Note: Descriptions are shown in the official language in which they were submitted.
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COMBINATORIAL SY~THESIS OF CARBOHYDRATE LIBRARIES
BACKGROUND OF THE INVENTION
Field of the Invention
S This invention is directed to methods for synthesi7in~ very iarge coll~ctionc Of
diverse thiosaccharide derivatives optionally attached to a solid support. This
invention is further directed to a library of diverse thios~ec~ride derivatives.
References
The following publications, patents and patent applications are cited in this
application as superscript numbers:
International Patent Application Publication No. WO 93/06121.
2 U.S. Patent No. 5,143,854, issued September 1, 1992.
3 Hol, W. G. J., et al., "Structure and Function of E. coli Heat-Labile
Enterotoxin and Cholera Toxin B Pent~mer", Bacterial Toxins and
Virulence Factors in Disease, Ed. by J. Moss et al., Marcel Deldcer,
Inc. (1995).
4 Spangler, B. D., "Structure and Function of Cholera Toxin and Related
Escherichia coli Heat-Labile Enterotoxin", Microbiological Reviews,
56(4):622-647 (1992).
Williams (ed.), Synthesis of Optically Active o~-Amino Acids, Pergarnon
Press (1989).
6 Evans et al., J. Amer. Chem. Soc., 112:4011-4030 (1990).
CA 02256694 1998-11-24 -
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7 PU et al., J. Amer. Chem. Soc., 56:128CL1283 (1991).
8 Williams et al., J. Amer. Chem. Soc., 113:9276-9286 (1991).
s
9 Ratcliffe, et al., U.S. Patent No. 5,079,353.
'~ J. Defaye, et al., "Thiooligosa~çh~rides: Their Synthesis and ~elcti~l~c
with Enzymes" in Studies in Natural Products Chemistry, Vol. 8, pp.
315-357, Elsevier Sci~n~es Publishers (1991).
" Kagen et al., Synlett, 1990, 643-650.
12 E. Hasegawa, K. Ishiyama, T. Horaguchi, T. Shimim, J. Org.
Chem. 1991, 56, 1631-1635.
3 H. Paulsen, K. Eberstein, W. Koebemick, Tetrahedron Letters,
4S-50, 4377-4380.
14 J.M. Kerr, S.C. Banville and R.N. Zuckermann, J. Am. Chem. Soc.,
5:2529 (1993).
V. Nikolaiev, A. Stierandova, V. Krchnak, B. Se~ m~nn~ K.S. Iam,
S.E. Salmon and M. Lebl, Pept. Res., 6:161 (1993)
6 M.C. Needels, D.G. Jones, E.M. Tate, G.L. ~einkPl, L.M.
Kochersperger, W.J. Dower, RW. Barrett and M.A. Gallop, Proc.
Natl. Acchl. Sci., USA, 90:10700 (1993)
17 M.H.J. Ohlmeyer, R.N. Swanson, L.W. Dillard, J.C. Reader, G.
Asouline, R. Kobayashi, M. Wigler and W.C. StiIl, Proc. Natl. Acad.
Sci. USA, ~2:10922 (1993)
18 U.S. Patent No. 4,137,401, issued January 30, 1979, tO R.
Lemieux et al.
9 H. H. Westal et al., "Methods of Enzymology," 34(b), 64
(1974).
T. Muk~iyama et al., Tetr~edron Letters, ~6, 5907-5908 (1968).
21 Svennerholm, A-M. et al., Current Microbiology, 1:19-23 (1978).
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All of the above publications, patents and patent appli~tiol-s are herein
incol~oldted by reference in their entirety to the same extent as if each individual
publication, patent or patent application was specifically and individually in(lic~ted to
be incorporated by reference in its entirety.
State of the Art
Compounds having biological activity can be identifiP~d by scr~ning diver_e
collections of compounds (i.e., libraries of compounds) produced through either
molecular biological or synthetic chernic~l techniques. Such screening metho~s
10 include methods wherein each member of the library is tagged with a unique identifi~r
tag to facilitate identification of compounds having biological activity' or where the
library comprises a plurality of compounds synthesi7e~ at spe~ific locations on the
surface of a solid substrate wherein a receptor is apl,lop~iately labeled to identify
binding to the compound, e.g., fluorescent or r~-lio~ctive labels. Correlation of the
15 labelled receptor bound to the substrate with its location on the substrate identifiP-c the
binding compound.2
Central to these methods is the screenhlg of a multiplicity of co..l~unds in thelibrary and the ability to identify the structures of the compounds which have arequisite biological activity. Preferably, in order to facilitate synthesis and
identific~tion, the compounds in the library are typically formed on solid SU~ swherein the compound is covalently ~tt~ehed to the support via a cleavable or non-
cleavable linking arm. In this regard, libraries of diverse compounds are plc~a~ed
and then screened to identify "lead compounds" having good binding affinity to the
receptor.
Pharm~euti~l drug discovery relies heavily on studies of structure-activity
relationships wherein the structure of "lead compounds" is typically altered to
determine the effect of the alteration on activity. Alteration of the structure of the
lead compounds permits evaluation of the effect of the structural alteration on activity.
Thus libraries of compounds derived from a lead compound can be created by
including derivatives of the lead compound and repeating the screening procedures.
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Ideally, the compounds are synthPci7ed in situ on the solid support so that the
support can be tagged to identify the synthetic steps employed and/or the derivatiw
incorporated onto the support. However, relatively simple synthetic metho l~ to
produce a diverse collection of such derivatives on the ~.lp~lL~ are often not
5 available.
One particular class of compounds which would be useful for inclucion in
screening libraries is thios~r~h~ride derivatives. It is well known that certain toxins
and organisms bind to oligos~crh~ride recel)tol~ on host cells as an initial step in the
pathological development of various disease conditions.3 For example, heat-labile
10 enterotoxin ("LT"), secreted by certain enterotoxigenic strains of Escherichia coli,
and cholera toxin ("CT"), produced by Vibrio cholerae, are known to bind to
g~nglis)~ e GMI. a glycosphingolipid situated in the outer leaflet of the host cell
membrane and which has a char~cteri~tic pentasaccharide structure, i.e.,
Gal(,~l ~3)GalNAc(,B1~4){NeuAc(cY2 3)}-
15 Gal(,B1~4)Glc, on its surface.3 LT has been i~lentified as one of the causative agentsof bacterial-induced traveller's diarrhea4 and CT is known to be the causative agent of
the severe diarrheal disease, cholera.4
Additionally, many virulent org~nism~ (e.g., bacteria, virus, fungi, and the
like) including enterovirulent org~nicm~ bind to cell surface receptors as part of the
20 disease process. For example, bacteria such as Vibrio cholerae and en~erotoAigenic
strains of Escherichia coli can directly bind to cell surface r~cepLo,~ forming a colony
at the point of ~tt~rhm~nt. Such binding is detrimental because it perrnits eApl..,3S~;
toxin to immeAi~t~ly interact with the cell surface.
Accordingly, in order to develop new pharm~reuti~i drugs to treat various
25 disease conditions, it would be highly desirable to be able to generate very large
libraries of diverse thiosaccharide derivatives.
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SIJMMARY OF THE INVENTION
This invention is directed to general synthetic m.otho ~s for gen~rating very
large libraries of diverse thiosarch~ride derivatives optionally ~tt~ched to a solid
support. The thio~cch~ride derivative libraries provided by this invention are
S synthesi~eci by reacting a thios~cch~ride with a Mich~l acceptor or an
a-halocarbonyl compound to provide for a thiosacch~ride carbonyl compound. The
carbonyl group of the thio~cch~ride carbonyl compound can optionally be reduced to
provide for a plurality of alcohol and/or amine thio~rch~ride derivatives. In one
embo lime-nt, the alcohol and/or amine group of the thiosacch~ri~e derivative is10 further derivatized to provide for a plurality of thiosaçch~ride derivatives.In one embodiment of this invention, the thiosaccharide derivatives are
covalently ~tt~che~ to a solid support. Solid SuppOll~ containing such thio~s~cch~ri~
derivatives preferably comprise a linking arm which links the solid support to the
thioc~cch~ride derivative. The linking arm can be either cleavable or non-cleavable
15 and when cleavable, can be used to prepare a library of either solid phase or soluble
thios~ch~ri~ie derivatives. The library of thiosacch~ride derivatives, whether soluble
or insoluble, can be screened to isolate individual compounds that possess some
desired biological activity. In a preferred embo~iment each compound in the library
is unique.
Accordingly, in one of its method ~Cpectc~ this invention is directed to a
method for synthesizing a thios~ch~ride derivative, which method comprises:
(a) providing a thios~cch~ride;
(b) providing at least a stoichiometric amount of a coupling reagent c~lectpd
from the group concictin~ of Michael acceptors and ~-halocarbonyl co.npoul~ds; and
(c) contacting the thioc~cch~ride and the coupling reagent under conditi~nc
which provide for a thiosaccharide carbonyl compound.
In another of its method aspects, this invention is directed to a method for
syntheci7ing a thio~crh~ride derivative on a solid support, which method compAses:
(a) providing a thiosacchaAde;
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(b) providing at least a stoichiometric amount of a coupling reagent s~l~ted
from Michael acceptors and cY-halocarbonyl compounds wherein either the
thio.cacc.h~ride or the coupling reagent is covalently ~tt~r-hed to a solid sllppo~l, and
(c) contacting the thios~cçh~ride and the coupling reagent under conciitiQnc
S which provide for a thio~rch~ride carbonyl compound covalently ~tt~ched to a solid
support.
In preferred embo~imentc of this invention, each of the above mPtho~35 for
synthesi7ing a thios~r.ch~nde derivative further comprises reducing the carbony} group
of the thio~cch~ride carbonyl compound to form a group sele~.ted from hydroxy and
10 amino derivatives. Optionally, the hydroxy or amino group can be further derivatized
to form a group selected from esters, substituted amines, ~midçs, carb~m~tes, ureas,
thiourea, thioesters and thiocarb~m~tçs.
In still another of its method aspects, this invention is directed to a method for
preparing a thio~rcharide derivative library produced by syntheci7ine on each of a
15 plurality of solid suppolls a single compound wherein each compound compr ~s a
thio~.ch~ride derivative, which library is synthesi7ed in a p-OCe5S comprising:
a) apportioning solid ~uppolLs among a plurality of reaction vessels which
supports comprise a reactive functional group covalently bound thereto which group is
capable of covalently binding a thiosaccharide at a position other than the thiol group;
b) contacting the supports in each reaction vessel with a unique thio~r.ch~n~e
under conditions wherein the thiosaccharide is covalently attached to the solid
supports through the reactive functional group;
c) pooling the ~.lp?olls;
d) apportioning the S~l~ Ol~ from (c) above among a plurality of reaction --
25 vessels; and
e) contacting the supports in each reaction vessel from (d) above with a
unique coupling reagent selected from the group concicting of Michael açc~tc,r~ and
a-halocarbonyl compounds under conditions which provide for a thios~cch~ride
carbonyl compound covalently bound to said support.
And, in yet another of its method ~Cpectc~ this invention is directed to a
method for preparing a thiosaccharide derivative library produced by syntheci7in~ on
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each of a plurality of solid supports a single compound wherein each con~pound
comprises a thios~ch~ridc dcrivative, which library is synthesi7~d in a plOCCS5
comprising:
a) apportioning solid supports arnong a plurality of reaction vessels which
S S~ S comprise.a reactive functional group covalently bound thereto which group is
capable of covalently binding a coupling reagent;
b) contacting the supports in each reaction vessel with a unique coupling
reagent sel~cted from the group conci.cting of Michael acceptols and a-halocarbonyl
com~ounds under conditions wherein the coupling reagent is covalently ~tt~che~ to the
10 solid supports through the reactive functional group;
c) pooling the supports;
d) apportioning the supports from (c) above among a plurality of reaction
vessels; and
e) contacting the supports in each reaction vessel from (d) above with a
15 unique thic s~ch~ride under conditions which provide for a thioc~cch~ride carbonyl
compound covalently bound to said support.
In prt:felled embo~iimçn~ of this invention, each of the above metho~3c for
prepa~ing a thiosaccharide derivative library exemplified in procedures (a) through (e)
further comprises: (f) pooling the supports from procedure (e);
20 (g) apportioning the supports from (f) above among a plurality of reaction ves~ls; and
(h) reJu~ing the carbonyl group of the thiosaccharide carbonyl compound to form a
group selected from hydroxy and amino derivatives. Still further, such methods
optionally include the further steps of: (i) pooling the s.lp~lls from procedure (h)
above; (j) apportioning the S~lppGlls from (i) above among a plurality of reaction
25 vessels; and (k) derivatizing the hydroxyl or amine groups to form a fun~ion~l group
selected from esters, substituted ~mineS, amides, carb~m~tes, ureas, thioureas,
thioesters and thiocarb~m~es.
The methods described above can be used to create a library of diverse
thios~cch~ride derivatives. Accordingly, in one its composition ~Cpectc~ this invention
30 is directed to a library of diverse thiosaccharide derivatives comprising a plurality of
solid supports having a plurality of covalently bound thios~rh~rides derivatives,
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wherein the thios~cch~ride derivative bound to each of said sU~p(JllSiS S~lbst~nt~ y
homogeneous and further wherein the thios~cch~ride derivative bound on one support
is different from the thios~rch~ride derivatives bound on the other Su~ and
further wherein said thios~rch~ride derivative is l~ ~nted by the formula (1):
s
R3
Saccharide Y ~R4
R' R2
15 wherein
Rl is s~Pl~Pcted from the group consi.cting of hydrogen, alkyl, su~ ul~d alkyl,
alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cyclo~l~P-nyl, heteroaryl, hel~ clic,
thio~lkt xyalkyl and a linlcing arm covalently linking the compound of formula I' to
the S~pOl~,
20. R2 is selected from the group consisting of hydrogen, alkyl, substituted alkyl,
alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cyclo~lk-Pnyl, heteroaryl, het~ clic,
thioalkoxyalkyl and a linking arm covalently linking the compound of formula I' to
the ~u~>poll;
R3 is sel~te~ from the group collcicting of hydrogen, alkyl, substitut~P l aIlyl,
alkenyl, alkaryl, alkoxyalkyl, aryl, cycloalkyl, cyclo~lkPnyl, hetef~yl, het~,oc~clic,
thioalkoxyalkyl and a linking arm covalently linking the compound of formula I' to
the support;
or Rl and R2, or R' and R3, or R2 and R3, or Rl, R2 and R3 can be joined,
together with the carbon atoms to which Rl and/or R2 and/or R3 are ~tt~che~ to form
a cycloalkyl, cycloalkenyl or heterocyclic ring;
R4 is selected from the group concicting of-XR5, -XC(W)R6,
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-XC(W)X'R7 and -C(W)XR8; wherein W is select~P~ from the group CQ. cicting of
oxygen, sulfur and NH; and X and X' are each independently SPlP~ct~P~ from the group
consisting of oxygen, sulfur and -NR9-, wherein R9 is sPlected from the group
concicting of hydrogen and alkyl; or when R4 is -XR5 and R5 is not hydrogen, X can
also be SPIPctP~ from the group Concictin& of -S(O)- and
-SO2-;
R5 is sele~tP~ from the group consisting of hydrogen, alkyl, alkenyl, alkaryl,
alkoxyalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thio~lkoxyalkyl
and a linking arm covalently linking the compound of formula I' to the support, and
when X is -NR9-, then R9 together with X can form an amino acid; or R5 and R~, or
R5 and R2, or R5 and R3 can be joined, together with X of the -XR5 group and thecarbon atoms to which R' and/or R2 and/or R3 are ~tt~- hP~l, to form a heterocyclic
ring;
R6 is selected from the group conciC~ing of alkyl, alkenyl, alkaryl, alkoxyalkyl,
aryl, cycloalkyl, cycloalkenyl, heteroaryl, hetèrocyclic, thio~lkoxyalkyl and a linking
arm covalently linking the compound of formula I' to the support; or R6 and R', or R6
and R2, or R6 and R3 can be joined, together with the -XC(W)- moiety of the -
XC(W)R6 group and the carbon atoms to which R' and/or R2 and/or R3 are ~tt~hPd,
to form a heterocyclic ring;
R7 is selected from the group con~i~ting of alkyl, alkenyl, alkaryl, alkoxyalkyl,
aryl, cycloalkyl, cyclo~lkPnyl, heteroaryl, heterocyclic, thioalkoxyalkyl and a linking
arm covalently linking the compound of formula I' to the sLIp~lL-, or R7 and R', or R~
and R2, or R7 and R3 can be joined, together with the -XC(W)X'- moiety of the -
XC(W)X'R7 group and the carbon atoms to which Rl and/or R2 and/or R3 are
~tt~hP~, to form a heterocyclic ring;
R8 is selected from the group consisting of alkyl, alkenyl, alkaryl, alkoxyallyl,
aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, thio~lkoxyalkyl and a linking
arm covalently linking the compound of formula I' to the support; or R8 and R', or R8
and R2, or R8 and R3 can be joined, together with the -C(W)X- moiety of the -
C(W)XR8 group and the carbon atoms to which Rl, R2 and/or R3 are ~s~chp~ to forma heterocyclic ring;
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Y is selected from the group consisting of sulfur, -S(O)- and -S(O)2-;
n is an integer equal to 0 or 1; and pharm~ceuti~lly ~rc~pt~hle salts ll.e,~or,
wherein the saccharide is selected from the group concictin~ of a
monos~cçh~nde, an oligos~çh~ride, monos~cch~ricle-Z- and oligoc~e~h~rid~Z-,
5 wherein Z is a linking arm covalently linking the compound of formula I to the solid
support;
with the proviso that only one of Rl, R2, R3, R4, R6, R', R8 and Z is linked to
the solid support.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates a preferred reaction scheme for synthç~i7ing a library of
diverse thios~cch~ride derivatives using an ~"B-unsatuldted carbonyl co-,-you- d, i.e.,
cyclohept-2-en- 1 -one.
Figure 2 illustrates a preferred reaction scheme for synthe~i7ing a library of
diverse thiosaccharide derivatives using an a-halocarbonyl compound, i.e, 2-
chlorocycll he~none.
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to libraries of diverse thio~cch~ndç derivatives
optionally ~tt~hPcl to a solid support and to methods for genel~Ling such libraries.
However, prior to describing this invention in further detail, the following terrns will
first be dçfinf?.i
Definitions
"Acyl" refers to the groups alkyl-C(O)-, aryl-C(O)-, and he~.~.a yl-C(O)-
where alkyl, aryl and heteroaryl are as defined herein.
"Acylamino" refers to the group -C(O)NRR where each R is in-:lepçnd~nt1y
hydrogen or alkyl.
"Acyloxy" refers to the groups alkyl-C(O)O-, aryl-C(O)O-, he~ro~yl-C(O)~,
and heterocyclic-C(O)O- where alkyl, aryl, heteloalyl and heterocyclic are as defined
herein.
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"Alkaryl" refers to -alkylene-aryl groups preferably having from 1 to 8 carbon
atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl moiety. Such
alkaryl groups are exemplified by benzyl, phenethyl and the like.
"Alkoxy'' refers to the group alkyl-O-. Such alkoxy groups include~ by way of
5 example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, ter~-butoxy, sec-
butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
"Alkoxyalkyl" refers to the group -alkylene-O-alkyl which incl~ldes by way of
example, methoxymethyl (CH30CH2-), methoxyethyl (CH3-O-CH2CH2-) and the like.
"Alkenyl" refers to alkenyl groups preferably having from 2 to 8 carbon atoms
10 and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-
2 sites of alkenyl unsaturation. Such alkenyl groups include ethenyl (-CH=CH2), n-
propenyl (i.e., allyl) (-CH2CH=CH2), iso-propenyl (-C(CH3)=CH2), and the like.
"Alkyl" refers to monovalent alkyl groups preferably having from 1 to 8
carbon atoms and more preferably 1 to 6 carbon atoms. This term is exemplified by
15 groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, and
the like.
"Substituted alkyl" refers to a branched or straight chain alkyl group of from 1to 8 carbon atoms having from 1 to 3 s~lbstit~le~ts se~e~t~ from the group con~i~t~
of hydroxy, acyl, acylamino, acyloxy, alkoxy, alkenyl, alkynyl, amino, ~minoacyl,
20 aryl, aryloxy, carboxy, carboxyalkyl, cyano, cycloalkyl, guanidino, halo, hetelu~yl,
heterocyclic, nitro, thiol, thioaryloxy, thioheteroaryloxy, and the like. Prefe~led
substituents include hydroxy and amino.
"Alkylene" or "alkyldiyl" refers to divalent alkylene groups preferably having
from 1 to 8 carbon atoms and more preferably 1 to 6 carbon atoms. This term is --
25 exemplified by groups such as methylene (-CH2-), ethylene (-CH2CHr), the propylene
isomers (e.g., -CH2CH2CH2- and -CH(CH3)CH2-) and the like.
"Alkynyl" refers to alkynyl groups preferably having from 2 to 8 carbon atoms
and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-
2 sites of alkynyl unsaturation. Such alkynyl groups include ethynyl ~-C 5 CH),
30 p~opalgyl (-CH2C--CH) and the like.
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"Amino acidN refers to any of the naturally occurring amino acids, as well as
synthetic analogs and derivatives thereof. c~-Amino acids comprise a carbon atom to
which is bonded an amino group, a carboxy group, a hydrogen atom, and a riictinctive
group referred to as a "side chain". The side chains of naturally occurring amino
S acids are well kno~n in the art and include, for example, hydrogen (e.g., as in
glycine), alkyl (e.g., as in ~l~nine, valine, leucine, isoleucine, proline), substituted
alkyl (e.g., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine,
glutamic acid, glutamine, arginine, and lysine), alkaryl (e.g., as in phenyl~l~nine and
tryptQphan), substituted arylalkyl (e.g., as in tyrosine), and heteroarylalkyl (e.g., as in
10 hicti~in~). One of skill in the art will appreciate that the term "amino acid" can also
include ,l~ , and ~-amino acids, and the like. Unnatural amino acids are also
known in the art, as set forth in, for example, Williams3, Evans et al.4, Pu et al.5,
Williams et al.6, and all references cited therein. Stereoisomers (e.g., D-arnino acids)
of the twenty conventional amino acids, unnatural amino acids such as ~
15 disubstituted amino acids and other unconventional amino acids may also be suitable
co..l~nents for compounds of the present invention. Examples of unconven~ign~l
amino acids include: 4-hydroxyproline, 3-methylhi~ti~ine, S-hydroxylysine, and other
similar amino acids and imino acids (e.g., 4-hydroxyproline).
"~minoacyl" refers to the group -NRC(O)R where each R is indepen~e-ntly
hydrogen or alkyl.
The term "amino derivative(s)" refers to a primary, secondary or tertiary
amine compound produced by reductive amination of a thioc~ch~ride carbonyl
compound in the presence of ammonia or an amine, including amino acids and
derivatives thereof.
"Aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to 14
carbon atoms having a single ring (e.g., phenyl) or multiple conden~ed rings (e.g.,
naphthyl or anthryl). ~lef~lled aryls include phenyl, naphthyl and the like.
Unless otherwise constrained by the definition for the aryl s~lbstituent, such
aryl groups can optionally be substituted with from 1- to 3 substituents selPct~ from
the group consisting of hydroxy, acyl, acyloxy, alkyl, substituted alkyl, alkoxy,
alkenyl, alkynyl, amino, aminoacyl, aryl, aryloxy, carboxy, carboxyalkyl, cyano,
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halo, nitro, heteroaryl, trihalomethyl and the like. Preferred subs~ituentc include
alkyl, alkoxy, halo, carboxy, cyano, nitro, trihalomethyl, and thio~lkoxy.
"Aryloxy" refers to the group aryl-O- where the aryl group is as defined
herein including optiona}ly substituted aryl groups as also defined herein.
"Carboxy" refers to the group -COOH.
"Carboxyalkyl" refers to the group -C(O)O-alkyl where alkyl is as defined
herein.
The term "coupling reagent" refers to Michael acceptola and a-haloc~lonyl
com~2ounds. "Michael acc~pto-s" refers to ~,~-unsaturated carbonyl compounds
10 having the general formula (II):
o
Il ~
R'-CH=C-C-R2 II
R3
wherein R', R2 and R3 are as defined herein; or R'CH=CR2-C(O)XR8, wherein R',
R2, R8 and X are as defined herein. Such Michael accepto~a include, by way of
20 example, ~"B-unsaturated aldehydes, ~,~-unsaturated ketones, a"B-unsaturated esters,
a"~-unsaturated thioesters, ~ -unsaturated amides and the like. "cr-Halocarbonylcompounds" refers to compounds having the general formula: W-CHRI-C(O)R2
wherein Rl and R2 are as defined herein, and W is chloro, bromo or iodo. Such a-halocarbonyl compounds include, by way of example, a-chloroaldehydes, a-
25 bromoaldehydes, a-iodoaldehydes, a-chloroketones, a-bromol~Ptones, a-ic~ ton~S
and the like.
"Cycloalkyl" refers to cyclic alkyl groups or cyclic alkyl rings of from 3 to 8
carbon atoms having a single cyclic ring or mnltiple conden~A rings which can beoptionally substituted with from 1 to 3 substituents sPle~ted from the group c~n~i~tin~
30 of hydroxy, acyl, acyloxy, alkyl, substituted alkyl, alkylene, alkoxy, alkenyl, allynyl,
amino, aminoacyl, aryl, aryloxy, carboxy, carboxyalkyl, cyano, halo, nitro,
heteroaryl, trihalomethyl and the like. Preferred substituents include alkyl, alkoxy,
halo, carboxy, cyano, nitro, trihalomethyl, and thioalkoxy. Such cycloalkyl groups
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include, by way of example, single ring structures such as cyclopropyl, cyclobut~
cyclopentyl, cyclooctyl, l-mothylcyclopropyl, 2-methylcyclopentyl, 2-
methylcyclooctyl, and the like, or multiple ring structures such as ~A~ nt~nyl and
the like, and spiro compounds. Examples of suitable cycloalkyl rings include single
5 ring structures such as cyclopentane, cycloheY~ne, cycloheptane, cyclooct~n~, and the
like, or multiple ring structures such as bicyclo[2.2. l]heptane, bicyclo[3.2. l]octane,
and the like. Preferred cycloalkyl rings include cyclopentane, cyclohPY~nP,
cycloheptane and bicyclo[3.2. l]octane.
"Cycloalkenyl" refers to cyclic alkenyl groups or cyclic alkenyl rings of from
10 4 to 8 carbon atoms having a single cyclic ring and at least one pcint of internal
unsaturation which can be optionally substituted with from 1 to 3 substihlentc SPlPct~p~d
from the group consisting of hydroxy, acyl, acyloxy, alkyl, substituted alkyl,
alkylene, alkoxy, alkenyl, alkynyl, amino, aminoacyl, aryl, aryloxy, carboxy,
carboxyalkyl, cyano, halo, nitro, h~elualyl, trihalomethyl and the like. Plere.led
15 substituents include alkyl, alkoxy, halo, carboxy, cyano, nitro, trihalomethyl, and
thio~lkoxy Examples of suitable cycloalkenyl groups include, for in~t~nce, cyclobut-
2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like. Such cyclo~lk~nyl rings
include, by way of example, cyclopentene, cyclohexene, and the like.
"Halo" or "halogen" refers to fluoro, chloro, bromo and iodo and preferably is
20 either chloro or bromo.
"a-Halocarbonyl compound" refers to a compound having the general forrnula:
Q-CHRI-C(O)R2 wherein R' and R2 are as defined herein, and Q is chloro, bromo oriodo. Such a-halocarbonyl co,l~poullds include, by way of example, a-
chloroaldehydes, a-bromo~ldehydes, ~x-iodoaldehydes, a-chloroketones, a-
25 bromoketones, a-iodoketones and the like.
"Heteroaryl" refers to a monovalent aromatic carbocyclic group of from 2 to 8
carbon atoms and 1 to 4 heteroatoms s~l~cted from oxygen, nitrogen and sulfur within
the ring.
Unless otherwise constrained by the definition for the heteloal~l subst~ ent,
30 such heteroaryl groups can be optionally substituted with 1 to 3 substituents sol~t~d
from the group consisting of alkyl, substituted alkyl, alkoxy, aryl, aryloxy, halo,
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nitro, heteroaryl, thio~lkoxy, thioaryloxy and the like. Such he~lo~yl groups can
have a single ring (e.g., pyridyl or furyl) or multiple con~enced rings (e.g.,
indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl and
furyl.
"Heterocycle" or "heterocyclic" refers to a monovalent saturated or
unsaturated group having a single ring or multiple condenced rings, from 1 to 8
carbon atoms and from 1 to 4 hetero atoms sel~cted from nitrogen, sulfur or oxygen
within the ring. For the purposes of this application, the terrn "heterocycle" or
"heterocyclic" does not include carbohydrate rings (i.e. mono- or oligos~çch~nde~).
Unless otherwise constrained by the definition for the heterocyclic substit~ent
such heterocyclic groups can be optionally substituted with 1 to 3 substituents sel~tçd
from the group consisting of alkyl, substituted alkyl, alkylene, alkoxy, aryl, aryloxy,
halo, nitro, heteroaryl, thioalkoxy, thioaryloxy and the like. Such heteroaryl groups
can have a single ring (e.g., pyrrolidinyl, piperidinyl, morpholinyl or
tetrahydrofuranyl) or multiple condence~ rings (e.g., indolinyl).
Fy~mples of nitrogen heterocycles and heleroa,yls inclu~e, but are not ~imited
to, pyrrole, imi~7ole, pyra_ole, pyridine, pyra_ine, pyrimi(~ine, pyri~7ine,
indoli7ine, isoindole, indole, inda_ole, purine, quinolizine, isoquinoline, qllinoline,
ph~h~l~7ine, naphthylpyridine, quinoxaline, quina_oline, cinnoline, pteridine,
carbazole, carboline, phen~nthridine, acridine, phen~nthroline, isothi~701e, ph~n~7ine,
isoxazole, phenoxa_ine, phenothia_ine, imidazolidine, imidazoline, piperidine,
pipera_ine, indoline and the like.
~ich~el acce~to-" refers to an a"~-unsaturated carbonyl col"~und having the
general formula (II):
O
R'-CH = C-C-R2 II
R3
wherein Rl, R2 and R3 are as defined herein; or R'CH=CR2-C(O)XR8, wherein R',
R2, R8 and X are as defined herein. Such Michael accepto,~ include, by way of
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example, cY,l~-unsaturated aldehydes, a,~-unsaturated ketones, a,l~-unsaturated esters,
a"~-unsaturated thioesters, ~"B-unsaturated amides and the like.
"Thioalkoxyalkyl~ refers to the group -alkylene-S-alkyl which includes by way
of example, thiomethoxymethyl (CH3SCH2-), thiomethoxyethyl (CH3-S-CH2CH2-) and
S the like.
"Thiol" refers to the group -SH.
"Thioalkoxy" refers to the group -S-alkyl wherein the alkyl group is as defined
herein.
"Thioaryloxy" refers to the group aryl-S- wherein the aryl group is as defined
herein, including optionally substituted aryl groups as also defined herein.
"Thioheteroaryloxy" refers to the group heteroaryl-S- wherein the heteroaryl
group is as defined herein, including optionally substituted heteroaryl groups as also
defined herein.
The term "thios~cch~ride" refers to a monosaccharide or oligo~cch~ride
having 2 to about 8 saccharide units wherein at least one, and preferably 1 or 2, of
the hydroxyl groups of the ca~ch~ride is replaced with a thiol group. Preferably, the
thiosaccharide is an animal c~ch~ride. The term "animal sacchariden refers to a
saccharide which is naturally e~p~ssed by one or more ~nim~ls, such as m~mm~l$,
birds or fish. Preferably, the animal saccharide is a m~mrn~ n ~rch~ride In
particular, preferred rn~mm~]i~n saccharides include D-galactose, D-glucose, D-
mannose, D-xylose, D-glucuronic acid, N-acetyl-D-glucos~mine, N-acetyl-D-
g~ to~mine, sialyic acid, iduronic acid, L-fucose, and the like. Included within the
definition of this terrn are acylated, phosphorylated and s--lf~t~d derivatives of animal
saccharides~
The term ''thiosaccharide carbonyl compound" refers to a compound having
the formula (III):
Saccharide--y ~ 111
R1 R2
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wherein R', R2, R3, n and saccharide are as defined herein.
Ihe term "substrate" or "solid support" refers to a material having a rigid or
semi-rigid surface which contains or can be derivatized to contain reactive
functionality which covalently links a compound to the surface thereof. Such
5 materials are well known in the art and include, by way of example, silicon ~ioYide
supports containing reactive Si-O~ groups, polyacrylamide supports, polystyrene
supports, polyethyleneglycol ~up~olls, and the like. Such ~uppolls will plefe.dbly
take the form of small beads, pellets, disks, or other conventional forms, although
other forms may be used. In some embodiments, at least one surface of the substrate
10 will be substantially flat.
In one embodiment, the activated ketone compound is covalently ~tt~ e~
directly to the solid support or is ~ttache~ to the support via a linking arm. ~ inkin~
arms are well known in the art and include, by way of example only, conventim-~llinking arms such as those comprising ester, amide, carb~m~te~ ether, thio ether,
15 urea, amine groups and the like. The linking arm can also be a covalent bond. The
linking arm can be cleavable or non-cleavable.
"Cleavable linking arms" refer to linking arms wherein at least one of the
covalent bonds of the linking arm which ~tt~hes the compound to the solid support
can be readily broken by specific chemical reactions thereby providing for co",~unds
20 comprising activated ketone groups free of the solid support ("soluble compoundsn).
The chemical reactions employed to break the covalent bond of the linking arm are
s~lP~ted so as to be specific for bond breakage thereby preventing unintended
reactions occurring elsewhere on the compound. The cleavable linking arm is
s~1Pxt~d relative to the synthesis of the compounds to be forrned on the solid support
25 so as to prevent premature cleavage of this compound from the solid support as well
as not to interfere with any of the procedures employed during compound synthesis on
the support. Suitable cleavable linking arms are well known in the art.
A particularly preferred linking arm is illustrated in the formula:
(saccharide)-NH-(CH2)m-NHC(O)NH-(support)
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wherein m is an integer of from 2 to about 10. Preferably, m is 6.
"Non-cleavable linking arms" refer to linking arms wherein the covalent
bond(s) linking the activated ketone compound to the solid support can only be
5 cleaved under conditions which chemically alters ~Inintende~ parts of the structure of
the compound ~tt~r~ed thereto.
The term "subst~nti~lly homogeneous" refers to collections of molecules
wherein at least 80%, preferably at least about 90% and more preferably at leastabout 95~ of the molecules are a single compound or stereoisomers thereof.
The term "stereoisomer" refers to a chemic~l compound having the same
molecular weight, chemical composition, and constitution as another, but with the
atoms grouped differently. That is, certain iden~ic~l chemi~l moieties are at different
orientations in space and, therefore, when pure, have the ability to rotate the plane of
polarized light. However, some pure stereoisomers may have an optical rotation that
15 is so slight that it is un~et~t~hle with present instrumentation. The compounds
described herein may have one or more asymmetrical carbon atoms and therefore
include various stereoisomers. A11 stereoisomers are included within the scope of the
invention.
When chiral centers are found in the thios~cr~ride derivatives of this
20 invention, it is to be understood that this invention encompasses all possible
stereoisomers. For example, when n is 0 in formula I, the carbon atoms to which R~
and R2 are attached may have an R,R or R,S or S,R or S,S configuration. Simil~rly,
when n is 1, the carbon atoms to which Rl, R2 and R3 are ~tt~rhed may have an
R,R,R or S,R,R or R,S,R or R,R,S or S,S,R or S,R,S or R,S,S or S,S,S
25 configuration.
The term "removable protecting group" or "protecting group" refers to any
group which when bound to a functionality such as hydroxyl, amino, or carboxyl
groups prevents reactions from occurring at these functional groups and which
~protecting group can be removed by conventional chemical or enzymatic steps to
30 reestablish the functional group. The particular removable protecLing group employed
is not critical.
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The term "toxin" refers to a compound produced by an organism which causes
or initi~tps the developln~nt of ~ noxious, poisonous or deleterious effect in a host
presented with the toxin. Such deleterious con-lition~ may include fever, nausea,
diarrhea, weight loss, neurologic disorders, renal disorders, hemorrhage, and the like.
S As used herein, the term "toxin" includes bacterial toxins, such as cholera toxin, heat-
liable and heat-stable toxins of E. coli, toxins A and B of Clostridium difficile,
aerolysins, hemolysins, and the like; toxins produced by protozoa, such as Giardia;
toxins produced by fungi; and the like. Included within this term are exotoxins, i.e.,
toxins secreted by an organism as an extracellular product, and enteroto"ins, i.e.,
10 toxins present in the gut of an organism.
The terms "heat-labile enterotoxin" or "LT" refer to an enterotoxin of
enterotoxigenic E. coli which initi~t~c traveller's diarrhea and related cQr-lition~. This
toxin has a lectin-like activity.
The term "traveller's diarrhea" refers to diarrhea of sudden onset, often
15 accompanied by abdominal cramps, vomiting and fever that occurs sporadically in
traveller's, usually during the first week of a trip. This diarrhea is most commonly
caused by enterotoxigenic E. coli.
The term "cholera" refers to an acute epidemic infectious disease caused by
Vibrio cholerae, wherein a soluble toxin elaborated in the inte5tin~1 tract by the Vibrio
20 alters the permeability of the mucosa, causing a profuse watery diarrhea, extreme loss
of fluid and electrolytes, and a state of dehydration and collapse, but no grossmorphologic change in the intestinal mucosa.
The terms "cholera toxin" or "CT" refer to an ente~ Lin of V. cholerae
which initi~t~s cholera and related contlit;ons. This toxin has a lectin-like activity.
The phrase "inhibit(s) the binding of a toxin to its receptor" means that a
compound inhibits the binding of a toxin to its recep~or by at least 20%. For
example, useful binding inhibition assays may measure inhibition of binding to
ganglioside GD,b or ganglioside GMI~ neutralization of cytotoxic activity, or the like.
Such binding is reported herein as percent toxin activity rem~ining so that those
30 compounds which result in about 80% or less toxin activity ~ ining under the
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bioassay conditions dicclose~ herein are deemed to inhibit the binding of a toxin to its
receptor.
The phrase "inhibit(s) the binding of heat-labile enterotoxin (LT~ andlor
cholera toxin (C~ to an LT and/or CT r~plor" means that a compound inhibits ~e
5 binding of LT andlor CT to an LT and/or CT receptor by at least 20 %.
The phrase "inhibit(s) the binding of an organism to its cell surface r~pt~l-
means that a çompound inhibits the binding of an organism, such as a bacterium, a
virus, a protozoan, a fungus, and the like, to its cell surface receptor. For e~c~mple,
for org~nicms such as Vibro cholera or enterotoxigenic strains of E. coli, a co".po~
10 is said to inhibit binding of an organism to a cell surface leceplor if it reduces binding
of a bacteri~l surface adhesion antigen, such as CFA I pili, by at least 10%.
The term "pharm~reutir~lly acceptable salt" refers to pharmaceuti~lly
acceptable salts of a compound of formula I which salts are derived from a variety of
organic and inorganic counter ions well known in the art and include, by way of
15 example only, sodium, potassium, calcium, m~gnecium, ammonium,
tetraalkylammonium, and the like; and when the mol~ le contains a basic
functionality, salts of organic or inorganic acids, such as hydrochloride,
hydroblolnide, tartrate, mesylate, ~cet~te, m~ tP, oxalate and the like.
For purpose of this application, all sugars are referenced using conven~ionql
20 three letter nomenclature. All sugars are assumed to be in the D-form unless
otherwise noted, except for fucose, which is in the L-forrn. Further, all sugars are in
the pyranose forrn.
General Synthetic Procedures
25 1. Method for Synthesizin~ Thiosaccharide Derivatives
In one aspect, the methods of this invention involve the novel ~ ition of a
thio~cch~ride to a coupling reagent selected from the group concictin~ of Michael
reagents and ~-halocarbonyl co~pollnds.
Specific~lly, the thios~rch~ride derivatives of this invention are typically
30 prepared by reaction of a suitably protected thios~rrh~ride interrn~i~te with an a"B-
unsaturated carbonyl compound or an a-halocarbonyl compound to provide for a
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thiosarch~ride carbonyl compound. The carbonyl group of the thios~cch~ride
carbonyl compound is then optionally reduced to provide for a plurality of alcohol
and/or amine thiosaccharide derivatives. In one embodiment, the alcohol and/or
amine group of the thios~cc~ride derivative is further derivatized to provide for a
5 plurality of thiosaccharide derivatives.
The a"B-unsaturated carbonyl compounds employed in prepa~ g the
thios~ch~ride derivatives of this invention preferably have the general formula (II):
o
Rl-CH=C-C-R2 II
R3
wherein R', R2 and R3 are as defined above; or RlCH=CR2-C(O)XR8, wherein Rl,
R2, R8 and X are as defined above. These compounds are either commercially
available or can be prepared from commercially available materials using art
recognized procedures. For example, such compounds can be pr~ed via a Wittig
reaction from an aldehyde, R'CHO, and a ~-carbonyl phosphorane, such as
(Ph)3PC(R3)C(O)R2.
Preferred ~Y"~-unsaturated carbonyl compounds for use in this invention
include, by way of example, cyclopent-2-en-1-one, 4,4-dimethylcyclopent-2-en-1-one,
cyclohex-2-en-1-one, 4,4-dimethylcyclohex-2-en-1-one, 6,6-dimethylcyclohex-2-en-1-
one, cyclohept-en-1-one, and 3-methylene-2-norbornanone.
The a-halocarbonyl compounds employed in preparing the thios~h~ e
derivatives of this invention preferably have the general formula: W-CHR'-C(O)R2wherein R' and R2 are as defined above, and W is chloro, bromo or iodo. Such
compounds are either commercially available or can be prepa-~d from commerciallyavailable materials using art recognized procedures. Preferred ~-halocarbonyl
compounds for use in this invention include, by way of example, 2-
chlorocyclopentanone and 2-chlorocyclohexanone. Alternatively, carbonyl co..,poul,ds
having a leaving group other than a halogen in the a-position may be employed.
Suitable leaving groups include, by way of illustration, various sulfonic ester groups,
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such as tosylate, mesylate, brosylate and nosylate groups and the like, and fluc..;n3
sulfonic ester groups, such as triflate, nonaflate and tresylate groups and the like.
The sugars employed in this invention are any thiol containing saccharides or
oligosaçch~ndP-s wherein the thiol substitution is at any position of the thioMc~h~ri~.
For example, thio!actose having a thiol (-SH) group at the 1, 2, 3, 6, 2', 3', 4' or 6'
can be used. Methods for chemically modifying saccharides to introduce suitable
substitution are well known in the art as illustrated in Ratcliffe, et al.9 and l~ere.~ces
cited therein as well as by Defaye'~. For example, 1-thiosaccharides can be p-epa~d
by reacting the saccharide with an acylating agent to convert all of the hydroxyl
groups to acyl groups. The 1-acyl group is then selectively converted to the 1-
thioacetyl group by reaction with an excess of thiolacetic acid. Hydrolysis thenprovides for the 1-thiosacch~ride.
Alternatively, selective protection of the hydroxyl groups of the saccharide
provides for one or more free hydroxyl groups which can be converted into
appro~liate leaving groups, such as mesyl or halo groups, by conventional çh~Pmis~ry
well known in the art. Such leaving groups can then be displaced to afford the
corresponding thiol groups. See, for example, International Patent Application Serial
No. PCT/CA92/00242. Specifically, a mesyl group is selectively introduced at one of
the hydroxyl groups and then reacted with a thioacetyl group (for eY~mple pot~ccillm
thio~cet~tP~) to provide for the corresponding thio~r-et~t~ derivative. Tr~tmçnt of this
compound with a-mild base provides for the collcs~nding thio group.
The resulting thiosaccharide is then reacted with a coupling reagent ~Ple~ted
from the group consisting of Michael acceptors and ~-halocarbonyl compounds.
~ypically, this reaction is conductP~ by contacting the thiosa(~ch~ride with at least one
equivalent, preferably 1 to 1.2 equivalents, of the coupling reagent in an inert diluent,
such as dichloromPth~nP, at a temperature of from about -40~C to about 50~C for
about l to about 6 hours to afford a thiosaccharide carbonyl compound. In a
preferred embodiment, when the thiosaccharide reagent is ~tt~chPfl to a solid support,
the coupling reagent is preferable used in excess to maximize the yield of the resulting
thiosaccharide carbonyl compound. Alternatively, when the the coupling reagent is
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?,tt~che~ to a solid support, the thios~ch~ride is preferably used in excess relative to
the coupling reagent.
The carbonyl group of the thiosaccharide carbonyl compound can then be
optionally reduced using a re~ucin~ agent to provide for an alcohol derivative.
S Preferably, this reduction is conducted by con~cting the thio~rch~ride carbonyl
compound with sodium borohydride, preferably about 1.2 to about 2.0 equivalents of
sodium borohydride based on the carbonyl compound. Generally, this reaction is
conducted in an inert diluent, such as tetrahydrofuran, isopropanol and mixture
thereof, at a temperature of about 25~C to about 30~C for about 0.5 to about 3.0hours, to afford the alcohol derivative.
Alternatively, the carbonyl group of the thiosaccharide carbonyl compound can
be reductively ~min~t~Pd to provide for an amine derivative. In this reaction, the
thiosaccharide carbonyl compound is contacted with an excess of ammonium acetateand at least one equivalent of sodium cyanoborohydride based on the carbonyl
compound. This reaction is typically con-lucted in an inert diluent, such as mPth~n- l,
tetrahydrofuran and mixtures thereof, at a temperature of about 25~C to about 30~C
for about 1 to about 72 hours.
The thiosaccharide carbonyl compound can also be reductively ~min~t~ in the
presence of a primary or secondary amine to provide for amine derivatives.
Preferably the amine used in the reductive amination is an amino acid or a derivative
thereof, such as amino acid esters. Typically, this reaction is conducted by collt~ctin~
the thiosaccharide carbonyl compound with a molar excess of an amino acid ester,such as the methyl ester or the te~-butyl ester, preferably with 10 equivalents based
on the carbonyl compound, in the presence of at least one molar equivalent,
preferably about 1.0 to about 1.2 equivalents, of sodium cyanoborohydride.
Typically, this reaction is conducted in an essenti~l]y anhydrous inert diluent, such as
acetonillile, at a ter,peldture of about 25~C to about 30~C for about 1 to about 72
hours. Subsequently, the ester group of the amino acid can be cle~aved using standard
conditions to provide the col~es~onding carboxylic acid.
In a preferred embodiment, the alcohol and/or amine derivatives ~re~ cd as
described above are further derivatized to form a group sPlerte~ from esters,
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substituted amines, amides, carb~m~tes, ureas, thioureas, thioesters and
thiocarb~m~t~s. Methods for derivatizing alcohols and/or amines to provide for such
functional groups are well known to those skilled in the art. For example, alcohols
and arnines can be reacted with acyl halides to form esters and amides, respectively.
Amines can also be reductively alkylated to form substituted ~mines. Similarly,
alcohols and amines can be reacted with isocyantes, among other reagents, to afford
carbarnates and ureas, respectively. Conditions for such reactions are well r~ni7~d
in the art.
Preferred embodiments of this invention are illustrated in Figures 1 and 2.
Figure 1 illustrates the synthesis of various 1-thiogalactose derivatives from cyclohept-
2-en-1-one. Figure 2 illustrates the synthesis of various 1-thiogalactose from 2-
chlorocyclohexanone. It will be readily apparent to those of ordinary shll in the art
that the synthetic procedure illustrated in Figures 1 and 2 and following reaction
conditions can be modified by selecting the applo~liate starting materials and reagents
to allow the preparation of a plurality of 1-thiogalactose derivatives.
As shown in Figure 1, D-galactose is perlauroylated by cont~ctin~ D-~ rt~se
with at least S equivalents, and preferably 10 equivalents, of lauroyl chlonde This
reaction is generally conducted in an inert diluent, such pentane, hexane,
dichloromethane and the like, using a tertiary amine such as pyridine or triethylamine
to neutralize the bydrochloric acid generated during the reaction. Preferably, acatalytic amount of 4-(N,N-dimethylamino)pyridine is added to the reaction mixture to
fa.~ilit~e this reaction. Typically, this reaction is conducted at a l~",l)el~ture of from
about -78~C to about 30~C for about 0.5 to about 96 hours to afford 1,2,3,4,6-penta-
O-lauroyl-a-D-galactopyranose, 1, in approximately 70% yield from D-~al~r-to~
Compound 1 is then converted into 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thi~-
D-galactopyranose, 2, by reaction of I with an excess of thiolacetic acid. In one
embodiment, this reaction is conducted in the presence of an excess of boron
trifluoride etherate, preferably using about 15 to 20 equivalents of boron trifluoride
etherate based on 1, in an inert diluent, such as dichloromethane and the like.
Typically, this reaction is conducted initially at about 0~C and then at about 20~C to
about 30~C for about 0.5 to about 48 hours.
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In another embodiment, compound 2 can be prep~ed from 1 by collt~ct;n~ 1
with at least one equivalent, preferably 1 to 1.2 equivalents, of benzylamine toselectively remove the 1-lauroyl group. This reaction is typically conducted at about
25~C to about 30~C for about l to about 96 hours to provide for 2,3,4,6-tetra-O-
5 lauroyl-(c~"B)-galactopyranoside. This intermediate is then converted into an O-
(2,3,4,6-tetra-O-lauroyl-(~Y"~)-galactopyranosyl) trichloroacetimid~te interme~ te by
contacting the tetralauroyl compound with an excess of trichloroacetonitrile,
preferably about 10 equivalents, and about 0.8 to about 1.0 equivalents, of 1,8-diaza~bicyclo[5.4.0]undec-7-ene (DBU) in an inert diluent, such as dichlorometh~n~.
10 The resulting O-trichloroacetidate intermeAi~te is then contacted with an excess of
thiolacetic acid in an inert diluent, such as dichlorometh~ne, at about 25~C to about
30~C for about 1 to about 96 hours to provide for 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-
1-thio-,B-D-galactopyranose, 2.
In still another embodiment, compound 2 can be prepared by contacting
compound 1 with about 1.5 to about 2.0 equivalents of thiolacetic acid and about 0.5
equivalents of trimethylsilyl trifluoromethanesulfonate based on 1 in an inert diluent,
such as dichloromethane and the like. Typically, this reaction is conducted initially at
about 0~C and then at about 20~C to about 30~C for about 0.5 to about 72 hours.
This method is especiaIly preferred since it provides the highest yield of compound 2
20 and produces no detectable traces of the coll~sponding a-isomer.
If desired, however, the ~-isomer, i.e., 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-
thio-~-D-galactopyranose, can be readily prepared by contacting compound 1 with an
excess, preferably about 20 equivalents, of thioacetic acid in the presence of about 1.0
to l.1 equivalents of tin (IV) chloride in an inert diluent, such toluene, at ambient
25 temperature for about 0.5 to about 2 hours. Alternatively, tre~tment of compound 1
with an excess, preferably about 3 to about 6 equivalents, of thioacetic acid in the
presence of about 2.0 to 3.0 equivalents of trimethylsilyl trifluorometh~nes~llfonate in
an inert diluent, such dichloromethane, at ambient temperature for about 12 to about
48 hours affords 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-l-thio-cY-D-galactopyranose.
.
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The Michael addition of compound 2 to cyclohept-2-en-1-one then affords
cycloheptanon-3-yl 2,3,4,6-tetra-O-lauroyl-1-thio-~-D-galactopyranoside, 3. Thisreaction is typically conducted by contacting 2 with at least one equivalent, ~ fe,ably
1.0 to 1.2 equivalents, of cyclohep-2-en-1-one in the presence of a molar excess of a
S dialkylamine, such as diethylamine.
Without being limited by any theory, it is believed that the dialkylamine first reacts
with the thio~cetyl of compound 2 thereby forming in situ the thiol derivative of
compound 2 which then reacts under basic conditions generated by the dialkylamine
with a Michael adduct.
Typically, this reaction is conducted in an inert diluent, such as
dichloromethane, at a temperature of from about -40~C to about 50~C for about 1 to
about 6 hours.
The carbonyl group of compound 3 can then reduced using a reducin~ agent to
provide for 3-hydroxycycloheptyl 2,3,4,~tetra-O-lauroyl-1-thio-~B-D-
15 galactopyranoside, 4. Preferably, this reduction is con~ucte~ by cont~rting 3 withsodium borohydride, preferably about 1.2 to about 2.0 equivalents of sodiumborohydride based on 3. Generally, this reaction is conducted in an inert diluent,
such as tetrahydrofuran, isopropanol and mixture thereof, at a temperature of about
25~C to about 30~C for about 0.5 to about 3.0 hours. The resulting alcohol, 4, is
20 readily purified by solid-phase extraction on Cl8 silica gel using pentane as an eluent.
Removal of the lauroyl groups from alcohol 4 is then accomplished by treating
4 with an excess of sodium methoxide in methanol and an inert diluent, such as
dichloromethane, at about 25~C to about 30~C for about 1 to about 24 hours.
Neutralization of the reaction mixture with Amberlite IR-50S (H+~ resin then provides
25 for 3-hydroxycycloheptyl 1-thio-,B-galactopyranoside, A5.
Alternatively, compound 3 can be reductively ~min~t~A to provide for 3-
aminocycloheptyl 2,3,4,6-tetra-O-lauroyl-l-thio-,B-D-galactopyr~nosi~e, 5. In one
embodiment of this reaction, compound 3 is contacted with an excess of ammonium
acetate and at least one equivalent of sodium cyanoborohydride based on 3. This
30 reaction is typically conducted in an inert diluent, such as met~nol, tetrahydrorul~n
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and mixtures thereof, at a temperature of about 25~C to about 30~C for about 1 to
about 72 hours.
In another preferred embo~iment, the reductive amination reaction is
accomplished by cont~rting compound 3 with an excess of ammonium acetate and an
5 excess of trimethyl orthoformate based on 3, in an inert ~iluent, such as 1,2-dichloroethane at a temperature of about 25~C to about 30~C for about 12 to about 72
hours to form an imine intermeAi~tP. The imine interrnedi~t~ is generally not j~l~tJ~
but is contacted in situ with an excess of sodium borohydride, preferably about 1.2 to
about 1.5 equiva}ents of sodium borohydride, based on 3. The resulting amino
10 compound 5 is then readily purified by solid-phase extraction on C18 silica gel using
pentane as an eluent.
Optionally, the amine group formed by reductive amination can be acylated
with conventional acylating agents under conventional conditions. The acylating agent
is generally of the formula L-C(O)R6 where L is a leaving group such as a halide, an
15 activated ester, and the like.
The lauroyl groups are removed from compound 5 by cont~tin~ 5 with an
excess of sodium methoxide in methanol and an inert diluent, such as
dichlorometh~ne, at about 25~C to about 30~C for about 1 to about 24 hours.
Neutralization of the reaction mixture with Amberlite IR-50S (H+) resin then provides
20 for 3-aminocycloheptyl 1-thio-,B-galactopyranoside, B5.
In one example, the primary amine group of compound B5 can optionally be
acylated by contacting B5 with an excess of acetic anhydride in meth~nQl containing a
trace of water. Generally, this reaction is conducted at about 25~C to about 30~C for
about 2 to about 24 hours to provide for 3-~rePmidocycloheptyl l-thio-~B-
25 galactopyranoside, CS.
Alternatively, the primary amine group of 5 can be acylated with phthalicanhydride before removal of the lauroyl groups to provide for 3-(2-
carboxybenzamido)cycloheptyl 2,3,4,6-tetra-O-lauroyl-1-thio-~-D-galactopyranoside,
6. This reaction is typically conducted by contacting compound 5 with at least one
30 molar equivalent, preferably with an excess of phthalic anhydride. Preferably, this
reaction is conducted in dry pyridine containing a catalytic amount of 4-(N,N-
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dimethylamino)pyridine. The reaction is typically conducted at about 25~C to about
30~C for about 12 to about 48 hours to provide for compound, 6. Removal of the
lauroyl groups from 6 is then ~ccomplished by treating 6 with sodium methoxide in
methanol and an inert diluent, such as dichlorometh~ne, at about 25~C to about 30~C
for about 1 to about 24 hours. Neutralization of the reaction mixture with ~mberlit~
IR-SOS (H+) resin then provides for 3-(2-carboxyben7~midQ)cycloheptyl l-thio-~-D-
galactopyranoside, DS.
As shown in Figure 1, compound 3 can also be reductively ~min~tçd with an
amino acid ester to provide for interm~Ai~t~s 7 or 8. Specifically, compound 3 is
10 contacted with a molar excess of ,~-alanine tert-butyl ester, preferably with 10
equivalents based on 3, in the presence of at least one molar equivalent, preferably
about 1.0 to about 1.2 equivalents, of sodium cyanoborohydride. Typically, this
reaction is conducted in an essentially anhydrous inert diluent, such as acetonitrile, at
a temperature of about 25~C to about 30~C for about 1 to about 72 hours. The
15 resulting intermeAi~te 7 is readily purified by solid-phase extraction on C18 silica gel
using pentane as the eluent.
The tert-butyl ester group of compound 7 is readily hydrolyzed to the
co~ onding carboxylic acid by treating 7 with an excess of trifluoroacetic acid in an
inert diluent such as dichlorometh~ne. This reaction is typically conducted at about
20 25~C to about 30~C for about 1 to about 10 hours. The lauroyl groups of the
rçslllting carboxylic acid intermçdi~te are then removed using sodium methoxide in
methanol as described above to provide for N,B-[l-(l-thio-,B-D-
galactopyranosyl)cyclohept-3-yl]-~ nine7 F5.
In a similar manner, compound 3 can be reductively ~ n~te~ using other
25 amino acid esters, such as glycine tert-butyl ester, L-leucine ter~-butyl ester, L-
histitline methyl ester, L-tryptophan methyl ester, and L-arginine methyl ester, to
provide for intermediate 8. In those cases where the amino acid ester employed is a
tert-butyl ester, the tert-butyl ester is cleaved as described above using trifluoroacetic
acid to afford N~-tl-(l-thio-,B-D-galactopyranosyl)cyclohept-3-yl]-glycine, E5, and
30 No~-[ 1-(1-thio-B-D-galactopyranosyl)cyclohept-3-yl]-L-leucine, G5. Alternatively, in
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those cases where an amino acid methyl ester is employed, the lauroyl groups of
interm~Ai~te 8 are preferably removed before cleaving the methyl ester by tre~trn~-nt
of 8 with sodium methoxide in methanol as described above. Subsequently, the
methyl ester of the amino acid moiety is cleaved to the corresponding carboxylic acid
5 by tre~tm~nt with an excess of aqueous lithium hydroxide for about 0.5 to about 2
hours. Neutralization of the reaction mixture with Amberlite IR-SOS (H+) resin then
provides for NcY-[l-(1-thio-1~-D-galactopyranosyl)cyclohept-3-yl]-L-hictilline, H5, Ncr-
[l-(1-thio-,~-D-galactopyranosyl)cyclohept-3-yl~-L-tryptophan, I5, and Nol-[1-(1-thio-,B-
D-galactopyranosyl)cyclohept-3-yl]-L-arginine, J5.
Additionally, if desired, the hydroxyl group of alcohol derivatives, such as
compound 4, can be converted into a leaving group, such as the mesylate, tosylate,
etc., and displaced with various nucleophiles. For example, treatment of an alcohol
derivative with an excess, preferably about 1.1 to about 1.5 equivalents, of
meth~nesulfonyl chloride in pyridine and an inert diluent, such as THF, affords the
corresponding mesylate. The mesylate group can then be ~ pl~ced with, for
example, sodium azide to provide the corresponding azido derivative. This reaction is
typically conducted by contacting the mesylate compound with an excess, preferably
about 5 to about 50 equivalents of sodium azide in an inert diluent, such as N,N-
dimethylformamide, THF and mixtures thereof, at a temperature of from about 50~Cto about 100~C for about 1 to about 6 hours. Preferably, a crown ether, such as 18-
crown-6, is added to the reaction mixture to promote the displ~cement re?~ction
The azido derivative can then be reduced with a reduçing agent to afford the
cGl-esponding primary amine, i.e., a compound such as 5. Preferably, this reaction
is conducted by contacting the azido compound with about 1.0 to about 1.1
equivalents of sodium borohydride and about 2.0 to about 2.2 equivalents of nickel
chloride (NiCl2) in an inert diluent, such as ethanol, isopropanol, or mixtures thereof,
at a temperature of from about 0~C to about 50~C for about 0.5 to about 6 hours.Removal of the lauroyl protecting groups can then be accomplished using the
procedures described above.
Additionally, the primary amine group of amino compounds such as 5 can be
further derivatized by reductive alkylation to afford a secondary amine. Typically,
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this reaction is conducted by contacting the primary amine with an excess, preferably
about 2 to about 500 equivalents of an aldehyde or a kefone in the presence of at least
one equivalent, preferably about 1.0 to about 10 equivalents, of a reduçin~ agent,
such as sodium triacetoxyborohydride. This reaction is typically conduct~ in an inert
S diluent, such as dichlorometh~ne, methanol, or mixtures thereof, at a te.~ dture of
about 0~C to about 50~C for about 10 to about 48 hours. In a prefel.~d embo~ en~the ketone employed in this reaction is a cyclic ketone including, by way of e~mp'~,
cyclobutanones, such as 3,3-dimethylcyclobutan-1-one; cyclopentanones, such as 3,3-
dimethylcyclopentan-1-one; cyclohexanones and cycloheptanones.
The lauroyl groups of the resulting secondary amine are then removed by
contacting the lauroyl-protected compound with an excess of sodium methoxide in
mPth~nol and an inert diluent, such as dichlorometh~ne, at about 25~C to about 30~C
for about 1 to about 24 hours. Neutralization of the reaction mixture with Amberlite
IR-SOS (H+) resin then provides the desired secondary amine compound.
As noted above, Figure 2 illustrates the synthesis of various 1-thio~ rtrse
derivatives using an a-halocarbonyl carbonyl compound, i.e., 2-chlorocycloh~T~n~As shown in Figure 2, 1-S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-,B-D-galactopyranose,
2, prepared as described above, reacts with 2-chlorocyclohexanone to give
cyclohexanon-2-yl 2,3,4,6-tetra-0-lauroyl-1-thio-~-D-galactopyranoside, 9. This
reaction is typically conducted by cont~cting 2 with at least one equivalent, preferably
1.0 to 1.2 equivalents, of 2-chlorocyclohexanone in the presence of an excess of a
dialkylamine, such as diethylamine. Typically, this reaction is conducted in an inert
diluent, such as dichloromPth~ne, at a te~-pe,dture of from about -40~C to about 50~C
for about 1 to about 6 hours to afford compound 9.
Compound 9 can then be reacted using the same reagents and condi~ions
described above for compound 3 to afford various l-thiogalactose derivatives.
Specifically, compound 9 is reduced with sodium borohydride to provide 10 which,after removal of the lauroyl groups, affords 2-hydroxycyclohexyl l-thio-,B-D-
galactopyranoside, A2.
Altematively, compound 9 is reductively ~min~t~d with ammonium acetate and
sodium cyanoborohydride to provide for interme~ te 11 which, upon removal of the
CA 02256694 1998-11-24
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lauroyl groups, affords 2-aminocyclohexyl l-thio-,B-D-galactopyranoside, B2.
Compound B2 can then be acylated with acetic anhydride to give 2-
~et~midocyclohexyl 1-thio-B-D-galactopyranoside, C2. Alternatively, interrneAi~
11 can be acylated with phthalic anhydride to provide for interme~ te 12 which
S affords 2-(2-carboxyben7~mitlocyclohexyl l-thio-~-D-galactopyr~noside, D2, by
removal of the lauroyl groups using the conditions described above.
Additionally, compound 9 can be reductively ~min~ted using an ,B-alanine tert-
butyl estèr to provide for intermediate 13 which then affords NB-tl-(l-thio-~-D-galactopyranosyl)cyclohex-2-yl]-~-alanine, F2, upon deprotection. Alternatively,10 compound 9 can be reductive ~min~tecl with other amino acid esters, such as glycine
tert-butyl ester, L-leucine tert-butyl ester, L-hi~tidine methyl ester, L-tr~lophan
methyl ester, and L-arginine methyl ester, to provide intermedi~t~ 14 which uponde~loteclion, affords NcY-[l-(1-thio-~-D-galactopyranosyl)cyclohex-2-yl]-glycine E2,
N~-[1-(1-thio-,~-D-galactopyranosyl)cyclohex-2-yl]-L-leucineG2, Na-tl-(l-thio-,B-D-
15 galactopyranosyl)cyclohex-2-yl]-L-histidine H2, Nc~-[1-(1-thio-,~-D-
galactopyranosyl)cyclohex-2-yl]-L-tryptophan ~2, and Na-[1-~1-thio-,~-D-
galactopyranosyl)cyclohex-2-yl]-L-arginine J2.
Optionally, the s~cch~ride derivatives of formula I wherein Y is a sulfide
linking group (-S-) can be oxidized using conventional reagents and con~litions to
20 provide the corresponding sulfoxide (Y = -S(O)-) and sulfone (Y = -SO2-)
derivatives. Suitable reagents for oxidizing a sulfide compound to a sulfoxide
include, by way of example, hydrogen peroxide, peracids such as 3-
chlolopeloxybenzoic acid (MCPBA), sodium periodate, sodium chlorite, sodium
hypochlorite, calcium hypochlorite, tert-butyl hypochlorite and the like. Chiral25 oxidizing reagents (optically active reagents) may also be employed to provide chi~al
sulfoxides. Such optically active reagents are well known in the art and include, for
example, the reagents described in Kagen et al.'l and references cited therein.
The oxidation reaction is typically conducted by contacting the s~ch~ri(le
derivative with about 0.95 to about 1.1 equivalents of the oxidizing reagent in an inert
30 diluent, such as dichloromethane, at a temperature ranging from about 0~C to about
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50~C for about 1 to about 48 hours. The resulsing sulfoxide can then be further
oxidized to the cor.. sponding sulfone by cont~Gting the sulfoxide with at least one
additional equivalent of an o~ in~ reagent, such as hydrogen peroxide, MCPBA,
potassium perm~n~n~tÇ and the like. Alternatively, the sulfone can be ~
S directly by cQnt~ting the sulfide with at least two equivalents, and preferably an
excess, of the oxidi7ing reagent.
In a similar manner, the ~ch~rlde of formula I, wherein R4 is -XR5, X is
sulfur and R5 is a defined substituent other than hydrogen, can be oxidized to afford
the co~,es~onding sulfoxide (X = -S(0)-) and sulfone (X = -S02-) derivatives.
Additionally, if desired, the hydroxyl groups of the saccharide moiety may be
readily acylated, sulfonylated- or phosphorylated using art recognized procedures and
reagents to provide compounds of formula I wherein at least one of the hydroxyl
groups of the saccharide is -O-SO2-OH, -C(O)RI~, -P(O)(ORIl)2 or pharn-~ceutir~lly
acceptable salts thereof, where Rl~ and Rll are as defined above. Such acylation15 re~ctions may occur as an initial step of the synthesis (i.e., using an acyl halide, such
as lauroyl chloride, as described above~ or as a post-synthetic transformation of
compounds of formula I using, for eY~mple, acyl h~lides, anhydrides, halophosph~ -s
sulfur trioxide, and the like.
For example, a de-blocked hydroxyl group can be sulfonylated by treating the
20 hydroxy-containing compound with an excess, preferably about 1.1 to about 1.2equivalents, of a pyridine:sulfur trioxide complex in an inert diluent, such as N,N-
dimethylform~mide, at ambient te,l.p~l~ture for about 1 to about 24 hours. Typically,
the resnlting sulfate (i.e., -O-SO2-OH) is isolated as its salt by tre~tnlent with, for
example, a Na+ resin in an inert ~iluent, such as m~th~nol. Further reaction --
25 conditions suitable for forming s-llf~tes and phosphates can be found, for eY~mrl~, in
U.S. Patent No. 5,580,858'2.
The methods illustrated in Figures 1 and 2 were con~ucted in a solution phase.
Surprisingly, these methods can also be con~ucted on the solid phase using reaction
~con-litions similar to those described above for the solution phase. When conduc~d
30 on the solid phase, one of the reagents employed is attached to a solid support via a
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cleavable or non-cleavable linking arm. Such linking arms are well known in the art
as well as their ~tt~chment to either the thios~cçh~ride or the coupling reagent.
Either of the reagents can be ~tt~hed to the solid support without criticality
provided that the attachment does not alter the reactivity of the reagent. For example,
S a linking arm may be covalently ~tt~ched to any position of the thio~ ch~ride other
than the thiol group. Such ~tt~rhm~nts are preferably made through, for example, an
ester or ether linkage to one the hydroxyl group of the thiosAc~h~ride. A pref~
linking arm is derived from succinic acid.
By way of example, 1-dithioethyl-~B-D-galactopyranoside is readily ~tt~hed to
a trityl chloride resin having about 0.80 to about 1.00 mmol/g of active chlorine by
contacting the resin with about 0.75 to about 2.0 equivalents of l-dithioethyl-~B-D-
galactopyranoside in pyridine containing a catalytic amount of 4-(N,N-
dimethylamino)pyridine at a telllpelature ranging from about 25~C to about 100~C for
about 12 to 48 hours. A free thiol group at the 1-position of the covalently bound
15 galactose is then generated by treating the resin with dithiothreitol (C'l~l~nd's reagent)
and triethylamine in an inert diluent, such as methanol, for about 6 to 24 hours at
ambient ten-pe,dture. The resulting l-thio-~B-D-galactopyranoside is then reacted as
described above to afford a 1-thiogalactose derivative of formula I covalently ~tt~Çh~d
to the solid support resin. If desired, the 1-thiog~l~ctQse derivative can be cleaved
20 from the solid support resin by cont~cting the resin with an excess of trifluoroacetic
acid and triisopropylsilane in an inert diluent, such as dichlorometh~ne, at ambient
te---pe,dture.
Similarly, a linking arm can be covalently ~t~hed to any position of the
coupling reagent provided that the point of ~t~-hment does not interfere with the
25 Michael addition of the thios~cch~ride to the a"B-unsaturated carbonyl group or with
the displacement of the halide from the ~-halocarbonyl compound by the
thiosacch~ride Accordingly, the linking arm is preferably ~tt~rh~d to the coupling
reagent through any one of substituents R'-Ra via a covalent bond. Such linkage can
be through, for example, an ester, ether, amine, amide, or urea functional group and
30 the like.
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By way of example, a carboxylic acid moiety can be covalently ~tt~hed to an
~min~ted solid support using convention~l coupling procedures and reagents.
Typically, such a coupling reaction will be conducte~d using well-known co.~.l;n~
reagents such as carbo~liimides, BOP reagent (benzotriazol-1-yloxy-
tris(dimethylamino)phosphonium hexafluorophosphonate) and the like. Suitable
carbo~iimides in~ de, by way of example, dicyclohexylcarbo~iimide (IDCC),
diisopropylcarbodiimide~ 1-(3-dimethylaminopropyl)-3-ethylcarbo liimi-~e (EDC) and
the like. Preferably, a well-known coupling promoter, such as N-hydroxysucrinimi~e,
1-hydroxybenzotriazole and the like, is also employed in the reaction IlliX~ c; to
f~ilit~te the coupling reaction.
The coupling reaction is typically conducted by contacting the solid support
with an excess, preferably about 1.1 to about 10 or more equivalents, of the
carboxylic acid-cont~ining compound (based on the number of equivalents of arnino
groups present on the solid support) and at least one equivalent, preferably about 1.5
to about 3.0 equivalents, of the coupling reagent (based on the carboxylic acid groups)
in an inert diluent, such N,N-dimethylformamide and the like. If desired, least one
equivalent, preferably about 1.5 to about 3.0 equivalents (based on the l-thiog~l~r~se
derivative), of a coupling promoter such as l-hydroxybenzotliazole may also be used
in the reaction. Generally, the coupling reaction is conducted at a le~ ture
ranging from about 0~C to about 50~C for about 24 to about 100 hours. Upon
completion of the reaction, the solid support is preferably co~t~cte~ with excess acetic
anhydride in methanol at a temperature ranging from about 0~C to about 40~C for
about 12 to about 24 hours to cap any unreacted amino groups present on the solid
support. The yield of inco.yGld~ion of a ~hio~-ch~ride onto the solid s~lppoll can be
determined using well-established procedures such as those described, for eY~mple~ by
M. Dubois et al.~3.
2. Method for Preparing A Thiosaccharide Derivative Library
In another aspect, the methods of this invention provide for a thioc~ch~ride
derivative library. Such libraries are produced by synthe-~i7in~ on each of a plurality
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of solid supports a single compound wherein each compound comprises a
thios~ch~ride derivative.
The thio~l~ch~ride derivative libraries provided by this invention are
syn~hesi7çd by first apportioning solid supports among a plurality of reaction vessels.
5 Such S~lppOll~ comprise a reactive functional group capable of covalently binding to
the solid support. The function~l group is one that is capable of covalently binding a
thios~ch~ride at a position other than the thiol group. Suitable functional groups
include, by way of example, alcohols, amines, isocyanates, carboxylic acid groups,
esters and the like. In one embo~iment this is accomplished by selectively blocking
10 the thiol group with a removable blocking group which, after coupling of the
thiosaccharide to the solid support, is removed thereby freeing the thiol group for
further reaction.
The supports in each reaction vessel are then cont~-t~d with a unique
thio~( ch~ride under conditions wherein the thio~ ch~ride is covalently ~t ~-hed to
15 the solid supports through the reactive filncti~n~l group. This reaction is typically
con~lucted by contacting the solid support with at least one equivalent, pf~feldbly 1 to
S equivalents, of the thiosaccharide based on the functional groups on the solidsupport.
After ~tt~hing the thiosaccharide to the solid support, the su~ s are then
20 pooled and the pooled supports are then apportioned among a plurality of reaction
vessels.
The supports having a thio~cch~ride covalently ~tt~chçd thereto are then
contacted in each reaction vessel with a unique coupling reagent sP-l~ct~ from the
group consi~tin~ of Michael accepto~ and ~-halocarbonyl compounds to provide for a
25 thio~h~ride carbonyl compound which covalently bound to the ~.lppoll. This
reaction is preferably conducted as described above.
The thios~cçh~ride carbonyl compound is then reduced as described above to
provide for an alcohol and/or an amine derivative. Optionally, the hydroxy or amino
group of these compounds can be further derivatized as described above to form a30 group selected from esters, substituted ~minçs, ~ çs, carb~m~tPs, ureas, thioesters
and thiocarb~m~t~s
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In an alternative embo~liment the thiosaccharide derivative libraries provide bythis invention are syntheci7ed by first apportioning solid suppo~Ls among a plurality of
reaction vessels wherein such S~pOlls comprise a reactive filnrtion~l group covalently
bound to the solid support such that the funrtio~ group one that is capable of
covalently binding a coupling reagent. Such functional groups include, by way ofexample, alcohols, ~minPs~ isocyanates, carboxylic acid groups, esters and the like.
The supports in each reaction vessel is then cont~-tPd with a unique coupling reagent
selP~cted from the group concicting of Michael acceptfjl~ and c~-halocarbonyl
compounds under conditions wherein the coupling reagent is covalently ~tt~rhed to the
10 solid supports through the reactive functional group. Typically, this reaction is
conducted by contacting the solid support with at least one equivalent of the coupling
reagent, preferably with about 1 to about 5 equivalents, based on the funrtio
groups on the solid support.
After ~tt~hing the coup}ing reagent to the solid support, the s~lppoll~ are then15 pooled and the pooled supports are then apportioned among a plurality of reaction
vessels.
The supports having a coupling reagent covalently ~ttach-P~ thereto are then
contacted in each reaction vessel with a unique thio~rçh~ride to provide for a
thios~cch~ride carbonyl compound which is covalently bound to the suppoll. This
20 reaction is preferably contiucte~ as described above. The thiosacch~ride carbonyl
compounds can then be reduced to provide for a plurality of alcohol and/or aminederivatives. As above, these alcohol and/or amine derivatives can optionally be
further derivatized to provide for a group SPl~Pc~f ~ from esters, substituted ~min
~mides, carb~m~tes, ureas, thioesters, and thiocarb~m~tes.
In a preferred embo~iment~ an identifier tag is employed in the metho~s of this
invention. The identifier tag has a recognizable feature that is, for eY~mp'e,
microscopically or otherwise distinguishable in shape, size, mass, charge, or color.
This recognizable feature may arise from the optical, çh~.mic~l, electronic, or
m~gmPtic properties of the tag, or from some combination of such pr~.Lies. In
30 essence, the tag serves to label a molecule and to encode information ~er;phf able at
the level of one (or a few) molecules or solid supports. By using i~PntifiPr tags to
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track the synthesis pathway that each rnember of a chemic-~l library has taken, one can
deduce the structure of ~ny chemi~l in the library by reading the id~ntifier tag.
The identifier tags identify each reagent or other reaction step that an
individual library member or solid support has experienced and record the step in the
5 synthesis series in which each reagent was added or other ch~mic~l reaction
performed. The tags may be ~tt~ched immedi~tely before, during, or after the reagent
addition or other reaction, as convenient and compatible with the type of idpntifi~or
tag, modes of ~t~ hment, and chemistry of activated ketone or other molecular
synthesis. The i~lentifi~r tag can be ~ccoci~ted with the thioc~cch~ride derivatives
10 through a variety of m~rh~ni~m~, either directly, through a linking mol~nle, or
through a solid support upon which the thiosaccharide derivative is synthesi7~d. In
the latter mode, one could also attach the tag to another solid support that, in turn, is
bound to the solid support upon which the thio~ch~-ide derivative is syn~h~i7~d
The identifier tag is added when the solid supports that have undergone a specific
15 reagent addition or other chemic~l reaction step are physically together and so can be
tagged as a group, i.e., prior to the next pooling step. P~felr~d iclentifi~- tags
include, by way of example, peptides'4 l5 oligonucleotidesl6 and halocarbon
derivatives'7.
20 3. Screening of Thiosaccharide Derivative Libraries
The libraries of thiosa~c~l~ride derivatives (e.g., compounds of forrnula I) maybe screened for biological activity. Generally the library to be screen is e.~l)osed to a
biological substance, usually a protein such as a r~ceptor, enzyme, ~ .llbld~ c binding
protein or antibody, and the presence or ~hsence of an interaction between the
25 thiosaccharide derivative and the biological substance is deterrnined. Typically this
will comprise determining whether the biological substance is bound to one or more
of the members of the library. Such binding may be determined by ~t~ching a label
to the biological substance. Commonly used labels include fluorescent labels. Other
methods of labeling may be used, such as radioactive labels. The degree of binding
30 affinity may be determined by quantitating the amount or intensity of the bound label.
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Thus, various lead compounds may be select~ by identifying which compounds bind
the particular biological substance most effectively.
In a preferred embo~limPnt, bead-based libraries are scr~n~d by assays in
which each different molecule in the library is assayed for its ability to bind to a
S receptor of interest. The recepto~ is contacted with the library of thio~r~h~n~e
derivatives, forming a bound member between the receptor and any thioc~-rh~nde
derivative in the library able to bind the l~ceptor under the assay con~itinnc. The
bound thiosaccharide derivative is then identified by eY~min~tion of the tag ~C~i~t~d
with that thio~ch~ride derivative. The receptor to which the library is e ~pos~
10 under binding conditions can be a mixture of receptors, each of which is ~soci~fd
with an idçntifi~r tag specifying the receptor type, and consequently two tags are
ex~mine~ after the binding assay. Specific beads can be isolated in a receptor
screening by a number of means, including infinite dilution, micromanipulation, or
preferably, flow cytometry (e.g., fluorescçnce activated cell sorting (FACS)). By
15 adopting cell-sized solid SUppOltS or beads, one can use flow cylolllc~ly for high
sensitivity receptor binding analysis and facile bead manipulation.
Thiosaccharide derivatives can be synthe~i7çd on beads and cleaved prior to
assay. Cleavage of the thiosaccharide derivatives from the beads may be
accomplished cleavable linker arms which are cleaved using conventional methods. In
20 either event, the thiosaccharide derivatives of interest are cleaved from the beads but
remain cont~ined within the colllp~lment along with the bead and the identifiP,~ tag(s).
Soluble tagged thiosacch~ e derivatives can also be screened using an
immobilized receptor. After cont~cting the tagged thiosa~ch~ride deAvatives with the
immobilized receptor and washing away non-spe~ific~lly bound mQIt-rlJl~, bound,
25 tagged thiosaccharide derivatives are released from the receptor by any of a wide
variety of methods. The tags are optionally amplified and then ex~min~ and ~ ed
to identify the structure of the molecules that bind spe~ific~lly to the ~ tor. A
tagged thiosaccharide derivative in solution can be assayed using a l~cep~or
immobilized by attachment to a bead, for example, by a co~ lition assay with a
30 fluorescently labeled ligand. One may recover the beads bearing immobilized
receptors and sort the beads using FACS to identify positives (~iminished fluo~ n~e
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caused by the library molecule competing with the labeled ligand). The ~c~ qt~d
identifier tag is then amplified and decoded.
Preferably, the libraries described herein will contain at least about 2
compounds, more preferably at least about 102 compounds, still more ~,ef~..bly from
S about 102 to about 101~ compounds and even still more preferably from about lO3 to
about 106 compounds.
Of particular interest is the identification of thios~ch~ride derivatives which
block binding of a toxin, such as heat-labile en~eiuto~in or cholera toxin, the toxin's
f~ceplor either in vitro or in vivo, and compounds which inhibit binding of or~nismS
(e.g., bacteria, virus, fungi, and the like), including enterovirulent organism such as
~brio cholerae and enterotoxigenic strains of Escherichia coli, to their cell surface
receptors.
The following synthetic and biological examples are offered to illustrate this
invention and are not to be construed in any way as limiting the scope of this
invention. Unless otherwise stated, all temperatures are in degrees CPI~;IIS.
EXAMPLES
In the e-~mples below, the following abbreviations have the following
me~ning~. If an abbreviation is not defined, it has its generally accepted rn~nine.
A = angstroms
bd = broad doublet
bs = broad singlet
d = doublet
dd = doublet of doublets
DMAP = dimethylaminopyridine
eq. = equivalents
g = grams
L = liter
m = multiplet
meq = milliequivalent
mg = milligram
mL = milliliter
mmol = millimol
N = normal
q = quartet
quint. = quintet
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s = singlet
t = triplet
TFA = - trlfluoroacetic acid
THF = tetrahydrofuran
TLC = thin layer chromatography
~L = microliter
IH-Nmr spectra were recorded with a Brueker AM-360 spe.;LIonleter and
MALDI-TOF mass spectra were recorded with a HP G2020A (LD-TOF) insL,u.ll.,.~t.
10 Optical rotations were measured with a Perkin-Elmer 241 pol~rimeter ~oactiorls
were monitored by TLC on Silica Gel FG254 (E. Merck, Darrnstadt, Ge~ any).
Example A
Solid-Phase Extraction of Lauroylated Intermediates
As indicated in the following examples, certain lauroylated reaction
intermediates were purifled by solid-phase extraction. In this purification procedure,
the reaction mixture is concentrated, re-dissolved in meth~nol, and applied onto C18
silica (Waters Prep C18, 125 A, 1 g per 20 mg lauroylated carbohydrate). The C18silica is then washed with mPth~nol (10 mLt g C18 silica) and the product is eluted
20 with pentane (10 mL/ g C18 silica). For L-arginine cont~ining colll~unds, thereaction mixture is concentrated, re-dissolved in 70% mPth~nol and applied onto C18
silica. The C18 silica is then washed with 70% methanol and the product is eluted
with meth~nol. The res~ltin~ product contains no residual reagents as deteln~ined'by
TLC, 'H-nmr, or MALDI-TOF mass speclroscopy.
Example B
Synthesis of
1.2~3~4~6-Penta-O-lauroyl-~Y-D-galactopvranose 1
To a suspension of galactose (3.78 g, 21.0 mmol), pyridine (50 mL), and 4-
30 dimethylaminopyridine (cat.) in pentane (150 mL) under argon ~tmosphPre, was added
lauroyl chloride (50 mL, 210 mmol) at -78~C. The mixture was allowed to reach
ambient l~ul~eldture~ The resulting white slurry slowly dissolved and a fine
precipit~tP of pyridinium hydrochloride formed~ After 40 h, the pyridinium
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hydrochloride was filtered off and the pentane solution was concentrated. Columnchromaeography (SiO2, pentane/EtOAc 9:1) gave 1 (16.0 g, 70% yield), ta~]D25 +39~
(c 0.9, CHCI3). 'H-Nmr data (CHC13): ~ 6.39 (d, lH, J 2.4 Hz, H-l), 5.51 (br s, lH,
H-4), 5.35 (m, 2H, H-2 and H-3), 4.32 (br t, lH, J 6.6 Hz, H-5), 4.08 (d, 2H, J
6.6 Hz, H-6a and H-6b), 2.39, 2.38, 2.30, 2.26 (4 t, 2H each, J 7.5 Hz, -CH2C~),2.21 (m, 2H, -CH2CO-), 0.88 (t, 15 H, J 7.0 Hz, -CH3). Anal. Calcd for C66H,22O~:
C, 72.2; H, 11.3. Found: C, 72.6; H, 11.5.
Example C
Synthesis of
l-S-Acetyl-2.3.4.6-tetra-O-lauroyl-l-thio-B-D-~alactopyranose (2)
Method 1: To compound 1 (from Fy~mple B, 1 g, 0.91 mmol) and thiollr~
acid (0.4 mL, 9.1 mmol) in dry dichlorometh~ne (5 mL) under argon at 0~C, was
added boron trifluoride etherate (1.7 mL, 13.6 mmol). The cold-bath was removed
after 10 min and after 24 h the mixture was diluted with dichlororneth~ne, washed
with saturated sodium bicarbonate, dried over sodium sulfate, and conc~ntrated.
Column chromatography (SiO2, pentane/Et20/EtOAc 9:1:1) gave 2 (0.60 g, 70%
yield).
Method 2: To compound 1 (from Example B, 276.5 mg, 0.253 mmol) in dry
tetrahydrofuran (2.0 mL) under argon, was added benzylamine (27.9 ~L, 0.255
mmol). The mixture was concen~rated after 70 h. The residue was dissolved in drydichloromethane (4.0 mL) under argon and then trichloroacetonitrile (250 ~LL, 2.5
mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (30 ~L, 0.2 mmol) were added. The
mixture was concentrated after 3 h and the residue was flashed through a short
column (SiO2, pentane/EtOAc 19:1), then concentrated. To the residue in dry
dichloromethane (3.5 mL) under argon, was added thiolacetic acid (1 mL). After 96
h, the reaction mixture was concentrated and the residue was purified by column
~ chromatography (SiO2, pentane, EtOAc 19: 1) to give 2 (90 mg, 37% yield), [~Y}D25
21~ (c 1, CHCl3). 'H-Nmr data (CHCl3): ~ 5.47 (d, lH, J 3.4 Hz, H-4), 5.32 (t, lH,
J 10.0 Hz, H-2), 5.25 (d, lH, J 10.0 Hz, H-l), 5.12 (dd, lH, J 3.4 and 10.0 Hz, H-
~ .... . . . ....
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3), 4.08 (m, 3H, H-S, H-6a and H-6b), 2.14-2.43 (m, 8H, -CH2CO-), 2.37 (s, 3H, -SAc), 0.88 (t, 15 H, J 7.0 Hz, -CH3). Anal. Calcd for C56HI02Olos C, 69.5; H,
10.6; S, 3.3. Found: C, 69.4; H, 10.8; S, 3.5.
S Method 3: To compound 1 (20.0 g, 18.2 mmol) and thio~ tic acid (5.0 mL,1.9 eq.) in dry dichlorome~h~ne (300 mL) under argon, was added trimethy}silyl
trifluorom~th~nesulfonate (5.0 mL, 0.5 eq.) at 0~C. The cold-bath was imm~i~ly
removed and after 48 h the mixture was diluted with dichlorometh~ne, washed withsaluldted sodium hydrogen carbonate, dried (Na2SO4), and concentrated. Column
chromatography (SiO2, pentane/EtOAc 20:1) gave 2 (13.7 g, 77%), [~]D25 ~21~ (c 1,
CHCI3). IH-Nmr data (CHCl3): ~ 5.47 (d, lH, J 3.4 Hz, H-4), 5.32 (t, lH, J 10.0
Hz, H-2), 5.25 (d, lH, J 10.0 Hz, H-1), 5.12 (dd, lH, J 3.4 and 10.0 Hz, H-3),
4.08 (m, 3H, H-5, H-6a and H-6b), 2.14-2.43 (m, 8H, -CH2CO-), 2.37 (s, 3H, -
SAc), 0.88 (t, 15 H, J 7.0 Hz, -CH3). Anal. Calcd for C56H,02OloS: C, 69.5; H, 10.6;
S, 3.3. Found: C, 69.4; H, 10.8; S, 3.5.
Fy~mplç C'
Synthesis of
1 -S-Acetyl-2.3.4.6-tetra-O-laurovl-l-thio-cY-D-galactopyranose
Method 1: To compound 1 (20.0 g, 18.2 mmol) and thioacetic acid (27.0 mL,
20 eq.) in dry toluene (80 mL) under argon was added tin (IV) chloride (21.3 mL)dropwise at room temperature. The reaction was monitored by Tlc carefully. After 1
h, 600 mL of lM aqueous HCl was added to the vigorously stirred mixture and the
res--lting mixture was filtered through Celite to remove the emulsion of tin salts. The
mixture was diluted with pentane (800 mL), washed with water (2 x 400 mL),
saturated sodium hydrogen carbonate (300 mL) and water (300 mL), dried with
Na2SO4 and concçntrated. The residue was purified by column chromatog~dphy threetimes (SiO2, pentane/EtOAc 20:1, 30:1, 40:1) to give 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thio-a-D-galactopyranose (3.65 g, 21 %). 'H-Nmr data (CHCl3): ~ 6.26 (d,
lH, J 5.5 Hz, H-1), 5.47 (dd, lH, J 11.0 Hz, 5.5 Hz, H-2), 5.46 (d, lH, J 3.5 Hz,
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H-4), 5.04 (dd, lH, J 11.0 Hz, 3.5 Hz, H-3), 4.17 (t, lH, J 6.5 Hz, H-5), 4.06 (d,
2H, J 6.~ Hz, H-6a and H-6b), 2.38 (t, ~H, J 7.0 Hz,
-COCH2-), 2.40 (s, 3H, -SAc), 0.87 (t, 15H, J 7.0 Hz, -CH3).
S Method 2: To compound 1 (25.0 g, 22.9 mmol) and thio~cetic acid (8.5 mL,
114.5 mmol) in dry dichlorometh~ne (100 mL) under argon, was added trimethylsilyl
trifluororneth~nesulfonate (5.6 mL, 45.8 mmol) at room t~ ture. After 20 h, the
mixture was diluted with dichloro-neth~ne (600 mL), washed with saturated sodiumhydrogen carbonate (250 mL) and water (2 x 200 mL), dried with Na2SO4 and
concentrated. The residue was purified by column chromatography three times (SiQ2,
pentane/EtOAc 20:1, 30:1, 40:1) to give 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thi~a-
D-galactopyranose (1.59 g, 7.2%).
Example D
General Procedure for
Michael Additions and c~!-Halocarbonyl Substitutions
To compound 2 (1 mmol) and an electrophile (1.2 mmol) in dry
dichlorometh~ne (~ mL) under argon, was added Et2NH (4 mL). After 1-3 h, the
mixture was concentrated and the residue was purified by column chromatography on
SiO2 by eluting with pentane/EtOAc. The products were characterized with 'H-nmr
spectroscopy and MALDI-TOF mass spectroscopy.
Example E
General Procedure for Reduction to Alcohols
To the product from Example D (100 ~mol) in dry tetrahydlorul~n (2.0 mL)
and isopropanol (0.7 mL) under argon atmosphere, was added NaBH4 (150 ~mol).
After 0.5-3 h, the mixture was concentrated (acetic acid (about 40 ~L) was addedprior to concentration in some cases) and the residue was purified according to the
solid-phase extraction procedure of Example A. The product alcohols were
characterized with 'H-nmr spectroscopy and MALDI-TOF mass spect~vsco~y.
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Example F
General Procedure for Reductive Amination to a Primary Amine
Method 1: To the product from Example D (ioo ~mol) and ~mmonillm acetate
(75 mg, 1 mmol) in dry methanol (2.3 mL) and tetrahydrofuran (0.9 mL) under
S argon, was added NaCNBH3 (100 ~mol). After 1-72 h, the mixture was con~ t~d
and the residue puAfied according to the solid-phase extraction procedure of Example
A. The product amines were char~cter 7e~ with 'H-nmr syecLIosco~y and MALDI-
TOF mass spectroscopy.
Method 2: The product from Example D (200 mg, 0.198 mmol) and dry
NH40Ac (30 mg, 0.4 mmol) were stirred in dry MeOH (6 mL), dry 1,2-
dichloroethane (6 mL), and trimethyl orthoformate (1 mL~ under argon for 24 h (un~l
TLC analysis showed that most of the starting material was con~umed). NaBH4 (10
mg, 0.26 mmol) was added and after 1 h the mixture was concentrated. The residue15 was purified according to the solid-phase extraction procedure of Example A to
provide the primary amine (containing traces of the co~ onding alcohol). This
mixture was dissolved in pentane/EtOAc (1:1) and applied onto a Waters Sep-Pak
Plus Longbody SiO2 cartridge. The cartridge was washed with pentane/EtOAc (1:1,
20 mL) (to remove the co~ ,uonding alcohol), followed by elution with toluene/EtOH
20 (9:1, 30 mL) to afford the primary amine.
Example G
General Procedure for
Acylation of Primary Amines with Phthalic Anhydride
The O-lauroylated primary amine from Example F (100 ~mol), phthalic
anhydride (2.7 mmol), and 4-(N,N-dimethylamino)pyridine (catalytic) were dissolved
in dry pyridine. The mixture was concentrated after 12-48 h and the residue purified
according to the solid-phase extraction procedure of Example A. The product 2-
carboxyben7~mides were characterized with 'H-nmr spect~uscopy and MALDI-TOF
30 mass spectroscopy.
.
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Example H
General Procedure for
Reductive Amination with Amino Acids
To the product from Example D (100 ~mol) and an amino acid te~t-butyl ester
5 hydrochloride or methyl ester hydrochloride (l mmol) in dry MeCN (2.25 mL) andTHF (0.75 mL), was added NaCNBH3 (100 ~mol). After 1-72 h, the ~ ul~ was
c~nc~ntrated and the residue was purified according to the solid-phase eYtrveti~n
procedure of Example A. The product N-alkylated amino acids were chata.;l~,iz~d
with IH-nmr spectroscopy and MALDI-TOF mass spectroscopy.
Example I
General Procedure for Deblocking of Alcohols
To the lauroylated alcohol from Example E (100 ~mol) in dry meth-s-nQl (7.1
mL) and dichlorornethsne (1.4 mL) under argon ~tmosphçre, was added ml;!l.sn~
sodium methoxide (1 M, 50 ~L). After 1-24 h, the mixture was neutralized with
Amberlite IR-50S (H+) resin, filtered and concentrated. The residue was dissolved in
water and applied onto a column of C18 silica (Waters Prep C18, 125 A, s g). TheC18 silica was washed with water (50 mL), and the product was then eluted with 70%
methanol (50 mL). The resulting alco,hDls were characterized with lH-nmr
spectroscopy and MALDI-TOF mass spectroscopy.
Example J
General Procedure for Deblocking of Primary Amines
To the O-lauroylated primary amine from FY~mple F (100 ,umol) in dry
meth~nol (7.1 mL) and dichloromethane (1.4 mL) under argon, was added methsn~
sodium methoxide (1 M, 50 ~LL). After 1-24 h, the mixture was neutralized with
Amberlite IR-50S (H+) resin, filtered and concentrated. The residue was dissolved in
dichloromethane/methanol 2:1 and applied to a Waters SepPak Plus Longbody SiO2
cartridge. The cartridge was washed with dichloromethane/me~h~nol (2:1) and thenthe product was eluted with dichloromethane/methanol/water (5:5:1) (20 mL) and
concentrated. The residue was dissolved in water and applied onto a column of C18
... ..... .. ..... ...... . .. .
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silica (Waters Prep C18, 125 A, s g). The C18 silica was washed with water (50
mL) and then the product was eluted with mçth~nol (50 mL). The res~ltin~
amines were characterized with lH-nmr sl,ecl,oscopy and MALDI-TOF mass
spec~l~,s~opy.
Example K
General Procedure for
N-Acetylation of Primary Amines
To the primary amine from Fl~mple J (100 ~mol) in moist meth~nol (4.4 mL)
was added acetic anhydride (0.4 mL). The mixture was col-centrated after 2-24 h, re-
dissolved in water and applied to a column of C18 silica (Waters Prep C18, 125 A, S
g). The C18 silica was washed with water (50 mL) and then the product was elutedwith meth~nol (50 mL). The r~s-llting ~et~mides were characterized with 'H-nmr
speclroscopy and MALDI-TOF mass ~e~ osco~y.
Example L
General Procedure for
Deblocking of 2-Carboxybenzamides
To the O-lauroylated 2-carboxybenzamide from Example G (100 ~mol) in dry
meth~nol (7.1 mL) and dichlororneth~ne (1.4 mL) under argon, was added meth~n5
sodium methoxide (1 M, 50 ~L). After 1-24 h, the mixture was neutralized with
Amberlite IR-50S (H+) resin, filtered and concentrated. The residue was dissolved in
dichlorometh~n~/meth~nol (8:1) and applied to a Waters SepPak Plus Longbody SiO2cartridge. The cartridge was washed with dichlorometh~n~/m~th~-lol (8:1) and then
the product was eluted with dichlorometh~ne/rneth~nol/water (65:35:5) (20 mL) and
concen~ ted. The residue was dissolved in water and applied to a column of C18
silica (Waters Prep C18, 125 ~, 5 g~. The C18 silica was washed with water (50
mL), and then the product was eluted with methanol (50 mL). The res~ in~ 2-
carbox~,l,e~,,..-.i~es were characterized with 'H-nmr ~peclrosco~y and MALDI-TOF30 mass speclfosC~l)Y-
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E,xample M
General Procedure for Deblocking of
N-Alkylated Glycine, ~-Alanine. and L-Leucine Com~ounds
The N-alkylated amino acid ten-butyl ester from Exarnple H (100 ~mol) was
treated with trifluoroacetic acid (3.5 mL) in dry dichlorometh~ne (3.5 mL) for 1-10 h.
n-Propyl acetate (8 mL) and toluene (16 mL) were added and the mixture was
concentrated, then co-concentrated twice with toluene. To the residue in dry
methanol (7.1 mL) and dichlorometh~ne (1.1 mL) under an argon ~tmosph~ore was
added methanolic sodium methoxide (1 M, 200 ~L). After 1-24 h, the l,li~lure was10 neutralized with Amberlite IR-SOS (H+) resin, filtered and concentrated. The residue
was dissolved in dichloromethane/methanol (9:1) and applied to a Waters SepPak Plus
Longbody SiO2 cartridge. The cartridge was washed with dichlorometh~ne/mPth~nol
(9:1) and then the product was eluted with dichlorome~h~ne/methanol/water (65:35:5)
(20 mL) and concentrated. The residue was dissolved in water and applied to a
15 column of C18 silica (Waters Prep C18, 125 A, s g). The C18 silica was washedwith water (50 mL) and then the product was eluted with 70% nleth~nol (50 mL).
The resulting N-alkylated glycine, ~-alanine, and L-leucine compounds were
characterized with 'H-nmr spectroscopy and MALDI-TOF mass a~ecLloscop~r.
Example N
General Procedure for Deblocking of
N-Alkylated L-Histidine and L-Tryptophan Compounds
To the N-alkylated amino acid methyl ester from Example H (100 ~Lmol) in dry
methanol (7.3 rnL) and dichlorometh~ne (1.1 mL) under an argon atm~hcr~ was
25 added methanolic sodium methoxide (1 M, 50 ~L). After 1-24 h, the .ni~lu~ wasneutralized with Amberlite IR-50S (H+) resin, filtered and concPntrated. The residue
was dissolved in 70% methanol and applied to a column of C18 silica (Waters PrepC18, 125 A, s g) and then the product was eluted with 70% mPth~nol (50 mL). To
the residue in water (3.7 mL) was added aqueous lithium hydroxide (lM, 0.3 mL).
30 After 0.5-2 h, the mixture was neutralized with Amberlite IR-SOS (H+) resin, filtered
and concent~ated. The residue was dissolved in dichloromethane/methanol (9:1) and
. _. . . ,,, ~ . .
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applied to a Waters SepPak Plus Longbody SiO~ cartridge. The cartridge was washed
with dichloromethane/methanol (9:1) and then the product was eluted with
dichlorometh~n~/meth~nol/water (65:35:5) (20 mL) and conc~ntrated. The residue
was dissolved in water and applied to a column of C18 silica (Waters Prep C18, 125
s A, 5 g). The C18 silica was washed with water (50 mL), and the product was eluted
with 70% methanol (50 mL). The resulting N-alkylated L-histidine and L-tr~p~ophan
compounds were characterized with IH-nmr spectroscopy and MALDI-TOF mass
spectroscopy.
Example O
General Procedure for
Deblocking of N-Alkylated L-Arginine Compounds
To the l~-alkylated arginine methyl ester from Example H (100 ~mol) in dry
meth~nQI (7.3 mL) and dichloromethane (1.1 mL) under an argon ~tmosph~re was
added m~th~nolic sodium methoxide (lM, 50 ~L). After 1-24 h, the mixture was
neutralized with Amberlite IR-SOS (H~) resin, filtered and concei,t.dted. The residue
was dissolved in 70% methanol and applied to a column of C18 silica and then theproduct was eluted with 70% methanol (50 mL). To the residue in water (3.7 mL)
wa then added aqueous lithium hydroxide (lM, 0.3 mL). After 0.5-2 h, the mixture20 was neutralized with Amberlite IR-SOs (H+) resin, filtered and concentrated. The
residue was dissolved in water and applied to column of C18 silica (Waters Prep C18,
125 A, s g). The C18 silica was washed with water (50 mL) and then the product was
eluted with 50% methanol (50 mL). The res~lting N-alkylated L-arginine cG...l~ou~ds
were characterized with 'H-nmr s~ecLloscopy and MALDI-TOF mass spe~ osco~.
Example P
General Procedure
for the P~el)aldlion of Mesylates
To the alcohol from Example D (0.3 mmol) in dry tetrahydrofuran (2 mL) and
30 dry pyridine (4 mL) under an argon atmosphere was added meth~n~sl-lfonyl chloride
(0.5 mL). After 12-24 h, the mixture was washed with O.5M HCI and extracted with
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pentane. The pentane extracts were concen~rated and the residue was purified on
C18-silica to afford the mesylate derivative.
Example Q
S General Procedure
for the Preparation of Azido Compounds
To the mesylate from FY~mple P (0.2 mmol) in dry DMF (8 mL) and dry THF
(3 mL) under an argon atmosphere at 60~C was added sodium azide (5 mmol) and 18-crown-6 (180 mg). After 2 hours, the reaction mixture was concentrated and the
10 residue was purified on C18-silica. In some cases, the product was re-
chromatographed with silica gel using pentane/EtOAc (9:1) as the eluant to afford the
azido derivative.
Example R
General Procedure
for Reduction of Azido Groups to Primary Amines
To a solution of the azido compound from Fy~mple S (15 ~lmol) in dry
isopro~anol (1 mL) and dry ethanol (1 mL) under an argon atmosphere, was added
NaBH4 (15 ~mol) and NiCl2 (30 ~mol). ARer 1 hour, the reaction mixture was
20 neutralized with acetic acid (1 drop), concentrated and purified on C18-silica to afford
the primary amine.
Example S
General P,ocedule
for Reductive Alkylation of Primary Amines
To the primary amine from Example F or S (6.8 ~mol) in dry m~th~nol (1 mL)
and dry dichlororne~h~n~ (1 mL) under an argon atmosphere was added an aldehyde
or ketone (3.4 mmol) and sodium triacetoxyborohydride (47 llmol). After 24~8
hours, toluene (1 mL) was added and the mixture was concentrated and the residue30 purified on C18-silica gel.
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Fy~mple T
- General Procedure for Reductive Amination
To the product from Example D (0.1 mmol) and a primary amine (0.45 mmol)
in dry dichloromethane (2 mL), meth~nol (2 mL) and triethylorthoformate (1 mL)
5 under argon, was added NaCNBH3 (1 mmol). After 24 h, the mixture was
concentrated and dissolved in toluene (1 mL) and purified on C18-silica gel (5 g).
Example U
General Procedure for Deblocking of Secondary Amines
To the O-lauroylated secondary amine from Example S or T (100 ~mol) in dry
methanol (7.1 mL) and dichloromethane (1.4 mL) under argon, was added meth~ns~
sodium methoxide (lM, 50 ~L). After 1-24 h, the mixture was neutralized with
Amberlite IR-50S (H+) resin, filtered and concentrated. The residue was dissolved in
dichloromethane/methanol 2:1 and applied to a Waters SepPak Plus Longbody SiO2
cartridge. The cartridge was washed with dichlorometh~nP/meth~ncl (2:1) and thenthe product was eluted with dichlorometh~ne/mPth~nol/water (5:5:1) (20 mL) and
concent-ated. The residue was dissolved in water and applied onto a column of C18
silica (Waters Prep C18, 125 A, 5 g). The C18 silica was washed with water (50
mL) and then the product was eluted with methanol (50 mL). The res--lting
secondary amines were characterized with lH-nmr spectroscopy and MALDI-TOF
mass spectroscopy.
Example Al
Sylllll~is of
2-Hy~l-ox~-;yclopent-1-yl 1-Thi~B-D-galactopyr~no~
The title compound was prepared according to procedures D, E and I above
using 2-chlorocyclopentanone as the electrophile. Mass spectra data was as follows:
M (calcd.): 280.34; M (found): 304.9 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D): ~ 4.44 (H-l), 4.42, 4.38, and 4.35.
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Example A2
Synthesis of
2-HydroA~c~clohex-1-yl l-Thi~-D-ga}actopyranoside
The title compound was plcpar~d according to procedures D, E and I above
5 using 2-chlorocyclohe~nQlle as the electrophile. Mass spectra data was as follows:
M (calcd.): 294.34; M (found): 318.8 ~M+Na+). Select~d nmr data was as follows:
H-nmr (CD30D): ~ 4.55 (H-l), 4.43, 4.39, and 4.34.
Example A3
Synthesis of
3-Hydroxy-l-phenylbut-1-yl 1-Thio-,15-D-~ ctopyr~n~cj~
The title compound was prepared according to procedures Dt E and I above
using 4-phenylbut-3-en-2-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 345.43; M (found): 368.0 (M+Na+). Selected nmr data was as follows:
15IH-nmr (CD30D): ~ 4.45 (H-l), 4.43, 4.31, and 4.25.
Example A4
Synthesis of
(3-Hydroxynorborn-2-yl)methyl 1-Thio-,B-D-~ ctopyranoside
20The title compound was prepared according to procedures D, E and I above
using 3-methylene-2-norbornanone as the electrophile. Mass spectra data was as
follows: M (calcd.): 320.41; M (found): 344.6 (M+Na+). Sel~c~ed nmr data was as
follows: 'H-nmr (CD30D~: ~ 4.30 (H-1) and 4.29.
25Exarnple A5
Synthesis of
3-Hydroxycyclohept-1-yl l-Thio-,B-D-~ ctopyranoside
- The title compound was plepa ed according to procedures D, E and I above
using cyclohept-en-l-one as the electrophile. Mass spectra data was as follows: M
30(calcd.): 308.40; M (found): 332.1 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D): â 4.394 (H-1), 4.389, and 4.381.
. . ..
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Exarnple AS'
_ Synthesis of
3-Hydrox~clQhept-l-yl 1-Thi~a-D-~ ctopyranoside
The title compound was ~l~ paled according to procedures D, E and I above
S using 1-S-acetyl-2,3,4,6-tetra-O-lauroyl- l-thio-a-D-galactopyranose (from Example C'
above) and cyclohépt-en-l-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 308.40; M (found): 331.3 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D): ~ 5.44 (d, J 5.8 Hz, H-1) and 5.45 (d, J 5.8 Hz, H-1).
Example A6
Synthesis of
2,2-Dimethyl-~hydrox~c~clopent-l-yl 1-Thio-~B-D-g~ topyr~
The title compound was prepared according to procedures D, E and I above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile. Mass spectra data was as
follows: M (calcd.): 308.40; M (found): 332.1 (M+Na+). Sel~t~ nmr data was as
follows: 'H-nmr (CD30D): ~ 4.34 (H-1), 4.315, 4.310, and 4.305.
Example A7
Synthesis of
3-Hydroxycyclopent-l-yl l-Thio-,B-D-g~lqctopyranoside
The title compound was prepared according to procedures D, E and I above
using cyclopent-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 280.34; M (found): 304.9 (M+Na+). Sele~ted nmr data was as follows:
'H-nmr (CD30D): ~ 4.36 (H-l), 4.355, and 4.34.
Example A8
Synthesis of
~Hydroxypent-2-yl l-Thi~,~-D-galactopyranoside
The title compound was prepared according to procedures D, E and I above
using pent-3-en-2-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 282.35; M (found): 305.3 (M+Na+). Select~l nmr data was as follows:
lH-nmr (CD30D): ~ 4.42 (H-l), 4.41, and 4.39.
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Fy~nlple A9
Synthesis of
2,2-Dimethyl-5-hylllo~cyclohex-1-yl l-Thio-~-D-gs~l~rtoFyr~nos~,de
The title compound was prepared according to procedures D, E and I above
S using 4,4-dimethylcyclohex-2-en-1-one as the electrophile. Mass spectra data was as
follows: M (calcd ): 322,42; M (found): 346.6 (M+Na+). S~lPct~d nmr data was as
follows: ~H-nmr (CD30D): ~ 4.34 (H-1), 4.33, and 4.32.
Example A10
Synthesis of
3-Hydroxycyclohex-1-yl l-Thi~,B-D-galactopyr~nos;~
The title compound was prepared according to procedures D, E and I above
using cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 294.37; M (found): 317.3 (M+Na+). S~le~ted nmr data was as follows:
'H-nmr (CD30D): ~ 4.422 (H-l), 4.417, and 4.38.
Example A11
Synthesis of
4,4-Dimethyl-3-hydroxycyclohex-1-yl l-Thio-,B-D-galactopyr~n~
The title compound was plc~ed according to procedures D, E and I above
using 6,6-dimethylcyclohex-2-en-1-one as the electrophile.
Exarnple B1
Synthesis of
2-Aminocyclor~nt-1-yl l-Thio-,B-D-galactopyranoside
The title compound was prepared according to procedures D, F and J above
using 2-chlorocyclopentanone as the electrophile. Mass spectra data was as follows:
M (calcd.): 279.36; M (found): 276.3 (M+H+). .~elec~d nmr data was as follows:
IH-nmr (CD30D): ~ 4.46 (H-l), 4.45, 4.37 and 4.27.
, . . ~ ~ . .. ... . . . ~
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Example B2
Synthesis of
2-Aminocyclohex-1-yl 1-Thio-,B-D-g~ topyranoside
The title compound was prepared according to procedures D, F and J above
5 using 2-chlorocycloheY~nonP as the electrophile. Mass spe~tra data was as follows:
M (calcd.3: 293.38; M (found): 295.8 (M+H+), and 319.7 (M+Na+). Sel~t~ nmr
data was as follows: IH-nmr (CD30D): ~ 4.48 (H-1), 4.44, 4.40 and 4.30.
Example B3
Synthesis of
3-Amino-1-phenyl~ut-1-yl 1-Thio-~-D-galactopyr~n~c;Ae
The title compound was prepared according to procedures D, F and J above
using 4-phenylbut-3-en-2-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 344.45; M (found): 345.1 (M+H+). Sele~t~d nmr data was as follows:
15IH-nmr (CD30D): ~ 4.41 (H-1), 4.12, and 3.90.
Example B4
Synthesis of
(3_~minrnQrborn-2-yl)methyl 1-Thio-,B-D-galactopyranoside
20The title compound was prepared according to procedures D, F and J above
using 3-methylene-2-norbomanone as the electrophile. Mass spectra data was as
follows: M (calcd.): 319.42; M (found): 321.6 (M+H+). Sele~ted nmr data was as
follows: 'H-nmr (CD30D): ~ 4.42 (H-1), 4.41, 4.38, and 4.35.
25Example B5
Synthesis of
3-Aminocy~ lohept-1-yl 1-Thio-,l~-D-galactopyranoside
The title compound was prepared according to procedures D, F and J above
using cyclohept-2-en-1-one as the electrophile. Mass spectra data was as follows: M
30(calcd.): 307.41; M (found): 333.0 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D): ~ 4.41 (H-1), 4.39, and 4.38.
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Example B6
Synthesis of
2,2-Dimethyl-~minocyclopent-l-yl 1-Thio~ D-g~ ctopyranoside
The title compound was pr~ed according to procedures D, F and J above
S using 4,4-dimethylcyclopent-2-en-1-one as the electrophile. Mass spectra data was as
follows: M (calcd;): 307.41; M (found): 307.2 (M+H+). SelP~ted nmr data was as
follows: ~H-nmr (CD30D): ~ 4.35 (H-1), 4.33, 4.32, and 4.30.
Example B6A
Synthesis of
2,2-Dimethyl-~(methylamino)-
cyclopent-1-yl l-Thio-,B-D-~ rtopyr~n~5i~1~
The title compound was prepared according to procedures D, T and U above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and methylamine as ~eprimary amine. Mass spectra data was as follows: M (calcd.): 321.43; M (found):
322.7 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.325 (H-
1), 4.315, 4.308, 4.304.
Example B6B
Synthesls of
2,2-Dimethyl~(isopropylamino)-
cyclopent-1-yl 1-Thio-~B-D-~ ~topyr~nos;~le
The title compound was prepared according to procedures D, T and U above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and isopropylamine as the
primary amine. Mass spectra data was as follows: M (calcd.): 349.48: M (found):
350.7 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 41460 (H- -
1), 4.401, 4.400, 4.391.
... .
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Example B6C
Synthesis of
2,2-Dimethyl-~(n-propylamino)-
cyclopent-l-yl 1-Thio-,B-D-galactopyr~nr~sid~
The title compound was prepared according to procedures D, T and U above
using 4,4-dimethyicyclopent-2-en-1-one as the electrophile and n-propylamine as the
primary amine. Mass spectra data was as follows: M (calcd.): 349.49; M (found):
350.5 (M+~+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.324 (H-
1), 4.317, 4.310, 4.307.~0
Example B6D
Synthesis of
2,2-Dimethyl~((R)-sec-butylamino)-
cyclopent-l-yl 1-Thio-,B-D-~ ctopyranoside
The title compound was prepa~ed according to procedures D, T and U above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and (R)-(-)-sec-butylan~ine
as the primary amine. Mass spectra data was as follows: M (calcd.): 364.52; M
(found): 364.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.328 (H-1), 4.319, 4.313, 4.311.
Example B6E
Synthesis of
2,2-Dimethyl-4((S)-sec-butylamino)-
cyrlopent-1-yl 1-Thi~,B-D-g~l~ctopyranoside
The title compound was prepared according to procedures D, T and U above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and (S)-(+)-sec-bu~lamine
as the primary amine. Mass spectra data was as fol}ows: M (calcd.): 364.52; M
(found): 364.6 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D):
4.333 (H-l), 4.330, 4.300, 4.290.
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Example B6F
Synthesis of
2,2-Dimethyl~(pent-3-ylamino)-
cyclopent-l-yl l-Thio-,li-D-~ ctopyrqncsi-le
The title compound was prepared according to procedures D, T and U above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and 3-pentylamine as the
primary amine. Mass spectra data was as follows: M (calcd.): 377.53; M (found):
376.7 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.333 (H-
1), 4.329, 4.300, 4.290.
Example B6G
Synthesis of
2,2-Dimethyl-4(n-hexylamino)-
cyclopent-l-yl l-Thio-,B-D-galactopyr~nosi~le
The title compound was prepared according to ~ ced-nes D, T and U above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and n-hexylamine as the
primary amine. Mass spectra data was as follows: M (calcd.): 391.57; M (found):
394.3 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.336 (H-
1), 4.332, 4.303, 4.291.
Example B6H
Synthesis of
2,2-Dimethyl-~cyclobut-1-ylamino)-
~clope--l-l-yl 1-Thio-~-D-~ ctopyranoside
The title compound was plepa,cd according to procedures D, T and U above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and cyclobutyl amine as
the primary arnine. Mass spectra data was as follows: M (calcd.): 361.50; M
(found): 361.6 (M+H+). Sçlçcted nmr data was as follows: 'H-nmr (CD30D):
4.315 (H-l), 4.300, 4.292, 4.290.
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Example B6I
Synthesis of
2,2-Dimethyl-4(3,3-dimethylcyclobut-1-ylamino)-
cyclopent-1-yl 1-Thi~B-D-galactopyranoside
S The title compound was prepared according to procedures D, T and U aboveusing 4,4-dimethyIcyclopent-2-en-1-one as the electrophile and 3,3-dimethylcyclobut-
1-ylamine as the primary amine. Mass spectra data was as follows: M (calcd.):
389.55; M (found): 392.2 (M+H+). Selected nmr data was as follows: 'H-nmr
(CD30D): ~ 4.324 (H-l), 4.311, 4.305, 4.294.
Example B6J
Synthesis of - _
2,2-Dimethyl-4-(cyclopent-1-ylamino)-
cyclopent-1-yl 1-Thio-,B-D-galactopyr~r~ ~s;d~
The title compound was prepared according to ~loced-lres D, T and U a'oove
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and cyclopentylamine as
the primary amine. Mass spectra data was as follows: M (calcd.): 375.52; M
(found): 376.6 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D):
4.322 (H-l), 4.310, 4.304, 4.295.
Example B6K
Synthesis of
2,2-Dimethyl-1 (cyclohex-1-ylamino)-
cyclopent-1-yl 1-Thio-,B-D-galactopyranoside
The title compound was p~epa ed according to procedures D, T and U a~ove
using 4,4-dimethylcyclopent-2-en-1-one as the elec~ophile and cyclohexylamine as the
primary amine. Mass spectra data was as follows: M (calcd.): 389.55; M (found):
391.2 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.319 (H-
1), 4.310, 4.307, 4.293.
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Examp}e B6L
Synthesis of
2,2-Dirnethyl-4-(~methylcyclohex-1-yla nino)-
cyclop~n~ yl l-Thi~,B-D-~ ctopyr~n~s;~le
The title compound was prepared according to procedures D, T and U above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and 4-methylcyclohex-1-
ylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 403.47;
M (found): 404.8 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.333 (H-1), 4.312, 4.300, 4.295.
Example B6Q
Synthesis of
2,2-Dimethyl-~(3-methylcyclopent-1-ylamino)-
cyclopent-1-yl 1-Thio-,B-D-galactopyr~n~-si~,~
The title compound was prepared according to procedures D, T and U above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and 3-methylcyclopent-1-
ylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 389.55;
M (found): 390.7 (M+H+). Sele~t~Pd nmr data was as follows: IH-nmr (CD30D):
4.383 (H-1), 4.325, 4.300, 4.292.
Example B6R
Synthesis of
2,2-Dimethyl-4(3,3-dilnethylcyrlorP.~t-l-ylamino)-
cyclopent-l-yl 1-Thio-,B-D-~ rtopyranoside
The title compound was p~ ed according to procedures D, T and U
above using 4,4-dimethylcyclopent-2-en- 1 -one as the electrophile and 3,3-
dimethylcyclopent-l-ylamine as the primary amine. Mass spectra data was as follows:
M (calcd.): 4.295; M (found): 404.3 (M+H+). SelP~ted nmr data was as follows:
'H-nmr (CD30D): ~ 4.322 (H-l), 4.305, 4.300, 4.295.
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Example B6T
Synthesis of
2,2-Dimethyl~(3-methylcyclohex-1-ylamino)-
cyclopent-l-yl l-Thio-,B-D-galactopyr~n~;de
S The title compound was plepar~d according to procedures D, T and U aboveusing 4,4-dimethylcyclopent-2-en-1-one as the electrophile and 3-methylcyclohex-1-
ylamine as the primary amine. Mass spectra data was as follows: M (calcd.): 403.57;
M (found): 404.8 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.326 (H-1), 4.313, 4.303, 4.294.
Example B7
Synthesis of
3-Aminocyclopent-l-yl l-Thio-~B-D-g~ topyr~r~os;de
The title compound was prepared according to procedures D, F and J above
15 using cyclopent-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 279.35; M (found): n.a. Selected nmr data was as follows: lH-nmr
(CD30D): ~ 4.46, 4.40, 4.38, and 4.34 (4 d, J 10 Hz), 3.88 (br s), 2.61, 2.27, 2.15,
1.82, and 1.64 (5 m).
Example B8
Synthesis of
~Aminopent-2-yl l-Thio-,B-D-galactopyr~n~s;-le
The title compound was prepared according to procedures D, F and J above
using pent-3-en-2-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 281.37; M (found): 283.4 (M+H+). Selectçd nmr data was as follows~
nmr (CD30D): ~ 4.41 (H-1), 4.40, and 4.36.
Example B10
Synthesis of
3-Aminocyclohex-l-yl l-Thio-~-D-gPl~rSopyranoside
The title compound was prepated according to procedure D, F and J above
using cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M
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(calcd.): 293.38; M (found): 317.9 (M+Na+). Selected nmr data was as follows:
H-nmr (CD30D): ~ 4.54 (H-1), 4.52, 4.49, and 4.47.
Example Bll
S Synthesis of
3-Amino-4,~dimethylcyclohex-1-yl 1-Thi~,B-D-gala~ to~ ~. snoside
The title compound was prepared according to procedure D, F and J above
using-6,6-dimethylcyclohex-2-en-1-one as the electrophile.
Example Cl
Synthesis of
2-Ac~t~mi~10cyclopent-l-yl l-Thio-,B-D-g~l~cto~yranoside
The title compound was prepared according to procedures D, F, J and K above
using 2-chlorocyclopent-1-one as the electrophile. Mass spectra data was as foIlows:
M (calcd.): 321.39; M (found): 345.8 (M+Na+). Selected nmr data was as follows:
H-nmr (CD30D): ~ 4.53 (H-l), 4.44, 4.32, and 4.24.
Example C2
Synthesis of
2-Acet~mirlocyclohex-1-yl 1-Thio-,B-D-galactopyr~noside
The title compound was prepared according to procedures D, F, J and K above
using 2-chlorocyclohexan-1-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 335.42; M (found): 359.4 (M+Na+). Selected nmr data was as follows:
~H-nmr (CD30D): ~ 4.43 (H-1), 4.42, 4.32, and 4.29.
Example C3
Synthesis of
3-Ac~t~ ~1-phenylbut-1-yl 1-Thi~,B-D-galactopyr~r~osi,d~
The title compound was prepared according to proced~res D, F, J and K above
using 4-phenylbut-3-en-2-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 386,48; M (found): 408.3 (M+Na+). Selected nmr data was as follows:
- lH-nmr (CD30D): ~ 4.32 (H-1), 4.25, 3.83, and 3.79.
,
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Example C5
Synthesis of
3-Acet~midocyrlQhel)t-l-yl l-Thi~,B-D-g~l~rtopyranoside
The title compound was prepared according to procedures D, F, J and K above
5 using cyclohept-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 349.42; M (found): 372.5 (M+Na+). .Sele~t~ nmr data was as follows:
'H-nmr (CD30D): ~ 4.403 (H-l), 4.397, 4.34, and 4.33.
Example C7
Synthesis of
3-Acetamidocyclopent-l-yl l-Thi~,B-D-galactopyr~nn~;de
The title compound was ~lel a~cd according to procedu~es D, F, J and K above
using cyclopent-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 321.39; M (found): 349.5 (M+Na+).
Example C8
Synthesis of
~Acet~midopent-2-yl l-Thio-,B-D-galactopyranoside
The title compound was prepared according to procedures D, F, J and K above
using pent-3-en-2-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 323.40; M (found): 347.7 (M+Na+). Selected nmr data was as follows:
H-nmr(CD30D):~ 4.42 (H-l), 4.38, 4.37, and 4.35.
Example C10
Synthesis of ~-
3-Acet~mi~ocyclohexyl l-Thi~-D-galactopyranoside
The title compound was prepared according to procedures D, F, J and K above
using cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 335.42; M (found): 373.7 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D):~ 4.52 (H-l), 4.464, and 4.455.
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Example C l l
Synthesis of
3-Ac~qmido~,~dimethylcyclohexyl l-Thio-,B-D-g,q~ ctopyrqn~
The title compound was prepared according to procedures D, F, J and K above
using 6,6-dimethylcyclohex-2-en-1-one as the electrophile.
Example Dl
Synthesis of
2-(2-Carboxyben7~mi~lc)cyclopent-l-yl l-Thio-,B-D-galactopyrqn~ e
The htle compound was prepared according to procedures D, F, G and L above
using 2-chlorocyclopentan-1-one as the electrophile. Mass spectra data was as
follows: M (calcd.): 427.47; M (found): 450.5 (M+H'). Sele~ted nmr data was as
follows: 'H-nmr (CD30D): ~ 4.69 (H-l), 4.58, 4.27, and 4.22.
Example D2
Synthesis of
2-(2-Carboxyben7~mido)cyclohex-1-yl l-Thi~,B-D-galactopyrqn~ e
The title compound was prepared according to procedures D, F, G and L above
using 2-chlorocyclohexan-1-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 441.50; M (found): 465.9 (M+Na+). .Sele~tçd nmr data was as follows:'H-nmr (CD30D): ~ 4.54 (H-1), 4.52, 4.50, and 4.35.
Exarnple D3
Synthesis of
3-(2-CarboA~l,c~ m;~lQ)-1-phenylbut-1-yl l-Thio-~B-~g,qlqctopyranos;de
The title compound was prepared according to procedures D, F, G and L above
using 4-phenylbut-3-en-2-one as the electrophile. Mass spectra data was as follows:
M (calcd.): 492.56; M (found): 513.0 (M+Na+). Selected nmr data was as follows:
'H-nmr (CD30D): ~ 4.41 (H-l), 4.115, 4.110, and 3.90.
~ . ,
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Example D4
S~ is of
[3-(Carboxyben7~mi~o)norborn-2-yl]methyl l-Thio-,B-~g~ topyr~nnsi l~
The title compound was prepared according to procedures D, F, G and L above
using 3-methylene-2-norbornanone as the electrophile. Mass spectra data was as
follows: M (calcd.): 467.54; M (found): 492.9 (M+Na+). SelPcted nmr data was as
follows: ~H-nmr (CD30D): ~ 4.39 (H-l), 4.34, 4.31, and 4.26.
Example D5
10- Synthesis of
3-(2-Carboxyben7~mido)cyclohept-l-yl l-Thio-,B-D-g~l~rtopyranoside
The title compound was prepared according to procedures D, F, G and L above
using cyclohept-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 453.52; M (found): 479.6 (M+Na+). Selected nmr data was as follows:
IH-nmr (CD30D): â 4.53 (H-l), 4.51, 4.42, and 4.40.
Example D8
Synthesis of
3-(2-Carboxyben7~mi~lo)pent-2-yl 1 Thio-,B-D-galactopyranoside
The title compound was prepared according to procedures D, F, G and L above
using pent-3-en-2-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 429.48; M (found): 452.7 (M+Na+). Selected nmr data was as follows:
IH-nmr (CD30D): ~ 4.42 (H-l), 4.41, 4.40, and 4.35.
Example D9
Synthesis of
5-(2-Carboxyben~mi~o)-2,2-dimethylcyclohex-1-yl
l-Thio-~B-D-galactopyr~n~s;~l~
The title compound was prepared according to procedures D, F, G and L above
using 4,4-dimethylcyclohex-2-en-1-one as the electrophile. Mass spectra data was as
follows: M (calcd.): 469.55, M (found): 492.4 (M+Na+).
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Example D10
Synthesis of
3-(2-Carboxyben7~mi~1o)cyclohex-l-yl
l-Thio-~B-D-g~lactopyr~nos;~l~
S The title compound was prepared according to procedures D, F, G and L above
using cyclohex-2-en-1-one as the electrophile. Mass spectra data was as follows: M
(calcd.): 441.50, M (found): n.a. Selected nmr data was as follows: IH-nmr
(CD30D): ~ 4.37 (H-l), 4.34, and 4.32.
Example El
Synthesis of
N~Y-[2-(1-Thio-,B-D-galactopyranosyl)cyclopent-l-yl]glycine
The title compound was prepared according to procedures D, H and M above
using 2-chlorocyclopentan-l-one as the electrophile and glycine tert-butyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 337.39; M (found):
359.8 (M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.44 (H-1),
4.41, 4.40, and 4.34.
Example E2
Synthesis of
Ncr-[2-(1-Thio-,B-D-galactopyranosyl)cyclohex-l-yl]glycine
The title compound was l)r~paled according to procedures D, H and M above
using 2-chlorocyclohexan-1-one as the electrophile and glycine tert-butyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 351.42; M (found):
353.5 (M+H+), 376.5 (M+Na+). Selected nmr data was as follows: IH-nmr
(CD30D): ~ 4.48 (H-1), 4.47, 4.36, and 4.29.
Example E3
Synthesis of
N~r-[~Phenyl-4-(1-thio-,B-D-galactopyranosyl)but-2-yl]glycine
The title compound was prepared according to procedures D, H and M above
using 4-phenylbut-3-en-2-one as the electrophile and glycine tert-butyl ester as the
~ ........ . . . . .
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amino acid ester. Mass spectra data was as follows: M (calcd.): 401.48; M (found):
403.1 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D): ~ 4.29 (H-l),
4.18, 3.92, and 3.91.
Exa nple E4
Synthesis of
Na-[3-((1-Thi~-D-~Pl ~ ~topyranosyl)methyl)nor~orn-2-yl]glycine
The title compound was pl~ared according to procedures D, H and M above
using 3-methylene-2-norbornanone as the electrophile and glycine tert-butyl ester as
the amino acid ester. Mass spectra data was as follows: M (calcd.~: 377.46; M
(found): 401.4 (M+Na+). Sele~tecl nmr data was as follows: 'H-nmr (CD30D):
4.42 (H-1), 4.40, 4.383, 4.377, and 4.35.
Example E5
Synthesis of
NcY-[3-(l-Thio-,B-D-~ ctopyranosyl)cyclQhept-l-yl]glycine
The title compound was prepared according to procedures D, H and M above
using cyclohept-2-en-1-one as the electrophile and glycine tert-butyl ester as the amino
acid ester. Mass spectra data was as follows: M (calcd.): 365.45; M (found): 367.4
(M+H+), 389.9 (M+Na+). Se1e~ted nmr data was as follows: 'H-nmr (CD30D):
4.46 (H-1), 4.45, 4.42, and 4.38.
Example E5'
Synthesis of
Nlx-[3-(1-Thio-'-D-galactopyranosyl)cycl~hept-1-yl]glycine
The title compound was prepa,ed according to procedures D, H and M above
using l-S-acetyl-2,3,4,6-tetra-O-lauroyl-l-thio-~-D-galactopyranose (from Fy~mpl~ C'
above), cyclohept-2-en-1-one as the electrophile and glycine tert-butyl ester as the
arnino acid ester. Mass spectra data was as follows: M (calcd.): 365.45; M (found):
366.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 5.51 (d, J
5.5 Hz, H-l (major), 5.46 (d, J 5.5 Hz, H-l), 5.47 (d, J 5.5 Hz, H-l (minor)), 5.48
(d, J 5.5 Hz, H-l).
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Example E6
Synthesis of
Na~-[4,~Dunethyl-3~ thio-,B-D-~Io~topyranosyl)
cyclopent-1-yl]glycine
The title compound was prepared according to procedures D, H and M above
using 4,4-dimethyicyclopent-2-en-1-one as the electrophile and glycine te~-butyl ester
as the arnino acid ester. Mass spectra data was as follows: M (calcd.): 365.44; M
(found): 368;0 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.330 (H-1), 4.325, 4.320, and 4.30.
Example E7
Synthesis of
N~-[3-(1-Thio-,B-D-galactopyranosyl)cyclopent-l-yl]glycine
The title compound was prepared according to procedures D, H and M above
15 using cyclopent-2-en-1-one as the electrophile and glycine te)t-butyl ester as the amino
acid ester. Mass spectra data was as follows: M (calcd.): 337.39; M (found): 360.9
(M+Na+). Selectçd nmr data was as follows: 'H-nmr (CD30D): ~ 4.38 (H-l),
4.375, 4.36, and 4.35.
Example E8
Synthesis of
Na-[~(l-Thio-,B-D-galactopyranosyl)pent-2-yl]glycine
The title compound was prepared according to procedures D, H and M above
using pent-3-en-2-one as the electrophile and glycine ~erl-butyl ester as the amino acid
25 ester. Mass spectra data was as follows: M (calcd.): 338.39; M (found): 363.9(M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.43, 4.42, 4.37
(H-l), and 4.36.
.. , . . ,, ,, .,, ...... ~.. _. . . ..
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Example E9
Syl~lLe~ls of
N~-[4,~Dimethyl-3-(1-thio-,B-D-galactopyranosyl)-
cyclohex-l -yl]glycine
5The title compound was piepal~d according to procedures D, H and M above
using 4,4-dimethyicyclohex-2-en-1-one as the electrophile and glycine tert-butyl ester
as the amino acid ester. Mass spectra data was as follows: M (calcd.): 379.47; M(found): 380.6 (M+H+), 403.5 ~M+Na+). Selected nmr data was as follows: 'H-
nmr (CD30D): ~ 4.38 (H-1), 4.36, 4.34, and 4.31.
Example E10
Synthesis of
Na-[3-(l-Thio-,B-D-~,q-l~rtopyranosyl)cyclohex-l-yl]glycine
The title compound was prel)ared according to procedures D, H and M above
15 using cyclohex-2-en-1-one as the electrophile and glycine tert-butyl ester as the amino
acid ester. Mass spectra data was as follows: M (calcd.): 351.42; M (found): 377.1
(M+Na+). Selecte~ nmr data was as follows: 'H-nmr (CD30D): ~ 4.46 (H-l), 4.44,
4.40, and 4.36.
20Example Ell
Synthesis of
Nc~-[5-(1-Thio-,B-D-galactopyranosyl)-
2,2-dimethylcyclohex-1-yl]glycine
The title compound was prepared according to procedures D, H and M above
25using 6,6-dimethylcyclohex-2-en-1-one as the electrophile and glycine tert-butyl ester
as the amino acid ester.
Exarnple F1
Synthesis of
30N~-[2-(1-Thio-,B-D-galactopyranosyl)cyclopent-l-yl]-,B qlqnine
The title compound was prepared according to~procedures D, H and M above
using 2-chlorocyclopentan-1-one as the electrophile and ,~-alanine tert-butyl ester as
the amino acid ester. Mass spectra data was as follows: M (calcd.): 351.42; M
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(found): 372.9 (M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.54 (H-l), 4.52, 4.36, and 4.35.
Example F2
Synthesis of
N~B-[2-(1-Thio-,B-D-galactopyranosyl)cyclohex l yl] ~ n;n~
The title compound was prepared according to procedures D, H and M above
using 2-chlorocyclohexan-1-one as the electrophile and ,B-alanine tert-butyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 365.45; M (found):
367.4 (M+H+), 389.9 (M+Na+), 412.0 (M+K+). Selected nmr data was as
follows: 'H-nmr (CD30D): ~ 4.47 (H-1), 4.42, 4.41, and 4.33.
Example F3
Synthesis of
N~B-[4-Phenyl-~(1-thio-~B-D-galactopyranosyl)but-2-yl]-~-alanine
The title compound was prepared according to procedures D, H and M above
using 4-phenylbut-3-en-2-one as the electrophile and ,B-alanine tert-butyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 415.50; M (found):
417.0 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.28 (H-l),
4.17, 3.97, and 3.96.
Example F4
Synthesis of
N~B-r3-((l-Thio-~-D-galactopyranosyl)methyl)norbol ll-2-yl]-~ n;n~
The title compound was prepared according to procedures D, H and M above
using 3-methylene-2-norbornanone as the electrophile and ~-alanine te~r-butyl ester as
the amino acid ester. Mass spectra data was as follows: M (calcd.): 391.48; M
(found): 393.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.40 (H-l), 4.37, 4.34, and 4.33.
~ ,
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Fy~mpl~ F5
~ - Synthesis of
N,B-[3-(1-Thio-,B-D-~ etopyranosyl)cyclQ~ept-l-yl]-,B~Iqn;rle
The title compound was prepared according to procedures D, H and M above
S using cyclohept-2-en-1-one as the electrophile and ,B-alanine tert-butyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 379.45; M (found):
381.7 (M+H+), 403.5 (M+Na+), 426.0 (M+K+). S~lected nmr data was as
follows: ~H-nmr (CD30D): ~ 4.46 (H-1), and 4.38.
Example F6
Synthesis of
N,B-[4,4-Dirnethyl-3~ thio-~-D-galactopyranosyl)-
cyclopent-l-yl]-~ nin~
The title compound was prepared according to procedures D, H and M above
15 using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and ~-alanine tert-butyl
ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 379.44;
M (found): 3B3.2 (M+H+). Sel~ted nmr data was as follows: 'H-nmr (CD30D): ô
4.34 (H-1), 4.33, 4.315, and 4.310.
Example F7
Synthesis of
N~ 3-(l-Thio-~-D-ga~actopyranosyl)cyclopent-l-y~ n;n~
The title compound was prepared according to procedures D, H and M above
using cyclopent-2-en-1-one as the electrophile and ,B-alanine tert-butyl ester as the
25 arnino acid ester. Mass spectra data was as follows: M (calcd.): 351.42; M (found):
375.1 (M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.41 (H-
1), and 4.40.
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Example F8
S~lltl~is of
[~(l-Thio-~B-D-~ ctopyranosyl)pent-2-yl~-,B-alal~ine
The title compound was prepared according to procedures D, H and M above
5 using pent-3-en-2-one as the electrophile and ~-alanine tert-butyl ester as the amino
acid ester. Mass spectra data was as follows: M (calcd.): 352.42; M (found): 356.0
(M+H+). SPlected nmr data was as follows: ~H-nmr (CD30D): ~ 4.49 (H-l), 4.440,
and 4.435.
Example F9
Synthesis of
N,B-[4,4-Dimethyl-3-(1-thio-~B-D-galactopyranosyl)-
cyclohex-l-yl]-~ n~
The title compound was prepared according to procedures D, H and M above
15 using 4,4-dimethylcyclohex-2-en-1-one as the electrophile and ,B-alanine tert-butyl
ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 393.50;
M (found): 399.3 (M+H+), 419.5 (M+Na+), 442.4 (M+K+). Sele~ted nmr data
was as follows: 'H-nmr (CD30D): ô 4.35 (H-l), 4.34, and 4.32.
Example F10
Synthesis of
N~ 3-(l-Thio-~-D-gs~l~ctopyranosyl)cyclohex~l-yl]-~- ~lqn;n~
The title compound was prepared according to procedures D, H and M above
using cyclohex-2-en-1-one as the electrophile and ,B-alanine tert-butyl ester as the
25 amino acid ester. Mass spectra data was as follows: M (calcd.): 365.45; M (found):
367.0 (M+H+), 389.9 (M+Na+). Selected nmr data was as follows: IH-nmr
(CD30D): ~ 4.46 (H-l~, 4.44, 4.43, and 4.36.
.... .. ..... . .. .
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Example F11
Synthesis of
N~B-[5-(1-Thio-~-D-~ topyranosyl)-
2,2-dimethylcyclohex-1-yl]-,B ql~ e
The title compound was p.epa~ed according to procedures D, H and M above
using 6,~dimethyrcyclohex-2-en-1-one as the electrophile and ,B-alanine tert-butyl
ester as the amino acid ester.
Example Gl
Synthesis of
Na-[2-(1-Thio-,B-D-g~ ctopyranosyl)cyclopent-1-yl]-L,leucine
The title compound was p~pa-ed according to procedures ~ H and M above
using 2-chlorocyclopentan-1-one as the electrophile and L-leucine tert-butyl ester as
the amino acid ester. Mass spectra data was as follows: M (calcd.): 393.50; M
(found): 396.4 (M+H+). Sele~t~d nmr data was as follows: IH-nmr (CD30D):
4.47 (H-1), 4.43, 4.36, and 4.34.
Example G2
Synthesis of
N~-[2-(1-Thio-~B-D-galactopyranosyl)cyclohex 1 yl] L~leucin~
The title compound was prepared according to procedures D, H and M above
using 2-chlorocyclohexan-1-one as the electrophile and L-leucine ten-butyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 407.53; M (found):
410.9, (M+H ' ), 435.5 (M+Na+). S~lect~d nmr data was as follows: ~H-nmr
(CD30D): ~ 4.49 (H-1), 4.44, 4.41, and 4.37.
Example G3
Synthesis of
N~-[~Phenyl-4,-(1-thi~B-D-galactopyranosyl)but-2-yl]-L,leuciDe
The title compound was prepared according to procedures D, H and M above
using 4-phenylbut-3-en-2-one as the electrophile and L-leucine tert-butyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 458.59; M (found):
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480.5 (M+Na+). Selected nmr data was as follows: IH-nmr (CD30D): ~ 4.39 (H-l),
4.36, 4.29, and 4.21.
Example G5
S Synthesis of
N~-[1-(1-Thio-~-D-galactopyranosyl)cyclohept-3-yl]-L,leucine
The title compound was prepared according to procedures D, H and M above
using cyclohept-2-en-1-one as the electrophile and L-leucine tert-butyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 421.55; M (found):
421.7 (M+H+), 448.0 (M+Na+). Selected nmr data was as follows: 'H-nmr
(CD30D): ~ 4.44 (H-1), 4.43, and 4.36.
Example G6
Synthesis of
Ncr-[4,4 Dimethyl-3-(1-thi~,B-D-galactopyranosyl)-
cyclopent-l-yl]-L,leucine
The title compound was ~-epared according to procedures D, H and M above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and L-leucine tert-butyl
ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 421.55;
M (found): 422.3 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D):
4.320 (H-l) and 4.315.
Example G7
Synthesis of
Nc~-t3-(1-Thio-,B-D-galactopyranosyl)cyclopent-l-yl]-L,Ieucine
The title compound was l~lc~aled according to procedures D, H and M above
using cyclopent-2-en-1-one as the electrophile and L-leucine tert-butyl ester as the
arnino acid ester. Mass spectra data was as follows: M (calcd.): 393.50; M (found):
393.6 (M+H+), 417.0 (M+Na+). Selected nmr data was as follows: IH-nmr
(CD30D): ~ 4.380 (H-l), 4.375, 4.370 and 4.367.
, .. ~ . . ..
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Example G8
Synthesis of
N~ ~(l-Thio-~-D-galactopyranosyl)pent-2-yl]-L~leucine
The title compound was prepared according to procedures D, H and M above
5 using pent-3-en-2-one as the electrophile and L-leucine tert-butyl ester as the amino
acid ester. Mass spectra data was as follows: M (calcd.): 395.51; M (found): 396.8
(M+H+), 419.1 (M+Na+), and 440.9 (M+K+). Sel~ct~ nmr data was as follows:
IH-nmr (CD30D): ~ 4.42 (H-l), 4.41, 4.405 and 4.40.
Example G9
- Synthesis of
N~-[4,4-Dimethyl-3-(1-thio-l~-D-g~lactQpyranosyJ)-
cyclohex-l-yl]-L,leucine
The title compound was prepared according to procedures D, H and M above
using 4,4-dimethylcyclohex-2-en-l-one as the electrophile and L-leucine tert-butyl
ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 436.58;
M (found): 438.0 (M+H+), 461.4 (M+Na+). Sele~t~ nmr data was as follows: IH-
nmr (CD30D): ~ 4.38 (H-l) and 4.34.
Example G10
Synthesis of
N~r-[3-(1-Thio-~B-D-galactopyranosyl)cyclohex-l-yl]-L~leucine
The title compound was prepared according to procedures D, H and M above
using cyclohex-2-en-1-one as the electrophile and L-leucine ten~-butyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 407.53; M (found):
408.4 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.46 (H-l),
4.42, 4.40, and 4.33.
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Example Hl
-- - Synthesis of
Na-[2-(1-Thio-~-D-galactopyranosyl)cyclopent-l-yl]-L,hi~;(l;ne
The title compound was prepared according to procedures D, H and N above
S using 2-chlorocyclopentan-1-one as the electrophile and L-his~i~line methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 417.48; M (found):
418.7 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ô 4.45 (H-l),
4.41, 4.40, and 4.29.
Example H2
Synthesis of
N~ 2-(1-Thio-,B-D-galactopyranosyl)cyclohex-l-yl]-L-hictillin~
The title compound was prepared according to procedures D, H and N above
using 2-chlorocyclohexan-1-one as the electrophile and L-hicti-line methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 431.50; M (found):
433.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.52 (H-1),
4.45, 4.40, and 4.28.
Example H3
Synthesis of
Na~-[~Phenyl-~(l-thio-~B-D-galactopyranosyl)but-2-yl]-L,histidine
The title compound was prepared according to procedures D, H and N above
using 4-phenylbut-3-en-2-one as the electrophile and L-hi~ti~ine methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 481.56; M (found):
482.8 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.38 (H-l),
4.36, 4.23, and 4.16.
Example H5
Synthesis of
Nlx-[3-(1-Thio-,~-D-galactopyranosyl)cyclohept-l-yl]-~hi~;din~
The title compound was prepared according to procedures D, H and N above
using cyclohept-2-en-1-one as the electrophile and L-histidine methyl ester as the
. , . . , .. .~ ~ ~ . . . ... ... .. .
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arnino acid ester. Mass spectra data was as follows: M (calcd.): 445.54; M (found):
448.0 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.50 (H-l),
4.44, 4.41, and 4.32.
Example H6
Synthesis of
Na-[4,~Dimethyl-3-(1-thio-~B-D-~ ctopyranosyl)-
cyclopent-l-yl]-~hicti-lin~
The title compound was prepared according to procedures D, H and N above
10 using-4,4-dimethylcyclopent-2-en-1-oneas the electrophile and L-histidine methyl
ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 445.54;
M (found): 447.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ô
4.33 (H-l), 4.32, 4.305, and 4.30.
1~ Example H7
Synthesis of
N~ 3-(~ o-~-D-~ ctopyranosyl)cyclopent-l-yl]-L~h~ ;r~
The title compound was plepared according to procedures D, H and N above
using cyclopent-2-en-1-one as the electrophile and L-hi~tidine methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 418.48; M (found):
418.0 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D): ~ 4.39 (H-l),
4.38, 4.36, and 4.32.
Example H8
Synthesis of
Na-[~(l-Thio-,B-D-~ rtopyranosyl)pent-2-yl]-L~hi.c~ te
The title compound was plcpared according to ~ ,cedures D, H and N above
using pent-3-en-2-one as the electrophile and L-hi~ti~line methyl ester as the amino
acid ester. Mass spectra data was as follows: M (calcd.): 419.49; M (found): 420.2
(M+Ht). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.44 (H-l), 4.41,
4.40, and 4.36.
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Example H9
Synthesis of
N~-[4,~Dimethyl-3-(1-thio-,B-D-~ ctopyranosyl)-
cyclohex-1-yl]-L~histidine
The title compound was prepared according to procedures D, H and N above
using 4,4-dimethylcyclohex-2-en-1-one as the electrophile and L-hi~tidine ethyl ester
as the amino acid ester. Mass spectra data was as follows: M (calcd.): 459.56; M(found): 462.2 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ô
4.364 (H-l), 4.357, and 4.34.
Example H10
Synthesis of
Nc~-[3-(1-Thio-~B-D-galactopyranosyl)cyclohex-l-yl]-L ~.;ct;~l;np
The title compound was prepared according to procedures D, H and N above
using cyclohex-2-en-1-one as the electrophile and L-histi~line methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 431.51; M (found):
433.2 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.43 (H-l),
4.425, 4.39 and 4.35.
Example I1
Synthesis of
N~-~2-(1-Thio-~B-D-galactopyranosyl)cyclopent-1-yl]-Irtryptophan
The title compound was prepared according to procedures D, H and N above
using 2-chlorocyclopentan-1-one as the electrophile and L-tryptophan methyl ester as
the amino acid ester. Mass spectra data was as follows: M (calcd.): 466.55; M
(found): 467.5 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.51 (H-1), 4.39, 4.28 and 4.27.
.... .. . . .. . ..
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Examp}e I2
Synthesis of
N~-[2-(l-Thio-~-D-gpl~ctopyranosyl)cyclohex-l-yl]-L~tryptorhql~
The title compound was prepared according to procedures D, H and N above
S using 2-chlorocyclohexan-1-one as the electrophile and L-try~.tophan methyl ester as
the amino acid ester. Mass spectra data was as follows: M (calcd.): 480.59; M
(found): 481.9 ~M+H+), 505.3 (M~Na+). Selected nmr data was as follows: 'H-
nmr (CD30D): ~ 4.46 (H-l), 4.40, 4.24 and 4.09.
Example I3
Synthesis of
N~-[4-Phenyl-~(l-thio-~-D-galactopyranosyl)but-2-yl]-L,tryptophan
The titIe compound was prcyared according to procedures D, H and N above
using 4-phenylbut-3-en-2-one as the electrophile and L-tryl)to~)han methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 531.64; M (found):
531.3 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.24 (H-l),
4.23, 4.14 and 4.09.
Example I5
Synthesis of
Ncr-[3-(1-Thio-,B-D-galactopyranosyl)cyclohept-l-yl]-L,tryptophan
The title compound was prepared according to procedures D, H and N above
using cyclohept-2-en-1-one as the electrophile and L-tryptophan methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 494.60; M (found):
495.9 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.50 (H-l),
4.44, 4.41 and 4.32.
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FY~mple I6
Synthesis of
Na-[4,~Di nethyl-3-(1-thio-,~-D~ ctopyranosyl)-
cyclopent-l-y~ tryptophan
S The title compound was prepared according to procedures D, H and N above
using 4,4-dimethylcyclopent-2-en-1-one as the electrophile and L-tryl~tophan methyl
ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 494.60;
M (found): 4gS.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ô
4.26 (H-1), 4.22, 4.20 and 4.13.
Example I7
Synthesis of
N~-[3-(1-Thio-,B-D-galactopyranosyl)cyclopent-l-yl]-L,tryptorhD~
The title compound was prepared according to procedures D, H and N above
15 using cyclopent-2-en-1-one as the electrophile and L-tryptophan methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 466.55; M (found):
467.9 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.33 (H-1),
4.32, 4.30 and 4.23.
Example I8
Synthesis of
N~-[~(l-Thio-,B-D-gPlncto~yranosyl)pent-2-yl]-Irtryptorh~-~
The title compound was prepared according to procedures D, H and N above
using pent-3-en-2-one as the electrophile and L-tryptophan methyl ester as the amino
25 acid ester. Mass spectra data was as follows: M (calcd.): 468.S7; M (found): 490.9
(M+Na+). S~ ted nmr data was as follows: 'H-nmr (CD30D): ~ 4.30 (H-l), 4.27,
4.22 and 4.09.
.. ... . .. . . ..
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Lxample I9
Synthesis of
N~Y-14,4-Dimethyl-3-(1-thi~,B-D-g~l~ctopyranosyl~-
cyclohex-l-yl~-L,tryptophan
5The title compound was prepared according to procedures D, H and N above
using 4,4-dimethylcyclohex-2-en-1-one as the electrophile and L-try~lo~han methyl
ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 508.63;
M (found): 512.1 (M+H+). Selecte4 nmr data was as ~ollows: 'H-nmr (CD30D):
4.30 (H-l), 4.26, and 4.21.
Example I10
Synthesis of
Na-[3-(1-Thio-,B-D-galactopyranosyl)cyclohex-1-yl]-L,tryptophan
The title compound was prepared according to procedures D, H and N above
15 using cyclohex-2-en-1-one as the electrophile and L-tr)~tophan methyl ester as the
arnino acid ester. Mass spectra data was as follows: M (calcd.): 480.59; M (found):
483.9 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.36 (H-l),
4.35, 4.33, and 4.24.
20Example Jl
Synthesis of
N~-[2-(1-Thi~,B-D-galactopyranosyl)cyclopent-1-yl]-L,arginine
The title compound was ~f~d according to procedures D, H and 0 above
using 2-chlorocyclopentan-1-one as the electrophile and L-arginine methyl ester as the
25amino acid ester. Mass spectra data was as follows: M (calcd.): 436.52; M (found):
436.2 (M+H+). SelPctP~ nmr data was as follows: 'H-nmr (CD30D): ~ 4.54 (H-l),
4.43, 4.41, and 4.28.
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Example J2
~ Synthesis of
N~-[2~ Thio-~B-D-g~ topyranosyl)cyclobex-l-yl]-L,arginine
- The title compound was prepared according to procedures D, H and O above
using 2-chlorocycloheY~n-1-one as the electrophile and L-arginine methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 450.56; M (found):
453.5 (M+H+). Select~d nmr data was as follows: 'H-nmr (CD30D): ~ 4.47 (H-1),
4.45, 4.44, and 4.38.
Example J3
Synthesis of
Na-[~pheny~ -Thio-~B-D-galactopyranosyl)but'-2-yl~-Irarginine
The title compound was prepared according to procedures D, H and O above
using 2-chlorocyclohexan-1-one as the electrophile and L-arginine methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 501.62; M (found):
503.8 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D): ~ 4.32 (H-l),
4.31, and 4.30.
Example JS
Synthesis of
N~r-[3-(1-Thio-,B-D-galactopyranosyl)cyclohept-1-yl]-I~arginine
The title compound was prepared according to procedures D, H and O above
using cyclohept-2-en-1-one as the electrophile and L-arginine methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 464.58; M (found):
467.1 (M+H+). Sçlecte~ nmr data was as follows: IH-nmr (CD30D): ~ 4.48 (H-l),
4.46, and 4.43.
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Example J6
~ - Synthesis of
N~Y-[4,~Dimethyl-3-(1-thio-~-D-g~l~~topyranosyl)-
cyclopent-l-yl]-L,arginine
S The title compound was prepared according to procedures D, H and 0 above
using 4,4-dimethylcyclopent-2-en-1-one as the e}ectrophile and L-arginine methyl ester
as the amino acid ester. Mass spectra data was as follows: M (calcd.): 464.58; M(found): 465.6 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.37 (H-1), 4.35, 4.34, and 4.30.
Example J7
Synthesis of
N~r-t3-(1-Thio-,B-D-galactopyranosyl)cy~lo~Je~ll-l-yl]-L~arginine
The title compound was prepared according to procedures D, H and 0 above
using cyclopent-2-en-1-one as the electrophile and L-arginine methyl ester as the
amino acid ester. Mass spectra data was as follows: M (calcd.): 436.53; M (found):
437.6 (M+H+). Sele~tecl nmr data was as follows: 'H-nmr (CD30D): ~ 4.37 (H-1),
4.35, and 4.34.
Example J8
Synthesis of
N~Y-[~(l-Thio-,B-D-galactopyranosyl)pent-2-yl]-L,arginine
The title compound was prepared according to procedures D, H and 0 above
using pent-3-en-2-one as the electrophile and L-arginine methyl ester as the amino
acid ester. Mass spectra data was as follows: M (calcd.): 438.54; M (found): 437.3
(M+H+). .Celecte~ nmr data was as follows: 'H-nmr (CD30D): ~ 4.46 (H-l), 4.41,
4.39, and 4.38.
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Example J9
Synthesis of
Na-[4,~Dimethyl-3-(1-thio-,B-D-~ ~topyranosyl)-
cyclohex-1-yl]-L,arginine
The title compound was prepared according to procedures D, H and O above
using 4,4-dimethylcyclohex-2-en-l-one as the electrophile and L-arginine methyl ester
as the arnino acid ester. Mass spectra data was as follows: M (calcd.): 478.60; M
(found): 479.0 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
4.43 (H-l), 4.41, 4.38, and 4.32.
Example J10
Synthe~sis of
Na-~3-(1-T;hio-~B-D-galactopyranosyl)cyclohex-1-yl]-L,arginine
The title compound was prepared according to procedures D, H and O above
using cyclohex-2-en-l-one as the electrophile and L-arginine methyl ester as the an~ino
acid ester. Mass spectra data was as follows: M (calcd.): 450.55; M (found): 451.8
(M+H+). .S~l~cted nmr data was as follows: 'H-nmr (CD30D): ~ 4.34 (H-l), 4.33,
4.32, and 4.29.
Example 1
Synthesis of the Individual Diastereomers of
2,2-Dimethyl-~(cyclobut-l-ylamino)-
cyclopent-1-yl 1-Thio-,B-D-galactopyranoside
This example illustrates the preparation of individual diastereomers of a
compound of formula I.
Step A--Syu~ is of (1R,S)-2,2-Dimethylcyclopentan-4On-1-yl2,3,4,6-
Tetra-O-lauroyl-1-thio-~B-D-galactopyr~noside: To l-S-acetyl-2,3,4,6-tetra-O-lauryl-
- 1-thio-,B-D-galactopyranose (5 g, 5 mmol) (from Example C above) and 4,4-dimethyl-
2-cyclopenten-1-one (500 mg, 4.45 mmol) in dry CH2Cl2 (10 mL) under argon, was
added Et2NH (6 mL). After 3 h, the mixture was concentrated and purified by
column chromatography (siO2, pentane/EtOAc, 9: l) to give the title co~ ound as a
mixture of diastereomers (3.54 g, 66%).
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Step B Separation of the Diastereomers of (lR,S)-2,2-
Dimethylcyclopentan-4-on-1-yl 2,3,4,~Tetra-O-lauroyl-1-thio-~B-D-
galactopyranoside: The two diastereomers from Step A (5 g, 4.8 mmol) were
separated by column chromatography (SiO2, pentanelEtOAc, 9:1) to give (lS)-2,2-
S dimethylcyclopentan-4-on-1-yl 2,3,4,6-tetra-0-lauroyl-1-thio-~-D-galactopyr~nosi~e
(428.8 mg, 8%) and (lR)-2,2-dimethylcyclopentan-4-on-1-yl2,3,4,6-tetra-O-lauroyl-
l-thio-,B-D-galactopyr~noci~e (373.8 mg, 6%) along with a mixture of unresolved
compounds (2.74 g, 52%).
Step C -- Synthesis of (lS, 4RS)- and (lR, 4RS)-2,2-Dimethyl~
hydroxycyclopent-1-yl2,3,4,~Tetra-O-lauroyl-l-thio-,B-D-galactopyr~n~si-le: To
each of the purified diastereomers from Step B (in separate reaction flasks) (320 mg,
0.3 mmol) in dry tetrahydrofuran (3 mL), methanol (0.5 mL) and isopropanol (2 mL)
under argon atmosphere, was added NaBH4 (0.12 mmol). After 30 min, AcOH (1
drop) is added and the mixtures were concentrated and the residues dissolved MeOH
(2 mL) and added to a column of C-18 silica (5 g). The columns were washed with
MeOH (50 mL) and products eluted pentane (50 mL) to give (lS, 41~S)-2,2-dime~yl-4-hydroxy-cyclopent-1-yl 2,3,4,6-tetra-O-lauroyl-l-thio-,~-D-galactopyr~nosi~e (281
mg, 88%) and (lR, 4RS)-2,2-dimethyl-4-hydroxy-cyclopent-1-y 2,3,4,~tet~a-~
lauroyl-l-thio-,B-D-galactopyranoside (297 mg, 93%).
Step D--Synthesis of (lS, 4RS)- and (lR, 4RS)-2,2-Dimethyl 4 O-
m_th~n~~-.lfonyloxycyclopent-1-yl 2,3,4,6-Tetra-O-lauroyl-l-thio-~B-D-
galactopyr~nos;~ To each of the (lS, 4RS) and (lR, 4RS) mixtures from Step C
(in separate reaction flasks) (280 mg, 0.3 mmol) in dry tetrahydrofuran (2 mL) and
dry pyridine (4 mL) under argon atmosphere, was added mPth~n~slllfonyl chloride
(0.5 mL). After 12 h, the mixtures were washed with 0.5 M HCl and extracted withpen~ne After concentration, the residues were purified on C18-silica (5 g) as
described in Step C to afford (lS, 4RS)-2,2-dimethyl-4-O-
mPth~nesulfonyloxycyclopent-l-yl 2,3,4,6-tetra-O-lauroyl-1-thio-~-D-
galactopyranoside (281 mg, 88%) and (5) (lR, 4RS)-2,2-dimethyl-4-O-
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me~h~n~suIfonyloxycyclopent-l-yl 2,3,4,6-tetra-O-lauroyl-l-thio-,~-D-
galactopyranoside (297 mg, 93 %) as white solids after pentane evaporation.
Step E--Synthesis of (lS, 4R)-, (lS, 4S)-, (lR, 4S)- and (lR, 4R)-2,~
Dimethyl-4azidocyclopent-1-yl 2,3,4,6-Tetra-O-lauroyl-1-thio-,B-
~
5 galactopyranoside: To the (IS, 4RS) and (lR, 4RS) mixtures from Step D (inseparate reaction flasks) (250 mg, 0.2 mmol) in dry DMF (8 mL) and dry THF (3
mL) under argon atmosphere at 60~C was adde~d NaN3 (340 mg, 5 mmol) and 18
crown-6 (180 mg). After 2 h, the mixtures were concentrated and purified on C18-silica (5 g) as described in Step C. Re-chromatography (SiO2, pentane/EtOAc, 9:1)
10 permitted the separation of diastereomers to give pure (lS, 4R)-2,2-dimethyl-4-
azidocyclopent-1-yl 2,3,4,6-tetra-O-lauroyl-l-thio-~-D-galactopyr~nosi~le (163 mg,
65 5~); (lS, 4S)-2,2-dimethyl-4-azidocyclopent-1-yl 2,3,4,6-tetla-O-lauroyl-l-thio-,B-D-
galactopyranoside (29 mg, 9%); (lR, 4S)-2,2-dimethyl-4-azidocyclopent-1-yl 2,3,4,~
tetra-O-lauroyl-l-thio-,B-D-galactopyranoside (68 mg, 28%); and (lR, 4R)-2,2-
dimethyl-4-azidocyclopent-1-yl 2,3,4,6-tetra-O-lauroyl-1-thio-~-D-galactopyr~nos;de
(21 mg, 9%).
Step F ~ Synthesis of (lS, 4R)-, (lS, 4S)-, (lR, 4S)- and (IR, 4R)-2,~
Dimethyl-4aminocyclopent-1-yl 2,3,4,~Tetra-O-lauroyl-1-thio-~B-D-
g;~l~ctopyranoside: To each of the four diastereomers of 2,2-dimethyl-4-azido-
cyclopent-1-yl l-thio-,B-D-galactopyranoside from Step E (5 mg, 15 ~umol) in dryisopropanol (1 mL) and dry ethanol (1 mL) under argon atmosphere, was added
NaBH4 (15 ~mol) and NiCl2 (30 ~Lmol). After 1 h, the mixtures were neutralized wi~
AcOH (1 drop), concentrated and purified on C18-silica (2 g) as described in Step C
to give (IS, 4R)-, (lS, 4S)-, (lR, 4S)- and (lR, 4R)-2,2-dimethyl-4-aminocyclopent- -
l-yl l-thio-,B-D-galactopyranoside (each S mg; quant.).
Step G--Synthesis of (1S, 4R)-, (lS, 4S)-, (lR, 4S)- and (lR, 4R)-2,2- -
Dimethyl-~(cyclobut-1-ylamino)cyclopent-1-yl 2,3,4,~Tctra-O-lauroyl-1-thio-,B-D-galactopyranoside: To each of four diastereomers of 2,2-dimethyl-4-amino-
cyclopent-1-yl l-thio~ D-galactopyranoside from Step F (in separate reaction flasks)
(2 mg, 6.8 ~Lmol) in dry methanol (1 mL) and dry dichlororn~th~ne (1 mL) under
argon atmosphere, was added cyclobutanone~(250 ~L, 3.4 mmol) and sodium
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triacetoxyborohydride (10 mg, 47 ~mol). After 24-48 h, toluene (1 mL) was added
and the mixture was concentrated and the residue purified on C18-silica as d~s.;libed
in Step C to give 2.1-2.4 mg (quant.) each of:
(lS, 4R)-2,2-dimethyl~-(cyclobut-1-ylamino)cyclopent-1-yl l-thio-,B-D-
5galactopyr~noside (B6HA); M (calcd.): 361.50; M (found): 361.6 (M+H+); ~H-nmr
(CD30D): ~ 4.292 (H-l);
(lS, 4S)-2,2-dimethyl-4-(cyclobut-1-ylamino)cyclopent-1-yl l-thio-,B-D-
galactopyranoside (B6HB); M (calcd.): 361.50; M (found): 361.6 (M+H+); IH-nmr
(CD30D): ~ 4.315 (H-1);
10(lR, 4S)-2,2-dimethyl-4-(cyclobut- 1-ylamino)cyclopent-1 -yl 1-thio-,B-D-
galactopyranoside (B6HC); M (calcd.): 361.50; M (found): 361.6 (M+H+); IH-nmr
(CD30D): ~ 4.300 (H-l);
(1 R, 4R)-2,2-dimethyl-4-(cyclobut- 1 -ylamino)cyclopent- 1 -yl 1 -thio-,B-D-
galactopyr~noside (B6HD); M (calcd.): 361.50; M (found): 361.6 (M+H+); IH-nmr
15(CD30D): ~ 4.290 (H-l).
Example 2
Synthesis of
3-Hydlo~-~c~clohex-1-yl 1-Thio-c~!-L,fucopyr~n~sid~
20The title compound was prepared according to procedures D, E and I above
using l-S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-a-L-fucopyranose (2') as the
thioc~ch~ ide and cyclohex-2-en-1-one as the electrophile. Mass spectra data was as
follows: M (calcd.): 278.37; M (found~: 302.5 (M+Na+). .S~le~te~ nmr data was asfollows: IH-nmr (CD30D): ~ 5.43 and 5.38 (H-1).
Example 3
Synthesis of
3-Aminocyclohex-1-yl 1-Thio-~-Irfucopyranoside
The title compound was prepared according to procedure D, F and J above
30using 1 -S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-a-L-fucopyranose (2') as the
thioc~ch~ride and cyclohex-2-en-1-one as the electrophile. Mass spectra data was as
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follows: M (calcd.): 277.38; M (found): 278.3 (M+H+). Sele~ted nmr data was as
follows: 'H-nmr (CD30D): ~ 5.43, 5.42, 5.36, and 5.34 (H-l).
Example 4
Synthesis of
3-~c~t~mid~cyclohexyl 1-Thio-~-l,fucopyranoside
The title compound was p~e~,ared according to procedures D, F, J and K above
using l-S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-a-L-fucopyranose (2') as the
thi~nch~ride and cyclohex-2-en-1-one as the electrophile. Mass spectra data was as
follows: M (calcd.): 319.42; M (found): 342.2 (M+Na+). Selected nmr data was as
follows: 'H-nmr (CD30D): ~ 5.43, 5.42, 5.38, and 5.37 (H-l).
Example 5
Synthesis of
3-(2-CarboxybPn7~mido)cyclohex-1-yl
l-Thi~-L,fucopyr~nos;~l~
The title compound was prepared according to procedures D, F, G and L above
using l-S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-~-L-fucopyranose (2') as the
t~hiosaccharide and cyclohex-2-en-1-one as the electrophile. Mass spectra data was as
follows: M (calcd.): 425.50, M (found): 448.7 (M+Na+). Sele~ted nmr data was as
follows: IH-nmr (CD30D): ô 5.48, 5.47, 5.45, and 5.40 ffI-1).
Example 6
Synthesis of
Nar-[3-(1-Th;o-~-~fucopyranosyl)cyclohex-l-yl]glycine
l he title compound was prepared according to procedures D, H and M above
using l-S-acetyl-2,3,4,6-tetra-0-lauroyl-1-thio-cY-L-fucopyranose (2') as the
thios~rh~.;de and cyclohex-2-en-1-one as the electrophile and glycine tert-butyl ester
as the amino acid ester. Mass spectra data was as follows: M (calcd.): 335.42; M~found): 336.4 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
5.48, 5.47, 5.39, and 5.36 (H-1).
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Example 7
Synthesis of
N,B-[3-(1-Thio-cY-L,fucopyranosyl)cyclohex-l-yl]~ lqni-~D
The title compound was prepared according to procedures D, H and M above
using 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-l-thio-a-L-fucopyranose (2') as the
thiosacch~ride and cyclohex-2-en-1-one as the electrophile and ,B-alanine tert-butyl
ester as the arnino acid ester. Mass spectra data was as follows: M (calcd.): 349.45;
M (found): 350.0 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D): ô
5.48, 5.47, 5.39 and 5.38 (H-l).
Example 8
Synthesis of
N~Y-[3-(1-Thio-a-L,fucopyranosyl)cyclohex-l-yl]-L,leucine
The title compound was prepaled according to procedures D, H and M above
using 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-l-thio-a-L-fucopyranose (2') as the
thiosaccharide and cyclohex-2-en-1-one as the electrophile and L-leucine tert-butyl
ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 391.53;
M (found): 392.6 (M+H+). Selected nmr data was as follows: IH-nmr (CD30D): ô
5.46, 5.40, and 5.35 (H-l).
Example 9
Synthesis of
Na-[3-(1-Thio-~ fucopyranosyl)cyclohex-l-yl]-L,hic~i lin-
~
The title compound was pl~pared according to procedures D, H and N above
using 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-l-thio-a-L-fucopyranose (2') as the
thio~cch~ride and cyclohex-2-en-1-one as the electrophile and L-hic~i~line methyl ester
as the amino acid ester. Mass spectra data was as follows: M (calcd.): 415.51; M(found): 418.0 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
5.44, 5.38, and 5.35 (H-l).
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Example 10
Synthesis of
Na-[3-(1-Thio-a-L,fucopyranosyl)cyclohex-l-yl~-L,tryptophan
The title compound was ~ ed according to procedures D, H and N above
S using l-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thio-cY-L-fucopyranose (2') as the
thio~ ~h~ride and cyclohex-2-en-1-one as the electrophi}e and L-tr~ opha~l methyl
ester as the amino acid ester. Mass spectra data was as follows: M (calcd.): 464.58;
M (found): 466.7 (M+Na+). Selected nmr data was as follows: 'H-nmr (CD30D):
5.35, 5.32, 5.27, and 5.22 (H-1).
Example 11
- Synthesis of
N~-~3-(1-Thio-~-L,fucopyranosyl)cyclohex-l-yll-L,arginine
The title compound was prepared according to procedures D, H and O above
using 1-S-acetyl-2,3,4,6-tetra-O-lauroyl-1-thio-a-L-fucopyranose (2') as the
thios~ch~ride and cyclohex-2-en-1-one as the electrophile and L-arginine methyl ester
as the amino acid ester. Mass spectra data was as follows: M (calcd.): 434.56; M(found): 435.4 (M+H+). Selected nmr data was as follows: 'H-nmr (CD30D):
5.433, 5.427, 5.38 and 5.32 (H-1).
~xample 12
Synthesis of
Nc~-[3-(5-AcePmi~1O-3,5-dideoxy-2-thio-D-glycero-cY-D-galacto-
2-nonulopyronosyl)cyclohex-1-yl]-L,hicti l;ne
The title compound was prepared according to procedures D, H and N abo~e
using methyl-5-ace~mido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-3,5-dideoxy-2-thi~D-
glycero-a-D-glacto-2-nonulopyranosonate'2 as the thio~ch~ride and cyclohex-2-en-1-
one as the electrophile and L-histi~ine methyl ester as the amino acid ester. Mass
spectra data was as follows: M (calcd.): 415.51; M (found): 418.0 (M+H+).
Selected nmr data was as follows: 'H-nmr (CD30D): ~ 5.44, 5.38, and 5.35 (H-1).
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Example 13
A~t~hment of
[3-(Carboxyben7~m;d~)norborn-2-yllmethyl
l-l'hi~,~-D-g~lqrtopyr~ e to a Solid Support
STo [3-(carboxyben7~mido)norborn-2-yl]methyl l-thio-,~-D-galaclop~ Q~i~e
(2.1 mg, 4.5 ~mor, from Example D4 above), silyl ~min~tP~ Chromosorb P (449 mg,
prepared as described in U.S. Patent No. 4,137,401l8 and Westal et al.'9), and
hydroxybenzotriazole (1.3 mg, 9.4 ~mol) in DMF (1 mL, dried over 4A molecular
sieves), was added diisopropylcarbodiimide (1.4 ~L, 9.0 ~mol). The beads were
10 filtered off after 75 hours, washed with water, DMF, MeOH, and CHtCl2. To theres~llting beads in MeOH (1.5 mL) was added acetic anhydride (0.5 mL) and after
16.5 hours, the beads were filtered and washed with water, DMF, MeOH, CH2Cl2,
and pentane. Fine particles were removed by suspending the beads in MeOH and
decanting the supernatant repeatedly. Drying under high-vacuum gave 433 mg of a
15 product having [3-(carboxybçn7~mido)norborn-2-yl]methyl 1-thio-,B-D-
galactopyranoside covalently ~tt~r~led to the Chromasorb P by formation of an amide
linkage between amine group of the chromasorb P and the carboxy group of the 1-
thiogalactose derivative as shown in formula III below. Phenol/H2SO4 assay using the
procedure described in M. Dubois et al.'3 showed an incorporation yield of 4.0
20 ~mol/g.
~ ~ ~ Ch.~ b~
t
HO ~ s ~
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Example 14
Att~rhmPnt of a Thios~c~h~ride to a Solid Support
To a solution of 1,2:3,4-di-0-isopropylidene-D-galactopyranose (1 eq.) in
pyridine at room temperature is added succinic anhydride (1.2 eq.). The reaction is
S stirred overnight then concentrated in vacuo to give 1,2:3,4-di-0-isopropylidene-~
(3-carboxy)propanoyl-D-galactopyranose. To the residue is added 80% aqueous acetic
acid to remove the isopropylidene groups. When this reaction is complete, the
reaction mixture is concentrated in vaCuo and to the residue is added excess 1:1 acetic
anhydride/pyridine to afford 1,2,3,4-0-acetyl-6-0-(3-carboxy)propanoyl-D-
galactopyranose. To this compound is then added excess thiolacetic acid in dry
dichloromethane under argon at 0~C and boron trifluoride etherate. The cold-bath is
removed after 10 min and after 24 h the mixture is diluted with dichlorometh~ne,washed with saturated sodium bicarbonate, dried over sodium sulfate, and
concentrated to afford l-S-acetyl-2,3,4-tri-0-acetyl-6-0-(3-carboxy)~lu~anoyl-1-thio-
~-D-galactopyranose. To this compound is added ~min7~ted Merrifield resin and a
carbodiimide coupling reagent to afford the 0,S-protected galactûpyranose coupled to
the resin through the 6-0-(3-carboxy)propanoyl group.
Example 15
Solid-Phase Synthesis of 1-Thio~ ctose Derivatives
The example illustrates the solid-phase synthesis of 1-thiog~l~rt( se derivatives
of formula I.
Step A Synthesis of 1-Di~hioe~hyl-2,3,4,~tetra-aacetyl-galactopyranoside:
1-Thio-2,3,4,6-tetra-0-acetyl-galactopyr~noside (500 mg, 1.37 mmol) and diethyl-N-
ethyl-sulfenylhydr~70rlic~rboxylate (360 mg, 2.0 mmol) (plep~ed as described in T.
Mukaiyama20) are dissolved in dichlorornPth~ne (14 mL) and stirred at room
~."pelature. After 10 min, the solution is concentrated and column chromatoE~a~hy
(siO2, hexane/ethylacetate 2:1) yields 1-dithioethyl-2,3,4,6-tetra-0-acetyl-
galactopyranoside (580 mg, quant) as a white solid (R~ 0.27 in hPY~ne~/ethyl acetate
(2: 1)).
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'H-NMR (360 MHz, CHC13): ~ 1.30 (dd, 3 H, J = 7.4 Hz, CH3), 1.96, 2.02, 2.03,
2.13 (4 s, 12 H, 4 CH3CO), 2.79 (ddd, 2 H, J = 7.4 Hz, J--7.4 Hz, J = 1.3 Hz,
CH2), 3.94 (ddd, 1 ~I, J45 = 1.0 Hz, J5.6. = 6.6 Hz, J5.6b = 7.6 Hz, 5-H), 4.10 (ddd,
2 H, 61-H, 6b-H), 4.51 (d, 1 H, J,.2 = 10.0 Hz, l-H), 5.05 (dd, 1 H, J2.3 = 10.0S Hz, J34 = 3.3 Hzj 3-H)), 5.38 (dd, 1 H, J~.2 = 10.0 Hz, J33= 10.0 Hz, 2-H), 5.40
(dd, 1 H, J3.4 = 3.3 Hz, J45 = 1.0 Hz, 4-H); m/z calcd. for C,6H2409S2 (M+Na)
447.1, found 447Ø
Step B--Synthesis of l-Dithioethyl-,~-D-galactopyranoside: l-Dithioethyl-
2,3,4,6-tetra-O-acetyl-galactopyranoside from Step A (500 mg, 1.18 mmol) was
dissolved in dry methanol (10 mL) and treated with methanolic sodium methoxide (1
M, 150 ~L). After 2 h, the solution was neutralized with Amberlite lR-120 (H+)
resin, filtered and concentrated to give l-dithioethyl-6-,~-D-galactopyranoside as a
white solid (300 mg, quant).
Step C--Coupling of l-Dithioethyl-,l5-D-galactopyr~nnc;~le to a Resin: 1-
Dithioet~yl-6-,~-D-galactopyranoside (200 mg, 780 ~mol) was dissolved in dry
pyridine (8 mL). Trityl chloride-resin (1 g, 950 ~mol trityl chloride resin, loading
0.95 mmol/g of active chlorine, polymer matrix: copolystyrene-l % DVB, 200~00
mesh, Novabiochem) and DMAP (5 mg) were added and the mixture was heated for
24 h at 60~C. The resin was filtered off, and washed succes~ively with meth~nnl,tetrahydrofuran, dichlorom~th~ne and diethyl ether (10 mL each) to afford 1-
~ithioethyl-~-D-galactopyranoside covalently linked to the trityl resin through the
hydroxyl group in the 6-position.
Step D--G~neration of the Free Thiol on the Resin: The resin from Step C
(50 mg) is swollen in dry tetrahydrofuran (1.5 mL). Dry meth~nol (300 ~L),
dithiothreitol (74 mg) and triethylamine (180 ~L) are added and the mixture is shaken
for 10 hours at room temperature. The resin is filtered off and washed succes~ively
with methanol, tetrahydrofuran, dichloromethane and diethyl ether (10 mLleach). IR
(of intact beads): 2565 cm~l (SH stretch).
Step E Michael Addition Re~ct;Qn: The resin from Step D (50 mg) was
swollen in dry N,N-dimethylformamide (1 rnL) and then cyclohept-2-en-1-one (70 ~1,
63 ~mol) was added and the mixture was shaken at room temperature. After 2 hours,
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the resin was filtered off and washed succescively with me~h~nol~ tetrahydrofuran,
dichlorometh~ne and ~iethyl eSher (10 mL each).
Step F ~ Reductive ~n;n~tion with an Amino Acid: The resin from Step E
(50 mg) was swollen in dichlororneth~n~ (1 mL). Glycine ten-butyl ester
hydrochloride (75.mg, 447 ~mol), sodium sulfate (100 mg), sodium
triacetoxyborohydride (63 mg, 297 llmol) and acetic acid (lO ILL) were added at room
temperature under argon atmosphere and the mixture shaken for 24 hours. The resin
was then filtered off and washed successively with water, methanol, tetrahydrofuran
and dichloromethane.
Step G -- Cleavage from the l-Thio~lact~se Derivative from the Resin and
Deblocking of the Amino Acid Ester: The resin from Step F (50 mg) was shaken
with trifluoroacetic acid (1 mL) and triisopropylsilane (20 ,uL) in dichlorol,.e~ ne (2
mL) at room temperature. After 3 hours, the resin was removed by filtration and
washed with dichlorometh~ne (10 mL). After adding toluene (10 mL), the solution
was concentrated, then co-evaporated twice with toluene. The residue was dissolved
in water (1 mL) and applied onto two C,g-Sep-Pak-cartridges (Waters Sep-Pak Plus).
The C18 silica was washed with water (4 mL) and the final product was eluted with
20% meth~nol and concentrated. After freeze drying from 5 mL of water, Na-[3-(1-thio-,B-D-galactopyranosyl)cyclohept-1-yl]glycine was obtained as a white powder (4.8
mg). The diastereomers ratio was 10:10:8:6 as determined by 'H-NMR.
'H-NMR (360 MHz, CD30D, anomeric protons): ~ 4.36 (d, J, 2 = 9.6 Hz), 4.40 (d,
Jl2 = 9.5 Hz), 4.44 (d, J~2 = 9.1 Hz), 4.45 (d, J, 2 = 9.2 Hz); m/z calcd. for
C,5H27NO7S (M+H), 366.2, found 366.1.
Example 16
Inhibition of Heat-Labile Enterotoxin Binding to GDlb
In this example, 1-thiog~lactose derivatives of forrnula I above were tested fortheir ability to inhibit the binding of heat-labile enterotoxin from E. coli to g~nglios~de
GDlb- This bioassay was conduct~l using the procedure described by A.-M.
Svennerholm2' except that ganglioside GDlb was used instead of ganglioside GMI- The
compounds of Examples A1, A2, A4-A7, A10, A11, B1, B2, B4-B7, B10, B11, C2,
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CS, C7, C10, Cll, D2, D4, D5, El, E2, E4, E10, Ell, Fl, F2, F5, F7, F10, ~11,
G2, G5, I2, I5, and J7 were tested in this bioassay. All of the compounds testedinhibited binding of heat-labile entcfotoAin to ganglioside GDIb by at least 20~, except
for the compounds of Examples A2, A5, A7, C10, D2 and G2, which did not inhibit
S binding by at least 20% at the concentration employed in the assay.
Example 17
Inhibition of Cholera Toxin Binding to GDlb
In this example, l-thiog~l~etose derivatives of formula I above were tested for
10 their ability to inhibit the binding of cholera toxin to ganglioside GD,b. This bioassay
was conducted using the following mo~ifi~tion of the procedure described by A.-M.
Svennerholm2l.
On day 1, microtiter plates (C96 Maxisorp) were coated with 100 ~LL of 1
mg/mL GDlb (disialoganglioside GDlb, MW = 2127, Fluka) in PBS per well and
15 incub~ted overnight at 37~C.
On day 2, the s~mples to be tested were diluted in BSA-Tween-PBS (0.1% BSA
and 0.05% Tween-20 in PBS; Sigma). A total of 500 ~L of each solution was
plep~ so that each point could be measured in quadruplicate. A conce~
curve of 10, 20 and 30 ng/mL of CTB5-HRP (CT-B5 conjugated to HRP, Sigma,
20 lyophilized, diluted in Tween-PBS) was prepared. For the inhibition experiment~, 20
ng/mL of CTB5-HRP was used. The samples were then incub~t~d for 2 hours at
room lem~ldture. After incub~tion~ the plates were emptied and un~tt~~h~d
ganglioside was removed by washing the plates 2 times with 200 ~LL PBS per well.Additi'~ binding sites on the plastic surface were then bloc~ed by in~;uh~l;n~ the
25 plates with 200 ,uL of 1% BSA in PBS per well for 30 minutes at 37~C. The plates
were then emptied and l-n~tt~hed BSA was removed by washing the plates 3 times
with 200 ~L of 0.05% Tween 20-PBS per well. Samples (100 ~L) were added to 4
different wells and incubated for 30 minutes at room le,llp~;ldture. The plates were
emptied and unat~che~ BSA was removed by washing the plates 3 times with 200 ~L
30 of 0.05% Tween 20-PBS per well.
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A substrate solution was freshly piepar~d for each ELISA. Each sol~tion
contained 10 mg of o-phenylene~ mine (Sigma), S mL of 0. lM sodium citrate (filter
sterile or autoclaved), 5 mL of 0. lM citric acid (filter sterile or autoclaved) and 4 mL
of 30% H2O2. (Gloves should be worn since ~phenyl~n~Ai~mine is carcinogenic).
S The substrate solution (100 ~LL) was then added to each well and inc~ t~ for 30
minutes at room temperature. After incub~tion, the OD450 was recorded. Under theco~litions of the assay, D-galactose had an IC50 of 30 mM.
The compounds of Examples Al-A10, Bl-B6, B~A-B6L, B6Q, B6T, B7-B8,
B10 C1-C3, C5, C7, C8, C10, Dl-D5, D8, El-E9, Fl-F10, G2, G3, G5-G10, H2,
H3, H5-H10, I1-I3, I5-I10, J1-J3 and J5-J10 were tested in this bioassay. All of the
compounds tested inhibited binding of cholera toxin to g~ iosi~e_GD,b by at least
20%, except for the compounds of Examples Al, A3, A4, A~A8, A10, Bl, B3, B4,
B10, Cl, C3, C8, D3, _5, E8, E9, Fl, F5-F7, F9, F10, G3, G7-G10, H2, H5, H8-
H10, I2, I8-I10, J5-J10, which did not inhibit binding by at least 20% at the
15 concentration employed in the assay (i.e., 1 mg/mL).
_xample 18
Neutralization of the Cytotonic Activity of CT and LT
In this example, the solid support material of Fy~mp]e 13 was tested for its
20 ability to neutralize the cytotonic activity of CT and LT. The cytotonic activity of CT
and LT was measured by the use of Chinese h~mster ovary cells (CHO) that were
maintained in Hams F12 media supplemented with 10% fetal bovine serum (FBS) in
an ~tmosphere of 5% CO2 at 37~C. Toxin samples were diluted 1:5 in Hams media
and filter sterilized through 0.22 micron syringe filters. Samples were then serial 5-
25 fold diluted in media and 100 ~L of each dilution was added to wells with co~fll~entmonolayers of CHO cells and incub~ for 24 h at 37~C (under 5% CO2). Each
sample was analyzed two times. Cytotonic effects were readily visible after 24 hincub?,tion by comparing wells with controls that do not contain toxin. After 24 h,
the cells were fixed with 95 % methanol and stained with Geimsa stain. Toxin
30 containing samples from neutralization experiments were treated in an analogous
fashion except that the percent neutralization was determined by co".p~ing the
. . -- . ., ~ , .
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endpoint dilutions of ~mples with and without the solid support m~teri~l of F~ rl,o
13. ~
A solution cont~ininE purified CT or LT (2, 10 or 20 ~g in 1 mL PBS) was
added to the solid support material of Example 13 (20 mg) in 1.5 mL mi.;ruc~ . ;fuge
S tubes and incub~ted at room te~ ture for 1 h on an end-over rotator. After
inc~lb~tion, the solid support m~tPri~l was allowed to settle to the bottom of the tubes
and the supernatants were carefully removed. The supernatants were added to CHO
cells and the cytotonic endpoint determined after incub~tion for 24 h as desrri~e~
above. The extent of reduction in the endpoint in the presence of the solid S.
10 material was determined by comparing with controls in which solid support nl~tf~r
was not added.
Results showed that the solid support material of F~mp1~ 13 neutralized more
than 90% of CT and LT activity, regardless of toxin concentration, i.e., less than
10% toxin activity rem~inçd.
Example 19
Inhibition of ColQr~i7~t;on Factor
Antigens (CFA pili) Binding to Glycophorin
In this example, l-thiog~l~et- se derivatives of formula I above were tested for20 their ability to inhibit CFA pili binding to glycophorin. Bacterial surface ~ll~si~n
antigens such as CFA pili are a virulence factor e,-~less~d by certain enteric
pathogens, including enterotoxigenic E. coli. These pili are inlpo~ factors in
bacterial ~tt~c~lm~nt to cell surface ~ecepto~. Accordingly, inhi~ition of CFA pili
binding is a useful test to determine whether a coml~o~ d will inhibit the binding of a
25 pathogenic microorganism to cell surface lecep~ol~.
Binding assays were done by coating microtitre wells with 50 ~L of glyc~ph~
(10 ~g/mL) in PBS for 2 h at 37~C. The solution was removed by aspiration and
replaced with 100 ~LL of 1% BSA in PBS cont~ining 0.05% Tween 20 (PBST) and
incllb~t~d at 37~C for an additional 1 h. The microtitre wells were washed three30 times with 200 ~L of PBST and then replaced with biotinylated CFA I (5 ~g/mL) in
50 ~L of PBS containing 0.05% BSA. After incub~ing for 2 h at 37~C the binding
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reaction was stopped by aspirating the solutions and the plate was washed with PBST
- (3 X 200 ~L). Avidin-peroxidase (50 ~L of a 1/3000 dilution of a 1 mg/mL solution
in PBST containing 0.05 % BSA) was added and the plates were incub~t~d for an
additional 1 h. After washing the wells as described above, 100 ~L of the s~bst~te
S solution (0.42 mM tetramethylben7idine (TMB) in 0.1 M sodium citrate buffer, pH
6.0, containing 0.5 ~LM urea peroxide) was added and the plates were inruh~te~ for
10 min at ambient temperature and the enzyme reaction stopped by adding 50 ~LL of
2N H2SO4. Binding assays were done in triplicate and background binding was
measured in wells coated with BSA only.
Binding inhibition assays were done using oligo~rc-h~ride analogs at a
concentration of 1 mg/mL in PBS. Inhibitors were preincub~ed with biotinyla~d
CFA I pili (5 /l/mL) for 1 h at 37~C prior to adding to glycophorin-coated microtitre
wells as outlined above. o-Nitrophenyl-,B-D-g~l~ctose was utilized as a control
inhibitor for these experiments.
The l-thiogalactose derivatives of Examples Al-A10, Bl-B8, B10, Cl-C3, C5,
C7, C8, C10, Dl-D5, D8, D10, El-E10, F1-F10, G1-G3, G5-G10, H1-H3, H5-H10,
I1-I3, I5-I10, J1-J3 and J5-J10 were tested. Of these compounds, the results showed
that the compounds of Examples B2, B5, H2, H3, H5, H6, H7, H8, H9, H10, I1, I2
and J9 inhibited CFA I pili binding to glycophorin, with the ~mollnt of inhibition
20 ranging from 13 to 71%. The compounds having a histidine or a tryptophan (Group
H and I) moiety were particularly good inhibitors in this experiment.
From the foregoing desc~ tion, various mo~lification~ and changes in the
col,lposiLion and method will occur to those skilled in the art. All such mo~ifi~tions
coming within the scope of the appended claims are intended to be included therein.
