Hyaluronic acid derivatives and their therapeutic use

Abstract

The present invention relates to hyaluronic acid derivatives of the general formula (I) to which anti-inflammatory or disease-modifying drug is attached through a biodegradable linker to treat chronic degenerative diseases such as osteoarthritis or related inflammatory disorders, their pharmaceutically acceptable salts, pharmaceutical compositions containing them, methods for their preparation, use of these compounds in medicine and the intermediates involved in their preparation.

Description

[0001] HYALURONIC ACID DERIVATIVES AND THEIR THERAPEUTIC USE

[0002] FIELD OF INVENTION

[0003] The present invention relates to hyaluronic acid derivatives of the general formula (I) to which anti-inflammatory or disease-modifying drug is attached through a biodegradable linker to treat chronic degenerative diseases such as osteoarthritis or related inflammatory disorders, their pharmaceutically acceptable salts, pharmaceutical compositions containing them, methods for their preparation, use of these compounds in medicine and the intermediates involved in their preparation.

[0004] BACKGROUND OF THE INVENTION

[0005] Osteoarthritis (OA) is the most common form of arthritis. Its prevalence is increasing markedly because of an ageing population. OA is a chronic degenerative disease that is characterized by the gradual and progressive destruction of the cartilage that coats the articular surface of knees, hips, shoulders, hands, ankles and the spinal column. Disease is further characterized by the inflammation of the synovial membrane as well as damage to menisci, tendons, muscles and nerves associated with the affected joint. The management of OA includes pharmacological therapies, which are mostly symptomatic. Paracetamol is the first- line oral analgesic, whilst oral non-steroidal anti-inflammatory drugs (NSAIDs), including selective cyclooxygenase-2 (COX-2) inhibitors, are the mainstay of therapy. These drugs control pain but do not have known disease-modifying effects. Treatment with NSAIDs is limited by their negative side effects on the gastrointestinal tract. Symptomatic slow-acting drugs for OA such as glucosamine, chondroitin sulphate and diacerein are used for non-acute treatment. US20090239822 described a process for the preparation of esters of diacerein with hyaluronic acid by means of which stable products are obtained, which are practically free of impurities and characterized by a prolonged anti-inflammatory activity. Also described are the pharmaceutical compositions having prolonged anti-inflammatory activity and suitable for intra-articular administration containing said esters.

[0006] W02012013670 discloses a process for providing esters of hyaluronic acid, hyaluronic acid salts or hyaluronic acid derivatives with hydrophobic organic compounds. US20210252025 describes synergic pharmaceutical combination of a selective inhibitor of COX-2 and anthraquinone derivative for the treatment of pain and inflammation in osteoarthritis, rheumatoid arthritis and / or degenerative joint disease caused by different etiologies. WO2021110177 describes rhein derivatives and antiviral application thereof. CN105884645 describes rhein compounds and application thereof. W02014011753 describes anthraquinone analogs for the treatment of proliferative disorders such as cancer. W02010124521 describes derivatives of rhein, preparation methods and uses in the preparation of medicaments for treating metabolic diseases. CN104892453 describes antimicrobial and anti-tumor emodin compound and application thereof.

[0007] IL-1 plays a major role in OA pathophysiology. Diacerein and its active metabolite Rhein have been shown to inhibit, in vitro and in vivo, the production and activity of IL-1 and the secretion of metalloproteinases, without affecting the synthesis of prostaglandins; therefore, diacerein and rhein do not have a deleterious effect on the upper gastrointestinal tract. Hyaluronic acid (HA) is a natural mucopolysaccharide having repeated units of N-acetyl glucosamine and D- glucuronic acid and represents a major component of articular cartilage and synovial fluid. Intraarticular HA has therapeutic value for the management of osteoarthritis. Diacerein is a drug with conditional approval in Europe and has been shown to be effective for the treatment of OA, however, because of its side effects such as liver toxicity and diarrhea, its use is highly restricted. As of now, OA is a condition that is lacking cost-effective interventions and has no cure. It has been surprisingly found that the product of the present invention represented by general formula (I) has shown significant effect in improving OA disease compared to individual Hyaluronic acid (HA) and diacerein. The therapeutic effectiveness of Hyaluronic acid (HA) is improved and reduces the side effects of diacerein by formulating them in an advantageous way.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention relates to hyaluronic acid derivatives of the general formula (I) having the potential to treat chronic degenerative diseases, especially osteoarthritis. The hyaluronic acid derivatives of the present invention are useful in the treatment of the human or animal body, by regulation of cytokine production. The hyaluronic acid derivatives of this invention are therefore suitable for the treatment of osteoarthritis.

[0010] EMBODIMENTS OF THE INVENTION

[0011] The main objective of the present invention is to provide novel hyaluronic acid derivatives of general formula (I) and their pharmaceutically acceptable salts.

[0012] An embodiment is provided with a process for the preparation of novel hyaluronic acid derivatives of general formula (I) and their pharmaceutically acceptable salts. Another embodiment provided pharmaceutical compositions containing novel hyaluronic acid derivatives of general formula (I) and their pharmaceutically acceptable salts, having pharmaceutically acceptable carriers, solvents, diluents, excipients and other media normally employed in their manufacture.

[0013] In a further embodiment is provided with the use of the novel hyaluronic acid derivatives of the present invention for the treatment of osteoarthritis, by administering a therapeutically effective & non-toxic amount of the novel hyaluronic acid derivatives of general formula (I), or their pharmaceutically acceptable compositions to the mammals.

[0014] In yet another embodiment is provided a method of treating osteoarthritis using novel hyaluronic acid derivatives of general formula (I) or their pharmaceutically acceptable salts, their pharmaceutically acceptable solvates, or their pharmaceutically acceptable compositions.

[0015] In another embodiment, the present invention provides a novel intermediates used for the preparation of formula (I) having chemical name 3-((3- aminopropoxy)carbonyl)-9, 10-dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate or its hydrochloride salt (Intermediate A), 3-(l l,l l-dimethyl-9-oxo-2,5,10-trioxa-8- azadodecanoyl)-9, 10-dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate (Intermediate B), 3-((2-(2-aminoethoxy)ethoxy)carbonyl)-9, 10-dioxo-9, 10- dihydroanthracene-l,8-diyl diacetate or its hydrochloride salt (Intermediate C), 3- (11,1 l-dimethyl-9-oxo-2, 10-dioxa-5-thia-8-azadodecanoyl)-9, 10-dioxo-9, 10- dihydroanthracene-l,8-diyl diacetate (Intermediate D), 3-((2-((2- aminoethyl)thio)ethoxy)carbonyl)-9, 10-dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate or its hydrochloride salt (Intermediate E), 3-(((4-

[0016] (aminomethyl)benzyl)oxy)carbonyl)-9, 10-dioxo-9, 10-dihydroanthracene- 1 ,8-diyl diacetate or its hydrochloride salt (Intermediate F), 3-(((l-

[0017] (aminomethyl)cyclopropyl)methoxy)carbonyl)-9, 10-dioxo-9, 10- dihydroanthracene- 1,8-diyl diacetate or its hydrochloride salt (Intermediate G), 3- ((((lr,4r)-4-aminocyclohexyl)oxy)carbonyl)-9,10-dioxo-9, 10-dihydroanthracene- 1,8-diyl diacetate or its hydrochloride salt (Intermediate H).

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows joint thickness difference of Hyaluronic acid-Diacerein derivative. FIG. 2 shows joint thickness difference of Hyaluronic acid-Rhein derivative.

[0020] DESCRIPTION OF THE INVENTION

[0021] Accordingly, the present invention relates to hyaluronic acid derivatives of general formula (I), or their pharmaceutically acceptable salts, their pharmaceutically acceptable solvates wherein, A-CO- represents one residue of the hyaluronic acid disaccharide unit or its derivatives or its salt, wherein the average molecular weight of hyaluronic acid ranges from 500000 to 3000000 Da;

[0022] L represents substituted or unsubstituted linear or branched hydrocarbon group having 2 to 10 carbon atoms or a 4-8 membered carbocyclic or heterocyclic group; the hydrocarbon group also contains heteroatom such as oxygen, sulfur and nitrogen, 6-10 membered aromatic ring system, 3-8 membered carbocyclic ring system, 3-6 membered spirocyclic ring system in the carbon chain;

[0023] -NH-L-O-, represents a spacer residue in a compound represented by NH2-L-OH;

[0024] Ri represents a carboxylic group containing drug residues selected from either Diacerein or its pharmaceutically acceptable salts, Rhein or its pharmaceutically acceptable salts, which are attached to the hydroxy group of L forming an ester linkage;

[0025] Diacerein Rhein

[0026] -CO-NH- represents an amide bond of the carboxylic group from hyaluronic acid (A-COOH) with an amino group of linker residue L; wherein the hyaluronic acid derivative has a degree of substitution of the drug residue from 5 to 50 mol % per repeating disaccharide unit of hyaluronic acid, and the carboxylic acid group of hyaluronic acid is either in free form i.e., not participating in bond formation or in the amide form with the amino group of linker, according to the degree of substitution of the drug residue;

[0027] In an embodiment, when L is substituted; the substitutions may be selected from hydrogen, (Ci-C 10) alkyl, (C2-Cio)alkenyl, (C2-Cio)alkynyl, cycloalkyl, aryl, alkoxy, acyl, carboxylic group, cyano, halo, oxo, heteroatoms such as -O-, -NR2-, -S(O)p-

[0028] R2 represents hydrogen or (Ci-C 10) alkyl; p represents integers from 0-2;

[0029] In a further embodiment the hydroxyl and acetamido group of hyaluronic acid may attach to the linker as described in specification.

[0030] As used herein, the term hyaluronic acid (HA) can mean any hyaluronic acid or salts thereof, and it includes, but it is not limited to them, sodium hyaluronate (NaHA), potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, and combinations thereof.

[0031] In an another embodiment, hyaluronic acid is bound to analogues of diacerein.

[0032] Analogues of diacerein is selected from WO2023199238.

[0033] A hyaluronic acid derivatives of general formula (I) may bound with Diacerein or its pharmaceutically acceptable salts or Rhein or its pharmaceutically acceptable salts or analogues of diacerein with suitable linker.

[0034] In a further embodiment provided novel hyaluronic acid derivatives of general formula (I) of the present invention are:

[0035] Aminoethanol-Diacerein-derivatised Sodium Hyaluronate;

[0036] Aminopropanol-Diacerein-derivatised Sodium Hyaluronate;

[0037] (2-hydroxyethoxy)ethylamine-Diacerein-derivatised Sodium Hyaluronate (2-hydroxyethyl)thio)ethyl amine-Diacerein-derivatised Sodium Hyaluronate; (Aminomethyl)benzyl)oxy)-Diacerein-derivatised Sodium Hyaluronate;

[0038] Aminomethyl cyclopropyl methanol -Diacerein-derivatised Sodium Hyaluronate; Aminocyclohexyl- 1-ol-Diacerein-derivatised Sodium Hyaluronate;

[0039] Aminoethanol-Rhein-derivatised Sodium Hyaluronate.

[0040] In a further embodiment provided novel intermediate for the preparation of novel hyaluronic acid derivatives of general formula (I) of the present invention are: 3-((3-aminopropoxy)carbonyl)-9,10-dioxo-9,10-dihydroanthracene-l,8-diyl diacetate or its hydrochloride salt, 3-(l l,l l-dimethyl-9-oxo-2,5,10-trioxa-8- azadodecanoyl)-9,10-dioxo-9,10-dihydroanthracene-l,8-diyl diacetate, 3-((2-(2- aminoethoxy)ethoxy)carbonyl)-9, 10-dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate or its hydrochloride salt, 3-(l l,l l-dimethyl-9-oxo-2,10-dioxa-5-thia-8- azadodecanoyl)-9,10-dioxo-9, 10-dihydroanthracene- 1,8-diyl diacetate, 3-((2-((2- aminoethyl)thio)ethoxy)carbonyl)-9, 10-dioxo-9, 10-dihydroanthracene- 1,8-diyl diacetate or its hydrochloride salt, 3-(((4-(aminomethyl)benzyl)oxy)carbonyl)- 9, 10-dioxo-9, 10-dihydroanthracene- 1,8-diyl diacetate or its hydrochloride salt, 3- (((l-(aminomethyl)cyclopropyl)methoxy)carbonyl)-9,10-dioxo-9, 10- dihydroanthracene- 1,8-diyl diacetate or its hydrochloride salt, 3-((((lr,4r)-4- aminocyclohexyl)oxy)carbonyl)-9,10-dioxo-9, 10-dihydroanthracene- 1,8-diyl diacetate or its hydrochloride salt.

[0041] In a further embodiment the groups, radicals described above may be selected from: the “alkyl” group used either alone or in combination with other radicals, denotes a linear or branched radical containing one to six carbons, selected from methyl, ethyl, n-propyl, z'.w-propyl, n-butyl, sec-butyl, tert-butyl, amyl, t-amyl, n-pentyl, zz-hexyl, and the like; the “alkenyl” group used either alone or in combination with other radicals, is selected from a radical containing from two to six carbons, more preferably groups selected from vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and the like; the “alkenyl” group includes dienes and trienes of straight and branched chains; the “alkynyl” group used either alone or in combination with other radicals, is selected from a linear or branched radical containing two to eight carbon atoms, more preferably thienyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3- butynyl, 1 -pentynyl, 2-pentynyl, 3 -pentynyl, 4-pentynyl, 1 -hexynyl, and the like. The term “alkynyl” includes di- and tri-ynes; the “aryl” or “aromatic” group used either alone or in combination with other radicals, is selected from a suitable aromatic system containing one, two or three rings wherein such rings may be attached together in a pendant manner or may be fused, more preferably the groups are selected from phenyl, naphthyl, tetrahydronaphthyl, indane, biphenyl and the like; the “alkoxy” group used either alone or in combination with other radicals, is selected from groups containing an alkyl radical, as defined above, attached directly to an oxygen atom, more preferably groups selected from methoxy, ethoxy, n-propoxy, z’so-propoxy, n-butoxy, / -butoxy, z’so-butoxy, pentyloxy, hexyloxy, and the like;the “acyl” group used either alone or in combination with other radicals, is selected from a radical containing one to eight carbons, more preferably selected from formyl, acetyl, propanoyl, butanoyl, z'.w-butanoyl, pentanoyl, hexanoyl, heptanoyl, benzoyl and the like, which may be substituted; the “carbocyclic” group used either alone or in combination with other radicals, is selected from a cyclic radical containing three to six carbons, more preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like;

[0042] “heterocyclic” group used either alone or in combination with other radicals, is selected from suitable saturated, partially saturated or unsaturated aromatic or non-aromatic mono, bi or tricyclic radicals, containing one or more heteroatoms selected from nitrogen, sulfur and oxygen; the “oxo” group used either alone or in combination with other groups represents radical of formula -C=O.

[0043] In one embodiment of the present invention is provided a process for the preparation of novel hyaluronic acid derivatives of compounds of the general formula (I).

[0044] The process for the preparation comprises the following steps:

[0045] Scheme: 1 Synthesis of compounds of general formula (I) when Ri is diacerein

[0046] PG = Protecting group The compound of the general formula (IV) can be prepared by reacting (II) with compounds of the general formula (III) using general esterification techniques. The most preferred technique is to prepare acid chloride of (II) using oxalyl chloride in dichloromethane and reacting it with (III) in the presence of bases such as triethyl amine, di isopropyl amine etc. and solvents such as dichloromethane, acetonitrile etc. The compound of the general formula (V) can be obtained by removing protecting group using the appropriate method for deprotection. For e.g., in the case of the text-butoxy carbonyl group, hydrochloric acid or TFA can be used and in the case of the benzyloxy carbonyl group, hydrogen in presence of palladium charcoal can be used. The compounds of the general formula (I) can be prepared by coupling (V) and sodium hyaluronate (VI) using various amide bond formation techniques known in the art such as described in Tetrahedron 2005, 61, 10827 with appropriate modifications as necessary. The preferred method for amide bond formation is using EDC: HC1 and N-hydroxy succinimide in a mixture of solvents such as dioxane and water.

[0047] Scheme 2: Synthesis of compounds of general formula (I) when Ri is Rhein

[0048] PG = Protecting group

[0049] The compound of the general formula (VII) can be prepared by reacting (IV) with inorganic base such as sodium carbonate in solvent such as dioxane, water and mixture thereof. The compound of the general formula (VIII) can be obtained by removing protecting group using the appropriate method for deprotection. For e.g., in the case of the text-butoxy carbonyl group, hydrochloric acid or TFA can be used and in the case of the benzyloxy carbonyl group, hydrogen in presence of palladium charcoal can be used. The compounds of the general formula (I) can be prepared by coupling (VIII) and sodium hyaluronate (VI) using various amide bond formation techniques known in the art such as described in Tetrahedron 2005, 61, 10827 with appropriate modifications as necessary. The preferred method for amide bond formation is using EDC: HC1 and N-hydroxy succinimide in a mixture of solvents such as dioxane and water.

[0050] The invention is explained in greater detail by the examples given below, which are provided by way of illustration only and therefore should not be construed to limit the scope of the invention.

[0051] EXAMPLE 1

[0052] Preparation of Aminoethanol-Diacerein-derivatised Sodium Hyaluronate

[0053] Step 1: Preparation of tert-butyl (2-hydroxy ethyl) carbamate

[0054] To a stirring solution of ethanolamine (10.0 g, 164 mmol) in DCM (100 ml) was added di-tert-butyl dicarbonate (35.7g, 164 mmol) at 20-25 °C and the reaction mixture was stirred for 2 h at 25-30°C. After completion of the reaction, water (50 ml) was added to the reaction mixture and the organic layer was separated. The organic layer was dried over anhydrous sodium sulphate and concentrated to get the title product. Step 2: Preparation of 3-(chlorocarbonyl)-9,10-dioxo-9, 10-dihydroanthracene- 1,8- diyl diacetate

[0055] To the solution of 4,5-diacetoxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid (8 g, 21.72 mmol) in DCM (70 ml) was added oxalyl chloride (15.21 ml, 174 mmol) followed by a catalytic amount of DMF (0.106 ml, 1.368 mmol). The resulting reaction mixture was stirred at 50°C for 2 h. The reaction mixture was concentered under nitrogen, stirred with n-hexane (50 mL) and decanted to get the solid product which was directly used for the next step.

[0056] Step 3: Preparation of 3-((2-((tert-butoxycarbonyl)amino)ethoxy)carbonyl)-9,10- dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate

[0057] To a stirring solution of the product of step 1 (5.00 g, 31.0 mmol) in DCM (70 ml) was added TEA (14.42 ml, 103 mmol) at 0°C. To this was added the product of step 2 (8 g, 20.69 mmol) in small portions. The reaction mixture was stirred at 25-30°C for 2 h. The reaction mixture was then concentrated and diluted with water. Filtered to get crude product which was purified by treatment with ethanol.

[0058] Step 4: Preparation of 3-((2-aminoethoxy)carbonyl)-9,10-dioxo-9, 10- dihydroanthracene- 1,8-diyl diacetate hydrochloride To a stirring solution of the product of step 3 (3 g, 5.87 mmol) in EtOAc (30 ml) was added Dioxane: HC1 (14.66 ml, 58.7 mmol) and the reaction mixture was stirred at 25-30°C for 16 h. The solid product obtained was filtered and washed with ethyl acetate. 'H NMR (DMSO-d6): 1H NMR (DMSO-d6): 8.65-8.65 (d, J = 1.6 Hz, 1H), 8.30 (s, 1H), 8.29-8.17 (bs, 3H), 8.17-8.15 (dd, J = 7.2 and 1.2 Hz, 1H), 7.99-7.95 (t, J = 8.0 Hz, 1H), 7.68-7.66 (m, 1H), 4.57-4.54 (t, J = 5.2 Hz, 2H), 3.36- 3.27 (m, 2H), 2.47 (s, 6H). ESI-MS (m / z): 412.12[M+H]+.

[0059] Step 5: Preparation of Aminoethanol-Diacerein-derivatised Sodium Hyaluronate

[0060] Sodium hyaluronate (A, 1 g, 2.38 mmol / disaccharide unit) with an average molecular weight of 1.46 MDa was dissolved in the solvent mixture of 1,4-Dioxane (125 ml) and water (125 ml). To this was added a 2M aqueous solution of N- hydroxy succinimide (1.19 ml, 2.38 mmol) followed by IM aqueous solution of EDC: HC1 (1.19 ml, 1.19 mmol) and the product of step 4 (0.53 g, 1.19 mmol) suspended in 15 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCCL (15 mL) solution was added, and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.5 ml (50%) of acetic acid followed by the addition of 4 g NaCl. The reaction mixture was diluted with ethanol (1000 mL). The precipitated product was filtered and washed with first 80% EtOH (50 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product.

[0061] Drug substitution by saponification:

[0062] The product’s fixed amount was stirred with 5% aq. NaOH (10 mL) solution at 25-30°C for 16 h. The reaction mixture was acidified using dil. HO and the product obtained was extracted with ethyl acetate. The organic layer was dried and evaporated to get a solid product. The structure of the product was confirmed by NMR and matched with Rhein (an active metabolite of Diacerein). The degree of substitution of diacerein based on Rhein’s recovery was around 13%. H NMR (DMSO- d ) 13.81 (bs, 1H), 11.91 (s, 2H), 8.146-8.142 (d, J = 1.6 Hz, 1H), 7.86-7.82 (m, 1H), 7.78-7.74 (m, 2H), 7.43-7.415 (d, J= 8 Hz, 1H). ESI-MS (m / z): 283.05 [M-H].

[0063] EXAMPLE 2

[0064] Preparation of Aminoethanol-Diacerein-derivatised Sodium Hyaluronate

[0065] (with improved drug substitution)

[0066] Sodium hyaluronate (A, 1 g, 2.38 mmol / disaccharide unit) with an average molecular weight of 1.46 MDa was dissolved in the solvent mixture of 1,4-Dioxane (125 ml) and water (125 ml). To this was added a 2M aqueous solution of N- hydroxy succinimide (1.19 ml, 2.38 mmol) followed by IM aqueous solution of EDC: HC1 (2.38ml, 2.38 mmol) and the product of step 4 from example 1 (1.06 g, 2.38 mmol) suspended in 15 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCCh (15 mL) solution was added and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.5 ml (50%) of acetic acid followed by the addition of 4 g NaCl. The reaction mixture was diluted with ethanol (1000 mL). The precipitated product was filtered and washed with first 80% EtOH (50 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product. Drug substitution using UV spectrophotometer was 16.6%.

[0067] EXAMPLE 3

[0068] Preparation of Aminoethanol-Diacerein-derivatised Sodium Hyaluronate (with different molecular weight hyaluronic acid)

[0069] Sodium hyaluronate (A, 1 g, 2.38 mmol / disaccharide unit) with an average molecular weight of 1.08 MDa was dissolved in the solvent mixture of 1,4-Dioxane (125 ml) and water (125 ml). To this was added a 2M aqueous solution of N- hydroxy succinimide (1.19 ml, 2.38 mmol) followed by IM aqueous solution of EDC: HC1 (2.38ml, 2.38 mmol) and the product of step 4 from example 1 (1.06 g, 2.38 mmol) suspended in 15 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCCh (15 mL) solution was added and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.5 ml (50%) of acetic acid followed by the addition of 4 g NaCl. The reaction mixture was diluted with ethanol (1000 mL). The precipitated product was filtered and washed with first 80% EtOH (50 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product. Drug substitution by saponification was around 17%.

[0070] EXAMPLE 4

[0071] Preparation of Aminopropanol-Diacerein-derivatised Sodium Hyaluronate

[0072] Step 1: Preparation of tert-butyl (3 -hydroxypropyl) carbamate

[0073] To a stirring solution of 3 -aminopropan- l-ol (10.0 g, 133 mmol) in DCM (100 ml) was added di-tert-butyl dicarbonate (32 g, 146 mmol) at 20-25°C and the reaction mixture was stirred for 2 h at 25-30°C. After completion of the reaction, water (50 ml) was added to the reaction mixture and the organic layer was separated. The organic layer was dried over anhydrous sodium sulphate and concentrated to get the title product. ESI-MS (m / z): 176 [M+H]+.

[0074] Step 2: Preparation of 3-((3-((tert-butoxycarbonyl)amino)propoxy)carbonyl)- 9, 10-dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate

[0075] To a stirring solution of the product of step 1 (5.00 g, 28.5 mmol) in DCM (70 ml) was added TEA (19.9 ml, 143 mmol) at 0°C. To this was added the product of Step- 2 from Example-1 (9.93g, 25.70 mmol) in small portions. The reaction mixture was stirred at 25-30°C for 2 h. The reaction mixture was then concentrated and diluted with water. Filtered to get crude product which was purified by treatment with ethanol.

[0076] Step 3: Preparation of 3-((3-aminopropoxy)carbonyl)-9,10-dioxo-9,10- dihydroanthracene-l,8-diyl diacetate hydrochloride

[0077] To a stirring solution of the product of step 2 (3 g, 5.86 mmol) in EtOAc (30 ml) was added Dioxane: HC1 (14.66 ml, 58.7 mmol) and the reaction mixture was stirred at 25-30°C for 16 h. The solid product obtained was filtered and washed with ethyl acetate. 'H NMR (DMSO-d6): 8.55-8.55 (d, J = 2.0 Hz, 1H), 8.15-8.13 (m, 2H), 8.11-7.99 (m, 2H), 7.97-7.93 (m, 2H), 7.68-7.66 (m, 1H), 4.43-4.40 (t, J = 6 Hz, 2H), 2.99-2.96 (m, 2H), 2.43 (s, 6H), 2.10-2.04 (m, 2H). ESI-MS (m / z): 426.11[M+H]+. Step 4: Preparation of Aminopropanol-Diacerein-derivatised Sodium Hyaluronate

[0078] Sodium hyaluronate (A, 1 g, 2.38 mmol / disaccharide unit) with an average molecular weight of 1.46 MDa was dissolved in the solvent mixture of 1,4-Dioxane (125 ml) and water (125 ml). To this was added a 2M aqueous solution of N- hydroxy succinimide (1.19 ml, 2.38 mmol) followed by IM aqueous solution of EDC: HC1 (2.38 ml, 2.38 mmol) and the product of step 3 (1.04 g, 2.38 mmol) suspended in 15 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCCh (15 mL) solution was added and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.5 ml (50%) of acetic acid followed by the addition of 4 g NaCl. The reaction mixture was diluted with ethanol (1000 mL). The precipitated product was filtered and washed with first 80% EtOH (50 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product. Drug substitution by saponification was around 16%

[0079] EXAMPLE 5

[0080] Preparation of (2-hydroxyethoxy)ethylamine-Diacerein-derivatised Sodium

[0081] Hyaluronate

[0082] Step 1: Preparation of 2-(2-hydroxyethoxy)ethyl 4-methylbenzene sulfonate

[0083] To a stirring solution of diethylene glycol (10.0 g, 94 mmol) in DCM (100 ml) was added triethyl amine (6.57 ml, 47.1 mmol) at 25-30°C and the reaction mixture was stirred for 15 min. at 25-30°C. To this added p-Toluene sulfonyl chloride (4.49 g, 23.56 mmol) in portions at 25-30°C and the reaction mixture was stirred for 4 h at 25-30°C. After completion of the reaction, water (50 ml) was added to the reaction mixture and the organic layer was separated. The organic layer was dried over anhydrous sodium sulphate and concentrated to get the crude product. The crude product was purified by column chromatography in DCM: Methanol to get the pure title product. ESI-MS (m / z): 261 [M+H]+.

[0084] Step 2: Preparation of 2-(2-azidoethoxy)ethan-l-ol

[0085] To a stirring solution of the product of step 1 (3.00 g, 11.52 mmol) in ethanol (30 ml) was added sodium azide (1.49 g, 23.05 mmol) at 25-30°C. The reaction mixture was stirred at 65-70°C for 16 h. The reaction mixture was then concentrated and diluted with water. Filtered to get crude product which was purified by treatment with ethanol.

[0086] Step 3: Preparation of 2-(2-aminoethoxy) ethan-l-ol

[0087] To a stirring solution of the product of step 2 (1.2 g, 9.15 mmol) in methanol (25 ml) was added Pd on carbon (10%, 50% wet) (0.097 g, 0.915 mmol) at 25-30°C. The reaction mixture was stirred under hydrogen atmosphere at 25-30°C for 16 h. The reaction mixture was filtered through celite pad and filtrate was concentrated to get the title product. ESI-MS (m / z): 106.09 [M+H]+.

[0088] Step 4: Preparation of tert-butyl (2-(2-hydroxyethoxy) ethyl) carbamate

[0089] To a stirring solution of the product of Step 3 (1.0 g, 9.51 mmol) in DCM (100 ml) was added di-tert-butyl dicarbonate (2.28 g, 10.46 mmol) at 25-30°C and the reaction mixture was stirred for 2 h at 25-30°C. After completion of the reaction, water (50 ml) was added to the reaction mixture and the organic layer was separated. The organic layer was dried over anhydrous sodium sulphate and concentrated to get the title product.

[0090] Step 5: Preparation of 3-(l l,l l-dimethyl-9-oxo-2,5,10-trioxa-8-azadodecanoyl)- 9, 10-dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate

[0091] To a stirring solution of the product of step 4 (1.50 g, 7.31 mmol) in DCM (50 ml) was added TEA (5.41 ml, 38.8 mmol) at 25-30°C. To this was added the product of

[0092] Step-2 from Example-1 (3.0 g, 7.76 mmol) in small portions. The reaction mixture was stirred at 25-30°C for 2 h. The reaction mixture was then concentrated and diluted with water. Filtered to get crude product which was purified by treatment with ethanol.

[0093] Step 6: Preparation of 3-((2-(2-aminoethoxy)ethoxy)carbonyl)-9,10-dioxo-9,10- dihydroanthracene- 1 ,8-diyl diacetate hydrochloride

[0094] To a stirring solution of the product of step 6 (1.2 g, 2.160 mmol) in EtOAc (12 ml) was added Dioxane: HC1 (10 ml, 21.60 mmol) and the reaction mixture was stirred at 25-30°C for 16 h. The solid product obtained was filtered and washed with ethyl acetate. 'H NMR (DMSO-d6): 8.57-8.57 (d, J = 1.6 Hz, 1H), 8.17-8.15 (m, 1H), 8.10-8.00 (m, 1H), 7.99-7.97 (m, 2H), 7.95-7.85 (m, 2H), 7.68-7.66 (m, 1H), 4.55- 4.52 (m, 2H), 3.86-3.84 (t, J = 4.4 Hz, 2H), 3.72-3.69 (t, J = 5.2 Hz, 2H), 3.02-2.98 (m, 2H), 2.43 (s, 6H). ESI-MS (m / z): 456.13[M+H]+.

[0095] Step 7: Preparation of (2-hydroxy ethoxy )ethylamine-Diacerein-derivatised Sodium Hyaluronate

[0096] Sodium hyaluronate (A, 0.5 g, 1.19 mmol / disaccharide unit) with an average molecular weight of 1.46 MDa was dissolved in the solvent mixture of 1,4-Dioxane (56 ml) and water (56 ml). To this was added a 2M aqueous solution of N-hydroxy succinimide (0.59 ml, 1.19 mmol) followed by IM aqueous solution of EDC: HC1 (1.19 ml, 1.19 mmol) and the product of step 6 (0.58 g, 1.19 mmol) suspended in 10 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCCh (7.5 mL) solution was added, and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.25 ml (50%) of acetic acid followed by the addition of 2 g NaCl. The reaction mixture was diluted with ethanol (500 mL). The precipitated product was filtered and washed with first 80% EtOH (30 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product. Drug substitution by saponification was around 10%.

[0097] EXAMPLE 6

[0098] Preparation of (2-hydroxyethyl)thio)ethyl amine-Diacerein-derivatised Sodium Hyaluronate

[0099]

[0100] Step 1: Preparation of 2-((2-aminoethyl)thio)ethan-l-ol

[0101] To a stirring solution of 2-mercaptoethan-l-ol (4.0 ml, 56.8 mmol) in water (25 ml) was added 30 % aqueous solution of NaOH (75 ml. 568 mml) at 20-25°C. To this was added 2-bromoethan-l -amine hydrobromide (11.64 g, 56.8 mmol) in small portions and the reaction mixture was stirred for 2 h at 50-55°C. After completion of the reaction, water (50 ml) was added to the reaction mixture and the product was extracted by 2x75 ml ethyl acetate. Then combined organic layer was washed by 25 ml brine solution. The organic layer was dried over anhydrous sodium sulphate and concentrated to get the title product. ESI-MS (m / z): 122.06 [M+H]+.

[0102] Step 2: Preparation of tert-butyl (2-(2-hydroxyethoxy) ethyl) carbamate

[0103] To a stirring solution of the product of Step 1 (1.80 g, 14.85 mmol) in DCM (100 ml) was added di-tert-butyl dicarbonate (3.24 g, 14.85 mmol) at 25-30°C and the reaction mixture was stirred for 2 h at 25-30°C. After completion of the reaction, water (50 ml) was added to the reaction mixture and the organic layer was separated. The organic layer was dried over anhydrous sodium sulphate and concentrated to get the title product. ESI-MS (m / z): 222.11 [M+H]+.

[0104] Step 3: Preparation of 3-(l l,l l-dimethyl-9-oxo-2,10-dioxa-5-thia-8- azadodecanoyl)-9, 10-dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate

[0105] To a stirring solution of the product of step 2 (0.687 g, 3.10 mmol) in DCM (50 ml) was added TEA (1.80 ml, 12.93 mmol) at 25-30°C. To this was added the product of Step-2 from Example- 1 (1.0 g, 2.59 mmol) in small portions. The reaction mixture was stirred at 25-30°C for 2 h. The reaction mixture was then concentrated and diluted with water. Filtered to get crude product which was purified by treatment with ethanol.

[0106] Step 4: Preparation of 3-((2-((2-aminoethyl)thio)ethoxy)carbonyl)-9,10-dioxo- 9,10-dihydroanthracene-l,8-diyl diacetate hydrochloride

[0107] To a stirring solution of the product of step 3 (0.75 g, 1.30 mmol) in EtOAc (7.5 ml) was added Dioxane: HC1 (3.2 ml, 13.12 mmol) and the reaction mixture was stirred at 25-30°C for 16 h. The solid product obtained was filtered and washed with ethyl acetate. 'H NMR (DMSO-de): 8.57-8.57 (d, J = 1.6 Hz, 1H), 8.16-8.14 (m, 1H), 8.08-7.99 (m, 4H), 7.97-7.95 (m, 1H), 7.68-7.65 (m, 1H), 4.55-4.52 (t, J = 6.4 Hz, 2H), 3.05-2.96 (m, 4H), 2.86-2.76 (m, 2H), 2.51 (s, 6H). ESI-MS (m / z): 472.11 [M+H]+.

[0108] Step 5: Preparation of (2-hydroxyethyl)thio)ethyl amine-Diacerein-derivatised Sodium Hyaluronate Sodium hyaluronate (A, 0.5 g, 1.19 mmol / disaccharide unit) with an average molecular weight of 1.46 MDa was dissolved in the solvent mixture of 1,4-Dioxane (56 ml) and water (56 ml). To this was added a 2M aqueous solution of N-hydroxy succinimide (0.59 ml, 1.19 mmol) followed by IM aqueous solution of EDC: HC1 (1.19 ml, 1.19 mmol) and the product of step 4 (0.6 g, 1.19 mmol) suspended in 15 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCCh (7.5 mL) solution was added, and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.25 ml (50%) of acetic acid followed by the addition of 2 g NaCl. The reaction mixture was diluted with ethanol (500 mL). The precipitated product was filtered and washed with first 80% EtOH (30 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product. Drug substitution by saponification was around 10%.

[0109] EXAMPLE 7

[0110] Preparation of (aminomethyl)benzyl)oxy)-Diacerein-derivatised Sodium Hyaluronate

[0111] Step 1: Preparation of (4-(aminomethyl)phenyl)methanol hydrochloride

[0112] Stirring solution of 4-formylbenzonitrile (1.5 g, 11.44 mmol) in THF (30 ml) was cooled to 0°C. To this added LAH (0.868 g, 22.88 mmol) in portions at 0-5°C. After the addition, ice bath was removed and reaction mixture was allowed to come at 25-30°C. Then reaction mixture was heated to 60°C and stirred for 2h at same temperature. After completion of the reaction, reaction mixture was cooled to 5- 10°C and 0.8 ml 2M aqueous solution of NaOH and 1.5 ml water was added. Resulting precipitate was filtered and filtrate was evaporated to get the residue. 3 ml 4M HC1 in ethyl acetate was added to the residue and stirred for 15 min. Resulting precipitate was filtered by suction and washed by 5 ml ethyl acetate. Off- white solid compound was collected as a pure product.

[0113] Step 2: Preparation of tert-butyl (4-(hydroxymethyl)benzyl)carbamate

[0114] To a stirring solution of the product of Step 1 (1.50 g, 8.64 mmol) in THF (15 ml) was added triethylamine (2.65 g, 19.00 mmol) followed by di-tert-butyl dicarbonate (2.08 g, 9.50 mmol) at 10-15°C and the reaction mixture was stirred for 16 h at 25- 30°C. After completion of the reaction, ethyl acetate (50 ml) was added to the reaction mixture. The reaction mixture was washed sequentially with NaHSCU solution (5%, 10ml), saturated NaHCCE solution (10 ml) and brine (10 ml). The organic layer was dried over anhydrous sodium sulphate and concentrated to get the title product.

[0115] Step 3: Preparation of 3-(l l,l l-dimethyl-9-oxo-2,10-dioxa-5-thia-8- azadodecanoyl)-9, 10-dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate

[0116] To a stirring solution of the product of step 2 (0.450 g, 1.896 mmol) in DCM (15 ml) was added TEA (1.20 ml, 9.05 mmol) at25-30°C. To this was added the product of Step-2 from Example-1 (0.733 g, 1.896 mmol) in small portions. The reaction mixture was stirred at 25-30°C for 2 h. The reaction mixture was then concentrated and diluted with water. Filtered to get crude product which was purified by treatment with ethanol. Step 4: Preparation of 3-(((4-(aminomethyl)benzyl)oxy)carbonyl)-9,10-dioxo- 9,10-dihydroanthracene-l,8-diyl diacetate hydrochloride

[0117] To a stirring solution of the product of step 3 (0.75 g, 1.19 mmol) in EtOAc (10 ml) was added Dioxane: HC1 (5 ml, 11.90 mmol)) and the reaction mixture was stirred at 25-30°C for 16 h. The solid product obtained was filtered and washed with ethyl acetate. 'H NMR (DMSO-d6): 8.54-8.54 (d, 7 =1.6 Hz, 1H), 8.39-8.15 (bs, 3H), 8.15-8.14 (m, 2H), 7.98-7.94 (t, 7 =8.0 Hz, 1H), 7.67-7.65 (m, 1H), 7.59-7.53 (m, 4H), 5.43 (s, 2H), 3.45-3.40 (t, 7 =7.2 Hz, 2H), 2.51 (s, 6H). ESI-MS (m / z): 488.13 [M+H]+.

[0118] Step 5: Preparation of (aminomethyl)benzyl)oxy)-Diacerein-derivatised Sodium Hyaluronate

[0119] Sodium hyaluronate (A, 0.5 g, 1.19 mmol / disaccharide unit) with an average molecular weight of 1.46 MDa was dissolved in the solvent mixture of 1,4-Dioxane (56 ml) and water (56 ml). To this was added a 2M aqueous solution of N-hydroxy succinimide (0.59 ml, 1.19 mmol) followed by IM aqueous solution of EDC: HC1 (1.19 ml, 1.19 mmol) and the product of step 4 (0.65 g, 1.19 mmol) suspended in 10 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCOa (7.5 mL) solution was added, and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.25 ml (50%) of acetic acid followed by the addition of 2 g NaCl. The reaction mixture was diluted with ethanol (500 mL). The precipitated product was filtered and washed with first 80% EtOH (30 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product. Drug substitution by saponification was around 15%.

[0120] EXAMPLE 8

[0121] Preparation of aminomethyl cyclopropyl methanol -Diacerein-derivatised

[0122] Sodium Hyaluronate

[0123] Step 1: Preparation of tert-butyl ((1-

[0124] (hydroxymethyl)cyclopropyl)methyl)carbamate

[0125] To a stirring solution of (l-(aminomethyl)cyclopropyl)methanol (1.8 g, 17.80 mmol) in DCM (40 ml) was added TEA (4.96 ml, 35.6 mmol) and di-tert-butyl dicarbonate (4.05 ml, 17.44 mmol) at 20-25°C and the reaction mixture was stirred for 16 h at 25-30°C. After completion of the reaction, water (50 ml) was added to the reaction mixture and the organic layer was separated. The organic layer was dried over anhydrous sodium sulphate and concentrated to get crude product. Which was purified by combi-flash column chromatography to get title product. ESI-MS (m / z): 202.14[M+H]+.

[0126] Step 2: Preparation of 3-(((l-(((tert-butoxycarbonyl) amino)methyl) cyclopropyl) methoxy)carbonyl)-9, 10-dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate

[0127] To a stirring solution of the product of step 1 (0.562 g, 2.79 mmol) in DCM (20 ml) was added TEA (1.622 ml, 11.64 mmol) at 0°C. To this was added the product of Step-2 from Example- 1 (0.90 g, 2.32 mmol) in small portions. The reaction mixture was stirred at 25-30°C for 2 h. The reaction mixture was then concentrated and diluted with water. Filtered to get crude product which was purified by treatment with ethanol.

[0128] Step 3: Preparation of 3-(((l-(aminomethyl)cyclopropyl)methoxy)carbonyl)-9,10- dioxo-9,10-dihydroanthracene-l,8-diyl diacetate hydrochloride

[0129] To a stirring solution of the product of step 2 (0.49 g, 0.888 mmol) in EtOAc (10 ml) was added Dioxane: HC1 (3.66 ml, 14.67 mmol) and the reaction mixture was stirred at 25-30°C for 16 h. The solid product obtained was filtered and washed with ethyl acetate. 'H NMR (DMSO-d6): 8.60-8.60 (d, J = 1.6 Hz, 1H), 8.18-8.15 (m, 2H), 8.00-7.96 (m, 4H), 7.68-7.66 (m, 1H), 4.30(s, 2H), 2.97-2.95 (d, J = 5.6 Hz, 2H), 2.50 (s, 6H), 0.80-0.78 (m, 4H). ESI-MS (m / z): 452.12 [M+H]+.

[0130] Step 4: Preparation of aminomethyl cyclopropyl methanol -Diacerein-Introduced Sodium Hyaluronate Sodium hyaluronate (A, 0.42 g, 1.00 mmol / disaccharide unit) with an average molecular weight of 1.46 MDa was dissolved in the solvent mixture of 1,4-Dioxane (44.5ml) and water (44.5 ml). To this was added a 2M aqueous solution of N- hydroxy succinimide (0.5ml, 1.00 mmol) followed by IM aqueous solution of EDC: HC1 (1.00 ml, 1.00 mmol) and the product of step 3 (0.48 g, 1.0 mmol) suspended in 15 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCCh (6 mL) solution was added, and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.2 ml (50%) of acetic acid followed by the addition of 1.6 g NaCl. The reaction mixture was diluted with ethanol (400 mL). The precipitated product was filtered and washed with first 80% EtOH (30 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product. Drug substitution by saponification was around 25%.

[0131] EXAMPLE 9

[0132] Preparation of Aminocyclohexyl-l-ol-Diacerein-derivatised Sodium Hyaluronate

[0133] Step 1: Preparation of tert-butyl ((lr,4r)-4-hydroxycyclohexyl) carbamate

[0134] To a stirring solution of (lr,4r)-4-aminocyclohexan-l-ol (2.0 g, 17.36 mmol) in DCM (40 ml) was added TEA (5.32 ml, 38.2 mmol) and di-tert-butyl dicarbonate (3.45 ml, 15.80 mmol) at 20-25°C and the reaction mixture was stirred for 16 h at 25-30°C. After completion of the reaction, water (50 ml) was added to the reaction mixture and the organic layer was separated. The organic layer was dried over anhydrous sodium sulphate and concentrated to get crude product. Which was purified by trituration with n-hexane to get desired product.

[0135] Step 2: Preparation of 3-((((lr,4r)-4-((tert-butoxycarbonyl)amino) cyclohexyl)oxy)carbonyl)-9,10-dioxo-9,10-dihydroanthracene-l,8-diyl diacetate

[0136] To a stirring solution of the product of step 1 (1.25 g, 5.82 mmol) in DCM (75 ml) was added TEA (2.70 ml, 19.39 mmol) at 0°C. To this was added the product of Step-2 from Example-1 (1.5 g, 3.88 mmol) in small portions. The reaction mixture was stirred at 25-30°C for 2 h. The reaction mixture was then concentrated and diluted with water. Filtered to get crude product which was purified by treatment with ethanol.

[0137] Step 3: Preparation of 3-((((lr,4r)-4-aminocyclohexyl)oxy)carbonyl)-9,10-dioxo- 9,10-dihydroanthracene-l,8-diyl diacetate hydrochloride

[0138] To a stirring solution of the product of step 2 (1.3 g, 2.29 mmol) in ethyl acetate (50 ml) was added 4M Dioxane: HC1 (5.75 ml, 22.99 mmol) and the reaction mixture was stirred at 25-30 °C for 16 h. The solid product obtained was filtered and washed with ethyl acetate. 'H NMR (DMSO-de): 8.56-8.53 (dd, J = 1.6 and 8.8 Hz, 1H), 8.16-8.14 (m, 1H), 8.06-7.97 (m, 3H), 7.96-7.94 (m, 2H), 7.68-7.64 (m, 1H), 4.95 (m, 1H), 3.21-3.11 (m, 1H), 2.49 (s, 6H), 2.20-1.95 (m, 4H), 1.64-1.55 (m, 4H). ESI-MS (m / z): 466.16 [M+H]+.

[0139] Step 4: Preparation of Aminocyclohexyl- 1-ol-Diacerein-derivatised Sodium Hyaluronate

[0140] Sodium hyaluronate (A, 1 g, 2.38 mmol / disaccharide unit) with an average molecular weight of 1.46 MDa was dissolved in the solvent mixture of 1,4-Dioxane (125 ml) and water (125 ml). To this was added a 2M aqueous solution of N- hydroxy succinimide (1.19 ml, 2.38 mmol) followed by IM aqueous solution of EDC: HC1 (2.38 ml, 2.38 mmol) and the product of step 3 (1.19 g, 2.38 mmol) suspended in 15 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCCh (15 mL) solution was added, and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.5 ml (50%) of acetic acid followed by the addition of 4 g NaCl. The reaction mixture was diluted with ethanol (1000 mL). The precipitated product was filtered and washed with first 80% EtOH (50 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product. Drug substitution by saponification was around 18%.

[0141] EXAMPLE 10

[0142] Preparation of Aminoethanol- Rhein-derivatised Sodium Hyaluronate Step 1: Preparation of 2-((tert-butoxy carbonyl) amino) ethyl 4,5-dihydroxy-9,10- dioxo-9,10-dihydroanthracene-2-carboxylate

[0143] To a stirring solution of the product of Step-3 from Example- 1 (1.5 g, 2.93 mmol) in Dioxane: water (20 ml) (1:1) was added Na2COs (0.93 g, 8.8 mmol) at 20-25°C and the reaction mixture was stirred for 16 h at 25-30°C. After completion of reaction, it was diluted with EtOAc and water. Organic layer was separated and dried over Na2SO4, concentrated under reduced pressure to get crude product which was purified by column chromatography to get desired product.

[0144] Step 2: 2-aminoethyl 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2- carboxylate hydrochloride

[0145] To a stirring solution of the product of step 1 (0.57 g, 1.3 mmol) in EtOAc (10 ml) was added Dioxane: HC1 (5 ml, 20.0 mmol) and the reaction mixture was stirred at 25-30°C for 16 h. The solid product obtained was filtered and washed with ethyl acetate. 'H NMR (DMSO-d6): 12.00 (s, 1H), 11.88 (s, 1H), 8.24-8.24 (d, J = 1.6 Hz, 1H), 8.17-8.07 (m, 3H), 8.06 (s, 1H), 7.88-7.84 (m, 1H), 7.78-7.76 (m, 1H), 7.46-7.44 (m, 1H), 4.54-4.52 (t, J = 4.8 Hz, 2H), 3.30-3.26 (m, 2H). ESI-MS (m / z): 328.07 [M+H]+.

[0146] Step 3: Preparation of Aminoethanol-Rhein-derivatised Sodium Hyaluronate Sodium hyaluronate (A, 0.5 g, 1.19 mmol / disaccharide unit) with an average molecular weight of 1.46 MDa was dissolved in the solvent mixture of 1,4-Dioxane (62.5 ml) and water (62.5 ml). To this was added a 2M aqueous solution of N- hydroxy succinimide (0.6 ml, 1.19 mmol) followed by IM aqueous solution of EDC: HC1 (1.19 ml, 1.19 mmol) and the product of step 2 (0.5 g, 1.19 mmol) suspended in 15 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCOa (7.5 mL) solution was added, and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.25 ml (50%) of acetic acid followed by the addition of 2 g NaCl. The reaction mixture was diluted with ethanol (500 mL). The precipitated product was filtered and washed with first 80% EtOH (50 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product. Drug substitution by UV is 10.4%.

[0147] EXAMPLE 11

[0148] Preparation of Aminoethanol- Rhein-derivatised Sodium Hyaluronate (with different molecular weight Hyaluronic acid)

[0149] Sodium hyaluronate (A, 0.5 g, 1.19 mmol / disaccharide unit) with an average molecular weight of 1.08 MDa was dissolved in the solvent mixture of 1,4-Dioxane (62.5 ml) and water (62.5 ml). To this was added a 2M aqueous solution of N- hydroxy succinimide (0.6 ml, 1.19 mmol) followed by IM aqueous solution of EDC: HC1 (1.19 ml, 1.19 mmol) and the product of step 2 from example 10 (0.5 g, 1.19 mmol) suspended in 15 ml water at 25-30°C. On completion of stirring for 16 h, 5% aq. NaHCO (7.5 mL) solution was added, and the reaction mixture was stirred for 3 h. The reaction mixture was neutralized by adding 0.25 ml (50%) of acetic acid followed by the addition of 2 g NaCl. The reaction mixture was diluted with ethanol (500 mL). The precipitated product was filtered and washed with first 80% EtOH (50 ml) and then with diethyl ether (25 mL). The product obtained was dried under a vacuum and purified by dialysis to get pure product. Drug substitution by saponification was around 16%

[0150] Activity Data

[0151] MIA-induced osteoarthritis model in rats

[0152] Male Sprague Dawley rats (7-8 weeks old) were acclimatized for three days in the incapacitance meter for measuring weight bearing. The rats were given monosodium iodoacetate MIA (2 mg / 50 pL / rat) by intraarticular route in one paw. Changes in hind paw weight distribution between the right (osteoarthritic) and left (contralateral control) paws were utilized as an index of joint discomfort in the osteoarthritic knee, which was measured by incapacitance meter. The next day, rats were randomized based on their weight distribution ratio (% of weight distribution in MIA-injected paw) and treated with a single injection of test compounds (dose as indicated by intraarticular route once,100pl per joint). The weight distribution ratio was measured daily for five days. MIA administration changes the weight bearing capacity of the injected paw, which was improved by the compound treatment. The data are expressed as a % improvement in weight distribution ratio against vehicle control as shown in Table 1.

[0153] The compound of the invention (Example 1) has shown greater improvement in the weight distribution than individual treatment with Diacerein (DCN) and Hyaluronic acid (average M. Wt. 1.46 MDa), which is indicative of a reduction in osteoarthritis in the joint. The compound of the invention (Example 1) has a greater effect on weight distribution than corresponding mixture of hyaluronic acid and diacerein.

[0154] Table 1: Improvement of % in weight distribution ratio against vehicle control

[0155] * Physical mixture of Hyaluronic acid and Diacerein

[0156] MIA-induced osteoarthritis model in rabbits

[0157] Male New Zealand White rabbits (<3-month-old) were acclimatized for a week and their joint thickness was measured. They were given monosodium iodoacetate MIA (6 mg / 50 pL / rabbit) by intraarticular route in left joint. The next day, rabbits were randomized based on their joint thickness difference (pre vs post MIA injection) and treated with a vehicle, hyaluronic acid (3.3 mg in 0.5 mL, once by intraarticular route, average M. Wt. 1.46 MDa), diacerein (5 mg / kg, twice a day, oral route), or intraarticular injection of Example 2 (Hyaluronic acid-Diacerein derivative) and Example 10 (Hyaluronic acid-Rhein derivative) once in a month. Saline injected treatment group was also maintained which serves as normal control animal (dose as indicated by intraarticular route once, 500pl per joint). The joint thickness was measured daily after treatment initiation. The data is expressed as a % change in joint thickness difference against vehicle control as shown in Figure 1 and 2. The compound of the invention (Example 2 and 10) have shown greater improvement in joint thickness than individual treatment with Diacerein and Hyaluronic acid on 28 days after treatment in rabbits.

[0158] Pharmacokinetic study to evaluate sustained release of drug

[0159] The synovial fluid was collected from the above experiment in rabbit and the amount of released compound (diacerein, measured as rhein) was estimated by LC- MS method. Data in table 2 indicates compound of the invention (Example 2) retained and caused sustained release of rhein in the joint cavity even after 28 days of treatment.

[0160] Table 2: Rhein levels in joints

[0161] Effect of compound of invention on ADAMTS5 Aggrecan is proteoglycan in articular cartilage, which helps to withstand compressive loads in joints. It is susceptible to cleavage by proteases, which leads to cartilage degradation in osteoarthritis. ADAMTS5 is an enzyme involved in degradation of aggrecan. ADAMTS5 is a major cause of cartilage breakdown and loss in osteoarthritis. Interleukin- 1 beta (IL- IB) increases ADAMTS5 and causes cartilage degradation in OA (Mol Med Rep. 2024 Jun 26;30(2): 149). Diacerein acts by preventing IL-1B signaling or secretion (Curr Drug Res Rev. 2022;14(3):215- 224). To measure the ADAMTS5, the synovial fluid was collected after 28 days of treatment as mentioned in the rabbit experiment above. Total protein was measured by Bradford method and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were measured by ELISA. The ADAMTS5 was normalized with total protein and data was shown in table 3.

[0162] Compound of the invention (Example 2) treated rabbit has reduced amount of ADAMTS5 (enzyme responsible for degradation of joint) than individual treatment with diacerein and hyaluronic acid. These data indicate compounds of the invention (Example 2) cause less degradation of joint than individual treatment with diacerein and hyaluronic acid.

[0163] Table 3: ADAMTS5 data

[0164] The novel compounds of general formula (I) and their pharmaceutically acceptable salts, of the present invention can be formulated into suitable pharmaceutically acceptable compositions by combining with suitable excipients by techniques and processes and concentrations as are well known.

[0165] The novel compounds of general formula (I) and their pharmaceutically acceptable salts, pharmaceutical compositions containing them are useful as a medicament for the treatment of osteoarthritis and suitable for humans and other warm blooded animals, and may be administered either by oral, topical or parenteral administration. The quantity of active component, that is, the novel compounds of general formula (I) and their pharmaceutically acceptable salts according to this invention, in the pharmaceutical composition and unit dosage form thereof may be varied or adjusted widely depending upon several factors such as the particular application method, the potency of the particular compound and the desired concentration.

[0166] The novel compounds of general formula (I) and their pharmaceutically acceptable salts, and pharmaceutical compositions of the present invention can be formulated into suitable pharmaceutically acceptable compositions by combining with suitable excipients by techniques and processes and concentrations as are well known. The pharmaceutical compositions further comprise an effective amount of an antiosteoarthritis agent. The dosage of anti-osteoarthritis agent may vary within wide limits and should be adjusted, in each particular case, to the individual conditions. Use of the novel compounds of general formula (I) and their pharmaceutically acceptable salts, or pharmaceutical compositions of the present invention use for the treatment of osteoarthritis.

[0167] The present invention includes a method for the treatment of osteoarthritis by administering to a subject in need thereof a therapeutically effective amount of a compound or salt of the novel compounds of general formula (I) and their pharmaceutically acceptable salts, or pharmaceutical compositions.

Claims

We claim:

1. A hyaluronic acid derivatives of general formula (I) or their pharmaceutically acceptable salts:wherein,A-CO- represents one residue of the hyaluronic acid disaccharide unit or its derivatives or its salt,L represents substituted or unsubstituted linear or branched hydrocarbon group having 2 to 10 carbon atoms or a 4-8 membered carbocyclic or heterocyclic group; wherein the hydrocarbon group also contains heteroatom such as oxygen, sulfur and nitrogen, 6-10 membered aromatic ring system, 3-8 membered carbocyclic ring system, 3-6 membered spirocyclic ring system in the carbon chain; wherein substitutions of L is selected from hydrogen, (Ci-Cio)alkyl, (C2-Cio)alkenyl, (C2-Cio)alkynyl, cycloalkyl, aryl, alkoxy, acyl, carboxylic group, cyano, halo, oxo, heteroatoms such as -O-, -NR.2-, -S(O)p-;Ri represents a carboxylic group containing residues selected from either Diacerein or its pharmaceutically acceptable salts, Rhein or its pharmaceutically acceptable salts;R2 represents hydrogen or (Ci-Cio)alkyl; p represents integers from 0-2;2. The hyaluronic acid derivatives of general formula (I) as claimed in claim, wherein the average molecular weight of hyaluronic acid ranges from 500000 to 3000000 Da.

3. The hyaluronic acid derivatives of general formula (I) as claimed in claim, wherein salts of hyaluronic acid are selected from sodium hyaluronate(NaHA), potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, and combinations thereof.

4. The hyaluronic acid derivatives of general formula (I) as claimed in claim 1, wherein L is selected from substituted or unsubstituted linear or branched hydrocarbon group having 2 to 10 carbon atoms or a 4-8 membered carbocyclic; the hydrocarbon group also contains heteroatom such as oxygen, and sulfur, 6-membered aromatic ring system, 3-6 membered carbocyclic ring system, 3-6 membered spirocyclic ring system in the carbon chain.

5. The hyaluronic acid derivatives of general formula (I) as claimed in claim 1:Aminoethanol-Diacerein-derivatised Sodium Hyaluronate; Aminopropanol-Diacerein-derivatised Sodium Hyaluronate; (2-hydroxyethoxy)ethylamine-Diacerein-derivatised Sodium Hyaluronate (2-hydroxyethyl)thio)ethyl amine-Diacerein-derivatised SodiumHyaluronate;(Aminomethyl)benzyl)oxy)-Diacerein-derivatised Sodium Hyaluronate;Aminomethyl cyclopropyl methanol -Diacerein-derivatised Sodium Hyaluronate;Aminocyclohexyl- 1-ol-Diacerein-derivatised Sodium Hyaluronate; Aminoethanol-Rhein-derivatised Sodium Hyaluronate.

6. Use of the hyaluronic acid derivatives of general formula (I) as claimed in claim 1 for the treatment of osteoarthritis.

7. Method of treating osteoarthritis in a subject comprising administering to a patient in need thereof a therapeutically effective amount of the hyaluronic acid derivatives of general formula (I) as claimed in claim 1.

8. A novel intermediate for the preparation of novel hyaluronic acid derivatives are: 3-((3-aminopropoxy)carbonyl)-9,10-dioxo-9,10-dihydroanthracene-l,8- diyl diacetate or its hydrochloride salt;3-( 11 , 1 l-dimethyl-9-oxo-2,5, 10-trioxa-8-azadodecanoyl)-9, 10-dioxo-9, 10- dihydroanthracene- 1 , 8 -diyl diacetate;3-((2-(2-aminoethoxy)ethoxy)carbonyl)-9, 10-dioxo-9, 10- dihydroanthracene-l,8-diyl diacetate or its hydrochloride salt;3-(l l,l l-dimethyl-9-oxo-2,10-dioxa-5-thia-8-azadodecanoyl)-9,10-dioxo-9, 10-dihydroanthracene- 1 , 8 -diyl diacetate;3-((2-((2-aminoethyl)thio)ethoxy)carbonyl)-9, 10-dioxo-9, 10- dihydroanthracene-l,8-diyl diacetate or its hydrochloride salt;3-(((4-(aminomethyl)benzyl)oxy)carbonyl)-9, 10-dioxo-9, 10- dihydroanthracene- 1,8-diyl diacetate or its hydrochloride salt;3-(((l-(aminomethyl)cyclopropyl)methoxy)carbonyl)-9,10-dioxo-9, 10- dihydroanthracene- 1,8-diyl diacetate or its hydrochloride salt;3-((((lr,4r)-4-aminocyclohexyl)oxy)carbonyl)-9,10-dioxo-9, 10- dihydroanthracene- 1,8-diyl diacetate or its hydrochloride salt.Dated this on 13thday of February 2025

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