WO2018166899A1 - Methods for production of pf1022a derivatives - Google Patents
Methods for production of pf1022a derivatives Download PDFInfo
- Publication number
- WO2018166899A1 WO2018166899A1 PCT/EP2018/055845 EP2018055845W WO2018166899A1 WO 2018166899 A1 WO2018166899 A1 WO 2018166899A1 EP 2018055845 W EP2018055845 W EP 2018055845W WO 2018166899 A1 WO2018166899 A1 WO 2018166899A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- formula
- compound
- substituted
- amino
- pf1022a
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 68
- 238000004519 manufacturing process Methods 0.000 title abstract description 12
- 108010004210 PF 1022A Proteins 0.000 title description 21
- -1 PF1022A derivative compounds Chemical class 0.000 claims abstract description 87
- 150000001875 compounds Chemical class 0.000 claims abstract description 65
- 239000001963 growth medium Substances 0.000 claims abstract description 30
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 24
- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 claims abstract description 22
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 21
- 244000005700 microbiome Species 0.000 claims abstract description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 13
- YJNUXGPXJFAUQJ-LYWANRAQSA-N PF1022A Chemical class C([C@@H]1C(=O)N(C)[C@H](C(O[C@H](C)C(=O)N(C)[C@@H](CC(C)C)C(=O)O[C@H](CC=2C=CC=CC=2)C(=O)N(C)[C@@H](CC(C)C)C(=O)O[C@H](C)C(=O)N(C)[C@@H](CC(C)C)C(=O)O1)=O)CC(C)C)C1=CC=CC=C1 YJNUXGPXJFAUQJ-LYWANRAQSA-N 0.000 claims description 40
- 125000005843 halogen group Chemical group 0.000 claims description 32
- 229910052739 hydrogen Inorganic materials 0.000 claims description 23
- 239000001257 hydrogen Substances 0.000 claims description 23
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 23
- 125000003118 aryl group Chemical group 0.000 claims description 20
- 125000000753 cycloalkyl group Chemical group 0.000 claims description 19
- 125000003282 alkyl amino group Chemical group 0.000 claims description 17
- 150000001412 amines Chemical class 0.000 claims description 17
- 230000002538 fungal effect Effects 0.000 claims description 17
- 125000000623 heterocyclic group Chemical group 0.000 claims description 17
- 125000000278 alkyl amino alkyl group Chemical group 0.000 claims description 16
- 125000004103 aminoalkyl group Chemical group 0.000 claims description 16
- 125000004181 carboxyalkyl group Chemical group 0.000 claims description 16
- 150000007942 carboxylates Chemical class 0.000 claims description 16
- 125000004985 dialkyl amino alkyl group Chemical group 0.000 claims description 16
- 125000004663 dialkyl amino group Chemical group 0.000 claims description 16
- 125000001072 heteroaryl group Chemical group 0.000 claims description 16
- 125000003545 alkoxy group Chemical group 0.000 claims description 15
- 125000006239 protecting group Chemical group 0.000 claims description 14
- NHFDIUPJVYYTLG-UHFFFAOYSA-N carbononitridic isocyanide Chemical compound [C-]#[N+]C#N NHFDIUPJVYYTLG-UHFFFAOYSA-N 0.000 claims description 13
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 11
- 125000001309 chloro group Chemical group Cl* 0.000 claims description 9
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 9
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 8
- 125000001246 bromo group Chemical group Br* 0.000 claims description 6
- 125000001153 fluoro group Chemical group F* 0.000 claims description 4
- 241001299709 Rosellinia Species 0.000 claims description 3
- 241001523965 Xylaria Species 0.000 claims description 3
- 150000001720 carbohydrates Chemical class 0.000 claims description 3
- 125000001477 organic nitrogen group Chemical group 0.000 claims description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 27
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 21
- 125000000217 alkyl group Chemical group 0.000 description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 13
- 150000002431 hydrogen Chemical class 0.000 description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 12
- 239000000126 substance Substances 0.000 description 11
- 239000000203 mixture Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 8
- 125000002911 monocyclic heterocycle group Chemical group 0.000 description 8
- ZMQMTKVVAMWKNY-YSXLEBCMSA-N emodepside Chemical compound C([C@@H]1C(=O)N(C)[C@@H](CC(C)C)C(=O)O[C@H](C)C(=O)N(C)[C@H](C(O[C@H](CC=2C=CC(=CC=2)N2CCOCC2)C(=O)N(C)[C@@H](CC(C)C)C(=O)O[C@H](C)C(=O)N(C)[C@@H](CC(C)C)C(=O)O1)=O)CC(C)C)C(C=C1)=CC=C1N1CCOCC1 ZMQMTKVVAMWKNY-YSXLEBCMSA-N 0.000 description 7
- 108010056417 emodepside Proteins 0.000 description 7
- 229960001575 emodepside Drugs 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 7
- 238000012809 post-inoculation Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 229920002472 Starch Polymers 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 125000002618 bicyclic heterocycle group Chemical group 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 235000019253 formic acid Nutrition 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 6
- 235000019341 magnesium sulphate Nutrition 0.000 description 6
- 239000003960 organic solvent Substances 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 239000008107 starch Substances 0.000 description 6
- 235000019698 starch Nutrition 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 125000004432 carbon atom Chemical group C* 0.000 description 5
- 210000004748 cultured cell Anatomy 0.000 description 5
- 239000000706 filtrate Substances 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 239000003643 water by type Substances 0.000 description 5
- HWVHXAYPEXNZSZ-UHFFFAOYSA-N 3-(4-chlorophenyl)-2-hydroxypropanoic acid Chemical compound OC(=O)C(O)CC1=CC=C(Cl)C=C1 HWVHXAYPEXNZSZ-UHFFFAOYSA-N 0.000 description 4
- UTTDJAPJOSBBIO-UHFFFAOYSA-N 3-(4-fluorophenyl)-2-oxopropanoic acid Chemical compound OC(=O)C(=O)CC1=CC=C(F)C=C1 UTTDJAPJOSBBIO-UHFFFAOYSA-N 0.000 description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 125000004429 atom Chemical group 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000012136 culture method Methods 0.000 description 4
- 125000004093 cyano group Chemical group *C#N 0.000 description 4
- 125000005842 heteroatom Chemical group 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 244000045947 parasite Species 0.000 description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 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 3
- 244000068988 Glycine max Species 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- 241000209140 Triticum Species 0.000 description 3
- 235000021307 Triticum Nutrition 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
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- 238000004440 column chromatography Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 238000006396 nitration reaction Methods 0.000 description 3
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- 238000001556 precipitation Methods 0.000 description 3
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- 238000000746 purification Methods 0.000 description 3
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- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 125000001424 substituent group Chemical group 0.000 description 3
- 239000006188 syrup Substances 0.000 description 3
- 235000020357 syrup Nutrition 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 125000000169 tricyclic heterocycle group Chemical group 0.000 description 3
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- HLLSOEKIMZEGFV-UHFFFAOYSA-N 4-(dibutylsulfamoyl)benzoic acid Chemical compound CCCCN(CCCC)S(=O)(=O)C1=CC=C(C(O)=O)C=C1 HLLSOEKIMZEGFV-UHFFFAOYSA-N 0.000 description 2
- 241000238876 Acari Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
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- 241000196324 Embryophyta Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
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- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 2
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- 125000002843 carboxylic acid group Chemical group 0.000 description 2
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- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- WDSQDRADSGGACS-UHFFFAOYSA-N methyl 3-(4-chlorophenyl)-2-hydroxypropanoate Chemical compound COC(=O)C(O)CC1=CC=C(Cl)C=C1 WDSQDRADSGGACS-UHFFFAOYSA-N 0.000 description 2
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- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 1
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
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- 241001508691 Martes zibellina Species 0.000 description 1
- AXFZADXWLMXITO-UHFFFAOYSA-N N-acetylcysteamine Chemical compound CC(=O)NCCS AXFZADXWLMXITO-UHFFFAOYSA-N 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
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- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
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- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
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- 239000008346 aqueous phase Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
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- NGPGDYLVALNKEG-UHFFFAOYSA-N azanium;azane;2,3,4-trihydroxy-4-oxobutanoate Chemical class [NH4+].[NH4+].[O-]C(=O)C(O)C(O)C([O-])=O NGPGDYLVALNKEG-UHFFFAOYSA-N 0.000 description 1
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- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
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- 125000001511 cyclopentyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
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- 125000005959 diazepanyl group Chemical group 0.000 description 1
- 238000006193 diazotization reaction Methods 0.000 description 1
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- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- ZKQFHRVKCYFVCN-UHFFFAOYSA-N ethoxyethane;hexane Chemical class CCOCC.CCCCCC ZKQFHRVKCYFVCN-UHFFFAOYSA-N 0.000 description 1
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- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 125000002541 furyl group Chemical group 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 244000000013 helminth Species 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 125000003707 hexyloxy group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])O* 0.000 description 1
- 150000002430 hydrocarbons Chemical group 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 125000002632 imidazolidinyl group Chemical group 0.000 description 1
- 125000003453 indazolyl group Chemical group N1N=C(C2=C1C=CC=C2)* 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 125000002346 iodo group Chemical group I* 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002527 isonitriles Chemical class 0.000 description 1
- 125000002183 isoquinolinyl group Chemical group C1(=NC=CC2=CC=CC=C12)* 0.000 description 1
- 125000004628 isothiazolidinyl group Chemical group S1N(CCC1)* 0.000 description 1
- 125000003965 isoxazolidinyl group Chemical group 0.000 description 1
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- 239000000594 mannitol Substances 0.000 description 1
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- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 125000002757 morpholinyl group Chemical group 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 125000005963 oxadiazolidinyl group Chemical group 0.000 description 1
- 125000000160 oxazolidinyl group Chemical group 0.000 description 1
- 125000002971 oxazolyl group Chemical group 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- KXFJZKUFXHWWAJ-UHFFFAOYSA-N p-hydroxybenzoylformic acid Natural products OC(=O)C(=O)C1=CC=C(O)C=C1 KXFJZKUFXHWWAJ-UHFFFAOYSA-N 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 150000002993 phenylalanine derivatives Chemical class 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 125000004193 piperazinyl group Chemical group 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- 239000010773 plant oil Substances 0.000 description 1
- 229930001118 polyketide hybrid Natural products 0.000 description 1
- 125000003308 polyketide hybrid group Chemical group 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 150000003141 primary amines Chemical group 0.000 description 1
- 208000019585 progressive encephalomyelitis with rigidity and myoclonus Diseases 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 125000004309 pyranyl group Chemical group O1C(C=CC=C1)* 0.000 description 1
- 125000003373 pyrazinyl group Chemical group 0.000 description 1
- 125000003072 pyrazolidinyl group Chemical group 0.000 description 1
- 125000003226 pyrazolyl group Chemical group 0.000 description 1
- 125000001725 pyrenyl group Chemical group 0.000 description 1
- 125000002098 pyridazinyl group Chemical group 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 125000000719 pyrrolidinyl group Chemical group 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 1
- 125000001567 quinoxalinyl group Chemical group N1=C(C=NC2=CC=CC=C12)* 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 125000005958 tetrahydrothienyl group Chemical group 0.000 description 1
- 125000005304 thiadiazolidinyl group Chemical group 0.000 description 1
- 125000001984 thiazolidinyl group Chemical group 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 125000004568 thiomorpholinyl group Chemical group 0.000 description 1
- 125000005455 trithianyl group Chemical group 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K11/02—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof cyclic, e.g. valinomycins ; Derivatives thereof
Definitions
- PF1022A is a fungally-derived non-ribosomal peptide natural product octadepsipeptide anthelmintic agent.
- Emodepside a complex semi-synthetic derivative of PF1022A, is a resistance breaking anthelmintic used exclusively for the more profitable companion animal market owing to high cost of production (Ohyama et al, Biosci., Biotechno., Biochem., 2011, 75,
- PF1022A The unique and highly complex core structure of the PF1022A natural product has provided challenging opportunities for synthesis.
- Conversion of PF1022A to the bis-4- morpholino derivative (emodepside) entails low-yielding chemistry such as nitration of the phenyl rings followed by reduction and subsequent functionalization.
- the generation of regioisomers further reduces the yield of useful intermediate and necessitates expensive purification of the desired para-regioisomers.
- Lower cost of goods for emodepside would enable the use of the compound in livestock herds, an application prohibited by its present high cost of manufacture.
- methods disclosed herein may be used in the preparation of new PF1022A derivatives, which may be useful as antihelmintic agents.
- X 1 , X 2 , X 3 and X 4 are independently selected from N and CH; and R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; comprising: incubating a microorganism strain capable of producing PF1022A derivative compounds in a culture medium to produce a resulting culture, said culture medium
- X 2 , X 3 , and X 4 are independently selected from N and CH;
- the feedstock comprises a compound of Formula II. In some embodiments, the feedstock comprises a compound of Formula II, and the compound of Formula II is a compound of Formula Ila:
- X 2 , X 3 , and X 4 are independently selected from N and CH;
- R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
- the feedstock comprises a compound of Formula III.
- the feedstock comprises a compound of Formula IV.
- the feedstock comprises a compound of Formula V.
- R 2 is H.
- R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino.
- R 1 is halo
- R 1 is fluoro
- R 1 is chloro
- R 1 is bromo
- the compound of Formula Ila is:
- the compound of Formula Ila is:
- the compound of Formula III is:
- the compound of Formula IV is:
- the process further includes recovering the PF1022A derivative compound from said culture.
- the carbon source comprises a carbohydrate.
- the nitrogen source comprises inorganic or organic nitrogen-containing compounds.
- the microorganism strain is a fungal strain.
- the fungal strain belongs to the genus Xylaria or the genus Rosellinia.
- the producing comprises accumulating said PF1022A derivative compound in the resulting culture.
- the compound of Formula I is enantiomerically pure.
- the com ound is a compound of Formula IV:
- R 1 is halo. In some embodiments, R 1 is bromo or chloro. In some embodiments, R 1 is fluoro. In some embodiments, R 1 is chloro.
- FIG. 1 depicts LC-MS chromatograms of the dichloro derivative of PF1022A prepared by the disclosed process.
- FIG. 2 depicts LC-MS chromatograms of the mono-chloro derivative of PF1022A prepared by the disclosed process.
- FIG. 3 depicts mass spectrometry patterns for the mono-chloro (left) and dichloro (right) derivatives of PF1022A prepared by the disclosed process.
- cyclooctadepsipeptide compounds such as the approved animal anthelmintic compound emodepside.
- alkyl or “alkyl group”, as used herein, means a straight-chain (i.e. , unbranched), or branched hydrocarbon chain that is completely saturated. In some embodiments the alkyl has 1 , 2, 3, 4, 5 or 6 carbon atoms. In certain embodiments, alkyl groups contain 1-6 carbon atoms (Ci_ 6 alkyl). In certain embodiments, alkyl groups contain 1-4 carbon atoms (C 1-4 alkyl).
- alkyl groups contain 1-3 carbon atoms (C 1 -3 alkyl). In still other embodiments, alkyl groups contain 2-3 carbon atoms (C 2-3 alkyl), and in yet other embodiments alkyl groups contain 1-2 carbon atoms (C 1-2 alkyl).
- Ar or aryl refer to an aromatic carbocyclic moiety having one or more closed rings. Examples include, without limitation, phenyl, naphthyl, anthracenyl, phenylanthracenyl, biphenyl, and pyrenyl.
- Heteroaryl refers to a cyclic moiety having one or more closed rings, with one or more heteroatoms (oxygen, nitrogen, or sulfur) in at least one of the rings, wherein at least one of the rings is aromatic, and wherein the ring or rings may independently be fused, and/or bridged. Examples include, without limitation, pyridyl, quinolinyl, isoquinolinyl, indolyl, furyl, thienyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, quinoxalinyl, pyrrolyl, indazolyl, thiazolyl, oxazolyl, and isoxazolyl.
- Alkoxy refers to an alkyl or cycloalkyl group, as herein defined, attached to the principal carbon chain through an oxygen atom.
- Representative examples of “alkoxy” include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, and hexyloxy.
- Halogen or “halo” refers to one or more of fluoro, chloro, bromo, and iodo.
- amine refers to a primary, secondary or tertiary amine.
- amino group refers to the primary amine group -NH 2 .
- secondary or tertiary amines include, but are not limited to, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, etc.
- Carboxylate refers to a salt or ester of a carboxylic acid moiety.
- Carboxyalkyl refers to a carboxylic acid group attached to the principal carbon chain or molecule through an alkyl group.
- the carboxylic acid group may be present as the free acid, salt, or an ester.
- Cycloalkyl refers to a saturated cyclic hydrocarbon group containing from 3 to 8 carbons or more.
- Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
- Heteroatom refers to O, S or N.
- Heterocycle or “heterocyclyl” as used herein, means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle containing at least one heteroatom in a ring.
- “Monocyclic heterocycle” means a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom, and which is not aromatic.
- Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3- dioxanyl, 1,3-dioxolanyl, dihydropyranyl (including 3,4-dihydro-2H-pyran-6-yl), 1,3- dithiolanyl, 1,3-dithianyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxadiazolidinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrrolidinyl, tetrahydrofuranyl, tetra
- Bicyclic heterocycle means a monocyclic heterocycle fused to an aryl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl or cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle.
- bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-pyranyl, 1,3- benzodioxolyl, 1,3-benzodithiolyl, 2,3-dihydro-l,4-benzodioxinyl, 2,3-dihydro-l- benzofuranyl, 2,3-dihydro-l-benzothienyl,2,3-dihydro-lH-indolyl, 3,4-dihydroquinolin- 2(lH)-one and 1,2,3,4- tetrahydroquinolinyl.
- Tricyclic heterocycle means a bicyclic heterocycle fused to an aryl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl or cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle.
- Representative examples of tricyclic heterocycles include, but are not limited to, 2,3,4,4a,9,9a-hexahydro- lH-carbazolyl, 5a,6,7,8,9,9a- hexahydro-dibenzo[b,d]furanyl, and 5a,6,7,8,9,9a-hexahydrodibenzo[b,d]thienyl.
- Cyano refers to the group -CN.
- Niro refers to the group -N0 2 .
- structures depicted herein are also meant to include all enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Tautomeric forms include keto-enol tautomers of a compound.
- “Isomers” refers to compounds having the same number and kind of atoms and hence the same molecular weight, but differing with respect to the arrangement or configuration of the atoms. It will be understood, however, that some isomers or racemates or others mixtures of isomers may exhibit more activity than others. "Stereoisomers” refers to isomers that differ only in the arrangement of the atoms in space. "Diastereoisomers” refers to stereoisomers that are not mirror images of each other. “Enantiomers” refers to stereoisomers that are non-superimpo sable mirror images of one another.
- enantiomeric compounds taught herein may be "enantiomerically pure" isomers that comprise substantially a single enantiomer, for example, greater than or equal to 90%, 92%, 95%, 98%, or 99%, or equal to 100% of a single enantiomer.
- enantiomeric compounds as taught herein may be stereochemically pure.
- “Stereochemically pure” as used herein means a compound or composition thereof that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound.
- An objective of the present invention is to provide a method for producing PF1022A derivatives, by a direct fermentation method. Accordingly, provided herein are methods for preparing cyclooctadepsipeptide compounds of Formula I:
- R 1 and R 2 are independently selected from the group consisting of hydrogen, halo, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
- one of R 1 and R 2 is hydrogen and the other is not hydrogen. In some embodiments, neither R 1 nor R 2 is hydrogen.
- the present invention provides for the biosynthetic production of cyclooctadepsipeptides of Formula I by providing a feedstock with a compound as taught herein to a microrganism strain capable of producing PF1022A derivative compounds.
- the microorganism strain may be cultured according to an ordinary method by appropriately selecting a medium, culture conditions, and the like.
- the medium may be supplemented with a carbon source and nitrogen source that can be utilized by the microorganism strain, inorganic salts, various vitamins, various amino acids such as glutamic acid and asparagine, trace nutrients such as nucleotides, selectable agents such as antibiotics, etc. See, e.g. , U.S.
- any kind of carbon source and nitrogen source may be used in the medium as long as they can be utilized by the microorganisms of the present invention.
- the carbon source for example, various carbohydrates, such as sucrose, glucose, starch, glycerin, fructose, maltose, mannitol, xylose, galactose, ribose, dextrin, animal and plant oils and the like, or hydrolysates thereof, can be used.
- the preferable concentration generally is from 0.1% to 5% of the medium.
- the utilizable nitrogen source for example, animal or plant components, or exudates or extracts thereof, such as peptone, meat extract, com steep liquor, and defatted soybean powder, organic acid ammonium salts such as succinic acid ammonium salts and tartaric acid ammonium salts, urea, and other various inorganic or organic nitrogen-containing compounds can be used.
- animal or plant components, or exudates or extracts thereof such as peptone, meat extract, com steep liquor, and defatted soybean powder
- organic acid ammonium salts such as succinic acid ammonium salts and tartaric acid ammonium salts, urea, and other various inorganic or organic nitrogen-containing compounds can be used.
- Inorganic salts for example, those which can produce sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphate, sulfate, and/or other ions may also be used.
- the pH of the medium may be, for example, about 6 to 8.
- Incubation may be carried out by a shaking culture method under an aerobic condition, an agitation culture method with aeration or an aerobic submerged culture method.
- An appropriate incubation temperature in some embodiments may be 15°C to 40°C, more preferably about 26°C to 37°C.
- Efficient production of the PF1022A derivative of the present invention may depend on the medium, culture conditions, or microorganism strain used. However, the maximum accumulation may generally be attained in 2 to 25 days by any culture method.
- the incubation may be terminated when the amount of the PF1022A derivative compound of the present invention in the medium reaches its peak, at which time the compound may be isolated from the culture and optionally further purified.
- the PF1022A derivative compound of the present invention accumulated in the culture thus obtained may be contained in the cells of the microorganism and/or in the culture filtrate. Accordingly, in some embodiments the PF1022A derivative compound may be recovered from both culture filtrate and microorganism cells by separating the culture into fractions, e.g., by centrifugation.
- the PF1022A derivative can be recovered from the culture filtrate according to ordinary procedures known to those skilled in the art.
- the procedures can be carried out singly, in combination in a certain order, or repeatedly.
- extraction, filtration, centrifugation, salting out, concentration, drying, freezing, adsorption, detaching, means for separation based on the difference in solubility in various solvents, such as precipitation, crystallization, recrystallization, reverse solution, counter- current distribution, and chromatography, can be used.
- the PF1022A derivative may be recovered from inside the cells of the microorganism, for example, by cell lysis (e.g., smashing or pressure disruption), cell recovery (e.g., filtration and centrifugation) and lysis, and purification (e.g., salting out and solvent precipitation), according to methods known in the art.
- cell lysis e.g., smashing or pressure disruption
- cell recovery e.g., filtration and centrifugation
- purification e.g., salting out and solvent precipitation
- the crude PF1022A derivative obtained may be further purified according to methods known in the art, for example, by column chromatography using a carrier such as silica gel and alumina or reverse-phase chromatography using an ODS carrier.
- the feedstock com rises a compound of Formula II:
- R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
- R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino.
- the feedstock comprises a compound of Formula Ila:
- R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
- the feedstock comprises a compound of Formula III:
- R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, R 1 is amino or nitro. In some embodiments, the carbon in Formula III marked with an asterisk has the R configuration. In some embodiments, the carbon in Formula III marked with an asterisk has the S configuration. In some embodiments, the compound of Formula III is a mixture of R and S isomers.
- R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
- R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, R 1 is amino or nitro. In some embodiments, R 1 is halo. In some embodiments, R 1 is bromo or chloro. In some embodiments, R 1 is chloro. In some embodiments, the carbon in Formula IV marked with an asterisk has the R configuration. In some embodiments, the carbon in Formula IV marked with an asterisk has the S configuration. In some embodiments, the compound of Formula IV is a mixture of R and S isomers.
- the feedstock com rises a compound of Formula V:
- R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; and R 3 is selected from the group consisting of hydrogen, amino, and sulfhydryl.
- R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino and R 3 is H. In some embodiments, R 1 is halo and R 3 is H.
- the microorganism strain is a fungal strain capable of producing compound PF1022A and derivatives thereof.
- the fungal strain is a fungal strain belonging to the genus Xylaria or the genus Rosellinia of the family Xylariaceae.
- One preferred example of a fungal strain capable of producing PF1022A substance or derivatives thereof is the PF1022 strain deposited at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Tsukuba-shi, Japan, under the accession number FERM BP-2671. The myco logical properties of BP-2671 are described for example in U.S. Patent No. 5,1 16,815 and the Journal of Antibiotics, 1992, Vol. 45, pg. 692. See also US Patent Application Publication No. 2011/0262969 to Harder et al.
- the PF1022A-producing strains may be labile in their properties.
- BP-2671 itself, or any mutant derived from this strain, phenotypic conjugation (either spontaneously generated or artificially induced), or genetic recombinant of said strain may be used in practicing the process of the invention, if it can produce PF1022A and the described derivatives thereof.
- the PF1022A-producing fungal strain is cultivated in a culture medium containing such ordinary carbon source and nitrogen source which can generally be utilized as nutrients for microorganisms, as noted above. Such nutrients may be used which are known to have been used for the cultivation of fungi.
- carbon sources which may be used with the PF1022A-producing fungal strain include glucose, sucrose, starch syrup, dextrin, starch, glycerol, molasses, animal oils, and vegetable oils.
- Non-limiting examples of nitrogen sources which may be used with the PF1022A-producing fungal strain include soybean flour, wheat germ, corn steep liquor, cotton seed oil, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, and urea.
- Inorganic salts may be added to the medium, as previously described herein.
- inorganic and organic substances capable of promoting the growth of the fungal strain may also be added in an appropriate amount.
- a cultivation method carried out under aerobic conditions is suitable. In some embodiments, a cultivation method under submerged conditions is preferable.
- a temperature range of 15-30°C may be suitable. In some embodiments, a temperature of about 26°C is preferable.
- PF1022A derivatives In shake- or tank-cultivation, the production of PF1022A derivatives typically arrives at a maximum accumulation in 2-10 days. The skilled worker will appreciate that the incubation period required for maximum accumulation may vary depending on the composition of the culture medium and the cultivation conditions employed. Cultivation may be discontinued when production of the PF1022A derivative substance has reached its peak. The resulting culture may then be separated by filtration or by centrifugation to provide a solid portion (pellet) including the cultured cells and other solid materials, and a broth filtrate. The filtering operation may be performed using filtering aids known in the art, such as diatomaceous earth.
- PF1022A derivative substances produced by the cultivation of microbial or fungal strains may be effected by the use of one or more techniques for the isolation of biological materials well known to those skilled in biochemistry or microbiology.
- suitable for use in the present invention include solvent extraction or adsorption, ion-exchange resin treatment, partition column chromatography, gel filtration, dialysis, and techniques of precipitation. These methods may be used alone or in appropriate combination.
- the PF1022A derivative substances are relatively insoluble in water and thus exist largely in the cultured cells rather than the broth filtrate.
- PF1022A derivative substances may be extracted from the cultured cells with an organic solvent or an aqueous organic solvent.
- Organic solvents which may be used for the purpose include by way of non- limiting examples methanol, ethanol, ethyl acetate, acetone, acetonitrile, and halogenated solvents such as dichloromethane and chloroform. Any of these organic solvents or similar organic solvents may be utilized as appropriate as solvent-water mixtures.
- Purification methods which may be used to isolate the PF1022A derivative substances of the invention include chromatographic methods with silica gel or alumina as an adsorbent, as well as chromatographic methods with a gel filtration agent such as Sephadex LH-20 (Pharmacia Co.).
- the PF1022A derivative substances of the present invention may be further purified by recrystallization. Recrystallization may be from single solvent or mixed solvents.
- Non-limiting examples of single solvents include methanol, ethanol, ethyl acetate, acetone, diethyl ether, methyl tert-butyl ether, and methylene chloride.
- mixed solvents include, without limitation, methanol-water, ethanol- water, ethyl acetate-hexanes, and diethyl ether-hexanes.
- the process disclosed herein comprises the steps of separating the cultured cells from the produced culture, and extracting the separated cultured cells with an organic solvent or an aqueous solvent, to obtain an extract containing the PF1022A derivative compound.
- the obtained extract is concentrated to provide the PF1022A derivative compound, which may be further purified as described herein.
- a process for the preparation of PF1022A derivatives comprising incubating a PF1022A-producing strain, in a culture medium containing carbon and nitrogen sources and in the presence of a 3-(4-substituted- phenyl)-2-oxopropanoic acid of Formula Ila, or a salt thereof.
- the 3-(4- substituted-phenyl)-2-oxopropanoic acid of Formula Ila is present as a sodium salt.
- the 3-(4-substituted-phenyl)-2-oxopropanoic acid of Formula Ila is present as a potassium salt.
- the 3-(4-substituted-phenyl)-2-oxopropanoic acid of Formula Ila is present as a free acid.
- the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium between 6 hours and 96 hours after inoculation of the culture medium with the microorganism. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 6 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 24 hours after inoculation of the culture medium.
- the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 48 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 72 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 96 hours after inoculation of the culture medium.
- the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium at more than one time point.
- the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V may be added to the culture medium at times 6 h and 24 h post-inoculation of the culture medium with the microorganism; 6h, 24h and 48h post inoculation; 6h, 24h, 48h and 72 h post-inoculation; 6h, 24h, 48h, 72h and 96 h post-inoculation; 48h and 72h post-inoculation, and 48h, 72h and 96h post-inoculation.
- time points may depend upon the particulars of the culture conditions.
- the microorganism is fungal strain FERM BP-2671 as the microorganism capable of producing PF1022A derivative compounds.
- Strain PERM BP-2671 (NBRC 33096) was inoculated from agar plugs and cultured in seed medium (2.0 % soluble starch, 1 .0 % glucose, 0.6 % wheat germ, 0.5 % polypeptone. 0.3 % yeast extract, 0.2 % soybean cake (Nutrisoy), 0.2 % calcium carbonate) in a baffled Erienmeyer flask at 120 rpm, 5 cm throw, 26°C for 3 days. A 5 % inoculum was transferred to production medium (6 % starch syrup ( maltose syrup), 2.6 % soluble starch.
- seed medium 2.0 % soluble starch, 1 .0 % glucose, 0.6 % wheat germ, 0.5 % polypeptone. 0.3 % yeast extract, 0.2 % soybean cake (Nutrisoy), 0.2 % calcium carbonate
- Mobile Phase Solution A was 10 mM ammonium formate / 0.2 % formic acid (pi 1 2.9), Mobile Phase Solution B was 95 % acetonitrile / 5 % water / 0.17 % formic acid.
- Sample injection volumes were typically 5 ⁇ L using the following gradient, 0.00 min, 5 % B, 1 .0 mL/min; 0.10 min, 5 % B, 1.0 mL/min; 0.20 min, 50 % B, 1.0 mL/min; 9.30 min, 80 % B, 1.0 mL/min; 9.50 min, 95 % B, 1.0 mL/min; 10.50 min, 95 % B, 1.0 mL/min; 10.60 min, 95 % B, 1.5 mL/min; 1 1.0 min, 95 % B, 1 .5 mL/min; 1 1 .05 min, 5 % B, 1 .5 mL/min; 1 1 .5 min, 95 % B, 1 .5 mL min and a 1 .5 min injection cycle for equilibration.
- the MS was operated in electrospray positive ion mode gathering data from 400- 1 300 m/z and UV data were gathered betw een 230
- Sample injection volumes were typically 5 ⁇ L using the following gradient, 0.00 min, 5 % B, 2.0 m I 'm in; 0.05 min, 5 % B, 2.0 ml/min; 0.1 min, 50 % B, 2.0 ml min; 3.9 min, 80 % B, 2.0 ml min; 4.0 min, 95 % B, 2.0 mi min; 5.0 min, 95 % B, 2.0 ml min, 5. 1 min, 5 % B, 2.0 ml min; 6.0 min, 5 % B, 2.0 ml min.
- the MS was operated in electrospray positive ion mode gathering data from 400- 1 00 m/z and UV data were gathered between 230 400 nm and 230 nm used for PF 1022A data analysis and calibration.
- cultures were grown as described as above.
- the feedstock compound (as set out in Table 1 below) was dissolved in methanol to a concentration of 280 mM and then added to the production medium at 2 mM final concentration at 72 hours post inoculation. Following the addition of feedstock, cultures were incubated at 200 rpm, 5 cm throw, 26°C for 7- 1 0 days.
- Substrate feeds utilized in the present invention may be prepared by methods known to those of skill in the art of organic synthesis.
- embodiments of substrate feeds may be prepared from substituted phenylalanine compounds as shown below in Scheme I.
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Abstract
Provided are processes for the production of PF1022A derivative compounds, including incubating a microorganism strain capable of producing such compounds in a culture medium to produce a resulting culture, said culture medium containing carbon and nitrogen sources, and further providing to said culture medium a feedstock comprising a compound of Formula II, Formula III, Formula IV or Formula V: (II), (III), (IV), (V) thereby producing said PF1022A derivative compound in the resulting culture. Compounds of Formula II, Formula III, Formula IV, or Formula V useful in such a feedstock are also provided.
Description
Methods for Production of PF1022A Derivatives
BACKGROUND
Every year the loss of valuable livestock/fishes to invertebrate parasites, both endoparasites (nematodes or helminths) and ectoparasites (flies, ticks, mites and sea lice) totals >£34 billion globally. This is despite the fact that farmers spend ~£4.5 billion globally on compounds to protect their animals from parasites (Parasitol Res (2005) 97:S11-S16, - Jeschke et a!. ). New products are continually needed as new parasites emerge and existing parasites evolve resistance to current treatments.
PF1022A is a fungally-derived non-ribosomal peptide natural product octadepsipeptide anthelmintic agent. Emodepside, a complex semi-synthetic derivative of PF1022A, is a resistance breaking anthelmintic used exclusively for the more profitable companion animal market owing to high cost of production (Ohyama et al, Biosci., Biotechno., Biochem., 2011, 75,
PF1022A Emodepside
The unique and highly complex core structure of the PF1022A natural product has provided challenging opportunities for synthesis. Conversion of PF1022A to the bis-4- morpholino derivative (emodepside) entails low-yielding chemistry such as nitration of the phenyl rings followed by reduction and subsequent functionalization. In addition to the poor chemical yields arising from nitration (or acetylation, another route), the generation of regioisomers further reduces the yield of useful intermediate and necessitates expensive purification of the desired para-regioisomers. Lower cost of goods for emodepside would enable the use of the compound in livestock herds, an application prohibited by its present high cost of manufacture. Additionally, with the increase of insect resistance, new PF1022A derived compounds and methods for their synthesis are needed. The recent demonstration that
emodepside may have utility in the treatment of African river disease in humans only sharpens the need for new methods for the preparation of emodepside and related structures.
Semisynthetic routes to the bis-hydroxy PF1022A derivative, PF1022H, have recently been described by Scherkenbeck et al. (Bioorg. Med. Chem. 2016, 24, 873-876). One route proceeds from PF1022A by nitration of the phenyl rings followed by reduction to the amine and diazotization followed by hydrolysis to the phenol. A second route utilizes Friedel-Crafts acylation of the phenyl rings followed by Baeyer-Villiger oxidation and subsequent ester cleavage. The nature of the electrophilic substitution chemistry results in mixtures of para and meta isomers, with para predominating. The para-bis- ydroxy compound PF1022H has been shown to be a useful intermediate for the preparation of lipophilic PF1022A derivatives. (Ohyama 2011).
There remains a need for new methods and compound useful for the preparation of PF1022A derivatives. SUMMARY
It is a purpose of the present invention to provide methods for the regioselective preparation of PF1022A derivatives. In addition, methods disclosed herein may be used in the preparation of new PF1022A derivatives, which may be useful as antihelmintic agents.
Disclosed herein is a process for the production of a PF1022A derivative compound of Formula I:
wherein X1, X2, X3 and X4, at each occurrence, are independently selected from N and CH; and
R1 and R2 are each independently selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; comprising: incubating a microorganism strain capable of producing PF1022A derivative compounds in a culture medium to produce a resulting culture, said culture medium containing carbon and nitrogen sources, and further providing to said culture medium a feedstock comprising a compound of Formula II, Formula III, Formula IV or Formula V:
II III IV V wherein the dotted line represents an optional double bond;
X2, X3, and X4 are independently selected from N and CH; and
R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; and R3 is selected from the group consisting of hydrogen, amino, and sulfhydryl, thereby producing said PF1022A derivative compound in the resulting culture.
In some embodiments, the feedstock comprises a compound of Formula II. In some embodiments, the feedstock comprises a compound of Formula II, and the compound of Formula II is a compound of Formula Ila:
I la
R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
In some embodiments, the feedstock comprises a compound of Formula III.
In some embodiments, the feedstock comprises a compound of Formula IV.
In some embodiments, the feedstock comprises a compound of Formula V.
In some embodiments of any of the above, R2 is H.
In some embodiments of any of the above, R1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino.
In some embodiments of any of the above, R1 is halo.
In some embodiments of any of the above, R1 is fluoro.
In some embodiments of any of the above, R1 is chloro.
In some embodiments of any of the above, R1 is bromo.
In some embodiments, the compound of Formula Ila is:
In some embodiments, the compound of Formula Ila is:
In some embodiments of any of the above, the process further includes recovering the PF1022A derivative compound from said culture.
In some embodiments of any of the above, the carbon source comprises a carbohydrate.
In some embodiments of any of the above, the nitrogen source comprises inorganic or organic nitrogen-containing compounds.
In some embodiments of any of the above, the microorganism strain is a fungal strain.
In some embodiments of any of the above, the fungal strain belongs to the genus Xylaria or the genus Rosellinia.
In some embodiments of any of the above, the producing comprises accumulating said PF1022A derivative compound in the resulting culture.
In some embodiments of any of the above, the compound of Formula I is enantiomerically pure.
Compounds of Formula II, Formula III, Formula IV, or Formula V taught herein that are useful for such feedstocks are also provided.
IV
or a salt thereof,
wherein R1 is halo. In some embodiments, R1 is bromo or chloro. In some embodiments, R1 is fluoro. In some embodiments, R1 is chloro.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts LC-MS chromatograms of the dichloro derivative of PF1022A prepared by the disclosed process.
FIG. 2 depicts LC-MS chromatograms of the mono-chloro derivative of PF1022A prepared by the disclosed process.
FIG. 3 depicts mass spectrometry patterns for the mono-chloro (left) and dichloro (right) derivatives of PF1022A prepared by the disclosed process.
DETAILED DESCRIPTION OF EMBODIMENTS
Provide herein are processes and methods useful for the preparation of cyclooctadepsipeptide compounds such as the approved animal anthelmintic compound emodepside.
A. DEFINITIONS
Processes and methods in accordance with the present disclosure include those generally described above and below, and are further illustrated by the embodiments, sub-embodiments, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated.
As described herein, compounds of the invention may be substituted with one or more substituents, such as those generally described herein, such as those illustrated generally herein, or as exemplified by particular classes, subclasses, and species of the invention. In general the term "substituted" refers to the replacement of hydrogen in a given structure with a specified substituent. When a structure is described as "substituted" without further specification, it is understood that said group may be substituted with one or more substituents chosen from the group consisting of hydrogen, halo, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl.
Unless indicated otherwise, nomenclature used to describe chemical groups or moieties as used herein follow the convention where, reading the name from left to right, the point of attachment to the rest of the molecule is at the right hand side of the name. For example, the group "alkylamino" is attached to the rest of the molecule at the amino end, whereas the group "aminoalkyl" is attached to the rest of the molecule at the alkyl end.
Unless indicated otherwise, where a chemical group is described by its chemical formula, including a terminal bond moiety indicated by it will be understood that the attachment is read from left to right. For example, -C(0)Ci_6alkyl is attached to the rest of the molecule at the carbonyl end.
"Alkyl" or "alkyl group", as used herein, means a straight-chain (i.e. , unbranched), or branched hydrocarbon chain that is completely saturated. In some embodiments the alkyl has 1 , 2, 3, 4, 5 or 6 carbon atoms. In certain embodiments, alkyl groups contain 1-6 carbon atoms (Ci_6 alkyl). In certain embodiments, alkyl groups contain 1-4 carbon atoms (C1-4 alkyl). In certain embodiments, alkyl groups contain 1-3 carbon atoms (C1 -3 alkyl). In still other embodiments, alkyl groups contain 2-3 carbon atoms (C2-3 alkyl), and in yet other embodiments alkyl groups contain 1-2 carbon atoms (C1-2 alkyl).
"Ar" or "aryl" refer to an aromatic carbocyclic moiety having one or more closed rings. Examples include, without limitation, phenyl, naphthyl, anthracenyl, phenylanthracenyl, biphenyl, and pyrenyl.
"Heteroaryl" refers to a cyclic moiety having one or more closed rings, with one or more heteroatoms (oxygen, nitrogen, or sulfur) in at least one of the rings, wherein at least one of the rings is aromatic, and wherein the ring or rings may independently be fused, and/or bridged. Examples include, without limitation, pyridyl, quinolinyl, isoquinolinyl, indolyl, furyl, thienyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, quinoxalinyl, pyrrolyl, indazolyl, thiazolyl, oxazolyl, and isoxazolyl.
"Alkoxy" refers to an alkyl or cycloalkyl group, as herein defined, attached to the principal carbon chain through an oxygen atom. Representative examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, and hexyloxy.
"Hydroxy" or "hydroxyl" refers to an -OH group.
"Halogen" or "halo" refers to one or more of fluoro, chloro, bromo, and iodo.
An "amine" as used herein refers to a primary, secondary or tertiary amine. An "amino" group refers to the primary amine group -NH2. Examples of secondary or tertiary amines include, but are not limited to, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, etc.
"Carboxylate" refers to a salt or ester of a carboxylic acid moiety.
"Carboxyalkyl" refers to a carboxylic acid group attached to the principal carbon chain or molecule through an alkyl group. The carboxylic acid group may be present as the free acid, salt, or an ester.
"Cycloalkyl" as used herein, refers to a saturated cyclic hydrocarbon group containing from 3 to 8 carbons or more. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
"Heteroatom" refers to O, S or N.
"Heterocycle" or "heterocyclyl" as used herein, means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle containing at least one heteroatom in a ring.
"Monocyclic heterocycle" means a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom, and which is not aromatic. Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3- dioxanyl, 1,3-dioxolanyl, dihydropyranyl (including 3,4-dihydro-2H-pyran-6-yl), 1,3- dithiolanyl, 1,3-dithianyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxadiazolidinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothienyl, thiadiazolidinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomor- pholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl.
"Bicyclic heterocycle" means a monocyclic heterocycle fused to an aryl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl or cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle. Representative examples of bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-pyranyl, 1,3- benzodioxolyl, 1,3-benzodithiolyl, 2,3-dihydro-l,4-benzodioxinyl, 2,3-dihydro-l- benzofuranyl, 2,3-dihydro-l-benzothienyl,2,3-dihydro-lH-indolyl, 3,4-dihydroquinolin- 2(lH)-one and 1,2,3,4- tetrahydroquinolinyl.
"Tricyclic heterocycle" means a bicyclic heterocycle fused to an aryl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl or cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle. Representative examples of tricyclic heterocycles include, but are not limited to, 2,3,4,4a,9,9a-hexahydro- lH-carbazolyl, 5a,6,7,8,9,9a- hexahydro-dibenzo[b,d]furanyl, and 5a,6,7,8,9,9a-hexahydrodibenzo[b,d]thienyl.
"Azido" refers to the group -N3.
"Cyano" refers to the group -CN.
"Isonitrile" refers to the group -NC.
"Nitro" refers to the group -N02.
"Vinyl" refers to the group -C=C.
Unless otherwise stated, structures depicted herein are also meant to include all enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all
tautomeric forms of the compounds of the invention are within the scope of the invention. Tautomeric forms include keto-enol tautomers of a compound. In addition, unless otherwise stated, all rotamer forms of the compounds of the invention are within the scope of the invention. Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.
"Isomers" refers to compounds having the same number and kind of atoms and hence the same molecular weight, but differing with respect to the arrangement or configuration of the atoms. It will be understood, however, that some isomers or racemates or others mixtures of isomers may exhibit more activity than others. "Stereoisomers" refers to isomers that differ only in the arrangement of the atoms in space. "Diastereoisomers" refers to stereoisomers that are not mirror images of each other. "Enantiomers" refers to stereoisomers that are non-superimpo sable mirror images of one another.
In some embodiments, enantiomeric compounds taught herein may be "enantiomerically pure" isomers that comprise substantially a single enantiomer, for example, greater than or equal to 90%, 92%, 95%, 98%, or 99%, or equal to 100% of a single enantiomer.
In some embodiments, enantiomeric compounds as taught herein may be stereochemically pure. "Stereochemically pure" as used herein means a compound or composition thereof that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound.
In some embodiments, "R" and "S" as terms describing isomers are descriptors of the stereochemical configuration at an asymmetrically substituted carbon atom. The designation of an asymmetrically substituted carbon atom as "R" or "S" is done by application of the Cahn-Ingold-Prelog priority rules, as are well known to those skilled in the art, and described in the International Union of Pure and Applied Chemistry (IUPAC) Rules for the Nomenclature of Organic Chemistry.
B. METHODS FOR PRODUCTION
An objective of the present invention is to provide a method for producing PF1022A derivatives, by a direct fermentation method. Accordingly, provided herein are methods for preparing cyclooctadepsipeptide compounds of Formula I:
I wherein X1, X2, X3 and X4, at each occurrence, are independently selected from N and
CH;
R1 and R2 are independently selected from the group consisting of hydrogen, halo, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, one of R1 and R2 is hydrogen and the other is not hydrogen. In some embodiments, neither R1 nor R2 is hydrogen.
In some embodiments, the present invention provides for the biosynthetic production of cyclooctadepsipeptides of Formula I by providing a feedstock with a compound as taught herein to a microrganism strain capable of producing PF1022A derivative compounds. The microorganism strain may be cultured according to an ordinary method by appropriately selecting a medium, culture conditions, and the like. The medium may be supplemented with a carbon source and nitrogen source that can be utilized by the microorganism strain, inorganic salts, various vitamins, various amino acids such as glutamic acid and asparagine, trace nutrients such as nucleotides, selectable agents such as antibiotics, etc. See, e.g. , U.S.
Patent Nos. 6,043,058 to Ohyama et al. and 7,285,404 to Midoh et al.
Any kind of carbon source and nitrogen source may be used in the medium as long
as they can be utilized by the microorganisms of the present invention. As the carbon source, for example, various carbohydrates, such as sucrose, glucose, starch, glycerin, fructose, maltose, mannitol, xylose, galactose, ribose, dextrin, animal and plant oils and the like, or hydrolysates thereof, can be used. The preferable concentration generally is from 0.1% to 5% of the medium. As the utilizable nitrogen source, for example, animal or plant components, or exudates or extracts thereof, such as peptone, meat extract, com steep liquor, and defatted soybean powder, organic acid ammonium salts such as succinic acid ammonium salts and tartaric acid ammonium salts, urea, and other various inorganic or organic nitrogen-containing compounds can be used.
Inorganic salts, for example, those which can produce sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphate, sulfate, and/or other ions may also be used.
Any medium which contains other components, such as cells, exudates or extracts of microorganisms such as yeasts, and fine plant powders, may be used as long as these other components do not unduly interfere with the growth of the microorganism and the production and accumulation of the PF1022A derivative of the present invention. When a mutant strain having a nutritional requirement is cultured, a substance to satisfy its nutritional requirement is added to the medium. However, this kind of nutrient may not necessarily be added when a medium containing natural substances is used.
The pH of the medium may be, for example, about 6 to 8. Incubation may be carried out by a shaking culture method under an aerobic condition, an agitation culture method with aeration or an aerobic submerged culture method. An appropriate incubation temperature in some embodiments may be 15°C to 40°C, more preferably about 26°C to 37°C.
Efficient production of the PF1022A derivative of the present invention may depend on the medium, culture conditions, or microorganism strain used. However, the maximum accumulation may generally be attained in 2 to 25 days by any culture method. The incubation may be terminated when the amount of the PF1022A derivative compound of the present invention in the medium reaches its peak, at which time the compound may be isolated from the culture and optionally further purified.
The PF1022A derivative compound of the present invention accumulated in the culture thus obtained may be contained in the cells of the microorganism and/or in the culture filtrate. Accordingly, in some embodiments the PF1022A derivative compound may be recovered from both culture filtrate and microorganism cells by separating the culture into fractions, e.g., by centrifugation.
The PF1022A derivative can be recovered from the culture filtrate according to
ordinary procedures known to those skilled in the art. The procedures can be carried out singly, in combination in a certain order, or repeatedly. For example, extraction, filtration, centrifugation, salting out, concentration, drying, freezing, adsorption, detaching, means for separation based on the difference in solubility in various solvents, such as precipitation, crystallization, recrystallization, reverse solution, counter- current distribution, and chromatography, can be used.
The PF1022A derivative may be recovered from inside the cells of the microorganism, for example, by cell lysis (e.g., smashing or pressure disruption), cell recovery (e.g., filtration and centrifugation) and lysis, and purification (e.g., salting out and solvent precipitation), according to methods known in the art.
The crude PF1022A derivative obtained may be further purified according to methods known in the art, for example, by column chromatography using a carrier such as silica gel and alumina or reverse-phase chromatography using an ODS carrier.
In some embodiments, the feedstock com rises a compound of Formula II:
I I
wherein the dotted line represents an optional double bond, X1, X2, X3, and X4 are independently chosen from N and CH; and R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, R1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino.
Ila wherein
X2, X3, and X4 are independently chosen from N and CH; and R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, R1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, the feedstock com rises a compound of Formula lib:
wherein
X2, X3, and X4 are independently chosen from N and CH; and R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, R1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, the carbon in Formula lib marked with an asterisk has the R configuration. In some embodiments, the carbon in Formula lib marked with an asterisk has the S configuration. In some embodiments, the compound of Formula lib is a mixture of R and S isomers.
In some embodiments of the formulas, X1, X2, X3 and X4 are CH. In some embodiments, R1 is methoxy or cyano. In some embodiments, X1, X2, X3 and X4 are CH and R1 is methoxy or cyano.
In some embodiments, the feedstock comprises a compound of Formula III:
wherein R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, R1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, R1 is amino or nitro. In some embodiments, the carbon in Formula III marked with an asterisk has the R configuration. In some embodiments, the carbon in Formula III marked with an asterisk has the S configuration. In some embodiments, the compound of Formula III is a mixture of R and S isomers.
In some embodiments, the feedstock com rises a compound of Formula IV:
wherein R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, R1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, R1 is amino or nitro. In some embodiments, R1 is halo. In some embodiments, R1 is bromo or chloro. In some embodiments, R1 is chloro. In some embodiments, the carbon in Formula IV marked with an asterisk has the R configuration. In some embodiments, the carbon in Formula IV marked with an asterisk has the S configuration. In some embodiments, the compound of Formula IV is a mixture of R and S isomers.
In some embodiments, the feedstock com rises a compound of Formula V:
V
wherein R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; and R3 is selected from the group consisting of hydrogen, amino, and sulfhydryl. In some embodiments, R1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, R1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino and R3 is H. In some embodiments, R1 is halo and R3 is H.
In some embodiments, the microorganism strain is a fungal strain capable of producing compound PF1022A and derivatives thereof. In some embodiments, the fungal strain is a fungal strain belonging to the genus Xylaria or the genus Rosellinia of the family Xylariaceae. One preferred example of a fungal strain capable of producing PF1022A substance or derivatives thereof is the PF1022 strain deposited at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Tsukuba-shi, Japan, under the accession number FERM BP-2671. The myco logical properties of BP-2671 are described for
example in U.S. Patent No. 5,1 16,815 and the Journal of Antibiotics, 1992, Vol. 45, pg. 692. See also US Patent Application Publication No. 2011/0262969 to Harder et al.
In some embodiments, the PF1022A-producing strains may be labile in their properties. Thus, for example, BP-2671 itself, or any mutant derived from this strain, phenotypic conjugation (either spontaneously generated or artificially induced), or genetic recombinant of said strain may be used in practicing the process of the invention, if it can produce PF1022A and the described derivatives thereof.
In an embodiment, the PF1022A-producing fungal strain is cultivated in a culture medium containing such ordinary carbon source and nitrogen source which can generally be utilized as nutrients for microorganisms, as noted above. Such nutrients may be used which are known to have been used for the cultivation of fungi. Non-limiting examples of carbon sources which may be used with the PF1022A-producing fungal strain include glucose, sucrose, starch syrup, dextrin, starch, glycerol, molasses, animal oils, and vegetable oils. Non-limiting examples of nitrogen sources which may be used with the PF1022A-producing fungal strain include soybean flour, wheat germ, corn steep liquor, cotton seed oil, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, and urea. Inorganic salts may be added to the medium, as previously described herein. In addition, inorganic and organic substances capable of promoting the growth of the fungal strain may also be added in an appropriate amount.
In some embodiments for the methods of cultivation of the PF1022A-producing fungal strain, a cultivation method carried out under aerobic conditions is suitable. In some embodiments, a cultivation method under submerged conditions is preferable. For cultivation of the PF1022A-producing fungal strain, a temperature range of 15-30°C may be suitable. In some embodiments, a temperature of about 26°C is preferable.
In shake- or tank-cultivation, the production of PF1022A derivatives typically arrives at a maximum accumulation in 2-10 days. The skilled worker will appreciate that the incubation period required for maximum accumulation may vary depending on the composition of the culture medium and the cultivation conditions employed. Cultivation may be discontinued when production of the PF1022A derivative substance has reached its peak. The resulting culture may then be separated by filtration or by centrifugation to provide a solid portion (pellet) including the cultured cells and other solid materials, and a broth filtrate. The filtering operation may be performed using filtering aids known in the art, such as diatomaceous earth.
The recovery of PF1022A derivative substances produced by the cultivation of microbial or fungal strains may be effected by the use of one or more techniques for the isolation of biological materials well known to those skilled in biochemistry or microbiology. Non-limiting
examples of such techniques suitable for use in the present invention include solvent extraction or adsorption, ion-exchange resin treatment, partition column chromatography, gel filtration, dialysis, and techniques of precipitation. These methods may be used alone or in appropriate combination.
In some embodiments, the PF1022A derivative substances are relatively insoluble in water and thus exist largely in the cultured cells rather than the broth filtrate. PF1022A derivative substances may be extracted from the cultured cells with an organic solvent or an aqueous organic solvent. Organic solvents which may be used for the purpose include by way of non- limiting examples methanol, ethanol, ethyl acetate, acetone, acetonitrile, and halogenated solvents such as dichloromethane and chloroform. Any of these organic solvents or similar organic solvents may be utilized as appropriate as solvent-water mixtures.
Purification methods which may be used to isolate the PF1022A derivative substances of the invention include chromatographic methods with silica gel or alumina as an adsorbent, as well as chromatographic methods with a gel filtration agent such as Sephadex LH-20 (Pharmacia Co.). The PF1022A derivative substances of the present invention may be further purified by recrystallization. Recrystallization may be from single solvent or mixed solvents. Non-limiting examples of single solvents include methanol, ethanol, ethyl acetate, acetone, diethyl ether, methyl tert-butyl ether, and methylene chloride. Examples of mixed solvents include, without limitation, methanol-water, ethanol- water, ethyl acetate-hexanes, and diethyl ether-hexanes.
In some embodiments the process disclosed herein comprises the steps of separating the cultured cells from the produced culture, and extracting the separated cultured cells with an organic solvent or an aqueous solvent, to obtain an extract containing the PF1022A derivative compound. In a further embodiment, the obtained extract is concentrated to provide the PF1022A derivative compound, which may be further purified as described herein.
In an embodiment of the present invention, there is provided a process for the preparation of PF1022A derivatives, the process comprising incubating a PF1022A-producing strain, in a culture medium containing carbon and nitrogen sources and in the presence of a 3-(4-substituted- phenyl)-2-oxopropanoic acid of Formula Ila, or a salt thereof. In some embodiments, the 3-(4- substituted-phenyl)-2-oxopropanoic acid of Formula Ila is present as a sodium salt. In some embodiments, the 3-(4-substituted-phenyl)-2-oxopropanoic acid of Formula Ila is present as a potassium salt. In some embodiments, the 3-(4-substituted-phenyl)-2-oxopropanoic acid of Formula Ila is present as a free acid.
In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium between 6 hours and 96 hours
after inoculation of the culture medium with the microorganism. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 6 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 24 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 48 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 72 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 96 hours after inoculation of the culture medium.
In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium at more than one time point. For example, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V may be added to the culture medium at times 6 h and 24 h post-inoculation of the culture medium with the microorganism; 6h, 24h and 48h post inoculation; 6h, 24h, 48h and 72 h post-inoculation; 6h, 24h, 48h, 72h and 96 h post-inoculation; 48h and 72h post-inoculation, and 48h, 72h and 96h post-inoculation. These examples are intended as non-limiting examples of embodiments of the present invention, and the skilled worker will understand that choice of time points may depend upon the particulars of the culture conditions.
In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium as a methanol solution feedstock. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium as a methanol solution feedstock at a concentration of between 2 and 10 mM. In some embodiments the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added at a concentration of 2mM; 4 mM; 6 mM; 8 mM; or 10 mM. Any of these concentrations of the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V may be added once, or more than once, to the culture medium following inoculation.
In some embodiments, the microorganism is fungal strain FERM BP-2671 as the microorganism capable of producing PF1022A derivative compounds.
The present invention is further illustrated by the following non-limiting examples.
EXAMPLES
Strain PERM BP-2671 (NBRC 33096) was inoculated from agar plugs and cultured in seed medium (2.0 % soluble starch, 1 .0 % glucose, 0.6 % wheat germ, 0.5 % polypeptone. 0.3 % yeast extract, 0.2 % soybean cake (Nutrisoy), 0.2 % calcium carbonate) in a baffled Erienmeyer flask at 120 rpm, 5 cm throw, 26°C for 3 days. A 5 % inoculum was transferred to production medium (6 % starch syrup ( maltose syrup), 2.6 % soluble starch. 2 % wheat germ, 1 % Pharma media (Archer Daniels Midland Co.), 0.2 % magnesium sulfate heptahydratc, 0.2 % calcium carbonate. 0.3 % sodium chloride) and incubated at 200 rpm, 5 cm throw, 26°C for 12-14 days. After incubation, samples were extracted with an equal volume of methanol, mixed for 30 mins, centrifuged and 150 μΐ aiiquots were transferred to HPLC vials for analysis.
Analysis method A :
Analyses were performed on an Agilent 1 1 00 HPLC connected to Waters ZQ single quadrupole MS. the HPLC column was a Waters XSelect CSH C 18 (2.1 mm x 50 mm. 3.5 μηι), fitted with a Waters VanGuard CSH CI 8 precolumn (2.1 mm x 5 mm, 3.5 μηι), and operated at
60 °C, 1.0 mL/min flow rate giving a system back pressure of 230 bar at injection. Mobile Phase Solution A was 10 mM ammonium formate / 0.2 % formic acid (pi 1 2.9), Mobile Phase Solution B was 95 % acetonitrile / 5 % water / 0.17 % formic acid. Sample injection volumes were typically 5 μ L using the following gradient, 0.00 min, 5 % B, 1 .0 mL/min; 0.10 min, 5 % B, 1.0 mL/min; 0.20 min, 50 % B, 1.0 mL/min; 9.30 min, 80 % B, 1.0 mL/min; 9.50 min, 95 % B, 1.0 mL/min; 10.50 min, 95 % B, 1.0 mL/min; 10.60 min, 95 % B, 1.5 mL/min; 1 1.0 min, 95 % B, 1 .5 mL/min; 1 1 .05 min, 5 % B, 1 .5 mL/min; 1 1 .5 min, 95 % B, 1 .5 mL min and a 1 .5 min injection cycle for equilibration. The MS was operated in electrospray positive ion mode gathering data from 400- 1 300 m/z and UV data were gathered betw een 230 - 400 nm and 230 nm used for PF1022A data analysis and calibration.
Analysis method B:
Analyses were performed on an Agilent 1 100 HPLC connected to Waters ZQ single quadrupole MS, the HPLC column was a Waters Atlantis dC 1 , (4.6 mm x 20 mm. 3 urn), fitted with an Acquity in-line column filter, 0.2 urn and operated at 60 °C, 2.0 ml min. Mobile Phase Solution A was 10 mM ammonium formate / 0.2 % formic acid (pH 2.9), Mobile Phase Solution B was 95 % acetonitrile 1 5 % water / 0. 1 7 % formic acid. Sample injection volumes were
typically 5 μ L using the following gradient, 0.00 min, 5 % B, 2.0 m I 'm in; 0.05 min, 5 % B, 2.0 ml/min; 0.1 min, 50 % B, 2.0 ml min; 3.9 min, 80 % B, 2.0 ml min; 4.0 min, 95 % B, 2.0 mi min; 5.0 min, 95 % B, 2.0 ml min, 5. 1 min, 5 % B, 2.0 ml min; 6.0 min, 5 % B, 2.0 ml min. The MS was operated in electrospray positive ion mode gathering data from 400- 1 00 m/z and UV data were gathered between 230 400 nm and 230 nm used for PF 1022A data analysis and calibration.
For directed biosynthesis experiments, cultures were grown as described as above. The feedstock compound (as set out in Table 1 below) was dissolved in methanol to a concentration of 280 mM and then added to the production medium at 2 mM final concentration at 72 hours post inoculation. Following the addition of feedstock, cultures were incubated at 200 rpm, 5 cm throw, 26°C for 7- 1 0 days.
After incubation, samples of broth and cells were extracted with an equal volume of methanol, mi ed for 30 mins, centrifuged and 1 50 μΐ aliquots were transferred to HPLC v ials for analysis. The samples were analysed using the LC-MS method described above (method A or method B), and the results are summarized in Table 1.
Table 1
3-(4-Hydroxyphenyl)-2-oxopropanoic acid
(R)-3-(4-Fiuorophenyl)-2-hydroxypropanoic acid
7.3 (Method A) 1002.6
OH
(R)-3-(4-Fluorophenyl)-2-hydroxypropanoic acid
7.3 (Method A) 984.5
3-(4-fluorophenyl)-2-oxopropanoic acid
7.3 (Method A) 1002.6
3-(4-fluorophenyl)-2-oxopropanoic acid
7.9 (Method A) I 1000.5 I Ci I H
These results shown in Table 1 demonstrate that the feedstock compounds can be incorporated into the final products of the fungal synthesis.
Substrate feeds utilized in the present invention may be prepared by methods known to those of skill in the art of organic synthesis. By way of non-limiting examples, embodiments of substrate feeds may be prepared from substituted phenylalanine compounds as shown below in Scheme I.
Scheme I
Synthesis of 3-(4-Chlorophenyl)-2-hydroxypropanoic acid
Sulfuric acid (34 mL) in water (230 mL) was added slowly to 4-chlorophenylalanine (10.0 g, 50 mmol) in water (200 mL) and cooled to 0 °C. A solution of sodium nitrite (11.75 g, 170 mmol) in water (82 mL) was then added, maintaining the internal temperature < 7 °C. The mixture was allowed to warm to room temperature overnight, diluted with ethyl acetate (500 mL) and the layers separated. The aqueous phase was extracted with ethyl acetate (2 x 500 mL), and the combined organic layers were dried over magnesium sulphate and concentrated under reduced pressure. The crude material was purified by reverse phase column chromatography (Biotage Isolera, C-18 3 x 120 g, 20-80% MeCN with formic acid in H20 0.1% formic acid). The relevant fractions were combined and extracted with dichloromethane (3 x 500 mL). The organics were dried over magnesium sulphate, and concentrated under reduced pressure to yield 3-(4-chlorophenyl)-2-hydroxypropanoic acid as a white powder, 1.74 g, 18%>.
UPLC (CSH C18, Short acid 2-95%): 0.62 min, 97.0 %, 199.0 (-ve, M-H)
1H NMR (CDCI3, 300 MHz): δ 7.32-7.17 (m, 4H), 4.51 (m, 1H), 3.23-2.90 (m, 2H)
Concentrated sulfuric acid (5 drops) was added to 3-(4-chlorophenyl)-2- hydroxypropanoic acid (650 mg, 3.2 mmol) in methanol (20 mL) and heated under reflux for 3 hours. The solution was cooled to room temperature then concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate (100 mL) and brine (100 mL), dried over magnesium sulphate, and concentrated under reduced pressure to yield methyl 3-(4-chlorophenyl)-2-hydroxypropanoate as a clear oil, 600 mg, 87 %.
UPLC (CSH_C18, Long acid 2-95 %): 0.72 min, 97.0 %
1H NMR (CDCI3, 300 MHz); δ 7.33-7.09 (m, 4H), 4.44 (t, 1H), 3.77 (s, 3H), 3.16-2.86 (m, 2H), 2.73 (s, 1H)
Synthesis of (2-Acetamidoethyl) 3-(4-chlorophenyl)-2-hydroxypropanethioate
Dicyclohexylcarbodiimide (660 mg, 3.2 mmol) and 1-hydroxybenzotriazole hydrate (432 mg, 3.2 mmol) were added to a solution of 3-(4-chlorophenyl)-2-hydroxypropanoic acid (650 mg, 3.2 mmol) and N-acetylcysteamine (1.16 g, 9.7 mmol) in acetonitrile (26 mL) and stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate (130 mL) and water (65 mL), filtered through celite, and the layers separated. The organics were washed with water (65 mL), then brine (65 mL), dried over magnesium sulphate and concentrated under
reduced pressure. The crude material was purified by normal phase chromatography (0-5% methanol in dichloromethane). The fractions containing the desired product were concentrated under reduced pressure, re-dissolved in ethyl acetate, washed with 1M HCl, then brine, dried over magnesium sulphate and concentrated under reduced pressure. The resulting solid was then triturated with diethyl ether to give (2-acetamidoethyl) 3-(4-chlorophenyl)-2- hydroxypropanethioate as a colourless solid, 387 mg, 40%.
UPLC (CSH_C18, Long acid 2-95 %): 1.54 min, 97.0 % 1H NMR (CDCls, 300 MHz); δ 7.32-7.26 (m, 2H), 7.20-7.15 (m, 2H), 5.66 (br s, 1H),
4.50-4.43 (m, 1H), 3.52-3.32 (m, 2H), 3.18-2.90 (m, 4H), 2.73 (d, 1H), 1.95 (s, 3H).
Claims
We claim:
1. A process for the roduction of a PF1022A derivative com ound of Formula I:
R1 and R2 are each independently selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; comprising: incubating a microorganism strain capable of producing PF1022A derivative compounds in a culture medium to produce a resulting culture, said culture medium containing carbon and nitrogen sources, and further providing to said culture medium a feedstock comprising a compound of Formula II, Formula III, Formula IV or Formula V:
I I I II IV V
wherein the dotted line represents an optional double bond;
X2, X3, and X4 are independently selected from N and CH; and
R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; and R3 is selected from the group consisting of hydrogen, amino, and sulfhydryl, thereby producing said PF1022A derivative compound in the resulting culture.
2. The process of claim 1 , wherein the feedstock comprises a compound of Formula II.
3. The process of claim 1 , wherein the feedstock comprises a compound of Formula II, and the compound of Formula II is a compound of Formula Ila:
Ila
R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
4. The process of claim 1 , wherein the feedstock comprises a compound of Formula III.
5. The process of claim 1 , wherein the feedstock comprises a compound of Formula IV.
6. The process of claim 1 , wherein the feedstock comprises a compound of Formula V.
7. The process of claim 6, wherein R2 is H.
8. The process of any one of claims 1-7, wherein R1 is selected from the group consisting halo, amino, hydroxyl, nitro, and morpholino.
9. The process of any one of claims 1-7, wherein R1 is halo.
10. The process of any one of claims 1-7, wherein R1 is fluoro.
11. The process of any one of claims 1-7, wherein R1 is chloro.
12. The process of any one of claims 1-7, wherein R1 is bromo.
13. The process of claim 3, wherein the compound of Formula Ila is:
The process of claim 3, wherein the compound of Formula Ila
The process of claim 4, wherein the compound of Formula III
17. The process of any one of claims 1-16, further comprising recovering said PF1022A derivative compound from said culture.
18. The process of any one of claims 1-17, wherein the carbon source comprises a carbohydrate.
19. The process of any one of claims 1-18, wherein the nitrogen source comprises inorganic or organic nitrogen-containing compounds.
20. The process of any one of claims 1-19, wherein said microorganism strain is a fungal strain.
21. The process of claim 20, wherein said fungal strain belongs to the genus Xylaria or the genus Rosellinia.
22. The process of any one of claims 1-21, wherein said producing comprises accumulating said PF1022A derivative compound in the resulting culture.
23. The process of any one of claims 1-22, wherein said compound of Formula I is enantiomerically pure.
IV
or a salt thereof,
wherein R is halo.
, ι
The compound of claim 24, wherein R is bromo or chloro.
26. The compound of claim 24, wherein R1 is chloro.
27. Use of a compound of Formula IV:
or a salt thereof,
wherein R1 is halo;
in a method of producing a PF1022A derivative compound.
28. The use of claim 27, wherein the PF1022A derivative compound is a compound of Formula I:
R1 and R2 are each independently selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.
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