NMR analysis of a new canthin-6-one alkaloid from Simaba bahiensis ( Simaroubacae )

Alkaloids of the canthi-6-one type have been reported in differents natural sources. These alkaloids have showed a wide range of pharmacological properties including cytotoxic, antibacterial, antifungal, antiparasict, antiviral, anti-inflammatory and beside it, some of them show excellent photophysical properties that gives an interesting use as fluorescent dye probe in florescent cellular microscopy. Because it, new studies have been carrying out in an attempt to identify new alkaloids with pharmacological properties. Some researchers have been trying to synthesize new derivatives or identify new compounds in natural sources. Among the >60 canthin-6-one alkaloids already reported in natural sources, more the half are present in plants of the family Simaroubaceae. In the Simaba genus, up to the present, only eighteen alkaloids was described in seven species, but other plants of the genus that occur in the brazilian’s flora haven’t been studied yet. In this work we describe the first phytochemical study of the specie Simaba bahiensis (Simaroubaceae), collected at the city of Camaçari, Bahia State, Brazil. Also, it’s related complete structural determination using differents NMR experiments and HRMS of a new canthin-6-one alkaloid in addition to two others already known MOL2NET, 2017, 3, doi:10.3390/mol2net-03-xxxx 2 Introduction Canthi-6-one alkaloids occur in plants from families Rutaceae, Simaroubareace, Amaranthaceae, Caryophyllaceae and Zygophylaceae [1-3]. More than 60 members of this type of alkaloid were isolated from natural sources since first report in 1952 [4]. These alkaloids have shown a wide range of pharmacological properties including cytotoxic, antibacterial, antifungal, antiparasict, antiviral, antiinflammatory and beside it, some of them show interesting photophysical properties that gives an interesting use the fluorescent dye probe in florescent cellular microscopy. New studies have been carring out in an attempt to identify new alkaloids with pharmacological properties by synthesis or isolation from different natural sources [2]. In family Simaroubaceae more than 30 canthi-6-one alkaloids have already been described with different substitution patterns and it shows that the investigation of new alkaloids in this family it’s an important way to search new chemical entities [2, 3]. In this work we describe the first phytochemical study of Simaba bahiensis (Simaroubaceae), collected at the city of Camaçari, Bahia State, Brazil. Also, it’s related complete structural determination using differents NMR experiments and HRMS of a new canthin-6-one alkaloid and two others already known. Materials and Methods Roots of S. bahiensis was collected in Arembepe, Bahia State, Brazil (S lat: -12.800833 long: 38.214444 WGS84 alt. 10m). Professor MSc Maria Lenise Silva Guedes performed the botanical identification and a voucher was deposited at Herbário Alexandre Leal Costa (ALCB) in Biology Institute of Federal University of Bahia with identification 1235018a. The yellower parts of S. bahiensis roots (17,57g), called roots deposits (RD) was manually separated and extracted with MeOH (750 mL) at ultrasound for 10 minutes. The methanol RD extract (0,77g) was produced by evaporation of alcoholic solution. The separation of RD extract was performed by preparative HPLC using: C18 column 250 mm; 21.0 mm; 5 μm; 100 °A, gradient elution with solvents A (H2O) and B (methanol), 0–35min 80 to 50% A, 35-70 min 50%A, flow 8,0 mL.min , diode array detector (DAD), injection volume was 100 μL, the total injection volume was 1,5 mL. Three fractions were collected, evaporated and then analyzed by NMR and HRMS. Results and Discussion The figure 1 shows the chromatogram of HPLC separation and the collected peaks. NMR 1H spectrum, of collected peak Rt(min) = 53.266, shows signals related to aromatic systems and one singlet that corresponds to methoxyl group. The 1H pattern observed for this substance indicates the existence of a canthi-6-one alkaloid substituted by a methoxyl. Four hydrogens (δH 8.62, 7.97, 7.69 and 7.50) of ring A of the canthi-6-ones were assigned considering multiplicity and chemical shifts. Hydrogens at positions 4 and 5 (δH = 7.74 and 6.93, J = 9.8 Hz) are present, the latter signal being a singlet for a hydrogen at ring C, which could be at position 1 or 2, another position would be the methoxyl group. The irradiation of hydrogen δH 7.30, by NOEDIFF experiment, resulted in an increase of the doublet signal δH 7.97. The signals at δH 7.50 and δH 7,30 were increased when δH 7.97 was irradiated, indicating the proximity between the H-11 position in ring A and the hydrogen δH 7,30 present in the Cring. NOEDIFF experiments suggest that the bound of the methoxyl group in this substance occurs in position 2. Irradiation of the hydrogens of the methoxyl group in δH 4.20 increased the signal of the H-4 δH 7.75. All NOE experiments was compared with previous reported data [5]. These NMR data led to 2-methoxy-canthi-6-one alkaloid, but HRMS shows ion [M + H] = 267.0733 m/z with molecular formula C15H10N2O3, and diverged 16 m/z from the proposed alkaloid 2MOL2NET, 2017, 3, doi:10.3390/mol2net-03-xxxx 3 methoxy-canthi-6-one. According to the NMR and MS data, the only possibility that justify this mass difference is the presence of an N-O group at the structure. The IR analysis confirms the N-O bond in the intense absorption at 1303 cm1. It is a first report of spectroscopic data that shows the presence of alkaloid 2-methoxy-canthi-6-one N-oxide (3). NOESY, HMBC and HMQC were performed and the results are described at table 1 and figures 2 and 3. Peaks that corresponds to compounds 1 and 2 were analyzed by NMR and HRMS all data were compared to previous described data [6, 7]. Other canthi-6-on alkaloids have been reported in the literature, among the structures most have substitutions at ring A, structural varieties with substitutions at the position 2 on ring C are uncommon. Although the substitution pattern is easily identified by 1H NMR and NOE experiments can allow unambiguous determination of the substitution pattern in ring A and C [5], the complementary spectroscopic techniques as IR and HRMS allow the determination of the presence of N-oxide groups. Conclusions The first phytochemical study of S. bahiensis were possible identify 3 canthi-6-one alkaloids and the new one 2-methoxy-canthi-6-one N-oxide, previous studies shows the occurrence of alkaloids with N-oxide but without substitution at position 2. Substitution pattern is easy to identify by 1H NMR, but is necessary use different experiments to correctly determine the position of substituents at rings A and C, since studies of structure relationships have determined that different substitution patterns in these rings influence the pharmacological activity of these substances. References 1. Dai, J., et al., Fruitful Decades for Canthin-6-ones from 1952 to 2015: Biosynthesis, Chemistry, and Biological Activities. Molecules, 2016. 21(4): p. 493. 2. Alves, I.A.B.S., et al., Simaroubaceae family: botany, chemical composition and biological activities. Revista Brasileira de Farmacognosia, 2014. 24: p. 481-501. 3. Barbosa, L.F., R. Braz-Filho, and I.J.C. Vieira, Chemical Constituents of Plants from the Genus Simaba (Simaroubaceae). Chemistry & Biodiversity, 2011. 8(12): p. 2163-2178. 4. Showalter, H.D.H., Progress in the Synthesis of Canthine Alkaloids and Ring-Truncated Congeners. Journal of Natural Products, 2013. 76(3): p. 455-467. 5. Zapesochnaya, G., et al., Canthin-6-one and β-Carboline Alkaloids from Aerva lanata. Planta Med, 1992. 58(02): p. 192-196. 6. Pettit, G.R., et al., Antineoplastic agents 157. Quassia kerstingII1. Tetrahedron, 1988. 44(11): p. 3349-3354. 7. Soriano-Agatón, F., et al., Extraction, Hemisynthesis, and Synthesis of Canthin-6-one Analogues. Evaluation of Their Antifungal Activities. Journal of Natural Products, 2005. 68(11): p. 1581-1587. MOL2NET, 2017, 3, doi:10.3390/mol2net-03-xxxx 4 Figure 1: Chromatogram (254 nm) of RD methanol extract obtained by preparative scale. Compound 1 (Rt(min) = 48.241) compound 2 (Rt(min) = 50.348) and compound 3 (Rt(min) = 53.266). Figure 2: HMBC (400MHz) observed correlations (arrows) of compound 3. Figure 3: NOESY (400MHz) and (*) NOEDIFF (500MHz) observed correlations (arrows) of compound 3. Table 1: NMR data 2-methoxy-N-oxide-canthi-6-one. 1H δ(ppm), J(Hz) 13C δ(ppm) 1 7,29 (1H, s) 114,13 a 2 ---157,64 * 4 7,74 (1H, d, J = 9,8) 125,48 a 5 6,93 (1H, d, J = 9,8) 127,85 a 6 --157,78 * 8 8,62 (1H, dl, J = 8,0) 117,39 a 9 7,69 (1H, ddd, J = 8,8; 8,0 ;1,2) 132,23 a 10 7,50 (1H, ddd, J = 8,8;7,6 ;1,2) 126,41 a 11 7,97 (1H, dl, J = 7,6) 123,92 a 12 --123,33 b 13 --142,15 b


Introduction
Canthi-6-one alkaloids occur in plants from families Rutaceae, Simaroubareace, Amaranthaceae, Caryophyllaceae and Zygophylaceae [1][2][3].More than 60 members of this type of alkaloid were isolated from natural sources since first report in 1952 [4].These alkaloids have shown a wide range of pharmacological properties including cytotoxic, antibacterial, antifungal, antiparasict, antiviral, antiinflammatory and beside it, some of them show interesting photophysical properties that gives an interesting use the fluorescent dye probe in florescent cellular microscopy.New studies have been carring out in an attempt to identify new alkaloids with pharmacological properties by synthesis or isolation from different natural sources [2].In family Simaroubaceae more than 30 canthi-6-one alkaloids have already been described with different substitution patterns and it shows that the investigation of new alkaloids in this family it's an important way to search new chemical entities [2,3].In this work we describe the first phytochemical study of Simaba bahiensis (Simaroubaceae), collected at the city of Camaçari, Bahia State, Brazil.Also, it's related complete structural determination using differents NMR experiments and HRMS of a new canthin-6-one alkaloid and two others already known.

Materials and Methods
Roots of S. bahiensis was collected in Arembepe, Bahia State, Brazil (S lat: -12.800833 long: -38.214444WGS84 alt.10m).Professor MSc Maria Lenise Silva Guedes performed the botanical identification and a voucher was deposited at Herbário Alexandre Leal Costa (ALCB) in Biology Institute of Federal University of Bahia with identification 1235018a.

Results and Discussion
The figure 1 shows the chromatogram of HPLC separation and the collected peaks.NMR ¹H spectrum, of collected peak Rt(min) = 53.266,shows signals related to aromatic systems and one singlet that corresponds to methoxyl group.The ¹H pattern observed for this substance indicates the existence of a canthi-6-one alkaloid substituted by a methoxyl.Four hydrogens (δH 8.62, 7.97, 7.69 and 7.50) of ring A of the canthi-6-ones were assigned considering multiplicity and chemical shifts.Hydrogens at positions 4 and 5 (δH = 7.74 and 6.93, J = 9.8 Hz) are present, the latter signal being a singlet for a hydrogen at ring C, which could be at position 1 or 2, another position would be the methoxyl group.
The irradiation of hydrogen δH 7.30, by NOEDIFF experiment, resulted in an increase of the doublet signal δH 7.97.The signals at δH 7.50 and δH 7,30 were increased when δH 7.97 was irradiated, indicating the proximity between the H-11 position in ring A and the hydrogen δH 7,30 present in the Cring.NOEDIFF experiments suggest that the bound of the methoxyl group in this substance occurs in position 2. Irradiation of the hydrogens of the methoxyl group in δH 4.20 increased the signal of the H-4 δH 7.75.All NOE experiments was compared with previous reported data [5].
These NMR data led to 2-methoxy-canthi-6-one alkaloid, but HRMS shows ion [M + H] + = 267.0733m/z with molecular formula C15H10N2O3, and diverged 16 m/z from the proposed alkaloid 2-methoxy-canthi-6-one.According to the NMR and MS data, the only possibility that justify this mass difference is the presence of an N-O -group at the structure.
The IR analysis confirms the N-O -bond in the intense absorption at 1303 cm -¹.It is a first report of spectroscopic data that shows the presence of alkaloid 2-methoxy-canthi-6-one N-oxide (3).NOESY, HMBC and HMQC were performed and the results are described at table 1 and figures 2 and 3. Peaks that corresponds to compounds 1 and 2 were analyzed by NMR and HRMS all data were compared to previous described data [6,7].
Other canthi-6-on alkaloids have been reported in the literature, among the structures most have substitutions at ring A, structural varieties with substitutions at the position 2 on ring C are uncommon.Although the substitution pattern is easily identified by ¹H NMR and NOE experiments can allow unambiguous determination of the substitution pattern in ring A and C [5], the complementary spectroscopic techniques as IR and HRMS allow the determination of the presence of N-oxide groups.

Conclusions
The first phytochemical study of S. bahiensis were possible identify 3 canthi-6-one alkaloids and the new one 2-methoxy-canthi-6-one N-oxide, previous studies shows the occurrence of alkaloids with N-oxide but without substitution at position 2.
Substitution pattern is easy to identify by ¹H NMR, but is necessary use different experiments to correctly determine the position of substituents at rings A and C, since studies of structure relationships have determined that different substitution patterns in these rings influence the pharmacological activity of these substances.