WO2016112513A1 - Compounds and methods for inducing mesenchymal stem cells into chondrocytes - Google Patents

Compounds and methods for inducing mesenchymal stem cells into chondrocytes Download PDF

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WO2016112513A1
WO2016112513A1 PCT/CN2015/070787 CN2015070787W WO2016112513A1 WO 2016112513 A1 WO2016112513 A1 WO 2016112513A1 CN 2015070787 W CN2015070787 W CN 2015070787W WO 2016112513 A1 WO2016112513 A1 WO 2016112513A1
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thiazol
oxopropyl
compound
acetylpiperazin
phenylureido
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PCT/CN2015/070787
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French (fr)
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Baishan JIANG
Sheng Ding
Langxi HU
Xiangnan Li
Chuang He
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Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/44—Acylated amino or imino radicals
    • C07D277/48—Acylated amino or imino radicals by radicals derived from carbonic acid, or sulfur or nitrogen analogues thereof, e.g. carbonylguanidines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

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  • the present invention relates to a compound for inducing mesenchymal stem cells into chondrocytes ex vivo and in vivo, the preparation method thereof, as well as a composition comprising the same. Further, the present invention relates to the use of the compound and the method to treat osteoarthritis and joint injuries.
  • Osteoarthritis is a degenerative joint disease that involves the destruction of articular cartilage and eventually leads to disability. Approximately 350 million people globally are currently affected as a result of the aging population and an increase in life expectancy. About 50%people over 60 years old have osteoarthritis, making it the number one disease for aged people and the fourth leading cause of disability.
  • Current OA therapies include pain relief with oral NSAIDs or selective cyclooxygenase 2 (COX-2) inhibitors, intra-articular (IA) injection with agents such as corticosteroids and hyaluronan, and surgical approaches. None of the therapy restores the lost cartilage.
  • MSCs Mesenchymal stem cells
  • Molecules that promote the selective differentiation of multipotent mesenchymal stem cells into chondrocytes may stimulate the repair of damaged cartilage. This may provide a new strategy of cell-based therapy for treating osteoarthritis, joint injuries and related diseases.
  • the present invention generally provides a compound and a pharmaceutical composition thereof, wherein the compound is used to induce mesenchymal stem cells (MSC) into chondrocytes ex vivo and in vivo, and the preparation method thereof, as well as the use of such compound and the method to treat osteoarthritis and joint injuries.
  • MSC mesenchymal stem cells
  • the present invention provides a compound for inducing mesenchymal stem cells into chondrocytes ex vivo and in vivo, which has the structure of Formula I:
  • R 1 is hydrogen, halogen or CR 6 ;
  • R 2 and R 3 may be taken together to form a three to seven membered heterocyclic ring containing 1-2 heteroatoms selected from N, O and S;
  • R 4 and R 5 may be taken together to form a three to seven membered heterocyclic ring containing 1-2 heteroatoms selected from N, O and S;
  • R 2 , R 3 , R 4 and R 5 could be independently and optionally substituted with 1-2 R 6 groups;
  • R 6 is hydrogen, halogen, C 1 -C 6 alkoxy, -S (O) 2 R 7 , -C (O) OR 7 , -C (O) R 7 , -C (O) NR 7 R 8 , C 1 -C 6 alkyl, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl, each of which can be optionally substituted with halogen, amino, hydroxyl, alkoxy or cyano;
  • R 7 , R 8 are independently hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl, each of which may be optionally substituted with halogen, amino, hydroxyl, alkoxy or cyano.
  • the above said 5 or 6 membered heteroaryl includes, but is not limited to, the following selected groups:
  • R 5 is defined as above.
  • R 1 is selected to be hydrogen.
  • R 4 and R 5 are defined as in formula I.
  • example of the compound of the invention includes, but is not limited to:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the compound of the present invention and at least one pharmaceutically acceptable carrier or diluent, wherein said compound is in free form or in a pharmaceutically acceptable salt or hydrate or solvate form.
  • Such composition may be for intra-articular delivery, an oral composition, injectable composition or suppository.
  • the composition may be manufactured in a conventional manner by mixing, granulating or coating methods.
  • the composition is an injectable composition, and may be an aqueous isotonic solution or suspension.
  • the composition is sterilized and/or contains adjuvant.
  • adjuvant can be preserving, stabilizing, wetting or emulsifying agent, solution promoter, salt for regulating the osmotic pressure, buffer and/or any combination thereof.
  • composition may further contain other therapeutically valuable substances for different applications, like solubilizers, stabilizers, tonicity enhancing agents, buffers and/or preservatives.
  • the present invention provides a method for inducing mesenchymal stem cells into chondrocytes ex vivo or in vivo by contacting cells, tissue or organism with the compounds or composition of the present invention.
  • the present invention also provides a method for treating osteoarthritis and joint injuries by administering an effective amount of the present compound or composition, by which mesenchymal stem cells are induced into chondrocytes ex vivo and in vivo, to the subject.
  • the compound of the present invention could be administered in a therapeutically effective amount via any acceptable way known in the art singly.
  • the therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. Generally, the satisfactory result is indicated to be obtained systemically at a daily dosage of about 0.001 to 1 mg/kg body weight of the subject.
  • the indicated daily dosage for larger mammal as human is in the range from about 0.1 mg to about 100 mg.
  • the compound of the present invention can be present in a concentration of 0.1 ⁇ M to 100 ⁇ M in an intra-articular injection to the knee.
  • the compound of the present invention may be administered in a therapeutically effective amount as the active ingredient in combination with one or more therapeutic agents, such as pharmaceutical combinations.
  • therapeutic agents such as pharmaceutical combinations.
  • the dosage of the co-administered compounds could vary depending on the type of co-drug employed, the specific drug employed, the condition being treated and so forth.
  • the invention also provides a pharmaceutical combination, preferably, a kit, comprising a) a first agent which is the compound of the present invention as disclosed herein, in free form or in pharmaceutically acceptable salt or hydrate or solvate form, and b) at least one co-agent.
  • a kit comprising a) a first agent which is the compound of the present invention as disclosed herein, in free form or in pharmaceutically acceptable salt or hydrate or solvate form, and b) at least one co-agent.
  • the kit may comprise instructions for its administration.
  • the combination of the present invention may be used in vitro or in vivo.
  • the desired therapeutic benefit of the administration may be achieved by contacting cell, tissue or organism with a single composition or pharmacological formulation that includes the compound of the present invention and one or more agents, or by contacting the cells, tissue or organism with two or more distinct compositions or formulations, wherein one composition includes one agent and the other includes another.
  • the agents of the combination may be administered at the same time or separately within a period of time.
  • the separate administration can result in a desired therapeutic benefit.
  • the present compound may precede, be co-current with and/or follow the other agents by intervals ranging from minutes to weeks.
  • a person skilled in the art could generally ensure the interval of the time of each delivery, wherein the agents administered separately could still be able to exert an advantageously combined effect on the cell, tissue or organism.
  • one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously as the candidate substance, i. e. , with less than about one minute.
  • one or more agents may be administered about between 1 minute and 14 days.
  • the present provides a process for preparing the compound of the present invention or the salts or other derivatives thereof.
  • the compound having Formula (I) may be prepared following any one of the synthetic methodologies described below.
  • reactive functional groups for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, may be protected to avoid their unwanted participation in the reactions.
  • Conventional protecting groups may be used in accordance with standard practice (see e.g., T.W. Greene and P.G.M. Wuts in “Protective Groups in Organic Chemistry” , John Wiley and Sons, 1991) .
  • Suitable leaving groups for use in the synthetic methodologies described include halogen leaving groups and other conventional leaving groups known in the art.
  • the leaving group is chloro or bromo.
  • the compound of the invention or the salts thereof may also be obtainable in the form of hydrates, or their crystals which may include for example the solvent used for crystallization (present as solvates) .
  • Salts can usually be converted to compounds in free form by treating with suitable basic agents, preferably with alkali metal carbonates, alkali metal hydrogen carbonates, or alkali metal hydroxides, more preferably with potassium carbonate or sodium hydroxide.
  • suitable basic agents preferably with alkali metal carbonates, alkali metal hydrogen carbonates, or alkali metal hydroxides, more preferably with potassium carbonate or sodium hydroxide.
  • a compound of the invention in a base addition salt form may be converted to the corresponding free acid by treating with a suitable acid, such as hydrochloric acid.
  • any reference to the free compounds is to be understood as referring also to the corresponding salts, as appropriate.
  • Salts of the present compound with a salt-forming group may be prepared in a manner known in the art. Acid addition salts of compound of Formula (I) may thus be obtained by treatment with an acid or with a suitable anion exchange reagent. Pharmaceutically acceptable salts of the compound of the invention may be formed as acid addition salts from compound of Formula (I) with a basic nitrogen atom with organic or inorganic acids.
  • suitable inorganic acids include, but are not limited to, halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid.
  • suitable organic acids include, but are not limited to, carboxylic, phosphoric, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, -malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4 aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane-or ethane-sulfonic acid, 2-hydroxyethanesulfonic acid, e
  • compound of the present invention in unoxidized form may be prepared from N-oxides of compound of the invention by treating with a reducing agent in a suitable inert organic solvent at 0 to 80°C.
  • a reducing agent is sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, or the like.
  • the invert organic solvent is acetonitrile, ethanol, aqueous dioxane, or the like.
  • prodrug derivatives of the compound of the present invention may be prepared by methods known in the art (for further details see Saulnier et al. , (1994) , Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985) .
  • an appropriate prodrug may be prepared by reacting a non-derivatized compound of the invention with a suitable carbamylating agent such as 1, 1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or the like.
  • protected derivatives of the compound of the present invention may be made by means known in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal may be found in T. W. Greene, “Protecting Groups in Organic Chemistry” , 3rd edition, John Wiley and Sons, Inc., 1999.
  • the present invention also includes isotopically-labeled compounds of the present invention, wherein one or more atoms are replaced by one or more atoms having specific atomic mass or mass numbers.
  • isotopes that can be incorporated into compounds of the invention include, but are not limited to,isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, sulfur, and chlorine (such as 2 H, 3 H, 11 C, 13 C, 14 C , 15 N, 17 O, 18 O, 35 S, 18 F, 36 Cl) .
  • Isotopically-labeled compounds of the present invention are useful in assays of the tissue distribution of the compounds and their prodrugs and metabolites; preferred isotopes for such assays include 3 H and 14 C.
  • Isotopically-labeled compounds of this invention can generally be prepared according to the methods described herein by substituting an isotopically-labeled reagent for a non-isotopically labeled reagent.
  • compound of the present invention may be prepared as their individual stereoisomers.
  • the process includes reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers.
  • Resolution of enantiomers may be carried out using covalent diastereomeric derivatives of the compound of the present invention, or by using dissociable complexes such as crystalline diastereomeric salts.
  • Diastereomers have distinct physical properties presented by melting points, boiling points, solubilities, reactivity, etc. , and may be readily separated by taking advantage of these dissimilarities.
  • the diastereomers may be separated by fractionated crystallization, chromatography, or by separation/resolution techniques based upon differences in solubility.
  • the optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization.
  • a more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture may be found in Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates and Resolutions” , John Wiley And Sons, Inc. , 1981.
  • the compound of the present invention could be made by the process described below; optionally a pharmaceutically acceptable salt may be converted from the compound of the present invention; optionally a pharmaceutically acceptable N-oxide may be converted from an unoxidized form of the compound the present invention; optionally an individual isomer of the compound of the present invention is resolved from a mixture of isomers; and optionally a pharmaceutically acceptable prodrug derivative may be converted from a non-derivatized compound of the present invention.
  • the present invention also provides the use of the compounds of formula I or the composition thereof in the preparation of medicines for inducing mesenchymal stem cells into chondrocytes, such as medicines for treating osteoarthritis and joint injuries.
  • Figure 1 Images of Nile Red stained chondrogenic nodules read on Acumen eX3, the left image represents typical positive hits (i.e., the one which has been treated with the compound of present invention) , the right image represents negative control without compound treatment.
  • Figure 2 Safranin O staining of cartilage injury mouse model treated with compound 16,on day 28.
  • the right image indicates increased cellularity and cartilage matrix and reduction of cartilage damage in mice left knees treated with selected compound of present invention compared with the control group with saline dosing control group (the left image) .
  • Figure 3 The joint severity scores of the lateral tibial plateau on day 28. Histomorphometric analyses and grading using a modified OARSI scoring system determined the joint severity scores of the lateral tibial plateau on day 28 after surgical induction. The right image represents that compound 16 can statistically improve in total joint scores comparing to PBS dosing control groups (the left image) .
  • the compounds of the present invention may be prepared by methods such as those illustrated in the following scheme I to II. Solvents, temperatures, pressures and other reaction conditions may readily be selected by one of ordinary skill in the art. Starting materials are commercially available or readily prepared using procedures generally known to those skilled in the art.
  • An optionally substituted Levulinate ester such as, but not limited to, ethyl levulinate was reacted with bromine in organic solvent such as, but not limited to, ethanol, methanol, toluene or carbon tetrachloride at elevated temperatures (60 ⁇ 150°C) to afford compound 1 of scheme I.
  • organic solvent such as, but not limited to, ethanol, methanol, toluene or carbon tetrachloride at elevated temperatures (60 ⁇ 150°C) to afford compound 1 of scheme I.
  • the compound 1 of scheme I was reacted with thiourea in organic solvents such as,but not limited to, ethanol, methanol, or isopropanol at elevated temperatures (60 ⁇ 150°C) to form a thiazole derivative 2 of scheme I.
  • organic solvents such as,but not limited to, ethanol, methanol, or isopropanol at elevated temperatures (60 ⁇ 150°C)
  • the thiazole compound 2 was reacted with substituted amine in organic solvents such as, but not limited to, ethanol, methanol, or isopropanol at elevated temperatures (100 ⁇ 150°C) to obtain an amide compound 3 of scheme I.
  • organic solvents such as, but not limited to, ethanol, methanol, or isopropanol at elevated temperatures (100 ⁇ 150°C)
  • the compound 2 obtained from scheme I was treated isocyanate in organic solvents such as, but not limited to, DCM, toluene or DMF at room temperature to provide compound 5 of Scheme II.
  • organic solvents such as, but not limited to, DCM, toluene or DMF at room temperature
  • the compound 5 was hydrolyzed in the presence of a base such as, but not limited to aqueous KOH, NaOH or LiOH in organic solvent such as, but not limited to, ethanol, methanol or THF to form an acid intermediate 6.
  • a base such as, but not limited to aqueous KOH, NaOH or LiOH
  • organic solvent such as, but not limited to, ethanol, methanol or THF
  • the acid intermediate 6 was coupled with substituted amine in the presence of classical coupling reagents in amide synthesis such as, but not limited to, EDCI/HOBt/DIPEA combination to afford the compounds of present invention, such as the compounds 7-17, 27-37 and 40-47 listed below.
  • classical coupling reagents in amide synthesis such as, but not limited to, EDCI/HOBt/DIPEA combination to afford the compounds of present invention, such as the compounds 7-17, 27-37 and 40-47 listed below.
  • the present invention is further exemplified, but not limited, by the following and examples that illustrate the preparation of the compounds of the invention.
  • Test Example 1 Evaluating compounds that induce chondrogenesis.
  • the unbiased cell-based assay used to evaluate the compounds was based on the development of chondrogenic nodules.
  • 5000 primary hMSCs (Lonza) were plated in monolayer conditions in a 96 well Greiner plates in serum free DMEM. The cells were respectively treated with 10 ⁇ M, 3.33 ⁇ M, 1.11 ⁇ M, 0.37 ⁇ M, 0.12 ⁇ M and 0.04 ⁇ M six concentration of each compound in DMSO and incubated for 4 days (37°C, in a humidified atmosphere of 5%CO 2 ) . During this period the chondrogenic nodule formed in wells were considered to be a positive hit (please see figure 1) and further calculated EC 50 .
  • Nile Red stained nodules were quantified on an Acumen eX3 (high content imaging device, TTP Labtech) by excitation with a 488 nm laser for rapid detection of the nodules
  • figure 1 shows one example of images of Nile Red stained chondrogenic nodules read on Acumen eX3
  • the left image represents typical positive hits (i. e. , the one which has been treated with the compound of present invention)
  • the right image represents negative control without treatment.
  • the positive hits were re-confirmed by 12 points titration from 10 ⁇ M to 0.005 ⁇ M with 2 folds dilution.
  • the EC 50 results are listed in the following table.
  • A:EC 50 ⁇ 0.1 ⁇ M
  • B: 0.1 ⁇ M ⁇ EC 50 ⁇ 1 ⁇ M
  • C: 1 ⁇ M ⁇ EC 50 ⁇ 10 ⁇ M
  • Test Example 2 Surgical Induction of Cartilage Injury mouse model and Treatment Surgical transection of the anterior crucial ligament (ACL) , medial collateral ligament (MCL) , and medial meniscotibial ligament (MMTL) was performed to induce cartilage injury in 8-weeks old female C57BL6 mice’s left knee (Vital River Lab. ) , 10 mice/group (the detailed procedures please see references 5, 6) .
  • the compound 16 was dissolved into DMSO to make 10 mM stock solution. Before injection, the compound stock solution was diluted into PBS to make 10 ⁇ M fimal drug concentration. Mice were dosed by intra-articular injection on days 7, 14 and 21 for 10 ⁇ M compound or PBS (10 ⁇ L) .
  • Intra-articular injection was performed under anesthesia (Isoflurane) .
  • Isoflurane anesthesia
  • a 1-2 mm incision on the surgical knee will be made.
  • a 30 gauge needle will be used to inject 10 ⁇ l of compound or PBS into the surgical knee.
  • the incision will be closed with Vetbond.
  • the medial tibial plateaus from representative mice were assessed on day 28. Histomorphometric analyses and grading using a modified OARSI scoring system determined the joint severity scores of the lateral tibial plateau on day 28 after surgical induction. It can be seen from figure 3 that compound 16 can statistically improve in total joint scores comparing to PBS dosing control groups.

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Abstract

This invention provides compounds and compositions for inducing mesenchymal stem cells into chondrocytes ex vivo and in vivo. The compound could lead to a stem cell-based therapy for osteoarthritis and joint injuries. The present invention also provides the preparation method of the compounds of the present invention, the use of the compound and the method to treat osteoarthritis and joint injuries.

Description

Compounds and Methods for Inducing Mesenchymal Stem Cells into Chondrocytes
Field of Invention
The present invention relates to a compound for inducing mesenchymal stem cells into chondrocytes ex vivo and in vivo， the preparation method thereof， as well as a composition comprising the same. Further， the present invention relates to the use of the compound and the method to treat osteoarthritis and joint injuries.
Background of the Invention
Osteoarthritis (OA) is a degenerative joint disease that involves the destruction of articular cartilage and eventually leads to disability. Approximately 350 million people globally are currently affected as a result of the aging population and an increase in life expectancy. About 50％people over 60 years old have osteoarthritis， making it the number one disease for aged people and the fourth leading cause of disability. Current OA therapies include pain relief with oral NSAIDs or selective cyclooxygenase 2 (COX-2) inhibitors， intra-articular (IA) injection with agents such as corticosteroids and hyaluronan， and surgical approaches. None of the therapy restores the lost cartilage. Mesenchymal stem cells (MSCs) are present in adult articular cartilage and can be differentiated into chondrocytes. Molecules that promote the selective differentiation of multipotent mesenchymal stem cells into chondrocytes may stimulate the repair of damaged cartilage. This may provide a new strategy of cell-based therapy for treating osteoarthritis， joint injuries and related diseases.
Summary of the Invention
The present invention generally provides a compound and a pharmaceutical composition thereof， wherein the compound is used to induce mesenchymal stem cells (MSC) into chondrocytes ex vivo and in vivo， and the preparation method thereof， as well as the use of such compound and the method to treat osteoarthritis and joint injuries.
In one aspect， the present invention provides a compound for inducing mesenchymal stem cells into chondrocytes ex vivo and in vivo， which has the structure of Formula I：
Figure PCTCN2015070787-appb-000001
or a physiologically acceptable salt or hydrate or solvate thereof， wherein：
R1 is hydrogen， halogen or CR6；
R2， R3， R4 and R5 are independently selected from the group consisting of H， C1-C6 alkyl， C2-C6 alkenyl， C2-C6 alkynyl， - (C1-C6 alkyl) -O- (C1-C6 alkyl) ， - (C1-C6 alkyl) -NR7R8， - (C1-C6 alkyl) -C (＝O) NR7R8， aryl， C3-C6 heterocycle， C3-C7 cycloalkyl， 5 or 6 membered heteroaryl containing 1-2 heteroatoms selected from N， O and S；
or R2 and R3 may be taken together to form a three to seven membered heterocyclic ring containing 1-2 heteroatoms selected from N， O and S；
or R4 and R5 may be taken together to form a three to seven membered heterocyclic ring containing 1-2 heteroatoms selected from N， O and S；
R2， R3， R4 and R5 could be independently and optionally substituted with 1-2 R6 groups；
R6 is hydrogen， halogen， C1-C6 alkoxy， -S (O) 2R7， -C (O) OR7， -C (O) R7， -C (O) NR7R8， C1-C6 alkyl， C2-C6 alkenyl or C2-C6 alkynyl， each of which can be optionally substituted with halogen， amino， hydroxyl， alkoxy or cyano；
R7， R8 are independently hydrogen， C1-C6 alkyl， C2-C6 alkenyl or C2-C6 alkynyl， each of which may be optionally substituted with halogen， amino， hydroxyl， alkoxy or cyano.
Preferably， the above said 5 or 6 membered heteroaryl includes， but is not limited to， the following selected groups：
Figure PCTCN2015070787-appb-000002
wherein， R5 is defined as above.
In a preferred embodiment of the invention， R1 is selected to be hydrogen.
In a preferred embodiment of the present invention， there is provided a compound of the formula II
Figure PCTCN2015070787-appb-000003
or pharmaceutically acceptable salt or hydrate or solvate thereof， wherein R2 and R3 are defined as in formula I.
In a preferred embodiment of the invention， there is provided a compound of the formula III or IV
Figure PCTCN2015070787-appb-000004
or pharmaceutically acceptable salt or hydrate or solvate thereof， wherein R4 and R5 are defined as in formula I.
In a preferred embodiment， example of the compound of the invention includes， but is not limited to：
1. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
2. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-methylurea；
3. 1- (4- (3- (4-acetylpiperazin-l-yl) -3-oxopropyl) thiazol-2-yl) -3-ethylurea；
4. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-isopropylurea；
5. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-cyclohexylurea；
6. N- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) morpholine-4-carboxa mide；
7. N-cyclopentyl-3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
8. N-methyl-3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
9. N-isopropyl-3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
10. N-phenyl-3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
11. N-benzyl-3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
12. N- (2-morpholinoethyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
13. 1- (4- (3- (4-methylpiperazin-1yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
14. 1- (4- (3-oxo-3- (piperazin-1-yl) propyl) thiazol-2-yl) -3-phenylurea；
15. tert-butyl 4-(3- (2- (3-phenylureido) thiazol-4-yl) propanoyl) piperazine-1-carboxylate；
16. 1- (4- (3-morpholino-3-oxopropyl) thiazol-2-yl) -3-phenylurea；
17. 1- (4- (3-oxo-3- (piperidin-1-yl) propyl) thiazol-2-yl) -3-phenylurea；
18. 1 - (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-o-tolylurea；
19. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-m-tolylurea；
20. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-p-tolylurea；
21. 1 - (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (4-phenoxyphenyl) ur ea；
22. l- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (4- (trifluoromethyl) p henyl) urea；
23. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (4-methoxyphenyl) ur ea；
24. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (4-fluorophenyl) urea ；
25. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (4- (trifluoromethoxy ) phenyl) urea；
26. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (pyridin-2-yl) urea；
27. 1- (4- (3- (4- (2-cyanoacetyl) piperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
28. 1- (4- (3- (4-benzoylpiperazin-l-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
29. 1- (4- (3- (4-acryloylpiperazin-l-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
30. 1- (4- (3- (4- (methylsulfonyl) piperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylure a；
31. 1- (4- (3-oxo-3- (4- (2-phenylacetyl) piperazin-1-yl) propyl) thiazol-2-yl) -3-phenylure a；
32. 1- (4- (3-oxo-3- (4-picolinoylpiperazin-1-yl) propyl) thiazol-2-yl) -3-phenylurea；
33. 1- (4- (3- (4- (2-chloroacetyl) piperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea ；
34. 1- (4- (3- (4- (2- (4-methylpiperazin-1-yl) acetyl) piperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
35. 1- (4- (3-oxo-3- (4- (phenylsulfonyl) piperazin-1-yl) propyl) thiazol-2-yl) -3-phenylure a；
36. benzyl 4- (3- (2- (3-phenylureido) thiazol-4-yl) propanoyl) piperazine-l-carboxylate；
37. 1- (4- (3- (4- (1H-pyrazole-4-carbonyl) piperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-p henylurea；
38. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) -5-fluorothiazol-2-yl) -3-phenylurea；
39. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) -5-ethylthiazol-2-yl) -3-phenylurea；
40. methyl 1- (3- (2- (3-phenylureido) thiazol-4-yl) propanoyl) azetidine-2-carboxylate；
41. methyl 1- (3- (2- (3-phenylureido) thiazol-4-yl) propanoyl) pyrrolidine-3-carboxylate；
42. methyl 1- (3- (2- (3-phenylureido) thiazol-4-yl) propanoyl) piperidine-4-carboxylate； 
43. N- (4-acetamidophenyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
44. N- (2-acetamidoethyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
45. N- (3-acetamidopropyl) -3 - (2- (3-phenylureido) thiazol-4-yl) propanamide
46. N- (2-methoxyethyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
47. N- (2- (2-acetamidoethoxy) ethyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
48. 1 - (4- (3- (4-acetylpiperazin-1 -yl) -3-oxopropyl) thiazol-2-yl) -3- (pyridin-3-yl) urea；
49. 1- (4- (3- (4-acetylpiperazin-1 -yl) -3-oxopropyl) thiazol-2-yl) -3- (pyridin-4-yl) urea；
50. 4- (3- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) ureido) pyridine 1-oxide；
51. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (pyrimidin-4-yl) urea；
52. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (isoxazol-4-yl) urea；
53. 1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (1H-pyrazol-4-yl) ure a.
In another aspect， the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of the present invention and at least one pharmaceutically acceptable carrier or diluent， wherein said compound is in free form or in a pharmaceutically acceptable salt or hydrate or solvate form. Such composition may be for intra-articular delivery， an oral composition， injectable composition or suppository. And the composition may be manufactured in a conventional manner by mixing， granulating or coating methods.
In another embodiment of the invention， the composition is an injectable composition， and may be an aqueous isotonic solution or suspension.
Preferably， the composition is sterilized and/or contains adjuvant. Such adjuvant can be preserving， stabilizing， wetting or emulsifying agent， solution promoter， salt for regulating the osmotic pressure， buffer and/or any combination thereof.
Alternatively or in addition， the composition may further contain other therapeutically valuable substances for different applications， like solubilizers， stabilizers， tonicity enhancing agents， buffers and/or preservatives.
In another aspect， the present invention provides a method for inducing mesenchymal stem cells into chondrocytes ex vivo or in vivo by contacting cells， tissue or organism with the compounds or composition of the present invention.
In another aspect， the present invention also provides a method for treating osteoarthritis and joint injuries by administering an effective amount of the present compound or composition， by which mesenchymal stem cells are induced into chondrocytes ex vivo and in vivo， to the subject.
For therapeutical use， the compound of the present invention could be administered in a therapeutically effective amount via any acceptable way known in the art singly. As used herein， the therapeutically effective amount may vary widely depending on the severity of the disease， the age and relative health of the subject， the potency of the  compound used and other factors. Generally， the satisfactory result is indicated to be obtained systemically at a daily dosage of about 0.001 to 1 mg/kg body weight of the subject. In one embodiment， the indicated daily dosage for larger mammal as human is in the range from about 0.1 mg to about 100 mg. In some embodiments， the compound of the present invention can be present in a concentration of 0.1 μM to 100 μM in an intra-articular injection to the knee.
Alternatively， the compound of the present invention may be administered in a therapeutically effective amount as the active ingredient in combination with one or more therapeutic agents， such as pharmaceutical combinations. There may be synergistic effects when the compound of the present invention is used with a chemotherapeutic agent known in the art. The dosage of the co-administered compounds could vary depending on the type of co-drug employed， the specific drug employed， the condition being treated and so forth.
In another aspect， the invention also provides a pharmaceutical combination， preferably， a kit， comprising a) a first agent which is the compound of the present invention as disclosed herein， in free form or in pharmaceutically acceptable salt or hydrate or solvate form， and b) at least one co-agent. In addition， the kit may comprise instructions for its administration.
The combination of the present invention may be used in vitro or in vivo. Preferably， the desired therapeutic benefit of the administration may be achieved by contacting cell， tissue or organism with a single composition or pharmacological formulation that includes the compound of the present invention and one or more agents， or by contacting the cells， tissue or organism with two or more distinct compositions or formulations， wherein one composition includes one agent and the other includes another. The agents of the combination may be administered at the same time or separately within a period of time. Preferably， the separate administration can result in a desired therapeutic benefit. The present compound may precede， be co-current with and/or follow the other agents by intervals ranging from minutes to weeks. A person skilled in the art could generally ensure the interval of the time of each delivery， wherein the agents administered separately could still be able to exert an advantageously combined effect on the cell， tissue or organism. In one embodiment， it is contemplated that one may contact the cell， tissue or organism with two， three， four or more modalities substantially simultaneously as the candidate substance， i. e. ， with less than about one minute. In another embodiment， one or more agents may be administered about between 1 minute and 14 days.
In another aspect， the present provides a process for preparing the compound of the present invention or the salts or other derivatives thereof.
In one embodiment， the compound having Formula (I) may be prepared following any one of the synthetic methodologies described below. In the reactions described， reactive functional groups， for example hydroxy， amino， imino， thio or carboxy groups， where these are desired in the final product， may be protected to avoid their unwanted participation in the reactions. Conventional protecting groups may be used  in accordance with standard practice (see e.g.， T.W. Greene and P.G.M. Wuts in “Protective Groups in Organic Chemistry” ， John Wiley and Sons， 1991) . Suitable leaving groups for use in the synthetic methodologies described include halogen leaving groups and other conventional leaving groups known in the art. Preferably， the leaving group is chloro or bromo.
In another embodiment， the compound of the invention or the salts thereof may also be obtainable in the form of hydrates， or their crystals which may include for example the solvent used for crystallization (present as solvates) . Salts can usually be converted to compounds in free form by treating with suitable basic agents， preferably with alkali metal carbonates， alkali metal hydrogen carbonates， or alkali metal hydroxides， more preferably with potassium carbonate or sodium hydroxide. A compound of the invention in a base addition salt form may be converted to the corresponding free acid by treating with a suitable acid， such as hydrochloric acid. In view of the close relationship between the novel compounds in free form and those in the form of their salts， including those salts that may be used as intermediates， for example in the purification or identification of the novel compounds， any reference to the free compounds is to be understood as referring also to the corresponding salts， as appropriate.
Salts of the present compound with a salt-forming group may be prepared in a manner known in the art. Acid addition salts of compound of Formula (I) may thus be obtained by treatment with an acid or with a suitable anion exchange reagent. Pharmaceutically acceptable salts of the compound of the invention may be formed as acid addition salts from compound of Formula (I) with a basic nitrogen atom with organic or inorganic acids.
Preferably， suitable inorganic acids include， but are not limited to， halogen acids， such as hydrochloric acid， sulfuric acid， or phosphoric acid.
Preferably， suitable organic acids include， but are not limited to， carboxylic， phosphoric， sulfonic or sulfamic acids， for example acetic acid， propionic acid， octanoic acid， decanoic acid， dodecanoic acid， glycolic acid， lactic acid， fumaric acid， succinic acid， adipic acid， pimelic acid， suberic acid， azelaic acid， -malic acid， tartaric acid， citric acid， amino acids， such as glutamic acid or aspartic acid， maleic acid， hydroxymaleic acid， methylmaleic acid， cyclohexanecarboxylic acid， adamantanecarboxylic acid， benzoic acid， salicylic acid， 4 aminosalicylic acid， phthalic acid， phenylacetic acid， mandelic acid， cinnamic acid， methane-or ethane-sulfonic acid， 2-hydroxyethanesulfonic acid， ethane-1， 2-disulfonic acid， benzenesulfonic acid， 2-naphthalenesulfonic acid， 1， 5-naphthalene-disuifonic acid， 2-， 3-or 4 methylbenzenesulfonic acid， methylsulfuric acid， ethylsulfuric acid， dodecylsulfuric acid， N cyclohexylsulfamic acid， N-methyl-， N-ethyl-or N-propyl-sulfamic acid， or other organic protonic acids， such as ascorbic acid.
Alternatively， it is also possible to use pharmaceutically unacceptable salts for isolation or purification， for example picrates or perchlorates. But for therapeutic use， only pharmaceutically acceptable salts or free compounds are employed， where  applicable in the form of pharmaceutical preparations.
In yet another embodiment， compound of the present invention in unoxidized form may be prepared from N-oxides of compound of the invention by treating with a reducing agent in a suitable inert organic solvent at 0 to 80℃. Preferably， the reducing agent is sulfur， sulfur dioxide， triphenyl phosphine， lithium borohydride， sodium borohydride， phosphorus trichloride， tribromide， or the like. Preferably， the invert organic solvent is acetonitrile， ethanol， aqueous dioxane， or the like.
In yet another embodiment， prodrug derivatives of the compound of the present invention may be prepared by methods known in the art (for further details see Saulnier et al. ， (1994) ， Bioorganic and Medicinal Chemistry Letters， Vol. 4， p. 1985) . In a preferable embodiment， an appropriate prodrug may be prepared by reacting a non-derivatized compound of the invention with a suitable carbamylating agent such as 1， 1-acyloxyalkylcarbanochloridate， para-nitrophenyl carbonate， or the like.
In yet another embodiment， protected derivatives of the compound of the present invention may be made by means known in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal may be found in T. W. Greene， “Protecting Groups in Organic Chemistry” ， 3rd edition， John Wiley and Sons， Inc.， 1999.
In yet another embodiment， the present invention also includes isotopically-labeled compounds of the present invention， wherein one or more atoms are replaced by one or more atoms having specific atomic mass or mass numbers. Examples of isotopes that can be incorporated into compounds of the invention include， but are not limited to，isotopes of hydrogen， carbon， nitrogen， oxygen， fluorine， sulfur， and chlorine (such as 2H， 3H， 11C， 13C， 14C ， 15N， 17O， 18O， 35S， 18F， 36Cl) . Isotopically-labeled compounds of the present invention are useful in assays of the tissue distribution of the compounds and their prodrugs and metabolites； preferred isotopes for such assays include 3H and 14C. In addition， in certain circumstances substitution with heavier isotopes， such as deuterium (2H or D) ， can provide increased metabolic stability， which offers therapeutic advantages such as increased in vivo half-life or reduced dosage requirements. Isotopically-labeled compounds of this invention can generally be prepared according to the methods described herein by substituting an isotopically-labeled reagent for a non-isotopically labeled reagent.
In yet another embodiment， compound of the present invention may be prepared as their individual stereoisomers. The process includes reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds， separating the diastereomers and recovering the optically pure enantiomers. Resolution of enantiomers may be carried out using covalent diastereomeric derivatives of the compound of the present invention， or by using dissociable complexes such as crystalline diastereomeric salts. Diastereomers have distinct physical properties presented by melting points， boiling points， solubilities， reactivity， etc. ， and may be readily separated by taking advantage of these dissimilarities. The diastereomers may be separated by fractionated crystallization，  chromatography， or by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered， along with the resolving agent， by any practical means that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture may be found in Jean Jacques， Andre Collet， Samuel H. Wilen， “Enantiomers， Racemates and Resolutions” ， John Wiley And Sons， Inc. ， 1981.
In conclusion， the compound of the present invention could be made by the process described below； optionally a pharmaceutically acceptable salt may be converted from the compound of the present invention； optionally a pharmaceutically acceptable N-oxide may be converted from an unoxidized form of the compound the present invention； optionally an individual isomer of the compound of the present invention is resolved from a mixture of isomers； and optionally a pharmaceutically acceptable prodrug derivative may be converted from a non-derivatized compound of the present invention.
Insofar as the production of the starting materials is not particularly described， the compounds are known or can be prepared analogously to methods known in the art or as disclosed in the examples hereinafter. One of skill in the art will appreciate that the above transformations are only representative of methods for preparation of the compounds of the present invention， and that other well-known methods can similarly be used.
In another aspect， the present invention also provides the use of the compounds of formula I or the composition thereof in the preparation of medicines for inducing mesenchymal stem cells into chondrocytes， such as medicines for treating osteoarthritis and joint injuries.
Description of the Figures
Figure 1： Images of Nile Red stained chondrogenic nodules read on Acumen eX3， the left image represents typical positive hits (i.e.， the one which has been treated with the compound of present invention) ， the right image represents negative control without compound treatment.
Figure 2： Safranin O staining of cartilage injury mouse model treated with compound 16，on day 28. The right image indicates increased cellularity and cartilage matrix and reduction of cartilage damage in mice left knees treated with selected compound of present invention compared with the control group with saline dosing control group (the left image) .
Figure 3： The joint severity scores of the lateral tibial plateau on day 28. Histomorphometric analyses and grading using a modified OARSI scoring system determined the joint severity scores of the lateral tibial plateau on day 28 after surgical induction. The right image represents that compound 16 can statistically improve in total joint scores comparing to PBS dosing control groups (the left image) .
Embodiments
Figure PCTCN2015070787-appb-000005
Methods of Preparation
The compounds of the present invention may be prepared by methods such as those illustrated in the following scheme I to II. Solvents， temperatures， pressures and other reaction conditions may readily be selected by one of ordinary skill in the art. Starting materials are commercially available or readily prepared using procedures generally known to those skilled in the art.
Figure PCTCN2015070787-appb-000006
Step 1
An optionally substituted Levulinate ester such as， but not limited to， ethyl levulinate was reacted with bromine in organic solvent such as， but not limited to， ethanol， methanol， toluene or carbon tetrachloride at elevated temperatures (60～150℃) to afford compound 1 of scheme I.
Step 2
The compound 1 of scheme I was reacted with thiourea in organic solvents such as，but not limited to， ethanol， methanol， or isopropanol at elevated temperatures (60～150℃) to form a thiazole derivative 2 of scheme I.
Step 3
The thiazole compound 2 was reacted with substituted amine in organic solvents such as， but not limited to， ethanol， methanol， or isopropanol at elevated temperatures  (100～150℃) to obtain an amide compound 3 of scheme I.
Step 4
The compound 3 of scheme I was then reacted with arylaniline/triphosgene or isocyanate in organic solvents such as， but not limited to， DCM， toluene or DMF at room temperature to provide the compounds of the present invention， such as the compounds 1-6， 18-26， 38-39 and 48-53 listed below.
Figure PCTCN2015070787-appb-000007
Step 1
The compound 2 obtained from scheme I was treated isocyanate in organic solvents such as， but not limited to， DCM， toluene or DMF at room temperature to provide compound 5 of Scheme II.
Step 2
The compound 5 was hydrolyzed in the presence of a base such as， but not limited to aqueous KOH， NaOH or LiOH in organic solvent such as， but not limited to， ethanol， methanol or THF to form an acid intermediate 6.
Step 3
The acid intermediate 6 was coupled with substituted amine in the presence of classical coupling reagents in amide synthesis such as， but not limited to， EDCI/HOBt/DIPEA combination to afford the compounds of present invention， such as the compounds 7-17， 27-37 and 40-47 listed below.
Detailed embodiments
The present invention is further exemplified， but not limited， by the following and examples that illustrate the preparation of the compounds of the invention.
The invention will now be further described by the following working examples， which are preferred embodiments of the invention. All temperatures are in degrees Celsius (℃) unless otherwise indicated. Preparative Reverse Phase (RP) HPLC purifications were done on C18 reverse phase (RP) columns using water/methanol mixtures. All the synthesized compounds were characterized by at least NMR or LC/MS. During work up of reactions， the organic extract was dried over sodium sulfate， purified by silica gel column chromatography or (RP) HPLC， unless  mentioned otherwise.
These examples are illustrative rather than limiting and it is to be understood that there may be other embodiments that fall within the spirit and scope of the invention as defined by the claims appended hereto.
Example 1
The rretaration of comround 1
Figure PCTCN2015070787-appb-000008
Intermediate 1-1
To a solution of ethyl levulinate (25g， 173.4mmol) in 250mL EtOH was added acetic acid (0.5mL， 7.8mmol) ， and the mixture was warmed to 50℃. Bromine (9.4mL， 1 82.1mmol) was added dropwise， and the reaction was stirred at room temperature for 6 hours. The mixture was then concentrated， and the residue was worked-up with Et2O and saturated aqueous NaHCO3. The organic phase was dried， filtered， and concentrated， and the crude material was purified by silica gel chromatography to give 14.7g of the title compound. 1H NMR (400 MHz， CDCl3) δ 4.1 8 (q， J＝7.5 Hz， 2H)， 4.01 (s， 2H) ， 2.98 (t， J＝7.0Hz， 2H) ， 2.60 (t， J＝7.0Hz， 2H) ， 1.21 (t， J＝7.5 Hz， 3H). ESI-MS m/z 224.1 [M+H] .
Intermediate 1-2
A solution of intermediate 1-1 (14.7g， 65.9mmol) in 50 mL ethanol was added to a solution of thiourea (6.0g， 79.08mmol) in 100mL ethanol. The mixture was then refluxed for 8 h. The reaction mixture was concentrated and the residue was dissolved in 1 00mL ethyl acetate， washed with saturated aqueous NaHCO3， brine， dried， filtered and concentrated to give the oily residue. This residue was purified by silica gel chromatography to afford 1 1.9g of the title compound. ESI-MS m/z 20 1.1 [M+H] .
Intermediate 1-3
In a 50mL sealed tube was added intermediate 1-2 (3.0g， 15.0mmol) ， N-aetylpiperazine (2.31 g， 18mmol) and 10mL anhydrous ethanol and the mixture was heated to 120℃for 12h. The mixture was allowed to cool to room temperature and evaporated under reduced pressure. This residue was purified by silica gel chromatography to afford 3.5g of the title compound. ESI-MS m/z 283.3 [M+H] .
Compound 1
To a solution of intermediate 1-3 (0.2g， 0.71mmol) in 5mL dry DCM was added a  solution of triphosgene (0.25g， 0.85mmol) in 5mL DCM， the mixture was stirred at room temperature for 2h， then aniline (80mg， 0.85mmol) was added， then kept stirring at room temperature for another 2h. The precipitate formed was collected by filtration， washed with DCM and dried to afford 0.25g of the title compound as a white solid. 1H NMR (500 MHz， DMSO) δ 10.44 (s， 1H) ， 8.93 (s， 1H) ， 7.46 (t， J＝ 8.9 Hz， 2H) ， 7.31 (t，J＝ 7.5 Hz， 2H) ， 7.03 (t， J ＝ 7.2 Hz， 1H) ， 6.71 (s， 1H) ， 3.66-3.61 (m， 4H) ， 3.45-3.39 (m， 4H) ， 2.80 (t， J ＝ 7.4 Hz， 2H) ， 2.68 (t， J ＝ 7.4 Hz， 2H) ， 2.01 (s， 3H) . ESI-MS m/z 402.1 [M+H] .
Example 2
The preparation of compound 7
Figure PCTCN2015070787-appb-000009
Intermediate 7-1
To a solution of intermediate 1-1 (3.0g， 15mmol) in 50mL dry DCM was added a solution of triphosgene (5.34g， 18mmol) in 50mL DCM， the mixture was stirred at room temperature for 2h， then aniline (1.68g， 18mmol) was added， then kept stirring for another 2h. The precipitate formed was collected by filtration， washed with DCM and dried to afford 4.1g of intermediate 7-1 as a white solid. ESI-MS m/z 320.3 [M+H] .
Compound 7
To a solution of Intermediate 7-1 (4.0g， 12.5mmol) in 50mL ethanol was added 15mL 1M aqueous KOH and stirred at room temperature for 3h. The pH value of the solution was then adjusted to 5-6 using 1M HCl， and the mixture was then evaporated， extracted， and dried to give an acid intermediate. ESI-MS m/z 292.3 [M+H] . The acid intermediate (50mg， 0.172mmol) was then added to a 25mL flask charged with EDCI (36， 1mg， 0.189mmol) ， HOBt (25.5mg， 0.189mmol) ， DIPEA (48.9mg， 0.378mmol) and 5mL DCM. The solution was stirred for 12h at room temperature， then evaporated under reduced pressure and the crude material was purified by silica gel chromatography to give 45mg of compound 7 as a white solid. 1H NMR (CDCl3) δ 10.41 (s， 1H) ， 8.90 (s， 1H) ， 7.52-7.45 (m， 2H) ， 7.41-7.33 (m， 2H) ， 7.11-7.01 (m， 1H) ， 6.79 (s， 1H) ， 4.11-4.01 (m， 1H) ， 2.85 (t， J ＝ 7.3 Hz， 2H) ， 2.66 (t， J ＝ 7.3 Hz， 2H) ， 2.10-2.00 (m， 2 H) ， 1.75-1.46 (m， 6 H) . ESI-MS m/z 359.1 [M+H] .
Example 3
The rretaration of comround 16
Figure PCTCN2015070787-appb-000010
The preparation of compound 16 is the same as compound 1 except using morpholine instead of N-aetylpiperazine. 1H NMR (500 MHz， DMSO) δ 10.44 (s， 1H) ， 8.95 (s， 1H)， 7.52-7.41 (m， 2H) ， 7.39-7.31 (m， 2H) ， 7.01 (t， J ＝ 7.1 Hz， 1H) ， 6.73 (s， 1H) ， 3.75-3.64 (m， 4H) ， 3.48-3.41 (m， 4H) ， 2.85 (t， J ＝ 7.3 Hz， 2H) ， 2.66 (t， J ＝ 7.3 Hz， 2H). ESI-MS m/z 361.1 [M+H] .
Other compounds listed below were prepared using a procedure similar to thatdescribed above.
Figure PCTCN2015070787-appb-000011
Figure PCTCN2015070787-appb-000012
Figure PCTCN2015070787-appb-000013
Figure PCTCN2015070787-appb-000014
Figure PCTCN2015070787-appb-000015
Figure PCTCN2015070787-appb-000016
Figure PCTCN2015070787-appb-000017
Figure PCTCN2015070787-appb-000018
Figure PCTCN2015070787-appb-000019
Figure PCTCN2015070787-appb-000020
Test Example 1： Evaluating compounds that induce chondrogenesis.
The unbiased cell-based assay used to evaluate the compounds was based on the development of chondrogenic nodules. 5000 primary hMSCs (Lonza) were plated in monolayer conditions in a 96 well Greiner plates in serum free DMEM. The cells were respectively treated with 10 μM， 3.33 μM， 1.11 μM， 0.37 μM， 0.12 μM and 0.04  μM six concentration of each compound in DMSO and incubated for 4 days (37℃， in a humidified atmosphere of 5％CO2) . During this period the chondrogenic nodule formed in wells were considered to be a positive hit (please see figure 1) and further calculated EC50. All populations of the chondrogenic nodules had high levels of cartilage specific matrix production (both proteoglycans and type II collagen expression) ， suggesting that chondrogenic differentiation has occurred. The wells could be fixed and stained with 1 μg/ml Rhodamine B where the nodules were easily detected by eye and images captured by light microscopy. But to speed up the capacity of compound evaluation， the chondrogenic nodules were stained with Nile red which binds non-specifically to collagens. The Nile Red stained nodules were quantified on an Acumen eX3 (high content imaging device， TTP Labtech) by excitation with a 488 nm laser for rapid detection of the nodules， figure 1 shows one example of images of Nile Red stained chondrogenic nodules read on Acumen eX3， the left image represents typical positive hits (i. e. ， the one which has been treated with the compound of present invention) ， the right image represents negative control without treatment. The positive hits were re-confirmed by 12 points titration from 10 μM to 0.005 μM with 2 folds dilution. The EC50 results are listed in the following table.
A：EC50 ＜＝ 0.1 μM， B： 0.1 μM ＜EC50＜＝1 μM， C： 1 μM ＜EC50＜＝10 μM
Compound No. Cbondrogenic EC50 (μM)
1 A
2 C
3 C
4 B
5 A
6 B
7 B
8 B
9 B
10 B
11 C
12 B
13 B
14 B
15 A
16 A
17 B
18 C
19 B
20 A
21 B
22 B
23 B
24 B
25 B
26 C
27 B
28 C
29 B
30 B
31 C
32 C
33 C
34 C
35 C
36 C
37 B
38 C
39 C
40 C
41 B
42 B
43 C
44 C
45 C
46 C
47 B
48 B
49 C
50 B
51 A
52 B
53 B
Test Example 2： Surgical Induction of Cartilage Injury mouse model and Treatment Surgical transection of the anterior crucial ligament (ACL) ， medial collateral ligament (MCL) ， and medial meniscotibial ligament (MMTL) was performed to induce cartilage injury in 8-weeks old female C57BL6 mice’s left knee (Vital River Lab. ) ， 10 mice/group (the detailed procedures please see references 5， 6) . The compound 16 was dissolved into DMSO to make 10 mM stock solution. Before injection， the compound stock solution was diluted into PBS to make 10 μM fimal drug concentration. Mice were dosed by intra-articular injection on days 7， 14 and 21 for 10 μM compound or PBS (10 μL) . Intra-articular injection was performed under anesthesia (Isoflurane) . A 1-2 mm incision on the surgical knee will be made. With a lateral approach， a 30 gauge needle will be used to inject 10 μl of compound or PBS into the surgical knee. The incision will be closed with Vetbond. The medial tibial plateaus from representative mice were assessed on day 28. Histomorphometric analyses and grading using a modified OARSI scoring system determined the joint severity scores of the lateral tibial plateau on day 28 after surgical induction. It can be seen from figure 3 that compound 16 can statistically improve in total joint scores comparing to PBS dosing control groups. The increasing in Safranin O staining following treatment with compound 16 (see figure 2) indicated increased cellularity and cartilage matrix and reduction of cartilage damage in mice left knees. The other compounds， such as compounds 1， 5 and 20 achieved the similar efficacy results in surgical induction of cartilage injury mouse models.
References：
1.COMPOUNDS AND METHODS FOR INDUCING CHONDROGENESIS. United States Patent Application 20140113012.
2.Kartogenin induces cartilage-like tissue formation in tendon-bone junction. Zhang J，et al. Bone Res. 2014， 2. pii： 14008.
3.Mouse limb skeletal growth and synovial joint development are coordinately enhanced by Kartogenin. Decker RS， et al. Dev Biol. 2014， 395 (2) ： 255-67.
4.A stem cell-based approach to cartilage repair. Johnson K， et al. Science. 2012， 336(6082) ： 717-21.
5.Osteoarthritis development in novel experimental mouse models induced by knee joint instability. S. Kamekura et al. Osteoarthritis Cartilage， 2005， 13： 632-41.
6.The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. S. S. Glasson， et al， Osteoarthritis Cartilage， 2007， 15： 1061-1069.

Claims (13)

  1. A compound having the structure of Formula I：
    Figure PCTCN2015070787-appb-100001
    or a physiologically acceptable salt or hydrate or solvate thereof， wherein：
    R1 is hydrogen， halogen or CR6；
    R2， R3， R4 and R5 are independently selected from the group consisting of H， C1-C6 alkyl， C2-C6 alkenyl， C2-C6 alkynyl， - (C1-C6 alkyl) -O- (C1-C6 alkyl) ， - (C1-C6 alkyl) -NR7R8， - (C1-C6 alkyl) -C (＝O) NR7R8， aryl， C3-C6 heterocycle， C3-C7 cycloalkyl， 5 or 6 membered heteroaryl containing 1-2 heteroatoms selected from N， O and S；
    or R2 and R3 may be taken together to form a three to seven membered heterocyclic ring containing 1-2 heteroatoms selected from N， O and S；
    or R4 and R5 may be taken together to form a three to seven membered heterocyclic ring containing 1-2 heteroatoms selected from N， O and S；
    R2， R3， R4 and R5 could be independently and optionally substituted with 1-2 R6 groups；
    R6 is hydrogen， halogen， C1-C6 alkoxy， -S (O) 2R7， -C (O) OR7， -C (O) R7， -C (O) NR7R8， C1-C6 alkyl， C2-C6 alkenyl or C2-C6 alkynyl， each of which can be optionally substituted with halogen， amino， hydroxyl， alkoxy or cyano；
    R7， R8 are independently hydrogen， C1-C6 alkyl， C2-C6 alkenyl or C2-C6 alkynyl， each of which may be optionally substituted with halogen， amino， hydroxyl， alkoxy or cyano.
  2. The compound or physiologically acceptable salt or hydrate or solvate thereof of claim 1， wherein the said 5 or 6 membered heteroarvl is selected from the following groups：
    Figure PCTCN2015070787-appb-100002
    wherein， R5 is defined as in claim 1.
  3. The compound or physiologically acceptable salt or hydrate or solvate thereof of claim 1， wherein R1 is hydrogen.
  4. The compound or physiologically acceptable salt or hydrate or solvate thereof of claim 1， wherein the compound is a compound of the formula II
    Figure PCTCN2015070787-appb-100003
    wherein R2 and R3 are defined as in formula I.
  5. The compound or physiologically acceptable salt or hydrate or solvate thereof of claim 1， wherein the compound is a compound of the formula III or IV
    Figure PCTCN2015070787-appb-100004
    wherein R4 and R5 are defined as in formula I.
  6. The compound or physiologically acceptable salt or hydrate or solvate thereof of claim 1， wherein the compound is selected from the following compounds：
    1 - (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-methylurea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-ethylurea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-isopropylurea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-cyclohexylurea；
    N- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) morpholine-4-carboxamide；
    N-cyclopentyl-3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    N-methyl-3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    N-isopropyl-3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    N-phenyl-3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    N-benzyl-3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    N- (2-morpholinoethyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    1- (4- (3- (4-methylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
    1- (4- (3-oxo-3- (piperazin-1-yl) propyl) thiazol-2-yl) -3-phenylurea；
    tert-butyl 4- (3- (2- (3-phenylureido) thiazol-4-yl) propanoyl) piperazine-1-carboxylate；
    1- (4- (3-morpholino-3-oxopropyl) thiazol-2-yl) -3-phenylurea；
    1- (4- (3-oxo-3- (piperidin-1-yl) propyl) thiazol-2-yl) -3-phenylurea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-o-tolylurea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-m-tolylurea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-p-tolylurea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (4-phenoxyphenyl) urea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (4- (trifluoromethyl) phen yl) urea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (4-methoxyphenyl) urea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (4-fluorophenyl) urea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (4- (trifluoromethoxy) phe nyl) urea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (pyridin-2-yl) urea；
    1- (4- (3- (4- (2-cyanoacetyl) piperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
    1- (4- (3- (4-benzoylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
    1- (4- (3- (4-acryloylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
    1- (4- (3- (4- (methylsulfonyl) piperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
    1 - (4- (3-oxo-3- (4- (2-phenylacetyl) piperazin-1-yl) propyl) thiazol-2-yl) -3-phenylurea；
    1- (4- (3-oxo-3- (4-picolinoylpiperazin-1-yl) propyl) thiazol-2-yl) -3-phenylurea；
    1- (4- (3- (4- (2-chloroacetyl) piperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3-phenylurea；
    1- (4- (3- (4- (2- (4-methylpiperazin-1-yl) acetyl) piperazin-1-yl) -3-oxopropyl) thiazol-2-yl  ) -3-phenylurea；
    1- (4- (3-oxo-3- (4- (phenylsulfonyl) piperazin-1-yl) propyl) thiazol-2-yl) -3-phenylurea；
    benzyl 4- (3- (2- (3-phenylureido) thiazol-4-yl) propanoyl) piperazine-1-carboxylate；
    1- (4- (3 - (4- (1H-pyrazo le-4-carbonyl) piperazin-1-yl) -3 -oxopropyl) thiazol-2-yl) -3-phen ylurea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) -5-fluorothiazol-2-yl) -3-phenylurea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) -5-ethylthiazol-2-yl) -3-phenylurea；
    methyl 1- (3- (2- (3-phenylureido) thiazol-4-yl) propanoyl) azetidine-2-carboxylate；
    methyl 1- (3- (2- (3-phenylureido) thiazol-4-yl) propanoyl) pyrrolidine-3-carboxylate；
    methyl 1- (3- (2- (3-phenylureido) thiazol-4-yl) propanoyl) piperidine-4-carboxylate；
    N- (4-acetamidophenyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    N- (2-acetamidoethyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    N- (3-acetamidopropyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    N- (2-methoxyethyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    N- (2- (2-acetamidoethoxy) ethyl) -3- (2- (3-phenylureido) thiazol-4-yl) propanamide；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (pyridin-3-yl) urea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (pyridin-4-yl) urea；
    4- (3- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) ureido) pyridine 1-oxide；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (pyrimidin-4-yl) urea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (isoxazol-4-yl) urea；
    1- (4- (3- (4-acetylpiperazin-1-yl) -3-oxopropyl) thiazol-2-yl) -3- (1H-pyrazol-4-yl) urea.
  7. A method for preparing the compound of any one of claims 1-6， wherein the compound is prepared by following scheme I or II，
    Figure PCTCN2015070787-appb-100005
    Step 1
    An optionally substituted Levulinate ester such as ethyl levulinate was reacted with bromine in organic solvent such as ethanol， methanol， toluene or  carbon tetrachloride at elevated temperatures (60～150℃) to afford compound 1 of scheme I，
    Step 2
    The compound 1 of scheme I was reacted with thiourea in organic solvents such  as ethanol， methanol， or isopropanol at elevated temperatures (60～150℃) to form a thiazole derivative 2 of scheme I，
    Step 3
    The thiazole compound 2 was reacted with substituted amine in organic solvents such as ethanol， methanol， or isopropanol at elevated temperatures (100～150℃) to obtain an amide compound 3 of scheme I，
    Step 4
    The compound 3 of scheme I was then reacted with arylaniline/triphosgene or isocyanate in organic solvents such as DCM， toluene or DMF at room temperature to provide the compounds of the present invention；
    Figure PCTCN2015070787-appb-100006
    Step 1
    The compound 2 obtained from scheme I was treated isocyanate in organic solvents such as DCM， toluene or DMF at room temperature to provide compound 5 of Scheme II，
    Step 2
    The compound 5 was hydrolyzed in the presence of a base such as aqueous KOH， NaOH or LiOH in organic solvent such as ethanol， methanol or THF to form an acid intermediate 6，
    Step 3
    The acid intermediate 6 was coupled with substituted amine in the presence of classical coupling reagents in amide synthesis such as EDCI/HOBt/DIPEA combination to afford the compounds of present invention.
  8. A pharmaceutical composition comprising a therapeutically effective amount of the compound or physiologically acceptable salt or hydrate or solvate thereof of any one of claims 1-6 and at least one pharmaceutically acceptable carrier or diluents.
  9. The pharmaceutical composition of claim 8， wherein the composition is for intra-articular delivery， an oral composition， injectable composition or suppository.
  10. A method for inducing mesenchymal stem cells into chondrocytes ex vivo or in vivo by contacting cells， tissue or organism with the compound or physiologically  acceptable salt or hydrate or solvate thereof of any one of claims 1-6 or pharmaceutical composition of any one of claims 8-9.
  11. A method for treating osteoarthritis or joint injuries by administering an effective amount of the compound or physiologically acceptable salt or hydrate or solvate thereof of any one of claims 1-6 or pharmaceutical composition of any one of claims 8-9 to the subject.
  12. Use of the compound or physiologically acceptable salt or hydrate or solvate thereof of any one of claims 1-6 or pharmaceutical composition of any one of claims 8-9 in the preparation of medicines for inducing mesenchymal stem cells into chondrocytes.
  13. The use of claim 12， wherein the said medicines are medicines for treating osteoarthritis or joint injuries. 
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