KR102091067B1 - Pharmaceutical composition for treatment of cancer comprising compound for inhibiting cell-specific viral defence as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for cancer treatment comprising a compound for inhibiting a virus cell-specific defense mechanism as an active ingredient, and more specifically, a next-generation cancer cell through inhibition of APOBEC3G (Apolipoprotein B mRNA Editing Catalytic Polypeptide-like 3G) It relates to the treatment of cancer by improving the infection efficiency of a specific replicable retroviral vector (spRRV).
The pharmaceutical composition for cancer treatment comprising the compound for inhibiting the virus cell-specific defense mechanism through the present invention as an active ingredient not only significantly increased virus infection efficiency through cell experiments, but also at the same time stable solubility, high metabolic safety, and pharmacokinetics It is expected to be able to dramatically improve the anti-cancer effect by significantly improving the infection efficiency of the next-generation cancer cell-specific replicable retrovirus vector (spRRV) for anti-cancer virotherapy.

Description

Pharmaceutical composition for treatment of cancer comprising compound for inhibiting cell-specific viral defense as active ingredient}

The present invention relates to a pharmaceutical composition for cancer treatment comprising a compound for inhibiting a virus cell specific defense mechanism as an active ingredient, and more specifically, a next-generation cancer cell through inhibition of APOBEC3G (Apolipoprotein B mRNA Editing Catalytic Polypeptide-like 3G) It relates to the treatment of cancer by improving the infection efficiency of a specific replicable retroviral vector (spRRV).

Existing anti-cancer virotherapy techniques can be largely divided into replication defective virus, oncolytic virus, and replicating retrovial vector, but in each technique It has the following limitations. The replication-defective virus has a low vector infection efficiency of less than 1%, and the anti-cancer virus has a possibility of virus modification, and its application is very limited due to its high immunity. In addition, due to the size of the vector, the first generation replicable retroviral vector has limited introduction of the therapeutic gene, so there is a limitation in the necessity of viral recombination and the efficiency due to the cell-specific defense mechanism. It is a desperate situation.

 Rheinbay E et al. Nature, 547, 55-60, 2017.  Roberts SA et al. Nature Genetics, 45, 970, 2013.  Sadler HA et al. Journal of Virology, 84, 7396-404, 2010.

In order to solve the problems of the existing anti-cancer virotherapy, in the present invention, the activity of APOBEC3G (Apolipoprotein B mRNA Editing Catalytic Polypeptide-like 3G) that performs a congenital antiviral immune function by inducing a lethal level mutation against a retrovirus It is a low-molecular compound that improves the infection efficiency of a next-generation cancer cell-specific replicable retroviral vector (spRRV) for anticancer virotherapy through the development of a compound for inhibiting a viral cell-specific defense mechanism that inhibits it and is effective. It was intended to provide a composition for treating cancer comprising as an ingredient.

The present invention relates to a pharmaceutical composition for the treatment of cancer comprising a compound represented by Formula 1, a prodrug thereof, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Chemical Formula 1,

R1 is hydrogen, (C1-C7) alkyl, or a 5- or 6-membered heterocyclic alkyl,

R2 is hydrogen or (C1-C7) alkyl when R1 is hydrogen or (C1-C7) alkyl, hydrogen when R1 is a 5- or 6-membered heterocyclic alkyl,

R3 is hydrogen when R1 is hydrogen or (C1-C7) alkyl, and when R1 is 5- or 6-membered heterocyclic alkyl, (C1-C7) alkyl or halo (C1-C7) alkyl,

R4 and R5 may combine to form a 5- or 6-membered ring, and the ring formed by combining R4 and R5 may be further substituted with (C1-C7) alkyl or halo (C1-C7) alkyl,

The heterocyclic alkyl includes at least one hetero atom selected from N, O and S.

The pharmaceutical composition for cancer treatment comprising the compound for inhibiting the virus cell-specific defense mechanism through the present invention as an active ingredient not only significantly increased virus infection efficiency through cell experiments, but also at the same time stable solubility, high metabolic safety, and pharmacokinetics It is expected to be able to dramatically improve the anti-cancer effect by significantly improving the infection efficiency of the next-generation cancer cell-specific replicable retroviral vector (spRRV) for anti-cancer virotherapy.

1 is a result of inhibiting retrovirus infection by APOBEC3G.
2 is a result of confirming whether or not endogenous A3G expression in various tumor cell lines.
Figure 3 is a result of the titer of the spRRVenvCMVGFP / sRRVgpCMVRFP virus suitable for screening APOBEC3G (A3G) inhibitors in the H9 cell line.
4 is a result of measuring the inhibitory effect of the APOBEC3G inhibitor using the H9 cell line.
(A) Measurement results for CIM885, CIM953, and CIM1110, (B) Measurement results for a control compound having a similar structure to the compound.

Hereinafter, the present invention will be described in detail with reference to the accompanying tables or drawings.

When the drawings are described, they are provided as an example to sufficiently convey the spirit of the present invention to those skilled in the art. Therefore, the present invention is not limited to the presented drawings and may be embodied in other forms, and the drawings may be exaggerated to clarify the spirit of the present invention.

At this time, unless there are other definitions in the technical terms and scientific terms to be used, those skilled in the art to which this invention pertains have the meanings commonly understood, and the subject matter of the present invention in the following description and accompanying drawings Descriptions of well-known functions and configurations that may be obscured are omitted.

The present invention relates to a pharmaceutical composition for the treatment of cancer comprising a compound represented by Formula 1, a prodrug thereof, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Chemical Formula 1,

R1 is hydrogen, (C1-C7) alkyl, or a 5- or 6-membered heterocyclic alkyl,

R2 is hydrogen or (C1-C7) alkyl when R1 is hydrogen or (C1-C7) alkyl, hydrogen when R1 is a 5- or 6-membered heterocyclic alkyl,

R3 is hydrogen when R1 is hydrogen or (C1-C7) alkyl, and when R1 is 5- or 6-membered heterocyclic alkyl, (C1-C7) alkyl or halo (C1-C7) alkyl,

R4 and R5 may combine to form a 5- or 6-membered ring, and the ring formed by combining R4 and R5 may be further substituted with (C1-C7) alkyl or halo (C1-C7) alkyl,

The heterocyclic alkyl includes at least one hetero atom selected from N, O and S.

The term “alkyl” of the present invention means a monovalent straight chain or ground saturated hydrocarbon radical consisting only of carbon and hydrogen atoms, examples of such alkyl radicals being methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl , Pentyl, hexyl, and the like.

The term “aryl” of the present invention is an aromatic ring monovalent organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, suitably containing 4 to 7, preferably 5 or 6 ring atoms in each ring. It includes a single or fused ring system, and includes a form in which multiple aryls are connected by a single bond. Specific examples include, but are not limited to, phenyl, naphthyl, biphenyl, indenyl, fluorenyl, and the like.

The term “heteroaryl” of the present invention is an aromatic ring backbone atom containing 1 to 4 heteroatoms selected from N, O and S, and the remaining aromatic ring backbone atom is a carbon group, an aryl group of 5 to 6 membered monocyclic heteroaromatic rings Means a monovalent radical. Examples of the heteroaryl group include pyrrolyl, pyrazolyl, pyridyl, pyrimidinyl, oxazolyl, thiazolyl, thiadiazolyl, triazolyl, imidazolyl, isooxazolyl, thiophenyl, furyl, etc. , But is not limited thereto.

The term “halo” or “halogen” of the present invention means a fluorine, chlorine, bromine or iodine atom.

The term “haloalkyl” of the present invention means alkyl substituted with one or more halogens, and examples thereof include trifluoromethyl and the like.

The term “cycloalkyl” of the present invention means a monovalent saturated carbocyclic radical consisting of one or more rings. Examples of cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

In the composition according to an embodiment of the present invention, the compound may be represented by the following Formula 2 or 3.

[Formula 2]

[Formula 3]

In Chemical Formulas 2 and 3,

R4 and R5 are the same as defined in Formula 1 of claim 1,

X is a heteroatom,

R1 and R2 are independently of each other hydrogen or (C1-C7) alkyl,

R3 is (C1-C7) alkyl or halo (C1-C7) alkyl.

In the composition according to an embodiment of the present invention, X may be NH or O.

In the composition according to an embodiment of the present invention, the 5-membered heterocyclic alkyl may have the following structure.

R11 is hydrogen or (C1-C7) alkyl,

R12 is hydrogen, (C1-C7) alkyl or halo (C1-C7) alkyl.

In the composition according to an embodiment of the present invention, the 6-membered heterocyclic alkyl may have the following structure.

R13 is hydrogen or (C1-C7) alkyl.

In the composition according to an embodiment of the present invention, the compound may be specifically selected from the following structures, but is not limited thereto.

In the composition according to an embodiment of the present invention, the composition is not limited unless it inhibits the object of the present invention, but may further include a pharmaceutically acceptable carrier as an active ingredient.

In the composition according to an embodiment of the present invention, the composition may be due to a mechanism for promoting retrovirus spread through weakening of the retroviral defense mechanism of cancer tissues due to APOBEC3 inhibition, but is not limited thereto.

In the composition according to an embodiment of the present invention, the cancer is mucus cell carcinoma expressing APOBEC3G, round cell carcinoma, locally advanced tumor, metastatic cancer, Ewing's sarcoma, cancer metastasis, lymphoid metastasis, squamous epithelium. Cell carcinoma, esophageal squamous cell carcinoma, oral carcinoma, multiple myeloma, acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, globular cellular leukemia, effusion lymphoma (celiac lymphoma), thymic lymphoma Lung cancer, small cell lung carcinoma, skin T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, adrenal cortical cancer, ACTH-producing tumor, non-small cell lung cancer, breast cancer, small cell carcinoma, catheter carcinoma, stomach cancer, colon cancer, colon Rectal cancer, polyps associated with colorectal tumor formation, pancreatic cancer, liver cancer, bladder cancer, primary surface bladder tumor, invasive metastatic cell bladder carcinoma of the bladder, muscle invasive bladder cancer, prostate cancer, colon cancer, kidney cancer, Cancer, esophageal cancer, ovarian carcinoma, cervical cancer, endometrial cancer, chorionic cancer, ovarian cancer, primary peritoneal epithelial neoplasm, cervical carcinoma, vaginal cancer, vulvar cancer, uterine cancer, solid tumor of follicle, testicular cancer, penile cancer, kidney cell carcinoma , Brain cancer, head and neck cancer, cervical cancer, neuroblastoma, brainstem glioma, glioma, metastatic tumor cell infiltration in the central nervous system, osteoma, osteosarcoma, malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, thyroid Cancer, retinoblastoma, neuroblastoma, mesothelioma, Wilms' tumor, gallbladder cancer, trophoblastic tumor, perivascular angioma, and Kaposi's sarcoma.

In the composition according to the present invention, the compound can be used in the form of a prodrug, a solvate and a pharmaceutically acceptable salt in order to enhance in vivo absorption or increase solubility. Also acceptable salts are also within the scope of the present invention. In addition, the compound has a chiral carbon, so stereoisomers thereof exist, and such stereoisomers are also included within the scope of the present invention.

In the composition according to the present invention, the compound may be prepared by various methods known according to the type of the substituent. For example, the following reaction schemes 1 to 2 are exemplified, and the following preparation method prepares the compound in the composition of the present invention. How to do is not limited. The preparation methods shown in Schemes 1 to 2 are only examples, and it will be apparent to those skilled in the art that they can be easily modified by those skilled in the art according to specific substituents. Same definition as in

[Scheme 1]

[Scheme 2]

In the composition according to the present invention, the compound can be used in the form of a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt can be prepared by a conventional method in the art, for example, hydrochloric acid, bromine. Salts with mineral acids such as acids, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, nitric acid, carbonic acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, succinic acid, benzoic acid, citric acid, maleic acid, malonic acid, mandelic acid, cin Salts with organic acids such as namic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid tartaric acid, gluconic acid, lactic acid, fumaric acid, lactobionic acid, ascorbic acid, salicylic acid, or acetylsalicylic acid (aspirin), glycine, alanine, vanillin, Salts with amino acids such as isoleucine, serine, cysteine, cystine, aspartic acid, glutamine, lysine, arginine, tyrosine, proline, methanesulfonic acid, e Includes sulfonic acid, benzene sulfonic acid, toluene sulfonic metal salt according to the sulfonic acid salt, the reaction with alkali metals such as sodium, potassium, such as acid, or such as a salt with an ammonium ion.

In the composition according to the invention, the compound may exist in solvated form, for example in hydrated and unsolvated form, in the composition according to the invention, the solvate of the compound is any solvated with pharmaceutical activity. It includes the old form.

In the composition according to the present invention, the compound may have a chiral center and may exist as a racemate, a racemic mixture and individual enantiomers or diastereomers. These isomers can be separated or resolved by conventional methods, and any desired isomer can be obtained by conventional synthetic methods or by stereospecific or asymmetric synthesis. All such isomeric forms and mixtures thereof are included within the scope of the present invention.

In the composition according to the invention, the compound can be administered in the form of a prodrug that degrades in the human or animal body to provide the compound of the invention. Prodrugs can be used to alter or improve the physical and / or pharmacokinetic profile of the parent compound and can be formed if the parent compound contains suitable groups or substituents that can be derived to form a prodrug.

In addition, the composition according to the present invention is an N- (1H-imidazol-2-yl) benzamide compound represented by Chemical Formula 1, a prodrug thereof, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. Conventional preparations in the pharmaceutical field by adding conventional non-toxic pharmaceutically acceptable carriers and excipients, such as tablets, pills, soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs It can be formulated as a formulation for oral administration such as or intravenously, subcutaneously, sublingually, intramuscularly, for sterile aqueous or oily formulations for parenteral administration.

Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention are those commonly used in formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin , Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and / or mineral oil, etc. , But is not limited thereto.

Excipients that can be used in the pharmaceutical composition of the present invention include sweeteners, binders, solubilizers, solubilizers, wetting agents, emulsifiers, isotonic agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, fragrances, etc. The proportions and properties of excipients can be determined by the solubility and chemical properties of the selected tablet, the route of administration chosen, and standard drug practice. Examples of excipients are lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, talc, stearic acid, sterin, magnesium stearate, magnesium aluminum silicate, starch, gelatin, tragacanth rubber, arginic acid, Sodium alginate, methylcellulose, sodium carboxymethylcellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, and vanilla flavor.

In addition, the pharmaceutical composition of the present invention may be formulated in a parenteral dosage form, in which case intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or local administration may be used, but is not limited thereto. At this time, in order to formulate the formulation for parenteral administration, the pharmaceutical composition is an active ingredient, that is, a compound of Formula 1, a prodrug thereof, a solvate thereof, a stereoisomer thereof or a pharmaceutically acceptable salt thereof is a stabilizer Alternatively, they can be mixed in water with a buffer to prepare a solution or suspension, and the solution or suspension can be prepared as a unit dosage form of an ampoule or vial.

In addition, the pharmaceutical composition of the present invention may be sterilized, or may further contain an auxiliary agent such as a preservative, stabilizer, hydration agent or emulsifying accelerator, salt and / or buffer for osmotic pressure control, and other therapeutically useful substances. It can also be formulated according to conventional methods of mixing, granulating or coating.

In addition, in the pharmaceutical composition according to the present invention, the compound represented by Chemical Formula 1, its prodrug, its solvate, its stereoisomer, or its pharmaceutically acceptable salt thereof, is administered to mammals including humans. It may vary depending on the patient's age, weight, gender, dosage form, health status and disease. In general, it may be included in the pharmaceutical composition in an effective amount of 0.001 to 500 mg / kg (body weight) per day, preferably 0.01 to 100 mg / kg (body weight) per day, and the pharmaceutical composition is once or twice a day The above can be divided and administered through an oral or parenteral route. However, since the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

Retroviruses (retrovirus, “retrovirus, retrovirus” (RNA tumor virus)) are a type of RNA virus. As a gene, it has RNA and a reverse transcriptase for inserting this RNA into the host's DNA. Because of this characteristic, it is classified as the most representative entity that does not follow the “Central Dogma”. Protein capsid is surrounded by an outer membrane with a membrane protein attached to the host cell when it leaves the host cell.

Retrovirus infection / proliferation occurs through the following process.

-Retroviral particles adhere to the surface of the host and cells. At this time, a special membrane protein plays a large role.

-The capsid penetrates into the “cytoplasm”.

-Reverse transcriptase reverse transcribes viral RNA into DNA.

-The reverse-transcribed DNA is inserted into the DNA of the host cell. (You will be interrupted anywhere.)

-The viral RNA made using the host cell system makes the proteins necessary for the virus (reverse transcriptase, capsid protein, membrane protein for penetration, etc.).

-The new assembled viral capsid extends out of the cell membrane.

Since most retroviruses use only the host cells without killing them, most of them progress to chronic infection because the "immune" system does not remove cells already infected with the retrovirus by only recognizing and attacking the virus. Therefore, the number of viruses in the blood is very small, but the infection itself continues to progress. Therefore, for treatment, a method of blocking the infection of the retrovirus itself or a method of destroying the cells infected with the retrovirus is needed. A representative retrovirus that infects humans is HIV (Human Immunodeficiency Virus).

The virus cell specific defense mechanism (APOBEC3G) of some cancer tissues is acting as a limiting factor in the development of anticancer virotherapy using spRRV. Administration of APOBEC3G-inhibited low-molecular compounds to cancer tissues may improve the infection efficiency of spRRV for anti-cancer virotherapy, but APOBEC3G inhibitors administered to cancer tissues can be transferred to systemic circulation through venous blood, which may be distributed outside cancer tissues. There is. It has been reported that adriamycin-infused liposomes are distributed in high concentrations in the liver and kidney when injected into cancer tissues, and it is also known that viral vectors also cause systemic migration to vascular sites damaged by injection. Therefore, it is advantageous that APOBEC3G inhibitory low-molecular compounds have a property of rapidly degrading in order to suppress innate immunity against viruses in the whole body other than cancer tissue, unlike general drugs.

When the distribution of replicable retroviruses on cancer tissue increases, it is possible to perform the recognition of the virus faster in the immune system in the human body, thereby promoting the activity of defense mechanisms against cancer cells, thereby improving the cancer treatment effect. Dramatic improvement is possible.

Hereinafter, the contents of the present invention will be described in more detail through examples. The examples are only intended to illustrate the present invention in more detail, and the scope of the present invention is not limited by them.

[Example 1] Cell-specific retrovirus protective factor inhibitory low molecular compound

Compounds having structures of the following Chemical Formulas 4, 5, and 6 were purchased or synthesized from commercial products (4; Enamie, USA, 5; Chemspace, USA), respectively.

[Formula 4]

(CIM885)

(CIM885)

[Formula 5]

(CIM953)

(CIM953)

[Formula 6]

(CIM1110)

(CIM1110)

[Scheme 3]

Compound a was first reacted with Cbz-protected piperazine compound to obtain b , then -NO ₂ was reduced to obtain -NH ₂ c , compound d was drawn from -NH2 acylation, and the piperidine function was inserted into the right part of the compound. After removing the Cbz group with Pd / C and H ₂ , the final compound CIM 1110 was obtained. 2-Piperazin-1-yl-N- (2-piperidin-4-yl-5-trifluoromethylphenyl) acetamide ( CIM 1110 ), ¹ H-NMR (300 MHz, CDCl ₃ ) δ 10.07 (s, 1H), 8.83 ( s, 1H), 7.34-7.20 (m, 2H), 3.16-3.13 (m, 6H), 2.89-2.87 (q, 4H), 2.58-2.65 (q, 4H), 1.71-1.69 (q, 4H), 1.54-1.52 (m, 2H).

[Example 2] Cell-specific retrovirus defense factor inhibition

1. In the cell APOBEC3G  Inhibited infection

To confirm infection inhibition by APOBEC3G (Apolipoprotein B mRNA Editing Catalytic Polypeptide-like 3G), 293T cells were first infected with LentiM1.4 and LentiM1.4-APOBEC3G, respectively, and then EQPAM and MFGmCMVGFP were secondary for 4 hours. Treatment produced virus. After 24 hours, virus was taken from 293T cells, infected with HeLa cells, and virus-labeled fluorescence was observed. The results are shown in FIG. 1. From this, it was confirmed that APOBEC3G reduces the infection of the virus.

2. Confirm whether endogenous A3G is expressed in various tumor cell lines

In the human lymphoma cell line H9 having a low retroviral infection efficiency, 100 μg of protein was confirmed by Western blot to show how much the endogenous APOBEC3G is expressed relative to other cell lines. More APOBEC3G is expressed in H9 than U-87 MG overexpressed by transfection of the APOBEC3G gene.

The results are shown in FIG. 2.

3. Endogenous APOBEC3G  Using the expressing human lymphoma cell line H9, APOBEC3G  Screening of inhibitors to increase virus propagation efficiency

end. APOBEC3G ( A3G ) Suitable for measuring inhibitor efficiency spRRVenvCMVGFP / sRRVgpCMVRFP  Viral Titer

The titer of spRRVenvCMVGFP / sRRVgpCMVRFP virus suitable for APOBEC3G (A3G) inhibitor screening using human H9 cell line was calculated by the following method.

In H9 cell line ^{10 10 gc, 10 9 gc,} 10 8 spRRVenvCMVGFP / sRRVgpCMVRFP of various titers of gc, 10 ⁷ gc and 10 ⁶ gc, treated with 2 ug / ml polybrene in order from low dose titer to high dose titer for 24 hours, then replaced with culture medium containing 1 ug / ml polybrene Culture was continued.

After infection, every 3 days, some samples measured the propagation efficiency of spRRVenvCMVGFP / sRRVgpCMVRFP, and the amount of GFP and RFP expressed through flow cytometry, and the remaining culture samples were passaged at a ratio of 1: 6.

The results are shown in FIG. 3.

As a result, when spRRVenvCMVGFP / sRRVgpCMVRFP of 10 ¹⁰ gc virus titer was infected, it was confirmed that fluorescence cells reached up to 44% on the 12th day after infection. On the other hand, when infected with 10 ⁸ gc or less, fluorescence expression did not increase.

I. Using the H9 cell line APOBEC3G  Inhibitory effect of inhibitors

Compounds of Formulas 4 to 6 (CIM885, CIM953, CIM1110) were treated with H9 cell lines at concentrations of 30 uM, 3 uM, and 0.3 uM 6 days before infection, respectively.

From the results of 4. above, a replicable retrovirus having a virus titer of 10 ⁸ gc was treated with 2 ug / ml polybrene for 24 hours, followed by 1 ug / ml polybrene and 30 uM, 3 uM or 0.3 uM The culture was maintained by replacing the APOBEC3G inhibitor with a culture solution.

After infection, the propagation efficiency of the replicable retrovirus was measured with a flow cytometer, and the cells were 1: 6 with a culture medium containing 1 ug / ml polybrene and 30 uM, 3 uM or 0.3 uM of the APOBEC3G inhibitor every 3 days. It was passaged at the rate of.

The results are shown in FIG. 4.

As a result, when the APOBEC3G inhibitor was treated according to the concentration change, it did not show cytotoxicity and showed significantly higher virus propagation, compared to the control (Control) DMSO treatment, on the 4th day of infection, CIM885 When treated with 30 uM, it showed significant viral propagation 3.8 times higher, and when treated with 30 uM, CIM953 showed 1.5 times higher viral propagation.

On the 6th day of infection, the virus spread increased rapidly to 3.2 times in the CIM885 treated group and 5.2 times in the CIM953 treated group. Finally, on the 8th day of infection, the virus infection efficiency was 3.8 times higher in the CIM885 treated group and 4.3 times higher in the CIM953 treated group.

The measurement was performed by the following statistical method.

-Two way ANOVA, date effect, F (3, 444) = 519.6, P <0.0001,

-APOBEC3G inhibitor effect, F (36, 444) = 48.31, P <0.0001,

-Interaction effect, F (108, 444) = 10.03, P <0.0001).

Using the Turkey post-test method, the calculation results for the virus infection rate between the experimental group and the control group are as follows.

-On the 4th day of infection, the 30 uM CIM885 and CIM1110 treated groups showed significantly higher viral infection cell proportions compared to the control DMSO treated group (Turkey post-test, 30 μM CIM885 P value = 0.0018, 30 μM CIM1110 P value <0.0001).

-On the 6th day of infection, 30 uM CIM885, CIM953 and CIM1110 treated groups showed significantly higher viral infection cell proportions compared to the control DMSO treated group (Turkey post-test, 30 μM CIM953 P value <0.0001, 30 μM CIM1110 P value <0.0001 , 30μM CIM885 P value <0.0001, Vif P value = 0.0022, 3μM CIM1110 P value = 0.0083, 3μM CIM885 P value = 0.0364).

-On the 8th day of infection, the 30 μM, 3 μM and 0.3 μM CIM1110 treated groups, the 30 μM and 3 μM CIM885 treated groups showed significantly higher rates of virus-infected cells compared to the control DMSO treated group (Turkey post-test, 30 μM CIM1110 P value <0.0001 , 3μM CIM1110 P value <0.0001, 30μM CIM953 P value <0.0001, 30μM CIM885 P value <0.0001, 3μM CIM885 P value = 0.0153, 0.3μM CIM1110 P value = 0.0181).

In the virus propagation rate, in the case of 30 μM CIM953, it did not show a significantly high virus propagation rate on the 4th day of the initial stage of infection, but rapidly increased on the 6th day of infection, indicating a maximum of 96.9% and an average of 70.98% of virus-infected cells.

In addition, CIM1110 showed a slightly lower virus propagation efficiency than CIM885 and CIM953, but showed a significantly higher proportion of virus-infected cells compared to other compounds having a similar chemical structure. The results can be confirmed by comparing FIGS. 4A and 4B.

[Example 3] Single dose administration of APOBEC3G inhibitor general toxicity

One. CIM885 Single dose  General toxicity

For the control group in which male ICR mice were administered with a solvent only and CIM885 intraperitoneally in mice at 30, 100 and 300 mg / 10mL / kg, respectively, the presence or absence of cytotoxicity was measured through observation for 5 days.

Table 1 shows the experimental conditions and results.

[Table 1]

From the above, as a result of administering CIM885 by concentration, the weight of the experimental mice showed a constant increase without a significant difference compared to the control group administered only with the solvent, and after 5 days of measurement, the mice were autopsied and observed with the naked eye, It was confirmed that no abnormalities were observed in the esophagus, lung, heart, stomach, liver, small intestine, and large intestine compared to the control group. From this, it was confirmed that CIM885 does not show toxicity up to 300 mg / kg upon intraperitoneal administration to mice.

2. CIM953 Single dose  General toxicity

In the same manner as in 1. above, for the control group in which only male ICR mice were administered with a solvent, and in the experimental group in which CIM953 was administered with 30, 100 and 300 mg / 10mL / kg mice, respectively, cytotoxicity was measured through observation for 5 days. Did.

Table 2 shows the experimental conditions and results.

[Table 2]

From the above, as a result of administering CIM953 by concentration, the weight of the experimental mice showed a constant increase without a significant difference compared to the control group administered only with the solvent, and after 5 days of measurement, the mice were autopsied and observed with the naked eye, It was confirmed that no abnormalities were observed in the esophagus, lung, heart, stomach, liver, small intestine, and large intestine compared to the control group. From this, it was confirmed that CIM953 does not show toxicity up to 300 mg / kg when administered intraperitoneally to mice.

[Example 4] Pharmacokinetic properties of APOBEC3G inhibitor

1. Measurement of blood protein binding rate

Blood protein binding rate affects the distribution, drug efficacy, and toxicity of the drug. Thermo Pierce, Inc. using the equilibrium dialysis method using the principle that only free drugs that are not bound to proteins can pass through the semipermeable membrane Blood protein binding rates of CIM885 and CIM953 were measured using a RED device.

Blood protein binding rate was measured while stirring and incubating at a concentration of 2 ug / mL at 37 ° C for 4 hours.

As a result, CIM885 and CIM953 showed blood protein binding rates of 99.07% and 80.81%, respectively.

2. Measurement of metabolic stability

Among the metabolic reactions that most affect the dynamics of the drug, phase 1 metabolism to cytochrome P (CYP) accounts for 75% of the total. Accordingly, CIM885 and CIM953 were added with coenzyme to the microsomes of the liver where CYP was most frequently expressed, and the rate of disappearance was measured to measure metabolic rate by CYP.

CIM885 and CIM953 were injected into rat and human liver microsome samples and cultured.

As a result, CIM885 showed half-lives of 26.3 minutes and 11.5 minutes in rat and human liver microsomes, respectively, and CIM953 showed 7.28 minutes and 7.33 minutes, respectively.

3. CYP  Inhibition measurement

As in 2. above, the CYP enzyme accounts for about 75% in drug metabolism, so a substance that inhibits CYP is very likely to cause drug interaction with other drugs.

To confirm whether it has such properties, the inhibition of 5 types of CYP (1A2, 2D6, 2C9, 2C19 and 3A4) was measured using recombinant human CYP (Promega, USA).

end. CIM885  One uM  And 10 uM By  Measurement of inhibition

As a result of treatment with CIM885 at concentrations of 1 uM and 10 uM to the five CYPs, no inhibition was observed for 5 CYPs at 1 uM, and trace inhibition of 4 1A2, 2C19, 2D6 and 3A4 at 10 uM was not observed. Was measured.

When this was calculated as the value of the inhibitory variable R = 1 + [I _un ] / Ki reflecting blood concentration and protein binding, it was calculated as R = 1.02, confirming that the possibility of drug interaction was low.

I. CIM953  One uM  And 10 uM By  Measurement of inhibition

As a result of treating CIM953 at concentrations of 1 uM and 10 uM to the CYP of the above 5 species, it was determined that there was a small amount of inhibition on 2C19 at 1 uM and 2C19, 2D6 at 10 uM.

When this was calculated as the value of the inhibitory variable R = 1 + [I _un ] / Ki reflecting blood concentration and protein binding, it was calculated as R = 1.02, confirming that the possibility of drug interaction was low.

From the results of the above examples, the compounds 4 to 6 of the present invention are inhibitors of APOBEC3G, which promote cancer cell infection and spread of the replicable retrovirus without showing toxicity, and can significantly improve the anti-cancer effect according to the immune system in vivo. It was confirmed that the present invention was completed.

Claims (9)

A pharmaceutical composition for the treatment of cancer comprising the compound represented by Formula 2, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 2]

In Chemical Formula 2,
R3 is (C1-C7) alkyl or halo (C1-C7) alkyl,
R4 and R5 are combined to form a 5- or 6-membered ring, and the ring formed by R4 and R5 bonding may be further substituted with (C1-C7) alkyl or halo (C1-C7) alkyl,
X is a heteroatom. delete delete delete delete According to claim 1,
The compound is a pharmaceutical composition for the treatment of cancer comprising a compound selected from the following structure, a solvate thereof, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition for treating cancer according to claim 1 or 6, wherein the composition further comprises a pharmaceutically acceptable carrier as an active ingredient. According to claim 1 or 6, wherein the composition is a pharmaceutical composition for the treatment of cancer, characterized in that by a mechanism that promotes the retrovirus spread through weakening of the retroviral defense mechanism of cancer tissues due to APOBEC3 inhibition. The method of claim 8,
These cancers are APOBEC3G mucinous cell carcinoma, round cell carcinoma, locally advanced tumor, metastatic cancer, Ewing's sarcoma, cancer metastasis, lymphoid metastasis, squamous cell carcinoma, esophageal squamous cell carcinoma, oral carcinoma , Multiple myeloma, acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, morphological cellular leukemia, effusion lymphoma, thymic lymphoma lung cancer, small cell lung carcinoma, skin T cell lymphoma, Hodgkin lymphoma, Non-Hodgkin's lymphoma, adrenal cortical cancer, ACTH-producing tumor, non-small cell lung cancer, breast cancer, small cell carcinoma, conduit carcinoma, gastric cancer, colon cancer, colorectal cancer, polyps related to colon tumor formation, pancreatic cancer, liver cancer, bladder cancer, Primary surface bladder tumor, invasive metastatic cell bladder carcinoma of the bladder, muscle invasive bladder cancer, prostate cancer, colon cancer, kidney cancer, liver cancer, esophageal cancer, ovarian carcinoma, cervical cancer, endometrial cancer, chorionic cancer, Ovarian cancer, primary peritoneal epithelial neoplasm, cervical carcinoma, vaginal cancer, vulvar cancer, uterine cancer, solid tumor among follicles, testicular cancer, penile cancer, kidney cell carcinoma, brain cancer, head and neck cancer, caliber cancer, neuroblastoma, brain stem glioma, nerve Gliomas, metastatic tumor cell infiltration of the central nervous system, osteoma, osteosarcoma, malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, thyroid cancer, retinoblastoma, neuroblastoma, mesothelioma, Wilms' tumor, gallbladder cancer, nutrition A pharmaceutical composition for the treatment of cancer, characterized in that one or more selected from the group consisting of subcellular tumors, perivascular hemangioma, and Kaposi's sarcoma.

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