Conjugate compounds
09764037 · 2017-09-19
Assignee
Inventors
- Regan James Anderson (Lower Hutt, NZ)
- Benjamin Jason Compton (Lower Hutt, NZ)
- Colin Malcolm Hayman (Lower Hutt, NZ)
- Ian Francis Hermans (Wellington, NZ)
- David Samuel Larsen (Dunedin, NZ)
- Gavin Frank Painter (Lower Hutt, NZ)
- Franca Ronchese (Wellington, NZ)
Cpc classification
A61P31/00
HUMAN NECESSITIES
C07D405/12
CHEMISTRY; METALLURGY
A61K47/64
HUMAN NECESSITIES
A61K47/646
HUMAN NECESSITIES
C07D309/12
CHEMISTRY; METALLURGY
A61K47/549
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
A61K2039/55572
HUMAN NECESSITIES
International classification
C07D309/12
CHEMISTRY; METALLURGY
C07D405/12
CHEMISTRY; METALLURGY
Abstract
The invention relates to sphingoglycolipid analogs and peptide derivatives thereof, which are useful in treating or preventing diseases or such as those relating to infection, atopic disorders, autoimmune diseases or cancer.
Claims
1. A compound of formula (I): ##STR00139## wherein: A is a self-immolative linker group; D is selected from the group consisting of: ##STR00140## wherein * denotes a point of attachment of group D to group A; R.sup.15 is a side chain of one of the following amino acids: L-lysine, L-citrulline, L-arginine, L-glutamine or L-threonine; R.sup.16 is a side chain of a hydrophobic amino acid; R.sup.19 is an alkylene group; R.sup.32 is an alkylene group or an O-alkylene group wherein the O is attached to the carbonyl group of D2; E is selected from the group consisting of: ##STR00141## ##STR00142## ##STR00143## wherein * denotes a point of attachment of group E to group D; R.sup.20 is H or lower alkyl; R.sup.21 is an alkylene group; g is 0 when R.sup.20 is H or g is 1 when R.sup.20 is lower alkyl; provided that E is E18 only when D is D1, D2 or D3 and provided that E is E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13, E15, E20, E21, E93, E94 or E96 only when D is D1, D2, D3 or D4; and provided that E is E91, E92 or E95 only when D is D5 and provided that E is E97 only when D is D2; G is absent or G is an amino acid sequence of up to 6 amino acids, attached through its N-terminus to group E and through its C-terminus to group J; J is a peptidic antigen, optionally substituted at its N and/or C-termini with up to 6 amino acids selected from the group of natural flanking residues for the antigen, and optionally terminated with NH.sub.2 at the C-terminus so as to provide a C-terminal amide, and attached to group G through its N-terminus or, wherein G is absent, attached to group E through its N-terminus; R.sup.1 is H or glycosyl, provided that if R.sup.1 is glycosyl then R.sup.2 and R.sup.3 are both OH and R.sup.4 is CH.sub.2OH; R.sup.2 is selected from the group consisting of H, OH, F and OR.sup.10; provided that if R.sup.2 is H, F or OR.sup.10, then R.sup.1 is H, R.sup.3 is OH and R.sup.4 is CH.sub.2OH; R.sup.3 is selected from the group consisting of H, OH, F and OR.sup.10; provided that if R.sup.3 is H, F or OR.sup.10, then R.sup.1 is H, R.sup.2 is OH and R.sup.4 is CH.sub.2OH; R.sup.4 is CH.sub.3, CH.sub.2OH, CH.sub.2OCOR.sup.11, CH.sub.2OR.sup.10, CH.sub.2OR.sup.11, CH.sub.2OSO.sub.3H, CH.sub.2SH, CH.sub.2SR.sup.11, CH.sub.2SOR.sup.11, CH.sub.2SO.sub.2R.sup.11, CH.sub.2PO.sub.3H2, CH.sub.2OP(O)(OH).sub.2, CH.sub.2OP(O)(OH)(OR.sup.11), CH.sub.2OP(O)(OR.sup.11).sub.2, CO.sub.2H, CH.sub.2NHCOR.sup.11, CH.sub.2NHCO.sub.2R.sup.11, CH.sub.2NHCONH.sub.2, CH.sub.2NHCONHR.sup.11, CH.sub.2NHCON(R.sup.11).sub.2, CH.sub.2N(R.sup.11).sub.2, CH.sub.2NHSO.sub.2R.sup.11; provided that if R.sup.4 is other than CH.sub.2OH, then R.sup.1 is H and R.sup.2 and R.sup.3 are OH; R.sup.6 is OR.sup.12, OH or H; R.sup.7 is OR.sup.12, OH or H; provided that at least one of R.sup.6 and R.sup.7 is OR.sup.12; wherein when R.sup.6 is OR.sup.12, R.sup.7 is H, R.sup.8 is C.sub.1-C.sub.15 alkyl and X is O, denotes an optional double bond linking the carbon adjacent to R.sup.7 with the carbon adjacent to R.sup.8; R.sup.8 is H or C.sub.1-C.sub.15 alkyl having a straight or branched carbon chain, wherein the carbon chain optionally incorporates one or more double bonds, one or more triple bonds, one or more oxygen atoms and/or a terminal or non-terminal optionally substituted aryl group; R.sup.10 is glycosyl; R.sup.11 is lower alkyl, lower alkenyl or aralkyl; R.sup.12 is C.sub.6-C.sub.30 acyl having a straight or branched carbon chain optionally substituted with one or more hydroxy groups at positions 2 and/or 3 of the acyl group and/or an optionally substituted chain terminating aryl group and which optionally incorporates one or more double bonds, one or more triple bonds, and/or one or more optionally substituted arylene groups and wherein the carbon chain is optionally substituted with one or more deuterium atoms; wherein the optional substituents on the aryl and arylene groups may be selected from halogen, cyano, dialkylamino, C.sub.1-C.sub.6 amide, nitro, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 acyloxy and C.sub.1-C.sub.6 thioalkyl; X is O, CH.sub.2 or S; n is 1 when X is O or S; or n is 0 or 1 when X is CH.sub.2; wherein where X is CH.sub.2 then the following must all be true: the stereochemistry of the 6-membered sugar ring in formula (I) is α-D-galacto; R.sup.1 is H; R.sup.2 and R.sup.3 are both OH; R.sup.4 is CH.sub.2OH, CH.sub.2OR.sup.10 or CH.sub.2OR.sup.11; and: either R.sup.6 is OH and R.sup.7 is OR.sup.12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R), (2S, 3S, 4S), (2R, 3S, 4S), (2R, 3S, 4R) or (2S, 3R, 4S); or R.sup.6 is OR.sup.12 and R.sup.7 is H, and R.sup.8 is C.sub.13H.sub.27 and the stereochemistry at carbon atoms 2 and 3 is (2S, 3S); wherein where X is S then the following must all be true: the stereochemistry of the 6-membered sugar ring in formula (I) is α-D-galacto; R.sup.1 is H; R.sup.2 and R.sup.3 are both OH; R.sup.4 is CH.sub.2OH, CH.sub.2OR.sup.10, CH.sub.2OR.sup.11 or CO.sub.2H; and: either R.sup.6 is OH and R.sup.7 is OR.sup.12 and the stereochemistry at carbon atoms 2, 3 and 4 is (2S, 3S, 4R); or R.sup.6 is OR.sup.12 and R.sup.7 is H and the stereochemistry at the carbon atoms 2 and 3 is (2S, 3S); or a pharmaceutically acceptable salt thereof.
2. A compound of formula (II): ##STR00144## wherein A, D, X, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.6, R.sup.7, R.sup.8, R.sup.10, R.sup.11, R.sup.12, R.sup.15, R.sup.16, R.sup.32, and n are all as defined in claim 1; Z is selected from the group consisting of: ##STR00145## ##STR00146## wherein * denotes a point of attachment of group Z to group D, except as defined for Z23; R.sup.20 is as defined above for formula (I); R.sup.23 is aryl, aralkyl or optionally substituted alkyl; R.sup.24 is lower alkyl; R.sup.25 is p-C.sub.6H.sub.4L wherein L is H, methoxy, COOH, C(O)NHCH.sub.2COOH or CH.sub.2CH.sub.2NMe.sub.2; R.sup.26 is aralkyl; R.sup.27 is H or lower alkyl; R.sup.28 is alkylene; R.sup.31 is (CH.sub.2CH.sub.2O).sub.k k is an integer from 2 to 100; W is an optionally substituted cyclooctynyl ring; or W is a fused bicyclic or tricyclic ring system comprising an optionally substituted cyclooctynyl ring fused to one or more aryl groups or one or more cycloalkyl groups; wherein the cyclooctynyl ring optionally contains a N atom within the ring, which N atom is optionally substituted with an acyl group; and wherein the cyclooctynyl ring is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, alkoxy and aralkyl wherein the aryl part of this group is optionally substituted with a carboxylic acid; and wherein * or one of the optional substituents comprises a point of attachment of Z23 to group D; provided that Z is Z1, Z2, Z3, Z4, Z7, Z8, Z9, Z10, Z11, Z13, Z15, Z16, Z17 or Z18 only when D is D1, D2, D3 or D4 and provided that Z is Z12 only when D is D1, D2 or D3 and provided that Z is Z5 or Z20 only when D is D5, and provided that Z is Z21, Z22 or Z23 only when D is D2; or a pharmaceutically acceptable salt thereof wherein the compound is not (2S, 3S, 4R)-1-O-α-D-Galactopyranosyl-4-hexacosanoyl-2-((4-oxopentanoyloxy)methoxycarbonylamino) octadecane-1,3, 4-triol.
3. The compound of claim 1, wherein A is selected from the group consisting of: ##STR00147## wherein * denotes a point of attachment of group A to group D; each Q.sup.1, the same or different, is independently selected from the group consisting of H, alkyl, alkoxy, halogen, nitro, aryl; or, together with the ring to which it is attached, forms a fused bicyclic aryl group; p is an integer from 1 to 4; Alk.sup.1 is C.sub.1-C.sub.4 straight chain alkyl; and R.sup.28 is H or lower alkyl; provided that A is A1 only when D is D1 and provided that A is A2 only when D is D2, D3 or D5 and provided that A is A3 only when D is D1, D3 or D4 and provided that A is A4 only when D is D2, D3 or D5 and provided that A is A5 only when D is D1, D3 or D4.
4. The compound of claim 3, wherein A is A1 or A2.
5. The compound of claim 1, wherein D is D1, D2 or D5.
6. The compound of claim 1, wherein E is E3, E4 or E97.
7. The compound of claim 1, wherein E is any one of E1 to E8, E93 or E94.
8. The compound of claim 1, wherein G is ##STR00148## wherein * denotes a point of attachment of group G to group E.
9. The compound of claim 1, wherein J is selected from the group consisting of: AMLGTHTMEV (SEQ ID NO: 1), MLGTHTMEV (SEQ ID NO:2), EAAGIGILTV (SEQ ID NO:3), AAGIGILTV (SEQ ID NO:4), AADHRQLQLSISSCLQQL (SEQ ID NO:5), AAGIGILTVILGVL (SEQ ID NO:6), AARAVFLAL (SEQ ID NO:7), ACDPHSGHFV (SEQ ID NO:8), ACYEFLWGPRALVETS (SEQ ID NO:9), ADHRQLQLSISSCLQQL (SEQ ID NO: 10), AEEAAGIGILT (SEQ ID NO: 11), AEEAAGIGIL (SEQ ID NO:12), AELVHFLLL (SEQ ID NO:13), AELVHFLLLKYRAR (SEQ ID NO:14), AEPINIQTW (SEQ ID NO:15), AFLPWHRLF (SEQ ID NO:16), AGATGGRGPRGAGA (SEQ ID NO:17), ALCRWGLLL (SEQ ID NO:18), ALDVYNGLL (SEQ ID NO:19), ALFDIESKV (SEQ ID NO:20), ALGGHPLLGV (SEQ ID NO:21), ALIHHNTHL (SEQ ID NO:22), ALKDVEERV (SEQ ID NO:23), ALLAVGATK (SEQ ID NO:24), ALLEIASCL (SEQ ID NO:25), ALNFPGSQK (SEQ ID NO:26), ALPYWNFATG (SEQ ID NO:27), ALSVMGVYV (SEQ ID NO:28), ALWPWLLMAT (SEQ ID NO:29), ALWPWLLMA (SEQ ID NO:30), ALYVDSLFFL (SEQ ID NO:31), ANDPIFVVL (SEQ ID NO:32), APPAYEKLSAEQ (SEQ ID NO:33), APRGPHGGAASGL (SEQ ID NO:34), APRGVRMAV (SEQ ID NO:35), ARGPESRLL (SEQ ID NO:36), ASGPGGGAPR (SEQ ID NO:37), ATGFKQSSKALQRPVAS (SEQ ID NO:38), AVCPWTWLR (SEQ ID NO:39), AWISKPPGV (SEQ ID NO:40), AYVCGIQNSVSANRS (SEQ ID NO:41), CATWKVICKSCISQTPG (SEQ ID NO:42), CEFHACWPAFTVLGE (SEQ ID NO:43), CLSRRPWKRSWSAGSCPGMPHL (SEQ ID NO:44), CMTWNQMNL (SEQ ID NO:45), CQWGRLWQL (SEQ ID NO:46), CTACRWKKACQR (SEQ ID NO:47), DPARYEFLW (SEQ ID NO:48), DTGFYTLHVIKSDLVNEEATGQFRV (SEQ ID NO:49), DVTFNIICKKCG (SEQ ID NO:50), EAAGIGILTV (SEQ ID NO:51), EADPTGHSY (SEQ ID NO:52), EAFIQPITR (SEQ ID NO:53), EDLTVKIGDFGLATEKSRWSGSHQFEQLS (SEQ ID NO:54), EEAAGIGILTVI (SEQ ID NO:55), EEKLIVVLF (SEQ ID NO:56), EFYLAMPFATPM (SEQ ID NO:57), EGDCAPEEK (SEQ ID NO:58), EIIYPNASLLIQN (SEQ ID NO:59), EKIQKAFDDIAKYFSK (SEQ ID NO:60), ELTLGEFLKL (SEQ ID NO:61), ELVRRILSR (SEQ ID NO:62), ESRLLEFYLAMPF (SEQ ID NO:63), ETVSEQSNV (SEQ ID NO:64), EVDPASNTY (SEQ ID NO:65), EVDPIGHLY (SEQ ID NO:66), EVDPIGHVY (SEQ ID NO:67), EVISCKLIKR (SEQ ID NO:68), EVYDGREHSA (SEQ ID NO:69), EYLQLVFGI (SEQ ID NO:70), EYLSLSDKI (SEQ ID NO:71), EYSKECLKEF (SEQ ID NO:72), EYVIKVSARVRF (SEQ ID NO:73), FIASNGVKLV (SEQ ID NO:74), FINDEIFVEL (SEQ ID NO:75), FLDEFMEGV (SEQ ID NO:76), FLEGNEVGKTY (SEQ ID NO:77), FLFLLFFWL (SEQ ID NO:78), FLIIWQNTM (SEQ ID NO:79), FLLHHAFVDSIFEQWLQRHRP (SEQ ID NO:80), FLLLKYRAREPVTKAE (SEQ ID NO:81), FLTPKKLQCV (SEQ ID NO:82), FLWGPRALV (SEQ ID NO:83), FMNKFIYEI (SEQ ID NO:84), FMVEDETVL (SEQ ID NO:85), FPSDSWCYF (SEQ ID NO:86), FRSGLDSYV (SEQ ID NO:87), FSWAMDLDPKGA (SEQ ID NO:88), GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPL (SEQ ID NO:89), GDNQIMPKAGLLIIV (SEQ ID NO:90), GELIGILNAAKVPAD (SEQ ID NO:91), GFKQSSKAL (SEQ ID NO:92), GLASFKSFLK (SEQ ID NO:93), GLCTLVAML (SEQ ID NO:94), GLPPDVQRV (SEQ ID NO:95), GLYDGMEHL1 (SEQ ID NO:96), GRAMLGTHTMEVTVY (SEQ ID NO:97), GVALQTMKQ (SEQ ID NO:98), GVGSPYVSRLLGICL (SEQ ID NO:99), AKFVAAWTLKAAA (SEQ ID NO: 100), GVLLKEFTVSGNILTIRLT (SEQ ID NO: 101), GVLVGVALI (SEQ ID NO: 102), GVYDGREHTV (SEQ ID NO:103), HLFGYSWYK (SEQ ID NO:104), HLIRVEGNLRVE (SEQ ID NO:105), HLSTAFARV (SEQ ID NO: 106), HLYQGCQVV (SEQ ID NO: 107), HQQYFYKIPILVINK (SEQ ID NO: 108), HTMEVTVYHR (SEQ ID NO: 109), IALNFPGSQK (SEQ ID NO: 110), IGRIAECILGMNPSR (SEQ ID NO: 111), IISAVVGIL (SEQ ID NO: 112), ILAKFLHWL (SEQ ID NO: 113), ILDSSEEDK (SEQ ID NO: 114), ILDTAGREEY (SEQ ID NO: 115), ILHNGAYSL (SEQ ID NO: 116), ILSRDAAPLPRPG (SEQ ID NO: 117), ILTVILGVL (SEQ ID NO: 118), IMDQVPFFS (SEQ ID NO: 119), IMDQVPFSV (SEQ ID NO: 120), IMIGVLVGV (SEQ ID NO: 121), INKTSGPKRGKHAWTHRLRE (SEQ ID NO: 122), ISGGPRISY (SEQ ID NO: 123), ISPNSVFSQWRVVCDSLEDYD (SEQ ID NO: 124), ISQAVHAAHAEINEAGR (SEQ ID NO: 125), ITDQVPFSV (SEQ ID NO: 126), ITKKVADLVGF (SEQ ID NO: 127), KASEKIFYV (SEQ ID NO: 128), KAVYNFATM (SEQ ID NO: 129), KCDICTDEY (SEQ ID NO:130), KEFTVSGNILT (SEQ ID NO:131), KEFTVSGNILTI (SEQ ID NO: 132), KELEGILLL (SEQ ID NO: 133), KHAWTHRLRERKQLVVYEEI (SEQ ID NO:134), KIFGSLAFL (SEQ ID NO:135), KIFSEVTLK (SEQ ID NO:136), KIFYVYMKRKYEAM (SEQ ID NO: 137), KIFYVYMKRKYEAMT (SEQ ID NO: 138), KILDAVVAQK (SEQ ID NO:139), KINKNPKYK (SEQ ID NO:140), KISQAVHAAHAEINEAGRESIINFEKLTEWT (SEQ ID NO:141), KKLLTQHFVQENYLEY (SEQ ID NO: 142), KMDAEHPEL (SEQ ID NO: 143), KNCEPVVPNAPPAYEKLSAE (SEQ ID NO: 144), KRYFKLSHLQMHSRKH (SEQ ID NO: 145), KSSEKIVYVYMKLNYEVMTK (SEQ ID NO: 146), KTWGQYWQV (SEQ ID NO: 147), KVAELVHFL (SEQ ID NO: 148), KVHPVIWSL (SEQ ID NO: 149), KVLEYVIKV (SEQ ID NO:150), KYDCFLHPF (SEQ ID NO:151), KYVGIEREM (SEQ ID NO:152), LAALPHSCL (SEQ ID NO:153), LAAQERRVPR (SEQ ID NO:154), LAGIGILTV (SEQ ID NO:155), LAMPFATPM (SEQ ID NO:156), LGFKVTLPPFMRSKRAADFH (SEQ ID NO:157), LGPGRPYR (SEQ ID NO:158), LHHAFVDSIF (SEQ ID NO:159), LIYRRRLMK (SEQ ID NO:160), LKEFTVSGNILTIRL (SEQ ID NO: 161), LKLSGVVRL (SEQ ID NO: 162), LLANGRMPTVLQCVN (SEQ ID NO: 163), LLDGTATLRL (SEQ ID NO: 164), LLEFYLAMPFATPM (SEQ ID NO:165), LLEFYLAMPFATPMEAELARRSLAQ (SEQ ID NO: 166), LLFGLALIEV (SEQ ID NO: 167), LLGATCMFV (SEQ ID NO: 168), LLGPGRPYR (SEQ ID NO:169), LLGRNSFEV (SEQ ID NO:170), LLKYRAREPVTKAE (SEQ ID NO:171), LLLDDLLVSI (SEQ ID NO: 172), LLLLTVLTV (SEQ ID NO:173), LLWSFQTSA (SEQ ID NO:174), LLYKLADLI (SEQ ID NO:175), LMLQNALTTM (SEQ ID NO:176), LPAVVGLSPGEQEY (SEQ ID NO:177), LPHSSSHWL (SEQ ID NO:178), LPRWPPPQL (SEQ ID NO:179), LPSSADVEF (SEQ ID NO:180), LSHLQMHSRKH (SEQ ID NO:181), LSRLSNRLL (SEQ ID NO:182), LTDLQPYMRQFVAHL (SEQ ID NO:183), LWWVNNQSLPVSP (SEQ ID NO:184), LYATVIHDI (SEQ ID NO:185), LYSACFWWL (SEQ ID NO:186), LYVDSLFFL (SEQ ID NO:187), MEVDPIGHLY (SEQ ID NO:188), MIAVFLPIV (SEQ ID NO:189), MIFEKHGFRRTTPP (SEQ ID NO:190), MKLNYEVMTKLGFKVTLPPF (SEQ ID NO: 191), MLAVISCAV (SEQ ID NO: 192), MLLAVLYCL (SEQ ID NO: 193), MLMAQEALAFL (SEQ ID NO: 194), MPFATPMEA (SEQ ID NO:195), MPREDAHFIYGYPKKGHGHS (SEQ ID NO:196), MSLQRQFLR (SEQ ID NO: 197), MVKISGGPR (SEQ ID NO: 198), NLVPMVATV (SEQ ID NO:199), NPPSMVAAGSVVAAV (SEQ ID NO:200), NSIVKSITVSASG (SEQ ID NO:201), NSNHVASGAGEAAIETQSSSSEEIV (SEQ ID NO:202), NSQPVWLCL (SEQ ID NO:203), NTYASPRFK (SEQ ID NO:204), NYARTEDFF (SEQ ID NO:205), NYKRCFPVI (SEQ ID NO:206), NYNNFYRFL (SEQ ID NO:207), PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:208), PFATPMEAELARR (SEQ ID NO:209), PGSTAPPAHGVT (SEQ ID NO:210), PGTRVRAMAIYKQ (SEQ ID NO:211), PGVLLKEFTVSGNILTIRLTAADHR (SEQ ID NO:212), PLLENVISK (SEQ ID NO:213), PLPPARNGGL (SEQ ID NO:214), PLQPEQLQV (SEQ ID NO:215), PLTSIISAV (SEQ ID NO:216), PRALAETSYVKVLEY (SEQ ID NO:217), PVTWRRAPA (SEQ ID NO:218), PYYFAAELPPRNLPEP (SEQ ID NO:219), QCSGNFMGF (SEQ ID NO:220), QCTEVRADTRPWSGP (SEQ ID NO:221), QGAMLAAQERRVPRAAEVPR (SEQ ID NO:222), QGQHFLQKV (SEQ ID NO:223), QLAVSVILRV (SEQ ID NO:224), QNILLSNAPLGPQFP (SEQ ID NO:225), QQITKTEV (SEQ ID NO:226), QRPYGYDQIM (SEQ ID NO:227), QYSWFVNGTF (SEQ ID NO:228), RAGLQVRKNK (SEQ ID NO:229), REPFTKAEMLGSVIR (SEQ ID NO:230), REPVTKAEML (SEQ ID NO:231), RIAECILGM (SEQ ID NO:232), RKVAELVHFLLLKYR (SEQ ID NO:233), RKVAELVHFLLLKYRA (SEQ ID NO:234), RLLEFYLAMPFA (SEQ ID NO:235), RLLQETELV (SEQ ID NO:236), RLMKQDFSV (SEQ ID NO:237), RLPRIFCSC (SEQ ID NO:238), RLSSCVPVA (SEQ ID NO:239), RLVDDFLLV (SEQ ID NO:240), RMPEAAPPV (SEQ ID NO:241), RMPTVLQCVNVSVVS (SEQ ID NO:242), RNGYRALMDKS (SEQ ID NO:243), RNGYRALMDKSLHVGTQCALTRR (SEQ ID NO:244), RPGLLGASVLGLDDI (SEQ ID NO:245), RPHVPESAF (SEQ ID NO:246), RQKRILVNL (SEQ ID NO:247), RSDSGQQARY (SEQ ID NO:248), RTKQLYPEW (SEQ ID NO:249), RVIKNSIRLTL (SEQ ID NO:250), RVRFFFPSL (SEQ ID NO:251), RYQLDPKFI (SEQ ID NO:252), SAFPTTINF (SEQ ID NO:253), SAWISKPPGV (SEQ ID NO:254), SAYGEPRKL (SEQ ID NO:255), SEIWRDIDF (SEQ ID NO:256), SELFRSGLDSY (SEQ ID NO:257), SESIKKKVL (SEQ ID NO:258), SESLKMIF (SEQ ID NO:259), SFSYTLLSL (SEQ ID NO:260), SHETVIIEL (SEQ ID NO:261), SIINFEKL (SEQ ID NO:262), SLADTNSLAV (SEQ ID NO:263), SLFEGIDIYT (SEQ ID NO:264), SLFPNSPKWTSK (SEQ ID NO:265), SLFRAVITK (SEQ ID NO:266), SLGWLFLLL (SEQ ID NO:267), SLLMWITQC (SEQ ID NO:268), SLLMWITQCFLPVF (SEQ ID NO:269), SLLQHLIGL (SEQ ID NO:270), SLPYWNFATG (SEQ ID NO:271), SLSKILDTV (SEQ ID NO:272), SLYKFSPFPL (SEQ ID NO:273), SLYSFPEPEA (SEQ ID NO:274), SNDGPTLI (SEQ ID NO:275), SPRWWPTCL (SEQ ID NO:276), SPSSNRIRNT (SEQ ID NO:277), SQKTYQGSY (SEQ ID NO:278), SRFGGAVVR (SEQ ID NO:279), SSALLSIFQSSPE (SEQ ID NO:280), SSDYVIPIGTY (SEQ ID NO:281), SSKALQRPV (SEQ ID NO:282), SSPGCQPPA (SEQ ID NO:283), STAPPVHNV (SEQ ID NO:284), SVASTITGV (SEQ ID NO:285), SVDYFFVWL (SEQ ID NO:286), SVSESDTIRSISIAS (SEQ ID NO:287), SVYDFFVWL (SEQ ID NO:288), SYLDSGIHF (SEQ ID NO:289), SYLQDSDPDSFQD (SEQ ID NO:290), TFPDLESEF (SEQ ID NO:291), TGRAMLGTHTMEVTVYH (SEQ ID NO:292), TLDSQVMSL (SEQ ID NO:293), TLDWLLQTPK (SEQ ID NO:294), TLEEITGYL (SEQ ID NO:295), TLMSAMTNL (SEQ ID NO:296), TLNDECWPA (SEQ ID NO:297), TLPGYPPHV (SEQ ID NO:298), TLYQDDTLTLQAAG (SEQ ID NO:299), TMKQICKKEIRRLHQY (SEQ ID NO:300), TMNGSKSPV (SEQ ID NO:301), TPRLPSSADVEF (SEQ ID NO:302), TSCILESLFRAVITK (SEQ ID NO:303), TSEKRPFMCAY (SEQ ID NO:304), TSYVKVLHHMVKISG (SEQ ID NO:305), TTEWVETTARELPIPEPE (SEQ ID NO:306), TVSGNILTIR (SEQ ID NO:307), TYACFVSNL (SEQ ID NO:308), TYLPTNASL (SEQ ID NO:309), TYYRPGVNLSLSC (SEQ ID NO:310), VAELVHFLL (SEQ ID NO:311), VFGIELMEVDPIGHL (SEQ ID NO:312), VGQDVSVLFRVTGALQ (SEQ ID NO:313), VIFSKASSSLQL (SEQ ID NO:314), VISNDVCAQV (SEQ ID NO:315), VLDGLDVLL (SEQ ID NO:316), VLFYLGQY (SEQ ID NO:317), VLHWDPETV (SEQ ID NO:318), VLLKEFTVSG (SEQ ID NO:319), VLLQAGSLHA (SEQ ID NO:320), VLPDVFIRCV (SEQ ID NO:321), VLPDVFIRC (SEQ ID NO:322), VLRENTSPK (SEQ ID NO:323), VLYRYGSFSV (SEQ ID NO:324), VPGVLLKEFTVSGNILTIRLTAADHR (SEQ ID NO:325), VPLDCVLYRY (SEQ ID NO:326), VRIGHLYIL (SEQ ID NO:327), VSSFFSYTL (SEQ ID NO:328), VVLGVVFGI (SEQ ID NO:329), VVPCEPPEV (SEQ ID NO:330), VVVGAVGVG (SEQ ID NO:331), VYFFLPDHL (SEQ ID NO:332), WEKMKASEKIFYVYMKRK (SEQ ID NO:333), WLPFGFILI (SEQ ID NO:334), WNRQLYPEWTEAQRLD (SEQ ID NO:335), WQYFFPVIF (SEQ ID NO:336), WRRAPAPGA (SEQ ID NO:337), YACFVSNLATGRNNS (SEQ ID NO:338), YFSKKEWEKMKSSEKIVYVY (SEQ ID NO:339), YLEPGPVTA (SEQ ID NO:340), YLEPGPVTV (SEQ ID NO:341), YLNDHLEPWI (SEQ ID NO:342), YLQLVFGIEV (SEQ ID NO:343), YLSGANLNL (SEQ ID NO:344), YLVPQQGFFC (SEQ ID NO:345), YMDGTMSQV (SEQ ID NO:346), YMIMVKCWMI (SEQ ID NO:347), YRPRPRRY (SEQ ID NO:348), YSVYFNLPADTIYTN (SEQ ID NO:349), YSWRINGIPQQHTQV (SEQ ID NO:350), YVDFREYEYY (SEQ ID NO:351), YYWPRPRRY (SEQ ID NO:352), IMDQVPFFS (SEQ ID NO:353), SVDYFFVWL (SEQ ID NO:354), ALFDIESKV (SEQ ID NO:355), NLVPMVATV (SEQ ID NO:356) and GLCTLVAML (SEQ ID NO:357), SVASTITGV (SEQ ID NO:358), VMAGDIYSV (SEQ ID NO:359), ALADGVQKV (SEQ ID NO:360), LLGATCMFV (SEQ ID NO:361), SVFAGVVGV (SEQ ID NO:362), ALFDGDPHL (SEQ ID NO:363), YVDPVITSI (SEQ ID NO:364), STAPPVHNV (SEQ ID NO:365), LAALPHSCL (SEQ ID NO:366), SQDDIKGIQKLYGKRS (SEQ ID NO:367), FLPSDFFPSV (SEQ ID NO:368), FLPSDFFPSV (SEQ ID NO:369), TLGEFLKLDRERAKN (SEQ ID NO:370), TFSYVDPVITSISPKYGMET (SEQ ID NO:371), AMTQLLAGV (SEQ ID NO:372), KVFAGIPTV (SEQ ID NO:373), AIIDGVESV (SEQ ID NO:374), GLWHHQTEV (SEQ ID NO:375), NLDTLMTYV (SEQ ID NO:376), KIQEILTQV (SEQ ID NO:377), LTFGDVVAV (SEQ ID NO:378), TMLARLASA (SEQ ID NO:379), IMDQVPFSV (SEQ ID NO:380), MHQKRTAMFQDPQERPRKLPQLCTELQTTIHD (SEQ ID NO:381), LPQLCTELQTTI (SEQ ID NO:382), HDIILECVYCKQQLLRREVY (SEQ ID NO:383), KQQLLRREVYDFAFRDLCIVYRDGN (SEQ ID NO:384), RDLCIVYRDGNPYAVCDKCLKFYSKI (SEQ ID NO:385), DKCLKFYSKISEYRHYCYSLYGTTL (SEQ ID NO:386), HYCYSLYGTTLEQQYNKPLCDLLIR (SEQ ID NO:387), YGTTLEQQYNKPLCDLLIRCINCQKPLCPEEK (SEQ ID NO:388), RCINCQKPLCPEEKQRHLDKKQRFHNIRGRWT (SEQ ID NO:389), DKKQRFHNIRGRTGRCMSCCRSSRTRRETQL (SEQ ID NO:390), MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEE (SEQ ID NO:391), LYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVT (SEQ ID NO:392), GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ ID NO:393), TLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (SEQ ID NO:394), ALPFGFILV (SEQ ID NO:395), TLADFDPRV (SEQ ID NO:396), IMDQVPFSV (SEQ ID NO:397), SIMTYDFHGA (SEQ ID NO:398), AQYIKANSKFIGITEL (SEQ ID NO:399), FLYDDNQRV (SEQ ID NO:400), YLIELIDRV (SEQ ID NO:401), NLMEQPIKV (SEQ ID NO:402), FLAEDALNTV (SEQ ID NO:403), ALMEQQHYV (SEQ ID NO:404), ILDDIGHGV (SEQ ID NO:405), KLDVGNAEV (SEQ ID NO:406), TFEFTSFFY (SEQ ID NO:407), SWPDGAELPF (SEQ ID NO:408), GILGFVFTL (SEQ ID NO:409), ILRGSVAHK (SEQ ID NO:410) SVYDFFVWLKFFHRTCKCTGNFA (SEQ ID NO:411), DLAQMFFCFKELEGW (SEQ ID NO:412), AVGALEGPRNQDWLGVPRQL (SEQ ID NO:413) and RAHYNIVTF (SEQ ID NO:414).
10. The compound of claim 1, wherein n is 1, the stereochemistry of the 6-membered sugar ring of formula (I) is α-D-galacto, R.sup.6 is OH and R.sup.7 is OR.sup.12.
11. The compound of claim 1, wherein n is 0, X is CH.sub.2, the stereochemistry of the 6-membered sugar ring is α-D-galacto, R.sup.6 is OH and R.sup.7 is OR.sup.12.
12. The compound of claim 1, wherein X is O, R.sup.6 is OR.sup.12, R.sup.7 is H, R.sup.8 is C.sub.1-C.sub.15 alkyl and is a double bond linking the carbon adjacent to R.sup.7 with the carbon adjacent to R.sup.8, and the stereochemistry at carbon atoms 2, 3 is (2S, 3S).
13. The compound of claim 1, wherein R.sup.8 is C.sub.1-C.sub.15 alkyl.
14. The compound of claim 1, wherein R.sup.11 is alkyl.
15. The compound of claim 1, wherein R.sup.12 is acyl having a straight carbon chain from 6 to 30 carbon atoms long.
16. The compound of claim 1, selected from the group consisting of: ##STR00149## ##STR00150## ##STR00151## ##STR00152## ##STR00153## ##STR00154## ##STR00155## ##STR00156## ##STR00157## ##STR00158## ##STR00159## or a pharmaceutically acceptable salt thereof.
17. The compound of claim 2, selected from the group consisting of: ##STR00160## ##STR00161## ##STR00162## ##STR00163## ##STR00164## or a pharmaceutically acceptable salt thereof.
18. A pharmaceutical composition comprising a pharmaceutically effective amount of the compound of claim 1 and optionally a pharmaceutically acceptable carrier.
19. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is a vaccine optionally comprising an antigen.
20. A method of treating or reducing a likelihood of occurrence of an infectious disease, an atopic disorder, an autoimmune disease, diabetes or cancer, comprising administering a pharmaceutically effective amount of the compound of claim 1 to a patient requiring treatment.
21. A compound selected from the group consisting of: ##STR00165## ##STR00166## ##STR00167## ##STR00168## or a pharmaceutically acceptable salt thereof.
Description
BRIEF DESCRIPTION OF THE FIGURES
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ABBREVIATIONS
(15) NMR Nuclear magnetic resonance spectrometry HRMS High resolution mass spectrometry ESI Electrospray ionisation Cbz Benzyloxycarbonyl RT Room temperature THF Tetrahydrofuran PBS Phosphate-buffered saline HPLC High performance liquid chromatography FCS Fetal calf serum MS Mass spectrometry LC-MS Liquid chromatography-mass spectrometry TFA Trifluoroacetic acid TLC Thin layer chromatography DMF Dimethylformamide DMSO Dimethylsulfoxide DCM Dichloromethane NMP N-methyl-2-pyrrolidone DDQ 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone PMB p-Methoxybenzyl DMAP 4-Dimethylaminopyridine TMS Trimethylsilyl DCC N,N′-dicyclohexylcarbodiimide DIPEA N,N-diisopropylethylamine TBDPS tert-Butyldiphenylsilyl TBAF Tetra-n-butylammonium fluoride THP Tetrahydropyranyl EEDQ 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide CAN Ceric ammonium nitrate Tbeoc-Thz N-(2-(tert-Butyldisulfanyl)ethoxycarbonyl)-L-thiazolidine-4-carboxylic acid HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflurophosphate. TCEP Tris(2-carboxyethyl)phosphine) TBTA Tris(benzyltriazolylmethyl)amine THPTA Tris(3-hydroxypropyltriazolylmethyl)amine Bim(Py).sub.2 ((2-Benzimidazolyl)methyl)-bis-((2-pyridyl)methyl)amine EDTA Ethylenediaminetetraacetic acid IPA isopropyl alcohol
EXAMPLES
(16) The examples described herein are for purposes of illustrating embodiments of the invention. Other embodiments, methods, and types of analyses are within the capabilities of persons of ordinary skill in the art and need not be described in detail herein. Other embodiments within the scope of the art are considered to be part of this invention. Anhydrous solvents are obtained commercially. Air sensitive reactions are carried out under Ar. Thin layer chromatography (TLC) is performed on aluminium sheets coated with 60 F.sub.254 silica. Flash column chromatography is performed on Merck or SiliCycle silica gel (40-63 μm) or SiliCycle reversed phase (C18) silica gel (40-63 μm). NMR spectra are recorded on a Bruker 500 MHz spectrometer. .sup.1H NMR spectra are referenced to tetramethylsilane at 0 ppm (internal standard) or to residual solvent peak (CHCl.sub.3 7.26 ppm, CHD.sub.2OD 3.31 ppm, CHD.sub.2S(O)CD.sub.3 2.50 ppm). .sup.13C NMR spectra are referenced to tetramethylsilane at 0 ppm (internal standard) or to the deuterated solvent peak (CDCl.sub.3 77.0 ppm, CD.sub.3OD 49.0 ppm, CD.sub.3S(O)CD.sub.3 39.52 ppm). CDCl.sub.3-CD.sub.3OD solvent mixtures are always referenced to the methanol peak. High resolution electrospray ionization mass spectra are recorded on a Q-T of Premier mass spectrometer.
Example 1.1—Synthesis of (2S,3S,4R)-2-Amino-1-O-α-D-galactopyranosyl-4-O-hexacosanoyl octadecane-1,3,4-triol (CN089) via hydrogenolysis of compound 1
(17) ##STR00084##
(18) A mixture of compound 1 (324 mg, 0.303 mmol) and 20% Pd(OH).sub.2/C (300 mg) in 3:7 CHCl.sub.3/MeOH (30 mL) is stirred under a hydrogen balloon at 35° C. for 21 h. The mixture is filtered through celite, washing with 3:1 CHCl.sub.3/MeOH (2×100 mL), and the filtrate is concentrated. The crude residue is purified by silica gel chromatography (1:4 i-PrOH/CHCl.sub.3 then 1:4 EtOH/CHCl.sub.3) to afford the title compound CN089 (45 mg, 17%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 2:1) δ 0.87-0.90 (m, 6H), 1.22-1.36 (m, 68H), 1.54-1.67 (m, 3H), 1.79-1.84 (m, 1H), 2.35-2.38 (m, 2H), 3.27-3.30 (m, 1H), 3.51-3.55 (m, 1H), 3.70-3.72 (m, 1H), 3.75 (dd, J=3.3, 10.0 Hz, 1H), 3.79-3.81 (m, 2H), 3.83-3.86 (m, 2H), 3.97 (d, J=3.3 Hz, 1H), 4.11 (dd, J=2.9, 10.8 Hz, 1H), 4.87 (d, J=3.8 Hz, 1H), 4.92 (dt, J=2.8, 8.8 Hz, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 2:1) δ 14.2, 23.0, 25.3, 25.4, 29.5, 29.65, 29.66, 29.68, 29.74, 29.9, 29.96, 29.99, 30.03, 31.5, 32.3, 34.8, 53.2, 62.2, 64.9, 69.3, 70.1, 70.3, 71.15, 71.18, 73.5, 99.9, 174.6; HRMS-ESI m/z calcd for C.sub.50H.sub.100NO.sub.9 [M+H].sup.+ 858.7398. found 858.7396.
Example 1.2—Synthesis of (2S,3S,4R)-2-Amino-1-O-α-D-galactopyranosyl-4-O-hexacosanoyl octadecane-1,3,4-triol (CN089) Via Isomerization of α-GalCer
(19) ##STR00085##
(20) α-GalCer (195 mg, 0.227 mmol) is heated under Ar in 10:1:2 1,4-dioxane/water/1 M HCl (61 mL) at 85° C. for 35 min, then cooled to 5° C. The collected precipitate is purified on silica gel (MeOH/CH.sub.2Cl.sub.2=10:90 to 20:80) to afford the title compound CN089 as a white solid (121 mg, 62%).
Example 2—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-hexacosanoyl-2-((4-oxopentanoyloxy)methoxycarbonylamino) octadecane-1,3,4-triol (CN146)
(21) ##STR00086##
Example 2.1—(4-Nitrophenoxy)carbonyloxymethyl 4-oxopentanoate (41)
(22) ##STR00087##
(23) The silver salt of levulinic acid is prepared by adding a solution of AgNO.sub.3 (700 mg, 4.1 mmol) in water (10 mL) to the sodium salt of levulinic acid (4.3 mmol in ˜10 mL water, prepared by basification of levulinic acid with 1 M aq NaOH to pH 7-8). After 30 min, the resultant precipitate is isolated by filtration and washed with cold water followed by Et.sub.2O. The product is dried under vacuum to afford the silver salt as a white solid (636 mg, 69%). A mixture of iodomethyl 4-nitrophenyl carbonate (40) (Gangwar, Pauletti et al. 1997) (105 mg, 0.325 mmol, dried by azeotropic distillation with toluene), 4 Å molecular sieves (˜250 mg) and silver levulinate (89 mg, 0.40 mmol) in dry toluene (1.5 mL) is protected from light and stirred at 40° C. After 4 h, the mixture is diluted with Et.sub.2O, filtered through celite, and concentrated under reduced pressure. The crude residue is purified by silica gel chromatography (30% to 40% EtOAc/petroleum ether) to afford the title compound (41) (85 mg, 84%) as a colourless oil. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 2.20 (s, 3H), 2.67-2.70 (m, 2H), 2.80-2.83 (m, 2H), 5.88 (s, 2H), 7.38-7.48 (m, 2H), 8.24-8.34 (m, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 27.7, 29.7, 37.6, 82.5, 121.8, 125.4, 145.7, 151.5, 155.1, 171.2, 206.0; HRMS (ESI): m/z calcd for C.sub.13H.sub.13NO.sub.8Na [M+Na].sup.+ 334.0539. found 334.0544.
Example 2.2—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-hexacosanoyl-2-((4-oxopentanoyloxy)methoxycarbonylamino) octadecane-1,3,4-triol (CN146)
(24) ##STR00088##
(25) To a solution of amine CN089 (22 mg, 0.026 mmol) in d.sub.5-pyridine (0.30 mL) is added a solution of (4-nitrophenoxy)carbonyloxymethyl 4-oxopentanoate (41) (8.0 mg, 0.026 mmol) in CDCl.sub.3 (0.15 mL). The progress of the reaction is followed in an NMR tube. After 3 h at rt, NEt.sub.3 (2.5 mg, 0.025 mmol) is added and the reaction is allowed to continue for a further 2.25 h, after which time >95% of the amine CN089 has been consumed. The volatiles are concentrated under reduced pressure and the crude residue is purified by silica gel chromatography (1.5:40:60 to 1.5:45:55 MeOH/dioxane/CHCl.sub.3) to afford the title compound CN146 (14.1 mg, 53%) as a white solid. .sup.1H NMR (500 MHz, 1:1 CDCl.sub.3/CD.sub.3OD) δ 0.88-0.90 (m, 6H), 1.24-1.34 (m, 68H), 1.60-1.72 (m, 4H), 2.21 (s, 3H), 2.31-2.42 (m, 2H), 2.62-2.64 (m, 2H), 2.80-2.83 (m, 2H), 3.71-3.83 (m, 8H), 3.88 (br d, J=10.1 Hz, 1H), 3.95 (br d, J=2.2 Hz, 1H), 4.86 (d, J=3.2 Hz, 1H) 4.94-4.98 (m, 1H), 5.68-5.76 (m, 2H); .sup.13C NMR (126 MHz, 1:1 CDCl.sub.3/CD.sub.3OD) δ 14.3, 23.2, 25.6, 25.9, 28.3, 29.3, 29.7, 29.79, 28.84, 29.86, 29.92, 30.0, 30.1, 30.15, 30.18, 30.21, 32.43, 32.44, 35.1, 38.1, 53.0, 62.3, 68.1, 69.7, 70.4, 70.8, 71.4, 72.1, 75.2, 80.7, 100.5, 155.6, 172.7, 175.0, 208.5; HRMS (ESI): m/z calcd for C.sub.57H.sub.107NO.sub.14Na [M+Na].sup.+ 1052.7589. found 1052.7578.
Example 3—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-hexacosanoyl-2-(6-(azido)hexanoylmethoxycarbonylamino) octadecane-1,3,4-triol (CN215)
Example 3.1—(4-Nitrophenoxy)carbonyloxymethyl 6-azidohexanoate (50)
(26) ##STR00089##
(27) A mixture of iodomethyl 4-nitrophenyl carbonate (40) (Gangwar, Pauletti et al. 1997) (340 mg, 1.05 mmol), 6-azidohexanoic acid (210 mg, 1.34 mmol), silver oxide (100 mg, 0.43 mmol) and 4 Å molecular sieves (˜500 mg) in dry acetonitrile (5 mL) is protected from light and stirred at rt. After 24 h, the mixture is filtered through celite, washed with EtOAc (20 mL) and concentrated under reduced pressure. The crude residue is purified by silica gel chromatography (EtOAc/toluene 0:10 to 1:4) to afford the title compound 50 as a colourless oil (150 mg, 40%). .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.42-1.48 (m, 2H), 1.60-1.66 (m, 2H), 1.68-1.74 (m, 2H), 2.45 (dd, J=7.4, 7.4 Hz, 2H), 3.28 (dd, 6.8, 6.8 Hz, 2H), 7.41 (dd, J=2.2, 9.2 Hz, 2H), 8.29 (dd, J=2.2, 9.2 Hz, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 23.9, 26.0, 28.4, 33.5, 51.6, 82.5, 121.6, 125.3, 145.6, 151.4, 155.0, 171.6; HRMS-ESI: m/z calcd for C.sub.14H.sub.16N.sub.4O.sub.7Na [M+Na].sup.+ 375.0917. found 375.0917.
Example 3.2—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-hexacosanoyl-2-(6-(azido)hexanoyloxymethoxycarbonylamino) octadecane-1,3,4-triol (CN215)
(28) ##STR00090##
(29) To a solution of amine CN089 (25 mg, 0.029 mmol) in pyridine (1 mL) is added a solution of (4-nitrophenoxy)carbonyloxymethyl 6-azidohexanoate (50) (20 mg, 0.056 mmol) in CH.sub.2Cl.sub.2 (0.15 mL) followed by Et.sub.3N (1 mL). After 0.5 h at rt, the mixture is diluted with MeOH and concentrated under reduced pressure. The crude residue is purified by silica gel chromatography (MeOH/CHCl.sub.30:10 to 2:8) to afford the title compound CN215 as a white solid (21 mg, 67%). .sup.1H NMR (500 MHz, 3:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 1.23-1.35 (m, 68H), 1.40-1.46 (m, 2H), 1.60-1.71 (m, 8H), 2.33-2.37 (m, 2H), 2.40 (dd, J=7.5, 7.5 Hz, 2H), 3.29 (dd, J=6.7, 6.7 Hz, 2H), 3.72-3.80 (m, 8H), 3.87 (dd, J=2.3, 10.3 Hz, 1H), 3.96 (d, J=2.9 Hz, 1H), 4.86 (d, J=3.7 Hz, 1H), 4.91-4.94 (m, 1H), 5.73 (s, 2H), 6.78 (d, J=8.5 Hz, 1H); .sup.13C NMR (126 MHz, 3:1 CDCl.sub.3/CD.sub.3OD) δ 13.6, 22.3, 23.7, 24.7, 25.0, 25.8, 28.2, 28.5, 28.9, 29.0, 29.1, 29.2, 29.3, 31.6, 33.4, 34.3, 50.9, 51.8, 61.5, 67.5, 68.7, 69.5, 70.0, 70.3, 71.3, 74.3, 79.8, 99.5, 154.5, 172.5, 174.2; HRMS-ESI: m/z calcd for C.sub.58H.sub.110N.sub.4O.sub.13Na [M+Na].sup.+ 1093.7967. found 1093.7972.
Example 4—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-hexacosanoyl-2-(6-(maleimido)hexanoylmethoxycarbonylamino) octadecane-1,3,4-triol (CN157)
Example 4.1—(4-Nitrophenoxy)carbonyloxymethyl 6-maleimidohexanoate (51)
(30) ##STR00091##
(31) To a mixture of iodomethyl 4-nitrophenyl carbonate (40) (Gangwar, Pauletti et al. 1997) (70 mg, 0.22 mmol), 6-maleimidohexanoic acid (40 mg, 0.19 mmol) and 4 Å molecular sieves (˜500 mg) in dry acetonitrile (5 mL) is added Ag.sub.2O (25 mg, 0.11 mmol) and the reaction is stirred, protected from light. After 3 h, the mixture is diluted with EtOAc, filtered through celite, and concentrated under reduced pressure. The crude residue is purified by silica gel chromatography (EtOAc/petroleum ether=0:1 to 4:6) to afford the title compound 51 as a colourless oil (25 mg, 33%). .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.32-1.38 (m, 2H), 1.59-1.65 (m, 2H), 1.67-1.73 (m, 2H), 2.42 (dd, J=7.3, 7.3 Hz, 2H), 3.52 (dd, J=7.3, 7.3 Hz, 2H), 5.88 (s, 2H), 6.69 (s, 2H), 7.40-7.44 (m, 2H), 8.28-8.31 (m, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 23.9, 26.0, 28.1, 29.7, 33.6, 37.5, 82.5, 121.7, 122.4, 125.4, 134.1, 145.7, 151.5, 155.1, 107.8, 171.7; HRMS (ESI) m/z calcd for C.sub.18H.sub.18N.sub.2O.sub.9Na [M+Na].sup.+: 429.0910. found 429.0905.
Example 4.2—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-hexacosanoyl-2-(6-(maleimido)hexanoylmethoxycarbonylamino) octadecane-1,3,4-triol (CN157)
(32) ##STR00092##
(33) To a solution of amine CN089 (21 mg, 0.024 mmol) in dry pyridine (3 mL) is added a solution of (4-nitrophenoxy)carbonyloxymethyl 6-maleimidohexanoate (51) (8.0 mg, 0.026 mmol) in CH.sub.2Cl.sub.2 (3 mL) followed by Et.sub.3N (2 mL). After 2 h the volatiles are concentrated under reduced pressure and the crude residue is purified by silica gel chromatography (MeOH/CHCl.sub.3=0:1 to 2:8) to afford the title compound CN157 as a white solid (14 mg, 23%). .sup.1H NMR (500 MHz, 3:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 1.23-1.34 (m, 70H), 1.58-1.70 (m, 8H), 2.33-2.39 (m, 4H), 3.52 (dd, J=7.3, 7.3 Hz, 2H), 3.71-3.79 (m, 8H), 3.88 (dd, J=2.5, 10.3 Hz, 1H), 3.96 (d, J=3.0 Hz, 1H), 4.86 (d, J=3.6 Hz, 1H), 4.93 (m, 1H), 5.70-5.75 (m, 1H), 6.73 (s, 2H); .sup.13C NMR (126 MHz, 3:1 CDCl.sub.3/CD.sub.3OD) δ 14.2, 22.9, 24.2, 25.3, 25.6, 26.3, 28.4, 29.1, 29.4, 29.6, 29.7, 29.9, 32.2, 33.9, 34.8, 37.8, 52.4, 62.1, 68.1, 69.3, 70.1, 70.5, 70.9, 72.0, 74.9, 80.4, 100.1, 134.4, 155.1, 171.4, 173.1, 174.8; HRMS (ESI) m/z calcd for C.sub.62H.sub.112N.sub.2O.sub.15Na [M+Na].sup.+: 1147.7960. found 1147.7960.
Example 5—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-Cbz-Phe-Lys-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN166)
Example 5.1—N-Cbz-Phe-Lys(Alloc)OH (42)
(34) ##STR00093##
(35) The title compound is synthesised in 59% yield, following the literature procedure (Dubowchik, Firestone et al. 2002). .sup.1H NMR (500 MHz, d6-DMSO) δ 1.26-1.45 (m, 4H), 1.57-1.65 (m, 1H), 1.69-1.77 (m, 1H), 2.73 (dd, J=11.1, 13.7 Hz, 1H), 2.92-3.04 (m, 3H), 4.16-4.20 (m, 1H), 4.28-4.33 (m, 1H), 4.44-4.49 (m, 2H), 4.94 (s, 2H), 5.15 (app dq, J=1.4, 10.4 Hz, 1H), 5.25 (app dq, J=1.7, 17.2 Hz, 1H), 5.85-5.93 (m, 1H), 7.13-7.34 (10H), 7.43 (d, J=8.9 Hz, 1H), 8.16-8.21 (m, 1H); .sup.13C NMR (126 MHz, d6-DMSO) δ 22.6, 29.0, 30.8, 37.4, 39.8 (obscured by solvent), 52.0, 55.9, 64.1, 65.2, 116.8, 126.2, 127.4, 127.6, 128.0, 128.2, 129.2, 133.8, 137.0, 138.1, 155.8, 155.9, 171.6, 173.5; HRMS-ESI [M+Na].sup.+ calcd for C.sub.27H.sub.33N.sub.3NaO.sub.7: 534.2216. Found 534.2209.
Example 5.2—N-Cbz-Phe-Lys(Alloc)-4-aminobenzyl alcohol (43)
(36) ##STR00094##
(37) A mixture of dipeptide 42 (243 mg, 0.475 mmol), 1-hydroxybenzotriazole hydrate (74 mg, 0.54 mmol) and 4-aminobenzyl alcohol (118 mg, 0.958 mmol) is dissolved in THF (5 mL) under Ar and cooled in an ice bath. N-Methyl morpholine (54 μL, 0.49 mmol) is added, followed by EDCI (97 mg, 0.51 mmol) and the mixture is stirred on ice for 2 h, then at rt for 2 h. The mixture is acidified to pH ˜3 with aq citric acid and extracted with EtOAc, and the extracts are dried (brine, MgSO.sub.4) and concentrated under reduced pressure. The solid residue is triturated with diethyl ether, and subsequently purified twice by column chromatography on silica gel (first column: MeOH/CH.sub.2C.sub.2=2:98 to 7:93; second column EtOAc/petroleum ether=8:2 to 1:0) to afford the title compound 43 (70 mg, 24%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3+3 drops CD.sub.3OD) δ 1.28-1.36 (m, 2H), 1.47-1.53 (m, 2H), 1.61-1.70 (m, 1H), 1.82-1.89 (m, 1H), 2.96-3.00 (m, 1H), 3.08-3.13 (m, 3H), 4.41-4.45 (m, 2H), 4.50-4.54 (m, 2H), 4.62 (s, 2H), 5.03-5.10 (m, 2H), 5.17-5.19 (m, 1H), 5.25-5.29 (m, 1H), 5.84-5.92 (m, 1H), 7.13-7.19 (m, 5H), 7.27-7.35 (m, 7H), 7.51 (d, J=8.5 Hz, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3+3 drops CD.sub.3OD) δ 22.0, 28.8, 30.9, 37.9, 39.8, 53.2, 55.9, 64.1, 65.2, 66.8, 117.2, 119.9, 126.7, 127.3, 127.5, 127.6, 127.9, 128.2, 128.3, 128.8, 132.5, 135.7, 136.7, 136.9, 155.9, 156.5, 169.4, 171.5; HRMS-ESI [M+Na].sup.+ calcd for C.sub.34H.sub.40N.sub.4NaO.sub.7: 639.2795. Found 639.2786.
Example 5.3—N-Cbz-Phe-Lys(Alloc)-4-aminobenzyl 4-nitrophenyl carbonate (44)
(38) ##STR00095##
(39) To an ice-cooled solution of alcohol 43 (70 mg, 0.11 mmol) in dry THF (5 mL) is added pyridine (46 μL, 0.57 mmol), followed by 4-nitrophenyl chloroformate (46 mg, 0.23 mmol) and the mixture is stirred at rt overnight. After diluting with EtOAc, the organic phase is washed with 10% aq citric acid and water, then dried (brine, MgSO.sub.4) and concentrated under reduced pressure. The solid residue is triturated with toluene, and subsequently purified column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=0:100 to 5:95) to afford the title compound 44 (63 mg, 71%) as a white solid. .sup.1H NMR (500 MHz, CDCl.sub.3+3 drops CD.sub.3OD) δ 1.27-1.37 (m, 2H), 1.48-1.54 (m, 2H), 1.61-1.70 (m, 1H), 1.83-1.91 (m, 1H), 2.98-3.03 (dd, J=7.2, 13.3 Hz, 1H), 3.09-3.16 (m, 3H), 4.41-4.46 (m, 2H), 4.50-4.57 (m, 2H), 5.07 (s, 2H), 5.18 (d, J=10.5 Hz, 1H), 5.25-5.29 (m, 3H), 5.84-5.92 (m, 1H), 7.13-7.19 (m, 5H), 7.27-7.42 (m, 9H), 7.61 (d, J=8.0 Hz, 2H), 8.27 (d, J=9.1 Hz, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3+3 drops CD.sub.3OD) δ 22.0, 28.8, 30.7, 37.8, 39.8, 53.2, 55.8, 65.2, 66.8, 70.3, 117.2, 119.8, 121.4, 124.9, 126.7, 127.6, 127.9, 128.2, 128.3, 128.8, 129.2, 129.6, 132.5, 135.6, 135.7, 138.3, 145.1, 152.1, 155.2, 155.9, 156.5, 169.5, 171.5; HRMS-ESI [M+Na].sup.+ calcd for C.sub.41H.sub.43N.sub.5NaO.sub.11: 804.2857. Found 804.2852.
Example 5.4—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-[N-Cbz-Phe-Lys(ε-N-Alloc)-4-aminobenzyloxycarbonylamino] octadecane-1,3,4-triol (45)
(40) ##STR00096##
(41) To a mixture of CN089 (18 mg, 0.021 mmol) in pyridine (0.25 mL) under Ar is added pNP-carbonate 44 (18 mg, 0.023 mmol) suspended in 17:1 CHCl.sub.3-MeOH (0.53 mL), followed by Et.sub.3N (4.5 μL, 0.032 mmol) and the mixture is stirred at rt. After 18 h, a further portion of Et.sub.3N (6 μL, 0.043 mmol) is added. After a further 16 h, the volatiles are gently concentrated on a rotary evaporator and more pyridine (0.25 mL) is added, followed by Et.sub.3N (4 μL, 0.029 mmol). After 24 h, the excess carbonate reagent is quenched with Et.sub.2NH (10 μL, 10 min) and the mixture is concentrated to dryness. The crude residue is purified by column chromatography on silica gel (MeOH/CHCl.sub.3=0:1 to 1:9) to afford the title compound 45 (16.4 mg, 52%) as a white solid. .sup.1H NMR (500 MHz, 1:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 1.15-1.42 (m, 70H), 1.48-1.55 (m, 2H), 1.60-1.74 (m, 5H), 1.84-1.91 (m, 1H), 2.31-2.41 (m, 2H), 2.94 (dd, J=8.4, 13.7 Hz, 1H), 3.10-3.16 (m, 3H), 3.68-3.81 (m, 8H), 3.86 (dd, J=2.2, 10.4 Hz, 1H), 3.89 (d, J=2.8 Hz, 1H), 4.42-4.46 (m, 2H), 4.50-4.51 (m, 2H), 4.85 (d, J=3.7 Hz, 1H), 4.96-5.00 (m, 1H), 5.03-5.10 (m, 4H), 5.15-5.18 (m, 1H), 5.25-5.29 (m, 1H), 5.85-5.92 (m, 1H), 7.14-7.23 (m, 5H), 7.27-7.35 (m, 7H), 7.56 (d, J=8.1 Hz, 2H); .sup.13C NMR (126 MHz, 1:1 CDCl.sub.3/CD.sub.3OD) δ 14.30, 14.32, 23.2, 25.6, 25.9, 29.2, 29.7, 29.88, 29.91, 29.93, 30.08, 30.13, 30.19, 30.22, 30.3, 32.2, 32.46, 32.48, 35.1, 38.6, 40.9, 52.9, 54.4, 57.1, 62.4, 65.9, 67.0, 67.4, 68.5, 69.7, 70.4, 70.9, 71.4, 72.3, 75.3, 100.6, 117.6, 120.8, 120.9, 127.4, 128.3, 128.6, 128.98, 129.02, 129.2, 129.3, 129.8, 133.3, 133.6, 137.0, 137.1, 138.4, 157.4, 158.0, 171.1, 173.0, 175.1; HRMS-ESI [M+Na].sup.+ calcd for C.sub.85H.sub.137N.sub.5NaO.sub.17: 1522.9907. Found 1522.9888.
Example 5.5—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-Cbz-Phe-Lys-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN166)
(42) To a mixture of compound 45 (16 mg, 0.011 mmol) and borane-dimethylamine complex (3.4 mg, 0.058 mmol) dissolved in freshly degassed 14:1 CH.sub.2Cl.sub.2-MeOH (0.16 mL), is added a catalytic amount of Pd(PPh.sub.3).sub.4 (approx 0.5 mg, 0.4 μmol) and the mixture is stirred at rt under Ar. After 80 min, the rxn mixture is filtered through a short plug of silica (0.15 g), washing with 50% to 75% MeOH/CH.sub.2Cl.sub.2 (12 mL of each). The washings are concentrated and purified by column chromatography on C18 silica gel (MeOH+0.5% TFA) to afford the TFA salt of the title compound CN166 (15.4 mg, 94%) as a colourless glass. .sup.1H NMR (500 MHz, 1:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 1.15-1.51 (m, 70H), 1.60-1.75 (m, 7H), 1.87-1.94 (m, 1H), 2.31-2.41 (m, 2H), 2.86-2.90 (br m, 2H), 2.95 (dd, J=8.4, 13.8 Hz, 1H), 3.13 (dd, J=6.1, 13.8 Hz, 1H), 3.67-3.80 (m, 8H), 3.85-3.87 (m, 2H), 4.38-4.41 (m, 1H), 4.46-4.49 (m, 1H), 4.85 (d, J=3.6 Hz, 1H), 4.97-5.13 (m, 5H), 7.12-7.16 (m, 1H), 7.19-7.20 (m, 4H), 7.28-7.36 (m, 7H), 7.56 (d, J=8.0 Hz, 2H); .sup.13C NMR (126 MHz, 1:1 CDCl.sub.3/CD.sub.3OD) δ 14.31, 14.33, 22.9, 23.23, 23.24, 25.7, 26.0, 27.4, 29.3, 29.8, 29.9, 30.0, 30.1, 30.18, 30.24, 30.26, 30.28, 30.31, 31.9, 32.52, 32.54, 35.2, 38.5, 40.0, 53.1, 54.2, 57.3, 62.4, 67.0, 67.4, 68.6, 69.7, 70.5, 70.9, 71.4, 72.4, 75.4, 100.8, 120.9, 127.5, 128.2, 128.7, 129.05, 129.10, 129.2, 129.8, 133.6, 137.0, 137.1, 138.4, 157.5, 157.6, 171.0, 173.3, 175.2; HRMS-ESI [M+Na].sup.+ calcd for C.sub.81H.sub.133N.sub.5NaO.sub.15: 1438.9696. Found 1438.9686.
Example 6—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-Cbz-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN165)
Example 6.1—N-Cbz-Val-Cit-4-aminobenzyl alcohol (47)
(43) ##STR00097##
(44) A mixture of acid 46 (Dubowchik, Firestone et al. 2002) (200 mg, 0.49 mmol) and 4-aminobenzyl alcohol (64 mg, 0.52 mmol) is stirred under Ar at 20° C. in 1:1 MeOH/1,4-dioxane (6 mL) until the starting materials are nearly dissolved (1 h). EEDQ (242 mg, 0.98 mmol) is added and stirring is continued at 20° C. for 3.5 d. The solvents are removed under reduced pressure and the solid residue is triturated with EtOAc, and subsequently purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=5:95 to 15:85) to afford the title compound 47 (106 mg, 42%) as a white solid. .sup.1H NMR (500 MHz, CD.sub.3OD) δ 0.95 (d J=6.8 Hz, 3H), 0.97 (d J=6.8 Hz, 3H), 1.52-1.63 (m, 2H), 1.71-1.78 (m, 1H), 1.87-1.94 (m, 1H), 2.03-2.11 (m, 1H), 3.07-3.12, (m, 1H), 3.15-3.21, (m, 1H), 3.98 (d, J=6.8 Hz, 1H), 4.50-4.52 (m, 1H), 4.55 (s, 2H), 5.10 (s, 2H), 7.26-7.36 (m, 7H), 7.54 (d, J=8.0 Hz, 2H); .sup.13C NMR (126 MHz, CD.sub.3OD) δ 18.6, 19.7, 27.8, 30.5, 31.9, 40.3, 55.0, 62.3, 64.8, 67.8, 121.3, 128.6, 128.8, 129.0, 129.5, 138.2, 138.6, 138.8, 158.8, 162.3, 172.2, 174.4; HRMS-ESI [M+Na].sup.+ calcd for C.sub.26H.sub.35N.sub.5NaO.sub.6: 536.2485. Found 536.2495.
Example 6.2—N-Cbz-Val-Cit-4-aminobenzyl 4-nitrophenyl carbonate (48)
(45) ##STR00098##
(46) To a solution of alcohol 47 (30 mg, 0.058 mmol) and bis(4-nitrophenyl) carbonate (23 mg, 0.076 mmol) in anhydrous DMF (0.5 mL) under Ar is added pyridine (0.10 mL), followed by i-Pr.sub.2NEt (10.5 μL, 0.060 mmol) and the reaction is stirred at rt. After 16 h, the mixture is concentrated under reduced pressure and the crude residue is purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=0:1 to 1:9), to afford the title compound 48 (30 mg, 76%) as a white solid. .sup.1H NMR (500 MHz, 5:1 CDCl.sub.3/CD.sub.3OD) δ 0.94 (d J=6.8 Hz, 3H), 0.98 (d J=6.8 Hz, 3H), 1.51-1.57 (m, 2H), 1.67-1.74 (m, 1H), 1.88-1.95 (m, 1H), 2.05-2.13 (m, 1H), 3.09-3.14, (m, 1H), 3.20-3.26, (m, 1H), 4.01 (d, J=6.4 Hz, 1H), 4.56 (dd, J=4.9, 9.0 Hz, 1H), 5.08-5.14 (m, 2H), 5.26 (s, 2H), 7.29-7.41 (m, 9H), 7.64 (d, J=8.0 Hz, 2H), 8.26-8.29 (m, 2H); .sup.13C NMR (126 MHz, 5:1 CDCl.sub.3/CD.sub.3OD) δ 17.8, 19.2, 26.3, 29.3, 31.0, 39.1, 53.3, 60.8, 67.2, 70.8, 120.2, 121.9, 125.4, 127.9, 128.3, 128.6, 129.7, 130.1, 136.3, 138.8, 145.5, 152.6, 155.7, 157.1, 160.5, 170.6, 172.6; HRMS-ESI [M+Na].sup.+ calcd for C.sub.33H.sub.38N.sub.6NaO.sub.10: 701.2536. Found 701.2540.
Example 6.3—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-Cbz-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN165)
(47) ##STR00099##
(48) To a mixture of CN089 (17 mg, 0.020 mmol) in pyridine (0.25 mL) under Ar is added pNP-carbonate 48 (15 mg, 0.022 mmol) dissolved in pyridine (0.25 mL), followed by Et.sub.3N (4.5 μL, 0.032 mmol) and the mixture is stirred at rt. After 18 h, a further portion of Et.sub.3N (3 μL, 0.022 mmol) is added and the reaction is stirred for a further 4 h before quenching excess carbonate reagent with Et.sub.2NH (10 μL, 10 min). The mixture is concentrated to dryness and the crude residue is purified by column chromatography on silica gel (MeOH/CHCl.sub.3=5:95 to 15:85), followed by column chromatography on C18 silica gel (MeOH), and finally by preparative HPLC (Phenomenex Luna C18(2), 5 μm, 30×250 mm, 35° C., 50 mL/min; Mobile phase A=80:20:0.05 MeOH/water/TFA; Mobile phase B=100:0.05 MeOH/TFA; 0-10 min: 0-100% B; 10-34 min: 100% B; 34-35 min: 100-0% B; 35-37 min: 100% A) to afford the title compound CN165 (21 mg, 76%) as a white solid. .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 0.94 (d J=6.7 Hz, 3H), 0.98 (d J=6.5 Hz, 3H), 1.20-1.40 (m, 68H), 1.50-1.76 (m, 7H), 1.87-1.96 (m, 1H), 2.05-2.13 (m, 1H), 2.29-2.41 (m, 2H), 3.07-3.15 (m, 1H), 3.18-3.26 (m, 1H), 3.63-3.81 (m, 8H), 3.83-3.90 (m, 2H), 4.01 (d, J=6.4 Hz, 1H), 4.52-4.57 (m, 1H), 4.80-4.86 (m, 1H), 4.91-5.00 (m, 2H), 5.04-5.16 (m, 3H), 7.27-7.37 (m, 7H), 7.57 (d, J=8.0 Hz, 2H); .sup.13C NMR (126 MHz, 2:1 CDCl.sub.3/CD3OD) δ 14.2, 18.1, 19.4, 23.0, 25.4, 25.6, 26.7, 29.2, 29.5, 29.65, 29.67, 29.7, 29.86, 29.88, 29.92, 29.95, 29.98, 30.02, 31.2, 32.2, 34.9, 39.3, 52.5, 53.7, 61.1, 62.3, 66.7, 67.4, 68.4, 69.3, 70.3, 70.6, 70.8, 72.3, 75.0, 100.3, 120.5, 128.2, 128.5, 128.8, 129.0, 133.0, 136.6, 138.1, 157.0, 157.6, 161.3, 171.0, 173.1, 174.9; HRMS-ESI [M+Na].sup.+ calcd for C.sub.77H.sub.132N.sub.6NaO.sub.16: 1419.9598. Found 1419.9584.
Example 7—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-(6-azidohexanoyl)-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN172)
Example 7.1—N-(6-Azidohexanoyl)-Val-Cit-4-aminobenzyl alcohol (53)
(49) ##STR00100##
(50) To a stirred solution of 6-azidohexanoic acid (85.0 mg, 0.541 mmol) in anhydrous CH.sub.2Cl.sub.2 (3.3 mL) at 0° C. is added by Et.sub.3N (80 μL, 0.57 mmol), followed by isobutyl chloroformate (68 μL, 0.52 mmol). After 30 min, the solution is transferred by cannula to a separate flask containing amine 52 (Dubowchik, Firestone et al. 2002) (166 mg, 0.438 mmol) dissolved in 3:1 CH.sub.2Cl.sub.2-MeOH (4 mL) at 0° C. The original flask is rinsed with CH.sub.2Cl.sub.2 (2×0.5 mL), which is transferred to the second flask. After 5 min, the reaction mixture is warmed to rt and stirred for 2.5 h. After concentration of the solvents under reduced pressure, the resulting solid is triturated successively with toluene, diethyl ether, acetone and MeCN, and purified by column chromatography on silica gel (MeOH/CHC.sub.3=10:90 to 14:86) to afford the title compound 53 as a white solid (160 mg, 71%). .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.95-97 (m, 6H), 1.39-1.45 (m, 2H), 1.53-1.77 (m, 7H), 1.88-1.95 (m, 1H), 2.04-2.11 (m, 1H), 2.29 (t, J=7.5 Hz, 2H), 3.09-3.15, (m, 1H), 3.20-3.26 (1H), 3.28 (t, J=6.9 Hz, 2H), 4.19, (d, J=7.3 Hz, 1H), 4.54 (dd, J=5.0, 8.8 Hz, 1H), 4.59 (s, 2H), 7.31 (d, J=8.5 Hz, 2H), 7.54 (d, J=8.5 Hz, 2H); .sup.13C NMR (126 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 18.4, 19.4, 25.6, 26.6, 28.9, 29.6, 31.0, 36.2, 39.4, 51.6, 53.6, 59.4, 64.3, 120.5, 127.9, 137.4, 137.7, 161.0, 170.9, 172.8, 174.9; HRMS-ESI [M+Na].sup.+ calcd for C.sub.24H.sub.38N.sub.8NaO.sub.5: 541.2863. found 541.2860.
Example 7.2—N-(6-Azidohexanoyl)-Val-Cit-4-aminobenzyl 4-nitrophenyl carbonate (54)
(51) ##STR00101##
(52) To a mixture of alcohol 53 (158 mg, 0.305 mmol) in anhydrous DMF (2.5 mL) is added N,N-diisopropylethylamine (66 μL, 0.38 mmol) followed by bis(4-nitrophenyl) carbonate (116 mg, 0.381 mmol) and the reaction is stirred under Ar at rt for 41 h. After concentrating the mixture under high vacuum, the crude product is purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=6:94 to 11:89) to afford the title compound 54 as an off-white solid (206 mg, 99%). .sup.1H NMR (500 MHz, d6-DMSO) δ 0.84 (d, J=6.8 Hz, 3H), 0.87 (d, J=6.7 Hz, 3H), 1.27-1.33 (m, 2H), 1.34-1.64 (m, 7H), 1.68-1.75 (m, 1H), 1.95-2.02 (m, 1H), 2.13-2.24 (m, 2H), 2.92-2.98 (m, 1H), 3.00-3.06, (m, 1H), 4.18-4.21 (m, 1H), 4.38-4.42 (m, 1H), 5.24 (s, 2H), 5.39 (s, 2H), 5.96 (t, J=5.7 Hz, 1H), 7.41 (d, J=8.4 Hz, 2H), 7.55-7.58 (m, 2H), 7.65 (d, J=8.4 Hz, 2H), 7.81 (d, J=8.6 Hz, 1H), 8.06 (d, J=7.5 Hz, 1H), 8.29-8.33 (m, 2H), 10.03 (s, 1H); .sup.13C NMR (126 MHz, d6-DMSO) δ 18.2, 19.2, 24.8, 25.7, 26.8, 27.9, 29.2, 30.3, 34.9, 38.5, 50.5, 53.1, 57.6, 70.2, 119.0, 122.5, 125.3, 129.3, 129.4, 139.3, 145.1, 151.9, 155.3, 158.8, 170.7, 171.3, 172.3; HRMS-ESI [M+Na]; calcd for C.sub.31H.sub.41N.sub.9NaO.sub.9: 706.2925. found 706.2913.
Example 7.3—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-(6-azidohexanoyl)-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN172)
(53) ##STR00102##
(54) To a mixture of CN089 (61 mg, 0.071 mmol) and pNP-carbonate 54 (54 mg, 0.079 mmol) in anhydrous pyridine (1.0 mL) under Ar is added Et.sub.3N (20 μL, 0.14 mmol) and the mixture is stirred at rt. After 26 h, the mixture is concentrated to dryness under high vacuum, and the crude residue is purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=5:95 to 20:80), followed by column chromatography on C18 silica gel (MeOH/CH.sub.2Cl.sub.2=100:0 to 90:10), to afford the title compound CN172 as a white solid (57 mg, 57%). .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 0.95-0.98 (m, 6H), 1.24-1.37 (m, 68H), 1.39-1.45 (m, 2H), 1.53-1.77 (m, 11H), 1.87-1.94 (m, 1H), 2.04-2.11 (m, 1H), 2.27-2.32 (m, 2H), 2.33-2.40 (m, 2H), 3.09-3.14 (m, 1H), 3.21-3.26 (m, 1H), 3.28 (t, J=6.8 Hz, 2H), 3.66-3.80 (m, 8H), 3.85-3.87 (m, 2H), 4.18 (d, J=7.3 Hz, 1H), 4.53 (dd, J=5.1, 8.6 Hz, 1H), 4.85 (d, J=3.7 Hz, 1H), 4.93-4.99 (m, 2H), 5.10-5.18 (m, 1H), 7.32 (d, J=8.3 Hz, 2H), 7.57 (d, J=8.3 Hz, 2H); .sup.13C NMR (126 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 14.2, 18.5, 19.4, 23.0, 25.4, 25.6, 25.7, 26.7, 28.9, 29.2, 29.6, 29.69, 29.72, 29.8, 29.90, 29.92, 29.96, 30.02, 30.06, 31.0, 32.3, 35.0, 36.2, 39.4, 51.6, 52.6, 53.7, 59.4, 62.3, 66.8, 68.4, 69.4, 70.2, 70.7, 71.0, 72.3, 75.1, 100.4, 120.5, 129.1, 133.0, 138.3, 157.1, 161.1, 171.0, 172.9, 175.0; HRMS-ESI [M+Na].sup.+ calcd for C.sub.75H.sub.135N.sub.9NaO.sub.15: 1424.9941. found 1424.9940.
Example 8—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-(5-hexenoyl)-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN173)
Example 8.1—N-(5-Hexenoyl)-Val-Cit-4-aminobenzyl alcohol (55)
(55) ##STR00103##
(56) To a stirred solution of 5-hexenoic acid (39 mg, 0.34 mmol) in anhydrous CH.sub.2Cl.sub.2 (2 mL) at 0° C. is added by Et.sub.3N (50 μL, 0.36 mmol), followed by isobutyl chloroformate (43 μL, 0.33 mmol). The solution is warmed to rt and stirred for 45 min, before transferring by cannula to a separate flask containing amine 52 (Dubowchik, Firestone et al. 2002) (100 mg, 0.264 mmol) in 5:1 CH.sub.2Cl.sub.2-MeOH (2.4 mL) at 0° C. The original flask is rinsed with CH.sub.2Cl.sub.2 (0.5 mL), which is transferred to the second flask. After 10 min, the reaction mixture is warmed to rt and MeOH (1 mL) is added to aid stirring of the heterogeneous mixture. After 85 min at rt, the reaction is quenched with Et.sub.2NH (25 μL) and the solvents are concentrated under reduced pressure. The resulting solid is triturated successively with diethyl ether and CH.sub.2Cl.sub.2 to afford the title compound 55 as an off-white solid (114 mg, 91%). .sup.1H NMR (500 MHz, d6-DMSO) δ 0.84 (d, J=6.7 Hz, 3H), 0.86 (d, J=6.7 Hz, 3H), 1.32-1.48 (m, 2H), 1.53-1.64 (m, 3H), 1.67-1.74 (m, 1H), 1.95-2.02 (m, 3H), 2.13-2.24 (m, 2H), 2.91-2.97 (m, 1H), 2.99-3.05, (m, 1H), 4.19 (dd, J=6.9, 8.5 Hz, 1H), 4.36-4.41 (m, 1H), 4.42 (d, J=5.7 Hz, 2H), 4.93-4.96 (m, 1H), 4.98-5.02 (m, 1H), 5.07 (t, J=5.7 Hz, 1H), 5.38 (s, 2H), 5.74-5.84 (m, 1H), 5.97 (t, J=5.6 Hz, 1H), 7.23 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H), 7.81 (d, J=8.5 Hz, 1H), 8.03 (d, J=7.6 Hz, 1H), 9.88 (s, 1H); .sup.13C NMR (126 MHz, d6-DMSO) δ 18.2, 19.2, 24.6, 26.7, 29.3, 30.3, 32.7, 34.6, 38.6, 53.0, 57.7, 62.6, 115.0, 118.9, 126.9, 137.4, 137.5, 138.3, 158.8, 170.3, 171.2, 172.3; HRMS-ESI [M+Na].sup.+ calcd for C.sub.24H.sub.37N.sub.5NaO.sub.5: 498.2692. found 498.2699.
Example 8.2—N-(5-Hexenoyl)-Val-Cit-4-aminobenzyl 4-nitrophenyl carbonate (56)
(57) ##STR00104##
(58) To a solution of alcohol 55 (110 mg, 0.231 mmol) in anhydrous DMF (2.0 mL) is added bis(4-nitrophenyl) carbonate (95 mg, 0.31 mmol) followed by N,N-diisopropylethylamine (48 μL, 0.28 mmol) and the reaction is stirred under Ar at rt for 7 h. The product is precipitated by the addition of diethyl ether and filtered, washing with diethyl ether and CH.sub.2Cl.sub.2. The crude product is purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=2:98 to 6:94) to afford the title compound 56 as a white solid (80 mg, 54%). .sup.1H NMR (500 MHz, d6-DMSO) δ 0.84 (d, J=6.8 Hz, 3H), 0.87 (d, J=6.7 Hz, 3H), 1.33-1.49 (m, 2H), 1.55-1.64 (m, 3H), 1.68-1.75 (m, 1H), 1.95-2.02 (m, 3H), 2.13-2.24 (m, 2H), 2.91-2.98 (m, 1H), 3.00-3.06, (m, 1H), 4.19 (dd, J=6.8, 8.6 Hz, 1H), 4.37-4.41 (m, 1H), 4.93-4.96 (m, 1H), 4.98-5.02 (m, 1H), 5.24 (s, 2H), 5.39 (s, 2H), 5.75-5.84 (m, 1H), 5.97 (t, J=5.6 Hz, 1H), 7.41 (d, J=8.5 Hz, 2H), 7.55-7.58 (m, 2H), 7.65 (d, J=8.5 Hz, 2H), 7.81 (d, J=8.6 Hz, 1H), 8.07 (d, J=7.5 Hz, 1H), 8.29-8.32 (m, 2H), 10.04 (s, 1H); .sup.13C NMR (126 MHz, d6-DMSO) δ 18.2, 19.2, 24.6, 26.8, 29.2, 30.3, 32.7, 34.6, 38.5, 53.1, 57.7, 70.2, 114.9, 119.0, 122.6, 125.4, 129.3, 129.4, 138.3, 139.3, 145.2, 151.9, 155.3, 158.9, 170.7, 171.3, 172.3; HRMS-ESI [M+Na].sup.+ calcd for C.sub.31H.sub.40N.sub.6NaO.sub.9: 663.2754. found 663.2764.
Example 8.3—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-(5-hexenoyl)-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN173)
(59) ##STR00105##
(60) To a solution of CN089 (17 mg, 0.020 mmol) in anhydrous pyridine (0.3 mL) is added pNP-carbonate 56 (16 mg, 0.025 mmol), Et.sub.3N (9 μL, 0.065 mmol) and MeOH (0.1 mL), and the mixture is stirred at rt. After 22 h, further Et.sub.3N (5 μL, 0.036 mmol) is added and stirring is continued for a further 19 h. The reaction is quenched with Et.sub.2NH (15 μL) and the mixture is concentrated to dryness under high vacuum. Purification by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=10:90 to 14:86), followed by trituration of the product with water, affords the title compound CN173 as a white solid (14.8 mg, 55%). .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 0.95-0.97 (m, 6H), 1.22-1.39 (m, 68H), 1.52-1.76 (m, 9H), 1.87-1.95 (m, 1H), 2.03-2.12 (m, 3H), 2.27-2.30 (m, 2H), 2.33-2.38 (m, 2H), 3.08-3.13 (m, 1H), 3.21-3.26 (m, 1H), 3.66-3.80 (m, 8H), 3.85-3.87 (m, 2H), 4.19 (d, J=7.3 Hz, 1H), 4.54 (dd, J=5.0, 8.7 Hz, 1H), 4.85 (d, J=3.7 Hz, 1H), 4.93-5.05 (m, 4H), 5.10-5.19 (m, 1H), 5.75-5.83 (m, 1H), 7.32 (d, J=8.1 Hz, 2H), 7.56 (d, J=8.1 Hz, 2H); .sup.13C NMR (126 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 14.2, 18.5, 19.4, 23.0, 25.2, 25.4, 25.7, 26.6, 29.2, 29.5, 29.66, 29.69, 29.8, 29.87, 29.89, 29.93, 29.96, 29.99, 30.04, 31.0, 32.3, 33.5, 34.9, 35.7, 39.3, 52.6, 53.7, 59.4, 62.3, 66.8, 68.4, 69.4, 70.2, 70.6, 70.9, 72.3, 75.0, 100.4, 115.5, 120.4, 129.1, 132.9, 138.1, 138.2, 157.0, 161.1, 171.0, 172.8, 174.9, 175.0; HRMS-ESI [M+Na].sup.+ calcd for C.sub.75H.sub.134N.sub.6NaO.sub.15: 1381.9829. found 1381.9825.
Example 9—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-levulinoyl-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN171)
Example 9.1—N-Levulinoyl-Val-Cit-4-aminobenzyl alcohol (57)
(61) ##STR00106##
(62) To a stirred solution of levulinic acid (40 mg, 0.34 mmol) in anhydrous CH.sub.2Cl.sub.2 (2.0 mL) at 0° C. is added Et.sub.3N (50 μL, 0.36 mmol), followed by isobutyl chloroformate (43 μL, 0.33 mmol). The solution is warmed to rt and stirred for 45 min, before transferring by cannula to a separate flask containing amine 52 (Dubowchik, Firestone et al. 2002) (100 mg, 0.264 mmol) in 5:1 CH.sub.2Cl.sub.2-MeOH (2.4 mL) at 0° C. The original flask is rinsed with CH.sub.2Cl.sub.2 (0.5 mL), which is transferred to the second flask. After 5 min, the reaction mixture is warmed to rt and MeOH (1 mL) is added to aid stirring of the heterogeneous mixture. After 85 min at rt, the reaction is quenched with Et.sub.2NH (25 μL) and the solvents are concentrated under reduced pressure. The resulting solid is triturated successively with diethyl ether and CH.sub.2Cl.sub.2, and purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=10:90 to 18:82) to afford the title compound 57 as a white solid (94 mg, 75%). .sup.1H NMR (500 MHz, d6-DMSO) δ 0.84 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H), 1.32-1.48 (m, 2H), 1.56-1.63 (m, 1H), 1.68-1.75 (m, 1H), 1.94-2.03 (m, 1H), 2.07 (s, 3H), 2.35-2.46 (m, 2H), 2.59-2.70 (m, 2H), 2.91-2.98 (m, 1H), 2.99-3.05, (m, 1H), 4.16 (dd, J=6.6, 8.4 Hz, 1H), 4.35-4.39 (m, 1H), 4.43 (d, J=5.7 Hz, 2H), 5.07 (t, J=5.7 Hz, 1H), 5.38 (s, 2H), 5.95 (t, J=5.7 Hz, 1H), 7.23 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H), 7.88 (d, J=8.4 Hz, 1H), 7.98 (d, J=7.7 Hz, 1H), 9.79 (s, 1H); .sup.13C NMR (126 MHz, d6-DMSO) δ 18.1, 19.2, 26.8, 29.0, 29.3, 29.6, 30.3, 38.1, 38.6 53.1, 57.8, 62.6, 118.8, 126.9, 137.4, 137.5, 158.8, 170.3, 171.1, 171.7, 207.5; HRMS-ESI [M+Na].sup.+ calcd for C.sub.23H.sub.35N.sub.5NaO.sub.6: 500.2485. found 500.2485.
Example 9.2—N-Levulinoyl-Val-Cit-4-aminobenzyl 4-nitrophenyl carbonate (58)
(63) ##STR00107##
(64) To a solution of alcohol 57(89 mg, 0.19 mmol) in anhydrous DMF (1.7 mL) is added bis(4-nitrophenyl) carbonate (67 mg, 0.22 mmol) followed by N,N-diisopropylethylamine (39 μL, 0.22 mmol) and the reaction is stirred under Ar at rt for 7 h. The product is precipitated by the addition of diethyl ether and filtered, washing with diethyl ether and CH.sub.2Cl.sub.2. The crude product is purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=4:96 to 8:92) to afford the title compound 58 as a white solid (70 mg, 58%). .sup.1H NMR (500 MHz, 2:3 CDCl.sub.3/CD.sub.3OD) δ 1.00-1.03 (m, 6H), 1.53-1.69 (m, 2H), 1.78-1.86 (m, 1H), 1.98-2.05 (m, 1H), 2.08 (s, 3H), 2.15-2.23 (m, 1H), 2.44-2.50 (m, 1H), 2.61 (ddd, J=5.1, 8.7, 15.6 Hz, 1H), 2.76-2.82 (m, 1H), 2.88 (ddd, J=5.4, 8.7, 18.6 Hz, 1H), 3.13-3.23 (m, 1H), 4.16 (d, J=6.1 Hz, 1H), 4.52 (dd, J=4.7, 9.7 Hz, 1H), 5.27 (s, 2H), 7.40-7.44 (m, 4H), 7.69 (d, J=8.6 Hz, 2H), 8.21-8.31 (m, 2H); .sup.13C NMR (126 MHz, 2:3 CDCl.sub.3/CD.sub.3OD) δ 18.3, 19.5, 27.3, 29.6, 29.76, 29.83, 30.7, 39.0, 39.9, 54.4, 60.6, 71.3, 120.9, 122.7, 125.9, 130.1, 131.1, 139.6, 146.3, 153.4, 156.5, 161.5, 171.7, 173.3, 175.2, 210.3; HRMS-ESI [M+Na].sup.+ calcd for C.sub.30H.sub.38N.sub.6NaO.sub.10: 665.2547. found 665.2553.
Example 9.3—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-levulinoyl-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN171)
(65) ##STR00108##
(66) To a mixture of CN089 (16 mg, 0.019 mmol) and pNP-carbonate 58 (14 mg, 0.022 mmol) in 10:3.5:1 pyridine/MeOH/CHC.sub.3 (0.58 mL) is added Et.sub.3N (5 μL, 0.036 mmol) and the mixture is stirred at rt. After 6 h, further Et.sub.3N (5 μL, 0.036 mmol) is added and stirring is continued for a further 15 h. The reaction is quenched with Et.sub.2NH (5 μL) and the mixture is concentrated to dryness under high vacuum. Purification by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=10:90 to 20:80), followed by preparative HPLC (Phenomenex Luna C18(2), 5 μm, 30×250 mm, 40° C., 50 mL/min; Mobile phase A=80:20:0.05 MeOH/water/TFA; Mobile phase B=100:0.05 MeOH/TFA; 0-10 min: 0-100% B; 10-29 min: 100% B; 29-30 min: 100-0% B; 30-31 min: 100% A) affords the 3-O-acyl regioisomer CN217 (3.2 mg, 13%) followed by the title compound CN171. A final trituration with water gives the product as a white solid (6.4 mg, 25%). .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 0.99-1.01 (m, 6H), 1.22-1.40 (m, 68H), 1.52-1.73 (m, 6H), 1.76-1.84 (m, 1H), 1.95-2.03 (m, 1H), 2.08 (s, 3H), 2.15-2.24 (m, 1H), 2.31-2.41 (m, 2H), 2.43-2.48 (m, 1H), 2.57-2.62 (m, 1H), 2.74-2.80 (m, 1H), 2.89 (ddd, J=5.3, 8.8, 18.7 Hz, 1H), 3.12-3.24 (m, 2H), 3.66-3.81 (m, 8H), 3.85-3.88 (m, 2H), 4.16 (d, J=6.1 Hz, 1H), 4.51 (dd, J=4.6, 9.4 Hz, 1H), 4.85 (d, J=3.6 Hz, 1H), 4.93-5.00 (m, 2H), 5.09-5.16 (m, 1H), 7.32 (d, J=8.3 Hz, 2H), 7.61 (d, J=8.3 Hz, 2H); .sup.13C NMR (126 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 14.2, 18.1, 19.4, 23.0, 25.4, 25.7, 26.8, 29.2, 29.56, 29.60, 29.69, 29.71, 29.74, 29.8, 29.90, 29.92, 29.95, 29.98, 30.01, 30.1, 30.4, 32.27, 32.29, 35.0, 38.8, 39.5, 52.6, 53.9, 60.1, 62.3, 66.8, 68.4, 69.4, 70.2, 70.7, 71.0, 72.3, 75.1, 100.4, 120.5, 129.0, 133.0, 138.3, 157.1, 161.0, 171.1, 172.9, 174.7, 175.0, 210.0; HRMS-ESI [M+Na].sup.+ calcd for C.sub.74H.sub.132N.sub.6NaO.sub.16: 1383.9598. found 1383.9594.
(67) ##STR00109##
(68) Data for CN217: .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 0.99-1.01 (m, 6H), 1.23-1.42 (m, 68H), 1.48-1.71 (m, 6H), 1.77-1.85 (m, 1H), 1.96-2.03 (m, 1H), 2.08 (s, 3H), 2.16-2.23 (m, 1H), 2.30-2.38 (m, 2H), 2.42-2.47 (m, 1H), 2.57-2.63 (m, 1H), 2.74-2.80 (m, 1H), 2.87-2.93 (m, 1H), 3.13-3.25 (m, 2H), 3.53-3.57 (m, 1H), 3.62-3.79 (m, 6H), 3.84 (d, J=2.7 Hz, 1H), 3.88 (dd, J=4.8, 10.8 Hz, 1H), 4.14-4.17 (m, 1H), 4.18-4.21 (m, 1H), 4.49-4.53 (m, 1H), 4.84-4.87 (m, 1H), 4.91-4.97 (m, 2H), 5.12-5.17 (m, 1H), 7.31 (d, J=8.3 Hz, 2H), 7.60 (d, J=8.3 Hz, 2H); .sup.13C NMR (126 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ selected peaks: 14.2, 18.1, 19.4, 23.0, 25.3, 25.8, 26.8, 29.6, 29.7, 30.0, 30.3, 32.3, 33.5, 34.7, 38.8, 39.4, 52.1, 54.0, 60.2, 62.3, 66.8, 69.3, 70.3, 70.6, 71.1, 76.6, 100.2, 120.5, 129.0; HRMS-ESI [M+Na].sup.+ calcd for C.sub.74H.sub.132N.sub.6NaO.sub.18: 1383.9598. found 1383.9586.
Example 10—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-maleimidohexanoyl-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN211)
Example 10.1—N-Fluorenylmethoxycarbonyl-Val-Cit-4-aminobenzyl 4-nitrophenyl carbonate (BJC209)
(69) ##STR00110##
(70) To a solution of alcohol 59 (Dubowchik, Firestone et al. 2002) (270 mg, 0.45 mmol) in DMF (4 mL) under Ar is added bis(4-nitrophenyl) carbonate (220 mg, 0.72 mmol), followed by i-Pr.sub.2NEt (90 μL, 0.51 mmol) and the reaction is stirred at rt. After 18 h, the mixture is diluted with MeOH (10 mL) then concentrated under reduced pressure and the residue is azeotroped with toluene (4×10 mL). The crude product is purified by column chromatography on silica gel (MeOH/CHCl.sub.3=0:1 to 1:4), to afford the title compound 60 as a yellow solid (219 mg, 64%). .sup.1H NMR (500 MHz, 3:1 CDCl.sub.3/CD.sub.3OD) δ 0.95 (d, J=6.8 Hz, 3H), 0.97 (d, J=6.8 Hz, 3H), 1.50-1.60 (m, 2H), 1.68-1.75 (m, 1H), 1.89-1.96 (m, 1H), 2.06-2.13 (m, 1H), 3.08-3.13, (m, 1H), 3.21-3.26, (m, 1H), 4.00 (d, J=6.5 Hz, 1H), 4.22 (dd, J=6.5, 6.5 Hz, 1H), 4.35-4.38 (m, 1H), 4.45-4.49 (m, 1H), 4.56-4.58 (m, 1H), 5.25 (s, 2H), 7.31 (dd, J=7.5, 7.5 Hz, 2H), 7.38-7.41 (m, 6H), 7.61-7.64 (m, 4H), 7.77 (d, J=7.7 Hz, 2H); .sup.13C NMR (126 MHz, 3:1 CDCl.sub.3/CD.sub.3OD) δ 18.1, 19.3, 26.6, 29.5, 31.2, 39.2, 53.5, 61.0, 67.3, 70.9, 120.2, 120.4, 122.1, 125.2, 125.3, 125.5, 127.3, 128.0, 129.8, 139.0, 141.6, 144.0, 144.1, 145.7, 152.8, 155.9, 157.4, 160.8, 170.9, 172.9; HRMS-ESI: m/z calcd for C.sub.40H.sub.42N.sub.6O.sub.10Na [M+Na].sup.+ 789.2860. found 789.2853.
Example 10.2—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-fluorenylmethoxycarbonyl-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (61)
(71) ##STR00111##
(72) To a mixture of CN089 (112 mg, 0.131 mmol) and pNP-carbonate 60 (138 mg, 0.180 mmol) in anhydrous pyridine (1.8 mL) under Ar is added Et.sub.3N (24 μL, 0.17 mmol) and the mixture is stirred at rt. After 23 h, the mixture is concentrated to dryness under high vacuum, and the crude residue is purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=5:95 to 13:87) to afford the title compound 61 as a white solid (122 mg, 63%). .sup.1H NMR (500 MHz, 2:3 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 0.95-0.98 (m, 6H), 1.24-1.37 (m, 68H), 1.51-1.78 (m, 7H), 1.89-1.96 (m, 1H), 2.07-2.13 (m, 1H), 2.32-2.42 (m, 2H), 3.07-3.13 (m, 1H), 3.20-3.25 (m, 1H), 3.66-3.81 (m, 8H), 3.84-3.87 (m, 2H), 3.99 (d, J=6.7 Hz, 1H), 4.24 (t, J=6.9 Hz, 1H), 4.37 (dd, J=6.9, 10.5 Hz, 1H), 4.45 (dd, J=6.9, 10.5 Hz, 1H), 4.54 (dd, J=5.2, 8.6 Hz, 1H), 4.84 (d, J=3.7 Hz, 1H), 4.97-5.03 (m, 2H), 5.06-5.10 (m, 1H), 7.30-7.33 (m, 4H), 7.38-7.41 (m, 2H), 7.58 (d, J=8.1 Hz, 2H), 7.63-7.65 (m, 2H), 7.78 (d, J=7.6 Hz, 2H); .sup.13C NMR (126 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 14.3, 18.2, 19.4, 23.0, 25.5, 25.7, 26.7, 29.2, 29.6, 29.27, 29.74, 29.8, 29.93, 29.95, 29.98, 30.02, 30.05, 30.08, 30.10, 31.4, 32.3, 35.0, 39.4, 47.6, 52.7, 53.8, 61.2, 62.3, 66.8, 67.4, 68.4, 69.4, 70.2, 70.7, 71.0, 72.3, 75.1, 100.4, 120.3, 120.5, 125.40, 125.44, 127.5, 128.2, 129.1, 133.0, 138.2, 141.7, 144.2, 144.3, 157.1, 157.6, 161.1, 171.1, 173.2, 175.0; HRMS-ESI m/z calcd for C.sub.84H.sub.137N.sub.6O.sub.16 [M+H].sup.+: 1486.0091. found 1486.0099.
Example 10.3—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (62)
(73) ##STR00112##
(74) To a mixture of compound 61 (125 mg, 0.0841 mmol) in DMF (2 mL) is added piperidine (0.2 mL) at 0° C. The mixture is stirred at 0° C. for 5 min, then at rt for 40 min. The solvents are concentrated to dryness under high vacuum, and the crude residue is purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=15:85 to 35:65) to afford the title compound 62 as a white solid (95 mg, 89%). .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.91 (m, 9H), 1.00 (d, J=6.9 Hz, 3H), 1.23-1.35 (m, 68H), 1.49-1.77 (m, 7H), 1.87-1.94 (m, 1H), 2.07-2.13 (m, 1H), 2.32-2.39 (m, 2H), 3.10-3.16 (m, 1H), 3.21 (d, J=4.9 Hz, 1H), 3.24-3.29 (m, 1H), 3.65-3.80 (m, 8H), 3.85-3.87 (m, 2H), 4.57 (dd, J=5.3, 8.5 Hz, 1H), 4.85 (d, J=3.7 Hz, 1H), 4.92-4.99 (m, 2H), 5.10-5.15 (m, 1H), 7.33 (d, J=8.3 Hz, 2H), 7.56 (d, J=8.3 Hz, 2H); .sup.13C NMR (75 MHz, 3:1 CDCl.sub.3/CD.sub.3OD) δ 14.1, 16.8, 19.5, 22.8, 25.2, 25.5, 26.4, 29.0, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30.0, 31.9, 32.1, 34.8, 39.2, 52.3, 53.1, 60.4, 62.1, 66.6, 68.2, 69.2, 70.0, 70.5, 70.6, 72.2, 74.9, 100.1, 120.3, 128.9, 132.9, 138.0, 156.8, 160.8, 171.1, 174.8, 175.7; HRMS-ESI m/z calcd for C.sub.69H.sub.127N.sub.6O.sub.14 [M+H].sup.+: 1263.9410. found 1263.9419.
Example 10.4—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-maleimidohexanoyl-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN211)
(75) ##STR00113##
(76) To a solution of amine 62 (10.4 mg, 0.00823 mmol) and 6-maleimidohexanoic acid NHS ester (Leonard and Brunckova 2010) (3.3 mg, 0.011 mmol) in DMF (80 uL) is added Et.sub.3N (0.9 mg, 0.009 mmol) and the mixture is stirred at rt. After 4 h, the mixture is concentrated under high vacuum, and the crude residue is purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=8:92 to 14:86) to afford the title compound CN211 as a white solid (11.2 mg, 93%). .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 0.94-0.97 (m, 6H), 1.23-1.36 (m, 70H), 1.49-1.77 (m, 11H), 1.87-1.94 (m, 1H), 2.03-2.10 (m, 1H), 2.24-2.30 (m, 2H), 2.31-2.41 (m, 2H), 3.09-3.14 (m, 1H), 3.20-3.26 (m, 1H), 3.51 (t, J=7.2 Hz, 2H), 3.66-3.81 (m, 8H), 3.85-3.87 (m, 2H), 4.17 (d, J=7.4 Hz, 1H), 4.53 (dd, J=5.1, 8.6 Hz, 1H), 4.85 (d, J=3.8 Hz, 1H), 4.92-4.98 (m, 2H), 5.10-5.15 (m, 1H), 6.74 (s, 2H), 7.31 (d, J=8.1 Hz, 2H), 7.56 (d, J=8.1 Hz, 2H); .sup.13C NMR (126 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 14.2, 18.5, 19.4, 23.0, 25.4, 25.5, 25.6, 26.6, 28.5, 29.2, 29.5, 29.58, 29.63, 29.65, 29.73, 29.84, 29.86, 29.90, 29.93, 29.97, 30.01, 31.0, 32.2, 34.9, 36.2, 37.9, 39.3, 52.5, 53.7, 59.4, 62.3, 66.7, 68.4, 69.4, 70.2, 70.6, 70.8, 72.3, 75.0, 100.3, 120.4, 129.0, 132.9, 134.5, 138.2, 157.0, 161.0, 171.0, 171.5, 172.8, 174.9; HRMS-ESI m/z calcd for C.sub.79H.sub.137N.sub.7NaO.sub.17 [M+Na].sup.+: 1478.9969. found 1478.9971.
Example 11—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-(bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl)-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN209)
(77) ##STR00114##
(78) To a solution of amine 62 (6.5 mg, 0.0051 mmol) in DMF (50 μL) is added bicyclo[6.1.0]non-4-yn-9-ylmethyl 4-nitrophenyl carbonate (Dommerholt, Schmidt et al. 2010) (2.0 mg, 0.0063 mmol) followed by Et.sub.3N (1.5 μL, 0.011 mmol) and the mixture is stirred at rt. After 20 h, the mixture is concentrated under high vacuum, and the crude residue is purified by column chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2=5:95 to 15:85) to afford the title compound CN209 as a white solid (6.4 mg, 86%). .sup.1H NMR (500 MHz, 2:3 CDCl.sub.3/CD.sub.3OD) δ 0.68-0.78 (m, 3H), 0.88-0.90 (m, 6H), 0.95 (d, J=6.8 Hz, 3H), 0.99 (d, J=6.8 Hz, 3H), 1.23-1.41 (m, 70H), 1.50-1.78 (m, 7H), 1.89-1.96 (m, 1H), 2.08-2.14 (m, 3H), 2.22-2.30 (m, 2H), 2.32-2.42 (m, 4H), 3.09-3.14 (m, 1H), 3.20-3.26 (m, 1H), 3.66-3.81 (m, 8H), 3.84-3.87 (m, 2H), 3.95-4.03 (m, 3H), 4.55 (dd, J=5.3, 8.3 Hz, 1H), 4.84 (d, J=3.7 Hz, 1H), 4.97-5.03 (m, 2H), 5.06-5.11 (m, 1H), 7.30-7.33 (m, 2H), 7.58 (d, J=8.2 Hz, 2H); .sup.13C NMR (126 MHz, 2:3 CDCl.sub.3/CD.sub.3OD) δ 14.34, 14.36, 18.3, 19.6, 21.7, 23.29, 23.31, 23.7, 23.8, 24.4, 25.8, 26.0, 27.2, 29.3, 29.9, 30.00, 30.03, 30.2, 30.25, 30.31, 30.34, 30.4, 31.6, 32.59, 32.62, 33.9, 35.2, 39.8, 53.2, 54.2, 61.5, 62.5, 67.0, 68.6, 69.8, 70.2, 70.6, 71.0, 71.6, 72.4, 75.4, 99.3, 100.8, 120.8, 129.3, 133.4, 138.7, 157.6, 158.5, 161.6, 171.5, 173.7, 175.2; HRMS-ESI m/z calcd for C.sub.80H.sub.138N.sub.6NaO.sub.16 [M+Na].sup.+: 1462.0067. found 1462.0061.
Example 12—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-(biotinoyl)-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN201)
(79) ##STR00115##
(80) To a solution of amine 62 (10.9 mg, 0.00862 mmol) in DMF (0.10 mL) is added D-(+)-biotin NHS ester (5.4 mg, 0.016 mmol) followed by Et.sub.3N (3.2 mg, 0.032 mmol) and the mixture is stirred at rt for 2 days. The heterogeneous mixture is diluted with MeOH (1 mL) and water (0.2 mL), and filtered, washing with MeOH. The collected precipitate is purified by column chromatography on silica gel (MeOH/CHCl.sub.3=10:90 to 30:70) to afford the title compound CN201 as a white solid (8.3 mg, 64%). .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 0.95-0.98 (m, 6H), 1.23-1.35 (m, 68H), 1.41-1.47 (m, 2H), 1.50-1.79 (m, 11H), 1.87-1.94 (m, 1H), 2.06-2.13 (m, 1H), 2.24-2.41 (m, 4H), 2.74 (d, J=12.8 Hz, 1H), 2.93 (dd, J=5.0, 12.8 Hz, 1H), 3.10-3.22 (m, 3H), 3.66-3.80 (m, 8H), 3.84-3.87 (m, 2H), 4.18 (d, J=6.8 Hz, 1H), ˜4.30 (m, 1H), 4.48-4.53 (m, 2H), 4.85 (d, J=3.7 Hz, 1H), 4.93-4.98 (m, 2H), 5.12-5.16 (m, 1H), 7.32 (d, J=8.3 Hz, 2H), 7.58 (d, J=8.3 Hz, 2H); .sup.13C NMR (126 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 14.2, 18.4, 19.5, 23.0, 25.4, 25.7, 26.0, 26.7, 28.34, 28.37, 29.2, 29.5, 29.6, 29.70, 29.72, 29.79, 29.91, 29.93, 29.96, 30.02, 30.8, 32.3, 35.0, 35.8, 39.5, 40.6, 52.6, 54.0, 55.8, 59.5, 60.6, 62.2, 62.3, 66.8, 68.4, 69.4, 70.3, 70.7, 70.9, 72.3, 75.1, 100.4, 120.5, 129.1, 133.0, 138.3, 157.1, 161.1, 164.9, 171.1, 173.0, 175.0, 175.3; HRMS-ESI m/z calcd for C.sub.79H.sub.140N.sub.8NaO.sub.16S [M+Na].sup.+: 1512.0006. found 1512.0006.
Example 13—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-(ω-mercapto(poly(ethyleneoxy))acetyl)-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN200)
Example 13.1—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-(ω-(isobutoxycarbonylthio)(poly(ethyleneoxy))acetyl)-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (63)
(81) ##STR00116##
(82) To a solution of ω-mercaptopoly(ethyleneoxy)acetic acid (average Mw 1,000) (5.9 mg, 0.0053 mmol) in anhydrous CH.sub.2Cl.sub.2 (0.2 mL) at 0° C. is added i-Pr.sub.2NEt (7.4 mg, 0.057 mmol), followed by isobutyl chloroformate (6.2 μL, 0.048 mmol). The solution is stirred at 0° C. for 45 min, then at rt for 15 min, before concentrating to dryness. The residue is co-evaporated twice with toluene to drive off excess isobutyl chloroformate reagent. The mixed anhydride intermediate is dissolved in 18:1 chloroform/MeOH (0.95 mL), and stirred with i-Pr.sub.2NEt (2.0 μL, 0.012 mmol) and amine 62 (3.1 mg, 0.0025 mmol) at rt for 2 days. After concentration of the solvents under reduced pressure, the residue is loaded (as a 1:1 MeOH/water solution) onto a cartridge containing 200 mg of end-capped cyclohexyl-bonded silica gel (Isolute CH(EC)). After removing more polar components, the product is eluted with MeOH/water (9:1 to 1:0). Further purification by preparative HPLC (Phenomenex Luna C18(1), 5 μm, 250×10 mm; 40° C.; 2.8 mL/min; mobile phase=80:20 IPA/MeOH) gives the title compound 63 as a colourless glass (3.2 mg, 53%). .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 0.95 (d, J=6.7 Hz, 6H), 0.97 (d, J=6.8 Hz, 3H), 0.99 (d, J=6.8 Hz, 3H), 1.23-1.35 (m, 68H), 1.49-1.77 (m, 7H), 1.88-2.02 (m, 2H), 2.11-2.18 (m, 1H), 2.31-2.40 (m, 2H), 3.07 (t, J=6.5 Hz, 2H), 3.10-3.15 (m, 1H), 3.21-3.26 (m, 1H), 3.61-3.81 (m, ˜110H), 3.85-3.88 (m, 2H), 4.01 (d, J=6.6 Hz, 1H), 4.07 (s, 2H), 4.25 (d, J=7.0 Hz, 1H), 4.55 (dd, J=5.1, 8.8 Hz, 1H), 4.85 (d, J=3.7 Hz, 1H), 4.93-4.99 (m, 2H), 5.10-5.15 (m, 1H), 7.32 (d, J=8.4 Hz, 2H), 7.57 (d, J=8.4 Hz, 2H); 13C NMR (126 MHz, 2:1 CDCl3/CD3OD) δ 14.2, 18.4, 19.1, 19.5, 23.0, 25.4, 25.7, 26.7, 28.3, 29.2, 29.6, 29.7, 29.8, 29.9, 30.0, 30.8, 31.3, 32.3, 35.0, 39.3, 52.6, 53.7, 58.9, 62.3, 66.8, 68.4, 69.4, 70.2, 70.3, 70.5, 70.6, 70.7, 70.8, 70.9, 71.3, 72.3, 74.0, 75.0, 100.4, 120.5, 129.1, 133.0, 138.3, 157.1, 161.1, 171.1, 171.4, 171.6, 172.1, 174.9; HRMS-ESI m/z calcd for C.sub.126H.sub.236N.sub.6Na.sub.2O.sub.42S [M(n=24)+2Na].sup.2+: 1291.8016. found 1291.7981.
Example 13.2—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-(ω-mercapto(poly(ethyleneoxy))acetyl)-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN200)
(83) ##STR00117##
(84) To a solution of thiocarbonate 63 (3 mg, 1.2 umol) in MeOH (0.5 mL) is added buffered hydroxylamine (0.5 mL, 0.5 M NH2OH.HCl, 25 mM EDTA dissolved in PBS and adjusted to pH 7.4 with 3.0 M NaOH). The reaction vial is incubated under Ar at 40° C. for 42 h. The mixture is purified by passage through a cartridge containing 1 g of endcapped C18 silica, (MeOH/IPA=1:0 to 1:1) to give the a mixture of the title compound CN200 and unreacted starting material. HRMS-ESI m/z calcd for C.sub.121H.sub.228N.sub.8Na.sub.2O.sub.40S [M(n=24)+2Na].sup.2+: 1241.7754. found 1241.7739.
Example 14—Synthesis of (2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-(4-fluorophenylundecanoyl)-2-(N-(6-azidohexanoyl)Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN213)
Example 14.1—(2S,3S,4R)-2-Amino-1-O-α-D-galactopyranosyl-4-O-(4-fluorophenylundecanoyl) octadecane-1,3,4-triol (65)
(85) ##STR00118##
(86) A solution of compound 64 (20 mg, 0.027 mmol) (Li, X., Fujio, M. et al. 2010) in 1,4-dioxane (3 mL) and 1 M HCl (0.6 mL) is heated at 80° C. for 1 h. The mixture is diluted with CHCl.sub.3/MeOH (1:1, 30 mL) and concentrated under reduced pressure. The crude residue is purified by column chromatography on silica gel (MeOH/CHCl.sub.3=0:10 to 3:7) to afford the title compound 65 as a white solid (14 mg, 70%). .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 5:1) δ 0.88 (t, J=6.9, 6.9 Hz, 3H), 1.24-1.32 (m, 38H), 1.54-1.64 (m, 5H), 1.76-1.83 (m, 1H), 2.34 (dd, J=7.5, 7.5 Hz, 2H), 2.57 (dd, J=7.7, 7.7 Hz, 2H), 3.24-3.27 (m, 1H), 3.54 (dd, J=9.7, 9.7 Hz, 2H), 3.76-3.87 (m, 6H), 3.97 (br d, J=2.8 Hz), 4.09 (dd, J=2.8, 10.6 Hz), 4.88 (d, J=3.7 Hz), 6.92-6.96 (m, 2H), 7.10-7.14 (m, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 5:1) δ 13.0, 21.9, 24.2, 24.4, 28.4, 28.58, 28.63, 28.67, 28.73, 28.77, 28.82, 29.0, 30.4, 30.9, 31.2, 33.7, 34.3, 52.2, 61.0, 64.1, 68.3, 68.6, 69.1, 69.3, 70.2, 70.4, 72.5, 99.0, 113.9, 114.1, 128.9, 129.0, 137.8, 159.6, 161.5, 173.4; .sup.19F NMR (470 MHz CDCl.sub.3/CD.sub.3OD 5:1) δ −118.68; HRMS-ESI m/z calcd for C.sub.41H.sub.73NO.sub.9F [M+H].sup.+ 742.5266. found 742.5269.
Example 14.2—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-(4-fluorophenyl undecanoyl)-2-(N-(6-azidohexanoyl)-Val-Cit-4-aminobenzyloxycarbonylamino) octadecane-1,3,4-triol (CN213)
(87) ##STR00119##
(88) To a mixture of amine 65 (14 mg, 0.018 mmol) and pNP-carbonate 54 (20 mg, 0.029 mmol) in anhydrous pyridine (0.26 mL) under Ar is added Et.sub.3N (3.6 μL, 0.026 mmol) and the mixture is stirred at rt. After 24 h, a further portion of Et.sub.3N (1.6 μL, 0.012 mmol) is added. After a further 8 h, the volatiles are removed under reduced pressure. The crude residue is purified by column chromatography on silica gel (MeOH/CHCl.sub.3=0:1 to 3:7) to afford the title compound CN213 as a white solid (17 mg, 71%). .sup.1H NMR (500 MHz, 5:1 CDCl.sub.3/CD.sub.3OD) δ 0.88 (t, J=6.9, 6.9 Hz, 3H), 0.94-0.97 (m, 6H), 1.25-1.35 (m, 38H), 1.39-1.45 (m, 2H), 1.52-1.76 (m, 12H), 1.86-1.93 (m, 1H), 2.03-2.10 (m, 1H), 2.29 (ddd, J=1.3, 7.5. 7.5 Hz, 2H), 2.33-2.37 (m, 2H), 2.57 (dd, J=7.6, 7.6 Hz, 2H), 3.08-3.13 (m, 1H), 3.20-3.24 (m, 1H), 3.28 (dd, J=7.0, 7.0 Hz, 2H), 3.65-3.77 (m, 8H), 3.84-3.87 (m, 2H), 4.52-4.55 (m, 1H), 4.84 (d, 3.8 Hz, 1H), 4.94-4.97 (m, 2H), 5.13-5.15 (m, 1H), 6.93-6.97 (m, 2H), 7.10-7.14 (m, 2H), 7.32 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.4 Hz, 2H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 5:1) δ 13.6, 17.8, 18.8, 22.3, 24.7, 24.9, 25.0, 26.0, 28.2, 28.6, 28.8, 28.9, 29.0, 29.1, 29.17, 29.23, 29.3, 30.4, 31.3, 31.6, 34.2, 34.7, 35.5, 38.6, 50.9, 51.8, 53.0, 58.7, 61.6, 66.1, 67.7, 68.7, 69.5, 69.9, 70.2, 71.7, 74.3, 99.7, 114.4, 114.5, 119.8, 128.4, 129.27, 129.33, 132.3, 137.5, 138.2, 156.3, 159.9, 160.3, 161.8, 170.3, 172.1, 174.1, 174.2; .sup.19F NMR (470 MHz CDCl.sub.3/CD.sub.3OD 5:1) δ −118.86; HRMS-ESI m/z calcd for C.sub.66H.sub.109N.sub.9O.sub.15F [M+H].sup.+ 1286.8022. found 1286.8027.
Example 15—Synthesis of (2S,3S,4R)-2-Amino-1-O-α-D-galactopyranosyl-4-O-hexacosanoyl nonane-1,3,4-triol (CN214)
Example 15.1—(2S,3S,4R)-2-Amino-1-O-α-D-galactopyranosyl-4-O-hexacosanoyl nonane-1,3,4-triol (67)
(89) ##STR00120##
(90) Compound 66 (Enzo Life Sciences, 10.2 mg, 0.014 mmol) is heated under Ar in 10:1:1.3 1,4-dioxane/water/1 M HCl (3.57 mL) at 83° C. for 30 min, then cooled to rt. After lyophilisation, the resulting solid is purified on silica gel (MeOH/CHCl.sub.3=15:85 to 25:75) to afford the title compound 67 as a white solid (6.1 mg, 60%). .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD 2:1) δ 0.87-0.91 (m, 6H), 1.22-1.40 (m, 50H), 1.54-1.67 (m, 3H), 1.78-1.84 (m, 1H), 2.35-2.38 (m, 2H), 3.26-3.29 (m, 1H), 3.51-3.55 (m, 1H), 3.71-3.73 (m, 1H), 3.76 (dd, J=3.3, 10.0 Hz, 1H), 3.79-3.81 (m, 2H), 3.83-3.86 (m, 2H), 3.97 (d, J=3.3 Hz, 1H), 4.11 (dd, J=3.0, 10.7 Hz, 1H), 4.88 (d, J=3.8 Hz, 1H), 4.93 (dt, J=3.0, 8.7 Hz, 1H); .sup.13C NMR (126 MHz, CDCl.sub.3/CD.sub.3OD 2:1) δ 14.1, 14.2, 22.8, 23.0, 25.0, 25.4, 29.5, 29.68, 29.71, 29.9, 30.00, 30.03, 30.05, 31.4, 31.9, 32.3, 34.8, 53.1, 62.2, 65.4, 69.4, 70.2, 70.4, 71.2, 71.6, 73.7, 100.0, 174.6; HRMS-ESI [M+H].sup.+ calcd for C.sub.41H.sub.82NO.sub.9: 732.5990. found 732.5984.
Example 15.2—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-O-hexacosanoyl-2-(N-(6-azidohexanoyl)-Val-Cit-4-aminobenzyloxycarbonylamino) nonane-1,3,4-triol (CN214)
(91) ##STR00121##
(92) To a mixture of amine 67 (6.1 mg, 0.0083 mmol) and pNP-carbonate 54 (10 mg, 0.015 mmol) in anhydrous pyridine (0.12 mL) under Ar is added Et.sub.3N (0.7 μL, 0.012 mmol) and the mixture is stirred at rt. After 24 h, further Et.sub.3N (0.7 μL, 0.005 mmol) is added and stirring is continued for a further 8 h. The mixture is concentrated to dryness under high vacuum, and the crude residue is purified by column chromatography on silica gel (MeOH/CHCl.sub.3=2:98 to 20:80) to afford the title compound CN214 as a white solid (7.0 mg, 66%). .sup.1H NMR (500 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 0.87-0.90 (m, 6H), 0.95-0.97 (m, 6H), 1.23-1.35 (m, 50H), 1.39-1.46 (m, 2H), 1.52-1.77 (m, 11H), 1.87-1.94 (m, 1H), 2.04-2.11 (m, 1H), 2.27-2.32 (m, 2H), 2.33-2.40 (m, 2H), 3.09-3.14 (m, 1H), 3.21-3.26 (m, 1H), 3.28 (t, J=6.9 Hz, 2H), 3.66-3.80 (m, 8H), 3.84-3.87 (m, 2H), 4.19 (d, J=7.3 Hz, 1H), 4.54 (dd, J=5.1, 8.7 Hz, 1H), 4.85 (d, J=3.8 Hz, 1H), 4.94-5.01 (m, 2H), 5.10-5.18 (m, 1H), 7.32 (d, J=8.5 Hz, 2H), 7.57 (d, J=8.5 Hz, 2H); .sup.13C NMR (126 MHz, 2:1 CDCl.sub.3/CD.sub.3OD) δ 14.1, 14.2, 18.5, 19.4, 22.8, 23.0, 25.3, 25.4, 25.6, 26.6, 28.9, 29.0, 29.5, 29.59, 29.63, 29.65, 29.8, 29.95, 29.98, 31.00, 31.9, 32.2, 34.9, 36.2, 39.3, 51.5, 52.5, 53.7, 59.4, 62.3, 66.7, 68.4, 69.4, 70.2, 70.6, 70.9, 72.2, 75.0, 100.3, 120.4, 129.0, 132.9, 138.2, 157.0, 161.0, 171.0, 172.8, 174.91, 174.95; HRMS-ESI [M+Na].sup.+ calcd for C.sub.66H.sub.117N.sub.9NaO.sub.15: 1298.8567 found 1298.8553.
Example 16—(2S,3S,4R)-1-O-α-D-Galactopyranosyl-4-hexacosanoyl-2-((4-(2-(FFRKSIINFEKL)-2-oxoethoxy)imino)pentanoyloxy)methoxycarbonylamino) octadecane-1,3,4-triol (CN152)
(93) ##STR00122##
(94) To a stirred suspension of peptide 2-(aminooxy)acetyl-FFRKSIINFEKL (in which residues 5-12 comprise SEQ ID NO: 262) (5.1 mg, 3.16 mmol) in THF/MeOH (2:1, 600 μL) is added an aqueous mixture of water/aniline/TFA (200:6:4, 300 μL). Once dissolved, a solution of ketone CN146 (2.5 mg, 2.4 mmol), dissolved in THF/MeOH (1:1, 600 μL) is added and the reaction mixture is stirred at 25° C. for 48 h. The solvent is removed and the crude product purified by preparative HPLC (Phenomenex Luna C18(2), 5 μm, 250×30 mm, 30° C., 40 mL/min; Mobile phase A=100:0.1 water/TFA; Mobile phase B=100:0.1 MeOH/TFA; 0-15 min: 50-100% B; 15-23 min: 100% B; 23-25 min: 100-50% B; 25-26 min: 50% B) to give the title compound CN152 (2.1 mg, 33%). .sup.1H NMR (500 MHz, d6-DMSO) δ 0.68-0.95 (m, 24H), 1.02-1.45 (m, 74H), 1.70-1.50 (m, 27H), 1.80 (s, 3H), 2.5-2.3 (m, 6H), 2.90-2.71 (m, 8H), 3.21-2.92 (m, 6H), 3.69-3.42 (m, 12H), 4.60-4.08 (m, 18H), 4.63 (s, 1H), 4.76 (brs, 1H), 5.02 (brs, 1H), 5.06 (brs, 1H), 5.68-5.61 (m, 2H), 8.39-7.32 (m, 22H), 7.42 (m, 15H), 6.92 (s, 1H); .sup.13C NMR (126 MHz, d6-DMSO) δ 73.1 (C-5′), 79.8 (C-2″), 99.5 (H-1); HRMS (ESI): m/z calcd for C.sub.134H.sub.226N.sub.20O.sub.32 [M+2H].sup.2+ 1313.8336. found 1313.8358.
Example 17—CN178
(95) ##STR00123##
(96) To a stirred suspension of peptide 2-(aminooxy)acetyl-FFRKKAVYNFATM (in which residues 5-13 comprise SEQ ID NO: 129) (2 mg, 1.17 μmol) in THF/MeOH (2:1, 600 μL) is added an aqueous mixture of water/aniline/TFA (200:6:4, 300 μL). Once dissolved, a solution of ketone CN146 (1 mg, 0.97 μmol) in THF/MeOH (1:1, 600 μL) is added and the reaction mixture stirred at 25° C. for 48 h. The solvent is removed and the crude product purified by preparative HPLC (Phenomenex Luna C18(2), 5 μm, 250×30 mm, 30° C., 40 ml/min; Mobile phase A=100:0.1 water/TFA; Mobile phase B=100:0.1 MeOH/TFA; 0-15 min: 50-100% B; 15-23 min: 100% B; 23-25 min: 100-50% B; 25-26 min: 50% B) to give the title compound CN178 (2.0 mg, 0.74 μmol, 76%, 94.0% pure by HPLC). .sup.1H NMR (500 MHz, d6-DMSO) δ 0.66-0.78 (m, 6H), 0.78-0.95 (m, 9H), 0.95-1.42 (m, 79H), 1.42-1.73 (m, 17H), 1.73-1.82 (m, 3H), 1.82-1.93 (m, 2H), 1.93-2.06 (m, 5H), 2.23-2.33 (m, 2H), 2.33-2.39 (m, 1H), 2.70-2.89 (m, 6H), 3.08-3.15 (m, 2H), 3.40-3.73 (m, 14H), 3.75-3.82 (m, 1H), 3.88 (t, J=8.1 Hz, 1H), 3.97-4.16 (m, 3H), 4.17-4.40 (m, 9H), 4.40-4.55 (m, 5H), 4.55-4.63 (m, 1H), 4.65 (d, J=3.1 Hz, 1H), 4.70-4.74 (m, 2H), 4.77 (t, J=6.4 Hz, 1H), 4.83-4.89 (m, 1H), 5.01 (d, J=6.2 Hz, 1H), 5.04-5.09 (m, 1H), 5.15 (d, J=5.6 Hz, 1H), 5.18 (d, J=3.6 Hz, 1H), 5.60-5.69 (m, 2H), 6.60 (d, J=8.3 Hz, 2H), 6.91-6.95 (m, 1H), 6.98 (d, J=8.3 Hz, 2H), 7.05-7.32 (m, 15H), 7.31-8.27 (m, 23H), 9.12 (br, s, 1H); HRMS-ESI m/z calcd for C.sub.137H.sub.225N.sub.21O.sub.32S [M+2H].sup.2+ 1354.3173. found 1354.3180.
Example 18—CN185
(97) ##STR00124##
(98) To a stirred solution of peptide 4-pentynoyl-FFRKSIINFEKL (in which residues 5-12 comprise SEQ ID NO: 262) (4.5 mg, 2.80 μmol) and CN215 (3.03 mg, 2.16 μmol) in DMSO (600 μL) and MeOH (280 μL) is added TBTA (0.33 mg, 0.6 μmol) in CHCl.sub.3 (280 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (100 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at rt for 18 h. The reaction mixture is concentrated by passing an Ar stream over the reaction mixture and the residue is centrifuged with an aqueous solution of 0.05 M EDTA (pH 7.7) (2×10 mL), water (2×10 mL) and the remaining pellet is lyophilized from water (3 mL). The crude product is purified by preparative HPLC (Phenomenex Luna C18(1), 5 μm, 250×10 mm, 40° C., 2.1 mL/min; Mobile phase A=100:0.05 water/TFA; Mobile phase B=100:0.0.05 MeOH/TFA; 0-7 min: 80-100% B; 7-14 min: 100% B; 14-15 min: 100-80% B; 15-20 min: 80% B) to give the title compound CN185 (2.55 mg, 44%, 97.8% pure by HPLC); HRMS-ESI m/z calcd for C.sub.138H.sub.232N.sub.22O.sub.31 [M+2H].sup.2+ 1347.3548. found 1347.3610.
Example 19—CN174
(99) ##STR00125##
(100) To a stirred suspension of peptide 2-(aminooxy)acetyl-FFRKSIINFEKL (in which residues 5-12 comprise SEQ ID NO: 262) (9 mg, 5.57 μmol) in THF/MeOH (2:1, 600 μL) is added an aqueous mixture of water/aniline/TFA (200:6:4, 300 μL). Once dissolved, a solution of ketone CN171 (5.7 mg, 4.2 μmol) in THF/MeOH (1:1, 600 μL) is added to the reaction mixture, followed by a further portion of water/aniline/TFA (200:6:4, 100 μL), and the mixture is stirred at 25° C. for 48 h. The solvent is removed and the crude product is purified by preparative HPLC (Phenomenex Luna C18(1), 5 μm, 250×10 mm, 40° C., 1.8 mL/min; Mobile phase A=100:0.05 water/TFA; Mobile phase B=100:0.0.05 MeOH/TFA; 0-5 min: 80-100% B; 5-15 min: 100% B; 15-16 min: 100-80% B; 16-20 min: 80% B) to give the title compound CN174 (0.3 mg, 2.5%, 95.1% pure by HPLC); HRMS-ESI m/z calcd for C.sub.151H.sub.251N.sub.25O.sub.34 [M+2H].sup.2+ 1479.9262. found 1479.9421.
Example 20—CN175
(101) ##STR00126##
(102) To a stirred solution of peptide 4-pentynoyl-FFRKSIINFEKL (in which residues 5-12 comprise SEQ ID NO: 262) (5.03 mg, 3.10 μmol), CN172 (3.03 mg, 2.16 μmol) and TBTA (0.80 mg, 1.5 mol) in DMSO (280 μL) is added CHCl.sub.3 (280 μL) and MeOH (280 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (107 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at 20° C. for 48 h. The volatiles are removed under reduced pressure to give a residue which is centrifuged with an aqueous solution of 0.1 M EDTA (pH 7.7) (2×10 mL), water (2×10 mL) and the remaining pellet is dried under high vacuum. The crude product is purified by preparative HPLC (Phenomenex Luna C18(1), 5 μm, 250×10 mm, 40° C., 1.8 mL/min; Mobile phase A=100:0.05 water/TFA; Mobile phase B=100:0.0.05 MeOH/TFA; 0-5 min: 80-100% B; 5-15 min: 100% B; 15-16 min: 100-80% B; 16-20 min: 80% B) to give the title compound CN175 (1.6 mg, 25%, 97.9% pure by HPLC); HRMS-ESI m/z calcd for C.sub.155H.sub.257N.sub.27O.sub.33 [M+2H].sup.2+ 1512.9553. found 1512.9609.
Example 21—CN194
(103) ##STR00127##
(104) To a stirred solution of peptide 4-pentynoyl-FFRKNLVPMVATV (in which residues 5-13 comprise SEQ ID NO: 199) (2.0 mg, 1.25 μmol), CN172 (1.0 mg, 0.71 μmol) and TBTA (0.29 mg, 0.55 μmol) in DMSO (93 μL) is added CHCl.sub.3 (93 μL) and MeOH (93 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (31 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at 20° C. for 15 h. The volatiles are removed under reduced pressure to give a residue which is centrifuged with an aqueous solution of 0.025 M EDTA (pH 7.7) (2×10 mL), water (2×10 mL) and the remaining pellet is dried under high vacuum. The crude product is purified by preparative HPLC (Phenomenex Luna C18(2), 5 μm, 250×30 mm, 30° C., 40 ml/min; Mobile phase A=100:0.1 water/TFA; Mobile phase B=100:0.1 MeOH/TFA; 0-15 min: 50-100% B; 15-23 min: 100% B; 23-25 min: 100-50% B; 25-26 min: 50% B) to give the title compound CN194 (1.65 mg, 77%, 94.2% pure by HPLC); HRMS-ESI m/z calcd for C.sub.152H.sub.256N.sub.27O.sub.32SNa [M+H+Na].sup.2+ 1513.9439. found 1513.9397.
Example 22—CN188
(105) ##STR00128##
(106) To a stirred solution of peptide 4-pentynoyl-ILARNLVPMVATV (in which residues 5-13 comprise SEQ ID NO: 199) (2.12 mg, 1.44 μmol), CN172 (0.99 mg, 0.71 μmol) and TBTA (0.22 mg, 0.41 μmol) in DMSO (93 μL) is added CHCl.sub.3 (93 μL) and MeOH (93 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (31 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at 20° C. for 15 h. The volatiles are removed under reduced pressure to give a residue which is centrifuged with an aqueous solution of 0.025 M EDTA (pH 7.7) (2×10 mL), water (2×10 mL) and the remaining pellet is dried under high vacuum. The crude product is purified by preparative HPLC (Phenomenex Luna C18(2), 5 μm, 250×30 mm, 30° C., 40 ml/min; Mobile phase A=100:0.1 water/TFA; Mobile phase B=100:0.1 MeOH/TFA; 0-15 min: 50-100% B; 15-23 min: 100% B; 23-25 min: 100-50% B; 25-26 min: 50% B) to give the title compound CN194 (1.00 mg, 50%, 94.8% pure by HPLC); HRMS-ESI m/z calcd for C.sub.143H.sub.253N.sub.26O.sub.32SNa [M+H+Na].sup.2+ 1451.4306. found 1451.4269.
Example 23—CN197
(107) ##STR00129##
(108) To a stirred solution of peptide 4-pentynoyl-FFRKAVGALEGPRNQDWLGVPRQL (in which residues 5-24 comprise SEQ ID NO: 413) (7.72 mg, 2.73 μmol), CN172 (2.02 mg, 1.44 μmol) and TBTA (0.42 mg, 0.79 μmol) in DMSO (186 μL) is added CHCl.sub.3 (186 μL) and MeOH (186 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (62 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at 20° C. for 13 h. The volatiles are removed under reduced pressure to give a residue which is centrifuged with an aqueous solution of 0.05 M EDTA (pH 7.7) (2×10 mL), water (3×10 mL) and the remaining pellet is dried under high vacuum. The crude product is purified by preparative HPLC (Phenomenex Luna C18(1), 5 μm, 250×10 mm, 40° C., 2.0 mL/min; Mobile phase A=100:0.05 water/TFA; Mobile phase B=100:0.0.05 MeOH/TFA; 0-8 min: 80-100% B; 8-15 min: 100% B; 15-16 min: 100-80% B; 16-20 min: 80% B) to give the title compound CN197 (4.90 mg, 80%, 95.1% pure by HPLC); HRMS-ESI m/z calcd for C.sub.206H.sub.338N.sub.47O.sub.48 [M+3H].sup.3+ 1413.5073. found 1413.4989.
Example 24—CN196
(109) ##STR00130##
(110) To a stirred solution of peptide 4-pentynoyl-FFRKDLAQMFFCFKELEGW (in which residues 5-19 comprise SEQ ID NO: 412) (7.07 mg, 2.80 μmol), CN172 (2.02 mg, 1.44 μmol) and TBTA (0.40 mg, 0.75 μmol) in DMSO (186 μL) is added CHCl.sub.3 (186 μL) and MeOH (186 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (62 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at 20° C. for 48 h. The volatiles are removed under reduced pressure to give a residue which is centrifuged with an aqueous solution of 0.05 M EDTA (pH 7.7) (2×10 mL), water (2×10 mL) and the remaining pellet is dried under high vacuum. The crude product is dissolved in DMSO (500 μL) and treated with TCEP-HCl (6 mg, 0.021 mmol) for 18 h then purified by preparative HPLC (Phenomenex Luna C18(1), 5 μm, 250×10 mm, 40° C., 2.0 mL/min; Mobile phase A=100:0.05 water/TFA; Mobile phase B=100:0.0.05 MeOH/TFA; 0-8 min: 80-100% B; 8-15 min: 100% B; 15-16 min: 100-80% B; 16-20 min: 80% B) to give the title compound CN197 (2.03 mg, 36%, 96.2% pure by HPLC); HRMS-ESI m/z calcd for C.sub.198H.sub.306N.sub.35O.sub.43S.sub.2[M+3H].sup.3+ 1309.0680. found 1309.0685.
Example 25—CN203
(111) ##STR00131##
(112) To a stirred solution of peptide 4-pentynoyl-FFRKSVYDFFVWLKFFHRTCKCTGNFA (in which residues 5-27 comprise SEQ ID NO:411) (5.1 mg, 1.5 μmol), CN172 (1.02 mg, 0.73 μmol) and TBTA (0.21 mg, 0.40 μmol) in DMSO (90 μL) is added CHCl.sub.3 (90 μL) and MeOH (90 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (30 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at 20° C. for 20 h. The volatiles are removed under reduced pressure to give a residue which is centrifuged with an aqueous solution of 0.05 M EDTA (pH 7.7) (2×10 mL), water (10 mL) and the remaining pellet is dried under high vacuum. An aqueous solution of TCEP-HCl (4.5 mg, 0.016 mmol) in water (90 μL) neutralized with K.sub.2CO.sub.3 (5 mg) is added to a solution of the crude product in HFIP (1.2 mL) and Et.sub.3N (60 μL). After 11 h the reduced product is purified by preparative HPLC (Phenomenex Luna C18(1), 5 μm, 250×10 mm, 40° C., 2.0 mL/min; Mobile phase A=100:0.05 water/TFA; Mobile phase B=100:0.0.05 MeOH/TFA; 0-10 min: 80-100% B; 10-13 min: 100% B; 13-13.5 min: 100-80% B; 13.5-17.5 min: 80% B) to give the title compound CN203 (1.1 mg, 31%, 94.8% pure by HPLC); HRMS-ESI m/z calcd for C.sub.245H.sub.370N.sub.49O.sub.51S.sub.2 [M+3H].sup.3+ 1626.9024. found 1626.9104.
Example 26—CN189
(113) ##STR00132##
(114) To a stirred solution of peptide 4-pentynoyl-SVYDFFVWLKFFHRTCKCTGNFA (SEQ ID NO:411) (1.8 mg, 0.62 μmol), CN172 (0.51 mg, 0.36 μmol) and TBTA (0.38 mg, 0.72 μmol) in DMSO (30 μL) is added MeOH (60 μL) and CHCl.sub.3 (45 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (15 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at 20° C. for 48 h. The volatiles are removed under reduced pressure to give a residue which is centrifuged with an aqueous solution of 0.05 M EDTA (pH 7.7) (2×10 mL), water (10 mL) and the remaining pellet is dried under high vacuum. The crude product is dissolved in DMSO (900 μL) and treated with TCEP-HCl (10 mg, 0.034 mmol) for 18 h then purified by preparative HPLC (Phenomenex Luna C18(1), 5 μm, 250×10 mm, 40° C., 2.0 mL/min; Mobile phase A=100:0.05 water/TFA; Mobile phase B=100:0.0.05 MeOH/TFA; 0-8 min: 80-100% B; 8-15 min: 100% B; 15-16 min: 100-80% B; 16-20 min: 80% B) to give the title compound CN189 (0.5 mg, 33%, 82% pure by HPLC); HRMS-ESI m/z calcd for C.sub.215H.sub.328N.sub.41O.sub.47S.sub.2 [M+3H].sup.3+ 1434.1248. found 1434.1223.
Example 27—CN191
(115) ##STR00133##
(116) To a stirred solution of peptide 4-pentynoyl-FFRKKISQAVHAAHAEINEAGRESIINFEKL-TEWT (in which residues 5-35 comprise SEQ ID NO: 141) (5.3 mg, 1.3 μmol), and CN172 (1 mg, 0.71 μmol) in DMSO (140 μL) and MeOH (140 μL) is added TBTA (0.26 mg, 0.49 μmol) in CHCl.sub.3 (140 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (50 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at rt for 18 h. The reaction mixture is concentrated by passing an Ar stream over the reaction mixture and the residue is centrifuged with an aqueous solution of 0.05 M EDTA (pH 7.7) (2×10 mL), water (10 mL) and the remaining pellet is lyophilized from water (3 mL). The crude product is purified by preparative HPLC (Phenomenex Luna C18(2), 5 μm, 250×30 mm, 30° C., 40 ml/min; Mobile phase A=100:0.1 water/TFA; Mobile phase B=100:0.1 MeOH/TFA; 0-15 min: 50-100% B; 15-23 min: 100% B; 23-25 min: 100-50% B; 25-26 min: 50% B) to give the title compound CN191 (2.7 mg, 69%, 97.6% pure by HPLC); HRMS-ESI m/z calcd for C.sub.264H.sub.426N.sub.61O.sub.69 [M+3H].sup.3+ 1852.3822. found 1852.3904.
Example 28—CN206
(117) ##STR00134##
(118) To a stirred solution of peptide 4-pentynoyl-FFRKSIINFEKL (in which residues 5-12 comprise SEQ ID NO: 262) (6.2 mg, 3.8 μmol), and CN213 (2.5 mg, 1.9 μmol) in DMSO (200 μL) and MeOH (200 μL) is added TBTA (0.74 mg, 1.4 μmol) in CHCl.sub.3 (200 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (50 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at rt for 18 h. The reaction mixture is concentrated by passing an Ar stream over the reaction mixture and the residue is centrifuged with an aqueous solution of 0.05 M EDTA (pH 7.7) (2×10 mL), water (10 mL) and the remaining pellet is lyophilized from water (3 mL). The crude product is purified by preparative HPLC (Phenomenex Luna C18(2), 5 μm, 250×30 mm, 30° C., 40 ml/min; Mobile phase A=40:60:0.05 water/MeOH/TFA; Mobile phase B=100:0.05 MeOH/TFA; 0-14 min: 0-100% B; 14-16 min: 100% B; 16-16.5 min: 100-0% B; 16.5-18 min: 0% B) to give the title compound CN206 (1.24 mg, 22%, 95.8% pure by HPLC); HRMS-ESI m/z calcd for C.sub.146H.sub.230FN.sub.27O.sub.33 [M+2H].sup.2+ 1454.8488. found 1454.8557.
Example 29—CN207
(119) ##STR00135##
(120) To a stirred solution of peptide 4-pentynoyl-FFRKSIINFEKL (in which residues 5-12 comprise SEQ ID NO: 262) (4.8 mg, 3.0 μmol), and CN214 (2.0 mg, 1.6 μmol) in DMSO (200 μL) and MeOH (200 μL) is added TBTA (0.59 mg, 1.1 μmol) in CHCl.sub.3 (200 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (50 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at rt for 18 h. The reaction mixture is concentrated by passing an Ar stream over the reaction mixture and the residue is centrifuged with an aqueous solution of 0.05 M EDTA (pH 7.7) (2×10 mL), water (10 mL) and the remaining pellet is lyophilized from water (3 mL). The crude product is purified by preparative HPLC (Phenomenex Luna C18(2), 5 μm, 250×30 mm, 30° C., 40 ml/min; Mobile phase A=40:60:0.05 water/MeOH/TFA; Mobile phase B=100:0.05 MeOH/TFA; 0-14 min: 0-100% B; 14-17 min: 100% B; 17-17.5 min: 100-0% B; 17.5-19 min: 0% B) to give the title compound CN207 (2.22 mg, 49%, 94.9% pure by HPLC); HRMS-ESI m/z calcd for C.sub.146H.sub.239N.sub.27O.sub.33 [M+2H].sup.2+ 1449.8849. found 1449.8951.
Example 30—CN212
(121) ##STR00136##
(122) Peptide CFFRKSIINFEKL (in which residues 6-13 comprise SEQ ID NO: 262) (1.4 mg, 0.85 mol) and CN211 (0.85 mg, 0.58 μmol) are dissolved in deoxygenated DMF (75 μL) under Ar and stirred at rt for 4 h. After concentration of the solvent, the crude product is purified by preparative HPLC (Phenomenex Luna C18(1), 5 μm, 250×10 mm, 40° C., 1.8 mL/min; Mobile phase A=100:0.05 water/TFA; Mobile phase B=100:0.0.05 MeOH/TFA; 0-5 min: 80-100% B; 5-15 min: 100% B; 15-16 min: 100-80% B; 16-20 min: 80% B) to give the title compound CN212. HRMS-ESI m/z calcd for C.sub.157H.sub.260N.sub.26O.sub.35S [M+2H].sup.2+: 1550.9542. found 1550.9521.
Example 31—CN210
(123) ##STR00137##
(124) To a stirred solution of peptide 5-azidopentanoyl-FFRKSIINFEKL (in which residues 5-12 comprise SEQ ID NO: 262) (0.43 mg, 0.26 μmol) in DMSO (22 μL) is added a solution of CN209 (0.25 mg, 0.17 μmol) in CHCl.sub.3/MeOH (1:1, 45 μL), followed by water (8.6 μL), and the reaction mixture is stirred at rt for 24 h. After concentration of the solvent, the crude product is purified by preparative HPLC (Phenomenex Luna C18(1), 5 μm, 250×10 mm, 40° C., 1.8 mL/min; Mobile phase A=100:0.05 water/TFA; Mobile phase B=100:0.0.05 MeOH/TFA; 0-5 min: 80-100% B; 5-15 min: 100% B; 15-16 min: 100-80% B; 16-20 min: 80% B) to give the title compound CN210. HRMS-ESI m/z calcd for C.sub.160H.sub.263N.sub.27O.sub.34 [M+2H].sup.2+: 1553.4840. found 1553.4850.
Example 32—CN205
(125) ##STR00138##
(126) To a stirred solution of peptide 4-pentynoyl-FFRKRAHYNIVTF (in which residues 5-13 comprise SEQ ID NO: 414) (4.6 mg, 2.6 μmol), CN172 (2 mg, 1.4 μmol) in DMSO (140 μL) and MeOH (140 μL) is added a solution of TBTA (0.54 mg, 0.99 μmol) in CHCl.sub.3 (140 μL) followed by an aqueous solution of 0.25 mM CuSO.sub.4 (50 μL). A small amount of copper foil (5 mm×2 mm) is added and the reaction mixture is stirred at rt for 18 h. The volatiles are removed under reduced pressure to give a residue which is centrifuged with an aqueous solution of 0.05 M EDTA (pH 7.7) (2×10 mL), water (10 mL) and the remaining pellet is dried under high vacuum. The crude product is purified by preparative HPLC (Phenomenex Luna C18(2), 5 μm, 250×30 mm, 30° C., 40 ml/min; Mobile phase A=40:60:0.05 water/MeOH/TFA; Mobile phase B=100:0.05 MeOH/TFA; 0-10 min: 0-100% B; 10-16 min: 100% B; 16-16.5 min: 100-0% B; 16.5-18 min: 0% B) to give the title compound CN205 (2.5 mg, 56%, 96.7% pure by HPLC). HRMS-ESI m/z calcd for C.sub.162H.sub.258N.sub.32O.sub.33 [M+2H].sup.2+ 1591.4747. found 1591.4823.
Example 33—Formulating Compounds of the Invention for Intravenous Injection
(127) Compounds of the invention are formulated analogously to reported methods for α-GalCer. Briefly, solubilisation of α-GalCer is based on excipient proportions described by Giaccone et al. (Giaccone, Punt et al. 2002). Thus, 100 μL of a 10 mg/mL solution of α-GalCer or a compound of the invention in 9:1 THF/MeOH is added to 1.78 mL of an aqueous solution of Tween 20 (15.9 mg), sucrose (177 mg) and L-histidine (23.8 mg). This homogeneous mixture is freeze dried and the resulting foam is stored under Ar at −18° C. This material is reconstituted with 1.0 mL of PBS or water prior to serial dilutions in PBS to achieve final injectable solutions of α-GalCer or compounds of the invention.
Example 34—HPLC-ESI-MSMS Quantification of α-GalCer
(128) Quantification of the amount of α-GalCer in various test samples of compounds of the invention is made by HPLC-ESI-MSMS analysis using a Waters 2795 HPLC and a Waters Q-TOF Premier™ Tandem Mass Spectrometer. The chromatography used a Phenomenex Kinetex C18 2.6 mm 3.0×50 mm column eluting with isocratic methanol containing 10 mM ammonium formate+0.5% formic acid at a flow rate of 0.2 mL/min. α-GalCer is monitored by selective reactant monitoring of 898.7 to 696.7 Da. The estimate of amount of α-GalCer is made by comparison of ion count integrals to a standard curve run on the same day or by comparison to test samples spiked with a known amount of α-GalCer. The level of α-GalCer is determined on freshly reconstituted formulated samples unless otherwise stated
(129) TABLE-US-00001 Compound α-GalCer/injection CN152 0.05 ng CN165 0.028 ng CN166 0.136 ng
Example 35—Biological Studies
(130) Mice. Breeding pairs of the inbred strains C57BL/6 (CD45.2.sup.+) and B6.SJL-Ptprc.sup.a Pepc.sup.b/BoyJ (CD45.1.sup.+) are obtained from Jackson Laboratories, Bar Harbor, Me., and from the Animal Resource Centre, Canning Vale, Western Australia. Also used are lang-DTREGFP and lang-EGFP knock in mice, which express the human diphtheria toxin (DT) receptor and/or enhanced green fluorescent protein (EGFP) under the control of the langerin promoter, CD1d.sup.−/− mice, which are devoid of Vα14 iNKT cells, TLR2.sup.−/− mice (17), OT-I mice, which are transgenic for a TCR recognizing an H-2K.sup.b-restricted epitope from chicken OVA (OVA.sub.257-264) and OT-Ill mice, with a TCR recognizing the I-A.sup.b-restricted epitope OVA.sub.323-339. For adoptive transfer experiments OT-I animals are crossed with B6.SJL-Ptprc.sup.a Pepc.sup.b/BoyJ animals, so that the congenic marker CD45.1 could be used to discriminate the transferred cells. All mice are maintained in the Biomedical Research Unit of the Malaghan Institute of Medical Research. Experiments are approved by a national Animal Ethics Committee and performed according to established national guidelines.
(131) Administration of Compounds of the Invention.
(132) Each compound of the invention is supplied as formulated product (see example 33), and diluted in phosphate-buffered saline (PBS) for injection (0-2.0 nmol/mouse) by intravenous injection into the lateral tail vein. In humans the expected therapeutic dose lies in the 50-4800 (μg/m.sup.2) range (Giaccone, Punt et al. 2002). Note, 0.23 nmol in a mouse is a human equivalent dose of 30 μg/m.sup.2 for α-GalCer. Higher doses are likely for other administration routes.
(133) All antibody labeling is performed on ice in FACS buffer (PBS supplemented with 1% FCS, 0.05% sodium azide, and 2 mM EDTA). Non-specific FcR-mediated antibody staining is blocked by incubation for 10 min with anti-CD16/32 Ab (24G2, prepared in-house from hybridoma supernatant). Flow cytometry is performed on a BD Biosciences FACSCalibur or BD LSRII SORP flow cytometer with data analysis using FlowJo software (Tree Star, Inc., OR, USA).
(134) Phenotyping DC from Spleen.
(135) Antibody staining and flow cytometry is used to examine the expression of maturation markers on dendritic cells in the spleen following injection of compounds of the invention. Splenocyte preparations are prepared by gentle teasing of splenic tissue through gauze in Iscove's Modified Dulbecco's Medium with 2 mM glutamine, 1% penicillin-streptomycin, 5×10-5 M 2-mercapto-ethanol and 5% fetal bovine serum (all Invitrogen, Auckland, New Zealand), followed by lysis of red blood cells with RBC lysis buffer (Puregene, Gentra Systems, Minneapolis, Minn., USA). Antibody staining is performed in PBS 2% fetal bovine serum and 0.01% sodium azide. The anti-FcgRII monoclonal antibody 2.4G2 is used at 10 mg/ml to inhibit non-specific staining. Monoclonal antibodies (all BD Biosciences Pharmingen, San Jose, Calif., USA) are used to examine expression of the maturation markers CD40, CD80 and CD86 on CD11c+ dendritic cells.
(136) Analysis of Peptide-Specific T Cell Proliferation In Vivo
(137) Pooled lymph node cell suspensions are prepared from animals of a cross between OT-I mice, which express a transgenic T cell receptor (TCR) specific for the ovalbumin epitope SIINFEKL in the context of H-2K.sup.b molecules, and B6.SJL-Ptprc.sup.a Pepc.sup.b/BoyJ mice, which are congenic with C57BL/6 mice for the CD45.1.sup.+ marker. The samples are enriched for CD8.sup.+ cells using antibody coated magnetic beads (Miltenyi), and then transferred into recipient mice (1×10.sup.4 per mouse). Groups of recipient animals (n=5) are immunized with compounds of the invention one day later. Doses are chosen to provide equivalent molar values of SIINFEKL peptide. Control animals receive phosphate-buffered saline. After seven days, blood samples are collected from the lateral tail vein and stained directly ex vivo with antibodies for TCR Vα2, CD45.1 and CD8 to detect the SIINFEKL-specific CD8.sup.+ T cells by flow cytometry.
(138) Analysis of Peptide-Specific T Cell-Mediated Cytotoxicity In Vivo
(139) The cytotoxic capacity of induced CD8+ T cell responses is measured by VITAL assay (Hermans, Silk et al. 2004). Mice are immunized with the compounds of the invention, or PBS, and then injected intravenously seven days later with two populations of syngeneic splenocytes; those loaded with 500 nM, SIINFEKL-peptide and labelled with 1.65 nM carboxyfluorescein succinimidyl ester (CFSE), or those loaded with peptide and labelled with 10 μM cell tracker orange (CTO). Specific lysis of the peptide-loaded targets is monitored by flow cytometry of blood or spleen samples 24 h later. Mean percent survival of peptide-pulsed (CFSE+) targets is calculated relative to that of the control population (CTO+), and cytotoxic activity is expressed as percent specific lysis (100−mean percent survival of peptide-pulsed targets).
(140) Analysis of Anti-Tumour Activity.
(141) Groups of C57BL/6 mice (n=5) receive a subcutaneous injection into the flank of 1×10.sup.5 B16.OVA melanoma cells, which express a cDNA encoding the chicken ovalbumin (OVA) sequence. The different groups are treated 7 days later, when tumours are fully engrafted, by intravenous injection of one of the following; 200 μg OVA protein together with 200 ng α-GalCer, 200 μg OVA protein together with 200 ng of a compound of the invention, or PBS. Mice are monitored for tumour growth every 3-4 days, and tumour size for each group calculated as the mean of the products of bisecting diameters (±SEM). Measurements are terminated for each group when the first animal develops a tumour exceeding 200 mm.sup.2.
(142) Assessment of Human T Cells.
(143) Peripheral blood mononuclear cells from CMV seropositive donors are cultured for 8 d in the presence of α-GalCer, NLVPMVATV peptide, admixed a-GalCer and peptide or the conjugate CN188 in complete medium (IMDM supplemented with 5% Human AB serum). α-GalCer 500 ng/ml (=582.5 nM), CN188 and NLVPMVATV used molar equivalent of 582.5 nM.
(144) Flow cytometry with fluorescent HLA-A2/NLVPMVATV tetramer (PE-conjugated, Immudex) and antibodies for CD3 (Alexa Fluor 700 CD3, Biolegend) and CD8 (APC-H7 CD8, BD) are used to detect peptide-specific T cells. Proportions of NLVPMVATV-specific CD8+ T cells are determined by gating out doublets and dead cells (with DAPI), by gating on lymphocyte population by forward and side scatter, and then by selecting for CD3 positive and CD19 (FITC CD19, BD) negative cells. A separate staining panel with PE-conjugated loaded CD1d tetramer is used to detect iNKT cells.
(145) Where the foregoing description reference has been made to integers having known equivalents thereof, those equivalents are herein incorporated as if individually set forth.
(146) Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.
(147) It is appreciated that further modifications may be made to the invention as described herein without departing from the spirit and scope of the invention.
INDUSTRIAL APPLICABILITY
(148) The invention relates to sphingoglycolipid analogues and peptide derivatives thereof, which are useful in treating or preventing diseases or such as those relating to infection, atopic disorders, autoimmune diseases or cancer.
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