WO1997029076A1

WO1997029076A1 - Compounds having dopamine beta-hydroxylase inhibitory activity

Info

Publication number: WO1997029076A1
Application number: PCT/KR1997/000021
Authority: WO
Inventors: Yong-Nam Han; Keum-Hee Hwang; Jung-Woo Kim; Sang-Joon Lee; Soon-Kil Ahn; Joon-Kyum Kim
Original assignee: Chong Kun Dang Corporation
Priority date: 1996-02-10
Filing date: 1997-02-06
Publication date: 1997-08-14
Also published as: AU1559597A

Abstract

The present invention relates to lettusic acid derivatives of formula (I), a preparation process thereof and a pharmaceutical composition comprising same as an active ingredient. Lettusic acid or its derivative of formula (I) has a dopamine β-hydroxylase inhibitory activity, wherein X and Y, which are the same or different, are OR1 or (II). R1 is a hydrogen atom, an optionally substituted lower alkyl group or an alkaline metal, R2 and R3 are independently a hydrogen atom or an optionally substituted lower alkyl group, or they are linked together to form a saturated or unsaturated ring having one or more N or O atoms.

Description

COMPOUNDS HAVING DOPAMINE BETA-HYDROXYLASE INHIBITORY ACTIVITY

FIELD OF THE INVENTION

The present invention relates to a novel compound having a dopamine β -hydroxylase (DBH) inhibitory activity and a process for the preparation thereof. More specifically, it pertains to N-[(carboxyacetyl-hydroxymethylamino)-methoxymethyl]-N-hydroxy methyl-malonamic acid (hereinafter, referred to as "lettusic acid") extracted from lettuce (Lactuca satiυa L.), its derivatives and a process for the preparation thereof.

BACKGROUND OF THE INVENTION

Dopamine β-hydroxylase("DBH") [E.G. 1.14.17.1] catalyzes the conversion of dopamine tυ norepinephrine, which is the final step of dopamine biosynthesis, in brain, peripheral sympathetic nerve and adrenal medulla [S. M. Schanberg, et al., Science 183, 523 (1974)].

DBH is a glycoprotein which requires vitamin C(ascorbic acid⁾ as a coenzyme and contains a copper atom [E. Spodine and J. anzur, Coord. Chem. Rew. 119. 171 (1992); and T. Skotland, et al.. Biochem. Biophysi. Res. Commu. 74, 1483 (1977)]. It has been reported that DBH activity can be increased by dicarboxylic acids such as fumaric acid [E. Levin and S. Kaufman, /. Biol Chem. 236. 2043 (1961)]. Further, DBH is a hydroxylase employing dopamine and phenylethyl amine derivatives as its substrates [C. R. Creveling, et al, Biochemica et Biophysica Acta. 64, 125 (1962)] and participates in the biosynthesis of physiologically active catecholamines. The DBH inhibitors are known to be useful in the treatment of hypertension and Parkinson' s disease.

The present inventors have reported that lettuce, gardenia seed, and the like herbal medicines exhibit strong DBH inhibitory activity [D. N. Tai, K. H. Hwang and Y. N. Han, Journal of Korea Society of Herbal Medicines 26, 62 (1995)].

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a novel dopamine β -hydroxylase (DBH) inhibitor and its derivatives.

Another object of the present invention is to provide a process for preparing said DBH inhibitor and its derivatives.

A further object of the present invention is to provide a pharmaceutical composition comprising said DBH inhibitor or its derivatives as an active ingredient, in combination with a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one aspect of the present invention, there is provided a novel compound of formula (I) having an DBH inhibitory activity, which is extracted from lettuce {Lactuca satiυa L.), and a process for the preparation thereof:

(I) wherein, X and Y, which may be the same or different, are

3 Ri is a hydrogen atom, an optionally substituted lower alkyl group or an alkaline metal, R2 and R3 are independently a hydrogen atom or an optionally substituted lower alkyl group, or they may be linked together to form a saturated or unsaturated ring having one or more

5 heteroatoms such as N or O.

Exemplary rings which may be formed by R2 and R3 include morpholine, pyrrole, pyrroline, pyrrolidine, imidazole, imidazoline, imindazolidine, pyrazole, pyrazoline, triazole, tetrazole, oxazine or piperazine. 0 All of the DBH inhibitory compounds of the present invention are novel compounds. Among them, the compound of formula (I) wherein Rj is a hydrogen atom is a novel compound extracted from lettus (Lactuca satiυa L.) and its chemical name is N-[(carboxyacetyl-hydroxymethylamino)-methoxymethyl]-N-hydroxy 5 methy 1-malonamic acid( which is named "lettusic acid"). The compound of formula (I) wherein Ri is an alkaline metal can be obtained by extracting it from lettuce or by reacting lettusic acid with an alkaline compounds such as hydroxide, carbonate and bicarbonate of alkaline metal. 0 On the other hand, the present invention also provides the derivatives of lettusic acid, which have enhanced pharmacological activities and physical properties. Such derivatives include the esters of lettusic acid, which are the compounds of formula (I) wherein Ri is an alkyl group, and lettusate amides. 5 Preferable compounds of the present invention are those of formula (I-l ):

(I-l) wherein, R , 0 o0c X and Y are independently ORi or, N «_^ R ^ Ri is a hydrogen atom, an alkaline metal or a C1-C4 alkyl group, R2 is a hydrogen atom and R3 is a benzyl group; or R2 and R3 are linked together to form a morpholino group.

Most preferable compounds of the present invention includes N-[(carboxyacetyl-hydroxymethylamino)-methoxymethyl]-N-hydroxy methyl-malonamic acid (lettusic acid), monosodium lettusate, disodium lettusate, monopotassium lettusate, dipotassium lettusate, lettusate methyl ester, lettusate ethyl ester, lettusate isopropyl ester, lettusate benzyl amide and lettusate morpholine amide. For the purpose of examining a solvent by which the DBH inhibitory component of lettuce is easily extractable, 50 g of dried lettuce powder was extracted with water and methanol, respectively, and then DBH inhibitory activities of the resulting extracts were determined. As a result, the total DBH inhibitory activity of the water extract was 2.11 x 10 units; and that of the methanol extract, 3.94 x 10^G units. This result shows that the DBH inhibitory component of lettuce is extracted more effectively with water than with methanol by a factor of 5.36.

In order to confirm the solubility of the DBH inhibitory component of lettuce, the water extract of dried lettuce was partitioned between n-butanol and water. Consequently, the water fraction(Fr. 1 ) exhibited a higher DBH inhibitory activity than the butanol extract by a factor of 43.8. This result shows that the DBH inhibitory component is water-soluble. When the water extract(Fr. 1) of dried lettuce was concentrated, potassium nitrate contained in the lettuce in a large amount was educed as crystals. The present inventors unexpectedly found in the course of methanol precipitation to remove the educts that the DBH inhibitory component co-precipitates with potassium nitrate. To remove potassium nitrate from the precipitate (Fr. 2), Fr. 2 was suspended in distilled water and then centrifuged to remove the insoluble materials. To the supernatant was added calcium chloride solution to obtain a white precipitate(Fr. 3). After confirming that the DBH inhibitory activity is present in Fr. 3, tetrasodium edetate(EDTA-Na4) solution was added to Fr. 3 to solubilize it and remove the calcium ion therefrom. Consequently, Fr. 4 was obtained, which was then subjected to a gel filtration using G-25(Sephadex G-25) column. As an eluent, 10 % ethanol was superior to other aqueous solution of inorganic salts. Specifically, since EDTA employed to remove calcium ion also has a DBH inhibitory activity, 10 % ethanol was appropriate to separate EDTA from the DBH inhibitory component of lettuce, while physiological saline or aqueous ammonium bicarbonate (NH4HCO3) solution were not appropriate for that purpose. Under these conditions, the DBH inhibitory component of lettuce was eluted, followed by the elution of EDTA. The fraction of DBH inhibitory component of lettuce eluted from Sephadex G-25 column contained peptide contaminants, which were then removed by passing the fraction through a cation exchange resin Dowex 50W x 2 column. Consequently, monosodium lettusate was educed as needle-like crystals from the non-adsorbed portion.

Monosodium lettusate was allowed to stand in 30% glacial acetic acid solution or 1 N hydrochloric acid solution to obtain lettusic acid as colorless needle-like crystals. The titration of lettusic acid with 1 N sodium hydroxide solution exhibited two pH jumps at pH 4.47 and pH 8.72. Further, the aqueous lettusic acid solution was adjusted to pH 10 by the addition of 1 N sodium hydroxide and ethanol was added thereto to a concentration of 70 %. The resulting mixture was allowed to stand at room temperature to obtain disodium lettusate as minute needle-like crystals.

Lettusic acid and its monosodium and disodium salts are not soluble in organic solvents such as ethanol, acetone, dimethylsulfoxide, chloroform and hexane. Lettusic acid itself is not soluble in water, while its sodium salts are water-soluble, the disodium salt being more soluble than the monosodium salt.

Based on this property, the present inventors have developed a process for purifying lettusic acid more effectively. Specifically, the methanol precipitate fraction(Fr. 2) of the water fraction(Fr. 1 ) of dried lettuce as explained above was dissolved again in water and the insoluble material was removed by centrifuge. The resulting supernatant was directly passed through Dowex 50W 2 column, with omitting the procedures for preparing Fr. 3 and Fr. 4, to obtain monopotassium salt of lettusic acid(monopotassium lettusate).

For the purpose of confirming whether lettusic acid is formed in the course of drying a fresh lettuce or is originally present in the fresh lettuce, a series of experiments including preparation of Fr. 1, Fr. 2, Fr. 3 and Fr. 4; gel filtration using Sephadex G-25 column; and ion -exchange chromatography using Dowex 50W x 2 column were conducted by employing a juice of fresh lettuce. Consequently, monosodium lettusate was obtained from the juice of fresh lettuce, and it was demonstrated that lettusic acid is present in fresh lettuce. The present inventors also developed a simple and quick process for the mass production of lettusic acid. Specifically, the juice of fresh lettuce was filtered through cellulose filter and the filtrate was concentrated to obtain a viscose crude extract. The extract was dissolved in 2 N or 6 N hydrochloric acid solution and then allowed to stand. Consequently, lettusic acid was educed together with potassium chloride as crystals. This mixed crystals were washed with distilled water to remove potassium chloride and a trace amount of pigment remained therein was removed by dissolving the resulting crystal with ethanol to obtain pure lettusic acid. The process is based on the property of lettusic acid that being very stable compound which does not decompose by heating at 100°C for 5 hours in 6 N hydrochloric acid.

When lettusic acid was dissolved in 1 N sodium hydroxide and then allowed to stand at room temperature, it was converted to its disodium salt by sodium hydroxide and the disodium salt was educed by the salting-out effect of sodium hydroxide. However, when the reaction mixture was heated at 100 °C for 30 minutes, it decomposes and, therefore, malonic acid was obtained.

When lettusic acid was subjected to cation FAB MS in the presence of sodium hydroxide and glycerol, a peak was detected at m/z 353[M+2Na-H]⁺. When monosodium and disodium salts were subjected to FAB MS, peaks were detected at m/z 331[M+H]⁺ and m/z 353[M+H]⁺, respectively. From these results, the molecular weight of lettusic acid, i.e., [M], was determined to be 308. Elemental analysis of lettusic acid exhibited that lettusic acid has a molecular formula of

^■ 2H2O.

Lettusic acid showed alcoholic OH peaks (3553, 3438, 924 cm^"3), carboxylic acid (COOH) peaks ( -3000, 1680, 1397, 1356 cm ), and tertiary acid amide peaks (1650— 1616 cm^"1) in IR spectrum. Lettusic acid is insoluble in water, and therefore,

H-NMRs of monosodium and disodium salts of lettusic acid were conducted respectively by using D2O. Consequently, CH2 peak of

COCH2CO was observed at δ 3.73 (br. s.) for monosodium salt, while it was observed at δ 2.64 (s.) for disodium salt. Further, CH₂ peak of NCH2O was observed at 54.02 (br. s.) for monosodium salt, while it was observed at δ 3.20 (dd., J= 16.1 and 22.1 Hz) for disodium salt. When ¹³C-NMR was conducted for disodium salt, only three carbon peaks were observed: namely, CH2 peak of COCH2CO was observed at δ 57.05 ( ), CH2 peak of NCH2O was observed at 5 62.94 (t.), and CO peak of COCH2CO was observed at δ 181.96 (s.).

From these results, chemical structure of lettusic acid was determined and chemical name thereof was determined to be N-[(carboxyacetyl-hydroxymethylamino)-methoxymethyl]-N-hydroxy methyl-malonamic acid.

On the other hand, the present inventors have synthesized novel derivatives of lettusic acid, which have enhanced DBH inhibitory activity and improved physical properties. For instance, lettusate esters of formula (I) wherein Ri is a alkyl group were prepared by reacting lettusic acid with alcohol in the presence of mild esterifying agent such as diethylcyanophosphonate. Further, lettusate amide was prepared by reacting lettusic acid with amine in the presence of diethylcyanophosphonate.

The following Examples are intended to further illustrate the present invention without limiting its scope. Further, percentages given below for solid in solid mixture, liquid in liquid, and solid in liquid are on a wt/wt, vol/vol and wt/vol basis, respectively, unless specifically indicated otherwise.

Example !■' Preparation of DBH Inhibitors from Lettuce

(Step 1) Extraction and isolation of monosodium lettusate

500 g of lettuce powder made by grounding dried lettuce was mixed with 3 L of water. The mixture was added to a column and then allowed to stand at room temperature overnight. Distilled water was added to the top of the column to obtain about 3 L of infusion, which was then concentrated under reduced pressure below 60 °C. Concentration was continued until potassium nitrate (KNO3) present in a large amount in lettuce was educed as crystals and the concentrate was collected on separatory funnel by using a small amount of distilled water. The concentrate was extracted twice with the same amount of n-butanol and the n-butanol layer was washed with a small amount of water. The aqueous layer was concentrated to 0.3 L to obtain fraction No. KFr. 1). To Fr. 1 was added slowly 0.7 L of methanol while stirring and the mixture was centrifuged to obtain 49 g of precipitate fraction(Fr. 2).

Fr. 2 was suspended in 0.2 L of distilled water and the suspension was centrifuged to remove insoluble materials. To the supernatant was added with stirring 0.5 M calcium chloride(CaCl2) solution until no white precipitate was formed. The resulting mixture was centrifuged to obtain a precipitate fraction(Fr. 3). Fr. 3 was dissolved in 0.5 M tetrasodium edetate(EDTA-Na4) solution(pH 9.0) and the mixture was centrifuged to obtain a supernatant (Fr. 4). Fr. 4 was divided into two portions and each of them was subjected to a gel filtration on Sephadex G-25 column (superfine, 5 x 100 cm, eluent: 10 % ethanol). The eluates were collected by 7.0 mi- per test tube and the DBH inhibitory activity of each fractions were determined in accordance with the method of Test Example 1. Consequently, Fr. A covering test tube Nos. 80 to 115 and Fr. B covering test tube Nos. 118 to 140 exhibited DBH inhibitory activities. Fr. B was identified to be tetrasodium edetate. Fr. A was collected and then concentrated and the concentrate was dissolved in pyridine/acetic acid buffer. 0.6 g of monosodium lettusate was obtained as colorless crystals from the resulting solution. The remaining solution was subjected to cation exchange chromatography on Dowex 50W x 2-100 column (1.2 x 22 cm, eluent-^' pyridine/acetic acid buffer, pH 3.1). The fractions containing non-adsorbed eluate was collected and then concentrated to obtain 0.6 g of monosodium lettusate (Total yield-^' 1.2 g).

m.p. 320 °C

[ Y D = 0 (concentration = 1%, water) UV : critical absorption (water) IR (cm^"1, KBr) : 3553, 3440 (-OH), -3000, 1680 (COOH), 1650-

1616 (tertiary acid amide), 1395 (0=C-OH), 1356 (0=C-Q). 1332 (C-N), 926 (C-OH) cation FAB MS m/z (relative intensity %) : 331[M+Hf (10.30),

423[M+G+H]⁺(6.03), 515[M⁺2G+HΓ (2.20)

Η-NMR(80MHz. D₂0, δppm) : 3.73 (2H x 2, br.s., COCH2CO),

4.02 (2H 4, br.s., NCH2O)

(Step 2) Purification of lettusic acid

1 g of monosodium lettusate was dissolved in 30 % glacial acetic acid or 0.1 N hydrochloric acid and the resulting solution was allowed to stand overnight to obtain 0.92 g of lettusic acid as needle-like crystals.

m.p. : 236 °C elemental analysis : C]oH_jCN2θ<r 2H2θ [calculated : C 34.98 %, H

5.86 %, N 8.14 %; measured : C 33.66 %, H 5.74 %, N 7.86 %] IR (cm^"', KBr) : 3553, 3438 (-OH), -3000, 1680 (COOH), 1650 - 1616 (tertiary acid amide), 1397 (0=C-OH), 1356 (OOP), 1321 (C-N), 924 (C-QH) cation FAB MS m/z(NaOH+glycerol) :

353[M+2Na-H]⁺, 397[M+4Na-3H]⁺, 445[M+2Na+G-H]⁺, 467[M+3Na+G-2Hf, 559[M+3Na+2G-2H]\581

[M+4Na+2G-3H]⁺

(Step 3) Preparation of disodium lettusate

0.82 g of lettusic acid was suspended in 30 m£ of distilled water and titrated with lN-NaOH (factor 1.026) with observing pH change. After observing a pH jump at pH 8.76(at this time, 8.4 mi? of 1 N-NaOH was consumed), the suspension was filtered and then concentrated. The concentrate was dissolved in a small amount of water and ethanol was added thereto to a concentration below 70 %. The mixture was mixed thoroughly and allowed to stand overnight to obtain 1.02 g of colorless minute needle-like crystals.

m.p. : 300 °C elemental analysis : C₁₀Hi4N₂θ9Na2- 8H2θ [calculated : C 24.20 %,

H 6.09 %, N 5.64 % measured : 23.46 %, H 5.73 %, N 5.25 %] [α]²⁰D = 0 (concentration = 1%, water) UV : critical absorption (water) IR (Cm^"1, KBr) : 3440 (-OH), 1591 (COO ) cation FAB MS m/z (relative intensity, %) :

353[M+Hf (100), 375[M+Naf (59), 467[M+G+Na]⁺(30), 559[M+2G+Na]⁺ anion FAB MS m/z (relative intensity, %) ^'■ 657[2M-H₂0-CHO]^" (8.24), 635[657-Na+H]^" (16.49),

565

(41.23), 543[565-Na+H]^" (35.05), 451[543-COCH₂CO] (100), 429[451-Na+H]^" (76.29) 1H-NMR(300MHz,D₂O,δppm) : 2.64 (2H x 2, s, COCH2CO), 3.20 (4 x

2H, dd, J=16.1 & 22.1Hz, NCH2OR) ^l3C-NMR(300MHz,D₂O,δppm) : 57.05 (COCH2CO, t), 62.94 (NCH2OR, t), 181.96 (COO, s)

Example 2: Extraction and Isolation of Lettusic Acid from Fresh Lettuce

4 kg of fresh lettuce was crushed by an electric juicer and then filtered through the filter of the juicer to obtain 3 L of filtrate. The residue remained in the filter was filtered under reduced pressure to obtain about 0.3 L of filtrate. The combined filtrate(about 3.3 L) was extracted with 1 L of n-butanol to remove green pigment and to obtain 2.8 L of residual aqueous layer. Treatment of the aqueous layer with 0.5 M calcium chloride(CaCl2) and the following procedure was carried out in accordance with the same procedure of (Step 1) of Example 1 to obtain 0.6 g of monosodium lettusate and 0.55 g of lettusic acid was obtained therefrom in accordance with the same procedure of (Step 2) of Example 1.

Example 3: Isolation of Monopotassium Lettusate from Lettuce

Lettusic acid was prepared from the precipitate fraction (Fr. 2) obtained in (Step 1) of Example 1 in accordance with the procedure other than that of (Steps 1 and 2) of Example 1. Specifically, Fr. 2 was suspended in 0.2 L of distilled water and the suspension was filtered to remove the insoluble materials. The supernatant was subjected to cation exchange chromatography on Dowex 50W x 2-100 column(3 x 22 cm, eluent: pyridine/acetic acid buffer, pH 3.1 ) and the eluates containing active materials were combined and concentrated to obtain 4.6 g of monopotassium lettusate.

m.p. : 318 °C

IR : identical with that of monosodium lettusate cation FAB MS m/z : 347 [M+H⁺], 439 [M+G+H]⁺, 531 [M+2G+H]⁺ H-NMR : identical with that of monosodium lettusate 1 g of monopotassium lettusate was dissolved in a small amount of water and cone. HCl was added thereto to obtain 1

N-HC1 solution. The solution was allowed to stand at room temperature to obtain 0.8 g of lettusic acid as colorless needle-like crystals.

Example 4: Simple Process for Isolating Lettusic Acid from Fresh Lettuce

4 kg of fresh lettuce was crushed by an electric juicer and then filtered through a cellulose filter to obtain about 3.6 L of filtrate. The filtrate was concentrated under reduced pressure to obtain 0.3 kg of viscous crude extract. The extract was dissolved in 0.3 L of 2 N HCL and the solution was allowed to stand overnight. The resultant was filtered to obtain crystals, which crystals were then washed successively with distilled water and ethanol and dried to obtain 1.6 g of lettusic acid.

Example 5 : Preparation of N-[(methoxycarbonylacetyl-hydroxy -methylamino)-methoxymethyl]-N-hydroxymethyl malonamate methyl ester

308 mg of lettusic acid and 0.5 ml of methanol were added to 10 ml of dimethylformamide, and the mixture was cooled to 0^°C . 358 mg of diethylcyanophosphonate and 0.3ml of triethylamine were added to the mixture and the resultant was stirred at 0'C for 90 minutes and then at room temperature for 3 hours. The reaction mixture was diluted with ethyl acetate and the organic layer was washed successively with saturated sodium bicarbonate solution, water, 10% citric acid, and water. The resultant was dried over anhydrous magnesium sulfate, filtered and then concentrated under reduced pressure. The residue was purified with silica gel column to obtain 84 mg of the title compound.

1H NMR (DMSO-dc) δ : 3.75 (4H, br, s), 4.02 (8H, br, s), 4.12 (6H, s)

Example 6 : Preparation of N- [(ethoxy carbonylacetyl-hydroxymethyl -amino)-methoxymethyl]-N-hydroxymethylmalonamate 5 ethyl ester

The same procedure as in Example 5 was repeated by using 308 mg of lettusic acid, 0.5 ml of ethanol, 10 ml of dimethylformamide, 358 mg of diethylcyanophosphonate, and 0.3ml 0 of triethylamine to obtain 105 mg of title compound.

^] NMR (DMSO-de) δ : 1.32 (6H, t), 3.74 (4H, br, s), 3.98 (4H, q),

4.02 (8H, br, s)

15 Example 7 Preparation of N-[(isopropoxycarbonylacetyl-hydroxy -methylamino)methoxymethyl]-N-hydroxymethyl -malonamate isopropyl ester

The same procedure as in Example 5 was repeated by using 20 308 mg of lettusic acid, 1 ml of isopropanol, 10 ml of dimethylformamide, 358 mg of diethylcyanophosphonate, and 0.3 ml of triethylamine to obtain 101 mg of title compound.

^JH NMR (DMSO-dc) δ : 0.83 (12H, d), 3.71 (4H, br. s), 4.01 (8H, 25 br, s), 4.86 (2H, m)

Examule 8 : Preparation of N-[(benzylaminocarbonylacetyl-hydroxy -methylamino)methoxymethyl]-N-hydroxymethyl -malonamate benzyl amide 30

308 mg of lettusic acid and 0.5 ml of benzylamine were added to 10 ml of dimethylformamide, and the mixture was cooled to CC . 358 mg of diethylcyanophosphonate and 222 mg of triethylamine were added to the mixture and the resultant was

35 stirred at 0^°C for 90 minutes and then at room temperature for 3 hours. The reaction mixture was diluted with ethyl acetate and the organic layer was washed successively with saturated sodium bicarbonate solution, water, 10% citric acid, and water. The resultant was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. The residue was purified with silica gel column to obtain 116 mg of the title compound.

1H NMR (DMSO-de) δ : 3.8 (4H, br, s), 4.0 (8H, br, s), 4.8 (4H, br, s), 7.2~ 7.6(10H,br, s)

Example 9 : Preparation of N-[(N-morpholinecarbonylacetyl- -hydroxymethylamino)methoxymethyl]-N -hydroxy -methylmalonamate morpholine amide

The same procedure as in Example 8 was repeated by using

308 mg of lettusic acid, 0.5 ml of morpholine, 10 ml of dimethylformamide, 358 mg of diethylcyanophosphonate, and 222 mg of triethylamine to obtain 120 mg of title compound.

1H NMR (DMSO-do) δ : 3.8 (4H, br, s), 4.0 (24H, m)

Test Example 1: DBH Inhibitory Activity υf Water and Methanol

Extracts of Lettuce

T e enzyme activity of DBH was determined in accordance with the method of Creveling et al. [C. R. Creveling, et al., Biochemica et Biophysica Acta 64. 125 (1962)]. Specifically, 5 mC υf 0.25 M sucrose solution was added to bovine adrenal gland per 1 g wet weight thereof and the mixture was ground with a Waring blender for 2 minutes and then centrifuged at 4 ^DC, 700^χg for 10 minutes. The supernatant was centrifuged again at 4 °C, 10,000^χg for 20 minutes to obtain a precipitate, which was then suspended in 2 m of 0.25 M sucrose solution. The resulting suspension was frozen and stored at -15 °C. Upon use as an enzyme source, the frozen suspension was thawed at room temperature and then diluted with 30-fold volume of 0.25 M sucrose solution. A solution prepared by dissolving sodium fumarate, N-ethylmaleimide, iproniazide phosphate and vitamin C to a concentration of 0.06 M, respectively, in distilled water was used as a reaction aid. To 0.3 mC of enzyme source prepared above were added 1.0 ml of sample, 0.2 m( of 0.4 % catalase, 0.5 ml of 1 M acetate buffer (pH 5.0) and 0.5 ml of a reaction aid, in this order, and reacted at 37 "C for 15 minutes. 0.5 ml of 0.12 M tyramine hydrochloride was added thereto and the resulting mixture was reacted for 90 minutes. 0.4 ml of 3 M trichloroacetic acid was added to the reaction mixture to terminate the enzyme reaction. Immediately thereafter, the resultant was centrifuged at 700χg for 10 minutes and 3.0 ml of the supernatant was poured onto Dowex 50W 8 column (0.8 3 cm, H⁺ form, 200-400 mesh) and the column was washed with 30 ml of distilled water. 3 ml of 4N ammonia solution was added to the column, the eluate was collected at a test tube and then 0.2 ml of 4 % sodium metaperiodate (NaI0₄) solution was added thereto. The test tube was allowed to stand for 10 minutes and 0.2 ml of 20 % sodium metabisulfite (Na2S2θs) solution was added thereto. UV absorption of the resulting mixture was determined at 330 nm.

The same procedure as above were repeated for a control group wherein the same amount of distilled water was added in place of the sample; for a blank group wherein the substrate solution was added at the finishing point of reaction in place of the initiation point thereof; and a sample compensation group wherein the same amount of distilled water was added in place of the substrate solution. The above experiment was repeated for each group and the enzyme inhibition rate of a sample was calculated by the following equation:

•AControl — v. -ASample ~ Asampie Compensation

Enzyme inhibition rate( % ) = — — x 100

Acσntrol ^~ Afilank

Wherein, Ac ntroi, Asa pie, Asampie compensation and Aπiank mean the UV absorbances at 330 nm of Control group, Sample, Sample Compensation group and Blank group, respectively.

The sample was serially diluted and the enzyme inhibitory activity of each dilution was calculated. The enzyme inhibition rate was dotted on a Logit-log paper with respect to the concentration of the sample and 50 % enzyme inhibitory concentration (IC™) was determined therefrom. The result is shown in Table 1.

Table 1. DBH inhibitory activities of water and methanol extracts υf dried lettuce

Extracting IC50 Total inhibitory activity Specific activity solvent^a) (g)^b) ( Units ) (Units/g)

water 2.37 x 10^"5 2.11 x 10⁶ 4.22 ^χl0⁴ methanol 1.27 x 10^"4 3.94 x 10⁵ 7.88 χl0³

IL of distilled water or methanol was added to 50 g of dried lettuce powder and the mixture was heated for 3 hours and then filtered. The residue was washed with distilled water or methanol and the combined volume of filtered solution and washed solution was adjusted to 1 L.

Amount that converted to that of dried lettuce. ^ l Unit is an amount that inhibits DBH bv 50 % (IC50).

Test Example 2- DBH Inhibitory Activity υf Solvent Fractions υf

Lettuce

To 100 g of dried lettuce powder was added 2 L of distilled water or methanol and the mixture was heated to reflux for 3 hours. The resultant was filtered and the residue was washed with distilled water or methanol. The combined volume υf filtered solution and washed solution was adjusted to 1 L. The water extract of lettuce was concentrated to a volume of about 500 mC and extracted twice with each 500 ml of n-butanol. The butanol layer was washed with 300 ml of water and then concentrated under reduced pressure to obtain 4.98 g of butanol fraction. The water layer and the washed solution of butanol layer was combined and then concentrated under reduced pressure to obtain 23.22 g . of water fraction.

The methanol extract was concentrated under reduced pressure and the concentrate was suspended in 100 mP of water.

The suspension was extracted three-times with the same a υunt of chloroform to obtain 15.02 g of chloroform fraction and the remaining water layer was concentrated to obtain 10.50 g of water fraction.

Each extract was properly diluted with distilled water and the DBH inhibitory activity was determined by using the dilutions as test samples. The result is shown in Table 2.

Table 2. DBH inhibitory activity of solvent extracts of lettuce

Solvent fraction Yield^* ICoo Total inhibitory activity Specific activity (g) (g) (Units)^* (Units/g⁾

Water extraction BuOH fraction 4.98 7.33 x 10^~r' 3.40 x 1& 1.36 10⁴

Water fraction 23.22 7.80 x 10^"c 1.49 x 10⁶ 1.28 x 10⁵

Methanol extraction

CCLj fraction 15.02 6.20 x 10^"4 1.20 x 10 1.60 x 10

Water fraction 10.50 1.97 x 10^"5 2.67 x lO^*"1 1.19 x 10'

^* Yield from 100 g of dried lettuce.

Test Example 3 •' DBH Inhibitory Activity of Disodium Lettusate

Disodium lettusate obtained as crystals in (Step 3) of Example 1 was dissolved in distilled water to various concentrations and the DBH inhibitory activity of disodium lettusate was determined by using the resulting solution in accordance with the procedure of Test Example 1. The result is shown in Table 3.

Table 3. DBH inhibitory activity of disodium lettusate

Concentration Inhibitory activity ICr>o

(μg/ml)^* ( % ) μg / 3 μ M

0.10 4.6 1.0 13.2 10.0 73.2 100.0 93.3

1.53 3.08

Final concentration in DBH enzyme reaction mixture.

Test Example 4 : DBH Inhibitory Activities of Lettusic Acid Derivatives

DBH inhibitory activities of lettusic acid derivatives prepared in Examples 5 to 9 were determined in accordance with the procedure of Test Example 1. The result is shown in Table 4.

Table 4. DBH inhibitory activity of lettusic acid derivatives

Compound ICr,o(uM)

lettusate methyl ester(Ex. 5) 3.12 lettusate ethyl ester(Ex. 6) 4.18 lettusate isopropyl ester(Ex. 7) 5.69 lettusate benzyl amide(Ex. 8) 11.19 lettusate morpholine amide(Ex. 9) 9.80 Test Example 5 : Test for Acute Toxicity of Disodium Lettusate

Disodium lettusate was dissolved in distilled water and three groups of mice each weighing 210 to 230 g, 6 mice consisting a group, were orally administered with the resulting solution in 1,250; 2,500; and 5,000 mg/kg body weight doses, respectively. Lethality of mice were observed after 3 days from the administration and the result is shown in Table 5.

Table 5. Acute toxicity of disodium lettusate

Doses Heads Deaths Death rate

(mgΛg) (%)

1,250 6 0 0

2,500 6 0 0

5,000 6 0 0

The above result demonstrates that acute toxicity level (LD50) of disodium lettusate is above 5,000 mg/kg body weight.

Therefore, the compounds of the present invention may be used as inhibitors of dopamine β -hydroxylase.

The compounds of the present invention may be administered in a dosage of 1.0 to 5000 mg/day in a single dose or divided doses. They may be prepared into the conventional, pharmaceutically acceptable formulations such as injections, tablets, capsules, solutions, ointments, creams or syrups by combining them with a conventional, pharmaceutically acceptable excipient, adjuvant, etc. Formulation Example 1: Preparation of an Injection

disodium lettusate 5 mg injection-grade distilled water 2 ml

The above ingredients were filled into a 2 ml ampule and then sterilized to obtain an injection.

Formulation Example 2- Preparation of a Tablet

monosodium lettusate 10 mg lactose 28 mg corn starch 10 mg talc 2 mg magnesium stearate trace amount

The above ingredients were mixed thoroughly and then subjected to a dry tabletting to obtain a tablet.

Formulation Example 3: Preparation of a Capsule

monopotassium lettusate 10 mg corn starch 38 mg talc 2 mg magnesium stearate trace amount

The above ingredients were mixed thoroughly and then filled into a gelatin capsule to prepare a capsule.

Other forms of formulation may be prepared in accordance with conventional, pharmaceutically acceptable methods.

Claims

What is claimed is:

1. Lettusic acid derivatives of formula (I) having a dopamine β -hydroxylase inhibitory activity:

(D

wherein,

R

X and Y, which are the same or different, are ORi or, N

"R

Ri is a hydrogen atom, an optionally substituted lower alkyl group or an alkaline metal, R2 and R3 are independently a hydrogen atom or an optionally substituted lower alkyl group, or they are linked together to form a saturated or unsaturated ring having one or more N or O atoms.

2. The lettusic acid derivatives of claim 1, which is the compound of formula (I-l):

(I-l ) wherein,

R 2 X and Y are independently ORi or, N ^*" Rι is a hydrogen atom, an alkaline metal or a C1-C alkyl group,

R2 is a hydrogen atom and R3 is a benzyl group; or R2 and R3 are linked together to form a morpholino group.

3. The lettusic acid derivatives of claim 2, which - is selected from the group consisting of: N-[(carboxy acetyl - hydroxymethylamino)-methoxymethyl]-N-hydroxymethyl-malonamic acid (lettusic acid), monosodium lettusate, disodium lettusate, monopotassium lettusate, dipotassium lettusate, lettusate methyl ester, lettusate ethyl ester, lettusate isopropyl ester, lettusate benzyl amide and lettusate morpholine amide.

4. A process for preparing lettusic acid derivatives of formula

(I) having a dopamine β -hydroxylase inhibitory activity, which comprises the steps of: filtering a crushed lettuce or extracting a dried lettuce powder with water to obtain a filtrate, concentrating the filtrate to obtain an extract, and acidifying the extract with a conventional organic acid or inorganic acid, optionally comprising a step of introducing an ester or amide group:

(I⁾

wherein,

^ 2

X and Y, which are the same or different, are OR1 or, N

5. A pharmaceutical composition prepared by mixing lettusic acid derivatives of formula (I), which has a dopamine β

-hydroxylase inhibitory activity, as an active ingredient with a pharmaceutically acceptable excipient and formulating the mixture

5 into a conventional pharmaceutically acceptable preparation form: .

(I)

- _ wherein,

15 R ₂

X and Y, which are the same or different, are OR] or, N "

^R ₃

Ri is a hydrogen atom, an optionally substituted lower alkyl group υr

20 an alkaline metal,

R2 and R3 are independently a hydrogen atom or an optionally substituted lower alkyl group, or they are linked together to foπn a saturated or unsaturated ring having one or more N or 0 atoms.

25

30

35