KR100348819B1 - vanilloid analogues containing resiniferatoxin pharmacophores as potent vanilloid receptor agonists and analgesics and the pharmaceutical compositions containing the same - Google Patents

Abstract

The present invention relates to novel vanilloid homologues containing resiniferatoxin pharmacophores and used as agonists and analgesics of potent vanilloid receptors and pharmaceutical compositions containing them. The present invention provides pharmaceutical compositions for the treatment of acute, chronic, inflammatory or neuropathic pain or for the treatment of bladder hypersensitivity.

Description

Vanilloid analogues containing resiniferatoxin pharmacophores as potent vanilloid receptor agonists and analgesics and the pharmaceutical compositions containing the same}

The present invention relates to novel vanilloid homologs for use as agonists and analgesics of potent vanilloid receptors containing resiniferatoxin pharmacophores and pharmaceutical compositions containing them. The present invention provides pharmaceutical compositions for the treatment of acute, chronic, inflammatory or neurological pain.

Capsaicin with the following structure stimulates sensory afferent nerve C-fibers to desensitize. Induced desensitization can be applied to allergic reactions including arthritis, asthma, rhinitis, fever, pain, bladder hypersensitivity, etc.

Moreover, vanilloid receptors (or capsaicin receptors) are specific neuronal membrane recognition sites for capsaicin and related stimulating compounds. It is expressed exclusively by the first neuronal neurons involved in nociception and neuronal inflammation. Receptor action as a cation-selective ion channel with an advantage over calcium and its action subtype VR1, activated by CAP and noxious heat, has recently been cloned. Its desensitization by specific ligands is recognized to allow therapeutic approaches to alleviate neuropathic pain and other pathological conditions in which neuropeptides released from primary neurons play an important role.

Most of the exogenous vanilloid receptor agonists developed or used as analgesics are structurally capsaicin (ie, Zostrix ^TM , Olvanil ^TM , SDZ-249482 ^TM and DA-5018 ^TM ) and Resinifera. It is related to toxins. Their structure usually includes a byloid ring that can exhibit activity as an agonist. However, recent reports suggest that compounds without vanilloid moieties, such as sesquaterpenoid unsaturated dialdehydes or triprenylphenols, can activate the receptor. Although there are few agonists of receptors, some such as capsazepine, channel blocker ruthenium red and capsazocaine that can compete competitively at capsaicin binding sites Compounds have been reported.

Resiniferatoxin (hereinafter referred to as 'RTX'), which has the following structure, is a tricyclic diterpene obtained from a medicinal plant called Euphorbia resinifera, which belongs to the cactus family, and is a powerful capsaicin homolog. It is a sieve.

In addition, RTX specific binding to capsaicin binding sites in the dorsal root ganglia is represented by labeled [ ³ H] RTX. RTX has been developed as a very powerful neuronal desensitizer for the treatment of pain associated with urinary urge incontinence and diabetic neuropathy. Recently, a full enantiomeric controlled synthesis and conformational analysis of RTX has been reported, however, in the structural activity studies, the C ₂₀ -homovanic moiety, C ₃ -keto group and ortho-ester phenyl group of the C ring have been described. Although it has been suggested that it is an important structural element in showing very potent efficacy, the pharmacophoric group of RTX is not yet clearly defined. On the other hand, the relatively lower efficacy of CAP than RTX can be explained by the lack of any of the above pharmacological groups, especially the C ₃ -keto group.

U.S. Patent No. 4,939,149 discloses desensitizing animals by administering RTX in an amount effective to treat desensitization to desensitize animals with neurological inflammation, chemically and thermally induced pain, and CAP-sensitive sensory afferent nerve pathways. The method of making is disclosed.

Many vanilloid agonists based on the structure of CAP and RTX have been reported as possible analgesics (eg, 12-dioxyphorball 13-phenylacetate 20-homovanylate and mesmerized with RTX in US Pat. No. 5,021,450). Homovanylylditerpene derivatives such as mezerein 20-homovanylate have been disclosed), these CAP type homologues are limited due to their low efficacy and narrow therapeutic index. On the other hand, since RTX is obtained from raw materials, there is a limit in usability, and due to their structural complexity, RTX has a problem that is difficult to be synthesized.

The inventors of the present invention sought to develop novel analgesics based on vanilloid receptors, which are simpler in structure than RTX or known RTX or CAP homologues. As a result, strong vanilloid agonists in receptor binding assays and CAP-active single channel assays have been studied. The present invention has been accomplished by discovering new compounds in which modifications have been made to the C ₂₀ -homovanic moiety, C ₃ -carbonyl and ortho-ester phenyl moieties as essential groups for recognition and binding to exhibit activity.

Therefore, the object of the present invention is a novel compound represented by the following general formula (I);

(Ⅰ)

(In the above meal,

X is an oxygen atom or a sulfur atom;

A is -NHCH ₂ -or -CH ₂ ;

R ₁ is an alkylaryl group having 1 to 4 carbon atoms or R ₄ CO—;

(In formula, R <_4> is a C1-C18 alkyl group, a C2-C18 alkenyl group, or a C6-C10 aryl group.)

R ₂ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, or a halogen atom;

R ₃ is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aminoalkyl group, a diacid monoester group, or an α-alkylacid group; And

* Represents a chiral carbon atom.)

And pharmaceutically acceptable salts thereof.

Another object of the present invention is to provide a method for preparing the compound (I).

It is another object of the present invention to provide a pharmaceutical composition containing, as an active ingredient, the compound of formula (I) together with a pharmaceutically acceptable carrier in an amount effective to alleviate pain.

The compound of the present invention is a compound represented by the following general formula (I);

(Ⅰ)

(In the above meal,

X is an oxygen atom or a sulfur atom;

A is -NHCH ₂ -or -CH ₂ ;

R ₁ is an alkylaryl group having 1 to 4 carbon atoms or R ₄ CO—;

(In formula, R <_4> is a C1-C18 alkyl group, a C2-C18 alkenyl group, or a C6-C10 aryl group.)

R ₂ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms or a halogen atom;

R ₃ is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aminoalkyl group, a diacid monoester group, or an α-alkylacid group; And

* Represents a chiral carbon atom.)

And pharmaceutically acceptable salts thereof.

Preferred compounds of the present invention are compounds represented by the following general formulas (I-a) and (I-b).

(Ia)

(Ib)

Wherein R ₁ , R ₂ and R ₃ are as defined above.

In a preferred embodiment of the compound of formula (Ia) or (Ib), R ₁ is R ₄ CO- (R ₄ is an alkyl group having 1 to 18 carbon atoms), R ₂ is an alkyl group having 1 to 6 carbon atoms, R ₃ is — (CH ₂ ) _n NH ₂ , —CO (—CH ₂ ) _n COOH or — (CH ₂ ) _n COOH (n is an integer from 1 to 4). On the other hand, when R ₁ or R ₄ is a substituted aryl group, the substituent is one or more lower alkyl groups or halogen atoms of 1 to 4 carbon atoms.

A more preferred embodiment is the compound of formula (Ia) or (Ib), wherein R ₁ is R ₄ CO- (R ₄ is an alkyl group having 1 to 6 carbon atoms), R ₂ is an alkyl group having 1 to 6 carbon atoms , R ₃ is — (CH ₂ ) ₂ NH ₂ , —CO (CH ₂ ) ₂ COOH or — (CH ₂ ) COOH.

In all cases, compound (I) is a racemic mixture or is a R or S type stereoisomer. The present invention also includes compound (I) in the form of a pharmaceutically acceptable salt.

The compounds of the present invention may be chemically synthesized by the method shown in the following schemes, but are not limited to these examples. The following schemes represent steps for the preparation of representative compounds of the present invention. Other compounds may be prepared by appropriate changes in the reagents and starting materials known to those skilled in the art.

[Common Synthesis Method]

The synthesis method of 3-acyloxy-2-benzylpropyl thiourea derivatives (7a-d) is represented by Schemes 1 and 2. Diethylmalonate is monoalkylated with various benzyl halides and then reduced with LiAlH ₄ to prepare diol (2a-d) compounds. Monoacetylation of compound (2), conversion of the alcohol remaining in the azide and deprotection of the acetyl group provide intermediate 3-azido-2-benzyl-1-propanol (5a-d). Compound (5a-d) was acylated with various acyl halides to prepare ester compound (6a-d), followed by reduction of the azide group, with 4-methoxymethyloxy-3-methoxybenzylisothiocyanate. Condensation and hydrogenation gave the final product (7a-d). Other ether homologues represented by 3-benzyloxy-2-benzylpropyl thiourea derivatives 9a, c are synthesized from compounds 5a, c, as shown in Scheme 2. Two chiral homologues of compound (7a-I) are enantioselective enzymatic by the selective isomeric enzyme of the lachemic of 2-benzyl-1,3-propanediol (Scheme 3) in the same manner as in the synthesis of 7a-I. It is prepared from (R) -3-acetoxy-2-benzyl-1-propanol ((R) -3a) obtained by acetylation).

O-acetic acid homologs (19a, b) of the thiourea derivatives are prepared by the method shown in Scheme 4. Compound 4-12 was prepared by protecting 4-hydroxy-3-methoxy-benzonitrile (11) with a methylmethylether (MOM) group, and reducing the cyano group of compound 12 to amine using LiAlH ₄ , followed by chlorobenzyl ( Compound 13 is prepared by protecting with a CBZ) group. Compound 14 is prepared by deprotecting MOM group of compound 13, and compound 15 is prepared by alkylating hydroxy group of compound 14 with methylbromoacetate. The methyl ester compound 15 was hydrogenated to prepare compound 16, and the CBZ group of compound 16 was deprotected to prepare compound 17. The amine compound 17 is condensed with isothiocyanate 18a, b to obtain the final product 19a, b, respectively.

O-succinic ester homologs (23a, b) of the thiourea derivatives are prepared by the method shown in Scheme 5. Compound 21 is prepared by acylating monobenzyl ester 20 of succinic acid with vanilloid 14, and compound 22 is prepared by deprotecting the benzyl group of compound 21 by hydrogenation. The amine compound 22 is condensed with isothiocyanate 18a, b to give 23a, b, the final product, respectively.

O-aminoethyl homologs (28a, b) of the amide derivatives are prepared by the method shown in Scheme 6. Compound 25 is prepared by reducing the azido group of compound 24 to amine followed by condensation of the amine with homovanic pentafluoro ester. Compound 26 is prepared by alkylating the hydroxy group of compound 25 with dibromoethane, and compound 27 is prepared by substituting the bromo group of compound 26 with an azido group. Compounds 27a, b are reduced to give 28a, b, the final product, respectively.

O-aminoethyl homologs (36a, b) of the thiourea derivatives are prepared by the method shown in Scheme 7. Compound 30 is prepared by protecting the amine group of 4-hydroxy-3-methoxybenzylamine compound 29 with a Boc group, followed by alkylation of the hydroxy group of compound 30 with dibromoethane. Compound 32 is prepared by replacing the bromo group of compound 31 with an azido group, and compound 33 is prepared by deprotecting the N-Boc group of compound 32. The amine group of the amine compound 33 is converted to isothiocyanate to prepare compound 34, and the compound 34 is condensed with azide compound 6 to prepare thiourea compounds 35a and b. The azido groups of compounds 35a, b are reduced to give 36a, b, the final product, respectively.

Compounds (I) of the present invention may provide a pharmaceutical composition with a pharmaceutically acceptable carrier, adjuvant or diluent. For example, the compounds of the present invention can be dissolved in oil, propylene glycol or other solvents commonly used in the preparation of injection solutions. Suitable carriers are not particularly limited, but examples thereof include physiological saline, polyethylene glycol, ethanol, vegetable oils, and isopropyl myristate. For topical application, the compounds of the present invention may be formulated in ointments or creams.

Pharmaceutical compositions comprising the compounds of the invention as active ingredients can be used in the following cases.

1) Posttherpetic neuralgia, diabetic neuropathy, postmastectomy pain syndrome, stem pain, reflex sympathetic dytrophy, trigeminal neuralgia ), Oral neuropathic pain, osteoarthritis, rheumatoid arthritis, fibromyalgia, Guillain-Barre syndrome, meralgia paraesthetica Relief of pain caused by burning mouth syndrome

2) amelioration of pain, such as difficult treatment, caused by bilateral peripheral neuropathy

Psoriasis, hemodiasis, aquagenic pruritus, vulvar vestibulitis, notalgia paraesthetica, brachioradial prutitus, schizophrenia Relief of itching caused by Lichen simplex chronicus)

4) Treatment of cluster headache, vasomotor rhinitis or perennial allergic rhinitis in the form of intranasal drip.

5) Treatment of bladder hypersensitivity or spinal detrusor hyperreflexia in the form of intra bladder solution.

The compounds of the present invention have analgesic and anti-inflammatory activity, and the pharmaceutical compositions of the present invention can be used to relieve or alleviate acute, chronic, inflammatory or neuropathic pain, to inhibit inflammation, or to treat urinary incontinence. .

Hereinafter, the formulation method and the excipient will be described, but are not limited only to these examples.

Pharmaceutically formulated forms of the compounds of the invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

The compounds of the present invention may be prepared by dissolving, suspending or emulsifying the compound in a conventional saline solution, a water-soluble solvent such as 5% textulose or a non-aqueous solvent such as vegetable oil, synthetic fatty acid glyceride, higher fatty acid ester or propylene glycol. It can be formulated as. Formulations of the present invention may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

Preferred dosages of the compounds of the invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg body weight per day, preferably at 0.001 to 100 mg / kg body weight. Administration may be administered once a day or may be divided several times. The compound of the present invention in the composition should be present in an amount of 0.0001 to 10% by weight, preferably 0.001 to 1% by weight based on the total weight of the total composition.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

EXAMPLE

General Synthesis of Compounds (Ia-d)

Diethylmalonate (6.4 g, 40 mmol) was dissolved in dimethylformamide (DMF, 20 mL), cooled to 0 ° C, and sodium hydride (60%, 1.92 g, 48 mmol) was added dropwise, at room temperature. Stir for 40 minutes. Benzyl chloride (48 mmol) was added to the reaction mixture, which was then stirred at room temperature for 24 hours, diluted with water, and extracted several times with ethyl acetate. The extracted organic layer was washed with water and brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (developing solvent: ethyl acetate / hexane = 1: 10) to give compound 1.

Example 1 Diethyl 3,4-dimethylbenzyl malonate (1b)

Yield: 70%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ6.9-7.05 (m, 3H), 4.14 (m, 4H, 2 × COOCH ₂ ), 3.61 (t, 1H, CH), 3.25 (d, 1H, CH ₂ Ar), 3.14 (d, 1H, CH ₂ Ar), 2.2-2.25 (m, 6H, 2 × CH ₃ ), 1.21 (m, 6H, 2 × COOCH ₂ CH ₃ )

Example 2 Diethyl 4-chlorobenzylmalonate (1c)

Yield: 74%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.25 (d, 2H), 7.16 (d, 2H), 4.14 (m, 4H, 2 × COOCH ₂ ), 3.60 (t, 1H, CH), 3.18 (d, 2H, CH ₂ Ar), 1.21 (t, 6H, 2 x COCH ₂ CH ₃ )

Example 3 Diethyl 4-t-butylbenzylmalonate (1d)

Yield: 74%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.29 (d, 2H), 7.13 (d, 2H), 4.16 (q, 4H, 2 × COOCH ₂ ), 3.63 (t, 1H, CH), 3.18 (d, 2H, CH ₂ Ar), 1.30 (s, 9H, C (CH ₃ ) ₃ ), 1.20 (t, 6H, 2 × COOCH ₂ CH ₃ )

General Synthesis of Compounds (2a-d)

Lithium aluminum hydride (3.64 g, 96 mmol) was suspended in diethyl ether (80 mL), cooled to 0 ° C., and a solution of compound 1 (24 mmol) dissolved in diethyl ether (20 mL) was added dropwise. After stirring at room temperature for 3 hours, the reaction mixture was cooled to 0 ° C., and stirred in an order of 3.5 ml of water, 7 ml of 15% sodium hydroxide, and 10.5 ml of water in that order. The mixture was washed with ethyl acetate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (developing solvent: ethyl acetate: hexane = 3: 1) to obtain compound 2.

Example 4 2-benzyl-1,3-propanediol (2a)

yield; 98%

Appearance; White solid

mp; 67 ℃

¹ H NMR (CDCl ₃ ) δ 7.15-7.30 (m, 5H, phenyl), 3.74 (dd, 2H, CH ₂ OH), 3.62 (dd, 2H, CH ₂ OH), 2.9-3.0 (bs, 2H , OH), 2.58 (d, 2H, CH ₂ Ph), 2.03 (m, 1H, CH).

Example 5 2- (3,4-dimethylbenzyl) -1,3-propanediol (2b)

yield; 95%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 6.88-7.06 (m, 3H, phenyl), 3.6-3.8 (m, 4H, 2 × CH ₂ OH), 2.5-2.7 (bs, 2H, OH), 2.60 (d, 1H, CH ₂ Ph), 2.52 (d, 1H, CH ₂ Ph), 2.2-2.28 (m, 6H, 2 x CH ₃ ), 2.02 (m, 1H, CH)

Example 6 2- (4-chlorobenzyl) -1,3-propanediol (2c)

yield; 75%

Appearance; White solid

mp = 64 ° C.

¹ H NMR (CDCl ₃ ) δ7.24 (d, 2H), 7.10 (d, 2H), 3.75 (dd, 2H, CH ₂ OH), 3.61 (dd, 2H, CH ₂ OH), 2.85 (bs, 2H , OH), 2.58 (d, 2H, CH ₂ Ph), 1.97 (m, 1H, CH)

Example 7 2- (4-t-butylbenzyl) -1,3-propanediol (2d)

yield; 80%

Appearance; White solid

mp = 74 ° C.

¹ H NMR (CDCl ₃ ) δ 7.31 (d, 2H), 7.11 (d, 2H), 3.81 (dd, 2H, CH ₂ OH), 3.68 (dd, 2H, CH ₂ OH), 2.58 (d, 2H , CH ₂ Ph), 2.23 (bs, 2H, OH), 2.05 (m, 1H, CH), 1.30 (s, 9H, C (CH ₃ ) ₃ )

General Synthesis of Compounds (3a-d)

Compound 2 (20 mmol), trimethylorthoacetate (3.6 g, 30 mmol) and a catalytic amount of p-toluenesulfonic acid were dissolved in methylene chloride (40 mL), stirred at room temperature for 2 hours, and then water (0.54 g, 30 mmol) was added thereto. gave. The reaction mixture was stirred at room temperature for 24 hours, concentrated under reduced pressure, and purified by column chromatography (developing solvent: ethyl acetate / hexane = 1: 2) to obtain compound 3.

Example 8 2-benzyl-3-hydroxypropyl acetate (3a)

yield; 97%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.1-7.25 (m, 5H, phenyl), 4.06 (ddd of AB, 2H, CH ₂ OAc), 3.48 (m, 2H, CH ₂ OH), 2.58 (ddd of AB, 2H, CH ₂ Ph), 2.10 (m, 1H, CH), 2.01 (s, 3H, COCH ₃ )

Example 9 (R) -2-benzyl-1,3-propanediol ((R) -3a)

This compound was prepared from Compound 2a by the method disclosed in Tetrahedron Letters 30, 6189-6192 (1989).

Example 10 2- (3,4-dimethylbenzyl) -3-hydroxypropyl acetate (3b)

yield; 90%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 6.88-7.06 (m, 5H, phenyl), 4.13 (m, 2H, CH ₂ OAc), 3.54 (m, 2H, CH ₂ OH), 2.62 (m, 2H, CH ₂ Ph), 2.2-2.3 (m, 6H, 2 x CH ₃ ), 2.10 (m, 1H, CH), 2.07 (s, 3H, COCH ₃ )

Example 11 2- (4-chlorobenzyl) -3-hydroxypropyl acetate (3c)

yield; 86%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.25 (d, 2H), 7.12 (d, 2H), 4.12 (ddd of AB, 2H, CH ₂ OAc), 3.53 (ddd of AB, 2H, CH ₂ OH), 2.63 (ddd of AB, 2H, CH ₂ Ph), 2.10 (m, 1H, CH), 2.06 (s, 3H, COCH ₃ )

Example 12 2- (4-t-butylbenzyl) -3-hydroxypropyl acetate (3d)

yield; 84%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.30 (d, 2H), 7.11 (d, 2H), 4.13 (ddd of AB, 2H, CH ₂ OAc), 3.56 (ddd of AB, 2H, CH ₂ OH), 2.62 (ddd of AB, 2H, CH ₂ Ph), 2.28 (s, 1H, OH), 2.10 (m, 1H, CH), 2.01 (s, 3H, COCH ₃ ), 1.30 (s, 9H, C (CH ₃₎ ) ₃ )

General Synthesis of Compounds (4a-d)

A solution of compound 3 (12 mmol) and triethylamine (3.64 g, 36 mmol) in methylene chloride (20 mL) was cooled to 0 ° C., and methanesulfonylchloride (2.06 g, 18 mmol) was added dropwise. After stirring at room temperature for 6 hours, the reaction mixture was diluted with methylene chloride, the organic layer was washed with 1N hydrochloric acid, water and brine, dried over magnesium sulfate, and concentrated under reduced pressure. The residue was column chromatographed (developing solvent: ethyl acetate / hexane = 1: 3) to give the mesylated compound in the form of an oil. This mesylate was dissolved in DMF (10 mL), and sodium azide (2.2 g, 34 mmol) was added thereto, followed by stirring at 80 ° C. for 8 hours. The reaction mixture was diluted with water and then extracted several times with ethyl acetate. The organic layer was washed with water, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (developing solvent: ethyl acetate / hexane = 1:15) to give compound 4.

Example 13 3-azido-2-benzylpropyl acetate (4a)

yield; 92%,

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.15-7.35 (m, 5H, phenyl), 4.05 (ddd of AB, 2H, CH ₂ OAc), 3.34 (m, 2H, CH ₂ N ₃ ), 2.68 (d, 2H , CH ₂ Ph), 2.22 (m, 1H, CH), 2.08 (s, 3H, COCH ₃ )

Example 14 3-azido-2- (3,4-dimethylbenzyl) propyl acetate (4b)

yield; 85%,

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ6.87-7.07 (m, 3H), 4.04 (m, 2H, CH ₂ OAc), 3.33 (m, 2H, CH ₂ N ₃ ), 2.69 (d, 1H, CH ₂ Ph ), 2.60 (d, 1H, CH ₂ Ph), 2.1-2.3 (m, 7H, 2 x CH ₃ & CH), 2.07 (s, 3H, COCH ₃ )

Example 15 3-azido-2- (4-chlorobenzyl) propyl acetate (4c)

yield; 88%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.26 (d, 2H), 7.12 (d, 2H), 4.03 (ddd of AB, 2H, CH ₂ OAc), 3.34 (m, 2H, CH ₂ N ₃ ), 2.65 ( d, 2H, CH ₂ Ph), 2.17 (m, 1H, CH), 2.07 (s, 3H, COCH ₃ )

Example 16 3-azido-2- (4-t-butylbenzyl) propyl acetate (4d)

yield; 92%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.32 (d, 2H), 7.08 (d, 2H), 4.05 (ddd of AB, 2H, CH ₂ OAc), 3.35 (ddd of AB, 2H, CH ₂ N ₃ ), 2.64 (d, 2H, CH ₂ Ph), 2.20 (m, 1H, CH), 2.07 (s, 3H, COCH ₃ ), 1.30 (s, 9H, C (CH ₃ ) ₃ )

General Synthesis of Compounds (5a-d)

Compound 4 (8 mmol) and a catalytic amount of potassium carbonate (K ₂ CO ₃ ) were dissolved in methanol (10 mL), followed by several drops of water, followed by stirring at room temperature for 2 hours. After 2 hours, a few drops of acetic acid were added to stop the reaction, and the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (developing solvent: ethyl acetate / hexane = 1: 3) to obtain compound 5.

Example 17 3-azido-2-benzyl-1-propanol (5a)

yield; 98%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.15-7.35 (m, 5H, phenyl), 3.65 (m, 2H, CH ₂ OH), 3.40 (ddd of AB, 2H, CH ₂ N ₃ ), 2.67 (ddd of AB , 2H, CH ₂ Ph), 2.04 (m, 1H, CH)

Example 18 3-azido-2- (3,4-dimethylbenzyl) -1-propanol (5b)

yield; 92%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 6.88-7.08 (m, 5H, phenyl), 3.65 (m, 2H, CH ₂ OH), 3.42 (m, 2H, CH ₂ N ₃ ), 2.68 (dd, 1H, CH ₂ Ph), 2.60 (dd, 1H, CH ₂ Ph), 2.2-2.3 (m, 6H, 2 x CH ₃ ), 2.03 (m, 1H, CH)

Example 19 3-azido-2- (4-chlorobenzyl) -1-propanol (5c)

yield; 88%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.25 (d, 2H), 7.10 (d, 2H), 3.58 (m, 2H, CH ₂ OH), 3.36 (m, 2H, CH ₂ N ₃ ), 2.63 (dd, 2H, CH ₂ Ph), 2.1 (bs, 1H, OH), 1.98 (m, 1H, CH)

Example 20 3-azido-2- (4-t-butylbenzyl) -1-propanol (5d)

yield; 98%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.32 (d, 2H), 7.10 (d, 2H), 3.63 (ddd of AB, 2H, CH ₂ OH), 3.39 (ddd of AB, 2H, CH ₂ N ₃ ), 2.62 (m, 2H, CH ₂ Ph), 2.02 (m, 1H, CH), 1.78 (bs, 1H, OH), 1.30 (s, 9H, C (CH ₃ ) ₃ )

General Synthesis of Compounds (6a-d)

Compound 5 (1 mmol), triethylamine (4 mmol) and a catalytic amount of DMAP (4-dimethylaminopyridine) were dissolved in methylene chloride (5 mL), cooled to 0 ° C., and then acyl chloride (2 mmol) was added thereto. After stirring at room temperature for 2 to 12 hours, the reaction mixture was diluted with methylene chloride, washed once with 1N hydrochloric acid, water and brine, and then dried over magnesium sulfate. The residue obtained by concentration under reduced pressure was purified by column chromatography (developing solvent: ethyl acetate / hexane = 1: 10 to 20) to obtain compound 6.

Example 21 3-Azido-2-benzylpropyl pivalate (6a-I)

yield; 92%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.15-7.35 (m, 5H, phenyl), 4.04 (ddd of AB, 2H, CH ₂ OCO), 3.34 (ddd of AB, 2H, CH ₂ N ₃ ), 2.68 (d , 2H, CH ₂ Ph), 2.21 (m, 1H, CH), 1.23 (s, 9H, CO (CH ₃ ) ₃ )

Example 22 3-Azido-2-benzylpropyl 2-methylpropanoate (6a-II)

yield; 93%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.15-7.35 (m, 5H, phenyl), 4.05 (ddd of AB, 2H, CH ₂ OCO), 3.34 (m, 2H, CH ₂ N ₃ ), 2.68 (d, 2H , CH ₂ Ph), 2.58 (m, CHMe ₂ ), 2.21 (m, 1H, CH), 1.18 (dd, 6H, 2 x CH ₃ )

Example 23 3-azido-2-benzylpropyl hexanoate (6a-III)

yield; 90%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.14-7.35 (m, 5H, phenyl), 4.05 (ddd of AB, 2H, CH ₂ OCO), 3.34 (m, 2H, CH ₂ N ₃ ), 2.68 (d, 2H , CH ₂ Ph), 2.32 (t, 2H, OOCCH ₂ ), 2.20 (m, 1H, CH), 1.64 (m, 2H), 1.2-1.4 (m, 4H), 0.88 (distorted t, 3H)

Example 24 3-azido-2-benzylpropyl stearate (6a-IV)

yield; 78%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.14-7.35 (m, 5H, phenyl), 4.05 (ddd of AB, 2H, CH ₂ OCO), 3.33 (m, 2H, CH ₂ N ₃ ), 2.68 (d, 2H , CH ₂ Ph), 2.32 (t, 2H, OOCCH ₂ ), 2.20 (m, 1H, CH), 1.2-1.7 (m, 30H), 0.88 (distorted t, 3H)

Example 25 3-azido-2-benzylpropyl benzoate (6a-V)

yield; 90%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 8.04 (m, 2H), 7.15-7.60 (m, 8H), 4.35 (dd, 1H, J = 5.1 and 11.2 Hz, BzOCH ₂ ), 4.26 (dd, 1H, BzOCH ₂ ), 3.43 (ddd of AB, 2H, J = 5.61, 5.85 and 12.42 Hz, CH ₂ N ₃ ), 2.78 (m, 2H, CH ₂ Ph), 2.35 (m, 1H, CH)

Example 26 3-azido-2- (3,4-dimethylbenzyl) propyl pivalate (6b-I)

yield; 94%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ6.86-7.07 (m, 3H), 4.04 (m, 2H, CH ₂ OCO), 3.36 (m, 2H, CH ₂ N ₃ ), 2.70 (d, 1H, CH ₂ Ph ), 2.61 (d, 1H, CH ₂ Ph), 2.1-2.3 (m, 7H, 2 × CH ₃ & CH), 1.23 (s, 9H, CO (CH ₃ ) ₃ )

Example 27 3-azido-2- (3,4-dimethylbenzyl) propyl benzoate (6b-V)

yield; 95%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 8.02 (m, 2H), 7.57 (m, 1H), 7.46 (m, 2H), 6.8-7.07 (m, 3H), 4.32 (m, 2H, CH ₂ OCO), 3.45 (m, 2H, CH ₂ N ₃ ), 2.80 (d, 1H, CH ₂ Ph), 2.71 (d, 1H, CH ₂ Ph), 2.2-2.4 (m, 7H, 2 x CH ₃ & CH)

Example 28 3-azido-2- (3,4-dimethylbenzyl) propyl 3,4-dimethylbenzoate (6b-VI)

yield; 84%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.77 (m, 2H), 7.21 (m, 1H), 6.9-7.07 (m, 3H), 4.30 (m, 2H, CH ₂ OCO), 3.44 (m, 2H, CH ₂ N ₃ ), 2.79 (d, 1H, CH ₂ Ph), 2.70 (d, 1H, CH ₂ Ph), 2.2-2.4 (m, 13H, 4 × CH ₃ & CH)

Example 29 3-azido-2- (4-chlorobenzyl) propyl pivalate (6c-I)

yield; 86%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.27 (d, 2H), 7.09 (d, 2H), 4.02 (ddd of AB, 2H, CH ₂ OCO), 3.33 (m, 2H, CH ₂ N ₃ ), 2.66 ( d, 2H, CH ₂ Ph), 2.18 (m, 1H, CH), 1.23 (s, 9H, CO (CH ₃ ) ₃ )

Example 30 3-azido-2- (4-chlorobenzyl) propyl benzoate (6c-V)

yield; 92%

Appearance; Colorless oil

¹ H HMR (CDCl ₃ ) δ 8.01 (m, 2H), 7.58 (m, 1H), 7.46 (m, 2H), 7.28 (d, 2H), 7.13 (d, 2H), 4.29 (ddd of AB, 2H, CH ₂ OCO), 3.43 (m, 2H, CH ₂ N ₃ ), 2.75 (d, 2H, CH ₂ Ph), 2.33 (m, 1H, CH)

Example 31 3-azido-2- (4-t-butylbenzyl) propyl pivalate (6d-I)

yield; 99%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.31 (d, 2H), 7.08 (d, 2H), 4.04 (ddd of AB, 2H, CH ₂ OCO), 3.35 (m, 2H, CH ₂ N ₃ ), 2.65 ( d, 2H, CH ₂ Ph), 2.20 (m, 1H, CH), 1.30 (s, 9H, C (CH ₃ ) ₃ ), 1.23 (s, 9H, CO (CH ₃ ) ₃ )

Example 32 3-azido-2- (4-t-butylbenzyl) propyl benzoate (6d-V)

yield; 88%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 8.04 (m, 2H), 7.57 (m, 1H), 7.45 (m, 2H), 7.32 (d, 2H), 7.12 (d, 2H), 4.31 (ddd of AB, 2H, CH ₂ OCO), 3.44 (m, 2H, CH ₂ N ₃ ), 2.75 (d, 2H, CH ₂ Ph), 2.35 (m, 1H, CH), 1.30 (s, 9H, C (CH ₃ ) ₃ )

General Synthesis of Compounds 7a-d

Compound 6 (0.5 mmol) and Lindler's catalyst (50 mg) were dissolved in ethanol (5 mL) and hydrogenated under a hydrogen balloon for 2 hours. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in methylene chloride and 4-[(methoxymethyl) oxy] -3-methoxybenzylisothiocyanate (0.5 mmol) was added thereto. After stirring for 24 hours at room temperature, the reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (developing solvent: ethyl acetate / hexane = 1: 1) to obtain thiourea. This thiourea was dissolved in methylene chloride (2 mL) and trifluoroacetic acid (1 mL) was added. After stirring for 1 hour at room temperature, the reaction was stopped by adding solid sodium bicarbonate to the mixture, filtered and the filtrate was concentrated. The residue was diluted with ethyl acetate, washed with sodium bicarbonate, water and brine and then concentrated under reduced pressure. The residue was purified by column chromatography (developing solvent: ethyl acetate / hexane = 1: 1) to give compound 7.

Example 33 2-benzyl-3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propyl pivalate (7a-I)

yield; 40%

Appearance: Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.1-7.32 (m, 5H, phenyl), 6.75-6.9 (m, 3H, Ar), 6.27 (bt, 1H, NH), 6.05 (bs, 1H, NH), 5.63 (s, 1H, OH), 4.40 (bd, 2H, J = 4.38 Hz, NHCH ₂ Ar), 4.17 (dd, 1H, J = 3.9 and 11.46 Hz, CH ₂ OCO), 3.87 (s, 3H, OCH ₃ ), 3.7-3.85 (m, 2H, CH ₂ OCO & CHCH ₂ NHC = S), 3.24 (ddd, 1H, J = 5.37, 8.07 and 13.89 Hz, CHCH ₂ NHC = S), 2.61 (ddd of AB, 2H) , CH ₂ Ph), 2.33 (m, 1H, CH), 1.23 (s, 9H, CO (CH ₃ ) ₃ )

IR (neat): 3362, 1715, 1278, 1157

MS m / e 445 (MH ⁺ )

analysis. (C ₂₄ H ₃₂ N ₂ O ₄ S) C, H, N, S

Example 34 (R) -2-benzyl-3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propyl pivalate ((R) -7a-I)

(R) -enantiomer of 7a-I: This compound was prepared from (R) -3a compound in the same manner as 7a-I.

[a] _d = −714 (c, 0.22, CHCl ₃ )

Example 34 (S) -2-benzyl-3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propyl pivalate ((S) -7a-I)

(S) -enantiomer of 7a-I: This compound was prepared from (R) -3a compound in the same manner as 7a-I.

[a] _d = + 695 (c, 0.08, CHCl ₃ )

Example 36 2-benzyl-3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propyl 2-methyl propanoate (7a-II)

yield; 33%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.13-7.30 (m, 5H, phenyl), 6.75-6.87 (m, 3H, Ar), 6.34 (bt, 1H, NH), 6.30 (bs, 1H, NH), 5.75 (s, 1H, OH), 4.40 (bs, 2H, NHCH ₂ Ar), 4.17 (dd, 1H, J = 3.9 and 10.95 Hz, CH ₂ OCO), 3.84 (s, 3H, OCH ₃ ), 3.65-3.85 (m, 2H, CH ₂ OCO & CHC H ₂ NHC = S), 3.28 (m, 1H, CHC H ₂ NHC = S), 2.60 (m, 3H, CH ₂ Ph & CHMe ₂ ), 2.30 (m, 1H , C H CH ₂ Ph), 1.18 (dd, 6H, 2 x CH ₃ )

IR (neat): 3361, 1715, 1273, 1157

MS m / e 430 (MH ⁺ )

analysis. (C ₂₃ H ₃₀ N ₂ O ₄ S) C, H, N, S

Example 37 2-benzyl-3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propylhexanoate (7a-III)

yield; 34%

Appearance: Colorless oil

¹ H NMR (CDCl ₃ ) δ7.1-7.3 (m, 5H, phenyl), 6.75-6.9 (m, 3H, Ar), 6.23 (bt, 1H, NH), 6.11 (bs, 1H, NH), 5.66 (s, 1H, OH), 4.40 (bs, 2H, NHCH ₂ Ar), 4.15 (dd, 1H, J = 3.9 and 11.43 Hz, CH ₂ OCO), 3.86 (s, 3H, OCH ₃ ), 3.65-3.85 (m, 2H, CH ₂ OCO & CHC H ₂ NHC = S), 3.29 (m, 1H, CHC H ₂ NHC = S), 2.61 (m, 2H, CH ₂ Ph), 2.30 (m, 3H, CH ₂ COO & CH), 1.65 (m, 2H), 1.2-1.4 (m, 4H), 0.88 (distorted t, 3H)

IR (neat): 3360, 1715, 1274, 1122

MS m / e 458 (M ⁺ )

analysis. (C ₂₅ H ₃₄ N ₂ O ₄ S) C, H, N, S

Example 38 2-benzyl-3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propylstearate (7a-IV)

yield; 30%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ7.1-7.3 (m, 5H, phenyl), 6.75-6.9 (m, 3H, Ar), 6.28 (bt, 1H, NH), 6.17 (bs, 1H, NH), 5.69 (s, 1H, OH), 4.39 (bs, 2H, NHCH ₂ Ar), 4.14 (dd, 1H, J = 3.9 and 11.67 Hz, CH ₂ OCO), 3.87 (s, 3H, OCH ₃ ), 3.65-3.85 (m, 2H, CH ₂ OCO & CHC H ₂ NHC = S), 3.30 (m, 1H, CHC H ₂ NHC = S), 2.60 (m, 2H, CH ₂ Ph), 2.30 (m, 3H, CH ₂ COO & CH), 1.2-1.7 (m, 30H), 0.88 (distorted t, 3H)

IR (neat): 3363, 1731, 1274, 1031

MS m / e 626 (M ⁺ )

analysis. (C ₃₇ H ₅₈ N ₂ O ₄ S)

Example 39 2-benzyl-3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propyl benzoate (7a-V)

yield; 50%

Appearance; White solid

mp = 90 ° C

¹ H NMR (CDCl ₃ ) δ 7.98-8.02 (m, 2H), 7.58 (m, 1H), 7.4-7.5 (m, 2H), 7.1-7.32 (m, 5H, phenyl), 6.72-6.85 (m , 3H, Ar), 6.42 (bt, 1H, NH), 6.31 (bs, 1H, NH), 5.73 (s, 1H, OH), 4.34-4.42 (m, 3H, NHCH ₂ Ar & CH ₂ OCO), 4.05 (dd, 1H, CH ₂ OCO), 3.7-3.85 (m, 4H, OCH ₃ & CHC H ₂ NHC = S), 3.38 (m, 1H, CHC H ₂ NHC = S), 2.6-2.78 (m, 2H, CH ₂ Ph), 2.46 (m, 1H, CH)

IR (neat): 3360, 1714, 1274, 1121

MS m / e 464 (M ⁺ )

analysis. (C ₂₆ H ₂₈ N ₂ O ₄ S) C, H, N, S

Example 40 2- (3,4-dimethylbenzyl) -3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propyl pivalate (7b-I)

Yield: 40%

Appearance; White solid

mp = 47 ° C.

¹ H NMR (CDCl ₃ ) δ 6.33-7.05 (m, 3H), 6.77-6.9 (m, 3H, Ar), 6.22 (m, 1H, NH), 5.98 (bs, 1H, NH), 5.61 (s , 1H, OH), 4.37 (bs, NHCH ₂ Ar), 4.17 (ddd of AB, 1H, CH ₂ OCO), 3.87 (s, 3H, OCH ₃ ), 3.7-3.85 (m, 2H, CH ₂ OCO & CHC H ₂ NHC = S), 3.27 (m, 1H, CHC H ₂ NHC = S), 2.60 (m, 2H, CH ₂ Ph), 2.2-2.32 (m, 7H, 2 x CH ₃ & CH), 1.22 (s, 9H, CO (CH ₃ ) ₃ )

IR (neat): 3360, 1714, 1279, 1159

MS m / e 474 (M ⁺ +2)

analysis. (C ₂₆ H ₃₆ N ₂ O ₄ S) C, H, N, S

Example 41 2- (3,4-dimethylbenzyl) -3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propylbenzoate (7b-V)

yield; 45%

Appearance; White solid

mp = 44 ° C

¹ H NMR (CDCl ₃ ) δ 8.02 (m, 2H), 7.59 (m, 1H), 7.46 (m, 2H), 6.9-7.06 (m, 3H), 6.72-6.85 (m, 3H, Ar), 6.33 (m, 1H, NH), 6.13 (bs, 1H, NH), 5.65 (s, 1H, OH), 4.35-4.44 (m, 3H, NHCH ₂ Ar & CH ₂ OCO), 4.05 (m, 1H, CH ₂ OCO), 3.7-3.85 (m, 4H, OCH ₃ & CHC H ₂ NHC = S), 3.40 (m, 1H, CHC H ₂ NHC = S), 2.6-2.78 (m, 2H, CH ₂ Ph) , 2.44 (m, 1H, CH), 2.2-2.3 (m, 6H, 2 × CH ₃ )

IR (neat): 3360, 1713, 1274, 1121

MS m / e 492 (M ⁺ )

analysis. (C ₂₈ H ₃₂ N ₂ O ₄ S) C, H, N, S

Example 42 2- (3,4-dimethylbenzyl) -3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propyl 3,4-dimethylbenzoate (7b-VI )

yield; 55%

Appearance; White solid

mp = 58 ° C.

¹ H NMR (CDCl ₃ ) δ7.75 (m, 2H), 7.20 (m, 1H), 6.9-7.06 (m, 3H), 6.74-6.85 (m, 3H, Ar), 6.39 (m, 1H, NH ), 6.07 (bs, 1H, NH), 5.63 (s, 1H, OH), 4.35-4.43 (m, 3H, NHCH ₂ Ar & CH ₂ OCO), 4.05 (m, 1H, CH ₂ OCO), 3.7- 3.85 (m, 4H, OCH ₃ & CHC H ₂ NHC = S), 3.37 (m, 1H, CHC H ₂ NHC = S), 2.6-2.74 (m, 2H, CH ₂ Ph), 2.41 (m, 1H, CH), 2.2-2.35 (m, 12H, 4 x CH ₃ )

IR (neat): 3373, 1711, 1266, 1124

MS m / e 520 (M ⁺ )

analysis. (C ₃₀ H ₃₆ N ₂ O ₄ S) C, H, N, S

Example 43 2- (4-chlorobenzyl) -3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propyl pivalate (7c-I)

yield; 43%

Appearance; White solid

mp = 62 ° C.

¹ H NMR (CDCl ₃ ) δ7.25 (d, 2H), 7.10 (d, 2H), 6.77-6.90 (m, 3H, Ar), 6.38 (t, 1H, NH), 6.25 (bs, 1H, NH ), 5.72 (s, 1H, OH), 4.42 (bs, 2H, NHCH ₂ Ar), 4.16 (dd of AB, 1H, CH ₂ OCO), 3.86 (s, 3H, OCH ₃ ), 3.7-3.85 (m , 2H, CH ₂ OCO & CHC H ₂ NHC = S), 3.19 (m, 1H, CHC H ₂ NHC = S), 2.59 (ddd of AB, 2H, CH ₂ Ph), 2.32 (m, 1H, CH) , 1.22 (s, 9H, CO (CH ₃ ) ₃ )

IR (neat): 3360, 1714, 1279, 1159

MS m / e 479 (M ⁺ )

analysis. (C ₂₄ H ₃₁ ClN ₂ O ₄ S) C, H, N, S

Example 44 2- (4-chlorobenzyl) -3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propylbenzoate (7c-V)

yield; 55%

Appearance; White solid

mp = 57 ° C.

¹ H NMR (CDCl ₃ ) δ8.00 (m, 2H), 7.60 (m, 1H), 7.47 (m, 2H), 7.27 (d, 2H), 7.15 (d, 2H), 6.76-6.90 (m, 3H, Ar), 6.36 (t, 1H, NH), 6.15 (bs, 1H, NH), 5.61 (s, 1H, OH), 4.40 (m, 3H, NHCH ₂ Ar & CH ₂ OCO), 4.00 (dd of AB, 1H, CH ₂ OCO), 3.86 (s, 3H, OCH ₃ ), 3.85 (m, 1H, CHC H ₂ NHC = S), 3.31 (m, 1H, CHC H ₂ NHC = S), 2.67 ( ddd of AB, 2H, CH ₂ Ph), 2.46 (m, 1H, CH)

IR (neat): 3373, 1711, 1266, 1124

MS m / e 499 (M ⁺ )

analysis. (C ₂₆ H ₂₇ ClN ₂ O ₄ S) C, H, N, S

Example 45 2- (4-t-butylbenzyl) -3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propylpivalate (7d-I)

yield; 40%

Appearance; Yellow solid

mp = 52 ° C.

¹ H NMR (CDCl ₃ ) δ7.30 (d, 2H), 7.09 (d, 2H), 6.75-6.90 (m, 3H, Ar), 6.27 (t, 1H, NH), 6.10 (bs, 1H, NH ), 5.66 (s, 1H, OH), 4.40 (bs, 2H, NHCH ₂ Ar), 4.15 (dd of AB, 1H, CH ₂ OCO), 3.86 (s, 3H, OCH ₃ ), 3.7-3.85 (m , 2H, CH ₂ OCO & CHC H ₂ NHC = S), 3.27 (m, 1H, CHC H ₂ NHC = S), 2.58 (ddd of AB, 2H, CH ₂ Ph), 2.30 (m, 1H, CH) , 1.29 (s, 9H, C (CH ₃ ) ₃ ), 1.22 (s, 9H, CO (CH ₃ ) ₃ )

IR (neat): 3360, 1714, 1277, 1158

MS m / e 500 (M ⁺ )

analysis. (C ₂₈ H ₄₀ N ₂ O ₄ S) C, H, N, S

Example 46 2- (4-t-butylbenzyl) -3-{[(4-hydroxy-3-methoxybenzyl) amino] carbothionyl} propyl benzoate [7d-V)

yield; 50%

Appearance; White solid

mp = 54 ° C

¹ H NMR (CDCl ₃ ) δ 8.02 (m, 2H), 7.60 (m, 1H), 7.46 (m, 2H), 7.32 (d, 2H), 7.14 (d, 2H), 6.75-6.90 (m, 3H, Ar), 6.28 (t, 1H, NH), 6.06 (bs, 1H, NH), 5.61 (s, 1H, OH), 4.40 (m, 3H, NHCH ₂ Ar & CH ₂ OCO), 4.08 (dd of AB, 1H, CH ₂ OCO), 3.86 (s, 3H, OCH ₃ ), 3.80 (m, 1H, CHC H ₂ NHC = S), 3.40 (m, 1H, CHC H ₂ NHC = S), 2.68 ( m, 2H, CH ₂ Ph), 2.46 (m, 1H, CHCH ₂ Ph), 1.29 (s, 9H, C (CH ₃ ) ₃ )

IR (neat): 3360, 1714, 1272, 1124

MS m / e 520 (M ⁺ )

analysis. (C ₃₀ H ₃₆ N ₂ O ₄ S) C, H, N, S

Example 47 N- [2-benzyl-3- (benzyloxy) propyl] -N '-(4-hydroxy-3-methoxybenzyl) thiourea (9a)

yield; 60%

Appearance; Colorless oil

¹ H NMR (CDCl ₃ ) δ 7.10-7.30 (m, 10H, 2 × Ph), 6.81 (d, 1H, Ar), 6.60 (bs, 1H, Ar), 6.56 (d, 1H, Ar), 6.54 (bs, 1H, NH), 5.64 (s, 1H, OH), 4.35 (m, 4H, NHCH ₂ Ar and PhCH ₂ O), 3.82 (s, 3H, OCH ₃ ), 3.50 (m, 2H, BnOCH ₂ ), 3.37 (bt, 2H, CHCH ₂ NHC = S), 2.64 (m, 2H, CH ₂ Ph), 2.20 (m, 1H, CH)

IR (neat): 3288, 1274, 1123

MS m / e 450 (M +)

analysis. (C ₂₆ H ₃₀ N ₂ O ₃ S) C, H, N, S.

Example 48 N- [3-benzyloxy-2- (4-chlorobenzyl) propyl] -N '-(4-hydroxy-3-methoxybenzyl) thiourea (9c)

Appearance; White solid

mp = 37.5 ° C.

¹ H NMR (CDCl ₃ ) δ 7.0-7.3 (m, 9H), 6.5-6.84 (m, 3H, Ar), 6.54 (bs, 1H, NH), 6.30 (bs, 1H, NH), 5.68 (s , 1H, OH), 4.32 (m, 4H, NHCH ₂ Ar and PhCH ₂ O), 3.82 (s, 3H, OCH ₃ ), 3.48 (m, 2H, BnOCH ₂ ), 3.34 (m, 2H, CHCH ₂ NHC = S), 2.58 (m, 2H, CH ₂ Ph), 2.16 (m, 1 H, CH)

IR (neat): 3340, 1273, 1153

MS m / e 484 (M +)

analysis. (C ₂₆ H ₂₉ ClN ₂ O ₃ S) C, H, N, S.

Example 49 3-methoxy-4-methoxymethoxy-benzonitrile (12)

4-Hydroxy-3-methoxy-benzonitrile (11, 1.5 g, 10 mmol) was dissolved in methylene chloride (50 mL), followed by diisopropylethylamine (2.7 mL, 15 mmol) and chloromethyl methyl ether (0.92 mL). , 12 mmol) was added and stirred for 3 hours. After dilution with methylene chloride, the mixture was washed with water, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (developing solvent: ethyl acetate / hexane = 1: 2) to give compound 12 as a white solid (1.54 g, 80%).

¹ H NMR (CDCl ₃ ) δ 7.27 (s, 1H, Ar), 7.24 (s, 1H, Ar), 7.11 (s, 1H, Ar), 5.29 (s, 2H, ArOCH ₂ ), 3.99 (s, 3H, ArOCH ₃ ), 3.51 (s, 3H, CH ₂ OCH ₃ )

Example 50 (3-methoxy-4-methoxymethoxybenzyl) carbamic acid benzyl ester (13)

Compound 12 (1.54 g, 8 mmol) was dissolved in THF (20 mL), and then added dropwise to a THF (30 mL) suspension in which a stirred lithium aluminum hydride (LiAlH ₄ ; 1.2 g, 32 mmol) was dissolved. After refluxing for 4 hours, the reaction mixture was cooled to 0 ° C. and the reaction was stopped with 5N sodium hydroxide to remove excess LiAlH ₄ . The precipitated aluminum salt was filtered off and THF was evaporated. The residue was precipitated with ether and water. The ether layer was washed with brine, dried over potassium carbonate, filtered and concentrated under reduced pressure to give an amine. The amine was dissolved in methylene chloride (15 mL) and triethylamine (1.16 mL, 8.3 mmol) and benzylchloroformate (1.18 mL, 8.3 mmol) were added. After stirring at room temperature for 2 hours, the reaction mixture was diluted with methylene chloride. The organic layer was washed with water and brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (developing solvent: ethyl acetate / hexane = 1: 2) to give compound 13 as a white solid (0.795 g, 30%).

¹ H NMR (CDCl ₃ ) δ 7.34 (m, 5H, phenyl), 7.08 (d, 1H, Ar), 6.81 (m, 2H, Ar), 5.21 (s, 2H, ArOC H ₂ ), 5.14 (s , 2H, OCH ₂ Ph), 4.32 (d, 2H, NH ₂ COO), 3.85 (s, 3H, ArOCH ₃ ), 3.50 (s, 3H, ArOCH ₂ OCH ₃ )

Example 51 (4-hydroxy-3-methoxybenzyl) carbamic acid benzyl ester (14)

Compound 13 (0.795 g, 2.4 mmol) was dissolved in methylene chloride (5 mL), trifluoroacetic acid (2.5 mL) was slowly added, and the mixture was stirred at room temperature for 5 minutes. After cooling to 0 ° C., the reaction mixture was neutralized with saturated sodium hydrogen carbonate solution. Then diluted with water and extracted several times with methylene chloride. The extracted organic layer was washed with water and brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (developing solvent: ethyl acetate / hexane = 1: 2) to give compound 14 as a white solid (680 mg, 99%).

¹ H NMR (CDCl ₃ ) δ7.34 (m, 5H, phenyl), 6.83 (m, 3H, Ar), 5.14 (s, 2H, OCH ₂ Ph), 4.30 (d, 2H, NH ₂ COO), 3.86 (s, 3H, ArOCH ₃ )

Example 52 [4- (benzyloxy-carbonylaminomethyl) -2-methoxy-phenoxy] -acetic acid methyl ester (15)

Compound 14 (0.287 g, 1 mmol) was dissolved in acetone (20 mL), and potassium carbonate (0.552 g, 4 mmol) and methylbromoacetate (0.14 mL, 1.5 mmol) were added thereto, and the mixture was refluxed for 3 hours. . After cooling, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (developing solvent: ethyl acetate / hexane = 3: 2) to give compound 15 as a white solid (0.323 g, 90%).

¹ H NMR (CDCl ₃ ) δ 7.28-7.33 (m, 5H, phenyl), 6.78 (m, 3H, Ar), 5.11 (s, 2H, COCH ₂ Ph), 4.76 (s, 2H, CH ₂ Ph) , 4.28 (d, 2H, CH ₂ NHCO), 3.81 (s, 3H, ArOCH ₃ ), 3.76 (s, 3H, COOCH ₃ )

Example 53 [4- (benzyloxycarbonylamino-methyl) -2-methoxy-phenoxy acetic acid (16)

Compound 5 (0.323 g, 0.9 mmol) was dissolved in THF (1 mL), 15% sodium hydroxide solution (1 mL) was added thereto, and stirred at room temperature for 30 minutes. The reaction mixture was neutralized with acetic acid, Diluted with water and extracted several times with methylene chloride. The extracted organic layer was washed with water, dried over magnesium sulfate, and concentrated under reduced pressure to obtain compound 16 as a white solid (0.276 g, 89%).

¹ H NMR (DMSO) δ7.72 (s, 1H, COOH), 7.17-7.33 (m, 5H, phenyl), 6.81 (s, 1H, Ar), 6.67 (s, 2H, Ar), 5.02 (s, 2H, COCH ₂ Ph), 4.16 (s, 2H, CH ₂ Ph), 4.09 (d, 2H, CH ₂ NHCO), 3.70 (s, 3H, ArOCH ₃ )

Example 54 (4-Aminomethyl-2-methoxy-phenoxy) acetic acid (17)

Compound 16 (0.276 g, 0.8 mmol) was dissolved in methanol (5 mL), 10% palladium catalyst (30 mg) was added, and then hydrogenated under a hydrogen balloon for 30 minutes. The reaction mixture was filtered and the filtrate was concentrated to give compound 17 as a white solid which could be used in the next step without further purification (0.093 g, 55%).

General Synthesis of Compound 19

Compound 17 (0.5 mmol) was dissolved in DMF (1 mL), triethylamine (1.0 mmol) was added, the mixture was stirred for 30 minutes, and isothiocyanate 18 (0.5 mmol) was added to DMF. The reaction mixture was stirred at room temperature for 24 hours, diluted with water and extracted several times with ethyl acetate. The extracted organic layer was collected, washed with water and brine, dried over magnesium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (methylene chloride / methanol / acetic acid = 100/10 / 0.5) to give compound 19.

Example 55 2,2-Dimethyl-propionic acid 2- [3- (4-carboxymethoxy-3-methoxy-benzyl) -thioureidomethyl] -3- (3,4-dimethyl-phenyl Propyl ester (19a)

yield; 27%

Appearance; White solid

¹ H NMR (CDCl ₃ ) δ 7.15-7.26 (m, 4H, Ar), 6.76 (m, 3H, Ar), 6.54 (m, 1H, NH), 4.53 (s, 2H, CH ₂ COOH), 4.45 (bs, 2H, NHCH ₂ Ar), 4.15 (ddd of AB, 1H, CH ₂ OC = O), 3.82 (s, 3H, OCH ₃ ), 3.6-3.85 (m, 2H, CH ₂ OCO and CHC H ₂ NHC = S), 3.26 (m, 1H, CHC H ₂ NHC = S), 2.60 (m, 2H, CH ₂ Ph), 2.20-2.28 (m, 7H, 2 × CH ₃ & CHCH ₂ Ph), 1.23 ( s, 9H, CO (CH ₃ ) ₃ )

Example 56 2,2-Dimethyl-propionic acid 3- (4-t-butylphenyl) -2- [3- (4-carboxymethoxy-3-methoxy-benzyl) -thioureidomethyl ] Propyl ester (19b)

yield; 52%

Appearance; White solid

¹ H NMR (CDCl ₃ ) δ 7.09-7.28 (m, 4H, Ar), 6.85 (m, 3H, Ar), 6.54 (m, 1H, NH), 4.58 (s, 2H, CH ₂ COOH), 4.46 (bs, 2H, NHCH ₂ Ar), 4.12 (ddd of AB, 1H, CH ₂ OC = O), 3.78 (s, 3H, OCH3), 3.7-3.85 (m, 2H, CH ₂ OC = O and CHCH ₂ NHC = S), 3.26 (m, 1H, CHCH ₂ NHC = S), 2.58 (m, 2H, CH ₂ Ph), 2.32 (m, 1H, CHCH ₂ Ph), 1.28 (s, 9H, ArC (CH ₃ ) ₃ ), 1.22 (s, 9H, CO (CH ₃ ) ₃ )

Example 57 Succinic Acid Monobenzyl Ester (20)

Succinic anhydride (5 mmol, 0.502 g), benzyl alcohol (5 mmol, 0.52 mL) and sodium hydride (60%, 5 mmol, 0.2 g) were dissolved in THF and refluxed for 4 hours. The reaction mixture was cooled to room temperature, diluted with water, and extracted several times with ethyl acetate. The extracted organic layer was collected, washed with water and brine, dried over magnesium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / hexane = 1: 1) to give compound 20 as a white solid (0.866 g, 57%).

¹ H NMR (CDCl ₃ ) δ7.38 (s, 5H, phenyl), 5.15 (s, 2H, PhCH ₂ ), 2.71 (m, 4H, COCH ₂ CH ₂ CO)

Example 58 Succinic acid benzyl ester 4- (benzyloxycarbonylamino-methyl) -2-methoxy-phenyl ester (21)

Compound 10 (0.866 g, 2.85 mmol), compound 14 (2.85 mmol), dicyclohexylcarbodiimide (4.2 mmol), and 4-dimethylaminopyridine (0.42 mmol) were dissolved in methylene chloride (10 mL), and the result was 24 at room temperature. Stir for hours. After diluting the reaction mixture with diethyl ether, the precipitates were filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / hexane = 2: 1) to give compound 21 as a white solid (1.15 g, 85%).

¹ H NMR (CDCl ₃ ) δ 7.30-7.36 (m, 10H, 2 × phenyl), 6.86 (m, 3H, Ar), 5.14 (d, 4H, PhCH ₂ ), 4.34 (d, 2H, CH ₂ NH ), 3.75 (s, 3H, ArOCH ₃ ), 2.93 (t, 2H, COCH ₂ CH ₂ CO), 2.79 (t, 2H, COCH ₂ CH ₂ CO)

Example 59 Succinic acid mono- (4-aminomethyl-2-methoxy-phenyl) ester (22)

It was prepared from compound 17 in the same manner as in the synthesis of compound 17.

yield; 99%

Appearance; Yellow solid

¹ H NMR (DMSO) δ 8.24 (t, 1H, COOH), 6.75 (m, 3H, Ar), 4.17 (d, 2H, CH ₂ NH), 3.73 (s, 3H, ArOCH ₃ ), 2.44 (t, 2H, COCH ₂ CH ₂ CO), 2.34 (t, 2H, COCH ₂ CH ₂ CO)

General Synthesis of Compounds 23

Prepared from Compounds 18 and 20 in the same manner as in the general synthesis of Compound 19.

Example 60 Succinic acid mono (4- {3- [3- (3,4-dimethylphenyl) -2- (2,2-dimethylpropionyloxymethyl) -propyl] -thioureidomethyl}- 2-methoxy-phenyl) ester (23a)

yield; 17%

Appearance; White solid

¹ H NMR (CDCl ₃ ) δ6.9-7.2 (m, 4H, Ar), 6.8 (m, 3H, Ar), 6.34 (m, 1H, NH), 4.56 (d, 2H, NHCH ₂ Ar), 4.04 (m, 1H, CH ₂ OCO), 3.84 (s, 3H, OCH ₃ ), 3.6-3.85 (m, 2H, CH ₂ OCO and CHCH ₂ NHC = S), 3.27 (m, 1H, CHCH ₂ NHC = S ), 2.65 (m, 2H, CH ₂ Ph), 2.50 (s, 4H, COCH ₂ CH ₂ CO) 2.18-2.29 (m, 7H, 2 × CH ₃ and CHCH ₂ Ph), 1.24 (s, 9H, CO (CH ₃ ) ₃ )

Example 61 Succinic Acid Mono (4- {3- [3- (4-t-Butylphenyl) -2- (2,2-dimethylpropionyloxymethyl) -propyl] -thioureidomethyl}- 2-methoxy-phenyl) ester (23b)

yield; 9%

Appearance; White solid

¹ H NMR (CDCl ₃ ) δ7.07-7.32 (m, 4H, Ar), 6.82 (m, 3H, Ar), 6.59 (m, 1H, NH), 4.87 (m, 3H, NHCH ₂ Ar and CH ₂ OCO), 4.12 (ddd of AB, 1H, CH ₂ OC = O), 3.85 (s, 3H, OCH ₃ ), 3.7-3.89 (m, 2H, CH ₂ OCO and CHCH ₂ NHC = S), 3.41 (m , 1H, CHCH ₂ NHC = S), 2.58 (m, 2H, CH ₂ Ph), 2.39 (t, 4H, COCH ₂ CH ₂ CO) 2.32 (m, 1H, CHCH ₂ Ph), 1.28 (s, 9H, ArC (CH ₃ ) ₃ ), 1.21 (s, 9H, CO (CH ₃ ) ₃ )

General Synthesis of Compound 25

Lindler's catalyst was added to compound 24 (1.15 mmol) and homovanyllic pentafluoro phenyl ester (0.4 g, 1.15 mmol), and ethyl acetate was added thereto. Reacted. The mixture was filtered, and the residue obtained by concentration under reduced pressure was purified by column chromatography (ethyl acetate / hexane = 1: 2) to obtain compound 25.

Example 62 2,2-Dimethyl-propionic acid 3- (3,4-dimethyl phenyl) -2-{[2- (4-hydroxy-3-methoxy phenyl) acetylamino] -methyl} propyl Ester (25a)

yield; 37%

Appearance; oil

¹ H NMR (CDCl ₃ ) δ6.68-7.02 (m, 6H), 5.96 (bs, 1H, NH), 4.00 (m, 1H, CH ₂ OCO) 3.87 (s, 3H, OCH ₃ ), 3.80 (m , 1H, CH ₂ OCO), 3.46 (s, 2H, COCH ₂ Ar), 3.32 (m, 1H, CH ₂ NH), 3.13 (m, 1H, CH ₂ NH), 2.50 (d, 2H, CH ₂ Ph ), 2.20 (m, 7H, 2 × CH ₃ and CH), 1.20 (s, 9H, CO (CH ₃ ) ₃ )

Example 63 2,2-Dimethyl-propionic acid 3- (4-t-butyl phenyl) -2-{[2- (4-hydroxy-3-methoxy phenyl) acetylamino] -methyl} propyl Ester (25b)

yield; 70%

Appearance; oil

¹ H NMR (CDCl ₃ ) δ 7.28-7.31 (d, 2H), 7.03-7.05 (d, 2H), 6.89 (d, 1H, Ar), 6.80 (d, 1H, Ar), 6.72 (d, 1H , Ar), 5.8 (bs, 1H, NH), 4.02 (m, 1H, CH ₂ OCO), 3.89 (s, 3H, OCH ₃ ), 3.83 (m, 1H, CH ₂ OCO), 3.49 (s, 2H , COCH ₂ Ar), 3.34 (m, 1H, CH ₂ NH), 3.12 (m, 1H, CH ₂ NH), 2.56 (d, 2H, CH ₂ Ph), 2.13 (m, 1H, CH), 1.31 ( s, 9H, C (CH ₃ ) ₃ ), 1.22 (s, 9H, CO (CH ₃ ) ₃ )

General Synthesis of Compound 26

To compound 25 (0.33 mmol), 40% potassium hydroxide (0.24 mL) and 40% tetrabutylammonium hydroxide (0.02 mL) was added 1,2-dibromoethane (0.96 mL) and heated at 50 ° C. for 12 h. It was. The reaction mixture was diluted with methylene chloride, washed with water and brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / hexane = 1: 2) to give 26.

Example 64 2,2-Dimethyl-propionic acid 2-({2- [4- (2-bromoethoxy) -3-methoxy phenyl] acetylamino} -methyl} 3- (3,4- Dimethyl phenyl) propyl ester (26a)

yield; 68%

¹ H NMR (CDCl ₃ ) δ6.74-7.03 (m, 6H), 5.75 (bs, 1H, NH), 4.32 (t, 2H, OCH 2), 4.01 (m, 1H, CH ₂ OCO), 3.86 (s , 3H, OCH ₃ ), 3.82 (m, 1H, CH ₂ OCO), 3.64 (t, 2H, CH ₂ Br), 3.47 (s, 2H, COCH ₂ Ar), 3.31 (m, 1H, CH ₂ NH) , 3.10 (m, 1H, CH ₂ NH), 2.50 (d, 2H, CH ₂ Ph), 2.20 (m, 7H, 2 × CH ₃ and CH), 1.21 (s, 9H, CO (CH ₃ ) ₃ )

Example 65 2,2-Dimethyl-propionic acid 2-({2- [4- (2-bromoethoxy) -3-methoxy phenyl] acetylamino} -methyl} 3- (4-t- Butyl phenyl) propyl ester (26b)

yield; 92%

¹ H NMR (CDCl ₃ ) δ 7.26-7.30 (d, 2H), 7.01-7.04 (d, 2H), 6.74-6.89 (m, 3H, Ar), 5.8 (bs, 1H, NH), 4.3 (t, 2H, OCH ₂ ), 4.01 (m, 1H, CH ₂ OCO), 3.86 (s, 3H, OCH ₃ ), 3.82 (m, 1H, CH ₂ OCO), 3.64 (t, 2H, CH ₂ Br), 3.47 (s, 2H, COCH ₂ Ar), 3.31 (m, 1H, CH ₂ NH), 3.10 (m, 1H, CH ₂ NH), 2.5 (d, 2H, CH ₂ Ph), 2.12 (m, 1H, CH ), 1.30 (s, 9H, C (CH ₃ ) ₃ ), 1.22 (s, 9H, CO (CH ₃ ) ₃ )

General Synthesis of Compounds 27

Compound 26 (0.22 mmol) was dissolved in DMF (2 mL), and sodium azide (0.044 g, 0.67 mmol) was added and heated at 100 ° C. for 8 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The obtained organic layer was washed with water and brine, dried over magnesium sulfate, and then concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / hexane = 1: 1) to give compound 27.

Example 66 2,2-Dimethyl-propionic acid 2-({2- [4- (2-azidoethoxy) -3-methoxy phenyl] acetylamino} -methyl} 3- (3,4 -Dimethyl phenyl) propyl ester (27a)

yield; 98%

¹ H NMR (CDCl ₃ ) δ6.74-7.03 (m, 6H), 5.74 (bs, 1H, NH), 4.19 (t, 2H, OCH ₂ ), 4.01 (m, 1H, CH ₂ OCO) 3.85 (s , 3H, OCH ₃ ), 3.82 (m, 1H, CH ₂ OCO), 3.62 (t, 2H, CH ₂ N ₃ ), 3.48 (s, 2H, COCH ₂ Ar), 3.29 (m, 1H, CH ₂ NH ), 3.10 (m, 1H, CH ₂ NH), 2.50 (d, 2H, CH ₂ Ph), 2.04-2.26 (m, 7H, 2 x CH ₃ and CH), 1.21 (s, 9H, CO (CH ₃₎ ) ₃ )

Example 67 2,2-Dimethyl-propionic acid 2-({2- [4- (2-azidoethoxy) -3-methoxy phenyl] acetylamino} -methyl} 3- (4-t -Butyl phenyl) propyl ester (27b)

yield; 81%

¹ H NMR (CDCl ₃ ) δ 7.26-7.30 (d, 2H), 7.01-7.04 (d, 2H), 6.75-6.91 (m, 3h, Ar), 5.76 (bs, 1H, NH), 4.17 ( t, 2H, OCH ₂ ), 4.00 (m, 1H, CH ₂ OCO), 3.85 (s, 3H, OCH ₃ ), 3.82 (m, 1H, CH ₂ OCO), 3.63 (t, 2H, CH ₂ N ₃ ), 3.48 (s, 2H, COCH ₂ Ar), 3.31 (m, 1H, CH ₂ NH), 3.06 (m, 1H, CH ₂ NH), 2.54 (d, 2H, CH ₂ Ph), 2.12 (m, 1H, CH), 1.29 (s, 9H, C (CH ₃ ) ₃ ), 1.20 (s, 9H, CO (CH ₃ ) ₃ )

General Synthesis of Compound 28

Compound 27 (0.05 mmol) and 10% palladium catalyst (0.02 g) were dissolved in methanol (5 mL), and then hydrogenated under a hydrogen balloon for 3 hours. After the reaction mixture was filtered, the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (chloroform / methanol / ammonium hydroxide = 9: 1: 0.1) to give compound 28.

Example 68 2,2-Dimethyl-propionic acid 2-({2- [4- (2-aminoethoxy) -3-methoxy phenyl] acetylamino} -methyl} 3- (3,4- Dimethyl phenyl) propyl ester (28a)

yield; 83%

Appearance; White solid

¹ H NMR (CDCl ₃ ) δ6.73-7.23 (m, 6H), 5.73 (bs, 1H, NH), 4.13 (m, 4H, OCH ₂ and CH ₂ OCO), 3.85 (s, 3H, OCH ₃ ) , 3.82 (m, 1H, CH ₂ OCO), 3.5 (m, 2H, COCH ₂ Ar), 3.23 (m, 2H, CH ₂ NH and CH ₂ NH ₂ ), 3.09 (m, 2H, CH ₂ NH and CH ₂ NH ₂ ), 2.50 (d, 2H, CH ₂ Ph), 2.04-2.26 (m, 7H, 2 × CH ₃ and CH), 1.20 (s, 9H, CO (CH ₃ ) ₃ )

Example 69 2,2-Dimethyl-propionic acid 2-({2- [4- (2-aminoethoxy) -3-methoxy phenyl] acetylamino} -methyl} -3- (4-t -Butyl phenyl) propyl ester (28b)

yield; 99%

Appearance; White solid

¹ H NMR (CDCl ₃ ) δ 7.26-7.29 (m, 2H), 7.01-7.04 (d, 2H), 6.76-6.89 (m, 3H, Ar), 5.84 (bs, 1H, NH), 4.05 (m , 1H, CH ₂ OCO), 3.85 (m, 3H, OCH ₃ ), 3.72 (m, 3H, CH ₂ OCO and OCH ₂ ), 3.47 (s, 2H, COCH ₂ Ar), 3.30 (m, 1H, CH ₂ NH), 3.10 (m, 1H, CH ₂ NH), 2.75 (bs, 2H, CH ₂ NH ₂ ) 2.52 (d, 2H, CH ₂ Ph), 2.12 (m, 1H, CH), 1.29 (s, 9H, C (CH ₃ ) ₃ ), 1.20 (s, 9H, CO (CH ₃ ) ₃ )

Example 70 (4-hydroxy-3-methoxy-benzyl) -carbamic acid-t-butyl ester (30)

4-hydroxy-3-methoxy benzylamine (Compound 29, 0.5 g, 2.64 mmol) and triethylamine (0.054 g, 5.29 mmol) were dissolved in distilled water. Di-t-butyldicarbonate (1.15 g 5.29 mmol) dissolved in dioxane (1.5 mL) was added thereto for 20 minutes, followed by stirring at room temperature for 24 hours. After the reaction mixture was extracted with methylene chloride, the extracted organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / nucleic acid = 1: 1) to give compound 30 as an oil (0.663 g, 99%).

¹ H NMR (CDCl ₃ ) δ6.74-7.00 (m, 3H, Ar), 5.65 (s, 1H, OH), 4.79 (bs, 1H, NH), 4.23 (d, 2H, NHCH ₂ ) 3.87 (s , 3H, OCH ₃ ), 1.43 (s, 9H, O (CH ₃ ) ₃ )

Example 71 (4- (2-Bromoethoxy) -3-methoxy-benzyl) -carbamic acid t-butyl ester (31)

Prepared from compound 30 in the same manner as in the synthesis of compound 26 (ethyl acetate / nucleic acid = 1: 3).

yield; 84%

¹ H NMR (CDCl ₃ ) δ6.78-6.89 (m, 3H, Ar), 4.81 (bs, 1H, NH), 4.29-4.33 (t, 2H, OCH ₂ ), 4.24 (d, 2H, NHCH ₂ ) , 3.90 (s, 3H, OCH ₃ ), 3.65 (t, 2H, CH ₂ Br), 1.46 (s, 9H, O (CH ₃ ) ₃ )

Example 72 [(4- (2-azido ethoxy) -3-methoxy-benzyl] -carbamic acid t-butyl ester (32)

Prepared from compound 31 in the same manner as in the synthesis of compound 27 (ethyl acetate / nucleic acid = 1: 3).

yield; 98%

Appearance; Yellow solid

¹ H NMR (CDCl ₃ ) δ6.78-6.87 (m, 3H, Ar), 4.83 (bs, 1H, NH), 4.24 (d, 2H, OCH ₂ ), 4.15 (t, 2H, NHCH ₂ ), 3.80 (s, 3H, OCH ₃ ), 3.62 (t, 2H, CH ₂ N ₃ ), 1.46 (s, 9H, (CH ₃ ) ₃ )

Example 73 4- (2-azidoethoxy) -3-methoxy-benzylamine trifluoroacetate (33)

Compound 32 (0.103 g, 0.32 mmol) was dissolved in methylene chloride (1 mL), trifluoroacetic acid was added dropwise, and heated at 100 ° C. for 1 hour, and concentrated under reduced pressure. The residue was diluted with toluene and concentrated under reduced pressure several times to obtain compound 33 as a brown solid (0.115 g, 100%).

¹ H NMR (CDCl ₃ ) δ 6.99-7.10 (m, 3H, Ar), 4.12 (t, 2H, OCH ₂ ), 3.93 (s, 2H, ArCH ₂ ), 3.76 (s, 3H, OCH ₃ ), 3.62 (m, 2H, CH ₂ N ₃ )

Example 74 1- (2-azidoethoxy) -4-isothiocyanatomethyl-2-methoxybenzene (34)

Compound 33 (0.115 g, 0.33 mmol) was dissolved in DMF (1.5 mL), triethylamine (0.037 g, 0.36 mmol) was added, and the mixture was stirred at room temperature for 1 hour. 1,1-thiocarbonyl di-2 (1H) -pyridone (0.084 g, 0.33 mmol) was added to the reaction and stirred at room temperature for 2.5 hours. The reaction mixture was diluted with water and extracted several times with ethyl ether. The extracted organic layer was washed with water and brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / nucleic acid = 1: 2) to give compound 34 as an oil (0.065 g, 74%).

¹ H NMR (CDCl ₃ ) δ 6.75-6.91 (m, 3H, Ar), 4.65 (s, 2H, CH ₂ N = C = S), 4.20 (t, 2H, OCH ₂ ), .3.90 (s, 3H , OCH ₃ ), 3.62 (t, 2H, CH ₂ N ₃ )

General Synthesis of Compound 35

Compound 6 (0.27 mmol) and Lindler catalyst (0.042 mg) were dissolved in ethanol (5 mL), and then hydrogenated under a hydrogen balloon for 2 hours. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, the residue was dissolved in methylene chloride, and compound 34 (0.27 mmol) was added. After stirring for 15 hours at room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / hexane = 1: 10) to give compound 35.

Example 75 2,2-dimethyl propionic acid 2- {3- [4- (2-azidoethoxy) -3-methoxybenzyl] thioureidomethyl} -3- (3,4-dimethyl Phenyl) propyl ester (35a)

yield; 39%

¹ H NMR (CDCl ₃ ) δ6.81-7.04 (m, 6H), 6.25 (m, 1H, NH), 6.02 (bs, 1H, NH), 4.41 (bs, 2H, NHCH ₂ Ar), 4.14-4.17 (m, 3H, CH ₂ OCO and OCH ₂ ), 3.85 (s, 3H, OCH ₃ ), 3.76-3.85 (m, 2H, CH ₂ OCO and CH C H ₂ NHC = S), 3.60-3.63 (t, 2H, CH ₂ N ₃ ), 3.21-3.28 (m, 1H, CH C H ₂ NC = S), 2.47-2.66 (m, 2H, CH ₂ Ph), 2.20-2.26 (m, 7H, 2 x CH ₃ and CH), 1.22 (s, 9H, CO (CH 3) ₃ )

Example 76 2,2-dimethyl propionic acid 2- {3- [4- (2-azidoethoxy) -3-methoxybenzyl] thioureidomethyl} -3- (4-t-butyl Phenyl) propyl ester (35b)

yield; 60%

¹ H NMR (CDCl ₃ ) δ 7.3 (d, 2H), 7.08 (d, 2H), 6.9 (m, 3H, Ar), 6.28 (m, 1H, NH), 6.10 (bs, 1H, NH), 4.43 (bs, 2H, NHCH ₂ Ar), 4.2 (m, 3H, CH ₂ OCO and OCH ₂ ) 3.85 (s, 3H, OCH ₃ ), 3.74-3.95 (m, 2H, CH ₂ OCO and CH C H ₂ NHC = S), 3.60-3.63 (t, 2H, CH ₂ N ³ ), 3.26 (m, 1H, CH C H ₂ NHC = S), 2.6 (m, 2H, CH ₂ Ph), 2.30 (m, 1H, CH), 1.29 (s, 9H, CO (CH ₃ ) 3)

General Synthesis of Compound 36

Compound 35 (0.068 g, 0.13 mmol) and triphenylphosphine (0.067 g, 0.25 mmol) were dissolved in THF, and water (4.5 mg, 0.25 mmol) was added thereto and stirred at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (chloroform / methanol / ammonium hydroxide = 9: 1: 0.1) to give compound 36.

Example 77 2,2-dimethyl propionic acid 2- {3- [4- (2-aminoethoxy) -3-methoxybenzyl] thioureidomethyl} -3- (3,4-dimethylphenyl ) Propyl Ester (36a)

yield; 49%

¹ H NMR (CDCl ₃ ) δ 6.77-7.04 (m, 6H), 6.47 (bs, 2H, NH), 4.43 (bs, 2H, NHCH ₂ Ar), 4.18 (ddd of AB, 1H, CH ₂ OCO), 3.98 (t, 2H, OCH ₂ ), 3.81 (s, 3H, OCH ₃ ), 3.71-3.77 (m, 2H, CH ₂ OCO and CH C H ₂ NHC = S), 3.06 (t, 2H, CH ₂ NH ₂ ), 2.48-2.68 (m, 2H, CH ₂ Ph), 2.20 (m, 7H, 2 × CH ₃ and CH), 1.23 (s, 9H, CO (CH ₃ ) ₃ )

Example 78 2,2-dimethyl propionic acid 2- {3- [4- (2-aminoethoxy) -3-methoxybenzyl] thioureidomethyl} -3- (4-t-butyl phenyl ) Propyl ester (36b)

Yield; 99%

¹ H NMR (CDCl ₃ ) δ 7.28-7.47 (m, 2H), 7.2 (d, 2H), 6.8-7.0 (m, 3H, Ar), 6.4 (bs, 1H, NH), 4.44 (bs, 2H, NHCH ₂ Ar), 4.18 (m, 1H, CH ₂ OCO), 3.82 (t, 2H, OCH ₂ ), 3.78 (s, 3H, OCH ₃ ), 3.66-3.82 (m, 2H, CH ₂ OCO and CH C H ₂ NHC = S), 3.09 (t, 2H, CH ₂ NH ₂ ), 2.48-2.68 (m, 2H, CH ₂ Ph), 2.20 (m, 1H, CH), 1.28 (s, 9H, C (CH) ₃ ) ₃ ), 1.22 (s, 9H, CO (CH ₃ ) ₃ )

Experimental Example 1 Receptor Binding Affinity and CAP-Active Channel Search

CAP-type activity of the compound of interest was determined via receptor binding affinity and CAP-active single channel search in vitro. In receptor binding screening, their ability is assessed by transferring bound [ ³ H] RTX to receptors. The results are expressed as K _i values (mean ± SEM, three experiments) expressed as the concentration of non-radioactive ligands that transfer half of the bound labeled RTX. In CAP-activated single channel activity assays, the increase in internal currents due to unselected cation introduction was found in cultured neonatal rat dorsal root ganglion neurons. It is measured by extracellular application of the compound. The activity of the compound is represented by the relative difference in ionic conductance compared to CAP as a control.

<Method>

1. [ ³ H] Resiniferatoxin Binding Assay

[ ³ H] RTX (37 Ci / mmol) was synthesized by a chemical synthesis and analysis laboratory, NCI-FCRDC. Non-radioactive RTX and capsazepine were purchased from LC lab (Woburn, MA).

Inhibition of [ ³ H] RTX binding in the presence of competing ligands was determined by membrane preparation from rat spinal cord. Membrane preparation was prepared as described in Neurosciences Vol 8, pp 368-380. That is, the animals were euthanized under general anesthesia, and the entire vertebral color was aseptically removed. Omni 2000 tissue homogenizer in ice-cold 10 mM HEPES at pH 7.4 containing 5 mM KCl, 5.8 mM NaCl, 2 mM MgCl ₂ , 0.75 mM CaCl ₂ , 12 mM D-glucose and 137 mM sucrose ('Buffer A') The sample was ground using an aid. Homogenize by centrifugation for 10 min at a temperature of 4 ° C. at 1,000 × g; The pellet was resuspended in Buffer A and centrifuged again at 35,000 x g for 40 minutes at a temperature of 4 ° C. The pellet recovered from the second centrifugation was resuspended in the same buffer at a protein concentration of about 2 mg / ml, and a small amount was rapidly lyophilized in dry ice and then stored at -70 ° C until analysis.

The experiment was designed to evaluate the inhibition of specific [ ³ H] RTX binding to the membrane by non-radioactive compounds. 60 min at 37 ° C. in Buffer A with 100 g membrane protein and 0.25 mg / ml bovine serum albumin (type V, Sigma) in the presence of a competitive ligand at 300: 1 over the total volume [ ³ H] RTX (100 pM) was incubated. Bovine serum albumin was included to reduce the nonspecific adsorption of RTX on the surface. Later incubation, the tubes were cooled with ice and 100 μg of 'α ₁ -acidglycoprotein (Sigma) was added to each tube in a volume of 50: 1 to reduce nonspecific binding. The membrane is pelleted and separated by centrifugation of bound and unbound [ ³ H] RTX at 10,000 × g for 15 minutes at a temperature of 4 ° C. The ends of the tubes containing the pellets were cut and bound radioactivity was determined by scintillation counting. Nonspecific binding was determined in the presence of 1 mM nonradioactive RTX. The measurement of binding is repeated three times in each experiment, and each experiment is repeated at least twice. Competitive curves in each experiment are usually determined by using 5 to 6 competing ligands. Binding is expressed as fmol / mg protein. Protein concentration was determined by bio-rad protein assay according to the manufacturer's manual (Bio-Rad Laboratories, CA). Samples were homogenized in the irradiation fluid for 10 hours before starting counting counts, and each sample was counted for 5 minutes. Binding data were analyzed by substituting the following equations:

B = ((L _H + L _C * K _d / K _I ) ^(n-1) / (K _d ⁿ + (L _H + L _C * K _d / K _I ) ⁿ )) / (L _H ^n-1 / (K _d ⁿ + L _H ⁿ ))

Wherein L _C is the concentration of the non-radioactive ligand;

K _I is the concentration of unbound inactive ligand that accounts for half of the binding site;

B represents specifically bound [ ³ H] RTX, Bmax is the receptor density, L _H is the concentration of unbound [ ³ H] RTX, and K _d is the concentration of [ ³ H] RTX Concentration, and n is the combined index referred to as the Hill coefficient.)

2. Capsaicin Active Single Channel Search

1) Cell Preparation

Cultured DRG neurons are prepared as described previously (J. Neuroscience 16, 1659-1667, (1996)). That is, DRGs were excised to all levels of the low cervical, thoracic and lumbar spinal cords of newborn mice 1 or 2 days old. DMEM / F-12 mixture (Gibco, Grand Island, NY), fetal bovine serum (10%, Gibco), 1 mM sodium pyruvate, 25 ng / ml nerve growth factor (Sigma, St. Louis, MO) and 100 units / ml penicillin DRGs were collected in cold culture medium (4 ° C.) containing streptomycin (Sigma). Ganglia were washed three times with DMEM / F-12 medium and then incubated for 30 minutes in DMEM / F-12 medium containing 1 mg / ml collagenase (Type II, Worthington Biomedical, Freehold, NJ). The ganglions were then washed three times with Mg ^{2 +} -and Ca ²⁺ -free Hank's solution and incubated with gentle shaking in a warm (37 ° C) Hank's solution containing 2.5 mg / ml trypsin (Gibco). The solution was centrifuged at 1,000 rpm for 10 minutes and the pellet was washed 2 or 3 times with culture medium to inhibit the enzyme. The pellet was suspended in culture medium and slowly ground with a Paster pipette. The suspension was applied to a circular glass coverslip (Fisher, Pittsburgh, PA) placed in a small Petri dish. Glass coverslips were treated with poly-L-lysine (Sigma) for one day and dried before use. Cells are incubated at 37 ° C. under 95% air / 5% CO ₂ conditions. Cells were used for 2-4 days after application.

2) Current record

A borosilicate glass pipette (Narishige Scientific Instrument Lab., Tokyo) was pulled and coated with Sylgard (Dow Corning Co., Midland, MI). The tip resistance is about 2 and 5 Mohms for the whole cell and single channel current recordings respectively. After the formation of gigaseal with a glass pipette, cell-attachment and internal and external patch centrifugation were used to study single-channel currents as described by Hamil et al. Salt bridges (1% agar in 300 mM potassium chloride) and a Ag / AgCl reference electrode in the pipette solution were used to minimize changes in junction potential. The bonding potential is eliminated before forming the giga chamber. The cell membrane on the glass pipette was ruptured with slight inhalation for recording of whole cells. After forming whole cells, the capacitative transients were eliminated.

Single-channel currents are recorded using a patch clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, Calif.) And filtered at 5 μs with an 8-pole, low-pass Bessel filter It was. Data was digitized at 37 Hz with a digital data recorder (VR-10B, Instrutech, Great Neck, NY) and stored on videotape for later analysis. For chart recording, the output of the amplifier was filtered at 500 kHz (Frequency Device, Havenhill, Mass.) And fed to a thermal array chart recorder (TA-240, Gould Instrument System, Valley View, OH). The digital data stored on the videotape was entered into a personal computer (IBM pentium-compatible) for computer analysis of single-channel currents.

3) Channel open probability (Po)

The amplitude and average open time of single channel currents were obtained with pCLAMP software (Version 6.02, Axon Instruments). The Po of a single channel was obtained by dividing the ratio of the area under the curve representing the open event by the sum of the areas under the curve representing the open and closed events. In FETCHAN (Axon Instruments) a half-amplitude algorithm is used for the inverse amplitude to detect an open event. Channel activity (NPo) was calculated as the product of the number of products N in the patch and Po. NPo or Po were collected from patches containing only 5 or less functional CAP active channels.

4) solution

The solution in a bath and pipette for single channel recording contains 140 mM Na ⁺ , 2 mM Mg ²⁺ , 144 mM Cl ⁻ , 5 mM EGTA and 10 mM HEPES and has a pH of 7.2. For whole cells the pipette solution contains 140 mM K ⁺ , 2 mM Mg ²⁺ , 144 mM Cl ⁻ , 5 mM EGTA, 10 mM HEPES and 4 mM ATP. Control perfusion solution for total cell recording contains 140 mM Na ⁺ , 5 mM K ⁺ , 2 mM Mg +, 1 mM Ca ⁺ , 151 mM Cl ⁻ and 10 mM HEPES. The synthesized compound was dissolved and stored in 100% ethanol to make a 10 mM stock solution. All other reagents used in cell culture or electrophysiological experiments were purchased from Sigma. All values are expressed as mean ± SE.

<Result>

The results are shown in Table 1 and can be summarized as follows.

(1) All compounds except 7b-Ia were tested and showed stronger agonist activity and receptor binding affinity than CAP; (2) Thiourea homolog 7a-I (Ki = 65.6 nM) showed stronger agonist activity than the CAP-associated amide homologue reported in earlier studies; (3) R ₁ [7a-V (Ki = 31.2 nM), in which the ester group at R ₁ designed to mimic the C ₃ -carbonyl group of RTX is suggested to play an important role for the carbonyl moiety in hydrogen bonding to the receptor And comparison of 7c-V (Ki = 148.7 nM) with 9a (Ki = 148 nM) and 9c (Ki = 659 nM), showing greater importance than benzylether homologs for potent agonist activity; (4) 7b-I and 7d-I (Ki = 19 nM and 10.8 nM), compounds in which pivaloyl group at R ₁ and 4-t-butylbenzyl group at R ₂ are about 280 and 490 times more potent than CAP , Respectively), which proved to be an effective hydrophobic group to obtain optimal agonist activity; (5) The binding of thiourea receptors was shown to differ significantly in receptor binding affinity compared to racemic bodies, two optically active enantiomers of 7a-I [7a-I (Ki = 65.6 nM) and (R ) -7a-I (Ki = 18.43 nM) and (S) -7a-I (Ki = 74 nM)].

Binding Affinity and Channel Activity R ₁ R ₂ R ₃ Affinity (nM) Channel activity ^a CAP 5,310 (± 370) RTX 0.023 amides (CH ₃ ) ₃ CCO H H 404 (± 37) ++ 7a-Ⅰ (CH ₃ ) ₃ CCO H H 65.6 (± 30.5) +++ (R) -7a-Ⅰ (CH ₃ ) ₃ CCO H H 18.43 (± 5.1) +++ 10 (CH ₃ ) ₃ CCO H CH ₂ OCH ₃ 146.2 (± 48) ++ (S) -7a-Ⅰ (CH ₃ ) ₃ CCO H H 74 (± 16) ++ 7a-Ⅱ (CH ₃ ) ₃ CHCO H H 132.9 (± 75) +++ 7a-Ⅲ CH ₃ (CH ₂ ) ₄ CO H H 632.3 (± 48) ++ 7a-Ⅳ CH ₃ (CH ₂ ) ₁₆ CO H H 1,873 (± 351) + 7a-Ⅴ PhCO H H 31.2 (± 9) ++ 9a PhCH ₂ H H 148 (± 11) ++ 7b-Ⅰ (CH ₃ ) ₃ CCO 3,4-diMe H 19 (± 4.3) ++ 7b-Ia (CH ₃ ) ₃ CCO 3,4-diMe CH ₂ OCH ₃ 876 (± 65) w 7b-Ⅴ PhCO 3,4-diMe H 409 (± 178) ++ 7b-Ⅵ (3,4-yaMe) PhCO 3,4-diMe H 183.5 (± 69) +++ 7c-Ⅰ (CH ₃ ) ₃ CCO 4-Cl H 54.4 (± 16) ++ 7c-Ⅴ PhCO 4-Cl H 148.7 (± 11) ++ 9c PhCH ₂ 4-Cl H 659 (± 286) ++ 7d-Ⅰ (CH ₃ ) ₃ CCO 4-t-Bu H 10.8 (± 4) ++ 7d-Ⅴ PhCO 4-t-Bu H 60.2 (± 10) ++ a; + = CAP, ++ = 10CAP, +++ = 100CAP

Experimental Example 2 Analgesic and Anti-inflammatory Activity

The analgesic activity of the most potent capsaicin agonists selected by receptor binding and single channel analysis was evaluated by PBQ-induced writing assay, and the results are shown in Table 2. As expected, the two compounds 7d-I and 7b-I, which were found to be the most potent compounds in the ex vivo assay, showed excellent analgesic activity with ED ₅₀ of 0.5 g / kg and 1 g / kg, respectively. This value indicates that analgesic activity is 300-600 times stronger than CAP. In addition, these two compounds are more potent than Albanyl or DA-5018 that are currently commercially available or have been clinically tested as topical analgesics.

In the TPA induced ear edema test, even the most potent agonists of the compounds of the present invention exhibit weak anti-inflammatory activity compared to CAP with potent anti-edema activity (Table 2). The inventors for the first time speculated that thiourea homologues dramatically reduce TPA-induced ear edema due to the high efficacy they inherently have. In this respect, their activity is similar to that of albanyl or DA-5018, and all show weak local activity in the TPA ear edema test. Weak bioavailability through transdermal administration can be explained from the weak local anti-inflammatory properties observed with this compound.

In vitro testing and analgesic activity of the compounds of the present invention can be assessed by calcium introduction assay and acetic acid induction writing assay, respectively. As shown in Table 3, all of these exhibit receptor agonist activity comparable to capsaicin. Among them, aminoethyl homologues exhibit relatively strong analgesic activity. In particular, compound 28a was found to exhibit stronger analgesic activity than DA-5018, which has been clinically tested as an analgesic agent in Korea.

<Method>

1. Mouse PBQ-induced writing analgesia analysis

Male ICR mice with an average body weight of 25 g were bred in a controlled environment with a 12-hour light cycle and used for experiments. 0.3 ml of a chemical stimulant, phenyl-p-quinone (dissolved in physiological saline containing 5% ethanol to a dosage of 4.5 mg / kg) was administered by intraperitoneal injection, and the number of abdominal contractions was measured after 6 minutes from 6 minutes. It was. Ten animals were used in one group, and the compound was dissolved in ethanol / twin-80 / saline (10/10/80) and administered intraperitoneally in a 0.2 ml dose 30 minutes before administration of phenyl-p-quinone. The reduction in the number of twisting reactions in the compound-treated group was used as an index of analgesic effect compared to the warping response of animals in the saline-only control group. The analgesic effect of each compound was represented by an ED ₅₀ value representing a 50% reduction in the number of twists.

2. Acetic acid-induced lighting test

ICR mice with an average body weight of 20 g were raised in a controlled environment with a 12 hour light and dark cycle and fasted one day before the experiment. 0.2 ml of 1.2% acetic acid solution was administered by intraperitoneal injection, and the number of abdominal contractions was measured from 6 minutes to 6 minutes. Ten animals were used in one group and drug or vesicle (10 mL / kg, i.p.) was administered 1 hour prior to the injection of acetic acid. The drug was dissolved in a solvent of ethanol / twitch-80 / physiological saline (10/10/80). Anti- analgesic activity is expressed as a decrease in the number of abdominal contractions in the drug-treated animals compared to the number of abdominal contractions in the control animals administered the vesicle.

3. TPA-induced Mouse Ear Edema Test

25 µl topical application of acetone itself or a drug dissolved in acetone was applied to the right ear of 10 male ICR mice (weight 25-30 g) per experimental group. After about 17 hours the same drug was retreated in the same manner. After 1 hour, 25 μl of 0.5 nmol of TPA dissolved in acetone was treated to the drug-treated ears. After 5 hours of TPA treatment, the animals were sacrificed and the ear tissue was cut out using a 6 mm diameter punch. Electronic scales weighing up to 0.1 mg were weighed and ear weights increased as a marker of inflammation.

The anti-inflammatory effect was expressed as% inhibition of swelling in the drug treatment group compared to the control group. % Inhibition is represented by the following formula (1).

In the formula, C and T represent an increase in ear weight in the TPA treated group and the TPA + drug treated group, respectively.

4. ⁴⁵ Ca introduction experiment

1) DRG neuron culture

DRG neurons were isolated from SD neonatal mice within 2 days of age with a slight modification of the method described above (Wood et al., 1988). In other words, DRG was isolated and collected from all the spinal cords under dissection. Ganglia were incubated for 30 minutes at 37 ° C. in 200 U / ml collagenase and 2.5 mg / ml trypsin. Digestion was stopped by adding equal amounts of DME / F12 medium containing 10% horse serum. Ganglia were pulverized by passing through a Paster pipette several times, filtered through a nylon membrane, and centrifuged. The isolated cells were plated to a density of 1500-1700neurons / well on a Terasaki plate coated with a concentration of 10 µg / ml of poly-D-ornithine. The cells were then 1: 1 with DME / F12 medium containing 1.2 g / L sodium bicarbonate, 15 mM HEPES, 50 mg / L gentamycin and 10% horse serum and C6-glioma cells grown in this medium for two days. Incubated for 3-4 days at 37 ℃ temperature in a humid atmosphere containing 5% CO ₂ in diluted media. Medium was supplemented with 250 ng / ml neuronal growth factor. Cytosine arabinoside (100 μM) was added during the first 2 days to kill divisible non-neuronal cells.

2) Introduction experiment

Terrasaki plates containing DRG neurons primary cultured for 3-4 days were washed four times with HEPES (10 mM, pH 7.4) -buffered Ca ²⁺ , Mg ²⁺ -free HBSS (H-HBSS) and equilibrated. It was done. The solution of each well was removed and medium (10 μl) containing test concentration of compound (drug) and ⁴⁵ Ca ²⁺ (10 μCi / mL) was added to each well. After incubating the nerve cells for 10 minutes at room temperature, the terasaki plates were washed six times with HEPES (10 mM, pH 7.4) -buffered Ca ²⁺ , Mg ²⁺ -free HBSS (H-HBSS) and dried in an oven. . 10 µl of 0.3% SDS was added to each well to lyse cells, and ⁴⁵ Ca ²⁺ was eluted. The contents of each well were transferred to the irradiation vial and 3 ml of Aquasol-2 scintillant were counted in. The efficacy of the test drug was calculated as a percentage using the maximum response of capsaicin at a concentration of 3 μM and the results are expressed in EC ₅₀ ± SEM.

<Result>

PBQ-Induced Lighting Test and Ear Edema Test Writhing TestED ₅₀ (㎍ / ㎏) RelativePotency Ear Edema Assay ID ₅₀ (µg / ear) RelativePotency CAP 300 One 3 One RTX 0.01 30,000 0.002 1,500 Albanil 30 10 30 0.1 DA-5018 ^* 3 100 200 0.015 Indomethacin 400 0.75 aspirin 3,500 0.086 Morphine 1,000 0.3 7a-Ⅰ 2 150 25 0.12 (R) -7a-Ⅰ 8 38 (S) -7a-Ⅰ 9 33 7a-Ⅱ 15 20 7a-Ⅲ 5 60 7a-Ⅳ 20 15 7a-Ⅴ 20 15 20 0.15 7b-Ⅰ 0.5 600 18 0.17 7b-Ⅴ 7 43 50 0.06 7b-Ⅵ 2 150 50 0.06 7c-Ⅰ 5 60 22 0.14 7c-Ⅴ 12 25 17 0.18 7d-Ⅰ One 300 5 0.6 7d-Ⅴ 1.5 200 30 0.1 DA-5018 (2- [4- (2-aminoethoxy) -3-methoxyphenyl] -N- [3- (3,4-dimethylphenyl) propyl] acetamide) C ₂₂ H ₃₀ N ₂ O ₃

Calcium introduction experiment and acetic acid induction lighting test (Ia) (Ib) R ₁ R ₂ R ₃ ⁴⁵ Ca Influx (EC ₅₀ = μM) Writhing Test (EC ₅₀ = μG / KG) I-a 19a COC (CH ₃ ) ₃ 3,4- (CH ₃ ) ₂ CH ₂ CO ₂ H 7.77 ± 0.80 431 19b COC (CH ₃ ) ₃ 4-C (CH ₃ ) ₂ CH ₂ CO ₂ H 12.3 ± 1.58 365 23a COC (CH ₃ ) ₃ 3,4- (CH ₃ ) ₂ COCH ₂ CH ₂ CO ₂ H 7.04 ± 1.3 53.1 23b COC (CH ₃ ) ₃ 4-C (CH ₃ ) ₂ COCH ₂ CH ₂ CO ₂ H 7.00 ± 1.2 60 36a COC (CH ₃ ) ₃ 3,4- (CH ₃ ) ₂ CH ₂ CH ₂ NH ₂ 0.083 ± 0.012 14.0 36b COC (CH ₃ ) ₃ 4-C (CH ₃ ) ₂ CH ₂ CH ₂ NH ₂ 0.377 ± 0.067 48.2 I-b 28a COC (CH ₃ ) ₃ 3,4- (CH ₃ ) ₂ CH ₂ CH ₂ NH ₂ 1.57 ± 0.23 0.960 28b COC (CH ₃ ) ₃ 4-C (CH ₃ ) ₂ CH ₂ CH ₂ NH ₂ 0.878 ± 0.17 8.12 Albanil 17.4 DA-5018 0.234 2.27 Capsaicin 0.435

Experimental Example 3 Pungency and Tactyphylaxis

In the development of vanilloid induction therapy, the focus of pharmacy chemistry is to improve bioavailability and reduce irritation. In connection with this purpose, a rat eye-wiping test was conducted as an in vivo stimulus test to observe the compound's pain-producing effects. As shown in Table 4, the stimulatory profiles of 7a-V, 7b-I and 7d-I selected as potent agonists do not follow the original tendency of analgesic activity measured in two in vitro assays. RTX, a capsaicin homologue that is very potent in this assay, is only three times more potent than CAP, and the compound selected in our invention is less effective in the development of acute pain. A possible explanation for the reduced stimulation of these synthetic compounds (7b-I and 7d-I) can be found in the rate of stimulation of sensory neurons.

Groups of rats treated with the selected synthetic compound to check for the occurrence of hypertolerance or cross-tolerance are treated with 0.001% CAP after 6 hours. Synthetic homologues with weak eye wiping reactions in CAP, RTX, and tests were treated in the same amount at 0.001% CAP, resulting in cross-tolerance between these classes of compounds. However, the occurrence of post-desensitization must precede the CAP homolog before the eye wiping reaction. In other words, the non-irritating dose of CAP homologue has no effect of preventing eye wiping migration by testing with 0.001% CAP.

<Method>

The onset effect of the compound was measured using an eye-wiping assay and expressed quantitatively as follows. Samples were prepared by dissolving the drug in physiological saline containing up to 5% ethanol and increasing the concentration by 10 times. The number of defense reactions (eye-wiping using the forefoot) that appeared after dropping them dropwise into rat eyes weighing 150 to 180 g was measured. Dose response correlations were derived from six groups, and moderate pain-producing potency (MPP) concentrations corresponding to 10 scratching responses were calculated. Based on this concentration, the stimulation of capsaicin was 100, and the irritability of each compound was expressed as a relative pain-producing potency (RPP).

To check for the occurrence of fast or cross fastness, 6 hours after each compound was treated, the same concentration of capsaicin at 0.001% was treated in the same eye. Mice treated with test compounds (drugs) were evaluated to be indicative of desensitization as a percentage of capsaicin at a concentration of 0.001% compared to mice treated with vesicles.

<Result>

Eye-wiping test MPP (%) ^a RPP ^b % Reduction Capsaicin 0.0003 100 55 (0.1) 35 (0.01) RTX 0.0001 300 85 (0.01) 20 (0.001) Albanil WP ^c - 0.1 DA-5018 0.003 10 90 (0.1) 35 (0.01) 7a-Ⅴ WP - 0 (0.1) 7b-Ⅰ 0.002 15 90 (0.1) 30 (0.01) 5 (0.001) 7d-Ⅰ 0.003 10 0 (0.1) a: MPP → moderate pain-producing potency b: RPP → Relative pain-producing potency c: WP → weakly pungent; weaker than 0.001% capsaicin at 0.01% concentration

As described above, the compounds of the present invention are useful for the treatment of acute, chronic, inflammatory or neuropathic pain or for the treatment of bladder hypersensitivity.

Claims (10)

New compounds represented by the following general formula (I); (Ⅰ) (In the above meal, X is an oxygen atom or a sulfur atom; A is -NHCH ₂ -or -CH ₂ ; R ₁ is an alkylaryl group having 1 to 4 carbon atoms or R ₄ CO—; (In formula, R <_4> is a C1-C18 alkyl group, a C2-C18 alkenyl group, or a C6-C10 aryl group.) R ₂ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, or a halogen atom; R ₃ is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an aminoalkyl group, a diacid monoester group, or an α-alkylacid group; And * Represents a chiral carbon atom.) And pharmaceutically acceptable salts thereof. 2. A compound according to claim 1, wherein R ₁ is a benzyl group or R ₄ CO- (wherein R ₄ is a phenyl group substituted with at least one alkyl group, phenyl group or alkyl group having 1 to 4 carbon atoms); R ₂ is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a haloalkyl group having 1 to 4 carbon atoms or a halogen atom; R ₃ is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms,-(CH ₂ ) _n NH _2- , -CO (CH ₂ ) _n CO ₂ H or-(CH ₂ ) _n CO ₂ H (wherein n is 1 or 2). A compound according to claim 1, wherein X is an oxygen atom and A is -CH _2- . A compound according to claim 1, wherein X is a sulfur atom and A is -NHCH _2- . A compound according to claim 1, wherein R ₃ is a hydrogen atom, a methoxymethyl group, -CH ₂ CH ₂ NH ₂ , -COCH ₂ CH ₂ CO ₂ H or -CH ₂ CO ₂ H. A compound according to claim 1, wherein R ₂ is selected from the group consisting of hydrogen atom, t-butyl, 3,4- (dimethyl) and chloro atom. A compound according to claim 1, wherein R ₄ is selected from the group consisting of t-butyl, i-propyl, pentyl, heptadecyl, phenyl and 3,4- (dimethyl) phenyl group. The compound according to claim 1, wherein the compound of general formula (I) 2,2-dimethylpropionic acid-2-({2- [4- (2-aminoethoxy) -3-methoxyphenyl] acetylamino} methyl) -3- (3,4-dimethylphenyl) propyl ester ; 2,2-dimethylpropionic acid-2-({2- [4- (2-aminoethoxy) -3-methoxyphenyl] acetylamino} methyl) -3- (4- t -butylphenyl) propyl ester ; 2,2-dimethylpropionic acid-2-benzyl 3- [3- (4-hydroxy-3-methoxybenzyl] thioureido} propyl ester; 2-methyl propionic acid 2-benzyl-3- [3- (4-hydroxy-3-methoxybenzyl) thioureido] propyl ester; 2,2-dimethylpropionic acid 2- (3,4-dimethylbenzyl) 3- [3- (4-hydroxy-3-methoxybenzyl) thioureido] propyl ester; And 1 type selected from the group consisting of 2,2-dimethyl propionic acid 2- (4- t -butylbenzyl) -3- [3- (4-hydroxy-3-methoxybenzyl) thioureido] propyl ester The compound characterized by the above. For ameliorating or alleviating acute, chronic, inflammatory or neuropathic pain, comprising as an active ingredient the compound (I) according to claim 1 as an active ingredient together with a pharmaceutically acceptable carrier; For inhibiting inflammation; Or a pharmaceutical composition for treating urgent incontinence. delete