HU211331A9 - Peptides ws-9326a and ws-9326b, derivatives thereof and uses thereof - Google Patents

Description

The present application is a continuation of the withdrawal of application filed on 05.10.1989, which is in part a continuation of the revoked application filed on 04.04.1989, and which is in part a continuing application. On 31.01.1989 to the withdrawn basic application.

The present invention relates to novel peptide derivatives having pharmaceutically active properties and their pharmaceutically acceptable salts.

More particularly, the present invention relates to novel peptide derivatives and pharmaceutically acceptable salts thereof having pharmacological effects, such as a substance antagonist, a neurokinin A (substance K) antagonist, analgesic or similar activity, and to novel peptide derivatives and pharmaceutical compositions containing them.

The present invention therefore relates to peptide derivatives and pharmaceutically acceptable salts thereof which are useful in the treatment and prevention of asthma, various pains (headaches, sore throats, cancer pains, etc.).

Another object of the present invention is a process for the preparation of the peptide derivatives and their pharmaceutically acceptable salts.

It is a further object of the present invention to provide a pharmaceutical composition comprising the peptide derivatives or pharmaceutically acceptable salts thereof as an active ingredient.

Yet another object of the present invention is the use of peptide derivatives and pharmaceutically acceptable salts thereof for the treatment and prevention of asthma and related diseases.

The peptide derivatives of the present invention are represented by formula (I) wherein R ¹ is hydrogen or acyl;

R ² is hydroxy;

R ³ is carboxyl or protected carboxyl; obsession

R ² and R ³ together form a -O - (= C) - group;

R ⁴ is hydroxy or protected hydroxy;

^R5 is hydroxy or protected hydroxy;

^R6 is hydroxy or protected hydroxy;

----- represents a single or double bond.

According to the invention, the novel peptide derivatives of formula I can be prepared in various ways (Figure 1220).

In the formulas in the figures, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , and ..... are as defined above;

R _a is acyl;

^R6 is acyloxy other class;

R ³ is an esterified carboxyl group;

R _{b is} lower alkenyl substituted aralkenoyl, wherein the alkenyl is lower;

R [alpha] -alkyl substituted aralkanoyl wherein the alkanoyl is lower and

R _{b is} lower alkoxy.

The starting materials of the formulas II and III can be prepared according to the novel reaction scheme shown in Figures 21 and 22. In the formulas in the figures

R ¹ , R ³ , R ⁵ , R ⁶ and ..... have the same meanings as defined above;

R ⁷ is a protected carboxyl group;

R ⁸ is a protected amino group;

R ⁹ is a protected amino group;

R ¹⁰ is a protected amino group;

R ¹¹ is a protected carboxyl group

R ¹² is a protected amino group;

R ¹³ is a protected amino group, and

R ¹⁴ is a protected amino group.

The procedures shown in the figures are detailed below:

Figure 12

The compound of formula (Ia) or a salt thereof may be prepared by ring closure of a compound of formula (II) or a salt thereof.

This reaction is carried out according to conventional methods for the synthesis of cyclic peptides, for example by the mixed acid anhydride, active ester, carbodiimide, etc.

The reaction is usually carried out in a conventional solvent, such as alcohol, tetrahydrofuran, ethyl acetate, Ν, Ν-dimethylformamide, dichloromethane, chloroform, or any other solvent which does not adversely affect the reaction.

The reaction temperature is not critical and may be carried out under cooling or heating.

Figure 13

The compound of formula (Ia) or a salt thereof is prepared by ring formation of a compound of formula (III) or a salt thereof.

The reaction is carried out according to conventional methods for the synthesis of cyclic peptides, for example by the mixed acid anhydride, active ester, carbodiimide, etc.

The reaction is generally carried out in a conventional solvent, such as alcohol, tetrahydrofuran, ethyl acetate, Ν, Ν-dimethylformamide, dichloromethane, chloroform or any other solvent which does not adversely affect the reaction.

The reaction temperature is not critical and may be carried out under cooling or heating.

Figure 14

The compound Nz (Ic) or a salt thereof can be prepared by deacylating the compound (Ib) or its salt. Suitable reactions include hydrolysis, reduction, and the like.

(i) Hydrolysis

The hydrolysis is preferably carried out in the presence of a base or an acid, including Lewis acids.

Suitable inorganic or organic bases are, for example, an alkali metal (sodium, potassium, etc.), an alkaline earth metal (magnesium, calcium, etc.), their hydroxide, carbonate or bicarbonate, trialkylamine (trimethylamine, triethylamine, etc.), picoline. , 1,5-diazabicyclo [4.3.0] non5-ene, 1,4-diazabicyclo [2.2.2] octane, 1,8-diazabicyclo [5.4.0] undec-7-ene and the like.

HU 211 331 A9

Suitable organic or inorganic acids are, for example, formic acid, acetic acid, propionic acid, trichloroacetic acid, trifluoroacetic acid or hydrochloric acid, hydrobromic acid, sulfuric acid, hydrochloric acid, hydrobromic acid and the like. Lewis acids used for hydrolysis such as trihaloacetic acids: trichloroacetic acid, trifluoroacetic acid, etc. preferably in the presence of cation-binding reagents (e.g. anisole, phenol, etc.).

The reaction solvent is usually water, alcohol (methanol, ethanol, etc.), methylene chloride, tetrahydrofuran or a mixture thereof, or any other solvent which does not adversely affect the reaction. The liquid base or acid itself can also be used as a solvent. The reaction temperature is not critical, and cooling or heating may also be employed.

(ii) Reduction

The reduction is carried out in the usual manner by chemical reduction or by catalysis.

A suitable reaction mixture for chemical reduction is metals (tin, zinc, iron, etc.) or metal compounds (e.g., chromium chloride, chromium acetate, etc.) and inorganic or organic acids (e.g., formic acid, acetic acid, propionic acid, trifluoroacetic acid, p-toluenesulfonic acid). , hydrochloric acid, hydrobromic acid, etc.).

Commonly used catalysts for catalytic reduction include platinum (platinum plate, platinum sponge, platinum carbon black, colloidal platinum, platinum oxide, platinum fiber, etc.), palladium (palladium sponge, palladium carbon black, palladium oxide, palladium on carbon, colloidal palladium, palladium on carbon) palladium on barium carbonate, etc.), nickel (e.g., reduced nickel, nickel oxide, Raney nickel, etc.), iron (e.g., reduced iron, Raney iron, etc.), copper catalysts (e.g., copper, Raney copper, Ullman copper, etc.). .) and the like. The reduction is carried out in a commonly used solvent which does not adversely affect the reaction, such as water, methanol, ethanol, propanes Ν, Ν-dimethylformamide, tetrahydrofuran or mixtures thereof. If the acids mentioned above are liquid in the case of chemical reduction, they may also be solvents themselves.

The reaction temperature is not critical and may be carried out under cooling or heating.

Figure 75. Reaction Process

The compound of formula (Ib) or a salt thereof is prepared by acylating the compound of formula (Ic) or a reactive derivative or salt thereof on its amino group.

An example of such a reactive derivative formed on the amino port is a Schiff base type isomer prepared by reacting a compound of formula Ic with a carbonyl compound such as an aldahyde, ketone or the like. The silyl derivatives may be prepared by reacting a compound of formula Ic with a silyl compound such as bis (trimethylsilyl) acetamide, mono (trimethylsilyl) acetamide, bis (trimethylsilyl) urea, and the like. reacted. Derivatives may also be formed by reacting a compound of formula (Ic) with phosphorus trichloride or phosgene.

The acylation agents used are the conventionally used acylating agents, which may be represented by the general formula R ' _a -OH (XIV) (as described above for R _a ), but also their reactive derivatives or salts thereof.

Suitable reactive derivatives of compound (XIV) include acid halide, acid anhydride, active amide, active ester, and the like.

Suitable acylating agents include, for example, acid chlorides; acid azides; mixed acid anhydrides in which substituted phosphoric acid (e.g., dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid, dibenzylphosphoric acid, halogenated phosphoric acid, etc.), dialkylphosphoric acid, sulfuric acid, thio-sulfonic acid, sulfonic acid, sulfuric acid, sulfuric acid, (e.g. pivalic acid, pentanoic acid, isopentanoic acid, 2-ethylbutyric acid or trichloroacetic acid, etc.); symmetric acid anhydrides; active amides with imidazole, 4-substituted imidazole, dimethylpyrazole, triazole or tetrazole, active esters (e.g., cyanomethyl, methoxymethyldimethyl-iminomethyl - [(CH ₃ ) ₂ N ⁺ = CH-], vinyl- , propargyl, p-nitrophenyl, 2,4-dinitrophenyl, trichlorophenyl, pentachlorophenyl, mesylphenyl, phenylazo-phenyl, phenylthio, p-nitro- phenylthio, ρ-cresylthio, carboxymethylthio, pyranyl, pyridyl, piperidyl, 8-quinolylthioester, etc.), or N-hydroxy compounds (e.g., Ν, Ν-dimethyl- hydroxylamine, 1-hydroxy-2- (1H) -pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxy-6-chloro-1H-benzothiazole, etc.). Thus, a derivative corresponding to the compound (XIV) to be used can be selected from the above.

The reaction is carried out in a commonly used solvent such as alcohol (methanol, ethanol, etc.), acetone in dioxane, acetonitrile, methylene chloride, ethylene chloride, tetrahydrofuran, N, N-dimethylformamide, pyridine or other solvent which does not adversely affect the reaction. These solvents may also be used as an aqueous mixture.

When compound (XIV) is used as the free acid or salt, it is preferred to carry out the reaction in the presence of a condensing agent. These include N, N'-dicyclohexylcarbodiimide; N-cyclohexyl-N'-morpholino-ethylcarbodiimide; N-cyclohexyl-N '- (4-diethyl-aminocyclohexyl) carbodiimide; Ν, Ν'-diethylcarbodiimide, N, N'-diisopropylcarbodiimide; N-ethyl-N '- (3-dimethylamino-propyl) carbodiimide; N, N-carbonyl-bis (2-methylimidazole); pentamethyleneketene-N-cyclohexylimine; diphenyl ketene-cyclohexylimine; ethoxyacetylene; 1-alkoxy-1-chloroethylene, trialkyl phosphite; ethyl polyphosphate; isopropyl polyphosphate; phosphorus oxychloride (phosphoryl chloride); phosphorus trichloride; thionyl chloride; oxalyl chloride; triphenylphosphine; 2-ethyl-7-hydroxybenzisoxazolium salt; 2-ethyl-5- (m-sulfophenyl) isoxazolium hydroxide inner salt; l- (p-chlorobenzenesulfonyl) -6-chloro-lH-benzotriazole; the so-called Vilsmeier reagent prepared by treating N, N-dimethylformamide with thionyl chloride, phosgene, trichloromethyl chloroformate or phosphorus oxychloride, etc. with.

The reaction may be carried out on inorganic or organic bases such as alkali metal hydrogencarbonate, lower alkyl trialkylamine, pyridine, N-

A9-alkyl morpholine, N, N-di (lower alkyl) benzylamine and the like. - even in his presence.

The reaction temperature is not critical, and the reaction may be carried out under cooling or heating.

Figure 16

The compound of formula (le) or salt thereof may be prepared by acylation of compound (Id) or salt thereof.

This reaction is illustrated by Examples 2, 4, 5, 7, 8, 17 and 18.

Figure 17 illustrates the reaction of the compound of formula (Ia) or its salt by hydrolysis of the compound of formula (Ia) or its salt.

The hydrolysis is carried out as described above for Reaction 3.

Figure 18 illustrates the reaction of the compound of formula (Ig) or its salt by esterification of compound (If) or its salt. The esterification is carried out using standard reagents, such as an alcohol or an active derivative thereof (e.g., halide, sulfonate, sulfate, diazo compound, etc.).

The reaction is carried out in one of the usual solvents, such as acetone, dioxane, alcohol, methylene chloride, ethylene chloride, n-hexane, tetrahydrofuran. ethyl acetate, N, N-dimethylformamide or other solvent which does not adversely affect the reaction.

The reaction temperature is not critical, and the reaction may be carried out under cooling and heating.

Figure 19

The compound of formula (II) or salt thereof may be prepared by reduction of compound (Ih) or salt thereof.

The reduction can be effected catalytically. Commonly used catalysts include platinum (platinum wire, platinum sponge, platinum carbon black, colloidal platinum oxide, platinum plate, etc.), palladium (palladium sponge, palladium carbon black, palladium oxide, palladium on carbon, colloidal palladium, palladium barium). palladium on barium carbonate, etc.), nickel (reduced nickel, nickel oxide, Raney nickel, etc.), cobalt (reduced cobalt, Raney cobalt, etc.), iron (reduced iron, Raney iron, etc.), copper (reduced copper, Raney copper, Ullmann copper, etc.) and other catalysts.

The reaction is carried out in conventional solvents such as acetone, dioxane, alcohol, tetrahydrofuran, ethyl acetate, Ν, Ν-dimethylformamide, dimethylsulfoxide or other solvents which do not adversely affect the reaction.

The reaction temperature is not critical, and the reaction may be carried out under cooling and heating.

Figure 20

The compound of formula (Ij) or its salt is prepared by alkylation of the compound of formula (Id) or its salt. This reaction is exemplified by Example 19.

Figure 21: Reaction process

The compound of formula (II) or salt thereof is prepared from the compound (IV) or salt thereof in the reaction steps shown in Figure 21. All reaction steps are commonly used in peptide synthesis. The preparation of the starting material of formula (IV) or its salt is detailed in the Examples.

Figure 22

The compound of formula (III) or salt thereof is prepared from compound (IX) or salt thereof by following the reaction steps shown in the figure. All reaction steps are commonly used in peptide syntheses. The preparation of the starting material of formula IX or its salt is described below.

WS-9326A and WS-9326B peptide derivatives are prepared by microbiological fermentation by culturing Streptomyces, such as Streptomyces violaceoniger No.9326, which produce said substances.

The Streptomyces used produce WS-9326A and / or WS-9326B peptide derivatives. Such is the case with Streptomyces violaceoniger 9326, which has recently been isolated from soil samples from Suwa, Japan (Nagano Prefecture).

Streptomyces violaceoniger strain 9326 was lyophilized under accession number FERM BP-1667 at the Farmentation Research Institute, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan (date of deposit: 20 January 1988).

It will be appreciated that the preparation of novel materials WS-9326A and 9326B is not limited to the production of the microorganisms described herein, but is provided for illustrative purposes only. The present invention also encompasses all mutants capable of producing WS-9326A or WS-9326B materials, whether natural or artificial, such as by X-ray irradiation, ultraviolet light irradiation, N-methyl-N'-nitro-N-nitrosoguanidine treatment, -aminopurine treatment, etc. - it's a mutant.

Streptomyces violaceoniger 9326 has the following morphological, cultural, biological and physiological properties:

[1] Morphological characteristics

For taxonomic studies, Shirling and Gottlieb (Methods for the Characterization of Streptomyces species). International Journal of Systematic Bacteriology, 16: 313-340 (1966).

Morphological observations were made using light and electron microscopy at 30 ° C on oat flour, yeast / malt extract agar or mineral salt / starch agar for 14 days.

The vegetative mycelium is well developed without ffagmentation. The aerial mycelia are monopodially branched to form a spiral strand of 10 to 30 spores. The spores have a smooth surface with an oval shape of 0.6-0.8x0.8-1.3 pm. No skeletal granules, sporangia and zoospores were observed.

EN 211 331 A9 [2] Breeding characteristics

Characteristic properties of the culture are described in Shirling and Gottlieb, cited above, and in Waksman, S.A., The actinomycetes, Vol. 2: Classification, Identification and Description of Genera and Species. The Williams and Wilkins Co., Baltimore (1961)] were tested on 10 media.

The cultures were grown for 21 days at 30 ° C. The colors are described in the Methuen Handbook of Color (Komerup, A. and J. H. Wansher, Methuen Handbook of Color, Methuen, London (1978)). The data are summarized in Table I below.

/. SPREADSHEET

broth No. 9326. strain culture description yeast / malt extract agar N: good L: Powerful oatmeal agar N: good L: medium mineral salt / starch agar N: good L: Powerful glycerol / asparagine agar N: good L: Powerful Peptone / yeast extract / iron agar N: good L: Thin tyrosine agar N: good L: Powerful glycose / asparagine agar N: good L: medium nutrient agar N: medium L: medium Bennet agar N: Weak L: Weak sucrose / nitrate agar N: Weak L: None F: Grayish brown (6E3) O: None.

N: growth.

L: Airfish.

F: back page,

O: soluble pigment /.

The aerial mycelium varies in color from gray to brownish gray. Part of the colony will become black and moist, showing a hygroscopic character on most agar media. The color of the back is tan, brown or dark brown. The color on the back is insensitive to changes in the pH. Melanoid and other soluble pigments are not formed.

Cell wall analysis was performed by Becker et al., Becker, B., M.P.Lechevalier, R.E.Gordon, and H.A. Lechevalier, "Rapid Differentiation between Nocardia and Streptomyces by Paper Chromatography of Whole Cell Hydrolysates", Appl. Microbiol., 12: 421-423 (1964)] and Yamaguchi's method [Comparison of cell wall composition of Yamaguchi]. morphologically distorting actinomycetes. ", J. Bacteriol., 89: 444-453 (1965)]. Analysis of whole cell hydrolyzate strain 9326 shows the presence of LL-diaminopimelic acid. Consequently, the cell wall of the strain is of type I.

[3] Biological and physiological features

The physiological properties of the strain and the utilization of carbon sources are shown in Figures Π and III. are summarized in Table.

Our investigations into the utilization of carbon sources were carried out according to the methods of Pridham and Gottlieb [Pridham, T.G. and Gottlieb, D., "The Utilization of Carbon Compounds by Somin Actinomycetes as an Aids for Species Determination", J. Bacteriol., 56: 107-114 (1948)].

The strain 9326 has clear morphological and chemical properties in the genus Streptomyces. The 9326 strain is in the Bergey Manual

Streptomyces species, Buchanan, R.E. and NE Gibbons: J. Bergey's Manual of Determinative Bacteriology, 8th Ed., The Williams and Wilkins Co., Baltimore (1974)] and compared with the Streptomyces species described by Shirling [(Shirling, EB and D. Gottlieb, Cooperative description). 2. Species description from first study. "Intern.J.Syst. Bacteriol., 18: 69-189 (1968)], [(Shirling, EB, and D. Gottlieb," Cooperative description of type culture "). 3. Additional species descriptions from first and second studies, "Int.J.Syst.Bacteriol., 18: 279-392 (1968)] and [(Shirling, EB and D. Gottlieb," Cooperative description of type culture of 4. Species description from the second, third and fourt studies, "Intern.J.Syst.Bacteriol., 19: 391-512 (1969)], the species is included in the" Approved lists of bacterial noble "[Skerman, VBD; V.McGowan and PHA Sneath, "Approved List of Bacterial Names," Intem.J.System.Bacteriol., 30: 225-420 ( 1980)] and is included in other species (Williams, S.T .: M.Goodfellow, G.Alerson,

E.M.H. Wellington, P.H.A. Sneath and M.J. Sackin, Numerical Classification of Streptomyces and Related Genera, J.Gen.Microbiol., 129: 1743-1813 (1983) and [Dietz.A: Criteria fór characterization of Hygroscopicus strains. In: Actinomycetes; The Boundary Microorganisms ”183-191. pp .; ed., T.Arai (1976)].

II. SPREADSHEET

Physiological properties of strain 9326

temperature range of growth 11-47 'C optimum temp. for growth 29-31 'C Liquefaction of gelatin + tejkoaguláció - milk peptonisation + starch hydrolysis + melanoid pigment production - cellulózbontás -

III. SPREADSHEET

Coal utilization of strain 9326

D-glucose + saccharose + D-xylose +

HU 211 331 A9

D-fructose + L-rhamnose + rafftnóz + L-arabinose + inositol + mannitol +

The results obtained show that strain 9326 is close to Streptomyces violaceoniger, therefore strain 9326 has been identified as Streptomyces violaceoniger and is designated Streptomyces violaceoniger 9326.

The novel peptide derivatives WS-9326A and Ws-9326B are prepared by culturing in culture medium the Streptomyces strain (e.g. Streptomyces violaceonigeer 9326 FERM BP-1667) producing WS-9326A and / or WS-9326B.

In general, the WS-93326A and WS-9326B peptide derivatives can be prepared by culturing the WS-9326A and / or WS-3926B producing strain in an aqueous medium containing assimilable carbon and nitrogen, preferably under aerobic conditions (shake culture). in culture, etc.).

Carbohydrates such as glucose, xylose, galactose, glycerol, starch, dextrin and the like are preferred carbon sources.

Other sources of carbon include maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate and the like.

A preferred source of nitrogen is yeast extract, peptone, gluten meal. cotton seed meal, soybean meal, corn jam, dried yeast, wheat germ, powdered flour, peanut meal, etc., and organic and inorganic nitrogenous compounds such as ammonium salts (ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, etc.

Preferably, the combined carbon and nitrogen source materials do not need to be in a pure state because less pure materials containing growth factors and significant amounts of mineral nutrients can also be used. If necessary, they may be added to the medium in the form of mineral salts, such as calcium carbonate, sodium or calcium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salt, copper salt, cobalt salt, etc. If necessary, especially if the medium is highly foaming, antifoaming agents such as liquid paraffin, fatty oils, vegetable oils, mineral oil or silicone may be added.

Submerged aerobic culture conditions are used to produce WS-9326A and WS-9326B in large quantities. Shake flask fermentation or surface culture is used to produce small amounts. If the fermentation is carried out in large tanks, it is preferable to use a vegetative inoculum to inoculate the production tank fermenter so that the lag phase is not produced in the production of WS-9326A and WS-3926B. Consequently, a smaller volume of the medium is first inoculated with the spores or mycelium of the microorganism of interest and a vegetative inoculum is prepared, then this vegetative culture is transferred to a large fermenter under sterile conditions. For the preparation of the vaccine, either the same medium as that used for the preparation of WS-9326A and WS9326B is used.

The culture can be mixed and aerated in a variety of ways. The agitation may be by means of a paddle stirrer or similar mechanical agitation, rotation or shaking of the fermentor, various pump systems, or by transferring sterile air into the culture. Aeration can be effected by passing sterile air through the fermentation broth.

The fermentation is usually carried out at a temperature in the range 20-40 ° C, preferably 25-35 ° C. The fermentation time is 50-150 hours, depending on the fermentation conditions and size.

Extraction of WS-9326A and WS-9326B from fermentation broth is accomplished by methods known per se which are commonly used to recover other known biologically active substances. The WS9326A and WS-9326B products are found in culture filtrate and nicelium, and WS-9326A and WS-9326B can be recovered and purified from the filtrate and mycelium. The mycelium is separated from the fermentation broth by filtration or centrifugation. Conventional recovery procedures may include evaporation under reduced pressure, freeze-drying, extraction with a suitable solvent, adjusting the pH, appropriate resins (e.g., anion or cation exchange resins, adsorption resins, etc.), commonly used adsorbents (e.g. , cellulose, alumina, etc.), crystallization, recrystallization, etc.

The figures show spectral data for WS-9326A, WS-9326B and their derivatives as follows:

Figure 1 shows the infrared absorption of compound WS-9326A in KBr;

Figure 2 shows the ¹³ C nuclear magnetic resonance spectrum of WS-9326A recorded in CD ₃ OD;

Figure 3 shows the * H nuclear magnetic resonance spectrum of WS-9326A in CD ₃ OD;

Figure 4 shows a ¹³ C nuclear magnetic resonance spectrum of triacetyl-WS-9326A in CDCl ₃ -CD ₃ OH (10: 1);

Figure 5 shows the nuclear magnetic resonance spectrum of triacetyl-WS-9326A ^] in CDCl ₃ CD ₃ OH (10: 1);

Figure 6 shows the ¹³ C nuclear magnetic resonance spectrum of WS-9326B recorded in CD ₃ OD;

Figure 7 shows the 'H nuclear magnetic resonance spectra of the WS-9326B compound was taken up in CD ₃ OD;

Fig. 8 shows the 'H' nuclear magnetic resonance spectrum of monoacetyl-WS-9326A in CDCl ₃ -CD ₃ OD (5: 1);

9 shows the diacetyl-WS-9326A Compound H nuclear magnetic resonance spectrum CDC1 _3-CD3OD (5: l) recorded in;

HU 211 331 A9

Figure 10 shows a ³ C nuclear magnetic resonance spectrum of tetrahydro-WS-9326A recorded in CD ₃ OD; and

11 illustrates Ή nuclear magnetic resonance spectrum of the tetrahydro-WS-9326A compound was taken up in CD ₃ OD.

The WS-9326A product prepared above has the following physical and chemical properties;

(1) Appearance and Color: Colorless Powder (2) Color Reactions: Cerium Sulfate Reaction, Iodine Vapor Reaction, Iron (III) Chloride / Potassium Iron (III) Cyanide Reaction, Negative Color Reactions: Ninhydrin Test: Molish Reaction III) chloride reaction, Ehrlich reaction, Pauli reaction.

(3) Solubility Soluble in: methanol, ethanol; poorly soluble in: acetone, ethyl acetate; insoluble in water, chloroform.

(4) Melting point: 187-190 [deg.] C. (5) Specific rotation, [[alpha]] D: -84 '(c = 1.0 methanol).

(6) Ultraviolet Spectrum: λ max: 280 nm (ε = 34700).

(7) Infrared Spectrum, γ {^ ® ® ³³ 3300, 3050, 2920, 2860, 1730, 1650, 1610, 1650, 1530, 1510, 1440, 1380, 1340, 1280, 1240, 1170, 1080, 1060, 1040, 970, 920, 860, 830 cm &^lt; -1 &^gt;, (see spectrum in Figure 1).

(8) Elemental analysis calculated for C ₅₄ H ₆₈ N ₈ O ₁₃ -2H ₂ O: C, 60.43; H, 6.76; N, 10.44.

Found: C, 60.18; H, 6.61; N, 10.32.

(9) TLC: _Rf value adsorbent mobile phase silica gel plate with chloroform / methanol. 5: 1 0.38 (Merck Art 5715)

RP-18 plate (Merck) methanol / water 8: 2 0.46 (10) Formula: C ₅₄ H ₆₈ N ₈ O ₁₃ (11) Molecular Weight: Based on FAB Mass Spec: m / z = 1037 (M + H) ⁺ (12) Chemical nature: acid type (13) ^l3 C. nuclear Magnetic resonance spectrum (100 MHz, CD ₃ OD)

delta: 175.69 (s), 174.70 (s), 173.73 (s), 173.38 (s), 172.89 (s), 171.04 (s), 170.45 (s), 167.79 (s), 167.15 (s), 159.20 (s), 140.05 (d), 139.12 (s), 138.71 (s), 135.27 (d), 134.85 (s), 132.11 (d), 132.03 (s), 131.69 (d) x 2 130.70 (d), 129.90 (d), 129.61 (d), x 2 129.22 (d) x 2, 128.55 (d), 128.04 (d), 127.99 (d) 127.38 (d), 126.09 (s), 123.70 (d), 115.63 (d) x 2, 73.46 (d), 71.34 (d), 62.80 (t), 59.53 (d), 56.91 (d), 56.76 (d), 55.55 (d), 53.64 (d), 52.10 (d), 39.85 (t), 37.18 (t);

37.09 (t), 34.58 (q),

31.37 (t), 24.56 (d),

23.63 (t), 22.71 (q),

22.52 (q), 21.17 (q)

17.19 (q), 14.13 (q).

The spectrum is shown in Figure 2.

(14) 1 H NMR (400 nm)

MHz, CD ₃ OD) delta: 7.80 (lH, d, J = 8Hz),

7.67 (1H, d, J = 16Hz),

7.45-7.14 (9 H, m),

7.06 (2H, d, J = 8Hz),

6.83 (1H, s),

6.65 (2H, d, J = 8Hz),

6.59 (1H, d, J = 12Hz),

5.88 (1H, dt, J = 12 and 7Hz),

5.55 (1H, m),

5.35 (1H, broad),

5.10 (1H, dd, J = 3 and 9.5Hz),

4.68 (1H, d, J = 10Hz),

4.55 (1H, t, J = 6Hz),

4.48 (1H, dd, J = 3 and 12Hz),

3.92 (2H, d, J = 6Hz),

3.70 (1H, t, J = 7.5Hz),

3.62 (lH, m);

3.46 (1H, dd, J = 3 and 14Hz),

2.94 (1H, dd, J = 3 and 16Hz),

2.89 (3H, s),

2.74 (1H, dd, J = 9.5 and 16Hz),

2.69 (1H, dd, J = 12 and 14Hz),

2.14 (2 H, m),

1.5-1.4 (2H, m), 1.2O (3H, d, J = 6Hz),

1.08 (3H, d, J = 6Hz)

1, O-O, 8 (2H, m),

0.91 (3H, t, J = 7Hz),

0.6 (1H, m),

0.53 (3H, d, J = 6Hz),

O, 51 (3H, d, J = 6Hz).

The spectrum is shown in Figure 3.

(15) Amino acid analysis: 5 mg of WS-9326A was hydrolyzed in 2 ml of hydrochloric acid in a sealed tube at 110 ° C for 20 hours. The hydrolyzate was evaporated to dryness and the evaporation residue analyzed on a Hitachi 835 automated amino acid analyzer. The result of the analysis was threonine (2), leucine (1), phenylalanine (1), aspartic acid (1), serine (1), methylamine (1), and ammonia (1).

With respect to the ¹³ C and * H nuclear resonance spectra (Figures 2 and 3), it is noted that WS-9326A is present in at least two stable conformations in deuterochloroform (CD ₃ OD) and is shown in Figures 13 and 14 respectively. points above chemical shift! data refers to the WS-9326A Main Conformer.

Physical and chemical properties of WS9326B material prepared by the above procedure:

(1) Appearance and color: Colorless amorphous powder.

(2) Color reactions, positive: cerium sulphate reaction, iodine vapor reaction; negative: ninhydride reaction.

(3) Solubility: soluble; slightly soluble in methanol:

A9 in ethanol, insoluble in water, acetone, ethyl acetate, chloroform.

(4) Melting point: 165-170 ° C (dec.).

(5) Specific rotation: [α] ρ: -64 'C (c = 1.0, methanol).

(6) Ultraviolet ^Spectrum : X ™ _a ' _x ^anol = 283 nm (£ = 27000) (7) Formula: C ₅₄ H ₇₀ N ₈ O | ₃ (8) Elemental analysis for C ₅₄ H ₇₀ N ₈ O _{3 - 2} H ₂ O:

Found: C, 60.32; H, 6.93; N, 10.42.

Found: C, 59.97; H, 6.87; N, 10.29.

(9) Molecular weight: Based on FAB mass spectrum, m / z = 1061.6 (M + Na) ⁺ (10) TLC:

mobile phase adsorbent ----- _Rf values on silica gel plates with chloroform / methanol, 0.38 (Merck

5715) 5: 1 RP-18 Plate Methanol / Water 8: 2 0.25 (Ratio of constituents of mobile phase to volume).

(11) Infrared spectrum: [X] = x ^_r ™ 33OO, 3050, 2950, 1735, 1660, 1530, 1510, 1450, 1400, 1380, 1340, 1260, 1220, 1080, 980. 920 ^cm-first

(12) ¹³ C nuclear magnetic resonance spectrum (100 MHz, CD 3 OD) δ: 174.99 (s), 174.54 (s),

173.60 (s), 173.41 (s),

173.30 (s), 171.27 (s),

170.74 (s), 170.19 (s),

168.68 (s), 157.59 (s),

140.53 (d), 139.35 (s),

139.18 (s), 135.76 (d).

134.17 (s), 131.15 (d) x2,

130.93 (d). 130.35 (d),

129.88 (d) x2.129.39 (d) x2,

128.70 (d), 128.58 (s),

128.13 (d), 127.64 (d),

127.53 (d), 121.99 (d),

116.45 (d) x2.72 (76) (d),

70.82 (d), 62.73 (t),

62.67 (d) .59.35 (d),

56.33 (d) x2.56.19 (d).

53.36 (d), 52.24 (d),

40.24 (t), 37.55 (t),

37.08 (t), 33.69 (t),

31.57 (t), 29.93 (q),

24.61 (d), 23.70 (q),

23.59 (t), 22.16 (q),

21.36 (q), 17.12 (q),

14.23 (q).

The spectrum is shown in Figure 6.

(13) 1 H-Nuclear Magnetic Resonance Spectrum (400 MHz, CD ₃ OD) δ: 7.86 (1H, J = 16Hz),

7.80 (1H, broad d, J = 8Hz),

7.12-7.42 (11H, m),

6.77 (2H, d, J = 28.5Hz),

6.61 (1H, d, J = 11, 5Hz),

5.88 (1H, dt, J = 7.5 and 11.5Hz),

5.08 (1H.dd, J = 3.5 and 10Hz),

5.04 (1H, q, J = 6.5Hz),

4.66 (1H, dd, J = 3.5 and 13Hz),

4.65 (1H, J = 11, 5Hz),

4.56 (1H, dd, J = 2.5 and 7Hz),

4.48 (1H, dd, J = 24.5 and 11 Hz),

4.46 (1H, s),

3.88 (2 H, m),

3.64 (2 H, m)

3.51 (1H, dd, J = 3.5 and 14Hz),

3.17 (1H, dd, J = 4.5 and 14Hz),

3.01 (1H, dd, J = 11 and 14 Hz),

2.94 (1H, dd, J = 3.5 and 16Hz),

2.71 (3H, s),

2.71 (1H, dd, J = 10 and 16Hz),

2.64 (1H, dd, J = 13 and 14Hz),

2.04 (2 H, m),

1.43 (2 H, m),

1.28 (2 H, m),

1.2O (3H, d, J = 6Hz),

0.95 (3H, d, J = 7.5Hz),

O, 87 (3H, t, J = 7.5Hz),

0.53 (1H, m),

0.52 (6H, d, J = 10.5Hz).

The spectrum is shown in Figure 7.

With respect to the ¹³ C and Ή nuclear resonance spectra, it is noted that the WS-9326B material has at least two stable conformations in the deuterochlorochloroform (CD ₃ OD) solution, and the chemical shift data for WS-9326B at points 12 and 13 above. their chief conformer.

The formulas established for the WS-9326A and WS-9326B based on the above physical and chemical data and further structural studies are shown in Figures 23 and 24.

Pharmaceutically acceptable salts of the compound of formula (I) are commonly used non-toxic salts. These base or acid addition salts may be formed with an inorganic base such as alkali metal salts (e.g., lithium salt, sodium salt, potassium salt, etc.), alkaline earth metal salts (e.g., calcium salt, magnesium salt, etc.), ammonium salts. organic base - such as the organic amine salts (e.g., triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, etc. ), inorganic acids (e.g. hydrochloride, hydroboride, sulfate, phosphate, etc.), organic carboxylic acids or sulfonic acids (e.g. formate, acetate, trifluoroacetate, meleate, tartrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, etc.), basic or acidic amino acids (e.g., arginine, aspartic acid, glutamic acid, etc.) and the like.

Suitable salts of the compounds of formula (Ia) - (Ij), (II) and (III) are of the same type as those listed for the compound of formula (I).

The terms used in the present invention are defined as follows.

The term "lower" refers to a chain of 1 to 6 carbon atoms, unless otherwise indicated.

The term "long chain", unless otherwise indicated, means carbon chains having from 7 to 20 carbon atoms.

Suitable "acyl" or "acyloxy" as used herein means carbamoyl, aliphatic acyl and acyl

A9 contains an aromatic ring - in this case aromatic acyl - or contains a heterocyclic ring, in which case the term heterocyclic acyl is used.

Exemplary acyl groups mentioned above include:

Aliphatic acyl groups which may be lower and lower alkanoyl groups (e.g. formyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl , dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl, icosanoyl, etc.);

lower or long-chain alkoxycarbonyl groups (e.g., methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, terpentyloxycarbonyl, heptyloxycarbonyl, etc.);

lower or long-chain alkanesulfonyl groups (e.g. methanesulfonyl, ethanesulfonyl, etc.);

lower or lower alkoxysulfonyl groups (e.g., methoxysulfonyl, ethoxysulfonyl, etc.) and the like;

Aromatic acyl groups which may be aroyl groups (e.g. benzoyl, toluoyl, naphthoyl, etc.); lower substituted lower alkanoyl groups (e.g., phenylalkanoyl such as phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutyl, phenylpentanoyl, phenylhexanoyl, etc .; naphthylalkanoyl such as naphthylacetyl, naphthylpropanoyl , naphthyl butanoyl, etc.);

aryl-substituted lower alkenoyl (e.g., phenylalkenoyl, such as phenylpropenoyl, phenylbutenoyl, phenylmethacryloyl, phenylpentenoyl, phenylhexenoyl, etc .; naphthylalkenoyl, such as naphthylpropenoyl, naphthylbutenoyl, naphthylpentenoyl); ., etc.); aryl-substituted lower alkoxycarbonyl (e.g., phenylalkoxycarbonyl, such as benzyloxycarbonyl, etc.);

aryloxycarbonyl (e.g., phenoxycarbonyl, naphthyloxycarbonyl, etc.);

lower alkanoyl substituted with aryloxy (e.g., phenoxyacetyl, phenoxypropionyl, etc.); aryl-substituted glyoxoyl (e.g., phenylglyoxoyl, naphthylglyoxoyl, etc.);

arenesulfonyl group (e.g., benzenesulfonyl, ptoluenesulfonyl, etc.) and the like;

Heterocyclic acyl groups which may be a heterocyclic-substituted carbonyl group (e.g., tenoyl, furoyl, nicotinoyl, etc.);

lower alkanoyl groups substituted with heterocyclic groups (e.g. thienylacetyl, thienylpropanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl, tetrazolylacetyl, etc.);

a heterocyclic-substituted glyoxoyl group (e.g. thiazolylglyoxoyl, thienylglyoxoyl, etc.) and the like.

The term "heterocyclyl-substituted carbonyl, lower alkanoyl, or glyoxoyl" is used herein to mean "heterocyclic acyl," more particularly a saturated or unsaturated, mono- or polycyclic heteroaryl structure having at least one heteroatom in the ring. -, sulfur or nitrogen, etc.

Particularly preferred are those groups having a heterocyclic structure wherein the heterocyclic ring:

unsaturated, 3-8, more preferably 5-6 membered, monocyclic and 1-4 nitrogen atoms, such as pyrrolyl, pyrrolidinyl, imidazolyl, pyrazolyl, pyridyl and N-oxide, dihydropyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g. -1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H1,2,3-triazolyl, etc.), tetrazolyl (e.g., 1H-tetrazolyl, 2H-tetrazolyl, etc.);

saturated, 3-8, more preferably 5-6 membered, monocyclic and containing 1-4 nitrogen atoms such as pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, etc .;

- unsaturated, condensed and containing from 1 to 4 nitrogen atoms, for example indolyl, isoindolyl, indolizinyl, benzoimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, etc .;

unsaturated, 3-8, more preferably 5-6 membered, monocyclic and containing 1-2 oxygen and 1-3 nitrogen atoms, such as oxazolyl, isoxazolyl, oxadiazolyl (e.g. 1,2,4-oxadiazolyl, 1,3,4- oxadiazolyl, 1,2,5-oxadiazolyl, etc.);

saturated, 3-8, more preferably 5-6 membered, monocyclic and containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as morpholinyl, sidnonyl, etc .;

unsaturated condensed and containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as benzoxazolyl, benzoxadiazolyl, etc .;

unsaturated, 3-8, more preferably 5-6 membered, monocyclic and containing 1-2 sulfur atoms and 1-3 nitrogen atoms such as thiazolyl, isothiazolyl, thiadiazolyl (e.g.

1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl,

1,2,5-thidiazolyl and the like. dihydrothiazinyl, etc .;

saturated, 3-8, more preferably 5-6 membered, monocyclic and containing 1-2 sulfur atoms and 1-3 nitrogen atoms such as thiazolidinyl, etc .;

unsaturated, 3-8, more preferably 5-6 membered, monocyclic and containing 1 to 2 sulfur atoms such as yenyl, dihydrodithinyl, dihydrodithionyl, etc .;

unsaturated, condensed and containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as benzothiazolyl, benzothiadiazolyl, etc .;

unsaturated, 3-8, more preferably 5-6 membered, monocyclic and containing an oxygen atom, such as furyl, etc .;

unsaturated, 3-8, more preferably 5-6 membered, monocyclic and containing one oxygen atom and one to two sulfur atoms, for example dihydroxyoxatin, etc .;

unsaturated, condensed and containing 1 to 2 sulfur atoms such as benzothienyl, benzodithinyl, etc .;

unsaturated, condensed and containing one oxygen atom and one to two sulfur atoms, e.g. benzoxathinyl, etc. and the like.

As mentioned above, the acyl group may have from 1 to 10, the same or different, suitable substituents, which may be lower alkyl groups (e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl hexyl, etc.), lower alkenyl groups (e.g. vinyl). , allyl, 1-propenyl, 1- or 2- or 3-butenyl, 1- or 2 or 3- or 4-pentyl, 1- or 2- or 3- or 4- or

5-hexenyl, etc.), lower alkoxy groups (e.g.

A9 methoxy, ethoxy, propoxy, etc.), lower alkylthio groups (e.g., methylthio, ethylthio, etc.), lower alkylamino groups (e.g., methylamino, etc.), lower cycloalkyl groups (e.g. e.g., cyclopentyl, cyclohexyl, etc.), lower alkynyl groups (e.g., cyclohexyl, etc.), halogen, amino, protected amino, hydroxyl, protected hydroxyl, cyano, nitro, carboxyl, protected carboxyl, sulfo, sulfamoyl, imino, oxo, lower aminoalkyl groups (e.g. aminomethyl, aminoethyl, etc.), carbamoyloxy groups, lower hydroxyalkyl groups (e.g., hydroxymethyl, 1- or 2-hydroxyethyl), or 2- or 3-hydroxypropyl, etc.), cyano (lower alkenylthio) groups (e.g., cyanovinylthio, etc.) and the like.

As used herein, the term "protected hydroxyl group" may include a phenyl (lower alkyl) group (e.g., benzyl, etc.), one of the aforementioned acyl groups, and the like.

A suitable "protected carboxyl group" may be an esterified carboxyl group.

Examples of the ester group of an esterified carboxyl group include:

lower alkyl group (e.g. methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, isobutyl ester, tert-butyl ester, pentyl ester, hexyl ester, 1-cyclopropylethyl ester, etc.) ), which may also have at least one suitable substituent, in which case the ester group may be, for example, a lower alkanoyloxy-lower alkyl group (e.g., acetoxymethyl ester, propionyloxymethyl ester, butyryloxymethyl ester). , valeryloxymethyl ester, pivaloyloxymethyl ester, hexanoyloxymethyl ester, 1- (or 2) -acetoxyethyl ester, 1- (or 2 or 3 or 4) -acetoxy butyl ester, 1- (or 2) -propionyloxyethyl ester, 1- (or 2 or 3) -propionyloxypropyl ester, 1- (or 2) -butyryloxyethyl ester, 1- ( or 2) -isobutyryloxyethyl ester, 1 (or 2) pivaloyloxyethyl ester, 1 (or 2) hexanoyloxyethyl ester. isobutyryloxymethyl ester, 2-ethylbutyryloxymethyl ester. 3,3-dimethyl-butyryloxy-methyl ester. 1 (or 2) pentanoyloxyethyl ester, etc.], a lower alkanesulfonyl (lower alkyl) ester group (e.g., 2-mesylethyl ester, etc.) substituted with one, two or three halogens. , lower alkyl group (e.g. 2-iodoethyl ester, 2,2,2-trichloroethyl ester, etc.), lower alkoxycarbonyloxy lower alkyl group (e.g. methoxycarbonyloxymethyl ester) , ethoxycarbonyloxymethyl ester, 2-methoxycarbonyloxyethyl ester, 1-ethoxycarbonyloxyethyl ester, 1-isopropoxycarbonyloxyethyl ester, etc.), phthalidylidene, (lower alkyl) or 5- (lower alkyl) -2-oxo-1,3-dioxol-4-yl (lower alkyl [e.g. (5-methyl-2-oxo-1,3-dioxol-4-yl) methyl) ester, (5-ethyl-2-oxo-1,3-dioxo-4-yl) methyl ester, (5-propyl-2-oxo-1,3-dioxol-4-yl) ethyl ester , etc.];

a lower alkenyl group (e.g., vinyl ester, allyl ester, etc.);

lower alkynyl group (e.g., ethynyl ester, propynyl ester, etc.);

aryl-substituted lower alkyl, which aralkyl may be substituted with at least one substituent; such as mono-, di- or triphenyl (lower alkyl) which may have at least one suitable substituent (e.g., benzyl ester, 4-methoxybenzyl ester, 4-nitrobenzyl ester, phenyl ester, trityl ester, benzhydryl ester, bis (methoxyphenyl) methyl ester, 3,4-dimethoxybenzyl ester, 4-hydroxy-3,5-di-tert-butylbenzyl ester, etc.);

- an aryl group which may also have at least one suitable substituent (e.g., phenyl ester, 4-chlorophenyl ester, tolyl ester, tert-butyl phenyl ester, xylyl ester, mesityl ester, cumenyl ester, etc.) .);

a phthalidyl group and the like.

Suitable "lower alkoxy" includes, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy and the like.

In the term "protected amino group", a suitable "amino protecting group" may be any of the above acyl groups, etc.

The term "aryl-substituted lower alkenyl substituted with aralkenoyl lower alkenyl" includes, for example, phenyl (lower alkenoyl) (e.g., phenylpropenoyl, phenylbutenoyl, phenylmethacryloyl, phenylpentenoyl, phenylpentenyl, etc.). lower alkenoyl (e.g. naphthylpropenoyl, naphthylbutenoyl, naphthylpentenoyl, etc.) and the like.

The term "lower alkenyl" as used in the aryl-substituted lower moiety is, for example, vinyl, allyl, 1-propenyl, 1- or 2- or 3-butenyl, 1 or 2- or 3- or 4-pentenyl, 1- or 2- or 3 or 4- or 5-hexenyl, etc. pose.

The term "aryl-substituted lower alkanoyl, which is aralkanoyl-substituted lower alkyl," includes, for example, phenyl-lower alkanoyl, phenyl-phenyl-propanoyl, phenyl-propanoyl, phenyl-propanoyl, phenyl-propanoyl, pentanoyl, phenylhexanoyl, etc.), naphthyl (lower alkanoyl) (e.g., naphthylacetyl, naphthylpropanoyl, naphthylbutanoyl, etc.) and the like.

The term "aryl-substituted lower alkanoyl substituted with lower alkyl aralkanoyl" is intended to include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, and the like. can be a group.

Preferred is the compound of formula I according to the invention, wherein

R ¹ is hydrogen, aryl-substituted lower alkoxycarbonyl (especially phenyl-lower alkoxycarbonyl), lower-alkanoyl, lower-alkanoyl, especially (C 15 -C 20) alkanoyl. aroyl (especially benzoyl), heterocyclic lower alkanoyl (especially thienyl (lower alkanoyl)), aryl-substituted lower alkenoyl, which aralkenoyl is lower

Substituted by A9 alkenyl (mainly phenyl (lower alkenyl) substituted by lower alkenyl) or aryl-substituted lower alkanoyl (lower alkenyl);

R ² is hydroxy;

R ³ is carboxyl or esterified carboxyl (especially lower alkoxycarbonyl) or

R ² and R ³ together form a -OC (= O) - group; R ⁴ is hydroxy, aryl-substituted lower alkoxy (especially phenyl-lower alkoxy) or acyloxy (especially lower-alkanoyloxy);

^R5 is hydroxy, lower alkoxy substituted by aryl (especially phenyl (lower) alkoxy, or acyloxy group (especially lower alkanoyloxy group);

^R6 is hydroxy, lower alkoxy, lower alkoxy substituted by aryl (especially phenyl (lower) alkoxy or acyloxy group (especially lower alkanoyloxy group), and

..... represents single or double bond.

Biological Properties of Peptide Derivatives: Peptide derivatives of formula (I) and pharmaceutically acceptable salts thereof have a substance P antagonist, neurokinin A (substance K) antagonist, analgesic and similar pharmacological action and are therefore useful in the treatment and prevention of asthma, various pains and the like. .

The following assays can be used to detect this type of pharmacological activity:

1. Radioligand binding assay

(a) Extraction of crude membrane from brain or lungs:

Female Wister rats (200 g) were used. The reagents are from Sigma Chemical Company. 4 g of whole brain were cut into small pieces and in 8 volumes of medium I (50 mM Tris-HCl, pH 7.5, 5 mM MnCl ₂ , 0.02% 5-calf serum albumin, 2 µg / ml chymostatin, 4 µg / ml leupeptin) and 40 μg / ml bacitracin) is homogenized with an Ultra-Disperser (Yamato Model LK-21) homogenizer. The homogenate is stored at -20 ° C or used immediately for assays.

600 g male albino male Hartley guinea pigs are killed by decapitation. The trachea and lungs were removed and stored at -80 ° C until use. 150 g of tissue was thawed and homogenized in 500 ml of buffer (0.25 M sucrose, 50 mM tris-HCl, pH 7.5, 0.1 mM EDTA) in a Matsuden MJ-761 compact mixer. The tissue was further homogenized in an Ultra-Dispetser (Yamato Model LK-21) with a maximum of 10 sec homogenisations separated by 10 sec cooling phases (total homogenization time: 60 sec). The tissue fragment was separated by centrifugation (900 g, 10 minutes), the supernatant was centrifuged at 14000 g for 20 minutes, and the resulting pellet was considered as the crude membrane preparation. This pellet was resuspended in Medium I, homogenized in a Teflon homogenizer, and centrifuged at 14000 g for 20 minutes. The pellet is stored at -20 ° C,

(b) Binding of tritium-labeled substance P to the membrane preparation:

nM tritium-labeled substance P (New England

Nuclear) incubated with 50 μΐ membrane preparation in Medium I at 4 ° C for 30 minutes; the final volume is 250 μΐ. At the end of the incubation time, the incubation mixture was rapidly filtered through a GF / B glass fiber filter, Brandel M-245, pretreated with 0.1% polyethyleneimine 3 hours prior to use. The filter was washed with 3 ml wash buffer (50 mM Tris-HCl, pH 7.5) at 0 ° C in 10 portions. Radioactive dispute was determined in 3 ml Aquazol-2 using a Packard TRICARB 4530 scintillation counter.

ARC. SPREADSHEET

Replacement of tritiated P material bound by rat brain and guinea pig lung membrane with WS-9326A, WS-9326B, triacetyl-WS-9326A or tetrahydro-WS-9326B

1C _SO (M) brain lungs WS-9326A 2,5x10 ^'5 3.8x1ο ^-6 triacetyl-WS- 9326A 9.4x10 ^-5 7.7x10 ^-5 WS-9326B 8.8x10 ⁵ tetrahydro-WS 9326A 4.2x10 ^-7

2. Effect of WS-9326A or Tetrahydro-WS-9326A on guinea pig trachea.

600 g of adult male albino male albino male guinea pigs are excised from the trachea using standard procedures and placed in a 30 ml glass jar with a thermostatic jacket fitted with a tissue bath. The tracheal tension is measured isometrically through a structure bound to a polygraph (Biophysiograph 180 system, San-Ei Instument). This transducer mediates the displacement. In a 30 ml 37 ° C tissue bath, a 2 mm wide 50 mm long trachea was suspended under 500 mg of rest tension. Tissue bath composition: 137 mM (8 g / l) sodium chloride,

2.7 mM (0.2 g / l) potassium chloride, 1.8 mM (0.264 g / l) calcium chloride-2 water, 1.02 mM (0.208 g / l) magnesium chloride6 water, 11, 9 mM (1 g / l) sodium bicarbonate, 0.42 mM (0.066 g / l) sodium dihydrogen phosphate, and

5.5 mM (1 g / L) glucose. The tissue bath was bubbled through with a gas mixture of 95% oxygen and 5% carbon dioxide. The tissues were incubated for 90 minutes to equilibrate the system and tested for WS-9326A or Tetrahydro-WS-9326A against various bronchoconstrictors. 10 ^-8 M P substance or 10 M neurokinin A were used to induce bronchoconstriction. The tension is fixed with a San-Ei Recúgraph85 writing device (San-Ei Instrument).

ll

HU 211 331 A9

TABLE V

Effect of WS-9326A or Tetrahydro-WS-9326A on Neurokinin A (NKA) and Substance P (SP) induced contraction in guinea pig trachea

WS-9326A / (Pg / ml) Inhibition"% NKA 10 ⁹ M SP10 ^-8 M 3 44% -10% 10 79% 50% 30 100% 63% WS-9326A # 1C (M) Tetrahydro-WS- 9326A 3,5x10- * 8.7xl (T ⁶ # 1C (M) LóxlO ⁶ 3.1x1ο ^-6

3. The effect of WS-9326A or tetrahydro-WS-9326A on bronchoconstriction induced by neurokinin A or capsaicin.

Male Hartley guinea pigs (300-500 g) were anesthetized with 10 mg of sodium pentobarbital intraperitoneally. A cannula is inserted into the cervical vein to deliver neurokinin A or capsaicin and the active ingredient. We breathe artificially through a catheter inserted into the trachea. Breathing is done with a small respiratory pump (Harvard B-34, 5 ml / stroke, 60 strokes / min). To measure the resistance to pulmonary dilatation, a modified Konzett-Rössler technique was used.

The antagonists and the antagonist drug (in saline containing 01% methylcellulose) are administered intravenously according to the schedule shown in Figure 25.

VI. SPREADSHEET

Inhibition of neurokinin A-induced bronchoconstriction with WS-9326A or tetrahydro-WS-9326A

Inhibition of neurokinin A (lmol / kg iv),%, n: 5 dose 2 minutes 17 minutes 32 minutes 47 minutes 62 minutes WS-9326A 10 mg / kg, iv. 16.5 ± 5.6 30.2 ± 12.9 45.8 + 13.8 55.4 ± 8.1 49.9 + 13.4 tetrahydro-WS-9326A 10 mg / kg, iv.41.0 ± 5.6 100.0 ± 0.0 99.2 + 0.8 98.4 ± 1.6 100.0 ± 0.0

VII. SPREADSHEET

Inhibition of Capsacin-Induced Bronchoconstriction with WS-9326A

Inhibition of capsaicin (10 nM / kg, iv),%, n: 4 dose 2 minutes 17 minutes 32 minutes 47 minutes 62 minutes 10 mg / kg, iv. ) 6.6 ± 5.1 40.6 ± 12.2 51.2 + 10.4 37.2 ± 19.7 47.1 + 16.7

4. Effect of intratracheally administered WS-9326A or tetrahydro-WS-9326A on neurokinin A-induced bronchoconstriction in guinea pigs: Examining the effect of inhalation of WS-9326A or tetrahydro-WS-9326A on bronchoconstriction. WS-9326A or tetrahidor-WS-9326A are dissolved in dimethyl sulfoxide and administered intratracheally. The method is almost the same as described above. As soon as Vili. and IX. As shown in Table II, WS-9326A and Tetrahydro-WS-9326A are highly effective.

Vili. SPREADSHEET

Inhibition of Neurokinin A ** '%

dose* 20 minutes 35 minutes 50 minutes 65 minutes n 0.03 mg / kg 32.3 28.4 35.6 35.5 4 0.3 50.6 42.4 44.9 42.0 4 3 73.4 79.4 81.7 77.7 4

ED ₅₀ mg / kg 0.23 0.29 0.19 0.24 * WS-9326A dissolved in dimethylsulfoxide.

nmol / kg. arc. 60

IX. SPREADSHEET

Neurokinin A-va! inhibition of induced bronchoconstriction with trachlor-delivered WS-9326A

Inhibition of neurokinin A ** '% dose* 20 minutes 35 minutes 50 minutes 65 minutes n 0,003 mg / kg 5.4 23.5 17.2 12.6 4 0.03 50.0 50.9 51.4 43.0 4 0.3 77.7 75.7 74.6 71.1 4

ED ₅₀ mg / kg 0.04? 0.030 0.037 0.059 * in tetrahydro-WS-9326 dissolved in dimethylsulfoxide.

lnmól / kg. arc.

5. Acute Toxicity: The acute toxicity of WS-9326A was determined in 5-week-old male ddY mice. 5 mice are dosed with a single intraperitoneal injection of the test substance in a stepwise dose. The LD # of WS-9326A is above 250 mg / kg and below 500 mg / kg.

(500 mg / kgLD ₅₀ 250 mg / kg).

6. Analgesic (analgesic effect) Acetic acid-induced writhing test

EN 211 331 A9 (i) Test method:

Ten male mice of ddY strain were used per group. To study the frequency of convulsive syndrome, the animals are observed for 3 to 13 minutes after intraperitoneal injection in which 0.6% acetic acid is administered at a dose of 0.2 ml / 10 g. Tetrahydro-WS-9326A to be tested is administered intraperitoneally 15 minutes prior to acetic acid injection. The frequency of tremor syndrome in treated animals is compared with that observed in untreated control animals.

(ii) Test result:

ED ₅₀ : 5.5 mg / kg.

The active ingredient of the present invention may be used in the form of pharmaceutical compositions. The compositions may be solid, semi-liquid or liquid. They contain as an active ingredient the peptide derivatives of formula (I) or pharmaceutically acceptable salts thereof in the experiment with organic or inorganic carriers or additives for external, enteral or parenteral administration. The active ingredient is used with conventional non-toxic pharmaceutically acceptable carriers to form tablets, pills, capsules, inhalers, solutions, emulsions, suspensions, and the like. The carrier may be water, glucose, lactose, acacia gum, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch. keratin, colloidal silicon oxide, potato starch, urea, and other carriers that allow the preparation of solid liquid formulations. Further, additives, stabilizers, thickeners, colorants, perfumes, etc. may be used. The active ingredient is contained in the pharmaceutical composition in an amount sufficient to produce the desired effect on the disease process or condition.

The pharmaceutically effective amount of the peptide derivative of formula (I) or a pharmaceutically acceptable salt thereof will vary and will vary with the age and condition of the subject being treated, with a daily dose of 0.01-1000 mg, preferably 0.1-500 mg, more preferably 0.5-100 mg. agent; the average single dose is usually 0.5 mg, 1 mg, 10 mg, 50 mg, 250 mg and 500 mg.

In the present specification, abbreviations such as IUPAC-IUB (Comission on Biological Nomenclature) are used for amino acids, peptides, protecting groups, etc. concerning. These are commonly used in the literature. In the examples, abbreviations other than IUPAC-IUB are also used.

Abbreviations used in the description of the invention:

Thr: L-threonine

Ser: L-serine

Tyr: L-tyrosine

Asn: L-asparagine allo-Thr: L-allotreonine D-Phe: D-phenylalanine Leu: L-leucine Z: Benzyloxycarbonyl Pac: phenacyl

Bzl: Benzyl

Boc: tert-butoxycarbonyl

Me: methyl

Tce: 2,2,2-trichloroethyl

Mmp: 4-methoxymethoxyphenyl

Si ': tert-butyldimethylsilyl

Ac: acetyl

Et: ethyl n-Hex: n-hexane

TLC: thin layer chromatography.

The foregoing is exemplified below. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

]. example

fermentation

In a 500 ml Erlenmeyer flask, sterilize 160 ml of inoculum medium with 1% starch, 1% sucrose, 1% glucose, 1% cotton seed meal, 0.5% peptone, 0.5% soy meal at 120 ° C for 30 minutes. and 0.2% calcium carbonate adjusted to pH 7.0 with 6N sodium hydroxide solution.

The medium is inoculated with cuckoo from a slant agar culture of Streptomyces violaceoniger strain 9326 and the flasks are shaken on a rotary shaker (220 rpm, 5.1 cm deflection) for 3 days at 30 ° C. The inoculum culture was used to inoculate 160 liters of fermentation broth with the following composition: 3% glycerol, 0.5% soybean meal, 1.5% soybean meal, 0.2% calcium carbonate, 0.001% sodium iodide. The 200 L stainless steel fermenter is fermented for 3 days at 30 ° C with aeration of 160 L of air per minute and stirred at 200 rpm. Quantification of WS9326A was performed by HPLC using a Hitachi Model 655 pump. The steel column used for chromatography (4.6 mm internal diameter, 250 mm length) was filled with R-ODS-5 (YMC-packed) at a flow rate of 1.0 ml / min. Mobile phase: methanol / water, 8: 2. For HPLC, the sample is prepared as follows: an equal volume of acetone is added to the culture broth sample, vigorously shaken, allowed to stand for 1 hour, and centrifuged. 5 μΐ of supernatant was applied to the column through a Hitachi Model 655 injector.

Isolation and cleaning

To 150 liters of fermentation broth is added an equal volume of acetone with stirring. Allow the mixture to stand at room temperature for one hour and then filter. The filtrate was concentrated under reduced pressure to 80 L, adjusted to pH 7.0 with IN hydrochloric acid and extracted with 80 L of ethyl acetate. The extract was evaporated to dryness under reduced pressure and applied to a silica gel column (Kieselgel 60, 70230 mesh, Merck, 3 L). The column was washed with 10 L of n-hexane, 10 L of n-hexane / ethyl acetate, 1: 1, 20 L of ethyl acetate and the active ingredient was eluted from the column with 6 L of acetone. The active fractions were evaporated to dryness under reduced pressure and the residue was chromatographed on a silica gel column (Kieselgel 60, 70-230 mesh, Merck, 1.2 L). The column is 5 liters

Wash A9 with chloroform / methanol 20: 1 and elute the desired material with 6 liters of chloroform / methanol 10: 1. The fractions were evaporated to dryness under reduced pressure to give a powder. This powder was dissolved in a small volume of methanol and applied to a NS gel column (Nihon Seimitsu, 500 mL). The desired material was eluted with 2 L of methanol / water 8: 2 and concentrated under reduced pressure to a volume of 300 mL and then extracted with 500 mL of ethyl acetate. The extract was evaporated to dryness under reduced pressure to give 5 g of powder. This was dissolved in 10 ml of methanol (500 mg / ml) and D-ODS-S (YMC-packed) HPLC column (stainless steel, 20 mm internal diameter, 250 mm long). The eluent was 8: 2 methanol / water, flow rate 9.9 ml / min. The resulting active fractions were evaporated under reduced pressure and extracted with ethyl acetate. Concentration of the extract under reduced pressure gave 150 mg of WS-9326A as a white powder.

Example 2

To a solution of WS-9326A (300 mg) in pyridine (4.5 ml) was added acetic anhydride (1.5 ml) and 4-dimethylaminopyridine (1 mg). The reaction mixture was allowed to stand at room temperature over a north. The reaction mixture was evaporated and the oily residue was purified by preparative thin layer chromatography eluting with chloroform / methanol (10: 1).

The resulting product was triturated with diethyl ether to give 332 mg of triacetyl WS-9326A as a colorless powder. Physical and chemical properties of triacetyl-WS-9326A:

1. Appearance and color: colorless powder

2. Color reactions: positive: cerium sulphate reaction, sulfuric acid reaction, iodine vapor reaction negative: ninhydrin reaction

3. Solubility: soluble in: methanol, dimethylsulfoxide, slightly soluble in: chloroform, diethyl ether; insoluble in n-hexane

4. Melting point: 141-143 ° C

5. Specific rotation, [α] β: -122 (c = 1.0, methanol).

6. Ultraviolet spectrum: γ ^ ₃ ^θ χ ^Η = 283 nm (£ 32,000).

8. Elemental analysis for C ₆₀ H ₇₄ N _g O | According to the formula ₆ -2H2O:

Found: C, 60.09; H, 6.56; N, 9.34.

Found: C, 60.19; H, 6.42; N, 9.77.

9. Molecular weight based on FAB mass spectrum: m / z 1133.6 (M + H) ⁺ .

10. Thin layer chromatogram

adsorbent mobile phase R _f value silica gel plate chloroform / methanol 0.50 (Merck Art 5715) 10: 1 (square). ethyl acetate 0.12

11. Infrared spectrum: y] _x = j ™ 3350, 3020, 2950, 2920, 2850, 1730, 1650, 1520, 1440, 1360, 1230, 1200, 1160, 1100, 1060, 1040, 910 cm in ^{the first}

12. Chemical nature: neutral.

13. ¹³ C nuclear magnetic resonance spectrum (100 MHz, CDCl ₃ / CD ₃ OH, 10: 1) delta: 174.20 (s), 173.23 (s),

173.06 (s), 171.32 (s),

171.02 (s), 170.84 (s),

169.79 (s), 169.59 (s),

169.55 (s), 168.52 (s),

167.03 (s), 166.36 (s),

151.02 (s), 140.74 (d),

138.82 (s), 138.74 (s),

137.12 (s), 135.23 (d),

133.75 (s), 131.31 (s),

130.20 (d), 129.96 (d) x2,

129.34 (d), 129.21 (d) x2,

128.56 (d) x2, 1.27.24 (d),

126.95 (d), 126.74 (d),

126.63 (d), 126.50 (d),

122.10 (d) x2.121.29 (d),

70.99 (d), 69.22 (d),

63.73 (t), 58.13 (d),

56.10 (d), 53.22 (d),

52.66 (d), 52.18 (d),

49.93 (d), 39.75 (t),

39.39 (q), 39.06 (t),

35.67 (t), 30.65 (t),

24.26 (d), 23.15 (q),

22.79 (1), 21.42 (q).

21.21 (q), 20.99 (q),

20.83 (q), 17.05 (q),

16.18 (q), 13.82 (q).

The spectrum is shown in Figure 4.

14. 1 H-nuclear magnetic resonance spectrum (400 MHz, CDCl ₃ / CD ₃ CH, 10: 1) delta 8.25 (1H, d, J = 8Hz),

8.0 (1H, d, J = 8Hz),

7.88 (1H, d, J = 16Hz),

7.86 (1AH, d, J = 8Hz),

7.70 (1H, d, J = 6Hz),

7.6K1H, d, J = 8Hz),

7.45 (1H, d, J = 7Hz),

7.32-7.15 (6 H, m),

7.03 (2H, d, J = 8Hz),

7.00-6.94 (3 H, m),

6.88 ~ 6.79 (4H, m),

6.7 (1H, s), 6.49 (1H, d, J = 12Hz),

5.76 (1H, dt, J = 12Hz & 7.5Hz),

5.54 (1H, broad, s),

5.50-5.45 (2H, m),

4.93 (1H, m),

4.75 (1H, m),

4.65-4.56 (2 H, m),

4.46 (1H, dd, J = 6 and 11 Hz),

4.31 (1H, t, J = 6Hz),

4.22 (1H, m),

4.18 (1H, dd, J = 8 and 11Hz),

3.56 (3H, s),

2.90 (1H, dd, J = 6 and 16Hz),

2.85-2.80 (2 H, m).

2.56 (1H, dd, J = 4 and 16Hz),

2.26 (3H, s);

HU 211 331 A9

2.00 (3H, s),

1.96-1.89 (2 H, m),

1.85 (3H, s),

1.58 (1H, m),

1.35 (3H, d, J = 6Hz),

1.32-1.2O (3 H, m),

1.07 (3H, d, J = 6Hz),

0.84 (1H, m),

0.72 (3H, d, J = 6Hz),

0.71 (3H, t, J = 7.5Hz),

0.65 (3H, d, J = 6Hz).

Example 3

fermentation

In a 500 ml Erlenmayer flask, 160 ml of inoculum medium with the following composition was sterilized at 120 ° C for 30 minutes: 1% peptone, 0.5% soybean meal, 0.2% calcium carbonate.

The medium was inoculated with Streptomyces violaceoniger 9326 strain from a slant culture scraper and shaken at 30 ° C for 3 days on a rotary shaker at 220 rpm and

With a deviation of 5.1 cm.

The above seeding culture was used to inoculate 160 liters of aqueous medium in a 500 liter stainless steel fermenter with 1% cotton seed meal, 0.5% peptone, 0.5% soybean meal, 0.2% calcium carbonate, 0.07% Adekanol LG-109 (Asahi Denka Co.) antifoam and 0.05% Silicone KM-70 (Shin-etsu Chemical Co.) antifoam sterilized at 120 ° C for 30 minutes. Fermentation is carried out for 1 day at 30 ° C with aeration of 160 liters of air per minute at 200 rpm.

60 liters of the above culture was inoculated into a 4,000 liter stainless steel fermenter with the following composition: 3% glycerol, 1% soybean meal, 1% chicken meat bone meal, 0.2% calcium carbonate, 0.001% sodium iodide, 0.07% Adecanol LG-109 and 0.05% Silicone KM-70. The medium was previously sterilized at 120 ° C for 30 minutes. Fermentation is carried out at 30 ° C for 3 days with 3000 liters of aeration and 100 rpm.

The fermentation was monitored by HPLC analysis (Hitachi Model 655 pump). The chromatographic steel column was packed with reverse phase silica gel ("YMC-packed column R-ODS-5", Yamamura Cheimical Institute); flow rate: 1.0 ml / min; eluent: 45% aqueous acetonitrile. Preparation of the sample for HPLC assays: an equal volume of acetone is added to the fermentation broth sample, shaken vigorously and allowed to stand for one hour and then centrifuged. Inject 5 μΐ of supernatant into the Hitachi Model 655.

Isolation and cleaning

The resulting fermentation broth was filtered with 15 kg of diatomaceous earth (Perlite Topko # 34, Showa Chemical Industry Co., Ltd.) with filtration aid. The filtered mycelium was extracted with 1600 L of ethyl acetate and the extract was filtered. The 1400 L filtrate was applied to a column of 200 L of activated carbon (Sirasagi KL, Takeda Pharmaceutical Co., Ltd.). The column was washed with ethyl acetate (200 L) and eluted with ethyl acetate / methanol (5: 1). The active fractions (50-1030 L) were combined and concentrated under reduced pressure to 45 L. After stirring, 120 L of n-hexane were added to the solution, allowed to stand at room temperature for one hour, and then co-seeded with 3 kg of Silika # 600 (Chuo Silika Co., Ltd.). The filtered material was washed with 15 L of n-hexane and the desired material was dissolved in 20 L of methanol.

The eluate was evaporated to dryness under reduced pressure. The residue (500 g) was dissolved in 4 liters of methanol / acetic acid / dichloromethane (1: 1: 2) and applied to a column of silica gel 60, 70-230 mesh (70 liters). The column was developed with 3.5 liters of methanol / acetic acid / dichloromethane, 1: 1: 2 and 25 liters of dichloromethane, the desired material was dichloromethane / methanol 10: 1 and dichloromethane / methanol 8: 1. elute with mixtures. The active phases are combined and concentrated under reduced pressure. The residue was dissolved in 1 L of methanol and 9 L of acetonitrile was added with stirring. The mixture was allowed to stand at room temperature for 1 hour and the precipitate was collected by filtration. This impact step is repeated three times. The resulting precipitate was washed with 1 L of acetonitrile and dried to give 190 g of WS-9326A as a white powder. The scalpels obtained in the precipitation steps were combined and concentrated under reduced pressure. The residue (11.7 g) was dissolved in 80% aqueous methanol and passed through a 300 mL column of activated carbon. The column was washed with 6 L of 80% aqueous methanol and eluted with 6 L of methanol. The active fractions were combined and evaporated to dryness under reduced pressure. The residue (3.4 g) was dissolved in methanol (12 ml) and applied to a reversed phase silica gel column (YMC packed column R-354 S-15/30 (OD) x 50 x 300 mm x 2); Yamamura Chemical Institute equilibrated with 50% aqueous acetonitrile. The column was developed with 50% aqueous acetonitrile using a Waters HPLC System 500. Fractions containing WS-9326B (3-3.5 L) were combined and evaporated to dryness to give 790 mg of WS-9326B as a white powder.

Example 4

To a solution of 100 mg of WS-9326A in 1 ml of pyridine was added 0.01 ml of acetic anhydride and the reaction mixture was allowed to stand at room temperature overnight. The reaction mixture was evaporated and the oily residue was purified by preparative thin layer chromatography; eluent: chloroform / methanol 9: 1. The product was triturated with diethyl ether to give 55 mg of monoacetyl-WS-9326A as a colorless powder. The chemical and physical properties of monoacetyl-WS9326A are as follows:

1. Appearance and color: colorless powder.

Formula 2: C ₅₆ H _70l Ngo] 4

3. Molecular weight based on FAB mass spectrum: m / z = 1079.4 (M + H) ⁺

HU 211 331 A9

4. TLC adsorbent stationary phase, mobile phase A, _Rf values on silica gel plates with chloroform / methanol = 0.17 (Merck

Art 5716) 10: 1 (vol.)

5. Infrared Absorption Spectrum, µ ™ / _χ = 33ΟΟ, 2920, 1730, 1650, 1500, 1360, 1190, 1170, 910 cm -1.

6. Chemical nature: neutral.

7. 1 H-Nuclear Magnetic Resonance Spectrum (400 MHz, CDCl 3 -CDOD, 5: 1) - Figure 8.

Example 5

To a solution of 100 mg of WS-9326A in 1 ml of pyridine was added 0.03 ml of acetic anhydride and the reaction mixture was allowed to stand at room temperature overnight. The reaction mixture was evaporated and the oily residue was purified by preparative thin layer chromatography; eluent: chloroform / methanol 9: 1. 72 mg of diacetyl-WS-9326A are obtained in the form of a colorless powder. Chemical and physical properties of Adiacetyl-WS-9326A:

1. Appearance and color: colorless powder.

2. Formula: C _5g H _7; N _g O | 5

3. Molecular weight based on FAB mass spectrum: m / z = 1121.4 (M + H) ⁺ .

4. TLC: adsorbent: silica gel plate (Merck Art 5715); eluent: chloroform / methanol, 10: 1 (v / v); _Rf: 0.35

5. IR absorption spectrum: y *), _the ^ ® 33OO, 3020, 2950, 1730, 1650, 1520, 1500, 1360, 1200, 1170, 1100, 1040, 980, 910 cm⁻¹.

6. Chemical nature: neutral.

7. Nuclear Magnetic Resonance Spectrum (400 MHz, CDCl3-CD3OD, 5: 1) - The spectrum is shown in Figure 9.

Example 6

WS-9326A (100 mg) was dissolved in methanol (2 mL) and hydrogenated in the presence of 25 mg of palladium on carbon (1 atm). hydrogen gas pressure at room temperature for 4 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The resulting product was triturated with diethyl ether to give 92 mg of tetrahydro-WS-9326A as a colorless powder. Physical and chemical properties of tetrahydro-WS-9326A:

1. Appearance and color: colorless powder.

2. Ultraviolet absorption spectrum, λ ^ ₃ ° _χ ^Η 287 nm (ε = 13000).

3. Formula: C ₅ 4 H ₇₂ N _g O ₁ 3

4. Molecular weight based on FAB mass spectroscopy: m / z = 1041.6 (M + H) ⁺ .

5. ¹³ C nuclear magnetic resonance spectrum (100 MHz, CD3OD); the spectrum is shown in Figure 10.

6.? Magnetic Resonance Spectrum (400 MHz, CD3OD); below the spectrum. is shown.

Example 7

To a solution of 1100 mg of tetrahydro-WS-9326A in 10 ml of pyridine was added 3 ml of acetic anhydride and 3 mg of 4-dimethylaminopyridine. The reaction mixture was allowed to stand at room temperature overnight. The solution was evaporated and the oily residue was purified on a silica gel column; eluent chloroform / methanol 20: 1. The resulting pure product was triturated with diethyl ether to give 998 mg of tetrahydro-triacetyl-WS-9326A as a colorless powder. Physical and chemical properties of tetrahydro-triacetyl-WS-9326A:

1. Appearance and color: colorless powder.

2. Ultraviolet Absorption Spectrum: µ θ ^Η 280 nm (= = 13000).

3. Formula: C ₆ O _{7 g} N _g 0 ₁₆

4. Elemental analysis based on C ₆₀ H ₇₈ N ₈ O ₁₆ H ₂ O:

Found: C, 60.80; H, 6.80; N, 9.45.

Found: C, 61.03; H, 6.70; N, 9.41.

5. Molecular weight by FAB mass spectroscopy: m / z = 167.6 (M + H) ⁺ .

6. ³ C nuclear magnetic resonance spectrum (100 MHz, CDCl 3): delta:

173.30 (s), 129.23 (d),

172.96 (s), 128.95 (d) x2,

172.90 (s), 128.61 (d) x2,

172.81 (s), 128.52 (d).

170.87 (s), 126.80 (d).

170.56 (s), 126.07 (d),

170.50 (s), 126.01 (d),

169.46 (s), 125.85 (d),

169.16 (s), 121.87 (d) x2,

168.48 (s), 70.46 (d),

167.99 (s), 69.10 (d),

165.52 (s), 63.32 (t),

150.70 (s), 58.28 (d),

140.84 (s), 56.19 (d),

138.93 (s), 52.63 (d),

138.58 (s), 52.07 (d),

136.83 (s), 51.63 (d),

131.04 (s), 49.23 (d),

129.71 (d) x2.39.3O (t),

39.17 (q), 22.50 (t),

38.31 (1), 21.46 (q),

36.52 (0.21.00 (q)).

35.22 (0.20.72 (q),

32.60 (0.20.63 (q),

31.77 (0.167.77 (q),

30.74 (0.16.22 (q),

27.66 (0.13,97 (q),

24.08 (d), 22.82 (q).

7. Nuclear Magnetic Resonance Spectrum (400 MHz, CDCl3): delta:

8.18 (1H, d, J = 8Hz),

7.66 (1H, d, J = 8Hz),

7.65 (1H, d, J = 8Hz),

7.29 (2H, d, J = 8Hz),

7.22 (1H, d, J = 8Hz),

7.15-7.02 (12H, m),

6.96 (1H, d, J = 7Hz),

6.67 (1H, s),

6.21 (1H, broad s),

5.51 (1H, broad s),

5.43 (lH, m);

5.36 (1H, broad, d, J = 8Hz),

HU 211 331 A9

4.85-4.75 (2 H, m),

4.66-4.58 (2 H, m),

4.40 (1H, dd, J = 11 and 6Hz),

4.34 (1H, m),

4.23 (1H, dd, J = 11 and 9Hz),

4.07 (1H, m),

3.53 (3H, s),

3.04-2.84 (5 H, m),

2.75-2.50 (4 H, m),

2.46 (1H, dd, J = 16 and 5Hz),

2.28 (3H, s),

1.99 (3H, s),

1.87 (3H, s),

1.66-1.50 (3 H, m),

1.37-1.27 (4 H, m),

1.27 (3H, d, J = 7Hz),

1.19 (1H, m), 1.03 (3H, d, J = 7Hz),

O, 88 (1H, m),

0.86 (3H, t, J = 6Hz),

0.72 (3H, d, J = 6Hz),

0.65 (3H, d. J = 6Hz).

Example 8

Triacetyl WS-9326A (100 mg) was dissolved in methanol (3 mL) and hydrogenated in the presence of 35 mg palladium on carbon (1 atm). hydrogen gas under pressure at room temperature for 3 hours.

The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was triturated with diethyl ether to give 90 mg of product as a colorless powder. This product is identical in every respect to the tetrahydro-triacetyl-WS-9326A obtained in Example 7.

Based on the above physical and chemical properties and further analysis of their structure, triacetylWS-9326A, monoacetyl WS-9326A, diacetyl-WS-9326A, ), (XVI), (XVII), or (XVIII).

Example 9

The reaction is illustrated in Figure 26. 3.24 g of starting material (XIX) is dissolved in 1000 ml of dichloromethane and 300 μΐ of triethylamine and 6.17 g of 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline are added. After stirring at room temperature for 24 hours, the solvent was evaporated. To the residue was added chloroform, and the chloroform solution was washed with water, hydrochloric acid, water, a saturated aqueous solution of sodium bicarbonate, and finally with water. It is dried over magnesium sulfate, filtered and the solvent is evaporated from the filtrate. The residue was chromatographed on a "lober" column (size C), eluting with 3% methanol in chloroform. The fractions containing the desired product were evaporated to give 1.06 g (XX) of the desired product.

[.alpha.] D @ 20 = -95.3 DEG (c = 0.33% chloroform).

IR (CHCl ₃ ): 1660, 1600, 1510 cm -1.

NMR (CDCl ₃ ), δ 0.88 (3H, d, J = 6Hz), 0.93 (3H, d,

J = 6Hz), 1.09 (3H, d, J = 6.5Hz), 1.37 (3H, d, J = 6.5

Hz), 2.82 (3H, s), 3.87 (2H, s), 6.93 (2H, d, J = 8Hz).

Example 10

The reaction is shown in Figure 27.

To a solution of 10 mg of the starting material (XXI) in 4 ml of dichloromethane and 0.1 m Ν, Ν-dimethylformamide is added 20.4 mg of N-hydroxysuccinimide and

8.2 mg of water-soluble carbodiimide hydrochloride.

After stirring for 1 hour at room temperature, 4 mg of carbodiimide hydrochloride are added every 1.5 hours until the starting material disappears.

The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (10 mL), washed with dilute hydrochloric acid and water.

After drying over magnesium sulfate, the solvent was evaporated under reduced pressure and the residue was dissolved in 1 ml of trifluoroacetic acid in 0.1 ml of anisole.

After stirring at room temperature for 30 minutes, the solvent was removed under reduced pressure. The residue was dissolved in N, N-dimethylformamide (2 mL) and pyridine (40 mL) was added.

After stirring for one hour at room temperature, the solvent was removed under reduced pressure. The residue was purified by thin layer chromatography (Merck 5744, eluent: chloroform / methanol, 10: 1).

15.2 mg (XXII) of product are obtained.

IR (KBr): 1365, 1510 cm -1.

NMR (CD ₃ OD) δ 6.24 (1H, s).

[α] D = + 18.0 ° (c = 0.1, methanol)

Example 11

The reaction is shown in Figure 28.

240 mg of starting material (XXIII) is hydrogenated in the presence of palladium in 10 ml of formic acid / methanol 1:24 at 0.028 bar (4 psi) for 7 hours. After filtration of the mixture, the filtrate was evaporated to give 140 mg of compound XXIV.

IR (KBr): 1730, 1650, 1510 cm -1.

[α] β = 21.04 ”(c = 0.1, methanol).

Example 12

The reaction sequence is shown in Figure 29. Under nitrogen, 22 mg (XXV) of the starting material was dissolved in 0.8 ml of hydrogen fluoride / pyridine and 0.2 ml of anisole was added. After stirring for 1 hour at room temperature, some ice was added and the pH of the solution was adjusted to pH 8 with aqueous sodium bicarbonate. The reaction mixture was applied to a Diaion HP-20 column (10 mL) and washed with water. The desired product was eluted with methanol and purified by TLC on a Merck 5715 plate using chloroform / methanol / water 3: 1: 0.1 (v / v) as eluent. 13.0 mg of compound (XXVI) are obtained.

IR (KBr): 1635, 1510 ^cm-first

NMR (CD ₃ OD), delta: 7.05 (1H, s).

[α] D = -90.6 ° (c = 0.1, methanol).

TLC: Merck 5715, chloroform = methanol / water, Rf = 0.35,

3: 1: 0.1).

Example 13

The reaction is shown in Figure 30.

6.0 mg (XXVI) of the starting material in 1.5 ml of dichloromethane

To a solution of A9 in Denmark containing 30 μΐ of bis (dimethylsilyl) acetamide and 0.3 ml of N, N-dimethylformamide, add 0.4 ml of a 0.02 M solution of starting material (XXVII). t. After stirring for one hour at room temperature, 0.1 mg of dimethylaminopyridine is added to the reaction mixture. The starting material (XXVII) is added to the reaction mixture every 30 minutes until starting material (XXVI) disappears. Diluted hydrochloric acid was added to the solution and the organic phase was washed with water. After evaporation under reduced pressure, the residue is purified by preparative thin layer chromatography on a Merck 5715 plate using chloroform / methanol / water 65: 25: 4 (v / v) as eluent. 92 mg of compound (XXIX) are obtained.

This product is identical to WS-9326A obtained in Example 1.

Example 14

The reaction is shown in Figure 31.

To a solution of the starting material (XXVI) in 1 ml of pyridine add 0.6 ml of a 0.02 M solution of the starting material (XXVII) in dichloromethane. After stirring for one hour at room temperature, starting material (XXVII) is added hourly to the reaction mixture until starting material (XXVI) disappears. Methanol (2 mL) was added to the reaction mixture and the solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (10 mL), washed with dilute hydrochloric acid and water. After the solution was dried over magnesium sulfate, the solvent was removed under reduced pressure. The evaporation residue was purified by preparative thin layer chromatography on a Merck 5715 plate using chloroform / methanol / water 3: 1: 0.1 (v / v) as eluent. 2.0 mg of compound (XXIX) are obtained.

IR (KBr): 1640, 1510 cm -1.

Example 75

The reaction is illustrated in Figure 32.

To a solution of 49.7 mg of starting material (XXIV) in 1 ml of pyridine is added 1.2 ml of a 0.1M solution of starting material (XXVII) in dichloromethane (1.2 ml) and stirred at room temperature for 3.5 hours. Ethyl acetate was added to the reaction mixture and the reaction mixture was washed with water, 7% acetic acid, water and then with a saturated aqueous solution of sodium chloride. After drying over magnesium sulfate, the solvent was evaporated and the residue was purified by preparative thin layer chromatography (0.5 mm x 2) using 20% methanol in chloroform as eluent. 20.6 mg of compound (XXX) are obtained. This compound is identical to WS9326B prepared in Example 3.

Example 76

The following compounds can be prepared as described in Example 15:

Compound (XXXI): R-: Benzoyl [α] 25 D-45.8 ° (c = 0.74, methanol).

176-178 ° C

TLC: Merck 5715, chloroform / methanol 5: 1 Rf = 0.48

IR (KBr) = 1720 (shoulder), 1655, 1640 cm -1.

Compound (XXXII): R-: 2- (2-Thienyl) -acetyl [α] 20 D = -16.8 ° (c = 0.73, methanol)

160-163 ° C

TLC: Rf = 0.24 (Merck 5715, chloroform / methanol, 5: 1) IR (KBr): 1720 (shoulder), 1650 cm @ -1.

Compound (ΧΧΧΙΠ): R-: acetyl [α] D = -37.4 ° (c = 0.72, methanol)

M.p. 231-233 ° C

TLC: Rf = 0.41 (Merck 5715, Chloroform / MeOH = 5: 1) IR (KBr): 1720 (shoulder), 1650 ^cm-first

/ Example 7

The reaction is shown in Figure 33.

To a solution of 100 mg (XXXIV) of the starting material in 1 ml of pyridine was added 11 μΐ of acetic anhydride and the reaction mixture was allowed to stand overnight at room temperature.

The reaction mixture was evaporated and the oily residue was purified by preparative thin layer chromatography (chloroform / methanol 9: 1) to give 52 mg (XXXV).

TLC: Merck 5715 plate, chloroform / methanol 10: 1 Rf = 0.17.

IR (Nujol): 3300, 1760, 1730, 1650, 1530, 1510, 1200,

1160, 1070, 910 cm -1.

Example 18

The reaction is shown in Figure 34.

To a solution of 100 mg (XXXVI) of the starting material in 1 ml of pyridine is added 25 μΐ of acetic anhydride and the mixture is allowed to stand overnight at room temperature.

The reaction mixture was evaporated and the oily residue was purified by preparative thin layer chromatography (chloroform: methanol = 9: 1) to give 78 mg (XXXVII).

TLC: Merck 5715 plate, chloroform / methanol 10: 1 R _t = 0.36.

IR (Nujol): 3300. 1740, 1650, 1540, 1510, 1300, 1220,

1200, 1170, 1050, 920 cm -1.

Figure 19

The reaction is illustrated in Figure 35.

To a solution of the starting material (0.21 g, XXXVI) in methanol (3 mL) was added diazomethane (3 mL) in diethyl ether. After stirring for 5 minutes, the solvent was evaporated under reduced pressure. The residue was purified by preparative thin layer chromatography (Merck 5744 plate, 20% methanol in chloroform) to give 45 mg (XXXVIII). IR (Nujol): 3300, 1730, 1645, 1530, 1510 cm -1.

Example 20

The reaction is shown in Figure 36.

To a solution of 1.0 g (XXXVI) of the starting material in 15 ml of methanol is added 5 ml of 1N sodium hydroxide solution at 0 ° C. After stirring for 1 hour, 5 ml of IN hydrochloric acid are added to the solution. The solvent

A9 was removed under reduced pressure, and the residue was dissolved in a mixture of 20 ml of ethyl acetate and 30 ml of dilute hydrochloric acid. The organic layer was washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The resulting solid was washed with ethyl acetate to give 0.95 g (XXXIX) of product.

IR (Nujol): 3300, 1710 shoulder), 1645, 1510 cm -1.

Example 21

The reaction is shown in Figure 37.

To a solution of 1.0 g of the starting material (XL) in a mixture of 2 ml of methanol and 20 ml of ethyl acetate is added 10 ml of a 10% solution of trimethylsilyl diazomethane in n-hexane. After stirring for 5 minutes, the solvent was evaporated under reduced pressure. The residue was applied to a 20 g silica gel column (Merck 7734) and eluted with chloroform / methanol (10: 1) to give 0.55 g of compound (XLI).

IR (Nujol): 3300, 1735, 1645, 1530, 1510 ^cm-first

Example 22

Example XXXIV was prepared as described in Example 14.

Molecular Weight FAB Mass Spec: m / z = 1041.6 (M + H) ⁺ .

The following examples illustrate the preparation of intermediates:

Example 23

The reaction sequence is shown in Figure 38.

A solution of 2.53 g of the starting material (XLII) in 10 ml of methanol / ml of water is added

1.63 g of cerium carbonate.

The solvent was evaporated and the residue was dissolved in Ν, Ν-dimethylformamide and 1.92 g of phenacyl bromide was added to the reaction mixture. The reaction mixture was stirred for 30 minutes at room temperature. The solvent was distilled off and the residue was dissolved in ethyl acetate and washed with water. After drying over magnesium sulfate and filtration, the solvent was evaporated to give 3.7 g of crystalline product (XLIII).

] ^Α ] ϋ ^{= -} 20.3 ° (c = 1, chloroform)

IR (Nujol): 1745, 1690, 1545 cm ^1st

Example 24

The reaction is shown in Figure 39.

To a solution of the starting material (XLIII) (1.48 g) in dichloromethane (80 mL) was added 0.764 g of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 0.488 g of 4-dimethylamino at 0 ° C. pyridine. After stirring for 6 hours, the solvent was evaporated.

The residue was dissolved in ethyl acetate and washed with water, 1N hydrochloric acid, saturated aqueous sodium bicarbonate, and water.

After drying over magnesium sulfate and filtration, the solvent was evaporated, yielding 2.58 g (XLIV). [.alpha.] D @ 20 = + 9.76 DEG (c = 0.3, chloroform)

IR (CHCl ₃ ): 1755, 1720, 1705, 1505 cm ^-1 .

NMR (CDCl 3, delta: 1.37 (3H, d, J = 6Hz), 1.45 (9H,

s), 3.70 (1H, m), 3.88 (1H, m), 4.52 (2H, m), 4.23 (1H, m), 5.17 (2H, s), 5, 37 (2H, s).

Example 25

The reaction is shown in Figure 40.

To a solution of starting material (XLIV) in 100 ml of 90% aqueous acetic acid was added 11 g of zinc powder with stirring and the reaction mixture was stirred under ice-cooling for 1 hour and then at room temperature for 2 hours.

The reaction mixture is filtered, the filtrate is concentrated, the pH is adjusted to pH 2 with citric acid and extracted with ethyl acetate.

The extract was dried over magnesium sulfate, filtered and the solvent was evaporated. The residue was washed with petroleum ether to give 5.4 g (XLV) product.

NMR (CDCl ₃ ), δ: 1.30 (3H, d, J = 6Hz), 1.40 (9H,

s), 3.60 (1H, m) 3.82 (1H, m).

Example 26

The reaction is shown in Figure 41.

To a solution of the starting material (XLVI) (7.7 g) in dichloromethane (60 ml) was added, with stirring at 0 ° C, 2.105 ml of 2,2-trichloroethanol, 4.2 g of 1-ethyl-3- (3-dimethyl- aminopropyl) carbodiimide hydrochloride and 244 µl of 4-dimethylaminopyridine. The reaction mixture was stirred at 0 ° C for 1 hour and then concentrated. The residue was dissolved in ethyl acetate, washed with water, 1N hydrochloric acid, water and then with a saturated aqueous solution of sodium bicarbonate.

The solution was dried over magnesium sulfate, filtered and evaporated to give 9.37 g (XLVII). [α] 25 D = - 29.84 ° (c = 0.4, chloroform).

IR (CHCl ₃ ): 1755, 1690, 1610, 1510 cm ^-1 .

Example 27

The reaction is shown in Figure 42.

The starting material (XLVII) (9.3 g) was cooled to 0 ° C and added to 15 ml of trifluoroacetic acid.

The reaction mixture was stirred at 0 ° C for 30 minutes and then concentrated. The residue was dissolved in ethyl acetate, washed with water, a saturated aqueous solution of sodium bicarbonate and water. After drying over magnesium sulfate and filtration, the solvent was evaporated to give 6 g (XLVIII).

NMR (CDCl ₃ ) delta: 2.45 (3H, s), 3.03 (2H, m), 3.62 (1H, t, J = 7Hz), 4.75 (2H, m), 5.06 (2H, s), 6.94 (2H, d, J = 8Hz), 7.15 (2H, d, J = 8Hz), 7.3-7.5 (5H, m).

Example 28

The reaction is shown in Figure 43.

To a solution of the starting material (4.07 g, XLIX) in dichloromethane (40 mL) was added 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (2.8 g) and the reaction mixture was stirred at room temperature for 24 hours. The solvent was evaporated and the residue was dissolved in ethyl acetate, washed with 1 N hydrochloric acid, water, a saturated aqueous solution of sodium bicarbonate and water.

The solution was dried over magnesium sulfate and the filtrate was evaporated to give 4.35 g (L). [α] D = -27.88 '(c = 0.12, chloroform).

IR (CHCl₃): 1750, 1710, 1655, 1510 ^cm-first

NMR (CDCl ₃ ) delta: 1.18 (3H, d, J = 6Hz), 1.36 (9H, s),

HU 211 331 A9

2.29 (3H, s), 4.69 (2H, s,), 4.91 (2H, s), 5.01 (2H, s), 6.80 (2H, d, J = 8Hz), 7.02 (2H, d, J = 8Hz).

Example 29

The reaction is shown in Figure 44.

The starting material (L) (4.35 g) was cooled to 0 ° C and added to trifluoroacetic acid (20 mL). The reaction mixture was stirred at 0 ° C for 45 minutes and then concentrated. The residue was dissolved in ethyl acetate and washed with a saturated aqueous solution of sodium bicarbonate and water. The solution was dried over magnesium sulfate, filtered and evaporated. The residue was dissolved in 100 ml of dichloromethane.

To the solution was added 1.2 g of Boc-Asn, 990 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 700 mg of 1-hydrobenzotriazole. The reaction mixture was stirred for 4 hours at 0 ° C, washed with IN hydrochloric acid, water, saturated aqueous sodium bicarbonate and finally water, and the solvent was evaporated, yielding 4.75 g of (LI).

[α] β = -15.7 '(c = 0.1, chloroform)

IR (KBr): 1740. 1690, 1645, 1510 cm -1.

NMR (CDCl 3 delta: 1.25 (3H, d, J = 6Hz), 1.43 (9H, s),

3.00 (3H, s), 2.55 (1H, m), 2.80 (1H, m), 3.05 (1H, m), 3.37 (1H, m), 3.62 (1H) , m), 3.82 (1H, m), 6.88 (2H, 2, J = 8.5Hz), 7.09 (2H, d, J = 8.5Hz).

Example 30

The reaction is shown in Figure 45.

Example 29 is prepared from compound (LII) from starting material (LI).

[α] D = -13.04 '(c = 0.11, CHCl ₃ ).

IR (KBr): 1740, 1700, 1655, 1510 ^cm-first

NMR (CDCl 3 delta: 1.18, 1.27 (each 3Η, d,

J = 6Hz), 1.43 (9Η, s), 2.52 (1Η, m), 2.80 (1H, m),

3.00 (3H, s), 3.36 (1H, m), 4.99 (2H, s), 6.87 (2H, d, J = 8Hz), 7.10 (2H, d, J = 8 Hz).

3J. example

The reaction flows are shown in Figure 46.

The desired compound (Lili) is prepared from starting material (LII) as described in Example 29. [α] 20 D = -15.19 ° (c = 0.1, CHCl 3).

IR (KBr): 1740, 1650, 1635, 1510 ^cm-first

NMR (CDCl 3) δ: 1.11 (3Η, d, J = 6Hz), 1.27 (3Η, d,

J = 6Hz). 1.33 (9H, s), 3.01 (3Η, s), 4.72 (2H, s),

4.98 (2H, s), 6.87 (2H, d, J = 8Hz), 7.10 (2H, d,

J = 8Hz).

Example 32

The reaction is shown in Figure 47.

The compound (LV) was prepared from the starting compound (LIV) as described in Example 29. [α] D 25 = -19.0 ° (c = 0.1, CHCl 3).

IR (KBr): 1740, 1635, 1510 cm ^-1 .

NMR (CDCl 3 delta: 0.81 (6H, m), 1.13, 1.28 (each 3H, d. J = 6Hz). 1.40 (9H, s), 3.02 (3H, s),

4.96 (2H, s), 6.87, 7.09 (each 2H, d, J = 8Hz).

Example 33

The reaction is shown in Figure 48.

To a solution of 4.2 g (LV) of the starting material in 80 ml of 90% aqueous acetic acid was added 9 g of zinc powder at 0 ° C and the reaction mixture was stirred at 0 ° C for 2 hours and then at room temperature for 1 hour.

The reaction mixture was filtered, the filtrate was evaporated, chloroform was added to the residue and the mixture was washed with 1 N hydrochloric acid and water.

The solution was dried over magnesium sulfate, the solvent was evaporated, and the residue was applied to a silica gel column (150 g) eluted with 2% methanol in chloroform followed by 8% methanol in chloroform.

The fractions containing the desired material were evaporated

3.4 g (LVI) of product are obtained.

[a] _D = -23.42 ° (c = 0.1, methanol).

IR (KBr): 1635. 1510 ^cm-first

Example 34

The reaction is shown in Figure 49.

3.4 g (LVI) of the starting material were cooled to 0 ° C and added to 20 ml of trifluoroacetic acid.

After stirring for 1 hour at 0 ° C, the reaction mixture was evaporated and the residue was dissolved in hydrochloric acid and evaporated. The residue was dissolved in chloroform and washed with water. The solution was dried over magnesium sulfate, filtered and evaporated to give 3.27 g (LVII).

[.alpha.] D @ 20 = -18.87 DEG (c = 0.12, methanol).

IR (KBr): 1650. 1510 cm ^-1 .

Example 35

To a solution of 26.8 g of potassium hydroxide in 500 ml of ethanol is added 14.6 g of glycine and 48.5 g of 4-methoxymethoxybenzaldehyde at room temperature. The reaction mixture was stirred for 19 hours and the solvent was evaporated under reduced pressure. The residue was dissolved in water and the solution acidified with hydrochloric acid. The solution was washed with ethyl acetate and adjusted to pH 6.0 with sodium bicarbonate. A white precipitate formed which was collected to give 9.2 g of o-methoxymethyl-p-hydroxytyrosine.

Mp: 164-166 ° C.

IR (KBr): 1610 cm ^-1 .

Example 36

To a solution of 21.0 g of o-methoxymethyl-p-hydroxytyrosine in 250 ml of 1N sodium hydroxide solution is added 16.5 g of dimethyl sulfate. After stirring at 90 ° C for 20 minutes, the solution is acidified with dilute hydrochloric acid in an ice bath. The acidic solution was washed with diethyl ether and adjusted to pH 6.0 with 1N sodium hydroxide solution. After evaporation, the precipitated solid was collected by filtration

5.2 g of o-methoxymethyl-N-methyl-β-hydroxytyrosine are obtained.

177-178 'C.

IR (KBr): 3100, 1600 cm -1.

HU 211 331 A9

Example 37

To a solution of 15.1 g of o-methoxymethyl-N-methyl-β-hydroxytyrosine and 25 ml of bis (trimethylsilyl) acetamide in 150 ml of dichloromethane are added 11.2 g of 2-nitrophenylsulfenyl chloride 50 ml of dichloromethane. After stirring for 2 hours at 0 ° C, 10 ml of bis (trimethylsilyl) acetamide and 5.6 g of 2-nitrophenylsulfenyl chloride are added. The reaction mixture is stirred at room temperature for 3 hours and 200 ml of 1N sodium hydroxide are added. The organic layer was washed with water (300 mL) and the aqueous solutions were combined. The aqueous solution was acidified with dilute hydrochloric acid, extracted with ethyl acetate (300 mL), and the extract was washed with water (100 mL). The solution was dried over magnesium sulfate and the solvent was evaporated under reduced pressure to give 20.5 g of o-methoxymethyl nitrophenylthio) - [1-hydroxytyrosine is obtained, m.p.

IR (KBr): 3400, 1700 ^cm-first

Example 38

To a solution of 20.0 g of o-methoxymethyl-N-methyl-N- (2-nitrophenylthio) -P-hydroxytyrosine in 100 ml of ethyl acetate is added 80 ml of diazomethane in diethyl ether. After stirring for 10 minutes, the solvent was evaporated under reduced pressure, the residue was applied to a column of 500 g of silica gel (Merck 7734) and eluted with chloroform, o-methoxymethyl-N-methyl-N- (2-nitrophenylthio) -? yielding tyrosine methyl ester in the form of 8.82 g of threo and 6.63 g of erythro isomer.

Threo isomer

IR (film): 3500, 2950, 1735 ^cm-first

TLC (Merck 5717 plate, ethyl acetate / n-hexane, 1: 1):

R (= 0.40

Erythro isomer

IR (film): 3500, 2950, 1735 cm ^-1 .

TLC (Merck 5715 plate, ethyl acetate / n-hexane, 1: 1):

Rf 0:31

Example 39

To a solution of 3.85 g of the erythro-isomer of o-methoxymethyl-N-methyl-N- (2-nitrophenylthio) -p-hydroxytyrosine methyl ester in 30 ml of dichloromethane is added 1.38 g of triethylamine, 0.45 g of 4-dimethylaminopyridine and 1.92 g of benzoyl chloride. After stirring for 16 hours at room temperature, 3.3 g of 3-dimethylaminopropylamine are added to the reaction mixture, and the solvent is evaporated under reduced pressure. The residue was dissolved in ethyl acetate (30 mL), washed with dilute hydrochloric acid, aqueous sodium bicarbonate and water. The residue obtained after evaporation was applied to a column of 150 g of silica gel (Merck 7734) and eluted with n-hexane / ethyl acetate (5: 2 (v / v)), 4.49 g of o-methoxymethyl-N-methylN- (2- Nitro-phenyl-lio) -N-benzoyloxy-tyrosine methyl ester is obtained as the erythro isomer.

IR (film): 2950, 1740 cm -1.

TLC: (Merck Art 5715 plate, ethyl acetate / n-hexane,

1: 2): R _f = 0.23.

Example 40

Preparation of O-Methoxy-N-methyl-N- (2-nitrophenylthio) -4-benzoyloxy-tyrosine methyl ester, threo isomer

Example 39.

114-115'C.

IR (CHCl ₃ ) delta: 2950, 1740 cm ^-1 .

TLC (Merck Art 5715, ethyl acetate / n-hexane, 1: 2),

Rf = 0.26.

Example 41

To a solution of 4.94 g of the threoisomer of o-methoxymethyl-N-methyl-N- (2-nitrophenylthio) -P-benzoyloxy-tyrosine methyl ester in 50 ml of dichloromethane was added at 0 ° C, 8 ml of thiophenol and 2.5 ml of trifluoroacetic acid. After stirring for 30 minutes, a solution of sodium bicarbonate is added. The organic layer was washed with aqueous sodium bicarbonate and brine. After evaporation, the residue was applied to a column of 100 g of silica gel (Merck 7734) and eluted with 5% methanol in chloroform to give 0.32 g of o-methoxymethyl-N-methyl-p-benzoyloxy-tyrosine methyl ester, yielding the threo isomer. .

TLC (Merck Art 5715, ethyl acetate / n-hexane, 1: 1),

Rf = 0.31

Example 42

The erythroisomer of o-methoxymethyl-N-methyl-β-benzoyloxy-tyrosine methyl ester was prepared as described in Example 41.

TLC (Merck Art 5715, ethyl acetate / n-hexane, 1: 1),

R (= 0.25.

Example 43

To a solution of 3.7 g of N-benzyloxycarbonyl-L-threonine and 3.11 gmethoxymethyl-N-methyl-4-benzoyloxy-tyrosine methyl ester in 50 ml of dichloromethane is added 2.9 g of , 2-dihydro-2-ethoxy-l-quinolinecarboxylate. After stirring for 20 hours at room temperature, the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (50 mL), washed with dilute hydrochloric acid, aqueous sodium bicarbonate and water. After evaporation, the residue was applied to a column of 100 g of silica gel (Merck 7734) and eluted with n-hexane / ethyl acetate (1: 1 v / v) to give 2.04 g of N- (N-benzoyloxycarbonyl-L-threonyl) The methoxy-methyl-N-methyl-P-benzyloxy-tyrosine methyl ester in threo isomer is obtained.

IR (film): 3400, 2950, 1740 (shoulder), 1720 ^cm-first

TLC (Merck Art 5715, methanol / chloroform, 3:97),

R (= 0.36.

Example 44

The erythro-isomer of N- (N-benzyloxycarbonyl-L-threonyl) -omethoxymethyl-N-methyl-β-benzoyloxy-tyrosine methyl ester was prepared as described in Example 43.

IR (film): 2950, 1740, 1730 (shoulder) cm -1.

TLC (Merck Art 5715, ethyl acetate / n-hexane, 1: 2),

R (= 0.23.

Example 45

To a solution of 1.20 g of p-benzoyloxy-N- (N-benzyloxycarbonyl-l-threonyl) -omethoxymethyl-N-methyl-tyrosine methyl ester, threoisomer in 20 ml of toluene21

0.8 g of 1.8 diazabicyclo [5.4.0] undec-7-ene are added. After stirring for half an hour at room temperature, 10 ml of 7% hydrochloric acid were added to the reaction mixture. The organic layer was washed with water and aqueous sodium bicarbonate, dried over magnesium sulfate, and the solvent was evaporated to give 0.95 g (LVIII).

IR (film): 3400, 2950, 1720 cm @ ^-1 .

[.Alpha.] D @ 20 = -7.7 DEG (c = 0.64 in methanol).

The product (LVIII) can also be obtained if a

P-benzoyloxy-N- (N-benzyloxycarbonyl-L-threonyl) -o-methoxymethyl-N-methyl-tyrosine methyl ester, starting from the erythro isomer instead of the threo isomer.

Example 46

The reaction is shown in Figure 50.

To a solution of 1.0 g (LVIII) of the starting material in 10 ml of Ν, Ν-dimethylformamide is added 0.75 g of tert-butyldimethylsilyl chloride and 0.34 g of imidazole. After stirring at room temperature for 16 hours, 30 ml of ethyl acetate and 50 g of ice were added. The organic layer was washed with dilute hydrochloric acid, aqueous sodium bicarbonate and water. The solvent was removed under reduced pressure and the residue was applied to a column of 30 g of silica gel (Merck 7734) and eluted with chloroform to give 1.21 g of product (LIX).

IR (film): 2950, 1720 ^cm-first

[α] D = -55.9 ° (c = 0.56, methanol).

Example 47

The reaction is shown in Figure 51.

To a solution of the starting material (0.95 g, LIX) is added 4.8 ml of an aqueous sodium hydroxide solution. After stirring for two days at 30 ° C, the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (20 mL), washed with dilute hydrochloric acid and water. The solution was evaporated to give 0.81 g of (LX).

IR (film): 3300, 2950, 1720, 1700 (shoulder) cm ^-1 .

[.alpha.] D @ 20 ⁼ -82.9 DEG (c = 1.06, methanol).

Example 48

The reaction is shown in Figure 52.

To a mixture of 1.60 g of (LX) starting material and 5.50 (LXI) of starting material in 50 ml of dichloromethane is added 3.04 g of 1,2-dihydro-2-ethoxy-1-quinolinecarboxylate. After stirring for 15 hours at room temperature, the white solid was filtered off and 2.23 g (LXI) starting material, 0.50 g triethylamine and 1.24 g ethyl-1,2-dihydro-2-ethoxy-1 were added to the filtrate. quinoline-carboxylate. The reaction mixture was stirred for 18 hours and then the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (50 mL), washed with dilute hydrochloric acid, aqueous sodium bicarbonate and water. Concentrate the solution under reduced pressure and apply the residue to a column of 100 g silica gel (Merck 7734) and elute with n-hexane / ethyl acetate 2: 1 (v / v) to give 0.87 g (LXII).

IR (film): 2950, 1760, 1740 (shoulder), 1720, 1660 cm ^1st [α] D = -31.5 ° (c = 1.07, methanol).

Example 49

The reaction is shown in Figure 53.

A solution of 0.85 g (LXII) starting material in 100 ml toluene and 10 ml acetone was irradiated with an ultraviolet lamp (100 V) for 1.5 hours at 0 ° C. The solution was evaporated and the residue was applied to a column of 50 g of silica gel (Merck 7734) and eluted with n-hexane / ethyl acetate 2: 1 (v / v) to give 0.18 g (LXIH).

TLC (Merck Art 5715, n-hexane / ethyl acetate 2: 1),

Rf = 0.22.

IR (KBr) 3300, 1740 (shoulder), 1640 ^cm-first

Example 50

The reaction sequence is shown in Figure 54.

0.17 g (LXIII) of the starting material is dissolved in 10 ml of 67% aqueous acetic acid. After stirring for 28 hours at 25 ° C, the solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (20 mL) and the solution was washed with aqueous sodium bicarbonate and water.

After concentration, the residue is washed with n-hexane and the solvent is evaporated under reduced pressure to give 0.15 g (LXIV).

IR (KBr): 3250, 1740 (shoulder), 1635 cm ^1st

TLC (Merck Art 5715, n-hexane / ethyl acetate 1: 1),

Rf = 0.18.

Example 51

The reaction is shown in Figure 55.

To a solution of 0.14 g (LXIV) starting material in 5 mL dichloromethane is added 0.10 g (XLIV) starting material. Water-soluble carbodiimide hydrochloride (65 g) and 4-dimethylaminopyridine (4 mg). Stir for 12 hours at room temperature. N, N-dimethylaminopropylamine (50 mg) was added to the reaction mixture and the solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (20 mL), washed with dilute hydrochloric acid and water. After evaporation, the residue was applied to a column of 10 g silica gel (Merck 7734) and eluted with n-hexane / ethyl acetate 1: 1 (v / v) to give 0.19 g (LXV).

IR (KBr): 3300. 1700, 1640, 1495 cm ^-1 .

TLC (Merck Art 5715, n-hexane / ethyl acetate 1: 1),

Rf 0.38.

Example 52

The reaction is shown in Figure 56.

145 mg (LXV) of the starting material is dissolved in 3 ml of 4N hydrochloric acid in dioxane containing 0.1 ml of anisole. After stirring for 30 minutes at room temperature, the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (3 mL). To the solution were added 35 mg of N-tert-butoxycarbonyl-L-asparagine, 13 mg of triethylamine, 18 mg of 1-hydroxybenzotriazole and 29 mg of water-soluble carbodiimide hydrochloride. After stirring for 1 hour at room temperature, 5 ml of 7% hydrochloric acid are added. The organic phase is washed with water. After evaporation, the residue was purified by preparative thin layer chromatography (Merck 5744) eluting with 6%

A9 using methanol containing chloroform. 110 mg (LXVI) of product are obtained.

IR (KBr): 3300, 1650, 1505 cm -1.

TLC (Merck Art 5715, chloroform / methanol, 10: 1),

Rf = 0.44.

Example 53

The reaction is shown in Figure 57.

105 mg (LXVI) of the starting material is dissolved in 3 ml of 4N hydrochloric acid in dioxane containing 0.1 ml of anisole. After stirring for 30 minutes at room temperature, the solvent was evaporated under reduced pressure. The residue was dissolved in dichloromethane (3 mL). To the solution were added 22 mg of N-tert-butoxycarbonyl-L-allotreonine, 9 mg of triethylamine, 12 mg of 1-hydroxybenzotriazole and 19 mg of water-soluble carbodiimide hydrochloride. After stirring the reaction mixture at room temperature for 8 hours, 5 ml of 7% hydrochloric acid was added. The organic phase is washed with water. After evaporation, the residue was purified by preparative thin layer chromatography (Merck 5744) using 6% methanol in chloroform as eluent.

Product (LXVII) Specifications:

TLC (Merck Art 5715, 5: 1 chloroform / methanol) Rf = 0.73.

IR (KBr) 3300, 1740 (shoulder), 1650, 1500 ^cm-first

Example 54

The reaction is shown in Figure 58.

58.5 mg (LXVII) of the starting material is dissolved in 1 ml of 90% aqueous acetic acid and 30 mg of zinc powder are added. After stirring for 9 hours at room temperature, an additional 30 mg of zinc powder is added at 1 hour intervals until starting material (LXVII) disappears. After filtration, the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (10 mL), washed with water and concentrated under reduced pressure. The residue was purified by preparative thin layer chromatography (Merck 5744) eluting with ethyl acetate / acetone / acetic acid / water 6: 3: 1: 1: v / v to give 43.5 mg (LXVIII).

IR (KBr): 3330, 1650, 1505 cm -1.

TLC (Merck Art 5715, chloroform / methanol / acetic acid, 10: 1: 0.1), _Rf = 0.16.

Example 55

To a solution of 6.7 g of phthalaldehyde in 30 ml of dichloromethane is added 17.42 g of ethoxycarbonylmethylene triphenylphosphorane and the reaction mixture is stirred for 30 minutes at room temperature. The solvent was evaporated and the residue was dissolved in diethyl ether. After the mixture was filtered, the filtrate was evaporated and the residue was distilled under vacuum at 125 ° C and 0.6 mm Hg. 6 g of (E) -3- (2-formylphenyl) propenoic acid ethyl ester are obtained.

NMR (CDCl 3) delta: 1.24 (3H, t, J = 6.5Hz), 4.19 (2H, q, J = 6.5Hz), 6.28 (1H, d, J = 15Hz), δ , Δ (3H, m),

7.77 (1H, m), 8.32 (1H, d, J = 15Hz), 10.18 (1H, s).

Example 56

To a solution of 3.2 g of butyl triphenylphosphonium bromide in 50 ml of tetrahydrofuran was added 900 mg of potassium tert-butoxide under nitrogen and stirred for 30 minutes at room temperature. A solution of (E) -3- (2-formylphenyl) propenoic acid ethyl ester (2.0 g) in tetrahydrofuran (30 mL) was added to the reaction mixture and stirred for 1 hour. The solvent was evaporated and the residue was dissolved in diethyl ether and washed with brine and water.

The solution was dried over magnesium sulfate, filtered and evaporated. The residue was applied to a silica gel column (100 g) and eluted with 3: 1 n-hexane / ethyl acetate. Fractions containing the desired product were evaporated to give (E) -3- [2 - ((Z) -1-pentenyl) phenyl] propenoic acid ethyl ester (2.00 g).

NMR (CDCl 3) δ: 0.88 (3H, t, J = 7Hz), 1.34 (3H, t,

J = 6.5Hz), 1.42 (2H, m), 2.05 (2H, m), 4.27 (2H, q,

J = 6.5Hz), 5.85 (1H, dt, J = 7 and 11Hz), 7.3 (3H, m),

7.61 (1H, m), 7.92 (1H, d, J = 16Hz).

Example 57 To a solution of (E) -3- [2 - ((N-pentenyl) phenyl] propenoic acid ethyl ester in 20% aqueous methanol was added 2.3 g of potassium hydroxide, and the reaction mixture was stirred for 2 hours. After stirring at 60 ° C, the pH was adjusted to pH 1 with hydrochloric acid and the mixture was extracted with ethyl acetate, dried over magnesium sulfate, filtered and the solvent was evaporated. the solution

Add to 1.63 ml of dicyclohexylamine. The resulting crystalline material was dissolved in ethyl acetate and the solution was washed with IN sulfuric acid. The solution was dried over magnesium sulfate, filtered and evaporated to give 0.92 g of (E) -3- [2 - ((Z) -1-pentenyl) phenyl] propenoic acid.

IR (Nujol): 1690, 1680, 1620 ^cm-first

Example 58

1.08 g of (E) -3- [2 - ((Z) -1-pentenyl) phenyl] propenoic acid are dissolved in 10 ml of dichloromethane, 0.5 ml of oxalyl chloride and 0.05 ml of Ν, Ν- dimethylformamide. The reaction mixture was stirred under nitrogen for 1 hour at room temperature and then the solvent was evaporated. The residue was dissolved in n-hexane and the mixture was filtered. The filtrate was evaporated to give (E) -3- [2 - ((Z) -1-pentenyl) phenyl] propenyl chloride (1.15 g).

IR (Neat): 1750, 1730, 1605, 1858 cm -1.

NMR (CDCl ₃ delta: 0.88 (3H, t, J = 6.5Hz), 1.45 (2H, m), 2.06 (2H, m), 5.95 (1H, dt, J and 7Hz), 6.58 (1H, d, J = 11Hz), 6.66 (1H, d, J = 16Hz), 7.4 (3H, m),

7.69 (1H, m), 8.12 (1H, d, J = 16Hz).

PATENT CLAIMS

Claims (10)

PATENT CLAIMS What is claimed is: 1. A compound of formula I wherein: R ¹ is hydrogen; C 1-20 alkanoyl; C2-C20 alkoxycarbonyl; C 1-20 alkanesulfonyl; C 1 -C 20 alkoxysulfonyl; benzoyl; toluolil-; naphthoyl; C 12 -C 16 naphthylalkanoyl; C 8 -C 12 phenylalkanoyl; 12-16 A9-C9 naphthylalkanoyl substituted with a substituent selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 carbon alkylamino, C3-6cycloalkyl, C3-6cycloalkenyl, halogen, amino, protected amino, hydroxy, protected hydroxy, cyano, nitro, carboxyl, protected carboxyl, sulfo, sulfamoyl, imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, C 1-6 hydroxyalkyl, and C 3-7 cyanoalkenylthio; C 8 -C 12 phenylalkanoyl substituted with one substituent selected from C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 1 -C 6 alkylamino, C3-6cycloalkyl, 3-6Cene cycloalkenyl, halogen. amino, protected amino, hydroxyl, protected hydroxyl, cyano. nitro, carboxyl, protected carboxyl. sulfo, sulfamoyl, imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, C 1-6 hydroxyalkyl, and C 3-7 cyanoalkenylthio; C 13 -C 16 naphthylalkenoyl; C 9 -C 12 phenylalkenoyl; C 13 -C 16 naphthylalkenoyl substituted with one substituent selected from the group consisting of C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 1 -C 6 alkylamino, C3-6cycloalkyl, C3-6cycloalkenyl, halogen, amino, protected amino, hydroxy, protected hydroxy, cyano, nitro, carboxyl, protected carboxyl, sulfo, sulfamoyl, imino, oxo, C 1-6 aminoalkyl, carbomoyloxy, C 1-6 hydroxyalkyl, and C 3-7 cyanoalkenylthio; C 9 -C 12 phenylalkenoyl substituted with one substituent selected from the group consisting of C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 1 -C 6 alkylamino -, C 3-6 cycloalkyl, C 36 cycloalkenyl, halogen, amino, protected amino, hydroxyl, protected hydroxyl, cyano, nitro, carboxyl, protected carboxyl, sulfo, sulfamoyl, imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, C 1-6 hydroxyalkyl, and C 3-7 cyanoalkenylthio; C2-C6alkoxycarbonyl substituted with phenyl; phenoxycarbonyl; naphthyloxy-carbonyl; fenilglioxiloil-; glioxiloil- naphthyl; benzenesulfonyl; p-toluenesulfonyl; or C 1 -C 6 alkanoyl or glyoxoyl group which is substituted by unsubstituted or substituted thienyl, pyrrolyl, pyrrolinyl selected from the group below. imidazolyl, pyrazolyl, pyridyl, dihydropyridyl, pyrimidyl, pyrazinyl. pyridazinyl, triazolyl, tetrazolyl, pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, , oxadiazolyl, morpholinyl, sidnonyl, benzoxazolyl, benzoxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, dihydrothiazinyl, thiazolidinyl, dihydrodithinyl, dihydrodithionyl, benzothiazolyl, benzothiazolyl, benzothiazolyl, benzothiazolyl, -, dihydro-oxathinyl, benzothienyl, benzodithinyl and benzoxathinyl, wherein the ring substituents are selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, C 6 alkylthio, C 1-6 alkylamino, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, halogen, amino, protected amino, hydroxy, protected hydroxy, cyano, nitro, carboxyl -, protected carboxyl, sulfo, sulfamoyl, imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, 1 C 6 -C 6 hydroxyalkyl and C 3 -C 7 cyanoalkenylthio; R ² is hydroxy and R ³ is carboxyl or protected carboxyl, or R ² and R ³ together are -OC (O) -; R ⁴ is hydroxy or protected hydroxy; ^R5 represents hydroxyl or protected hydroxyl; ^R6 is hydroxy, protected hydroxy or lower alkoxy; and ..... is a single or double bond, or a pharmaceutically acceptable salt thereof. A compound according to claim 1, wherein R ¹ is hydrogen; C 1-20 alkanoyl; benzoyl; C 8 -C 12 phenylalkanoyl; C 8 -C 12 phenylalkanoyl substituted with C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 1 -C 6 alkylamino, C3-6cycloalkyl, C3-6cycloalkenyl, halogen, amino, protected amino, hydroxy. protected hydroxy, cyano, nitro, carboxyl, protected carboxyl, sulfo, sulfamoyl, imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, C 1-6 hydroxyalkyl, and 3-7 Cyanoalkenylthio; C 9 -C 12 phenylalkenoyl; C 9 -C 12 phenylalkenoyl substituted with C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 1 -C 6 alkylamino, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkenyl-; halogen, amino, protected amino, hydroxy, protected hydroxy, cyano, nitro, carboxyl, protected carboxyl, sulfosulfamoyl, imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, C 1-6 hydroxyalkyl and C 3-7 cyanoalkenylthio; C2-C6alkoxycarbonyl substituted with phenyl; or C 1-6 alkanoyl substituted with unsubstituted or substituted thienyl, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl, pyrimidyl, pyrazinyl, pyridazinyl selected from the group consisting of -, triazolyl, tetrazolyl, pyrrolidinyl, imidazolidinyl, EN 211 331 A9 Piperidino, piperazinyl, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, oxazolyl, isoxazolyl, oxadiazolyl, morpholinyl, morpholinyl, -, benzoxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, dihydrothiazinyl, thiazolidinyl, dihydrodithinyl, dihydrodithionyl, benzothiazolyl, benzothiadiazolyl, furyl, dihydrooxathinyl, benzothienyl, benzothienyl, - and benzoxanthynyl, wherein said substituents are selected from C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkylamino, C3-6cycloalkyl, C3-6cycloalkenyl, halogen, amino, protected amino, hydroxy, protected hydroxyl, cyano, nitro, carboxyl, protected carboxyl, sulfo, sulfamoyl , imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, C 1-6 hydroxyalkyl, and C 37 cyano keniltio group. A compound according to claim 1, wherein R ¹ is hydrogen; C 1-20 alkanoyl; C2-C20 alkoxycarbonyl; C 1 -C 20 alkanesulfonyl; C 1 -C 20 alkoxysulfonyl; benzoyl; toluoyl; naphthoyl; C 12 -C 16 naphthylalkanoyl; C 8 -C 12 phenylalkanoyl; C 12 -C 16 naphthylalkanoyl substituted with one substituent selected from the group consisting of C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 1 -C 6 alkylamino, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkenyl, halogen, amino, protected amino -, hydroxy, protected hydroxy, cyano, nitro, carboxyl, protected carboxyl, sulfo, sulfamoyl, imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, C 1-6 hydroxyalkyl and C 3-7 cyanoalkenylthio; C 8 -C 12 phenylalkanoyl substituted with a substituent selected from the group consisting of C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 1-6 alkylamino, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, halogen, cyano, nitro, carboxyl, protected carboxyl, sulfo, sulfamoyl, imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, C 1-6 hydroxyalkyl, and C 3-7 cyanoacenylthio; C 13 -C 16 naphthylalkenoyl; C 9 -C 12 phenylalkenoyl; C 13 -C 16 naphthylalkenoyl substituted with C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 1 -C 6 alkylamino, C3-6cycloalkyl, C3-6cycloalkenyl, halogen, amino, protected amino, hydroxy, protected hydroxy, cyano, nitro, carboxyl, protected carboxyl, sulfo, sulfamoyl, imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, C 1-6 hydroxyalkyl, and C 3-7 cyanoalkenylthio; C 9 -C 12 phenylalkenoyl substituted with one of the following substituents: C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkylamino, C3-6cycloalkyl, C3-6cycloalkenyl, halogen, amino, protected amino, hydroxy, protected hydroxy, cyano, nitro, carboxyl, protected carboxyl, sulfo, sulfamoyl - imino, oxo, C 1-6 aminoalkyl, carbamoyloxy, C 1-6 hydroxyalkyl and C 3-7 cyanoalkenylthio; C2-C6alkoxycarbonyl substituted with phenyl; phenoxycarbonyl; naphthyloxy-carbonyl; phenylglyoxyloyl; glioxiloil- naphthyl; benzenesulfonyl; or p-toluenesulfonyl. A compound according to claim 2, wherein R ¹ is hydrogen, phenyl (C 2 -C 6) alkoxy-carbonyl, C 1 -C 6 alkanoyl, C 15 -C 20 alkanoyl, benzoyl, thienyl, (C 2 -C 6) alkanoyl, 2-6 phenyl (C3-C6 alkenoyl) substituted by (C1 -C6) alkenoyl or phenyl (C2 -C6 alkanoyl) substituted by (C1 -C6) alkyl. A compound according to claim 4, wherein R ¹ is phenyl (C 2 -C 6) alkanoyl substituted with C 1-6 alkyl, R ² and R ³ together are -OC (O) -, R ⁴ is hydroxy, R ⁵ is hydroxy, R ⁶ is hydroxy, and ----- double bond. A compound according to claim 5, wherein R ¹ is 3- (2-pentylphenyl) propanoyl. A compound according to claim 6, which is a It has the general formula LXIX, wherein R ¹ is as defined in claim 6. A pharmaceutical composition comprising an effective amount of a compound of claim 1 for the treatment or prevention of pain or asthma, together with pharmaceutically acceptable, substantially non-toxic carriers and excipients. A method of treating or preventing asthma or pain, comprising administering to a subject in need thereof an effective dose of a compound of claim 1. The method of claim 9, wherein said therapeutically effective dose is 0.01 to 1000 mg / day. HU 211 331 A9 Int.CL ⁶ : A 61 K 37/02