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Chemico-Biological Interactions 180 (2009) 220–225

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

tudies on the cytotoxic activity of synthetic 2H-azirine-2-azetidinoneompounds

aniel Pinheiro Maiac, Diego Veras Wilkea, Jair Mafezolib, José Nunes da Silva Júniorc,anoel Odorico de Moraesa, Claudia Pessoaa, Letícia Veras Costa-Lotufoa,∗

Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Caixa Postal 3157, 60430-270 Fortaleza, Ceará, BrazilDepartamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Caixa Postal 6044, 60455-970 Fortaleza, Ceará, BrazilCurso de Farmácia, Centro de Ciências da Saúde, Universidade de Fortaleza, Caixa Postal 1258, 60.811-905, Fortaleza, Ceará, Brazil

r t i c l e i n f o

rticle history:eceived 21 November 2008eceived in revised form 26 February 2009ccepted 27 February 2009vailable online 11 March 2009

a b s t r a c t

2-Azetidinones and 2H-azirines show antibacterial and cytotoxic activities, however the biological prop-erties of molecules containing both 2H-azirine and 2-azetidinone functions in the same structure hadnever been evaluated before. In the present study, two 2H-azirine-2-azetidinones (1 and 2) and three 2H-azirines (3–5) were synthesized from 2-formyl-3-phenyl-2H-azirine-N-arylimines with diphenylketene.

eywords:H-azirine-2-azetidinoneytotoxicitypoptosisL-60 cells

The compounds were assayed for antibacterial and cytotoxic activities. None of them showed antibacte-rial activity on the tested strains, but both 2H-azirine-2-azetidinones showed cytotoxicity against fourtumor cell lines (HL-60, leukemia; HCT-8, colon cancer; MDA-MB-435, melanoma; and SF-295, CNS). TheIC50 values of 1 ranged from 1.1 to 10.5 �M and from 3.8 to 26.6 �M for 2. The mechanism of cell growthinhibition of 1 and 2 towards HL-60 cell line was also investigated. Membrane damage, cell viability, DNAsynthesis inhibition and morphological changes were evaluated. The preliminary findings suggested that1 and 2 induce apoptosis.

. Introduction

Aware of the increasing chemical and pharmacological inter-sts in molecules derived from 2H-azirines [1–6] and especiallyrom 2-azetidinones [7–11] in the development of novel drugs,e have previously suggested a viable and convenient syn-

hetic route to the yet unknown 2H-azirinyl-2-azetidinone system12]. The reaction of 2-formyl-3-phenyl-2H-azirine-N-aryliminesith diphenylketene demonstrated an enhanced reactivity of the

xo-imine group of an intermediate compound originating, as prod-cts, 2H-azirinyl-2-azetidinones and N-aryldiphenylacetamides inhighly diastereoselective way [12].

Compounds with 2-azetidinone or 2H-azirine functions showedainly antibacterial [7–11,13] and cytotoxic [13–22] activities, but,

o far, no molecules containing both 2H-azirine and 2-azetidinone

unctions, such as the case of 1 (1-(4-chloride)-3,3-diphenyl--(3-phenyl-2H-azirinyl)-2-azetidinone) and 2 (1-(4-methyl)-3,3-iphenyl-4-(3-phenyl-2H-azirinyl)-2-azetidinone) (Fig. 1), haveeen recognized for their potential biological properties. Therefore,

∗ Corresponding author at: Departamento de Fisiologia e Farmacologia, Univer-idade Federal do Ceará, Rua Cel. Nunes de Melo, 1127, 60430-270 Fortaleza, Ceará,razil. Tel.: +55 85 3366 8255; fax: +55 85 3366 8333.

E-mail address: lvcosta@secrel.com.br (L.V. Costa-Lotufo).

009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2009.02.015

© 2009 Elsevier Ireland Ltd. All rights reserved.

this work aims to estimate for the first time the antibacterial andcytotoxic activities of these compounds. Some additional studieswere conducted in HL-60 leukemia cell lines to evaluate the abilityof these compounds to inhibit cell growth and DNA synthesis andalso to induce apoptosis.

2. Materials and methods

2.1. Chemicals and reagents

BrdU and Doxorubicin (Doxolem) were purchased fromSigma–Aldrich Inc. and Zodiac Produtos Farmacêuticos, respec-tively. All reagents used were of analytical grade.

2.2. Preparation of 1–5 (general procedure)

The syntheses of compounds 1–5 were performed as describedby Nunes and Kascheres [12] and Padwa et al. [23] and were brieflydescribed in this section. Further details, regarding the syntheticroute or organic mechanisms, may be found in Refs [12,23]. Thestructures of the final products 1 and 2, as well as that of the inter-mediate compounds (including 3–5), were confirmed by IR, 1H NMRand 13C NMR spectroscopies.

D.P. Maia et al. / Chemico-Biological Interactions 180 (2009) 220–225 221

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.2.1. 1-(4-Chloride)-3,3-diphenyl-4-(3-phenyl-2H-azirinyl)-2-zetidinone (1)

To a stirring solution of 2-formyl-3phenyl-2H-azirine (4)107 mg, 0.737 mmol) in dry benzene (15 mL) was added p-hloroaniline (94 mg, 0.737 mmol) and the resulting solutionas stirred for 42 h at 65 ◦C. Diphenyldiazoethanone (313 mg,

.47 mmol) was added to the reaction and the resulting solutionas stirred for 6 h at 70 ◦C. The solvent was evaporated under vac-um, and the crude product was immediately purified by flashhromatography (dichloromethane as eluent) to yield 1 (50 mg,7%, Rf = 0.63) as a colorless solid. Mp: 204–206 ◦C (hexane–ethylcetate); IR (KBr) � 3057, 1751, 1594, 1493, 1447, 1382 cm−1; 1HMR (500 MHz, CDCl3) ı 7.99 (2H, d, J = 6.8 Hz), 7.65–7.25 (17H, m),.15 (1H, d, J = 8.0 Hz), 2.26 (1H, d, J = 8.0 Hz); 13C NMR (125 MHz,DCl3) ı 167.1 (C), 166.7 (C), 160–114 (C–Ar), 70.2 (C), 69.7 (CH),1.8 (CH).

.2.2. 1-(4-Methyl)-3,3-diphenyl-4-(3-phenyl-2H-azirinyl)-2-zetidinone (2)

The synthesis of 2 was similar to that of 1. To a stirring solution of-formyl-3phenyl-2H-azirine (4) (110 mg, 0.760 mmol) in dry ben-ene (15 mL) was added p-chloroaniline (121 mg, 1,13 mmol) andhe resulting solution was stirred for 96 h at 70 ◦C. Diphenyldia-oethanone (288 mg, 1.357 mmol) was added to the reaction andhe resulting solution was stirred for 24 h at 70 ◦C. The solvent wasvaporated under vacuum, and the crude product was immediatelyurified by flash chromatography (20:80 (v/v) EtOAc in hexane) toield 2 (120 mg, 38%, Rf = 0.34) as a colorless solid. Mp: 210–212 ◦Chexane–ethyl acetate); IR (KBr) � 3028, 1748, 1600, 1495, 1448,371 cm−1; 1H NMR (500 MHz, CDCl3) ı 7.87 (2H, d, J = 8.4 Hz),.65–7.24 (17H, m), 4.11 (1H, d, J = 7.9 Hz), 2.36 (3H, s), 2.25 (1H, d,= 7.9 Hz); 13C NMR (125 MHz, CDCl3) ı 167.1 (C), 166.7 (C), 160–114C–Ar), 70.2 (C), 69.8 (CH), 31.8 (CH), 21.2 (CH3).

.2.3. 2-Dimetoximethyl-3-phenyl-2H-azirine (3)This compound was obtained from intermediate l-azido-3,3-

imethoxy-l-phenyl-l-propene and followed the same procedureescribed for compound 5. Product 3 was obtained in 40% yield. IRneat) � 2992, 2832, 1746, 1599, 1451, 1061 cm−1; EI-MS m/z (%) 191M+•

, 1), 75 (100); 1H NMR (500 MHz, CDCl3) ı 7.88 (2H, d, J = 6.9 Hz),.57–7.50 (3H, m), 4.38 (1H, d, J = 2.5 Hz), 3.45 (3H, s), 3.34 (3H, s),

of tested compounds.

2.36 (1H, d, J = 2.5 Hz); 13C NMR (125 MHz, CDCl3) ı 166.1 (C), 133.1(CH), 129.7 (2 CH), 129.1 (2 CH), 125.0 (C), 54.2 (CH3), 53.5 (CH3),32.9 (CH).

2.2.4. 2-Formyl-3-phenyl-2H-azirine (4)A solution of 2.141 g (11.194 mmol) of 2-dimetoximethyl-3-

phenyl-2H-azirine (3) in 46 mL of dioxane and 40 mL of 20%aqueous acetic acid was stirred and heated at reflux for 6 h.The solution containing the crude product was extracted withdichloromethane (30 mL). The organic layer was dried (anhydrideNa2SO4), filtered and concentrated under vacuum. The crude prod-uct was purified by flash chromatography (dichloromethane aseluent) to yield 4 (790 mg, 49%) as a crystalline solid. Mp: 45–47 ◦C;IR (KBr) � 2848, 2750, 1774, 1703, 1594, 1487, 1449 cm−1; EI-MS m/z(%) 145 (M+•

, 40), 51 (100); 1H NMR (300 MHz, CDCl3) ı 9.01 (1H, d,J = 6.5 Hz), 7.96 (2H, dd, J = 2.0, 6.9 Hz), 7.77–7.63 (3H, m), 2.93 (1H,d, J = 6.5 Hz); 13C NMR (75 MHz, CDCl3) ı 200.0 (CH), 159.6 (C), 134.6(CH), 130.8 (2 CH), 129.7 (2 CH), 122.8 (C), 39.2 (CH).

2.2.5. 2-Dimetoximethyl-2-methyl-3-phenyl-2H-azirine (5)A solution of the intermediate l-azido-3,3-dimethoxy-2-methyl-

l-phenyl-l-propene (3.41 g, 15 mmol), obtained according to Padwaet al. [23], in 40 mL of chloroform was stirred and heated at refluxfor 24 h. The solvent was evaporated under vacuum and the crudeproduct was purified by flash chromatography (dichloromethaneas eluent) to yield 5 (0.766 g, 26%, Rf = 0.12) as a colorless liquid. IR(neat) � 2931, 2832, 1451, 1372, 1110, 1072 cm−1; EI-MS m/z (%) 205(M+•

, 2), 75 (100); 1H NMR (500 MHz, CDCl3) ı 7.84 (2H, dd, J = 1.5,8.1 Hz), 7.56–7.49 (3H, m), 4.39 (1H, s), 3.42 (3H, s), 3.35 (3H, s), 1.38(3H, s); 13C NMR (125 MHz, CDCl3) ı 171.3 (C), 132.9 (CH), 129.4 (2CH), 129.1 (2 CH), 125.3 (C), 106.3 (CH), 54.5 (CH3), 54.4 (CH3), 37.9(C), 18.1 (CH3).

2.3. Preparation of stocks and cell culture

Stock solutions of compounds 1 and 2 were diluted in DMSO to

5 mg/ml (11.40 and 11.96 mM, respectively) and frozen at −20 ◦C.Prior to assays, the compounds were diluted in culture medium toyield a final concentration of DMSO lower than 0.5%. All cell lines(National Cancer Institute, Bethesda, MD, USA) were maintainedat 37 ◦C, under 5% CO2, in RPMI 1640 media supplemented with

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22 D.P. Maia et al. / Chemico-Biolog

0% fetal bovine serum, 10 mM penicillin (100 U/ml), streptomycin100 �g/ml) and glutamine (2 mM).

.4. In vitro growth inhibition assay

The effects of compounds on growth of tumor cells were evalu-ted in vitro by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-etrazolium bromide (MTT) assay, as described by Mosmann [24],fter 72 h incubation. Four human tumor cell lines were used: HL-60promielocytic leukemia), MDA-MB-435 (melanoma), SF-295 (CNSlioblastoma) and HCT-8 (colon carcinoma). Doxorubicin was useds positive control.

.5. Hemolytic assay

This test was performed in 96-well plates using a 1% mouserythrocytes (Mus musculus Swiss) suspension in 0.85% NaClontaining 10 mM CaCl2, following the method described inosta-Lotufo et al. [25]. Compounds 1 and 2 were assayed at con-entrations ranging from 17.4 to 557 �M and 18.2 to 583.9 �Mespectively. After 1 h incubation, the plates were centrifuged andhe supernatant containing hemoglobin was measured spectropho-ometrically for the absorbance at 450 nm (Multimode DetectorTX 880, Beckman Coulter).

.6. Cellular effects on HL-60 cells

HL-60 cells (plated at 3 × 105 cells/ml) were used to evaluateome cellular effects of products 1 and 2 at concentrations of 0.5, 1nd 2 �M, after 24 h incubation. Untreated cells were used as nega-ive control and doxorubicin at 0.5 �M was used as positive controlor all experiments.

.6.1. Cell viability assayCell viability was determined by the Trypan blue dye exclusion

est. After the incubation period, cells were differentially countedy using a haemocytometer [26].

.6.2. Inhibition of DNA synthesisTen microliters of 5-bromo-2′-deoxyuridine (BrdU, final con-

entration of 10 mM) were added to each well and incubatedor 3 h at 37 ◦C before the completion of the incubation period.

o access the BrdU incorporation on DNA, cells were harvested,laced on a glass slide using cytospin, and left to dry for 2 ht room temperature. Cells that incorporated BrdU were labelledy direct peroxidase immunocytochemistry using the chromogeniaminobenzidine [27]. Slides were counterstained with hema-

able 1H-azirine intermediate products and its 2H-azirine-2-azetidinone final products testedresented as inhibitory concentration mean values (IC50) with their respective 95%confide

ompounds IC50 �M (CI 95%)

HL-60 HCT-8

oxorubicin 0.03 0.07(0.02–0.04) (0.06–0.09)

1.14 10.53(0.82–1.60) (9.25–11.96)

3.78 26.58(1.58–9.03) (18.98–37.19

144.89 105.25(111.21–188.76) (76.08–145.

>689.06 >689.06

405.40 289.99(329.70–498.42) (146.64–573

teractions 180 (2009) 220–225

toxylin, mounted, and coverslipped. Evaluation of BrdU uptake wasaccomplished by light microscopy. Two hundred cells were countedper sample to calculate the percentage of positive cells.

2.6.3. Morphological change analysisTo access the nuclear and membrane morphology, the cells

were harvested, placed on a glass slide using cytospin, fixed withmethanol for about 1 min and stained with hematoxylin and eosin.Visual analysis was accomplished using light microscopy [28].

2.7. Antimicrobial assay

For evaluation of antimicrobial activity of compounds 1–5, theKirby–Bauer Disk Diffusion Test was chosen [29]. The compoundsdiluted in chloroform (30 and 60 �g/25 �l) were applied to 6 mmsterile filter-papers (Newprov Prod. Lab. Ltd., Pinhais, Brazil). Aftersolvent evaporation at 37 ◦C, the filter-papers containing 30 �g or60 �g of each substance were tested, in duplicates, against Staphy-lococcus aureus ATCC 6538; Pseudomonas aeruginosa ATCC 9027and Escherichia coli ATCC 8739. Other disks, from the same man-ufacturer, already containing 30 �g of cephalotine, were used as apositive control and disks containing only the solvent, chloroform,as the negative control.

2.8. Statistical analysis

For the MTT assay, IC50 values and their 95% confidence intervals(CI 95%) were obtained by nonlinear regression using GRAPHPADPRISM software version 5 (Intuitive Software for Science, San Diego,CA, USA). For cell viability, the differences between experimen-tal groups were compared by ANOVA followed by Dunnet test. Toevaluate the effect on cell DNA synthesis, the differences betweenexperimental groups were compared by �2. The minimal signifi-cance level used was p < 0.05.

3. Results

3.1. In vitro growth inhibition assay

The inhibition growth effects of intermediate products 3–5 andfinal products 1 and 2 were evaluated against four tumor cell linesin the presence of a range of concentrations after 72 h incubation.

As shown in Table 1, the 2H-azirines did not show cytotoxicityor showed only weak potency, however, 2H-azirine-2-azetidinonesstrongly inhibited the growth of all the tested tumor cell lines, withIC50 values ranging from 1.1 to 10.5 �M for 1 and from 3.8 to 26.6 �Mfor 2 (Table 1).

against 4 tumor cell lines, using the MTT assay and 72 h incubation. The effects arence intervals of (CI 95%). Doxorubicin was used as positive control. N.T.—not tested.

MDA-MB-435 SF-295

0.81 0.27(0.62–1.20) (0.24–0.42)

2.57 4.01(2.32–2.85) (2.94–5.47)

8.39 5.54) (5.83–12.07) (4.02–7.70)

N.T. 483.3972) (454.79–513.77)

N.T. >689.06

N.T. >487.21.93)

D.P. Maia et al. / Chemico-Biological Interactions 180 (2009) 220–225 223

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ig. 2. Viability of non-treated HL-60 cells or treated with vehicle (DMSO), doxorell count; B, non-viable cell count (results on A and B were assessed by the Trypncorporation. *p < 0.01.

.2. Membrane damage

The hemolytic activity of the cytotoxic compounds was evalu-ted on mouse suspension erythrocytes to investigate if cytotoxicityn tumor cell lines was related directly to membrane damage. Nei-her 1 nor 2 showed hemolytic activity at the highest assayedoncentration (557.9 and 583.9 mM respectively).

.3. Cellular effects on HL-60 cells

The ability to exclude the Trypan blue dye is restricted to viableells, while necrotic or late apoptotic cells are incapable of dyexclusion and are stained blue. Compound 1 decreased viable cellsount starting at 0.5 �M and, at 1 �M, it also increased non viableells. Compound 2 decreased the number of viable cells only at�M without increasing non-viable cells, as shown in Fig. 2A and. The antiproliferative activity was confirmed by the evaluationf BrdU incorporation, whereas both 1 and 2 significantly inhibitedNA synthesis at 1 �M (see results in Fig. 2C). At 2 �M cell countingas not possible due to an extensive cellular disruption.

Morphological changes in cells were accessed by nuclear stain-ng with hematoxylin and cytoplasm counter-staining with eosin.his process allows the observation of nuclear and membrane alter-tions under light microscopy. Morphological analysis of treatedells (Fig. 3) revealed characteristics, which were compatible withpoptosis for both 1 and 2 treatments, mainly at intermediate andigh concentrations. At high concentrations, dead cells could beeen, as well. Compound 1, at 1 �M, decreased cellular volumend reduced cell quantity, while inducing pyknosis and a modestlasmatic membrane disruption. At 2 �M, pyknosis and membraneisruption were more intense. The cells treated with 2, differently,howed mainly perinuclear chromatin condensation at 1 �M. At�M this compound also induced, rather in a weaker manner,yknosis and membrane disruption.

.4. Antimicrobial effects

Neither 1 nor 2 showed antimicrobial activity, at the tested con-entrations (30 �g or 60 �g), against any of the tested bacterialpecies by the methodology described.

. Discussion

The chemistry of 2H-azirines has been widely explored due tohe high reactivity of this ring system with nucleophilic and elec-

rophilic reagents [6]. Concerning their biological properties, theatural products azirinomycin and dysidazirine are examples ofntibiotics, and the latter one is also cytotoxic [4–6]. For this study,ytotoxic and antibacterial activities of 2H-azirines 3–5 and 2H-zirine-2-azetidinones 1 and 2 were evaluated.

(Dox) at 0.5 �M and 1 and 2 at 0.5; 1 and 2 �M after 24 h incubation. A, viablee exclusion assay); and C, percentage of proliferating cells assessed by the BrDU

The 2H-azirine intermediates assayed here showed only weakcytotoxicity. However, 2H-azirine-2-azetidinones inhibited thegrowth of the four tested tumor cell lines (Table 1). The absenceof hemolytic activity, performed with mouse erythrocytes suspen-sion, suggests that the cytotoxicity of these compounds was notrelated to membrane damage.

2-Azetidinone rings are useful for improving properties andactivities of natural compounds [30]. A recent report [19] revealedsome cytotoxic compounds amongst several 1,3,4-trisubstituted 2-azetidinones with p-aryl substitutions at position 1, however noneof them contained 2H-azirine or phenyl groups in their structures.The presence of –Cl or –CH3 at the p-aryl position is the only dif-ference between the tested products. The –Cl substituent seems topositively influence cytotoxicity, as 1 potency ranged from 1.4 to3.3-fold higher than that of 2 on the tumor cell lines assayed.

Investigations during the last two decades convincingly demon-strated the prospects of structural modifications with differentsubstituents in monocyclic �-lactams as an effective procedurefor the detection and improvement of pharmacological effectsnot related to antibacterial proprieties [19]. A variety of 1,3,4-trisubstituted 2-azetidinones, such as 1 and 2, was producedand showed a diverse range of biological properties, includinganticancer [19]. However, none of the compounds, 2H-azirinesor 2H-azirine-2-azetidinones, showed any antimicrobial activityagainst S. aureus, P. aeruginosa and E. coli (data not shown).

Cytotoxic effects of compounds 1 and 2 were studied on HL-60 cells at 0.5, 1 and 2 �M after 24 h incubation. Compound 1decreased viable cell count at 1 and 2 �M, while also increasing nonviable cells at 2 �M. Compound 2, on the other hand, only decreasedthe viable cell number at 2 �M, without interfering with the nonviable count. In addition, both 1 and 2 significantly reduced DNAsynthesis at 1 �M.

Subsequently, morphological alterations due to 2H-azirine-2-azetidinones treatment were investigated in order to evaluatewhether apoptosis and/or necrosis induction is related to the mech-anism of 2H-azirine-2-azetidinones cytotoxicity. Cells undergoingapoptosis activate several pathways that cause a number of struc-tural alterations and those may be observed under microscopy[31,32]. Morphological analysis revealed characteristics compati-ble with 1 and 2 treated cells undergoing an apoptotic process.Compound 1 caused cell shrinkage and pyknosis in most cells butfewer membrane disruptions could also be seen. This is characteris-tic of late apoptotic or dead cells. Compound 2, differently, showedmainly perinuclear chromatin condensation and some pyknosis.

The synthesis and bioactivity of 2-azetidinones have previ-

ously been investigated by other research groups, however thereare not many studies directed to defining the mechanisms ofaction of such compounds [33–37]. Several 1,3,4-trisubstituted2-azetidinones with anti-inflammatory, anticoagulant, anticancerand antiviral properties were synthesized [19], and these biological

224 D.P. Maia et al. / Chemico-Biological Interactions 180 (2009) 220–225

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ig. 3. Morphological changes. Photomicrography of HL-60 cells treated with 1 atreated with doxorubicin at 0.5 �M (B). NC, perinuclear condensation; P, picnosis; a

ctivities are due, mainly, to their capability of inhibiting serine-roteases (elastase [35,36], thrombine [38], specific antigens ofrostate [39], and protease of human cytomegalovirus [40,41]). Aeport described G2 cell cycle arrest without apoptosis inductionor 1,4-disubstituted azetidinones [37]. Our findings suggest that

poptosis induction does occur with the 2H-azirine-2-azetidinonesnvestigated.

In summary, two cytotoxic 2H-azirine-2-azetidinone com-ounds, lacking antibacterial properties, were synthesized. Theesults suggest that these products induce apoptosis on HL-60 cell

2 �M (C and D) and 2 at 1 and 2 �M (E and F) after 24 h. Non-treated cells (A) and, membrane disruption.

line. Intermediate products, with only 2H-azirine function, showednone or merely a residual activity. Further studies are needed inorder to improve the knowledge on this cytotoxic mechanism ofaction. However, it is made clear that the 2H-azirine-2-azetidinonecompounds reported here represent new leads for anticancer drug

development.

Conflict of interest statement

None.

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D.P. Maia et al. / Chemico-Biolog

cknowledgments

The authors would like to acknowledge the financial support inhe form of grants and/or fellowships from the following Brazil-an agencies: Conselho Nacional de Desenvolvimento Científico eecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP)nd Instituto Claude Bernard (InCB). The technical assistance ofilvana Franca and Ângela Maria Veras Muniz is gratefully acknowl-dged.

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