CA2379321A1 - Pseudomycin amide and ester analogs - Google Patents
Pseudomycin amide and ester analogs Download PDFInfo
- Publication number
- CA2379321A1 CA2379321A1 CA002379321A CA2379321A CA2379321A1 CA 2379321 A1 CA2379321 A1 CA 2379321A1 CA 002379321 A CA002379321 A CA 002379321A CA 2379321 A CA2379321 A CA 2379321A CA 2379321 A1 CA2379321 A1 CA 2379321A1
- Authority
- CA
- Canada
- Prior art keywords
- alkyl
- alkoxy
- aromatic ring
- membered aromatic
- hydrogen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 150000001408 amides Chemical class 0.000 title claims description 23
- 150000002148 esters Chemical class 0.000 title description 18
- 150000001875 compounds Chemical class 0.000 claims abstract description 90
- 238000000034 method Methods 0.000 claims abstract description 48
- -1 a-acetoacetate Chemical group 0.000 claims description 68
- 125000000217 alkyl group Chemical group 0.000 claims description 60
- 125000003118 aryl group Chemical group 0.000 claims description 52
- 239000001257 hydrogen Substances 0.000 claims description 50
- 229910052739 hydrogen Inorganic materials 0.000 claims description 50
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 46
- 125000003545 alkoxy group Chemical group 0.000 claims description 44
- 150000002431 hydrogen Chemical class 0.000 claims description 37
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 29
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 23
- 241001465754 Metazoa Species 0.000 claims description 22
- 125000000753 cycloalkyl group Chemical group 0.000 claims description 21
- 125000003282 alkyl amino group Chemical group 0.000 claims description 16
- 150000001412 amines Chemical class 0.000 claims description 16
- 229910052736 halogen Inorganic materials 0.000 claims description 15
- 150000002367 halogens Chemical class 0.000 claims description 15
- 125000000229 (C1-C4)alkoxy group Chemical group 0.000 claims description 14
- 125000003277 amino group Chemical group 0.000 claims description 14
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 13
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 claims description 13
- 230000000843 anti-fungal effect Effects 0.000 claims description 13
- 150000003839 salts Chemical class 0.000 claims description 13
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical compound C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 claims description 12
- 239000012453 solvate Substances 0.000 claims description 12
- 239000007822 coupling agent Substances 0.000 claims description 11
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 9
- 235000001014 amino acid Nutrition 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 8
- 125000006732 (C1-C15) alkyl group Chemical group 0.000 claims description 7
- 229940121375 antifungal agent Drugs 0.000 claims description 7
- 239000000651 prodrug Substances 0.000 claims description 7
- 229940002612 prodrug Drugs 0.000 claims description 7
- 239000004305 biphenyl Substances 0.000 claims description 6
- 235000010290 biphenyl Nutrition 0.000 claims description 6
- 208000015181 infectious disease Diseases 0.000 claims description 6
- 125000001072 heteroaryl group Chemical group 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 4
- 206010017533 Fungal infection Diseases 0.000 claims description 3
- 208000031888 Mycoses Diseases 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 125000006273 (C1-C3) alkyl group Chemical group 0.000 claims description 2
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 2
- 125000005913 (C3-C6) cycloalkyl group Chemical group 0.000 claims description 2
- 206010017543 Fungal skin infection Diseases 0.000 claims description 2
- 125000005599 alkyl carboxylate group Chemical group 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000009885 systemic effect Effects 0.000 claims description 2
- 125000000008 (C1-C10) alkyl group Chemical group 0.000 claims 13
- 125000006314 C5-C8 alkoxy group Chemical group 0.000 claims 6
- 125000006374 C2-C10 alkenyl group Chemical group 0.000 claims 1
- 150000007854 aminals Chemical class 0.000 claims 1
- YDBVAWZTOAZPTJ-REOHCLBHSA-N (2s)-2-(hydroxyamino)butanedioic acid Chemical group ON[C@H](C(O)=O)CC(O)=O YDBVAWZTOAZPTJ-REOHCLBHSA-N 0.000 abstract description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 abstract description 5
- 230000002538 fungal effect Effects 0.000 abstract description 5
- 238000012986 modification Methods 0.000 abstract description 4
- 235000003704 aspartic acid Nutrition 0.000 abstract description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 description 31
- 108010033821 pseudomycin B Proteins 0.000 description 30
- 241000589615 Pseudomonas syringae Species 0.000 description 29
- NYRWZRIVFVWNTD-SGRJACMKSA-N (2s)-2-[(3s,6s,9z,12s,15s,18s,21s,24r,27s)-18-(4-aminobutyl)-15,24-bis(2-aminoethyl)-21-(carboxymethyl)-3-[(1s)-2-chloro-1-hydroxyethyl]-9-ethylidene-12-[(1s)-1-hydroxyethyl]-27-[[(3r)-3-hydroxytetradecanoyl]amino]-2,5,8,11,14,17,20,23,26-nonaoxo-1-oxa-4, Chemical compound CCCCCCCCCCC[C@@H](O)CC(=O)N[C@H]1COC(=O)[C@H]([C@H](O)CCl)NC(=O)[C@H]([C@H](O)C(O)=O)NC(=O)\C(=C\C)NC(=O)[C@H]([C@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CCN)NC1=O NYRWZRIVFVWNTD-SGRJACMKSA-N 0.000 description 27
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 26
- 239000000203 mixture Substances 0.000 description 25
- 230000015572 biosynthetic process Effects 0.000 description 24
- 238000004128 high performance liquid chromatography Methods 0.000 description 22
- 239000000243 solution Substances 0.000 description 21
- 238000003786 synthesis reaction Methods 0.000 description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 15
- 125000006239 protecting group Chemical group 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 13
- 239000002253 acid Substances 0.000 description 13
- 230000000694 effects Effects 0.000 description 13
- 239000000047 product Substances 0.000 description 12
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 241000222122 Candida albicans Species 0.000 description 10
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 10
- 229930014626 natural product Natural products 0.000 description 10
- 239000002904 solvent Substances 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 229910052799 carbon Inorganic materials 0.000 description 9
- 238000004108 freeze drying Methods 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- XDPWWEOIDYYUDN-HCEIJDMSSA-N 2-[(9z)-18-(4-aminobutyl)-15,24-bis(2-aminoethyl)-21-(carboxymethyl)-3-(2-chloro-1-hydroxyethyl)-27-(3,4-dihydroxytetradecanoylamino)-9-ethylidene-12-(1-hydroxyethyl)-2,5,8,11,14,17,20,23,26-nonaoxo-1-oxa-4,7,10,13,16,19,22,25-octazacyclooctacos-6-yl]-2-h Chemical compound CCCCCCCCCCC(O)C(O)CC(=O)NC1COC(=O)C(C(O)CCl)NC(=O)C(C(O)C(O)=O)NC(=O)\C(=C\C)NC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCCCN)NC(=O)C(CC(O)=O)NC(=O)C(CCN)NC1=O XDPWWEOIDYYUDN-HCEIJDMSSA-N 0.000 description 8
- 239000012317 TBTU Substances 0.000 description 8
- 125000003368 amide group Chemical group 0.000 description 8
- 108010033876 pseudomycin A Proteins 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 239000007858 starting material Substances 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- 229940024606 amino acid Drugs 0.000 description 7
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 6
- 238000002953 preparative HPLC Methods 0.000 description 6
- 125000001424 substituent group Chemical group 0.000 description 6
- 241001225321 Aspergillus fumigatus Species 0.000 description 5
- 241000233866 Fungi Species 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 230000002411 adverse Effects 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 5
- 229940095731 candida albicans Drugs 0.000 description 5
- 125000001559 cyclopropyl group Chemical group [H]C1([H])C([H])([H])C1([H])* 0.000 description 5
- 238000010511 deprotection reaction Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 5
- DBGVGMSCBYYSLD-UHFFFAOYSA-N tributylstannane Chemical compound CCCC[SnH](CCCC)CCCC DBGVGMSCBYYSLD-UHFFFAOYSA-N 0.000 description 5
- 210000003462 vein Anatomy 0.000 description 5
- KZSKIDBMTSZKLE-NDWYDANWSA-N (2s)-2-[(3s,6s,9e,12s,15r,18s,21s,24r,27s)-18-(4-aminobutyl)-15,24-bis(2-aminoethyl)-21-(carboxymethyl)-3-[(1s)-2-chloro-1-hydroxyethyl]-9-ethylidene-12-[(1s)-1-hydroxyethyl]-27-[[(3r)-3-hydroxyhexadecanoyl]amino]-2,5,8,11,14,17,20,23,26-nonaoxo-1-oxa-4,7 Chemical compound CCCCCCCCCCCCC[C@@H](O)CC(=O)N[C@H]1COC(=O)[C@H]([C@H](O)CCl)NC(=O)[C@H]([C@H](O)C(O)=O)NC(=O)\C(=C/C)NC(=O)[C@H]([C@H](C)O)NC(=O)[C@@H](CCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CCN)NC1=O KZSKIDBMTSZKLE-NDWYDANWSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 125000003342 alkenyl group Chemical group 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 239000003429 antifungal agent Substances 0.000 description 4
- 125000004122 cyclic group Chemical group 0.000 description 4
- 230000020176 deacylation Effects 0.000 description 4
- 238000005947 deacylation reaction Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Substances C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
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- WSNMPAVSZJSIMT-UHFFFAOYSA-N COc1c(C)c2COC(=O)c2c(O)c1CC(O)C1(C)CCC(=O)O1 Chemical compound COc1c(C)c2COC(=O)c2c(O)c1CC(O)C1(C)CCC(=O)O1 WSNMPAVSZJSIMT-UHFFFAOYSA-N 0.000 description 3
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- 125000001931 aliphatic group Chemical group 0.000 description 3
- 125000000304 alkynyl group Chemical group 0.000 description 3
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- 125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 3
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 2
- LKIUUVZMMOKAMF-UHFFFAOYSA-N 2-(2,6-difluorophenyl)-2-methylpropanoic acid Chemical compound OC(=O)C(C)(C)C1=C(F)C=CC=C1F LKIUUVZMMOKAMF-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
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- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229960001153 serine Drugs 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- OGNAOIGAPPSUMG-UHFFFAOYSA-N spiro[2.2]pentane Chemical compound C1CC11CC1 OGNAOIGAPPSUMG-UHFFFAOYSA-N 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000008227 sterile water for injection Substances 0.000 description 1
- 150000003890 succinate salts Chemical class 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical class [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229930185586 syringostatin Natural products 0.000 description 1
- 229930194835 syringotoxin Natural products 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 150000003567 thiocyanates Chemical class 0.000 description 1
- 125000003396 thiol group Chemical class [H]S* 0.000 description 1
- 125000004055 thiomethyl group Chemical group [H]SC([H])([H])* 0.000 description 1
- 229930192474 thiophene Natural products 0.000 description 1
- 231100000440 toxicity profile Toxicity 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
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- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Communicable Diseases (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Saccharide Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Acid-modification of the aspartic acid and/or hydroxyaspartic acid units of naturally occurring or semi-synthetic pseudomycin compounds is described as well as methods of treatment against fungal activities.
Description
PSEUDOMYCIN AMIDE & ESTER ANALOGS
FIELD OF THE INVENTION
The present invention relates to pseudomycin compounds, in particular, acid-modified, semi-synthetic pseudomycin compounds.
BACKGROUND OF THE INVENTION
Pseudomycins are natural products isolated from liquid cultures of Pseudomonas syringae (plant-associated bacterium) and have been shown to have antifungal activities. (see i.e., Harrison, L., et al., "Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity," J. Gen.
Microbiology, 137(12), 2857-65 (1991) and US Patent Nos.
5,576,298 and 5,837,685) Unlike the previously described antimycotics from P. syringae (e. g., syringomycins, syringotoxins and syringostatins), pseudomycins A-C contain hydroxyaspartic acid, aspartic acid, serine, dehydroaminobutyric acid, lysine and diaminobutyric acid.
The peptide moiety for pseudomycins A, A', B, B', C, C' corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-C1) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N-terminal Ser. The analogs are distinguished by the N-acyl side chain, i.e., pseudomycin A is N-acylated by 3,4-dihydroxytetradeconoyl, pseudomycin A' by 3,4-dihydroxypentadecanoyl, pseudomycin B by 3-hydroxytetradecanoyl, pseudomycin B' by 3-hydroxydodecanoyl, pseudomycin C by 3,4-dihydroxyhexadecanoyl and pseudomycin C' by 3-hydroxyhexadecanoyl. (see i.e., Ballio, A., et al., "Novel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins," FEBS Letters, 355(1), 96-100, (1994) and Coiro, V.M., et al., "Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from NMR data,"
Eur. J. Biochem., 257(2), 449-456 (1998).) Pseudomycins are known to have certain adverse biological effects. For example, destruction of the endothelium of the vein, destruction of tissue, inflammation, and local toxicity to host tissues have been observed when pseudomycin is administered intraveneously.
Since the pseudomycins have proven antifungal activity and relatively unexplored chemistry, there is a need to explore this class of compounds for other potential compounds that may be useful as antifungal agents having less adverse side affects.
FIELD OF THE INVENTION
The present invention relates to pseudomycin compounds, in particular, acid-modified, semi-synthetic pseudomycin compounds.
BACKGROUND OF THE INVENTION
Pseudomycins are natural products isolated from liquid cultures of Pseudomonas syringae (plant-associated bacterium) and have been shown to have antifungal activities. (see i.e., Harrison, L., et al., "Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity," J. Gen.
Microbiology, 137(12), 2857-65 (1991) and US Patent Nos.
5,576,298 and 5,837,685) Unlike the previously described antimycotics from P. syringae (e. g., syringomycins, syringotoxins and syringostatins), pseudomycins A-C contain hydroxyaspartic acid, aspartic acid, serine, dehydroaminobutyric acid, lysine and diaminobutyric acid.
The peptide moiety for pseudomycins A, A', B, B', C, C' corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-C1) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N-terminal Ser. The analogs are distinguished by the N-acyl side chain, i.e., pseudomycin A is N-acylated by 3,4-dihydroxytetradeconoyl, pseudomycin A' by 3,4-dihydroxypentadecanoyl, pseudomycin B by 3-hydroxytetradecanoyl, pseudomycin B' by 3-hydroxydodecanoyl, pseudomycin C by 3,4-dihydroxyhexadecanoyl and pseudomycin C' by 3-hydroxyhexadecanoyl. (see i.e., Ballio, A., et al., "Novel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins," FEBS Letters, 355(1), 96-100, (1994) and Coiro, V.M., et al., "Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from NMR data,"
Eur. J. Biochem., 257(2), 449-456 (1998).) Pseudomycins are known to have certain adverse biological effects. For example, destruction of the endothelium of the vein, destruction of tissue, inflammation, and local toxicity to host tissues have been observed when pseudomycin is administered intraveneously.
Since the pseudomycins have proven antifungal activity and relatively unexplored chemistry, there is a need to explore this class of compounds for other potential compounds that may be useful as antifungal agents having less adverse side affects.
BRIEF SLTt~lARY OF THE INVENTION
The present invention provides pseudomycin compounds represented by the following structure which are useful as antifungal agents or in the design of antifungal agents.
O
O
OH
O~N H
NH H N OH
HO O
NH SCI
O
R' O O O
NH
O O O N~R
H
N NH
O
RZ R' R' O
I
wherein R is b b~ d R R R
r R
a a, ~ ~
R R c e R R
where Ra and Ra~ are independently hydrogen or methyl, or either Ra or Ra~ is alkyl amino, taken together with Rb or Rb~ forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with R° forms a six-membered aromatic ring;
Rb and Rb~ are independently hydrogen, halogen, or methyl, or either Rb or Rb~ is amino, alkylamino, a-acetoacetate, methoxy, or hydroxy;
R° is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, CS-C14 alkoxy substituted six-membered aromatic ring, or CS-C14 alkyl substituted six-membered aromatic ring, and Rf is C6-C18 alkyl, C5-C11 alkoxy, or biphenyl;
R is Rn where Rg is hydrogen, or C1-C13 alkyl, and Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-Clo alkyl ) phenyl , - ( CH2 ) n-aryl , or - ( CHZ ) n- ( C5-Cs cycloalkyl), where n = 1 or 2; or R is R
m where R1 is a hydrogen, halogen, or CS-C8 alkoxy, and m is 1, 2 or 3;
R is OH
p R' where R' is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is -N
Rk where Rk is CS-C14 alkoxy; or R is - (CHZ) -NRm- (C13-C1$ alkyl) , where Rm is H, -CH3 or -C ( 0 ) CH3 ;
R1 is independently -NHz or -NHp-Pg, where p is 0 or 1;
RZ and R3 are independently -ORza, or -N(R2b) (R2~) , where Rza and R2b are independently hydrogen, C1-Clo alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-amyl, i-amyl, n-hexyl, n-heptyl, n-octyl, n-nonanyl, n-decyl, etc.), C3_C6 cycloalkyl (e. g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentylmethyl, methylcyclopentyl, cyclohexyl, etc.) haloalkyl ( a . g . , CF3CH2- ) , hydroxy ( C1-Clo ) alkyl , alkoxy ( C1-C1o ) alkyl ( a . g, methoxyethyl ) , al lyl , CZ-Clo alkenyl , amino ( C1-Clo ) alkyl , mono- or di-alkylamino ( C1-Clo ) alkyl , aryl ( C1-Clo ) alkyl ( a . g . , benzyl ) , heteroaryl(C1-Clo)alkyl (e.g., 3-pyridylmethyl, 4-pyridylmethyl), or cycloheteroalkyl(C1-Clo)alkyl (e. g., N-tetrahydro-1,4-oxazinylethyl and N-piperazinylethyl), or R2b is an alkyl carboxylate residue of an aminoacid alkyl ester ( a . g . , -CHZCOZCH3 , -CH ( COzCH3 ) CH ( CH3 ) 2 , -CH ( C02CH3 ) CH ( phenyl ) , -CH ( COzCH3 ) CH20H, -CH ( COZCH3 ) CH2 (p-hydroxyphenyl ) , -CH ( COZCH3 ) CH2SH, -CH ( COZCH3 ) CHZ ( CHZ ) 3NH2 , -CH (COZCH3 ) CH2 (4- or 5-imidazole) , -CH (COZCH3 ) CHZCOzCH3, -CH ( COZCH3 ) CH2C02NH2 , and the 1 ike ) , and RZ° is hydrogen or C1-C6 alkyl, provided that both Rz and R3 are not -OH; and pharmaceutically acceptable salts and solvates thereof.
The present invention provides pseudomycin compounds represented by the following structure which are useful as antifungal agents or in the design of antifungal agents.
O
O
OH
O~N H
NH H N OH
HO O
NH SCI
O
R' O O O
NH
O O O N~R
H
N NH
O
RZ R' R' O
I
wherein R is b b~ d R R R
r R
a a, ~ ~
R R c e R R
where Ra and Ra~ are independently hydrogen or methyl, or either Ra or Ra~ is alkyl amino, taken together with Rb or Rb~ forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with R° forms a six-membered aromatic ring;
Rb and Rb~ are independently hydrogen, halogen, or methyl, or either Rb or Rb~ is amino, alkylamino, a-acetoacetate, methoxy, or hydroxy;
R° is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, CS-C14 alkoxy substituted six-membered aromatic ring, or CS-C14 alkyl substituted six-membered aromatic ring, and Rf is C6-C18 alkyl, C5-C11 alkoxy, or biphenyl;
R is Rn where Rg is hydrogen, or C1-C13 alkyl, and Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-Clo alkyl ) phenyl , - ( CH2 ) n-aryl , or - ( CHZ ) n- ( C5-Cs cycloalkyl), where n = 1 or 2; or R is R
m where R1 is a hydrogen, halogen, or CS-C8 alkoxy, and m is 1, 2 or 3;
R is OH
p R' where R' is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is -N
Rk where Rk is CS-C14 alkoxy; or R is - (CHZ) -NRm- (C13-C1$ alkyl) , where Rm is H, -CH3 or -C ( 0 ) CH3 ;
R1 is independently -NHz or -NHp-Pg, where p is 0 or 1;
RZ and R3 are independently -ORza, or -N(R2b) (R2~) , where Rza and R2b are independently hydrogen, C1-Clo alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-amyl, i-amyl, n-hexyl, n-heptyl, n-octyl, n-nonanyl, n-decyl, etc.), C3_C6 cycloalkyl (e. g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentylmethyl, methylcyclopentyl, cyclohexyl, etc.) haloalkyl ( a . g . , CF3CH2- ) , hydroxy ( C1-Clo ) alkyl , alkoxy ( C1-C1o ) alkyl ( a . g, methoxyethyl ) , al lyl , CZ-Clo alkenyl , amino ( C1-Clo ) alkyl , mono- or di-alkylamino ( C1-Clo ) alkyl , aryl ( C1-Clo ) alkyl ( a . g . , benzyl ) , heteroaryl(C1-Clo)alkyl (e.g., 3-pyridylmethyl, 4-pyridylmethyl), or cycloheteroalkyl(C1-Clo)alkyl (e. g., N-tetrahydro-1,4-oxazinylethyl and N-piperazinylethyl), or R2b is an alkyl carboxylate residue of an aminoacid alkyl ester ( a . g . , -CHZCOZCH3 , -CH ( COzCH3 ) CH ( CH3 ) 2 , -CH ( C02CH3 ) CH ( phenyl ) , -CH ( COzCH3 ) CH20H, -CH ( COZCH3 ) CH2 (p-hydroxyphenyl ) , -CH ( COZCH3 ) CH2SH, -CH ( COZCH3 ) CHZ ( CHZ ) 3NH2 , -CH (COZCH3 ) CH2 (4- or 5-imidazole) , -CH (COZCH3 ) CHZCOzCH3, -CH ( COZCH3 ) CH2C02NH2 , and the 1 ike ) , and RZ° is hydrogen or C1-C6 alkyl, provided that both Rz and R3 are not -OH; and pharmaceutically acceptable salts and solvates thereof.
In another embodiment of the present invention, a prodrug of a pseudomycin compound is provided having structure I represented above wherein Rz and R3 are represented by -OR2a, where R2a is C1-C3 alkyl.
In yet another embodiment of the present invention, a 3-amido derivative of a pseudomycin compound is provided where the compound is prepared by the steps of (i) providing a compound having structure I above wherein R1 is -NHz and R2 and R3 are both -OH; (ii) protecting the amino groups, R1, at positions 2, 4 and 5 with an amino-protecting group;
(iii) forming an amide linkage at position 3 using an o-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate as a coupling agent; and (iv) removing the amino-protecting groups. An 8-amido derivative is also provided where the derivative is prepared using the steps described above except using benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate as the coupling agent.
In another embodiment of the present invention, a pharmaceutical formulation is provided which includes the pseudomycin compound represented by structure I above and a pharmaceutically acceptable carrier.
In yet another embodiment of the present invention, a method is provided for treating an antifungal infection in an animal in need thereof, which comprises administering to the animal the pseudomycin compound I described above.
Definitions As used herein, the term "alkyl" refers to a hydrocarbon radical of the general formula CnH2n+i containing from 1 to 30 carbon atoms unless otherwise indicated. The alkane radical may be straight (e. g. methyl, ethyl, propyl, butyl, etc.), branched (e. g., isopropyl, isobutyl, tertiary butyl, neopentyl, etc.), cyclic (e. g., cyclopropyl, cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl, etc.), or multi-cyclic (e. g., bicyclo[2.2.1]heptane, spiro[2.2]pentane, etc.). The alkane radical may be substituted or unsubstituted. Similarly, the alkyl portion of an alkoxy group, alkanoyl, or alkanoate have the same definition as above.
The term "alkenyl" refers to an acyclic hydrocarbon containing at least one carbon carbon double bond. The alkene radical may be straight, branched, cyclic, or multi-cyclic. The alkene radical may be substituted or unsubstituted. The alkenyl portion of an alkenoxy, alkenoyl or alkenoate group has the same definition as above.
The term "alkynyl" refers to an acyclic hydrocarbon containing at least one carbon carbon triple bond. The alkyne radical may be straight, or branched. The alkyne radical may be substituted or unsubstituted. The alkynyl portion of an alkynoxy, alkynoyl or alkynoate group has the same definition as above.
The term "aryl" refers to aromatic moieties having single (e. g., phenyl) or fused ring systems (e. g., naphthalene, anthracene, phenanthrene, etc.). The aryl groups may be substituted or unsubstituted.
The term "heteroaryl" refers to aromatic moieties containing at least one heteratom within the aromatic ring system (e. g., pyrrole, pyridine, indole, thiophene, furan, benzofuran, imidazole, oxazine, pyrimidine, purine, benzimidazole, quinoline, etc.). The aromatic moiety may consist of a single or fused ring system. The heteroaryl groups may be substituted or unsubstituted.
"NHp-Pg" and °'amino protecting group" refer to a substituent of the amino group (Pg) commonly employed to block or protect the amino functionality while reacting other functional groups on the compound. When p is 0, the amino protecting group, when taken with the nitrogen to which it is attached, forms a cyclic imide, e.g., phthalimido and tetrachlorophthalimido. When p is 1, the protecting group, when taken with the nitrogen to which it is attached, can form a carbamate, e.g., methyl, ethyl, and 9-fluorenylmethylcarbamate; or an amide, e.g., N-formyl and N-acetylamide.
Within the field of organic chemistry and particularly within the field of organic biochemistry, it is widely understood that significant substitution of compounds is tolerated or even useful. In the present invention, for example, the term alkyl group allows for substitutents which is a classic alkyl, such as methyl, ethyl, propyl, hexyl, isooctyl, dodecyl, stearyl, etc. The term "group"
specifically envisions and allows for substitutions on alkyls which are common in the art, such as hydroxy, halogen, alkoxy, carbonyl, keto, ester, carbamato, etc., as well as including the unsubstituted alkyl moiety. However, it is generally understood by those skilled in the art that the substituents should be selected so as to not adversely affect the pharmacological characteristics of the compound or adversely interfere with the use of the medicament.
Suitable substituents for any of the groups defined above include alkyl, alkenyl, alkynyl, aryl, halo, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, mono- and di-alkyl amino, quaternary ammonium salts, aminoalkoxy, hydroxyalkylamino, aminoalkylthio, carbamyl, carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl, and combinations thereof.
The term "solvate" refers to an aggregate that comprises one or more molecules of the solute, such as a compound of structure I, with one or more molecules of a pharmaceutical solvent, such as water, ethanol, and the like.
The term "pharmaceutically acceptable salt" refers to organic or inorganic salts of the compounds represented by structure I that are substantially non-toxic to the recipient at the doses administered.
The term "prodrug" refers to a class of drugs which result in pharmacological action due to conversion by metabolic processes within the body (i.e., biotransformation). In the present invention, the pseudomycin prodrug compounds contain ester functionalities that can be cleaved by esterases in the plasma to produce the active drug.
The term "animal" refers to humans, companion animals (e. g., dogs, cats and horses), food-source animals (e. g., cows, pigs, sheep and poultry), zoo animals, marine animals, birds and other similar animal species.
DETAILED DESCRIPTION OF THE INVENTION
Applicants have discovered that modification of the acid functionality attached to the hydroxyaspartic acid and/or aspartic acid units of a pseudomycin natural product or semi-synthetic derivative provides compounds having in vitro indications which suggest that the new compounds may be active against C. albican, C, neoformans, and/or A.
fumigatus. Some bis-esters have been shown to act as a prodrug; therefore, these particular compounds have reduced in vitro activity but show in vivo efficacy.
Scheme I below illustrates the general procedures for synthesizing Compound I from any one of the naturally occurring pseudomycins or N-acyl modified derivatives. In general, three synthetic steps are used to produce Compound I: (1) selective amino protection; (2) condensation with the appropriate alcohol or amine to produce the respective ester or amide; and (4) deprotection of the amino groups.
HO O
O OH HO
O OH
$ N OH O NH H N OH
HO,~
/ -NH O ,-,~CI (1~ HO~H O CI
_ ~ O
H PgHN ~O O
H
~N R O O H 0~~~~N R
N N~NH
H
NHZ
OH NHPg H2N U PgHN O
(2) R
O OH p OH
O NH H N OH O~H N OH
HO~ O CI HO~ NH O CI
~NH O~ ~ (3) _ ~ O
H N~O O ~ PgHN~O O
NH O O "'N R O NH O H O ~~~N R
O
H
H N~--!NH O
H N~-!NH O
~NH2 RZ ~NHPg H2N O PgHN O
Scheme I
The pendant amino groups at residues 2, 4 and 5 may be protected using any standard means known to those skilled in the art for amino protection. The exact genus and species of amino protecting group employed is not critical so long as the derivatized amino group is stable to the conditions of subsequent reactions) on other positions of the intermediate molecule and the protecting group can be selectively removed at the appropriate point without disrupting the remainder of the molecule including any other amino protecting group(s). Preferred amino protecting groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyl, phthalimido, and benzyloxycarbonyl (CBZ). Most preferred is allyloxycarbonyl (Alloc) and benzyloxycarbonyl (CBZ).
Further examples of suitable protecting groups are described in T.W. Greene, "Protective Groups in Organic Synthesis,"
John Wiley and Sons, New York, N.Y., (2nd ed., 1991), at chapter 7.
Formation of the ester groups may be accomplished using standard esterification procedures well-known to those skilled in the art. Esterification under acidic conditions typically includes dissolving or suspending the pseudomycin compound in the appropriate alcohol in the presence of a protic acid (e. g., HC1, TFA, p-toluenesulfonic acid, etc.).
Under basic conditions, the pseudomycin compound is generally reacted with the appropriate alkyl halide in the presence of a weak base (e. g., sodium bicarbonate under anhydrous conditions).
Formation of the amide groups may be accomplished using standard amidation procedures well-known to those skilled in the art. However, the choice of coupling agents provides selective modification of the acid groups. For example, the use of benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate(PyBOP) as the coupling agent allows one to isolate pure mono-amides at residue 8 and (in some cases) pure bis amides simultaneously. Whereas, coupling agents such as o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) and 2(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) favor formation of monoamides at residue 3.
Applicants also discovered that the addition of a bulky amine enhances the ratio of monoamides at residue 3. The ratio of amidation at residue 3 vs. residue 8 increased from about 1:1 to about 6:1 and the amount of bis-amides was reduced through the addition of a bulky amine. The term "bulky amine" refers to an amine having multiple and/or large substituents on the nitrogen atom. Any tertiary amine may be used that is compatible with the reaction conditions.
Preferred bulky amines include N,N-diisopropylethylamine (DIEA) and N-ethyldicyclohexylamine. The amount of bulky amine added is generally from about 1 to 10 equivalents, preferably 3 to 8 equivalents, more preferably 5 to 6 equivalents. The reaction is generally ran at temperatures from about room temperature (25°C) to about -20°C. However, Applicants discovered that lower temperatures (from about 0°C to about -20°C) further enhance the formation of monoamides at residue 3. The ratio of amidation at residue 3 vs. residue 8 increased as much as 20:1 by adding a bulky amine and lowering the temperature of the reaction.
However, it will be understood by those skilled in the art that the lower temperature limit will depend upon the solubility of the reactive components.
Once the acid groups) are modified, then the amino protecting groups (at positions 2, 4 and 5) may be removed using standard procedures appropriate for the specific protecting group used. For example, CBZ groups are removed by hydrogenation in the presence of a hydrogenation catalyst (e.g., 10o Pd/C). When the amino protecting group is allyloxycarbonyl, then the protecting group may be removed using tributyltinhydride and triphenylphosphine palladium dichloride. This particular protection/deprotection scheme has the advantage of reducing the potential for hydrogenating the vinyl group of the Z-Dhb unit of the pseudomycin structure.
As discussed earlier, pseudomycins are natural products isolated from the bacterium Pseudomonas syringae that have been characterized as lipodepsinonapetpides containing a cyclic peptide portion closed by a lactone bond and including the unusual amino acids 4-chlorothreonine (ClThr), 3-hydroxyaspartic acid (HOAsp), 2,3-dehydro-2-aminobutyric acid (Dhb), and 2,4-diaminobutyric acid (Dab). Methods for growth of various strains of P. syringae to produce the different pseudomycin analogs (A, A', B, B', C, and C') are described below and described in more detail in PCT Patent Application Serial No. PCT/US00/08728 filed by Hilton, et al. on April 14, 2000 entitled "Pseudomycin Production by Pseudomonas Syringae," incorporated herein by reference, PCT
Patent Application Serial No. PCT/US00/08727 filed by Kulanthaivel, et al. on April 14, 2000 entitled "Pseudomycin Natural Products," incorporated herein by reference, and U.S. Patent Nos. 5,576,298 and 5,837,685, each of which are incorporated herein by reference.
Isolated strains of P. syringae that produce one or more pseudomycins are known in the art. Wild type strain MSU 174 and a mutant of this strain generated by transposon mutagenesis, MSU 16H are described in U.S. Patent Nos.
5,576,298 and 5,837,685; Harrison, et al., "Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity," J. Gen.
Microbiology, 137, 2857-2865 (1991); and Lamb et al., "Transposon mutagenesis and tagging of fluorescent pseudomonas: Antimycotic production is necessary for control of Dutch elm disease," Proc. Natl. Acad. Sci. USA, 84, 6447-6451 (1987).
A strain of P. syringae that is suitable for production of one or more pseudomycins can be isolated from environmental sources including plants (e. g., barley plants, citrus plants, and lilac plants) as well as, sources such as soil, water, air, and dust. A preferred stain is isolated from plants. Strains of P. syringae that are isolated from environmental sources can be referred to as wild type. As used herein, "wild type" refers to a dominant genotype which naturally occurs in the normal population of P. syringae (e.g., strains or isolates of P. syringae that are found in nature and not produced by laboratory manipulation). Like most organisms, the characteristics of the pseudomycin-producing cultures employed (P. syringae strains such as MSU
174, MSU 16H, MSU 206, 25-B1, 7H9-1) are subject to variation. Hence, progeny of these strains (e. g., recombinants, mutants and variants) may be obtained by methods known in the art.
P. syringae MSU 16H is publicly available from the American Type Culture Collection, Parklawn Drive, Rockville, MD, USA as Accession No. ATCC 67028. P. syringae strains 25-B1, 7H9-1, and 67 H1 were deposited with the American Type Culture Collection on March 23, 2000 and were assigned the following Accession Nos.:
25-B1 Accession No. PTA-1622 7H9-1 Accession No. PTA-1623 67 H1 Accession No. PTA-1621 Mutant strains of P. syringae are also suitable for production of one or more pseudomycins. As used herein, "mutant" refers to a sudden heritable change in the phenotype of a strain, which can be spontaneous or induced by known mutagenic agents, such as radiation (e. g., ultraviolet radiation or x-rays), chemical mutagens (e. g., ethyl methanesulfonate (EMS), diepoxyoctane, N-methyl-N-nitro-N'-nitrosoguanine (NTG), and nitrous acid), site-s specific mutagenesis, and transposon mediated mutagenesis.
Pseudomycin-producing mutants of P. syringae can be produced by treating the bacteria with an amount of a mutagenic agent effective to produce mutants that overproduce one or more pseudomycins, that produce one pseudomycin (e. g., pseudomycin B) in excess over other pseudomycins, or that produce one or more pseudomycins under advantageous growth conditions. While the type and amount of mutagenic agent to be used can vary, a preferred method is to serially dilute NTG to levels ranging from 1 to 100 ~,g/ml. Preferred mutants are those that overproduce pseudomycin B and grow in minimal defined media.
Environmental isolates, mutant strains, and other desirable strains of P. syringae can be subjected to selection for desirable traits of growth habit, growth medium nutrient source, carbon source, growth conditions, amino acid requirements, and the like. Preferably, a pseudomycin producing strain of P. syringae is selected for growth on minimal defined medium such as N21 medium and/or for production of one or more pseudomycins at levels greater than about 10 ~g/ml. Preferred strains exhibit the characteristic of producing one or more pseudomycins when grown on a medium including three or fewer amino acids and optionally, either a lipid, a potato product or combination thereof.
Recombinant strains can be developed by transforming the P. syringae strains, using procedures known in the art.
Through the use of recombinant DNA technology, the P.
syringae strains can be transformed to express.a variety of gene products in addition to the antibiotics these strains produce. For example, one can modify the strains to introduce multiple copies of the endogenous pseudomycin-biosynthesis genes to achieve greater pseudomycin yield.
To produce one or more pseudomycins from a wild type or mutant strain of P. syringae, the organism is cultured with agitation in an aqueous nutrient medium including an effective amount of three or fewer amino acids, preferably glutamic acid, glycine, histidine, or a combination thereof.
Alternatively, glycine is combined with one or more of a potato product and a lipid. Culturing is conducted under conditions effective for growth of P. syringae and production of the desired pseudomycin or pseudomycins.
Effective conditions include temperatures from about 22°-C to about 27°-C, and a duration of about 36 hours to about 96 hours. Controlling the concentration of oxygen in the medium during culturing of P. syringae is advantageous for production of a pseudomycin. Preferably, oxygen levels are maintained at about 5 to 50o saturation, more preferably about 30o saturation. Sparging with air, pure oxygen, or gas mixtures including oxygen can regulate the concentration of oxygen in the medium.
Controlling the pH of the medium during culturing of P.
syringae is also advantageous. Pseudomycins are labile at basic pH, and significant degradation can occux if the pH of the culture medium is above about 6 for more than about 12 hours. Preferably, the pH of the culture medium is maintained between 6 and 4. P. syringae can produce one or more pseudomycins when grown in batch culture. However, fed-bath or semi-continuous feed of glucose and optionally, an acid or base (e.g., ammonium hydroxide) to control pH, enhances production. Pseudomycin production can be further enhanced by using continuous culture methods in which glucose and ammonium hydroxide are fed automatically.
Choice of P. syringae strain can affect the amount and distribution of pseudomycin or pseudomycins produced. For example, strains MSU 16H and 67 H1 each produce predominantly pseudomycin A, but also produce pseudomycin B
and C, typically in ratios of 4:2:1. Strain 67 H1 typically produces levels of pseudomycins about three to five fold larger than are produced by strain MSU 16H. Compared to strains MSU 16H and 67 H1, strain 25-B1 produces more pseudomycin B and less pseudomycin C. Strain 7H9-1 are distinctive in producing predominantly pseudomycin B and larger amount of pseudomycin B than other strains. For example, this strain can produce pseudomycin B in at least a ten fold excess over either pseudomycin A or C.
Each pseudomycin, pseudomycin intermediate and mixtures can be detected, determined, isolated, and/or purified by any variety of methods known to those skilled in the art.
For example, the level of pseudomycin activity in a broth or in an isolate or purified composition can be determined by antifungal action against a fungus such as Candida and can be isolated and purified by high performance liquid chromatography.
Alternatively, the amido or ester derivative can be formed from an N-acyl semi-synthetic compound. Semi-synthetic pseudomycin compounds may be synthesized by exchanging the N-acyl group on the L-serine unit. Examples of various N-acyl derivatives are described in PCT Patent Application Serial No. , Belvo, et al., filed evendate herewith entitled "Pseudomycin N-Acyl Side-Chain Analogs" and incorporated herein by reference. In general, four synthetic steps are used to produce the semi-synthetic compounds from naturally occurring pseudomycin compounds:
(1) selective amino protection; (2) chemical or enzymatic deacylation of the N-acyl side-chain; (3) reacylation with a different side-chain; and (4) deprotection of the amino groups. The aspartic acid and/or hydroxyaspartic acid units can be modified prior to deprotecting the amino groups.
The deacylation of an N-acyl group having a gamma or delta hydroxylated side chain (e. g., 3,4-dihydroxytetra-deconoate) may be accomplished by treating the amino-protected pseudomycin compound with acid in an aqueous solvent. Suitable acids include acetic acid and trifluoroacetic acid. A preferred acid is trifluoroacetic acid. If trifluoroacetic acid is used, the reaction may be accomplished at or near room temperature. However, when acetic acid is used the reaction is generally ran at about 40°C. Suitable aqueous solvent systems include acetonitrile, water, and mixtures thereof. Organic solvents accelerate the reaction; however, the addition of an organic solvent may lead to other by-products. Pseudomycin compounds lacking a delta or gamma hydroxy group on the side chain (e. g., Pseudomycin B and C') may be deacylated enzymatically. Suitable deacylase enzymes include Polymyxin Acylase (164-16081 Fatty Acylase (crude) or 161-16091 Fatty Acylase (pure) available from Wako Pure Chemical Industries, Ltd.), or ECB deacylase. The enzymatic deacylation may be accomplished using standard deacylation procedures well known to those skilled in the art. For example, general procedures for using polymyxin acylase may be found in Yasuda, N., et al, Agric. Biol. Chem., 53, 3245 (1989) and Kimura, Y., et al., Agric. Biol. Chem., 53, 497 (1989).
The deacylated product (also known as the pseudomycin nucleus) is reacylated using the corresponding acid of the desired acyl group in the presence of a carbonyl activating agent. "Carbonyl activating group" refers to a substituent of a carbonyl that promotes nucleophilic addition reactions at that carbonyl. Suitable activating substituents are those which have a net electron withdrawing effect on the carbonyl. Such groups;include, but are not limited to, alkoxy, aryloxy, nitrogen containing aromatic heterocycles, or amino groups (e. g., oxybenzotriazole, imidazolyl, nitrophenoxy, pentachlorophenoxy, N-oxysuccinimide, N,N'-dicyclohexylisoure-O-yl, and N-hydroxy-N-methoxyamino);
acetates; formates; sulfonates (e. g., methanesulfonate, ethanesulfonate, benzenesulfonate, and p-tolylsulfonate);
and halides (e. g., chloride, bromide, and iodide).
A variety of acids may be used in the acylation process. Suitable acids include aliphatic acids containing one or more pendant aryl, alkyl, amino(including primary, secondary and tertiary amines), hydroxy, alkoxy, and amido groups; aliphatic acids containing nitrogen or oxygen within the aliphatic chain; aromatic acids substituted with alkyl, hydroxy, alkoxy and/or alkyl amino groups; and heteroaromatic acids substituted with alkyl, hydroxy, alkoxy and/or alkyl amino groups.
Alternatively, a solid phase synthesis may be used where a hydroxybenzotriazole-resin (HOBt-resin) serves as the coupling agent for the acylation reaction.
The acid-modification of the protected N-acyl semi-synthetic compound is then accomplished by reacting at least one of the pendant carboxyl groups attached to. the aspartic or hydroxyaspartic peptide units of the N-acyl modified semi-synthetic pseudomycin compound to form the desired amide or ester linkage(s). The protecting groups are then removed as described earlier.
The pseudomycin compound may be isolated and used per se or in the form of its pharmaceutically acceptable salt or solvate. The term "pharmaceutically acceptable salt" refers to non-toxic acid addition salts derived from inorganic and organic acids. Suitable salt derivatives include halides, thiocyanates, sulfates, bisulfates, sulfites, bisulfites, arylsulfonates, alkylsulfates, phosphonates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphonates, alkanoates, cycloalkylalkanoates, arylalkonates, adipates, alginates, aspartates, benzoates, fumarates, glucoheptanoates, glycerophosphates, lactates, maleates, nicotinates, oxalates, palmitates, pectinates, picrates, pivalates, succinates, tartarates, citrates, camphorates, camphorsulfonates, digluconates, trifluoroacetates, and the like.
The term "solvate" refers to an aggregate that comprises one or more molecules of the solute (i.e., pseudomycin compound) with one or more molecules of a pharmaceutical solvent, such as water, ethanol, and the like. When the solvent is water, then the aggregate is referred to as a hydrate. Solvates are generally formed by dissolving the compound in the appropriate solvent with heat and slowing cooling to generate an amorphous or crystalline solvate form.
The active ingredient (i.e., pseudomycin compound) is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient, physician or veterinarian an elegant and easy to handle product. Formulations may comprise from 0.10 to 99.90 by weight of active ingredient, more generally from about 10o to about 30o by weight.
As used herein, the term "unit dose" or "unit dosage"
refers to physically discrete units that contain a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect. When a unit dose is administered orally or parenterally, it is typically provided in the form of a tablet, capsule, pill, powder packet, topical composition, suppository, wafer, measured units in ampoules or in multidose containers, etc.
Alternatively, a unit dose may be administered in the form of a dry or liquid aerosol which may be inhaled or sprayed.
The dosage to be administered may vary depending upon the physical characteristics of the animal, the severity of the animal's symptoms, the means used to administer the drug and the animal species. The specific dose for a given animal is usually set by the judgment of the attending physician or veterinarian.
Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like. The particular carrier, diluent or excipient used will depend upon the means and purpose for which the active ingredient is being applied. The formulations may also include wetting agents, lubricating agents, surfactants, buffers, tonicity agents, bulking agents, stabilizers, emulsifiers, suspending agents, preservatives, sweeteners, perfuming agents, flavoring agents and combinations thereof.
A pharmaceutical composition may be administered using a variety of methods. Suitable methods include topical (e. g., ointments or sprays), oral, injection and inhalation.
The particular treatment method used will depend upon the type of infection being addressed.
In parenteral iv applications, the formulations are typically diluted or reconstituted (if freeze-dried) and further diluted if necessary, prior to administration. An example of reconstitution instructions for the freeze-dried product are to add ten ml of water for injection (WFI) to the vial and gently agitate to dissolve. Typical reconstitution times are less than one minute. The resulting solution is then further diluted in an infusion solution such as dextrose 5% in water (D5W), prior to administration.
Pseudomycin compounds have been shown to exhibit antifungal activity such as growth inhibition of various infectious fungi including Candida spp. (i.e., C. albicans, C. parapsilosis, C. krusei, C. glabrata, C. tropicalis, or C. lusitaniaw); Torulopus spp.(i.e., T. glabrata);
Aspergillus spp. (i.e., A. fumigatus); Histoplasma spp.
(i.e., H. capsulatum); Cryptococcus spp. (i.e., C.
neoformans); Blastomyces spp. (i.e., B. dermatitidis);
Fusarium spp.; Trichophyton spp., Pseudallescheria boydii, Coccidioides immits, Sporothrix schenckii, etc.
Consequently, the compounds and formulations of the present invention are useful in the preparation of medicaments for use in combating either systemic fungal infections or fungal skin infections. Accordingly, a method is provided for inhibiting fungal activity comprising contacting the pseudomycin compound of the present invention with a fungus. A preferred method includes inhibiting Candida albicans or Aspergillus fumigatus activity. The term "contacting" includes a union or junction, or apparent touching or mutual tangency of a compound of the invention with a fungus. The term does not imply any further limitations to the process, such as by mechanism of inhibition. The methods are defined to encompass the inhibition of fungal activity by the action of the compounds and their inherent antifungal properties.
A method for treating a fungal infection which comprises administering an effective amount of a pharmaceutical formulation of the present invention to an animal host in need of such treatment is also provided. A
preferred method includes treating a Candida albicans or Aspergillus fumigatus infection. The term "effective amount" refers to an amount of active compound which is capable of inhibiting fungal activity. The dose administered will vary depending on such factors as the nature and severity of the infection, the age and general health of the host, the tolerance of the host to the antifungal agent and species of the host. The particular dose regimen likewise may vary according to these factors.
The medicament may be given in a single daily dose or in multiple doses during the day. The regimen may last from about 2-3 days to about 2-3 weeks or longer. A typical daily dose (administered in single or divided doses) contains a dosage level between about 0.01 mg/kg to 100 mg/kg of body weight of an active compound. Preferred daily doses are generally between about 0.1 mg/kg to 60 mg/kg and more preferably between about 2.5 mg/kg to 40 mg/kg. The host may be any animal including humans, companion animals (e. g., dogs, cats and horses), food-source animals (e. g., cows, pigs, sheep and poultry), zoo animals, marine animals, birds and other similar animal species.
EXAMPLES
Unless indicated otherwise, all chemicals can be acquired from Aldrich Chemical (Milwaukee, WI). The following abbreviations are used through out the examples to represent the respective listed materials:
ACN - acetonitrile TFA - trifluoroacetic acid DMF - dimethylformamide EDCI - 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride BOC = t-butoxycarbonyl, (CH3)3C-0-C(0)-2 5 CBZ = benzyloxycarbonyl , C6HSCH2-O-C ( O ) -PyBOP = benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate TBTU = o-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate DIEA = N,N-diisopropylethylamine HPLC Conditions Unless indicated otherwise, analytical reverse-phase HPLC work was done using the Waters 600E systems equipped with Waters ~,Bondapak (C18, 3.9 X 300 mm) column. The eluent used was 65:35 acetonitrile/0.1o aqueous TFA solvent system to 100% acetonitrile over 20 minutes with a flow rate of 1.5 ml/minute and using UV detection at 230 nm.
Preparative HPLC work was performed with a Waters Prep 2000 system using Dynamax 60 angstrom C18 column and identical solvent systems as used in the analytical HPLC
system but with a flow rate of 40 ml/min.
Biological Analysis Detection and Quantification of An ti fungal Activity:
Antifungal activity was determined in vitro by obtaining the minimum inhibitory concentration (MIC) of the compound using a standard agar dilution test or a disc-diffusion test. A typical fungus employed in testing antifungal activity is Candida albicans. Antifungal activity is considered significant when the test sample (50 ~,1) causes 10-12 mm diameter zones of inhibition on C.
albicans x657 seeded agar plates.
Tail Vein Toxicity:
Mice were treated intravenously (IV) through the lateral tail vein with 0.1 ml of testing compound (20 mg/kg) at 0, 24, 48 and 72 hours. Two mice were included in each group. Compounds were formulated in 5.0o dextrose and sterile water for injection. The mice were monitored for 7 days following the first treatment and observed closely for signs of irritation including erythema, swelling, discoloration, necrosis, tail loss and any other signs of adverse effects indicating toxicity.
The mice used in the study were outbred, male ICR mice having an average weight between 18-20 g (available from Harlan Sprangue Dawley, Indianapolis, IN).
General Procedures CBZ-Protected Pseudomycin: General procedures used to protect the pendant amino groups at positions 2, 4 and 5 of Pseudomycin A, A' , B, B' , C or C' wi th CBZ.
Dissolve/suspend pseudomycin compound (Rl=H) in DMF (20 mg/ml, Aldrich Sure Seal). V~hile stirring at room temperature add N-(Benzyloxycarbonyloxy)succinimide (6 eq).
Allow to stir at room temperature for 32 hours. Monitor reaction by HPLC (4.6x50 mm, 3.5 E,tm, 300-SB, C8, Zorbax column). Concentrate reaction to 10 ml on high vacuum rotovap at room temperature. Put material in freezer until ready to prep by chromatography. Reverse phase preparative HPLC yields an amorphous, white solid after lyophilization (R1 - CBZ in structure II below).
Alloc-Protected Pseudomycin: General procedures used to protect the pendant amino groups at positions 2, 4 and 5 of Pseudomycin A, A', B, B', C or C' with Alloc.
Diallyl pyrocarbonate (558 mg, 3.0 mmol) was added to a solution of Pseudomycin A (1.22 g, 1.0 mmol) ixi 600 ml DMF.
The reaction was stirred at room temperature overnight. The solvent was removed in vacuo to afford an oily residue which was washed with ether three times. The oily residue was redissolved in a mixture of water and ACN (~1:1) and lyophilized to provide an alloc-protected psuedomycin A
compound in 90o yield.
The alloc-protected pseudomycin B compound was prepared using the same procedures in 90o yield (R1 - alloc in structure II below).
General procedures used to remove CBZ protecting groups at position 2, 4 and 5 by hydrogenation.
Dissolve CBZ-protected acylated-derivative in a cold 10 to 10o acetic/methanol solution (5 mg/ml) and add an equivalent amount of 10% Pd/C. Charge the reaction with hydrogen by degassing reaction and replacing volume with HZ
,4-7 times. Allow reaction to proceed at room temperature.
Monitor the reaction by HPLC every hour until starting material is consumed. When the reaction is complete, remove balloon and filter reaction with 0.45 ~,m filter disk (Acrodisk GHP, GF by Gelman). Concentrate to about 1/l0th volume and prep by HPLC. Lyophilize fractions containing product.
General procedures used to remove Alloc protecting groups at position 2, 4 and 5 with tributyltinhydride and triphenylphosphine palladium dichloride.
Acetic acid (1 ml) was added to a suspension of alloc-protected pseudomycin B (0.05 mmol) in 5 ml methylene chloride. After degassing under vacuum, the solution was treated with 6.0 mg PdCl2(PPH3)2 (0.008 mmol) and 0.40 ml tri-n-butyltin hydride (1.5 mmol)at room temperature for 2 hours. The solvent was evaporated in vacuo and the residue dissolved in water/ACN (~1:1) and filtered. The resulting solution was purified by preparative HPLC to afford the desired pseudomycin B compound in 93o yield. Alternatively, 5 ml tetrahydrofuran and 0.1 ml acetic acid may be used as the solvent instead of 5 ml methylene chloride and 1.0 ml acetic acid.
The following structure II will be used to describe the products observed in Examples 1 through 27. Although a specific pseudomycin natural product (pseudomycin B) was used in the Examples below, those skilled in the art will appreciate that other pseudomycin natural products or semi-synthetic derivatives may be used as starting materials.
r, ~n OH
H N _ R O O
O ...H~(CHzyoCHa H
1NHR' II
Examples 1-3 illustrate the formation of bis-esters at residues 3 and 8.
Example 1 Synthesis of Bis-Ethyl ester 1-1:
A 50 ml round bottom flask was charged with 10 ml of absolute ethanol and CBZ-protected pseudomycin B(251.7 mg, 0.156 mmol). To this mixture was added ~ 1 ml of acidified ethanol (previously acidified using HC1 gas) and the reaction was allowed to stir at room temperature overnight.
HO,. / O
The solvent was then removed in vacuo and the residue was carried on to the next step without further purification by dissolving it in a solution of 10 ml MeOH/1.5 ml glacial AcOH. Standard hydrogenolysis using 249.7 mg of 10o Pd/C
for 30 minutes, removal of the catalyst via filtration and purification via preparatory HPLC led to Compound 1-1 (120.9 mg) after lyophilization. MS (Ionspray) calcd for C55H96C1N1zOlg (M+H)+ 1264.89, found 1264.3.
The mono-esters may be isolated by following the reaction carefully by HPLC. The reaction is stopped at the appropriate time when the ratio of starting material: mono ester(s): bis ester is greatest. The methodology remains the same. The resulting mixture of mono esters is isolated where some ester is formed on the aspartic acid residue and some on the hydroxy aspartic acid residue. This mixture of CBZ-protected, mono esters is hydrogenated using standard methodology to yield a mixture of mono ethyl esters of Pseudomycin B.
Compounds 1-2 and 1-3 were synthesized using the same procedures described above.
R = -H R = -H
R2 - -OCH3 Rz - -OCH ( CH3 ) 2 R3 = -OCH3 R3 - -OCH ( CH3 ) z Example 2 illustrates the synthesize of bis-esters using basic conditions.
Example 2 Synthesis of Bis-propyl ester 2-1:
R = -H
R2 - -OCHzCH2CH3 R3 - -OCH2CHzCH3 CBZ-protected pseudomycin B (247.3 mg, 0..154 mmol) was dissolved in 5 ml DMF. A large excess of propyl iodide and an excess of NaHC03 were then added. The reaction was allowed to stir for 10 h at room temperature. Purification via preparatory HPLC followed by lyophilization provided 147.6 mg of the protected bis ester. Hydrogenolyis of this compound under standard condition using 149.3 mg of 10% Pd/C
yielded 78.9 mg of Compound 2-1 after HPLC purification and lyophilization.
Exantpl a 3 R = -H R = -H
2 0 RZ - -0 ( CHz ) 4CH3 RZ - -OH
R3 - -OH R3 - -O ( CHZ ) 4CH3 CBZ-protected pseudomycin B (282.3 mg, 0.175 mmol) was dissolved in 5 ml DMF. A large excess of n-pentyl iodide and an excess of NaHC03 were then added. The reaction was allowed to stir for 10 h at room temperature. Purification via preparatory HPLC followed by lyophilization provided 49.1 mg of the mixture of protected mono pentyl esters.
Hydrogenolyis of this mixture under standard condition using 47.3 mg of 10o Pd/C yielded 30.6 mg of Compounds 3-1 and 3-2 after HPLC purification and lyophilization.
R = -H R = -H R = -H
R2 - -O ( CHZ ) 3CH3 RZ= -O ( CHz ) 3CH3 Rz - -OH
R3 - -O ( CHZ ) 3CH3 R3 - -OH R3 - -0 ( CHZ ) 3CH3 Substitution of the propyl iodide with n-butyl iodide afforded the bis-butyl ester (3-3), a mixture of mono esters (3-4 + 3-5) and a mixture of mono ester + the following cyclic imide compound 3-6:
o O O
O O NH H ~ OH
O ~CI
,~~ O O
HZN~O
NH
O O H O~"'H (CH2)~oCH3 N NH
O
Example 4 Synthesis of cyclopentylmethyl ester 4-1:
R = -H
Rz - -OCHZ(cyclopentyl) CBZ-protected pseudomycin B, a large excess of p-toluenesulfonic acid and cyclopentanemethanol are mixed and allowed to stir overnight. An additional 10 equivalents of alcohol was added the next day. The CBZ-protected ester was isolated via preparatory HPLC and then hydrogenated using standard methodology to produce Compound 4-1.
Each of the compounds synthesized in Examples 1-4 showed measurable activity against Candida Albicans, Cryptococcus neoformans, Aspergillus Fumigatus, Candida Parapsilosis, or Histoplasma capsulatum. However, the following basic trends in activity were observed based on the compounds synthesized. Simple esters (bis-methyl, bis-ethyl and mono-ethyl) were active and efficacious; however, the larger esters exhibited less efficacy (e. g., propyl esters and larger). ADME has shown that Compounds 1-1 and 2-1 quickly cleave to the parent pseudomycin B compound.
Examples 5-11 illustrate the synthesis of amide derivatives at residue 3.
Exasnpl a 5 Synthesis of Compound 5-1:
R = -H
Rz _ -NHz CBZ-protected pseudomycin B (1.12 g) and 224 mg TBTU, 0.56 ml DIEA and 1.0 g deprotected rink amide resin (4-(2',4'-dimethoxyphenyl-aminomethyl)-phenoxy resin, available from Advance ChemTech, Inc., Louisville, KY) were mixed for 3 days. The mixture was filtered and the resin washed 3x with DMF and 3x with dichloromethane. The resin was treated with 5o water in 1:1 TFA/CHZC12 for 3 hours. The mixture was filtered and the resin washed 3x with TFA. The filtrate was collected and concentrated in vacuo. Upon purification by HPLC, 60 mg (5.30) of the CBZ-protected amido product was isolated.
The protected amido compound (60 mg) was dissolved in 6 ml of 1o AcOH in methanol and 60 mg of 10o Pd/C was added.
The mixture was stirred for 30 minutes under hydrogen at room temperature. After filtering, the solution was concentrated in vacuo. The residue was dissolved in 500 ACN/water and lyophilized to yield 45 mg (900) yield of Compound 5-1.
Example 6 Synthesis of Compound 6-1:
R = -H
R2 - -NH(cyclopropyl) CBZ-protected pseudomycin B (400 mg, 0.25 mmol) is dissolved in 4 ml dry DMF. TBTU (79 mg, 0.25 mmol), DIEA
(200 ~,1, 6 equivalents) and cyclopropylamine (14.2 mg, 0.25 mmol) were added sequentially. The reaction was stirred at room temperature under nitrogen while being monitored by HPLC. Upon completion the reaction was concentrated in vacuo. The crude product purified by preparative HPLC.
Lyophilization yielded 209.2 mg (51.10) of a colorless powder.
The 3-amido compound (279.1 mg, 0.169 mmol) was hydrogenated under hydrogen balloon catalyzed by 10% Pd/C in 1o HOAc/MeOH for 45 minutes. The reaction was filtered and concentrated in vacuo. The residue was picked up in a 1:1 mixture of water:ACN and then lyophilized to give 208.3 mg (98.60) of a colorless powder. The structure was verified by Hl-NMR.
Compound 6-1 can also be made from the Alloc-protected pseudomycin B using the following procedures.
1-Hydroxybenzotriazole hydrate (136 mg, 1.0 mmol) and EDCI (211 mg, 1.1 mmol) was added to a solution of alloc-protected pseudomycin B (730 mg, 0.50 mmol) in 7 ml of DMF.
After stirring overnight, cyclopropylamine (85.6 mg, 1.5 mmol) was added. The progress of the reaction was monitored by HPLC. Upon completion, the alloc-protected pseudomycin derivative (334 mg, 50o yield) was isolated via preparative HPLC and lyophilization.
The alloc-protected intermediate (117 mg, 0.078 mmol) was dissolved in 15 ml of methylene chloride and 1 ml of acetic acid. After degassing the reaction mixture with dry nitrogen, 30 mg of (PPh3)ZPdCl2 and 1 ml of tributyltinhydride was added to the mixture. The progress of the reaction was monitored by HPLC. Upon completion, the reaction mixture was purified by reverse phase preparative HPLC to provide 88 mg (91o yield) of Compound 6-1.
Table I below lists other 3-amido derivatives that were synthesized using the same general procedures described above using the appropriate corresponding amine starting material.
Table I
Example # R R R
6-3 -H -NHCHzCH3 -OH
6-5 -H -NH (CHZ) zCH3 -OH
6 - 6 -H -NHCH2 ( CH3 ) 2 -OH
6-6 -H -NH(cyclopropyl) -OH
6-7 -H -NHCHZCH=CHz -OH
6-8 -H -NH (CHz ) 4CH3 -OH
6-9 -H -NHCH ( CH3 ) ( CHZ -OH
) ZCH3 6-10 -H -NH (CHZ) SCH3 -OH
6-11 -H -NH(cyclohexyl) -OH
6-12 -H -NH(CHZ)6CH3 -OH
6-13 -H -NH (CHZ) 7CH3 -OH
6-14 -H -NH (CHZ) 8CH3 -OH
6 -15 -H -NH ( CHZ ) 9CH3 -OH
~N
H
6-17 -H -NH ( CHZ ) ZN ( CH3 -OH
) 2 6-18 -H -NH ( CH2 ) 2N ( CHZCH3-OH
) 2 6-19 -H -NH ( CHz ) 3N ( CH3 -OH
) 2 6-2 0 -H -NH ( CH2 ) 3N ( CHZCH3-OH
) z 6-21 -H -NH ( CH2 ) 4N ( CH3 -OH
) 2 6-22 -H -NH ( CH2 ) 6N ( CH3 -OH
) 2 6-2 3 -H -NH ( CH2 ) 7N ( CH3 -OH
) 2 '~N~N~
H
H
~N //~
Exampl a 7 Synthesis of 3-amido compound 7-1:
R = -H
R2 - '~N \
H ~.~~~~
i N
In a 500 mL oven dried round bottom flask, CBZ-protected Pseudomycin B(0.5 g, 0.311mmo1) was dissolved in 25 mL of DMF. To this solution was added TBTU(0.2 g, 0.622 mmol), 3-(aminomethyl)pyridine(0.067 g, 0.622mmo1), and N-ethyldicyclohexylamine(0.391 g, 1.87 mmol). The solution was stirred for three hours and then concentrated down. The product was isolated by reverse-phase preparatory HPLC, and lyophilized to yield, (96 mg, 18o yield) CBZ-protected amide. The deprotection of the CBZ groups was performed by adding slowly an equivalent mass of lOoPd/C to a cold 10 acetic/methanol solution of CBZ-protected amide. The solution was allowed to warm to rt and stirred rapidly for 3.5 hours under 1 atm H2. After removal of the catalyst via filtration, purification on reverse phase HPLC and lyophilization yielded 40 mg , 55o yield of Compound 7-1.
MS data Calculated for C57 H93 Cl N14 018 Mol. Wt. - 1296.6 Found ES+ 1297.15, ES- 1294.95 Example 8 Synthesis of 3-amido compound 8-1:
R = -H
Rz = ~N~N~
H
The same general procedures as described in Example 7 may be used. When no base is added, a mixture of 8 and 3 amido substituted compounds are observed.
Example 9 Synthesis of 3-amido compound 9-1:
R = -H R = -H
Rz - -NH(benzyl) RZ - -NH(benzyl) R3 - -OH R3 - -NH(benzyl) The same general procedures as described in Example 7 may be used. When no base is added, a mixture of Compounds 9-1 and 9-2 are observed.
Example 10 Synthesis of 3-amido compound 10-1:
R = -H
~NH
R2 = ~N~N~
H
In yet another embodiment of the present invention, a 3-amido derivative of a pseudomycin compound is provided where the compound is prepared by the steps of (i) providing a compound having structure I above wherein R1 is -NHz and R2 and R3 are both -OH; (ii) protecting the amino groups, R1, at positions 2, 4 and 5 with an amino-protecting group;
(iii) forming an amide linkage at position 3 using an o-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate as a coupling agent; and (iv) removing the amino-protecting groups. An 8-amido derivative is also provided where the derivative is prepared using the steps described above except using benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate as the coupling agent.
In another embodiment of the present invention, a pharmaceutical formulation is provided which includes the pseudomycin compound represented by structure I above and a pharmaceutically acceptable carrier.
In yet another embodiment of the present invention, a method is provided for treating an antifungal infection in an animal in need thereof, which comprises administering to the animal the pseudomycin compound I described above.
Definitions As used herein, the term "alkyl" refers to a hydrocarbon radical of the general formula CnH2n+i containing from 1 to 30 carbon atoms unless otherwise indicated. The alkane radical may be straight (e. g. methyl, ethyl, propyl, butyl, etc.), branched (e. g., isopropyl, isobutyl, tertiary butyl, neopentyl, etc.), cyclic (e. g., cyclopropyl, cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl, etc.), or multi-cyclic (e. g., bicyclo[2.2.1]heptane, spiro[2.2]pentane, etc.). The alkane radical may be substituted or unsubstituted. Similarly, the alkyl portion of an alkoxy group, alkanoyl, or alkanoate have the same definition as above.
The term "alkenyl" refers to an acyclic hydrocarbon containing at least one carbon carbon double bond. The alkene radical may be straight, branched, cyclic, or multi-cyclic. The alkene radical may be substituted or unsubstituted. The alkenyl portion of an alkenoxy, alkenoyl or alkenoate group has the same definition as above.
The term "alkynyl" refers to an acyclic hydrocarbon containing at least one carbon carbon triple bond. The alkyne radical may be straight, or branched. The alkyne radical may be substituted or unsubstituted. The alkynyl portion of an alkynoxy, alkynoyl or alkynoate group has the same definition as above.
The term "aryl" refers to aromatic moieties having single (e. g., phenyl) or fused ring systems (e. g., naphthalene, anthracene, phenanthrene, etc.). The aryl groups may be substituted or unsubstituted.
The term "heteroaryl" refers to aromatic moieties containing at least one heteratom within the aromatic ring system (e. g., pyrrole, pyridine, indole, thiophene, furan, benzofuran, imidazole, oxazine, pyrimidine, purine, benzimidazole, quinoline, etc.). The aromatic moiety may consist of a single or fused ring system. The heteroaryl groups may be substituted or unsubstituted.
"NHp-Pg" and °'amino protecting group" refer to a substituent of the amino group (Pg) commonly employed to block or protect the amino functionality while reacting other functional groups on the compound. When p is 0, the amino protecting group, when taken with the nitrogen to which it is attached, forms a cyclic imide, e.g., phthalimido and tetrachlorophthalimido. When p is 1, the protecting group, when taken with the nitrogen to which it is attached, can form a carbamate, e.g., methyl, ethyl, and 9-fluorenylmethylcarbamate; or an amide, e.g., N-formyl and N-acetylamide.
Within the field of organic chemistry and particularly within the field of organic biochemistry, it is widely understood that significant substitution of compounds is tolerated or even useful. In the present invention, for example, the term alkyl group allows for substitutents which is a classic alkyl, such as methyl, ethyl, propyl, hexyl, isooctyl, dodecyl, stearyl, etc. The term "group"
specifically envisions and allows for substitutions on alkyls which are common in the art, such as hydroxy, halogen, alkoxy, carbonyl, keto, ester, carbamato, etc., as well as including the unsubstituted alkyl moiety. However, it is generally understood by those skilled in the art that the substituents should be selected so as to not adversely affect the pharmacological characteristics of the compound or adversely interfere with the use of the medicament.
Suitable substituents for any of the groups defined above include alkyl, alkenyl, alkynyl, aryl, halo, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, mono- and di-alkyl amino, quaternary ammonium salts, aminoalkoxy, hydroxyalkylamino, aminoalkylthio, carbamyl, carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl, and combinations thereof.
The term "solvate" refers to an aggregate that comprises one or more molecules of the solute, such as a compound of structure I, with one or more molecules of a pharmaceutical solvent, such as water, ethanol, and the like.
The term "pharmaceutically acceptable salt" refers to organic or inorganic salts of the compounds represented by structure I that are substantially non-toxic to the recipient at the doses administered.
The term "prodrug" refers to a class of drugs which result in pharmacological action due to conversion by metabolic processes within the body (i.e., biotransformation). In the present invention, the pseudomycin prodrug compounds contain ester functionalities that can be cleaved by esterases in the plasma to produce the active drug.
The term "animal" refers to humans, companion animals (e. g., dogs, cats and horses), food-source animals (e. g., cows, pigs, sheep and poultry), zoo animals, marine animals, birds and other similar animal species.
DETAILED DESCRIPTION OF THE INVENTION
Applicants have discovered that modification of the acid functionality attached to the hydroxyaspartic acid and/or aspartic acid units of a pseudomycin natural product or semi-synthetic derivative provides compounds having in vitro indications which suggest that the new compounds may be active against C. albican, C, neoformans, and/or A.
fumigatus. Some bis-esters have been shown to act as a prodrug; therefore, these particular compounds have reduced in vitro activity but show in vivo efficacy.
Scheme I below illustrates the general procedures for synthesizing Compound I from any one of the naturally occurring pseudomycins or N-acyl modified derivatives. In general, three synthetic steps are used to produce Compound I: (1) selective amino protection; (2) condensation with the appropriate alcohol or amine to produce the respective ester or amide; and (4) deprotection of the amino groups.
HO O
O OH HO
O OH
$ N OH O NH H N OH
HO,~
/ -NH O ,-,~CI (1~ HO~H O CI
_ ~ O
H PgHN ~O O
H
~N R O O H 0~~~~N R
N N~NH
H
NHZ
OH NHPg H2N U PgHN O
(2) R
O OH p OH
O NH H N OH O~H N OH
HO~ O CI HO~ NH O CI
~NH O~ ~ (3) _ ~ O
H N~O O ~ PgHN~O O
NH O O "'N R O NH O H O ~~~N R
O
H
H N~--!NH O
H N~-!NH O
~NH2 RZ ~NHPg H2N O PgHN O
Scheme I
The pendant amino groups at residues 2, 4 and 5 may be protected using any standard means known to those skilled in the art for amino protection. The exact genus and species of amino protecting group employed is not critical so long as the derivatized amino group is stable to the conditions of subsequent reactions) on other positions of the intermediate molecule and the protecting group can be selectively removed at the appropriate point without disrupting the remainder of the molecule including any other amino protecting group(s). Preferred amino protecting groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyl, phthalimido, and benzyloxycarbonyl (CBZ). Most preferred is allyloxycarbonyl (Alloc) and benzyloxycarbonyl (CBZ).
Further examples of suitable protecting groups are described in T.W. Greene, "Protective Groups in Organic Synthesis,"
John Wiley and Sons, New York, N.Y., (2nd ed., 1991), at chapter 7.
Formation of the ester groups may be accomplished using standard esterification procedures well-known to those skilled in the art. Esterification under acidic conditions typically includes dissolving or suspending the pseudomycin compound in the appropriate alcohol in the presence of a protic acid (e. g., HC1, TFA, p-toluenesulfonic acid, etc.).
Under basic conditions, the pseudomycin compound is generally reacted with the appropriate alkyl halide in the presence of a weak base (e. g., sodium bicarbonate under anhydrous conditions).
Formation of the amide groups may be accomplished using standard amidation procedures well-known to those skilled in the art. However, the choice of coupling agents provides selective modification of the acid groups. For example, the use of benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate(PyBOP) as the coupling agent allows one to isolate pure mono-amides at residue 8 and (in some cases) pure bis amides simultaneously. Whereas, coupling agents such as o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) and 2(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) favor formation of monoamides at residue 3.
Applicants also discovered that the addition of a bulky amine enhances the ratio of monoamides at residue 3. The ratio of amidation at residue 3 vs. residue 8 increased from about 1:1 to about 6:1 and the amount of bis-amides was reduced through the addition of a bulky amine. The term "bulky amine" refers to an amine having multiple and/or large substituents on the nitrogen atom. Any tertiary amine may be used that is compatible with the reaction conditions.
Preferred bulky amines include N,N-diisopropylethylamine (DIEA) and N-ethyldicyclohexylamine. The amount of bulky amine added is generally from about 1 to 10 equivalents, preferably 3 to 8 equivalents, more preferably 5 to 6 equivalents. The reaction is generally ran at temperatures from about room temperature (25°C) to about -20°C. However, Applicants discovered that lower temperatures (from about 0°C to about -20°C) further enhance the formation of monoamides at residue 3. The ratio of amidation at residue 3 vs. residue 8 increased as much as 20:1 by adding a bulky amine and lowering the temperature of the reaction.
However, it will be understood by those skilled in the art that the lower temperature limit will depend upon the solubility of the reactive components.
Once the acid groups) are modified, then the amino protecting groups (at positions 2, 4 and 5) may be removed using standard procedures appropriate for the specific protecting group used. For example, CBZ groups are removed by hydrogenation in the presence of a hydrogenation catalyst (e.g., 10o Pd/C). When the amino protecting group is allyloxycarbonyl, then the protecting group may be removed using tributyltinhydride and triphenylphosphine palladium dichloride. This particular protection/deprotection scheme has the advantage of reducing the potential for hydrogenating the vinyl group of the Z-Dhb unit of the pseudomycin structure.
As discussed earlier, pseudomycins are natural products isolated from the bacterium Pseudomonas syringae that have been characterized as lipodepsinonapetpides containing a cyclic peptide portion closed by a lactone bond and including the unusual amino acids 4-chlorothreonine (ClThr), 3-hydroxyaspartic acid (HOAsp), 2,3-dehydro-2-aminobutyric acid (Dhb), and 2,4-diaminobutyric acid (Dab). Methods for growth of various strains of P. syringae to produce the different pseudomycin analogs (A, A', B, B', C, and C') are described below and described in more detail in PCT Patent Application Serial No. PCT/US00/08728 filed by Hilton, et al. on April 14, 2000 entitled "Pseudomycin Production by Pseudomonas Syringae," incorporated herein by reference, PCT
Patent Application Serial No. PCT/US00/08727 filed by Kulanthaivel, et al. on April 14, 2000 entitled "Pseudomycin Natural Products," incorporated herein by reference, and U.S. Patent Nos. 5,576,298 and 5,837,685, each of which are incorporated herein by reference.
Isolated strains of P. syringae that produce one or more pseudomycins are known in the art. Wild type strain MSU 174 and a mutant of this strain generated by transposon mutagenesis, MSU 16H are described in U.S. Patent Nos.
5,576,298 and 5,837,685; Harrison, et al., "Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity," J. Gen.
Microbiology, 137, 2857-2865 (1991); and Lamb et al., "Transposon mutagenesis and tagging of fluorescent pseudomonas: Antimycotic production is necessary for control of Dutch elm disease," Proc. Natl. Acad. Sci. USA, 84, 6447-6451 (1987).
A strain of P. syringae that is suitable for production of one or more pseudomycins can be isolated from environmental sources including plants (e. g., barley plants, citrus plants, and lilac plants) as well as, sources such as soil, water, air, and dust. A preferred stain is isolated from plants. Strains of P. syringae that are isolated from environmental sources can be referred to as wild type. As used herein, "wild type" refers to a dominant genotype which naturally occurs in the normal population of P. syringae (e.g., strains or isolates of P. syringae that are found in nature and not produced by laboratory manipulation). Like most organisms, the characteristics of the pseudomycin-producing cultures employed (P. syringae strains such as MSU
174, MSU 16H, MSU 206, 25-B1, 7H9-1) are subject to variation. Hence, progeny of these strains (e. g., recombinants, mutants and variants) may be obtained by methods known in the art.
P. syringae MSU 16H is publicly available from the American Type Culture Collection, Parklawn Drive, Rockville, MD, USA as Accession No. ATCC 67028. P. syringae strains 25-B1, 7H9-1, and 67 H1 were deposited with the American Type Culture Collection on March 23, 2000 and were assigned the following Accession Nos.:
25-B1 Accession No. PTA-1622 7H9-1 Accession No. PTA-1623 67 H1 Accession No. PTA-1621 Mutant strains of P. syringae are also suitable for production of one or more pseudomycins. As used herein, "mutant" refers to a sudden heritable change in the phenotype of a strain, which can be spontaneous or induced by known mutagenic agents, such as radiation (e. g., ultraviolet radiation or x-rays), chemical mutagens (e. g., ethyl methanesulfonate (EMS), diepoxyoctane, N-methyl-N-nitro-N'-nitrosoguanine (NTG), and nitrous acid), site-s specific mutagenesis, and transposon mediated mutagenesis.
Pseudomycin-producing mutants of P. syringae can be produced by treating the bacteria with an amount of a mutagenic agent effective to produce mutants that overproduce one or more pseudomycins, that produce one pseudomycin (e. g., pseudomycin B) in excess over other pseudomycins, or that produce one or more pseudomycins under advantageous growth conditions. While the type and amount of mutagenic agent to be used can vary, a preferred method is to serially dilute NTG to levels ranging from 1 to 100 ~,g/ml. Preferred mutants are those that overproduce pseudomycin B and grow in minimal defined media.
Environmental isolates, mutant strains, and other desirable strains of P. syringae can be subjected to selection for desirable traits of growth habit, growth medium nutrient source, carbon source, growth conditions, amino acid requirements, and the like. Preferably, a pseudomycin producing strain of P. syringae is selected for growth on minimal defined medium such as N21 medium and/or for production of one or more pseudomycins at levels greater than about 10 ~g/ml. Preferred strains exhibit the characteristic of producing one or more pseudomycins when grown on a medium including three or fewer amino acids and optionally, either a lipid, a potato product or combination thereof.
Recombinant strains can be developed by transforming the P. syringae strains, using procedures known in the art.
Through the use of recombinant DNA technology, the P.
syringae strains can be transformed to express.a variety of gene products in addition to the antibiotics these strains produce. For example, one can modify the strains to introduce multiple copies of the endogenous pseudomycin-biosynthesis genes to achieve greater pseudomycin yield.
To produce one or more pseudomycins from a wild type or mutant strain of P. syringae, the organism is cultured with agitation in an aqueous nutrient medium including an effective amount of three or fewer amino acids, preferably glutamic acid, glycine, histidine, or a combination thereof.
Alternatively, glycine is combined with one or more of a potato product and a lipid. Culturing is conducted under conditions effective for growth of P. syringae and production of the desired pseudomycin or pseudomycins.
Effective conditions include temperatures from about 22°-C to about 27°-C, and a duration of about 36 hours to about 96 hours. Controlling the concentration of oxygen in the medium during culturing of P. syringae is advantageous for production of a pseudomycin. Preferably, oxygen levels are maintained at about 5 to 50o saturation, more preferably about 30o saturation. Sparging with air, pure oxygen, or gas mixtures including oxygen can regulate the concentration of oxygen in the medium.
Controlling the pH of the medium during culturing of P.
syringae is also advantageous. Pseudomycins are labile at basic pH, and significant degradation can occux if the pH of the culture medium is above about 6 for more than about 12 hours. Preferably, the pH of the culture medium is maintained between 6 and 4. P. syringae can produce one or more pseudomycins when grown in batch culture. However, fed-bath or semi-continuous feed of glucose and optionally, an acid or base (e.g., ammonium hydroxide) to control pH, enhances production. Pseudomycin production can be further enhanced by using continuous culture methods in which glucose and ammonium hydroxide are fed automatically.
Choice of P. syringae strain can affect the amount and distribution of pseudomycin or pseudomycins produced. For example, strains MSU 16H and 67 H1 each produce predominantly pseudomycin A, but also produce pseudomycin B
and C, typically in ratios of 4:2:1. Strain 67 H1 typically produces levels of pseudomycins about three to five fold larger than are produced by strain MSU 16H. Compared to strains MSU 16H and 67 H1, strain 25-B1 produces more pseudomycin B and less pseudomycin C. Strain 7H9-1 are distinctive in producing predominantly pseudomycin B and larger amount of pseudomycin B than other strains. For example, this strain can produce pseudomycin B in at least a ten fold excess over either pseudomycin A or C.
Each pseudomycin, pseudomycin intermediate and mixtures can be detected, determined, isolated, and/or purified by any variety of methods known to those skilled in the art.
For example, the level of pseudomycin activity in a broth or in an isolate or purified composition can be determined by antifungal action against a fungus such as Candida and can be isolated and purified by high performance liquid chromatography.
Alternatively, the amido or ester derivative can be formed from an N-acyl semi-synthetic compound. Semi-synthetic pseudomycin compounds may be synthesized by exchanging the N-acyl group on the L-serine unit. Examples of various N-acyl derivatives are described in PCT Patent Application Serial No. , Belvo, et al., filed evendate herewith entitled "Pseudomycin N-Acyl Side-Chain Analogs" and incorporated herein by reference. In general, four synthetic steps are used to produce the semi-synthetic compounds from naturally occurring pseudomycin compounds:
(1) selective amino protection; (2) chemical or enzymatic deacylation of the N-acyl side-chain; (3) reacylation with a different side-chain; and (4) deprotection of the amino groups. The aspartic acid and/or hydroxyaspartic acid units can be modified prior to deprotecting the amino groups.
The deacylation of an N-acyl group having a gamma or delta hydroxylated side chain (e. g., 3,4-dihydroxytetra-deconoate) may be accomplished by treating the amino-protected pseudomycin compound with acid in an aqueous solvent. Suitable acids include acetic acid and trifluoroacetic acid. A preferred acid is trifluoroacetic acid. If trifluoroacetic acid is used, the reaction may be accomplished at or near room temperature. However, when acetic acid is used the reaction is generally ran at about 40°C. Suitable aqueous solvent systems include acetonitrile, water, and mixtures thereof. Organic solvents accelerate the reaction; however, the addition of an organic solvent may lead to other by-products. Pseudomycin compounds lacking a delta or gamma hydroxy group on the side chain (e. g., Pseudomycin B and C') may be deacylated enzymatically. Suitable deacylase enzymes include Polymyxin Acylase (164-16081 Fatty Acylase (crude) or 161-16091 Fatty Acylase (pure) available from Wako Pure Chemical Industries, Ltd.), or ECB deacylase. The enzymatic deacylation may be accomplished using standard deacylation procedures well known to those skilled in the art. For example, general procedures for using polymyxin acylase may be found in Yasuda, N., et al, Agric. Biol. Chem., 53, 3245 (1989) and Kimura, Y., et al., Agric. Biol. Chem., 53, 497 (1989).
The deacylated product (also known as the pseudomycin nucleus) is reacylated using the corresponding acid of the desired acyl group in the presence of a carbonyl activating agent. "Carbonyl activating group" refers to a substituent of a carbonyl that promotes nucleophilic addition reactions at that carbonyl. Suitable activating substituents are those which have a net electron withdrawing effect on the carbonyl. Such groups;include, but are not limited to, alkoxy, aryloxy, nitrogen containing aromatic heterocycles, or amino groups (e. g., oxybenzotriazole, imidazolyl, nitrophenoxy, pentachlorophenoxy, N-oxysuccinimide, N,N'-dicyclohexylisoure-O-yl, and N-hydroxy-N-methoxyamino);
acetates; formates; sulfonates (e. g., methanesulfonate, ethanesulfonate, benzenesulfonate, and p-tolylsulfonate);
and halides (e. g., chloride, bromide, and iodide).
A variety of acids may be used in the acylation process. Suitable acids include aliphatic acids containing one or more pendant aryl, alkyl, amino(including primary, secondary and tertiary amines), hydroxy, alkoxy, and amido groups; aliphatic acids containing nitrogen or oxygen within the aliphatic chain; aromatic acids substituted with alkyl, hydroxy, alkoxy and/or alkyl amino groups; and heteroaromatic acids substituted with alkyl, hydroxy, alkoxy and/or alkyl amino groups.
Alternatively, a solid phase synthesis may be used where a hydroxybenzotriazole-resin (HOBt-resin) serves as the coupling agent for the acylation reaction.
The acid-modification of the protected N-acyl semi-synthetic compound is then accomplished by reacting at least one of the pendant carboxyl groups attached to. the aspartic or hydroxyaspartic peptide units of the N-acyl modified semi-synthetic pseudomycin compound to form the desired amide or ester linkage(s). The protecting groups are then removed as described earlier.
The pseudomycin compound may be isolated and used per se or in the form of its pharmaceutically acceptable salt or solvate. The term "pharmaceutically acceptable salt" refers to non-toxic acid addition salts derived from inorganic and organic acids. Suitable salt derivatives include halides, thiocyanates, sulfates, bisulfates, sulfites, bisulfites, arylsulfonates, alkylsulfates, phosphonates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphonates, alkanoates, cycloalkylalkanoates, arylalkonates, adipates, alginates, aspartates, benzoates, fumarates, glucoheptanoates, glycerophosphates, lactates, maleates, nicotinates, oxalates, palmitates, pectinates, picrates, pivalates, succinates, tartarates, citrates, camphorates, camphorsulfonates, digluconates, trifluoroacetates, and the like.
The term "solvate" refers to an aggregate that comprises one or more molecules of the solute (i.e., pseudomycin compound) with one or more molecules of a pharmaceutical solvent, such as water, ethanol, and the like. When the solvent is water, then the aggregate is referred to as a hydrate. Solvates are generally formed by dissolving the compound in the appropriate solvent with heat and slowing cooling to generate an amorphous or crystalline solvate form.
The active ingredient (i.e., pseudomycin compound) is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient, physician or veterinarian an elegant and easy to handle product. Formulations may comprise from 0.10 to 99.90 by weight of active ingredient, more generally from about 10o to about 30o by weight.
As used herein, the term "unit dose" or "unit dosage"
refers to physically discrete units that contain a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect. When a unit dose is administered orally or parenterally, it is typically provided in the form of a tablet, capsule, pill, powder packet, topical composition, suppository, wafer, measured units in ampoules or in multidose containers, etc.
Alternatively, a unit dose may be administered in the form of a dry or liquid aerosol which may be inhaled or sprayed.
The dosage to be administered may vary depending upon the physical characteristics of the animal, the severity of the animal's symptoms, the means used to administer the drug and the animal species. The specific dose for a given animal is usually set by the judgment of the attending physician or veterinarian.
Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like. The particular carrier, diluent or excipient used will depend upon the means and purpose for which the active ingredient is being applied. The formulations may also include wetting agents, lubricating agents, surfactants, buffers, tonicity agents, bulking agents, stabilizers, emulsifiers, suspending agents, preservatives, sweeteners, perfuming agents, flavoring agents and combinations thereof.
A pharmaceutical composition may be administered using a variety of methods. Suitable methods include topical (e. g., ointments or sprays), oral, injection and inhalation.
The particular treatment method used will depend upon the type of infection being addressed.
In parenteral iv applications, the formulations are typically diluted or reconstituted (if freeze-dried) and further diluted if necessary, prior to administration. An example of reconstitution instructions for the freeze-dried product are to add ten ml of water for injection (WFI) to the vial and gently agitate to dissolve. Typical reconstitution times are less than one minute. The resulting solution is then further diluted in an infusion solution such as dextrose 5% in water (D5W), prior to administration.
Pseudomycin compounds have been shown to exhibit antifungal activity such as growth inhibition of various infectious fungi including Candida spp. (i.e., C. albicans, C. parapsilosis, C. krusei, C. glabrata, C. tropicalis, or C. lusitaniaw); Torulopus spp.(i.e., T. glabrata);
Aspergillus spp. (i.e., A. fumigatus); Histoplasma spp.
(i.e., H. capsulatum); Cryptococcus spp. (i.e., C.
neoformans); Blastomyces spp. (i.e., B. dermatitidis);
Fusarium spp.; Trichophyton spp., Pseudallescheria boydii, Coccidioides immits, Sporothrix schenckii, etc.
Consequently, the compounds and formulations of the present invention are useful in the preparation of medicaments for use in combating either systemic fungal infections or fungal skin infections. Accordingly, a method is provided for inhibiting fungal activity comprising contacting the pseudomycin compound of the present invention with a fungus. A preferred method includes inhibiting Candida albicans or Aspergillus fumigatus activity. The term "contacting" includes a union or junction, or apparent touching or mutual tangency of a compound of the invention with a fungus. The term does not imply any further limitations to the process, such as by mechanism of inhibition. The methods are defined to encompass the inhibition of fungal activity by the action of the compounds and their inherent antifungal properties.
A method for treating a fungal infection which comprises administering an effective amount of a pharmaceutical formulation of the present invention to an animal host in need of such treatment is also provided. A
preferred method includes treating a Candida albicans or Aspergillus fumigatus infection. The term "effective amount" refers to an amount of active compound which is capable of inhibiting fungal activity. The dose administered will vary depending on such factors as the nature and severity of the infection, the age and general health of the host, the tolerance of the host to the antifungal agent and species of the host. The particular dose regimen likewise may vary according to these factors.
The medicament may be given in a single daily dose or in multiple doses during the day. The regimen may last from about 2-3 days to about 2-3 weeks or longer. A typical daily dose (administered in single or divided doses) contains a dosage level between about 0.01 mg/kg to 100 mg/kg of body weight of an active compound. Preferred daily doses are generally between about 0.1 mg/kg to 60 mg/kg and more preferably between about 2.5 mg/kg to 40 mg/kg. The host may be any animal including humans, companion animals (e. g., dogs, cats and horses), food-source animals (e. g., cows, pigs, sheep and poultry), zoo animals, marine animals, birds and other similar animal species.
EXAMPLES
Unless indicated otherwise, all chemicals can be acquired from Aldrich Chemical (Milwaukee, WI). The following abbreviations are used through out the examples to represent the respective listed materials:
ACN - acetonitrile TFA - trifluoroacetic acid DMF - dimethylformamide EDCI - 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride BOC = t-butoxycarbonyl, (CH3)3C-0-C(0)-2 5 CBZ = benzyloxycarbonyl , C6HSCH2-O-C ( O ) -PyBOP = benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate TBTU = o-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate DIEA = N,N-diisopropylethylamine HPLC Conditions Unless indicated otherwise, analytical reverse-phase HPLC work was done using the Waters 600E systems equipped with Waters ~,Bondapak (C18, 3.9 X 300 mm) column. The eluent used was 65:35 acetonitrile/0.1o aqueous TFA solvent system to 100% acetonitrile over 20 minutes with a flow rate of 1.5 ml/minute and using UV detection at 230 nm.
Preparative HPLC work was performed with a Waters Prep 2000 system using Dynamax 60 angstrom C18 column and identical solvent systems as used in the analytical HPLC
system but with a flow rate of 40 ml/min.
Biological Analysis Detection and Quantification of An ti fungal Activity:
Antifungal activity was determined in vitro by obtaining the minimum inhibitory concentration (MIC) of the compound using a standard agar dilution test or a disc-diffusion test. A typical fungus employed in testing antifungal activity is Candida albicans. Antifungal activity is considered significant when the test sample (50 ~,1) causes 10-12 mm diameter zones of inhibition on C.
albicans x657 seeded agar plates.
Tail Vein Toxicity:
Mice were treated intravenously (IV) through the lateral tail vein with 0.1 ml of testing compound (20 mg/kg) at 0, 24, 48 and 72 hours. Two mice were included in each group. Compounds were formulated in 5.0o dextrose and sterile water for injection. The mice were monitored for 7 days following the first treatment and observed closely for signs of irritation including erythema, swelling, discoloration, necrosis, tail loss and any other signs of adverse effects indicating toxicity.
The mice used in the study were outbred, male ICR mice having an average weight between 18-20 g (available from Harlan Sprangue Dawley, Indianapolis, IN).
General Procedures CBZ-Protected Pseudomycin: General procedures used to protect the pendant amino groups at positions 2, 4 and 5 of Pseudomycin A, A' , B, B' , C or C' wi th CBZ.
Dissolve/suspend pseudomycin compound (Rl=H) in DMF (20 mg/ml, Aldrich Sure Seal). V~hile stirring at room temperature add N-(Benzyloxycarbonyloxy)succinimide (6 eq).
Allow to stir at room temperature for 32 hours. Monitor reaction by HPLC (4.6x50 mm, 3.5 E,tm, 300-SB, C8, Zorbax column). Concentrate reaction to 10 ml on high vacuum rotovap at room temperature. Put material in freezer until ready to prep by chromatography. Reverse phase preparative HPLC yields an amorphous, white solid after lyophilization (R1 - CBZ in structure II below).
Alloc-Protected Pseudomycin: General procedures used to protect the pendant amino groups at positions 2, 4 and 5 of Pseudomycin A, A', B, B', C or C' with Alloc.
Diallyl pyrocarbonate (558 mg, 3.0 mmol) was added to a solution of Pseudomycin A (1.22 g, 1.0 mmol) ixi 600 ml DMF.
The reaction was stirred at room temperature overnight. The solvent was removed in vacuo to afford an oily residue which was washed with ether three times. The oily residue was redissolved in a mixture of water and ACN (~1:1) and lyophilized to provide an alloc-protected psuedomycin A
compound in 90o yield.
The alloc-protected pseudomycin B compound was prepared using the same procedures in 90o yield (R1 - alloc in structure II below).
General procedures used to remove CBZ protecting groups at position 2, 4 and 5 by hydrogenation.
Dissolve CBZ-protected acylated-derivative in a cold 10 to 10o acetic/methanol solution (5 mg/ml) and add an equivalent amount of 10% Pd/C. Charge the reaction with hydrogen by degassing reaction and replacing volume with HZ
,4-7 times. Allow reaction to proceed at room temperature.
Monitor the reaction by HPLC every hour until starting material is consumed. When the reaction is complete, remove balloon and filter reaction with 0.45 ~,m filter disk (Acrodisk GHP, GF by Gelman). Concentrate to about 1/l0th volume and prep by HPLC. Lyophilize fractions containing product.
General procedures used to remove Alloc protecting groups at position 2, 4 and 5 with tributyltinhydride and triphenylphosphine palladium dichloride.
Acetic acid (1 ml) was added to a suspension of alloc-protected pseudomycin B (0.05 mmol) in 5 ml methylene chloride. After degassing under vacuum, the solution was treated with 6.0 mg PdCl2(PPH3)2 (0.008 mmol) and 0.40 ml tri-n-butyltin hydride (1.5 mmol)at room temperature for 2 hours. The solvent was evaporated in vacuo and the residue dissolved in water/ACN (~1:1) and filtered. The resulting solution was purified by preparative HPLC to afford the desired pseudomycin B compound in 93o yield. Alternatively, 5 ml tetrahydrofuran and 0.1 ml acetic acid may be used as the solvent instead of 5 ml methylene chloride and 1.0 ml acetic acid.
The following structure II will be used to describe the products observed in Examples 1 through 27. Although a specific pseudomycin natural product (pseudomycin B) was used in the Examples below, those skilled in the art will appreciate that other pseudomycin natural products or semi-synthetic derivatives may be used as starting materials.
r, ~n OH
H N _ R O O
O ...H~(CHzyoCHa H
1NHR' II
Examples 1-3 illustrate the formation of bis-esters at residues 3 and 8.
Example 1 Synthesis of Bis-Ethyl ester 1-1:
A 50 ml round bottom flask was charged with 10 ml of absolute ethanol and CBZ-protected pseudomycin B(251.7 mg, 0.156 mmol). To this mixture was added ~ 1 ml of acidified ethanol (previously acidified using HC1 gas) and the reaction was allowed to stir at room temperature overnight.
HO,. / O
The solvent was then removed in vacuo and the residue was carried on to the next step without further purification by dissolving it in a solution of 10 ml MeOH/1.5 ml glacial AcOH. Standard hydrogenolysis using 249.7 mg of 10o Pd/C
for 30 minutes, removal of the catalyst via filtration and purification via preparatory HPLC led to Compound 1-1 (120.9 mg) after lyophilization. MS (Ionspray) calcd for C55H96C1N1zOlg (M+H)+ 1264.89, found 1264.3.
The mono-esters may be isolated by following the reaction carefully by HPLC. The reaction is stopped at the appropriate time when the ratio of starting material: mono ester(s): bis ester is greatest. The methodology remains the same. The resulting mixture of mono esters is isolated where some ester is formed on the aspartic acid residue and some on the hydroxy aspartic acid residue. This mixture of CBZ-protected, mono esters is hydrogenated using standard methodology to yield a mixture of mono ethyl esters of Pseudomycin B.
Compounds 1-2 and 1-3 were synthesized using the same procedures described above.
R = -H R = -H
R2 - -OCH3 Rz - -OCH ( CH3 ) 2 R3 = -OCH3 R3 - -OCH ( CH3 ) z Example 2 illustrates the synthesize of bis-esters using basic conditions.
Example 2 Synthesis of Bis-propyl ester 2-1:
R = -H
R2 - -OCHzCH2CH3 R3 - -OCH2CHzCH3 CBZ-protected pseudomycin B (247.3 mg, 0..154 mmol) was dissolved in 5 ml DMF. A large excess of propyl iodide and an excess of NaHC03 were then added. The reaction was allowed to stir for 10 h at room temperature. Purification via preparatory HPLC followed by lyophilization provided 147.6 mg of the protected bis ester. Hydrogenolyis of this compound under standard condition using 149.3 mg of 10% Pd/C
yielded 78.9 mg of Compound 2-1 after HPLC purification and lyophilization.
Exantpl a 3 R = -H R = -H
2 0 RZ - -0 ( CHz ) 4CH3 RZ - -OH
R3 - -OH R3 - -O ( CHZ ) 4CH3 CBZ-protected pseudomycin B (282.3 mg, 0.175 mmol) was dissolved in 5 ml DMF. A large excess of n-pentyl iodide and an excess of NaHC03 were then added. The reaction was allowed to stir for 10 h at room temperature. Purification via preparatory HPLC followed by lyophilization provided 49.1 mg of the mixture of protected mono pentyl esters.
Hydrogenolyis of this mixture under standard condition using 47.3 mg of 10o Pd/C yielded 30.6 mg of Compounds 3-1 and 3-2 after HPLC purification and lyophilization.
R = -H R = -H R = -H
R2 - -O ( CHZ ) 3CH3 RZ= -O ( CHz ) 3CH3 Rz - -OH
R3 - -O ( CHZ ) 3CH3 R3 - -OH R3 - -0 ( CHZ ) 3CH3 Substitution of the propyl iodide with n-butyl iodide afforded the bis-butyl ester (3-3), a mixture of mono esters (3-4 + 3-5) and a mixture of mono ester + the following cyclic imide compound 3-6:
o O O
O O NH H ~ OH
O ~CI
,~~ O O
HZN~O
NH
O O H O~"'H (CH2)~oCH3 N NH
O
Example 4 Synthesis of cyclopentylmethyl ester 4-1:
R = -H
Rz - -OCHZ(cyclopentyl) CBZ-protected pseudomycin B, a large excess of p-toluenesulfonic acid and cyclopentanemethanol are mixed and allowed to stir overnight. An additional 10 equivalents of alcohol was added the next day. The CBZ-protected ester was isolated via preparatory HPLC and then hydrogenated using standard methodology to produce Compound 4-1.
Each of the compounds synthesized in Examples 1-4 showed measurable activity against Candida Albicans, Cryptococcus neoformans, Aspergillus Fumigatus, Candida Parapsilosis, or Histoplasma capsulatum. However, the following basic trends in activity were observed based on the compounds synthesized. Simple esters (bis-methyl, bis-ethyl and mono-ethyl) were active and efficacious; however, the larger esters exhibited less efficacy (e. g., propyl esters and larger). ADME has shown that Compounds 1-1 and 2-1 quickly cleave to the parent pseudomycin B compound.
Examples 5-11 illustrate the synthesis of amide derivatives at residue 3.
Exasnpl a 5 Synthesis of Compound 5-1:
R = -H
Rz _ -NHz CBZ-protected pseudomycin B (1.12 g) and 224 mg TBTU, 0.56 ml DIEA and 1.0 g deprotected rink amide resin (4-(2',4'-dimethoxyphenyl-aminomethyl)-phenoxy resin, available from Advance ChemTech, Inc., Louisville, KY) were mixed for 3 days. The mixture was filtered and the resin washed 3x with DMF and 3x with dichloromethane. The resin was treated with 5o water in 1:1 TFA/CHZC12 for 3 hours. The mixture was filtered and the resin washed 3x with TFA. The filtrate was collected and concentrated in vacuo. Upon purification by HPLC, 60 mg (5.30) of the CBZ-protected amido product was isolated.
The protected amido compound (60 mg) was dissolved in 6 ml of 1o AcOH in methanol and 60 mg of 10o Pd/C was added.
The mixture was stirred for 30 minutes under hydrogen at room temperature. After filtering, the solution was concentrated in vacuo. The residue was dissolved in 500 ACN/water and lyophilized to yield 45 mg (900) yield of Compound 5-1.
Example 6 Synthesis of Compound 6-1:
R = -H
R2 - -NH(cyclopropyl) CBZ-protected pseudomycin B (400 mg, 0.25 mmol) is dissolved in 4 ml dry DMF. TBTU (79 mg, 0.25 mmol), DIEA
(200 ~,1, 6 equivalents) and cyclopropylamine (14.2 mg, 0.25 mmol) were added sequentially. The reaction was stirred at room temperature under nitrogen while being monitored by HPLC. Upon completion the reaction was concentrated in vacuo. The crude product purified by preparative HPLC.
Lyophilization yielded 209.2 mg (51.10) of a colorless powder.
The 3-amido compound (279.1 mg, 0.169 mmol) was hydrogenated under hydrogen balloon catalyzed by 10% Pd/C in 1o HOAc/MeOH for 45 minutes. The reaction was filtered and concentrated in vacuo. The residue was picked up in a 1:1 mixture of water:ACN and then lyophilized to give 208.3 mg (98.60) of a colorless powder. The structure was verified by Hl-NMR.
Compound 6-1 can also be made from the Alloc-protected pseudomycin B using the following procedures.
1-Hydroxybenzotriazole hydrate (136 mg, 1.0 mmol) and EDCI (211 mg, 1.1 mmol) was added to a solution of alloc-protected pseudomycin B (730 mg, 0.50 mmol) in 7 ml of DMF.
After stirring overnight, cyclopropylamine (85.6 mg, 1.5 mmol) was added. The progress of the reaction was monitored by HPLC. Upon completion, the alloc-protected pseudomycin derivative (334 mg, 50o yield) was isolated via preparative HPLC and lyophilization.
The alloc-protected intermediate (117 mg, 0.078 mmol) was dissolved in 15 ml of methylene chloride and 1 ml of acetic acid. After degassing the reaction mixture with dry nitrogen, 30 mg of (PPh3)ZPdCl2 and 1 ml of tributyltinhydride was added to the mixture. The progress of the reaction was monitored by HPLC. Upon completion, the reaction mixture was purified by reverse phase preparative HPLC to provide 88 mg (91o yield) of Compound 6-1.
Table I below lists other 3-amido derivatives that were synthesized using the same general procedures described above using the appropriate corresponding amine starting material.
Table I
Example # R R R
6-3 -H -NHCHzCH3 -OH
6-5 -H -NH (CHZ) zCH3 -OH
6 - 6 -H -NHCH2 ( CH3 ) 2 -OH
6-6 -H -NH(cyclopropyl) -OH
6-7 -H -NHCHZCH=CHz -OH
6-8 -H -NH (CHz ) 4CH3 -OH
6-9 -H -NHCH ( CH3 ) ( CHZ -OH
) ZCH3 6-10 -H -NH (CHZ) SCH3 -OH
6-11 -H -NH(cyclohexyl) -OH
6-12 -H -NH(CHZ)6CH3 -OH
6-13 -H -NH (CHZ) 7CH3 -OH
6-14 -H -NH (CHZ) 8CH3 -OH
6 -15 -H -NH ( CHZ ) 9CH3 -OH
~N
H
6-17 -H -NH ( CHZ ) ZN ( CH3 -OH
) 2 6-18 -H -NH ( CH2 ) 2N ( CHZCH3-OH
) 2 6-19 -H -NH ( CHz ) 3N ( CH3 -OH
) 2 6-2 0 -H -NH ( CH2 ) 3N ( CHZCH3-OH
) z 6-21 -H -NH ( CH2 ) 4N ( CH3 -OH
) 2 6-22 -H -NH ( CH2 ) 6N ( CH3 -OH
) 2 6-2 3 -H -NH ( CH2 ) 7N ( CH3 -OH
) 2 '~N~N~
H
H
~N //~
Exampl a 7 Synthesis of 3-amido compound 7-1:
R = -H
R2 - '~N \
H ~.~~~~
i N
In a 500 mL oven dried round bottom flask, CBZ-protected Pseudomycin B(0.5 g, 0.311mmo1) was dissolved in 25 mL of DMF. To this solution was added TBTU(0.2 g, 0.622 mmol), 3-(aminomethyl)pyridine(0.067 g, 0.622mmo1), and N-ethyldicyclohexylamine(0.391 g, 1.87 mmol). The solution was stirred for three hours and then concentrated down. The product was isolated by reverse-phase preparatory HPLC, and lyophilized to yield, (96 mg, 18o yield) CBZ-protected amide. The deprotection of the CBZ groups was performed by adding slowly an equivalent mass of lOoPd/C to a cold 10 acetic/methanol solution of CBZ-protected amide. The solution was allowed to warm to rt and stirred rapidly for 3.5 hours under 1 atm H2. After removal of the catalyst via filtration, purification on reverse phase HPLC and lyophilization yielded 40 mg , 55o yield of Compound 7-1.
MS data Calculated for C57 H93 Cl N14 018 Mol. Wt. - 1296.6 Found ES+ 1297.15, ES- 1294.95 Example 8 Synthesis of 3-amido compound 8-1:
R = -H
Rz = ~N~N~
H
The same general procedures as described in Example 7 may be used. When no base is added, a mixture of 8 and 3 amido substituted compounds are observed.
Example 9 Synthesis of 3-amido compound 9-1:
R = -H R = -H
Rz - -NH(benzyl) RZ - -NH(benzyl) R3 - -OH R3 - -NH(benzyl) The same general procedures as described in Example 7 may be used. When no base is added, a mixture of Compounds 9-1 and 9-2 are observed.
Example 10 Synthesis of 3-amido compound 10-1:
R = -H
~NH
R2 = ~N~N~
H
The same general procedures as described in Example 7 are used to synthesize Compound 10-1 using the appropriate corresponding amine starting material.
Example Z1 Synthesis of 3-amido compound I1-2:
R = -H
Rz = ~N \
H
iN
Example Z1 Synthesis of 3-amido compound I1-2:
R = -H
Rz = ~N \
H
iN
The same general procedures as described in Example 7 are used to synthesize Compound 11-1 using 4-(aminomethyl) pyridine as the amine starting material.
Example Z2 Synthesis of 3-amido Compound 12-1:
R = -H
Rz - -N ( CH3 ) 2 CBZ-protected pseudomycin B (260 mg, 0.16 mmol), 51.8 mg TBTU and 152 ~l DIEA were dissolved in 3 ml DMF and 320 ml dimethylamine (0.16 mmol) in THF (2 molar solution). The reaction was stirred at room temperature for 20 minutes and the then purified via HPLC. The product was lyophilized to give 172,mg (66% yield) of the desired CBZ-protected amide.
The CBZ-protected amide was hydrogenated using the general procedure described above to provide Compound 12-1.
Example 13 illustrates the synthesis of pseudomycin compounds where the carboxylic acid group is reacted with a variety of amino acid alkyl esters.
Example 13 Synthesis of 3-amido Compound 13-1:
R = -H
Rz - -NHCH (COZCH3 ) CHZCHZCHzCH2NHz CBZ-protected Lysine methyl ester (164 mg, 0.49 mmol) was added to a solution of CBZ-protected pseudomycin B (800 mg, 0.49 mmol), TBTU (158 mg, 0.49 mmol) and 400 ml DIEA
(2.51 mmol) in 8 ml DMF. The reaction was allowed to stir at room temperature for 20 minutes and then purified via HPLC to yield 260 mg (32o yield) of the CBZ-protected amide.
The CBZ-protected amide was hydrogenated using the general procedures described above to produce Compound 13-1.
The compounds 13-2 through 13-4 listed in Table II may be synthesized using the same general procedures as described above using the appropriate corresponding aminoacid ester.
Table II
Example # R R R
13-2 -H -NHCH2COzCH3 -OH
13-3 -H O O,CH -OH
~N
H
13 - 4 -H O 0 , CH -OH
~N
H
OH
Examples 14-16 illustrate the synthesis of amide derivatives at residue 8.
Example 14 Synthesis of 8-amido Compound 14-1:
R = -H
R3 _ -NH2 Compound 14-1 is synthesized using the same procedures as described for compound 6-1 using a rink amide resin with the exception that PyBOP is used as the coupling agent instead of TBTU.
Example 15 Synthesis of 8-amido Compound 15-1:
R = -H
R3 - -NH ( CHZ ) 3CH3 n-Butyl amine (45.4 mg, 0.62 mmol) was added to a solution of CBZ-protected pseudomycin B (1000 mg, 0.62 mmol) and PyBop (323 mg, 0.62 mmol) dissolved in 10 ml of DMF.
The reaction was stirred at room temperature for 1 hour and then purified via HPLC. The product was lyophilized to give 280 mg (27% yield) of the CBZ-protected amide.
The CBZ-protected amide (280 mg, 0.17 mmol) was hydrogenated under hydrogen catalyzed by 10o Pd/C in 10 acetic acid/methanol for 45 minutes. The reaction mixture was filtered and the solvent removed in vacuo. The residue was dissolved in 50o ACN in water and lyophilized to give 189 mg (89o yield) of Compound 15-1.
The 8-amido compounds listed in Table III may be synthesized using the same general procedures described above using the appropriate corresponding amine starting material.
Table III
Example # R R R
15-4 -H -OH -NH (CHZ ) ZCH3 15-5 -H -OH -NH(cyclopropyl) 15-6 -H -OH -NH(cyclobutyl) 15 - 8 -H -OH -NHCHZCH2N ( CH3 ) 2 15-9 -H -OH -NHCHzCH2CH2N ( CH-~ ) Example 16 Synthesis of 8-amido Compound 26-1:
R = -H
Rz - -OH
R3._ ~N~N~
H
Example Z2 Synthesis of 3-amido Compound 12-1:
R = -H
Rz - -N ( CH3 ) 2 CBZ-protected pseudomycin B (260 mg, 0.16 mmol), 51.8 mg TBTU and 152 ~l DIEA were dissolved in 3 ml DMF and 320 ml dimethylamine (0.16 mmol) in THF (2 molar solution). The reaction was stirred at room temperature for 20 minutes and the then purified via HPLC. The product was lyophilized to give 172,mg (66% yield) of the desired CBZ-protected amide.
The CBZ-protected amide was hydrogenated using the general procedure described above to provide Compound 12-1.
Example 13 illustrates the synthesis of pseudomycin compounds where the carboxylic acid group is reacted with a variety of amino acid alkyl esters.
Example 13 Synthesis of 3-amido Compound 13-1:
R = -H
Rz - -NHCH (COZCH3 ) CHZCHZCHzCH2NHz CBZ-protected Lysine methyl ester (164 mg, 0.49 mmol) was added to a solution of CBZ-protected pseudomycin B (800 mg, 0.49 mmol), TBTU (158 mg, 0.49 mmol) and 400 ml DIEA
(2.51 mmol) in 8 ml DMF. The reaction was allowed to stir at room temperature for 20 minutes and then purified via HPLC to yield 260 mg (32o yield) of the CBZ-protected amide.
The CBZ-protected amide was hydrogenated using the general procedures described above to produce Compound 13-1.
The compounds 13-2 through 13-4 listed in Table II may be synthesized using the same general procedures as described above using the appropriate corresponding aminoacid ester.
Table II
Example # R R R
13-2 -H -NHCH2COzCH3 -OH
13-3 -H O O,CH -OH
~N
H
13 - 4 -H O 0 , CH -OH
~N
H
OH
Examples 14-16 illustrate the synthesis of amide derivatives at residue 8.
Example 14 Synthesis of 8-amido Compound 14-1:
R = -H
R3 _ -NH2 Compound 14-1 is synthesized using the same procedures as described for compound 6-1 using a rink amide resin with the exception that PyBOP is used as the coupling agent instead of TBTU.
Example 15 Synthesis of 8-amido Compound 15-1:
R = -H
R3 - -NH ( CHZ ) 3CH3 n-Butyl amine (45.4 mg, 0.62 mmol) was added to a solution of CBZ-protected pseudomycin B (1000 mg, 0.62 mmol) and PyBop (323 mg, 0.62 mmol) dissolved in 10 ml of DMF.
The reaction was stirred at room temperature for 1 hour and then purified via HPLC. The product was lyophilized to give 280 mg (27% yield) of the CBZ-protected amide.
The CBZ-protected amide (280 mg, 0.17 mmol) was hydrogenated under hydrogen catalyzed by 10o Pd/C in 10 acetic acid/methanol for 45 minutes. The reaction mixture was filtered and the solvent removed in vacuo. The residue was dissolved in 50o ACN in water and lyophilized to give 189 mg (89o yield) of Compound 15-1.
The 8-amido compounds listed in Table III may be synthesized using the same general procedures described above using the appropriate corresponding amine starting material.
Table III
Example # R R R
15-4 -H -OH -NH (CHZ ) ZCH3 15-5 -H -OH -NH(cyclopropyl) 15-6 -H -OH -NH(cyclobutyl) 15 - 8 -H -OH -NHCHZCH2N ( CH3 ) 2 15-9 -H -OH -NHCHzCH2CH2N ( CH-~ ) Example 16 Synthesis of 8-amido Compound 26-1:
R = -H
Rz - -OH
R3._ ~N~N~
H
In a 100 ml round bottom flask, alloc-protected Pseudomycin B(0.25 g, 0.171 mmol) was dissolved in 25 ml of DMF. To this solution was added Pybop (0.0898, 0.171 mmol)and 4-(2-Aminoethyl)morpholine (0.022 g, 0.171 mmol).
The solution was stirred rapidly overnight under 1 atm N2, The solution was concentrated down, and the product was isolated by reverse-phase HPLC, and lyophilized to yield (140 mg, 0.089 mmol, 520) alloc-protected Psuedomycin B
Morpholine derivative. The deprotection of the alloc groups was performed by adding Bu3SnH(0.648 g, 2.23 mmol), and (Ph3P)ZPdCl2(0.009g, 0.013 mmol) to a 1o acetic/
The solution was stirred rapidly overnight under 1 atm N2, The solution was concentrated down, and the product was isolated by reverse-phase HPLC, and lyophilized to yield (140 mg, 0.089 mmol, 520) alloc-protected Psuedomycin B
Morpholine derivative. The deprotection of the alloc groups was performed by adding Bu3SnH(0.648 g, 2.23 mmol), and (Ph3P)ZPdCl2(0.009g, 0.013 mmol) to a 1o acetic/
dichloromethane solution of alloc-protected Psuedomycin B
Morpholine derivative (10 mg/mL). Reaction time was 30 minutes. Reaction was monitored by HPLC. The solution was concentrated down, and the product was isolated by reverse phase HPLC prep, and lyophilized to yield 38 mg, 320 of Compound 16-1. MS data: Calculated for C57 H99 C1 N14 019 Mol. Wt. 1318.7 Found ES+ 1320.0, ES- 1318.0 The 8-amido compounds listed in Table IV were synthesized using the same general procedures described above using the appropriate corresponding amine starting material.
Table IV
Example # R R R
16-2 -H -OH -NH(benzyl) 16-3 -H -OH ~NH
'~N~N~
H
N
H
~
N
Each of the compounds synthesized in Examples 5-16 showed measurable activity against Candida Albicans, Cryptococcus neoformans, Aspergillus Fumigatus, Candida Parapsilosis, or Histoplasma capsulatum. However, the following basic trends in activity were observed based on the compounds synthesized.
When the 8-amido derivatives were assayed against C.
albicans, several trends were apparent from the data. The in vitro potency decreases in the following order of R3 substitution: -NHz > -NHCH3 > -NHCHZCH3 > -NH (CHZ) 2CH3 >
-NH ( CH2 ) 3CH3 ; -NHCH2CHZN ( CH3 ) 2 > -NH ( CH2 ) 3IV ( CH3 ) 2 ; and -NH(GIyOMe) > -NH(PheOMe). In general, better activities were realized with amido groups having smaller alkyl groups.
The free amide group was found to be the most active of the series. In addition, the cycloalkyl amides demonstrated better activity than the corresponding straight chain alkyl groups. Alkyl groups having a polar substitution on the end of the alkyl chain showed less activity than the corresponding natural product. Unlike the parent natural product, none of the 8-amido derivatives showed tail vein irritation.
The 3-amido derivatives demonstrated a similar trend as observed with the 8-amido derivatives in comparison with the parent natural product (e.g., amide substituents at R2 having shorter alkyl chains were more active than longer alkyl chains). Unlike the 8-amido derivatives, the 3-amido derivatives did not show a significant decrease in in vitro activity against C. albicans until the alkyl chain reached 7-carbons or longer (3-amido PSB compound where RZ --NH(CHz)6CH3 had a MIC = 20 ~,g/ml) versus 4-carbons or longer for the 8-amido derivatives (8-amido PSB compound where R3 --NH(CHz)3CH3 had a MIC = 20 ~g/ml). Most of the 3-amido derivatives tested showed an improvement in tail vein irritation. The exceptions being Rz - -NH(iso-amyl), -NH(n-hexyl ) , -NH ( CHz ) zN ( CHZCH3 ) z , and -NH ( CHz ) 3N ( CH3 ) z .
Although formation of an amide bond at residues 3 and 8 demonstrated an improved toxicity profile in comparison with the corresponding natural product (Pseudomycin B), in vivo efficacy generally decreased.
Morpholine derivative (10 mg/mL). Reaction time was 30 minutes. Reaction was monitored by HPLC. The solution was concentrated down, and the product was isolated by reverse phase HPLC prep, and lyophilized to yield 38 mg, 320 of Compound 16-1. MS data: Calculated for C57 H99 C1 N14 019 Mol. Wt. 1318.7 Found ES+ 1320.0, ES- 1318.0 The 8-amido compounds listed in Table IV were synthesized using the same general procedures described above using the appropriate corresponding amine starting material.
Table IV
Example # R R R
16-2 -H -OH -NH(benzyl) 16-3 -H -OH ~NH
'~N~N~
H
N
H
~
N
Each of the compounds synthesized in Examples 5-16 showed measurable activity against Candida Albicans, Cryptococcus neoformans, Aspergillus Fumigatus, Candida Parapsilosis, or Histoplasma capsulatum. However, the following basic trends in activity were observed based on the compounds synthesized.
When the 8-amido derivatives were assayed against C.
albicans, several trends were apparent from the data. The in vitro potency decreases in the following order of R3 substitution: -NHz > -NHCH3 > -NHCHZCH3 > -NH (CHZ) 2CH3 >
-NH ( CH2 ) 3CH3 ; -NHCH2CHZN ( CH3 ) 2 > -NH ( CH2 ) 3IV ( CH3 ) 2 ; and -NH(GIyOMe) > -NH(PheOMe). In general, better activities were realized with amido groups having smaller alkyl groups.
The free amide group was found to be the most active of the series. In addition, the cycloalkyl amides demonstrated better activity than the corresponding straight chain alkyl groups. Alkyl groups having a polar substitution on the end of the alkyl chain showed less activity than the corresponding natural product. Unlike the parent natural product, none of the 8-amido derivatives showed tail vein irritation.
The 3-amido derivatives demonstrated a similar trend as observed with the 8-amido derivatives in comparison with the parent natural product (e.g., amide substituents at R2 having shorter alkyl chains were more active than longer alkyl chains). Unlike the 8-amido derivatives, the 3-amido derivatives did not show a significant decrease in in vitro activity against C. albicans until the alkyl chain reached 7-carbons or longer (3-amido PSB compound where RZ --NH(CHz)6CH3 had a MIC = 20 ~,g/ml) versus 4-carbons or longer for the 8-amido derivatives (8-amido PSB compound where R3 --NH(CHz)3CH3 had a MIC = 20 ~g/ml). Most of the 3-amido derivatives tested showed an improvement in tail vein irritation. The exceptions being Rz - -NH(iso-amyl), -NH(n-hexyl ) , -NH ( CHz ) zN ( CHZCH3 ) z , and -NH ( CHz ) 3N ( CH3 ) z .
Although formation of an amide bond at residues 3 and 8 demonstrated an improved toxicity profile in comparison with the corresponding natural product (Pseudomycin B), in vivo efficacy generally decreased.
Claims (13)
1. A pseudomycin compound having the following structure I
wherein R is where Ra and Ra' are independently hydrogen or methyl, or either Ra or Ra' is alkyl amino, taken together with Rb or Rb' forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with Rc forms a six-membered aromatic ring;
Rb and Rb' are independently hydrogen, halogen, or methyl, or either Rb or Rb' is amino, alkylamino, a-acetoacetate, methoxy, or hydroxy;
Rc is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and Rf is C8-C18 alkyl, or C5-C11 alkoxy;
R is where Rg is hydrogen, or C1-C13 alkyl, and Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10) alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R1 is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where Rj is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where Rk is C5-C14 alkoxy; or R is -(CH2)-NRm-(C13-C18 alkyl), where Rm is H, -CH3 or -C(O)CH3;
R1 is independently -NH2 or -NHp-Pg, where p is 0 or 1;
R2 and R3 are independently -OR2a, or -N(R2b) (R2c), where R2a and R2b are independently hydrogen, C1-C10 alkyl, C3-C6 cycloalkyl, hydroxy(C1-C10)alkyl, alkoxy (C1-C10)alkyl, C2-C10 alkenyl, amino(C1-C10)alkyl, mono- or di-alkylamino(C1-C10)alkyl, aryl(C1-C10)alkyl, heteroaryl(C1-C10)alkyl, or cycloheteroalkyl (C1-C10)alkyl, or R2b is an alkyl carboxylate residue of an aminoacid alkyl ester, and R2c is hydrogen or C1-C6 alkyl, provided that both R2 and R3 are not -OH; and pharmaceutically acceptable salts and solvates thereof.
wherein R is where Ra and Ra' are independently hydrogen or methyl, or either Ra or Ra' is alkyl amino, taken together with Rb or Rb' forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with Rc forms a six-membered aromatic ring;
Rb and Rb' are independently hydrogen, halogen, or methyl, or either Rb or Rb' is amino, alkylamino, a-acetoacetate, methoxy, or hydroxy;
Rc is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and Rf is C8-C18 alkyl, or C5-C11 alkoxy;
R is where Rg is hydrogen, or C1-C13 alkyl, and Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10) alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R1 is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where Rj is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where Rk is C5-C14 alkoxy; or R is -(CH2)-NRm-(C13-C18 alkyl), where Rm is H, -CH3 or -C(O)CH3;
R1 is independently -NH2 or -NHp-Pg, where p is 0 or 1;
R2 and R3 are independently -OR2a, or -N(R2b) (R2c), where R2a and R2b are independently hydrogen, C1-C10 alkyl, C3-C6 cycloalkyl, hydroxy(C1-C10)alkyl, alkoxy (C1-C10)alkyl, C2-C10 alkenyl, amino(C1-C10)alkyl, mono- or di-alkylamino(C1-C10)alkyl, aryl(C1-C10)alkyl, heteroaryl(C1-C10)alkyl, or cycloheteroalkyl (C1-C10)alkyl, or R2b is an alkyl carboxylate residue of an aminoacid alkyl ester, and R2c is hydrogen or C1-C6 alkyl, provided that both R2 and R3 are not -OH; and pharmaceutically acceptable salts and solvates thereof.
2. A pseudomycin prodrug having the following structure wherein R is where Ra and Ra' are independently hydrogen or methyl, or either Ra or Ra' is alkyl amino, taken together with Rb or Rb' forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with Rc forms a six-membered aromatic ring;
Rb and Rb' are independently hydrogen, halogen, or methyl, or either Rb or Rb' is amino, alkylamino, .alpha.-acetoacetate, methoxy, or hydroxy;
Rc is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and Rf is C8-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is where Rg is hydrogen, or C1-C13 alkyl, and Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R1 is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where Rj is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where Rk is C5-C14 alkoxy; or R is -(CH2)-NRm-(C13-C18 alkyl), where Rm is H, -CH3 or -C(O)CH3;
R1 is independently -NH2 or -NHp-Pg, where p is 0 or 1;
R2 and R3 are -OR2a, where R2a is C1-C3 alkyl; and pharmaceutically acceptable salts and solvates thereof.
Rb and Rb' are independently hydrogen, halogen, or methyl, or either Rb or Rb' is amino, alkylamino, .alpha.-acetoacetate, methoxy, or hydroxy;
Rc is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and Rf is C8-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is where Rg is hydrogen, or C1-C13 alkyl, and Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R1 is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where Rj is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where Rk is C5-C14 alkoxy; or R is -(CH2)-NRm-(C13-C18 alkyl), where Rm is H, -CH3 or -C(O)CH3;
R1 is independently -NH2 or -NHp-Pg, where p is 0 or 1;
R2 and R3 are -OR2a, where R2a is C1-C3 alkyl; and pharmaceutically acceptable salts and solvates thereof.
3. A 3-amido derivative of a pseudomycin compound prepared by the steps of (i) providing a pseudomycin compound having the following structure wherein R is where Ra and Ra' are independently hydrogen or methyl, or either Ra or Ra' is alkyl amino, taken together with Rb or Rb' forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with Rc forms a six-membered aromatic ring;
Rb and Rb' are independently hydrogen, halogen, or methyl, or either Rb or Rb' is amino, alkylamino, oc-acetoacetate, methoxy, or hydroxy;
Rc is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and Rf is C6-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is where Rg is hydrogen, or C1-C13 alkyl, and Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R1 is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where Rj is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where Rk is C5-C14 alkoxy; or R i s -(CH2) -NRm-(C13-C18 alkyl), where Rm i s H, -CH3 or -C(C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and pharmaceutically acceptable salts and solvates thereof;
(ii) protecting the amino groups, R1, at positions 2, 4 and 5 with an amino-protecting group;
(iii)forming an amide linkage at position 3 using o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate or 2(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate as a coupling agent;
(iv) removing said amino-protecting groups.
Rb and Rb' are independently hydrogen, halogen, or methyl, or either Rb or Rb' is amino, alkylamino, oc-acetoacetate, methoxy, or hydroxy;
Rc is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and Rf is C6-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is where Rg is hydrogen, or C1-C13 alkyl, and Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R1 is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where Rj is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where Rk is C5-C14 alkoxy; or R i s -(CH2) -NRm-(C13-C18 alkyl), where Rm i s H, -CH3 or -C(C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and pharmaceutically acceptable salts and solvates thereof;
(ii) protecting the amino groups, R1, at positions 2, 4 and 5 with an amino-protecting group;
(iii)forming an amide linkage at position 3 using o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate or 2(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate as a coupling agent;
(iv) removing said amino-protecting groups.
4. The 3-amido derivative of Claim 3 wherein step (iii) forming an amide linkage is accomplished in the presence of a bulky amine.
5. The 3-amido derivative of Claim 3 wherein step (iii) forming an amide linkage is accomplished in the presence of a bulky amine and at a temperature between about 0°C and -20°C .
6. An 8-amido derivative of a pseudomycin compound prepared by the steps of (i) providing a pseudomycin compound having the following structure I
wherein R is where Ra and Ra' are independently hydrogen or methyl, or either Ra or Ra' is alkyl amino, taken together with Rb or Rb' forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with Rc forms a six-membered aromatic ring;
Rb and Rb' are independently hydrogen, halogen, or methyl, or either Rb or Rb' is amino, alkylamino, .alpha.-acetoacetate, methoxy, or hydroxy;
Rb is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and Rf is C6-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is where Rg is hydrogen, or C1-C13 alkyl, and Rh i s C1-C15 alkyl , C4-C15 alkoxy, ( C1-C10 alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R1 is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where R j is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where R k is C5-C14 alkoxy; or R is -(CH2)-NR m-(C13-C18 alkyl), where R m is H, -CH3 or -C (C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and pharmaceutically acceptable salts and solvates thereof;
(ii) protecting the amino groups at positions 2, 4 and 5 with an amino-protecting group;
(iii) forming an amide linkage at position 8 using benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate as a coupling agent;
(iv) removing said amino-protecting groups.
wherein R is where Ra and Ra' are independently hydrogen or methyl, or either Ra or Ra' is alkyl amino, taken together with Rb or Rb' forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with Rc forms a six-membered aromatic ring;
Rb and Rb' are independently hydrogen, halogen, or methyl, or either Rb or Rb' is amino, alkylamino, .alpha.-acetoacetate, methoxy, or hydroxy;
Rb is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and Rf is C6-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is where Rg is hydrogen, or C1-C13 alkyl, and Rh i s C1-C15 alkyl , C4-C15 alkoxy, ( C1-C10 alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R1 is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where R j is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where R k is C5-C14 alkoxy; or R is -(CH2)-NR m-(C13-C18 alkyl), where R m is H, -CH3 or -C (C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and pharmaceutically acceptable salts and solvates thereof;
(ii) protecting the amino groups at positions 2, 4 and 5 with an amino-protecting group;
(iii) forming an amide linkage at position 8 using benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate as a coupling agent;
(iv) removing said amino-protecting groups.
7. The use of a compound as claimed in any one of the preceding claims in the preparation of a medicament for use in combating either systemic fungal infections or fungal skin infections.
8. A process for making a 3-amido derivative of a pseudomycin compound comprising the steps of (i) providing a pseudomycin compound having the following structure wherein R is where R a and R a' are independently hydrogen or methyl, or either R a or R a' is alkyl amino, taken together with R b or R b' forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with R c forms a six-membered aromatic ring;
R b and R b' are independently hydrogen, halogen, or methyl, or either R b or R b' is amino, alkylamino, .alpha.-acetoacetate, methoxy, or hydroxy;
R c is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with R e forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and R f is C6-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is where R g is hydrogen, or C1-C13 alkyl, and R h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R i is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where R j is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where R k is C5-C14 alkoxy; or R is -(CH2)-NR m-(C13-C18 alkyl), where R m is H, -CH3 or -C(C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and pharmaceutically acceptable salts and solvates thereof;
(ii) protecting the amino groups, R1, at positions 2, 4 and 5 with an amino-protecting group;
(iii)forming an amide linkage at position 3 using o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate or 2(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate as a coupling agent in the presence of a bulky amine and at a temperature between about 0°C and -20°C;
(iv) removing said amino-protecting groups.
R b and R b' are independently hydrogen, halogen, or methyl, or either R b or R b' is amino, alkylamino, .alpha.-acetoacetate, methoxy, or hydroxy;
R c is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with R e forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and R f is C6-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is where R g is hydrogen, or C1-C13 alkyl, and R h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R i is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where R j is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where R k is C5-C14 alkoxy; or R is -(CH2)-NR m-(C13-C18 alkyl), where R m is H, -CH3 or -C(C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and pharmaceutically acceptable salts and solvates thereof;
(ii) protecting the amino groups, R1, at positions 2, 4 and 5 with an amino-protecting group;
(iii)forming an amide linkage at position 3 using o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate or 2(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate as a coupling agent in the presence of a bulky amine and at a temperature between about 0°C and -20°C;
(iv) removing said amino-protecting groups.
9. A process for making an 8-amido derivative of a pseudomycin compound comprising the steps of (ii) providing a pseudomycin compound having the following structure wherein R is where R a and R a' are independently hydrogen or methyl, or either R a or R a' is alkyl amino, taken together with R b or R b' forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with R c forms a six-membered aromatic ring;
R b and R b' are independently hydrogen, halogen, or methyl, or either R b or R b' is amino, alkylamino, .alpha.-acetoacetate, methoxy, or hydroxy;
R c is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with R e forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and R f is C6-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is where R g is hydrogen, or C1-C13 alkyl, and R h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R i is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where R j is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where R k is C5-C14 alkoxy; or R is -(CH2)-NR m-(C13-C18 alkyl), where R m is H, -CH3 or -C(C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and pharmaceutically acceptable salts and solvates thereof;
(ii) protecting the amino groups at positions 2, 4 and 5 with an amino-protecting group;
(iii) forming an amide linkage at position 8 using benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate as a coupling agent;
(iv) removing said amino-protecting groups.
R b and R b' are independently hydrogen, halogen, or methyl, or either R b or R b' is amino, alkylamino, .alpha.-acetoacetate, methoxy, or hydroxy;
R c is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-C4)alkoxy, or taken together with R e forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C5-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and R f is C6-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is where R g is hydrogen, or C1-C13 alkyl, and R h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6 cycloalkyl), where n = 1 or 2; or R is where R i is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is where R j is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where R k is C5-C14 alkoxy; or R is -(CH2)-NR m-(C13-C18 alkyl), where R m is H, -CH3 or -C(C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and pharmaceutically acceptable salts and solvates thereof;
(ii) protecting the amino groups at positions 2, 4 and 5 with an amino-protecting group;
(iii) forming an amide linkage at position 8 using benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate as a coupling agent;
(iv) removing said amino-protecting groups.
10. A pharmaceutical formulation comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.
11. A pharmaceutical formulation comprising a prodrug of Claim 2 and a pharmaceutically acceptable carrier.
12. A method for treating an antifungal infection in an aminal in need thereof, which comprises administering to said animal a pseudomycin compound of Claim 1.
13. A method for treating an antifungal infection in an animal in need thereof, which comprises administering to said animal a prodrug of Claim 2.
Applications Claiming Priority (3)
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US14398199P | 1999-07-15 | 1999-07-15 | |
US60/143,981 | 1999-07-15 | ||
PCT/US2000/015021 WO2001005817A1 (en) | 1999-07-15 | 2000-06-08 | Pseudomycin amide and ester analogs |
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CA2379321A1 true CA2379321A1 (en) | 2001-01-25 |
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CA002379321A Abandoned CA2379321A1 (en) | 1999-07-15 | 2000-06-08 | Pseudomycin amide and ester analogs |
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EP (1) | EP1198473A1 (en) |
JP (1) | JP2003505399A (en) |
CN (1) | CN1362966A (en) |
AU (1) | AU5725000A (en) |
BR (1) | BR0013163A (en) |
CA (1) | CA2379321A1 (en) |
EA (1) | EA200200165A1 (en) |
HU (1) | HUP0202267A3 (en) |
MX (1) | MXPA02000312A (en) |
NO (1) | NO20020186L (en) |
WO (1) | WO2001005817A1 (en) |
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AU3071801A (en) * | 1999-12-13 | 2001-06-18 | Eli Lilly And Company | Pseudomycin phosphate prodrugs |
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US5576298A (en) * | 1992-11-30 | 1996-11-19 | Research And Development Institute, Inc. At Montana State University | Peptides from pseudomonas syringae possessing broad-spectrum antibiotic activity |
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2000
- 2000-06-08 HU HU0202267A patent/HUP0202267A3/en unknown
- 2000-06-08 JP JP2001511474A patent/JP2003505399A/en not_active Withdrawn
- 2000-06-08 AU AU57250/00A patent/AU5725000A/en not_active Abandoned
- 2000-06-08 MX MXPA02000312A patent/MXPA02000312A/en unknown
- 2000-06-08 EP EP00942656A patent/EP1198473A1/en not_active Withdrawn
- 2000-06-08 CA CA002379321A patent/CA2379321A1/en not_active Abandoned
- 2000-06-08 CN CN00810330A patent/CN1362966A/en active Pending
- 2000-06-08 EA EA200200165A patent/EA200200165A1/en unknown
- 2000-06-08 BR BR0013163-6A patent/BR0013163A/en not_active Application Discontinuation
- 2000-06-08 WO PCT/US2000/015021 patent/WO2001005817A1/en not_active Application Discontinuation
-
2002
- 2002-01-14 NO NO20020186A patent/NO20020186L/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
MXPA02000312A (en) | 2002-06-21 |
BR0013163A (en) | 2002-04-02 |
WO2001005817A1 (en) | 2001-01-25 |
JP2003505399A (en) | 2003-02-12 |
NO20020186L (en) | 2002-03-04 |
AU5725000A (en) | 2001-02-05 |
HUP0202267A2 (en) | 2002-10-28 |
EP1198473A1 (en) | 2002-04-24 |
EA200200165A1 (en) | 2002-06-27 |
CN1362966A (en) | 2002-08-07 |
HUP0202267A3 (en) | 2002-11-28 |
NO20020186D0 (en) | 2002-01-14 |
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Legal Events
Date | Code | Title | Description |
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FZDE | Discontinued |