FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
'TLR SIGNALING ANTAGONIST
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:-
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FIELD OF THE INVENTION
The present invention relates to novel molecules, compositions and methods for preparation and modulation of immune functions mediated through Toll-like receptor (TLR) signaling.
BACKGROUND OF THE INVENTION:
The innate or natural immune system recognizes a wide spectrum of pathogens without a need for prior exposure. Cells of the innate immune system effectively prevent free growth of bacteria within the body; however, many pathogens have evolved mechanisms allowing them to evade the innate immune system, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. Innate immune systems provide immediate defense against infection, and are found in all classes of plant and animal life. The main cells responsible for innate immunity are monocytes /macrophages and neutrophils, phagocytose microbial pathogens and are responsible for triggering 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 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
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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' antimicrobial killing mechanisms and antigen presenting capacity. The function of the TLRs was discovered by Beutler and colleagues (Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B. Science. 1998 Dec 11;282(5396):2085-8.) 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 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. (Du,X., Poltorak,A., Wei,Y., and Beutler,B. 2000. Three novel mammalian toll-like receptors: gene structure, expression, and evolution. Eur. Cytokine Netw. 11:362-371; Chuang,T.H., and Ulevitch,R.J. 2000. Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur. Cytokine Netw. 11:372-378 ; Tabeta,K., Georgel,P., Janssen,E., Du,X., Hoebe,K., Crozat,K., Mudd,S., Shamel,L., Sovath,S., GoodeJ. et al 2004. Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc. Natl Acad. Sci. U. S. A 101:3516-3521.)
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.
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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 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);
The US patent application number 20070167409 is based on discovery that animals that do not express Toll-like receptor 2 (TLR2) are protected from dextran sulfate sodium (DSS) induction of colitis, a model for inflammatory bowel disease (IBD, the invention relates to the agents that block activation of TLR2 to treat or to prevent colitis and related diseases or conditions, as well as other diseases or conditions characterized by activation of TLR2.
The European application EP1635846 methods and compositions useful for modulating signaling through Toll-like receptors that involve contacting a TLR-expressing cell with a small molecule having a core structure including at least two rings. Certain of the compounds are 4-primary amino quinolines.
The US patent application US20060058365 relates to the treatment of inflammatory bowel disease (IBD) and related gastrointestinal pathologies that are cytokine-mediated or associated with Toll-like receptor 4 using methimazole derivatives and tautomeric cyclic thiones.
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Further US patent application 20050004144 provides a broad-spectrum, long-lasting, and non-toxic combination of synthetic immunostimulatory agents, which are useful for activating the immune system of a mammal and treating diseases such as cancer and autoimmune disease involving 7-substituted, 8-substituted and 7,8-di substituted 7-deazaguanosines.
The US patent application 20030139364 involves administration of an imidazoquinoline agent in combination with another therapeutic agent in synergistic amounts to enhance ADCC, stimulate immune responses and/or patient and treat certain disorders.
The inventors of the present invention have designed novel phenanthrene derivatives that act as antagonist of Toll like receptor and its methods and compositions for modulating the immune functions through Toll like receptor.
The novel phenanthrene derivatives of the present invention have found its potential in inhibiting signaling of toll like receptor.
Different phenanthrene derivatives that have been used in the prior art are discussed
below:
The PCT patent application WO 2006027345 discloses novel 3-Thia-10-aza-
phenanthrene derivatives as novel effective PDE4 inhibitors.
The PCT application WO2006089881 has described novel phenanthrene derivatives as antiinflammatory agents and WO 9113855 has proivided a new phenanthrene derivative having IL-1 inhibiting activity and useful for the treatment of chronic inflammatory diseases.
US patent 3683091 specifically provides di-7-hydroxy or methyl-2,3,4,4a,9,10-hexahydrophenanthren-2-one and 4a-alkyl derivatives, useful as specific anti-acne agents.
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UK patent GB1069067 provides novel phenanthrene derivatives having analgesic and morphine antagonistic activity.
US patent 476678 discloses phenathrene derivatives possessing valuable fungicidal properties useful in agriculture, horticulture and other antifungal compositions.
US patent 4808625 discloses aminoalkanol derivatives of containing a polycarbocyclic aromatic ring system such as phenanthrene, as biocidal agents, particularly antitumor agents.
Japanese patent JP 8067626 discloses hydrogenated condensed ring hydrocarbons such as hydrogenated phenanthrene (e.g. 1,2,3,4,5,6,7,8-octahydrophenanthrene), a hydrogenated anthracene (e.g. 1,2-dihydroanthracene), a hydrogenated naphthalene (e.g. 1,2- dihydronaphthalene), etc as capable of inhibiting the carcinogenesis induced by a carcinogenic organic compound without accompanied by side effects
UK patent GB2186570 provides 9, 10-dihydrophenanthrene derivatives which are useful in treating diseases characterised by an immunological imbalance and bacterial and viral infections in mammals.
Certain phenanthrene derivatives from plants have also been used in the prior art for immune system disorders such as 9,10- dihydrophenanthrene called eulophiol isolated from the tubers of Eulophia nuda [ Bhandari , et.al. A 9,10-dihydrophenanthren from tubers of Eulophia nuda; Phytochemistry (oxford) 22(3): 747-748, 1983} and [Tuchina et al, Phenanthrenes fo Eulophia nuda , Phytochemistry, Vol 27, No 10, 3267-3271, 1988]
Japanese patent JP 7267895 has provided plant extracts containing phenanthrene derivatives from Raikoutou ( a root or leaf of Tripterygium wilfordii Hook. F.) a Chinese herbal drug. These compounds have been found to be useful as a therapeutic agent for diseases owing to leukotriene of pollinosis, bronchial asthma, arthritis etc.
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A number of phenanthrene derivatives have been extracted as a principal constituent from plant species Orchidaceae family has been reported [Majumdar et.al, Pendulin, a polyoxygenated phenanthrene derivative from the Orchidaceae Cymbidum pendulum, Phytochemistry Vol 30, 2432-2434, 1991], [Shimuzu et.al. , Anti-inflammatory constituents of topically applied crude drugys III Constituents and anti-inflammatory effect of Paraguayan crude drug of Tamada cuna, Chem Pharm. Bulletin., 36 (11), 447-4452, 1988], these extracts have been uses as antipyretic, antioxidant and antispasmodic Agents.
The PCT application WO 2006089881 has focused on methoxy phenanthrene derivatives from Tamus communis as anti-inflammatory agents.
The past decade has seen an explosion in TLR antagonist research, including their potential implication in auto-immune and chronic inflammatory diseases. The present invention focuses on novel derivatives of phenanthrenes for potential use in inhibition of immune stimulation involving toll like receptor ligands. These molecules have been developed for potential use in treatment of inflammation, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer and immunodeficiency. 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
OBJECT OF THE INVENTION
It is the object of the present invention to provide small molecules for modulation of immune functions through Toll like receptors.
It is the object of this invention to provide small molecules for inhibitions of TLR signaling in response to TLR ligands.
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It is the object of the present invention to provide phenanthrene derivatives and its analogs for modulation of the immune functions through toll like receptors.
It is the object of the present invention to provide methods of preparation or isolation of these phenanthrene derivatives and its analogs.
It is the object of the present invention to provide compositions of the phenanthrene derivatives and its analogs.
It is the object of the present invention to provide compositions useful for the prevention or treatment of inflammation, wounds, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer and immunodeficiency.
It is also the object of this invention to provide a method and composition for affecting the TLR mediated signaling in response to a TLR ligand.
It is the object of the present invention to provide phenanthrene based molecules for inhibition of TLR ligand, which can be used in combinations with other agents.
SUMMARY OF THE INVENTION
The present invention relates to phenanthrene derivatives; 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) molecules.
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 or the compositions are useful as for inhibition of TLR signaling in response to TLR ligands.
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In the preferred embodiments the compositions for inhibition of TLR signaling as described in the present invention 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 and the method of manufacture 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.
In yet other embodiments, the compositions as described in the present invention can be used to prevent or treat clinical manifestations and diseases caused by microbial pathogens
In yet other embodiments on basis of inhibition of TLR signaling properties, the composition can be used in veterinary medicine to prevent or treat clinical manifestations and diseases caused by microbial pathogens.
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.
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
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immunity and acquired immunity. Since innate immunity is believed to initiate and support acquired immunity, the combination intervention is synergistic
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.
In another embodiment, the invention provides a method of affecting TLR-mediated immunostimulation in a subject.
Methods of treatment for variety of conditions involving autoimmunity, inflammation, allergy, asthma, graft rejection, graft-versus-host disease (GvHD), infection, sepsis, cancer, and immunodeficiency. Generally, methods useful in the treatment of conditions involving infection, cancer, and immunodeficiency will employ 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.
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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 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, the present invention provides compounds which can be isolated from plant species such as Eulophia. Preferably these base compound eulophiol (RSCL- 0520) is extracted and further derivatives can be prepared by synthetic routes.
In another aspect the present invention also provides compounds, which can be prepared by synthetic routes.
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

in which R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are identical or different and may each be hydrogen, hydroxy, C1-6 alkoxy, C1-6 alkyl, halogen, haloalkyl, acyloxy, hydroxyalkyl, alkenyl, alkenyloxy, carboxyl, carbalkoxy, carbamido, a conjugated group, substituted or
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unsubstituted phenyl, substituted or unsubstituted heterocyclic group, nitro, amino, acylamino, dialkylamino, nitric oxide ( NO ) - releasing moiety, pharmaceutically acceptable salts, amides and esters thereof. Ring A, Ring B, and Ring C may be aliphatic or aromatic.
Some of the compounds of the present invention but not limited to the above general formula are listed below :
1. 2, 7-dihydroxy-3,4-dimethoxyphenanthrene.
2. 2, 7-diacetoxy-3,4-dimethoxyphenanthrene
3. 2,3,4,7-tetramethoxyphenanthrene.
4. 1,5-dihydroxy-2,7-dimethoxy-9,10-dihydrophenanthrene
5. l,5-diacetoxy-2,7-dimethoxy-9,10-dihydrophenanthrene (RSCL 0520)
6. 3,4-dimethoxyphenanthrene-2,7-bis-[(2E)-3-[3,4-bis(acetyloxy)phenyl]acrylate.
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: illustrates that RSCL-0520 inhibits TLR-4 induced TNF-(secretion Cells untreated with RSCL-0520 served as controls
Briefly, THP-1 cells (2 x l05 cells/well) were pretreated with RSCL-0520(50uM) 1hr prior to TLR ligand treatment. Following pre-treatment, the cells were treated with various TLR ligands at different concentrations (TLR-1/2- 75ng/ml, TLR-3- 75(g/ml, TLR-4 750ng/ml, TLR-5, 75ng/ml, TLR-6, 75ng/ml, TLR-7/8-7.5(g/ml and TLR-9-7.5(g/ml) for 24hr. The culture supernatant was then assayed for TNF-( secretion. Cells untreated with RSCL-0520 served as controls. Treatment with RSCL-0520 inhibited TNF-( secretion by -50%.
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Figure 2: illustrates the inhibition of TNF-( secretion in THP-1 cells by RSCL-0520 is dose- dependent
THP-1 cells (2 x105 cells/well) pretreated with RSCL-0520 in increasing concentrations (1uM, 10uM, 50uM and 100uM) lhr prior to LPS stimulation. Cells were treated with LPS served as controls group. TNF-( secretion following 24 hr LPS stimulation was assayed in culture supernatants using a Duoset ELISA detection Kit. LPS induced TNF-( secretion was inhibited by RSCL-0520 in a concentration dependent manner with viability of the cells remaining unaffected indicating that RSCL-0520 is non-toxic to cells.
Figure 3: illustrates LPS dose dependent effect on RSCL-0520
THP-1 cells were stimulated with increasing concentrations on LPS (31.25ng/ml -1000ng/ml) with and without pre-treatment of RSCL-0520 (1uM-100uM) for 24hr. Supernatent was then assayed for ability of cells to release TNF-(. RSCL-0520 suppresses TNF secretion even from cells stimulated with high dose of LPS (1000ng/ml) exhibiting its high potency.
Figure 4: illustrates that RSCL- 0520 inhibits LPS induced TNF-( release in PBMC) PBMCs isolated from human donors were stimulated with LPS in the presence or absence of RSCL-0520. TNF-( secretion in culture supernatants was estimated as before. Results obtained were similar to our observations in THP-1 cells. RSCL-0520 inhibited TNF-( in PBMCs (-63% inhibition), with TNF levels undetectable in PBMCs without LPS and with RSCL-0520 treatment alone, indicating its specificity in inhibiting LPS induced TNF-( through a TLR mediated process.
Figure 5: illustrates that RSCL- 0520 suppresses LPS induced TNF- ( release in Balb/c
mice)
Balb/c (5 animals/group, -20-30g) were intraperitoneally injected with LPS (225ug/ml)
with and without pretreatment of RSCL-0520 (l00mg/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-( secretion.
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Pretreatment with RSCL-0520 showed -30% in TNF-( secretion as against untreated mice, indicating its efficiency to effectively inhibit TLR induced inflammation.
Figure 6: illustrates that RSCL-0520 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. RSCL-0520 (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 (1) was then calculated in control and in drug-treated animals. Inhibition was then derived through comparison with the vehicle control group. Mice pretreated with RSCL-0520, showed reduced the carrageenan-induced inflammation (-80% - 24hr and - 100% - 48hr) and the reduction was effective over a 120hr time period indicating its long term effectiveness.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term "synthetic small molecules" as used herein refers to molecules with basic
phenanthrene backbone.
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.
The term "pharmaceutically acceptable salt," as use herein, refers to those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues
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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, suberate, 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. Amides and esters could also be prepared by coupling the compounds of the present invention with phenocli acids such as Non- steroidal anti-inflammatory drugs (NSAIDs) etc.
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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 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.
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COMPOUNDS OF THE PRESENT INVENTION:
The present invention relates to the compounds of formula (I) and derivatives thereo including but not limited to polymorphs, isomers and prodrugs thereof, geometric or optical isomers thereof, and pharmaceutical ly acceptable esters, ethers, carbamates of such compounds, all solvates and hydrates thereof and all salts thereof.
Particularly the present invention provides the compounds of formula I which are represented by structure numbers as follows
A compound represented by the formula (I):

in which R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10 are identical or different and may each be hydrogen, hydroxy, C1-6 alkoxy, C1-6 alkyl, halogen, haloalkyl, acyloxy, hydroxyalkyl, alkenyl, alkenyloxy, carboxyl, carbalkoxy, carbamido, a conjugated group, substituted or unsubstituted phenyl, substituted or unsubstituted heterocyclic group, nitro, amino, acylamino, dialkylamino, nitric oxide ( NO ) - releasing moiety, pharmaceutical^ acceptable salts, amides and esters thereof. Ring A, Ring B, and Ring C may be aliphatic or aromatic.
The compounds of the present invention may contain asymmetric or chiral centers, and therefore may exist in different stereoisomeric forms. All suitable optical isomers and stereoisomeric forms of the compounds of the present invention as well as mixtures thereof, including racemic mixtures, form part of the present invention. In addition, the present invention embraces all geometric and positional isomers. Moreover, some
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compounds of the present invention may exhibit polymorphism. The present invention includes all polymorphic forms of the compounds according to the invention, which forms the further aspect of the invention. It is to be understood that the present invention encompasses any and all racemic, optically-active, polymorphic and stereoisomeric forms, or mixtures thereof, which form or forms possess properties useful in the treatment of the conditions indicated herein.
Furthermore, the present invention also include isotopically-labeled compounds of the present invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
PREPARATION:
All of the starting materials used in any of these methods are commercially available
from chemical vendors such as Aldrich, Sigma, Nova Biochemicals, Bachem
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:
Synthesis of TLR antagonists:
A general method of synthesis includes the preparation of phenanthrene molecules by any of the following approaches:
A mixture of substituted benzaldehyde, 3,4,5-trimethoxyphenyl acetic acid, acetic anhydride and triethylamine is refluxed and then acididied to obtain acid (A) which is crystallized from suitable solvent. The acid (A) obtained is mixed with quinoline and copper chromite and refluxed under inert atmosphere and then precipitated with Ethyl
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acetate. After filtration, the filtrate is washed with dilute hydrochloric acid, water, and brine, dried (sodium sulphate), and evaporated under reduced pressure to isolate the product. (B) A stirred solution of the product obtained (B) and iodine in ethanol is irradiated at 254 nm. The solution is then evaporated under reduced pressure and the product (C) is isolated by silica gel column chromatography. (Pet-ether-ethyl acetate gradient) SCHEME 1:

2. An alternate approach to the above reaction is suzuki coupling of the appropriate reactants 1 and 2 gives a biaryl dialdehyde 3. The compound 3 on wittig olefination, and ring closing metathesis of the resultant diene 4 with the Grubbs second-generation ruthenium catalyst gives 5. SCHEME II



CHO R

3. An alternate approach is Pschorr synthesis wherein substituted nitroaldehyde, 3,4,5-trimethoxyphenyl acetic acid, acetic anhydride and triethylamine are refluxed. The mixture is then acidified and stirred. The precipitated solid is filtered off and washed with hot water, leaving acid (A). A solution of the acid (A) in 5N ammonium hydroxide
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is added to a slurry of iron (II) sulphate, and concentrated ammonium hydroxide at 80-90°C. After heating, the product is filtered and the precipitate is washed with 5N-ammonium hydroxide. The cooled filtrate is acidified with glacial acetic acid and the precipitate is filtered off and vacuum dried (B). To a mixture of amine (B), ethanol and 5N-hydrochloric acid at 0°C is added a 50% ethanolic solution of isopentyl nitrite. The mixture stirred and then diluted with water, further copper powder is added and stirred at 50°C. The cooled mixture is then extracted with ethyl acetate, which on concentration under reduced pressure gives acid (C). A mixture of acid (C), quinoline and basic copper carbonate is refluxed, and extracted with ethyl acetate. The ethyl acetate layer is washed with acid, alkali, and evaporated. The product (P) is purified by column chromatography on silica ( Pet-ether-ethyl acetate gradient). SCHEME III

R, R2

MeO
MeO

Isolation of RSCL-520 (Eulophial) from plant material:
Alternatively the compounds may be isolated from plants species such as Eulophia. Eulophias are distributed in tropics, numerous in Africa. There are about 50 species occurring all over the world. Eulophia flava (Lindl.) Hook.f. (Synonym: Crytopera flava Lindl.) Family: Orchidaceae, distributed in west to Central tropical Himalaya, Palamau, Ranchi, Singhbhum, Dehra Dun and Satpuda mountain ranges of Nandurbar district, Maharashtra. Normally, it grows as undergrowth in dry deciduous forest associated with Dendrocalamus strictus Nees in huge patches & on slope where soil is deep and moist. E. flava are terrestrial herbs with scapes attaining over 45-60 cm. Rhizomes tuberous, growing in horizontal chain up to 20, ca 4.8 x 3.5 cm ovoid, conical and white-green in
20

color with horizontal irregular markings. Pseudostem 10-22 cm long green. Leaves 2-5, arising along with scape, mature leaves are always 3, largest at the top, ca 3-10 x 15-20 cm, oblong-lanceolate, acute, entire, many nerved, glabrous. Racemes with 12-20 flowers clustered along the end of the scape. Bracts linear-lanceolate, acute, membranous, whitish. Flowers yellowish-green, 1-1.5 cm across. Lip 3 lobed with wavy margin. Anthers with bifid top and long anterior process. Capsule ca 3.0 x 0.8 cm, broadly ovoid, deflexed, ridged, green. Seeds black. Flowering and fruiting time is June to July. Pawra tribals of Toranmal region, Nandurbar, Maharashtra eat raw tubers for rejuvenating and aphrodisiac properties and tuber juice is also used for curing rheumatism.
The tubers from Eluphia flava is collected and powedered, subjected to cold extraction using various solvents from non polar to polar. Further the extracts on evaporation yields various residues which is subjected to chromatographic separation to islated eulophiol and nudol
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 intlammatory bowel disease (IBD), sepsis, periodontal disease, mucositis, acne, cardiovascular disease, chronic obstructive pulmonary disease, arthritis, cystic fibrosis, bacterial-induced infections, viral-induced infections, mycoplasma-associated diseases, post herpetic neuralgia, ischemia/reperfusion injury, asthma, stroke, brain injury, necrotizing enterocolitis, bed sores, leprosy, atopic dermatitis, psoriasis, trauma, neurodegenerative disease, amphotericin B-induced fever and nephritis, coronary artery bypass grafting, and atherosclerosis.
Delivery and dosage of the TLR antagonist:
The present invention provides compositions comprising carbohydrate based molecules
21

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.
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.
22
The present invention provides compositions of phenanthrene derivatives being 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 pharmaceutically 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
23

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 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
24

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.
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
25

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 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
26

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 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.
The invention will now be illustrated with the aid of following non-limiting examples. It should be understood, however, that the invention is not limited to the solely to the particular examples given below. It will be apparent that those skill in the art that any modifications, both to the materials and methods, may be practiced without departing from the purpose and interest of this invention
a) all operations which were carried out at room temperature or ambient temperature were in the range of 18 to 25 degree C.
b) Evaporation of the solvent was carried out under reduced pressure (600-4000 pascals;4.5-30mm Hg)with a bath temperature of upto 40 degree C.
c) The course of the reaction was monitored by thin layer chromatography (TLC) and reaction times are given for illustration only.
27

d) Melting points are uncorrected, the melting points are given for the materials prepared as described, polymorphism may result in isolation of materials with different melting points in some preparations.
e) The structure and purity of all final products were assured by at least one of the
following techniques: TLC, NMR(nuclear magnetic resonance)spectroscopy, IR(lnfrared spectroscopy), or microanalytical data, and HPLC
f) Yields are given for illustration only.
g) When given, NMR data is in the form of delta (.delta.)values for major diagnostic protons given in parts per million ( ppm) relative to tetramefhylsilane (TMS) as internal standard determined at 300 MHz or 400 MHz using the indicated solvent.
h) Chemical symbols have their usual meanings; the following abbreviations have also been used: v( volume ), w(weight), B.P.( boiling point), M. R. (Melting range), M.pt.(melting point), L(liters), ml(milliliters),gms(grams), mg(milligrams), mol (moles), mmol(millimoles) eq (equivalents) deg C (degree centigrade), cone. HC1( concentrated hydrochloric acid) any other
EXAMPLE 1:
GENERAL PROCEDURE FOR PREPARATION OF TLR ANTAGONISTS
1. A mixture of substituted benzaldehyde ( 0.6g, 4.42mmol), 3,4,5-trimethoxyphenyl acetic acid ( l.0g, 4.42mmol ), acetic anhydride ( 2 mL ), and triethylamine ( 1 mL) is heated under reflux for two hour. The mixture is then acidified with cone, hydrochloric acid (3 mL) and stirred. The solid, which is separated out, is filtered off and crystallized from aqueous ethanol. (A).
The acid (A) obtained in the above step is mixed with quinoline (5 mL) and copper chromite (0.1 g) and heated under reflux for 30 min under nitrogen. Ethyl acetate is added and the product is filtered off through Celite. The filtrate is washed with dilute hydrochloric acid, water, and brine, dried (sodium sulphate), and evaporated under reduced pressure to isolate the product. (B)
28

A stirred solution of the product obtained (B) in the above step (0.2 g) and iodine (0.02 g) in ethanol (500 mL) is irradiated for 14 hour at 254 nm. The solution is then evaporated under reduced pressure and the product (C) is isolated by silica gel column chromatography. (Pet-ether-ethyl acetate gradient)

OMe OMe 0Me
A B C
2. An alternate approach to the above reaction is suzuki coupling of the appropriate reactants 1 and 2 gives a biaryl dialdehyde 3. The compound 3 on wittig olefination, and ring closing metathesis of the resultant diene 4 with the Grubbs second-generation ruthenium catalyst gives 5.



CHO R

12 3 4 5
3. An alternate approach is Pschorr synthesis wherein substituted nitroaldehyde ( 1 g ), 3,4,5-trimethoxyphenyl acetic acid ( 1 g ) , acetic anhydride ( 2 mL ) and triethylamine ( 1 mL ) are refluxed for 1 hour. The mixture is then acidified with concentrated hydrochloric acid ( 3 mL ) and stirred. The solid which precipitated out is filtered off and washed with hot water, leaving acid ( A ). A solution of the acid ( A ) ( 1 g ) in 5N ammonium hydroxide ( 10 mL ) is added to a slurry of iron ( II ) sulphate ( 7 g ), water 10 mL, and concentrated ammonium hydroxide ( 18 mL ) at 80-90°C. After one hour, the product is filtered through Celite and the precipitate is washed with 5N-ammonium hydroxide ( 5 mL ) . The cooled filtrate is acidified with glacial acetic acid and the precipitate is filtered off and vacuum dried ( B ) . To a mixture of amine ( B ) ( 0.5 g ) , ethanol and 5N-hydrochloric acid ( 2 mL ) at 0 C is
29

added a 50% ethanolic solution of isopentyl nitrite ( 0.5 mL ). The mixture stirred for 1 hour, diluted with water ( 50 mL ); copper powder ( 0.2 g ) is added and stirred for two hour at 50°C. The mixture cooled, extracted with ethyl acetate, which on concentration under reduced pressure gives acid ( C ). A mixture of acid ( C ) ( 0.5 g ) , quinoline ( 3 mL ) and basic copper carbonate ( 0.3 g ) is refluxed for 3 hour, cooled and extracted with ethyl acetate. The ethyl acetate layer washed with dilute hydrochloric acid, followed by dilute aqueous alkali, and evaporated. The product ( P ) is purified by column chromatography on silica ( Pet-ether-ethyl acetate gradient).

The following compounds were prepared by one of the above techniques using appropriate intermediates:
EXAMPLE 2: Compound 1: 2, 7-dihydroxy-3,4-dimethoxyphenanthrene

State: solid, amorphous M.pt: 253, MS: m/z 271 ( M+l ),
NMR: ( 400MHz, CDC13, 5 ppm ) 5 3.95 ( S, 3H, 0Me), 8 4.05 ( S, 3H, 0Me), 8 5.08 ( br
s, IH, exch.D2O, OH), 8 6.01 (br s, IH, exch.D20, OH), 8 7.08 ( S, IH, H-1), 8 7.18 ( dd,
1H,J=9.2 Hz, J=2.8Hz, ortho and meta coupled, H-6 ), 5 7.22 (d, IH, J=2.8Hz, meta coupled, H-8 ), 5 7.53 ( AB-quartet, 2H, J=9.0Hz, H-9 and H-10), 5 9.33 ( d, IH, J=9.2Hz, ortho coupled, H-5 )
EXAMPLE 3 : Compound 2 : 2, 7-diacetoxy-3,4-dimethoxyphenanthrene.
OAc
MeO^J^^

OAc

State: solid, amorphous ,M.pt. 148, MS: m/z 355 ( M+l ),
'HNMR: ( 400MHz, CDC13, d ppm) 5 2.37 ( S, 3H, OAc), 5 2.46 ( S, 3H, OAc ), 5 3.92 ( S, 3H, OMe), 5 3.98 ( S, 3H, OMe ), 5 7.15 ( S, IH, H-l), 5 7.36 ( dd, IH, J=9.2Hz, J=2.8Hz, ortho and meta coupled, H-6 ), 5 7.58 ( d, IH, J=2.8Hz,meta coupled, H-8 ), 6 7.64 ( apparent singlet (AB-quartet), 2H, H-9 and H-10), 5 9.42 ( d, IH, J=8.8Hz, ortho coupled , H-5 )
EXAMPLE 4: Compound 3: 2,3><4,7-tetramethoxyphenanthrene
OMe


MeO
UeO^Jl^
OMe

State: solid, amorphous, M.pt. 148, MS: m/z 299 ( M+l ),
31

NMR: ( 400MHz, CDC13, d ppm) d 3.95 ( S, 3H, OMe), d 4.00 ( S, 3H, OMe ), d 4.03 ( S, 3H, OMe), d 4.05( S, 3H, OMe ), d 7.00 ( s, 1H, H-l ), 6 7.16 ( dd, J=9.2Hz, J=2.8Hz, ortho and meta coupled, H-6), d 7.19 ( d, 1H, J=2.8Hz, meta coupled, H-8 ), d 7.51 (apparent singlet (AB-quartet), 2H, H-9 and H-10 ), d 9.34 ( d, 1H, J=9.2Hz, ortho coupled , H-5 )
EXAMPLE 5: Compound 4: l,5-dihydoxy-2, 7-dimethoxy-9,10-dihydrophenanthrene.

State: solid, crystalline, M.pt. 201 - 203, MS: m/z 273 ( M+l ),
NMR: ( 400MHz, CD3COCD3, d ppm ) 8 2.59-2.75 ( m, 4H, H-9 and H-10 ), d 3.82 ( S, 3H, OMe ), 8 3.85 ( S, 3H, OMe), d 6.39 ( d, 1H, J=2.4Hz, meta coupled, H-6 ), d 6.45 (d, 1H, J=2.4Hz, meta coupled, H-8 ), d 6.78 ( d, 1H, J=8.4Hz, ortho coupled, H-3 ), d 7.28 ( br s, 1H, exch.D20, OH ), 8 7.72 ( d, 1H, J=8.4Hz, ortho coupled, H-4 ), d 8.37 ( br s, lH,exch.D20, OH)
32
EXAMPLE 6: Compound 5: l,5-diacetoxy-2,7-dimethoxy-9,10-dihydrophenanthrene.


State: solid, crystalline , M.pt. 143, MS: m/z 257 ( M+l ),
NMR: ( 400MHz, CD3COCD3, d ppm ) d 2.25 ( S, 3H, OAc ), d 2.29 ( S, 3H, OAc ), d 2.59-2.70 ( m, 4H, H-9 and H-10 ), d 3.84 ( S, 3H, OMe), 8 3.89 ( S, 3H, OMe), d 6.66 ( d, 1H, J=2.4Hz, meta coupled, H-6 ), d 6.78 (d, 1H, J=2.4Hz, meta coupled, H-8 ), d 6.96 ( d, 1H, J=9.2Hz, ortho coupled, H-3 ), d 8.16 ( d, 1H, J=8.4Hz, ortho coupled, H-4),

EXAMPLE 7: Compound 6: 3,4-dimethoxyphenanthrene-2, 7-bis-[ (2E)-3-[ 3,4-bis
(acetyloxy) phenyl ] acrylate.
O
,0.
State: solid, amorphous , MS: m/z 785 ( M+Na ),
NMR: ( 400MHz, CDCI3, d ppm ) d 2.33 ( S, 12H, OAc), d 3.95 ( S, 3H, OMe ), d 3.99 ( S, 3H, OMe), d 6.65 ( d, lH,J=16Hz ), 8 6.76 ( d, 1H, J=16Hz ), d 7.19 ( S, 1H ), d 7.28 ( d, 1H ), d 7.30 ( d, 1H ), d 7.40-7.55 ( m, 5H ), d 7.68 ( d, 3H ), d 7.87 ( d, 1H, J=16Hz ), d 7.93 ( d, 1H, J=16.4Hz ), d 9.47 ( d, 1H, J=9.2Hz )
EXAMPLE 8: Isolation of RSCL-520 (Eulophial) from plant material:
Tubers of Eulophia flava were collected in the month of October 2006 from Toranmal, Dhule district, Maharashtra, India. Herbarium voucher specimen (Accession No. 157) was authenticated and deposited at herbarium of Reliance Life Sciences, Dhirubhai
33

Ambani Life Sciences Center, and Navi Mumbai. Tubers were chopped in small pieces, shade dried and pulverized.
Preparation of extract from plant material:
Powdered tubers were subjected to sequential cold extraction by using solvents viz. Petroleum ether (60-80), Dichloro Methane, Ethyl acetate, Methanol and water. The selection of solvents was done in order to get all the compounds (Nonpolar to polar) extracted in solvents. Solvent is added to material in 1:6 proportions, stirred for 6-7 hours in stainless steel vessels at room temperature and filtered. The process repeated for thrice and accumulated solution from pet ether (60-80), Dichloro Methane, Ethyl acetate, Methanol were concentrated to dryness under reduced pressure and controlled temperature (42-45 c) using rotary evaporator. Aqueous extract was prepared by using RO water (1:10) with stirring, generated filtrate was lyophilized.
Yield (in percentage) & colour of each extract observed was;

Extract Color of each extract Yield %
Pet ether Dark yellow 0.46
Dichloromethane Dark brown to chocolate 2.40
Ethyl acetate Dark black 1.64
Methanol Dark brown to blackish 0.49
Water Yellowish to white powder 10.88
Isolation of RSCL-520 (Eulophiol) and Nudol:
Column chromatographic fractionation by using silica gel (60-120 mesh) (Acme synthetic chemicals) of dichloro methane extract with pet ether and ethyl acetate (89:11) and (85:15) as an eluent afforded eulophiol and nudol respectively.
Isolated eulophiol showed 98.89 % purity by HPLC analysis under chromatographic conditions as follows: Column: Hypersil GOLD CI8, 5 (.im, 4.6 x 250 mm, Flow rate: 1 ml / minute, Wave length: 265 nm, Mobile phase: Buffer : Acetonitrile, Buffer: 2 ml TEA = 1 litre water. Adjust pH to 3.0 with phosphoric acid, Run time: 60 minutes, Injection
34

volume: 20m.1, Sample preparation: 0.1 mg/ml, Diluent: Acetonitrile, Retention time: about 17.3 minutes

Time Buffer Acetonitrile
0.01 70 30
35 60 40
40 30 70
50 30 70
52 70 30
60 70 30
Isolated nudol showed 97.02 % purity by HPLC analysis under chromatographic conditions as follows: Column: Hypersil GOLD C18, 5 mm, 4.6 x 250 mm, Flow rate:
1 ml / minute, Wave length: 257, 275 nm, Mobile phase: Buffer : Acetonitrile, Buffer:
2 ml TEA = 1 litre water. Adjust pH to 3.0 with phosphoric acid, Run time: 35 minutes, Injection volume: 20 ul, Sample preparation: 0.1 mg/ml, Diluent: Acetonitrile, Retention time: about 15.32 minutes

Time Buffer Acetonitrile
0.01 30 70
5 35 65
10 40 60
15 40 60
20 50 50
25 50 50
30 40 60
35 30 70
EXAMPLE 9: BIOLOGICAL DATA:
A) Effect of RSCL- 0520 (Eulophial) on TNF-D release in response to TLR ligands:
35

Toll-like receptors (TLRs) play an important role in innate immunity in mammals. Activation of these TLRs leads to the activation of many inflammatory responses and leads to recruitment of numerous signaling molecules. The present invention has studied effect of various TLR ligands on THP-1 monocytes and their ability to activate them and release TNF-a. The present invention has used 9 TLR ligands (1-9: Apotech Cat; APO-54N-018-KI01) and assayed for TNF-a release in culture supernatants.
THP-1 cells (2 x105 cells/well) were plated in 96-well plate. The cells were pretreated with RSCL-0520 (50uM) in DMSO 1hr prior to TLR ligand treatment. Following 1hr pre-treatment, the cells were treated with TLR ligands (TLR-1/2- 75ng/ml, TLR-3-75mg/ml, TLR-4 750ng/ml, TLR-5, 75ng/ml, TLR-6, 75ng/ml, TLR-7/8-7.5mg/ml and TLR-9-7.5mg/ml) 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 RSCL-0520 and only RSCL-0520 without ligand treatment.
Results were observed for TNF-a secretion following stimulation with TLR-1/2, TLR-4 and TLR-6 ligands. It was fond that treatment with RSCL-0520 inhibited the TNF-a secretion in only in TLR-4 (-50% inhibition). There was however no effect on cells treated with TLR-1/2 and TLR-6 ligands, indicating that RSCL-0520 is a potential inhibitor for TLR-4 signaling mechanisms induced by LPS.
B) Effect of RSCL- 0520 on LPS Induced TNF-a release in THP-1 cells
To confirm whether the inhibitory effect of RSCL-0520 is dose-dependent, the present invention has checked its ability to inhibit TNF-a secretion from LPS (250ng/ml) induced THP-1 monocytes.
THP-1 cells (2 x105 cells/well) were plated in 96-well plate. The cells were pretreated with RSCL-0520 at various concentrations (luM, 10uM, 50uM and 100uM) 1 hr prior to LPS stimulation. As a control group, cells were treated with just LPS and cells treated
36

with only RSCL-0520 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 RSCL-0520 was also analysed in tandem by treating cells with RSCL-0520 with and without LPS by MTT assay.
LPS induced TNF-a secretion was inhibited by RSCL-0520 in a concentration dependent manner. The viability of the cells was not affected by RSCL-0520 indicating its non-toxic nature.
C) LPS dose dependent effect on RSCL-0520
To check the ability of RSCL-0520 to inhibit LPS induced TNF-a secretion from THP-1 cells, the present invention stimulated THP-1 cells with increasing concentrations of LPS (31.25 ng/ml to 1000ng/ml) with and without pre-treatment of cells with different concentrations of RSCL-0520.
It was clearly seen that an increased secretion of TNF-a with increasing concentration of LPS is inhibited by RSCL-0520 to varied extents in a concentration dependent manner. It was observed that RSCL-0520 has the ability to inhibit LPS (1000ng/ml) induced TNF-a secretion at concentration as low as lOuM, but it was observed that -100% inhibition at 100uM indicating its inhibitory potential.
D) Effect of RSCL- 0520 on LPS induced TNF-a release in PBMC
The present invention has also evaluated the ability of RSCL-0520 to inhibit LPS induced TNF-a secretion in a physiological scenario, by testing the same in PBMC isolated from human blood.
The results indicated that similar inhibitory effects observed in PBMCs also. The TNF levels were not detectable in PBMCs without LPS and with RSCL-0520 treatment alone, indicating its specificity in LPS induced TNF-a through a TLR mediated process.
Example 7: In vivo Results:
A) Effect of RSCL- 0520 on LPS induced TNF- a release in Balb/c mice
37

The present invention has further checked the ability of RSCL-0520 to exert protection
against inflammatory agents in a mice (Balb/c) model.
Balb/c (5 animals per group) mice was injected intraperitoneally with LPS (225ug/ml)
with and without pretreatment of RSCL-0520.
RSCL-0520 (100mg/kg body weight) given intraperitoneal 15minutes before LPS
treatment. Blood collection was done retro-orbitally under anesthesia. Serum collected
was analysed for TNF-a secretion.
lhr post LPS injection it was seen that secretion of large amounts on TNF-a in the serum.
The secretion of TNF-a is inhibited to significant levels (-30%) when the mice were pre-
treated with RSCL-0520 indicating its efficiency to effectively inhibit TLR induced
inflammation.
B) Inhibition of carrageenan induced foot pad edema
The anti-inflammatory effect of RSCL-0520 was checked in mice by inducing local inflammatory reaction by footpad injection of carrageenan into the left hind paw. Balb/c mice of 5-6 weeks age weighing ~ 20-30g were taken for the experiment. Carrageenan(l%) in saline (50ul) was injected to the mouse footpad to induce edema. As a vehicle control, 50ul of vehicle without carrageenan was injected. RSCL-0520 (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 (.il) was then calculated in control and in drug-treated animals. Inhibition was then derived through comparison with the vehicle control group.
Analysis revealed that in the mice pretreated with RSCL-0520. the carrageenan-induced inflammation was reduced (-80% - 24hr and ~ 100% - 48hr) and the reduction was effective over a 48hr period.
Further the present invention has also checked for its ability to reduce over 120hr time period and it was observed that inhibition of footpad edema was over the entire period of time indicating its effectiveness for long durations.
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All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the 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.
Dated this 24th day of September , 2007

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ABSTRACT
The present invention relates to novel synthetic toll like receptor antagonist. The present invention in particular provides compounds, methods and compositions for specifically inhibiting immune stimulation involving TLR ligands, especially TLR-4. The compounds are potentially useful in treatment of inflammation, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer and immunodeficiency.
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