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Nitrophenyl dihydropyridine-derivatives from Seriphidium oliverianum
Phytochemistry Letters 21 (2017) 226–229
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Nitrophenyl dihydropyridine-derivatives from Seriphidium oliverianum a
b
a
c
MARK d
Liaquat Ali , Muhammad Imran Tousif , Naheed Riaz , Mamona Nazir , Hidayat Hussain , ⁎ Nusrat Shafiqe, Abdul Jabbara, Rasool Bakhsh Tareenf, Muhammad Saleema, a
Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, 63100-Bahawalpur, Pakistan Department of Chemistry, Dera Ghazi Khan Campus, University of Education Lahore, 32200-Dera Ghazi Khan, Pakistan Department of Chemistry, Government Sadiq College Women University, Bahawalpur, 63100-Bahawalpur, Pakistan d UoN Chair of Oman’s Medicinal Plants and Marine Natural Products, University of Nizwa, P.O. Box 33, Postal Code 616, Birkat Al Mauz, Nizwa, Oman e Department of Chemistry, Government College women University, Madina Town, Faisalabad-3800, Pakistan f Department of Botany, Baluchistan University Quetta, Pakistan b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Seriphidium oliverianum Secondary metabolites Dihydropyridine derivatives Structure elucidation
The chromatographic purification of the n-hexane soluble part of methanolic extract of Seriphidium oliverianum (J. Gay ex Besser) yielded six secondary metabolites that includes two new natural products; diethyl-2,6-diphenyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (1) and 2,6-diacetyl-3,5-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine (2) along with four known phytochemicals; tetradecyl ferulate (3), octadecyl coumarate (4), methyl coumarate (5) and methyl grevillate (6). The new compounds were characterized with the help of 1D-, 2D-NMR and high resolution mass spectrometric techniques, whereas, the known compounds were identified through 1D-NMR and mass spectrometry, and in comparison with the literature values. Compound 1 is synthetically known, however, it is first time reported from any natural source, whereas, compound 2 has never been reported from any other source before. All the isolates were screened in anti-urease and anti-glucosidase bioassay but were found inactive.
1. Introduction Seriphidium plants are hardy herbs and shrubs growing in temperate climates (Watson et al., 2002) and are used in folk medicine as antihelminthics (Deng et al., 2004). Strong aroma and bitter taste of several Seriphidium species have been attributed to the presence of sesquiterpene lactones and other terpenoids that discourage herbivores, however some Seriphidium plants are used as food (Shultz, 2006). Besides their use in medicine and as flavoring agents, they are also used to repel fleas and moths, and in brewing (Shultz, 2006). Antioxidant flavonoids and flavonoid glycosides have been reported as main secondary metabolites of various Seriphidium species (Shultz, 2006; Markham et al., 1978; Wagner et al., 1976; Kupchan et al., 1969; Mohamed et al., 2010). Seriphidium oliverianum is an important relative of this group of plants growing in Baluchistan, Pakistan. The leave extract of this plant is reported to contain several important antioxidants like gallic acid, kaempferol-3-O-β-D-glucopyranoside and 1,2,4,6-O-tetra-galloyl-β-Dglucopyranoside as major constituents (Ho et al., 2012). In continuation of our studies on the species of the genus Seriphidium (Shafiq et al., 2014, 2013, 2015), herein we report the isolation of six secondary
⁎
metabolites (Fig. 1) that includes two new natural products; diethyl2,6-diphenyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (1) and 2,6-diacetyl-3,5-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine (2) along with four known phytochemicals; tetradecyl ferulate (3) (Nidiry et al., 2016), octadecyl coumarate (4) (He et al., 2015), methyl coumarate (5) (Hooper et al., 1984) and methyl grevillate (6) (Ara et al., 1989) from the methanolic extract of Seriphidium oliverianum. 2. Results and discussion Pale yellow amorphous solid of compound 1 displayed IR absorption bands due to secondary amine (3355 cm−1), carbonyl group (1710 cm−1), olefinic system (1640 cm−1), aromatic moiety (1590, 1520, 1475 cm−1) and nitro group (1510, 1380 cm−1). Presence of the nitro group in 1 was substantiated through its reaction with ferrous hydroxide to get red brown precipitates of ferric hydroxide (Cekavicus et al., 1985), for nitro group oxidizes Fe(II) to Fe(III). The ESIMS in negative mode of 1 showed a pseudo-molecular ion peak at m/z 497 [MH]−, while the high resolution analysis of the same ion (m/z 497.1721 [M-H]−) depicted the molecular formula C29H26N2O6 with 18 DBE. The 1H NMR spectrum (Table 1) of 1 displayed discrete resonances
Corresponding author. E-mail addresses: [email protected], [email protected] (M. Saleem).
http://dx.doi.org/10.1016/j.phytol.2017.07.007 Received 22 March 2017; Received in revised form 20 June 2017; Accepted 7 July 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 21 (2017) 226–229
L. Ali et al.
Fig. 1. Structures of the compounds 1-6 isolated from hexane fraction of the methanolic extract of Seriphidium oliverianum. Table 1 1 H (dH) and Hz).
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attributed to two identical mono-substituted benzene rings. A broad singlet due to an exchangeable proton at δH 6.05 was assigned to a secondary amine, whereas, another proton resonance at δH 5.32 (1H, s) was correlated in HSQC spectrum with the carbon at δC 40.0. The NMR shifts of this methine revealed that it may be connected with several sp2 hybridized carbon atoms. Other proton resonances in the same spectrum at δH 3.90 (4H, q, J = 7.0 Hz) and 0.88 (6H, t, J = 7.0 Hz) could be assigned to chemically equivalent two ethoxy groups. This data indicated that compound 1 could have a part with symmetrical structural features, which was further substantiated due to 13C NMR spectral analysis (Table 1) of 1. The 13C NMR spectrum of 1 showed 16 signals at δC 166.4 (C), 149.3 (C), 148.5 (C), 146.4 (C), 136.1 (C), 134.3 (CH), 129.5 (CH), 129.0 (CH), 128.5 (CH), 128.0 (CH), 122.7 (CH), 121.7 (CH), 103.4 (C), 60.0 (CH2), 40.0 (CH) and 13.6 (CH3). Based on the COSY and HSQC experiments, the carbon resonances at δC 149.3, 148.5, 134.3, 129.0, 122.7 and 121.7 were indicative of 1,3-disubstituted benzene ring, whereas, the signals at δC 136.1, 129.5, 128.5 and 128.0 were diagnostic of two chemically equivalent mono-substituted benzene rings. Other carbon resonances at δC 166.4, 60.0 and 13.6 were attributed to two ethyl carboxylate units. The above identified parts (one nitro group, three aryl units and two carboxylate moieties) of compound 1 accommodated fifteen DBE. The remaining three DBE could be satisfied for a 1,4-dihydropyridine ring, which could be substantiated due to the carbon resonances at δC 146.4, 103.4 and 40.0. The HMBC correlation (Fig. 2) of the aromatic H-15 (δH 8.41) and H-19 (δH 7.92) with that of C-4 (δC 40.0) determined the attachment of nitrophenyl moiety at C-4 of the dihydropyridine system,
C (dC) NMR data of 1 and 2 (600 and 150 MHz respectively in CDCl3, J in
Position
1
1 2, 6 3, 5 4 7, 7′ 8, 8′ 9, 9′ 10, 10′ 11, 11′ 12, 12′ 13, 13′ 1′, 1′′ 2′, 2′′ 14 15 16 17 18 19
6.05, – – 5.32, – – 7.35, 7.37, 7.40, 7.37, 7.35, 3.90, 0.88, – 8.41, – 8.08, 7.49, 7.92,
2 s
s
dd (7.6, 1.7) m (overlapped) m (overlapped) m (overlapped) dd (7.6, 1.7) q (7.0) t (7.0) t (1.8) br. dd, (7.9, 1.6) t (7.9) br. d; (7.6)
– 146.4 103.4 40.0 166.4 136.1 128.0 128.5 129.5 128.5 128.0 60.0 13.6 148.5 122.7 149.3 121.7 129.0 134.3
5.91, – – 5.26, 2.35, – 2.25, – 8.01, – 7.99, – – 7.39, 7.65, – – – –
s
s s s d (1.6) dd (8.0, 1.8)
t (7.9) d (7.6)
– 143.6 113.6 39.5 20.6 196.9 30.4 147.9 122.3 148.6 121.7 – – 129.1 134.1 – – – –
for two aromatic systems; one set of signals at δH 8.41 (1H, t, J = 1.8 Hz), 8.08 (1H, br dd, J = 7.9, 1.6 Hz), 7.92 (1H, brd, J = 7.6 Hz) and 7.49 (1H, t, J = 7.9 Hz) was attested for a 1,3-disubstituted benzene ring due to the splitting pattern and COSY correlations, while other set of proton signals at δH 7.40 (2H, m, overlapped), 7.37 (4H, m, overlapped) and 7.35 (4H, dd, J = 7.6, 1.7 Hz) could be
Fig. 2. HMBC Correlations observed in the spectra of compounds 1 and 2.
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whereas, HMBC correlation of H-4 (δH 5.32) with the carbons at δC 146.4 (C-2, 6), 103.4 (C-3, 5) and 166.4 (C-7, 7′) helped to fix ethyl carboxylate units at C-3 and C-5 of dihydropyridine ring. The HMBC interaction of the singlet proton at δH 6.05 (NH) with the carbons at δC 146.4 (C-2, 6), 136.1 (C-8, 8′) and 103.4 (C-3, 5) and that of aromatic protons of mono-substituted benzene rings (δH 7.35, H-9, 9′, 13, 13′) with C-2 and C-6 confirmed the attachment of the mono-substituted benzene rings at C-2 and C-6 respectively. The nitro group could be fixed at C-16 due to the splitting pattern of the aromatic protons of disubstituted benzene ring and downfield shift (δC 149.3) of C-16. The above discussed data finally led to the structure of 1 as diethyl2,6-diphenyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate, which has been reported as a synthetic compound (Rowan and Holt, 1996; Sundar et al., 2006) but this is first report on its discovery as natural product. The IR spectrum of 2 was nearly identical to that of compound 1, whereas, the HR-ESIMS (m/z 313.1165 [M-H−]) of 2 depicted the molecular formula as C17H18N2O4 with 10 DBE, which indicated that compound 2 could be an analogue of 1 missing the two phenyl ring systems. The 1H NMR of 2 (Table 1) displayed signals in the aromatic region only for 1,3-disubstituted benzene ring that substantiated the above deduction of missing the two aromatic rings. Among other nuclei, the resonances of secondary amine and aliphatic methine proton were observed nearly at the same position (δH 5.91 and 5.26 respectively). However, the 1H NMR spectrum of 2 exhibited a different signal pattern in the upper filed region as it displayed only two methyl signals at δH 2.35 (6H, s) and 2.25 (6H, s) indicating the presence of symmetric part in 2. The 13C NMR spectrum (Table 1) of 2 displayed 12 carbon signals, which could be declared as two methyl, four methine and six quaternary carbon resonances. Although the chemical shifts of the benzene ring carbons were observed nearly at the same positions but there was significant difference in the carbon shifts of dihydropyridine ring (δC 143.6, 113.6 and 39.5) and in carbonyl carbon (δC 196.9). This data revealed that compound 2 is missing ethyl carboxylate moieties, and instead afforded acetyl groups (δC 196.9, 30.4), besides, it has two allelic methyl units (δC 20.6). The HMBC correlation (Fig. 2) of singlet methine proton (δH 5.26, H-4) with the carbons at δC 143.6 (C-2, 6), 113.6 (C-3, 5) and 20.6 (CH3-7, 7′) fixed the positions of two allelic methyl at C-3 and C-5 of dihydropyridine ring. Therefore, the two acetyl groups were placed at C-2 and C-6, which could further be confirmed with the HMBC correlations between the amine proton (δH 5.91) and C-8, 8′ (δC 196.9) and C-9, 9′ (δC 30.4). Above discussion and the combination of COSY, HSQC and HMBC spectral data led to the structure of compound 2 as 2,6diacetyl-3,5-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine, which is also a new natural product. Due to limited lab facilities, we could only perform urease and glucosidase enzyme inhibitory activities, where the compounds 1-6 were found inactive. However, in literature dihydropyridine derivatives (analogues of compounds 1 and 2) are known to possess calcium antagonistic activity as they inhibit the influx of Ca+2 ions through plasma membrane channels. Moreover, it is reported that compounds of this class are being used in the treatment of angina and hypertension (Rowan and Holt, 1996; Sundar et al., 2006). This is the first report on the discovery of nitophenyl dihydropyridine derivatives from any natural source. This discovery may prompt the medicinal chemists to investigate the species of the genus Seriphidium for their use as anti-hypertension and in coronary heart disease related treatments.
460 spectrophotometer. ESIMS, HR-ESIMS spectra were recorded on Jeol JMS-HX 110 spectrometer with data system. The 1H NMR, COSY, HSQC and HMBC spectra were recorded on Bruker AMX-600 instrument using TMS as an internal reference. The chemical shifts are reported in ppm (δ) while coupling constants (J) in Hz. The 13C NMR spectra were recorded on the same machine operating at a frequency of 150 MHz. Column chromatography was carried out using silica gel (EMerck, 70–230 and 230–400 mesh, Darmstadt, Germany). Aluminium sheets precoated with silica gel 60 F254 (0.2 mm thick; E-Merck, Darmstadt, Germany) were used for TLC to check the purity of the compounds. The TLC chromatograms were visualized under UV light (254 and 366 nm) followed by heating with ceric sulfate as spraying reagent. 3.2. Plant material The whole plant of Seriphidium oliverianum (J. Gay ex Besser) was collected from Quetta, Baluchistan in September 2012, and was identified by Prof. Dr. Rasool Bakhsh Tareen, Department of Botany, Baluchistan University, Quetta, where a voucher specimen (SO/RBT211-12) is deposited in the herbarium. 3.3. Extraction and isolation The shade dried plant (10 kg) was ground into coarse powder and was extracted (twice) with MeOH for one week. The extract was concentrated under reduced pressure to get a dark brown gummy mass (175 g), which was suspended in water (1.0 L) and was extracted with n-hexane followed by its extraction with EtOAc. The n-hexane fraction was chromatographed on silica gel column eluting with a gradient of nhexane, n-hexane:EtOAc, EtOAc, EtOAc:MeOH and MeOH to get six fractions (SO-1 to SO-6). The fraction SO-3 was further chromatographed on silica gel column eluting with a gradient of n-hexane: EtOAc to get five sub-fractions SO-3b1-SO-3b5. The sub-fraction SO-3b2 was further subjected to silica gel column chromatography eluting with nhexane:EtOAc (6:4) that yielded compounds 1 (25 mg) and 3 (51 mg). The sub-fraction SO-3b3 was further purified on silica gel column eluting with n-hexane:EtOAc (6:4) to get compound 2 (30 mg). The main fraction SO-4 was subjected to chromatography on silica gel column eluting with gradient of n-hexane:EtOAc to get 4 sub-fractions SO-4a-SO-4d. The sub-fraction SO-4a was further purified on silica gel column eluting with n-hexane:EtOAc (7:4) to get compound 4 (60 mg), while the sub-fraction SO-4d yielded compound 5 (48 mg) when passed with CHCl3:MeOH (5:5) through Sephadex LH-20 column. Another main fraction SO-2 was also subjected to silica gel column chromatography eluting with a gradient of n-hexane:EtOAc, that yielded a semipure fraction, which was washed with MeOH to get compound 6 (45 mg). 3.3.1. Diethyl-2,6-diphenyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5dicarboxylate (1) Yellow amorphous powder (25 mg); UV (Chloroform) λmax (log ε): 310 (2.88); IR (KBr pellet) νmax 3355, 1710 and 1640, 1590, 1520, 1510 and 1380 cm−1; for 1H and 13C NMR spectral data, see Table 1; ESI–MS: m/z 497 [M-H−]; HRESI–MS: m/z 497.1721 [M-H−] (calcd. 497.1713 for C29H25N2O6, led to the formula as C29H26N2O6). 3.3.2. 2,6-Diacetyl-3,5-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine (2) Yellowish amorphous powder (30 mg); UV (Chloroform) λmax (log ε): 265 (2.56); IR (KBr pellet) νmax 3365, 1680, 1635, 1605, 1525, 1512, 1386 and 1475 cm−1; for 1H and 13C NMR spectral data, see Table 1; ESI–MS: m/z 313 [M-H−]; HRESI–MS: m/z 313.1165 [M-H−] (calcd. 313.1188 for C17H17N2O4 led to the formula as C17H18N2O4).
3. Experimental 3.1. General experimental procedures The UV spectra were recorded in ethanol on a Hitachi UV-3200 Spectrometer, while IR experiments were performed on Shimadzu IR228
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1603–1615. Markham, K.R., Ternai, B., Stanley, R., Geiger, H., Mabry, T.J., 1978. Carbon-13 NMR studies of flavonoids-III: naturally occurring flavonoid glycosides and their acylated derivatives. Tetrahedron 34, 1389–1397. Mohamed, A.E.-H.H., Magdi, A.E.-S., Hegazy, M.E., Soleiman, E.H., Esmail, A.M., Naglaa, S.M., 2010. Chemical constituents and biological activities of Artemisia herba-alba. Rec. Nat. Prod. 4, 1–25. Nidiry, E.S.J., Ganeshan, G., Lokesha, A.N., Ramachandran, N., 2016. Additional studies on the antifungal activity of a methanol extract of Ipomoea carnea subsp. stulosa and octadecyl p-coumarates. Pharmacogn. Commn. 6, 148–151. Rowan, K.R., Holt, E.M., 1996. Dibutyl 2,6-dimethyi-4-(3-nitrophenyl)-l,4- dihydropyridine-3,5-dicarboxylate, diisobutyl 2,6-dimethyl-4-(3-nitrophenyl)-l,4-dihydropyridine-3,5-dicarboxylate and di-tert-butyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate. Acta Cryst. C52, 2207–2212. Shafiq, N., Riaz, N., Ahmed, S., Ashraf, M., Ejaz, S.A., Ahmed, I., Saleem, M., Tousif, M.I., Tareen, R.B., Jabbar, A., 2013. Bioactive phenolics from Seriphidium stenocephalum. J. Asian Nat. Prod. Res. 15, 286–293. Shafiq, N., Ali, L., Riaz, N., Yaqoob, A., Tareen, R.B., Saleem, M., Nasim, F.U.-H., Jabbar, A., Tousif, M.I., 2014. Isolation, characterization of flavonoids from Seriphidium oliverianum and their antioxidant and anti-urease activities. J. Chem. Soc. Pak. 36, 517–523. Shafiq, N., Ali, L., Riaz, N., Khatoon, T., Touseef, M.I., Jabbar, A., Tareen, R.B., Saleem, M., 2015. Further secondary metabolites from Seriphidium stenocephalum. J. Chem. Soc. Pak. 37, 704–712. Shultz, L.M., 2006. Artemisia linnaeus, sp. Flora of North America, FNA 19–21, 503–504 (http://www.efloras.org/florataxon.aspx?flora_id=1 & taxon_id=102682). Sundar, T.V., Parthasarathi, V., Narang, G., Bansal, R., Sridhar, B., 2006. Dimethyl 4-(4isopropylcarbonyloxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate. Acta Cryst. E62, o4001–o4003. Wagner, H., Chari, V.M., Sonnenbichler, J., 1976. 13C NMR-spektren natürlich vorkommender flavonoide. Tetrahedron Lett. 17, 1799–1802. Watson, L.E., Bates, P.L., Evans, T.M., Unwin, M.M., Estes, J.R., 2002. Molecular phylogeny of Subtribe Artemisiinae (Asteraceae), including Artemisia and its allied and segregate genera. Biomed. Centr. Evol. Biol. 2, 17–28.
Acknowledgement Muhammad Saleem and Naheed Riaz are highly obliged of the Alexander von Humboldt Foundation, Germany for its generous equipment support to their lab in Pakistan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.07.007. References Ara, I., Siddiqui, B.S., Faizi, S., Siddiqui, S., 1989. Diterpenoids from the stem bark of Azadirachta indica. Phytochemistry 28, 1177–1180. Cekavicus, B., Sausins, A., Zolotyabko, R.M., Duburs, G., 1985. From Latvijas PSR Zinatnu Akademijas Vestis. Kimijas Serija 1, 77–85. Deng, Y.R., Song, A.X., Wang, H.Q., 2004. Chemical components of Seriphidium santolium poljak. J. Chin. Chem. Soc. 51, 629–636. He, D., Simoneit Jernd, R.T., Jara, B., Jaffe, R., 2015. Gas chromatography mass spectrometry based profiling of alkyl coumarates and ferulates in two species of cattail (Typha domingensis P., and Typha latifolia L.). Phytochem. Lett. 13, 91–98. Ho, S.T., Tung, Y.T., Chen, Y.L., Zhao, Y.Y., Chung, J., Wu, J.H., 2012. Antioxidant activities and phytochemical study of leaf extracts from 18 indigenous tree species in Taiwan. Evid. Based Complement. Alternat. Med. 1–8. http://dx.doi.org/10.1155/ 2012/215959. Hooper, S.N., Jurgens, T., Chandler, R.F., Stevens, M.F.G., 1984. Methyl p-coumarate a cytotoxic constituent from comptonia peregrine. Phytochemistry 23, 2096–2097. Kupchan, S.M., Sigel, C.W., Hemingway, R.J., Knox, J.R., Udayamurthy, M.S., 1969. Tumor inhibitors-XXXII: cytotoxicflavones from Eupatorium species. Tetrahedron 25,
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Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Nitrophenyl dihydropyridine-derivatives from Seriphidium oliverianum a
b
a
c
MARK d
Liaquat Ali , Muhammad Imran Tousif , Naheed Riaz , Mamona Nazir , Hidayat Hussain , ⁎ Nusrat Shafiqe, Abdul Jabbara, Rasool Bakhsh Tareenf, Muhammad Saleema, a
Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, 63100-Bahawalpur, Pakistan Department of Chemistry, Dera Ghazi Khan Campus, University of Education Lahore, 32200-Dera Ghazi Khan, Pakistan Department of Chemistry, Government Sadiq College Women University, Bahawalpur, 63100-Bahawalpur, Pakistan d UoN Chair of Oman’s Medicinal Plants and Marine Natural Products, University of Nizwa, P.O. Box 33, Postal Code 616, Birkat Al Mauz, Nizwa, Oman e Department of Chemistry, Government College women University, Madina Town, Faisalabad-3800, Pakistan f Department of Botany, Baluchistan University Quetta, Pakistan b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Seriphidium oliverianum Secondary metabolites Dihydropyridine derivatives Structure elucidation
The chromatographic purification of the n-hexane soluble part of methanolic extract of Seriphidium oliverianum (J. Gay ex Besser) yielded six secondary metabolites that includes two new natural products; diethyl-2,6-diphenyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (1) and 2,6-diacetyl-3,5-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine (2) along with four known phytochemicals; tetradecyl ferulate (3), octadecyl coumarate (4), methyl coumarate (5) and methyl grevillate (6). The new compounds were characterized with the help of 1D-, 2D-NMR and high resolution mass spectrometric techniques, whereas, the known compounds were identified through 1D-NMR and mass spectrometry, and in comparison with the literature values. Compound 1 is synthetically known, however, it is first time reported from any natural source, whereas, compound 2 has never been reported from any other source before. All the isolates were screened in anti-urease and anti-glucosidase bioassay but were found inactive.
1. Introduction Seriphidium plants are hardy herbs and shrubs growing in temperate climates (Watson et al., 2002) and are used in folk medicine as antihelminthics (Deng et al., 2004). Strong aroma and bitter taste of several Seriphidium species have been attributed to the presence of sesquiterpene lactones and other terpenoids that discourage herbivores, however some Seriphidium plants are used as food (Shultz, 2006). Besides their use in medicine and as flavoring agents, they are also used to repel fleas and moths, and in brewing (Shultz, 2006). Antioxidant flavonoids and flavonoid glycosides have been reported as main secondary metabolites of various Seriphidium species (Shultz, 2006; Markham et al., 1978; Wagner et al., 1976; Kupchan et al., 1969; Mohamed et al., 2010). Seriphidium oliverianum is an important relative of this group of plants growing in Baluchistan, Pakistan. The leave extract of this plant is reported to contain several important antioxidants like gallic acid, kaempferol-3-O-β-D-glucopyranoside and 1,2,4,6-O-tetra-galloyl-β-Dglucopyranoside as major constituents (Ho et al., 2012). In continuation of our studies on the species of the genus Seriphidium (Shafiq et al., 2014, 2013, 2015), herein we report the isolation of six secondary
⁎
metabolites (Fig. 1) that includes two new natural products; diethyl2,6-diphenyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (1) and 2,6-diacetyl-3,5-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine (2) along with four known phytochemicals; tetradecyl ferulate (3) (Nidiry et al., 2016), octadecyl coumarate (4) (He et al., 2015), methyl coumarate (5) (Hooper et al., 1984) and methyl grevillate (6) (Ara et al., 1989) from the methanolic extract of Seriphidium oliverianum. 2. Results and discussion Pale yellow amorphous solid of compound 1 displayed IR absorption bands due to secondary amine (3355 cm−1), carbonyl group (1710 cm−1), olefinic system (1640 cm−1), aromatic moiety (1590, 1520, 1475 cm−1) and nitro group (1510, 1380 cm−1). Presence of the nitro group in 1 was substantiated through its reaction with ferrous hydroxide to get red brown precipitates of ferric hydroxide (Cekavicus et al., 1985), for nitro group oxidizes Fe(II) to Fe(III). The ESIMS in negative mode of 1 showed a pseudo-molecular ion peak at m/z 497 [MH]−, while the high resolution analysis of the same ion (m/z 497.1721 [M-H]−) depicted the molecular formula C29H26N2O6 with 18 DBE. The 1H NMR spectrum (Table 1) of 1 displayed discrete resonances
Corresponding author. E-mail addresses: [email protected], [email protected] (M. Saleem).
http://dx.doi.org/10.1016/j.phytol.2017.07.007 Received 22 March 2017; Received in revised form 20 June 2017; Accepted 7 July 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 21 (2017) 226–229
L. Ali et al.
Fig. 1. Structures of the compounds 1-6 isolated from hexane fraction of the methanolic extract of Seriphidium oliverianum. Table 1 1 H (dH) and Hz).
13
attributed to two identical mono-substituted benzene rings. A broad singlet due to an exchangeable proton at δH 6.05 was assigned to a secondary amine, whereas, another proton resonance at δH 5.32 (1H, s) was correlated in HSQC spectrum with the carbon at δC 40.0. The NMR shifts of this methine revealed that it may be connected with several sp2 hybridized carbon atoms. Other proton resonances in the same spectrum at δH 3.90 (4H, q, J = 7.0 Hz) and 0.88 (6H, t, J = 7.0 Hz) could be assigned to chemically equivalent two ethoxy groups. This data indicated that compound 1 could have a part with symmetrical structural features, which was further substantiated due to 13C NMR spectral analysis (Table 1) of 1. The 13C NMR spectrum of 1 showed 16 signals at δC 166.4 (C), 149.3 (C), 148.5 (C), 146.4 (C), 136.1 (C), 134.3 (CH), 129.5 (CH), 129.0 (CH), 128.5 (CH), 128.0 (CH), 122.7 (CH), 121.7 (CH), 103.4 (C), 60.0 (CH2), 40.0 (CH) and 13.6 (CH3). Based on the COSY and HSQC experiments, the carbon resonances at δC 149.3, 148.5, 134.3, 129.0, 122.7 and 121.7 were indicative of 1,3-disubstituted benzene ring, whereas, the signals at δC 136.1, 129.5, 128.5 and 128.0 were diagnostic of two chemically equivalent mono-substituted benzene rings. Other carbon resonances at δC 166.4, 60.0 and 13.6 were attributed to two ethyl carboxylate units. The above identified parts (one nitro group, three aryl units and two carboxylate moieties) of compound 1 accommodated fifteen DBE. The remaining three DBE could be satisfied for a 1,4-dihydropyridine ring, which could be substantiated due to the carbon resonances at δC 146.4, 103.4 and 40.0. The HMBC correlation (Fig. 2) of the aromatic H-15 (δH 8.41) and H-19 (δH 7.92) with that of C-4 (δC 40.0) determined the attachment of nitrophenyl moiety at C-4 of the dihydropyridine system,
C (dC) NMR data of 1 and 2 (600 and 150 MHz respectively in CDCl3, J in
Position
1
1 2, 6 3, 5 4 7, 7′ 8, 8′ 9, 9′ 10, 10′ 11, 11′ 12, 12′ 13, 13′ 1′, 1′′ 2′, 2′′ 14 15 16 17 18 19
6.05, – – 5.32, – – 7.35, 7.37, 7.40, 7.37, 7.35, 3.90, 0.88, – 8.41, – 8.08, 7.49, 7.92,
2 s
s
dd (7.6, 1.7) m (overlapped) m (overlapped) m (overlapped) dd (7.6, 1.7) q (7.0) t (7.0) t (1.8) br. dd, (7.9, 1.6) t (7.9) br. d; (7.6)
– 146.4 103.4 40.0 166.4 136.1 128.0 128.5 129.5 128.5 128.0 60.0 13.6 148.5 122.7 149.3 121.7 129.0 134.3
5.91, – – 5.26, 2.35, – 2.25, – 8.01, – 7.99, – – 7.39, 7.65, – – – –
s
s s s d (1.6) dd (8.0, 1.8)
t (7.9) d (7.6)
– 143.6 113.6 39.5 20.6 196.9 30.4 147.9 122.3 148.6 121.7 – – 129.1 134.1 – – – –
for two aromatic systems; one set of signals at δH 8.41 (1H, t, J = 1.8 Hz), 8.08 (1H, br dd, J = 7.9, 1.6 Hz), 7.92 (1H, brd, J = 7.6 Hz) and 7.49 (1H, t, J = 7.9 Hz) was attested for a 1,3-disubstituted benzene ring due to the splitting pattern and COSY correlations, while other set of proton signals at δH 7.40 (2H, m, overlapped), 7.37 (4H, m, overlapped) and 7.35 (4H, dd, J = 7.6, 1.7 Hz) could be
Fig. 2. HMBC Correlations observed in the spectra of compounds 1 and 2.
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whereas, HMBC correlation of H-4 (δH 5.32) with the carbons at δC 146.4 (C-2, 6), 103.4 (C-3, 5) and 166.4 (C-7, 7′) helped to fix ethyl carboxylate units at C-3 and C-5 of dihydropyridine ring. The HMBC interaction of the singlet proton at δH 6.05 (NH) with the carbons at δC 146.4 (C-2, 6), 136.1 (C-8, 8′) and 103.4 (C-3, 5) and that of aromatic protons of mono-substituted benzene rings (δH 7.35, H-9, 9′, 13, 13′) with C-2 and C-6 confirmed the attachment of the mono-substituted benzene rings at C-2 and C-6 respectively. The nitro group could be fixed at C-16 due to the splitting pattern of the aromatic protons of disubstituted benzene ring and downfield shift (δC 149.3) of C-16. The above discussed data finally led to the structure of 1 as diethyl2,6-diphenyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate, which has been reported as a synthetic compound (Rowan and Holt, 1996; Sundar et al., 2006) but this is first report on its discovery as natural product. The IR spectrum of 2 was nearly identical to that of compound 1, whereas, the HR-ESIMS (m/z 313.1165 [M-H−]) of 2 depicted the molecular formula as C17H18N2O4 with 10 DBE, which indicated that compound 2 could be an analogue of 1 missing the two phenyl ring systems. The 1H NMR of 2 (Table 1) displayed signals in the aromatic region only for 1,3-disubstituted benzene ring that substantiated the above deduction of missing the two aromatic rings. Among other nuclei, the resonances of secondary amine and aliphatic methine proton were observed nearly at the same position (δH 5.91 and 5.26 respectively). However, the 1H NMR spectrum of 2 exhibited a different signal pattern in the upper filed region as it displayed only two methyl signals at δH 2.35 (6H, s) and 2.25 (6H, s) indicating the presence of symmetric part in 2. The 13C NMR spectrum (Table 1) of 2 displayed 12 carbon signals, which could be declared as two methyl, four methine and six quaternary carbon resonances. Although the chemical shifts of the benzene ring carbons were observed nearly at the same positions but there was significant difference in the carbon shifts of dihydropyridine ring (δC 143.6, 113.6 and 39.5) and in carbonyl carbon (δC 196.9). This data revealed that compound 2 is missing ethyl carboxylate moieties, and instead afforded acetyl groups (δC 196.9, 30.4), besides, it has two allelic methyl units (δC 20.6). The HMBC correlation (Fig. 2) of singlet methine proton (δH 5.26, H-4) with the carbons at δC 143.6 (C-2, 6), 113.6 (C-3, 5) and 20.6 (CH3-7, 7′) fixed the positions of two allelic methyl at C-3 and C-5 of dihydropyridine ring. Therefore, the two acetyl groups were placed at C-2 and C-6, which could further be confirmed with the HMBC correlations between the amine proton (δH 5.91) and C-8, 8′ (δC 196.9) and C-9, 9′ (δC 30.4). Above discussion and the combination of COSY, HSQC and HMBC spectral data led to the structure of compound 2 as 2,6diacetyl-3,5-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine, which is also a new natural product. Due to limited lab facilities, we could only perform urease and glucosidase enzyme inhibitory activities, where the compounds 1-6 were found inactive. However, in literature dihydropyridine derivatives (analogues of compounds 1 and 2) are known to possess calcium antagonistic activity as they inhibit the influx of Ca+2 ions through plasma membrane channels. Moreover, it is reported that compounds of this class are being used in the treatment of angina and hypertension (Rowan and Holt, 1996; Sundar et al., 2006). This is the first report on the discovery of nitophenyl dihydropyridine derivatives from any natural source. This discovery may prompt the medicinal chemists to investigate the species of the genus Seriphidium for their use as anti-hypertension and in coronary heart disease related treatments.
460 spectrophotometer. ESIMS, HR-ESIMS spectra were recorded on Jeol JMS-HX 110 spectrometer with data system. The 1H NMR, COSY, HSQC and HMBC spectra were recorded on Bruker AMX-600 instrument using TMS as an internal reference. The chemical shifts are reported in ppm (δ) while coupling constants (J) in Hz. The 13C NMR spectra were recorded on the same machine operating at a frequency of 150 MHz. Column chromatography was carried out using silica gel (EMerck, 70–230 and 230–400 mesh, Darmstadt, Germany). Aluminium sheets precoated with silica gel 60 F254 (0.2 mm thick; E-Merck, Darmstadt, Germany) were used for TLC to check the purity of the compounds. The TLC chromatograms were visualized under UV light (254 and 366 nm) followed by heating with ceric sulfate as spraying reagent. 3.2. Plant material The whole plant of Seriphidium oliverianum (J. Gay ex Besser) was collected from Quetta, Baluchistan in September 2012, and was identified by Prof. Dr. Rasool Bakhsh Tareen, Department of Botany, Baluchistan University, Quetta, where a voucher specimen (SO/RBT211-12) is deposited in the herbarium. 3.3. Extraction and isolation The shade dried plant (10 kg) was ground into coarse powder and was extracted (twice) with MeOH for one week. The extract was concentrated under reduced pressure to get a dark brown gummy mass (175 g), which was suspended in water (1.0 L) and was extracted with n-hexane followed by its extraction with EtOAc. The n-hexane fraction was chromatographed on silica gel column eluting with a gradient of nhexane, n-hexane:EtOAc, EtOAc, EtOAc:MeOH and MeOH to get six fractions (SO-1 to SO-6). The fraction SO-3 was further chromatographed on silica gel column eluting with a gradient of n-hexane: EtOAc to get five sub-fractions SO-3b1-SO-3b5. The sub-fraction SO-3b2 was further subjected to silica gel column chromatography eluting with nhexane:EtOAc (6:4) that yielded compounds 1 (25 mg) and 3 (51 mg). The sub-fraction SO-3b3 was further purified on silica gel column eluting with n-hexane:EtOAc (6:4) to get compound 2 (30 mg). The main fraction SO-4 was subjected to chromatography on silica gel column eluting with gradient of n-hexane:EtOAc to get 4 sub-fractions SO-4a-SO-4d. The sub-fraction SO-4a was further purified on silica gel column eluting with n-hexane:EtOAc (7:4) to get compound 4 (60 mg), while the sub-fraction SO-4d yielded compound 5 (48 mg) when passed with CHCl3:MeOH (5:5) through Sephadex LH-20 column. Another main fraction SO-2 was also subjected to silica gel column chromatography eluting with a gradient of n-hexane:EtOAc, that yielded a semipure fraction, which was washed with MeOH to get compound 6 (45 mg). 3.3.1. Diethyl-2,6-diphenyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5dicarboxylate (1) Yellow amorphous powder (25 mg); UV (Chloroform) λmax (log ε): 310 (2.88); IR (KBr pellet) νmax 3355, 1710 and 1640, 1590, 1520, 1510 and 1380 cm−1; for 1H and 13C NMR spectral data, see Table 1; ESI–MS: m/z 497 [M-H−]; HRESI–MS: m/z 497.1721 [M-H−] (calcd. 497.1713 for C29H25N2O6, led to the formula as C29H26N2O6). 3.3.2. 2,6-Diacetyl-3,5-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine (2) Yellowish amorphous powder (30 mg); UV (Chloroform) λmax (log ε): 265 (2.56); IR (KBr pellet) νmax 3365, 1680, 1635, 1605, 1525, 1512, 1386 and 1475 cm−1; for 1H and 13C NMR spectral data, see Table 1; ESI–MS: m/z 313 [M-H−]; HRESI–MS: m/z 313.1165 [M-H−] (calcd. 313.1188 for C17H17N2O4 led to the formula as C17H18N2O4).
3. Experimental 3.1. General experimental procedures The UV spectra were recorded in ethanol on a Hitachi UV-3200 Spectrometer, while IR experiments were performed on Shimadzu IR228
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