Journal of Medicinal Chemistry, 1977, Vol. 20, No. 3
Antitumor Agents
333
Antitumor Agents. 21. A Proposed Mechanism for Inhibition of Cancer Growth by Tenulin and Helenalin and Related Cyclopentenones Iris H. Hall,' Kuo-Hsiung Lee,* Eng Chun Mar, Charles 0. Starnes, Department of Medicinal Chemistry, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27514
and Thomas G . Waddell Department of Chemistry, University of Tennessee a t Chattanooga, Chattanooga, Tennessee 37401. Received July 9, 1976 Evidence is presented that sesquiterpene lactones or ketones containing the O=CC=CH2 moiety, e.g., tenulin and helenalin, alkylate the thiol group of reduced glutathione and L-cysteine in vitro. A proposal is offered that this mechanism of action is responsible for the observed potent in vivo antitumor activity of these agents in the Ehrlich ascites and Walker 256 carcinosarcoma and to a lesser extent in the P388 leukemic screen. Inhibition of tumor growth is thought to occur due to the O=CC=CH2 system alkylating by rapid Michael addition the SH biological nucleophiles of key regulatory enzymes of nucleic acid and chromatin metabolism. This proposition is in accord with the ability of these agents to inhibit DNA synthesis and gene activity of Ehrlich ascites cells.
Previously it has been demonstrated that sesquiterpene lactones or ketones containing an O=CC=CH2 system had significant antitumor e.g., helenalin (1) caused 99% inhibition of Ehrlich ascites cell growth and produced a T/C = 316 for the survival of rats with Walker 256 ascites carcinosarcoma.6 Structures containing the a-methylene-y-lactone moiety have been reported to attack biological nucleophiles, e.g., L-cysteine8 and the enzymes phosphofructokinaseg and glycogen synthetase,1°by rapid Michael addition. Other related sesquiterpene lactones, tenulin (2)" and plenolin (3),7which have been characterized previously, were found to have potent cytotoxic activity; however, these compounds contain only an amethyl-y-lactone. Compounds 1, 2, and 3 contain the cyclopentenone ring system 4. Previously it has been demonstrated that 2,3,11,13-tetrahydrohelenalinwhich possessess the a-methyl-y-lactonic moiety is i n a ~ t i v e .At ~ this time we are reporting the in vivo antitumor activity of these compounds and their effect on DNA and chromatin protein metabolism. H I
H
H I
!
.,
I
1
HO
0 0
0
3
2 COOH
COOH
6
4 HO
5
Experimental Section Source of Compounds. Helenalin (1) was isolated from the extraction of Texas Helenium microcephalum according to the literature procedure." Ten& (2) was isolated from the extraction of Helenium amarum. A 476-g sample of the leaves and stems of Helenium amurum (Raf.) H. Rock (collected in Bledsoe County, Tenn.) was cut into 0.5-in. pieces and extracted with CHCl3 for 4 days at room temperature. After decanting, the plant material was extracted (twice) in the same manner. The combined extracts were evaporated to dryness leaving a dark green tar (48.6 g). The crude extract was stirred for 30 min with 400 ml of water-EtOH (3:l)and filtered through Celite. The aqueous filtrate was extracted with five 100-ml portions of CHC13 and the combined
organic phase was dried over anhydrous MgS04. Filtration and evaporation in vacuo left 19.4 g of the final extract as an amber oil. Trituration of thisoil with benzene gave 6.27 g of crude ten& (mp 181-183 "C) which was recrystallized from EtOH-water, mp 186-188 "C (lit.13mp 184-186 "C). A direct comparison (melting point, mixture melting point, IR, TLC) of this material with authentic tenulin established its identity. Tenulin used in the experiments described in this paper showed one spot on TLC (Rf 0.50,20% acetone in chloroform, Merck silica gel G, and visualized by spraying with concentrated HzSO4 and heating). Plenolin (3) was isolated from the extraction of Florida Helenium autumnale according to the procedure previously outlined.7 Cyclopentenone (4) was purchased from Aldrich Chemical Co., Milwaukee, Wis., and used after the NMR spectrum indicated that the compound was homogeneous. Radioactive substrates for biochemical assays were purchased from New England Nuclear, Boston, Mass.; all other reagents were purchased from Calbiochem, La Jolla, Calif. Melting points were determined on a Thomas-Hoover melting point apparatus and are corrected. IR spectra were determined in Nujol with a Perkin-Elmer 257 grating IR or Model 700 recording spectrophotometer. W spectra were obtained with a Cary 15 spectrophotometer. NMR spectra were measured in D20 with a Jeolco C-60 HL spectrometer (Me,%) and chemical shifts were reported in 6 units: s, singlet; d, doublet: t, triplet; and J values in hertz. Elemental analyses were performed by Integral Microanalytical Laboratories, Inc., Raleigh, N.C., or Galbraith Laboratories, Inc., Knoxville, Tenn. Formation of the Tenulin-L-Cysteine Adduct (5). T o deoxygenated 50 mmol of phosphate buffer (pH 7.4) (0.5 ml) was added 0.306 g (1 mmol) of tenulin (2)and 0.177 g (1mmol) of L-cysteine hydrochloride monohydrate. Deoxygenated EtOH was added to obtain a total volume of 3 ml. High purity nitrogen was deoxygenated by bubbling it through a solution of pyrogallol [KOH (25 g), pyrogallol (100g), water (250 ml)] and then through a solution of sodium vanadate (vide infra).14 The sodium vanadate was obtained by the following procedure. Zinc amalgam (125 g) was obtained by treatment with 10% mercurous nitrate (125 ml) containing 5-10 ml of concentrated nitric acid. The zinc was washed with water and placed in a gas washing bottle. A solution of 0.1 mol of sodium metavanadate in 2 mol of H2S04was added. Nitrogen was bubbled through the yellow-green solution which became dark blue in a few minutes. A color change to muddy brown indicated a loss of effectiveness. A stream of oxygen-free nitrogen was bubbled through the reaction mixture of tenulin and cysteine for 1 h. The flask was sealed and stored in the dark at room temperature for 5 days. The precipitated reaction product was separated by filtration and washed thoroughly with distilled water (in which cysteine is soluble) and CHC1, (in which tenulin is soluble). This reaction product (5,0.135 g) was dried in vacuo at 78 "C for 33 h. A paper chromatogram [Whatman No. 1, 1-butanol-acetic acid-water (3:1:1)] showed a single elongated, ninhydrin-positive spot with a higher Rf value than L-cysteine. The IR spectrum demonstrated the following peaks in Nujol: 3200-2400 (NH3+),1775 (y-lactone), 1730 (cyclopentanone), 1600 cm-' (carboxylate): mp 231 "C dec.
Hall, Lee et al.
334 Journal of Medicinal Chemistry, 1977, Vol. 20, No. 3
Anal. Calcd for C2&29N07S:C, 56.21; H, 6.79. Found: C, 55.96; H, 6.83. Formation of the CyclopentenoneL-Cysteine Adduct (6).40 T o a stirring solution of L-cysteine (84.2 mg, 0.695 mmol) (2 ml) was added cyclopentenone (4, 57 mg, 0.695 mmol). The mixture was stirred at room temperature for 24 h. Evaporation of the water in vacuo at 35 "C yielded a yellow solid (6) which was purified by recrystallization from 80% hot EtOH or by preparative TLC (cellulose F) using the solvent system 1-BuOH-HOAc-H20 (3:l:l). This product showed a positive ninhydrin test and a strong absorption band a t 1742 c m ~ cor' responding to the carbonyl of cyclopentanone. Compound 6 also exhibited NMR (D20) signals at 6 3.12 (2 H, d, J = 8 Hz, SCH2CHCOOH)and 3.93 (1H, t, J = 8 Hz, SCH,CHCOOH); mp 168-170 "C dec. Anal. Calcd for C8Hl3NO3S.0.25HzO:C, 46.27; H, fi.50; N, 6.74. Found: C, 46.13; H? 6.37; N, 7.06. Reaction of Helenalin (1) with L-Cysteine a n d Glutathione. Helenalin (1, 10 mg, 0.038 mmol) was treated with l.-cysteine (10 mg, 0.082 mmol), reduced glutathione (10 mg, 0.032 mmol), or oxidized glutathionine (10 mg, 0.016 mmol), separately, in deuterated water by homogenation (drill press). After the mixture was allowed to stand 4 h at room temperature, NMR spectra were obtained. Reaction of Cyclopentenone (4) with Reduced Glutathione and Histidine. Cyclopentenone (4, 28 mg, 0.35 mmol) was treated with reduced glutathione (110 mg, 0.36 mmol) for 16 h or histidine (75mg, 0.37 mmol) for 7 days in deuterated water (2 ml) and NMR spectra were obtained. UV Absorption Spectra. Compounds for spectral absorption studies were suspended in 0.1 mol of phosphate buffer pH 7.2 by homogenization. Nucleic acid binding was determined using helenalin or tenulin (0.015pg) incubated with DNA, d4TP, dGTP. dGMP, or dAMP ( 7 5 p g ) . These concentrations gave approximately 50--7570deflection of the UV absorption scale between 200 and 300 nm for each compound. The maximum absorption of helenalin is 220 nm, of tenulin is 225 nm: of guanosine is 248 nm, and of DNA is 258 nm. Helenalin and tenulin had a second absorption peak at 310 and 340 nm, respectively. Binding with dGMP and DNA was also examined at this peak. Solvents other than phosphate buffer were also used, i.e., 0.1 N HCl and EtOH. I n Vivo T u m o r Screens. In the Ehrlich ascites screen, lo6 cells were implanted on day 0. Test compounds were suspended in 0.05% Tween 80-water and homogenized to obtain a fine suspension. Each compound was injected ip (1 mg/day) 33.3 mg/kg/day into CF1 male mice (-30 g). On day 7 the mice were sacrificed and the total volume of ascites fluid and packed cell volume (ascites-crit) was determined in order to calculate the percent inhibition.'% 6-Mercaptopurine was used as a positive standard. In the Walker 256 ascites carcinosarcoma screen, 10' tumor cells were implanted in 80 f 10 g Sprague-Dawley male rats. In the P388 lymphocytic leukemia screen, lo6 cells were implanted into DRA/2 male mice (-18 g) on day 0. Test compounds were injected ip at 2.5 mg/kg/day for the rats and 25 mg/kg/day for the mice. The treated/control (T/C) values for the average days survived were calculated according to NIH protocol 1.500." Phenylalanine mustard and 5-fluorouracil were used as positive standards, respectively, in these screens. Tissue cytotoxic activity was determined with the H.Ep.-Z cell line using the microtechnique of Huang.'' Biochemical Mechanisms of Action. All biochemical studies were carried out on 8-day Ehrlich ascites tumor bearing CF, male mice which had been treated with helenalin (0.125 mg/day) or tenulin (0.25 mg/day) on days, 5,6, and 7. The number of ascites cells per milliliter of fluid was determined utilizing an hemocytometer and the percent of nonviable cells was determined after incubating with 0.4% trypan blue. [I4C]Thymidineincorporation into DNA was determined by the method of Chae.'* One hour prior to sacrifice of the animal, 10 pCi of [methy2-I4C]thymidine (54 mCi/mmol) in 0.1 cmRof isotonic NaCl was injected ip. The DNA was isolated and 'C content was determined by placing an aliquot in 10 ml of scintillation fluid: 2 parts of toluene, 1part of Triton X-100, 0.4% PPO, and 0.1% POPOP. The DNA concentration per aliquot was determined by the diphenylamine reactionIg or by UV spectrophotometry a t 260 nm. DNA polymerase activity was determined on washed (three times) isolated
Table I. Effects of Sesquiterpene Lactones o n Inhibition of Ehrlich Ascites Tumor Growth Survival % at 7 AsciCompd N C days tocrit Vol inhibn Helenalin (1) Tenulin(2) Plenolin ( 3 ) Cyclopentenone ( 4 ) Tenulin- L cysteine adduct 5 6-Mercaptopurineb 0.05% Tween SO-H,O
6 5 5 6
4/6 515 616
0 17 0 31
0.1 0.2 0.0 1.0
100.0 97.3 100.0 74.8
6
616
37
2.8
15.8
6
616
2
0.4
99.4
6
516
30a
4.1'
515
' Standard deviation o n the control ascitocrit was 11.5 and o n the volume it was 1.2. Sigma Chemical Co. N is the number of animals in the group. Table 11. Effects of Sesquiterpene Lactones on Walker 256 Ascites Carcinosarcoma and P388 Leukemic and H.Ep.-2 Tumor Growth
Compd 1
2 3 4
5 Standard
'N =
8.
activity. activity.
Walker 256a TICb 316 266 207 188
P388," TICb 127 131
590
186
134
HEP,, EDWc 0.100 4.500 0.814 50.000 13.700
TIC i125% for minimum significant ED,, < 4 pglml for minimum significant
nuclei from ascites cells prepared by the method of Hymer.20 The incubation medium was that described by Sawada" except [rnethyZ-3H]-d'M'P (10 Ci/mmol) was used. The insoluble nucleic acids were precipitated with 1 mol of PCA containing 10 mmol of Na4PzO7.10H20and were collected on glass fiber paper GF/F (Whatman) by vacuum suction, which were washed and counted. 'j2Pincorporation into histones was determined by the technique of Raineri." One hour prior to sacrifice, 10 pCi of [ Y - ~ ~ P ] - A T P (8.32 Ci/mmol) was injected ip. A nuclear fraction was isolated and the histone extracted and counted. 32Pincorporation into acidic or nonhistones (protein kinase) was determined by the method of KishZ3on isolated nuclei.m The chromatin was collected on nitrocellulose membrane filters B6 (Schleicher and Schuell) and counted. 3',5'-CAMP was measured by the Schwarz-Mann radioimmunoassay using IO6 cells.24 Free DNase activity was determined on ascites fluid homogenized (1:l) in 0.25 mol of ~ ~ acsucrose + 1 mmol of EDTA as outlined p r e v i o u ~ l y .The cumulation of tumor tissue sulhydryl groups was measured in vitro using 0.6 mg of helenalin or tenulin and 0.1 ml of Ehrlich ascites fluid (1:4) in sucrose EDTA by the method of Ellman." After incubation for 20 min at 25 "C, 80% EtOH was added, followed by centrifugation. The supernatant was read at 419 nm.
+
Results Compounds 1-4 were found to be active in the Ehrlich ascites (Table I) and Walker 256 ascites carcinosarcoma. Compounds 1-3 were marginally active in the lymphocytic leukemia screen. Compounds 1-4 possessed cytotoxic activity against the H.Ep.-2 cells (Table 11). No interaction was observed spectrophotometrically between dGMP, dGTP, dAMP, dATP, or DNA and tenulin or helenalin over the range of 210-270 or 300-360 nm. NMR data confirmed this finding, i.e., no reaction between helenalin and dGMP. Furthermore, NMR studies revealed no reaction between helenalin and oxidized glutathione but
Antitumor Agents
Journal of Medicinal Chemistry, 1977, Vol. 20, No. 3 335
Table 111. Effects of Helenalin and Tenulin Dosed on Days 5, 6, and 7 on Mousea Ehrlich Ascites Chromatin Metabolism on Day 8 % control
In vivo DNA Protein [ 14C]Thymidineincorpn into DNA DNA polymerase act. Free DNase act. 32Pincorpn into histones 32Pincorpn into nonhistone-CAMP dependent protein kinase 3',5'-CAMPlevels Ascitocrit
N = 8.
p = 0.001.
Control, 0.05ETween 80, x i SD
0.122mg/ day,
Helenalin,
1ooi 9
6 6 + 13b
1002 9 1ooi 21
83 i 3b 12i 2b
x i SD
l o o + 25
73 f 12b 92k 2 9 + 2b 71 + 17c 29i 15b 17 i 4b
52+ loh 25 t 8 b 3 0 2 13b 6 3 i 14b
1OOi 8 100 t 6 lOOt 6
loot
49 100 * 3
292+ 14Sb 7 0 k 3b
Tenulin, 0.25mg/ day, x i SD
236 43
* 92b t
126
p = 0.050 determined by Student's t test calculation^.^^
helenalin did react with reduced glutathione and L-cysteine after 4 h. This was observed as the disappearance of olefinic protons (two H-13) of the lactone and the H-2 and H-3 of the cyclopentenone. Studies showed that cyclopentenone also reacted with reduced glutathione and L-cysteine but not histidine. IR data confirmed that the carbonyl group of the lactone ring of helenalin was intact after interaction with L-cysteine. The isolation and analysis of the tenulin and cyclopentenone adduct with L-cysteine demonstrated that this reaction did occur in vitro. In 8-day Ehrlich ascites tumor cells the normal DNA content per milliliter of ascites fluid was 1.32 mg. Administration of helenalin or tenulin resulted in a reduction of DNA content by 34 and 27%, respectively (Table 111). The number of ascites cells per milliliter of fluid for the control was 158 X lo6 cells/m13. Tenulin treatment caused 34% reduction in cell numbers whereas helenalin caused 63% reduction. Drug treatment resulted in no significant difference in the percent of nonviable cells, dry weight (mg/ml), or the gross microscopic examination of the size of the cells or morphology. For the control animal, [rnethyl-'4C]thymidine incorporation into DNA was 263948 dpm/mg of DNA. Helenalin caused a drastic inhibition, i.e., 88% while tenulin caused 91% inhibition. Examination of nuclear DNA polymerase activity resulted in 47424 dpm/mg of DNA for the control group. Helenalin and tenulin caused 48 and 39% inhibition, respectively. Free lysosomal DNase activity, for the control, was 1.43 mg of DNA hydrolyzed per gram of dry weight of ascites fluid. DNase activity was inhibited 75% by helenalin and 71% by tenulin. The [y3'P]-ATP incorporation into crude histones for the control was 7989 dpm/mg of chromatin protein. Helenalin caused a 70% reduction and tenulin an 83% reduction. The [y-?P]-ATP incorporation into nonhistone protein for the control was 97 238 dpm/mg of chromatin protein. This was depressed 37% by helenalin. Chromatin protein was 1.086 mg/ml of ascites fluid. Helenalin caused a 12% reduction in chromatin protein whereas tenulin resulted in only an 8% reduction. 3',5'-CAMP level for the control was 3.48 pmol/ lo6 cells. Helenalin administration resulted in a 193% increase while tenulin caused a 136% increase. In vitro helenalin produced an 83% increase in SH group accumulation in ascites cells and tenulin caused a 103% increase (Table IV). Discussion The accepted mechanism of action of antitumor agents, e.g., phenylalanine mustard, is the alkylation of the N-7 nucleophile of guanosine and to a lesser extent the N-1 and
Table IV. In Vitro Effects of Helenalin and Tenulin (0.6 mg)on Day 8 of Mouse Ehrlich Ascites Tumora
Enzyme
Control, 0.05% Tween - 80, Hglenalin, xiSD xtSD
Tenulin, xkSD
DNA polymerase act. 100 t 5 35 t 4b 88 t 9 TissueSHaccumulation 1 O O t 12 1 8 3 t 15b 203 + 22b
N = 6.
p = 0.001.
N-3 position of adenosine.27 We utilized a number of methods for intercalation, DNA binding, N-7 attack, etc., as o ~ t l i n e d ~ and ~ - ~ finally O attempted to measure binding to DNA or guanosine in a phosphate buffer, pH 7.2, over a period of 24 h at room temperature and at a concentration of nucleophile and alkylating agent which was appropriate for cellular reactions. The maximum absorption of nucleophile, e.g., guanosine, was examined to determine (1)if the peak had increased or decreased in magnitude, (2) if the peak shifted toward longer wavel e n g t h ~or , ~ (3) ~ if a methylated purine30 had been generated with a maximum absorption of 280 nm. Taking advantage of the second absorption peak of helenalin (320-360 nm), we tried to determine if the absorption spectrum of the drug was altered by the nucleophile. No interaction was observed for tenulin of helenalin with DNA, dGMP, or dGTP under any of the above in vitro conditions. This was subsequently confirmed with NMR data on the interaction with helenalin and dGMP. However, this does not exclude the possibility of a drug metaboliteb) interacting with purines. Helenalin did interact with reduced glutathione and cysteine supposedly by a rapid Michael type addition of the O=CC=CH2 moiety with the SH groups of these compounds. Kupchan demonstrated that vernolepin and euparotin acetate, and moieties containing an a-methylene-y-lactone, alkylate sulfhydryl-bearing enzymes e.g., phosphofructokinaseg and glycogen synthetase'O and L-cysteine.E The cyclopentenone ring is a component of both tenulin and helenalin. NMR studies have demonstrated that this ring structure also interacts with reduced glutathione and L-cysteine but not L-histidine. The isolation of the cyclopentenone cysteine adduct adds further proof that the reaction is feasible and the O=CC=C system of the cyclopentenone ring represents a major alkylating center of antitumor agents 2-4 which is particularly reactive with thiol groups of proteins. The rapid SH groups accumulation in the tumor cells after helenalin or tenulin treatment suggests that reoxidation of these groups was prevented, thereby making more S H groups available for Michael addition by the O=CC=CH2
Hall. Lee et
336 Journal of Mediccnal Chemistry, 1977, Vol. 20, N o . 3
system in the ketone as well as the lactone. P388, EL-H, and L1210 leukemia cells require sulfhydryl compounds for tissue cell proliferation.” Both tenulin and helenalin reduced DNA synthesis, DNA levels, and cell number in Ehrlich ascites fluid. Since DNase activity was reduced, this was not due to accelerated DNA catabolism but rather due to suppression of DNA polymerase activity by these agents. Loeb claims that nuclear DNA polymerase I1 and I11 possess an SH group which is susceptible to inhibition by thiol reagents (p-mercurochlorobenzoate).32 This SH could be subject to Michael addition by helenalin or tenulin in a similar manner as demonstrated with cysteine and reduced glutathione. Thymidine kinase, and microtubular protein of the mitotic apparatus, contains exposed SH groups which could account for additional depression of DNA content in Ehrlich ascites fluid treated with tenulin or helenalin. DNase endonuclease activity is accelerated in rapid proliferating tumor cellsz4and functions also in normal cells to introduce a nick in DNA, so that new fragments can be added to the 3‘-hydroxy end of the strand.74 The alkylating agent, phenylalanine mustard, caused an increase in DNase activity of Ehrlich ascites cells24which supposedly aids in the hydrolysis of DNA so that crosslinking, apurination, or hydrolysis of the imidazole ring of purine can occur.27 Tenulin and helenalin, rather than causing an increase in DNase activity, caused a reduction in Ehrlich ascites fluid. The fact that no interaction of these drugs occurred with the bases of DNA may suggest why DNase activity was not elevated with these types of alkylating agents. Cellular replication is also regulated by phosphorylation of histone protein by phosphoprotein kinases of the f l fraction. This occurs when cGMP is high, cAMP is decreasing, and during the GI and S phases of the cell cycle3j when DNA polymerase enzyme concentration is increasing. Helenalin and tenulin treatment suppressed histone phosphorylation, again indicating that cellular proliferation and gene activity had slowed due to drug treatment. Nonhistone chromatin protein phosphorylation fluctuates during the cell cycle and parallels RNA transcription and cellular differentiation.q6 In proliferating lymphocytes the phosphorylation of nonhistone proteins is dependent on high cGMP; however, a particular fraction (mol wt 52 O00) is phosphorylated with high cGMP levels and inhibited with high cAMP levels. The latter correlated with inhibition of phosphorylation of chromatin protein. Helenalin and tenulin treatment resulted in a twofold increase in cAMP level in the tumor cells and helenalin caused a significant reduction in nonhistone protein phosphorylation. Energy for phosphorylation of chromatin protein and macromolecule synthesis is derived from Preliminary data have oxidative phosphorylation. demonstrated that in vitro 0.2 mg of tenulin or helenalin drastically suppresses basal respiration and ADP-stimuATP) of Ehrlich ascites lated respiration (ADP + Pi and Walker 256 cells using sodium succinate as substrate.% Thus the sesquiterpene lactones, helenalin and tenulin, do not alkylate nucleophiles of purine bases but rather appear to be reactive with thiol groups of enzymes necessary for DNA replication. Furthermore, these agents cause an increase in cAMP levels of tumor cells which may be correlated with the suppression of chromatin protein phosphorylation necessary for cell replication and differentation.
’’
’’
-
Acknowledgment. We thank Dr. Werner Herz of the Department of Chemistry, Florida State TJniversity, for
a/.
a gift of authentic tenulin. This research was supported by grants from the University of North Carolina, Institutional American Cancer Society IN-15P, and the American Cancer Society No. CH-19. References and Notes K. H. Lee, E. S. Huang, C. Piantadosi, J. S.Pagano, and T. A. Geissman, Cancer Res., 31, 1649 (1971). K. H. Lee, H. Furukawa, and E. S.Huang, J . Med. Chem., 15, 609 (1972).
K. H. Lee, S. H. Kim, C. Piantadosi, E. S. Huang, and T. A. Geissman, J . Pharm. Sei., 63, 1162 (1974). K. H. Lee, R. Meck, C. Piantadosi, and E. S.Huang, J. Med. Chem., 16, 229 (1973). K. H. Lee, T. Ibuka, and R. Y. Wu, Chem. Pharm. Bull., 22, 2206 (1973). K. H. Lee, T. Ibuka, S. H. Kim, B. R. Vestal, I. H. Hall, and E. S. Huang, J . Med. Chem., 18, 812 (1975). K. H. Lee, T. Ibuka, A. T. MePhail, K. D. Onan, T. A. Geissman, and T. G. Waddell, Tetrahedron Lett., No. 13, 1149 (1974). S. M. Kupchan, L). C. Fessler, M. A. Eakin. and T. J. Giacobbe, Science, 168, 376 (1970). R. I,. Hanson, H. A. Lardy, and S.M. Kupchan, Science, 168, 378 (1970). C. H. Smith, J. Larner, A. M. Thomas, and S. M. Kupchan, Biochim. Biophys. Acta, 276, 94 (1972). T. G. Waddell and T. A. Ceissman, Phytochemistry, 8, 2371 11969). K. H. Lee and T. A. Geissman, Phytochemistry, 9, 403 (1970). €3. A. Parker and T. A. Geissman, J . Org. Chem., 27,4127 (1962). S. M. Kupchan, T. J. Giacobbe, I. S. Krull, A. M. Thomas, M. A. Eakin, and D. C. Fessler, J . Org. Chem., 35, 3539 (1970). c‘. Piantadosi, C. S. Kim, and J. L. Irvin, J . Pharm. Sci., 58, 821 (1969). R. I. Geran, N. H. Greenberg, M. M. MacDonald, A. M. Schumacher, and B. J. Abbott, Cancer Chemother. Rep., Part 3, 3 (21, 9, 15 (1972). E. S. Huang, K. H. Lee, C. Piantadosi, T. A. Geissman, and J. S. Pagano, J . Pharm. Sei., 61, 12 (1972). C. B. Chae, J. L. Irvin, and C. Piantadosi, Proc. Am. Rssoc. Cancer Res., 9, 44 (1968). K. Burton, Biochem. J., 62, 315 (1956). W.C. Hymer and E. I,.Kuff, J. Histochem. Cytochem., 12, 359 (1964). H. Sawada, K. Tatsumi, M. Sasada, S. Shirakawa, T. Nakumura, and G. Wakisaka, Cancer Res., 34,3341 (1974). A. Raineri, R. C. Simsiman, and R. K. Boutwell, Cancer Rex, 33, 134 (1973). Y. M. Kish and I,. J. Kleinsmith, Methods Enzymol., 40, 201 (1975). I. H. Hall, K. S. lshaq, and C. Piantadosi, J . Phnrm. Sei., 63, 625 (1974). A. L.Steiner, D. M. Kipnis, R. Utiger, and C. Parker, Proc. .Vat/. h a d . Sci. U.S.A., 64, 367 (1969). G. L. Ellman, Arch. Biochem. Biophys., 82, 70 (1959). P. Calabresi and R. E. Parks in “The Pharmacological Basis of Therapeutics“, 5th ed, L. S. Goodman and A. Gilman, Ed.. Macmillan, New York, N.Y., 1975, Chapter 62, p 1254. 0. M. Friedman, G. N . Mahapatra, B. Dash, and R. Stevenson, Biochim. Biophys. Acta, 103, 286 (1965). K. Y. Zee-Cheng and C. C. Cheng, J . Pharrn. Sei., 62,1572 (1973). A. Loveless, Nature (London),223, 206 (1969). J. I. Toohey, Proc. Natl. Acad. Sei. U.S.A., 72, 73 (1975). I,. A. Loeb in “The Enzymes”, 3d ed, P. D. Boyer, Ed., Academic Press, New York, N.Y., 1974, p 173. M. Friedkin, Adu. Enzymol., 38, 235 (1973). A. Kornberg, Science, 163, 1410 (1969). C1. S. Rubin and 0. M. Roseu, Annu. Rel;. Biochem., 44,831 (1975). E. M. Johnson and J. W. Hadden, Science, 187,1198 (1975). .I. D. Roos and .J. A. I m x , E x p . C ~ l lRes.. . 77, 121 (1973).
Journal of Medicinal Chemistry, 1977, Vol. 20, No, 3 337
Pyrimidinylpropenamides (38) Results to be published. State 4 basal respiration of Ehrlich ascites cells was depressed 38% by helenalin and 48% by tenulin at 0.2 mg. State 3 ADP stimulated respiration was reduced 40% in the presence of helenalin and 30% of tenulin (p = 0.001 for both drugs).
(39) G. W. Snedecor in "Statistical Methods", The Iowa State College Press, Ames, Iowa, 1956, Section 2. (40) Reaction of equimolar cyclopentenone and L-cysteine in 0.067 phosphate buffer-D20 (pH 7.4) for 20 min gave the same product (6)as analyzed by NMR.
Pyrimidinylpropenamides as Antitumor Agents. Analogues of the Antibiotic Sparsomycin Chie-Chang L. Lin and Ronald J. Dubois' Cancer Chemotherapy Division, Microbiological Associates, Bethesda, Maryland 20016. Received August 6, 1976
A series of pyrimidinylpropenamides 9 and their oxidation products 10 was prepared, as analogues of sparsomycin (I), for antitumor evaluation. Syntheses involved condensation of the appropriate amino alcohol 5 with acid 8. The resulting sulfides 9 were then oxidized with NaI04 or H202 to sulfoxides 10. Activity was studied in lymphocytic leukemia P-388 and KB cell culture. With the exception of the n-decyl analogue, all of the deoxygenated compounds 9 were inactive regardless of the stereochemical form. In the sulfoxide series 10, those compounds prepared with an L configuration at the asymmetric carbon were also inactive. The completely racemic sulfoxides, on the other hand, displayed substantial antitumor activity (ILS = 37-61% in P-388; ED50= 1.2-2.4 fig/ml in KB) suggesting that both the presence of a sulfoxide moiety and a D configuration at the chiral carbon atom were structural requirements for a positive antitumor response. There appeared to be a large tolerance for the group substituted at the sulfoxide moiety, however.
Sparsomycin (1) is a fermentation product of Streptomyces sparsogenes, first isolated in 1962.' Soon after this, 1 was subjected to several preliminary biological tests where it displayed a broad spectrum of moderate in vitro activity against bacteria and moderate antifungal activity.2 I t also showed moderate to high inhibition in several in vivo tumor systems such as the Walker carcinosarcoma 256 and the sarcoma 180 solid tumor.2 Its biological activity appears to be primarily due to inhibition of protein synthesis, and this inhibition has been s ~ b s t a n t i a t e d . ~Further ~~ work5 indicated that its mechanism of action in the Escherichia coli system is on the 50s ribosome subunit, where it prevents peptide transfer by interfering with the function of the enzyme peptidyl transferase. Clinical testing of sparsomycin, however, revealed a severe eye toxicity, limiting the
Scheme I NH,CHCH,SSCH,CHNH, I
I
CO,H
CO,H
Socl,, R'OH or +
HCl(g), R'OH
1. Na. ",(I)
2. RX
NH,CH(CO,H)CH,SR 3
HCl.NH,CH(CO,R')CH,SR 4, R' = CH, or CH,CH,
-
(see text for appropriate group) HCl.NH,CH(CH,OH)CH,SR
1. LiBH,
2. HCl(g)
5
a, R = CH,SCH, L stereoisomer b, R = (CH,),CH, L stereoisomer c , R = CH, L stereoisomer d , R = CH,C,H, L stereoisomer e, R = CH(CH,), L stereoisomer
f, R = CH,SCH, DL mixture g, R = (CH,),CH, DL mixture h, R = CH, DL mixture i, R = (CH,),CH, DL mixture j, R = CH,SCH, D stereoisomer
1
usefulness of 1 in cancer chemotherapy.6 Consequently, a synthetic program was initiated in our laboratories in an attempt to exploit the antitumor activity of sparsomycin in a molecule presenting more selective pharmacological properties while determining the minimum structural and stereochemical requirements necessary for antitumor activity. Our initial investigations involved the synthesis of several N-substituted 3-aryl-2-propenamides 2 which proved to be essentially i n a ~ t i v e .In ~ this report, we wish trans-ArCH=CHCONHCH( CH,OH)CH,SCH ,SCH, n
1
to relate the synthesis and biological activity of various stereochemical forms of the pyrimidinylpropenamides 9 and their oxidation products 10. Chemistry. The synthetic route employed for the preparation of the intermediate amino alcohols 5 is out-
lined in Scheme I. Reduction of cystine in the appropriate stereochemical form with Na and liquid ammonia followed by addition of the substituted chloride (iodide for 3h and bromide for 3e) generated the amino acids (3a,b,d-j). Compound 3c was purchased from the Aldrich Chemical Co. These materials were converted into their methyl or ethyl ester hydrochlorides 4 by thionyl chloride in methanol (4a-c) or ethanol (4d-h) or by ethanol saturated with gaseous HC1(4i,j). Lithium borohydride treatment conveniently led to the desired amino alcohols 58-j isolated as the hydrochloride salts. Oxidation of 5-hydroxymethyl-&methyluracil with potassium persulfate and silver nitratea (Scheme 11) gave the 5-formyl derivative 6. Treatment with carbethoxymethylenetriphenylphosph~rane~ in DMF at 90 "C followed by KOH-EtOH hydrolysisg of the resulting ester 7 yielded the uracilacrylic acid 8 exclusively in the trans configuration.
