WO1994008563A9 - Novel class of phosphocholine derivatives having antifungal activity - Google Patents
Novel class of phosphocholine derivatives having antifungal activityInfo
- Publication number
- WO1994008563A9 WO1994008563A9 PCT/US1993/009623 US9309623W WO9408563A9 WO 1994008563 A9 WO1994008563 A9 WO 1994008563A9 US 9309623 W US9309623 W US 9309623W WO 9408563 A9 WO9408563 A9 WO 9408563A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- alkyl
- alkynyl
- alkenyl
- branched
- radicals
- Prior art date
Links
- YHHSONZFOIEMCP-UHFFFAOYSA-O Phosphocholine Chemical class C[N+](C)(C)CCOP(O)(O)=O YHHSONZFOIEMCP-UHFFFAOYSA-O 0.000 title claims abstract description 52
- 230000000843 anti-fungal Effects 0.000 title claims abstract description 21
- 241001635594 Chelonanthus alatus Species 0.000 claims abstract description 16
- 206010017533 Fungal infection Diseases 0.000 claims abstract description 12
- 125000000217 alkyl group Chemical group 0.000 claims description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 43
- 150000001875 compounds Chemical class 0.000 claims description 41
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 36
- 125000003342 alkenyl group Chemical group 0.000 claims description 32
- 125000000304 alkynyl group Chemical group 0.000 claims description 27
- 125000006239 protecting group Chemical group 0.000 claims description 27
- 239000000203 mixture Substances 0.000 claims description 24
- 229910052760 oxygen Inorganic materials 0.000 claims description 24
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims description 23
- 239000001301 oxygen Substances 0.000 claims description 23
- 125000004432 carbon atoms Chemical group C* 0.000 claims description 22
- 235000000346 sugar Nutrition 0.000 claims description 22
- 229910052717 sulfur Inorganic materials 0.000 claims description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- 125000000753 cycloalkyl group Chemical group 0.000 claims description 21
- NINIDFKCEFEMDL-UHFFFAOYSA-N sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 21
- 239000011593 sulfur Substances 0.000 claims description 21
- 241000196324 Embryophyta Species 0.000 claims description 20
- 229910052739 hydrogen Inorganic materials 0.000 claims description 20
- 239000001257 hydrogen Substances 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 19
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 claims description 18
- 229910052799 carbon Inorganic materials 0.000 claims description 17
- -1 poly(alkenyl) Chemical group 0.000 claims description 15
- 125000000392 cycloalkenyl group Chemical group 0.000 claims description 14
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 14
- 239000000727 fraction Substances 0.000 claims description 13
- 239000003429 antifungal agent Substances 0.000 claims description 12
- 150000002431 hydrogen Chemical class 0.000 claims description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 11
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 10
- 230000002538 fungal Effects 0.000 claims description 10
- 230000002194 synthesizing Effects 0.000 claims description 10
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- LRHPLDYGYMQRHN-UHFFFAOYSA-N n-butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 8
- 239000008194 pharmaceutical composition Substances 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 7
- 229910052736 halogen Inorganic materials 0.000 claims description 7
- 150000002367 halogens Chemical group 0.000 claims description 7
- 125000002252 acyl group Chemical group 0.000 claims description 6
- 238000001819 mass spectrum Methods 0.000 claims description 6
- 230000003287 optical Effects 0.000 claims description 6
- 125000003282 alkyl amino group Chemical group 0.000 claims description 5
- 125000005210 alkyl ammonium group Chemical group 0.000 claims description 5
- 125000004663 dialkyl amino group Chemical group 0.000 claims description 5
- 125000005131 dialkylammonium group Chemical group 0.000 claims description 5
- OAKJQQAXSVQMHS-UHFFFAOYSA-O hydrazinium(1+) Chemical compound [NH3+]N OAKJQQAXSVQMHS-UHFFFAOYSA-O 0.000 claims description 5
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 claims description 5
- 125000004954 trialkylamino group Chemical group 0.000 claims description 5
- 125000005208 trialkylammonium group Chemical group 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 4
- 125000003545 alkoxy group Chemical group 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 125000004435 hydrogen atoms Chemical group [H]* 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 125000001820 oxy group Chemical group [*:1]O[*:2] 0.000 claims description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- GZCGUPFRVQAUEE-KCDKBNATSA-N D-(+)-Galactose Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-KCDKBNATSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 3
- PNNNRSAQSRJVSB-BXKVDMCESA-N Rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 claims description 3
- 125000000089 arabinosyl group Chemical class C1([C@@H](O)[C@H](O)[C@H](O)CO1)* 0.000 claims description 3
- 125000001589 carboacyl group Chemical group 0.000 claims description 3
- 150000002243 furanoses Chemical class 0.000 claims description 3
- 238000002523 gelfiltration Methods 0.000 claims description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N hydrogen Chemical group [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
- 238000007327 hydrogenolysis reaction Methods 0.000 claims description 3
- 238000002329 infrared spectrum Methods 0.000 claims description 3
- 125000000286 phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])* 0.000 claims description 3
- 150000004291 polyenes Polymers 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 150000008163 sugars Chemical class 0.000 claims description 3
- WQZGKKKJIJFFOK-PHYPRBDBSA-N α-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 claims description 3
- 230000035693 Fab Effects 0.000 claims description 2
- 206010061217 Infestation Diseases 0.000 claims description 2
- 229920000126 Latex Polymers 0.000 claims description 2
- 238000005481 NMR spectroscopy Methods 0.000 claims description 2
- 125000003302 alkenyloxy group Chemical group 0.000 claims description 2
- 125000004390 alkyl sulfonyl group Chemical group 0.000 claims description 2
- 125000004414 alkyl thio group Chemical group 0.000 claims description 2
- 125000005133 alkynyloxy group Chemical group 0.000 claims description 2
- 125000004391 aryl sulfonyl group Chemical group 0.000 claims description 2
- 125000005110 aryl thio group Chemical group 0.000 claims description 2
- 150000001793 charged compounds Chemical class 0.000 claims description 2
- 238000004440 column chromatography Methods 0.000 claims description 2
- 238000005227 gel permeation chromatography Methods 0.000 claims description 2
- 150000002314 glycerols Chemical class 0.000 claims description 2
- 230000012010 growth Effects 0.000 claims description 2
- 239000004816 latex Substances 0.000 claims description 2
- 125000003396 thiol group Chemical class [H]S* 0.000 claims description 2
- 230000000865 phosphorylative Effects 0.000 claims 3
- YAMUFBLWGFFICM-PTGWMXDISA-N 1-O-oleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C YAMUFBLWGFFICM-PTGWMXDISA-N 0.000 claims 2
- 241001465754 Metazoa Species 0.000 claims 1
- 230000002152 alkylating Effects 0.000 claims 1
- 150000001412 amines Chemical class 0.000 claims 1
- 239000012223 aqueous fraction Substances 0.000 claims 1
- 230000001279 glycosylating Effects 0.000 claims 1
- 150000004820 halides Chemical class 0.000 claims 1
- 125000001188 haloalkyl group Chemical group 0.000 claims 1
- 238000001228 spectrum Methods 0.000 claims 1
- 230000001225 therapeutic Effects 0.000 abstract 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 77
- 239000000243 solution Substances 0.000 description 55
- XEKOWRVHYACXOJ-UHFFFAOYSA-N acetic acid ethyl ester Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 51
- RTZKZFJDLAIYFH-UHFFFAOYSA-N diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 48
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 48
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene dichloride Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 48
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 42
- 239000011541 reaction mixture Substances 0.000 description 31
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 29
- 239000008079 hexane Substances 0.000 description 28
- 239000000741 silica gel Substances 0.000 description 23
- 229910002027 silica gel Inorganic materials 0.000 description 23
- 238000005160 1H NMR spectroscopy Methods 0.000 description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 22
- 239000010410 layer Substances 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 19
- 239000003921 oil Substances 0.000 description 19
- 238000003818 flash chromatography Methods 0.000 description 18
- 239000007787 solid Substances 0.000 description 18
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 17
- 238000003786 synthesis reaction Methods 0.000 description 16
- WVDDGKGOMKODPV-UHFFFAOYSA-N benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 15
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 14
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
- 239000012044 organic layer Substances 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- FPGGTKZVZWFYPV-UHFFFAOYSA-M Tetra-n-butylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- 238000007792 addition Methods 0.000 description 11
- OKKJLVBELUTLKV-MZCSYVLQSA-N cd3od Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 10
- 239000012230 colorless oil Substances 0.000 description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L mgso4 Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 10
- BZKBCQXYZZXSCO-UHFFFAOYSA-N sodium hydride Chemical compound [H-].[Na+] BZKBCQXYZZXSCO-UHFFFAOYSA-N 0.000 description 9
- RAXXELZNTBOGNW-UHFFFAOYSA-N Imidazole Chemical compound C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 229960000583 Acetic Acid Drugs 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000002829 reduced Effects 0.000 description 7
- 238000004679 31P NMR spectroscopy Methods 0.000 description 6
- UHOVQNZJYSORNB-UHFFFAOYSA-N benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- 239000003054 catalyst Substances 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- SJRJJKPEHAURKC-UHFFFAOYSA-N n-methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium on carbon Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 5
- 235000019445 benzyl alcohol Nutrition 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 5
- 239000012141 concentrate Substances 0.000 description 5
- 150000002009 diols Chemical class 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 description 5
- AFABGHUZZDYHJO-UHFFFAOYSA-N 2-Methylpentane Chemical class CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 4
- 241000222122 Candida albicans Species 0.000 description 4
- 241001661836 Irlbachia Species 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- 230000002255 enzymatic Effects 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 235000019341 magnesium sulphate Nutrition 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000010189 synthetic method Methods 0.000 description 4
- OGOMAWHSXRDAKZ-BKJHVTENSA-N (3R,4S,5R,6R)-3,4,5-tris(phenylmethoxy)-6-(phenylmethoxymethyl)oxan-2-ol Chemical compound C([C@H]1OC([C@@H]([C@@H](OCC=2C=CC=CC=2)[C@@H]1OCC=1C=CC=CC=1)OCC=1C=CC=CC=1)O)OCC1=CC=CC=C1 OGOMAWHSXRDAKZ-BKJHVTENSA-N 0.000 description 3
- UPQQXPKAYZYUKO-UHFFFAOYSA-M 2,2,2-trichloroethanimidate Chemical compound [O-]C(=N)C(Cl)(Cl)Cl UPQQXPKAYZYUKO-UHFFFAOYSA-M 0.000 description 3
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 3
- 241001225321 Aspergillus fumigatus Species 0.000 description 3
- 241000221204 Cryptococcus neoformans Species 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- PSHKMPUSSFXUIA-UHFFFAOYSA-N N,N-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 3
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- 239000012507 Sephadex™ Substances 0.000 description 3
- 210000003491 Skin Anatomy 0.000 description 3
- GETQZCLCWQTVFV-UHFFFAOYSA-N Trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 201000009910 diseases by infectious agent Diseases 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- BDAGIHXWWSANSR-UHFFFAOYSA-N formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000012362 glacial acetic acid Substances 0.000 description 3
- 238000005858 glycosidation reaction Methods 0.000 description 3
- QWZBEFCPZJWDKC-UHFFFAOYSA-N hexadecanoyl hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OC(=O)CCCCCCCCCCCCCCC QWZBEFCPZJWDKC-UHFFFAOYSA-N 0.000 description 3
- 238000005984 hydrogenation reaction Methods 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N iso-propanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000012452 mother liquor Substances 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L na2so4 Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 3
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- 238000001556 precipitation Methods 0.000 description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 3
- 235000011152 sodium sulphate Nutrition 0.000 description 3
- BCNZYOJHNLTNEZ-UHFFFAOYSA-N tert-butyldimethylsilyl chloride Chemical compound CC(C)(C)[Si](C)(C)Cl BCNZYOJHNLTNEZ-UHFFFAOYSA-N 0.000 description 3
- WJKHJLXJJJATHN-UHFFFAOYSA-N triflic anhydride Chemical compound FC(F)(F)S(=O)(=O)OS(=O)(=O)C(F)(F)F WJKHJLXJJJATHN-UHFFFAOYSA-N 0.000 description 3
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 2
- NYKRTKYIPKOPLK-UHFFFAOYSA-N 1-bromo-2-dichlorophosphoryloxyethane Chemical compound ClP(Cl)(=O)OCCBr NYKRTKYIPKOPLK-UHFFFAOYSA-N 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L Copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
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- 102000004357 Transferases Human genes 0.000 description 2
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical group [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
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- 230000000875 corresponding Effects 0.000 description 2
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- KZMGYPLQYOPHEL-UHFFFAOYSA-N ethoxyethane;trifluoroborane Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 description 2
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- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
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- 150000003839 salts Chemical class 0.000 description 2
- 150000003333 secondary alcohols Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- YXFVVABEGXRONW-UHFFFAOYSA-N toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 2
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- YCNUIKXUDLHAPA-GICYAKJESA-N 3-[(2R,3R,4S,5R,6R)-3,4,5-tris(phenylmethoxy)-6-(phenylmethoxymethyl)oxan-2-yl]oxypropane-1,2-diol Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H](OCC=2C=CC=CC=2)[C@@H]1OCC=1C=CC=CC=1)OCC=1C=CC=CC=1)OCC(O)CO)OCC1=CC=CC=C1 YCNUIKXUDLHAPA-GICYAKJESA-N 0.000 description 1
- YCNUIKXUDLHAPA-ZXWXWWAESA-N 3-[(2R,3S,4S,5R,6R)-3,4,5-tris(phenylmethoxy)-6-(phenylmethoxymethyl)oxan-2-yl]oxypropane-1,2-diol Chemical compound C([C@H]1O[C@H]([C@H]([C@@H](OCC=2C=CC=CC=2)[C@@H]1OCC=1C=CC=CC=1)OCC=1C=CC=CC=1)OCC(O)CO)OCC1=CC=CC=C1 YCNUIKXUDLHAPA-ZXWXWWAESA-N 0.000 description 1
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- 206010040943 Skin ulcer Diseases 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N Tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
- JBWKIWSBJXDJDT-UHFFFAOYSA-N Triphenylmethyl chloride Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(Cl)C1=CC=CC=C1 JBWKIWSBJXDJDT-UHFFFAOYSA-N 0.000 description 1
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- CGWVAQMLSHXXQE-GASJEMHNSA-N [(3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] 2,2,2-trichloroethanimidate Chemical compound OC[C@H]1OC(OC(=N)C(Cl)(Cl)Cl)[C@H](O)[C@@H](O)[C@@H]1O CGWVAQMLSHXXQE-GASJEMHNSA-N 0.000 description 1
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- 238000009903 catalytic hydrogenation reaction Methods 0.000 description 1
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- 238000009792 diffusion process Methods 0.000 description 1
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- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
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- 238000002955 isolation Methods 0.000 description 1
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- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
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- 125000001117 oleyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000001312 palmitoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000001717 pathogenic Effects 0.000 description 1
- 239000000546 pharmaceutic aid Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 150000003138 primary alcohols Chemical class 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001681 protective Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
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- 238000000844 transformation Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
Definitions
- This invention relates to new classes of
- phosphocholine derivatives as well as to various methods for preparing these compounds -- including synthetic, enzymatic and extractive using certain plants.
- inventions are non-toxic and exhibit substantial antifungal activity in slowing fungal growth and in killing fungi.
- the plant species Irlbachia alata has been used as an anti-infective agent in the Peruvian Amazon region.
- the leaves are squeezed and the liquid is applied to infected skin sores.
- the same liquid from the leaves is applied to skin problems and skin fungal infections. It is utilized to treat vaginal yeast infections.
- Irlbachia alata is one species of 10-12 species of the plant family Gentianaceae. These species occur in tropical South America especially in the Amazon and Negro River basins.
- the plants in the genus Irlbachia are generally low herbs characteristically with 3-5 plinerved leaves. The most consistent diagnostic feature for the genus is the pollen morphology.
- these compounds are phosphocholine derivatives (1 or 2-deacyl-phosphatidyl cholines) in which the 1 or 2-OH-group of the glycerol moiety has been glycosylated with glucose, galactose, arabinose, mannose, rhamnose or another sugar.
- R or R' is a sugar moiety and the other is an acyl or sugar moiety.
- the molecular backbone common to all members of this class of compounds is drawn above.
- the acyl- group can be any long-chain fatty acid, while the sugar unit can be any of the sugars commonly found in plants, including but not limited to glucose,
- galactose arabinose, mannose, rhamnose, or another naturally occurring sugar.
- Q is C2 to C30 alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, or branched alkynyl;
- Z is oxygen or sulfur;
- X and Y are independent oxygen, sulfur, CH 2 , CF 2 , or N-R 1 ;
- A, B, and T are independently alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, or branched alkynyl radicals of C1 to C20 chain lengths; are independently or together cycloalkyl or bridged cycloalkyl radicals of ring size C3 to C20, or
- D is oxygen, sulfur, CH 2 , CF 2 , or N-R 2 ;
- F is alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl or cycloalkynyl radicals containing C1 to C20 carbon atoms;
- R 1 and R 2 are independently hydrogen, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl, bridged cycloalkenyl or cycloalkynyl radicals containing C1 to C20 carbon atoms, or any protecting group described in the book "Protecting Groups in Organic Synthesis" by Theodora Greene and Peter G.M. Wuts.
- the central carbon atom can be either the R and S optical stereoisomer or a mixture of R and S
- AA is A-J with A being attached to the carbon atom of the three carbon central unit and J is defined below;
- BB is B-Y, with B being attached to the carbon atom of the three carbon central unit and Y is defined below:
- A is oxygen, sulfur, CH 2 , CF 2 or N-R 1 ;
- B is oxygen, sulfur, CH 2 , CF 2 or N-R 2 ;
- D is oxygen, sulfur, CH 2 , CF 2 or N-R 3 ;
- Y is alkyl, alkenyl, alkynyl, poly(alkenyl), poly(alkynyl), or poly(alkenoalkynyl) radicals
- poly(alkyn)oyl or poly(alkenoalkyn)oyl radicals comprised of C2 to C20 chain lengths or alkyloxy, alkenyloxy, alkynyloxy, poly(alkenyl)oxy,
- X is oxygen, sulfur, CH 2 , CF 2 or N-R 4 ;
- F, K, L and M are independently hydrogen, hydroxyl, protected hydroxyl (as described in the book “Protecting Groups in Organic Synthesis” by Theodora Greene and Peter G.M. Wuts), alkyloxy, thiol,
- alkylthio arylthio, alkylsulfonyl, arylsulfonyl, amino, ammonium, alkylamino, alkylammonium,
- dialkylamino dialkylammonium, trialkylamino
- trialkylammonium where the alkyl chain on nitrogen is comprised of C1 to C20 carbon atoms; or alkyl, alkenyl, or alkynyl radicals comprised of C1 to C20 carbon atoms.
- Z is oxygen or sulfur
- E is oxygen, sulfur, CH 2 CF 2 or N-R 5 ;
- G is alkyl, branched alkyl, cycloalkyl or bridged cycloalkyl radicals of C1 to C20 chain lengths;
- Q is halogen, hydroxyl, protected hydroxyl utilizing any protecting groups described in the book “Protecting Groups in Organic Synthesis” by Theodora Greene and Peter G.M. Wuts, O-arylsulfonyl-, O- alkylsulfonyl- or O-(perfluoroalkyl)sulfonyloxy, amino, ammonium, alkylamino, alkylammonium,
- dialkylamino dialkylammonium, trialkylamino
- R 1 , R 2 , or R 3 can independently or together be a mixture of alkyl groups of C1 to C20 in chain length and a protecting group described in the book "Protecting Groups in Organic Synthesis" by Theodora Green and Peter G.M. Wuts, and R 1 can equal R 2 , R 2 can equal R 3 , or R 1 can equal R 3 which can equal R3;
- R 1 , R 2 , R 3 , R 4 and R 5 are independently alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl or cycloalkynyl radicals of C1 to C20 chain lengths, or any protecting group described in the book "Protecting Groups in Organic Synthesis" by Theodora Greene and Peter G.M. Wuts;
- Class III of phosphocholine derivatives have the following structures:
- R 1 is phenyl or phenylmethyl, hydrogen, or nil
- R 2 is hydrogen, phenylmethyl, or any protecting group described in the book "Protecting Group in
- AA, BB, and Q are as defined above
- R 1 is phenyl or phenylmethyl, hydrogen, or nil
- R 2 is hydrogen, phenyl methyl or any protecting group described in the book “Protecting Groups in Organic Synthesis” by Theodora Greene and Peter G.M. Wuts which can be cleaved by hydrogenolysis;
- R 3 is hydrogen or a protecting group as described in the book “Protecting Groups in Organic Synthesis” by Theodora Greene and Peter G.M. Wuts.;
- central carbon atom of the three carbon unit is either the R optical isomer, the S optical isomer, or any mixture of the two optical isomers thereof; and Q is defined above.
- Still another preferred subgroup of the above- described Class III of phosphocholine derivatives have the following structures: where R 1 is phenyl or phenylmethyl, hydrogen, or nil;
- R 2 is a protecting group as described in the book
- Fig. 1 is the FTIR spectrum of the composition comprising a phosphocholine derivative obtained from
- Fig. 2 is the proton NMR spectrum of the
- composition comprising a phosphocholine derivative obtained from Irlbachia alata in D 2 O at 400 mHz.
- Fig. 3 is the FAB-/MB mass spectrum of the composition comprising a phosphocholine derivative obtained from Irlbachia alata .
- the glysosylated lysolecithins of the invention can be prepared by synthetic methods or by enzymatic methods.
- the phosphocholine derivatives can be prepared either by synthetic methods or by methods entailing extraction from plant materials.
- benzyl ethers or the benzilidine moiety are the preferred protecting group, since they can be selectively removed by catalytic hydrogenation, while leaving the sensitive acyl-glycerol linkage intact.
- the glycosidation requires silver, mercury (Helferich modification), or cadmium salts as catalytic halogen abstractor, in the presence of a dehydrating agent (Timell, T.E., Can .J. Chem . 1964, 42., 1456; Dejter-Juszynsky, M. and
- 2,3,4,6-Tetrabenzyl- 2,3-dibenzyl-4,6-benzylidene- glucose can be converted into the 1-bromo- or 1-O- triflate compound by standard methodology.
- the corresponding phosphocholine derivatives are available through AVANTI POLAR LIPIDS, Inc. All other reagents are available from ALDRICH.
- the methodology outlined above is also applicable to either 1-acyl or 2-acyl (1-acyl detailed above). 5.2.
- glycosidation catalysts are glycosyltransferases.
- a general synthetic method of synthesizing phosphocholine derivatives of the various structures described in section 3 is outlined as follows.
- deprotected product is then alkylated or esterified to produce the phosphocholine derivatives.
- the general scheme for this outlined synthetic method is shown below.
- Glycerol Derivative can be either the R or S optical isomer, racemic, or a mixture of R and S isomers Implies that a number of synthetic transformations are required
- R 1 Sugar, carbocyclic sugar, functionalized sugar derivative, etc.
- R 2 Phosphate or phosphate isostere moiety
- R 3 alkyl, alkanoyl, alkenyl, alkenoyl, etc.
- X, Y, and Z can be C, O, N, S independently or equal to each other 5.4.
- the phosphocholine derivative in Classes I, II and III are all useful in treating fungal infection by the administration to a warm-blooded animal of a therapeutically effective amount of a phosphocholine derivative.
- the pharmaceutical composition comprising the phosphocholine derivative used for such
- administration may also contain pharmaceutically acceptable excipients and carriers.
- antifungal agent of Classes I, II and III may be administered to a warm-blooded animal intravenously, intraperitoneally, subcutaneously, intramuscularly, orally, topically, by aerosol, or combinations
- the fungal agent of Class II can be administered intraperitoneally in a range of about 0.1 to about 10 mg/kg.
- the fungal agent of Class II can be administered subcutaneously in a range of about 1 to about 20.
- the fungal agent of Class II can be administered intramuscularly in a range of about 1 to about 20.
- the fungal agent of Class II can be administered orally in a range of about 5.0 to about 30 mg/kg.
- the fungal agent of Class II can be administered topically in a range of about.5.0 to about 15% by weight.
- the fungal agent of Class II can be administered by aerosol in a range of about 5.0 to about 30
- Plants are not known to contain phosphocholine derivatives.
- the plant source material such as the whole plant, the roots, leaves, stem and/or latex of the plant, is extracted with water and/or a water miscible solvent.
- the preferred solvents are alcohol of 1-3 carbon atoms or acetone.
- the aqueous extract is extracted with butanol.
- the butanol-soluble fraction is subjected to gel filtration (e.g., over Sephadex), reversed-phase column chromatography (e.g., C-8), or gel-permeation chromatography (e.g., divinyl benzene cross-linked gels) such as PL-GEL or membranes (e.g., an Amicon membrane) using water or water and a water miscible solvent, with or without a buffer, as the mobile phase.
- the water miscible solvent is
- the useful phosphocholine derivatives containing compound is the fraction detected by NMR spectroscopy.
- a specific member of the class of phosphocholine derivatives of the present invention is 2-palmitoyl-1-O-glycopyranosyllysolecithin shown below:
- 2-palmitoyl-1-O- glucopyranosyllysolecithin is a relatively active antifungal agent similar in activity to
- the extracted material was separated from the marc (i.e., residual of the plant after solvent extraction) and discarded.
- the marc was then extracted with 1.5L of isopropanol/water (1:1 v/v) at room temperature for 24 hours. The marc was separated from the extract and discarded.
- isopropanol/water (1:1 v/v) soluble extract was partitioned between water and ethyl acetate.
- the ethyl acetate phase was separated and discarded.
- the water soluble phase after extraction with n-butanol, was then discarded.
- the n-butanol phase was subjected to filtration over two Sephadex LH-20 gel columns using 90% aqueous ethanol (for first filtration) and 20% aqueous acetone (for second filtration) as the mobile phases.
- 1,22-docosandiol bisphosphocholine ester was collected from the early fractions of each gel filtration.
- the isolated phosphocholine derivative fraction containing 1,22-docosandiol bisphosphocholine ester has the characteristic IR, proton NMR and FAB- mass spectra shown in Figs. 1, 2 and 3, respectively.
- the IR spectrum has peaks at approximately 1060, 1220, 1475, 1600-1700, 2850, 2950 and 3400 cm -1 .
- the 1 H NMR spectrum has major peaks at ⁇ 1.2, 1.4, 1.7, 3.1, 3.5, 3.7 and 4.3.
- the FAB-/MB mass spectrum has major peaks (>40%) at m/z 657, 612, 587, 586, 555, 493, 491, 475, 403, 277, 233, 201, 194, 179, 168, 165 and 163.
- Tetrahydrofuran was distilled from potassium/benzophenone; benzene, triethylamine, and methylene chloride, N-methylmorpholine, and benzyl alcohol were distilled from calcium hydride; 2- bromoethylphosphorodichloridate was prepared according to the procedure reported by Baumann et al Lipids, 17, 453 (1982) and was freshly distilled prior to use;
- NMR coupling constants are reported in Hertz. Melting points were determined using a Buchi model 535 melting point apparatus and are uncorrected.
- Trifluoromethanesulfonic anhydride (1.25 mL, 7.6 mmol) was then added, and the reaction was stirred for 30 minutes with the temperature maintained at 5°C. The solution was then filtered through a bed of silica. The filtrate was concentrated under reduced pressure at 30°C to give an orange/brown oil (1.84 g, 7.0 mmol) in 92% yield which was used directly for the next step.
- 2,3,4,6-Tetra-O-benzyl-D-glucopyranose 100 g, 0.182 mol was dissolved in THF (1.4 L) and chilled to -10°C in a nitrogen-purged 3-L three-necked morton flask fitted with a thermometer, stopper, and
- Procedure B Compound 4 (500 mg, 0.52 mmol) was dissolved in THF (20 mL) in a 100-mL three-necked round-bottomed flask fitted with two stoppers and a septum. Glacial acetic acid (9.5 mL) was added, and the solution was chilled to 0°C. A solution of TBAF (5.16 mL, 1.0 M in THF) was syringed into the chilled solution, and stinting was continued at 0°C for 8 h and then at rt for 25 hours.
- Procedure A In a nitrogen-purged 100-mL three-necked round-bottomed flask fitted with two stoppers and a septum was dissolved freshly distilled 2- bromoethylphosophorodichloridate (1.72 g, 7.11 mmol) in diethyl ether (20 mL). The solution was chilled to 0°C, and triethylamine (8.15 mL, 58.5 mmol) was injected into the solution which caused precipitation of a white solid. A solution of compound 5 (1.0 g, 1.17 mmol) in anhyd diethyl ether (55 ml) was injected into the chilled reaction mixture, and the ice bath was removed. The reaction was stirred for 30 minutes after which benzyl alcohol (1.21 mL, 11.7 mmol) was injected into the reaction mixture. Stirring was continued at rt for 5 d. The reaction was then
- reaction mixture was concentrated to a viscous oil and triturated with ethyl ether, upon which a white precipitate formed, the precipitate was filtered off, washed with ether, and then the combined ethereal solutions were concentrated to a glassy solid.
- Glacial acetic acid (13.8 mL 241 mmol) was then poured into the reaction mixture to quench the reaction, and the resulting solution was stirred for approximately 30 minutes.
- the reaction mixture was poured into a separatory funnel containing ice water (500 mL) and methylene chloride (200 mL). The layers were separated, and aqueous layer was extracted twice more with methylene chloride (100 mL portions) and then the combined organic layer was washed with brine (400 mL). Following dring (MgS04), filtration, and then concentration, purification by flash
- reaction was stirred at 55°C in an oil bath for 24 hours behind a blast shield.
- the bomb vessel was then cooled to -78°C, opened, and left in a hood to
- reaction mixture was evaporated to a small volume and separated by flash chromatography (silica gel, diethyl ether/hexane, 1:3) to give compound 17 as a colorless oil (120 mg, 22%), which was identical to the material described earlier.
- phosphocholine fraction was determined in vitro by using three fungal cultures -- Candida albicans,
- Cryptococcus neoformans and Aspergillus fumigatus .
- MIC minimum inhibitory concentration
- MFC minimum fungicidal concentration
- R is the group identified in table 2.
- the toxicity of the isolated phosphocholine derivative fraction is low, based on tests with Hep 2 cells indicating an ID 50 of greater than 1000 ug/ml.
- the method used in determining cytotoxicity is discussed in Mosmann, T., "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays", J. Immun . Methods, 65, 55-63, 1986.
- the isolated fraction having the above-described in vitro antifungal activity and low toxicity is expected to similarly exhibit significant in vivo antifungal activity against fungal infections which are dermatophytic, systemic, ophthalmic and vaginal.
- fungal infections which are dermatophytic, systemic, ophthalmic and vaginal.
- other human and animal infections treatable with the compounds of the present invention include
Abstract
Certain phosphocholine derivatives having substantial antifungal therapeutic activity are disclosed. The phosphocholine derivatives may be chemically synthesized, enzymatically prepared or extracted from the plant Irlbachia alata. The phosphocholine derivatives are useful in treating fungal infections including those which are dermatophytic, systemic, ophthalmic and vaginal.
Description
NOVEL CLASS OF PHOSPHOCHOLINE DERIVATIVES
HAVING ANTIFUNGAL ACTIVITY
This is a continuation-in-part application of U.S. patent application Serial No. 07/958,416, filed October 8, 1993, the entire disclosure of which is incorporated by reference.
1. Field of the Invention
This invention relates to new classes of
phosphocholine derivatives as well as to various methods for preparing these compounds -- including synthetic, enzymatic and extractive using certain plants. The phosphocholine derivatives of the
invention are non-toxic and exhibit substantial antifungal activity in slowing fungal growth and in killing fungi.
2. Background of the Invention
The plant species Irlbachia alata has been used as an anti-infective agent in the Peruvian Amazon region. The leaves are squeezed and the liquid is applied to infected skin sores. The same liquid from the leaves is applied to skin problems and skin fungal infections. It is utilized to treat vaginal yeast infections.
Irlbachia alata is one species of 10-12 species of the plant family Gentianaceae. These species occur in tropical South America especially in the Amazon and Negro River basins. The plants in the genus Irlbachia are generally low herbs characteristically with 3-5 plinerved leaves. The most consistent diagnostic feature for the genus is the pollen morphology.
A reference to Irlbachia alata and related species was made in 1775 by the French scientist Fusee
Aublet (Aublet, F. 1775, Histoire des Plantes de la Guiane Francoise, Didot, Paris). The ethnobotanical notes from this reference were subsequently compiled and republished in English. Aublet noted the
following about two species in the genus Irlbachia :
Irlbachia alata The entire plant is bitter.
It is used to clear obstructions; I (Aublet) have used it with good results. The species is called "Bois creux" (Hollow wood) by the Creoles.
Irlbachia pururascens All parts of this plant are bitter. It is used as an apertif and to reduce fever.
3. Summary of the Invention
We have discovered a class of phosphocholine derivatives (Class I) having extraordinary antifungal activity.
Structurally, these compounds are phosphocholine derivatives (1 or 2-deacyl-phosphatidyl cholines) in which the 1 or 2-OH-group of the glycerol moiety has been glycosylated with glucose, galactose, arabinose, mannose, rhamnose or another sugar. The basic
chemical structure may be drawn as follows:
The molecular backbone common to all members of this class of compounds is drawn above. The acyl- group can be any long-chain fatty acid, while the sugar unit can be any of the sugars commonly found in plants, including but not limited to glucose,
galactose, arabinose, mannose, rhamnose, or another naturally occurring sugar.
We have additionally found a structurally related class of phosphocholine derivatives of similar or greater antifungal activity than the above-discussed class of phosphocholine derivatives (i.e., Class I).
One novel class of phosphocholine derivatives (Class II) having antifungal activity has the basic structure shown below:
where Q is C2 to C30 alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, or branched alkynyl;
Z is oxygen or sulfur; X and Y are independent oxygen, sulfur, CH2, CF2, or N-R1;
A, B, and T are independently alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, or branched alkynyl radicals of C1 to C20 chain lengths; are independently or together cycloalkyl or bridged cycloalkyl radicals of ring size C3 to C20, or
cylcoalkenyl, bridged cycloalkenyl or
cyclo(polyene) radicals of ring size C4 to C20, cycloalkynyl, bridged cycloalkeynl or
cyclo(polyalkynyl) radicals of ring size C8 to C20;
D is oxygen, sulfur, CH2, CF2, or N-R2;
F is alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl or cycloalkynyl radicals containing C1 to C20 carbon atoms;
R1 and R2 are independently hydrogen, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl, bridged cycloalkenyl or cycloalkynyl radicals containing C1 to C20 carbon atoms, or any protecting group described in the book "Protecting Groups in Organic Synthesis" by Theodora Greene and Peter G.M. Wuts.
Another class of phosphocholine derivatives
(Class III) having antifungal activity has the
following structures:
where AA, BB, DD are independent of each other, equal to each other, or interchanged as shown above, the central carbon atom can be either the R and S
optical stereoisomer or a mixture of R and S
stereoisomers, and where AA, BB, and CC are defined as follows:
where AA, is A-J with A being attached to the carbon atom of the three carbon central unit and J is defined below;
BB is B-Y, with B being attached to the carbon atom of the three carbon central unit and Y is defined below:
DD is
A is oxygen, sulfur, CH2, CF2 or N-R1;
B is oxygen, sulfur, CH2, CF2 or N-R2;
D is oxygen, sulfur, CH2, CF2 or N-R3;
Y is alkyl, alkenyl, alkynyl, poly(alkenyl), poly(alkynyl), or poly(alkenoalkynyl) radicals
comprised of C1 to C20 carbon atoms chain lengths, or alkanoyl, alkenoyl, alkynoyl, poly(alken)oyl,
poly(alkyn)oyl or poly(alkenoalkyn)oyl radicals comprised of C2 to C20 chain lengths or alkyloxy, alkenyloxy, alkynyloxy, poly(alkenyl)oxy,
poly(alkynyl)oxy, poly(alkenoalkynyl)oxy radicals comprised of C1 to C20 carbon atoms;
J is a furanose or pyranose radical of the type:
where X is oxygen, sulfur, CH2, CF2 or N-R4;
F, K, L and M are independently hydrogen, hydroxyl, protected hydroxyl (as described in the book "Protecting Groups in Organic Synthesis" by Theodora Greene and Peter G.M. Wuts), alkyloxy, thiol,
alkylthio, arylthio, alkylsulfonyl, arylsulfonyl, amino, ammonium, alkylamino, alkylammonium,
dialkylamino, dialkylammonium, trialkylamino,
trialkylammonium where the alkyl chain on nitrogen is comprised of C1 to C20 carbon atoms; or alkyl, alkenyl, or alkynyl radicals comprised of C1 to C20 carbon atoms.
Z is oxygen or sulfur
E is oxygen, sulfur, CH2 CF2 or N-R5;
G is alkyl, branched alkyl, cycloalkyl or bridged cycloalkyl radicals of C1 to C20 chain lengths;
Q is halogen, hydroxyl, protected hydroxyl utilizing any protecting groups described in the book "Protecting Groups in Organic Synthesis" by Theodora Greene and Peter G.M. Wuts, O-arylsulfonyl-, O- alkylsulfonyl- or O-(perfluoroalkyl)sulfonyloxy, amino, ammonium, alkylamino, alkylammonium,
dialkylamino, dialkylammonium, trialkylamino,
trialkylammonium where the alkyl chains on nitrogen are C1 to C20, or Q=NR1R2R3, where R1, R2, or R3 can independently or together be a mixture of alkyl groups
of C1 to C20 in chain length and a protecting group described in the book "Protecting Groups in Organic Synthesis" by Theodora Green and Peter G.M. Wuts, and R1 can equal R2, R2 can equal R3, or R1 can equal R3 which can equal R3;
R1, R2, R3, R4 and R5 are independently alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl or cycloalkynyl radicals of C1 to C20 chain lengths, or any protecting group described in the book "Protecting Groups in Organic Synthesis" by Theodora Greene and Peter G.M. Wuts;
where W1 and W2 are P(-OR) (with R being phenyl, phenylmethyl, or negatively-charged oxygen), S=O, carbon, or sulfur, provided that if W1 is not P(-OR) W2 is P(-OR) and provided that if J is a furanose or pyranose radical then W1 is P(-OR).
A preferred subgroup of the above-described
Class III of phosphocholine derivatives have the following structures:
R2 is hydrogen, phenylmethyl, or any protecting group described in the book "Protecting Group in
Organic Synthesis" by Theodora Green and Peter G.M. Wuts which can be cleaved by hydrogenolysis;
AA, BB, and Q are as defined above
where the central carbon atom of the three carbon unit is either the R optical isomer, the S optical isomer, or any mixture of the two optical isomers thereof;
Another preferred subgroup of the above-described Class III of phosphocholine derivatives have the following structures:
where R1 is phenyl or phenylmethyl, hydrogen, or nil; R2 is hydrogen, phenyl methyl or any protecting group described in the book "Protecting Groups in Organic Synthesis" by Theodora Greene and Peter G.M. Wuts which can be cleaved by hydrogenolysis;
R3 is hydrogen or a protecting group as described in the book "Protecting Groups in Organic Synthesis" by Theodora Greene and Peter G.M. Wuts.;
where the central carbon atom of the three carbon unit is either the R optical isomer, the S optical isomer, or any mixture of the two optical isomers thereof; and Q is defined above.
Still another preferred subgroup of the above- described Class III of phosphocholine derivatives have the following structures:
where R1 is phenyl or phenylmethyl, hydrogen, or nil;
R2 is a protecting group as described in the book
"Protecting Groups in Organic Synthesis" by Theodora
Greene and Peter G.M. Wuts, or hydrogen if R1 is not hydrogen;
and Q is defined above.
We have further found a novel, generally
applicable method for the synthesis of the above described broad classes of phosphocholine derivatives
(Classes I, II and III). 4. Brief Description of the Drawings
Fig. 1 is the FTIR spectrum of the composition comprising a phosphocholine derivative obtained from
Irlbachia alata.
Fig. 2 is the proton NMR spectrum of the
composition comprising a phosphocholine derivative obtained from Irlbachia alata in D2O at 400 mHz.
Fig. 3 is the FAB-/MB mass spectrum of the composition comprising a phosphocholine derivative obtained from Irlbachia alata .
5. Detailed Description of the Invention
The glysosylated lysolecithins of the invention can be prepared by synthetic methods or by enzymatic methods. The phosphocholine derivatives can be
prepared either by synthetic methods or by methods entailing extraction from plant materials.
5.1. Chemical Synthesis of phosphocholine derivatives A wide variety of compounds having accessible alcoholic functionalities can be glycosylated
following the classic Koenigs-Knorr methodology.
Bochkov, A.F. and Zaikov. G.E., Chemistry of the O- Glycosidic Bond. Pergamon Press, 1979. As part of the synthetic route to phosphocholine derivatives with sugar, all but the anomeric hydroxyl group of the sugar to be introduced are protected either as esters or ethers, while the anomeric hydroxyl is being replaced by a halogen. The aglycon-sugar linkage is then formed via alcoholysis. Finally, the protective groups are selectively removed.
In the present invention, benzyl ethers or the benzilidine moiety are are the preferred protecting group, since they can be selectively removed by catalytic hydrogenation, while leaving the sensitive acyl-glycerol linkage intact. The glycosidation requires silver, mercury (Helferich modification), or cadmium salts as catalytic halogen abstractor, in the presence of a dehydrating agent (Timell, T.E., Can .J. Chem . 1964, 42., 1456; Dejter-Juszynsky, M. and
Flowers, H.M., Carbohydr. Res . 1973, 30, 287;
Marousek, V., Lucas, T.J., Wheat, P.E., and Schuerch, C., Carbohydr. Res . 1978, 6 0, 85), and with or without auxiliaries such as crown-ethers. (Knöchel,A. Ger, R., and Thiem, J. Tetrahedron Letters 1974, 551) More recent methodology makes use of the halogen- abstracting power of non-nucleophilic bases such as diisopropylethylamine and/or of molecular sieves in an anhydrous media. (Garegg, P.J. and Norberg, T., Carbohydr. Res . 1976, 52, 235) The following
synthetic scheme is based on the latter reaction sequence:
2-O-β-D-Glucopyranosyl lysolecithin
The synthetic two-step scheme outlined above can be conducted with commercially available materials.
2,3,4,6-Tetrabenzyl- 2,3-dibenzyl-4,6-benzylidene- glucose can be converted into the 1-bromo- or 1-O- triflate compound by standard methodology. Leroux, J. and Perlin, A.S. Carbohydr. Res . 1976, 47 , C8. The corresponding phosphocholine derivatives are available through AVANTI POLAR LIPIDS, Inc. All other reagents are available from ALDRICH. The methodology outlined above is also applicable to either 1-acyl or 2-acyl (1-acyl detailed above).
5.2. Enzymatic preparation of 1 or 2 glvcosylated lysolecithins
As an alternative to the synthetic sequence outlined above, an in vitro enzymatic glycosidation simulating the biosynthetic process will produce the desired compounds in comparable yields. The natural glycosidation catalysts are glycosyltransferases.
These enzymes operate with uridinediphospho-glycosides (UDP-sugars) as substrates and ATP as the energy source. While the enzymes have to be prepared from fresh plant material, UDP-sugars, ATP, as well as the respective phosphocholine derivatives are commercially available. This synthesis has the advantage of being essentially a one-step process with the high
selectivity and yields expected from an enzymatic reaction. The following scheme describes the
preparation of a glucoside. Other transferases, not specific to glucose, could be applied in the
preparation of glycosylated lysolecithins with other sugars as well:
R = Palmitoyl-, Oleyl-, Palmitoleyl-, etc.) Uridine-5 '-diphospho-glucose
(=UDP-glucose)
1)1-Glucosyl Transferase/ATP;Phosphate Buffer
2-O-β-D-Glucopyranosyl lysolecithin
5.3. Total Svnthesis of Phosphocholine Derivatives
A general synthetic method of synthesizing phosphocholine derivatives of the various structures described in section 3 is outlined as follows.
An alcohol is phosphorylated or glycosylated. The product is subsequently deprotected. The
deprotected product is then alkylated or esterified to produce the phosphocholine derivatives. The general
scheme for this outlined synthetic method is shown below.
General Scheme for the Synthesis of Known and Novel Lysolecithins
V
Glycerol Derivative
can be either the R or S optical isomer, racemic, or a mixture of R and S isomers Implies that a number of synthetic transformations
are required
R1 = Sugar, carbocyclic sugar, functionalized sugar derivative, etc. R2 = Phosphate or phosphate isostere moiety
R3 = alkyl, alkanoyl, alkenyl, alkenoyl, etc.
X, Y, and Z can be C, O, N, S independently or equal to each other
5.4. Methods of Use
The phosphocholine derivative in Classes I, II and III are all useful in treating fungal infection by the administration to a warm-blooded animal of a therapeutically effective amount of a phosphocholine derivative. The pharmaceutical composition comprising the phosphocholine derivative used for such
administration may also contain pharmaceutically acceptable excipients and carriers.
Phosphocholine derivatives in Classes I and
II are believed to be novel compositions.
In order to treat a fungal infection, the
antifungal agent of Classes I, II and III may be administered to a warm-blooded animal intravenously, intraperitoneally, subcutaneously, intramuscularly, orally, topically, by aerosol, or combinations
thereof.
The antifungal agent of phosphocholine
derivatives in Class II can be administered
intravenously in a range of about 0.1 to about 10 mg/kg.
The fungal agent of Class II can be administered intraperitoneally in a range of about 0.1 to about 10 mg/kg.
The fungal agent of Class II can be administered subcutaneously in a range of about 1 to about 20.
The fungal agent of Class II can be administered intramuscularly in a range of about 1 to about 20.
The fungal agent of Class II can be administered orally in a range of about 5.0 to about 30 mg/kg.
The fungal agent of Class II can be administered topically in a range of about.5.0 to about 15% by weight.
The fungal agent of Class II can be administered by aerosol in a range of about 5.0 to about 30
mg/kg/day.
The above dosage ranges may need to be doubled for those phosphocholine derivatives in Class I and III with lower antifungal activity which are identical or similar to those in table 2 (see below).
6. Extraction of phosphocholine derivatives
from plants
Plants are not known to contain phosphocholine derivatives.
The general manner of chemical extraction from the plants can be summarized as follows.
The plant source material, such as the whole plant, the roots, leaves, stem and/or latex of the plant, is extracted with water and/or a water miscible solvent. The preferred solvents are alcohol of 1-3 carbon atoms or acetone. The aqueous extract is extracted with butanol. The butanol-soluble fraction is subjected to gel filtration (e.g., over Sephadex), reversed-phase column chromatography (e.g., C-8), or gel-permeation chromatography (e.g., divinyl benzene cross-linked gels) such as PL-GEL or membranes (e.g., an Amicon membrane) using water or water and a water miscible solvent, with or without a buffer, as the mobile phase. The water miscible solvent is
preferably a 1-3 carbon alcohol, acetone or
acetonitrile.
The useful phosphocholine derivatives containing compound is the fraction detected by NMR spectroscopy.
A specific member of the class of phosphocholine derivatives of the present invention is
2-palmitoyl-1-O-glycopyranosyllysolecithin shown below:
2 -PaImitoyI-1-O-glucopyranosyllysolecithin
We have found that 2-palmitoyl-1-O- glucopyranosyllysolecithin is a relatively active antifungal agent similar in activity to
L-a-Lysophosphatidyl inositol, discussed in Table 2 below.
We have found that one of the most active antifungal compounds has the following structure.
1,22-docosan diol bisphosphocholine ester.
6.1. Extraction
We have isolated by chemical extraction 1,22- docosan diol bisphosphocholine ester, the active antifungal compound contained in the plant Irlbachia alata . The leaves of Irlbachia alata were milled and 200g of the milled leaves was extracted with 1L of dichloromethane/isopropanol (1:1 v/v) at room
temperature for 24 hours. The extracted material was separated from the marc (i.e., residual of the plant after solvent extraction) and discarded. The marc was then extracted with 1.5L of isopropanol/water (1:1 v/v) at room temperature for 24 hours. The marc was separated from the extract and discarded. The
isopropanol/water (1:1 v/v) soluble extract was partitioned between water and ethyl acetate. The ethyl acetate phase was separated and discarded. The water soluble phase, after extraction with n-butanol, was then discarded. The n-butanol phase was subjected to filtration over two Sephadex LH-20 gel columns using 90% aqueous ethanol (for first filtration) and 20% aqueous acetone (for second filtration) as the mobile phases. 1,22-docosandiol bisphosphocholine ester was collected from the early fractions of each gel filtration.
We believe that several related genera are the same and/or closely related to the genus Irlbachia , and would have similar medicinal properties. One species from a closely related genus, Lisianthus nigrens is used in Mexico. The leaves are applied as a poultice to treat fungal infections of the skin, feet, ankles and hands. A decoction of the root is also taken orally as a "bitter" and as a febrifuge. Another species Lisianthus alatus is considered to be the same as Irlbachia alata . Another species and genus of interest is Chelonanthus alatus . There are
several uses described for Chelonanthus alatus , including oral decoctions to treat smallpox, fevers and for gastric disturbances. 6.2. Spectral Characteristics
The isolated phosphocholine derivative fraction containing 1,22-docosandiol bisphosphocholine ester has the characteristic IR, proton NMR and FAB- mass spectra shown in Figs. 1, 2 and 3, respectively.
The IR spectrum has peaks at approximately 1060, 1220, 1475, 1600-1700, 2850, 2950 and 3400 cm-1.
The 1H NMR spectrum has major peaks at δ 1.2, 1.4, 1.7, 3.1, 3.5, 3.7 and 4.3.
The FAB-/MB mass spectrum has major peaks (>40%) at m/z 657, 612, 587, 586, 555, 493, 491, 475, 403, 277, 233, 201, 194, 179, 168, 165 and 163.
The high resolution mass spectrum (FAB+) has a molecular ion at 673.4669 amu. 6.3. Total Synthesis of 2-palmitoyl-1-O- glucopyranosyllysolecithin Experimental Section
General. Tetrahydrofuran (THF) was distilled from potassium/benzophenone; benzene, triethylamine, and methylene chloride, N-methylmorpholine, and benzyl alcohol were distilled from calcium hydride; 2- bromoethylphosphorodichloridate was prepared according to the procedure reported by Baumann et al Lipids, 17, 453 (1982) and was freshly distilled prior to use;
trifluromethanesulfonic anhydride was freshly
distilled under inert atmosphere; O-α-D-
(Glucopyranosyl) trichloroacetimidate was prepared by the method of Schmidt. (a) R. R. Schmidt, J. Michael, Angew. Chem . Int . Ed Engl . (1980), 19, 731; (b) R. R. Schnmidt, J. Michael, Tetrahedron Lett . (1984), 25, 821. Anhydrous dimethylformamide (DMF) was obtained
from Aldrich. S-(+)-1,2-0-isopropylidene glycerol and R-(-)-1,2-O-isopropylidene glycerol were obtained from Lancaster. 2,3,4, 6-Tetra-O-benzyl-D-glucopyranose was obtained from Sigma. Preparative thin layer
chromatography plates was performed on Whatman 2000 μ TLC silica gel plates. Flash column chromatography was performed on Whatman 230-400 mesh silica gel using nitrogen pressure. 1H and13C NMR were provided by using a Varian 400 MHz spectrometer with chloroform as an internal reference unless otherwise noted. NMR shifts were expressed in ppm downfield from internal
tetramethylsilane. Carbon 13 multiplicities as determined by DEPT experiments are reported in
parentheses following the chemical shift value
according to the following format: (0) for quaternary carbon, (1) for methine carbon, (2) for methylene carbon, and (3) for methyl carbons. NMR assignments were determined on the basis of COSY, HMQC, and HMBC and DEPT experiments performed on selected
intermediates. NMR coupling constants are reported in Hertz. Melting points were determined using a Buchi model 535 melting point apparatus and are uncorrected.
The synthetic routes for the total synthesis of 2-palmitoyl-1-0-glucopyranosyllysolecithin are
outlined in the following diagrams and detailed in the subsequent discussion that refer to these diagrams.
Scheme 1. Synthesis of the (S) SP-19501 : Preparation of the Regicisomeric Glycerol Alcohols
Regioisomeric Glycerol Alcohols
S
(R) 2,3-O-Isopropylidene-1-O-trifluromethylsulfonyl— glycerol. A nitrogen-purged 250-mL three-necked roundbottomed flask fitted with a thermometer, stopper, and septum was charged with S-(+) -1,2-0- isopropylidene glycerol (1.0 g, 7.6 mmol) dissolved in benzene (75 ml). Triethylamine (1.25 mL, 9.0 mmol) was injected into the solution, and the reaction mixture was chilled until a cloudy solution appeared. Trifluoromethanesulfonic anhydride (1.25 mL, 7.6 mmol) was then added, and the reaction was stirred for 30 minutes with the temperature maintained at 5°C. The solution was then filtered through a bed of silica. The filtrate was concentrated under reduced pressure at 30°C to give an orange/brown oil (1.84 g, 7.0 mmol) in 92% yield which was used directly for the next step.
(2R) [1-O-(2,3,4,,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2,3-0-isopropylidene] glycerol 1
2,3,4,6-Tetra-O-benzyl-D-glucopyranose (100 g, 0.182 mol) was dissolved in THF (1.4 L) and chilled to -10°C in a nitrogen-purged 3-L three-necked morton flask fitted with a thermometer, stopper, and
mechanical stirrer. Sodium hydride 60% in oil (16.1 g, 0.403 mol) was added in 4 increments over 10 minutes, and the solution was stirred for 30 minutes. (R) 2,3-O-Isopropylidene-1-O- trifluoromethylsulfonylglycerol (60.0 g, 0.227 mol) dissolved in THF (500 mL) was then dropped via an addition funnel into the reaction mixture over a 30 minute period. The solution was stirred at -10°C for 7 hours. Methanol (200 mL) was added dropwise to quench excess sodium hydride, the resulting brown solution was rotary evaporated under reduced pressure
and then the residue redissolved in chloroform (750 mL). The organic layer was washed with water (2 x 750 mL). The combined aqueous layers were washed with chloroform (3 x 500 mL). Organic layers were pooled and rotary evaporated under reduced pressure to give a white solid which contained both α and β-epimers of the desired product. The solid was triturated with diethyl ether to give a white solid of purely β-- product and a mother liquor which contained α and β- epimers. The mother liquor was concentrated and flash chromatographed (silica gel, 20% ethyl
acetate/hexane). Yield of the solid white β-epimer product (81 g, 0.123 mol) was 68%, mp 91-91.7°C (lit 83-84°C);1H-NMR (CDCl3) δ7.4-7.29 (m, 18H), 7.20 (m, 2H), 4.96 (d, 2H J=10.8), 4.84 (t, 2H, J=10.8), 4.75 (d, 1H, J=10.8), 4.65 (d, 1H, J=12.4), 4.6-4.54
(overlapping dd, 2H, J=12H, J=10.4), 4.46 (d, 1H, J=7.2, H1'), 4.38 (p, 1H, H2) , 4.12-4.02 (m, 2H, H1a, H2), 3.89 (pseudo t, 1H, J=7.2, H1b), 3.79-3.6 (m, 5H), 3.50 (pseudo t, 2H), 1.46 (s, 3H), 1.40 (s, 3H); 13C- NMR (CDCl3) 5138.529 (0), 138.370 (0), 138.066 (0), 138.013 (O), 128.432, 128.409, 128.129 , 128.015,
127.901, 127.810, 127.734, 127.666, 109.399 (0),
103.824 (C1'), 84-631 (C3'), 82.120 (C2'), 77.713
(C4'), 75.748 (2), 75.058 (2), 74.891, 74.853, 74.315
(2), 73.495 (C1), 70.317 (2), 68.762 (C6'), 66.896 (C3), 26.880 (3), 25.386(3). Yield of the colorless, oily α-epimer (23 g, 0.035 mol) was 19%; 1H NMR (CDCI3) 57.4-7.24 (m, 18H), 7.14 (m, 2H), 4.98 (d, 1H,
J=10.8), 4.88-4.78 (m, 3H), 4.67 (d, 1H, J=12), 4.62 (d, 1H, J=11.6), 4.47 (d, 2H J=11.6), 4.37 (t, 1H, J=6.4), 4.07 (pseudo pentet, 1H), 3.96 (t, 1H, J=8.8), 3.8-3.54 (m, 9H), 1.43 (s, 3H), 1.37 (s, 3H); 13C NMR (CDCl3) δ138.764 (0), 138.203 (0), 138.165 (0), 137.81) (0), 128.440, 128.387, 128.364, 128.030, 127.947,
127.916, 127.886, 127.696, 127.590, 109.422 (O),
97.482 (C1'), 81.885 (1), 79.890 (1), 77.508 (1), 75.703 (2), 75.088 (2), 74.535 (1), 73.457 (2), 73.108 (2), 70.279 (1), 69.020 (2), 68.314 (2), 67.040 (2), 26.827 (3), 25.424 (3).
(2R) 1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)- glycerol 2. A 5-L three-necked morton flask fitted with a mechanical stirrer, condenser, and stopper was charged with compound 1 (50 g, 76.2 mmol) in 60% aqueous acetic acid (2.5 L). The acidic solution was refluxed for 1.5 hours at 103°C and then cooled to room temperature. Distilled water (1.5 L) was added to the solution causing precipitation of a white solid. The acidic solution was extracted with
methylene chloride (4 x 1 L) which was subsequently neutralized with sodium bicarbonate solution and concentrated to a white solid. Trituration with diethyl ether gave white product. The remaining mother liquor was flash chromatographed (silica gel, 50% ethyl acetate/hexane) to give white solid product. The combined yield (61.9 g, 0.101 mol) was 83 %, mp 101.5-102.4°C (lit 76-78°C); 1H NMR (CDCl3) 5.40-7.26 (m, 18H), 7.19 (t, J=3.5, 2H), 5.0-4.7 (m, 5H), 4.64- 4.5 (m, 3H), 4.46 (d, 1H, J=8.0, H1 ' ), 4.0-3.60 (m,
11H, H1's, H2, H3, H3', H6b', H6a', H4', H5',H2, '), 2.55 (s, 2H, OH'S); 13C NMR (CDC13) 38.529 (0), 138.332 (0), 137.952 (0), 137.740 (0), 128.531, 128.550, 128.478, 128.189, 128.114, 128.091, 127.931, 127.871,
127.749,104.279 (C1'), 84.654 (C3'), 82.158 (C2'), 77.819 (C4'), 75.779 (2), 75.081 (2) , 75.028 (2), 74.527 (C5'), 73.571 (2), 72.207 (C1 ), 71.204 (C2), 68.883, (C6'), 63.353 (C3).
(2S) [1-O-(2113,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-3-O-tert-butyldimethylsilyl] glycerol
3. In a nitrogen-purged 100-mL round-bottomed flask fitted with a septum was dissolved diol 2 (9.0 g, 14.7 mmol), imidazole (2.05 g, 30.2 mmol), and t-butyl dimethylsilylchloride (2.28 g, 15.1 mmol) in anhyd DMF (45 mL). The reaction mixture was stirred under nitrogen for 2.5 days, transferred to a 1-L separatory funnel, and methylene chloride (250 mL) and water (250 mL) were added. The aqueous layer was extracted with methylene chloride (2 x 250 mL) and then the combined organic layers were washed with water (2 x 100 mL). After drying and concentration, purification by flash chromatography (silica gel , 33% ethyl acetate/hexane) gave a colorless oil (9.2 g, 12.6 mmol) in 88% yield; 1H NMR (CDCl3) 57.48-7.3 (m, 18H), 7.25-7.21 (m, 2H), 5.00 (d, 2H, J=11.2), 4.89 and 4.88 (overlapping doublets, 2H, J=10.8, J=10.4), 4.83 (d, 1H, J=11.2), 4.67 (d, 1H, J=12.4), 4.60 and 4.59 (overlapping doublets, 2H, J=12.4, J=10.8); 4.50 (d, 1H, J=7.6 H1 ' ), 4.06-3.92 (m, 2H), 3.9-3.62 (m, 7H), 3.6-3.52 (m, 2H), 3.04 (s, 1H, OH), 0.978 (s, 9H), 0.142 (s, 6H); 13C NMR (CDCl3) 5188.552 (0), 138.385 (0), 138.005 (0), 137.960 (0), 128.455, 128.440, 128.121, 128.060, 127.931, 127.863, 127.772, 127.734, 127.696, 104.377 (C1'),
84.692 (C31), 82.219 (C2'), 77.804 (C4'), 75.771 (2), 75.073 (2), 74.959,(2), 74.717 (C5'), 73.541 (2), 73.078 (2), 71.060 (C2), 68.785 (C6'), 63.998 (C3), 25.970 (3), 18.668 (0), -5.299 (3).
(2S) [1-0-(2,3,4,6-T6tra-O-benzyl-β-D-glucopyranosyl)-2-O-palmitoyl-3-0-t-butyldimethylsilyl] glycerol 4. A nitrogen purged 500-mL round-bottomed flask fitted with a septum was charged with compound 3 (9.3 g, 12.8 mmol) and palmitic anhydride (6.94 g, 14.0 mmol) in
dry THF (200 mL). Dimethylaminopyridine (316 mg, 2.6 mmol) and triethylamine (2.04 mL, 14.7 mmol) were added, and the reaction was stirred under nitrogen for 12 h. The mixture was then transferred to a 2-L separatory funnel, and diethyl ether (500 mL) and water (500 mL) were added. The aqueous layer was filtered through Whatman No. 1 paper and extracted with diethyl ether (2 x 500 mL). After drying over magnesium sulfate, the combined organic layers were concentrated and purified by flash chromatography
(silica gel, 14% ethyl acetate/hexane) to give a light yellow oil (12.1 g, 12.5 mmol) in 97% yield; 1H NMR (CDCl3) 57.40 (br. S, 20H), 5.15 (5, 1H), 4.98 (t, 2H), 4.84 (t, 2H), 4.76 (d, 1H), 4.67 (d, 1H), 4.59 (dd, 2H), 4.52 (d, 1H), 4.13 (dd, 1H), 3.84 (m, 6H), 3.67 (dd, 2H), 3.49 (t, 2H), 2.32 (t, 2H), 1.61 (m, 2H), 1.25 (br. s, 24H), 0.98 (s, 9H), 0.97 (s, 3H), 0.14 (s, 6H). 13C NMR (CDCl3) 573.280, 138.597, 138.438, 138.127, 138.096, 128.379, 128.356, 128.333, 128.083, 127.977, 127.863, 127.780, 127.605, 127.582, 103.831, 84.556, 81.984, 77.721, 75.695, 75.020, 74.876,
74.603, 73.488, 72.904, 68.754, 67.821, 61.661,
34.428, 33.950, 31.941, 29.717, 29.687, 29.649,
29.619, 29.497, 29.459, 29.384, 29.300, 29.148,
25.826, 24.953, 22.716, 18.268, 14.159, -5.375.
(2R) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)- 2-Opalmitoyl] glycerol 5.
Procedure A. Compound 4 (34.0 g, 35.1 mmol) was dissolved in THF (1.4 L) in a 3-L three-necked Norton flask fitted with a mechanical stirrer, thermometer, and a 500-mL addition funnel. The solution was chilled to 0°C, and a solution of tetrabutylammonium fluoride (TBAF) (520 mL, 1.0 M in THF) which was buffered to pH=6.5 with acetic acid was added dropwise
through the addition funnel. The reaction mixture was stirred for 11 h at 0°C, left to sit at -15°C for 12 h, and stirred again for 4 h at rt. Water (100 mL) was added, and the solution was concentrated to 200 mL of solution. The concentrate was redissolved in methylene chloride (750 mL) in a 3-L separatory funnel and washed with water three times (750 mL, 2 x 500 mL). The combined aqueous layers were extracted with diethyl ether (500 mL). The combined organic layers were concentrated to give a red oil which was purified by flash chromatography (silica gel, 33-40% gradient of ethyl acetate/hexane). A white solid (28.0 g, 32.8 mmol) was obtained in 93% yield. 1H NMR (CDCl3) 57.36 (br. s, 20H), 5.06 (t, 1H), 4.96 (dd, 2H), 4.84 (dd, 2H), 4.75 (d, 1H) 4.59 (m, 2H), 4.53 (dd, 1H), 4.45 (dd, 1H), 4.14 (m, 2H), 3.91 (m, 2H), 3.78 (m, 6H), 2.80 (s, 1H), 1.64 (m, 2H), 1.27 (br. s, 26H), 0.90 (t, 3H).
Procedure B. Compound 4 (500 mg, 0.52 mmol) was dissolved in THF (20 mL) in a 100-mL three-necked round-bottomed flask fitted with two stoppers and a septum. Glacial acetic acid (9.5 mL) was added, and the solution was chilled to 0°C. A solution of TBAF (5.16 mL, 1.0 M in THF) was syringed into the chilled solution, and stinting was continued at 0°C for 8 h and then at rt for 25 hours. Methylene chloride (50 mL) was added, and the entire solution was transferred to a 250-ml separatory funnel where it was neutralized with 1M disodium phosphate solution (2 x 75 mL). The combined organic layers were rotary evaporated under reduced pressure and the concentrate was purified by flash chromatography (silica gel, 25-40% gradient of ethyl acetate/hexane), yielding a colorless oil (424 mg, 0.497 mmol, 95%) which later solidified upon standing; 1H NMR (CDCl3) 57.36 (br. s, 20H), 5.06 (t,
1H), 4.96 (dd, 2H), 4.84 (dd, 2H), 4.75 (d, 1H), 4.59 (m, 2H), 4.53 (dd, 1H), 4.45 (dd, 1H), 4.14 (m, 2H), 3.91 (m, 2H), 3.78 (m, 6H), 2.80 (s, 1H), 1.64 (m, 2H), 1.27 (br. S, 26H), 0.90 (t, 3H) .
(2S) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-3-O-palmitoyl] glycerol 6. Compound 4 (3.0 g, 3.1 mmol) was dissolved in THF (120mL) in a 250-mL three- necked round-bottomed flask fitted with a 60-mL addition funnel, glass stopper, and septum. TBAF (54 mL, 1.0 M in THF) was added through the addition funnel over a 15 minute period. Glacial acetic acid (18 mL) measured in a graduated cylinder was then poured into the reaction mixture, and the solution was stirred for 45 minutes. The solution was concentrated under reduced pressure to approximately 30 mL of liquid and then redissolved in methylene chloride (150 mL). The organic layer was washed with water (3 x 120 mL) and neutralized with sodium bicarbonate solution (2 x 150 mL). The combined aqueous layers were extracted with methylene chloride (100 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated. The resulting dark red concentrate was purified by flash
chromatography (silica gel, 25% ethyl acetate/hexane) to give 6 a colorless oil which corresponded to an upper TLC spot (1.3 g, 1.52 mmol) in 46% yield. 1H NMR (CDCl3) 57.36 (br. s, 20H), 4.95 (m, 2H), 4.86 (m, 3H), 4.64 (d, 1H), 4,58 (m, 2H), 4.47 (d, 1H), 4.16 (m, 1H), 3.96 (dd, 1H), 3.68 (m, 8H), 2.38 (t, 2H), 1.62 (m, 2H), 1.27 (br. s, 24H), 0.96 (t, 3H). Isolation of a lower TLC spot gave a white solid (400 mg, 0.469 mmol) in 15% yield which corresponded to compound 5; 1H NMR (CDCl3) 57.36 (br- s, 20H), 5.06, (t, 1H), 4.96 (dd, 2H), 4.84 (dd, 2H), 4.75 (d, 1H), 4.59 (m, 2H),
4. 53 (dd, 1H) , 4 . 45 (dd, 1H) , 4 . 14 (m, 2H) , 3 . 91 (m, 2H) , 3 . 78 (m, 6H) , 2 . 80 ( s , 1H) , 1. 64 (m, 2H) , 1. 27 (br . s , 26H) , 0. 90 (t, 3H) . Resilation of (2R) [1-O-(2,3,4,6-Tetra-O-benzyl-β-Dglucopyranosyl)-2-O-palmitoyl] glycerol 5. In a nitrogen-purged 50-mL round-bottomed flask fitted with a septum was placed compound 5 (318 mg, 0.373 mmol) dissolved in DMF (8 mL). tert-Butyl-dimethylsilyl chloride (281 mg, 1.86 mmol) and imidazole (254 mg, 3.73 mmol) were added, and the solution was stirred for 22 h. Methylene chloride (50 mL) was added, and the reaction mixture was transferred to a 250-mL separatory funnel. The organic layer was washed with water (50 ml), and then the aqueous layer was
extracted with methylene chloride (2 x 50 mL). The pooled methylene chloride layers were washed with water (2 x 75 mL), dried over magnesium sulfate, and the filtered. The filtrate was concentrated and purified by flash chromatography (silica gel, 14% ethyl acetate/hexane) to give 4 as a yellow oil (239 mg, 0.247 mmol) in 66% yield.
Resilation of (2S) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-3-O-palmitoyl] glycerol 6. In a nitrogen-purged 25-mL round-bottomed flask fitted with a septum was placed compound 6 (176 mg, 0.206 mmol) dissolved in anhyd DMF (5 mL). tert- Butyldimethylsilyl chloride (155 mg, 1.03 mmol) and imidazole (140 mg, 2.06 mmol) were added, and the solution was stirred for 43 h. Methylene chloride (50 mL) was added, and the reaction mixture was
transferred to a 250-mL separatory funnel. The organic layer was washed with water (50 mL). The aqueous layer was extracted with methylene chloride (2
x 50 ml). The methylene chloride layers were pooled methylene and washed with water (2 x 75 mL), dried over magnesium sulfate, and then filtered. The filtrate was concentrated and flash chromatographed (silica gel, 14% ethyl acetate/hexane) to give 7 as a light yellow oil (190 mg, 0.223 mmol) in 95% yield; 1H NMR (CDCl3) 57.38 (br. s, 20H), 4.99 (dd, 2H), 4.85 (t, 2H), 4.78 (d, 1H), 4.68 (d, 1H), 4.61 (dd, 2H), 4.48 (d, 1H), 4.37 (d, 1H), 4.13 (s, 2H), 3.98 (m, 1H), 3.77 (m, 2H), 3.67 (m, 3H), 3.52 (m, 2H) 2.35 (t, 2H), 1.67 (m, 2H), 1.31 (br. s, 24H), 0.93 (s, 12H), 0.14 (s, 6H).
(28) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-O-palmitoyl 3-O-(2-bromoethyl)benzylphosphoryl] glycerol 15.
Procedure A. In a nitrogen-purged 100-mL three-necked round-bottomed flask fitted with two stoppers and a septum was dissolved freshly distilled 2- bromoethylphosophorodichloridate (1.72 g, 7.11 mmol) in diethyl ether (20 mL). The solution was chilled to 0°C, and triethylamine (8.15 mL, 58.5 mmol) was injected into the solution which caused precipitation of a white solid. A solution of compound 5 (1.0 g, 1.17 mmol) in anhyd diethyl ether (55 ml) was injected into the chilled reaction mixture, and the ice bath was removed. The reaction was stirred for 30 minutes after which benzyl alcohol (1.21 mL, 11.7 mmol) was injected into the reaction mixture. Stirring was continued at rt for 5 d. The reaction was then
filtered through a fritted glass funnel, and the filtrate was concentrated. The orange concentrate was purified by flash chromatography (0-33% ethyl
acetate/hexane) to give 15 as a light yellow oil (566 mg, 0.501 mmol) in 43% yield; 1H NMR (CDCl3) 57.38-7.25
(br. s, 23H), 7.16 (m, 2H), 5.26 (m, 1H), 5.10 (t, 2H), 4.94 (m, 2H), 4.81 (t, 3H), 4.71 (d, 1H), 4.61 (d, 1H), 4.55 (d, 2H), 4.39 (d, 1H), 4.25 (m, 4H), 4.08 (dd, 1H), 3.73 (m, 3H), 3.64 (dd, 2H), 3.42 (m, 4H), 2.27 (t, 2H), 1.58 (m, 2H), 1.25 (br. d, 24H), 0.89 (t, 3H).; 13C NMR (CDCl3) δ173.210, 138.559,
138.362, 138.096, 138.074, 128.667, 128.622, 128.333, 128.318, 128.296, 127.962, 127.878, 127.757, 127.734, 127.696, 127.605, 127.522, 103.862, 84.540, 81.969, 71.652, 75.589, 74.937, 74.906, 74.686, 73.480,
70.469, 70.385, 69.680, 69.619, 68.777, 67.283,
66.099, 66.069, 34.170, 31.887, 29.657, 29.619,
29.596, 29.452, 29.315, 29.239, 29.080, 24,802,
22.647, 14.050.
Procedure B. In a nitrogen-purged 100-mL three-necked roundbottomed flask fitted with a thermometer,
stopper, and septum was dissolved freshly distilled 2- bromoethylphosphorodichloridate (1.42 g, 5.85 mmol) in methylene chloride (15 mL). The solution was chilled to 0°C, and compound 5 (1.0 g, 1.17 mmol) and a solution of N-methylmorphiline (1.28 mL, 11.7 mmol) dissolved in methylene chloride (35 mL) was injected into the solution over a 10 minute period. The reaction mixture was stirred at 0°C for 5.5 h at which point a new TLC spot which co-spotted with secondary alcohol 6 appeared. Stirring was continued for another 30 minutes, and benzyl alcohol (1.21 ml, 11.7 mmol) was injected into the reaction. After 6 days of stirring, the reaction mixture was transferred to a 500-mL separatory funnel, and methylene chloride (150 mL) and water (200 ml) were added. The layers were separated, and the organic layer was rotary evaporated under reduced pressure. The resulting oil was flash chromatographed (silica gel, 33% ethyl acetate/hexane). to give 15 as a yellow oil (2 50 mg, 19%); 1H NMR
(CDCl3) δ7.38 8-7.2 5 (br. s, 23H), 7.16 (m, 2H), 5.26 (m, 1H), 5.10 (t, 2H), 4.94 (m, 2H), 4.81 (t, 3H), 4.71 (d, 1H), 4.61 (d, 1H), 4.55 (d, 2H), 4.89 (d, 1H), 4.25 (m, 4H), 4.08 (dd, 1H), 3.73 (m, 3H), 3 . 64 (dd, 2H) , 3.42 (m, 4H) , 2.27 (t, 2H) , 1. 58 (m, 2H), 1.25 (br. d, 24H), 0.89 (t, 3H).; 13C NMR (CDCl3) δ 173.210, 138.491, 138.286, 137.990, 137.975, 128.720, 128.652, 128.387, 128.364, 128.015, 127.954, 127.878, 127.810, 127.780, 127.727, 127.681, 127.613, 103.854, 84.495, 81.923, 77.781, 77.546, 75.688, 75.020,
74.808, 74.747, 73.473, 70.438, 69.642, 68.633,
67.322, 66.759, 66.129, 34.178, 31.925, 29.702,
29.664, 29.641, 29.490, 29.422, 29.368, 29.285,
29.103, 24.802, 22.700, 14.198.
(2S) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-O-palmitoyl-3-O-phosphatidylcholine] glycerol 16. A 45 mL Parr bomb equipped with a magnetic stirring bar was charged with a solution of phosphate 15 in toluene (10 mL). Condensed anhydrous trimethylamine (12 mL) was added quickly in one portion, and then the vessel was sealed and heated in an oil bath at 55°C for 24 h. The reaction mixture was concentrated to a viscous oil and triturated with ethyl ether, upon which a white precipitate formed, the precipitate was filtered off, washed with ether, and then the combined ethereal solutions were concentrated to a glassy solid.
Purification of this residue using preparative TLC (2000 μ double elution with 75%,12.5%,12.5% methylene chloride/reethanol/ hexanes gave inner salt 16 as a glassy solid;
(2S) [β-D-glucopyranos-1-yl-2-O-palmitoyl-3-O- phosphatidylcholine] glycerol SP-19501. A solution of phosphatidylcholine 16 (200.4 mg, 0.197 mmol) in
reagent grade methanol (25 mL) was hydrogenated at 60 psi over 10% Pd/C (40 mg, 20 wt%). After 30 h, the catalyst was ffltered off through celite and the methanol washing were combined and concentrated. The residue was dissolved in fresh methanol (25 mL) and resubjected to hydrogenation at 60 psi over 80 mg (40 wt%) of 10%Pd/C. After 48 h, the reaction was still incomplete. After filtration, washing of the
catalyst, and concentration, the residue was subjected to hydrogenation using 400 mg (200 wt%) of Pd/C at 60 psi in methanol (25 mL). After 22h, the catalyst was filtered off through celite and the methanol filtrate and washings were combined and concentrated to afford 92.8 mg (71.6%) of (S) SP-19501 as a white solid; 1H NMR (CD3OD) δ5.12 (br t, 0.5 H), 4.88 (br m, 4.5 H), 4.25 (br m, 2H), 4.12-3.57 (M, 12H), 3.4-3.1 (m
containing singlet at 3.18, 12H), 2.3 (m, 2H), 1.55(m, 2H), 1.24 (m, 22H), 0.86 (br t, 3H); 13C NMR (CD3OD) δ 74.93, 104.80, 78.02, 77.93, 75.19, doublet at 71.53 and 71.49, doublet at 70.80 and 70.73, doublet at
67.79 and 67.74, multiplet at 67.50, 62.53, doublet at 60.56 and 60.52, triplet at 54.79, 34.88, 33.15,
30.85, 30.85, 30.66, 30.56, 30.46, 30.26, 26.10 and 26.03, 23.82, 14.56; 31P NMR (CD3OD) 51.65.
(2S) 2,3-O-Isopropyiidene-1-O-trifluromethylsulfonyl- glycerol was prepared according to the method
described for the corresponding (R) isomer in 92% yield and used immediately.
(2S) [1-O-(2,3,4,6-Tetra-O-benzyl-O-D-glucopyranosyl)-2,3-O-isopropylidene] glycerol 8. 2,3,4,6-Tetra-O- benzyl-D-glucopyranose (65 g, 0.12 mol) was dissolved in THF (800 mL) and chilled to -10°C in a nitrogen- purged 3-L three-necked morton flask fitted with a
thermometer, stopper, and mechanical stirrer. Sodium hydride 60% in oil (33 g, 0.825 mol) was added in 4 increments over 10 minutes, and the solution was stirred for lh. (S) 2,3-O-Isopropylidene-1-O- trifluoromethylsulfonylglycerol (0.15 mol) dissolved in THF (200 mL) was then dropped via an addition funnel into the reaction mixture over a 20 minute period at -10 to -15°C. The solution was stirred at - 10 to -15°C for 6 hours. The reaction mixture was filtered through a short plug of silica gel and concentrated to an orange brown oil, 114 g.
Purification of the crude by flash chromatography using 50% ethyl ether/hexanes gave 39.8 g (67.6%) of βepimer 8 as a white solid, along with 4 g (5.1 %) of a mixture of α and β epimers; mp of β anomer 85.7-
87.2°C; 1H NMR of β epimer (CDCl3) δ 7.4-7.2 (m, 18H), 7.19-7.14 (m, 2H), 4.98-4.92 [overlapping doublets at 4.97 (J=10.8) and 4.94 (J=10.8), 2H], 4.82 (t, 2H, J- 10.8), 4.73 (d, 1H, J=10.4), 4.63 (d, 1H, J=12.4), 4.58-4.51 [overlapping doublets at 4.55 (J=12) and
4.53 (J=10.8), 2H] 4.45 (dy 1H, J=7.2), 4.36 (p, 1H, H2), 4.08 (pseudo triplet, 1H), 3.94-3.89 [overlapping doublets at 3.92 (J=10) and 3.91 (J=9.6), 1H] , 3.82- 3.57 (m, 6H), 3.47 (pseudo triplet, 2H), 1.44 (s, 3H), 1.38 (S, 3H); 13C NMR (CDCl3) δ 138.569 (0), 138.384 (0), 138.006 (0), 138.021 (0), 128.341, 128.258,
127.962, 127.856, 127.765, 127.696, 127.620, 127.605, 109.467 (0), 103.869 (C1'), 84.586 (C3'), 82.075
(C2'), 77.705 (C4'), 75.680 (2), 75.005 (2), 74.815 (2 carbons, C2, C5'), 74.512 (1), 73.457 (C1), 71.151 (2), 68.785 (C6'), 67.017 (C3), 26.895 (3), 25.393 (3).
(2S) 1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)--glycerol 9. A suspension of 8 (20 g, 30.5 mmol) in
60% acetic acid (800) was heated to reflux for 1h. Workup was similar to that described for the (R) diol 2, providing 18 g (96% yield) of 9 as a white solid, which was of sufficient purity after trituration with ether for the subsequent step. Diol 9 could be recrystallized from ether/hexane, mp 89.6-90.90C; 1H NMR (CDCl3) δ7.38-7.27 (m, 18H), 7.16 (t, J=3.5, 2H), 4.98-4.74 (m, 5H), 4.61-4.5 (m, 3H), 4.42 (d, 1H, J=8.0, H1'), 3.89-3.80 (m, 3H, H1's, H2), 3.72-3.63 (m, 4H, H3 H3', H6b'), 3.62-3.44 (m, 4H, H6b ', H4' , H6 ', H2'), 2.59 (s, 2H, OH's); 13C NMR (CDCl3) δ138.370 (0),
138.119 (0), 137.78 (0), 137.69 (0), 128.462, 128.447, 128.432, 128.060, 128.038, 127.962, 127.894, 127.848, 127.810, 127.704, 104.195 (C1'), 84.616 (C3'), 82.037 (C2'), 77.736 (C4'), 75.733 (2), 75.043 (2, 2 carbons), 74.466 (C5'), 73.480 (2), 72.312 (C1), 70.772 (C2), 68.731, (C6 '), 63.355 (C3).
(2R) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-3-O-tert-butyldimethylsilyl] glycerol 10. In a nitrogen-purged 100-mL round-bottomed flask fitted with a septum was dissolved diol 9 (28.0 g, 45 mmol), imidazole (5.71 g, 90 mmol), and t-butyl
dimethylsilylchloride (6.92 g, 45.3 mmol) in anhyd DMF (75 mL). The reaction mixture was stirred under nitrogen overnight, transferred to a 1-L separatory funnel, and chloroform (300 mL) and water (300 mL) were added. The aqueous layer was extracted with chloroform (2 x 100 mL) and then the combined organic layers were washed with water (3 x 100 mL). After drying (Na2SO4) and concentration, purification by flash chromatography (silica gel, 50% ethyl
ether/hexanes) gave 10 as a colorless oil (29.5 g) in
90% yield;
(2R) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-Opalmitoyl-3-O-t-butyldimethylsilyl] glycerol 11. A mixture of 10 (22.2 g, 3 0.4 mmol), palmitic anhydride (16.5 g, 33.4 mmol), dimethylaminopyridine (741 mg, 6.08 mmol), triethylamine (3.78 g, 5.2 mL, 37.3 mmol) and anhyd THF (250 mL) was stirred under nitrogen at rt overnight. The mixture was poured into a 2-L separatory funnel, diluted with diethyl ether (500 mL) and water (500 mL), and the layers separated. The aqueous layer was filtered through Whatman No. 1 paper and extracted with more diethyl ether (2 x 500 mL). The combined ether layer was washed with water (3 x 200 mL) and then dried (MgSO4). Following filtration, purification by flash chromatography (silica gel, 33% ethyl ether/hexane) gave 11 as a light yellow oil, 28.2 g, 96% yield);
Compound 11 could be carried on to the next
transformation without chromatographic purification. (28) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-Opalmitoyl] glycerol 12. Crude 11 (30.4 mmol based on 10) was dissolved in THF (100 mL) and the solution was chilled to 0°C. A premixed solution of TBAF (520 mL, 1.0 M in THF) which was buffered to pH=6.37 with acetic acid was added dropwise via an addition funnel at 0°C for 1 h, and then at -15°C overnight. The reaction mixture was concentrated, water (100 mL) was added, and the resulting mixture was extracted with chloroform (3 x 300 mL). The combined chloroform layer was washed with water (4 x 500 ML), and then the combined aqueous layer was backextracted with diethyl ether (500 mL). After drying the combined organic layer over Na2SO4, concentration gave a red oil which was purified by flash chromatography (50% ethyl ether/hexane). Evaporation of the product containing
fractions afforded 12 as a white solid (24.6 g, 94.6% yield for two steps);
(2R) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-3-Opalmitoyl] glycerol 13. Compound 11 (2.33 g, 2.41 mmol) was dissolved in THF (150 mL) in a 250-mL three- necked round-bottomed flask fitted with a 60-mL addition funnel, glass stopper, and septum. After cooling the solution to OoC, TBAF (24.1 mL, 1.0 M in THF) was added through the addition funnel over a 5 minute period. Glacial acetic acid (13.8 mL 241 mmol) was then poured into the reaction mixture to quench the reaction, and the resulting solution was stirred for approximately 30 minutes. The reaction mixture was poured into a separatory funnel containing ice water (500 mL) and methylene chloride (200 mL). The layers were separated, and aqueous layer was extracted twice more with methylene chloride (100 mL portions) and then the combined organic layer was washed with brine (400 mL). Following dring (MgS04), filtration, and then concentration, purification by flash
chromatography using 1/5 EtoAc/ hexanes gave secondary alcohol 13, 0.96 g (46.8%), as a colorless oil;
Further elution gave 238 mg (11.6%) of primary alcohol 12; Also isolated was a mixture of the two alcohols in 5.3% yield.
Resilation of (2S) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D glucopyranosyl)-2-O-palmitoyl] glycerol 12. The identity of 12 was established by resilylation of 12 according to the procedure described above for the (R) isomer, compound 5.
(2R) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-O-palmitoyl-3-O-(2-bromoethyl)benzylphosphoryl] glycerol 17. In a nitrogen-purged 1-L three-necked morton flask fitted with two stoppers and a septum was dissolved freshly distilled
2-brorhoethylphosphorodichloridate (17.2 g, 71.1 mmol) in anhyd diethyl ether (500 mL). The solution was chilled to 0°C and triethylamine (81.5 mL, 0.585 mol) was injected into the solution, causing precipitation of a white solid. A solution of 12 (10.0 g, 11.7 mmol) dissolved in diethyl ether (250 mL) was
cannulated into the morton flask, and the solution was stirred for 1.5 hours. TLC showed disappearance of 12. Benzyl alcohol (12.1 mL, 0.117 mol) was injected into the reaction mixture, and stirring was continued at rt for 16 h. The reaction mixture was then filtered through a fretted glass funnel. Filtrate was then concentrated and purified by flash chromatography twice. First chromatography (silica gel, 33% ethyl acetate/hexane) and second chromatography (silica gel, 25% ethyl acetate/hexane) gave 17 as a light oil (5.5 g) in 42% yield;
(28) [1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)- 2-Opalmitoyl-3-O-phosphatidylcholinel glycerol 18. A
45 mL Parr bomb was equipped with a magnetic stir bar and then charged with a solution of 17 (1.17 g, 1.04 mmol) in benzene (15 mL).
Anhyd trimethylamine (15 mL, 0.145 mmol) which had been condensed at -78°C was quickly poured into the reaction vessel, and the bomb was sealed. The
reaction was stirred at 55°C in an oil bath for 24 hours behind a blast shield. The bomb vessel was then cooled to -78°C, opened, and left in a hood to
evaporate trimethylamine. The remaining solution was
rotary evaporated under reduced pressure, and the oily concentrate was dissolved in methylene chloride and purified by preparative TLC (2000 μ). Double elution with 75%; 12.5%: 12.5% methylene
chloride/methanol/hexane gave inner salt 18 as an opaque glassy solid (223 mg, 21%). Η-NMR (CDCl3) δ.32 (br. s, 20H), 5.21 (m, 1H), 4.90 (dd, 2H), 4.82 (m, 2H), 4.64 (m, 2H), 4.50 (t, 2H), 4.42 (d, 1H), 4.22 (br. S, 3H), 3.95 (S. 2H), 3.72 (s, 2H), 3.62 (t, 2H), 3.55 (S, 1H), 3.40 (m, 4H), 3.10 (s, 9H), 2.19 (m, 2H), 1.47 (m, 2H), 1.20 (br. d, 24H), 0.87 (t, 3H). 13C-NMR (CDCl3) δ73.393, 138.453, 138.377, 138.051, 137.998, 128.470, 128.417, 128.356, 128.318, 128.235, 128.053, 128.007, 127.947, 127.856, 127.780, 127.719, 127.636, 127.567, 103.899, 84.472, 81.984, 77.478, 77.394, 77.311, 77.190, 75.672, 74.944, 74.550,
73.336, 68.663, 68.489, 59.158, 59.135, 54.409,
54.349, 34.246, 31.902, 29.710, 29.664, 29.535,
29.353, 29.330, 29.148, 24.7871 22.678, 14.121.
(2R) [β- D-glucopyranos-1-yl-2-O-pslmiyoyl-3-O- phosphatidylcholine] glycerol SP-19501. A solution of phosphatidylcholine 18 (130 mg, 0.127 mmol) in reagent grade methanol (25 mL) was hydrogenated at 60 psi over 10% Pd/C (52 mg, 40 wt%). After 23 h, TLC showed an incomplete reaction. The catalyst was filtered off through celite and the methanol washings were combined and concentrated. The residue was dissolved in fresh methanol (25 mL) and resubjected to hydrogenation at 60 psi over 240 mg (185 wt%) of 10% Pd/C. After 20 h, the reaction was complete by TLC. The catalyst was filtered off through celite and the methanol filtrate and washings were combined and concentrated to afford 64.0 mg (76.6%) of (R) SP-19501 as a white solid; 1H NMR (CDCl3) δ 5.19 (m, 1H), 4.97 (s, OH + HDO), 4.34-
4.26 (br m, 2H), 4.16-3.95 (m, 3H), 3.9-3.6 (m, 6H), 3.42-3.14 (multiplet containing singlet at 3.24, 13H), 2.37 (t, J=7.6, 2H), 1.62 (pseudo t, 2H), 1.31 (m, 24H), 0.92 (t, J=7.2, 3H) ; 13C NMR (CD3OD) δ175.02, 104.88, 78.07, 78.04, 75.04, 72.97, 72.89, 71.52, 68.56, multiplet at 67.50, doublet at 64.99 and 64.94, 62.65, doublet at 60.52 and 60.48, triplet at 54.74 (J=3.1), 35.14, 33.13, 30.85, .30.69, 30.54, 30.29, 26.01, 23.79, 14.50; 31P NMR (CD3OD) δ 1.35
(2R) 1-[Benzyl-(2'bromoethyl)-phosphproyl]-2,3-isopropylidene glycerol (19).
2-Bromoethylphosphodichloridate (20.0 g, 0.08 mol) was dissolved in CC14 (50 ml) in a nitrogen-purged 0.5 L three-necked flask fitted with a magnetic stir bar, thermometer, and a 125-ml addition funnel. The solution was chilled to 0°C, and to this stirred solution was added dropwise the solution of (S)-form solketal (10.7 g, 98 mol %) and N-methyl-morpholine (8.22 g,98 mol %) in CCL4 (25 ml). After 2 hours TLC showed disappearance of solketal. To the reaction mixture was added dropwise the solution of benzyl alcohol (44.6 g, 500 mol %) and N-methylmorpholine (8.38 g, 100 mol %). The reaction mixture was stirred under nitrogen for 60 hours at room temperature. TLC showed the complete reaction. The reaction mixture was filtered through Shott filter #C, and the solution was rotary evaporated to volume near 70 ml and
purified by flash chromatography (silica gel, diethyl ether) to give colorless oil (15.1 g, 0.04 mol) in 45 % yield; 1HNMR (CDCL3) δppm: 7.40 (br. s 5 H), 5.2 (d, 2 H), 4.3 (br.s, 3 H), 4.0 (br.s, 3 H), 3.85 (br.s,1 H), 3.2 (s, 2 H), 1.4 (d, 6H); 13C NMR (CDCL3):
128.743, 128.682, 128.645, 128.114, 128.076, 109.885, 77.364, 77.046, 76.727, 73.920, 73.837, 69.771,
69.710, 67.760, 67.707, 67.654, 66.774, 66.721,
65.955, 29.353, 29.277, 26.683, 25.204; 31P NMR
(CDCL3):-1.108. (2R) 1-[Benzyl-(2'-bromoethyl)-phosphoroyl] 1-2,3-dihydroxy glycerol (20). A nitrogen purged 1 L round- bottomed flask fitted with septum was charged with compound 19 (19.5 g, 0.048 mol) in dry THF (50 ml) and the solution of 1 M H3PO4 (800ml) was added. The reaction mixture was stirred under nitrogen by room temperature for 15 hours. TLC showed the completness of the reaction. Then the reaction mixture was transferred to a 2 L sepapatory funnel. The acidic layer was extracted with ethyl acetate (7 x 450 ml). The combined organic extract was washed with water (2 x 850 ml). After drying over sodium sulfate it was rotary evaporated and dryed in high vacuo for 10 hours to give a colorless oil (14 g, 0.04 mol %) in 80 % yield; 1H NMR (CDCL3), δppm: 7.38 (br.s, 5H), 5.2 (d, 2H), 4.25-3.8 (multiplet, 6 H), 3.7-3.25 (br.m 5 H) 13C NMR (CDCL3): 77.789, 77.774, 77.349, 77.030, 76.712, 70.522, 70. 491, 70.461, 70.431, 70.097, 70.044, 68.898, 68,883, 68.822,
67.085, 67.032, 62.617, 62.496, 42.363, 42.280; 31P NMR (CDCL3): -0.485 (85 % H3PO4).
(2R) 1-[Benzyl-(2'-bromoethyl)-phosphoroyl-2-hydroxy-3-O-triphenylmethyl glycerol (21). To a stirred
solution of diol 20 (8.0 g, 21.6 mmol) in DMF (16 nil) was added diisopropylethylamine (4 ml, 105 mol %) followed by addition of trityl chloride ( 6.4 g, 105 mol %). After 40 hours at room temperature under nitrogen the reaction was complete by TLC. The reaction mixture was diluted twice with water and extracted with diethyl ether (4 x 100 ml). The
combined extract was dryed over sodium sulfate, concentrated and purified by flash chromatography silica gel, ethyl acetate:hexane, 1:1) to give 21 as a light oil 6.9 g (52.1 %); 1H NMR (CDCL3) 5.2-7.5 (br.m, 20 h), 5.07 (t, 2H), 4.12-4.26 (m,4H), 3.44 (dd 2H), 2.05 (S, 1H), 1.26 (t, 1H). 13C NMR (CDCL3): 138.772, 124.017, 123.911, 123.820, 123.342, 123.266, 123-152, 122.462, 122.417, 72.593, 72.274, 71.955, 65.105, 65.090, 65.030, 65.014, 64.954, 64.893, 62.109,
62.056, 58.877, 24.680, 24.604. 31P NMR (CDCL3) -0.158 (s).
(2R) 1-[Benzyl-(2'-bromoethyl)-phosphoroyl-2-O-palmitoyl-3-O-triphenyl methyl glycerol (22). To a stirred solution of compound 21 (6.9 g, 11.3 mmol) in dry THF (90 ml) was added triethylamine (1.79 ml, 1 10,mol %), palmitic anhydride (6.13 g, 1 10 mol%) and dimethylaminopyridine (276 mg, 20 mol%). The reaction was stirred under nitrogen for 3 hours untill TLC showed disappearance of the starting material 21. The reaction mixture was rotary evaporated to a small volume and purified by flash chromatography (silica gel, diethyl ether:hexane, 1:3 to elute UV-nonactive impurities, diethyl ether:hexane, 1 : 1 to elute compound 22). Yield 8.8 g (92.6%0, colorless oil, 1HNMR (CDCL3) 7.41-7.22 (m, 20 H), 5.20 (d, 1H), 5.04 (t, 2H), 4.23 (m, 4H), 3.58 (S, 1H), 3.41 (s, 1H), 3.23 (s, 2H), 2.33 (t, 2H), 1.62 (m, 3H), 1.24 (s, 24H), 0.88 (t, 3H). 13C NMR 172.984, 143.407, 143.285, 128.675,
128.607, 128.576, 128.523, 127.985, 127.848, 127.180, 127.135, 86.672, 77.319, 77.000, 76.681, 70.901,
70.818, 69.604, 69.581, 66.463, 66.440, 61.828,
34.284, 31.894, 29.672, 29.634, 29.611, 29.437,
29.346, 29.285, 29.247, 29.232, 29.141, 29.095,
24.832, 22.678, 14.121. 32p NMR (CDCL3) -1.327.
(2R) 1-[Benzyl(2'- bromoethyl)-phosphoro]-yl-2-O-palmtoyl-3-hydroxy glycerol 23. Procedure A To a stirred solution of compound 22 (3.2 g, 3.76 mmol) in 45 ml THF was added 45 ml 96% formic acid. After 2 hours qt room temperature the reaction was coplete by TLC. The reaction mixture was diluted twice with water, neutralized with sodium bicarbonate (3 x 300 ml). The combined extract was washed with water, dryed over sodium sulfate, rotary evaporated to a small volume and purified by flash chromatography
(silica gel, ethyl acetat: hexane, 1:3 to. elute less polar impurities, ethyl acetat:hexane, 1:1 to elute compound 23. Yield 1.65 g (72.7 %), colorless oil. Procedure B. A nitrogen purged 0.5 L round-bottomed flask fitted with condenser was charged with compound 22 (1 g, 1.17 mmol) in dry benzene (230 ml) in the presence of anhydrous CuSO4 (17.6 g). The reaction mixture was stirred at room temperature for 15 hours and then reflux for 2 hours untill the reaction was complete by TLC. The CuSO4 was filtered off through Shott filter #C and concentrated in vacuo and purified by flash chromatography (silica gel, ethyl acetate/ hexane, 1:1) to give a light yellow oil (0.47 g, 0.77 mmol) in 66 % yield. 1HNMR (CDCL3) : α7.40 (br. s, 5H), 5.2 (d, 2H), 4.2 (mult, 8H), 2.32 (t, 1H), 1.62
(pseudo t, 2H ), 1.31 (m, 24H), 0.88 (t, 3H). 13C NMR (CDCL3): 130.898, 128.872, 128.789, 128.698, 128.538, 128.516, 128.114, 127.886, 126.968, 77.326, 77.008, 76.689, 70.097, 70.074, 69.012, 68.951, 68.633,
68,604, 68.572, 68.542, 67.085, 67.047, 67.032,
66.994, 65.272, 64.195, 62.731, 62.716, 34.041,
31.902, 29.672, 29.588, 29.505, 29.444, 29.338,
29.239, 29.103, 24.749, 22.670, 14.113. 31P NMR: - 3.069 (85% H3PO4).
(2R)-1-O-(2,3.4.6-Tetra-O-benzyl-β-D-gluco-pyranosyl)- 2'-O-palmitoyl-3'-O-[benzyl(2"-bromoethyl)-phosphoril]-glycerol (17). To a stirred solution of 0-(α -D-glucopyranosyl) trichloroacetimidate (24) (390 mg, 115 mol %) in dry methylene chloride (3 ml) was added dropwise a solution of compound 6 (300 mg, 0.49 mmol) and boron trifluoride etherate (70 mg, 100 mol %) in dry methylene chloride (3 ml). The reaction mixture was stirred under nitrogen for 2 hours at room temperature, then more compound 24 (100 mg, 35 mol %) was added to bring the reaction to the end. After 4h, the reaction mixture was evaporated to a small volume and separated by flash chromatography (silica gel, diethyl ether/hexane, 1:3) to give compound 17 as a colorless oil (120 mg, 22%), which was identical to the material described earlier.
6.4. Antifungal Activity
The antifungal activity of the isolated
phosphocholine fraction was determined in vitro by using three fungal cultures -- Candida albicans,
Cryptococcus neoformans and Aspergillus fumigatus .
The method used to determine in vitro antifungal activity is discussed in McGinnis, M.R., Laboratory Handbook of Medical Mycology, Academic Press, New York, London, p661 (1980); and Droughet E., Dupont, B., Improvisi, L., Vivian, M.A. and Tortorano, A.M., "Disc agar diffusion and microplate automatized technics for in vitro evaluation of antifungal agents on yeast and sporulated pathogenic fungi" in In Vitro and In Vivo Evaluation of Antifungal Agents , Eds.
Iwata, K. and Vanden Bossche, H., Elsevier Science Publishers, New York, Oxford p303 (1986).
The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) are summarized in the table 1 below. Fungus Culture MIC (ug/ml) MFC (ug/ml)
C. albicans 0.8
C. neoformans < 0.1
A. fumigatus < 0.1 <0.4-0.8 These results clearly indicate the significant antifungal activity of the isolated fraction
containing against a variety of fungal cultures.
6.5. Antifungal Activities of the Phosphocholine
Derivatives Class
A series of related analogs to 2-palmitoyl-1-0- glucopyranosyllysolecithin obtained commercially from Avanti Biolipids have also been found to have high antifungal activities. A summary of the antifungal screening test is shown in table 2. The analog compounds were tested for their activity against
C. albicans, C. neoformans, A. fumigatus and T.
rubrum. Partial inhibition of the fungus of between 25 to 75% was measured along with the total inhibition (MIC) by these anolog compounds. A description of the partial inhibition measurement can be found in R. L. Stiller, et al The Journal of Infectious Diseases , 147, No. 6 (1983). The structure of these analog compounds is as follows.
wherein R is the group identified in table 2.
6. 6. Toxicity
The toxicity of the isolated phosphocholine derivative fraction is low, based on tests with Hep 2 cells indicating an ID50 of greater than 1000 ug/ml. The method used in determining cytotoxicity is discussed in Mosmann, T., "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays", J. Immun . Methods, 65, 55-63, 1986.
The isolated fraction having the above-described in vitro antifungal activity and low toxicity is expected to similarly exhibit significant in vivo antifungal activity against fungal infections which are dermatophytic, systemic, ophthalmic and vaginal. other human and animal infections treatable with the compounds of the present invention include
aspergilliosis, candidiasis, and cryptococcus
infections.
It is expected that the same isolated fraction would be useful in treating fungal infestation in plants as well.
It is apparent that many modifications and variations of this invention may be made without departing from the spirit and scope thereof. The specific embodiments described are given by way of example only and the invention is limited only by the terms of the appended claims.
A number of references are cited in the present specification, the entire disclosure of each of which is incorporated by reference herein, in its entirety.
Claims
1. A phosphocholine derivative having the structure of:
2. The phosphocholine derivative according to claim 1 wherein the sugar moiety is selected from the group consisting of glucose, galactose, arabinose, mannose, rhamnose, and other naturally occurring sugars.
3. The phosphocholine derivative according to claim 1 having the structure of:
2-PaImitoyI-1-O-glucopyranosyllysolecithin
4. An antifungal composition capable of inhibiting fungal growth when administered to warm blooded animals or plants comprising an effective amount of a phosphocholine derivatives having the structure of:
5. A composition comprising a phosphocholine derivative obtained from Irlbachia alata characterized by: (a) IR spectrum having peaks at
approximately 1060, 1220, 1475, 1600-1700, 2850, 2950, and 3400 cm-1;
(b) 1H NMR spectrum having major peaks at δ 1.2, 1.4, 1.7, 3.1, 3.5, 3.7 and 4.3; and
(c) FAB-/MB mass spectrum having major peaks (>40%) at m/z 657, 612, 587, 586, 555, 493, 491, 475, 403, 277, 233, 201, 194, 179, 168, 165 and 163.
6. A composition comprising a phosphocholine derivative according to claim 5, characterized by:
(a) IR spectrum substantially illustrated in Fig. 1;
(b) 1H NMR spectrum substantially illustrated in Fig. 2; and
(c) FAB-/MB mass spectrum substantially illustrated in Fig. 3.
(d) HRMS (FAB+) spectrum having a molecular ion at 673.4669 amu.
7. A composition comprising a phosphocholine derivative obtained from Irlbachia alata by a method which comprises:
(a) extracting the whole plant, the leaves, the stems, the roots or the latex of the plant Irlbachia alata with a lower alcohol of about 1-3 carbons, acetone, water or other water miscible solvent or combinations thereof to obtain an aqueous soluble fraction;
(b) subjecting the aqueous fraction to
butanol extraction and the butanol- soluble fraction to gel filtration using water and/or water and a water miscible solvent with or without a buffer as the mobile phase; or to reversed phase column chromatography using water, and/or water and a water miscible solvent as the mobile-phase; or to gel permeation chromatography using water and/or water and water miscible solvent and acetonitrile with or without a buffer as the mobile phase; or combination thereof and
(c) collecting the fractions detected by
NMR spectroscopy.
8. A pharmaceutical composition which is useful in treating a fungal infection when administered to a warm-blooded animal a therapeutically effective amount of an antifungal agent comprising a phosphocholine derivative having the structure of:
wherein one of R or R' is a sugar moiety, and the other is an acyl or sugar moiety.
9. A pharmaceutical composition which is useful in treating a fungal infestation when administered to a plant comprising an effective amount of an
antifungal agent comprising a phosphocholine
derivative having the structure of:
wherein one of R or R' is a sugar moiety, and the other is an acyl moiety or sugar moiety.
10. A pharmaceutical composition which is useful in treating a fungal infection when administered to a warm-blooded animal comprising a therapeutically effective amount of an antifungal agent comprising a compound having the structure of:
where Q is C2 to C30 alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, or branched alkynyl;
Z is oxygen or sulfure; X and Y are independent oxygen, sulfur, CH2, CF2, or N-R1;
A, B, and T are independently alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, or branched alkynyl radicals of C1 to C20 chain lengths; are independently or together cycloalkyl or bridged cycloalkyl radicals of ring size C3 to C20, or
cylcoalkenyl, bridged cycloalkenyl or
cyclo(polyene) radicals of ring size C4 to C20, cycloalkynyl, bridged cycloalkenyl or
cyclo(polyalkynyl) radicals of ring size C8 to C20;
D is oxygen, sulfur, CH2, CF2, or N-R2;
F is alkyl, alkenyl, alkynyl, branched alkyl, branced alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl or cycloalkynyl radicals containing C1 to C20 carbon atoms;
R1 and R2 are independently hydrogen, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl, bridged cycloalkenyl, or cycloalkynyl radicals containing C1 to C20 carbon atoms, or a protecting group.
11. A pharmaceutical composition which is useful in treating a fungal infection when administered to a warm-blooded animal comprising a therapeutically effective amount of an antifungal agent comprising a compound having the structure of:
where AA, BB, DD are independent of each other or equal to each other, the central carbon atom can be either the R and S optical stereoisomer or a mixture of R and S stereoisomers, and where AA, BB, and CC are as follows:
AA is A-J with A attached to the carbon atom of the three carbon central unit and J is defined below; BB is B-Y, with B attached to the carbon atom of the three carbon central unit and Y is hydrogen, alkyl, alkenyl, alkynyl, poly(alkenyl), poly(alkynyl), or poly(alkenoalkynyl) radicals comprised of C1 to C20 carbon atoms; chain lengths or alkanoyl, alkenoyl, alkynoyl. poly(alken)oyl, poly(alkyn)oyl,
poly(alkenoalkyn)oyl radicals of C2 to C20 chain lengths, alkyloxy, alkenyloxy, alkynyloxy,
poly(alkenyl)oxy, or poly(alkynyl)oxy,
poly(alkenoalkynyl)oxy radicals comprised of C1 to C20 carbon atoms;
DD is
; wherein
A is oxygen, sulfur, CH2, CF2 or N-R1;
B is oxygen, sulfur, CH2, CF2 or N-R2;
D is oxygen, sulfur, CH2, CF2 or N-R3;
J is a furanose or pyranose radical of the type:
where X is oxygen, sulfur, CH2, CF2 or N-R4;
F, K, L and M are independently hydrogen,
hydroxyl, a protected hydroxyl, alkyloxy, thiol, alkylthio, arylthio, alkylsulfonyl, arylsulfonyl, amino, ammonium, alkylamino, alkylammonium, dialkylamino, dialkylammonium, trialkylamino,
trialkylammonium where the alkyl chain on nitrogen is comprised of C1 to C20 carbon atoms; or alkyl, alkenyl, or alkynyl radicals comprised of C1 to C20 carbon atoms;
Z is oxygen or sulfur;
E is oxygen, sulfur, CH2 CF2 or N-R5;
G is alkyl, branched alkyl, cycloalkyl or bridged cycloalkyl radicals of C1 to C20 chain lengths;
Q is halogen, hydroxyl, protected hydroxyl utilizing a protecting group, O-arylsulfonyl-, O- alkylsulfonyl- or O-(perfluoroalkyl)sulfonyloxy, amino, ammonium, alkylamino, alkylammonium,
dialkylamino, dialkylammonium, trialkylamino, or trialkylammonium where the alkyl chains on nitrogen are C1 to C20; or Q=NR1R2R3, where R1, R2, or R3 can independently or together be a mixture of alkyl groups of C1 to C20 in chain length and a protecting group and R1 can equal R2, R2 can equal R3, or R1 can equal R3 which can equal R3;
R1, R2, R3, R4 and R5 are independently alkyl, alkeny, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl or cycloalkynyl radicals of C1 to C20 chain lengths, or any protecting group;
and where W1 and W2 are P(-OR), (with R being phenyl, phenylmethyl, or negatively-charged oxygen), S=O, carbon, or sulfur, provided that if W1 is not P(-OR) W2 is P(-OR) and provided that if J is furnose or pyranose radical then W2 if P(-OR).
12. A pharmaceutical composition which is useful in treating a fungal infection according to claim 11 , comprising a therapeutically effective amount of an antifungal agent comprising a compound having the structure of :
R2 is hydrogen phenylmethyl or any protecting group which can be cleaved by hydrogenolysis;
Q is halogen, hydroxyl, protected hydroxyl utilizing a protecting group, O-arylsulfonyl-, O- alkylsulfonyl- or O-(perfluoroalkyl)sulfonyloxy, amino, ammonium, alkylamino, alkylammonium,
dialkylamino, dialkylammonium, trialkylamino, or trialkylammonium where the alkyl chains on nitrogen are C1 to C20; or Q=NR1R2R3, where R1, R2, or R3 can independently or together be a mixture of alkyl groups of C1 to C20 in chain length and a protecting group and R1 can equal R2, R2 can equal R3, or R, can equal R3 which can equal R3;
13. A pharmaceutical composition which is useful in treating a fungal infection according to claim 11 , comprising a therapeutically effective amount of an antifungal agent comprising a compound having the structure of:
where R1 is phenyl, phenylmethyl, hydrogen, or nil;
R2 is hydrogen, phenymethyl or a protecting group cleavable by hydrogenolysis;
R3 is hydrogen or a protecting group;
and Q is a halogen, hydroxyl, O-arylsulfonyl-, O- alkylsulfonyl- or O-(perfluoroalkyl)sulfonyloxy;
14. A pharmaceutical composition which is useful in treating a fungal infection according to claim 11, comprising a therapeutically effective amount of an antifungal agent comprising a compound having the structure of:
where R1 is phenyl, phenylmethyl, hydrogen or nil; R2 is a protecting group, or hydrogen if R1 is not hydrogen;
Q is a halogen, hydroxyl, O-arylsulfonyl-, O- alkylsulfonyl- or O-(perfluoroalkyl)sulfonyloxy.
15. A compound having the structrure of:
where Q is C2 to C30 alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, or branched alkynyl;
Z is oxygen or sulfure; X and Y are independently oxygen, sulfur, CH2, CF2, or N-R1;
A, B, and T are independently alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, or branched alkynyl radicals of C1 to C20 chain lengths; are independently or together cycloalkyl or bridged cycloalkyl radicals of ring size C3 to C20, or
cylcoalkenyl or cyclo (polyene) radicals of ring size C4 to C20, cycloalkynyl or cyclo(polyalkynyl) radicals of ring size C8 to C20;
D is oxygen, sulfur, CH2, CF2, or N-R2;
F is alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl or cycloalkynyl radicals containing C1 to C20 carbon atoms;
R1 and R2 are independently hydrogen, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl, bridged cycloalkenyl or cycloalkynyl radicals containing Cl to C20 carbon atoms, or a protecting group.
16. A compound according to claim 6 having the structure of :
17. A method of synthesizing a phosphocholine derivative comprising the steps of:
(a) phosphorylating an alcohol with a halo- alkyl containing phosphorylating agent; and
(b) displacing the halide by an amine to produce the phosphocholine derivative.
18. A method of synthesizing a lysolecithin comprising the steps of:
(a) phosphorylating a glycosylating an acetenide derivative of glycerol;
(b) deprotecting the phosphorylated or glycosylated product; and
(c) alkylating or esterifying the deprotected product to form a lysolecithin.
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---|---|---|---|---|
US4684625A (en) * | 1982-07-08 | 1987-08-04 | Syntex (U.S.A.) Inc. | Method for enhancing the anti-infective activity of muramyldipeptide derivatives |
DE3829899C2 (en) * | 1988-09-02 | 1994-12-15 | Reutter Werner | Glycerine glycophosphatides and pharmaceutical compositions containing them for combating skin diseases |
-
1993
- 1993-10-08 EP EP93923809A patent/EP0663816A4/en not_active Withdrawn
- 1993-10-08 CA CA002146639A patent/CA2146639A1/en not_active Abandoned
- 1993-10-08 AU AU53544/94A patent/AU5354494A/en not_active Abandoned
- 1993-10-08 WO PCT/US1993/009623 patent/WO1994008563A1/en not_active Application Discontinuation
- 1993-10-08 JP JP6510144A patent/JPH08502298A/en active Pending
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