Rubrolide analogues and their derived lactams as potential anticancer agents

Article abstract of DOI:10.1039/c5md00459d

Seven β-aryl-substituted γ-alkylidene-γ-lactone analogues of rubrolides were synthesized from mucochloric acid and converted into their corresponding γ-hydroxy-γ-lactams (76-85%) by a reaction with isobutylamine and propylamine. Further dehydration of the

Full text of DOI:10.1039/c5md00459d

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RESEARCH ARTICLE
Rubrolide analogues and their derived lactams as
potential anticancer agents†‡
Cite this: DOI: 10.1039/c5md00459d
a
a
ab
a
c
U. A. Pereira, T. A. Moreira, L. C. A. Barbosa,* C. R. A. Maltha, I. S. Bomfim,
c
c
cd
e
S. S. Maranhão, M. O. Moraes, C. Pessoa and F. W. A. Barros-Nepomuceno
Seven β-aryl-substituted γ-alkylidene-γ-lactone analogues of rubrolides were synthesized from
mucochloric acid and converted into their corresponding γ-hydroxy-γ-lactams (76–85%) by a reaction with
isobutylamine and propylamine. Further dehydration of the γ-hydroxy-γ-lactams led to the corresponding
(Z)- and (E)-γ-alkylidene-γ-lactams (23–45%). All compounds were fully characterized by spectroscopic
methods. These 14 compounds, together with 32 other rubrolide analogues, were assayed against four
human tumor cell lines (HL-60, leukaemia; HCT-116, colon; SF-295, central nervous system; and OVCAR-8,
ovarian). Of the 46 compounds assayed, 7 caused a large reduction in cell viability (% RCV > 80%) in the
tested cell lines and the most active compounds had halogen substituents on the aromatic ring. Com-
pounds 10a and 14i were the most active (RC50 = 3.00 and 3.58 μM, respectively) against HL-60 and were
not cytotoxic to L929 normal cells at the concentrations tested (RC50 > 50 μM). To further understand the
mechanism underlying the cytotoxicity of 10a and 14i, studies involving DNA fragmentation, cell cycle anal-
ysis, phosphatidyl serine externalization and mitochondrial depolarization were performed in the HL-60
cells, using doxorubicin as a positive control. The results indicated that the cytotoxicity of 10a and 14i
involved the induction of cell death by apoptosis. The cell cycle analysis showed that 14i caused the
accumulation of cells in the G0/G1 phase at 2.5 and 5 μM.
Received 6th October 2015,
Accepted 9th December 2015
DOI: 10.1039/c5md00459d
www.rsc.org/medchemcomm
disease. Although enormous efforts have been dedicated to
the development of new drugs for cancer treatment, there is
Introduction
Transformation of a single cell into a tumorigenic phenotype
is defined as cancer, where the balance between cell prolifera-
tion and cell death is disrupted. As a result of cancer,
still an urgent need to find better cures for this health
problem.
For the development of more potent drugs against cancer,
natural products have served as important chemical proto-
types for the discovery of new molecules. Since the use of
plant and microbial secondary metabolites has aided in dou-
bling our life span in the 20th century, these compounds
continue to be the most promising source of drug leads, es-
1
uncontrolled growth occurs, with potential cell metastasis.
Cancer is the leading cause of death in modern society. The
International Agency for Research on Cancer estimated 14.1
million new cancer cases and 8.2 million deaths from cancers
2
occurred in 2012, which makes it a major life-threatening
3
,4
pecially in the anticancer field.
5
According to Newman and Cragg, over the time frame
from around the 1950s to 2010, the utility of natural products
as sources of novel structures is still alive and well. Of the
175 small molecules approved for use as anticancer agents,
a
Department of Chemistry, Federal University of Viçosa, Viçosa, MG, Brazil
b
Department of Chemistry, Universidade Federal de Minas Gerais, Av. Pres.
Antônio Carlos, 6627, CEP 31270-901, Belo Horizonte, MG, Brazil.
E-mail: lcab@ufmg.br; Fax: +55 31 3899 3065; Tel: +55 31 3899 3068
4
8.6% are either natural products or directly derived
therefrom.
Rubrolides are a class of γ-alkylidene-γ-lactones isolated
c
Center for Research and Drug Development, Federal University of Ceará, Rua
Coronel Nunes de Melo, 1000, CEP 60430-275, Fortaleza, CE, Brazil
d
Oswaldo Cruz Foundation, Av. Santos Dumont, 5753, Torre Saúde, Sala 1303,
from ascidian species, as illustrated by rubrolides K (1) and
M (2) (Fig. 1). Several rubrolides and synthetic analogues
are endowed with a large array of bioactivities, includ-
Papicu, Fortaleza, CE, Brazil
e
Institute of Health Sciences, University of International Integration of the
Afro-Brazilian Lusophony, Rodovia CE 060, Km 51, CEP 62785-000, Acarape,
CE, Brazil
6–10
ing antibiotic, anti-inflammatory, cytotoxic,
inhibition of
†
‡
The authors declare no competing interest.
1
1,12
bacterial biofilm formation,
phytotoxicity and inhibi-
The research work by Ortega
et al. showed that rubrolides I–N were active against four
Electronic supplementary information (ESI) available: Additional details on the
1
3,14
tion of photosynthesis.
experimental part of the synthesis and biological assays. Selected spectra are in-
cluded. See DOI: 10.1039/c5md00459d
7
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Results and discussion
Synthesis
The lactone 3,4-dichlorofuranone (9) was prepared from
commercially available mucochloric acid (8), with a yield of
88%. The subsequent step involved an aldol condensation
between 9 and aromatic aldehydes in the presence of tert-
butyldimethylsilyltrifluoromethanesulfonate (TBDMSOTf) and
diisopropylethylamine (DIPEA), followed by treatment of the
silyl ether generated in situ with DBU. Such a reaction
afforded the rubrolide analogues (10a–10h), stereoselectively
formed in yields ranging from 10 to 58%. All compounds
were fully characterized by detailed IR, NMR, and MS analy-
1
ses. The H NMR spectra of all the products showed signals
related to aromatic H ranging from 6.5 to 8.0 ppm. Although
some products were obtained in low yields, no effort to opti-
mize the reaction conditions was made, since at this stage,
we focused our attention in obtaining the final products for
biological evaluation. However, from these reactions, we ob-
served that lower yields were obtained with benzaldehydes
substituted with electron-withdrawing groups, while those
bearing electron-donating groups led to higher yields of the
desired products. The low yields in such reactions were due
in part to a rearrangement that occurred in the case of alde-
hydes bearing electron-withdrawing groups, resulting in
Fig. 1 Structures of rubrolide K (1), rubrolide M (2), γ-hydroxy-γ-
lactams (3) and γ-alkylidene-γ-lactams (4), myceliothermophins A (5)
and F (6) and pyridazin-3IJ2H)-ones (7).
2
1
tumour cell lines, with rubrolide M (2) being the most
cyclopentenediones in large amounts. According to previ-
ous studies, the compounds 10a–10h have an exocyclic
double bond with a Z stereochemistry. These results are
due to the steric hindrance of the β substituent in the
lactone ring, as observed in the synthesis of other
−
1
active (ED50 = 1.2 μg mL ) against all the tumour cell lines
tested.
Other classes of natural products bearing a five or six
membered heterocycle, such as the γ-hydroxy-γ-lactams (3)
and γ-alkylidene-γ-lactams (4) have also received attention
from the chemical community due to their biological proper-
ties, including anticancer activities. The myceliothermophins
A (5) and F (6), isolated from the thermophilic fungus
Myceliophthora thermophila, were cytotoxic in the concentra-
2
2
γ-alkylidenebutenolides. While this is a possible explana-
tion, other factors can also be involved since a preferential Z
stereochemistry in the products has also been reported for
2
3
similar structures without β substituent.
In a further step, lactones 10a and 10b were treated with
excess amine to produce the corresponding γ-hydroxy-γ-
−
1
15
tion range of 0.2–1.3 μg mL to various cancer cell lines.
2
4
Besides the lactams, some pyridazin-3IJ2H)-ones have also
lactams 11a and 11b, in good yields (70–84%). An impor-
tant feature of these compounds is the presence of a hy-
droxyl, which was confirmed by the absorption bands at 3306
been highlighted as promising lead structures for the devel-
1
6
opment of new cancer treatment agents.
−
1
Accordingly and in line with the continuous effort from
our group in the search for bioactive molecules with antican-
and 3290 cm for 11a and 11b, respectively, in the IR spec-
trum. The hydroxylactams 11 were then dehydrated with
PTSA under reflux affording γ-alkylidene-γ-lactams. As re-
vealed by the TLC analysis of the reaction mixture, two prod-
ucts were always formed. After purification by column chro-
matography, these were identified as the Z and E isomers.
The stereochemistry of the exocyclic double bond was con-
firmed by NOE experiments, where decoupling of H-6 caused
enhancement of the H-7/H-8/H-9/H-10 absorptions, in the
case of compound 13a. Such results confirm that such hydro-
gen atoms are in close proximity in space, as expected for the
isomer E. In general, the Z-compounds were obtained in bet-
ter yields (56–71%) than the E-form (27–36%).
1
7–20
cer activity,
in the present work, a series of rubrolide an-
alogues and their corresponding lactams, totalling 14 com-
pounds, were synthesized.
These new compounds together with 32 other compounds
1
1,12
previously synthesized,
including lactones as rubrolide
analogues and their derived lactams, which had not been
previously analysed for their anticancer properties, were eval-
uated in the present study for their cytotoxic potential
against different human cancer cell lines. Also, the anti-
tumor effects of the most active derivatives were assessed
using the HL-60 cell line. Therefore, studies involving DNA
fragmentation, cell cycle analysis, phosphatidyl serine (PS)
externalization and mitochondrial depolarization were
performed with the objective of understanding their mecha-
nism of action.
Previous work in this area led us to prepare compounds
14–18 (Fig. 2), which were shown to be able to inhibit bacte-
1
2,13
rial biofilm formation.
For the present investigation, the
synthesis was repeated to produce the rubrolide analogues
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Fig. 2 Analogues of rubrolides (14a–14d and 14f–14k) and their corresponding γ-hydroxy-γ-lactams (15a–15e), (Z)-γ-alkylidene-γ-lactams
16a–16e) and (E)-γ-alkylidene-γ-lactams (17a–17e) and pyridazin-3IJ2H)-ones (18f, 18h–18m).
(
(
14a–14d and 14f–14k) and their corresponding γ-hydroxy-γ-
lactams (15a–15e) and γ-alkylidene-γ-lactams (16a–16e and
7a–17e) and pyridazin-3IJ2H)-ones (18f, 18h–18m).
To carry out a preliminary structure–activity relationship
study, the cytotoxicity of the compounds mentioned in
Scheme 1 and Fig. 2 was evaluated against three human tu-
mour cell lines (HCT-116, colon; SF-295, central nervous sys-
tem; and OVCAR-8, ovarian) at 25 μg mL with 72 h of incu-
bation, using the MTT assay.
The results of the antitumor activity for the 45 compounds
tested are presented in Table 1 as percentage reduction in
cell viability (% RCV). Compound 12b was not evaluated due
to its limited amount in our laboratory. A large reduction in
cell viability was achieved when the compounds had % RCV
greater than 75%. Thus, for the HCT-116 cells, 27 compounds
1
−
1
Biological assay
2
5–27
The MTT assay is widely used in cytotoxicity analysis.
is a fast, sensitive and inexpensive method, described for the
first time by Mosman (1983) and subsequently modified by
Alley et al. (1988). This evaluation allows one to easily deter-
mine the cytotoxicity of a particular compound, but it does
not provide any insight into the mechanism of action.
It
2
8
2
9
3
0
Scheme 1 Preparation of γ-alkylidene-γ-lactones (10a–10h) and their corresponding γ-hydroxy-γ-lactams (11a–11b) and γ-alkylidene-γ-lactams
12a–12b and 13a–13b).
(
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Table 1 Percent reduction in cell viability (% RCV) caused by compounds
lactones had better anticancer activity compared with the
lactams examined and that the pyridazinones were ineffective
against the tumour cell lines used in the current study. It
is also important to note that the most active compounds
had halogen substituents such as F, Cl and Br on the
aromatic ring, attached at carbon 3 of the lactone or
lactam ring. Moreover, only one of the 7 compounds was a
γ-alkylidenelactam. Similarly, Bellina et al. demonstrated
that some aryl-halogenated-furanones, including rubrolide M,
possess anticancer activity against three tumour cell lines
1
0a–10h, 11a, 11b, 12a, 13a, 13b, 14a–14d, 14f–14k, 15a–15e, 16a–16e,
−1
17a–17e, 18f, 18h–18m at a single concentration (25 μg mL ) against hu-
man tumour cells after 72 h of incubation, using MTT assay
a
Cell lines – % RCV
Compound
HCT-116
OVCAR-8
SF-295
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
8
1
1
0a
0b
0c
0d
0e
0f
0g
0h
1a
1b
2a
3a
3b
4a
4b
4c
4d
4f
98.27
97.68
76.92
93.05
95.81
62.17
97.58
53.64
49.89
25.82
97.93
57.48
64.83
84.46
97.53
97.09
35.84
55.49
92.20
91.81
98.22
94.87
74.40
78.84
82.39
67.25
78.50
92.75
79.09
44.22
38.65
77.02
86.98
88.80
97.98
75.51
19.23
93.44
14.71
97.73
98.03
98.03
59.80
38.10
10.78
31.84
98.99
98.99
98.20
98.20
0.00
99.86
93.14
94.90
13.42
9.25
54.45
52.94
99.07
98.85
98.28
97.92
26.18
32.6
53.71
96.84
99.50
94.68
57.61
21.61
32.10
27.00
44.39
98.49
47.91
25.49
42.28
20.46
52.15
75.07
90.66
55.28
34.80
93.39
42.17
53.87
46.18
46.18
29.08
27.93
10.69
37.42
94.12
68.44
92.21
84.12
48.84
86.08
92.81
87.84
42.06
10.95
48.29
58.59
97.74
71.31
75.68
48.09
2.02
84.72
82.86
80.10
91.06
82.06
49.40
67.94
56.18
69.20
63.57
78.09
51.01
30.25
25.57
61.86
72.36
84.72
53.77
79.36
6.91
3
1
(
NCI-H460, lung; MCF-7, breast and SF-268, CNS), in agree-
ment with our results.
−1
The RC50 data (μg mL ) for the antitumour activity of the
7
selected compounds are presented in Table 2. In this step
of the study, doxorubicin was used as the positive control
and the tumour cell line HL-60 (human leukaemia) was
added as the tumour biological model, which is commonly
3
2–34
used in experimental anticancer research.
After 72 h of incubation, all the compounds tested showed
moderate cytotoxic activity against the four tumor cell lines
used. It is worth noting that lactones 10a and 14i were the
4g/14g′
4h
4i
−
1
most cytotoxic, with RC50 < 4 μg mL against all the cell
lines used. Analyzing each cell line separately, compound 14i
was the most active against the cell lines OVCAR-8, SF-295
and HCT-116, with RC values ranging from 1.8 to 3.2 μg
4j
4k
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8f
5
0
−
1
mL , and compound 10a was the most active against HL-60
(0.6 μg mL ). These results are in accordance with National
−
1
Cancer Institute (NCI) protocols, where compounds
−1
35
exhibiting IC50 values <4 μg mL are considered active.
As a comparison of the cytotoxicity assay results of the
rubrolide analogues presented in this work with the cytotox-
icity of rubrolides reported in the literature, it was found that
rubrolides M and K (Fig. 1) were also active against some of
the tumor cell lines tested. The best results showed that
rubrolides K and M were very active against HT-29 human co-
−1
lon carcinoma (ED50 = 1.2 μg mL ) among the other cancer
90.65
18.95
73.87
74.92
74.92
48.14
47.29
30.95
47.34
7
cell lines tested.
8h
8i
8i
8j
k
The cytotoxicity of 10a and 14i was also evaluated against
L929 normal cells (mouse fibroblasts). The results presented
in Table 2 show that the cytotoxic effects of these compounds
seemed to be selective for tumour cells.
8l
8m
Based on these results, all subsequent experiments were
conducted to understand the mechanism underlying the cyto-
toxicity of compounds 10a and 14i on the HL-60 cell line.
Initially, the morphology of untreated and treated HL-60
cells was analysed by light microscopy after 24 h of incuba-
tion. The negative control cells showed a typical non-
adherent morphology (Fig. 3). On the other hand, HL-60 cells
treated with 10a showed intense chromatin condensation,
intranuclear vacuoles, cytoplasmic budding, reduction in the
cellular and nuclear volumes, and chromatolysis, which be-
came more evident for incubations at higher concentrations
(Fig. 3E). Additionally, an increasing number of cells with
round shape were observed, suggesting a preserved mem-
brane, but smaller than a viable cell and with an intense
staining, different from the viable ones (Fig. 3C and D),
a
1% < % RCV < 50% = low activity; 50% < % RCV < 75% = RCV
medium activity; and 75% < % RCV < 100% = high activity.
were very active, whereas this number of compounds was less
for the OVCAR-8 cells (18) and SF-295 cells (17). Accordingly,
the ones with the highest % RCV against the three tested cell
lines (% RCV > 80%) were selected, totalling 7 compounds,
for determination of the concentration able to reduce cell via-
bility by 50% (RC50) by the MTT test. Among the 7 com-
pounds, 6 of these were lactones (10a, 10d, 10g, 14h–14j),
only one was a lactam (17e) and none of them had the
pyridazin-3IJ2H)-one nucleus. These results showed that the
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Table 2 Cytotoxic effect of the compounds synthesized on four human tumour cell lines and normal cells
a
RC50 (μM)
Compound
OVCAR-8
SF-295
HCT-116
HL-60
L929
1
1
1
1
1
1
1
0a
0d
0g
4h
4i
13.68 ± 1.68
9.51 ± 1.89
11.64 ± 5 .54
7.51 ± 1.41
2.74 ± 1.11
5.17 ± 0.22
4.18 ± 0.15
0.62 ± 0.09
14.68 ± 9.50
29.37 ± 9.1
32.64 ± 7.41
14.12 ± 2.02
4.90 ± 1.05
15.44 ± 2.55
8.03 ± 0.13
0.44 ± 0.28
8.18 ± 5.91
22.76 ± 5.63
32.22 ± 13.95
12.66 ± 1.09
2.98 ± 1.84
13.06 ± 0.55
5.52 ± 0.66
0.04 ± 0.03
3.00 ± 1.04
8.98 ± 2.48
10.01 ± 2.23
5.50 ± 1.91
3.58 ± 2.36
9.80 ± 2.95
3.46 ± 0.04
0.02 ± 0.02
>50
ND
c
ND
ND
>50
ND
ND
ND
4j
7e
b
Doxorubicin
a
Data are presented as RC50 ± SEM for ovarian (OVCAR-60), central nervous system (SF-295), colon (HCT-116) and leukaemia (HL-60) tumour
b
cells and normal cells (L929), obtained from at least three independent experiments performed in triplicate. Doxorubicin was used as the
positive control. Not determined.
c
which were considered apoptotic cells. Compound 14i also
promoted these apoptotic features in the HL-60 cells, but
with lower intensity (Fig. 3F–H) compared to 10a. Doxorubi-
cin (0.5 μM) induced cell shrinkage, chromatin condensation
and nuclear fragmentation in the HL-60 cell line, which are
features of apoptosis (Fig. 3B).
5 and 10 μM, viable cells were reduced to 67.1, 38.8 and
11.8%, respectively.
Apoptosis started occurring significantly at a low concen-
tration of 2.5 μM (31.3%), and it was extensive at 10 μM
(71.5%). Necrotic cells occurred just at 10 μM (16.7%). These
data show that compounds 10a and 14i may have a potential
lead structure to develop new drugs to overcome cancer by in-
The results reported above were confirmed by fluorescence
microscopy. After 24 h of incubation, compounds 10a and
3
6,37
duction of apoptosis, necrosis or necroptosis.
On the ba-
1
4i caused a decrease in HL-60 cell viability followed by
sis of the morphological results suggestive of apoptosis, we
used flow cytometry to evaluate DNA fragmentation, cell cy-
cle, PS externalization and mitochondrial depolarization in
HL-60 cells incubated with compounds 10a and 14i. After 24
h, DNA fragmentation of the HL-60 cells was significantly in-
creased by the compounds in a concentration-dependent
manner (Table 3).
Doxorubicin at 0.5 μM, used as a positive control, induced
DNA fragmentation in 46.25% of the cells, comparable with
the data from compound 10a (17.50, 29.63 and 51.26%, re-
spectively) and 14i (15.27, 23.17 and 48.52%, respectively) at
an increase in the apoptotic and necrotic cells in
a
concentration-dependent manner (Fig. 4). Compound 10a sig-
nificantly reduced the number of viable cells just at concen-
trations of 5 and 10 μM with values of 17.7 and 47.3%, respec-
tively. Also, apoptotic cells were observed at 5 μM (24.1%)
and 10 μM (46.8%) as previously described. With respect to
necrosis, it was observed only at 10 μM (12.7%). Treatment
with compound 14i showed a significant reduction in cell
viability at all concentrations tested. At concentrations of 2.5,
2.5, 5 and 10 μM. Regarding cell cycle analysis (Table 3), no
changes were observed after incubation with compound 10a.
On the other hand, compound 14i caused accumulation of
Fig. 4 Effect of 10a and 14i on HL-60 cell death pattern determined
by acridine orange- and ethidium bromide-staining (AO/EB) after 24 h
of incubation. The negative control (C) was treated with the vehicle
used for diluting the test compound. Doxorubicin (0.5 μM) was used as
the positive control (D). The results are expressed as mean ± standard
error of mean (SEM) for three independent experiments performed in
triplicate (n = 2). *p < 0.05, compared to the control by ANOVA
followed by Newman–Keuls test.
Fig. 3 Microscopic analysis (May-Grünwald/Giemsa-stained) of the
effect of 10a and 14i on the HL-60 cells after 24 h of incubation.
Untreated cells (A) or cells treated with 10a (2.5 μM, C; 5 μM, D; or 10
μM, E) and 14i (2.5 μM, F; 5 μM, G; or 10 μM, H) were analysed by light
microscopy (200×). Doxorubicin (0.5 μM) was used as the positive con-
trol (B). Black arrows show reduction in the cell volume, nuclear frag-
mentation and cellular debris.
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Table 3 Effect of 10a and 14i on the nuclear DNA content of HL-60 cells determined by flow cytometry after 24 h of incubation
b
Cell cycle phase (%)
Concentration
Compound
(μM)
Sub-G0/G1
G0/G1
S
G2/M
Control
Doxorubicin
—
0.5
2.5
6.10 ± 0.28
46.25 ± 0.81*
17.50 ± 1.11*
29.63 ± 4.10*
51.26 ± 2.45*
15.27 ± 1.10*
23.17 ± 4.15*
48.52 ± 2.23*
45.81 ± 3.16
33.78 ± 1.10*
45.29 ± 0.22
43.19 ± 2.15
33.59 ± 1.15*
72.81 ± 3.16*
72.43 ± 1.17*
31.64 ± 1.25*
27.64 ± 2.15
15.50 ± 1.45*
24.16 ± 1.45
14.77 ± 0.10*
7.26 ± 2.33*
13.75 ± 2.45*
12.93 ± 0.10*
11.81 ± 0.11*
19.04 ± 1.10
2.59 ± 0.21*
7.39 ± 0.25*
4.68 ± 1.10*
2.71 ± 0.50*
5.92 ± 3.17*
2.86 ± 1.15*
9.12 ± 4.11*
a
10a
5
1
.0
0.0
14i
2.5
5
1
.0
0.0
a
b
*
p < 0.05, compared to the control by ANOVA followed by Dunnett's test. Doxorubicin (0.5 μM) was used as the positive control. Results are
expressed as mean ± standard error of mean (SEM) for two independent experiments performed in triplicate (n = 2).
cells in the G0/G1 phase at 2.5 and 5 μM (72.81 and 72.43%,
respectively) (p < 0.05). Cell cycle arrest could have been re-
lated to an attempt by the cell to repair the DNA damage
caused by 14i. Since the damage appeared to be very intense,
that 10a and 14i were also able to cause mitochondrial depo-
larization in a significant and concentration-dependent man-
ner (Fig. 5A). After incubation with 10a, the results showed
that 9.5, 19.0 and 29.0% of the cells displayed PS exter-
nalization and 21.7, 37.1 and 56.2 of the cells showed mito-
chondrial depolarization at 2.5, 5 and 10 μM, respectively
(Fig. 5B). Compound 14i promoted PS externalization in 16.2,
33.3 and 48.1% of the cells and mitochondrial depolarization
in 35.1, 47.0 and 68.0% of the cells at 2.5, 5 and 10 μM, re-
spectively. All results presented herein suggested that com-
pounds 10a and 14i induce cell death by apoptosis in HL-60
3
8
repair did not occur and apoptosis was triggered. The
induction of apoptosis is one of the main mechanisms that
inhibit cancer growth and proliferation and is used by several
3
9,40
antitumor agents.
In addition, the detection of PS externalization showed
that both compounds significantly induced apoptosis
(Fig. 5A) at all concentrations tested. In parallel, it was found
4
1
human leukaemia cells.
Conclusions
We synthesized new rubrolide analogues and converted them
into their corresponding lactam and pyridazin-3IJ2H)-one de-
rivatives. All compounds tested showed moderate cytotoxic
activity against the four human cancer cell lines used. In gen-
eral, the presence of Br and F substituents on the aromatic
ring linked to the γ position of the lactone core increased the
activity. Compared to the lactones and lactams, the pyridazin-
3IJ2H)-ones did not show good activity. Moreover, compounds
10a and 14i displayed superior cytotoxicity against all cell
lines tested. Interestingly, these compounds were not cyto-
toxic towards normal cells at the concentrations tested. Re-
garding the mechanism of action, 10a and 14i induced the
death of HL-60 cells by induction of apoptosis, which was
demonstrated by morphological analysis (volume reduction,
maintenance of cell membrane integrity and nuclear frag-
mentation), DNA fragmentation and PS externalization.
Additionally, compound 14i caused a large increase in the
number of HL-60 cells in the G0/G1 phase. Accordingly,
additional studies are needed to investigate the specific mo-
lecular mechanism of cell death induction and cell cycle
arrest.
Fig. 5 Effect of 10a and 14i on PS externalization (panel A) and
mitochondrial depolarization (panel B) in the HL-60 cells by flow cy-
tometry after 24 h of incubation. The negative control (C) was treated
with the vehicle used for diluting the test compound. Doxorubicin (0.5
μM) was used as the positive control (D). The results are expressed as
mean ± standard error of mean (SEM) for two independent experi-
Acknowledgements
We are grateful to the following Brazilian agencies: Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
ments performed in triplicate (n = 2). *p < 0.05, compared to the con-
trol by ANOVA followed by Newman–Keuls test.
Med. Chem. Commun.
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for research fellowships (CRAM, LCAB), Fundação de Amparo
à Pesquisa de Minas Gerais, Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES) and FINEP for financial
support. Dr. A. Leyva helped with the English editing of this
manuscript.
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