Phytochemistry, Vol. 28, No. 12, pp. 35 13 3517, 1989 003 I 9422.89 $3.00+ 0.

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Printed in Great Britain. c 1989 Pergamon Press plc

ALKALOIDS FROM TYLOPHORA INDICA”

M. ALI and K. K. BHUTANI?

Regional Research Laboratory (C.S.I.R.) Jammu Tawi-180001, India

(Received 20 February 1989)

Key Word Index~~~$ophora indica; Asclepiadaceae; rare phenanthroindolizidine alkaloids; tyloindicines A-E;
(+)-14-hydroxyisotylocerebrine; 4,6-desdimethylisotylocrebrine; structural determination.

Abstract-Seven rare and four known alkaloids have been isolated from two varieties of Tq’lophora indica. The new
alkaloids include tyloindicines A-E, (+)-14-hydroxyisotylocrebrine and 4,6-desdimethylisotylocrebrine. Tylophorine,
6-desmethyltylophorine, tylophorinidine and 5-hydroxy-0-methyltylophorinidine were the known alkaloids. Struc-
tural studies indicate that apart from tyloindicine B all alkaloids possess the dibenzo-[f, h] pyrrolo [ 1,2h] isoquinoline
skeleton but differ in the number, nature and distribution of the oxygen-bearing substituents, in the presence or
absence of C-13a or benzylic hydroxyls and an angular methyl function. Tyloindicine B possesses a cleaved substituted
phenanthrene moiety nucleus and an angular methyl group on the indolizidine portion.

INTRODUCTION obtained from the benzene-ethyl acetate (3 : 1) and (1: 1)

In a recent communication [Z], we have ascribed antiam- eluates, respectively.
oebic activity to various phenanthroindolizidine alka- The total alkaloidal fraction (0.1% yield) isolated from
loids isolated by us earlier [3-51 from Tylophora and the aerial parts of the tropical plant was also subjected to
deduced many interesting structure-activity relationships CC over basic alumina with solvents of increasing polar-
[2]. In addition to amoebicidal properties, these alkaloids ity. (+)-14-Hydroxyisotylocrebrine (7,0.002% yield) and
have been found to possess anticandidal activity [6]. The tylophorine (3, 0.03% yield) were obtained from the
demonstration of these pharmacological properties fur- benzene-ethyl aceiate (9: 1) and (3: 1) eluates, respect-
ther prompted us to extend our isolation work from T. ively. 4,6-Desdimethylisotylocrebrine (8) and tyloindicine
hirsuta to T. indicn species. The aerial parts of the latter
C (9) were obtained as a mixture from the benzene-ethyl
have been extensively studied for their alkaloids by many acetate (1: 1) eluants. Alkaloid 8 (0.002% yield) was
workers [7-121. However, during our bulk isolations of separated from 9 by fractional crystallization from
two varieties of 7. in&a, one cultivated in our sub- methanollacetone (1: 1). The mother liquor upon further
tropical campus, the other collected from tropical region purification afforded 9 (0.001% yield). Further elution of
of Madras, we encountered seven new alkaloids named as the main column with ethyl acetate afforded tyloindicine
tyloindicines A-E, (+)-14-hydroxyisotylocrebrine and D (10, 0.005% yield). Tyloindicine E (11, 0.0002% yield)
4,6-desdimethylisotylocrebrine in addition to the four was obtained in ethyl acetate-methanol (9: 1) eluates.
Compound 1 possessed a different substitution pattern
known alkaloids tylophorine, 6-desmethyltylophorine,
tylophorinidine of the four methoxy groups from that observed in tylo-
and 5-hydroxy-O-methyltylophorini-
dine. The structural elucidations of these alkaloids are phorine [9, 121 tylocrebrine [13, 141 and isotylocrebrine
described herein. [lo] containing the same number of methoxyls. Alkaloid
6 had a cleaved phenanthroindolizidine nucleus as en-
countered earlier in the characterization of (+ tsepticine
RESULTS AND DISCUSSION [lo] C-13a-hydroxysepticine [3] and tylohirsuticine [S],
with an angular methyl function on the indolizidine
The total alkaloidal mixture (0.17% yield) isolated
moeity. The base 6 contained one methoxy and one
from the aerial parts of the sub-tropical plant was chro-
hydroxyl function on C-3 and C-4, respectively, as detec-
matographed on basic alumina with solvents of increas-
ted in tylohirsuticine [S], but differed from this alkaloid
ing polarity. Tyloindicine A (1) and 6-desmethyltylopho-
in the attachment of an acetoxy group at C-7 whose
rine (2) were obtained from benzene eluant fractions in
presence was characterized for the first time in Tylophora
0.013 and 0.003% yields, respectively. Further elution of
alkaloids. Compound 7 resembled the alkaloid A of Rao
the column with benzene-ethyl acetate (9: 1) afforded
[15] in the substitution pattern offour methoxyls and one
tylophorine (3, 0.05% yield) and tylophorinidine (4,
benzylic hydroxyl but was of opposite specific rotation.
0.003% yield). 5-Hydroxy-0-methyltylophorinidine (5,
Alkaloids 8 and 9 had isotylocrebrine type substituents
0.006% yield) and tyloindicine B (6, 0.017% yield) were
arrangement on a phenanthrene nucleus with the ex-
ception of an extra angular methyl group. Compound 10
*Part 11in the series ‘Investigation of Medicinal Plants’. For resembled closely 5-hydroxy-O-methyltylophorinidine
part 10 see ref. [l]. (5) [S] reported earlier by us from T. hirsutu but differed
tAuthor to whom correspondence should be addressed. from it in the absence of a 14.hydroxyl group.

3513
3514 M. ALI and K. K. BHUTAN]

The physical and spectroscopic data for 6-desmethyl- of seven aromatic protons indicated 6 to be a cleaved
tylophorine (2) [S], tylophorine (3) [9, 121 tylophorini- phenanthrene analogue like d-septicine [lo], 13a-
dine (4) [lo, 123 and Shydroxy-0-methyltylophorinidine hydroxysepticine [3] and tylohirsuticine [S]. However,
(5) [S] were in agreement with those previously reported the position of the aromatic signals in 6 differed consider-
in the literature. ably from these alkaloids indicating a different substitu-
Tyloindicine A (l), mp 1999201”, [M]’ m/z 393 tion pattern. The seven aromatic protons appearing at
(C,,H,,NO,), [XI;’ + 7.2 (MeOH; c 2.1) had a different 68.14 (d, 1 H, J = 3 Hz), 7.90 (dd, 2H, J = 3 Hz and 9 Hz),
UV and IR spectral characteristics from the other alka- 7.70 (m lH), 7.16 (m, 2H) and 6.97 (d, 1H, J = 3 Hz) fitted
loids indicating a different substitution pattern of meth- well with the substitution arrangement shown in the
oxy groups. The compound did not respond to ferric cleaved form. In the light of earlier findings [3, lo] the
chloride, oxidizing reagents such as ceric sulphate, nitric shielded signal at 66.97 has been assigned to C-8 in 6.
acid, bromine water or chromic acid, acetic anhydride in Because this proton appeared as doublet with a m-
pyridine and to diazomethane showing the absence of an coupling constant (J = 3 Hz), the acetoxyl function could
hydroxyl group. Its IR spectrum did not show any band therefore be placed at C-7. The deshielded signal at 68.14
beyond 2900 cm- ‘. The presence of a phenolic hydroxyl could be assigned to the proton adjacent to the phenohc
was also ruled out due to the absence of any shift in its UV hydroxyl at C-4a. The doublets at 67.90 and 7.16 integrat-
spectrum on addition of sodium hydroxide. In its mass ing for two protons each have been assigned to the C-l,
spectrum the base peak at m/z 323, arising from the C-5a and C-2, C-5 protons, respectively. The methoxy
cleavage of the characteristic pyrrolidine fragment by a protons appeared at 63.80 along with a D,O exchange-
retro-Diels-Alder reaction from the [M]’ peak, sup- able (phenolic OH) proton at 66.30. The acetoxy group,
ported the absence of any functional group in ring D or E. encountered for the first time in phenanthroindolizidine
The ‘H NMR spectrum of 1 showed the presence of four alkaloids, appeared as a singlet at 62.52. Another singlet
aromatic protons at 6 7.94 (d, J = 9 Hz), 7.70 (d, J = 9 Hz) at 6 1.60 integrating for three protons could be assigned to
7.36 (d, J = 9 Hz) and 7.10 (d, J = 9 Hz) assigned to C-l, C- an angular methyl function as described in earlier work
7, C-2 and C-8, respectively. The ortho value of the same [S, 161. The cleaved phenanthrene alkaloid 6, for which
coupling constants (J=9 Hz) for all the four aromatic the structure was deduced from the above data, has been
protons suggested the presence of two pairs of methoxy named as tyloindicine B.
groups on adjacent positions in ring A and C. Out of the Alkaloid 7, mp 240-242”, [M]’ at m/z 409
four possible combinations, three were ruled out on the (CZ4H2,NOs), [a];” f30” (MeOH; ~0.3) showed one
basis of known spin-spin interactions for the aromatic broad hydroxyl band 3450 cm- ’ in its IR spectrum. The
protons in tylophorine [l l] (C-l, C-4, C-S and C-8 as s), hydroxyl function was not phenolic, the compound giv-
tylocrebrine [14] (C-l, C-4, C-7 and C-8) as two sand two ing no reaction with ferric chloride and diazomethane
d) and isotylocrebrine [lo] (C-l, C-2, C-5 and C-8 as two and no bathochromic shift in the UV spectrum with
s and two d). Thus structure 1 has been assigned to alkali. Its mass spectrum showed a weak peak at m/z 391
tyloindicine A. (3.5% due to loss of H,O from [Ml’). A peak at m/z 339
Tyloindicine B (6) mp 183-185”, [M]’ at m/z 393 (4.7%) arose due to loss of a pyrrohdine ring at m/z 70
(C2,H,,N0,), [%]A5 + 14.30” (MeOH; c 1.05) had UV (67.6%) from [M]’ by a retro-Diels-Alder reaction. The
maxima at 220,230,260,3 10 and 355 nm. Its IR spectrum base peak at m/z 310, which resulted from expulsion of
showed hydroxyl and carbonyl stretchings at 3370 and CHO from m/z 339, indicated the hydroxyl function to be
1710 cm _ ‘, respectively. The hydroxyl group is phenolic on C-14 [3, 131. The ‘HNMR spectrum of 7 showed the
in nature as indicated by a green coloration with ferric presence of four methoxy groups at 64.00, 3.92, 3.90 and
chloride and by the presence of a shift in its UV maxima 3.80; four aromatic protons at 68.24 (s, lH), 8.0 (d, J
at 230,280, and 320 nm on addition of sodium hydroxide. = 9 Hz), 7.28 (d, J = 9 Hz) and 7.10 (s, 1H) assigned to C-5,
Acetylation of 6 gave a monoacetylated product further C-l, C-2, and C-8, respectively, one D,O exchangeable
confirming the presence of one hydroxyl group. A mono- proton at 64.60 and signals at 66.04 (brs) and 63.40 (br s)
methyl ether was formed on treatment of 6 with dia- due to protons attached to the hydroxyl carbon at C-14
zomethane which could not be further acetylated proving and C-13a, respectively. The deshielding (by 0.21 ppm) of
the existence of one phenohc hydroxyl group. Deacetyl- the aromatic proton at C-l in 7 from the similar structure
ation of the monomethyl ether afforded 7_deacetyltyloin- of isotylocrebrine [lo] could be easily postulated due to
dicine B. The hydroxyl function of this product was the presence of a benzylic hydroxyl at C-14.
shown to be phenolic in nature by a green coloration Acetylation of 7 yielded a monoacetylated product
with ferric chloride, a shift in its UV spectrum from 228, (vKBr
max 1735 cm- ‘). The placement of the hydroxyl at C-14
275 and 335 nm to 235 and 280 nm on addition of sodium instead of C-9 in isotylocrebrine alkaloids has been
hydroxide and by remethylation with diazomethane. The established conclusively by Rao [15]. These data led to
mass spectrum of 6 showed a [M -Me]+ ion at m/z 378 structure 7 for the base which was identical to alkaloid A
(4.4%) and a [M -AC] + ion at m/z 349 (1.3%) indicating of Rao isolated from T. crebrijora [15]. However, alka-
the presence of an angular methyl function as well as loid A possessed a (-)-rotation. It was concluded that
acetyl group, respectively. The fragement at m/z 310 the base 7 and Rao’s alkaloid A were related to each other
(10.6%) arose due to loss of 83 mass units in the form of as diastereoisomers. As this new stereoisomer with (+)-
methylpyrrolidine from [M] + by retro-DielssAlder reac- rotation has been isolated from a natural source, it has
tion as encountered earlier in phenanthroindolizine alka- been named (+)-hydroxyisotylocrebrine (7) in which
loids [16]. An intense peak at m/z 295 (67%) resulted OH-14 and H-13a are trans diaxially disposed. The
from the fragment at m/z 310 due to loss of 15 mass units. hydroxyl is below the plane of the molecule and H-13a
The base peak at m/z 252 arising from the loss of an acetyl above it as observed for (+)-0-methyltylophorinidine
group from m/z 295 was further indicative of the presence c51.
of an acetyl group. In the ‘H NMR spectrum the presence Alkaloid (8) mp 1777179“, [M]’ at m/z 365
 Alkaloids from Tylophora 3515

(C,,H,,NO,), [a];’ + 7.3” (MeOH; c 1.75) had UV maxi- named tyloindicine C was assigned.
ma at 215, 228, 270, 310, 335 and 355nm. The IR Tyloindicine D (lo), mp 229-231” (decomp), [M] + at
spectrum showed two hydroxyl bands at 3515 and m/z 409 (C,,H,,NO,), [alA + 1.6” (MeOH; c 0.06) close-
3405 cm-‘. The hydroxyl functions were shown to be ly resembled the isotylocrebrine-type (7, 8 and 9) and
phenolic by a green coloration with ferric chloride and tylophorinidine-type (5), alkaloids in UV and IR spectral
the presence of a shift in its UV maxima at 230, 280 and behaviour indicating the same substitution pattern of the
315 nm on addition of sodium hydroxide. Its mass spec- methoxy and hydroxyl groups. The presence of a phenolic
trum showed an intense [M]’ peak at m/z 365 (51.8%) hydroxyl was indicated by the shift in its UV maxima at
with a base peak at m/z 295 which arose due to cleavage of 222, 230,272, 318, 332 and 350 nm to 230, 280, 320 and
the pyrrolidine fragment of m/z 70 (99%) from [M] ’ by a 350 nm on addition of sodium hydroxide. The IR spec-
retro-Diels-Alder reaction. A prominent peak at m/z 280 trum showed a strong band at ca 3250cm-‘. Its mass
(28.9%) was due to the loss of 15 mass units (Me) from the spectrum showed a peak at m/z 339 (7.5%) arising from
base peak. The ‘HNMR spectrum of 8 showed the the expulsion of a neutral pyrrolizidine ring at m/z 70
presence of two methoxy groups at 64.00 and 3.92; four (100%) by a characteristic Diels-Alder reaction. The
aiomatic protons at 68.33 (s, lH), 8.04 (d, lH, J=9 Hz), peaks at m/z 324 (11.1%) 309 (14.7%) and 294 (13.5%)
7.26 (d, lH, J = 9 Hz) and 7.10 (s, 1H), assigned to C-5, C- arose by cleavage of methyl fragments in a stepwise
l, C-2 and C-8, respectively, two D,O-exchangeable manner from m/z 339. The ‘HNMR spectrum of 10
phenolic hydroxyls at 67.83 and 6.96 and a broad signal showed the presence of one methoxy group at 64.10, three
at 64.70 due to a proton attached to C-13a. The de- methoxy groups at 6 3.92, three aromatic protons at 6 8.00
shielded singlet at 68.33 could be conveniently assigned (d,1H,J=9Hz),7.20(d,1H,J=9Hz)and7.16(s,1H)
to the proton on C-5 placed in between two phenolic assigned to C-l, C-2 and C-8, respectively, and one D,O-
hydroxyls, whereas the shielded singlet at 67.10 was exchangeable proton at 67.80 (phenolic OH). The
assigned to C-8. The shielded (67.26) and the deshielded ‘H NMR values of the aromatic protons at C-l, C-2 and
(68.04) doublets integrating for one proton each have C-8 in 10were in agreement with corresponding values of
been assigned to C-2 and C-l protons, respectively. Shydroxy-O-methyltylophorinidine [S] isolated from
Acetylation of 8 gave a diacetylated product. Methyl- wild population of T. hirsuta. Compound 10 on acetyl-
ation of 8 with diazomethane formed isotylocrebrine. The ation gave a monoacetylated product. A monomethyl
‘HNMR spectra of 8, its diacetylated product, its di- ether was formed on treatment with diazomethane which
methyl ether and isotylocrebrine were compared. There in turn could not be acetylated proving the presence of
was a large deshielding (by 6 1.00) of the signal due to the only one phenolic hydroxy (further proof of the phenolic
proton at C-5 in isotylocrebrine. The diacetylated prod- hydroxyl at C-4 came from the positive Gibb’s test for a
uct showed no such effect. The effect appears to be due to free-hydroxyl group). These data led to structure 10 for
the increased electron as a result of its close proximity to the alkaloid named tyloindicine D.
the phenolic hydroxyls. Base 8 also responded to the Tyloindicine E (II), mp 350” (decomp), [M] + at m/z
Gibb’s test. From these data structure 8 for the alkaloid 349 (C,, H,,NO,), [a];” - 12” (MeOH; ~0.25) had UV
named 4,6-desdimethylisotylocrebrine is suggested. maxima at 210, 232, 277, 310 sh, 338 and 357 nm. The
Tyloindicine C (9), mp 223-225”, [M]’ at m/z 379 presence of a phenolic hydroxyl was indicated by the shift
(C,,H,,NO,), [a]:‘+ 16” (MeOH; c 0.25), had UV maxi- in its UV maxima at 226,284 and 322 nm on addition of
ma at 210, 230, 270, 310, 335 and 355nm. The IR sodium hydroxide. The IR spectrum showed a band at
spectrum showed two hydroxyl bands at 3515 and 3450 cm- ‘. Acetylation of 11 yielded a monoacetyl de-
34OOcm-‘. The presence of phenolic hydroxyls was rivative. Methylation of 11 with diazomethane formed a
confirmed by a green coloration with ferric chloride and a monomethyi ether, desoxypergularinin [9], which could
shift in its UV maxima at 225, 278 and 317 nm on not be acetylated further. Its mass spectrum showed a
addition of sodium hydroxide. Its mass spectrum showed weak [M]’ at m/z 349 (4.6%) with a more pronounced
a peak at m/z 364 (14.9%) due to loss of 15 mass units peak at m/z 279 (16.4%) arising from elimination of the
from the [M]’ which indicated the presence of an characteristic pyrrolidine nucleus at m/z 70 (15%). The
angular methyl group. The peaks at m/z 310 (8.0%) and base peak at m/z 265 resulted from expulsion of 14 mass
296 (93.8%) originated from [M - 69]+ and [M - 83]+ units from the fragment at m/z 279. The ‘H NMR spec-
ions, respectively by retro-Diels-Alder reactions charac- trum of 11showed the presence of two methoxy functions
teristic of phenanthroindolizidine alkaloids. The loss of at 3.94 and 3.42; five aromatic protons at 67.96 (d, J
methylpyrrolidine at m/z 83 (6.6%) along with a pyrroli- =9 Hz), 7.72 (s), 7.62 (d, 5=3 Hz), 7.20 (d, J=9 Hz) and
dine fragment at m/z 69 (100%) further confirmed the 7.04 (s) assigned to C-l, C-5, C-4, C-2 and C-8, respect-
presence of an angular methyl function. The ‘HNMR ively and one D,O exchangable proton, at 68.14 (phenol-
spectrum of 9 was almost identical to that of 8 in the ic OH). These data led to structure 11 for the alkaloid
aromatic region. It showed the presence of two methoxy tyloindicine E.
groups at 63.80 and 3.70, four aromatic protons at 68.03
(s, lH), 7.90 (d, lH, J=9 Hz), 7.00 (d, lH, J=9 Hz) and EXPERIMENTAL
6.79 (s, lH), assigned to C-5, C-l, C-2 and C-8, respect-
ively, two D,O exchangeable phenolic protons at 67.66 Mps: uncorr. ‘H NMR 6values are given in ppm downfield
and 6.60 and a methyl singlet at 61.60 indicative of an from TMS. TLC was carried out on silica gel in
angular methyl function. The arguments given for the C,H,-EtOAc-Et,NH (6:3: l), spots were detected with
structure elucidation of 8 also holds good for 9 with an DragendroR’s reagent.
addition of an angular methyl function. Acetylation of 9 Isolation of alkaloids,from sub-tropical plants. Air-dried aerial
yielded a diacetylated product. Because methylation of 9 parts of T. indica (7.0 kg), grown at RRL campus, were collected
with diazomethane afforded a dimethyl ether identical to during October 1984 and extd with MeOH. The EtOAc-sol.
13a-methyltylohirsutine, the structure 9 for the alkaloid brown coloured crude total alkaloids (12 g, 0.17%) were sepd as
3516 M. ALI and K. K. BHUTANI

described in refs [335]. On TLC these showed seven major and deacetylated product gave a mono Me ether (confirmed by TLC
two minor components. The EtOAc-sol. portion was coned in comparison).
uacuo and subjected to CC over basic Al,O, after the formation Isolation of alkaloidsfrom tropical plants. Air-dried aerial parts
of a slurry. The column was eluted with mixts of C,H,, EtOAc T. indica (25 kg) supplied by M/s Pharmaproducts, Madras,
and MeOH of increasing polarity. India were extd exhaustively by hot percolation with EtOH and
Tyloindicine A. (1). Fr. 1 eluted with C,H, gave 1 (0.9 g, 0.013% the EtOAc-sol. brown coloured alkaloids (24 g, 0.096% yield)
yield; from Me&O), mp 199-201 O, [ali + 7.2 (MeOH; c 2. I); sepd as described in refs [3-51. On TLC it showed six major and
UV 2:::” nm: 228, 280, 335, 355 and 375 (logs 4.09, 4.85, 1.72, one minor components. The EtOAc-sol. portion was coned in
1.69 and 1.66). IR Y:!: cm -I: 2900, 1600, 1510, 1460,1410, 1290, uacuo and subjected to CC over basic Al,O, after the formation
1205,1160,1110,1025 and 780. ‘H NMR (DMSO-d, + ldrop of of a slurry. The column was eluted with mixts of C,H,, EtOAc
TFA): 67.94 (d, J = 9 Hz, o-coupled H, C-l), 7.70 (d, J = 9 Hz, o- and MeOH of increasing polarity.
coupled H, C-7), 7.36 (d, J = 9 Hz, o-coupled H, C-2), 7.10 (d, J 14-Hydroxyisotylocrebrine (7). Fr 1, eluted with C,H,-EtOAc
= 9 Hz, o-coupled H, C-8), 5.40 (m, C-13a H), 4.00 (br s, 12 H, (9: 1) on crystallization from Me,CO gave a light yellow powder
40Me); MS m/z (rel.int.): 393 [M]’ (C,,H,,NO,, 32.1), 377 of 7 (0.5 g, 0.002% yield), mp 24&242”, [ali + 30” (MeOH;
(0.2), 363 (7.1) 323 (lOO), 308 (16.9) 293 (26.8), 85 (24.2), 83 (72.7), cO.3); uv 1::;” nm: 210, 275, 335 and 350 (logs 4.66, 5.42, 2.50
70 (7.9). and 2.45); IR Y!,!,!;cm-‘: 3450,2910,1615,1505,1475,1420,1255,
6-Desmethylty/ophorine (2). Frs 2-3 from the main column 1205, 1160,1115, 1035 and 830. ‘H NMR (DMSO-d,): 68.24 (s,
eluted with C,H, gave 2 (0.2 g, 0.003% yield; from p-coupled H, C-5), 8.00 (d, J = 9 Hz, o-coupled H, C-l), 7.28 (d, J
CHCI,-MeOH (1: l), mp 234236” (decomp.), (ht. mp. 235-237” = 9 Hz, o-coupled H, C-2), 7.10 (s, p-coupled H, C-8), 6.04 (br s,
decomp), [z]:’ - 166.6” (MeOH; ~0.12); UV;.E:p nm; 210,225, lH, C-14) 4.60 (brs, D,O exchangeable, OH), 4.0 (s, 3H, OMe),
270, 305, 330 and 350 (log& 6.06, 7.58, 6.61, 2.87, 3.1 and 2.04); 3.92 (s, 3H, OMe), 3.90 (s, 3H, OMe), 3.80 (s, 3H, OMe), 3.40 (m,
A!$‘” (on addition of NaOH) nm: 230, 280 and 320 (logs 9.52, lH, C-13a), 2.60 (m, CH,), 2.40 (m, CH,), 1.80 (m, CH,). MS m/z
9.29 and 4.20). MS m/z (rel. int.): 379 [M]’ (C,,H,,NO,, 30.8), (rel. int.): 409 [M]’ (C,,H,,NO,, 17.0) 391, (3.5) 377 (13.8) 339
364(11.8), 348 (28.1), 309(26.5), 295 (76.0), 279 (100) and 70(88.1). (4.7), 310 (lOO), 295 (5.6j. 280 (5.0), 265 (3.6) 70 (67.6), 29 (6.7) and
Acetylation of 2 with Ac,O-pyridine at room temp for 24 hr and 18 (99). Acetylation of 7 with Ac,O-pyridine at room temp. for
usual work-up gave a monoacetylated product, mp 212-213”. 24 hr and usual work-up gave a monoacetylated product, mp
IR i.KB’
max 1725 and 1225 cm-‘. Methylation of2 with CH,N, gave 211-212”. IRvif: cm-‘: 1735.
tylophorine, mp and mmp 29G292” (decomp.). Tylophorine (3). Frs 223 from the main column eluted with
Tylophorine (3). Frs 45 from the main column eluted with C,H,-EtOAc (3: 1) afforded light yellow crystals of 3 (7.5 g,
C,H,-EtOAc (9: 1) afforded 3 (3.6 g, 0.05% yield, from 0.03% yield, from CHCl,-MeOH), mp and mmp 291-292”
CHCI,-MeOH), mp and mmp 29G292” (decomp.) [lit mp Cdecomp), [xl? -21.05” (MeOH; ~0.7); MS m/z (rel. int): 393
290-292” (decomp)]. [u]i’-22.60” (MeOH; c 1.2). MS, m/z (rel. WI + K,,H,,NO,, 82.4).
int.) 393 [M] + (C,,H,,NO,, 82.6). 4,6-Desmethyhotylocrebrine (8). Frs 445 from the main col-
Tylophorinidine (4). Fr. 6 from the main column eluted with umn, eluted with C,H,-EtOAc (1: I) on crystallization from
C,H,-EtOAc (9: 1) gave 4 (0.22 g, 0.003% yield, from MeJO); Me,CO (1 : l), gave colourless beads of 9 (0.5 g, 0.002% yield),
mp 215-217” (lit. mp 213-214”) [ml;‘+ 122” (MeOH; c 0.48), (ht. mp 177-179”, [r]i5 + 7.3” (MeOH; c 1.75) UVJ.“,$‘” nm: 215,
[a],+125” (MeOH; ~0.1). MS m/z (rel. int.); 365 [M]’ 228, 270, 310, 335 and 355 (logs 11.68, 13.0, 13.87, 4.0, 2.0 and
(C,,H,,NO,, 15.5). 1.80), i::;” (on addition of NaOH) nm; 230,280, and 3 15 (log E
5-Hydroxy-0-methyltylophorinidine (5). Fr 7 from the main 18.25, 19.15 and 5.87), IRvkt; cm-‘: 3515, 3405, 2910, 1615,
column eluted with C,H,-EtOAc (3: 1) gave 5 (0.4 g, 0.006% 1525, 1510, 1465, 1420, 1260, 1230, 1200, 1160, 1100, 1035 and
yield, from Me,CO), mp and mmp 243-245” (decomp) [lit. mp 820. ‘H NMR (DMSO-d,): 68.33 (s, p-coupled H, C-5), 8.04(d, J
245-247”] [,%]A*+ 55.85” (MeOH; c 0.65), (lit [alA + 58.97” =9 Hz, o-coupled H, C-l), 7.83 (br s, D,O exchangeable, OH),
(MeOH; cO.8), MS m/z (rel. int.): 395 [M]’ (C,,H,,NO,, 5.2). 7.26 (d, J = 9 Hz, o-coupled H, C-2), 7.10 (s, p-coupled H, C-8),
Tyloindicine B (6). Frs 8-9 eluted with C,H,-EtOAc (1: 1) gave 6.96 (s, D,O exchangeable, OH), 4.70 (brs, lH, C-13a), 4.00 (s,
beads of 6 (0.75 g, 0.017% yield, from CHCl,), mp 1833185”, 3H, OMe), 3.92 (s, 3H, OMe), 2.52 (m, CH,), 2.20 (m, CH,), 1.80
[cx];~+ 14.3” (MeOH; c 1.05), UVAL::” nm; 220, 230, 270, 310 (m, CH,): MS m/z (rel. int.): 365 [Ml+ (C,,H,,NO,, 51.8), 295
and 355 (logs 1.96, 1.72, 0.32, 5.61, 21.81 and 20.63), i,$f” (on (lOO), 281 (22.5) 280 (28.9), 267 (19.6) 265 (9.0), 253 (27.9), 252
addition of NaOH) nm: 230, 288 and 320 (loge 0.59, 0.35 and (15.0), 225 (25.1), 224 (12.5) 70 (99.0). Acetylation of 8 with
7.03); IR r,:z; cm -‘: 3370, 2950, 1710, 1615, 1520, 1465, 1420, Ac,O-pyridine at room temp. for 24 hr and usual work-up gave
1230, 1195, 1160, 1105, 1035 and 815. ‘H NMR (DMSO-d,): a diacetylated product, mp 1655167”, IRvEF: 1760 and
68.14 (d, J = 3 Hz, m-coupled H, C-4a), 7.90 (dd, J =9 and 3 Hz, 1755 cm-‘. MS m/z (rel. int.): 449 [Ml+ (C,,H*,NO,, 15.0).
ZH, o-, m-coupled H, C-l, C-5a), 7.70 (m, 1H C-6), 7.16 (m, 2H, C- Methylation of 8 with CH,N, gave isotylocrebrine, mp and
2, C-5), 6.97 (d, 5=3 Hz, m-coupled H, C-8), 6.30 (brs, D,O mmp 21 l-213’ (lit mp 212-214”).
exchangable, OH), 3.80 (s, 3H, OMe), 2.52 (s, 3H, OAc), 1.60 (s, 3H, Tyloindicine C (9). The dried mother liquor of 8 was chromato-
C-13a Me). MS m/z (rel. int.): 393 [M]’ (CZ4H2,N04, 0.2), 378 graphed over basic Al,O, in EtOAc to obtain 9 (0.25 g, 0.001%
(4.4), 365 (10.5), 349 (1.3), 329 (0.2). 311 (10.8), 310(10.6), 295 (67), yield) as brown beads after crystallization from Me&O, mp
252 (IOO), 83 (6.2), 69 (60.3) 43 (79.7). Acetylation of 6 with 223-225’; [z]A5+ 16” (MeOH; ~0.25); UVAE$‘” nm: 210, 230,
Ac,O-pyridine at room temp gave a monoacetylated product, 270, 310, 335 and 355 (logs 13.15, 15.62, 18.25, 3.95, 2.12 and
mp 165-167”, IR Y,,, KBr 1745 and 1715 cm-‘, MS m/z(rel. int.): 435 2.04). n/:;” (On additon of NaOH) nm: 225,278 and 317 (log c
[M]’ (C,,H,,NO,, 12). Methylation of 6 with CH,N, gave a 18.65, 20.15 and 6.12). IRvty: cm-‘: 3510, 3405, 2925, 1615,
mono Me ether, mp 206-209”. C,,H,,NO, ([Ml’ 407). Deace- 1525, 1465, 1425, 1260, 1235, 1195, 1160 and 812. ‘HNMR
tylation of the mono Me ether with dil HCI (10%) yielded 7- (DMSO-d,); 68.03 (s, p-coupled H, C-5), 7.90 (d, J=9 Hz, o-
deacetyltyloindicine B 4-Me ether, mp 218-219”, UVI.i:p nm: coupled H, C-l).; 7.66 (br s, D,O exchangeable, OH), 7.00 (d, J
228, 275 and 335 (logs 3.65, 4.38 and 2.26), UVnE:p (on = 9 Hz. o-coupled H, C-2), 6.78 (s, p-coupled H, C-8), 6.60 (br s
addition of NaOH) nm: 235 and 280 (logs 1.2 and 1.09), D,O-exchangeable, OH), 3.80 (s, 3H, OMe). 3.70 (s, 3H, OMe),
IRvtt; 3430cm-‘, C,,H,,NO,([M]+ 365). Methylation ofthe and 1.60 (s, 3H, C-Me). MS m/z (ml. int.): 379 [M]’
 Alkaloids from Tylophora 3517

6
1 R,=R<R,=” ; i-12= RJ=R,, = Us= OMc

2 RJ=R,,=R,:H; R, = R2=R6 = OHc;Rs=OH

3 Rj=RL=R,=H; R1 = R2=R6 = R6= OMe

4 R,= Rj = R&=“; R2 = R6=OHc ; R5 = R,= OH

5 R,=RJ=H; R2=Rs= R6=Ot&; R/,= R,= OH

7 R,= R&z)+; R2=R3 = R5=R6 = Oh@; R,=OH

8 R,= R,,= R,=H ; R2 = R6=OHe; R3 = Rs=OH
10 R,=R,=H;R2=,+3= Rr,=R6 = OMe; R/, =OH

11 R,= R-J= R&=R?=H ; R2=R6 = 0,&Z; R6 = OH

OMe
9

(C,,H,SNO,, 12.2), 364 (14.9), 349 (5.9), 334 (11.6), 310 (8.0), 296 CH,N, gave desoxypergularinin, mp 209-210” (decomp.) (lit. mp
(93.8), 281 (6.4). 267 (5.8), 83 (6.6), 69 (100). Acetylation of 9 with 208-209”), [a]? - 12.8” (MeOH, ~0.2) (lit [ali - 13.6, CHCI,;
Ac,O-pyridine at room temp for 24 hr and usual work-up gave a c 0.25), C,,H,,NO, ( [M] + 363, 90%)
diacetylated product, mp 176-177”; IR v cm-‘: 1760 and 1755,
MS m/z (rel. int.): 463 [Ml’ (C,,H2,N0,, 35.5). Methylation of
Acknowledgements-The authors gratefully thank Mrs S. Gupta,
9 with CH,N, gave a diMe ether, mp 198-200”. This product
(R.R.L., Jammu) and M/s Pharmaproducts, Madras for suppl-
was comparable (mmp, TLC, co-TLC, UV and IR) with 13a-
ying cultivated and wild populations of T. indica. respectively.
methyltylohirsutine [7].
Tyloindicine D (10). Further elution of the main column with
EtOAc gave a light yellow powder of 10 (0.125 g, 0.005% yield) in
REFERENCES
frs 6 and 7 after crystallization from Me&O; mp 229-231”
(decomp.), [a];’ + 1.6’ (MeOH; c 0.6); UVAE,Sz” nm: 222, 230, 1. Bhutani, K. K., Ali M., Sharma, S. R., Vaid, R. M. and
272,318,332and350(logs8.2, 10.2, 10.5,4.1,2.4and2.2):R~:PH Gupta, D. K. (1988) Phytochemistry 27, 925.
(on addition of NaOH) nm: 230,280,320 and 350 (log E 20.5,21.2, 2. Bhutani, K. K., Sharma, G. L. and Ali M. (1987) Planta Med.
6.76 and 4.3) IR v:fi cm- l., 3250, 2930, 1615, 1510, 1462, 1420, 53, 532.
1260. 1225, 1190, 1145, 1040 and 815. ‘HNMR (DMSO-d,): 3. Bhutani, K. K., Ali. M. and Atal, C. K. (1984) Phytochemistry
68.00 (d, J = 9 Hz, o-coupled H, C-l), 7.80 (br s, D,O-exchan- 23, 1765.
geable, OH), 7.20 (d, J = 9 Hz, o-coupled H, C-2), 7.16 (s, C-8), 4. Bhutani, K. K., Ali M. and Atal, C. K. (1985) Phytochemistry
4.60 (brs, lH, C-13a), 4.10 (s, 3H, OMe), 3.96 (s, 9H, 3 OMe). MS 24, 2778.
m/z (rel. int.): 409 [M]’ (C,,H,,NO,, 3.2), 394 (7.1), 379 (10.5), 5. Ali M. and Bhutani, K. K. (1987) Phytochemistry 26, 2089.
364 (13.8), 339 (7.5), 324 (11.19, 309 (14.7), 296 (17.6), 295 (86.3), 6. Annual Report, Regional Research Laboratory (CSIR),
294 (13.5), 280 (5.6), 266 (5.3), 252 (5.8) and 70 (100). Acetylation Jammu, 1987-1988, p. 7.
of 10 with Ac,O-pyridine at room temp. for 24 hr and usual 7. Govindachari, T. R., Pai, B. R. and Nagarajan, K. (1954) J.
work-up gave a monoacetylated product, mp 193-194”; Chem. Sot. 2801.
IR vtt; cm-‘: 1755. Methylation of 10 with CH,N, gave a mono 8. Rao, K. V., Wilson, R. A. and Cummings, B (1971) J. Pharm.
Me ether, mp 217-219”; C,SH,,NO, ([Ml’ 423). Sci. 60, 1725.
Tyloindicine E (11). Frs 8-10 eluted with EtOAc-MeOH (9: 1) 9. Mulchandani, N. B., Iyer, S. S. and Badheka, L. P. (1971)
gave a brown coloured powder of 11 (0.05 g, 0.0002% yield) on Chem. Ind. 505.
crystallization from MeOH, mp 350” (decomp.), [a]:‘- 12” 10. Govindachari, T. R., Vishwanathan, N., Radhakrishnan, J.,
(MeOH; cO.25), UV ,?z$“’ nm: 210,232,277,310 sh, 338 and 357 Pai, B. R., Natarajan, S. and Subramaniam, P. S. (1973)
(loge 2.44, 2.83, 2.40, 0.68, 0.23 and 0.22), 1:::” on addition of Tetrahedron 29, 891.
NaOH) nm: 226, 284 and 322 (log& 3.4, 3.2 and 1.0); 11. Govindachari, T. R., Vishwanathan, N., Radhakrishnan, J.,
IRvifi cm-‘: 3450, 2925, 1610. 1520, 1460, 1405, 1265, 1240, Rao, N. N. and Pai, B. R. (1973) Indian J. Chem. 11, 1215.
1190, 1155 and 815. ‘HNMR (DMSO-d,): 68.14 (brs, D,O 12. Phillipson, J. D.. Tezcan, I. and Hylands, P. J. (1974) PIanta
exchangeable, OH), 7.96 (d, .J = 9 Hz, o-coupled H, C-l), 7.72 (s, Med. 25, 301.
p-coupled H, C-5), 7.62 (d, J = 3 Hz, m-coupled H, C-4), 7.20 (d, J 13. Rao, K. V., Wilson,, R. and Cummings, B. (1970). J. Pharm.
= 9 Hz, o-coupled H, C-2), 7.04 (s p-coupled H, C-8), 4.23 (br s, C- Sci. 59, 1501.
13a), 3.94 (s, 3 H, OMe) and 3.42 (s, 3H, OMe). MS m/z (rel. int.): 14. Chauncy, B and Gallert, E. (1970) Aust. J. Chem. 23, 2503.
349 (C,,H,,NO,, 4.6), 320 (2.6), 279 (16.4), 265 (100), 250 (10.9). 15. Rao, K. V. (1970) J. Pharm. Sci. 59, 1608.
and 70 (15.0). Acetylation of 11 with Ac,O-pyridine at room 16. Pettit, G. R., Goswami, A., Cragg, G.M., Schmidt, J. M. and
temp gave a monoacetylated product, mp 281&282”, Irving Zou, J. C. (1984) J. Nat. Prod. 47, 913.
1735 cm~‘, C,,H,,NO, ([Ml’ 391). Methylation of 11 with 17. Herbert, R. B. and Moody, C. J. (1970) Chem. Commun. 121.