Synthesis and Biological Evaluation of 14-Methoxy Digitalis Derivatives*

M. Gobbini,** N. Almirante, A. Cerri, G. Padoani and P. MelloniPrassis Istituto di Ricerche Sigma-Tau, Via Forlanini, 3, 20019 Settimo Milanese (MI), Italy. Tel +39 2 33500388, Fax +392 33500408, Email: MC3405@mclink.itReceived: 11 November 1997 / Accepted: 15 January 1998 / Published: 25 January 1998Abstract: The synthesis and biological evaluation of 14 β-methoxy derivatives of digitoxigenin and of otherdigitalis-like compounds are reported. These compounds have a 14β-oxygen, which can be a hydrogenbonding acceptor, as is the case of 14 β,15 β -epoxide derivatives, but not a hydrogen bonding donor as is thecase of 14 β-hydroxy derivatives. All the new 14 β-methoxy derivatives show a considerable reduced bindingaffinity on Na


Introduction
Digitalis cardiac glycosides are well known drugs clinically used for treatment of congestive heart failure [1].Their action is mainly due to inhibition of Na + ,K + -ATPase, an enzyme located in the cell membrane and promoting the outward transport of Na + and the inward transport of K + [2].Recently the existence of endogenous digitalis-like factors that may be responsible for essential hypertension [3] has opened a new field in the study of compounds acting on the Na + ,K + -ATPase.The most potent inhibitors of Na + ,K + -ATPase are cardenolides such as digitoxigenin (Figure 1) with the following structural characteristics: 17β-unsaturated lactone, 3β-and 14β-hydroxy substituents and A/B and C/D cis ring junctions.The 14β-hydroxy group is involved in hydrogen bonding with the receptor and plays an important role in binding digitalis compounds to Na + ,K + -ATPase receptor; in fact compounds in which this group is absent show very low binding affinity or no affinity at all [4].However, the known derivatives with a 14β,15β-epoxy group (Figure 1) show high binding affinities although not as high as the 14β-hydroxy analogues (Table 1).
Herein, we report the synthesis and biological evaluation of novel 14β-methoxy derivatives of digitoxigenin and of other digitalis-like compounds.These compounds have a 14β-oxygen, which can be a hydrogen bonding acceptor, as is the case of 14β,15β-epoxide derivatives, but not a hydrogen bonding donor as is the case of 14β-hydroxy derivatives.Comparison of the binding values of these three classes of compounds could allow more insight into the requirements necessary for recognition by the receptor.Only a 3β-glucoside derivative of digitoxigenin-14β-methoxy has been described [10], for which the inotropic activity was reported to be marginal, but no synthetic route was given.Isodigitoxigenin [6] 17β-(4-Pyridazynil) [7] Digitoxigenin 17β-(3-Furyl) 14β,15β-epoxy [8] 14β,15β-epoxy [9] Figure 1.Parent compounds.

Results and Discussion
Attemps to introduce a methyl on the 14β-hydroxy group of digitoxigenin, with the secondary 3β-hydroxy protected, using diazomethane or dimethyl sulfate failed; diazomethane failed also when applied on the 17β-(3furyl) analogue, while dimethyl sulfate gave a low yield.We then turned our attention to a Williamson reaction with MeI and, since the strongly basic reaction conditions proved incompatible with the presence of the α,β-unsaturated lactone of digitoxigenin, we tried the reaction on the 17β-furyl derivative 2 (Scheme 1).The known 17β-(3-furyl)-5β-androstane-3β,14β-diol 1 [11] was reacted with tert-butyldimethylsilyl chloride in DMF in the presence of triethylamine to give the protected derivative 2 (90%); this TBS derivative and KH were kept at reflux temperature for one hour in dry THF, the addition of MeI instantaneously gave the desired 14β-methoxy derivative 3a.The crude 3a was deprotected with n-Bu 4 NF in THF at reflux temperature to give 3b in quantitative yield from 2. From the 14β-methoxy derivative 3a the 14β-methoxydigitoxigenin 6b could be obtained by the oxidative/reductive procedure [6] shown in Scheme 2. The crude 3a was reacted with m-chloroperbenzoic acid in CHCl 3 in the presence of AcOH and AcONa; the crude hydroxy lactone intermediates 4 and 5 were reduced with NaBH 4 in CH 2 Cl 2 /water to give a mixture of the desired digitoxigenin derivative 6a and of the isomeric isodigitoxigenin derivative 7a in a 8:2 ratio.The two compounds were separated by flash chromatography to give 6a (49% from 2) and 7a (13% from 2) and then deprotected by acidic hydrolysis with HCl in a CHCl 3 /MeOH mixture (6b 81%; 7b 58%) [12].

Scheme 2. Synthesis of 14β-methoxy-digitoxigenin and 14β-methoxy-isodigitoxigenin.
The 17β-(4-pyridazinyl) derivative 8a (Scheme 3) was prepared by reacting the crude 17β-(3-furyl) derivative 3a with NBS in dioxane/water in the presence of AcONa and then with hydrazine [7] to give, after chromatographic purification, the desired 8a (24% from 2) and the N-amino lactam derivative 9a as a side product (20% from 2); 8a was deprotected with n-Bu 4 NF in THF at reflux temperature (81% yield), while 9a degraded to a complex mixture under the same conditions.The synthesized compounds were evaluated, in comparison with 14β,15β-epoxy and/or 14β-hydroxy analogues, for displacement of the specific [ 3 H]-ouabain binding from the Na + ,K + -ATPase receptor [13a] isolated from dog kidney and purified according to Jørghensen [13b].The biological data are showed in Table 1.

Conclusion
All the new 14β-methoxy derivatives show a considerable reduced binding affinity when compared with the 14β-hydroxy analogues and also with the 14β,15βepoxy derivatives; the reduction in the affinity varies from 65 times for 6b, the most potent 14β-methoxy derivative, to 200 times for 3b; the 14 β-methoxy derivative of isodigitoxigenin 7b was almost devoid of any affinity.
These results could mean that the digitalis receptor does not permit the presence of a bulky substituent in the 14β region, even of relatively small volume like the methyl group.In fact the reduced binding affinities of the 14βmethoxy derivatives do not seem to depend on the impossibility of being hydrogen donors since the two epoxy derivatives reported in Table 1 show high binding affinity although lower than that of the 14β-hydroxy analogues.

General
Melting points were measured on a capillary melting point apparatus and are uncorrected.Elemental analyses were performed by Redox, Cologno Monzese, Italy. 1 H-NMR spectra were recorded on a Bruker AC-300 spectrometer at 300.13 MHz.Chemical shifts (δ) are given in ppm downfield from tetramethylsilane as internal standard.Chromatography was carried out on silica gel (Baker 7024-02).Solvents and reagents (Aldrich) were used as purchased.
The residue obtained above was dissolved in 1.6 L of CH 2 Cl 2 and 320 mL of water and to the well stirred biphasic mixture NaBH 4 (6.0 g, 158.72 mmol) was added in two portions, the second after 4 hrs.After another 18 hrs the reaction mixture was added to a 5% aq.solution of citric acid (1 L) and the organic layer extracted, separated, washed with a 5% aq.solution of NaHCO 3 and a saturated aq.solution of NaCl.The organic layer was dried over Na 2 SO 4 , evaporated to dryness under reduced pressure and purified by flash-chromatography (CH 2 Cl 2 ) to give the protected derivatives 6a (3.3 g, 49% from 2) and 7a (0.88 g, 13% from 2).
The silyloxy derivative 6a (2.4 g, 4.78 mmol) was dissolved in 100 mL of CHCl 3 /MeOH 1:1, few drops of conc.HCl were added and the reaction mixture was stirred at room temperature for 24 h.The acidic solution was neutralized with solid NaHCO 3 and evaporated at reduced pressure; the residue was extracted with CH 2 Cl 2 and water; the organic layer was dried over Na 2 SO 4 , evaporated to dryness under reduced pressure and crystallized from Et 2 O to give 6b (1.5 g, 81%) as a white solid: m.p.: 179-182 °C; The silyloxy derivative 7a (0.54 g, 1.07 mmol) were dissolved in 25 mL of CHCl 3 /MeOH 1:1, few drops of conc.HCl were added and the reaction mixture was stirred at room temperature for 24 h.The acidic solution was neutralized with solid NaHCO 3 and evaporated at reduced pressure; the residue was extracted with CH 2 Cl 2 and water; the organic layer was dried over Na 2 SO 4 , evaporated to dryness under reduced pressure and purified by flashchromatography (CH 2 Cl 2 /AcOEt 90:10 v/v) to give 7b (0.24 g, 58%) as a white solid: m.p.: 163-166 °C;
A solution of 8a (0.24 g, 0.48 mmol) in 5 mL of a 1.1 M solution of n-Bu 4 NF (5.5 mmol) in THF was heated at 70 °C under nitrogen for 1 hr and then poured into a saturated aq.solution of NaCl and extracted with EtOAc.The organic layer was dried over Na 2 SO 4, evaporated to dryness under reduced pressure and purified by flashchromatography (AcOEt) to give 8b (0.16 g, 81%) as a white foam.An analytical sample was obtained as the hzdrogen fumarate, a white solid, m.p. 157-159 °C (dec);