Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 92/22564 PCl /CA92/00245
2 1 1 0 ~
I1~08~ 8I~5 aND ~rO~æROG2~IC
~ODIFI~D I~I~3X I~ID I.EWI8~ C0~5POV~
BAC~CGR~9~D OF ~ ~ ~IO~
' , ~
S T~ presenlt invent:ion is directed t~ novel
~` Lewi~;X and Lewis8 analogues, pharmaceutical
c:os~po it~or~s ~ont;aining such an~loguel~:, an~thods for
their pr~para l:ion and m~khods ~or their use .
2. ~e~en~es
1~ Th~ following referenc:es are Gited in this
application a~; supersc:rip~ numbers a~ ~he relevant
po~ion of the appl ication:
::
Horowitz, the Glycoconj uga~:es I Vols . I-V,
Pigman, Editor, New York Ac:ad2mic Pres~; (1977,
is lg78, l9B2, 1983~.
: 2 Ippolito et al., U.S. Patent ~pplication 5erial
No. 07~714,161, filed June 10~ l99I for
"Tmmunosuppresive ~nd Tolerogenic
Oligosaccharide Glycosides.
~o 3 Sialic Acids in "Cell Biology Monographsl'
Schauer, ~ditor, Vol. 10 ~1982~.
4 Lowe et al., Cell, 63:47~-485 (1990).
W092/22564 Pr/CA92/00245
~ 0 6 __ 2 --
Phillips et al., Science, 250:1130-1132 (1~90).
6 Walz et alO, Science 250:1132 et seq. (1990).
7 Larsen et a}., Cell, 63:467-47~ (19903.
8 Ratcli~e et al., U.S. Patent No. 5,07~,353,
i~sued January 7, 1992, for "Sialic Acid
Glycosides, Antigens, Immunoadsorbents, and
Methods for their Preparation".
9 Ratcliffe et al~, U.S. Patent Application
Serial No. 07/278,106, filed November 30, 1988,
~10 for "Sialic Acid ~lyc~sides, ~n~igens,
Immunoadsorbents t and Methods for their
Preparation".
Venot et al., ~.SO Patent Applica~ion S~rial
No. 07/771,007, "~ethods for the Enzymatic
~5 Synthe~is o Alpha-sialylated Oligosaccharide
Glycosidess~, iled October 2, 1991.
11 Wein~tein et al~, J~ Biol. Che~., 257:13845-
13~53 (lg8~).
12 Reuter et al., Glycoconjugate J. 5:133-135
~19~8).
13 Palcic et al., Caxbohydx~ ~e~
:~ (19893.
14 Prieels et al., J. Biol. ~hem., 56:10456 10463
(19~
Eppenberger-C~stori et alO, Glycocon;. J9
6:101~ (1989).
16 Go~hale et al., ~an. J. Chem., 68:10~3-1071
( 1990~ .
17 Jiang et al., U.S. Patent Application Serial
No. 07/848,223 filed March 9, 19929 for
"Chemical Synthesis of GDP Fucosel'.
18 Ekberg et al., Carbohydr. Res. 110:55-67
(19~2~.
19 Dahmen et al~, Carbohydr~ Res. 1~8:~92-301
t1983)~
Rana et al., Carbohydr. Res. ~1:149-157 ~1981).
W092/225~ 2 1 1 ~ 7 0 7 PC~tCA92/~45
21 Amvam-Zollo et al., Car~ohydr. Res. 150:~99-212
(1986).
22 Paulsen et al., Carbohydr. Res. 104:19S 219
(1982).
23 Chernyak et al., Carbohydr. Res. 128:269-282
(1984).
24 Fernandez-Santana et al., J. Carbohydr. Chem.
8:531-537 (19~9).
Lee ~t al., Carbohydr. Res., 37:193 et seq.
~1~ (1974~
2S Norberg et al., Carbohydr. Res. 183-71 ~t seq.
~: (1988).
27 ~atta et alO, Carbohydro. Res. 208:51-58
(1980).
~: 28 ~handras~kar~n et al., Ab~tracts of ~he 11th
I~ternational Symposium on Glycoconjugates,
~une 30, 1991.
29 ~kowska-Latallo et al., Genes and Development,
0 ~-1288-1303 ~199~).
Duma et al., Bioorg. Med. Letters, ~:425-428
(1991).
31 Okamoto et al.,:Tetrahedron, Vol. 46~ No. 17,
pp. 5835-5837 (1990)~
32 Abbas et al., Proc. JapanesQ-G~nman Symp.
Berlin, pp. ~0-21 (l9&B~.
33 Paulsen, ~gne~. Chem. Int. Ed. Eng., ~1:155-173
(1982).
34 Schmidt, Agnew. Chem. Int. Ed. Eng., 25:~12-23S
3~ (lg86).
~jedi et al., Glycoconj. J., 4:97-108 (1987~.
: 36 Kameyama et al., Carbohydr. R~s., ~2 Cl-C4
.. ~1991).
37 Inazu et al., Bull. Soc. Chim~ Jap., 611:44S7
3~ (lg88).
38 Bernotas et al., ~iochem. J., 2?0:539-540
(1990).
W092/22564 PCT/CA92/00245
~ Q~ ~7 4 __
39 Wollenberg et al., U.S. Patent NoO 4,612,132,
for Issued September 21, l9B6 for "Modified
Succinimides".
Greig et al., J. Chem. Soc., p. 879 ~1961).
~1 Piekarska-Bartowzewicz et al., Carbohydr. Res.,
2.03:302-307 (1990~.
42 Petitou et a~., Carbohydr. Res., 1472210~36
(198~).
43 Trumtez et al., Carbohydr. Res., 191:29_52
~10 (19893.
~ 44 ~emieux et al., J. Amer. Chem~ Soc., g7:4076-
:` 40~3 (1975).
Ogaw~ et al., Tet. Letters, ~2:4759-4761
(198~).
~ 5 46 Lemieux et al., canO J. ~hem. ~Q:63-67 (1982).
: 47 ~icolaou et al., JACS, 112:3693-3695 (1990).
48 Hindsgaul e al O ~ J. Org. Chem~ 2:2869-2875
: (1975)
49 Richard~on et al., Carbohydr. Reæ. ~ 271-287
~1991~
~; 50 Smith et al., Immunology, 58:245 (1986).
~: ~ 51 Sleytr et al~, ~rch. Microbiol., ~ 19 (1986).
: 52 Ziola et al., J. Neuroimm~nol., 7:315--330
(I985).
53 ~okhale et al., Can. J. Chem., 68:1063-1071
~1990)
54 Schmidt, et al., Liebigs Ann. Ch~mO, 121-124
( 1991~
Nu~e~ , et al O, Can. J . Chem., ~9 : 2086-2095
(1981)
5Ç Veeneman, et al., Tetrahedron Lett., 32 : 6175-
6178 (1991)
W~92/22564 2 ~ i PCT/CA92/00245
__ 5 __
All publications and patent applications
mentioned in this specification are indicative of
the level of skill of tho e skilled in the art to
which this invention pertains. ~11 publications and
S patent applications are herein incorporat~d by
reference in their entir~ty to ~he same exten as if
each individual publication or patent application
was specifically and individually indicated to be
incorporated by r~ference in its entirety.
3. ~ z~JYh~
Carbobydratas and/or oligosac harides are
present on a variety of natural and pathological
glycoconjugates1. Of particular inter~t are
: carbohydrates and oligosaccharides containing sialyl
~: 15 and/or fucosyl residues3. Such sialyl and/or
: fuco~yl ~arbohydrates and oligosaccharides are
:~ pre~en in a n~mber of products which ha~e been
~: implicated in a wid~ range of biological phen~mena
~: bAsed, in part, on the conc~pt of recognition
sign~ls carried by the car~ohydrate structures and
by their bi~ding to ~pecific ligands.
8pecifically, a numb~r of sialylat~d and
: ~ialylated/fucosylated oligosaccharide glycoside
:~ ha~e be~n proposed as mediators of cell adhesion in
: 25 that they are ligands ~or selectins tor LEC-
~AM's)4,5,6~7 Sialylat~d, fucosylated, and
sialylated and fucosylated oligosaccharide
structures relating to blood group determinants,
including LewisX, Lewisa, sialyl LewisX and sialyl
Lewis~, have also been shown by Ippolito et al.2 to
po~sess in ivo im~unomodulating and tolerogenic
properties in mammals including anti-inflammatory
immunomodulating properties. In this regard, the
DTH anti-inflammatory immunomodulating properties of
W092/22564 PCT/CA92/00245
C~`
LewisX and sialyl LewisX reported by Ippolito et al. 2
demon~trate that the presence of the sialyl residue
on sialyl LewisX re~ults in enhanced anti-
in~lammatory acti~ity as compared to LewisX.
Contrarily, sialyl LewisX, and sialyl Lewis~ and
related compounds are dif f icult to chemically
synthesize in high yield with anomeric specificity
for the a~2~3) linkag~ of NeuSAc to galactose.
~nown chemical methodologies include a multistep
`10 synthesis which fir~t generatas a blocked
~Neu5Ac(2~3)Gal disaccharide having a suitable
leaving ~roup at the reducing sugar tsrminus of the
galactose8~9. This disaccharide is then reacted with
a suitably protected GlcNAc-O~ saccharida glycoside
lS and then ~ suitably protected L-fucose d~rivative
which, after d~protection, pr~ides for the ~ialyl
~: L~wisX g}yco~ide ~Neu5Ac(2~3~Gal(~4)t~uc~1~3)]
~GlcNAc-OR~ or the ~ialyl L~wis~ glyaoside ti~e.~
~N~u5Ac(2~3)0Gal~1-3)~uc(1~4)]~1eN~c-OR] where R
is an aglycon of at lea~t one carbon ~to~
Additionally, sialyl LewisX, sialyl lewisa and
relat~d co~pound~ can al80 be synthesi2ed via
~hemical/enzy~atic synthesi~2~9~l0. In ~neral, the
~Gal(1~4~GlcNAc-OR, the derivatiz~d
~al(1~4)~GlcN~c-OR, the ~al(1~33~GlcN~c-OR, or the
deri~tized ~Gal(1~3~GlcNAc-QR backbone is f irst
~ynthesized chemically and then the ~ialic acid
r~sidue (e.g., Neu5Ac) is attached to th~ galactose
to form the aNeuSAc(2~3)~Gal(1~3)~GlcNA~OR or the
~Neu5Ac(2~3~Gal~1~4~GlcNAc-OR structures by use of
a compatible sialyltransferase and the fuco~e
residue is then attached to the 4-hydroxyl position
of the N-acetylglucosamine residue by use of a
compatib~e fucosyltransferase.
W092/22564 21 1 0 7 ~ 7 PCT/CA92/00245
However, the enzymatic synthesis of sialyl
LewisX and related compounds is restricted by the
availabilîty of compatibl~ sialyltransferases. For
example, the ~Gal(1~3J4~GlcNAc a(2~3)sialyltrans-
ferase disclosed in the art for sialylating the
~al(1~4)~GlcNAc backbone is currently recovered
from ra~ livers11.
In any event, the inclusion of a sialyl residue
on LewisX, Lewis~ and related compounds so as to
`~0 pro~ide for sialyl LewisX, ~ialyl Lewis~ and relat~d
compounds results in a more c~mplex and costly
synthesis.
8~a~Y OF T~ INVE~TIO~
The pre~ent inv~n~ion is directed, in part, t~
: 15 the discov2ry that modified L~wisX and modified
; ~ Lewi~a compounds having a sulfate, a phosphate or a
~: carboxylate containing group posse~s enhanc~d
mun~uppressive and tolerogenic propert~e as
;~` ; oompared to imilar co~pounds lacking such
8 ~ ~titution. ~oreover, the 3-sulfa~e LewisX
compound posse~se8 at least eg~iva~ent
immunosuppressive and tolerogenic properti~s as
:~ :
compared to sialyl LewisX. This result is
~:~ particularly surprising insofar as unmodif i~d LewisX
25 po~;~esse inferior immunosuppressive and tol ~rogenic
prcperties as co~pared to sialyl LewisX.
Additionally, bec:ause these modif ied LewisX and
: ~ ~ modified Lewis~ compolmds do not contain a sialyl
residue at thç~ 3-position of the g~lac~ose so as to
30 ~brm an ~ (2~3) linkage, the problems inherent with
f orming such a 1 inkag~ are avoided .
W092/22564 PCT/CA92/00245
~t~ 8 --
Accordingly, in one of its composition aspects,
the present invention is directed to compounds of
Formula I:
XQ
IOH
C~3 ~ OH
HO ~ OXl ¦ ~
X2O ~ O ~ Y-R
R3 Rl
: S where R is ~elected from the group eonsi~ting
~: of hydrogen, a ~accharide ORl4, an oligosaccharide-
ORl4, or an aglycon having at le~st 1 carbon atom
~: where R14 is hydrogen or an aglycon of at least one
: carbon ato~; :~
O Y i~ sel~cted;from ~he group consisting of
o~ygen, ~ulfur, and -NH-:
R1 is selected from the group con~i~tin,g of
hydrogen, -NH2, -N3, -NHS03H, -N~C (O j R~,
-NzC(R~)2, -NHC~ NHR6, N(R~)2, OH,- OR~,
3, -S(O)R~ -S(O)~and sulfate,
whereîn R4 is~selected from the group
: consistin,g of
~: :hydrogen,
: ~ :
: alkyl of from 1 to 4 carbon atom~,
: -OR7 whereîn R7 i5 alkyl of from 1 to 4
~: : carbon ato~s, or alkyl of from 2 to 4 carbon atoms
substîtuted with a hydroxyl group, and
-NR~R~ wherein R8 and Rg are îndependently
selected from the group consîstîng of hydrogen and
al~yl of from 1 to 4 carbon atoms,
each R~ is selected from the group consi~ting
: of hydrogen and alkyl of from 1 to 4 carbon atoms,
each R6 îs alkyl of from 1 to 4 carbon at~ms,
W(~ ~2/22564 PCr/C~92/0024S
21 ~ D 7 (3 i
R2 is selected fxom the group consisting of
hydrogen, -N3, -NH2, NHS03H, -NRl~C (O) R10 ~
-N=C (R71 ) 2 ~ (Rl1 ) 21 ~ 2 ~ -N (R12) 2~ OH and
--ORl 2 ~
wherein R~o is selected from the group
con~;isting of
hydrogen,
alkyl o~ f rom 1 to 4 carbon atoms,
-O~R13 wherein R13 is alkyl of from 1 to 4
car!bon atoms, or alkyl of from 2 to 4 car~on atoms
substitut~d with a hydroxyl group, and
4Rls wherein R14 and R15 are
independç~ntly seleated îrom t:he group consistinS~ of
hydrogen and alkyl of frs~ 1 to 4 carbon atc~ms,
~: ~ 15 ~ each R11 is select~d from the group consisting
of hydrogen and alkyl of from 1 to 4 carbon ~toms;
~ach R1;! is alkyl oP îrom 1 to 4 carbon a~o~as,
R3 is ~elec:ted from the group c:onsisting of
hydrogen, fluoro, sulfate and ~ydroxy;
~: 20 X is s~lected from the group consisting of
hydrogen, sulfate, ansl phosphate;
X1 is select~d i~rom ~ group consistirlg of
~: hydrogen, sulfat~, phosphate, and CHR18COOH wh~re
R18 is selected rom the group con~;isting of
hydrcgen, alkyl of from 1 to 7 carbon atoms and
~COOH;
X2 is selected from the group con~isting of
~, hydrogen, sul~a~e, phosphate, and -CHRl3COOH where
R~8 is ~:elect~d ~rom the group consisting of
hydrogen, alkyl of from 1 to 7 c:arbon atoms ~nd
-COO~I: and
pharmaceutically ac~eptable salts thereof;
and with the proviso that either at least one
of X, X1 0 or X2 is sulfate or phosphate or at least
or~e of X1 or X2 is -CHR1~,COOH.
WO 92/22564 PC~/CA92/00245
r~ 10 --
The present invention is also directed to
compounds of Formula II:
X~
IOH
CH~ ~ OH , R1
II
where R is selected from the group consisting
s of hydrogen, a saccharide-OR1~, an oligo-
: ~ saccharide-OR14, or an aglycon having at least l
carbon ~tom where R14 is hydrogen or an aglycon ~f at
: least one carbon atom;
Y is selected from the group consisting of
oxygen, sulfur, and -NH-;
R1 is selected from the group consisting of
hydrog~n, -NH2, -N3~ -NHS~H, -NR~C(O)R~,
N=C~R~2~ -N~C~(R~)2, -NHR6, -N(R~32~ -OH~ OR~
: S(O~R~, -S(0~2R~ and sulfate,
; wherein h~is selected from the group
consisting of :~ ~
: hydrogen,
alkyl of from 1 to 4 carbon atoms;
OR7 wh~rein R7 is alkyl of from l to 4
carbon ato~s, or alkyl of from 2 to 4 carbon at~ms
~: substitut~d with a hydroxyl group, and
:: N~bR~ wherein Rb and R9 ar~ independently
selected from the group consisting of hydrogen and
alkyl of from l to 4 carbon atoms,
: 25 each ~ is selected from the group consisting
:: ~ of hydrogen and alkyl of from l to 4 carb~n atoms,
each R6 is alkyl of from l to 4 carbon atoms,
R2 i5 select~d from the group consisting of
hydrogen, -N3, -NH2, -NHS03H, -NR~1C(O)R10,
~092/22564 PCT/CA92/00245
~11 1) 7 1,' ~
-N=C(R11)2, -NHCH(R1~)2, -NHR12, -N (R12) 2, -OH and
-R12 ~
wherein R1~ is selected from the grsup
cvnsisting of
hydrogen,
alkyl of from 1 to 4 carbon atoms,
-OR13 wherein Rl3 is alkyl of from 1 to 4
~ carbon a~oms, or alkyl of from 2 to 4 carbon atoms
; ~ubstituted with a hydroxyl group, and
-N~14~5 wh~rein R14 and R15 are
ind~pendently sele~ted from th~ group consisting of
hydrogQn and alkyl of fro~ l to 4 carbon atoms,
ea~h R~ s~lected from the sroup consisting
o~ hydrogen and alkyl of from 1 to 4 carbon atoms;
~ch R12 is al ~ l Qf fr~m 1 to 4 carbon atom~,
:~ R3 i. elect~d from th~ group consi~ting of
hydrog~n, fluoro, sulfa~e and hydroxy;
X i~ ~elect~d from the grou~ consisting of
h~ rogsn, ~ulfate, and ph~sphate;
20:: X1 is selected ~rom the ~roup consisting of
: hydrogen, sulfa~e,:phoæphate~ and -CHRl8COOH where
R18 is seleated from the group aonsiæting of
hydrogen,:al ~ l of ~r~ 1 to 7 caxb~n a~oms a~d
:-COO~
~ X2 i~ ~elected from the group consis~ing o~
hydrogen, sulfate, phosphate, and -CHR18COQH where
. elected ~frs~m the group consisting o~
hydrogen, alkyl of fxom 1 to 7 carbon atoDI~; and
-COOH; and
3~ pharmaceutically acc~ptabla salts thereof;
and with the proviso that sithQr at least one
of X, X1, or X2 is sulfate or phospha e or at least
one of Xl or X2 is -CHR,8COOH.
The compounds of Formula I and II are
particularly useful in modulating a cell-mediated
W092J22564 PCT/CA92fO0245
~ 12 --
immune response to an antigen and in particular a
cell-mediated immune inflammatory response to an
antigen. In this regardl when the compounds of
: Formula I and II are administered to a mammal in
response to an antigen challenge, such
administration induces tolerance to later challenges
from this same antigen.
In another of its composition aspects, the
present invention is directed to a pharmaceutical
~10 composition suitable for administration to a mammal
; (e.g., human) which comprises a pharmaceutically
inert carrier~and an effective amount of the
compound of Formula I or Formula II to modulate a
cell-mediated immune re~ponse in said ~ammal.
~5 In still another of its composition aspects,
the present i m ention is directed to novel
intermediates~useful in preparing the compounds of
Formula I and~Formula II. In this regard, so~e of
the:intermediate~mono accharides disclosed herein
20: are:highly:crystalline and can be produced in large
quantities in~high~purity without the n~ed for
chromatography~;to separa~e these compounds.
In~:one of its method aspects, the present
invention is directed to a method for ~odulating a
:cell-mediated~immune response to an antigen in a
: : mammal:which~method comprises administering to said
mammal an amount of a compound of Formula I or
Formula II effective in modulating said i~mune
response.
~:: 30 In another:of its method aspects, the present
::
~ : invention is directed to a me~hod for preparing the
:~: c~mpounds o~ Formula I and II above and to the
: ~ preparation of intermediates useful in preparin~ the
compoumds of Formula I and II.
~'
W092~22564 PCT/CA92J00245
-- 13 -- 2 ~ 1 0 7 ~ ~
BRI~F D~gcRIpTIsN OF T~ DRA~I~G8
Figure 1 illustrates reaction scheme~ for the
synthesis of partially blocked N-ace~yl glucosamine
d~ri~atives which are then used to prepare either
S modified Lewis~ compounds or modified Lewisa
co~pounds.
Figure 2 illustrates reaction schemes for the
synthesis of blocked fucose derivative~ which are
~hen used to prepare either modified LewisX or
10 ~odified L~wis compounds.
Figure 3 illustrates r~action schemes for the
~ynthesis of partially blocked galactose derivatives
: which are then u~ed to prepare either modi~ied
: L~ X co~pounds or modified Lewi5a co~pound~.
: 15 Figure 4 illustrates the synthesi~ of modifi~d
~ewisX ~ompound~ having a sulga~e substi~uent in the
3 po~ition of the galactose unit. In thi~ scheme,
~he 2,3 positions of galaotose are diffexentially
: blocked ~o that th~ 3-po~i~ion c~n be ~electi~ely
20: deblock~d and ~hen s~lec~i~ely con~e~t~d to the
sulfate æ~bstituent.
::
Figure 5 illustrates the ~ynthesis o~ modified
LQwisX compounds having a sul~ate substituent in the
3 po ition of the galactose unit~ In this scheme,
the 2,3 positions of galactose are not
di~ferentially blocked. Accordingly, deprotection
of the 3-position of the galactose unit also results
in deprQtection of the 2-position and subsequent
reaction to form the sulfate at the 3-position does
not proceed with 100% yield but rather some of the
product has a sulfate substituent at the 2-position
WO 92/?2~i64 PCI'/CAg2/00245
of the galactose which is then separated by
chromatoqraphy.
Fi~ure 6 illustrates the synthesis of modified
LewisX derivatives bearing a sulfate ~ubstituent at
S the 3-position of the galactose and which utiliæe a
6-benzyl and 2-N-ph~haloyl blocked glucosa~ine which
can be later deblocked to provide for a glucosamine
derivative .
In this figure, because the 3,4-dihydroxyl
:~ 10 groups of th~ 6-benzyl and 2-N-phthaloyl blocked
glucosamine 15 are not blocked, reaction with 1-
bromo-2j3 9 4 ~ 6-tetraacetyl galactose will result in
fo~mation of both the blocked ~Gal(1~4)~GlcNH2
d~rivative 48 and the block~d ~Gal(1~3)~GlcNH2
~5 derivatîve ~n~t sho~n).
In turn, these materials can be further
:~ derivatîzQd ~t an appropriate point in t~e æynthesis
so as to provide for N-functionalized derivatives of
L2WisX and Lewîs~O
~O Figure 7 illustrates a second synthe is of
m~difigd Lewi~X co~pounds bearing a sulfat~
sub~tituent at the 3-position of the galactose and
which utilize a different N-phthaloyl blocked
glucosamine intermediate that allow~ for th~
selecti~e preparation of 2-amino or N-functionalized
~, LewisX derivatives. In this figure, only the 4-
pocition of the glucosamine is not ~locked ~o that
only the blocked ~al(1~4)~GlcNH2 derivativ~ 68 is
formed.
:
Figure 8 illustrates the preparation of
modified Lewis~ analogues having a sulfate
substituent in the 3 position of the galactose unit.
WO 92/22564 PCI /CA92/00245
211~7i37
---- 15 ----
In this scheme, the 2, 3 positis:~ns of galactose are
differentially blocked so that the 3-position c:an be
sel . ctively deblocked and then selectively converted
to the sul f ate substituent .
Figure 9 illustrates the synthesis o~E the 6
azido derivative of GlcNAc-OR.
Figure 10 illustrates the synthesi~; of the 6-
alkoxy derivatives and the 6-deoxy derivatives of
Glc:NAc: .
Figure 11 illustrates the increase in footpad
2~w~ of immunized mic~ arising from a DTH
infla~atory resporlse mea~ursd ~4 hours ~fter
chall¢Iage with 10 ~g of th~ L~ll S-Layer protein
a~ti~en wh~2rein ~;ome: of the mice ha~e been treated
at 5 hours after ~he challenge with 100 ~g of
l~ewi~;X-OR and ~ialyl LewisX-OR (R= -O (CR2) 8COOC~I3) .
Fis~ure 12 illustrates the increa~e in f ootpad
swelling of immunized mice arising from a DTH
inflasamato~y r~ ponse measure~ 24 houxs after
challenge with 20 ,ug o~ Super~:arrier gfro~ Pierc~,
~ckford, IL 61105) antigen wher~in some o~ the mice
have been treat~d at 5 hours after the challenge
with lQ0 ~g of sialyl LewisX-OR (S}.eX) and with 3-O-
sulphate (on the gal~ctose moiety) of the ~wisX OR -
(SULF9NATE:-LeX~ whexe R is -o (CH2) 8COOCH3 in both
cas~s .
Figure 13 demonstrates the ef~Ee~t that sulfated
LewisX-OR (SULFONATE-LeX3 has on the induction of an
immune response t~ an antigen where R i5 ;IS de~ined
3 0 in Figure 12 .
WO 92/22564 PCr/C~92/n0245
1 6 ----
Figure 14 illustrates the effect that sulfated
LewisX-OR (LeXsulf~ and sialyl I.ewisX-OR (SleX) on
lung injury arising f rom the intranasal
administration of LP5 to mice ~R is as def ined in
S Figure 12 ) .
Figure 15 demonstrates the long term
tolerogenic effec:t of sulfated IRwisX-OR tSULEONATE
LeX~ and sialyl IA~wisX-OR (SLeX) on an i~rmune
re~ponse to an antigen w~ere R is as def ined in
Figure 12 ) .
I:U3 g!~II.13D D~8C~Pq!IO~ OF ~E P~ D ~l~ODTNE5~!8
P.s noted above, the present in~rention is
direeted, in pa~, to t:he dise:ov~ry oi~ novel Lewi~;
and novel Lewi~i" a~alogues whiah, in mammals,
inc1udirlg hu~a~s, ~ are useful for ~ v .ro modulaltion
(eSg. ~ suppression) of a c:ell mediated immune
response inc1uding cell-mediated arld immune directed
inf1amma~ory respons~ to an antigen in a m ~al
~e.g., a DTH response).
- 20 Additiona11y, th~ present in rent~on is
direated, in part, to nc~vel methods for the
~ynthesis of L~wi~;X and for the synthesi~ of L@wis~
ana1Ogue~; and to novel inter~edial:es useful in these
syntheæes .
However, prior to discussin~ t~is in~ ntion in
further detail, the following ~erms will f ~ rst be
def ined .
Def initions
As used herein, the f ol1Owing terms have the
de~initions given be1Ow:
W092/22~64 2 t 1 ~ 7 ~ 7 PCT/CAg2/00245
-- 17 --
The term "cell-mediated immune response to an
antigen in a mammal" refers to ~hose mammalian
immune responses which are mediated by cell-cell
interactions~ Included within this term are cell
mediated inflammatory responses to an antigen such
~s DTH responses as well as cell-mediated
inflammatory responses arising from myocardial
infarction, virus-induced pneumonia, shock and
:~ s~quelae (e.g., multiple organ failure), adult
`10 respirato~y distress syndrome, psoriasis, arthritis,
~: and the like. Preferably, the cell-medi~ted immune
: response is a leucocyte-mediated response.
The term "LacNAc" refers to the disaccharide
~Gal(1~4)~GlcNAc which is repr~sented by the
lS ~or~ula:
~: HO OH ~Hac
~ ~ OH
OH oH
: ~
he term "LacNH2" refers to the LacNAc
der~vative wherein the N-acetyl group of LacNAc has
been repl~ced wi~h an amine (-NH2).
The te~m "LacN3~l refers to the LacNAc
d~riYative wh rein the N-acetyl group of LacNAc has
~: been replaced with an azido (-N3~.
The term "~ewisX" (sometimes referred to "LeX")
refers to *he trisaccharide having the following
: structure:
W092/225~ PCT/CA92/00245
18 --
OH
H~OH
OH
0~
Because of its relation~hip to blood group
de~e ~inants, th~ core ~Gal(1-43~GlcNAc struckure of
Lewi5X i5 often referred a~ a "type II structure~' or
"LacNAc structure".
Th~ term ~'Le~i~n" (sometimes ref~rred to IlLe~
re~rs to the tri~accharide hav.ing the followi~g
~: struc~ure: :
HO
30H
C H3 S~0H ~ OH
H~\~l ~ OH
Becauss of its relationship to blood group
dete~minants, the core ~Gal(1~3)~GlcNAc structure of
10 Lewis3 is often referrad as a "type I structure'~.
The term ~modified LRwisX glycosides and
deri~ati~es thereof" refer to deri~atives of the LeX
modified in one or more of the fucose, galactose and
N-acetylglucosamine saccharid~ units of ~ewisX and
which have an -YR substituent as defi~ed above.
When the R subs~ituent is an aglycon yroup, this
W092/22564 PCT/CA92/00245
3 7 0 7
19 ----
group has at least one carbon a~om, but neverthel2ss
are different from glycoconjugates because such
aglycon moieties are neither a protein nor a lipid
capable ~f forming a micelle or other large
aggregate stru~ture.
The term "modified Lewis~ glycosides and
derivatives thereof" refer to derivatives of the Le~
modified in one or more of the fucose, galaatose and
N-acetylglucosamine saccharide units of Lewi8~ and
which have an -YR substituent as defined abov~.
When t~e R ~ub~tituent is an aglycon group, this
group has at least one carbon atom, but ne~ertheless
are different ~rom glycoconjugat~s because such
aglyc~n moieties are neither a pro~ein ~or a lipid
aapable of forming a micelle or o~her large
aggr~gate structure.
The term "aglycon of at leas~ one carbon a~om~'
xefers to non-saccharide co~taining residues having
~: a~ lea~t one carbon atsm. Preferably, the aglycon
20 i8 ~elected fro~ th~ gro~p con~isting of ~ Z
wherein A represents a bond, an ~lkylene group of
f~om 2 to lO carbon atoms, and a moiety of the form
. - (~H~-~5G~ n~ wher~in n i~ an integer equa~ tv 1 to
~ 5~: Rl5 is ~elocted ~rom th~ group aonsisti~g o~
hydrogen, methyl, or ethyl; and G i5 s~lected from
the group consisting of hydrogen, haloge~, oxygen,
sulphur, nitrogen, phenyl and phenyl s~bstituted
with 1 to 3 substituents selected from t~e group
consisting o~ amine, hydroxyl, halo, alk~l of from 1
to 4 carbon atom~ and alkoxy of ~r~m 1 to 4 carbon
at~ms. and Z is s~lected from the group consisting
of hydrogen, methyl, phenyl, nitrop~enyl and, when G
is not oxygen, sulphur or nitrogen and ~ is not a
bond, the~ Z is also selected ~rom the group
consi5ting of -OH, -SH, -~H2, -NHR16, -N(R16) 2
W O 92/22564 PC~r/CA92/00245
~rl Q~ - 20 --
-C(O)OH, -C(O~OR21, -C(O)~nH-NH2, C(O)NnH2,
( ) 16~ C()N(R16)2~ and -OR17 wherein each R16
is independently alkyl of from 1 to 4 carbon atoms
and R17 is an alkenyl group of from 3 to 10 carbon
atoms.
Nu~nerous aglycons are hno~nn in the a ~ . For
example, an aglycon comprising a para-nitrophenyl
group (i.e., -YR = -OC6H4pN02) has been disclosed by
Ekberg et al-18 At the appropriat~ time during
~10 synthesist the nitro ~roup is reduced to an amino
group which can ~e protected as N-~ri~luoroacet-
amido. When desir~d, the trifluoroace amido group
i~ remov~d thereby unmasking the amino group.
~n aglycon containing sulfur is di~closed by
~ ~5 D~h~en et al.19. Sp~ci~ically, the aglycon i~
: deriv~d fro~ a 2-bromoe~hyl group which, in a
ubstitution reaction with thionucleophiles, has
b~en h~wn to lead to aglycons pos~es~ing a variety
ten~inal functional groups ~uch as
~: 20 O~H2cH2s~H2co2c~3 and -OCH2c~2sc6H~-pNH2
ana et al.~ di d o es a 6-trifluoroacetamido-
h~xyl aglycon ~-0-(CH2)6-N~COCF3~ i~ which th~
~ri~luoroacet~mido protecting group can b~ removed
unmasking the pri~ary amino group.
~5 Oth~r exemplification of kn~wn aglycon~ include
the 7~methoxycarbonyl-3,6,dioxaheptyl a~lycon~l
( OCH2-~H2~ zOCH2CO2C~3; the 2-~4-methoxyc~rbonyl-
butancarboxamido)ethyl22 ~-O~H2CH~NHC (O) (C~2~ bC2~H3);
and th@ allyl aglycon23 ~O~H2CH~CH2) which, by
r~dical co-polymerization with an appropriate
monomer,leads to co-polymers; other allyl linking
aglycons24 are known [e~g., -O(CH2~H20)2CH2CH=CH2].
Additionally, allyl linkin~ arms can be derivati~ed
in the presence of 2-aminoethanekhiol25 to provide
W092/22~64 PCT/CAg2/0024~
i O ~r~7 ~ t ri
-- 21 --
for aglycons -OCH2CHzCH2SCH2CH2NH2~ Still other
aglycons are illustrated hereinbelow.
Additionally, as shown by Ratcli~fe et al. 9,
the R group can be an additional saccharide OR14 or
5 an oligosaccharide-OR14 at the reducing sugar
term,inus (where R14 is as defined above).
Preferably, the aglycon moiety is a hydrophobic
group and most preferably, ~he aglycon moiety is a
hyd~ophobic group selected from the group consi~ting
f -(CH2)8COOCH3, -(CH2)sOCH2CHzC~2 and ~(CH2)sCH
The t~rm, "oligosaccharide" re~ers to a
carbohydrate ~t~ucture having from 2 to about lO
saccharide units. The particular saccharide units
: ~mployed are not critical and include, by way of
~5 exam~le, ~ll natural and ~ynthetic deriv~t.ives o~
glucoæe, galactose t N-acetylgluco~amine, N-acetyl-
~galactosamin~, fucos~, sialic acid, 3-deoxy-D,L-
oatulo onic acid, and the like.
In addition ~o b~ing in ~heir pyranose ~orm~
all saccharide units described herein are in their D
fo ~ except for fuc~se which is in its L fonmO
The term 3'sialic acid~' or ~'sialyl" me~ns all
naturally oc~urring structures of sialic acid and
analo~ue~ of sialic acid. Naturally occuxring
~tructures o~ sialic acid include, by way o~
exa~ple, 5-acetamido-3,5-dideoxy-D-glycero-D-
galactononulo-pyranosylonic acid ("N~uSAc3a), N-
glycoyl neuraminic acid (NeuSGc~ and 9-O acetyl
neuraminic acid ~Neu5,9Ac~)O Analogues of sialic
acid r~fers to analo~ues of naturally ocGurring
struc~ur~s of sialic acid including those wherein
~ t~e sial ic acid unit has been ch~mically modified so
as to introduce and/or remove one or more
functionalities from such st~uctures~ For example,
such modi~ication can result in ths removal of an
W092/22564 PCT/CA92/00245
22 --
-OH functionality, the introduction of an amine
functionality, the introduction of a halo
functionality, and the like.
Certain analo ~ es of sialic acid are known in
S the art and include, by way of example, S-azido-
NeuSAc, 9-amino-NeuSAc, 9-deoxy-NeuSAc, 9-fluoro-
NeuSAc, 9-bromo-Neu5Ac, 7-deoxy-NeuSAc, ~ epi-
Neu5Ac, 7,8-bis-epi-Neu5Ac, 4-O-methyl-Nsu5Ac, 4-N-
acetyl-Neu5Ac, 4,7-di-deoxy-Neu5Ac, ~-oxo-Neu5Ac, as
`10 well as the 6-thio analogues of NeuSAc. The
: nomenclature employed herein in describing analogues
of sialic acid is as set forth by Reuter et al.12
The term "fucose" or "fucosyl" refers to
~-fucose and~analogues thereof. The ~ucose
: substituent ca~:be:attached to the derivatized
Gal~ 3)GlcNAc~-disaccharide (type I) or the -^
derivatized;Ga1~ 4)GlcNAcO-disaccharide (type II)
so as to f~orm the Lewis' or LewisX molety by eithar
chemical ox:;enzymatic means. While che~ical means
~are illustrsted:~in the figures, enzymatic means for
attaching L-fucose to~th~ 3-position of the GlcNAc
~; unit~of ~Gal(1-4)~GlcNAcO-Iipid containing a sulfate
: on the 3-po~ition;:~of the galactose unit has been
: repo ~ ed~. In~uch~ca~es, ~he fucose e~ployed,~- as
its GDP derivative,~is one which is compatib~e with
the fucosyltransferase (e.g., ~Gal(l~3j4)~GlcNAc
a(l-3/4)fucosyltransferase). The ~Gal(l~3/4~GlcNAc
4(1 ~3~4~fucosyltransferase is readily isolated from
human milkl3~l4~15. : M ditionally, it is contemplated
that these fucose or fucosyl compounds will also be
:~ compatible with other furosyltransferases of
::: appropriate specificity such as cloned
fucosyltransferases~30.
: In regard to the absve, any fucose compound
~: 35 which, as its GDP-derivative, is recognized by the
:::
W092/225~ PCTJCA92/00245
2~ 7 ,~ ~
-- 23 --
~Gal~l~3/4)-~GlcNAc ~ 3/4)fucosyltransferase so as
to bind to the enzyme and is then available for
transfer to the compound of Formula III or IV:
Y-R III
R~
S s:~
I10 0~
X2 ~ ~ Y-R IV
R~ Rl
where R, R1, R2, R3, Xl and X~ are as defined abov~,
is said to be compatible with this
fucos~1~ransf~rase.
An2logues of ~ucose refer to naturally
occurring and synthetic analogues of fucose
: including those where the Pucose unit has b~en
che~ically modified 80 as to introduce and~or remove
~: o~e or ~ore ~unctionalities from this structure.
lS For example, such modification ~an result in the
r~mo~al of an -OH functionality, the introduction of
~an ami~e functionality, ~he introduc~ion of a halQ
~unc~ionality, a~d ~h~ like.
: Certain compatible analogues o~ fucose are
known in the art and include, by way of example, 3-
deoxy-ucose, arabinose, and the like .16
The GDP--derivative of fucose refer ~o
guanosine 5'~ L fucopyranosyl)diphospha~e and any
and ~11 compatible salts thereof which has the
- 25 formula:
W092/22564 PCT/CA92/0024~
O.
~Z~ o~ O~
~ethods ~or preparing GDP-fucose are kn~wn in the
art. However, GDP-fucose is preferably prepared by
the m~thod described by.Jiang et al.l7 in U.S~ Patent
Application Serial No. 07/848,223 which is
~; 5 incorpor~t~d herein ~y re~erence in i~s entirety.
he texm "co~patlbl~ salts" as it îs u~ed in
rela~ion to guanosine 5'~ L-fucopyranosyl~-
: ~ ~ip~osphate refers to those salts of guanosine 5'-
L-~ucopyranosyl)diphosphate which readily form
10~ count~r ions (i-eO ~ cations) and which are
co~pa$ibl~ with~the i~tended reactions and/or
pu~ifications. ~Suitable co~pati~le alts include
:~ tho~e prepared fr~m counter ion~ ~uch as ~odiu~,
pota~ium~ lithium,~:calcium, magnesium, ammonium,
mono-, di-, tri- or te~ra-alkylammonium, iro~, zinc,
and t~e lik~
: The term "pharmaceutically acceptable salts3'
; includes the pharmaceutically acceptable addition
salts of the co~pounds of F~rmula I derived from a
variety of organic and inorganic counter salts well
:: Xnown in the art and include, by way of example
only, sodium, potassium, calcium, magnesium,
ammonium, tetraalkylammonium, and the like,
The term "sulfate" such as used to define the
: 25 substituents -OX, -OX~, and -OX2 refers to
WQ92/22564 PCT/CA92~00245
-- 25 -- 2~ 7
substituents which, with the oxygen of a hydroxyl
group of the galactose unit and/or fucose group,
form a sulfate group (i.e., -0-S(0)2-OH). Thus,
when X, X1 or X2 is a sulfa~el the resulting -OX,
-OX1 and/or -OX2 group is -0-S(Q)2-OH, which readily
forms pharmaceutically acceptable salts thereof
(e-g-, -0-S(0)2-O~Na~,
The term "phosphate" such as used to define the
substituents -OX, -X1, and -OX2 refers to
substituents which, with the oxygen of a hydroxyl
group of the gal~ctose unit and/or fucose group,
form a phosphate group (i.e., -O-P(0)2-OH). Thu.,
when X, X1 or X2 i~ a phosphate, t~e resulting -OX,
-X1 and/or -OX2 group is -0-P(0)2-OH~ which readily
~5 form~ pharmaceutiGally acceptable salts ther~of
(e.g~ -0-P~0)2-0-N~
The term '~re~ovable blocking group'~ refers to
any group which when bound to one or more hydroxyl
groups of the galactosa 9 the N-acetylglucosamine,
and/or the fucose units of L~wi X and ~ewis moieties
: ~ prevents reactions ~rom occurring at ~hese hydroxyl
groups and which protecting group ~an ba removed ~y
con~entional ~ iaal or e~zym~tic ~eps to
xeestabli h the hydroxyl group. The particular
r~movable ~locking group employed is not critical
and pr~ferred re~ovable hydroxyl bloc~ing groups
include conventq nal substituants such as benzyl,
benzoyl acPtyl, chloroacetyl, benzylldine,
t-butyldiphenylsilyl and any o~her group that can be
introduced either enzymatically or chemically onto a
hydroxyl functionality and later selectively removed
either by e~zymatic or chemical methods in mild
conditions compa~ible with the nature of the
product. One such additional contemplated blocking
WO 92/22564 P~/CA92/00245
2 6 --
group is a ~-galactose which can be remo~ed
enzymatically with an ~-galactosidase.
2. ~hQ~
The modif ied LewisX and modif ied Lewis~
co~pounds disc10s2d herein are readily prepared
either by complete chemic:al syntheses or by
chemical/enzymatic: syntheses wherein
glycosyltransferase~ are employed to effect the
æaquent~al addition of one or more of sugar units
onto a GlcN~c OR saccharide strurture, a deriv~tized
Glc:NAc~-C)R saccharide structure, a Lac~Ac OR
disac:charide structure, a derivatized L~acNAc-OR
saccharide ~ tru~::ture, a ~Gal ( 1~3 ) ~GlcNAc-OR
dis;lccharide struct;ure, or a derivatized
~Gal ( 1~3 ) ~GlcNAc-OR disaccharide struoture .
Th~ f i~uras a~ached hereto elabc~rate on a
variety o~ complete che~aical synthetic schemes which
re~ult in ~he :pr~paration of modified LewisX an~
modi~i~d Lewi~8 co~pounds.
Enzyma~ic means to prepare modified LswisX
compounds and modi~ied Lewis~ compounds can be used
at di~fer@nt~;teps. For ~xample~ L-fucos~ c:an be
n~ymatically transferr~d on~o ~h~ d~bloak~d
~ul~ate, p~osph~t~, or carboxylat~ containing
: 25 derivatives of:
a LacNAc-OR structure or a derivatized
LacNAc OR structure (modified ~ewisX compounds); or
a ~Gal(~33~GlcNAc-OR structura or a
der vatized ~Gal(1-33~GlcNAc-OR ~tructure (modifiad
Lewisa compound )
by an appropriate fucosyltran ferase such as
: the ~Gal(1~3/4)~GlcNAc ~(1~3/4)fucosyltransferase
which is readily obtained from human milkt3~l4~5.
W092/22564 PCT/CA92/00245
7 ~ 7
-- 27 --
The LacNAc-OR disaccharide can be made
enzymatically from an N-acetyl glucosamine glycoside
and the known ~-galactose~1~4)transferase. The
~Gal(1~3)~GlcNAc-OR disaccharide glycoside can be
made chemically.
Additio~ally, it is con~emplated that
sulfotransferases may be used to effect sulfation at
the 3-position of galactose on either the type I or
type II structures~ As is apparent, this can be
followed by transfer of fucose using an appropriate
fucosyl~ransferase as de cribed above.
Alte~natively, chemical and enz~matic means can
be coupled wherein, for example, the sulfated,
phospho ~ lated, or ~ 1~COOH substituted Laa~c-OR
: 15 ~ructure or ~ulfated, phosphorylated, or -CHR1aCOOH
: substituted ~&al(1~3)~GlcNAc-OR structure is made
chemically and the fucosyl group transferred
enzyma~ically.
Chemic~l syn~hesis is a convenient method for
: 20 ~ preparing either the complete oligosaccharide
~lycoside; for chemically modifying a saccharide
unit which can then be ch~ically or enzymatically
coupled to an oligosaccharide glycoside; or ~or
chemically preparing an olig~saccharide glycosi~e ~o
which can be enzy~a~lcally coupled one or more
: saccharide unit
Several chemical syntheses of blocked
intermediates exist46~47~48. These intermediat~s are
~uitable for the modifications described herein.
30 Syntheses of the~;e int rmediates or similar ones
utilizing methods known in the art allow the
synthesis of the modified L~wisX and modifie~ I.ewisa
compounds contained herein.
Chemical modif ications include introduc:tion of
s the sulphate or phosphate group or a -oCHR18COOH at
WO ~2/22564 P~/CA92/00245
c~ 28 --
the 3 and/or 6 position of the terminal galactc~se.
Additionally the 3 or 4 position of the fucose can
be sulphated or phosphorylated if the fucose
intermediate 25 of Figure 2 is employed rather than
S fucose ~ntermediate 20.
2Ao CHE~fICAL SYNTHESIS OF SACCHARIDE MONOMERS
Chemical methods f or the synthesis of LewisX
and Lewis~ and some analogues thereof are known in
the art. The~e materials are generally assembled
.0 using suitably protected individual monosa ::charides
including the desirable glu~osamine, fucose and
galacto~e, lactose or ~Gal(1 ~3),8GlcNAc:
intermediate~. The modifications to the final
structures are acromplished using kno~n methods or
15 ~;ul~atic~n or pho~;phorylation after ~ppropriate
s~lec:tive d~blocking of the to-be functionalized
;: hydroxyl group (s) of the fully blocked I,ewisX or
I~di8a o
The s~ec:if i c method~ employed are genérally
20 adapl:ed and opti~Qized for each individual structure
to be synth~sized. In general, the chemical
synthesis of all or part of the oligo~acc~aride
glycosides first involves formation of a glycosidic
linkage on the anomeric carbon atom of the reducing
sugar. Specifically, an appropriately protected
form of a naturally occurring or of a chemically
modified saccharide structure (the glycosyl donor)
is selectiYely modified at the an~meric center of
the reducing unit so as to introduce a leaving group
comprising halides, trichloroacetimidate, acetyl,
thioglycoside, etc. The donor is then reacted under
catalytic conditivns well known in the art with an
aglycon or an appropriate form of a car~ohydrate
acceptor which possess one free hydroxyl group at
WO 92f22564 PCr/CA92/00245
-- 29 -- ~11 1)70 ~
the position whexe the glycosidic linkage is to be
established. A large variety of aglycon moieties
are known in the art and can be attached with the
proper conf iguratio~ to the anomeric center of the
S reducing unit. Appropriate use of compatible
blocking groups, well knowrl in the art of
carbohydra~te synthesis, will allow selective
modif icatîon of the ~;ynthesized structures or the
further attachment of additional sugar units or
10 ugar blocks to t:he acceptor s~ructures.
After formatiorl of the glyc:c~sidic linkage, the
sacchaL~ide glycoside can be used to effect coupling
of additional saccharide unit ~s) or chemically
Dlodif i~d at sel~cted positions or, a~ter
15 conventional deprotection, used in an enzymatic
`~ ~ ynthesis. In g~neral, chemical coupling of a
naturally occurrins~ or chemically modi~ied
sac: charide unit to ~e saccharide glycoside is
acc~mpli~hed by employing established c:hemistry well
2 0 doaw~l~nt~d in the l ~ tQrature . See, f or ~xample,
Oka~oto ~t al.3l, Ratcliffe et al.8, Abbas et al.32,
Paulson33, Schmidt34, Fugedi et al . 35, and Kameyama
al.36
With referenc:e to the figures, Figure 1
illus . rates the synthesis of numerous blocked
derivatives of glucosamine and N-acetylgluc:osamine
whic:h are then useful in the preparation of blocked
LacNH2-OR, LacN~c-OR, ,BGal(l ~33,~GlcNAc:-OR;
,~lGal ( 1 ~ 3 ) ~Gls:NH2-OR, etc . structures . Spec:if ically,
~: 30 in Figure 1, glucosa~mine hydrochloride is slurried
in dichloroethane containing an equivalent of
a~hydrous sodium acetate to whic:h acetic anhydride
is added dropwise and, after addition is completa~d,
the solution is refluxed for a period of from about
W092/22~64 PCT/CA92/0024~
~ 91~ ~ 30 __
12-16 hours to provide for the peracylated compound
10 (about 3:1 ratio of a/~).
Alternatively, the glucosamine hydrochloride is
first taken up in methanol and then treated with 1
eguivalent of metallic fiodium to neutralize the HClo
Phthali~ anhydride is then quickly to the reaction
mix~ure followed shortly thereafter ~y triethylamine
to provide for the phthalimido derivative. This
compound is then isolated and acetylated with acetic
~10 a~hydride/pyridine using conventional techni~ues to
provide for peracylated compound 1 having a
phthalimide blocking group protecting ~he amine.
Afterwards, th~ aglycon is formed by
conventional techni~ue~. For example, compound 10
lS is converted to l-a-chloro compound ~ by w~ll known
chemiætry which involves~ bubbling ~aturating a~un~s
:of~hydrogen chloride directly into a di~hloroethane
solution of ~co~pound 10. In thi~ rega~, the
solution used to~prepare c:ompound 10 ~an be used in
: 20 ~his reaction after that solution has been guenc:~ed
~into water to ;remove ac:etic anhydride and sodium
acetate, dried and r~covered. Th~ reaction
genera~lly proceeds~ ov~r a~period ~of about 4-6 day~
arld::hydrogen chloride is bu}~bled ~nto the ~;olution
2S perios~ic~lly ~.g., about o~ce: ever 1-2 days).
Aft~r reaction ~coBpl~tiont the solution is quenched
in aqueous :sodiumi :bicar~onate at abo~lt
.
0-5 c C and the product is recoveredi after drying the
organic layer and tripping the solution to providl3
0 for compound 2 (one spot on t~I~co)
Compound 2 is then converted ts: the
(CHz~8COOCH3 açllycon by well known ch~imiætry
which involves reaction of compound 2 with
H0(~2)8C:OOCH3 in anhiydrous dichIoromethane
- 35 containirlg molecular sieves in the presence of an
~092f~2564 PCT/CA92/00245
-- 31 __ ~I10~7
equivalent amount of mercuric cyanide. The reaction
is generally conducted at room temperature for a
period of about 12 to 24 h~urs. Upon reaction
completion (as evidenced by t.l.c.), the reaction
solu~ion is filter through silica and the resulting
solution i5 quenched by adding the reaction solution
to cold wat~r. Th~ organic layer is recovered and
the washed twice with an aqueous potassium iodide (5
weight/vol percent) solution and then with a
`10 saturat~d aqueous sodium bicarbonate solution. The
re8ulting organic ~olution is then dried and the
solv~nt removed by stripping to pro~ide for
co~pou~d 3.
The 3, 4, and 6 hydroxyl groups of compound 3
ar then deprotected by reaction with sodium
methoxide in met~anol to prc~vide for N-ace~yl-
gluco~;amine-0~, compound 4. This compouIld s::an
r~acted with Cs~5CH:(OCH3)2 in, for example, an acidic
~: me~ium in arl appropriate solvent ~t around ~10-50C
for about 4-6 hours to provide for the 4, 6-0-
dipxotected b~nzylidine co~pQund 5. In turn,
compound S can be reacted wi~h p-metho~ybenzyl
:: ~ : t~ichlo~oacetiiaidate in an appropriate olvent
e.g. ,- DMF, dich~oromethane) in the pre~;enc:e oi~ a
catalytic amount:of aal acid (e.g., p-tolu~nesul~onic
acid) to provide for the p-methoxybenzyl protec:~ed
3-hydroxy compound 6. Trea~ment of compound 6 with
sodium cyanoborohydride in acetonitrile followed by
he dropwise additio~ of HCl saturated ether at
about 0C leads to~compound 7.
: Alternatively~ compound S can be blocked at the
3-hydroxyl group by reaction with, for example,
allyl bro~ide and base (e.g., barium
hydroxide/barium oxide) to provide for comp~und ~.
3~ Treatment of compound 8 with sodium cyanoborohydride
W092/22564 PCT/CA92/002~45
c~ 32 --
in tetrahydrofuran followed by the dropwise addition
of HCl saturated ether at about 0C lead~ to
compound ~.
Because compoundæ 7 and ~ contain only a freo
hydroxyl ~roup at the 4-position of the blocked
GlcNAc-OR saccharide, subseguent reaction with an
appropriately blocked galactose will result in
for~ation of a type II LacNAc-OR structure
t~Ga~ 4)~GlCNAc O~3.
~ cause compound 5 contains a free hydroxyl
group only at the 3-po~ition of the hlocked GlcNAc-
0~ saccharide, subsequent reaction with an
appropriately blocked galactose will resu}t in
formation of ~ type I structure t,BGal ( ~--3 ~ ,BGlcNAc-
15 OR].
:~ ~lternatively, compound 1 can be converted to ^
co~pourld 11 by reaction of compound 1 with an
eguivalent of p-c:hlorothiophenol in dichloromethane
at room te~perature in the presen ::e of 2 equi~ral~nts
20 o~ boron trifluoride etherate BF3-~therate ~ to provide
for co~apound 1~.
In yet another embodiment, compound 1 is
ao~rt~d to c~mpound 12 (or th~ bromo analogu~ by
follow~ng simi~ar procedures s~t forth above ~or
compound 2.
Compound I2 is con~erted to compound 13 (R =
~C~H3~ by reac~ion wi~h ethanol in manner similar
to that of compound 3 wi~h the exception that
ethanol replaces HO(~H2) 8COOCH3. Compound 13 is then
conYerted to compound 1~ with sodium
methoxide/me~hanol and is then con~erted to c~mpound
lS by reaction with bis~tributyltin] oxide in
refluxing toluene containing tetraethylammonium
bromide followed by reaction with benzyl bromide.
W092/22564 PCT/CA92/00245
3 7 ~3 ,'
- 33 --
Because compound 15 contains free hydroxyl
groups at the 3- and 4-positions of the blocked
Glc~A~-OR saccharide, subsequent reaction with an
appropriately blockad galactose will result in
S formation of both a type I structure
~Gal(1~3)~GlcNAc-OR] and type II structure
~Gal(1~4~GlcNAc-OR~ which are readily separated by
: co~entional technique~ including chromatography.
Com~ound a6 is prepared by treating p-chloro-
thiophenol with 0.95 equivalents of potassiumhydroxide in ethanol followed by heating the
solution to abou~ 4OA-5O-C and then adding about 0-5
equivalents of c~mpound 2 to the reaction solution.
The reaction is ~aintained at 40-50-C for about 1-2
: 15 hours and the product 16 precipitates upon cooling
the ~olution and is reco~ered by filtration.
In Figure 2, the synthesis o~ c~poun~s 17 - 20
are set ~o ~ h in the ex~m~les hereinbelow. ~he
: process to prod~ce ~he highly crystalline fucose
intermediate 20~ from L-fucose as shown in Fisure 2
i o~el. This procedure opti~izes the production
: of ~-~ucopyra~ose tetraacetate 17 by adding acetic
anhydride (AcOAc~dropwi~e to ~ slurry of fucose and
about ~quLmolar ~mounts ~e~g., abo~t 1.1
2S ~ui~alents) of ~odium acetate tNaO~c) maintain~d at
bout 50-55 C in~dichloroethane (DCE3 and stirred at
thi te~perature for a suffici~nt period of ti~e to
result in ~ormation of compound 17 ~e.g., for about .
2-3 days)O The reaction mixture is treated with
:: 30 water, quenched into ice water, extracted with
additional dichloromethane and dried and partially
concentrated to provide ~he peracylated compound 17
(about 4~ ratio of l-acetate~.
Compound 17 is then r~acted with an
approximately equivalent amount of p-chl~rothio-
W092/22~ PCT/CA92/0024
~ - 34 --
~,~'
phenol (p-Cl-Ph-SH) and approximately 1 to 3
(preferably 2) equivalents of boron trifluoride
etherate (BF3-OEt2) in a suitable solvent (e.g.,
dichloromethane) to provide the p-chlorophenyl
2,3,4-tri-O-acetyl-B-thiofucopyranoside, compound
18. The reaction conditions employed are not
critical and temperatures of from about O~ to about
:~: 25-C (preferably at room temperature) and reaction
times of about 3 to about 16 hours can be used.
`10 Compound 18 is quickly deacetylated under
Zemplen conditions:(NaOMe, MeOH) to yield p-
chlorophenyl ~-thiofucopyranoside 19 as a
crystalline product in S5-65% overall yield from
fucose after recrstal}ization from an appropriate
l5~ solvent (e~g., isobutanol). Again, the reaction
: conditions employed are not critical and
: ~emperatures of from about 15- to about 30-C and
reaction times of about 1 to about 10 hours can be
:; used.
Compound~a9 is, in turn, readily benzylated
with benzyl chloride:or benzyl bromide to yield p-
chlorophenyl~2,3,4-tri-O-benzyl-B-thiofuco-
pyranoside, compound 2Q, in 45-50% overall yield
~:~ from fucose. ~As before, the reaction conditions
~: ~ 25 employed are not~critical and temperatures of from
: about 15- to about 3 a ~ c and reaction times of about
~ 4 to about 48~hours can be used. In general, at
~:
least 3 equivalents of benzyl chloride or bromide
are employed and the reaction is generally conducted
in the presence~of at least about 4-5 equivalents of
a ~uitable base (e.g., potassium hydroxide -- K~H)
in a suitable inert solvent (e.g., dimethoxy-
sulfoxide -- DMSO).
In a preferred embodiment, about 3 equivalent
3s of base are added to the reaction system prior to
W092/22564 PCT/CA92/00~4
__ 35 __ h 1, ~ ri ~j~ r~
addition of about 3 equivalents benzyl chloride or
benzyl bromide. ~fter about 18 hours, an additional
1.5 equivalents of b~se and an additional equivalent
of benzyl chloride are addad.
The simple reagents, easy processing and highly
crystalline products eliminate the chromatography
that frequently has been required using heretofore
described methodology.
Thus, in this aspect, the present invention
`10 relates to ~ method for the preparation of p-
chlorophenyl 2,3,4-tri-O-benzyl-B-thiofucopyranoside
which co~prises ~he ~teps:
(a) contacting L-fucose wi~h sodium ac~tate in
dichloroethane maintained at a temperature of from
about 50-55CC while adding dropwise at least 4
equivalents of acetic anhydride;
(b) ~aintaining the ~olutio~ pro~uced in ~tep
(a) above at about 50-55-C for a suf~icient p~riod
of time o as to produce the 1,2,3,4 tetraacetylated
~0 deriv~tive of L-~ucose;
(c) re~cting the product produced in step ~b)
: abo~e wi~h at lea t one equivale~t of p- hlorothio~
phenol and Pxom about 1 ~o about 3 equivalents of
boron trifluoride eth~ra~e under conditions
suf~icient to prsvide the p chlorophenyl 2,3,4-tri-
O ac~tyl-B-~hiofucopyranosid~;
~ d) remuving ~he acs yl blocking groupC by
contact~ng the p-chloropheny} 2,3,4-tri-O-acetyl-B
thiofucopyranoside wi~h sodium methoxide and
m~thanol under conditions sufficient to provide for
p-chlorophenyl B-thiofucopyranoside; and
(e) contacting the p-chlorophenyl B~thiofuco-
pyranoside produced in step (d~ with benzyl c~loride
or benzyl bromide in the presence of a bace and
W092/22564 PCT/CA92/00245
9 ~ 36 --
under conditions sufficient to provide for p-chloro-
phanyl 2,3,4-tri-O-benzyl-B-thiofucopyranoside.
The synthesis of compounds 21-24 are conducted
by following known techniques, for example those
S described by Matta et al. 27 In the procedure of
Matta et al., compound 23 can be con~erted to either
a 3-acetyl (co~pound 24~ or the 4-acetyl blocking
group (not shown). In turn, both of theee compounds
are then blocked at the remaining hydroxyl group
` 10 with a chloroacetyl blocking group by acetylation
with chloraacetylchloride in pyridine/dichloro-
methane at about O-C a
This resul~s in compounds which have
di~ferentially protected 3,4-hydroxy groups. The
: 15 chloroaaetyl blooking group in either co~pound can
be lectively re~ov~ at ~he appropria~e point in^
ths æynth~ by tr~atment with thiourea in
pyridine/ethanol (6:1) anà then reac~ed to form the
sul~ate or phosphate in the manner describad }: elow.
l~e ~synthe~is of compounds 26 - 31 are depicted
~ in Fi~ure 3 a~d are 5~t forth in the exa~ples
:~ ~ herei~elou n In ~is f igure, ~he synthesis o~
ao~pou~ds ~6, 27, 28, and 3C parallels that of
ce~pounds ~7,: ~8:, 19, and 20 as se~ ~orth above and
illustrated in Figure 20 In this regard, benzyl
4, 6-0-be~azyliderle-2 0-benzoyl-3 0-chloroacetyl-B D-
thiogalactopyranoside ~compound 31) ha~: be~n
produced wilthout the necessity of chromatography.
D Galac~ose pentaacetate ~6 is produs::ed by slurring
D-galartose and about an equimolar amount (e.g.,
abou~ 1.1 equivalents) of sodium acetate tNa0~c:~ in
dichloroethane ~DC~:), heating to reflux and addirlg
at least 5 equivalents of acetic anhydride ~cOAc~
dropwise to the refluxing solution (about ~0-85 C)
3s and then maintaining the reaction system at this
W092/22564 PCT/CA92/00245
2 1 ~` O r~ (?
temperature for a sufficient period of time (about
16-32 hour~) to result in formation of compound 26.
This procedure optimizes the yield of ~-D-galactose
pentaacetate 2~ and controls the exotherm of
heretofore known procedures.
After workup of the solution in a similar
~anner to that described above for c~mpound 17, the
product is treated with approximately e~uimolar
amounts of benzyl mercaptan (Ph-CH2-SH) and from
`10 about 1-3 ~preferably ~wo) eguivalent sf boron
trifluorida eth~rate (BF30Et2) in dichloromethane.
The reaction conditi ~ ~ are not critical and the
reaction is preferably conducted at from about O-C
to about 30-C for a period a~ouut 6 to 16 hou~s to
yieId after crystallization from hot m~thanol or hot
: isopropanol 55-65% of benzyl 2,3,4,6-tetra-O-acetyl^
~-D-t~iogalaato-pryanoside, compound Z7O
Deacetyla~ion under Z~mplen conditions (sodium
~ethoxide/meth~nol) leads to compound 28~
Deacetylation reaction conditions are not critical
and ~he reaction is generally conduct~d at roo~
temp~rature for a period of ~rom about 2 to ab~ut 15
hours. ~fter th~ deacetylation reac~ion is complete
: (a. judged by t.l.c~), the solution is neutralized
with an acid ion exchange resin7 filtered and
e~ap~rated to dryness to provide for compound 28.
The r~sidue is crystallized from hot acetone ~nd the
product is taken up in dim~thylformamide or
acetonitrile and treated with from 1 to 2
equi~alents (preferably 1.4 equivalents) of
benzaldehyde dimethyl acetal and about 0.25 to 3
weight percent of p-toluenesulphonic acid (based on
compound 28). The reaction conditions are not
critical and preferably the reaction is conducted at
~5 room temperature and is generally complete in about
W0~2/2~564 PCT/C~9 /00245
t
38
12 to 24 hours. After neutralization, the benzyl
4,6-O-benzylidene B-D-thiogalactopyranoside, 29, is
isolated and crystallized from hot isopropanol.
Benzyl 4,6-O benzylidene-3-o-chloroac~tyl-
B-D-thiogalactopyranoside 30 is prepared by
chloroacetylation using from about 1 to 3
~preferably 2) equivalents of chloroacetylchloride
which is added to a dimethylformamide (D~F) solution
containing benzyl 4,6-O-benzylidene B-D-thiogalacto-
~0 pyranoside 29. The chloroacetylchlorid~ is add~ddropwi~e while maintaining the DMF solution at from
about 40- to about -lS-C (preferably at -25C~.
Under these conditions, it is unexpectedly been
found that the use of D~F permits selective
chloroacetylation of compound 29 without the need
for additional basè. The reaction is generally
complete in about 10-2~ hour~.
Benz~l 4 r ~-O-benzylidene-3-o-chloroacetyl-B-D-
thiogalactopyranosi~e (compound 30) i5 ben~ylated
~ 20 with at least 1 equivalent (and preferably~about 2
: : e~uivalent ) of benzoyl chloride in a suitable
~olvent co~taining a ba~e (e.g., pyridine/methyl ne
chloride) wi~ fr~m about 0~1 to about 1 weight
p~rcent of dimethylaminopyridine ~DM~P~ as a
cataly~t. Th~reaction conditions are not critical
and preferably th~ reaction is conducted at from
about O~C to about 30C and for.about 1 to about 4
hours tpreferably room temperature for 2 hours) to
give cryst~lline benzyl 4,6-0-benzylidene 2-O-
benzoyl-3-O-chloroacetyl-B-D-thio-galacto-
pyranoside, compound 31, in approximately 10-20%
overall yield from galactose.
The advantag of this approach is that after
subsequent assenbly the block d intermedia~es will
35 be simply deblocked and modified by sulfation or
W09~/22564 2 1 1 ~ 7 ~J r/ PCT/CAg2/00245
-- 39 --
phosphorylation. The mat rial is crystalline and the
process obviates the need for chromatography. The
sulfates and phosphates of the galactose moiety of
blocked Lewisa and LewisX can also be made using
compound 32 in the synthesis of these compound.
This compound is made by direct benzoylation of both
the 2,3-hydroxyl groups of compound 29. Howe~er,
after deblocking, both the 2 and 3 hydroxyl groups
of galactose are then available for sulfation and
phosphorylation and the selectivity is not as
e~fici~n~0 Compound 29 can be converted to the 2,3-
dibenzoyl prot@cted compound 32 in a manner similar
to that described above for the preparation of
compound 3~. In this case, 3-5 equiv~lents of
benzoyl chloride are generally 2mployed.
~: Co~pounds 3~ and 32 are convert~d to compounds
~: 33 and:32~ (shown in Figure 5~ via known methodology
Norberg et al~ 26) U ing bromine tetra~thylammonium
~: bromide.
Alternatively, compound 3~ can be converted to
~;~ compound 3~ by contacting compound 31 with 80%
:~ acetic acid~water at approxLm~tely 50~C for about 1-
hours. Compound~34 is th~n conver~ed to compound
35 ~y *rea ~ en~ with acetic anhydride/pyridine in
di~hloro~ thane. ~
In ano~h~r embodiment, ;ompound 32 is treat~d
with sodium cyanoborohydride and ceric chloride to
~, provide for the benzyl-2,3-0-dibenzoyl-4-o-ben2yl-~-
D-thiogalactopyranoside (not shown~. In turn, this
compound is chloroacetylated at the 6-hydroxyl
group. After formation of the LewisX or ~ewis~
: struc~ures, the ~hloroacetyl group can be
electively removed (as described above) and then
: either phosphorylated or sulphonated so as to
provide for the 6-phosphate or 6 sulfate deriYative.
W092/22564~ P~T/C~92/~0245
-- 40 --
Thus, in ano~her of i~s me~hod aspects, the
pr~sent in~ention relates to a method for the
preparation of benzyl 4,6-di-0-benzylidene-2-0-
benzoy~-3-0-chloroacetyl-B-D-thiogalactopyranoside
which comprises the step~:
(a) contacting D-galactose wi~h sodium ac~tate
in dichloroethane maintained at a temperature of
from about 75-~5-C while adding dropwise at least 5
~uivalents of acetic anhydride;
(b) maintaining the solution produced in step
~) a~ove at about 7~-85-C for a suffici~nt period
of time so as to produce the 1,2,3,4,6-penta-
acetylated derivative of D-galactose;
(c~ reacting the product produced in step (b)
lS ab~ve with at least one ~uivalent of benzyl
~; ~ercaptan and about 1-3 equivalents of boron -^
trifluorid~ etherate under conditions suf~icient to
pro~ide the benzyl 2,3,4,~-tetra 0-acetyl-B-
thiogalactopyranoside;
(d~ remo~ing ~he ace~yl blocking groups by
contacting the b~nzyl 2,3~4,6-t~tra--O-acetyl-~
thiogalactopyranoside with sodium methoxide and
methanol under conditions sufficien* to provide ~or
phenyl B-thiogalactopyxanoside;
~e) contacting ~he benzyl ~-thiogalaG~o-
pyranoside produced in step (d) aboYe with
:: benzylaldehyde dimethyl acetal and p-toluene~u}fonic
acid under conditions sufficisnt to provide the
benzyl 4,6-di-O-benzylidene-B~D-thiogalacto
~0 pyrano~ide;
(f) adding chloroacetylchloride to a
dime~hylformamide (DMF) solution containing the
benzyl 4,6-di-O-benzylidene-B-D-thiogalacto-
pyranoside produced in step (e) above maintain~d at
a temperature of from about -40C to about -15C for
W092/22~64 PCT/CA92/00245
-- 41 __ ~Jl 1 ~ 7~ 1~
a sufficient period of time so as to provide benzyl
4,6-di-O benzylidene 3-O-chloroacetyl-B-D-
thiogalactopyranoside; and
(g) adding benzoyl chloride or other suitable
~enzoylating agent to a solution of benzyl 4,6-di-O-
benzylidene-3-Ochloroacetyl-B-D-thiogalacto-
pyranoside in a suitable solvent containing a base
and dimethylaminopyridine under conditions
sufficient to provide for benzyl 4,6-di-O-
~enzylidene-2-O-benzoyl-3-O-chloroacetyl-~-D-
thiogalactopyranoside.
2B. S~NTHESIS OF TYPE LEWISX STRUC~URES
[~Gal(1-4)~Fuc(1~3)]~GlcNAc-OR]
-~ Fiyure 4 illustrates one m~thod for
lS synthesizlng blocked type II b~ckbones and
conv~rsion to blocked LewisX struatu~esO In ~hi8
~igureO the 2,3 hydroxyl groups of the galactose are
di~ferentially blocked ~o tha . at the appropriate
point in the synthesis of LewisX and L2wisa
derivative~, the chloroacetyl protecting group at
~he 3-position of galactose is selectively removed
and then co~verted to the sulfate, pho~phate or
OoH~8~OO~ group. ~1BO~ as noted above, ~he
chloroac~tyl protQcting ~roup can be selectiv~ly
placed a~ the 6-position o~ the galactose and then
.elQctively removed ~o as to allow for the formation
of the sulfate, phosphate or -OCHRl8COOH group at th~
~-position of galactose~
Specifically, in Figure 4, ¢ompound 7 and
co~pound 33 are co~bined to form compound 37. ~his
is acc~mplished by dissolving co~pound 7 and
approximately 1.5 equivalents of compound 33 in
dichloromethane containing molecular sieves to which
is added about 1 equivalent (based on compound 7~ of
W092/72~64 PCT/CA92/00245
c; ~
-- 42 --
2,6-di-t-butyl-4-methylpyridine. The reaction is
stirred for 30 minutes at room temperature and then
cooled to -50~C. An anhydrous toluene solution
containing approximately a slight excess (e.g.,
S about l.~ equivalents) of silver trifluoromethane
sulfonate is then ~dded to the solution and the
reaction is allowed to warm to -15C o~er 2 hours
and maintained at that temperature fDr an additional
5 hours.
At thi~ time, the molecular sieves are removed
by ~iltration by passing t~rough celite a~d the
recovered solution is guenched by addition to a
saturated sodium bicarbonate solution. The organic
extract is then wash~d with water, with aqueous O.SN
:~ 15 ~Cl, and then wi~h water~ The organic solution is
~ ~hen dri~d and conce~trated in vacuo to provide a
;~ ~ c~uds product of compound 37. This is then purified
: by conven~ional techniqu~s such as colu~n
~:~: chr~matography using silica gel and hexane~ethyl
acetate (1:1) as~the eluant.
To a dichloromethane solution containing
compound 37 is added an excess of
dichlorodicyano ~ inone ~DDQ) which selectively
:: r~moves the p-methoxybenzyl protecting group to
~ 25 ~provide compo~nd 38. This co~pound is fucosylated
; ~ wi*h an ~xc~ss of ~ompound 20 ~about 1. 3-1r 5
e~ui~alents) in dichloromethane containing mercuric
bromide or cupric bromide and about 1-1.5 volume
perce~t DMF to give blocked LewisX compound 39.
After work-up and chromatography compound 39 is
treat~d with thiourea to removP the chloroacetyl
group and the compound is sulphonated with sulphur
: trioxide/pyridine complex in DMF at 0C for 2 hours
to provide compound ~1. The blocking groups on
35 compound 41 are then removed by conventional
W092/22564 PCT/CA92/00245
-- 43 --
techniques to provide for the LewisX analogue having
a sulfate group at the 3-position of the galactose
unit.
Alternatively, compound 25 (or the 3-
chloroacetyl analo~ue of compound 25 describ~dabove--not shown3 can be used in place o~ compound
20 in the above synthesis. Removal of the
chloroacetyl blocking groups on the 3-hydroxyl of
the galactose and the 4-hydroxyl of the fucose
pr~vides an facile route to the preparation of a
~isulfated, diphosphQrylated ~wisX derivatives.
In another embodiment, compound 40 can then be
alkylated by ~irst adding an appropriated ba~e
(e.g., silv~r oxide, barium hydroxide, ~odium
hydrid~) and then adding benzyl bromide acetate
(BrCH2COO8n) or other ~imilar acetate~ (e.g.,
:~ BrCHR18,COOBn - where R18, is alkyl of from 1 to 7
¢arbon ato~s or -COOBn) to the reaction m~dium in an
appropria e solvent such a~ DMF. After reaction
co~ple~ion, the be~zyl e~ter(s~ ic (ar~) readily
:: r~moved by conven~ional hydrogenation techniques
which additionally removes the other be~zyl
prot~cting groups and the benzylidine protecting
grsupO Trea~menk with sodium methoxide/methanol
pr~vides for a OCH2COOH (or -OC~R1~COOH wh~re R18 i~
alkyl of fr5m 1 to 7 carbon atoms or -CQO~)
substîtuted to the 3-position of galactose. Similar
type chemistry can be performed at the &-hydro~yl
group of the galactos~ or at the 4-hydroxyl group of
the ~ucose by use o appropriate blocking groups.
In another embodiment, compound 40 can be
treated by known methods48 t~ pro~ide for the 3-
phosphate compound~ Specifically, compound 40 can
be treated with diphenylphosphorochloridate and ~-
dimethylaminopyridine (1:1) in pyridin~ at O~C. The
W092/22564 PCT/CA9~/00245
?~ 44 --
solution is allowed to warm to room t~mpera~ure over
0.5 hours and stirred for 15 hours. The resulting
compound is then hydrogenated under conv~ntional
conditions (first with H2 in EtOH with Pd on carbon
for 15 hours and then with H2 in EtO~ with PtO2 for 3
hours~ to provide for the phosphate derivative at
the 3-position of galactose. Further deprotection
leads to the modified LewisX compound having a
pho~phate substituent at the 3-position of
~0 galacto e) which is purified and converted to its
di~odium salt by contac*ing a solution of ~his
compound with a sodium form of Dowex 50 x 8.
As is apparent, t~e procedures set forth above
can also be used to introduce a phosphate or a
-OCH~8COOH group at the 6-position of galactose or a
phosphate group on the fucose. -
~
Figure 5 illustrates another method foryn~hesizing bl~cked type II ~ackbones and~
con~ersion ko blocked Lewis~ structur~s. In this
fi ~ e, th~ 2,3 hydroxyl gr~ups of the galactose ~re
not di~f~rentïally blocked and, accordinglyd while
the resulting com~ound ~5 tand ~he ~yp~ ~ ~nalogu~)
i~ u~ul ~or preparing the 3-sul~ate (as p rt of a
:~ mixture with the ~-sulfate which can be purifi~d by
chromatography) t is not as ver~akile as the
synthetic scheme set forth in Figure 4.
In any eYent, in Figure 5, compound 7 and
approximately 1O6 1.7 equivalents of compound 32
are dissolved in dichloromethane containing
molecular sieves to which is added about 1
eguivalant (based on compound 7~ of 2,6 di-t-butyl-
4-methylpyridine. The reaction is stirred for 30
minutes at room temperature and then cooled to -
~092/225~ PCT/CA92/00245
-- 4S -- 2 11 0 7~l~
50C. An anhydrous toluene solution containing
approximately a slight excess (e.g., about 1.2
equivalents) of silver trifluoromethane sulfonate is
then added to the solution and the reaction was
S allowed to warm to -15-C over 2 hours and maintained
at that temperature ~or an addi~ional 5 hours.
After reaction completion, the reaction system
was worked up to provide a crude product of compound
: ~2. This is then purified by conventional
:~ 10 te¢hnigues such as column chromatography using
~ ~ silica gel and toluene-ethyl acetate (1:1) as the
: eluant. : ~
To a dichlorame~hane solution containing
:~ ~ compound ~2 i ~added an exce s of dichlorodicyano-
15: guinone ~DDQ) which celectively ramoves the
p-methoxybenzyl protectins group to pro~ide compoun~
3. This compound is;fucosylat~d with an excess of
compound 20 (about 1-3 equivalents and pre~erably
about 1.3-1.5~equivalents) in dichlor~methane
: 20~ con~aining mercuric bromide vr cupric bromid~ and
:abo~t 1~2 volume percent DNF to give blocked LewisX
co~pound A4. ; After work-up and chromatography
compound ~: is~treated with sodium
:: ~
~ :~ethoxide/methanol:to remove the benzoyl blocking
: : : 25 ~group~ a~ the 2,3-positions of the galactose o as
to provide for co~pound 45. This compound is th~n
sulphonated with~sulphur trioxide/pyridine complex
in DMF at 0C for Z hours to pro~ide compound 46.
Compound ~6 is produced as a mixture o~ the 3-
: 30 sulfate and the 2-sulfate (or 2,3-disulfate) which
:: : is separated by chromatography (e.g., column
: chromatography on silica). Conventional
: : deprotection of the removable protecting groups
provides for the sulfate derivative at the 3~
position of galactose for LewisX, compound 47, which
W092/22564 ~, PCT/CA92/0024S
i ~-'
-- ~6 --
can be passed onto an anion exchange resin (sodium
form) to generate the sodium salt.
Additionally, lactose can be used in the
methods of this invention in place LacNAc by merely
a suitable b~ocking group at the 2-hydroxy of the
glucos moiety of the lactose structure49.
Dif~erential blocking of the lactose provides for a
compo~ition having a electively removable blocking
group at the 3 a~d/or 6 position of the galactose.
~10 Thi8 compound is th~n selecti~ely deblocked at the 3
and/or 6 po~ition and then derivatized to the 3
and/or 6 sul~ate, phosphate or -OCHR~8COOH.
Afterwards, the remaining blocking groups are
removed and the fucosyl unit added enzymatically
s ( ~ee below) .
2C. S~NTHESIS OF LEWISA STRUCTURES
~Gal(1~3j[~uc(1~4)]~GlcNAc-OR]
While Figures 4 and 5 illustrzte ~he synthesis
of LewisX struc~ures, ~ewisa structures are readily
prep~red in a ~imilar manner, a~ illustrated in
Figure 8, using appropriately blocked GlcN~c-OR
structures. The ~Gal(1~3)~Glc~c-~R structures can
be prepar~d, for example, ~rom c3mpounds 5 and 35.
Specifically, compound 3~ is first conver~ed to the
2S l-~-bro~o derivative via known methodology (Norberg
et al.26) using bromine (Br2~ and ~etraethylammonium
bromide (Et4~Br ) at about O~C. About 1.5
equivalent~ of this compound and compound 5 are
dissolved in dichloromethane (Cl2CH2~ containing
molecular sieves to which is added about 1
equivalent (based on compound 5) of 2,6-di-t-butyl-
~-methylpyridin~. The reaction is stirred for 30
minutes at room temperature and then cooled ko
~092/22564 2 ¦ PCT/CA92/0024
47 __
-50C. An anhydrous toluene solution containing
approximately a slight excess (e.g., about 1~2
equivalents) of sil~er trifluoromethane sulfonate
~silver triflate) is then added to the solution and
the reaction is allowed to warm to -~5-C over 2
hours and maintained at that temperature f or an
additional 5 hours. Afterwards, the solution is
allowed t~ come to room temperatur~ and ~îrred
o~ernight.
; ~10 At this time, pyridine and dichloromethane are
:: ad~ed and the molecular sieves are remo~ed by
filtration by pa~sing through celite and the
recovered solution is quenched by addition to a
saturated sodium bicarbonate solutio~. The organic
15 extract is then washed with water, with asIueous 0.5N
HCl, and then~ with water~, The organic ~olution is ^
then dried and concentrat~d in vacuo ~o provide a
; arude product which; is then purified by conventional
techni~ues ~uGh a~; column chromatography usirlg
20: ~ silica gel and hexane-ethyl ac~at. (1~ as the
eluant to pro~ride :for compQund 100. The benzylidine
protecting ~ro~p of c~mpound 100 is then selecti~ely
remo~red by tre~ nt;~with 80% ac:etic acid ~AcOH~ in
water ~3~2) t~ proYide for aompound ~01. Compound
25 :01 . is selectively acetylated at th~ 6-hydroxy group
of the Glc~Ac unit by trea~ment with acetic
, ~
anhydride (AcOAc~ in pyridine at about -20-C to
provide for co~pound 102. (i.e., 8-methoxy-
~, ;
carbonyloctyl-2-~cetamido-3(2-O-benzoyl-3-
~: 30 chloroacetyl-4,6-di-O-acetyI-~-D galactopyranosyl)-
6-O-acetyl-2 deoxy-~-D-glucopyranoside~ This
compound is th~n fucosylated in the manner similar
to compound 38 as described above to provide for
compound 103 and ~hen deblocked and sulfa~ed in the
W092/22~64 PCT/CA92/00245
48 --
manner described above ~or compounds 40, 41, and ~7
to provide for compounds 104, 105, and 106.
Alternatively, compound 32 is converted to the
~ bromo derivatiYe via known methodology ~Norberg
S et al. 26) as described above and the resulting
compound is then treated with sodium
cyanoborohydride and ceric chloride to provide for
the benzyl-2~3-O-dibenzoyl-4-O-benzyl-~-D-
thiogalactopyranoside (not shown). In turn, this
compound is chloroacetylated at the 6-hydroxyl group
and then rsacted with compound 5 in the manner
: d~cribed above to provide for the 8 metho~y-
carbonyloctyl-2-acetamido-3(4-O benzoyl-6-
chloroacetyl 2,3-di O-benzoyl-~-D-galactopyranosyl)-
~5 6-O-ac~tyl-2-deoxy-~-D-glucopyranoside. T~is
compound is th~n tr~ated in the manner de~cribed
above for compound 102 so as to provide for a Lewi~
derivative having a sulfate at ~he 6-position of the
galacto~.
In yet another embodiment, both type I and t~pe
II st ~ ctures ~an be made simultan@ou~ly by
combining compound 15 and compound 33 under
appropriate condition~ well known in the art~ For
example, compound 15 and approximately 1.5
equivalents of compound 33 ar~ added to
dichloromethane containing molecular sieves to whiah
is added about 1 equivalent ~based on compound 15)
of 2,6-diot-butyl-4-methylpyridine~ The reactlon is .
stirr d for 30 minutes at room temperature and then
cooled to -50C. An anhydrous toluene solution
contai~ing approximately a slight excess (e.g.,
a~out 1~2 equivalent~) of silver trifluoromethane
sulfonate is then added ~o the solution and the
reaction is allowed to warm to -15C over 2 hours
and maintained at that temperature for an additional
W092/22564 PCT/CA92/00245
2 ~ 1 0 7 ~ J
-- 4g --
5 hours. Afterwards, the solution is allowed to
come to room temperature and stirred overnight.
At this time, pyxidine and dichloromethane are
added and the molacular sieves are removed by
filtration by passing through celite and the
r~covered solution is quenched by addition to a
saturated sodium bicarbonate solution. The or~anic
: extract is then ~ashed with water, with aqueous 0.5N
~Cl, and then with water. The organic solution is
then dried and concentrated in vacuo to provide a
crude pro~uct which contains both the type I and
type II structures which are separated and purîfied
by conv~ntional techniques such as column
chro~atography using ~ilica gel and hexane-ethyl
lS acetate (1:1) as th~ ~luant.
The ratio o~ type I structure to type II
:~ ~ structur~ rssulting from this rea~tion can be
imprQv~d by using the 2-NAc derivati~e of ~lcNH2
co~pound 5. This c~mpound can be readily prepared
20 by reac~ing co~ound 15 with hydrazine, ac~tylating
the resulting product with asetic anhydrid~/pyridine
:an~ ~ ~n deacetylating the 3,4-hydroxyl groups by
treatment with ~odium methoxid~/m~thanol.
2D. ENZY~ATIC RE~CTIONS
In addition to th~ chemical ynth~ses of the
Lewis~ and LewisX analogues described above, the
appropriately blocked type I t~al(l-3)~Gl~NAc-OR]
and type II t~Gal(1~4)~GlcN~c-ORJ structures (e~g.,
: compound 37) can be selectively deblocked ~o proYide
3~ for a hydroxyl group at the 3-position of galactose
(or at the 6-position) and then sulfonated,
phosphorylated, or converted to OCHR~8COOH (each of
which are described above). Th~ resulting compou~d
is then totally deblocked and fucosylated by using,
W092/22~64 PCT/CA92/00245
50 --
for example, ~Gal(1-3/~3~GlcNAc ~ 3/4)
fucosyltransferase~. The enzymatic transfer of
fucose onto the 4-position of &lcNAc to form Lewisa
and to the 3-position of GlcNAc to form LewisX
structur~s requires the prior synthesis of its
nucleotide (GDP) derivatives. Synthesis of G~P-
fucose is preferably accomplished in the manner
recited by Jiang et al.17 and which is exemplified in
the exampl~ hereunder.
GDP-fucos2 (GDP-Fuc) is then combined with the
d~rivatiz~d ~Galtl~4)~1cNAc-oR compound or the
derivatized ~Gal(1~3)~GlcNAc-OR compound in the
pr~ence of a ~uitable ~ucosyltra~s~erase ~e.g.,
~Gal(1~3/4)~GlcNAc ~ 3/4)~ucosyltrans~rase~ under
~5 conditlons wherein fucose is transferred to ~he 3
po5ition of GieN~c of ~he deri~atiz~d
~Gal(1~4~0GlcN~c-OR or the 4-posi~ion of the
deri~atized ~Gal(1~3)~GlcNAc-OR compound to form a
Lewi~X or Lewis~ ~txuctures respectively.
: 20 Suitable fucosylations conditions, ~nown in the
: art, include the addition of ~he fucosyltran~erase
to a mix~ure of the derivatized ~Gal(1~4~GlcN~c-OR
(or al~ernatively~the d~rivatized ~Galtl~3)~GlcNAc-
OR compound) and the GDP-fucos~ in a appropria~e
~S buffer s~c~ as 50 mM sodium cacodylate in
appropriate conditions of pH and temperature such as
at a pH of 6.5 and a temperatuxe between 30 and
45C, preferably 35 to 40-C while incubating for
about 12 hours to 4 days. The re~ulting ~ucosylated
produ t can be isolated and purified using
conventional methodology comprising HPLC, ion
exchange-, gel-, reverse-phase- or adsorption
: chro~atography.
W092/22564 PCT/CA92/00245
2 1 ~ ~ 7 ~ ~
~- 51 --
It is also contemplated that the deblocked t~peI and II structures can be sulfated by u e of an
appropriate sulfotransferase.
i
2E, M~DIFICATION ON THE 2 ~ND/OR 6 POSITIONS OF
GlcNAc
Figure~ 6 and 7 illustrate two different
synthese~ for the retention of the 2-amino
s~b~tituent on the glucosamine (i.e., a derivati~e
:~ o~ ~Gal(1~3)~GlcN~c-OR or ~Gal(1~4)~Glc~Ac-OR where
~:~ 10 ~he N~c group of GlaNAc has been converted to an
amine). As shown b~low, the retention of ~he amino
~roup on the glucosamine unit allows ~or the ~acile
preparation of di~fere~t 2-substituted derivati~esu
In F~gure 6~ compounds 1, 13, 14, and lS are
~pr~pared in ~he manner described above and
: illustrated~in Figure 1~ Likewi~e, l-~bromo-
2~3,4,6~tetracetyl-galactose i prepared by ~ir~t
~fonmi~g th~ perace~ylated derisrati~re o~ galactose,
compourld ~6. Co~pou~d 26 i~ then convert~d to the
20 1; a-b~omo deri~ative via known methodology
IBr/~c2tic acid --: at about 0C ~o 20C) ~o as to
:provide for l-~-bromo-2, 3, 4, 6~e . rac:etylated
~: : galactos~.
The l-a-bro~ao-2,3,4,6-~etracetylated galactose
2S (about 1. 2 to about 1. 5 equi~Jalent~;) is add~d
dropwise to a solution of c:ompound 15 in
dichloro~nethane at abou~ 50 ' C in the pr~sencs of
~: excess calc::ium su}fate, about 4 equivalents of
silver carbonate and about o . 5 equivalents of sil~er
30 triflate. The~ rea~::tion is then a}lowed to warm to
-30-C and maintained there for-about 1-3 days. The
reaction is then quenched by the addition of
methanol, warmed to room temperature, and f iltered
W092t22564 PCT/CA92/00245
-- 52 --
through celite. The filtrate is washed with aqueous
sodi~m bicarbonate and aqueous ethylene diamine
t~traacetic acid (EDTA). The recovered solution is
dried and then stripped in vacuo to provide a crude
product containing both the type I structure (not
shown~ and the type II structure (compound 48). The
residue is chromatographed on a silica gel column
eluted with toluene:acetone:methanol (20:3:1) to
give compound ~8 as well as the type I analogue (not
`10 sho~n).
For convenience sake, further reactions are
~hown on compound ~8, it being understood however,
that the same reactions could be conducted on the
type I analogue to provide m~dified Lewisa
compounds.
Compound 20 is then reacted with one equivalent
of bromine in dichloromethane at -20-C for about 1
hour to pro~ide for the 1 ~-bromo d~rivative of
: compound 20. The solution is then quenched with a
: 20 cold agueous sodium bicarbonate solution. -The
~: : organic solution is dried and concentrated to
: approximately half the original volume in ~acuo at
room t~mperature. About 2 equivalen~s of this
compound are then add to a dichloromethane solution
of compound ~8 ~hat contains about 2 egui~al~nt~ of
~:~ mercuric bromide (HgBr2), molecular si~ves and
tetraethylammonium bromide. The reaction iæ stirred
at room temp~rature for approximately 48 hour~ and
the solution is filtered through celite and the
3D ~iltrate washed with water, a 5% EDTA solution,
~aturated a~ueous sodium bicarbonate, and then
water. The organic layer is then dried and the
solve~t removed in vacuo to provide for compound ~9
which is purified by chromatography on silica gel.
wo g2,22564 2 1 1 ~ 7 0 PCT/CA9210024$
-- 53 --
Compound 49 is converted to compound 5Q by
conventional Z~mplen condi~ions and compound S0 is
then con~erted to compound 5l by conventional
methodology (e.g., benzaldehyde dimethylacetal, D~F,
S pTSA). In turn, compound 51 is treated with
hydrazine acetate in methanol at room temperature
for about l - 5 hours to provide for ccmpound 52
which is co~erted to compound 53 by contacting with
trifluoroacetic anhydride in methanol.
Alternatively, compound 52 serves as a convenient
point in the synthesis to convert this amine to an
amide, a carbamate, an urea, a -NHS03H ~roup, etc.
in the manner described below.
Compound S3 can then be sulfated in the same
~anner as described above *or compound ~S.
Alt~rnatiYely, c~pound 53 can ~e differentially
blocked at the 2,3 hydroxyl groups of the galacto~e
in ~h~ same set forth above for co~pound~ 29 - 31 so
as to pro~ide for compounds 54 and 55. In ~urn,
compound 55 is ~electively deblock~d with thiourea
to provide for c~mpound 5S in the s~e manner
escribed above for compound 39 (to provide compound
~ 40). ~ompound 56 is then ~electiYely sulfated in
;~ the mann~r descri~ed above to pro~id~ for compound
57. Alternativ~ly, compound 56 can be converted to
the 3~phosphate group on the galactose by reaction
with diphenylphosphorochloridate an~ 4-dim~thyl-
aminopyridine (l:l) in pyridine at O-C~ The
solution is allowed to warm to ro~m temperature ~ver
O~S hours and stirred for lS hours. The resulting
compound is then hydrogenated under conventional
conditions (first with H2 in EtO~ with Pd on carbon
for 15 hours and then with H2 in EtOH with P*02 for 3
hours) to provide for the phosphate derivative at
the 3-position of galactose. Further deprotection
WO g2/22564 PCI/CA92/lD024~i
r l
leads to the modified LewisX compound having a
phosphate substituent at the 3-position of
galactose) which is purified and converted to its
disodium salt by contacting a solution of this
compound with a sodium form of Dowex S0 x 8.
Compound S6 can also be converted to the -CHR~8COOH
in the manner described above.
Lastly, co~pound 57 is deblocked ~y
conventional techniques to provide for co~pound 60
which is a LewisX analogue having a 2-amino glucose
~accharide unit in~tead of a GlcNAc saccharide and
further having a sulfate at the 3-position of the
galactose saccharide unit.
Figure 7 parallels somewhat the chemistry
; ~ 15 depicted in Figure :6 but, because the 3-hydroxyl
group of the GlcN~2 derivati~e is blocked (compound^
69), this synthe~ic results only in type II
:stru~tures. ~n particular, in Figure 7, compound 13
prepared by th~ ~sthods described above~ Thi~
a~mpound is then deacety~ated by conven~io~al
techni ~ es (sodiu~ ~ethoxide/methanol) to provide
for c~mpound l~ which i~ then benzylidenated under
co~ventional technigues to provide co~pound 66~
Compound 6C is~th~n treat~d with benzyl chlorids and
~: ~5 ~odium ~ydride in di~ethylformamide at about -20~C
to 20-C to pr~vide fo~ co~pound 670 ~e benzylidine
~:~ group of compo~nd 67 is then rem~ved with 80~
aqueous acetic acid at about 80C for about 1~4
, ~
hours to provide for compound 68. This co~pound is
: ~ 30 then selectively acetylated at the 6-position by use
of approximately e~uimolar amounts of acetyl
chloride/pyridine in dichloromethan at about -10C
~o provide for compound 69. Approximately 1.2 - 1.5
equivalents of the l-a-bromo-2,3,4,6-tetraacetylated
galactose (described above) is added dropwise ~o a
W092~22564 2 ~ I PCr/CA92/00245
---- 55 ----
solution of compound 69 in dichloromethane
maintained at about -30-C in the presence of about
1.3 equivalents of 2,6,-di-t-butyl-4-methyl-pyridine
and about 1.3 eguivalents of silver triflate. The
S reaction i~ then maintained at -30-C for 1 hour and
then allowed to warm to S-C and maintained there for
about 2 hours. The reaction is then quenched by the
addition of nethanol, warmed to room temperature,
and filtered through celite. The filtrate is washed
~0 with aqueous sodium bicarbonate. The recovered
solution is dried and then stripped in vacuo to
provide a csude product containing compound 70 which
is purified by chromatography on a 8iliC8 gel column
eluted with ethyl acetate:hexanes (~:2) to give
lS compound 70.
$he b nzyl protecting group is then renoved by^
hydrog nolysi~ (H~/Pd on C) to provide fos compound
7~. Co~pound 71, in turn, is fucosylated in the
~ane manner as described above for compound ~8 (to
provide for compound ~9 as illustrated in Figure 6)
so as to provide for co~pound 72. Compound 72 is
deacetylated by conve~tional techniques described
above to provide for compound 73. Compound 73 is
then converted to compound 7~ by conventional
methodology (e.g., benzaldehyde dimethylacetal, DMF,
pTSA), followed by selective acetylation at the 6-
position of the partially protected GlcNH2
derivative by an approximately equivalent amount of
acetyl chloride/pyridine in dichloromethane
maintained at about -50 to about -20-C). Compound
7~ is then converted to compound 79 in the same
manner (described above) as compound 53 was treated
to provide for compound 60.
Alternatively, the free hydroxyl groups of
compound 7~ can be acetylated with acetyl
W092/22564 PCT/CA92/00245
56 --
chloride~pyridine in the manner described above and
the benzylidine group selectively opened by sodium
cyanoborohydride and ceric or aluminum chloride to
give the 2,3-diacetyl-4-benzyl-6-hydroxy deri~ative
on the galactose moiety (not shown). This compound
is then functionalized at the 6-position of the
galactose so as to contain a sulfate, phosphate or
-CHR~8COOH group at this position.
` In addition to the above, th~ 2,6 positions of
the GlcNAc unit can be modified prior ~o coupling so
as to provide for type I and type II structures
~odified at these positions.which are then further
modifiad in th~ manner described above to prepare
:the sul~at~d, phosphorylated or -CHR18COOH
subctitut~d ~wi~ and LewisX structures. As shown
~- by Venot et al., U.S. Patent Application Serial NoO
a7/~ filed ~ay 22, 1992 as Attorney Docket
No. 000475-011 and~entitled l'MODIFIE~ SIALYL LEWISA
COMPOUNDS" and by Venot et al., U.S. Patent~
Appl cation Serial No~ 07/ , , filed ~ay 22,
~ ~; 1992 as ~ttorney~Docket No. 900475-029 and entitled
: ~ "M~DIFIED SIALYL ~EWISX CONP~UNDS'I, modification at
~; : ~ e 2 and/or 6-positions of ~he GlcN~ moi~ty of
typ~ I structures ~ ,~Gàl ( 1~3 ) ~GlcNAc-OR~ and on type
25 II stxuctures ~:,BGal ( ~--3 ) ~GlcNAc OR -- LacNAc-OR3
s~ill permit the use of the ~BGal(1~3/4)~GlcNAc
3/4~fu~osyltransferase on the deblocked
compound. The disclos~res of both of these
applications are incorporated herein by reference in
their en~irety.
i. Modification at the 2-position of GlcNAc
W092/22564 P~T/CA92/00245
? l ~ 3,
-- 57 --
~ odification at the 2-position of GlcNAc can be
accomplished by a variety of ways. For example, the
known8 2-azido-2-deoxy-glucose-OR compound
(prepared, for example, by azi~onitration of 4,5,6-
triacetylglucal) can be protected at the 6 positionwith a removable protecting group (i.e.,
Si(C6H5)2tBu) by conventional techniques8 and then
combined with an appropriate blocked galactos~
compound in the manner descri~ed above to provide a
mixtur@ of blocked ~Gal(1-3)GlcN3-OR and
~Gal(1~4~GlcN3-OR derivatives which are readily
separated by conventional techniques.
At the appropriat~ tim~ during synthesis of the
Lewis~ or Lewi~ structures, the azido group i~
reduced to ~n a~ino group which can be protected as
N-tri~luoxoacztamido~ In turn, the
trifluoroacetamido group is remov~d a~ the
~ppropriate point in the ~ynthesis thereby unmasking
th~ a~ino group.
The amino group can also be derivatized by
con~entional m~thods to provide f~r -NR1~C(O~R~or
( 11 ) 2 ~ ~HCH((~l)zi -NHR12~ a~d ~N(R~)2 groups
by co~entio~al me~hods. :For example, ~he -N~2
group can be re~ated, using con~entional
t~hniques, with:
a carboxylic acid, anhydride or chloride to
provide for amides. Alternatively/ the de~ired acid
can be actiYatad, as reported ~y In~zu et al37 and
then reac~ed with the amino group~ The carbo~ylic
39 acid, anhydride, chloride, or activated acid is
selected so as to provide for an Rto group (i~eO ~ as
part of the ~NRl1C(O~R1~ substituent) which is
hydrogen or alkyl of from 1 to 4 carbon atoms,
with an aldehyde or ketone (of from 1 to 4
3S carbon atoms~ at controlled pH to form an imine
W092/22564 PCr/CA92/00245
-- 58
[-N=C(Rl1)2] which upon reduction (e.g., with sodium
cyanoborohydride) provides for an alkylamine
substituent ~i.e., -NHCH(R~,~2~ as reported by
Bernotas et al.~,
with a cyclic car~onate such as ethylene
carbonate or propylene carbonate which ring opens
upon reaction with the amine to form a carbamate
group having an HO-alkylene-OC(O~NH- substi~uent
where alkyl~ne is from 2 to 4 carbon atoms as
reported by Wollenberg et al.39, U.S. Patent No.
4,612,13Z,
with a chloroformate ti.e., ClC(O)OR~3~ in the
manner di~clo ed by Greig et al.~~ In thi5 case,
the chloroformate has an Rl3 group which i~ alkyl of
lS from 1 t~ 4 carbon ato~s,
Wit~ O=C ~-C~ pN2) 2 which le ds to an -~
activated intermediate which is then re~cted with an
a~ine (HNR14R15) to provide for ur~as r-N~C(O)NR,4R15
as de~cribed by Piekarska-Bartosz~wicz et al.~l,
wikh trime~hylamine, sulfur trioxide~(SO3) so
as to form the -NHSO3H ~roup as d~scribed by
Petitou42, and
with derivatized formic acid or other materials
to form a fonmamîde (-NH-CHO)43 which can be further
25 functionalized to the isocyano (-NzC=O~ and reduced
to the deoxy derivati~e by tributyltin hydride
(Bu3SnH) 43 .
Alternatively, the 2-deoxy (R2 = H) and 2-
alkoxy glucose derivatiYes [ i ~ e., derivatives of
30 GlcNAc where the N~c has been replac:ed by -H (deoxy
or by an -~12 (alkoxy~ ] are prepared using a
synthetic scheme similar to that rec:i~ed by Trumtez
et al . 43 Specif ically, the known 3, 4, 6-triacylated
1, 2-ortho ester of glucose is deacylated under
3S conventional conditions to give the 1, 2-ortho ester
W092/225~ PCT/CA92/00245
7 ~
-- 59 --
of glucose. This compound is then converted to the
3,4,6-tribenzyl 1,2-ortho ester of glucose using
conventional techniques. The 1,2-ortho ester of the
resulting compound is then opened by conventional
techniques to provide a protected gl~cosyl donor
such as the 1 ~-bromo-2-acetyl-3,4,6-tribenzyl
deriYati~e of glucose. This 1 ~-bromo derivative is
the~ converted to the glycoside (-OR) by
conventional techniques and the 2-acetyl group is
`10 then removed. ~he 2-position is now ready for
formation of the 2-deoxy by conventional methods
such as firs~ treating with carbon disulfide an~
methyl iodide in the presence of one equivalent of a
base to form the -C~S)SCH3 deri~ative, followed by
75 reaction with ~ributyltin hydride~ or for the
preparation of the 2-~lkoxy. The remaining
~; ~ proteating s~roups are removed æo as to prc~vide ~or
2-deoxy-glucose glycoside or a 2-alkoxygluco~;e
: glycoside which c:an then be deriva~ized in ~he
20 nlanne:r described above and illustrated in~- Figure 1
~:~ without t~e need to form the aglycon.
: :
~: ii. ~odific~tion at the 6-Po~ition of GlcN~c
m~ 6-deoxy deri~atiYe of ~lcNAc-OR is
: ~ynthe~ized from a known benzylidene ring blocked
sa~charid~ (~-methoxycarbonyloctyl-2-acetamido-4,6-
O-benzylidene-2-deoxy-~-D-glucopyranoside)~ which is
protected at the 3-hydroxy position with a removable
benzoyl blocking group (Bz) by reaction with be~zoic
: anhydride in pyridine. Further conversion of this
aompound by reaction with N-bro~osuccinimide and
barium car~onate in car~on tetrachloride ( CC14) at
65C leads to the 3,4-dibenzoyl-6-bromo-GlcNAc-OR
compound. This compound is, in turn, converted to
the 3,4-dibenzyl-6-d~oxy-GlcNAc-OR by reaction with
W092/22564 PCT/C~9~/00245
60 --
(c4Hg)3snH in the presence of AIBN ~azo bis-
isobutyronitrile~ a~ 110C followed by treatment
with me~hanol/sodium methoxide. This compound can
then be d protected by conventional techniques to
pro~ide for the 6-deoxy~lcNAc-OR glycoside which can
then be derivatized in the manner described a~ove
and illustrated in Figure 1 withou~ the n~ed to form
the aglycon.
The 6-azido derivatives of GlcNAc-OR can be
prepared in the manner described in Figure 9.
Specifically, GlcN~c-OR, compound 1~0, is converted
o the pmethoxybenzylidine blocked compound 141 by
reaCtiQn wi~h ( CH30) 2CH-C6H4-p-OCH3. This compound
is th~n protected at the 3-hydroxyl position by
reaction with 4-~H3O-C6H~CH2Br to pro~ide for
compound 1~2 where X' i~ 4-CH3O-C6H4-CH2-. Compound~
14~ is partially deprotecte~ at the 4 and 6
positions by reaction with acetic acid ~AcOH) in
water at about 45C to provide for co~pound 1~3.
:~:2~ The 6-mecylate, c~mpound 144, is prepared by
~:~; reacting compound 1~3 with mesyl chIoride in
pyridine (~sCl/py). The 6-azido derivative,
c~mpo~ d 1~5, is then formed ~y reaction with sodium
agide in dime~hylformamide (DMF) and removal of th~
3-blocking group with dichlorodicyanoquinone (DDQ)
yields comp~und 1~6.
The 6-mesyl compound ~ can also be
derivatized to any o~ a number of 6-sub tituents
including alkoxy s~bsti~uent~, and the like by well
known chemistry.
The 6-azido compound 1~5 can be derivatized to
the 6-amino at an appropriate point in the synthesis
of the ~ewisA or LewisX analogues in the manner
described above. The 6-amino deriva~ive can then be
W092/22~64 PCT/CA92/00245
2l~ 07~ ;i
-- 61 --
further functionalized by conventional me~hods to
provide for -NF~C~O)R4, -NHSO3H, -N=C(R~)2,
NHCH(R~)2, -NHR6 and -N(R6) 2. For example, the -NH2
group can be reacted, using conventional techniques,
with:
a carboxylic acid, a~hydride or chloride to
provide ~or amides. Alternatively, the de~ired acid
can be acti~ated, as report~d by Inazu et al37 and
then reacted with ~he amino group. The carboxylic
acid, anhydride, ch~oride, or activ2ted acid i~
: s~lected ~o as to provide or an R4 group ti.e. f a~
part of the ~NR~C(O)R~ substituent) which is hydrogen
or alkyl of from 1 to 4 car~on atoms,
with an aldehyde or ketone (of from 1 to 4
car~Dn ato~s) at co~troll~d p~ to form an imine
t~ (F~)23 which upon reduction (e.g.~ with sodium
cyanoborohydride) pr~vides for an alkylamine
5ub8tituent ~i.a., NHCH~R~)2] as reported by
rno~as et alO~
with a cyclic carbonate such as ethylene
carbonat~ or propylene carbonat@ which ring opens
upon reaction with the amine ~o form a ¢arbamate
group having a~ HO-alkylene-OC(O)N~ su~sti~u~nt
~ where alkylene i~ fr~m 2 to 4 car~on ato~s 2S
: 25 reported by Woll~berg e~ al.39, U.S. Pat~n~ No.
: 4,612,132,
with a chloro~onmate ~i.e., ClC(O)OR7] in the
manner disclosed by Greig et al. 40 . In this case,
the hloroPormate has an R7 group which is alkyl of
from 1 to 4 car~on atoms,
with O=C(O-C6H4-pNO2)2 which laads to an
activated intermediate which is then reacted with an
amine (HNE~F~) to provîde for ureas ~-NHC(O~NR~R~) as
described by Piekarska-Bartoszewic2 et al . 41,
W092/22564 PCT/CA92/~0245
62 --
with trimethylamine, sul~ur trioxide (S03~ at
pH 9.5 so as to form the -NHS03H group as described
by Petitou42~ and
with derivatized formic acid or other materials
S to form a formamide (NH-CHO) 43 which can b~ further
functionalized to the isocyano (-N=C=O) and reduced
to the deoxy d~rivative by tributyltin hydride
(Bu3Sn~)43.
The 6-alkoxy derivat~ves of GlcNAc c~n be
prepared in the manner descri~ed in Figure 10.
Sp~cificallyf GlcNAc-OR, compound 1~0 t is reacted
with C6H5CH(OCH3~2 in an acidic medium in
acetonitrile to provide for the 4,6-diprotected
benzylidine compound 1~7. Ih turn, compound 147 can
I5 be reacted with benzy~ (Bn) bromide and sodium
hydride in ~he presence of dLme~hyl~ormamide at
around 0C to provide for a benzyl prokec~ing group
at ~he 3 po ition, i.e., compound 148. Deprotection
at ~ e 4,6 positions by contacting co~pound 1~8 with
ac~tic acid a~d water at about 80-~O-C provides for
compound 14~. R~action of compound 1~9 with
dibutyltin oxide t~Bu~2SnO3 and R~Br pro~ide~ for the
: 6-alkoxy com~ound 150. Conven~ional deprotection of
~h~ benæyl group with hydrogan in palladium/carbon
yield~ compound 151.
In anoth@r embodiment, compound ~47 can ~P
react~d with ~C6H~C(0)320 in pyridine to provide for
a benzoyl protecting group (Bz~ at the 3-poaition,
i~e., compound 152. ~eaction of compound ~52 wi~h
N-bromosuccinimide in carbon tetrachlorid~ yields
~he 6-bromo compound 153. Compound 153 can be
reacted with tributyltin hydride ~(Buj3SnH] in
toluene to provide for the 6-deoxy compound 155
which after conventional deprotection of the benzoy
W092/22564 21 1 0 7 O ~ PCT/CA92/00245
-- 63 --
groups with sodium methoxide in methanol gives the
6-deoxy compound 156.
Th~ 6-SR6 compounds are prepared from the 6-
mesyl derivative, compound 1~4, by reaction with
potassium thioacetate, CH3C(O)S ~, to give the
thioace~ate derivative at the 6-position. This
deri~ative is then treated with mild base to produce
the 6-SH derivativ~ The 6-SH can be reacted with
an alkyl halide (e.g., CH3Br) to provide the -SR~
derivatives which, in turn, can be partially or
fully oxidized to the 6-sulfone or the 6 sulfoxide
d~rivatives, -S(O)R6 and -S(0)~ where
R6 is alkyl of from 1 to 4 carbon atoms.
c. ~L~Y
Without being limited to any theory, it is
believed that the modified L~wisX and Lewi5a
glycoside~ disclo~ed herein affect the c~ll mediated
mune response in a number of ways. Specifically,
~he~Q compounds can inhibit the ability o~ the
immune response to become edu~ated about a specific
aN~igen when the compound is adminis~ered
simultaneously with ~he first exposure of the immun~
~ystem to the antigen. Also, the modified LewisX
and Lewi glyco ide~ disclosed herein can inhibit
~he ef~ector phase of a cell-mediated i~mun~
response (eg., the inflammatory component of a DTH
re~ponse~ when administered after second or later
exposures of the iDune system to the same antigen.
A, dditionally, the modif ied LewisX and Lewis~
30 glycosides disclosed herein ran induce tolerance to
antigens when administered at the time of sacond or
later exposures of the immune syst m to the antigen.
W092/~2564 PCT/CA92/00245
- 64 --
The suppression of the inflammatory component
of the immune response by the modified ~ewisX and
~ewisa glycosides disclosed herein is ~elieved to
require the initiation of a secondary immune
response (i.e., a response to a se ond exposure to
antigen~. The modified LewisX and Lewis~ glycoside
is generally administered to the patient at least
about O.S hours after an inflammatory episode,
preferably, at least ab~ut l hour after, and most
~: 10 preferably, ~t least about 5 hours after an
inflammatory episode or exacerbation.
The modified LewisX and Lewis~ glyco~ides
disclosed h~rein are ef~ective in suppressing cell-
: mediated immune responses to an antigen (eg. the
inflammatory co~ponent of a DTH response) when~ad~inistered at a dosage range of from about O.5
to about SO mg/kg of body weight, and preferably
from about 0.5 tc ~bou~ 5 mgJkg of body weight. The
: : : speoif ic dose e~ployed is regulated 3~y the
20 particular cell-mediated im~une response ~eing
reated as well as :by the judgment of the attending
: cIinician depending upon factors such as the
seYerity of the: adverse i~amune response, the age and
g eneral condition of the patient, and ~he like. ~he
25 dified I.swis~ or I,ewi nalogue is gen2rally
dministered par~nterally, such as intranasally,
intrapulmonarily, transdermally and intravenously,
alths)ugh other forms of administration are
contemplated . Pref erably, the suppression of a
30 cell-mediated immune response t eg . the inflammatory
component of a DTH response, is reduced by at least
abou~ IO% as oppc~sed to control measurç~d 24 hours
after administration of the challenge to the mammal
and 19 hours after administration of the modif ied
35 LewisX or Lewisa glycoside as per this invention.
W092/22~64 PCT/CA92/00245
2~ 7 (~ r~;
-- 6~ --
In addition to providing supp ession of the
inflammatory component of the cell-mediated immune
response to an antigen, administration of the
modified LewisX and Lewis~ glycosides disclosed
s herein also impa ~ s a tolerance to additional
challenges from the same antigen. In this regard,
re-challenge by the ame antigen weeks after
administration of the modified LewisX or Lewisa
~lycoside results in a significantly reduced immune
response.
Administration of the modified LewisX and Lewisa
glycosides disclosed herein simultaneously with
f irst exposure to an antigen imparts suppression of
a cell-mediated immune response to the antigen and
tolera~ce to future challenges with that antigen.
In this regard the term "reducing censi~iz~tio~"
means that the modified LewisX or Lewisa glycoside,
when administered to a mammal in an ef~ect~ve amount
along with a suffieient amount of antigen to in~uce
an immune response, reduces the ability ofYth~
mune system~of the mammal to become educated and
thus sensitized~to:the antigen administered at the
same~time as the~:~odified LewisX or Lewis~ glycoside
compound. An :"effective amount" of ~his co~pound i~
:: : 25 ~ at amount which~will reduce sensitiza~ion
(immunological~education) of a mammal, including
humans, to an antigen admînistered simultaneously as
determined by a reduction in a cell-mediated
response to the antigen such as DTH responses as
~ested by the footpad challenge test. Pr~er~bly
the reduction in sensitization will be at least
a~out 20% and more preferably at least about 30% or
more. Generally, modified LewisX and Lewis~
glycoside compounds related to blood group
~5 determinants are effective in reducing sensitization
WO 92/22564 PCI/CA92/00245
6 6 --
when administPred at a dosage range of from about
O. 5 mg to about 50 mg/kg of body weight, and
preferably from about 0.5 mg to about S mg/kg of
body weight. The specific dose employed is
regulated by th~ sen~itization being treated as well
as the judgement of thOE attending clinician
depending upon the age and general condition of the
patient and the like. "Simultaneous" administration
of the compound with the antigen with regard to
i~hibiting sensitization means that the compound is
administered once or continuously throughout a
period o~ time within 3 hours of the administration
of an antigen, ~ore preferably the compound is
administered within 1 hour of the antigen.
~ The methods of thiæ invention are generally
:; achi~ved by use of a pbarmaceutical composition
uitable for use in the parenteral administration of
: an:e~fective amount of an oligosaccharide glycoside
: : related to a blood group determinant. These
compositions çomprise a pharmaceutically inert
~-: carrier such as water, buffered saline, ~tco and an
effective amount of a modified LewisX or Lewi~
~ :glycoside co~pound 50 as to provide the abo~e-noted
: ~ do~age of ~he oligosaccharide glycoside when
administered~to a patient. It i~ contemplated that
: :suitable pha~maceutical compositions can
additionally contain optional components such as an
adjuvant, a preservative, etc.
It is also contemplated that other suitable
pharmaceutical compositions can include oral
compositions, transdermal compositions or bandages
etc., which are well known in the art~
The following examples are offered to
illustrate this invention and are not to be
construed in any way as limiting the scope of this
W092/22564 P~CT/CA92/#0245
B7 jf~
-- 67 --
inventionO Unlfess otherwise stated, all temperatures
are in degrees Celsius. Also, in these example~,
unless otherwise defined bfelow, the abbreviations
femployed have their generally accepted meaning:
s A = ~ngstroms
AB - AB pattern
ax = axial
bs = broad singlet
BSA = ~fofvine serum albumin
~10 13C-n.m.r = C'''3 nuclear magnetic resonance
~ = doublet
dd = doublet of doubflets
ddd = doublet of doublets of doublffPts
DDQ = dichlorodicyano~uinone
15 DTH = fdelayed-typfe hypfersensitivity
eq = efquatorial
: f~ = gram
r~-n.m.r. = proton nufclear magnetic resonance
i.r. f- infra red
: 20 kg = kilogram
L = liter
m - muItiplet
mL - milliliter
~ a ~uartet
: 25 s = singlet
t - triplet
t.l.c. = thin layer chromatofgraphy
U = Unitfc
fum = microns
:
AG 1 ~ 8 ~ormate form) = ion exchanffge resin AG 1 x
8 (formate form) available from Bio-Rad
Lahoratoriffes, Richmond, '{~A
Dowex SOW x B (H~ form~ = ion exchange resin Dowex
f 50 x 8 (H~ form) available from Df~W
Chemical, Midland, MI
IR-120 resin (H~ form) - amberlite resin a~fallable
from Rohm f~ Haas, Philadelphia, PA
IR-C50 resin (H~ form3 = ion exchange resin IR-C50
(H~ form) available from Rohm & Haas,
Philadelphia, PA
WO 92/22~i64 r. ~ PCI'/CA92/00~45
~8 --
Commercial~y availa~le components are listed by
manufacturer and~where appropriate, the order
number. Some of the recited manufacturers are as
follow~:
S Merck = E. ~erck AG, Darmstadt, Germany
Millipore 3 Millipore Corp., Bedford, MA.
Waters = Waters Associates, Inc., Milford, MA.
The following examples are divided into two
parts. The first par* (part I) relates to the
~ynthetic proceduras to make the recited compounds
whereas the second part (part II) relates to the
biologica} results.
Part I -- Synthetic Procedures
~ Examples 1-24 illustrate syntheses of the
:: lS described compounds.
EXAMPLE 1 - Syn~hesis of Benzyl-2-0-benzoyl-4,6-
0-benzylidene-3-0-chloroacetyl-~-D-
thiogalactopyranoside (compound 31~
:: Dry a 20 L stirred reactor equipped with reflux
condenser, heating mantle and 1 L addition funnel.
Charge to thi. reactor 10 L of dichloroethane. Begin
to stir the reactor then charge 1 kg D-g~lactose and
500 g anhydrous sodium acetate to the
d1chloroethane. Heat this slurry to reflux. Add
~5 dropwise 4 L of acetic anhydride to he rea~tion
mixture using the 1 L addition funnel on the
reactor. Reflux is to ~e maintained during the 2-4
W092/22~64 PCT/CA92tO0245
-- 69 _~ 7~ j
hour addition period. Continue to stir and heat the
mixture at reflux overnight.
When the reac~ion is complete as determined by
t.l.c., t~rn off the heat to the reactor a~d add 250
mL of water in slow dropwise fashion using the
addition funnel. ThiC ~eaction is quite vigorous
but is controlled by slowing the addition of the
water. Stir the reaction for 1-2 hours. Charge 30
L of cold water ~o a 50 L stirred reactor and begin
~0 stirxing. Drain the co~tents of the 20 L reactor
into a 20 ~ polyethylene pail and pour into the
~tirring ice water in the 50 ~ react~r. Stir this
mixture for twenty minutes. Drain the lower organic
layer into a 20 L polyethylene pail. Extract the
agu~ous layer in the 50 L r~actor with an additional
5 L of dichloro~ethane. ~ombine the dichlorome*hane
extraet with the first organic layer. Drain th~
agueous la~er to polyethylene pails and discard as
a~ueou~ waste.
Return the combined organic layers to the 50 L
reactor and extract t~ice with S L portions of ic~
water for 10 ~inutes. Drain th~ organic layer to a
clean 20 L polyethylene pail. Drain the a~u~ous to
wa~tP, return the organic layer to the 50 L reactor,
stir and add 1 kg of anhydrous sodium sul~ate. Stir
for 1-2 hours ~nd then drain the ~olution into a
clean 20 L polyethylene pail and ~ilter the solution
using a 4 L ~acuum filtration ~et ~or large Buchner
attached to a collector~.
Concentrate the filtrate to 8 L then ~ransfer
into a cl~an 20 L reactor equipped with stirrer, 1 L
addition funnel and cooling bath. Additional
solvent can be added if the level of the solutiDn is
below the thermowell. Cool the organic solution to
0 G C using a cooling bath. Charge to this cool
W092/22~64 ~ PCT/CA92/00245
- 70 --
solution 724 g of benzyl mercaptan. Add a total of
1.1 L of colorless boron trifluoride etherate in
slow dropwise fashion over 2 hsurs using the 1 L
addition funnel. Stir the reaction 3-4 hours after
S the addition is complete maintaining the temperature
at 0C. The reaction is checked for comp}etion by
t.l~c. on silica gel. ~The reaction can be left to
~it ov8rnight].
The reaction mixture is drained into a clean 20
L polyethylene pail. The 50 L reac~or is charged
wikh lS L of fiaturated sodium carbonate solution.
T~e 20 L polyethylene pail is slowly transferred
into the slowly stirring ~arbonate solution at such
a rate that the gas evolution is not ~verly
lS vigorous. Stir the ~olu~ion for 20 minutss then
increase the rate of stirring. When ga~ evolution -
~C . S88 bubble air through the entire ~olution for
~-36 hours.~ Drain ~he organic layer into a clean
20 L polyethylene pail and store in a hood. Extract
the sodium carbonate solution with 3-5 L of
dichloromethane and :drain this solution into the
same 20 L polyethylene pail.
Once th~ ~mell has be~n reduced the organic
~olution can be filt~red u ing a 4 L vacuum
~ltration set and the filtrate evaporated under
: r~duced pressure on the 20 L rotovap. 7 L of
methanol is introduced into the rotavap flask and
the residue heated with the rotavap bath till the
re~idue dissolves in the warm methanol. The flask
is rotated and allowed to cool. Cool ice water is
added to the ro avap bath and t~ flask slowly
rotated for several hours. The flask is removed
from the rotovap and ~he white crystalline product
filtered using a 4 L vacuum filtration set.
W092/225~4 PCT/CA92fO0245
-- 7 1 -- 2 f ~ O ^.~
The benzyl 2,3,4,6-tetra-0-acetyl-B-D-
thiogalactopyranoside (-1.3 kg~ is charged into a
clean dry 20 L reactor with stirring motor and 7 L
of dry methanol is added to dissolve the material.
The solution is treated with 3 g of freshly surfaced
sodium and stirred for two hours. The reaction is
checked by t.l.c~ on silica gel using a retained
~ample of the benzyl 2,3,4,6-tetra-0-acetyl-B-D-
thiogalactopyranoside with 80:20 ethyl acetate:
m~thanol (v/v) the eluant. The absence of starting
material indicates the reac~ion is compl~te.
50 g of fresh methanol washed H~ ion exchange
resin is added, the reac on stirred for 15 minutes.
The pH is ch~cked using pH paper to ensure a ne~tral
~: 15 .olution. The r~sin is filtered off under r~duced
prassure and the methanol is remov~d under reduced ^
pr~s~ure using the 20L roto~ap. To the r~sidue, 5 L
o~ aceton~ is added to the 20 ~ flask and the
solution warmed to reflux. The residue dis~ol~es
and is allowed to cool to room temperatur~at which
::~ tim~ ice is added to the bath, the solution ro~at~d
with cooling ov@rnight. 800-9OOg o~ benzyl B-D-
thiogalactopyr noside cry~tallizes and is filt~red
and dried under va~uum.
To 8 L of dry acetonitrile is added 800 g of
benzyl B-D-galactopyrano~e, 600 g of ~enzaldehyde
: dimethyl acetal and 2-5 g of p-toluenesulphonic
acid. The solution is stirred at room temperature
ove ~ ight. The reaction pro~ress is checked by
t.l.c. When complete, the reaction is brought to pH
7 by ~he addition of triethy}amine. The volume of
acetonitrile is reduced to a minimum, 7 L of
isopropanol is added and the mixture is heated to
near reflux. Most of the product gQeS into the hot
isopropanol after ~arming for several hours. The
W092/22564 PCTlCA92/00245
- 72 --
mixture is cooled and ice added to the bath and
cooling continued overnight to give a precipitate.
After filtering and drying the precipitate, 760 g of
benzyl-4,6-O-benzylidene-B-D-thiogalactopyranoside
S is obtained.
: 180 g of benzyl-4,6-O-benzylidene-~-D-
thiogalactopyranoside was dissol~ed in dry DMF and
placed in a jacketed reactor. The reactor was
: cooled using a recirculating cooling bath maintained
~10 at a temperature of -25-C and treated dropwise with
108 g of chloroacetyl chloride over 3 hours while
stirring:the:reaction mixture. Stirring was
continued 24 hours at this temperature then the
reaction was quenched into se~eral vo}umes of cold
bicarbonate solution. The product was extracted
into methylene chloride~ water washed several times,
dried over sodium~:sulphate and evaporat d to
: dryness. The product was crystallized from
isopropanol. Yield: lZ5 g of benzyl 4,6-O-
20~ enzyl:idene-3-O-chloroacetyl-B-~-thiogalac~o-
pyranoside. ~
S g Benzyl~4,6-O-benzylide~e-3-O-chloroacetyl-
,~:
:B-D-thiogalac~opyranoside was benzoylated at room
temperature~in mothylene chloride/pyridine using 3
e~ui~alents ~benzoyl chloride and a catalytic
amount of dimethylaminopyridine. The solution is
quenched into cold sodium bicarbonate solution, the
organic layer is washed with saturated copper
sulphate solution to remoYe the pyridine the organic
layer dried and evaporated. Th~ residue is taken up
in hot isopropanol~ and benzyl 4,6-O-benzylidene-2 O-
bénzoyl-3-O-chloroacetyl-B-D-thiogalactopyranoside
crystallizes from solution. 1H-n.m.r. (CDCl3): ~ =
7.96, 7.4 (2m, lSH, aromatic, 5.79 (t~ lHr H 2~1 5.5
(s, lH, CH), 5.2 (q, lH, H-4, J23 9 9 HZr J3,4 3-3
W092/225~ PCT/CA92/00245
__ 73 __ 2 ~
Hz), 4.5 ~m, 2H), 4.4 (d, lH), 3.99 (m, 5H), 3.~5
~s, lH).
Example 2 -- Synthesis of 4,6-O-benzylidene-2,3-
di-O-benzoyl-B-D~galactopyranosyl
bromide (compound 32A)
Benzyl-4,6-O-benzylidene ~-D-thiogalacto-
pyranoside (10 g) was dissolved in 100 mL
dichloromethane and 6.35 g of pyridine was added.
To the solution was added 9 g o~ benzoyl chloride in
dropwi~e fashion and after 1 hour, 50 mg of
dimethylaminopyridine was added to the solution and
the mix~ure was stirred for an addition 2 to 4
hours. The progress of the reaction was checked by
; to l-c- on silica gel. Benzyl-~,6-O benzylidene-2,3-
di-O-benzoyl-~-D-thiogalactopyranoside (compound 3O
waæ i~olat~d by ~uenching the reaction mixture into
~ ~,
satura~ed sodium bicar~onate solution and wa~hing
the organic extract with water, 5% copper sulfate
solution, water, drying and e~apoxating the solvent.
: ~ 20 The residue was crystallized from isopropanol ~o
~ ~ give 10.7 g of compound 32.
: Compound 32, benzyl-4,6-O-b~nzylidene-2,3-di-O
::~ benzoyl-~-D-thiogalactopyranoside (~.89 g), was
di$solved i:n lOO ~L of dichloromethane, cooled to
O~C, and treated wi ~ a solution of bromine (2.85 g)
in 10 mL of dichloromethan After 15 minutes, 1.8
grams of tetraethylammonium bromide was added to the
mixtur~ and the mix~ure stirred for 2-3 hours at
room t~mperature (~ollowed by t.l.c. on silica gel).
30 A smal l quantity o~ cyclohexane was added to ~uench
excess bromine and the reaction mixture wa~ quenched
into cold saturate sodium bicarbona~e solution,
washed with water, dried and volume of the solution
reduced to 30 mL. This dichloromethane solution of
3~ compound 32a was used directly in the synthesis of
W092/22~64 PCT/CA92/OQ24~
?~ G1 74
compound 42 without further isolation and/or
purification.
Examp}e 3 -- Synthesis of p-Chlorophenyl 2,3,4-
tri-O-benzyl-~-L thiofucopyranoside
S (compound 20)
Dry a 2 L thr~e neck round bottomed flask,
r~flux condenser and 500 mL addition funnel. Then
cool under a fIow of nitrogen. Charge to the flask
1000 g of L-fucose, 500 g of anhydrous sodium
acatate and 800 mL of dxy dichloroethane. ~eat the
mixture with stirring to 50-C. Charge to the
addition fu~nel 400 mL of acetic anhydride. Add t~e
ace~ic anhydrid~ to the stirring, warm ~ 50 o _55 o C)
: ~lu~ry in dropwi e fa.hion at a rate that does not
: 15 cool the reaction appreciably. Upon completion of
; the additiv~ stir the mixture for 72 hours at thi~
t~mp~rature, removi~g aliquots from the reaction
mixture ev~ry 24 hours to check the progr~s of the
r~action by t.l.c~
: :~ 20 Wh~n the reaction appears to be complete add
200 m~ of water to he warm stirring mixture
dropwi~ over 30 min. and stir for 1 hour at this
: temper~ure. This converts the remaining acetic
an~ydride to acetic acid. The reaction mixture is
quenched int~ 3-4 volumes of water. T~e organic
layer is removed and the aqueous layer is extr~cted
with 4 L dichloromethane. The combined orga~ic
: layers are backwashed three times with 2 L portions
of wat~r~ The organic layers ara dried o~er sodium
sulphate and concentrated under reduced pressure to
approximately 5 L.
To the organic layer is added 925 g of
p-chlorothiophenol. The organic layer is cooled
with cold water. To the mixture of p-chloro-
W092/22~64 PCT/CA92/00245
2 ~ 7 (~
-- 75
thiophenol and fucose acetates is added 1.72 kg of
boxon trifluoride etherate in dropwise fashion. The
mixture is then stirred for 6 hours (overnight is
~cceptable~ allowing the reaction mixture to come to
S ambient temperature. A small aliquot is rem~ved
from the rea~tion mixture and quenched into sodium
bicarbonate solution. Once CO2 evolution has ceased,
the reaction is checked for completion by t.l.c. If
complete, the whole reac~ion mixture is quench2d
into 1 L of satura~ed sodium bicarbonate and the
organic layer separated after CO2 evolution has
fini~hed. The organic layer is separated and air
bubbled through this layer for 1 hour.
me separated organic layer is then dried over
sodium sulphate and eYaporated to dryness. The
; re~i~ue is;taken up in 1 L of dry methanol in a 2 L^
round ~ottom flask and treated with 1 g of freshly
~ su~faced sodium. The reaction i~ kept under
:~ nitrogen for se~eral hours then checked by t.l.c.
2~ for r moval of the acetate groups. The reaction is
ne~tralized with H~ ion exchange r~sin and filtered
and evaporated under reduced pr~ssure. The re~idue
is taken up in a minim ~ of hot isobuta~ol and the
: p-chloroph~nyl-8-~-thiofucopyranQside crystallizes
from solution aft~r cooling overnight at O-C.
~: Yield: 1060 g.
: p-Chlorophenyl-B-L-~hiofucopyranoside is
dissolved in 7 L of dry dimethylsulphoxide. To the .
solution is added 600 g of powdered KOH and the
reaction mixture tirred for 30 minutes. Benzyl
chloride (1.275 L) is added dropwise to the stirring
solution and the mixture stirred o~ernight at room
temperature. T.l.c. indicates incomplete reaction
so an additional 300 g of powdared KOH is added to
the reaction mixture followed 30 minutes later by
W092/22564 PCT/CA92/00245
~ 91 -- 76 --
425 mL of benzyl chloride. The solution is stirred
at room temperature until t.l.c. indicates the
reaction is complete. If the reaction is not
c~mplete after 24 hours, powdered KOH is added
followed by 200 mL of benzyl chloride. The reaction
is quenched in~o several volumes of water, extracted
with methylene chloride, backwashed ~wice with
wat~r, dried and evaporated. The residue is taken
up in hot hexanes. p-Chlorophenyl-2,3,4-tri-O-
~enzyl-~-L-thiofu o-pyranoside crystallizes and is
filtered and dried under vacuum. Yield: 1.3 kg.
H-n.m.r. (CDCl3): ~ = 7.57 (m, l9H, aromatic), 4.99
(d, lH), 4.65 (m, SH), 4.55 (d, lH, J12 9 5 Hz)~
3.9B ~t, 1~), 3.55 ~m, 3H), 1.26 (d, 3~ Js6 6-2
~ 6).
Example 4 -- Synthesi~ of 8-methoxycarbonyloctyl-
2-acetamido-4,6-di-O-benzylidene 2-
deoxy-B-D-glucopyranoside
Compound 5)
A 20L glass reactor was charged with 8 L of
dichloroethane, 1 L of acetic anhydride and 1 kg of
anhydrous ~odium acetate. To the skirring mixture
Was added 1 kg of glucosamine hydrochloride and the
~ixture was brought to reflux. ~ further 3.5 L of
acetic anhydride~was added dropwise to the refluxing
solution over 3-4 hours and the solution maintained
at reflux for 36 hours. During the last hour of
re~lux 200 mL of water was added dropwise to the
solution. The reaction was ~hen cooled and added to
35 L o~ ice water in a 50 L stirr d reactor. The
organic layer was removed and then water washed a
second time with an additional 20 L of water. The
organic layer was dried over sodium sulphate,
filtered, and saturated with anhydrous ~aseous HCl
WO 92/22S64 ~ 1 L 0 7 o r;
~~ 7 7 ~~
for 2 hours. The reaction was allowed to sit for 6
days being saturated with ~Cl for 1 hour every
second day. 2-acetamido-2-deoxy-3,4,6-tri-0-acetyl-
B-D-glucopyranosyl chloridc was isolated by
S quenching into ice cold sodium bicarbonate solution.
The organic layer was dried over sodium sulphate and
evaporated to a brown solid.
: Four hundred grams of 2-acetamido-2-deoxy-
3,4,6-tri-O~acetyl-B-D-glucopyranosyl chloride was
dissolved in 2 L of anhydrous dichloromethane
~ containing 200 g of activated molecular sieves. 266
: g of 8-methoxycarbonyloctanol was charged to the
reaction mixture along with 317 g of mercuric
cyanide. The solution was stirred rapidly at room
~: 15 temperature ~or 24 hours. A ter checking for
~: rea~tion completion by t.l.c. ~he reaction mixture ^
was filtered through a buchner funnel of silica and
the organic lay~r~washed twic~ with water, twice
wi~h a 5% ~olution of potassium iodide and twice
20:~: with a satura~ed solution of sodium bica~bonate.
The soIutio~ was dried over sodium sulphate and
~: ~ :ev~porated to dryne s.~ The residue was taken up in
anhydrous methanol and treated with 1 g of freshly
cut sodium t~n stirred at roo~ temperature
~ 25 o~ernight. The solution of 8-methoxycarbonyloc~yl
;;~ 2-acetamido-2-deoxy-B-D-gl~copyranoside was
neutralized with~acid ion exchange resin and
, . filtered and evaporated to yield 218 ~ o~ product
: after crystallization from isopropanol /diisopropyl
ether.
Two hundred grams of 8-me~oxy arbonyloctyl 2-
acetamido-2-deoxy-~-D-glucopyranoside was dissolved
in 1.2 L of anhydrous dimethylformamide and treated
with 169 mL of dimethoxytoluene (benzylaldehyde
dimethyl acetal) and 1-2 g of p toluenesulphonic
W092/22564 PCT/CA92/00245
~r`~ ~~ 7 B ~~
acid. The reaction was stirred and heated to 40C
for 5 hours, then chec~ed for completion by t.l.c.
When the reaction appears complete the mixture was
neutralized with triethylamine and quenched into
S several volume~ of ice water, extracted into
dichlorome~hane and backwashed several times with
water. The organic layer was dried over sadium
~ulphate, evaporated to dryness and taken up in hot
isopropanol. After cooling 8-me~hoxycar~onyloctyl-
2-acetamido~4,6-O benzyliden~-2-deoxy B-D-
glucopyranoside precipitates. It is filtered and
dried to yi~ld 106 g of product. 1H-n.m.r. (CDCl3):
~ = 7.41 ~m,SH, aromatic), 6.11 (d, lH, NH), 5.5 (s,
lH, C~), 4.63 (d~ lH, H l, J-2 7.4 Hz), 2.29 (t,
~5 2H~, 1.99 ~s, 3H, Ac), 1.58 Im, 4H), 1~29 (bs, 8H).
..
: ~xample 5 -- Synthesis of 8-Methoxycarbonyloctyl-
2 acetamido-3-O-p-methoxyb~nzyl~4,6-
O benzylidene-B-D~glucopyranoside
~co~pound ~).
To a stirred solution of compound 5 (17.5 g, -3
mmol) in dry dichloromethane (100 mL~ and catalytic
amount of p-toluen sulfonic acid (0.2~ to 3 weight
perc~nt ba5e~ on compound S~ was added dropwi~e a
solution of p-~ethoxybenzyl trichloroacetimide (10 g
Z5 in 25 mL CH2C12). The reaction ~ixture was stirred
at room temperature overnight. Triethylamine was
added to quench ~he reaction, the organic layer was
washed with sodium bicarbonate solution and the
organic layer dried and evaporated to dryness;
Crystallization in hot ethanol gave 20 g of the
desired product. 1H-n.m.r. (CDCl3): ~ 7~56-6.90 (m,
9H, aromatic~, 5.60 (d, lH, NH), 5.3~ (s, lH, PhCH),
4.94 (d~ lH~ J12 8.0Hz, H-1), 3.80 (s, 3H, CH3),
WO 9~/22564 n PCr/CA92/00245
~1~o~
-- 79 --
3.60 (s, 3H, CH3Ph), 2.30 (t, 2H, CH2C0), 1.90 (s,
3H, AcNH), 1.80-1.10 (m, 12H, (CH2)b).
Example 6 -- Syn~hesis of 8-~ethoxycarbonyloctyl-
2-acetamido-2-deoxy-3-0-p
methoxybenzyl-Ç-0-benzyl-2-B-D-
glucopyranoside (compound 7~
To a stirred solution of compound 6 (15.0 g, -3
mmol) in 200 m~ of dry T~F were added, 11.0 g of
sodium cyanoborohydride, 10 g of molecular sieves 4A
and 5 mg of methyl ora~ge~ The solution w~s cooled
to -lO^C and then ethereal hydrochloric acid was
added dropwîse un~il the solution remain~d acidic.
on completion of the r~action, i~ was diluted with
:; dichlorome ~ ane ~200 mL), filtered through celite
lS~ and washed successive~y with aqueous sodium
bicarbonate (2 x 100 mL) and water ~2 x 100 mL) and
t~en the solvent dried and evaporated to give a
yrup. Purification of the mixture on column
chroma~ography using silica gel as adsorbent and
eluting with hexane:ethyl acetate:ethanol ~20:10:1)
~ave 7~ in 70% yield. 1H-n.m.r. (CDCl3~: ~ 7.40-
6.90(m, 9H~ aromaticj, 5.70(d, lH, NH), 4.64(d, 1~,
J12 8.0Hz, H-l), 3.86(s, 3H, CH30~, 3.68~s, 3Hp
C~30Ph), 2.30(t, 2~, CH2CO), 1~90~s, 3H, N~Ac), 1.80-
l~lO(m, 12~, t~H2)s)-
pl~ 7 -- Synthesis of 8-Methoxycarbonyloctyl
2-acetamido-2-deoxy-3-0-p-
methoxybenzyl-4-0-~4,6-o-benzylidene-
2~3-0-dibenzoyl-B-D-
galactopyranosyl)~6-0-benzyl-2-deoxy-
~-D-glucopyranoside (compound 42)
A solution of compound 7 (10.61 g, 19.7 mmol)
and compound 32A (1.6-1.7 equivalents based on
W092/225~ PCT/CA92/00245
80 --
compound 7~ and 2,6-di-t-butyl-4-methyl pyridine
(3.11 g, 15~2 mmol) in 250 L of dichloromethane and
40 g of molecular sieves (4A) was stirred at room
temperature for 30 minutes, and then coole~ to -50~C
under nitrogen. A dry so$ution of silver triflate
(4.47 g, 17.3 mmol) in toluene (40 mL) was added to
the stirred mixture. Th~ mixture was warmed ~o -
15-C during two hours and kept at -15-C for an
: additional 5 hours. At the end of which the mixture
~ ~o w~s warmed to room temperature and stirred
: : overnight. 3 mL of pyridine and 250 mL of
dichloromethane~were added to the mixture and was
filtered over celite, filtrate was washed with
saturated aqueouC sodium hydrogen carbonate (200 mL)
and then with water (200 m~), aqueous hydrogen
chloride (0.5N, 200 mL) and water (200 m~
` concentrated in vacuo. 6.0 g of compound ~ was
crystallized as white crystals from ethyl acetate-
diethyl ether-hexane. ;The moth~r liquor was
; :20 concentrated, purified over chromatography-(300 g
silica gel, toluene:ethyl acetate, (1:1) to gi~e 4.5
; g pure compound~:~2.~ Total yield was 10.5 g (68%).
Rf~0.~48 (methanol~:dichloromethane, 4:96). ~H-n.m.r.
(CDCl3]: ~ 5.80(t,~1H,~J2 3 ll.O~z, H-2 ), 5.S2(~,
; 25 lN~, CNPhj, 5.25(dd,:1H, J3~ 4.0Hz, ~-3'), 4.88(d,
~ : : lH, Jl 2 ll.OEz,~H-l ), 4.70(d, lH, J12 9 OHz~ ~~
:5~ ~ 1), 3.78~s, 2~,~CH30j, 3.64(s, 3H, CH30Ph).
~: ,.
Example 8 -- Synthesis of 8-Methoxycarbonyloctyl
:- 2-ace~amido-4-0-~4,6-0-benzylidene-
2 3 -di-O-benzoyl-B-D-yalacto-
: pyranosyl)-6-0-benzyl-2-deoxy-B-D-
glucopyranoside (compound 43)
DDQ (126 mg, 0.5 mmol) was added to a stirred
~ solution of compound ~2 (350 mg, 185 ~mol~ in
:; 35 dichloromethane (10 mL) saturated with water. After
W092/22564 2 l 1 ~ 7 o ~ PCT/CA92~00245
-- 81 --
2 hours at room temperature, the reaction was
complete, and organic layer was successi~ely washed
with aqu~ous sodium bicarbonate and water, dried and
concentrated. Col~mn chromatography gave the
desired compound ~3 in 85~ yield. 1~-n.m.r. (CDCl3~:
~ 5.65(dd, lH, J2 3- 10.8Hæ, H-2 ), 5.61(d, lH,
33 $ 4.0Hz, H-3 ), 4.68~d, lH, Jl 2- ll.OHz, H~
4.62(d, lH, Jl2 lOHz, H-l), 3.60(s, 3H, COOCH3).
Example 9 - Synthesis of 8-methoxycarbonyloctyl
2-ac~tamido-3-0-~2,3,4-tri-o-benzyl-
-fucopyranosyl)-4-0-(4~6-0-
benzylidene-2,3-di-o-benzoyl-B-D-
galactopyranosyl)-6-o-benzyl-2-deoxy-
B-D-glucopyr~no~ide~cQmpound 44)
~5 ~o a mixture o~ copper (cupric) bromide (40 g,^
17.~ ~mol) and 5 g of molecular sieves 4A in lo mL
sf dry dichloromethane were added 1.2 mL of dry DMF
and tetraethylammonium bromide (1.85 g, 8.8 mmol).
The ~ixture was stirred at room temperature for 1
hour and th~n a sclution of compound 43 (5.0 g, 5.7S
mmol) and the thiofucoside 20 (7.5 g, 11.8 mmol) in
30 ~L dry dichloromethane was adde~ dropwise at 0-C
fox 30 minutes. The mixture was stixred at room
te~p~rature for 48 hours, at t~e end of which time 5
mL o~ methanol wa~ added and tirred for 30 minutes.
Further, 3 mL of pyridine, 100 mL of ethyl acetate
and 100 mL of toluene were zdded to the reaction
mixture. The mixture was filtered over celite pad
and the ~olvent evaporated to give a brown syrup.
Purification over column chromatography with silica
gél and eluted with toluene:ethyl acetate (2:1) gave
the compound 4~ in 86% yield. 1H-n.m.r. (CDCl3):
~ 5.BO(dd, lH, J2 3- ll.OHz, H-2 ~, 5.60(s, lH,
W092~22564 PCT/CA92/00245
` 9 ~ 82 --
CHPh), 5.50~d, lH, NH), 5.10(dd, lH, 33 4. 4.0Hz,
H-3 ), 3.60~s, 3H, OCH3~ 1.20(d, 3H, CH3, ucose)O
Example 10 -- Synthesis of 8-methoxycarbonyloctyl
2-acetamido-3~0-(2,3,4,-tri-D-benzyl-
~-L-fucopyranosyl~-4-0-(4,6-O~
benzylidene-B-D-galactopyranosyl)-6-
O-benzyl-2-deoxy-B-D-glucopyranoside
(compou~d 45~
Compound 44 (200mg) was treated with 20 mL of
sodium methoxide in methanol. After 3 hours, t.l.cO
(tolu~ne-ethyl acetate, 1:1) indicated the
disappaarance of the star ing material and the
: appearance of a slower movin~ spot. The solution was
neutralized with ~Qrli~e re~;in IR-120 H~ and th~
:; 15 solvent evaporated under reduced pressure to gi~e a
uantitative yield of crude compound 45. ~he
product wa~ purif ied on silica gel using toluene-
et~yl acetate (2 : 1) as ~luant. ~H-n.m. r. (CDCl3): ~
7.15-7.55 (aro~atic, 25H), 5.62 ~d, lH,N~I), 5.58 (s,
lH, ~I benzylidene), 5.06(d, lH, J1.. ;2u 7.0Hz9 H-1"3,
4O95 ~d, l~t Jl~,2~: 3.8Hz, ~ ) 4.85 ~d, 1~,
Jl 2=9.0~1z, H ) 3.62 ~s, 3~, COO~I3) 1.O(d, 2H,
Fu~-C~3)
~:
:E5xample 11 -- Synthesis of B-methoxycarbonyloctyl
2-acetamido-3-0- ~oL-fucopyrano~yl) -
4-0- ( 3-O-slllphate-B-D-galacto
pyranosyl ) 2-deoxy-B-D-gluc:o-
pyranoside ( compound 47 )
Diol (100 mg -- compound 45~ was dissolved in
39 5mL dry dimethylformamid~. Pyridine: sulfur trioxide
complex ( 120 mg) was addPd to the solution and the
reaction mixture stirred ar room temperature for 1
hour. The reaction was followed by t.l.c. to
monitor the disappearance of the diol (Rf =0.28 in
W092/22564 ~ ~ O PCT/CA92/0024
- 83 --
EtO~c: MeOH 80:20). Solvent was evaporated to
dryness and taken up in 50mL of methanol then
treated with Na' resin to convert it to the sodium
~alt. Purification by column chromatography on
silica gel gave 65 mg of compound ~6 which was
immediately hydrogenated with 10% Pd(OH)z on carbon
to give 35 mg of compound 47. 13C-n.m.r. (DzO): ~
103.94(C-l, Gal), 103.4~(Cl, GlcNAc), 101.07(C-1,
Fuc~, 82.7(C-3, Gal), 63.83~C-6, Gal), 62.~(C~,
GlcNAc), 54.55(C-N, Glc~Ac), 17.75(C6-Fuc).
Example 12 -- Synthesis of 2-0-benzoyl-4 9 6-0-
b~nzylidene-3-0-chloroacetyl-~-D
galactopyranosyl bromide (compound
~ 3)
: 15 C~mpo~nd 32, benzyl 4,6-0-benzylid~ne-2-0-
benzQyl-3-chloroacet~ D-thiogalactopyranoside
: (8.87 g) wa di~olvad in 100 mL of dichloromethane,
~ ~ co~led to O-C and ~reated with a ~olution.of br~mine
: ~ t 2 . 7 g j in 10 mL of dichlorome~hane. A~ter 15
minutes, 1.7 g o~ tetraethy~ammonium bromide was
add~d to the mixture and th mixture ~tirred for 2
to 3 h~urs at room temperature (follo~ed by t.l.c.
on silica gel). A small quantity of cyclohexene was
added to ~uench excess bromine and the reaction
: 25 ~ixture was quenched into cDld saturate sodium
bicarbonate solution, washed with water, dried, and
the volume of the solution reduced to 30 mL so as to
provide a dichloromethane solution of compound 330
This solution was used directly in the synthesis of
com~ound 38.
W09~/22564 PCT~CA92/00245
84 --
Example 13 -- Synthesis o* 8-methoxycarbonyloctyl
2-acetamido-4-0-(2'-o-benzoyl-4',6'-
O~benzylidena-3'-0-chloroacetyl-~-D-
galactopyranosyl)-6-o-benzyl-2-deoxy-
3 0-p-m~thoxybenzyl-B-D-gluco-
pyranosid~ ~compound 37)
A solution of the compound 7 (5.0 g, 0.9 mmol~
and compound 33 (1.4 to 1.5 equivalents -- from
example 12) and 2,6-di t~butyl-4-methyl pyridine
(1.78 g, 1.0 ~mol~ in 50 mL of dichloromethane and
20 g of molecular sieves ~4A) was stirred at room
tQmperature for 30 minutes, and then cooled to -50'C
und~r nitrogen. A dry solution of silver triflate
: (3.3 g~ 1.5 mLj in toluena (10 mL) was added to the
stirred ml~ture. The miXture was warmed to -15-C
over two hour~ and kept at -15~C for an additional
5 hour~, then allo~ed ~o warm to roo~ temperature
and stirred overni~ht. 1 mL of pyridine and 100 mL
of dichlorc)~thane ~were ~dd~d to the mixture and it
2 0 w~æ filtered o~er c:elite, the filtrate wa~ washed
wlt~ a~eous sodium bicarbonate ( 100 mL) and then
with water ( 10~0 mL), aqueous hydrogen chloride ( 0 O 5
N , 100 mI-) and water ( 100 ~L~, then cor~centrated in
vac:uo~ Purii~lcation of the crude mixture on colu~
25 chromatography WIth silica gel as adso~ent eluted
with h~xane:~yl :ac~tate (1:1) gave 5.2 g of pure
ompound 37. lH ~mOr. ~C~DCl3) ~ 85(d, lh, NH),
5.62(t, lH, 32 3 10.8Hz, H-2 3, 5~52ts, lH-C:H
benzylidene~, 5.0B ~dd, lH, J3. ,~ 4H2:r H-3 ) ~ 4~85
3~ (d, lH, J1 2 11.0Hz, H-l ), 4.Ç8 (d, lH, Jl 2 9.0Hz,
:: H 1), 3~72 and 3.64 (2s, 6~, OCH3 and C:OOCH3); 13c-
n .m~ r~: 15g ~ 0 (aromatic c-p-methoxyl ) 165 . 15 ~c:~0 ,
chloroacetyl) ~ 167 . 12 (c-o, acetyl), 174 . 2 ~c=0,
COQC~13), 99.64(c 1), 100.26(c-1 ), 101.0(PhCH).
W092/22564 PCT/CA92/0024
21-i~7~
-- 85 --
Compound 37 was then treated with DDQ in th~
same manner as Example 8 to give co~pound 38 in near
quantitative yields.
Example 14 -- Synthesi~ of 8~methoxycarbonyloctyl
S 2-acetamido-3-0-(2,3,4-tri-0-benzyl-
~-L-fucopyranosyl-4-0-(2-0-benz~yl-
4,6-0-benzylidene-3-0-chloroacetyl-B-
D-galactopyranosyl)-6-0-benzyl-2
deoxy-B-D glucopyranoside (compound
~10 39)
Thiofucoside 20 (4 g) was stirred in dry
dichloromethane ~50mL) and bromine (0.60g) wa~
added. The mixture was cooled to -~0-C~ The
conversion to the bromide was complete in 1 hour and
th~ reaction mixtur~ was washed with cold a~ueous
sodiu~ bicaEbonate, dried and concentrated to lOmh
and syringed i~to a fla~k cDntaining the alcohol 3
: (2.97 g, 3~56 mmol), CuBr2 (2.39 g)~ tetraethyl
ammonium bromide (2.24 g), molecular ~ieves 4 A (4g)
:in dimethylfonmamide (1 mL) in dry dichloxomethane
(75 mL). The mixture wa stirred at room
te~p~rature for 48 hours after which the t~l.c~
: .ho~ed the disappearanc~ of ~h~ alcohol 38 and a
~s~er ~oving ~pot ~f 0.5S -- toluene:e~yl acetate
2:1). ~fter the usual work up, th~ crude mixture
was purified ~y colu~n chromatography to gi~e
compo~nd 39 (4~2 g, about 80% yield). 1H-n.m.r.
(CDC13): ~ 7.1-8.0 ~m,aromatic-30H), 5.53, 5.61 (m~
2~, NH and ~H-benzylidene, overlapping), 5~56 (d,
1~, J1u2" 7.0 Hz, H~ , 4.98 (d, lH, J12 8.0 Hz, H-
1), 4.95 (d, lH, J~2~ 3~ Hz, H-1'), 3.6S (s, 3H,
COOCH3~ a~d ~ d, 3H, C~3-Fuc).
Compound 39 i5 then dechloroacetyla~ed by
treatment with thiourea and the compund is sulphated
W092/22564 PCT/CA92/00245
~ - 86
with sulfur trioxide/pyridine complex in
dimethylformamide at 0C for 2 hours to provide for
compound 41. The blocking groups on compound 41 are
then remcved by conventional techniques to pr~vid~
for co~pound ~7.
Example 15 -- Synthesis of 2-deoxy-2-phthalimido-
1,3,4,6 tetra-0-acetyl-~-D-
glucopyranoside (compound 1)
(D+) Glucosamine hydrochlorid~ (100 g, 0.46
mol) was added to a solution of sodium methoxide in
methanol which was prepar~d from equimolar amount of
sodium metal i~ methanol (O.5 L). The resultant
mixture was treated with equimolar equivalent of
phthalic a~hydrid2 and triethylamine (80 mL). The
mi~ture was then s~irred for 2 hours, filtered and
:~ the ~olid was dried in ~acuum for 12 hours. The dry
- solid wa~ dissolved in pyridine (300 ~h) and treated
~` ~ with acetic a~hydride ~200 mL, 2.1 mol). m e
: mixture was then stirred at room temperatùre for 48
20 ~hours. The reaction mixture was then treated with
~:` an ice-water mixture, and the resultant precipitate
was filtered, con~ntrated and crys~allized fro~
di~thylether to 98.3 g (4~%3 of the title compou~d.
~ : 7H-n.m~r. (CDC13): ~ 7.75 ~m, 4h, aromatic)l 6.45 (d,
:: 25 lH, H-1~ J12 9.0H2), 5.85 (t, lH), 5.15 ~t, lH), 4~4
~t, lH), 4.3 ~q, lH), 4.1 ~q,. lH), 4~00 (m, lH),
2.05, 2.00, 1.95, 1.80-(4s, 12H, 4Ac). 13C-n.m.r.
~CDCl3) ~ 89.7 (C-1), 72.6, 70.5, 68.3 (3C, C-3, C-
4, C-5), 61.45 (~-6~, 53.42 ~C-2).
: .
W092/22S64 PCT/~92/00245
2l~7~
-- 87 --
Example 16 -- Synthesis o~ 2-deoxy-2-phthalamido-
3,4,6-tri-0-acetyl-~-D-glucQpyranosyl
bromide (c~mpound 12)
2-deoxy-2 phthalamido-1,3,~,6-tetra-0-
acetyl-~-D~glucopyranoside 1 (20g, 41.9mmol) was
treated with hydrogen bromide solution in acetic
acid ~30%, 200mL) and stirred at room temperature
for 2 hrs. The mixture was then poured into an ice
water mixture and extracted with dicloro~ethane.
`10 The ~xtract was washed with ~aHCO3 solution and
water followed by ~gS04 drying. The mixture is
:~ ~iltered, dried and concentrated in vacuo to give
compound 12 as a dry syrup (compound 12)
~xample ~7 - Synthesi~ of E~hyl 2-deoxy-2-
phthalimido-3,4,6-tri 0-acetyl-B-D-
glucopyranoside (compound 13)
2-Deoxy-o2-phthalamido-3,4,6-tri-0-acetyl-~-D~
glucopyranosyl bromide (co~pound 12) fro~ example 16
was taken up in dry ethanol and treat~d ~irectly
with dry etha~ol (200 mL), mercuric ~yanide (1307 g~
~ :5~ ~mol) and stirred at room ~emperat~re for 48 hr.
: i The mixture was then filtered and concen~ra~ed~ ~he
~: residu~ was takèn up in 200 mL of dichloromethane
and wa~hed with a ~olution of 10~ potas~ium iodide,
5% sodium bicarbonate, water, dried over MgSC~ and
concentrated to a syxupO
Example 18 -- Synthesis of Ethyl 2-deoxy 2-
phthalimido-B-D-glucopyranoside
. ~Compound 143
Ethyl 2-deoxy-2-phthalamido-3,4,~-tri-o-acetyl-
~-D-glucopyra~osyide (compound 133 from example 17
was taken up in 100 mL of dry methanol and treated
W0~2/22564 PCT/CA92J00245
88 --
with 100 mg of sodium metal. The solution was
stirred at room temperature for 24 hours and then
neutralized with Am~erlite ER-120(H+)] resin,
filter~d, and evaporated to dryness in vacuo. This
compound was used in the preparation of compound 15
and compound 66.
Example 19 -- Synthesis of Ethyl 2-deoxy-2-
: ~ phthalimido-6-O-benzyl-B-D-
glucopyranoside (Compound 15)
Compound 1~ (2.:1 g, 6.23 mmol~ was taken up in
100 mL of toluene. To it waC ~dded bis(tributyl
tin) oxide (2.22 mL,-4.35 mmol) and
tetrabutylammonium bromide (0.983 g, 3.05 mmol~.
The mixture was~heated at 150C for 4 hours and the~
toluene ~50 m$): was~distilled off from the mixture.
The:reaction mixture was eooled to:room temperature
and benzyl bro~ de ~2.17 mL, 18.27 mmol) was added
and the reaction heated to llO-C for 36 hours.
Toluene was ev~porated and the residue taken up ino ~: ~ 20 ethyl acetate ~:22 mL),:washed successively with
aqueous sodium bicarbona~e, saturated sodium
: chloride solution~and water. The organic layer wa~
dried and e~aporated~to dryness to giv~ a crude
solid. Purification~by column chroma~ography on
:: 25 silica gel gave a:crystalline solid 15 gl.4 g, 70%).
H-n.m.r.(CDCl3) ~ 7.~3-8.1 (9H, aromatic), 4.5 (dd,
i 2H, Ca2Ph), 5.18 (d, lH, J12 lO.OHz, H-l), 4.36 ~dd,
; lH, H-3), 4.25 (dd, H, J21 lO.OHz, J23 8.0Hz, H-2)
and l.O(t, 3H,~ CH~).
:
~ ~ :
:
W092/22564 PCTJCA92/00245
3 7 ~ !
Example 20 -- Synthesis of Ethyl 6-O-benzyl-2-
deoxy-2-phthalimido-3 0-(2,3,~,-tri-
O benzyl-~-L-fucopyranosyl)-4-Oo
(2,3,4,6-tetra O-ac tyl-B-D-galacto-
pyranosyl)-B-D-glucopyranoside
(Compound 49~
To a stirred solution of compound 15 (2.49,
5.71 mmol) in dry dichloromethane (50 mL~ was added
dry CaSO4 (7.5 g), sllver triflate (0.73 g, 2.8
m~ol) and silver carbonate (7.0 g, 2S.7 ~mol) and
the reaction mixture cooled to 50-C. 2,3,4,6~
t~traacetyl~ bromogalactose (3OS g, 8~5 ~mol) in
dry dichloromethane (15 mL) was added dropwise
througk a dropping funnel. The reaction mixt~re was
lS warmed to -3~-C and stirred .for 48 hours and then
m~thanol (S m~) was adde~ to cease the reaction and
the mixture allowed to warm to room e~perature.
Aft~-r filtration th~ough a celite p~d and the
filtrat~ was wa~h~d with agueous ~i~ar~o~ate and S%
: 20 ~DT~ ~olution. Evaporation of the solvent in vacuo
gave a r*ddish bro ~ syrup which was chromatographed
on:silica with toluene: acetone:~eOH (20:3:1~ as
elu~nt o give compound 48 (Rf O.S28) as the major
co~pound~ I
Thiofuco~ide 20 ~1.5 g, 2.8 mmol3 was stirred
in dry dichloro~ethane (S0 mL) cooled to -20c and
br~mine (0.40 y) was added. The co~version to
br~mide w~ c~m~lete in 1 hour a~d the reaction
mixture was washed with cold aqueous bicaxbonate,
dried a~d concentrated to 50 ml and syring~d into a
~lask containing compound ~8 (1 g, 1.4 ~mol), H~r2
~1~08 g, 3 mmol), molecular sieves 4A ~2g) and
tetraethylammonium bromide (1 g~ in dry
dichloromethane (50 mL). The mixture was stirred at
room temperature for 48 hours. T.l.c. showed a
~as~er moving spot. The reaction mixture was
filtered through celite, and the filtrate washed
W092/22564 PCT/CA92/00245
with water, 5% EDTA, saturated aqueous sodium
bicarbonate, water, then dried over sodium sulphate,
filtered and evaporated to dryness in vacuo.
Purification of the crude product by silica gel
chromatography gave the title compound 4Q (1.2 g,
70%, Rf 0.669 in toluene; acetone; MeOH 20:3:1).
1~ n.m.r.(CD~13): ~ 7.00 - 7.8 (aromatic 24 H)
5.35(d,1H, J-2 9.0Hz, H-l), 5.15 (d, lH, J-2 3.8Hz,
~ Fuc~, 4.35(dd, lH, J2 ,3 10.0Hz, H-3 ) 2.1(s,
3H, ace~yl CH3~ 1.95(s, 6H, acetyl CH3), l.9O(S, 3H,
acetyl CH3), l.l(t, 3H, C 3~, and 0.S ~d, 3H, CH3-
Fuc). 13C-n.m.r.: ~ 168, 170 (C=O, ph~halimido and
aee~yl), 101.0 (C-l, Gal), 100.0(C-1, GlcNPhth)
97.7(C-l-Fuc), 2O.6(CH2~ ) and 15.98(C-6-Fuc).
~; :
:Example 2~ -- Synthesis of Ethyl 2-acetamido-6-O-
acetyl 3-O-benzyl-2-deoxy-B-D-
: glucopyranoside
~ A ~olution of compound 1 (2 g, 4.68 mmol3 in
:~ agueous acetic a~id (80%r 150 mL) was heated at 80-C:~ 20 for 2 hour~. The:mixture then was evaporated and
the r~ulta~t solid was dried over P205 in high
: ~ ~ vacuu~. The dry solid was selectively acetylated
with acetyl chloride (0.33 mL, 4O7 mmol) and
:~ pyridine ~10 mh) in dichloromethane (~00 mL) at ~
:~ 25 ~:0C to SC. The mixture was then diluted with
diehloromethane (50 m~), washed with a~ueous
NaHCO3, dried ~ver MgSQ4 and evaporated. The residue
was chro~tographed on a silica gel colu~n using
ET~Ac: hexanes, 3:1 (v:v) as eluant to give 0.82 g
(46%) of the title compound: 1H-n.m.r. (300 MHz,
CDC13): ~7.3(m, 5H, aromatic), 5.67(bs, lH, NH~,
4.86(d, lH, H-l), 4.75(m, 2H), ~.48(q, lH), ~.27(d,
lH), 4.1(t, lHj, 3.85(m, lH), 3.5(m, 3H), 3.16(m,
lH), 2.70(bs, lH, OH), 2.1(s, 3H, Ac), l.9(s, 3H,
SIJBST~TI~TE SI~E~T
~V0~2/22564 PCT/C~92/~0245
~~ g 1 ~~ ~ 1
Ac), 1.18(t, 3H, CH3), 13C-n.m.r. (CDC13): ~
99.45(C-1), 79.85, 74.5(CH2ph~, 73.7, 71.09, 65.25
(C-6), 63.36(CH2-), 57.7(C-2~, 23.6(Ac), 20.86(Ac),
15.06(CH3)~
Exa~ple 22 -- Synthesis of Ethyl 6-O-acetyl-3-O-
: benzyl-2-deoxy-2-phthalimido-B-D-
glucopyranoside (compound 69)
A solution of ethyl ~-deoxy-2-phthalimido-~-D-
` glucopyranoside (compound 14) from Example 18 was
taken up in dry acetonitrile (100 mL) and ~reated
~:~ with benzyaldehyde dimethylacetal (9.6 g) and a
catalyt~c amount of p-toluenesulphonic acid tlO0
mg). The mixture was stirred for 17 hours at room
temperature and then neutralized to pH 7 with
~15 triethyla~ine. The mixture was evaporated and
:~: crystalli~ed ~rom hot hexanes to give 12.7 grams of
ethyl 4~6-0-benzylidene-2-deoxy-2-phthalimido-B-D-
glucopyranoside compound 66.
Compound 66 ( 10 g) was dissolved in d~y
: ~ ~ 20 ~dimethylformamide (D~qF) at -5 - C and treated with 1.1
:~ : g ~:46. 6 mol) ~odium hydride arld benzoyl bro~ide
(5.46 mL, 22 mmol).~ The mixture was stirred a~ 0C
or 2 hours and then treated slowly with 20 mL
methanol then slowly brought to room temperature and
trea~ed with ~ICl (lN) to pH 7 and then extracted
three times with dichlor~me~ane. The organic layer
was dried ~rer anhydrous magnesium sulfate then
filtered, concentrated to dryness and taken up in
: hot ethanol to give 7.2 g of compound 67~ Compound
67 (5.43 g, 10.50 mmol) in aqueous acetic acid ~80%,
200 mL) w~s heated at 80C for 2 hours. The mixture
was evaporated and the resultant solid was dried
over P205 in high vacuum. The dry solid was
selectively acetylated with acetyl chloride (0.8 mL,
3S 11.0 mmol) and pyridine (lo mL) in dichloromethane
WO 92/22564 ~r~, PCI`/CA92/00245
Grl
-- 92 --
(200 mL) at -10C to 0C. The mixture was then
diluted with dichloromethane (1 0 m~), washed with
aqueous NaHCO3, dried over MgSO4 and evaporated. The
residue was chromatographed on a silica gel column
using EtOAc:hexane, 1:2 (v:v) as eluant to give 3.5
g (71%~ of the compound 69: lH-n.m.r. (300 MHz,
CDCl3): ~ 7.7(ml 4H, aromatic), 7.0(m, 5H,
aromatic), 5.16(d~ lH, H~ .7(d, lH), 4.5(m, 2H),
4.2(m, 3H), 3.8(m, lH), 3.6(m, 2H), 3.45~m, lH),
1~0 2.9(bs, lH, OH), 2.1(s, 3H, Ac), 1.95(t, 3H, CH3).
3C-n.m.r. (CDCl3): ~ g8.09(C-1), 78.45, 7~.5, 73.9,
71.7, 65.1, 63.1, 55.5, 20.87 (Ac), 14.92 (CH3~.
Example 23 -- Sy~thesis of Ethyl 6-0-acety'-3-
benzy}-2-deoxy-2-phthalimido-~0
~2,3,4,6-tetra-0-acetyl-B-D-
galactosyl)-B-D-glucopyranoside
o~pound 70)
: To a stirred solution of c~mpound 9 (80 mg,
:: O.17 mmol) in dichloromethane (10 m~) containing
; 20 molecular sie~es (3A, 1 g), 2,6-di-tert-butyl-4-
: methyl-pyridine ~45 ~g, 0.22 mmol~ and silver
triflate (57 mg, 0.22 mmol) was added, at -30-C
under nitrogen, 2,3,4~6-tetra O-acetyl-~-D-
ga~actosyl bromide in dichloromethane (5 mL). The
mixture wa~ stirrcd at this temperature for 1 h and
: then warmed up to 5~C over 2h. The mixture was then
diluted with d~hloromethane (10 m~), filtered and
the insoluble material was washed with
dichloromethane (5 mL). The combined filtrates were
washed with saturate~ aqueous sodium hydrogen
carbonate and water, dried over MgSO4, and
concentrated. The residue was chromatographed on a
silica gel column using ethyl acetate: hexanes, 1:2
~v:v) as eluant to give 120 mg (80~) of the title
W092/22~64 PCT/CA92/00245
21t ~Brl
-- ~3 --
compound: ~H-n.m.r. (300 MHz, CDC13): ~ 7.68,
6.96(2m, 9H, aromatic), 5.3(m, 2H), 5.13(d, lH, H-
1~, J1 z 8.0Hz), 4.99(q, lH), 4.82(d, lH), 4.62~d~
lH, H-l, J12 7.7Hz), 4~54(d, lH), 4.42(d, lH),
4.3~q, 1~), 4.15(m ,2H), 3.99(m, 2H), 3.87(m, 2H),
3.72(m, 2H), 3.46~m, lh), 2.15, 2.12, 2.09, 2.00,
1.98(5s, 15H, 5X~c), l.OO(t, 3H, C~3).
3C-n.m.r. (CDCl3): ~ 101.2, 97.8(C-1, C-l ),
14.85(~H3)-
The 2-amine of Compound 67 above can be
r~generated by contacting this compound with
hydrazine acetate and then acetylated with acetic
anhydride pyridine or other acetylating agents to
provide for a disaccharide (90)
5 Example 24 ~- Synthesis of Ethyl 2-acetamido-Ç-O-
acetyl-2-deoxy-4-0-(2,3,4,6-tetra-0-
acetyl-B-D-galacto~yl)-3-0-(2,3,4-
tri-O-benzyl ~-h-fuco yl)-B-D-
glucopyranoside
?o To a stirred solution o,f the disaccharidQ 90
(80 mg, 0.129 mmol) in dichloromethane (2 mL)
conSaîning molecular sîev~s (3A-, 1 g),
tetra~thylammonî~m bromîde (41 mg, 0.195 mmol),
dimethylformamîde (0.1 mL) and diisopropylethylamine
(0.087 mL, 0.5 mmol~ was added, at room t~mperature
under nitrogen~ a solution of 2,3,4-tri-0-~enzyl
fucosyl bromîde (130 mg, 0.26 mmol -- as per Example
9) în dichloromethane (2 mL~. The mixture was
stirred at room temperature under nitrogen for 72 h
and then filtered, and the insoluble material was
washed with dichloromethane (10 mL). The combined
filtratPs were washed with saturated aqueous sodium
hydrogen carbonate and water, dried over M~SO4, and
concentrated~ The residue was chromatographed on a
WO 92/22~64 . PCr/CA92/00245
J I
~7,~
---- 94 ----
silica gel column using ethyl acetate:hexanes, 3: 1
(v:~r) as eluant to give 115 mg (90%) of the title
trisaccharide 6: ~H-n.m.r. ~300 M~Iz, CDCl3): S
7 . 30 (m, 15H, aroma~ic), 6 ~ 00 (d, lH, NH, J 8 . 0Hz),
5.38(d, lH, H-l Fuc, J1 2 3.3Hz), 5.14(d, lH, H 1
Glc, J1 2 7.8Hz~, 5.1(m, lH), 4.98(m, 2H), 4.80(m,
6~), 4 . 40 (m, 2H), 4 . 33 ~q, lH), 4 . 1 (m, 5H), 3 . 77 (m,
7~I), 3.48(m, 1~). 2.09, ~.07, 2.01, 2.00,
1.97(5XAc, lSH), 1.80(s, 3H, NAc), 1.18(d, 3H, H--6
EUC, J5 6 6.~Hz), 10087(t,, 3H, CH30fEt) . 13C-n.m.r.
(C:~C13): ~ 99 0 ~ (C-l ~;al), 99.19 (C-l Glc~,
97.,18(C-l Fuc~, 16.67(C--6 Fuc:), 14.79(CH30fEt).
1 :xample 2 5 -- Synthe~;is of GDP-Fucose
As not~d above, fucosylation of the sulfated
15 LewisX and L~wi~ ;tructure~ c:an ba ac:~ie~red by use
o:l~ an approp:riate fucosyltransf eras~ which ar~ well
known in the art. Enzymatic fucosylation requires
the use of GDP-fucose. Accordingly, the purpose of
l:his e~am~le i8 to illustrate the synthesi~: of GDP-
20 fucos~. This is achieved in a 3 step pros~ess asillustrated below:
A. Preparation of Bis (tetra n-butylammoniu~)
~5 Tetra-n-butyla~nonium hydroxide (40% aq. w/w,
abo~t 150g) was added dropwise to a ~;olution of
phosphoric acid (85% aq, w/w, 18g~ 0. lS5 mmol) in
water ( 15 0 mL) until the pH reached 7 . Water was
t~ien evaporated in vacllo to give a syrup which was
30 s::o-evaporated with dry aceto-nitrile (2 x ~Q0 mL~
followed by dry toluene (2 x 400 mL). The resulting
`~092/22564 PCT/CA92/00245
211~
-- 95 --
white solid (75g) was dried in ~acuo and stored over
phosphoru~ pentoxide under vacuum until used.
B. Preearation of_~-L-Fuco~ranosyl-l-~hQsPhate
A solution of bis(tetra-n-bu~ylammonium)
hydrogen phosphate (58g, 127.8 mmol) in dry
acetonitrile (300 mL) was stirred at room
temperature under nitrogen in the presence of
mole ular sieves (4~, 20g) for about one hour. A
solution of tri-0-acetyl fucosyl-l-bromide (freshly
: 10 prepared ~r~m 31g, 93 mmol of L-fucose tetraacetate
in the man~er of Nunez et al.53) in dry toluene (lO0
mL3 was added dropwise in about 0.5 hour to the
a~oYe solution, cooled at 0-C. After one more hour
at 0-C~ the ~ixtur~ was brought to ro~m temperature
and stirred for 3 hour. Tlc ~l:l toluene:~thyl
acet~te) indicated a main spot on the base line and
sev~ral faster moving smaller spots.
The mix~ure was filt~red o~er a pad of Celite
: (which was further washed with acetonitrilè) and the
~olvent~ e~aporated in vacuo to give a red syrup.
~his material was dissolved in water (400 mL) a~d
; ~ ~ racted with eth~l acetate (250 mL, twi~e). The
aqueous layer was then evaporated in vacuo leaving a
yellowish syrup to which ~ solution of a~monium
2S hydroxide ~2~% aqO, 200 mL) was added. The mix~ure
w s stirred at room temperakure for 3 hours after
which tlc (6~:35:8 chloroform:methanol:water)
indicated a baseline spot. The solve~t was
evaporat~d in vacuo to give a yellowish syrup which
was diluted with water ~400 mL). The pH of this
solution was checked and brought to 7, if necessary,
by addition of a small amount of hydrochloric acid.
The solution was slowly absorbed onto a column of
ion exchange resin Dowex 2 X 8 ~200-40Q mesh, 5 x 45
W092/~2564 PCT/CA9~/00245
~ - g6 --
cm, bicarbonate form which had been prepared by
sequential washing of the resin with methanol (800
mL~, water (1200 mL), ammonium bicarbonate (1 M,
1600 mL) and water (1200 mL)]. Water (1000 mL) was
then run through the column followed by a solution
of ammonium bicarbonate (O.5 M; 2.3 mL/minute,
overnight). The eluate was collected in fractions
(15 m~ and the product detected by charring after
spotting on a tlc plate. Fractions 20 to 57 were
pooled and vaporated in vacuo leaving a white solid
which was further co-evaporated with water (3 x 300
mL) and freeze drying of the last 50 mL and then
dryin~ of the residue with a vacuum pump to give ~-
LDfucopyransyll-phocphate (9O5g, 40%) as a 12:1
mixkure of ~ and ~ ano~ers containing some ammonium
a¢et~te identified by a singlet at ~=1.940 in the
1H-n.m.~. spectrum. This product was slowly run
through a col~mn o~ Dow~x S X 8 resin tlOO-200 mesh,
tri~thylammonium fo ~) and eluted with water to
provide the bis triethylammonium salt of ~-L-
: fucopyransyl-l-phosphate as a sticky gum after
reeze d ~ ing of the eluate. lHDn,m~r~
~ 4-84g ~dd, J12 ~ J1P = 7~5 Hz, H-l), 3.82 (q, lH,
J56 6.5 Nz, H-5~, 3.750 (dd, lH, J34 3.5, J45 1.0
Hz, H-4), 3.679 (dd, lH, J23 10.0 Hz, H-3), 3.S20
~ddt lH, H-2), 1.940 (s, acetate), 1.26 (d, ~ 6).
Int~gral of th~ sign~ls at 3.20 (q, J 7~4 Hz, NCH2)
and 1.280 and 1.260 ~NCH2CH3 and H-6) indicates that
the product is the bis-triethyl-ammonium salt which
may loose some triethylamine upon extensive drying.
13C-n.m.r. ~:98.3 (d, J lP 3.4 Hz, C-l), 72.8 (d,
JC 2P 7-5 HZ, C_2), 16.4(C-6); 31P_nmr ~ +2.6~S) .
~ -L-fucopyransyl-1-phosphate appears to slowly
degrade upon prolong~d storage (l+ days) in water at
22C and, accordingly, the material should not be
~092/22564 PCT/CA92/00245
-- g7 - ~ 7 ~
left, handled or stored as an aqueous solution at
22C or higher temperatures. In the present case,
this material was kept at -18C and dried in vacuo
o~er phs~phoru~ pentoxide prior to being used in the
next step.
C. Preparation of Guanosine 5'~ fucopy-
ranosyl)-diphosphate
: Guanosine S'~ fucopyranosyl)-diphosphate
was prepared from ~-L-fucopyranosyl-l-phosphate
10 using two different a ~ recognized procedures as set
forth below:
,
PROCBD~RB #
~ ~ L-fuc~pyranosyl-1-phosphate and guanosiné 5'-
:~ mono~pho~phomorpholidate (4-msrpholine-N,N'-di-
cy d ~hexyl~carboxamidine salt, available from Si~ma,
St. Louis, ~issouri, t'G~P-~orpholidate") were
reacted a~ described in a recent modification54~56 of
Nunez's original procedureS5. Accordingly, tri~n
: o~tylamine (0.800g, available from Aldrich Chemical
Company, ~ilwaukee, Wisconsin) was added to a
mixture of ~ -~ucopyranosyl-1-p~osphate (triethyl-
ammonium salt, 1.00~, about 2~20 mmol) in dry
pyridine (10 ~) under nitrogen the sol~ent removed
in vacuo. The process was repeat~d three times with
care to allow only dry air to enter the flaskO GMP
morpholidate (2O4g, about 3.30 mmol) was dissolved
in a 1:1 mixture of dry dimethylformamide and
pyridine (10 mL). The solvents wexe evaporated in
vacuo and the proce~ure repeated three times as
abo~e. The re idue was dissolved in the same
mixture of solvents ~20 mL) and the solu~ion added
to the reaction flask accompanied by crushed
molecular sieves (2g, 4A). The mixture was stirred
W092/22564 PCT/CA92/00245
q~ 98 --
at room temperature under nitrogen. Tlc (3:5:2 25%
aq. ammonium hydroxide, isopropanol and water)
showed spots corresponding to the starting GMP-
morpholidate (Rf-0.8, U.V.), guanosine S'~
fucopyranosyl)-diphosphate (Rf-0.5, U.V. and
charring), followed by the tailing spot of the
starting fucose-1-phosphate (Rf-0.44, charring).
Additional ~.V. active minor spots were also
present. After stirring for 4 days at room
io temperature, the yellowish mixture was co-evaporated
in vacuo with toluene and the yellowish residue
further dried overnight at the vacuum pump leaving a
thick residue (2.43g). Water (10 mL) was then added
into the flask to give a yelIow cloudy solution
which was added on top o~ a column of AG 50W-X12
~:: (fro~ Biorad) resin~100-200 mesh, 25 x 1.5 cm, Na~ ^
: form). ~The product eluted with water after the void
:volume. The ~ractions which were active, both by
U.V. and charring:after ~potting on a tlc plate,
20 ~ were recovered and the solution freeze-dried
overnight in vacuo providing a crude material
~:
(1.96g). ~ : :
This residue was dissolved in water (10 mL
- ~ :
overall) and slowly absorbed on*o a column of
hydrophobic C18 :~silica gel (Wateræ, 2.5 x 30 cm~
: which had been conditioned by washing with water,
~: methanol and water (250 mL each). Water was then
run through ihe column tO.4 mL~min) and the eluate
collected in fractions (0.8 m~) which wer~ checked
: : 30 by klc (3:~:2 25%~aq. ammonium hydroxide,
isopropanol and water). ~ fucopyranosyl 1-
p~o~phate, (Rf-0.54, charring) was eluted in
fractions 29 to 45. A product showing a strongly
~ U.V. active spot (Rf~0.51) eluted mainly in
: 35 fractions 46 to 65. Other minor U.V. active spots
~92/22564 PCT/C~92/00245
~.llG~o~
__ 99 __
of higher or lower Rf were observed. Fractions 59
to 86, which contained guanosine 5'~
fucopyranosyl)-diphosphate ~Rf~0.~2), also showed a
narrow U.V. active spot (Rf-0.57~. Fractions 59 to
86 were pooled and freeze-dried overnight providing
O.353g of material enriched in guanosine 5'-(~
fucopyranosyl)-diphosphate. 1H-n.m.r. indicated
that this material was contaminated by a small
amount of impurities giving signals at ~ = 4.12 and
~ = 5.05.
Fractions 29 to 45 and 47 to 57 were separately
pooled and freeze-dried pro~iding recovered ~ L-
~uco-pyranosyl-l-phosphate (0.264g and 0.223g,
re~pectively, in which the seco~d fraction contains
so~e impuriti~s). Qcca~ionally, pooling of
appropriate fractions provid@d some amou~t of
guanosine 5'o(~-1 fucopyranosyl)-diph~sphate in good
purity (1H-n.m.r.). Generally, all the material
enriched in guanosine 5'~ l fuco-pyranosyl)-
~: ~0 diphosphate wa~ dissol~ed i~ a mini~um a~ount of
water and run on the same column which had been
regenerated ~y washing with large amounts of
methanol follcwed by water. The fractions
containing the purified guanosine S'~
fucopyranosyl~-diphosphate (tlc) were pooled and
freezed dried in vacuo leaving a white fluf*y
material (187 mg, 16%). ~-n.m.r~ was iden$ical to
the previously reported data53.
PROCED~R$ #2
. ~-L-fucopyranosyl-l-phosphat and ~uanosine
5/-monophosphomorpholidate (4-morpholine-N,N'-di
cyclohexyl-carboxamidine salt -- ''GMP-morpholidate~)
were reacted in dry pyridine as indica ed in the
original procedure55. Accordingly, the ~
W092J22~64 PCT/CA92/00245
100 --
~i
fucopyranosyl~l-phosphate (triethyl-ammonium salt,
o.528g, about 1~18 mmol) was dissolved in dry
pyridine (20 mL) and the solvent removed in vacuo.
The process was repeated three times with care ~o
allow only dry air to enter the flask. GMP-
morpholidate (1.2g, 1.65 mmol3 and pyridine (20 mL)
were added into the reaction flask, the solvent
: evaporated in vacuo and the process repeated three
times as abo~e. Pyridine ~20 mL) was added to the
final residue and the heterogeneous mixture was
stirred for 3 to 4 days at room temperature under
nitrogen. An insoluble mass was formed which had to
be occasionally broken down by sonication~
The reaction was followed by tlc and worked up
as indicated in the first procedure to provide the
GDP-fucose (120~mg, 16%).
~:
:, .
II. Biological Results
Examples:26-29~illustrate the immunomodulatory,
antii~flammatoryt ahd tolerogenic properties of
20 ~ c~mpounds disclosed herein.
: ~ Examp~e 26 - Inhibition of ~ Inflammatory Response
: DTH infl~matory~responses were measured using
:~ the mouse footpad:swélling assay as described by
,
Smith and Ziola55. Briefly, groups of Balb/c mice
were immunized with S-layer protein, a bacterial
surface protein5l from Clostridium
hermohvdrosulfur um Ll11-69 ~Llll] or with
~ SuperCarrier (SC) which have been shown to induce a
: strong inflammatory DTH response. Seven days later,
each group of mice was footpad-challenged with 10 ~g
of L-lll S-Layer protein or with 20 ~g of SC. The
resulting inflammatory footpad swelling was m~asured
:: ~
WO9~/22564 ~ 7~ 7 PCT/~A9~/00245
----101 ----
with a Mitutoyo Engine~ring micrometer 24 hours
after challenge.
To assess the effect on the DTH inflammatory
response by sialyl LewisX-OR and sulfated IRwisX-OR
(compound ~7), groups of mice challenged with 10 ~g
of the same anti~en 7 d~ys after immunization with
the antigen received 100 ~g of sialyl L2wisx-oR or
sulfated LewisX-OR tR = -(CH2)8COOCH3] injected into
the tail vein, 5 hours after challenge. Control
`~0 groups were left untreated. The results of this
experiment are shown in FIG. 11 which demonstrates
that while mice injected with either sialyl LewisX-
OR or sulfat~d LRwisX OR exhibited reduced
inflammation, those injected with sialyl LewisX
~xhibited the greater reduc~ion in in~lammation.
- To ass~ss the e~fect on the DTH infl~mmatory
: : ~r~æponse by the modified LewisX-OR c~pound
containing a sulfate substituent at the 3-position
o~ galactose (c~m~ound ~7), group; of mice
challenged with either 10 ~g of the L111 antigen
~Figure 11) or with 20 ~g of the SC antigen (Figure
12~ 7 days after i ~ unization with the antigen
received 100 ~g of sialyl LewisX or sul ated LewisX
i~ject d into the tail vein, S hours after
~5 challenge. Control groups were l~ft untrea~ed or
~: received 100 ~L of phosphate-buffered saline (PBS).
The results of this part of this example are set
~orth in Figure 12 which illustrates that the
sulfated LewisX provided roughly e~uivalent
reduction in inflammation. This result is
particularly surprising when compared to Figure 11
and dem~nstrates that substitution of the sulfat
group at the 3-position of the galactose of LewisX
significantly enhances the anti-inflammatory
3S properties of these compounds.
WO 92/22S64 PCI/CA92/01)245
~?~r~
/~ 3 ---- 102 ~--
Example 27 -- Effect of Oligosaccharide G-ycosides
on LPS Caused Lung Injury
LPS (lipopolysaccharide) caused lung injury is
measured by weighing the lungs of sacrificed mice 24
S hours after mice are given LPS intranasally.
Briefly, groups of 8-10 weeX old Balb/c mice were
s~nsitized with S ~g /mouse of ~PS in 50 ~1 of PBS
intranasally under light anesthesia.
The method of administering compound
~0 intranasally ic descr~bed in Smith et al., Infection
and Immunity, 31: 129 (1980), which is incorporated
by reference. Briefly, mice are anethesitized with
Metofane (Pitman-~oore Ltd., Mississauga, Ontario,
Canada) and a 50 ~1 drop of compound is placed on
~5 the nares o~ the mouse and is inhaled.
Five hours later, 100 ~g/mouse of sialyl LewisX
or ~ul~ated LewisX in 200 ~1 o~ PBS are given to the
: mouse in~ravenously. After 24 hours, the mice are
sacrificed and ~ e lungs removed and weighed. The
weight of the lungs of mice treated with either
sialyl L~wisX or ~ulfated Lswis~ are c:ompared against
control ( i . ~ ., mice treaked with LPS but to which
nt3ither sialyl LewisX or I,ewisX has been
administered~. Th~ percent reduction is measured by
25 s~btracting f rom 100 the following:
The fraction derived by a num~rator whose value
is the weight of the treated lungs subtracted f rom
the weight of normal lungs ~lungs from mice not
exposed to LPS, sialyl LewisX or sulfated LRwisX),
and whose denominator whose value is the weight of
the control lungs (mice that received only LPS)
subtracted from he weight of normal lun~s and
multiplying the resulting fraction by 100.
The greater the percent reduction, the better
the compound is in alle~ating lung damage.
~092/22564 PCT/CA92/00245
~s ~ 7 ~ 7
-- 103 --
The results of this test are set forth in
Figure 13 which illustrates that sulfated LewisX
provid~s about 50~ reduction whereas sialyl Lewisa
provides only about a 30% reducti~n in the DTH
inflammatory response in lungs. This suggests that
not only do both sialyl LewisX and sulfated LewisX
can b~ use~ul in reducing inflamation in lungs
exposed to antigen, for example Acute Respiratory
Distress Syndrome but that sulfated LewisX actually
provides significantly enhanced results.
Exa~le 28 -- ~ffect of Adminis~ration of Sialyl
Lewis~ and Sulfated LewisX at the Time
of Immunization on the Induction of
an Immune Response to an ~ntigen
Groups of Balb/c female mice were immunized
: with 20 ~g/mouse SC in 100 ~1 of PBS in~ramuscularl~
i ~ o the hind leg muscle which formulation also
contained 100 ~g/mouse of sialyl LewisX or ~ulfated
~ewisX. SeYen day later ~he mice were ~ootpad
challenged with 20 ~g/mouse of 5C in 20 ~L of PBS.
Control groups were ei~her not immunized or received
20 ~ of phosphat~-buffered salin~ (PBS) in place of
either sialyl LewisX or sulfated LewisX. The
footpad swelling was measured 24 hours later with a
2S Mitutoyo Engineering micrometer.
FIG. 14 shows that admini~tering sialyl LewisX
or sulfated LewisX to the mice at the ti~e of
immunization reduces ths induction of an immune
response to an antigen as compared to PBS control.
This suggests that administration of a compound of
thi invention a~ the tim~ of antigen immunization
will reduce the ability sf the mammal to become
educated concerning this antigen.
W092/22564 PCT/CA92/00~45
104 --
Example 29 -- Persistence of Suppression of the DTH
Inflammatory Response at 6 Weeks
After Challenge
i. The identical groUps of mice treated with
S sialyl LewisX and sul~ated LRwisx in Example 26 were
re-challenged 6 weeks after primayr immmuniZation
with 20 ~g/mouse with SC. Untreated controls
r~spond~d with the usual degree of footpad swelling
wherea~ all other groups showed reduced footpad
swelling as shown in Figure 15.
In addition to providing an anti-inflammatory
effect as well as modulation of a cell-mediated
immune response, the abo~e data demonstrate that
treatment with sul~ated LewisX as per this invention
; ~5 also imparts tolerance to additional challenges from
t~e same antigen.
~ ditionally, o ~ er compounds disclosed herein
can be used in place of the sulfated LewisX
: including the following ~alartose substitu~ed
~0 materials: 6-sulfated ~RWiSX, 3- or 6-sulfated
Lewisa, 3- or 6-pho phated LewisX, 3-6-phosphated
i. 3t and th~ following fuco~ed . ubstituted
ma~ rials: 3 :3r 4- sulfated or phospha~ed ~uc:o e
groups on Le~isX or L~wis~ and the 1 ike .
