β-Lapachone analogs with enhanced antiproliferative activity

European Journal of Medicinal Chemistry 53 (2012) 264e274

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Original article

b-Lapachone analogs with enhanced antiproliferative activity Carla Ríos-Luci a,1, Evelyn L. Bonifazi b,1, Leticia G. León a, Juan C. Montero c, Gerardo Burton b, Atanasio Pandiella c, Rosana I. Misico b, **, José M. Padrón a, * a

BioLab, Instituto Universitario de Bio-Orgánica “Antonio González”, Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain Departamento de Química Orgánica/UMYMFOR (CONICET-UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EHA Buenos Aires, Argentina c Centro de Investigación del Cáncer, IBMCC/CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 February 2012 Received in revised form 30 March 2012 Accepted 7 April 2012 Available online 19 April 2012

In this study, we describe the synthesis of a series of a- and b-lapachone containing hydroxyl or methoxyl groups on the benzene ring, by means of the selective acid promoted cyclization of the appropriate lapachol analog. The evaluation of the antiproliferative activity in human solid tumor cell lines provided 7-hydroxy-b-lapachone as lead with enhanced activity over the parent drug b-lapachone. Cell cycle studies, protein expression experiments, and reactive oxygen species analysis revealed that, similarly to b-lapachone, ROS formation and DNA damage are critical factors in the cellular toxicity of 7hydroxy-b-lapachone. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Antitumor agents Cell cycle b-Lapachone Naphthoquinone Structureeactivity relationships

1. Introduction Lapachol (1), and its derivatives a-lapachone (1a) and b-lapachone (ARQ 501, 1b) are natural naphthoquinones (NQs) isolated from the lapacho tree (Tabebuia avellanedae) (Fig. 1) [1]. There is great interest in this family of compounds as a result of their significant biological activities like antibacterial and antifungal [2], antiplasmodial [3], trypanocidal [4], anti-inflammatory [5], antiangiogenic [6], antimetastatic and anti-invasive [7], and anti-cancer [8]. Unlike conventional chemotherapeutic agents, b-lapachone (1b) has been reported to selectively induce cell death in human cancer cells, but not in normal cells [9]. In this particular context, b-lapachone (1b) resulted a promising treatment in phase I clinical trials of pancreatic cancer, head and neck cancer and

* Corresponding author. BioLab, Instituto Universitario de Bio-Orgánica “Antonio González”, Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain. Tel.: þ34 922316502x6126; fax: þ34 922318571. ** Corresponding author. Departamento de Química Orgánica/UMYMFOR (CONICET-UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EHA Buenos Aires, Argentina. E-mail addresses: [email protected] (R.I. Misico), [email protected] (J.M. Padrón). 1 These authors contributed equally. 0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2012.04.008

leiomyosarcoma [10]. b-Lapachone (1b) has gone through multiple phase II clinical trials as monotherapy and in combination with other cytotoxic drugs. However, the exact mechanism of cell death triggered by b-lapachone (1b) remains unknown. Indeed, apoptosis [11], necrosis [12] or autophagy [13] have been observed in cells treated with the drug. Although NAD(P)H quinone oxidoreductase (NQO1) [14] appears as the most reliable biological target for blapachone (1b), DNA topoisomerase I [8], DNA topoisomerase II [15], and MAP kinase [16] have been identified also as possible targets. As part of our program directed at the discovery of new antitumor agents, we have reported earlier the synthesis and antiproliferative activities of 5-hydroxylapachol (2) and related NQs [17]. The structureeactivity relationship (SAR) study revealed that the biological activity in that set of NQs was favored by the presence of the hydroxyl group at position 5 of the benzene ring (Fig. 1). This result encouraged us to study in further detail the effect on the antiproliferative activity of hydroxylated analogs of a- (1a) and blapachone (1b). Herein, we describe the synthesis of a- (1a) and blapachone (1b) analogs bearing hydroxyl or methoxyl substituents on the benzene ring. The compounds were prepared by means of the acid promoted cyclization of the appropriate lapachol precursor, and the conditions for such reaction were explored. In vitro antiproliferative studies of these analogs in the human solid

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Fig. 1. Structures and atom numbering of lapachol (1), a- (1a) and b-lapachone (1b).

tumor cells HBL-100, HeLa, SW1573 and WiDr, provided a lead, which was tested to investigate its mechanism of action. The results were compared to those obtained for b-lapachone (1b). 2. Results and discussion 2.1. Chemistry The most common route to a- (1a) and b-lapachone (1b) is the cyclization of lapachol (1) catalyzed by HCl and H2SO4, respectively (Scheme 1) [18]. Accordingly, the cyclization of lapachol under acidic conditions may follow two possible pathways to give either p- (a-lapachone series) or o-pyranonaphthoquinones (b-lapachone series) [19]. The proposed mechanism for these transformations is depicted in Fig. 2. Treatment of 1 with acid produces the protonation of the double bond of the side chain generating a stable tertiary carbocation. The carbocation is intramolecularly trapped either by the hydroxyl group at C-2 yielding the linear tricyclic derivative a-lapachone (1a) or by the quinonic oxygen group at C-4 giving the angular tricyclic derivative b-lapachone (1b). Indeed, Hooker [18] established a quantitative isomerization of a-lapachone (1a) to b-lapachone (1b) in H2SO4 and the reverse in HCl, and hence a probable inversion of equilibrium. The study was followed by Ettlinger [20], who found that b-lapachone (1b) is a base stronger that a-lapachone (1a). For this reason, although a-lapachone (1a) is more stable than b-lapachone (1b) in conditions of not ionization, the b-oxonium ion is more stable that the corresponding a-oxonium ion. In concentrated H2SO4 the equilibrium favors the b-oxonium, and the b-lapachone (1b) precipitates after dilution with water. The ionization in HCl is not strong enough to displace the equilibrium, and the more stable a-lapachone (1a) is separated when water is added gradually. At this point, it was envisioned that the cyclization of lapachol analogs 2e5 would proceed in a similar fashion. In order to study

the effects of the substituents in this reaction, we first synthesized compounds 2e5. Briefly, 5-hydroxylapachol (2) and 8-hydroxylapachol (3) were obtained by direct prenylation of 2-hydroxyjuglone and 3-hydroxyjuglone as reported earlier [17], and in 57% and 55% yield, respectively. The synthesis of compounds 4 and 5 was accomplished in two steps as shown in Scheme 2. First, the reaction of the appropriate commercially available tetralones 6 and 7 with oxygen and potassium t-butoxide [21] afforded the corresponding 2-hydroxynaphthoquinones 8 and 9 that were prenylated to give the desired methoxylapachols 4 and 5, respectively. Thus, 5-methoxylapachol (4) and 7-methoxylapachol (5) were obtained from 5-methoxytetralone (6) and 7-methoxytetralone (7) in 17% and 14% overall yield, respectively. The results of the cyclization of lapachol analogs 2e5 under diverse acidic conditions are shown in Table 1. At variance with lapachol (1), the reaction of 5-hydroxylapachol (2) with concentrated H2SO4 afforded exclusively the a-lapachone isomer 2a (100%; Table 1, entries 1e2) instead of the expected b-lapachone isomer 2b [17]. The 1H NMR spectrum allows unambiguous assignment of the p-quinone structure as the signal corresponding to the phenolic hydrogen of 2a appears at d 12.37 ppm due to the formation of an intramolecular hydrogen bond with the quinonic oxygen (Fig. 3). This effect is not present in the b-lapachone isomer 2b. In view of these results, different conditions were needed to obtain compound 2b. It is well known that TiCl4 stabilizes the o-quinone moiety via formation of a complex with the quinone oxygens [22]. Thus, blapachone (1b) was obtained upon treatment of a-lapachone (1a) with TiCl4. However, our attempts to isomerize compound 2a using the same reaction conditions were unsuccessful. We speculate that in this case TiCl4 may also form a stable complex with the hydroxyl group and the neighboring quinonic oxygen stabilizing the p-quinone moiety. Recently, it was reported the cyclization of lapachol (1) under microwave irradiation using different solid

Scheme 1. Acid catalyzed cyclization of lapachol and its hydroxy and methoxy analogs: a) See Table 1 for conditions.

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elucidation was obtained from the analysis of the corresponding HMBC spectra, which for 2b showed correlations between the signals of C-10b (d 164.0) and the H-9 (d 7.17) (Fig. 3). We next examined the cyclization of 8-hydroxylapachol (3) (Table 1, entries 8e10). As in the previous case, a mixture of a- and b-lapachone analogs 3a and 3b was obtained, respectively. The synthesis of the natural compound 3b had been reported previously in an overall yield of 6e16% [24e28]. The procedure described here, affords 3b from commercially available juglone in four steps and 44% overall yield. To the best of our knowledge, this is the most efficient synthetic route available to obtain 3b. The cyclization reaction of 5-methoxylapachol (4) afforded derivative 4a as the sole isomer regardless of the acid used when this reaction was carried out at room temperature and long reaction times (Table 1, entries 11e14). As in the previous cases, the b-isomer 4b could be obtained only when the reaction was carried out with MsOH in dichloromethane, at low temperature and short reaction times (Table 1, entries 15e17) although the yield of 4b was much lower. The correlation between the signals at d 7.23 (H-9) and d 165.7 (C-10b) in the HMBC spectrum of the b-lapachone analog 4b support the proposed structure (Fig. 3). Finally, the reaction of compound 5 with MsOH in dichloromethane at 15  C gave the bisomer 5b as the major product in 62% yield, and only 26% yield of the a-isomer 5a. In addition to b-lapachone (1b) and compounds 2be5b, we prepared three extra b-lapachone analogs for biological testing. Scheme 3 shows the synthesis of compounds 10e12. Briefly, methylation of 3b under standard conditions led to 7-methoxy-blapachone (10) in 90% yield. Treatment of 3b with pivaloyl chloride led to 7-pivaloyl-b-lapachone (11) in 40% yield. Demethylation of 5b with BBr3 gave 8-hydroxy-b-lapachone (12) in 53% yield. 2.2. Antiproliferative activity

Fig. 2. The proposed mechanism for the acid catalyzed reaction of lapachol and its analogs.

supports [23]. Based on those results, we reacted under microwave irradiation 5-hydroxylapachol (2) with several acidic solid supports such as MK-10, acid alumina, and silica gel, without satisfactory results. Alternatively, treatment of 5-hydroxylapachol (2) with HClO4 or methanesulfonic acid (MsOH) led to compound 2b, in mixture with compound 2a (Table 1, entries 3e7). Lower temperatures and a short reaction time favored formation of compound 2b when using an excess of MsOH in dichloromethane (Table 1, entries 5e7). Derivatives 2a and 2b may be separated easily from the mixture by column chromatography on silica gel. The unambiguous structural

The biological evaluation of the compounds started with the study of the in vitro antiproliferative activity in order to obtain drug leads and to establish some SARs. Hence, we evaluated compounds 1e5 and 10e12 was studied against a panel of human solid tumor cells HBL-100, HeLa, SW1573, and WiDr. The results expressed as GI50 were obtained using the SRB assay [29] and are shown in Table 2. Overall, the data show a clear difference in activity between the three series of compounds, being the order established as blapachone analogs > a-lapachone analogs z lapachol analogs. Additionally, the sensitivity of the cell lines was in the order HBL100 > SW1573 > HeLa > WiDr. The b-lapachone analog 3b was found to be the most active product of the series with GI50 values in the range 0.029e2.0 mM. With the exception of WiDr cells, compound 3b showed enhanced activity in cell lines when compared to the parent drug b-lapachone (1b). The analysis of the data allowed us to establish some SARs. When compared to the respective parent natural compounds, the presence of a hydroxyl group in position R1 led to the more active

Scheme 2. Synthesis of 5-methoxylapachol (4) and 7-methoxylapachol (5): a) O2, t-BuOK, t-BuOH; b) 1-bromo-3-methylbut-2-ene, NaI, Et3N, DMF.

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Table 1 Acid promoted cyclization of lapachol analogs 2e5. Reaction 2 / 2a þ 2b

3 / 3a þ 3b

4 / 4a þ 4b

5 / 5a þ 5b a b c

Entry c

1 2c 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Acid H2SO4 H2SO4 HClO4 MsOH MsOH MsOH MsOH H2SO4 MsOH MsOH H2SO4 H2SO4 HClO4 MsOH MsOH MsOH MsOH MsOH

(c) (c)

(c)

(c) (c)

Solvent

Temperature ( C)

Time

Selectivity (a:b)a

Isolated yield (%)b

Neat Neat Neat Neat CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 Neat Neat Neat Neat CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2

0-rt 0-rt rt rt 15 25 45 0-rt 15 45 0-rt 0-rt rt rt 15 25 45 15

1h 24 h 24 h 24 h 3 min 3 min 3 min 1h 3 min 3 min 1h 24 h 24 h 24 h 1 min 1 min 1 min 3 min

1:0 1:0 1:0.09 1:0.22 0.63:1 1:1 0.45:1 0.23:1 0.08:1 0.85:1 1:0 1:0 1:0 1:0 1:0.11 1:0.01 1:0.03 0.4:1

Quant. Quant. Quant. 96 Quant. Quant. Quant. 79 Quant. Quant. Quant. Quant. Quant. Quant. 79 86 Quant. 88

Determined by 1H NMR of the reaction product (2, 3 and 5) or from the isolated compounds (4). Total yield of a þ b isomers. Ref. [17].

compounds 2 and 2a, whilst no improvement occurred in 2b. Replacement of the hydroxyl group by a methoxyl substituent produced the less active analogs 4 and 4a. The same replacement in the b-lapachone series (compound 4b) did not have a significant effect, with an antiproliferative profile comparable to 1b and 2b. These findings indicate that the enhancement of the biological activity observed in 2a may be related to the hydrogen bond between the phenolic hydrogen and the quinonic oxygen (Fig. 3), which cannot occur in the methoxy derivatives 4 and 4a, or in the b-lapachone analogs 1b, 2b, and 4b. The introduction of hydroxyl or methoxyl groups in position R2 resulted in loss of activity for lapachol and a-lapachone analogs 5 and 5a, respectively. No improvement in activity was observed for derivatives 5b and 12, when compared to the reference compound b-lapachone (1b). However, analog 3b, which has a hydroxyl group in position R3, gave the best results of all the series in three of the cell lines tested. Once again, a hydrogen bond between the phenolic hydrogen and the quinonic oxygen might explain these findings, as deduced from the loss in activity exhibited by the methoxyl derivative 10. It has been shown that the introduction of hydroxyl groups at 5- and 8positions of the 1,4-naphthoquinone nucleus increases its reduction potential by means of the formation of hydrogen bond with semiquinone radical and quinone dianion [30]. This effect might be responsible for the observed enhancement in the biological activity. In WiDr cells, the influence of a hydroxyl or a methoxyl group in the biological activity was related to its position within the aromatic ring. Thus, in position R1 (2b) and R3 (3b) a hydroxyl group

does not influence the activity when compared to the parent drug b-lapachone (1b), whilst in position R2 (11) the activity is clearly reduced. A methoxyl group in position R1 (4b) does not influence the activity, whilst in position R2 (5b) and R3 (6b) produces significant loss of activity. Our findings are comparable to those reported for 7-hydroxy-b-lapachone analogs against a panel of melanoma (MDA-MB435), leukemia (HL-60), colon (HCT-8), and central nervous system (SF-295) cell lines [25]. Interestingly, said 7hydroxy-b-lapachone analogs (including 3b) did not exhibit lytic effects against mouse erythrocytes. The above results prompted us to perform further biological studies on analog 3b that would shed some light on the mechanisms involved. b-Lapachone (1b) and 2b were used for comparative purposes. 2.3. Cell cycle In a second step, our biological evaluation approach involves the analysis of possible cell cycle disturbances. Cancer is a condition caused by the uncontrolled growth of cells. Hence, the search for new anti-cancer therapies focuses on the discovery of agents that target dividing cells. To determine whether the antiproliferative activity of b-lapachone (1b) and its analogs 2b and 3b was due to cell cycle blockade, we analyzed changes in the cell cycle profiles using flow cytometry. Cells were exposed to each agent at two different concentrations which were chosen based on the GI50 values and the cell line sensitiveness, taking into account that all compounds induced considerably cell death (>20%) at doses higher

Fig. 3. Chemical shifts of the phenolic hydrogen in compounds 2aeb and HMBC correlations in 2b and 4b.

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Scheme 3. Synthesis of b-isomers 10e12: a) MeI, K2CO3, DMF, rt, 22 h; b) pivaloyl chloride, DMPA, Et3N, CH2Cl2, rt, overnight; c) BBr3, CH2Cl2, e40 C-rt, 1 h.

than reported. As shown in Fig. 4, treatment of cells for 24 h with blapachone (1b), 2b or 3b produced accumulation of cells in S or G2/ M phase. The cell cycle alterations were concentration dependent for the three compounds. These results are consistent with previous reports indicating that b-lapachone (1b) and related NQs induced a cell cycle delay in S phase in diverse cancer cell lines [31]. 2.4. Protein expression The third stage of our evaluation protocol comprises diverse protein expression studies that are carefully selected on the basis of results obtained from the cell cycle analysis. In the particular case of compound 3b, which caused cell cycle delay in S phase, we next investigated the expression of proteins involved in cell cycle progression, apoptosis and DNA damage. To get insights into the mechanism of action, we analyzed protein expression at

different time intervals after exposure of HeLa cells to a 3 mM single dose of 3b. The results are shown in Fig. 5. Since we observed death in cell cycle studies, we first looked at the expression of proteins that are affected by apoptotic cell death. Caspases are crucial mediators of apoptosis. Among them, caspase 3 is one of the key executors, which is activated after cleavage of procaspase 3. Caspase 3 is responsible either partially or totally for the proteolytic cleavage of many important proteins, such as PARP [32]. Treatment of HeLa cells with 3 mM 3b did not provoke PARP and procaspase 3 cleavage (Fig. 5). These results indicate that 3b did not activate an apoptotic cell death response, suggesting that a mechanism other than apoptosis may be responsible for the induction of cell death by 3b. Several studies point out that b-lapachone (1b) triggers either apoptotic or necrotic cell death in various human carcinoma and leukemia cells [33,34], suggesting a wide spectrum of activity.

Table 2 GI50 values of naphthoquinones against human solid tumor cells.a Scaffold

a

Comp.

R1

R2

R3

1 2 3 4 5

H OH H OMe H

H H H H OMe

H H OH H H

7.8 0.60 21 22 19

(4.6) (0.12) (4.1) (2.9) (0.6)

2.3 0.42 2.6 30 18

1a 2a 3a 4a 5a

H OH H OMe H

H H H H OMe

H H OH H H

14 2.6 3.4 28 28

(4.5) (0.8) (0.2) (3.1) (4.1)

15 (2.6) 12 (1.2) 1.8 (0.2) >100 40 (8.6)

3.1 (0.1) 2.7 (0.7) 19 (0.9) >100 27 (3.0)

26 (0.6) 18 (1.6) 14 (2.2) >100 82 (13)

1b 2b 3b 4b 5b 10 11 12

H OH H OMe H H H H

H H H H OMe H H OH

H H OH H H OMe OPiv H

1.0 (0.4) 1.1 (0.2) 0.21 (0.04) 1.8 (0.4) 1.7 (0.2) 5.1 (0.4) 1.6 (0.03) 2.5 (0.3)

0.84 (0.32) 0.36 (0.01) 0.029 (0.007) 0.46 (0.01) 0.45 (0.15) 1.4 (0.5) 0.23 (0.01) 2.3 (0.1)

2.0 (0.2) 1.9 (0.1) 2.0 (0.2) 2.1 (0.2) 27 (0.8) 86 (25) 2.1 (0.03) 21 (1.6)

HBL-100 (breast)

0.38 (0.14) 0.62 (0.16) 0.036 (0.009) 0.69 (0.03) 0.41 (0.16) 2.7 (0.4) 0.18 (0.05) 2.3 (0.1)

Values are given in mM and are means of three to six experiments (standard deviation).

HeLa (cervix)

(0.8) (0.11) (0.4) (13) (0.8)

SW1573 (lung)

34 (6.5) 0.70 (0.23) 18 (2.7) 26 (4.5) 17 (2.0)

WiDr (colon)

36 (9.8) 6.3 (1.9) 19 (2.4) >100 68 (16)

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Fig. 4. Cell cycle phase distribution in untreated cells (C) and cells treated for 24 h with b-lapachone (1b), 2b and 3b at two drug doses. a) HBL-100; b) HeLa; c) SW1573; d) WiDr. Areas: black ¼ G0/G1, white ¼ S, gray ¼ G2/M.

We next examined alterations in the expression of proteins that regulate or are regulated by cell cycle. Molecules that play key roles in controlling cell cycle progression are cyclins and the cyclindependent kinases (CDKs). Most cyclins show dramatic fluctuations in their expression during the cell cycle [35]. The expression of cyclin A and E peak at the onset of S phase and during the G1 to S transition, respectively, which is followed by their degradation [36]. As shown in Fig. 5, the levels of cyclin A moderately increased after

3 h of treatment, while cyclin E decreased by the end of treatment. These results are in agreement with the observed S-phase accumulation of HeLa cells treated with compound 3b (Fig. 3). Accumulation of cells in the S phase led us to explore whether treatment with 3b activates the S-phase checkpoint. The S-phase checkpoint decreases the rate of DNA synthesis after induction of DNA damage, mainly associated to double strand breaks (DSBs), and controls the activation of DNA repair pathway [36]. The DNA damage response pathway is a signal transduction pathway consisting of sensors, transducers and effector proteins. Once DSBs are generated the MRE11A-RAD50-NBS1 complex (MRN) acts as a damage sensor and facilitates the activation of ataxia telangiectasia mutated (ATM) kinase. Under these circumstances, ATM phosphorylates downstream substrates such as histone H2AX, a surrogate marker of DSBs, and checkpoint protein 2 (Chk2) [37]. Our time-course experiments indicate that exposure of HeLa cells to 3b increased the levels of MRE11A, which was followed by the phosphorylation of Chk2 (p-Chk2) and H2AX (g-H2AX). These results show that 3b induced in treated HeLa cells a strong DNA damage response, possibly due to generation of DSBs. 2.5. Reactive oxygen species

Fig. 5. Immunoblotting of protein extracts from untreated HeLa cells (C) and HeLa cells treated at different time intervals with compound 3b at 3 mM.

Since b-lapachone (1b) has been suggested to exert its cytotoxicity by the generation of superoxide and reactive oxygen species (ROS) within mitochondria [38], we decided to evaluate whether its analog 3b was able to induce ROS generation. ROS are traditionally regarded as toxic metabolic byproducts that have the potential to cause direct damage to biological macromolecules such as DNA. ROS formation has been implicated in the antitumor effects of some anti-cancer drugs [39]. The generation of intracellular ROS has been widely applied to measure oxidant levels in living cells by flow cytometry using the probe 20 ,70 -dichlorodyhydrofluorescein diacetate (DCFH-DA). Membraneepermeable DCFH-DA enters cells where it is deacetylated by cytosolic esterases to the membraneeimpermeable

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Fig. 6. Generation of intracellular ROS in untreated HeLa cells (C) and HeLa cells exposed to derivative 3b for 3 h at 3 mM. H2O2 (500 mM) was used as positive control. Oxidant levels were evaluated by flow cytometry using the probe 20 ,70 -dichlorodyhydrofluorescein diacetate (DCFH-DA) which ends as dichlorofluorescein (DCF) upon oxidation by ROS.

dichloro-dihydrofluorescin (DCFH). DCFH accumulates inside the cell, mostly in the cytosol [40]. Once in cells, DCFH is oxidized by ROS into dichlorofluorescein (DCF), which can be visualized easily by flow cytometry [41]. When compared to untreated cells, a sixfold increase in fluorescence intensity was observed in HeLa cells exposed to compound 3b for 3 h at 3 mM (Fig. 6), indicating a raise in ROS levels. Consistent with the results reported for b-lapachone (1b) [13,42], our data shows that the b-lapachone analog 3b also induced the generation of ROS. 2.6. Topoisomerase II inhibition Previous studies have reported on related pyranonaphthoquinones that inhibit the human DNA topoisomerase IIa (TOP2) [43,44]. However, the cell cycle (Fig. 4) and immunoblotting (Fig. 5) results obtained in this study did not provide evidence for a possible inhibition of TOP2. To verify this extent, we tested the capacity of b-lapachone (1b), 2b and 3b to inhibit TOP2-mediated kDNA decatenation, the so-called decatenation assay. This assay is considered to be the most specific one to detect TOP2 activity [45]. The decatenation assay showed unequivocally that b-lapachone (1b), 2b and 3b are not TOP2 inhibitors (Supporting information). 3. Conclusion In summary, we have described the synthesis of a series of aand b-lapachones containing hydroxyl or methoxyl groups on the benzene ring, by means of the acid promoted cyclization of the appropriate lapachol analog. The method constitutes the first synthesis of b-lapachone analogs 2b and 4b. The conventional SAR indicates that the antiproliferative activity is favored by NQs possessing a hydroxy group in the aromatic ring close to the quinonic oxygen. The evaluation of the antiproliferative activity in human solid tumor cell lines provided 7-hydroxy-b-lapachone (3b) as lead, with an enhanced activity over the parent drug b-lapachone (1b). Taken together, our findings show that ROS formation and DNA damage are critical factors in the cellular toxicity of cells exposed to the b-lapachone analog 3b, and this may be the leading mechanism of action of this compound. 4. Experimental 4.1. Chemistry Melting points were taken on a FishereJohns apparatus and are uncorrected. IR spectra were recorded in thin films using KBr disks on a Nicolet Magna 550 FT-IR spectrophotometer, values are given

in cm1. 1H and 13C NMR spectra were recorded on a Bruker Avance II 500 at 500.13 and 125.77 MHz respectively, in deuterochloroform unless indicated otherwise. Chemical shifts are given in ppm downfield from TMS as internal standard, J values are given in Hz. Multiplicity determinations and 2D spectra (COSY, HSQC and HMBC) were obtained using standard Bruker software. Highresolution mass spectra (HRMS) were measured on an Agilent LCTOF, high resolution TOF analyzer or on a Bruker microTOF-Q II spectrometer with ESI ionization. Elemental analysis was performed on an EAI Exeter Analytical, Inc. CE-440 apparatus. Reactions were monitored using thin-layer chromatography (TLC) on aluminum backed precoated Silica Gel 60 F254 plates (E Merck). In general naphthoquinones are highly colored and were visible on a TLC plate; colorless compounds were detected using UV light. Flash chromatography was carried out using Silica Gel 60 (230e400 mesh) with the solvent system indicated in the individual procedures. All solvent ratios are quoted as v/v. Solvents were evaporated at reduced pressure and ca. 40e50  C. 5-Methoxytetralone (6) and 7-methoxytetralone (7) were purchased from SigmaeAldrich (St Louis, MO). 5-Hydroxylapachol (2) and 8-hydroxylapachol (3) were prepared from commercially available juglone [17]. Lapachol (1) was isolated by extraction of the powdered wood of Tabebuia impetiginosa (Bignoniaceae) with a cold solution of sodium carbonate [46]. a-Lapachone (3,4dihydro-2,2-dimethyl-2H-benzo-[g]chromene-5,10-dione, 1a) and b-lapachone (3,4-dihydro-2,2-dimethyl-2H-benzo[h]chromene5,6-dione, 1b) were obtained by intramolecular cyclization of lapachol (1) using concentrated hydrochloric acid and concentrated sulfuric acid, respectively [18]. 4.1.1. General procedure for the acid promoted cyclization reactions Methanesulfonic acid (0.3 mL, 5 mmol), H2SO4 (c) (0.3 mL, 6 mmol) or HClO4 (0.3 mL, 5 mmol) was added to the appropriate quinone (2e5) (0.04 mmol) neat or dissolved in CH2Cl2 (0.1 mL, 0.4 M). The reaction mixture was stirred at the indicated temperature for a variable period of time (see Table 1) and was monitored by TLC. The reaction was quenched with water and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated under vacuum. The residue was analyzed by 1H NMR and then was purified by column chromatography on silica gel (hexanes/EtOAc, gradient). 4.1.1.1. 3,4-Dihydro-6-hydroxy-2,2-dimethyl-2H-benzo[g]chromene5,10-dione (6-hydroxy-a-lapachone, 2a). Compound 2a (20 mg, 100%) was obtained as yellow crystals from 5-hydroxylapachol (2) (20 mg, 0.08 mmol) following the general procedure for cyclization reactions with sulfuric acid (Table 1, entry 1): Rf ¼ 0.71 (hexanes/ EtOAc, 7:3); mp: 179e180  C; 1H NMR: 1.43 (s, 6H, CH3), 1.82 (t, J ¼ 6.6 Hz, 2H, H-3), 2.59 (t, J ¼ 6.6 Hz, 2H, H-4), 7.21 (dd, J ¼ 8.4, 1.0 Hz, 1H, H-7), 7.51 (t, J ¼ 7.9 Hz, 1H, H-8), 7.62 (dd, J ¼ 7.5, 1.0 Hz, 1H, H-9), 12.37 (s, 1H, OH) [47]; 13C NMR: 16.1 (C-4), 26.5 (CH3), 31.3 (C-3), 78.6 (C-2), 114.1 (C-5a), 119.1 (C-9), 119.5 (C-4a), 124.7 (C-7), 131.2 (C-9a), 135.0 (C-8), 155.3 (C-10a), 160.9 (C-6), 179.3 (C-10), 190.1 (C-5); IR (KBr): 2972, 2935, 2869, 2857, 1719, 1679, 1622, 1608 cm1; HRMS-ESI m/z [M þ Na]þ calcd for C15H14NaO4: 281.0784, found: 281.0790. 4.1.1.2. 3,4-Dihydro-10-hydroxy-2,2-dimethyl-2H-benzo[h]chromene5,6-dione (10-hydroxy-b-lapachone, 2b). Compound 2b (7.1 mg, 68.5%) was obtained as red crystals from 5-hydroxylapachol (2) (10 mg, 0.04 mmol) as described in the general procedure for cyclization reactions using methanesulfonic acid (Table 1, entry 7): Rf ¼ 0.42 (hexanes/EtOAc, 7:3); mp: 133e134  C; 1H NMR: 1.56 (s, 6H, CH3), 1.92 (t, J ¼ 6.7 Hz, 2H, H-3), 2.59 (t, J ¼ 6.7 Hz, 2H, H-4), 7.17 (dd, J ¼ 8.4, 1.3 Hz, 1H, H-9), 7.38 (dd, J ¼ 8.2, 7.6 Hz, 1H, H-8), 7.71

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(dd, J ¼ 7.4, 1.3 Hz, 1H, H-7), 9.08 (s, 1H, OH); 13C NMR: 16.1 (C-4), 26.9 (CH3), 31.2 (C-3), 81.9 (C-2), 112.3 (C-4a), 114.6 (C-10a), 122.9 (C7), 126.1 (C-9), 131.0 (C-6a), 132.1 (C-8), 155.8 (C-10), 164.0 (C-10b), 178.4 (C-5), 179.3 (C-6); IR (KBr): 3351, 2978, 1646, 1602, 1394 cm1; HRMS-ESI m/z [M þ H]þ calcd for C15H15O4: 259.0965, found: 259.0972. 4.1.1.3. 3,4-Dihydro-9-hydroxy-2,2-dimethyl-2H-benzo[g]chromene5,10-dione (9-hydroxy-a-lapachone, 3a). Compound 3a (94.8 mg, 46%) was obtained as yellow needles from 8-hydroxylapachol (3) (20 mg, 0.08 mmol) following the general procedure for cyclization reactions with methanesulfonic acid (Table 1, entry 10): Rf ¼ 0.45 (hexanes/EtOAc, 8:2); mp: 115e116  C (from methanol) (lit. [47] 114.5e115  C); 1H NMR: 1.44 (s, 6H, CH3), 1.82 (t, J ¼ 6.6 Hz, 2H, H-3), 2.61 (t, J ¼ 6.6 Hz, 2H, H-4), 7.19 (dd, J ¼ 8.2, 1.4 Hz, 1H, H-8), 7.58 (td, J ¼ 7.5, 0.5 Hz, 1H, H-7), 7.62 (dd, J ¼ 7.4, 1.4 Hz, 1H, H-6), 11.85 (d, J ¼ 0.4 Hz, 1H, OH); 13C NMR: 16.8 (C-4), 26.5 (CH3), 31.3 (C-3), 78.4 (C-2), 114.2 (C-9a), 118.7 (C-6), 121.1 (C-4a), 123.5 (C-8), 132.1 (C-5a), 136.6 (C-7), 154.1 (C-10a), 161.6 (C-9), 183.5 (C-5), 184.9 (C-10); IR (KBr): 2971, 2931, 1638, 1610 cm1; HRMS-ESI m/z [M þ H]þ calcd for C15H15O4: 259.0965, found: 259.0957. 4.1.1.4. 3,4-Dihydro-7-hydroxy-2,2-dimethyl-2H-benzo[h]chromene5,6-dione (7-hydroxy-b-lapachone, 3b). Compound 3b (9.6 mg, 93%) was obtained as reddish orange needles from 8-hydroxylapachol (3) (10 mg, y 0.04 mmol) following the general procedure for cyclization reactions with methanesulfonic acid (Table 1, entries 9e10):Rf ¼ 0.37 (Hexane/EtOAc 7:3); mp: 183e184  C (from methanol) (lit. [26] 184185  C from methanol); 1H NMR: 1.45 (s, 6H, CH3), 1.84 (t, J ¼ 6.7 Hz, 2H, H-3), 2.55 (t, J ¼ 6.7 Hz, 2H, H-4), 7.05 (dd, J ¼ 8.6, 1.0 Hz, 1H, H-8), 7.35 (dd, J ¼ 7.5, 1.0 Hz, 1H, H-10), 7.53 (ddd, J ¼ 8.5, 7.5, 0.3 Hz, 1H, H-9), 11.98 (s, 1H, OH); 13C NMR: 16.2 (C-4), 26.7 (CH3), 31.5 (C-3), 79.2 (C-2), 112.7 (C-4a), 113.6 (C6a), 116.8 (C-10), 121.4 (C-8), 132.4 (C-10a), 137.9 (C-9), 161.5 (C10b), 164.3 (C-7), 178.1 (C-5), 183.2 (C-6); IR (KBr): 2969, 1653, 1636, 1585, 1570 cm1; HRMS-ESI m/z [M þ H]þ calcd for C15H15O4: 259.0965, found: 259.0970; Anal. calcd for C15H14O4: C 69.76, H 5.46, found: C 69.59, H 5.06. 4.1.1.5. 3,4-Dihydro-6-methoxy-2,2-dimethyl-2H-benzo[g]chromene5,10-dione (6-methoxy-a-lapachone, 4a). Compound 4a (11 mg, 100%) was obtained as yellow crystals from 5-methoxylapachol (4) (11 mg, 0.04 mmol) following the general procedure for cyclization reactions with sulfuric acid (Table 1, entry 11): Rf ¼ 0.63 (CH2Cl2/ MeOH, 4:0.1); mp: 145e146  C; 1H NMR: 1.41 (s, 6H, CH3), 1.79 (t, J ¼ 6.6 Hz, 2H, H-3), 2.59 (t, J ¼ 6.6 Hz, 2H, H-4), 3.99 (s, 3H, CH3O), 7.27 (dd, J ¼ 8.5, 0.9 Hz, 1H, H-7), 7.60 (dd, J ¼ 8.4, 7.6 Hz, 1H, H-8), 7.77 (dd, J ¼ 7.6, 1.1 Hz, 1H, H-9); 13C NMR: 16.9 (C-4), 26.4 (CH3), 31.6 (C-3), 56.5 (CH3O), 77.5 (C-2), 118.2 (C-7), 119.3 (C-9), 119.5 (C5a), 121.9 (C-4a), 133.4 (C-9a), 133.8 (C-8), 152.8 (C-10a), 159.2 (C-6), 180.2 (C-10), 184.1 (C-5); IR (KBr): 3002, 2981, 2928, 2837, 1668, 1640, 1625, 1582, 1276 cm1; HRMS-ESI m/z [M þ H]þ calcd for C16H17O4: 273.1121, found: 273.1129. 4.1.1.6. 3,4-Dihydro-10-methoxy-2,2-dimethyl-2H-benzo[h]chromene5,6-dione (10-methoxy-b-lapachone, 4b). Compound 4b (0.9 mg, 7.9%) was obtained as red crystals from 5-methoxylapachol (4) (11 mg, 0.04 mmol) following the general procedure for cyclization reactions with methanesulfonic acid (Table 1, entries 15e17): Rf ¼ 0.50 (CH2Cl2/MeOH, 4:0.1); mp: 102e103  C; 1H NMR: 1.46 (s, 6H, CH3), 1.83 (t, J ¼ 6.7 Hz, 2H, H-3), 2.57 (t, J ¼ 6.8 Hz, 2H, H-4), 3.90 (s, 3H, CH3O), 7.23 (dd, J ¼ 8.5, 1.2 Hz, 1H, H-9), 7.44 (dd, J ¼ 8.4, 7.5 Hz, 1H, H-8), 7.75 (dd, J ¼ 7.5, 1.2 Hz, 1H, H-7); 13C NMR: 16.5 (C4), 26.7 (CH3), 31.2 (C-3), 57.1 (CH3O), 79.0 (C-2), 112.1 (C-4a), 120.2 (C-10a), 121.4 (C-9), 122.4 (C-7), 131.7 (C-8), 132.2 (C-6a), 157.9 (C-10),

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165.7 (C-10b), 178.1 (C-5), 180.5 (C-6); IR (KBr): 2971, 2929, 2843, 1693, 1634, 1589, 1552, 1279 cm-1; HRMS-ESI m/z [M þ H]þ calcd for C16H17O4: 273.1121, found: 273.1127. 4.1.1.7. 3,4-Dihydro-8-methoxy-2,2-dimethyl-2H-benzo[g]chromene5,10-dione (8-methoxy-a-lapachone, 5a). Compound 5a (2.8 mg, 25%) was obtained as yellow crystals from 7-methoxylapachol (5) (11 mg, 0.04 mmol) following the general procedure for cyclization reactions with methanesulfonic acid (Table 1, entry 18): Rf ¼ 0.53 (hexanes/EtOAc, 7:3); mp: 155e156  C; 1H NMR: 1.43 (s, 6H, CH3), 1.81 (t, J ¼ 6.6 Hz, 2H, H-3), 2.60 (t, J ¼ 6.6 Hz, 2H, H-4), 3.93 (s, 3H, CH3O), 7.15 (dd, J ¼ 8.6, 2.7 Hz, 1H, H-7), 7.52 (d, J ¼ 2.7 Hz, 1H, H-9), 8.00 (d, J ¼ 8.6 Hz, 1H, H-6); 13C NMR: 16.7 (C-4), 26.5 (CH3), 31.4 (C3), 55.9 (CH3O), 77.9 (C-2), 109.9 (C-9), 120.0 (C-4a), 120.0 (C-7), 125.5 (C-5a), 128.3 (C-6), 133.0 (C-9a), 154.3 (C-10a), 163.4 (C-8), 180.0 (C-10), 183.8 (C-5); IR (KBr): 2966, 1673, 1597, 1335 cm1; HRMS-ESI m/z [M þ H]þ calcd for C16H17O4: 273.1121, found: 273.1122. 4.1.1.8. 3,4-Dihydro-8-methoxy-2,2-dimethyl-2H-benzo[h]chromene5,6-dione (8-methoxy-b-lapachone, 5b). Compound 5b (8.4 mg, 62%) was obtained as red crystals from 7-methoxylapachol (5) (13 mg, 0.05 mmol) as described in the general procedure for cyclization reactions using methanesulfonic acid (Table 1, entry 18): Rf ¼ 0.29 (hexanes/EtOAc, 7:3); mp: 166e167  C (lit. [24] 162e163  C); 1H NMR: 1.46 (s, 6H, CH3), 1.84 (t, J ¼ 6.7 Hz, 2H, H-3), 2.54 (t, J ¼ 6.7 Hz, 2H, H-4), 3.90 (s, 3H, CH3O), 7.12 (dd, J ¼ 8.6, 2.8 Hz, 1H, H-9), 7.55 (d, J ¼ 2.8 Hz, 1H, H-7), 7.72 (d, J ¼ 8.5 Hz, 1H, H-10); 13C NMR: 16.0 (C-4), 26.8 (CH3), 31.7 (C-3), 55.8 (CH3O), 79.2 (C-2), 110.6 (C-4a), 112.5 (C-7), 121.0 (C-9), 125.4 (C-10a), 125.9 (C-10), 131.7 (C-6a), 161.6 (C-8), 162.8 (C-10b), 178.2 (C-5), 179.9 (C-6); IR (KBr): 2975, 2935, 1693, 1634, 1601, 1594 cm1; HRMS-ESI m/z [M þ H]þ calcd for C16H17O4: 273.1121, found: 273.1118. 4.1.2. Synthesis of lapachol analogs 4.1.2.1. 2-Hydroxy-5-methoxynaphthalene-1,4-dione (2-hydroxy-5methoxy-1,4 naphthoquinone, 8). Compound 8 was obtained as orange crystals (64% yield) from 5-methoxytetralone (6) following the procedure described by W. Steglich [48]; mp: 176e177  C (lit. [49] 176e177  C); 1H NMR (500.13 MHz, DMSO-d6): 3.92 (s, 3H, OCH3), 6.05 (s, 1H, H-3), 7.57 (dd, J ¼ 8.5, 0.9 Hz, 1H, H-8), 7.66 (dd, J ¼ 7.6, 1.1 Hz, 1H, H-6), 7.77 (dd, J ¼ 8.4, 7.6 Hz, 1H, H-7); 13C NMR (125.77 MHz, DMSO-d6) 57.2 (CH3O), 114.2 (C-3), 119.5 (C-6), 119.7 (C-4a), 120.7 (C-8), 133.7 (C-8a), 135.2 (C-7), 158.0 (C-2), 159.8 (C-5), 182.6 (C-4), 185.1 (C-1); HRMS (ESI): calcd for m/z C11H9O4 [M þ H]þ 205.0495; found 205.0494. 4.1.2.2. 2-Hydroxy-7-methoxynaphthalene-1,4-dione (2-hydroxy-7methoxy-1,4-naphthoquinone, 9). Compound 9 was obtained as red crystals (90% yield) from 7-methoxytetralone (7), following the general procedure described by Thomson and Baillie [21]; mp: 216e217  C (lit. [50] 216  C); 1H NMR (200.13 MHz, DMSO-d6): 3.95 (s, 3H, OCH3), 6.12 (s, 1H, H-3), 7.37 (dd, J ¼ 8.4, 2.6 Hz, 1H, H-6), 7.44 (d, J ¼ 2.6 Hz, 1H, H-8), 7.91 (d, J ¼ 8.4 Hz, 1H, H-5), 11.61 (s, 1H, OH); 13 C NMR (50.32 MHz, DMSO-d6): 56.9 (CH3O), 110.9 (C-8), 111.8 (C3), 121.0 (C-6), 126.1 (C-4a), 128.8 (C-5), 133.3 (C-8a), 160.1 (C-2), 163.9 (C-7), 182.2 (C-1), 185.0 (C-4); HRMS (ESI): calcd for m/z C11H8NaO4 [M þ Na]þ 227.0320; found 227.0322. 4.1.2.3. 2-Hydroxy-5-methoxy-3-(3-methylbut-2-enyl)naphthalene1,4-dione (5-methoxylapachol, 4). Compound 4 was obtained as yellow crystals (26% yield) from 8 following the procedure previously described previously [17] for the synthesis of 5hydroxylapachol (2). Rf ¼ 0.46 (hexanes/EtOAc, 6:4); mp:

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131e132  C (lit. [24] 122e124  C); 1H NMR: 1.67 (d, J ¼ 1.1 Hz, 3H, CH3-50 ), 1.78 (d, J ¼ 0.9 Hz, 3H, CH3-40 ), 3.28 (dsept, J ¼ 7.3, 0.9 Hz, 2H, H-10 ), 4.00 (s, 3H, OCH3), 5.23 (tsept, J ¼ 7.4, 1.4 Hz, 1H, H-20 ), 7.09 (s, 1H, OH-2), 7.33 (dd, J ¼ 8.5, 0.8 Hz, 1H, H-6), 7.61 (dd, J ¼ 8.5, 7.6 Hz, 1H, H-7), 7.75 (dd, J ¼ 7.6, 1.1 Hz, 1H, H-8); 13C NMR: 17.9 (C40 ), 22.8 (C-10 ), 25.7 (C-50 ), 56.5 (OCH3), 118.9 (C-8), 119.4 (C-6), 119.8 (C-4a), 119.9 (C-20 ), 125.0 (C-3), 131.6 (C-8a), 133.5 (C-30 ), 133.9 (C-7), 150.9 (C-2), 159.6 (C-5), 181.9 (C-1), 184.3 (C-4); IR (KBr) 3388, 2978, 2931, 2847, 1769, 1725, 1645 cm1; HRMS (ESI): calcd for m/z C16H17O4 [M þ H]þ 273.1121; found 273.1129. 4.1.2.4. 3-Hydroxy-6-methoxy-2-(3-methylbut-2-en-1-yl)naphthalene1,4-dione (7-methoxylapachol, 5). Compound 5 was obtained as yellow crystals (16% yield) from 9 following the procedure previously described previously for the synthesis of 5-hydroxylapachol (2) [17]; mp: 112e113  C (lit. [24] 113e115  C); 1H NMR: 1.69 (d, J ¼ 1.1 Hz, 3H, CH3-50 ), 1.79 (d, J ¼ 1.0 Hz, 3H, CH3-40 ), 3.29 (dsept, J ¼ 7.1, 0.9 Hz, 2H, H-10 ), 3.93 (s, 3H, OCH3), 5.20 (tsept, J ¼ 7.4, 1.4 Hz, 1H, H-20 ), 7.19 (s, 1H, OH-3), 7.20 (dd, J ¼ 8.6, 2.7 Hz, 1H, H-7), 7.51 (d, J ¼ 2.7 Hz, 1H, H5), 8.05 (dd, J ¼ 8.6, 0.3 Hz, 1H, H-8); 13C NMR: 17.9 (C-40 ), 22.6 (C-10 ), 25.8 (C-50 ), 55.9 (OCH3), 109.8 (C-5), 119.7 (C-20 ), 120.7 (C-7), 123.2 (C2), 126.2 (C-8a), 129.1 (C-8), 131.1 (C-4a), 133.8 (C-30 ), 152.4 (C-3), 163.3 (C-6), 181.9 (C-1), 184.0 (C-4); IR (KBr) 3352, 2958, 2910, 1658, 1640, 1597 cm1. HRMS (ESI): calcd for m/z C16H17O4 [M þ H]þ 273.1121; found 273.1119. 4.1.3. Synthesis of naphthoquinones 10e12 4.1.3.1. 3,4-Dihydro-7-methoxy-2,2-dimethyl-2H-benzo[h]chromene5,6-dione (7-methoxy-b-lapachone, 10). To a stirred solution of 3b (10 mg, 0.04 mmol) and K2CO3 (27 mg, 0.2 mmol) in dry DMF (0.7 mL) was added methyl iodide (97 mL, 1.6 mmol) and was stirred at rt for 22 h. The reaction mixture was diluted with water and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by column chromatography (hexanes/EtOAc, gradient) to give 10 (9.8 mg, 90%) as orange crystals: Rf ¼ 0.76 (hexanes/EtOAc, 2:8); mp: 155e156  C (lit. [24] 156157  C); 1H NMR: 1.45 (s, 6H, CH3), 1.83 (t, J ¼ 6.7 Hz, 2H, H-3), 2.54 (t, J ¼ 6.7 Hz, 2H, H-4), 3.98 (s, 3H, CH3O), 7.09 (dd, J ¼ 8.5, 0.5 Hz, 1H, H-8), 7.48 (dd, J ¼ 7.7, 0.9 Hz, 1H, H-10), 7.58 (dd, J ¼ 8.4, 7.8 Hz, 1H, H-9); 13C NMR: 16.1 (C-4), 26.7 (CH3), 31.5 (C-3), 56.3 (CH3O), 79.0 (C-2), 112.2 (C-4a), 115.0 (C-8), 116.7 (C-10), 118.2 (C-6a), 134.6 (C-10a), 135.8 (C-9), 161.4 (C-7), 161.5 (C-10b), 178.9 (C-5), 179.0 (C-6); IR (KBr): 3468, 2963, 2925, 1674, 1641, 1606, 1575 cm1; HRMS-ESI m/z [M þ Na]þ calcd for C16H16NaO4: 259.0941, found: 259.0950. 4.1.3.2. 3,4,5,6-Tetrahydro-2,2-dimethyl-5,6-dioxo-2H-benzo[h]chromen-7-yl pivalate (7-pivaloyl-b-lapachone, 11). To a solution of 3b (21.8 mg, 0.08 mmol) in dry CH2Cl2 (0.3 mL) under argon atmosphere was added DMPA (1 mg), Et3N (13 mL) and pivaloyl chloride (12 mL) and the mixture was stirred overnight. The reaction was quenched with water and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated under vacuum. The residue was purified by column chromatography on silica gel (hexane/EtOAc, gradient) to give 11 (10.9 mg, 40%) as orange crystals; mp 171172  C. 1H NMR: 1.44 (s, 9H, C(CH3)3), 1.46 (s, 6H, CH3), 1.84 (t, J ¼ 6.7 Hz, 2H, H-3), 2.55 (t, J ¼ 6.7 Hz, 2H, H-4), 7.09 (dd, J ¼ 8.1, 1.2 Hz, 1H, H-8), 7.63 (t, J ¼ 8.0 Hz, 1H, H-9), 7.76 (dd, J ¼ 7.9, 1.2 Hz, 1H, H-10), 13C NMR: 16.2 (C-4), 26.7 (CH3), 27.2 (C(CH3)3), 31.5 (C-3), 39.2 (C(CH3)3), 79.4 (C-2), 112.8 (C-4a), 122.2 (C-6a y 10), 126.4 (C-8), 134.3 (C10a), 135.4 (C-9), 152.0 (C-7), 161.3 (C-10b), 176.5 (CO C(CH3)3), 177.7 (C-6), 177.9 (C-5). IR (KBr) 2968, 2930, 2870, 1754, 1645, 1604, 1583 cm1. HRMS (ESI): calcd for m/z C20H23O5 [M þ H]þ 343.1540; found 343.1535.

4.1.3.3. 3,4-Dihydro-8-hydroxy-2,2-dimethyl-2H-benzo[h]chromene5,6-dione (8-hydroxy-b-lapachone, 12). To a solution of 5b (23.3 mg, 0.09 mmol) in dry CH2Cl2 (1.5 mL) at 40  C under an argon atmosphere was added BBr3 (20 mL). The reaction mixture was stirred at rt for 1 h. The mixture was cooled at 0  C and EtOH was added dropwise, then water and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by column chromatography (hexanes/ EtOAc, gradient) to give 11 as red crystals (12 mg, 53%): Rf ¼ 0.43 (hexanes/EtOAc, 4:6); mp: 125e126  C; 1H NMR: 1.46 (s, 6H, CH3), 1.85 (t, J ¼ 6.7 Hz, 2H, H-3), 2.51 (t, J ¼ 6.7 Hz, 2H, H-4), 7.06 (dd, J ¼ 8.5, 2.7 Hz, 1H, H-9), 7.42 (d, J ¼ 2.6 Hz, 1H, H-7), 7.67 (d, J ¼ 8.5 Hz, 1H, H-10); 13C NMR: 15.7 (C-4), 26.5 (CH3), 31.5 (C-3), 79.3 (C-2), 109.9 (C-4a), 115.1 (C-7), 121.4 (C-9), 123.9 (C-10a), 126.3 (C-10), 131.5 (C-6a), 160.0 (C-8), 164.3 (C-10b), 178.7 (C-5), 180.4 (C6); IR (KBr): 3332, 2979, 2923, 2850, 1733, 1693, 1639, 1601, 1552, 1314 cm1; HRMS-ESI m/z [M þ H]þ calcd for C15H15O4: 259.0965, found: 259.0967. 4.2. Biology All starting materials were commercially available researchgrade chemicals and used without further purification. RPMI 1640 medium was purchased from Flow Laboratories (Irvine, UK), fetal calf serum (FCS) was from Gibco (Grand Island, NY), trichloroacetic acid (TCA) and glutamine were from Merck (Darmstadt, Germany), and penicillin G, streptomycin, DMSO and sulforhodamine B (SRB) were from Sigma (St Louis, MO). 4.2.1. Cells, culture and plating The human solid tumor cell lines HBL-100, HeLa, SW1573, and WiDr were used in this study. These cell lines were a kind gift from Prof. G. J. Peters (VU Medical Center, Amsterdam, The Netherlands). Cells were maintained in 25 cm2 culture flasks in RPMI 1640 supplemented with 5% heat inactivated fetal calf serum and 2 mM L-glutamine in a 37  C, 5% CO2, 95% humidified air incubator. Exponentially growing cells were trypsinized and resuspended in antibiotic containing medium (100 units penicillin G and 0.1 mg of streptomycin per mL). Single cell suspensions displaying >97% viability by trypan blue dye exclusion were subsequently counted. After counting, dilutions were made to give the appropriate cell densities for inoculation onto 96-well microtiter plates. Cells were inoculated in a volume of 100 mL per well at densities 10 000 (SW1573 and HBL-100) of 15 000 (HeLa), and 20 000 (WiDr) cells per well, based on their doubling times. 4.2.2. Chemosensitivity testing Compounds were initially dissolved in DMSO at 400 times the desired final maximum test concentration. Control cells were exposed to an equivalent concentration of DMSO (0.25% v/v, negative control). Each agent was tested in triplicate at different dilutions in the range of 1e100 mM. The drug treatment was started on day 1 after plating. Drug incubation times were 48 h, after which time cells were precipitated with 25 mL ice-cold TCA (50% w/v) and fixed for 60 min at 4  C. Then the SRB assay was performed [29]. The optical density (OD) of each well was measured at 492 nm, using BioTek’s PowerWave XS Absorbance Microplate Reader. Values were corrected for background OD from wells only containing medium. 4.2.3. Cell cycle analysis Cells were seeded in six well plates at a density of 2.5e5  105 cells/well. After 24 h the products were added to the respective well and incubated for an additional period of 24 h. Cells

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were trypsinized, harvested, transferred to test tubes (12  75 mm) and centrifuged at 1500 rpm for 10 min. The supernatant was discarded and the cell pellets were resuspended in 200 mL of cold PBS and fixed by the addition of 1 mL ice-cold 70% EtOH. Fixed cells were incubated overnight at 20  C after which time were centrifuged at 1500 rpm for 10 min. The cell pellets were resuspended in 500 mL of PBS and 5 mL of DNAse-free RNAse solution (10 mg/mL) were added. The mixture was incubated at 37  C for 30 min. Finally, 5 mL of PI (0.5 mg/mL) were added. Flow cytometric determination of DNA content (20 000 cells/sample) was analyzed on a LRSII Flow Cytometer (Becton Dickinson, San José, CA, USA). The fractions of the cells in G0/G1, S, and G2/M phase were analyzed using FACS Diva 6.0 (BD Software San José, CA, USA) software. 4.2.4. Immunoblotting Protein extracts were prepared using a lysis buffer freshly supplemented with protease and phosphatase inhibitors (20 mM Tris [pH 8.0], 140 mM NaCl, 1% NP-40, 10% glycerol, 5 mM EDTA, 2 mM Na3VO4, 10 mM Na2P2O7, 10 mM NaF, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 0.5 mg/mL pepstatin, and 1 mM PMSF). Cells were incubated on ice for 10 min and then cell debris was spun down at 10 000 g for 10 min. Proteins were separated by SDSePAGE and electrotransferred to ImmunoBlot PVDF membrane (BioRad). Poly(ADP-ribose) polymerase-1 (Santa Cruz, USA), procaspase 3 (BD Bioscience, USA), cyclin A (Santa Cruz, USA), cyclin E (Santa Cruz, USA), MRE11 (Santa Cruz, USA), phospho checkpoint kinase 2 (p-CHK2) (Cell Signalling, USA), phospho histone H2AX (g-H2AX) (Cell Signalling, USA), and a-tubulin (BD Bioscience, USA) primary antibodies were used. After being washed with Tris-buffered saline with Tween (TBST) (100 mM Tris [pH 7.5], 150 mM NaCl, 0.05% Tween 20), membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min and bands were visualized by a luminal-based detection system with p-iodophenol enhancement. 4.2.5. Measurement of reactive oxygen species (ROS) Intracellular accumulation of ROS was determined using 20 ,70 dichlorodihydrofluorescein diacetate (DCFH-DA) [51]. HeLa cells were seeded in a density of 3  105 cells/well. After 24 h the compounds were added and coincubated with 5 mM of DCFH-DA for 1 h at 37  C. Cells were collected, transferred to test tubes (12  75 mm) and centrifuged at 200 g for 5 min. Cells were resuspended in 500 mL of PBS. Flow cytometric determination of ROS content (10 000 cells/sample) was analyzed on a LRSII Flow Cytometer (Becton Dickinson, San José, CA, USA). Fluorescent emission was analyzed using WinMDI 2.8 software.

Acknowledgements Co-financed by the European Social Fund (FEDER), the Spanish Instituto de Salud Carlos III (PI11/00840), the Spanish MSC (RTICC RD06/0020/1046 and RD06/0020/0041), the Canary Islands ACIISI (PI 2007/021), the Canary Islands FUNCIS (PI 43/ 09), and by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET PIP 0447), and Universidad de Buenos Aires (UBACyT 20020090200697). E.L.B. thanks CONICET for a fellowship. L.G.L. thanks ACIISI for a postdoctoral contract (SE10/19).

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejmech.2012.04.008.

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References [1] H. Hussain, K. Krohn, V.U. Ahmad, G.A. Miana, I.R. Green, Lapachol: an overview, ARKIVOC (2007) 145e171. [2] P. Guiraud, R. Steiman, G.M. Campos-Takaki, F. Seigle-Murandi, M. Simeon de Buochberg, Comparison of antibacterial and antifungal activities of lapachol and beta-lapachone, Planta Med. 60 (1994) 373e374. [3] E. Pérez-Sacau, A. Estévez-Braun, A.G. Ravelo, D. Gutiérrez Yapu, A. Giménez Turba, Antiplasmodial activity of naphthoquinones related to lapachol and beta-lapachone, Chem. Biodivers. 2 (2005) 264e274. [4] C. Salas, R.A. Tapia, K. Ciudad, V. Armstrong, M. Orellana, U. Kemmerling, J. Ferreira, J.D. Maya, A. Morello, Trypanosoma cruzi: activities of lapachol and alpha- and beta-lapachone derivatives against epimastigote and trypomastigote forms, Bioorg. Med. Chem. 16 (2008) 668e674. [5] D.O. Moon, Y.H. Choi, N.D. Kim, Y.M. Park, G.Y. Kim, Anti-inflammatory effects of beta-lapachone in lipopolysaccharide-stimulated BV2 microglia, Int. Immunopharmacol. 7 (2007) 506e514. [6] H.N. Kung, C.L. Chien, G.Y. Chau, M.J. Don, K.S. Lu, Y.P. Chau, Involvement of NO/cGMP signaling in the apoptotic and anti-angiogenic effects of betalapachone on endothelial cells in vitro, J. Cell. Physiol. 211 (2007) 522e532. [7] S.O. Kim, J.I. Kwon, Y.K. Jeong, G.Y. Kim, N.D. Kim, Y.H. Choi, Induction of Egr-1 is associated with anti-metastatic and anti-invasive ability of beta-lapachone in human hepatocarcinoma cells, Biosci. Biotechnol. Biochem. 71 (2007) 2169e2176. [8] A.B. Pardee, Y.Z. Li, C.J. Li, Cancer therapy with beta-lapachone, Curr. Canc. Drug Targets 2 (2002) 227e242. [9] Y. Li, X. Sun, J.T. LaMont, A.B. Pardee, C.J. Li, Selective killing of cancer cells by b-lapachone: direct checkpoint activation as a strategy against cancer, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 2674e2678. [10] N.E. Mealy, B. Lupone, Drugs under development for the treatment of head and neck cancer, Drugs Future 31 (2006) 627e639. [11] H. Lee, M.-T. Park, B.-H. Choi, E.-T. Oh, M.-J. Song, J. Lee, C. Kim, B.U. Lim, H.J. Park, Endoplasmic reticulum stress-induced JNK activation is a critical event leading to mitochondria-mediated cell death caused by b-lapachone treatment, PLoS One 6 (2011) e21533. [12] X. Sun, Y. Li, W. Li, B. Zhang, A.J. Wang, J. Sun, K. Mikule, Z. Jiang, C.J. Li, Selective induction of necrotic cell death in cancer cells by beta-lapachone through activation of DNA damage response pathway, Cell Cycle 5 (2006) 2029e2035. [13] E.J. Park, K.S. Choi, T.K. Kwon, b-Lapachone-induced reactive oxygen species (ROS) generation mediates autophagic cell death in glioma U87 MG cells, Chem. Biol. Interact. 189 (2011) 37e44. [14] L.S. Li, E.A. Bey, Y. Dong, J. Meng, B. Patra, J. Yan, X.J. Xie, R.A. Brekken, C.C. Barnett, W.G. Bornmann, J. Gao, D.A. Boothman, Modulating endogenous NQO1 levels identifies key regulatory mechanisms of action of beta-lapachone for pancreatic cancer therapy, Clin. Canc. Res. 17 (2011) 275e285. [15] P. Krishnan, K.F. Bastow, Novel mechanism of cellular DNA topoisomerase II inhibition by the pyranonaphthoquinone derivatives a-lapachone and blapachone, Canc. Chemother. Pharmacol. 47 (2001) 187e198. [16] Y.C. Lien, H.N. Kung, K.S. Lu, C.J. Jeng, Y.P. Chau, Involvement of endoplasmic reticulum stress and activation of MAP kinases in beta-lapachone-induced human prostate cancer cell apoptosis, Histol. Histopathol. 23 (2008) 1299e1308. [17] E.L. Bonifazi, C. Ríos-Luci, L.G. León, G. Burton, J.M. Padrón, R.I. Misico, Antiproliferative activity of synthetic naphthoquinones related to lapachol. First synthesis of 5-hydroxylapachol, Bioorg. Med. Chem. 18 (2010) 2621e2630. [18] S.C. Hooker, The constitution of lapachol and its derivatives. Part V. the structure of Paternò’s “isolapachone”, J. Am. Chem. Soc. 58 (1936) 1190e1197. [19] R.H. Thomson, Naturally Occurring Quinones, Academic Press, London and New York, 1971, 206e209. [20] M.G. Ettlinger, Hydroxynaphthoquinones. II. cyclization and the basicity and interconversion of ortho and para quinones, J. Am. Chem. Soc. 72 (1950) 3090e3095. [21] A.C. Baillie, R.H. Thomson, Quinones. Part VII. new routes to 2-hydroxy-1,4naphthaquinones, J. Chem. Soc. Sec. C (1966) 2184e2186. [22] P.H. Di Chenna, V. Benedetti-Doctorovich, R.F. Baggio, M.T. Garland, G. Burton, Preparation and cytotoxicity toward cancer cells of mono(arylimino) derivatives of beta-lapachone, J. Med. Chem. 44 (2001) 2486e2489. [23] P. Singh, K. Natani, S. Jain, K. Arya, A. Dandia, Microwave-assisted rapid cyclization of lapachol, a main constituent of Heterophragma adenophyllum, Nat. Prod. Res. 20 (2006) 207e212. [24] R.-Y. Yang, D. Kizer, H. Wu, E. Volckova, X.-S. Miao, S.M. Ali, M. Tandon, R.E. Savage, T.C.K. Chan, M.A. Ashwell, Synthetic methods for the preparation of ARQ 501 (beta-Lapachone) human blood metabolites, Bioorg. Med. Chem. 16 (2008) 5635e5643. [25] D.R. da Rocha, A.C.G. de Souza, J.A.L.C. Resende, W.C. Santos, E.A. dos Santos, C. Pessoa, M.O. de Moraes, L.V. Costa-Lotufo, R.C. Montenegro, V.F. Ferreira, Synthesis of new 9-hydroxy-a- and 7-hydroxy-b-pyran naphthoquinones and cytotoxicity against cancer cell lines, Org. Biomol. Chem. (2011) 4315e4322. [26] H. Inouye, T. Okuda, T. Hayashi, Quinones and related compounds in higher plants. II. on the naphthoquinones and related compounds from catalpa wood, Chem. Pharm. Bull. 23 (1975) 384e389. [27] A. Boveris, A.O.M. Stoppani, R. Docampo, F.S. Cruz, Superoxide anion production and trypanocidal action of naphthoquinones on Trypanosoma cruzi, Comp. Biochem. Physiol. C 61 (1978) 327e329.

274

C. Ríos-Luci et al. / European Journal of Medicinal Chemistry 53 (2012) 264e274

[28] T. Matsumoto, C. Mayer, C.H. Eugster, a-Caryopteron, ein neues Pyrano-juglon aus Caryopteris clandonensis, Helv. Chim. Acta 52 (1969) 808e812. [29] P.O. Miranda, J.M. Padrón, J.I. Padrón, J. Villar, V.S. Martín, Prins-type synthesis and SAR study of cytotoxic alkyl chloro dihydropyrans, ChemMedChem 1 (2006) 323e329. [30] P.S. Guin, S. Das, P.C. Mandal, Electrochemical reduction of quinones in different media: a review, Int. J. Electrochem 2011 (2011) Article ID 816202. [31] D. Gupta, K. Podar, Y. Tai, B. Lin, T. Hideshima, M. Akiyama, R. LeBlanc, L. Catley, N. Mitsiades, C. Mitsiades, D. Chauhan, N.C. Munshi, K.C. Anderson, Beta-lapachone, a novel plant product, overcomes drug resistance in human multiple myeloma cells, Exp. Hematol. 30 (2002) 711e720. [32] G.M. Cohen, Caspases: the executioners of apoptosis, Biochem. J. 326 (1997) 1e26. [33] Y.Z. Li, C.J. Li, A.V. Pinto, A.B. Pardee, Release of mitochondrial cytochrome C in both apoptosis and necrosis induced by beta-lapachone in human carcinoma cells, Mol. Med. 5 (1999) 232e239. [34] S.M. Planchon, S. Wuerzberger, B. Frydman, D.T. Witiak, P. Hutson, D.R. Church, G. Wilding, D.A. Boothman, Beta-lapachone-mediated apoptosis in human promyelocytic leukemia (HL-60) and human prostate cancer cells: a p53-independent response, Canc. Res. 55 (1995) 3706e3711. [35] G. Brooks, N.B. La Thangue, Drug discovery and the cell cycle: the promise and the hope, Drug Discov. Today 4 (1999) 455e464. [36] R.A. Woo, R.Y.C. Poon, Bullet cyclin-dependent kinases and S phase control in mammalian cells, Cell Cycle 2 (2003) 316e324. [37] B.S. Zhou, S.J. Elledge, The DNA damage response: putting checkpoints in perspective, Nature 408 (2000) 433e439. [38] T. Liu, S. Lin, Y. Chau, Inhibition of poly(ADP-ribose) polymerase activation attenuates beta-lapachone-induced necrotic cell death in human osteosarcoma cells, Toxicol. Appl. Pharmacol. 182 (2002) 116e125. [39] H. Lu, H. Zhu, M. Huang, Y. Chen, Y. Cai, Z.-H. Miao, J. Zhang, J. Ding, Reactive oxygen species elicit apoptosis by concurrently disrupting topoisomerase II and DNA-dependent protein kinase, Mol. Pharmacol. 68 (2005) 983e994. [40] M. Forkink, J.A.M. Smeitink, R. Brock, P.H.G.M. Willems, W.J.H. Koopman, Detection and manipulation of mitochondrial reactive oxygen species in mammalian cells, Biochim. Biophys. Acta 1797 (2010) 1034e1044.

[41] B. Halliwell, M. Whiteman, Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br. J. Pharmacol. 142 (2004) 231e255. [42] E.A. Bey, M.S. Bentle, K.E. Reinicke, Y. Dong, C. Yang, L. Girard, J.D. Minna, W.G. Bornmann, J. Gao, D.A. Boothman, An NQO1- and PARP-1-mediated cell death pathway induced in non-small-cell lung cancer cells by beta-lapachone, Proc. Natl. Acad. Sci. 104 (2007) 11832e11837. [43] S. Jiménez-Alonso, H.C. Orellana, A. Estévez-Braun, A.G. Ravelo, E. Pérez-Sacau, F. Machín, Design and synthesis of a novel series of pyranonaphthoquinones as topoisomerase II catalytic inhibitors, J. Med. Chem. 51 (2008) 6761e6772. [44] J. Sperry, I. Lorenzo-Castrillejo, M.A. Brimble, F. Machín, Pyranonaphthoquinone derivatives of eleutherin, ventiloquinone L, thysanone and nanaomycin A possessing a diverse topoisomerase II inhibition and cytotoxicity spectrum, Bioorg. Med. Chem. 17 (2009) 7131e7317. [45] M.T. Muller, K. Helal, S. Soisson, J.R. Spitzner, A rapid and quantitative microtiter assay for eukaryotic topoisomerase II, Nucleic Acids Res. 17 (1989) 9499. [46] A.G. Ravelo, A. Estévez-Braun, E. Pérez-Sacau, The chemistry and biology of lapachol and related natural products: a- and b-lapachones, Stud. Nat. Prod. Chem. 29 (2003) 719e760. [47] T. Matsumoto, A. Ichihara, M. Yanagiya, T. Yuzawa, A. Sannai, H. Oikawa, S. Sakamura, C.H. Eugster, Two new syntheses of the pyranojuglone pigment a-caryopterone, Helv. Chim. Acta 68 (1985) 2324e2331. [48] L. Kopaski, D. Karbach, G. Selbitschka, W. Steglich, Pilzfarbstoffe, 53. vesparion, ein naphtho[2,3-b]pyrandion-derivat aus dem Schleimpilz Metatrichia vesparium (Myxomycetes), Liebigs Ann. Chem. 1987 (1987) 793e796. [49] R.G. Cooke, W. Segal, Colouring matters of Australian plants. I. the structure of droserone, Aust. J. Sci. Res. Ser. A: Phys. Sci. 3 (1950) 628e634. [50] D.R. Buckle, B.C.C. Cantello, H. Smith, R.J. Smith, B.A. Spicer, Synthesis and antiallergic activity of 2-hydroxy-3-nitro-1,4-naphthoquinones, J. Med. Chem. 20 (1977) 1059e1064. [51] C.P. LeBel, H. Ischiropoulos, C.C. Bondy, Evaluation of the probe 2’,7’-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress, Chem. Res. Toxicol. 5 (1992) 227e231.