Picranitine, A New Indole Alkaloid from Picralima Nitida (Apocynaceae) By Prof. EDET M. ANAM Dept. of Chemical Sciences Cross River University of Technology, CRUTECH P.M.B. 1123, Calabar, And E. O. E. EYAMBA Dept. of Chemical Sciences Cross River University of Technology, CRUTECH P.M.B. 1123, Calabar. Abstract A new indole alkaloid, picranitine, has been isolated from the seeds of Picralima nitida along with five known indole alkaloids, picratidine, akuammine, pseudoakuammine, akuamminicine and akuamidine previously isolated from the same source. Structures of these compounds were determined using spectral measurements including 1-D (1H and 1C NMR) and 2D-NMR HMQC, HMBC and NOESY) Picralima nitida (Staf.) TH S H. Durant (Apocynaceae) is a medium sized tree growing in the Western and Central Zones of Africa and are used in folk medicine to treat diverse ailments (Irvine and Walker, 1961). In Eniong Abatim, Odukpani Local Government Area, Cross Rive State, Nigeria, the seeds of this plant find extensive exploitation in the treatment of malaria and abdominal pains. Medicinal potency of P. ntida has been the impetus for its scientific investigation in order to establish the natural product(s) responsible for curing malaria fever and abdominal pains. Hitherto, a number of indole alkaloids from this plant have been characterized and some of such alkaloids have demonstrated affinity for opioid receptors (Mezies, Peterson, Duwiedjua and Corbett, 1998; Corbett, Mezies, Macdonald, Perterson and Duwiedjua, 1996). 1 The Coconut This work reports the isolation and characterization of a new indole alkaloid, picranitine 1 along with five known alkaloids, picratidine 2 akuammine 3 (Lewin, Le Menez, Roland, and Giesen, 1992), pseudoakuammine, 4 (Moeller, Seedorff and Nartey, 1972) 5 Moeller, Seedorff and Nartey, 1972 and akuammidine (Janot, 1966) Results and Discussion Air-dried ground seeds of P. nitida were extracted, at room temperature, with ethanol. Concentration of the extract under reduced pressure, gave a crude product which was subjected to alkaline fractionation which yielded a mixture of alkaloids. The alkaloid mixture was column chromatographed over silica gel and six alkaloids 1-6 were obtained. Compound 1 Yellow crystals from EtOAc. Mp 215-2170c. The molecular ion peak at m/z 384 (HRHMS) was consistent with the formula, C12H24N2O5 which agreed with the 21 carbon signals observed in the 13C NMR spectrum (Table 1a) which showed resonances of two carbonyl ester signals at 175.1 and 174.8 and those of two oxygenated carbons at 65.2 and 60.2. Acetylation of 1 yielded a monoacetate derivative thus indicating the presence of one hydroxyl group in the structure. Its IR spectrum showed hydroxyl absorption at 3450 cm-1, two carbonyl functions at 1735 cm-1, (ester) and 1745 cm-1, (lactone). The 1H NMR spectrum (table 1) was very close to that of 2 (Mezies, Peterson, Duwiedjue and Corbett, 1998) and showed four aromatic protons in an ABCD splitting pattern between 7.24 and 6.62, but only two methyl functions were observed in 1. The methyl group obtained as a doublet and the olefinic proton, also a doublet in the 1H NMR of 2 were respectively, displayed as triplet at 0.92 and singlet at 6.01 in the 1H NMR spectrum of 1 suggesting the migration of the double bond. The proton and 13carbon spectra were unambiguously assigned by analysis of the 2D-NMR experiment. 1H and 13C Chemical Shifts were correlated in inverse mode two dimensional HMQC and HMBC and proton chemical Shifts correlation COSY was recorded. The signal at 65.5 corresponded to a quaternary oxygenated carbon while the one at 60.1 was assigned to an oxymethylene carbon in the HMQC spectrum. Analysis of the COSY and HMBC spectra (Table 1b) led to the overall construction of the skeleton of the molecule. Pertinent correlation, in the HMBC spectrum, were observed between proton H-6 and C-2, C-5, C-7 and C-16 and also between H-3 and C-2, C-14 and C-21. The relative stereochemistry at C-3 andC-5 of 1 was determined with the help of NOESY experiment and the coupling constants observed in the 1H NMR spectrum. 2 Picranitine, A New Indole Alkaloid from Picralima Nitida (Apocynaceae) Prof. Edet M. Anam and E. O. E. Eyamba Significant correlation in the NOESY spectrum between H-3 and H-15 indicated the same orientation for the two protons. Table 1a: NMR assignments of compounds 1, 2 and 2a in CD3COCD3. Chemical Shifts () are given in ppm; multiplicities and coupling constants, J (parenthesis) in Hz. C/H 1 H 2 2a C - C H H 2 163.4 (s) 6.58(s) 160.3(s) - 164(s) - 3 102.7 (d) - 106.0(s) 6.62(s) 103.5(s) 6.69(s) 4 182.1 (s) - 176.2(s) - 182.7(s) - 4a 102.7(s) - - - 104.2(s) - 5 159.0 (s) - 158.1(s) - 159.5(s) - 6 104.5 (s) - 107.2(s) - 105.3(s) - 7 163.2 (s) - 160.2(s) - 160.6(s) 6.47(s) 8 93.4 (d) 6.48(s) 94.2(d) 6.44(s) 95.1(s) - 8a 156.1 (s) - 156.4(s) - 155.9(s) - 1 121.8 (s) - 123.7(s) - 123.2(s) 7.60(d,J=2.1) 2 128.3 (s) 7.85(brd, J=8.8) 109.5(d) 7.51(d,J=2.1 109.9(s) - ) 3 116.0(d) 6.94(brd,J=8.8 148.2(s) - 148.3(s) - 4 160.7 (s) - 150.0(s) - 150.9(s) 7.01(d,J=83) 5 116.0(d) 6.94(brd,J=8.8 115.7(d) 6.98(d,J=8.3 115.9(d) 7.64(dd,J=8.3 ,2.1) ) 6 128.3(d) 7.85(brd,J=8.8 120.0(d) 7.48(dd,J=8. 120.8 - 3, 2.1) 1 30.2(t) 3.00(dd,J=13.2, - - - - 5.9) 2.90(dd,J=13.4, - - - 7.3) 2 87.8(d) 4.66(t,J=7.2) 75.1(s) 1.83(t,J=6.8) 76.3(s) - 3 147.4(s) - 17.3(t) 2.70(t,J=6.9) 16.2 2.69(t,J=6.8) 4 110.5(t) 4.82;4.75(brs) 31.6(t) 1.38(s) 31.7(t) 1.88(t,J=6.8) 5 165.5(q) 1.85(s) 26.4(q) 1.38(s) 26.4(q) 1.38(s) 6 16.5(q) - 26.4(q) 1.38(s) 56.0(q) 1.38(s) OMe - - 56.0(q) 3.97(s) 4.01(s) 5- - 13.34(s) OH 3 The Coconut The structures of the five known compounds were determined in a similar manner using spectroscopic methods. Their NMR and MS data were in agreement with those previously reported. Table 1b: 1H and 13C NMR Chemical Shifts assignments and HMBC Correlations for Compounds 1. Multiplicities and Coupling Constants, J, in Parentheses Position c H HMBC 1 2 109.8 3 48.2 3.75 (brs) C-2, C-14, C-21 4 5 175.2 or 174.8 6 39.3 3.19 (d, J=17.0HZ) C-5, C-7, C-8, C-2 2.93 (d, J=17.0Hz) 7 53.0 8 131.5 9 126.2 7.24(dd, J=7.7 and 1.5Hz) C-7, C-11, C-13 10 120.0 6.72(td, J=7.7 and 1.1Hz) C-8, C-12 11 129.0 7.04(dd, J=7.7 and 1.1Hz) C-9, C-13 12 110.1 6.62(d,J=7.7) C-8, C-10 13 147.2 14 23.6 153(dt,J=13.2 and 3.5Hz) C-2, C-16, C-20 15 32.1 3.05(m) C-2, C-16, C-20 16 60.2 17 65.6 3.96(s) C-7, C-15, C-16 18 13.4 0.92(t,J=7.4Hz) C-19, C-20 19 28.0 2.05(dq, J=14.0and7.4Hz) C-15, C-18, C-20, C-21 20 108.4 1.97(dq, J=14.0and7.4Hz) C-15, C-18, C-20, C-21 21 127.3 6.01(s) C-3, C-15, C-19, C-20 COOCH3 175.1 or 174.8 OCH3 51.3 3.00(s) Experimental Section General All melting points are uncorrected. 1H NMR (125 MHz) were recorded in CDCl3 or (CD3)2SO with an inverse 5mm probe equipped with a shielded gradient coil. COSY, HMQC and HMBC experiments were performed with gradient enhancement using shaped gradient pulse. In the 2D heteronuclear correlation 1 2 spectroscopy, the refocusing delays were optimized for JCH = 145Hz and JCH = 10Hz. The raw data were transformed and the spectra were evaluated with the standard Bruker UXNMR soft ware (rev. 941001). Chemical shifts are given in ppm with solvent signals as reference. EIMS was recorded by Jeol SX102 spectrometer at 4 Picranitine, A New Indole Alkaloid from Picralima Nitida (Apocynaceae) Prof. Edet M. Anam and E. O. E. Eyamba 70ev. Column chromatography was performed on Merck silica gel 60 and TLC on silica gel GF254 precoated plates and detection accomplished by spraying with 50% H2SO4, followed by heating. Plant Materials The seeds of the plants were collected at Ntan Obu, Eniong Abatim, Odukpani Local Government Area, Cross River State, Nigeria. The plant was identified by the Botany Division of the Biological Sciences department of the University of Calabar, Calabar.