Synthetic cyclopenta[b]indoles exhibit antineoplastic activity by targeting microtubule dynamics in acute myeloid leukemia cells

Article abstract of DOI:10.1016/j.ejphar.2021.173853

Acute promyelocytic leukemia (APL) is associated with PML-RARα oncogene, which is treated using all-trans retinoic acid (ATRA)-based chemotherapy. However, chemoresistance is observed in 20–30% of treated patients and represents a clinical challenge, rais

Full text of DOI:10.1016/j.ejphar.2021.173853

European Journal of Pharmacology 894 (2021) 173853
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Synthetic cyclopenta[b]indoles exhibit antineoplastic activity by targeting
microtubule dynamics in acute myeloid leukemia cells
a
a
b
b,c
Hugo Passos Vicari , Keli Lima , Ralph da Costa Gomes , Daniara Cristina Fernandes
,
a
b
Jean Carlos Lipreri da Silva , Manoel Trindade Rodrigues Junior ,
b
d
Aline Silva Barroso de Oliveira , Ricardo Nascimento dos Santos , Adriano
d
b
a
Defini Andricopulo , Fernando Coelho , Leticia Veras Costa-Lotufo , Jo a˜ o Agostinho Machado-
a,*
Neto
a
Department of Pharmacology, Institute of Biomedical Sciences, University of S a˜ o Paulo, S a˜ o Paulo, SP, 05508-900, Brazil
Department of Organic Chemistry, Chemistry Institute, University of Campinas, Campinas, S a˜ o Paulo, SP, 13083-970, Brazil
Currently at Instituto Federal de Educaç a˜ o Ci ˆe ncia e Tecnologia de S a˜ o Paulo, Mat a˜ o, SP, 15991-502, Brazil
Physics Institute of S a˜ o Carlos, University of S a˜ o Paulo, S a˜ o Carlos, SP, 13565-905, Brazil
b
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
Acute promyelocytic leukemia (APL) is associated with PML-RAR oncogene, which is treated using all-trans
α
cyclopenta[b]indoles
Acute promyelocytic leukemia
Microtubule dynamics
Chemotherapy resistance
retinoic acid (ATRA)-based chemotherapy. However, chemoresistance is observed in 20–30% of treated patients
and represents a clinical challenge, raising the importance of the development of new therapeutic options. In the
present study, the effects of three synthetic cyclopenta[b]indoles on the leukemia phenotype were investigated
using NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Among the tested synthetic cyclopenta[b]in-
doles, compound 2, which contains a heterocyclic nucleus, was the most active, presenting time-dependent
cytotoxic activity in the
for further characterization. Compound 2 significantly decreased clonogenicity, increased apoptosis, and caused
cell cycle arrest at S and G /M phases in a drug concentration-dependent manner. Morphological analyses
μM range in APL cells, without cytotoxicity for normal leukocytes, and was selected
2
indicated aberrant mitosis and diffuse tubulin staining upon compound 2 exposure, which corroborates cell cycle
findings. In the molecular scenario, compound 2 reduced STMN1 expression and activity, and induced PARP1
cleavage and H2AX and CHK2 phosphorylation, and modulated CDKN1A, PMAIP1, GADD45A, and XRCC3 ex-
pressions, indicating reduction of cell proliferation, apoptosis, and DNA damage. Moreover, in the in vivo tubulin
polymerization assay, NB4 and NB4-R2 cells showed a reduction in the levels of polymerized tubulin upon
compound 2 exposure, which indicates tubulin as a target of the drug. Molecular docking supports this hy-
pothesis. Taken together, these data indicated that compound 2 exhibits antileukemic effects through disrupting
the microtubule dynamics, identifying a possible novel potential antineoplastic agent for the treatment of ATRA-
resistant APL.
1
. Introduction
Acute myeloid leukemia (AML) is a heterogeneous hematopoietic
subtypes (M3v) according to the French-American-British classification,
and AML subtype associated with a translocation between chromosomes
15 and 17 [t (15; 17)] and variants according to the World Health Or-
ganization classification (Arber et al., 2016; Bennett et al., 1976; Van
den Berghe, 1988). Indeed, approximately 90% of APL cases are asso-
ciated with t(15; 17) (q22; q21) translocation, resulting in the fusion of
system-related cancer characterized by abnormal clonal proliferation
and impaired differentiation of myeloid precursors (De Kouchkovsky
and Abdul-Hay, 2016; Short et al., 2018). Acute promyelocytic leukemia
(
APL) is AML subtype characterized by bone marrow infiltration by
the PML (promyelocytic leukemia) and RAR
α
(alpha retinoic acid re-
(Melnick
promyelocyte-like blasts, which corresponds to the M3 and M3 variant
ceptor) genes and creating the hybrid oncogene PML-RAR
α
*
Corresponding author. Department of Pharmacology Institute of Biomedical Sciences of University of S ˜a o Paulo Av. Prof. Lineu Prestes, 1524CEP 05508-900, S a˜ o
Paulo, SP, Brazil.
E-mail address: jamachadoneto@usp.br (J.A. Machado-Neto).
https://doi.org/10.1016/j.ejphar.2021.173853
Received 26 August 2020; Received in revised form 11 December 2020; Accepted 5 January 2021
Available online 8 January 2021
0
014-2999/© 2021 Elsevier B.V. All rights reserved.

H.P. Vicari et al.
European Journal of Pharmacology 894 (2021) 173853
and Licht, 1999; Rowley et al., 1977), which has successfully been tar-
geted by all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO)
compounds in solid tumors showed the ability to disrupting tubulin
microtubules and to inhibit cell migration (Santos et al., 2016). In the
present study, we characterized the antileukemia activity of a novel
series of three synthetic enantiomerically pure cyclopenta[b]indoles in
ATRA-sensitive and -resistant APL cellular models.
(
Avvisati et al., 2001; Reiter et al., 2004; Sanz et al., 2009; Wang and
Chen, 2008).
The main mechanisms related to ATRA resistance in APL patients are
genetic mutations in the RAR domain, fusions of the RAR gene with
α
α
other genes limiting the therapeutic effectiveness of ATRA, increased
ATRA catabolism, presence of the acid-binding protein cytoplasmic
retinoic (CRABP), and the abnormal passage from ATRA to the cell
nucleus (Duprez et al., 1992; Kitamura et al., 1997; Nason-Burchenal
et al., 1997). Although the good clinical response of most ATRA-treated
APL patients, relapse still occurs in about 20–30% of these patients and
clinical resistance to ATRA represents a therapeutic challenge.
2. Material and methods
2.1. Cell culture and reagent chemicals
Acute myeloid leukemia cell lines sensitive to ATRA (NB4) and
resistant to ATRA (NB4-R2) were kindly provided by Prof. Dr. Eduardo
Magalh ˜a es Rego (University of S ˜a o Paulo, Ribeir a˜ o Preto, Brazil). Cell
lines were cultured in RPMI supplemented with 10% fetal bovine serum
Therefore, the discovery and development of new therapeutic mol-
ecules become of great importance in the field. Cyclopenta[b]indoles are
present in several biologically active natural and synthetic compounds,
being directly responsible for their biological effects (Qiao et al., 2010;
Ratni et al., 2009; Roll et al., 2009; Talaz et al., 2009). Recently, our
research group synthesized new molecules with the structure based on
cyclopenta[b]indole, and the analysis of the cytotoxic activity of these
(FBS) and 1% penicillin/streptomycin and maintained at 5% CO
2
and
◦
37 C. The authenticity of the cells was determined by Short Tandem
Repeat (STR). The compounds 1, 2, and 3 were obtained as described in
Supplementary Information and prepared as a 10 mM stock solution in
dimethyl sulfoxide (Me
2 4
SO ; DMSO). Of note, all compounds are en-
antiomers that present >95% of purity and absolute stereochemistry (R,
Fig. 1. Characterization of the cytotoxic activity of synthetic cyclopenta[b]indoles in acute myeloid leukemia cell lines. (A) The synthetic cyclopenta[b]indoles were
arbitrarily named as compound 1 (molecular weight: 397.43), 2 (molecular weight: 351.36), and 3 (molecular weight: 365.39), and their molecular chemical
structures are illustrated. (B) Dose-response curves for compound 1, 2, and 3 (0.8–50
μ
M) after 72 h of exposure in NB4 and NB4-R2 cells. The IC50 values of each
M) after 24, 48, and 72 h of exposure in NB4 and NB4-R2
compound are shown. (C) Bar graph represents to dose- and time-responses for compound 2 (0.8–12.5
μ
cells. Values were expressed as a percentage of DMSO-treated (Ø) cells. The results are presented as mean ± S.D. of at least three independent experiments. The P
values and cell lines are shown in the graphs: *P < 0.05; **P < 0.01; ***P < 0.001 for cells treated with compound 2 compared to DMSO-treated (Ø) cells; ANOVA
and Bonferroni post-test.
2

H.P. Vicari et al.
European Journal of Pharmacology 894 (2021) 173853
R) for the two chiral centers. Molecular structures of the cyclopenta[b]
indoles used are illustrated in Fig. 1A. Colchicine was obtained from
Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) and prepared as a
annexin V-FITC. All specimens were analyzed by flow cytometry
(FACSCalibur; Becton Dickinson) after incubation for 15 min at room
temperature in a light-protected area. Ten thousand events were ac-
quired for each sample, using FlowJo software (Treestar, Inc., San
Carlos, CA, USA).
1
0 mM stock solution in DMSO.
2
.2. Cell viability assay
2
.6. Western blot analysis
To evaluate the effects on cell viability of synthesized compounds,
methylthiazoletetrazolium (MTT) assays were performed. Briefly, a total
NB4 and NB4-R2 were treated with compound 2 (0.8, 1.6 or 3.2 M)
μ
4
of 2 × 10 cells per well were seeded in a 96-well plate and exposed to
or vehicle for 48 h and submitted for total protein extraction using a
buffer containing 100 mM Tris (pH 7.6), 1% Triton X-100, 150 mM
increasing concentrations of the compounds (Ø, 0.8, 1.6, 3.2, 6.3, 12.5,
2
5 and 50
μ
M) in RPMI 10% FBS for 24, 48 and/or 72 h. To evaluated
NaCl, 2 mM PMSF, 10 mM Na
3
VO
4
, 100 mM NaF, 10 mM Na
4 2 7
P O , and
cytotoxicity in normal leukocytes, peripheral blood mononuclear cells
4 mM EDTA. Equal amounts of protein (30
μg) of the samples were then
(
(
PBMC) were obtained by Ficoll–Hypaque gradient centrifugation
Sigma-Aldrich, St Louis, MO, USA) from three healthy donors (3 males,
subjected to electrophoresis on SDS-PAGE polyacrylamide gel in an
electrophoresis device, followed by electrotransfer of the gel proteins to
the nitrocellulose membrane. The membrane was blocked with 5% milk
and then incubated with specific primary antibodies diluted in blocking
buffer and then with secondary antibodies conjugated to HRP (horse-
radish peroxidase). Western blot analysis with the indicated antibodies
aged 26–33 years). Informed consent was obtained from all healthy
donors and the Ethics Committee of the Institute of Biomedical Sciences
of the University of S ˜a o Paulo approved this study (Protocol number:
4
423074; CAAE: 39510920.1.0000.5467). Primary PBMC were cultured
TM
in RPMI-1640 medium containing 30% FBS, penicillin/streptomycin
and recombinant cytokines (PeproTech, USA) (30 ng/ml IL3, 100 ng/ml
was performed using the SuperSignal West Dura Extended Duration
Substrate system (Thermo Fisher Scientific, San Jose, CA, USA) and G:
BOX Chemi XX6 gel document system (Syngene, Cambridge, UK
6
IL7, 100 ng/ml FLT3-ligand, and 30 ng/ml SCF), at a density of 2 × 10
cells/ml, in presence of vehicle or compound 2 (Ø, 0.8, 1.6, 3.2, 6.3,
United). The antibodies against Stathmin 1 (OP18; sc-55531), p-Stath-
S16
1
2.5, 25 and 50
μ
M) for 72 h. Then, after incubation, 10
μ
l MTT solution
min 1
(p-OP18 Ser16; sc-12948-R), and γH2AX (sc-517348) were
◦
(
5 mg/ml) was added and incubated at 37 C, 5% CO
2
for 4 h. The re-
obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The
T68
action was stopped by using 100
propanol. Cell viability was evaluated by spectrophotometry measuring
the absorbance at 570 nm. The inhibitory concentration of 50% (IC50
μ
l of 0.1N HCl in anhydrous iso-
antibodies against PARP1 (#9542), GAPDH (#5174), p-CHK2
(#2197), CHK2 (#6334) and
α
-tubulin (#2144) were from Cell
)
Signaling Technology (Danvers, MA, USA).
values was calculated using non-linear regression analysis on GraphPad
Prism 5 (GraphPad Software, Inc., San Diego, CA, USA).
2.7. Quantitative RT-PCR (qRT-PCR)
2
.3. Colony formation assay
NB4 and NB4-R2 were treated with compound 2 (3.2 M) or vehicle
μ
for 48 h and total RNA was obtained using TRIzol reagent (Thermo
Fisher Scientific). cDNA was synthesized from 1 g of RNA using a High-
Colony formation was carried out in semisolid methylcellulose me-
μ
3
dium (0.5 × 10 cells/ml; MethoCult 4230; StemCell Technologies Inc.,
Vancouver, BC, Canada) in the presence, or not, of compound 2 (0.8, 1.6
Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).
Quantitative PCR (qPCR) was performed using a QuantStudio 3 Real-
Time PCR System in conjunction with a SybrGreen System for the
expression of DNA damage-related genes (Supplementary Table 1).
HPRT1 and ACTB were used as reference genes. Relative quantification
and 3.2
μM) for 8 days (long-term exposure). Colonies were detected
after 8 days of culture by adding 150
μ
l (5 mg/ml) of MTT reagent and
scored by Image J quantification software (U.S. National Institutes of
Health, Bethesda, MD, USA).
values were calculated using the 2-
001). A negative ‘No Template Control’ was included for each primer
pair. Data were illustrated using multiple experiment viewer (MeV)
.9.0 software (http://www.tm4.org/mev/).
ΔΔCT
equation (Livak and Schmittgen,
2
2
.4. Cell cycle analysis
4
5
A total of 6 × 10 cells per well were seeded in six-well plates in the
presence, or not, of compound 2 (0.8, 1.6 and 3.2
μM), harvested at 48 h,
2.8. Cell morphology analysis
◦
fixed with 70% ethanol and stored at 4 C for at least 2 h before analysis.
Alternatively, NB4 and NB4-R2 cells were synchronized by the double
thymidine block method. In brief, cells were incubated in 10% FBS RPMI
plus 2 mM thymidine for 16 h (first block). Thymidine was removed by
washing with PBS and fresh 10% FBS RMPI was add to release cells for 8
h. Then, cells were incubated in 10% FBS RPMI plus 2 mM thymidine for
additional 16 h (second block). To release cells for cell cycle progression,
thymidine was removed by washing with PBS and fresh 10% FBS RMPI
5
An amount of 1 × 10 cells was seeded per well in 24-well plates,
with vehicle or compound 2 (1.6 M) for 48 h. After the incubation, the
μ
treated cells were adhered to microscopic slides using cytospin (Sero-
cito, Model 2400, FANEM, Brazil), and subsequent hematoxylin and
eosin staining (rapid panotic). The morphological analyzes of the nu-
cleus and cytoplasm of the treated cells were made from the visualiza-
tion of them in a Leica DM 2500 optical microscope and acquisition of
the photos performed by the LAS V4.6 software (Leica, Germany).
with vehicle or compound 2 (3.2
fixed with 70% ethanol at indicated time points. Fixed cells were stained
with 20 g/ml propidium iodide (PI) containing 10 g/ml RNase A for
0 min at room temperature in a light-protected area. DNA content
μ
M) was add. Cells were harvested and
μ
μ
2.9. Immunofluorescence microscopy
3
distribution was acquired in a FACSCalibur cytometer (Becton-Dick-
inson) and analyzed using FlowJo software (Treestar, Inc.).
Thymidine-synchronized NB4 and NB4-R2 cells, treated with vehicle
or compound 2 were attached on coverslips coated with poly-L-lysine (1
mg/ml) 6 h after thymidine release, fixed with 3.7% formaldehyde,
permeabilized with 0.5% Triton X-PBS and blocked with 3% bovine
2
.5. Apoptosis assessment by annexin V and 7AAD staining
serum albumin (BSA) PBS. Cells were then incubated with anti- -tubulin
α
5
A total of 1 × 10 cells per well were seeded in a 24-well plate in
Alexa Fluor® 488 conjugate (1:200 in 3% BSA PBS, Thermo Fisher
Scientific) for 12 h, and followed by PBS wash. The slides were mounted
in ProLong Gold Anti-Fade Mounting Medium with DAPI (Thermo
Fisher Scientific). Images were generated using fluorescence microscopy
RPMI 10% FBS in the presence, or not, of compound 2 (0.8, 1.6 and 3.2
μ
M) for 48 h. Then, cells were washed twice with ice-cold PBS and
resuspended in binding buffer containing 1 g/ml 7AAD and 1 g/ml
μ
μ
3

H.P. Vicari et al.
European Journal of Pharmacology 894 (2021) 173853
(
LionHeart FX, BioTek, Winooski, VT, USA).
2.12. Statistical analysis
Statistical analysis was performed using GraphPad Instat 5 (Graph-
Pad Software, Inc., San. Diego, CA, USA). For comparisons, ANOVA and
Bonferroni post-test or Student t test were used. A P-value < 0.05 was
considered as statistically significant.
2
.10. Microtubule polymerization in vivo analysis
The intracellular levels of microtubule polymerization were evalu-
ated as previously described (Mistry and Atweh, 2001) with minor
modifications. Briefly, an equal number of NB4 and NB4-R2 cells (1 ×
6
3. Results
1
0 ) treated with vehicle, compound 2 (1.6
μ
M) or paclitaxel (5 nM) for
4
8 h were lysed in microtubule-stabilizing lysis buffer containing 0.1 M
3
.1. Cyclopenta[b]indoles cytotoxicity in acute myeloid leukemia cells
PIPES pH 6.9, 2 M glycerol, 5 mM MgCl2, 2 mM EGTA, 0.5% Triton
X-100, 4
μ
M Taxol, and 5
μ
g/ml leupeptin and submitted to centrifu-
◦
Firstly, three synthetic cyclopenta[b]indoles 1, 2, and 3 were tested
gation at 20,000×g for 45 min at 22 C. Supernatants containing soluble
tubulin (soluble (S) fraction) were separated from the pellets containing
the polymerized tubulin (polymerized (P) fraction). Then, pellets were
solubilized in the microtubule-stabilizing buffer and subjected to ultra-
sonic waves for 20 min. S and P fractions were submitted to Western blot
in ATRA-sensible and ATRA-resistant acute myeloid leukemia cellular
models. Among the evaluated compounds, compound 2 presented
greater cytotoxic potential for both leukemia cell lines. However, the
same cytotoxic potential was not observed for compounds 1 and 3,
which showed less potency and/or efficacy (Fig. 1B). To further char-
acterize the pharmacological proprieties of compound 2, a dose and time
concentration analysis was performed. In both, NB4 and NB4-R2 cells,
compound 2 exerts dose- and time-dependent reduction of cell viability
at similar extension between ATRA-sensitive and ATRA–resistance cells,
analysis for
α
-tubulin detection. GAPDH expression, which is presented
only in S fraction, was used as control of fractionation.
2
.11. Molecular modeling
as evidenced by the similar IC50 (NB4: 1.3 and 1.4
μ
M; NB4-R2: 1.3 and
For docking analysis the GOLD 5.1 software (Cambridge Crystallo-
1
.3 M, upon 48 and 72 h of exposure, respectively; P < 0.05) (Fig. 1C).
μ
graphic Data Centre, Cambridge, UK), crystallographic model of
Colchicine was used as a reference compound in initial assays and its
dose and time concentration curves for NB4 and NB4-R2 cells are shown
tubulin/colchicine (PDB 1SA0) and the structure of compounds 1, 2 and
3
were used.
Fig. 2. Compound 2 inhibits autonomous clonal growth and induces apoptosis in NB4 and NB4-R2 cells. (A) Colonies containing viable cells were detected by the
addition of MTT after 8 days of culture of NB4 and NB4-R2 cells treated with compound 2 (0.8, 1.6, 3.2 M) for 24 h and normalized to the corresponding DMSO-
treated controls (Ø). Colony images are shown for one experiment and the bar graphs show the mean ± S.D. of at least three independent experiments. *P < 0.05;
**P < 0.0001; ANOVA and Bonferroni post-test. (B) Apoptosis was detected by flow cytometry in NB4 and NB4-R2 cells treated with graded concentrations of
compound 2 (0.8, 1.6 and 3.6 M) for 48 h using an annexin V/7AAD staining method. Representative dot plots are shown for each condition; the upper and lower
μ
*
μ
right quadrants (Q2 plus Q3) cumulatively contain the apoptotic population (annexin V+ cells). Bar graphs represent the mean ± S.D. of at least four independent
experiments quantifying apoptotic cell death. The P values and cell lines are indicated in the graphs; *P < 0.05, **P < 0.01; ***P < 0.0001 for cells treated with
compound 2 compared to DMSO-treated (Ø) cells; ANOVA and Bonferroni post-test.
4

H.P. Vicari et al.
European Journal of Pharmacology 894 (2021) 173853
in Supplementary Fig. 1. Of note, compound 2 did not reduce the
during the early progression of the cell cycle. Notably, compound 2
viability of normal leukocytes, suggesting selectivity for malignant
induces a strong cell arrest at G
Supplementary Fig. 3).
2
/M from 6 h of treatment (Fig. 3D and
blood cells (IC50 > 50 M; Supplementary Fig. 2).
μ
3
.3. Compound 2 reduces STMN1 expression/activity, and induces
3
.2. Compound 2 decreases autonomous clonal growth and promotes
apoptosis and DNA damage markers in NB4 and NB4-R2 cells
apoptosis and cell cycle arrest in NB4 and NB4-R2 cells
In the molecular analysis, STMN1 expression and its inactive form
(phospho-STMN1S16) (proliferation marker), γH2AX and CHK2 (DNA
damage marker) and cleaved PARP1 (apoptosis marker) were investi-
gated by Western blot and a panel of 14 DNA damage-related genes
(BAX, BBC3, CDKN1A, CDKN1B, ERCC1, GADD45A, MSH3, PCNA,
PMAIP1, RNF168, RNF8, RPA1, XPC, and XRCC3) were investigated by
quantitative RT-PCR. Increased doses of compound 2 significantly re-
duces STMN1 expression and increases STMN1 phosphorylation, as
well, induces H2AX and CHK2 phosphorylation, and PARP1 cleavage in
both acute myeloid leukemia cell lines (all P < 0.05) (Fig. 4A and B).
Among the DNA damage-related genes investigated, 4 out of 14
(CDKN1A, PMAIP1, GADD45A, and XRCC3) were significantly modu-
lated by compound 2 in NB4 and NB4-R2 cells (all P < 0.05, Fig. 5 and
Supplementary Table 2).
Next, to define the cellular events involved in the reduction of cell
viability upon compound 2 treatment, multiple cellular assays were
performed. Long-term exposure to compound 2 strongly decreased
autonomous clonal growth in both AML cell lines (Fig. 2A). In NB4 and
NB4-R2 cells, 48 h of exposure to compound 2 induces significant levels
of apoptosis (P < 0.05, Fig. 2B).
1
In the cell cycle analysis, a significant increase in cells in subG was
observed in NB4 an NB4-R2 upon compound 2 treatment, which cor-
roborates the findings of apoptosis (all P < 0.05, Fig. 3A–B). Addition-
ally, a significant reduction in a concentration-dependent manner of
cells at G
0
/G
1
was observed (P < 0.05), indicating an accumulation of
/M phases, and failure in the cell cycle progression
Fig. 3C). Due to the large proportion of cells in sub after treatment for
4 h with compound 2 that impacts the distribution of the other cell
cells in S and G
2
(
G
1
2
cycle phases, NB4 and NB4-R2 cells were synchronized with double-
thymidine block and the impact of the compound 2 was evaluated
Fig. 3. Compound 2 leads to cell cycle arrest at S and G
content through staining with propidium iodide and flow cytometry in NB4 or NB4-R2 cells treated with compound 2 (0.8, 1.6 or 3.2
representative histogram for each condition is illustrated. (B) The bar graph represents the mean ± S.D. of the percentages of cells in Sub
/G , S and G /M phases of the cell cycle (excluding subG
1
2
/M phases in acute leukemia cells. (A) The phases of the cell cycle were determined by analyzing the DNA
M) or vehicle for 48 h. A
from at least three
) of at least three
μ
G
1
independent experiments. (C) The mean ± S.D. of the cell distributions that are in the G
0
1
2
independent experiments are represented in the bar graph. The values of P and cell lines are indicated in the graphs; *P < 0.05, **P < 0.01, ***P < 0.001 for DMSO-
treated (Ø) cells vs. compound 2; ANOVA and Bonferroni post-test. (D) NB4 and NB4-R2 cells were synchronized by a double-thymidine block. After thymidine
release, cells were treated with vehicle or compound 2 (3.2
μ
M), collected every 2 h (five time points) and DNA content distribution was evaluated by FACS
as indicated.
5

H.P. Vicari et al.
European Journal of Pharmacology 894 (2021) 173853
Fig. 4. Compound 2 induces molecular markers of
DNA damage and apoptosis in NB4 and NB4-R2
cells. (A) Western blot analysis for levels of pho-
S16
T68
pho (p)-Stathmin 1 , STMN1, γH2AX, p-CHK2
,
CHK2, and PARP1 (total and cleaved) in total cell
extracts of NB4 and NB4-R2 cells treated with
compound 2 (0.8, 1.6 or 3.2 M) or vehicle for 48 h;
μ
The membranes were incubated with the indicated
antibodies and developed with the SuperSignal ™
West Dura Extended Duration Substrate and Gel Doc
XR + system. (B) Bar graphs represent the mean ±
S.D. of three independent experiments quantifying
band intensities of indicated proteins. *P < 0.05,
**P < 0.01, ***P < 0.001; ANOVA and Bonferroni
post-test. Note that compound 2 treatment induces
Stathmin 1 phosphorylation at serine 16 site (an
inhibitory site), H2AX phosphorylation at serine
1
39 and CHK2 at threonine 68 (DNA damage
markers) and PARP1 cleavage (an apoptosis marker)
in NB4 and NB4-R2 cells.
6

H.P. Vicari et al.
European Journal of Pharmacology 894 (2021) 173853
Fig. 5. Compound 2 modulates CDKN1A, PMAIP1, GADD45A, and XRCC3 in NB4 and NB4-R2 cells. (A) The heatmap illustrates the quantitative RT-PCR analysis of
the DNA damage-related genes in NB4 and NB4-R2 cells upon treatment with compound 2 (3.2
M; mean; n = 3) for 48 h. The data are represented as fold-change of
μ
vehicle-treated cells, down and upregulated genes are given by blue and red, respectively. (B) The bar graph represents mean ± S.D. of the fold-change of vehicle-
treated cells for CDKN1A, PMAIP1, GADD45A, and XRCC3 in NB4 and NB4-R2 cells upon compound 2 (3.2
lines are indicated in the graphs; *P < 0.05; **P < 0.01; Student t test.
μM; mean; n = 3) exposure for 48 h. The p values and cell
3
.4. Compound 2 causes microtubule instability and generates mitotic
Cyclopenta[b]indoles are present in several biologically active natural
and synthetic compounds, being directly responsible for their biological
effects (Baran and Richter, 2005; Richter et al., 2008; Uhlig et al., 2009).
Among biological functions described for compounds that contain the
cyclopenta[b]indole portion have been reported abortive and
anti-estrogenic activities, prostaglandin D2 receptor antagonist activity,
progesterone receptor agonist activity, antioxidant and insecticidal ac-
tivity (Levesque et al., 2007; Nicoll-Griffith et al., 2007; Qiao et al.,
2010; Ratni et al., 2009; Roll et al., 2009; Sturino et al., 2007; Talaz
et al., 2009; Wong et al., 1998).
aberrations in NB4 and NB4-R2 cells
NB4 and NB4-R2 cells stained with H&E and analyzed by optical
microscopy showed a high frequency of mitotic aberrations after treat-
ment with 1.6
μ
M of compound 2 for 48 h (Fig. 6A). Since compound 2
induces a strong G
2
/M arrest after 6 h of thymidine release in syn-
chronized NB4 and NB4-R2 cells, we evaluated nuclear and microtu-
bules morphology through immunofluorescence at this time point. In
NB4 and NB4-R2 cells, compound 2 induced diffuse tubulin staining,
indicating instability of microtubules, also, cells with condensed chro-
mosomes or large nuclei were observed, indicating failure to complete
mitosis, which corroborates the data obtained in flow cytometry
Due to the great relevance of these class of compound, several syn-
thetic protocols have been described to synthesize this heterocyclic
nucleus, such as Fischer indolization, Friedel-Crafts intramolecular re-
action and palladium-catalyzed cyclization, but many of these protocols
have low yield and low stereoselectivity (Sorensen and Pombo-Villar,
2004; Xu et al., 2012). The cyclopenta[b]indoles used for the present
(
Fig. 6B). Quantitative data analysis confirms that 6 h after the thymi-
dine block release, vehicle-treated cells present a higher proportion in
interphase (G /G ), which indicates the ability of these cells to complete
0
1
the mitosis. In contrast, the proportion of compound 2-treated NB4 and
NB4-R2 cells with large nuclei or aberrant mitotic spindle were signifi-
cantly increased (P < 0.05; Fig. 6B). Moreover, in vivo tubulin assay
indicates that compound 2 leads to reduction of microtubule polymer-
ization (Fig. 6C). In docking analysis, two main interactions for com-
study were obtained using
a
sequence of reactions using
Morita-Baylis-Hillman (MBH) adducts as building blocks and Indium
Chloride III as an acid catalyst (Supplementary Information). The dif-
ferential of this synthesis is that it is a diastereoselective synthesis,
already reported by our research group (Santos et al., 2016), which also
evaluated the effects of some cyclopenta[b]indoles against a panel of
human tumor cell lines. Some of the tested cyclopenta[b]indoles showed
antitumor activity like doxorubicin, but with better selectivity. For
instance, the cyclopenta[b]indole derivative substituted by a 4-methox-
yphenyl group at C3 (methyl-7-hydroxy-3-(4-methoxyphenyl)-1,2,3,
2
pound 2 and tubulin colchicine site were observed: one with the sp
3
oxygen atom of the carboxyl group and other with a sp oxygen atom of
the dioxolane five-membered ring, indicating that compound 2 has more
efficient interactions than compounds 1 and 3 at the tubulin colchicine
site (Fig. 6D).
4
-tetrahydrocyclopenta[b]indole-2-carboxylate) presented 12 times
4
. Discussion
more potency than doxorubicin to completely inhibit the cell growth in
ovarian cancer cell lines, OVCAR-3 (Santos et al., 2016).
Herein, the anti-leukemia effects of novel synthetic cyclopenta[b]
In the present study, among the three pure enantiomer of novel
synthetic cyclopenta[b]indoles tested, compound 2 displayed higher
indoles in ATRA-sensitive and -resistant APL cell lines were investigated.
7

H.P. Vicari et al.
European Journal of Pharmacology 894 (2021) 173853
Fig. 6. Compound 2 treatment generates aberrant mitoses and microtubule instability in leukemia cell lines. (A) NB4 and NB4-R2 cells were treated with vehicle or
compound 2 (1.6
M) for 48 h, fixed and stained with hematoxylin and eosin (H&E). 400 × and 1000 × magnification images are illustrated. (B) Nuclear and
microtubules morphology was evaluated by immunofluorescence analysis in synchronized NB4 and NB4-R2 cells treated with vehicle or compound 2 after 6 h of
thymidine release. The zoomed area displays details in compound 2-treated NB4 or NB4-R2, -Tubulin (green) and DAPI (blue). Scale bars are shown in figure (200
m). Bar graphs represent the mean ± S.D. of the proportion of NB4 or NB4-R2 treated with vehicle or compound 2 from three independent experiments (at least 150
cells per condition), which were classified according to morphology into interphase, cells with a condensed nucleus (mitosis), large nuclei or aberrant mitotic spindle.
P < 0.05, **P < 0.01, ***P < 0.001; Student t test. (C) NB4 and NB4-R2 cells were treated with vehicle or compound 2 (1.6 M) for 48 h and submitted extraction of
μ
α
μ
*
μ
the soluble (S) and precipitated (P) cellular fractions and Western blot. Free tubulin is present in fraction S, while polymerized tubulin is present in fraction P. The
expression of GAPDH, which is present only in fraction S, was used to verify the efficiency of fractionation. Paclitaxel was used as a control for the polymerization of
microtubules. The ratio of -tubulin present in fractions S and P are described. The membranes were incubated with the indicated antibodies and developed with the
α
SuperSignal ™ West Dura Extended Duration Substrate and Gel Doc XR + system. (D) Conformation proposal of the interaction compound 2 (panel I) on the binding
site of colchicine of the tubulin compared with the interactions of compounds 1 and 3 (panel II).
cytotoxic effects, reduced autonomous clonal growth, which is usually
associated with aggressiveness in leukemia (Lowenberg et al., 1993),
and induced apoptosis and cell cycle arrest in ATRA sensible and resis-
tant APL cells. Additionally, compound 2 did not exhibit cytotoxicity in
normal leukocytes, which suggest selectivity for malignant hematopoi-
etic cells. Of note, initial observations using NB4 as a cellular model of
APL generate evidence of the therapeutic potential of ATRA and arsenic
trioxide (Idres et al., 2001; Shao et al., 1998), which are currently the
first line of treatment in this disease with clinical outcomes superior to
directly inducing microtubule-catastrophe, and high STMN1 expression
has been associated with acceleration in mitosis, migration, and inva-
sion (Belletti and Baldassarre, 2011). Despite the importance of STMN1
in the malignant type of several types of hematological and solid can-
cers, effective pharmacological inhibitors of STMN1 have not yet been
described (Biaoxue et al., 2016). PARP1 is one of several known cellular
substrates of caspases and be cleaved and inactivated by active caspases
3 and 7 and this cleavage is considered as a hallmark of apoptosis
forming fragments of 24 kDa and 89 kDa (Castri et al., 2014; Desroches
and Denault, 2019).
previous therapeutic options (Rego et al., 2013). NB4-R2 is
a
ATRA-resistant subclone of the NB4 APL cell line, which changes the
amino acid Gln903 to an in-phase stop codon, generating a truncated
H2AX is a variant of the H2A protein family, which is a component of
the histone octamer in nucleosomes. In the presence of DNA damage,
H2AX is phosphorylated at the serine 139, being called γH2AX (Kuo and
Yang, 2008; Rogakou et al., 1998, 1999). Similarly, CHK2 is also
phosphorylated upon DNA damage (Zannini et al., 2014). In the in vivo
tubulin polymerization assay, NB4 and NB4-R2 cells showed a great
reduction in the levels of polymerized tubulin. The accumulation of DNA
damage is one of the hallmarks of the mitotic catastrophe, a type of cell
death that occurs during mitosis (Castedo et al., 2004). Among the DNA
damage-related genes investigated, CDKN1A, GADD45A, PMAIP1, and
XRCC3 were modulated by compound 2 in NB4 and NB4-R2 cells.
CDKN1A and GADD45A expressions are induced by DNA damage and
associated with growth arrest and apoptosis (Kreis et al., 2019; Salvador
form of PML-RAR
α
which has lost 52 amino acids at its C-terminal
(
Duprez et al., 2000). In the clinic, ATRA resistance represents a ther-
apeutic challenge in the treatment of APL and new therapeutic options
are of interest (Baljevic et al., 2011; Noguera et al., 2019).
In the molecular scenario, compound 2 reduced STMN1 expression,
and induced PARP1 cleavage, H2AX and CHK2 phosphorylation. In
acute leukemia cells, STMN1 plays an important role in cell cycle pro-
gression and clonogenicity, being a proliferation marker of normal and
malignant hematopoietic cells (Machado-Neto et al., 2014a, 2014b). In
malignant tumors, STMN1 integrates multiple signaling pathways by
regulation of microtubule dynamics, sequestering free tubulin dimers or
8

H.P. Vicari et al.
European Journal of Pharmacology 894 (2021) 173853
et al., 2013). PMAIP1 (also known as NOXA) is a BH3-only member of
the BCL2 family that acts as a pro-apoptotic protein, and its expression is
also activated in response to DNA damage (Morsi et al., 2018). XRCC3
plays a role in DNA double-strand break/recombinational repair to
maintain chromosome stability, and its downregulation have been
associated with increased chemotherapy-induced apoptosis (Roos et al.,
References
Arber, D.A., Orazi, A., Hasserjian, R., Thiele, J., Borowitz, M.J., Le Beau, M.M.,
Bloomfield, C.D., Cazzola, M., Vardiman, J.W., 2016. The 2016 revision to the World
Health Organization classification of myeloid neoplasms and acute leukemia. Blood
127, 2391–2405.
Avvisati, G., Lo Coco, F., Mandelli, F., 2001. Acute promyelocytic leukemia: clinical and
morphologic features and prognostic factors. Semin. Hematol. 38, 4–12.
Baljevic, M., Park, J.H., Stein, E., Douer, D., Altman, J.K., Tallman, M.S., 2011. Curing all
patients with acute promyelocytic leukemia: are we there yet? Hematol. Oncol. Clin.
N. Am. 25, 1215–1233 viii.
2
018). Taken together, these data indicated that the compound 2 dis-
turbs microtubule dynamics, which reduces cell viability by cell cycle
arrest and DNA damage, ultimately, triggering apoptosis.
Baran, P.S., Richter, J.M., 2005. Enantioselective total syntheses of welwitindolinone A
and fischerindoles I and G. J. Am. Chem. Soc. 127, 15394–15396.
Belletti, B., Baldassarre, G., 2011. Stathmin: a protein with many tasks. New biomarker
and potential target in cancer. Expert Opin. Ther. Targets 15, 1249–1266.
Bennett, J.M., Catovsky, D., Daniel, M.T., Flandrin, G., Galton, D.A., Gralnick, H.R.,
Sultan, C., 1976. Proposals for the classification of the acute leukaemias. French-
American-British (FAB) co-operative group. Br. J. Haematol. 33, 451–458.
Biaoxue, R., Xiguang, C., Hua, L., Shuanying, Y., 2016. Stathmin-dependent molecular
targeting therapy for malignant tumor: the latest 5 years’ discoveries and
developments. J. Transl. Med. 14, 279.
Castedo, M., Perfettini, J.L., Roumier, T., Andreau, K., Medema, R., Kroemer, G., 2004.
Cell death by mitotic catastrophe: a molecular definition. Oncogene 23, 2825–2837.
Castri, P., Lee, Y.J., Ponzio, T., Maric, D., Spatz, M., Bembry, J., Hallenbeck, J., 2014.
Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate
cellular protection through NF-kappaB-dependent signaling. Biochim. Biophys. Acta
1843, 640–651.
Regarding structure-activity, our results suggest that the trimethoxy
that has the freest O-methyl groups presented the lower cytotoxic ac-
tivity, and when their freedom were restricted using the O-CH
the cytotoxic activity increased, being even improved by restricting it
even more with O-CH -O. These data suggest that the restriction of the
2 2
-CH -O,
2
freedom groups favors interactions of hydrogen bonding with functional
groups in the active site. Molecular docking experiments support this
2
hypothesis, since the main interactions, one with the sp oxygen atom of
the carboxyl group and other with a sp3 oxygen atom of the dioxolane
five-membered ring, were observed for compound 2.
In summary, the present study identified a novel synthetic cyclo-
penta[b]indole that display antineoplastic effects, inducing cell cycle
deregulation and apoptosis in NB4 and NB4-R2 cell lines. Our explor-
atory molecular analysis identified reduction of proliferation markers
and tubulin stability, and induction of DNA damage and apoptosis
markers. These results shed light on the structure-activity relationship of
cyclopenta[b]indoles allowing new molecular modifications to improve
potency and selectivity, increasing the antineoplastic arsenal.
De Kouchkovsky, I., Abdul-Hay, M., 2016. ’Acute myeloid leukemia: a comprehensive
review and 2016 update. Blood Canc. J. 6, e441.
Desroches, A., Denault, J.B., 2019. Caspase-7 uses RNA to enhance proteolysis of poly
(ADP-ribose) polymerase 1 and other RNA-binding proteins. Proc. Natl. Acad. Sci. U.
S. A. 116, 21521–21528.
Duprez, E., Benoit, G., Flexor, M., Lillehaug, J.R., Lanotte, M., 2000. A mutated PML/
RARA found in the retinoid maturation resistant NB4 subclone, NB4-R2, blocks
RARA and wild-type PML/RARA transcriptional activities. Leukemia 14, 255–261.
Duprez, E., Ruchaud, S., Houge, G., Martin-Thouvenin, V., Valensi, F., Kastner, P.,
Berger, R., Lanotte, M., 1992. A retinoid acid ’resistant’ t(15;17) acute
promyelocytic leukemia cell line: isolation, morphological, immunological, and
molecular features. Leukemia 6, 1281–1287.
CRediT authorship contribution statement
Hugo Passos Vicari: Data curation, Formal analysis, Investigation,
Methodology, Writing - original draft. Keli Lima: Investigation, Meth-
odology, Writing - review & editing. Ralph da Costa Gomes: Investi-
gation, Methodology, Writing - review & editing. Daniara Cristina
Fernandes: Investigation, Methodology, Writing - review & editing.
Jean Carlos Lipreri da Silva: Investigation, Methodology, Writing -
review & editing. Manoel Trindade Rodrigues Junior: Investigation,
Methodology, Writing - review & editing. Aline Silva Barroso de Oli-
veira: Investigation, Methodology, Writing - review & editing. Ricardo
Nascimento dos Santos: Investigation, Methodology, Writing - review
Idres, N., Benoit, G., Flexor, M.A., Lanotte, M., Chabot, G.G., 2001. Granulocytic
differentiation of human NB4 promyelocytic leukemia cells induced by all-trans
retinoic acid metabolites. Canc. Res. 61, 700–705.
Kitamura, K., Kiyoi, H., Yoshida, H., Saito, H., Ohno, R., Naoe, T., 1997. Mutant AF-2
domain of PML-RARalpha in retinoic acid-resistant NB4 cells: differentiation
induced by RA is triggered directly through PML-RARalpha and its down-regulation
in acute promyelocytic leukemia. Leukemia 11, 1950–1956.
Kreis, N.N., Louwen, F., Yuan, J., 2019. The multifaceted p21 (Cip1/Waf1/CDKN1A) in
cell differentiation, migration and cancer therapy. Cancers (Basel) 11.
Kuo, L.J., Yang, L.X., 2008. Gamma-H2AX - a novel biomarker for DNA double-strand
breaks. In Vivo 22, 305–309.
Levesque, J.F., Day, S.H., Chauret, N., Seto, C., Trimble, L., Bateman, K.P., Silva, J.M.,
Berthelette, C., Lachance, N., Boyd, M., et al., 2007. Metabolic activation of indole-
containing prostaglandin D2 receptor 1 antagonists: impacts of glutathione trapping
and glucuronide conjugation on covalent binding. Bioorg. Med. Chem. Lett 17,
&
editing. Adriano Defini Andricopulo: Funding acquisition, Investi-
gation, Writing - review & editing. Fernando Coelho: Conceptualiza-
tion, Formal analysis, Funding acquisition, Supervision, Writing -
original draft. Leticia Veras Costa-Lotufo: Conceptualization, Formal
analysis, Funding acquisition, Supervision, Writing - original draft. Jo a˜ o
Agostinho Machado-Neto: Conceptualization, Data curation, Funding
acquisition, Project administration, Supervision, Writing - original draft.
3
038–3043.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-
time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408.
Lowenberg, B., van Putten, W.L., Touw, I.P., Delwel, R., Santini, V., 1993. Autonomous
proliferation of leukemic cells in vitro as a determinant of prognosis in adult acute
myeloid leukemia. N. Engl. J. Med. 328, 614–619.
Machado-Neto, J.A., de Melo Campos, P., Favaro, P., Lazarini, M., Lorand-Metze, I.,
Costa, F.F., Olalla Saad, S.T., Traina, F., 2014a. Stathmin 1 is involved in the highly
proliferative phenotype of high-risk myelodysplastic syndromes and acute leukemia
cells. Leuk. Res. 38, 251–257.
Declaration of competing interest
Machado-Neto, J.A., Saad, S.T., Traina, F., 2014b. Stathmin 1 in normal and malignant
hematopoiesis. BMB Rep. 47, 660–665.
The authors declare no conflict of interest.
Acknowledgments
Melnick, A., Licht, J.D., 1999. Deconstructing a disease: RARalpha, its fusion partners,
and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 93,
3167–3215.
Mistry, S.J., Atweh, G.F., 2001. Stathmin inhibition enhances okadaic acid-induced
mitotic arrest: a potential role for stathmin in mitotic exit. J. Biol. Chem. 276,
This study was supported by the grant #2017/24993-0, #2019/
3
1209–31215.
2
3864-7 #2019/01700-2, and #2015/17177-6, S a˜ o Paulo Research
Morsi, R.Z., Hage-Sleiman, R., Kobeissy, H., Dbaibo, G., 2018. Noxa: role in cancer
pathogenesis and treatment. Curr. Cancer Drug Targets 18, 914–928.
Nason-Burchenal, K., Maerz, W., Albanell, J., Allopenna, J., Martin, P., Moore, M.A.,
Dmitrovsky, E., 1997. Common defects of different retinoic acid resistant
promyelocytic leukemia cells are persistent telomerase activity and nuclear body
disorganization. Differentiation 61, 321–331.
Foundation (FAPESP), and grant #402587/2016-2, Conselho Nacional
de Desenvolvimento Científico e Tecnol o´ gico (CNPq), and Coordenaç a˜ o
de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). F.C. and A.D.
A. thank also FAPESP for financial support (#2013/07600-3). F.C.
thanks also to CNPq for grants #422890/2016-2 and #301330/2018-2.
Nicoll-Griffith, D.A., Seto, C., Aubin, Y., Levesque, J.F., Chauret, N., Day, S., Silva, J.M.,
Trimble, L.A., Truchon, J.F., Berthelette, C., et al., 2007. In vitro biotransformations
of the prostaglandin D2 (DP) antagonist MK-0524 and synthesis of metabolites.
Bioorg. Med. Chem. Lett 17, 301–304.
Appendix A. Supplementary data
Noguera, N.I., Catalano, G., Banella, C., Divona, M., Faraoni, I., Ottone, T., Arcese, W.,
Voso, M.T., 2019. Acute promyelocytic leukemia: update on the mechanisms of
leukemogenesis, resistance and on innovative treatment strategies. Cancers (Basel)
11.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejphar.2021.173853.
9

H.P. Vicari et al.
European Journal of Pharmacology 894 (2021) 173853
Qiao, M.F., Ji, N.Y., Liu, X.H., Li, K., Zhu, Q.M., Xue, Q.Z., 2010. Indoloditerpenes from
an algicolous isolate of Aspergillus oryzae. Bioorg. Med. Chem. Lett 20, 5677–5680.
Ratni, H., Blum-Kaelin, D., Dehmlow, H., Hartman, P., Jablonski, P., Masciadri, R.,
Maugeais, C., Patiny-Adam, A., Panday, N., Wright, M., 2009. Discovery of
tetrahydro-cyclopenta[b]indole as selective LXRs modulator. Bioorg. Med. Chem.
Lett 19, 1654–1657.
Sanz, M.A., Grimwade, D., Tallman, M.S., Lowenberg, B., Fenaux, P., Estey, E.H.,
Naoe, T., Lengfelder, E., Buchner, T., Dohner, H., et al., 2009. Management of acute
promyelocytic leukemia: recommendations from an expert panel on behalf of the
European LeukemiaNet. Blood 113, 1875–1891.
Shao, W., Fanelli, M., Ferrara, F.F., Riccioni, R., Rosenauer, A., Davison, K., Lamph, W.
W., Waxman, S., Pelicci, P.G., Lo Coco, F., et al., 1998. Arsenic trioxide as an inducer
of apoptosis and loss of PML/RAR alpha protein in acute promyelocytic leukemia
cells. J. Natl. Cancer Inst. 90, 124–133.
Rego, E.M., Kim, H.T., Ruiz-Arguelles, G.J., Undurraga, M.S., Uriarte Mdel, R.,
Jacomo, R.H., Gutierrez-Aguirre, H., Melo, R.A., Bittencourt, R., Pasquini, R., et al.,
2
013. Improving acute promyelocytic leukemia (APL) outcome in developing
Short, N.J., Rytting, M.E., Cortes, J.E., 2018. Acute myeloid leukaemia. Lancet 392,
593–606.
countries through networking, results of the International Consortium on. APL.
Blood 121, 1935–1943.
Sorensen, U.S., Pombo-Villar, E., 2004. Synthesis of cyclopenta[b]indol-1-ones and
carbazol-4-ones fromN-(2-Halophenyl)-substituted enaminones by intramolecular
heck reaction. Helv. Chim. Acta 87, 82–89.
Reiter, A., Lengfelder, E., Grimwade, D., 2004. Pathogenesis, diagnosis and monitoring of
residual disease in acute promyelocytic leukaemia. Acta Haematol. 112, 55–67.
Richter, J.M., Ishihara, Y., Masuda, T., Whitefield, B.W., Llamas, T., Pohjakallio, A.,
Baran, P.S., 2008. Enantiospecific total synthesis of the hapalindoles, fischerindoles,
and welwitindolinones via a redox economic approach. J. Am. Chem. Soc. 130,
Sturino, C.F., O’Neill, G., Lachance, N., Boyd, M., Berthelette, C., Labelle, M., Li, L.,
Roy, B., Scheigetz, J., Tsou, N., et al., 2007. Discovery of a potent and selective
prostaglandin D2 receptor antagonist, [(3R)-4-(4-chloro-benzyl)-7-fluoro-5-
(methylsulfonyl)-1,2,3,4-tetrahydrocyclopent a[b]indol-3-yl]-acetic acid (MK-
0524). J. Med. Chem. 50, 794–806.
1
7938–17954.
Rogakou, E.P., Boon, C., Redon, C., Bonner, W.M., 1999. Megabase chromatin domains
involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916.
Talaz, O., Gulcin, I., Goksu, S., Saracoglu, N., 2009. Antioxidant activity of 5,10-dihy-
droindeno[1,2-b]indoles containing substituents on dihydroindeno part. Bioorg.
Med. Chem. 17, 6583–6589.
Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S., Bonner, W.M., 1998. DNA double-
stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem.
2
73, 5858–5868.
Uhlig, S., Botha, C.J., Vralstad, T., Rolen, E., Miles, C.O., 2009. Indole-diterpenes and
ergot alkaloids in Cynodon dactylon (Bermuda grass) infected with Claviceps
cynodontis from an outbreak of tremors in cattle. J. Agric. Food Chem. 57,
11112–11119.
Roll, D.M., Barbieri, L.R., Bigelis, R., McDonald, L.A., Arias, D.A., Chang, L.P., Singh, M.
P., Luckman, S.W., Berrodin, T.J., Yudt, M.R., 2009. The lecanindoles, nonsteroidal
progestins from the terrestrial fungus Verticillium lecanii 6144. J. Nat. Prod. 72,
1
944–1948.
Van den Berghe, H., 1988. Morphologic, immunologic and cytogenetic (MIC) working
classification of the acute myeloid leukaemias. Second MIC Cooperative Study
Group. Br. J. Haematol. 68, 487–494.
Roos, W.P., Frohnapfel, L., Quiros, S., Ringel, F., Kaina, B., 2018. XRCC3 contributes to
temozolomide resistance of glioblastoma cells by promoting DNA double-strand
break repair. Canc. Lett. 424, 119–126.
Wang, Z.Y., Chen, Z., 2008. Acute promyelocytic leukemia: from highly fatal to highly
curable. Blood 111, 2505–2515.
Rowley, J.D., Golomb, H.M., Vardiman, J., Fukuhara, S., Dougherty, C., Potter, D., 1977.
Further evidence for a non-random chromosomal abnormality in acute
promyelocytic leukemia. Int. J. Canc. 20, 869–872.
Wong, D.C., Fong, W.P., Lee, S.S., Kong, Y.C., Cheng, K.F., Stone, G., 1998. Induction of
estradiol-2-hydroxylase and ethoxyresorufin-O-deethylase by 3-substituted indole
compounds. Eur. J. Pharmacol. 362, 87–93.
Salvador, J.M., Brown-Clay, J.D., Fornace Jr., A.J., 2013. Gadd45 in stress signaling, cell
cycle control, and apoptosis. Adv. Exp. Med. Biol. 793, 1–19.
Xu, B., Guo, Z.L., Jin, W.Y., Wang, Z.P., Peng, Y.G., Guo, Q.X., 2012. Multistep one-pot
synthesis of enantioenriched polysubstituted cyclopenta[b]indoles. Angew Chem.
Int. Ed. Engl. 51, 1059–1062.
Santos, M.S., Fernandes, D.C., Rodrigues Jr., M.T., Regiani, T., Andricopulo, A.D.,
Ruiz, A.L., Vendramini-Costa, D.B., de Carvalho, J.E., Eberlin, M.N., Coelho, F.,
2
016. Diastereoselective synthesis of biologically active cyclopenta[b]indoles.
Zannini, L., Delia, D., Buscemi, G., 2014. CHK2 kinase in the DNA damage response and
beyond. J. Mol. Cell Biol. 6, 442–457.
J. Org. Chem. 81, 6626–6639.
1
0