WO2022014433A1 - Analgesic drug using nr4a1 antagonist - Google Patents

Abstract

The present invention pertains to: a compound represented by formula (I) (in the formula, R1 represents a hydroxyl group, a mercapto group, a C1-6-alkoxy group, a C1-6-haloalkoxy group, an amino group, a C1-6-alkyl amino group, or a di-C1-6-alkyl amino group, R2 represents a hydroxyl group, a mercapto group, a C1-6-alkoxy group, a C1-6-haloalkoxy group, an amino group, a C1-6-alkyl amino group, or a di-C1-6-alkyl amino group, R3 represents a halogen atom, a nitro group, a carboxyl group, a C1-6-alkoxy-carbonyl group, or a carboxy-C1-6-alkyl group, and R4 represents a substituted or unsubstituted C3-12-hydrocarbon group) or a salt thereof, or a solvate thereof; and an analgesic drug that contains the compound or a salt thereof, or a solvate thereof.

Description

Analgesics with Nr4a1 antagonists

The present invention relates to an analgesic using an Nr4a1 antagonist.

Cancer has been the leading cause of death in Japan since 1981. The number of cancer patients increases with age, and it is said that one in three to four people over the age of 70 is a cancer patient.

Bone marrow suppression, vomiting, hair loss, etc., which are common side effects of administration of anticancer drugs, have resulted in suffering for cancer patients. Some anticancer drugs cause peripheral neuropathic pain such as pain, numbness, and cold sensation in the limbs, such as taxanes such as paclitaxel and platinum such as oxaliplatin. Furthermore, in addition to mental and social pain, physical pain, so-called "cancer pain," has become a major problem in cancer patients. The term "cancerous pain" as used herein includes pain in the primary tumor, pain due to bone metastasis, and neuropathic pain associated with infiltration of cancer cells into nerves.

Nearly 70% of cancer patients experience pain. Cancer pain relief follows the three-step pain relief ladder defined by WHO, mainly non-steroidal anti-inflammatory drugs (NSAIDs) and non-opioid analgesics such as acetaminophen and opioids such as codeine, morphine, and fentanyl. Treatment is done in combination with analgesics. While 80% of cancer pain has been shown to be analgesic in many patients with the use of appropriate analgesics, neuropathic pain due to infiltration of cancer cells into the nerve, bone metastatic pain, It is said that opioids are not effective for gastrointestinal obstruction. In addition, side effects such as constipation, vomiting, addiction, and tolerance due to opioids further afflict patients. Therefore, the development of analgesics effective for these pains is desired.

Thus, at present, the administration of two types of drugs, an anticancer drug and an opioid analgesic for cancer and cancer pain, and the strong side effects associated therewith have resulted in distress to cancer patients. Therefore, the development of an "anticancer analgesic" that has both analgesic and anticancer effects with one drug and has fewer side effects than conventional analgesics and anticancer drugs is for cancer patients. It has the potential to be a breakthrough treatment that greatly enhances the quality of life. However, at present, there is no single drug that has both analgesic and anticancer effects.

On the other hand, regarding "Nuclear receptor subfamily 4, group A member 1, Nr4a1" which is one of the nuclear receptors, Patent Document 1 discloses a method for identifying an agent that regulates the activity and expression of Nr4a1. .. Patent Document 1 describes that it inhibits the growth of cancer cells, but does not describe any analgesic effect.

The present inventors first aimed at acquiring an Nr4a1 antagonist, and based on the complex structure (PDB; 3V3Q) of the Nr4a1 protein and a known antagonist TMPA, "three-dimensional pharmacohore-based narrowing down" and "molecular docking simulation" A multi-step in-cilico screening consisting of "narrowing down by" and "molecular similarity analysis based on chemical properties" was performed, and 10 compounds were identified as novel Nr4a1 antagonist candidate compounds from the existing compound database, and one of them was used. It was reported that a certain NRA-8 suppresses the pain caused by PACAP (Non-Patent Document 1). However, Non-Patent Document 1 does not disclose the structure of any of the compounds, and does not mention any anticancer activity.

Special Table 2018-536384 Gazette

It is an object of the present invention to provide a pain therapeutic drug having an action mechanism different from that of NSAIDs and opioids and having both analgesic action and anticancer action with one drug.

In order to solve the above-mentioned problems, the present inventors searched for a molecule common to chronic pain and proliferation / infiltration of cancer cells in order to select a target molecule, and found that it is one of the nuclear receptors as a candidate molecule. A certain Nr4a1 was identified. That is, when gene expression in the posterior horn of the spinal cord of neuropathy pain model mice and gene expression in various cancer cell lines were comprehensively analyzed using the GeneChip microarray method, they were common to pain model mice and cancer cells. As a result, Nr4a1 mRNA could be identified as a gene whose expression is increased.

Therefore, the present inventors have the following equation:

Since the Nr4a1 inhibitory effect of NRA-8 shown in 1 is equivalent to or stronger than that of the already reported Nr4a1 antagonists TMPA and DIM-C-pPhOH, the activity is stronger than that of NRA-8. Aiming at the acquisition of the compound showing the above, the structure was developed based on the structure of NRA-8. In screening using the NBRE-Luc reporter vector (NBRE; Nr4a1 binding DNA domain), many 1,2,3,4-tetrahydroquinazoline derivatives have the same or higher efficacy as the original compound NRA-8. Succeeded in the acquisition of, and came to complete the present invention.

That is, the gist of the present invention is as follows.
(1) The following equation (I):

(In the formula, R ¹ is a hydroxyl group, a mercapto group, a C _1-6 -alkoxy group, a C _1-6 -haloalkoxy group, an amino group, a C _1-6 -alkylamino group or a diC _1-6 -alkylamino group. R ² is a hydroxyl group, a mercapto group, a C _1-6 -alkoxy group, a C _1-6 -haloalkoxy group, an amino group, a C _1-6 -alkylamino group or a diC _1-6 -alkylamino group. Yes; R ³ is a halogen atom, nitro group, carboxyl group, C _1-6 -alkoxy-carbonyl group or carboxy-C _1-6 -alkyl group; R ⁴ is substituted or unsubstituted C _3-12 -carbonated It is a hydrogen group.)
A compound represented by (1) or a salt thereof or a solvate thereof.
(2) In the formula (I), the ^{compound according to the above (1) in which R 1} and R ² are hydroxyl groups, a salt thereof, or a solvate thereof.
(3) In the above formula (I), the above (1) or (2), wherein ^{R 4} is a substituted or unsubstituted C _3-12 -alkyl group or a substituted or unsubstituted C _{7-12-aralkyl group.} The compound described or a salt thereof or a solvate thereof.
(4) An analgesic containing the compound according to any one of (1) to (3) above, a salt thereof, or a solvate thereof.
(5) The analgesic according to (4) above, which is applied to a cancer patient.
(6) The following equation (II):

^(Wherein, R 1, ^{R 2} and ^{R 3} are as defined in the formula of claim ^{1 (I); R 4 '} C 2-12 substituted or unsubstituted is - hydrocarbon group.)
An analgesic containing the compound represented by (1) or a salt thereof or a solvate thereof.
(7) The analgesic according to (6) above, which is applied to a cancer patient.

The compound of the present invention is a compound having a phenyl group substituted with a halogen atom at the 2-position of the 1,2,3,4-tetrahydroquinazoline-4-one skeleton and a hydrocarbon group substituted or unsubstituted at the 3-position. There is, and it is useful as an analgesic, and the analgesic is a new pain therapeutic agent having a mechanism of action different from that of NSAIDs and opioids.

Further, since the compound of the present invention has both analgesic action and anticancer action, cancer treatment that can avoid side effects such as myelosuppression by conventional anticancer drugs becomes possible. In addition, pain treatment that avoids adverse effects such as digestive ulcers seen in NSAIDs and constipation seen in opioids becomes possible. Since therapeutic effects can be expected in both cancer and pain, the need for polypharmacy is reduced, and the appearance of unexpected adverse effects can be avoided, and the quality of patients with intractable cancer pain.・ Improvement of life can be expected.

FIG. 1 shows the results of the reporter assay and the structure of NRA-8. FIG. 2 shows the effect of the compound on the PACAP-induced long-term allodynia reaction. FIG. 3 shows the effect of NRA-8 on allodynia in neuropathic pain model mice. FIG. 4 shows the effect of NRA-8 on allodynia in bone cancer pain model mice. FIG. 5 shows the effect of NRA-8 on the proliferation, survival and migration of Panc1 cells. FIG. 6 shows the effect of existing analgesics on the proliferation of Panc1 cells. FIG. 7 shows the evaluation of a compound of the present invention by a reporter assay. FIG. 8 shows the analgesia evaluation of the compound of the present invention. FIG. 9 shows an anticancer activity evaluation (cell line) of the compound of the present invention. FIG. 10 shows an anticancer activity evaluation (transplant model) of the compound of the present invention.

Hereinafter, the present invention will be described in detail.

_{Examples of the C 1-6} -alkoxy group represented by ^{R 1} or R ² in the formulas (I) and (II) include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group and sec. -Butoxy group, tert-butoxy group, pentyloxy group, isopentyloxy group, hexyloxy group, cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, cyclohexyloxy group can be mentioned.

_{Examples of the C 1-6} -haloalkoxy group represented by ^{R 1} or R ² in the formulas (I) and (II) include a trifluoromethoxy group.

Formula (I) and C in (II) represented by R ¹ or R ² _{1-6 -} The alkylamino group, for example methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group , Isobutylamino group, sec-butylamino group, tert-butylamino group, pentylamino group, isopentylamino group, hexylamino group, cyclopropylamino group, cyclobutylamino group, cyclopentylamino group, cyclohexylamino group. ..

Di-C _1-6 represented by R ¹ or R ² in the formula (I) and (II) _- The alkylamino group, such as dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, methyl ethyl Amino groups can be mentioned.

The halogen atom represented by R ³ in the formula (I) and (II), for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom.

_{Examples of the C 1-6} -alkoxy-carbonyl group represented by ^{R 3} in the formulas (I) and (II) include a methoxycarbonyl group, an ethoxycarbonyl group, a propoxycarbonyl group, an isopropoxycarbonyl group, and a butoxycarbonyl group. Isobutoxycarbonyl group, sec-butoxycarbonyl group, tert-butoxycarbonyl group, pentyloxycarbonyl group, isopentyloxycarbonyl group, hexyloxycarbonyl group, cyclopropyloxycarbonyl group, cyclobutyloxycarbonyl group, cyclopentyloxycarbonyl group, Examples include cyclohexyloxycarbonyl groups.

Carboxy -C _1-6 represented by R ³ in the formula (I) and (II) _- Examples of the alkyl group include a carboxymethyl group, a carboxyethyl group, and a carboxypropyl group.

_{C 3-12} represented by ^{R 4} in the above Formulas (I) - As the hydrocarbon group, for example, propyl group, isopropyl group, butyl group, isobutyl group, sec- butyl group, tert- butyl group, a pentyl group, _{Linear or branched C 3-12} -alkyl groups such as isopentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group; cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl, _{C 3-12} such as cycloheptyl group - a cycloalkyl group; a 1-propenyl group, allyl group, 1-butenyl, 2-butenyl, pentenyl, hexenyl, _{C 3-12,} such as oleyl group - _{Alkenyl group; C 3-12} -alkynyl group such as 1-propynyl group, 2-propynyl (propargyl) group, 3-butynyl group, pentynyl group, hexynyl _{group; C 6} H ₅ (CH ₂ ) _n- (here, n = 1 ~ is an integer of 6), a naphthylmethyl group, C _7-12 such naphthylethyl group _- aralkyl;. bridged cyclic hydrocarbons such as adamantyl group; a phenyl group, an aromatic hydrocarbon group such as a naphthyl group Hydrogen group; Spiro hydrocarbon group and the like can be mentioned.

The hydrocarbon group, C _3-12 of the _{- -} C _2-12 represented by R ^{4 'in} the formula (II) include those obtained by adding in addition to ethyl hydrocarbon group.

The C _3-12- hydrogen group and the C _2-12- hydrogen group are, for example, C _1-6 -alkoxy groups (eg, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, isobutoxy group). , Sc-butoxy group, tert-butoxy group, pentyloxy group, isopentyloxy group, hexyloxy group, cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, cyclohexyloxy group), methylenedioxy group, C _{2 -6} -alkenyloxy group, aralkyloxy group (eg benzyloxy group, 4-methylbenzyloxy group, 3-methylbenzyloxy group, 2-methylbenzyloxy group, 4-fluorobenzyloxy group, 3-fluorobenzyloxy group) , 4-Chlorobenzyloxy group, 3-chlorobenzyloxy group), halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom), C _1-6 -haloalkyl group, C _1-6 -haloalkoxy group, acyl _{Group (for example, C 1-6} -aliphatic acyl group such as formyl group, acetyl group, propanoyl group, butanoyl group, pentanoyl group, hexanoyl group; alloyl group such as benzoyl group and toluoil group), acyloxy group (for example, formyloxy group). _{, C 1-6} -aliphatic acyloxy group such as acetoxy group, propanoyloxy group, butanoyloxy group, pentanoyloxy group, hexanoyloxy group; alloyloxy group such as benzoyloxy group and toluoiloxy group), hydroxyl group , A carboxyl group, an acetamide group, a carbamoyl group, a cyano group, a nitro group and the like, and may be substituted with one or more substituents.

The substituted hydrocarbon group represented by ^{R 4} or R ^{4 'in} the formula (I) or (II), 2-hydroxyethyl group, 2-methoxyethyl group, 3-hydroxypropyl group, 3-methoxy Propyl group, 4-hydroxybutyl group, 4-methoxybutyl group, 3- (p-methoxyphenyl) propyl group, 3- (p-fluorophenyl) propyl group, 3- (p-chlorophenyl) propyl group and the like. However, it is not limited to these.

As the compound represented by the formula (I) or (II), R ¹ and R ² are hydroxyl groups, R ³ is a halogen atom, for example, a chlorine atom, and R ⁴ is a substituted or unsubstituted C _3-8 -alkyl group. Alternatively, a compound which is a substituted or unsubstituted C _{7-11-aralkyl group is preferable.}

The compound represented by the formula (I) is a novel compound, and the compound represented by the formula (II) includes a known compound in addition to the novel compound represented by the formula (I).

As the salt of the compound represented by the formula (I) or (II), a pharmaceutically acceptable salt is preferable, and for example, hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, nitric acid, and pyro. Inorganic acids such as sulfuric acid and metaphosphoric acid, or organic acids such as citric acid, benzoic acid, acetic acid, propionic acid, fumaric acid, maleic acid and sulfonic acid (eg, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid). And salt can be mentioned.

Examples of the solvate of the compound represented by the formula (I) or (II) or a salt thereof include hydrates.

In the formula (I), the ^{compound in which R 1} and R ² are hydroxyl groups and R ³ is a halogen atom, for example, a chlorine atom can be produced, for example, as shown below.

(In the formula, R ⁴ is synonymous with the above formula (I).)

Namely, amine compound ^(R 4 -NH ₂₎ and the o- nitrobenzoic acid by condensation, converted into o- nitrobenzoic acid amides, reduction, and 3,4-di (benzyloxy) -5-chloro-benzaldehyde After the reaction is carried out to obtain a benzyl form, the desired product can be obtained by debenzylation.

R ¹ and ^{R 2} are _{C 1-6} - alkoxy or _{C 1-6} - as haloalkoxy group, when there is no need to be protected with a benzyl group, 3,4-di (benzyloxy) -5- The desired product can be directly obtained by using 3,4-di (C _1-6 -alkoxy or C _{1-6-haloalkoxy) -5-chlorobenzaldehyde instead of chlorobenzaldehyde.}

The compound represented by the above formula (II) can also be obtained in the same manner as described above.

In order to purify the product obtained as described above, commonly used methods such as column chromatography using silica gel or the like as a carrier, methanol, ethanol, chloroform, dimethylsulfoxide, n-hexane-ethyl acetate, water and the like can be used. It may be according to the recrystallization method using. Examples of the elution solvent for column chromatography include methanol, ethanol, chloroform, acetone, hexane, dichloromethane, ethyl acetate, and a mixed solvent thereof.

The above compound can be formulated as an analgesic and / or an anticancer drug in combination with a conventional pharmaceutical carrier. The dosage form is not particularly limited and is appropriately selected and used as necessary, and is used as tablets, capsules, granules, fine granules, powders, sustained release preparations, liquids, suspensions, emulsions and syrups. , Oral agents such as elixirs, parenteral agents such as injections and suppositories.

Oral preparations are conventionally produced using, for example, starch, lactose, sucrose, mannitol, carboxymethyl cellulose, inorganic salts and the like. In addition to these, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a fragrance and the like can be appropriately added.

Examples of the binder include starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, macrogol and the like.

Examples of the disintegrant include starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose and the like.

Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80 and the like.

Examples of the lubricant include talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like.

Examples of the fluidity accelerator include light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like.

The injection is produced according to a conventional method, and distilled water for injection, physiological saline, aqueous glucose solution, olive oil, sesame oil, lacquer oil, soybean oil, corn oil, propylene glycol, polyethylene glycol and the like can be generally used as the diluent. .. Further, if necessary, a bactericide, a preservative, a stabilizer, an tonicity agent, a painless agent and the like may be added. Further, from the viewpoint of stability, the injection can be filled in a vial or the like and then frozen, water is removed by a usual freeze-drying technique, and the liquid preparation can be reprepared from the freeze-dried product immediately before use. The proportion of the compound of formula (I) or (II) in the injection may vary from 5 to 50% by weight, but is not limited thereto.

Examples of other parenteral preparations include suppositories for intrarectal administration, which are manufactured according to a conventional method.

The formulated analgesic and / or anticancer drug varies depending on the dosage form, administration route, etc., but for example, it is possible to administer 1 to 4 times a day for a period of 1 week to 3 months.

In order to exert the desired effect as an oral preparation, it depends on the age, body weight, and degree of disease of the patient, but in the case of an adult, the weight of the compound of the above formula (I) or (II) is, for example, 0. It is appropriate to take 1 to 1000 mg, preferably 1 to 500 mg in several divided doses a day.

In order to exert the desired effect as a parenteral preparation, it depends on the age, body weight, and degree of disease of the patient, but usually in the case of an adult, the weight of the compound of the above formula (I) or (II) is used, for example. It is appropriate to administer 0.1 to 1000 mg, preferably 1 to 500 mg by intravenous injection, intravenous drip infusion, subcutaneous injection, or intramuscular injection.

The compound of the present invention has both analgesic and anticancer effects, and the difference in dose required for analgesic and anticancer effects is small. Therefore, when applied to cancer patients, the need for multidrug combination is reduced. Therefore, it is possible to avoid the appearance of unexpected adverse effects, and further, it is expected that the quality of life of patients with intractable cancer pain will be improved. The compound of the present invention has an analgesic effect on a neuropathic pain model and an osteocancer pain model, and suppresses the growth, survival, and migration of various cancer cells. As a drug, it can be expected to have a therapeutic effect on various cancers as an anticancer drug alone. In particular, it showed a good analgesic effect on the pain response of bone cancer pain model mice (Fig. 8). It has already been reported that opioids are less effective against the pain response of this model mouse (Minami et al., J Pharmacol Sci., 111 (1), 60-72, 2009), and clinically, cancer cells It is said that opioids are not effective for bone metastatic pain. Therefore, the compound of the present invention can be expected to have a therapeutic effect on pain associated with metastatic bone tumors to which opioids are difficult to respond.

The 5-year survival rate after being diagnosed with cancer is 8.9% for pancreatic cancer, 29.5% for lung cancer, and 34.1% for the brain / central nervous system, which are significantly higher than other tumors. Low (according to National Cancer Center statistics). The low 5-year survival rate is due to "recurrence" in which tumors that have not been removed by surgery re-grow or "metastasis" in which cancer is found elsewhere, but recurrence / metastasis of these tumors It shows that the drug treatment for the disease is not perfect at present. The compound of the present invention was able to strongly suppress proliferation, survival, and migration in various cancer cells including pancreatic cancer cell Panc1, lung cancer cell line A549, and brain tumor (glioma) cells U87 and U251 (Fig.). 5 and FIG. 9). Also, in the Xenograft model, the compound of the present invention showed a tumor growth inhibitory effect by oral administration (FIG. 10). From the above, it can be expected to be used to prevent cancer recurrence / metastasis after tumor removal in surgical operation, and to lead to improvement in 5-year survival rate.

In recent years, the nuclear receptor Nr4a1, which is the target molecule of the present invention, has "expression of PD-L1" (Karki et al., Cancer Res 80 (5), 1011-1023, 2020) and "depletion of T cells". (Liu et al., Nature 567, 525-529, 2019; Chen et al., Nature 567, 530-534, 2019) has been reported to be involved, and when Nr4a1 is inhibited by the compound of the present invention, " It is expected that cancer immunotherapy may be enhanced through "decreased expression of PD-L1 in cancer cells" and "activation of T cells".

This specification includes the contents described in the specification and drawings of Japanese Patent Application No. 2020-120069, which is the basis of the priority of the present application.

Hereinafter, the present invention will be described in more detail by way of examples, but the scope of the present invention is not limited thereto.

[Example 1] Synthesis of 3-substituted-2-phenyl-1,2,3,4-tetrahydroquinazoline-4-one derivative

Under Ar atmosphere, commercially available amine 2a-i (0.60 mmol), 1- (3-dimethylaminopropyl) -3-ethyl in a dichloromethane solution (3 mL) of commercially available o-nitrobenzoic acid (100 mg, 0.60 mmol) Carbodiimide hydrochloride (149 mg, 0.78 mmol), 1-hydroxybenzotriazole (92 mg, 0.60 mmol) and triethylamine (0.08 mL, 0.60 mmol) were added sequentially under ice-cooling, and then stirred at room temperature for 24 hours. .. Then, a 10% aqueous HCl solution (3 mL) was added, and the mixture was extracted with dichloromethane (1 mL × 3 times). After combining the organic layers and drying over anhydrous sodium sulfate, the solvent was distilled off using a rotary evaporator, and the obtained residue was purified by silica gel column chromatography (n-hexane: acetone = 3: 1 to 1: 1). By doing so, amide 3a-i was obtained.

10% palladium activated carbon (5 mg) was added to an ethyl acetate solution (3 mL) of amide 3a-i (0.48 mmol) at room temperature, hydrogen substitution was performed, and the mixture was stirred at room temperature for 2 hours. Then, the catalyst was removed by filtration through Celite, and the solvent was distilled off using a rotary evaporator to obtain aniline. The obtained aniline was used in the next reaction without purification. Under Ar atmosphere, 5-chloro-3,4-dibenzyloxybenzaldehyde (168 mg, 0.48 mmol) and cyanuric chloride (18 mg, 0.10 mmol) were sequentially added to the obtained acetonitrile solution of aniline (3 mL) at room temperature. After addition, the mixture was stirred at room temperature for 24 hours. Then, the solvent was distilled off using a rotary evaporator, and the obtained residue was purified by silica gel column chromatography (n-hexane: acetone = 2: 1 to 1: 1) to 1,2,3,4-. A tetrahydroquinazoline-4-one derivative 4a-i was obtained.

After adding 20% palladium hydroxide activated carbon (3 mg) to a methanol solution (3 mL) of 1,2,3,4-tetrahydroquinazoline-4-one derivative 4a-i at room temperature, hydrogen substitution is performed at room temperature. The mixture was stirred for 4 hours. Then, the catalyst was removed by filtration through Celite, and the solvent was distilled off using a rotary evaporator. The obtained residue was purified by silica gel column chromatography (dichloromethane: methanol = 50: 1 to 10: 1) to obtain the desired NRA-807 to NRA-815.

3-Butyl-2- (5-chloro-3,4-dihydroxyphenyl) -1,2,3,4-tetrahydroquinazoline-4-one (NRA-807)
¹ H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.4 Hz), 1.32 (2H, sext, J = 7.4 Hz), 1.46-1.64 (2H, m), 2.75-2.82 (1H, m), 3.94-4.04 (1H, m), 5.78 (1H, d, J = 2.4 Hz), 6.41 (1H, s), 6.65 (1H, d, J = 7.6 Hz), 6.70 (1H, m) td, J = 7.6, 1.3 Hz), 6.82 (1H, d, J = 2.4 Hz), 6.90 (1H, d, J = 2.4 Hz), 7.19 (1H, td, J = 7.6, 1.3 Hz), 7.75 ( 1H, dd, J = 7.6, 1.3 Hz), 8.49 (1H, s)

2- (5-Chloro-3,4-dihydroxyphenyl) -3-isopropyl-1,2,3,4-tetrahydroquinazoline-4-one (NRA-808)
¹ H-NMR (400 MHz, DMSO-d6) δ: 1.00 (3H, d, J = 6.9 Hz), 1.26 (3H, d, J = 6.9 Hz), 4.73 (1H, sept, J = 6.9 Hz), 5.82 (1H, d, J = 2.0 Hz), 6.40 (1H, s), 6.58 (1H, d, J = 7.6 Hz), 6.73 (1H, t, J = 7.6 Hz), 6.81 (1H, d, J) = 2.0 Hz), 6.89 (1H, d, J = 2.0 Hz), 7.14 (1H, td, J = 7.6, 2.0 Hz), 7.43 (1H, dd, J = 7.6, 2.0 Hz), 8.43 (1H, br) )
3-Benzyl-2- (5-chloro-3,4-dihydroxyphenyl) -1,2,3,4-tetrahydroquinazoline-4-one (NRA-809)
¹ H-NMR (400 MHz, DMSO-d6) δ: 3.78 (1H, d, J = 15.6 Hz), 5.48 (1H, d, J = 15.6 Hz), 5.63 (1H, d, J = 1.8 Hz), 6.37 (1H, s), 6.67 (1H, d, J = 7.2 Hz), 6.75 (1H, t, J = 7.2 Hz), 6.79 (1H, d, J = 1.8 Hz), 6.83 (1H, d, J) = 1.8 Hz), 7.20-7.32 (6H, m), 7.84 (1H, dd, J = 7.2, 1.8 Hz), 8.48 (1H, br)

2- (5-Chloro-3,4-dihydroxyphenyl) -3-phenethyl-1,2,3,4-tetrahydroquinazoline-4-one (NRA-810)
¹ H-NMR (400 MHz, DMSO-d6) δ: 2.76-2.79 (1H, m), 2.92-3.07 (2H, m), 4.05-4.11 (1H, m), 5.71 (1H, d, J = 2.0) Hz), 6.34 (1H, s), 6.66 (1H, d, J = 7.5 Hz), 6.72 (1H, t, J = 7.5 Hz), 6.82 (1H, d, J = 2.0 Hz), 6.89 (1H, 1H, d, J = 2.0 Hz), 7.15-7.28 (6H, m), 7.79 (1H, dd, J = 7.5, 2.0 Hz), 8.54 (1H, br)

2- (5-Chloro-3,4-dihydroxyphenyl) -3- (3-phenylpropyl) -1,2,3,4-tetrahydroquinazoline-4-one (NRA-811)
¹ H-NMR (400 MHz, DMSO-d6) δ: 1.81-2.00 (2H, m), 2.62 (2H, t, J = 7.6 Hz), 2.84-2.91 (2H, m), 3.92-3.99 (1H, m), 5.78 (1H, d, J = 1.8 Hz), 6.66 (1H, d, J = 7.7 Hz), 6.71 (1H, t, J = 7.7 Hz), 7.13 (1H, t, J = 7.7 Hz) , 7.17-7.26 (5H, m), 7.78 (1H, d, J = 7.7 Hz), 8.56 (1H, br)

2- (5-Chloro-3,4-dihydroxyphenyl) -3- (4-phenylbutyl) -1,2,3,4-tetrahydroquinazoline-4-one (NRA-812)
¹ H-NMR (400 MHz, DMSO-d6) δ: 1.62-1.65 (5H, m), 2.03-2.08 (1H, m), 2.60-2.61 (1H, m), 3.98-4.05 (1H, m), 5.75 (1H, J = 2.7 Hz), 6.64 (1H, J = 7.7 Hz), 6.79 (1H, d, J = 1.8 Hz), 6.87 (1H, d, J = 1.8 Hz), 7.10-7.25 (6H, m), 7.77 (1H, dd, J = 7.7, 1.8 Hz), 8.52 (2H, br)

2- (5-Chloro-3,4-dihydroxyphenyl) -3-(3- (p-methoxyphenyl) propyl) -1,2,3,4-tetrahydroquinazoline-4-one (NRA-813)
¹ H-NMR (400 MHz, Acetone-d6) δ: 1.79-1.98 (2H, m), 2.55 (2H, t, J = 8.0 Hz), 2.82-2.90 (1H, m), 3.73 (3H, s) , 3.88-3.96 (1H, m), 5.77 (1H, d, J = 1.6 Hz), 6.66 (1H, d, J = 8.0 Hz), 6.70 (1H, t, J = 8.0 Hz), 6.78-6.82 ( 3H, m), 6.87 (1H, d, J = 2.4 Hz), 7.08 (1H, d, J = 8.0 Hz), 7.20 (1H, td, J = 1.6 Hz), 7.77 (1H, dd, J = 8.0) , 1.6 Hz)

2- (5-Chloro-3,4-dihydroxyphenyl) -3-(3- (p-fluorophenyl) propyl) -1,2,3,4-tetrahydroquinazoline-4-one (NRA-814)
¹ H-NMR (400 MHz, Acetone-d6) δ: 1.80-2.00 (2H, m), 2.63 (2H, t, J = 8.0 Hz), 2.82-2.92 (1H, m), 3.91-3.98 (1H, m), 5.78 (1H, d, J = 2.4 Hz), 6.66 (1H, d, J = 8.0 Hz), 6.72 (1H, t, J = 8.0 Hz), 6.81 (1H, d, J = 2.4 Hz) , 6.88 (1H, d, J = 2.4 Hz), 7.00 (2H, t, J = 8.0 Hz), 7.19-7.24 (3H, m), 7.79 (1H, dd, J = 8.0, 1.2 Hz), 8.12 ( 1H, s), 8.76 (1H, s)

2- (5-Chloro-3,4-dihydroxyphenyl) -3- (3- (p-chlorophenyl) propyl) -1,2,3,4-tetrahydroquinazoline-4-one (NRA-815) ¹ H- NMR (400 MHz, Acetone-d6) δ: 1.80-2.00 (2H, m), 2.63 (2H, t, J = 8.0 Hz), 2.83-2.94 (1H, m), 3.88-3.96 (1H, m), 5.79 (1H, d, J = 1.6 Hz), 6.40 (1H, s), 6.67 (1H, t, J = 8.0 Hz), 6.72 (1H, t, J = 8.0 Hz), 6.82 (1H, d, J) = 2.4 Hz), 6.88 (1H, d, J = 2.4 Hz), 7.18-7.28 (5H, m), 7.78 (1H, dd, J = 8.0, 1.2 Hz), 8.56 (1H, br)

[Example 2] Identification of a novel Nr4a1 antagonist candidate compound The present inventors aimed at acquiring a novel Nr4a1 antagonist, and based on the complex structure (PDB; 3V3Q) of Nr4a1 protein and a known antagonist TMPA, "3D Pharmaco". A multi-step in-silico screening consisting of "narrowing down based on hoa", "narrowing down by molecular docking simulation", and "molecular similarity analysis based on chemical properties" was performed, and 10 compounds (Namiki Shoji Co., Ltd.) were used from the existing compound database. (Purchased from) was identified as a novel Nr4a1 antagonist candidate compound. Then, primary screening was performed using the NBRE-Luc reporter vector (NBRE; Nr4a1 binding DNA domain). That is, 1 × 10 ⁵ CHO cells were seeded on a 96-well plate and incubated for 24 hours. The NBRE-Luc reporter vector and Nr4a1 expression vector were transfected and incubated for 6 hours. After the medium exchange, the candidate compound (10 compounds) and the already reported Nr4a1 antagonists TMPA and DIM-C-pPhOH were added to 20 μM and incubated for 18 hours. Then, luciferin was added, and the luminescence was measured with a microplate reader. In such a reporter assay, one species could be identified as a compound with antagonistic properties (named NRA-8). The Nr4a1 inhibitory effect of NRA-8 was comparable to or stronger than the previously reported Nr4a1 antagonists TMPA and DIM-C-pPhOH. The results of the reporter assay and the structure of NRA-8 are shown in FIG.

[Example 3] Drug efficacy evaluation 1 using a mouse pain model (mechanical allodynia induced by intrathecal administration of PACAP)
A single intraspinal spinal subarachnoid space (it) administration of PACAP (100 pmol / 5 μL) to mice caused hypersensitivity to mechanical stimuli (mechanical allodynia) over a long period of time (at least 3 months after administration) (at least 3 months after administration). Yokai et al. Mol. Pain 2016. 12, 1-13.). The onset of this PACAP-induced long-term mechanical hypersensitivity phenomenon was almost completely suppressed by co-administration of NRA-8 (1 nmol) (Fig. 2). The known Nr4a1 antagonists TMPA and DIM-C-pPhOH used as controls also suppressed the onset, but the effect was most potent with NRA-8 (Fig. 2). In the example in which a solvent (VEH: 0.2% DMSO-containing artificial cerebrospinal fluid) was used instead of the compound, the PACAP-induced mechanical hypersensitivity phenomenon was not suppressed.

Mice used male ddY (6-12 weeks old) and the threshold for mechanical stimulation was evaluated by the vonFrey test. That is, the 50% escape reflex threshold (Threshold) was calculated according to the method of Chaplan et al. (Chaplan et al., J. Neurosci. Meth. 53, 55-63, 1994). The compound was prepared by dissolving in 99.7% DMSO and then diluting with artificial cerebrospinal fluid (final concentration of DMSO is 0.2%).

[Example 4] Drug efficacy evaluation 2 using a mouse pain model (peripheral neuropathic pain model: mechanical allodynia induced by spinal nerve injury)
A spinal nerve ligation (SNL) model was used in which the fourth lumbar spinal nerve of male ddY (6-12 weeks old) was ligated with silk thread. The efficacy evaluation was performed 14 days after the nerve ligation. NRA-8 (100 pmol and 1 nmol) i. t. Administration dose-dependently suppressed mechanical allodynia induced by nerve ligation (Fig. 3). On the other hand, in the example using the solvent (VEH: artificial cerebrospinal fluid containing 0.2% DMSO), mechanical allodynia was not suppressed.

[Example 5] Drug efficacy evaluation 3 using a mouse pain model (bone cancer pain model: mechanical allodynia induced by transplantation of cancer cells into the femur)
The effect of the drug on mechanical allodynia evoked by transplanting NCTC2472 cells into the femoral bone marrow (from the knee joint site) of male C3H / HeN mice (6-12 weeks old) was investigated. NRA-8 was orally administered on the 10th day of cancer cell transplantation. Oral administration of NRA-8 (10 and 30 mg / kg) dose-dependently suppressed the mechanical allodynia induced by cancer cell transplantation (FIG. 4). On the other hand, in the example using the solvent (VEH: distilled water containing 0.1% DMSO), mechanical allodynia was not suppressed.

[Example 6] Anti-cancer activity against human pancreatic cancer cell line Panc1 When the anti-cancer activity against human pancreatic cancer cell line Panc1 was investigated, the novel Nr4a1 antagonist NRA-8 was found to be the proliferation and survival of Panc1 cells. The migration was suppressed (Fig. 5). On the other hand, the existing analgesics (aspirin (acetylsalicylic acid), diclofenac, pregabalin) did not have anticancer activity (cell proliferation inhibitory effect) (Fig. 6). NRA-8 also has similar anti-cancer in various cancer cell lines such as A549 cells (human lung cancer), U87 and U251 (human brain tumor), LNCaP (human prostate cancer), MCF7 (human breast cancer). Showed activity.

Furthermore, the present inventors have developed the structure based on the structure of NRA-8 with the aim of obtaining a compound showing stronger activity than NRA-8. In the screening using the NBRE-Luc reporter vector (NBRE; Nr4a1 binding DNA domain), NRA-811 was successfully obtained as a compound having an efficacy equal to or higher than that of the original compound NRA-8. Further, when the structure of the compound having stronger activity was developed based on the structure of NRA-811 and the reporter assay was performed, the compounds NRA-813, 814 and 815 having stronger activity than NRA-811 were obtained. succeeded in.

Figure 7 shows the evaluation of the compound of the present invention by the reporter assay.

It showed a stronger analgesic effect than NRA-8 on the PACAP-induced long-term pain model, sciatic nerve ligation neuropathic pain model, and osteocancer pain model (Fig. 8).

In addition, NRA-811 and 815 showed suppression of cell proliferation, survival, and migration in the human pancreatic cancer cell line Panc1, and the effects were stronger than those of the original compound (NRA-8) (FIG. 9). NRA-811 and 815 also have similar anti-cancer cells in various cancer cell lines such as A549 cells (human lung cancer), U87 and U251 (human brain tumor), LNCaP (human prostate cancer), MCF7 (human breast cancer). Showed cancer activity.

[Example 7] Anticancer activity in a Xenograft model (cancer cell transplantation model) Using male nude mice (Balb / c nu / nu, 6 weeks old), human pancreatic cancer cell line Panc1 (6 weeks old) was subcutaneously placed on the right flank. 1 × 10 ⁶ ) were transplanted. The NRA compound was suspended in water containing 0.3% methylcellulose and orally administered once a day for 7 days from the 7th day of cancer cell transplantation. The size of the tumor was measured by measuring the major axis (mm) and the minor axis (mm) of the tumor using a caliper, and the volume was calculated as major axis (mm) × minor axis (mm) × minor axis (mm).

The tumor growth inhibitory effect of NRA-8 was not strong in the Xenograft model, but the compounds of the present invention, NRA-811 and 815, showed a stronger tumor growth inhibitory effect than NRA-8 (FIG. 10).

All publications, patents and patent applications cited in this specification shall be incorporated herein by reference as they are.

Claims (7)

1. Equation (I):
   

   

   (In the formula, R ¹ is a hydroxyl group, a mercapto group, a C _1-6 -alkoxy group, a C _1-6 -haloalkoxy group, an amino group, a C _1-6 -alkylamino group or a diC _1-6 -alkylamino group. R ² is a hydroxyl group, a mercapto group, a C _1-6 -alkoxy group, a C _1-6 -haloalkoxy group, an amino group, a C _1-6 -alkylamino group or a diC _1-6 -alkylamino group. Yes; R ³ is a halogen atom, nitro group, carboxyl group, C _1-6 -alkoxy-carbonyl group or carboxy-C _1-6 -alkyl group; R ⁴ is substituted or unsubstituted C _3-12 -carbonated It is a hydrogen group.)
   A compound represented by (1) or a salt thereof or a solvate thereof.
2. The compound according to claim 1, a salt thereof, or a solvate thereof, ^{wherein R 1} and R ² are hydroxyl groups in the formula (I).
3. In the formula (I), ^{R 4} is a substituted or unsubstituted _{C 3-12} - alkyl group, or a substituted or unsubstituted _{C 7-12} - compound of claim 1 or 2, wherein an aralkyl group or a salt thereof, or Those solvates.
4. An analgesic containing the compound according to any one of claims 1 to 3, a salt thereof, or a solvate thereof.
5. The analgesic according to claim 4, which is applied to a cancer patient.
6. Equation (II):
   

   

   ^(Wherein, R 1, ^{R 2} and ^{R 3} are as defined in the formula of claim ^{1 (I); R 4 '} C 2-12 substituted or unsubstituted is - hydrocarbon group.)
   An analgesic containing the compound represented by (1) or a salt thereof or a solvate thereof.
7. The analgesic according to claim 6, which is applied to a cancer patient.

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