1222
J . Med. Chem. 1982,25, 1222-1226
with CHC13. The extract was washed with water, dried over Na2S04, and evaporated. The residual oil was solidified on trituration with ether to give Nl-phthalidyl-N3-o-toluyl-5fluorouracil (15): IR (KBr) 1780, 1740, 1710, 1680, 1660 cm-I; NMR (CDC13) 6 2.72 (3 H, S, CH3), 6.70 (1H, d, J = 6 Hz, C&), 7.17-8.33 (9 H, m, Ar H and Ar CH). Reaction of 5-Fluorouracil with 3-Bromophthalide (Method C). 5-FU (1.30 g, 10 mmol) was reacted with 3-bromophthalide (4.69 g, 22 mmol) in the presence of sodium hydride (0.80 g, 20 mmol) as described for the preparation of 8. The product was crystallized from CHC13 to yield Nl-phthalidyl-5fluorouracil (6) as a colorless solid (0.26 g, lo%), which showed identical spectra (IR and NMR) with those of compound 6 prepared by method D. The above CHCl, filtrate was evaporated, and the residue was chromatographed on silica gel with CHC13 as an eluent, yielding one of the diastereomers of 12 (12a;0.23 g, 6%): IR (KBr) 1785,1730, 1690,1670 cm-l; NMR (CDC13+ Me2SO-d6)6 7.00 (1 H, d, J = 6 Hz, C6 H). Subsequent elution with CHC13gave 1212 (2.53 g, 64%) and the following elution afforded the other diastereomer of 12 (12b; 0.28 g, 7%): IR (KBr) 1770, 1730,1680 cm-'; NMR (CDC13 Me2SO-d6)6 6.95 (1 H, d, J = 6 Hz, c6 H). Deacylation of N-Acyl-N-phthalidyl-5-fluorouracils. (a) Solvolysis in AcOH-EtOH (Method D). A mixture of 8 (3.00 g), EtOH (450 mL), and AcOH (25 mL) was refluxed for 35 h. After the mixture was cooled, the resulting precipitate was collected and washed with ether tQ give 6: IR (KBr) 1790,1700,1660 cm-'; NMR (CDC13+ Me2SO-d6)6 7.43-8.17 (6 H, m, c6 H, Ar H and Ar CH). (b) Hydrolysis in Dilute HCl-EtOH (Method E). NlAcetyl-N3-phthalidyl-5-fluorouracil (11;" 4.38 g) was heated with a mixture of 0.05 N HCl(50 mL) and EtOH (50 mL) under reflux, and the solvent was removed. To the residue was added water, and the precipitated crystals were collected and washed with water. The dried crystals were treated with hot CHCl, (15 mL) for 3 min. The insoluble crystals in CHC1, were collected to give 10: IR (KBr) 1780, 1725,1675 cm-l; NMR (CDC13 + Me2SO-d6) 6 7.38-8.05 (6 H, m, c6 H, Ar H and Ar CH). Antitumor Activity against P-388 Leukemia (Table 11). Female BDFl mice (Charles River, Japan) weighing 17-23 g were used. Five mice for each test group were implanted intraperi' ~ administered toneally with lo6 cells of P-388.13 Test s ~ l u t i o nwas
+
(12) A mixture of diastereomers of 12a and 12b was formed in a ratio of approximately 1:1, mp 214-217 "C (AcOEt). (13) R. I. Geran, H. H. Greenberg, M. M. Macdonald, A. M. Schumacher, and B. J. Abbott, Cancer Chernother. Rep., Part 3,3, 8 (1972).
orally once daily for 9 days, starting 1day after implantation. The antitumor activity of the compounds was expressed by the ratio of the median survival time of the treated mice (T)to that of the control mice (C). Antitumor Activity of Compounds 6 and 10 and Tegafur (2) against MH 134 and Meth A Tumors. Five female mice (C3H for MH 134 and BALB/c for Meth A) for each group, weighing 17-23 g, were implanted with one of the tumors: lo6 cells of MH 134 and 5 X lo6 cells of Meth A were implanted subcutaneously in the side of the abdominal region of the mouse. Test solution14 was administered orally twice daily for 20 days, starting 1 day after implantation. On 21st day, the mice were killed, and the tumor weight was assessed. The antitumor activity of compounds was expressed by the ratio of the median weight of tumors of the treated mice (T)to that of the control mice (C). Acute Toxicity of Compounds 6 and 10 and Tegafur (2). Ten female ICR mice for each group, weighing 22-30 g, were used. Test solution14 was administered orally. The LD, value was calculated by the Litchfield and Wilcoxon method15 from the mortality of mice for 21 days after the administration. In addition, the general behavior of the mice was observed after the administration. Serum Concentration of 5-FU for Mice Treated with Compounds 6 and 10 and Tegafur (2). Female BDFl mice weighing 20-26 g (Charles River, Japan) were subjected to this study. Test solution14 was administered orally a t a dose of 1 mmol/kg (dosage of 6 and 10,260 mg/kg). After 0.5, 1, 2,4, and 7 h, the mice were killed by bleeding under ether anesthesia. The collected blood was centrifuged, and the sera were decanted into tubes. The sera were diluted with saline, acidified, and extracted with CHC1,. In a similar way, 5-FU (aqueous layer) was separated from 6, 10, and tegafur (organic layer). 5-FU and tegafur concentrations in the sera were determined biologically by the antibacterial activity against Staphylococcus aureus P-2O9,I6while the concentrations of compounds 6 and 10 were determined by high-pressure liquid chromatography (Waters Associates, Inc.).
Acknowledgment. We thank President A. Yanagisawa and Director H. 0. Takagi, Grelan Pharmaceutical Co., Ltd., for t h e i r encouragement. We also thank the Pharmaceutical Institute, Tohoku University, for microanalyses. (14) Test solution was prepared by dissolving or suspending the test compound in 0.3% sodium carboxymethylcellulose (CMC). (15) J. T. LitcMield and F. Wilcoxon, J. Pharrnacol. Exp. Ther., 96, 99 (1949). (16) Y. Yasuda, T. Togo, N. Unemi, S. Watanabe, K. Harima, and T. Suzue, J. Jpn. SOC.Cancer Ther., 21, 1711 (1973).
Cardenolide Analogues. 14. Synthesis and Biological Activity of Glucosides of Cl7P-Modified Derivatives of Digitoxigenin Phillipa S m i t h , Lindsay Brown, John Boutagy, and Richard Thomas* Department of Pharmacy, University of Sydney, Sydney, Australia 2006. Received September 10, 1981 An improved method for the synthesis of cardiac glycosides was used to prepare 3P-glucosides of digitoxigenin derivatives in which the 17p side chain was CH=CHX (X = COOH, CONHp,COCH3, CN, or COOR). We compared the inotropic
activity of the compounds with that of digitoxigenin glucoside using guinea pig left atria. All compounds were active except for the acid (7)and the amide (8). The inactivity of the amide, in spite of its favorable shape and high capacity for forming intermolecular hydrogen bonds, is incompatible with some previous structure-activity relationship theories. Of the active genins, glucosidation enhanced activity by a factor of about 2. All glucosides, including those with high potency, showed rapid onset and offset of action. The stepwise fall in potency that occurred when the ester group (CH=CHCOOR) was increased in bulk supported previous suggestions that the portion of the digitalis receptor that interacts with the C17 side chain lies within a cleft.
In 1971 we described' a simple route whereby the lactone of digitoxigenin could be replaced with a variety of open0022-2623/82/1825-1222$01.25/0
chain moieties to produce compounds of the t y p e shown i n Scheme I (4a-h). In subsequent publications we de@ 1982 American Chemical Society
Glucosides of Digitoxigenin Derivatives
Journal of Medicinal Chemistry, 1982, Vol. 25, No. 10 1223
Scheme I. Synthesis of Cl7P-Modified Derivatives of Digitoxigenin and the Subsequent Conversion of These to Glucosidesa
hence, permit the SAR significance of increasing the size of the C17 side chain to be evaluated. Chemistry. We prepared compounds as shown in Scheme I using published methods. The etianaldehyde (3) was prepared by degradation of the lactone of digitoxigenin.l Compounds 4a-h were prepared by condensing gluco3 with appropriate phosphonate r e a g e n t ~ . ~ The J sides 6a-h were prepared by reacting 4a-h with acetobromoglucose in the presence of a mixture of catalystss to give the tetraacetyl glucosides (5a-h), which were converted to the free glucosides by mild deacetylation with triethylamineas New compounds described in this report include the glucosides 6a-h and the aglycons 4f-h and 8. Rigorous proof of structure is essential in all SAR studies, and considerable attention was given to this question in our papers cited above. Chemical and spectroscopic evidence were used to establish that the side chain a t C17 was 6 oriented and that the configuration of about the 20,21 double bond was t r a n ~ . l > ~Proof J structure of glycosides presents a number of problems, since EIMS does not give the molecular ion and lH NMR provides limited information about the configuration of the glycosidic linkage. To overcome these problems, we used ammonia CIMSg and 13CNMR,'O together with more traditional methods of structure determination.8 Ammonia CIMS of the glucosides gave the molecular ion as the base peak, as well as useful information about purityaQ With the aid of appropriate reference compounds, it was possible, using lH and 13C NMR spectroscopy, to establish that all 36-glucosides had the P-D configuration.8J0 Biological Activity. We tested the compounds for inotropic activity using the isolated left atria of the guinea pig. The concentrations of drug required to increase the force of contraction by 75% (AF75) were obtained from the log dose-response curves. Half-times for onset and offset of inotropy were measured after the addition of a single AF75dose. These data are shown in Table I, together with corresponding values for a selection of reference compounds. Visual inspection indicated that the dose-response curves were roughly parallel over the initial parts of the curve (including the points a t which relative potencies were measured). However, the higher esters did not show the same maximum increase as shown by other active compounds. No reason for this was found, except that it was not due to solubility being exceeded. The importance of the carbohydrate residue in the inhibition of (Na+,K+)ATPaseby cardiac glycosides was first investigated by Yoda et al.ll Although these authors examined a number of sugar residues, glucose was not included, nor was the work extended to inotropic activity. In a previous publication,s we drew attention to the range of the contribution that the carbohydrate can make to inotropic activity. For example, we found that digitoxigenin rhamnoside was 22 times more potent than digitoxigenin, digitoxigenin galactoside was equipotent, and digitoxigenin rhamnoside 2'-acetate was half as potent. In the present series, it was not possible to determine the
P-lHO
v
FHO
OH
R (a) (b) (c) (d) (e)
(f ) OH
6 !a-h)
(g) (h)
CH =C HCOO COOH H 4
( C I
(@
A Iy1,
OH 7
IYI1
-CH=CH-CN -CH=CH-COOCH, -CH=CH-COOCHzCH3 -CH=CH-COOCH,CHZCH, -CH=CH-COOCH (cH3)2 -CH=CH-COOCH2CH2CH2CH3 -CH=CH-COOCH2CH(CH3)2 -CH=CH-COCH3
- (@
CHECHCONH,
OH 8
a I = (a) (Ac),O/pyridine, ( b ) 0,, (c) NaBH,, (d) NaOH, (e) NaIO,; I1 = (R'O),P(O)CH-R"; I11 = Ag,CO, (Celite)/ HgBr,/Hg(CN),; IV = Et,N/MeOH/H,O; V = OH-; VI = Et,N, ClCOOCH,CH(CH,),, NH,OH.
scribed the application of this reaction to the study of digitalis SAR.2 The route has been used by Eberlein and Heider3 and by Fullerton et aL4to prepare C17 analogues of digitoxin and digitoxigenin and by Beard and coworkers5 to prepare C17 analogues of strophanthidol. Our earlier work was limited by the fact that certain key compounds had very low water solubility. The compounds included the esters 4b-e (Scheme I). The low potency and low water solubility of these compounds meant that it was not possible to obtain concentrations high enough to measure the full dose-response curve. To overcome this problem, we synthesized the glucosides of these and related compounds and tested them for inotropic activity using guinea pig left atria. The results of this work are described in this paper. The advantages of testing glycosides rather than genins are several. Glycosides are usually (but not always) more potent than their corresponding genins. They are also more water soluble. These two sets of properties should enable the full dose-response curves to be obtained and, ~~
(1) J. Boutagy and R. Thomas, Aust. J. Chem., 24, 2723 (1971).
R. Thomas, L. Brown, J. Boutagy, and A. Gelbert, Circ. Res., 46, 1-167 (1980). (3) W. Eberlein and J. Heider, US Patent 3752803 (1973). (4) D. S. Fullerton, M. C. Pankaskie, K. Ahmed, and A. H. L. From, J. Med. Chem., 19, 1330 (1976). ( 5 ) N. A. Beard, W. Rouse, and A. R. Somerville, Br. J. Pharmacol., 54, 65 (1975). (2)
(6) J. Boutagy and R. Thomas, Aust. J. Pharm. Sci., NS1, 67 (1972). (7) J. Boutagy and R. Thomas, Aust. J. Pharm. Sci., NS2, 9 (1973). (8) L. Brown, J. Boutagy, and R. Thomas, Arzneim.-Forsch., 31, 1059 (1981). (9) J. Vine, L. Brown, J. Boutagy, R. Thomas, and D. Nelson, Biomed. Mass. Spectrom., 6,415 (1979). (10) H. T. Cheung, L. Brown., J. Boutagy, and R. Thomas, J. Chem. SOC.,Perkin Trans. 1 , 1773 (1981). (11) A. Yoda, Ann. N.Y. Acad. Sci., 242, 598 (1974).
1224 Journal of
Medicinal Chemistry, 1982, Vol. 25, No. 10
Smith et al.
Table I. Inotropic Effects on Guinea Pig Left Atria of Genins and Glucosides of Cl7P-Modified Derivatives of Digitoxigenin
Genins 12,75 i 19 1 2 , 7 51 16 9, 7 5 i 30 11, 75 i 24
1.1 x
digitoxigenin (1)
nitrile (4a) Me ester (4b) Me ketone (4h)
1.6 X 3.8 X 5.6 X
acid (7 ) amide ( 8 )
inactive
