FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
"CARBOHYDRATE BASED TOLL-LIKE RECEPTOR (TLR) ANTAGONISTS"
RELIANCE LIFE SCIENCES PVT.LTD
an Indian Company having its Registered Office at
Dhirubhai Ambani Life Sciences Centre,
R-282, TTC Area of MIDC,
Thane Belapur Road, Rabale,
Navi Mumbai - 400 701
Maharashtra India.
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-

FIELD OF THE INVENTION
The present invention relates to novel carbohydrate based molecules, compositions and methods for preparation and modulation of immune functions mediated through Toll-like receptor (TLR) molecules.
BACKGROUND OF THE INVENTION
The innate or natural immune system recognizes a wide spectrum of pathogens without a need for prior exposure. The main cells responsible for innate immunity are monocytes /macrophages and neutrophils, phagocytose microbial pathogens that trigger the innate, inflammatory, and specific immune responses.
Toll-like receptor are a family of receptors involved in the recognition of a wide range of microbial molecules e.g. Lipopolysaccharides (LPS) from Gram- negative bacteria and peptidoglycan from Gram- positive bacteria. The prototype receptor Toll was first identified in the fruit fly Drosophila but several TLR was found in mammals, particularly on mononuclear phagocytes. Toll-like receptors (TLRs) are a class of single membrane-spanning non-catalytic which are designated TLR2, TLR 4, TLR5. etc., and each receptor recognizes a small range of structurally conserved molecules once they have breached physical barriers such as the skin or intestinal tract mucosa, and activate immune cell responses. They are believed to play a key role in the innate immune system. TLRs are a type of pattern recognition receptors (PRRs) and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs).
The discovery of the Toll-like receptors finally identified the innate immune receptors that were responsible for many of the innate immune functions that had been studied for many years. Interestingly, TLRs seem only to be involved in the cytokine production and cellular activation in response to microbes, and do not play a significant role in the adhesion and phagocytosis of microorganisms. Binding of TLR leads to the production of inflammatory cytokines, including TNF-alpha and IL-12 and enhances the cells'
2

antimicrobial killing mechanisms and antigen presenting capacity. The function of the TLRs was discovered by Beutler and colleagues. These workers used positional cloning to prove that mice that could not respond to LPS had mutations that abolished the function of TLR4. This identified TLR4 as a key component of the receptor for LPS, and strongly suggested that other Toll-like receptors might detect other signature molecules of microbes, such as those mentioned above.
The first microbial product discovered to be a toll-like receptor agonist was LPS, an abundant outer cell-wall constituent from gram - negative bacteria that is recognised by the innate immune system. The chemical structure and the molecular basis of the recognition of the LPS by serum proteins has gained attention in recent years, which has lead to the discovery of a family of receptors like Toll like receptors. It has been estimated that most mammalian species have between ten and fifteen types of Toll-like receptors. Thirteen TLRs (named simply TLR1 to TLR13) have been identified in humans and mice together, and equivalent forms of many of these have been found in other mammalian species.
The significance of the toll like receptors in the immune response to LPS has further demonstrated specifically two receptors TLR 2 and TLR 4. (Yang et.al Nature 395:284-288 1998: Krishning et.al. J.Exp Med. 11:2091-97, 1998). Further reports (Protorak et.al. Science 282:2085-2088 (1988); Quereshi et al. J.Exp. Med 4:615-625 (1999)) concluded that TLR4 was required for a response to LPS.
Although LPS is an immunomodulatroty agent, its medicinal use is limited due to its extreme toxicity including the induction of systemic inflammatory response syndrome. The biologically active endotoxio sub-structural moeity of LPS is a lipid-A, a phosphorylated , multiple fatty acid acylated glucosamine disaccharide that serves to anchor the entire structure in the outer membrane of the gram - negative bacteria. The toxic effects of the lipd A was addressed by selective chemical modification of the lipid A to produce monophosphoryl lipid A compounds (MPL® : immunostimulant from Corixa and structurally like MPL® compounds) which is described in U.S. Patent
3

Numbers : 4,436,727; 4,877,611; 4,866,034; 4,912,094; 4,987,237; and Johnson et.al. J.Med Chem 42: 4640-4649 (1999);
Considering the basis of the MPL® immunostimulant and other bacterial cell wall components, a family of novel synthetic compounds, the aminoalkyl glucosaminide phosphates (AGPs) were developed. AGP compounds were found to be TLR-4 agonists and antagonists. These ABGP compounds belong to the class of synthetic mono and disaccharide mimetics of monophosphoryl lipid A as described in U.S. Patent number 6,303,347 and in WO 98/50399). These compounds besides being synthetic in nature has improved toxicity profiles as compared to monophosphoryl lipid A. These compounds have been used in combination with antigens in vaccine formulations ( U. S. Patent Number 6,113,918) and also as monotherapies in absence of antigens ( WO 01/90129).
Based on the above various aminoalkyl glucosaminide phosphate compounds have been described in U.S. Patent publication number 20050227943. Cyclic AGPS have been described in PCT Patent application number PCT/US01/24284 and azacycloalkyl AGPs have been described in U.S. Patent numbers 6,911,434 and 6,800,613. Further specific glycosaminoglycan polymers (GAG molecules) have been disclosed in U.S. Patent Publication number 20050272696 which have differential effects on cancer and hence have been designed for "personalised medicine".
Further reports also have suggested that non sulfated glycosaminoglycans such as Hyaluronan play a role in innate immunity. Evidence support that hyaluronan degradation products transduce their inflammatory signal through Toll-like receptor 2 (TLR2), TLR 4 or both in macrophages and dendritic cells.
A large number of synthetic lipid A analogs have been prepared. Lien te.al. 2001 described the agonist ER-112022, in which the disaccharide backbone of lipid A is replaced with -CH2CFI2-NHCO- (CH2)4-CONH- (CH2)2. The two phosphate groupd linkthis substitutre backbone to the lipid chains.
4

Christ et.al 1995 prepared the lipid A antagonist E5531 derived by modification of the structure of the endotoxin- antagonist Rhodobacter Capsulatus lipid A, in which naturally occuring acyl linkages at the C-3 and C-3' carbons are replaced by ether linkages, and the C-6' hydrozyl group was blocked which has resulted in increased stability and purity.
Ribi et al 1982 showed the minimal structure required for toxicity was a bisphosphorylated p- 1,6- linked di glucosamine core to which long fatty acid chains are attached. The reports suggested that an opitimal number of lipid chains in the form of acyl or acyloxyacyl groups are required on the dissacharide backbone in order to exert strong endotoxic and related biological activities of Lipid A. (kotani 1986a).
Werner 1996 has suggested that removal of either phosphate group resulted in significant loss of toxicity without compromising on adjuvant activity. Bioassays on monophosphoryl Lipid A showed that although 1000 times less potent in eliciting toxic properties, it was comparable to diphoshoryl Lipid A in immunostrimulating activities.
Further studies by Seydel et. al 2000 and Schromm et al 2000 suggested that the agonistic and antagonistic activity of Lipid A were governed by instrinsic conformation of Lipid A which in turn was defined by the number of charges, the number and distribution of acyl chains in the molecule.
Thus in view of the prior literature of synthetic lipid A derivative behaving as TLR-4 antagonist such as reported by T Kawata, et. al. (J. Pharmacology and Expt. Therapeutics, 304, 1093-1102, 2003) relating to,E5564 [a-D-glucopyranose,3-0-decyl-2-deoxy-6-0-[2-deoxy-3-0-[(3R)-3-mefhoxydecyl]-6-0-mefhyl-2-[[(l 1Z)-1 -oxo-11-octadecenyl] amino]-4-0-phosphono-P-D-glucopyranosyl]-2-[(l ,3-dioxotetradecyl)amino]-1 -(dihydrogen phosphate), tetrasodium salt] tetrasodium salt wherein the lipodisaccharide has a complicated structure posed more problems in synthesis and the chemical synthesis involves multisteps.The inventors have developed an alternate synthetic TLR-4 TLR-4 antagonists for the effective treatment of TLR-4 ligand/signaling /LPS associated disorders and which can be used as monotherapies in the absence of antigen.
5

The present invention provides novel carbohydrate based disaccharides for modulation of immunity with substantial antagonistic activity in vitro and in-vivo system and offers a simple synthetic process of preparation which results in substantially pure, reproducible, and stable molecules for efficient inhibition of TLR-4 ligand mediated signaling events and consequences . Moreover, the present invention provides monotherapies or combinations thereof formulated and administered in the absence of exogenous antigens for the therapeutic/ prophylactic treatment of the plant and animal diseases.
OBJECT OF THE INVENTION:
It is the object of the present invention to provide carbohydrate based molecules for modulation of immunity.
It is the object of the present invention to provide carbohydrate based molecules for inhibition of TLR ligands.
It is the object of the present invention to provide methods of preparation of the carbohydrate based molecules.
It is the object of the present invention to provide compositions of the carbohydrate based molecules.
It is the object of the present invention to provide compositions useful for the treatment of prevention or treatment of inflammation, wounds, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer, and immunodeficiency
It is the object of the present invention to provide compositions for inhibition of TLR mediated conditions.
6

It is the object of the present invention to provide carbohydrate based molecules for inhibition of TLR ligands which can be used in combinations with other agents.
SUMMARY OF THE INVENTION
The present invention relates to carbohydrate-based molecules, methods of preparations, compositions for use in TLR mediated immune conditions. The present invention also relates to compositions and methods for modulating immune functions mediated through Toll-like receptor (TLR) for efficient inhibition of TLR-4 ligand mediated signaling events and consequences. Moreover, the present invention provides monotherapies or combinations thereof formulated and administered in the absence of exogenous antigens for the therapeutic/ prophylactic treatment of the plant and animal diseases.
In one embodiment the present invention provides compositions that are useful for the prevention or treatment of inflammation, wounds, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer, and immunodeficiency. In the preferred embodiments the compositions as described in the present invention are useful for inhibition of TLR signaling in response to TLR ligands.
In the preferred embodiments the present invention provides the compositions for inhibition of TLR signaling in a therapeutically effective amount and pharmaceutically inert adjuvants, diluents or carriers.
In one embodiment the compositions as described in the present invention or composition comprising the same is believed to have the ability to inhibit inhibition of TLR signaling under physiological conditions, and thereby would have corresponding effectiveness for prevention or treatment of inflammation, wounds, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer, and immunodeficiency.
7

In yet another embodiment on basis of inhibition of TLR signaling properties, the composition can be used in veterinary medicine for the prevention and treatment of inflammation, wounds, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer, and immunodeficiency.
In preferred embodiments the present invention also provides the pharmaceutical formulations either alone or a suitable pharmaceutically acceptable adjuvant useful in inhibition of TLR mediated clinical manifestations.
The compositions as described in the present invention are useful for the prevention or treatment of inflammation, wounds, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer, and immunodeficiency. As a feature of the present invention, the methods of the invention can be combined with administration of additional agents to achieve synergistic effect on TLR-mediated immunostimulation. More specifically, whereas the agents described herein have been discovered to affect TLRs directly and thus directly affect TLR-bearing cells, e.g., antigen-presenting cells (APCs), such agents can be used in conjunction with additional agents which affect non-APC immune cells, e.g., T lymphocytes (T cells). Such an approach effectively introduces an immunomodulatory intervention at two levels: innate immunity and acquired immunity. Since innate immunity is believed to initiate and support acquired immunity, the combination intervention is synergistic
The present invention also provides the manner of manufacture of compositions as described in the present invention in a therapeutically effective amount either alone or in combination with pharmaceutically acceptable adjuvant.
In another embodiment of the invention, a method of affecting TLR-mediated signaling in response to a TLR ligand is provided.
In one embodiment of the invention, a method of inhibiting TLR-mediated immunostimulatory signaling is provided.
8

In another embodiment, the invention provides a method of modulating TLR-mediated immunostimulation in a subject.
The carbohydrate-based molecule of the present invention can be used in the treatment for variety of conditions involving autoimmunity, inflammation, allergy, asthma, graft rejection, graft-versus-host disease (GvHD), infection, sepsis, cancer, and immunodeficiency. Generally, for treating conditions involving infection, cancer, and immunodeficiency employs small molecules that augment TLR-mediated signaling in response to a suitable TLR ligand. In some instances the methods can be used to inhibit or promote TLR-mediated signaling in response to a TLR ligand or TLR signaling agonist. In some instances the methods can be used to inhibit TLR-mediated immunostimulatory signaling in response to a TLR ligand or TLR signaling agonist. In some instances the methods can be used to inhibit or promote TLR-mediated immunostimulation in a subject. In some instances the methods can be used to inhibit TLR-mediated immunostimulation in a subject. In some instances the methods can be used to inhibit an immunostimulatory nucleic acid-associated response in a subject.
In one embodiment, the present invention provides molecules and methods useful for modulating TLR-mediated signaling. The molecules of the present invention are applicable to alter any TLR - mediated signaling in response to a suitable ligand or signaling agonist.
In one embodiment the present invention also provides methods for identifying agents that decrease or inhibit activation of Toll-like receptor 2. These methods involve (i) contacting a cell expressing the receptor with a candidate agent in the presence of an activator of the receptor (in vitro or in vivo) and (ii) determining the effect of the agent on activation of the receptor. Detection of a decrease in activation of the receptor by the activator in the presence of the agent indicates the identification of agent that can be used to decrease or inhibit activation of the receptor. In these methods, the effect of the agent on the activation of the receptor can be determined by analysis of the expression of a
9

reporter gene that is under the control of a promoter that is induced in a signaling pathway triggered by activation of the receptor.
In one aspect of the invention, a method of affecting TLR-mediated signaling in response to a TLR ligand is provided. The method according to this aspect involves contacting a cell expressing a TLR with an effective amount of a compound of Formula I: P-D-gIucopyranosyl-(l-4)- 2-deoxy-2-amino-P-D-glucopyranoside

OR

X'
OR.;

O
OR4 ORr OOR;

A
O
NHR,

wherein all or at least one Rl, R2, R3 or R4 is;

0

O

CH,

bH^CH3

|CH)TCH3

O

CCL

O
L

c-ci
H,

o
o

(CH2)rrC=C-{CH2)-CH;
CH2)-CH3
(CH^C^C— (CH2)-CH2

10

M—( 0^0=0 (CH2)-q"CH,
SCH2)m (CH2hrCH3


o
o

l/\(CH2^-C=CH—(CH2}q-CH3

CH2y^(CH2hrCH;


o
0 L/\(CH2}rC=C—(CH2)-q-CH3
v(CH2)m (CH2hrCH3

o

^ (CH2 ^C=CH-tCH2)-q-CH2

'CH,)-^"^[CH2y— CH,
\ 2/ m \ 2 / n 2

12

o

L^(CH2hrC=(^-(cH2HrCH;

(ChL)^^^(CH2y— CH,
\ 2/m \ 2/n ^
wherein each L is O, N or C; each M is O or N; each E, independently, is an integer between 0 and 14 inclusive; each G, independently, is N, O, S, SO, or SO2; each m, independently, is an integer between 0 and 14 inclusive; each n, independently, is an integer between 0 and 14 inclusive; each p, independently, is an integer between 0 and 10 inclusive; each r, independently is an integer between 0 and 20 inclusive and each q, independently, is an integer between 0 and 10 inclusive;
each of the remaining Rl, R2, R3, and R4, independently, is:

CH2)fNcH2)xCH,
O O OH
CH2CH3

O
0 /V^lyCHs
^CH2)fNCH2)xCH3
0 0-fCH2)CH3 Q 0-(CH2)yCH3
(CH2)fNCH2) XCH3 ^(CH2)^(CH2) XCH3
13

y~"3
fCHjyCH
O^/
o
^tCH2)z (CH2>XCH

o
\(CH2^G—(CH2)XCH2

CH2[Z ^CH2)xCHa

0

o

O
(CH2)CNCH2)XCH;

OH

0^fCH2)yCH2

(CH2L>H2)XCH;

(CH2)C>(CH2)xCH:

O

O

l/T^ly0^

,/^M—(CHjyCH,

(CH2)^+>(CH2)XCH3

, or

(CH2L>H2)XCH:

wherein each L is O, N or C; each M is O or N; each x, independently, is an integer between 0 and 14 inclusive; each y, independently, is an integer between 0 and 14 inclusive; each z, independently, is an integer between 0 and 10 inclusive and each G, independently, is N, O, S, SO, S02;
each A and X, independently, is H, OH, OCH3> C6H5OCH3,

O
IL

CH,

O

"C-CI H,

O
II -O-P-OH I OH

O
II —S-OH
II
o

14

O 0 0
(CH^O-P-OH (CH2^0-P-OH — 0-jj-OH
OH OH O
o o
(CH2)T-P-°H O-(CH2)^P-OH
OH OH
(CH^COOH , O—(CH^COOH ,
(CH^—A1 /(CH^—A1

^fCH^A1 XfCH^A

wherein each d, independently, is an integer between 0 and 5 inclusive; each f, independently, is an integer between 0 and 5 inclusive; each g, independently, is an integer between 0 and 5 inclusive and each A , independently, is
O O
(CH2}-0-P-OH , (CH^O-P-OH
OH OH
O O
(CH2)-P-OH 0-(CH2)-P-OH
OH OH
(CK)—-COOH , O—(CHJ—COOH ,
wherein each j, independently, is an integer between 0 and 14 inclusive; X is OH, CI, 0(CH2)tCH3, 0(CH2)tOH, 0(CH2)tO(CH2)vCH3
15

o
11
0^\(CH2)-CH3
wherein i, independently, is an integer between 0 and 20 inclusive; each t and v, independently, is an integer between 0 and 14 inclusive;
R5 and R6 is any possibilities listed above for Ri - R4; in addition R5 and R6 are H, benzylidene, acetonide ;
Most preferably, the above compounds are formulated as a lysine salt, a TRIS salt, a potassium or a sodium salt.
In a second aspect, the invention features a compound II of the formula:

OR,
O
A
OR6

5^ .0
o
OR
NHFT, 3 OR_
Formula II : 2-Deoxy-2-amino-b-D-glucopyranosyl-(l-4)-P-D-glucopyranoside
wherein all or at least one RI, R2, R3 or R4 is;

I
C CI ^CH^-CH3 HCH)TCH
3

o
o o
CCL CH,
16

o
(CH^=C-(CH2)-CH3 (CH2)-CH3
o
(CH2}^C=C-(CH2)-CH;


O

^g^-tCH^CH,

0

o
p H H
CHJ™ 1CH2^C=C— (CH2VCH

O

O

CH.L >tcH2}-C=C—(CH2)q-CH3
O
CH2)m+^CH2}?-C=C—(CH2VCH
p H H
O
(CH2)m+1 \CH2}FC=CH—(CH2)-q-CH,
O
\ ICH2)^-C=CH—(CH2)^CH;
ChL) (CH2)—CH3
2/m+1 \ ^/n 3
17

o

MCH^CEC-

•(CH2}q-CH,

O
CH2 G— (CH2}^-C=CH CH2—CH;
0
^(CH2^rG-(CH2[rC=C-(cH2)^-CH3
O

o

H
L M-(CH2^C

CH—(CH2)^-CH;

^(CH2)m (CH2hrCH;
o
0 L M—(CH2V-C=C (CH2)q-CH,
NCH2)m (CH2hrCH3


o
O U \-"2/p |_|
L X(CH2^C=CH— (CH2)q-CH,
18


o
o /NCH2hrc=c—(CH2)^CH;
\^2ym lCH2hrCH;

o

L^(CH2^C=CH-(CH2)T-CH3

\CH2

CH2^-CH2

o

CH,

•(CH2^-C=(3—(CH2}q-CH2 CH2^CH3

wherein each L is O, N or C; each M is O or N; each E, independently, is an integer between 0 and 14 inclusive; each G, independently, is N, O, S, SO, or SO2; each m, independently, is an integer between 0 and 14 inclusive; each n, independently, is an integer between 0 and 14 inclusive; each p, independently, is an integer between 0 and 10 inclusive; each r, independently, is an integer between 0 and 20 inclusive; and each q, independently, is an integer between 0 and 10 inclusive;
each of the remaining Rl, R2, R3, and R4, independently, is:

O

CH2CH3

O

OH CH2)fNcH2)xCH2

19

o-
y 3
CH2)fNcH2)xCH,
O

(CH2)CH

O 0-(CH2)CH3
H2)XCH3

(CH2)C>(CH2)XCH2

20

wherein each L is O, N or C; each M is O or N; each x, independently, is an integer between 0 and 14 inclusive; each y, independently, is an integer between 0 and 14 inclusive; each z, independently, is an integer between 0 and 10 inclusive and each G, independently, is N, O, S, SO, SO2;
each A and X, independently, is H, OH, OCH3, QH5OCH3,

Q Q 0 0
L„ ^r-ri —O-P-OH —O-S-OH
O Ol 1 ' 11
"CH
H2 OH 6
O 0 0
/ \ II , II II
CHj^-O-P-OH , CH0h^O-P-OH —S-OH
\ a a 1 , lwll2/d+i | ||
OH OH O
o o
(CHJ-P-OH o-fCHj^-P-OH
OH OH
(CH^-COOH , O—(CH-^-COOH ,
(CH^-A1 /(CH^-A1
(CH^X |0r o-fcH^-Y
X(CH^A1 XfCH^A1
wherein each d, independently, is an integer between 0 and 5 inclusive; each f, independently, is an integer between 0 and 5 inclusive; each g, independently, is an integer between 0 and 5 inclusive and each A1, independently, is

v 2/J I ' 1^ 2jj +1 |
OH OH
21

0II
(CHJr -P-OH1
OH
(c% -COOH

o
'J
OH
°-H2hrCH3

o

H
CH2)rC=CH—(CH2)q-CH,

o

fCH.

CH2y7rCH3
(CH2^—C=C (CH2)-q-CH,

[ChU^ ^(CH2V-CH,
\ 2/ m \ z I n -
wherein each L is O, N or C; each M is O or N; each E, independently, is an integer between 0 and 14 inclusive; each G, independently, is N, O, S, SO, or SO2; each m, independently, is an integer between 0 and 14 inclusive; each n, independently, is an integer between 0 and 14 inclusive; each p, independently, is an integer between 0 and 10 inclusive; each r, independently, is an integer between 0 and 20 inclusive and each q, independently, is an integer between 0 and 10 inclusive;
each of the remaining Rl, R2, R3, and R4, independently, is:
25

o

CH2CH3

O OH
\fCH2)f^(CH2)xCH3

O
o

L lCH2)yCH3
CH2)f SCH2)XCH3

o-
o-
y 3
y 3
CH2)fNcH2)xCH,
CH2)fNcH2)xCH,
O

(CH2)CH

O

(CH2)CH

yCH3
Q iCH2)
^CH2)f^(CH2)xCH

O ^SCH^G—(CH2)XCH,

CH2[Z ^CH2)XCH,

O

o

O
(CH2^>(CH2) XCH3
wherein each L is O, N or C; each M is O or N; each x, independently, is an integer between 0 and 14 inclusive; each y, independently, is an integer between 0 and 14 inclusive; each z, independently, is an integer between 0 and 10 inclusive and each G, independently, is N, O, S, SO, S02;
each A and X, independently, is H, OH, OCH3, C6H5OCH3,
0 0 0 0
^ou ^r_n —0-P-OH —0-S-OH
CH3 g CI | n
H2 OH O
O O ' O
(CH2)^-0-P-OH , (CH^O-P-OH — S-OH
OH O
o o
(CH2)-P-0H o- OH
OH OH
(CH^COOH , O—{CH-j-^-COOH ,
/M-A1 /(CH^-A1
(CH^ , or O^CHW
XfCH^-A1 XfCH^A1
27

wherein each d, independently, is an integer between 0 and 5 inclusive; each f, independently, is an integer between 0 and 5 inclusive; each g, independently, is an integer between 0 and 5 inclusive and each A1, independently, is

011 011
(CH2)-0-P-OH , (CH2)i7r ■0—P-OHI
OH OH
0II 0II
(CHJ-P-OH 0-(CH2): — P-OHI
OH OH
(CHJ—COOH , OHCHJ- -COOH ,
wherein each j, independently, is an integer between 0 and 14 inclusive; X is OH, CI, 0(CH2)tCH3, 0(CH2),OH, 0(CH2)tO(CH2)vCH3 0
o^(CH2)r-cH3
wherein i, independently, is an integer between 0 and 20 inclusive; each t and v, independently, is an integer between 0 and 14 inclusive;
R5 and R(, is any possibilities listed above for Ri - R4; in additionR5 and R6 are H, benzylidene, acetonide ;
Most preferably, the above compounds are formulated as a lysine salt, a TRIS salt, a potassium salt or a sodium salt.
28

In a fourth aspect, the invention features a compound IV of the formula:

A
X
OR

3 OR

ORc
O

ORc
O

O
. 0
NHR0R3

4
o
OR
OR

Formula IV : (P-D-Glucopyranosyl)-(l-3)-0-(2-deoxy-2-amino-P-D-glucopyranosyl)-(l-4)-p-D-glucopyranoside
wherein all or at least one Rl, R2, R3 or R4 is;

O
IL

C-CI

O

tCH^-CH3

PH^"CH2

O

CCL

O

CH,

O
o
0

(CH2)rrC=C-(CH2)-CH3 (CH2)-CH3
(cH^CEEC—(CH2)-CH3
(CH2)nr^c-HcH2)-cH3

29

o

o

CH2)m lCH2brC=c—(CH2VCH3

o

o

CHjm \CH2}-C=C— (CH2}q-CH
o
CH2) m+^t CH2 ^C=C—(CH^CH
p H H
o
CH2)m+1 \CH2)i-C=CH—(CH2yq-CHa
o

2> H wl '3
Jr ICH2^C=CH-
CH2)m+1 (CH2hrCH3 o

CH2)-q-CH

\ 1CH2^C=C (CH2h-CH,
CH2J;+1 (GH2hrCH3
o

CH,—G—(CH2)^C=CH-

-CH—CH3

o

\ CH^G—(CH2 ^C=C-fcHj^ CH3
30

o

o

L M—(CH2)^-C=CH—(CH2Vq-CH,

'CH2)m ""(CH^CH, O
O L M—(CH2)^C=C (cH2)-q-CH;
SCH2)m (CH2hrCH3
o

2/q v"3
o

CH—(CH9)F-CH

^(CK

CH2^CH3

o
o

/NcH^C^C—(CH2)—CH3

^(CH2)m (CH2hrCH;

o

L/"(CH2^C=CH—(CH2)—CH3

^(CH2

CH2hrCH2

o

•(CH^C^C—(CH2)—CH,

CH2l^T^(CH2hrCH3

31

wherein each L is O, N or C; each M is O or N; each E, independently, is an integer between 0 and 14 inclusive; each G, independently, is N, O, S, SO, or SO2; each m, independently, is an integer between 0 and 14 inclusive; each n, independently, is an integer between 0 and 14 inclusive; each p, independently, is an integer between 0 and 10 inclusive; each r, independently, is an integer between 0 and 20 inclusive and each q, independently, is an integer between 0 and 10 inclusive;
each of the remaining Rl, R2, R3, and R4, independently, is:

O

CrLCrL

O OH
^SCH2)^(CH2)XCH3


O
CH2)CH3
O L 1 «y
^CH2)fNCH2)xCHa


o-
CH2)fNcH2)xCH,
O

0-(CH2)yCH3 CH2)^(CH2)XCH3

O

(CH2)CH;

yCH3
iCH2)
D
^\(CH2)fNcH2)xCH

O
\(CH2^G—(CH2)XCH=

32



CH2fz XfCH2)xCH3 (CH2)^+1X(CH2)XCH3
Q °

OH 0^CHJyCH3
(CH^CHj XCH3 (CH2)C^CH2) XCH3
o o
L^\(CH2) y CH3 L/^M-fCHj y CH3
(CH2)A(CH2) XCH3 (CH2^CH2) XCH3
wherein each L is O, N or C; each M is O or N; each x, independently, is an integer between 0 and 14 inclusive; each y, independently, is an integer between 0 and 14 inclusive; each z, independently, is an integer between 0 and 10 inclusive and each G, independently, is N, O, S, SO, SO2;
each A and X, independently, is H, OH, OCH3, C6H5OCH3,

0 0^CH, ^C-CIH2 0II -O-P-OH 1 OH 0II —O-S-OHII 05
011 011 0II—S-OHII
(CH2hrO-p-OH , (CH2fc -O-P-OH1
OH OH O
33

o o
(CHjj-P-OH 0-(CH2)^P-OH
OH OH
(CK^-COOH , O—(CH^COOH ,
(CKJ-A1 /MrA'
(CH^H ,or o-(CHW
XfCH^-A1 XfCH^-Ai
wherein each d, independently, is an integer between 0 and 5 inclusive; each f, independently, is an integer between 0 and 5 inclusive; each g, independently, is an integer between 0 and 5 inclusive and each A1, independently, is
O O
(CH2)-0-P-OH , (CHJ-^O-P-OH
OH OH
0 O
(CHJ-P-OH 0-(CH2)-P-OH
OH OH
(CHJ— COOH , O—(CHJ—COOH ,
wherein each j, independently, is an integer between 0 and 14 inclusive; X is OH, CI, 0(CH2)tCH3, 0(CH2)tOH, 0(CH2)lO(CH2)vCH3 O
u
0^\(CH2)-CH3
34

wherein i, independently, is an integer between 0 and 20 inclusive; each t and v, independently, is an integer between 0 and 14 inclusive;
R5 and R6 is any possibilities listed above for Ri - R4; in addition, R5 and R6 are H, benzylidene, acetonide ;
Most preferably, the above compounds are formulated as a lysine salt, a TRIS salt, a potassium salt or a sodium salt.
In a fifth aspect, the invention features a compound V of the formula:

4 O
o-
OR
OR

X
OR

OR
3 OR

OR;

O

O
,OR4 OR£ OOR,

A
O
NHR,

Formula V : (P-D-GIucopyranosyl)-(l-3)-0-(2-deoxy-2-amino-P-D-glucopyranosyl)-(l-4)-0-(P-D-glucopyranosyl)-(l-3)-0-2-deoxy-2-amino-P-D-glucopyranoside
wherein at least one RI, R2, R3 or R4 is;

PH)TCH=
O

O
IL
C-CI
CCL

CH
O

O

35

o
(CH^-C=C—(CH2)-CH2
o
(CH2)— C=C—(CH2)—CH,


o

o
CHjm \CH2}p-C=C—(CH2}q-CHa

o

o
CH^ \CH2}-C=C— (CH2}q-CH

O
(CH2)m+iVtCH2)FC=C—(CH2h-CH3 n n
o
(CH2)m+1 IC^^g^H-tCHj-CH,
O
V ICH2}F-C=CH-(CH2VCH3
CH2i;+1 (CH2hrCH3 o
jr. ICH2)y-c=c—(cH2)q-cH, (CH2i;+i (CH2hrCH3
36

o

^Ch%—G-(CH2^C=CH-

-CH—CH3

O
; CH^GH CH2 ^c=c—(CH2) q-CH,
o
O L M— (CH2y-C=CH— (CH2)q-CH,
Mlm (CH2hrCH2
o
O L M— (CH2y—CEEC (CH2h-CH,
fCH2)m lCH2hrCH3
o

o

L ^(CH2^C:

CH—(CH2Vq-CH;

(CH9\-""^(CH2}— CH,
\ 2/m \ 2/n ^
O
Q L .(0^0=0— (CH2)T-CH;
SCH2)m (CH2hrCH3
37

o

L^(CH2hrS=CH-(cH2hrCH3

rCH.

cH2y7rcH3

o

^ (CH2 \vC=C (CH2)^CH3

CH.

CH2hrCH2

wherein each L is O, N or C; each M is O or N; each E, independently, is an integer between 0 and 14 inclusive; each G, independently, is N, O, S, SO, or SO2; each m, independently, is an integer between 0 and 14 inclusive; each n, independently, is an integer between 0 and 14 inclusive; each p, independently, is an integer between 0 and
10 inclusive; each r, independently, is an integer between 0 and 20 inclusive and each q, independently, is an integer between 0 and 10 inclusive;
each of the remaining Rl, R2, R3, and R4, independently, is:

O

CrLCH,

O OH
^CH2)fNcH2)xCH3

o
CH2)

O

CH2)yCH3 (CH2)XCH3

O

0-(CH2)CH3

,

O

0-(CH2)yCH3
(CH2)fNCH2)xCH3

38

y~"3
^CHjLCH

O
^CH2)f^(CH2)xCH3

O
\(CH2}^G—(CH2)XCH3

CH2[Z ^CHjxCHg

O

o

O
(CH2)C>(CH2)XCH3

OH (CH2AcH2)xCH3

0^fCH2)yCH3
(CH2rz+i^CH2)xCH3

0

0

/NCH2)yCH= (CH2L>(CH2)XCH3

,/^M— (CHjyCH3
(CH2)C>H2)XCHG

wherein each L is O, N or C; each M is O or N; each x, independently, is an integer between 0 and 14 inclusive; each y, independently, is an integer between 0 and 14 inclusive; each z, independently, is an integer between 0 and 10 inclusive and each G, independently, is N, O, S, SO, SO2;
each A and X, independently, is H, OH, OCH3, C6H5OCH3,
39

0CH3 0 H2 0 II -CI -O-P-OHOH i9 0 II —0-S-OHII 0
011 011 011
(CH2hr0-p-0H , (cH2fcr° i °» —S-OHII
OH 1 1OH 1 10
011 011
(CHJ-P-OH O-tCHJT-P-OH
OH OH
(CHJ^-COOH , 0—(CH.J-J-COOH ,
(CH^-A1 \CH^-A , or1 /Mr -A1 -A1
wherein each d, independently, is an integer between 0 and 5 inclusive; each f, independently, is an integer between 0 and 5 inclusive; each g, independently, is an integer between 0 and 5 inclusive and each A , independently, is
O O
(CH2}-0-P-OH , (CH2)—pO-P-OH
OH OH
O O
II
(CHJ-P-OH 0-{CH2)-P-OH
OH OH
(CHJ— COOH , O—(CH^—COOH
40

wherein each j, independently, is an integer between 0 and 14 inclusive; X is OH, CI, 0(CH2)tCH3, 0(CH2)tOH, 0(CH2)tO(CH2)vCH3
O
II °^(CH2)-CH3
wherein i, independently, is an integer between 0 and 20 inclusive; each t and v, independently, is an integer between 0 and 14 inclusive;
Rs and R6 is any possibilities listed above for R| - R4; in addition, R5 and R6 are H, benzylidene, acetonide ;
Most preferably, the above compounds are formulated as a lysine salt, a TRIS salt, a potassium salt or a sodium salt.
In a sixth aspect, the invention features a compound VI of the formula:

V1 - ~,,2
Formula VI : (2-Deoxy-2-amino-P-D-glucopyranosyl)-(l-4)-0-( p-D-
glucopyranosyl)-(l-3)-0-(2-deoxy-2-amino-P-D-glucopyranosyl)-(l-4)-0-P-D-
glucopyranoside
wherein all or at least one Rl, R2, R3 or R4 is;

O
L

c-ci
H„

o

CH^rCH3

CH)TCH3

41


CH,
o

CCL

o

CH,

o

O

O
(CH2t-^cHCH2)-CH3 (CH2)—CH3

CH^-C=C—(CH2)—CH,
O


CH2)^C=C-{CH2)-CH3
O


O

o
Ch)m 1CH2}^C=C—(CH2)-q-CH:

0

H
O
42

o

H
L ^CH2}rC=CH—(CH2h-CH,

;CH2)m+1 (CH^CH3
o
^ (CH2)^CEEC (CH2hr CH,
;CH2);+1 (CH^CH,
o
CH2—G— (CH2)^C=CH CH—CH;
0
^(CH2}ls-G-(CH2}rC=C-(cH2)^CH2 O

o

|/ XM—(CH2)—C=CHHCH2)^CH3

^(CH2)m (CH^C^
O
O L M—(CH^CEEC (CH2h-CH2
^(CH2)m (CH2^CH3


o
o

L/\(CH2}rC=CH-(CH2VCH2

CH2y^(CH2hrCH3

43

o
0 L ^(CH2^C=C—(CH2)T-CH3
^(CH2)m (CH2^H3

0

L/(CH2}rC=CH—(CH2yq"CH,

2/ m \ 2 / n 3

0

CH.

■(CH2^-C=C (cH2Vq-CH2
CH2^CH3

wherein each L is O, N or C; each M is O or N; each E, independently, is an integer between 0 and 14 inclusive; each G, independently, is N, O, S, SO, or SO2; each m, independently, is an integer between 0 and 14 inclusive; each n, independently, is an integer between 0 and 14 inclusive; each p, independently, is an integer between 0 and
10 inclusive; each r, independently, is an integer between 0 and 20 inclusive and each q, independently, is an integer between 0 and 10 inclusive;
each of the remaining Rl, R2, R3, and R4, independently, is:

O

CH2CH3

O OH
^CH2)^(CH2)XCH3

CH2)CH3 (CH2)XCH3

44


o-
y~"3
CH2)fScH2)xCH2
o

(CH2)CH

O 0-(CH2)CH3
(CH2)f^(CH2)xCH3

iCHj

yCH3

o
\(CH2)^NCH2)XCH

O
^SCH^G—(CH2)XCH2

CH2fz NICH2)XCH3

O

o

O
(CH2)C>H2)XCH;

y~' '3
(CH2[2+11CH2)XCH3
OH

O
-{CH2) CH
CH2)C>(CH2)XCH;

L

O

wherein each L is O, N or C; each M is O or N; each x, independently, is an integer between 0 and 14 inclusive; each y, independently, is an integer between 0 and 14 inclusive; each z, independently, is an integer between 0 and 10 inclusive and each G, independently, is N, O, S, SO, S02;
45

each A and X, independently, is H, OH, OCH3, C6H5OCH3,

0CH3 0 H2 0II-ci -°-prOH OH1 9 0II—o-s-II0 -OH
011 011 011
(CH^O-P-' OH j (CH2fc7° P « 3H —S-OH11
OH 1 1OH ? 1 10
011 011
(CHJ7-P-OH O-fcHj-P-OH
OH OH
(CH^COOH 0—(CH^COOH ,
(CHJ—A1 /(CHo)—A1
(CHh-\ •or o-(CH^rwherein each d, independently, is an integer between 0 and 5 inclusive; each f, independently, is an integer between 0 and 5 inclusive; each g, independently, is an integer between 0 and 5 inclusive and each A1, independently, is
O O
, . II II
CHJ—0-P-OH /cH,h 0-P-OH
^ zl\ I ' > 2/j +1 |
OH OH
O O
(CH2)-P-OH 0^CH2)-[i-OH
OH OH
46

(CK)—-COOH , O—(CHJ—COOH ,
wherein each j, independently, is an integer between 0 and 14 inclusive; X is OH, CI, 0(CH2)tCH3, 0(CH2)tOH, 0(CH2)tO(CH2)vCH3
0
JJ 0^-(CH2)-CH3
wherein i, independently, is an integer between 0 and 20 inclusive; each t and v, independently, is an integer between 0 and 14 inclusive;
R> and R& is any possibilities listed above for Ri - R4; in addition Rs and R6 are H, benzylidene, acetonide ;
Most preferably, the above compounds are formulated as a lysine salt, a TRIS salt, a potassium or a sodium salt.
Accordingly, the invention features compounds of formula I-IV and a pharmaceutical^ acceptable salt or prodrug thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Figure 1: shows that RSCL- 0409 inhibits TLR2, TLR4 induced TNF-a secretion Briefly, THP-1 cells (2 xlO5 cells/well) were pretreated with RLS-0409(50uM) lhr prior to TLR ligand treatment. The pre-treated cells were stimulated with various TLR ligands
47

at different concentrations (TLR-1/2- 75ng/ml, TLR-3- 75|ag/ml, TLR-4 750ng/ml, TLR-5, 75ng/ml, TLR-6, 75ng/ml, TLR-7/8-7.5(.ig/ml and TLR-9-7.5(.ig/ml) for 24hr. The culture supernatant was then assayed for TNF-a secretion. Cells untreated with RLS-0409 served as controls.
Figure 2a: shows inhibition of TNF-a secretion in THP-1 cells by RLS-0409 is dose-dependent
THP-1 cells (2 xlO5 cells/well) pretreated with RLS-0409 in increasing concentrations (luM, lOuM, 50uM and 100uM) lhr prior to LPS stimulation. Cells were treated with LPS served as controls group. TNF-a secretion following 24 hr LPS stimulation was assayed in culture supernatants using a Duoset ELISA detection Kit.
Figure 2b: RSCL- 0409 is not toxic to THP-1 cells
THP-1 cells (2 xlO5 cells/well) pretreated with RLS-0409 in increasing concentrations (luM, lOuM, 50uM and 100uM) lhr prior to LPS stimulation. The viability(checked by MTT) of the cells remained unaffected indicating that RLS-0409 is non-toxic to cells.
Figure 3: LPS dose dependent effect on RSCL- 0409
THP-1 cells were stimulated with increasing concentrations on LPS (62.5ng/ml -1000ng/ml) with and without pre-treatment of RLS-0409 (luM-100uM) for 24hr. Supernatent was then assayed for ability of cells to release TNF-a. RLS-0409 suppresses TNF secretion even from cells stimulated with high dose of LPS (1000ng/ml).
Figure 4: RSCL- 0409 inhibits LPS induced TNF-a release in PBMC
PBMCs isolated from human donors were stimulated with LPS in the presence or absence of RSCL-0520. TNF-a secretion in culture supernatants was estimated as before. RSCL-0520 inhibited TNF-a in PBMCs (-57% inhibition), with TNF levels undetectable in PBMCs without LPS and with RSCL-0520 treatment alone.
Figure 5: RSCL- 0409 suppresses LPS induced TNF- a release in Balb/c mice
48

Balb/c (5 animals/group, ~20-30g) were intraperitoneally injected with LPS (225ug/ml) with and without pretreatment of RLS-0409 (100mg/kg body weight) also injected intraperitoneal 15minutes before LPS injection, lhr post LPS injection, b100d was collected retro-orbitally under anesthesia and serum analysed for TNF-a secretion. Pretreatment with RLS-0409 showed -32% in TNF-a secretion as against untreated mice, indicating its efficiency to effectively inhibit TLR induced inflammation.
Figure 6: RLS-0409 inhibits carrageenan induced foot pad edema in Balb/c
The footpads of Balb/c mice (5-6 weeks, weighing ~ 20-30g) were injected with 1% carrageenanin saline (50ul) to induce edema. 50ul of vehicle (PBS) without carrageenan served as a vehicle control. RLS-0409 (100mg/kg body weight) was injected intraperitoneally 15 minutes prior to carrageenan injection in the test animals. The footpad volume using a plethysmometer was measured after 3, 6, 24 and 48hour interval and compared with the pre-injection volume of the same paw with the untreated foot of the same mice which served as a reference. Swelling (in ul) was then calculated in control and in drug-treated animals. Inhibition was then derived through comparison with the vehicle control group. Mice pretreated with RLS-0409, showed reduced the carrageenan-induced inflammation (-57% - 24hr and - 47% - 48hr).
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term "carbohydrate based molecules" as used herein refers to molecules with basic carbohydrate backbone in pyranose ( six membered) configuration linked through a glycosidic bond.
The term "LPS" as used herein refers to Lipopolysaccharide. Lipopolysaccharide (LPS), which is contained in the outer membrane of the cell wall of various gram-negative bacteria, consists of a glycolipid called "Lipid A" to which various saccharides are bonded. It has been known for along time that LPS is the main component of endotoxins.
The term" TLR" as used herein refers to Toll like receptor.
49

The term "pharmaceutically acceptable salt," as use herein, refers to those salts which are, within the scope of sound medical, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences 66:1 19, 1977. The salts can be prepared in situ during the final isolation and purification of a compound of the invention or separately by reacting the free base group with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphersulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The term "pharmaceutically acceptable ester," as used herein, represents esters that hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic, and alkanedioic acids, in which each alkyl or alkenyl group preferably has not more than 6 carbon atoms. Examples of particular esters include formates, acetates, propionates, butyates, acrylates, and ethylsuccinates.
The term "pharmaceutically acceptable prodrugs," as used herein, means prodrugs of the compounds of the present invention which are, within the scope of sound medical
50

judgement, suitable for use in contact with the tissues of humans and animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
The term "prodrug," as used herein, represents compounds that are transformed in vivo into a parent compound of the above formula, for example, by hydrolysis in b100d. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, Edward B. Roche, ed., "Bioreversible Carriers in Drug Design," American Pharmaceutical Association and Pergamon Press, 1987, and Judkins et al., Synthetic Communications 26(23):4351 4367, 1996, each of which is incorporated herein by reference.
Asymmetric or chiral centers may exist in the compounds of the present invention. The present invention includes the various stereoisomers and mixtures thereof. Individual stereoisomers of compounds or the present invention may be prepared synthetically from commercially available starting materials that contain asymmetric or chiral centers or by preparation of mixtures of enantiometic compounds followed by resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a racemic mixture of enantiomers, designated (+/-), to a chiral auxiliary, separation of the resulting diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Enantiomers are designated herein by the symbols "R" or "S," depending on the configuration of substituents around the chiral carbon atom, or are drawn by conventional means with a bolded line defining a substituent above the plane of the page in three-dimensional space and a hashed or dashed line defining a substituent beneath the plane of the printed page in three-dimensional space. If no stereochemical designation is made, it is to be assumed that the structure definition includes both stereochemical possibilities. All of the starting materials used in any of these methods are commercially available from chemical vendors such as Aldrich, Sigma, Nova Biochemicals, Bachem
51

Biosciences, Advanced ChemTech, and the like, or may be readily synthesized by known procedures.
The reaction products are isolated and purified by conventional methods, typically by solvent extraction into a compatible solvent. The products may be further purified by column chromatography or other appropriate methods, including medium pressure or high pressure liquid chromatography.
The compounds and methods of the invention are described in further detail, as follows.
Therapeutic Use of TLR antagonist
The present invention provides agents that can be used to prevent or to treat LPS
mediated diseases or conditions that are characterized by TLR activation. The conditions that are prevented or treated but are not limited to inflammation, wounds, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer, and immunodeficiency
Delivery and dosage of the TLR antagonist:
The present invention provides compositions comprising carbohydrate based molecules
in an effective amount that achieves the desired therapeutic effect for a particular condition, patient and mode of administration. The dosage level selected depends on the route of administration and the severity of the condition being treated.
For example: For adults, the doses are generally from about 0.01 to about 100 mg/kg, desirably about 0.1 to about 1 mg/kg body weight per day by inhalation, from about 0.01 to about 100 mg/kg, desirably 0.1 to 70 mg/kg, more desirably 0.5 to 10 mg/kg body weight per day by oral administration, and from about 0.01 to about 50 mg/kg, desirably 0.1 to 1 mg/kg body weight per day by intravenous administration. Doses are determined for each particular case using standard methods in accordance with factors unique to the patient, including age, weight, general state of health, and other factors that can influence the efficacy of the compound(s) of the invention.
52

Further the administration of the compounds of the present invention is not limited to mammal, including humans, be limited to a particular mode of administration, dosage, or frequency of dosing.
The present invention encompasses all modes of administration, including oral, intraperitoneal, intramuscular, intravenous, intraarticular, intralesional, subcutaneous, or nasally, rectally, buccally, or any other route sufficient to provide a dose adequate to prevent or treat excess or undesired TLR activity.
The present invention also contemplates that one or more compounds may be administered to a mammal in a single dose or multiple doses. When multiple doses are administered, the doses may be separated from one another by, for example, several hours, one day, one week, one month, or one year. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of a pharmaceutical composition that includes a compound of the invention
The present invention provides compositions of carbohydrate based TLR antagonists which may be prepared by conventional methods using one or more pharmaceutically acceptable excipients or adjuvants which may comprise inert diluents, sterile aqueous media and/or various non toxic solvents. The pharmaceutically acceptable carrier or diluents may be used as described in literature such as Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988 1999, Marcel Dekker, New York
The compositions may be presented in the form of tablets, pills, granules, powders, aqueous solutions or suspensions, injectable solutions, elixirs, or syrups, and the compositions may optionally contain one or more agents chosen from the group comprising sweeteners, flavorings, colorings, and stabilizers in order to obtain
53

pharmaceutical^ acceptable preparations.
The choice of vehicle and the content of active substance in the vehicle are generally determined in accordance with the solubility and chemical properties of the product, the particular mode of administration, and the provisions to be observed in pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, and dicalcium phosphate and disintegrating agents such as starch, alginic acids, and certain complex silicates combined with lubricants (e.g., magnesium stearate, sodium lauryl sulfate, and talc) may be used for preparing tablets. To prepare a capsule, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used, they may contain emulsifying agents that facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol, chloroform, or mixtures thereof may also be used.
For parenteral administration, emulsions, suspensions, or solutions of the compositions of the invention in vegetable oil (e.g., sesame oil, groundnut oil, or olive oil), aqueous-organic solutions (e.g., water and propylene glycol), injectable organic esters (e.g., ethyl oleate), or sterile aqueous solutions of the pharmaceutically acceptable salts are used. The solutions of the salts of the compositions of the invention are especially useful for administration by intramuscular or subcutaneous injection. Aqueous solutions that include solutions of the salts in pure distilled water may be used for intravenous administration with the proviso that (i) their pH is adjusted suitably, (ii) they are appropriately buffered and rendered isotonic with a sufficient quantity of glucose or sodium chloride, and (iii) they are sterilized by heating, irradiation, or microfiltration. Suitable compositions containing a compound of the invention may be dissolved or suspended in a suitable carrier for use in a nebulizer or a suspension or solution aerosol, or may be absorbed or adsorbed onto a suitable solid carrier for use in a dry powder inhaler. Solid compositions for rectal administration include suppositories formulated in accordance with known methods and containing at least one compound of formula I or II.
Dosage formulations of a compound of the invention to be used for therapeutic
54

administration must be sterile. Sterility is readily accomplished by filtration through
sterile membranes (e.g., 0.2 micron membranes) or by other conventional methods.
Formulations typically are stored in lyophilized form or as an aqueous solution. The pH
of the compositions of this invention is typically between 3 and 11, more desirably
between 5 and 9, and most desirably between 7 and 8, inclusive. While a desirable route
of administration is by injection such as intravenously (bolus and/or infusion), other
methods of administration may be used. For example, compositions may be administered
subcutaneously, intramuscularly, colonically, rectally, nasally, or intraperitoneally in a
variety of dosage forms such as suppositories, implanted pellets or small cylinders,
aerosols, oral dosage formulations, and topical formulations such as ointments, drops,
and dermal patches. A compound of the invention is desirably incorporated into shaped
articles such as implants, including but not limited to valves, stents, tubing, and
prostheses, which may employ inert materials such as synthetic polymers or silicones,
(e.g., Silastic, silicone rubber, or other commercially available polymers). Such polymers
can include polyvinylpyrrolidone, pyran copolymer, polyhydroxy-propyl-
methacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-
polylysine substituted with palmitoyl residues. Furthermore, a TLR2 inhibitor of the
invention may be coupled to a class of biodegradable polymers useful in achieving
controlled release of a drug, for example polylactic acid, polyglycolic acid, copolymers
of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross linked or
amphipathic block copolymers of hydrogels.
A compound of the invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of lipids, such as cholesterol, stearylamine, or phosphatidylcholines. A compound of the invention may also be delivered using antibodies, antibody fragments, growth factors, hormones, or other targeting moieties to which the compound molecules are coupled (e.g., see Remington: The Science and Practice of Pharmacy, vide supra), including in vivo conjugation to b100d components of a compound of the formula I or II, as described herein.
55

In vitro application in identification of TLR Antagonists:
Pharmaceutical agents that can be used in the therapeutic methods of the invention can be identified in screening methods. For example, cell-based screening methods can be used, in which cells expressing TLR are contacted with a candidate agent and the impact of the agent on the activation of TLR in the cells is determined. In one example of such a method, the effect of an agent on the activation of TLR by a known ligand (e.g., a lipopeptide,) is determined. Agents that are found to decrease or to block activation of the receptor by the ligand can then be considered for further analysis and/or for use as TLR inhibitors in therapeutic methods. Activation of TLR in these methods can be measured using, for example, a reporter system. For example, cells used in the screening assay can include a reporter gene that is under the control of a promoter that is inducible by a signaling pathway triggered by TLR activation.
In addition to cell-based methods, candidate agents can be tested in animal model systems. This may be desirable, for example, if an agent has been found to have antagonist activity in a cell-based assay or to bind to TLR in an in vitro assay (see below). For example, in animal studies, test agents can be administered to an animal model concurrently with a molecule known to activate TLR (e.g., lipopeptide), and the impact of the agent on a response in the animal that is normally triggered by activation of the receptor (e.g., cytokine induction) can be determined. Further, in vitro methods can be used. For example, a candidate compound can be assayed for whether it binds to TLR or a fragment of the receptor that includes at least a portion of the ligand binding site. Such assays can be carried out using, for example, columns or beads to which the receptor or fragment is bound.
In addition to the methods described above, additional TLR antagonists can be identified in methods in which candidate compounds are compared for TLR antagonist activity with any of the TLR antagonists described herein. Further, in addition to being compared for TLR antagonist activity, the candidate compounds can be compared with TLR2 antagonists with respect to specificity for TLR versus other receptors. Candidate
56

compounds identified as having TLR antagonist activity that is, for example, similar to or greater than the activity of the antagonists described herein (and/or with similar or greater levels of specificity for TLR2 versus TLR4) in these assays can be tested further, for example, in appropriate animal model assays for any of the diseases or conditions described herein, as well as in human clinical studies.
Also included in the invention are compounds that are selective for TLR2 over TLR4, as well as compounds that are dual antagonists (i.e., antagonists of both TLR2 and TLR4). A compound that is selective for TLR2 over TLR4 is one that has, for example, an IC50 value in a TLR2 antagonist assay, such as is described herein, that is less than that found in a TLR4 antagonist assay, such as is described herein. For example, the IC50 in the TLR2 assay can be at least 5, 10, 25, or 50-fold less than the value for the same compound tested in the TLR4 assay. Compounds that are dual antagonists are those that have, for example, IC50 values that are within a 5-fold range of one another using, e.g., the assays described herein. Thus, dual antagonists include those that have activities that are 1:5 5:1 with respect to one another (e.g., 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1). The invention also includes the use of TLR2 antagonists such as those described herein in the study of physiological and molecular pathways involved in or affected by TLR2 activation (or inactivation).
Agents that can be screened using the methods of the invention include, for example, compounds that are present in compound libraries (e.g., random libraries), as well as analogs of known TLR2 ligands (e.g., lipopeptides) that are modified to prevent rather than activate TLR2. Further, peptides that correspond to the binding site of TLR2 for its ligands, which can competitively inhibit ligand binding to the binding site, can be tested. Further, antibodies or antibody fragments to the ligand or the ligand binding site of the receptor can be screened.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor
57

to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1 : GENERAL PROCEDURE FOR PREPARATION OF MONOSACCHARIDE AND DISACCHARIDE BUILDING BLOCKS FOR ALL THE SIX FORMULAS:
Synthesis of l-0-(para-methoxy phenyl)-2,3,4,6-tetra-0-acetyl-P-D-glucopyranoside
0)
Stepl
OAc
/
0Ac OAc
To a cooled solution of P-D-Glucose-pentaacetate (10g, 25.6 mmol) and 4-methoxy
phenol (4.8 g, 38.7 mmol) in dichloroethane (80 mL) was added trimethylsilyltrifluoro
-methane sulfonate (50 mL). The reaction was stirred at room temperature for four hours.
Reaction mixture was diluted with dichloromethane (50 mL), washed with saturated
aqueous sodium bicarbonate solution (2 X 100 mL) and water (2 X 100 mL). The organic
layer was dried (Na2S04), concentrated and column purified (ethyl acetate: hexane, 1:3)
to provide product 1 (11 g, 95 %); Rf 0.51 (ethyl acetate: hexane, 2:3); 'H NMR (CDC13)
5 2.03, 2.04, 2.07, 2.08 (4s, 12H), 3.77 (s, 3H), 4.16 (dd, 1H, J = 12 Hz), 4.27 (dd, 1H, J
= 5.1 Hz), 4.93 (d, 1H, J = 7.2 Hz), 6.81 (d, 1H), 6.94 (2d, 1H); ESMS m/z 477 (M+Na)+.
Synthesis of l-0-(para-methoxy phenyl)-P-D-glucopyranoside (2)
Step2
OH
/
HO^^A/OpMP HO^
OH
2
58

To a stirred solution of compound 1 (12 g, 24.2 mmol) in methanol (25 mL) was gradually added 2 mL of a 25 % (wt/v) sodium methoxide/methanol solution. The resulting solution was stirred at room temperature overnight. The mixture was neutralized by Amberlite IR 120 (H+) resin. The neutralized reaction mixture was filtered, concentrated and purified (methanol: dichloromethane, 1:4) to provide compound 2 (6.5 g, 94 %); Rf 0.55 (methanol: dichloromethane, 1: 9); 'H NMR (CD3OD) 6 3.77 (s, 3H), 4.06 (dd, 1H, J = 12 Hz), 4.27 (dd, 1H, J - 5.1 Hz), 4.52 (d, 1H, J = 7.2 Hz), 6.81 (d, 1H), 6.94 (2d, 1H); ESMS m/z 309 (M+Na)+.
Synthesis of l-0-(para-methoxy phenyl)-4,6-0-benzylidene-P-D-glucopyranoside (3)
Step3
PhMU OH
Compound 3 (6.5 g, 23 mmol) was dissolved in anhydrous N, N-dimethylformamide (100 mL). To this solution was first added p-toluene sulphonic acid (2 g) and next slowly benzaldehyde dimethyl acetal (6 mL). The reaction was stirred at room temperature overnight. N, N-dimethylformamide was removed on high vacuum and the residue was column purified (methanol: dichloromethane, 1:9) to provide compound 3 (6.5 g, 77 %); Rf 0.45 (Dichloromethane: Acetone, 5: 1); 'H NMR (CDC13) 5 3.79 (s, 3H), 4.90 (d, 1H, J = 7.7 Hz), 5.58 (s, 1H), 6.81 (d, 1H), 6.94 (2d, 1H), 7.36-7.37 (m, 2H), 7.48-7.52 (m, 3H); ESMS m/z 397 (M+Na)+.
Synthesis of l-0-(para-methoxy phenyl)-2,3-di-0-acetyl-4,6-0-benzylidene-P-D-glucopyranoside (4)
Step4

Ph.

-o-
o-
OAc-

O
OAc

OpMP

59

To a solution of 3 (19 g, 0.050 mol) in pyridine (25 mL) at 0 °C was added acetic anhydride (25 mL) dropwise. The reaction mixture was stirred at room temperature overnight and the reaction was quenched by adding ice. Ethyl acetate was added, organic layer was washed with 1 N HC1 (100 mL X 2), dried, concentrated and purified through silica gel column to give compound 4 (21 g, 91%) R(0.62 (40% ethyl acetate in hexane); 'H NMR (CDC13) 8 2.02 (s, 3H), 2.04 (s, 3H), 3.59-3.63 (m, 1H), 3.77 (s, 3H), 4.37-4.41 (dd, 1H, J = 5.2 Hz & 4.8 Hz), 5.05-5.07 (d, 1H, J = 7.7 Hz), 5.22-5.26 (t, 1 H, J =9.2, 8 Hz ), 5.36-5.41 (t, 1H, J =9.2, 9.6 Hz ), 5.53 (s, lH),6.82-6.84 (d, 2H, J = 9.2 Hz), 6.94-6.96 (d, 2H, J = 9.2 Hz), 7.35-7.37 (m, 1H), 7.43-7.47 (m, 6H); ESMS m/z 481 (M+Na)+.
Synthesis of l-0-(para-methoxy phenyI)-2,3-di-0-acetyl-P-D-glucopyranoside (5)

OpMP
HO OAc-

/

OH
O
OAc

Step5

To 4 (31.5 g, 0.068 mol) in CH2C12 (250 mL) and water (6.5 mL) was added trifluoroacetic acid (56 mL) dropwise with vigorous stirring at room temperature. The reaction mixture was stirred for 3.5 h, diluted with CH2CI2 (200 mL), washed with 10 % NaHCCh (100 mL X 2) and water (100 mL X 2), dried and concentrated. The residue was purified by column chromatography (1-3 % methanol/ CH2CI2) to give viscous product 5, (20 g, 80 %) Rf 0.26 (5:1 CH2C12: Acetone); 'H NMR (CDCI3) 5 2.07 (s, 3H), 2.11 (s, 3H), 3.51-3.55 (m, 1H), 3.77 (s, 3H), 3.82-3.88 (m, 2H), 3.93-3.97 (dd, 1H, J = 2.8 Hz & 3.2 Hz), 4.99-5.01 (d, 1H, J = 7.3 Hz), 5.08-5.18 (m, 2H), 6.81-6.83 (d, 2H, J = 8.8 Hz), 6.91-6.93 (d, 2H, J = 8.8 Hz); ESMS m/z 393 (M+Na)+.
60

Synthesis of l-0-(para-methoxy phenyl)-2,3-di-0-acetyl-6-0-levulinoyl-P-D-glucopyranoside (6)

/
OpMP
HO OAc

.OLev O
OAc

Step 6

To a solution of 5 (15 g, 0.04 mol) in CH2CI2 (150 mL) added levulinic acid (8.2 mL, 0.08 mol) and 2-chloro-l-methyl-pyridinium iodide (26.5 g, 0.102 mol). The reaction mixture was stirred at room temperature for 15 min. To it was added 1,4-diazabicyclo [2.2.2] octane (17.5 g, 0.15 mol) and the reaction was stirred for an hour. Reaction mixture was filtered through celite, diluted with CH2CI2 (200 mL) and washed with saturated NaHCCh (150 mL X 4), dried, concentrated and column purified (1-3 % methanol/ CHC13) to give product 6, (17.4 g, 90 %) Rf 0.67 (6:1 CH2C12: Acetone); lH NMR (CDCI3) 5 2.08 (s, 3H), 2.12 (s, 3H), 2.19 (s, 3H), 2.60-2.63 (m, 2H), 2.77-2.80 (m, 2H), 3.55 (s, 2H), 3.60-3.65 (m, 1H), 3.77 (s, 3H), 4.30-4.34 (dd, 1H, J = 2.4 Hz, 2.0 Hz), 4.55-4.59 (dd, 1H, J = 4 Hz, 4.4 Hz), 4.93-4.95 (d, 1H, J = 7.2 Hz), 5.09-5.20 (m, 2H), 6.80-6.83 (d, 2H, J = 9.2 Hz), 6.93-6.96 (d, 2H, J = 8.8 Hz); ESMS m/z 491 (M+Na)+.

Synthesis of l-0-(para-methoxy phenyl)-2,3-di-0-acetyl-4-0-chloroacetyl-6-0-levulinoyl-P-D-glucopyranoside (7)
/
OpMP
OLev O
OAc
ClAcO OAc-

Step7

61

To a stirred solution of 6 (2.4 g, 5.12 mmol) in dry CH2CI2 (20 mL) and dry pyridine (3.8 mL) was added chloroacetic anhydride (1.31 g, 7.69 mmol) at 0 °C. The reaction mixture was stirred at this temperature for 0.5 h and then quenched with ice. The reaction was diluted with CH2C12 (50 mL), washed with saturated NaHC03 solution (20 mL) and water (20 mL), dried, concentrated and column purified (1-2 % Acetone/ CH2CI2) to give viscous product which was crystallized from hexane/ ethyl acetate to furnish compound 7 (2.1 g, 78 %) Rf 0.74 (9:1 CH2C12: Acetone); 'H NMR (CDC13) 5 2.08 (s, 3H), 2.12 (s, 3H), 2.19 (s, 3H), 2.60-2.63 (m, 2H), 2.77-2.80 (m, 2H), 3.55 (s, 2H), 3.60-3.65 (m, 1H), 3.77 (s, 3H), 4.30-4.34 (dd, 1H, J = 2.4 Hz, 2.0 Hz), 4.55-4.59(dd, 1H, J = 4 Hz, 4.4 Hz), 4.93-4.95 (d, 1H, J = 7.2 Hz), 5.09-5.20 (m, 2H), 6.80-6.83 (d, 2H, J = 9.2 Hz), 6.93-6.96 (d, 2H, J = 8.8 Hz), ESMS m/z 568 (M+Na)+.
Synthesis of 2,3-Di-0-acetyl-4-0-chloroacetyl 6-O-levulinoyl-p-D-glucopyranoside
(8)

Step 8
OLev /
ClAcO^^ ^ OAc-

To a stirred solution of 7 (2.1 g, 3.85 mmol) in toluene: acetonitrile: water (1: 1: 1, 312 mL) was added eerie ammonium nitrate (21 g, 38.56 mmol). The reaction mixture was stirred at room temperature for 0.5 h and diluted with ethyl acetate. Organic layer was washed with NaHC03 solution (40 mL), water (50 mL), dried, concentrated and column purified (5-10 % Acetone/ CH2C12) to furnish compound 8 (1.2 g, 70 %) Rf 0.25 (9:1 CH2C12: Acetone); 'H NMR (CDCI3) 5 2.08 (s, 3H), 2.12 (s, 3H), 2.19 (s, 3H), 2.60-2.63 (m, 2H), 2.77-2.80 (m, 2H), 3.55 (s, 2H), 3.60-3.65 (m, 1H), 3.77 (s, 3H), 4.30-4.34 (dd, 1H, J = 2.4 Hz, 2.0 Hz), 4.55-4.59 (dd, 1H, J = 4 Hz, 4.4 Hz), 4.93-4.95 (d, 1H, J = 7.2 Hz), 5.09-5.20 (m, 2H), ESMS m/z 461 (M+Na)+.
62

Synthesis of 2,3-Di-O-acetyl-4-0-chIoroacetyl-6-0-levulinoyl-a-D-glucopyranosyl trichloroacetimidate (9)
Step 9
/
ClAcO' OAc-
OAc
OLev O
O
.CCL

NH
To a solution of 8 (1.2 g, 2.73 mmol) in dry CH2CI2 (14 mL) and trichloroacetonitrile (2.6 mL, 27 mmol) was added 1,8-diazabicylco [5.4.0]undec-7-ene (103 mL). The reaction was stirred at room temperature for 3 h, concentrated and the residue purified by column chromatography (1-5 % Acetone/ CH2C12) to yield donor 9 (1.27 g, 80 %) Rf 0.37 (35 % Ethyl acetate/Hexane); 'H NMR (CDCI3) 5 2.08, (s, 3H), 2.12 (s, 3H), 2.19 (s, 3H), 2.60-2.63 (m, 2H), 2.77-2.80 (m, 2H), 3.55 (s, 2H), 3.60-3.65 (m, IH), 3.77 (s, 3H), 4.30-4.34 (dd, IH, J = 2.4 Hz, 2.0 Hz), 4.55-4.59 (dd, IH, J = 4 Hz, 4.4 Hz), 4.93-4.95 (d, IH, J = 7.2 Hz), 5.09-5.20 (m, 2H), 8.72 (s, IH, NH).
Synthesis of 1,3,4,6-Tetra-0-acetyl-2-deoxy-2-trichloroacetamido-a/p-D-glucopyra-noside (10)
63
Step 10


Trichloroacetyl chloride (8.4 mL, 75 mmol) was added dropwise at room temperature to a vigorously stirred solution of D-glucosamine hydrochloride (10.78 g, 50 mmol) and sodium bicarbonate (12.6g, 150 mol) in water (100 mL). The mixture was stirred for 1 hour, neutralized with M HC1, concentrated, and dried in vacuo. The residue was stirred for 2 hours at 0 °C with methanol (100 mL), the salts were filtered off, and the filtrate was concentrated. Crystallization of the residue from cold water afforded 8 g of 2-deoxy-2-trichloroacetamido-D-glucopyranose, which was dissolved in pyridine (80 mL). The solution was cooled to 0 °C, acetic anhydride (50 mL0 was added dropwise. The reaction was stirred at room temperature overnight, then concentrated. A solution of the residue was filtered through celite and concentrated to give 9 g of compound 10 as a mixture of a and p anomers in the ration of 9: 1 Rf 0.42 (35 % Ethyl acetate/Hexane); 'H NMR (CDC13) 5 2.04 (s, 3H), 2.10 (s, 3H), 2.18 (s, 3H), 4.04(m, 1H), 4.08 (dd, 1H, J = 2.4 Hz), 4.28 (dd, 1H, J = 4.5 Hz & 13 Hz), 4.34 (m, 1H), 4.35-4.41 (m, 1H), 5.24 (t, 1H, J = 9.5 Hz), 5.36 (dd, 1H, J = 10.5 Hz & 9.5 Hz), 5.81 (d, 1H, J = 8.5 Hz), 6.31 (d, 1H, J = 3.6 Hz), 6.80 (d, 2H, J = 8.5 Hz)
Synthesis of l-0-(p-methoxy phenyl)-2-Deoxy-2-trichloroacetamido-3,4,6-tri-0-
acetyl-a-D-glucopyranoside (11)
Stepl

OAc O

O
OpMP CCL
'3
To a stirred solution of l,3,4,6-tetra-0-acetyl-2-deoxy-2-trichloroacetamido-a/(3-D-glucopyranoside3'4 10 ( 10 g, 20.32 mmol) and p-methoxy phenol (7.56 g, 60.97 mmol) in dry CH2CI2 at 0 °C was added TMSOTf (4.4 mL, 1.2 eq). The reaction was stirred at room temperature overnight, diluted with CH2CI2. Organic layer was washed with saturated NaHC03 (200 mL X 2) and water (200 mL X 2), dried, concentrated and purified by column chromatography to give compound 11 (9.1 g, 80 %) Rf 0.47 (35 %
64

Ethyl acetate/Hexane); 'H NMR (CDC13) S 2.06 (s, 6H), 2.08 (s, 1H), 3.78 (s, 3H), 4.09-4.1 (d, 1H, J = 2.4Hz), 4.13-4.12 (d, 1H, J = 2.4 Hz), 4.17-4.21 (m, 1H), 4.26-4.30 (dd, 1H, J = 4Hz, 4.4Hz), 4.35-4.41 (m, 1H), 6.83-6.85 (d, 2H, J = 9.2 Hz), 7.01-7.03 (d, 2H, J = 9.2 Hz), 7.09-7.07 (d, NH, J = 9.2 Hz); ESMS m/z 575 (M+NH4)+.
Synthesis of l-0-(p-methoxy phenyl)-2-deoxy-2-trichloroacetamido-a-D-glucopyra-noside (12)
Step 12
OH
"A
0=y OPMP cci3 12
A solution of compound 11 (9 g, 16.10 mmol) in dry methanol (100 mL) was treated with methanol sodium methoxide (0.1 N, 10 mL) for 4 h at room temperature. The mixture was then neutralized with Amberlite IR-120 (H+) resin, filtered, concentrated and column purified (10 % methanol/ CH2C12) to give compound 12 (6.6 g, 95 %), Rf 0.23 (10 % methanol/ CH2C12); *H NMR (CD3OD) 5 3.74 (s, 3H), 3.75-3.84 (m, 4H), 3.96-3.95 (d, 1H, J = 3.6 Hz), 3.99-3.98 (d, 1H, J = 3.2 Hz), 4.02-4.07 (m, 1H), 5.38-5.39 (d, 1H, J = 3.2 Hz), 6.82-6.84 (d, 2H, J = 9.2 Hz), 7.05-7.07 (d, 2H, J = 9.2 Hz); ESMS m/z 454 (M+Na)+.
Synthesis of l-0-(p-methoxy phenyl)-2-deoxy-2-trichloroacetamido-4,6-0-benzyIid-ene-a-D-glucopyranoside (13)
65
Step 13


13
To a solution of 12 (5 g, 11.62 mmol) in dry DMF (15 mL0 and dry THF (47 mL) was added Benzaldehyde dimethyl acetal (3.5 mL, 23.25 mmol) and p-toluene sulphonic acid (catalytic amount). The reaction mixture was stirred at room temp overnight. The mixture was neutralized with triethylamine, concentrated and purified using column chromatography to give compound 13 (5.4 g, 90 %) Rf 0.64 (40 % ethyl acetate/hexane); 'H NMR (CDC13) 5 3.67-3.72 (t, 1H, J = 9.2 Hz), 3.78 (s, 1H), 4.02-4.09 (m, 1H), 4.22-4.34 (m, 2H), 5.50-5.49 (d, 1H, J = 3.2 Hz), 5.59 (s, 1H), 6.83-6.85 (d, 2H, J = 9.2 Hz), 6.98-7.00 (d, 2H, J = 9.2 Hz), 7.04-7.06 (d, NH, J = 8.4 Hz), 7.39-7.66 (m, 5H); ESMS w/z519(M+H)+.
Synthesis of p-Methoxy phenyl-0-(2,3-di-0-acetyl-4-0-chloroacetyl-6-0-levu!inoyI-p-D-glucopyranosyl)-(l-3)-2-deoxy-2-trichloroacetamido-4,6-0-benzylidene-a-D-glucopyranoside (14)
Step 14


-o-
o o-
O
O

ClAcO OAc-

Ph\ OLev
OAc

O
NH

OpMP

14
Compound 13 (600 mg, 1.15 mmol) and 9 (1.17 g, 2.02 mmol, 1.75 eq,) were dissolved in anhydrous CH2CI2 (14 mL). Activated powdered molecular sieves were added and the reaction mixture was stirred at RT for 15 min. The temperature was lowered to 0 C, TMSOTf solution (10.5 mL, 0.057mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 30 min after which TLC showed compete disappearance of the donor. The reaction was quenched with Et3N and filtered through celite and the filtrate concentrated. The residue was purified using silica gel column (40% ethyl acetate in
66

hexane) to give disaccharide 14 (975 mg, 91 %) Rr 0.62 (50% ethyl acetate/ hexane); 'H NMR(CDC13) 5 1.97 (s, 3H), 2.03 (s, 3H), 2.14 (s, 3H), 2.40-2.48 (m, 211), 2.55-2.65 (m, 2 H), 2.74-2.82 (m, 2H), 3.63-3.68 (m, 2H), 3.76 (s, 3H), 3.82-3.84 (m, 3H), 3.95 (s, 1H), 3.97 (s, 1H), 4.00-4.14 (m, 3H), 4.19-4.18 (d, 1H, J = 2.8 Hz), 4.21-4.26 (m, 2H), 4.39-4.46 (m, 3H), 4.84-4.86 (m, 1H), 5.00-5.05 (m, 2H), 5.10-5.15 (m, 1H), 5.37 (d, 1H, J = 2.8 Hz), 5.60 (s, 1H), 6.80-6.83 (d, 2H, J = 9.2 Hz), 6.91-6.95 (d, 2H, J = 9.2 Hz), 7.34-7.37 (m, 3H), 7.50-7.53 (m, 2H); ESMS m/z 957 (M+NH4)+.
EXAMPLE 2: In vitro testing:
The inhibition of TNF release in response to TLR ligand binding was studied. The compounds of the present invention further selectively blocks TLR 4 and TLR2 mediated TNF release.
a) Effect of RSCL- 0409 (compound 14)on various TLR ligands:
Toll-like receptors (TLRs), which have pathogen-associated molecular patterns, play important roles in innate immunity in mammals. Recently, at least 10 members of the TLR family have been identified, and several ligands recognized by TLRs have been reported. Activation of these TLRs leads to the activation of many inflammatory responses and as results recruitment of numerous signaling molecules which further activate a range of transcription factors.
The present invention has checked the effect of various TLR ligands on THP-1 mono cytes and their ability to activate and release TNF-a. For this purpose about 9 TLR ligands were used. These ligands were obtained from Apotech Cat; APO-54N-018-KI01 and assayed for TNF-a release in culture supernatants.
THP-1, 2 xlO5 cells/well was plated in 96-well plate. The cells were pretreated with RLS-0409(50uM) in DMSO lhr prior to TLR ligand treatment. Following lhr pre-treatment, the cells were treated with TLR ligands (TLR-1/2- 75ng/ml, TLR-3- 75|.ig/ml, TLR-4 750ng/ml, TLR-5, 75ng/ml, TLR-6, 75ng/ml, TLR-7/8-7.5ug/ml and TLR-9-7.5|.ig/ml)
67

for 24hrs. The culture supernatant were collected after the stipulated time and assayed for TNF-a release using a Duoset ELISA detection Kit (R&D systems; Cat: DY-210 E). Simultaneously supernatants were collected from cells treated with ligands without pre-treatment with RLS-0409 and only RLS-0409 without ligand treatment.
It was observed that ((Figure 1) TNF-a secretion following stimulation with TLR-1/2, TLR-4 and TLR-6 ligands. Treatment with RLS-0409 inhibited the TNF-a secretion in TLR-1/2 treated cells (~ 54% inhibition) and TLR-4(~100% inhibition). However, there was no effect in cells treated with TLR-6 ligand, indicating that RLS-0409 is a potential inhibitor for signaling mechanisms induced by LPS or lipoproteins which signal through the above mentioned TLRs.
b) Effect of RSCL- 0409 on LPS Induced TNF-a release in THP-1 cells
To confirm whether the inhibitory effect of RLS-0409 is dose-dependent, the present invention has studied the ability of RLS-0409 to inhibit TNF-a secretion from LPS (250ng/ml) induced THP-1 monocytes.
THP-1, 2 xlO5 cells/well was plated in 96-well plate. The cells were pretreated with RLS-0409 at various concentrations (100um, 50uM, lOuM and luM) lhr prior to LPS stimulation. As a control group, cells were treated with LPS alone and cells treated with RLS-0409 alone were used. TNF-a secretion was estimated in the culture supernatants following 24hr LPS stimulation using Duoset ELISA detection Kit (R&D systems). The toxicity of RLS-0409 was also analysed in tandem by treating cells with RLS-0409 with and without LPS by MTT assay.
LPS induced TNF-a secretion was inhibited by RLS-0409 in a concentration dependent manner. The viability of the cells was not affected by RLS-0409 indicating its non-toxic nature.
68

c) EFFECT OF RLS-0409 ON TNF RESPONSE TO INCREASING
CONCENTRATION OF LPS
The present invention has also conducted experiments to check the ability of RLS-0409 to inhibit LPS induced TNF-a secretion from THP-1 cells, wherein the studies involved the stimulation of THP-1 cells with increasing concentrations of LPS (62.5ng/ml to 1000ng/ml) with and without pre-treatment of cells with different concentrations of RLS-0409.
It is clearly evident ( figure 2) that with an increased secretion of TNF-a with increasing concentration of LPS is inhibited by RLS-0409 to varied extents on a concentration dependent manner. Further RLS-0409 has the ability to inhibit LPS (1000ng/ml) induced TNF-a secretion at concentration as low as lOuM, but -100% inhibition was observed at 100uM indicating its inhibitory potential.
d) Effect of RSCL- 0409 on LPS induced TNF- release in human PBMC
In order to evaluate the ability of RLS-0409 to inhibit LPS induced TNF-a secretion in a physiological scenario, the present invention has tested the same in PBMC isolated from human b100d.(figure 3)
The results indicated similar inhibitory effects observed in PBMCs also. The TNF levels were not detectable in PBMCs without LPS and with RLS-0409 treatment alone, indicating its specificity in LPS induced TNF-a through a TLR mediated process.
EXAMPLE 5 : In vivo testing
a) Effect of RSCL- 0409 on LPS induced TNF- a release in Balb/c mice
The present invention has studied the ability of RLS-0409 to exert protection against inflammatory agents in a mice (Balb/c) model. Balb/c (5 animals per group) mice were injected with LPS (225ug/ml) intraperitoneally with and without pretreatment of RLS-0409 (100mg/kg body weight) 15minutes before LPS treatment. B100d collection was
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done retro-orbitally under anesthesia. Serum collected after cells were allowed to settle was analysed for TNF-a secretion.
lhr post LPS injection the secretion of large amounts on TNF-a was seen in the serum. The secretion of TNF-a is inhibited to significant levels (-32%) when the mice were pre-treated with RLS-0409 indicating its efficiency to effectively inhibit TLR induced inflammation.(figure 4)
b) RLS-0409 inhibits carrageenan induced foot pad edema in Balb/c
The anti-inflammatory effect of RLS-0409 was checked in mice by inducing local inflammatory reaction by footpad injection of carraggenan into the left hind paw. Balb/c mice of 5-6 weeks age weighing ~ 20-30g are taken for the experiment. Carrageenan( 1 %) in saline (50)ul was injected to the mouse footpad to induce edema. As a vehicle control, 50ul of vehicle without carrageenan was injected. RLS-0409 (100mg/kg body weight) was injected intraperitoneally 15 minutes prior to carrageenan injection. The footpad volume was measured after 3, 6, 24 and 48hour interval using a plethysmometer (Ugo Basile Model 7140) and compared with the pre-injection volume of the same paw. The non-treated foot of the same mice is used as a reference. Swelling (in ul) was then calculated in control and in drug-treated animals. Inhibition was then derived through comparison with the vehicle control group.
Analysis revealed that (figure 5) in the mice pretreated with RLS-0409, the carrageenan-induced inflammation was reduced (~57.5%-24hr and ~47%-48hr) and the reduction was effective over a 48hr period. Further the present invention has also studied the ability of RSCL to reduce inhibition of footpad edema.over 120hr time period and found similar results as above.
REFERENCES:
1. Slaghek, T.M. et al. "Synthesis of Hyaluronic acid-related di-, tri-, and tetra-saccharides having an N-acetylglucosamine residue at the reducing end," Carbohydrate Research 1994, pp. 61-85, vol. 255.
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2. Jacquinet, J.C. et al. "The use of 2-deoxy-2-trichloroacetamido-D-glucopyranose derivatives in synthesis of oligosaccharides," Carbohydrate Research 1994, pp. 189-202, vol. 260.
3. Slaghek, T.M. et al. "Synthesis of Hyaluronic acid-related di- and tetra-saccharides having a glucuronic acid at the reducing end," Tetrahedron Asymmetry 1994, pp. 2291-2301, vol. 5.
4. Linhardt, R.J. et al. "Recent chemical and enzymatic approaches to the synthesis of glycosaminoglycan oligosaccharides," Current Medicinal Chemistry 2003, pp. 1993-2031, vol. 10.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

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ABSTRACT:
The invention provides carbohydrate based molecules, methods of preparation, and composition, useful for modulating signaling through Toll-like receptors. The methods involve contacting a TLR-expressing cell with a carbohydrate based molecules having a core structure comprising of one or more sugar molecules. These carbohydrate based moleculesare useful specifically for inhibiting immune stimulation involving TLR ligands, especially TLR-4, 6 and 2. The methods may have use in the treatment of inflammation, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer, and immunodeficiency.
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