- Email: [email protected]
Structural modifications on the phenazine N,N′-dioxide-scaffold looking for new selective hypoxic cytotoxins
European Journal of Medicinal Chemistry 45 (2010) 5362e5369
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structural modifications on the phenazine N,N0 -dioxide-scaffold looking for new selective hypoxic cytotoxins María Laura Lavaggi a, Marcos Nieves a, Mauricio Cabrera a, Claudio Olea-Azar b, Adela López de Ceráin c, Antonio Monge c, Hugo Cerecetto a, *, Mercedes González a, * a b c
Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile Centro de Investigación en Farmacobiología Aplicada, Universidad de Navarra, Pamplona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2010 Received in revised form 28 July 2010 Accepted 26 August 2010 Available online 16 September 2010
We have identified phenazine 5,10-dioxides as prodrugs for antitumour therapy that undergo hypoxicselective bioreduction to form cytotoxic species. Here, we investigated some structural modifications in order to find new selective hypoxic cytotoxins and to establish the structural requirements for adequate activity. Three different chemical-series were prepared and the clonogenic survival of V79 cells on aerobic and anaerobic conditions was determined. Electrochemical- and DNA-interaction studies were done for the most relevant derivatives. The new fluoro-derivative 7-fluoro-2-aminophenazine 5,10dioxide displayed selective toxicity towards hypoxic V79 cells having adequate hypoxic cytotoxicity ratio (HCR ¼ 6.8) and being the most potent hypoxic cytotoxins (P ¼ 2.5 mM) described for this family of bioreductive agents. The reduction potential of the N-oxide moiety in this new fluoro-derivative was in the range for adequate bioreduction property. According to the fluorescence studies, the DNA-interaction mechanism was especially operative in the phenazine drugs more than in the corresponding prodrugs, phenazine dioxides. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Phenazine N,N0 -dioxides Bioreductive agents Cancer
1. Introduction Since the 1960’ it is well known the existence of hypoxic and necrotic regions in solid tumours [1] as consequence of the rapid growth of cancerous cells and their deficient vascularisation that produces molecular oxygen diffusion decreasing [2]. The hypoxic regions, associated with increased resistance to radiation [3], are refractory to conventional anticancer drugs because they are antiproliferative agents that kill dividing cells and these tumour cells are not dividing rapidly. In addition, it has also been demonstrated that hypoxia in tumours alters cellular metabolism tending to select for a more malignant phenotype, increasing mutation rates. It also increases expression of genes associated with angiogenesis and tumour invasion, and is associated with a more metastatic phenotype of human cancers [4]. By enhancing metastasis, hypoxia can compromise curability of tumours by surgery. This common feature of cancerous cells, hypoxia, is used for the development of a distinct therapy for treating cancer.
* Corresponding author. Tel.: þ598 2 5258618; fax: þ598 2 5250749. E-mail addresses: [email protected] (H. Cerecetto), [email protected] (M. González). 0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2010.08.061
Based on using prodrugs, capable to be bioreduced under hypoxic conditions (bioreductive antitumour agents, [5] BAA) to further drugs that produce cytotoxic events causing different degrees of cancerous cells damage, hypoxic-selective cytotoxins have been developed. Molecular oxygen reverts the bioreductive process in normal oxygenated tissues giving the selectivity to this process. This selectivity is governed by the redox properties of the drugs and the reductases enzymes levels [6]. N-oxides derivatives have been described among the compounds classified as BAA [7,8]. We have reported phenazine N,N0 -dioxides (PDOs) [9] as a new group of hypoxia-activated prodrugs that potentially interact with DNA after the corresponding bioreduction in hypoxic conditions [10]. The PDOs 1, 2, and 5 displayed excellent selective cytotoxic properties being phenazine 5,10-dioxides 3 and 4 toxics in both conditions at the assayed dose (20 mM) (Fig. 1a) [11]. On the other hand, the benzo[a]phenazine 7,12-dioxides analogues, i.e. 6 and 7 (Fig. 1a) exhibited very particular cytotoxic profiles having high oxic-cytotoxicities without toxicity in hypoxia, probably as result of the aerobic futile cycle [12]. The selective anaerobic-reduction and its relation to bioreductive activity were proved using enzymatic mammal systems [13]. Furthermore, some benzo[a]phenazine 7,12dioxide prodrugs were able to interact with DNA, i.e. 6 (Fig. 1a) and produce some degree of DNA-damage in oxic conditions [12].
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
5363
Fig. 1. First and second generations of PDOs and designed new generation of potential selective hypoxic cytotoxins.
Besides, some of these derivatives displayed in vitro aerobic-antitumour activity against Caco-2 cells [12,14]. To complete the necessary amassed structural-information refer to the potential PDO-behaviours as selective hypoxic cytotoxins we planned three different chemical modifications (series a, b, and c, Fig. 1b). The rationality was to: i) explore 7- and 8-substitutions by halo/alkyl groups, using PDOs 1e5 as parent compounds (series a); ii) explore aza analogues of parent compounds 6 and 7 (series b); iii) block OH-functionality for parent compound 5 (series c). Additionally, the biological effects of all the new compounds were analyzed by use of a clonogenic assay with V79 cells in simulated oxic and hypoxic conditions determining the hypoxic potency, P, and hypoxic cytotoxicity ratio, HCR. The PDOs electrochemical behaviour and DNA-interaction capability of some of these prodrugs and drugs were also studied by UV- and fluorescencespectroscopy.
2. Methods and results 2.1. Chemistry For the preparation of PDOs belonging to series a we have selected one of the best described synthetic methodology that involves the reaction between the corresponding benzofuroxan and different phenols (Scheme 1a) [15]. The designed derivatives 8e11 were generated with the original described methodology, MeONa/MeOHeTHF/5 C, in good yields however in this condition we were unable to obtain derivatives 12e15 (Scheme 1a). As it was previously observed for similar reactions [16], the preparation of these derivatives implicated as the main process the undesirable nucleophilic substitution of one fluorine by the reactant phenol. From the messy crude of reaction, it was possible to detect, by 1H NMR and MS, products like (I) and (II) (Fig. 2). Softer experimental conditions were probed for the preparation of the desired PDO 12, as Et3N/THF/5 C and Et3N/DMF/5 C. This compound was obtained in good yield, 55%, using the last one. However, our efforts to obtain derivatives 13e15 in this condition were unsuccessful. For the preparation of PDOs belonging to series b the corresponding benzofuroxans were reacted with 8-hydroxyquinoline in basic milieu (MeONa/MeOH) [17]. This procedure can yield derivatives
16e19 (Scheme 1b), produced by ortho (pyrido[2,3-a]phenazines 16 and 17) and para (pyrido[3,2-a]phenazines 18 and 19) coupling procedures through the corresponding phenolate anion. In our cases, we were able to obtain only derivatives 18 and 19 after pH change. Maybe, the generation of derivatives 16 and 17 is less favourable for the potential stereo-electronic hindrance promoted by 1-nitrogen. The reaction of 5-substituted-benzofuroxans with phenols produced the mixtures of the 7- and 8-, or 9and 10-, substituted-phenazine isomers as result of the well known tautomerism of benzofuroxan reactant at room temperature [18,19]. Although, the carbanion could react unselectively and with similar probability with each tautomeric form, some isomeric preferences were observed according to the proportion of each tautomer at the temperature of reaction [9]. In example, when 5-fluorobenzofuroxan reacted with p-aminophenol the isolated product corresponded almost exclusively to 7-fluoro-isomer (see Experimental section). In all cases, the products were characterized and evaluated as a non-separable mixture of 7- and 8-, or 9- and 10-, isomers because they could not be separated neither by crystallization nor by chromatographic methods. Derivatives 20 and 21, belonging to series c, were prepared by reaction between parent compound 5 and the two electrophilic reactants shown in Scheme 1c. In these reactions phenazine N-deoxygenation processes took place extensively producing the desired products in modest yields. All the proposed structures were established by 1H, and 13C NMR spectroscopies (using COSY, HMQC, and HMBC experiments) and MS. The purity was analyzed and established by TLC and microanalysis, respectively.
2.2. Biological characterization 2.2.1. In vitro normoxic and hypoxic cytotoxicity All the PDOs were examined for their selective hypoxic cytotoxicities in a pre-established model of V79 cells [10e12,20,21]. They were analyzed as a non-separable mixture of 7- and 8-isomers, in the cases of 10e12, 20 and 21, and 9- and 10-isomers, in the case of 18. Previous results for similar chemical systems (quinoxaline 1,4-dioxides) demonstrated that no difference between both positional isomers was observed in the selective hypoxic cytotoxicities against V79 cells [20]. The percentages of
5364
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
Scheme 1. Synthetic procedures used to prepare the designed PDO derivatives.
survival fractions (SF, Table 1) were measured at 20.0 mM while derivatives 8, 9, and 12 were also evaluated at 10.0 mM. The phenol 11, the pyrido[3,2-a]phenazines 18 and 19, and the O-substitutedderivatives 20 and 21 were non-selective exhibiting similar survival fraction in normoxia and hypoxia. The methyl- and fluorosubstituted-aminophenazine dioxide 10 showed a modest degree of selectivity similar to that of parent compounds, the methoxy-, 1,
Table 1 PDO cytotoxic effects in normoxia and hypoxia on V79 cells. Compd.
8 9 10 11 12 18 19 20 21
Norm
Hypox
2 (3) 1 (0) 88 100 0 (30) 49 58 84 86
0 (18) 0 (8) 15 95 0 (0) 46 53 62 90
SF norm ¼ survival fraction in normoxia at 20.0 mM. SF hypox ¼ survival fraction in hypoxia at 20.0 mM. c Values are means of two different experiments. The assays were done by duplicate and using at least three repetitions, standard errors were not greater than 2% for most assays. d Values in parenthesis are at 10.0 mM. a
b
Fig. 2. Products detected in the expansion of benzofuroxans bfx3 and bfx4 to PDOs 12e15, using traditional conditions [16].
SFa,b,c,d
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369 Table 2 Potencies (P) and hypoxic cytotoxicity ratios (HCR) on V79 cells for selected PDOs. Compd.
P (mM)
HCR
1 2 3 4 5a 8 12
35.0 25.0 18.0 12.0 10.0 10.0 2.5
>3.0 >2.5 1.1 5.0 >10.0 0.9 6.8
a
From Ref. [11].
and methyl-substituted-aminophenazine dioxide, 2. On the other hand, the dichloro-derivatives 8 and 9, and the fluoro-derivative 12 showed high cytotoxicity in both conditions, normoxia and hypoxia. Derivative 12 displayed some degree of selectivity at lower doses (10.0 mM). In order to complete the information the amino derivatives 8 and 12, together with the parent compounds 1e5, were tested at different doses to obtain doseeresponse curves in air and hypoxia. Potency (P) and hypoxic cytotoxicity ratio (HCR) were calculated from these curves (Table 2). P is defined as the dose, in micromolar, which gives 1% of control cell survival in hypoxia and HCR as ratio between the dose in air divided by the dose in hypoxia giving 1% of control cell survival. The dichloro-amine 8 was more cytotoxic in oxia than hypoxia in all the studied dose-range (Fig. 3a). However, the fluoro-amine 12 showed the best hypoxic potency, P ¼ 2.5 mM (Fig. 3b), between all the studied PDOs with HCR value, on V79 cells (Table 2), in the same order that others BAA [6e8]. 2.3. Mechanism of action studies To gain insight into PDO mechanism of toxicity, first of all we studied the PDO electrochemical behaviour in term of cyclic voltammetry in organic aprotic solvent (DMSO) [10e12]. The new developed PDO displayed comparable voltammetric behaviour in this condition showing two to three reduction peaks and the anodic counterparts. Especially, we focus our attention in the first reduction potential because it could be used as a descriptor of the PDO feasibility to be reduced in hypoxic conditions. Table 3 lists the values of the first N-oxide cathodic peak, Epc, which correspond to
5365
Table 3 Reduction potentials and DNA-interacting capability by UV studies of PDO derivatives. Compd.
Epc vs. SCEa (V)
a24/a0
1 2 3 4 5 6 7 8 9 10 11 12 18 21 m-AMSA Ethidium bromide NSC 322921 (bis-benzimide) Mitoxantrone
1.03b 1.00b 0.96b 0.92b 1.14b 1.36c 1.22c 1.08 0.96
0.89b 0.98b 0.89b 1.00b 1.00b 0.47c 0.94c 0.98 0.96 0.85 0.83 0.88 nse 0.88 0.30 0.50 0.57 0.00
a b c d e
d d
0.83 1.63 1.28 e e e e
Peaks potentials (w0.01 V) measured at a scan rate of 2.00 V/s. From Ref. [10]. From Ref. [12]. Solubility problems in the assay milieu do not allow to perform the study. ns: not studied.
quasireversible process. This value seems to be correlated to the hypoxic toxicity since the lowest hypoxic-SF values correspond to compounds with the lowest reduction potentials, lower than 1.14 V (Fig. 4a), consequently compounds with more favourable reduction processes. The same relationship was observed between P and Epc for the studied amino derivatives 1e4, and 12 (Fig. 4b) outlying the parent compound 5 that belongs to the phenol family and derivative 8 which possessed a particular biological behaviour (Tables 1 and 2). The new derivative 12 has the lowest N-oxide Epc value, i.e. 0.83 V (Table 3), showing the relevance of the fluorine substituent, at the benzo-ring, in the modulation of the reduction potential. Secondly, we analyze the PDO-prodrugs’ capability to interact with DNA, in oxic conditions, by measuring the hypochromic and bathochromic effect of prodrugs- and drugs-absorbances in the UV spectra, in a 20 nm band centered on the maximal absorbance value of each compound, at 0 and 24 h [22,23]. Additionally, in
Fig. 3. Doseeresponse curves in air and hypoxia for the new derivatives 8 (a) and 12 (b). Black squares and dotted lines: oxic conditions.
5366
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
Fig. 4. (a) Epc vs. SFhypoxia for PDOs. In red parent compounds. (b) Epc vs. hypoxic potency (P) for PDOs. In red outliers. The dotted line is only indicative.
order to confirm our hypothesis we evaluated the DNA-interaction capability of the drugs (phenazines or phenazine mono-oxides produced during the hypoxic conditions) choosing previously developed compounds 22e25 [11,13] (Fig. 5). The degree of interaction was expressed by the ratio between the final absorbance area (a24) and the absorbance of the compound (a0). Values of 1.00 or superior to 1.00 indicate a total lack of affinity and a value of 0 indicates binding of the entire compound to DNA (Table 3). No relevant interactions with DNA in oxic conditions, using this methodology, were observed for any of the studied compounds (Table 4). However, in the UV-experiments after 24 h of incubation some curious findings were observed. Together with the absence of hypochromic or bathochromic effects it was observed the apparition of some new bands or shoulders near the absorption maximums (data not shown). These findings could be indicating formation of productsaggregates or products-DNA-aggregates. [24,25]. For this reason, another technique and conditions were employed. The intrinsic changes on the fluorescence of phenazine while varying DNA concentrations after 30 min of incubation were monitored [26]. The sensitive of this assay allowed us to evidence interactions after this incubation time. Because it is the static quenching of DNA to compounds (Fig. 6a), the quenching constant is considered as the formation constant of compounds and DNA [27], i.e., the binding constant (Kq) of compounds with DNA. The SterneVolmer plots (Fig. 6b) provided the Kq of studied compounds at pH 6.0 [28] (Table 4). The studied compounds were able, in different degrees, to interact with DNA but lesser than toluene blue (TB, Table 4). Additionally, it is clear the relationship between the level of phenazine oxygenation and DNA-interaction capability, i.e. the
Fig. 5. Phenazines and phenazine mono-oxides herein evaluated as DNA-interacting agents.
reduced derivates, PDO 24 and 25, interacted better than the PDO parent compound 4 (Table 4). Finally, no correlation was observed between oxic-cytotoxicities (see values of SF, Table 4) and DNAinteraction abilities. For example, the best DNA-interacting PDOs, 2 and 12, displayed very different oxic-cytotoxicities. 3. Discussion We report the study of new series of PDOs with potential use as bioreductive antitumour agents. Among the new developed PDOs different features could be stated. Firstly, the hydroxy-phenazine 5,10-dioxides, 9 and 11, are no selective. In the first case cytotoxic in both conditions and the contrary for the second one. The aminoanalogue of 9, derivative 8, showed the same biological profile and when it was studied at a lower dose it resulted more toxic in oxic conditions than in hypoxic ones (Fig. 3a). Secondly, the 4-aza analogues of the oxic-cytotoxic 6 and 7, derivatives 18 and 19, were not cytotoxic in oxia as the parent compounds however they were also non-selective. Thirdly, modifications at OH-level in parent compound 5, derivatives 20 and 21, conduct to inactive and nonselective agents. Fourthly, the fluoro-amino derivatives 10 and 12 displayed hypoxic-cytotoxic selectivity (Table 1) being derivative 12
Table 4 DNA-interacting capability by fluorescence-studies of PDOs, phenazines monooxides and phenazines. Compd.
a24/a0
Kqa
SFb,c,d Norm
Hypox
2 4 12 22 23 24 25 TBf
0.98 1.00 0.88 0.92 0.85 0.92 0.90 e
7.7 1.0 2.6 0.1 7.8 2.7 5.3 0.6 6.1 0.6 4.2 0.8 7.3 3.9 12.6 1.5
61 12 0 100 98 0e 0 e
11 0 0 100 100 0e 0 e
a Kq ¼ SterneVolmer fluorescence quenching constant Experimental section). b SF norm ¼ survival fraction in normoxia at 20 mM. c SF hypox ¼ survival fraction in hypoxia at 20 mM. d From Ref. [10] except for compound 24. e It was determined herein. f TB: toluene blue.
(for
details
see
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
5367
Fig. 6. (a) Fluorescence spectra of parent compound 4 (left) and the corresponding reduced derivative 25 (right) in PBS (50 mM, pH 6.0) after addition of CT DNA. (b) SterneVolmer quenching plot of parent compound 2 with increasing concentrations of DNA in PBS (50 mM, pH 6.0).
the most hypoxic-potent cytotoxin with a potency of 2.5 mM (P, Table 1) and adequate HCR value (6.8, Table 1). The profile of hypoxic cytotoxicity of PDOs seemed to be governed by the bioreduction process. This fact could be realized in Table 3 and Fig. 4 where compounds with the lowest hypoxic survival fractions (SFhypoxia, Fig. 4a) have reduction potential (Epc) between 0.83 and 1.14 V. It was possible to observe for aminophenazines 1e4 and 12 that hypoxic potency, P, was linearly related to Epc observing better P for lowest reduction potential (Fig. 4b). It was not possible to find relationships between neither hypoxicselectivity, expressed as HCR, nor oxic-cytotoxicity, expressed as SFnorm, and Epc (data not shown). The DNA-interaction studies showed that the PDO prodrugs interact with this biomolecule, in oxic conditions, less than the well-established DNA-interacting agent toluene blue (Table 4). On the other hand, studying the DNA-interaction capability of the potential drugs, phenazine and phenazine mono-oxide, we confirmed that the proposed extra-mechanism of PDOs, interaction with this biomolecule after the corresponding bioreduction in hypoxic conditions, could be operative (compare Kq values for prodrug 4, and potential drugs 24 and 25, Table 4). The structural changes done to obtain derivatives belonging to series c (Fig. 1) modify favourably the DNA-capability of the parent compound 5 (Fig. 1). Given that the OH-blocking derivative 21, with an extra-pinteracting moiety, has little better improved ability to interact with DNA according to UV studies (compare a24/a0 values for 5 and 21, Table 3). However, this change did not modify the oxic/hypoxic biological profile.
were determined on an MSD 5973 Hewlett-Packard spectrometer using electronic impact ionization. Microanalyses were performed on a Fisons EA 1108 CHNS-O instrument and were within (0.4% of the calculated compositions). Column chromatography was carried out using Merck silica gel (60e230 mesh). Most chemicals and solvents were analytical grade and used without further purification. 5.1. General procedure for the preparation of the phenazine 5,10dioxide derivatives 8e11 To a solution of 3.9 mmol of metallic sodium in anhydrous MeOH (25.0 mL), at 5 C and under nitrogen atmosphere, was added a solution of 20.0 mmol of the corresponding phenol (p-aminophenol or p-hydroquinone) and 20.0 mmol of the corresponding benzofuroxan, bfx1 or bfx2, in 5.0 mL of anhydrous MeOH and 30.0 mL of anhydrous THF. After stirring at room temperature for 24 h and maintaining at 20 C for 24 h, the resulting precipitate was filtered, washed with petroleum ether, THF, and ethyl ether yielding the desired product. 5.1.1. 2-Amino-7,8-dichlorophenazine 5,10-dioxide (8) Black-violet solid (50%); mp >240.0 C. 1H NMR (DMSO-d6:D2O, 1:1) dH: 7.35 (1H, d, J ¼ 2.2 Hz), 7.38 (1H, dd, J1 ¼ 9.5, J2 ¼ 2.2 Hz), 8.30 (1H, d, J ¼ 9.4 Hz), 8.60 (2H, s). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6:D2O, 1:1) dC: 94.3, 121.9, 122.0, 126.2, 131.3, 132.2, 132.5, 135.0, 135.3, 138.8, 153.4. EI-MS, m/z (abundance, %): 295 (Mþ , 14), 279 (100), 263 (29). Found: C, 48.5; H, 2.2; N, 13.9. C12H7Cl2N3O2 requires C, 48.7; H, 2.4; N, 14.2%.
4. Conclusions We have identified the new derivative 7-fluoro-2-aminophenazine 5,10-dioxide, 12, as promising bioreductive antitumour agent. Further studies, like QSAR, and in vivo activities are currently in progress. 5. Experimental Some starting materials were commercially available researchgrade chemicals and used without further purification. All solvents were dried and distilled prior to use. All the reactions were carried out in a nitrogen atmosphere. Starting materials bfx1ebfx5 and 5, and derivative 19 were prepared following synthetic procedures previously reported [11,16,29]. Melting points were determined with an electrothermal melting point apparatus (Electrothermal 9100) and are uncorrected. Proton and carbon NMR spectra were recorded on a Bruker DPX-400 spectrometer at 298 K. The chemical shifts values are expressed in parts per million (d) relative to tetramethylsilane as internal standard and the J in Hertz. Mass spectra
5.1.2. 7,8-Dichloro-2-hydroxyphenazine 5,10-dioxide (9) Black-violet solid (48%); mp >240.0 C. 1H NMR (DMSO-d6) dH: 6.78 (1H, br s), 7.36 (1H, dd, J1 ¼ 9.4, J2 ¼ 2.1 Hz), 7.54 (1H, d, J ¼ 2.2 Hz), 8.45 (1H, d, J ¼ 9.4 Hz), 8.65 (2H, s). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6) dC: 100.1, 116.5, 121.4, 122.0, 122.4, 128.1, 133.1, 135.1, 135.3, 138.7, 150.6, 170.1. EI-MS, m/z (abundance, %): 296 (Mþ , 17), 279 (100), 264 (30). Found: C, 48.6; H, 1.9; N, 9.4. C12H6Cl2N2O3 requires C, 48.5; H, 2.0; N, 9.4%. 5.1.3. 2-Amino-7(8)-fluoro-8(7)-methylphenazine 5,10-dioxide (10) Red solid (51%). 7-Fluoro- and 8-fluoro-isomer ratio 41:59. 1H NMR (DMSO-d6) dH: 7-fluoro-isomer, 2.40 (3H, s), 6.62 (2H, br s), 7.31 (1H, d, J ¼ 2.2 Hz), 7.38 (1H, dd, J1 ¼ 8.0, J2 ¼ 2.2 Hz), 7.71 (1H, s), 8.31 (2H, m); 8-fluoro-isomer, 2.40 (3H, s), 6.80 (2H, br s), 7.35 (1H, d, J ¼ 2.2 Hz), 7.40 (1H, dd, J1 ¼ 8.2, J2 ¼ 2.3 Hz), 8.14 (1H, d, J ¼ 9.5 Hz), 8.30 (1H, s), 8.44 (1H, d, J ¼ 7.9 Hz). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6) dC: 7- and 8-isomers, 17.6, 96.3, 97.4, 104.2, 121.3, 122.1, 122.5, 124.9, 125.6, 129.5, 133.6, 137.9, 152.6. EI-MS, m/z (abundance, %): 7- and 8-isomers, 259 (Mþ ,
5368
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
2), 243 (6), 227 (8). Found: C, 60.0; H, 4.0; N, 16.0. C13H10FN3O2 requires C, 60.2; H, 3.9; N, 16.2%. 5.1.4. 7(8)-Fluoro-2-hydroxy-8(7)-methylphenazine 5,10-dioxide (11) Orange solid (46%). 7 and 8-isomer ratio 37:63. 1H NMR (DMSOd6:D2O, 1:1) dH: 7-isomer, 2.38 (3H, s), 7.38 (1H, d, J ¼ 2.1 Hz), 7.58 (1H, dd, J1 ¼ 7.8, J2 ¼ 2.1 Hz), 7.66 (1H, s), 8.30 (2H, m); 8-isomer, 2.38 (3H, s), 7.55 (1H, d, J ¼ 2.2 Hz), 7.72 (1H, dd, J1 ¼ 8.0, J2 ¼ 2.2 Hz), 8.27 (1H, d, J ¼ 9.5 Hz), 8.44 (1H, s), 8.56 (1H, d, J ¼ 7.9 Hz). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6:D2O, 1:1) dC: 7- and 8-isomers, 15.4, 99.5, 104.0, 121.5, 122.7, 122.8, 126.8, 128.9, 129.0, 132.0, 132.1, 132.8, 155.1. EI-MS, m/z (abundance, %): 7- and 8 isomers, 260 (Mþ , 5), 244 (10), 228 (3). Found: C, 59.8; H, 3.4; N, 10.4. C13H9FN2O3 requires C, 60.0; H, 3.5; N, 10.8%. 5.2. 2-Amino-7-fluorophenazine 5,10-dioxide (12) To a solution of Et3N (0.1 mL) in anhydrous DMF (0.48 mL), at 5 C and under nitrogen atmosphere, was added a solution of p-aminophenol (177 mg, 1.6 mmol) and 1.6 mmol of bfx3 in 0.15 mL of anhydrous DMF. After stirring at room temperature for 24 h and maintaining at 20 C for 24 h, the resulting precipitate was filtered, washed with petroleum ether, THF, and ethyl ether yielding the desired product. Brown-orange solid (50%). 7- and 8-isomer 97:3 ratio. 1H NMR (DMSO-d6) dH: 7-isomer, 6.80 (2H, br s), 7.36 (1H, d, J ¼ 9.9 Hz), 7.39 (1H, d, J ¼ 2.4 Hz), 7.78 (1H, dt, J1 ¼ 8.8, J2 ¼ 2.7 Hz), 8.18 (1H, dd, J1 ¼ 9.4, J2 ¼ 2.7 Hz), 8.32 (1H, d, J ¼ 10.1 Hz), 8.54 (1H, dd, J1 ¼ 9.7, J2 ¼ 5.5 Hz). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6) dC: 7-isomer,100.5,122.1,122.5,122.6,122.9, 124.3, 126.3, 126.5, 133.7, 135.0, 135.4, 161.8. EI-MS, m/z (abundance, %): 7- and 8-isomers, 245 (Mþ , 100), 229 (45), 213 (12). Found: C, 58.7; H, 3.1; N, 17.4. C12H8FN3O2 requires C, 58.8; H, 3.3; N, 17.1%. 5.3. General procedure for the preparation of the 5-hydroxypyrido [3,2-a]phenazine 7,12-dioxide derivatives 18 and 19 To a hot solution of 8-hydroxyquinoline (7.3 mmol), NaOMe (7.3 mmol) and MeOH (1.1 mL) was added a hot solution of the corresponding benzofuroxan (7.3 mmol) in 7.3 mL of MeOH. The reaction mixture was heated at reflux for 2.5 h, and after cooling, filtration gave the sodium salt of phenol that was acidified, with excess of AcOH, to give the desired product. 5.3.1. 5-Hydroxy-9(10)-methylpyrido[3,2-a]phenazine 7,12-dioxide (18) Orange solid (28%). 9- and 10-isomer ratio 50:50. 1H NMR (DMSO-d6:D2O) dH: 9-isomer, 2.65 (3H, s), 7.70 (1H, m), 7.85 (1H, s), 7.95 (1H, m), 7.97 (1H, s), 8.45 (1H, m), 9.18 (1H, m), 10.95 (1H, dd, J1 ¼ 8.8, J2 ¼ 1.6 Hz); 10-isomer, 2.60 (3H, s), 7.65 (1H, s), 7.70 (1H, m), 7.95 (1H, m), 8.05 (1H, s), 8.45 (1H, m), 9.19 (1H, m), 10.92 (1H, dd, J1 ¼ 8.8, J2 ¼ 1.6 Hz). 13C NMR (DMSO-d6:D2O) dC: 9- and 10-isomers, 23.0, 108.5, 111.6, 118.6, 119.7, 120.5, 124.4, 125.6, 130.0, 131.5, 131.8, 132.3, 133.7, 136.6, 150.0, 152.3. EI-MS, m/z (abundance, %): 293 (Mþ , 4), 277 (Mþ 16, 21), 261 (60). Found: C, 65.3; H, 3.5; N, 14.1. C16H11N3O3 requires C, 65.5; H, 3.8; N, 14.3%. 5.3.2. (5,10-Dioxide-7(8)-bromophenazin-2-yl) 2chloroetanoate (20) To a stirred solution of 5 (100 mg, 0.32 mmol) and Et3N (60 mL) in toluene (5.0 mL), chloracetylchloride (30 mL) was added dropwise. The mixture was maintained at 50 C until phenazine 5 was not present (SiO2, CH2Cl2:MeOH (98:2)). The mixture of reaction was partitioned between EtOAc and saturated aqueous solution of NaHCO3. After the work up the organic layer was evaporated in
vacuo and the residue was purified by column chromatography (SiO2, CH2Cl2:MeOH (0e5%)). Red solid (51%). 7 and 8-isomer ratio 64:36. 1H NMR (DMSO-d6) dH: 7-isomer, 4.80 (2H, s), 7.82 (1H, dd, J1 ¼ 9.6, J2 ¼ 2.3 Hz), 8.09 (1H, m), 8.41 (1H, d, J ¼ 2.6 Hz), 8.49 (1H, dd, J1 ¼ 9.4, J2 ¼ 3.4 Hz), 8.65 (1H, dd, J1 ¼ 9.6, J2 ¼ 3.4 Hz), 8.72 (1H, d, J ¼ 2.2 Hz). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6) dC: 7-isomer, 42.3, 100.6, 122.2, 122.6, 122.9, 124.4, 126.4, 132.4, 133.9, 135.1, 135.5, 137.9, 161.7, 169.4. EI-MS, m/z (abundance, %): 7- and 8-isomers, 384 (Mþ , 1), 368 (1), 352 (0.5). Found: C, 44.0; H, 2.4; N, 7.4. C14H8BrClN2O4 requires C, 43.8; H, 2.1; N, 7.3%. 5.4. 2-Benzyloxy-7(8)-bromophenazine 5,10-dioxide (21) To a solution of 5 (100 mg, 0.03 mmol) in DMF (5.0 mL) were added benzyl chloride (40 mg, 0.03 mmol), 18-crown-6 ether (87 mg, 0.03 mmol), K2CO3 (45 mg, 0.03 mmol), and KI (1 mg, 0.015 mmol). The mixture was heated at 40 C for 24 h. Then the crude of reaction was partitioned between EtOAc and aqueous HCl (10%). After the work up the organic layer was evaporated in vacuo and the residue was purified by column chromatography (SiO2, petroleum ether:EtOAc (4:6)). Orange solid (31%). 7 and 8-isomer ratio 64:36. 1H NMR (DMSO-d6) dH: 7-isomer, 5.42 (2H, s), 7.35 (2H, d, J ¼ 7.8 Hz), 7.49 (1H, m), 7.50 (1H, d, J ¼ 7.9 Hz), 7.60e7.70 (3H, m), 8.03 (2H, m), 8.50 (2H, m); 8-isomer, 5.40 (2H, s), 7.30 (2H, d, J ¼ 7.70 Hz); 7.48 (1H, s), 7.62e7.65 (2H, m), 8.10 (2H, m), 8.45 (2H, m), 8.70 (2H, m). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6) dC: 7- and 8-isomers, 69.1, 98.9, 108.2, 121.6, 122.8, 123.0, 123.6, 124.8, 126.8, 128.9, 129.3, 130.0, 134.5, 135.3, 136.8, 138.6, 138.8. EI-MS, m/z (abundance, %): 7- and 8-isomers, 396 (Mþ , 0.5), 380 (2), 364 (7). Found: C, 57.5; H, 3.4; N, 6.8. C19H13BrN2O3 requires C, 57.4; H, 3.3; N, 7.0%. 5.5. Biology. Bioreductive activity [10e12,20,21] Cells: V79 cells (Chinese hamster lung fibroblasts) were obtained from ECACC (European Collection of Animal Cell Cultures) and maintained in logarithmic growth as subconfluent monolayer by trypsinization and subculture to (1e2) 104 cells/cm2 twice weekly. The growth medium was EMEM (Eagle’s Minimal Essential Medium), containing 10% (v/v) foetal bovine serum (FBS) and penicillin/streptomycin at 100 U/100 mg/mL. Aerobic and hypoxic cytotoxicity: suspension cultures. Monolayers of V79 cells in exponential growth were trypsinized, and suspension cultures were set up in 50 mL glass flasks: 2 104 cells/mL in 30 mL of EMEM containing 10% (v/v) FBS and HEPES (10 mM). The glass flasks were submerged and stirred in a water bath at 37 C, where they were gassed with humidified air or pure nitrogen. Treatment: compound solutions, 8e12, 18e21, and 24, were prepared just before dosing. Stock solutions, 150-fold more concentrated, were prepared in pure DMSO (Aldrich) or sterilized distilled water. Thirty minutes after the start of gassing, 0.2 mL of the stock compound solution was added to each flask, two flasks per dose. In every assay there was one flask with 0.2 mL of DMSO (negative control) and another with 7-chloro-3-[3-(N,N-dimethylamino)propylamino]-2-quinoxalinecarbonitrile 1,4-dioxide hydrochloride (positive control). Cloning: after 2 h exposure to the compound, the cells were centrifuged and resuspended in plating medium (EMEM plus 10% (v/v) FBS and penicillin/streptomycin). Cell numbers were determined with a haemocytometer and 102e103 cells were plated in 6-well plates to give a final volume of 2 mL/30 mm of well. Plates were incubated at 37 C in 5% CO2 for 7 days and then stained with aqueous crystal violet. Colonies with more than 64 cells were counted. The plating efficiency (PE) was calculated by dividing the number of colonies by the number of cells seeded. The percent of control cell survival for the compound-treated cultures (SFnormoxia and
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
SFhypoxia) was calculated as PEtreated/PEcontrol 100. The compounds were tested at 20 mM in duplicate flasks both in aerobic and hypoxic conditions. 5.6. Cyclic voltammetric studies [12,30,31] DMSO (spectroscopy grade) was obtained from Aldrich. Tetrabutylammonium perchlorate (TBAP), used as supporting electrolyte, was obtained from Fluka. Cyclic voltammetry was carried out using a Metrohm 693 VA instrument with a 694 VA Stand convertor and a 693 VA Processor, in DMSO (ca. 1.0 mM), under a nitrogen atmosphere at room temperature (TBAP, ca. 0.1 mM) as supporting electrolyte. A three-electrode cell configuration was used, a mercury dropping working electrode, a platinum wire auxiliary electrode, and a saturated calomel reference electrode. Voltage scan rates ranged from 0.10 to 2.00 V/s. 5.7. DNA-interaction studies 5.7.1. UVevisible spectroscopy [10e12] DNA solution: calf thymus DNA (CTDNA, 12.5 mg, Sigma Chemical Co., USA) was slowly magnetically stirred in 5 mL TriseHCl buffer (10 mM, pH 7.4) for 24 h at 4 C. From this solution, 0.6 mL was diluted with the same buffer to 25 mL. Test compound solution: it was prepared at 104 M concentration using a minimal volume of adequate solvent, no more than 5%, and then diluted adding water to 2 105 M. No effect on DNA was observed by these concentrations of solvents. Study: a 3.0 mL sample of this resulting solution was mixed with 3.0 mL of DNA solution described above. The mixtures were slowly rotated for 24 h and subsequently their UV spectra were recorded using a 1-cm cell at 20 C on a Jasco V-570 UV/VIS/NIR spectrophotometer. Areas were calculated automatically by the apparatus. 5.8. Fluorescence spectroscopy [25,26] DNA solutions: CTDNA (12.5 mg, Sigma Chemical Co., USA) was slowly magnetically stirred in 5 mL phosphate buffer solution (PBS) (50 mM, pH 6.0) for 24 h at 4 C. From this solution, the further necessary dilutions were performed using the same buffer. Test compound solution: it was prepared at 20 mM concentration using PBS (50 mM, pH 6.0) and 20% of DMSO. No effect on DNA was observed by these concentrations of solvents. Study: fluorescence spectra were recorded on a Varioskan Flash 2.4.1 spectrometer. The fluorescence quenching titrations with CTDNA were performed by keeping the compounds concentrations constant (20 mM) and varying the nucleic acid concentrations (0e400 mM) maintaining the total volume of the solution constant. Fluorescence emission spectra were recorded in the wavelength range of 425e825 nm by exciting the compounds at the corresponding wavelength with the excitation slit widths of 12 nm. The intrinsic binding constants of compounds with CTDNA were determined by fluorescence titrations. The data were plotted according to the SterneVolmer equation [32]:
fluorescence quenching constant. [Q] is the concentration of quencher. Acknowledgements Financial supports from Comisión Honoraria de Lucha contra el Cáncer (Uruguay) and from the project of the Work Community of Pyrenees (CTP 07-P11 Ref. IIQ011698.R11) is acknowledged. We thank PEDECIBA-ANII for scholarships to MLL and MC, ANII for scholarship to MN, and PEDECIBA and CSIC-Universidad de la República for fellowships. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
[11] [12]
[13] [14]
[15] [16] [17] [18] [19] [20]
[21]
[22] [23] [24] [25] [26] [27] [28] [29]
[30]
I0/I ¼ 1 þ Kq [Q] where, I0 and I are the fluorescence intensities in the absence and presence of CTDNA, respectively. Kq is the SterneVolmer
5369
[31]
[32]
S. Rockwell, J.E. Moulder, Int. J. Radiat. Oncol. Biol. Phys. 19 (1990) 197e202. W.A. Denny, Eur. J. Med. Chem. 36 (2001) 577e595. K. Ruan, G. Song, G. Ouyang, J. Cell. Biochem. 107 (2009) 1053e1062. J.M. Brown, W.R. Wilson, Nat. Rev. Cancer 4 (2004) 437e447. A.J. Lin, L.A. Cosby, C.W. Shansky, A.C. Sartorelli, J. Med. Chem. 15 (1972) 1247e1252. P. Wardman, Curr. Med. Chem. 8 (2001) 739e761. H. Cerecetto, M. González, Minirev. Med. Chem. 1 (2001) 219e231. H. Cerecetto, M. González, M.L. Lavaggi, Med. Chem. 2 (2006) 315e327. H. Cerecetto, M. González, M.L. Lavaggi, W. Porcal, J. Braz. Chem. Soc. 6 (2005) 1290e1296. H. Cerecetto, M. González, M.L. Lavaggi, M.A. Aravena, C. Rigol, C. Olea-Azar, A. Azqueta, A. López de Ceráin, A. Monge, A.M. Bruno, Med. Chem. 2 (2006) 511e521. H. Cerecetto, M. González, M.L. Lavaggi, A. Azqueta, O. Ezpeleta, A. López de Ceraín, A. Monge-Vega, J. Med. Chem. 48 (2005) 21e23. M.L. Lavaggi, M. Cabrera, M.A. Aravena, C. Olea-Azar, A. López de Ceráin, A. Monge, G. Pachón, M. Cascante, A.M. Bruno, L.I. Pietrasanta, M. González, H. Cerecetto, Bioorg. Med. Chem. 18 (2010) 4433e4440. M.L. Lavaggi, M. Cabrera, M. González, H. Cerecetto, Chem. Res. Toxicol. 21 (2008) 1900e1906. O.G. Pachón, A. Azqueta, M.L. Lavaggi, A. López de Ceraín, E. Creppy, A. Collins, H. Cerecetto, M. González, J.J. Centelles, M. Cascante, Chem. Res. Toxicol. 21 (2008) 1578e1585. G.-W. Ludwig, H. Baumgartel, Chem. Ber. 115 (1982) 2380e2383. E. Vicente, R. Villar, A. Burguete, B. Solano, S. Ancizu, S. Pérez-Silanes, I. Aldana, A. Monge, Molecules 13 (2008) 86e95. M.J. Abu El-Haj, B.W. Dominy, J.D. Johnston, J. Org. Chem. 37 (1972) 589e593. A.J. Boulton, A.R. Katritzky, M.J. Sewell, B. Wallis, J. Chem. Soc. B 9 (1967) 914e917. A.J. Boulton, P.J. Halls, A.R. Katritzky, J. Chem. Soc. B (1970) 636e640. A. Monge, J.A. Palop, A. López de Ceráin, V. Senador, F.J. Martínez-Crespo, Y. Sáinz, S. Narro, E. García, C. de Miguel, M. González, A.J. Barker, E. Hamilton, E.D. Clarke, D.T. Greenhow, J. Med. Chem. 38 (1995) 1786e1792. C. Urquiola, M. Vieites, M.H. Torre, M. Cabrera, M.L. Lavaggi, H. Cerecetto, M. González, A. López de Cerain, A. Monge, P. Smircich, B. Garat, D. Gambino, Bioorg. Med. Chem. 17 (2009) 1623e1629. J.D. Aguirre, A.M. Angeles-Boza, A. Chouai, J.P. Pellois, C. Turro, K.R. Dunbar, J. Am. Chem. Soc. 131 (2009) 11353e11360. J. Wang, X. Yang, Spectrochim. Acta A Mol. Biomol. Spectrosc. 74 (2009) 421e426. P.U. Maheswari, V. Rajendiran, M. Palaniandavar, R. Parthasarathi, V. Subramanian, J. Inorg. Biochem. 100 (2006) 3e17. X. Jiang, L. Shang, Z. Wang, S. Dong, Biophys. Chem. 118 (2005) 42e50. Y.N. Ni, X.Z. Zhong, Chin. Chem. Lett. 18 (2007) 569e572. H. Heli, S.Z. Bathaie, M.F. Mousavi, Electrochem. Commun. 6 (2004) 1114e1118. Y. Li, X.J. Yao, J. Jin, X.G. Chen, Z.D. Hu, Biochim. Biophys. Acta 1774 (2007) 51e58. H. Cerecetto, R. Di Maio, M. González, M. Risso, P. Saenz, G. Seoane, A. Denicola, G. Peluffo, C. Quijano, C. Olea-Azar, J. Med. Chem. 42 (1999) 1941e1950. L.A. Fernández, M.R. Santo, M. Reta, L. Giacomelli, R. Cattana, J.J. Silber, M. Risso, H. Cerecetto, M. González, C. Olea-Azar, Molecules 10 (2005) 1197e1208. J. Rodríguez, A. Gerpe, G. Aguirre, U. Kemmerling, O.E. Piro, V.J. Arán, J.D. Maya, C. Olea-Azar, M. González, H. Cerecetto, Eur. J. Med. Chem. 44 (2009) 1545e1553. Y. Cao, X.W. He, Spectrochim. Acta, Part A 54 (1998) 883e892.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structural modifications on the phenazine N,N0 -dioxide-scaffold looking for new selective hypoxic cytotoxins María Laura Lavaggi a, Marcos Nieves a, Mauricio Cabrera a, Claudio Olea-Azar b, Adela López de Ceráin c, Antonio Monge c, Hugo Cerecetto a, *, Mercedes González a, * a b c
Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Chile Centro de Investigación en Farmacobiología Aplicada, Universidad de Navarra, Pamplona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2010 Received in revised form 28 July 2010 Accepted 26 August 2010 Available online 16 September 2010
We have identified phenazine 5,10-dioxides as prodrugs for antitumour therapy that undergo hypoxicselective bioreduction to form cytotoxic species. Here, we investigated some structural modifications in order to find new selective hypoxic cytotoxins and to establish the structural requirements for adequate activity. Three different chemical-series were prepared and the clonogenic survival of V79 cells on aerobic and anaerobic conditions was determined. Electrochemical- and DNA-interaction studies were done for the most relevant derivatives. The new fluoro-derivative 7-fluoro-2-aminophenazine 5,10dioxide displayed selective toxicity towards hypoxic V79 cells having adequate hypoxic cytotoxicity ratio (HCR ¼ 6.8) and being the most potent hypoxic cytotoxins (P ¼ 2.5 mM) described for this family of bioreductive agents. The reduction potential of the N-oxide moiety in this new fluoro-derivative was in the range for adequate bioreduction property. According to the fluorescence studies, the DNA-interaction mechanism was especially operative in the phenazine drugs more than in the corresponding prodrugs, phenazine dioxides. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Phenazine N,N0 -dioxides Bioreductive agents Cancer
1. Introduction Since the 1960’ it is well known the existence of hypoxic and necrotic regions in solid tumours [1] as consequence of the rapid growth of cancerous cells and their deficient vascularisation that produces molecular oxygen diffusion decreasing [2]. The hypoxic regions, associated with increased resistance to radiation [3], are refractory to conventional anticancer drugs because they are antiproliferative agents that kill dividing cells and these tumour cells are not dividing rapidly. In addition, it has also been demonstrated that hypoxia in tumours alters cellular metabolism tending to select for a more malignant phenotype, increasing mutation rates. It also increases expression of genes associated with angiogenesis and tumour invasion, and is associated with a more metastatic phenotype of human cancers [4]. By enhancing metastasis, hypoxia can compromise curability of tumours by surgery. This common feature of cancerous cells, hypoxia, is used for the development of a distinct therapy for treating cancer.
* Corresponding author. Tel.: þ598 2 5258618; fax: þ598 2 5250749. E-mail addresses: [email protected] (H. Cerecetto), [email protected] (M. González). 0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2010.08.061
Based on using prodrugs, capable to be bioreduced under hypoxic conditions (bioreductive antitumour agents, [5] BAA) to further drugs that produce cytotoxic events causing different degrees of cancerous cells damage, hypoxic-selective cytotoxins have been developed. Molecular oxygen reverts the bioreductive process in normal oxygenated tissues giving the selectivity to this process. This selectivity is governed by the redox properties of the drugs and the reductases enzymes levels [6]. N-oxides derivatives have been described among the compounds classified as BAA [7,8]. We have reported phenazine N,N0 -dioxides (PDOs) [9] as a new group of hypoxia-activated prodrugs that potentially interact with DNA after the corresponding bioreduction in hypoxic conditions [10]. The PDOs 1, 2, and 5 displayed excellent selective cytotoxic properties being phenazine 5,10-dioxides 3 and 4 toxics in both conditions at the assayed dose (20 mM) (Fig. 1a) [11]. On the other hand, the benzo[a]phenazine 7,12-dioxides analogues, i.e. 6 and 7 (Fig. 1a) exhibited very particular cytotoxic profiles having high oxic-cytotoxicities without toxicity in hypoxia, probably as result of the aerobic futile cycle [12]. The selective anaerobic-reduction and its relation to bioreductive activity were proved using enzymatic mammal systems [13]. Furthermore, some benzo[a]phenazine 7,12dioxide prodrugs were able to interact with DNA, i.e. 6 (Fig. 1a) and produce some degree of DNA-damage in oxic conditions [12].
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
5363
Fig. 1. First and second generations of PDOs and designed new generation of potential selective hypoxic cytotoxins.
Besides, some of these derivatives displayed in vitro aerobic-antitumour activity against Caco-2 cells [12,14]. To complete the necessary amassed structural-information refer to the potential PDO-behaviours as selective hypoxic cytotoxins we planned three different chemical modifications (series a, b, and c, Fig. 1b). The rationality was to: i) explore 7- and 8-substitutions by halo/alkyl groups, using PDOs 1e5 as parent compounds (series a); ii) explore aza analogues of parent compounds 6 and 7 (series b); iii) block OH-functionality for parent compound 5 (series c). Additionally, the biological effects of all the new compounds were analyzed by use of a clonogenic assay with V79 cells in simulated oxic and hypoxic conditions determining the hypoxic potency, P, and hypoxic cytotoxicity ratio, HCR. The PDOs electrochemical behaviour and DNA-interaction capability of some of these prodrugs and drugs were also studied by UV- and fluorescencespectroscopy.
2. Methods and results 2.1. Chemistry For the preparation of PDOs belonging to series a we have selected one of the best described synthetic methodology that involves the reaction between the corresponding benzofuroxan and different phenols (Scheme 1a) [15]. The designed derivatives 8e11 were generated with the original described methodology, MeONa/MeOHeTHF/5 C, in good yields however in this condition we were unable to obtain derivatives 12e15 (Scheme 1a). As it was previously observed for similar reactions [16], the preparation of these derivatives implicated as the main process the undesirable nucleophilic substitution of one fluorine by the reactant phenol. From the messy crude of reaction, it was possible to detect, by 1H NMR and MS, products like (I) and (II) (Fig. 2). Softer experimental conditions were probed for the preparation of the desired PDO 12, as Et3N/THF/5 C and Et3N/DMF/5 C. This compound was obtained in good yield, 55%, using the last one. However, our efforts to obtain derivatives 13e15 in this condition were unsuccessful. For the preparation of PDOs belonging to series b the corresponding benzofuroxans were reacted with 8-hydroxyquinoline in basic milieu (MeONa/MeOH) [17]. This procedure can yield derivatives
16e19 (Scheme 1b), produced by ortho (pyrido[2,3-a]phenazines 16 and 17) and para (pyrido[3,2-a]phenazines 18 and 19) coupling procedures through the corresponding phenolate anion. In our cases, we were able to obtain only derivatives 18 and 19 after pH change. Maybe, the generation of derivatives 16 and 17 is less favourable for the potential stereo-electronic hindrance promoted by 1-nitrogen. The reaction of 5-substituted-benzofuroxans with phenols produced the mixtures of the 7- and 8-, or 9and 10-, substituted-phenazine isomers as result of the well known tautomerism of benzofuroxan reactant at room temperature [18,19]. Although, the carbanion could react unselectively and with similar probability with each tautomeric form, some isomeric preferences were observed according to the proportion of each tautomer at the temperature of reaction [9]. In example, when 5-fluorobenzofuroxan reacted with p-aminophenol the isolated product corresponded almost exclusively to 7-fluoro-isomer (see Experimental section). In all cases, the products were characterized and evaluated as a non-separable mixture of 7- and 8-, or 9- and 10-, isomers because they could not be separated neither by crystallization nor by chromatographic methods. Derivatives 20 and 21, belonging to series c, were prepared by reaction between parent compound 5 and the two electrophilic reactants shown in Scheme 1c. In these reactions phenazine N-deoxygenation processes took place extensively producing the desired products in modest yields. All the proposed structures were established by 1H, and 13C NMR spectroscopies (using COSY, HMQC, and HMBC experiments) and MS. The purity was analyzed and established by TLC and microanalysis, respectively.
2.2. Biological characterization 2.2.1. In vitro normoxic and hypoxic cytotoxicity All the PDOs were examined for their selective hypoxic cytotoxicities in a pre-established model of V79 cells [10e12,20,21]. They were analyzed as a non-separable mixture of 7- and 8-isomers, in the cases of 10e12, 20 and 21, and 9- and 10-isomers, in the case of 18. Previous results for similar chemical systems (quinoxaline 1,4-dioxides) demonstrated that no difference between both positional isomers was observed in the selective hypoxic cytotoxicities against V79 cells [20]. The percentages of
5364
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
Scheme 1. Synthetic procedures used to prepare the designed PDO derivatives.
survival fractions (SF, Table 1) were measured at 20.0 mM while derivatives 8, 9, and 12 were also evaluated at 10.0 mM. The phenol 11, the pyrido[3,2-a]phenazines 18 and 19, and the O-substitutedderivatives 20 and 21 were non-selective exhibiting similar survival fraction in normoxia and hypoxia. The methyl- and fluorosubstituted-aminophenazine dioxide 10 showed a modest degree of selectivity similar to that of parent compounds, the methoxy-, 1,
Table 1 PDO cytotoxic effects in normoxia and hypoxia on V79 cells. Compd.
8 9 10 11 12 18 19 20 21
Norm
Hypox
2 (3) 1 (0) 88 100 0 (30) 49 58 84 86
0 (18) 0 (8) 15 95 0 (0) 46 53 62 90
SF norm ¼ survival fraction in normoxia at 20.0 mM. SF hypox ¼ survival fraction in hypoxia at 20.0 mM. c Values are means of two different experiments. The assays were done by duplicate and using at least three repetitions, standard errors were not greater than 2% for most assays. d Values in parenthesis are at 10.0 mM. a
b
Fig. 2. Products detected in the expansion of benzofuroxans bfx3 and bfx4 to PDOs 12e15, using traditional conditions [16].
SFa,b,c,d
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369 Table 2 Potencies (P) and hypoxic cytotoxicity ratios (HCR) on V79 cells for selected PDOs. Compd.
P (mM)
HCR
1 2 3 4 5a 8 12
35.0 25.0 18.0 12.0 10.0 10.0 2.5
>3.0 >2.5 1.1 5.0 >10.0 0.9 6.8
a
From Ref. [11].
and methyl-substituted-aminophenazine dioxide, 2. On the other hand, the dichloro-derivatives 8 and 9, and the fluoro-derivative 12 showed high cytotoxicity in both conditions, normoxia and hypoxia. Derivative 12 displayed some degree of selectivity at lower doses (10.0 mM). In order to complete the information the amino derivatives 8 and 12, together with the parent compounds 1e5, were tested at different doses to obtain doseeresponse curves in air and hypoxia. Potency (P) and hypoxic cytotoxicity ratio (HCR) were calculated from these curves (Table 2). P is defined as the dose, in micromolar, which gives 1% of control cell survival in hypoxia and HCR as ratio between the dose in air divided by the dose in hypoxia giving 1% of control cell survival. The dichloro-amine 8 was more cytotoxic in oxia than hypoxia in all the studied dose-range (Fig. 3a). However, the fluoro-amine 12 showed the best hypoxic potency, P ¼ 2.5 mM (Fig. 3b), between all the studied PDOs with HCR value, on V79 cells (Table 2), in the same order that others BAA [6e8]. 2.3. Mechanism of action studies To gain insight into PDO mechanism of toxicity, first of all we studied the PDO electrochemical behaviour in term of cyclic voltammetry in organic aprotic solvent (DMSO) [10e12]. The new developed PDO displayed comparable voltammetric behaviour in this condition showing two to three reduction peaks and the anodic counterparts. Especially, we focus our attention in the first reduction potential because it could be used as a descriptor of the PDO feasibility to be reduced in hypoxic conditions. Table 3 lists the values of the first N-oxide cathodic peak, Epc, which correspond to
5365
Table 3 Reduction potentials and DNA-interacting capability by UV studies of PDO derivatives. Compd.
Epc vs. SCEa (V)
a24/a0
1 2 3 4 5 6 7 8 9 10 11 12 18 21 m-AMSA Ethidium bromide NSC 322921 (bis-benzimide) Mitoxantrone
1.03b 1.00b 0.96b 0.92b 1.14b 1.36c 1.22c 1.08 0.96
0.89b 0.98b 0.89b 1.00b 1.00b 0.47c 0.94c 0.98 0.96 0.85 0.83 0.88 nse 0.88 0.30 0.50 0.57 0.00
a b c d e
d d
0.83 1.63 1.28 e e e e
Peaks potentials (w0.01 V) measured at a scan rate of 2.00 V/s. From Ref. [10]. From Ref. [12]. Solubility problems in the assay milieu do not allow to perform the study. ns: not studied.
quasireversible process. This value seems to be correlated to the hypoxic toxicity since the lowest hypoxic-SF values correspond to compounds with the lowest reduction potentials, lower than 1.14 V (Fig. 4a), consequently compounds with more favourable reduction processes. The same relationship was observed between P and Epc for the studied amino derivatives 1e4, and 12 (Fig. 4b) outlying the parent compound 5 that belongs to the phenol family and derivative 8 which possessed a particular biological behaviour (Tables 1 and 2). The new derivative 12 has the lowest N-oxide Epc value, i.e. 0.83 V (Table 3), showing the relevance of the fluorine substituent, at the benzo-ring, in the modulation of the reduction potential. Secondly, we analyze the PDO-prodrugs’ capability to interact with DNA, in oxic conditions, by measuring the hypochromic and bathochromic effect of prodrugs- and drugs-absorbances in the UV spectra, in a 20 nm band centered on the maximal absorbance value of each compound, at 0 and 24 h [22,23]. Additionally, in
Fig. 3. Doseeresponse curves in air and hypoxia for the new derivatives 8 (a) and 12 (b). Black squares and dotted lines: oxic conditions.
5366
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
Fig. 4. (a) Epc vs. SFhypoxia for PDOs. In red parent compounds. (b) Epc vs. hypoxic potency (P) for PDOs. In red outliers. The dotted line is only indicative.
order to confirm our hypothesis we evaluated the DNA-interaction capability of the drugs (phenazines or phenazine mono-oxides produced during the hypoxic conditions) choosing previously developed compounds 22e25 [11,13] (Fig. 5). The degree of interaction was expressed by the ratio between the final absorbance area (a24) and the absorbance of the compound (a0). Values of 1.00 or superior to 1.00 indicate a total lack of affinity and a value of 0 indicates binding of the entire compound to DNA (Table 3). No relevant interactions with DNA in oxic conditions, using this methodology, were observed for any of the studied compounds (Table 4). However, in the UV-experiments after 24 h of incubation some curious findings were observed. Together with the absence of hypochromic or bathochromic effects it was observed the apparition of some new bands or shoulders near the absorption maximums (data not shown). These findings could be indicating formation of productsaggregates or products-DNA-aggregates. [24,25]. For this reason, another technique and conditions were employed. The intrinsic changes on the fluorescence of phenazine while varying DNA concentrations after 30 min of incubation were monitored [26]. The sensitive of this assay allowed us to evidence interactions after this incubation time. Because it is the static quenching of DNA to compounds (Fig. 6a), the quenching constant is considered as the formation constant of compounds and DNA [27], i.e., the binding constant (Kq) of compounds with DNA. The SterneVolmer plots (Fig. 6b) provided the Kq of studied compounds at pH 6.0 [28] (Table 4). The studied compounds were able, in different degrees, to interact with DNA but lesser than toluene blue (TB, Table 4). Additionally, it is clear the relationship between the level of phenazine oxygenation and DNA-interaction capability, i.e. the
Fig. 5. Phenazines and phenazine mono-oxides herein evaluated as DNA-interacting agents.
reduced derivates, PDO 24 and 25, interacted better than the PDO parent compound 4 (Table 4). Finally, no correlation was observed between oxic-cytotoxicities (see values of SF, Table 4) and DNAinteraction abilities. For example, the best DNA-interacting PDOs, 2 and 12, displayed very different oxic-cytotoxicities. 3. Discussion We report the study of new series of PDOs with potential use as bioreductive antitumour agents. Among the new developed PDOs different features could be stated. Firstly, the hydroxy-phenazine 5,10-dioxides, 9 and 11, are no selective. In the first case cytotoxic in both conditions and the contrary for the second one. The aminoanalogue of 9, derivative 8, showed the same biological profile and when it was studied at a lower dose it resulted more toxic in oxic conditions than in hypoxic ones (Fig. 3a). Secondly, the 4-aza analogues of the oxic-cytotoxic 6 and 7, derivatives 18 and 19, were not cytotoxic in oxia as the parent compounds however they were also non-selective. Thirdly, modifications at OH-level in parent compound 5, derivatives 20 and 21, conduct to inactive and nonselective agents. Fourthly, the fluoro-amino derivatives 10 and 12 displayed hypoxic-cytotoxic selectivity (Table 1) being derivative 12
Table 4 DNA-interacting capability by fluorescence-studies of PDOs, phenazines monooxides and phenazines. Compd.
a24/a0
Kqa
SFb,c,d Norm
Hypox
2 4 12 22 23 24 25 TBf
0.98 1.00 0.88 0.92 0.85 0.92 0.90 e
7.7 1.0 2.6 0.1 7.8 2.7 5.3 0.6 6.1 0.6 4.2 0.8 7.3 3.9 12.6 1.5
61 12 0 100 98 0e 0 e
11 0 0 100 100 0e 0 e
a Kq ¼ SterneVolmer fluorescence quenching constant Experimental section). b SF norm ¼ survival fraction in normoxia at 20 mM. c SF hypox ¼ survival fraction in hypoxia at 20 mM. d From Ref. [10] except for compound 24. e It was determined herein. f TB: toluene blue.
(for
details
see
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
5367
Fig. 6. (a) Fluorescence spectra of parent compound 4 (left) and the corresponding reduced derivative 25 (right) in PBS (50 mM, pH 6.0) after addition of CT DNA. (b) SterneVolmer quenching plot of parent compound 2 with increasing concentrations of DNA in PBS (50 mM, pH 6.0).
the most hypoxic-potent cytotoxin with a potency of 2.5 mM (P, Table 1) and adequate HCR value (6.8, Table 1). The profile of hypoxic cytotoxicity of PDOs seemed to be governed by the bioreduction process. This fact could be realized in Table 3 and Fig. 4 where compounds with the lowest hypoxic survival fractions (SFhypoxia, Fig. 4a) have reduction potential (Epc) between 0.83 and 1.14 V. It was possible to observe for aminophenazines 1e4 and 12 that hypoxic potency, P, was linearly related to Epc observing better P for lowest reduction potential (Fig. 4b). It was not possible to find relationships between neither hypoxicselectivity, expressed as HCR, nor oxic-cytotoxicity, expressed as SFnorm, and Epc (data not shown). The DNA-interaction studies showed that the PDO prodrugs interact with this biomolecule, in oxic conditions, less than the well-established DNA-interacting agent toluene blue (Table 4). On the other hand, studying the DNA-interaction capability of the potential drugs, phenazine and phenazine mono-oxide, we confirmed that the proposed extra-mechanism of PDOs, interaction with this biomolecule after the corresponding bioreduction in hypoxic conditions, could be operative (compare Kq values for prodrug 4, and potential drugs 24 and 25, Table 4). The structural changes done to obtain derivatives belonging to series c (Fig. 1) modify favourably the DNA-capability of the parent compound 5 (Fig. 1). Given that the OH-blocking derivative 21, with an extra-pinteracting moiety, has little better improved ability to interact with DNA according to UV studies (compare a24/a0 values for 5 and 21, Table 3). However, this change did not modify the oxic/hypoxic biological profile.
were determined on an MSD 5973 Hewlett-Packard spectrometer using electronic impact ionization. Microanalyses were performed on a Fisons EA 1108 CHNS-O instrument and were within (0.4% of the calculated compositions). Column chromatography was carried out using Merck silica gel (60e230 mesh). Most chemicals and solvents were analytical grade and used without further purification. 5.1. General procedure for the preparation of the phenazine 5,10dioxide derivatives 8e11 To a solution of 3.9 mmol of metallic sodium in anhydrous MeOH (25.0 mL), at 5 C and under nitrogen atmosphere, was added a solution of 20.0 mmol of the corresponding phenol (p-aminophenol or p-hydroquinone) and 20.0 mmol of the corresponding benzofuroxan, bfx1 or bfx2, in 5.0 mL of anhydrous MeOH and 30.0 mL of anhydrous THF. After stirring at room temperature for 24 h and maintaining at 20 C for 24 h, the resulting precipitate was filtered, washed with petroleum ether, THF, and ethyl ether yielding the desired product. 5.1.1. 2-Amino-7,8-dichlorophenazine 5,10-dioxide (8) Black-violet solid (50%); mp >240.0 C. 1H NMR (DMSO-d6:D2O, 1:1) dH: 7.35 (1H, d, J ¼ 2.2 Hz), 7.38 (1H, dd, J1 ¼ 9.5, J2 ¼ 2.2 Hz), 8.30 (1H, d, J ¼ 9.4 Hz), 8.60 (2H, s). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6:D2O, 1:1) dC: 94.3, 121.9, 122.0, 126.2, 131.3, 132.2, 132.5, 135.0, 135.3, 138.8, 153.4. EI-MS, m/z (abundance, %): 295 (Mþ , 14), 279 (100), 263 (29). Found: C, 48.5; H, 2.2; N, 13.9. C12H7Cl2N3O2 requires C, 48.7; H, 2.4; N, 14.2%.
4. Conclusions We have identified the new derivative 7-fluoro-2-aminophenazine 5,10-dioxide, 12, as promising bioreductive antitumour agent. Further studies, like QSAR, and in vivo activities are currently in progress. 5. Experimental Some starting materials were commercially available researchgrade chemicals and used without further purification. All solvents were dried and distilled prior to use. All the reactions were carried out in a nitrogen atmosphere. Starting materials bfx1ebfx5 and 5, and derivative 19 were prepared following synthetic procedures previously reported [11,16,29]. Melting points were determined with an electrothermal melting point apparatus (Electrothermal 9100) and are uncorrected. Proton and carbon NMR spectra were recorded on a Bruker DPX-400 spectrometer at 298 K. The chemical shifts values are expressed in parts per million (d) relative to tetramethylsilane as internal standard and the J in Hertz. Mass spectra
5.1.2. 7,8-Dichloro-2-hydroxyphenazine 5,10-dioxide (9) Black-violet solid (48%); mp >240.0 C. 1H NMR (DMSO-d6) dH: 6.78 (1H, br s), 7.36 (1H, dd, J1 ¼ 9.4, J2 ¼ 2.1 Hz), 7.54 (1H, d, J ¼ 2.2 Hz), 8.45 (1H, d, J ¼ 9.4 Hz), 8.65 (2H, s). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6) dC: 100.1, 116.5, 121.4, 122.0, 122.4, 128.1, 133.1, 135.1, 135.3, 138.7, 150.6, 170.1. EI-MS, m/z (abundance, %): 296 (Mþ , 17), 279 (100), 264 (30). Found: C, 48.6; H, 1.9; N, 9.4. C12H6Cl2N2O3 requires C, 48.5; H, 2.0; N, 9.4%. 5.1.3. 2-Amino-7(8)-fluoro-8(7)-methylphenazine 5,10-dioxide (10) Red solid (51%). 7-Fluoro- and 8-fluoro-isomer ratio 41:59. 1H NMR (DMSO-d6) dH: 7-fluoro-isomer, 2.40 (3H, s), 6.62 (2H, br s), 7.31 (1H, d, J ¼ 2.2 Hz), 7.38 (1H, dd, J1 ¼ 8.0, J2 ¼ 2.2 Hz), 7.71 (1H, s), 8.31 (2H, m); 8-fluoro-isomer, 2.40 (3H, s), 6.80 (2H, br s), 7.35 (1H, d, J ¼ 2.2 Hz), 7.40 (1H, dd, J1 ¼ 8.2, J2 ¼ 2.3 Hz), 8.14 (1H, d, J ¼ 9.5 Hz), 8.30 (1H, s), 8.44 (1H, d, J ¼ 7.9 Hz). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6) dC: 7- and 8-isomers, 17.6, 96.3, 97.4, 104.2, 121.3, 122.1, 122.5, 124.9, 125.6, 129.5, 133.6, 137.9, 152.6. EI-MS, m/z (abundance, %): 7- and 8-isomers, 259 (Mþ ,
5368
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
2), 243 (6), 227 (8). Found: C, 60.0; H, 4.0; N, 16.0. C13H10FN3O2 requires C, 60.2; H, 3.9; N, 16.2%. 5.1.4. 7(8)-Fluoro-2-hydroxy-8(7)-methylphenazine 5,10-dioxide (11) Orange solid (46%). 7 and 8-isomer ratio 37:63. 1H NMR (DMSOd6:D2O, 1:1) dH: 7-isomer, 2.38 (3H, s), 7.38 (1H, d, J ¼ 2.1 Hz), 7.58 (1H, dd, J1 ¼ 7.8, J2 ¼ 2.1 Hz), 7.66 (1H, s), 8.30 (2H, m); 8-isomer, 2.38 (3H, s), 7.55 (1H, d, J ¼ 2.2 Hz), 7.72 (1H, dd, J1 ¼ 8.0, J2 ¼ 2.2 Hz), 8.27 (1H, d, J ¼ 9.5 Hz), 8.44 (1H, s), 8.56 (1H, d, J ¼ 7.9 Hz). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6:D2O, 1:1) dC: 7- and 8-isomers, 15.4, 99.5, 104.0, 121.5, 122.7, 122.8, 126.8, 128.9, 129.0, 132.0, 132.1, 132.8, 155.1. EI-MS, m/z (abundance, %): 7- and 8 isomers, 260 (Mþ , 5), 244 (10), 228 (3). Found: C, 59.8; H, 3.4; N, 10.4. C13H9FN2O3 requires C, 60.0; H, 3.5; N, 10.8%. 5.2. 2-Amino-7-fluorophenazine 5,10-dioxide (12) To a solution of Et3N (0.1 mL) in anhydrous DMF (0.48 mL), at 5 C and under nitrogen atmosphere, was added a solution of p-aminophenol (177 mg, 1.6 mmol) and 1.6 mmol of bfx3 in 0.15 mL of anhydrous DMF. After stirring at room temperature for 24 h and maintaining at 20 C for 24 h, the resulting precipitate was filtered, washed with petroleum ether, THF, and ethyl ether yielding the desired product. Brown-orange solid (50%). 7- and 8-isomer 97:3 ratio. 1H NMR (DMSO-d6) dH: 7-isomer, 6.80 (2H, br s), 7.36 (1H, d, J ¼ 9.9 Hz), 7.39 (1H, d, J ¼ 2.4 Hz), 7.78 (1H, dt, J1 ¼ 8.8, J2 ¼ 2.7 Hz), 8.18 (1H, dd, J1 ¼ 9.4, J2 ¼ 2.7 Hz), 8.32 (1H, d, J ¼ 10.1 Hz), 8.54 (1H, dd, J1 ¼ 9.7, J2 ¼ 5.5 Hz). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6) dC: 7-isomer,100.5,122.1,122.5,122.6,122.9, 124.3, 126.3, 126.5, 133.7, 135.0, 135.4, 161.8. EI-MS, m/z (abundance, %): 7- and 8-isomers, 245 (Mþ , 100), 229 (45), 213 (12). Found: C, 58.7; H, 3.1; N, 17.4. C12H8FN3O2 requires C, 58.8; H, 3.3; N, 17.1%. 5.3. General procedure for the preparation of the 5-hydroxypyrido [3,2-a]phenazine 7,12-dioxide derivatives 18 and 19 To a hot solution of 8-hydroxyquinoline (7.3 mmol), NaOMe (7.3 mmol) and MeOH (1.1 mL) was added a hot solution of the corresponding benzofuroxan (7.3 mmol) in 7.3 mL of MeOH. The reaction mixture was heated at reflux for 2.5 h, and after cooling, filtration gave the sodium salt of phenol that was acidified, with excess of AcOH, to give the desired product. 5.3.1. 5-Hydroxy-9(10)-methylpyrido[3,2-a]phenazine 7,12-dioxide (18) Orange solid (28%). 9- and 10-isomer ratio 50:50. 1H NMR (DMSO-d6:D2O) dH: 9-isomer, 2.65 (3H, s), 7.70 (1H, m), 7.85 (1H, s), 7.95 (1H, m), 7.97 (1H, s), 8.45 (1H, m), 9.18 (1H, m), 10.95 (1H, dd, J1 ¼ 8.8, J2 ¼ 1.6 Hz); 10-isomer, 2.60 (3H, s), 7.65 (1H, s), 7.70 (1H, m), 7.95 (1H, m), 8.05 (1H, s), 8.45 (1H, m), 9.19 (1H, m), 10.92 (1H, dd, J1 ¼ 8.8, J2 ¼ 1.6 Hz). 13C NMR (DMSO-d6:D2O) dC: 9- and 10-isomers, 23.0, 108.5, 111.6, 118.6, 119.7, 120.5, 124.4, 125.6, 130.0, 131.5, 131.8, 132.3, 133.7, 136.6, 150.0, 152.3. EI-MS, m/z (abundance, %): 293 (Mþ , 4), 277 (Mþ 16, 21), 261 (60). Found: C, 65.3; H, 3.5; N, 14.1. C16H11N3O3 requires C, 65.5; H, 3.8; N, 14.3%. 5.3.2. (5,10-Dioxide-7(8)-bromophenazin-2-yl) 2chloroetanoate (20) To a stirred solution of 5 (100 mg, 0.32 mmol) and Et3N (60 mL) in toluene (5.0 mL), chloracetylchloride (30 mL) was added dropwise. The mixture was maintained at 50 C until phenazine 5 was not present (SiO2, CH2Cl2:MeOH (98:2)). The mixture of reaction was partitioned between EtOAc and saturated aqueous solution of NaHCO3. After the work up the organic layer was evaporated in
vacuo and the residue was purified by column chromatography (SiO2, CH2Cl2:MeOH (0e5%)). Red solid (51%). 7 and 8-isomer ratio 64:36. 1H NMR (DMSO-d6) dH: 7-isomer, 4.80 (2H, s), 7.82 (1H, dd, J1 ¼ 9.6, J2 ¼ 2.3 Hz), 8.09 (1H, m), 8.41 (1H, d, J ¼ 2.6 Hz), 8.49 (1H, dd, J1 ¼ 9.4, J2 ¼ 3.4 Hz), 8.65 (1H, dd, J1 ¼ 9.6, J2 ¼ 3.4 Hz), 8.72 (1H, d, J ¼ 2.2 Hz). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6) dC: 7-isomer, 42.3, 100.6, 122.2, 122.6, 122.9, 124.4, 126.4, 132.4, 133.9, 135.1, 135.5, 137.9, 161.7, 169.4. EI-MS, m/z (abundance, %): 7- and 8-isomers, 384 (Mþ , 1), 368 (1), 352 (0.5). Found: C, 44.0; H, 2.4; N, 7.4. C14H8BrClN2O4 requires C, 43.8; H, 2.1; N, 7.3%. 5.4. 2-Benzyloxy-7(8)-bromophenazine 5,10-dioxide (21) To a solution of 5 (100 mg, 0.03 mmol) in DMF (5.0 mL) were added benzyl chloride (40 mg, 0.03 mmol), 18-crown-6 ether (87 mg, 0.03 mmol), K2CO3 (45 mg, 0.03 mmol), and KI (1 mg, 0.015 mmol). The mixture was heated at 40 C for 24 h. Then the crude of reaction was partitioned between EtOAc and aqueous HCl (10%). After the work up the organic layer was evaporated in vacuo and the residue was purified by column chromatography (SiO2, petroleum ether:EtOAc (4:6)). Orange solid (31%). 7 and 8-isomer ratio 64:36. 1H NMR (DMSO-d6) dH: 7-isomer, 5.42 (2H, s), 7.35 (2H, d, J ¼ 7.8 Hz), 7.49 (1H, m), 7.50 (1H, d, J ¼ 7.9 Hz), 7.60e7.70 (3H, m), 8.03 (2H, m), 8.50 (2H, m); 8-isomer, 5.40 (2H, s), 7.30 (2H, d, J ¼ 7.70 Hz); 7.48 (1H, s), 7.62e7.65 (2H, m), 8.10 (2H, m), 8.45 (2H, m), 8.70 (2H, m). 13C NMR (from the HMQC and HMBC experiments) (DMSO-d6) dC: 7- and 8-isomers, 69.1, 98.9, 108.2, 121.6, 122.8, 123.0, 123.6, 124.8, 126.8, 128.9, 129.3, 130.0, 134.5, 135.3, 136.8, 138.6, 138.8. EI-MS, m/z (abundance, %): 7- and 8-isomers, 396 (Mþ , 0.5), 380 (2), 364 (7). Found: C, 57.5; H, 3.4; N, 6.8. C19H13BrN2O3 requires C, 57.4; H, 3.3; N, 7.0%. 5.5. Biology. Bioreductive activity [10e12,20,21] Cells: V79 cells (Chinese hamster lung fibroblasts) were obtained from ECACC (European Collection of Animal Cell Cultures) and maintained in logarithmic growth as subconfluent monolayer by trypsinization and subculture to (1e2) 104 cells/cm2 twice weekly. The growth medium was EMEM (Eagle’s Minimal Essential Medium), containing 10% (v/v) foetal bovine serum (FBS) and penicillin/streptomycin at 100 U/100 mg/mL. Aerobic and hypoxic cytotoxicity: suspension cultures. Monolayers of V79 cells in exponential growth were trypsinized, and suspension cultures were set up in 50 mL glass flasks: 2 104 cells/mL in 30 mL of EMEM containing 10% (v/v) FBS and HEPES (10 mM). The glass flasks were submerged and stirred in a water bath at 37 C, where they were gassed with humidified air or pure nitrogen. Treatment: compound solutions, 8e12, 18e21, and 24, were prepared just before dosing. Stock solutions, 150-fold more concentrated, were prepared in pure DMSO (Aldrich) or sterilized distilled water. Thirty minutes after the start of gassing, 0.2 mL of the stock compound solution was added to each flask, two flasks per dose. In every assay there was one flask with 0.2 mL of DMSO (negative control) and another with 7-chloro-3-[3-(N,N-dimethylamino)propylamino]-2-quinoxalinecarbonitrile 1,4-dioxide hydrochloride (positive control). Cloning: after 2 h exposure to the compound, the cells were centrifuged and resuspended in plating medium (EMEM plus 10% (v/v) FBS and penicillin/streptomycin). Cell numbers were determined with a haemocytometer and 102e103 cells were plated in 6-well plates to give a final volume of 2 mL/30 mm of well. Plates were incubated at 37 C in 5% CO2 for 7 days and then stained with aqueous crystal violet. Colonies with more than 64 cells were counted. The plating efficiency (PE) was calculated by dividing the number of colonies by the number of cells seeded. The percent of control cell survival for the compound-treated cultures (SFnormoxia and
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 45 (2010) 5362e5369
SFhypoxia) was calculated as PEtreated/PEcontrol 100. The compounds were tested at 20 mM in duplicate flasks both in aerobic and hypoxic conditions. 5.6. Cyclic voltammetric studies [12,30,31] DMSO (spectroscopy grade) was obtained from Aldrich. Tetrabutylammonium perchlorate (TBAP), used as supporting electrolyte, was obtained from Fluka. Cyclic voltammetry was carried out using a Metrohm 693 VA instrument with a 694 VA Stand convertor and a 693 VA Processor, in DMSO (ca. 1.0 mM), under a nitrogen atmosphere at room temperature (TBAP, ca. 0.1 mM) as supporting electrolyte. A three-electrode cell configuration was used, a mercury dropping working electrode, a platinum wire auxiliary electrode, and a saturated calomel reference electrode. Voltage scan rates ranged from 0.10 to 2.00 V/s. 5.7. DNA-interaction studies 5.7.1. UVevisible spectroscopy [10e12] DNA solution: calf thymus DNA (CTDNA, 12.5 mg, Sigma Chemical Co., USA) was slowly magnetically stirred in 5 mL TriseHCl buffer (10 mM, pH 7.4) for 24 h at 4 C. From this solution, 0.6 mL was diluted with the same buffer to 25 mL. Test compound solution: it was prepared at 104 M concentration using a minimal volume of adequate solvent, no more than 5%, and then diluted adding water to 2 105 M. No effect on DNA was observed by these concentrations of solvents. Study: a 3.0 mL sample of this resulting solution was mixed with 3.0 mL of DNA solution described above. The mixtures were slowly rotated for 24 h and subsequently their UV spectra were recorded using a 1-cm cell at 20 C on a Jasco V-570 UV/VIS/NIR spectrophotometer. Areas were calculated automatically by the apparatus. 5.8. Fluorescence spectroscopy [25,26] DNA solutions: CTDNA (12.5 mg, Sigma Chemical Co., USA) was slowly magnetically stirred in 5 mL phosphate buffer solution (PBS) (50 mM, pH 6.0) for 24 h at 4 C. From this solution, the further necessary dilutions were performed using the same buffer. Test compound solution: it was prepared at 20 mM concentration using PBS (50 mM, pH 6.0) and 20% of DMSO. No effect on DNA was observed by these concentrations of solvents. Study: fluorescence spectra were recorded on a Varioskan Flash 2.4.1 spectrometer. The fluorescence quenching titrations with CTDNA were performed by keeping the compounds concentrations constant (20 mM) and varying the nucleic acid concentrations (0e400 mM) maintaining the total volume of the solution constant. Fluorescence emission spectra were recorded in the wavelength range of 425e825 nm by exciting the compounds at the corresponding wavelength with the excitation slit widths of 12 nm. The intrinsic binding constants of compounds with CTDNA were determined by fluorescence titrations. The data were plotted according to the SterneVolmer equation [32]:
fluorescence quenching constant. [Q] is the concentration of quencher. Acknowledgements Financial supports from Comisión Honoraria de Lucha contra el Cáncer (Uruguay) and from the project of the Work Community of Pyrenees (CTP 07-P11 Ref. IIQ011698.R11) is acknowledged. We thank PEDECIBA-ANII for scholarships to MLL and MC, ANII for scholarship to MN, and PEDECIBA and CSIC-Universidad de la República for fellowships. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
[11] [12]
[13] [14]
[15] [16] [17] [18] [19] [20]
[21]
[22] [23] [24] [25] [26] [27] [28] [29]
[30]
I0/I ¼ 1 þ Kq [Q] where, I0 and I are the fluorescence intensities in the absence and presence of CTDNA, respectively. Kq is the SterneVolmer
5369
[31]
[32]
S. Rockwell, J.E. Moulder, Int. J. Radiat. Oncol. Biol. Phys. 19 (1990) 197e202. W.A. Denny, Eur. J. Med. Chem. 36 (2001) 577e595. K. Ruan, G. Song, G. Ouyang, J. Cell. Biochem. 107 (2009) 1053e1062. J.M. Brown, W.R. Wilson, Nat. Rev. Cancer 4 (2004) 437e447. A.J. Lin, L.A. Cosby, C.W. Shansky, A.C. Sartorelli, J. Med. Chem. 15 (1972) 1247e1252. P. Wardman, Curr. Med. Chem. 8 (2001) 739e761. H. Cerecetto, M. González, Minirev. Med. Chem. 1 (2001) 219e231. H. Cerecetto, M. González, M.L. Lavaggi, Med. Chem. 2 (2006) 315e327. H. Cerecetto, M. González, M.L. Lavaggi, W. Porcal, J. Braz. Chem. Soc. 6 (2005) 1290e1296. H. Cerecetto, M. González, M.L. Lavaggi, M.A. Aravena, C. Rigol, C. Olea-Azar, A. Azqueta, A. López de Ceráin, A. Monge, A.M. Bruno, Med. Chem. 2 (2006) 511e521. H. Cerecetto, M. González, M.L. Lavaggi, A. Azqueta, O. Ezpeleta, A. López de Ceraín, A. Monge-Vega, J. Med. Chem. 48 (2005) 21e23. M.L. Lavaggi, M. Cabrera, M.A. Aravena, C. Olea-Azar, A. López de Ceráin, A. Monge, G. Pachón, M. Cascante, A.M. Bruno, L.I. Pietrasanta, M. González, H. Cerecetto, Bioorg. Med. Chem. 18 (2010) 4433e4440. M.L. Lavaggi, M. Cabrera, M. González, H. Cerecetto, Chem. Res. Toxicol. 21 (2008) 1900e1906. O.G. Pachón, A. Azqueta, M.L. Lavaggi, A. López de Ceraín, E. Creppy, A. Collins, H. Cerecetto, M. González, J.J. Centelles, M. Cascante, Chem. Res. Toxicol. 21 (2008) 1578e1585. G.-W. Ludwig, H. Baumgartel, Chem. Ber. 115 (1982) 2380e2383. E. Vicente, R. Villar, A. Burguete, B. Solano, S. Ancizu, S. Pérez-Silanes, I. Aldana, A. Monge, Molecules 13 (2008) 86e95. M.J. Abu El-Haj, B.W. Dominy, J.D. Johnston, J. Org. Chem. 37 (1972) 589e593. A.J. Boulton, A.R. Katritzky, M.J. Sewell, B. Wallis, J. Chem. Soc. B 9 (1967) 914e917. A.J. Boulton, P.J. Halls, A.R. Katritzky, J. Chem. Soc. B (1970) 636e640. A. Monge, J.A. Palop, A. López de Ceráin, V. Senador, F.J. Martínez-Crespo, Y. Sáinz, S. Narro, E. García, C. de Miguel, M. González, A.J. Barker, E. Hamilton, E.D. Clarke, D.T. Greenhow, J. Med. Chem. 38 (1995) 1786e1792. C. Urquiola, M. Vieites, M.H. Torre, M. Cabrera, M.L. Lavaggi, H. Cerecetto, M. González, A. López de Cerain, A. Monge, P. Smircich, B. Garat, D. Gambino, Bioorg. Med. Chem. 17 (2009) 1623e1629. J.D. Aguirre, A.M. Angeles-Boza, A. Chouai, J.P. Pellois, C. Turro, K.R. Dunbar, J. Am. Chem. Soc. 131 (2009) 11353e11360. J. Wang, X. Yang, Spectrochim. Acta A Mol. Biomol. Spectrosc. 74 (2009) 421e426. P.U. Maheswari, V. Rajendiran, M. Palaniandavar, R. Parthasarathi, V. Subramanian, J. Inorg. Biochem. 100 (2006) 3e17. X. Jiang, L. Shang, Z. Wang, S. Dong, Biophys. Chem. 118 (2005) 42e50. Y.N. Ni, X.Z. Zhong, Chin. Chem. Lett. 18 (2007) 569e572. H. Heli, S.Z. Bathaie, M.F. Mousavi, Electrochem. Commun. 6 (2004) 1114e1118. Y. Li, X.J. Yao, J. Jin, X.G. Chen, Z.D. Hu, Biochim. Biophys. Acta 1774 (2007) 51e58. H. Cerecetto, R. Di Maio, M. González, M. Risso, P. Saenz, G. Seoane, A. Denicola, G. Peluffo, C. Quijano, C. Olea-Azar, J. Med. Chem. 42 (1999) 1941e1950. L.A. Fernández, M.R. Santo, M. Reta, L. Giacomelli, R. Cattana, J.J. Silber, M. Risso, H. Cerecetto, M. González, C. Olea-Azar, Molecules 10 (2005) 1197e1208. J. Rodríguez, A. Gerpe, G. Aguirre, U. Kemmerling, O.E. Piro, V.J. Arán, J.D. Maya, C. Olea-Azar, M. González, H. Cerecetto, Eur. J. Med. Chem. 44 (2009) 1545e1553. Y. Cao, X.W. He, Spectrochim. Acta, Part A 54 (1998) 883e892.