Alkaloids of Alstonia scholaris from Thailand.

Postgraduate School of Studies in Pharmacy, University of Bradford, Bradford, West Yorkshire BD7 IDP,England

ALKALOIDS OF ALSTONlA SCHOLARIS FROM THAILAND

E. COURT By (Miss) WARANK BOONCHUAY and WILLIAM

The indole alkaloids akuammicine, akuammicine-Na-oxide, akuammicine-Namethiodide, q-akuammicirte, Nb-demethyl-ecbitamine and tubotaiwine were isolated from the roots of A 1s t o n i a s c h o 1 a r i s together with echitamine, the principal alkaloid, and some echitamidine isomers.. Na-demethylechitamine, tubotaiwine and picrinine were found in the stems associated with echitamine and some echitamidine isomers. Alstonia scholaris R. Br. (Apocynaceae) is a tall evergreen tree occurring widely in India, in sub-Himalayan areas and through S.E. Asia to Indochina. Known as Dita Bark or Devil's Bark, A.scholaris has been widely used in folk medicine as an astringent, alterative, tonic, anthelmintic and antiperiodic in fevers and has been claimed to be useful in restoring stomach muscle tone after debility due to fevers and in the treatment of some forms of rheumatism (WAIT, 1889; WREN, 1956). Although popular in Europe in the early part of this century (GREENISH, 1920), dita bark declined in popularity although no real pharmacological evidence was available (MARTINDALE, 1967). The bark was also a source of poor quality caoutchouc and the wood was used in the manufacture of furniture, boxes, coffins, etc. (WAIT, 1889). The use of A. scholaris in folk medicine and interest in the indigenous plants of Thailand stimulated a re-appraisal of the alkaloids present in the stems and roots. Early work on the chemistry of A. scholaris was confused but the principal alkaloids echitamine (HESSE,1880) and echitamidine (GOODSON, 1932) were isolated from the bark. Subsequently picrinine (CHAITERJEEet al., 1965) and picralinal '(RASTOGI et al., 1970) were discovered in the leaves and akuarnmidine (16carbomethoxy-10-deoxysarpagine) in the fruits and trunk (CHA~TERJEE et al., 1969). Quantitative data is limited but GOODSON (1932) reported 0.16-0.27 per cent total alkaloids, 0.08-0.1 per cent echitamine and 0.008 per cent echitamidine as picrate in Indian Alstonia scholaris stems. In Mysore A. scholaris SIDDAPPA. (1945)

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Abstract

Alkaloids of Alstonia scholaris

381

estimated 0.18 per cent total alkaloids and 0.05 per cent echitamine as hydro(1952) recorded chloride. Assaying an Indochinese sample, CHABASSE-MASSONEAN 0.03 per cent echitamine and 0.001 per cent echitamidine.

Plant Material A.scholaris roots collected from and vouched for by the Botanical Garden, Department of Medical Sciences, Thailand in 1973. A. scholaris stems collected from and vouched for by the Forestry Garden, Sraburee, Thailand in 1972. Specimcns were compared with authentic samples in the Pharmaceutical society of Great Britain Collection of Crude Drugs and Herbaria in the University of Bradford.

Analytical Methods Thin-layer chromatography (TLC)and Preparative layer chromatography (PLC) Silica Gel G and GF,,,; Alumina GF,,,. The plates (20 x 2 0 cm with layers 250 pm thick) were activated (60 minutes) a t 110° C and stored in a desiccator. Spectroscopy Ultraviolet (UV)spectra were determined in methanol using a Unicam SP800 recording spectrophotometer. Infrared (IR) spectra were obtained using potassium bromide discs and a Unicam SP200 spectrophotometer. Nuclear Magnetic Resonance (NMR)spectra were determined in deuterochloroform o r 4-dimethylsulphoxide a t 60 mHz using a Perkin-Elmer R12 instrument and tetramethylsilane as an internal standard. Mass spectra (MS) were determined using an A. E. I. MS902 mass spectrometer (direct inlet, 70 eV, 100 pA, 200-250° C). Physical constants Melting points Melting points were determined ,using closed capillaries and a GaIlenkamp melting point apparatus. Results were uncorrected. Microanalysis Elemental analysis was undertaken by Dornis u. Kolbe, Microanalytisches Laboratorium, Miilheim a. d. Ruhr, West Germany. Detection of alkaloids on chromatographic plates Plates were viewed in screened ultraviolet light at 254 nm for fluorescence quenching spots or 366 nm for fluorescent spots. Chromogenic reagents employed were: Ceric sulphate reagent - 1 per cent ceric sulphate in 10 per cent sulphuric acid solution.

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Experimental

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Dragendorff's reagent - Munier-Macheboeuf nlodification. Ferric chloride-percbloric acid reagent - 5 per cent ferric chloride in 35 per cent vlv aqueous perchloric acid solution.

Extraction of alkaloids Powdered plant material (root o r stem bark) was defatted with petroleum ether (b.p. 40-60°). T h e marc was extracted with 2 per cent ammoniated methanol and the filtered extract evaporated t o dryness under reduced pressure. The residue was dissolved in 1. ON hydrochloric acid solution and the solution filtered. The filtrate was fractionated into 3 parts. The acid solution was extracted with chloroform to yield the weakly basic fraction. The aqueous phase was adjusted to pH8 using ammonia and the solution again extracted with chloroform to give the intermediately basic fraction. Finally the aqueous phase was adjusted t o pH11 using sodium hydroxide and extracted with chloroform to yield the strongly basic fraction. Isolation of alkaloids (a) Root bark 400 g root bark yielded 1.2 g weakly basic fraction, 0.8 g intermediately basic fraction and 1.4 g strongly basic fraction. Separation of weak bases PLC on alumina using chloroform : acetone : methanol (11:8:1) as solvent system yielded four bands, A, B, C and D. Band A, the least polar, was further separated by PLC on alumina using chloroform : acetone (5:4) as solvent system and yielded two alkaloids. ?'he uppermost band eluted in methanol yielded a white amorphous powder AS1 (1.6 mg). The lower band similarly gave AS2 (1.8 mg). Band B was subjected t o PLC on silica gel G (solvent system, chloroform: methanol (19:l) in a tank atmosphere of ammonia) and yielded a white amorphous powder after elution (AS3, 2 mg). Band C, separated by PLC on silica gel G (solvent systenl, chloroform : isopropanol (4:1) in an atmosphere of ammonia), yielded an off-white, amorphous powder (AS4,1.6 mg). Band D was purified by double development PLC on alumina using the solvent system chloroform : methanol (3:2) and yielded an off-white amorphous powder (ASS, 1.4 mg). Separation of intermediate bases PLC o n alumina using the solvent system chloroform: methanol (9:l) gave two prominent bands. The upper band after rechromatography using the same solvent system gave an off-white amorphous compound AS6 (1.8 mg). The lower band yielded, on crystallisation from methanol : ether, a white compound AS7 (9mg). Separation of strong bases The strongly basic fraction was crystallised out as chloride salt from anhydrous methanol : ether using hydrochloric acid gas. Excess hydrogen chloride was removed by successive evaporation with benzene. The residue was recrystallised from petroleum ether: methanol to yield a white crystalline compound AS8 (830 mg).


Preparation of acetyl derivatives Derivatives were prepared by acetylation with 10 per cent acetic anhydride in pyridine at room temperature for 24 hours and purified by PLC.


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The mother liquor was concentrated and a compound isolated by PLC on silica gel G in the solvent system acetone : petroleum ether : carbon tetrachloride : isooctane (7:6:4:3) as yellow needles (AS9, 15 mg).

(b) Stem bark 1.5 kg stem bark yielded 6.5 g weak bases, 3 g intermediate bases and 1.2 g strong bases. Separation of weak bases PLC on silica gel C (solvent system, chloroform: methanol (19:l) in an atmosphere of ammonia) yielded 3 major bands, A, B and C. Band A was purified by PLC on silica gel G (solvent system, chloroform : acetone : methanol, 5:4:1) to give an off-white amorphous powder AS11 (1 mg). Band B was separated by PLC on silica gel G (solvent system, chloroform : acetone : methanol : ammonia, 5:4:1:0.01) into 3 zones, Ba, Bb and Bc. Ba yielded an off-white powder AS12 (1.5 mg). Bb was further purified by PLC on silica gel G (chloroform: methanol (99:l) in an atmosphere of ammonia) to give a white amorphous powder AS13 (1.5 mg). Bc, after purification by PLC on alumina (solvent system, chloroform: acetone, 5:4), yielded a white amorphous powder AS14 (1 mg). Table I Chromatographic and chromogenic test data for A. scholaris root bark alkaloids Isolate

Solvent System

Chromogenic reagents

1

2

3

4

AS1 AS2 AS3

41 36 15

85 87 65

27 24 24

52 53 47

84 82 64

Red Violet-Blue Blue

Faint pink Violet-Blue Blue

AS4 ASS AS6

21 0 11

0 0 0

37 10 40

64 32 55

33 0 21

Blue Blue Blue

Blue Blue Blue

AS7 AS8 AS9 AS10

12 5 2 37

37 6 12 84

57 18 28 25

61 46 28 60

67 0 7 86

Pink Pink Orange Blue

Pink Pink Orange Blue

5 Ceric sulphate

Identity

Ferric chloride: perchloric acid

tubotaiwine hydroxv-19,20-dihydroakuammicine a kuammicine-Nb-oxide akuammicine-Nb-methyl 18 o r 19-hydroxy-19,20dihydroakuammicine Nb-demethylechitamine echitamine unidentified alkaloid akuammicine


Separation of akuammicine 50 g powdered root bark was fractionated as before. The weak base fraction was separated by PLC on silica gel % using the solvent system chloroform: methanol (19:l) in an atmosphere of ammonia. Several bands were located under screened ultraviolet light. The third band from the solvent front, which yielded a blue fluorescence and approximate hRr = 80, was removed and further purified by PLC o n silica gel G (solvent system, ethyl acetate: diethylamine, 36:l). An amorphous residue (AS10) was obtained after elution of the uppermost band and removal of solvent.

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Band C yielded by PLC o n silica gel G (solvent system, chloroform: acetone: methanol ammonia, 5:4:1:0.01) a white a i o r p h o u s powder AS15 (17 mg).

:

Separation of intermediate bases PLC separation, as in the case of root bark, yielded two alkaloids, AS16 (10 mg), a white crystalline compound and AS17 (9 mg), an off-white amorphous compound.

Characterisation of the alkaloids Co-TLC was employed; hR, values and chromogenic tests are recorded in ~ a b l e Is and 11. ASI, white amorphous powder, UV Amax 217, 245, 291, Amin 230, 269 nm; IRyKBr 2950 s, 1730 s, 1605 m, 1465 s, 1270 s, 1070 s, 740 s cm-I; MS mle 366 (100 per cent relative intensity), 352 (8), 336 (40), 321 (12), 277 (35), 266 (16), 263 (IS), 222 (lo), 184 ( l l ) , 170 (lo),

T a b l e I1 Chromatographic and chromogenic test data for A. scholaris stem bark alkaloids Isolate

Solvent System

Chromogenic reagents

1

2

3

4

5 Ceric sulphate

AS11 AS12 AS13 AS14

36 46 27 35

87 85 89 82

24 55 28 35

53 56 55 46

82 79 .87 76

Violet-blue Yellow Grey Blue

Violet-blue Yellow Grey Blue

AS15

15

65

24

47

64

Blue

Blue

AS16 AS17

12 11

37 0

57 40

61 55

67 21

Pink Blue

Pink Blue

AS18 AS19

5 2

6 12

18 28

46 26

0 8

Pink Orange

Pink Orange

Identity

Ferric chloride: perchloric acid tubotaiwine picrinine unidentified alkaloid hydroxy-19,20-dihydro. akuammicine hydroxy-19,20-dihydroakuammicine Nb-demethylechitamine 18 o r 19-hydroxy-19,20dihydroakuammicine echitamine unidentified alkaloid

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Solvent systems (Tables I and 11) Adsorbent - Silica gel G 1 - n-Butanol : ethyl acetate : ethylene dichloride (1:3:6) 2 - Acetone :petroleum ether (b.p. 40-60° C) : carbon tetrachloride : isooctane (7:6:4:3) 3 - Acetone : petroleum ether (b.p. 40-60° C) :diethylamine (2:7:1) 4 - Chloroform :methanol (19:l) (tank atmosphere saturated with ammonia) 5 - n-Bucanol : glacial acetic acid : water (4:l:l) hRf values recorded are the mean determinations based on at least five observations (mean


Separation of strong bases PLC separation, as in the case of root bark, yielded an off-white crystalline compound AS18 (620 mg) and a yellow compound occurring as short needles (AS19,4 mg).

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158 (12), 144 (13), 121 (13). Agrees with y-akuammigine (confirmed by direct comparison [UV, IR, co-TLC] with an authentic sample). AS2, pale yellow amorphous powder; UV Amax 230, 295, 327, hmin 263, 306 nm; IRy KBr 3400 m, 2950 m, 1670 s, '1630 s, 1465 .m, 1440 m, 1275 m, 1230 s, 1145 s, 740 s cm-l; MS m/e 324 (l1per cent), 307 (3), 293 (3), 281 (2), 267 (7), 265 (6), 253 (S), 240 (6), 229 (27), 194 (lo), 182 (14), 181 (12), 180 (20), 167 (16), 95 (60), 71 (100). TLC data and MS agrees with tubotaiwine (19,20-dihydrocondylocarpine). (PINAR,RENNER,HESSEand SCHMID, 1972). AS3 was found to be identical (UV, IR, MS, co-TLC) with AS15 which was available in larger yield. AS4, off-white amorphous powder; UV Amax 228,294,327, htnin 260, 307 nm; JRyKBr 3400 m, 2930 m, 1655 s, 1600 s, 1464 s, 1430 m, 1225 s, 1100 m, 755 m cml; MS m/e 338 (31 per cent), 336 (26), 322 (loo), 320 (46), 307 (14), 291 (3), 280 (16), 263 (34), 252 (24), 249 (17), 239 (17),234 (25), 226 (26),220 (22), 156 (39,144 (16), 130 (14), 121 (67), 115 (17), 92 (45). The IR spectrum was almost identical with the spectrum of an authentic sample of akuammicine. MS was similarly almost identical but M + was 16 mass units larger. The data agrees with akuammicine-Nb-oxide. AS4 reduced b y treatment for 12 hours with dilute sulphurous acid solution yielded akuammicine (confirmed by co-TLC, 8 systems). ASS, off-white amorphous powder; UV hmax 228,294,328, hmin 260,307 nm; IRyKBr 3400 m, 2950 m, 1660 s, 1605 s, 1470 m, 1435 m, 1230 s, 1100 s, 750 s cm-l; MS m/e 336 (15 per cent), 322 (39), 307 (6), 291 (6), 277 (16), 264 (37), 252 (ll),246 ( l l ) , 234 (22), 220 (16), 206 (16), 195 (IS), 180 (27), 167 (19), 158 (30), 142 (loo), 127 (29), 121 (loo), 115 (16), 106 (16), 93 (20). MS was identical with akuammicine with an added peak a t m/e 336 (M+ 14 units). Peaks at m/e 142 (CH,I) and m/e 127 ( I + ) were prominent. Agrees with akuammicine-Nb-methiodide (confirmed by comparison [UV, IR, MS, co-TLC] with a sample prepared from akuammicine and methyl iodide). AS6 was identical (UV, IR, MS, co-TLC) with AS17 which was available in greater yield. AS7, off-white crystals; m.p. 238-240° C; UV hmax 215, 243, 302, hmin 227, 268 nm; IRyKBr 3450 s, 3350 s, 2975 s, 1735 s, 1605 m, 1485 m, 1470s, 1450m, 1210 m, 1110s, 1070 m, 805 m, 750 s cm-l; MS mle 370 (100 per cent), 353 (84), 326 (18), 267 (9), 249 (8), 221 (8), 214 (6), 207 (6), 180 (9), 171 ( l l ) , 157 (24), 154 (18), 144 (14), 143 (13), 130 (28); N M R t 2.3-3.6 (multiplet, five aromatic protons, integrates t o four protons after shaking with D,O and rerunning the spectrum; the only species absorbing in this region which would undergo H+ exchange is aromatic >NH), quartet centred at 4.5 (one proton, J = 6.5 Hz), 6.15 ('singlet, three protons), 6.8 (broad signal approximately 1-2 protons), 8.5 (doublet, three protons, J = 6.5 Hz). Acetyl derivative, UV as before; IR as before with added ester peak at 1720 cm-1 as a shoulder o n the 1735 cm-I peak; MS m/e 412 (20 per cent), 395 (IS), 370 (67), 353 (67), 341 (lo), 326 (18), 307 (lo), 295 (40), 281 (39), 279 (41), 267 (IS), 249 (12). 221 (loo), 207 (15), 194 (13), 180 (13), 167 (46), 157 (24), 154 (26), 147 (SO), 130 (34). Methiodide derivative - 5 mg alkaloid methylated overnight a t 4' C with methyl iodide. UV hmax 210, 223, 235 (sh), 292, Lmin 217, 258 nm; IRyKBr 3350 s, 2950 m, 1735 s, 1610 m, 1595 m, 1485 s, 1440 m, 1210 s, 755 s cmP; MS m/e 384 (35 per cent), 293 (9), 232 (lo), 194 (IS), 180 (IS), 168 (15), 167 (17), 152 (18), 142 (loo), 127 (82). UV spectrum in strong acid showed a hypsochromic shifl of about 8 nm suggesting a Ph-N-C-N chromophore similar to echitamine but not quaternary. IR spectrum resembled echitamine but was not'completely superimposable. N M R spectrum was almost superimposable on that of echitamine but the N-CH, peak was absent. AS7 was identified as Nb-demethylechitamine. Methylation with methyl iodide yielded a compound identical (UV, IR, MS,

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co-TLC) with echitamine iodide prepared from echitamine chloride previously isolated from the plant material. AS8, white, short needle crystals; m.p. 274O C (dec); elemental analysis, calculated for C,H,,N,O,+ C1-, C 62.77 per cent, H. 6.65 per cent, N.6.65 per cent, 0 15.20 per cent and C1 8.42 per cent, found C 62.90 per cent, H 6.93 per cent, N 6.57 per cent, 0 15.26 per cent, C1 8.34 per cent; UV l m a x 214,237,294, hmin 224,258 nm; IRyKBr 3500 m, 3280 s, 3000 mi 2930 m, 1735 s, 1608 m, 1595 m, 1485 s, 1445 m, 1305 m, 1270 m, 1210 s, 1085 s, 1055 s, 1040 s, 755 s cm-I; MS mle 384 (100 per cent), 383 (lo), 369 (6), 353 ( 6 ) , 194 (6), 152 (7), 144 (lo), 130 (lo), 124 (8); NMR r (CDCI,, free base) 2.35-3.45 (multiplet, aromatic protons, integrates to five including >NH as in AS7), 4.3-4.8 (quartet, one proton, J = 6.5 Hz), 5.15 (singlet, one proton, disappears on deuteration), 6.25 (singlet, approximately two protons), 6.3 (singlet, three protons), doublet centred at 8.35 (three protons, J = 6.5 Hz). The spectral data, melting point, and polar nature of the compound agreed with its identity as echitamine chloride. Echitamine was previously shown to be the major base in A. scholaris (HESSE,1880). AS9, yellow needle crystals; m.p. 210" C (softens and slowly decomposes without completely melting); UV hmax 212, 225 (sh), 246, 295, hmin 232, 270 nm; hmax (SN HCI) 215, 240 (sh), 267, hrnin 250 nm; IRyKBr 3450 m, 2950 s, 1720 m, 1600 m, 1465 m, 1380 m, 1260 m, 1120 m, 1100 m, 740 s cm-I; MS m/e 382 (3 per cent), 352 (9), 340 (6), 322 (28), 279 (19), 265 (IS), 247 (15), 213 (20), 194 (20), 185 (19), 167 (16), 157 (23), 143 (loo), 130 (55), 122 (IS), 115 (25). Acetyl derivative. IRyKBr 3450 s, 3000 s, 1730 m, 1710 w, 1650 m, 1490 w, 1400 m, 1260 s, 1100 s, 1030 s, 810 s cm-'; MS mle 468 (30 per cent), 425 (9), 411 (7), 395 (15). 296 (loo), 269 (39, 236 (IS), 216 (16), 144 (16), 143 (16), 130 (16), (121 (IS), 97 (16), 95 (18). Insufficient data was available for identification. AS10, white amorphous powder. Confirmed as akuammicine by co-TLC (8 systems) with an authentic sample. (b) Stem bark AS11 was found to be identical with AS2. AS12, yellow powder. Chrornogenic reactions and spectral data indicated the known Alstonia alkaloid picrinine. Confirmed by direct comparison (UV, IR, MS and co-TLC) with an authentic sample. AS13, white amorphous powder; UV l m a x 220,283,291, hmin 248 nm; IRyKBr 3400 m, 2950 m, 1725 s, 1595 m, 1470 m, 1440 m, 1330 m, 1230 m, 1210 m, 1100 m, 1065 m, 740 s cm-I; MS m/e 354 (6 per cent), 340 (20), 310 (31), 201 (12), 194 (13), 180 (lo), 169 (28), 154 (19), 144 ( l l ) , 143 (12), 130 (28), 122 (200), 108 (40), 84 (13). Yield 0.0001 per cent. Insufficient data was available for identification. ~ s, AS14, white amorphous powder; UV hmax 225, 292, 326, lmin 260, 305 nm; I R ~ K ' B3400 2960 s, 1660 s, 1600 s, 1470 s, 1230 m, 1150 s, 1100 s, 750 s cm-I; MS m/e 340 (18 per cent), 295 (6), 281 (4), 267 (3), 241 (loo), 227 (25), 225 (27), 214 (12), 208 (14), 194 (21), 180 (31), 167 (31), 156 (13), 154 (IS), 139 (23), 130 (7), 94 (55). MS in the range below m/e 220 resembled published data for 19, 20-dihydroakuammicine (SCOTT and YEH,1974) but the base peak at m/e 225 was shifted to m/e 241. The presence of an hydroxy group and a conjugated carbomethoxy group was indicated by IR and MS (3400 cm-I, mle 322 and 1660 cm-I, m/e 281 respectively). Spectral data and fragmentation pattern evidence showed that the hydroxyl group was nor attached to the aromatic ring or


386

387

at positions C-3, C-5, C-6, C-14 o r C-21. The compound was identified as hydroxy-19,20dihydroakuammicine but the exact position of the hydroxyl group was not confirmed. AS15, white amorphous powder; UV Amax 225, 297, 328, l m i n 265, 305 nm; IRyKBr 3400s, 2910 s, 1665 s, 1460 s, 1435 m, 1230 m, 1190 m, 1090 m, 740 s cmP; MS rnle 340 (100 per cent), 322 (16), 309 (16). 295 (29), 281 (17), 241 (30), 225 (loo), 214 (33, 209 (23), 194 (38). 180 (53), 167 (44), 156 (X),154 (23), 149 (lo), 139 (53), 115 (25), 109 (21), 94 (72). Acetyl deriw~tive.MS m/e 382 (43 per cent), 351 (6), 323 (26), 295 (9), 283 (16), 268 (20), 225 (80), 214 (24), 208 (X), 194 (26), 180 (29), 167 (29), 156 (lo), 154 (ll), 149 (22), 121 (52), 98 (loo), 94 (5). Data suggests hydroxy-19,20-akuammicine, AS15 being isomeric with AS17 but less polar (TLC). AS16 was found to be identical with AS7. AS17, off-white amorphous powder; UV Amax 225,291,325, Amin 260,307 nm; IRyKBr 3400 m, 2950 s, 1670 m, 1605 s, 1460 rn, 1430 m, 1250 s, 1080 s, 1020 s, 800 s cm-I; MS rnle 340 (63 per cent), 338 (40), 322 (12), 295 (21), 281 (IS), 265 (13), 2.53 (ll),241 (30), 22.5 (89), 215 (34), 214 (34), 208 (33). 194 (44), 180 (65). 167 (53), 156 (44), 154 (39, 139 (52), I30 (15), 115 (33), 107 (16), 94 (100). Acetyl derivative MS d e 382 (66 per cent), 323 (36), 296 ( l l ) , 295 ( l l ) , 283 (21), 268 (25), 263 (ll),252 (15), 235 (12), 22.5 (loo), 214 (26), 194 (28), 180 (28), 167 (29), 154 ( l l ) , 145 (lo), 130 (6), 121 (41), 98 (76), 94 (35). Redrrction product of 0-acetyl AS17 (reduced by heating under reflux for 80 min. in 10 per cent methanolic sdlphuric acid with zinc dust; purified by PLC). UV Amax 217, 247, 297, Amin 232, 270 nm; MS m/c 342 (46 per cent), 311 (9), 297 (8), 225 (7), 212 (100 x 3), 199 (43), 168 (16), 156 (14), 154 (14), 144 (93), 130 (53), 123 (ll),115 (13), 111 (12), 109 (13), 97 (18), 95 (20). Spectral data and colour reactions indicated the presence of a 2-methylene-indoline chromophore and -COOCH,, >NH and -OH groups. T h e intense MS peak at m/e 225 resembled that of 19, 20-dihydroakuammicine. The MS fragmentation pattern of the reduction product resembled the pattern of tetrahydroakuammicine with ions at d e 130, 144 and 199 but the m/e 196 ion was shifted t o d e 212 indicating a difference due to an oxygen atom. The fragmentation pattern indicated the absence of substitution in the indole moiety o r a t C-3, C-5, C-6, C-14 o r C-21. Formation of the 0-acetate compound eliminated possible substitution at C-15 or C-20 as tertiary hydroxyl groups lose water on attempted et al., 1962). Therefore AS17 is 18- o r 19-hydroxy-19,20-dihydroacetylation (NAKAGAWA akuammicine. AS18, white short needles and identical with AS8. AS19, yellow needle crystals and identical with AS9.

Results and Discussion Akuammicine, akuammicine-Nb-oxide, akuammicine-Nb-methiodide,q-akuammigine and tubotaiwine have not previously been reported in A. scholaris roots. The principal alkaloid of the roots was echitamine and three echitamidine (hydroxy-19,20-dihydroakuammicine)isomers were isolated but in insufficient yield for adequate differentiation. The stems yielded echitamine, Nb-demethylechitamine, tubotaiwine, picrinine and some echitamidine isomers. Picrinine, known to occur in the leaves, has not previously been isolated from the stem bark.



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Fig. 1. Alkaloids of A. scholaris. I-Akuamrnicine, 11-Echitamidine, 111-Tubotaiwine, IV-y-Akuarnrnigine, V-Picrinine, VI-Picralinal, VII-Echitamine, VIII-Akuarnmidine.

These alkaloids are characterised by the lack of a carbocyclic o r heterocyclic ring E and are regarded as E-seco derived alkaloids. SHARP (1934) divided the Alstonia species into three groups on a chemical basis. T h e echitamine group containing A. scholaris yielded echitamine as the principal alkaloid whilst the other groups were characterised by alstonine-type anhydronium bases and bisindoles respectively. T h e present work supports this classification, the echitamine group being more correctly described as E-seco derived alkaloids.


T h e alkaloids so far isolated from A. scholaris can be shown to comprise four groups: a) the akuammicine group (akuammicine, akuammicine-Nb-oxide, akuammicineNb-methiodide, echitamidine isomers and tubotaiwine), b) q-akuammigine, picrinine and picralinal, c) echitamirie and Nb-demethylechitamine, d) akuammidine (rhazine) (Fig. 1).

389

CORDELL (1974) in his scheme for the biosynthesis of the main indole skeleton suggested strictosidine/vincoside as the probable starting nitrogenous glycosidal intermediate leading to corynantheine aldehyde and geissoschizine. From geissoschizine at least four routes are possible: i) via geissoschizine oxindole and preakuammicine to akuammicine and its derivatives; ii) via precondylocarpine to condylocarpine and its isomers, iii) via the formyl strictamines to picrinine, 11-akuammigine and akuammidine, iv) via ajrnalicine to the anhydronium bases. A. scholaris probably employs at least three of these routes and the apparent absence of the fourth may be of chemotaxonomic significance. The distribution of alkaloids within the plant (Table 111) does not at this stage permit any obvious conclusions concerning the biosynthetic cycle. Echitamine is the dominant alkaloid of root and stem and the yield of related alkaloids is very Iow. Thus the pool size of intermediates may be too low for isolation using current techniques.

T a b l e I11 Distribution of alkaloids in A. scholaris

Fruits

Leaves

Stems

Roots

Akuammicine Akuammicine-Nb-oxide Akuammicine-Nb-methiodide Echitamidine Tubotaiwine ?I-Akuarnrnigine Picrinine Picrilinal Echitamine N,,-Demethylechitamine Akuammidine

Acknowledgements The authors are grateful to Dr. A. BANERJEE, Dr. J. A. JOULE,Professor J. LE MEN and Profor the gifls of authentic alkaloids. fessor R. N. FARNSWORTH



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CHABASSE-MASSONEAN, J.: Trav. Lab. Matihe Med. Pharrn. Galenique, Fac. Pharm., Paris, 37, 1 (1952) B., GHOSAL,S. and BANERJEE, P. K.:J. Indian Chem. Soc., 46,635 CHATTERJEE, A., MUKHERJEE, (1969) CHA~TERJEE, A:, MUKHERJEE, B., RAY,A. B. and DAS,B.: Tetrahedron Letters, 3633 (1965) CORDELL,G. A.: Lloydia, 37,219 (1974) J. A.: J. Chem. Soc., 2626 (1932) GOODSON, GREENISH, H. G.: Materia Medica (London: J. and A. Churchill) 268 (1920) HESSE, 0.: Ann., 205,360 (1880) MARTINDALE: Extra Pharmacopoeia (London: Pharmaceutical Press) 323 (1967) PINAR,M., RENNER,U., HESSE,M. and SCHMID,H.: Helv. Chim. Acta, 55,2972 (1972) RASTOGI,R. C., KAPJL,R. S. and POPLI,S. P.: Experientia, 26,1056 (1970) SHARP,T. M.: J. Chem. Soc., 1227 (1934) SIDDAPPA, S.: J. Mysore Univ., 5,63 (1945) WAIT, G.: A Dictionary of the Economic Products of India (Calcutta: Superintendent of Government Printing, India) Vol. I., 197 (1889) WREN,R. C.: Potter's Cyclopaedia of Botanical Drugs and Preparations (London: Pitrnan and Sons, Ltd.) 14 (1956) Addresses: Dr. W.E. Court, Postgraduate School of Studies in Pharmacy, University of Bradford, Bradford, West Yorkshire BD7 1 DP, England. Miss W . Boonchuay, Division of Drug Analysis, Department of Medical Sciences, Yod-se, Bangkok 1 , Thailand


References

Flower alkaloids of Alstonia scholaris.

Monoterpenoid Indole Alkaloids from Inadequately Dried Leaves of Alstonia scholaris.

Antiviral Cystine Knot α-Amylase Inhibitors from Alstonia scholaris.

Macroline, akuammiline, sarpagine, and ajmaline alkaloids from Alstonia macrophylla.

Study on Phytochemical Composition, Antibacterial and Antioxidant Properties of Different Parts of Alstonia scholaris Linn.

Ethanolic extracts of Alstonia Scholaris and Bacopa Monniera possess neuroleptic activity due to anti-dopaminergic effect.

The role of pentacyclic triterpenoids in the allelopathic effects of Alstonia scholaris.

Preliminary evaluation of extracts of Alstonia scholaris bark for in vivo antimalarial activity in mice.

Quinine and Alstonia Scholaris (Chhatim) in Malaria: A Study of Possible Synergy.

[Alstonia scholaris: the structure of the indole alkaloid nareline (author's transl)].

Oxidized derivatives of macroline, sarpagine, and pleiocarpamine alkaloids from Alstonia angustifolia.

Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities.

Ameliorative potential of Alstonia scholaris (Linn.) R. Br. against chronic constriction injury-induced neuropathic pain in rats.

Ethanolic extracts of Tinospora cordifolia and Alstonia scholaris show antimicrobial activity towards clinical isolates of methicillin-resistant and carbapenemase-producing bacteria.

Biologically active vallesamine, strychnan, and rhazinilam alkaloids from Alstonia: Pneumatophorine, a nor-secovallesamine with unusual incorporation of a 3-ethylpyridine moiety.

Alkaloids from Dehaasia incrassata.

Amide alkaloids from Scopolia tangutica.

Pyrrolizidine alkaloids from Werneria nubigena.

Pyrrolizidine alkaloids from Melampyrum pratense.

New Alkaloids from Aconitum stapfianum.

Catharanthus alkaloids XXXII: isolation of alkaloids from Catharanthus trichophyllus roots and structure elucidation of cathaphylline.

New potently bioactive alkaloids from Crinum erubescens.

Indole alkaloids from Balansia epichloë (Weese).

[Alkaloids and glyco-alkaloids from leaves of Nauclea latifolia SM (author's transl)].