Phymchemistry, Vol. 33, No. 2, pp.497-500, 1993 0031-9422/93 $6.00+0.

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Printed in Great Britain. @ 1993 Pergamon Press Ltd

CYTOTOXIC ALKALOIDS OF -ANNONA MONTANA

YANG-CHANG WV,* GWO-YUAN CHANG, CHANGYIH DUH and SHANG-KWEIWANG~

Graduate Institute of Natural Products and $Department of Microbiolo~, Kaohsiung Medical College, Kaohsiung 807, Taiwan,
Republic of China

(Received in revised firm 24 1992)

September

Key Word Index-Annona montana; Annonaceae; alkaloids; annoretine; annolatine; cytotoxicity.

Abstract-The leaves of Annona montana afforded one novei cytotoxic phenanthrene alkaloid, annoretine [Zhydroxy-
4-methoxy-N-methyl-tetrahydropyrido(4,3-a)phenanthrene], together with two cytotoxic alkaloids, argentinine and
liriodenine, as well as a new oxoaporphine alkaloid, annolatine, and one steroid, sitosterol+D-glucoside, which are
inactive. The structures of annoretine and annolatine were elucidated from spectral data and chemical evidence.

INTRODUCl’lON mass spectrometry (found 293.3371, calcd 293.3660). Its

Annona montana is a small evergreen tree which is widely
distributed from west India to southern Brazib it is
cultured for its fruit in Taiwan El]. Several alkaloids
[2-51, one anthraq~one c6] and two bioactive com-
pounds, annoquinone-A and j?-sitostenone [6] were pre-
viously reported from the leaves, stems, stem barks and
root barks of this plant. As a result of our continuing
searches for novel plant antitumour agents, the meth-
anolic extract of the leaves of A. montana was found to
show significant cytotoxicity against in vitro tissue culture
cells of human KB, A-549 lung ~rc~noma and HT-29
colon tumour as well as in murine P-388 and L-1210
lymphocytic leukemia. Bioassay-directed fractionation
traced the active fractions to alkaloid components. We
report herein on the isolation and ch~acte~tion of one
novel cytotoxic phe~n~~ne alkaloid, annoretine (I),
together with two known cytotoxic alkaloids, Argentine
(2) and liriodenine (3), in addition to a new oxoaporphine
alkaloid, annolatine (4), which is not bioactive. Annore-
tine (1) is the second example of a naturally occurring
phenanthrene alkaloid possessing an isoquinoline moiety
[73. Moreover, 1 is isolated for the first time from the
family Annona~ae.
RESULTS AND DiSCUSXON
The methanolic extract of A. montuna leaves was
f~~ionated by in vitro KB cell ~otoxi~ty tests. Further
separation and pu~fi~tion by chromato~phy fur-
nished the principal active components, annoretine (I),
argentinine (2) and liriodenine (3).
Annoretine (1) was isolated as an oily base. Its molecu-
lar formula was established as C,,H1,N02 by HREI
*Author to whom correspondence should be addressed.
UV absorption maxima at 205, 231, 255, 302, 343 and
361 nm are characteristic of a phenanthrene 18-l 11, The
IR absorption at 3350 cm-’ and a bathochromic shift of
UV absorption while adding alkaline solution indicate
the presence of a phenolic function. The ‘H NMR spec-
trum (in CDCI,) of 1 shows six aromatic proton signals at
H-6, I-I-7, H-8, H-9 and H-10 [67.57-7.90 (.5H, m)] and
H-5 C69.39 (lH, m), a characteristic signal of H-5 of
phenanthrenes] [S-113, three methylenes (63.81, s; 3.32, t,
J = 6.0 Hz; and 2.90, t, f ==6.0 Hz), one N-Me group
C62.61 (3H, s)] and one DzO exchangeable signal (66.45).
The latter indicates one phenolic function which was
supported by the UV and IR spectra. The 13C NMR
spectrum of 1 shows signals of 14 aromatic carbons,
constituting the phenanthrene moiety, one OMe, three
CH,, of which two carbons (653.1 and 53.6) are N-linked,
and one N-Me. The formula 1 indicates 11 double bond
and ring equivalents of which the phenanthrene moiety
contains 10. Subtracting two oxygenated carbons
(6, > 140 ppm) and four ring-bound quaternary carbons
of the phenanthrene from the eight quaternary carbons
observed in the ‘jC NMR spectrum leaves two quatern-
ary aromatic carbons. The unlocated three methylenes
and a nitrogen together with these two carbons, hence
constitute the remaining ring, i.e. a ~t~ydropy~dine
ring. The location of this ring, which fuses with phenan-
threne, is in full agreement with litebamine [7]. Recently,
we proposed that litebamme could be biogenetically
derived from boldine via secoboldine as an in~~~ia~
[7], and we conkmed it by ~m~synthesis [ 121. Therefore,
on the basis of the above results and argentinine (2)
existing in the same plant which can act as an inter-
mediate, the substitution pattern is determined and the
structure of annoretine (1) should be represented as 1
[3-hydroxy-4-methoxy-N-methyl-tetrahydropyrido (4,3-
a) phen~threne]. Annoretine (1) showed significant cyto-
toxicity against RB, P-388, A-549 and HT-29 cells with

497
498 Y.-C. Wu et al.

(1) (2) R=H

(h) R=Ac

(2) R,+R2=-OCH,O-, R,=R,=R,=R,=H

(4) R,=R3=R,=OMe, R2=OH, RS=R6=H
(&) R,=R3=R4=OMe, Rz=OAc, RS=R6=H
0
($J RI=R&R6=OMe, R,=OH, R,=R,=H
(6) R,=OH, R2=R3=R4=OMe, R,=R,=H
(z> R,=OH, R2=RS=R6=OMe, R3=R4=H

R4

ED,, values of 14.47, 3.33, 4.46 and 2.51 pg ml-‘, re-
spectively.
The identities of 2 and 3 were confirmed by direct
comparison with authentic samples and by spectral ana-
lyses. Moreover, acetylation of 2 with acetic anhydride
and pyridine gave 0-acetyl argentinine (2a) which again
indicated that the phenolic group was at C-3. Argentinine
(2) [8-l l] demonstrated significant cytotoxicity against
KB, P-388, A-549 and HT-29 cells with ED5,, values of
4.58, 4.42, 9.89 and 2.93 pg ml-‘, respectively, while
0-acetyl argentinine (2a), also with ED,, values of 11.03,
4.68, 10.22 and 2.72 /Ig ml-‘, was demonstrated to
possess cytotoxic activity against KB, P-388, A-549 and
HT-29 cells, respectively. The cytotoxicity of compound 2
and its 0-acetyl derivative (2a) are reported for the first
time. Liriodenine (3) [8-l l] demonstrated potent cytoto-
xicity against KB, A-549, P-388 and L-1210 cells with
ED,, values of 1.00, 0.72, 0.70, 0.57 and 2.33 pg ml-‘,
respectively. This compound has recently been shown to
be the main cytotoxic alkaloid in Thalictrum sessile [13],
Artabotrys uncinatus [ 141 and Polyalthia longifolia [ 151.
Annolatine (4) was obtained as a green amorphous
powder, [a]g4 _+O”(CHCI,; ~0.1). Its molecular formula
was established as C,gH,,N05 by HREI mass spectro-
metry (found 337.5073, calcd 337.5063). The presence of
an oxoaporphine skeleton in the molecule was easily
deduced by the UV spectrum (absorption maxima at 205,
218sh, 249, 268sh, 3OOsh, 348, 406 nm), along with a
conjugated carbonyl group absorption band at
1650 cm-’ in the IR spectrum. The presence of a phenolic
hydroxyl group in the molecule was indicated by the IR
absorption at 3400 cm- ’ and a bathochromic shift of UV
absorption on adding an alkaline solution. The ‘H NMR
spectrum (in CDCI,) of 4 revealed the presence of two
AB-quartets. One of them at 67.79 and 8.86 (J= 5.0 Hz)
was assigned to H-4 and H-5 [8-l 11, while the other at
67.24 and 8.60 (J = 8.8 Hz) was attributed to two mutual-
ly ortho-located protons on the aromatic ring. The other
‘H NMR signals which appeared at 6 3.86,4.09 and 4.13
(3H each, s each) and 67.24 (lH, s) were assigned to three
methoxyls and H-3, respectively. The above data led us to
propose the structure of annolatine either as 4,5,6 or 7. A
comparison of the ‘H NMR spectra of 4, oxo-O-meth-
ylbulbocapnine, oxocrebanine and kuafumine [ll, 161
clearly ruled out the possibility of 5 or 7 as the coupling
constants of H-10 (J=8.8 Hz) and H-11 (J=8.8 Hz) as
well as the chemical shifts of two of the three methoxyl
groups at C-8 (64.13) and C-9 (64.09) of 4 are comparable
to those of oxocrebanine and kuafumine instead of those
of oxo-0-methylbulbocapnine. The latter showed a J
value of 9.0 Hz each for H-8 and H-9 as well as 6 3.78 and
63.98 for the methoxyl groups at C-10 and C-l 1, respect-
ively. This evidence also confirmed the assignment of two
of the three methoxyl groups of 4 at C-8 and C-9 instead
of at C-10 and C-l 1 as found in oxo-O-methylbulboca-
pine [16]. Further evidence to support the assignment of
4 for annolatine was sought in an acetylation experiment.
The ‘H NMR spectrum (in CDCI,) of the acetyl derivat-
ive (4a) of annolatine exhibited the presence of two AB-
quartets. One of them was at 67.80 and 8.88 (1H each, d, J
=5.0 Hz), the other at 67.19 and 8.34 (1H each, d, J
= 8.8 Hz), with one singlet aromatic proton at 67.26 (lH,
s) and three methoxyl signals at 63.52,4.03 and 4.04 (3H
each, s), in addition to one acetyl signal at 62.39 (3H, s),
 Cytotoxic alkaloids of Annona montana
respectively, demonstrating that the phenolic hydroxyl
group was situated at C-l owing to the shielding effect of
the C-2 OMe (63.86+3.52) and H-11 (68.6O-r8.34) which
was shielded by the C-l OCOMe. On the basis of these
results, annolatine should be represented by formula 4.
EXPERIMENTAL
Mps: uncorr. Optical rotation: MeOH. MS: 70 eV
direct inlet. UV: MeOH. IR: Nujol. ‘H NMR and
13CNMR: CDCl,, chemical shifts reported in 6 from
TMS as int. standard. CC: Merck 9385 silica gel (230-400
mesh). TLC: silica gel GF-254 TLC, spots detected under
UV (254 and 365 nm), and by heating the plates to 100
after spraying with 60% HzSO,.
Plant material. Leaves of A. montana Mad. used in this
investigation were collected from Chie Shan, Kaohsiung
Hsien, Taiwan in December 1990. A voucher specimen is
deposited in the School of Pharmacy, Kaohsiung Medical
College, Kaohsiung, Taiwan, Republic of China.
Extraction and isolation. Fresh leaves (8 kg) were ex-
tracted repeatedly with MeOH at room temp. The comb.
MeOH extracts were evapd and partitioned to yield n-
hexane, CHCl, and aq. extracts as guided by bioassays in
KB and P-388 cells. The final, active CHCl, extract (67 g)
was chromatographed on a silica gel (2 kg, 60 x 6 cm)
column using CHCl,-MeOH mixts of increasing polarity
to yield 80 frs of 100 ml, each of which was monitored by
TLC and KB cell bioassay. Further purification of the
active frs 21-37 (37 mg) by CC and prep. TLC
(CHCl,-MeOH, 20: 1) yielded 3 (1.6 mg). Compound 4
(3.3 mg) was obtained in a like manner from active
frs 44-50 (43 mg). Frs 58-75 (241 mg) eluting with
CHCl,-MeOH (10: 1) were further sepd and purified by
silica gel CC and prep. TLC (CHCl,-MeOH, 10: 1) to
give compounds 1 (0.6 mg) and 2 (45 mg), respectively.
Annoretine (1). Purple oily base. IR v~!~’ cm-‘: 3350
(-OH). UV AK:” (log E):205 (4.08), 231 (3.99), 255 (4.35),
302 (4.08), 343 (3.52) and 361 (3.46) nm; UV cEH+NaoH
(log a): 205 (4.19), 258 (4.11), 312 (3.71), 345 (3.70) and 367
(3.64). EIMS 70 eV m/z (rel. int.): 293 [M]’ (86.36), 292
(27.73), 277 (19.05), 250 (lOO), 235 (38.64), 207 (31.36);
HREIMS: found 293.3371, calcd for Cr9H,,N0,,
293.3660. ‘H NMR (200 MHz, CDCl,): 62.61 (3H, s, N-
Me),290(2H, t,J=6 Hz, H-12), 3.32(2H, t,5=6Hz,H-
ll), 3.81(2H,s, H-14), 3.83 (3H,s, OMe), 7.57-790(5H, m,
H-6, H-7, H-8, H-9 and H-lo), 9.39 (lH, m, H-5).
13C NMR (50.3 MHz, CDCl,): 6: 27.2 (t, C-11), 46.5 (q,
N-Me), 53.1 (t, C-12), 53.6 (t, C-14), 60.7 (q, C-4 OMe),
123.0 (d), 126.1 (d), 127.4 (d), 127.5 (d), 128.1 (Q 129.4 (d),
124.6 (a), 128.5 (s), 126.8 (s), 124.3 (s), 130.1 (s), 133.6 (s),
141.8 (s, C-4) and 145.2 (s, C-3).
Argentinine (2). Prisms, mp 112” (lit. 110-111”) [2],
[a]k4 f0” (MeOH; c O.l), identified by spectral com-
parison (IR, UV and ‘H NMR) with lit. [23.
Argentinine acetate (h). Argentinine (2) (2mg) was
acetylated (Ac,O-pyridine, 20 hr, room temp.) and the
mixt. partitioned between Hz0 and CHCI,. The CHCl,
extract on concn and chromatography afforded O-acetyl-
argentinine (b) (1.8 mg). ‘H NMR (200 MHz, acetone-
499
ds) 6: 2.35 [6H, s, N(Me),], 2.42 (3H, s, OCOMe), 2.67
(2H, t, J=8.0Hz, H-12), 3.31 (2H, t, J=&OHz, H-11),
3.89 (3H, s, OMe), 7.40 (lH, s, H-2), 7.67-8.07 (5H, m, H-6,
H-7, H-8, H-9 and H-lo), 9.57 (lH, m, H-5). EIMS m/z
(rel. int.) 337 [M]’ (13.33), 295 (7.65), 237 (14.41), 222
(5.59), 194 (20.29) 178 (16.37), 165 (60).
Liriodenine (3). Yellow needles, mp 286-288” (lit. 286-
288”) [17, 181, [r]i4 +_O”(MeOH; cO.l), identified by
direct comparison with an authentic sample available in
our laboratory (mmp, IR and TLC).
Annolatine (4). Green amorphous powder. [a]k4 +O
(MeOH; c 0.1). UV 1ziI;‘” (log E): 205 (4.44), 218sh (4.40),
249 (4.22), 268sh (4.13), 300sh (3.83), 348 (3.78) and 406
(3.73) nm; UV 1 :::H+NaoH (log E): 205 (4.37) 227 (4.36),
263 (4.14), 294 (4.19), 326 (3.97), 370sh (3.65) and 534
(3.82) nm. IR v ~~‘crn- r: 3400 (-OH), 1650 (C=O).
EIMS 70 eV m/z (rel. int.): 337 [M]’ (6X2), 322 (3.40), 311
(2&l), 279 (2.11), 251 (2.47), 207 (3.08), 189 (2.44), 161
(3.73); HR-EIMS: found 337.5073, calcd for C,,H,,NOS,
337.5063. ‘H NMR (200 MHz, CDCl,) 6: 3.86 (3H, s,
OMe), 4.09 (3H, s, OMe), 4.13 (3H, s, OMe), 7.24 (lH, s,
H-3),7.24(1H,d,J=8.8Hz,H-10),7.79(1H,d,J=5.0Hz,
H-4),8.60(1H,d,J=8.8Hz,H-11),8.86(1H,d,J=5.0Hz,
H-5).
Annolatine acetate (4a). Annolatine (4) (2 mg) was
acetylated (AczO-pyridine, 20 hr, room temp.) and the
mixt. partitioned between Hz0 and CHCI,. The CHCl,
extract on concn and chromatography afforded O-acetyl-
annolatine (4a) as yellow prisms (2.4 mg). ‘H NMR
(200 MHz, CDCl,) 6: 2.39 (3H, s, OCOMe), 3.52 (3H, s,
OMe), 4.03 (3H, s, OMe), 4.04 (3H, s, OMe), 7.19 (lH, d, J
=8.8 Hz, H-lo), 7.26(1H, s, H-3), 7.80(1H, d,J=5.0Hz,
H-4),8.34(1H,d,J=8.8Hz,H-ll),8.88(1H,d,J=5.0Hz,
H-5).
Cytotoxicity testing. Carried out according to ref. [ 191.
Acknowledgement-This investigation was supported
by the National Science Council of the Republic of
China (Grant No. NSC 81-0208-M037-505) awarded to
Y.-C.W.
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