Accepted Manuscript Hybrid Compounds with Two Redox Centres: Modular Synthesis of ChalcogenContaining Lapachones and Studies on their Antitumor Activity André A. Vieira, Igor R. Brandão, Wagner O. Valença, Carlos A. de Simone, Bruno C. Cavalcanti, Claudia Pessoa, Teiliane R. Carneiro, Prof. Antonio L. Braga, Prof. Eufrânio N. da Silva, Júnior PII:
S0223-5234(15)30119-7
DOI:
10.1016/j.ejmech.2015.06.044
Reference:
EJMECH 7971
To appear in:
European Journal of Medicinal Chemistry
Received Date: 11 February 2015 Revised Date:
20 June 2015
Accepted Date: 22 June 2015
Please cite this article as: A.A. Vieira, I.R. Brandão, W.O. Valença, C.A. de Simone, B.C. Cavalcanti, C. Pessoa, T.R. Carneiro, A.L. Braga, E.N. da SilvaJúnior, Hybrid Compounds with Two Redox Centres: Modular Synthesis of Chalcogen-Containing Lapachones and Studies on their Antitumor Activity, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.06.044. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical abstract
Chalcogen-containing β-lapachone derivatives were designed and synthesized using a straightforward methodology and evaluated against several human cancer cell lines
AC C
EP
TE D
M AN U
SC
RI PT
showing, in some cases, IC50 values below 1 µM.
ACCEPTED MANUSCRIPT Hybrid Compounds with Two Redox Centres: Modular Synthesis of ChalcogenContaining Lapachones and Studies on their Antitumor Activity André A. Vieira,a,f,1 Igor R. Brandão,a Wagner O. Valença,b,1 Carlos A. de Simone,c Bruno C. Cavalcanti,d Claudia Pessoa,d,e Teiliane R. Carneiro,d Antonio L. Bragaa* and
a
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Eufrânio N. da Silva Júniorb* Departamento de Química, UFSC, 88040-900, Florianópolis, SC, Brazil; bInstituto de
Ciências Exatas, Departamento de Química, UFMG, 31270-901, Belo Horizonte, MG, Brazil; cDepartamento de Física e Informática, Instituto de Física, USP, 13560-160, São
SC
Carlos, SP, Brazil; dDepartamento de Fisiologia e Farmacologia, UFC, 60430-270,
Fortaleza, CE, Brazil; eFiocruz – Ceará, 60180-900, Fortaleza, CE, Brazil; fInstituto de
M AN U
Química, Departamento de Química Orgânica, UFBA, 40170-290, Salvador, BA, Brazil.
1. These authors contributed equally to this work. Corresponding authors:
Prof. Eufrânio N. da Silva Júnior - Tel.: 55 31 34095720; Fax: 55 31 34095700.
TE D
E-mail address: [email protected]; Prof. Antonio L. Braga - Tel.: 55 48 37216427; Fax: 55 48 3721 6427; E-mail address: [email protected]
EP
Abstract: Chalcogen-containing β-lapachone derivatives were synthesized using a straightforward methodology and evaluated against several cancer cell lines (leukaemia, human colon carcinoma, prostate, human metastatic prostate, ovarian, central nervous
AC C
system and breast), showing, in some cases, IC50 values below 1 µM. The cytotoxic potential of the lapachones evaluated was also assayed using non-tumor cells: human peripheral blood mononucluear cells, two murine fibroblast lines (L929 and V79 cells) and MDCK (canine kidney epithelial cells). These compounds could provide promising new lead derivatives for anticancer drug development. This manuscript reports important findings since few authors have described C-3 substituted β-lapachone with potent antitumor activity. The methodology employed allowed the preparation of the compounds from lapachol within a few minutes in a green approach.
Keywords: β-Lapachone; Quinone; Selenium; Sulfur; Antitumor; Selenide; Anticancer. 1
ACCEPTED MANUSCRIPT 1. Introduction
In medicinal chemistry, within a broad range of privileged structures, naphthoquinones (NQs) could be considered as a special class due to their notable biological activity against various diseases, including cancer [1], and unique properties,
RI PT
for instance, their characteristics as oxidants and electrophiles [2]. Within the context of cancer, the cytotoxicity of quinones is mainly related to the generation of reactive oxygen species (ROS) and the alkylation of crucial proteins and nucleic acids which provoke cell damage [3].
SC
The naturally occurring naphthoquinone lapachol (1) was isolated for the first time by Arnaudon in 1858 [4] and synthesized in 1927 by Fieser [5]. It is the most abundant naphthoquinoidal compound isolated from the core of trees of the
M AN U
Bignoniaceae family, popularly known in Brazil as ipê. This natural product has been extensively studied due to its important biological activities, for instance, trypanocidal [6], leishmanicidal [7] and antitumoral [8,9,10]. From lapachol (1), β-lapachone (β-lap) can be easily obtained by simple acid cyclization. β-lap (also known as ARQ 501) has been extensively studied in recent years [11,12] and is currently in multiple phase II
[13,14].
TE D
clinical trials as a monotherapy and applied in combination with other cytotoxic drugs
Besides the biological activity presented by β-lap, this compound can be used as a prototype for preparing new bioactive substances [6]. Recently, through the strategy of
EP
the redox centre modification of β-lap, imidazoles with trypanocidal and antimycobacterial activities have been described [15,16]. Di Chenna and coworkers [17] reported the synthesis and antitumor activity of imine derivatives obtained from β-lap
AC C
(Scheme 1). Methodologies for the synthesis of lapachones have also recently emerged, for instance, the use of asymmetric organocatalysis [18,19]. da Silva Júnior and Namboothiri et al. showed the potential of chiral squaramide-catalyzed asymmetric synthesis via cascade reactions of 1,3-dicarbonyls with Morita-Baylis-Hillman acetates of nitroalkenes for the preparation of asymmetric lapachones [18]. Rueping et al. described the synthesis of chiral lapachones from the reaction of lawsone with α,βunsaturated aldehydes in the presence of an organocatalyst to generate α-lapachone derivatives, and after isomerization C-ring-substituted β-lapachones were obtained [19]. Jimenez-Alonso and collaborators described other examples of C-ring-modified lapachones derived from the Knoevenagel condensation of lawsone with unsaturated 2
ACCEPTED MANUSCRIPT aldehydes (Scheme 1). Authors have discussed the action of these compounds as in vitro inhibitors of human topoisomerase II [20]. In addition, β-lapachone-based 1,2,3-triazoles with activity against cancer cells, with IC50 values below 2 µM, have been described by our group [21]. We gained some insights related to the generation of reactive oxygen species (ROS) and carried out some
RI PT
preliminary studies on the mechanism of action in tumor cells. Investigations on the formation of thiobarbituric acid reactive substances (TBARS) and oxidative DNA damage after treatment, detected by the comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase and endonuclease III, were also conducted
SC
[21].
Organoselenium compounds have been widely studied due to their recognized biological activity against several major diseases, including diabetes, Alzheimer’s
M AN U
disease and strokes, and they play an important role in cancer prevention and treatment. [22]. Several mechanisms have been proposed regarding the anticancer activity of these compounds, such as antioxidant protection by selenoenzymes, specific inhibition of tumor cell growth by Se metabolites, modulation of the cell cycle and apoptosis, and an effect on DNA repair, but the specific mode of action is still not fully understood.
TE D
Several kinds of organoselenium compounds are able to mimic the activity of some selenoenzymes, and antioxidant properties are recognized features of these compounds [23]. In the past few years, Jacob and co-workers have conducted studies aimed at the synthesis and antitumor evaluation of chalcogen-containing quinones able
EP
to disturb ROS in cancer cell lines to deadly levels [24]. The strategy employed was based on a combination of ROS generators, for instance, a quinoidal system, and ROS users, exemplified by organochalcogens. In general terms, ROS in cancer cells are
AC C
elevated to levels close to the critical redox, resulting in the induction of apoptosis [24,25]. ROS-generating quinones efficiently enhance the formation of, for example, H2O2, O2•- and •OH radicals, but interesting effects can be observed by diminishing the capacity of the cell to eliminate these species (antioxidant defense). Pre-existing ROS can be used by organochalcogen compounds, accelerating reactions with redox-sensitive proteins and enzymes, and this process can cause malfunction of the cell and finally cell death [26]. Following this approach, 1,4-naphthoquinone-based chalcogens and peptidomimetic compounds with the presence of chalcogen and quinone redox centres and notable antitumor activity have been reported [24-27] (Figure 1).
3
ACCEPTED MANUSCRIPT Figure 1.
Based on recent findings reported by our group and other authors, we envisioned a straightforward synthesis of chalcogen-containing β-lapachone. The compounds were evaluated against ten types of cancer cell lines, HL-60 (leukaemia), MOLT-4
RI PT
(leukaemia), HCT-116 (human colon carcinoma), HCT-8 (colon), PC3 (prostate), PC3M (human metastatic prostate), OVCAR-3 (ovarian), OVCAR-8 (ovarian), SF295 (central nervous system) and MDA-MB-435 (breast), and normal cell lines, exemplified by peripheral blood mononuclear cells (PBMC), V79 and L929. The preparation of this
SC
class of β-lapachones by exploiting the electrophilic reactivity of different dichalcogenides and studies on their antitumor properties are as yet unreported.
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Scheme 1.
2. Results and discussion 2.1 Chemistry
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The chemical reactivity of lapachol (1) is mainly related to the isoprenyl lateral chain which is susceptible to reaction with different electrophiles. As previously reported, the reactions with sulfuric acid [28], bromine [29], iodine [30] and metachloroperoxybenzoic acid [31], to name a few, can provide the respective substituted
EP
bioactive β-lapachone derivatives [32] (Scheme 1). Bearing in mind the nucleophilic characteristic of lapachol (1), the selenium-containing β-lapachone derivatives 2-8 and the sulfur analogous 9-13 were synthesized using a recently described methodology [33]
AC C
through reaction with electrophilic species RYI (Y = S or Se). The chalcogenylation of lapachol (1) using molecular iodine as a catalyst, DMSO as a stoichiometric oxidant and different nucleophiles under microwave irradiation afforded the selenium- and sulfurcontaining β-lapachone derivatives (Schemes 2 and 3). This methodology represents a rapid, green and efficient method to prepare chalcogen-containing β-lapachone, with a reaction time of only 10 min.
Scheme 2. Scheme 3. 4
ACCEPTED MANUSCRIPT A proposed reaction pathway for the chalcogen functionalization of lapachol (1), based on the information reported above, is illustrated in Scheme 4. Initially, the electrophilic specie RYI (Y = S or Se) is formed, probably through the reaction of diorganyl dichalcogenide with the catalyst. The electrophile generated then undergoes
RI PT
nucleophilic attack of the double bond of the isoprenyl lateral chain of the lapachol (1) leading to the formation of the intermediate chalcogeniranium ion. In the next step, a cyclization and the elimination of H+ lead to the formation of chalcogen-containing βlapachone (Scheme 4). Only the formation of the ortho-quinone was observed in the
evidenced.
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Scheme 4.
SC
reaction. The other possible product, para-quinone (α-lapachone derivative), was not
The chemical shift of the hydrogens of the pyranic ring of β-lap derivatives appears in the same region (δ 2.5-3.5). In general, the signals of the hydrogens of the two methyl groups appear at δ 1.6 and 1.5. The other signals corresponding to the substitution pattern observed for each substance are totally in accordance with those
TE D
expected for the prepared compounds. For all compounds, the signals consistent with the proposed structures can be observed on the 13C NMR spectra. The unpublished compounds 3-8 and 9-13 were obtained as orange and red solids, respectively, in moderate to high yields, and their structures were determined by
EP
IR, 1H and 13C NMR. Electrospray ionization mass spectra were also obtained. Suitable crystals of compound 5 were obtained, as an example, and the structure was confirmed
AC C
by crystallographic methods
2.2. X-ray analysis
The bond lengths and angles are in good agreement with the expected values, as reported in the literature [34]. The atoms of the rings (C1-C10) are coplanar and the largest deviation [0.072(2) Å] from the least-squares plane is exhibited by atom C9. Atoms O1, O2 and O3 are in the mean least-squares plane of the rings with deviations of -0.112(2), 0.199(2) and -0.132(3) Å, respectively. Regarding the pyran ring, the atoms C12 and C13 are out of the least-squares plane, giving this ring the conformation 5
ACCEPTED MANUSCRIPT of a half chair. The puckering parameters calculated for this conformation were: q2 = 0.178(6) Å, q3 = 0.459(6) Å, Q = 0.492(3) Å, θ = 21.2(7)° and φ = 132.1(3)° [35]. The bond angles between atoms are 96.6° for C16-Se-C12 and 109.9° for C11-C12-Se. The dihedral angle between the least-squares plane calculated through the atoms [C1-C10] and [C16-C21] is 141.37(1)° (Figure 2). All H atoms were located by geometric
RI PT
considerations placed (C-H = 0.93-0.97 Å) and refined using a riding model with Uiso(H) = 1.5Ueq(C-methyl) or 1.2Ueq(other). An Ortep-3 diagram of the molecule is shown in Figure 2 and Table S1 shows the main crystallographic data.
SC
Figure 2.
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2.3. Biological Activity
All of the substances described (Schemes 2 and 3) were evaluated in vitro using the MTT assay against ten cancer cell lines: HL-60, MOLT-4, HCT-116, HCT-8, PC3, PC3M, OVCAR-3, OVCAR-8, SF295 and MDA-MB-435. β-Lapachone and doxorubicin were used as positive controls (Table 1). Normal cells, human peripheral
TE D
blood mononucluear cells (PBMC), murine fibroblasts cell lines (L929 and V79) and canine kidney epithelial cells (MDCK) were used to evaluate the selectivity of the compounds. As previously described [36], the compounds were classified according to their activity as highly active (IC50 < 2 µM), moderately active (2 µM < IC50 < 10 µM),
EP
or inactive (IC50 >10 µM).
Chalcogen-containing β-lapachone (compounds 2, 4-10 and 12-13) were considered moderately active, with IC50 values in the range of 2.03-10.0 µM. However,
AC C
high activity was observed in some lineages evaluated. For HL-60, compounds 6 (IC50 = 0.94 µM) and 7 (IC50 = 0.53 µM) were considered to be highly active. Compounds 4, 910, 12-13 with IC50 values in the range of 1.22-1.92 µM were also promising against HL-60 (Table 1). Another lineage sensitive to the compounds described herein was MOLT-4 (leukaemia) with four active compounds 6-7 and 12-13 (IC50 values between 0.73-1.89 µM). Recently, we described the synthesis of nor-β-lapachone-based 2,1,3benzothiadiazole-linked-triazole with potent activity against MOLT-4 [37], showing the importance of this class of compounds against leukaemia cancer cell lines. Into the new compounds prepared, we inserted phenylselenyl and phenylthio substituents with the presence of electron-withdrawing (chlorine, bromine and 6
ACCEPTED MANUSCRIPT trifluoromethyl) and electron-donating (methyl and methoxy) groups. Phenylselenyl (unsubstituted), butylselanyl, benzylthio, phenylthio and ethylthio substituents were also considered. In general terms, the choice of the groups was based on two important characteristics: diselenides and disulfides able to generate electrophilic species in the reaction medium and the presence of groups capable of modifying the electronic nature
RI PT
of the molecules studied. Regarding 3-phenylseleno-β-lapachone (2), potent antitumor activity was observed against PC3, OVCAR-3 and OVCAR-8 (IC50 = 0.55, 1.93 and 1.76 µM, respectively). The insertion of a bromine group (electron-withdrawing group in the
SC
para-position) intensified the activity and compound 6 was active against all cancer cell lines evaluated, with IC50 values in the range of 0.94 to 1.72 µM. However, this compound presented cytotoxicity against the four normal cell lines used in the study,
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and the selectivity index was low (Table 2). We considered compound 7, with the presence of a trifluoromethyl group, one of the most promising of this series. This compound was very active against HL-60 and MOLT-4, as discussed previously, but when evaluated against the normal cell line PBMC the IC50 value was 6.36 µM. The selectivity index for 7 was 12.0. A comparison with doxorubicin (selectivity index =
TE D
10.0) indicates that 7 should be considered for further studies.
Compounds 12 and 13, with the presence of a sulfur atom, were active against seven cancer lineages with IC50 values < 2 µM. For these substances moderate cytotoxicity was observed against the normal cell line PBMC, with IC50 values between
EP
3.40 and 3.94 µM.
Lapachones represent an important family of compounds with relevant antitumor activity [38]. Since Pink and co-workers reported the use of β-lapachone against tumors overexpress
NAD(P)H:quinone
AC C
that
oxidoreductase
(NQO1),
this
ubiquitous
flavoprotein remains an important intracellular target of β-lap in tumor cells [39]. β-Lap is able to kill different cancer cells that overexpress endogenous NQO1 [40], for instance, solid tumors, and their mechanism of action is related to NQO1-dependent futile redox cycling, which consumes O2 and generates ROS, as recently described by Bey and co-workers [41]. As discussed by the authors, in a redox cycle of β-lap in the presence of NQO1, the hydroquinone form of β-lap is unstable. Through 2 one-electron oxidations, probably using O2, at equilibrium the hydroquinone can be bio-oxidized to β-lap. In this type of redox cycle superoxide is formed [41].
7
ACCEPTED MANUSCRIPT The presence of two individual redox centres in tellurium-containing naphthoquinoidal compounds was confirmed by electrochemical studies carried out by Jacob et al. [42]. Their results indicated the presence of multifunctional properties related to the generation of ROS (quinoidal moiety) and GPx-like peroxidation catalysis by the chalcogenium atom, which provokes cell damage. Recently, Braga and co-
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workers shed light on the mechanism of the GPx-like activity of selenides and selenoxides [43] and demonstrated the participation of hydroxy perhydroxy selenane, as a stronger oxidizing agent in the oxidation of PhSH with H2O2 catalyzed by selenoxides.
SC
In view of the above, the possible mechanism of action involving chalcogencontaining β-lapachone is shown in Scheme 5. In compounds with two redox centres, that is, a quinoidal moiety able to generate ROS and selenium or sulfur redox centres
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for GPx-like peroxidation catalysis, both being biologically important in cellular redox balance, either centre can be responsible for the cell damage [44]. In general, naphthoquinones
are
able
to
generate
ROS
and
the
catalytically
active
organochalcogens are usually capable to use ROS. With the chalcogen-containing βlapachones herein described, the aim was explore simultaneously these different
TE D
properties presented by the quinoidal system and chalcogens in order to obtain more active compounds, which are able to act as redox modulator derivatives, since lethal cocktails of reactive species can push cancer cell lines over a critical redox threshold
EP
and finally kill them through apoptosis as recently proposed by Jacob et al. [24].
Scheme 5.
AC C
Studies on the mechanism of action of selected compounds are currently
underway in our laboratories and will be reported in due course. The aim is to describe, in a concise and complete manner, the proposed mode of action of β-lap derivatives. Initially, to determine the involvement of ROS in the antitumor activity, we performed experiments to measurement the protein oxidation and lipid peroxidation through protein carbonylation and TBARS assays, respectively, using selected compounds 6, 12 and 13 and ovarian OVCAR-3 cancer cells. Compounds 6, 12 and 13 presented IC50 in the range of 1.22 to 2.81 µM for the cancer cell lines evaluated and are among the most active substances of this class. Besides the biological aspects, these compounds
8
ACCEPTED MANUSCRIPT represent three distinct organochalcogen groups, exemplified by phenylselenyl, phenylthio and ethylthio substituents. Proteins are major targets for ROS, and ROS-induced protein modifications can result in the unfolding, or other alteration, of the protein structure. The carbonylation of proteins is an irreversible type of oxidative damage, which is considered to be a
RI PT
widespread indicator of severe oxidative damage, often leading to a loss of protein function [45,46]. Also, lipid peroxidation has been shown to be involved in oxidative stress. Thus, the extent of compound-induced lipid peroxidation was determined by the reaction of thiobarbituric acids (TBARS) which are formed as a byproduct of lipid
SC
peroxidation (i.e. as fat degradation products). As shown in Figure 3 A and B, after 12 h of exposure, OVCAR-3 cells treated with compounds 6, 12 and 13 resulted in a
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significant (p2sigma(I)]
R1 = 0.0557, wR2 = 0.0794
R indices (all data)
R1 = 0.1420, wR2 = 0.1548
Largest diff. peak and hole
0.52 and -0.84 e.Å-3
S19
S0223-5234(15)30119-7
DOI:
10.1016/j.ejmech.2015.06.044
Reference:
EJMECH 7971
To appear in:
European Journal of Medicinal Chemistry
Received Date: 11 February 2015 Revised Date:
20 June 2015
Accepted Date: 22 June 2015
Please cite this article as: A.A. Vieira, I.R. Brandão, W.O. Valença, C.A. de Simone, B.C. Cavalcanti, C. Pessoa, T.R. Carneiro, A.L. Braga, E.N. da SilvaJúnior, Hybrid Compounds with Two Redox Centres: Modular Synthesis of Chalcogen-Containing Lapachones and Studies on their Antitumor Activity, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.06.044. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical abstract
Chalcogen-containing β-lapachone derivatives were designed and synthesized using a straightforward methodology and evaluated against several human cancer cell lines
AC C
EP
TE D
M AN U
SC
RI PT
showing, in some cases, IC50 values below 1 µM.
ACCEPTED MANUSCRIPT Hybrid Compounds with Two Redox Centres: Modular Synthesis of ChalcogenContaining Lapachones and Studies on their Antitumor Activity André A. Vieira,a,f,1 Igor R. Brandão,a Wagner O. Valença,b,1 Carlos A. de Simone,c Bruno C. Cavalcanti,d Claudia Pessoa,d,e Teiliane R. Carneiro,d Antonio L. Bragaa* and
a
RI PT
Eufrânio N. da Silva Júniorb* Departamento de Química, UFSC, 88040-900, Florianópolis, SC, Brazil; bInstituto de
Ciências Exatas, Departamento de Química, UFMG, 31270-901, Belo Horizonte, MG, Brazil; cDepartamento de Física e Informática, Instituto de Física, USP, 13560-160, São
SC
Carlos, SP, Brazil; dDepartamento de Fisiologia e Farmacologia, UFC, 60430-270,
Fortaleza, CE, Brazil; eFiocruz – Ceará, 60180-900, Fortaleza, CE, Brazil; fInstituto de
M AN U
Química, Departamento de Química Orgânica, UFBA, 40170-290, Salvador, BA, Brazil.
1. These authors contributed equally to this work. Corresponding authors:
Prof. Eufrânio N. da Silva Júnior - Tel.: 55 31 34095720; Fax: 55 31 34095700.
TE D
E-mail address: [email protected]; Prof. Antonio L. Braga - Tel.: 55 48 37216427; Fax: 55 48 3721 6427; E-mail address: [email protected]
EP
Abstract: Chalcogen-containing β-lapachone derivatives were synthesized using a straightforward methodology and evaluated against several cancer cell lines (leukaemia, human colon carcinoma, prostate, human metastatic prostate, ovarian, central nervous
AC C
system and breast), showing, in some cases, IC50 values below 1 µM. The cytotoxic potential of the lapachones evaluated was also assayed using non-tumor cells: human peripheral blood mononucluear cells, two murine fibroblast lines (L929 and V79 cells) and MDCK (canine kidney epithelial cells). These compounds could provide promising new lead derivatives for anticancer drug development. This manuscript reports important findings since few authors have described C-3 substituted β-lapachone with potent antitumor activity. The methodology employed allowed the preparation of the compounds from lapachol within a few minutes in a green approach.
Keywords: β-Lapachone; Quinone; Selenium; Sulfur; Antitumor; Selenide; Anticancer. 1
ACCEPTED MANUSCRIPT 1. Introduction
In medicinal chemistry, within a broad range of privileged structures, naphthoquinones (NQs) could be considered as a special class due to their notable biological activity against various diseases, including cancer [1], and unique properties,
RI PT
for instance, their characteristics as oxidants and electrophiles [2]. Within the context of cancer, the cytotoxicity of quinones is mainly related to the generation of reactive oxygen species (ROS) and the alkylation of crucial proteins and nucleic acids which provoke cell damage [3].
SC
The naturally occurring naphthoquinone lapachol (1) was isolated for the first time by Arnaudon in 1858 [4] and synthesized in 1927 by Fieser [5]. It is the most abundant naphthoquinoidal compound isolated from the core of trees of the
M AN U
Bignoniaceae family, popularly known in Brazil as ipê. This natural product has been extensively studied due to its important biological activities, for instance, trypanocidal [6], leishmanicidal [7] and antitumoral [8,9,10]. From lapachol (1), β-lapachone (β-lap) can be easily obtained by simple acid cyclization. β-lap (also known as ARQ 501) has been extensively studied in recent years [11,12] and is currently in multiple phase II
[13,14].
TE D
clinical trials as a monotherapy and applied in combination with other cytotoxic drugs
Besides the biological activity presented by β-lap, this compound can be used as a prototype for preparing new bioactive substances [6]. Recently, through the strategy of
EP
the redox centre modification of β-lap, imidazoles with trypanocidal and antimycobacterial activities have been described [15,16]. Di Chenna and coworkers [17] reported the synthesis and antitumor activity of imine derivatives obtained from β-lap
AC C
(Scheme 1). Methodologies for the synthesis of lapachones have also recently emerged, for instance, the use of asymmetric organocatalysis [18,19]. da Silva Júnior and Namboothiri et al. showed the potential of chiral squaramide-catalyzed asymmetric synthesis via cascade reactions of 1,3-dicarbonyls with Morita-Baylis-Hillman acetates of nitroalkenes for the preparation of asymmetric lapachones [18]. Rueping et al. described the synthesis of chiral lapachones from the reaction of lawsone with α,βunsaturated aldehydes in the presence of an organocatalyst to generate α-lapachone derivatives, and after isomerization C-ring-substituted β-lapachones were obtained [19]. Jimenez-Alonso and collaborators described other examples of C-ring-modified lapachones derived from the Knoevenagel condensation of lawsone with unsaturated 2
ACCEPTED MANUSCRIPT aldehydes (Scheme 1). Authors have discussed the action of these compounds as in vitro inhibitors of human topoisomerase II [20]. In addition, β-lapachone-based 1,2,3-triazoles with activity against cancer cells, with IC50 values below 2 µM, have been described by our group [21]. We gained some insights related to the generation of reactive oxygen species (ROS) and carried out some
RI PT
preliminary studies on the mechanism of action in tumor cells. Investigations on the formation of thiobarbituric acid reactive substances (TBARS) and oxidative DNA damage after treatment, detected by the comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase and endonuclease III, were also conducted
SC
[21].
Organoselenium compounds have been widely studied due to their recognized biological activity against several major diseases, including diabetes, Alzheimer’s
M AN U
disease and strokes, and they play an important role in cancer prevention and treatment. [22]. Several mechanisms have been proposed regarding the anticancer activity of these compounds, such as antioxidant protection by selenoenzymes, specific inhibition of tumor cell growth by Se metabolites, modulation of the cell cycle and apoptosis, and an effect on DNA repair, but the specific mode of action is still not fully understood.
TE D
Several kinds of organoselenium compounds are able to mimic the activity of some selenoenzymes, and antioxidant properties are recognized features of these compounds [23]. In the past few years, Jacob and co-workers have conducted studies aimed at the synthesis and antitumor evaluation of chalcogen-containing quinones able
EP
to disturb ROS in cancer cell lines to deadly levels [24]. The strategy employed was based on a combination of ROS generators, for instance, a quinoidal system, and ROS users, exemplified by organochalcogens. In general terms, ROS in cancer cells are
AC C
elevated to levels close to the critical redox, resulting in the induction of apoptosis [24,25]. ROS-generating quinones efficiently enhance the formation of, for example, H2O2, O2•- and •OH radicals, but interesting effects can be observed by diminishing the capacity of the cell to eliminate these species (antioxidant defense). Pre-existing ROS can be used by organochalcogen compounds, accelerating reactions with redox-sensitive proteins and enzymes, and this process can cause malfunction of the cell and finally cell death [26]. Following this approach, 1,4-naphthoquinone-based chalcogens and peptidomimetic compounds with the presence of chalcogen and quinone redox centres and notable antitumor activity have been reported [24-27] (Figure 1).
3
ACCEPTED MANUSCRIPT Figure 1.
Based on recent findings reported by our group and other authors, we envisioned a straightforward synthesis of chalcogen-containing β-lapachone. The compounds were evaluated against ten types of cancer cell lines, HL-60 (leukaemia), MOLT-4
RI PT
(leukaemia), HCT-116 (human colon carcinoma), HCT-8 (colon), PC3 (prostate), PC3M (human metastatic prostate), OVCAR-3 (ovarian), OVCAR-8 (ovarian), SF295 (central nervous system) and MDA-MB-435 (breast), and normal cell lines, exemplified by peripheral blood mononuclear cells (PBMC), V79 and L929. The preparation of this
SC
class of β-lapachones by exploiting the electrophilic reactivity of different dichalcogenides and studies on their antitumor properties are as yet unreported.
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Scheme 1.
2. Results and discussion 2.1 Chemistry
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The chemical reactivity of lapachol (1) is mainly related to the isoprenyl lateral chain which is susceptible to reaction with different electrophiles. As previously reported, the reactions with sulfuric acid [28], bromine [29], iodine [30] and metachloroperoxybenzoic acid [31], to name a few, can provide the respective substituted
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bioactive β-lapachone derivatives [32] (Scheme 1). Bearing in mind the nucleophilic characteristic of lapachol (1), the selenium-containing β-lapachone derivatives 2-8 and the sulfur analogous 9-13 were synthesized using a recently described methodology [33]
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through reaction with electrophilic species RYI (Y = S or Se). The chalcogenylation of lapachol (1) using molecular iodine as a catalyst, DMSO as a stoichiometric oxidant and different nucleophiles under microwave irradiation afforded the selenium- and sulfurcontaining β-lapachone derivatives (Schemes 2 and 3). This methodology represents a rapid, green and efficient method to prepare chalcogen-containing β-lapachone, with a reaction time of only 10 min.
Scheme 2. Scheme 3. 4
ACCEPTED MANUSCRIPT A proposed reaction pathway for the chalcogen functionalization of lapachol (1), based on the information reported above, is illustrated in Scheme 4. Initially, the electrophilic specie RYI (Y = S or Se) is formed, probably through the reaction of diorganyl dichalcogenide with the catalyst. The electrophile generated then undergoes
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nucleophilic attack of the double bond of the isoprenyl lateral chain of the lapachol (1) leading to the formation of the intermediate chalcogeniranium ion. In the next step, a cyclization and the elimination of H+ lead to the formation of chalcogen-containing βlapachone (Scheme 4). Only the formation of the ortho-quinone was observed in the
evidenced.
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Scheme 4.
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reaction. The other possible product, para-quinone (α-lapachone derivative), was not
The chemical shift of the hydrogens of the pyranic ring of β-lap derivatives appears in the same region (δ 2.5-3.5). In general, the signals of the hydrogens of the two methyl groups appear at δ 1.6 and 1.5. The other signals corresponding to the substitution pattern observed for each substance are totally in accordance with those
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expected for the prepared compounds. For all compounds, the signals consistent with the proposed structures can be observed on the 13C NMR spectra. The unpublished compounds 3-8 and 9-13 were obtained as orange and red solids, respectively, in moderate to high yields, and their structures were determined by
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IR, 1H and 13C NMR. Electrospray ionization mass spectra were also obtained. Suitable crystals of compound 5 were obtained, as an example, and the structure was confirmed
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by crystallographic methods
2.2. X-ray analysis
The bond lengths and angles are in good agreement with the expected values, as reported in the literature [34]. The atoms of the rings (C1-C10) are coplanar and the largest deviation [0.072(2) Å] from the least-squares plane is exhibited by atom C9. Atoms O1, O2 and O3 are in the mean least-squares plane of the rings with deviations of -0.112(2), 0.199(2) and -0.132(3) Å, respectively. Regarding the pyran ring, the atoms C12 and C13 are out of the least-squares plane, giving this ring the conformation 5
ACCEPTED MANUSCRIPT of a half chair. The puckering parameters calculated for this conformation were: q2 = 0.178(6) Å, q3 = 0.459(6) Å, Q = 0.492(3) Å, θ = 21.2(7)° and φ = 132.1(3)° [35]. The bond angles between atoms are 96.6° for C16-Se-C12 and 109.9° for C11-C12-Se. The dihedral angle between the least-squares plane calculated through the atoms [C1-C10] and [C16-C21] is 141.37(1)° (Figure 2). All H atoms were located by geometric
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considerations placed (C-H = 0.93-0.97 Å) and refined using a riding model with Uiso(H) = 1.5Ueq(C-methyl) or 1.2Ueq(other). An Ortep-3 diagram of the molecule is shown in Figure 2 and Table S1 shows the main crystallographic data.
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Figure 2.
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2.3. Biological Activity
All of the substances described (Schemes 2 and 3) were evaluated in vitro using the MTT assay against ten cancer cell lines: HL-60, MOLT-4, HCT-116, HCT-8, PC3, PC3M, OVCAR-3, OVCAR-8, SF295 and MDA-MB-435. β-Lapachone and doxorubicin were used as positive controls (Table 1). Normal cells, human peripheral
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blood mononucluear cells (PBMC), murine fibroblasts cell lines (L929 and V79) and canine kidney epithelial cells (MDCK) were used to evaluate the selectivity of the compounds. As previously described [36], the compounds were classified according to their activity as highly active (IC50 < 2 µM), moderately active (2 µM < IC50 < 10 µM),
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or inactive (IC50 >10 µM).
Chalcogen-containing β-lapachone (compounds 2, 4-10 and 12-13) were considered moderately active, with IC50 values in the range of 2.03-10.0 µM. However,
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high activity was observed in some lineages evaluated. For HL-60, compounds 6 (IC50 = 0.94 µM) and 7 (IC50 = 0.53 µM) were considered to be highly active. Compounds 4, 910, 12-13 with IC50 values in the range of 1.22-1.92 µM were also promising against HL-60 (Table 1). Another lineage sensitive to the compounds described herein was MOLT-4 (leukaemia) with four active compounds 6-7 and 12-13 (IC50 values between 0.73-1.89 µM). Recently, we described the synthesis of nor-β-lapachone-based 2,1,3benzothiadiazole-linked-triazole with potent activity against MOLT-4 [37], showing the importance of this class of compounds against leukaemia cancer cell lines. Into the new compounds prepared, we inserted phenylselenyl and phenylthio substituents with the presence of electron-withdrawing (chlorine, bromine and 6
ACCEPTED MANUSCRIPT trifluoromethyl) and electron-donating (methyl and methoxy) groups. Phenylselenyl (unsubstituted), butylselanyl, benzylthio, phenylthio and ethylthio substituents were also considered. In general terms, the choice of the groups was based on two important characteristics: diselenides and disulfides able to generate electrophilic species in the reaction medium and the presence of groups capable of modifying the electronic nature
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of the molecules studied. Regarding 3-phenylseleno-β-lapachone (2), potent antitumor activity was observed against PC3, OVCAR-3 and OVCAR-8 (IC50 = 0.55, 1.93 and 1.76 µM, respectively). The insertion of a bromine group (electron-withdrawing group in the
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para-position) intensified the activity and compound 6 was active against all cancer cell lines evaluated, with IC50 values in the range of 0.94 to 1.72 µM. However, this compound presented cytotoxicity against the four normal cell lines used in the study,
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and the selectivity index was low (Table 2). We considered compound 7, with the presence of a trifluoromethyl group, one of the most promising of this series. This compound was very active against HL-60 and MOLT-4, as discussed previously, but when evaluated against the normal cell line PBMC the IC50 value was 6.36 µM. The selectivity index for 7 was 12.0. A comparison with doxorubicin (selectivity index =
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10.0) indicates that 7 should be considered for further studies.
Compounds 12 and 13, with the presence of a sulfur atom, were active against seven cancer lineages with IC50 values < 2 µM. For these substances moderate cytotoxicity was observed against the normal cell line PBMC, with IC50 values between
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3.40 and 3.94 µM.
Lapachones represent an important family of compounds with relevant antitumor activity [38]. Since Pink and co-workers reported the use of β-lapachone against tumors overexpress
NAD(P)H:quinone
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that
oxidoreductase
(NQO1),
this
ubiquitous
flavoprotein remains an important intracellular target of β-lap in tumor cells [39]. β-Lap is able to kill different cancer cells that overexpress endogenous NQO1 [40], for instance, solid tumors, and their mechanism of action is related to NQO1-dependent futile redox cycling, which consumes O2 and generates ROS, as recently described by Bey and co-workers [41]. As discussed by the authors, in a redox cycle of β-lap in the presence of NQO1, the hydroquinone form of β-lap is unstable. Through 2 one-electron oxidations, probably using O2, at equilibrium the hydroquinone can be bio-oxidized to β-lap. In this type of redox cycle superoxide is formed [41].
7
ACCEPTED MANUSCRIPT The presence of two individual redox centres in tellurium-containing naphthoquinoidal compounds was confirmed by electrochemical studies carried out by Jacob et al. [42]. Their results indicated the presence of multifunctional properties related to the generation of ROS (quinoidal moiety) and GPx-like peroxidation catalysis by the chalcogenium atom, which provokes cell damage. Recently, Braga and co-
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workers shed light on the mechanism of the GPx-like activity of selenides and selenoxides [43] and demonstrated the participation of hydroxy perhydroxy selenane, as a stronger oxidizing agent in the oxidation of PhSH with H2O2 catalyzed by selenoxides.
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In view of the above, the possible mechanism of action involving chalcogencontaining β-lapachone is shown in Scheme 5. In compounds with two redox centres, that is, a quinoidal moiety able to generate ROS and selenium or sulfur redox centres
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for GPx-like peroxidation catalysis, both being biologically important in cellular redox balance, either centre can be responsible for the cell damage [44]. In general, naphthoquinones
are
able
to
generate
ROS
and
the
catalytically
active
organochalcogens are usually capable to use ROS. With the chalcogen-containing βlapachones herein described, the aim was explore simultaneously these different
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properties presented by the quinoidal system and chalcogens in order to obtain more active compounds, which are able to act as redox modulator derivatives, since lethal cocktails of reactive species can push cancer cell lines over a critical redox threshold
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and finally kill them through apoptosis as recently proposed by Jacob et al. [24].
Scheme 5.
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Studies on the mechanism of action of selected compounds are currently
underway in our laboratories and will be reported in due course. The aim is to describe, in a concise and complete manner, the proposed mode of action of β-lap derivatives. Initially, to determine the involvement of ROS in the antitumor activity, we performed experiments to measurement the protein oxidation and lipid peroxidation through protein carbonylation and TBARS assays, respectively, using selected compounds 6, 12 and 13 and ovarian OVCAR-3 cancer cells. Compounds 6, 12 and 13 presented IC50 in the range of 1.22 to 2.81 µM for the cancer cell lines evaluated and are among the most active substances of this class. Besides the biological aspects, these compounds
8
ACCEPTED MANUSCRIPT represent three distinct organochalcogen groups, exemplified by phenylselenyl, phenylthio and ethylthio substituents. Proteins are major targets for ROS, and ROS-induced protein modifications can result in the unfolding, or other alteration, of the protein structure. The carbonylation of proteins is an irreversible type of oxidative damage, which is considered to be a
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widespread indicator of severe oxidative damage, often leading to a loss of protein function [45,46]. Also, lipid peroxidation has been shown to be involved in oxidative stress. Thus, the extent of compound-induced lipid peroxidation was determined by the reaction of thiobarbituric acids (TBARS) which are formed as a byproduct of lipid
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peroxidation (i.e. as fat degradation products). As shown in Figure 3 A and B, after 12 h of exposure, OVCAR-3 cells treated with compounds 6, 12 and 13 resulted in a
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significant (p2sigma(I)]
R1 = 0.0557, wR2 = 0.0794
R indices (all data)
R1 = 0.1420, wR2 = 0.1548
Largest diff. peak and hole
0.52 and -0.84 e.Å-3
S19
