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Haplophytin-A and B: the alkaloidal constituents of Haplophyllum acutifolium
Phytochemistry 57 (2001) 1277–1280 www.elsevier.com/locate/phytochem
Haplophytin-A and B: the alkaloidal constituents of Haplophyllum acutifolium Muhammad Shaiq Alia,*, Muhammad Kashif Perveza, Muhammad Saleema, Rasool Bakhsh Tareenb a
H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan b Department of Botany, Baluchistan University, Quetta (Baluchistan), Pakistan Received 13 September 2000; received in revised form 30 November 2000
Dedicated to my PhD research supervisor, Professor Viqar Uddin Ahmad on his retirement
Abstract During the phytochemical investigation of Haplophyllum acutifolium, two alkaloids named haplophytin-A (1) and B (2) have been obtained. In addition, some known constituents: flindersine (3), kusunokinin (4), b-sitosterol, oleanolic acid, cholesterol and hexadecanoic acid, have also been obtained. The known constituents have never been obtained from this source. The structures of all the isolated constituents were elucidated by spectroscopic means. However, the structures of constituents 1 and 2 were further confirmed through HMBC technique. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Haplophyllum acutifolium; Rutaceae; Haplophytin-A; Haplophytin-B; Structure elucidation; Spectroscopy
1. Introduction The genus Haplophyllum belongs to the family Rutaceae having about 70 species distributed in the Mediterranean countries. Out of 70, seven are found in Pakistan. Chemical literature survey reveals the presence of lignans (Gozler et al., 1996; Evcium et al., 1986; Gozler and Gozler, 1984), lignan-glycosides (Gozler et al., 1994), coumarins (Ulubelen et al., 1993), sterols, flavonoids (Yuldasher et al., 1987), several classes of alkaloids [furoquinoline (Gozler et al., 1996), quinoline (Sheriha et al., 1987), pyranoquinoline (Ulubelen, 1985) and tetrahydrofuroquinoline (Rozsa et al., 1986)] in various species of the genus Haplophyllum. Several biologically active secondary metabolites have been detected from various Haplophyllum species (Sheriha et al., 1987). The presence of variety of biologically active chemical constituents form the members of the genus Haplophyllum motivated us to start our phytochemical investigation on H. acutifolium. In the present communication, we wish to describe the isolation and characterization of two alkaloids named haplophytin-A and B, and several * Corresponding author. Tel.: +92-21-496-8497; fax: +92-21-4963373. E-mail address: [email protected] (M.S. Ali).
known constituents [flindersine (3), kusunokinin (4), bsitosterol, oleanolic acid, cholesterol and hexadecanoic acid] from the titled source.
2. Results and discussion Haplophytin-A (1) was isolated from the methanol soluble part of Haplophyllum acutifolium. Lack of optical rotation means that there is probably no chirality in the molecule. The compound 1 gave positive response with UV light (254 nm) and Dragendroff’s reagent confirming the presence of conjugated/aromatic system and nitrogen atom, respectively. The presence of unsaturation was further determined through infra-red spectrum which showed an absorption at 1500 cm1 (C¼C) together with other absorption bands at 3260 and 1660 cm1 due to the N–H and conjugated amide-carbonyl, respectively. The molecular weight of 1 was confirmed with the help of FDMS and found 257 a.m.u. The formula of the corresponding peak was depicted from HR– EIMS as C15H15NO3. The proton NMR of 1 showed the presence of three quaternary methyl signals, out of them one appeared at 3.90 and the remaining two at 1.51 as a common signal of six protons. The spectrum did not contain any
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00188-1
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methylene however, the same spectrum showed five downfield methine signals at 7.26 (dd, J=8.8, 2.8 Hz, H-6), 7.10 (d, J=8.8 Hz, H-7), 7.25 (dd, 8.8, 2.8 Hz, H8), 5.54 (d) and 6.76 (d). The two upfield methine signals at 5.54 and 6.76 having the same magnitude of coupling constant (J=9.9 Hz) confirming the vicinal orientation attested for C-11 and C-12. The chemical shifts of remaining three methine signals at 7.26, 7.10 and 7.25 concluded the presence of aromatic ring in the molecule. The multiplicities of these three methine signals and their corresponding coupling constants showed that they are arranged in the manner that the H-6 and H-8 are ortho to H-7. The methoxyl moiety could be fixed either at C-5 or C-8 which was partially confirmed through HMBC experiment (Fig. 1) and finally, by compression with the data of closely related published compounds (Huffman and Hau, 1972). The broad-band spectrum of 1 displayed 15 carbon signal which were sorted out into three methyls, five methines and remaining seven quaternary carbons. The chemical shift of methoxyl moiety ( 55.7) inferred the direct attachment of this moiety to aromatic ring. The position of this methoxyl moiety at C-5 was determined through NOE experiment. Upon irradiation at 1.51 (H-14, H-15), a 33 and 16% NOE effect of the signals of H-12 and OMe, respectively, was observed. This 16% NOE effect would not have been observed if the methoxyl moiety had been situated at C-8. The other two quaternary methyls (C-14 and C-15) appeared at 28.0, found to be attached with a quaternary carbon connected to the electron withdrawing hetero-atom and thus assigned to C-13 ( 79.0). The most downfield signal at 162.4 was due to the conjugated amide whereas the second quaternary downfield signal at 155.0 was due to the carbon at which methoxyl moieties is attached (C-5). The two methine signals of the ring having the oxygen as an hetero-atom appeared at 126.2 and 117.5 due to C-11 and C-12, respectively. The remaining three aromatic methine signals were observed in the carbon spectrum at their usual positions ( 103.5, 120.5 and 117.4). With the help of spectral evidences given earlier, the structure of the discussed compound was assigned as 1
Fig. 1. HMBC connectivities.
and named haplophytin-A which is a new addition in the alkaloidal constituents of Haplophyllum acutifolium. Although similar types of compounds are already reported in the literature (Huffman and Hau, 1972) but having no methoxyl at C-5. Haplophytin-B (2) was obtained from the same extract with the elution of 45% ethyl acetate in hexane. It showed positive UV response confirming the presence of conjugated/aromatic system in the molecule. The presence of unsaturation was supported by the absorption at 1500 cm1 (C¼C) in the IR spectrum which also showed an intense absorption band at 3320 cm1 due to the hydroxyl function in the molecule. The molecular mass of 2 was determined through FDMS and found 347 a.m.u. The formula of this peak was observed as C18H21NO6 with the aid of HRMS. The proton spectrum of 2 exhibited four methyl resonances, out of them two at 4.47 and 4.10 due to the methoxyl moieties and their chemical shifts suggested direct attachment to the aromatic system. The remaining two quaternary methyl signals appeared at 1.29 and 1.25 (H-40 and H-50 ). The molecule contains only one methylene appeared as two separate sets of doubledoublets at 4.13 (J=9.9, 8.0 Hz, Ha-0 1) and 3.81 (J=8.0, 2.5 Hz, Hb-0 1). The same spectrum showed four downfield methine signals at 8.01 (d, J=9.4 Hz, H-5), 7.34 (d, J=9.4 Hz, H-6), 7.73 (d, J=2.8 Hz, H-a) and 7.30 (d, J=2.8 Hz, H-b) and an upfield carbinylic proton at 4.49 (H-0 2) as a double-doublet (J=9.9, 2.5 Hz). The signals at 8.01 and 7.34 and their same coupling constant confirmed their ortho arrangement and thus assigned to H-5 and H-6, respectively. The methine signal at 7.73 showed the presence of an electron withdrawing hetero-atom in the vicinity and attested for H-a. With the help of coupling constant of H-a (J=2.8 Hz) the H-b was located in the spectrum at 7.30. The broad-band spectrum of haplophytin-B exhibited 18 carbon signals which were resolved into four methyls, one methylene, five methines and remaining eight quaternary carbon atoms. Out of four methyl signals, two were at 60.0 and 61.9 due to the methoxyl moieties in the molecule and their positions were fixed through HMBC experiment (Fig. 2) and literature values (Bessonova and Yunusov, 1986; Rasulova et al., 1987). The remaining two methyl signals appeared at 25.0 and 26.9 due to the C-40 and C-50 , respectively. The only one methylene (C-10 ) present in the molecule resonated at 72.5. An upfield methine signal resonated at 77.8 corresponded to the carbinylic carbon. The molecule contains four downfield methine signals at 119.5 (C-5), 114.0 (C-6), 144.3 (C-a) and 106.5 (C-b). The most downfield methine signal at 144.3 was due to the direct attachment to an hetero-atom (oxygen). A hydroxyl function attached to the quaternary carbon at which two quaternary methyls are attached appeared at 72.8.
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Fig. 2. HMBC connectivities.
Three more quaternary carbons resonated at 153.9 (C-4), 142.1 (C-8) and 142.9 (C-7) were due to the two methoxyl and a side chain containing carbons, respectively. The position of side chain at C-7 was determined through HMBC connectivities (Fig. 2) and n.O.e measurements. Upon irradiation at 4.10 (C-8 OMe), a 29 and 13% NOE effect of the signals of H-20 and H-10 , respectively, was observed suggested the attachment of side chain at C-3. Such a type of skeleton is already reported in literature with either different side chains or sugar units (Bessonova and Yunusov, 1986; Rasulova et al., 1987) and therefore, the previously discussed compound is assigned as 2 and named haplophytin-B. This compound is also a new addition in the alkaloidal constituents of H. acutifolium. In addition to 1 and 2, six more known constituents have been detected which have never been reported so far from our investigated source and their data are given in Section 3. The new constituents, haplophytin-A and B were found totally inactive against various tested bacteria and fungi.
3. Experimental 3.1. General 3.3. Extraction and isolation The 1H and 13C NMR spectra were recorded at 500, 400, 300 and 75,100, 125 MHz, respectively, on Bru¨ker AM-500, AM-400 and AM-300 in CDCl3. 3.2. Collection and identification The plant material was collected from Quetta (Pakistan) in May 1999 and identified by Dr. R.B. Tareen, Department of Botany, Baluchistan University, Quetta (Baluchistan), where the voucher specimen has been deposited in the herbarium.
The collected plant material (15 kg) was dried under shade and chopped into small pieces. The dried and chopped material (8 kg) was soaked in MeOH (14 l) for a period of 10 days. The percolation was repeated three times. The MeOH was evaporated under reduced temperature (28 C) and the gummy material thus obtained (276 g) was subjected to silica gel column chromatography using hexane, hexane–ethyl acetate, ethyl acetate, ethyl acetate–methanol and finally, pure methanol as mobile phase.
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The fraction obtained with 15% ethyl acetate in hexane showed a strong UV-active spot on TLC with some minor impurities which on washing with methanol yielded 1 as a white powder (13 mg). The same UV active spot on spraying with Dragendroff’s reagent changed into an orange spot confirming the presence of nitrogen in the molecule. Haplophytin-A (1): m.p.: 209–210.5 C; [a]D: 0 (CHCl3, c 0.506 ); UV lmax CHCl3 nm (log "): 244 (7.92); IR (CHCl3): 3260 (N–H), 1660 (C¼O), 1500 (C¼C) cm1; EI–MS: m/z 257 [M+], 242 [M–Me]+ (100%), 199, 121, 106; FDMS: m/z 257; HR–MS: m/z 257.9968 (C15H15 NO3 requires m/z 257.10518); 1H NMR (CDCl3, 400 MHz): 7.26 (1H, dd, J=8.8, 2.8 Hz, H-6), 7.25 (H, dd, J=8.8, 2.8 Hz, H-8), 7.10 (1H, d, J=8.8 Hz,H-7), 6.76 (1H, d, J=9.9 Hz, H-12), 5.54 (1H, d, J=9.9 Hz, H-11), 3.90 (3H, s, OMe) and 1.51 (6H, s, H-14, 15); 13C NMR (CDCl3, 100 MHz): 162.4 (C-2), 115.7 (C-3), 156.0 (C-4), 155.0 (C-5), 103.5 (C-6), 120.5 (C-7), 117.4 (C-8), 132.9 (C-9), 106.5 (C-10), 126.2 (C-11), 117.5 (C-12), 79.0 (C13), 28.0 (C-14, 15) and 55.7 (OMe); HMBC: see Fig. 1. Elution with 45% ethyl acetate in hexane from the same column afforded another UV and Dragendroff’s reagent positive sample as a dirty white powder (16.5 mg) which on washing with a 1:1 mixture of chloroform and hexane yielded 2. Haplophytin-B (2): mp: 151.5–153 C; [a]D: +63.6 (MeOH, c 0.33 ); UV lmax CD3OD nm (log "): 251 (5.16); IR (KBr): 3320 (OH), 1500 (C=C) cm1; EI– MS: m/z 347 [M]+, 287, 227 [M–side chain+] (100%), 216; FDMS: m/z 347; HR–MS: m/z 347.13711 (C18H21N O6 requires m/z 347.13687); 1H NMR (CD3OD, 500 MHz): 8.01 (1H, d, J=9.4 Hz, H-5), 7.73 (1H, d, J=2.8 Hz, H-a), 7.34 (1H, d, J=9.4 Hz, H6), 7.30 (1H, d, J=2.8 Hz, H-b), 4.49 (1H, dd, J=9.9, 2.5 Hz, H-0 2), 4.47 (3H, s, C-4 OMe), 4.13 (1H, ds, J=9.9, 8.0 Hz, Ha-0 1), 4.10 (3H, s, C-8 OMe), 3.81 (1H, ds, J=8.0, 2.5 Hz, Hb-0 1), 1.29 (3H, s, H-0 4), 1.25 (3H, s, H-0 5); 13C NMR (CD3OD, 125 M Hz): 165.8 (C-2),
115.9 (C-3), 153.9 (C-4), 119.5 (C-5), 114.0 (C-6), 142.9 (C-7), 142.1 (C-8), 159.0 (C-9), 103.3 (C-10), 144.3 (Ca), 106.5 (C-b), 61.9 (C-8 OMe), 60.0 (C-4 OMe), 72.5 (C-0 1), 77.8 (C-0 2), 72.8 (C-0 3), 25.0 and 26.9 (C-0 4, C-0 5); HMBC: see Fig. 2.
References Bessonova, I.A., Yunusov, S.Yu., 1986. Alkaloids of the roots of the Haplophyllum obtusifolium. Chemistry of Natural Products 22 (6), 684–686. Evcium, U., Gozler, B., Freyer, A.J., Shamma, M., 1986. Haplomyrtin and () haplomyrfolin: two lignans from Haplophyllum myrtifolium. Phytochemistry 25, 1949–1951. Gozler, B., Gozler, T., Saglam, H., Hesse, M., 1996. Minor lignans from Haplophyllum cappadocicum. Phytochemistry 42 (3), 689–693. Gozler, B., Onur, M.A., Gozler, T., Kadan, G., Hesse, M., 1994. Lignans and lignan glycosides from Haplophyllum cappadocicum. Phytochemistry 37 (6), 1693–1998. Gozler, B., Rentsch, D., Gozler, T., Unver, N., Hesse, M., 1996. Lignans, alkaloids and coumarins from Haplophyllum vulcanicum. Phytochemistry 42 (3), 695–699. Gozler, T., Gozler, B., 1984. Konyanin: a new lignan from Haplophyllum vulcanicum. Tetrahedron 40, 1145–1150. Huffman, J.W., Hsu, T.M., 1972. A one step synthesis of flindersine. Tetrahedron Lett., 141–143. Rasulova, Kh.A., Bessonova, I.A., Yagudaev, M.R., Yunusov, S.Yu, 1987. Haplosinine — a new furanoquinoline glycoalkaloid. Chemistry of Natural Products 23 (6), 731–734. Rozsa, Zs., Rabik, M., Szendrei, K., Kalman, A., Argay, Gy., Pelczer, I., Aynechi, M., Meste, M., Reisch, J., 1986. Dihydroperfamine, an alkaloid from Haplophyllum glabrinum. Phytochemistry 25 (8), 2005–2007. Sheriha, G.M., Abouamer, K., Elshtaiwi, B.Z., Ashour, A.S., Abed, F., Alhallaq, H.H., 1987. Quinoline alkaloids and cytotoxic lignans from Haplophyllum tuberculatum. Phytochemistry 26, 3339–3341. Ulubelen, A., Mericli, A.H., Mericli, F., Sonhez, U., Ilarslan, R., 1993. Alkaloids and coumarins from Haplophyllum thesioides. Nat. Prod. Lett. 1 (4), 269–272. Ulubelen, A., 1985. Alkaloids from Haplophyllum buxbaumii. Phytochemistry 24 (2), 372–374. Yuldasher, M.P., Batirov, E.Kh., Malikov, V.M., 1987. Flavonoids of some plants of the genus Haplophyllum. Kim. Prir. Soendin 3, 452– 453.
Haplophytin-A and B: the alkaloidal constituents of Haplophyllum acutifolium Muhammad Shaiq Alia,*, Muhammad Kashif Perveza, Muhammad Saleema, Rasool Bakhsh Tareenb a
H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan b Department of Botany, Baluchistan University, Quetta (Baluchistan), Pakistan Received 13 September 2000; received in revised form 30 November 2000
Dedicated to my PhD research supervisor, Professor Viqar Uddin Ahmad on his retirement
Abstract During the phytochemical investigation of Haplophyllum acutifolium, two alkaloids named haplophytin-A (1) and B (2) have been obtained. In addition, some known constituents: flindersine (3), kusunokinin (4), b-sitosterol, oleanolic acid, cholesterol and hexadecanoic acid, have also been obtained. The known constituents have never been obtained from this source. The structures of all the isolated constituents were elucidated by spectroscopic means. However, the structures of constituents 1 and 2 were further confirmed through HMBC technique. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Haplophyllum acutifolium; Rutaceae; Haplophytin-A; Haplophytin-B; Structure elucidation; Spectroscopy
1. Introduction The genus Haplophyllum belongs to the family Rutaceae having about 70 species distributed in the Mediterranean countries. Out of 70, seven are found in Pakistan. Chemical literature survey reveals the presence of lignans (Gozler et al., 1996; Evcium et al., 1986; Gozler and Gozler, 1984), lignan-glycosides (Gozler et al., 1994), coumarins (Ulubelen et al., 1993), sterols, flavonoids (Yuldasher et al., 1987), several classes of alkaloids [furoquinoline (Gozler et al., 1996), quinoline (Sheriha et al., 1987), pyranoquinoline (Ulubelen, 1985) and tetrahydrofuroquinoline (Rozsa et al., 1986)] in various species of the genus Haplophyllum. Several biologically active secondary metabolites have been detected from various Haplophyllum species (Sheriha et al., 1987). The presence of variety of biologically active chemical constituents form the members of the genus Haplophyllum motivated us to start our phytochemical investigation on H. acutifolium. In the present communication, we wish to describe the isolation and characterization of two alkaloids named haplophytin-A and B, and several * Corresponding author. Tel.: +92-21-496-8497; fax: +92-21-4963373. E-mail address: [email protected] (M.S. Ali).
known constituents [flindersine (3), kusunokinin (4), bsitosterol, oleanolic acid, cholesterol and hexadecanoic acid] from the titled source.
2. Results and discussion Haplophytin-A (1) was isolated from the methanol soluble part of Haplophyllum acutifolium. Lack of optical rotation means that there is probably no chirality in the molecule. The compound 1 gave positive response with UV light (254 nm) and Dragendroff’s reagent confirming the presence of conjugated/aromatic system and nitrogen atom, respectively. The presence of unsaturation was further determined through infra-red spectrum which showed an absorption at 1500 cm1 (C¼C) together with other absorption bands at 3260 and 1660 cm1 due to the N–H and conjugated amide-carbonyl, respectively. The molecular weight of 1 was confirmed with the help of FDMS and found 257 a.m.u. The formula of the corresponding peak was depicted from HR– EIMS as C15H15NO3. The proton NMR of 1 showed the presence of three quaternary methyl signals, out of them one appeared at 3.90 and the remaining two at 1.51 as a common signal of six protons. The spectrum did not contain any
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methylene however, the same spectrum showed five downfield methine signals at 7.26 (dd, J=8.8, 2.8 Hz, H-6), 7.10 (d, J=8.8 Hz, H-7), 7.25 (dd, 8.8, 2.8 Hz, H8), 5.54 (d) and 6.76 (d). The two upfield methine signals at 5.54 and 6.76 having the same magnitude of coupling constant (J=9.9 Hz) confirming the vicinal orientation attested for C-11 and C-12. The chemical shifts of remaining three methine signals at 7.26, 7.10 and 7.25 concluded the presence of aromatic ring in the molecule. The multiplicities of these three methine signals and their corresponding coupling constants showed that they are arranged in the manner that the H-6 and H-8 are ortho to H-7. The methoxyl moiety could be fixed either at C-5 or C-8 which was partially confirmed through HMBC experiment (Fig. 1) and finally, by compression with the data of closely related published compounds (Huffman and Hau, 1972). The broad-band spectrum of 1 displayed 15 carbon signal which were sorted out into three methyls, five methines and remaining seven quaternary carbons. The chemical shift of methoxyl moiety ( 55.7) inferred the direct attachment of this moiety to aromatic ring. The position of this methoxyl moiety at C-5 was determined through NOE experiment. Upon irradiation at 1.51 (H-14, H-15), a 33 and 16% NOE effect of the signals of H-12 and OMe, respectively, was observed. This 16% NOE effect would not have been observed if the methoxyl moiety had been situated at C-8. The other two quaternary methyls (C-14 and C-15) appeared at 28.0, found to be attached with a quaternary carbon connected to the electron withdrawing hetero-atom and thus assigned to C-13 ( 79.0). The most downfield signal at 162.4 was due to the conjugated amide whereas the second quaternary downfield signal at 155.0 was due to the carbon at which methoxyl moieties is attached (C-5). The two methine signals of the ring having the oxygen as an hetero-atom appeared at 126.2 and 117.5 due to C-11 and C-12, respectively. The remaining three aromatic methine signals were observed in the carbon spectrum at their usual positions ( 103.5, 120.5 and 117.4). With the help of spectral evidences given earlier, the structure of the discussed compound was assigned as 1
Fig. 1. HMBC connectivities.
and named haplophytin-A which is a new addition in the alkaloidal constituents of Haplophyllum acutifolium. Although similar types of compounds are already reported in the literature (Huffman and Hau, 1972) but having no methoxyl at C-5. Haplophytin-B (2) was obtained from the same extract with the elution of 45% ethyl acetate in hexane. It showed positive UV response confirming the presence of conjugated/aromatic system in the molecule. The presence of unsaturation was supported by the absorption at 1500 cm1 (C¼C) in the IR spectrum which also showed an intense absorption band at 3320 cm1 due to the hydroxyl function in the molecule. The molecular mass of 2 was determined through FDMS and found 347 a.m.u. The formula of this peak was observed as C18H21NO6 with the aid of HRMS. The proton spectrum of 2 exhibited four methyl resonances, out of them two at 4.47 and 4.10 due to the methoxyl moieties and their chemical shifts suggested direct attachment to the aromatic system. The remaining two quaternary methyl signals appeared at 1.29 and 1.25 (H-40 and H-50 ). The molecule contains only one methylene appeared as two separate sets of doubledoublets at 4.13 (J=9.9, 8.0 Hz, Ha-0 1) and 3.81 (J=8.0, 2.5 Hz, Hb-0 1). The same spectrum showed four downfield methine signals at 8.01 (d, J=9.4 Hz, H-5), 7.34 (d, J=9.4 Hz, H-6), 7.73 (d, J=2.8 Hz, H-a) and 7.30 (d, J=2.8 Hz, H-b) and an upfield carbinylic proton at 4.49 (H-0 2) as a double-doublet (J=9.9, 2.5 Hz). The signals at 8.01 and 7.34 and their same coupling constant confirmed their ortho arrangement and thus assigned to H-5 and H-6, respectively. The methine signal at 7.73 showed the presence of an electron withdrawing hetero-atom in the vicinity and attested for H-a. With the help of coupling constant of H-a (J=2.8 Hz) the H-b was located in the spectrum at 7.30. The broad-band spectrum of haplophytin-B exhibited 18 carbon signals which were resolved into four methyls, one methylene, five methines and remaining eight quaternary carbon atoms. Out of four methyl signals, two were at 60.0 and 61.9 due to the methoxyl moieties in the molecule and their positions were fixed through HMBC experiment (Fig. 2) and literature values (Bessonova and Yunusov, 1986; Rasulova et al., 1987). The remaining two methyl signals appeared at 25.0 and 26.9 due to the C-40 and C-50 , respectively. The only one methylene (C-10 ) present in the molecule resonated at 72.5. An upfield methine signal resonated at 77.8 corresponded to the carbinylic carbon. The molecule contains four downfield methine signals at 119.5 (C-5), 114.0 (C-6), 144.3 (C-a) and 106.5 (C-b). The most downfield methine signal at 144.3 was due to the direct attachment to an hetero-atom (oxygen). A hydroxyl function attached to the quaternary carbon at which two quaternary methyls are attached appeared at 72.8.
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Fig. 2. HMBC connectivities.
Three more quaternary carbons resonated at 153.9 (C-4), 142.1 (C-8) and 142.9 (C-7) were due to the two methoxyl and a side chain containing carbons, respectively. The position of side chain at C-7 was determined through HMBC connectivities (Fig. 2) and n.O.e measurements. Upon irradiation at 4.10 (C-8 OMe), a 29 and 13% NOE effect of the signals of H-20 and H-10 , respectively, was observed suggested the attachment of side chain at C-3. Such a type of skeleton is already reported in literature with either different side chains or sugar units (Bessonova and Yunusov, 1986; Rasulova et al., 1987) and therefore, the previously discussed compound is assigned as 2 and named haplophytin-B. This compound is also a new addition in the alkaloidal constituents of H. acutifolium. In addition to 1 and 2, six more known constituents have been detected which have never been reported so far from our investigated source and their data are given in Section 3. The new constituents, haplophytin-A and B were found totally inactive against various tested bacteria and fungi.
3. Experimental 3.1. General 3.3. Extraction and isolation The 1H and 13C NMR spectra were recorded at 500, 400, 300 and 75,100, 125 MHz, respectively, on Bru¨ker AM-500, AM-400 and AM-300 in CDCl3. 3.2. Collection and identification The plant material was collected from Quetta (Pakistan) in May 1999 and identified by Dr. R.B. Tareen, Department of Botany, Baluchistan University, Quetta (Baluchistan), where the voucher specimen has been deposited in the herbarium.
The collected plant material (15 kg) was dried under shade and chopped into small pieces. The dried and chopped material (8 kg) was soaked in MeOH (14 l) for a period of 10 days. The percolation was repeated three times. The MeOH was evaporated under reduced temperature (28 C) and the gummy material thus obtained (276 g) was subjected to silica gel column chromatography using hexane, hexane–ethyl acetate, ethyl acetate, ethyl acetate–methanol and finally, pure methanol as mobile phase.
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The fraction obtained with 15% ethyl acetate in hexane showed a strong UV-active spot on TLC with some minor impurities which on washing with methanol yielded 1 as a white powder (13 mg). The same UV active spot on spraying with Dragendroff’s reagent changed into an orange spot confirming the presence of nitrogen in the molecule. Haplophytin-A (1): m.p.: 209–210.5 C; [a]D: 0 (CHCl3, c 0.506 ); UV lmax CHCl3 nm (log "): 244 (7.92); IR (CHCl3): 3260 (N–H), 1660 (C¼O), 1500 (C¼C) cm1; EI–MS: m/z 257 [M+], 242 [M–Me]+ (100%), 199, 121, 106; FDMS: m/z 257; HR–MS: m/z 257.9968 (C15H15 NO3 requires m/z 257.10518); 1H NMR (CDCl3, 400 MHz): 7.26 (1H, dd, J=8.8, 2.8 Hz, H-6), 7.25 (H, dd, J=8.8, 2.8 Hz, H-8), 7.10 (1H, d, J=8.8 Hz,H-7), 6.76 (1H, d, J=9.9 Hz, H-12), 5.54 (1H, d, J=9.9 Hz, H-11), 3.90 (3H, s, OMe) and 1.51 (6H, s, H-14, 15); 13C NMR (CDCl3, 100 MHz): 162.4 (C-2), 115.7 (C-3), 156.0 (C-4), 155.0 (C-5), 103.5 (C-6), 120.5 (C-7), 117.4 (C-8), 132.9 (C-9), 106.5 (C-10), 126.2 (C-11), 117.5 (C-12), 79.0 (C13), 28.0 (C-14, 15) and 55.7 (OMe); HMBC: see Fig. 1. Elution with 45% ethyl acetate in hexane from the same column afforded another UV and Dragendroff’s reagent positive sample as a dirty white powder (16.5 mg) which on washing with a 1:1 mixture of chloroform and hexane yielded 2. Haplophytin-B (2): mp: 151.5–153 C; [a]D: +63.6 (MeOH, c 0.33 ); UV lmax CD3OD nm (log "): 251 (5.16); IR (KBr): 3320 (OH), 1500 (C=C) cm1; EI– MS: m/z 347 [M]+, 287, 227 [M–side chain+] (100%), 216; FDMS: m/z 347; HR–MS: m/z 347.13711 (C18H21N O6 requires m/z 347.13687); 1H NMR (CD3OD, 500 MHz): 8.01 (1H, d, J=9.4 Hz, H-5), 7.73 (1H, d, J=2.8 Hz, H-a), 7.34 (1H, d, J=9.4 Hz, H6), 7.30 (1H, d, J=2.8 Hz, H-b), 4.49 (1H, dd, J=9.9, 2.5 Hz, H-0 2), 4.47 (3H, s, C-4 OMe), 4.13 (1H, ds, J=9.9, 8.0 Hz, Ha-0 1), 4.10 (3H, s, C-8 OMe), 3.81 (1H, ds, J=8.0, 2.5 Hz, Hb-0 1), 1.29 (3H, s, H-0 4), 1.25 (3H, s, H-0 5); 13C NMR (CD3OD, 125 M Hz): 165.8 (C-2),
115.9 (C-3), 153.9 (C-4), 119.5 (C-5), 114.0 (C-6), 142.9 (C-7), 142.1 (C-8), 159.0 (C-9), 103.3 (C-10), 144.3 (Ca), 106.5 (C-b), 61.9 (C-8 OMe), 60.0 (C-4 OMe), 72.5 (C-0 1), 77.8 (C-0 2), 72.8 (C-0 3), 25.0 and 26.9 (C-0 4, C-0 5); HMBC: see Fig. 2.
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