U.S. patent application number 15/181725 was filed with the patent office on 2016-12-15 for heterobifunctional pan-selectin inhibitors.
The applicant listed for this patent is GLYCOMIMETICS, INC.. Invention is credited to Beat Ernst, John L. Magnani, John T. Patton, Arun K. Sarkar, Sergei A. Svarovsky.
Application Number | 20160361332 15/181725 |
Document ID | / |
Family ID | 37603124 |
Filed Date | 2016-12-15 |
United States Patent
Application |
20160361332 |
Kind Code |
A1 |
Magnani; John L. ; et
al. |
December 15, 2016 |
HETEROBIFUNCTIONAL PAN-SELECTIN INHIBITORS
Abstract
Compounds and methods are provided for modulating in vitro and
in vivo processes mediated by selectin binding. More specifically,
selectin modulators and their use are described, wherein the
selectin modulators that modulate (e.g., inhibit or enhance) a
selectin-mediated function comprise particular glycomimetics alone
or linked to a member of a class of compounds termed BASAs (Benzyl
Amino Sulfonic Acids) or a member of a class of compounds termed
BACAs (Benzyl Amino Carboxylic Acids).
Inventors: |
Magnani; John L.;
(Gaithersburg, MD) ; Patton; John T.; (Angola,
IN) ; Sarkar; Arun K.; (North Potomac, MD) ;
Svarovsky; Sergei A.; (San Diego, CA) ; Ernst;
Beat; (Magden, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLYCOMIMETICS, INC. |
Rockville |
MD |
US |
|
|
Family ID: |
37603124 |
Appl. No.: |
15/181725 |
Filed: |
June 14, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14106662 |
Dec 13, 2013 |
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15181725 |
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13165609 |
Jun 21, 2011 |
8633303 |
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14106662 |
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12764027 |
Apr 20, 2010 |
7989601 |
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13165609 |
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11515343 |
Sep 1, 2006 |
7728117 |
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12764027 |
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60713994 |
Sep 2, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 19/10 20180101; A61P 1/00 20180101; A61P 31/00 20180101; A61P
3/10 20180101; A61P 37/02 20180101; A61P 17/04 20180101; A61P 29/00
20180101; A61P 37/08 20180101; A61P 17/02 20180101; A61P 35/00
20180101; A61P 21/04 20180101; A61P 1/04 20180101; A61P 7/02
20180101; A61P 31/04 20180101; A61P 11/16 20180101; A61P 17/06
20180101; A61P 37/06 20180101; A61P 7/00 20180101; A61P 35/02
20180101; A61P 43/00 20180101; A61P 9/00 20180101; C07H 15/22
20130101; A61P 11/00 20180101; C07H 17/00 20130101; A61P 13/12
20180101; A61P 9/10 20180101; C07H 15/26 20130101; A61K 31/706
20130101; A61P 9/14 20180101; A61P 35/04 20180101; A61P 19/08
20180101; A61K 47/549 20170801; A61P 37/00 20180101; A61P 25/00
20180101; A61P 11/06 20180101 |
International
Class: |
A61K 31/706 20060101
A61K031/706; A61K 47/48 20060101 A61K047/48 |
Claims
1-25. (canceled)
26. A method of inhibiting rejection of a transplanted tissue in a
patient wherein said patient is a recipient of the transplanted
tissue, comprising administering to the patient a compound of
formula (I): ##STR00037## or a physiologically acceptable salt
thereof, wherein: ##STR00038## where n=0-2, and each occurrence of
R.sup.8 is independently selected where n=2: R.sup.2=H,
--C(.dbd.O)OX where X is C.sub.1-C.sub.8 alkanyl, C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.8 alkynyl or C.sub.6-C.sub.14 aryl;
--C(.dbd.O)NH(CH.sub.2).sub.nNH.sub.2,
--[C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)].sub.m(L).sub.mZ, where
n=0-30, m=0-1, L is a linker, and Z is a benzyl amino carboxylic
acid or a polyethylene glycol; ##STR00039## --O--C(.dbd.O)--X,
--NH.sub.2, --NH--C(.dbd.O)--NHX, or --NH--C(.dbd.O)--X where n=0-2
and X is independently selected from C.sub.1-C.sub.8 alkanyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, ##STR00040##
where n=0-10, and any of the above ring compounds may be
substituted with one to three independently selected of Cl, F,
C.sub.1-C.sub.8 alkanyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.6-C.sub.14 aryl, or OY where Y is H, C.sub.1-C.sub.8
alkanyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, or
C.sub.6-C.sub.14 aryl; ##STR00041## 6'sulfated GlcNAc,
6'carboxylated GlcNAc, 6'sulfated GalNAc, 6'sulfated galactose, or
6'carboxylated galactose; R.sup.5=H; R.sup.6=H, fucose, mannose,
arabinose, galactose or polyols; R.sup.7=H, C.sub.1-C.sub.8
alkanyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl or
##STR00042## and R.sup.8=H, C.sub.1-C.sub.8 alkanyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, ##STR00043##
where n=0-3 and X is independently selected from H, OH, Cl, F,
N.sub.3, NH.sub.2, C.sub.1-C.sub.8 alkanyl, C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.6-C.sub.14 aryl,
OC.sub.1-C.sub.8 alkanyl, OC.sub.2-C.sub.8 alkenyl,
OC.sub.2-C.sub.8 alkynyl, and OC.sub.6-C.sub.14 aryl, and any of
the above ring compounds may be substituted with one to three
independently selected of Cl, F, C.sub.1-C.sub.8 alkanyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.6-C.sub.14
aryl or OY where Y is H, C.sub.1-C.sub.8 alkanyl, C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.8 alkynyl, or C.sub.6-C.sub.14 aryl.
27. A method of targeting an agent to a selectin-expressing cell,
comprising contacting said selectin-expressing cell with a compound
of formula (I): ##STR00044## or a physiologically acceptable salt
thereof, wherein: ##STR00045## where n=0-2, and each occurrence of
R.sup.8 is independently selected where n=2; R.sup.2=H,
--C(.dbd.O)OX where X is C.sub.1-C.sub.8 alkanyl, C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.8 alkynyl or C.sub.6-C.sub.14 aryl;
--C(.dbd.O)NH(CH.sub.2)--NH.sub.2,
--[C(.dbd.O)NH(CH.sub.2)--NHC(.dbd.O)].sub.m(L).sub.mZ, where
n=0-30, m=0-1, L is a linker, and Z is a benzyl amino carboxylic
acid or a polyethylene glycol; ##STR00046## --O--C(.dbd.O)--X,
--NH.sub.2, --NH--C(.dbd.O)--NHX, or --NH--C(.dbd.O)--X where n=0-2
and X is independently selected from C.sub.1-C.sub.8 alkanyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, ##STR00047##
where n=0-10, and any of the above ring compounds may be
substituted with one to three independently selected of C1, F,
C.sub.1-C.sub.8 alkanyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8
alkynyl, C.sub.6-C.sub.14 aryl, or OY where Y is H, C.sub.1-C.sub.8
alkanyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, or
C.sub.6-C.sub.14 aryl; ##STR00048## 6'sulfated GlcNAc,
6'carboxylated GlcNAc, 6'sulfated GalNAc, 6'sulfated galactose, or
6'carboxylated galactose; R.sup.5=H; R.sup.6=H, fucose, mannose,
arabinose, galactose or polyols; R.sup.7=H, C.sub.1-C.sub.8
alkanyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl or
##STR00049## and R.sup.8=H, C.sub.1-C.sub.8 alkanyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, ##STR00050##
where n=0-3 and X is independently selected from H, OH, Cl, F,
N.sub.3, NH.sub.2, C.sub.1-C.sub.8 alkanyl, C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.6-C.sub.14 aryl,
OC.sub.1-C.sub.8 alkanyl, OC.sub.2-C.sub.8 alkenyl,
OC.sub.2-C.sub.8 alkynyl, and OC.sub.6-C.sub.14 aryl, and any of
the above ring compounds may be substituted with one to three
independently selected of Cl, F, C.sub.1-C.sub.8 alkanyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.6-C.sub.14
aryl or OY where Y is H, C.sub.1-C.sub.8 alkanyl, C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.8 alkynyl, or C.sub.5-C.sub.14 aryl; wherein
said compound further comprises a diagnostic or therapeutic
agent.
28. The method according to claim 26 or 27, wherein R.sup.6 is
fucose.
29. The method according to claim 26 or 27, wherein R.sup.7 is
H.
30. The method according to claim 26 or 27, wherein R.sup.4 is
##STR00051##
31. The method according to claim 26 or 27, wherein R.sup.6 is
galactose.
32. The method according to claim 26 or 27, wherein R.sup.2 is
--[C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)].sub.m(L).sub.mZ, where
n, m, L and Z are defined according to claim 26 or 27.
33. The method according to claim 26 or 27, wherein L is
##STR00052## and wherein the terminal carbonyl group of said L is
attached to Z.
34. The method according to claim 26 or 27, wherein R.sup.3 is
--O--C(.dbd.O)--X or --NH--C(.dbd.O)--X, where X is defined
according to claim 26 or 27.
35. The method according to claim 34, wherein X is ##STR00053##
36. The method according to claim 26 or 27, wherein R.sup.8 is:
##STR00054##
37. The method according to claim 26 or 27, wherein said
physiologically acceptable salt is a sodium salt or a potassium
salt.
38. The method according to claim 26 or 27, wherein the compound is
administered in combination with a pharmaceutically acceptable
carrier or diluent.
39. The method according to claim 26 or 27, wherein the compound is
attached to a diagnostic or therapeutic agent.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application No. 60/713,994
filed Sep. 2, 2005, which application is incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Field of the Invention
[0003] The present invention relates generally to compounds,
compositions and methods for modulating processes mediated by
selectin binding, and more particularly to selectin modulators and
their use, wherein the selectin modulators that modulate a
selectin-mediated function comprise particular glycomimetics alone
or linked to a member of a class of compounds termed BACAs (Benzyl
Amino Sulfonic Acids) or a member of a class of compounds termed
BACAs (Benzyl Amino Carboxylic Acids).
[0004] Description of the Related Art
[0005] When a tissue is infected or damaged, the inflammatory
process directs leukocytes and other immune system components to
the site of infection or injury. Within this process, leukocytes
play an important role in the engulfment and digestion of
microorganisms. Thus, the recruitment of leukocytes to infected or
damaged tissue is critical for mounting an effective immune
defense.
[0006] Selectins are a group of structurally similar cell surface
receptors that are important for mediating leukocyte binding to
endothelial cells. These proteins are type 1 membrane proteins and
are composed of an amino terminal lectin domain, an epidermal
growth factor (EGF)-like domain, a variable number of complement
receptor related repeats, a hydrophobic domain spanning region and
a cytoplasmic domain. The binding interactions appear to be
mediated by contact of the lectin domain of the selectins and
various carbohydrate ligands.
[0007] There are three known selectins: E-selectin, P-selectin and
L-selectin. E-selectin is found on the surface of activated
endothelial cells, which line the interior wall of capillaries.
E-selectin binds to the carbohydrate sialyl-Lewis.sup.x
(SLe.sup.x), which is presented as a glycoprotein or glycolipid on
the surface of certain leukocytes (monocytes and neutrophils) and
helps these cells adhere to capillary walls in areas where
surrounding tissue is infected or damaged; and E-selectin also
binds to sialyl-Lewis.sup.a (SLe.sup.a), which is expressed on many
tumor cells. P-selectin is expressed on inflamed endothelium and
platelets, and also recognizes SLe.sup.x and SLe.sup.a, but also
contains a second site that interacts with sulfated tyrosine. The
expression of E-selectin and P-selectin is generally increased when
the tissue adjacent to a capillary is infected or damaged.
L-selectin is expressed on leukocytes. Selectin-mediated
intercellular adhesion is an example of a selectin-mediated
function.
[0008] Modulators of selectin-mediated function include the PSGL-1
protein (and smaller peptide fragments), fucoidan, glycyrrhizin
(and derivatives), anti-selectin antibodies, sulfated lactose
derivatives, and heparin. All have shown to be unsuitable for drug
development due to insufficient activity, toxicity, lack of
specificity, poor ADME characteristics and/or availability of
material.
[0009] Although selectin-mediated cell adhesion is required for
fighting infection and destroying foreign material, there are
situations in which such cell adhesion is undesirable or excessive,
resulting in tissue damage instead of repair. For example, many
pathologies (such as autoimmune and inflammatory diseases, shock
and reperfusion injuries) involve abnormal adhesion of white blood
cells. Such abnormal cell adhesion may also play a role in
transplant and graft rejection. In addition, some circulating
cancer cells appear to take advantage of the inflammatory mechanism
to bind to activated endothelium. In such circumstances, modulation
of selectin-mediated intercellular adhesion may be desirable.
[0010] Accordingly, there is a need in the art for identifying
inhibitors of selectin-mediated function, e.g., of
selectin-dependent cell adhesion, and for the development of
methods employing such compounds to inhibit conditions associated
with excessive selectin activity. The present invention fulfills
these needs and further provides other related advantages.
BRIEF SUMMARY OF THE INVENTION
[0011] Briefly stated, this invention provides compounds,
compositions and methods for modulating selectin-mediated
processes. In the present invention, the compounds that modulate
(e.g., inhibit or enhance) a selectin-mediated function comprise a
particular glycomimetic alone or linked to a BASA or a BACA. Such
compounds may be combined with a pharmaceutically acceptable
carrier or diluent to form a pharmaceutical composition. The
compounds or compositions may be used in a method to modulate
(e.g., inhibit or enhance) a selectin-mediated function, such as
inhibiting a selectin-mediated intercellular adhesion.
[0012] In one aspect of the present invention, compounds are
provided having the formula:
##STR00001##
wherein:
##STR00002##
[0013] where n=0-2, and R.sup.8 are independently selected where
n=2; [0014] R.sup.2=H, --C(.dbd.O)OX where X is C.sub.1-C.sub.8
alkanyl, C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl or
C.sub.1-C.sub.14 aryl, --C(.dbd.O)NH(CH.sub.2).sub.nNH.sub.2,
--[C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)].sub.m(L).sub.mZ, where
n=0-30, m=0-1, L is a linker, and Z is a benzyl amino sulfonic
acid, a benzyl amino carboxylic acid, a polyethylene glycol, or a
second compound or salt thereof having the above formula to form a
dimer where R.sup.2 of the second compound or salt thereof has m=0,
no Z, and is the point of attachment;
[0014] ##STR00003## --O--C(.dbd.O)--X, --NH.sub.2,
--NH--C(.dbd.O)--NHX, or --NH--C(.dbd.O)--X where n=0-2 and X is
independently selected from C.sub.1-C.sub.8 alkanyl,
C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl,
##STR00004##
where n=0-10, and any of the above ring compounds may be
substituted with one to three independently selected of Cl, F,
C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8
alkynyl, C.sub.1-C.sub.14 aryl, or OY where Y is H, C.sub.1-C.sub.8
alkanyl, C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl, or
C.sub.1-C.sub.14 aryl;
##STR00005## 6'sulfated GlcNAc, 6'carboxylated GlcNAc, 6'sulfated
GalNAc, 6'sulfated galactose, 6'carboxylated galactose or
##STR00006##
where R.sup.9 is aryl, heteroaryl, cyclohexane, t-butane,
adamantane, or triazole, and any of R.sup.9 may be substituted with
one to three independently selected of Cl, F, C.sub.1-C.sub.8
alkanyl, C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl or OY
where Y is H, C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8 alkenyl,
C.sub.1-C.sub.8 alkynyl or C.sub.1-C.sub.14 aryl; [0015] R.sup.5=H,
or R.sup.4 and R.sup.5 are taken together to form
[0015] ##STR00007## where R.sup.10 is aryl, heteroaryl,
##STR00008## where n=0-10, and any one of the above ring compounds
may be substituted with one to three independently selected of Cl,
F, C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8 alkenyl,
C.sub.1-C.sub.8 alkynyl or OY where Y is H, C.sub.1-C.sub.8
alkanyl, C.sub.1-C.sub.8 alkenyl or C.sub.1-C.sub.8 alkynyl; [0016]
R.sup.6=H, fucose, mannose, arabinose, galactose or polyols; [0017]
R.sup.7=H, C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8 alkenyl,
C.sub.1-C.sub.8 alkynyl or
##STR00009##
[0017] and [0018] R.sup.8=H, C.sub.1-C.sub.8 alkanyl,
C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl,
[0018] ##STR00010## [0019] where n=0-3 and X is independently
selected from H, OH, Cl, F, N.sub.3, NH.sub.2, C.sub.1-C.sub.8
alkanyl, C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl,
C.sub.1-C.sub.14 aryl, OC.sub.1-C.sub.8 alkanyl, OC.sub.1-C.sub.8
alkenyl, OC.sub.1-C.sub.8 alkynyl, and OC.sub.1-C.sub.14 aryl, and
any of the above ring compounds may be substituted with one to
three independently selected of Cl, F, C.sub.1-C.sub.8 alkanyl,
C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl, C.sub.1-C.sub.14
aryl or OY where Y is H, C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8
alkenyl, C.sub.1-C.sub.8 alkynyl, or C.sub.1-C.sub.14 aryl.
[0020] A compound of the present invention includes physiologically
acceptable salts thereof. A compound of the present invention in
combination with a pharmaceutically acceptable carrier or diluent
provides a composition of the present invention. In the chemical
formulae herein, a line extending from an atom depicted or a carbon
implied by the intersection of the two other lines, represents the
point of attachment (and does not represent a methyl group).
[0021] In an embodiment of the present invention, R.sup.6 is
fucose:
##STR00011##
[0022] In an embodiment, R.sup.7 is H.
[0023] In an embodiment, R.sup.4 is
##STR00012##
##STR00013##
[0024] In an embodiment, R.sup.4 is where R.sup.9 is defined as for
the general formula above.
[0025] In an embodiment, R.sup.9 is cyclohexane.
[0026] In an embodiment, R.sup.6 is galactose.
[0027] In an embodiment, R.sup.8 is
##STR00014##
[0028] In an embodiment, R.sup.2 is
--[C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)].sub.m(L).sub.mZ, where
n, m, L and Z are defined as for the general formula above.
[0029] In an embodiment, Z is a benzyl amino sulfonic acid, a
benzyl amino carboxylic acid or a polyethylene glycol.
[0030] In an embodiment, R.sup.3 is --O--C(.dbd.O)--X, where X is
defined as for the general formula above.
[0031] In an embodiment, X is
##STR00015##
[0032] In an embodiment, R.sup.5 is H.
[0033] In an embodiment, L is a polyethylene glycol or a
thiadiazole.
[0034] In an embodiment, a compound comprises a compound according
to the present invention, further comprising a diagnostic or
therapeutic agent. Such a compound may be combined with a
pharmaceutically acceptable carrier or diluent to form one
embodiment of a composition of the present invention.
[0035] In another aspect of the present invention, methods are
provided for using a compound or composition of the present
invention to modulate a selectin-mediated function. Such a compound
or composition can be used, for example, to inhibit or enhance a
selectin-mediated function, such as selectin-mediated intercellular
interactions. A compound or composition can be used in a method to
contact a cell expressing a selectin in an amount effective to
modulate the selectin's function. A compound or composition can be
used in a method to administer to a patient, who is in need of
having inhibited the development of a condition associated with an
excessive selectin-mediated function (such as an excessive
selectin-mediated intercellular adhesion), in an amount effective
to inhibit the development of such a condition. Examples of such
conditions include inflammatory diseases, autoimmune diseases,
infection, cancer, shock, thrombosis, wounds, burns, reperfusion
injury, platelet-mediated diseases, leukocyte-mediated lung injury,
spinal cord damage, digestive tract mucous membrane disorders,
osteoporosis, arthritis, asthma and allergic reactions. A compound
or composition can be used in a method to administer to a patient
who is the recipient of a transplanted tissue in an amount
effective to inhibit rejection of the transplanted tissue. A
compound or composition can be used in a method in an amount
effective to target an agent (e.g., a diagnostic or therapeutic
agent) to a selectin-expressing cell by contacting such a cell with
the agent linked to the compound or composition. A compound or
composition can be used in the manufacture of a medicament, for
example for any of the uses recited above.
[0036] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings. All references disclosed herein are hereby
incorporated by reference in their entirety as if each was
incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIGS. 1A and 1B are diagrams illustrating the syntheses of
BASAs.
[0038] FIG. 2 is a diagram illustrating the synthesis of a
BACA.
[0039] FIG. 3 is a diagram illustrating the synthesis of a
glycomimetic (XIX).
[0040] FIG. 4 is a diagram illustrating the synthesis of a
glycomimetic (XXVIII).
[0041] FIG. 5 is a diagram illustrating the synthesis of a
PEGylated glycomimetic (XXX).
[0042] FIG. 6 is a diagram illustrating the synthesis of a
PEGylated glycomimetic (XXXI).
[0043] FIGS. 7A, 7B, and 7C are diagrams illustrating the syntheses
of PEGylated BASAs (XXXII and XXXIII) and PEGylated BACAs (XXXVI
and XXXVIa).
[0044] FIGS. 8A, 8B and 8C are diagrams illustrating the syntheses
of Glycomimetic-BASA (FIGS. 8A and 8C) and Glycomimetic-BACA (FIG.
8B).
[0045] FIGS. 9A, 9B and 9C are diagrams illustrating the syntheses
of Glycomimetic-BASA (FIGS. 9A and 9C) and Glycomimetic-BACA (FIG.
9B).
[0046] FIG. 10 is a diagram illustrating the synthesis of a
glycomimetic.
[0047] FIG. 11 is a diagram illustrating the synthesis of a
glycomimetic.
[0048] FIG. 12 is a diagram illustrating the synthesis of a
glycomimetic.
[0049] FIG. 13 is a diagram illustrating the synthesis of a
glycomimetic-BASA.
[0050] FIG. 14 is a diagram illustrating the synthesis of a
glycomimetic-BASA.
[0051] FIG. 15 is a diagram illustrating the synthesis of a
glycomimetic-BASA.
[0052] FIG. 16 is a diagram illustrating the synthesis of
glycomimetics.
[0053] FIGS. 17A and 17B show a comparison of the activity of
glycomimetic inhibitors in binding assays for E-selectins (FIG.
17A) and P-selectins (FIG. 17B) in vitro. A and B are
glycomimetic-BASAs other than the present invention. C is the
glycomimetic-BASA of FIG. 8C.
[0054] FIG. 18 shows the effect of glycomimetic-BASA of FIG. 8C on
neutrophil migration. A is a vehicle only and E is a positive
control (mixed antibodies). B, C and D are the glycomimetic-BASA of
FIG. 8C at a dose of 5 mg/kg, 10 mg/kg, and 20 mg/kg,
respectively.
[0055] FIG. 19 shows a comparison of the effects of glycomimetic
inhibitors on neutrophil migration in murine air pouch model. A is
IL-1.beta.+vehicle. B is IL-1.beta.+the glycomimetic-BASA of FIG.
13. C is IL-1.beta.+the glycomimetic-BASA of FIG. 8C. D is
IL-1.beta.+mixed antibodies (positive control). *P<0.05 vs. A
(vehicle group).
DETAILED DESCRIPTION OF THE INVENTION
[0056] As noted above, the present invention provides selectin
modulators, compositions thereof and methods for modulating
selectin-mediated functions. Such modulators may be used in vitro
or in vivo, to modulate (e.g., inhibit or enhance)
selectin-mediated functions in a variety of contexts, discussed in
further detail below. Examples of selectin-mediated functions
include intercellular adhesion and the formation of new capillaries
during angiogenesis.
Selectin Modulators
[0057] The term "selectin modulator," as used herein, refers to a
molecule(s) that modulates (e.g., inhibits or enhances) a
selectin-mediated function, such as selectin-mediated intercellular
interactions. A selectin modulator may consist entirely of a
glycomimetic compound of the present invention, or may consist of
such a glycomimetic linked to a BASA (Benzyl Amino Sulfonic Acid)
or a BACA (Benzyl Amino Carboxylic Acid), or may comprise one or
more additional molecular components to any of the above.
[0058] A selectin modulator of the present invention which does not
possess a BASA or a BACA is preferably used to inhibit an
E-selectin-mediated function. With the addition of a BASA or BACA
to a glycomimetic of the present invention, the selectin modulator
has increased ability to modulate P- and L-selectin-mediated
functions as well.
[0059] A selectin modulator of the present invention is a compound
or physiologically acceptable salt thereof, having the formula:
##STR00016##
wherein:
##STR00017## where n=0-2, and R.sup.8 are independently selected
where n=2; [0060] R.sup.2=H, --C(.dbd.O)OX where X is
C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8
alkynyl or C.sub.1-C.sub.14 aryl,
--C(.dbd.O)NH(CH.sub.2).sub.nNH.sub.2, [0061]
[C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)].sub.m(L).sub.mZ, where
n=0-30, m=0-1, L is a linker, and Z is a benzyl amino sulfonic
acid, a benzyl amino carboxylic acid, a polyethylene glycol, or a
second compound or salt thereof having the above formula to form a
dimer where R.sup.2 of the second compound or salt thereof has m=0,
no Z, and is the point of attachment;
[0061] ##STR00018## [0062] --O--C(.dbd.O)--X, --NH.sub.2,
--NH--C(.dbd.O)--NHX, or --NH--C(.dbd.O)--X where n=0-2 and X is
independently selected from C.sub.1-C.sub.8 alkanyl,
C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl,
##STR00019##
[0062] where n=0-10, and any of the above ring compounds may be
substituted with one to three independently selected of Cl, F,
C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8
alkynyl, C.sub.1-C.sub.14 aryl, or OY where Y is H, C.sub.1-C.sub.8
alkanyl, C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl, or
C.sub.1-C.sub.14 aryl;
##STR00020## 6'sulfated GlcNAc, 6'carboxylated GlcNAc, 6'sulfated
GalNAc, 6'sulfated galactose, 6'carboxylated galactose or
##STR00021##
where R.sup.9 is aryl, heteroaryl, cyclohexane, t-butane,
adamantane, or triazole, and any of R.sup.9 may be substituted with
one to three independently selected of Cl, F, C.sub.1-C.sub.8
alkanyl, C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl or OY
where Y is H, C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8 alkenyl,
C.sub.1-C.sub.8 alkynyl or C.sub.1-C.sub.14 aryl; [0063] R.sup.5=H,
or R.sup.4 and R.sup.5 are taken together to form
[0063] ##STR00022## where R.sup.10 is aryl, heteroaryl,
##STR00023## [0064] where n=0-10, and any one of the above ring
compounds may be substituted with one to three independently
selected of Cl, F, C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8
alkenyl, C.sub.1-C.sub.8 alkynyl or OY where Y is H,
C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8 alkenyl or C.sub.1-C.sub.8
alkynyl; [0065] R.sup.6=H, fucose, mannose, arabinose, galactose or
polyols; [0066] R.sup.7=H, C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8
alkenyl, C.sub.1-C.sub.8 alkynyl or
##STR00024##
[0066] and [0067] R.sup.8=H, C.sub.1-C.sub.8 alkanyl,
C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl,
[0067] ##STR00025## [0068] where n=0-3 and X is independently
selected from H, OH, Cl, F, N.sub.3, NH.sub.2, C.sub.1-C.sub.8
alkanyl, C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl,
C.sub.1-C.sub.14 aryl, OC.sub.1-C.sub.8 alkanyl, OC.sub.1-C.sub.8
alkenyl, OC.sub.1-C.sub.8 alkynyl, and OC.sub.1-C.sub.14 aryl, and
any of the above ring compounds may be substituted with one to
three independently selected of Cl, F, C.sub.1-C.sub.8 alkanyl,
C.sub.1-C.sub.8 alkenyl, C.sub.1-C.sub.8 alkynyl, C.sub.1-C.sub.14
aryl or OY where Y is H, C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8
alkenyl, C.sub.1-C.sub.8 alkynyl, or C.sub.1-C.sub.14 aryl.
[0069] As used herein, a "C.sub.1-C.sub.8 alkanyl" refers to an
alkane substituent with one to eight carbon atoms and may be
straight chain or branched. Examples are methyl, ethyl, propyl,
isopropyl, butyl and t-butyl. A "C.sub.1-C.sub.8 alkenyl" refers to
an alkene substituent with one to eight carbon atoms, at least one
carbon-carbon double bond, and may be straight chain or branched.
Examples are similar to "C.sub.1-C.sub.8 alkanyl" examples except
possessing at least one carbon-carbon double bond. A
"C.sub.1-C.sub.8 alkynyl" refers to an alkyne substituent with one
to eight carbon atoms, at least one carbon-carbon triple bond, and
may be straight chain or branched. Examples are similar to
"C.sub.1-C.sub.8 alkanyl" examples except possessing at least one
carbon-carbon triple bond. An "aryl" refers to an aromatic
substituent with one to fourteen carbon atoms in one or multiple
rings which may be separated by a bond or fused. A "heteroaryl" is
similar to an "aryl" except the aromatic substituent possesses at
least one heteroatom (such as N, O or S) in place of a ring carbon.
Examples of aryls and heteroaryls include phenyl, naphthyl,
pyridinyl, pyrimidinyl, triazolo, furanyl, oxazolyl, thiophenyl,
quinolinyl and diphenyl. As used herein, the term "independently
selected" refers to the selection of identical or different
substituents.
[0070] As used herein, polyethylene glycol ("PEG") refers to
multiple units of ethylene glycol, as well as those with one or
more substituents (e.g., dicarboxylated PEG). PEGs with and without
substituents are well known to those in the art. Within the present
invention, PEG can serve as a substituent on a selectin modulator,
or as a linker to attach other groups or compounds to a selectin
modulator, or a selectin modulator may possess more than one
PEG.
[0071] Where a second selectin modulator is linked to a first
selectin modulator, a dimer of selectin modulators (i.e., a
divalent molecule) is formed. A variety of linkers may be used to
join the two selectin modulators. For example, PEG may be used as
the linker to prepare a dimer. As used herein, a "dimer" can be a
homodimer or a heterodimer. A homodimer refers to a dimer where the
two selectin modulators joined together are identical (independent
of the substituents for the linking to one another). A heterodimer
refers to a dimer where the two selectin modulators (independent of
the linkage substituents) are not identical.
[0072] A selectin modulator of the present invention may possess,
at R.sup.4 of the above formula, sialic acid or a sialic acid mimic
as set forth above. For example, the hexose ring of sialic acid may
be replaced with cyclohexane. The presence of sialic acid in the
selectin modulator enhanced P-selectin binding. Where only
E-selectin binding (and not both E- and P-selecting binding) is
desired, a sialic acid mimic replaces sialic acid in the selectin
modulator.
[0073] Alternative to (or in combination with) the replacement of a
sialic acid mimic with sialic acid, P-selectin binding may be
enhanced by the addition of a BASA or a BACA. As disclosed above,
the selectin modulator compounds of the present invention may
possess a "Z" at R.sup.2, and Z may be a BASA or a BACA. The
addition of a BASA or BACA to a selectin modulator compound of the
present invention that lacks sialic acid, can convert the selectin
modulator from a compound that is selective for binding to
E-selectin to one that binds both E- and P-selectin. BASA or BACA
includes a portion or an analogue of a BASA or BACA or portion of
either, provided that the compound retains the ability to modulate
a selectin-mediated function. PEG may be added to a selectin
modulator with or without a BASA (or BACA). PEG may also be used to
link a BASA or BACA to a selectin modulator.
[0074] Within the present invention, BASAs are low molecular weight
sulfated compounds which have the ability to interact with a
selectin. The interaction modulates or assists in the modulation
(e.g., inhibition or enhancement) of a selectin-mediated function
(e.g., an intercellular interaction). They exist as either their
protonated acid form, or as a sodium salt, although sodium may be
replaced with potassium or any other pharmaceutically acceptable
counterion. A representative BASA has the following structure:
##STR00026##
[0075] Portions of BASA that retain the ability to interact with a
selectin (which interaction modulates or assists in the modulation
of a selectin-mediated function as described herein) are also a
BASA component of the selectin modulators of the present invention.
Such portions generally comprise at least one aromatic ring present
within the BASA structure. Within certain embodiments, a portion
may comprise a single aromatic ring, multiple such rings or half of
a symmetrical BASA molecule.
[0076] As noted above, analogues of BASA and portions thereof (both
of which possess the biological characteristic set forth above) are
also encompassed, e.g., by the BASA component of the selectin
modulators, within the present invention. As used herein, an
"analogue" is a compound that differs from BASA or a portion
thereof because of one or more additions, deletions and/or
substitutions of chemical moieties, such that the ability of the
analogue to inhibit a selectin-mediated interaction is not
diminished. For example, an analogue may contain S to P
substitutions (e.g., a sulfate group replaced with a phosphate
group). Other possible modifications include: (a) modifications to
ring size (e.g., any ring may contain between 4 and 7 carbon
atoms); (b) variations in the number of fused rings (e.g., a single
ring may be replaced with a polycyclic moiety containing up to
three fused rings, a polycyclic moiety may be replaced with a
single unfused ring or the number of fused rings within a
polycyclic moiety may be altered); (c) ring substitutions in which
hydrogen atoms or other moieties covalently bonded to a carbon atom
within an aromatic ring may be replaced with any of a variety of
moieties, such as F, Cl, Br, I, OH, O-alkyl (C1-8), SH, NO.sub.2,
CN, NH.sub.2, NH-alkyl (C1-8), N-(alkyl).sub.2, SO.sub.3M (where
M=H.sup.+, Na.sup.+, K.sup.+ or other pharmaceutically acceptable
counterion), CO.sub.2M, PO.sub.4M.sub.2, SO.sub.2NH.sub.2, alkyl
(C1-8), aryl (C6-10), CO.sub.2-alkyl (C1-8), --CF.sub.2X (where X
can be H, F, alkyl, aryl or acyl groups) and carbohydrates; and (d)
modifications to linking moieties (i.e., moieties located between
rings in the BASA molecule) in which groups such as alkyl, ester,
amide, anhydride and carbamate groups may be substituted for one
another.
[0077] Certain BASA portions and analogues contain one of the
following generic structures:
##STR00027##
Within this structure, n may be 0 or 1, X.sup.1 may be --PO.sub.2M,
--SO.sub.2M or --CF.sub.2-- (where M is a pharmaceutically
acceptable counterion such as hydrogen, sodium or potassium),
R.sup.1 may be --OH, --F or --CO.sub.2R.sup.4 (where R.sup.4 may be
--H or --(CH.sub.2).sub.m--CH.sub.3 and m is a number ranging from
0 to 3, R.sup.2 may be --H, --PO.sub.3M.sub.2, --SO.sub.3M.sub.2,
--CH.sub.2--PO.sub.3M.sub.2, --CH.sub.2--SO.sub.3M.sub.2,
--CF.sub.3 or --(CH.sub.2).sub.m--C(R.sup.6)H--R.sup.5 or
R.sup.9--N(R.sup.10)--, R.sup.3 may be --H,
--(CH.sub.2).sub.m--C(R.sup.6)H--R.sup.5 or R.sup.9--N(R.sup.10)--
(where R.sup.5 and R.sup.6 may be independently selected from --H,
--CO.sub.2--R.sup.7 and --NH--R.sup.8, R.sup.7 and R.sup.8 may be
independently selected from hydrogen and moieties comprising one or
more of an alkyl group, an aromatic moiety, an amino group or a
carboxy group, and R.sup.9 and R.sup.10 may be independently
selected from --H, --(CH.sub.2).sub.m--CH.sub.3; --CH.sub.2-Ar,
--CO-Ar, where m is a number ranging from 0 to 3 and Ar is an
aromatic moiety (i.e., any moiety that comprises at least one
substituted or unsubstituted aromatic ring, wherein the ring is
directly bonded to the --CH.sub.2-- or --CO-- group indicated
above)).
[0078] Other portions and analogues of BASA comprise the generic
structure:
##STR00028##
Within this structure, R.sub.1 and R.sub.2 may be independently
selected from (i) hydrogen, (ii) moieties comprising one or more of
an alkyl group, an aromatic moiety, an amino group or a carboxy
group, and (iii) --CO--R.sub.3 (where R.sub.3 comprises an alkyl or
aromatic moiety as described above) and M is a pharmaceutically
acceptable counterion.
[0079] The individual compounds, or groups of compounds, derived
from the various combinations of the structures and substituents
described herein, are disclosed by the present application to the
same extent as if each compound or group of compounds was set forth
individually. Thus, selection of particular structures and/or
particular substituents is within the scope of the present
invention.
[0080] Representative BASA portions and analogues are included in
the compounds shown in FIGS. 1A-1B. It will be apparent to those of
ordinary skill in the art that modifications may be made to the
compounds shown within these figures, without adversely affecting
the ability to function as selectin modulators. Such modifications
include deletions, additions and substitutions as described
above.
[0081] A BACA is similar to a BASA, except instead of sulfonic acid
groups, the compound possesses carboxylic acid groups. For example,
the sulfonic acid groups of the above BASA compounds may be
replaced with carboxylic acid groups. Thus, the above disclosure to
BASAs is incorporated by reference into this description of
BACAs.
[0082] Examples of BACAs include:
##STR00029##
where X is F or Cl; Y is H, --C(.dbd.O)(O--CH.sub.2CH.sub.2).sub.n
or --C(.dbd.O)(CH.sub.2).sub.n wherein n=0-8; and Z is H,
C.sub.1-C.sub.8 alkanyl, C.sub.1-C.sub.8 alkenyl or C.sub.1-C.sub.8
alkynyl.
[0083] As described above, a BASA or BACA may be joined to a
compound of the present invention at R.sup.2 via a linker ("L").
Typically a linker is first attached to one of a glycomimetic or a
BASA/BACA, which is then reacted with the other. The attachment of
a BASA or BACA to a particular glycomimetic can be accomplished in
a variety of ways to form a selectin modulator. A linker possessed
by (or added to) a BASA or BACA or a glycomimetic may include a
spacer group, such as --(CH.sub.2).sub.n-- or --O(CH.sub.2).sub.n--
where n is generally about 1-20 (including any whole integer range
therein). An example of a linker is --NH.sub.2 on a glycomimetic,
e.g., --CH.sub.2--NH.sub.2 when it includes a short spacer group.
In an embodiment, --CH.sub.2--NH.sub.2 is attached to a
glycomimetic at R' which may then be used to attach a BASA or BACA.
The simplest attachment method is reductive amination of the BASA
or BACA to a glycomimetic containing a reducing end (an anomeric
hydroxyl/aldehyde). This is accomplished by simple reaction of the
BASA or BACA to the reducing end and subsequent reduction (e.g.,
with NaCNBH.sub.3 at pH 4.0) of the imine formed. The most general
approach entails the simple attachment of an activated linker to
the glycomimetic via an O, S or N heteroatom (or C atom) at the
anomeric position. The methodology of such attachments has been
extensively researched for carbohydrates and anomeric selectivity
is easily accomplished by proper selection of methodology and/or
protecting groups. Examples of potential glycosidic synthetic
methods include Lewis acid catalyzed bond formation with halogen or
peracetylated sugars (Koenigs Knorr), trichloroacetamidate bond
formation, thioglycoside activation and coupling, glucal activation
and coupling, n-pentenyl coupling, phosphonate ester homologation
(Horner-Wadsworth-Emmons reaction), and many others. Alternatively,
linkers could be attached to positions on the moieties other than
the anomeric. The most accessible site for attachment is at a six
hydroxyl (6-OH) position of a glycomimetic (a primary alcohol). The
attachment of a linker at the 6-OH can be easily achieved by a
variety of means. Examples include reaction of the oxy-anion
(alcohol anion formed by deprotonation with base) with an
appropriate electrophile such as an alkyl/acyl bromide, chloride or
sulfonate ester, activation of the alcohol via reaction with a
sulfonate ester chloride or POCl.sub.3 and displacement with a
subsequent nucleophile, oxidation of the alcohol to the aldehyde or
carboxylic acid for coupling, or even use of the Mitsunobu reaction
to introduce differing functionalities. Once attached the linker is
then functionalized for reaction with a suitable nucleophile on the
BASA or BACA (or vice versa). This is often accomplished by use of
thiophosgene and amines to make thiourea-linked heterobifunctional
ligands, diethyl squarate attachment (again with amines) and/or
simple alkyl/acylation reactions. Additional methods that could be
utilized include FMOC solid or solution phase synthetic techniques
traditionally used for carbohydrate and peptide coupling and
chemo-enzymatic synthesis techniques possibly utilizing
glycosyl/fucosyl transferases and/or oligosaccharyltransferase
(OST).
[0084] Embodiments of linkers include the following:
##STR00030##
Other linkers (e.g., PEG) will be familiar to those in the art or
in possession of the present disclosure.
[0085] A compound, or physiologically acceptable salt thereof, of
the present invention has the formula:
##STR00031##
wherein R.sup.1-R.sup.9 are defined as set forth above.
[0086] In a preferred embodiment, R.sup.6 is fucose:
##STR00032##
In a preferred embodiment, R.sup.7 is H. In a preferred embodiment,
R.sup.4 is
##STR00033##
In a preferred embodiment, R.sup.4 is
##STR00034##
where R.sup.9 is defined as above. In a preferred embodiment,
R.sup.9 is cyclohexane. In a preferred embodiment. R.sup.6 is
galactose. In a preferred embodiment, R.sup.8 is
##STR00035##
In a preferred embodiment, R.sup.2 is
--[C(.dbd.O)NH(CH.sub.2)--NHC(.dbd.O)].sub.m(L).sub.mZ, where n, m,
L and Z are defined as above. In a preferred embodiment, Z is a
benzyl amino sulfonic acid, a benzyl amino carboxylic acid or a
polyethylene glycol. In a preferred embodiment, R.sup.3 is
--O--C(.dbd.O)--X, where X is defined as above. In a preferred
embodiment, X is
##STR00036##
In a preferred embodiment, R.sup.5 is H. In a preferred embodiment,
L is a polyethylene glycol or a thiadiazole.
[0087] Although selectin modulators as described herein may
sufficiently target a desired site in vivo, it may be beneficial
for certain applications to include an additional targeting moiety
to facilitate targeting to one or more specific tissues. As used
herein, a "targeting moiety," may be any substance (such as a
compound or cell) that, when linked to a modulating agent enhances
the transport of the modulator to a target tissue, thereby
increasing the local concentration of the modulator. Targeting
moieties include antibodies or fragments thereof, receptors,
ligands and other molecules that bind to cells of, or in the
vicinity of, the target tissue. Linkage is generally covalent and
may be achieved by, for example, direct condensation or other
reactions, or by way of bi- or multi-functional linkers.
[0088] For certain embodiments, it may be beneficial to also, or
alternatively, link a drug to a selectin modulator. As used herein,
the term "drug" refers to any bioactive agent intended for
administration to a mammal to prevent or treat a disease or other
undesirable condition. Drugs include hormones, growth factors,
proteins, peptides and other compounds. Examples of potential drugs
include antineoplastic agents (such as 5-fluorouracil and
distamycin), integrin agonist/antagonists (such as cyclic-RGD
peptide), cytokine agonist/antagonists, histamine
agonist/antagonists (such as diphenhydramine and chlorpheniramine),
antibiotics (such as aminoglycosides and cephalosporins) and redox
active biological agents (such as glutathione and thioredoxin). In
other embodiments, diagnostic or therapeutic radionuclides may be
linked to a selectin modulator. In many embodiments, the agent may
be linked directly or indirectly to a selectin modulator.
[0089] Modulators as described herein may be present within a
pharmaceutical composition. A pharmaceutical composition comprises
one or more modulators in combination with one or more
pharmaceutically or physiologically acceptable carriers, diluents
or excipients. Such compositions may comprise buffers (e.g.,
neutral buffered saline or phosphate buffered saline),
carbohydrates (e.g., glucose, mannose, sucrose or dextrans),
mannitol, proteins, polypeptides or amino acids such as glycine,
antioxidants, chelating agents such as EDTA or glutathione,
adjuvants (e.g., aluminum hydroxide) and/or preservatives. Within
yet other embodiments, compositions of the present invention may be
formulated as a lyophilizate. Compositions of the present invention
may be formulated for any appropriate manner of administration,
including for example, topical, oral, nasal, intravenous,
intracranial, intraperitoneal, subcutaneous, or intramuscular
administration.
[0090] A pharmaceutical composition may also, or alternatively,
contain one or more active agents, such as drugs (e.g., those set
forth above), which may be linked to a modulator or may be free
within the composition.
[0091] The compositions described herein may be administered as
part of a sustained release formulation (i.e., a formulation such
as a capsule or sponge that effects a slow release of modulating
agent following administration). Such formulations may generally be
prepared using well known technology and administered by, for
example, oral, rectal or subcutaneous implantation, or by
implantation at the desired target site. Carriers for use within
such formulations are biocompatible, and may also be biodegradable;
preferably the formulation provides a relatively constant level of
modulating agent release. The amount of modulating agent contained
within a sustained release formulation depends upon the site of
implantation, the rate and expected duration of release and the
nature of the condition to be treated or prevented.
[0092] Selectin modulators are generally present within a
pharmaceutical composition in a therapeutically effective amount. A
therapeutically effective amount is an amount that results in a
discernible patient benefit, such as increased healing of a
condition associated with excess selectin-mediated function (e.g.,
intercellular adhesion), as described below.
[0093] In general, the modulating agents and compositions described
herein may be used for enhancing or inhibiting a selectin-mediated
function. Such enhancement or inhibition may be achieved in vitro
and/or in vivo in a warm-blooded animal, preferably in a mammal
such as a human, provided that a selectin-expressing cell is
ultimately contacted with a modulator, in an amount and for a time
sufficient to enhance or inhibit selectin-mediated function.
[0094] Within certain aspects, the present invention provides
methods for inhibiting the development of a condition associated
with a selectin-mediated function, such as intercellular adhesion.
In general, such methods may be used to prevent, delay or treat
such a condition. In other words, therapeutic methods provided
herein may be used to treat a disease, or may be used to prevent or
delay the onset of such a disease in a patient who is free of
disease or who is afflicted with a disease that is not associated
with a selectin-mediated function. For example, the therapeutic
methods have uses that may include the arrest of cell growth, the
killing of cells, the prevention of cells or cell growth, the delay
of the onset of cells or cell growth, or the prolongation of
survival of an organism.
[0095] A variety of conditions are associated with a
selectin-mediated function. Such conditions include, for example,
tissue transplant rejection, platelet-mediated diseases (e.g.,
atherosclerosis and clotting), hyperactive coronary circulation,
acute leukocyte-mediated lung injury (e.g., adult respiratory
distress syndrome (ARDS)), Crohn's disease, inflammatory diseases
(e.g., inflammatory bowel disease), autoimmune diseases (MS,
myasthenia gravis), infection, cancer (and metastasis), thrombosis,
wounds (and wound-associated sepsis), burns, spinal cord damage,
digestive tract mucous membrane disorders (gastritis, ulcers),
osteoporosis, rheumatoid arthritis, osteoarthritis, asthma,
allergy, psoriasis, septic shock, traumatic shock, stroke,
nephritis, atopic dermatitis, frostbite injury, adult dyspnoea
syndrome, ulcerative colitis, systemic lupus erythematosus,
diabetes and reperfusion injury following ischaemic episodes.
Selectin modulators may also be administered to a patient prior to
heart surgery to enhance recovery. Other uses include pain
management, prevention of restinosis associated with vascular
stenting, and for undesirable angiogenesis, e.g., associated with
cancer.
[0096] Selectin modulators of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). Appropriate dosages and a suitable duration and
frequency of administration may be determined by such factors as
the condition of the patient, the type and severity of the
patient's disease and the method of administration. In general, an
appropriate dosage and treatment regimen provides the modulating
agent(s) in an amount sufficient to provide therapeutic and/or
prophylactic benefit. Within particularly preferred embodiments of
the invention, a selectin modulator may be administered at a dosage
ranging from 0.001 to 1000 mg/kg body weight (more typically 0.01
to 1000 mg/kg), on a regimen of single or multiple daily doses.
Appropriate dosages may generally be determined using experimental
models and/or clinical trials. In general, the use of the minimum
dosage that is sufficient to provide effective therapy is
preferred. Patients may generally be monitored for therapeutic
effectiveness using assays suitable for the condition being treated
or prevented, which will be familiar to those of ordinary skill in
the art.
[0097] Selectin modulators may also be used to target substances to
cells that express a selectin. Such substances include therapeutic
agents and diagnostic agents. Therapeutic agents may be a molecule,
virus, viral component, cell, cell component or any other substance
that can be demonstrated to modify the properties of a target cell
so as to provide a benefit for treating or preventing a disorder or
regulating the physiology of a patient. A therapeutic agent may
also be a prodrug that generates an agent having a biological
activity in vivo. Molecules that may be therapeutic agents may be,
for example, polypeptides, amino acids, nucleic acids,
polynucleotides, steroids, polysaccharides or inorganic compounds.
Such molecules may function in any of a variety of ways, including
as enzymes, enzyme inhibitors, hormones, receptors, antisense
oligonucleotides, catalytic polynucleotides, anti-viral agents,
anti-tumor agents, anti-bacterial agents, immunomodulating agents
and cytotoxic agents (e.g., radionuclides such as iodine, bromine,
lead, palladium or copper). Diagnostic agents include imaging
agents such as metals and radioactive agents (e.g., gallium,
technetium, indium, strontium, iodine, barium, bromine and
phosphorus-containing compounds), contrast agents, dyes (e.g.,
fluorescent dyes and chromophores) and enzymes that catalyze a
colorimetric or fluorometric reaction. In general, therapeutic and
diagnostic agents may be attached to a selectin modulator using a
variety of techniques such as those described above. For targeting
purposes, a selectin modulator may be administered to a patient as
described herein. Since selectins are expressed on endothelial
cells involved in the formation of new capillaries during
angiogenesis, a selectin modulator may be used to target a
therapeutic agent for killing a tumor's vasculature. A selectin
modulator may also be used for gene targeting.
[0098] Selectin modulators may also be used in vitro, e.g., within
a variety of well known cell culture and cell separation methods.
For example, modulators may be linked to the interior surface of a
tissue culture plate or other cell culture support, for use in
immobilizing selectin-expressing cells for screens, assays and
growth in culture. Such linkage may be performed by any suitable
technique, such as the methods described above, as well as other
standard techniques. Modulators may also be used, for example, to
facilitate cell identification and sorting in vitro, permitting the
selection of cells expressing a selectin (or different selectin
levels). Preferably, the modulator(s) for use in such methods are
linked to a detectable marker. Suitable markers are well known in
the art and include radionuclides, luminescent groups, fluorescent
groups, enzymes, dyes, constant immunoglobulin domains and biotin.
Within one preferred embodiment, a modulator linked to a
fluorescent marker, such as fluorescein, is contacted with the
cells, which are then analyzed by fluorescence activated cell
sorting (FACS).
[0099] Modulating agents as described above are capable, for
example, of inhibiting selectin-mediated cell adhesion. This
ability may generally be evaluated using any of a variety of in
vitro assays designed to measure the effect on adhesion between
selectin-expressing cells (e.g., adhesion between leukocytes or
tumor cells and platelets or endothelial cells). For example, such
cells may be plated under standard conditions that, in the absence
of modulator, permit cell adhesion. In general, a modulator is an
inhibitor of selectin-mediated cell adhesion if contact of the test
cells with the modulator results in a discernible inhibition of
cell adhesion. For example, in the presence of modulators (e.g.,
micromolar levels), disruption of adhesion between leukocytes or
tumor cells and platelets or endothelial cells may be determined
visually within approximately several minutes, by observing the
reduction of cells interacting with one another.
[0100] All compounds of the present invention or useful thereto,
include physiologically acceptable salts thereof.
[0101] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Synthesis of BASA (FIG. 1A)
[0102] Synthesis of Compound 4:
[0103] Nitration of commercially available 2 (1 g) is according to
the procedure described (for literature conditions see U.S. Pat.
No. 4,534,905; Allison, F. et al. Helv. Chim. Acta 4:2139
(1952)).
[0104] The crude product 3 is dissolved in water (40 mL) and 10%
Pd/C (0.3 g) added. The mixture is hydrogenated (.about.45 psi) at
room temperature for 48 h. The catalyst is filtered through Celite
and the filter bed is washed with water. The filtrate is
concentrated under vacuum to afford a pink solid. After removal of
the catalyst, the filtrate is concentrated to 15 mL and an equal
volume of ethanol is added. The precipitate is collected by
filtration to give compound 4 with very little impurity.
[0105] Synthesis of Compound 7a:
[0106] A solution of 5 (5 g) and 8 (4.45 g, 24.7 mmol), and
K.sub.2CO.sub.3 (2 M in H.sub.2O, 24.7 mL, 49.4 mmol) in 10:1
toluene/ethanol (70 mL) is treated with Pd(PPh.sub.3).sub.4 (1.43
g, 1.24 mmol) and the mixture is refluxed for 20 h. After work up,
recrystallization of the crude product in EtOH and chromatographic
purification of the recrystallization filtrate affords compound 9
(2.9 g, 46%, >90% HPLC) and 2.2 g of recovered 5. The product is
characterized by .sup.1H NMR.
[0107] A mixture of 9 (2.9 g, 11.3 mmol) and LiOH--H.sub.2O (1.43
g, 34.1 mmol) in 1:1 THF/H.sub.2O (250 mL) is stirred at RT for 21
h. The reaction affords 7 (2.58 g, 94%, >90% HPLC) after work
up. The product is characterized by .sup.1H NMR.
[0108] DMF (20 .mu.l) is added to a suspension of 7 (500 mg, 1.94
mmol), SOCl.sub.2 (0.23 mL, 3.10 mmol) and toluene (3 mL) and then
heated to 80.degree. C. After 20 h the reaction is worked up to
afford the acid chloride (640 mg). The product 7a is characterized
by IR and .sup.1H NMR.
[0109] Synthesis of Compound 10:
[0110] To a solution of amine 4 (268 mg, 0.641 mmol) in H.sub.2O (2
mL) and dioxane (18 mL) is added a solution of 7a (273 mg, 0.99
mmol) in dioxane (16 mL) dropwise over 30 min. The pH of the
reaction mixture is adjusted to 8.5 with 0.25 M NaOH as the
addition progresses. The reaction is stirred at room temperature
for 2.5 h after the addition. Purification by column chromatography
(methanol/toluene 1:1) followed by prep. TLC (methanol/toluene 1:1)
affords 50 mg of compound 10, which is characterized by .sup.1H NMR
and MS.
[0111] Hydrogenation of Compound 10:
[0112] A suspension of 10 (30 mg, 0.049 mmol) and 10% Pd on carbon
(50 mg) in H.sub.2O (20 mL) is hydrogenated (55 psi) at room
temperature for 4 h to yield the BASA of FIG. 1A.
Example 2
Synthesis of BASA (FIG. 1B)
[0113] Synthesis of Compound 4:
[0114] 3-nitro-benzyl iodide (1) (48.3 g) is added to an aqueous
solution (pH 11) of commercially available,
8-aminonaphthalene-1,3,5-trisulfonic acid (2) (29.5 g) with
stirring at room temperature. pH of the solution is adjusted to 1
and after evaporation of the solvent, the product 3 (6.4 g) is
precipitated out from ethanol.
[0115] Platinum catalyzed hydrogenation of compound 3 affords
compound 4 (the BASA of FIG. 1B) in 96% yield.
Example 3
Synthesis of BACA (FIG. 2)
[0116] A suspension of 1 (8.9 g), paraformaldehyde (8.9 g), and
H.sub.2SO.sub.4 (125 mL) is heated to 90.degree. C. for 14 h and
affords crude 2 (7.8 g) after work up. The crude product is 77%
pure by HPLC and characterized by .sup.1H NMR.
[0117] To a solution of 2 (1.0 g) in acetone (30 mL) is added
K.sub.2CO.sub.3 (3.1 g) and dimethylsulfate (1.4 mL) and the
reaction is heated to reflux for 24 h. The reaction is combined
with the next batch for work up and purification.
[0118] To a solution of 2 (7.5 g) in acetone (225 mL) is added
K.sub.2CO.sub.3 (23.2 g) and dimethylsulfate (10.8 mL) and the
reaction is heated to reflux for 16 h. The reaction, combined with
the previous batch, affords 3 (7.3 g, 74%) after work up and column
chromatographic purification (ethyl acetate/heptane 1:9). The
product is 80% pure by HPLC and characterized by .sup.1H NMR.
[0119] Chromic anhydride (6.94 g) is added to a suspension of 3
(7.16 g) in acetic anhydride (175 mL) at 3.degree. C. and then
stirred at room temperature for 15 h. The reaction affords 4 (5.89
g) after work up and column purification (100% dichloromethane).
The product is 90% pure by HPLC and characterized by .sup.1H
NMR.
[0120] To a suspension of 4 (5.89 g) in THF/H.sub.2O (300 mL, 1:1)
is added LiOH H.sub.2O (1.74 g) at room temperature and the
resulting mixture is stirred for 14 h. After an acid/base work up,
the product is obtained as a white solid. The product is dried
under high vacuum and characterized by nmr and mass
spectroscopy.
Example 4
Synthesis of Glycomimetic (FIG. 3)
[0121] Synthesis of Intermediate II:
[0122] (-)-Shikimic acid (20 g) in MeOH (200 ml) and sulfuric acid
(2 ml, 98%) are stirred at rt for 50 h. The reaction mixture is
neutralized with 2N aqueous NaOH in the cold. After evaporation to
dryness, the residue is purified by silica gel chromatography to
afford II (19.2 g).
[0123] Synthesis of Intermediate (III):
[0124] Methyl shikimate (II, 10 g), 2,2 dimethoxypropane (10 ml)
and p-TsOH (0.8 g) are dissolved in acetonitrile (125 ml) and
stirred at rt for 1 h. The reaction mixture is then neutralized
with triethylamine (2 ml) and evaporated to dryness. The residue is
chromatographed on silica gel to yield III (11 g).
[0125] Synthesis of Intermediate IV:
[0126] The shikimic acid derivative III (10 g) and PtO.sub.2/C
(10%, 250 mg) in MeOH (40 ml) are hydrogenated at rt under vigorous
stirring. After 16 h the reaction mixture is filtered over celite
and evaporated to dryness. The residue is chromatographed on silica
gel to yield IV.
[0127] Synthesis of Intermediate V:
[0128] To a solution of IV (8 g) in DCM (100 ml) at 0.degree. C.
are added pyridine (12 ml), acetic anhydride (7 ml) and a DMAP (25
mg). The reaction mixture is stirred at rt for 1 h, and diluted
with EtOAc (250 ml). After washing with 0.5 M aqueous HCl
(3.times.50 ml), saturated solution of KHCO.sub.3 (3.times.50 ml)
and brine (3.times.50 ml), the combined organic layers are dried
(Na.sub.2SO.sub.4) and evaporated to dryness. The residue is
purified by chromatography on silica gel to yield V (6.8 g).
[0129] Synthesis of Intermediate VI:
[0130] A solution of V (6.0 g) in acetic acid (30 ml, 80%) is
stirred at 80.degree. C. for 1 h. Solvent is evaporated off and the
residue is purified by chromatography on silica gel (DCM/MeOH 14:1)
to yield VI (3.6 g).
[0131] Synthesis of Intermediate (VII):
[0132] A solution of VI (3 g) and p-TsCl (3.5 g) in pyridine (30
ml) is stirred at rt for 6 h. MeOH (5 ml) is added and the solvent
is evaporated at reduced pressure, the residue dissolved in EtOAc
(3.times.150 ml) and the organic layers are washed with 0.5 M
aqueous HCl (0.degree. C.), water (cold) and brine (cold). The
combined organic layers are dried (Na.sub.2SO.sub.4), filtered on
Celite and evaporated to dryness. The residue is purified by
chromatography on silica gel (toluene/EtOAc 4:1) to yield VII (3.7
g).
[0133] Synthesis of Compound VIII:
[0134] A solution of VII (3 g) and NaN.sub.3 (2.5 g) in DMF (20 ml)
is stirred at 80.degree. C. The reaction mixture is cooled to rt
and diluted with EtOAc (200 ml) and water (50 ml). The organic
layer is additionally washed twice with water (2.times.50 ml) and
once with brine (50 ml). All aqueous layers are extracted twice
with EtOAc (2.times.50 ml). The combined organic layers are dried
with Na.sub.2SO.sub.4, filtered and the solvent is evaporated off.
The residue is purified by chromatography on silica gel (petroleum
ether/EtOAc 5:2) to give VIII (2.2 g).
[0135] Synthesis of Compound X:
[0136] To a solution of ethyl
2,3,4-tri-O-benzyl-.alpha.-L-fucothiopyanoside IX (1.5 g) in DCM (3
ml), bromine (150 .mu.l) is added at 0.degree. C. under argon.
After 5 min the cooling bath is removed and the reaction mixture is
stirred for additional 25 min at rt. Cyclohexene (200 .mu.l) is
added and the reaction mixture is added to a solution of VIII (400
mg), (Et).sub.4NBr (750 mg) and powdered 4 .ANG. molecular sieves
in DCM (10 ml) and DMF (5 ml). After 16 h, triethylamine (1.5 ml)
is added and stirred for an additional for 10 min, diluted with
EtOAc (50 ml) and washed with sat. aqueous NaHCO.sub.3, water and
brine. The aqueous layers are extracted twice with EtOAc
(2.times.50 ml). The combined organic layers are dried
(Na.sub.2SO.sub.4), filtered and evaporated to dryness. The residue
is purified by chromatography on silica gel (toluene/EtOAc 9:1) to
yield X (700 mg).
[0137] Synthesis of Compound XI:
[0138] To a solution of X (1.5 g) in MeOH (20 ml) is added freshly
prepared NaOMe (80 mg) and the reaction mixture is stirred in a
pressure tube at 80.degree. C. for 20 h. The reaction mixture is
cooled to rt and neutralized with acetic acid. Solvent is
evaporated to dryness and the residue is dissolved in ether.
Freshly prepared diazomethane is added and the excess diazomethane
is neutralized with acetic acid. Solvent is evaporated off to give
XI (1.25 g).
[0139] Synthesis of Building Block XV:
[0140] This synthesis is done exactly in same way as described
previously (Helvetica Chemica Acta 83:2893-2907 (2000)).
[0141] Synthesis of Compound XVI:
[0142] A mixture of XI (1.6 g), XV (3 g) and activated powdered
molecular sieves 4 .ANG. (1 g) in DCM (17 ml) is stirred at rt
under argon for 2 h. Then DMTST (2 g) is added in 4 equal portions
over a period of 1.5 h. After 24 h the reaction mixture is filtered
over Celite and the filtrate is diluted with DCM (100 ml). The
organic layer is washed with sat. aqueous NaHCO.sub.3 and brine and
the aqueous layers are extracted twice with DCM. The combined
organic layers are dried (Na.sub.2SO.sub.4), filtered and
evaporated to dryness. The residue is purified by chromatography on
silica gel (toluene/EtOAc 8:1) to yield XVI (1.5 g).
[0143] Synthesis of Compound XVII:
[0144] To a solution of XVI (500 mg) and orotic acid chloride (500
mg) in dichloromethane (10 ml) is added a solution of
triphenylphosphine (500 mg in 5 ml dichloromethane) dropwise during
10 min. The reaction mixture is stirred at rt for 25 h and the
solvent is evaporated off. The residue is purified (chromatography
on silica gel DCM/MeOH 19:1) to give XVII (250 mg).
[0145] Synthesis of Compound XVIII:
[0146] To a solution of XVII (200 mg) in dioxane-water (5:1, 12 ml)
is added 10% Pd--C (100 mg) and the reaction mixture is stirred
vigorously under hydrogen (55 psi) for 24 h. Catalyst is filtered
through a bed of celite and the solvent is evaporated off. Residue
is purified by silica gel chromatography to give compound XVIII
(150 mg).
[0147] Synthesis of XIX:
[0148] To a solution of compound XVIII (145 mg) in MeOH (5 ml) is
added a solution of NaOMe in MeOH (25%, 0.025 ml) and the reaction
mixture is stirred at rt for 4 h, neutralized with acetic acid and
the solvent is evaporated off. Residue is dissolved in water and
passed through a bed of Dowex 50wX-8 (Na-form) resin. Water wash is
evaporated off to afford compound XIX (100 mg).
[0149] Synthesis of EDA-XIX:
[0150] XIX (80 mg) is heated at 70.degree. C. with ethylenediamine
(EDA) (1 ml) with stirring for 5 h. Solvent is evaporated off and
the purified by sephadex G-25 column to give EDA-XIX (82 mg).
Example 5
Synthesis of Glycomimetic (FIG. 4)
[0151] Synthesis of Compound XXI:
[0152] To a solution of compound XX (1.5 g, synthesized according
to previously published procedure Carbohydrate Chemistry and
Biochemistry, 2000, vol. 1, page 345-365) in pyridine (60 ml) is
added benzoic anhydride (0.73 g) and dimethyl amino pyridine (0.02
g). The reaction mixture is stirred at room temperature for 20 h.
Solvent is evaporated off and the residue is dissolved in
dichloromethane. The solution is washed successively with cold 1N
HCl and water. The solution is dried (sodium sulfate) and
concentrated to dryness. Residue is purified by column
chromatography (silica gel) to give compound XXI (1 g).
[0153] Synthesis of Compound XXII:
[0154] To a solution of compound XXI in dichloromethane (20 ml) is
added trifluoroacetic acid (20 ml) at 0.degree. C. and the reaction
mixture is stirred at the same temperature for 1 h. Solvent is
evaporated off and the residue is purified by column chromatography
(silica gel) to give compound XXII (0.6 g).
[0155] Synthesis of Compound XXIII:
[0156] Toa solution of compound XXII (1 g) in dichloromethane (40
ml) is added DBU (0.05 ml) and tricholroacetonitrile (0.4 g) at
0.degree. C. The solution is stirred at the same temperature for
1.5 h. Solvent is evaporated off and purified by column
chromatography (silica gel) to give compound XXIII (0.6 g).
[0157] Synthesis of Compound XXIV:
[0158] To a mixture of compound XI (0.7 g), compound XXIII (0.5 g)
in dichloromethane (40 ml) and molecular sieves (4 .ANG., 5 g) is
added a solution of TMSOTf (0.15 ml in dichloromethane (5 ml))
dropwise at 0.degree. C. with stirring. Stirring is continued at
the same temperature for 2 h. Triethylamine (0.2 ml) is added and
the reaction mixture is filtered through a bed of celite. Reaction
mixture is washed with cold saturated solution of sodium
bicarbonate and water. Dried (sodium sulfate) and concentrated to
dryness. The residue is purified by column chromatography (silica
gel) to give compound XXIV (0.7 g).
[0159] Synthesis of Compound XXV:
[0160] To a solution of compound XXIV (0.6 g) in DMF (30 ml) is
added DBU (30 drops) and dl-dithio-threitol (DTT, 0.28 g). The
reaction mixture is stirred at room temperature for 1 h. Solvent is
evaporated off, residue is dissolved in dichloromethane and washed
with water. Organic layer is dried (sodium sulfate) and
concentrated to dryness. The residue is purified by column
chromatography (silica gel) to give compound XXV (0.45 g).
[0161] Synthesis of Compound XXVI:
[0162] To a solution of compound XXV (0.4 g) in DMF (10 ml) is
added orotic acid (0.14 g), EEDQ (0.19 g), 4-methyl-morpholine
(0.09 g) and the reaction mixture is stirred at 70.degree. C. for
20 h. Solvent is evaporated off and residue is dissolved in
dichloromethane. The solution is washed with cold saturated sodium
bicarbonate solution and water. Organic layer is dried (sodium
sulfate) and concentrated to dryness. The residue is purified by
column chromatography (silica gel) to give compound XXVI (0.2
g).
[0163] Synthesis of Compound XXVII:
[0164] Compound XXVII (0.2 g) is hydrogenated exactly in the same
condition as described and the intermediate is partially
debenzoylated using NaOMe in MeOH as described to give compound
XXVII (0.050 g) after chromatographic purification.
[0165] Synthesis of Compound XXVIII:
[0166] Compound XXVII is treated with ethylenediamine as described
and purified by column chromatography (silica gel and gel
filtration sephadex G-25) to give compound XXVIII (25 mg).
Example 6
Synthesis of Pegylated BASA (FIG. 7B)
[0167] To a solution of 3,6-dioxaoctanedioic acid (PEG, 200 mg,
available commercially) in DMF (1 ml) is added Hunig base (0.4 ml),
and then HATU (0.35 g) is added after 5 min. The solution is
stirred at RT for 10 min. and then a solution of the BASA of
Example 2 (50 mg) in DMF (0.1 ml) is added. The reaction mixture is
stirred for 4 h at rt and the solvent is evaporated off. The
residue is purified by hplc (reverse-phase C18 column) to give
XXXIII (40 mg).
Example 7
Synthesis of Pegylated BASA (FIG. 7A)
[0168] This synthesis is performed in the same way as described in
Example 6, except using the BASA of Example 1 to give XXXII (50
mg).
Example 8
Synthesis of Pegylated BACA (FIG. 7C)
[0169] Synthesis of Intermediate XXXIV (Method 1):
[0170] The BACA of Example 3 (0.5 g) is suspended in methanol-water
(1 ml, 9:1) and the pH is adjusted to 8.2 by the addition of an
aqueous solution of Cs.sub.2CO.sub.3. The solvent is removed and
then coevaporated with toluene. The residue is dissolved in DMF (1
ml). Benzylbromide (0.5 ml) is added and stirred for 20 h at room
temperature. Dichloromethane (15 ml) is added washed with cold
water. Organic layer is dried (anhydrous sodium sulfate) and
solvent is evaporated off. The residue is purified by column
chromatography (silica) to give XXXIV (0.48 g).
[0171] Synthesis of Intermediate XXXIV (Method 2):
[0172] To a solution of the BACA of Example 3 (1 g) in DMF is added
N,N-diisopropyl ethylamine (1.5 g) and Benzylbromide (1.5 g). The
reaction mixture is stirred at 50.degree. C. for 20 h. Solvent is
evaporated off and the residue is purified by column chromatography
(silica) to give XXXIV.
[0173] Synthesis of Intermediate of XXXV:
[0174] To a solution of XXXIV (0.2 g) in MeOH (10 ml) is added
sodiumborohydride (0.070 g) at 0.degree. C. and the reaction
mixture is stirred at 0.degree. C. for 1 h. The reaction is
quenched by addition of acetic acid and concentrated to dryness.
Residue is purified column chromatography (silica) to afford XXXV
(0.16 g).
[0175] Synthesis of Intermediate of XXXVb:
[0176] To a solution of XXXV (0.36 g) in dichloromethane is added
triethylamine (0.6 ml) and MeSO.sub.2Cl (0.29 ml). The reaction
mixture is stirred at RT for 21 h. Reaction mixture is diluted with
dichloromethane, washed with water, 1 M HCl, and brine. Organic
layer is dried (Na.sub.2SO.sub.4) and concentrated to dryness to
give crude XXXVa. To a solution of crude XXXVa in DMF (5 ml) is
added NaN.sub.3 (0.18 g). The reaction mixture is stirred at
100.degree. C. for 4 h and the solvent is evaporated off. The
residue is dissolved in CH.sub.2Cl.sub.2 and washed with cold
brine, cold 1 M HCl, cold NaHCO.sub.3 solution, and cold water.
Organic layer is dried (Na.sub.2SO.sub.4) and concentrated to
dryness. The residue is purified by column chromatography (silica)
to give XXXVb (0.14 g).
[0177] Synthesis of Intermediate XXXVc:
[0178] To a solution of XXXVc (0.08 g) in DMF (3 ml) is added DTT
(0.04 g) and DBU (0.02 ml) and stirred at RT for 1 h. Solvent is
evaporated off and the residue is dissolved in EtOAc, washed with
H.sub.2O and the organic layer is concentrated to dryness. The
residue is purified by column chromatography (silica) to afford
intermediate XXXVc (0.061 g).
[0179] Synthesis of Intermediate XXXVd:
[0180] To a solution of monoprotected PEG-dicarboxylic acid (0.6 g)
in DMF (3 ml) is added diisopropylethylamine (0.24 ml) and HATU
(0.513 g) with stirring. A solution of intermediate XXXVc (0.185 g)
in DMF (3 ml) is added into the above solution. The reaction
mixture is stirred at RT for 1 h. Solvent is evaporated off and the
residue is dissolved in EtOAc. EtOAc layer is washed with water and
purified by column chromatography to give intermediate XXXVd.
[0181] Synthesis of XXXVI:
[0182] To a solution of XXXVd (0.185 g) in glacial AcOH (3 ml) is
added Zn dust (0.1 g) and the reaction is stirred at 40.degree. C.
for 30 min. Reaction mixture is filtered through a celite bed and
Zn cake is washed with MeOH. The filtrate is concentrated to
dryness and purified by sep-pak C18 column to afford XXXVI (0.050
g).
[0183] Synthesis of Intermediate XXXIVa:
[0184] A suspension of TiCl.sub.4-Tetrahydrofuran complex (0.705 g)
and Zn-dust (0.28 g) in THF (25 ml) is refluxed for 2 h at
75.degree. C. for 2 h with stirring under inert atmosphere. To this
mixture is added a solution of the BACA of Example 3 (0.3 g) and
N-fluorenylmethoxycarbonyl-3-aminopropanol (0.312 g, prepared as
described in the literature Casimiro-Garcia et al, Bioorg. Med.
Chem., 1979 (2001) 2827). The reaction mixture is stirred at
75.degree. C. for 2.5 h (McMurray coupling) under inert atmosphere.
The reaction is cooled down to RT and H.sub.2O (30 ml) is added,
filtered through a celite bed and the filtrate is washed three
times with EtOAc (30 ml each). Organic layer is collected together
and dried (Na.sub.2SO.sub.4), filtered and concentrated to dryness.
Residue is purified by column chromatography to yield XXXIVa.
[0185] Synthesis of Intermediate XXXIVb:
[0186] To a solution of XXXIVa (0.444 g) in anhydrous THF (21 ml)
is added piperidine (6.25 ml) and the reaction mixture is stirred
at RT for 3 h. The solvent is evaporated off and the residue is
purified by column chromatography (silica) to give intermediate
XXXIVb (0.26 g).
[0187] Solid Phase Synthesis of Intermediate XXXIVc:
[0188] A mixture of PS-Carbodiimide resin (0.200 g), HOBt (0.030
g), and mono protected PEG-COOH (0.080 g) in CH.sub.2Cl.sub.2 (3
ml) is stirred for 5 min at RT in a syringe reactor. To the above
mixture is added a solution of intermediate XXXIVb (0.080 g) in
CH.sub.2Cl.sub.2 (3 ml) and the reaction mixture is stirred at RT
for 3 h. MP-carbonate resin (0.216 g) is added and stirred for 2 h
at RT. Resin is filtered off and then the resin is washed 5 times
with CH.sub.2Cl.sub.2. Filtrate is combined and concentrated to
dryness to afford intermediate XXXIVc.
[0189] Synthesis of Intermediate XXXVIa:
[0190] XXXIVc (0.12 g) is treated with Zn/AcOH exactly in the same
as described for the synthesis of intermediate XXXVI to yield
intermediate XXXVIa (0.104 g).
Example 9
Synthesis of Pegylated BACA (FIG. 7C)
[0191] Synthesis of Intermediate XXXIVa:
[0192] A suspension of TiCl.sub.4-Tetrahydrofuran complex (0.705 g)
and Zn-dust (0.28 g) in THF (25 ml) is refluxed for 2 h at
75.degree. C. for 2 h with stirring under inert atmosphere. To this
mixture is added a solution of the BACA of Example 3 (0.3 g) and
N-fluorenylmethoxycarbonyl-3-aminopropanol (0.312 g, prepared as
described in the literature Casimiro-Garcia et al, Bioorg. Med.
Chem., 1979 (2001) 2827). The reaction mixture is stirred at
75.degree. C. for 2.5 h (McMurray coupling) under inert atmosphere.
The reaction is cooled down to RT and H.sub.2O (30 ml) is added,
filtered through a celite bed and the filtrate is washed three
times with EtOAc (30 ml each). Organic layer is collected together
and dried (Na.sub.2SO.sub.4), filtered and concentrated to dryness.
Residue is purified by column chromatography to yield XXXIVa.
[0193] Synthesis of Intermediate XXXIVb:
[0194] To a solution of XXXIVa (0.444 g) in anhydrous THF (21 ml)
is added piperidine (6.25 ml) and the reaction mixture is stirred
at RT for 3 h. The solvent is evaporated off and the residue is
purified by column chromatography (silica) to give intermediate
XXXIVb (0.26 g).
[0195] Solid Phase Synthesis of Intermediate XXXIVc:
[0196] A mixture of PS-Carbodiimide resin (0.200 g), HOBt (0.030
g), and mono protected PEG-COOH (0.080 g) in CH.sub.2Cl.sub.2 (3
ml) is stirred for 5 min at RT in a syringe reactor. To the above
mixture is added a solution of intermediate XXXIVb (0.080 g) in
CH.sub.2Cl.sub.2 (3 ml) and the reaction mixture is stirred at RT
for 3 h. MP-carbonate resin (0.216 g) is added and stirred for 2 h
at RT. Resin is filtered off and then the resin is washed 5 times
with CH.sub.2Cl.sub.2. Filtrate is combined and concentrated to
dryness to afford intermediate XXXIVc.
[0197] Synthesis of XXXVIa:
[0198] XXXIVc (0.12 g) is treated with Zn/AcOH exactly in the same
as described for the synthesis of intermediate XXXVI to yield
intermediate XXXVIa (0.104 g).
Example 10
Synthesis of Glycomimetic-BASA (FIG. 8A)
[0199] To a solution of XXXII from Example 7 (0.015 g) in DMF (0.1
ml) is added Hunig base (0.015 ml) and then HATU (0.007 g). The
reaction mixture is stirred for 10 min at RT. A solution of EDA-XIX
from Example 4 (0.010 g in DMF ml) is added and the reaction
mixture is stirred at RT for 8 h. Solvent is evaporated off and the
residue is purified by sephadex G-25 chromatography to give
Glycomimetic-BASA of FIG. 8A (0.008 g).
Example 11
Synthesis of Glycomimetic-BACA (FIG. 8B)
[0200] Coupling Between EDA-XIX and XXXVI:
[0201] To a solution of XXXVI from Example 8 in DMF is added
diisopropylethylamine and then HATU. The solution is stirred for 3
min at RT. The above solution is then added to EDA-XIX with
stirring in a conical vial. Reaction mixture is stirred for 2 h at
RT. Solvent is evaporated off to give crude intermediate XXXVIb and
is used for the next step without further purification.
[0202] The crude XXXVIb is treated with NaOMe-MeOH--H.sub.2O for 2
h and then purified by gel filtration to give a
Glycomimetic-BACA.
[0203] Coupling Between EDA-XIX and XXXVIa from Example 9:
[0204] This coupling reaction is performed exactly in the same way
as described for the synthesis of XXXVIb to give crude product
XXXVIc.
[0205] XXXVIc is treated with NaOMe-MeOH--H.sub.2O exactly in the
same way as described to afford a Glycomimetic-BACA.
Example 12
Synthesis of Glycomimetic-BASA (FIG. 8C)
[0206] This synthesis is performed in the same way as described in
Example 10 using XXXIII from Example 6 and EDA-XIX from Example 4
to give Glycomimetic-BASA of FIG. 8C. Alternatively, XXXIII can be
replaced with XLV from Example 18 for reaction with EDA-XIX.
Example 13
Synthesis of Glycomimetic-BASA (FIG. 9A)
[0207] This synthesis is performed in the same way as described in
Example 10 using XXXII from Example 7 and EDA-XXVIII from Example 5
to give Glycomimetic-BASA of FIG. 9A.
Example 14
Synthesis of Glycomimetic-BACA (FIG. 9B)
[0208] Coupling Between EDA-XXVIII and XXXVI:
[0209] This coupling reaction is performed exactly in the same way
as described for the synthesis of XXXVIb to give crude product
XXXVId.
[0210] XXXVId is treated with NaOMe-MeOH--H.sub.2O exactly in the
same way as described to afford a Glycomimetic-BACA.
[0211] Coupling Between EDA-XXVIII and XXXVIa:
[0212] This coupling reaction is performed exactly in the same way
as described for the synthesis of XXXVIb to give crude product
XXXVIe.
[0213] XXXVIe is treated with NaOMe-MeOH--H.sub.2O exactly in the
same way as described to afford a Glycomimetic-BACA.
Example 15
Synthesis of Glycomimetic-BASA (FIG. 9C)
[0214] This synthesis is performed in the same way as described in
Example 10 using XXXIII from Example 6 and EDA-XXVIII from Example
5 to give Glycomimetic-BASA of FIG. 9C. Alternatively, XXXIII can
be replaced with XLV from Example 18 for reaction with
EDA-XXVIII.
Example 16
Synthesis of Glycomimetic (FIG. 12)
[0215] Synthesis of Intermediate XXXVII:
[0216] A solution of intermediate XXIV (0.1 g) from Example 5 in
pyridine is treated with nicotinyl chloride (0.08 ml) in pyridine
and dimethylaminopyridine (0.04 g) in the same way as described in
Example 5 for the synthesis of intermediate XXV to give
intermediate XXXVII (0.075 g).
[0217] Synthesis of Intermediate XXXVIII:
[0218] Intermediate XXXVII (0.07 g) is treated with
triphenylphosphine and orotic acid chloride in the same way as
described in Example 4 for the synthesis of compound XVII to give
XXXVIII (0.048 g).
[0219] Synthesis of Intermediate XXXIX:
[0220] Hydrogenation of intermediate XXXVIII (0.04 g) with Pd/C as
described gives compound XXXIX (0.02 g).
[0221] Synthesis of Compound XL:
[0222] Intermediate XXXIX (0.015 g) is treated with 0.5N NaOH for
10 min at 60.degree. C. to afford compound XL (0.010 g) after
purification by sephadex G-25 gel filtration.
Example 17
Synthesis of Glycomimetic (FIG. 13)
[0223] Synthesis of XLI:
[0224] To a suspension of compound XVI (0.1 g) in t-BuOH-water (4
ml, 1:1) is added 1-ethynyl-3-fluorobenzene (0.9 g), 1% CuSO4 (0.1
ml), and Na--I-ascorbate (4 mg). The mixture is heated (70.degree.
C.) with stirring for 20 h. Solvent is evaporated off and the
residue is dissolved in dichloromethane. Organic layer is washed
with water dried (anhydrous sodium sulfate) and concentrated to
dryness. The residue is purified by column chromatography (silica
gel) to give compound XLI (0.08 g).
[0225] Synthesis of XLII:
[0226] Compound XLI (0.25 g) is dissolved in dioxane-water (4:1,
7.5 ml). 10% Pd--C (0.25 g) is added, followed by AcOH (7 drops).
The mixture is hydrogenated for 15 h at 40 psi. The reaction
mixture is filtered through a celite bed and concentrated to
dryness to give compound XLII (0.2 g).
[0227] Synthesis of XLIII:
[0228] To a solution of compound XLII (0.2 g) in MeOH (5 ml) is
added a solution of NaOMe in MeOH (0.05 ml) and the reaction
mixture is stirred at room temperature for 2 h. The reaction
mixture is neutralized with few drops of acetic acid and
concentrated to dryness. Residue is purified by column
chromatography (silica gel) to give compound XLIII (0.15 g).
[0229] Synthesis of XLIV:
[0230] Compound XLIII (0.15 g) is dissolved in ethylenediamine (7
ml) and the reaction mixture is stirred at 70.degree. C. for 9 h.
Solvent is evaporated off and the residue is first purified by
column chromatography (silica gel) and then by reverse phase C18 to
give compound XLIV (0.11 g).
[0231] Synthesis of Glycomimetic-BASA (Compound XLV):
[0232] This synthesis is performed in the same way as described in
example 10 using XXXIII from example 6 and XLIV to give compound
XLV.
Example 18
Synthesis of Glycomimetic-BASA (FIG. 14)
[0233] Synthesis of Compound XLV:
[0234] To a solution of 3,6-dioxaoctanedioic acid (PEG, 200 mg,
available commercially) in DMF (1 ml) is added Hunig base (0.4 ml)
and then HATU (0.35 g) is added after 5 min. The solution is
stirred at RT for 10 min and then solution of
8-aminonaphthalene-1,3,6-trisulfonic acid (50 mg, available
commercially) in DMF is added. The reaction mixture is stirred for
4 h at RT and the solvent is evaporated off. The residue is
purified by hplc (reverse-phase C18 column) to give XLV (25
mg).
[0235] Synthesis of Compound XLVI:
[0236] This synthesis is performed in the same way as described in
example 10 using XLV and EDA-XIX from example 4 to give compound
XLVI (4 mg).
Example 19
Synthesis of Glycomimetic-BASA (FIG. 15)
[0237] Synthesis of XLVII:
[0238] Starting with compound XXIV, this synthesis is performed in
the same way as described for the synthesis of XLI to give compound
XLVI.
[0239] Synthesis of XLVIII:
[0240] Starting with compound XLVII, this synthesis is performed in
the same way as described for compound XLIII (from XLI) to give
compound XLVIII.
[0241] Synthesis of XLIX:
[0242] Starting with compound XLVIII, this synthesis is performed
in the same way as described for the synthesis of XLIV to give
compound XLIX.
[0243] Synthesis of Glycomimetic-BASA (Compound L):
[0244] This synthesis is performed in the same way as described in
example 10 using XXXIII from example 6 and XLIX to give compound L.
Alternatively, XXXIII can be replaced with XLV from Example 18 for
reaction with XLIX.
Example 20
Synthesis of Glycomimetics (FIG. 16)
[0245] Synthesis of Compound I:
[0246] as described in the literature [J. Org. Chem. 54, 3738-3740
(1989); Liebigs Annalen der Chemie 575, 1 (1952)]
[0247] Synthesis of Intermediate II:
[0248] I (2.8 g, 15.04 mmol), pyridine (4.8 ml, 60.15 mmol),
benzoylchloride (3.5 ml, 30.07 mmol) and a catalytical amount of
dimethyl aminopyridine are stirred in dichloromethane (6 ml) at
room temperature ("r.t."). After 2 h, TLC control shows completion
of the reaction. The reaction mixture then is diluted with ethyl
acetate (200 ml) and washed with water, 1N aqueous HCl
(ice-cooled), saturated aqueous NaHCO.sub.3 and brine (each 50 ml).
The aqueous layers are washed twice with ethyl acetate (2.times.150
ml), combined and dried with Na.sub.2SO.sub.4. After filtration and
evaporation of the solvent the residue is purified by
chromatography on silica gel (PE/EtOAc 4:1) to yield compound II
(3.78 g, 86%).
[0249] Synthesis of Intermediate III:
[0250] 11 (3.78 g, 13.02 mmol) and NaBH.sub.4 are stirred in
methanol (35 ml) at 0.degree. C. After 30 min. the reaction mixture
is quenched with water (15 ml) and neutralized with 1N aqueous
AcOH. Again water (10 ml) is added and the mixture is extracted 3
times with dichloromethane (3.times.150 ml). The combined organic
layers are dried with Na.sub.2SO.sub.4, filtered and evaporated.
Chromatography of the residue on silica gel (PE/EtOAc 3.2) gives
compound III (3.7 g, 97%). [.alpha.].sub.D+79.78.degree. (c=0.940,
CH.sub.2Cl.sub.2);
[0251] Synthesis of Intermediate IV:
[0252] To a solution of III (3.4 mg, 11.64 mmol) in CHCl.sub.3 (50
ml, filtered on basic Alox) at r.t. under argon is added
1-chloro-N,N,2-trimethylpropenylamine (4.94 ml, 34.93 mmol) by a
syringe. The reaction mixture is stirred at reflux until TLC
control indicated completion of the reaction (30 min.) After
cooling to r.t. the reaction mixture is quenched with triethylamine
(6 ml) and evaporated (bath temperature 30.degree. C.) to dryness.
The residue is purified by chromatography on silica gel (PE/EtOAc
9:1) to yield compound IV (3.3 g, 92%).
[0253] Synthesis of Intermediate V:
[0254] To a solution of IV (3.25 g, 10.47 mmol) in dry toluene (40
ml) under argon are added freshly distilled Bu.sub.3SnH (30.58 ml,
115.14 mmol) and AIBN (1.7 g, 10.47 mmol). The reaction is
refluxed. After 75 min., when TLC shows completion of the reaction,
the reaction mixture is cooled to r.t. and then diluted with
acetonitrile (50 ml). The solution is washed with hexane (50 ml)
and the hexane layer is extracted again with acetonitrile (50 ml).
The combined acetonitrile layers are evaporated. Chromatography of
the residue on silica gel (toluene/EtOAc 14:1) yields compound V
(2.66 g, 92%). [.alpha.].sub.D-117.79.degree. (c=1.810,
CH.sub.2Cl.sub.2).
[0255] Synthesis of Intermediate VI:
[0256] A solution of V (2.63 g, 9.54 mmol) in aqueous AcOH 80% is
stirred at 80.degree. C. When TLC control indicates completion of
the reaction (30 min.), reaction mixture is cooled to r.t. After
neutralizing with aqueous NaOH, the mixture is extracted 3 times
with dichloromethane (3.times.200 ml). The combined organic layers
are dried with Na.sub.2SO.sub.4, filtered and evaporated to
dryness. The residue is purified by chromatography on silica gel
(PE/EtOAc 3:2) to yield compound VI (2.01 g, 89%).
[.alpha.].sub.D-54.86.degree. (c=1.420, CH.sub.2Cl.sub.2).
[0257] Synthesis of Intermediate VII:
[0258] A solution of VI (1.98 g, 8.39 mmol), freshly recrystallized
toluene-4-sulfonylchloride (1.9 g, 10.07 mmol), Bu.sub.2SnO (2.09
g, 8.39 mmol) and triethylamine (1.8 ml, 16.78 mmol) in dry
dichloromethane (40 ml) is stirred at r.t. under argon. After 20 h,
TLC control indicated completion of the reaction. The reaction
mixture is then quenched with methanol (15 ml) and then evaporated
to dryness. Chromatography of the residue on silica gel
(toluene/EtOAc 8:1) gives compound VII (2.65 g, 85%).
[.alpha.].sub.D-68.08.degree. (c=0.448, CH.sub.2Cl.sub.2).
[0259] Synthesis of Intermediate VIII:
[0260] A mixture of VII (640 mg, 1.71 mmol) and NaN.sub.3 (555 mg,
8.55 mmol) in DMF (30 ml) is stirred under argon at 80.degree. C.
When TLC control shows completion of the reaction (after 1 h), the
reaction mixture is cooled to r.t., diluted with dichloromethane
(50 ml) and washed with water (50 ml). The aqueous layer is then
extracted twice with dichloromethane (2.times.50 ml) and the
combined organic layers are dried with Na.sub.2SO.sub.4, filtered
and evaporated. The residue is purified by chromatography on silica
gel (toluene/EtOAc 6:1) to yield compound VIII (391 mg, 87%).
[.alpha.].sub.D-69.39.degree. (c=2.330, CH.sub.2Cl.sub.2).
[0261] Synthesis of Intermediate IX:
[0262] To a stirred solution of ethyl
2,3,4-tri-O-benzyl-.alpha.-L-fucothiopyranoside (223 mg, 0.47 mmol)
in dry dichloromethane (1 ml) at 0.degree. C. under Ar atmosphere,
bromine (48.mu., 0.54 mmol) is added. After 5 min. the cooling bath
is removed and the reaction mixture is stirred for additional 40
min. at r.t. To remove the excess of bromine, cyclohexene (50
.mu.l) is added, leading to a decolorization of the reaction
mixture. Then the reaction mixture is added to a pre-stirred
solution (1 h, r.t.) of VIII (61 mg, 0.23 mmol), (Et).sub.4NBr (98
mg, 0.47 mmol) and powdered 4 .ANG. molecular sieves (100 mg) in
dichloromethane (1.6 ml) and DMF (1 ml). After 18 h, the reaction
is quenched with pyridine (2 ml) and stirred for additional for 15
min., before it is diluted with EtOAc (50 ml) and washed with sat.
aqueous KHCO.sub.3, water and brine (each 50 ml). The aqueous
layers are extracted twice with EtOAc (2.times.50 ml). The combined
organic layers are dried with Na.sub.2SO.sub.4, filtered and
evaporated to dryness. The residue is purified by chromatography on
silica gel (toluene/EtOAc 9:1) to yield IX (116 mg, 73%).
[.alpha.].sub.D-95.96.degree. (c=1.040, CH.sub.2Cl.sub.2).
[0263] Synthesis of Intermediate X:
[0264] IX (530 mg, 0.78 mmol) in MeOH (10 ml) and a catalytical
amount of freshly prepared NaOMe are stirred at r.t. under Ar for
48 h. The reaction mixture is neutralized with powdered
Amberlyst-15 and filtered over Celite. The filtrate is evaporated
to dryness and purified by chromatography on silica gel
(toluene/EtOAc 9:1) to give compound X (380 mg, 85%).
[.alpha.].sub.C-76.425.degree. (c=4.00, CH.sub.2Cl.sub.2).
[0265] Synthesis of Intermediate XI:
[0266] A mixture of X (184 mg, 0.32 mmol), the galactose building
block [375 mg, 0.4811 mmol, synthesis as described previously,
Helv. Chim. Acta 83: 2893-2907 (2000)] and activated powdered
molecular sieves 4 .ANG. (200 mg) in dichloromethane (3 ml) is
stirred at r.t. under argon for 4 h. Then DMTST [B] (165 mg, 0.64
mmol) is added in 4 equal portions over a period of 1.5 h to a
pre-stirred mixture (4 h, r.t.) of activated powered molecular
sieves 4 .ANG. (100 mg) in dichloromethane (3 ml). After 92 h, when
TLC control shows completion of the reaction, the reaction mixture
is filtered over Celite and the filtrate is diluted with
dichloromethane (50 ml). The organic layer is washed with sat.
aqueous NaHCO.sub.3 and brine (each 20 ml) and the aqueous layers
are extracted twice with dichloromethane (2.times.50 ml). The
combined organic layers are dried with Na.sub.2SO.sub.4, filtered
and evaporated to dryness. The residue is purified by
chromatography on silica gel (toluene/EtOAc 9:1) to yield compound
XI (310 mg, 75%). [.alpha.].sub.C-36.98.degree. (c=2.32,
CH.sub.2Cl.sub.2).
General Procedure A
[0267] Synthesis of Intermediate XII:
[0268] To a stirred solution of XI (100 mg, 0.077 mol) in dry
dichloromethane (3 ml), acetic acid chloride (27 .mu.l, 0.387 mmol)
is added. After 5 min. PPh.sub.3 (101 mg, 0.387 mmol) is added and
the solution is stirred at r.t. When TLC after 22 h shows
completion of the reaction, the solvent is removed and the residue
chromatographed on silica gel (toluene/EtOAc 2:1) yielding compound
XII (43 mg, 43%). [.alpha.].sub.D-55.33.degree. (c=2.185,
CH.sub.2Cl.sub.2).
[0269] Synthesis of Intermediate XIII:
[0270] According to procedure A: XI (80 mg, 0.062 mol) with orotic
acid chloride (59 mg, 0.310 mmol) and PPh.sub.3 (81 mg, 0.310
mmol), 4 h, (dichloromethane/MeOH 25:1) gives compound XIII (35 mg,
40%). [.alpha.].sub.D-26.08.degree. (c=1.48, CH.sub.2Cl.sub.2).
[0271] Synthesis of Intermediate XIV:
[0272] According to procedure A: XI (70 mg, 0.054 mmol) with
biphenyl-4-carboxylic acid chloride (58 mg, 0.271 mmol) and
PPh.sub.3 (71 mg, 0.271 mmol), 4 h, (toluene/EtOAc 6:1) gives XIV
(22 mg, 28%). [.alpha.].sub.D-32.11.degree. (c=1.065,
CH.sub.2Cl.sub.2).
[0273] Synthesis of Intermediate XV:
[0274] According to procedure A: XI (45 mg, 0.035 mmol) with
biphenyl-2-carboxylic acid chloride (125 mg, 0.577 mmol) and
PPh.sub.3 (151 mg, 0.577 mmol), 5 h, (toluene/EtOAc 6:1), gives
compound XV (22 mg, 44%). [.alpha.].sub.D-19.54.degree. (c=1.10,
CH.sub.2Cl.sub.2).
General Procedure B
[0275] Synthesis of Intermediate XVI:
[0276] A mixture of XI (120 mg, 0.093 mmol) and PPh.sub.3 (30 mg,
0.116 mmol) in CH.sub.2Cl.sub.2 (2 ml) and water (100 .mu.l) is
stirred for 44 h at r.t. Then the solvents are removed and the
residue dissolved in CH.sub.2Cl.sub.2 (2 ml) and DIC (11.8 mg,
0.094 mmol) and the vanillic acid (24 mg, 0.139 mmol) are added.
After the mixture is stirred at r.t. for additional 62 h, the
solvents are removed and the crude product is purified by
chromatography on silica gel (toluene/EtOAc 2.5:1) to give compound
XVI (62 mg, 47%). [.alpha.].sub.D-29.58.degree. (c=2.86,
CH.sub.2Cl.sub.2).
General Procedure C
[0277] Synthesis of Intermediate XVII:
[0278] A mixture of XII (40 mg, 0.0306 mmol), Pd(OH).sub.2 (30 mg),
dioxane (2 ml) and water (0.4 ml) is hydrogenated in a Parr-shaker
under 5 bar at r.t. After 20 h TLC control indicated completion of
the reaction. The reaction mixture is filtered over Celite and
evaporated to dryness. Purification of the crude product by
chromatography (CH.sub.2Cl.sub.2/MeOH 9:1) yielded XVII (20 mg,
69%). [.alpha.].sub.D-43.50.degree. (c=1.00, MeOH).
[0279] Synthesis of Intermediate XVIII:
[0280] According to procedure C: XIV (26 mg, 0.018 mmol),
Pd(OH).sub.2 (11 mg), dioxane (1.2 ml), water (0.25 ml), 50 h,
(CH.sub.2Cl.sub.2/MeOH 2:1) gives XVIII (10 mg, 50%).
[0281] Synthesis of Intermediate XIX:
[0282] According to procedure C: XV (22 mg, 0.015 mmol),
Pd(OH).sub.2 (20 mg), dioxane (1 ml), water (0.25 ml), 22 h,
(CH.sub.2Cl.sub.2/MeOH 15:1) gives compound XIX (13 mg, 82%).
[.alpha.].sub.D-9.50.degree. (c=1.21, MeOH).
[0283] Synthesis of Intermediate XXI:
[0284] According to procedure C: XIII (21 mg, 0.015 mmol),
Pd(OH).sub.2 (20 mg), dioxane (1 ml), water (0.25 ml), 22 h,
(CH.sub.2Cl.sub.2/MeOH 15:1) gives compound XXI (13 mg, 86%).
[0285] Synthesis of Intermediate XX:
[0286] A mixture of XVI (20 mg, 0.0141 mmol), 10% Pd/C (20 mg) MeOH
(2 ml) and AcOH (50 .mu.l) is stirred under a hydrogen atmosphere.
When TLC control indicated completion of the reaction (after 22 h),
the mixture is filtered over celite and evaporated to dryness.
Chromatography of the residue on silica gel (CH.sub.2Cl.sub.2/MeOH
2:1) gives XX (15 mg, quant.). [.alpha.].sub.D-29.32.degree.
(c=1.105, MeOH).
General Procedure D
[0287] Synthesis of Product XXII:
[0288] A solution of XVII (27 mg, 0.0278 mmol) in dry MeOH (1 ml)
and a catalytic amount of freshly prepared NaOMe is stirred at r.t.
under argon. After 1 h, TLC control shows completion of the
reaction. The reaction mixture is neutralized with powdered
amberlyst-15, filtered over Celite and the filtrate evaporated to
dryness. The residue is dissolved in MeOH (20 ml) and filtered over
ion-exchange Dowex Na.sup..sym.. The filtrate is evaporated to
dryness and chromatography (CH.sub.2Cl.sub.2/MeOH/water 10:4:0.8)
of the crude product gives XXII (12 mg, 56%).
[.alpha.].sub.D-97.49.degree. (c=0.546, MeOH).
[0289] Synthesis of Product XXIII:
[0290] According to procedure D: XVIII (9.0 mg, 0.0083 mmol), 2 h,
(CH.sub.2Cl.sub.2/MeOH/water 10:3:0.5) gives compound XXIII (7.5
mg, quant.). [.alpha.].sub.D-79.50.degree. (c=0.400, MeOH).
[0291] Synthesis of Product XXIV:
[0292] According to procedure D: XIX (23 mg, 0.0212 mmol), MeOH (2
ml), 2.5 h, (CH.sub.2Cl.sub.2/MeOH/water 10:3:0.5) gives compound
XXIV (7.0 mg, 39%). [.alpha.].sub.C-54.54.degree. (c=0.586,
MeOH).
[0293] Synthesis of Product XXV:
[0294] According to procedure D: XX (19 mg, 0.0180 mmol), MeOH (2
ml), 4 h, (CH.sub.2Cl.sub.2/MeOH/water 10:3:0.5) gives compound XXV
(10.5 mg, 70%.) [.alpha.].sub.D-69.96.degree. (c=0.793, MeOH).
[0295] Synthesis of Product XXVI:
[0296] According to procedure D: XXI (19 mg, 0.0180 mmol), MeOH (2
ml), 4 h, (CH.sub.2Cl.sub.2/MeOH/water 10:3:0.5) gives compound
XXVI (10 mg, 65%.).
Example 21
Assay for E-Selectin Antagonist Activity (FIG. 17A)
[0297] Wells of a microtiter plate (plate 1) are coated with
E-selectin/hlg chimera (GlycoTech Corp., Rockville, Md.) by
incubation for 2 hr at 37.degree. C. After washing the plate 5
times with 50 mM TrisHCl, 150 mM NaCl, 2 mM CaCl.sub.2, pH 7.4
(Tris-Ca), 100 .mu.l of 1% BSA in Tris-Ca/Stabilcoat (SurModics,
Eden Prairie, Minn.) (1:1, v/v) are added to each well to block
non-specific binding. Test compounds are serially diluted in a
second low-binding, round bottomed plate (plate 2) in Tris-Ca (60
.mu.l/well). Preformed conjugates of SLea-PAA-biotin (GlycoTech
Corp., Rockville, Md.) mixed with Streptavidin-HRP (Sigma, St.
Louis, Mo.) are added to each well of plate 2 (60 .mu.l/well of 1
.mu.g/ml). Plate 1 is washed several times with Tris-Ca and 100
.mu.l/well are transferred from plate 2 to plate 1. After
incubation at room temperature for exactly 2 hours the plate is
washed and 100 .mu.l/well of TMB reagent (KPL labs, Gaithersburg,
Md.) is added to each well. After incubation for 3 minutes at room
temperature, the reaction is stopped by adding 100 .mu.l/well of 1
M H.sub.3PO.sub.4 and the absorbance of light at 450 nm is
determined by a microtiter plate reader.
Example 22
Assay for P-Selectin Antagonist Activity (FIG. 17B)
[0298] The neoglycoprotein, sialylLe.sup.a-HSA (Isosep AB, Sweden)
is coated onto wells of a microtiter plate (plate 1) and the wells
are then blocked by the addition of 2% bovine serum albumin (BSA)
diluted in Dulbecco's phosphate-buffered saline (DPBS). In a second
microtiter plate (plate 2), test antagonists are serially diluted
in 1% BSA in DPBS. After blocking, plate 1 is washed and the
contents of plate 2 are transferred to plate 1. Pselectin/hlg
recombinant chimeric protein (GlycoTech Corp., Rockville, Md.) is
further added to each well in plate 1 and the binding process is
allowed to incubate for 2 hours at room temperature. Plate 1 is
then washed with DPBS and peroxidase-labelled goat anti-human
Ig(.gamma.) (KPL Labs, Gaithersburg, Md.) at 1 .mu.g/ml is added to
each well. After incubation at room temperature for 1 hour, the
plate is washed with DBPS and then TMB substrate (KPL Labs) is
added to each well. After 5 minutes, the reaction is stopped by the
addition of 1 M H.sub.3PO.sub.4. Absorbance of light at 450 nm is
then determined using a microtiter plate reader.
Example 23
Anti-Inflammatory Mouse Model
Effects of Test Compound on IL-1.beta.-Induced Neutrophil Migration
to an Air Pouch In Vivo. (FIG. 19)
Animals.
[0299] Male outbred Swiss albino mice (15-18 g body weight) are
purchased from Bantin and Kingman (T.O. strain; Hull, Humberside)
and maintained on a standard chow pellet diet with tap water ad
libitum and a 12:00 h light/dark cycle. All animals are housed for
7 days prior to experimentation to allow body weight to reach
.about.25 g on the day of the experiment (day 6; see below)
Experimental Design.
[0300] Air-pouches are formed on the back of mice by air injection
(2.5 ml s.c.) on day 0 and day 3 (Perretti & Flower, 1993). A
homogenous suspension of carboxymethylcellulose (CMC) is made at
0.5% w/v in PBS and murine recombinant IL-1.beta. added to it at a
concentration of 20 ng/ml.
[0301] Test Compound is given at time 0 just before IL-1.beta.
administration. An extra group is also added, in which a group of
mice received CMC only (no IL-1.beta.) to provide a basal negative
control.
[0302] In all cases, air-pouches are washed 4 h after IL-1.beta.
with 2 ml of PBS containing 3 mM EDTA, and the number of migrated
leukocytes (90% polymorphonuclear leukocytes, PMN) determined, by
taking an aliquot (100 .mu.l) of the lavage fluid and diluting 1:10
in Turk's solution (0.01% crystal violet in 3% acetic acid). The
samples are then vortexed and 10 .mu.l of the stained cell solution
are placed in a Neubauer haematocymometer and neutrophils numbers
counted using a light microscope (Olympus B061).
Compound Administration.
[0303] On the day of the experiment, a fresh solutions of Test
Compound is prepared in PBS Dulbecco's buffer supplemented with 1
mM CaCl.sub.2 and MgCl.sub.2. Monoclonal antibodies (mAb) against
mouse P- or E-selectin are purchased from BD Pharmingen, whereas
the anti-L-selectin mAb is from Serotec:
[0304] Rat anti-mouse L selectin (clone MEL-14): 1 mg
[0305] Rat anti-mouse P-selectin (clone RB40.34): 0.5 mg/ml
[0306] Rat anti-mouse E-selectin (clone 10E9.6): 0.5 mg/ml
FIG. 19.
[0307] Mouse 6-day old air-pouches are inflamed with IL-1.beta. (10
ng) at time 0; Example 17 glycomimetic-BASA (FIG. 13) and Example
12 glycomimetic-BASA (FIG. 8C) are given I.v. at time 0 and 4 h;
and the mix of anti-selectin mAb is given I.v. at time 0.
[0308] Air-pouches are washed at the 8 h time-point and the number
of migrated PMN determined by staining and light microscopy.
[0309] The n number is 9, 8, 7 and 8 mice per group A, B, C and D,
respectively, in FIG. 19.
[0310] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0311] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
* * * * *