Immune stimulating macrolides

Abstract

The present invention provides immune stimulating macrolides of formula (I), wherein the substituents are as defined in the claims. The macrolides have utility in treating viral diseases and cancer. ##STR00001##

Claims

1. A compound of Formula (I), wherein the compound has the following structure: ##STR00054## wherein: X is selected from NR.sub.3CH.sub.2, CH.sub.2NR.sub.3, NR.sub.3(CO), (CO)NR.sub.3, and CNOH; R.sub.2 is a sugar of Formula (II) or Formula (III): ##STR00055## R.sub.1 is selected from an alkyl, heteroalkyl, cycloalkyl, aryl, and heteroaryl moiety, wherein the alkyl moiety is selected from C.sub.1-C.sub.6 alkyl groups that are optionally branched, the heteroalkyl moiety is selected from C.sub.1-C.sub.6 alkyl groups that are optionally branched or substituted and that optionally comprise one or more heteroatoms, the cycloalkyl moiety is selected from a C.sub.1-C.sub.6 cyclic alkyl groups that are optionally substituted and that optionally comprise one or more heteroatoms, the aryl moiety is selected from optionally substituted C.sub.6 aromatic rings, the heteroaryl moiety is selected from optionally substituted C.sub.1-C.sub.5 aromatic rings comprising one or more heteroatoms, wherein the one or more heteroatoms are selected from O, N, P, and S, and the heteroalkyl, cycloalkyl, aryl, and heteroaryl moieties, when substituted, independently, are substituted with one or more groups selected from alkyl, OH, F, Cl, NH.sub.2, NH-alkyl, NH-acyl, S-alkyl, S-acyl, O-alkyl, and O-acyl, wherein acyl is selected from C.sub.1-C.sub.4 optionally branched acyl groups; R.sub.3 is selected from H and Me; R.sub.4 is selected from H and Me; R.sub.a is selected from H and CR.sub.21R.sub.22R.sub.23; R.sub.21, R.sub.22, R.sub.23, and R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, and R.sub.10, independently, are selected from H, Me, NR.sub.11R.sub.12, and OR.sup.11, wherein R.sub.23 together with R.sub.4 in Formula (II), R.sub.4 together with R.sub.5 in Formula (II), R.sub.5 together with R.sub.7 in Formula (II), and R.sub.7 together with R.sub.9 in Formula (II), independently, may be joined to represent a bond to leave a double bond between the carbon atoms that each group is connected to, and R.sub.21 together with R.sub.22, R.sub.5 together with R.sub.6, R.sub.7 together with R.sub.8, or R.sub.9 together with R.sub.10 may be replaced with a carbonyl; R.sub.11 and R.sub.12, independently, are selected from H and alkyl; R.sub.13 is selected from H, OH, and OCH.sub.3; R.sub.14 is selected from H and OH; and one of R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9 or R.sub.10 is NR.sub.11R.sub.12; or a pharmaceutically acceptable salt, hydrate, solvate, tautomer, enantiomer or diastereomer thereof.

2. The compound according to claim 1, wherein: X is selected from NR.sub.3CH.sub.2 and CH.sub.2NR.sub.3; and R.sub.2 is a sugar of Formula (II).

3. The compound according to claim 1, wherein R.sub.1 is methyl or ethyl.

4. The compound according to claim 1, wherein one of R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is NR.sub.11R.sub.12.

5. The compound according to claim 1, wherein R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, and R.sub.10, independently, are selected from H, Me, NR.sub.11R.sub.12, and OR.sub.11.

6. The compound according to claim 1, wherein R.sub.13 and R.sub.14 are OH.

7. The compound according to claim 1, wherein X is selected from NR.sub.3CH.sub.2 and CH.sub.2NR.sub.3; R.sub.2 is a sugar of Formula (II); R.sub.1 is methyl or ethyl; R.sub.3 is selected from H and Me; R.sub.4 is H; R.sub.a is CR.sub.21R.sub.22R.sub.23; R.sub.21, R.sub.22, R.sub.23, and R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, and R.sub.10, independently, are selected from H, Me, NR.sub.11R.sub.12, and OR.sub.11; R.sub.11 and R.sub.12, independently, are selected from H and an alkyl moiety, wherein the alkyl moiety is selected from C.sub.1-C.sub.6 alkyl groups that are optionally branched; R.sub.13 is selected from H, OH, and OCH.sub.3; R.sub.14 is selected from H and OH; and one of R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, or R.sub.10 is NR.sub.11R.sub.12.

8. The compound according to claim 1, wherein R.sub.2 is a sugar according to Formula (II), R.sub.a is H, R.sub.4 is Me, R.sub.5 is H, R.sub.6 is OH, R.sub.7 is H, R.sub.8 is NR.sub.11R.sub.12, R.sub.9 is H, and R.sub.10 is H.

9. The compound according to claim 1, wherein R.sub.11 and R.sub.12, independently, are selected from H, Me, and Et.

10. The compound according to claim 1, wherein X is NR.sub.3CH.sub.2.

11. The compound according to claim 1, wherein R.sub.1 is Et.

12. A compound selected from: ##STR00056## or a pharmaceutically acceptable salt thereof.

13. The compound according to claim 12, wherein the compound is: ##STR00057##

14. A pharmaceutical composition comprising the compound of claim 1.

15. A method for treating a viral disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the compound of claim 1.

16. The method of claim 15, wherein the viral disease is HIV/AIDS.

17. A method for treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the compound of claim 1.

18. A method for preparing a compound according to claim 1, the method comprising adding an aglycone of Formula (IV): ##STR00058## to a culture of a biotransformation strain which glycosylates at the 3-hydroxyl position of a compound of Formula (IV), wherein the biotransformation strain expresses glycosyltransferases with 70% or more homology to AngMII (SEQ ID NO: 1) or AngMIII (SEQ ID NO: 2).

19. A compound selected from: ##STR00059## or a pharmaceutically acceptable salt thereof.

20. The compound according to claim 12, wherein the compound is a pharmaceutically acceptable salt of: ##STR00060##

Description

LEGENDS TO FIGURES

(1) FIG. 1. The structures of the macrolides Erythromycin A, Compound 1, Compound A, compound B and EM703.

(2) FIG. 2. CD69 upregulation on T- and B-cells. PBMC were treated for 24 h with compound 1, compound A and activation controls LPS and IFN-gamma. The expression of the early activation marker CD69 was measured on the CD4+ T cell population (left) and CD19+ B cell population (right) with flow cytometry. Values represents mean fluorescent intensity, MFI, and error bars standard deviation in the triplicate samples.

(3) FIG. 3. HLA-A,B,C upregulation on T- and B-cells. PBMC were treated for 24 h with compounds 1 or A and activation controls LPS and IFN-. The expression of HLA-A,B,C was measured on the CD4+ T cell population (left) and CD19+ B cell population (right) with flow cytometry. Values represents mean fluorescent intensity, MFI, and error bars standard deviation in the triplicate samples.

(4) FIG. 4. CD80 and HLA-DR upregulation on blood monocytes. PBMC were treated for 24 h with compounds 1 or A as well as activation controls LPS and IFN-gamma. The expression of CD80 and HLA-DR was measured on the monocyte cell population with flow cytometry. Values represents mean fluorescent intensity, MFI, and error bars standard deviation in the triplicate samples.

(5) FIG. 5. CD80 upregulation on blood monocytes. PBMC were treated for 24 h with compounds 1 or A as well as activation control IFN-gamma. The expression of CD80 was measured on the monocyte cell population with flow cytometry. Values represents mean fluorescent intensity, MFI, and error bars standard deviation in the triplicate samples.

(6) FIG. 6. Production of IL-10 from PBMCs after stimulation with compound 1 for 48 h or 1 week, measured with ELISA.

(7) FIG. 7. CD4 T cell proliferation after 6 days stimulation with compound 1, measured with proliferation dye Celltrace violet (Invitrogen) and flow cytometry. Untreated cells (UNT) or compound A were used as controls.

(8) FIG. 8. Upregulation of IL-7 receptor (CD127) on CMV specific CD8 T cells after incubation with compound 1, measured with flow cytometry.

(9) FIG. 9: Interferon-gamma secretion (as measured by cytometric bead assay) from PBMCs (from a CMV+ donor) grown with CMV peptides in the presence or absence of compound 1 or A for 5 days.

(10) FIG. 10: Interferon-gamma secretion (as measured by cytometric bead assay) from macrophages stimulated with indicated compound for 48 h.

(11) FIG. 11: Chemokine RANTES secretion (as measured by cytometric bead assay) from PBMC or macrophages stimulated with indicated compound for 48 h.

(12) FIG. 12: IL12p70 secretion (as measured by cytometric bead assay) from PBMC or macrophages stimulated with indicated compound for 48 h.

(13) FIG. 13: IL1b secretion (as measured by cytometric bead assay) from PBMC, macrophages or CD4 T cells stimulated with indicated compound for 48 h.

(14) FIG. 14: % CD25high cells in blood of C57bl/6 mice injected 24 h previously with indicated dose of compound 1. CD25 expression was measured by flow cytometry.

(15) FIG. 15: % MHC class I high CD11 b+ cells in spleen of 3 individual C57bl/6 mice injected 24 h previously with indicated compound. MHC class I and CD11b expression was measured by flow cytometry.

EXPERIMENTAL

Materials

(16) Unless otherwise indicated, all reagents used in the examples below are obtained from commercial sources. Example suppliers of Azithromycin B include Santa Cruz Biotechnology (Texas, USA) and Toronto Research Chemicals (Toronto, Canada).

Antibodies

(17) Anti-CD80 V450, anti-CD69 PE, anti HLA-DR APC-R700, anti CD127-APC, and antiAnti-HLA-A,B,C FITC were purchased from BD Biosciences. Celltrace violet for T cell proliferation assay was purchased from Invitrogen. ELISA antibodies were purchased from BD Biosciences.

Media

(18) RPMI-1640 (Invitrogen) supplemented with 25 mM HEPES, L-glutamine, Sodium pyruvate, 10% fetal bovine serum (Gibco), 100 g/mL penicillin and 100 g/mL streptomycin

General Biology Methods

(19) The effect of the compounds of the invention on immune stimulation may be tested using one or more of the methods described below:

General Compound Method

(20) Compound AnalysisSolubility and Stability in Solution

Analysis of Fermentation Broths and Compounds

(21) An aliquot of fermentation broth obtained as described below was shaken vigorously for 30 minutes with an equal volume of ethyl acetate, and then separated by centrifugation, or the already isolated compounds were dissolved in methanol:water (9:1, 0.1 mg/ml), and then separated by centrifugation. Supernatants were analysed by LC-MS and LC-MS/MS and chromatography was achieved over base-deactivated Luna C18 reversed-phase silica (5 micron particle size) using a Luna HPLC column (2504.6 mm; Phenomenex (Macclesfield, UK)) heated at 40 C. Agilent 1100 HPLC system comprising of quaternary pump, auto sampler, column oven and diode array detector coupled to a Bruker Esquire ion trap MS.

(22) Mobile phase A=0.1% formic acid in water

(23) Mobile phase B=0.1% formic acid in acetonitrile

(24) Gradient: T=0 min, B=50%; T=4.5 min, B=50%; T=7 min, B=100%; T=10.5 min, B=100%; T=10.75 min, B=50%; T=13 min, B=50%.

(25) Compounds were identified by LC-MS and LC-MS/MS and quantified by LC-MS/MS against an internal standard.

Analysis of Marker Expression by Flow Cytometry

(26) Human peripheral blood mononuclear cells (PBMCs) were purified from healthy donors with Ficoll-Paque density centrifugation. Cells were cultured in complete RPMI-1640 media (Invitrogen) supplemented with 25 mM HEPES, L-glutamine, Sodium pyruvate (Sigma), 10% fetal bovine serum, 100 g/mL penicillin and 100 g/mL streptomycin (Hyclone) for 24-72 hours in 37 C., 5% CO.sub.2 and stimulated with and increasing concentrations of compound 1 and 2. Cells were then washed in PBS and stained with monoclonal antibodies specific for cell surface markers (BD Pharmingen) and analysed with flow cytomtetry using a BD FACS Canto II flow cytometer. All samples were tested in duplicates.

Cytomegalovirus (CMV) Cultures

(27) Human peripheral blood mononuclear cells (PBMCs) were purified from healthy CMV positive donors with Ficoll-Paque density centrifugation. The PBMC were labeled with 5 M celltrace violet (Invitrogen) in PBS for 15 minutes and then washed with complete cell culture medium. The labeled PBMC was cultured in the presence of a peptide library spanning the CMV pp65 protein (1 g peptide/ml, JPT) in AIM-V media (Invitrogen) supplemented with L-glutamine, Sodium pyruvate (Sigma), 10% fetal bovine serum, 100 g/mL penicillin and 100 g/mL streptomycin (Hyclone) for 6-8 days in 37 C., 5% CO.sub.2. Cell proliferation was assessed with flow cytomtery using a BD FACS Canto II flow cytometer.

ELISA

(28) Supernatant IL-10 was measured with a standard sandwich ELISA (all antibodies from BD Biosciences) after 48 hours and 7 days incubation with 2.5 M of compound 1 and 100 U/mL IL-2 (Miltenyi Biotechnologies) in complete RPMI media, 37 C., 5% CO.sub.2

TLR.SUB.2 .Assay

(29) Samples and controls were tested in duplicate on recombinant HEK-293-TLR cell lines using a cell reporter assay at Invivogen using their standard assay conditions. These cell lines functionally over-express human TLR.sub.2 protein as well as a reporter gene which is a secreted alkaline phosphatase (SEAP). The production of this reporter gene is driven by an NFkB inducible promoter. The TLR reporter cell lines activation results are given as optical density values (OD).

(30) 20 l of each test article were used to stimulate the hTLR.sub.2 reporter cell lines in a 200 l of final reaction volume. Samples were tested in duplicate, with at least two concentrations tested20 uM and 10 uM.

Assessment of Cell Permeability (Bidirectional)

(31) 10 M Test article was added to the apical (A) surface of Caco-2 cell monolayers (in HBSS buffer with 0.3% DMSO and 5 M LY at 37 degrees C.) and compound permeation into the basolateral (B) compartment measured following 90 minutes incubation. This was also performed in the reverse direction (basolateral to apical) to investigate active transport. LC-MS/MS is used to quantify levels of both the test and standard control compounds. Efflux ratio was calculated by dividing the B to A permeability by the A to B permeability.
Drug permeability: Papp=(VA/(Areatime))([drug]accepter/(([drug]initial, donor)Dilution Factor)

Assessment of Metabolic Stability (Microsome Stability Assay)

(32) Rate of metabolism in microsomes was tested as follows:

(33) Human liver microsomes were diluted with buffer C (0.1 M Potassium Phosphate buffer, 1.0 mM EDTA, pH 7.4) to a concentration of 2.5 mg/mL. Microsomal stability studies were carried out by adding 30 L of 1.5 M compound spiking solution to wells (1.5 L of 500 M spiking solution (10 L of 10 mM DMSO stock solution into 190 L ACN to eventually generate final test concentration of 1 uM) and 18.75 L of 20 mg/mL liver microsomes into 479.75 L of Buffer C). All samples were pre-incubated for approximately 15 minutes at 37 C. Following this, the reaction was initiated by adding 15 L of the NADPH solution (6 mM) with gentle mixing. Aliquots (40 L) were removed at 0, 5, 15, 30 and 45 minutes and quenched with ACN containing internal standard (135 L). Protein was removed by centrifugation (4000 rpm, 15 min) and the sample plate analysed for compound concentration by LC-MS/MS. Half-lives were then calculated by standard methods, comparing the concentration of analyte with the amount originally present.

EXAMPLES

Example 1Preparation of Compound 1

(34) ##STR00022##

Preparation of Azithromycin Aglycone (Az-AG) (1a)

(35) Azithromycin aglycone (1a) was generated using methods described in the literature (Djokic et al. 1988). In brief, azithromycin is converted to azithromycin aglycone by the acidic removal of the 3-O and 5-O sugars. The 5-O amino sugar is first oxidised and pyrolyzed to facilitate cleavage.

Generation of Biotransformation Strains Capable of Glycosylating Erythromycin Aglycones (Erythronolides)

Generation of S. erythraea 18A1 (pAES52)

(36) pAES52, an expression plasmid containing angAI, angAII, angCVI, ang-orf14, angMIII, angB, angMI and angMII along with the actII-ORF4 pactI/III expression system (Rowe et al. 1998) was generated as follows.

(37) The angolamycin sugar biosynthetic genes were amplified from a cosmid library of strain S. eurythermus ATCC23956 obtained from the American Type Culture Collection (Manassas, Va., USA). The biosynthetic gene cluster sequence was deposited as EU038272, EU220288 and EU232693 (Schell et al. 2008).

(38) The biosynthetic gene cassette was assembled in the vector pSG144 as described previously (Schell et al. 2008, ESI), adding sequential genes until the 8 required for sugar biosynthesis were obtained, creating plasmid pAES52.

(39) pAES52 was transformed into strain 18A1 (WO2005054265).

Transformation of pAES52 into S. erythraea 18A1

(40) pAES52 was transformed by protoplast into S. erythraea 18A1 using standard methods (Kieser et al. 2000, Gaisser et al. 1997). The resulting strain was designated ISOM-4522, which is deposited at the NCIMB on 24 Jan. 2017 with Accession number: NCIMB 42718.

Generation of S. erythraea SGT2 (pAES54)

(41) pAES54, an expression plasmid containing angAI, angAII, angCVI, ang-orf14, angMIII, angB, angMI and angMII along with the actII-ORF4 pactI/III expression system (Rowe et al., 1998) was generated as follows

(42) The angolamycin sugar biosynthetic genes were amplified from a cosmid library of strain S. eurythermus ATCC23956 obtained from the American Type Culture Collection (Manassas, Va., USA). The biosynthetic gene cluster sequence was deposited as EU038272, EU220288 and EU232693 (Schell et al. 2008)

(43) The biosynthetic gene cassette was assembled in the vector pSG144 as described previously (Schell et al. 2008, ESI), adding sequential genes until the 8 required for sugar biosynthesis were obtained, creating plasmid pAES52.

(44) Plasmid pAES54 was made by ligating the 11,541 bp SpeI-NheI fragment containing the actII-ORF4 pactI/III promotor system and the 8 ang genes was excised from pAES52 with the 5,087 bp XbaI-SpeI fragment from pGP9, containing an apramycin resistance gene, oriC, oriT for transfer in streptomycetes and phiBT1 integrase with attP site for integrative transformation. (The compatible NheI and XbaI sites were eliminated during the ligation.)

(45) pAES54 was then transformed into S. erythraea SGT2 (Gaisser et al. 2000, WO2005054265).

(46) Transformation of pAES54 into S. erythraea SGT2

(47) pAES54 was transferred by conjugation into S. erythraea SGT2 using standard methods. In brief, E. coli ET12567 pUZ8002 was transformed with pAES54 via standard procedures and spread onto 2TY with Apramycin (50 g/mL), Kanamycin (50 g/mL), and Chloramphenicol (33 g/mL) selection. This plate was incubated at 37 C. overnight. Colonies from this were used to set up fresh liquid 2TY cultures which were incubated at 37 C. until late log phase was reached. Cells were harvested, washed, mixed with spores of S. erythraea SGT2, spread onto plates of R.sub.6 and incubated at 28 C. After 24 hours, these plates were overlaid with 1 mL of sterile water containing 3 mg apramycin and 2.5 mg nalidixic acid and incubated at 28 C. for a further 5-7 days. Exconjugants on this plate were transferred to fresh plates of R.sub.6 containing apramycin (100 g/mL).

Alternative Biotransformation Strain

(48) Alternatively, BIOT-2945 (Schell et al. 2008) may be used as the biotransformation strain, as this also adds angolosamine to erythronolides.

Biotransformation of Azithromycin Aglycone to Prepare Compound 1

(49) Erlenmeyer flasks (250 mL) containing SV2 medium (40 mL) and 8 uL thiostrepton (25 mg/mL) were inoculated with 0.2 mL of spore stock of strain ISOM-4522 and incubated at 30 C. and shaken at 300 rpm with a 2.5 cm throw for 48 hours.

(50) SV2 Media:

(51) TABLE-US-00001 Ingredient Amount glycerol 15 g glucose 15 g soy peptone A3SC 15 g NaCl 3 g CaCO.sub.3 1 g RO water To final volume of 1 L Pre-sterilisation pH adjusted to pH 7.0 with 10M HCl Sterilised by autoclaving @ 121 C., 30 minutes

(52) Sterile bunged falcon tubes (50 mL) containing EryPP medium (7 mL) were prepared and inoculated with culture from seed flask (0.5 mL per falcon tube) without antibiotics. The falcons were incubated at 30 C. and shaken at 300 rpm with a 2.5 cm throw for 24 hours.

(53) ERYPP Medium:

(54) TABLE-US-00002 Ingredient Amount toasted soy flour (Nutrisoy) 30 g glucose 50 g (NH.sub.4).sub.2SO.sub.4 3 g NaCl 5 g CaCO.sub.3 6 g RO water To final volume of 1 L Pre-sterilisation pH adjusted to pH 7.0 with 10M HCl Sterilised in situ by autoclaving @ 121 C., 30 minutes Post sterilisation 10 ml/L propan-1-ol added

(55) After 24 hours, azithromycin aglycone (0.5 mM in DMSO, 50 uL) was added to each falcon tube and incubation continued at 300 rpm with a 2.5 cm throw for a further 6 days.

(56) Isolation of Compound 1

(57) Whole broth was adjusted to pH 9.5 and extracted twice with one volume of ethyl acetate. The organic layers were collected by aspiration following centrifugation (3,500 rpm, 25 minutes). The organic layers were combined and reduced in vacuo to reveal a brown gum that contained compound 1. This extract was partitioned between ethyl acetate (200 ml) and aqueous ammonium chloride (20 ml of a 50% concentrated solution). After separation, the organic layer was extracted with a further volume (200 ml) of the ammonium chloride aqueous solution. The combined aqueous layers were then adjusted to pH 9.0 with aqueous sodium hydroxide and then extracted twice with one volume equivalent of ethyl acetate. The organic layers were combined and reduced in vacuo to a brown solid. This extract was then applied to a silica column and eluted step wise (in 500 ml lots) with:

(58) TABLE-US-00003 Solvent Hexanes EtOAc MeOH Aq. NH.sub.4OH A 0.499 0.499 0 0.002 B 0.250 0.748 0 0.002 C 0 0.998 0 0.002 D 0 0.988 0.01 0.002 E 0 0.978 0.02 0.002 F 0 0.968 0.03 0.002 G 0 0.958 0.04 0.002

(59) Compound 1 was predominantly in F and G. These solvents were combined and reduced in vacuo to yield a brown solid containing compound 1. This material was then purified by preparative HPLC (C18 Gemini NX column, Phenomenex with 20 mM ammonium acetate and acetonitrile as solvent). Fraction containing the target compound were pooled and taken to dryness followed by desalting on a C18 SPE cartridge.

Example 2Preparation of Compound 3_(Known CompoundCorresponds to Compound 17 in Schell et al., 2008)

(60) ##STR00023##

(61) Erythronolide B (3a) can be generated by fermentation of strains of S. erythraea blocked in glycosylation, such as strains and processes described, for example, in U.S. Pat. No. 3,127,315 (e.g. NRRL2361, 2360, 2359 and 2338), Gaisser et al 2000 (e.g. S. erythraea DM BV CIII.

(62) Erythronolide B (3a) was then fed to a biotransformation strain capable of adding angolosamine to the 3-hydroxyl (such as NCIMB 42718) and compound 3 was isolated from the fermentation broth by standard methods.

Example 3Preparation of Compound 4

(63) ##STR00024##

(64) Azithromycin B aglycone (4a) was generated by hydrolysis of the sugars from azithromycin B in the same way as for azithromycin A.

(65) Azithromycin B aglycone (4a) was then fed to a biotransformation strain capable of adding angolosamine to the 3-hydroxyl (such as NCIMB 42718) and isolated from the fermentation broth using standard methods.

Example 4Preparation of Compound 5

(66) ##STR00025##

(67) Cyclobutyl erythronolide B (5a) was generated using methods described in WO98/01571. In brief, S. erythraea DM BV CIII (Gaisser et al. 2000) was transformed with pIG1 (Long et al., 2002, WO98/01571). Fermentation of the resulting strain with addition of cyclobutene carboxylic acid led to production of Cyclobutyl erythronolide B (5a). This was isolated from fermentation broths using standard methods. Cyclobutyl erythronolide B (5a) was then fed to a biotransformation strain capable of adding angolosamine to the 3-hydroxyl (such as NCIMB 42718) and compound 5 isolated from the fermentation broth using standard methods.

Example 5Preparation of Compound 6

(68) ##STR00026##

(69) A methyl group was removed from the aminosugar of compound 3 (see example 2) by adding it to a fermentation of ATCC 31771 and isolating compound 6 from the fermentation broth using standard methods.

Example 6Preparation of Compound 7

(70) ##STR00027##

(71) Compound 3 was treated with sodium borohydride in solvent. Following standard reaction work up compound 7 was purified by standard methods.

Example 7Preparation of Compound 8

(72) ##STR00028##

(73) 14-desmethyl erythronolide B (8a) was generated using methods described in WO2000/00618. In brief, S. erythraea DM BV CIII (Gaisser et al. 2000) was transformed with pPFL43. The resulting strain was fermented using typical methods and compound 8a was isolated using chromatography.

(74) 14-desmethyl erythronolide B (8a) was then fed to a biotransformation strain capable of adding angolosamine to the 3-hydroxyl (such as NCIMB 42718) and isolated from the fermentation broth using standard methods.

Example 8Preparation of Compound 9

(75) ##STR00029##

(76) 14-hydroxy angolosamine erythronolide B (9) was generated by feeding compound 3 (see example 2) to a fermentation of S. rochei ATCC 21250, which adds the hydroxyl group. Compound 9 was then isolated from the fermentation broth using standard methods.

Example 9Preparation of Compound 10

(77) ##STR00030##

(78) Compound 6 (6.0 mg, 0.01 mmol) was dissolved in dichloromethane (1 mL) and acetaldehyde (1.0 L, 0.02 mmol) was added. The reaction was stirred at room temperature and sodium triacetoxyborohydride (2.1 mg, 0.01 mmol) was added. The reaction was stirred for 30 minutes and then quenched by the addition of concentrated aqueous sodium bicarbonate (25 mL). The aqueous extract was extracted with ethyl acetate (325 mL). The organic extracts were combined, washed with concentrated brine solution and the solvent was removed in vacuo. The target compound 10 was then purified by preparative HPLC.

Example 10Preparation of Compound 12

(79) ##STR00031##

(80) Compound 3 (see example 2) was biotransformed to remove both methyl groups from the aminosugar by adding it to a fermentation of ATCC 31771 and compound 11 was isolated from the fermentation broth using standard methods.

(81) Compound 11 is dissolved in THF and acetaldehyde is added. The reaction is stirred at room temperature and sodium cyanoborohydride is added. The reaction is stirred further and the reaction is quenched by the addition of aqueous sodium bicarbonate. The aqueous extract is extracted with EtOAc (3vol equivalent). The organic extracts are combined, washed with brine and the solvent is removed in vacuo. The target compound 12 is then purified using standard methods.

Example 11Preparation of Compound 14

(82) ##STR00032##

(83) Compound 1 (see example 1) is biotransformed to remove a methyl group from the aminosugar by adding it to a fermentation of ATCC 31771 and compound 13 is isolated from the fermentation broth using standard methods.

(84) Compound 13 is dissolved in THF and acetaldehyde is added. The reaction is stirred at room temperature and sodium cyanoborohydride is added. The reaction is stirred further and the reaction is quenched by the addition of aqueous sodium bicarbonate. The aqueous extract is extracted with EtOAc (3vol equivalent). The organic extracts are combined, washed with brine and the solvent is removed in vacuo. The target compound 14 is then purified using standard methods.

Example 12Preparation of Compound 16

(85) ##STR00033##

(86) Compound 1 (see example 1) is biotransformed to remove both methyl groups from the aminosugar by adding it to a fermentation of ATCC 31771 and compound 15 is isolated from the fermentation broth using standard methods.

(87) Compound 15 is dissolved in THF and acetaldehyde is added. The reaction is stirred at room temperature and sodium cyanoborohydride is added. The reaction is stirred further and the reaction is quenched by the addition of aqueous sodium bicarbonate. The aqueous extract is extracted with EtOAc (3vol equivalent). The organic extracts are combined, washed with brine and the solvent is removed in vacuo. The target compound 16 is then purified using standard methods.

Example 13Assessment of Direct Antibacterial Activity

(88) The bioactivity of macrolide compounds against 4 strains of common gut bacteria (Escherichia coli, Streptococcus salivarius subsp. salivarius, Lactobacillus casei and Bifidobacterium longum subsp. infantis) and common mammalian skin isolate Micrococcus luteus, was assessed using the Minimum Inhibitory Concentration (MIC) assay. Bacterial strains were purchased from DSMZ (Brunswick, Germany) except M. luteus which was obtained from NCIMB, and stored in 20% glycerol at 80 C.

(89) Stock solutions (100% DMSO) of positive controls (azithromycin and erythromycin), and of test compounds 1 and 2 were diluted in broth to working stock concentrations of 256 g/ml (final assay testing concentration range 128 g/ml to 0.00391 g/ml). Stock solutions of all other compounds were diluted in broth to working stock concentrations of 128 g/ml (final assay testing concentration range 64 g/ml to 0.00195 g/ml).

(90) Bacterial strains were cultivated in appropriate broth in an anaerobic chamber at 37 C., except for M. luteus which was incubated aerobically at 37 C. 18 h cultures were diluted in broth to an OD.sub.595 of 0.1 and then further diluted 1:10. In 96-well plates, in duplicate, 200 l working stock of test compound was transferred to well 1 and serially diluted (1:2) in broth. 100 l bacterial suspension was aliquoted into each well and mixed thoroughly. Appropriate sterility controls were included and plates were incubated in an anaerobic chamber, or aerobically (M. luteus) at 37 C. for 18 h. The MIC was determined to be the concentration of test compound in the first well with no visible growth.

(91) TABLE-US-00004 TABLE 1 Escherichia Streptococcus Lactobacillus Bifidobacterium Micrococcus coli salivarius casei longum luteus Azithromycin <8 g/ml <0.5 g/ml <1.0 g/ml >64 g/ml 0.125 g/ml Erythromycin >64 g/ml <0.06 g/ml <0.25 g/ml >64 g/ml <0.0625 g/ml Compound 1 >64 g/ml >64 g/ml >64 g/ml >64 g/ml >256 g/ml Compound 4 >64 g/ml >64 g/ml >64 g/ml >64 g/ml Compound 5 >64 g/ml >64 g/ml >64 g/ml >64 g/ml Compound 6 >64 g/ml >64 g/ml >64 g/ml >64 g/ml Compound 7 >64 g/ml >64 g/ml >64 g/ml >64 g/ml Compound 8 >64 g/ml >64 g/ml >64 g/ml >64 g/ml Compound 9 >64 g/ml >64 g/ml >64 g/ml >64 g/ml EM703 64-128 g/ml

(92) As can be seen from the data presented in Table 1, compounds 1, 3, 4, 5, 6, 7, 8 and 9 show no antibacterial activity against any of the bacterial strains tested, whilst erythromycin and azithromycin show potent activity against a number of the strains.

Example 14Assessment of Immune Stimulatory Activity

(93) Human peripheral blood mononuclear cells (PBMCs) were purified from healthy donors with Ficoll-Paque density centrifugation. Cells were cultured in complete RPMI-1640 medium (Invitrogen) supplemented with 25 mM HEPES, L-glutamine, Sodium pyruvate (Sigma), 10% fetal bovine serum, 100 g/mL penicillin and 100 g/mL streptomycin (Hyclone). Cells were stimulated for 24 h (study 1-4) or 48 h to 1 week (study 5) in 37 C., 5% CO.sub.2 with increasing concentrations of compound 1 and 2 in tissue culture plates. The cells were removed from the plate, washed in PBS and analysed for expression of cell specific surface markers and MHC class I with flow cytomtery using monoclonal antibodies from BD Pharmingen and a FACS Canto II flow cytometer.

(94) Supernatant IL-10 was measured with a standard sandwich ELISA (all antibodies from BD Biosciences) after 48 hours and 7 days incubation with 2.5 uM of compound 1 and 100 U/mL IL-2 (Miltenyi Biotechnologies) in complete RPMI media, 37 C., 5% CO.sub.2.

(95) Study 1: After 24 h of in vitro stimulation of peripheral blood mononuclear cells (PBMC) with 1 M compound 1 (FIG. 8) the activation marker CD69 was upregulated on CD4+ T cells and B cells (FIG. 1).

(96) Study 2: We also observed upregulation of the molecule MHC class I (HLA-ABC) on T- and B-cells (FIG. 2), indicating an effect on antigen presentation of viral antigens.

(97) Study 3: Stimulation of PBMC with compound 1 led to the upregulation of the co-stimulatory molecule CD80 as well as the antigen presenting molecule MHC class II (HLA-DR) on monocytes (FIG. 3).

(98) Study 4: Monocytes differentiated into macrophages also upregulated CD80 in response to stimulation by compound 1 (FIG. 4).

(99) Study 5: PBMCs stimulated with compound 1 for 48 h and 7 days expressed an altered cytokine profile with increased production of the immunosuppressive cytokine IL-10, measured with sandwich ELISA. This indicate an immune inhibitory effect under certain conditions (FIG. 5).

(100) Study 6: PBMC were stimulated with compound 1 and cultured in RPMI media for 6 days in the presence of IL-2 (Miltenyi Biotechnologies) and Cell Trace Violet Dye (Invitrogen). Proliferation was measured with flow cytometry. Analysis of the immunological effect of compound 1 revealed an altered cytokine driven proliferation profile of T cells (FIG. 6).

(101) Study 7: Virus specific T cell proliferation was also affected by compound 1. PBMCs from cytomegalovirus (CMV) infected donors cultured in the presence of CMV antigen and compound 1 for 6 days displayed an altered phenotype of activated CMV specific CD8+ T cells with an increased expression of IL-7 receptor a (CD127), measured with flow cytometry (FIG. 7). CD127 is crucial for T cell homeostasis, differentiation and function, and reduced expression correlates with disease severity in HIV and other chronic viral diseases (Crawley et al. 2012).

(102) As can be seen, compound 1 has a surprising ability to specifically activate and modify an immune response by affecting antigen presentation, co-stimulation and T cell activation and proliferation. In many of these studies, compound 2, another related macrolide erythromycin analogue with altered glycosylation, previously published in Schell et al, 2008 (as compound 20), was included and showed little or no activity in the assays.

(103) Study 8: PBMCs from CMV infected donors cultured in the presence of CMV antigen where either untreated or exposed to compound 1 or compound 2 for 3 days. Exposure to compound 1 induced secretion of high levels of IFN-gamma, whereas antigen culture alone or antigen together with compounds A did not induce IFN-gamma secretion (FIG. 9).

(104) Study 9: Macrophages from healthy donors where exposed to compounds 1 or 2 for 48 hours. Only macrophages exposed to compound 1 secreted IFN-gamma whereas untreated macrophages and macrophages exposed to compound A did not secrete IFN-gamma (FIG. 10). Compound 1 is therefore able to induce IFN-gamma secretion in macrophages from healthy donors.

(105) Study 10: PBMCs and macrophages where exposed to compounds 1 or 2 for 2 days (FIG. 11). Basal expression of RANTES in PBMCs was unaffected by compound 2, whereas compound 1 induced a twofold upregulation of expression. Expression of RANTES was miniscule in macrophages, and compound 1 induced a high expression.

(106) Study 11: PBMCs and macrophages where exposed to compounds 1 and 2 for 2 days. PBMCs and macrophages secreted IL-12p70 in response to compound 1, whereas compound 2 failed to induce secretion over untreated cells (FIG. 12).

(107) Study 12: PBMCs, macrophages and CD4+ T cells where exposed to compounds 1 and 2 for 2 days. IL-1beta secretion was increased by compound 1 in macrophages and slightly in PBMCs while no IL-1beta was induced in CD4 + T cells (FIG. 13).

(108) Study 13: Compound 1 was administered i.v. to C57bl/6 mice at 0.165 mg/kg to 5 mg/kg. CD25+ cell abundance was increased in animals receiving the highest dose of 5 mg/kg (FIG. 14), as was body weight in the same group (not shown).

(109) Study 14: Compound 1 or 2 was administered i.v. to C57bl/6 mice. 24 h later the spleen was removed and MHC class I expression on CD11b+ splenocytes was assessed Compound 1 induced an increase in splenocyte cells with high MHC I expression, whereas no effect was observed in splenocytes from mice injected with compound A.

Example 15Assessment of Metabolic Stability

(110) The metabolic stability of the compounds of the invention was assessed in a standard human microsome stability assay (see general methods). Compounds with longer half-lives would be expected to have longer half-lives following dosing, which can be useful to allow less frequent dosing. Compounds with shorter half-lives could be useful for use as soft drugs where the active entity degrades rapidly once entering the patient's system. The half-life of the compounds assessed in shown in table 2 below:

(111) TABLE-US-00005 TABLE 2 T (minutes) Azithromycin 245 Erythromycin 31 Compound 1 108 Compound 3 35 Compound 4 160 Compound 5 83 Compound 6 109 Compound 7 56 Compound 8 33 Compound 9 100 Compound 10 31 Compound 17 151 Compound 18 25 Compound 19 18 EM703 97

(112) As can be seen, many of the compounds of the invention have increased or decreased metabolic stability as compared to azithromycin, erythromycin and EM703 (e.g. see EP1350510).

Example 16Assessment of Caco-2 Permeability

(113) The permeability of the compounds of the invention was assessed in a standard caco-2 bidirectional permeability assay (see general methods). Compounds with increased permeability would be expected to have better cell penetration and potential for effect, those with improved permeability and/or reduced efflux would be expected to have increased oral bioavailability. The permeability and efflux of the compounds is shown in table 3 below:

(114) TABLE-US-00006 TABLE 3 P.sub.app 10.sup.6/cm .Math. s.sup.1 Efflux ratio Azithromycin <0.14 >78 Compound 1 0.32 63 Compound 3 0.27 166 Compound 4 0.38 49 Compound 5 0.47 81 Compound 8 0.46 56 Compound 10 1.23 26 Compound 17 0.5 39 Compound 18 9.44 3.5 EM703 <0.15 >108

(115) As can be seen, many of the compounds of the invention have improved cell permeability and/or reduced efflux as compared to azithromycin and EM703 (e.g. see EP1350510).

Example 17Assessment of TLR.SUB.2 .Stimulation

(116) Compounds were tested using a TLR.sub.2 reporter assay (see general methods) that measured for stimulation of the TLR.sub.2 receptor. Stimulatory effect was measured as an increase in optical density (OD) due to release of secreted alkaline phosphatase (SEAP) and is shown in table 4:

(117) TABLE-US-00007 TABLE 4 OD after OD after OD after addition of addition of addition of 20 M 10 M 5 M test article test article test article Azithromycin 0.031 0.045 0.029 Erythromycin 0.045 0.065 0.035 Compound 1 0.458 0.202 0.111 Compound 2 0.044 0.010 0.046 Compound 3 0.026 0.015 0.043 Compound 17 0.234 0.155 0.054 EM703 0.033 0.024 0.040

(118) As can be seen, compound 1 stimulated TLR.sub.2 at concentrations down to 5 uM, compound 17 stimulated TLR.sub.2 at concentrations down to 10 uM, whilst erythromycin A, azithromycin and compounds 2 and 3, related macrolide erythromycin analogues with altered glycosylation, previously published in Schell et al, 2008 (as compounds 17 and 20), showed little or no stimulation at concentrations up to 20 uM.

Example 18Preparation of Compound 17

(119) ##STR00034##

(120) The aglycone 17a was generated from 9-deoxo-8a-aza-8a-methyl-8a-homoerythromycin (Wilkening 1993) followed by hydrolysis of the sugars. 17a was then fed to a biotransformation strain capable of adding angolosamine to the 3-hydroxyl (such as NCIMB 42718) and compound 17 isolated from the fermentation broth using standard methods.

Example 19Preparation of Compound 18

(121) ##STR00035##

(122) 6-deoxy erythronolide B (6-DEB, 18a) was fed to a biotransformation strain capable of adding angolosamine to the 3-hydroxyl (such as NCIMB 42718) and isolated from the fermentation broth using standard methods.

REFERENCES

(123) Kieser et al. 2000 Practical Streptomyces Genetics, Published by the John Innes Foundation

(124) Crawley et al. The influence of HIV on CD127 expression and its potential implications for IL-7 therapy. Semin Immunol. 2012 June; 24(3):231-40.

(125) Gaisser et al. Analysis of seven genes from the eryAl-eryK region of the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea. Mol. Gen. Genet., 1997 October; 256(3):239-51.

(126) Gaisser et al. A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea Mol. Micro., 2000; 36(2):391-401

(127) Schell et al. Engineered biosynthesis of hybrid macrolide polyketides containing D-angolosamine and D-mycaminose moieties Org. Biomol. Chem., 2008; 6:3315-3327

(128) LeMahieu et al. Glycosidic Cleavage Reactions on Erythromycin A. Preparation of Erythronolide A, J. Med. Chem., 1974, 17(9):953-956

(129) Djokic et al. Erythromycin Series. Part 13. Synthesis and Structure Elucidation of 10-Dihydro-10-deoxo-11-methyl-11-azaerythromycin A J. Chem. Res. (S), 1988; 5:152-153

(130) Glansdorp et al. Using Chemical Probes to Investigate the Sub-Inhibitory Effects of Azithromycin, Org. Biolmol. Chem., 2008; 208(6): 4120-4124

(131) Rowe et al. Construction of new vectors for high-level expression in actinomycetes. Gene. 1998 Aug. 17; 216(1):215-23.

(132) Long et al. Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase. Mol. Microbiol. 2002 March; 43(5):1215-25.

(133) Wilkening et al. The synthesis of novel 8a-aza-8a-homoerythromycin derivatives via the Beckmann rearrangement of (9Z)-erythromycin A oxime, Bioorg. Med. Chem Lett. 1993, 3 (6), p 1287-1292

(134) All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.

(135) Specific embodiments of the invention are given in the following list of items.

(136) Item 1

(137) A compound of Formula I

(138) ##STR00036##

(139) wherein X is selected from CO, NR.sub.3CH.sub.2, CH.sub.2NR.sub.3, NR.sub.3(CO), (CO)NR.sub.3, CNOH, and CH(OH), and R.sub.2 is a sugar of Formula (II) or Formula (III):

(140) ##STR00037##

(141) wherein R.sub.1 is selected from an alkyl, heteroalkyl, cycloalkyl, aryl, and heteroaryl moiety,

(142) wherein heteroatoms are selected from O, N, P, and S,

(143) wherein alkyl moiety is selected from C.sub.1-C.sub.6 alkyl groups that are optionally branched,

(144) wherein heteroalkyl moiety is selected from C.sub.1-C.sub.6 alkyl groups that are optionally branched or substituted and that optionally comprise one or more heteroatoms,

(145) wherein cycloalkyl moiety is selected from a C.sub.1-C.sub.6 cyclic alkyl groups that are optionally substituted and that optionally comprise one or more heteroatoms,

(146) wherein aryl moiety is selected from optionally substituted C.sub.6 aromatic rings,

(147) wherein heteroaryl moiety is selected from optionally substituted C.sub.1-C.sub.5 aromatic rings comprising one or more heteroatoms,

(148) wherein substituents, independently, are selected from alkyl, OH, F, Cl, NH.sub.2, NH-alkyl, NH-acyl, S-alkyl, S-acyl, O-alkyl, and O-acyl,

(149) wherein acyl is selected from C.sub.1-C.sub.4 optionally branched acyl groups,

(150) wherein R.sub.3 is selected from H and Me,

(151) wherein R.sub.4 is selected from H and Me,

(152) wherein R.sub.a is selected from H and CR.sub.21R.sub.22R.sub.23,

(153) wherein R.sub.21, R.sub.22, R.sub.23, and R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, and R.sub.10, independently, are selected from H, Me, NR.sub.11R.sub.12, NO.sub.2, and OR.sub.11,

(154) wherein R.sub.23 together with R.sub.4 in Formula (II), R.sub.4 together with R.sub.5 in Formula (II), R.sub.5 together with R.sub.7 in Formula (II), and R.sub.7 together with R.sub.9 in Formula (II), independently, may be joined to represent a bond to leave a double bond between the carbon atoms that each group is connected to,

(155) wherein R.sub.21 together with R.sub.22, R.sub.5 together with R.sub.6, R.sub.7 together with R.sub.8, or R.sub.9 together with R.sub.10 may be replaced with a carbonyl,

(156) wherein R.sub.11 and R.sub.12, independently, are selected from H and alkyl,

(157) wherein R.sub.13 is selected from H, OH, and OCH.sub.3,

(158) wherein R.sub.14 is selected from H and OH,

(159) and wherein one of R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9 or R.sub.10 is selected from NR.sub.11R.sub.12 and NO.sub.2,

(160) with the proviso that when R.sub.1 is Et, R.sub.2 is a sugar of Formula (II), R.sub.13 is OH, R.sub.14 is H, R.sub.a is H, R.sub.4 is Me, R.sub.5 is H, R.sub.6 is OH, R.sub.7 is H, R.sub.8 is NR.sub.11R.sub.12, R.sub.9 is H, and R.sub.10 is H, X may not be CO.

(161) Item 2

(162) A compound of item 1

(163) with the proviso that when R.sub.1 is Et, R.sub.2 is a sugar of Formula (II), R.sub.13 is H or OH, R.sub.14 is H or OH, R.sub.a is H, R.sub.4 is Me, R.sub.5 is H, R.sub.6 is OH, R.sub.7 is H, R.sub.8 is NR.sub.11R.sub.12, R.sub.9 is H, and R.sub.10 is H, X may not be CO.

(164) with the proviso that when R.sub.1 is Et, R.sub.2 is a sugar of Formula (II), R.sub.13 is H or OH, R.sub.14 is H or OH, R.sub.a is H, R.sub.4 is Me, R.sub.5 is OH, R.sub.6 is H, R.sub.7 is OH, R.sub.8 is Me, R.sub.9 is H, and R.sub.10 is H, X may not be CO.

(165) with the proviso that when R.sub.1 is Et, R.sub.2 is a sugar of Formula (II), R.sub.13 is H or OH, R.sub.14 is H or OH, R.sub.a is H, R.sub.4 is Me, R.sub.5 is OH, R.sub.6 is H, R.sub.7 is H, R.sub.8 is NR.sub.11R.sub.12, R.sub.9 is H, and R.sub.10 is OH, X may not be CO.

(166) Item 3

(167) A compound according to Item 1 or 2,

(168) wherein R.sub.23 together with R.sub.4 in Formula (II), R.sub.4 together with R.sub.5 in Formula (II), R.sub.5 together with R.sub.7 in Formula (II), and R.sub.7 together with R.sub.9 in Formula (II), independently, may be joined to represent a bond to leave a double bond between the carbon atoms that each group is connected to, so that

(169) wherein if R.sub.23 and R.sub.4 are joined to forma double bond, then Formula (II) can be represented by:

(170) ##STR00038##

(171) wherein if R.sub.4 and R.sub.5 are joined to form a double bond, then Formula (II) can be represented by:

(172) ##STR00039##

(173) wherein if R.sub.5 and R.sub.7 are joined to form a double bond, then Formula (II) can be represented by:

(174) ##STR00040##

(175) wherein if R.sub.7 and R.sub.9 are joined to form a double bond, then Formula (II) can be represented by:

(176) ##STR00041##

(177) wherein R.sub.4 together with R.sub.5 in Formula (III), R.sub.4 together with R.sub.7 in Formula (III), and R.sub.7 together with R.sub.9 in Formula (III), independently, may be joined to represent a bond to leave a double bond between the carbon atoms that each group is connected to, so that

(178) wherein if R.sub.4 and R.sub.5 are joined to form a double bond, then Formula (III) can be represented by:

(179) ##STR00042##

(180) wherein if R.sub.4 and R.sub.7 are joined to form a double bond, then Formula (III) can be represented by:

(181) ##STR00043##

(182) wherein if R.sub.7 and R.sub.9 are joined to form a double bond, then Formula (III) can be represented by:

(183) ##STR00044##

(184) Item 4

(185) A compound according to Formula (I)

(186) ##STR00045##

(187) wherein X is selected from CO, NR.sub.3CH.sub.2, and CH(OH), and R.sub.2 is a sugar of Formula (II):

(188) ##STR00046##

(189) wherein R.sub.1 is selected from and alkyl or cycloalkyl moiety,

(190) wherein alkyl moiety is selected from C.sub.1-C.sub.6 alkyl groups that are optionally branched and, independently, optionally hydroxylated,

(191) wherein cycloalkyl moiety is selected from C.sub.1-C.sub.6 optionally substituted cyclic alkyl groups,

(192) wherein substituents are selected from alkyl and OH,

(193) wherein R.sub.3 is selected from H and Me,

(194) wherein R.sub.4 is selected from H and Me,

(195) wherein R.sub.a is selected from H and CR.sub.21R.sub.22R.sub.23,

(196) wherein R.sub.21, R.sub.22, R.sub.23, and R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, and R.sub.10, independently, are selected from H, Me, NR.sub.11R.sub.12, NO.sub.2, and OR.sub.11,

(197) wherein R.sub.23 together with R.sub.4 in Formula (II), R.sub.4 together with R.sub.5 in Formula (II), R.sub.5 together with R.sub.7 in Formula (II), and R.sub.7 together with R.sub.9 in Formula (II), independently, may be joined to represent a bond to leave a double bond between the carbon atoms that each group is connected to, so that

(198) wherein if R.sub.23 and R.sub.4 are joined to forma double bond, then Formula (II) can be represented by:

(199) ##STR00047##

(200) wherein if R.sub.4 and R.sub.5 are joined to form a double bond, then Formula (II) can be represented by:

(201) ##STR00048##

(202) wherein if R.sub.5 and R.sub.7 are joined to form a double bond, then Formula (II) can be represented by:

(203) ##STR00049##

(204) wherein if R.sub.7 and R.sub.9 are joined to form a double bond, then Formula (II) can be represented by:

(205) ##STR00050##

(206) wherein R.sub.21 together with R.sub.22, R.sub.5 together with R.sub.6, R.sub.7 together with R.sub.8, or R.sub.9 together with R.sub.10 may be replaced with a carbonyl,

(207) wherein R.sub.11 and R.sub.12, independently, are selected from H and alkyl,

(208) wherein R.sub.13 is selected from H, OH, and OCH.sub.3,

(209) wherein R.sub.14 is selected from H and OH,

(210) and wherein one of R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9 or R.sub.10 is selected from NR.sub.11R.sub.12 and NO.sub.2,

(211) with the proviso that when R.sub.1 is Et, R.sub.2 is a sugar of Formula (II), R.sub.13 is OH, R.sub.14 is H, R.sub.a is H, R.sub.4 is Me, R.sub.5 is H, R.sub.6 is OH, R.sub.7 is H, R.sub.8 is NR.sub.11R.sub.12, R.sub.9 is H, and R.sub.10 is H, X may not be CO.

(212) Item 5

(213) A compound according to any one of the preceding items, wherein R.sub.2 is selected from L-daunosamine, L-acosamine, L-ristosamine, D-ristosamine, 4-oxo-L-vancosamine, L-vancosamine, D-forosamine, L-actinosamine, 3-epi-L-vancosamine, L-vicenisamine, L-mycosamine, D-mycosamine, D-3-N-methyl-4-O-methyl-L-ristosamine, D-desosamine, N,N-dimethyl-L-pyrrolosamine, L-megosamine, L-nogalamine, L-rhodosamine, D-angolosamine, L-kedarosamine, 2-N-methyl-D-fucosamine, 3-N,N-dimethyl-L-eremosamine, D-ravidosamine, 3-N,N-dimethyl-D-mycosamine/D-mycaminose, 3-N-acetyl-D-ravidosamine, 4-O-acetyl-D-ravidosamine, 3-N-acetyl-4-O-acetyl-D-ravidosamine, D-glucosamine, N-acetyl-D-glucosamine, L-desosamine, D-amosamine, D-viosamine, L-avidinosamine, D-gulosamine, D-allosamine, and L-sibirosamine.

(214) Item 6

(215) A compound according to any one of the preceding items, wherein R.sub.2 is selected from D-angolosamine, N-desmethyl D-angolosamine, N-didesmethyl D-angolosamine, N-desmethyl N-ethyl D-angolosamine, and N-didesmethyl N-diethyl D-angolosamine.

(216) Item 7

(217) A compound according to any one of the preceding items, wherein R.sub.2 is a sugar according to Formula (II).

(218) Item 8

(219) A compound according to any one of the preceding items, wherein R.sub.2 is a sugar according to formula II wherein R.sub.a is H, R.sub.4 is Me, R.sub.5 is H, R.sub.6 is OH, R.sub.7 is H, R.sub.8 is NR.sub.11R.sub.12, R.sub.9 is H and R.sub.10 is H.

(220) Item 9

(221) A compound according to any one of the preceding items, wherein R.sub.11 is selected from H, Me, and Et, and R.sub.12 is selected from H, Me, and Et.

(222) Item 10

(223) A compound according to any one of the preceding items, wherein R.sub.11 is Et and R.sub.12 is Et.

(224) Item 11

(225) A compound according to any one of items 1-87, wherein R.sub.11 is Me and R.sub.12 is Et.

(226) Item 12

(227) A compound according to any one of the preceding items, wherein X is selected from CO, NR.sub.3CH.sub.2 and CH(OH)

(228) Item 13

(229) A compound according to any one of the preceding items, wherein R.sub.1 is selected from Me, Et, and cycloalkyl.

(230) Item 14

(231) A compound according to any one of the preceding items, wherein R.sub.1 is selected from Me and Et.

(232) Item 15

(233) A compound according to any one of the preceding items selected from:

(234) ##STR00051## ##STR00052##

(235) Item 16

(236) A compound as defined in any one of the preceding items for use in medicine.

(237) Item 17

(238) A compound as defined in any one of items 1-15 for use in the treatment of viral disease.

(239) Item 18

(240) A compound as defined in any one of items 1-15 for use in the treatment of HIV/AIDS.

(241) Item 19

(242) A method for preparing a compound as defined in any one of items 1-15, the method comprising addition of an aglycone with Formula (IV)

(243) ##STR00053##
to a culture of a biotransformation strain which glycosylates at the 3-hydroxyl position.

(244) Item 20

(245) A method according to item 19, wherein the biotransformation strain expresses glycosyltransferases with >70% homology to AngMII and AngMIII