jection of the phlogistic agent. This was done to offset any possible Exp. Ther., 141,369(1963).

error caused by the difference in the size of the animals used. The (3) C. V. Winder, J. Wax, L. Scotti, R. A. Schemer, E. M.
standard error of the mean (SEM) was calculated according to the Jones, and F. W. Short, ibid., 138,405(1962).
formula given by Burn et al. (10): (4) C . V. Winder, J. Wax, B. Serrano, E. M. Jones, and M.
McPhee, Arthritis Rheum., 6, 36(1963).
sji = p-N(N
- Y)’
1)
(5) K.J. Rorig and H. A. Wagner, U. S. pat. 3,149,109 (Sept.
15, 1964).
(6) B. H.Chase and W. T. Weller, J. Phurm. Phurmacol., 16,
where s j = SEM, = mean value, y = any single value, N = 163(1964).
total number of animals used in the assay, and S = sum. (7) W. B. Deichman and T. J. LeBlanc, J. Ind. Hyg. Toxicol.,
The method and techniques developed for the anti-inflammatory 25,415(1943).
screening carried out in this investigation proved to be quite satis- (8) M. W. Jordin and W. D. Easterly, Jr., J. Phurm. Sci., 54,
factory. The method was simple and quick, and the apparatus was 1190(1965).
sufficiently sensitive for the determination of therapeutic levels (9) C.A. Winter, E. A. Risley, and G. Nuss, Proc. Soc. Exp.
of antiedema activity. Results with the hydrocortisone acetate and Biol. Med., 111, 544(1962).
indomethacin, the control compounds, compared favorably with (10) J. H. Burn, D. J. Finney, and L. G. Goodwin, “Biological
the results of others using antiedema assay procedures (2). Carra- Standardization,” 2nd ed., Oxford University Press, London,
geenin seemed to possess a distinct advantage as a phlogistic agent England, 1960, p. 33.
because it produced an edema effectively controlled by the single,
oral, nontoxic doses of the known anti-inflammatory agents used in ACKNOWLEDGMENTS AND ADDRESSES
the studies.
Received March 5,1971, from the School ofPhurmacy, Uniuersity
REFERENCES of Arkansas, Little Rock, AR 72201
Accepted for publication July 30,1971.
(1) T.Y.Shen, T. B. Windholz, A. Rosegay, B. E. Witzel, A. N. Abstracted in part from a thesis presented by W. S. Dorsey in
Wilson, J. D. Willett, W. J. Holtz, R. L. Ellis, A, R. Matzuk, S. partial fulfillment of Master of Science degree requirements.
Lucas, C. H. Stammer, F. W. Holly, L. H. Sarett, E. A. Risley, Supported in part by Grant GM-10371 from the National
G. W. Nuss, and C. A. Winter, J. Amer. Chem. SOC.,85,488(1963). Institutes of Health, U. S. Public Health Service, Bethesda, MD
(2) C. A. Winter, E. A. Risley, and G. Nuss, J. Pharmacol. 20014 .

Alkaloids of Tylophora 111: New Alkaloids of

Tylophora indica (Burm) Merrill and
Tylophora dalzellii Hook. f .

KOPPAKA V. RAO*, RICHARD A. WILSON, and BERNICE CUMMINGS

Abstract During a reexamination of the alkaloidal constituents

of Tylophora indicu, three new alkaloids (designated A, B, and C )
were isolated. Analytical and spectral data indicate that these
alkaloids are related to tylophorine and tylophorinine, which are
also present in the plant. Alkaloid B is shown to be a desmethyl-
tylophorine, and Alkaloid C is shown to be a desmethyltylophori-
nine. T. dalzellii has a relatively low alkaloid content, and desmethyl-
tylophorinine is the major constituent. A brief description of the
antileukemic activity of desmethyltylophorinine is presented. 6CH3
bCH3
Keyphrases 0 Tylophora indica-isolation of three new alkaloids 0 I II
Tylophora dalzellii-desmethyltylophorinine determined as major
constituent 0 Desmethyltylophorinine-isolated and identified
from Tylophora indica and Tylophora dalzellii JI Desmethyltylo- species T. indica (Burm) Merrill. A related species,
phorine-isolated and identified from Tylophora indicu T. dalzellii Hook. f., had not been examined, so a
brief study of it also was undertaken.
DISCUSSION
In a recent publication, isolation of six new alkaloids
from Tylophora crebriJlora, together with the known From T. indicu, Govindachari et al. (3) isolated two alkaloids
members tylocrebrine, tylophorine, and septicine, was tylophorine and tylophorinine. They assigned Structures I and TI
described (1). Structures for the new members were for these compounds, respectively, and later recorded their syn-
proposed based on the dibenzo[f,h]pyrrolo[ 1,2b]iso- theses (4-7).
The crude alkaloid from T . indicd was isolated by the following
quinoline skeleton with four or five oxygen-bearing steps: extraction with 0.5 methanolic acetic acid, concentration,
substituents (2). Interest in these alkaloids arose be-
cause of the antitumor activity shown by some members ~ ~~

of this group. In this connection, it seemed worthwhile 1The plant material used was received from India. It was identified
and a voucher specimen was preserved at Chas. Pfizer & Co., Inc.,
to reexamine the alkaloidal constituents of the related Maywood, N. J.

Vol. 60, No. iI, November 1971 [7 1725

Table I-Properties of the Alkaloids of T. iridica
Alkaloid A Alkaloid B Alkaloid C
Melting point 250-252" dec. 235-237' dec. 253-255" dec.
Analysis ----CzaHzsNOs .HzO---. -'--(;0Hz6N04- --CBH~~NOI. '/&LO-
Calc. Found Calc. Found Calc. Found
C 66.15 66.89 72.80 72.48 70.61 10.65
H 6.31 6.45 6.64 6.45 6.46 6.31
N 3.50 3.45 3.68 3.56 3.74 3.76
OCHI 15.52(2) 15.28 24.51 (3) 23.95 16.56(2) 16.85
Molecular weight, m/ea 381 379 365
U V spectrum Amax. log E L.X. log E Amsx. log E
290 4.48 290 4.30 288 4.32
257 4.70 257 4.80 258 4.65
Paper chromatographic behavior, 0.2 0.5 0.1
R j : formamideb-chloroform
6 Mass spectra were obtained on a Hitachi-Perkin-Elmer single-focusing instrument wjth 70 ev. as the ionizing roltage. Whatman No. 1 Nter
paper moistened with a 30% solution of-formamide in acetone, blotted, and develo ed with chloroform saturated with formamide, after the samples
are spotted. Tylophorine and tylophorlnlne show &values of 0.7 and 0.3, respectweyy.

partition of the concentrate between 0.1 N HC1 and ethyl acetate, Anal.-Calc. for GeHnNOe:C, 69.47;H, 6.05;N, 3.11. Found:
extraction of the aqueous layer with chloroform at pH 9,concentra- C,69.05;H,6.22; N, 3.25.
tion of the extract, and crystallization from methanol. It was freed Its IR spectrum (bands at 1725 and 1760 cm.-') and NMR spec-
from most of the tylophorine in the form of its slightly soluble trum (3H peaks at r 7.87 and 7.49) support the presence of an
hydrochloride. The mother liquors were then subjected to counter- alcoholic and a phenolic acetyl group, respectively. Methylation
current distribution in the following system: 3% aqueous acetic with diazomethane leads to tylophorinine. Because of the low
acid-chloroform-n-butanol(3:2:1). The presence of five alkaloids solubility in organic solvents, satisfactory NMR spectra could not
was revealed, two of which were identified as tylophorine and be obtained. A comparison of the NMR spectra of the acetates
tylophorinine. The remaining three appeared to be new and were of Alkaloid C and of tylophorinine is shown in Table 11.
designated as Alkaloids A, B, and C in the order of increasing The spectra suggest that the phenolic hydroxyl is at either 3 or 6.
polarity in the distribution. Together, these three alkaloids repre- A choice between the two is not possible from these data, and
sented approximately l0-15% of the total alkaloid content; of further work is planned.
the three, Alkaloid C was the major component. The analytical Unlike the related species, T. crebripora and T. indica, T. dalzelliiii
and physical data are given in Table I. is rather low in its alkaloid content. After routine extraction, the
Alkaloid A has two phenolic groups and two methoxyls. The crude alkaloid was subjected to countercurrent distribution in the
general similarity of its UV and IR spectra with those of tylo- system already described. One major peak represented nearly all
phorine suggests that the substitution pattern of tylophorine may be of the alkaloid. It was crystallized from chloroform-methanol,
present, with the fifth oxygen presumably being located at 14, in m.p. 253-255 O.

analogy with other members of this group. Anal.-Calc. for C&H23N04.HzO: C, 68.91;H, 6.57; N, 3.65;
Alkaloid B has three methoxyls. Its UV spectrum is very similar OCHs (2), 16.20.Found: C, 68.64;H, 6.37;N, 3.37;OCHs, 15.60.
to that of tylophorine but shifts in basic solution, thus suggesting Its melting point and analytical, spectral, and chromatographic
the presence of a phenolic hydroxyl. Methylation with diazo- data showed that it was identical with Alkaloid C of T. indica,
methane yields tylophorine. Alkaloid B is thus desmethyltylo- desmethyltylophorinine.
phorine, although the position of the phenolic group cannot be Of the five alkaloids of T. indica, only Alkaloid C showed signif-
located unequivocally at this time. icant activity in murine leukemia (G1210system) (Table 111).
Alkaloid C has two methoxyls, one phenolic hydroxyl, and one
benzylic hydroxyl. It forms a diacetate, m.p. 225-227".
REFERENCES
Table 11-NMR Spectra of Acetates of
Tylophorinine and Alkaloid C
(1) K.V. Rao, J. Pharm. Sci.,59,1501(1970).
Proton Tylophorinine Alkaloid C Remarks (2) Ibid., 59, 1608(1970).
(3) T. R. Govindachari, B. R. Pai, and K. Nagarajan, J. Chem.
1 r 2.32,2.17 2.35,2.20 ABpattern Soc., 1954,2669.
2 2.79, 2.94 2.79,2.94 AB pattern, (4) T.R.Govindachari, M. V. Lakshmikantham, K. Nagarajan,
split (J = 2) and B. R.Pai, Chem. I d . , 1957,1484.
4 2 24 2 z5 Split (J = 2) ( 5 ) T. R.Govindachari, B. R. Pai, S . Rajappa, and N. Viswana-
5 2.24 1.77 Singlet than, Ibid., 1959,950.
8 2.97 2.97 Singlet (6) T. R. Govindachari, M. V. Lakshmikantham, B. R. Pai, and
S. Rajappa, Tetrahedron, 9, 53(1960).
(7) T. R. Govindachari, B. R. Pai, S. Prabhakar, and T. S.
Table III-Antileukemic Activity of Desmethyltylophorinine" Savitri, ibid., 21,2573(1965).
Change in Increase in
Dose, mg./kg. Body Weight Survival Time, ACKNOWLEDGMENTS AND ADDRESSES
~ ~~ ~ ~~~

12 -2.2 135
8 -1.5 149 Received April 5, 1971, from the John L. Smith Memorial for
6 +o. 2 145 Cancer Research, Maywood, NJ 07607
4 +1.3 138
2.7 +2.3 123 Accepted for publication July 30,1971.
Supported by Contract PH43-68-45from the Cancer Chemo-
0 The author is indebted to Dr. T. J. McBride of the John L. Smith therapy National Service Center, National Institutes of Health,
Memorial for Cancer Research, Chas. Pfizer & Co. Inc., Maywood. U. S. Public Health Service, Bethesda, MD 20014
N. J., for the data in this table. The assay was carded out according * To whom correspondence should be addressed: College of
to the protocols given in Cancer Chemotherapy Report No..25, Dec.
1962. An increase in survival rate of 125% or higher is considered as Pharmacy, J. Hillis Miller Health Center, University of Florida,
positive activity. Gainesville, FL 32601

1726 0 Journal of Pharmaceutical Sciences