A new synthetic resorcinolic lipid 3-Heptyl-3,4,6-trimethoxy-3H- isobenzofuran-1-one: Evaluation of toxicology and ability to potentiate the mutagenic and apoptotic effects of cyclophosphamide

Article abstract of DOI:10.1016/j.ejmech.2014.01.057

Resorcinolic lipids have important biological actions, including anti-carcinogenic activity. Therefore, we evaluated the mutagenic, genotoxic, immunomodulatory and apoptotic potential and the biochemical and histopathological changes caused by the synthetic resorcinolic lipid 3-Heptyl-3,4,6-trimethoxy-3H-isobenzofuran-1-one, (AMS35AA; 10, 20 and 40 mg/kg) alone or in combination with cyclophosphamide (100 mg/kg) in Swiss mice. The results indicated that AMS35AA is not genotoxic or mutagenic and does not alter liver or kidney histology. However, the compound does cause an increase (p < 0.05) in the levels of glutamic-oxaloacetic transaminase and creatinine and in splenic phagocytosis and liver and kidney apoptosis. When combined with cyclophosphamide, AMS35AA caused increased (p < 0.05) mutagenic damage (although the compound had anti-genotoxic activity), splenic phagocytosis, neutropenia and glutamic-oxaloacetic transaminase and creatinine levels (even in the absence of histological damage) and induced liver and kidney apoptosis. We conclude that this resorcinolic lipid may be an important chemotherapy adjuvant that can potentiate mutagenic damage and increase apoptosis caused by cyclophosphamide without causing adverse effects. In addition, the immunomodulatory activity of the compound should be noted, which counters reductions in lymphocyte number, a primary side effect of cyclophosphamide in cancer therapy.

Full text of DOI:10.1016/j.ejmech.2014.01.057

European Journal of Medicinal Chemistry 75 (2014) 132e142
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A new synthetic resorcinolic lipid 3-Heptyl-3,4,6-trimethoxy-3H-
isobenzofuran-1-one: Evaluation of toxicology and ability to
potentiate the mutagenic and apoptotic effects of cyclophosphamide
,
,
,d
Stephanie Dynczuki Navarro a b, Adilson Beatriz b c, Alisson Meza c, João Renato Pesarini a
Roberto da Silva Gomes c, Caroline Bilhar Karaziack c, Andréa Luiza Cunha-Laura e,
Antônio Carlos Duenhas Monreal e, Wanderson Romão f, Valdemar Lacerda Júnior f,
,
Mariana de Oliveira Mauro a g, Rodrigo Juliano Oliveira a
,
,b,d,
*
a Centro de Estudos em Células Tronco, Terapia Celular e Genética Toxicológica e CeTroGen, Núcleo de Hospital Universitário e NHU,
Universidade Federal de Mato Grosso do Sul e UFMS, Campo Grande, MS, Brazil
b Programa de Mestrado em Farmácia, Centro de Ciências Biológicas e da Saúde e CCBS, Universidade Federal de Mato Grosso do Sul e UFMS,
Campo Grande, MS, Brazil
c Instituto de Química, Universidade Federal de Mato Grosso do Sul e UFMS, Campo Grande, MS, Brazil
d Programa de Pós-graduação em Saúde e Desenvolvimento na Região Centro-Oeste, Faculdade de Medicina “Dr. Hélio Mandetta” e FAMED,
Campo Grande, MS, Brazil
e Centro de Ciências Biológicas e da Saúde e CCBS, Universidade Federal de Mato Grosso do Sul e UFMS, Campo Grande, MS, Brazil
f Departamento de Química, Universidade Federal do Espírito Santo e UFES, Vitória, ES, Brazil
g Programa de Doutorado em Biotecnologia e Biodiversidade e Rede Pró Centro-Oeste, Universidade Federal de Mato Grosso do Sul,
Campo Grande, MS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Resorcinolic lipids have important biological actions, including anti-carcinogenic activity. Therefore, we
evaluated the mutagenic, genotoxic, immunomodulatory and apoptotic potential and the biochemical
and histopathological changes caused by the synthetic resorcinolic lipid 3-Heptyl-3,4,6-trimethoxy-3H-
isobenzofuran-1-one, (AMS35AA; 10, 20 and 40 mg/kg) alone or in combination with cyclophosphamide
(100 mg/kg) in Swiss mice. The results indicated that AMS35AA is not genotoxic or mutagenic and does
not alter liver or kidney histology. However, the compound does cause an increase (p < 0.05) in the levels
of glutamic-oxaloacetic transaminase and creatinine and in splenic phagocytosis and liver and kidney
apoptosis. When combined with cyclophosphamide, AMS35AA caused increased (p < 0.05) mutagenic
damage (although the compound had anti-genotoxic activity), splenic phagocytosis, neutropenia and
glutamic-oxaloacetic transaminase and creatinine levels (even in the absence of histological damage)
and induced liver and kidney apoptosis. We conclude that this resorcinolic lipid may be an important
chemotherapy adjuvant that can potentiate mutagenic damage and increase apoptosis caused by
cyclophosphamide without causing adverse effects. In addition, the immunomodulatory activity of the
compound should be noted, which counters reductions in lymphocyte number, a primary side effect of
cyclophosphamide in cancer therapy.
Received 12 July 2013
Received in revised form
27 January 2014
Accepted 29 January 2014
Available online 30 January 2014
Keywords:
Immunomodulation
Micronucleus
Comet assay
Cytosporone
Chemotherapy
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
molecular and in vivo experimental studies, can contribute to un-
derstanding the activity of a given compound. Mutagenicity assays
The study of compounds that can interfere with processes
responsible for the development of cancer has been an important
trend in current research. Many approaches, including in vitro
are an important starting point to evaluate compounds that can
cause DNA damage, particularly those compounds with few reports
available [1]. Once the interactions between the compound and
DNA are known, designing and directing studies aimed at pre-
venting and/or treating cancer becomes possible.
Non-isoprenic resorcinolic lipids are agents that could poten-
tially prevent and repair DNA damage [2]. These compounds are
produced by bacteria, protozoans, algae, fungi, plants and animals.
* Corresponding author. Faculdade de Medicina, Universidade Federal do Mato
Grosso do Sul, Cidade Universitária, S/N, 79070-900 Campo Grande, MS, Brazil.
E-mail address: rodrigo.oliveira@ufms.br (R.J. Oliveira).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.057

S.D. Navarro et al. / European Journal of Medicinal Chemistry 75 (2014) 132e142
133
The general chemical structure of non-isoprenic resorcinolic lipids
consists of a long carbon chain (saturated or unsaturated) linked to
a dihydroxybenzene ring (resorcinol ring), with one or both hy-
droxyls methylated [3]. Because the carbon chain and resorcinol
ring have opposite polarities, these lipids can have amphiphilic
properties [4,5]. Therefore, the lipids can exert a variety of biolog-
ical activities [6], including interactions with phospholipid bilayers
[7], liposome formation [8], protection against oxidative stress [2]
and inhibition of bacterial [9e11] and tumor cell growth [12,13].
In addition, the resorcinolic lipid cytosporone B has recently been
shown to be able to interact with the orphan nuclear receptor
Nur77 and thereby trigger cancer cell apoptosis [14,15].
as white solid. M.p. ¼ 85 ꢀC. Yield 93%. 1H NMR (CDCl3, 300 MHz).
d
(ppm): 6.90 (d, J ¼ 3 Hz,1H); 6.67 (d, J ¼ 3 Hz,1H); 5.77 (t, J ¼ 9 Hz,
1H); 3.92 (s, 3H); 3.85 (s, 3H); 2.42 (q, J ¼ 9 Hz e 6 Hz. 2H); 1.28 a
1.54 (m, 8H) e 0.87 (t, J ¼ 6 Hz, 3H). 13C NMR (CDCl3, 75 MHz).
d
(ppm): 14.07 (CH3); 22.59 (CH2); 26.20 (CH2); 29.04 (CH2); 29.48
(CH2); 31.85 (CH2); 55.77 (CH3); 55.95 (CH3); 98.18 (CH); 105.15
(CH); 112.55 (CH); 121.68 (C); 127.17 (C); 143.94 (C); 155.40 (C);
162.07 (C) and 171.53 (C). ESI(þ)-FT-ICR MS provides an unambig-
uous molecular formula (M) of C17H22O4, where ions [MþH]þ and
[MþNa]þ with m/z of 291.1592 and 313.1410, respectively, are
identified. Both have DBE ¼ 7 and error ꢂ0.37 and ꢂ0.32 ppm.
The goal of this study was to evaluate the mutagenic, genotoxic,
immunomodulatory and apoptotic potential and the biochemical
and histopathological changes caused by 3-Heptyl-3,4,6-
trimethoxy-3H-isobenzofuran-1-one (Compound 4, AMS35AA,
Fig. 1), a new synthetic resorcinolic lipid that belongs to the cyto-
sporone family, alone or in combination with cyclophosphamide in
Swiss mice.
2.1.2. Synthesis of the resorcinolic lipid 4 (3-Heptyl-3,4,6-
trimethoxy-3H-isobenzofuran-1-one)
To synthesize the compound, 0.432 mmol (125.0 mg) of ftalide 3
was dissolved in 20 mL of methanol with 0.449 mmol (17.0 mg) of
NaBH4 and 0.2 mL of distilled water. The mix was stirred for 72 h at
room temperature, then 0.2 mL of glacial acetic acid was added. The
methanol was evaporated under reduced pressure. Ten milliliters of
distilled water were added to the product, and extraction was
performed with ethyl acetate (2 ꢁ 20 mL). The organic phase was
dried with MgSO4, and the solvent was evaporated. Two products
(compounds 4 and 5) were detected by TLC (6 elutions with the
hexane/AcOEt, 10:1, v/v). The products were separated by liquid
chromatography using a column packed with silica gel of the flash
type, with hexane/AcOEt (10:1, v/v) as the eluent.
2. Materials and methods
2.1. Chemistry
1H and 13C NMR spectra were obtained at 300 and 75 MHz,
respectively, using
a Bruker Avance DPX-300 spectrometer.
Compound 4. Yellowish oil; 42.7 mg (0.133 mmol), 31% yield; 1H
Chemical shifts are reported relative to TMS; coupling constants are
provided in hertz. Infrared (IR) spectra were recorded on a Bomen
FT-IR-MB100 Spectrometer. DBE1 and m/z values were confirmed
from ultra-high resolution and accuracy mass spectrometer (model
9.4 T Solarix, Bruker Daltonics, Bremen, Germany), operated in
positive ionization mode (ESI(þ)) over a mass range of m/z 200e
2000. Mass spectra are shown in Supplementary material, Fig. S1.
Purifications were performed by flash chromatography using silica
from Merck. All compounds and reagents were commercially ac-
quired from Acros, Synth and Merck.
NMR (CDCl3, 300 MHz).
d
(ppm)- 0.81 (t, J ¼ 6.7 Hz, 3H); 1.18 (m,
10H); 2.06 ppm (m, 1H); 2.25 (m, 1H); 3.04 (s, 3H); 3.85 (s, 3H); 3.86
(s, 3H); 6.66 (d, J ¼ 1.8 Hz, 1H); 6.88 (d, J ¼ 1.8 Hz, 1H). 13C NMR
(CDCl3, 75 MHz).
d (ppm): 14.0 (CH3); 22.6 (CH2); 23.1 (CH2); 29.0
(CH2); 29.3 (CH2); 31.6 (CH2); 36.8 (CH2); 51.2 (CH3); 55.9 (CH3);
55.9 (CH3); 98.8 (CH); 104.9 (CH); 111.2 (C); 126.2 (C); 131.0 (C);
155.5 (C); 163.6 (C); 168.4 (C). IR (KBr pellet). nmax/cmꢂ1: 2955,
2928, 2851,1771. EI-MS fragments: 322 (Mþ.), 291, 281, 253, 223
(base peak), 207, 195, 165, 150, 135,106, 77, 55, 45. ESI(þ)-FT-ICR MS
provides M of C18H26O5, where ions [MþH]þ, [MþNa]þ and [MþK]þ
with m/z of 323.1854, 345.1673 and 361.1412, respectively, are
identified. Both have DBE ¼ 6 and mass error ꢂ0.22, ꢂ0.13 and
ꢂ0.11 ppm.
2.1.1. Synthesis of ftalide 3 (3-Heptylidene-4,6-dimethoxy-3H-
isobenzofuran-1-one)
To a solution of the acid 1 (55 mmol) in dichloromethane
(75 mL), cooled to 0 ꢀC, was added AlCl3 (110 mmol) and octanoyl
chloride (55 mmol). The mixture was stirred for 6 h at room tem-
perature. Aqueous solution of HCl 18% was poured to the mixture to
consume the remaining AlCl3. The resultant mixture was trans-
ferred to a dropping funnel and extracted with dichloromethane
(4 ꢁ 20 mL). The organic layer was washed with aqueous solution of
NaOH 5% (2 ꢁ 15 mL), water (2 ꢁ 15 mL), brine (3 ꢁ 15 mL), dried
over MgSO4 and the solvent was evaporated at reduced pressure
and the resulting material was submitted to flash chromatography
using ethyl acetate:n-hexane (1:10) as eluent to afford compound 3
Compound 5. (3,5-Dimethoxy-2-octanoyl-benzoic acid methyl
ester). Yellowish oil; 55.5 mg (0.164 mmol), 40% yield; 1H NMR
(CDCl3, 300 MHz).
d
(ppm)- 0.85 (t, J ¼ 6.7 Hz, 3H); 1.29 (m, 8H);
1.72 ppm (m, 2H); 2.74 (t, J ¼ 7.5 Hz, 2H); 3.79 (s, 3H); 3.82 (s, 3H);
3.82 (s, 3H); 6.60 (d, J ¼ 2.1 Hz, 1H); 7.00 (d, J ¼ 2.1 Hz, 1H). 13C NMR
(CDCl3, 75 MHz).
d (ppm)- 14.1 (CH3); 22.6 (CH2); 23.5 (CH2); 29.1
(CH2); 29.2 (CH2); 31.8 (CH2); 44.3 (CH2); 52.5 (CH3); 55.7 (CH3);
56.0 (CH3); 103.0 (CH); 105.3 (CH); 127.1 (C); 129.4 (C); 157.1 (C);
160.6 (C); 166.4 (C); 205.8 (C). IR (Nujol oil). nmax/cmꢂ1: 1601, 1724,
2854, 2924, 2951. EI-MS fragments: 322 (Mþ.), 313, 291, 263, 238,
223 (base peak), 195, 165, 150, 135, 106, 77, 55. ESI(þ)-FT-ICR MS
provides M of C18H26O5, where ion [MþK]þ with m/z of 361.1412,
DBE ¼ 6 and mass error ꢂ0.59 ppm is detected.
2.2. Chemical agents, animals and experimental design
A single dose of 100 mg/kg body weight (bw) cyclophosphamide
(FosfaseronÒ, Ítaca Laboratories, REG. M.S. No. 1.2603.0056.002-1;
Batch 063020, Brazil) diluted in physiological solution (0.9 g NaCl;
100 mL H2O) was administered by intraperitoneal (ip) injection as
the positive control.
Fig. 1. Chemical structure of AMS35AA (3-Heptyl-3,4,6-trimethoxy-3H-isobenzofuran-
1-one).
The resorcinolic lipid 4 (AMS35AA) was diluted first in ethanol
(1%) and then in Milli-Q water, then administered at the 10 mg/kg
(bw, ip) dose suggested by Chen [16] and two additional doses (20
and 40 mg/kg bw, ip).
1
DBE (double bond equivalent) ¼ c ꢂ h/2 þ n/2 þ 1, where c, h, and n are the
number of carbon, hydrogen, and nitrogen atoms in the molecular formula.

134
S.D. Navarro et al. / European Journal of Medicinal Chemistry 75 (2014) 132e142
Eighty-five male, 8-week-old, Swiss mice (Mus musculus) were
harvesting. For the second set, the animals were anesthetized
(50 mg/kg of ketamine and 10 mg/kg of xylazine as intramuscular
injection), and blood was immediately collected for the biochem-
ical assays by exsanguination through the orbital sinus [17]. The
animals were then euthanized by cervical dislocation for organ
harvesting.
obtained from the Central Animal Facility of the Center for Bio-
logical and Health Sciences from the Federal University of Mato
Grosso do Sul and divided into 2 groups for the biological assays
(Fig. 2). The animals were housed in propylene boxes covered with
wood shavings in ventilated shelves under a 12-h light/12-h dark
photoperiod at 22 ꢃ 2 ꢀC and 55 ꢃ 10% relative humidity. The an-
imals were fed commercial feed (NuvitalÒ) and filtered water ad
libitum. The experiment was conducted according to the Universal
Declaration of Animal Rights and with the approval of the UFMS
Ethics Committee on Animal Use (Protocol No. 399/2012).
The animals were divided into the following experimental
groups (Fig. 3):
2.3. Biological assays
2.3.1. Comet assay
The comet assay was performed according to Singh [18] and
modifications by Oliveira [19]. Twenty microliters of peripheral
blood was homogenized with 120 mL of LMP agarose (0.5%). The mix
was placed on glass slides that had previously been coated with
agarose (5%), and the slides were covered with coverslips. The
slides were cooled at 4 ꢀC for 20 min. The coverslips were removed,
and the slides were placed in a freshly prepared lysis solution
(89.0 mL lysis stock containing 2.5 M NaCl, 100 mM EDTA, 10 mM
Tris, pH 10.0 [corrected with solid NaOH], 1 mL Triton X-100 and
10 mL DMSO) and incubated for 1 h at 4 ꢀC in the dark. The slides
were then placed in an electrophoresis tank, with buffer at
pH > 13.0 (300 mM NaOH and 1 mM EDTA, prepared from 10 N
NaOH and 200 mM EDTA, pH 10.0 stock solutions) for 20 min at 4 ꢀC
for DNA denaturation. Electrophoresis was performed at 25 V and
300 mA (1.25 V/cm) for 20 min. The slides were then neutralized in
0.4 M TriseHCl, pH 7.5 in 3 cycles of 5 min each, air dried and fixed
in absolute ethanol for 10 min. The material was stained a posteriori
Group 1 e negative control: The animals were administered
one injection of physiological solution (the vehicle for cyclo-
phosphamide, 0.1 mL/10 g bw, ip) and one injection of Milli-Q
water to which 1% ethanol had been added (the vehicle for
compound 4, 0.1 mL/10 g bw, ip).
Group 2 e positive control e cyclophosphamide: The animals
received one injection of cyclophosphamide (100 mg/kg bw, ip)
and one injection of the vehicle for resorcinolic lipid in a volume
of 0.1 mL/10 g (bw).
Group 3, 4 and 5 e AMS35AA: The animals received one in-
jection of physiological solution (0.1 mL/10 g bw, ip) and one
injection of AMS35AA (10, 20 or 40 mg/kg bw, ip).
Group 6, 7 and 8 e combination: The animals simultaneously
received one injection of AMS35AA (10, 20 or 40 mg/kg, bw, ip)
and one injection of cyclophosphamide (100 mg/kg bw, ip).
Group 9 e Naive: The animals were not treated.
(100
m
L ethidium bromide, 20 ꢁ 10ꢂ3 mg/mL) and analyzed under
40ꢁ magnification with an epifluorescence microscope (BiovalÒ,
Model L 2000A) equipped with a 420e490 nm excitation filter and
520 nm barrier filter according to Kobayashi [20]. The total score
was calculated by adding the values resulting from the multipli-
cation of the total number of observed cells from each lesion
category by the value of the category.
Twenty-four (T1), fourty-eight (T2) and seventy-two hours (T3)
after administration of the compounds, samples of peripheral blood
(20
mL) were collected for use in the micronucleus assay. Samples of
peripheral blood (20
mL) were also collected at T1 and T3 for use in
the comet assay and differential blood cell count, respectively.
Seventy-two hours after the beginning of the experiment, the first
set of animals was euthanized by cervical dislocation for organ
2.3.2. Micronucleus assay in peripheral blood
The micronucleus assay in peripheral blood was modified from
Hayashi [21] and Oliveira [22]. A 20 mL aliquot of peripheral blood
Fig. 2. Experimental design e biological assays.

S.D. Navarro et al. / European Journal of Medicinal Chemistry 75 (2014) 132e142
135
Fig. 3. Experimental design e animal treatments.
was placed on a slide that had been coated with 20 mL of acridine
orange (1 mg/mL), and the slide was covered with a coverslip. The
slide was placed at ꢂ20 ꢀC for a minimum of 15 days. Two thousand
cells per animal were analyzed under 400ꢁ magnification with an
epifluorescence microscope (BiovalÒ, Model L 2000A) equipped
with a 420e490 nm excitation filter and 520 nm barrier filter.
2.3.3. Apoptosis assay
One hundred microliters of a solution of macerated liver or
kidney was smeared on a slide. The slide was then fixed in Carnoy’s
solution for 5 min, subjected to a gradient of decreasing ethanol
concentrations (95%e25%), washed with McIlvaine buffer for 5 min,
stained with acridine orange (0.01%, 5 min) and washed again with
the buffer. Apoptotic cells were identified by analysis of DNA
fragmentation patterns according to Rovozzo and Burke [23] and
Mauro et al. [24] (Fig. 4). One thousand cells per animal were
analyzed under 400ꢁ magnification with an epifluorescence mi-
croscope (BiovalÒ, Model L 2000A) equipped with a 420e490 nm
excitation filter and 520 nm barrier filter.
Fig. 4. Morphological changes induced by AMS35AA assessed using Acridine Orange
staining 1) live cells and 2) apoptotic cell.
2.3.4. Splenic phagocytosis assay
The spleen was ground in physiological solution. One hundred
microliters of the cellular suspension was placed on a slide that had
been previously stained with 20 mL of acridine orange (1 mg/mL),
and the slide was covered with a coverslip. The slides were kept in a
freezer until analysis. The analysis was performed using 400ꢁ
magnification on a fluorescence microscope (BiovalÒ, Model L
2000A) equipped with a 420e490 nm excitation filter and 520 nm
barrier filter [25]. Two hundred cells per animal were analyzed. The
absence or presence of phagocytosis was identified according to
Hayashi [21].
field microscopy using 1000ꢁ magnification. A total of 100 cells/
animal were analyzed and classified as lymphocytes, neutrophils,
monocytes, eosinophils or basophils.
2.3.6. Biochemical assays
Following blood harvesting and sedimentation at 4 ꢀC, serum
was prepared and frozen at ꢂ20 ꢀC until analysis. Biochemical
parameters (glutamic-oxaloacetic transaminase (GOT), glutamate-
pyruvate transaminase (GPT), urea, creatinine and Naþ, Kþ, Ca2þ
and Mg2þ ions) were measured using a Cobas 6000 (Roche Diag-
nosticsÒ) analyzer according to the manufacturer’s instructions.
For the free hemoglobin assay, blood was collected in tubes with
spray-coated EDTA (BDÒ) and the analysis was performed accord-
ing to the manufacturer’s recommendations.
2.3.5. Differential blood cell count
Twenty microliters of peripheral blood was smeared on a his-
tology slide. The slides were dried in open air and stained with
Giemsa (10%) for 10 min. The slides were analyzed under bright

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S.D. Navarro et al. / European Journal of Medicinal Chemistry 75 (2014) 132e142
2.3.7. Histopathological analysis
13C NMR analysis of the 4 revealed a C-1 lactone carbonyl at
168.4 ppm. In addition, a chiral carbon C-3 was detected at
111.2 ppm (ketal carbon), and the methoxyl group linked to that
carbon atom was detected at 51.2 ppm. We have recently reported
the synthesis of an analog of 4 possessing hydroxyl group at C-3
instead of methoxyl group, with promising allelopathic activity
[29]. The 1H NMR spectra from compound 5 shows a triplet at dH
2.77 ppm, which is characteristic of the homotopic methylenic
hydrogens (H-9) adjacent to a ketone carbonyl, and 2 duplets at dH
6.60 ppm (1H) and 7.00 ppm (1H), J 2.1 Hz, which are consistent
with aromatic hydrogens with a meta relationship. 13C chemical
shifts and DEPT-135 data confirmed the presence of 4 methyl, 6
methylenic, 6 aromatic and 1 carboxylic carbon (at 166.4 ppm) and
a carbonyl carbon from an aromatic ketone (at 205.7 ppm).
Liver, spleen and kidneys were sectioned, fixed in 10% neutral
buffered formalin and prepared following routine practices for
histological analysis. Briefly, after fixation, the tissue fragments
were dehydrated, cleared and paraffin embedded. The samples
were sectioned into 6
mm slices and stained with hematoxylin-
eosin for histopathological analysis.
2.3.8. Calculation of percent damage reduction (DR%) and damage
increase (DI%)
The calculation of the percent decrease in damage as a method
to evaluate the chemopreventive capacity of a compound in com-
bination with a known mutagenic agent was proposed by Man-
oharan and Beneriee [26] and Waters [27]. In the present study, the
tested compound was found to have anti-genotoxic activity but not
anti-mutagenic activity. An increase in the frequency of DNA
damage was observed in the assessment of anti-mutagenic activity.
For this reason, both the DR% and DI% were calculated using the
same formula.
The 1H and 13C NMR data for the two compounds are close, with
the exception of the C-3 and H-10 chemical shifts of the lactone 4
(C-8 and H-9 at compound 5, respectively). The H-10 is unfolded in
2 multiplets at dH 2.06 and dH 2.2 ppm, indicating diastereotopic
methylenic hydrogens (Table 1). Two-dimensional NMR experi-
ꢀ
ꢁ
Mean of positive control ꢂ Mean of combination group
Mean of positive control ꢂ Mean of negative control
DR% or DI% ¼
ꢁ 100
2.3.9. Statistical analysis
ments (Heteronuclear Multiple-Bond Coherence and Heteronuclear
Single Quantum Coherence) corroborated the structure assign-
ments of the compounds.
The compound 4 has a chiral lactone ring moiety in their
structure, which is present also in the bioactive cytosporones C, D
and E, isolated from the endophytic fungi Cytospora sp. and Dia-
porthe sp. [30].
The averages ꢃ standard error of the average (SE) were calcu-
lated, and ANOVA/Tukey’s tests were performed at p < 0.05 using
GraphPad Prism software (version 3.02; Graph-Pad Software Inc.,
San Diego, CA, USA).
3. Results
3.2. Biological assays
3.1. Synthesis
3.2.1. Biometric parameters
The new synthetic resorcinolic lipid 4 was synthesized from
ftalide 3, which was obtained by FriedeleCrafts acylation of 3,5-
dimetoxibenzoic acid (1) with octanoyl chloride (2) according to
Zamberlam [28] with a 93% yield (Fig. 5).
The average values for the initial and final weights and weight
gain are presented in Table 2, and the absolute and relative organ
weights are presented in Table 3. A decrease (p < 0.05) in the ab-
solute and relative spleen weights were observed in the groups of
The treatment of ftalide 3 with methanol in the presence of
NaBH4 at room temperature for 72 h resulted in the formation of
the 4 and 5 isomers, with 31 and 40% yield, respectively. The iso-
mers were separated by chromatography using a silica gel column.
Compounds 4 and 5 were fully characterized by IR spectroscopy,
1H and 13C NMR and high-resolution mass spectrometry. Table 1
shows the chemical shift values for both compounds.
Table 1
NMR data related to compounds 4 and 5 (1H at 300 MHz, 13C at 75 MHz; CDCl3).
Position
4
Position
5
dC
dH mult. (J in Hz)
dC
dH mult. (J in Hz)
1
2
3
4
5
6
7
8
168.4
e
e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
129.4
127.1
157.1
103.0
160.6
105.3
166.4
205.7
44.3
23.5
29.2
29.1
31.8
22.6
14.1
52.5
55.7
56.0
e
e
e
e
111.2
155.5
104.0
163.6
98.8
131.0
126.2
36.8
23.1
29.3
29.0
31.6
22.6
14.0
51.2
55.9
55.9
e
e
e
6.60 d (2.1)
6.66 d (1.8)
e
7.00 d (2.1)
e
6.88 d (1.8)
e
e
9
e
2.74 t (7.5)
1.72 m
1.29 m
1.29 m
1.29 m
1.29 m
0.85 t (6.7)
3.80 s
10
11
12
13
14
15
16
17
18
19
2.2 and 2.06 m
1.18 m
1.18 m
1.18 m
1.18 m
1.18 m
0.81 t (6.7)
3.04 s
3.86 s
3.85 s
3.86 s
3.86 s
e
Fig. 5. Synthesis of the compound 4 and 5.

S.D. Navarro et al. / European Journal of Medicinal Chemistry 75 (2014) 132e142
137
Table 2
frequency of apoptosis (p < 0.05) in the liver and kidneys. The
administration of cyclophosphamide in combination with the
resorcinolic lipid resulted in an increase (p < 0.05) in the frequency
of apoptosis by 23.81-, 27.26- and 27.59-fold in the liver and 13.79-,
18.95- and 23.01-fold in the kidneys at the 10, 20 and 40 mg/kg
doses, respectively (Table 6).
Mean ꢃ SE of initial weight, final weight and weight gain during the experimental
period of the animals.
Experimental groups Initial weight (g) Final weight (g) Weight gain (g)
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
29.90 ꢃ 0.43a
30.60 ꢃ 0.48a
31.00 ꢃ 2.26a
29.50 ꢃ 0.91a
30.70 ꢃ 0.94a
29.10 ꢃ 0.91a
28.20 ꢃ 0.84a
29.60 ꢃ 1.01a
32.00 ꢃ 1.14a
30.50 ꢃ 0.37a
30.20 ꢃ 0.59a
31.00 ꢃ 0.71a
29.00 ꢃ 0.88a
30.90 ꢃ 1.02a
29.80 ꢃ 0.84a
29.00 ꢃ 0.67a
29.90 ꢃ 0.82a
32.00 ꢃ 0.95a
0.60 ꢃ 0.30a
ꢂ0.40 ꢃ 0.33a
0.00 ꢃ 0.39a
ꢂ0.50 ꢃ 0.37a
0.20 ꢃ 0.36a
0.70 ꢃ 0.42a
0.80 ꢃ 0.65a
0.30 ꢃ 0.61a
0.00 ꢃ 0.32a
3.2.5. Splenic phagocytosis assay
The administration of cyclophosphamide caused a 38.82-fold
reduction (p < 0.05) in splenic phagocytosis, whereas the resorci-
nolic lipid alone increased (p < 0.05) splenic phagocytosis by 18.43-
, 20.24- and 15.26-fold at the 10, 20 and 40 mg/kg doses, respec-
tively. The frequency of splenic phagocytosis was higher in the
group that received the lipid in combination with cyclophospha-
mide than in the group that received cyclophosphamide alone
(p < 0.05), but lower than in the groups treated solely with the
lipids (p < 0.05), with 33.83-, 39.75- and 44.94-fold increases
compared with cyclophosphamide at the 10, 20 and 40 mg/kg
doses, respectively (Table 7).
SE: Standard error.
Different letters indicate statistically significant differences (p < 0.05; ANOVA/
Tukey).
animals treated with cyclophosphamide (both alone and in com-
bination with the lipid).
3.2.2. Comet assay
The administration of the resorcinolic lipid alone did not result
in genotoxic activity (Table 4). When analyzing the damage classes
and damage score, it was observed a tendency for increasing the
genotoxic damage (p > 0.05), mainly in the combination groups.
The resorcinolic lipid had anti-genotoxic activity when admin-
istered in combination with cyclophosphamide (p < 0.05) and the
results did not show doseeresponse relation, since the middle dose
had the lower damage reduction percentage.
3.2.6. Differential blood cell count
The frequencies of monocytes, eosinophils and basophils were
similar in all groups, and eosinophilia was not present in only
groups 6, 7 and 8 (Table 8). The administration of the resorcinolic
lipid alone reduced (p < 0.05) the neutrophil frequencies in groups
3, 4 and 5; however, the values remained within the reference
range, which was not observed in groups 7 and 8, where the
reduction was more pronounced. The lymphocyte frequencies
increased (p < 0.05) in groups 3, 4 and 5, but a more pronounced
increase was observed in the combination groups (6, 7 and 8).
However, the values were within the reference range.
3.2.3. Micronucleus assay in peripheral blood
The micronucleus frequency indicated that the resorcinolic lipid
did not have mutagenic activity; however, when combined with
cyclophosphamide, the lipid caused an increase in the mutagenic
effect at T1 (Table 5). Evaluations at T2 and T3 were not possible
due to a reduction in the erythrocytes that could be analyzed.
3.2.7. Biochemical assays
The biochemical profile of the peripheral blood indicated no
significant differences in the levels of GPT, urea, Naþ, Kþ, Ca2þ
,
3.2.4. Apoptosis assay
Mg2þ ions and free hemoglobin. The intermediate dose of the
resorcinolic lipid did not affect the GOT levels, whereas the other
two doses and cyclophosphamide (either alone or in combination
with the resorcinolic lipid) caused increases in these levels
(p < 0.05). The resorcinolic lipid, either alone or in combination
The resorcinolic lipid increased (p < 0.05) the frequency of
apoptosis by 16.35-, 17.51- and 15.55-fold in the liver and 10.41-,
10.67- and 12.42-fold in the kidneys at the 10, 20 and 40 mg/kg
doses, respectively. Cyclophosphamide alone also increased the
Table 3
Mean ꢃ SE of absolute weight and relative weight of the animals’ organs after the experimentation period.
Experimental groups
Heart
Lungs
Liver
Kidneys
Spleen
Absolute weight (g)
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
Relative weight (g)
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
,b
0.1632 ꢃ 0.0219a
0.1543 ꢃ 0.0095a
0.1590 ꢃ 0.0107a
0.1614 ꢃ 0.0077a
0.1925 ꢃ 0.0140a
0.1614 ꢃ 0.0080a
0.1347 ꢃ 0.0071a
0.1560 ꢃ 0.0161a
0.1508 ꢃ 0.0041a
0.1932 ꢃ 0.0116a
0.2232 ꢃ 0.0175a
0.2256 ꢃ 0.0097a
0.2181 ꢃ 0.0086a
0.2339 ꢃ 0.0070a
0.2182 ꢃ 0.0096a
0.2147 ꢃ 0.0076a
0.1943 ꢃ 0.0071a
0.1954 ꢃ 0.0066a
1.4257 ꢃ 0.0838a,b
1.5502 ꢃ 0.0844b
1.4707 ꢃ 0.0427a,b
1.4660 ꢃ 0.0558a,b
1.5728 ꢃ 0.0504b
1.3455 ꢃ 0.0605a,b
1.4238 ꢃ 0.0889a,b
1.2367 ꢃ 0.0649a
0.3983 ꢃ 0.0139a
0.1157 ꢃ 0.0057b
0.0786 ꢃ 0.0042a
0.1354 ꢃ 0.0049b
0.1266 ꢃ 0.0059b
0.1329 ꢃ 0.0078b
0.0849 ꢃ 0.0035a
0.0737 ꢃ 0.0030a
0.0803 ꢃ 0.0022a
0.1273 ꢃ 0.0047b
0.4103 ꢃ 0.0138a,b
0.4277 ꢃ 0.0122b
0.3888 ꢃ 0.0184a,b
0.4375 ꢃ 0.0093b
0.3820 ꢃ 0.0161a,b
0.3529 ꢃ 0.0220a,b
0.3435 ꢃ 0.0225a
0.3917 ꢃ 0.0252a,b
,b
1.2365 ꢃ 0.0551a
0.0054 ꢃ 0.0008a
0.0051 ꢃ 0.0004a
0.0051 ꢃ 0.0004a
0.0055 ꢃ 0.0001a
0.0066 ꢃ 0.0005a
0.0054 ꢃ 0.0003a
0.0046 ꢃ 0.0003a
0.0052 ꢃ 0.0006a
0.0047 ꢃ 0.0002a
0.0063 ꢃ 0.0003a
0.0073 ꢃ 0.0004a
0.0072 ꢃ 0.0003a
0.0074 ꢃ 0.0002a
0.0075 ꢃ 0.0006a
0.0073 ꢃ 0.0003a
0.0074 ꢃ 0.0003a
0.0067 ꢃ 0.0003a
0.0061 ꢃ 0.0003a
0.0468 ꢃ 0.0028a
0.0515 ꢃ 0.0032a
0.0475 ꢃ 0.0016a
0.0459 ꢃ 0.0047a
0.0514 ꢃ 0.0026a
0.0457 ꢃ 0.0030a
0.0493 ꢃ 0.0030a
0.0416 ꢃ 0.0025a
0.0391 ꢃ 0.0014a
0.0133 ꢃ 0.0003a
0.0135 ꢃ 0.0005a
0.0138 ꢃ 0.0004a
0.0134 ꢃ 0.0004a
0.0131 ꢃ 0.0013a
0.0015 ꢃ 0.0012a
0.0122 ꢃ 0.0009a
0.0115 ꢃ 0.0007a
0.0123 ꢃ 0.0010a
0.0037 ꢃ 0.0001b
0.0026 ꢃ 0.0002a
0.0043 ꢃ 0.0002b
0.0049 ꢃ 0.0003b
0.0042 ꢃ 0.0002b
0.0028 ꢃ 0.0001a
0.0025 ꢃ 0.0001a
0.0027 ꢃ 0.0001a
0.0040 ꢃ 0.0002b
Group 8
Group 9
SE: Standard error.
Different letters indicate statistically significant differences (p < 0.05; ANOVA/Tukey).

138
S.D. Navarro et al. / European Journal of Medicinal Chemistry 75 (2014) 132e142
Table 4
Mean ꢃ SE frequency of damaged cells, distribution between damage classes, and scores related to anti-genotoxicity tests of AMS35AA through the comet assay in mice
peripheral blood cells.
Experimental groups
Damaged cells
Damage classes
0
Score
DR%
1
2
3
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
22.80 ꢃ 5.04a,b
86.40 ꢃ 3.15e
30.80 ꢃ 5.64a,b,c
38.60 ꢃ 4.74b,c,d
36.80 ꢃ 3.80b,c
45.20 ꢃ 3.83c,d
57.40 ꢃ 3.07d
49.40 ꢃ 4.26c,d
NA
77.40 ꢃ 5.07
13.20 ꢃ 3.38
69.20 ꢃ 5.64
63.40 ꢃ 3.89
63.20 ꢃ 3.80
54.80 ꢃ 3.84
42.60 ꢃ 3.07
50.60 ꢃ 4.26
NA
20.00 ꢃ 4.86
64.00 ꢃ 2.02
28.40 ꢃ 5.48
35.00 ꢃ 4.63
33.60 ꢃ 2.66
34.80 ꢃ 3.65
46.60 ꢃ 2.27
40.20 ꢃ 4.02
NA
2.80 ꢃ 0.80
0.00 ꢃ 0.00
5.20 ꢃ 1.39
0.00 ꢃ 0.00
0.00 ꢃ 0.00
0.20 ꢃ 0.20
2.40 ꢃ 0.81
3.20 ꢃ 0.97
3.40 ꢃ 1.14
NA
25.60 ꢃ 5.34a
114.00 ꢃ 7.45d
33.20 ꢃ 5.86a
42.20 ꢃ 4.97a,b
40.20 ꢃ 5.29a,b
58.00 ꢃ 4.52c,b
71.40 ꢃ 5.33c
62.00 ꢃ 4.90c,b
NA
e
17.20 ꢃ 1.88
2.40 ꢃ 0.60
3.60 ꢃ 0.81
3.00 ꢃ 1.26
8.00 ꢃ 1.04
7.60 ꢃ 1.89
5.80 ꢃ 1.50
NA
e
e
e
e
64.78
45.60
58.18
e
SE: Standard error.
DR%: Damage reduction percentage.
NA: Not analyzed.
Different letters indicate statistically significant differences (p < 0.05; ANOVA/Tukey).
Table 5
Total frequency and mean ꢃ SE of the micronucleus assay in mice peripheral blood cells.
Experimental groups
Micronucleus frequency
Mean ꢃ SE
DI%
24 h (T1)
48 (T2)
72 (T3)
24 h (T1)
48 h (T2)
72 h (T3)
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
100
504
90
110
100
582
544
542
NA
82
350
60
82
110
e
64
222
52
68
86
e
10.0 ꢃ 1.14a
50.4 ꢃ 4.16b
9.0 ꢃ 1.14a
11.0 ꢃ 1.70a
10.0 ꢃ 0.84a
58.2 ꢃ 5.17b
54.4 ꢃ 4.52b
54.2 ꢃ 4.89b
NA
8.2 ꢃ 0.96a
35.0 ꢃ 2.38b
6.0 ꢃ 0.70a
8.2 ꢃ 0.96a
11.0 ꢃ 2.07a
NA
NA
NA
NA
6.4 ꢃ 1.17a
22.2 ꢃ 1.85b
5.2 ꢃ 0.96a
6.8 ꢃ 1.02a
8.6 ꢃ 1.16a
NA
NA
NA
NA
e
e
e
e
e
19.31
9.91
9.41
NA
e
e
e
e
NA
NA
SE: Standard error.
DI%: Damage increase percentage.
NA: Not analyzed.
Different letters indicate statistically significant differences (p < 0.05; ANOVA/Tukey).
with cyclophosphamide, increased the creatinine levels (p < 0.05).
Cyclophosphamide alone did not affect the creatinine levels
(Table 9).
preserved architecture in the cortical region. The medullary region
exhibited tubules with a finely eosinophilic material. The histology
of groups 2, 3, 4 and 5 was similar to the negative control groups. In
groups 6, 7 and 8, the liver histological sections presented a rare-
faction of the cytoplasm with loss of the cytoplasmatic eosinophilia.
The results for the kidneys were similar to those found for the
negative controls.
3.2.8. Histopathological analysis
The histopathological assay indicated that the animals in group
1 had normal hepatic lobe morphology; the hepatic lobes were
contained hepatocytes with normal and preserved morphology,
and the vascular portal triad was observed in places. The histo-
logical sections of the kidneys showed the presence of clusters with
4. Discussion
Multiple assays and experimental designs have shown that
resorcinolic lipids, from either synthetic or natural origin, do not
cause DNA damage at low concentrations and may possess anti-
carcinogenic activity [12,13,31e43]. These observations are in
agreement with the present study, in which no genotoxicity or
mutagenicity was observed. Furthermore, the results support
additional studies to understand the chemotherapeutic and/or
chemotherapy adjuvant effects of these compounds. The present
study was also innovative in being the first to combine a resorci-
nolic lipid with a drug used in cancer treatment.
The evaluation of anti-genotoxicity and anti-mutagenicity ac-
tivities did not confirm a chemopreventive profile because the lipid
possesses only anti-genotoxic activity. This result has previously
been reported by Parikka [41]. These data indicate that the lipid is
potentially active only in repairing primary lesions that involve
single-stranded DNA breaks, double-stranded DNA breaks, cross-
links, base excision repair sites and/or alkali/labile lesions; these
lesions can be assessed using the comet assay [18], but chromo-
somal level lesions are assessed using the micronucleus assay.
Table 6
Apoptosis evaluation on mice’ kidneys and liver.
Experimental
groups
Liver
Kidneys
Number of
apoptotic cells
Mean ꢃ SE
Number of
apoptotic cells
Mean ꢃ SE
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
74
1843
1210
1296
1151
1762
2017
2042
NA
14.8 ꢃ 3.69a
81
16.2 ꢃ 4.42a
201.6 ꢃ 7.92b
168.6 ꢃ 15.24b
173.0 ꢃ 11.07b
201.2 ꢃ 18.70b
223.4 ꢃ 15.93b
307.0 ꢃ 22,65c
372.8 ꢃ 17.52c
NA
368.6 ꢃ 34.12c 1008
242.0 ꢃ 21.65b 843
259.2 ꢃ 22.06b 865
230.2 ꢃ 23.05b 1006
352.4 ꢃ 20.99c 1117
403.4 ꢃ 10.09c 1535
408.4 ꢃ 11.51c 1846
NA
NA
SE: Standard error.
NA: Not analyzed.
Different letters indicate statistically significant differences (p < 0.05; ANOVA/
Tukey).

S.D. Navarro et al. / European Journal of Medicinal Chemistry 75 (2014) 132e142
139
Table 7
Number of analyzed cells, mean frequency ꢃ SE, and cell percentage with or without splenic phagocytosis evidence in mice.
Experimental groups
Number of analyzed cells
Total of cells without phagocytosis evidence
Total of cells with phagocytosis evidence
Absolute values
Average ꢃ SE
Percentage (%)
Absolute values
Average ꢃ SE
Percentage (%)
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
1000
1000
1000
1000
1000
1000
1000
1000
NA
338
595
216
204
237
458
434
403
NA
67.6 ꢃ 4.28b
119.0 ꢃ 3.57d
43.2 ꢃ 3.39a
40.8 ꢃ 2.92a
47.4 ꢃ 3.20a
91.6 ꢃ 2.62c
86.9 ꢃ 2.94c
80.6 ꢃ 5.63b,c
NA
33.8
59.5
21.6
20.4
23.7
45.8
43.4
40.3
NA
662
405
784
796
763
542
566
587
NA
132.4 ꢃ 4.28c
81.0 ꢃ 3.57a
156.8 ꢃ 3.40d
159.2 ꢃ 2.92d
152.6 ꢃ 3.20d
108.4 ꢃ 2.62b
113.2 ꢃ 2.94b
117.4 ꢃ 4.81a,b
NA
66.2
40.5
78.4
79.6
76.3
54.2
56.6
58.7
NA
SE: Standard error.
NA: Not analyzed.
Different letters indicate statistically significant differences (p < 0.05; ANOVA/Tukey).
In a simultaneous 24 h protocol, the lipid was observed to
reduce the genotoxic damage and increase the mutagenic damage
induced by cyclophosphamide. This effect may be relevant in can-
cer treatment if the resorcinolic lipid is used as an adjuvant,
although no significant difference was observed in the micronu-
cleus increment rate because chemotherapy drugs (such as cyclo-
phosphamide) induce micronuclei and activate cascades, including
caspases and cysteine proteases [44].
observed greater anti-mutagenic activity for the compounds with
direct action. These results were confirmed by George and Kuttan
[35]. In a sister chromatid exchange assay, Gasiorowski [34] have
reported that the phenolic lipids reduce mutations in human
lymphocytes by 50%. The anti-mutagenic activity was confirmed in
additional experiments using the cytokinesis-block micronucleus
assay and the thioguanine resistance test, and the phenolic lipids
were found to significantly decrease the mutation frequency in
lymphocytes cultured with benzo[a]pyrene and mitomycin C [36].
However, this report conflicts with the lack of anti-mutagenic ac-
tivity of the resorcinolic lipids observed in the present study.
Notably, although the previous studies have used phenolic
lipids, different approaches were used in the synthesis of the lipids,
including structural modifications. In addition, a resorcinolic lipid
with greater similarity to the lipid tested in the present study was
found to inhibit the in vitro proliferation of pulmonary (H1299),
hepatic (HepG2), gastric (BGC823) and colon (SW620) cancer cells.
However, the effect was smaller in healthy cells, including mouse
fibroblasts (NIH3T3) and human hepatocytes (HL-7702) and hep-
atoblasts (ME-Hep4), indicating that the activity is selective for
cancer cells [43] and/or cells with DNA lesions. Therefore, the lack
of observed anti-mutagenic activity of this compound may favor
the accumulation of genetic damage and lead to apoptosis in the
targeted cells. In the present study, the cells from the animals
treated with the chemotherapy agent were not cancer cells but
possessed mutations that could lead to their transformation. The
identity of the target cell may determine the selectivity of the
compound and its ability to cause apoptosis.
The micronucleus frequency could not be evaluated at 48 and
72 h due to the decrease in the number of erythrocytes that could
be analyzed. This decrease may have been due to the interaction of
the resorcinolic lipid with the erythrocyte plasma membrane,
which may have induced spacing and rupture of the lipid bilayer
[45,46] followed by an increase in permeability to small non-
electrolytes (molecular diameter of up to 1.4 nm) and water [4].
The sum of these factors may have resulted in hemolysis of the
erythrocytes [47,48]. Although the literature allows considering the
hypothesis of hemolysis, the results obtained in this research are
contradictory, since the hemolysis assay indicated no statistically
significant differences among the experimental groups. However,
increased levels of GOT may indicate hemolysis [49e51] and this
fact was verified in the combination groups, exactly those groups
where the erythrocytes could not be analyzed, suggesting hemo-
lysis caused by the combination of the tested compound and
chemotherapy. Based on these reports, there is a need for more
detailed studies, which will be the next steps of our research group.
A relationship between the hemolytic capacity of the homolo-
gous resorcinolic lipids and the length and degree of unsaturation
of the aliphatic side chain was also observed [52], which could
explain the absence of analyzable erythrocytes in the peripheral
blood micronucleus assay.
The apoptosis assay indicated that AMS35AA caused an increase
in the number of apoptotic cells, both alone and in combination
with cyclophosphamide. These results suggest strong apoptosis
induction capacity and, as reported, this capacity could correlate
with increased DNA damage, as determined by the micronucleus
assay. This increase in apoptosis was expected because synthetic
analogs of compound 4, such as cytosporone B [43] and amoitone B
The mutagenic activity of phenolic lipids has previously been
assessed by other authors; all results have indicated that the lipids
have no toxicity. Gasiorowski [34] have tested phenolic lipids for
mutagens with direct and indirect action using the Ames test and
Table 8
Reference values and mean ꢃ SE of the differential blood cell count.
Cell types
Reference values Experimental groups
Group 1 Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
Lymphocyte
Neutrophil
Monocyte
Eosinophil
Basophil
55e95%
10e40%
0.1e3.5%
0e0.4%
46.8 ꢃ 1.96a 40.4 ꢃ 3.21a 71.6 ꢃ 2.42b 69.0 ꢃ 1.92b 71.6 ꢃ 2.63b 87.2 ꢃ 0.96c 84.8 ꢃ 2.51c 87.4 ꢃ 1.43c NA
48.8 ꢃ 1.93c 55.2 ꢃ 1.68c 25.4 ꢃ 1.78b 26.4 ꢃ 2.42b 24.2 ꢃ 3.20b 7.80 ꢃ 1.02a 11.6 ꢃ 1.56a 8.80 ꢃ 1.46a NA
2.20 ꢃ 0.73a 2.40 ꢃ 1.16a 1.40 ꢃ 0.50a 3.20 ꢃ 0.91a 2.40 ꢃ 0.87a 4.60 ꢃ 0.67a 2.80 ꢃ 0.58a 3.40 ꢃ 0.51a NA
2.20 ꢃ 0.58a 1.80 ꢃ 0.58a 1.60 ꢃ 0.51a 1.40 ꢃ 0.51a 1.60 ꢃ 0.40a 0.20 ꢃ 0.20a 0.60 ꢃ 0.60a 0.40 ꢃ 0.40a NA
0.00 ꢃ 0.00a 0.20 ꢃ 0.20a 0.00 ꢃ 0.00a 0.00 ꢃ 0.00a 0.20 ꢃ 0.20a 0.20 ꢃ 0.20a 0.20 ꢃ 0.20a 0.00 ꢃ 0.00a NA
0e0.3%
SE: Standard error.
Different letters indicate statistically significant differences (p < 0.05; ANOVA/Tukey).

140
S.D. Navarro et al. / European Journal of Medicinal Chemistry 75 (2014) 132e142
Table 9
Biochemical evaluation of mice’ peripheral blood.
Experimental GOT (U/L)
groups
GPT (U/L)
Urea
(mg/dL)
Creatinine
(mg/dL)
Sodium
(mEq/L)
Potassium
(mmol/L)
Calcium
(mg/dL)
Magnesium Free
(mg/dL) hemoglobin
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
114.2 ꢃ 6.53a
59.4 ꢃ 8.86a
64.3 ꢃ 5.88a 0.28 ꢃ 0.06a
125.0 ꢃ 7.59a 67.4 ꢃ 10.79a,b
134.2 ꢃ 1.93a 83.6 ꢃ 4.41b
134.4 ꢃ 7.52a 44.9 ꢃ 10.26a
138.0 ꢃ 6.15a 41.6 ꢃ 9.90a
9.86 ꢃ 0.18a,b 0.38 ꢃ 0.06a 0.076 ꢃ 0.004a
,b
158.2 ꢃ 8.32a
127.8 ꢃ 8.06a
89.2 ꢃ 12.74a 58.2 ꢃ 4.62a 0.26 ꢃ 0.02a
66.8 ꢃ 13.42a 56.0 ꢃ 5.90a 0.48 ꢃ 0.02a,b
98.4 ꢃ 26.33a 60.6 ꢃ 3.61a 0.38 ꢃ 0.05a,b
9.70 ꢃ 0.21a,b 0.32 ꢃ 0.02a 0.114 ꢃ 0.021a
9.10 ꢃ 0.24a
9.66 ꢃ 0.36b
10.24 ꢃ 0.13b
10.56 ꢃ 0.29b
0.45 ꢃ 0.05a 0.144 ꢃ 0.011a
0.43 ꢃ 0.05a 0.096 ꢃ 0.016a
0.36 ꢃ 0.02a 0.066 ꢃ 0.008a
0.45 ꢃ 0.02a 0.172 ꢃ 0.067a
170.0 ꢃ 26.67a,b,c
194.0 ꢃ 9.39b,c
154.2 ꢃ 16.39a,b
223.0 ꢃ 18.6c
98.8 ꢃ 21.64a 67.2 ꢃ 2.53a 0.50 ꢃ 0.04a,b,c 137.0 ꢃ 5.94a 59.2 ꢃ 3.47a
45.8 ꢃ 12.23a 68.1 ꢃ 3.92a 0.50 ꢃ 0.10a,b,c 145.0 ꢃ 3.01a 40.1 ꢃ 11.71a
109.4 ꢃ 25.66a 65.1 ꢃ 3.66a 0.62 ꢃ 0.09b,c
141.2 ꢃ 2.13a 40.2 ꢃ 7.34a
142.0 ꢃ 2.16a 39.8 ꢃ 13.88a
9.98 ꢃ 0.15a,b 0.42 ꢃ 0.01a 0.100 ꢃ 0.017a
202.2 ꢃ 6.24b,c
94.6 ꢃ 8.72a
63.2 ꢃ 7.28a
52.2 ꢃ 2.06a 0.74 ꢃ 0.06c
57.0 ꢃ 2.90a 0.28 ꢃ 0.04a
10.26 ꢃ 0.21b
9.86 ꢃ 0.09a
0.43 ꢃ 0.00a 0.06 8 ꢃ 0.008a
0.38 ꢃ 0.05a NA
118.0 ꢃ 6.63a
122.8 ꢃ 2.35a 50.8 ꢃ 5.91a
,b
,b
SE: Standard error.
NA: Not analyzed.
GOT: Glutamic-oxaloacetic transaminase.
GPT: Glutamate-pyruvate transaminase.
Different letters indicate statistically significant differences (p < 0.05; ANOVA/Tukey).
[53], induce apoptosis through Nur77 activation. Other cytotoxicity
assays using analogs of the resorcinolic lipid have also shown a
capacity for apoptosis induction in cancer cells [14,16,54,55].
Furthermore, compounds with similar structure have been re-
ported to possess anti-cancer activity in xenograft tumors that is
mediated by Nur77 activation [14].
Nur77, also known as TR3 or NGFI-B, is a unique transcription
factor that belongs to the orphan nuclear receptor superfamily [56].
Nur77 contributes to the regulation of several biological processes,
including cell proliferation, differentiation and apoptosis [57,58]. In
cancer cells, Nur77 acts as an oncogenic survival factor in the nu-
cleus and becomes a strong activator of apoptosis when certain
death stimuli promote its migration to the mitochondria. At the
mitochondria, Nur77 interacts with and induces conformational
changes in Bcl-2, resulting in cytochrome c release and apoptosis
[59e61].
Results by Liu [14] have indicated that cytosporone B analogs
can activate Nur77 due to the structure-dependent affinity of
cytosporone B with Nur77, in which the ester radical plays a
particularly important role. These results support the hypothesis
that AMS35AA may act through activation of Nur77, leading to
apoptosis due to the ester radical in the structure of the compound.
In previous studies, analogs have been found to promote the
anti-tumor activity of Nur77 through cross-talk between Nur77 and
BRE (a protein associated with the cell death receptor), which is
specifically mediated by the repression of BRE transcriptional ac-
tivity through N-CoR corepressor recruitment [62,63]. Although
BRE is known as an anti-apoptotic protein that acts by inhibiting
the mitochondrial apoptosis pathway [62,63], the authors suggest
that Nur77 can associate with the promoter and that Nur77 tran-
scription can be regulated by BRE. Due to these unique properties,
Nur77 is an attractive molecular target for the development of
novel cancer therapies. In addition, the structural modification of
natural products can result in molecules with improved properties,
such as greater production quantity, reduced toxicity or enhanced
efficacy.
The splenic phagocytosis assay was proposed and performed by
the research group to complement the results of the present study.
The results obtained at 72 h after the administration of AMS35AA
and/or cyclophosphamide indicated that the compounds generally
cause a statistically significant increase in splenic phagocytosis.
These data indicate that AMS35AA increases splenic phagocytosis,
which is responsible for removing lesions or cancer cells from the
organism. This cell phagocytosis can be mediated by more than one
signaling pathway. The cells are believed to be signalized upon the
induction of apoptosis [64] or after cell lysis has occurred. There-
fore, splenic phagocytosis would be effective in the removal of
anucleated and/or micronucleated cells and cells with membrane
lesions [65].
At 72 h, cyclophosphamide alone caused an increase in the
micronucleus frequency, despite the downward trend throughout
the experiment. However, the lowest splenic phagocytosis values
were also observed in this group, indicating hypoactivity of the
spleen upon administration of cyclophosphamide. Furthermore, an
increase in the frequency of apoptosis was also observed in both the
liver and kidneys. Together, these data indicate an acute toxicity of
cyclophosphamide, which is not necessarily observed in histolog-
ical studies of short duration (which is the case in the present
study). The data further indicate that in addition to causing DNA
damage, an increase in apoptosis can correlate with decreased
spleen activity, which are adverse effects of chemotherapy. In
contrast, the combination with AMS35AA indicated that the cells
that have a micronucleus (a cancer biomarker) can be removed
from circulation, such as through membrane lysis. Furthermore, the
resorcinolic lipid increases apoptosis in cells that may eventually be
targets of therapy in solid tissues and may have suffered damage
caused by chemotherapy (although the chemotherapy may not
necessarily have impaired the function of the organ). Finally, the
phagocyte activity, which is responsible for removing damaged
cells or cellular remains from the circulation, seems to be
preserved.
These conclusions are supported by the histological observa-
tions, in which cytoplasm rarefaction and eosinophilia, character-
istic apoptotic events [66], were observed only in the livers of the
animals that received cyclophosphamide in combination with
AMS35AA. These results are supported by the data from the
apoptosis assay.
The biochemical analysis indicated an increase in GOT, an
enzyme associated with hepatic activity, in response to the resor-
cinolic lipid. Significant increases were observed upon adminis-
tration of the 2 highest doses of AMS35AA, both alone and in
combination with cyclophosphamide. Although this enzyme can be
released upon hepatic damage [67], the histological analysis did not
reveal such lesions, indicating that tissue damage did not occur.
The biochemical parameters for the kidneys showed an increase
in creatinine, particularly in the 2 groups that received AMS35AA in
combination with cyclophosphamide. However, although an in-
crease in the plasma concentration was observed, no histological
damage was present. Apoptotic events observed in this organ may
explain this observation. Together, the histological observations
and biochemical data therefore support a lack of toxicity of
AMS35AA under the experimental conditions tested.
Previous reports have shown that resorcinolic lipids have the
capacity to form liposomes [8]. These structures are

S.D. Navarro et al. / European Journal of Medicinal Chemistry 75 (2014) 132e142
141
microscopically stable vesicles formed by phospholipids and
similar amphipathic lipids; in aqueous medium, these structures
form lipid bilayers that are structurally similar to cellular biological
membranes [68]. Since Gregoriadis [69] described the ability of li-
posomes to transport and release therapeutic agents, several clin-
ical studies have demonstrated that the incorporation of
compounds (such as resorcinolic lipids) into liposomes can result in
increased efficacy and reduced side effects of chemotherapy drugs.
The resorcinolic lipids and their semi-synthetic derivatives are
more stable at neutral pH, and the liposome drug encapsulation
efficiency is increased under this condition [70]. Therefore, the
liposome formation capacity may have potentiated and prolonged
the duration of the effects of cyclophosphamide. Small changes in
the biochemical tests may reflect the potentiation of the cyclo-
phosphamide effect, as shown in previous studies [71].
biochemical assays. We also thank GG Facco for his support in the
histopathological assay.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.057.
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This project was supported by FUNDECT (Edital Chamada
FUNDECT Nꢀ 09/2008 e Universal e Processo Nꢀ 23/200.117/2009
e Termo de Outorga Nꢀ 0093/09; Nꢀ Siafem: 014987. Edital Cha-
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Termo de Outorga Nꢀ 0207/12; Nꢀ Siafem: 020937). We would like
to thank the following people for their contribution: MS Torres, VR
Verardino e S Baldissera (HEMOSUL e HEMORREDE, Campo
Grande, Mato Grosso do Sul, Brasil), TM Sakamoto and PC de
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