KR20240001072A - Novel pomalidomide derivative and manufacturing method thereof - Google Patents

Abstract

The present invention relates to novel pomalidomide derivatives and methods for their preparation. The compound according to the present invention may be developed as a drug that can replace pomalidomide by increasing cereblon binding ability and reducing teratogenic side effects compared to pomalidomide.

Description

Novel pomalidomide derivative and manufacturing method thereof}

The present invention relates to novel pomalidomide derivatives and methods for their preparation.

Thalidomide was a drug sold from the late 1950s to the 1960s to prevent morning sickness in pregnant women, but as teratogenic side effects were reported, its use and handling was prohibited for pregnant women, as well as people of childbearing age or those likely to become pregnant (Lancet) .2004 May 29;363(9423):1802-11). However, interest in the drug increased again after thalidomide was found to be clinically effective in the treatment of erythema nodosum (ENL), HIV wasting syndrome, and various cancers.

Anti-tumor necrosis factor alpha (anti-TNF-α) activity was confirmed from mechanistic studies on ENL activity. Specifically, thalidomide increases the degradation of TNF-α RNA, thereby reducing its synthesis and secretion. Further studies have shown that it is a known co-stimulator of both CD8+ and CD4+ T cells and may also act as an angiogenesis inhibitor as an inhibitor of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and the transcription factor NFκB. It turns out.

TNF-α and its family members play a pivotal role in various physiological and pathological processes, including cell proliferation and differentiation, apoptosis, regulation of immune responses, and induction of inflammation. TNF-α acts through two receptors, TNFR1 and 2, the former of which is expressed in all tissues and is the main signaling receptor for TNF-α. The latter is mainly expressed in immune cells and mediates more limited biological responses. When cells are exposed to TNF-α, the caspase cascade can be activated, leading to cell death through apoptosis. In fact, the main cell surface molecules that can initiate apoptosis are the ligands and receptors of TNF family members. For example, each death-inducing member of the TNF receptor family contains a cytoplasmic 'death domain' (DD), a protein-protein interaction motif that is important for binding to downstream components of the signaling machinery.

Recently, TRAIL, a tumor necrosis factor-related apoptosis-inducing ligand, was shown to selectively induce apoptosis of tumor cells but not most normal cells. TRAIL is known to mediate apoptosis of thymocytes and plays an important role in the induction of autoimmune diseases. More often, however, TNF-α receptor binding leads to activation of the transcription factors, AP-1 and NFκB, and subsequently genes involved in acute and chronic inflammatory responses. Therefore, overproduction of TNF-α has been implicated in many inflammatory diseases, such as rheumatoid arthritis, graft-versus-host disease, and Crohn's disease, and is known to further exacerbate dementias associated with ENL, septic shock, AIDS, and Alzheimer's disease (AD). .

Commercialized thalidomide-based treatments include thalidomide, bortezomib, lenalidomide, and pomalidomide. The injectable drug bortezomib is steadily maintaining its market and growing, and the oral drug is taking over the existing thalidomide position. Domide has replaced it and is recording high growth in the market. Lenalidomide is the next-generation drug of thalidomide and shows better therapeutic effects than thalidomide through more powerful cancer cell killing and immune regulation. In cases of recurrence or refractoriness to existing treatment, the combined treatment of lenalidomide and dexamethasone is known to be quite effective, with a disease-free survival of 13.4 months and an overall survival of 38 months. As for side effects, problems such as peripheral neuropathy that occurred with existing thalidomide have almost disappeared, and the side effects of bone marrow suppression have become slightly more severe, but it is known that there is no major problem if leukocyte stimulating factors are administered. Pomalidomide was approved by the U.S. Food and Drug Administration in 2013 for the treatment of relapsed and refractory multiple myeloma, making it possible for people who have received at least two prior treatments, including lenalidomide and bortezomib, to experience disease progression within 60 days of completing the last treatment. It is used if applicable. Pomalidomide directly inhibits angiogenesis and myeloma cell growth, and this dual effect is centered on its activity in myeloma rather than on other pathways such as TNF-α inhibition. Through enhancing the expression of IFN-γ, IL-2 and IL-10 as well as inhibiting the expression of IL-6, pomalidomide exhibits anti-angiogenic and anti-myeloma activities.

The present inventors have made diligent efforts to develop a new derivative of pomalidomide with improved cereblon binding properties, and as a result, a novel derivative with enhanced cereblon binding ability and reduced reproductive toxicity such as myeloid cell suppression and teratogenic side effects compared to pomalidomide has been developed. The present invention was completed by developing a new compound.

Lancet. 2004 May 29;363(9423):1802-11.

The purpose of the present invention is to provide a novel pomalidomide derivative with improved cereblon binding ability and a method for producing the same.

In order to achieve the above object, the present invention provides a compound of [Formula 1]:

[Formula 1]

.

In the present invention, the compound may be characterized as having improved cereblon binding ability compared to pomalidomide.

In the present invention, the compound may be characterized as being non-teratogenic compared to pomalidomide.

The present invention also aims to provide a method for producing a compound [Formula 1] comprising the following steps:

(a) reacting salicylic acid with potassium carbonate (K ₂ CO ₃ ) and benzyl bromide to prepare a compound of [Formula I];

[Formula I]

(b) reacting the compound of [Formula I] with oxaline chloride to prepare a compound of [Formula II];

[Formula II]

(c) preparing a compound of [Formula III] by reacting the compound of [Formula II] with pomalidomide; and

[Formula III]

(d) Preparing a compound of [Formula 1] by reacting the compound of [Formula III] with palladium/carbon (Pd/C).

In the present invention, step (a) may be characterized in that salicylic acid is reacted with potassium carbonate and benzyl bromide and then further reacted with potassium hydroxide (KOH) to prepare a compound of [Chemical Formula I].

In the present invention, step (a) may be characterized in that benzyl bromide is added to a suspension of salicylic acid and potassium carbonate dissolved in dimethylformamide (DMF) for reaction.

In the present invention, the compound of [Formula I] prepared in step (a) may be purified and then reacted in solid form in step (b).

In the present invention, step (b) may be characterized by reacting the compound of [Chemical Formula I] with oxaline chloride by additionally adding dimethylformamide.

In the present invention, step (b) may be characterized in that the compound of [Formula I] is dissolved in dichloromethane (DCM) and then reacted with oxaline chloride and dimethylformamide.

In the present invention, step (c) may be characterized in that the compound of [Formula II] is reacted with pomalidomide dissolved in dioxane to prepare the compound of [Formula III].

In the present invention, the compound of [Formula III] prepared in step (c) may be purified and reacted in solid form in step (d).

In the present invention, step (d) may be characterized in that the compound of [Formula 1] is prepared by reacting the compound of [Formula III] with palladium/carbon and then purifying the filtered filtrate.

The compound according to the present invention may be developed as a drug that can replace pomalidomide by increasing cereblon binding ability and reducing teratogenic side effects compared to pomalidomide.

Figure 1 shows the results of comparing the cereblon binding activity of the compound of Formula 1 with pomalidomide through competitive inhibition reaction in vitro .
Figure 2 shows the results of comparing the cytotoxicity of the compound of Formula 1 to the MM.1S cell line with pomalidomide.
Figure 3 shows the results of treating the compound of Formula 1 on the MM.1S cell line and comparing the intracellular Ikaros and Aiolos decomposition effects with pomalidomide by immunoblotting.
Figure 4 shows the results of treating Tera-1 cells with the compound of Formula 1 and comparing the degree of intracellular SALL4 degradation with pomalidomide by immunoblotting.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

In the present invention, a novel pomalidomide derivative with enhanced cereblon binding ability and reduced reproductive toxicity compared to pomalidomide was prepared.

Accordingly, the present invention, in one aspect, relates to the compound [Formula 1]:

[Formula 1]

.

In the present invention, the compound of [Formula 1] may be characterized as having improved cereblon binding ability compared to pomalidomide.

Specifically, the compound of [Formula 1] may be characterized as having a cereblon binding ability that is more than two times improved compared to pomalidomide.

In the present invention, the compound [Formula 1] may be characterized as being non-teratogenic compared to pomalidomide.

In the present invention, the compound of [Formula 1] can be characterized as being non-teratogenic due to its inhibition of SALL4 decomposition ability compared to pomalidomide.

From another aspect, the present invention relates to a method for producing a compound [Formula 1] comprising the following steps:

(a) reacting salicylic acid with potassium carbonate (K ₂ CO ₃ ) and benzyl bromide to prepare a compound of [Formula I];

[Formula I]

(b) reacting the compound of [Formula I] with oxaline chloride to prepare a compound of [Formula II];

[Formula II]

(c) preparing a compound of [Formula III] by reacting the compound of [Formula II] with pomalidomide; and

[Formula III]

(d) Preparing a compound of [Formula 1] by reacting the compound of [Formula III] with palladium/carbon (Pd/C).

In the present invention, salicylic acid in step (a) may be dissolved in dimethylformamide and reacted with potassium carbonate (K ₂ CO ₃ ) and benzyl bromide.

In the present invention, salicylic acid in step (a): potassium carbonate (K ₂ CO ₃ ) : Benzyl bromide may be reacted at a molar ratio of 1:2 - 3: 2 -3.

In the present invention, step (a) may be characterized by stirring at 15 to 30°C, preferably at room temperature, for about 24 to 72 hours, for example, about 48 hours.

In the present invention, step (a) may further include, after the stirring, dissolving the stirred suspension in ethyl acetate and water, and then extracting with ethyl acetate.

In the present invention, step (a) may further include drying with sodium sulfate after the extraction process.

In the present invention, step (a) may further include filtration and concentration processes after the drying process.

In the present invention, step (a) may be characterized by dissolving the intermediate obtained through filtration and concentration in ethanol and water, and then reacting the dissolved intermediate with potassium hydroxide (KOH).

In the present invention, the potassium hydroxide (KOH) may be added to the salicylic acid at a molar ratio of 1:3 to 5 for reaction.

In the present invention, the intermediate is reacted with potassium hydroxide (KOH) at 80 to 120°C, such as 90 to 110°C, preferably at about 100°C for 30 minutes to 5 hours, such as about 1 hour to 3 hours, preferably. may be characterized by secondary stirring for about 1 hour and 30 minutes.

In the present invention, step (a) may further include, after secondary stirring, dissolving the secondary stirring suspension in ethyl acetate and water, and then performing secondary extraction with ethyl acetate.

In the present invention, step (a) may further include secondary drying with sodium sulfate after the secondary extraction process.

In the present invention, step (a) may further include secondary filtration and concentration processes after the secondary drying process.

In the present invention, step (a) may further include a purification process after secondary filtration and concentration.

In the present invention, the chromatography may be column chromatography, preferably silica gel (SiO ₂ ) chromatography, and dichloromethane: methanol = 50 to 25: 1 (v/v) as the mobile phase. It may be tube chromatography.

That is, in the present invention, step (a) is characterized in that salicylic acid is reacted with potassium carbonate (K ₂ CO ₃ ) and benzyl bromide and then further reacted with potassium hydroxide (KOH) to prepare the compound of [Chemical Formula I] You can do this.

In the present invention, the compound of [Formula I] prepared in step (a) may be purified and then reacted in solid form in step (b).

In the present invention, step (b) may be characterized by reacting the compound of [Chemical Formula I] with oxaline chloride by additionally adding dimethylformamide.

In the present invention, step (b) may be characterized in that the compound of [Formula I] is dissolved in dichloromethane and then reacted with oxaline chloride and dimethylformamide.

In the present invention, step (b) involves reacting the compound of [Formula I] with oxaline chloride at a molar ratio of 1:1 to 3, for example, 1:1.5 to 2.5 molar ratio, preferably about 1:5. It can be characterized.

At this time, dimethylformamide may be included in a catalytic amount.

In the present invention, step (b) may be characterized by adding oxaline chloride and dimethylformamide to the compound of [Formula I] at 0°C and then reacting at room temperature.

In the present invention, step (b) may be characterized by stirring and reacting for about 10 minutes to about 1 hour, for example, about 20 minutes to about 40 minutes, preferably about 30 minutes.

In the present invention, step (b) may be characterized by concentration after stirring.

In the present invention, step (c) may be characterized in that the compound of [Formula II] is reacted with pomalidomide dissolved in dioxane to prepare the compound of [Formula III].

In the present invention, in step (c), the compound of [Formula II] is reacted with pomalidomide at a molar ratio of 1:1 to 5, for example, 1:1 to 4 molar ratio, preferably about 1:3.75. It can be characterized.

In the present invention, step (c) may be characterized by reacting the compound of [Formula II] with pomalidomide by additionally adding diisopropylethylamine.

In the present invention, in step (c), pomalidomide and diisopropylethylamine are reacted at a molar ratio of 1:2 to 8, for example, 1:3 to 6 molar ratio, preferably about 1:4 molar ratio. can do.

In the present invention, step (c) may be characterized by stirring overnight at 80 to 120°C, for example, 90 to 110°C, preferably about 100°C.

In the present invention, step (c) may further include the process of dissolving the stirred suspension in ethyl acetate and water after the stirring, and then extracting it with ethyl acetate.

In the present invention, step (c) may further include drying with sodium sulfate after the extraction process.

In the present invention, step (c) may further include filtration and concentration processes after the drying process.

In the present invention, the compound of [Formula III] prepared in step (c) may be purified and reacted in solid form in step (d).

That is, in the present invention, step (c) may further include a purification process after the filtration and concentration processes.

In the present invention, the chromatography may be column chromatography, preferably silica gel (SiO ₂ ) chromatography, and the mobile phase may be chloroform: acetone = 5 to 15: 1 (v/ It may be tube chromatography using v).

In the present invention, step (d) is characterized in that the compound of [Formula 1] is prepared by reacting the compound of [Formula III] with palladium/carbon (Pd/C) and then purifying the filtered filtrate. You can.

In the present invention, step (d) may be characterized in that the compound of [Formula III] is dissolved in methanol and chloroform and reacted with palladium/carbon.

In the present invention, step (d) may be characterized in that the compound of [Formula III] and palladium/carbon are reacted at a mass ratio of 5:1 to 3.

In the present invention, step (d) may be characterized in that the reaction is carried out by stirring overnight at room temperature under a hydrogen atmosphere.

In the present invention, step (d) may further include filtration and concentration after stirring.

In the present invention, step (d) may further include a purification process after the filtration and concentration processes.

In the present invention, the chromatography may be column chromatography, preferably silica gel (SiO ₂ ) chromatography, and the mobile phase may be chloroform: acetone = 5 to 15: 1 (v/ It may be tube chromatography using v).

As used herein, "chromatography" means a method for separating and purifying the compounds of the invention, wherein a solution containing the compounds of the invention is passed using a liquid stream over a stationary phase, and the components of the mixture are fractionated. I get angry. The stationary phase is preferably the packing material in the chromatography column. The filling material, hereinafter also referred to as the so-called gel material, preferably consists of a solid support material consisting of porous or non-porous particles of approximately equal size, on which functional groups that establish the mode of separation are covalently bonded. Support materials can be biopolymers such as, for example, agarose, cellulose and dextran (preferably Sepharose and Sephadex), or polymers such as, for example, methacrylates, polyvinylbenzene, polystyrene and polyacrylamide. It may be a synthetic polymer or an inorganic polymer such as, for example, silica or porous glass beads.

In the present invention, the chromatography may be selected from the group consisting of hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), size exclusion chromatography (SEC), affinity chromatography, and reversed phase chromatography. , but is not limited to this.

Meanwhile, in the present invention, in order to further purify and/or concentrate the compound, it can be selectively subjected to ultrafiltration (UF), diafiltration (DF), or concentrated under reduced pressure.

In the present invention, the compound may be used as an active ingredient in a pharmaceutical composition such as a cereblon combination therapeutic agent, depending on its ability to bind to cereblon and its properties to reduce reproductive toxicity such as myeloid cell suppression or teratogenic side effects.

As used herein, the term “pharmaceutical composition” or “pharmaceutical formulation” refers to a pharmaceutical agent, such as a diluent or carrier, that makes the novel compound of the present invention particularly suitable for in vivo or in vitro diagnostic or therapeutic use. It refers to a mixture containing acceptable excipients. According to some embodiments, a pharmaceutical composition containing the composition of the present invention may be provided by administering a therapeutically effective amount to a subject according to need. In some embodiments, compositions of the present invention can be administered to humans.

As used herein, “effective amount” or “therapeutically-effective amount” refers to the amount of a compound or composition (e.g., a compound or composition of the invention) sufficient to achieve a beneficial or desired result. Refers to an amount. An effective amount may be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular agent or route of administration.

Although the pharmaceutical compositions provided by the present invention relate in principle to pharmaceutical compositions for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. That is, the pharmaceutical composition according to the present invention is suitable for use in animals requiring veterinary treatment, such as livestock (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.) and laboratory animals. It can also be administered to other mammals such as rats, mice, guinea pigs, etc. A skilled veterinary pharmacologist with a good understanding of the modifications of pharmaceutical compositions for administration to various animals can design and/or perform such modifications, if necessary, simply by routine experimentation.

The pharmaceutical compositions described in the present invention may be prepared by any method known in the field of pharmacology or as discussed later in the text. Generally, these methods for tableting involve the steps of associating the active ingredient with excipients and/or one or more other auxiliary ingredients, followed by shaping and/or packaging the product into the desired single- or multi-dose units, if necessary or desired. Includes steps.

The pharmaceutical compositions of the present invention may be manufactured, packaged, and/or sold unpackaged as a single unit dose and/or multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dosage of active ingredient administered to the subject and/or a convenient fraction of such dosage such as, for example, one-half or one-third of the dosage.

The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical compositions of the invention will vary depending on the identity, size, and/or disorder of the subject being treated and the route by which the composition is administered. . By way of example, the composition may include 0.001% to 100% (w/w) active ingredient.

As used herein, a pharmaceutically acceptable excipient is any solvent, dispersion medium, diluent, or other liquid vehicle, dispersion or suspension aid, surface active agent, isotonic agent, thickener or emulsifier suitable for the purpose of the particular dosage form; Contains preservatives, solid binders, lubricants, etc. Remington's (The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) describes various excipients used in the preparation of pharmaceutical compositions and known methods for their preparation. The technology is disclosed, except that any conventional carrier medium is incompatible with the substance or derivative thereof, for example by providing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition. and their uses are considered to be within the scope of the present invention. Pharmaceutically acceptable excipients are at least 95%, 96%, 97%, 98%, 99%, or 100% pure.

The excipients are approved for human and veterinary use. In some embodiments, the excipient is approved by the Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and/or International Pharmacopoeia (EP).

Pharmaceutically acceptable excipients used in the preparation of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and/or granulating agents, surface active agents and/or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and/or oils. Not limited.

Such excipients may optionally be included in the formulations of the present invention. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavors, and perfumers may be present in the composition at the discretion of the formulator.

Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, Including, but not limited to, corn starch, powdered sugar, and combinations thereof.

Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation-exchange resins. , Calcium Carbonate, Silicate, Sodium Carbonate, Cross-Linked Poly(vinyl-pyrrolidone) (Crospovidone), Sodium Carboxymethyl Starch (Sodium Starch Glycolate), Carboxymethyl Cellulose, Cross-Linked Sodium Carboxymethyl Cellulose ( croscarmellose), methylcellulose, pregelatinized starch (Starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (vegeum), sodium lauryl sulfate, quaternary ammonium compounds, and these. Including, but not limited to, combinations of.

Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, waxes, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and beegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol) , triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymers), carrageenans, cellulose derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxymethylcellulose) Ethylene Sorbitan Monolaurate [Tween 20], Polyoxyethylene Sorbitan [Tween 60], Polyoxyethylene Sorbitan Monooleate [Tween 80], Sorbitan Monopalmitate [Span 40], Sorbitan Monostearate [ span 60], sorbitan tristearate [span 65], glyceryl monooleate, sorbitan monooleate [span 80]), polyoxyethylene ester (e.g. polyoxyethylene monostearate [mirz 45]) , polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. cremophor), polyoxyethylene ethers, ( For example, polyoxyethylene lauryl ether [Brise 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl. Including, but not limited to, lauray, sodium lauryl sulfate, pluronic F 68, poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or combinations thereof. No.

Exemplary binders include starches (e.g. corn starch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); Natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, Farnwer gum, Shatty gum, Isapol Husk's slime, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl) cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (bigum), and lachi arabogalactan); alginate; polyethylene oxide; polyethylene glycol; inorganic calcium salt; silicic acid; polymethacrylate; wax; water; Alcohol; and combinations thereof.

Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, bisulfite. Including, but not limited to, sodium sulfate, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, disodium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate. . Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, Includes, but is not limited to, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include tocopherol, tocopherol acetate, deteroxyme mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluende (BHT), ethylenediamine, sodium lauryl sulfate (SLS), Sodium Lauryl Ether Sulfate (SLES), Sodium Bisulfite, Sodium Metabisulfite, Potassium Sulfite, Potassium Metabisulfite, Glydant Plus, Fenonib, Methylparaben, Low Mol 115, Germaben II, Neolon, Katon, and E Including, but not limited to, Uxil. In certain embodiments, the preservative is an antioxidant. In another embodiment, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluvionate, calcium gluceptate, calcium gluconate, D-gluconic acid, glyceroside. Calcium phosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic Basic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen -Includes, but is not limited to, free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium. Including, but not limited to, lauryl sulfate, and combinations thereof.

Exemplary oils include almond, apricot kernel, avocado, babassu palm, bergamot, black currant seed, borage, cayenne, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee. , corn, cotton seed, emu, eucalyptus, evening primrose, fish, flax seed, geraniol, pumpkin, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litthea cucumber. beba, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange-orange rappi, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower. , sandalwood, camellia, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut, and wheat germ oil. Exemplary oils include butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil. , and combinations thereof.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents such as, for example, water or other solvents, solubilizers and emulsifiers commonly used in the art such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl. Alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, sprout, olive, castor, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, It may also include polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the novel compounds of the invention are mixed with solubilizing agents such as cremophor, alcohols, oils, denatured oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous or oily suspensions, may be prepared according to the known art using dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, such as solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. Additionally, sterile, fixed oils are commonly employed as solvents or suspending media. For this purpose any bland fixed oil may be employed, including synthetic mono- or diglycerides. Additionally, fatty acids such as oleic acid have been used in the preparation of injectables.

Injectable preparations may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. .

To prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This is achieved by using liquid suspensions of crystalline or amorphous substances with low water solubility. The absorption rate of the drug then depends on the dissolution rate, which may ultimately depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug is achieved by dissolving or suspending the drug in an oil vehicle.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient may be combined with one or more inert pharmaceutically acceptable excipients or carriers such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b ) binders such as, for example, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca starch. , alginic acid, certain silicates, and sodium carbonate, e) dissolution retarders such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) humectants such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof.

In the case of capsules, tablets and pills, dosage forms may contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk sugar as well as high molecular weight polyethylene glycols and the like as such excipients. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may be compositions that release the active ingredient only, or preferentially, in a specific part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk sugar as well as high molecular weight polyethylene glycols and the like as such excipients.

The active ingredient may be in micro-encapsulated form with one or more of the excipients described above. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings, and other coatings well known in the pharmaceutical formulation art. In these solid dosage forms the active ingredient may be mixed with one or more inert diluents such as sucrose, lactose or starch. These dosage forms may contain additional substances other than inert diluents as in common practice, such as tablet lubricants and other tablet auxiliaries such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, dosage forms may also contain buffering agents. They may optionally contain opacifying agents and may be compositions that release the active ingredient only, or preferentially, in a specific part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of the novel compounds of the present invention or pharmaceutical compositions containing them may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. . Generally, the active ingredient is admixed under conditions of sterility with a pharmaceutically acceptable carrier and/or any necessary preservatives and/or buffering agents that may be required. Additionally, the present invention contemplates the use of transdermal patches, which often have the additional advantage of providing controlled delivery of the active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispersing the active ingredient in a suitable medium. Alternatively or additionally, the rate may be controlled by providing a rate controlling membrane and/or dispersing the active ingredient in a polymer matrix and/or gel.

Formulations for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments/or pastes, and/or solutions and/or suspensions. Although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent, topically-administrable formulations may also comprise, for example, from about 1% to about 10% (w/w) of the active ingredient. Formulations for topical administration may further comprise one or more additional ingredients described herein.

The novel compounds of the invention described herein or pharmaceutical compositions containing them are typically prepared in dosage unit form for easy administration and uniform administration. However, it will be understood that the total daily dosage of the composition of the present invention will be determined by the attending physician within the scope of sound medical judgment. The particular therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disease, disorder, or disorder being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; The subject's age, weight, general health, gender, and diet; the time of administration, route of administration, and rate of excretion of the particular active ingredient employed; duration of treatment; Drugs used in combination or simultaneously with the specific active ingredients employed; and factors well known in the medical field.

The novel compound of the present invention, its salt, or pharmaceutical composition may be administered by any route. In some embodiments, the novel compound, salt thereof, or pharmaceutical composition thereof may be administered orally, intravenously, intramuscularly, intraarterially, intramedullarily, intrathecally, subcutaneously, intracerebroventricularly, transdermally, intradermally, rectally, intravaginally, intraperitoneally, Topically (by powder, ointment, cream, and/or drop), mucosal, nasal, oral, enteral, sublingual; endotracheal instillation, bronchial instillation, and/or inhalation; and/or administered by various routes, including oral spray, nasal spray, and/or aerosol. Particularly contemplated routes are permeable intravenous injection, local administration via the blood and/or lymphatic supply, and/or direct administration to the affected area. In general, the most appropriate route of administration will depend on a variety of factors, including the properties of the agent (e.g., stability in the environment of the gastrointestinal tract), and the disorder of the subject (e.g., whether the subject can tolerate oral administration). will be.

The novel compound of the present invention or its salt may be included in an amount of 0.001 to 99.9% of the total weight of the pharmaceutical composition.

In certain embodiments, the novel compound of the present invention, salt thereof, or pharmaceutical composition thereof is administered in an amount of about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 50 mg/kg, or about 0.1 mg/kg of the subject's body weight per day. mg/kg to about 40 mg/kg, about 0.5 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, or about 1 mg. /kg to about 25 mg/kg may be administered at a dosage level sufficient to deliver once or more per day to achieve the desired therapeutic effect. The intended dosage may be delivered three times a day, twice a day, daily, every two days, every three days, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered via multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations). .

It will be understood that the dosage ranges described herein provide guidance for administration of pharmaceutical compositions provided to adults. For example, the amount administered to a child or adolescent may be determined by a physician or person skilled in the art, and may be less or the same as that administered to an adult. The exact amount of a peptide according to the invention required to achieve an effective amount will vary from subject to subject, depending, for example, on the subject's species, age, and overall disorder, severity of side effects or disorders, identity of the specific compound, mode of administration, etc.

It will be appreciated that the novel compounds and pharmaceutical compositions of the present invention can be used in combination therapy. The particular combination of treatments (treatments or procedures) for use in combination therapy will take into account the desired therapeutic effect to be achieved and the suitability of the desired treatments and/or procedures.

The pharmaceutical composition of the present invention can be administered alone or in combination with one or more therapeutically active agents. As for "combination", it is not intended to imply that the agents must be administered at the same time and/or be formulated for delivery together, although the following delivery methods are within the scope of the present invention. The composition may be administered concurrently with, prior to, or subsequent to one or more other therapeutic agents or medical procedures. Generally, each agent will be administered at a dose and/or time schedule established for that agent. Additionally, the present invention provides a pharmaceutical form of the present invention in combination with agents capable of improving its bioavailability, reducing and/or modifying its metabolism, inhibiting its secretion, and/or modifying its distribution within the body. It encompasses delivering the composition. It will be further understood that the novel compound of the invention and the therapeutically active agent used in this combination may be administered together in a single composition or may be administered separately in different compositions.

The particular combination used in combination therapy will take into account the desired therapeutic effect to be achieved and/or the suitability of the procedure and/or therapeutically active agent comprising the peptide of the invention. The combination used may achieve the desired effect for the same disorder (e.g., a novel compound of the invention may be administered in combination with another therapeutically active agent (e.g., a second therapeutic agent) used to treat the same disorder. It will be understood that they may achieve different effects (e.g., control of any side effects), and/or they may achieve different effects (e.g., control of any side effects).

As used herein, “therapeutically active agent” refers to any substance used as a medicine to treat, prevent, delay, reduce or ameliorate a disorder, and refers to substances used in treatment, including prophylactic and curative treatments. .

In some embodiments, the pharmaceutical compositions of the invention treat, alleviate, ameliorate, alleviate, delay the onset, inhibit the progression, reduce the severity, and/or the incidence of one or more symptoms or characteristics. Can be administered in combination with any therapeutically active agent or procedure (e.g., surgery, radiation therapy) useful for reducing .

In one aspect, the compound of the present invention can be provided as a kit for treating diseases.

In some embodiments, the kit comprises i) instructions for administering the novel compound or pharmaceutical composition according to the invention to a subject suffering from a disease, and ii) the novel compound or pharmaceutical composition according to the invention. . In some embodiments, the kit may include one or more unit dosage forms containing a dose of the novel compound or pharmaceutical composition as described herein that is effective for treating a disease in a subject. In some embodiments, the subject is a human patient.

In some embodiments, the kit further includes one or more selected from the group comprising a sterile syringe, a sterile needle, a sterile IV bag, an infusion pump, or any combination thereof.

The term “about” used in the present invention may be interpreted to mean approximately, approximately, approximately, or to a certain extent. When the term "about" is used with a numeric range, it is interpreted as modifying that range by extending the boundaries above and below the specified numeric value. Generally, the term "about" is used herein to modify a numerical value above or below a specified value by a variance of 10%.

“Individual,” “patient,” and “subject” are used interchangeably and refer to mammals, such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, and sheep. , horses, or any animal, including primates, including humans.

In the present invention, unless otherwise stated, "treatment" means reversing, alleviating, inhibiting the progression of, or preventing the disease or disease to which the term applies, or one or more symptoms of the disease or disease. means, and the term treatment as used herein refers to the act of treating when “treating” is defined as above. Accordingly, “treatment” or “therapy” of a disease in a mammal may include one or more of the following:

(1) arresting the development of the disease;

(2) preventing the spread of disease;

(3) alleviating disease;

(4) preventing disease recurrence and

(5) Alleviating symptoms of disease (palliating)

In the present invention, “prevention”, unless otherwise stated, means any action that suppresses or delays the onset of a disease by administering the pharmaceutical composition according to the present invention.

Pomalidomide used in the present invention is known as (R,S)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione, [Chemical formula] IV].

[Formula IV]

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Manufacturing example

Preparation of Compound 1 (Formula I Compound)

Salicylic acid (Daejeong Chemical, 7503-1405) (1.38g, 9.99mmol) dissolved in dimethylformamide (DMF) (Samchun, D0552) (40.0mL), potassium carbonate (K ₂ CO ₃ ) (Samchun, Benzyl bromide (BnBr) (Daejeong Chemical, 2084-4440) (4.27g, 25.0mmol) was added to the suspension of P0795) (3.45g, 25.0mmol) and stirred at room temperature for 48 hours. The stirred suspension was dissolved in ethyl acetate (Samchun, E0191) (100mL) and water (Samchun, W0003) (100mL), and then dissolved in ethyl acetate (Samchun, E0191) (100mL × 3). Extracted three times, dried with sodium sulfate (Na ₂ SO ₄ ) (Daejeong Chemical, 7630-4400), filtered through a paper filter (Advantec, F12-510-221), and rotary evaporator (EYELA, N-1300). ) to obtain an intermediate. Potassium hydroxide (KOH) (Samchun, P1446) (2.24 g, 40.0 mmol) was added to the intermediate solution dissolved in ethanol (EtOH) (Samchun, E1095) (36.0 mL) and water (Samchun, W0003) (4.00 mL). , and stirred at 100°C for 1.5 hours. Afterwards, ethyl acetate (Samchun, E0191) (40.0mL) and water (Samchun, W0003) (20.0mL) were additionally added, and ethyl acetate (Samchun, E0191) (100mL × 3) After extraction three times, it was dried with sodium sulfate (Na ₂ SO ₄ ) (Daejeong Chemical, 7630-4400), filtered with a paper filter (Advantec, F12-510-221), and rotary evaporator (EYELA, N-1300). Concentrated. The residue was purified by column chromatography using silica gel (SiO ₂ ) (MERCK, 1.09385.2500) [Ethyl acetate (Samchun, E0191) : Hexane (Samchun, H0109) = 1:4) to obtain Compound 1. (1.05 g, 46%, T0069-03-05) was obtained as a white solid.

Preparation of Compound 2 (Formula II Compound)

Oxalyl chloride (TCI, O0082) (879mg, 6.92mmol) in a solution of compound 1 (790mg, 3.46mmol, T0069-03-05) dissolved in dichloromethane (DCM) (Samchun, M0822) (20.0mL). and a catalytic amount of dimethylformamide (DMF) (Samchun, D0552) (3 drops) were added at 0°C and stirred at room temperature for 0.5 hours. The stirred mixture was concentrated using a rotary evaporator (EYELA, N-1300) to obtain compound 2 (crude, T0069-03-07), which was used in the next step without further purification.

Preparation of Compound 3 (Formula III Compound)

Compound 2 (1.1g, 1.1g, T0069-03-07) and diisopropylethylamine (DIPEA) (TCI, D1599) (1.34g, 10.4mmol) were added and stirred at 80°C overnight. The stirred mixture was diluted with H ₂ O (Samchun, W0003) (15.0 mL) and ethyl acetate (Samchun, E0191) (20.0 mL), and ethyl acetate (Samchun, E0191) (20.0 mL) × 3), dried with sodium sulfate (Na ₂ SO ₄ ) (Daejeong Chemical, 7630-4400), filtered with a paper filter (Advantec, F12-510-221), and rotary evaporator (EYELA, N). -1300). The residue was purified using silica gel (SiO ₂ ) (MERCK, 1.09385.2500) [chloroform (Daejeong Chemical, 2548-4480) : acetone (Samchun, A0098) = 10:1)] column chromatography. Compound 3 (726 mg, 43%, T0069-03-08) was obtained as a white solid.

POM-2HBA Manufacturing

Compound 3 (726mg, 1.50mmol, T0069) in methanol (MeOH) (Daejeong Chemical, 5558-4410) (15.0mL) and chloroform (CHCl ₃ ) (Daejeong Chemical, 2548-4480) (15.0mL) 10% palladium on carbon (Pd/C) (Alfa, A12012) (140mg, 20wt%) was added to the suspension in which -03-08) was dissolved, then degassed three times and left at room temperature under a hydrogen atmosphere overnight. It was stirred. The stirred mixture was filtered through a Celite pad (Samchun, C1243), the filtrate was concentrated with a rotary evaporator (EYELA, N-1300), and the residue was subjected to silica gel (SiO ₂ ) (MERCK, 1.09385.2500) tube chromatography [ Purified with chloroform (Daejeong Chemical, 2548-4480): acetone (Samchun, A0098) = 10:1), POM-2HBA (132 mg, 22 %, T0069), a compound of formula 1, is obtained as an off-white solid. -03-09) was obtained.

Example 1. In vitro CRBN binding assay

Thalidomide-based compounds are known to regulate the function of immune cells and exert various pharmacological effects by binding to Cereblon (CRBN), an E3 ligase, and decomposing the transcription factors Aiolos and Ikaros as its substrates. Accordingly, the cereblon binding ability of the compound of Formula 1 was compared with pomalidomide in vitro .

The binding force to Cereblon was measured using the 'AlphaScreen' method based on Fluorescence Resonance Energy Transfer (FRET), and the test was performed using the PROTAC Optimization kit for BET Bromodomain-Cereblon binding kit (#79770, BPS bioscience, CA USA). It was carried out according to the 'Competitive Inhibition of the PROTAC assay' written in the manual in the kit. All reagents except the candidate substance, DMSO, and Flag/Glutathione beads (#6765300, PerkinElmer, USA) were used as those included in the kit.

As a result, as shown in FIG. 1, it was confirmed that the compound of Formula 1 according to the present invention had significantly better binding ability to cereblon than its parent pomalidomide.

Example 2. Cytotoxicity evaluation

The MM.1S cell line, a human multiple myeloma cell line, was treated with compounds of Formula 1 and Formula 2, respectively, at different concentrations, and cytotoxicity was evaluated.

MM.1S (CRL-2974), a human multiple myeloma cell line, was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), 10% fetal bovine serum (Corning, MD, USA), 100 U/ Cultivated for 24 hours in an incubator at 37°C and 5% CO ₂ using RPMI1640 medium (Corning, MD, USA) supplemented with ml penicillin (Corning, MD, USA) and 100 ㎍/ml streptomycin (Corning, MD, USA). did. Cells were distributed in a 96- ^well plate at 1 The analysis was conducted according to. As a control group, pomalidomide was treated at the same concentration.

As a result, as shown in Figure 2, it was confirmed that the compound of Formula 1 did not exhibit cytotoxicity in the MM.1S cell line.

Example 3. Mechanism of reducing cytotoxicity

Thalidomide-based compounds are known to regulate the function of immune cells by binding to Cereblon (CRBN), an E3 ligase, and decomposing the transcription factors Aiolos and Ikaros as its substrate. We wanted to check whether expression decreased.

For this purpose, the MM.1S cell line was supplemented with 10% fetal bovine serum (Corning, MD, USA), 100 U/ml penicillin (Corning, MD, USA), and 100 μg/ml streptomycin (Corning, MD, USA). RPMI medium (Corning, MD, USA) was used and cultured at 37°C in a 5% CO ₂ incubator. ¹ RIPA buffer was added, disrupted, and cell extract was obtained by centrifugation at 4°C at 14,000 rpm for 15 minutes. The same amount of cell extract was loaded, separated using SDS-PAGE, and transferred to a PVDF membrane. The protein-transferred membrane was blocked using skim milk, incubated with primary antibody at room temperature for 3 hours, and then incubated with HRP-attached secondary antibody at room temperature for 1 hour. Washed three times with TBS-T between each step. Detection was performed by applying a chemoluminescence reagent (Thermo Fisher Scientific) and confirmed using Chemidoc (iBright CL1500, Invitrogen, CA, USA). Aiolos (#15103), Ikaros (#9034), and GAPDH (#2118S) antibodies used as primary antibodies and secondary antibodies were purchased from Cell signaling technology (Danvers, MA, USA).

As a result, as shown in Figure 3, it was confirmed that the compound of Formula 1 did not effectively reduce the expression of Aiolos and Ikaros in MM.1S cells.

Experimental Example 4. Reproductive toxicity reduction mechanism

It has been reported that cereblon (CRBN)-binding treatments, such as thalidomide-based compounds, can cause serious congenital deformities such as forelimb shortening or brachydactyly, and that this is closely related to the degradation of SALL4. Therefore, the effect of the compound of Formula 1 on decomposing SALL4 was confirmed.

For this purpose, Tera-1 cell line (HTB-105), a human embryonic carcinoma cell line, was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), 10% fetal bovine serum (Corning, MD, USA), DMEM medium (Corning, MD, USA) supplemented with 100 U/ml penicillin (Corning, MD, USA) and 100 ㎍/ml streptomycin (Corning, MD, USA) was used at 37°C in a 5% CO ₂ incubator. Cultured.

Cells were distributed at 5 × 10 ⁵ cells/well in a 12-well plate and cultured for 24 hours, then treated with 1 or 10 μM of the compound of Formula 1 and cultured for an additional 4 hours. Cells were disrupted by adding RIPA buffer containing protease inhibitor cocktail (Thermo Fisher Scientific), and centrifuged at 14,000 rpm for 15 minutes at 4°C to obtain cell extract. The same amount of cell extract was loaded, separated using SDS-PAGE, and transferred to a PVDF membrane. The protein-transferred membrane was blocked using skim milk, incubated with primary antibody at room temperature for 3 hours, and then incubated with HRP-attached secondary antibody at room temperature for 1 hour. Washed three times with TBS-T between each step. Detection was performed by applying a chemoluminescence reagent (Thermo Fisher Scientific) and confirmed using Chemidoc (iBright CL1500, Invitrogen, CA, USA). SALL4 (#5850) and GAPDH (#2118S) antibodies used as primary antibodies and secondary antibodies were purchased from Cell signaling technology (Danvers, MA, USA).

As a result, as shown in FIG. 4, it was confirmed that the compound of Formula 1 according to the present invention in Tera-1 cells reduced the SALL4 protein less than pomalidomide, thereby reducing teratogenic side effects.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

Compound represented by formula 1:
[Formula 1]
.
The compound according to claim 1, wherein the cereblon binding ability is improved compared to pomalidomide.
The compound of claim 1, wherein the compound is non-teratogenic compared to pomalidomide.
A method for preparing the compound of claim 1 comprising the following steps:
(a) reacting salicylic acid with potassium carbonate (K ₂ CO ₃ ) and benzyl bromide to prepare a compound of [Formula I];
[Formula I]

(b) reacting the compound of [Formula I] with oxaline chloride to prepare a compound of [Formula II];
[Formula II]

(c) preparing a compound of [Formula III] by reacting the compound of [Formula II] with pomalidomide; and
[Formula III]

(d) Preparing a compound of [Formula 1] by reacting the compound of [Formula III] with palladium/carbon (Pd/C).
The preparation method according to claim 4, wherein step (a) is performed by reacting salicylic acid with potassium carbonate and benzyl bromide and then further reacting with potassium hydroxide (KOH) to prepare the compound of [Chemical Formula I].
The manufacturing method according to claim 4, wherein step (a) is performed by adding benzyl bromide to a suspension of salicylic acid and potassium carbonate dissolved in dimethylformamide (DMF).
The preparation method according to claim 4, wherein the compound of [Formula I] prepared in step (a) is purified and then reacted in solid form in step (b).
The preparation method according to claim 4, wherein in step (b), dimethylformamide is further added to react the compound of [Chemical Formula I] with oxaline chloride.
The method of claim 8, wherein step (b) is performed by dissolving the compound of [Chemical Formula I] in dichloromethane (DCM) and then reacting it with oxaline chloride and dimethylformamide.
The preparation method according to claim 4, wherein step (c) is characterized in that the compound of [Formula III] is prepared by reacting the compound of [Formula II] with pomalidomide dissolved in dioxane.
The preparation method according to claim 4, wherein the compound of [Formula III] prepared in step (c) is purified and then reacted in solid form in step (d).
The preparation method according to claim 4, wherein step (d) is performed by reacting the compound of [Formula III] with palladium/carbon and then purifying the filtered filtrate to prepare the compound of [Formula 1].