Full text of DOI:10.1016/S0031-9422(00)89897-0

2511
Press. Printed in England.
RHYNCHOPHYLLINE AND ISORHYNCHOPHYLLINE
N-OXIDES FROM SPECIES OF MITRAGYNA
E.J.
D. PHILLIPSON and K.
Pharmacognosy Research Laboratories, Department of Pharmacy, Chelsea College, University of London,
Manresa Rd., London S.W.3
(Received 31
1970)
Abstract-The ‘base-line’ alkaloid previously isolated from leaves of Mitragyna
(Roxb.)
0. Kuntze and Mitrugyna
(Willd.) 0. Kuntze, has been identified as isorhynchophylline N-oxide.
Rhynchophylline N-oxide has also been isolated from the leaves of M. inermis.
INTRODUCTION
THE ALKALOIDS obtained from species of Mitrugyna
are indolic or oxindolic
oxindole alkaloids being
with closed or open E rings (E
shown in I.
the structure of the E
Stereoisomerism of E
normal type alkaloids having
oxindole alkaloids occurs with
type having
type alkaloids having
do not exist]. Isomerism also occurs
and
and
and
and
[pseudo type oxindole alkaloids
and
at to give A and B series oxindoles while substitution may occur at
in the aromatic
ring.
The earlier examinations of the leaves of
(Roxb.) 0 Kuntze,
indicated the presence of rhynchophylline (I, normal,
B series), rotundifoline
which was later shown to be identical with
Later investigations showed the absence of
(I, normal,
isorotundifoline (I, normal
A series) and mitragynol
B
series).
rotundifoline and isorotundifoline and the presence of rhynchophylline, isorhynchophylline
A series) and a third alkaloid referred to as the ‘base-line’ alkaloid because
it remained on the base line during TLC with various solvent This alkaloid was
(I, normal
similar to an alkaloid isolated from the leaves of Mitrugyna inermis (Willd.) 0.
The structure of this alkaloid is now discussed, together with a new
obtained from the leaves of M. inermis.
alkaloid
G. BARGER, E. DYER and L. J. SARGENT, J. Org.
4,418 (1939).
867 (1950).
G. M. BADGER, J. W.
and P. A. ONGLEY, J.
A. H.
and A. N. TACKIE, 1122 (1963).
G. M. BADGER, L, M.
R.
and E. WENKERT
,
Chem.
Lond. 206 (1963).
E. J.
SHELLARD and J. D.
12, 27 (1964).
A. N. TACKIE, Ph.D. Thesis. University of London (1963).
E. J. SHELLARD and K. SARPONG, J. 21, (Suppl.),
(1969).
2505

J . SH E L L AR D , J . D.
a n d K.
2506
R E S U L T S A N D D I S C U S S I O N
The ‘base-line’ alkaloid from
m.p.
analysed for
(mol. wt.
while the equivalent weight was determined by non-aqueous titration as 399;
the indication being that the alkaloid contains one additional oxygen atom to that present in
rhynchophylline (mol. wt. 384). The UV spectrum
(log 225 nm (log 278 nm (log
243 nm (log
is identical with that of
283 nm
phylline and isorhynchophylline. In the IR spectrum a broad band occurs at 2500 cm-’
similar to that given with rotundifoline, suggesting the possible presence of hydrogen
bonding. An imino group (3350
ester and oxindole carbonyls
and 1685 cm-‘)
and a double bond (1625 cm-‘) are also indicated to be present. The NMR (60 MHz) shows
a three-proton triplet at S
for the
confirming that the alkaloid is an E
and
not closed E ring type. Two three-proton singlets at S
and
corresponding to two
methoxyl groups and a signal at S
belonging to an olefinic proton also points to the
presence of the ester/vinyl ether grouping,
appear as a four-proton multiplet at S
The aromatic protons
confirming that the oxindole moiety is
substituted in the aromatic ring. The imino group is revealed by a one-proton singlet at
S
which disappears on deuteration,
A similar ‘base-line’ alkaloid was obtained recently from the leaves of
inermis’ as
needle crystals (13 mg), m.p.
‘base-line’ alkaloid; the UV and IR spectra and TLC with several systems
were identical. The mass spectrum of the alkaloid was also very similar to that of the
rotundifolia ‘base-line’ alkaloid which showed a molecular ion peak at m/e 400
with a prominent peak at m/e 384 molecular ion peak for rhynchophylline) and at
m/e 239 (II, also found in the mass spectrum of rhynchophylline). Peaks associated with
fragment II were also present at m/e 224 (loss of Me), 210 (loss of Et), 208 (loss of and
69; these peaks are characteristic for rhynchophylline-type Peaks belonging
to the oxindole portion of the molecule were noted at m/e 130, 144, 145, 146 and The
undepressed by admixture with the
mass spectrum therefore, indicated that the ‘base-line’ alkaloid has a rhynchophylline-type
structure which readily loses 16 mass units. Hence, it is considered to be an N-oxide of one
of the rhynchophylline-type alkaloids, i.e. isorhynchophylline (normal, A), rhynchophylline
(normal, B), corynoxine
A), corynoxine B
B)
-Me
- - - - m / e 2 2 4
2 1 0
2 0 8
When the
inermis ‘base-line’ alkaloid was reduced with sulphurous acid, the reaction
mixture made alkaline with ammonia, extracted with chloroform, and examined by TLC, the
results clearly indicated that only one alkaloidal spot was present, corresponding to
rhynchophylline, and that rhynchophylline, corynoxine and corynoxine B were absent.
A. H.
B. GI L B E R T, J . A.
Am.
D. DW U M A- BAD U a n d R. E. HA D D O C K,
N. F I N C H , W. I. TAYL O R , H.
85, 1523 (1963).
25, 5961 (1969).
J . M. WILSON and C.

Rhynchophylline and isorhynchophylline N-oxides from species of Mitragyna
2507
When isorhynchophylline was treated with 15 hydrogen peroxide at room temperature,
crystals separated out of the reaction mixture, which were identified as isorhynchophylline
N-oxide and had
mixed
UV, IR, NMR and mass spectra identical with those of
the ‘base-line’ alkaloid. Rhynchophylline N-oxide was similarly prepared from rhyncho-
phylline, isolated as an amorphous solid and shown to differ from isorhynchophylline
N-oxide by TLC. TLC comparison of rhynchophylline N-oxide with the mother-liquors
from which the ‘base-line’ alkaloid of
inermis was obtained, showed that the major spot
in the mother-liquors corresponded to rhynchophylline N-oxide. This major component was
isolated from the ‘base-line’ alkaloid mother-liquors by preparative TLC to yield an
amorphous solid (2 mg), identical with rhynchophylline N-oxide (by mass spectrum and
TLC behaviour in three systems). Reduction of a trace of the new alkaloid with sulphurous
acid, followed by extraction and TLC examination, showed only one alkaloid to be present,
namely, rhynchophylline, and that isorhynchophylline, corynoxine and corynoxine B were
absent.
In order to check whether the N-oxides were reduced to the corresponding bases without
isomerization at
samples of the prepared rhynchophylline and isorhynchophylline
N-oxides were similarly treated with sulphurous acid, the reaction mixtures made alkaline
with ammonia and extracted with chloroform. TLC examination confirmed that the
oxides had been reduced to the corresponding alkaloids and that no isomerization had taken
place.
Further evidence for the structures and, more particularly, the conformations of
rhynchophylline N-oxide (III) and rhynchophylline N-oxide (IV) was obtained from a study
of their NMR spectra. The 100 MHz spectrum of prepared isorhynchophylline N-oxide
showed a poorly resolved three-proton triplet at
methylene protons are not affected by the oxygen at
which established that the
This suggests that the con-
formation of the N-oxide is the same as that of the parent base. Support for this is also
provided by the signals for the aromatic protons which appear as a three-proton multiplet
at
with the NMR spectrum of isorhynchophylline, the signal for the
field by
and
and as a one-proton doublet at
Compared
proton is shifted down-
ppm and it is evident that this shift must be due to deshielding by the oxygen on Nb.
The 100 MHz NMR spectrum of rhynchophylline N-oxide (IV) is similar to that of
rhynchophylline N-oxide (III) but differs in that the signal for the
the other aromatic protons in the four-proton multiplet at
proton appears with
Hence, in rhyncho-
phylline N-oxide the
proton is not affected by the oxygen at Nb and this is further
evidence for no conformational changes.
W. F.
T
R AGER
,
M. LE E, J . D.
R. E. H ADDOCK , D.
and A. H. BECKETT ,
Tetrahedron 24, 523 (1968).

2508
E. J . SH E L L AR D . J . D. P H I L L I P S O N a n d K. SAR P O N G
on the basis of the CD curves of
It has been proposed,”
phylline and of
(the corresponding normal
and normal A
closed E-ring oxindole alkaloids), that a negative Cotton effect in the 280-290 nm region can
be attributed to the A oxindole configuration whereas alkaloids with the B configuration
show in the same region a positive Cotton effect. A negative Cotton effect in the 250-270 nm
region has been attributed to a
establishing the structures of the four closed E-ring oxindole alkaloids, uncarines C, D, E
and F (i.e. A and B, epiallo A and B). However, this is not always the case, because
B alkaloid, corynoxine B, shows a positive Cotton effect between 250 and 270 nm,
Similar arguments have been used for
the
when it does, in fact, possess a
suggesting a
Apparently anomalous behaviour is shown by rotundifoline (9-hydroxyisorhynchophylline)
which has a positive Cotton effect and by isorotundifoline (9-hydroxyrhynchophylline) which
has a negative Cotton effect, in the 280-290 nm region. Although the structure of
foline (partial V) is similar to that of isorhynchophylline N-oxide (III), in that an oxygen
atom is present between Nb and
there is no indication of anomalous behaviour and the
CD curves of isorhynchophylline N-oxide and isorhynchophylline both show a negative
Cotton effect between 280 and 290 nm; rhynchophylline N-oxide and rhynchophylline curves
have a positive Cotton effect in the same region (Fig. 1). The fact that the isorhynchophylline
N-oxide prepared from isorhynchophylline and the naturally-occurring isorhynchophylline
N-oxide have identical CD curves demonstrates that they both possess the same absolute
configuration.
- - - - - - - - Rhynchophylline
8
Rhynchophylline -N-oxide
Isorhynchophylline
Is orhynchophylline-N-oxide
F I G. 1. C.D. C U R VE S O F R H YN C H O P H YL L I N E , I S O R H YN C H O P H YL L I N E AN D T H E I R N -O XI D E S.
J . P OISSON a n d M.
J . L.
A. F .
A. F .
Tetrahedron Letters 6283 (1966).
N. K. H AR T, S. R. J OH NS and J . A. LAMBE R TON , Tetrahedron Letters 991 (1967).
21,491 (1968).
N. K. H A R T, S. R. J OH NS a n d J . A. L AM B E R T O N ,

Rhynchophylline and isorhynchophylline N-oxides from species of Mitragyna
2509
Mass spectrometry has been shown to reflect stereochemical differences in the
yohimbine alkaloids.* However, it has been stated that the abundance of the m/e 239 frag-
ment in the oxindole alkaloids seems to be independent of the stereochemistry at
perhaps due to thermal isomerizations at the high temperature of the mass spectrometer
Nevertheless, if the relative abundances reported for the m/e 239 peak are compared
for the four unsubstituted normal and
that the relative abundance is higher for the B isomers than for the corresponding A isomers,
i.e. 239 relative abundance for isorhynchophylline (normal A) rhynchophylline
(normal B) 82 corynoxine A) 46 corynoxine B B) 71%.
A similar relationship exists for the two N-oxides i.e. m/e 239 relative abundance for
rhynchophylline N-oxide (normal A) 43 rhynchophylline N-oxide (normal B) 78 Hence,
A and B, E-seco oxindole alkaloids, it can be seen
it would appear that if unsubstituted alkaloids are compared, the relative abundance of the
m/e 239 peak could well be a reflection of their stereochemistry. This, however, is not the
case with the
It is tempting to ascribe the broad band at 2500 cm-’ in the
phylline N-oxide to the hydrogen bonded since a Dreiding model shows the close
substituted oxindole alkaloids.
spectrum of
proximity of this hydrogen to the oxygen at Nb. However, this seems unlikely since there is
no evidence that aromatic C-H takes part in such bonding. We are not therefore in a position
to assign the band at 2500
The naturally-occurring oxindole alkaloids can be readily isomerized about the
and/or
centres by heating with either pyridine or acetic acid. It has been suggested that
bond
such isomerization involves scission and reformation of the
In order to establish what would happen to oxindole N-oxides under such conditions,
rhynchophylline N-oxide and isorhynchophylline N-oxide were each refluxed for 48 hr with
both pyridine and 50% aqueous acetic acid. TLC examinations of the reaction mixtures
showed that with pyridine the oxygen at Nb was lost and the parent oxindole alkaloid pro-
duced, whether rhynchophylline or isorhynchophylline, isomerized in the expected manner
to give a mixture of isorhynchophylline (major) and rhynchophylline (minor). With acetic
acid it was also noted that both N-oxides readily lost the oxygen at Nb.
There is a possibility that the N-oxides are formed during the isolation procedure,
particularly as the N-oxides were isolated after the tertiary bases had been obtained. In order
to establish whether the N-oxides are natural products, a small quantity of
inermis leaves
was extracted by maceration with 96 ethanol. The concentrated alcoholic extract proved
too crude for TLC examination, but after shaking with chloroform: 5% ammonia the
chloroform layer could be examined by TLC, and showed that rhynchophylline N-oxide and
isorhynchophylline N-oxide were present in the extract together with rhynchophylline and
isorhynchophylline. As similar treatment with rhynchophylline and isorhynchophylline did
E. WEN KERT, J. H. UDELHOFEN and N. K.
Am.
(1959).
J. C.
J. L.
M. D.
and J. PO ISSO N , C.R.
0. E. ED WARD S and L. MARIO N , Can.
Sci., Paris 259, 597 (1964).

E. J. SHELLARD, J. D.
and K.
2510
not give the N-oxides nor the other isomer, it is considered that rhynchophylline and
rhynchophylline and their N-oxides are natural products and not
From the biosynthetic point of view it might be envisaged that A and B oxindole tertiary
alkaloids are first formed and that the N-oxides are formed subsequently. This would be
supported by the fact that the N-oxides of both A and B isomers were isolated. Once formed,
it may be that the parent alkaloids and corresponding N-oxides are interconvertible.
The isolation of isorhynchophylline N-oxide and rhynchophylline N-oxide from two
species of
of
yohimbine
would suggest that these alkaloids may also be present in other species
and it could be anticipated that other N-oxides of oxindole and
and E-closed alkaloids will also be isolated.
EXPERIMENTAL
are uncorrected; IR spectra were in (Nujol); NMR spectra in
with TMS internal reference;
CD curves, Roussel-Jouan Dichrograph, methanol solutions, 1
ml; mass spectra were determined on
AEI MS902 high resolution mass spectrometer at 70
titration of 5 mg samples with N/50 perchloric acid in
G (Merck) with A,
Range of
inlet temperature, 285”; equivalent weight by
using Oracet blue indicator; TLC, silica gel
(60: 35
(5 4); B,
C,
values:
C
B
A
isorhynchophylline
rhynchophylline
68-75
60-64
84-89
69-72
70-72
24-33
isorhynchophylline N-oxide 0
rhynchophylline N-oxide
5-6
0
Isolation of isorhynchophylline N-oxide. The alkaloid was isolated from M.
and from
M, inermis leaves as previously described.
M. rotundifolia alkaloid. Colourless needle crystals from light
petroleum-ethanol,
C, 65.99; H, N,
4.09). IR 3350
242-243”. Found: C,
eq. wt. 400. UV
H, 7.37; N, 7.47; eq. wt. 399
required,
225 nm
ester and
243 nm (log
283 nm (log
(NH, weak), 2500
(broad), 1700
(broad CO and 1675
oxindole
ester
950
s, vinyl
(N-O) NMR signals
60 MHz,
3.63
s,
s, NH, dis-
210 (13
3.67
m, aromatic), 7.29
384 (78
130 (100%) 69 (61%).
UV, IR, TLC (systems B and C) with synthetic isorhynchophylline N-oxide.
mixed UV, IR, TLC (systems B and C) identical with
s, olefinic), 8.25
239 (43
appears on deuteration). Mass spectrum m/e
8
224 (37
208
159
146
145
144
Identical m.p., mixed
M. inermis alkaloid.
‘base-line’ alkaloid and synthetic isorhynchophylline N-oxide, Mass spectrum,
400
384,239, 224,
210, 208, 159, 146, 145, 144, 130, 69.
Isolation of
N-oxide. The mother liquor of isorhynchophylline N-oxide, isolated from
M. inermis, was subjected to preparative TLC (silica gel/methanol), the major alkaloidal band extracted with
methanol, concentrated to dryness and extracted with Concentration to dryness yielded 2 mg of an
amorphous residue which was found to be identical in UV, IR, and TLC (systems A, B and C) with synthetic
rhynchophylline N-oxide. Mass spectrum, m/e 400
384 (66
130
239 (100
69 (96%).
224 (56
2
(42
208
159
Preparation of
ml) and 15
146
145
144
N-oxide. Isorhynchophylline (150 mg) was dissolved in 96
ethanol
added
ml). The mixture was allowed to stand at room temp. overnight and then
heated on a boiling water bath for 30 min. On cooling colourless needle crystals were obtained, dissolved in
water (3 ml) and boiled with
needle crystals (110 mg, 70%)
wire for 5 min. Two
242”. UV
from
245 nm (log
ethanol yielded colourless
286 nm (log
226 nm (log 4.07). IR 3400 cm-’ (NH, weak), 2500 cm-’ (broad), 1700 cm-’ (broad CO with inflexion
at 1675 cm-‘), 950
(N-O). NMR
s, vinyl
100 MHz,
m, aromatic protons
Mass spectrum m/e
146 145
t, poorly resolved,
s, ester
vinyl), 8.22
384
3.68
d,
239
69
Preparation of rhynchophylline N-oxide. Rhynchophylline (150 mg) was treated with
7.23
s,
144
11.43
224
broad, NH, disappears with
210 208 159
as described
above. Synthetic rhynchophylline N-oxide could not be obtained crystalline but only as an amorphous
powder (61 mg, 38%). UV
245 nm (log
287 nm (log
226 nm (log 3.85).
100 MHz,
IR 3400 (NH, weak), 1700 and 1710
(CO, broad doublet). NMR

Rhynchophylline and isorhynchophylline N-oxides from species of
2511
t, poorly resolved,
s, ester
s, NH disappears with
210 208 159
3.75
s, vinyl
m, aro-
matic protons), 7.22
s, vinyl),
224
Mass spectrum m/e 400
146 14.5
384
130
239
144
(180%).
Reduction of N-oxides. Natural and synthetic isorhynchophylline N-oxide and synthetic rhynchophylline
N-oxide were reduced as follows. Sulphurous acid
ml, 5 % w/v
was added to the N-oxide (1 mg),
(3 x 1 ml). The com-
allowed to stand overnight, made alkaline with ammonia and extracted with
bined extracts were concentrated and examined by TLC. Natural rhynchophylline N-oxide was reduced by
the same procedure using only a trace of material and 1 drop of sulphurous acid. TLC examination (systems
A, B and C) showed that isorhynchophylline N-oxide (natural and synthetic) was completely reduced to
isorhynchophylline only whilst rhynchophylline N-oxide (natural and synthetic) was partially reduced
(estimated 50%) to rhynchophylline only.
Aclcnowledgements-We thank Mr. E.
(then) Head of the Department of Pharmacy, University of
Science Technology, Kumasi, Ghana, and Professor A. N.
macy at Kumasi, for providing the plant material, Mr. G.
present Dean of the Faculty of Phar-
for the 60 MHz NMR spectra,
Mr. A. Saunders for C.D. determinations and Mr. R. Brown for the figures. 100 MHz NMR spectra were
determined at the Physico-Chemical Measurements Unit, Harwell, Berks. for which we thank the
Director, and the mass spectra were determined at the School of Pharmacy, University of London, and we
acknowledge the help of Dr. B. J. Millard and Mr. D. Carter. We are also grateful to Mr. R. F. Branch for
discussions of IR spectra and Dr. N. G. Bisset (both of this College) for helpful comments during the pre-
paration of the manuscript. One of us (KS) wishes to thank the
Government and the University of
Science Technology, Kumasi, for a scholarship enabling him to undertake research work in the
Research Laboratories at Chelsea College, University of London.