Immune stimulating macrolide

Abstract

The present invention provides immune stimulating macrolide of formula (I). The macrolide has utility in treating viral diseases and cancer. ##STR00001##

Claims

1. A compound having the structure of Formula (I): ##STR00004##

2. A pharmaceutical composition comprising the compound according to claim 1 and one or more pharmaceutically acceptable excipients, diluents, or carriers.

3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is formulated for parenteral, oral, topical, or mucosal administration or administration by inhalation.

4. A method for treating a viral infection, the method comprising administering to a human or animal subject in need thereof a therapeutically effective amount of the compound according to claim 1.

5. A method for treating a disease caused by a viral infection, the method comprising administering to a human or animal subject in need thereof a therapeutically effective amount of the compound according to claim 1; wherein the disease is selected from AIDS, Borna Disease, Condyloma Acuminata, Dengue fever, Contagious Ecthyma, Erythema Infectiosum, Viral Hemorrhagic Fever, Viral Hepatitis, Herpes Simplex, Infectious Mononucleosis, Influenza, Lassa Fever, Measles, Mumps, Molluscum Contagiosum, Phlebotomus fever, Rift Valley Fever, Rubella, Smallpox, Subacute Sclerosing Panencephalitis, Tumor Virus Infections, West Nile Fever, Yellow Fever, and Rabies.

6. A method for treating cancer, the method comprising administering to a human or animal subject in need thereof a therapeutically effective amount of the compound according to claim 1; wherein the cancer is selected from Adrenal Cancer, Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNS Tumors, Breast Cancer, Castleman Disease, Cervical Cancer, Colon/Rectum Cancer, Endometrial Cancer, Esophagus Cancer, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Acute Myeloid Leukemia, Chronic Lymphocytic Leukemia, Acute Lymphocytic Leukemia, Chronic Myeloid Leukemia, Chronic Myelomonocytic Leukemia, Liver Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, Lung Carcinoid Tumor, Lymphoma, Malignant Mesothelioma, Multiple Myeloma, Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Basal and Squamous Cell Skin Cancer, Melanoma, Merkel Cell Skin Cancer, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.

7. The method according to claim 4, wherein the viral infection is caused by human immunodeficiency deficiency virus (HIV), Adenovirus, Alphavirus, Arbovirus, Bunyavirus, Calicivirus, Coronavirus, Coxsackievirus, Cytomegalovirus, Dengue fever virus, Epstein-Barr virus, Hantavirus, Herpes Simplex Virus, Herpes Zoster virus, Lassa Fever virus, Paramyxovirus, Polyoma-virus, Slow Disease Virus, West Nile Virus, Yellow Fever Virus, Rabies Virus, and Respiratory Syncitial Virus.

8. The method according to claim 4, wherein the viral infection is caused by HIV.

9. The method according to claim 4, wherein the viral infection is caused by Coronavirus.

10. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is in the form of a tablet, capsule, film, ovule, elixir, solution, emulsion, suspension, cachet, powder, granule(s), bolus, electuary, or paste.

11. A compound that is a pharmaceutically acceptable salt of the compound having the structure of Formula (I): ##STR00005##

12. The compound according to claim 11, wherein the pharmaceutically acceptable salt is a hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, or tannic acid salt.

13. A pharmaceutical composition comprising the compound according to claim 11 and one or more pharmaceutically acceptable excipients, diluents, or carriers.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is formulated for parenteral, oral, topical, or mucosal administration or administration by inhalation.

15. A method for treating a viral infection, the method comprising administering to a human or animal subject in need thereof a therapeutically effective amount of the compound according to claim 11.

16. The method according to claim 15, wherein the viral infection is caused by human immunodeficiency deficiency virus (HIV), Adenovirus, Alphavirus, Arbovirus, Bunyavirus, Calicivirus, Coronavirus, Coxsackievirus, Cytomegalovirus, Dengue fever virus, Epstein-Barr virus, Hantavirus, Herpes Simplex Virus, Herpes Zoster virus, Lassa Fever virus, Paramyxovirus, Polyoma-virus, Slow Disease Virus, West Nile Virus, Yellow Fever Virus, Rabies Virus, and Respiratory Syncitial Virus.

17. The method according to claim 15, wherein the viral infection is caused by HIV.

18. The method according to claim 15, wherein the viral infection is caused by Coronavirus.

19. A method for treating a disease caused by a viral infection, the method comprising administering to a human or animal subject in need thereof a therapeutically effective amount of the compound according to claim 11; wherein the disease is selected from AIDS, Borna Disease, Condyloma Acuminata, Dengue fever, Contagious Ecthyma, Erythema Infectiosum, Viral Hemorrhagic Fever, Viral Hepatitis, Herpes Simplex, Infectious Mononucleosis, Influenza, Lassa Fever, Measles, Mumps, Molluscum Contagiosum, Phlebotomus fever, Rift Valley Fever, Rubella, Smallpox, Subacute Sclerosing Panencephalitis, Tumor Virus Infections, West Nile Fever, Yellow Fever, and Rabies.

20. A method for treating cancer, the method comprising administering to a human or animal subject in need thereof a therapeutically effective amount of the compound according to claim 11; wherein the cancer is selected from Adrenal Cancer, Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNS Tumors, Breast Cancer, Castleman Disease, Cervical Cancer, Colon/Rectum Cancer, Endometrial Cancer, Esophagus Cancer, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Acute Myeloid Leukemia, Chronic Lymphocytic Leukemia, Acute Lymphocytic Leukemia, Chronic Myeloid Leukemia, Chronic Myelomonocytic Leukemia, Liver Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, Lung Carcinoid Tumor, Lymphoma, Malignant Mesothelioma, Multiple Myeloma, Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Basal and Squamous Cell Skin Cancer, Melanoma, Merkel Cell Skin Cancer, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.

Description

LEGENDS TO FIGURES

(1) FIG. 1. The structures of the macrolides Erythromycin A, Compound 1, Compound 2, compound 3 and EM703.

(2) FIG. 2. CD69 upregulation on T- and B-cells. PBMC were treated for 24 h with compound 1, compound 2 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 2 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 2 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 2 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 2 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 2 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: % CD25 high 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 CD11 b expression was measured by flow cytometry.

EXPERIMENTAL

(16) Materials

(17) Unless otherwise indicated, all reagents used in the examples below are obtained from commercial sources.

(18) Antibodies

(19) Anti-CD80 V450, anti-CD69 PE, anti HLA-DR APC-R700, anti CD127-APC, and anti-Anti-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.

(20) Media

(21) 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

(22) General Biology Methods

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

(24) General Compound Method

(25) Compound analysissolubility and stability in solution

(26) Analysis of Fermentation Broths and Compounds

(27) 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.

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

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

(30) 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%.

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

(32) Analysis of Marker Expression by Flow Cytometry

(33) 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 cytomtery using a BD FACS Canto II flow cytometer. All samples were tested in duplicates.

(34) Cytomegalovirus (CMV) Cultures

(35) 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.

(36) ELISA

(37) 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

(38) TLR2 Assay

(39) 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 TLR2 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).

(40) 20 l of each test article were used to stimulate the hTLR2 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.

(41) Assessment of Cell Permeability (Bidirectional)

(42) 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 B to A permeability.
Drug permeability: Papp=(VA/(Areatime))([drug]accepter/(([drug]initial,donor)Dilution Factor).
Assessment of Metabolic Stability (Microsome Stability Assay)

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

(44) 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 1Generation of Compound 1

(45) Generation of az-AG

(46) Azithromycin aglycone was generated using methods described in the literature (Djokic, S., 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.

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

(48) Generation of S. erythraea 18A1 (pAES52)

(49) 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.

(50) 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, 2008).

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

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

(53) Transformation of pAES52 into S. erythraea 18A1

(54) 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.

(55) Generation of S. erythraea SGT2 (pAES54)

(56) 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

(57) 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, 2008).

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

(59) 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.)

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

(61) Transformation of pAES54 into S. erythraea SGT2

(62) 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 R6 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 R6 containing apramycin (100 g/mL).

(63) Alternative Biotransformation Strain

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

(65) Biotransformation of Azithromycin Aglycone

(66) 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.

(67) SV2 Media

(68) 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

(69) 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.

(70) ERYPP Medium

(71) 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

(72) 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.

(73) Isolation of Compound 1

(74) 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:

(75) 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
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 2Assessment of Direct Antibacterial Activity

(76) 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. 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). 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.

(77) 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 EM703 64-128 g/ml

(78) As can be seen from the data presented in Table 1, compound 1 shows no antibacterial activity against any of the bacterial strains tested, whilst erythromycin and azithromycin show potent activity against a number of the strains.

Example 3Assessment of Immune Stimulatory Activity

(79) 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 cytometry using monoclonal antibodies from BD Pharmingen and a FACS Canto II flow cytometer.

(80) 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.

(81) Study1: After 24 h of in vitro stimulation of peripheral blood mononuclear cells (PBMC) with 1 M compound 1 (FIG. 1) the activation marker CD69 was upregulated on CD4+ T cells and B cells (FIG. 2).

(82) Study 2: We also observed upregulation of the molecule MHC class I (HLA-ABC) on T- and B-cells (FIG. 3), indicating an effect on antigen presentation of viral antigens.

(83) 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. 4).

(84) Study 4: Monocytes differentiated into macrophages also upregulated CD80 in response to stimulation by compound 1 (FIG. 5).

(85) 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. 6).

(86) 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. 7).

(87) 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 (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 Sem Imm 2012).

(88) 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.

(89) 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 2 did not induce IFN-gamma secretion (FIG. 9).

(90) 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 2 did not secrete IFN-gamma (FIG. 10). Compound 1 is therefore able to induce IFN-gamma secretion in macrophages from healthy donors.

(91) 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.

(92) 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).

(93) 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).

(94) 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).

(95) 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 2.

Example 4Assessment of Activity Against TLR2

(96) Compounds were tested using a TLR2 reporter assay (see general methods) that measured for stimulation of the TLR2 receptor. Stimulatory effect was measured as an increase in optical density, as compared to the negative control (OD) due to release of secreted alkaline phosphatase (SEAP) and is shown in table 2.

(97) TABLE-US-00005 TABLE 2 OD after OD after OD after addition addition of 20 M addition of 10 M of 5 M test test article test article article Erythromycin 0.045 0.065 0.035 A Azithromycin 0.031 0.045 0.029 Compound 2 0.044 0.010 0.046 Compound 1 0.458 0.202 0.111 EM703 0.033 0.024 0.040 Compound 3 0.026 0.015 0.043

(98) As can be seen, compound 1 stimulated TLR2 at concentrations down to 5 uM, whilst erythromycin A, azithromycin, EM703 (e.g. see EP1350510) and compounds 2 and 3, related macrolide erythromycin analogue 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 5Assessment of Caco-2 Permeability

(99) Compounds were tested using a standard caco-2 bidirectional permeability assay (see general methods). The data generated is shown in table 3.

(100) TABLE-US-00006 TABLE 3 A to B permeability (Papp 10.sup.6/cm .Math. s1) Efflux ratio Azithromycin <0.14 >77.6 Compound 1 0.32 63.4 EM703 <0.15 >108

(101) As can be seen from the data in table 3, Compound 1 is more cell permeable and has a lower efflux ratio than both Azithromycin and EM703 (e.g. see EP1350510).

Example 6Assessment of Metabolic Stability

(102) The metabolic stability of the compound 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 4 below:

(103) TABLE-US-00007 TABLE 4 T (minutes) Azithromycin 245 Erythromycin 31 Compound 1 108 EM703 97

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