JP2010111628A - Actinonin analog - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new compound that is synthesized more simply than an existing actinonin from actinonin as a lead compound and has physiological activity equal to or more than that of an existing actinonin compound. <P>SOLUTION: The new actinonin analog is obtained by substituting an asymmetric succinic acid structure in actinonin with an amino acid, etc. The compound has protease inhibitory activity, concretely, peptide deformylase (PDF) inhibitory activity and aminopeptidase (APN) inhibitory activity. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to novel actinonin analogs. Furthermore, it is related with the synthesis method.

  Actinonin is an antibiotic isolated and purified from actinomycete culture in 1962 and found to exhibit high antibacterial activity against many bacteria. The antibacterial activity exhibited by actinonin has been studied in detail, and it has been revealed that it is expressed by inhibiting peptide deformylase (PDF) (Non-patent Document 1).

In 2001, genetic analysis of malaria revealed that P. falciparum had PDF, (Non-patent document 2) Actinonin had an IC ₅₀ value of 2.5 μM against P. falciparum. (Non-Patent Document 3).

  Furthermore, it has been discovered that actinonin inhibits aminopeptidase N (APN) (Non-patent Document 4). APN is an enzyme involved in invasion and motility (metastasis) of cancer cells and inflammatory gastrointestinal diseases such as ulcerative colitis. Therefore, anticancer activity and further anti-inflammatory activity by actinonin have been reported. (Non-Patent Documents 5 and 6).

The molecular structure of actinonin is as shown in Formula IV.

  Actinonin has hydroxyproline and valine as constituent elements, but additionally contains an asymmetric succinic acid structure having a pentyl group in the side chain. Prolinol, valine, and the like are amino acids and can be obtained at low cost. However, asymmetric succinic acid compounds require several steps of asymmetric synthesis. Therefore, structural conversion is not easy, and conversion to a compound that exceeds actinonin and creation of a novel compound that separates the physiological activity of actinonin has been difficult.

Actinonine analogs have been reported by several groups, and there is a report regarding the conversion of the hydroxyproline site to benzimidazole and the discovery of antibacterial activity (Non-patent Document 6). However, there is no report in which the portion corresponding to the amino group of valine and the hydroxylamine in the actinonin molecule, that is, the asymmetric succinic acid structural site is replaced with an amino acid.
Biochemistry, 39 (6), 1256 -1262, 2000 Archives of Biochemistry and Biophysics, 396 (12), 162-170, 2001 Antimicrob Agents Chemother. 47 (8), 2545-2550, 2003 J. Clin. Invest. 114 (8), 1107-1116, 2004 Journal of Investigative Dermatology 127, 1042-1051, 2007 International Immunopharmacology, 6, 1935-1942, 2006

  The present invention uses a known actinonin as a compound having an anticancer action, an anti-inflammatory action, an antibacterial action, and an antimalarial action as a lead compound, and can be synthesized more easily than an existing actinonin, and is equivalent to an existing actinonin compound. Another object is to provide a novel compound having a physiological activity higher than that.

  As a result of intensive research, the inventors have replaced the asymmetric succinic acid structure in actinonin with amino acids and the like, and further substituted valine with various amino acids to have a novel physiological activity similar to that of actinonin. Actinonin derivatives were synthesized to complete the present invention.

（式中、Ａは、Ｃ1-3のアルキル、アミノ基等から選択され、Ｒ^1, Ｒ²は、各々直鎖アルキル、分枝アルキル、及び芳香環を有するアルキル、ヒドロキシル基、アミノ基、カルボキシル基、アミド基、チオール基、グアニジノ基などを有するアルキルから選択され、Ｗは、カルボン酸エステル、並びにカルボキシル基、ヒドロキサム酸及びそれらの塩から選択される化合物。但し、ＡがCH₂、Ｒ¹がC₅H₁₁、Ｒ²がi-Pr及びＷがCONHOHの場合を除く。）
２．下記の一般式IIで表される化合物。
下記の一般式IIで表される化合物。
一般式II：

（式中、Ｒ^1, Ｒ²は、各々直鎖アルキル、分枝アルキル、及び芳香環を有するアルキル、ヒドロキシル基、アミノ基、カルボキシル基、アミド基、チオール基、グアニジノ基などを有するアルキルから選択され、Ｗは、アルキルチオール、カルボン酸エステル、並びにカルボキシル基、ヒドロキサム酸及びそれらの塩から選択される化合物。）
３．下記の一般式IIIで表される化合物。
一般式III：

（式中、Ｒ^1, Ｒ²は、各々直鎖アルキル、分枝アルキル、及び芳香環を有するアルキル、ヒドロキシル基、アミノ基、カルボキシル基、アミド基、チオール基、グアニジノ基などを有するアルキルから選択される化合物。）
４．不斉合成工程含まない工程により合成される前項１〜３のいずれか１に記載の化合物の合成方法。
５．以下の式IVで表される化合物のうち、不斉コハク酸構造部位をアミノ酸により代替する工程を含む、前項４に記載の合成方法。
式IV：

６．前項１〜３のいずれか１に記載の化合物を有効成分として含有する薬剤。
７．有効成分が、プロテアーゼ阻害活性を有する化合物である前項６に記載の薬剤。
８．前項６又は７に記載の薬剤、並びに薬理学的及び製剤学的に許容される担体を含む医薬組成物。 That is, this invention consists of the following.
1. A compound represented by the following general formula I:
Formula I:

Wherein A is selected from C1-3 alkyl, amino group, etc., and R ^{1 and} R ² are each linear alkyl, branched alkyl, and alkyl having an aromatic ring, hydroxyl group, amino group, carboxyl Selected from alkyl having a group, an amide group, a thiol group, a guanidino group, etc., and W is a compound selected from carboxylic acid esters, and carboxyl groups, hydroxamic acids and salts thereof, provided that A is CH ₂ , R ¹ There C ₅ H _11, R ² is i-Pr and W except in the case of CONHOH.)
2. A compound represented by the following general formula II.
A compound represented by the following general formula II.
Formula II:

Wherein R ^{1 and} R ² are each selected from linear alkyl, branched alkyl, and alkyl having an aromatic ring, hydroxyl group, amino group, carboxyl group, amide group, thiol group, guanidino group, etc. W is a compound selected from alkylthiols, carboxylic acid esters, and carboxyl groups, hydroxamic acids and their salts.)
3. A compound represented by the following general formula III.
Formula III:

Wherein R ^{1 and} R ² are each selected from linear alkyl, branched alkyl, and alkyl having an aromatic ring, hydroxyl group, amino group, carboxyl group, amide group, thiol group, guanidino group, etc. Compound.)
4). 4. The method for synthesizing a compound according to any one of items 1 to 3, which is synthesized by a step not including an asymmetric synthesis step.
5). 5. The synthesis method according to item 4 above, comprising a step of substituting an asymmetric succinic acid structure site with an amino acid among the compounds represented by the following formula IV.
Formula IV:

6). A drug comprising the compound according to any one of items 1 to 3 as an active ingredient.
7). 7. The drug according to 6 above, wherein the active ingredient is a compound having protease inhibitory activity.
8). 8. A pharmaceutical composition comprising the drug according to item 6 or 7, and a pharmacologically and pharmaceutically acceptable carrier.

  The actinonin analog compound of the present invention, unlike actinonin itself, does not require a separate synthesis of succinic acid having an asymmetric carbon, so it can be expected to greatly reduce the number of synthesis steps, and further, natural amino acids are synthesized from main raw materials. Is possible. Some have PDF inhibitory activity, and some have APN inhibitory activity. Among the compounds of the present invention, compounds having PDF inhibitory activity can be expected to be applied as new antibacterial agents and antimalarial agents. Moreover, the compound which has APN inhibitory activity can anticipate the application as an anticancer agent and an anti-inflammatory agent.

（式中、Ａは、Ｃ1-3のアルキル、アミノ基等から選択され、Ｒ^1, Ｒ²は、各々直鎖アルキル、分枝アルキル、及び芳香環を有するアルキル、ヒドロキシル基、アミノ基、カルボキシル基、アミド基、チオール基、グアニジノ基などを有するアルキルから選択され、Ｗは、カルボン酸エステル、並びにカルボキシル基、ヒドロキサム酸及びそれらの塩から選択される化合物。但し、ＡがCH₂、Ｒ¹がC₅H₁₁、Ｒ²がi-Pr及びＷがCONHOHの場合を除く。） The compound of the present invention is represented by the following general formula I.
Formula I:

(In the formula, A, alkyl of C1-3, selected from amino group, R ^1, R ² are each straight-chain alkyl, branched alkyl, and alkyl having an aromatic ring, a hydroxyl group, an amino group, a carboxyl Selected from alkyl having a group, an amide group, a thiol group, a guanidino group, etc., and W is a compound selected from carboxylic acid esters, and carboxyl groups, hydroxamic acids and salts thereof, provided that A is CH ₂ , R ¹ There C ₅ H _11, R ² is i-Pr and W except in the case of CONHOH.)

（式中、Ｒ^1, Ｒ²は、各々直鎖アルキル、分枝アルキル、及び芳香環を有するアルキル、ヒドロキシル基、アミノ基、カルボキシル基、アミド基、チオール基、グアニジノ基などを有するアルキルから選択され、Ｗは、アルキルチオール、カルボン酸エステル、並びにカルボキシル基、ヒドロキサム酸及びそれらの塩から選択される化合物。） Moreover, the compound of this invention can also be represented by the following general formula II.
Formula II:

Wherein R ^{1 and} R ² are each selected from linear alkyl, branched alkyl, and alkyl having an aromatic ring, hydroxyl group, amino group, carboxyl group, amide group, thiol group, guanidino group, etc. W is a compound selected from alkylthiols, carboxylic acid esters, and carboxyl groups, hydroxamic acids and their salts.)

（式中、Ｒ^1, Ｒ²は、各々直鎖アルキル、分枝アルキル、及び芳香環を有するアルキル、ヒドロキシル基、アミノ基、カルボキシル基、アミド基、チオール基、グアニジノ基などを有するアルキルから選択される化合物。） Furthermore, the compound of this invention can also be represented by the following general formula III.
Formula III:

(Wherein, R ^1, R ² is selected from alkyl having each linear alkyl, branched alkyl, and alkyl having an aromatic ring, a hydroxyl group, an amino group, a carboxyl group, an amide group, a thiol group, a guanidino group and the like Compound.)

Specifically, the compound of the present invention is represented by the structural formula shown in the previous section, and specifically represented by the structural formula shown in Table 1.

  More specifically, among the compounds shown in the following examples, compounds shown as compounds 9a and 9b can be mentioned. Among the compounds of the present invention, examples of the PDF inhibitor include compounds shown as compounds 9a and 9b.

  The compounds of the present invention can be synthesized by the methods shown in the following examples. The method for synthesizing the actinonin analog, which is the compound of the present invention, differs from the conventional method for synthesizing actinonin. The feature is that it does not need to be synthesized separately. That is, the compound of the present invention can be synthesized without including an asymmetric synthesis step. Specifically, it can be synthesized by replacing the asymmetric succinic acid structure site with an amino acid without including an asymmetric synthesis step in the conventional actinonin synthesis method.

  In the present invention, the compound represented by any one of the general formulas I to III may be a pharmaceutically acceptable salt. In addition, in the compound of any one of the general formulas I to III or a salt thereof, when isomers (for example, optical isomers, geometric isomers, and compatible isomers) exist, the present invention includes those isomers. And also solvates, hydrates and crystals of various shapes.

In the present invention, the pharmaceutically acceptable salt includes general pharmacologically and pharmaceutically acceptable salts. Specific examples of such salts are as follows.
Examples of basic addition salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; trimethylamine salts and triethylamine salts; dicyclohexylamine salts and ethanolamines. Aliphatic amine salts such as salts, diethanolamine salts, triethanolamine salts and brocaine salts; Aralkylamine salts such as N, N-dibenzylethylenediamine; and heterocyclic aromatics such as pyridine salts, picoline salts, quinoline salts and isoquinoline salts For example, tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, Quaternary ammonium salts such as tiger butyl ammonium salt; arginine; basic amino acid salts such as lysine salts.

  Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, hydrogen carbonate, perchlorate; for example, acetate, propionate, lactate, maleate , Organic acid salts such as fumarate, tartrate, malate, citrate and ascorbate; sulfonic acids such as methanesulfonate, isethionate, benzenesulfonate, p-toluenesulfonate Salts; for example, acidic amino acids such as aspartate and glutamate.

  In the present invention, the compound represented by any one of the general formulas I to III has an antagonistic action against PDF and APN. Which of these enzyme inhibitory actions the compound of the present invention has can be easily assayed according to the method specifically shown in the experimental examples of the present specification. Specifically, for example, it can be tested by the following method.

As for the enzyme inhibitory activity, for example, APN inhibitory activity can be performed using animal cells, for example, HL-60 cells, with the enzymatic hydrolysis of the fluorescent substrate (Ala-MCA) by the compound of the present invention as an index. Specifically, dispensed 790μL amount of 50mM Tris-HClpH7.4 in 5mL plastic tube, after a DMSO solution of compound was added to ^{10μL, 2 × 10 6 cells /} mL were suspended in 50mM Tris-HClpH7.4 After adding 100 μL of the HL-60 PBS (−) cell suspension adjusted to 50 ° C., incubation was performed for 10 minutes in a constant temperature water bath adjusted to 37 ° C., and 50 mM Tris-HCl pH 7 of the fluorescent substrate Ala-MCA adjusted to 1 mM. After adding 100 μL of 4 and incubating for 30 minutes in a constant temperature bath adjusted to 37 ° C., 3 mL of 1M AcONa-AcOH pH 4.0 was added to stop the enzyme reaction, and the fluorescence intensity at an excitation wavelength of 380 nm and a fluorescence wavelength of 420 nm Can be measured.

  In the present invention, the compound represented by any one of the general formulas I to III has protease inhibitory activity. The protease inhibitory activity specifically refers to peptide deformylase (PDF) inhibitory activity or aminopeptidase N (APN) inhibitory activity.

  The scope of the present invention also includes a reagent such as a reagent or a medicine containing the compound of the present invention as an active ingredient. When used as a pharmaceutical, it can be used for diseases that can exert a therapeutic effect by protease inhibitory activity, for example, PDF inhibitory activity or APN inhibitory activity. Examples of such diseases include cancer and inflammation.

  When used as a pharmaceutical comprising the compound of the present invention as an active ingredient, the dosage is not particularly limited. For example, when the agent of the present invention is administered as a PDF inhibitory active agent or APN inhibitory active agent, an appropriate dose can be easily selected in any administration method. For example, in the case of oral administration, the active ingredient can be used in the range of about 0.01 to 1000 mg per adult day. When a drug containing the anticancer agent or anti-inflammatory agent as an active ingredient is used in combination with the drug of the present invention, the drug is administered during the administration period of these drugs and / or before or after that period. It is possible to administer the medicament of the invention.

  As the drug of the present invention, one or two or more substances selected from the compounds represented by any one of the above general formulas I to III may be administered as they are, but preferably one of the above substances or It is preferably administered as an oral or parenteral pharmaceutical composition containing two or more. Oral or parenteral pharmaceutical compositions can be prepared using pharmaceutical additives available to those skilled in the art, that is, pharmacologically and pharmaceutically acceptable carriers.

  Examples of the pharmaceutical composition suitable for oral administration include tablets, capsules, powders, fine granules, granules, liquids, and syrups. The pharmaceutical composition suitable for parenteral administration includes For example, injections, drops, suppositories, inhalants, eye drops, nasal drops, ointments, creams, patches and the like can be mentioned. Examples of pharmacologically and pharmaceutically acceptable carriers used in the production of the above pharmaceutical composition include, for example, excipients, disintegrating agents or disintegrating aids, binders, lubricants, coating agents, dyes, Diluents, bases, solubilizers or solubilizers, isotonic agents, pH adjusters, stabilizers, propellants, adhesives, and the like can be mentioned.

  In the examples of the present specification, a method for producing a preferred compound represented by the formula I of the present invention will be specifically described. Any of the compounds included in the scope of the present invention can be produced by appropriately modifying or altering the starting materials and reagents used in these production methods and reaction conditions. The manufacturing method of the compound of this invention is not limited to what was specifically demonstrated by the Example.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the scope of the following examples.

[Examples] Synthesis of target compounds FIGS. 1 to 4 show schemes of production methods for obtaining final products 9a and 9b in this example.

1) Synthesis of intermediate N-Bock-L-valine O-nitrophenol ester (2)

  N-Bock-L-valine (1) (18.8 g, 87.5 mmol) and O-nitrophenol (21.5 g, 157.5 mmol) were dissolved in pyridine (87 mL), and DCC (19.5 mg, 87. 5 mmol) was added and stirred at room temperature for 2 hours, and the completion of the reaction was confirmed by TLC plate (colored with ethyl acetate: hexane = 1: 10 ninhydrin reagent). The precipitated DCU was removed by suction filtration. The filtrate was acidified with 1N HCl solution (170 mL) and extracted with diethyl ether (200 mL). The organic layer was washed with saturated aqueous bicarbonate (100 mL × 2), water (100 mL × 2) and saturated brine (100 mL), dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure to give a yellow oily residue. . This yellow oily residue was isolated by flash column chromatography (ethyl acetate: hexane = 1: 7) and crystallized from hexane to give the title compound (pale yellow crystals). Yield 87%.

Melting point 53.6-55.0 ° C. IR (KBr) cm < ^-1 >: 3360 (NH), 1755 (C = O), 1690 (C = O). ^{[Α] D 23 -39.4 (c0.15} , CHCl 3).
_{^{1 H NMR (CDCl 3, 300MHz}} ) δ: 8.07 (1H, dd, Jo = 8.0Hz, Jm = 1.5Hz, ArH ortho to NO 2), 7.66 (1H, td, Jo = 8. 0 Hz, Jm = 1.5 Hz, ArH para to NO ₂ ), 7.41 (1H, td, Jo = 8.0 Hz, Jm = 1.0 Hz, ArH para to O), 7.28 (1H, d, Jo = 8.0 Hz, ArH ortho to O), 5.04 (1H, d, J = 8.5 Hz, NH), 4.37 (1H, q, J = 9.0 Hz, J = 4.5 Hz, NHCHCO ), 2.45 (1H, m, CHMe ₂ ), 1.47 (9H, s, CMe ₃ ), 1.11 (3H, d, J = 7.0 Hz, CHMeMe), 1.04 (3H, d , J = 7.0 Hz, CHMeMe).

2) Synthesis of intermediate N-Bock-L-valylprolinol (3)

  (S)-(+)-2-pyrrolidinemethanol (505 mg, 5.0 mmol) and triethylamine (3.0 mL) dissolved in anhydrous tetrahydrofuran (70 mL) were mixed with N-Boc-L-valine O-nitrophenol. What melt | dissolved ester (2) (1776 mg, 5.25 mmol) in anhydrous tetrahydrofuran (15 mL) was dripped, and it stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off under reduced pressure and poured into ethyl acetate (500 mL). The organic layer was washed with 2N HCl solution (200 mL), water (200 mL), saturated aqueous bicarbonate (200 mL), saturated brine (200 mL) and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure to give a yellow oil A residue was obtained. The oily residue was isolated by flash column chromatography (ethyl acetate: hexane = 2: 1) to give the title compound (yellow oil). Yield 98%.

^{[Α] D 24 -32.2 (c} 0.42, CHCl 3). IR (neat) cm ⁻¹ : 3400 br (OH), 1700 (C═O), 1630 (C═O).
¹ H-NMR (300 MHz, CDCl ₃ ): δ 5.23 (1H, d, J = 9.5 Hz, NH), 4.62 (1H, d, J = 5.5 Hz, OH), 4.28-4 _{.23 (2H, m, NHCHCO,} NCHCH 2 OH), 3.84 3.79 (1H, M, NHH '), 3.66~3.63 (1H, m, NHH'), 3.56~3 .53 (1H, m, CHH ′), 3.48 to 3.43 (1H, m, CHH ′), 2.06 to 1.85 (4H, m, NCH ₂ CH ₂ CH ₂ ), 1.62 _{~1.52 (1H, m, CHMe 2} ), 1.41 (9H, s, CMe 3), 0.97 (3H, d, CHMeMe), 0.91 (3H, d, CHMeMe)

3) Synthesis of intermediate O-benzyl-N-bock-L-valylprolinol (4)

Synthesis method (I)
N-bock-L-valylprolinol (3) (1408 mg, 4.69 mmol), sodium hydride (50% in oil, 270 mg, 5.62 mmol) and benzyl bromide (0.55 mL) in anhydrous tetrahydrofuran (34 mL) And stirred at room temperature for 36 hours. After completion of the reaction, the solvent is distilled off under reduced pressure and poured into ethyl acetate (400 mL). The organic layer was washed with 2N HCl solution (160 mL), water (160 mL), saturated aqueous hydrogen carbonate (160 mL) and saturated brine (160 mL) and then dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. A pale yellow oily residue was obtained. The residue was isolated by flash column chromatography (ethyl acetate: hexane = 1: 1) to give the title compound (yellow oil). Yield 84%.

Synthesis Method (II)
To (S)-(+)-2-pyrrolidinemethanol (549.2 mg, 5.4 mmol) and triethylamine (3.0 mL) dissolved in anhydrous tetrahydrofuran (70 mL), N-Boc-L-valine O- A solution of nitrophenol ester (3) (1839.8 mg, 5.4 mmol) dissolved in anhydrous tetrahydrofuran (15 mL) was added dropwise and stirred at room temperature for 2 hours.

  After completion of the reaction, sodium hydride (50% in oil 628 mg, 13 mmol) and benzyl bromide (1.6 mL, 13 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 36 hours. After completion of the reaction, the solvent was distilled off under reduced pressure and poured into ethyl acetate (50 mL). The organic layer was acidified with 2N HCl (10 mL), washed with saturated aqueous bicarbonate (30 mL × 2), water (30 mL × 2) and saturated brine (30 mL), dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. Distilled off. A yellow oily residue (2810 mg) was obtained. The yellow oily residue (2810 mg) was subjected to flash column chromatography. The title compound (yellow oil) was obtained from ethyl acetate: hexane = 1: 1. Yield 65%.

^{[Α] D 26 -60.0 (c} 1.0, CHCl 3). ^{IR (neat) cm -1: 3300} (NH), 1750 (C = O), 1640 (C = O).
¹ H-NMR (300 MHz, CDCl ₃ ): δ 7.33-7.27 (5H, m, ArH), 5.28 (1H, d, J = 9.3 Hz, NH), 4.49 (2H, d , J = 2.4 Hz, CH ₂ Ph), 4.35 to 4.24 (2H, m, NHCHCO, NCHCH ₂ O), 3.68 to 3.46 (4H, m, NCH ₂ , NCHCH ₂ O) _{, 2.04~1.88 (5H, m, NCH} 2 CH 2 CH, CHMe 2), 1.42 (9H, s, CMe 3), 0.95 (3H, d, J = 7.0Hz, CHMeMe ), 0.89 (3H, d, J = 7.0 Hz, CHMeMe).

4) Synthesis of intermediate O-benzyl-N-bock-L-methionylvalylprolinol (6a)

  O-Benzyl-N-Bock-L-valylprolinol (4) (1543 mg, 3.95 mmol) was dissolved in methylene chloride (7.11 mL), trifluoroacetic acid (4.3 mL) was added dropwise, and 1 at room temperature. Stir for hours. After completion of the reaction, the reaction mixture was poured into ethyl acetate (400 mL), washed with saturated aqueous bicarbonate and water (160 mL each), dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. A colorless oily residue of O-benzyl-L-valylprolinol (1166 mg) was obtained.

  After dissolving O-benzyl-N-Bock-L-valylprolinol (1166 mg), N-Bock-L-methionine (1083 mg, 4.34 mmol) and HOBt (587 mg, 4.34 mmol) in dimethylformide (27 mL). , EDC (830 mg, 4.34 mmol) is added and stirred for 2 hours. After completion of the reaction, the reaction mixture was poured into ethyl acetate (400 mL), washed with saturated aqueous bicarbonate, water and saturated brine (160 mL each), dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. A colorless oily residue was obtained. Flash column chromatography was performed, and the title compound was obtained from the fraction eluted with ethyl acetate: hexane = 2: 3. Yield 76%.

[Α] D ²⁶ 56.1 (c 1.0, CHCl ₃ ). ^{IR (neat) cm -1 3300 (} NH), 1640 (C = O).
¹ H-NMR (300 MHz, CDCl ₃ ): Store δ 732 to 7.29 (5H, m, ArH), 6.81 (1H, d, J = 10.8 Hz, NH), 5.16 (1H, d, J = 8.4 Hz, NH), 4.59-4.50 (1H, m, NHCHCO), 4.48 (2H, d, J = 2.1 Hz, CH ₂ Ph), 4.30 to 4.26. (2H, m, NHCHCO, NCHCH ₂ O), 3.69 to 3.46 (4H, m, NCH ₂ , NCHCH ₂ O), 2.55 (2H, t, J = 6.9 Hz, SCH ₂ CH _{2 ), 2.10 (1H, s,} CH 3 S), 2.08~1.89 (7H, m, NCH 2 CH 2 CH, CHMe 2, SCH 2 CH 2), 1.44 (9H, s, _{CMe 3), 0.94 (3H,} d, J = 7.0Hz, CHMeMe), 0. 9 (3H, d, J = 7.0Hz, CHMeMe).

¹³ C-NMR (75 MHz, CDCl ₃ ): 171.4, 170.1, 155.5, 138.4, 128.4, 127.6, 127.5, 80.0, 73.2, 70.1 , 56.8, 56.8, 55.9, 53.6, 47.8, 31.9, 31.6, 30.2, 28.4, 27.4, 24.6, 19.5.

5) Synthesis of intermediate O-benzyl-O-benzyloxycarbonyl-L-methionylvalylprolinol (8a)

  O-Benzyl-N-Bock-L-methionylvalylprolinol (6a) (624 mg, 1.2 mmol) was dissolved in methylene chloride (2 mL), trifluoroacetic acid (1.3 mL) was added dropwise, and 1 mL was added at room temperature. Stir for hours. After completion of the reaction, the reaction mixture was poured into ethyl acetate (150 mL), washed with saturated aqueous bicarbonate (60 mL) and saturated brine (30 mL), dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. A colorless oily residue, O-benzyl-L-methionylvalylprolinol, was obtained.

O-benzyl-L-methionylvalylprolinol (459 mg), 4-nitrophenyl-N- (O-benzylhydroxy) carbamate (345 mg, 1.2 mmol), triethylamine (0.167 mL) was added to CH ₂ Cl ₂ (14 ) And stirred for 1.5 hours. After completion of the reaction, the mixture was washed with 1M sodium hydroxide solution (30 mL × 3), 1M HCl (30 mL) and water (30 mL), and then dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. A colorless oily residue was obtained. The title compound was obtained by flash column chromatography with ethyl acetate: hexane = 1: 1. Yield 73%.

IR (CHCl ₃ ) cm ⁻¹ : 3400 (NH), 1640 (C═O), 1680 (C═O).
¹ H-NMR (300 MHz, CDCl ₃ ): 7.38-7.29 (10H, m, ArH).
7.04 (1H, d, J = 9.0 Hz, NH), 6.33 (1H, d, J = 9.0 Hz, NH), 4.80 (2H, d, J = 5.1 Hz, CH _{2 Ph), 4.56~4.51 (2H, m} , NHCHCO, NHCHCO), 4.47 (2H, s, CH 2 Ph), 4.30 (1H, s, NCHCH 2 O), 3.70~ 3.33 (4H, m, NCH ₂ , NCHCH ₂ O), 2.44 (2H, t, J = 7.2 Hz, SCH ₂ CH ₂ ), 2.07 (3H, s, CH ₃ S), 2 _{.04~1.86 (7H, m, NCH 2} CH 2 CH 2, CHMe 2, SCH 2 CH 2), 0.93 (3H, d, J = 6.6Hz, CHMeMe), 0.87 (3H, d, J = 6.6 Hz, CHMeMe).

¹³ C-NMR (75 MHz, CDCl ₃ ): 171.3, 170.3, 159.6, 138.5, 135.6, 129.4, 128.8, 128.4, 127.8, 127.5 78.7, 73.3, 70.1, 56.8, 56.1, 52.4, 47.9, 32.5, 31.5, 30.1, 27.4, 24.6, 19 4, 17.9, 15.4.

6) O-Benzyl-N- (N- (O-benzylhydroxycarbonyl) -L-methionyl-valyl) (9a) Synthesis of L-prolinol

  O-benzyl-O-benzyloxycarbonyl-L-methionylvalylprolinol (8a) (228 mg, 0.4 mmol) was dissolved in anhydrous tetrahydrofuran (8 mL), and liquid ammonia (about 45 mL) was added while cooling at −78 ° C. Dripping. Add sodium a little at a time until the color of the reaction mixture turns blue. Add ammonium chloride until the blue color of the reaction solution disappears. After liquid ammonium is distilled off, the temperature is raised to room temperature. Pour the residue into ethyl acetate and filter. Dry over anhydrous magnesium sulfate and evaporate the solvent under reduced pressure. A colorless oily residue was obtained. The title compound was obtained by dissolving in methylene chloride and performing flash column chromatography with ethyl acetate: 2-propanol = 20: 1. Yield 39%.

IR (CHCl ₃ ) cm ⁻¹ : 3400 (OH), 1720 (C═O), 1670 (C═O), 1620 (C═O). ¹ H-NMR (300 MHz, CDCl ₃ ): 8.48 (1H, s, OH), 7.89 (1H, s, OH), 7.74 (1H, d, J = 8.7 Hz, NH), 6.76 (1H, d, J = 8.4 Hz, NH), 4.70 to 4.52 (2H, m, NHCHCO, NHCHCO), 4.42 (1H, brs, NH), 4.24 ( 1Hbr s, NCHCH ₂ OH), 3.90 to 3.86, 3.64 to 3.49 (4H, m, NCH ₂ , NCHCH ₂ OH), 2.55 (2H, m, SCH ₂ CH ₂ ), _{2.08 (3H, s, CH 3} S), 2.14~1.73 (7H, m, NCH 2 CH 2 CH 2, CHMe 2, SCH 2 CH 2), 0.96~0.92 (6H , M, CHMeMe).

¹³ C-NMR (75 MHz, CDCl ₃ ): 172.5, 172.2, 161.8, 65.1, 60.8, 56.5, 53.2, 48.4, 32.2, 31. 4, 30.3, 27.7, 24.3, 19.4, 18.3.

7) Synthesis of intermediate O-benzyl-N- (N-bock-L-valyl-L-norleucyl) -L-prolinol (6b)

O-benzyl-N- (N-bock-L-valyl) -L-prolinol (4) (301.1 mg, 0.77 mmol) was dissolved in methylene chloride (1.4 mL) and trifluoroacetic acid (0. 85 mL) was added dropwise and stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was poured into ethyl acetate (30 mL), washed with saturated aqueous bicarbonate, water (each 30 mL × 2) and saturated brine (30 mL), dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. A colorless oily residue was obtained.
After dissolving O-benzyl-L-valyl-L-prolinol (200 mg, 0.68 mmol), norleucine (175 mg, 0.76 mmol) and HOBt (101.1 mg, 0.74 mmol) in dimethylformamide (5 mL), EDC (142.9 mg, 0.75 mmol) was added and stirred for 1.5 hours. After completion of the reaction, the reaction mixture was poured into ethyl acetate (20 mL), washed with saturated aqueous hydrogen carbonate, water (each 20 mL × 2) and saturated brine (20 mL), dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. A colorless oily residue was obtained. The colorless oily residue was subjected to flash column chromatography. The title compound was obtained from the eluate of ethyl acetate: hexane = 1: 1. Yield 68%.

^{1 H-NMR (300MHz, CDCl} 3) δ: 7.29~7.23 (5H, m, ArH '), 6.80 (1H, d, J = 8.7Hz, NH), 5.06 (1H , D, J = 8.4 Hz, NH), 4.45 (1H, d, J = 1.8 Hz, CH _A H _B Ph), 4.45 (1H, d, J = 1.8 Hz, CH _A H _B Ph), 4.31 to 4.27 (1H, m, NHCHCO), 4.10 to 4.05 (1H, m, NCH ₂ CH ₂ CH ₂ CH), 3.67 to 3.44 (4H , M, CH ₂ , OCH ₂ Ph, NCH ₂ CH ₂ CH ₂ CH), 2.01 to 1.55, 1.27 to 1.22 (11H, m, CH (Me) ₂ , CHCH ₂ CH ₂ CH _{2 CH 3, NCH 2 CH 2} CH 2 CH), 1.42 (9H, s, CMe 3), 0.92~0. _{85 (9H, m, CHMe 2} , CHCH 2 CH 2 CH 2 CH 3). ^{IR (KBr) cm -1: 3300} (NH), 1710 (C = O), 1630 (C = O).

8) Synthesis of intermediate O-benzyl-O-benzyloxycarbamoyl-L-norleucylvalylprolinol (8b)

  O-benzyl-N- (N-Bock-L-valyl-L-norleucyl) -L-prolinol (6b) (262.3 mg, 0.52 mmol) was dissolved in methylene chloride (1 mL) and trifluoroacetic acid ( 0.6 mL) was added dropwise and stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was poured into ethyl acetate (30 mL), washed with saturated aqueous hydrogen carbonate, water (each 30 mL × 2) and saturated brine (30 mL), dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. A colorless oily residue was obtained. O-benzyl-N- (L-valyl-L-norleucyl) -L-prolinol (228.4 mg, 0.57 mmol), 4-nitrophenyl-N- (O-benzyloxy) carbamate (63.5 mg) , 0.57 mmol) and triethylamine (0.08 mL, 0.57 mmol) were dissolved in methylene chloride (7 mL) and stirred for 2 hours. After completion of the reaction, the mixture was washed with 1M sodium hydroxide (20 × 3 mL), 1M HCl aqueous solution (20 mL) and water (20 mL), dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. A colorless oily residue was obtained. The title compound was obtained from the colorless oily residue by flash column chromatography from an eluate of ethyl acetate: hexane = 1: 1. Yield 71%.

¹ H-NMR (300 MHz, CDCl ₃ ) δ: 8.21 (1H, d, J = 9.0 Hz, NH), 7.89 (1H, d, J = 7.5 Hz, NH), 7.40 to 7.26 (10H, m, ArH ′), 6.12 (1H, d, J = 8.7 Hz, NH), 4.75 (4H, d, J = 1.8 Hz, CH ₂ Ph, CH ₂ Ph _{), 4.28~4.29 (1H, m,} NHCHCO), 3.57~3.55 (4H, m, CH 2, OCH 2 Ph, NCH 2 CH 2 CH 2 CH), 2.44~2 _{.43 (1H, m, NHCHCO)} , 2.07~1.88 (4H, m, NCH 2 CH 2 CH 2 CH), 1.83~1.14 (6H, m, CHCH 2 CH 2 CH 2 CH _{3), 0.87~0.83 (9H, m} , CHMe 2, CHCH 2 CH 2 CH 2 CH ₃ ).

¹³ C-NMR (75 MHz, CDCl ₃ ) δ: 172.3, 170.5, 159.8, 138.4, 135.7, 129.3, 128.3, 128.3, 127.5, 127. 4, 129.3, 128.6, 128.4, 128.3, 127.5, 127.4, 78.5, 73.1, 70.0, 56.7, 56.0, 53.0, 47.8, 33.0, 31.3, 28.3, 27.5, 27.3, 24.4, 22.4, 19.3, 18.1, 14.0.

9) Synthesis of O-benzyl-N- (N- (O-benzylhydroxycarbonyl) -L-norleucyl-valyl) -L-prolinol (9b)

  O-benzyl-O-benzyloxycarbamoyl-L-norleucylvalylprolinol (8b) was dissolved in methanol (5 mL), acetic acid (0.6 mL) was added, and 10% palladium / carbon (27 mg) was added. In addition, the mixture was stirred at room temperature for 6 hours under a hydrogen (1 atm) atmosphere. After completion of the reaction, 10% palladium / carbon was filtered, extracted with water (20 mL × 2), washed with aqueous hydrogen carbonate (30 mL), dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. A colorless oily residue was obtained. Perform flash column chromatography. The title compound was obtained from the eluate of ethyl acetate: isopropyl alcohol = 1: 1.

[Experimental Example] Measurement of Enzyme Inhibitory Activity Regarding enzyme inhibitory activity, for example, APN inhibitory activity was performed using HL-60 cells and using enzymatic hydrolysis of a fluorescent substrate (Ala-MCA) as an index. Specifically, 790 μL of 50 mM Tris-HCl pH 7.4 was dispensed into a 5 mL plastic tube, 10 μL of a DMSO solution of a synthetic compound was added, and then 2 × 10 ⁶ cells / mL suspended in 50 mM Tris-HCl pH 7.4. After adding 100 μL of the HL-60 PBS (−) cell suspension adjusted to 50 ° C., incubation was performed for 10 minutes in a constant temperature water bath adjusted to 37 ° C., and 50 mM Tris-HCl pH 7 of the fluorescent substrate Ala-MCA adjusted to 1 mM. After adding 100 μL of 4 and incubating for 30 minutes in a constant temperature water bath adjusted to 37 ° C., 3 mL of 1M AcONa-AcOH pH 4.0 was added to stop the enzyme reaction, and the fluorescence intensity at an excitation wavelength of 380 nm and a fluorescence wavelength of 420 nm was measured. Can be measured.

  As described above in detail, since the compound of the present invention (actinonin analog) has protease inhibitory activity, specifically, PDF inhibitory activity and APN inhibitory activity, it exhibits therapeutic effects by PDF inhibitory activity and APN inhibitory activity. Can be used against possible diseases. Examples of such diseases include cancer and inflammation, and can be used as a medicine for such diseases. It can also be used as a biochemical test reagent.

FIG. 3 is a diagram showing a synthesis scheme of a compound of Intermediate 6. FIG. 3 is a diagram showing a synthesis scheme of a compound of Intermediate 7. FIG. 3 is a diagram showing a synthesis scheme of a compound of Intermediate 8. 2 is a diagram illustrating a synthesis scheme of a target compound 9. FIG.

Claims (8)

A compound represented by the following general formula I:
Formula I:

Wherein A is selected from C1-3 alkyl, amino group, etc., and R ¹ and R ² are each linear alkyl, branched alkyl, and alkyl having an aromatic ring, hydroxyl group, amino group, carboxyl Selected from alkyl having a group, an amide group, a thiol group, a guanidino group, etc., and W is a compound selected from carboxylic acid esters, and carboxyl groups, hydroxamic acids and salts thereof, provided that A is CH ₂ , R ¹ There C ₅ H _11, R ² is i-Pr and W except in the case of CONHOH.) A compound represented by the following general formula II.
Formula II:

(Wherein, R ^1, R ² is selected from alkyl having each linear alkyl, branched alkyl, and alkyl having an aromatic ring, a hydroxyl group, an amino group, a carboxyl group, an amide group, a thiol group, a guanidino group and the like W is a compound selected from alkylthiols, carboxylic acid esters, and carboxyl groups, hydroxamic acids and their salts.) A compound represented by the following general formula III.
Formula III:

Wherein R ^{1 and} R ² are each selected from linear alkyl, branched alkyl, and alkyl having an aromatic ring, hydroxyl group, amino group, carboxyl group, amide group, thiol group, guanidino group, etc. Compound.) The method for synthesizing a compound according to any one of claims 1 to 3, which is synthesized by a step not including an asymmetric synthesis step. The synthesis | combining method of Claim 4 including the process of substituting an asymmetric succinic-acid structure site | part with an amino acid among the compounds represented by the following formula IV.
Formula IV:

The chemical | medical agent containing the compound of any one of Claims 1-3 as an active ingredient. The drug according to claim 6, wherein the active ingredient is a compound having protease inhibitory activity. A pharmaceutical composition comprising the drug according to claim 6 or 7, and a pharmacologically and pharmaceutically acceptable carrier.

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