Pharmacological and physicochemical profile of arylacetamides as tools against human cancers

Article abstract of DOI:10.1016/j.taap.2019.114692

Arylacetamides are widely used as synthetic intermediates to obtain medicinal substances. This work evaluated in vitro antiproliferative activity of ten 2-Chloro-N-arylacetamides on human normal and cancer cells and detailed in vivo toxicological and anticancer investigations. Initially, cytotoxic colorimetric assays were performed using tumor lines, peripheral blood mononuclear cells (PBMC) and erythrocytes. Compounds 2, 3 and 4 were tested for acute toxicity (50, 150 and 300 mg/kg) and for subacute antitumoral capacity in HCT-116 colon carcinoma-bearing xenograft mice for 15 days at 25 mg/kg/day. Most compounds revealed cytotoxic action on tumor lines and PBMC, but activity on human erythrocytes were not detected. Molecular dipole moment, lipophilicity and electronic constant of aryl substituents had effects upon in vitro antiproliferative capacity. More common in vivo acute behavioral signals with compounds 2, 3 and 4 were muscle relaxation, reduction of spontaneous locomotor activity and number of entries in closed arms and increased number of falls andtime spent in open arms, suggesting diazepam-like anxiolytic properties. Decrease of grabbing strength and overall activity were common, but palpebral ptosis and deaths occurred at 300 mg/kg only. Compounds 2 and 3 reduced colon carcinoma growth (21.2 and 27.5%, respectively, p < 0.05) without causing apparent signals of organ-specific toxicity after subacute exposure. The structural chemical simplicity of arylacetamides make them cost-effective alternatives and justifies further improvements to enhance activity, selectivity and the development of pharmaceutical formulations.

Full text of DOI:10.1016/j.taap.2019.114692

Accepted Manuscript
Pharmacological and physicochemical profile of arylacetamides
as tools against human cancers
Paulo Michel Pinheiro Ferreira, Kátia da Conceição Machado,
Stefânia Neiva Lavorato, Fátima de Cássia Evangelista de
Oliveira, Jurandy do Nascimento Silva, Antonia Amanda Cardoso
de Almeida, Luciano de Souza Santos, Valdenizia Rodrigues
Silva, Daniel Pereira Bezerra, Milena Botelho Pereira Soares,
Cláudia Pessoa, Manoel Odorico de Moraes Filho, José Roberto
de Oliveira Ferreira, João Marcelo de Castroe Sousa, Vinícius
Gonçalves Maltarollo, Ricardo José Alves
PII:
DOI:
Article Number:
Reference:
S0041-008X(19)30300-X
https://doi.org/10.1016/j.taap.2019.114692
114692
YTAAP 114692
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Toxicology and Applied Pharmacology
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Revised date:
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2 January 2019
22 July 2019
23 July 2019
Please cite this article as: P.M.P. Ferreira, K. da Conceição Machado, S.N. Lavorato,
et al., Pharmacological and physicochemical profile of arylacetamides as tools against
human cancers, Toxicology and Applied Pharmacology, https://doi.org/10.1016/
j.taap.2019.114692
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ACCEPTED MANUSCRIPT
Pharmacological and physicochemical profile of arylacetamides as tools
against human cancers
Paulo Michel Pinheiro Ferreiraa,b,*, Kátia da Conceição Machadoa,b, Stefânia Neiva
Lavoratoc,d, Fátima de Cássia Evangelista de Oliveirae, Jurandy do Nascimento
Silvaa,b, Antonia Amanda Cardoso de Almeidaa,b, Luciano de Souza Santosf,
Valdenizia Rodrigues Silvaf, Daniel Pereira Bezerraf, Milena Botelho Pereira Soaresf,
Cláudia Pessoae, Manoel Odorico de Moraes Filhoe, José Roberto de Oliveira
Ferreirag, João Marcelo de Castro e Sousab,h, Vinícius Gonçalves Maltarolloc,
Ricardo José Alvesc
a Department of Biophysics and Physiology, Laboratory of Experimental Cancerology,
Federal University of Piauí, 64049-550 Teresina, Brazil
b
Postgraduate Programs in Pharmaceutical Sciences and Biotechnology, Federal
University of Piauí, 64.049-550 Teresina, Brazil
c Department of Pharmaceutical Products, Faculty of Pharmacy, Federal University of
Minas Gerais, 31270-901 Belo Horizonte, Brazil
d
Center of Biological Sciences and Health, Federal University of Western Bahia,
47808-021 Barreiras, Brazil
e Department of Physiology and Pharmacology, Faculty of Medicine, Federal
University of Ceará, 60430-270 Fortaleza, Brazil
f Oswaldo Cruz Foundation, 40296-710 Salvador, Brazil
g School of Medical Sciences, State University of Alagoas, 57010-382 Maceió, Brazil
h Department of Biology, Federal University of Piauí, 64067-670 Picos, Piauí, Brazil
*Corresponding author at: Department of Biophysics and Physiology, Laboratory of Experimental
Cancerology, Center for Health Sciences, Federal University of Piauí, Teresina, Brazil.
E-mail address: pmpf@ufpi.edu.br (P.M.P. Ferreira)
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ABSTRACT
Arylacetamides are widely used as synthetic intermediates to obtain medicinal
substances. This work evaluated in vitro antiproliferative activity of ten 2-Chloro-N-
arylacetamides on human normal and cancer cells and detailed in vivo toxicological
and anticancer investigations. Initially, cytotoxic colorimetric assays were performed
using tumor lines, peripheral blood mononuclear cells (PBMC) and erythrocytes.
Compounds 2, 3 and 4 were tested for acute toxicity (50, 150 and 300 mg/kg) and for
subacute antitumoral capacity in HCT-116 colon carcinoma-bearing xenograft mice
for 15 days at25 mg/kg/day. Most compounds revealed cytotoxic action on tumor
lines and PBMC, but activity on human erythrocytes were not detected. Molecular
dipole moment, lipophilicity and electronic constant of aryl substituents had effects
upon in vitro antiproliferative capacity. More common in vivo acute behavioral signals
with compounds 2, 3 and 4 were muscle relaxation, reduction of spontaneous
locomotor activity and number of entries in closed arms and increased number of
falls andtime spent in open arms, suggesting diazepam-like anxiolytic properties.
Decrease of grabbing strength and overall activity were common, but palpebral ptosis
and deaths occurred at 300 mg/kg only. Compounds 2 and 3reducedcolon carcinoma
growth (21.2 and 27.5 %, respectively, p < 0.05) without causing apparent signals of
organ-specific toxicity after subacute exposure. The structural chemical simplicity of
arylacetamides make them cost-effective alternatives and justifies further
improvements to enhance activity, selectivity and the development of pharmaceutical
formulations.
Keywords: colon carcinoma; xenograft model; physiological parameters; anxiolytic-
like effects; behavioral animal.
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1. Introduction
Cancer is a leading cause of death worldwide and the number of new cases is
expected to increase considerably over the next decades, according to World Health
Organization (WHO, 2017). There are many types of cancer treatment and the best
selection depend on the cancer type and stage (Pazdur et al., 2002). Among them,
chemotherapy is one of the most important and recommended treatments. However,
several drawbacks, like drug resistance and adverse side effects, habitually limit
adherence and treatment success (Carlotto et al., 2013; Willyard, 2016; Rapoport,
2017). On the other hand, these drawbacks have stimulated the search for new and
secure anticancer drugs.
In this context, 2-Chloro-N-arylacetamidesbelongs to a chemical class widely
used as synthetic intermediates to obtain bioactive substances for medicinal and
chemical purposes. Chloroacetamides produce pharmaceutical intermediates during
organic synthesis and are used as preservative of shampoos, cutting oils, plastics,
coatings slab and shower gel (Amrutkar et al., 2012; Jain et al., 2013; Harkovet al.,
2013). Reports have also demonstrated their potential as herbicidal, antimicrobial
action against filamentous fungi (Aspergillus niger), yeasts (Candida albicans),
bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus),
and on drug-sensitive strain of Mycobacterium tuberculosis (Hamm and Speziale,
1956; Marco-Contelles and Gómez-Sánchez, 2005; Katke et al., 2011; Aschale et al.,
2012).
However, to the best of our knowledge, their antiproliferative activity has not
been evaluated so far. Studies on the mechanism of action of chloroacetanilides
have indicated that the biological activity of this class could be attributed to its ability
to alkylate important bionucleophiles (Jablonkai, 2003; Helleday et al., 2008; Singh et
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al., 2011; Swift and Golsteyn, 2014). These features make chloroacetanilides an
interesting subject of study as potential anticancer agents. Thus, in this work, we
evaluated the in vitro antiproliferative activity of ten 2-Chloro-N-arylacetamides,
differing in aromatic substituents, against human normal and cancer cells,
Subsequently, in vivo toxicological investigations and analysis of anticancer action
using a xenograft model of human carcinoma were performed to assess
chemotherapeutic applications and toxic profile.
2. Material and Methods
2.1 Chemistry
Compounds 1-10 were synthesized (Table 1) and fully characterized by their
1
melting points and IR, H and 13C NMR spectra as described by Lavorato et al.
(2017).All compounds were solubilized in sterile dimethylsulfoxide (DMSO, Vetec,
Brazil).
2.2 Cell culture and animals’ facilities
Human leukemia (HL-60), ovarian (OVCAR-8), glioblastoma (SF-295), colon
(HCT-116), andliver (HEPG-2) tumor lines andperipheral blood mononuclear cells
(PBMC)were maintained in RPMI-1640 medium supplemented with 10% fetal bovine
serum, 2 mM glutamine, 100 U/mL penicillin and 100 g/mL streptomycin, at 37C in
a 5% CO2 atmosphere (Shel Lab CO2 Incubator, USA).
For PBMC isolation, heparinized human blood samples (from healthy, non-
smoker donors who had not taken any drug for at least 15 days prior to sampling,
aged 18-35 years old) were collected. Then, PBMC were isolated by the standard
method of density-gradient centrifugation over Ficoll-Hypaque (Cultilab, Campinas,
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Brazil). After some days, extra blood collection was performed and a suspension of
red blood cells (2 %) was prepared. All studies were executed in accordance with
Brazilian guidelines (Law 466/2012, National Council of Health), the Declaration of
Helsinki and with the Universal Declaration on Bioethics and Human Rights of
UNESCO.
For in vivo studies, Swiss (Mus musculus) andCB17 severe combined
immunodeficiency (SCID) female mice were obtained from the animal facilities at
Universidade Federal do Piauí (UFPI, Teresina, Brazil) and at Fundação Oswaldo
Cruz (FIOCRUZ, Salvador, Brazil), respectively. Swiss mice were kept in ventilated
racks (AlescoTM, Brazil), while CB17-SCID animals were maintained in well-ventilated
sterile cages (TecniplastTM, Germain), according to international standards for
production and maintaining of germ-free animals. All animals were housed under
standard conditions of light (12:12 hlight/dark cycle) and temperature (22 ± 1°C), with
access to sterile commercial rodent stock diet (Nutrilabor, Campinas, Brazil) and water
ad libitum. All procedures were approved by the Committee on Animal Research at
FIOCRUZ (#006/2015) and UFPI (#202/2016) and followed Brazilian (Colégio
Brasileiro de Experimentação Animal - COBEA) and International rules on the care
and use of experimental animals (Directive 2010/63/EU of the European Parliament
and of the Council on the protection of animals used for scientific purposes).
2.3 Cytotoxicity analysis
The cytotoxic action was assessed by colorimetric assays after 72 h exposure.
Cell proliferation was determined spectrophotometrically using a multiplate reader
(DTX 880 Multimode Detector, Beckman Coulter). Control groups (negative and
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positive) received the same amount of solvent (DMSO 0.1%). Doxorubicin (0.005 - 5
μg/mL) was used as positive control.
2.3.1 Antiproliferative assays with human tumor cells
The cytotoxicity on HL-60, OVCAR-8, SF-295, HCT-116 and HEPG-2 was
determined by the MTT assay (Mosmann, 1983), which determines the ability of living
cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) to a purple formazan product. Line cells were plated in 96-well plates
(0.3-0.7 x 105 cells/well) and incubated to allow cell adhesion or equilibration
(suspension cultures). Twenty-four hours later, compoundswere added to each well
(0.2 - 10μg/mL). After 69 h of incubation, the supernatant was replaced with fresh
medium containing 10% MTT, and the cells incubated for an additional 3 h. The plates
were centrifuged, formazan product was dissolved in DMSO and absorbance was read
at 595 nm.
2.3.2 Antiproliferative study with human normal peripheral blood mononuclear cells
All compounds were also investigated on human PBMC using the Alamar
Blue™ assay. PBMC were washed and resuspended (3 x 105 cells/mL) in
supplemented RPMI-1640 medium plus 4% phytohemagglutinin for growth stimulation.
PBMC were then plated in 96-well plates (3 x 105 cells/well in 100 µL of medium). After
24 h, compounds dissolved in DMSO were added to each well (0.2 - 25μg/mL) and
cells were incubated for 72 h. Twenty-four hours before the end of the incubation, 20
μL of resazurin (Alamar Blue™) stock solution (0.156 mg/mL) (Sigma Aldrich Co.,
USA) were added to each well. The absorbance was read at 570 and 595 nm and the
drug effect was expressed as the percentage of the control (Ferreira et al., 2015).
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2.3.3 Hemolytic assay
Molecules were tested for hemolytic activity according to Santos et al. (2010)
at 250μg/mL in 96-well plates during 60 min at room temperature (25oC) using
suspension of human erythrocytes (2 % in 0.85 % NaCl containing 10 mM CaCl2).
After centrifugation, hemoglobin levels in the supernatants were determined at 540
nm.
2.4. Acute toxicity and behavior analysis
Taking into consideration to minimize pain and suffering as well as ensuring
the robustness and reproducibility of the experiments, it was adopted a methodology
recommended by the acute toxic class method - Guideline 423 – described in the
"Guideline for Testing of Chemicals" from OECD to evaluate the acute
toxicity(OECD, 2001). It described that testing in one sex (usually females) is now
considered sufficient because, although there is little difference in sensitivity between
the sexes, in those cases where differences are observed females are generally
slightly more sensitive. Since the compounds 2, 3 and 4 were not tested previously,
and their toxicity was not described yet, the initial dose administered to animals was
300 mg/kg. It is important to note that before administrations, all animals were
acclimatized for 5 days. Administrations were performed and mice were observed for
14 days. Negative groups received DMSO 5% in distilled water since compounds
were dissolved in pure DMSO.
Following the administration, the animals were fed restricted for 2 hours and
observed after 60 min and 24 h. Thereafter, animals were observed daily until the
14th day. According to the daily Hippocratic screening, the following signs were
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assessed: general activity, irritability, touch response, response to tail clamping,
writhing, righting reflex, grip strength, auricular reflex, corneal reflex, tremors,
convulsions, ptosis, piloerection, cyanosis, and death. It was also evaluated the
variation of body weight, food and water consumption, and production of excretions
(urination and defecation) using metabolic cages (Lucio et al., 2000). Due to the
occurrence of deaths, it was proceeded a new administration at lower doses
according to OECD 423. Doses observed the limit of 0.1 mL/10 g of body weight.
Range of the LD50 was estimated according to the Globally Harmonized System
(GHS) (Brazil, 2013).
For behavior investigations, experiments were performed as described below.
Diazepam (2 mg/kg, oral by gavage) was administered as standard drug. Doses
were chosen base on the toxicity studies and OECD guidelines. After each animal,
the cleaning of the field was performed with a paper towel humidified with alcohol 70
% to remove excreta left by prior animals. All analyzes were conducted with each
animal singly.
2.4.1 Open field test
The exploratory activity was verified using an open field made of acrylic
(transparent walls and black floor, with dimensions of 30 x 30 x 15 cm) divided into 9
quadrants and based on the model described by Archer (1973) and Araújo et al.
(2017). Thirty minutes after the treatment, animals were placed in the center of the
open field. Afterwards, the number of intersections or crossings with four legs
(spontaneous locomotor activity - SLA), number of self-cleaning behavior (grooming)
and number of lifting without lean against the walls (rearing) were accounted for 5
minutes.
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2.4.2 Rota rod test
The test route rod assesses the degree of muscle relaxation or motor
incoordination induced by bioactive substances (Araújo et al., 2017). Each mouse
was placed with all four feet onto a bar of 2.5 cm diameter, 25 cm high from the floor,
in a rotation of 17 rpm for a period of 3 minutes. The duration of permanence in the
swivel bar, in seconds (s), and the number of falls, with three renewals at maximum,
was recorded.
2.4.3 Elevated plus maze test
The elevated plus maze apparatus consists of two open arms (30 x 5 cm) and
two closed arms (30 x 25 x 5 cm) crossed perpendicularly. In this frame, the animal is
placed 60 cm above the ground exactly on the intersection of the arms (central
platform, 5 x 5 cm) with its head turned to the entry of closed arms (Lister, 1987). The
animals were placed on the intersection of the arms 30 min after treatment and
observed for 5 minutes. The parameters quantified in this test were number of entries
into the open arms (NEOA) and time spent in the open arms (TSOA).
2.5 In vivo xenograft assay with human colon carcinoma
HCT-116 cells were maintained in supplemented RPMI-1640 medium,
counted in Neubauer chamber and subcutaneously implanted into the left hind
axillary of CB-17mice (2 × 107 cells/mL/animal). On the next day, animals were
randomly divided into five groups (n=12 each) and the substances (2, 3 and 4)
dissolved in DMSO 5% were intraperitoneally administered for 15 days at25
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mg/kg/day. Negative and positive controls received DMSO 5% (i.p.) and 5•FU (15
mg/kg/day, i.p.), respectively.
On the 16thday, animals were anaesthetized with ketamine (90 mg/kg) +
xylazine (4.5 mg/kg) for blood collection from each animal via retrorbital plexus
(Waynforth, 1980) using sterile tubes and heparinize pipettes to determine the profile
of circulating peripheral leukocytes and analyzed at 400x magnification in May-
Grünwald-Giemsa-stained blood smears (two per animal) to obtain differential
amount of white blood cells (WBC).The absolute count of a leukocyte subtype was
calculated as the product of its respective differential percentage and total leukocyte
count (Biermann et al., 1999).
Afterwards, all mice were sacrificed by cervical dislocation and tumors, livers,
kidneys, hearts and lungs were dissected out, weighed and fixed in 10%
formaldehyde for examination of size, color changes and hemorrhages. The inhibition
ratio of tumor growth (%) was calculated as follows: inhibition ratio (%) = [(A - B)/A] x
100, where A is the average tumor weight in the negative control, and B is the
average in each separately treated group.
2.6 Evaluation of intestinal motility
Female Swiss mice were randomly divided into 6 groups (n = 8
animals/group): negative control (DMSO 5%, i.p.), positive control for increased
intestinal transit (Bisacodil, 10 mg/kg, oral), positive control for reduced intestinal
transit (Atropine sulfate, 5 mg/kg, i.p.) and the substances (2, 3 and 4) (50 mg/kg,
i.p.). Thirty minutes later, animals received 0.3 mL of activatedcharcoal10 % in
carboxymethylcellulose1.5 % by gavage. After additional 30 min, mice were
euthanized by dislocation cervical and the small intestines were withdrawn (from the
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pylorus to the beginning of the cecum). Outcomes were expressed as a percentage
of the total length of the small intestine (Harrison et al., 2004) as follows: Intestinal
Transit (%) = Distance journeyed by activated carbon/Total length of small intestine x
100.
2.7 Statistical analysis
Values of IC50 and their 95% confidence intervals were obtained by nonlinear
regression using the GraphPad program (Intuitive Software for Science, San Diego,
CA). Differences were evaluated by comparing data using one-way analysis of
variance (ANOVA) followed by the Newman-Keuls test (p < 0.05). All in vitro studies
were carried out in duplicate and represent independent biological evaluations.
3 Results
3.1 Acute toxicity and behavioral changes
The compound 2 reduced grabbing strength and overall activity at 150 mg/kg
and increased defecation, decreased general activity and grabbing strength, and
presence of palpebral ptosis at the highest dose (300 mg/kg) (Table 1). Two animals
died at 300 mg/kg after 24 h. Compound 3, which has bromine in its chemical
structure, decreased overall activity (apathy and absence of body tonus), and all
animals died at 300 mg/kg after 24 h exposure. At 150 mg/kg, death was not
detected but decreasing of general activity was obvious. Compound 4 diminished
general activity, corneal reflex and grabbing strength, and increased urination and
defecation at 300 mg/kg, and one death was noticed. At 150 mg/kg, death was not
detected but decreasing of overall activity and grabbing strength were apparent.
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Insert Table 1 here
The open field test (Fig. 1) revealed reduction in the number of crossings after
administration of compound 3 at 150 mg/kg (23.8 ± 6.8) and compound 4 at 300
mg/kg (18.8 ± 4.3) when compared to the negative group (41.3 ± 6.9 crossings) (p <
0.05).
Insert Figure 1 here
The elevated plus maze, an experimental model used to investigate the
modulation of anxiety status and exploratory activity of animals, showed that
compounds 2 at 300 mg/kg and 4 at 150 and 300 mg/kg increased TSOA (179.0 ±
16.7, 196.2 ± 15.3 and 269.0 ± 10.4 s) and reduced TSCA (121.0 ± 17.0, 103.8 ±
15.3 and 38.8 ± 9.0 s) in a similar way displayed with diazepam (199.6 ± 27.3 s and
125.5 ± 14.0 s), if compared to the negative control (100.2 ± 8.4 e 199.8 ± 8.4 s),
respectively (p< 0.05, Table 2). Moreover, the compound 4 changed NEOA for
values (1.0 ± 0.3 entries) lower than those seen in the negative control group (3.6 ±
0.4 entries, p< 0.05). Meanwhile, the total number of entries was reduced by
compounds 3 (150 mg/kg: 3.2 ± 1.5) and 4 (2.2 ± 0.5 entries, p < 0.05).
Insert Table 2 here
Compounds 2 (300 mg/kg: 2.8 ± 0.2) and 4 (150 mg/kg: 2.4 ± 0.2; 300 mg/kg:
2.8 ± 0.2 falls) increased number of falls in the rota rod device (Table 3) when
compared to the negative control (1.0 ± 0.4) (p < 0.05), but only compound 2 at 300
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mg/kg diminished permanence time in the swivel bar for 46.8 ± 10.6 s, a value nearly
4-fold lower than that found for negative group (173.8 ± 2.9 s).
Deaths and/or behavior changes were noted with doses of 150 and 300
mg/kg, we also analyzed single doses at 50 mg/kg as suggested by OECD (2001). At
this dose, significant outcomes and deaths were not found when compared to
negative control (details not showed).
3.2 In vitro cytotoxicity on tumor and normal cells and toxicity
A series of 2-chloro-N-arylacetamides (1-10) was evaluated for their
cytotoxicity against five tumor cell lines. These compounds were obtained by the
reaction of ten para-substituted aniline derivatives with 2-chloroacetic anhydride at
room temperature (Lavorato et al., 2017). With exception of compounds 6 and 9, all
compounds displayed IC50 values ranging from 4.9 to 50.1 μM (Table 3). Compound
2 (R = Cl) was the most active against three of the five tumor cell lines studied in this
work (HL-60, SF295 and HCT-116 cell lines). Additionally, compounds 3 (R = Br) and
4 (R = NO2) were the only active substances against the most resistant cell line SF-
295.
Interestingly, the compounds did not cause significant in vitro cytolytic action
against human erythrocytes until the concentration tested (250μg/mL). On the other
hand, most molecules were also cytotoxic on proliferating human PBMC, and IC50
values ranged from 3.4 to 7.6 μg/mL (Table 3).
Insert Table 3 here
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Physicochemical data, including the influence of van der Waals volume
(vdWV), Hammet’s electronic constant (σ), lipophilicity, and dipole moment and their
relationship are described in Table 4 and Fig. 2, 3, 4 and 5. They are examined in the
Discussion section.
Insert Table 4 here
Insert Figure 2, 3, 4 and 5 here
3.3 In vivo antitumor action and subacute effects
Compounds 2, 3 and 4 were tested for in vivo antitumor activity using a
xenograft model of colon carcinoma. Only compounds 2 and 3significantly reduced
tumor growth in 21.2 % (0.57 ± 0.02 g) and 27.5 % (0.53 ± 0.04 g) at 25 mg/kg/day
(Table 5)when compared to the negative control (0.73 ± 0.04 g respectively, p <
0.05). Comparably, 5-FU, as positive control, also cause tumor reduction.
Insert Table 5 here
Deaths or changes in relative weight of organs and in hematological
parameters were not detected in arylacetamides-treated animals (p >0.05) but livers
increased in 5-FU-treated mice (6.18 ± 0.39 g, p < 0.05) in comparison with negative
control animals (4.95 ± 0.14 g) (Table 6). Similarly, only animals receiving 5-FU
revealed decreasing of erythrocytes, leukocytes, hemoglobin content and hematocrit
levels (p < 0.05, Table 6).
Insert Table 6 here
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The compounds 2, 3 and 4 were not able to cause diarrhea. Moreover, they
did not alter the intestinal transit (71.1 ± 2.3, 57.9 ± 4.6 e 72.2 ± 4.9 %, respectively)
when compared to the negative group (67.3 ± 4.0 %). On the other hand, Bisacodil
increased the distance travelled by activated charcoal for85.1 ± 3.7 % and atropine
(muscarinic blocker) reduced the intestinal transit for 39.8 ± 4.7 % (p < 0.05).
4. Discussion
The interest in molecular modeling, combinatorial chemistry and other
techniques of chemical synthesis in medicinal chemistry is responsible for the newest
therapeutic agents against parasites and cancers, and to treatinflammatory,
neurodegenerative and sensory disorders(Soares et al., 2009; Oliveira et al., 2013;
Ferreira et al., 2015; Lopes et al., 2015; Araújo et al., 2016; Almeida et al., 2017).
Despite some studies relating bioactivity of synthetic arylacetamides, this is the first
work focusing on the antiproliferative properties of 2-chloro-N-arylacetamides.
Subsequently, we also performed in vivo preclinical evaluation about their
toxicological and antitumoral capacity using xenograft model of colon carcinoma.
Herein, synthetic arylacetamides presented different antiproliferative activity
according to the tumor cell line tested and the nature of the aryl substituent at the
para position. Compound 6 (R = OCH3) and 9 (R = COOH) were considered inactive
against all cell lines they were evaluated whereas only compounds 3 (R = Br) and 4
(R = NO2) were active against all tumor cells. We also investigated the role of several
physicochemical properties of the evaluated compounds, like lipophilicity, dipole
moment and van der Waals volume, as well as electronic effects of the aryl
substituent in antitumor potential of this chemical class. To this analysis, IC50 values
in micromolar (µM) were used.
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Although we could not clearly establish the effect of aryl substituent to
antiproliferative activity of 2-chloro-N-arylacetamides, some aspects should be
highlighted. The unsubstituted compound 1 was one of the least active compounds,
indicating that the presence of a substituent at para position is a contributing factor to
activity. Among para-substituted compounds, only 2 (R = Cl), 3 (R = Br) and 4 (R =
NO2) were active against glioblastoma SF-295 cells. These compounds present van
der Waals volumes ranging from 157.61 to 166.65 Å3. This may indicate that volume
occupied by such compounds can be an important feature to the cytotoxic activity.
Nevertheless, based only in this parameter, we would expect compound 5 (R = CH3),
whose van der Waals volume is 160.45 Å3, it would be also active on SF-295 line.
So, besides steric aspects, it is likely that the electronic effect of the aryl substituent
may also interfere on the activity against this cell line. The electronic effect of a
substituent can be measure by its Hammet’s electronic constant (σ). A positive σ
indicates that the substituent is an electron-withdrawing group, whereas a negative σ
is related to an electron-donating group (Tavares et al., 2004). According to their σ
values, chloro, bromo and nitro play an electron-withdrawing effect, whereas methyl
group plays an electron-donating effect, which can contribute to explain the
bioactivity differences of these compounds. Thus, this information indicates that the
compounds with an electron-withdrawing substituent present
antiproliferative activity against glioblastoma SF-295 cells.
a
potential
The antiproliferative activity of 2-chloro-N-arylacetamides against OVCAR-8
cell line can also be partially explained by Hammet’s electronic constant of ring
substituents (σ). We observed that the most potent compound against OVCAR-8 cell
line, compound 4 (R = NO2), presents a substituent with the highest and positive σ of
the series, indicating its high electron-withdrawing character. On the other hand, the
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least active compound 1 (R = H) have no substituent, so the σ value attributed to
hydrogen is zero. Accordingly, we would expect compound 8 (R = SO2NH2), with the
second highest σ value (0.57), to be more active against tumor lines. However,
outcomes frustrate this possibility. Probably, this occurred because this compound
has the lowest lipophilicity among active compounds (ClogP = 0.35). A compound
with low lipophilicity is less able to cross cell membranes, which reduces its ability to
act into tumor cells (Rutkowska et al., 2013). Although possessing a lower value of σ
for bromine, compound 3 (R= Br) present higher cytotoxic potential than 8 (R =
SO2NH2), probably due to its higher ClogP. Indeed, we found a negative correlation
among IC50 values and σ e ClogP, as described in the following equation:
IC50 = - 9.2985 ClogP – 27.7010 σ + 59.8736 (n = 6; R = 0.901)
The correlation model indicates that both physicochemical properties can
influence the antiproliferative activity of these compounds against OVCAR-8 cells,
increasing the activity or reducing IC50 values as the values of these two parameters
increase. Although it is not a predictive model, it is in accordance with our qualitative
analysis of active compounds in the present study. The high electron-donating
character of methoxy group in compound 6and the low lipophilicity of 9 (R = COOH)
could explain the absence of activity of these compounds. However, based only on
the physiochemical characteristics, we could not find a logical reason for the
unexpected inactivity of compounds 2 (R = Cl) and 7 (R = COCH3) against this cell
line.
On the other hand, compound 2 (R = Cl) presented a potent antiproliferative
effect against HL-60 cells, with an IC50 significantly lower than the IC50 of the other
compounds. We were not able to propose any structure-activity relationship
regarding this cell line with the data available thus far. In this case, we cannot
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establish a correlation among the antiproliferative profile of active compounds against
HL-60 cells and their electronic and lipophilicity features. Compounds 4 (R = NO2), 7
(R = COCH3) and 8 (R = SO2NH2), with electron-deficient aromatic rings, present
different activity profiles, since 4 and 8 are almost twice active than 7. Compounds 4
and 8 are also more active than 3 (R = Br), which is the one whose physicochemical
properties presented in Table 4 most resemble the properties of compound 2, the
most active compound against this cell line. Although the lipophilicity of 2 can justify
its better activity compared to 4 and 8, the same property cannot explain why 3 has a
higher IC50 value than 5 (R = CH3), even though it has an electron-deficient aromatic
ring.
The electronic constant of the ring substituent (σ) seems also to affect the
antiproliferative activity of these compounds against HCT-116 cells. We observe a
tendency towards the increase of the activity as σ increases, i.e., as the ring
electronic density decreases. However, compounds 2 (R = Cl) and 3 (R = Br) do not
follow this trend. We believe that the high lipophilicity of 2 and 3, represented by their
high ClogP values, can be a positive contribution to their antitumor activity.
Regarding the activity against HepG2 cell line, the compound’s dipole moment
seems to influence the antiproliferative action of 2-chloro-N-arylacetamides,
increasing their activity as its value increases. Compounds 4 (R = NO2), 7 (R =
COCH3) and 8 (R = SO2NH2) present the highest values of dipole moment among the
evaluated compounds and were also the most active against HepG2 cells, whereas
compound 1 (R = H), the least active against HepG2, displayed the lowest dipole
moment.
In MTT assays, compound 9 (R = COOH) was inactive against all tumor cell
lines. Interestingly, compound 10 (R = COOCH2CH3), the ethyl ester of 9, was active
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against two lines only. Since bioassays were run at pH~7.4, compound 9 was in its
ionized form. Then, its antiproliferative potential may be reduced since it cannot
permeate membranes and reach inside tumor cells (Lima et al., 2007). If this is true,
the low antiproliferative activity of 10 may be explained by its in-situ hydrolysis and
conversion to 9.
The initial analysis of structure-activity relationship indicates that
physicochemical features work jointly to affect antitumor effects of this series. By the
way, the compounds that stood out in the studies – 2 (R = Cl), 3 (R = Br) and 4 (R =
NO2) – have lipophilic character and lower van der Waals volumes. These qualities
contribute to the ability to cross cell membranes and to accumulate inside tumor
cells. Moreover, these three compounds have electron-withdrawing aromatic
substituents and a dipole moment that contribute to accentuate electrophilic
properties of chloroacetamide derivatives. Since biological activities of this chemical
class have been attributed to the ability to alkylate nucleophiles, like protein and
nucleic acids (Jablonkai, 2003; Helleday et al., 2008; Singh et al., 2011; Swift and
Golsteyn, 2014), these findings suggest that the antiproliferative activity described
here may also be attributed to the same mechanism of action.
In the present work, only a small set of compounds was tested in order to
assess whether this chemical class has a potential use as antitumor agents. So, the
small dataset limits the performance of conclusive QSAR analysis. Moreover, the
antiproliferative activity was determined against tumor cells, not against an isolated
specific target. This could explain why we have not found a linear correlation
between the activity and a specific physicochemical property (Scior et al., 2009). In
this case, the activity-correlated properties lipophilicity and van der Waals volume are
likely to influence the antitumor action by affecting the ability of compounds to
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permeate cell membranes to reach this target. On the other hand, the properties
dipole moment and electronic effect of aromatic substituent may have a greater
influence on the compound recognition by macromolecules inside cell, its interaction
with its molecular target and its alkylating profile, directly affecting the biological
activity.
When assayed on human erythrocytes, none of the compounds showed
critical lytic action, suggesting cytotoxicity is not related to the direct action on cellular
membrane disruption. On the other hand, only compound 5 (R = CH3) was non-toxic
towards PBMC cells (IC50 > 25 µg/mL). So, most compounds did not reveal selective
cytotoxicity only upon tumor cells. Interestingly, 5 is the only evaluated compound
with an electron-donating aromatic substituent (Table 4), which contributes to reduce
the electrophilicity of the compound and consequently its ability to alkylate
bionucleophiles. This suggests that the cytotoxicity may also be modulate by the
electrophilic character of the compound in the same way as the antiproliferative
activity previously discussed.
Given that the toxic profile of a probable drug must be part of early steps for
the development of new medications (Brazil, 2013; Ferreira et al., 2015; Araújo et al.,
2018), we also analyzed the acute toxicity of the compounds that stood out to
determine toxic effects and to establish safe dosages for subsequent
pharmacological studies.
According to the guideline OECD 423 and based on the Globally Harmonised
System, compounds 2, 3 and 4 presented intermediate toxicity, since all of them are
classified in the Category 3 (LD50 value: > 50 mg/kg < 300 mg/kg) (OECD, 2001).
Indeed, studies have suggested that some 2-chloroacetamides present LD50 values
ranging from about 30 to 300 mg/kg of body weight, and that such toxicity apparently
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depends neither on routes of administration nor species-specific results (CDC, 2018;
National Library of Medicine, 2018).
In the hippocratic screening study, the most common events were reduction of
general activity and loss of grapping strength. These effects suggest central nervous
system depressant activity despite loss of the righting reflex had not been observed.
Such findings indicate these compounds may have central depressant action or
selective sedative activity. The loss of grapping strength is an indication for skeletal
muscle relaxant activity, which may be peripherally (at the neuromuscular junction) or
centrally located (Carlini and Burgos, 1979; Kanjanapothiet al., 2004; Araújo et al.,
2017). With this in mind, motor effects using a revolving bar were evaluated, and it
was detected that compounds 2 (by reducing time on the revolving bars and
increasing falls at 300 mg/kg) and 4 (by increasing falls at 150 and 300 mg/kg) have
a myorelaxant activity and cause light but significant psychomotor retardation. It is
likely that motor effects are associated with ataxia caused by some2-
Chloroacetamides (CDC, 2018).
Reduction ofcrossingswas noticed for compounds 3 and 4 suggesting
modifications in SLA of treated animals, but none of the three compounds altered the
number of groomings and rearings, indicating such compounds do not interfere in the
exploratory activity of mice subjected to open spaces nor affect the motor
coordination. On the other hand, compounds 2, 3 and 4 reduced the NECA and
compounds 2 and 4 increased TSOA (and reduced TSCA) in a very similar way seen
for diazepam (standard drug).These conclusions were found using the plus maze
test, which consists of a more specific anxiety model for the assessment of anxiolytic
drugs with capacity to reduce the rejection animals present to walk to the open arms,
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since this behavior is conditioned by the fear or stress in aversive environments (Walf
and Frye, 2007; Neumann et al., 2011).
In an overview, the acute behavioral signals more commonly associated with
compounds 2, 3 and 4 with were muscle relaxation and reduction of locomotor
activity in the revolving bar with consequent increase in the number of falls, decrease
of NECA and increase of TSOA, suggesting sedative actions. Such actions
surprisingly exhibit characteristics of anxiolytic properties related to the diazepam,
mainly for compound 2 (R = Cl) and 4 (R = Br). These behavioral data confirm the
reduction of general activity, coordination of the motor system (grabbing strength)
and muscle tonus from Hippocratic screening tests (Walf and Frye, 2007; Almeida et
al., 2012).
Ash (2004) demonstrated that the LD50 of some 2-chloroacetamides on mice is
around 100mg/kg body weight, which could justify, at least in part, the presence of
toxicity signs at 150mg/kg. Behavioral effects analyzed in glyphosate-based
herbicide-treated mice showed impairment effects upon the central nervous system
probably due to alterations in neurotransmission pathways that participate or regulate
locomotor activity, anxiety and memory involving GABAergic, dopaminergic,
serotonergic and/or cholinergic systems (Baier et al., 2017).
Benzodiazepines, as diazepam and alprazolam, present a range of actions –
sedative/hypnotic, anxiolytic, anticonvulsant and muscle relaxant – and most of them
were found in arylacetamides-treated animals. It is likely such synthetic compounds
do not activate GABAA receptors directly but, instead, are positive allosteric
modulators of the effects of GABA and allow lower concentrations of this
neurotransmitter to open the Cl- channels, like most benzodiazepines. As a
consequence of the enhancement of GABA’s inhibitory activity caused by
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benzodiazepines, the brain’s output of excitatory neurotransmitters including
norepinephrine, serotonin, dopamine and acetylcholine is reduced (Sigel and
Steinmann, 2012; Miller and Aricescu, 2014). Further pharmacological investigations
are in progress to confirm the mechanism(s) of action because this was not the main
focus of this research.
Based on the absence of toxicity with doses of 150 mg/kg and considering that
a drug has a good safety profile if its therapeutic index exceeds the value of 8-10
(Tamargo et al., 2015), we elected the dose of 25 mg/kg/day for efficacy assays. As
in vivo models, xenograft animals are technically represented by athymic nude mice,
severely compromised immunodeficient (SCID) mice. They have been extensively
used to monitor tumorigenicity and tumor growth, can simulate the complexity of
genetic and epigenetic abnormalities in human tumors and aid in the development of
individualized molecular approaches (Morton and Houghton, 2007; Jung et al., 2014).
In the present study, we chose colon carcinoma as preclinical cancer model
due to its epidemiological importance: a) colorectal neoplasm is the third mostly
common occurring cancer in the world; b) causes about 694,000 deaths a year (10 %
of all cancer deaths); c) presents wide geographical variation in incidence with rates
varying ten-fold in both sexes, and d) 95 % of them are adenocarcinomas, especially
in countries characterized by high or very high indices of development and/or income
(two thirds of cases), which strongly demonstrates how certain lifestyle factors affect
the risk of developing colorectal carcinomas (Ferlay et al., 2014; Mármol et al., 2017).
We showed that compounds 2 and 3 significantly reduced colon carcinoma
tumor growth. We believe that their higher lipophilicities, represented by high Clog P
values, has positive (but partial) role in this antitumor activity. Furthermore, the
electronic constant of the ring substituent (σ) seems also to affect the antiproliferative
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activity of the series against in vitro HCT-116 cells, being observed a tendency
towards the increase of bioactivity as σ increases, i.e., as the ring electronic density
decreases. However, compounds 2 (R = Cl) and 3 (R = Br) do not follow this trend,
and in vivo tumor inhibition rates were equivalent (p> 0.05).
Some 2-chloro-N-arylacetamides behave as alkylating agents, especially
those molecules containing sulfhydryl groups (Jablonkai and Hatzios, 1991;
Jablonkai, 2003). It is also worth to note that classical classes of antitumor
molecules, like nitrogen mustards, nitrosoureas and quinones causes cell death
clearly associated with electronic and lipophilic properties (Hansch et al., 1972;
Denny and Wilson, 1986; Gourdie et al., 1990; Driebergen et al., 1993).
Compounds tested in vivo neither caused macroscopical/morphological
damage on key organs, affected intestinal transit nor altered figurative elements of
the peripheral blood, which suggest that subacute exposure for 15 days at
25mg/kg/day neither promoted direct action on blood cells/hematopoiesis nor
showed target organ toxicity, a great advantage if we take into consideration that
most antineoplasic drugs clinically available cause myelosuppression (anemia,
leucopenia with neutropenia), hepatotoxicity, diarrhea, and alopecia, besides
cardiotoxicity, opportunistic infections, peripheral neuropatia, nausea, vomiting,
anorexia, fatigue and tiredness (Carlotto et al., 2013; Ferreira and Pessoa,
2017;Rapoport, 2017;Nurgali et al., 2018).
In summary, most of 2-chloro-N-arylacetamide derivatives showed moderate
to high antiproliferative action on tumor lines, cell toxicity on normal dividing
leukocytes, and in vivo investigations demonstrated diazepam-like anxiolytic
properties of compounds 2 and 4, including muscle relaxation and reduction
ofspontaneous locomotor activity, general mobility and muscle tonus.
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Physicochemical characters as van der Waals volume, molecular dipole moment,
lipophilicity and electronic constant may work together to alter the anticancer
capacity, but the evaluation of more compounds of this chemical class is imperative
to better understanding their structure-activity relationship. Compounds 2 and 3
reduced tumor growth in xenograft colon carcinoma-bearing mice without causing
apparent signals of organ-specific toxicity after subacute exposure. The structural
chemical simplicity of these arylacetamides make them cost-effective alternatives
and justifies further improvements to enhance activity and selectivity and the
development of pharmaceutical formulations.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This study was financed in part by the Brazilian agency “Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior” – Brasil (CAPES) – Finance code
001. This study was financed in part by the Brazilian agency “Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior” – Brasil (CAPES) – Finance code
001. Dr. Paulo Michel Pinheiro Ferreira is grateful to the Conselho Nacional de
Desenvolvimento Científico e Tecnológico” [CNPq (#305086/2016-2)] for the
personal scholarship.
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