NEOSORAPHENS

Abstract

The present invention relates to compounds according to general formula (I); to compositions, including a pharmaceutical formulation and a combination preparation comprising one or more of the compound (s); to a process for their preparation; to uses thereof, including the use in the treatment or prevention of a viral infection or a Th17-associated inflammatory and/or autoimmune disease.

##STR00001##

Claims

1. A compound of the general formula (I): ##STR00017## or a pharmacologically acceptable salt thereof, wherein A represents a group of the formula: ##STR00018## R.sup.1, R.sup.2 and R.sup.3, in each case, independently represents a hydrogen atom, a halogen atom, CN, CF.sub.3, NO.sub.2, NH.sub.2, NHC(O)R.sup.4, NHSO.sub.2R.sup.4, (CH.sub.2).sub.m-L.sup.1-W, O(CH.sub.2).sub.m-L.sup.2-W, C(O)(CH.sub.2).sub.m-L.sup.3-W, OC(O)(CH.sub.2).sub.m-L.sup.3-W, OC(O).sub.2(CH.sub.2).sub.m-L.sup.3-W, (C.sub.5-6 heterocyclyl)-L.sup.4-W, SOCH.sub.3, SOCN, SOCF.sub.3, SO.sub.2CH.sub.3, SO.sub.2CN, SO.sub.2CF.sub.3, SO.sub.2NR.sup.5R.sup.6, C(O)NR.sup.5R.sup.6, COCH.sub.3, COCF.sub.3, or C(CN).sub.3; R.sup.4, in each case, independently represents a hydrogen atom; an alkyl; alkenyl; alkynyl; heteroalkyl; cycloalkyl; heterocycloalkyl; aryl; heteroaryl; aralkyl; or heteroaralkyl group; R.sup.5 and R.sup.6 each independently represents a hydrogen atom; or (CH.sub.2).sub.m-L.sup.5; or R.sup.5 and R.sup.6 are taken together to form a 5- to 8-membered saturated, unsaturated or aromatic heterocycle containing 1 to 4 N atoms or 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur, which heterocycle may be unsubstituted or mono-, di or trisubstituted by halogen atom or L.sup.5; or R.sup.5 and R.sup.6 are taken together to form a 5- to 8-membered saturated, unsaturated or aromatic heterocycle containing 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur, which is fused to one or two rings selected from the group consisting of cycloalkyl; heterocycloalkyl; alkylcycloalkyl; heteroalkylcycloalkyl; aryl; heteroaryl; aralkyl; and heteroaralkyl; L.sup.1, L.sup.2, L.sup.3 and L.sup.4, in each case, independently represents a single bond, O, S, NH, SO, SO.sub.2, O(CH.sub.2CH.sub.2O).sub.n, (OCH.sub.2CH.sub.2).sub.n, or [C(O)CH.sub.2(OCH.sub.2CH.sub.2).sub.2NH].sub.n; L.sup.5 represents H; (CH.sub.2).sub.p-L.sup.6; (CH.sub.2).sub.pOL.sup.6; a C.sub.1-6 heteroalkyl; a cycloalkyl; a heterocycloalkyl; an alkylcycloalkyl; a heteroalkylcycloalkyl; an aryl; a heteroaryl; an aralkyl; or a heteroaralkyl group; L.sup.6 represents a C.sub.1-6 alkyl, C.sub.3-6 alkenyl, or C.sub.3-6 alkynyl group, in which 1 to 5 H atoms may, independently of each other, be replaced by halogen atom, CN, CF.sub.3, NO.sub.2, OR or NHR, and/or in which one or two non-adjacent CH.sub.2 group(s) may be replaced by O, NH, S, SO, SO.sub.2, or C.sub.3-7 cycloalkyl; W represents a hydrogen atom; a C.sub.1-6 alkyl, C.sub.3-6 alkenyl, or C.sub.3-6 alkynyl group, in which 1 to 5 H atoms may, independently of each other, be replaced by halogen atom, CN, CF.sub.3, NO.sub.2, OR or NHR, and/or in which one or two non-adjacent CH.sub.2 group(s) may be replaced by O, NH, S, SO, SO.sub.2, or C.sub.3-7 cycloalkyl; NR.sup.5R.sup.6, NHC(O)R.sup.4, NHSO.sub.2R.sup.4, C(O)R.sup.5, (CH)[(CH.sub.2).sub.mC(O)OR.sup.4].sub.2; or a 5, 6, or 7-membered saturated, unsaturated or aromatic carbocyclic or heterocyclic ring, optionally substituted by 1, 2, or 3 R.sup.7; R.sup.7, in each case, independently represents a halogen atom, an oxygen atom, a sulphur atom, CN, CF.sub.3, NH.sub.2, or NO.sub.2; X.sup.1, X.sup.2, and X.sup.3 each independently represents H or CH.sub.3; m each independently is 0, 1, 2, 3, or 4; n each independently is an integer from 1 to 40; and p each independently is 0, 1, 2 or 3.

2. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.1, R.sup.2 or R.sup.3 represents a group selected from the groups: ##STR00019## ##STR00020## wherein n and W are defined as in claim 1.

3. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.1 represents OH or OMe.

4. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein X.sup.1 is CH.sub.3 and X.sup.2 is H.

5. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein A represents a group of the formula: ##STR00021##

6. The compound according to claim 5, or a pharmacologically acceptable salt thereof, wherein R.sup.3 represents a hydrogen atom or a hydroxyl group.

7. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein the compound is represented by one of the following formulas: ##STR00022## wherein R.sup.2 is defined as in claim 1 or 2.

8. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein the compound is: ##STR00023##

9. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein A represents a group of the formula: ##STR00024##

10. A pharmaceutical composition comprising at least one compound according to claim 1 and, optionally, one or more carrier substance(s), excipient(s) and/or adjuvant(s).

11. A combination preparation containing at least one compound according to claim 1, and at least one further active pharmaceutical ingredient.

12. The combination preparation of claim 11, wherein the further active pharmaceutical ingredient is selected from (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone (Rapamycin); AP-23481, (1R,2R,4S)-4-{(2R)-2-[(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H-23,27-epoxypyrido[2,1-c][1,4]oxazacyclohentriacontin-3-yl]propyl}-2-methoxycyclohexyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate (Temsirolimus), dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0.sup.4,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone (Everolimus); and (1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0.sup.4,9]hexa-triaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate (Ridaforolimus); and antiviral drugs.

13. The compound according to claim 1, the pharmaceutical composition according to, or the combination preparation according to for use as a medicament.

14. The compound according to claim 1, the pharmaceutical composition according to, or the combination preparation according to for use in the treatment or prophylaxis of a Th17-associated inflammatory and/or autoimmune disease.

15. The compound, the pharmaceutical composition, or the combination preparation for use as in claim 14, wherein the Th17-associated inflammatory and/or autoimmune disease is selected from: rheumatoid arthritis, psoriasis, systemic sclerosis, systemic lupus erythematosus, asthma, atopic dermatitis, contact hypersensitivity, multiple sclerosis, autoimmune myocarditis, type I diabetes, autoimmune thyroiditis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and transplant rejection.

16. The compound according to claim 1, the pharmaceutical composition according to, or the combination preparation according to for use in the treatment or prophylaxis of a viral infection.

17. A method for the preparation of a compound of formula (I), the method comprising the steps of: (a) fermenting Sorangium cellulosum (DSM 28093); and (b) separating and retaining the compound from the culture broth; wherein the compound is a compound according to claim 8.

18. A method for treating a subject suffering from or susceptible to a Th17-associated inflammatory and/or autoimmune disease, comprising administering to the subject an effective amount of a compound of claim 1.

19. The method of claim 18 wherein the Th17-associated inflammatory and/or autoimmune disease is selected from: rheumatoid arthritis, psoriasis, systemic sclerosis, systemic lupus erythematosus, asthma, atopic dermatitis, contact hypersensitivity, multiple sclerosis, autoimmune myocarditis, type I diabetes, autoimmune thyroiditis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and transplant rejection.

20. A method for treating a subject suffering from or susceptible to a viral infection comprising administering to the subject an effective amount of a compound of claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0106] FIG. 1: Inhibition of Th17 and induction of Treg differentiation by the compounds of the present inventionNeosoraphene DC, 476, 478, 462 and 464, respectivelyin vitro. A: Formation of IL17A+ cells within a live CD4+ T cell pool in the presence of varying concentrations of the compounds of the present invention compared to the known ACC inhibitor Soraphen A (SorA, 2.sup.nd bar from the left) and DMSO (left bar). Mean of triplicates+s.d. B: Formation of Foxp3+ Treg cells within a live CD4+ T cell pool in the presence of varying concentrations of the compounds of the present invention compared to the known ACC inhibitor Soraphen A (SorA, 2.sup.nd bar from the left) and DMSO (left bar). Mean of triplicates+s.d.

[0107] As can be taken from FIG. 1, addition of the ACC inhibitor Soraphen A as well as of the compounds of the present invention decreases the percentage of IL-17A producing T-cells in a dose dependent manner (EC.sub.50=40 nM) and induces the reciprocal differentiation of Foxp3+ cells.

EXAMPLES

Materials and Methods

[0108] In general, standard bioassays and protocols were used in the following examples. Materials were normally purchased from commercial suppliers and used according to respective manuals supplied therewith.

Media for the Cultivation of Sorangium cellulosum Strain MSr1424 (DSM 28093): [0109] CY+H-Medium: 0.15% casitone, 0.15% yeast extract (Marcor typ 9000), 0.1% glucose (monohydrate), 0.1% soy flour (degreased), 0.4% starch (Cerestar), 0.1% CaCl.sub.2 2H.sub.2O, 0.05% MgSO.sub.47H.sub.2O, 0.0004% NaFe-EDTA, 50 mM HEPES (11.8 g/l); pH 7.2 [0110] M-Medium: 1.0% peptone (soy), 1.0% maltose monohydrate, 0.1% CaCl.sub.22H.sub.2O, 0.1% MgSO.sub.44H.sub.2O, 50 mM HEPES (11.9 g/l), 8 mg/l Fe-EDTA; pH 7.2 [0111] P-Medium: 0.2% peptone (Marcor M), 0.8% starch (Cerestar), 0.4% Probion, 0.2% yeast extract (Marcor typ 9000), 0.1% CaCl.sub.22H.sub.2O, 0.1% MgSO.sub.44H.sub.2O, 100 mM HEPES (23.8 g/l), 8 mg/l Fe-EDTA; pH 7.5 [0112] S-Medium: 0.4% soy flour (degreased), 0.2% glucose monohydrate, 0.8% starch (Cerestar), 0.1% CaCl.sub.22H.sub.2O, 0.1% MgSO.sub.44H.sub.2O, 50 mM HEPES (11.9 g/l), 8 mg/l Fe-EDTA; pH 7.4 [0113] Vitamin solution (Schlegel): 0.2% Biotin, 2.0% nicotinic acid, 1.0% Thiamin, 1.0% 4-Aminobenzoe acid, 0.5% Pantothenat, 5.0% Pyridoxamine, 2.0% Cyanocobalamine [0114] Vitamin B12 solution: 0.5 mg/mL [0115] Add vitamins after autoclaving (100 L in 100 mL medium) [0116] The vitamin solutions is sterilized by filtration and stored at 4 C. [0117] Adjust pH of medium with KOH. [0118] Cy+H-medium+Vit.+Vit. B12 [0119] Cy+H-medium+5 mL P-medium+Vit.+Vit. B12 [0120] S-medium+5 mL P-medium+Vit.+Vit. B12 [0121] M-medium+Vit.+Vit. B12

In Vitro Th17 Differentiation

[0122] Single cell suspensions were prepared and pooled from the spleen, mesenteric and peripheral lymph nodes of adult C57BL/6 mice by filtering the crushed organs through a 70 m cell-strainer. Nave CD4+CD25.sup.CD62L+ T cells were subsequently isolated from these suspensions using the CD4+CD25.sup.CD62L+ T cell isolation Kit II and AutoMACS according to the manufacturer's protocol (Miltenyi Biotec) or by FACS sorting. Purified T cells were cultured under classic Th17 inducing conditions (Bettelli et al., Nature 441(7090): 235-8 (2006)). In detail, the cells were cultured in 96 well flat bottom plates (310.sup.5 cells per well), that were precoated with 10 g/ml anti-CD3 Antibody (clone 145-2C11, BioXcell) in the presence of antibodies against CD28 (1 g/ml, clone 37.51, BioXcell), IFN- (5 g/ml, clone XMG1.2, BioXcell) and IL-4 (5 g/ml, clone 11B11, BioXcell) as well as in the presence of TGF1 (2 ng/ml, R&D), IL-6 (5 ng/ml, R&D) and IL-1 (50 ng/ml, R&D) in IMDM Medium (supplemented with 10% FCS+500 U PenStrep+50 M -Mercaptoethanol). To these cultures, increasing concentrations of ACC Inhibitors Soraphen A or 5-(Tetradecyloxy)-2-furoic acid (TOFA) (both dissolved in DMSO) were added on day 0 and cultured for up to 4 days in an cell culture incubator (37 C. and 5% CO2). As control, cells were cultured in the presence of DMSO only.

In Vitro Restimulation and Flow Cytometry (FACS)

[0123] Were indicated, cells from Th17 cultures were restimulated with 100 ng/ml phorbol 12-myristate 13-acetate (Sigma) and 1 g/ml Inonomycin (Sigma) for 4 h and Brefeldin A (1:1000, Sigma) was added for the last 2 h of restimulation. After restimulation, cells were harvested for flow cytometry. For surface staining: fluorophore-labelled mAbs aginst CD3 and CD4 were obtained from eBiosciences. For intracellular staining, cells were fixed with Fix/Perm (eBioscience) according to the manufacturer's protocol, and stained intracellular with fluorophore-labelled mAbs directed against IL-17A and Foxp3 (eBiosciences). Cells were run on a flow cytometer (CyAn, Beckman Coulter) and data was analysed on FlowJo (TreeStar) software.

[0124] To isolate naive CD4+CD25.sup.CD62L+ T cells by FACS-sorting, lymphocyte suspensions were surface-stained with fluorophore-labelled mAbs aginst CD4, CD25 and CD62L (all from ebioscience) and live CD4+CD25.sup.CD62L+ T cells were sorted to >98% purity using a Moflo (Beckman Coulter) or FACS ARIAII (BD Biosciences) cell sorter.

Example 1

Biosynthesis of Neosoraphens

[0125] Cultivation of Sorangium cellulosum Strain MSr1424 (DSM 28093):

[0126] 2 ml of cryo stock culture of strain MSr1424 (DSM28093) was carefully defrosted at room temperature and subsequently cultivated for 7 days at 30 C. in a 100 mL shake flask containing 20 mL of medium S supplemented with 5 mL medium P, vitamin solution (Schlegel) and vitamin B12. After 7 days of cultivation, the 20 mL seed culture was transferred in a 250 mL shake flask containing the same medium.

[0127] A 70 L fermentation of strain MSr1424 (DSM 28093) was performed in the presence of 0.7 L adsorber resin (Amberlite XAD-16). At the end of the fermentation (after 14 days) the adsorber resin was harvested by sieving with a process filter.

Isolation of Neosoraphenes:

[0128] After sieving the adsorber resin (Amberlite XAD-16) from the culture broth, the adsorber resin was washed in an open glass column with 3 L methanol/water 2:8 to remove polar substances. Subsequently the adsorber resin was extracted with 6 L of methanol to yield raw Neosoraphens. After evaporation of the methanol, the residual waterphase was extracted three times with ethylacetate. The organic layer was dried, filtrated and concentrated in vacuo to give a crude extract of 9.8 g.

[0129] Initial separation was achieved in two steps by silica gel column chromatography on a Reveleris Flash chromatography System. Column: Reveleris Silica 80 g, Grace; Elution solvent A: dichloromethane, solvent B: acetone, gradient: 11% B 3 min. isocratic, in 12 min. up to 20% B, in 6 min. to 29% B, isocratic for 13 min. at 29% B, in 2 min. to 100% B. Flow rate: 60 mL/min. Detection at 254 nm. Based on TLC analyses the chromatography gave 8 fractions. To separate the Neosoraphenes the fractions were further chromatographed on Nucleosil Si100, 7 m, 25016 mm column (Macherey+Nagel); Solvent: t-Butylmethylether: n-Heptane 25:75+1% Methanol; Flow rate: 20 mL/min; Detection: 217 nm. In some cases a additional separation on Phenomenex Gemini column C18, 10 m, 25021 mm was used. Solvent A: acetonitrile:water=2:8+0.1% acetic acid; Solvent B: acetonitrile:water=4:6+0.1% acetic acid, Gradient: 80% B in 30 min. up to 100% B than 20 min. 100% B; Flow rate: 20 mL/min; Detection. 215 nm. Using this procedure, 95 mg Neosoraphen M462, 340 mg Neosoraphen M464, 600 mg Neosoraphen M476, 200 mg Neosoraphen M478, and 19 mg Neosoraphen M480 were obtained.

Physico-Chemical Characterisation of Neosoraphen M462

[0130] ##STR00010##

[0131] Formula: C.sub.26H.sub.38O.sub.7

[0132] Molecular weight: 462.58

[0133] HR ESI MS: [M+Na].sup.+ calcd.: 485.2509. found: 485.2504.

[0134] IR (KBr): =3423, 2920, 2850, 1736, 1436, 1222, 1164, 1091, 1053 cm.sup.1

[0135] UV (MeOH): .sub.max (lg)=endabsorption

[0136] HPLC: column: Waters ACQUITY UPLC BEH C18 Column, 2.150 mm, 1.7 m; Solvent A: H2O+0.1% HCOOH; Solvent B: ACN+0.1% HCOOH; Gradient: 5% B(0.5)-->5% B(19.5)-->100% B(10)-->100% B; Flow: 0.6 ml/min. Detection: dioden array 200-600 nm; Retention time: 9.7 min.

TABLE-US-00001 TABLE 1 .sup.1H (600 MHZ) and .sup.13C (150 MHz) NMR data of Neosoraphen M462 in CDCL.sub.3 Proton (ppm) Multipl J [Hz] No. C (ppm) 1 167.5 2Ha 2.66 d 12.8 2 47.6 2Hb 2.55 d 12.8 3OH 4.75 s 3 96.2 4Ha 1.83 m 4 35.1 4Hb 1.83 m 5H 3.89 dq 7, 3, 3, 2.9 5 71.3 5OH 3.52 d 7.5 6H 1.88 m 6 35.8 6 Me 0.90 d 7.0 6 Me 10.3 7H 3.82 dd 10.3, 2.2 7 72.6 8H 2.40 m 8 35.4 8 Me 1.00 d 6.6 8 Me 12.9 9H 6.13 dd 16.1, 4.4 9 137.6 10H 5.45 ddd 16.1, 9.0, 1.7 10 124.9 11H 4.18 td 9.0, 8.6, 2.6 11 74.8 11OH 2.47 d 8.4 12H 3.34 dt 10.6, 3.3, 2.6 12 84.2 12OMe 3.42 s 12 OMe 57.7 13Ha 1.73 m 13 29.7 13Hb 1.25 m 14Ha 1.49 m 14 23.3 14Hb 1.14 m 15Ha 1.51 m 15 25.9 15Hb 1.46 m 16Ha 2.11 ddt 14.8, 11.4, 3.8 16 36.0 16Hb 1.67 17H 5.88 dd 11.6, 3.1 17 74.6 17 141.1 17H* 7.32 m 17* 126.2 17H* 7.30 m 17* 128.6 17H 7.27 m 17 128.0 *double

Physico-Chemical Characterisation of Neosoraphen M464

[0137] ##STR00011##

[0138] Formula: C.sub.26H.sub.40O.sub.7

[0139] Molecular weight: 464.59

[0140] HR ESI MS: [M+Na].sup.+ calcd.: 487.2666. found: 487.2656.

[0141] IR (KBr): =3429, 2934, 1738, 1456, 1220, 1167, 1097, 1053 cm.sup.1

[0142] UV (MeOH): .sub.max (lg)=endabsorption

[0143] HPLC: column: Waters ACQUITY UPLC BEH C18 Column, 2.150 mm, 1.7 m; Solvent A: H.sub.2O+0.1% HCOOH; Solvent B: ACN+0.1% HCOOH; Gradient: 5% B(0.5)-->5% B(19.5)-->100% B(10)-->100% B; Flow: 0.6 ml/min; Detection: dioden array 200-600 nm; Retention time: 9.8 min.

TABLE-US-00002 TABLE 2 .sup.1H (600 MHZ) and .sup.13C (150 MHz) NMR data of Neosoraphen M464 in CDCL.sub.3 Proton (ppm) Multipl. (Hz) No. C (ppm) 1 168.0 2Ha 2.67 d 13.9 2 47.1 2Hb 2.54 d 13.6 3OH 4.85 s 3 96.2 4Ha 1.97 dd 14.1, 3.1 4 35.0 4Hb 1.76 dd 14.3, 2.2 5H 3.95 dd 5.9, 2.9 5 71.8 5OH 3.05 d 5.9 6H 1.80 m 6 35.3 6Me 0.88 d 7.3 6Me 10.2 7H 3.93 dd 10.3, 2.2 7 69.0 8H 1.85 m 8 31.9 8Me 0.85 d 7.0 8Me 14.6 9Ha 1.69 m 9* 28.1 9Hb 1.56 m 10Ha 1.55 m 10 24.8 10Hb 1.46 m 11H 3.80 m 11 70.9 11OH 2.14 d 6.6 12H 3.32 td 7.0, 7.0, 2.9 12 82.8 12OMe 3.40 s 12OMe 57.4 13Ha 1.73 m 13* 28.0 13Hb 1.37 m 14Ha 1.51 m 14 23.1 14Hb 1.28 m 15Ha 1.58 m 15 25.4 15Hb 1.45 m 16Ha 2.07 m 16 35.7 16Hb 1.70 m 17H 5.93 dd 11.0, 3.7 17 74.6 17 141.1 17H 7.33* m 17* 126.4 17H 7.32* m 17* 128.5 17H 7.27 m 17 128.0 *double

Physico-Chemical Characterisation of Neosoraphen M476

[0144] ##STR00012##

[0145] Formula: C.sub.27H.sub.40O.sub.7

[0146] Molecular weight: 476.60

[0147] HR ESI MS: [M+Na].sup.+ calcd.: 499.2666. found: 499.2661.

[0148] IR (KBr): =3423, 2920, 2850, 1736, 1436, 1222, 1164, 1091, 1053 cm.sup.1

[0149] UV (MeOH): .sub.max (lg)=endabsorption

[0150] HPLC: column: Waters ACQUITY UPLC BEH C18 Column, 2.150 mm, 1.7 m; Solvent A: H.sub.2O+0.1% HCOOH; Solvent B: ACN+0.1% HCOOH; Gradient: 5% B(0.5)-->5% B(19.5)-->100% B(10)-->100% B; Flow: 0.6 ml/min; Detection: dioden array 200-600 nm; Retention time: 10.3 min.

TABLE-US-00003 TABLE 3 .sup.1H (600 MHZ) and .sup.13C (150 MHz) NMR data of Neosoraphen M476 in CDCL.sub.3 Proton (ppm) Multipl. (Hz) No. C (ppm) 1 167.7 2Ha 2.67 d 12.5 2 47.7 2Hb 2.54 d 12.5 3 96.2 3OH 4.77 s 4Ha 1.83 m 4 35.3 4Hb 1.82 m 5H 3.90 sbr 5 71.3 5OH d 6H 1.88 m 6 35.8 6Me 0.91 d 7.3 6Me 10.2 7H 3.82 dd 10.3, 2.2 7 72.6 8H 2.44 m 8 35.3 8Me 1.02 d 7.0 8Me 12.7 9H 6.20 dd 16.1, 4.4 9 140.1 10H 5.44 ddd 16.1, 9.4, 1.3 10 122.5 11H 3.68 dd 9.2, 1.8 11 84.8 11OMe 3.29 s 11OMe 56.3 12H 3.41 m 12 83.1 12OMe 3.43 s 12OMe 58.0 13Ha 1.65 m 13 30.6 13Hb 1.28 m 14Ha 1.47 m 14 23.4 14Hb 1.11 m 15Ha 1.54 m 15 25.9 15Hb 1.47 m 16Ha 2.10 m 16 35.9 16Hb 1.67 m 17H 5.91 dd 11.7, 3.3 17 74.3 17 141.2 17H* 7.33 m 17* 126.2 17H* 7.32 m 17* 128.5 17H 7.27 m 17 128.0 *double

Physico-Chemical Characterisation of Neosoraphen M478

[0151] ##STR00013##

[0152] Formula: C.sub.27H.sub.42O.sub.7

[0153] Molecular weight: 478.62

[0154] HR ESI MS: [M+Na].sup.+ calcd.: 501.2823. found: 501.2820.

[0155] IR (KBr): =3423, 2920, 2850, 1736, 1436, 1222, 1164, 1091, 1053 cm.sup.1

[0156] UV (MeOH): .sub.max (lg)=endabsorption

HPLC: column: Waters ACQUITY UPLC BEH C18 Column, 2.150 mm, 1.7 m; Solvent A: H.sub.2O+0.1% HCOOH; Solvent B: ACN+0.1% HCOOH; Gradient: 5% B(0.5)-->5% B(19.5)-->100% B(10)-->100% B; Flow: 0.6 ml/min; Detection: dioden array 200-600 nm; Retention time: 11.6 min.

TABLE-US-00004 TABLE 4 .sup.1H (600 MHZ) and .sup.13C (150 MHz) NMR data of Neosoraphen M478 in CDCL.sub.3 Proton (ppm) Multipl. (Hz) No. C (ppm) 1 168.1 2Ha 2.67 d 13.9 2 47.3 2Hb 2.55 d 13.6 3 96.2 3OH 4.70 s 4Ha 1.93 m 4 35.1 4Hb 1.79 m 5H 3.94 sbr 5 71.7 5OH 2.94 d 13.9 6H 1.82 m 6 35.4 6Me 0.88 d 7.0 6Me 10.2 7H 3.93 m 7 69.0 8H 1.81 m 8 32.3 8Me 0.86 d 7.0 8Me 14.3 9Ha 1.73 m 9 28.4 9Hb 1.50 m 10Ha 1.55 m 10 23.1 10Hb 1.55 m 11H 3.35 m 11 81.5 11OMe 3.42 s 11OMe 57.7 12H 3.40 m 12 80.3 12OMe 3.42 s 12OMe 57.6 13Ha 1.73 m 13 28.8 13Hb 1.43 m 14Ha 1.55 m 14 23.4 14Hb 1.30 m 15Ha 1.54 m 15 25.0 15Hb 1.43 m 16Ha 2.06 m 16 35.5 16Hb 1.71 m 17H 5.92 dd 11.3, 4.4 17 74.8 17 141.0 17H* 7.33 m 17* 126.5 17H* 7.32 m 17* 128.5 17H 7.27 m 17 128.0 *double

Physico-Chemical Characterisation of Neosoraphen M480

[0157] ##STR00014##

[0158] Formula: C.sub.26H.sub.40O.sub.8

[0159] Molecular weight: 480.59

[0160] HR ESI MS: [M+Na].sup.+ calcd.: 503.2615. found: 503.2611.

[0161] IR (KBr): =3423, 2920, 2850, 1736, 1436, 1222, 1164, 1091, 1053 cm.sup.1

[0162] UV (MeOH): .sub.max (lg)=endabsorption

[0163] HPLC: column: Waters ACQUITY UPLC BEH C18 Column, 2.150 mm, 1.7 m; Solvent A: H.sub.2O+0.1% HCOOH; Solvent B: ACN+0.1% HCOOH; Gradient: 5% B(0.5)-->5% B(19.5)-->100% B(10)-->100% B; Flow: 0.6 ml/min; Detection: dioden array 200-600 nm; Retention time: 8.9 min.

TABLE-US-00005 TABLE 5 .sup.1H (600 MHZ) and .sup.13C (150 MHz) NMR data of Neosoraphen M480 in CDCL.sub.3 Proton (ppm) Multipl. (Hz) No. C (ppm) 1 167.60 2Ha 2.63 d 12.5 2 47.4 2Hb 2.49 d 12.5 3 96.2 3OH 5.17 s 4Ha 1.82 m 4 35.7 4Hb 1.82 m 5H 4.02 ddd 2.9, 2.9, 2.9 5 71.9 5OH 6H 1.79 m 6 35.5 6 Me 0.91 d 7.0 6 Me 10.2 7H 3.98 dd 11.0, 2.6 7 68.1 8H 2.05 m 8 39.3 8 Me 0.84 d 7.0 8 Me 8.8 9H 4.39 ddd 11.2, 4.2, 2.2 9 72.2 9 OH 3.17 sbr 10Ha 1.67 m 10 27.5 10Hb 1.44 m 11H 4.09 d 9.2 11 73.7 11 OH 3.08 sbr 12H 3.36 ddd 10.6, 3.0, 3.0 12 84.2 12 OMe 3.43 s 12 OMe 57.8 13Ha 1.84 m 13 29.8 13Hb 1.27 m 14Ha 1.49 m 14 23.3 14Hb 1.18 m 15Ha 1.64 m 15 25.4 15Hb 1.44 m 16Ha 2.11 m 16 35.8 16Hb 1.66 m 17H 6.02 Dd 11.4, 2.8 17 73.4 17 141.4 17H* 7.33 M 17* 126.5 17H* 7.32 M 17* 128.5 17H 7.33 M 17 127.9 *double

Example 2

Chemical Synthesis of 5-Substituted Neosoraphens

[0164] A 5-substituted compound of formula (I) can be obtained by chemical synthesis in a number of ways well known to one skilled in the art of organic synthesis using usual chemical reaction and synthesis methods. For example, 5-substituted compounds of formula (I) can be obtained according to Reaction Scheme 1 shown below.

##STR00015##

[0165] 1 eq of biotinylated linker (IRIS Biotech), dissolved in DMF (0.1 M), and 0.2 equivalents of EDCI*HCl were stirred for ten min at room temperature. After this time 0.2 eq DMAP and after five min 0.11 eq of Neosoraphen M476 were added. After ca. 12 h 0.1 additional eq of EDCI*HCl and DMP were added. The mixture was stirred at room temperature for eight additional hours before NH.sub.4Cl solution was added for hydrolysis (2 volume of DMF) and the mixture was extracted with EtOAc. The organic phase was dried with MgSO.sub.4, filtered and dried in vacuo. The crude product was purified by HPLC using a Waters auto purifier system equipped with a Waters XBridge column (prep C18, 5 m, 19150 mm) applying a H.sub.2O+0.1% HCO.sub.2H(A)/MeOH+0.1% HCO.sub.2H(B) gradient from 10% B to 90% B in 30 min to thereby yield 31% of the 5-substituted Neosoraphen M476.

Example 3

Chemical Synthesis of a Neosoraphen M476-Epoxide Derivative

[0166] A Neosoraphen M476-epoxide derivative has been synthezised according to Reaction Scheme 2 shown below.

##STR00016##

Example 4

Inhibition of Th17 and Induction of Treg Differentiation (In Vitro)

[0167] Naive mouse CD4.sup.+ T cells were cultured for 4 days under Th17 conditions in the presence of varying concentrations of the compounds of the invention. As control, cells were cultured in the presence of DMSO only (negative control) or ACC inhibitor Soraphen A (positive control). After 4 days, cells were restimulated with PMA/Ionomycin for 4 h and Brefeldin A for 2 h and further analysed by flow cytometry. The results are shown in FIGS. 1A and 1B. These results show that addition of Soraphen A as well as of the compounds of the invention decreases the percentage of IL-17A producing T-cells in a dose dependent manner (FIG. 1A; EC.sub.50=40 nM) and induces the reciprocal differentiation of Foxp3+ Treg cells (FIG. 1B). Thus, the results indicate that inhibition of ACC significantly lowers formation of pro-inflammatory Th17 cells and favours formation of Foxp3+ Tregs that are crucial for the induction and maintenance of self-tolerance and for the prevention of autoimmunity.

Example 5

Inhibition of HCV Infection (In Vitro)

[0168] To assess the inhibitory effects of Neosoraphens on HCV infection, a HCV cell-culture (HCVcc) system.sup.[1-3] was used. Unless otherwise stated, Huh7/Scr cells (provided by F. Chisari, The Scripps Research Institute, La Jolla, Calif.) which are highly permissive for HCV propagation in vitro were employed. The experiments were performed in the context of genotype 2a (Jc1 chimera.sup.[4]) HCVcc virus. To facilitate the quantification of infection, the bicistronic Jc1 luciferase reporter construct, designated Luc-Jc1.sup.[5], was used. The commercial ACC inhibitor 5-(Tetradecyloxy)-2-furoic acid (TOFA), which has been previously described to inhibit HCV replication.sup.[6], was chosen for comparison of the antiviral activities of the Neosoraphens. The NS3-4A serine protease inhibitor VX-950.sup.[7], a known HCV replication inhibitor, was used as positive control. Briefly, Huh7/Scr cells were seeded at a density of 1.210.sup.4 cells/well in 96-well plates. One day later cells were pre-incubated for 1 h at 37 C. with the compounds and then inoculated with the virus and the compounds for 4 h at 37 C. Finally, virus-containing media was replaced by a fresh media-compounds mix. Firefly luciferase activities were assayed 72 h post infection with the Dual-Glo Luciferase Assay System while cytotoxicity (viability) assays were carried out with the CytoTox-Glo Cytotoxicity Assay (both purchased from Promega Corporation, Madison, Wis.), according to manufacturer's instructions, using a plate luminometer FLUOstar OPTIMA (BMG LABTECH, Ortenberg, Germany). Mean relative light units (RLU) were plotted as percentage relative to control infections (solvent without compounds) for both infectivity and cell viability. Infections were carried out in triplicates and measured in duplicates (meanSEM; n=6). Half maximal effective concentration (EC.sub.50) and half maximal cytotoxic concentration 50 (CC.sub.50) were estimated by non-linear regression of log inhibitor vs. normalized response and used to calculate the Selectivity Index (SI) value. The results of the experiments are summarized in Table 6 below.

TABLE-US-00006 TABLE 6 In vitro antiviral activity (EC.sub.50, CC.sub.50, SI). Compound EC.sub.50 (nM) CC.sub.50 (M) SI Neosoraphen M462 7.74 13.56 1752 Neosoraphen M464 46.00 52.24 1136 Neosoraphen M476 2.09 198.7 95072 Neosoraphen M478 1.52 98.74 64961 Neosoraphen M480 65.73 45.52 693 TOFA 1010 33.87 34 VX-950 18.92 94.51 4995

REFERENCES

[0169] [1] Zhong J et al., PNAS 2005; 102:9294-9299. [0170] [2] Lindenbach B D et al., Science 2005; 309:623-626. [0171] [3] Wakita et al., Nature medicine 2005; 11:791-796. [0172] [4] Pietschmann T et al., PNAS 2006; 103:7408-7413. [0173] [5] Koutsoudakis G et al., J Virol. 2006; 80:5308-5320. [0174] [6] Kapadia S B, Chisari F V, PNAS 2005; 102:2561-2566. [0175] [7] Lin C, Kwong A D, Perni R B, Infectious disorders drug targets 2006; 6:3-16.

[0176] The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof. Especially preferred are combinations of preferred embodiments of the invention.