13.
`
`MBA (rel ICSO)
`IL-6 dep. prot, (rel. ICSO)
`MLR (rel ICSO)
`
`2.2
`28
`3;4
`
`5,665,772
`
`14
`{[M-{0CH3]1, 952 ([M-{0CH3+H20)]1. 934 ([M-{OCH;+
`2H20)J1. 920 ([M-(2CH30H+oH)n, 902 ([M-{OCH3f.
`CH30H+~O)J~.
`
`MBA (rel. ICS>)
`IIAi d.cp. pro!. (rd. ICSO)
`MLR (rel IC50)
`
`1.2
`3.2
`2
`
`MBA (rel. ICSO)
`IL-6 d.cp. prot (rd. JCSO)
`MLR (n:LIC50)
`
`0.9
`8
`29J
`
`Example 9: 40-0-(3-Hydroxy)propyl-rapamycin
`a) 40-0-!3-{t-Butyldimclhylsilyl)oxy )propyl-rapamycin
`The same procedure as dcsaibed in example 8, step a)
`.
`.
`·
`·
`using 3-{t-butyldimcthylsilyl)oxyprop-1-yl triflate affords 10 Example 12: 40~0-[(3S)-2.2-Dimethyldioxolao-3-yl]
`40-0-[3~t-bUtyldimcthylsilyl)oxy]propyl-rapamycin: 1H
`~::!:"::C':re as described in example 8. step a)
`NMR. (CDCl3) 80.05 (6H. s), 0.72 (IH, dd), 0.90 <98• s),
`using the triflate of glycerol acctooidc affords the title
`compouod: iH NMR (~) li0.72 (lH. dd), 1.36 (3H, s),
`1.65 (3H. s). 1.74 (311. s). 1.77 (2H, m), 3.03 (lH. m),
`3.52-3.73 (7H. m); MS (FAB) m/z 1108 ([M+Naf), 1036
`1.42 (311. s). 1.65 (3H. s), 1.75 (311. s), 3.06 (IH, m), 3.55
`ts (2H. m), 3.69 (3H, m), 4.06 (lH, dd), 4.26 (lH, m); MS
`([M-{OCH,+H20)rJ.
`(FAB) m/z 1050 ([M+NaJ1, 996 ([M-{OCH3]1. 978 ([M-
`b) 4~(3-Hydroxy)JXOPYl-rapamycin
`(OCH3+"20>J+), 960 ([M-(OCH3+2H20)11.
`'IIcatment of the compound obtained in step a) in the
`.
`.
`cooditioos ~din example 8, step b) yields the title
`comJ>oond: 1H NMR (CDC13 ) 50.72 (lH, dd), 1.65 (3H, s),
`l. 75 (3H. s). 1.80 (2H, m), 3.05 (lH, m), 3.55-3.91 (8H, m); 20
`MS (FAB) m/z 994 ([M+Na]1. 940 ([M-{OCH3n, 922
`([M-(OCH3+H20)]~. 904 ([M-(OCH3+2H20)f). 872 ([M(cid:173)
`(0CH3+Cll30H+2H20)J+).
`
`- - - - - - - - - - - - - - - - - 2s
`MBA(n:L~) ..
`1.6
`JL..6 dep. pro!. (n:l Oil)
`27
`MLlt (rd.ICSO)
`11
`
`Example 13: 40-0-{(2S)-2,3-Dihydroxyprop-l-ylJ-
`rapamycin
`Treatment of the compouod obtained io the previous
`example in the cooditions desail>cd in example 3 yields the
`· title compound: 1H NMR (CDCl3) 50.72 (lH, dd), 1.65 (3H,
`s), 1.75 (3H. s), 3.07 (lH. m), 3.68 (8H. m); MS (FAB) m/z
`30 1010 ([M+NaJ'), 956 ([M-(OCH5J'), 938 ({M-(OCH3+
`H20))"'"). 920 ([M-(0CH,+2H20)]~. 888 ([M-(OCH3+
`CH30H 2H20)r').
`
`MBA (i:d. lCSO)
`IL-6 dep. pro!. (.rd. ICSO)
`MU (n:I. ICSO)
`
`. 0.67
`9
`JO
`
`~
`
`3
`
`3
`
`2
`
`MBA (n:L ICSO)
`II,(; dep. proL (rel. ICSQ)
`MLR (ml ICSO)
`
`2
`7.6
`3.6
`
`&ample 10: 40-0-(6-Hydroxy)hcxyl-rapamycin
`a) 40-0-f 6{t-Butyldimclhylsilyl)oxy Jbexyl-rapamycin
`The same procedure as described in example 8, step a)
`usiog 6-(t-butyldimcthylsilyl)oxyhcx-l-yl triilate affords
`40-0-f 6-(t-Butyldimcthylsilyl)oxy Jhexyl-rapamycin: MS
`(FAB) mlz 1150 ([M+NaJ+).
`b) 40-0-(6-Hydroxy)hcxyl-rapamycin
`Trcatmeat of the compouod obtained in step a) in the
`conditions desaibed in example 8, step b) yields the title
`Example 14: 40-0-(2-Acctoxy)ethyl-rapamycin
`compound: 1H NMR (CDCIJ &l.72 (lH, dd), 1.38 (2H. m),
`To a stincd, cooled (0° C.) solution of 53 mg (0.055
`1.57 (4H, m), 1.65 (38. s), 1.74 (3H, s), 3.02 (lH, m); 40 mmol) of 40-0-hydroxyclhyl-rapamycin in 2 mL of meth~
`yleoe dtlorideis added 0.2 ml.of pyridine followed by 0.02
`3.49-3.72 (8H, m); MS (FAB) mlz 1036 ([M+Na]1, 982
`([M-(OCHJ1, 964 ([M-(OCH3+H20)J+), 946 ([M-{OCH3+ mL (0.281 mmol) of acetyl dtloride. The mixture is stim:d
`for 3 hand diluted with ethyl acetate, theo washed with aq.
`2}40)]), 914 (IM-{OCH3-+Cll30H+2H20)t).
`sodium bii:arbooate. cold IN HO and again with aq. sodium
`_____________ o_.s ___ 45 biaubooate. The argaoic solutioo is dried o\tc:r anhydrous
`MBA(n:L~)
`sodiilm sulfate, filtered and con~ntratcd. The residue is
`JL..6 dep. prol. (Id. &:::SO)
`8.5
`purified by column chromatography OD silica gd (30:70
`MLR (n:l ICSO)
`18
`hexane-ethyl acetate) to afford the title COIDpCIWld !IS a white
`solid: 1H NMR (~) 00.72 (ui, dd), 1.6.5 (3H, s). 1.75
`.
`Example 11: 40-0-(2-(2-Hydroxy~oxy]clhyl-rapamycin so (3H, s). 2.08 (3H, s), 3.07 (lH, m), 3.78 (2H. dd), 4.20(2H,
`dd); MS (FAB) m/z 1022 ([M+Naf), 999 (M}, 982 ((M-
`a) 40-0-(_2-t-Butyldimethylsilyl)oxyethoxy]etbyl-
`(OHh, 968 (lM-(OCH3h, 950 ([M-{OCH3+H2+H,O)]~.
`rapamyan
`.
`.
`932 ([M-(OCH +2H O)r) 918 ([M-(2CH OH+oH)J~
`The same iroccdure as desaibed to example 8, step a)
`3
`900 ((M-(OCH ~ OH+2H O)t).
`'
`usiog 2-12-(t'-butyldimctb>:lsilyl)oxy-ethoxy]elhyl triftate
`affords 40-0-(2-(t-butyldimethylsilyl)oxyethoxy]ethyl- "
`rapamycin: 1H NMR. (CDCIJ &l.06 (6H, s), 0.71 (lH. dd),
`0.88 (9H, s), 1.65 (38, s), 1.74 (3H, s), 3.07 (lH, m).
`3.51-3.79 (UH. m); MS (FAB) m/z 1138 ([M+Na]~; 1115
`(M'), 1097 ([M-H,0)1. 1084 ([M-(OCH3J1, 1066 ([M(cid:173)
`(OCH3+H20)]1. 1048 ([M-{OCH3+2"20)J'), 1034 ([M- 1iO
`Example 15: 40-0-(2-Nicotinoyloxy)ethyl-rapamycin
`(2CH30H+OH)f). 1016 ([M-(OCH3-+CH30ff.+.2H20}]1.
`The same procedure as described in the previous example
`b) 40-0-{2-(2-Hydroxy)cthoxy]ethyl-rapamycin
`Tn:atmcot of the compound obtained in step a) in the
`using nicotinoyl chlcride hydrocblcridc affords the title
`compouod: 1H NMR (C00.3) li0.72 (lH. dd). 1.65 (3H, s),
`cooditions dcsaibcd lo example 8, step b) yields the title
`compound: 1H NMR (CDCIJ 00. 72 (lH, dd), 1.65 (3H, s), 6S 1. 75 (3H, s). 3.07 (lH, m), 3.94 (2H, dd). 4.49 (2H, t), 7 39
`1.74 (3H. s). 3.05 (18, m), 3.51-3.77 (llH, m); MS (FAB)
`(lH. dd). 8.31 (lH,; ddd), 8.78 (lH.ddd). 9.24 (lH, dd);MS
`m/z 1024 ([M+Na)1. 1001 (M"). 983 ([M-H20)1. 970
`_(FAB) m/z 1085 ([M+Na]+), 1063 ([M+HJ"1. 1045
`
`
`
`

`
`15
`([M-OHJ'), 1031 ([M-(OCH3t). 1013 ({M-(OCH3+H2
`O)t).
`
`MBA (rel. ICSO)
`JL.6 dcp. pmL (rel ICSO)
`MLR (rel lCSO)
`
`I.I
`6.9
`s
`
`5,665,772
`
`16
`
`MBA (rel JCSO)
`IL-6 dcp. pmL (rel. ICSO)
`MLR (rel lCSO)
`
`2.6
`10.3
`s
`
`s
`
`Example 19: 39-0-Desmetbyl-39,40-0,0-ethylene(cid:173)
`rapamycin
`To a stirred, cooled (-20° C.) solution of 48 mg (0.05
`Example 16: 40-0-[2-(N-Morpholino)acetoxy]ethyl-
`mmol) of 40-0-hydroxyethyl-rapamycin and 0.023 mL
`.
`10 (0.20 mmol) of 2,6-lutidine in 0.S mL of methylene cbloride
`rapamycm
`is added 0.008 mL (0.05 mJilol) of trifilc anhydride. The
`a) 40-0-(2-Bromoacetaxy)ethyl-rapamyci.n
`mixture is stirred at this temperature for 2 !J. then allowed to
`The same procedure as descnlx:d in example 14 using
`warm to room temperature and still'ed for one more hour.
`bromoacc:tyl chloride affords 40..0-(2-bromoacetoxy)ethyl-
`rapamyci.n: 1H NMR. (CDC13) 00.72 (lH, dd), 1.67 (3H, s),
`The reaction is quenched with aq. sodium bicarbonate and
`1.76 (3H; s), 3.03 (lH. m), 3.82 (2H, m), 3.87 (2H, s), 4.31 15 the resulting mixture is extracted with three portions of ethyl
`acetate. The organic solution is washed with brine, dried
`(2H, m); MS (FAB) mlz 1100, 1102 ([M+Naf), 1077 (M'),
`over aiihydrous sodium sulfate. filtered and concentrated.
`1061 (fM-H20]"'). 1046, 1048 ([M-(OCH3J'), 1028, 1030
`The residue is purified by colmnn cbromatography on silica
`([M-(0CH3+H20>J+), 1012 ([M-(OCH3+2H20W), 996
`gel (30:70 hexane-ethyl acetate) to afford the title compound
`([M-(2CH30H+oH>J+). 980 ([M-(OCH3+cH30H+2H2 20
`as a white solid: 1H NMR. (CDCl,) Sl.66 (3H, s), 1.75 (3H,
`0)]"'),
`.
`b) 40-0-[2-(N-Marpholino)acetoxy]ethyl-rapamycin
`s). 3.14 (3H, s), 3.35 (3H, .s), 3.76 (4H, s); MS (PAB) m/z
`To a stirred, cooled (-4.5° C.) solution of S4 mg (0.05
`948 ([M+Na]'), 925. (M"'), 908 ([M-OBJ'), 894 · ([M-
`(OC"3]"'), 876 ([M-(OCH3+H20)]"'), 858 ([M-(OCH3+
`mmol) of 40-0-(2-bromoacetoxy)elhyl-rapamyci.n in O.S
`mL of DMF is added a solution of 0.022 mL (0.25 mmol) of 2S 2H20)]"'), 844 ([M-(2CH30H+QH)T'), 826 ([M-(OCH,+
`morpholine in 0.2 mL of DMP and the resulting mixture is
`CH30H+2H20>J').
`stirred at that ·temperature for 1 h, then treated with aq:
`sodium bicarbonate. tbis mixture is extracted three times
`1.6
`MBA (rcl. ICSO)
`with ethyl acetate. The organic solution is washed with
`22.9
`JL6 dcp. pmL (rel.ICSO)
`brine, dried over anhydrous sodium sulfate, filtered and 30 ___ MLR __ <re_i_
`. .rc_SO)_. ____ ___ 16 ___ _
`concentrated. The residue is purified hy column chromatog(cid:173)
`raphy on silica gel (95:5 ethyl acetate-methanol) yielding the
`title compound as an am.mphous white solid: 1H NMR
`(CDC13) B0.72 (lH, dd), 1.67 (3H, s), 1.76 (3H, s), 2.60 (3H,
`m), 3.07 (lH, m); 3.24 (2H, s), 3.78 (8H, m), 4.27 (2H; t); 35
`MS (FAB) mlz 1107 ([M+NaJ"'), 1085 ([M+H]"'), 1067
`([M-OHj+), 1053 ([M-(OCH3 )"'), 1035 ([M-(OCH_,+
`H20)f).
`
`Example 20: (26R)-26-Dihydro-40-0-(2-hydroxy)ethyl(cid:173)
`rapamycin
`a) (26R)-26-Dihydro-40-0-[2-(t-Butyldimethylsilyloxy)J
`ethyl-rapamyci.n
`.
`Jn·4.5 mL of2:1 accton.itrilc-acetic acid is dissolved315
`mg (1.2 mmol) of tetrlimethylammonium(cid:173)
`triacetoxyborohydrlde. The resulting solution is stiired for 1
`at room temperature and cooled to -35° C, then 161 mg
`40 (0.15 mmol) of 40-0-[2-(t-butyldimethylsilyloxy)Jethyl-
`rapamydn is added. The resulting mixture is stirred at the
`same temperature overnight aDd is quenched by tbe addition
`of aq. sodium biairbonate. The m.ixiure is extracted· with
`three portioos of ethyl acetate. The oigaaic solution is
`Example 17: 40-0-(2-N-Imidazolylacetoxy)ethyl- 45 washed with aq. sodium bicarbonate, two portions of 30%
`aq. Rochelle's salt and brine, dried over anhydrous sodium
`rapamycin
`The same procedure as described in example 16, step b)
`sulfate, filtered and concentrated. The residue is pudfi.ed by
`using imidazole affords the title coropound: 1H NMR
`column chromatography on silica gel (40:60 hexane-ethyl
`acetate) to.afford the title compound as a while solid: 1H
`(CDCl~B0'.72(1H,dd), 1,67 (3H, s), 1.78 (3H, s),3.06(3H,
`m);3.80(2H, m),4.32 (2H, m), 4.73 (2H, s),6.97 (lH,dd), '° NMR. (CDQ~ 00.06 (6H, s), 0.73 (lH, dd), 0.90 (9H, s),
`7.09 (lH, dd), 7.52 (lH, dd); MS (PAB) m/z 1066
`1.64 (3H, s), 1.67 (3H, s), 3.02 (lH, m), 3.15 (lH, m), 3.64
`([MtNaJ'), 1048 ([M+QH]"'), 1034 ([M-(OCH3]"'), 1016
`(3H, m), 3.71 (2H, dd). 3.91 (lH, s); MS (FAB) m/z 1096
`([M-(0CH3+H20)J).
`([M+Na]"'), 1041 ([M-HOCH3J'), 1024. ([M-(0CH3+H2
`.
`0)1'), 1006 ([M-(0CH3+2H20)]"'), 974 ([M-(OCH3+
`----------------- s' CH30H+2H20)J"').
`
`- - - - - - - - - - - - - - - - -
`MBA (rcL ICSO)
`1.3
`JL6 dcp. pmL (rel ICSO)
`4
`3.s
`MLR (ml IC$O)
`
`MllA (reL ICSO)
`JL6 dep. proL (mL ICSO)
`MLR (rel 100)
`
`I
`7.6
`3.4
`
`3.9
`MBA (rel ICSO)
`'3
`II..6 dep. pmL (rel. JCSO)
`Example 18: 40-0-(2-(N-Methyl-N'-piperazinyl)acetoxyJ 60 ---MLll--(lel.-IC$0) ________ 1s ___ _
`ethyl-rapamycin
`The same procedilre as descnbed in example 16, step b)
`Example 21: 28-0-Methyl-rapamycin
`using N-methylpiperazine affords the title compOWld: 1H
`Toa stined solution of 103 mg (0.1mmol)of40-0-TBS-
`NMR. (CD03) 60.72 (lH. dd), 1.67 (3H. s), 1.77 (3H, s),
`rapamyci.n (obtained hy silylation of rapamycin with 1 eq. of
`2.78 (4H, sand m), 3.02 (4H, bs). 3.08 (lH, m), 3.32 (2H, 6S TBS tri1late in methylene chloride in the presence of 2 eq.
`of 2,6-lutidine at 0° C.) in 0.5 mL of niethylene chloride iS
`s), 3.80 (2H, dd). 4.27 (2H. t); MS (PAB) m/z 1098
`([M+Na]~, 1066 .([M-(OCH3n.
`added 85.B mg (0:40 mmol) of proton sponge followed hy 44
`
`
`
`

`
`5,665,772
`
`1.!18
`1240
`1300
`
`MBA (n:L IC.50)
`IL-6 dcp. pmL (n:L ICSO)
`MLR (n:I. ICSO)
`
`17
`18
`exlraCted 3x with 5 mL ethyl acetate. the organic phases arc
`mg (030 mmol) of trimcthyloxonium tctrafluoroborate. The
`combined and dried ovc.r sodium sulfate. The solvent is
`.resulting lrown heterogeneous mixture is s~cd overnight.
`evaporated and the residue chromatographcd on 10 g silica .
`quenched with aq. sodium bicarbonate and extracted with
`ethyl accta1c. The organic solution is washed with lN HO,
`gel eluting first with cd!yl acetate followed by ethyl acetatd
`aq; sodiwn bicarbonate and brine. then dried over anhydrous s methanol/acetic acid 50/50/0.S to afford the title product:
`sodium sulfate, filtered and concentrated.. The residue is MS (FAB) m/z 1021 (20%, M+Na); 967 (28%, M-MeOH);
`pwiftcd by column chromatography on silica gel (60:40
`949 (100%, M-(Me0H+H20)
`·
`hexane-ethyl acetate) to afford 40-0.t-butyldimelhylsilyl-
`H-NMR (CDCl3) d: 0.71 (lH. q, J=l2 Hz); 1.98 (3H. s);
`28-0-metbyl-rapamycin. The latter compound is dcsilylated
`3.13 (3H, s); 3.34 (3H, s); 3.44 (3H, s); 4.75 (lH, s)
`in the conditions descnbed in example 10. step b) to affard. 10 MBA (rel IC50): 1.1
`·
`·
`after PnC (ethyl acetate), the title compound as a white
`IlAi dep. prol. (rel ICSO): 23
`solid: 18 NMR (CDCl,) 00.70 (lH, dd). 1.68 (6H, 2s), 2.95
`&ample 24: 40-0-(2~11icotinamldoethyl}rapamycin
`101 mg of 40-0-(2-aminocthyl}-rapamycin acetate arc
`(lH. m), 3.13 (3H, s), 3.14 (3H, s). 3.28 (3H. s), 3.41 (3H,
`dissolved in S ml ethyl acetate and extracted 2x with
`s); MS (FAB) m/z 950 ([M+Naj+), 927 (W), 909
`([M-H20t'). 896 ([M-OCH3l}, 878 ([M-(OCH3+H 20))1. is saturated sodium bicarbonate. The organic phase is dried
`864 ([M-(OCH3-+Cll30H)]j, 846 ([M-(2CH30H+oH)T').
`·over sodium sulfate and the solvent evaporated. The residue
`is dissolved in 2 mL 11IP and treated with 22 mg DCC and .
`832 ([M-(OCH3+2CH30H)l'), 814 ([M-(3CH30H+oH)f).
`15 mg nieotinic acid. After 15 h at room temperature the
`reaction mixture is evaporated and the residue chromato-
`20 graphed on silica geL eluting with cd!yl acetate followed by
`ethyl acetate/methanol 9/1. to afford the title product: MS
`(FAB) mJz 1084 (80%, M+Na); 1062 (40%. Mii); 1038
`(100%, M-McOH); 1012 (50'16. M-(Mc0H+H20)
`H-NMR (CD03} d: 0.72 (lH. q, 1=12 Hz); 3.13 (3B. s);
`Bltamplc 22: 40-0-{2-aminocthyl)-.rapamyciD
`25 3.33 (3H. s); 337 (3H. s); 739 (lH, dd); J=6 Hz, J=8 Hz),
`a) 40-0-(2-bromoetbyl)-rapam.ycin
`A solution of 914 mg rapamycin in 5 mL toluene con-
`8.19 (lH, d, J=8 Hz); 8.7.5 (lH, d. ]=6 Hz); 9;04 (lH, broad
`taining 0.64 ml of 2,6-lutidine and l.28 g of 2-bromoethyi
`s)
`trifiate is heated at 65° C. for 18 h. The reaction mixture is MBA (rel ICSO): 1.2
`IL-6 dep. proL (rel ICSO): 2.8
`then cooled to room temperature, poured on 20 ml of a
`saturated bicarbonate solution and extracted with 3x20 mL 30 Example 2.5: 40-0-(2-(N-Methyl-imidazo-2' -
`ethyl acetate. The OJganic J,ilases arc dried over sodium
`ylcarbcthoxamido)ethyl)-rapamycin
`To a solution of 30 mg N-mcthyl-imidazol-2-carboxylic
`carbonate and the solvent removed at reduced pressure on
`acid in 1 mL DMF are added 58 mg DCC and 58 mg HOBT.
`the rotary evap<rat<r. The residue is chromatographed on
`100 g silica gel. cluting with hexane/ethyl acetate 3fl to
`After 2 h, 150 mg 40-0-(2-aminoethyl)-rapam.ycin arc
`aff<rd 40-0-(2-lromoethyl}-rapam.ycin as an amorphons 3.5 added and the reaction mixture is stirred for 18 at room
`temperature. The suspension is then rutacd, the .filttate
`solid: MS (FAB) m/z 1044 and 1042 (100%; M+Na}; 'I72
`diluted with 5 mL ethyl acetate and washed with 2x2 mL of
`and 970 (.5.5%, M-(Mc0H+H20)).
`H·NMR (CDCl3) d: 0.72 (IH, q, J=l2 Hz); 3.13 (3H, s);
`a samrated aqueous bicarboilate solution. The organic phase
`is dried over sodium sulfate and the solvent evaporated
`3.33 (3H, s); 3.45 (3H,s);.3.9 (4H, m); 4.78 (lH, s)
`40 under reduced pressure. The residue is ch.romatographed
`b) 40-0-(2-azidocthyl)-rapamycin
`A solution of 2.4 g of 40-0-(2«omoelhyl)-rapamycin In
`over 10 silica gel; eluting with hexane/ethyl acetate 1A and
`40 mL DMF is treated with 0.19 g sodium arlde at room
`then.ethyl acetate to aff<rd the tide product:
`tcmpaaturc. After 2 h, the mixture is poured on 100 mL of MS (FAB) m/z 1087 (36%, M+Na); 1065 (57%,MH); 1033
`saturated sodium bica1bonatc and extracted with 3xl00 mL
`(lOO'li, M-MeOH); 1015 (46%; M-(Me0H+H20)
`ediyl acetate. The OJganic phases are i:ombined, dried over 4.5 H-NMR {CDCl3) d: 0.72 (lH, q, J=l2 H7.); 3.13 {3H, s);
`sodium sulfate and the solvent removed undu reduced
`3.33 (3H, s); 3.46 (3H. s); 4.03 {3H, s): 6.93 (lH, broad s);
`pressure. The aude product is puiificd by chromatography
`6.98 (lH, broads); 7.78 {lH, m);
`MBA (rel ICSO): 1.1
`oo silica gel eluting. with hcxandcd!yl to afford 40-0-(2-
`azidoethyl)-rapamycin: MS (FAB): 1005 (100%, M+Na); ~ dep. prol (rel ICSO): 7
`so Example 26: 40-0-(2-ethoxycarbonylaminoethyl)-
`951 (24%, M-McOH); 933 (57%, M{MCOH+H20)
`rapamycin
`c) 40-0-(2-aminoethyl)-rapamycin
`To a solution of 230 mg 40-0-(azidocthyl)-rapamycin in
`A solution of 200 mg 40-0-(2-azidoetbyl)-rapam.ycin in 3
`3 mL ofTIIF/watcr 5/1 at room temperature lire added 307
`mL THF/watcr 5/1 is treated With 267 mg tripbenylphos-
`mg of triphenylphosrb.ine. The reaction mixture is becomes
`phine for 7 h at room temperature. Then 0.4 mL pyridine are
`yellow. After 7 h. the reaclion mixture is loaded on x g silica ss added followed by 194 µL ethyl chloroformiatc. After 2 h.
`the reaction mixture is poured on 5 mL ethyl acetate and
`gel and chromatognipbed with ethyl acetak/methanol/acetic
`acid 5CYS0/0.5 to aff<rd the tit.le product in the fOim of its
`washed successively with 10 mL saturated sodium
`bicarbonate, S mL water and 5 ml 10% citric acid. The
`acetate:MS(FAB)m/z979(45%,M~a);957(100%MH);
`925 (63%, M-MeOH); 9<J7 (25%. M-(Me0H+B20)
`organic phase is dried over sodium sulfate and the solvent
`MBA(rcl ICSO): 0.7
`& evaporated. Theresiducischromatographedover20gsilica
`IL-6 dep. prol.Jrcl ICSO): 10
`gel, eluting with ethyl acetate followed by cd!yl acdAt.e/
`me.thauol 9/1, to afford the title product.: MS (FAB) rDlz
`Example 23: 40-0-(2-acetaminocthyl)-rapamycin
`To a solution of 101 mg of the acetate of 40-0-(2-
`1051 (35%, M+Na); 997 (30%. M-McOH); 979 {lOO'ii,
`aminocthyl)-rapamycin in 2 mL THF arc added 0.02 ~ M-(Me0H+H20)
`pyridine and 0,07 mL acctyl chloride. The .reaction mixttuc 6.5 H-NMR (CDCl3) d: 0.71 ( JH. q. J=l2 Hz); 1.24 (3H, t. .J=8
`is kept at room temperature for 18 hand then poured on 7
`Hz), 3.13 (3H,s); 3;34 (3H, s); 3.43 (3H. s);4.10(2H.q.J=8
`mL saturated sodium bicart>onate. The aqueous phase is
`Hz); (lH, m)
`
`
`
`

`
`5,665,772
`
`20
`
`.
`
`19
`
`20
`following charactcrlsti.c physical data: 1H NMR (CDCL,)
`MBA {rel. ICSO): 1.1
`Il.-6 dcp. prol. (rel. ICSO): 1.7
`51.61 (3H,dJ=l Hz, C17-CH~, 1.76 (3H,d.l==l.2 Hz, C29-
`Eumple 'n: 40-0-(2-tolylsulfonamidoelhyl)-rapamycin
`CH3), 2.42 (lH,dJ=l4.5 Hz, H-9). 2.74 {lH,d,1=14.5 Hz,
`A solutitin of 200 mg 40-0-(2-aminoethyl)-rapamydn in
`H-9), 3.13 (3H,s,Cl6-0CH3) 3.5 (3H,s,C27-0CH3), 3.40
`3 mL THF is treated with 0.4 mL pyridine and 390 mg tosyl s (3H,s,C39-0CH~, 5.40 (lH,d,J=lO Hz. H-30), 5.57 (lH,
`chloride and the reaction mixture is stiired for 12 hat room
`ddJi.=8.6 Hz, 12=15 Hz, H-22), 5.96 (lH.dJ=9 Hz, H-18),
`temperature. The solution is then poured onto 5 ml of a
`6.09 (111.dJ=l.7 Hz, 10-011), 6.15(1H,dd,J1=10Hz,12=15
`Hz, H-21), 6.37 {1H,dd,J1=1.5 Hz, l:z=.5 Hz. H-19), 6.38
`sawrated bicarbonate solution and the aque0us phase is
`(1HJ=9.5 Hz, H-20). 13C NMR (ffiCI:i) 538.5 (C-9), 98.0
`extracted with 2x5 mLethyl acetate. The combined organic
`phases are washed with 5 mL of 10% cilric acid and 5 ml. 10 (C-10), 170.7 {C-1), 173.0 (C-8), 208.8 (C-32), 216.9
`water. After drying on sodium sulfate the solvent is evap<>-
`(C-26).
`rated and the residue chromatographed on 20 g silica gel,
`MS(FAB) m/z 922 8[M+Na1), 899 ~. 881 ([M-B20]~.
`868 ([M-OCHJ"'). 850 ([M-(H2o+ocH3)]).
`eluting wilh hcxandcthyl acetate 111 to afford the title
`JR {major peaks)(cm-1
`product as a white foam: MS (FAB) m/z .1133 {lOO'J&,
`) 987, 1086, 1193, 1453, 1616, 1717,
`M+Na); 1078 (25%, M-MeOH); 1061 (85%, M-(MeOH+ JS 1739, 3443.
`H20))
`MBA (rel. I~: 1
`H-NMR (CDCI.3) d: 0.68 (lH, q, 1=12 Hz); 2,43 (3H, s);
`MLR (rel. I~: 14
`Il.-6 dep. proL (rcL IC'°): 9
`3,13 (JH, s); 3.35 (3H, s); 3,41 (JH, s); 4.76 (lH, s); 5.85
`(lH, t. J=6 Hz); 7.30 (2H, cl. J=8 Hz); 7.75 (2H, cl. 1=8 Hz).
`Example 30: Dihydrogenation of keto at C26
`To a stiired solution of 421 mg (1.6 mmol) of tetram-
`MBA (rel. ICSO): 15.9
`Il.-6 dcp. pro!. {rel. ICSO): 14
`ethylamnionfum triacetoxybarobydride in 2 ml of acetoni-
`Example 28: 40-0-[2-(4' ,5'-dicarboethoxy-1' ,2' .3'-triazol-1'-,
`trile is added 2 ml of acetic acid. The resulting mixture is
`stirred for JO minutes at room temperature and coOied to
`yl)-ethyl]-rapamydn
`-35° C. At this temperature a solution of 180 mg (0.2 mmol)
`98 mg of 40-0-(2-aiidoethyl)-rapamydn and 32 mg
`--diethyl.acetylene dicarboxylate are suspended in 0.5 ml zs of 9-deoxo-rapamydn in 1 ml of acetonitrile is added and
`toluene and heated at 65° C. for S h. The reaction mixture is
`the resulting mixture is allowed to stir for 24 hours. The
`mixture is quenched with a saturated solution potassimn
`then cooled at room temperature, loaded on 10 g silica gel
`and eluted with hexane/ethyl acetate 1/1 to afford the title
`tartrate solution and allowed to warm to room temperature.
`Stining is continued until both layers are clear and ethyl
`product: MS (FAB) mJz 1175 (20%, M+Na); 1121 (15%,
`30 acetate is added. The layers are separated and the aqueous
`M-MeOH); 1103 (60%, M-(Me0H+H20))
`H-NMR. (CDC13) d: 0.62 (IH, q, 1=12 Hz); 1.40 (JH, t, J=8
`layer is extracted twice with ethyl acetate. The resulting
`Hz); 1.42 (3B, t, J:z8 Hz); 3.13 (JH, s); J.2S (JH, s); 3.33
`organic solution is washed once with a 10% sodium bicar-
`(JH, s)
`·
`.bonate solution and twice with saturated brine, then dried
`MBA (rel. ICSO): 2.7
`over anhydrous sodium sulfate, filtered and concentrated
`IL-6 dep. prol. {rel. ICSO): 12
`3' under reduced pressure. The residue is purified by column
`chromatography on silica gel (90: 10 AcOEt-heune ). As the
`The previous examples may also be made using as
`starling material in this case was 9-deoxorapamycin, the
`starting material instead of rapamycin, ·9-deoxo-rapamycin,
`26-dihydro rapamycin, or 9-deoxo-, 26-dihydro-rapamycia.
`fulaJ. compound is 9-dcoxarapamycin, 26-dihydrorapamydn
`Alternatively, and prcf erably, as described e.g., in example
`is produced as a colorless foam, having the following
`20, the rapamydn compounds of the above examples may be 40 charactcrutic spectroscopic data: 1H NMR (CDCL.,) (major
`isomer) B0.9 (3H,dJ=6.9 Hz, CH!Jb), 0.93 (JH,d,1=6.9
`hydrogenated or reduced, using suitable protecting groups
`Hz, CH.CIL), 1.00 (3H.dJ=6.9 Hz, CJIQ!,), 1.07 (JH,dJ=
`where oeressary. The following novel methods for reducing
`6.9 Hz, CHCII,), 1.17 (JH,d,1=6.9 -Hz. CHQL), 1:61
`the keto at C9, or hydrogenating the keto at C26 are
`(3B,dJ=l Hz, Cl7-CH3 ), 1.73 (3H,d,1=1.2 Hz, C29-CH3),
`provided:
`4S 2.43 (1~!.ddJ==4.l and 16.0 Hz, H-33), 2.46 (IH,dd.1=13.8
`Example 29: Removal of keto at C9
`A stream of hydrogen suliide is passed at room tcmpe(a-
`Hz, H-9), 2.58 (1H,m,H-2S), 2.77 (1H,dd,Jml3.8 Hz, H-9),
`turc through a stil:rcd solution of 3.2 g (3.5 mmol) of
`2.82 (lH,ddJ=83 and 16;0 Hz, H-33), 3.17 (lH,ddJ==4,l
`and 9.2 Hz, H-27), 3.61 (211,m, H-14 and ms), 5.19
`rapamycin in SO ml pyridine and 2.S ml DMF. The solution
`turns from colorless to yellow. After two hours, the intro-
`(lH,dddJ=4.l, 4.6 and 8.3 .tiz, H-34), S.49 (lH, broad
`dudion of hydrogen si.ilfide is stopped and stirring is eon- so d.i=S.O Hi, H-2), 5.S6 (1H,dJ=9.1 Hz, H-30), 5.75 (IH,dd,
`tinoed far five days, during which .time the solution turns
`1==6.9 and 14.7 Hz, H-22), 5.76 (lH,s,lO-OH), S.99 (lH,
`gradually orange. TLC and HPLC analysis verifies complete
`broad d,1=92 Hz, H-18), 6.10 (lH,m,H-21), 6.36 (2H,mJI-
`comumptioa of the starting maleri.al and the presence of a
`19 and H-20);
`single new compound. The solution is purged with nitrogen MS (FAB) m/z 924 ([M+Naj), 852 ([M-(B20iCH30)J1.
`for one hotD' and concentrated under reduced JXCSSUl'e. The ss MBA (reL IC,o): 47
`residue is taken up in ethyl acetate, washed with cold IN MLR (rel. IC,J: 134
`Il.-6 dep. proL (iel. I~): 78
`HO solution (Jx), saturated sodium bicarbonate solution
`26-dihydrorapamycin is prepared in the same manner,
`and saturated brine. The organic layer is dried over anhy- ·
`using mpamydn in place of 9-deoxorapam.ydn. This p-od-
`drous sodium sulfate and filtered and conceatrated _under
`n:duced pzusure. The residue is taken up in ether and 60 uct has the following characteristic spectroscopic data: 13C-
`precipitated sulfur is filtered off. Concentration of the ethe-
`NMR {C003 ) (major' isomer) d=208.3 (C-32); 194.0 (C-9);
`real solution followed by column chioniatography on silica
`169.J {C-1); 166.6 (C-8); 140.9 (C-22); 136.S (C-29); 136.2
`gel (10:4:1 CH 2 Cl 2 /i-Pr 2 0/Me0B) yields
`(C-17); 133.5 (C-20); 129.1 (C-21); 128.7 (C-18); 126.2
`9-deoxorapamycin as a colorless foam. The identity of lhe
`{C-30); 125.3 (C-19); 98.6 (C-10); 84.4 (C-39); 83.9 (C-16;
`product is confimled by nuclear magnetic resonance spec- 6!I 81.6 (C-27); 75.4 (C-34); 74.3 (C-28); 73.9 (C40); 72.9
`troscopy (NMR), mass spectrometry (MS); and/or infrared
`(C-26); 67.4 (C-14); 59.1 (27-00l3); 56.6 (39-00I,); SS.9
`spectrosopy {JR). 9-deoxarapamycin is found to exhibit the
`(16-0Di3); Sl.J (C-2); 46.8 '(C-31); 44.3 (C-6); 40.4
`
`
`
`

`
`5,665,772
`
`21
`(C-33); 40.4 (C-25); 39.5 (C-24); 38.8 (C-15); 38.0 (C-36);
`343 (C-23); 34.2 (C-38); 33.5 (C-11); 33.3 (C-37); 33.2
`(C-35); 31.5 (C-42); 31.3 (C-41); 30.9 (C-13); 27.1 (C-12);
`27.0 (C-3); 25.2 (C-5); 21.4 (23-.CH,,); 20.7 (C-4); 17.3 (ll(cid:173)
`.CH~; 16.1 (31-,CH3); 15.9 (35-CU3); 14.4 (25-013 ); 14.2
`(29-,CH,,); 10.3 (17-0f,,).
`MS (FAB) m/z: 884 (M-0CH3 , 35%); 8(,6 (M-[OCH3+
`H20J~ 1Cl0%; 848 (M-[0CH3+2 H20J, 40%).
`MBA (rel ICso): 1.7
`MlR (rel. IC'°): 1
`Il..-6 dep. prol. (rel IC30): 7 .5
`.
`We claim:
`I. A cOmPoond of the foonula
`
`24
`
`10
`
`22
`wherein R1 is bydroxy(C1-.s)alkyl or
`hydroxy(C1-3)al.koxy(C 1-3)alkyl.
`2. A compound according to claim 1 in which R1 is
`5 hydroxy(C1_,,)alk:yl or hydroxy(C1_ 3)al.koxy(C1-3)alkyl.
`3. A compound according to claim 1 in which R1 is
`.
`hydroxy(C 1...,,}ali:yL
`4. A compound according to claim 1 in which R 1 is
`hydroxy(C1_j)alkoxy(C1_ 3)alkyl.
`5. The compound according to claim 1 which is 40-0-
`(3-bydroxypropyl)-rapamycin.
`6. The compound according to claim 1 which is 40-0-
`[2-(2-hydroxyethoxy)ethyl]-rapamycin.
`7. Aphamw:eulical composition comprising a therapeu(cid:173)
`tically effective amount of a compound according to claim
`1 and a pharmaceutically acceptable caaicr thcrctor.
`8. A method of inducing an immunosuppressant e1fect in
`20 a subject in need of immllnosuppression, which comprises
`administering to said ~ubjcct an immunosuppressant effec(cid:173)
`tive amount of a compound according to claim 1.
`9. A method of preventing allograft rejection in a subject
`25 in need of such treatment, which conlprlses administering to
`said subject a compound according to clairn 1 m an amount
`effective to prevent allograph rejection.
`10. The compound according to claim 1 which is 40-0-
`30 (3-hydroxyethyl)-rapamycin.
`
`15
`
`* * * * *
`
`
`
`

`
`UNITED STA TES PA TENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`
`: 5,665, 772
`PATENT NO.
`: Septeaber 9, 1997
`DATED
`INVENTOR(S) : Sylvain Cottens and Richard Sedrani
`
`It is ceriffled that error appears in the above-Identified patent and that said Letters Patent is hereby
`conected as shown below:
`
`Claim 10, lines 1-2, delete •40-Q-(3-hydroxyethyl)-rapamycin" ~ replace
`
`it with ~ 40-0-(2-hydroxyethyl)-rapamycin ~.
`
`Signed and Sealed this
`
`Thirtieth Day of June, 1998
`
`Aue.rt:
`
`Arresting Officer
`
`ComlJfission~r nf Pal,nts and Tradrntarb
`
`BRUCE LEHMAN
`
`
`
`

`
`I
`
`Appendix F
`
`lJNHl!;JJ'~lA'IE~ UEYAKTMENT Uff t:UMM.1£KCI£
`Patent and Trademark Office
`Address: COMMISSIONER o:'
`rENTS AND TRADEMARKS
`Washingtcin, o:c:"2fi131' .
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`000000
`
`MI23K
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`below.
`If a maintenance fee payment is defective, the reason is indicated by code in column 11, "ST AT" below. TIMELY
`CORRECTION IS REQUIRED IN ORDER TO AVOID EXPIRATION OF THE PATENT. NOTE 37 CFR 1.377.
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`If the statement of small entity status is defective the reasori ·is indicated below in column I 0 for the related patent number.
`THE STATEMENT OF SMALL ENTITY STATUS WILL BE ENTERED UPON RECEIPT OF ACCEPTABLE
`CORRECTION.
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`ITEM PATENT
`NBR
`NUMBER
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`SERIAL
`NUMBER
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`PATENT
`DATE
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`FILE
`DATE
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`1
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`5,665,772 183
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`850
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`08/416,673 09/09/97 04/07/95 04 NO
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`PAID
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`UNIT.KU STATl!:S ll~AKTMl!:NT 014' LUMMl!:KLI!:
`Pat~nt and Trad~.:.ifrth ·ce
`·
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`. COMMISSI6N£r.l'A TENTS AND TRADEMARKS
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`
`If a maintenance fee payment is defective, the reason is indicated by code in column 10, "STAT" below. TIMELY
`CORRECTION IS REQUIRED IN ORDER TO AVOID EXPIRATION OF THE PATENT. NOTE 37CFR 1.377. THE
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`STATEMENT OF SMALL ENTITY STATUS WILL BE ENTERED UPON RECEIPT OF ACCEPTABLE CORRECTION.
`
`PATENT
`NUMBER
`5,665,772
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`FEE
`CODE
`I 552
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`FEE
`AMT
`$2,300.0o
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`SUR
`CHARGE
`$0.00
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`APPLICATION
`NUMBER
`08/416,673
`
`PATENT
`DATE
`09109191
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`FILE
`DATE
`04101195
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`YR ENT STAT
`08 NO PAID
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`DIRECT YOUR RESPONSE TOGETHER WITH ANY QUESTIONS ~BOUTTHIS NOTICE TO:
`Mail Stop: M. Correspondence, Director of the United States Patent and Trademark Office
`P.O. Box 1450
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`
`
`

`
`Appendix G
`
`.
`
`·
`
`"" \
`
`-o
`~
`
`loo-7 f?J~-jec_T
`case No.
`. .
`I
`I
`a<?! 41.L, 4 I. 7 3
`ApplicabUl/Serial No.
`.
`,
`'--......T
`Malling Date: JUo tv=J. t >? o> I 9'f g
`6J&Daia=~------------­
`ihe Patent & Trademark Office acknowledges. and has stamped
`.. tieraon the date of 111C81pt of the items ~ ~-..#-
`/¥ ~ ~
`• Amendment. Fee$
`a ~Brief. Fees
`37 <!.EK /. J1>--(3-1-,-"':)
`a Appllcatlon Fiiing Papers • Fee s --..--
`£Jr&., th f_,
`a PCT national Stage
`C Provisional Application
`a
`CJ Assignment Recordation - Fee S
`C As8ociate Power of Attomey
`0 · Clilm of Priority
`a Certified Copy(ies)
`Q Declaration and Power of Attomey ~4
`0 cd.ciaration
`C Rule 131
`a Forsign Fiiing license request
`~Formal Drawings
`C•-lnfq~ttd";'~~Sta~~~ ..... , __ _
`0 lss\J&•f~.!~lttal-.~ Sc!
`J, •: :'1 ii
`.. 'Ji i
`\) Lei;;~
`d No~~,Oti~RFe ~
`; i U '
`a Pe1ltiOA·lor
`s __ _
`a Pet. ·on rtr_~!l .. cm .. ~ttl~;-=-=.iu--+--
`c Re Briaf ur:t'' ... . . \_LL
`-
`0 Be~t.ieeHer O'iaf
`'
`. ..~• - - - -
`1(" Request for Certification of Correctl·:.n • HK> $ /()0. gr)
`a
`
`~rR - 3 \998 ~J
`~:
`~
`t-.~
`O~
`
`0 Ru
`
`9:'·7~ i~\
`
`(~ ,•.:;_•
`
`83048187
`
`
`
`

`
`I •
`
`, .. t
`I
`
`CERTIFICATE OF MAILING
`
`CASE 100-7932/PCT
`
`Type or print name
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`IN RE APPLICATION OF
`
`COTTENS ET AL.
`
`U. S. Patent No. 5,665,772
`
`Certificate of Correction Branch
`
`APPLICATION NO: 08/416,673
`
`FILED: APRIL 7, 1995
`
`FOR: 0-ALKYLATED RAPAMYCIN DERIVATIVES AND THEIR USE,
`PARTICULARLY AS IMMUNOSUPPRESSANTS
`
`Assistant Commissioner for Patents
`Washir.gton, D.C. 20231
`
`Sir:
`
`REQUEST FOR CERTIFICATE OF CORRECTION
`
`An error has been noted in the above-identified United States Patent, and a Certificate of
`
`Correction is hereby requested.
`
`In particular, the error resides in claim 10 ofthe issued patent. This claim was presented as
`
`new claim "20" of applicants' "Amendment Under 37 CFR 1.3.12" mailed Mar.ch 14, 1997 (copy
`
`appended). At page 3 of said Amendment. applicants indicated that said claim 20 was intended to
`
`replace claim 4 of the application as filed, which applicants indicated may have been erroneously
`
`cancelled by the Office during prosecution.
`
`However, through applicants' inadvertent error, said claim 20 was incorrectly drawn to the
`
`compound "40-0-(~hydroxyethyl)-r