CARDIAC STEROID DERIVATIVES

Abstract

The technology disclosed herein concerns de-hydroxylated cardiac steroid of formula (I) and uses thereof in medicine.

##STR00001##

Claims

1-54. (canceled)

55. A compound of general formula (I): ##STR00015## wherein R.sub.1 is a lactone, each of R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7, independently of the other, is selected from H, OR, C.sub.1-C.sub.5alkyl, C.sub.1-C.sub.5alkylene-hydroxyl and C(O)R, each of R and R, independently of the other, is selected from H, C.sub.1-C.sub.5alkyl, C.sub.1-C.sub.5alkylene-hydroxyl and C(O)C.sub.1-C.sub.5alkyl; and a bond designated is a double bond or a single bond, such that one of the bonds designated is a double bond and the other is a single bond.

56. The compound according to claim 55, wherein the lactone is lactone (i) or lactone (ii): ##STR00016## wherein designates a bond of connectivity.

57. The compound according to claim 56, being a compound of the general formulae (Ia) or (Ib) ##STR00017## wherein each of each of R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R, R and is as defined in claim 1.

58. The compound according to claim 57, wherein in each of compound of general formula (Ia) and (Ib), R.sub.2 is selected from H and OR, wherein R is as defined in claim 55.

59. The compound according to claim 60, wherein R is H or is C(O) C.sub.1-C.sub.5alkyl.

60. The compound according to claim 59, wherein the C(O)C.sub.1-C.sub.5alkyl is C(O)CH.sub.3.

61. The compound according to claim 57, wherein in each compound of general formula (Ia) and (Ib), R.sub.3 is selected from H and OH.

62. The compound according to claim 57, wherein in each compound of general formula (Ia) and (Ib), R.sub.4 is selected from H and OH.

63. The compound according to claim 57, wherein R.sub.5 is C.sub.1-C.sub.5alkyl, being optionally a methyl group.

64. The compound according to claim 57, wherein in each compound of general formula (Ia) and (Ib), R.sub.6 is selected from H and OH.

65. The compound according to claim 57, wherein in each compound of general formula (Ia) and (Ib), R.sub.7 is selected from H and OR, wherein R is selected from H and C.sub.1-C.sub.5alkyl.

66. The compound according to claim 57, wherein in each compound of general formula (Ia) and (Ib), the bond between C3 and C2 is a double bond.

67. The compound according to claim 57, wherein in each compound of general formula (Ia) and (Ib), the bond between C3 and C4 is a double bond.

68. A compound having structure (IIa) or (IIb): ##STR00018##

69. A pharmaceutical composition comprising a compound according to claim 55.

70. A method of treatment or prophylaxis of a disease or disorder associated with Na.sup.+/K.sup.+ ATPase activity, the method comprising administering to a subject (human or non-human) in need thereof an effective amount of a compound according to claim 55.

71. The method according to claim 70, wherein the disease or disorder is selected from heart failure, atrial fibrillation, fetal tachycardia, supraventricular tachycardia, cor pulmonale, pulmonary hypertension, cancers, neurological diseases and psychiatric diseases.

72. A method of treatment or prophylaxis of cancer, the method comprising administering to a subject (human or non-human) in need thereof an effective amount of a compound according to claim 55.

73. A method of treatment or prophylaxis of a disease or disorder associated with a viral infection, the method comprising administering to a subject (human or non-human) in need thereof an effective amount of a compound according to claim 55.

74. A de-hydroxy cardiac steroid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0139] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0140] FIG. 1 shows the X-Ray crystallography of bufalin dehydroxy-3,4-ene (2).

[0141] FIG. 2 depicts effect of Compound 1 on Brain Microsomal Na.sup.+, K.sup.+-ATPase activity. Na.sup.+, K.sup.+-ATPase activity in rat brain microsomal fraction was determined as previously described (Lichtstein, D et al. Hypertension 7:729-733, 1985) by the colorimetric determination of inorganic phosphate after the incubation of microsomes (30 g protein/reaction) at 37 C. in a solution (final volume 500 l) containing (final concentrations): Tris-HCl (50 mM, pH 7-4), NaCl (100 mM), KCl (10 mM), MgCl.sub.2 (4 mM) and ATP (2 mM, Tris, vanadium free). After 10-min pre-incubation, the ATP was added to initiate the reaction. Reactions were terminated by the addition of 100 l 5% trichloroacetic acid and the precipitate was removed by centrifugation.

[0142] FIG. 3 depicts the effect of Compound 2 on brain Microsomal Na.sup.+, K.sup.+-ATPase activity.

[0143] FIG. 4 depicts the effect of Acetylcholine and Norepinephrine on Cardiomyocytes Contractility.

[0144] FIG. 5 depicts the effects of Digoxin and New Synthetic-Steroids on Cardiomyocytes Contractility (10 minutes).

[0145] FIG. 6 depicts the effects of Digoxin and New Synthetic-Steroids on Cardiomyocytes Contractility (30 minutes).

[0146] FIG. 7. depicts the effects of compound 1 on SARS-Cov-2 viral infection and toxicity in vero cells.

[0147] FIG. 8 depicts the effects of compound 2 on SARS-Cov-2 viral infection and toxicity in vero cells.

[0148] FIG. 9 depicts the effects of bufalin on SARS-Cov-2 viral infection and toxicity in vero cells.

[0149] FIG. 10 depicts the effects of digoxin on SARS-Cov-2 viral infection and toxicity in vero cells.

[0150] FIG. 11 depicts the effects of ouabain on SARS-Cov-2 viral infection and toxicity in vero cells.

[0151] FIG. 12 depicts the effects of remdesivir on SARS-Cov-2 viral infection and toxicity in vero cells.

[0152] FIG. 13 depicts the effects of bufalin on growth of different cancer cell.

[0153] FIG. 14 depicts the effects of compound 1 on growth of different cancer cells.

[0154] FIG. 15 depicts the effects of compound 2 on growth of different cancer cells.

DETAILED DESCRIPTION OF EMBODIMENTS

[0155] Bufalin, the major component of the traditional Chinese medicine Chan-Su, is an extract of the skin and parotid venom glands of a toad of the Bufo family. Chan-Su has been widely used in China and other Asian countries to treat cancer and additional ailments. Bufalin belongs to the cardiac steroid family and, like other members such as ouabain and digoxin, increases the force of contraction of heart muscle, thus improving circulation in cases of insufficient cardiac output. However, the toxicity and the small therapeutic window of this family of steroids limits their use as cardiotonic drugs. A similar problem is encountered in the use of these steroids for the treatment of cancer: Although bufalin has been shown to kill various tumor cells in vitro, it produced unsatisfactory results when administered in vivo. Because of its fast metabolism, toxicity, insolubility in water and short half-life, its application in the clinical setting is limited. In addition, bufalin and other cardiac steroids have been shown to have potent anti-inflammatory and anti-viral activities. However, all these beneficial properties are obtained at concentrations higher than the toxic effects of these compounds. Therefore, the development of bufalin derivatives with lower toxicity is of great importance.

[0156] The plasma membrane Na.sup.+ and K.sup.+ transporter Nat, K.sup.+-ATPase is an established receptor for cardiac steroids. The interaction of these steroids with Na.sup.+, K.sup.+-ATPase results in inhibition of the ion-pumping function and, in addition, causes the activation of several signal transduction cascades, including mitogen-activated protein kinase: extracellular signal-regulated kinase: proto-oncogene tyrosine-protein kinase (Src): PI3K/Akt, Ca.sup.++ signaling, and reactive oxygen species generation pathways. It is well established that the toxicity of cardiac steroids in the heart is due to calcium overload, produced by excessive inhibition of the Nat, K.sup.+-ATPase in the myocytes, leading to arrhythmia and lethality. Conversely, the positive inotropic effect, as well as the anti-cancer and anti-viral effects, are largely a result of the CS-induced signaling activation. Indeed, the inhibition of ERK activation totally prevented the bufalin and other CS-induced increase in heart contractility: the bufalin anti-cancer effect was shown repeatedly to be mediated by ERK and AKT signaling, as was the anti-viral activity of cardiac steroids. It is reasonable to suggest, therefore, that differences in cardiac steroids-induced signaling by various CS will have a profound effect on their pharmacological profiles.

[0157] Cardiac steroids are composed of three major structural components: a steroid core, in which rings AB and CD are cis-fused, whereas rings BC are trans-fused: a 5- and 6-membered lactone ring at position 17 (cardenolides and bufadienolides, respectively); and a variable number of sugar residues at position 3. The significance of the moiety orientation at position 3 of the steroid for its biological activity was studied extensively, especially in relation to the nature of the sugar bound at this position. Furthermore, the inventors' previous study showed that the a/B orientation of the 3-OH group may have a substantial effect on biological activity. Whereas the 3-OH isomer displayed the standard capability of increasing heart contractility, an a 3-OH isomer did not boost the force of contraction, but actually inhibited the contractility induced by digoxin.

[0158] In the present application, two isomers lacking an OH at the 3 position, bufalin-2,3-ene and bufalin-3,4-ene, were synthesized and studied as examples of de-hydroxylated cardiac steroids. The biological activities of these compounds were evaluated by testing their ability to inhibit Nat, K.sup.+-ATPase activity in a pig brain microsomal fraction, to induce ERK and AKT phosphorylation in human neuroblastoma LAN-5 cells, to cause cytotoxicity in human cancer cells and to prompt positive inotropy in quail cardiac cells in culture.

Materials and Methods

Materials

[0159] Solvents were purchased from Romical (Jerusalem, Israel), bufalin and Ishikawa's reagent were purchased from Chengdu Biopurify Phytochemicals Ltd. Wenjiang, Chengdu, China and Sigma Aldrich Co. (St. Louis MO, USA), respectively. TLC Silica Gel 60 F254 Aluminum Sheets were purchased from Merck, (Darmstadt, Germany) and Sep-pak, C18 columns from Waters (Milford, MA, USA). A549, alveolar basal epithelia cell carcinoma, HCT 116, colonorectal carcinoma, and HFF-1, human fibroblasts were obtained from ATCC, (Manassas, VA. USA). HaCaT, immortalized keratinocytes were obtained from AddexoBio (San Diego, CA, USA) and U251 glioblastoma cells were from ECACC General Cell Collection (Salisbury, United Kingdom). Serum, DMEM cell culture medium, antibiotics and a chemiluminescence kit were acquired from Biological Industries (Beit Ha emek, Israel). ATP and protease inhibitor cocktail were purchased from Sigma-Aldrich, (St. Louis, MO, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, West Grove, PA, USA. Bradford reagent and Laemmli sample buffer were provided by Bio-Rad Laboratories, Hercules, CA, USA. Polyvinylidene fluoride membranes were purchased from Pall Corporation, Pensacola, FL, USA and a Pierce primary cardiomyocyte isolation kit was obtained from Thermo Scientific, Rockford, Il, USA.

High Performance Liquid Chromatography

[0160] HPLC was performed with a Hewlett-Packard (HP) 1050 Series chromatograph with a HP 1010 detection system and an Agilent computer system. A 50 l volume of each sample was passed through a Luna C-18, 5 m column (2504.6 mm) Phenomenex, Torrance, CA, USA), provided with a pre-column. Elution was performed with a 35 min, 68% CH.sub.3CN/water isocratic system with a flow rate of 1.0 ml/min.

NMR and Mass Spectroscopy

[0161] The 1H NMR (500 MHZ) measurements were performed with a Bruker AVANCE III HD 500 Mhz spectrometer in CDCl3. Electron spray mass spectra were obtained with a Quadrupole LCMS mass spectrometer system (Thermo Scientific, USA).

Crystallographic Structure Analysis

[0162] Crystallography was performed on an ENRAF-NONIUS CAD-4 computer-controlled diffractometer, and all crystallographic computing was made with a VAX9000 computer at the Hebrew University of Jerusalem.

ATPase Activity

[0163] A pig brain microsomal fraction was prepared as previously described. Na.sup.+, K.sup.+-ATPase activity in the microsomal fraction was determined by the amount of inorganic phosphate released during incubation at 37 C. In brief: 480 l of the microsomal preparation (60 g protein) was added to 3520 l reaction buffer (50 mM Tris-Base, 120 mM NaCl, 10 mM KCl, 4 mM MgCl.sub.2, pH 7.4) in the presence of varying concentrations of an inhibitor (bufalin or bufalin derivatives). Following 20 min. incubation, 10 l of ATP (2.5 mM final concentration) was added and the incubation was allowed to proceed for an additional 30 min. The reaction was terminated by the addition of 1 ml 16% trichloroacetic acid and placing the tubes on ice for 10 min. Following centrifugation (500g, 10 min, 4 C.), 50 l of the supernatant was removed for determination of inorganic phosphate according to a colorimetric method, as described previously.

ERK and AKT Phosphorylation in LAN-5 Cells:

[0164] Human neuroblatoma LAN-5 cells were grown in RPMI 1640 supplemented with penicillin-streptomycin (PS) (100)-1%, l-glutamine (100) 200 mM-1%, and FBS-10%-50 ml, in an incubator maintained at 37 C., with 5% CO.sub.2. Before starting the experiment, the cells were transferred to 6 well plates at a density of 100,000 cells/well and grown for 24 hrs in serum-free medium. The steroids (10 nM final concentration) were then added and the wells were incubated for 10 min. The proteins were then extracted by adding RIPA lysis buffer and protease inhibitor (1:100) and centrifuged (14,000g). The protein content of the supernatants was determined according to Bradford. Then the supernatants were diluted in Laemmli sample buffer and incubated at 95 C. for 5 min. A total 30 g of each sample was loaded onto a 12% sodium dodecyl sulfate polyacrylamide gel and electrophoresed. The proteins were transferred to polyvinylidene fluoride membranes which were blocked with Tris-buffered saline containing 0.1% (v/v) Tween and 5% (w/v) skim milk and incubated overnight at 4 C. with PathScan Multiplex Western Cocktail I containing phospho-p44/42 (ERK1/2 Tyr204) (D13.14.4E) XP rabbit mAb for ERK, and Phospho-Akt (Ser473) (D9E) XPR rabbit mAb for AKT. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Antibodies were detected with an enhanced chemiluminescence kit, according to the manufacturer's instructions. Signals were visualized on film (Kodak: BioMax, Wellsville, NY, USA) and quantified densitometrically (Fluro-s Multilmager: Bio-Rad, Hercules, CA, USA).

Cytotoxicity Against Cancer and Normal Cells

[0165] The anti-proliferative activity of the target compounds was tested against the NCI-60 cell line panel. The screening was performed at the National Cancer Institute (NCI), Bethesda, Maryland, USA (www.dtp.nci.nih.gov), according to their standard protocol (https://dtp.cancer.gov/discovery_development/nci-60/methodology.htm).

[0166] All cell lines were grown at at 37 C. in 5% CO.sub.2. A549 cell line were grown in F-12 (Ham's) media with L-Glutamine supplemented with 10% FBS, 1% sodium pyruvate and 1% Penicillin-Streptomycin. HCT 116 cell line were grown in McCoy's 5A Medium with L-Glutamine, supplemented with 10% FBS, 1% sodium pyruvate and 1% Penicillin-Streptomycin. U-251 cell line were grown in MEM-NEAA, Earle's Salts Base, with Non-Essential Amino Acids, supplemented with 10% FBS, 1% sodium pyruvate, 1% L-Alanyl-L-Glutamine, 0.01 mg/ml human recombinant insulin and 1% Penicillin-Streptomycin. HaCaT and HFF-1 cell lines were grown in DMEM with L-Glutamine, supplemented with 10% FBS, 1% sodium pyruvate and 1% Penicillin-Streptomycin.

[0167] One day before the experiments cells from all cell lines were seeded into 96-well plates at a density of 110.sup.4 cells/well in 100 l of appropriate growth media. Plates were incubated overnight in 37 C. to allow attachment. Growth media (100 l) containing the tested steroids were added to obtain the desired concentration and the cells were incubated for 48 hours in 37 C. Cells incubated in growth media containing 0.33% DMSO (vehicle) served as control. At the end of incubation period, cell viability evaluation was performed using VisionBlue Quick Cell Viability Fluorometric Assay Kit, according to manufacturer's instructions. Fluorescent signal by was detected by TECAN spark 10M microplate reader, Excitation/Emission 53525/59020 nm. The fluorescence data is expressed as percentage of cell viability (%) compared to vehicle control.

Quail Cardiac Muscle Cell Contractility

[0168] Quantification of cardiomyocytes contractility was performed as previously described. Since avian embryos (quail included) are not considered animals, their use is exempt at the Hebrew University, like in all other academic institutions, of the need for ethical approval. Quail (Coturnix japonica) cardiomyocytes were prepared from E4 embryos with a Pierce Primary Cardiomyocyte Isolation Kit (Thermo Scientific). Contractility at 37 C. was measured 30 min after drug addition. Cells were photographed for 15 sec, with an Olympus CKX41 (Japan) upright microscope (20 magnification), and integrated incandescent illumination. A FastCam imi-tech (Korea) high speed digital camera with a 640480 pixel gray scale image sensor was mounted on the microscope with ImCam software (IMI Tecnology, Co. Ltd Gangnam-gu, Seoul, South Korea). Changes in cell contraction were deduced from the mean difference in area change between relaxation and the contraction peaks. Three cell clusters in 3 wells were photographed and measured under each experimental condition.

Statistical Analysis

[0169] All the data are expressed as the meanstandard error (SEM). Significance was determined according to the independent Student's t-test: p<0.05 was considered significant.

The Synthesis of Novel Dehydroxy Bufalin

[0170] Ishikawa's reagent ((CH.sub.3CH.sub.2).sub.2NCF.sub.2CHFCF.sub.3) was used to produce the new compounds.

Experimental:

Synthesis

[0171] In a 2 ml glass vial provided with a magnetic stirrer a suspension of bufalin (15 mg) in diethylether (1.0 ml was prepared. The vial was cooled at 10 C, wrapped in Aluminium foil and Ishikava's reagent (35-40 mg, 50-60 l) was slowly added. The mixture was stirred overnight at room temperature. Next day, the product was monitored by TLC and HPLC for the new peaks that were formed (RT 26-28 min). In order to separate the product from the excess of the reagent, the solution was loaded on a silica gel TLC plate and eluted with 70% diethyl ether and hexane for 4 min. Following visualization with a UV lamp at 260 nm, the silica gel plate at 2 cm was scraped into a small vial and the organic material dissolved in methanol (0.3 ml). From the filtrated solution, 50 l was injected in to the HPLC system. Two products (at RT 27 and 28.5) were detected. The first product bufalin 2,3-ene is herein designated Compound 1 and the second product bufalin 3,4-ene is herein designated Compound 2.

[0172] HPLC was performed with a Hewlett-Packard (HP) 1050 Series chromatograph provided with a HP 1010 detection system and an Agilent ChemStation (Waldbron, Germany). The detector was set to 220 and 260 nm and the sample was injected to a Luna C-18, 5 m column (2504.6 mm) Phenomenex, Torrance, CA) provided with a pre-column. The elution was performed with a 35 min CH.sub.3CN/water system with a flow rate of 1.0 ml/min. 50 l of sample was injected into HPLC, the elution was carried out with 68% CH.sub.3CN in water.

[0173] The Electron spray mass spectrum was obtained using a Quadrupole LCMS mass spectrometer system (Thermo scientific, USA).

[0174] The two compounds were collected, dried, and subjected to mass spectra, NMR and X Ray crystallographic measurements. The first product Compound 1, appears an amorphous solid powder and the second solid Compound 2, formed large crystals.

[0175] Both compounds showed identical mass spectrum with a protonated molecular mass at 369 that can correspond to isomers of dehydroxy bufalin formed by elimination of one of the OH groups from either C3 or C14 position with a hydrogen atom from a nearby position (bufalin H.sub.2O).

[0176] Compound 2 was recrystallized from acetonitrile/water 70% and subjected to X ray crystallography measurements that produced the unexpected structure, bufalin dehydroxy-3,4-ene (FIG. 1). In other words, suggesting an unusual catalytic effect induced by the Ishikawa's reagent to produce the elimination of the 30H group leading selectively to a double bounds in ring A, the dehydroxy bufalin isomers are shown below:

##STR00014##

[0177] Examination of the proton NMR of compound 1 showed in addition to the vinyl hydrogens of the diolone ring, two symmetrical vinyl hydrogens at 5.3 and 5.5 ppm, corresponding to a bufalin dehydroxy-2,3-ene isomer. Further, examination of NMR spectrum of the 1:2 mixture of Compound 1 and Compound 2 isomers shows clearly the doublets of the vinyl hydrogens at 5.4 and 5.7 ppm, corresponding the 3,4-ene double bound as an additional prove of the structure of Compound 2.

Biological Effects

Na.sup.+, K.sup.+-ATPase Activity Inhibition

[0178] The only established receptor for cardiac steroids including bufalin is the plasma membrane enzyme, the Nat, K.sup.+-ATPase. The inhibition of Na.sup.+, K.sup.+-ATPase activity by bufalin is the underlying mechanism for the increase in force of contraction of heart muscle as well as other biological effects of the steroid (for review see Clausen M V. Et a1. Front Physiol. 2017: Schoner W and Scheiner-Bobis G. Am J Physiol. Cell Physiol. 293: C509-536, 2007). The a2 and a3 isoforms of the Nat, K.sup.+-ATPase are more sensitive to the cardiac steroids and are those that participate in many of their pharmacological effects. Hence, the ability of novel bufalin derivatives to inhibit Na.sup.+, K.sup.+-ATPase activity is a measure for their potency to increase cardiac contractility and affect other biological processes (Blaustein M P. Am J Physiol. 309: H958-968, 2015; Yuen G K et al. J Mol. Cell Cardiology 108:158-169, 2017).

[0179] The inhibition of brain microsomal Na.sup.+, K.sup.+-ATPase activity by Compounds 1 and 2 in comparison to that of Bufalin are depicted in FIGS. 2 and 3, respectively. It can be seen that while Compound 1 has approximately the same potency as Bufalin, Compound 2 is a more potent inhibitor. Importantly, Compound 2 inhibit Na.sup.+, K.sup.+-ATPase activity better than Bufalin even at the very low (nanomolar) concentrations, relevant to inhibitions of the a2 and a3 isoforms of the Na.sup.+, K.sup.+-ATPase.

[0180] Na.sup.+, K.sup.+-ATPase activity in rat brain microsomal fraction was determined as previously described (Lichtstein, D et al. Hypertension 7:729-733, 1985) by the colorimetric determination of inorganic phosphate after the incubation of microsomes (30 g protein/reaction) at 37 C. in a solution (final volume 500 l) containing (final concentrations): Tris-HCl (50 mM, pH 7-4), NaCl (100 mM), KCl (10 mM), MgCl.sub.2 (4 mM) and ATP (2 mM, Tris, vanadium free). After 10-min pre-incubation, the ATP was added to initiate the reaction. Reactions were terminated by the addition of 100 l 5% trichloroacetic acid and the precipitate was removed by centrifugation.

Increase of Heart Cells Contractility

[0181] To test for the potential effect of the synthetic compounds on heart muscle contractility, this was examined in quail cardiomyocytes. Basic characterization of this system was performed by testing the effects of the classical neurotransmitters acetylcholine and nor-adrenalin cardiomyocyte's contractility. As can be seen in FIG. 4, as expected, acetylcholine and noradrenaline, dose-dependently, decreased and increased, respectively, cardiomyocytes contractility.

[0182] Quails cardiomyocytes were prepared from quail embryos at E4. Contractility at 37 C. was measured 10 minutes after drug addition. Cells were photographed for 15 seconds, and the change in cell contraction was deduced from the mean difference in area change between relaxation and the contraction peaks. 3 cell clusters in 3 wells were photographed and measured for each experimental condition. Quantification of motility is displayed as the cell Area Change.

[0183] The effects of the cardiac steroid digoxin and the two new synthetic compounds on quail cardiomyocytes contractility is depicted in FIG. 5. Most notably, compound 1 and compound 2 increase dramatically cardiomyocytes contractility compared to the effect of the known steroid. At all concentrations tested, compound 1 and most so, compound 2 had a stronger effect on contractility than digoxin. This was seen when contractility was measured 10 minutes (FIG. 5) and 30 minutes (FIG. 6) after the steroids addition.

[0184] Quails cardiomyocytes were prepared from quail embryos at E4. Contractility at 37 C. was measured 10 minutes after drug addition. Cells were photographed for 15 seconds, and the change in cell contraction was deduced from the mean difference in area change between relaxation and the contraction peaks. 3 cell clusters in 3 wells were photographed and measured for each experimental condition. Quantification of motility is displayed as the cell Area Change. New synthetic steroids are: Bufalin deoxy-2,3-ene (Compound 1) and Bufalin deoxy-3,4-ene (Compound 2).

Cytotoxic Effect of the Synthetic Compounds in Comparison to that of Other Cardiac Steroids.

[0185] The effect of compound 1 and compound 2 on cell viability was determined using neuroblastoma SH-SY5Y cells. As seen in Table 1 below, ouabain, the most studied cardiac steroid, and digoxin, the cardiac steroid that used most frequently used in the clinic caused a complete cell death already at 5-25 M. Digoxin, Importantly, both compounds 1 and 2 exhibit a lower cytotoxic potency at all concentrations tested, in comparison to the other steroids.

TABLE-US-00001 TABLE 1 Effect of Cardiac Steroids and New Synthetic Compounds on Neuroblastoma (SH-SY5Y) cells Viability. Compound Concentration (M) Cell Viability (%) Control 100 Ouabain 5 6.26 25 1.92 50 0.51 100 4.09 Digoxin 5 23.37 25 1.02 50 0.26 100 2.55 Compound 1 5 32.21 25 13.79 50 5.62 100 1.28 Compound 2 5 53.83 25 24.71 50 11.49 100 6.13

[0186] Neuroblastoma SH-SY5Y cells (ATCC, Manassas, VA, USA) were grown in 96 wells plate in DMEM and Ham's F12 growth media supplemented with 10% fetal calf serum containing 100 g/ml streptomycin and 100 U/ml penicillin at 37 C. and 5% CO2. Cardiac steroids were added in a media without 10% fetal calf serum FCS and cell viability was measured after 16 hr. using the conventional MTT assay. MTT was dissolved in above mentioned media at a concentration of 5 mg/ml and 50 L of the solution was added to each well and plates were incubated at 37 C. for 2 h. After incubation media was dispensed and 200 L of DMSO was added to each well to dissolve the MTT formazan, with 30 min incubation at room temperature. Absorbance was measured with an ELISA-plate reader (Bio-Tek Instruments) at 570 nm to quantify the amount of formazan product, which reflects the number of viable cells in culture and the percent viability was calculated with respect to control untreated cells.

Anti-Viral Activity of Compounds of the Invention

[0187] Recently, Na.sup.+, K.sup.+-ATPase a1 subunit mediated Src signaling was reported to be involved in viruse entry into cells (Burkard, C., Verheije, M. H., Haagmans, B. L., van Kuppeveld, F. J., Rottier, P. J., Bosch, B. J., de Haan, C. A., 2015. ATPIA1-mediated Src signaling inhibits coronavirus entry into host cells. J. Virol. 89, 4434-4448). This study showed that ouabain and bufalin at nM concentrations inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV.

[0188] In a recent study ouabain was found to diminish anti-transmissible gastroenteritis coronavirus (TGEV) activity in swine testicular (ST) cells titers and inhibit the TGEV-induced production of IL-6 in a dose dependent manner (Yang, C.W., Chang, H. Y., Hsu, H. Y., Lee, Y. Z., Chang, H. S., Chen, I. S., Lee, S. J., 2017a. identification of anti-viral activity of the cardenolides, Na+/K+-ATPase inhibitors, against porcine transmissible gastroenteritis virus. Toxicol. Appl. Pharmacol. 332, 129-137).

[0189] The effect of cardiac steroids on viral infection was also demonstrated for other viruses. Ouabain and other related steroids inhibited Human respiratory syncytial virus (RSV) entry into human small airway epithelial cells (Lingemann M. The alpha-1 subunit of the Na.sup.+, K.sup.+-ATPase (ATP1A1) is required for macropinocytic entry of respiratory syncytial virus (RSV) in human respiratory epithelial cells. PLOS Pathog 15 (8): e1007963, 2019). Treatment of human cells with digoxin or ouabain, resulted in a dose-dependent decrease in infection by Chikungunya virus (CHIKV) (Ashbrook A W et al. Antagonism of the Sodium-Potassium ATPase Impairs Chikungunya Virus Infection. mBio. 2016 7 (3)). The effect was cell type-specific, as the steroid treatment of either murine or mosquito cells did not diminish CHIKV infection. Screening for anti-Japanese Encephalitis Virus infection identified ouabain and digoxin having robust efficiency against the virus (Guo J. et al. Screening of natural extracts for inhibitors against Japanese Encephalitis Virus infection. Antimicrob Agents Chemother 64: e02373-19). Ouabain was shown to impair herpes simplex virus infection. In contrast to the effects described above, in this study, the steroid did not inhibit viral attachment or entry, but did affect replication, reducing the expression of viral immediate-early and early genes by at least 5-fold (Dodson A W. Et al. Inhibitors of the sodium potassium ATPase that impair herpes simplex virus replication identified via a chemical screening approach. Virology 366 (2007) 340-348). Recently it was demonstrated that ouabain decrease influenza virus replication by inhibiting cell protein translational machinery (Amarella L. et al. Cardiac glycosides decrease influenza virus replication by inhibiting cell protein translational machinery. Am J Physiol Lung Cell Mol Physiol. 2019 Jun. 1; 316 (6): L1094-L1106).

Experimental

[0190] Vero E6 cells were pre-treated for 2 h with increasing concentrations of the indicated compound and then infected with SARS-COV-2. Forty eight hours after infection, cells were fixed and analyzed by immunofluorescence imaging as described previously (Riva, L. et al. Discovery of SARS-COV-2 antiviral drugs through large-scale compound repurposing. Nature volume 586, pages113-119, 2020). SARS-COV-2 Nucleocapsid protein (NP) was used to quantify number of infected (NP-positive) cells and DAPI to quantify total number of cells. For each condition, the percentage of infection was calculated as the ratio of the number of infected cells stained for coronavirus NP to number of cells stained with DAPI. Cell viability was measured with MTT assay and viral infection was measured when cells were pre-incubated with the compounds for 2 hours prior to infection and infection was over 48 hours.

Anti-Cancer Activity of Compounds of the Invention

[0191] The effects of compound 1, compound 2 and bufalin on the growth of different cancer cells are depicted in FIGS. 13-15. One dose effect of Compounds 1 and 2 of the present invention and bufalin on cancer cell viability, as determined at the NCI, NIH, is shown in FIGS. 13-15 provide evidence for the anti-cancer effects of the synthetic compound. The most sensitive line to the compounds 1 and 2 seem to be SNB-19 and the least sensitive was K562 (see materials and methods for data interpretation).

Experimental:

[0192] In the anti-cancer studies were performed at the NCI, NIH in the one-dose testing of the NCI-60 project (https://dtp.cancer.gov/discovery_development/nci-60/methodology.htm). The numbers reported are growth relative to the no-drug control, and relative to the time zero number of cells. This allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). For example, a value of 100 means no growth inhibition. A value of 40 would mean 60% growth inhibition. A value of 0 means no net growth over the course of the experiment. A value of 40 would mean 40% lethality. A value of 100 means all cells are dead. Hence, the most sensitive line to the two compounds seem to be SNB-19 and the least sensitive was K562.