Synthesis and evaluation of nitrostyrene derivative compounds, new snake venom phospholipase A2 inhibitors

ARTICLE IN PRESS Toxicon 51 (2008) 1467– 1478

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Synthesis and evaluation of nitrostyrene derivative compounds, new snake venom phospholipase A2 inhibitors$ J.A.F.P. Villar a, F.T.D. Lima b, C.L. Veber a, A.R.M. Oliveira a, A.K. Calgarotto c, S. Marangoni c, S.L. da Silva b, a b c

´—UFPR, Curitiba, PR 81531-990, Brazil Depto de Quı´mica, ICE, Universidade Federal do Parana Depto de Quı´mica, ICE, Universidade Federal do Amazonas—UFAM, Manaus, AM 69077-000, Brazil Depto de Bioquı´mica, IB, Universidade Estadual de Campinas—UNICAMP, Campinas, SP 131000-000, Brazil

a r t i c l e in fo

abstract

Article history: Received 4 January 2008 Received in revised form 19 March 2008 Accepted 20 March 2008 Available online 26 March 2008

Several nitrostyrene derivatives were synthesized and their inhibitive activities on phospholipase A2 (PLA2) from Bothrops jararacussu venom were evaluated. Some compounds were very efficient as inhibition agents against edema-inducing, enzymatic and myotoxic activities. Data revealed that the size of the substitute and substitution position in the nitrostyrene moiety had important influence on the inhibition capacities. The enzymatic kinetic studies show that the nitrostyrene derivatives compounds inhibit PLA2 in a non-competitive manner. The electronic, molecular and topologic parameters were calculated using ab initio quantum calculations (density functional theory—DFT) and analyzed by chemometric methods (principal component analysis (PCA) and hierarchical cluster analysis (HCA)) in order to build models able to establish relationships between the electronic features and the structure–activity presented by the target compound. Compounds with the nitro group in the ortho, meta and para position (compounds 2–4) on the aromatic ring were more efficient in the inhibition of PLA2 activity in all tests. These results indicate that the influence of the nitro group in the aromatic ring is, in fact, important. In addition, quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA2 present lower values of highest occupied molecular orbital (HOMO) energy and polarizability, suggesting the formation of a charge-transferring complex between the nitrostyrene compounds and PLA2. & 2008 Elsevier Ltd. All rights reserved.

Keywords: Nitrostyrene Bothrops jararacussu DFT Chemometrics PLA2

1. Introduction Phospholipases A2 (EC 3.1.1.4) are proteins that hydrolyze glycerophospholipid membranes (PL) in the sn-2

$ Ethical statement: The authors declare that this manuscript was not submitted to any journal and that it was read and approved by all the authors. They also declare that this study was made according to all national, international and institutional bioethics rules.  Corresponding author. Rua Fortunato Badan, 103, Jardim Silva ˆ nia, Moji Mirim, SP, CEP 13806-679, Brazil. Tel.: +55 92 36474034; fax: +55 92 36474027. E-mail address: [email protected] (S.L. da Silva).

0041-0101/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2008.03.023

position, releasing lysophospholipids and, among other fatty acids, the arachidonic acid (AA). AA is the substrate for other enzymes involved in the inflammatory process (COX-1, COX-2 and 5-LO), producing the pro-inflammatory prostaglandins (PGs) and leukotrienes (LTs) (also named eicosanoids) (Ikai, 2001). Even though the PGs and LTs are physiologically involved in homeostasis, the excessive production of these compounds is associated with many physiopathological processes such as asthma, cerebral illnesses, cancers, cardiovascular disorders and inflammation (Wijkander et al., 1995; Funk, 2001). The inhibition of phospholipase A2 (PLA2) can prevent the excessive production of PGs and LTs, since the formation of AA is avoided (Yedgar et al., 2000; Balsinde et al., 2002).

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Snake venoms are complex mixtures of proteins, including phospholipases A2, myotoxins, proteolytic enzymes, neurotoxins, cytotoxins and cardiotoxins, among others. Intense local inflammation is associated with snake bite due to the presence of sPLA2 in the venom (Teixeira et al., 2003). Venom from different snake specimens is utilized as a PLA2 source, due to the abundance of these enzymes in snake venoms and the lack of difficulty in the isolation process. Thus, the enzyme is utilized as a tool for several pharmacological studies (Diaz and Arm, 2003; Teixeira et al., 2003; Singh et al., 2004, 2005; Jabeen et al., 2005; Yedgar et al., 2006; Souza et al., 2008; Da Silva et al., 2008). Moreover, it has been reported that transb-nitrostyrene (TBNS) is a potent enzymatic inhibitor. Studies have shown that TBNS inhibits the protein phosphatases, PTB1 (Park and Pei, 2004) and PP2A (Fathi et al., 2000) and displays an associated pro-apoptotic effect even in some multidrug-resistant tumor cells (Fathi et al., 2000; Kaap et al., 2003). TBNS is also a potential antibacterial agent (Milharez et al., 2006). Quercetin is a flavonoid compound able to inhibit different types of PLA2. Studies have shown that hydroxyls bound to C3 and C7 are essential for enzyme inhibition (Fig. 1) (Lindahl and Tagesson, 1997; Kim et al., 2001; Gil et al., 1997; La¨ttig et al., 2007). Thus, based on the structure of quercetin, the synthesis and analysis of the action of ten nitrostyrene derivative compounds (Fig. 2) on some biological effects caused by PLA2, purified from Bothrops jararacussu venom were proposed. The inhibitive capacity of these synthetic nitrostyrene compounds on the edema-inducing, enzymatic and myotoxic activities provoked by PLA2 was analyzed. After the experiments were performed, all the nitrostyrene compounds were submitted to ab initio quantum calculations (density functional theory (DFT)—UB3LYP/6-31G*) and the values of electronic, molecular and topological properties were analyzed through the utilization of chemometric methods (principal component analysis (PCA) and hierarchical cluster analysis (HCA)) in order to recognize patterns able to correlate the nitrostyrene structures with their biological activities. The results obtained might be useful in the

development of new selective inhibitors for phospholipases A2. 2. Material and methods 2.1. Chemicals B. jararacussu venoms were purchased from a private serpentarium (Batatais, SP, Brazil). B. jararacussu PLA2 was isolated on Sephadex G-75, followed by cation-exchange chromatography. The column was previously equilibrated with 0.05 M ammonium bicarbonate buffer, pH 8.0. Elution was carried out with a continuous gradient up to a concentration of 0.5 M ammonium bicarbonate. Absorbance of the effluent solution was recorded at a wavelength of 280 nm (Souza et al., 2008; Da Silva et al., 2008). PLA2 homogeneity was assessed by native and SDS-PAGE and reverse-phase HPLC. In this study the faction II known as Asp49 BthTX-II was utilized. This phospholipase will be denominated in this paper as just PLA2. All reagents are purchased from Aldrich or Sigma Co. (USA). 2.2. Animals The mice used in this study were kept under specific pathogen-free conditions. The animals were housed in laminar-flow cages maintained at a temperature of 2272 1C and a relative humidity of 50–60%, under a 12:12 h light–dark cycle. The animals were kept under these conditions for at least 1 week before the experiment. Male BALB/c mice, 6–8 weeks old, were matched for body weight (18–22 g). The animal experiments were done with the approval of the institutional committee of ethics in accordance with protocols following the recommendations of the Canadian Council on Animal Care. 2.3. Synthesis of nitrostyrene derivatives Nitrostyrenes were prepared by procedures described in the literature, 1–5 (Vogel, 1989), 6–9 (Ford et al., 1994)

Fig. 1. The ten nitrostyrene derivatives were based on the quercetin structure. In the quercetin molecule, the oxygens (O30 and O70 ) present structural correlation with nitrogens (N1 and N10) from compound 4 (1-nitro-4-((E)-2-nitrovinyl)benzene—see Fig. 2). In both compounds, quercetin and compound 4, both the oxygen and the hydrogen atoms are separated by seven double conjugated bonds. Thus, N1 and N10 of the nitro groups from compound 4 are placed in positions equivalent to the ones of the oxygens of the hydroxyls in C3 and C7 from quercetin, which are essential for the enzyme inhibition. The other compounds synthesized were variations of compound 4. The numbering used in the structure of compound 4 does not correspond to the one recommended by IUPAC and was elaborated aiming to facilitate the analysis of partial charge on all the atoms of carbon, nitrogen, oxygen and chloride. The numbering and nomenclature suggested by IUPAC are described in item 2.3 (material and methods).

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Fig. 2. The nitrostyrene compounds synthesized in this study.

and 10 (Oliveira et al., 2007). IR spectra were recorded on a Bomem MB100 spectrometer, KBr, nmax, cm1. 1H and 13C NMR spectra were obtained on a Bruker AVANCE 400 spectrometer, operating at 9.4 T, observing 1H at 400.13 MHz and 13C at 100.61 MHz and a Bruker AC-200 spectrometer operating at 4.7 T, observing 1H at 200.13 MHz and 13C at 50.61 MHz. All 1H and 13C spectra were taken in CDCl3 or CDCl3/dimethyl sulfoxide (DMSO)d6 mixture and the chemical shifts are given in ppm related to tetramethylsilane, as an internal reference. All reagents and solvents used were previously purified and dried as reported in the literature (Perrin et al., 1980).

1-((E)-2-nitrovinyl)benzene (1): IR nmax 1632, 1513, 1496, 1344 cm1; 1H NMR (CDCl3, 200 MHz) d 7.4–7.55 (m, 4 H), 7.6 (d, J ¼ 13.5 Hz, 1 H), 7.8 (d, J ¼ 13.5 Hz, 1 H); 13C NMR (CDCl3, 50 MHz) d 129.19, 129.45, 130.13, 132.19, 137.19, 139.15.

1-nitro-2-((E)-2-nitrovinyl)benzene (2): IR nmax 1553, 1522, 1348 cm1; 1H NMR ( CDCl3, 400 MHz) d 7.44 (d, J ¼ 13.5 Hz, 1 H), 7.61–7.65 (m, 1 H), 7.68–7.74 (m, 1 H), 7.75–7.8 (m, 1 H), 8.22 (dd, J ¼ 8.13 Hz, J ¼ 1.3 Hz, 1 H), 8.53 (d, J ¼ 13.5 Hz, 1 H); 13C NMR (CDCl3, 100 MHz) d 125.7, 126.3, 129.6, 132.0, 134.2, 135.4, 139.9, 148.2. 1-nitro-3-((E)-2-nitrovinyl)benzene (3): IR nmax 1640, 1553, 1527, 1509, 1351 cm1; 1H NMR (CDCl3/DMSO-d6, 400 MHz) d 7.7 (t, J ¼ 8.0 Hz, 1 H), 7.89 (d, J ¼ 13.7 Hz, 1 H); 7.96–8.02 (m, 1 H), 8.1 (d, J ¼ 13.7 Hz, 1 H), 8.3–8.37 (m, 1 H), 8.51 (t, J ¼ 1.8 Hz, 1 H); 13C NMR (CDCl3/DMSO-d6, 100 MHz) d 123.64, 126.03, 130.57, 131.9, 134.8, 136.4, 139.5, 148.61. 1-nitro-4-((E)-2-nitrovinyl)benzene (4): IR nmax 1640, 1604, 1531, 1345 cm1; 1H NMR (CDCl3/DMSO-d6, 400 MHz) d 8.0 (d, J ¼ 8.92 Hz, 2 H), 8.15 (d, J ¼ 13.7 Hz, 1 H), 8.2 (d, J ¼ 13.7 Hz, 1 H), 8.27 (d, J ¼ 8.92 Hz, 2 H); 13 C NMR (CDCl3/DMSO-d6, 100 MHz) d 123.94, 130.52, 136.2, 136.6, 140.68, 148.9.

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1-chloro-4-((E)-2-nitrovinyl)benzene (5): IR nmax 1631, 1517, 1500, 1491, 1340 cm1; 1H NMR (CDCl3, 200 MHz) d 7.32–7.42 (m, 4 H), 7.5 (d, J ¼ 13.68 Hz, 1 H), 7.9 (d, J ¼ 13.68 Hz, 1 H); 13C NMR (CDCl3, 50 MHz) d 128.52, 129.73, 130.24, 137.41, 137.66, 138.32. N,N-dimethylamine-4-((E)-2-nitrovinyl)benze (6): IR nmax 1598, 1535, 1485, 1325 cm1; 1H NMR (CDCl3, 200 MHz) d 3.0 (s, 6 H), 6.6 (d, J ¼ 8.54 Hz, 2 H), 7.35 (d, J ¼ 8.54 Hz, 2 H), 7.42 (d, J ¼ 13.6 Hz, 1 H), 7.89 (d, J ¼ 13.6 Hz, 1 H); 13C NMR (CDCl3, 50 MHz) d 40.02, 111.90, 117.19, 131.45, 132.05, 140.30, 152.98. 1-methoxy-4-((E)-2-nitrovinyl)benzene (7): IR nmax 1625, 1603, 1573, 1516, 1499, 1328, 1310, 1254 cm1; 1H NMR (CDCl3, 200 MHz) d 3.86 (s, 3 H), 6.94 (d, J ¼ 8.54 Hz, 2 H), 7.5 (d, J ¼ 13.7 Hz, 1 H), 7.5 (d, J ¼ 9.04 Hz, 2 H), 7.96 (d, J ¼ 13.7, 1 H); 13C NMR (CDCl3, 50 MHz) d 55.50, 114.91, 122.55, 131.14, 135.04, 139.01, 162.95. 2-methoxy-4-((E)-2-nitrovinyl)phenol (8): IR nmax 3473, 1603, 1519, 1489, 1359, 1291 cm1; 1H NMR (CDCl3, 400 MHz) d 3.96 (s, 3 H), 6.98 (d, J ¼ 8.2 Hz, 1 H), 7.0 (d, J ¼ 1.96, 1 H), 7.14 (dd, J ¼ 8.2 Hz, J ¼ 1.96 Hz, 1 H), 7.52 (d, J ¼ 13.56 Hz, 1 H), 7.96 (d, J ¼ 13.56 Hz, 1 H); 13C NMR (CDCl3, 100 MHz) d 56.1, 110.14, 115.3, 122.43, 124.91, 135.01, 139.46, 147.06, 149.75. 2-methoxy-4-((E)-2-nitrovinyl)phenyl acetate (9): IR nmax 1745, 1520, 1503, 1348, 1307, 1232 cm1; 1H NMR (CDCl3, 200 MHz) d 2.34 (s, 3 H), 3.88 (s, 3 H), 7.08 (d, J ¼ 1.71 Hz, 1 H), 7.13 (s, 1 H), 7.16 (d, J ¼ 1.71 Hz, 1 H), 7.55 (d, J ¼ 13.4, 1 H), 7.97 (d, J ¼ 13.4 Hz, 1 H); 13C NMR (CDCl3, 50 MHz) d 20.8, 56.27, 112.3, 122.7, 124.1, 129.1, 137.4, 138.6, 143.2, 152.07, 168.7. 2-(cyclopentyloxy)-1-methoxy-4-((E)-2-nitrovinyl)benzene (10): IR nmax 1627, 1594, 1511, 1490, 1438, 1332, 1263 cm1; 1H NMR (CDCl3, 200 MHz) d 1.5–1.65 (m, 2 H), 1.7–2 (m, 6 H), 3.84 (s, 3 H), 4.73 (m, 1 H), 6.83 (d, J ¼ 8.3 Hz, 1 H) 6.94 (d, J ¼ 2.2 Hz, 1 H), 7.0 (dd, J ¼ 8.5 Hz, J ¼ 2.2 Hz, 1 H), 7.4 (d, J ¼ 13.6 Hz, 1 H), 7.8 (d, J ¼ 13.6 Hz, 1 H); 13C NMR (CDCl3, 50 MHz) d 153.86, 148.20, 139.54, 134.99, 124.23, 122.63, 113.83, 111.79, 80.74, 56.07, 32.75, 24.02.

2.4. Edema-inducing activity Edema was induced in the right foot pad of male Swiss mice (18–22 g) by i.d. injection of 50 mL of a solution containing 50 mg of PLA2 purified from B. jararacussu venom dissolved in 1% DMSO in PBS (phosphate-buffered saline—pH 7.2). Inhibition studies were performed by i.d. injection of 50 mL of a solution containing a mixture of 50 mg of PLA2 and 25 mg of each nitrostyrene derivative compound dissolved in 1% DMSO in PBS (pH 7.2). Prior to the injections, the mixtures containing PLA2 and the inhibitors were pre-incubated for 10 min at 37 1C. Negative control groups were injected with 50 mL of 1% DMSO in PBS (pH 7.2). Control groups for each nitrostyrene compound obtained through the i.d. injection of 50 mL of a solution containing only 25 mg of each nitrostyrene

derivative compound dissolved in DMSO in PBS (pH 7.2). The progression of edema was evaluated with a lowpressure pachymeter (Mitutoyo, Japan) at various time intervals after injection (0.5, 1, 2, 4, 6, 24 h) (Soares et al., 2000; Souza et al., 2008). 2.5. Myotoxic activity Swiss male mice (18–22 g) were intramuscularly injected in the right gastrocnemius muscle with 50 mL of a solution containing 25 mg of PLA2 purified from B. jararacussu. Inhibition studies were performed by injecting 50 mL of a mixed solution composed of 25 mg of PLA2 and 25 mg of each nitrostyrene derivative compound dissolved in 1% DMSO in PBS (pH 7.2). Prior to the injections, the mixtures containing PLA2 and the inhibitors were pre-incubated for 10 min at 37 1C. Negative controls received 50 mL of 1% DMSO–PBS alone. The control group for each nitrostyrene compound received an intramuscular injection of 50 mL of a solution containing just 25 mg of each nitrostyrene derivative compound dissolved in 1% DMSO in PBS (pH 7.2). Mice were bled from the tail 3 h after injections and blood was collected into heparinized capillary tubes. Plasma creatine kinase activity was determined using the Kit 47-UV (Sigma Chemical Co.). Activity was expressed in units/L, one unit corresponding to the production of 1 mmole of NADH per min at 30 1C (Souza et al., 2008; Da Silva et al., 2008). 2.6. Enzymatic activities The measurement of the enzymatic activity using the micellar substrate, 1-hexadecanoyl-2-(1-pyrenedecanoyl)sn-glycero-3-phosphoglycerol (HPGP), was performed through the microtiter plate assay (Souza et al., 2008; Da Silva et al., 2008). Each nitrostyrene compound was tested in final concentrations of 0.0, 0.1, 0.2, 0.3 and 0.4 mM. Seven wells of a 96-well microtiter plate were used for each assay, resulting in six measurement repetitions of the enzymatic activity for each final concentration of the inhibitor. Thus, for each assay with different concentrations of the inhibitor, 100 mL of solution A in assay buffer (27 mM bovine serum albumin, 50 mM KCl, 1 mM CaCl2, 50 mM Tris–HCl, pH 8.0) were added to seven wells, followed by the addition of a volume of 26.5 mM nitrostyrene derivative compounds (1–4 mL, according to the assay concentration) dissolved in DMSO. For control reactions, 1–4 mL of DMSO were used alone. Solution B had the same composition of Solution A, however, with the presence of PLA2 (0.5 mg/mL) and was delivered in 100 mL volumes to seven wells, except for the first well. Instead of Solution B, an additional 100 mL portion of Solution A was added to the first of the seven wells in the assay. Solution B was prepared immediately before to each set of assays to avoid loss of enzymatic activity. Quickly after the addition of Solution B, the assay was initiated by the addition of 0.5–50 mL of Solution C (53 mM HPGP vesicles in assay buffer) with a repeating pipettor to the seven wells. The final concentration of HPGP varied from 0.125 to 10 mM. The final volume of the

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assay was 265 mL and, when necessary, the wells received an extra volume of solution A in order to complete this volume. The fluorescence (excitation ¼ 342 nm, emission ¼ 395 nm) was read with a microtiter plate spectrophotometer (Fluorocount, Packard Instruments). Control reactions without enzyme or inhibitor were run for all assays and the initial velocity was calculated from the initial slope of fluorescence versus time, for each concentration of the substrate used. The significance of differences between groups was evaluated using the Student’s t-test. A P-value o0.05 was considered to be significant. 2.7. Ab initio quantum calculations The structures of nitrostyrene derivative compounds were submitted to ab initio quantum calculations. In order to select the best conformations, the HyperChem 7.51 software was utilized. The final geometry optimization was carried out using the GAUSSIAN03 package, applying the DFT methodology with the use of UB3LYP functional with 6-31G* basis set (Hariharan and Pople, 1973). The following molecular and electronic properties (descriptors) were calculated: total non-relativistic electronic energy (ET), dipole moment (m), highest occupied molecular orbital energy (HOMO), lowest occupied molecular orbital energy (LUMO), surface area (A), molecular volume (VOL), logarithm of partition coefficient (Log P), polarizability (POL), molecular refractivity, the difference between the energy values of HOMO and LUMO (GAP), Mullikan electronegativity (x—Eq. (1)), hardness (Z—Eq. (2)), electronegativity (w—Eq. (3)), softness (S—Eq. (4)), electrophilicity index (o—Eq. (5)), ionization potential (IP—Eq. (6)), electron affinity (AE—Eq. (7)), partial atomic charges (Qn, where n corresponds to the atom number, according to Fig. 1) on the carbon, nitrogen, oxygen and chlorine atoms. The atom numbering shown in Fig. 1 does not correspond to the one recommended by IUPAC, and it was elaborated aiming to standardize the chemometric analysis of the partial atomic charge (Qn). The numbering in agreement with the UIPAC is the one used in item 2.3 (material and methods) and reports the structural elucidation of the compounds synthesized x¼

ðHOMO  LUMOÞ 2

ðLUMO  HOMOÞ Z¼ 2

(1)

(2)

w¼

ðIP=EAÞ 2

(3)

S¼

1 2Z

(4)

m2 2Z

(5)

o¼

EA ¼ ½ðTENEUTRAL þ TCENEUTRAL  0:9806Þ  ðTEANION þ TCEANION  0:9806Þ27:2114

(7)

where TE is the total electronic energy and TCE is the total energy corrected for zero-point vibrational energy (ZPVE) for both neutral and ionic (positive and negative) species. The correction factor of the ZPVE is 0.9806 for model B3LYP/6-31G* and 1 Hartree ¼ 27.2114 eV (Parr and Pearson, 1983; Chattaraj et al., 1991; Scott and Radom, 1996; Kohn et al., 1996; Da Silva et al., 1998; Parr et al., 1999; Sinha et al., 2004). In addition to the molecular and electronic descriptors, 16 topological descriptors were also calculated through the use of the program Dragon: total structure connectivity index, polarity number log of product of row sums, average vertex distance degree, mean square distance index (Balaban), Schultz Molecular Topological Index, Wiener-type index from van der Waals weighted distance matrix, Wiener-type index from electronegativity weighted distance matrix, Wiener-type index from polarizability weighted distance matrix, Balaban distance connectivity index, Balaban-type index from electronegativity weighted distance matrix, Balaban-type index from polarizability weighted distance matrix, Balaban-type index from mass weighted distance matrix, Balaban-type index from van der Waals weighted distance matrix, maximal electrotopological negative variation, maximal electrotopological positive variation, molecular electrotopological variation, and sum of topological distances between different heteroatom pairs. The chemometric methods are particularly appropriate to provide insight into the structure–activity relationships (SAR) when one is dealing with systems depending on many variables (Beebe and Pell, 1988). PCA and HCA are statistical methods used in the recognition of standards in multivaried studies (Da Silva et al., 2004; Weber et al., 2005; Calgarotto et al., 2007). The properties calculated by DFT were auto-scaled and, through the use of Fisher weight (Costa and Takahata, 2003), the properties with differences able to better discriminate the relationship between the structures of nitrostyrene derivative compounds and their biological activity were selected. The Pirouette program was used to perform the PCA and HCA. 2.8. Statistical analysis Results are presented as the means values 7S.D. obtained with the indicated number of tested animals. The statistical significance of differences between groups was evaluated using Student’s unpaired t-test. A P-value o0.05 was considered to indicate significance. 3. Results and discussion

IP ¼ ½ðTECATION þ TCECATION  0:9806Þ  ðTENEUTRAL þ TCENEUTRAL  0:9806Þ27:2114

1471

(6)

Edema-inducing activity is a multifactorial pharmacological activity and depends on the combined action of various toxins, including PLA2 (Soares and Giglio, 2003). Fig. 3 shows that after a 2-h period, nitrostyrene 1–5 reduces the levels of edema-inducing activity of PLA2 to 10%–30% when compared to the PLA2 control experiment. In this same period, compounds 6–8 reduce the edema

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Fig. 3. Effect of nitrostyrene compounds on edema induced by PLA2 purified from B. jararacussu venom. The PBS control solution contains 1% of DMSO. Results are expressed as the means7S.D. (n ¼ 6).

Fig. 4. Effect of nitrostyrene compounds on the myotoxic activity induced by purified PLA2 from B. jararacussu venom. The PBS control solution contains 1% of DMSO. Results are expressed as the means7S.D. (n ¼ 6).

levels to 85–95% while compounds 9 and 10 reduce them to only 65–75%. In compounds 2, 3 and 4, the influence of the NO2 group on the increased inhibition of edema formation is clear when compared with compound 1. However, the presence of nitrostyrene in other functional groups, as seen in compounds 5–10, leads to a reduction in the inhibition activity of PLA2-induced edema. Muscle tissue damage, myonecrosis, is also a common consequence of envenoming by snakes of the genus Bothrops (Gutie´rrez, 2002). The muscle damaging activity of Bothrops venoms is partially caused by a group of highly basic proteins with PLA2 structure. Compounds 1–5 reduced myotoxicity to 45–55%, when compared to the PLA2 control assay (Fig. 4). Compounds 8–10 reduced the myotoxic activity by around 30%, while compounds 6 and 7 did not demonstrate any activity against myotoxic effects.

The kinetic behavior study of the PLA2 from B. jararacussu in the presence and absence of nitrostyrene derivative compounds is shown in Table 1. The kinetic parameters (and the curves—data not shown) indicate that, in the substrate concentration range used in this study (0–10 mM), the action of the enzyme on the micellar substrate HPGP reflects the classical behavior of a Michaelian enzyme, not only in the presence of small concentrations of the nitrostyrene compounds (0.1–0.4 mM), but also in their absence. Table 1 demonstrates that in all the tests, the maximum velocity of the enzyme (VMAX) varied in function of the presence of growing concentrations of compounds 1–10 and its values decreased from around 18 nmols/min (inhibitor absence) to 1.4 nmols/min (0.4 mM of compound 4). On the other hand, the presence of the inhibitors did not induce any alteration in KM values.

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Table 1 Kinetic parametersa obtained on the reaction of PLA2 from B. jararacussu in the presence and absence of nitrostyrene derivative compounds using the substrate HPGP Inhibitor concentrations (mM) 0.0

0.1

0.2

0.3

0.4

Com 1 Vmax Km KI

18.1570.32 0.6970.07

13.1171.01 0.7070.12 260.071.0

10.1571.00 0.6970.09 254.370.9

8.3270.73 0.5170.10 253.971.4

6.9570.39 0.6970.14 248.271.1

Com 2 Vmax Km KI

18.3370.51 0.6870.08

6.2771.01 0.7070.10 52.070.4

3.7470.31 0.7270.08 51.271.9

2.6570.3 0.7070.06 50.671.5

2.0970.3 0.6870.12 51.571.0

Com 3 Vmax Km KI

18.1370.68 0.7170.11

7.1270.65 0.6870.05 64.771.3

4.3370.71 0.7070.07 62.870.9

3.2070.39 0.7070.11 64.271.4

2.4770.67 0.7170.08 63.171.0

Com 4 Vmax Km KI

17.9971.03 0.7070.14

4.6571.11 0.7270.04 34.971.3

2.7271.03 0.6870.01 35.671.9

1.8770.83 0.6970.11 34.770.4

1.4270.99 0.7170.07 34.371.8

Com 5 Vmax Km KI

18.0071.02 0.7270.09

13.7070.71 0.7770.12 318.172.0

11.1270.91 0.6970.09 323.570.9

9.2271.03 0.7070.11 315.571.4

7.9970.83 0.7070.06 319.272.1

Com 6 Vmax Km KI

17.9971.32 0.6970.13

17.6970.41 0.7170.16 5972.571.0

17.4270.90 0.7070.10 6124.271.9

17.1370.62 0.7070.10 6011.171.4

16.8570.38 0.7270.02 5911.271.9

Com 7 Vmax Km KI

18.1970.32 0.7070.12

17.8171.01 0.7170.10 4631.271.7

17.4570.31 0.7070.08 4711.870.8

17.1270.3 0.6970.11 4785.170.7

16.7670.3 0.6970.06 4690.571.8

Com 8 Vmax Km KI

18.3071.32 0.7170.04

16.4970.21 0.6970.09 913.171.9

15.0370.61 0.6870.10 921.670.9

13.7771.32 0.7070.02 911.471.4

12.6771.11 0.6970.03 901.071.8

Com 9 Vmax Km KI

18.2071.02 0.6970.03

15.8270.87 0.7070.10 709.372.0

14.0870.54 0.6970.11 716.571.9

12.6871.03 0.6870.08 712.471.4

11.5370.42 0.7170.03 711.471.9

Com 10 Vmax Km KI

17.9970.56 0.7170.02

16.3871.89 0.7170.07 1039.171.5

15.0771.31 0.6870.03 1034.471.9

13.9070.76 0.6970.08 1021.570.4

12.8971.03 0.6870.11 1012.771.1

The Vmax values are given in nmols/min. KM values are given in mM. KI values are given in nM. a Average of three independent determinations; six replicates; values are mean7S.D.

In the presence of growing concentrations of compounds 1–10, the KM values of PLA2 were kept at around 0.7 mM. In addition, the inhibition constant values (KI) for compounds 2, 3 and 4 were, respectively, around 51, 64 and 35 nM and were considered the lowest values of all the tested compounds. Nevertheless, compounds 6 and 7 presented much higher KI, with values of around 6000 and 4700 nM,

respectively. In addition, compounds 1 and 5 presented KI values of, respectively, around 260 and 318 nM while compounds 8–10 varied from 900 to 1000 nM (see Table 1). Thus, since KI reflects the dissociation of the enzyme–inhibitor complex the lower its value, the higher the inhibitorbinding capacity, it may be observed that compounds 2–4 presented the best capacity to inhibit the enzymatic activity

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of B. jararacussu PLA2. However, compounds 6 and 7 presented a very low inhibition capacity and almost did not change VMAX values (Table 1). This set of results shows that the enzymatic inhibition provoked by nitrostyrene derivatives is non-competitive and that these compounds might be bound to a site different from the enzyme active site and do not compete with HPGP. Thus, these studies oppose the initial idea of drawing compound 4 in function of quercetin (Fig. 1), since we believed that the similarity in the distances between the nitro groups (compound 4) and the hydroxyls from quercetin may facilitate the binding of our nitrostyrene derivatives at the active site of the PLA2. After the assays, the structures were submitted to quantum chemistry calculations and chemometric studies. PCA is a multivariate statistical technique that reduces the data dimensionality by the linear transformation of the original data set in a new and smaller set of uncorrelated variables (Principal Components or PCs) (Beebe and Pell, 1988). This technique has been widely applied in the chemometric studies of bioactive compounds (Da Silva et al., 2004; Weber et al., 2005; Calgarotto et al., 2007). The auto-scaled values for all the calculated properties (molecular, electronic and topological) were analyzed according to Fisher weight. Nine descriptors, whose variances may be responsible for the differences observed in the biological activity of the nitrostyrene, were indicated (HOMO, VOL, POL, electronegativity (w), IP, partial atomic charges (net charge on C5, C7 and C9 atoms—according Fig. 1), Wiener-type index from electronegativity weighted distance matrix, Balabantype index from polarizability weighted distance matrix, sum of topological distances between different heteroatom pairs). The PCA algorithm used left open the possibility for the selection of different combinations of these nine descriptors in order to discover which variance could better describe this multivariate system. Thus, when the PCA technique was applied to the auto-scaled values of the selected properties obtained from the ab initio quantum calculations (DFT-UB3LYP/6-31G*) of the nitrostyrene derivative compounds, the best separation was obtained using the values of three variables (HOMO energy, net charge in the C9 and polarizability—Table 2 and Fig. 5A). Fig. 5B shows that, utilizing values of three properties selected by PCA, all these nitrostyrene derivative compounds may be grouped in three distinct regions: Group 1 (compounds 1–5, high activity in all tests); Group 2 (Compounds 8–10, intermediate activity in all tests); Group 3 (compounds 6 and 7, low activity in all tests). PCA results show that the first component (PC1) is responsible for 65.78% of the data variance and the second one (PC2) is responsible for 27.29%. Considering the first and second principal components (PC1 and PC2), the accumulated variance increased to 93.07%. The HCA technique verifies the distance among the samples in a data set and is a tool for preliminary data analysis that can be used to inspect data sets for expected or unexpected clusters (Beebe and Pell, 1988; Da Silva et al., 2004; Weber et al., 2005; Calgarotto et al., 2007). In the HCA analysis, the same nine descriptors selected according to Fisher weight and used in the PCA were

Table 2 Nitrostyrene compound property values selected by PCA and calculated by ab initio method quantum chemistry (DFT-UB3LYP/6-31G*) Compounds

HOMO energy (eV)

C9 net charge (e.u.)

Polarizability (A˚3)

1 2 3 4 5 6 7 8 9 10

–8.4464 –8.9335 –8.7730 –8.9607 –8.4736 –7.2953 –7.3688 –7.9998 –8.1417 –8.0110

–0.1810 –0.1960 –0.1918 –0.2000 –0.1909 –0.2301 –0.2231 –0.1950 –0.1918 –0.2006

15.75 17.59 17.59 17.59 16.08 21.08 21.05 25.62 25.20 27.26

utilized. Similar to the PCA, the HCA algorithm also permits different combinations among the descriptors selected to describe the best multivariate system based on the degree of similarity of their variances. The HCA indicated that the best similarity degree among the most active and less active compounds is reached through the combination of values of HOMO energy, net charge on the C9 atom and the polarizability. It is possible to see, from Fig. 5C, that HCA separates the nitrostyrene compounds in two major blocks with zero similarity. One branch of the dendrogram contains the compounds of Groups 1 (active) and 2 (intermediate activity). Another distinct branch grouped the two compounds with low activity inside the concentration range used in the tests (compounds 6 and 7—Group 3). This classification confirms the same pattern observed in PCA and indicates that HOMO energy, net charge on the C9 atom and the polarizability could potentially be responsible for the biological activity shown by the nitrostyrene compounds.

3.1. Proposed model for PLA2 nitrostyrene compound binding sites Table 2 shows that the more active compounds (1–5) present lower HOMO energy and polarizability values. The selection of these two properties by PCA, confirmed by HCA, indicates the ability of the nitrostyrene compounds to form transference charge complexes during the inhibition process of PLA2. Lower HOMO values indicate that compounds 1–5 might be receiving electrons from PLA2 amino acids in an easier manner than the compounds 6–10. However, compounds 8–10 present some response in the inhibition of biological effects provoked by PLA2 (edema-inducing activity, enzymatic activities and myotoxic activity) and this might be related to its HOMO values, which are much closer to the values of compounds 1–5 (higher activity) than to the inactive ones (6 and 7—low activity). This proximity of the HOMO values of compounds 8–10 and 1–5 probably allows the electron transference from the PLA2 to compounds 8–10. From the results of the SARs (Figs. 3 and 4 and Table 1) and chemometric studies (Fig. 5 and Table 2), it is possible to infer that the binding site of the nitrostyrene compounds does not support molecules with great electronic

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Fig. 5. Chemometric results: (A) Loading plot for the three variables responsible for the classification of the ten nitrostyrene derivative compounds: HOMO energy (highest occupied molecular orbital energy); C9 (net charge on carbon atom 9—see Fig. 1); POL (molecular polarizability). (B) Score plot for the ten nitrostyrene derivative compounds. (C) Dendrogram obtained for all the nitrostyrene derivative compounds.

cloud distortion capacity (high values of polarizability) as do those from the less active compounds (6–10). However, the high values of polarizability presented by compounds 8–10 may apparently be compensated by the HOMO energy and the net charge on the C9 atom values. In these compounds (8–10), the values of these properties are closer to the values of the active compounds (compounds 2–4). In compounds 6 and 7, higher values of negative net charge on C9 might be inducing a reduction in the

inhibition of PLA2. It is possible that, inside the PLA2 binding site of the nitrostyrenes derivative compounds, the atom C9 is close to a region with accentuated negative character. Thus, compounds with higher negative net charge on the C9 atom could destabilize the formation of the PLA2 complex with nitrostyrene. The kinetic parameter values found in the enzymatic assays (Table 1) show that the nitrostyrene derivative compounds inhibit PLA2 in a non-competitive manner,

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Fig. 6. Proposed model for the principal topologic aspects of the nitrostyrene compound binding site in the PLA2. This model was established using compound 4, which was the compound with the highest PLA2 inhibition capacity. Numbering is based on Fig. 1.

signifying that, the binding site of these inhibitors might be different from the active site of the enzyme. The set of experimental evidences, as well as the structural information obtained with the ab initio calculations and the chemometric studies allow the proposition of a model of the binding site of the nitrostyrene compounds to PLA2. This site must be small and it is not able to support large molecules. Compound 4 was the most active of all the analyzed molecules and can serve as the basis to propose a binding site model (Fig. 6). In the binding site, there is a region that adequately accommodates the nitro group bound to the C7 position (Fig. 1) of the aromatic ring. The nitro groups in the C5 and C6 positions are also able to be bound in the binding site; however, there are slight grades of steric hindrance on these positions, explaining the minor reduction on the activity of compounds 2 and 3 compared to compound 4. Compounds 1 and 5 adjust well inside the binding site and they also present some significant inhibition capacity of the PLA2 effects. However, these compounds (1 and 5) present slightly different HOMO and polarizability values from the values presented by compounds 2–4. These facts reduce the capacity of PLA2 inhibition by compounds 1 and 5. Compounds 6 (group dimethylamine in C7) and 7 (group metoxyl in C7) were inactive in all the assays performed. The dimethylamine and methoxy groups occupy the same region as the nitro group in compound 4, but the characteristics of these groups, mainly charge distribution and group volume, are the difficult binding of the compounds 6 and 7 to PLA2. In addition, both nitrostyrene derivative compounds present very different properties responsible for the inhibition of PLA2 (HOMO energy, net charge on C9 atom and polarizability) from those presented by the active compounds (compounds 1–5). Thus, the impediment provoked by the volume in the C7 position and the high HOMO energy values make the formation of the charge-transferring complex between inactive compounds (6 and 7) and the enzyme more difficult. Therefore, the net charge on C9 is much more negative than those verified in the active compounds.

4. Conclusions An efficient and selective PLA2 inhibitor must prevent not only PGs, but also LTs formation as well all their side effects (Yedgar et al., 2000, 2006). In the development of new PLA2 inhibitors, many chemical substances have been tested (Binisti et al., 1997, 2001; Sekar et al., 1997). Natural compounds such as quercetin, rosmarinic acid, aristolochic acid and a-tocoferol (vitamin E) also present high capacities of PLA2 inhibition (Lindahl and Tagesson, 1997; Kim et al., 2001; Gil et al., 1997; Chandra et al., 2002a., 2002b; Ticli et al., 2005; La¨ttig et al., 2007). In this study, we verified that the nitrostyrenes are able to inhibit several biological effects provoked by PLA2 from B. jararacussu venom. Our studies of SAR showed that the most active compounds in the inhibition of edemainducing activity, enzymatic activities and myotoxic activity provoked by PLA2 purified from venom of B. jararacussu are those that present a nitro group in the ortho, meta and para positions in the aromatic ring. Other substitution patterns in the aromatic ring also affect the inhibition activity, but to a lesser extent. The electronic, molecular and topologic properties of all nitrostyrene derivatives were calculated using ab initio quantum calculations (DFT) and analyzed by chemometric methods (PCA and HCA). It was verified that the proprieties of HOMO energy, net charge on the C9 atom and polarizability are probably responsible for the differences between the most and the less active compounds. One possible explanation for the inhibition effects on PLA2 is the formation of transfer charge complexes between PLA2 and the nitrostyrene derivative compounds. Thus, the most active compounds (2, 3 and 4) present low HOMO energy values, which are favorable for PLA2 electrons reception. Our strategy for the drawing of nitrostyrene derivative compounds was not shown to be valid, since according to the enzymatic kinetic studies, the nitrostyrene binding site in the PLA2 is different from the enzyme active site and does not compete for the substrate, even when the nitro (compound 4) and hydroxyl (quercetin) groups are separated by an equivalent distance (Fig. 1). This fact might be related to the available charge amount on the

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nitro group of the nitrostyrene derivatives in relation to the hydroxyl group from quercetin, causing the binding of compounds 1–10 to different regions of the active site. This nitrostyrene derivatives binding site allows the binding of molecules with a low capacity of electronic cloud distortion (low polarizability); furthermore, the net charges on the C9 atom must present adequate values. In deep structural studies, it is necessary to define the conditions of the nitrostyrene compounds binding to PLA2. However, the results obtained herein will be useful for the development of new molecules used in the production of a specific PLA2 inhibitor.

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