WO2009090063A1 - Steroid sapogenin, androstane and triterpenoid sapogenin derivatives for the treatment and prevention of infectious diseases - Google Patents

Abstract

The present invention relates to steroid sapogenin, androstane and triterpenoid sapogenin derived amines or ammonium derivatives in the medical intervention of infectious diseases, in particular diseases caused by a virus or a bacterium, including drug-resistant strains.

Description

Steroid Sapogenin, Androstane and Triterpenoid Sapogenin Derivatives for the Treatment and Prevention of Infectious Diseases

The present invention relates to steroid sapogenin, androstane and triterpenoid sapogenin derived amines or ammonium derivatives in the medical intervention of infectious diseases, in particular diseases caused by a virus or a bacterium, including drug-resistant strains.

The lipid bilayer that forms cell membranes is a two-dimensional liquid the organization of which has been the object of intensive investigations for decades by biochemists and biophysicists. Although the bulk of the bilayer has been considered to be a homogeneous fluid, there have been repeated attempts to introduce lateral heterogeneities, lipid microdomains, into our model for the structure and dynamics of the bilayer liquid (Glaser, Curr. Opin. Struct. Biol. 3 (1993), 475-481 ; Jacobson, Comments MoI. Cell Biophys. 8 (1992), 1-144; Jain, Adv. Lipid Res. 15 (1977), 1-60;

Winchil, Curr. Opin. Struct. Biol. 3 (1993), 482-488).

The realization that epithelial cells polarize their cell surfaces into apical and basolateral domains with different protein and lipid compositions in each of these domains, initiated a new development that led to the "lipid raft" concept (Simons, Biochemistry 27 (1988), 6197-6202; Simons, Nature 387 (1997), 569-572). The concept of assemblies of sphingolipids and cholesterol functioning as platforms for membrane proteins was promoted by the observation that these assemblies survived detergent extraction, and are referred to as detergent resistant membranes, DRM (Brown, Cell 68 (1992), 533- 544). This was an operational break-through where raft-association was equated with resistance to Triton-X100 extraction at 4°C. The addition of a second criterion, depletion of cholesterol using methyl-β-cyclodextrin (llangumaran, Biochem. J. 335 (1998), 433- 440; Scheiffele, EMBO J. 16 (1997), 5501-5508), leading to loss of detergent resistance, prompted several groups in the field to explore the role of lipid microdomains in a wide spectrum of biological reactions. There is now increasing support for a role of lipid assemblies in regulating numerous cellular processes including cell polarity, protein trafficking and processing, as well as signal transduction.

Hence, lipid rafts are lipid platforms of a special chemical composition (rich in sphingomyelin and cholesterol in the outer leaflet of the cell membrane) that function to segregate membrane components within the cell membrane. Rafts are understood to be relatively small (30 to 50 nm in diameter, estimates of size varying considerably depending on the probes used and cell types analyzed) but they can coalesce under certain conditions. Their specificity with regard to lipid composition is reminiscent of phase separation behavior in heterogeneous model membrane systems.

Several groups of pathogens, bacteria, prions, viruses, and parasites hijack lipid rafts for their purposes (Simons, J. Clin. Invest. 110 (2002), 597-603; Van der Goot, Semin. Immunol. 13 (2001), 89-97) to infect host cells. For example viral infections caused by influenza virus, HIV-1 , measles virus, respiratory syncytial virus, filoviridae such as Ebola virus and Marburg virus, papillomaviridae and polyomaviridae, Epstein-Barr virus, Hepatitis C virus, and Echovirus 1 represent diseases for which rafts and/or raft proteins are targets. Moreover, in bacterial infections caused by Escherichia coli, Mycobacterium tuberculosis and bovis, Campylobacter jejuni, Vibrio cholerae, Clostridium difficile, Clostridium tetani, Streptococci species, Salmonella, Shigella, Chlamydia and uropathogenic bacteria, the organism is taken up into the cell in a raft- dependent internalization process.

The first example to be characterized was influenza virus (Scheiffele, J. Biol. Chem. 274 (1999), 2038-2044; Scheiffele, EMBO J. 16 (1997), 5501-5508). The virus contains two integral glycoproteins, hemagglutinin and neuraminidase, both of which are raft- associated as judged by cholesterol-dependent detergent resistance (Zhang, J. Virol. 74 (2000), 4634-4644). Cholesterol and the integrity of rafts are essential to the transport of hemagglutinin to the plasma membrane (Keller, J. Cell Biol. 140 (1998), 1357-1367). Influenza virus buds out from the apical membrane of epithelial cells, which is enriched in lipid rafts. The influenza virus envelope is formed from coalesced rafts during budding, a process in which assemblies of M proteins form a layer at the cytosolic leaflet of the nascent viral envelope which drives raft clustering (Zhang, J. Virol. 74 (2000), 4634-4644). According to a recent model, the viral M2 protein, a peripheral raft protein, promotes the pinching-off of mature influenza virus particles (Schroeder, Eur. Biophys. J. 34 (2005), 52-66).

HIV-1 , which likewise incorporates host raft lipids and proteins into its envelope, employs rafts for at least four key events in its life cycle: passage across a new host's mucosa, viral entry into immune cells, signaling of changes in host cell functions as well as viral exit from cells, and dispersion through the host's vascular system.

Viruses, bacteria, and parasites may enter or interact with a host cell by changing the cellular state of signaling. This is also the case during HIV infection. Nef, an early HIV gene product, promotes infectivity of the virus via lipid rafts (Zheng, Curr. Biol. 11 (2001 ), 875-879), and infection with HIV-1 virions lacking Nef does not progress to AIDS (Kirchhoff, N. Engl. J. Med. 332 (1995), 228-232). By clustering lipid rafts carrying relevant host cell surface proteins, Nef oligomerization may aid in organizing the T-cell signaling complex and the HIV budding site (Zheng, Curr. Biol. 11 (2001 ), 875-879; Wang, Proc. Natl. Acad. Sci. USA 97 (2000), 394-399). HIV exit from the cell, another raft-dependent step, depends critically on the viral Gag protein (Ono, Proc. Natl. Acad. Sci. USA 98 (2001), 13925-13930; Lindwasser, J. Virol. 75 (2001), 7913-7924): Gag proteins specifically bind to rafts containing HIV spike proteins, which cluster rafts together to promote virus assembly. The interaction between HIV-1 protein and lipid rafts may cause a conformational change in Gag required for envelope assembly (Campbell, J. Clin. Virol. 22 (2001), 217-227).

Another example of raft clustering is the pathogenic mechanism of pore-forming toxins, which are secreted by Clostridium, Streptococcus, and Aeromonas species, among other bacteria (Lesieur, MoI. Membr. Biol. 14 (1997), 45-64). These toxins may cause diseases ranging from mild cellulites to gaseous gangrene and pseudomembranous colitis. Best studied is the toxin aerolysin from the marine bacterium Aeromonas hydrophila. Aerolysin is secreted and binds to a GPI-anchored raft protein on the surface of the host cell. The toxin is incorporated into the membrane after proteolysis and then heptamerizes in a raft-dependent manner to form a raft-associated channel through which small molecules and ions flow to trigger the pathogenic changes. The oligomerization of aerolysin can be triggered in solution but occurs at more than 10⁵- fold lower toxin concentration at the surface of the living cell. This enormous increase in efficiency is due to activation by raft binding and by concentration into raft clusters, which is driven by the oligomerization of the toxin. Again, a small change can lead to a huge effect by amplification of raft clustering (Lesieur, MoI. Membr. Biol. 14 (1997), 45- 64; Abrami, J. Cell Biol. 147 (1999), 175-184).

Tuberculosis is an example of a bacterial infectious disease involving rafts. Complement receptor type 3 (CR3) is a receptor able to internalize zymosan and C3bi-coated particles and is responsible for the nonopsonic phagocytosis of Mycobacterium kansasii in human neutrophils. In these cells CR3 has been found associated with several GPI-anchored proteins localized in lipid rafts of the plasma membrane. Cholesterol depletion markedly inhibits phagocytosis of M. kansasii, without affecting phagocytosis of zymosan or serum-opsonized M. kansasii. CR3, when associated with a GPI protein, relocates in cholesterol-rich domains where M. kansasii are internalized. When CR3 is not associated with a GPI protein, it remains outside of these domains and mediates phagocytosis of zymosan and opsonized particles, but not of M. kansasii (Peyron, J. Immunol. 165 (2000), 5186-5191).

Membrane heterogeneity in bacteria has been described in the prior art, e.g. in Fishov, MoI. Microbiol. 32 (1999), 1 166-1172 and Mileykovskaya, J. Bacterid. 182 (2000), 1172-1175. Different proteins in bacterial membranes displayed variable sensitivity to lipid fluidity, suggesting the coexistence of physically separated lipid domains of diverse fluidity and composition (Linden, Proc. Natl. Acad. Sci. USA 70 (1973), 2271- 2275; Morrisett, J. Biol. Chem. 250 (1975), 6969-6976), and there is also evidence for the organization of a bacterial cytoplasmic membrane into functional domains in which cytoplasmic membrane components are found to be differentially localized (Myers, Curr. Microbiol. 19 (1989), 45-51) and, moreover, membrane phospholipids are segregated into distinct domains that differ in composition, proteo-lipid interaction and degree of order (Vanounou, MoI. Microbiol. 49 (2003), 1067-1079). In this respect, bacterial cell membrane domains are functionally and to an extent structurally analogous to mammalian lipid rafts. Most recently it was reported that antimicrobial peptides were differentially toxic to bacteria with a high phosphoethanolamine content in their membranes, emphasizing the potential importance of the lipid composition of the cell surface in determining selective toxicity of antimicrobial agents (Epand, Biochim. Biophys. Acta. 1758 (2006), 1343-1350).

Wang, Biochemistry 43 (2004), 1010-1018 investigates the relationship between sterol/steroid structures and participation in lipid rafts. These authors consider this question of interest, since sterols may be used to distinguish biological processes dependent on cholesterol in cells from those processes that can be supported by any raft environment. Interestingly, Wang and colleagues have found steroids which promoted the formation of ordered domains in biological membranes.

WO 01/22957 describes the use of gangliosides for the modulation of sphingolipid/cholesterol microdomains.

WO 01/23406, WO 01/23407 and WO 01/49703 disclose the use of certain substituted sapogenins in the treatment of cognitive dysfunction and similar conditions. WO 02/79221 discloses certain steroidal sapogenins and derivatives thereof, and their use in the treatment of cognitive dysfunction, non-cognitive neurodegeneration, non- cognitive neuromuscular degeneration, and receptor loss in the absence of cognitive, neural and neuromuscular impairment. WO 03/082893 describes therapeutic methods and uses of certain steroidal sapogenins, related compounds and derivatives thereof, in the treatment of non-cognitive neurodegeneration, non-cognitive neuromuscular degeneration, motor-sensory neurodegeneration or receptor dysfunction or loss in the absence of cognitive, neural and neuromuscular impairment.

WO 02/26762 describes certain triterpenes having antibacterial activity, wherein said triterpenes include betulin, allobetulin and lupeol. WO 03/62260 provides certain quarternary amine derivatives of betulin and other triterpenes having antibacterial, antifungal and surfactant properties. WO 07/101873 describes the use of certain betulonic and betulinic acid derivatives for the treatment of cancer or a viral infection.

A problem underlying the present invention is the provision of means and methods for clinical and/or pharmaceutical intervention in infectious diseases/disorders, in particular those linked to and/or associated with biological/biochemical processes regulated by lipid rafts.

The solution to this technical problem is achieved by providing the embodiments characterized herein below as well as in the claims.

In the context of this invention, it was found that amino-substituted steroid sapogenin, androstane and triterpenoid sapogenin derivatives are suitable for the medical management of infectious diseases, in particular disorders or diseases caused by viral agents and bacteria.

Accordingly, the present invention provides a compound of one of the following formulae 1 , 2 or 3:

or a pharmaceutically acceptable salt, derivative, solvate or prodrug thereof for the treatment, prevention and/or amelioration of an infectious disease/disorder. The disorders or diseases may be caused by a virus or bacterium, including drug-resistant bacteria.

The following numbering of the carbon atoms and denotation of the rings of the steroid sapogenin (compounds of general formula 1), androstane (compounds of general formula 2) and triterpenoid sapogenin (compounds of general formula 3) scaffolds will be adhered to throughout the description:

Furthermore, the general formulae given in the present invention are intended to cover all possible stereoisomers and diastereomers of the indicated compounds. Unless indicated differently, the stereochemical configuration of naturally occurring steroid sapogenin, androstane and triterpenoid sapogenin derivatives as shown in the exemplified compounds is preferred.

In formula 1 , 2 or 3, one of R¹, R², R³, if R³ is present, R⁴, if R⁴ is present, and R⁵ is a linear amine-containing group selected from X(CH₂)_nNH₂, X(CH₂)_HNH(Ci_-4 alkyl), X(CH₂)_ON(Ci_-4 alkyl)₂ or X(CH₂)_nN(Ci-4 alkyl)₃ ⁺, or a cyclic amine-containing group selected from piperidin-1-yl, 1-(Ci_-4 alkyl)⁺-pipehdin-1-yl, morpholin-4-yl or 4-(Ci_-4 alkyl)⁺-morpholin-4-yl. In one embodiment, one of R¹, R², R³, if R³ is present, R⁴, if R⁴ is present, and R⁵ is the linear amine-containing group, preferably X(CH₂)_nNH₂. In another embodiment, one of R¹, R², R³, if R³ is present, R⁴, if R⁴ is present, and R⁵ is the cyclic amine-containing group, preferably piperidin-1-yl or morpholin-4-yl. In most of the embodiments provided herein, R⁴ is not the amine-containing group and one of R¹, R², R³, if R³ is present, and R⁵ is the amine-containing group. Yet, in most embodiments of the compounds described herein, only R⁵ is the amine-containing group.

In formula 1 or 3, X is a direct bond or a phosphorus-containing group selected from OP(O)(CT)O, OP(O)(OC_1-4 alkyl)O, OP(O)(O^")CH₂O or OP(O)(OCi_-4 alkyl)CH₂O. In the phosphonate containing groups, P is attached to the A ring via O and to the amine-containing moiety (i.e. to (CH₂)nNH₂, (CH₂)HNH(Ci_-4 alkyl), (CH₂J_nN(Ci_-4 alkyl)₂ or (CH₂)nN(Ci-₄ alkyl)₃ ⁺) via CH₂O. In one preferred embodiment of formula 1 or 3, X is a direct bond. In one embodiment of formula 1 or 3, X is OP(O)(O-)O. In another embodiment of formula 1 or 3, X is OP(O)(OCi_-4 alkyl)O. In yet another embodiment of formula 1 or 3, X is OP(O)(O-)CH₂O. In a further embodiment of formula 1 or 3, X is OP(O)(OC₁^ alkyl)CH₂O.

In formula 2, X is a direct bond or a phosphorus-containing group selected from OP(O)(OC_1-4 alkyl)O, OP(O)(O-)CH₂O or OP(O)(OC_1-4 alkyl)CH₂O. In the phosphonate containing groups, P is attached to the A ring via O and to the amine- containing moiety (i.e. to (CH₂)_nNH₂, (CH₂)_nNH(d^ alkyl), (CH₂)_nN(C_1-4 alkyl)₂ or (CH₂)_PiN(C_1-4 alkyl)₃ ⁺) via CH₂O. In one preferred embodiment of formula 2, X is a direct bond. In another embodiment of formula 2, X is OP(O)(OCi_-4 alkyl)O. In yet another embodiment of formula 2, X is OP(O)(O-)CH₂O. In a further embodiment of formula 2, X is OP(O)(OC₁^ alkyl)CH₂O.

When X is a direct bond, n is an integer from O to 2. In one embodiment, n is O. In another embodiment, n is 1. In yet another embodiment, n is 2. In one preferred group of compounds, n is O or 1. In another preferred group of compounds, n is 1 or 2.

When X is the phosphorus-containing group, n is an integer from 2 to 6, preferably 2.

When X is OP(O)(O-)O or OP(O)(O-)CH₂O, the compound of formula 1 , 2 or 3 can exist as a salt. Suitable counterions are listed below as "pharmaceutically acceptable salts". The corresponding free base, i.e. wherein X is OP(O)(OH)O or OP(O)(OH)CH₂O is also within the scope of the present invention.

In the embodiment, wherein R⁵ is the amine-containing group, ==■ is a single bond or a double bond, preferably a single bond. When == is a double bond, R⁴ is absent. R¹, R², R³, if R³ is present, and R⁴, if R⁴ is present, are independently H or OH. R⁶ is H or, when X is a direct bond and n is 1 or 2, R⁶ can also be OH. Preferably, R⁶ is H. In the embodiment, wherein R¹ is the amine-containing group, R² and R⁶ are independently H or OH. R³, if R³ is present, R⁴, if R⁴ is present, and R⁵ are H. == is a single bond.

In the embodiment, wherein R² is the amine-containing group, R³, if R³ is present, and R⁶ are independently H or OH. R¹, R⁴, if R⁴ is present, and R⁵ are H. ^=r= is a single bond.

In the embodiment, wherein R³ is the amine-containing group, R² and R⁶ are independently H or OH. R¹, R⁴ and R⁵ are H. == is a single bond.

In the embodiment, wherein R⁴ is the amine-containing group, R² and R⁶ are independently H or OH. R¹, R³ and R⁵ are H. == is a single bond.

When X is a direct bond, in a preferred embodiment, the compound of formula 1 , 2 or 3 contains one to four hydroxy! groups. The hydroxyl groups increase solubility of the material in an amphiphilic or polar medium which can be advantageous in medical applications.

When one of R¹, R², R³, R⁴ and R⁵ is the linear amine-containing group, R⁷ is a C_6-H aliphatic group, such as C₆-n alkyl, C_6-n alkylidene or C_6-n alkenyl, provided that R⁷ is not the branched C₈ alkyl occurring in natural cholesterol, i.e. 2-methyl-hept-6-yl. Preferably, R⁷ is linear C₅-₁₁ alkyl, linear C_6-n alkylidene or linear C₆-n alkenyl, in which one or more (preferably one) hydrogens are optionally replaced by a methyl group (the total number of carbon atoms after replacement of hydrogen by methyl is to be 6 to 11). In another preferred embodiment, R⁷ is C_7-I2 alkyl, in which one or two (preferably one) CH₂ is/are replaced by oxygen (the total number of carbon atoms after replacement of the CH₂ by O is to be 6 to 11). In yet another preferred embodiment, R⁷ is a C_9-H aliphatic group comprising a linear C₆ alkyl or a linear C₆ alkenyl main chain and three or four side chains independently selected from methyl or ethyl (the total number of carbon atoms is to be 9 to 11 ). When one of R¹, R², R³, R⁴ and R⁵ is the cyclic amine-containing group, R⁷ is the branched C₈ alkyl occurring in natural cholesterol, i.e. 2-methyl-hept-6-yl.

In formula 3, R⁸, R⁹ and R¹⁰ are independently C_1-4 alkyl, C_1-4 alkenyl or H. Preferably, at least one of R⁸, R⁹ and R¹⁰ is H.

m is 1 or 2. In one embodiment, m is 1 and R⁹ and R¹⁰ are preferably H. In another embodiment, m is 2 and R⁹ is preferably C_1-4 alkyl, more preferably CH₃.

R¹¹ is H or 0(C_1-4 alkyl). Preferably, R¹¹ is H or OCH₃.

In a preferred embodiment of formula 3, m is 2, R⁸ and R⁹ are both CH₃, R¹⁰ is H and R¹¹ is OCH₃. In another preferred embodiment of formula 3, m is 2, R⁸ is H, R⁹ and R¹⁰ are both CH₃ and R¹¹ is OCH₃. In a third preferred embodiment of formula 3, m is 1 , R⁸ is prop-2-yl or propen-2-yl, R⁹ and R¹⁰ are both H and R¹¹ is H or OCH₃.

In one embodiment, the compound of formula 1 , 2 or 3 contains 0 or 1 hydroxyl groups. In another embodiment, the compound of formula 1 , 2 or 3 does not contain any hydroxyl group.

In a preferred embodiment of formula 1 , 2 or 3, R⁵ is the linear amine-containing group; R¹, R², R³, if R³ is present, and R⁴, if R⁴ is present, are independently H or OH;

R⁶ is H; X is a direct bond; == is a single bond or a double bond, preferably a single bond; R⁷ is linear Cβ-n alkyl, linear Cβ-n alkylidene or linear C_6-11 alkenyl, in which one or more hydrogens are optionally replaced by a methyl group (the total number of carbon atoms after replacement of hydrogen by methyl is to be 6 to 11); C_7-12 alkyl, in which one or two CH₂ is/are replaced by oxygen (the total number of carbon atoms after replacement of the CH₂ by O is to be 6 to 1 1); or a Cg--_H aliphatic group comprising a linear C₆ alkyl or a linear C₆ alkenyl main chain and three or four side chains independently selected from methyl or ethyl (the total number of carbon atoms is to be 9 to 11 ). In another preferred embodiment of formula 1, 2 or 3, R⁵ is the linear amine-containing group; R¹, R², R³, if R³ is present, and R⁴, if R⁴ is present, are independently H or OH; R⁶ is H; X is the phosphorus-containing group; === is a single bond or a double bond, preferably a single bond; R⁷ is linear C_6--_{I 1} alkyl, linear C_6-11 alkylidene or linear C_6--_I1 alkenyl, in which one or more hydrogens are optionally replaced by a methyl group (the total number of carbon atoms after replacement of hydrogen by methyl is to be 6 to 11); C_7-i₂ alkyl, in which one or two CH₂ is/are replaced by oxygen (the total number of carbon atoms after replacement of the CH₂ by O is to be 6 to 11 ); or a C_9-11 aliphatic group comprising a linear C₆ alkyl or a linear C₆ alkenyl main chain and three or four side chains independently selected from methyl or ethyl (the total number of carbon atoms is to be 9 to 11).

The following compounds of formulae 1a to 1d are preferred examples of the compound of formula 1.

The following compounds of formulae 2a to 2g are preferred examples of the compound of formula 2.

Among compounds 2a to 2g, compounds 2a, 2c, 2e and 2g are preferred.

The following compounds of formulae 2h and 2i are further examples of the compound of formula 2.

Among compounds 2h and 2i, compound 2h is preferred.

The following compounds of formulae 3a to 3d are preferred examples of the compound of formula 3.

The compounds to be used in accordance with the present invention can be prepared by standard methods known in the art.

Compounds of the general formula 1 can be prepared starting from commercially available steroid sapogenin derivatives, e.g. diosgenin, sarsasapogenin, smilagenin, and the corresponding stereoisomers thereof.

Compounds of the general formula 2 can be prepared starting from commercially available steroid derivatives, e.g. androsterone, pregnolone, stigmasterol, 3- cholestanone, and isomers thereof. Various side chains can be introduced via the 17- or 20-keto derivatives using Wittig- or Wittig-type reactions. Alternatively, O-alkylations can be used to generate various ether-decorated side chain motifs. The corresponding alkyl precursors for Wittig or alkylation reactions are either commercially available or can be easily prepared by standard techniques.

Compounds of the general formula 3 can be prepared starting from commercially available triterpenoid sapogenin derivatives, e.g. ursolic acid, oleanolic acid, betulinic acid, betulin or lupeol, and the corresponding stereoisomers thereof.

The various A-ring substitution patterns of compounds of the general formula 1 , 2 or 3 can be prepared from the corresponding hydroxy or keto derivatives using methods, protocols and/or synthetic strategies described and/or developed for cholesteryl or cholestane derivatives or similar steroid derivatives.

Compounds of the general formula 1 , 2 or 3 wherein X is a direct bond and which have a primary amino function at position 3, can be prepared from the corresponding alcohol via the sequence sulfonate-azide-amine. Alternatively, a ketone, e.g. 3- androstanone, can be used as substrate for reductive aminations with a plethora of (commercially available) amines. For swapping the stereochemistry at a given position standard strategies can be used, such as Mitsunobu reactions or stereoselective reductions of ketones using L-selectride followed by the sequence sulfonate-azide- amine.

If the amine-containing group is attached to the C2-position of the steroid scaffold, the corresponding ketone can be used as synthetic precursor, which can be produced similar to cholestane derivatives as described by various alternative literature-known procedures (Barillier, Tetrahedron 50 (1994), 5413-5424; Penz, Monatshefte fuer Chemie 112 (1981), 1045-1054; Lightner, Steroids 35 (1980), 189-207; Nakai, Tetrahedron Lett. (1979), 531-534). Subsequent introduction of the amine-containing group can be achieved by the general strategies described above for 3-androstanone. The same principle can be used for 4-androstanone (available as described for cholestanone derivatives in Nakai, Tetrahedron Lett. (1979), 531-534; Sondheimer, J. Org. Chem. 26 (1961 ), 630-631 ; Shoppee, J. Chem. Soc. (1959), 630-636) as substrate or employed on 1-androstanone (available as described for cholestanone derivatives in Shoppee, J. Chem. Soc. C (1968), 245-249) to provide the corresponding derivatives having the amine-containing function attached to C4 or C1 of the androstane scaffold, respectively.

Compounds of the general formula 1, 2 or 3 wherein X is a direct bond and which have a primary amino function at position 1 , 2 or 4 can be prepared from the corresponding allyl amines. Alternatively, epoxidation and subsequent opening or bishydroxylation of the double bond would result in the described hydroxy-decorated compounds. The allyl amines can be prepared from a suitable 2α,3α-epoxy-5α- androstane derivative via ring opening of the epoxy moiety with benzylamine followed by debenzylation or via treatment with mesylchloride followed by treatment with azide and subsequent lithium aluminium hydride reduction. 2α,3α-Epoxy-5α-androstane derivatives are, for example, available via meta-chloroperbenzoic acid mediated epoxidation of the corresponding 5α-androst-2-ene derivative which itself can be prepared as described in the literature for cholestane derivatives (Cruz Silva, Tetrahedron 61 (2005), 3065-3073).

Compounds of the general formula 1 , 2 or 3 wherein X is an O-alkylphosphate, or compounds of the general formula 1 or 3 wherein X is a phosphate, can be prepared from the corresponding alcohols (available as described above) using the literature- known phosphoramidite methodology (Beaucage, S. L.; J. Org. Chem. 2007, 72(3), 805-815; Noyori, R.; J. Am Chem. Soc. 2001 , 723(34), 8165-8176; Hayakawa, Y.; Bull. Chem. Soc. Jpn. 2001 , 74(9), 1547-1565; Noyori, R.; Tetrahedron Lett. 1986, 27(35), 4191-4194; Reviews: Bannwarth W.; HeIv. Chim. Acta 1987, 70, 175-186 and Beaucage, S. L; Tetrahedron 1993, 49(10), 1925-1963). As compared to the classical strategy using phosphorylchloride, the phosphoramidite protocol is more robust and provides for more reliable results.

Compounds of the general formula 1 , 2 or 3 wherein X is a phosphonate or O- alkylphosphonate can be prepared from the corresponding alcohols (available as described above) using strategies described in the literature (Rejman, D.; Nucleosides Nucleotides Nucleic Acids 2001 , 20, 1497-1522 and Holy, A.; J. Med. Chem. 2001 , 44, 4462-4467). The resulting phosphonates can be O-alkylated using C_1-4 alkyl halides in standard protocols known to the skilled person.

The compounds provided herein are useful in the treatment (as well as prevention and/or amelioration) of infectious diseases or disorders, like viral diseases or bacterial infections. Compounds provided herein have been evaluated in corresponding cell- based disease/disorder models. In accordance with the data and information provided herein the present invention provides in particular for the use of the compounds as shown in formulae 1a to 1d as well as 2a to 2i and 3a to 3d in a medical setting for the treatment of disorders and diseases which are caused by a viral or bacterial infection. Of particular interest in this context are, however, mycobacterial infections, such as tuberculosis, and influenza infections. In the following, more detailed information on diseases and disorders is given. These diseases and disorders may be prevented, ameliorated and/or treated by using the steroid sapogenin, androstane and triterpenoid sapogenin derivatives provided herein. In particular, the experimental data provided herein document that compounds 1a to 1d, 2a, 2c, 2e, 2g and 2h, 3a to 3d are particularly preferred compounds in distinct medical interventions or preventions. Particularly preferred compounds in this regard are compounds 1a to 1c, 2e and 2g. Corresponding experimental evidence is provided in the appended examples.

The appended examples document that biological and/or biochemical processes involved in infectious diseases and disorders may be influenced by modulating the assembly of lipid rafts when the herein defined and described steroid sapogenin, androstane and triterpenoid sapogenin derivatives are employed. Without being bound by theory, the specific amines and ammonium derivatives disclosed herein are believed to be capable of interfering with the partitioning of regulatory molecules within lipid rafts. Accordingly, the formation of protein complexes with lipid rafts and/or the clustering of lipid rafts may be modified and the diseased status is interfered with or even prevented. Provided herein are specific molecules, namely steroid sapogenin, androstane and triterpenoid sapogenin derivatives as defined herein above which are believed to be capable of interfering with biological processes, in particular pathological processes taking place in, on, or within lipid rafts of cells, preferably diseased cells. These molecules may be considered as "raft modulators".

Without being bound by theory, the compounds described herein are in particular useful in the treatment, prevention and/or amelioration of an infectious disease/disorder caused by (a) biochemical/biophysical pathological process(es) occurring on, in or within lipid rafts. Corresponding examples of such diseases/disorders as well as of such biochemical/biophysical processes are given herein. The term biochemical/biophysical pathological process occurring on, in or within lipid rafts, accordingly, means for example, pathogen-induced abnormal raft clustering upon viral or bacterial infections, the formation of oligomeric structures of (bacterial) toxins in lipid rafts upon infection with pathogens, or the enhanced activity of signaling molecules in lipid rafts caused by a virus or bacterium. Also a tighter than normal packing of lipid rafts/lipid raft components or a direct bacteriocidal effect on the bacterial membrane is considered a "biochemical/biophysical pathological process" in accordance with this invention.

For example, in bacterial infections such as tuberculosis, shigellosis and infection by Chlamydia and uropathogenic bacteria the organism is taken up into the cell in a raft- dependent internalization process often involving caveolae. Prevention of localization of the bacteria in rafts, blockage of bacterial phagocytosis or internalization of bacteria would prevent infection. In the case of caveolae, which depend on a cholesterol binding protein, caveolin, exclusion of cholesterol from the raft with steroid sapogenin, androstane or triterpenoid sapogenin derivatives may prevent uptake of the pathogen.

Bacterial infections to be treated in accordance with the present invention include in particular infections induced by clinically and medically relevant bacteria and corresponding bacterial strains. Such bacteria or strains comprise, for example, Borrelia spp., Bartonella quintana, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila felis, Chlamydophila psittaci, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium ulcerans, Mycobacterium kanasasii, Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium scrofulaceam, Mycobacterium microti, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium intracellular, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium xenopi, Mycobacterium fortuitum, Mycobacterium chelonei, Mycobacterium marinum, Legionella pneumophila, Rickettsia spp. and Treponema spp. As documented in the appended examples and as described herein the compounds of the present invention are in particular of medical relevance in a treatment and medical intervention of mycobacterial disorders/diseases, like tuberculosis and leprosis.

In the context of this invention, a "mycobacteria-induced disease" may comprise a disorder/disease elucidated and/or related to an infection with, inter alia, M. tuberculosis, M. bovis, M. avium, M. africanum, M. kanasasii, M. intracellular, M. ulcerans, M. paratuberculosis, M. simiae, M. scrofulaceam, M. szulgai, M. xenopi, M. fortuitum, M. chelonei, M. leprae, M. marinum, M. microti and M. canettii. The appended examples provide in particular data for the inventive use of the compounds disclosed herein in the treatment and/or prevention of tuberculosis, i.e. an infection caused by M. tuberculosis. As mentioned above in the context of "infections to be treated and/or prevented", the present invention is not limited to the treatment/prevention of a disorder/disease caused by the pathogen agent (e.g. bacterium) per se, but comprises also the medical amelioration of a disorder/disease caused by products produced by the pathogens, like, e.g. toxins.

Mycobacteria-induced diseases to be treated in accordance with the present invention include, inter alia, tuberculosis, leprosy, tropical skin ulcer, ulceration, abscess, pulmonary disease, granulomatous (skin) disease, opportunistic infections with non- tuberculous mycobacteria as well as diseases elicited by atypical mycobacteria such as M. avium including pulmonary disease, lymphadenitis, cutaneous and disseminated diseases, e.g. in immunocompromised patients. The use is not restricted to mycobacteria-induced diseases in humans, but comprises also the use of the present invention in animal diseases, like bovine tuberculosis. In a preferred embodiment, the mycobacteria-induced disease is tuberculosis as also documented in the appended examples. In a further preferred embodiment, the mycobacteria-induced disease is caused by bacterial strains resistant to standard drugs for the treatment of tuberculosis as described in more detail below.

Accordingly, the present invention also provides for the use of the compounds disclosed herein in the treatment and/or amelioration of a Mycobacterium infection, preferably of a Mycobacterium tuberculosis infection.

As documented in the present invention and described in the appended examples, preferred compounds for the medical intervention or prevention of bacterial infections caused by Mycobacteria (e.g. infections caused by M. tuberculosis and/or tuberculosis itself), including drug resistant and multi-drug resistant bacterial strains, are compounds 1a, 1c and 1d, 2a to 2c, 2e, 2f, 2h, 2i and 3a to 3d; particularly preferred compounds are 1a, 1c and 1d as well as 2a, 2c, 2h and 3a to 3d. Due to their nanomolar potency compounds 1a and 2a are an even more preferred embodiment of the present invention, namely these compounds are particularly useful in the medical intervention of bacterial infections, preferably in mycobacteria-induced infections. However, the present invention also provides for the medical use of these compounds in pharmaceutical and medical interventions of other bacterial or viral infections.

The cell wall of M. tuberculosis, in its full structural and functional integrity, is essential for growth and survival of the bacterial cell in the infected host. Hence, some of the most effective antituberculosis agents including isoniazid (INH) act on cell wall targets, for instance by inhibiting the biogenesis of cell wall components (Chatterjee, D.; Curr. Opin. Chem. Biol. 1997, 1, 579-588; and literature cited therein). Rifamycins form another class of drugs used for standard treatment of tuberculosis. They act via inhibition of bacterial RNA synthesis by binding to the beta-subunit of the DNA- dependent polymerase. Remarkably, rifamycins are the only clinically used antibiotics with this mechanism, and rifampin (RMP) is the most prominent example of the rifamycins.

Rifampin and isoniazid are the primary drugs for treatment of tuberculosis, and emerging resistance against first-line antituberculosis drugs is among the most serious worldwide health threats. Drug resistance may, in the context of this invention, be defined as a decrease in the in vitro susceptibility of infectious agents, e.g. bacteria like M. tuberculosis, of sufficient degree to be reasonably certain that the strain concerned of said infectious agent is different from a corresponding wild strain that has never come into contact with the drug, and it comprises primary drug resistance and acquired drug resistance (see, e.g. "Anti-tuberculosis Drug Resistance in the World. The WHO/IUATLD Global Project on Anti-tuberculosis Drug Resistance Surveillance", WHO Global Tuberculosis Programme. World Health Organization, Geneva, Switzerland, 1997. WHO/TB/97.229). Following the WHO/IUATLD Guidelines, primary drug resistance is understood as the presence of drug-resistant infectious agent, like M. tuberculosis, in a patient with no, or less than one month of, previous medical intervention, like (in case of a mycobacterial (M. tuberculosis) infection) anti- tuberculosis drug treatment (WHO/IUATLD Global Working Group on Antituberculosis Drug Resistance Surveillance. Guidelines for surveillance of drug resistance in tuberculosis. World Health Organization, Geneva, Switzerland, 1997. WHO/TB/96.216). Acquired drug resistance is found in a patient who has previously received at least one month of, e.g., anti-tuberculosis drug treatment. Combined drug resistance is the prevalence of drug resistance among all cases of tuberculosis, regardless of prior drug treatment, in a given year and country. The various forms of drug resistance, in particular in the context of tuberculosis, are defined as follows: single drug-resistant tuberculosis is a form of tuberculosis that is resistant to one of the primary drugs (i.e. first line drugs, such as rifampin (= rifampicin), isoniazid, pyrazinamide, ethambutol), to one of the secondary drugs or to one of the injectable drugs (i.e. second line drugs, such as ethionamide, thiacetazone, para-aminosalicylic acid, cycloserine, streptomycin, kanamycin, capreomycin, or the fluoroquinolones, e.g. ofloxacin, moxifloxacin) used for the treatment of tuberculosis. Multidrug-resistant tuberculosis is a form of tuberculosis (TB) that is resistant to two or more of the primary drugs used for the treatment of tuberculosis. Extensively drug-resistant TB is as form of tuberculosis resistant to at least isoniazid and rifampin among first-line anti-TB drugs and among second-line drugs, is resistant to any fluoroquinolone and at least one of the injectable drugs (Raviglione, M.; N. Engl. J. Med. 2007, 356, 7).

The occurrence of multidrug resistant strains of M. tuberculosis, at least resistant to the action of isoniazid and rifampin, implies recourse to other compounds. In 2000, an estimated 3.2% of all new tuberculosis cases (between 185,000 and 414,000) were multiresistant (Dye, C; J. Infect. Dis. 2002, 185, 1 197-1202). Further first-line agents are pyrazinamide and ethambutol. This is followed by the group of injectable drugs as second-line drugs, e.g. streptomycin and kanamycin, and the antibacterial fluoroquinolones, e.g. moxifloxacin (Janin, Y. L.; Bioorg. Med. Chem. 2007, 15, 2479- 2513). Resistant M. tuberculosis strains have been isolated for all these drugs, in particular because of their use in monotherapy. Thus, development of pharmaceutical compositions capable of inhibiting the growth of drug-resistant and multidrug-resistant strains of M. tuberculosis and clinical isolates of Mycobacteria addresses an important and yet unmet medical need.

Examples for M. tuberculosis strains, in particular said drug-resistant or multidrug- resistant strains and clinical isolates, to be treated with the compounds described herein are, inter alia, NCTC 8337 (OV 185/10; ATCC® 25584), H₃₇Rv (ATCC® 25618), H₃₇Rv(mma1), H₃₇Ra, HN34, HN35, HN40, HN59, HN88, HN93, HN224, HN386, NHN5, NHN50, Aoyama B (ATCC® 31726), TMC 102 (H₃₇Rv; ATCC® 27294), TMC 106 (Summit Park; ATCC® 35800), TMC 107 (Erdman; ATCC® 35801), TMC 108 (Campbell; ATCC® 35802), TMC 109 (NIH-199RB; ATCC® 35803), TMC 110 (Amerzaga; ATCC® 35804), TMC 111 (Kerrigan; ATCC® 35805), TMC 112 (Hand; ATCC® 35806), TMC 113 (Fick; ATCC® 35807), TMC 1 16 (C; ATCC® 35808), TMC 117 (PN; ATCC® 35809), TMC 119 (DT; ATCC® 35810), TMC 120 (Indian 79157; ATCC® 3581 1 ), TMC 124 (Kurono; ATCC® 35812), TMC 125 (ATCC® 35813), TMC 132 (Jamaica 22; ATCC® 35814), TMC 202 (JH6Ra; ATCC® 35815), TMC 203 (JH16Ra; ATCC® 35816), TMC 204 (H4Ra; ATCC® 35817), TMC 205 (R1 Rv; ATCC® 35818), TMC 206 (H₃₇Rv-SM-R; ATCC® 35820), TMC 302 (H₃₇Rv-PAS-R; ATCC® 35821), TMC 303 (CIP 105794; H₃₇Rv-INH-R; ATCC® 35822), TMC 304 (H₃₇Rv- SM/INH-R; ATCC® 35823), TMC 305 (H₃₇Rv-PAS/SM-R; ATCC® 35824), TMC 306 (H₃₇Rv-PAS/SM/INH-R; ATCC® 35825), TMC 307 (H₃₇Rv-CS-R; ATCC® 35826), TMC 309 (H₃₇Rv-KM-R; ATCC® 35827), TMC 311 (H₃₇Rv-PZA-R; ATCC® 35828), TMC 313 (H₃₇Rv-TAC-R; ATCC® 35829), TMC 314 (H₃₇Rv-ETA-R; ATCC® 35830), TMC 320 (Ameraga-SM-R; ATCC® 35831), TMC 321 (Fick-SM-R; ATCC® 35832), TMC 322 (Hand-SM-R; ATCC® 35833), TMC 323 (Kerrigan-SM-R; ATCC® 35834), TMC 326 (H₃₇Ra-INH-R; ATCC® 35835), TMC 327 (H₃₇Ra-SM-R; ATCC® 35836), TMC 330 (CIP 105793; H₃₇Rv-EMB-R; ATCC® 35837), TMC 331 (CIP 105795; H₃₇Rv-RIF-R, ATCC® 35838), pi (ATCC® 51910), H₃₇Rv-RPT-R (ATCC® 700457), CDC-MPEP 200206 (H; ATCC® BAA-812), H₃₇Ra (ATCC® 25177). It is of note that these drug- resistant or multidrug-resistant strains and clinical isolates are not limiting for the present invention and also other/further infections with drug-resistant or multidrug- resistant strains and clinical isolates may be treated, prevented and/or ameliorated by the use of the compounds described herein.

When testing the compounds of the present invention, which provided for particularly good results in the tuberculosis assay model, in a similar assay setting using bacterial strains being resistant against the standard treatment employing rifampin, isoniazid, pyrazinamide, ethambutol, kanamycin, streptomycin and moxifloxacin, compounds 1a, 1c and 1d as well as 2a, 2h and 2i showed particularly strong growth inhibition of these drug-resistant strains. Hence, compounds 1a, 1c, 1d, 2a, 2h and 2i offer an opportunity for a pharmaceutical composition for a medical intervention or prevention of tuberculosis caused by drug-resistant bacteria. Corresponding experimental evidence is provided in the appended examples.

Bacterial infections to be treated in accordance with the present invention also include infections induced by, inter alia, Gram-positive bacilli, Gram-positive cocci, Gram- negative bacilli and Gram-negative cocci. Gram-positive bacilli are, for example, Clostridium spp., Bacillus anthracis, Erysipelothrix rhusiopathiae, Listeria monocytogenes, Nocardia spp., Corynebactehum diphtheriae and Propionibactehum acnes. Gram-positive cocci are, for example, Staphylococcus aureus and Streptococcus spp. Gram-negative bacilli are, for example, Escherichia coli, Heliobacter pylori, Brucella spp., Aeromonas hydrophila, Shigella spp., Vibrio spp., Yersinia pestis, Salmonella spp., Klebsiella pneumoniae, Burkholderia cepacia, Enterobacter spp., Pseudomonas aeruginosa, Campylobacter jejuni and Legionella pneumophila. Gram-negative cocci are, for example, Neisseria gonorrhoeae and Moraxella catarrhalis.

As pointed out above, the compounds described herein may also be employed in the treatment of bacterial infections or toxicoses induced by secreted bacterial toxins. Bacterial toxins are known in the art and comprise, inter alia, toxins as produced by Vibrio cholerae, aerolysin as, inter alia, produced by Aeromonas spp., anthrax as produced by Bacillus anthracis or helicobacter toxin. These toxins may form oligomeric structures in the raft, crucial to their function. The raft is targeted by binding to raft lipids such as ganglioside GM1 for cholera. Without being bound by theory, in the context of this invention, prevention of oligomerization is considered to be equivalent to prevention of raft clustering, hence the same or similar compounds as those used for viral infection should be able to inhibit the activity of bacterial toxins. The person skilled in the art, in particular an attending physician is readily in a position to adopt the treatment regime with the herein defined steroid sapogenin, androstane and triterpenoid sapogenin derived amines or ammonium derivatives in the treatment of a bacterial infection per se and/or in the amelioration of disorders and diseases caused by the corresponding toxins. Diseases/disorders to be treated in accordance with the present invention also include, inter alia, borreliosis, relapsing fever, trench fever, endocarditis, cervicitis, conjunctivitis, diseases of the thyphos group, diseases of the spotted fever group, pinta and syphilis.

Viral diseases to be treated in accordance with the present invention include diseases induced by a virus selected from the group consisting of influenza virus (A, B, C), HIV, Hepatitis virus (A, B, C, D, E), Rotavirus, Respiratory syncytial virus, Herpetoviridae (e.g. Herpes simplex virus, Epstein-Barr virus), Echovirus 1 , measles virus, Picornaviridae (e.g. Enterovirus, Coxsackie virus), Filoviridae (e.g. Ebola virus, Marburg virus), Papillomaviridae (e.g. human papilloma viruses HPV-1 , HPV-2, HPV- 3, HPV-4, HPV-5, HPV-6, HPV-7, HPV-10, HPV-11 , HPV-13, HPV-16, HPV-18, HPV- 31 , HPV-32, HPV-33, HPV-35, HPV-39, HPV-42, HPV-43, HPV-44, HPV-45, HPV-51 , HPV-52, HPV-55, HPV-56, HPV-58, HPV-59, HPV-68, HPV-73, HPV-82) and Polyomaviridae. In certain embodiments the virus to be treated is influenza virus or HIV.

The steroid sapogenin, androstane and triterpenoid sapogenin derived amines or ammonium derivatives described in this invention can be applied to 1) modulate raft formation and interfere with the transport of hemagglutinin and neuraminidase to the cell surface, 2) prevent the clustering of rafts containing the spike glycoproteins induced by M proteins and, thus, interfere with virus assembly, or 3) by increasing the size/volume of lipid rafts or 4) prevent the fission of the budding pore (pinching-off) which occurs at the phase boundary of raft (viral membrane) and non-raft (plasma membrane) or 5) disrupt the envelope of the free virus to render it non-infectious or of reduced infectivity (virucidal effect).

In viral infections, raft clustering is involved in the virus assembly process. The compounds 1a to 1d as well as 2b to 2g have an effect in a virus replication assay. Preferred in this context are the compounds 1a to 1d as well as 2b, 2e and 2g; particularly preferred are compounds 1a, 1 b, 1c, 2e and 2g. Without being bound by theory, the structural feature underlying this effect is thought to be represented by the combination of an amine-substitution at the steroidal A ring and the presence of a steroid-type B, C, D ring system having a hydrocarbon side chain, including hydrocarbon side chains comprising an oxa function, or having an additional E, F ring system displaying a spiroacetal system as provided by the steroid sapogenin scaffold. Additional decoration with hydroxy functions inside the A ring might provide compounds of increased solubility, thus enhancing bioavailability. As demonstrated by the results obtained in the viral replication assay described in the experimental part, these compounds may be useful for pharmaceutical intervention.

As the mechanism of virus release for HIV-1 is similar to that of influenza virus, with respect to raft involvement, the above compounds can also be used in the treatment of HIV infections and in the medical management of HIV-related diseases, in particular AIDS.

Further viral diseases (as non-limiting examples) which may be approached with the above compounds or derivatives thereof are herpes, ebola, enterovirus, coxsackie virus, hepatitis C, rotavirus and respiratory syncytial virus. Accordingly, particularly preferred compounds as well as preferred compounds provided herein in the context of a specific (viral) assay or test system may also be considered useful in the medical intervention and/or prevention of other infectious diseases, in particular viral infections.

Besides the disease-inhibitory activity of the steroid sapogenin, androstane and thterpenoid sapogenin derivatives to be employed in the context of this invention, the compounds are also evaluated in several toxicity assays. Toxicity assays are well known in the art and may, inter alia, comprise lactate dehydrogenase (LDH) or adenylate kinase (AK) assays or an apoptosis assay. In addition, cytotoxicity of test compounds was evaluated in mammalian Vero cells and in J774A.1 macrophages, which are a widely used assay model particularly useful for the evaluation of toxicity of lipophilic compounds showing limited solubility. Yet, these (cyto)-toxicity assays are, as known by the skilled artisan, not limited to these assays.

The compounds described herein may be administered as compounds per se in their use as pharmacophores or pharmaceutical compositions or may be formulated as medicaments. Within the scope of the present invention are pharmaceutical compositions comprising as an active ingredient a compound of formula 1 , 2 or 3, in particular one of the formulae 1a to 1d, 2a to 2i as well as 3a to 3d as defined above. The pharmaceutical compositions may optionally comprise pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives or antioxidants. The compounds described herein may be administered in the context of a monotherapy or in combination with one or more other anti-infective pharmaceutical agents, in particular for the treatment of a primary infection complicated by one or more co- infections. Such a combination therapy might be of particular use for the treatment or prevention of infections caused by drug resistant or multi-drug resistant bacterial strains. The same applies mutatis mutandis for infections caused by viral agents. For example, the compounds of the present invention may be employed in co- therapeutical medical interventions in viral infections like the HAART therapy in HIV infections.

The pharmaceutical compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in Remington's Pharmaceutical Sciences, 20^th Edition. The pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, rectal, nasal, topical, aerosol or vaginal administration. Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets. Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration. Dosage forms for rectal and vaginal administration include suppositories and ovula. Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler. Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems. Pharmaceutically acceptable salts of compounds that can be used in the present invention can be formed with various organic and inorganic acids and bases. Exemplary base addition salts comprise, for example, alkali metal salts such as sodium or potassium salts; alkaline-earth metal salts such as calcium or magnesium salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, diethanol amine salts or ethylenediamine salts; aralkyl amine salts such as N,N-dibenzylethylenediamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts or tetrabutylammonium salts; and basic amino acid salts such as arginine salts or lysine salts. Exemplary acid addition salts comprise acetate, adipate, alginate, ascorbate, benzoate, benzenesulfonate, hydrogensulfate, borate, bromide, butyrate, chloride, citrate, caphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylsulfonate, phosphate, hydrogenphosphate, dihydrogenphosphate, picrate, pivalate, propionate, salicylate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, such as tosylate, undecanoate and the like, as well as salts with amino acids.

Pharmaceutically acceptable solvates of compounds that can be used in the present invention may exist in the form of solvates with water, for example hydrates, or with organic solvents such as methanol, ethanol or acetonitrile, i.e. as a methanolate, ethanolate or acetonitrilate, respectively.

Pharmaceutically acceptable prodrugs of compounds that can be used in the present invention are derivatives which have chemically or metabolically cleavable groups and become, by solvolysis or under physiological conditions, the compounds of the invention which are pharmaceutically active in vivo. Prodrugs of compounds that can be used in the present invention may be formed in a conventional manner with a functional group of the compounds such as with an amino or hydroxy group, e.g. as carbamates esters or glycosides. The prodrug derivative form often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgaard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985).

The pharmaceutical compositions described herein can be administered to the subject/patient at a suitable dose. The dosage regiment will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 0.1 μg to 15000 mg units per day. If the regimen is a continuous infusion, it may also be in the range of 0.1 ng to 10 μg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Preferably, the subject/patient is a mammal; more preferably, the subject/patient is a human.

The uses as described herein, i.e. the use of the steroid sapogenin, androstane and triterpenoid sapogenin derived amines or ammonium derivatives for the treatment, amelioration and/or prevention of (an) infectious disease(s)/disorder(s) is of medical as well as pharmaceutical interest.

Accordingly, the present invention also relates to a method of treating a subject in need of such a medical treatment, said method comprising the administration of (a) compound of one of the formulae 1 , 2 or 3 as defined herein or a pharmaceutically acceptable salt, derivative, solvate or prodrug thereof in an amount sufficient to elucidate a pharmaceutical effect, i.e. to ameliorate or cure the medical conditions said subject is suffering from, in particular to counter-act the infectious diseases/disorders. In a preferred embodiment, the subject to be treated is a mammal. In a more preferred embodiment, the subject to be treated is a human. The present invention further relates to the use of a compound of one of the formulae 1 , 2 or 3 as defined herein or a pharmaceutically acceptable salt, derivative, solvate or prodrug thereof for the preparation of a medicament for the treatment, prevention and/or amelioration of an infectious disease/disorder.

Due to the medical importance of the steroid sapogenin, androstane and triterpenoid sapogenin derived amines or ammonium derivatives described in the context of the present invention, the invention also provides for a method for the preparation of a pharmaceutical composition which comprises the admixture of the herein defined compound with one or more pharmaceutically acceptable excipierits. Corresponding excipients are mentioned herein above and comprise, but are not limited to lipid derivatives used for liposome formation. As pointed out above, should the pharmaceutical composition of the invention be administered by injection or infusion, it is preferred that the pharmaceutical composition is an emulsion.

As mentioned above and as documented in the appended examples, preferred compounds for the medical intervention in tuberculosis and/or an infection with M. tuberculosis are compounds of formula 1a, 1c, 1d, 2a, 2b, 2c, 2e, 2f, 2h, 2i, 3a, 3b, 3c or 3d. These compounds are also useful in the medical intervention of (drug-) resistant strains of mycobacteria. Again, selective examples are provided in the appended experimental data and results. Similarly, preferred compounds for the medical intervention in influenza infection and/or viral hepatitis are compounds of formula 1a, 1b, 1c, 1d, 2b, 2c, 2d, 2e, 2f or 2g. Also for these selective compounds experimental data and non-limiting examples are provided below.

The following examples illustrate this invention.

All following chemical reactions were carried out in dry solvents under an inert gas atmosphere (argon or nitrogen). THF (tetrahydrofuran) and diethyl ether were dried using a solvent purification system (MBraun-SPS). Chemicals and solvents were used as received from commercial sources. TLC: Merck TLC aluminium sheets Silica gel 60 F₂₅₄. Flash chromatography: Merck silica gel (0.040 - 0.063 mm). Mass spectra: Bruker HP-Esquire LC. NMR spectra: Bruker DRX 500 or DRX 300; δ in ppm, J in Hz. Al! synthetic transformations described below for the synthesis of the exemplary compounds follow standard protocols used in synthetic chemistry and can easily be carried out by the skilled artisan. The structures of the obtained compounds were confirmed using mass spectrometry and NMR spectroscopy.

Examples 1 , 2, 3 and 4: Preparation of 3β-amino-5α,6-dihydrodiosgenin (1a), 3α- amino-5α,6-dihydrodiosgenin (1b), 3β-methylamino-5α,6-dihydrodiosgenin (1c), and 3β-aminomethyl-5α,6-dihydrodiosgenin (1d) Commercially available diosgenin was transformed to 5,6-dihydrodiosgenin by hydrogenation with hydrogen and palladium on charcoal. Subsequent oxidation of the alcohol by treatment with pyridinium dichromate (PDC) provided the corresponding 3- ketone which was used as substrate for synthesis of compounds 1a to 1d. Compounds 1a and 1b were obtained by reductive amination of the 3-ketone using hydroxylamine hydrochloride and hydrogen with Raney nickel. The resulting epimers 1a and 1b were chromatographically separated.

Compound 1c was obtained by reductive amination of the 3-ketone using a solution of methylamine in tetrahydrofuran and sodium trisacetoxy borohydride. Compound 1d was prepared via a two-step process using treatment with tosylmethylisocyanide followed by reduction of the resulting isocyanide with hydrogen and Raney nickel.

Examples 5 and 6: Preparation of 3β-amino-17β-hexyl-5α-androstane (2a) and 3β-amino-c/s-Δ¹⁷⁽²⁰⁾-17-hexylidene-5α-androstane (2c) Starting from commercial androsterone, Wittig reaction with n-hexyl triphenylphosphonium bromide provided 17-hexylidene-3α-androstanol, which was transformed to the corresponding 3β-azide by mesylation followed by treatment with sodium azide in dimethylsulfoxide. Then, reaction with lithium aluminium hydride afforded amine 2c. Subsequent hydrogenation using palladium on charcoal as catalyst provided saturated amine 2a.

Example 7: Preparation of 3α-aminomethyl-17β-hexyl-5α-androstane (2b) Oxidation of the above mentioned 17-hexylidene-3α-androstanol to the corresponding 3-keto derivative with pyridinium chlorochromate (PCC) followed by treatment with tosylmethylisocyanide (TosMIC), chromatographic separation of the obtained epimers and subsequent reactions with lithium aluminium hydride and hydrogen in the presence of palladium on charcoal provided compound 2b.

Example 8: Preparation of 3α-amino-17β-(oct-2-yl)-5α-androstane (2d)

Commercially available pregnolone was employed in a Wittig reaction with n-hexyl triphenylphosphonium bromide followed by hydrogenation of all double bonds using hydrogen and palladium on charcoal. Subsequent formation of the corresponding mesylate with mesyl chloride followed by preparation of the azide using sodium azide in dimethylsulfoxide and reduction of the azide with lithium aluminium hydride afforded compound 2d.

Example 9: Preparation of 3β-methylaminostigmastane (2e)

Commercially available stigmasterol was completely saturated by hydrogenation using hydrogen and palladium on charcoal. Subsequent oxidation of the alcohol to the 3- ketone with PCC followed by reductive amination with methylamine and sodium trisacetoxy borohydride provided compound 2e.

Example 10: Preparation of 3β-methylamino-17β-(6-oxadodec-2-yl)-5β- androstane (2f)

Commercial lithocholic acid was subsequently treated with te/t-butyldiphenylsilyl chloride and tetramethylguanidine followed by reduction of the carboxylic acid to the corresponding alcohol using lithium aluminium hydride. Alkylation with sodium hydride and n-hexyl iodide in tetrahydrofuran followed by desilylation with tetra- butylammonium fluoride provided 17β-(6-oxadodec-2-yl)-coprostan-3α-ol, which was oxidized to the corresponding 3-ketone using PCC. Subsequent reductive amination using methylamine and sodium trisacetoxy borohydride afforded compound 2f.

Example 11 : Preparation of 3β-aminoethyl-17β-(6-oxadodec-2-yl)-5α-androstane (2g) Commercial pregnolone was transformed to the corresponding terf-butyldimethylsilyl ether followed by Wittig reaction with commercial (3-benzyloxypropyl)triphenyl- phosphonium bromide. After concomitant hydrogenation of all double bonds and removal of the benzyl group with hydrogen and palladium(ll)-hydroxide (Pearlman's catalyst), O-alkylation with sodium hydride and n-hexyl iodide established the 17β-(6- oxadodec-2-yl) side chain. Desilylation with tetrabutylammonium fluoride and oxidation with PCC provided the corresponding 3-keto derivative as substrate for a Horner- Wadsworth-Emmons reaction to introduce the aminoethyl group at position 3. Thus, reaction of the ketone with commercial diethyl cyanomethylphosphonate followed by hydrogenation of the formed double bond with hydrogen and palladium on charcoal and lithium aluminium hydride reaction of the nitrile provided compound 2g.

Example 12 and 13: Preparation of 3β-(piperidin-1-yl)-5α-cholestane (2h) and 3β- (morpholin-4-yl)-5α-cholestane (2i) Compounds 2h and 2i were prepared from commercially available 3-cholestanone and piperidine (for 2h) or morpholine (for 2i) using a standard protocol for reductive amination.

Example 14 and 15: Preparation of 28-methoxy-3β-methylamino-urs-12-en (3a) and 28-methoxy-3β-methylamino-olean-12-en (3b)

Commercially available ursolic acid was transformed to the corresponding methyl ester by treatment with trimethylsilyldiazomethane followed by Dess-Martin oxidation to the 3-keto derivative. Reductive amination with methylamine and sodium cyanoborohydride followed by reduction of the methyl ester using lithium aluminium hydride provided the corresponding alcohol. Boc-protection of the methylamino group, O-methylation with sodium hydride and methyl iodide and subsequent Boc- deprotection by treatment with trifluoroacetic acid afforded compound 3a. Performing the same synthetic sequence starting from commercial oleanolic acid provided compound 3b.

Example 16: Preparation of 3β-amino-28-methoxylupen (3c)

Commercially available betulinic acid was transformed to the corresponding methyl ester by treatment with trimethylsilyldiazomethane followed by Dess-Martin oxidation to the 3-keto derivative. Reductive amination by subsequent treatment with hydroxylamine and lithium aluminium hydride followed by Raney-nickel-mediated reduction of the methyl ester to the corresponding carbinol provided the 3β-amino intermediate after chromatographic separation of the 3-epimers. Λ/,Λ/-Bis-Boc- protection followed by O-methylation with sodium hydride and methyl iodide and final Boc-deprotection by treatment with trifluoroacetic acid provided compound 3c.

Example 17: Preparation of 3β-methylaminolυpan (3d)

Commercially available lupeol was oxidized to the corresponding 3-keto derivative using PCC. Subsequent reductive amination using methylamine and sodium cyanoboro-hydride, chromatographic separation of the 3-epimers and hydrogenation of the propen-2-yl to the prop-2-yl side chain employing hydrogen and palladium on charcoal provided compound 3d.

Examples 18, 19 and 20: Antimicrobial Activity Assays

The aim of this assay is the identification of compounds having antituberculosis activity, as evaluated using the strain M. tuberculosis H₃₇Rv (ATCC® 27294; ATCC® stands for American Type Culture Collection®, and various M. tuberculosis strains are commercially available from that source) as disease model for tuberculosis. Potency in antimicrobial assays (MIC₉₀, i.e. the minimum concentration at which microbial growth or non-replicating persistence, respectively, is inhibited by 90%) was evaluated as described below and compared to toxicity independently in mammalian Vero cells and murine J774A.1 macrophages.

Microplate Alamar Blue Assay (MABA) used as aerobic replication assay

Determination of growth inhibition of Mycobacterium tuberculosis H₃₇RV by test compounds was carried out as described in the literature (Collins, Antimicrob. Agents Chemother. 41 (1997) 1004-1009; Franzblau, J. Clin. Microbiol. 36 (1998), 362-366; Pauli, Life Sci. 78 (2005), 485). The percent inhibition was defined as 1 - (test well fluorescence units/mean fluorescence units of triplicate wells containing only bacteria) x 100. The lowest drug concentration effecting an inhibition of >90% was considered the MIC₉₀. The values presented herein are means of three replicate experiments. Determination of growth inhibition of rifampin-resistant M. tuberculosis strain ATCC® 35838 and of isoniazid-resistant M. tuberculosis strain ATCC® 35822 was done as described above for the H₃₇Rv strain.

Low Oxygen Recovery Assay (LORA) used as non-replicating persistence assay

A physiological state of non-replicating persistence (NRP) is responsible for antimicrobial tolerance in many bacterial infections. In tuberculosis, it is essential to target this NRP subpopulation in order to shorten the 6-months regimen. A high- throughput, luminescence-based low oxygen-recovery assay (LORA) was used to screen test compounds against NRP or stationary-phase Mycobacterium tuberculosis as described in the literature (Cho, S. H.; Antimicrob. Agents Chemother. 2007, 57(4), 1380-1385). M. tuberculosis H₃₇Rv containing a plasmid with an acetamidase promoter driving a bacterial luciferase gene was adapted to low oxygen conditions by extended culture in a fermentor and MICgo was determined in microplate cultures maintained under anaerobic conditions for 10 days. Percent inhibition was determined as for MABA.

Determination of the cytotoxic activity

Evaluation of the cytotoxic activity of test compounds using Vero cells was performed as described earlier (Cantrell, J. Nat. Prod. 59 (1996), 1131-1136) using the CellTiter

96 aqueous non-radioactive cell proliferation assay (Promega Corp., Madison, Wl).

Evaluation of cytotoxicity using J774A.1 macrophage cells was done as described in the literature (Falzari, K.; Antimicrob. Agents Chemother. 2005, 49, 1447-1454).

No toxicity was observed within the concentration range in which the compounds showed activity.

Results

When using the above discussed MABA as a model for an infection by M. tuberculosis strain H₃₇Rv for evaluation of the inhibitory effect of various steroid sapogenin, androstane and triterpenoid sapogenin derivatives, it was found that the compounds of the present invention also inhibit bacterial growth. Accordingly, compounds 1a, 1c and 1d, 2a to 2c, 2e, 2f, 2h and 2i as well as 3a to 3d inhibited growth as well as non- replicating persistence of M. tuberculosis effectively, as can be seen from the results shown in Table 1. The combination of the steroid sapogeniπ, androstane or triterpenoid sapogenin scaffold with an amino function directly or indirectly attached to the steroidal A-ring are preferred structural motifs for the inhibition of mycobacterial growth, in particular Mycobacterium tuberculosis. However, frans-2-aminomethyl-1- cyclohexanol does not show any inhibitory effect. This demonstrates that an amino or aminoalcohol moiety attached to a cyclic hydrocarbon motif is not the sole reason for antituberculosis activity.

Table 1. Inhibition of replication of M. tuberculosis (strain H₃₇RV) by compounds provided herein.

Compound MIC₉₀ [μM] MABA MIC₉₀ [μM] LORA

1a 0.75 1.97

1c 1.24 3.01

1d 1.55 1.46

2a 0.37 1.4

2b 2.95 3.35

2c 4.99 1.48

2e 3.0 2.89

2f 5.27 2.82

2h 2.92 1.73

2i 2.9 4.66

3a 2.75 0.77

3b 2.77 2.8

3c 1 .37 1.44

3d 2.97 2.85

Compounds 1a, 1c and 1d, 2a to 2c, 2e, 2f, 2h, 2i and 3a to 3d are preferred compounds for the pharmaceutical intervention of mycobacterial diseases, in particular of tuberculosis. Ten of these compounds, i.e. compounds 1a, 1c, 1d, 2a, 2c, 2h and 3a to 3d, provided for particularly good results in the mycobacteria-assays as demonstrated by the remarkably low MIC₉₀ values. Hence, compounds 1a, 1c, 1d, 2a, 2c, 2h and 3a to 3d represent even more preferred compounds to be used in pharmaceutical compositions for the treatment of mycobacterial diseases, like tuberculosis.

When evaluating the potential of test compounds to target the persistent bacterial subpopulation, it was found that compounds 1a and 1d, 2a, 2c and 2h as well as 3a and 3c provided for particularly good results in the LORA model. Thus, compounds 1a and 1d, 2a, 2c and 2h as well as 3a and 3c represent particularly preferred compounds to be used in pharmaceutical compositions for treating persistent Mycobacterium tuberculosis phenotypes.

When using the above discussed MABA as a model for an infection by rifampin- resistant M. tuberculosis strain ATCC® 35838 for evaluation of the inhibitory effect of various steroid sapogenin and androstane derivatives, it was found that the compounds to be used in accordance with the present invention also inhibit the growth of rifampin-resistant bacteria. For example, compounds 1a, 1c and 1d as well as 2a, 2h and 2i provided particularly good results (Table 2).

Table 2. Inhibition of replication of rifampin-resistant strains of M. tuberculosis (strain ATCC® 35838) by compounds provided herein. (RMP = rifampin, used as control)

Compound MIC₉₀ [μM] MABA

1a 0.71

1c 1.29

1d 1 .34

2a 0.36

2h 1.43

2i 1.40

RMP >128

Furthermore, the MABA described herein was used to assess the potential of various steroid sapogenin and androstane derivatives for inhibiting the replication of isoniazid- resistant M. tuberculosis strain ATCC® 35822. In that context, compounds 1a, 1c and 1d as well as 2a, 2h and 2i provided particularly good results for stopping the growth of isoniazid-resistant bacteria (Table 3).

Table 3. Inhibition of replication of an isoniazid-resistant strain of M. tuberculosis (strain ATCC® 35822) by compounds provided herein. (INH = isoniazid, used as control)

Compound MIC₉₀ [μM] MABA

1a 0.74

1c 1.32

1d 1.20

2a 1.27

2h 1.48

2i 2.10

INH >128

The steroid sapogenin and androstane derivatives described herein were also found to be highly potent growth inhibitors of various further drug resistant strains . of M. tuberculosis. Evaluation was performed in the experimental setting of the MABA as described above using the pyrazinamide-resistant (r-pza) strain ATCC® 35828, the ethambutol-resistant (r-emb) strain ATCC® 35837, the kanamycin-resistant (r-km) strain ATCC® 35827, the streptomycin-resistant (r-sm) strain ATCC® 35834 and the moxifloxacin-resistant (r-mox) strain. The moxifloxacin-resistant strain of M. tuberculosis used herein is not commercially available, but was prepared in analogy to literature reports (Matrat, Antimicrob. Agents Chemother. 2006, 50(12), 4170-4173 and literature cited therein). In this evaluation, for example, compounds 1a, 1c and 1d as well as 2a, 2h and 2i provided particularly good results (Table 4).

Table 4. Inhibition of replication of a pyrazinamide-resistant strain of M. tuberculosis (strain ATCC® 35828), of an ethambutol-resistant strain (ATCC® 35837), of a kanamycin-resistant strain (ATCC® 35827), of a streptomycin-resistant strain (ATCC® 35834) and of a moxifloxacin-resistant strain by compounds provided herein, (r-pza = pyrazinamide-resistant, r-emb = ethambutol-resistant, r-km = kanamycin-resistant, r- sm = streptomycin-resistant, r-mox = moxifloxacin-resistant)

MIC₉₀ [μM] MABA Compound r-pza r-pza (pH 6) r-emb r-km r-sm r-mox

1a 0.76 0. 82 0 .67 0 .72 0 .74 0.73

1c 0.74 1. 28 1 .01 1 .11 1 .47 1.18

1d 1.50 1. 84 1 .48 1 .48 1 .37 1.44

2a 0.74 0. 68 0 .31 0 .37 0 .73 0.36

2h 1.49 1. 25 1 .47 1 .44 1 .48 1.43

2i 2.69 1. 27 1 .33 1 .43 1 .52 1.47

Hence, compounds 1a, 1c, 1d, 2a, 2h and 2i represent preferred compounds to be used in pharmaceutical compositions for the treatment of mycobacterial diseases, like tuberculosis, caused by bacterial strains which are resistant against standard drugs such as rifampin, isoniazid, pyrazinamide or ethambutol used for first-line treatment of tuberculosis or standard drugs such as kanamycin, streptomycin or moxifloxacin used for second-line treatment of tuberculosis. Two of these compounds, i.e. 1a and 2a, inhibited the growth of the majority of drug-resistant strains of M. tuberculosis very effectively when applied in nanomolar concentration and thus represents a most preferred embodiment of the present invention (Tables 2, 3 and 4).

Example 21 : Virus Reproduction and Infectivity Assay (Focus Reduction Assay) In the following, the above identified compounds were evaluated for their potential in inhibiting virus replication and/or lowering virus infectivity. As corresponding viral agent, influenza A virus was employed. Antiviral effects were evaluated by virus titration, equivalent to a traditional plaque reduction assay. The present assay was carried out on microtiter plates and developed as a cell ELISA. Cells (Madin-Darby canine kidney cells, MDCK) were preincubated for 5 min with serial dilutions of test compound and then infected with serially diluted virus. Potency in the virus reproduction and infectivity assay (characterized by IC₅₀ and IC₉₀ values, i.e. the concentrations at which 50% or 90% of viral reproduction is inhibited) was evaluated as described below and compared to toxicity in a cell model also used for determination of potency. Compounds were tested for inhibition of viral reproduction and infectivity in the concentration range in which toxicity was not observed. Quantification was done by calculating the concentration at which 50% or 90% of viral reproduction was inhibited. The corresponding values are denoted "IC₅₀" or "ICgo", respectively. In that context, "no inhibition" (i.e. zero) is defined as inhibition resulting from solvent vehicle alone, i.e. solvent without test compound. "Complete inhibition" or 100% inhibition is defined by the absence of foci.

The following materials are used for the Focus Reduction Assay: low retention tubes and glass dilution plate (from 70% ethanol, dried under hood); two thermomixers, 1.5 mL Eppendorf and 96-well blocks; 96-well glass plates or glass-coated plates (Zinsser or Lab Hut) to prepare test compounds dilutions; Costar 96-well plates (black) or glass-coated Lab Hut plates containing MDCK cells 1-2 days of age; virus aliquots with known titer; IM (infection medium) supplemented with bovine serum albumin (BSA) (commercial from Celliance, catalogue number 82-046-4); 2 mg/mL stock solution of trypsin, stored in aliquots at -80⁰C; 0.05% solution of glutaraldehyde (25% in water, Sigma catalogue number G 5882, kept at -20 "C) in PBS (phosphate- buffered saline, dilution 1 :500), which is freshly prepared in an amount of 250 mL per plate; antibodies for cell Elisa development; Pierce SuperSignal (West Dura) substrate. MDCK cells and human influenza A virus (strain A/PR8/34 (H1 N1)) were obtained from American Type Culture Collection® (Rockville, MD, USA).

The following working routine is used to perform the Focus Reduction Assay:

Step 1 : Preparation of test compound solution

The test compounds, which are stored at -20⁰C as 1 OmM, 5mM or 3mM stock solutions in DMSO, are thawed out at 37°C and sonicated, if necessary, in order to obtain a clear solution. The IM is preheated in low retention tubes at 37°C in a thermomixer, and test compound stock solutions are added in the following manner (example calculated for a 1OmM test compound stock solution): for a 100μM test compound solution: 1078 μL IM + 22 μL test compound stock solution; for a 50μM test compound solution: 1089 μl_ IM + 11 μl_ test compound stock solution; for a 25μM test compound solution: 1094 μl_ IM + 5.5 μl_ test compound stock solution; for a 10μM test compound solution: 1098 μl_ IM + 2.2 μl_ test compound stock solution. The resulting test compound solutions are shaken for 30 to 60 min and transferred into a 96-well glass plate, which was preheated in a thermomixer microplate block at 37⁰C. For two titration plates one glass plate is used, the left half receives the test media for plate 1 , the right half for plate 2. Each well receives 250 μl_ test compound solution or control medium (see template below). Finally, the test compound dilutions (100 μL each) are transferred using a multichannel pipette from the glass dilution plate to the MDCK cell culture plate.

Step 2: Infection

The edge columns of a 96-well plate with MDCK cell monolayers are treated with test compound solution and are mock-infected; they serve as background controls (well a) for densitometric evaluation (see below "Quantification of Assay Results"). Three further wells b, c and d are charged with virus dilutions, e.g. 2 x 10^~6 foci forming units, 1 x 10^"6 foci forming units or 5 x 10^~7 foci forming units, so that the 2 x 10^~6 foci forming units dilution will generate 50 to 100 foci. Suitable dilutions were determined by virus titration. All virus dilutions are prepared in IM. The virus is prediluted 1 :64 in IM (i.e. 630 μl_ IM + 10 μL virus solution). The 1 :64 virus dilution is diluted into cold IM 1 :2000 (= 1 ) followed by two further 2-fold dilutions. For one 96-well plate 3 ml_, 1.5 mL, and 1.5 ml_ of such solutions are prepared, for two plates 6 ml_, 3 ml_, and 3 ml_, and these solutions are kept at 4°C. A 20 μg/mL solution of trypsin is prepared and passed through a 0.2 μm sterile syringe filter, and then diluted to 4 μg/mL in IM. 10 min before infection, an equal volume, 3 mL or 6 mL of trypsin dilution (4 μg/mL) is added to the virus dilutions or to IM (for mock infection) and kept at 4⁰C until infection. The cell monolayers are washed with 2 x 200 μL IM. 100 μL test compounds or solvent controls in IM are added with a multichannel pipette, so that each column (2 to 1 1) contains a single test compound dilution. Columns 1 and 12 receive IM and serve as solvent-free controls if edge effects are minimal. 100 μL IM and 2 μg trypsin/mL are added to rows A and H (mock infection) with a multichannel pipette. To the other rows virus dilutions are added, whereby the pipette tips are changed every time. After each addition, the well content is pipetted up and down. The plate is incubated at 37°C for 16 h. Toxicity/cell morphology/precipitation in mock-infected wells is assessed by microscopy. The infection is terminated by fixing and immersing/filling the whole plate with 250 ml_ of a 0.05% glutaraldehyde solution in PBS for at least 20 min at room temperature.

Step 3: Detection

The glutaraldehyde solution is shaken off and the plate is rinsed with PBS₁ permeabilized with 50 μl_ of 0.1 % Triton X-100 in PBS for 30 min and rinsed again with PBS. The wells are blocked on a rocker for 1 h at room temperature or overnight at 4°C with 200 μl_ per well of a mixture of PBS + 10% heat-inactivated fetal calf serum (block), followed by 1 h treatment with 50 μl_ per well antibody to viral nucleoprotein (ATCC® HB65) diluted 1 :2000 in block. The antibody is removed by three times 5 min washes with TBS (tris-buffered saline) + 0.1% Tween. A 1 h incubation follows with 50 μl_ per well of a secondary anti-mouse antibody, conjugated to horseradish peroxidase, which is 1 :2000 diluted in block. The plate is put on a rocker for 1 h at room temperature, washed three times with TBS/0.1 % Tween and once with TBS.

Step 4: Imaging Following removal of the last wash, microtiter wells are filled with 50 μl_ substrate solution (SuperSignal West Dura, Pierce 34076) which is prepared just before use by mixing equal volumes of the two components. The plates are then placed in the

Fresnel lense rack of the CCD camera LAS 3000 (Fuji/Raytest) and exposed at high resolution for 10 min.

Step 5: Quantification of Assay Results

Images are evaluated densitometrically. Initially the background is subtracted (well a, see above). The densitometric intensity is calculated as follows:

I = [0.25 x i(well b) + 0.5 * i(well c) + i(well d)] / 1.75 wherein i is defined by 10000 times the intensity per area measured for the relevant well b, c or d. This calculation corresponds to the classical plaque assay. The factors represent the weighting of the individual values.

Results are expressed as % inhibition defined as follows: % inhibition = 100 - % control wherein % control is calculated by multiplying a given I for test compound by 100 and dividing by I for the appropriate solvent control. If I is a control or solvent control, its value is set as 100 %. This evaluation to quantify the assay results is made for a series of different test compound concentrations, e.g. 100 μM, 50 μM, 25 μM, 10 μM, 2.5 μM, 0.25 μM, 0.1 μM, whereby it is ensured that the highest concentration used in this series is nontoxic, as evaluated in a toxicity assay using MDCK Il cells prior to IC50/IC90 evaluation. Values for each concentration are the mean of three replicate experiments. The obtained dose-response results are processed using the software Sigmaplot 9.0 (Systat Software Inc.) based on a four parameter logistic function to provide IC50 and IC90 values.

Results Various amino-substituted steroid sapogenin and androstane derivatives showed strong inhibitory effects in the PR8 (H1 N1) virus replication assay (as a cellular model for influenza infection). In particular, compounds 1a to 1d and 2b to 2g yielded good results (Table 5). Without being bound by theory, it appears that the steroid sapogenin and androstane scaffold in combination with an amino function directly or indirectly attached to the steroidal A-ring represent structural motifs leading to inhibition of viral replication. Moreover, the presence of an amino function appears to be beneficial to obtain a particular high activity of the compounds. However, fraπs-2-aminomethyl-i- cyclohexanol does not show any inhibitory effect. This demonstrates that an amino or aminoalcohol moiety attached to a cyclic hydrocarbon motif is not the sole reason for anti-influenza activity.

Table 5. Virus replication results (IC₅₀ values) of compounds provided herein.

Compound IC₅₀ [μM]

1a 4.9

1b 2.8

1c 3.8

1d 5.4 2b 7.3

2c 16.4

2d 19.3

2e 3.4

2f 12.1

2g 3.1

As demonstrated by the data presented herein, compounds 1a to 1d and compounds 2b, 2e and 2g are preferred compounds for the pharmaceutical intervention in influenza infection. Five of these compounds, i.e. compounds 1a, 1b, 1c, 2e and 2g provided for particularly good results in the influenza virus replication assay. Furthermore, these compounds showed good results in solubility tests and provided for therapeutic indices making them particularly suitable for pharmaceutical compositions for the treatment of viral infections, in particular influenza infections.

Claims

Claims

1. A compound of one of the following formulae 1 , 2 or 3:

wherein

one of R¹, R², R³, if R³ is present, R⁴, if R⁴ is present, and R⁵ is a linear amine- containing group selected from X(CH₂)nNH₂, X(CH₂)_nNH(Ci-₄ alkyl), X(CH₂)_nN(Ci-₄ alkyl)₂ or X(CH₂)_nN(C₁^ alkyl)₃ ⁺; or a cyclic amine-containing group selected from piperidin-1-yl, 1-(Ci_-4 alkyl)⁺-piperidin-1-yl, morpholin-4-yl or 4-(Ci_-4 alkyl)⁺-morpholin-4-yl;

X is a direct bond or a phosphorus-containing group selected from OP(O)(OCi- ₄ alkyl)O, OP(O)(OlCH₂O, OP(O)(Od_-4 alkyl)CH₂O or, when the compound is of formula 1 or 3, OP(O)(CT)O;

when X is a direct bond, n is an integer from O to 2; when X is the phosphorus- containing group, n is an integer from 2 to 6;

when R⁵ is the amine-containing group, then

R¹, R², R³, if R³ is present, and R⁴, if R⁴ is present, are independently H or OH; and R⁶ is H or, when X is a direct bond and n is 1 or 2, R⁶ is H or OH;

== is a single bond or a double bond, wherein when == is a double bond, R⁴ is absent;

when R¹ is the amine-containing group, then

R² and R⁶ are independently H or OH;

R³, if R³ is present, R⁴ if R⁴ is present, and R⁵ are H; and

== is a single bond;

when R² is the amine-containing group, then

R³, if R³ is present, and R⁶ are independently H or OH,

R¹ , R⁴, if R⁴ is present, and R⁵ are H, and == is a single bond; when R³ is the amine-containing group, then

R² and R⁶ are independently H or OH,

R¹ , R⁴ and R⁵ are H, and

== is a single bond; when R⁴ is the amine-containing group, then

R² and R⁶ are independently H or OH₁ R¹, R³ and R⁵ are H, and

== is a single bond;

when one of R¹, R², R³, R⁴ and R⁵ is the linear amine-containing group, R⁷ is a

C_δ-n aliphatic group, provided that R⁷ is not 2-methyl-hept-6-yl;

when one of R¹ , R², R³, R⁴ and R⁵ is the cyclic amine-containing group, R⁷ is 2- methyl-hept-6-yl;

R⁸, R⁹ and R¹⁰ are independently Ci_-4 alkyl, C_1-4 alkenyl or H;

m is 1 or 2; and

R¹¹ is H or 0(C_1-4 alkyl);

or a pharmaceutically acceptable salt, derivative, solvate or prodrug thereof

for the treatment, prevention and/or amelioration of an infectious disease/disorder.

2. The compound according to claim 1 , wherein == is a single bond.

3. The compound according to claim 1 or 2, wherein R⁵ is the linear amine- containing group; X is a direct bond; R¹ , R², R³, if R³ is present, and R⁴, if R⁴ is present, are independently H or OH; and R⁶ is H.

4. The compound according to claim 1 or 2, wherein R⁵ is the linear amine- containing group; X is the phosphorus-containing group; R¹ , R², R³, if R³ is present, and R⁴, if R⁴ is present, are independently H or OH; and R⁶ is H.

5. The compound according to claim 3 or 4, wherein R⁷ is linear C_6-11 alkyl, linear C_6-I₁ alkylidene or linear C_6-n alkenyl, in which one or more hydrogens are optionally replaced by a methyl group (the total number of carbon atoms after replacement of hydrogen by methyl is to be 6 to 11); C_7--_I2 alkyl, in which one or two CH₂ is/are replaced by oxygen (the total number of carbon atoms after replacement of the CH₂ by O is to be 6 to 11 ); or a Cg_-H aliphatic group comprising a linear C₆ alkyl or a linear C₆ alkenyl main chain and three or four side chains independently selected from methyl or ethyl (the total number of carbon atoms is to be 9 to 11).

6. The compound according to claim 1 or 2, wherein R⁵ is the cyclic amine- containing group; X is a direct bond; R¹, R², R³, if R³ is present, and R⁴, if R⁴ is present, are independently H or OH; and R⁶ is H.

7. The compound according to claim 1 , wherein the compound of formula 1 is a compound of one of the following formulae 1a to 1d:

8. The compound according to claim 1 , wherein the compound of formula 2 is a compound of one of the following formulae 2a to 2i:

9. The compound according to claim 1, wherein the compound of formula 3 is a compound of one of the following formulae 3a to 3d:

10. The compound according to any of claims 1 to 9, wherein the infectious disease/disorder is caused by a virus or a bacterium.

11. The compound according to claim 10, wherein the bacterium is a mycobacterium, a chlamydia/chlamydophila, a borrelia, a bartonella, a legionella, a rickettsia or a treponema.

12. The compound according to claim 11 , wherein the bacterium is a mycobacterium selected from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium ulcerans, Mycobacterium kanasasii, Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium scrofulaceam, Mycobacterium microti, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium intracellulare, Mycobacterium simiae, Mycobacterium szulgai,

Mycobacterium xenopi, Mycobacterium fortuitum, Mycobacterium chelonei or Mycobacterium marinum.

13. The compound according to claim 11 , wherein the bacterium is a chlamydia/chlamydophila selected from Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila felis or Chlamydophila psittaci.

14. The compound according to claim 11 , wherein the bacterium is Bartonella quintana.

15. The compound according to claim 11 , wherein the bacterium is Legionella pneumophila.

16. The compound according to any of claims 10 to 15, wherein the infectious disease/disorder is selected from the group consisting of tuberculosis, leprosy, tropical skin ulcer, abscess, pulmonary disease, cutaneous and disseminated disease, borreliosis, relapsing fever, trench fever, endocarditis, cervicitis, conjunctivitis, diseases of the thyphos group, diseases of the spotted fever group, pinta and syphilis.

17. The compound according to claim 16, wherein the compound has the formula 1a, 1c, 1d, 2a, 2b, 2c, 2e, 2f, 2h, 2i, 3a, 3b, 3c or 3d and the infectious disease/disorder is tuberculosis and/or an infection with M. tuberculosis.

18. The compound according to claim 17, wherein the compound has the formula 1a, 1c, 1d, 2a, 2h or 2i and the bacterium causing the infectious disease/disorder is resistant to one or more of rifampin, isoniazid, pyrazinamide, ethambutol, kanamycin, streptomycin and moxifloxacin.

19. The compound according to claim 10, wherein the bacterium is selected from the group consisting of Gram-positive bacilli, Gram-positive cocci, Gram-negative bacilli and Gram-negative cocci.

20. The compound according claim 10, wherein the virus is selected from the group consisting of influenza virus, HIV, Hepatitis virus, Rotavirus, Respiratory syncytial virus, Herpetoviridae, Echovirus 1 , measles virus, Picomaviridae, Filoviridae, Papillomaviridae and Polyomaviridae.

21. The compound according to claim 20, wherein the influenza virus is selected from influenza A virus, influenza B virus or influenza C virus.

22. The compound according to claim 20, wherein the Hepatitis virus is selected from Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus or Hepatitis E. virus.

23. The compound according to claim 20, wherein the Herpetoviridae are selected from Herpes simplex virus or Epstein-Barr virus.

24. The compound according to claim 20, wherein the Picornaviridae are selected from Enterovirus or Coxsackie virus.

25. The compound according to claim 20, wherein the Filoviridae are selected from Ebola virus or Marburg virus.

26. The compound according to claim 20, wherein the Papillomaviridae are selected from human papilloma virus HPV-1 , HPV-2, HPV-3, HPV-4, HPV-5, HPV-6,

HPV-7, HPV-10, HPV-1 1 , HPV-13, HPV-16, HPV-18, HPV-31 , HPV-32, HPV-33, HPV-35, HPV-39, HPV-42, HPV-43, HPV-44, HPV-45, HPV-51 , HPV-52, HPV-55, HPV-56, HPV-58, HPV-59, HPV-68, HPV-73 or HPV-82.

27. The compound according to any of claims 20 to 26, wherein the compound has the formula 1a, 1 b, 1c, 1d, 2b, 2c, 2d, 2e, 2f or 2g and the infectious disease/disorder is an influenza infection or a viral hepatitis.