Composition for preventing or treating uveitis
阅读说明：本技术 用于预防或治疗葡萄膜炎的组合物 (Composition for preventing or treating uveitis ) 是由 崔泳一 河妮娜 申宅桓 于 2019-02-19 设计创作，主要内容包括：本发明涉及一种用于预防或治疗葡萄膜炎的药物组合物,其包含由式I表示的化合物、其旋光异构体或其药学可接受的盐作为有效成分,并且本发明还涉及一种使用该化合物的治疗方法以及该化合物在制备用于治疗葡萄膜炎的药物中的用途。本发明的药物组合物显示出预防或治疗葡萄膜炎的优异效果。(The present invention relates to a pharmaceutical composition for preventing or treating uveitis, which comprises a compound represented by formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, and a therapeutic method using the same and use of the same in the preparation of a medicament for treating uveitis. The pharmaceutical composition of the present invention shows an excellent effect of preventing or treating uveitis.)
1. A pharmaceutical composition for preventing or treating uveitis, comprising a compound represented by the following formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an effective ingredient;
[ formula I ]
Wherein
A is
Xa and Xb are each independently CH or N,
L1and L2Each independently of the others being hydrogen, halogen, -CF3or-C1-3A linear or branched alkyl group,
q is C (O), S (O)2S (═ O) or C (═ NH),
y is selected from:
m is C, N, O, S or S (═ O)2Wherein at this time, if M is C, then l and M are 1; if M is N, then l is 1 and M is 0; and if M is O, S or S (═ O)2And then l and m are 0,
Ra1and Ra2Each independently is hydrogen; a hydroxyl group; -C1-4Straight-chain or branched alkyl which is unsubstituted or substituted by at least one halogenGeneration; -C1-4A straight or branched chain alcohol; a benzhydryl group; -C1-4A linear or branched alkyl substituted with a saturated or unsaturated 5 to 7 membered heterocyclic compound containing from 1 to 3 heteroatoms being N, O or S as ring members, wherein in this case the heterocyclic compound may be unsubstituted or at least one hydrogen may optionally be replaced by OH, OCH3、CH3、CH2CH3Or halogen substitution; saturated or unsaturated 5-to 7-membered heterocyclic compounds containing 1 to 3 heteroatoms which are N, O or S as ring members, where in this case the heterocyclic compound may be unsubstituted or at least one hydrogen may optionally be replaced by OH, OCH3、CH3、CH2CH3Or halogen substitution; phenyl, wherein the phenyl is unsubstituted or at least one hydrogen is replaced by halogen, C1-4Alkoxy radical, C1-2Alkyl or hydroxy substitution; benzyl, wherein the benzyl is unsubstituted or at least one hydrogen is replaced by halogen, C1-4Alkoxy radical, C1-2Alkyl or hydroxy substitution; -S (═ O)2CH3(ii) a Halogen; -C1-6A linear or branched alkoxy group; -C2-6An alkoxyalkyl group; -C (═ O) RxWherein R isxIs straight or branched C1-3Alkyl or C3-10A cycloalkyl group;wherein R iscAnd RdEach independently is hydrogen, C1-3A linear or branched alkyl group; and
n is an integer of 0, 1 or 2,
Rbis hydrogen; a hydroxyl group; -C1-6A linear or branched alkyl group, wherein said-C1-6Straight or branched chain alkyl is unsubstituted or at least one hydrogen is substituted by halogen; -C (═ O) CH3；-C1-4Straight or branched chain hydroxyalkyl; -C1-6A linear or branched alkoxy group; -C2-6Linear or branched alkoxyalkyl; -CF3(ii) a Halogen; or
ReAnd RfEach independently is hydrogen or-C1-3A linear or branched alkyl group,
z is selected from:
Paand PbEach independently isHydrogen; a hydroxyl group; -C1-4A linear or branched alkyl group, wherein said-C1-4Straight or branched chain alkyl is unsubstituted or at least one hydrogen is substituted by halogen; halogen; -CF3；-OCF3；-CN；-C1-6A linear or branched alkoxy group; -C2-6A linear or branched alkylalkoxy group; -CH2F; or-C1-3The alcohol is added into the mixture of the alcohol,
wherein
x, y and z are each independently an integer of 0 or 1,
Rg1、Rg2and Rg3Each independently is hydrogen; a hydroxyl group; -C1-3An alkyl group; -CF3；-C1-6A linear or branched alkoxy group; -C2-6A linear or branched alkylalkoxy group; -C (═ O) CH3；-C1-4Straight or branched chain hydroxyalkyl; -N (CH)3)2(ii) a Halogen; a phenyl group; -S ((═ O)2)CH3(ii) a Or is selected from:
2. the pharmaceutical composition according to claim 1, wherein the compound represented by the formula I above is a compound represented by the following formula Ia:
[ formula Ia ]
Wherein
L1And L2Each independently of the other being hydrogen or halogen,
y is
Z is phenyl or pyridyl, wherein at least one hydrogen of the phenyl or pyridyl may be replaced by halogen, CF3Or CF2And H is substituted.
3. The pharmaceutical composition according to claim 2, wherein the compound represented by formula Ia above is a compound described in the following table:
4. the pharmaceutical composition of claim 1, wherein the uveitis is anterior uveitis, intermediate uveitis, posterior uveitis, or panuveitis.
5. The pharmaceutical composition of claim 1, wherein the uveitis is infectious uveitis or non-infectious uveitis.
6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered parenterally.
7. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered by eye drop.
8. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered intraperitoneally.
9. A method of treating uveitis comprising administering a therapeutically effective amount of a compound represented by formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof according to claim 1.
10. Use of a compound represented by formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof according to claim 1 for the manufacture of a medicament for the treatment of uveitis.
Technical Field
The present invention relates to a pharmaceutical composition for preventing or treating uveitis, which comprises a compound represented by formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, and a therapeutic method using the same and use of the same in the preparation of a medicament for treating uveitis.
Background
The uvea, the intermediate layer inside the outermost layers of the eye (cornea and sclera), consists of the iris, ciliary body and choroid, and the inflammation occurring within it is defined as uveitis. Since the uvea has abundant blood vessels and many connective tissues, it is easily inflamed, wherein various symptoms are presented to the uvea according to various causes and degrees of inflammation. As representative symptoms of uveitis, visual loss, muscae volitantes, pain, bleeding, lacrimation, and blepharitis are known.
Uveitis can be classified into anterior uveitis, intermediate uveitis, posterior uveitis, and panuveitis, depending on the location of its inflammation. In addition, uveitis is also classified into infectious uveitis caused by viruses or germs and non-infectious uveitis caused by abnormalities of the autoimmune system, most of which are caused by immune factors. However, to date, the precise pathogenesis of uveitis has not been disclosed.
Steroids or immunosuppressive agents are employed as representative therapeutic agents for uveitis. However, steroid eye drops often cause serious side effects such as cataract, increased intraocular pressure, increased inflammation, etc., because they are used at such a high dose that the drugs may penetrate into the uveal tissue. In addition, long-term use of oral steroids can cause various side effects such as osteoporosis, osteonecrosis, hypothalamic-pituitary-adrenal axis inhibition, increased infection, and abnormalities in metabolism, electrolytes, and digestive system. On the other hand, immunosuppressive agents, which have been used as replacement therapies for steroids, may also cause side effects such as bone marrow suppression, renal function, and liver function impairment. Therefore, although adequate treatment can be achieved using steroids and immunosuppressive agents, it poses many problems to the life of the patient. For example, blindness occurs in 5% to 10% of patients after all. Therefore, there is an urgent need to develop a therapeutic agent for uveitis that has good penetration into target tissues with less side effects.
Under these circumstances, the present inventors made an effort to develop a therapeutic agent for uveitis, and thus identified that the compound of the present invention can be effectively used for preventing or treating uveitis, and completed the present invention.
[ Prior art reference ]
[ patent document ]
Korean patent application laid-open No. 2014-0128886
DISCLOSURE OF THE INVENTION
Technical problem
An object of the present invention is to provide a pharmaceutical composition for preventing or treating uveitis, which comprises a compound represented by the following formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an effective ingredient.
It is another object of the invention to provide a method of treating uveitis wherein the method comprises administering a therapeutically effective amount of the compound.
It is another object of the invention to provide the use of said compounds in the manufacture of a medicament for the treatment of uveitis.
Means for solving the problems
This will be described in detail below. Also, each of the descriptions and implementation forms disclosed in the present invention can be applied to other descriptions and implementation forms of the present invention, respectively. In other words, all combinations of the various elements disclosed in this invention are within the scope of the invention. Also, the scope of the present invention is not limited to the specific description set forth below.
The present invention provides a pharmaceutical composition for preventing or treating uveitis, comprising a compound represented by the following formula I:
[ formula I ]
Wherein in the formula I, the compound has the structure shown in the specification,
a is
Xa and Xb are each independently CH or N,
L1and L2Each independently of the others being hydrogen, halogen, -CF3or-C1-3A linear or branched alkyl group,
q is C (O), S (O)2S (═ O) or C (═ NH),
y is selected from:
m is C, N, O, S or S (═ O)2Wherein at this time, if M is C, then l and M are 1; if M is N, then l is 1 and M is 0; and if M is O, S or S (═ O)2And then l and m are 0,
Ra1and Ra2Each independently is hydrogen; a hydroxyl group; -C1-4A linear or branched alkyl group, which is unsubstituted or substituted by at least one halogen; -C1-4A straight or branched chain alcohol; a benzhydryl group; -C1-4A linear or branched alkyl substituted with a saturated or unsaturated 5 to 7 membered heterocyclic compound containing from 1 to 3 heteroatoms being N, O or S as ring members, wherein in this case the heterocyclic compound may be unsubstituted or at least one hydrogen may optionally be replaced by OH, OCH3、CH3、CH2CH3Or halogen substitution; comprises 1 to 3 ofN, O or S as ring members, wherein in this case the heterocyclic compound may be unsubstituted or at least one hydrogen may optionally be replaced by OH, OCH3、CH3、CH2CH3Or halogen substitution; phenyl, wherein the phenyl is unsubstituted or at least one hydrogen is replaced by halogen, C1-4Alkoxy radical, C1-2Alkyl or hydroxy substitution; benzyl, wherein the benzyl is unsubstituted or at least one hydrogen is replaced by halogen, C1-4Alkoxy radical, C1-2Alkyl or hydroxy substitution; -S (═ O)2CH3(ii) a Halogen; -C1-6A linear or branched alkoxy group; -C2-6An alkoxyalkyl group; -C (═ O) RxWherein R isxIs straight or branched C1-3Alkyl or C3-10A cycloalkyl group;wherein R iscAnd RdEach independently is hydrogen, C1-3A linear or branched alkyl group; and
n is an integer of 0, 1 or 2,
Rbis hydrogen; a hydroxyl group; -C1-6A linear or branched alkyl group, wherein said-C1-6Straight or branched chain alkyl is unsubstituted or at least one hydrogen is substituted by halogen; -C (═ O) CH3；-C1-4Straight or branched chain hydroxyalkyl; -C1-6A linear or branched alkoxy group; -C2-6Linear or branched alkoxyalkyl; -CF3(ii) a Halogen; or
ReAnd RfEach independently is hydrogen or-C1-3A linear or branched alkyl group,
z is selected from:
Paand PbEach independently is
wherein
x, y and z are each independently an integer of 0 or 1,
Rg1、Rg2and Rg3Each independently is hydrogen; a hydroxyl group; -C1-3An alkyl group; -CF3；-C1-6A linear or branched alkoxy group; -C2-6A linear or branched alkylalkoxy group; -C (═ O) CH3；-C1-4Straight or branched chain hydroxyalkyl; -N (CH)3)2(ii) a Halogen; a phenyl group; -S ((═ O)2)CH3(ii) a Or
Selected from:
the compound represented by formula I according to the present invention may be a compound represented by the following formula Ia:
[ formula Ia ]
Wherein
L1And L2Each independently of the other being hydrogen or halogen,
y is
Z is phenyl or pyridyl, wherein at least one hydrogen of the phenyl or pyridyl may be replaced by halogen, CF3Or CF2And H is substituted.
According to a particular embodiment of the invention, the compound represented by formula Ia above is a compound described in table 1 below:
[ Table 1]
In the present invention, the compound represented by the above formula I can be prepared by the method disclosed in Korean unexamined patent application publication No. 2014-0128886, but is not limited thereto.
In the present invention, pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, such as inorganic ionic salts prepared from calcium, potassium, sodium, magnesium, and the like; inorganic acid salts prepared from hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, sulfuric acid, and the like; organic acid salts prepared from acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like; sulfonates prepared from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like; amino acid salts prepared from glycine, arginine, lysine, etc.; amine salts prepared from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc.; and the like, but the types of salts involved in the present invention are not limited to those listed.
As used herein, the term "uveitis" refers to inflammation that occurs within the interior of the eye, particularly in the middle layer of the eye (uvea). More specifically, uveitis according to the invention comprises: anterior uveitis, an inflammation of the anterior portion of the uveal system, such as inflammation of the iris (iritis) and inflammation of the iris and ciliary body (ciliitis); intermediate uveitis, which is an inflammation of the vitreous (peripheral uveitis or chronic ciliary inflammation); and posterior uveitis, which is an inflammation in the portion of the uveal system behind the lens of the eye, such as inflammation of the choroid (choroiditis) and inflammation of the choroid and retina (chorioretinitis); and panuveitis, which is uveitis that affects the entire uveal system. In addition, according to the present invention, uveitis includes infectious uveitis caused by viruses or germs and non-infectious uveitis caused by autoimmune diseases.
In an embodiment of the present invention,
In addition, compound 374 of the present invention was identified to have excellent effects in the following respects: reducing uveitis lesions in animal models inducing uveitis disease (fig. 4-6), inhibiting infiltration of inflammatory cells (fig. 7-10), inhibiting expression of inflammatory cytokines in the spleen and inflammatory areas (fig. 11 and 12), and reducing the number of immune cells in the retina and lymph nodes (fig. 13-21).
For the purpose of administration, the pharmaceutical composition of the present invention may further comprise at least one type of pharmaceutically acceptable carrier in addition to the compound represented by the above formula I, its optical isomer, or a pharmaceutically acceptable salt thereof. As a pharmaceutically acceptable carrier, a combination of saline solution, sterile water, ringer's solution, buffered saline, dextrose (dextrose) solution, maltodextrin solution, glycerin, ethanol, and at least one component thereof may be used, wherein other conventional additives such as antioxidants, buffer solutions, bacteriostats, and the like may also be added to the pharmaceutical composition, if desired. Also, the pharmaceutical composition of the present invention may be formulated into injectable dosage forms (such as aqueous solutions, suspensions, emulsions, etc.), pills, capsules, granules, or tablets by further adding diluents, dispersants, surfactants, binders, and lubricants to the pharmaceutical composition of the present invention. Thus, the composition of the present invention may be a patch, a liquid medicine (liquid medicine), a pill, a capsule, a granule, a tablet, a suppository, or the like. These preparations can be formulated according to each disease and/or ingredient by a conventional method for preparation in the art to which the present invention pertains, or by a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack publishing company, Easton PA.
Non-limiting examples of formulations for oral administration using the pharmaceutical composition of the present invention may be tablets, troches (troche), troches (lozenge), aqueous suspensions, oil suspensions, prepared powders (prepard powder), granules, emulsions, hard capsules, soft capsules, syrups, elixirs and the like. In order to formulate the pharmaceutical composition of the present invention into a formulation for oral administration, binders such as lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose, gelatin, etc.; excipients such as dicalcium phosphate and the like; disintegrants such as corn starch, sweet potato starch, and the like; lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, polyethylene glycol waxes, and the like; and sweeteners, aromatics, syrups, and the like may also be used. Further, in the case of capsules, a liquid carrier such as fatty oil may be used in addition to the above materials.
Non-limiting examples of parenteral formulations using the pharmaceutical composition of the present invention may be injectable solutions, suppositories, powders for respiratory inhalation, aerosols for spraying, ointments, powders for application, oils, creams and the like. In order to formulate the pharmaceutical composition of the present invention into a formulation for parenteral administration, a sterile aqueous solution, a nonaqueous solvent, a suspension, an emulsion, a lyophilized formulation, an external preparation, etc. may be used. As the nonaqueous solvent and suspension, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate, and the like can be used, and for example, ophthalmic solutions or emulsions, ophthalmic gels, ophthalmic ointments or oily lotions containing eye drop compositions can be used, but not limited thereto.
The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally, for example, by eye drop administration or intraperitoneal administration, but is not limited thereto.
If the pharmaceutical composition according to the present invention is used in the form of an eye-drop composition, the eye-drop composition may be prepared by suspending the compound of formula I of the present invention, its optical isomer, or a pharmaceutically acceptable salt thereof in a sterile aqueous solution (e.g., saline, buffer, etc.), or by mixing the above-mentioned composition in the form of a soluble powder in a sterile aqueous solution before use.
Other additives may be included in the eye drop composition, for example, isotonic agents (e.g., sodium chloride, etc.), buffers (e.g., boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.), thickeners (e.g., sugars such as lactose, mannitol, maltose, etc.; e.g., hyaluronic acid or salts thereof such as sodium hyaluronate, potassium hyaluronate, etc.; e.g., mucopolysaccharides such as chondroitin sulfate, etc.; e.g., sodium polyacrylate, carboxyvinyl polymer, crosslinked polyacrylate salt, etc.).
According to exemplary embodiments of the present invention, it was identified that the compound represented by formula I has an excellent inhibitory effect on inflammatory regions and systemic inflammation when dropped into the eye of a mouse with induced uveitis disease, and thus shows an effective therapeutic effect on uveitis.
The daily dose of the compound represented by formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof of the present invention may be, for example, in the range of about 0.1 to 10000mg/kg, in the range of about 1 to 8000mg/kg, in the range of about 5 to 6000mg/kg, or in the range of about 10 to 4000mg/kg, preferably in the range of about 50 to 2000mg/kg, but is not limited thereto, wherein such a dose may also be administered once per day or divided into several administrations per day.
The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present invention may be varied by methods for formulating the pharmaceutical composition into a preparation, administration manner, administration time and/or administration route, etc., and may be varied according to various factors including the type and degree of reaction to be achieved by administration of the pharmaceutical composition, the type of subject to be administered, age, body weight, general health status, symptoms or severity of disease, sex, daily diet, excretion, components of other pharmaceutical compositions used together at the same time or at different times for the respective subjects, etc., and other similar factors well known in the pharmaceutical art, wherein a person skilled in the art can easily determine and prescribe a dose effective for targeted therapy.
In the case of administering the pharmaceutical composition of the present invention, it may be administered once a day or divided into several administrations per day. The pharmaceutical compositions of the present invention may be administered as the sole therapeutic agent or in combination with other therapeutic agents, and may also be administered sequentially or simultaneously with conventional therapeutic agents. In view of all of the above, the pharmaceutical composition of the present invention may be administered in an amount that may exhibit the maximum effect achieved at the minimum amount without any side effects, wherein such an amount may be easily determined by those skilled in the art to which the present invention pertains.
The pharmaceutical composition of the present invention can exhibit excellent effects even when used alone, but can be further used in combination with various methods such as hormone therapy, drug therapy, and the like in order to improve the therapeutic efficiency.
The present invention also provides a method for treating uveitis, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound represented by formula I above, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.
As used herein, the term "therapeutically effective amount" refers to an amount of a compound represented by formula I above, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, that is effective in treating uveitis.
In the method of treatment of the present invention, a suitable total daily dose of the compound represented by the above formula I, an optical isomer thereof or a pharmaceutically acceptable salt thereof may be determined by the attending physician within the scope of sound medical decision, and such a dose, which may be in the range of, for example, about 0.1 to 10000mg/kg, about 1 to 8000mg/kg, about 5 to 6000mg/kg, or about 10 to 4000mg/kg, and preferably, in the range of about 50 to 2000mg/kg, may be administered once per day or divided into several administrations per day. However, for the purposes of the present invention, it is preferred that a specific therapeutically effective amount for a certain patient be administered in different ways depending on a variety of factors including the type and extent of the response to be achieved, the specific composition (including whether or not other agents are used in some cases), the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration and rate of secretion of the composition, the treatment period, and the drugs used together or simultaneously with the specific composition, and other similar factors well known in the pharmaceutical art.
The method of treating uveitis according to the present invention includes not only treating the disease itself before manifestation of symptoms of the disease, but also inhibiting or avoiding such symptoms by administering a compound represented by formula I described above, an optical isomer thereof, or a pharmaceutically acceptable salt thereof. In treating diseases, the prophylactic or therapeutic dose of an active ingredient may vary depending on the nature and severity of the disease or condition and the route of administration of the active ingredient. The dosage and frequency may vary according to the age, weight and response of the individual patient. Naturally, in view of such factors, the skilled person can easily select the appropriate dosage and use. Also, the method for treating uveitis according to the present invention may further comprise administering a therapeutically effective dose of an additional active agent useful for treating the disease together with the compound represented by the above formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, wherein the additional active agent may exhibit a synergistic or additive effect together with the compound of the above formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.
The present invention also provides the use of a compound represented by formula I above, an optical isomer thereof, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of uveitis.
The compound represented by the above formula I, its optical isomer or a pharmaceutically acceptable salt thereof for the preparation of a medicament may be combined with pharmaceutically acceptable adjuvants, diluents, carriers, etc., and may be prepared as a complex medicament together with other active agents, thereby having a synergistic effect.
The contents mentioned in the pharmaceutical composition, the method of treatment and the use of the present invention are equally applicable if they are not contradictory to each other.
Advantageous effects of the invention
The pharmaceutical composition comprising the compound represented by formula I, an optical isomer thereof, or a pharmaceutically acceptable salt thereof of the present invention may exhibit an excellent effect of treating uveitis, so that the pharmaceutical composition may be widely used for preventing or treating uveitis.
Brief Description of Drawings
Fig. 1 shows the results of identifying the effect of the compounds of the present invention on the inhibition of TNF α secretion.
FIG. 2 shows the results of identifying the effect of the compounds of the present invention on the inhibition of the proliferation of responding T cells.
Figure 3 shows the results of identifying the effect of compounds of the invention on modulating the function of regulatory T cells.
Figure 4 shows a graph evaluating the clinical grade of uveitis on the retina of Experimental Autoimmune Uveitis (EAU) mice (p < 0.05;. p < 0.01;. p < 0.001, by Tukey's multiple comparative test).
Figure 5 shows a picture identifying clinical changes in the retina of an EAU mouse.
Fig. 6 shows hematoxylin and eosin (H & E) staining results on retinas of EAU mice.
Figure 7 shows the results of immunofluorescent staining for CD3 and B220 in the retina of EAU mice.
Figure 8 shows the results of immunofluorescent staining for HDAC6 and CD4 in the retina of EAU mice.
Figure 9 shows the results of immunofluorescent staining for HDAC6 and B220 in the retina of EAU mice.
Figure 10 shows the results of immunofluorescent staining for HDAC6 and a-tubulin in the retina of EAU mice.
FIG. 11 shows the results of ELISA on IFN-. gamma.and IL-17A in spleen tissue of EAU mice (p < 0.05;. p < 0.01;. p < 0.001, by Tukey's multiple comparative experiment).
FIG. 12 shows the results of real-time PCR of HDAC, IL- β, IFN- γ, IL-17 and TNF- α in the eye tissues of EAU mice (V: vehicle; C: CKD 4; p: 0.05; p: 0.01; p: 0.001, by Tukey's multiple comparative experiment).
FIG. 13 shows the results of FACS on IFN-. gamma. (+)/CD4(+) immune cells in retinal tissues of EAU mice to which the compound of the present invention was administered by eye-dropping.
FIG. 14 shows the results of FACS on IFN-. gamma. (+)/CD4(+) immune cells in retinal tissues of EAU mice to which the compound of the present invention was administered intraperitoneally.
FIG. 15 shows the results of FACS on IFN-. gamma. (+)/CD4(+) immune cells in lymph nodes of EAU mice to which the compound of the present invention was administered by eye drop.
FIG. 16 shows the results of FACS on IFN-. gamma. (+)/CD4(+) immune cells in lymph nodes of EAU mice to which the compound of the present invention was intraperitoneally administered.
FIG. 17 shows the results of FACS on IL-1. beta. (+) immune cells in lymph nodes of EAU mice to which the compound of the present invention was administered by eye drop.
FIG. 18 shows the results of FACS on IL-1. beta. (+) immune cells in retinal tissues of EAU mice to which the compound of the present invention was administered by eye-dropping.
Fig. 19 shows the results of FACS performed on CD11b (+) immune cells in lymph nodes of an EAU mouse to which the compound of the present invention was intraperitoneally administered.
Fig. 20 shows the results of FACS performed on CD19(+) immune cells in lymph nodes of an EAU mouse to which a compound of the present invention was intraperitoneally administered.
FIG. 21 shows the results of FACS on F4/80(+) immune cells in lymph nodes of EAU mice to which the compounds of the present invention were administered intraperitoneally.
MODE OF THE INVENTION
Hereinafter, the present invention will be described in more detail based on preparation examples and embodiments. However, these preparation examples and embodiments are only for the purpose of illustrating the present invention, and thus the present invention is not limited thereto.
The
Preparation example 1 Synthesis of Compound 255{ N- (3-bromophenyl) -N- (4- (hydroxycarbamoyl) benzyl) morpholine-4-carboxamide }
[ step 1] Synthesis of methyl 4- ((N- (3-bromophenyl) morpholine-4-carboxamido) methyl) benzoate
Methyl 4- (((3-bromophenyl) ((4-nitrophenoxy) carbonyl) amino) methyl) benzoate (1.5g, 3.09mmol) was dissolved in acetonitrile (50mL), to which was then slowly added potassium carbonate (1.28g, 9.3mmol) and morpholine (0.40mL, 4.64 mmol). Thereafter, the temperature of the resulting mixture was slowly raised to 80 ℃, and then the resulting mixture was stirred at that temperature for three hours. The temperature was cooled to room temperature, and then dimethylformamide (50ml) was further added to the resultant mixture, and then the temperature was again raised to 80 ℃, followed by stirring the resultant mixture at that temperature for five hours. After the completion of the reaction, the organic layer was washed three times with a saturated aqueous ammonium chloride solution, then dried over sodium sulfate, and filtered, and then the filtrate was concentrated under reduced pressure. The concentrate was purified by column chromatography (silica; ethyl acetate/hexane ═ 0-50%) to obtain the title compound (0.45g, 33.6%) as a clear oil.
[ step 2] Synthesis of N- (3-bromophenyl) -N- (4- (hydroxycarbamoyl) benzyl) morpholine-4-carboxamide
Methyl 4- ((N- (3-bromophenyl) morpholine-4-carboxamido) methyl) benzoate (0.05g, 0.12mmol) was dissolved in methanol (2ml), to which was then slowly added hydroxylamine hydrochloride (0.040g, 0.58 mmol). Thereafter, potassium hydroxide (0.065g, 1.15mmol) was added to the resulting mixture and stirred at room temperature for ten minutes, and then hydroxylamine (50.0 wt% aqueous solution, 0.14mL, 2.31mmol) was added thereto. The resulting mixture was stirred at room temperature for one day, after which the organic solvent was concentrated under reduced pressure and then neutralized by adding 2N hydrochloric acid. Then, the organic layer was washed three times with a saturated aqueous sodium chloride solution, followed by drying over anhydrous sodium sulfate and filtration. Thereafter, the filtrate was concentrated under reduced pressure, and then the concentrate was purified by column chromatography (silica; ethyl acetate/hexane ═ 0 to 80%), to obtain the title compound (0.036g, 72%) as a white solid.
1H NMR(400MHz,CDCl3-d6)7.63(d,2H,J＝7.8Hz)，7.27-7.20(m,4H)，7.13(t,1H,J＝7.8Hz)，6.96(d,1H,J＝7.1Hz)，4.83(s,2H)，3.49(brs,4H)，3.23(brs,4H)；MS(ESI)m/z436(M++H)。
Preparation example 2 Synthesis of Compound 280{ N- (4- (hydroxycarbamoyl) benzyl) -N- (pyridin-2-yl) morpholine-4-carboxamide }
[ step 1] Synthesis of methyl 4- ((pyridin-2-ylamino) methyl) benzoate
Pyridin-2-amine (0.2g, 2.13mmol) was dissolved in methanol (10mL) and methyl 4-formylbenzoate (0.35g, 2.13mmol) was added thereto. The resulting mixture was stirred at room temperature for 20 minutes, after which sodium cyanoborohydride (0.13g, 2.13mmol) and acetic acid (0.12mL, 2.13mmol) were slowly added thereto, followed by stirring at room temperature for five hours. The resulting mixture was washed three times with a saturated aqueous sodium chloride solution, and then the organic layer was dried over sodium sulfate and filtered, followed by concentrating the filtrate under reduced pressure. The concentrate was purified by column chromatography (silica; ethyl acetate/hexane ═ 0-30%) to obtain the title compound (0.10g, 19%) as a clear oil.
1H NMR(400MHz,CDCl3)8.17(d,1H,J＝5.8Hz)，8.06(d,2H,J＝8.4Hz)，7.66(t,1H,J＝7.8Hz)，7.44(d,2H,J＝8.0Hz)，6.76(t,1H,J＝6.7Hz)，6.58(d,1H,J＝8.6Hz)，4.67(d,2H,J＝6.0Hz)，3.92(s,3H)。
[ step 2] Synthesis of methyl 4- ((((4-nitrophenoxy) carbonyl) (pyridin-2-yl) amino) methyl) benzoate
Methyl 4- ((pyridin-2-ylamino) methyl) benzoate (0.040g, 0.16mmol) was dissolved in dimethylformamide (3mL), to which was then slowly added potassium carbonate (0.046g, 0.33 mmol). Thereafter, 4-nitrophenyl chloroformate (0.037g, 0.18mmol) was added to the resulting mixture, and then the temperature of the resulting mixture was slowly raised to 50 ℃, followed by stirring the resulting mixture at that temperature for two days. After the completion of the reaction, the ethyl acetate layer was washed three times with a saturated aqueous ammonium chloride solution, and then the organic layer was dried over sodium sulfate and filtered, followed by concentrating the filtrate under reduced pressure. The concentrate was purified by column chromatography (silica; ethyl acetate/hexane ═ 0-50%) to obtain the title compound (0.048g, 71%) as a yellow oil.
1H NMR(400MHz,CDCl3)8.49-8.48(m,1H)，8.24(dd,2H,J＝7.0,2.2Hz)，8.17(dd,2H,J＝7.2,2.0Hz)，8.00(d,2H,J＝8.4Hz)，7.78(t,1H,J＝3.8Hz)，7.44(d,2H,J＝8.0Hz)，6.91(dd,2H,J＝7.3,2.1Hz)，5.39(brs,2H)，3.92(s,3H)；MS(ESI)m/z 408(M++H)。
[ step 3] Synthesis of methyl 4- ((N- (pyridin-2-yl) morpholine-4-carboxamido) methyl) benzoate
Methyl 4- (((((4-nitrophenoxy) carbonyl) (pyridin-2-yl) amino) methyl) benzoate (0.040g, 0.098mmol) was dissolved in dimethylformamide (5mL), to which was then slowly added potassium carbonate (0.040g, 0.30mmol) and morpholine (0.013mL, 0.15 mmol). Thereafter, the temperature of the resulting mixture was slowly raised to 80 ℃, and then the resulting mixture was stirred at that temperature for three hours. After the completion of the reaction, the resultant mixture was washed three times with a saturated aqueous ammonium chloride solution, and then the organic layer was dried over sodium sulfate and filtered, followed by concentrating the filtrate under reduced pressure. The concentrate was purified by column chromatography (silica; ethyl acetate/hexane ═ 0-50%) to obtain the title compound (0.022g, 63%) as a pale yellow solid.
1H NMR(400MHz,CDCl3)8.37-8.35(m,1H)，7.95(d,2H,J＝8.4Hz)，7.60-7.58(m,1H)，7.47(d,2H,J＝8.4Hz)，6.94-6.89(m,2H)，5.13(s,2H)，3.89(s,3H)，3.53-3.51(m,4H)，3.31-3.29(m,4H)。
[ step 4] Synthesis of N- (4- (hydroxycarbamoyl) benzyl) -N- (pyridin-2-yl) morpholine-4-carboxamide
Methyl 4- ((N- (pyridin-2-yl) morpholine-4-carboxamido) methyl) benzoate (0.022g, 0.062mmol) was dissolved in MeOH (2ml) to which hydroxylamine hydrochloride (0.022g, 0.31mmol) was then slowly added. Thereafter, potassium hydroxide (0.035g, 0.62mmol) was added to the resulting mixture, followed by stirring at room temperature for ten minutes, followed by addition of hydroxylamine (50.0 wt% aqueous solution, 0.082mL, 1.24mmol) thereto. The resulting mixture was stirred at room temperature for one day, after which the organic solvent was concentrated under reduced pressure, then neutralized by adding 2N HCl, then washed three times with saturated aqueous sodium chloride solution, and then the organic layer was dried over anhydrous sodium sulfate and filtered, thereby obtaining the title compound (0.007g, 32%) as a white solid.
1H NMR(400MHz,MeOD-d3)8.32(d,1H,J＝3.6Hz)，7.72(t,1H,J＝6.6Hz)，7.67(d,2H,J＝8.2Hz)，7.48(d,2H,J＝8.2Hz)，7.08-7.01(m,2H)，5.08(s,2H)，3.52(t,4H,J＝4.8Hz)，3.29(t,4H,J＝4.8Hz)；MS(ESI)m/z 357(M++H)。
Preparation example 3 Synthesis of Compound 374(CKD4) { N- (4- (hydroxycarbamoyl) benzyl) -N- (3- (trifluoromethyl) phenyl) morpholine-4-carboxamide }
[ step 1] Synthesis of methyl 4- ((3- (trifluoromethyl) phenylamino) methyl) benzoate
3- (trifluoromethyl) aniline (0.30g, 1.84mmol) and potassium carbonate (0.76g, 5.53mmol) were dissolved in Dimethylformamide (DMF) (5mL), to which was then added methyl 4- (bromomethyl) benzoate (0.42g, 1.84 mmol). The resulting mixture was allowed to react at room temperature for one day and diluted with ethyl acetate. The reaction mixture was washed with water and saturated aqueous sodium chloride solution, and then dried over anhydrous magnesium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by column chromatography (silica; ethyl acetate/hexane ═ 20%) to obtain the title compound (0.37g, 65%).
1H NMR(400MHz,DMSO-d6)7.93(d,2H,J＝8.3Hz)，7.49(d,2H,J＝8.3Hz)，7.24(t,1H,J＝7.9Hz)，6.88-6.78(m,4H)，4.42(d,2H,J＝6.1Hz)，3.83(s,3H)，MS(ESI)m/z 310(M++H)。
[ step 2] Synthesis of methyl 4- ((((4-nitrophenoxy) carbonyl) (3- (trifluoromethyl) phenyl) amino) methyl) benzoate
Methyl 4- ((3- (trifluoromethyl) phenylamino) methyl) benzoate (0.26g, 0.82mmol) and 4-nitrophenyl chloroformate (0.33g,1.65mmol) were dissolved in acetonitrile (10mL), to which was then added potassium carbonate (0.34g, 2.47 mmol). The resulting mixture was allowed to react at room temperature for one day and diluted with ethyl acetate. The reaction mixture was washed with a saturated aqueous solution of sodium chloride, then dried over anhydrous sodium sulfate, and filtered, followed by concentration under reduced pressure. The residue was purified by column chromatography (silica; ethyl acetate/hexane ═ 20%) to obtain the title compound (0.35g, 89%) as a colorless oil.
1H NMR(400MHz,CDCl3)8.20(d,2H,J＝10.2Hz)，8.01(d,2H,J＝7.8Hz)，7.56-7.46(m,3H)，7.35(d,3H,J＝8.0Hz)，7.26(d,2H,J＝8.1Hz)，5.01(bs,2H)，3.90(s,3H)。
[ step 3] Synthesis of methyl 4- ((N- (3- (trifluoromethyl) phenyl) morpholine-4-carboxamido) methyl) benzoate
Methyl 4- ((((4-nitrophenoxy) carbonyl) (3- (trifluoromethyl) phenyl) amino) methyl) benzoate (0.29g, 0.60mmol) was dissolved in dimethylformamide (10mL), and then potassium carbonate (0.25g, 1.81mmol) and morpholine (0.05mL, 0.60mmol) were added thereto. The resulting mixture was allowed to react at 60 ℃ for two days and then diluted with saturated ammonium chloride solution. Extraction was performed by ethyl acetate, and then the extract was dried over anhydrous sodium sulfate and filtered, followed by concentration under reduced pressure. The residue was purified by column chromatography (silica; ethyl acetate/hexane ═ 50%) to obtain the title compound (0.15g, 60%).
1H NMR(400MHz,DMSO-d6)7.97(d,2H,J＝8.2Hz)，7.43-7.32(m,5H)，7.20(d,1H,J＝8.0Hz)，4.94(s,2H)，3.90(s,3H)，3.50(t,4H,J＝4.8Hz)，3.25(t,4H,J＝4.8Hz)；MS(ESI)m/z 423(M++H)。
[ step 4] Synthesis of N- (4- (hydroxycarbamoyl) benzyl) -N- (3- (trifluoromethyl) phenyl) morpholine-4-carboxamide
Methyl 4- ((N- (3- (trifluoromethyl) phenyl) morpholine-4-carboxamido) methyl) benzoate (0.15g, 0.36mmol) was dissolved in methanol (5mL), to which was then added an aqueous hydroxylamine solution (50% by weight, 1mL) and potassium hydroxide (0.10g, 1.81mmol), followed by stirring overnight. After completion of the reaction, methanol was distilled and removed under reduced pressure, and then extracted by ethyl acetate and water, followed by workup. The resulting extract was dried over anhydrous sodium sulfate, and filtered, followed by concentration under reduced pressure. The residue was stirred in ether, then a solid product was prepared, which was filtered and dried to obtain the title compound (0.082g, 54%) as a white solid.
1H NMR(400MHz,MeOD-d3)11.14(brs,1H)，8.99(brs,1H)，7.85(d,2H,J＝8.0Hz)，7.66-7.27(m,6H)，4.94(s,2H)，3.41(s,2H)，3.15(s,2H)。MS(ESI)m/z 424(M++H)。
Preparation example 4 Synthesis of Compound 416{ N- (2, 4-difluorophenyl) -N- (4- (hydroxycarbamoyl) benzyl) -4-methylpiperazine-1-carboxamide }
[ step 1] Synthesis of methyl 4- ((N- (2, 4-difluorophenyl) -4-methylpiperazine-1-carboxamido) methyl) benzoate
Methyl 4- (((2, 4-difluorophenyl) ((4-nitrophenoxy) carbonyl) amino) methyl) benzoate (0.50g, 1.13mmol) and 1-methylpiperazine (0.126mL, 1.13mmol) were dissolved in dimethylformamide (10mL), then heated and stirred at 60 ℃ for two days. Dimethylformamide was removed under reduced pressure, then water was poured into the resulting reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, and then dehydrated over anhydrous magnesium sulfate, followed by concentration under reduced pressure. The residue was purified by column chromatography (silica; methanol/dichloromethane ═ 5%) and concentrated to give the title compound (0.46g, 101%) as a yellow oil.
[ step 2] Synthesis of N- (2, 4-difluorophenyl) -N- (4- (hydroxycarbamoyl) benzyl) -4-methylpiperazine-1-carboxamide
Methyl 4- ((N- (2, 4-difluorophenyl) -4-methylpiperazine-1-carboxamido) methyl) benzoate (0.22g, 0.545mmol) was dissolved in methanol (20mL), and then hydroxylamine hydrochloride (0.189g, 2.73mmol) and potassium hydroxide (0.306g, 5.45mmol) were added thereto and stirred, and then hydroxylamine (50 wt% aqueous solution; 0.701mL, 10.9mmol) was added dropwise thereto, followed by stirring at room temperature for three hours. After completion of the reaction, methanol was removed under reduced pressure, and then water was poured into the resulting reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, and then dehydrated over anhydrous magnesium sulfate, followed by concentration under reduced pressure. Thereafter, the resulting concentrate was dissolved in dichloromethane, and then hexane was added thereto, followed by precipitation of a solid, filtration and drying, to obtain the title compound (0.154g, 70%) as a yellow solid.
1H NMR(400MHz,MeOD-d3)7.65(d,2H,J＝8.2Hz)，7.40(d,2H,J＝8.2Hz)，7.26-7.25(m,1H)，7.04-6.96(m,2H)，4.79(s,2H)，3.25-3.23(m,4H)，2.24-2.21(m,7H)；MS(ESI)m/z 405.1(M++H)。
Preparation example 5 Synthesis of Compound 461{ 4-Ethyl-N- (4- (hydroxycarbamoyl) benzyl) -N- (3- (trifluoromethyl) phenyl) piperazine-1-carboxamide }
[ step 1] Synthesis of methyl 4- ((4-ethyl-N- (3- (trifluoromethyl) phenyl) piperazine-1-carboxamido) methyl) benzoate
Methyl 4- ((((4-nitrophenoxy) carbonyl) (3- (trifluoromethyl) phenyl) amino) methyl) benzoate (0.346g, 0.73mmol) was dissolved in dimethylformamide (10mL), and then potassium carbonate (0.30g, 2.19mmol) and 1-ethylpiperazine (0.09mL,0.73mmol) were added thereto. The resulting mixture was allowed to react at 60 ℃ for one day, then diluted with ethyl acetate, followed by washing with a saturated ammonium chloride solution. The resulting mixture was dried over anhydrous magnesium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by column chromatography (silica; ethyl acetate/hexane ═ 50%) to obtain the title compound (0.15g, 46%).
[ step 2] Synthesis of 4-Ethyl-N- (4- (hydroxycarbamoyl) benzyl) -N- (3- (trifluoromethyl) phenyl) piperazine-1-carboxamide
Methyl 4- ((4-ethyl-N- (3- (trifluoromethyl) phenyl) piperazine-1-carboxamido) methyl) benzoate (0.15g, 0.33mmol) was dissolved in methanol (10mL), and then hydroxylamine (50% by weight aqueous solution, 0.20mL) and potassium hydroxide (0.09g, 1.67mmol) were added thereto, followed by stirring overnight.
After completion of the reaction, methanol was distilled and removed under reduced pressure, and then extracted by ethyl acetate and water, followed by workup. The obtained extract was dried over anhydrous magnesium sulfate, and filtered, followed by concentration under reduced pressure. The residue was stirred in ether and then made solid, which was filtered and dried to obtain the title compound (0.09g, 61%) as a yellow solid.
1H NMR(400MHz,DMSO-d6)11.1(brs,1H)，7.65(d,2H,J＝8.2Hz)，7.51(t,1H,J＝7.9Hz)，7.41-7.36(m,5H)，4.92(s,2H),3.17-3.14(m,4H)，2.25,2.22(ABq,2H,J＝12.4,7.2Hz)，2.18-2.15(m,4H)，0.92(t,3H,J＝7.2Hz)；MS(ESI)m/z 451.1(M++H)。
Preparation example 6 Synthesis of Compound 476{3, 3-difluoro-N- (4- (hydroxycarbamoyl) benzyl) -N- (3- (trifluoromethyl) phenyl) azetidine-1-carboxamide }
[ step 1] Synthesis of methyl 4- ((3, 3-difluoro-N- (3- (trifluoromethyl) phenyl) azetidine-1-carboxamido) methyl) benzoate
Methyl 4- ((((4-nitrophenoxy) carbonyl) (3- (trifluoromethyl) phenyl) amino) methyl) benzoate (0.24g, 0.51mmol) was dissolved in dimethylformamide (5ml), to which was then added potassium carbonate (0.21g, 1.52mmol) and 3, 3-difluoroazetidine hydrochloride (0.13g, 1.10 mmol). The resulting mixture was allowed to react at 60 ℃ for two days and then diluted with saturated ammonium chloride solution. Extraction was performed by ethyl acetate, and then the obtained extract was dried by anhydrous sodium sulfate, and filtered, followed by concentration under reduced pressure. The residue was purified by column chromatography (silica; ethyl acetate/hexane ═ 30%) to obtain the title compound (0.14g, 63%).
[ step 2] Synthesis of 3, 3-difluoro-N- (4- (hydroxycarbamoyl) benzyl) -N- (3- (trifluoromethyl) phenyl) azetidine-1-carboxamide
Methyl 4- ((3, 3-difluoro-N- (3- (trifluoromethyl) phenyl) azetidine-1-carboxamido) methyl) benzoate (0.14g, 0.32mmol) was dissolved in methanol (10mL), and then an aqueous hydroxylamine solution (50% by weight, 0.2mL) and potassium hydroxide (0.09g, 1.60mmol) were added thereto, followed by stirring overnight. After completion of the reaction, methanol was distilled and removed under reduced pressure, and then extracted by ethyl acetate and water, followed by workup. The resulting extract was dried over anhydrous sodium sulfate, and filtered, followed by concentration under reduced pressure. The residue was stirred in ether, then a solid product was prepared, which was filtered and dried to obtain the title compound (0.072g, 52%) as a white solid.
Preparation example 7 Synthesis of Compound 500{ N- (3- (fluoromethyl) phenyl) -N- (4- (hydroxycarbamoyl) benzyl) morpholine-4-carboxamide }
[ step 1] Synthesis of methyl 4- ((N- (3- (fluoromethyl) phenyl) morpholine-4-carboxamido) methyl) benzoate
4- ((N- (3- (hydroxymethyl) phenyl) morpholine-4-carboxamido) methyl) benzoic acid (1.25g, 3.25mmol) was dissolved in dichloromethane (20mL), diethylaminosulfur trifluoride (DAST, 0.424mL, 3.58mmol) was then added thereto at 0 ℃ and then stirred at the same temperature for one hour, and then a saturated aqueous sodium bicarbonate solution was poured into the resulting reaction mixture, followed by extraction with dichloromethane. The organic layer was washed with a saturated aqueous sodium chloride solution, and then dehydrated over anhydrous magnesium sulfate, followed by concentration under reduced pressure. The residue was purified by column chromatography (silica; ethyl acetate/hexane ═ 30-50%) and concentrated to obtain the title compound (0.617g, 49%) as a colorless liquid.
[ step 2] Synthesis of N- (3- (fluoromethyl) phenyl) -N- (4- (hydroxycarbamoyl) benzyl) morpholine-4-carboxamide
Methyl 4- ((N- (3- (fluoromethyl) phenyl) morpholine-4-carboxamido) methyl) benzoate (0.100g, 0.259mmol) was dissolved in methanol (10mL), and hydroxylamine (50.0 wt% aqueous solution, 1.11mL, 18.1mmol) was added thereto at room temperature. Then, potassium hydroxide (0.145g, 2.59mmol) was added to the resultant mixture, and stirred at the same temperature for 30 minutes. After that, the solvent was removed from the resulting reaction mixture under reduced pressure, and then a saturated aqueous sodium hydrogencarbonate solution was poured into the resulting concentrate, followed by extraction with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, and then dehydrated over anhydrous magnesium sulfate, followed by concentration under reduced pressure. To the resulting concentrate were added dichloromethane (5mL) and hexane (30mL) and stirred, followed by filtration and drying of the precipitated solid, thereby obtaining the title compound (0.089g, 89%) as a white solid.
1H NMR(400MHz,DMSO-d6)11.12(brs,1H)，8.98(brs,1H)，7.64(d,2H,J＝8.3Hz)，7.36-7.32(m,3H)，7.20(s,1H)，7.15(d,1H,J＝7.5Hz)，7.09(d,1H,J＝7.4Hz)，5.36(d,2H,J＝47.5Hz)，4.87(s,2H)，3.39(t,4H,J＝4.6Hz)，3.13(t,4H,J＝4.6Hz)。MS(ESI)m/z 388(M++H)。
Preparation example 8 Synthesis of Compound 530{ N- (3-fluorophenyl) -N- (4- (hydroxycarbamoyl) benzyl) morpholine-4-carboxamide }
[ step 1] Synthesis of methyl 4- ((3-fluorophenylamino) methyl) benzoate
Methyl 4-formylbenzoate (1.47g, 8.99mmol) was dissolved in methanol (50mL), and 3-fluoroaniline (1.0g, 8.99mmol) was then added thereto. The resulting mixture was allowed to react at room temperature for three hours, and then sodium cyanoborohydride (NaCNBH) was added thereto3) (0.56g, 8.99mmol) and acetic acid (1.03mL, 17.99 mmol). The reaction was allowed to react at room temperature for one day, after which the reaction solvent was left under reduced pressure and removed, and then a saturated aqueous sodium hydrogencarbonate solution was poured thereinto, followed by extraction with ethyl acetate. The organic layer was dehydrated over anhydrous magnesium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography (silica; ethyl acetate/hexane ═ 20%) to obtain the title compound (1.84g, 79%).
[ step 2] Synthesis of methyl 4- (((3-fluorophenyl) ((4-nitrophenoxy) carbonyl) amino) methyl) benzoate
Methyl 4- ((3-fluorophenylamino) methyl) benzoate (2.7g, 10.4mmol) and 4-nitrophenyl chloroformate (4.20g, 20.8mmol) were dissolved in acetonitrile (100mL), to which was then added potassium carbonate (4.32g, 31.2 mmol). The resulting mixture was allowed to react at room temperature for one day and diluted with ethyl acetate. The reaction mixture was washed with a saturated aqueous solution of sodium chloride, then dried over anhydrous sodium sulfate, and filtered, followed by concentration under reduced pressure. The residue was purified by column chromatography (silica; ethyl acetate/hexane ═ 20%) to obtain the title compound (2.65g, 60%) as a colorless oil.
[ step 3] Synthesis of methyl 4- ((N- (3-fluorophenyl) morpholine-4-carboxamido) methyl) benzoate
Methyl 4- (((3-fluorophenyl) ((4-nitrophenoxy) carbonyl) amino) methyl) benzoate (0.32g, 0.75mmol) was dissolved in dimethylformamide (5mL), to which was then added potassium carbonate (0.31g, 2.24mmol) and morpholine (0.13mL, 1.49 mmol). The resulting mixture was allowed to react at 60 ℃ for one day and then diluted with a saturated ammonium chloride solution. Extraction was performed by ethyl acetate, and then the obtained extract was dried by anhydrous sodium sulfate, and filtered, followed by concentration under reduced pressure. The residue was purified by column chromatography (silica; ethyl acetate/hexane ═ 30%) to obtain the title compound (0.13g, 45%).
[ step 4] Synthesis of N- (3-fluorophenyl) -N- (4- (hydroxycarbamoyl) benzyl) morpholine-4-carboxamide
Methyl 4- ((N- (3-fluorophenyl) morpholine-4-carboxamido) methyl) benzoate (0.108g, 0.290mmol) was dissolved in methanol (10mL), and hydroxylamine (50.0 wt% aqueous solution, 1.19mL, 19.4mmol) was added thereto at room temperature. Then, potassium hydroxide (0.156g, 2.78mmol) was added to the resultant mixture, and stirred at the same temperature for 16 hours. After that, the solvent was removed from the resulting reaction mixture under reduced pressure, and then a saturated aqueous sodium hydrogencarbonate solution was poured into the resulting concentrate, followed by extraction with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, and then dehydrated over anhydrous magnesium sulfate, followed by concentration under reduced pressure. The precipitated solid was filtered and dried to obtain the title compound (0.062g, 57%) as a white solid.
1H NMR(400MHz,DMSO-d6)11.14(brs,1H)，8.99(brs,1H)，7.65(d,2H,J＝7.0Hz)，7.38-7.30(m,3H)，7.05-6.85(m,3H)，4.89(s,1H)，3.44-3.42(m,4H)，3.18-3.15(m,4H)，2.08(s,3H)。MS(ESI)m/z 374(M++H)。
Preparation example 9 Synthesis of Compound 532{ N- (2-fluoro-4- (hydroxycarbamoyl) benzyl) -N- (3- (trifluoromethyl) phenyl) morpholine-4-carboxamide }
[ step 1] Synthesis of 3-fluoro-4- (((3- (trifluoromethyl) phenyl) amino) methyl) benzonitrile
3- (trifluoromethyl) aniline (0.998mL,8.068mmol) was dissolved in acetonitrile (60mL), and then 4- (bromomethyl) -3-fluorobenzonitrile (2.072g, 9.682mmol) and DIPEA (2.143mL, 12.102mmol) were added thereto at room temperature, followed by stirring at the same temperature for one day. After that, a saturated aqueous sodium hydrogencarbonate solution was poured into the resulting reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, and then dehydrated over anhydrous magnesium sulfate, followed by concentration under reduced pressure. The resulting concentrate was purified by column chromatography (silica; ethyl acetate/hexane ═ 5-20%) and concentrated to obtain the title compound (2.380g, 64.4%) as a yellow liquid.
[ step 2] Synthesis of 3-fluoro-4- (((3- (trifluoromethyl) phenyl) amino) methyl) benzoic acid
3-fluoro-4- (((3- (trifluoromethyl) phenyl) amino) methyl) benzonitrile (2.310g, 7.850mmol) and lithium hydroxide (3.294g, 78.505mmol) in methanol (40mL)/H2O (20mL), followed by heating the resulting reaction mixture at reflux for 16 hours, then cooling to room temperature, followed by concentration of the resulting reaction mixture under reduced pressure. To obtainTo the mixture was added 2M aqueous hydrochloric acid solution to reach pH 1, and then the precipitated solid was filtered and dried, thereby obtaining the title compound (1.700g, 69.1%) as a white solid.
[ step 3] Synthesis of methyl 3-fluoro-4- (((3- (trifluoromethyl) phenyl) amino) methyl) benzoate
3-fluoro-4- (((3- (trifluoromethyl) phenyl) amino) methyl) benzoic acid (1.700g, 5.427mmol), methanol (4.402mL, 108.540mmol), EDC (2.081g, 10.854mmol), HOBt (1.467g, 10.854mmol) and DIPEA (2.883mL, 16.281mmol) were dissolved in tetrahydrofuran (50mL) at room temperature, and then the resulting reaction solution was stirred at the same temperature for 16 hours, and then a saturated aqueous sodium bicarbonate solution was poured into the resulting reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, and then dehydrated over anhydrous magnesium sulfate, followed by concentration under reduced pressure. The concentrate was purified by column chromatography (silica; ethyl acetate/hexane ═ 10-40%) and concentrated to obtain the title compound (1.500g, 84.5%) as a colorless liquid.
[ step 4] Synthesis of methyl 3-fluoro-4- (((((4-nitrophenoxy) carbonyl) (3- (trifluoromethyl) phenyl) amino) methyl) benzoate
Methyl 3-fluoro-4- (((3- (trifluoromethyl) phenyl) amino) methyl) benzoate (1.500g, 4.583mmol), 4-nitrophenylchloroformate (1.848g, 9.167mmol) and potassium carbonate (1.900g, 13.750mmol) were dissolved in acetonitrile (80mL) at room temperature, and then the resulting reaction solution was stirred at the same temperature for 16 hours, and then a saturated aqueous sodium bicarbonate solution was poured into the resulting reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, and then dehydrated over anhydrous magnesium sulfate, followed by concentration under reduced pressure. The resulting concentrate was purified by column chromatography (silica; ethyl acetate/hexane ═ 10 to 40%) and concentrated to obtain the title compound (0.927g, 41.1%) as a colorless liquid.
[ step 5] Synthesis of methyl 3-fluoro-4- ((N- (3- (trifluoromethyl) phenyl) morpholine-4-carboxamido) methyl) benzoate
Methyl 3-fluoro-4- ((((4-nitrophenoxy) carbonyl) (3- (trifluoromethyl) phenyl) amino) methyl) benzoate (0.129g, 0.262mmol), morpholine (0.046mL, 0.524mmol) and potassium carbonate (0.109g, 0.786mmol) were dissolved in N, N-dimethylformamide (5mL) at 60 ℃, then the resulting reaction solution was stirred at the same temperature for two days, and then a saturated aqueous sodium bicarbonate solution was poured into the resulting reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with a saturated aqueous sodium chloride solution, and then dehydrated over anhydrous magnesium sulfate, followed by concentration under reduced pressure. The resulting concentrate was purified by column chromatography (silica; ethyl acetate/hexane ═ 30 to 60%) and concentrated to obtain the title compound (0.094g, 81.5%) as a colorless liquid.
[ step 6] Synthesis of N- (2-fluoro-4- (hydroxycarbamoyl) benzyl) -N- (3- (trifluoromethyl) phenyl) morpholine-4-carboxamide
Methyl 3-fluoro-4- ((N- (3- (trifluoromethyl) phenyl) morpholine-4-carboxamido) methyl) benzoate (0.094g, 0.213mmol) and hydroxylamine (50.0 wt% aqueous solution, 0.071g, 2.134mmol) were dissolved in methanol (5mL), and then potassium hydroxide (0.060g, 1.067mmol) was added thereto at room temperature, followed by stirring at the same temperature for two hours, followed by concentrating the resulting reaction mixture under reduced pressure. Ether (10mL) was added to the resulting concentrate and stirred, followed by filtration and drying of the precipitated solid, thereby obtaining compound 532(0.068g, 72.2%) as a bright yellow solid.
1H NMR(400MHz,DMSO-d6)11.2(brs,1H)，9.13(brs,1H)，7.57-7.42(m,7H)，4.94(s,2H)，3.44-3.34(m,4H)，3.18-3.12(m,4H)；MS(ESI)m/z 442.1(M++H)。
Example 1 identification of inhibitory Effect on TNF α secretion in immune cell lines (in vitro)
To identify the efficacy of the compounds of the invention in inhibiting TNF α secretion in an immune response, the inhibition of TNF α production by enzyme immunoassay (ELISA) was quantified by treatment with the
Specifically, the THP-1 cell line (ATCC) was cultured in RPMI-1640 medium containing 10% FBS. At
The results demonstrate that the level of TNF α secretion, in which an inflammatory response is elicited by LPS, is reduced in all experimental groups compared to the control group. In fig. 1, the compounds denoted as SM374, SM461, SM500, SM530, and SM532 are
The above experimental results demonstrate that the compounds of the present invention are very effective in inhibiting the secretion of TNF α (i.e., inflammatory response factor) which is typically elevated in uveitis, and thus effectively inhibit the inflammatory response induced in uveitis.
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