1979, Vol. 35, pp. 19-30, O Hippokrates Verlag GmbH
plants-
medii
aant-
Alkaloids and Biological Activity of Strychnos angolensis Department of Pharmacognosy, Faculty of Pharmacy, University of Uppsala, Biomedical Center, Uppsala, Sweden. *" Department of Pharmacognosy, University of Leyden, Gorlaeus Laboratories, Leyden, The Netherlands.
Key Word Index: Strychnos angolensis; Loganiaceae; (f)-Tubotaiwine; 11-methoxy-Wieland-Gumlich aldehyde; 17-0-methyl-1l-methoxy-wielandGumlich aldehyde; 11-methoxy-diaboline; epi-17-0-methyl-11-methoxydiaboline; Caracurine V; lndole alkaloids; Pharmacology. Abstract
tree, found on river banks in rain forests or in gallery forests up to an altiA weak convulsant effect is found in tude of 1500 m. It is found in Nigeria, the nonpolar part of the tertiary alkaCentral and East Africa. S. angolensis loid extract of Strychnos angolensis and S. malacoclados C. H. WRIGHTare GILG, while the polar part shows a closely allied and belongs to the same strong muscle-relaxant activity. botanical section, Breviflorae [I]. Five indole alkaloids have been isoBISSETand LEEUWENBERG have related from the nonpolar part of the extract. One is the known (+)-tubotai- ported the use of S. angolensis in ordeal wine while the others are derivatives of poisons [ 2 ] . Investigations by DENOEL et al. [3,4] Wieland Gumlich aldehyde (WGA): 11-methoxy-WGA, 17-0-methyl-1 1- of S. angolensis from Congo (Kinshasa) methoxy-WGA, 11-methoxy-diaboline have shown a rather variable alkaloid and epi-17-0-methyl-1 I-methoxy-dia- content, particularly in the root bark. They found that aqueous alcoholic tincboline. Caracurine V, a tertiary dimeric al- tures and crude alkaloid extract had a kaloid, is also found to be present in a curarizing effect when tested in frog toluene extract of bark of S. angolensis. and cat. However, MARINI-BETT~LO [5] found no curarizing action and a very small amount of quaternary alkaIntroduction loids in a study of the same species. Strychnos angolensis GILGis a climBISSETand PHILLIPSON [6]have screebing shrub or liana or a semi-scandent ned several specimen of S. angolensis
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Lars Bohlin*, Wenche Rolfsen*, Jan Strombom* and Robert Verpoorte**
Bohlin et al.
Results and Discussion
Phytochemistry From the nonpolar part of the tertiary alkaloid fraction of S. angolensis several alkaloids were isolated (Table 111). The formulae of the isolated alkaloids are given in Fig. 1, 2 and 4. (+)-Tubotaiwine (I) showed the typical a-methylenindoline chromophore of the akuammicine/condylocarpine type in the UV spectrum [ l 11. Because of the strong specific optical rotation,
+
[a] 2,2 = 638' (0.02, CHCl,), the akuammicine type is excluded [12]. The colour reaction with ceric sulphate, Rrvalues (systems F, G, H) and the spectral data established the identity, when compared to authentical (+)-tubotaiwine1 (I) (Fig. 1). Tubotaiwine has not
Fig. I . (+)-Tubotaiwine.
Kindly provided by Prof. P. POTIER,Gifsur-Yvette, France.
+ Fig. 2. The relationship between the WielandGumlih aldehyde derivatives. 1. R = H WG A 2. R=OCH3 11-methoxy-WGA (11) 3. R = H 17-0-methyl-WGA 4. R=OCH3 17-0-methyl-11-methoxy-WGA (111) 5. R = H epi-17-0-methyl-WGA 6. R=OCH3 epi-17-0-methyl-11-methoxy-WGA 7. R = H Diaboline 8. R=OCH3 11-methoxy-diaboline (IV) 9. R = H 17-0-methyl-diaboline 10. R=OCH3 17-0-methyl-1 1-methoxy-diaboline 11. R = H epi-17-0-methyl-diaboline 12. R=OCH, epi-17-0-methyl-11-methoxy-diaboline (V)
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for its alkaloid content, using herbarium samples. Three of the four investigated samples of leafy twigs contained alkaloids, whereas the fourth probably contained only traces of alkaloids. TLC of the extracts indicated the presence of at least 8 components a. o. a yellow alkaloid, which also has been observed in several other Strychnos species. Variation in the alkaloid content and pharmacological activity were also found for six materials of S. angolensis tested in the screening programme of African Strychnos species for convulsant and muscle-relaxant effects [7, 8,9, 101 (Table 11). The aim of further investigations on this species was therefore to locate the pharmacological activity in the extracts and isolate and elucidate the structures of the alkaloids. This study deals with the nonpolar fractions of the dichloromethane extract of S. angolensis GILG[8], whereas the polar fractions (alkaloids A,-A,,), which are responsible for the musclerelaxant activity, will be subject for further studies.
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Alkaloids of Strychnos angolensis 21
Bohlin et al.
are also shifted 14 mass units compared to (11) (Table I). Furthermore the presence of a fragment at 322 mle (fragment a in fig. 3) in the MS points to the axial position of the proton in the 17 position [20]. 11-methoxy-diaboline ( I V ) was identified by comparing its chromatographical- and spectral data with an authentic sample of this alkaloid*.
Epi-17-0-methyl-11-methoxy-diaboline ( V ) .showed similar spectral data to (IV). The absence of a peak at 3450 cm-I and the molecular peak at 396 instead of 382 compared to (IV) point to the presence of a methoxy group instead of a hydroxy group. The peaks in the MS corresponding to the non-aromatic part are also shifted 14 units compared to (IV) (Table 11). The isolated 17-0methyl ether (V) of I l-methoxy-diaboline has its methoxy group in the axial positions as it can be concluded from the 365 and 323 mle fragments (resp. c and d in fig. 3) in the MS 1201 and the 4.70 ppm signal for H-17 (equatorial) 11-methoxy-WGA (11) was identified in the 'H-NMR [17, 181. The structure by its UV and MS [19]. The identity is further confirmed by the treatment of was confirmed by converting ll-me- 11-methoxy-diaboline with methanol thoxy-diaboline (IV) into this alka- acidified with HC1-gas, which accorloid, by treating it with dilute sulphuric ding to TLC and MS gives a product similar to V. acid. Because of the strong muscle-relaxant 17-0-methyl-11-methoxy-WGA ( I I I ) effect found in the tertiary alkaloid had similar spectral data as (11). The fraction, the presence of tertiary dimeric molecular peak for (111) in t.he mass alkaloids of the toxiferine type was exspectrum at 354 is situated 14 units pected. Toluene has been found to be a higher than (11) (M+ = 340) which sug- suitable solvent for the extraction of gests a methoxy group instead of a hydroxy group. The peaks in the MS Kindly provided by Prof. Dr. C. GALEFFI, corresponding to the non-aromatic part Rome.
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been isolated from Strychnos species before, previously it has been found in several plant species belonging to the plant family, Apocynaceae [ l l , 13, 141. HEIMBERGER and SCOTT 1151 found condylocarpine to be an intermediate in the biosynthesis of strychnine in Strychnos nux vomica, but only very small amounts were present in the plant material. Hence it is interesting that this type of alkaloid is found as one of the major components in Strychnos species (tubotaiwine is also present in S. dolichothyrsa [I 61). The other identified alkaloids are derivatives of Wieland Gumlich aldehyde (WGA) (Fig. 2). The cyclic hemiacetal form of WGA has two possible epimeres, which can be converted to the corresponding acetals when treated with alcohols [I 7, 181. Diaboline (NBacetyl-WGA) behaves similar to WGA in this respect [17, 181. Several alkaloids isolated from S. angolensis fit into this general scheme, although they belong to the 11-methoxy series.
23
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Alkaloids of Strychnos angolensis
Fig. 3. Mass fragments of the Wieland-Gumlich aldehyde derivatives [19, 201.
Bohlin et al.
24
Table I Mass fragments of the Wieland-Gumlich aldehyd derivatives
M+ M+-R'OH
340 322 F 311 E 297 M+-R'COOH 294 .+-
46 5 40 15 3
17-0-methyl-1111-methoxymethoxy-WGA (111) diaboline (IV) R=H, R'=CH, R=COCH,, R'=H m/e O/o m/e O/o 354 322
100 8
epi-17-0-methyl-11methoxy-diaboline (V) R=COCH,, R'=CH, mfe Olo
{tIOH
D C B C-CH2C0 B-R'OH A-CH2C0
229 174 180
13 45 100
174 194
31 83
162
11
162
12
such alkaloids [16].Hence this solvent was chosen for the extraction of a small sample of the bark of S. angolensis. According to TLC (+)-tubotaiwine was the major component of the basic toluene extract. The WGA-type of alkaloids were only present in relatively small amounts. Bisnor-dihydrotoxiferine and bisnor-C-alkaloid H could not be detected in the toluene extract. The
Fig. 4. Caracurine V.
dimeric alkaloid caracurine V (VI) was, however, present as another major component in the toluene extract. By means of preparative TLC this alkaloid could be isolated and identified as caracurine V by comparison with a synthesized reference compound [21, 221 on TLC and its UV and MS data. I t should be noted that this alkaloid was not found in the dichloromethane- and chloroform-fractions. In fraction 4 of the column chromatographic separation, WGA could be detected by means of TLC. The isolated methyl ethers of I 1-methoxy-WGA and 11-methoxy-diaboline are probably artefacts due to the purification method used. In the conversion of rhe complex of Mayers reagent with the alkaloids, the complex is dissolved in a mixture of acetone, methanol and water and run over a ionexchange resin in the chloride
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11-methoxyWGA (11) R=H, R'=H mfe O/o
form. The alkaloids are eluted as chlorides. Under these conditions the methyl ethers may be formed from the hemiacetals. The fact that the monomeres of the WGA-type mainly belong to 11-methoxy series and the only dimeric alkaloid found so far is of the non-aromatic substituted type, needs further study. Dimeric alkaloids with 11-methoxy substitution might be present as well. However, the combination of 11-methoxydiaboline with non-aromatic substituted dimeric alkaloids is also found in S. dolichothyrsa and S. urceolata [16]. 11-methoxy-diaboline has already been found in the African species S. malacoclados [22, 231 and is probably present in S. henningsii [24] as well. S. angolensis is hence the fourth African species in the section Breviflorae of the genus Strychnos which certainly contains 11-methoxy-diaboline. . Interesting is the comparison with S. malacoclados is which according t o LEEUWENBERG closely related to and difficult to distinguish botanically from S. angolensis. The phytochemical investigations of these two species have so far shown several differences: The alkaloid content of the sternbark of S. angolensis is much higher than of S. malacoclados. In S. malacoclados the alkaloids normacusine B and 1I-methoxy-diaboline are found as major alkaloids [22]. In S. angolensis only the latter alkaloid has been found, the former alkaloid could not be detected so far in any fraction. Of the other alkaloids found in S. angolensis none could be detected in S. malacoclados. TLC in combination with the selective spray reagent ceric sulphate in sulphuric acid shows clearly the
difference in the alkaloid content of the two species. In S. angolensis a number of alkaloids are observed which give various colours with this reagent, ranging from blue to orange, a. o. caracurin V and (+)-tubotaiwine, (resp. blueviolet and blue) whereas in S. malacoclados only orange coloured spots are observed. The number of specimen investigated so far (S. angolensis 5, S. malacoclados 3) is, however, too small to draw more than preliminary conclusions from the observed differences. Pharmacology In the pharmacological screenings [7, 8, 9, 101 six materials of S. angolensis GILGhave been tested. Both convulsive and muscle-relaxant effects are found in varying degrees. Muscle-relaxant activity is found in all the tested materials with strongest activity in the tertiary alkaloid fractions (Table 11). Because of the different extraction methods and also some differences in the pharmacological method used, it is not possible to draw any further conclusions. Fractionation of the tertiary alkaloid fraction by means of column chromatography and testing of these fractions on their pharmacological activity, leads to the results as summarized in Table 111. Obviously the muscle-relaxant activity is connected with the more polar alkaloid fractions, whereas weak clonic convulsions were found in fraction 2, in which the major component was found to be (+)-tubotaiwine. The isolated (+)-tubotaiwine also showed weak clonic convulsions at the same dose. In fraction 3 and 4 neither muscle-relaxant activity nor convulsant activity were found in the dose levels tested.
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Alkaloids of Strychnos angolensis
Specimen Collection Time of Collec- site the year tion No.
Part of plant
Alkaloid Fraction dontent
0 I1 I11 IV
Lg 6014 Nachtigal November Bark Cameroun 1965
v Lg 6014 Nachtigal November Top Cameroun 1965 branches
Lg 7340 Bertoua December Cameroun 1965
0 I1 I11 IV
Stem bark
Lg 7844Kribi June-July Cameroun 1970
Bark
Lg 10651 West October Cameroun 1972
Bark
Lg 11404 Makokou Gabon
0 I1 I11
October- Stem November bark 1977
++
++ + ++
v
C A D C A D C A CHCI,
Muscle-relaxant effect Convulsive effect Screen-grip in mice Rat dia- Clonic conv. Tonic conv. Graded Dose phragm. Res- Dose Res- Dose resp. mg/kg 50°/o ponse mg/kg ponse mg/kg inhibition mdml
-
X X
-
- 200 - -
-
(+)
500 250
-
-
-
-
-
- - (+)
200
- -
-
-
-
-
(+)
1000
(+)
1000
- - .-
300 300
X X
-
-
-
(+)
300
X XXX X XX
1000 275 75
(+)
275 7 5 - -
-
-
X X X X X
- - -
0.10
XX X X X
500 50 1000 350 50
0.13
0.12
+
- -
Minimum Reference lethal dose , mg/kg
(+)
-
300
-
500 500
-
-
-
-
-
-
-
500 100
- -
-
181
100
- -
-
[TI
300 300
- - - - - - 275 -
[TI
[91
75 [lo]
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Pharmacological summary of the specimen of S. angolensis
Alkaloids of Strychnos angolensis
27
Table 111 The pharmacological effects of the separated fractions of S. angolensis.
1 2
3
4
5
Muscle-relaxant Clonic Tonic effect convulsions convulsions Screen-grip Graded dose Graded dose Graded dose response mglkgresponse mg/kg response mg/kg
coloured material coloured material (+)-tubotaiwin (I) 17-0-methyl-11-methoxy-WGA (111), epi-17-O-methyl-11methoxy-diaboline (V) 11-methoxy-WGA (11) I 1-methoxy-diaboline (IV) A,-A,,'
-
-
-
-
(+)
-
-
X
-
Minimum lethal dose mg/kg
-
-
> 200
-
-
> 200
-
-
-
> 200
-
-
-
100
200
-
-
-
-
-
75
-
-
Alkaloids ArA,,
have not yet been isolated and identified.
These results were also confirmed by testing a basic toluene extract of the plant material, which mainly consists of the less polar alkaloids. Despite the presence of caracurine V, which exhibits muscle-relaxant activity [21, 221, the toluene extract showed clonic convulsions at a dose of 100 mg/kg. Dichloromethane may lead to the formation of quaternary chlorometho compounds. In the case of strychnine this compound does not show any convulsive activity but instead shows weak muscle relaxant activity 1251. Therefore also a purification of the alkaloids was carried out using chloroform instead of dichloromethane. Indeed a much weaker muscle-relaxant activity was found for the chloroform fraction. Further studies of the alkaloids present (A6-A13) in the polar fractions, which are responsible for the musclerelaxant activity, are to be carried out.
Experimental Plant Material The plant material, collection number Lg 7844, was collected by Professor F. SANDBERG and Dr. A. J. M. LEEUWENBERG during JuneJuly 1970 in Kribi, Cameroun. It was identified by Dr. A. J. M. LEEUWENBERG as S. angolensis GILG; a herbarium specimen is kept a t the herbarium in Wageningen, The Netherlands. Apparatus Melting points were determined with a Leitz Mikroskopheiztisch 350. UV spectra were recorded on a Shimadzu NPS 5000 instrument. IR spectra were obtained with a Perkin Elmer 157 G spectrophotometer. Mass spectra were carried out with an LKB 9000 instrument a t 70 eV with direct inlet. NMR spectra were measured in CDCI, on a Varian 100 MHz instrument with TMS as internal standard. The high performance liquid chromatograms were run on an instrument having a Waters model 6000 pump, U6 K injector and a Varian Variscan 635 UV detector. The optical rotation was determined on a Perkin Elmer 241 polarimeter.
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Frac- Main alkaloid content tion
Bohlin et al.
28
Column chromatography The dichloromethane extract (19.7 g) of S. angolensis was chromatographed over alumina oxide (Merck, activity 111). The column was eluted with ethylacetate containing an increasing amount of ethanol. The eluate was collected in 20 ml portions and after TLC comparison, combined into fractions (Table 111). TLC The following systems were used: Ethylacetate-2-propanol- 25 O10 ammonia, A. (100: 2:1), B. (70:25:5), C. (40:55:5), D. (45:35:10); E. Ethylacetate-diethylamine (36:l); F. Chloroform-cyclohecane-diethylamine (70:20:10); G . Toluene-ethylacetate-diethylamine (40:40:20), with 0.25 and 0.5 mm Merck Silica gel 60 F,,, precoated plates in saturated chambers. The systems A and F were run twice. A 10/o ceric sulphate solution in 10010 sul-
phuric acid was the spray reagent used .to locate the alkaloids. HPLC The solvent systems used were: H. Chloroform-methanol (containing 2 010 ammonia) (89:ll); I. Chloroform-methanol (containing 2 O/o ammonia) (85:15); J. Chloroform-methano1 (containing 4'10 ammonia) (96:4) with a stainless steel column (30 cm X 4.5 mm ID) filled with y-Porasil, at a flow rate of 1 ml/ min. with UV-detection at 254 nm. Isolation o f the alkaloids With preparative TLC six alkaloids were isolated from the fractions of S. angolensis. The solvent systems used were B, C, D and G dependent on the type of alkaloid. Semi-preparative HPLC was used to purify the isolated alkaloids. Physical data of the isolated alkaloids (+)-Tubotaiwin ( I ) was isolated as an amourphous powder with [a] = 638O (0.02; 231, 298 and 327 CHCI,). UV (EtOH): A,:, nm (log E 3.85, 3.80 and 4.00). IR (CHCI,): r,,,: 3400, 2950, 1680, 1615, 1475, 1250, 1160 and 1040 cm-'. IH-NMR: 6: 8.89 ( l H , s, N H , dissappears at deuteration), 7.20-6.75 (4H, m, aromatic protons), 3.75 (3H, s, -COOCH,), 3.15-2.75 (5H, m), 2.10-1.70 (4H, m), 1.0-0.6 (5H, m, -CH,CH,). MS (mle, Ole): 324 (M+; 25), 267 (13), 265 ( l l ) , 253 (lo), 240 (8), 229 (41), 209 (lo), 197 (17), 194 (15), 182 (19), 181 (16), 180 (26), 167 (28), 71 (100). The UV, IR, MS, NMR data and the Rf-value were identical to authentic (+)-tubotaiwin, showing blue colour with ceric sulphate spray reagent.
+
>
11-methoxy-Wieland-Gumlich aldehyde (II). UV (EtOH): A,,: 262 and 302 nm (log E 3.75 and 3.65). For mass spectra see Table I. On TLC, orange colour is observed with ceric sulphate spray reagent. 17-0-methyl-11-methoxy-Wieland-Gumlichaldehyde (III). UV (EtOH): l,,,: 255 and 300 nm (log E 3.80 and 3.70). IR (KBr): v,,,: 3380 (: NH), 2920, 2850, 1740, 1610, 1570, 1440, 1380, 1150 and 1030 cm-I. For mass spectra see
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Extraction 16 kg ground root- and stem bark of S. angolensis was extracted twice with 65 1 1010 acetic acid, at room temperature, stirring over night. The filtrate was acidified with 5010 hydrochloric acid to p H 2. Mayer's reagent was then added unril no more precipitate was formed. The precipitate formed was filtered and dissolved in acetone-methanol-water (6:2:1) and filtered again. The solution was run through a column packed with anion exchange resin Amberlite IRA 400, 20-50 mesh in the chloride form and the acetone-methanol-water mixture as eluant. The alkaloid solution, after removal of the acetone and methanol, was basified with 10OIo ammonia and extracted with dichloromethane. Both the aqueous and the dichloromethane phase were evaporated in vacuo to dryness. The yield of alkaloids was 0.7OIo. Another extract was done but instead of dichloromethane, chloroform was used, for a small amount of the same material. The third extraction method used for the bark of S. angolensis was carried out as follows: A small amount of ground plant material, basified with 10°/o NaHCO,, was extracted with toluene, which then was shaken with 10/o acetic acid. The water phase was then basified with NaHCO, and again extracted with toluene.
Alkaloids of Strychnos angolensis
11-methoxy-diaboline (IV). Crystallized from chloroformlpet. ether (4040') m.p. 210-14' C (lit. 214-16O) [18]. UV (EtOH: A,:, 252, 292 and 296 (log 4.02, 3.77 and 3.73). IR (KBr): v,,,: 3450, 2930,2850, 1660, 1610, 1500, 1460, '1400,1320,1300,1140,1020, 875, 830 and 770 cm-I. 'H-NMR: 6: 7.60 ( l H , d, J,,,,, = 3Hz, H-12), 6.97 ( l H , d, J,,,, = 9HZ, H-9), 6.64 ( l H , q, Jlo,l, = 3Hz and JlO,,= 9Hz, H-101, 5.80 ( l H , m, H-19), 5.30 ( l H , d, J = 2Hz), 3.82 (3H, s, 0-CH,), 2.38 (3H, s, CH3-CON). For mass spectra see Table I. On TLC orange colour is observed with ceric sulphate spray reagent.
Epi-17-0-methyl-11-methoxy-diaboline(V). Crystallized as needles from ethylacetateldiethy1 ether m.p. 190-92' C. UV (EtOH): A,:, 251, 292 and 297 (log E 4.00, 3.75 and 3.70). IR (KBr): v,,,: 2925, 2855, 1665, 1610, 1495, 1450, 1390,1305, 1140, 1015, 840 and 810 cm-I. 'H-NMR: 6: 7.61 (lH, dl Jle,lo = ~ H z H-12), , 6.96 (IH, d, J,,,, = 8Hz, H-9), 6.61 ( l H , q, Jlo,le = 2Hz and J,,,, = 8Hz), 5.80 ( l H , m, H-19), 4.70 (lH, d, J = 2Hz), 3.80 (3H, s, 0-CH,), 3.38 (3H, s, 0-CH,), 2.36 (3H, S, CH,-CON). For mass spectra see Table I. The same colour reaction with ceric sulphate on TLC as (IV). Caracurine V (VI). The alkaloid was isolated from the toluene extract by means of preparative TLC (0.5 mm Merck Silica gel 60 F,,,) with the solvent system D. UV and MS data were identical to the synthesized caracurine V [20]. TLC also showed the same Rf-values in the systems D and G. Decomposition experiments with the isolated alkaloids 11-methoxy-diaboline (IV) was treated with 1 N sulphuric acid using the method described by MARINI-BETT~LO et al. [19]. Preparative TLC was used to purify the major product which was identified as Il-methoxy-WielandGumlich aldehyde by means of TLC and MS. 11-methoxy-diaboline (IV) was also treated with absolute methanol (acidified with hydro-
gen chloride gas) in the way described by HYMON et al. [17] and DEYRUP et al. [la], which yielded the methyl ether of (IV). Preparative TLC was used to purify the formed products. Their identity was established by TLC and MS. Pharmacology The different fractions were weighed as bases, an equivalent amount of citric acid was added and the mixture was dissolved in saline (0.9 O/o NaC1). Female mice of the NMRI strain, weighing 18-25 g, were injected intraperitoneally with the solutions of the fractions, using between 2-10 mice for each fraction tested. The mice were observed for at least one hour for the occurrence of clonic and tonic convulsions [8]. The screen-grip test was used to determine the musclerelaxant effect [8].
Acknowledgements A grant from IF'S Stiftelse for Farmaceutisk Forskning is gratefully admowledged. We thank Prof. F. SANDBERG for discussion of the manuscript. Miss Karin HOLM and Mrs. Cilly STOLThave given skilful assistance in the technical and animal experiments respectively.
References 1. Leeuwenberg, A. J. M.: Medel. Landbouwhogeschool, Wageningen 69, 1 (1969). 2. Bisset, N. G. and A. J. M. Leeuwenberg: Lloydia 31, 208 (1968). 3. DenoEl, A., F. Jaminet, E. Philippot and M. J. Dallemagne: Arch. Int. Physiol. 59, 341 (1951). 4. DenoSl, A., F. Jaminet, G. Detilleux, M. van Sumsen and L. Merveille: Contribution A 1'Ctude chimique des Strychnos du Congo belge. Minisdre des Colonies, Direction de l'Agriculture, Bruxelles. pp. 208. 1953. 5. Marini-Bettblo, G. B.: Curare and Curarelike agents. Edited by D. Bovet, F. BovetNitti and G. B. Marini-Bettblo: Elsevier Publishing Co., Amsterdam. Curarizing alkaloids of Strychnos. pp. 138-148, 144 (1958).
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Table I. The same colour reaction on TLC as (11).
29
6. Bisset, N.G. and J. D. Phillipson: Lloydia 34, 1 (1971). 7. Sandberg, F., E. Lunell and K. J. Ryrberg: Acta Pharm. Suecica 6, 79 (1969). 8. Sandberg, F. R. Verpoorte and A. Cronlund: Acta Pharm. Suecica 8, 341 (1971). 9. Bohlin, L., Y. Ali and F. Sandberg: Acta Pharm. Suecica 11, 233 (1974). 10. Rolfsen, W., 2. M. Hakizadeh, F. Sandberg and J. Strombom: Acta Pharm. Succ. (in press). 11. Kump, W. G.,M. B. Patel, J. M. Rowson and H. Schmid: Helv. Chim. Acta 47, 1497 (1964). 12. Schumann, D. and H. Schmid: Helv. Chim. Acta 46, 1996 (1963). 13. Pinar, M. and H. Schmid: Liebigs Ann. Chem. 668, 97 (1963). 14. Pinar, M., U. Renner, M. Hesse and H. Schmid: Helv. Chim. Acta 55, 2972 (1972). 15. Heimberger, S. I. and A. I. Scott: JCS Chem. Comm. No. 6, 217 (1973). 16. Verpoorte, R.: unpublished results. 17. Hymon, J. R., H. Schmid, P. Karrer, A. Boller, H. Els, P. Fahrni and A. Fiirst: Helv. Chim. Acta 52, 1564 (1969).
18. Deyrup, J. A., H. Schmid and P. Karrer: Helv. Chim. Acta 45, 2266 (1962). 19. Marini-Bettblo, G. B., E. Miranda Delle Monache, S. Erazo Giuffra and C. Galeffi: Gazz. Chim. Ital. 101, 971 (1971). 20. Hesse, M.: Indolalkaloide Teil 1 und 2, Fortschritte der Massenspektrometrie, Vol. 1, Weinheim 1974, Verlag Chemie. 21. Verpoorte, R. and A. Baerheim Svendsen: J. Pharm. Sci. 67, 171 (1978). 22. Verpoorte, R.: Ph. D. Thesis. Leiden 1976. 23. Verpoorte, R. and A. Baerheim Svendsen: Phytochemistry 13, 2011 (1974). 24. Spiteller-Friedmann, M. and G. Spiteller: Liebigs Ann. Chem. 712, 179 (1968). 25. Phillipson, J. D. and N. G. Bisset: Phytochemistry 11, 2547 (1972).
Address: Wencbe Rolfsen Department o f Pharmacognosy Faculty of Pharmacy University of Uppsala, Biomedical Center Box 579, S-75123 Uppsala Sweden
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Bohlin et al.
plants-
medii
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Alkaloids and Biological Activity of Strychnos angolensis Department of Pharmacognosy, Faculty of Pharmacy, University of Uppsala, Biomedical Center, Uppsala, Sweden. *" Department of Pharmacognosy, University of Leyden, Gorlaeus Laboratories, Leyden, The Netherlands.
Key Word Index: Strychnos angolensis; Loganiaceae; (f)-Tubotaiwine; 11-methoxy-Wieland-Gumlich aldehyde; 17-0-methyl-1l-methoxy-wielandGumlich aldehyde; 11-methoxy-diaboline; epi-17-0-methyl-11-methoxydiaboline; Caracurine V; lndole alkaloids; Pharmacology. Abstract
tree, found on river banks in rain forests or in gallery forests up to an altiA weak convulsant effect is found in tude of 1500 m. It is found in Nigeria, the nonpolar part of the tertiary alkaCentral and East Africa. S. angolensis loid extract of Strychnos angolensis and S. malacoclados C. H. WRIGHTare GILG, while the polar part shows a closely allied and belongs to the same strong muscle-relaxant activity. botanical section, Breviflorae [I]. Five indole alkaloids have been isoBISSETand LEEUWENBERG have related from the nonpolar part of the extract. One is the known (+)-tubotai- ported the use of S. angolensis in ordeal wine while the others are derivatives of poisons [ 2 ] . Investigations by DENOEL et al. [3,4] Wieland Gumlich aldehyde (WGA): 11-methoxy-WGA, 17-0-methyl-1 1- of S. angolensis from Congo (Kinshasa) methoxy-WGA, 11-methoxy-diaboline have shown a rather variable alkaloid and epi-17-0-methyl-1 I-methoxy-dia- content, particularly in the root bark. They found that aqueous alcoholic tincboline. Caracurine V, a tertiary dimeric al- tures and crude alkaloid extract had a kaloid, is also found to be present in a curarizing effect when tested in frog toluene extract of bark of S. angolensis. and cat. However, MARINI-BETT~LO [5] found no curarizing action and a very small amount of quaternary alkaIntroduction loids in a study of the same species. Strychnos angolensis GILGis a climBISSETand PHILLIPSON [6]have screebing shrub or liana or a semi-scandent ned several specimen of S. angolensis
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Lars Bohlin*, Wenche Rolfsen*, Jan Strombom* and Robert Verpoorte**
Bohlin et al.
Results and Discussion
Phytochemistry From the nonpolar part of the tertiary alkaloid fraction of S. angolensis several alkaloids were isolated (Table 111). The formulae of the isolated alkaloids are given in Fig. 1, 2 and 4. (+)-Tubotaiwine (I) showed the typical a-methylenindoline chromophore of the akuammicine/condylocarpine type in the UV spectrum [ l 11. Because of the strong specific optical rotation,
+
[a] 2,2 = 638' (0.02, CHCl,), the akuammicine type is excluded [12]. The colour reaction with ceric sulphate, Rrvalues (systems F, G, H) and the spectral data established the identity, when compared to authentical (+)-tubotaiwine1 (I) (Fig. 1). Tubotaiwine has not
Fig. I . (+)-Tubotaiwine.
Kindly provided by Prof. P. POTIER,Gifsur-Yvette, France.
+ Fig. 2. The relationship between the WielandGumlih aldehyde derivatives. 1. R = H WG A 2. R=OCH3 11-methoxy-WGA (11) 3. R = H 17-0-methyl-WGA 4. R=OCH3 17-0-methyl-11-methoxy-WGA (111) 5. R = H epi-17-0-methyl-WGA 6. R=OCH3 epi-17-0-methyl-11-methoxy-WGA 7. R = H Diaboline 8. R=OCH3 11-methoxy-diaboline (IV) 9. R = H 17-0-methyl-diaboline 10. R=OCH3 17-0-methyl-1 1-methoxy-diaboline 11. R = H epi-17-0-methyl-diaboline 12. R=OCH, epi-17-0-methyl-11-methoxy-diaboline (V)
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for its alkaloid content, using herbarium samples. Three of the four investigated samples of leafy twigs contained alkaloids, whereas the fourth probably contained only traces of alkaloids. TLC of the extracts indicated the presence of at least 8 components a. o. a yellow alkaloid, which also has been observed in several other Strychnos species. Variation in the alkaloid content and pharmacological activity were also found for six materials of S. angolensis tested in the screening programme of African Strychnos species for convulsant and muscle-relaxant effects [7, 8,9, 101 (Table 11). The aim of further investigations on this species was therefore to locate the pharmacological activity in the extracts and isolate and elucidate the structures of the alkaloids. This study deals with the nonpolar fractions of the dichloromethane extract of S. angolensis GILG[8], whereas the polar fractions (alkaloids A,-A,,), which are responsible for the musclerelaxant activity, will be subject for further studies.
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Alkaloids of Strychnos angolensis 21
Bohlin et al.
are also shifted 14 mass units compared to (11) (Table I). Furthermore the presence of a fragment at 322 mle (fragment a in fig. 3) in the MS points to the axial position of the proton in the 17 position [20]. 11-methoxy-diaboline ( I V ) was identified by comparing its chromatographical- and spectral data with an authentic sample of this alkaloid*.
Epi-17-0-methyl-11-methoxy-diaboline ( V ) .showed similar spectral data to (IV). The absence of a peak at 3450 cm-I and the molecular peak at 396 instead of 382 compared to (IV) point to the presence of a methoxy group instead of a hydroxy group. The peaks in the MS corresponding to the non-aromatic part are also shifted 14 units compared to (IV) (Table 11). The isolated 17-0methyl ether (V) of I l-methoxy-diaboline has its methoxy group in the axial positions as it can be concluded from the 365 and 323 mle fragments (resp. c and d in fig. 3) in the MS 1201 and the 4.70 ppm signal for H-17 (equatorial) 11-methoxy-WGA (11) was identified in the 'H-NMR [17, 181. The structure by its UV and MS [19]. The identity is further confirmed by the treatment of was confirmed by converting ll-me- 11-methoxy-diaboline with methanol thoxy-diaboline (IV) into this alka- acidified with HC1-gas, which accorloid, by treating it with dilute sulphuric ding to TLC and MS gives a product similar to V. acid. Because of the strong muscle-relaxant 17-0-methyl-11-methoxy-WGA ( I I I ) effect found in the tertiary alkaloid had similar spectral data as (11). The fraction, the presence of tertiary dimeric molecular peak for (111) in t.he mass alkaloids of the toxiferine type was exspectrum at 354 is situated 14 units pected. Toluene has been found to be a higher than (11) (M+ = 340) which sug- suitable solvent for the extraction of gests a methoxy group instead of a hydroxy group. The peaks in the MS Kindly provided by Prof. Dr. C. GALEFFI, corresponding to the non-aromatic part Rome.
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been isolated from Strychnos species before, previously it has been found in several plant species belonging to the plant family, Apocynaceae [ l l , 13, 141. HEIMBERGER and SCOTT 1151 found condylocarpine to be an intermediate in the biosynthesis of strychnine in Strychnos nux vomica, but only very small amounts were present in the plant material. Hence it is interesting that this type of alkaloid is found as one of the major components in Strychnos species (tubotaiwine is also present in S. dolichothyrsa [I 61). The other identified alkaloids are derivatives of Wieland Gumlich aldehyde (WGA) (Fig. 2). The cyclic hemiacetal form of WGA has two possible epimeres, which can be converted to the corresponding acetals when treated with alcohols [I 7, 181. Diaboline (NBacetyl-WGA) behaves similar to WGA in this respect [17, 181. Several alkaloids isolated from S. angolensis fit into this general scheme, although they belong to the 11-methoxy series.
23
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Alkaloids of Strychnos angolensis
Fig. 3. Mass fragments of the Wieland-Gumlich aldehyde derivatives [19, 201.
Bohlin et al.
24
Table I Mass fragments of the Wieland-Gumlich aldehyd derivatives
M+ M+-R'OH
340 322 F 311 E 297 M+-R'COOH 294 .+-
46 5 40 15 3
17-0-methyl-1111-methoxymethoxy-WGA (111) diaboline (IV) R=H, R'=CH, R=COCH,, R'=H m/e O/o m/e O/o 354 322
100 8
epi-17-0-methyl-11methoxy-diaboline (V) R=COCH,, R'=CH, mfe Olo
{tIOH
D C B C-CH2C0 B-R'OH A-CH2C0
229 174 180
13 45 100
174 194
31 83
162
11
162
12
such alkaloids [16].Hence this solvent was chosen for the extraction of a small sample of the bark of S. angolensis. According to TLC (+)-tubotaiwine was the major component of the basic toluene extract. The WGA-type of alkaloids were only present in relatively small amounts. Bisnor-dihydrotoxiferine and bisnor-C-alkaloid H could not be detected in the toluene extract. The
Fig. 4. Caracurine V.
dimeric alkaloid caracurine V (VI) was, however, present as another major component in the toluene extract. By means of preparative TLC this alkaloid could be isolated and identified as caracurine V by comparison with a synthesized reference compound [21, 221 on TLC and its UV and MS data. I t should be noted that this alkaloid was not found in the dichloromethane- and chloroform-fractions. In fraction 4 of the column chromatographic separation, WGA could be detected by means of TLC. The isolated methyl ethers of I 1-methoxy-WGA and 11-methoxy-diaboline are probably artefacts due to the purification method used. In the conversion of rhe complex of Mayers reagent with the alkaloids, the complex is dissolved in a mixture of acetone, methanol and water and run over a ionexchange resin in the chloride
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11-methoxyWGA (11) R=H, R'=H mfe O/o
form. The alkaloids are eluted as chlorides. Under these conditions the methyl ethers may be formed from the hemiacetals. The fact that the monomeres of the WGA-type mainly belong to 11-methoxy series and the only dimeric alkaloid found so far is of the non-aromatic substituted type, needs further study. Dimeric alkaloids with 11-methoxy substitution might be present as well. However, the combination of 11-methoxydiaboline with non-aromatic substituted dimeric alkaloids is also found in S. dolichothyrsa and S. urceolata [16]. 11-methoxy-diaboline has already been found in the African species S. malacoclados [22, 231 and is probably present in S. henningsii [24] as well. S. angolensis is hence the fourth African species in the section Breviflorae of the genus Strychnos which certainly contains 11-methoxy-diaboline. . Interesting is the comparison with S. malacoclados is which according t o LEEUWENBERG closely related to and difficult to distinguish botanically from S. angolensis. The phytochemical investigations of these two species have so far shown several differences: The alkaloid content of the sternbark of S. angolensis is much higher than of S. malacoclados. In S. malacoclados the alkaloids normacusine B and 1I-methoxy-diaboline are found as major alkaloids [22]. In S. angolensis only the latter alkaloid has been found, the former alkaloid could not be detected so far in any fraction. Of the other alkaloids found in S. angolensis none could be detected in S. malacoclados. TLC in combination with the selective spray reagent ceric sulphate in sulphuric acid shows clearly the
difference in the alkaloid content of the two species. In S. angolensis a number of alkaloids are observed which give various colours with this reagent, ranging from blue to orange, a. o. caracurin V and (+)-tubotaiwine, (resp. blueviolet and blue) whereas in S. malacoclados only orange coloured spots are observed. The number of specimen investigated so far (S. angolensis 5, S. malacoclados 3) is, however, too small to draw more than preliminary conclusions from the observed differences. Pharmacology In the pharmacological screenings [7, 8, 9, 101 six materials of S. angolensis GILGhave been tested. Both convulsive and muscle-relaxant effects are found in varying degrees. Muscle-relaxant activity is found in all the tested materials with strongest activity in the tertiary alkaloid fractions (Table 11). Because of the different extraction methods and also some differences in the pharmacological method used, it is not possible to draw any further conclusions. Fractionation of the tertiary alkaloid fraction by means of column chromatography and testing of these fractions on their pharmacological activity, leads to the results as summarized in Table 111. Obviously the muscle-relaxant activity is connected with the more polar alkaloid fractions, whereas weak clonic convulsions were found in fraction 2, in which the major component was found to be (+)-tubotaiwine. The isolated (+)-tubotaiwine also showed weak clonic convulsions at the same dose. In fraction 3 and 4 neither muscle-relaxant activity nor convulsant activity were found in the dose levels tested.
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Alkaloids of Strychnos angolensis
Specimen Collection Time of Collec- site the year tion No.
Part of plant
Alkaloid Fraction dontent
0 I1 I11 IV
Lg 6014 Nachtigal November Bark Cameroun 1965
v Lg 6014 Nachtigal November Top Cameroun 1965 branches
Lg 7340 Bertoua December Cameroun 1965
0 I1 I11 IV
Stem bark
Lg 7844Kribi June-July Cameroun 1970
Bark
Lg 10651 West October Cameroun 1972
Bark
Lg 11404 Makokou Gabon
0 I1 I11
October- Stem November bark 1977
++
++ + ++
v
C A D C A D C A CHCI,
Muscle-relaxant effect Convulsive effect Screen-grip in mice Rat dia- Clonic conv. Tonic conv. Graded Dose phragm. Res- Dose Res- Dose resp. mg/kg 50°/o ponse mg/kg ponse mg/kg inhibition mdml
-
X X
-
- 200 - -
-
(+)
500 250
-
-
-
-
-
- - (+)
200
- -
-
-
-
-
(+)
1000
(+)
1000
- - .-
300 300
X X
-
-
-
(+)
300
X XXX X XX
1000 275 75
(+)
275 7 5 - -
-
-
X X X X X
- - -
0.10
XX X X X
500 50 1000 350 50
0.13
0.12
+
- -
Minimum Reference lethal dose , mg/kg
(+)
-
300
-
500 500
-
-
-
-
-
-
-
500 100
- -
-
181
100
- -
-
[TI
300 300
- - - - - - 275 -
[TI
[91
75 [lo]
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Pharmacological summary of the specimen of S. angolensis
Alkaloids of Strychnos angolensis
27
Table 111 The pharmacological effects of the separated fractions of S. angolensis.
1 2
3
4
5
Muscle-relaxant Clonic Tonic effect convulsions convulsions Screen-grip Graded dose Graded dose Graded dose response mglkgresponse mg/kg response mg/kg
coloured material coloured material (+)-tubotaiwin (I) 17-0-methyl-11-methoxy-WGA (111), epi-17-O-methyl-11methoxy-diaboline (V) 11-methoxy-WGA (11) I 1-methoxy-diaboline (IV) A,-A,,'
-
-
-
-
(+)
-
-
X
-
Minimum lethal dose mg/kg
-
-
> 200
-
-
> 200
-
-
-
> 200
-
-
-
100
200
-
-
-
-
-
75
-
-
Alkaloids ArA,,
have not yet been isolated and identified.
These results were also confirmed by testing a basic toluene extract of the plant material, which mainly consists of the less polar alkaloids. Despite the presence of caracurine V, which exhibits muscle-relaxant activity [21, 221, the toluene extract showed clonic convulsions at a dose of 100 mg/kg. Dichloromethane may lead to the formation of quaternary chlorometho compounds. In the case of strychnine this compound does not show any convulsive activity but instead shows weak muscle relaxant activity 1251. Therefore also a purification of the alkaloids was carried out using chloroform instead of dichloromethane. Indeed a much weaker muscle-relaxant activity was found for the chloroform fraction. Further studies of the alkaloids present (A6-A13) in the polar fractions, which are responsible for the musclerelaxant activity, are to be carried out.
Experimental Plant Material The plant material, collection number Lg 7844, was collected by Professor F. SANDBERG and Dr. A. J. M. LEEUWENBERG during JuneJuly 1970 in Kribi, Cameroun. It was identified by Dr. A. J. M. LEEUWENBERG as S. angolensis GILG; a herbarium specimen is kept a t the herbarium in Wageningen, The Netherlands. Apparatus Melting points were determined with a Leitz Mikroskopheiztisch 350. UV spectra were recorded on a Shimadzu NPS 5000 instrument. IR spectra were obtained with a Perkin Elmer 157 G spectrophotometer. Mass spectra were carried out with an LKB 9000 instrument a t 70 eV with direct inlet. NMR spectra were measured in CDCI, on a Varian 100 MHz instrument with TMS as internal standard. The high performance liquid chromatograms were run on an instrument having a Waters model 6000 pump, U6 K injector and a Varian Variscan 635 UV detector. The optical rotation was determined on a Perkin Elmer 241 polarimeter.
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Frac- Main alkaloid content tion
Bohlin et al.
28
Column chromatography The dichloromethane extract (19.7 g) of S. angolensis was chromatographed over alumina oxide (Merck, activity 111). The column was eluted with ethylacetate containing an increasing amount of ethanol. The eluate was collected in 20 ml portions and after TLC comparison, combined into fractions (Table 111). TLC The following systems were used: Ethylacetate-2-propanol- 25 O10 ammonia, A. (100: 2:1), B. (70:25:5), C. (40:55:5), D. (45:35:10); E. Ethylacetate-diethylamine (36:l); F. Chloroform-cyclohecane-diethylamine (70:20:10); G . Toluene-ethylacetate-diethylamine (40:40:20), with 0.25 and 0.5 mm Merck Silica gel 60 F,,, precoated plates in saturated chambers. The systems A and F were run twice. A 10/o ceric sulphate solution in 10010 sul-
phuric acid was the spray reagent used .to locate the alkaloids. HPLC The solvent systems used were: H. Chloroform-methanol (containing 2 010 ammonia) (89:ll); I. Chloroform-methanol (containing 2 O/o ammonia) (85:15); J. Chloroform-methano1 (containing 4'10 ammonia) (96:4) with a stainless steel column (30 cm X 4.5 mm ID) filled with y-Porasil, at a flow rate of 1 ml/ min. with UV-detection at 254 nm. Isolation o f the alkaloids With preparative TLC six alkaloids were isolated from the fractions of S. angolensis. The solvent systems used were B, C, D and G dependent on the type of alkaloid. Semi-preparative HPLC was used to purify the isolated alkaloids. Physical data of the isolated alkaloids (+)-Tubotaiwin ( I ) was isolated as an amourphous powder with [a] = 638O (0.02; 231, 298 and 327 CHCI,). UV (EtOH): A,:, nm (log E 3.85, 3.80 and 4.00). IR (CHCI,): r,,,: 3400, 2950, 1680, 1615, 1475, 1250, 1160 and 1040 cm-'. IH-NMR: 6: 8.89 ( l H , s, N H , dissappears at deuteration), 7.20-6.75 (4H, m, aromatic protons), 3.75 (3H, s, -COOCH,), 3.15-2.75 (5H, m), 2.10-1.70 (4H, m), 1.0-0.6 (5H, m, -CH,CH,). MS (mle, Ole): 324 (M+; 25), 267 (13), 265 ( l l ) , 253 (lo), 240 (8), 229 (41), 209 (lo), 197 (17), 194 (15), 182 (19), 181 (16), 180 (26), 167 (28), 71 (100). The UV, IR, MS, NMR data and the Rf-value were identical to authentic (+)-tubotaiwin, showing blue colour with ceric sulphate spray reagent.
+
>
11-methoxy-Wieland-Gumlich aldehyde (II). UV (EtOH): A,,: 262 and 302 nm (log E 3.75 and 3.65). For mass spectra see Table I. On TLC, orange colour is observed with ceric sulphate spray reagent. 17-0-methyl-11-methoxy-Wieland-Gumlichaldehyde (III). UV (EtOH): l,,,: 255 and 300 nm (log E 3.80 and 3.70). IR (KBr): v,,,: 3380 (: NH), 2920, 2850, 1740, 1610, 1570, 1440, 1380, 1150 and 1030 cm-I. For mass spectra see
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Extraction 16 kg ground root- and stem bark of S. angolensis was extracted twice with 65 1 1010 acetic acid, at room temperature, stirring over night. The filtrate was acidified with 5010 hydrochloric acid to p H 2. Mayer's reagent was then added unril no more precipitate was formed. The precipitate formed was filtered and dissolved in acetone-methanol-water (6:2:1) and filtered again. The solution was run through a column packed with anion exchange resin Amberlite IRA 400, 20-50 mesh in the chloride form and the acetone-methanol-water mixture as eluant. The alkaloid solution, after removal of the acetone and methanol, was basified with 10OIo ammonia and extracted with dichloromethane. Both the aqueous and the dichloromethane phase were evaporated in vacuo to dryness. The yield of alkaloids was 0.7OIo. Another extract was done but instead of dichloromethane, chloroform was used, for a small amount of the same material. The third extraction method used for the bark of S. angolensis was carried out as follows: A small amount of ground plant material, basified with 10°/o NaHCO,, was extracted with toluene, which then was shaken with 10/o acetic acid. The water phase was then basified with NaHCO, and again extracted with toluene.
Alkaloids of Strychnos angolensis
11-methoxy-diaboline (IV). Crystallized from chloroformlpet. ether (4040') m.p. 210-14' C (lit. 214-16O) [18]. UV (EtOH: A,:, 252, 292 and 296 (log 4.02, 3.77 and 3.73). IR (KBr): v,,,: 3450, 2930,2850, 1660, 1610, 1500, 1460, '1400,1320,1300,1140,1020, 875, 830 and 770 cm-I. 'H-NMR: 6: 7.60 ( l H , d, J,,,,, = 3Hz, H-12), 6.97 ( l H , d, J,,,, = 9HZ, H-9), 6.64 ( l H , q, Jlo,l, = 3Hz and JlO,,= 9Hz, H-101, 5.80 ( l H , m, H-19), 5.30 ( l H , d, J = 2Hz), 3.82 (3H, s, 0-CH,), 2.38 (3H, s, CH3-CON). For mass spectra see Table I. On TLC orange colour is observed with ceric sulphate spray reagent.
Epi-17-0-methyl-11-methoxy-diaboline(V). Crystallized as needles from ethylacetateldiethy1 ether m.p. 190-92' C. UV (EtOH): A,:, 251, 292 and 297 (log E 4.00, 3.75 and 3.70). IR (KBr): v,,,: 2925, 2855, 1665, 1610, 1495, 1450, 1390,1305, 1140, 1015, 840 and 810 cm-I. 'H-NMR: 6: 7.61 (lH, dl Jle,lo = ~ H z H-12), , 6.96 (IH, d, J,,,, = 8Hz, H-9), 6.61 ( l H , q, Jlo,le = 2Hz and J,,,, = 8Hz), 5.80 ( l H , m, H-19), 4.70 (lH, d, J = 2Hz), 3.80 (3H, s, 0-CH,), 3.38 (3H, s, 0-CH,), 2.36 (3H, S, CH,-CON). For mass spectra see Table I. The same colour reaction with ceric sulphate on TLC as (IV). Caracurine V (VI). The alkaloid was isolated from the toluene extract by means of preparative TLC (0.5 mm Merck Silica gel 60 F,,,) with the solvent system D. UV and MS data were identical to the synthesized caracurine V [20]. TLC also showed the same Rf-values in the systems D and G. Decomposition experiments with the isolated alkaloids 11-methoxy-diaboline (IV) was treated with 1 N sulphuric acid using the method described by MARINI-BETT~LO et al. [19]. Preparative TLC was used to purify the major product which was identified as Il-methoxy-WielandGumlich aldehyde by means of TLC and MS. 11-methoxy-diaboline (IV) was also treated with absolute methanol (acidified with hydro-
gen chloride gas) in the way described by HYMON et al. [17] and DEYRUP et al. [la], which yielded the methyl ether of (IV). Preparative TLC was used to purify the formed products. Their identity was established by TLC and MS. Pharmacology The different fractions were weighed as bases, an equivalent amount of citric acid was added and the mixture was dissolved in saline (0.9 O/o NaC1). Female mice of the NMRI strain, weighing 18-25 g, were injected intraperitoneally with the solutions of the fractions, using between 2-10 mice for each fraction tested. The mice were observed for at least one hour for the occurrence of clonic and tonic convulsions [8]. The screen-grip test was used to determine the musclerelaxant effect [8].
Acknowledgements A grant from IF'S Stiftelse for Farmaceutisk Forskning is gratefully admowledged. We thank Prof. F. SANDBERG for discussion of the manuscript. Miss Karin HOLM and Mrs. Cilly STOLThave given skilful assistance in the technical and animal experiments respectively.
References 1. Leeuwenberg, A. J. M.: Medel. Landbouwhogeschool, Wageningen 69, 1 (1969). 2. Bisset, N. G. and A. J. M. Leeuwenberg: Lloydia 31, 208 (1968). 3. DenoEl, A., F. Jaminet, E. Philippot and M. J. Dallemagne: Arch. Int. Physiol. 59, 341 (1951). 4. DenoSl, A., F. Jaminet, G. Detilleux, M. van Sumsen and L. Merveille: Contribution A 1'Ctude chimique des Strychnos du Congo belge. Minisdre des Colonies, Direction de l'Agriculture, Bruxelles. pp. 208. 1953. 5. Marini-Bettblo, G. B.: Curare and Curarelike agents. Edited by D. Bovet, F. BovetNitti and G. B. Marini-Bettblo: Elsevier Publishing Co., Amsterdam. Curarizing alkaloids of Strychnos. pp. 138-148, 144 (1958).
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Table I. The same colour reaction on TLC as (11).
29
6. Bisset, N.G. and J. D. Phillipson: Lloydia 34, 1 (1971). 7. Sandberg, F., E. Lunell and K. J. Ryrberg: Acta Pharm. Suecica 6, 79 (1969). 8. Sandberg, F. R. Verpoorte and A. Cronlund: Acta Pharm. Suecica 8, 341 (1971). 9. Bohlin, L., Y. Ali and F. Sandberg: Acta Pharm. Suecica 11, 233 (1974). 10. Rolfsen, W., 2. M. Hakizadeh, F. Sandberg and J. Strombom: Acta Pharm. Succ. (in press). 11. Kump, W. G.,M. B. Patel, J. M. Rowson and H. Schmid: Helv. Chim. Acta 47, 1497 (1964). 12. Schumann, D. and H. Schmid: Helv. Chim. Acta 46, 1996 (1963). 13. Pinar, M. and H. Schmid: Liebigs Ann. Chem. 668, 97 (1963). 14. Pinar, M., U. Renner, M. Hesse and H. Schmid: Helv. Chim. Acta 55, 2972 (1972). 15. Heimberger, S. I. and A. I. Scott: JCS Chem. Comm. No. 6, 217 (1973). 16. Verpoorte, R.: unpublished results. 17. Hymon, J. R., H. Schmid, P. Karrer, A. Boller, H. Els, P. Fahrni and A. Fiirst: Helv. Chim. Acta 52, 1564 (1969).
18. Deyrup, J. A., H. Schmid and P. Karrer: Helv. Chim. Acta 45, 2266 (1962). 19. Marini-Bettblo, G. B., E. Miranda Delle Monache, S. Erazo Giuffra and C. Galeffi: Gazz. Chim. Ital. 101, 971 (1971). 20. Hesse, M.: Indolalkaloide Teil 1 und 2, Fortschritte der Massenspektrometrie, Vol. 1, Weinheim 1974, Verlag Chemie. 21. Verpoorte, R. and A. Baerheim Svendsen: J. Pharm. Sci. 67, 171 (1978). 22. Verpoorte, R.: Ph. D. Thesis. Leiden 1976. 23. Verpoorte, R. and A. Baerheim Svendsen: Phytochemistry 13, 2011 (1974). 24. Spiteller-Friedmann, M. and G. Spiteller: Liebigs Ann. Chem. 712, 179 (1968). 25. Phillipson, J. D. and N. G. Bisset: Phytochemistry 11, 2547 (1972).
Address: Wencbe Rolfsen Department o f Pharmacognosy Faculty of Pharmacy University of Uppsala, Biomedical Center Box 579, S-75123 Uppsala Sweden
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Bohlin et al.
