RESEARCH
Plants supply promising antitumor agents Active compounds derived from plants may also serve as templates for new synthetic approaches to cancer chemotherapy Growing confidence marks the search for plants containing substances which are potential antitumor agents. The degree of confidence among scientists was characterized by Dr. S. Morris Kupchan of the University of Wisconsin, who told the Seventh Pan American Congress of Pharmacy and Biochemistry, in Buenos Aires, that studies of plant-derived agents have revealed several new types of compounds that inhibit tumor growth to some degree. Some of the new natural products
sin began in 1959 with studies of plants collected in Wisconsin and India. Two of the plants have yielded alkaloidal inhibitors of tumor growth. Thalicarpine, from the purple meadow rue of Wisconsin, significantly inhibits the growth of Walker 256 intramuscular tumor in rats over a wide dosage range. The dimeric alkaloid, dl-tetrandine, also shows significant activity against the Walker 256 tumor. This derivative of bisbenzylisoquinoline was isolated by the Wisconsin group from the roots of Stephania hernandifolia
THALICARPINE. Dr. S. Morris Kupchan of the University of Wisconsin points out the unusual alkaloidal structure of thalicarpine, an antitumor agent extracted from the purple meadow rue
may show sufficient promise in further animal studies to be evaluated clinically. Furthermore, the new and unusual types of compounds may serve as chemical templates for new synthetic approaches to cancer chemotherapy. Meanwhile, to give Dr. Kupchan and other chemists material to work with, botanists from the U.S. Department of Agriculture's Agricultural Research Service have been combing forests and fields the world over for plant sources of antitumor agents. The total number of known plant species that contain active agents is currently about 750. But as active agents are isolated from plants, duplication and advanced testing of the compounds may eliminate many of them. Dr. Kupchan's program at Wiscon64 C&EN DEC. 12, 1966
Walp., a plant that grows in India. Structural studies of thalicarpine show that the compound has an unusual alkaloidal structure; it is the first recognized dimeric aporphinebenzylisoquinoline alkaloid. Further studies are now under way at the National Cancer Institute to evaluate thalicarpine's suitability for clinical trial. The two major groups of scientists that screen compounds for antitumor activity are at the National Cancer Institute'and at Eli Lilly & Co. Dr. J. L. Hartwell coordinates the natural products program at NCI's Cancer Chemotherapy National Service Center (CCNSC). USDA supplies about half of the plant samples (about 2000 annually) for this program. Other plant collectors associated with the program are the University of Arizona
and the Commonwealth Scientific and Industrial Research Organization (CSIRO), Melbourne, Australia. There are also several other contributors to the program. Dr. R. E. Perdue, Jr., head of USDA's plant-collection program, has gathered at least 11,000 random samples of plants from about 5000 species. About 450 of these have yielded extracts possessing confirmed activity in experimental tumor systems. All USDA samples are first inspected in Washington, D.C. In addition, samples from foreign countries are inspected for insects. All samples are then sent to the Wisconsin Alumni Research Foundation, Madison, where they are extracted with aqueous alcohol. The extracts are then sent for testing to any one of eight laboratories. Each sample is tested for cytotoxicity in KB cell culture (derived from a carcinoma of the human nasopharynx). Besides the Wisconsin laboratory, others cooperating in the testing program are Hazleton Laboratories, Battelle Memorial Institute, Arthur D. Little, Southern Research Institute, Charles Pfizer & Co., the University of Miami, and IIT Research Institute. The most important in vivo tumors used for testing in the NCI program are the Walker 256 carcinosarcoma and the lymphoid leukemia L-1210. Among some of the other types of tumors formerly used in the government program are sarcoma 180, hepatoma 129, adenocarcinoma 755, and Cloudman melanoma (S91). As of September 1966, NCI had published data on 10,237 plant extracts tested mostly in sarcoma 180, carcinoma 755, leukemia L-1210, and KB cell culture. Dr. Kupchan and his group have studied a number of antitumor agents. They extracted two growth inhibitors, elephantopin and elephantin (C&EN, Aug. 8, page 15), from the Florida plant Elephantopus elatus Bertol. The two compounds were isolated in a systematic study directed toward identifying the factors which impart activity against cancer cells in tissue culture. After isolation, the compounds showed significant activity against Walker 256 tumor in rats. Dr. Kupchan also found that withaferin A, derived from Acnistus arborcsccns Schlechtd, shows activity
Wisconsin, Lly compounds typical of structural diversity
against Walker 256 (the plant has been popularly used to treat cancer in Costa Rica for years). The growth inhibitor possesses an unusual steroidal unsaturated lactone structure. In addition, he isolated /3-solamarine, a steroid alkaloid glycoside, from Solanum dulcamara, a plant used by physicians to treat cancer from the time of Galen, about 180 A. D. References to the use of S. dulcamara in treating cancer have appeared in the literature of at least 10 countries. Solamarine shows activity against the sarcoma-180 tumor system in mice as well as against Walker 256 in rats. Last June at a meeting sponsored by the International Union of Pure and Applied Chemistry in Stockholm, Sweden, Dr. Kupchan disclosed structural data showing that both elephantopin and elephantin possess unusual sesquiterpene dilactone structures. Dr. A. T. McPhail and Dr. G. A. Sim at the University of Illinois provided the x-ray analyses which helped determine the structures of these compounds. Independent of NCI, Eli Lilly & Co. set up a program in 1956, under the direction of Dr. Irving S. Johnson, for testing antitumor agents with tumor systems which differed from those set
up at NCI. Among the tumors used at Lilly are C-1498 leukemia, P-1534 leukemia, sarcoma 180, and X-5563 myeloma. Lilly's program is older than the expanded NCI program under CCNSC (although first systematic activity in this field in the U.S. was started by NCI in about 1939). According to Lilly's Dr. Gordon H. Svoboda, "The purpose of this [Lilly's] program was hopefully to uncover unique agents which the synthetic organic chemist would not attempt? either because of novelty or complexity." Among the first antitumor agents of botanical origin to find success in Lilly's program were the alkaloids isolated from the apocynaceous plant, Catharanthus roseus G. Don (Vinca rosea L.). Out of more than 60 alkaloids extracted from this plant, six are active. Workers at Lilly found that leurosine, vincaleukoblastine (vlb.), leurocristine, leurosidine, leurosivine, and rovidine are capable of curing a particular lymphocytic leukemia or inhibiting its growth. Vincaleukoblastine, marketed for five years by Lilly as Velban, is effective in treating chorioepithelioma, Hodgkin's disease, and other lympho-
mas. In addition, a number of beneficial results have been obtained with it in treating breast cancer. Another Lilly anticancer agent, Oncovin (vincristin), is used to treat acute leukemia in children. Acronycin, isolated from Australian scrub ash (Acronychia Baueri Schott), has shown the broadest experimental antitumor activity of any alkaloid studied at Lilly, Dr. Svoboda told the Division of Medicinal Chemistry during the 152nd ACS National Meeting in September. Acronycin was effective against 12 of the 17 neoplasms tested. The alkaloid shows activity with the C-1498 leukemia neoplasm, against which no agent has previously shown activity. An attempt was made to test acronycin on mice with tumor systems comparable to human cancer. Dr. Svoboda chose the X-5563 plasma cell myeloma as a model for human multiple myeloma. But the results of the test were inconclusive. y-Globulin appears to be directly related to the growth of this tumor and a decrease in the level of this protein is an indication of a mouse's positive response to acronycin. However, a depression of growth of the tumor in humans does not necessarily mean a drop in the level of y-globulin. Acronycin is also effective against the tumor Shionogi Carcinoma 115. This is an androgen-dependent tumor system which may be valuable as a model for activity against cancer of the prostate. Only minimum activity was observed when acronycin was administered intravenously to the animals, according to Dr. Svoboda. The low activity probably stems from the compound's insolubility; therefore, sufficiently high blood levels could not be attained. In several instances, blockage of the circulatory system showed up with the autopsy revealing an actual aortic block by the compound itself. An equally promising antitumor agent, camptothecin (C&EN, July 11, page 19), comes from the Chinese tree Camptotheca acuminata Decne. It inhibits the growth of tumors in mice infected with lymphoid leukemia L-1210 and in rats bearing the Walker 256 tumor. The first announcement DEC.
12, 1966 C&EN
65
CAMPTOTHECIN. Dr. Monroe E. Wall, seated at center, Dr. M. C. Wani, Dr. Keith Palmer, and Dr. Clarence E. Cook (left to right) study a model of the camptothecin molecule at Research Triangle Institute, where the compound was first isolated
of the substance was made at the June IUPAC meeting by Dr. Monroe E. Wall of Research Triangle Institute of Durham, N.C. He is head of a program affiliated with NCI. U.S. chemists have had trouble obtaining camptothecin for years, perhaps because very few of the trees containing it exist in this country. In addition, it gives no positive response to standard alkaloid tests, making its detection difficult. Last year, though,
USDA planted a number of the trees at its Plant Introduction Station, in Chico, Calif. It won't be long before these trees can be harvested, because the Camptotheca reaches a height of 12 feet in two years. Camptothecin's insolubility made its isolation easier. It was isolated by silica gel chromatography of the methanol-insoluble material from the chloroform extract. This was followed by crystallization from methanol-acetoni-
New and old compounds being studied for antitutmor action
66
C&EN DEC. 12, 1966
trile to give pale yellow needles. Camptothecin is active against leukemia L-1210 in mice. It prolongs their lives by as much as 100% on a daily dose of 0.25 to 1.0 mg. per kg. of animal weight. Also, concentrations as low as 1.25 mg. per kg. significantly inhibit the growth of Walker 256 tumor in rats. Two more common substances that have retained recognition as potential antitumor agents are colchicine and podophyllotoxin. These two agents have been known for years to have antimitotic activity and even some use in cancer therapy. Among the natural analogs of colchicine, demethylcolchicine shows significantly better activity against mouse leukemia than does colchicine. Demethylcolchicine was isolated from Colchicum luteum Baker by Dr. Kappaka V. Rao at Pfizer's John L. Smith Memorial for Cancer Research. Comparative tests by Dr. Rao of the more promising derivatives of colchicine show even better activity. These derivatives include demecolcine, acetylcolchinol, and trimethylcolchicinic acid methyl ether. The last two were originally developed and tested at NCI. These examples show that relatively slight changes in structure can quantitatively change the pattern of activity. The podophyllum resin derived from either Podophyllum peltatum L. or Podophyllum emodi Wall, was studied by Dr. J. L. Hartwell and others at NCI between 1947 and 1952. The resin yielded a group of compounds somewhat resembling colchicine in their antimitotic activity, though not closely in structure. The chief active components are podophyllotoxin, demethylpodophyllotoxin, a-peltatin and /?-peltatin. Chemists at Switzerland's Sandoz, Ltd., have prepared a variety of new compounds structurally related to podophyllotoxin. The aim of the study, directed by Dr. Emil Schreier, is to get a more complete picture of the relationship between chemical structure and antimitotic activity. Dr. A. von Wartburg, at Sandoz, isolated four lignan glucosides of podophyllotoxin and its derivatives. Three of the lignans inhibit mitosis significantly, but lignan J is by far the best of the group as an inhibitor. Other alkaloids, such as olivacine and the synthetic demethylolivacine, show activity against leukemia L-1210 transplanted in mice, according to Dr. Leon Goodman of Stanford Research Institute. The benzo and phenyl derivatives are inactive. Olivacine itself is also active against KB cells. Dr. Goodman, director of an NCIaffiliated program at Stanford Research, has also devised a new syn-
REDUCTION COMBUSTION
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Helium is the d r i v i n g gas a n d pure O2 is used for combustion. The combustion a n d r e d u c t i o n furnaces, c o n t a i n e d in o n e housing, a r e indep e n d e n t l y heated t o 9 0 0 ° C a n d 6 5 0 ° C respectively. Halogens a n d sulfur a r e r e m o v e d by silver; other 'scrubbing*' materials m a y be a d d e d as r e q u i r e d . Excess o x y g e n is r e m o v e d a n d oxides of nitrogen a r e converted t o nitrogen in the reduction furnace. The combustion gases a t t a i n e q u i l i b r i u m in a glass mixing v o l u m e . The sample v o l u m e is a coil of t u b i n g 12 feet l o n g , a l l o w i n g a m p l e time for the detectors t o measure the steady-state gas concentration. Chemical traps a r e used to remove CO2 a n d H2O between thermal conductivity cells. The n i t r o g e n is measured by reference to pure helium in the third pair of thermal conductivity cells. Each d e t e c t o r s o u t p u t is electrically i n d e p e n d e n t a n d is r e c o r d e d separately.
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thesis of the naturally occurring antitumor alkaloid ellipticine, which is also active in the L-1210 tumor. Moreover, he has synthesized isoellipticine, a closely related but inactive compound. Dr. J. W. Loder, of Australia's Commonwealth Scientific and Industrial Research Organization, isolated ellipticine and methoxyellipticine from native plants. Ellipticine, the structure of which was proved by Nobel Laureate R. B. Woodward at Harvard, and the naturally occurring 6-methoxyellipticine have also been synthesized by Dr. C. C. J. Culvenor of CSIRO, in collaboration with NCI. The screening program by CCNSC shows that another alkaloid found in Australia, tylocrebrine, has antitumor activity against leukemia L-1210. The alkaloid was isolated and its structure established by Dr. E. Gellert at CSIRO. Since a large amount of this material was needed for clinical evaluation, NCI contracted with Dr. Rao at Pfizer to scale up the preparation of the alkaloid. During this work with the genus Tylophora, Dr. Rao isolated several other new alkaloids that have antitumor activity. Most of the alkaloids isolated from Tylophora, which retain the phenanthroindolizidine skeleton, also exhibit cytotoxic activity against HeLa cells (a type of human tumor), Dr. Rao says. The activity seems to vary with the nature of the substituents. For example, tylophorine with methoxyl groups at C-2, C-3, C-6, and C-7 is much less active than the isomeric tylocrebrine with the C-2, C-3, C-5, and C-6 arrangement. Also, compounds with a benzylic hydroxyl are more active than those without. Similarly, members with both the phenolic and benzylic hydroxyls are the most active, according to Dr. Rao. The University of Arizona group, under the direction of Dr. Willis R. Brewer, has found about 77 extracts which he says have confirmed activity. Among the plants yielding antitumor substances in this study are Maytenus trichotomus Turcz., Gutierrczia sarothrac Britt and Rushy, and Rumex hymcnoscpalus Torr. The relationship between structure and antitumor activity is elusive, but scientists are deriving systematic views and methods from an empirical approach. "Several of the new growth inhibitors are now being studied for possible effects on a variety of biochemical systems," Dr. Kupchan said in Buenos Aires at the Pan American Congress. "The structures and biochemical modes of action of these empirically derived growth inhibitors could provide important basic knowledge for new approaches to cancer chemotherapy."
