Cholesterylamines for the Treatment and Prevention of Infectious Diseases

The present invention relates to the use of cholesterylamines in the preparation of pharmaceutical compositions. These pharmaceutical compositions are to be used in the medical intervention of infectious diseases, in particular diseases caused by a virus or a bacterium.

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.

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 analysed) but they can be coalesced 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 (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, bacterial infections caused by Escherichia coli, Mycobacterium tuberculosis and bovis, Campylobacter jejuni, Vibrio cholerae, Clostridium difficile, Clostridium tetani, Streptococci species, Salmonella, and Shigella involve pathological processes related to rafts (Simons, J. Clin. Invest. 110 (2002), 597-603).

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 10 ⁵ - 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).

Membrane heterogeneity in bacteria has been described in the prior art, see Fishov, MoI. Microbiol. 32 (1999), 1166-1172; Mileykovskaya, J. Bacteriol. 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.

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 context of this invention, it was found that cholesterylamines are surprisingly superior to other cholesteryl derivatives, in particular cholesteryl sulfate, in the medical management of infectious diseases, in particular disorders or diseases caused by viral agents and bacteria. In the context of the present invention, the term "cholesterylamine" is intended to also cover compounds which are sometimes referred to as "aminocholesteryl derivatives" and compounds which are sometimes referred to as "aminocholestane and aminocholestene derivatives". Further, in the context of the present invention, the term "cholesterylamine" is intended to also cover derivatives of cholesterol, wherein the A ring has been replaced by a nitrogen-containing heterocycle as shown in formula 2 below.

Accordingly, in one embodiment, the present invention provides for the use of a compound of the following formula 1:

or a pharmaceutically acceptable salt, derivative, solvate or prodrug thereof for the preparation of a pharmaceutical composition for the treatment, prevention and/or amelioration of an infectious disease/disorder. Said disorders or diseases may be caused by a virus or bacterium.

The following numbering of the carbon atoms and denotation of the rings of the steroid scaffold 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.

In formula 1, one of R ¹ , R ² , R ³ , R ⁴ and R ⁵ is an amine-containing group selected from X(CH ₂ ) _n NH ₂ , X(CH ₂ J _n NH(C _1-4 alkyl), X(CH ₂ J _n N(C _1-4 alkyl) ₂ , X(CH ₂ J _n N(C _1-4 alkyl) ₃ ⁺ . Preferably, R ⁵ is the amine-containing group.

X is a direct bond or a phosphorous-containing group selected from OP(O)(OC _1-4 alkyl)O, OP(O)(O-)CH ₂ O or OP(O)(OC _1-4 alkyl)CH ₂ O. In one preferred embodiment, X

is a direct bond. In one embodiment, X is OP(O)(OC _1-4 alkyl)O. In another embodiment, X is OP(O)(O-)CH ₂ O. In yet another embodiment, X is OP(O)(OCi _-4 alkyl)CH ₂ O.

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-)CH ₂ O, the compound of formula 1 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)CH ₂ O is also within the scope of the present invention.

In the embodiment, wherein R ⁵ is the amine-containing group, == is a single bond or a double bond, preferably a single bond. When == is a double bond, R ⁴ is absent. R ¹ , R ² , R ³ and R ⁴ are independently H or OH. R ⁶ is H. When X is a direct bond and n is 1 or 2, R ⁶ can also be OH. Preferably, R ⁶ is H.

In the embodiment, wherein R ¹ is the amine-containing group, R ² and R ⁶ are independently H or OH. R ³ , R ⁴ and R ⁵ are H. == is a single bond.

In the embodiment, wherein R ² is the amine-containing group, R ³ and R ⁶ are independently H or OH. R ¹ , R ⁴ and R ⁵ are H. == is a single bond.

In the embodiment, wherein R ³ is the amine-containing group, R ² and R ⁶ are independently H or OH. R ¹ , R ⁴ and R ⁵ are H. == is a single bond.

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

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

The following compounds of formulae 1a to 11 are preferred examples of the compound of formula 1.

Compounds 1e and 11 represent cations, which can be used in combination with any pharmaceutically acceptable anion, such as e.g. halogenides, phosphate, sulfate and acetate.

Among compounds 1a to 11, compounds 1a, 1g, 1i, 1j and 1k are preferred.

In another embodiment, the present invention provides for the use of a compound of the following formula 2:

or a pharmaceutically acceptable salt, derivative, solvate or prodrug thereof for the preparation of a pharmaceutical composition for the treatment, prevention and/or amelioration of an infectious disease/disorder. Said disorders or diseases may be caused by a virus or bacterium.

In formula 2, Y is NH, N(Ci _-4 alkyl) or N(C _1-4 alkyl) ₂ ⁺ , preferably NH.

p is an integer from 0 to 2 and q is an integer of 1 or 2, provided that if p is 2, then q is 1. Preferably, p is 0 and q is 2.

== is a single bond or a double bond, preferably a single bond.

The following compounds of formulae 2a and 2b are preferred examples of the compound of formula 2.

2a 2b

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, wherein X is a direct bond and which have a primary amino function at position 3, can be prepared from commercial 5-cholesten- 3β-ol or 5α-cholestan-3β-ol using the synthetic sequence alcohol-mesylate-azide- amine. The stereochemistry of the final amine depends on the stereochemistry of the starting alcohol: a 3β-alcohol will provide a 3α-amine and vice versa. 5-cholesten-3α- ol or 5α-cholestan-3α-ol are available via literature protocols (Boonyarattanakalin, J. Am. Chem. Soc. 126 (2004), 16379-16386). Alternatively, such amines can be prepared via reductive amination strategies: e.g. substituted amines by using alkylamines or dialkylamines as reagents or primary amines by using hydroxylamine as reagent followed by treatment with Raney-Nickel. Amines can be obtained either as free bases or as the corresponding hydrochlorides by precipitation with HCI as solution in diethyl ether. The corresponding hydroxy-decorated derivatives can be prepared from 4-cholesten-3-one or 1 ,4-cholestadien-3-one, which are both commercially available, using epoxidations followed by ring-opening with hydride or by osmium-mediated bishydroxylation strategies. Aminomethyl derivatives are available by treatment of a ketone with tosylmethylisocyanide (TosMIC) as described in Oldenziel, J. Org. Chem. 42 (1977), 3114-3118. Aminoethyl-decorated cholesteryl derivatives can be prepared starting from the corresponding ketone using a Horner- Wadsworth-Emmons (HWE) approach with commercial cyanomethylphosphonate as reagent according descriptions documented in various literature protocols (Drefahl, Chem. Ber. 97 (1964), 2011-2013; Kargiozov, Synth. Commun. 34 (2004), 871-888; Shen, Bioorg. Med. Chem. Lett. 15 (2005), 4564-4569). Subsequent hydrogenation of the formed double bond followed by hydride-mediated reduction of the nitrile provides the aminoethyl-substituted cholesteryl derivatives.

If the amine-containing group is attached to the C2-position of the cholestane scaffold, the corresponding ketone can be used as synthetic precursor, which can be produced 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-cholestanone. The same principle can be used for 4- cholestanone (available as described 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-cholestanone (available as described 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 cholesteryl scaffold, respectively.

Compounds of the general formula 1, 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 ally) amines. Alternatively, epoxidation and subsequent opening or bishydroxylation of the double bond would result in the described hydroxy-decorated compounds. Said allyl amines can be prepared from 2α,3α-epoxy-5α-cholestane 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-cholestane is, for example, available via meta- chloroperbenzoic acid mediated epoxidation of 5α-cholestan-2-ene which itself can be prepared as described in the literature (Cruz Silva, Tetrahedron 61 (2005), 3065- 3073).

Compounds of the general formula 1 , wherein X is a O-alkylphosphate can be prepared from the corresponding cholesterols or dihydrocholesterols (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, wherein X is a phosphonate or O- alkylphosphonate can be prepared from the corresponding cholesterols or

dihydrocholesterols (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 Ci _-4 alkyl halides in standard protocols known to the skilled person.

Compounds of the general formula 2 can be prepared as outlined in the literature. For instance, Shoppee et al. describe synthetic methods to prepare 3- and 4-aza-A- cholesteryl derivatives using Beckmann rearrangements for ring expansion with concomitant incorporation of nitrogen (Shoppee, CW. ; J. Chem. Soc. 1962, 105). Alternatively, cholesteryl derivatives displaying an expanded, nitrogen-containing A- ring can be generated using Schmidt-type rearrangements (Doorenbos, NJ. ; J. Org. Chem. 1961, 26, 2548-2549). Synthetic methods to prepare 4-aza-cholesteryl derivatives without expanding the steroidal A-ring utilize a twofold strategy of oxidative A-ring opening (Boύcza-Tomaszewski, Z.; Tetrahedron Lett. 1986, 27, 3767-3770) to obtain the corresponding seco-cholesteryl derivatives and subsequent ring closing in the presence of a nitrogen source (Doorenbos, N. J.; J. Org. Chem. 1961 , 26, 2546- 2548) followed by reduction of the resulting lactame derivatives to the corresponding amines (Shoppee, C.W.; J. Chem. Soc. 1962, 2275-2285 and Kim, J.C.; Bull. Korean Chem. Soc. 1993, 14(2), 176). A-Nor-azasteroids can be obtained, for example, by a Favorski-type ring contraction of the corresponding 4-aza-steroids (Edwards, O. E.; Can. J. Chem. 1997, 75(6), 857-872) or by an amination-cyclocondensation sequence using seco-cholesteryl derivatives described above as substrates (Chupina, L.N.; Khimiko-Farmatsevticheskii Zhurnal 1982, 16(5), 563-567 and Rulin, V.A.; Zhumal Organicheskoi Khimii 1975, 11(8), 1763-1766).

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.

Viral diseases to be treated in accordance with the present invention include diseases induced by a virus selected from the group consisting of influenza, HIV, Hepatitis virus

(A, B, C, D), Rotavirus, Respiratory syncytial virus, Herpetoviridae (e.g. Herpes simplex virus, Epstein-Barr virus), Echovirus 1 , measles virus, Picornaviridae (e.g. Enterovirus, Coxsackievirus), Filoviridae (e.g. Ebolavirus, Marburgvirus), Papillomaviridae and Polyomaviridae. In a preferred embodiment the virus is influenza virus.

Bacterial infections to be treated in accordance with the present invention 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., Corynebacterium diphtheriae and Propionibacterium 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, Burkhoideria cepacia, Enterobacter spp., Pseudomonas aeruginosa, Campylobacter jejuni and Legionella pneumophila. Gram-negative cocci are, for example, Neisseria gonorrhoeae and Moraxella catarrhalis

Bacterial infections to be treated in accordance with the present invention also include infections induced by clinical bacteria for which the Gram stain is not applicable. These bacteria are, for example, Borrelia spp., Bartonella Quintana, Chlamydia pneumoniae, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium ulcerans, Mycobacterium kanasasii, Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium scrofulaceam, Rickettsia spp. and Treponema spp.

In context of this invention, a "mycobacteria-induced disease" may comprise an 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 and M. marinum. 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 with M. tuberculosis. As mentioned above in context of "infections to be treated and/or prevented", the present invention is not limited to the treatment/prevention of a disorder caused by the pathogen agent (e.g. bacterium) per se, but comprises also the medical amelioration of a disorder caused by products produced by said 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.

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 is considered a "biochemical/biophysical pathological process" in accordance with this invention.

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 cholesterylamines are

employed. Without being bound by theory, the specific cholesterylamines 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 cholesterylamines 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".

Besides the disease-inhibitory activity of the cholesterylamines to be employed in context of this invention, said 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. Yet, these (cyto)-toxicity assays are, as known by the skilled artisan, not limited to these assays.

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 11 as well as 2a and 2b 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, influenza infections and tuberculosis.

In the following more detailed information on diseases and disorders are given. These diseases and disorders may be prevented, ameliorated or treated by using the cholesterylamines provided herein. In particular, the experimental data provided herein document that 1a, 1 b, 1f, 1g, 1h, 1i, 1j, 1k, 2a and 2b are particularly preferred compounds in distinct medical interventions or preventions.

The cholesterylamines 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).

Particularly preferred compounds in this regard are compounds 1a, 1b, 1g, 1h, 1i, 1j, 1k, 2a and 2b, and compounds 1a, 1i, 1k and 2b represent an even more preferred embodiment within the context of the present invention. Corresponding experimental evidence is provided in the appended examples.

In viral infection, raft clustering is involved in the virus assembly process. The compounds 1a to 11 as well as 2a and 2b have an effect in a virus replication assay. Without being bound by theory, the structural feature underlying this effect is thought to be represented by the combination of an amine-substitution inside the steroidal A ring and the presence of cholesteryl-type B, C, D ring including the cholesteryl-type side chain. Using a 3-aminomethy! or 3-aminoethy! substitution in the A-ring results in increased potency of compound 1i or 1k, thus indicating the 3-aminomethyl or 3- aminoethyl substitution pattern as an even more preferred embodiment. 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 provided in the experimental part, these compounds may be useful for pharmaceutical intervention. In contrast trans-2- aminomethyl-1-cyclohexanol, cholesteryl sulfate and cholesterol do not show significant activity in a model assay for influenza infection.

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, coxsackievirus, hepatitis C, rotavirus and respiratory syncytial virus. Accordingly, particularly preferred compounds as well as preferred compounds provided herein in

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.

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 such as cholera, aerolysin, anthrax and helicobacter toxin form oligomeric structures in the raft, crucial to their function. The raft is targeted by binding to raft lipids such as ganglioside GM 1 for cholera. Without being bound by theory, in 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 ready in a position to adopt the treatment regime with the herein defined choiesteryiamines in the treatment of a bacterial infection per se and/or in the amelioration of disorders and diseases caused by the corresponding toxins.

In bacterial infection 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 bacterial receptor in rafts or blockage of internalization would prevent infection. In the case of caveolae, which depend on a cholesterol binding protein, caveolin, exclusion of cholesterol from the raft with steroid derivatives may prevent uptake of the pathogen.

Tuberculosis is an example of a bacterial infectious disease involving rafts. First, 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 isopentenyl pyrophosphate (IPP), a mycobacterial antigen that specifically stimulates Vgamma9Vdelta2 T cells. Accordingly, the present invention also provides for the use of the compounds disclosed herein in the treatment and/or amelioration of an Mycobacterium infection, preferably of a Mycobacterium tuberculosis infection.

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 or 2, in particular one of the formulae 1a to 11 as well as 2a and 2b 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 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 pyridin salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammoniurn 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 or esters. 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 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 depends 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.

The uses as described herein, i.e. the use of cholesterylamines for the treatment, amelioration and/or prevention of (an) infectious disease(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) cholesterylamine(s) as defined herein 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. In a preferred embodiment, the subject to be treated is a human.

Due to the medical importance of the cholesterylamines described in 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 excipients. 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.

For example, an emulsion can be made of 67 w-% lipoid S 100 (Lipoid catalog number 790), ethanol (25 w-%) and glycerol (8.33 w-%) by stirring for 60 min at 37°C or until homogeneity. Test compound, e.g. 14.1 mg in the case of compound 1i, is dissolved in 185.9 mg formulation by mixing for 90 min at 37°C in Agilent glass tubes (volume 1.7 mL) in a thermomixer (Eppendorf). This lipid emulsion represents a pro-liposomal concentrate of the test compound. Thereafter 200 mg lipid emulsion is diluted in 1.04 g of 0.536% NaCI solution, giving an isotonic solution, and vortexed for 20 s. This liposome suspension (11.35 mg/mL test compound) is further diluted in 0.9% NaCI solution to achieve desired concentrations.

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 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 F _2S4 - 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\n ppm, J in Hz.

Examples 1, 2 and 3: Preparation of 3β-Amino-5α-cholestane 1a, 3α-Amino-5α- cholestane 1b and 3β-Aminocholest-5-ene 1f

Synthesis of compound 1a started from dihydroepicholesterol, which was prepared from commercial 3-cholestanone using the L-selectride protocol described by Boonyarattanakalin, J. Am. Chem. Soc. 126 (2004), 16379-16386.

Dihydroepicholesterol was then transformed to the corresponding mesylate followed by substitution with azide (Davis, Tetrahedron Lett. 38 (1997), 4305-4308) and subsequent reduction of the azide to amine by treatment with lithium aluminium hydride. Synthesis of compound 1b started from commercial dihydrocholesterol, which was transformed to the corresponding mesylate followed by substitution with azide (Davis, Tetrahedron Lett. 38 (1997), 4305-4308) and subsequent reduction of the azide to amine by treatment with lithium aluminium hydride.

Synthesis of compound 1f started from epicholesterol, which was prepared in two steps from commercial cholesterol according to a literature procedure (Boonyarattanakalin, J. Am. Chem. Soc. 126 (2004), 16379-16386). Epicholesterol was transformed to the corresponding mesylate followed by substitution with azide (Davis, Tetrahedron Lett. 38 (1997), 4305-4308) and subsequent reduction of the azide to amine by treatment with lithium aluminium hydride.

In the following typical procedures are described, which were used in the syntheses of compounds 1a, 1b and 1f.

Typical procedure for preparing a mesylate from an alcohol: Cholest-5-en-3β-ol methane-sulfonate

Epicholesterol (1.32 g, 3.42 mmol) and 4-dimethylamino pyridine (42 mg, 0.34 mmol) were dissolved in dry dichloromethane (25 mL) under an argon atmosphere. Diisopropylethylamine (486 mg, 3.76 mmol) and methanesulfonyl chloride (431 mg, 3.76 mmol) were added with stirring. After 100 min TLC analysis in petrol ether / dichloromethane (1 :1) + 1 % ethanol showed complete conversion. The mixture was partitioned between dichloromethane and water (each 100 mL). The organic layer was washed successively with dilute NaHSO ₄ , water, saturated NaHCO ₃ , water and brine, dried over Na ₂ SO ₄ , filtered, evaporated to dryness and dried in vacuo overnight. Yield: 1.54 g (96%) of cholest-5-en-3β-ol methane-sulfonate as an off-white solid. This material was used in the next step without further purification. Further purification can be accomplished by flash chromatography using petrol ether / ethyl acetate (4:1). MS (ESI): 482.3 (M+NH ₄ ) ⁺ .

¹ H-NMR (300 MHz, CDCI ₃ ): δ= 0.67 (s, 3 H), 0.81 - 2.07 (m, 38 H), 2.36 (d, J = 15.6, 1 H) ₁ 2.56 (Cl ₁ J = 15.6, 1 H), 2.98 (s, 3 H), 4.55 (m, 1 H), 5.35 (d, J = 5.1, 1 H).

Typical procedure for preparing an azide from a mesylate: 3β-Azido-5α- cholestane

Cholestan-3β-ol methane-sulfonate (17.2 g, 37.0 mmol) and powderized anhydrous sodium azide (12.03 g, 185 mmol) were placed under an atmosphere of argon and suspended in dry N.N'-dimethylpropylene urea (240 ml_). The mixture was heated to 46°C with efficient stirring for 24 h. The crude product was partitioned between water (1 L) and diethyl ether/petrol ether mixture 1 :1 (500 ml_). The aqueous layer was washed with the same mixture again. The combined organic layers were washed with water (4 x 1 L), dried over Na ₂ SO ₄ , filtered, evaporated to dryness and dried in vacuo. Flash chromatography on silica (stepwise elution by petroleum ether containing 0, 5, 10, 15 and 20% dichloromethane) yielded 14.9 g (97%) of 3β-azido-5α-cholestane as a colourless solid.

MS (ESI): 414.5 (M+H) ⁺ .

¹ H-NMR (500 MHz, CDCI ₃ ): S= 0.64 (s + m, 4 H), 0.79 (s, 3 H), 0.81 - 1.61 (m, 34 H),

1.62 - 1.70 (m, 1 H), 1.72 - 1.86 (m, 3 H), 1.96 (d m, J = 12.6, 1 H), 3.24 (m, 1 H).

Typical procedure for preparing an amine from an azide: 3β-Amino-5α- cholestane 1a

In an inert atmosphere of argon, a suspension of lithium aluminium hydride (5.0 g, 131.6 mmol) in anhydrous diethyl ether (250 mL) was heated to reflux with stirring. A solution of 3β-azido-5α-cholestane (14.9 g, 36.04 mmol) in anhydrous diethyl ether (110 mL) was added dropwise at such a rate that the reaction was kept at gentle reflux. Upon completed addition, the mixture was refluxed for 1 h, after which TLC analysis in petrol ether / dichloromethane (85:15) showed complete consumption of the starting material. The mixture was cooled in an ice bath and 40 mL of methanol were added dropwise, followed by 200 mL of 2 M NaOH. The mixture was diluted with water (around 500 mL), the organic layer was separated and the turbid aqueous phase was extracted with dichloromethane and diethyl ether several times. The combined organic layers were washed with water and brine, dried over Na ₂ SO ₄ ,

filtered, evaporated to dryness and dried in vacuo overnight. Yield: 12.1 g (87%) of 3β- Amino-5α-cholestane 1a as a white solid. MS (ESI): 388.3 (M+H) ⁺ .

¹ H-NMR (300 MHz, CDCI ₃ ): S= 0.63 (s + m, 4 H) ₁ 0.76 (s, 3 H), 0.70 - 1.87 (m, 40 H), 1.94 (d m, J = 12.2, 1 H), 2.62 (m, 1 H).

Preparation of 3α-Amino-5α-cholestane 1b

Compound 1b was prepared as described above and outlined in the typical procedures. MS (ESI): 388.3 (M+H) ⁺ .

¹ H-NMR (300 MHz, CDCI ₃ ): δ= 0.64 (s, 3H), 0.66 - 1.90 (m, 44 H), 1.95 ((J m ₁ J = 12.1, 1 H), 3.16 (m, 1 H).

Preparation of 3β-Aminocholest-5-ene 1f Compound 1f was prepared as described above and outlined in the typical procedures.

MS (ESI): 386.4 (M+H) ⁺ .

¹ H-NMR (300 MHz, CDCI ₃ ): δ= 0.66 (s, 3 H), 0.77 - 2.21 (m, 42 H), 2.59 (m, 1 H),

5.31 (Cl ₁ J = 5.2, 1 H).

Examples 4 and 5: Preparation of 3β-Methylamino-5α-cholestane 1c and 3β-

Dimethylamino-5α-cholestane 1d

Compounds 1c and 1d were prepared in one step from commercial 5α-cholestan-3- one via the reductive amination strategy using methylamine or dimethylamine as their 2M solutions in THF according the following general protocol. Conveniently, the amines were obtained in pure form as their corresponding hydrochlorides.

A solution of 5α-cholestan-3-one (1.0 eq) and amine (1.0 eq, used as 2M solution in

THF as commercially available) in dry 1 ,2-dichloroethane was treated with sodium triacetoxyborohydride (1.4 eq) and glacial acetic acid (1.0 eq). The mixture was stirred at room temperature under an argon atmosphere for 18h until the reactants were consumed as determined by TLC analysis. The reaction mixture was quenched by adding water, and the product was extracted with diethyl ether. The organic layer was

washed with brine and dried over MgSO ₄ . The solvent was evaporated to give the crude product (i.e. free amine). The hydrochloride salt was prepared by addition of a 1 M solution of HCI in diethyl ether to the amine solution in diethyl ether and subsequent washing with diethyl ether. This protocol provided white crystals of hydrochloride salts of compounds 1c and 1d in yields of 60 to 70%, respectively.

3β-Methylamino-5α-cholestane 1c: MS (ESI): m/z = 402.5 (M+)

¹ H-NMR (300 MHz, CDCI ₃ ): δ= 0.62 (s, 3 H) ₁ 0.79 (s, 3 H), 0.84 - 0.88 (m, 12 H), 0.96 - 1.32 (m, 18 H) ₁ 1.45 - 1.80 (m, 12 H), 1.90 (br d, J = 10.4 Hz, 1 H), 1.97 (br d, J = 15.5 Hz, 1 H), 2.64 (br s, 3 H), 3.20 (br s, 1 H), 9.30 (br s, 1 H).

3β-Dimethylamino-5α-cholestane 1 d:

MS (ESI): m/z = 416.4 (M+) ¹ H-NMR (300 MHz, CDCI ₃ ): δ= 0.61 (d, J = 5.5 Hz, 3 H), 0.81 (m, 10 H), 0.97 - 1.31 (m, 18 H), 1.42 - 2.02 (m, 15 H), 2.73 (br s, 3 H), 2.82 (br s, 3 H), 3.03 (br m, 1 H), 11.33 (br s, 1 H).

Example 6: Preparation of 3β-Trimethylammonium-5α-cholestane chloride 1e A suspension of 3β-Methylamino-5α-cholestane 1c (1.0 eq) and sodium hydride (8.0 eq as 60% suspension in mineral oil) in dry dichloromethane was heated to reflux for 30 min. Neat methyl iodide (20 eq) was added and the reaction mixture was heated at 45°C for further two days. The reaction mixture was cooled and diluted with dichloromethane (50 ml_), washed repeatedly with brine and extracted with dichloromethane (3 x 100 ml_). The combined organic layers were dried over sodium sulfate and concentrated in vacuo. Washing of the crude product with diethyl ether provided 3β-trimethyl-ammonium-5α-cholestane 1e as the corresponding chloride as white crystals. MS (ESI): m/z = 430.5 (M+) ¹ H-NMR (300 MHz, CDCI ₃ ): δ= 0.67 (m, 3 H), 0.88 (m, 12 H), 1.13 - 2.01 (m, 31 H), 3.17 (br s, 6 H) ₁ 3.36 (br s, 1 H), 3.72 (br s, 1 H), 4.48 (br s, 2 H).

Example 7: Preparation of 2β-Amino-3α-hydroxy-5α-cholestane 1g

Compound 1g was prepared from 2α,3α-epoxycholestane, which is available by a known procedure involving dehydration of dihydrocholesterol to 2-cholestene followed by epoxidation (Cruz Silva, Tetrahedron 61 (2005), 3065-3073). As outlined in the following, epoxide opening with benzylamine and debenzylation with hydrogen and palladium on charcoal provided aminoalcohol 1g.

Preparation of 2/?-(tø-benzylamino)-3fl«-hydroxy-5ocholestane

In an argon atmosphere, 2α,3α-epoxycholestane (217 mg, 0.56 mmol) was suspended in benzylamine (3 mL). The mixture was stirred at 120 ⁰ C for three days. Then, TLC in petrol ether / ethyl acetate (10:1) indicated consumption of about 90% of the starting material. Most of the benzyl amine was removed in vacuo. The residue was taken up in THF / isopropanol (2:1), acidified with trifluoroacetic acid (TFA) and submitted to purification by preparative HPLC. (Column: Vydac 208TP1050 (RP-C8), detector: UV (215 nm), flow rate: 50 mUmin, eluent A: water/acetonitrile (85:15) + 0.1% TFA, eluent B: THF + 0.1 % TFA, gradient from 38% to 59% B over 21 min, RT = 19.3 min.) Fractions containing the product were collected. Most of the THF was removed under reduced pressure. The remaining solution was basified with saturated aqueous Na ₂ CO ₃ and extracted with dichloromethane. The organic layers were dried with Na ₂ SO ₄ , filtered, evaporated to dryness and dried in vacuo. The product was further purified by chromatography with petrol ether / ethyl acetate (4:1) + 2% ethanol on Alox-N (activity 4). Yield: 27 mg (10%) of 2/?-(/V-benzylamino)-3«-hydroxy-5a- cholestane as colourless oil. MS (ESI): 494.4 (M+H) ⁺ . ¹ H-NMR (500 MHz, CDCI ₃ ): S= 0.63 (s, 1 H), 0.65 - 0.75 (m, 1 H), 0.80 - 1.40 (m, 32 H) 1.45 - 1.70 (m, 8 H), 1.71 - 1.91 (m, 3 H), 1.95 (d m, J = 12.6, 1 H), 2.84 (d m, J = 4.6, 1 H), 3.75 (d, J = 13.2, 1 H), 3.83 (d m, J = 4.2, 1 H), 3.87 (d, J = 13.4, 1 H), 7.25 (m, 1 H), 7.32 (m, 4 H).

Preparation of 2β-Amino-3α-hydroxy-5α-cholestane 1g as its hydrochloride

2/?-(λ/-Benzylamino)-3αr-hydroxy-5α'-cholestane (25 mg, 50.6 μmol) was dissolved in dichloromethane (3 mL) and hydrogenated at atmospheric pressure overnight in the presence of 20 mg of palladium (10%) on carbon. Mass spectroscopic analysis of the

crude mixture showed no conversion. Another portion of 50 mg of catalyst and 3 ml_ of dichloromethane / methanol (4:1) were added and hydrogenation was continued for another 24h. Then, TLC analysis using petrol ether / ethyl acetate (4:1 ) + 10% dichloromethane + 3% methanol indicated complete conversion. The catalyst was removed by filtration through a short pad of celite 577. Washing with dichloromethane / methanol (70:30) (300 ml_) was employed to ensure full recovery of the material. Removal of the solvents under reduced pressure and drying in vacuo yielded 18.4 mg (90%) of a colourless solid, which was taken up in diethyl ether (4.5 ml.) and a few drops of isopropanol. Addition of a solution of 1M HCI in diethyl ether (60 μl_), dilution with diethyl ether (4 ml_) and cooling led to precipitation of the hydrochloride, which was collected by membrane filtration, washed with diethyl ether and dried in vacuo. Yield: 17 mg (77%) of 2β-amino-3α-hydroxy-5α-cholestane hydrochloride as a colourless solid. MS (ESI): 404.4 (M+H) ⁺ . ¹ H-NMR (500 MHz, DMSO-d ₆ ): S= 0.62 (s, 3 H), 0.62 - 0.69 (m, 1 H), 0.76 - 1.80 (m, 39 H), 1.92 (d m, J = 12.3, 1 H), 3.14 (m, 1 H, H-2), 3.72 (m, 1 H, H-3), 5.17 (d, J = 3.7, 1 H, OH), 8.04 (br s, 3 H ₁ NH ₃ ⁺ ).

¹ H-NMR (500 MHz, CDCI ₃ / CD ₃ OD 8:2): S= 0.53 (s, 3 H), 0.64 (m, 1 H), 0.69 - 1.60 (m, 40 H), 1.65 - 1.78 (m, 3 H), 1.85 (d m, J = 12.6, 1 H), 3.15 (d m, J = 6.1 , 1 H), 3.84 (br s, OH, NH ₂ , H-3).

Example 8 and 9: Preparation of 3β-Aminomethyl-5α-cholestane 1h and 3α- Aminomethyl-5α-cholestane 1i

Compounds 1h and 1i were prepared by converting commercial 5oc-cholestane-3-one into a mixture of epimeric nitriles, i.e. 3α- and 3β-cyanocholestane, by treatment with commercial tosylmethylisocyanide (TosMIC) according to literature precedence (Oldenziel, J. Org. Chem. 42 (1977), 3114-3118). Subsequent chromatographic separation of the epimers and lithium aluminium hydride reduction of the isolated nitriles afforded then compounds 1h and 1i. The separation of 3α- and 3β-cyanocholestane was accomplished by flash chromatography on silica using either petrol ether / ethyl acetate (10:1) or petrol ether / dichloromethane (2:1) as eluent, or by preparative HPLC on a reverse phase C8

column using water as eluent A, THF as eluent B and a gradient of 40 to 70% B over 40 min.

Preparation of 3α-Aminomethyl-5α-cholestane 1i In an inert atmosphere, lithium aluminium hydride (1.68 g, 44.3 mmol) was suspended in dry THF (200 mL) and the mixture was heated to reflux with stirring. A solution of 3«-cyano-5cι?-cholestane (7.28 g, 18.3 mmol) in dry THF (200 mL) was added dropwise over a period of 15 min. The mixture was stirred at reflux for another 90 min, after which TLC analysis in petrol ether / dichloromethane (1 :1) indicated complete consumption of the starting material. The reaction was quenched by dropwise addition of water. The mixture was partitioned between water and diethyl ether and the aqueous layer was extracted with diethyl ether and ethyl acetate several times. The combined organic layers were dried over Na ₂ SO-I, filtered and evaporated to dryness. The residue was taken up in dichloromethane / methanol (9:1) and filtered through a short plug of Celite 577 to remove inorganic material. Removal of the solvents and drying in vacuo yielded 6.60 g (90%) of compound 1i as colourless solid. MS (ESI): 402.5 (M+H) ⁺ .

¹ H-NMR (500 MHz ₁ CDCI ₃ / CD ₃ OD 8:2): S= 0.67 (s, 3 H), 0.70 - 0.73 (m, 1 H), 0.80 - 1.72 (m, 41 H), 1.75 - 1.87 (m, 2 H), 1.99 {d m, J = 12.5, 1 H), 2.08 (m, 1 H), 3.00 (m, 2 H).

Preparation of 3β-Aminomethyl-5α-cholestane 1h as its hydrochloride

Compound 1h was prepared using the same protocol as described above for compound 1i. The crude product was dissolved in diethyl ether (10 mL) and the corresponding hydrochloride of 1 h was precipitated by addition of 1 M HCI in diethyl ether (35 mL). The formed solid was filtered off, washed with diethyl ether and dried at high vacuum to provide 1h as its hydrochloride as colourless solid (0.36 g, 68%).

MS (ESI): 402.4 (M+H) ⁺ .

¹ H-NMR (500 MHz, CDCI ₃ / CD ₃ OD 8:2): S= 0.67 (s, 3 H), 0.70 (m, 1 H), 0.79 (s, 3 H), 0.86 - 1.38 (m, 31 H), 1.49 - 1.58 (m, 3 H), 1.67 (m, 3 H), 1.77 - 1.85 (m, 2 H), 1.99 (d m, J = 12.6, 1 H), 2.77 (d, J = 5.2, 1 H). NH ₃ ⁺ not clearly visible.

Example 10: Preparation of 3α-Aminomethyl-3β-hydroxy-5α-cholestane 1j as its hydrochloride

Compound 1j was prepared as its hydrochloride salt from commercial 5α-cholestane- 3-one via the corresponding O-trimethylsilyl-protected cyanhydrin derivative (Evans, J. Org. Chem. 39 (1974), 914-917) and subsequent treatment with lithium aluminium hydride followed by precipitation with HCI. The cyanhydrin intermediate was not purified, but subjected as crude material to the hydride reduction. Preparation of compound 1j by reduction of cyanhydrin derivative with lithium aluminium hydride was achieved as previously described for compound 1i, and the formation of the corresponding hydrochloride as previously described for compound 1h.

MS (ESI): 418.4 (M+H) ⁺ .

¹ H-NMR (300 MHz, CDCI ₃ ): δ= 0.48 (s, 3 H), 0.59 - 1.52 (m, 42 H), 1.58 - 1.71 (m, 1 H), 1.76 - 1.84 (m, 1 H), 2.84 (br s, 2 H). NH ₃ ⁺ not clearly visible.

Example 11 : Preparation of 3β-Aminoethyl-5α-cholestane 1k as its hydrochloride

Compound 1k was prepared starting from commercially available 3-cholestanone and diethylcyanomethyl phosphonate using a Horner-Wadsworth-Emmons protocol as described in the literature (Karagiozov, S. K.; Synth. Commun. 2004, 34(5), 871-888). The resulting α,β-unsaturated nitrile was transformed to the corresponding nitrile by standard hydrogenation with palladium on charcoal as catalyst. Subsequent reduction of the nitrile to the corresponding amine was achieved with lithium aluminium hydride in diethyl ether. Finally, compound 1k was obtained in very pure form as its hydrochloride salt by precipitation using a commercial solution of hydrochloric acid in diethyl ether. MS (ESI): 416.4 (M+H) ⁺ .

¹ H-NMR (300 MHz, CDCI ₃ ): δ= 0.67 (s, 3 H), 0.71 - 1.69 (m, 44 H), 1.78 - 1.85 (m, 1 H), 2.61 (m, 2 H). NH ₃ ⁺ not clearly visible.

Example 12: Preparation of (5α-Cholestan-3β-ol) methyl [2-(trimethylammonio)- ethyl] phosphate 11 as its trifluoroacetate

Compound 11 was prepared from commercial dihydrocholesterol in a one-pot procedure using the phosphoramidite method employing the commercial reagents methyl λ/,N,/V,/V-tetraisopropylphosphorodiamidite, tetrazole, choline tosylate, N- phenylimidazolium trifluoromethanesulfonate and terf-butylperoxide as described in the general synthetic discussion above. The crude product was then purified by preparative HPLC resulting in isolation of the corresponding trifluoroacetate salt. MS (ESI): 568.4 M ⁺ . ¹ H-NMR (300 MHz, CDCI ₃ ): δ= 0.63 (s, 3 H), 0.63 (m, 1 H), 0.70 - 2.00 (m, 42 H), 3.22 (s, 9 H), 3.71 (d, J = 11.3, 3 H), 3.76 (m, 2 H), 4.23 (m, 1 H), 4.39 (m, 2 H).

Example 13: Preparation of 3-Aza-A-homo-5α-cholestane 2a as its hydrochloride

Compound 2a was prepared from commercial cholestan-3-one via the corresponding oxime in a Beckmann-type rearrangement reaction as described by Doorenbos et al. (Doorenbos, N. J.; J. Org. Chem. 1961, 26, 2548-2549).

Melting point and ¹ H-NMR spectrum of the obtained material were in agreement with published data (Takahashi, T.T.; J. Chem. Soc. Perkin 1 1980, 1916-1919).

Example 14: Preparation of 4-Aza-5α-cholestane 2b as its hydrochloride

Compound 2b was prepared from commercial cholest-4,5-en-3-one via the sequence 4,5-bishydroxylation, reduction of 3-keto to 3-hydroxy, oxidative cleavage of the 3,4,5- triol using either lead tetraacetate or perchloric acid in methanol, saponification of the resulting methyl ester, condensation to 4-azacholest-4,5-en-3-one by treatment with ammonia under pressure, hydrogenation of the 5,6-double bond and reduction of the resulting lactam to 2b using lithium aluminium hydride (Boύcza-Tomaszewski, Z.; Tetrahedron Lett. 1986, 27, 3767-3770; Doorenbos, NJ. ; J. Org. Chem. 1961, 26, 2546-2548; Shoppee, CW. ; J. Chem. Soc. 1962, 2275-2285). An alternative method for the preparation of 2b is described by McKenna and Tulley (McKenna, J.; J. Chem. Soc. 1960, 945). Melting point and ¹ H-NMR spectrum of the obtained material were in agreement with published data (Pradhan, S. K.; Heterocycles 1989, 28(2), 813-839).

Example 15: 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 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) are 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 IC ₅₀ and IC ₉₀ 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 are 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 is inhibited. The corresponding values are denoted "IC ₅₀ ^" or "IC ₉₀ , 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 -8O ⁰ C; 0.05% solution of glutaraldehyde (25% in water, Sigma catalogue number G 5882, kept at -20 ⁰ 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 (H1N1)) 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 -2O ⁰ C as 1OmM, 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 37 ⁰ C. 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, I x IO ^"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 4°C 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 11) 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 ⁰ 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 PBS, 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 1h at room temperature or overnight at 4 ⁰ 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 (MAb pool 5, US Biological I7650-04A) diluted 1 :1000 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 IC ₅₀ /IC ₉₀ 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 IC ₅₀ and IC ₉₀ values.

Results

Various cholesterylamines showed strong inhibitory effects in the PR8 (H1 N1) virus replication assay (as a cellular model for influenza infection). In particular, compounds 1a to 11, 2a and 2b yielded good results (Table 1). Without being bound by theory, it appears that the combination of the cholestane scaffold with an amino function either attached to the steroidal A-ring (cholesterylamines and derivatives thereof) or being part of it (azacholestanes, azahomocholestanes or derivatives thereof) is a structural motif leading to inhibition of viral replication. In contrast, cholesteryl sulfate provided no inhibition of viral replication when tested at concentrations of 10, 20 and 50 μM, whereby at 50 μM and higher concentrations toxicity was observed. Also cholesteryl- 3β-glycolic acid, displaying a negatively charged function under assay conditions, and 3-oximocholestane, showing a nitrogen atom attached to the A-ring of the cholesteryl

core structure, provided no inhibition in the assay setting described herein. Furthermore, 3-keto-4α,5α-cholestanediol (IC ₅₀ 16.0 μM) and 3β,4α,5α-cholestanetriol (IC _5O 16.1 μM) provided weak potency in the viral replication assay used as disease model for influenza infection. It appears that strong polarity localized at the steroidal A- ring does not suffice to provide for antiviral activity. Moreover, the presence of an amino function appears to be necessary to obtain a particular high activity of the compounds. 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 anti-influenza activity.

Table 1. Virus replication results (IC ₅ Q and IC ₉₀ ) values of examples provided herein.

Compound IC ₅₀ [μM] IC ₉₀ [μM]

1a 1.7 9.0

1b 1.9 16.2

1c 3.3 17.5

1d 3.7 15.1

1e 3.5 17.0

1f 8.6 11.4 ig 4.7 62.0

1h 4.0 11.6

1i 3.7 9.8

1j 3.1 25.1

1k 2.1 13.4

11 5.3 10.1

2a 3.3 9.7

2b 5.9 30.1

As demonstrated by the data presented herein, compounds 1a to 11 and compounds 2a and 2b are preferred compounds for the pharmaceutical intervention in influenza infection. Eight of these compounds, i.e. compounds 1a, 1b, 1g, 1h, 1i, 1j, 1k and 2a provided for particularly good results in the influenza virus replication assay.

Furthermore, these compounds showed good results in solubility tests and therapeutic indices. The solubility of cholesterylamines in a polar medium increases with increasing substitution of hydrogen atoms attached to the steroidal A-ring by hydroxy functions, so that a slight decrease of potency is counterbalanced by an increased solubility, which may lead to increased bioavailability. In this context, compounds 1g and 1i are preferred molecular entities for the treatment of viral infections, in particular influenza infections.

Example 16: Antimicrobial Activity Assay The aim of this assay is the identification of compounds having antituberculosis activity, as evaluated using the strain M. tuberculosis H ₃₇ RV as disease model for tuberculosis. Potency in antimicrobial assays (MIC ₉₀ ) was evaluated as described below and compared to toxicity in mammalian Vera cells.

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

Determination of growth inhibition of Mycobacterium tuberculosis H ₃₇ RV by test compounds was carried out as described in 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 MIC ₉₀ . The values presented herein are means of three replicate experiments.

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 H ₃₇ Rv 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 MIC ₉₀ 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 Vera 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). 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 for evaluation of the inhibitory effect of various cholestane and cholestene derivatives, it was found that the cholesterylamine derivatives to be used in accordance with the present invention also inhibit bacterial growth. For example, compounds 1a, 1f, 1h, 1i, 2a and 2b provided good results (Table 2). The combination of the cholestane scaffold with an amino function attached to the steroidal A-ring is a preferred structural motif in the inhibition of mycobacterial growth, in particular Mycobacterium tuberculosis. In contrast, cholesteryl sulfate or -τaπs-2-aminomethyl-i-cyclohexanol provided no corresponding effect on mycobacteria when tested at concentrations up to 100 μM. It was also found that incorporation of the amino moiety into the steroidal A-ring provides azacholesteryl derivatives which efficiently inhibit bacterial growth as exemplified by the MABA assay (Table 2). Hence, the combination of the cholestane scaffold with a nitrogen which is incorporated into the steroidal A-ring or analogue derivatives with an expanded or constricted A-ring are preferred structural motifs for the inhibition of mycobacterial growth, in particular Mycobacterium tuberculosis, as exemplified by compounds 2a and 2b.

Table 2. Inhibition of replication of M. tuberculosis (strain H ₃₇ Rv) by examples provided herein.

Compound MIC ₉₀ [μM] MABA MIC ₉₀ [μM] LORA

1f 1.3 3.01

1i 2.6 2.73

1h 1.5 2.63

1a 2.5 1.33

2a 2.82 1.51

2b 3.01 5.03

Compounds 1a, 1f, 1h, 1i, 2a and 2b are preferred compounds for the pharmaceutical intervention of mycobacterial diseases, in particular of tuberculosis. Two of these compounds, i.e. compounds 1f and 1h, provided for particularly good results in the mycobacteria-assays as demonstrated by the remarkably low MIC ₉₀ values. Hence, compounds 1f and 1h represent even more preferred compounds to be used in the 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 2a provided for particularly good results in the LORA model. Thus, compounds 1a and 2a represent particularly preferred compounds to be used in pharmaceutical compositions for treating persistent Mycobacterium tuberculosis phenotypes.