JPH0539266A - New tryptophan derivative - Google Patents

Abstract

PURPOSE:To obtain a new compound useful for treating hypertension and ache, specifically inhibiting the actions of angiotensin converting enzymes, enkephalinase and endothelin converting enzyme. CONSTITUTION:A compound shown by formula I (A is single bond or lower alkylene; R1 and R2 are H or aralkyl; Trp is tryptophan residue) such as N-(RS)-[2-(hydroxyaminocarbonyl)-3-methyl-1-oxobutyl]-L-tryptophan. The compound is obtained by amidating a carboxylic acid shown by formula II or a reactive derivative thereof with an amine shown by formula III in a solvent (e.g. pyridine). this compound shows hypotensive action, inhibitory action on bradykinin inactivation, inhibitory action on ANP inactivation and inhibitory action on cerebrovascular contraction and coronary blood vessel contraction. The compound suppresses decomposition of enkephalin and raises opioid activity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel tryptophan derivative which specifically inhibits the actions of angiotensin converting enzyme, enkephalinase and endothelin converting enzyme.

[0002]

BACKGROUND OF THE INVENTION Angiotensin-converting enzyme and endothelin-converting enzyme are mainly present in vascular endothelial cells, and angiotensin II and endothelin produced by them are both in vivo substances that strongly contract blood vessels. .. On the other hand, enkephalinase is present in the striatum of the brain, spinal cord, kidney, etc., and is involved in the inactivation of the endogenous morphine-like substance enkephalin activity, and is also referred to as Neutral Endopeptide Ace (EC3.4.24.11). Angiotensin converting enzyme is abundantly present in lung vascular endothelial cell membrane, renal tubular and brush green membrane of small intestine, Asp ^1-
Arg ² -Val ³ -Tyr ⁴ -Ile ⁵ -His ⁶ -Pro ⁷ -Phe ⁸ -His ⁹ -Le
Angiotensin that acts on angiotensin I, which is a decapeptide composed of u ^10, and cleaves and releases the His ⁹ -Leu ¹⁰ dipeptide from its C terminal, and has a strong pressor action
It is an enzyme that produces II. It is also known that this enzyme has a function of destroying and inactivating Bradyginin, which is an antihypertensive substance in vivo, and is strongly involved in the pressor system.

It has been conventionally considered that inhibition of the activity of angiotensin converting enzyme acts on blood pressure reduction and is clinically effective for the prevention and treatment of hypertension. Also, in the past research, it has been known that several kinds of peptides extracted from biological components have angiotensin converting enzyme inhibitory activity, but Cheung et al. Further focused on the affinity site between the peptide and the enzyme, As a result of an inhibition experiment using various dipeptides and the like, it was found that Val-Trp and N-mercaptopropanoyltryptophan show particularly excellent inhibitory activity (J. Biol. Chem., 255 , 401, 198).
0).

In addition, endothelin is a potent vasoconstrictor peptide isolated from the culture supernatant of vascular endothelial cells (Ya
nagisawa et al., Nature, 332 , 411-415, 1988). Big endothelin (39 amino acids) is first excised from the endothelin precursor, and then the endothelin-converting enzyme specifically cleaves the Trp (21) -Val (22) bond of the big endothelin to endothelin (21 amino acids). To be converted. Big endothelin, compared to endothelin
Endothelin-converting enzyme inhibitors are effective for cardiovascular diseases, such as hypertension, ischemic heart disease, and renal disease, because vasoconstrictor activity is low and it is thought that big endothelin is converted to endothelin in the body to act. It is expected.

Endothelin was most recently (1988)
Since it was discovered in this study, the research on inhibitors is new, and there are few reports on inhibitors of endothelin converting enzyme. The only known phosphoramidon inhibits endothelin converting enzyme by the action of metal chelates.

Enkephalin is a morphine-like substance in vivo isolated from the brains of pigs and cows (Hughes, J. Smith.
Et al., Nature, 258 , 579, 1975). The structure of enkephalin is composed of 5 amino acids, and it is said that enkephalinase specifically cleaves Gly ³ -Phe ⁴ to lose its activity. Therefore, many inhibitors specific to enkephalinase have been investigated, and thiorphan, keratorphan, etc. have been reported as candidates for central analgesics (Dickenson, AH Nature, 320 , 681, 1986).

On the other hand, enkephalinase cleaves the Cys ⁷ -Phe ⁸ and Ser ²⁵ -Phe ²⁶ bonds of natriuretic hormone Atrial natriuretic peptide aka ANP,
Known to be involved in inactivating ANP (No
rthridge, DG Lancent, Sep, 9, 591 , 1989). Therefore, enkephalinase (also known as atriopeptidase) inhibitors have been studied as experimental drugs for heart failure and hypertension in experimental animals, clinical trials, etc. (Sybertz, EJ and others, Hypertens
ion, 15 , 152, 1990. Margulies, KB Other, Kidney In
t, 38 , 67, 1990).

The present inventors have synthesized a novel tryptophan derivative, and have conducted diligent research on its angiotensin-converting enzyme, endothelin-converting enzyme and enkephalinase inhibitory activity. As a result, the compound represented by the following general formula is known. The present invention has been completed by discovering that it has a significantly superior inhibitory activity than the compound.

[0009]

The present invention provides a general formula,

[0010]

[Chemical 2]

(Where A is a single bond or a lower alkylene group, R ¹ and R ² are hydrogen atoms or aralkyl groups, and T
rp means tryptophan residue. ) Relating to a novel tryptophan derivative.

The compound of the present invention will be described in detail below. In the definition of the general formula in the present specification, the term “lower” means a straight or branched carbon chain having 1 to 6 carbon atoms unless otherwise specified.

Therefore, the "lower alkyl group" constituting the above "aralkyl group" is specifically, for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert. -Butyl group, pentyl group, isopentyl group, neopentyl group,
Examples thereof include tert-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 1,2-dimethylpropyl group, hexyl group and isohexyl group.

The "lower alkylene group" is preferably an alkylene group having 1 to 6 carbon atoms, specifically,
Methylene group, ethylene group, methylmethylene group, trimethylene group, propylene group, 2-propylene group, dimethylmethylene group, tetramethylene group, 1-methyltrimethylene group, 2-methyltrimethylene group, 3-methyltrimethylene group, 1-ethylethylene group, 2-ethylethylene group,
2,2-dimethylethylene group, 1,1-dimethylethylene group, ethylmethylmethylene group, pentamethylene group, 1
-Methyltetramethylene group, 2-methyltetramethylene group, 3-methyltetramethylene group, 4-methyltetramethylene group, hexamethylene group, 1-methylpentamethylene group and the like.

The "aralkyl group" means a group in which any hydrogen atom of the above "lower alkyl group" is substituted with an aryl group such as a phenyl group and a naphthyl group. For example, when an aryl group is exemplified by a phenyl group, Specific examples thereof include a benzyl group, a phenethyl group, a 1-phenylethyl group, a benzhydryl group and a trityl group.

The compound represented by the general formula (I) contains two asymmetric carbon atoms and has optical isomers such as diastereomers. The present invention includes all the isolated forms of these isomers and their mixtures.
The compound (I) of the present invention may form an acid addition salt. Also, depending on the type of substituent, it may form a salt with a base. Specific examples of such salt include hydrochloric acid,
Hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, mineral acids such as phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid,
Succinic acid, fumaric acid, maleic acid, lactic acid, malic acid,
Organic acids such as tartaric acid, citric acid, methanesulfonic acid and ethanesulfonic acid, acid addition salts with acidic amino acids such as aspartic acid and glutamic acid, inorganic bases such as sodium, potassium, magnesium and aluminum, methylamine,
Examples thereof include organic bases such as ethylamine and ethanolamine, salts with basic amino acids such as lysine and ornithine, and ammonium salts. Further, the present invention also includes hydrates of the compound (I) of the present invention, various solvates and polymorphic substances.

[0017]

[Production Method] The production method of the compound (I) of the present invention is described below.

[0018]

[Chemical 3]

(In the formula, A, R ¹ and R ² have the same meanings as described above.) The amide compound represented by the general formula (I) which is the compound of the present invention is represented by the formula (II). Carboxylic acid or a reactive derivative thereof, represented by the general formula (II
It can be produced by amidating the amine represented by I) by a conventional method.

Examples of the reactive derivative of the compound (II) include acid halides such as acid chloride and acid bromide; acid azides;
Examples thereof include active esters such as N-hydroxybenzotriazole and N-hydroxysuccinimide; symmetrical acid anhydrides; mixed acid anhydrides such as alkyl carbonic acid and p-toluenesulfonic acid.

When the compound (II) is reacted with a free carboxylic acid, dicyclohexylcarbodiimide (DC
It is advantageous to carry out in the presence of a condensing agent such as C) or 1,1'-carbonyldiimidazole.

The reaction conditions are the starting compounds, especially the compound (II)
Pyridine, tetrahydrofuran, dioxane, ether, N,
N-dimethylformamide, benzene, toluene, xylene, methylene chloride, dichloroethane, chloroform, ethyl acetate, acetonitrile and the like in an organic solvent inert to the reaction, the starting compounds (II) and (III) are equimolar to the starting compounds (III) It is advantageous to react with a slight molar excess.

Depending on the kind of the reactive derivative, or when a salt of the starting compound (III) is used, trimethylamine, triethylamine, pyridine,
It is advantageous to carry out in the presence of an organic base such as picoline, lutidine, dimethylaniline and N-methylmorpholine, and a base such as an inorganic base such as potassium carbonate, sodium carbonate, sodium hydrogen carbonate, sodium hydroxide and potassium hydroxide. There are cases. Pyridine can also serve as a solvent.

The reaction temperature varies depending on the kind of the reactive derivative and is set appropriately. R ¹ and R ² of compound (I)
The compound in which is an aralkyl group can be reduced to a compound (I) in which R ¹ and / or R ² is a hydrogen atom, if necessary. Reduction is R ¹ and R
^It is preferable to simultaneously remove the ^two aralkyl groups, and catalytic reduction using palladium-carbon as a catalyst is advantageous. The catalytic reduction can be carried out by applying a conventional method.

The starting compound (II) is obtained by hydrolyzing one of the dicarboxylic acid diesters under controlled conditions, and adding aralkyloxyamine to the obtained monocarboxylic acid to amidate the amide compound. It can be easily obtained by hydrolyzing a. At this time, the amidation can be carried out in the same manner as the amidation. The reaction product obtained by each of the above production methods is isolated and purified as a free compound, its salt or various solvates. The salt can be produced by subjecting it to an ordinary salt-forming reaction. Isolation and purification include extraction, concentration, evaporation, crystallization, filtration,
It is carried out by applying ordinary chemical operations such as recrystallization and various kinds of chromatography.

[0026]

INDUSTRIAL APPLICABILITY The compound (I) of the present invention has excellent angiotensin-converting enzyme inhibitory activity, endothelin-converting enzyme inhibitory activity and enkephalinase (neutral endopeptidase) inhibitory activity, and has an antihypertensive effect and a bradyginin-inhibiting effect. Inhibition of activation, inhibition of ANP inactivation, inhibition of cerebral and coronary spasm, essential hypertension,
It is useful as a prophylactic and therapeutic agent for pathological conditions such as hypertension such as renal hypertension and adrenal hypertension, congestive heart failure, and vasospasm, as a diagnostic agent for these diseases and as a cardiovascular agent used in various pathological conditions. .. In addition, the compound (I) of the present invention has a strong enkephalinase inhibitory activity, and enhances the endogenous opioid activity by suppressing the degradation of enkephalin, and is useful as a diagnostic agent, preventive agent, or therapeutic agent for chronic pain diseases. Is.

The above-mentioned enzyme inhibitory activities of the compound (I) of the present invention were confirmed by the following methods.

I. Measurement of angiotensin-converting enzyme inhibitory activity Experimental materials: (1) Angiotensin-converting enzyme from pig serum [Arch.Biochem.Biophys., 240 , 330-33
6 (1985)] Purified according to the method described. (2) Enzyme substrate hippril-histidyl-leucine (manufactured by Protein Research Institute)

[0029]

[Chemical 4]

(3) Test compound A compound represented by the following formula, obtained in Example 2.

[0031]

[Chemical 5]

Method for measuring inhibitory activity: The enzyme activity of angiotensin converting enzyme in the presence and absence of a test compound was measured by an enzyme assay method, and the inhibitory activity of the sample was determined from the inhibition rate of the test compound determined from the enzyme activity. It was calculated the IC _50.

The angiotensin converting enzyme activity is measured by
Modified method of HAYAKARI et al. [Anal. Biochem., 84 , 361-369.
(1978)].

That is, 10 mg of hippril-histidyl-leucine (enzyme substrate) in 0.5M Tris-HCl buffer (pH 7.4) containing 3M sodium chloride.
Dissolve it in 1 ml / ml and put it in a 250 μl reaction tube,
The test compound, angiotensin converting enzyme and water were added thereto to make a final volume of 250 μl, and the mixture was incubated at 37 ° C. for 10 minutes.

Next, a 1N sodium hydroxide aqueous solution 15 μ
The reaction was stopped by adding 1 and left at room temperature for 30 minutes.
1 ml of 0 mM sodium phosphate buffer (pH 7.2),
1 ml of methyl cellosolve containing 1% cyanuric acid chloride was added, and 15 minutes later, the absorbance was measured with visible light at 382 nm.

Measured value of absorbance in a reaction solution containing no test compound (amount of hippuric acid liberated from the enzyme substrate due to enzyme activity. Incidentally, hippuric acid reacts with cyanuric chloride to give a yellow coloration) Then, the enzyme activity was calculated by the following formula.

Enzyme activity (mU / ml) = 595 × A₃₈₂ * Enzyme activity 1 mU is 1 nmole of enzyme substrate change per minute
Activity that catalyzes oxidization * A₃₈₂ At 382 nm for the reaction solution containing no sample
Absorbance

Further, the absorbance (a) of the reaction solution containing the test compound, the absorbance (b) not containing the test compound, and the blind absorbances (a 'and b) which do not react to each of them
The inhibition rate was calculated from the following equation.

[0039]

[Equation 1]

The inhibitory activity IC ₅₀ of the angiotensin converting enzyme inhibitor was defined as the concentration (ng / ml) of the test compound required to inhibit the enzyme activity of the angiotensin converting enzyme by 50% (inhibition rate).

Experimental results: The inhibitory activity (IC ₅₀ value) of the test compound against angiotensin converting enzyme was 4.5 ng / ml.

II. Measurement of endothelin-converting enzyme inhibitory activity Experimental materials: (1) Endothelin-converting enzyme Human umbilical vein derived vascular endothelial cells Reference [Biochem. Bio
phys. Res. Commun., 171 , 1192-1198 (1990)]. (2) Enzyme substrate Human Big Endothelin-1 (Peptide Institute)

[0043]

[Chemical 6]

(3) Test compound The compound obtained in Example 2.

Method for measuring inhibitory activity: The inhibitory activity for endothelin-converting enzyme was determined by measuring the endothelin produced by endothelin-converting enzyme in the presence and absence of a test compound by an enzyme assay method. From this, the inhibitory activity IC ₅₀ of the test compound was calculated.

Endothelin can be measured by a modified method of OKADA et al. [Biochem. Biophys. Res. Commun., 171 , 1192-1198 (199
0)].

That is, human big endothelin-1
(Enzyme substrate) is dissolved in 0.1% acetic acid aqueous solution to 100 μM, put into a 5 μl reaction test tube, and the test compound, endothelin converting enzyme and 0.2 M Tris-HCl buffer (pH 7.2) are added thereto. To a final volume of 100 μl, 3
Incubate at 7 ° C for 3 hours.

Then, 100 μl of 1% acetic acid aqueous solution was added to stop the reaction, and the mixture was centrifuged at 4 ° C. for 30 minutes, and the supernatant 10
A reverse phase ODS column (Capc) in which an aqueous solution containing 28.5% acetonitrile, 10% tetrahydrofuran, 0.18% trifluoroacetic acid was equilibrated at a flow rate of 1 ml / min.
ell-pak C 18, AG-120, SHISEIDO), and the excitation wavelength was 290 nm and the fluorescence wavelength was 35 by an on-line fluorometer.
The fluorescence that appeared at the retention time of endothelin at 0 nm was measured.

The amount of endothelin released was determined from the measured value of fluorescence in the reaction solution containing no test compound (proportional to the amount of endothelin released from the enzyme substrate due to the enzyme activity).

Further, the inhibition rate was determined from the fluorescence degree (a) of the reaction solution containing the test compound and the fluorescence degree (b) not containing the test compound by the following formula.

[0051]

[Equation 2]

Inhibitory activity of endothelin converting enzyme inhibitors I
The C ₅₀ value indicates the enzyme activity of the endothelin converting enzyme at 50
% (Inhibition rate) It was defined as the concentration (nM) of the test compound necessary for inhibition.

Experimental results: The inhibitory activity (IC ₅₀ value) of the test compound against endothelin converting enzyme was 13 nM. The inhibitory activity of captopril against endothelin converting enzyme is 25 at 100 μM.
Since it was% inhibition, it became clear that the compound of the present invention has a very strong endothelin converting enzyme inhibitory activity as compared with captopril.

III. Measurement of Enkephalinase Inhibitory Activity Experimental Materials: (1) Enkephalinase From monkey brain, human spinal cord and pig kidney Article (Gorenstein, C
Others, Life Sci., 25, 2065, 1979). (2) Enzyme substrate

Glutaryl-alanyl-alanyl-phenylalanyl-betanaphthylamide (Vega, Switzerland)

(3) Test Compound A compound represented by the following formula, which is contained in the compound (I) of the present invention and is obtained as the previous component by Example 2 described later.

[0057]

[Chemical 7]

Method for measuring inhibitory activity (enzyme assay method for inhibitory activity): Enkephalinase In the enzyme assay for inhibitory activity, the enzyme activity of enkephalinase in the presence and absence of a sample is measured and obtained. The inhibitory activity IC ₅₀ value of the sample was calculated from the obtained inhibition rate.

That is, the enkephalinase activity was measured according to the modified method of KOEHN et al. [Peptides., 9 , 173 (1988)] as follows. Enzyme substrate Glutaryl-Al
a-Phe-β-Naphtylamide was dissolved in water to 1 mM, placed in a 250 μl reaction tube, and the compound obtained as a pre-component of Example 2 below, enkephalinase and water were added to the solution to give a final volume of 250 μl. And incubate at 37 ° C for 2 hours.

Then, the reaction was terminated by incubating at 80 ° C. for 10 minutes, and left at room temperature for 30 minutes. In addition, Aminopeptidice M (porcine kidney, microsome.
(Manufactured by Sigma) is added and reacted for 30 minutes to produce a reaction product P
β-Naphtylamide is produced from he-β-Naphtylamide. Its β-Naphtylamide contains 10% Tween 1
0.1% dissolved in M acetate buffer (PH4.2)
After adding 750 μl of Fast Garnet (manufactured by FAST GARNET GBC SALT Sigma) and performing diazo coupling at room temperature for 15 minutes (showing a red color), the absorbance was measured with visible light at 525 nm.

Measurement of absorbance in a reaction solution containing no sample
Fixed value (β-Naphty released from enzyme substrate by enzyme activity
The amount of lamide. In addition, β-Naphtylamide is
-It reacts with the net and shows a red coloration. ) From
The enzyme activity was calculated. Enzyme activity (mU / ml) = 595 × A ₅₂₅ * Enzyme activity 1 mU is 1 nmole of enzyme substrate change per minute
Activity that catalyzes oxidization * A₅₂₅At 525 nm of the reaction solution containing no sample
Absorbance

Also, the absorbance (a) of the reaction solution containing the sample
Then, the inhibition rate was calculated from the absorbance (b) containing the above sample and the blind absorbance (a ′ and b ′) that did not react to each of them, by the following formula.

[0063]

[Equation 3]

Inhibitory activity IC of enkephalinase inhibitors
_A value of ₅₀ indicates that the enkephalinase enzyme activity is 50%.
(Inhibition rate) Concentration of sample required for inhibition (ng / m
l).

Experimental results: The inhibitory activity values (IC ₅₀ values) of the compound obtained as the pre-component of Example 2 for enkephalinase were 0.8 nM for monkey brain-derived enkephalinase and human spinal cord-derived one, respectively. 1 nM for enkephalinase and 6 nM for enkephalinase from pig kidney.

[0066]

EXAMPLES Next, the compounds of the present invention and the method for producing the same will be described in more detail with reference to Examples. In addition, a method for producing a raw material compound of the compound of the present invention will be described as a reference example. Reference example 1

[0067]

[Chemical 8]

(A) 10.1 g (50 mmol) of isopropylmalonic acid diethyl ester was dissolved in 50 ml of acetone, and 1M aqueous sodium hydroxide solution 50 was added thereto under ice cooling.
ml was added, and the mixture was stirred at the same temperature for 15 minutes and further at room temperature for 2 hours. Acetone was distilled off under reduced pressure, the remaining aqueous phase was washed with ether, acidified with citric acid and extracted several times with ether. The extract was washed with water and saturated saline and dried over anhydrous sodium sulfate. .. The crystals obtained by removing the drying agent and the solvent were dissolved again in ether, 9.7 ml of dicyclohexylamine was added, and the precipitated crystals were collected by filtration,
It was washed with ether and dried to obtain isopropylmalonic acid monoethyl ester dicyclohexylamine salt. Physicochemical properties Melting point 131-132 ° C, Rf value (chloroform: methanol = 9: 1) 0.32

(B) 8.87 g (2) of the compound obtained in (a)
5 mmol) was dissolved in 25 ml of anhydrous chloroform, and 3.21 g (25 mg of dimethylphosphinothioyl chloride) was dissolved in this solution.
mmol) was added and the mixture was stirred at room temperature for 30 minutes. Then, 3.99 g (25 mmo of benzylhydroxylamine hydrochloride to which 7.0 ml (50 mmol) of triethylamine was added.
An anhydrous chloroform (25 ml) solution of 1) was added, and the mixture was stirred at room temperature for 20 hours. Chloroform was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, and water, 5% citric acid aqueous solution, 5%
It was washed with a sodium hydrogen carbonate solution and saturated saline. The crystals obtained by drying over anhydrous sodium sulfate and evaporating the solvent were extracted several times with hot petroleum ether, and the extract was cooled to give colorless crystals of isopropylmalonic acid ethyl ester N-.
Benzyloxyamide was obtained.

Physicochemical properties Melting point 65-68 ° C., Rf value (hexane: ethyl acetate = 1: 1) 0.61 Nuclear magnetic resonance spectrum ( ¹ H-NMR (CDCl ₃ )) δ: 0.8-1. 4 (m, 9H, CH ( C H 3) 2, C
_{H 2 C H 3), 1.9-2.6} (m, 1H, C H (CH 3) 2), 2.9-3.1 (d, 1H, α-CH), 4.0-4 .4 (q, 2H, C H 2 CH 3), 4.9 (s, 2H, C H 2 C 6 H 5), 7.4 (s, 5H, CH 2 C 6 H 5), 8.7 -9.6 (br, 1H, CON H )

8.96 g (3) of the compound obtained in (c) and (b)
2 mmol) was dissolved in 32 ml of acetone and cooled under ice to 32
After adding 2 ml of 2M sodium hydroxide solution and stirring for 15 minutes, the temperature was returned to room temperature, and 2M sodium hydroxide solution was added little by little until the reaction was completed (total 8-24 ml (0.5
~ 1.5 eq)) stirred. After completion of the reaction, acetone was distilled off under reduced pressure, the aqueous phase was washed with ether, acidified with citric acid, extracted with ether, and the extract was washed with saturated brine and dried. The crystals obtained by distilling off the ether were recrystallized from ethyl acetate-petroleum ether to obtain isopropylmalonic acid mono (N-benzyloxy) amide.

Physicochemical properties Melting point 126.5-127.5 ° C., Rf value (chloroform: methanol: acetic acid = 85: 1)
0: 5) 0.63 Nuclear magnetic resonance spectrum _{^{(1 H-NMR (CDCl 3}} )) δ: 0.8-1.2 (d, 6H, CH (C H 3) 2), 2.0-2. 7 (m, 1H, C H (CH 3) 2), 2.8-3.1 (d, 1H, α-C H), 4.9 (s, 2H, C H 2 C 6 H 5), _{7.4 (s, 5H, CH 2} C 6 H 5), 8.7-9.9 (br, 2H, CON H, COO H) example 1

[0073]

[Chemical 9]

15 ml of 3.02 g (12 mmol) of isopropylmalonic acid mono (N-benzyloxy) amide
Dissolved in anhydrous chloroform, and 1.68 ml (12 mmol) of triethylamine and 1.54 g (12 mmol) of dimethyl phosphinothioyl chloride under ice cooling.
Was added and stirred for 30 minutes, and then 3.36 g (24 mmol) of triethylamine and 3.97 g (12 mmol) of L-tryptophan benzyl hydrochloride were added and stirred for 19 hours. The solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed with water, 5% aqueous citric acid solution, 5% aqueous sodium hydrogen carbonate solution and saturated brine, and then dried over anhydrous sodium sulfate. Adsorb the crystals obtained by removing the desiccant and solvent onto a silica gel column,
Elution with hexane-ethyl acetate (5: 2). The eluate was concentrated, and the crude crystals were recrystallized from chloroform-petroleum ether to give colorless crystals of N- (RS)-[2- (benzyloxyaminocarbonyl) -3-methyl-1-oxobutyl] -L-tryptophan. 3.15 g of benzyl ester (mixture of diastereomers) was obtained (yield 50
%).

Physicochemical properties Melting point 168 to 173 ° C., specific optical rotation [α] D ³⁰ + 2.67 ° (c0.5, CH ₃
OH) Rf value (hexane: ethyl acetate = 1: 1) 0.32, Rf value (chloroform: methanol = 20: 1) 0.5
6, nuclear magnetic resonance spectra _{^{(1 H-NMR (CDCl 3}} )) δ: 0.7-0.9 (t, 6H, (C H 3) 2 CH
-), 2.0-2.2 (m, 1H , (CH 3) 2 C H -), 2.5-2.7 (dd, 1H, α-CH), 3.2-3.3 ( d, 2H, Trp β-CH ₂ ), 4.7-4.9 (m, 3H, Trp α-CH, PhC
H ₂ ), 5.0 (s, 2H, PhCH ₂ ), 6.9 (s, 1H, Trp NH), 7.0-7.2 (m, 4H, indole 5H, 6
H, 2H, 7H), 7.3 (s, 10H, Ph), 7.5 (br, 1H, indole 4H), 8.3-8.4 (d, 1H, indole NH), 10-12 ( br, 1H, NH) Example 2

[0076]

[Chemical 10]

N- (RS)-[2- (benzyloxyaminocarbonyl) -3-methyl-1-oxobutyl]-
211 g of L-tryptophan benzyl ester (0.
40 mmol) was dissolved in 8 ml of methanol, 31 mg of 10% palladium / activated carbon was added, and hydrogen gas was bubbled through for 2 hours. The catalyst was filtered off, methanol was removed under reduced pressure, and N-
(RS)-[2- (hydroxyaminocarbonyl) -3
-Methyl-1-oxobutyl] -L-tryptophan 3
Obtained 8.5 mg (quantitative).

High Performance Liquid Chromatography Method (HPL
Separation of diastereomers was performed according to C). HPLC conditions Column: WAKOSIL 5C18-200 (4.0 m
mφ × 150 mm) Mobile phase: 0.01N HCl / MeOH (27% → 32
%, 15 min) Detection wavelength: 254 nm Flow rate: 1.0 ml / min

Previous component (elution time: 8.95 minutes) Physicochemical properties Elemental analysis value Experimental value: C, 58.96; H, 5.90; N, 12.3
3 Calculated _{(C 17 H 21 N 3 O} 5): C, 58.78; H,
6.09; N, 12.10 Nuclear magnetic resonance spectrum ( ¹ H-NMR (270 MHz,
_{DMSO-d 6)) δ:} 0.79-0.89 (6H, (C H 3) 2 CH
-), 2.16 (1H, ( CH 3) 2 C H -), 2.60 (1H, C H CH (CH 3) 2), 3.15 (2H, Trp β-CH 2), 4. 57 (1H, Trp α-CH), 6.96-7.12 (3H, indole 2H, 5
H, 7H), 7.35 (1H , indole 7H), 7.54 (1H, indole 4H), 7.76 (1H, Trp NH), 10.54 (1H, N H OH), 10.77 ( 1H, indole NH)

After component (elution time: 10.17 minutes) Physicochemical properties Elemental analysis value Experimental value: C, 53.49; H, 6.45; N, 10.9
1 Calculated _{(C 17 H 21 N 3 O} 5 · 2H 2 O): C, 53.2
6; H, 6.57; N, 10.96 Nuclear magnetic resonance spectrum ( ¹ H-NMR (270 MHz,
DMSO-d ₆ )) δ: 0.78-0.88 (6H, (C H ₃ ) ₂ CH
-), 2.10 (1H, ( CH 3) 2 C H -), 2.57 (1H, C H CH (CH 3) 2), 3.15 (2H, Trp β-CH 2), 4. 54 (1H, Trp α-CH), 6.93-7.09 (3H, indole 2H, 5
H, 7H), 7.32 (1H , indole 7H), 7.52 (1H, indole 4H), 7.87 (1H, Trp NH), 10.52 (1H, N H OH), 10.73 ( 1H, indole NH)

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Kazuo Honda 5097-2 Shiba, Kawaguchi City, Saitama Minami Urawa Park Heights No. 315

Claims (1)

[Claims] 1. A general formula: (In the formula, A is a single bond or a lower alkylene group, R ¹ ,
R ² means a hydrogen atom or an aralkyl group, and Trp means a tryptophan residue. ) A novel tryptophan derivative.