
values (in module) are a topological descriptor (Randic index),
in PC1, and an electronic descriptor as frontier molecular
orbital energy (Elumo), in PC2. This electronic property can be
graphically visualized through the LUMO map distribution.
The LUMO maps for compounds 5f (active) and 6a (inactive)
are displayed in Fig. 5. The active compound 5f had a 10-fold
more negative value of Elumo (ꢁ20.90 kcal/mol) than 6a
(ꢁ2.13 kcal/mol). Additionally, the LUMO map distribution
was in opposite regions regarding the two molecules,
corroborating with the experimental findings.
Experimental
Chemistry
All reagents used are of analytical grade having >97% purity. For
the chemical synthesis and analysis of capsaicin analogues the
following compounds were used: Piperonylamine, piperonyl
alcohol, propionic acid, butanoic acid, hexanoic acid, non-anoic
acid, isovaleric acid, benzoic acid, 4-methylbenzoic acid, 3,4-
chlorobenzoic acid, 4-methoxybenzoic acid, 3-nitrobenzoic acid,
and oxalyl chloride (Sigma–Aldrich Inc., St. Louis, MO, USA).
Melting points were recorded on a Buchi M-565 apparatus and
were uncorrected. TLC was carried out using silica gel plates (TLC
silica gel 60 F254 aluminum sheets, Merck Darmstadt, Germany)
and the spots were visualized under UV light at 254 nm. Flash
chromatography was performed for purification of the com-
pounds on silica gel 60 (70–230 mesh ASTM) (Merck Darmstadt).
The structure of each compound was confirmed by 1H NMR,
13C NMR and elemental analysis. 1H and 13C NMR spectra were
recorded on Bruker AC-300 at 300 and 75 MHz, respectively, and
chemical shifts were expressed as d units, using tetramethylsilane
(TMS) as an internal standard. The spectral splitting patterns were
described as follows: s, singlet; d, doublet; dd, double doublet;
Conclusions
A series of twelve novel benzo[d][1,3]dioxol-5-ylmethyl func-
tional analogues of capsaicin were designed through LBDD
strategy, synthesized in a facile acylation method, and
experimentally evaluated. Three compounds (5f, 6c, and 6e)
exhibited significant in vitro cytotoxicity at micromolar (mM)
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ddd, double double doublet; t, triplet; m, multiplet; bs, broad
singlet, and bt, broad triplet peak.
J ¼ 8.4 Hz, 14-ArH), 6.85 (3H, m, 4, 5, 7-ArH), 5.98 (2H, s, 1-OCH2O–),
4.38 (2H, d, J ¼ 5.9 Hz, 8-CH2); 13C NMR (DMSO-d6, 75 MHz) d: 163.8
(C10), 147.2 (C3), 146.1 (C2), 134.6 (C16), 134.0 (C11), 133.0 (C15),
131.2 (C14), 130.7 (C13), 129.2 (C12), 127.6 (C6), 120.6 (C7), 108.0
(C4), 108.0 (C5), 100.8 (C1), 42.7 (C8). Anal. calcd. for
C15H11O3NCl2: C, 55.58; H, 3.42; N, 4.32. Found: C, 55.46;
H, 3.53; N, 4.28.
General procedure for the synthesis of compounds 5a–f
and 6a–e
In different 100 mL flasks, the carboxylic acids 3a–j (5 mmol) were
dissolved in dichloromethane (CH2Cl2) (50 mL). Next, oxalyl
chloride (0.63 g, 458 mL, 5 mmol) and three drops of N,N-
dimethylformamide were added. The resulting solution was
stirred at room temperature for 2 h. Subsequently, the excess of
oxalyl chloride was removed under high vacuum and the second
step was performed in one pot through the addition of 10 mL of
CH2Cl2, 1 (622 mL, 0.755 g, 5 mmol) (5a–f synthesis) or 2 (0.760 g,
5 mmol) (6a–e synthesis), triethylamine (Et3N) (723 mL, 0.525 g,
5.2 mmol), and 4-dimethylaminopyridine (DMAP) (catalytic). The
reaction mixture was stirred for 4 h under nitrogen atmosphere
at 0°C. The organic layer was washed with 5% HCl aqueous
solution, water, brine and dried over MgSO4. The solvent was
removed under high vacuum and the products were purified by
flash column chromatography with hexanes/ethyl acetate system
as solvent.
N-(Benzo[d][1,3]dioxol-5-ylmethyl)-3-nitrobenzamide (5e)
Yield: 63%; Yellow solid, m.p. 111–112°C; 1H NMR (DMSO-d6;
300 MHz) d: 8.60 (1H, t, 13-ArH), 8.37 (1H, ddd, 16-ArH), 8.19
(1H, m, 12-ArH), 7.66 (1H, t, 14-ArH), 6.84 (3H, m, 4, 5, 7-ArH), 6.58
(1H, bs, 9-NH), 5.97 (2H, s, 1-OCH2O–), 4.59 (2H, d, J ¼ 5.6 Hz,
8-CH2); 13C NMR (DMSO-d6, 75 MHz) d: 164.8 (C10), 148.2 (C15),
148.1 (C3), 146.4 (C2), 136.0 (C11), 133.3 (C12), 131.3 (C6), 129.9
(C14), 126.1 (C16), 121.7 (C13), 121.5 (C6), 108.6 (C4), 108.5 (C5),
101.2 (C1), 44.3 (C8). Anal. calcd. for C15H12N2O5: C, 60.00; H, 4.03;
N, 9.33. Found: C, 59.83; H, 3.79; N, 9.02.
N-(Benzo[d][1,3]dioxol-5-ylmethyl)-4-methoxybenzamide
(5f)
Yield: 73%; White solid, m.p. 113–114°C; 1H NMR (CDCl3;
300 MHz) d: 7.74 (2H, d, J ¼ 8.9 Hz, 12, 13-ArH), 6.91 (2H, d,
J ¼ 8.9 Hz, 14, 15-ArH), 6.81 (3H, m, 4, 5, 7-ArH), 6.33 (1H, bt, 9-NH),
5.94 (2H, s, 1-OCH2O–), 4.52 (2H, d, J ¼ 5.6 Hz, 8-CH2), 3.84 (3H, s,
14-OCH3); 13C NMR (CDCl3, 75 MHz) d: 166.8 (C10), 162.3 (C16),
148.0 (C3), 147.0 (C2), 132.3 (C12, C13), 128.8 (C6), 126.7 (C11),
121.2 (C7), 113.8 (C14, C15), 108.5 (C4), 108.3 (C5), 101.1 (C1), 55.4
(C17), 43.9 (C8). Anal. calcd. for C16H15NO4: C, 67.36; H, 5.30;
N, 4.91. Found: C, 66.86; H, 5.53; N, 4.84.
N-(Benzo[d][1,3]dioxol-5-ylmethyl)propionamide (5a)
Yield: 68%; White solid, m.p. 97–98°C; 1H NMR (CDCl3; 300 MHz)
d: 6.77 (3H, m, 4, 5, 7-ArH), 5.97 (2H, s, 1-OCH2O–), 5.63 (1H, bs,
9-NH), 4.37 (2H, d, 8-CH2), 2.26 (2H, q, J ¼ 7.6 Hz, 11-CH2), 1.20 (3H,
t, J ¼ 7.6 Hz, 12-CH3); 13C NMR (CDCl3, 75 MHz) d: 173.5 (C10), 147.9
(C3), 147.0 (C2), 132.3 (C6), 121.1 (C7), 108.4 (C4), 108.3 (C5), 101.1
(C1), 43.4 (C8), 29.7 (C11), 9.8 (C12). Anal. calcd. for C11H13O3N:
C, 63.76; H, 6.32; N, 6.76. Found: C, 63.82; H, 6.41; N, 6.58.
N-(Benzo[d][1,3]dioxol-5-ylmethyl)nonanamide (5b)
Benzo[d][1,3]dioxol-5-ylmethyl butanoate (6a)
Yield: 70%; White solid, m.p. 90–91°C; 1H NMR (CDCl3; 300 MHz)
d: 6.77 (3H, m, 4, 5, 7-ArH), 5.96 (2H, s, 1-OCH2O–), 5.70 (1H, bs,
9-NH), 4.36 (2H, d, 8-CH2), 2.21 (2H, t, J ¼ 7.6 Hz, 11-CH2), 1.68
(2H, m, 12-CH2), 1.30 (10H, m, 13, 14, 15, 16, 17-CH2), 0.90 (3H, t,
J ¼ 6.7 Hz, 18-CH3); 13C NMR (CDCl3, 75 MHz) d: 172.9 (C10), 147.9
(C3), 147.0 (C2), 132.4 (C6), 121.1 (C7), 108.3 (C4), 108.3 (C5), 101.1
(C1), 43.4 (C8), 36.8 (C11), 31.8 (C12), 29.3 (C13), 29.3 (C14) 29.1
(C15), 25.7 (C16), 22.6 (C17), 14.0 (C18). Anal. calcd. for
Yield: 63%; Yellow liquid; 1H NMR (CDCl3; 300 MHz) d: 6.80
(3H, m, 4, 5, 7-ArH), 5.95 (2H, s, 1-OCH2O–), 5.01 (2H, s, 8-CH2),
2.31 (t, 2H, J ¼ 7.4 Hz, 11-CH2), 1.66 (m, 2H, J ¼ 7.4 Hz, 14.9 Hz, 12-
CH2), 0.84 (t, 3H, 14.8 Hz, 13-CH3); 13C NMR (CDCl3, 75 MHz)
d: 173.4 (C10), 147.8 (C3), 147.6 (C2), 130.0 (C6), 122.1 (C7), 108.9
(C4), 108.2 (C5), 101.1 (C1), 66.0 (C8), 36.2 (C11), 18.4 (C12), 13.6
(C13). Anal. calcd. for C12H14O4: C, 64.85; H, 6.35. Found: C,
64.85; H, 6.35.
C
17H25O3N: C, 70.07; H, 8.65; N, 4.81. Found: C, 69.83; H, 8.72;
N, 4.81.
Benzo[d][1,3]dioxol-5-ylmethyl 3-methyl butanoate (6b)
Yield: 70%; Yellow liquid; 1H NMR (CDCl3; 300 MHz) d: 6.80
(3H, m, 4, 5, 7-ArH), 5.94 (2H, s, 1-OCH2O–), 5.00 (2H, s, 8-CH2), 2.15
(m, 3H, 11,12-CH2), 0.94 (d, 6H, 13,14-CH2); 13C NMR (CDCl3,
75 MHz) d: 172.9 (C10), 147.8 (C3), 147.6 (C2), 130.0 (C6), 122.2 (C7),
109.0 (C4), 108.2 (C5), 101.1 (C1), 65.9 (C8), 43.4 (C11), 25.7 (C12),
22.4 (C13, C14). Anal. calcd. for C13H16O4: C, 66.09; H, 6.83.
Found: C, 65.85; H, 6.71.
N-(Benzo[d][1,3]dioxol-5-ylmethyl)-4-methylbenzamide (5c)
Yield: 72%; White crystal, m.p. 131–132°C; 1H NMR (DMSO-d6;
300 MHz) d: 8.87 (1H, bt, J ¼ 5,8 Hz, 9-NH), 7.79 (2H, d, J ¼ 8.2 Hz,12,
13-ArH), 7.27 (2H, d, J ¼ 8.2 Hz, 14, 15-ArH), 6.83 (3H, m, 4, 5,
7-ArH), 5.98 (2H, s, 1-OCH2O–), 4.38 (2H, d, 8-CH2), 2.36 (3H, s,
17-CH3); 13C NMR (DMSO-d6, 75 MHz) d: 165.9 (C10), 147.2 (C3),
146.0 (C2), 141.0 (C16), 133.7 (C11), 131.7 (C6), 128.8 (C14, C15),
127.2 (C12, C13), 120.4 (C7), 107.9 (C5), 107.9 (C4), 100.7 (C1), 42.4
(C8), 21.0 (C17). Anal. calcd. for C16H15O3N: C, 71.36; H, 5.61;
N, 5.20. Found: C, 71.30; H, 5.74; N, 5.17.
Benzo[d][1,3]dioxol-5-ylmethyl hexanoate (6c)
Yield: 68%; Yellow liquid; 1H NMR (CDCl3; 300 MHz) d: 6.85 (2H, d,
J ¼ 7.4 Hz, 4, 5-ArH), 6.80 (1H, d, J ¼ 7.4 Hz, 7-ArH), 5.97 (2H, s,
1-OCH2O–), 5.03 (2H, s, 8-CH2), 2.35 (2H, t, J ¼ 7.5 Hz, 11-CH2), 1.66
(2H, m, 12-CH2), 1.33 (4H, m, 13,14-CH2), 0.91 (3H, t, 15-CH3);
13C NMR (CDCl3, 75 MHz) d: 173.5 (C10), 147.8 (C3), 147.6 (C2),
130.0 (C6), 122.1 (C7), 108.9 (C4), 108.2 (C5), 101.1 (C1), 66.0 (C8),
34.3 (C11), 31.3 (C12), 24.6 (C13), 22.3 (C14), 13.8 (C15). Anal. calcd.
for C14H18O4: C, 67.18; H, 7.25. Found: C, 67.54; H, 7.46.
N-(Benzo[d][1,3]dioxol-5-ylmethyl)-3,4-dichlorobenzamide
(5d)
Yield: 60%; White solid, m.p. 125–126°C; 1H NMR (DMSO-d6;
300 MHz) d: 9.13 (1H, bt, J ¼ 5.8Hz, 9-NH), 8.12 (1H, d, J ¼ 2.0 Hz,
13-ArH), 7.86 (1H, dd, J ¼ 8.4 Hz, J ¼ 2.1 Hz, 12-ArH), 7.76 (1H, d,
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Novel Capsaicin Analogues as Potential Anticancer Agents
9
Benzo[d][1,3]dioxol-5-ylmethyl benzoate (6d)
implemented in HyperChem 7.0 [30]. The MOLSIM 3.2 soft-
ware [34] was used to perform energy-minimization (steepest
descent and conjugate gradient methods; convergence criterion
of 0.01 kcal/mol) and molecular dynamics (MD) simulations. MD
simulations of 1 ns (step size of 1 fs) at 310 K were performed for
each molecular model. Dielectric constant of 3.5 was used to
simulate the environment of biological membranes and enzyme
or receptor binding sites [35]. It was assigned a fictitious
atomic mass of 5000 u.m.a. to some atoms position in order to
maintain the structural integrity of the molecular models during
simulation. Output trajectory file was recorded every 20 ps steps
resulting in a conformational ensemble profile (CEP) of 50,000
conformers for each investigated molecule. After equilibration,
conformers were selected, for each 100 ps, in order to find the
lowest-energy conformation more frequent. Then, care was taken
in the conformation selection process. The intramolecular
solvation and hydrogen bonding energy contributions were
computed for each selected conformer from MD simulations
scheme. The hydration shell model proposed by Forsythe and
Hopfinger [36] was employed to estimate the solvation energy
contribution. Total potential energy (ETOT) of each selected
conformation corresponds to the summation of the following
intramolecular energy contributions: stretching (ESTRETCH), bend-
ing (EBEND), torsional (ETORS), Lennard–Jones or 1–4 interactions
(E1,4), van der Waals (EvdW), electrostatic (ECHARGE), hydrogen
bonding (EHb), and solvation (ESOLV).
Yield: 74%; White solid, m.p. 63–64°C; 1H NMR (CDCl3; 300 MHz)
d: 7.98 (2H, d, J ¼ 7.6 Hz, 12, 13-ArH), 7.48 (1H, t, J ¼ 7.6, 16-ArH),
7.35 (2H, t, J ¼ 7.6 Hz, 14, 15-ArH), 6.85 (2H, d, 4, 5-ArH), 6.73 (1H,
d, 7-ArH), 5.89 (2H, s, 1-OCH2O–), 5.19 (2H, s, 8-CH2); 13C NMR
(CDCl3, 75 MHz) d: 166.4 (C10), 147.9 (C3), 147.7 (C2), 133.0 (C16),
130.2 (C6), 129.9 (C11), 129.7 (C14, C15), 128.4 (C12, C13), 122.2
(C7), 109.0 (C4), 108.3 (C5), 101.2 (C1), 66.7 (C8). Anal. calcd. for
C15H12O4: C, 70.31; H, 4.72. Found: C, 70.24; H, 4.93.
Benzo[d][1,3]dioxol-5-ylmethyl 3,4-dichlorobenzoate (6e)
Yield: 61%; White solid, m.p. 98–99°C; 1H NMR (DMSO-d6;
300 MHz) d: 8.07 (1H, d, J ¼ 2.0 Hz, 13-ArH), 7.90 (1H, dd, J ¼ 2.0 Hz,
J ¼ 8.4 Hz, 12-ArH), 7.78 (1H, d, J ¼ 8.4 Hz, 14-ArH) 7.06 (1H, d,
J ¼ 1.5 Hz, 4-ArH), 6.98 (1H, dd, J ¼ 7.9 Hz, J ¼ 1.5 Hz, 7-ArH), 6.91
(1H, d, J ¼ 7.9 Hz, 5-ArH), 6.03 (2H, s, 1-OCH2O–), 5.25 (2H, s, 8-CH2);
13C NMR (DMSO-d6, 75 MHz) d: 163.8 (C10), 147.4 (C3), 147.3 (C2),
136.3 (C16), 131.7 (C11), 131.2 (C6), 130.8 (C14), 130.1 (C13), 129.2
(C15), 129.2 (C12), 122.3 (C7), 109.0 (C4), 108.1 (C5), 101.1 (C1), 66.9
(C8). Anal. calcd. for C15H10O4Cl2: C, 55.41; H, 3.10. Found: C,
55.58; H, 3.29.
Cytotoxic activity
All cell lines studied were purchased from the American Type
Tissue Culture Collection (ATCC, Rockville, MD, USA). Murine
melanoma (B16F10), human melanoma (SK-MEL-28), non-small
cell lung cancer (NCI-H1299 and NCI-H460), breast adenocarcino-
ma (SK-BR-3 and MDA-MB-231), and human fetal lung fibroblast
(MRC-5), were routinely cultured in Modified Dulbecco’s Medium
or RPMI supplemented with 2 mM L-glutamine, 5% (v/v) FCS and
maintained at 37°C in 95% humidified atmosphere, containing
5% CO2. Asynchronous cell cultures in the exponential phase of
growth.
The lowest-energy conformation of each molecule from MD
simulations was energy-minimized [34] and, subsequently, used
as starting geometry to calculate physicochemical and structural
properties of distinct nature (thermodynamic, hydrophobic,
electronic, topological, geometric, and steric), which were
explored in this study. Electronic properties, such as partial
atomic charges from electrostatic potentials using a grid based
method (CHELPG) [37], dipole moment (m) (total and x, y, z) and
frontier molecular orbital energies (EHOMO, energy of the highest
Cytotoxicity activities of compounds 5a–f, 6a–e, and capsaicin
were evaluated against seven different cell lines. Cells were plated
in 96-well plates at a concentration of 1 ꢂ 104 cells per well. The
cells were allowed to grow for 24 h, then, treated with the
compounds 5a–f, 6a–e, or capsaicin, at concentration of 200, 175,
150, 100, and 50 mM. After 24 h of treatment, cell viability was
determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide) (Sigma, St Louis, MO, USA) [29]. Briefly, MTT
solution (20 mL) was added to each well (final concentration
¼ 0.5 mg/mL) and the plate was incubated for additional 3 h.
Finally, DMSO (200 mL) was added to each well, and the
absorbance was measured in a plate reader (Thermoplate Q4
TP Reader, Tokay Hit, Japan) at 540 nm. The 50% inhibitory
concentration (IC50) values were calculated using a proper
analysis (non-linear regression-curve fit). Each experiment was
performed using six replicates for each compound concentration,
and repeated in triplicate.
occupied molecular orbital; ELUMO
, energy of the lowest
unoccupied molecular orbital), were computed with the B3LYP
(Becke, three-parameter, Lee–Yang–Parr) [38] hybrid functional
and 6–31(d,p) basis [39].
MarvinView program [40] was used to calculate steric, topologi-
cal/geometric, and lipophilic properties. ClogP was calculated by
the weighted method, assigning equal weight for each method [41–
43]. Further detailed information regarding all descriptors,
methods, and respective software used to perform these calcu-
lations are listed in the Supplementary Information (Table S1).
A table containing 12 rows, which correspond to the samples
(analogues) and capsaicin, and 48 columns, which correspond to
the descriptors or calculated molecular properties (see Supple-
mentary Information, Table S2), was used as input for
the exploratory data analysis. Due to distinct magnitude orders
among the calculated variables, an autoscaling procedure was
applied as a preprocessing method [22].
Molecular modeling approach
Exploratory data analysis
The three-dimensional (3D) molecular models of capsaicin and its
derivatives were built up, in their neutral forms, using the
HyperChem 7.0 MM þ force field [30] without any constrains. The
crystal data from 3-chloro-N-(4-hydroxy-3-methoxy-benzyl)-2,2-
diethylpropanamide [31] was employed as the starting geometry
for capsaicin and N-[(1,3-benzodioxol-5-yl)methyl]-4-methylbenz-
amide [32] for compounds 5a–f and 6a–e. Partial atomic charges
were computed with AM1 semi-empirical method [33], also
The exploratory data analysis, which comprises the principal
component analysis (PCA) and hierarchical cluster analysis (HCA)
was carried out employing the Pirouette 3.11 software [27].
Principal component analysis (PCA)
PCA is a data compression method based upon the correlation
among variables or descriptors. Its purpose is to reduce the
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10
M. C. F. C. B. Damião et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
dimensionality of a data set by grouping the correlated variables
and replacing the original descriptors with a new set called
factors or principal components (PCs), onto which the data is
projected. PCs or factors are completely uncorrelated variables
built up as simple linear combinations from the original
variables, containing most of the variability in the data set even
though in a much lower dimensional space.
The data matrix X(I ꢂ J) corresponding to I compounds and J
descriptors, was decomposed into two matrices, T and L, such
that X ¼ TLT. The T matrix, which is known as the score matrix,
represents the positions of the compounds in the new coordinate
system where the PCs are the axes. Scores are integral to
exploratory analysis because they show intersample relation-
ships. L is the matrix of loadings whose columns describe how the
new axes (the PCs) are built from the old axes, and indicates the
variables contribution to each PC [20, 21, 27]. In this exploratory
data analysis, PCA was run up to ten factors or PCs. The outliers’
diagnosis, implemented in Pirouette 3.11 software, was also
performed through the Mahalanobis distance [26].
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Hierarchical cluster analysis (HCA)
HCA was also carried out using the Pirouette 3.11 program,
employing the complete linkage method and Euclidean distance.
In HCA, distances between pairs of samples (or variables) are
calculated and compared. When distances between samples
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distances between samples or variables were calculated and
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The authors have declared no conflict of interest.
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