Hydroxybisphosphonic derivatives of meloxicam for the treatment of osteoarticular inflammatory diseases

Abstract

The invention relates to the field of targeted treatments for inflammatory osteoarticular diseases. More particularly, the invention relates to novel water-soluble hydroxybisphosphonic derivatives of meloxicam, their use as a medicinal product, particularly for the treatment of inflammatory osteoarticular diseases, a pharmaceutical composition containing them, and their synthesis process.

Description

Title of the invention: Hydroxybisphosphonic derivatives of meloxicam for the treatment of osteoarticular inflammatory diseases

[0001] The invention relates to the field of targeted treatments for inflammatory osteoarticular diseases. More particularly, the invention relates to novel water-soluble hydroxybisphosphonic derivatives of meloxicam, their use as a medicinal product, particularly for the treatment of inflammatory osteoarticular diseases, a pharmaceutical composition containing them, and their synthesis process. Field of the invention

[0002] Bone tissue is a connective tissue composed of a mineral fraction consisting of calcium phosphate in the form of hydroxyapatite crystals and an organic fraction containing an extracellular matrix and specialized cells. Bone tissue is constantly being remodeled through a process called "bone remodeling." This process is characterized by an apposition phase due to the activity of osteoblasts, which synthesize a new organic matrix and induce its mineralization (Owen et al., Curr. Opin. Nephrol. Hypertens. 1998, 7, 363), and a degradation phase carried out by osteoclasts, which resorb the organic matrix and dissolve the mineral (Roodman et al., Endocr. Rev. 1996, 17, 308). This physiological process helps to maintain phosphocalcic homeostasis and bone mass (Manologas et al., Endocriv. Rev. 2000, 21, 115) and to adapt to mechanical stresses.Disruption of this balance due to inflammation can lead to the development of osteosclerotic or osteolytic pathologies. In both humans and animals, these bone disorders cause chronic pain (lower back pain) and weaken the patient's skeleton, leading to fractures. The pathology can then progress to temporary or permanent immobilization of the patient. Treatment of these conditions requires the chronic administration of anti-inflammatory drugs.

[0003] Non-steroidal anti-inflammatory drugs (NSAIDs) are the standard treatment for inflammatory and painful conditions, including chronic osteoarticular diseases such as arthritis and osteoarthritis. Regular and frequent administration is often necessary for optimal effectiveness. However, prolonged use of these products can cause serious renal and gastrointestinal side effects, which is a significant problem.

[0004] To limit side effects, it has been proposed to conjugate NSAIDs to bisphosphonic molecules. Bisphosphonic molecules are known for their affinity for bone hydroxyapatite (HA) and can be used as molecules targeting, which helps limit the systemic effects of NSAIDs. Furthermore, these targeting molecules can be cleaved at the bone level to release the active ingredient; this in situ release near the bone can be accompanied by activation of the molecule whose activity was previously inhibited. This two-step activation is also advantageous for limiting distant systemic side effects.

[0005] Inflammatory osteoarticular diseases cause both osteolytic damage by releasing bone surface tissue and osteosclerotic damage by inducing bone growths. These two types of bone changes are attractive to bisphosphonic molecules, which increases the selectivity of targeting to pathological joints compared to normal bone. One variety of bisphosphonates (BPs) are hydroxybisphosphonates (HBPs), known for their superior affinity for bone.

[0006] Sporadic studies report trials of conjugating anti-inflammatory molecules with bisphosphonates (BPs). The aim of this approach is to use BPs as a targeting molecule for the anti-inflammatory active ingredient at the bone tissue level, limiting the systemic dispersion of NSAIDs. Furthermore, local cleavage of the active ingredient allows for targeted action. For example, an initial study reported the conjugation of cortisone with bisphosphonic derivatives (Guervenou et al., Phosphorus Sulfur and Silicon, 1994, 88, 1-13), but it did not confirm the ability to release the active ingredient, and no anti-inflammatory effect was documented.A second study presents the use of bisphosphonic derivatives of an NSAID, diclofenac; the results show a concentration of diclofenac at the bone site followed by the release of the active ingredient, allowing for a reduction in the effective dose accompanied by the disappearance of the classic gastrointestinal side effects of NSAIDs (H. Hirabayashi et al., Journal of Controlled Release 70, 2001, 183-191; H. Hirabayashi et al., Pharmaceutical Research, 18(5), 2001, 646-651). This work confirms the value of the localized targeting strategy using bisphosphonic molecules; however, the choice of diclofenac remains questionable due to its higher risk of gastrointestinal toxicity compared to other NSAIDs such as meloxicam (C. Hawkey et al., British Journal of Rheumatology, 1998, 37, 937-945). Furthermore, in this study, the BP vector used exhibits a lower affinity for bone than that of HBP derivatives.

[0007] Finally, a recently published study describes the use of HBP derivatives of another NSAID, ibuprofen. However, the ability of ibuprofen to release itself in situ and its anti-inflammatory efficacy are not mentioned (Aoun et al., Synthesis 2019, 51, AK).

[0008] Apart from these few studies, the field of vectorization of anti-inflammatory molecules using bisphosphonic vectors has not been truly explored and data relating to the effectiveness of these molecules in the treatment of osteoarthritis and osteoarthritis are lacking.

[0009] Meloxicam is an NSAID commonly used in veterinary medicine. It is also used in humans (Mobic) to relieve acute flare-ups of osteoarthritis, rheumatoid arthritis and ankylosing spondylitis, but with the current formulation, treatment must be short-term and at the lowest possible dose, due to the systemic side effects it causes.

[0010] In parallel, the conjugation of anticancer and antibacterial molecules to bisphosphonic derivatives has been more widely explored, both from the point of view of the synthesis of the conjugates and of the biological activity of the latter (Farrell et al., Bone Reports 9, 2018, 47-60; Xing et al., Bone 138, 2020, 115492).

[0011] To date, the need for anti-inflammatory treatments for osteoarticular diseases that are effective and free of long-term side effects is not met. Description of the invention

[0012] The anti-inflammatory molecules of the present invention consist of a delivery system for a non-steroidal anti-inflammatory drug (NSAID), Meloxicam (MLX) – a preferential COX-2 inhibitor – using a hydroxybisphosphate (HBP) vector that targets bone tissue. These bifunctional molecules are proposed for the treatment of osteoarticular inflammation and consist of three parts: (i) an HBP vector that targets bone and delivers the active ingredient, (ii) the active ingredient, Meloxicam (MLX), and (iii) a linker connecting the HBP and MLX parts, capable of ensuring the release of MLX.

[0013] Thus, the invention relates to new hydroxybisphosphonic derivatives of Meloxicam of formula (I) as defined below, or a pharmaceutically acceptable salt thereof.

[0014] The invention also relates to the use of a hydroxybisphosphonic derivative of Meloxicam of formula (I) as a drug and more particularly for the treatment of inflammatory osteoarticular diseases.

[0015] The invention also relates to a method for synthesizing hydroxybisphosphonic derivatives of Meloxicam of formula (I), or a pharmaceutically acceptable salt thereof.

[0016] Finally, the invention relates to a pharmaceutical composition comprising a hydroxybisphosphonic acid derivative of formula (I) or a pharmaceutically acceptable salt thereof, and at least one excipient. Advantages of the invention

[0017] The present invention opens up new perspectives in the treatment of Inflammatory osteoarticular diseases are being treated with an innovative approach to effectively target NSAIDs to the inflamed bone area. This approach involves delivering NSAIDs, in this case Meloxicam, via HBP derivatives.

[0018] By targeting MLX to the osteoarticular area, the local concentration of MLX can be increased without inducing systemic side effects. Anti-inflammatory efficacy is thus enhanced. This strategy has the advantage of broadening the therapeutic window of MLX and providing stronger and longer-lasting pain relief of inflammatory origin after administration, thanks to the gradual release of meloxicam. Treatment can also be considered long-term, with repeated administrations, due to reduced systemic toxicity. Furthermore, the gradual release allows the anti-inflammatory effect to be maintained for at least two weeks, whereas with meloxicam, only daily administration is necessary for pain relief.This significantly improves the treatment of inflammation (reduced side effects and increased efficacy) by offering administrations spaced out over time, and whose deleterious effects are significantly attenuated.

[0019] The vectorization strategy can be adapted according to the therapeutic approach envisaged by choosing the linker.

[0020] In an advantageous embodiment of the invention, MLX is linked to the HBP vector by a cleavable linker. The HBP-MLX molecule is then in the form of a prodrug which will be converted into an active molecule at the pathological site by cleavage and release of MLX.

[0021] In another embodiment, the HBP-MLX molecules are not cleavable. This results in lower efficacy of MLX but opens the possibility of oral administration. Indeed, in this case, the molecules must be stable in the extremely acidic environment of the stomach.

[0022] The hydroxybisphosphonic vector (HBP Vector) associated with Meloxicam in the present invention is described in patent (WO2016079327) where it was used to vector the anticancer molecule Doxorubicin by forming an imine bond with its ketone group. Here, Meloxicam (MLX) is linked to the HBP Vector by a linker; this linker forms a bridge between, on one side, MLX via a potentially cleavable bond and, on the other side, the vector via a stable imine bond. This strategy facilitates synthesis, allowing the addition of the vector (HBP Vector) during the final step. Free HBP derivatives are highly polar, hydrophilic, chelating, and mostly insoluble in conventional organic solvents, with water being the best solvent. For this reason, the best strategy is to introduce this grouping at the end of the synthesis, which is made possible by the imine strategy chosen in the present invention.Patents WO2012130911 and . WO2016079327 exemplify an equivalent approach using other vectors; this approach makes it possible to vectorize complex polyfunctional molecules, which would be more complicated, or even impossible, by linear synthesis routes, as described in patent application FR 2 926 081. Thus, the synthesis process proposed here is more universal, economical, ecological and transferable than linear processes.

[0023] Finally, the HBP portion gives the final molecules good solubility in water, whereas MLX is very poorly soluble in it. Vectorization therefore contributes to improving the bioavailability of the active ingredient. DETAILED DESCRIPTION OF THE INVENTION

[0024] A first object of the invention relates to a hydroxybisphosphonic derivative of Meloxicam of general formula (I) or a pharmaceutically acceptable salt thereof,

[0025] [Chem.l]

[0026] wherein one and only one of the radicals RI and R2 represents an H (hydrogen) group or is absent and

[0027] - when R2 is absent then X is chosen from the following groups Rl-A, Rl-B and Rl-C:

[0028] [Chem.2] (Rl-A) [Chem. 3]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] (Rl-B) [Chem. 4] (Rl-C) - when RI = H then X is chosen from the following groups R2-D, R2-E, R2-F, R2-G and R2-H: [Chem. 5] » (R2-D) [Chem. 6] K (R2-E) [Chem. 7] " (R2-F) [Chem. 8] (R2-G) [Chem.9] (R2-H)

[0037] In a particular embodiment of the invention, the hydroxy-bisphosphonic acid derivatives described above can be in their tautomeric form and are represented by the general formula (II)

[0038] [Chem. 10] R X O OH (he)

[0039] in which R2 is chosen from the groups R2-D, R2-E, R2-F, R2-G and R2-H as described above.

[0040] The corresponding molecules are, in the order of presentation of the compounds: A (18A166), B (19A143), C (19A115), D (18A135), E (18A184), F (18A182), G (19A4) and H (19A22). These molecules are represented below.

[0041] Pharmaceutically acceptable salts of these compounds are obtained by association with organic or mineral bases. In a preferred embodiment of the invention, these are sodium salts or meglumine salts.

[0042] A second object of the invention relates to the use of a hydroxybisphos-phonic derivative of Meloxicam as described above as a medicinal product.

[0043] This medicinal product can be used in veterinary and human medicine. Due to its targeted action on the osteoarticular areas, the frequency of administration can be reduced to a single injection with a prolonged effect thanks to the gradual release of Meloxicam (over a minimum period of 2 weeks). It can be administered long-term or repeatedly while limiting systemic side effects. themic.

[0044] Since the HBP vector has a tropism for bone and MLX possesses anti-inflammatory properties, the hydroxybisphosphonic derivatives of meloxicam according to the invention can be advantageously used in the treatment of inflammatory osteoarticular diseases. It is particularly suitable for treating pain associated with chronic inflammatory osteoarticular diseases such as osteoarthritis and arthritis. In a preferred embodiment of the invention, hydroxybisphosphonic derivatives of meloxicam are used in veterinary medicine, particularly in dogs and cats. This treatment is nevertheless suitable for animals suffering from inflammatory osteoarticular diseases.

[0045] In a preferred embodiment, the linker X enables cleavage of MLX at the osteoarticular level. The molecule of formula (I) then exists as a prodrug that can be activated by cleavage of MLX. In this configuration, X is selected from among the RrA, RrB, and RrC, R2-E, and R2-G groups. Conversely, molecules incorporating the R2-D and R2-F groups are non-cleavable, while the one incorporating the R2-H group is theoretically cleavable in vivo.

[0046] In a most preferred embodiment, the HBP-MLX molecule is compound C (19A115) or compound E (18A184).

[0047] A third object of the invention relates to a process for synthesizing a hydroxybisphosphonic derivative of Meloxicam of formula (I) as defined above, comprising the steps of:

[0048] - Coupling of Meloxicam to a linker X chosen from the groups RrA, Ri-B and RrC, R2-D, R2-E, R2-F, R2-G and R2-H as defined previously; - Coupling of the HBP derivative to the opposite end of the linker relative to the Meloxicam.

[0049] In this process, the coupling of Meloxicam to the linker is done via a potentially and preferentially cleavable bond, while the coupling of the linker to the HBP is done via a cleavage-resistant bond of the stable -C=N- imine type.

[0050] The compounds obtained can be purified by chromatography or precipitation. The purification method will be chosen by those skilled in the art based on the efficiency of each approach for each molecule and according to its form. In particular, it should be noted, for example, that compound E (18A184) can be obtained by chromatographic purification as a sodium salt and by precipitation as sodium and meglumine salts. The number of base equivalents can vary from 0, which corresponds to free hydroxybisphosphonic acids, to 4 and even 5 for molecule C (19A115). In a preferred embodiment, it is 2 to 3 base equivalents.

[0051] The strategy for synthesizing molecules according to the invention consists first of synthesis

[0052]

[0053]

[0054] of Meloxicam-Linker from Meloxicam, then coupling it with the vector to obtain the following final product: Meloxicam + Linker —> Meloxicam-Linker —[+HBP Vector]—> Compounds A, B, C, D, E, F, G or H. Compounds A, B, C, D, E, F, G and H are as represented below: [Chem. 11] ohP h HO O'

[0055] (HAS) [Chem. 12] NaO o HO— L-OH OH OO (B) [Chem. 13] (C)

[0057] And tautomers for the following compounds:

[0058] [Chem. 14] i-tr P PH q oh N

[0059]

[0061]

[0062] [Chem. 17] (H)

[0063] A fourth object of the invention relates to a pharmaceutical composition comprising a hydroxybisphosphonic derivative of meloxicam as defined above or a pharmaceutically acceptable salt thereof and at least one excipient. The salts may be chosen from those obtained either with mineral bases such as sodium salts, and those obtained with organic bases such as meglumine salts.

[0064] The hydroxybisphosphonic derivative of such a composition is preferably chosen from compounds A, B, C, D, E, F, G and H.

[0065] In a most preferred embodiment, the composition comprises compound C (19A115) or compound E (18A184) in the form of free acid or salts, such as sodium or meglumine salts.

[0066] This composition can be formulated to allow administration, in particular, by subcutaneous, intravenous, oral, intramuscular or transdermal route, that is to say preferably in the form of an injectable solution or in the form of a patch, and is intended for use in humans and animals. The dosage will be adapted according to the individual (weight, age, etc.) and the disease.

[0067] The compounds according to the invention can be used at doses ranging from 0.01 mg to 100 mg per day, administered as a single dose once daily or in multiple doses, for example, two equivalent doses. The daily dose is advantageously between 5 mg and 100 mg, and even more advantageously between 10 mg and 200 mg. It may be necessary to use doses outside these ranges; those skilled in the art will be able to assess this need.

[0068] The present invention will be better understood upon reading the following examples, which are provided by way of illustration and should in no way be considered as limiting the scope of the present invention. DESCRIPTION OF THE FIGURES

[0069] [fig.1][fig.1]: Synthesis of compound A: preparation of intermediate compound 2 from compound 1 and Meloxicam

[0070] [fig.2][fig.2]: Synthesis of compound A: preparation of compound A from the composed of 2 and the vector HBP

[0071] [fig.3][fig.3]: Synthesis of compound B: preparation of intermediate compound 5 including Meloxicam

[0072] [fig.4][fig.4]: Synthesis of compound B: preparation of compound B from the compound 5 and the vector HBP

[0073] [fig.5][fig.5]: Synthesis of compound C: preparation of intermediate compound 7 at starting from compound 6

[0074] [fig.6][fig.6]: Synthesis of compound C: preparation of intermediate compound 9 at starting from compound 7 and meloxicam

[0075] [fig.7] [fig.7]: Synthesis of compound C: preparation of compound C from the compound 9 and the vector HBP

[0076] [fig.8] [fig.8]: Synthesis of compound D: preparation of intermediate compound 11

[0077] [fig.9][fig.9]: Synthesis of compound D: preparation of intermediate compound 12 at starting from compound 11

[0078] [fig. 10] [fig. 10]: Synthesis of compound D: preparation of intermediate compound 13 from compound 12 and Meloxicam

[0079] [fig. 11][fig. 11]: Synthesis of compound D: preparation of compound D from compound 13 and the HBP vector

[0080] [fig. 12] [fig. 12]: Synthesis of compound E: preparation of intermediate compound 16

[0081] [fig. 13] [fig. 13]: Synthesis of compound E: preparation of intermediate compound 17 from compound 16 and Meloxicam

[0082] [fig. 14] [fig. 14]: Synthesis of compound E: preparation of compound E from compound 17 and the HBP vector

[0083] [fig. 15] [fig. 15]: Synthesis of compound F: preparation of intermediate compound 20 from compounds 18 and 19

[0084] [fig. 16] [fig. 16]: Synthesis of compound F: preparation of intermediate compound 21 from compound 20 and Meloxicam

[0085] [fig. 17] [fig. 17]: Synthesis of compound F: preparation of compound F from compound 21 and the HBP vector

[0086] [fig. 18] [fig. 18]: Synthesis of compound G: preparation of intermediate compound 23 from compounds 15 and 22

[0087] [fig. 19] [fig. 19]: Synthesis of compound G: preparation of intermediate compound 24 from compound 23 and Meloxicam

[0088] [fig.20][fig.20]: Synthesis of compound G: preparation of compound G from the composed of 24 and the vector HBP

[0089] [fig.21] [fig.21]: Synthesis of compound H: preparation of intermediate compound 25 from compound 11

[0090] [fig.22][fig.22]: Synthesis of compound H: preparation of intermediate compound 26 from compound 25 and Meloxicam

[0091] [fig.23][fig.23]: Synthesis of compound H: preparation of compound H from the compound 26 and the vector HBP

[0092] [fig.24][fig.24]: In vitro evaluation of the binding capacity of HBP- molecules Meloxicam with hydroxyapatite

[0093] [fig.25][fig.25]: Evaluation of the Meloxicam release capacity by the cleavable HBP-Meloxicam molecules

[0094] [fig.26][fig.26] : ELISA analysis of the anti-COX2 activity of HBP- molecules Meloxicam on chondrocytes

[0095] [fig.27] [fig.27]: Toxicity evaluation of HBP-Meloxicam molecules on the stomach, duodenum and kidneys after administration in rats

[0096] [fig.28][fig.28]: Therapeutic effect of HBP-Meloxicam compounds on the knee swelling caused by arthritis.

[0097] [fig.29][fig.29]: Therapeutic effect of HBP-Meloxicam compounds on lameness caused by arthritis. EXAMPLES

[0098] The HBP vector is synthesized according to the process described in document WO2016 / 079327. EXAMPLE 1: Synthesis of compound A (18A166)

[0099] The synthesis of compound A is shown in Figures 1 and 2.

[0100] Compound 1 (1.088 g, 4.98 mmol, 1.17 eq) was added to a solution of Meloxicam (1.5 g, 4.27 mmol, 1 eq) and triethanolamine (TEA) (0.75 mL, 5.4 mmol, 1.26 eq) were reacted in dichloromethane (DCM) (6 mL). The reaction mixture was stirred for 2 h at room temperature. The resulting solid was centrifuged, washed with DCM (2 x 7 mL), and dried under vacuum at room temperature. Compound 2 was obtained (a clear yellow solid, 1.867 g, 3.5 mmol, yield 82%, UPLC-MS: 100%) and characterized by 1H NMR, MS, and UV spectra ([Fig. 1]).

[0101] A solution of compound 2 (1.5 g, 2.81 mmol, 1 eq) and HBP vector (70 wt., 1 g, 2.95 mmol, 1.05 eq) in 5% TFA / DMSO (15 mL) was stirred for 16 h at room temperature. Then, an aqueous solution of NaHCO3 (0.5 M, 46 mL) was added, followed by 350 mL of water. The resulting colloidal solution was introduced into a C18 column (4 x 40 g) and eluted in a gradient from 3% EtOH to 50% EtOH. The fractions (HPLC >90%) were pooled. EtOH was evaporated under vacuum, and the resulting solution was lyophilized. Compound A (18A166) was obtained (a clear yellow solid, 1.11 g, 1.39 mmol, yield 50%), HPLC: 95% and characterized by 1H NMR, 31P NMR, MS and UV spectra ( [fig-2]). EXAMPLE 2: Synthesis of compound B (19A143)

[0102] The principle is the same as for compound A.

[0103] The synthesis of compound B is shown in Figures 3 and 4.

[0104] Meloxicam (1 eq., 686 mg, 1.95 mmol) was solubilized in DCM (3 mL) and Et3N (1.3 eq., 350 pL, 2.54 mmol) under argon, forming a yellow solution. Then, 3-Acetylbenzenesulfonyl chloride (4) (1.17 eq., 500 mg, 2.29 mmol) was added, and the reaction mixture was stirred for 15 min at room temperature. The reaction mixture was evaporated under vacuum with silica gel, introduced into a silica gel column, and eluted in a 9:1 to 5:5 cHex / EtOAc gradient. The fractions have were evaporated under vacuum, TLC: 4 / 6 cHex / EtOAc. A compound 5 was obtained (a yellow solid, 530 mg, 79% UPLC-MS, yield 40%). UPLC-MS: 60-80% and characterized by 1H NMR, MS and UV spectra ([fig.3]).

[0105] Compound 5 (1 eq., 530 mg, 0.785 mmol) was added to the HBP vector solution (1.25 eq., 314 mg, 0.994 mmol) in 1% TFA / DMSO (4 mL). The reaction mixture was stirred for 4 h at room temperature under argon and monitored by HPLC. A 0.5 M NaHCO3 solution was added to the reaction mixture until a neutral pH was reached (precipitation), followed by the addition of MeOH. The solid was filtered, washed with MeOH, and dried under vacuum at room temperature. The resulting yellow solid was solubilized in MilliQ water and purified on a C18 column with a gradient from 3% EtOH to 30% EtOH. The HPLC fractions >90% were pooled and lyophilized. Compound C (19A143) was obtained (a yellow solid, 226 mg, yield 36%), HPLC: 95%, and characterized by 1H NMR, 31P NMR, MS, and UV spectra ([Fig. 4]). EXAMPLE 3: Synthesis of compound C (19A115)

[0106] The principle is the same as for compound A.

[0107] The synthesis of compound C is shown in Figures 5, 6 and 7.

[0108] A solution of TEA (5.7 mL, 41 mmol, 1 eq) in DCM (10 mL) was added dropwise to a solution of compound 6 (5 g, 41 mmol, 1 eq) and POC13 (38 mL, 408 mmol, 9.96 eq) in DCM (100 mL) at 0 °C for 8 min, during which a precipitate formed. The reaction mixture was stirred for 1 h at 0 °C and then concentrated under vacuum. Ether (50 mL) was added, the solid was filtered, and the resulting solution was concentrated under vacuum for 5 h. Crude compound 7 was obtained (a viscous orange oil, 8.5 g, 35.6 mmol, yield 87%) and characterized by 1H NMR, 31P NMR, MS and UV spectra ([fig.5]).

[0109] Compound 7 (1.5 g, 6.28 mmol, 2.21 eq) was added to the solution of meloxicam (1 g, 2.85 mmol, 1 eq) and TEA (1 ml, 7.19 mmol, 2.53 eq) in DCM (8 ml) at room temperature. The reaction mixture was stirred for 45 min at room temperature and then added to the pentane / 0.5 M HCl mixture, shaken, filtered, the solid was washed with 0.5 M HCl and dried under vacuum. A compound 9 was obtained (a yellow solid, 1.84 g, 81% purity, 92% yield for the pure composite) and characterized by 1H NMR, 31P NMR, MS and UV spectra ([Fig. 6]).

[0110] Compound 9 (2.84 g, 4.97 mmol, 1 eq) was added to the solution of HBP Vector (1.8 g, 5.47 mmol, 1.1 eq) in 5% TFA / DMSO (8 mL), and the resulting viscous solution was stirred and vortexed for 20 min at room temperature. The reaction mixture was then solubilized in phosphate buffer, forming a colloidal solution, which was introduced into a Cl8 column and eluted using a gradient from 3% EtOH to 20% EtOH. The fractions, yielding over 90% of the HPLC, were pooled and lyophilized. Compound C (19A115) was obtained (a clear yellow solid, 1.205 g, yield 34%), HPLC: 98% and characterized by 1H NMR, 31P NMR, MS and UV spectra ([fig.7]). EXAMPLE 4: Synthesis of compound D (18A135)

[0111] The principle is the same as for compound A.

[0112] The synthesis of compound D is shown in Figures 8, 9, 10 and 11.

[0113] 1,4-Phthalaldehyde 10 (4 eq., 10 g, 74.6 mmol) was solubilized in DCM (25 mL) and MeOH (25 mL) at 40 °C, forming a yellow solution. The resulting solution was cooled in a cold water bath, and NaBH4 (1 eq., 0.705 g, 18.6 mmol) was added for 5 min. An opaque yellow solution was obtained. The reaction was immediate. A TLC control, DCM: 100%, was used. Silica gel (20 g) was added, the solvent was completely evaporated under vacuum, and the mixture was eluted on a silica gel column in a gradient from 100% DCM to 10% MeOH / DCM. The pure fractions were concentrated under vacuum. Product 11 is obtained at 5.3 g, a yellow oil which crystallizes (yield 52%, UPLC-MS: 100%) and is characterized by 1H NMR, MS and UV spectra ([fig.8]).

[0114] 4-(Hydroxymethyl)benzaldehyde 11 (1 eq., 1.0 g, 7.34 mmol) was solubilized in toluene (7 mL). Then, HBr 48 wt.% in H2O (3.3 eq., 4.0 g, 2.7 mL, 24.24 mmol) was added, and the reaction mixture was refluxed for 3 h. The reaction was monitored by UPLC-MS. The reaction was complete. Chloroform (60 mL) was added, and the resulting solution was extracted with aqueous NaHCO3 until complete acid neutralization. The organic phase was dried with anhydrous Na2SO4 and concentrated at reduced pressure. Beige crystals of compound 12 were obtained, 1.69 g (yield 100%, UPLC-MS: 97%) and characterized by 1H NMR, MS and UV spectra. ([fig.9])

[0115] Meloxicam (1.1 eq., 0.50 g, 1.42 mmol) was solubilized in DMF (20 mL) under argon. Then, NaH (2.3 eq., 0.13 g, 3.25 mmol) was added, and the reaction mixture was stirred for 5–10 min at room temperature. The reaction mixture was cooled with an ice bath, and 4-bromomethylbenzaldehyde 12 (1 eq., 0.255 g, 1.2 mmol) was added. The reaction was quenched for 5–10 min with NH4Cl(aq), resulting in the formation of a precipitate. After extraction of the reaction mixture with EtOAc, the organic phase was washed with NaCl(sat), dried with anhydrous Na2SO4, concentrated at reduced pressure, and used in the next step. Product 13 was characterized by 1H NMR, MS and UV spectra ([fig.10]).

[0116] The HBP vector (1 eq., 0.35 g, 1.11 mmol) was solubilized in DMSO (20 mL) with a few drops of TFA. The crude product 13 from the previous step (1 eq., 1.20 g, 1.11 mmol) was added to this colorless mixture. The resulting yellow solution was stirred at room temperature for 1 h. A 0.5 M NaHCO3 aqueous solution (30 mL) was added, followed by MeOH. The solid was filtered, washed with MeOH, and dried under vacuum. The resulting yellow solid (500 mg) was solubilized in MilliQ water and purified. On a C18 column: gradient at 20 ml / min (3% → 25% EtOH), the >90% HPLC fractions (HPLC, UV at 360 nm) were concentrated under vacuum and lyophilized. Compound D (18A135) was obtained, 150 mg (a yellow solid, 20% yield) and characterized by 1H NMR, 31P NMR, MS, and UV spectra ([Fig. 11]). EXAMPLE 5: Synthesis of compound E (18A184)

[0117] The principle is the same as for compound A.

[0118] The synthesis of compound E is shown in Figures 12, 13 and 14.

[0119] Compound 15 (1.31 ml, 12.73 mmol, 1.21 eq) was added to the compound solution Compound 14 (1.58 g, 10.52 mmol, 1 eq), tetrabutylammonium hydrosulfate (0.353 g, 1.04 mmol, 0.1 eq), and NaHCO3 (3.53 g, 42 mmol, 4 eq) were mixed with water (22 mL) and DCM (22 mL) under vigorous stirring at room temperature. After 3 h and extraction with DCM / 0.5 M NaHCO3 followed by water, the organic phase was dried with anhydrous Na2SO4 and concentrated. Compound 16 was obtained (a clear oil that solidifies at +4 °C to form a colorless solid, 2.12 g, 10.67 mmol, 100% yield) and was used in the next step without further purification. The product was characterized by UPLC and 1H NMR, MS and UV spectra ([fig. 12]).

[0120] Compound 16 (2.12 g, 10.67 mmol, 1.25 eq) was added to a solution of meloxicam (3 g, 8.54 mmol, 1 eq) and TEA (2.4 mL, 17.27 mmol, 2 eq) in DCM (12 mL). The reaction mixture was stirred for 3 days at room temperature. After extraction with a DCM / MeOH / H2O solution, the organic phase was dried with anhydrous Na2SO4 and concentrated under vacuum. Compound 17 was obtained (a yellow solid, 4.44 g, 8.65 mmol, 100% yield, UPLC-MS: 100%) and characterized by 1H NMR, MS, and UV spectra. ([Fig. 13])

[0121] The solution of compound 17 (1.66 g, 3.23 mmol, 1 eq), the HBP vector (78%, 1.11 g, 3.66 mmol, 1.13 eq), and TFA (0.1 mL, 1.35 mmol, 0.42 eq) in DMSO (15 mL) were stirred for 20 min at room temperature. Then, 0.5 M aqueous NaHCO3 solution was added, followed by water. The resulting colloidal solution was introduced into a C18 column and eluted in a gradient from 3% EtOH to 50% EtOH. The HPLC fractions >90% were pooled, the EtOH was evaporated under vacuum, and the resulting solution was lyophilized. Compound E (18A184) was obtained (a yellow solid, 0.78 g, 1 mmol, yield 31%), HPLC: 98%, and characterized by 1H NMR, 31P NMR, MS, and UV spectra. ([Fig. 14]) EXAMPLE 6: Synthesis of compound F (18A182)

[0122] The principle is the same as for compound A.

[0123] The synthesis of compound F is shown in Figures 15, 16 and 17.

[0124] The solution of 3-hydroxybenzaldehyde (compound 18) (1 eq., 2.0 g, 16.38 mmol), 1,3-dibromopropane (compound 19) (4 eq., 6.6 ml, 65.51 mmol) and K2CO3 (1.3 eq., 2.9 g (21.29 mmol) in ethylene glycol (15 mL) was stirred for 5 h at 80 °C. The reaction was monitored by UPLC-MS. The reaction mixture was diluted with water and an extract with ether. The organic phase was dried with Na₂SO₄ and concentrated under vacuum at 40 °C. The resulting colorless oil (UPLC-MS 69%) was purified on a silica gel column, gradient elution 100% cHex → 6% EtOAc / cHex, TLC: cHex / EtOAc = 9:1. The solvent was evaporated under reduced pressure. Compound 20 was obtained, 1 g (yield 26%), and characterized by 1H NMR, MS, and UV spectra ([Fig. 15]).

[0125] Meloxicam (1 eq., 0.5 g, 1.42 mmol) was solubilized in DMF (25 mL) under argon. Then NaH (4.4 eq., 0.15 g, 6.25 mmol) and Nal (1 eq., 0.21 g, 1.42 mmol) were added. The reaction mixture was stirred at room temperature for 5–10 min. Compound 20 (2 eq., 0.7 g, 2.88 mmol) was added, and after 1 h, a SAT NH4Cl solution was added.After extraction with EtOAc, the organic phase was washed with NaCl(sat), dried with Na2SO4, and concentrated under vacuum at 40 °C. Compound 21 was obtained, 1.65 g, yield 65%, and characterized by MS and UV spectra ([fig. 16]).

[0126] Vector BP (1 eq., 0.45 g, 1.42 mmol) was solubilized in DMSO (10 mL) with a few drops of TFA. Compound 21 (1 eq., 1.65 g, 1.42 mmol) was added to the resulting colorless solution, and the yellow solution was stirred at room temperature for 1 h. 0.5 M NaHCO3 (20 mL) was added, followed by MeOH. The solid was filtered, washed with MeOH, and dried under vacuum. The resulting yellow solid (1 g) was solubilized in MilliQ water and purified on a C18 column with a gradient from 3% EtOH to 25% EtOH. The HPLC fractions >90% were concentrated under vacuum and lyophilized. Compound F (18A182) was obtained, 350 mg (a yellow solid, yield 30%), HPLC: 95% and characterized by 1H NMR, 31P NMR, MS and UV spectra ([fig. 17]). EXAMPLE 7: Synthesis of compound G (19A4)

[0127] The principle is the same as for compound G.

[0128] The synthesis of compound G is shown in Figures 18, 19 and 20.

[0129] Compound 15 (1.81 ml, 17.58 mmol, 1 eq) was added to the solution of compound 22 (2.92 ml, 28.42 mmol, 1.62 eq), tetrabutylammonium hydrosulfate (0.78 g, 2.3 mmol, 0.13 eq), and NaHCO3 (7.84 g, 93 mmol, 5.3 eq) were mixed with water (50 ml) and DCM (50 ml) under vigorous stirring at room temperature. After 3 h and extraction with DCM / 0.5M NaHCO3, the organic phase was dried with anhydrous Na2SO4 and concentrated under vacuum. Compound 23, a colorless oil (2.68 g, 16.28 mmol, 93% yield), was obtained and used in the next step without further purification. It was characterized by 1H NMR spectra ([Fig. 18]).

[0130] Compound 23 (2.3 g, 14 mmol, 1.4 eq) was added to the solution of Meloxicam (3.5 g, 10 mmol, 1 eq) and TEA (2.8 mL, 20.14 mmol, 2 eq) in DCM (14 mL). The reaction mixture was stirred for 48 h at room temperature and introduced into a column with a silica gel and eluted in a cHex—> EA gradient, TLC: DCM / EA=3 / 1. The solvent was evaporated under reduced pressure. Compound 24 was obtained (a yellow solid, 1.782 g, 3.716 mmol, yield 37%), UPLC-MS: 85% and characterized by 1H NMR, MS and UV spectra ([fig. 19]).

[0131] Compound 24 (1.66 g, 3.46 mmol, 1 eq) was added to the HBP (1.11 g, 3.65 mmol, 1.06 eq) and TFA (0.1 mL, 1.35 mmol, 0.39 eq) vector solution in DMSO (15 mL) and stirred for 24 h at room temperature. Then, 0.5 M aqueous NaHCO3 solution was added, followed by water. The resulting colloidal solution was introduced into a C18 column and eluted in a gradient from 3% EtOH to 50% EtOH. The HPLC fractions >90% were pooled, the EtOH was evaporated under vacuum, and the resulting solution (-100 mL) was lyophilized. Compound G (19A4) was obtained (a yellow solid, 0.82 g, 1.1 mmol, yield 32%), HPLC: 95% and characterized by 1H NMR, 31P NMR, MS and UV spectra ([fig.20]). EXAMPLE 8: Synthesis of compound H (19A22)

[0132] The principle is the same as for compound A.

[0133] The synthesis of compound H is shown in Figures 21, 22 and 23

[0134] The mixture of compound 11 (4.08 ml, 30 mmol, 1 eq), paraformaldehyde (0.9 g, 30 mmol, 1 eq) and TMSC1 (1.5 ml, 117 mmol, 3.92 eq) were stirred at room temperature for 50 min and concentrated under vacuum at room temperature for 50 min. The crude product obtained (a brown oil, 5.53 g, -100%) was used immediately in the next step ([fig.21]).

[0135] Compound 25 (5.53 g, 30 mmol, 1.63 eq) was added to the solution of Meloxicam (6.45 g, 18.36 mmol, 1 eq) and TEA (6 mL, 43.17 mmol, 2.35 eq) in DCM (30 mL). The reaction mixture was stirred for 150 min at room temperature, then introduced into a silica gel column and eluted with a cHex → cHex / EtOAc = 2 / 3 gradient, TLC: DCM / EtOAc = 3 / l. Compound 26 was obtained: a yellow solid, 1.73 g, 3.46 mmol, yield 19%, UPLC-MS: 81%, and characterized by 1H NMR, MS, and UV spectra ([Fig. 22]).

[0136] Compound 26 (1.727 g, 3.46 mmol, 1 eq) was added to a solution of HBP vector (1.27 g, 4.18 mmol, 1.21 eq) and TFA (0.1 mL, 1.35 mmol, 0.39 eq) in DMSO (15 mL). The resulting mixture was stirred for 20 min at room temperature, then poured into NaHCO3 solution and sonicated for 5 min. The resulting turbid colloid was introduced into a C18 column and eluted using a gradient from 3% EtOH to 50% EtOH. The EtOH from the HPLC >90% was evaporated under vacuum, and the aqueous solution was lyophilized. Compound H (19A22) was obtained, 1.314 g, yellow solid, yield 50%, HPLC: 94-95% and characterized by 1H NMR, 31P NMR, MS and UV spectra ( [fig-23]).

[0137] EXAMPLE 9: Evaluation of the binding capacity of HBP- molecules Meloxicam with hydroxyapatite

[0138] Solutions of molecules A, B, C, D, E, F, G and H were placed in the presence of hydroxyapatite (1 ml at 1 mM with 10 mg of HA) and the fixation rate was estimated after 30 minutes of stirring at room temperature by HPLC; the tests were carried out in triplicate.

[0139] The molecules obtained bind rapidly to Idihydroxyapatite (HA), as illustrated in [fig.24].

[0140] EXAMPLE 10: Evaluation of the release capacity of Meloxicam by clivable HBP-Meloxicam molecules

[0141] The molecules obtained have different capacities for releasing Meloxicam in solution. The compounds in solution at 37°C in rat serum or in 3 buffers at pH 7.4, 5 and 3 were analyzed by HPLC. The amount of MLX released in 48 h is shown in [Fig. 25].

[0142] EXAMPLE 11: In vitro evaluation of the anti-inflammatory activity of HBP-Meloxicam molecules

[0143] The molecules obtained were tested for their ability to inhibit the activity of the pro-inflammatory enzyme COX2 on primary rat articular chondrocytes. COX2 activity was induced by stimulating the chondrocytes for 24 h with LPS at a concentration of 1 pg / mL. During LPS stimulation, the chondrocytes were cultured in serum-free DMEM medium with 25 mM HEPES and 0.5% BSA. The molecules 18A135, 18A166, 18A182, 18A184, 19A4, 19A22, 19A115, 19A143 or Meloxicam were added for 24 h at the same time as LPS at concentrations of 0.1 pM, 0.5 pM, 1 pM, 5 pM or 10 pM.

[0144] HBP-Meloxicam molecules were solubilized in sterile water and Meloxicam was solubilized in PBS with 10% DMSO (0.1% DMSO at the final cell contact point). COX2 activity was measured by ELISA. The results are shown in [Fig. 26].

[0145] The cleavable molecules 18A166, 18A184, 19A4, 19A115 and 19A143 show COX2 inhibitory activity similar to Meloxicam. The non-cleavable molecules show lower COX2 inhibitory activity than the cleavable molecules and Meloxicam, but it is present.

[0146] EXAMPLE 12: Evaluation of maximum tolerable dose and toxicity in rats

[0147] These tests were carried out in two separate studies. The molecules 18A135, 18A166, 18A182, 18A184, 19A22, 19A115, and 19A143 were solubilized in 5% glucose and then administered by single intravenous injection (caudal vein, 5-minute infusion, 10 mL / kg volume) to male and female Sprague Dawley rats at least 16 weeks old. The molecules HBP-Meloxicam The doses were administered at 30, 45, 67.5, and 101.25 pmol / kg. Acute toxicity was determined by monitoring weight change over 7 days. The dose was considered tolerable if the animal did not exhibit continuous weight loss for 7 days or if its weight loss fell below 90% of its body weight on the day of treatment for 3 days.

[0148] In controlling the toxicity of Meloxicam in the second study, one male rat and one female rat were treated with Meloxicam orally daily for 28 days at a dose of 1 mg / kg - 2.85 pmol / kg, for a cumulative dose of 79.8 pmol / kg.

[0149] The maximum tolerated dose (MTD) value for each molecule is indicated in Table 1 below:

[0150] [Tables 1] MTD molecule (pmol / kg) 18A135 67.5 18A166 67.5 18A182 45 18A184 45 19A4 45 19A115 45 19A143 45

[0151] Table 1: Maximum tolerated dose values ​​for each of the molecules HBP-Meloxicam

[0152] Molecules 18A135 and 18A166 have the highest MTD values ​​(67.5pmol / kg) while the other molecules have an MTD of 45pmol / kg.

[0153] The animals that received their MTD dose (3 males and 3 females) were sacrificed 4 weeks after administration. Organs characteristic of Meloxicam toxicity (stomach, duodenum, kidneys) were collected, fixed in formalin, prepared histologically, and analyzed by a veterinary pathologist to assess overall toxicity to these organs (scale of 0 to 12.5 in the first study and 0 to 13.5 in the second study).

[0154] The histological analyses corresponding to each study are presented in [fig.27]. The median value is represented by the horizontal bar.

[0155] HBP-Meloxicam molecules at their MTD dose exhibit toxicity values ​​identical (molecule 18A166) or lower (molecules 18A135, 18A182, 18A184, 19A4, 19A115 and 19A143) to Meloxicam administered daily.

[0156] EXAMPLE 13: Evaluation of the anti-inflammatory efficacy of molecules HBP-Meloxicam in a rat model of monoarthritis.

[0157] Monoarthritis was induced in 14-week-old male Sprague Dawley rats by intra-articular (knee) injection of 100 pg of mBSA after two prior sensitizations (2 and 3 weeks before induction) by subcutaneous injection of a CFA emulsion containing 500 pg of mBSA. Allocation was performed on day 2, 24 hours after induction, based on the severity of swelling to ensure homogeneity between groups. After allocation, the animals were treated with 18A184 and 19A115 by single intravenous injection (caudal vein, 5-minute infusion, 10 mL / kg volume) at an identical dose of 30 pmol / kg. HBP-Meloxicam was solubilized in 5% glucose. In the control group, the diseased animals (mBSA) received a single injection (caudal vein, 5 min infusion, volume of 10 mL / kg) of 5% glucose.

[0158] In reference treatment, the animals received daily oral administration of Meloxicam for 28 days at the NOEL dose of 0.2 mg / kg - 0.57 pmol / kg, i.e. a cumulative dose of 15.96 pmol / kg.

[0159] The progression of arthritis was monitored by measuring the swelling of the pathological knee as well as the lameness of the animals.

[0160] Fig. 28 shows the evolution of the swelling after administration of treatments on day 2.

[0161] Molecules 18A184 and 19A115 show better efficacy on the acute phase of arthritis (first week) than Meloxicam administered daily.

[0162] Molecule 18A184 shows prolonged efficacy compared to molecule 19A115.

[0163] Fig. 29 shows the evolution of the lameness from the induction of arthritis on day 1 and after the administration of treatments on day 2.

[0164] Molecules 18A184 and 19A115 show better efficacy on the acute phase of arthritis (first week) than Meloxicam administered daily.

[0165] This efficacy is detectable earlier: cessation of lameness 24 h after treatment 19A115 or 48 h after treatment 18A184 versus 9 days after Meloxicam treatment.

[0166] Molecule 18A184 shows prolonged efficacy compared to molecule 19A115.

Claims

Claims

1. Hydroxybisphosphonic derivative of Meloxicam of formula (I) or a pharmaceutically acceptable salt thereof, [Chem.l] in which one and only one of the radicals RI and R2 represents an H group or is absent and when R2 is absent then X is chosen from the following groups Rl-A, Rl-B and Rl-C: [Chem. 2] (Rl-A) [Chem. 3] (Rl-B) [Chem. 4] (Rl-C) when RI = H then X is chosen from the following groups R2-D, R2-E, R2-F, R2-G and R2-H: [Chem. 5] has (R2-D) [Chem. 6] (R2-E) [Chem. 7] has (R2-F) [Chem. 8] (R2-G) [Chem.9] (R2-H)

2. A hydroxybisphosphonic derivative of Meloxicam according to claim 1 wherein RI = H, said compound may be in its tau- tomeric and being represented by the general formula (II): [Chem. 10] E £5 In which R2 is chosen from the following groups R2-D, R2-E, R2-F, R2-G and R2-H: [Chem. 5] » s (R2-D) [Chem. 6] (R2-E) [Chem. 7] has (R2-F) [Chem. 8] ​​XK Ô (R2-G) [Chem.9] r "y ■ •x - <MV (R2-H) ^Revendication 3] Dérivé hydroxybisphosphonique de Méloxicam tel que défini à l’une des revendications 1 ou 2 pour son utilisation en tant que médicament. ^Revendication 4] Dérivé hydroxybisphosphonique de Méloxicam tel que défini à la revendication 3 pour son utilisation selon la revendication 3, dans le traitement des maladies inflammatoires ostéoarticulaires. ^Revendication 5] Dérivé hydroxybisphosphonique de Méloxicam tel que défini à la revendication 4 pour son utilisation selon la revendication 4 choisi parmi les composés C ou E. ^Revendication 6] Procédé de synthèse d’un dérivé hydroxybisphosphonique de Méloxicam tel que défini aux revendications 1 à 3 comprenant les étapes de : • Couplage du Méloxicam à un linker X choisi parmi les groupements RrA, RrB et RrC, R2-D, R2-E, R2-F, R2-G et R2 -H tels que définis précédemment ; • Couplage du dérivé HBP à l’extrémité opposée du linker par rapport au Méloxicam.^Claim 7] Method according to claim 6 in which the linker X is chosen from the groups RrA, RrB, RrC, R2-E, R2-G and R2-H so as to obtain a cleavable prodrug allowing the release of meloxicam at the osteoarticular level. ^Claim 8] Pharmaceutical composition comprising a hydroxybisphosphonic derivative of Meloxicam as defined in one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and at least one excipient. ^Claim 9] Composition according to claim 8 in which said hydroxybisphosphonic derivative is chosen from compounds C or E.

10. Composition according to one of claims 8 or 9 in which said hydroxybisphosphonic derivative is in the form of a sodium salt or a meglumic salt.

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