160
Ind. Eng. Chem. Prod. Res. Dev., Vol. 17, No. 2, 1978
as possible during the first step of bishophite drying is fully justified. Conclusions 1. By chlorination of partially dried bishophite with HCl gas a t 400 "C a practically pure and dried MgC12 with only traces of MgO and HzO was obtained. 2. The specific rates of dehydration and chlorination of partially dried MgClz are as follows: R D = 0.013 g/min cm2 HzO; R c = 0.02 g/min cm2 MgClZ. 3. The kinetics of dehydration and chlorination of partially dried bishophite can be represented by the equations 1
In K c = 0.614 - 1.33
T
x 103
4. The energies of activation for chlorination and dehydration are 2700 cal/deg mol and 4700 cal/deg mol, respectively.
Literature Cited Blaker, I., Basyum, O . , German Patent 2 053 189 (May 13. 1971). Buchmann, F. J., U S . Patent 3 352 634. Dow Chemical Co., French Patent 2 067 216. Frantasiev, N. A . , Shcegolov, V. I., Tsvetn. Met., 46(7), 54-58 (1967). Lystsov, A. I., Pechenik, T. S.. Timofeeva, 0. I., Tsvetn. Met., 38(1), 62-66 (1965). Nadler, M., U.S. Patent 3 395 977. Nightingale, E. R., Jr., US. Patent 3 354 128. Shamir, I., Research Thesis, Technion. Israel Institute of Technology, Haifa, 1973. Streletz, H.L., "Metallurghia Magnia Metallurghizdat" (in Russian), 1960.
1
Received for reuieu: July 7, 1977 Accepted February 14, 1978
In KD = 1.552 - 2.365 - X lo3
T
Isolation of Camptothecin and 10-Methoxycamptothecinfrom Camptofheca Acumhafa by Gel Permeation Chromatography J. George Buta' and Michael J. Novak Beltsville Agricultural Research Center, Beitsville. Maryland 20705
Camptothecin and 10-methoxycamptothecin have been isolated semipreparatively from Camptotheca acuminata for study as plant growth regulators. Extraction of the woody plant material with ethanol was followed by partition between CH2CI2and H20.The organic-soluble portion was fractionated by gel permeation chromatography using Biobeads S-X1 or 2. The camptothecin alkaloids, recrystallized from methanol, were obtained in 35 mg yield from 69 g of dry plant material in a typical small-scale separation.
Introduction Camptothecin has shown significant activity as a plant growth inhibitor (Buta and Worley, 1976) and much promise as a tumor inhibitor in mammalian cancer investigations (Schaeppi et al., 1974). Preliminary findings indicate that the alkaloid inhibits cell division in meristematic areas of tobacco. T o extend the study of camptothecin as a plant growth regulator of other plant species, it became necessary to develop a rapid preparatory-scale process for obtaining biologically active camptothecin alkaloids. The gel permeation chromatographic (GPC) process described here yields the alkaloids (camptothecin and its 10-methoxy analogue) in milligram quantities in purity sufficient for studies on plants without further column chromatographic purification. Experimental Section Procedure. Air-dried stemwood of Camptotheca acuminata Decaisne was ground in a hammer mill to pass through a 5-mm screen. The wood chips (69 g) were extracted for 24 h with 95% EtOH in a Soxhlet extractor. The extract was evaporated under reduced pressure and then partitioned between dichloromethane and water. The aqueous portion was extracted twice with dichloromethane. The combined dichloromethane extracts were evaporated and the concentrate (ca. 500 mg) was dissolved in a minimum of EtOAc-CHZC12 (4:l) before filtration. The filtrate was applied to a gel per-
meation column (52 X 6 cm) packed with Biobeads S-X1 (370g) in EtOAc-CH2C12 (4:l). The camptothecin alkaloids were observed as a bright blue fluorescent band when a hand-held ultraviolet (365 mm) lamp was used. Collection of fractions was begun when elution of the blue fluorescent band from the column occurred. Ten fractions of 50 mL each were collected and monitored by TLC using the solvent, benzene-EtOAc-MeOH ( 1 O : l O : l v/v) (Tafur et al., 1976). As indicated in Figure 1, a mixture of camptothecin and 10methoxycamptothecin was present only in the first four fractions collected during a typical separation, whereas camptothecin was present throughout the band. After removal of solvent from the combined fractions and recrystallization from methanol, 35 mg of camptothecin alkaloids were obtained. Melting points were comparable to that of camptothecin, 260-262 "C (Wall et al., 1966). Ultraviolet spectra of the isolated camptothecin alkaloids were taken to determine the purity of various fractions. The relative ratios of camptothecin and 10-methoxycamptothecin could be determined because of the A, for camptothecin a t 253 nm ( c 29 000) and 10-methoxycamptothecin at 265nm (e 23800). The pure camptothecin obtained by this chromatographic procedure was compared by melting point, TLC, and UV with an authentic sample obtained by the silica column chromatographic procedure of Wall et al. (1966). No 10-hydroxycamptothecin was found by TLC analysis although it had been
This article not subject to U. S. Copyright. Published 1978 by the American Chemical Society.
Ind. Eng. Chem. Prod. Res. Dev., Vol. 17, No. 2, 1978
161
Table I. Isolation of Camptothecin Alkaloids from C. acuminata by Gel Permeation Chromatography with Biobeads S-X1. Solvent, EtOAc-CHZC12 (4:l) Wt of
W t of alkaloids,
no.
plant material, g
1
77
2
62 69 68 750
Batch
3 4
5 and 6 Figure 1. Thin-layer chromatography of C. acuminata CHZC12 extract and fractions after GPC chromatography: A, Carnptotheca extract; I, camptothecin; I1,lO-methoxycamptothecin.
isolated as a minor component in Camptotheca (Wani and Wall, 1969). The yields from four gel permeation separations on a single column are presented in Table I. The Biobeadpacked column could be used for four successive separations and then regenerated by washing with 95% EtOH. A larger scale separation was done using an 8.2 X 22.5 cm column packed with Biobeads S-X1 following the same procedure. Two fractions (Batch No. 5 and 6) of the CHZC12 portion of the extract from 750 g of plant material yielded a total of 290 mg of camptothecin alkaloids of sufficient purity (mp and TLC) for further study of plant growth regulating activity.
mg 13 32
35 31 290
regulating activity, it was not considered necessary to separate the two camptothecin alkaloids. The 10-methoxycamptothecin represents approximately 10% of the total camptothecin alkaloids isolated. The two alkaloids are obtained in preparations of greater than 90% purity as determined by UV, TLC, and melting point. Acknowledgments We thank Dr. J. F. Worley for the bioassay study of 10methoxycamptothecin growth activity. The plant material was obtained from Dr. R. E. Perdue, Jr., Medicinal Plant Resources Laboratory, BARC, Beltsville, Md. The material was collected as part of the USDA program developed with the Cancer Chemotherapy National Service Center, NIH. Literature Cited
Discussion Camptothecin has been obtained as a pure compound in small quantities by silica column chromatography (Wall et al., 1966). The gel permeation procedure described here is an attempt to obtain larger quantities of camptothecin alkaloids by a less time-consuming technique. It is to be noted that the 10-methoxy analogue of camptothecin is not completely separated from the major component of interest, camptothecin, by the gel permeation chromatographic technique. 10-Methoxycamptothecin was isolated by silica column chromatography and was found to have plant growth-inhibiting activity at a M concentration essentially comparable to camptothecin. Therefore, for further studies of plant growth
Buta, J. G., Worley, 3. F., J. Agric. FoodChern., 24, 1085 (1976). Schaeppi, U., Fleischrnan, R. W., Cooney, D. A,, Cancer Cbemother. Rep., 3 , 25 (1974).
Tafur, S . , Nelson, J. D., DeLong, D. C., Svoboda, G. H., Lloydia, 39, 261 (1976).
Wall, M. E., Wani, M. C., Cook, C. E., Palmer, K. H.. McPhail, A. T., Sim, G. A,, J. Am. Chern. SOC.,08,3888 (1966).
Wani, M. C., Wall, M. E.. J. Org. Chem., 34, 1364, (1969).
Receiued for review January 9, 1978 Accepted February 16,1978 Mention of a trade mark or proprietary product does not constitute a guarantee or warranty of the product by the US.Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
