1979, Vol. 35, pp. 235-241, O Hippokrates Verlag GmbH
Phnta
medii
F w d ~ b nResearch t
Cytotoxic Activity of 6.20-Epoxylathyrol and its Aliphatic Diesters G. Schroeder*, M. Rohmer', J. P. Beck**, and R. Anton*
Key Word Index: Euphorbia lathyris L.; Diterpene; 6,20-Epoxylathyrol; Cytotoxicity; HTC Hepatoma Cells.
Abstract
6,20-epoxylathyrol,
isolated
from
Euphorbia lathyris L., is a pharmacologically inactive lathyrane type diterpene. After alkaline hydrolysis and reesterification of this natural product with aliphatic acids of various chain lengths, some 3,5-aliphatic diesters showed interesting cytotoxic properties. The influence of liposolubility on cytotoxic activity of 6,20-epoxylathyrol diesters is discussed.
Introduction
Numerous cytotoxic andlor irritant diterpenes have been isolated from various species of Euphorbiaceae. These diterpenes are of four types, the tigliane, ingenane, daphnane and lathyrane types. So far the lathyrane type diterpenes have been considered to have no cytotoxic or irritant activity [I].
The cytotoxic or irritant properties depend on the lipos~lubilit~ of the diterpenes. Thus, study of the aliphatic phorbol diesters (from phorbol 12,13diacetate to phorbol12,13-dipalmitate), has shown that phorbol is most irritant when it has a medium liposolubility (phorbol 12,13-dioctanoate or phorbol 12,13-didecanoate) [2]. Some authors suggest that esterification of these molecules increases their liposolubility, which allows them to penetrate into the cells and reach their activity site. This hypothesis has been confirmed for certain cytotoxic diterpenes [2, 3,4, 51. 6,20-epoxylathyrol has been isolated from Euphorbia lathyris L. [6, 71. It is an important by-product of oil production from the seeds of Euphorbia lathy-
ris L. We have attempted the following experimental steps: 1) to determine whether a lathyranetype diterpene, 6,20-epoxylathyrol, isolated as a triester (3-phenylacetate 5,10-diacetate 6,20-epoxylathyrol)
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' Laboratoire de Pharmacognosie, Facult6 de Pharmacie, Strasbourg, France.
" lnstitut de Physiologie et de Chimie Biologlque, Strasbourg, France.
Schroeder, Beck, Anton
Material and Methods Chemical methods Melting points were taken o n a Reichert microscope and are corrected. Infrared absorption spectra were recorded over the range of 4 0 0 4 0 0 0 cm-I with a Beckmann I.R. 8 spectrophotorneter, using CHC13 as a solvent. Nuclear magnetic resonance spectra in CDC1, solutions on a Perkin Elmer R12B (60 MHz) spectrometer or on a Brucker (90 MHz) spectrometer. Chemical shifts were recorded in parts per million downfield from an internal reference of tetramethylsilane. The following abbreviations are used: - s (singlet), - d (doublet), - t (triplet), - m multiplet). Mass spectra were obtained by direct introduction on a Thompson T H N o r on a LKB 9000 S spectrometer (ionisation energy = 70 eV). Ultraviolet absorption spectra were obtained on a Beckmann D. B. spectrometer. 3-Phenylacetate 5,lO-diacetate 6,20-epoxylathyrol, 1, was submitted to alkaline hydrolysis ( K O H 1 O/o/MeOH). After extraction with CH,CI, and recrystallization in MeOWH,O, we obtained 6,20epoxylathyrol, 2, whose physical properties (mp, IR, UV, NMR, MS) were identical to those already reported [8]. The aliphatic diesters of 6,20-epoxylathyrol were prepared as follows: 6,20-epoxylathyrol (1 mmole) was dissolved in anhydrous pyridine (20 ml) and treated with the corresponding aliphatic acid chloride (6 mmoles) for 24 hours a t room temperature under N p atmosphere.
After acidification with HC1 N, extraction with CH,Cl,, washing of the organic phase with saturated solutions of NaHCO, and NaC1, and drying on anhydrous Na,SO,, the aliphatic diesters were purified by silica gel column &tomatography using cyclohexane-ethylacetate (8:2) as the solvent. Since this method does not esterify tertiary alcohols, the tertiary alcohol a t C-10 was not esterified. We thus obtained nine 3,s-aliphatic diesters of 6,20-epoxylathyrol (see Table I).
Table I Molecular formulas of natural triesterified epoxylathyrol, I , hydrolysed epoxylathyrol, 2, and aliphatic diesters of epoxylathyrol, 3-11.
As an example, we indicate the detailed physical data for 3,s-dihexanoate 6,20-epoxylathyrol (c32H600,) : MS: M+ = 546; M+-CH, = 531; M+-H,O = 528; M+-C,H,,O, = 430; M+-(C,H,20,)2 = 314. I R (CHC1,): v (OH) = 3450 cm"; v (-CH) aliphatic = 2950-2920 cm-l; v (-C=O) aliphatic ester = 1730 cm-'; (-C=O) a$-ethylenic ketone = 1671 cm"; v (C(CH,),) = 1452 cm-l.
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from Euphorbia lathyris L. has cytotoxic properties. 2) to synthesize the series of aliphatic diesters (from 3,5-diacetate to 3,5distearate 6,20-epoxylathyrol) from the trio1 diterpene and to test these compounds for cytotoxicity in vitro, in order to ascertain whether changing the lipos~lubilit~ of 6,20epoxylathyrol could change its cytotoxic effects.
Cytotoxic Activity of 6.20-Epoxylathyrol
g-:-CO-R,
Biological assays For the study of the cytotoxic activity of 6,20-epoxylathyrol and its aliphatic diesters we used the in vitro cultured H T C cells [9] derived from clone 7288 of a Morris rat hepatoma. These cells have retained the sensitivity to glucocorticoid hormones and respond like normal hepatocytes by induction of the enzyme tyrosine-aminotransferase. The cells were grown as a n homogeneous suspension in SWIM'S-S-77 medium supplemented with 10°/o calf serum. Their mean generation time is approximately 28 hours, and they grow exponentially for 3 t o 4 days, before reaching the stationary phase. Assays were performed on 75 ml samples of cell suspension incubated a t 37O in Bellco spinner flasks with magnetic stirring. The initial concentration of the cell suspensions was adjusted to about 1.5X 105 cells/ml. Compounds 1 to 11 were dissolved in ethanol to give concentrations of 25, 50, and 100 mglml. 100 p l of these solutions was added t o the 75 ml culture media. T o the control culture media was added 100 p l of pure ethanol. Thus we obtained final concentrations of 33, 66, and 132 ,ug/ml of cell culture. Every 24 hours, 1 ml samples were taken from each cell culture and further incubated for 15 minutes with Trypan Blue. Growth control was then recorded by counting the cells of these samples in a Neubauer microcytometer.
We thus evaluated the viability total cells - dead cells) X 100010 total initial cells as reported in Figs. 1, 2, and 3. For the study of the irritant activity of 6,20-epoxylathyrol and its aliphatic diesters, we used the mouse-ear test developed by HECKER 1101. - The products t o be analyzed were applied in acetone solution (0.006 ml) to one ear of the mice; the second ear was used as a control. The irritation which becomes visible in the form of a redness on the skin of the ear was evaluated 24 hours after the application of the product. The amount (&ear) of diterpene which produced an average irritation after 24 hours was defined as the 50°/o-inflammation unit (IU,,). The control substance used was croton oil DAB7, the IU,, of which is 2.4 pglear. Products 1 to 11 were applied a t three different concentrations (2.4, 4.8, and 9.6 pglear) and five mice were used for each concentration.
[(
I
Results Fig. 1 shows the effect of 6,20-epoxylathyrol, 2, at three different concentrations on HTC cell cultures. At 33 and 66 pglml of culture medium, the growth of the cells was only weakly inhibited. At 132 ,uglml of culture medium, the 6,20-epoxylathyrol was clearly cytotoxic. At this concentration, after three days, only 30 O/o of viable cells remained, whereas the number of viable cells in the control culture reached 400°/o. The percentages of inhibition of cell were not directly proportional to the concentrations of 6,20-epoxylathyrol used, but suggest a threshold effect. Fig. 2 shows the comparative viablity of the HTC cells in the presence of various by-products of 6,20-epoxylathyrol. The natural triesterified epoxylathyrol isolated from Euphorbia lathy-
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U V (MeOH): Rmax = 273,208 nm (e = 15000, 5100) N M R (CDCI,): C_H3-CH,, 0.89 ppm (6H, t, J = 6 Hz), X c r l t , 1.10 ppm (3H, d, J = 6.5 H Hz); :;X : ,1.14 ppm (6H, s); -CH,-, 1.20 pprn (12H, m); = -CH3, 1.65 ppm (3H, s large); -CH,-COO-, 1.98 ppm (4H, m); y-;, 2.34 and 2.42 ppm (2H, m); X 5.50 ppm and 6.42 ppm ( l H , m); = -H, 6.60 ppm (1H, m). The 3,5-aliphatic diesters of 6,20-epoxylathyrol are colourless resins. The presence of two aliphatic ester functions was confirmed by MS, NMR, IR. The UV spectral data (Imax = 273, 208 nm, s = 15000, 5100) indicate that the chromophore group of 6,20-epoxylathyrol was not altered. The supplementary spectral data (IR, NMR, MS) were identical to those already published [6, 81.
Schroeder, Beck, Anton
238 viable cell number
%
Fig. I . Hepatoma tissue culture viability in presence of three doses of hydrolysed ep~x~lathyrol, 2.
ris L. 1 had no cytotoxic activity, whereas hydrolysed epoxylathyrol, 2, was cytotoxic at the same concentration. The cytotoxic activity of the 3,5-aliphatic diesters of 6,20-epoxylathyrol depended on the length of the aliphatic acid chains. 3,5-dihexanoate 6,20-epoxylathyrol, 4, had the greatest cytotoxic activity. From 3,5-dihexanoate 6,20epoxylathyrol, 5, the cytotoxic activity declined regularly with the increasing length of the aliphatic acid chains, and 3,5-distearate 6,20-epoxylathyrol, 11, was practically inactive. Fig. 3 shows the action of 3,5-dibutyrate 6,20-epoxylathyrol, 4, on HTC
cells. At 66 pg/ml of culture medium, it was only cytostatic, and no Trypan Blue stained dead cells could be observed, .but at 132 pg/ml of culture medium, the cytotoxicity of 3,5-dibutyrate 6,20-epoxylathyrol, 4, became evident. At this concentration 4 killed all the HTC cells within 48 hours as shown by the Trypan Blue staining procedure. The irritant activity tests were negative for 6,20-epoxylat-hyrol as well as for its aliphatic diesters. Even a t a very high concentration (9.6 pglear) products I to 11 did not visibly irritate the ears. Thus, we did not try to investigate a possible cocarcinogenic activity of these
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
initial total cell number
Cytotoxic Activity of 6,20-Epoxylathyrol
viable cell n u m b e r t o t a l init iat cell number
239
%
I
,0 c o n t r o l
.
/ lo
07
v8 v
loo-
\
\ 9
2
'
3
days
Fig. 2. Hepatoma tissue culture viability in presence of natural triesterified epoxylathyrol, 1, hydrolysed epoxylathyrol, 2, and aliphatic diesters of epoxylathyrol, 3-11.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
)0
Schroeder. Beck. Anton
240 viable
" "1
c e l l number
i n i t i a l t o t a l cell number
%
m m,,
4004 -
H
control control
0
0 200-
o-O-o
Fig. 3. Hepatoma tissue culture viability in presence of three doses of 3,5-dibutyrate 6,20-epoxylathyrol, 4.
products, because an irritant activity is a necessary although not sufficient condition for a cocarcinogenic activity [2].
Discussion \
3,s-dibutyrate 6,20-epoxylathyrol, 4, is the first lathyrane-type diterpene of Euphorbiaceae that has been found to be cytotoxic. Although its cytotoxicity is evident only at a relatively high concentration, we nevertheless attribute a cytotoxic activity to it for two reasons. First, at 132 ,uglml of culture medium, this diterpene inhibited the
growth of H T C cells, whereas at the same concentration, the more liposoluble 3,s-aliphatic diesters of 6,20-epoxylathyrol and the naturally triesterified epoxylathyrol isolated from Euphorbia lathyris L. showed no inhibition on cell growth. Secondly, the rat hepatoma H T C cells still have a certain detoxication capacity, which is characteristic for normal hepatic cells. Therefore, it will be necessary to analyze the action of 3,5-dibutyrate 6,20-epoxylathyrol, 4, on different cell systems. These studies will perhaps permit demonstration of a specific action of this diterpene on tumor-cell systems; if such
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
6 6 ug/rnl
3 days
100.
Cytotoxic Activity of 6,PO-Epoxylathyrol
Acknowledgments We thank Professor Dr. G. OURISSONfor generous gifts of 6,20-epoxylathyrol.
References 1. Hecker, E.: 2. Krebsforscfiung 78,99 (1972). 2. Thielmann, H. W. and E. Hecker: Fortschritte der Krebsforschung 7, 161 (1969). 3. Sakata, K., K. Kawazu, T. Mitsui and N. Masaki: Tetrahedron Letters 16, 1141 (1971). 4. Kinghorn, A. D. and F. J. Evans: J. Pharm. Pharmac. 27, 329 (1975). 5. Kupchan, S. M., J. G. Sweeny, R. L. Baxter, T. Murae, V. A. Zimmerly and B. R. Sickles: J. Am. Chem. Soc. 97, 672 (1975). 6. Adolf, W., E. Hecker, A. Balmain, M. F. Lhomme, Y. Nakatani, G. Ourisson, G. Ponsinet, R. J. Pryce, T. S. Santhanakrishnan, L. G. Matyukhina and I. A. Saltikova: Tetrahedron Letters 26, 2241 (1970). 7. Zechmeister, K., M. RGhrl, F. Brandl, S. Hechtfischer, W. Hoppe, E. Hecker, W. Adolf and H. Kubinyi: Tetrahedron Letters 35, 3071 (1970). 8. Balrnain, A.: J. &em. Soc. 1253 (1975). 9. Thompson, E. B., G. M. Tomkins and J. F. Curran: Proc. Natl. Acad. Sci. USA 56, 296 (1966). 10. Hecker, E.: Z. Krebsfor~chun~65, 325 (1963). 11. Kupchan, S. M., I. Ucfiida, A. R. Branfman, R. G. Dailey, Jr. and B. Yu Fei: Science 191, 571 (1976). 12. Kupchan, S. M. and R. L. Baxter: Science 187, 652 (1975). 13. Furstenberger, G and E. Hecker: Planta Med. 22, 241 (1972). 14. Warri, M. C., H. L. Taylor, M. E. Wall, P. Coggon, A. P. McPhail: J. Am. Chem. SOC.93, 2325 (1971). 15. Kupchan, S. M. and R. M. Scfiubert: Science 185, 791 (1974). 16. Monroe, E. and M. C. Walland: Ann. Rev. Pharmacol. Toxicol. 17, 117 (1977).
Address: Prof. Dr. R. Anton, Laboratoire de Pharmacognosie, Faculte de Phaumacie. 7 4 , route du Rhin, 67400 Illkirch, France
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an action is found, it would be possible to attribute an antitumor activity to the substance. The results concerning the cytotoxic activity of the aliphatic diesters of 6,20epoxylathyrol - demonstrate the part played by liposolubility in the cytotoxic activity of Euphorbia diterpenes. They confirm the results of studies concerning the irritant and cocarcinogenic activity of the 12,13-aliphatic diesters of phorbol [2]. We have studied both the cytotoxic and irritant activities of the 3,5-aliphatic diesters of 6,20-epoxylathyrol, because of the paradoxical similarity of structure of cocarcinogenic and/or antileukemic compounds of Euphorbiaceae and Thymeleaceae [11, 131. 6,20-epoxylathyrol, 1, is a commercially available product known to be pharmacologically inactive. By means of some simple structural transformations, we were able to demonstrate the appearance of cytotoxic properties. The action of a certain number of antitumor substances has been explained by the presence of functional groups that can act as alkylating agents [14, 161. We intend to study the influence of the a,ðylenic ketone and the oxirane ring of the 6,20-epoxylathyrol molecule. Indeed, these functional groups may confer to 6,20-epoxylathyrol alkylating properties, which might explain its cytotoxic activity against the in vitro cultured HTC cells.
Phnta
medii
F w d ~ b nResearch t
Cytotoxic Activity of 6.20-Epoxylathyrol and its Aliphatic Diesters G. Schroeder*, M. Rohmer', J. P. Beck**, and R. Anton*
Key Word Index: Euphorbia lathyris L.; Diterpene; 6,20-Epoxylathyrol; Cytotoxicity; HTC Hepatoma Cells.
Abstract
6,20-epoxylathyrol,
isolated
from
Euphorbia lathyris L., is a pharmacologically inactive lathyrane type diterpene. After alkaline hydrolysis and reesterification of this natural product with aliphatic acids of various chain lengths, some 3,5-aliphatic diesters showed interesting cytotoxic properties. The influence of liposolubility on cytotoxic activity of 6,20-epoxylathyrol diesters is discussed.
Introduction
Numerous cytotoxic andlor irritant diterpenes have been isolated from various species of Euphorbiaceae. These diterpenes are of four types, the tigliane, ingenane, daphnane and lathyrane types. So far the lathyrane type diterpenes have been considered to have no cytotoxic or irritant activity [I].
The cytotoxic or irritant properties depend on the lipos~lubilit~ of the diterpenes. Thus, study of the aliphatic phorbol diesters (from phorbol 12,13diacetate to phorbol12,13-dipalmitate), has shown that phorbol is most irritant when it has a medium liposolubility (phorbol 12,13-dioctanoate or phorbol 12,13-didecanoate) [2]. Some authors suggest that esterification of these molecules increases their liposolubility, which allows them to penetrate into the cells and reach their activity site. This hypothesis has been confirmed for certain cytotoxic diterpenes [2, 3,4, 51. 6,20-epoxylathyrol has been isolated from Euphorbia lathyris L. [6, 71. It is an important by-product of oil production from the seeds of Euphorbia lathy-
ris L. We have attempted the following experimental steps: 1) to determine whether a lathyranetype diterpene, 6,20-epoxylathyrol, isolated as a triester (3-phenylacetate 5,10-diacetate 6,20-epoxylathyrol)
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
' Laboratoire de Pharmacognosie, Facult6 de Pharmacie, Strasbourg, France.
" lnstitut de Physiologie et de Chimie Biologlque, Strasbourg, France.
Schroeder, Beck, Anton
Material and Methods Chemical methods Melting points were taken o n a Reichert microscope and are corrected. Infrared absorption spectra were recorded over the range of 4 0 0 4 0 0 0 cm-I with a Beckmann I.R. 8 spectrophotorneter, using CHC13 as a solvent. Nuclear magnetic resonance spectra in CDC1, solutions on a Perkin Elmer R12B (60 MHz) spectrometer or on a Brucker (90 MHz) spectrometer. Chemical shifts were recorded in parts per million downfield from an internal reference of tetramethylsilane. The following abbreviations are used: - s (singlet), - d (doublet), - t (triplet), - m multiplet). Mass spectra were obtained by direct introduction on a Thompson T H N o r on a LKB 9000 S spectrometer (ionisation energy = 70 eV). Ultraviolet absorption spectra were obtained on a Beckmann D. B. spectrometer. 3-Phenylacetate 5,lO-diacetate 6,20-epoxylathyrol, 1, was submitted to alkaline hydrolysis ( K O H 1 O/o/MeOH). After extraction with CH,CI, and recrystallization in MeOWH,O, we obtained 6,20epoxylathyrol, 2, whose physical properties (mp, IR, UV, NMR, MS) were identical to those already reported [8]. The aliphatic diesters of 6,20-epoxylathyrol were prepared as follows: 6,20-epoxylathyrol (1 mmole) was dissolved in anhydrous pyridine (20 ml) and treated with the corresponding aliphatic acid chloride (6 mmoles) for 24 hours a t room temperature under N p atmosphere.
After acidification with HC1 N, extraction with CH,Cl,, washing of the organic phase with saturated solutions of NaHCO, and NaC1, and drying on anhydrous Na,SO,, the aliphatic diesters were purified by silica gel column &tomatography using cyclohexane-ethylacetate (8:2) as the solvent. Since this method does not esterify tertiary alcohols, the tertiary alcohol a t C-10 was not esterified. We thus obtained nine 3,s-aliphatic diesters of 6,20-epoxylathyrol (see Table I).
Table I Molecular formulas of natural triesterified epoxylathyrol, I , hydrolysed epoxylathyrol, 2, and aliphatic diesters of epoxylathyrol, 3-11.
As an example, we indicate the detailed physical data for 3,s-dihexanoate 6,20-epoxylathyrol (c32H600,) : MS: M+ = 546; M+-CH, = 531; M+-H,O = 528; M+-C,H,,O, = 430; M+-(C,H,20,)2 = 314. I R (CHC1,): v (OH) = 3450 cm"; v (-CH) aliphatic = 2950-2920 cm-l; v (-C=O) aliphatic ester = 1730 cm-'; (-C=O) a$-ethylenic ketone = 1671 cm"; v (C(CH,),) = 1452 cm-l.
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from Euphorbia lathyris L. has cytotoxic properties. 2) to synthesize the series of aliphatic diesters (from 3,5-diacetate to 3,5distearate 6,20-epoxylathyrol) from the trio1 diterpene and to test these compounds for cytotoxicity in vitro, in order to ascertain whether changing the lipos~lubilit~ of 6,20epoxylathyrol could change its cytotoxic effects.
Cytotoxic Activity of 6.20-Epoxylathyrol
g-:-CO-R,
Biological assays For the study of the cytotoxic activity of 6,20-epoxylathyrol and its aliphatic diesters we used the in vitro cultured H T C cells [9] derived from clone 7288 of a Morris rat hepatoma. These cells have retained the sensitivity to glucocorticoid hormones and respond like normal hepatocytes by induction of the enzyme tyrosine-aminotransferase. The cells were grown as a n homogeneous suspension in SWIM'S-S-77 medium supplemented with 10°/o calf serum. Their mean generation time is approximately 28 hours, and they grow exponentially for 3 t o 4 days, before reaching the stationary phase. Assays were performed on 75 ml samples of cell suspension incubated a t 37O in Bellco spinner flasks with magnetic stirring. The initial concentration of the cell suspensions was adjusted to about 1.5X 105 cells/ml. Compounds 1 to 11 were dissolved in ethanol to give concentrations of 25, 50, and 100 mglml. 100 p l of these solutions was added t o the 75 ml culture media. T o the control culture media was added 100 p l of pure ethanol. Thus we obtained final concentrations of 33, 66, and 132 ,ug/ml of cell culture. Every 24 hours, 1 ml samples were taken from each cell culture and further incubated for 15 minutes with Trypan Blue. Growth control was then recorded by counting the cells of these samples in a Neubauer microcytometer.
We thus evaluated the viability total cells - dead cells) X 100010 total initial cells as reported in Figs. 1, 2, and 3. For the study of the irritant activity of 6,20-epoxylathyrol and its aliphatic diesters, we used the mouse-ear test developed by HECKER 1101. - The products t o be analyzed were applied in acetone solution (0.006 ml) to one ear of the mice; the second ear was used as a control. The irritation which becomes visible in the form of a redness on the skin of the ear was evaluated 24 hours after the application of the product. The amount (&ear) of diterpene which produced an average irritation after 24 hours was defined as the 50°/o-inflammation unit (IU,,). The control substance used was croton oil DAB7, the IU,, of which is 2.4 pglear. Products 1 to 11 were applied a t three different concentrations (2.4, 4.8, and 9.6 pglear) and five mice were used for each concentration.
[(
I
Results Fig. 1 shows the effect of 6,20-epoxylathyrol, 2, at three different concentrations on HTC cell cultures. At 33 and 66 pglml of culture medium, the growth of the cells was only weakly inhibited. At 132 ,uglml of culture medium, the 6,20-epoxylathyrol was clearly cytotoxic. At this concentration, after three days, only 30 O/o of viable cells remained, whereas the number of viable cells in the control culture reached 400°/o. The percentages of inhibition of cell were not directly proportional to the concentrations of 6,20-epoxylathyrol used, but suggest a threshold effect. Fig. 2 shows the comparative viablity of the HTC cells in the presence of various by-products of 6,20-epoxylathyrol. The natural triesterified epoxylathyrol isolated from Euphorbia lathy-
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U V (MeOH): Rmax = 273,208 nm (e = 15000, 5100) N M R (CDCI,): C_H3-CH,, 0.89 ppm (6H, t, J = 6 Hz), X c r l t , 1.10 ppm (3H, d, J = 6.5 H Hz); :;X : ,1.14 ppm (6H, s); -CH,-, 1.20 pprn (12H, m); = -CH3, 1.65 ppm (3H, s large); -CH,-COO-, 1.98 ppm (4H, m); y-;, 2.34 and 2.42 ppm (2H, m); X 5.50 ppm and 6.42 ppm ( l H , m); = -H, 6.60 ppm (1H, m). The 3,5-aliphatic diesters of 6,20-epoxylathyrol are colourless resins. The presence of two aliphatic ester functions was confirmed by MS, NMR, IR. The UV spectral data (Imax = 273, 208 nm, s = 15000, 5100) indicate that the chromophore group of 6,20-epoxylathyrol was not altered. The supplementary spectral data (IR, NMR, MS) were identical to those already published [6, 81.
Schroeder, Beck, Anton
238 viable cell number
%
Fig. I . Hepatoma tissue culture viability in presence of three doses of hydrolysed ep~x~lathyrol, 2.
ris L. 1 had no cytotoxic activity, whereas hydrolysed epoxylathyrol, 2, was cytotoxic at the same concentration. The cytotoxic activity of the 3,5-aliphatic diesters of 6,20-epoxylathyrol depended on the length of the aliphatic acid chains. 3,5-dihexanoate 6,20-epoxylathyrol, 4, had the greatest cytotoxic activity. From 3,5-dihexanoate 6,20epoxylathyrol, 5, the cytotoxic activity declined regularly with the increasing length of the aliphatic acid chains, and 3,5-distearate 6,20-epoxylathyrol, 11, was practically inactive. Fig. 3 shows the action of 3,5-dibutyrate 6,20-epoxylathyrol, 4, on HTC
cells. At 66 pg/ml of culture medium, it was only cytostatic, and no Trypan Blue stained dead cells could be observed, .but at 132 pg/ml of culture medium, the cytotoxicity of 3,5-dibutyrate 6,20-epoxylathyrol, 4, became evident. At this concentration 4 killed all the HTC cells within 48 hours as shown by the Trypan Blue staining procedure. The irritant activity tests were negative for 6,20-epoxylat-hyrol as well as for its aliphatic diesters. Even a t a very high concentration (9.6 pglear) products I to 11 did not visibly irritate the ears. Thus, we did not try to investigate a possible cocarcinogenic activity of these
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
initial total cell number
Cytotoxic Activity of 6,20-Epoxylathyrol
viable cell n u m b e r t o t a l init iat cell number
239
%
I
,0 c o n t r o l
.
/ lo
07
v8 v
loo-
\
\ 9
2
'
3
days
Fig. 2. Hepatoma tissue culture viability in presence of natural triesterified epoxylathyrol, 1, hydrolysed epoxylathyrol, 2, and aliphatic diesters of epoxylathyrol, 3-11.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
)0
Schroeder. Beck. Anton
240 viable
" "1
c e l l number
i n i t i a l t o t a l cell number
%
m m,,
4004 -
H
control control
0
0 200-
o-O-o
Fig. 3. Hepatoma tissue culture viability in presence of three doses of 3,5-dibutyrate 6,20-epoxylathyrol, 4.
products, because an irritant activity is a necessary although not sufficient condition for a cocarcinogenic activity [2].
Discussion \
3,s-dibutyrate 6,20-epoxylathyrol, 4, is the first lathyrane-type diterpene of Euphorbiaceae that has been found to be cytotoxic. Although its cytotoxicity is evident only at a relatively high concentration, we nevertheless attribute a cytotoxic activity to it for two reasons. First, at 132 ,uglml of culture medium, this diterpene inhibited the
growth of H T C cells, whereas at the same concentration, the more liposoluble 3,s-aliphatic diesters of 6,20-epoxylathyrol and the naturally triesterified epoxylathyrol isolated from Euphorbia lathyris L. showed no inhibition on cell growth. Secondly, the rat hepatoma H T C cells still have a certain detoxication capacity, which is characteristic for normal hepatic cells. Therefore, it will be necessary to analyze the action of 3,5-dibutyrate 6,20-epoxylathyrol, 4, on different cell systems. These studies will perhaps permit demonstration of a specific action of this diterpene on tumor-cell systems; if such
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
6 6 ug/rnl
3 days
100.
Cytotoxic Activity of 6,PO-Epoxylathyrol
Acknowledgments We thank Professor Dr. G. OURISSONfor generous gifts of 6,20-epoxylathyrol.
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Address: Prof. Dr. R. Anton, Laboratoire de Pharmacognosie, Faculte de Phaumacie. 7 4 , route du Rhin, 67400 Illkirch, France
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an action is found, it would be possible to attribute an antitumor activity to the substance. The results concerning the cytotoxic activity of the aliphatic diesters of 6,20epoxylathyrol - demonstrate the part played by liposolubility in the cytotoxic activity of Euphorbia diterpenes. They confirm the results of studies concerning the irritant and cocarcinogenic activity of the 12,13-aliphatic diesters of phorbol [2]. We have studied both the cytotoxic and irritant activities of the 3,5-aliphatic diesters of 6,20-epoxylathyrol, because of the paradoxical similarity of structure of cocarcinogenic and/or antileukemic compounds of Euphorbiaceae and Thymeleaceae [11, 131. 6,20-epoxylathyrol, 1, is a commercially available product known to be pharmacologically inactive. By means of some simple structural transformations, we were able to demonstrate the appearance of cytotoxic properties. The action of a certain number of antitumor substances has been explained by the presence of functional groups that can act as alkylating agents [14, 161. We intend to study the influence of the a,ðylenic ketone and the oxirane ring of the 6,20-epoxylathyrol molecule. Indeed, these functional groups may confer to 6,20-epoxylathyrol alkylating properties, which might explain its cytotoxic activity against the in vitro cultured HTC cells.
