BACKGROUND ART Phosphodiesterase inhibitors have been developed as an anti-heart failure drug. Clinical trials performed in the <BR> <BR> <BR> <BR> United States have proven, however, that a long term use of these drugs does not improve a prognosis of heart failure (Packer M. et al.: Effect of oral milrinone on mortality in severe chronic heart failure. New Engl. J. Med. 325, 1468-1475, 1991). It has been thought that an increase in cyclic AMP leads to deteriolation of a long term prognosis because cyclic AMP enhances automaticity of heart muscles, increases oxygen consumption of the muscles, and can be a cause of ventricular arrhythmia.

Although digitalis has been widely used for treatment of heart failure, the agent causes a wide variety of side effects including arrhythmias, safe range being narrow (Hoffman B. F. and Bigger J. T. Jr.: Digitalis and allied cardiac glycosides.

In Goodman and Gilman s "The pharmacological basis of therapeutics. eds.: Gilman A. G. et al., Pergamon Press, pp.814-839, 1990.; Poole-Whilson P. A. and Robinson K.: Digoxin-A redundant drug in congestive cardiac failure.

Cardiovasc. Drug Ther. 2, 733-741, 1989).

Accordingly, the present inventors have made attempts to find a novel agent that has a strong cardiotonic activity with few side effects. As a result, they have found that a culture broth of fungi of Trichothecium sp. contains the cyclodepsipeptides which have such characteristics. and based on this finding, have filed the patent applications (Japanese laid-open patent application (kokai) No. 7-138290 and No.

7-188286). However, a need continues to exist for further compounds that have cardiotonic activity.

DISCLOSURE OF THE INVENTION In view of the above circumstances, the present inventors have synthesized analogs of cyclodepsipeptides and examined their cardiotonic effects as well as the side effects. It has been consequently found that the new cyclodepsipeptides represented by the formula (1) shown below possess strong cardiotonic, antiarrhythmic and vasodilating effects without increasing cyclic AMP in heart tissues and having side effects such as arrythmia, and that the cyclodepsipeptides are useful as a medicine, the findings leading to the completion of this invention.

Specifically, the present invention provides cyclodepsipeptides represented by the formula (1): wherein, Rla, Rlb and Rip are each a hydrogen atom or a methyl group , R2 and R4 are each a lower alkyl group, R3 is a lower alkyl group or aralkyl group, R5 and R6 each are a hydrogen atom, an alkyl group or aralkyl group having 1 to 8 carbon atoms, both of them being not hydrogen atoms at the same time, A and B are each an ethylene or a methylene group substituted by a lower alkyl group, X and Y represent each -0- or -N(Rld)- in which Rld is a hydrogen atom or a methyl group, R2 and Rid of X, and/or R4 and Ric being capable of forming a nitrogen-containing ring together with the contiguous carbon and nitrogen atoms; and a medicine containing the same as an effective ingredient.

The present invention also provides a pharmaceutical composition containing the above cyclodepsipeptide and a pharmaceutically acceptable carrier.

The present invention further provides use of the above cyclodepsipeptide as a medicine.

The present invention still further provides a method of treatment of a patient suffering from heart failure, arrhythmias, hypertension, angina pectoris or myocardial infarctions by administering an effective amount of the above cyclodepsipeptide to the patient.

Since the cyclodepsipeptides (1) of the present invention <BR> <BR> <BR> <BR> <BR> have many asymmetric carbon atoms, there are many stereoisomers.

The present invention includes all of these stereoisomers and mixtures thereof. Moreover, the cyclodepsipeptides (1) of the present invention may be present in the form of a solvate such as a hydrate. This invention includes all these forms of the cyclodepsipeptieds.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows 1H-NMR spectrum of the cyclodepsipeptide (TK22-4) obtained in Example 1.

Fig. 2 shows IR spectrum of the cyclodepsipeptide (TK22-4) obtained in Example 1.

Fig. 3 shows 1H-NMR spectrum of the cyclodepsipeptide (TK330-6) obtained in Example 2.

Fig. 4 shows 1H-NMR spectrum of the cyclodepsipeptide (TK340-6-A) obtained in Example 3.

Fig. 5 shows 1H-NMR spectrum of the cyclodepsipeptide (TK340-6-B) obtained in Example 3.

Fig. 6 shows 1H-NMR spectrum of the cyclodepsipeptide (TK350-6-A) obtained in Example 4.

Fig. 7 shows 1H-NMR spectrum of the cyclodepsipeptide (TK350-6-B) obtained in Example 4.

Fig. 8 shows 1H-NMR spectrum of the cyclodepsipeptide (TK70-5) obtained in Example 5.

Fig. 9 shows 1H-NMR spectrum of the cyclodepsipeptide (TK32-5) obtained in Example 6.

Fig. 10 shows 1H-NMR spectrum of the cyclodepsipeptide (TK33-5) obtained in Example 7.

Fig. 11 shows 1H-NMR spectrum of the cyclodepsipeptide (TK50-8) obtained in Example 8.

Fig. 12 shows 1H-NMR spectrum of the cyclodepsipeptide (TK510-7) obtained in Example 9.

Fig. 13 shows 1H-NMR spectrum of the cyclodepsipeptide (TK610-7) obtained in Example 10.

Fig. 14 shows 1H-NMR spectrum of the cyclodepsipeptide (TK620-9) obtained in Example 11.

Fig. 15 shows IR spectrum of the cyclodepsipeptide (TK620-9) obtained in Example 11.

Fig. 16 shows 1H-NMR spectrum of the cyclodepsipeptide (TK690-4) obtained in Example 12.

Fig. 17 shows 1H-NMR spectrum of the cyclodepsipeptide (TK660-7) obtained in Example 13.

Fig. 18 shows the correlation between TK22-4 concentration and the relative contractile force of right atrial muscles of rats.

Fig. 19 shows the correlation between TK22-4 concentration and the relative intercontraction interval of right atrial automatic contractions of rats.

Fig. 20 shows the correlation between TK620-9 concentration and the relative contractile force of right atrial muscles of rats.

Fig. 21 shows the correlation between TK620-9 concentration and the relative intercontraction interval of right atrial automatic contractions of rats.

Fig. 22 shows the inhibitory effect of TK620-9 on aberrant contractions of a right atrium of the guinea pig. (A) indicates amplitude and rhythm of contraction before digoxin application (Control), (B) indicates those during digoxin application and (C) indicates those during simultaneous application of digoxin and TK620-9.

Fig. 23 shows the vasodilating effect of TK22-4 on a rat aorta. TK22-4 was applied during the bar.

Fig. 24 shows the effect of TK22-4 on a maximal rising rate of the left ventricular pressure(LVdP/dt). The values are relative to the control before the drug application.

Fig. 25 shows the effect of TK610-7 on a maximal rising rate of the left ventricular pressure (LVdP/dt). The values are relative to the control before the drug application.

Fig. 26 shows the effects of TK22-4, TK610-7 and disopyramide on times (minutes) for ouabain-induced premature ventricular contraction, ventricular fibrillation and cardiac arrest to occur.

BEST MODE FOR CARRYING OUT THE INVENTION In formula (1), R2, R3 and R4 preferably represent linear or branched alkyl groups consisting of 1 to 5 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and pentyl groups, and A-and B preferably represent either simple methylene group or methylene group in which hydrogen substituted with linear or branched alkyl groups consisting of 1 to 5 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and pentyl groups.

Examples of the aralkyl group represented by R3, R5 and R6 include benzyl, and the alkyl groups having 1 to 8 carbon atoms represented by R5 and R6 include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, hexyl, heptyl and octyl groups. Nitrogen-containing rings formed by R2 and Rld of X, and/or R4 and Ric together with the adjacent carbon atom and nitrogen atom include a pyrrolidine ring and an octahydroindole ring.

The cyclodepsipeptide (1) of this invention is a compound that consists of amino acids and hydroxy acids, as shown by the following formula: The compounds are classified into the following three types in terms of chemical structure.

The first type (type-l), as shown by formula (1A), is a compound wherein X and Y in the formula (1) are both -N(R1d)-.

In this case, the two Rld'sR1d's Rld'sR1d's may be the same or different. wherein the meaning of each symbol has the same meaning as mentioned above.

The second type (type 2), as shown in formula (1B), is a compound wherein X in formula (1) is N(Rid)-N(R1d)- N(Rid)-N(R1d)- , and Y is -O-. wherein the meaning of each symbol has the same meaning as mentioned above.

The third type (type 3), as shown in formula (1C), is a compound wherein both of X and Y in formula (1) are -0-. wherein the meaning of each symbol has the same meaning as mentioned above.

In cyclodepsipeptides (1A) of type 1, each of (a), (b)(b), (b)(b), (c), (d) and (e) is an amino acid or an N-methyl amino acid, and (f) is a hydroxy acid.

Preferable examples of Type 1 compounds are as follows: TK22-4 in which (a) is -alanine, (b) methylalanine, (c) methylvaline, (d) isoleucine, (e) proline and (f) phenyllactic <BR> <BR> <BR> <BR> <BR> acid; TK32-5 in which (a) is B -alanine, (b) methylalanine, (c) methylvaline, (d) isoleucine, (e) methylalanine and (f) leucic <BR> <BR> <BR> <BR> <BR> acid; TK33-5 in which (a) is 8 -alanine, (b) methylalanine, (c) methylvaline, (d) isoleucine, (e) methylvaline and (f) leucic <BR> <BR> acid; TK50-8 in which (a) is B B -alanine, (b) methylalanine, (c) methylvaline, (d) N-methylisoleucine, (e) proline and (f) leucic acid; Tk70-5 in which (a) is -alanine, (b) methylalanine, (c) methylvaline, (d) isoleucine, (e) octahydroxyindole-2-carboxylic acid and (f) leucic acid; <BR> <BR> <BR> <BR> <BR> TK330-6 in which (a) is B -alanine, (b) methylalanine, (c) methylvaline, (d) isoleucine, (e) proline and (f) 2-ethyl- <BR> <BR> <BR> <BR> <BR> 2-hydroxybutyric acid; TK340-6 in which (a) is B -alanine, (b) methylalanine, (c) methylvaline, (d) isoleucine, (e) proline and (f) 2-hydroxyoctanoic acid;and TK350-6 in which in which (a) is -alanine, (b) methylalanine, (c) methylvaline, (d) isoleucine, (e) proline and (f) 2-hydroxyhexanoic acid.

In cyclodepsipeptides (1B) of type 2, (a), (b), (d) and (e) are amino acids or N-methyl amino acids, (c) and (f) are hydroxy acids. Among cyclodepsipeptides (1B), preferred are the compounds wherein each of (b) and (e) are a nitrogen- containing ring, each compound having a point symmetrical structure.

Preferred examples of the type 2 compound include: TK510-7 in which (a) and (d) are -alanine, (b) and (e) proline, (c) and (f) phenyllactic acid; TK610-7 in which (a) and (d) are alanine, (b) and (e) proline, (c) and (f) phenyllactic acids; TK620-9 in which (a) and (d) are valine, (b) and (e) proline, (c) and (f) phenyllactic acid; and TK690-4 in which (a) and (d) are isoleucine, (b) and (e) proline, (c) and (f) phenyllactic acid.

In cyclodepsipeptides (1C) of type 3, (a), (d) and (e) are amino acids or N-methyl amino acids, (b), (c) and (f) are hydroxy acid.

Preferred examples of type 3 compounds are as follows: <BR> <BR> <BR> <BR> <BR> <BR> TK660-7 in which (a) is B -alanine, (b) lactic acid, (c) leucic acid, (d) isoleucine, (e) proline, and (f) phenyllactic acid.

Synthesis of cyclodepsipeptides C1: Amino acids, N-methyl amino acids and hydroxy acids, which correspond to the above-mentioned (a)-(f), respectively, of cyclodepsipeptides (1), are coupled with each other sequentially by stepwise condensation, and finally a ring can be formed by an intramolecular condensation. In case of cyclodepsipeptides (1) with a point symmetry in their three amino acids and/or hydroxy acids which are successive in a ring can be coupled with each other sequentially, and finally the same two molecules can be coupled with each other by condensation, resulting in formation of a ring.

Condensation of amino acids or N-methyl amino acids can be achieved by activating a carboxylic acid of one amino acid and then making the acid react with an amino acid of the other amino acid after protecting an amino group of the former amino acid and a carboxylic acid of the latter. Condensation between a carboxyl group of a hydroxycarboxylic acid and an amino group of an amino acid can be achieved by activating the carboxyl group of the hydroxycaboxylic acid and making it react with the amino group of the amino acid after protecting the carboxylic group of the amino acid.

The amino group of amino acids can be protected by a t-butoxycarbonyl group (Boc) or a benzyloxycarbonyl group (Z), and the carboxyl group can be protected by phenacyl ester (Pac) or a benzyl ester (Bzl). Protection of amino acid by Boc can be done by reaction with t-butylchloroformate and t- butylazidoformate. Removal of protecting groups can be done by reaction with trifluoroacetic acid, HCl/acetic acid and HCl/dioxane. Protection of amino group by Z can be done by reaction with benzylchloroformate in a weak basic solution or dioxane, and removal of protecting groups can be done by catalytic reduction with palladium catalyst, reaction with phosphonium iodide/acetic acid and reaction with hydrogen halide/acetic acid. Protection of carboxyl group by Pac can be done by reaction with phenacyl bromide in the presence of triethylamine, and removal of the protecting groups can be done by reaction with acetic acid-zinc powder, catalytic reduction with palladium catalyst and reaction with thiophenol sodium salt/dimethylsulfoxide. Protection of carboxyl group by Bzl can be done by reaction with triethylamine after reaction with benzylalcohol/polyphosphoric acid or benzylalcohol/bezenesulfonic acid . Removal of Bzl can be done by catalytic reduction with palladium catalyst or reduction by NaOH/dioxane. Protected amino acids commercially available can also be utilized in this invention.

Activation of carboxyl group can be done by using an azide method , a mixed anhydride- method, an activated ester method, or a dicyclohexylcarbodiimide (DCC) method, and a carbodiimide method using water-soluble carbodiimide (WSCD).

Reaction between the hydroxyl group of hydroxycarboxyl acids and the carboxyl group of amino acids can be done in the presence of 4-pyrrolidinopylidine and dicyclohexycarbodiimide.

The final condensation which forms a ring can be done in the presence of diisopropylethylamine (DIEA) and 0-(7- azabenzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate (HATU).

Since cyclodepsipeptides (1) obtained as mentioned above have excellent cardiotonic, antiarrhythmic and vasodilating effects without an increase in cyclic AMP level in cardiac muscles, they are useful as cardiotonic, antiarrhythmic and vasodilating drugs without side effects. The target diseases include acute and chronic heart failures, a variety of arrhythmias, hypertensions, angina pectoris and myocardiac infarctions.

In usage of the cyclodepsipeptides (1) as a cardiotonic, an antiarrhythmic and a vasodilating drug, the cyclodepsipeptides (1) can be administered solely, or in combination with an excipient, thickner, binder, moisturizing agent, disintegrator, surfactant, smoothing agent, dispersing agent, buffer, preservative, flavoring agent, perfume, or coating agent. For preparations, the cyclodepsipeptides can be administered, orally or párenterally, in various forms of preparations, including oral administration agents such as powders, granules, tablets, capsules, ampoules; subcutaneous, intramuscular or intravenous injection; and suppository.

The dosage may vary depending upon site of administration or severity of a disease. Preferred dosage for adults is 0.6-300 mg/day, suitably 5-200 mg/day, which dosage should preferably be administered by dividing it 3-4 times per day.

Examples: The present invention is described in more detail by use of Examples, which however should not be construed to limit the invention. Abbreviations used in the Examples exept for general amino acids are as follows: Pac: phenacyl group, Boc: t-butoxycarbonyl group; Z, bezyloxycarbonyl group; Bzl: benzyl group; Me: methyl group; HOBt: l-hydroxybenzotriazole; HOAt: l-hydroxy-7-azabenzotriazole; HATU: O-(7-azabenzotriazol-1- yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; DIEA: diisopropylethylamine; DCC: dicyclohexylcarbodiimide; WSCD: water-soluble carbodiimide; H-OBut-OH: 2-ethyl-2- hydroxybutyric acid; H-OHex-OH: 2-hydroyhexanic acid; H- OOct-OH: 2-hydroxyoctanic acid; Oleu: leucic acid; Ophe: phenyllactic acid; Lac: lactic acid; and Oic: octahydroindole-2-carboxylic acid.

Example 1 <BR> <BR> <BR> Synthesis of Cyclo-(Ile-Metral -MeAla-8 -Ala-OPhe Pro)(TK22-4) :Synthesis of Cyclo-(Ile-MeValMeAla -Ala-OPhe Pro) (TK22-4): Synthesis of Cyclo-(Ile-Metral -MeAla-8 -Ala-OPhe Pro)(TK22-4) :Synthesis of Cyclo-(Ile-MeValMeAla -Ala-OPhe Pro) (TK22-4): PacBr HCUdioxane Boc-NtAIa-OH Boc- -Ala-OH r Boc- -Ala-OPac » HCl-H-B-Ala-OPac NE:3 WSCDlHOAt I) Boc-MeVal-OH WSCD/HOAt Boc-MeAla- -Ala-OPac - HCI-H-MeAla- -Ala-OPac a-OPac 2) HClldioxane Z-lle-OH Zn/AcOH HCI-H-MeVal-MeAla- -Ala-OPac ac Z-lle-MeVal-lvleAla- -Ala-OPac WSCD/HOAt Z-lle-MeV al-hleAla- -Ala-OH 0 WSCD!KOAt Ho 9 DCC HCI.H-Pro-OBz{0 CH3 H O pyrolidinopyridine N+00 NH-Z OH OBzI N-CH H O Md N/90 X N-CH3 H ° o X 04 0 N m ¼3L\Mi' O[3zI O 9 DIEA/HATU N~CHz H 0 ° ( o H N <F8C X X (1) Boc-B-Ala-Opac Phenacyl bromide (11.57g) was added to a solution of Boc-B-Ala-OH (10.0g) and triethylamine (8.1ml) in acetone (100ml) on ice-cooling, which was then stirred for Smin and then further stirred at room temperature for 24h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was crystallized in hexane to yield 14.7g of the title compound.

(2) HCl H-B-Ala-Opac Hydrogen chloride (5.ON) in 1,4-dioxane solution (189ml) was added to Boc-B-Ala-Opac (14.54g) obtained in (1). The solution was allowed to stand at room temperature for lh. After removal of the solvent by concentration under reduced pressure, the residue was washed with ether to obtain 11.2g of the title compound.

(3) Boc-MeAla-B-Ala-Opac.

The HClH-B-Ala-Opac (5.55g) obtained in (2) was dissolved in DMF (25ml) containing Boc-MeAla-OH (4.86g) and HOAt (3.42g) under ice-cooling. WSCD (5.48m1) was added to the solution, which was then stirred for 30min, and then further stirred at room temperature for 18 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a toluene-ethyl acetate mixture in a ratio of 2 : 1 to give 8.92 g of the title compound in a form of a solid.

(4) HClH-MeAla-B-Ala-Opac Hydrogen chloride (4.5N) in 1,4-dioxane solution (101ml) was added to Boc-MeAla- -Ala-Opac (8.92g) obtained in (3). The solution was allowed to stand at room temperature for ih.

Removal of the solvent was done by concentration under reduced pressure and washing with ether; yield 7.32g.

(5) HCl H-MeVal-MeAla-B-Ala-Opac The HClH-MeAla-B-Ala-Opac (7.00g) obtained in (4) was dissolved in DMF (35ml) containing Boc-MeVal-OH (5.18g) and HOAt (3.20g) in under ice-cooling. WSCD (5.15ml) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was dissolved in 4.5 N hydrogen chloride in 1,4-dioxane solution (95.1 ml). The solution was allowed to stand at room temperature for ih. Removal of the solvent was done by concentration under reduced pressure and washing with ether; yield 8.71g.

(6) Z-Ile-MeVal-MeAla-B-Ala-Opac The HCl.H-Meval-MeAla-B-Ala-Opac (8.50g) obtained in (5) was dissolved in DMF (45ml) containing Z-Ile-OH (5.37g) and HOAt (2.89g) under ice-cooling. WSCD (4.64ml) was added to the solution, which was then stirred for 1 h, and further stirred at room temperature for 17 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a chloroform- methanol mixture in a ratio of 100 : 1 gave an oil; this was washed by hexane; yield 10.7g.

(7) Z-Ile-MeVal-MeAla-B-Ala-OH Z-Ile-MeVal-MeAla-B-Ala-Pac (10.7g) obtained in (6) was dissolved in 90% aqueous acetic acid (200ml) containing zinc dust (53.7g). The mixture was vigorously stirred for 2 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid.

The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a chloroform -methanol mixture in a ratio of 19 : 1 gave a oil; this was washed by hexane; yield 6.05g.

(8) H-OPhe-Pro-Obzl The HCl Pro-OBzl (0.763g, 3.16mmol) was dissolved in DMF (10ml) containing phenyl lactic acid (0.50g, 3.0lmmol) andHOAt (0.451g, 3.31mmol)under ice-cooling. WSCD (0.724ml, 3.3irnmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification performed by silica gel column chromatography using a chloroform -methanol mixture in a ratio of 100 : 1 gave a solid; yield 1.03g (97%).

(9) Z-Ile-MeVal-MeAla-B-Ala-OPhe-Pro-Obzl The Z-Ile-MeVal-MeAla- -Ala-OH (0.151g,0.283mmol) obtained in (7) was dissolved in DMF (2ml) containing H- OPhe-Pro-OBzl (0.100g, 0.283mmol) obtained in (8) and 4- pyrrolidinopyridine (12.6mg, 0.085mmol) under ice-cooling.

DCC (64.6mg, 0.313mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 15 h. The ethyl acetate was added in the reaction mixture and then the insoluble material was filtered off, and the filtrate was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a chloroform -methanol mixture in a ratio of 30 : 1; yield 78.2mg (31.8%).

(10) H-Ile-MeVal-MeAla-B-Ala-OPhe-Pro-OH Z-Ile-MeVal-MeAla-B-Ala-OPhe-Pro-OBzl (78.2mg, 0.090mmol) obtained in (9) was dissolved in MeOH-acetic acid-water with ratio of 8:2:1 (30ml). The solution was bubbled with hydrogen in the presence of Pd-black for lh. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure, followed by lyophilization; yield 48.8mg(84.1%).

(11) Cyclo-(Ile-MeVal-MeAla-B-Ala-OPhe-Pro) (TK22-4) H-Ile-MeVal-MeAla-B-Ala-OPhe-Pro-OH (40.Omg,0.062mmol) obtained in (10) and DIEA (0.0648ml,0.372mmol) dissolved in dichloromethane (25ml) was dropped at a rate of 12.5ml per hour into dichloromethane (37ml) containing HATU (70.7mg, 0.186mmol), which was then stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a chloroform -methanol mixture in a ratio of 19:1; yield 21.Omg (54.1%). mp : 252.80C MS : m/z 628.0(M+H+) calculated as C33H49N507 : 627. 4 UV Amax(MeOH) nm (£):208(22750.7) H-NMR: Fig.1 IR(KBr): Fig.2 Example 2 Synthesis of Cyclo-(Ile-MeVal-MeAla-B-Ala-OBut-Pro) (TK330- 6) H 0 + HO OPac DCC ~ N JWHH-Z DX pylolldin pyndine °~ tNr / WSCD/HOAt MW LA-CON NoCH3 H ° og HCIH-L-Pro-OBzl N-CH3 H O t NH aH3C X oX (1) Z-Ile-MeVal-MeAla-B-Ala-OBut-Opac The Z-Ile-MeVal-MeAla-B-Ala-OH (0.500g,0.937mmol) was dissolved in DMF(3ml) containing H-OBut-OPac (0.234g, 0.937mmol) and 4-pyrrolidinopyridine (41.7mg, 0.281mmol) under ice-cooling. DCC (268mg, 1.03mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h. The ethyl acetate was added in the reaction mixture and then the insoluble material was filtered off, and the filtrate was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the title compound.

(2) Z-Ile-MeVal-MeAla- -Ala-OBut-OH Z-Ile-MeVal-MeAla-B-Ala-OBut-OPac (717mg,0.937mmol) obtained in (1) was added to 90% aqueous acetic acid (30ml) containing zinc dust (3.06g,46.9mmol). The mixture was vigorously stirred for 1 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure.

The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification performed by silica gel column chromatography using a chloroform-methanol mixture in a ratio of 50 : 1 gave an oil.

(3) Z-Ile-MeVal-MeAla-B-Ala-OBut-Pro-Obzl The HCl Pro-OBzl (0.217g, 0.898mmol) was dissolved in DMF (15ml) containing Z-Ile-MeVal-MeAla- -Ala-OBut-OH (0.582g, 0.898mmol) obtained in (2) and HOAt (0.134g, 0.988mmol) under ice-cooling. WSCD (0.216ml, 0.988mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 671mg.

(4) H-Ile-MeVal-MeAla- -Ala-OBut-Pro-OH Z-Ile-MeVal-MeAla-B-Ala-OBut-Pro-OBzl (671mg, 0.804mmol) obtained in (3) was dissolved in MeOH-acetic acid-water with ratio of 8:2:1 (100ml). The solution was bubbled with hydrogen in the presence of Pd-black for lh. The catalyst was filtered off, and the filtrate concentrated under reduced pressure, followed by lyophilization.

(5) Cyclo-(Ile-MeVal-MeAla-B-Ala-OBut-Pro) (TK330-6) H-Ile-MeVal-MeAla- -Ala-OBut-Pro-OH (100.Omg,0.159mmol) obtained in (4) and DIEA (0.166ml,0.954mmol) dissolved in dichloromethane (50ml) was dropped at a rate of 12.5ml per an hour into dichloromethane (100ml) containing HATU (181.Omg, 0.477mmol), which was then stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a chloroform-methanol mixture in a ratio of 19:1 to yield 33.2mg of the title compound (yield: 35.0%).

MS : m/z 594.2(M+H+) calculated as C30H51N507 : 593. 4 1H-NMR: Fig.3 Example 3 Cyclo-(Ile-MeVal-MeAla-B-Ala-L-OOct-Pro) (TK340-6-A) and Cyclo-(Ile-MeVal-MeAla- -Ala-D-OOct-Pro) (TK340-6-B): H 0 + HO DCC X DCC ;¼¼;NH-z oW; ,N/ rOX WSCD/HOAt ¼%)MwO H N 00N% N-CH3 H 0 0 N2/Pd N-CH3 H 0: 0 g~ N ~ o DIEA/HATU N-CH, H ° o0 XHO (1) Z-Ile-MeVal-MeAla-B-Ala-D,L-OOct-Opac The Z-Ile-MeVal-MeAla-B-Ala-OH (0.500g,0.937mmol) was dissolved in DMF(3ml) containing H-D,L-OOct-OPac (0.261g, 0.937mmol) and 4-pyrrolidinopyridine (41.7mg, 0.281mmol) under ice-cooling. DCC (268mg, 1.03mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h. The ethyl acetate was added in the reaction mixture and then the insoluble material was filtered off, and the filtrate was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure, thereby producing the title compound.

(2) Z-Ile-MeVal-MeAla-B-Ala-D,L-OOct-OH Z-Ile-MeVal-MeAla-B-Ala-D,L-OOct-OPac (744mg,0.937mmol) obtained in (1) was added to 90% aqueous acetic acid (30ml) containing zinc dust (3.06g,46.9mmol). The mixture was vigorously stirred for 1 h. The insoluble material was filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a chloroform -methanol mixture in a ratio of 50 : 1 gave an oil.

(3) Z-Ile-MeVal-MeAla-B-Ala-D,L-OOct-Pro-Obzl The HCl-Pro-OBzl (0.226g, 0.937mmol) was dissolved in DMF (15ml) containing Z-Ile-MeVal-MeAla- -Ala-D,L-OOct-OH (0.635g, 0.937mmol) obtained in (2) and HOAt (0.140g, 1.03mmol) in under ice-cooling. WSCD (0.225m1, 1.03mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure to yield 800mg of the title compound.

(4) H-Ile-MeVal-MeAla-B-Ala-D,L-OOct-Pro-OH Z-Ile-MeVal-MeAla-B-Ala-D,L-OOct-Pro-OBzl (800mg, 0.937mmol) obtained in (3) was dissolved in MeOH-acetic acid-water in aratio of 8:2:1 (100ml) . The solution was bubbled with hydrogen in the presence of Pd-black for lh. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure, followed by lyophilization.

(5) Cyclo-(Ile-MeVal-MeAla- -Ala-L-OOct-Pro) (TK340-6-A) and Cyclo-(Ile-MeVal-MeAla- -Ala-D-OOct-Pro) (TK340-6-B) H-Ile-MeVal-MeAla- -Ala-D,L-OOct-Pro-OH (100.Omg, 0.152mmol) obtained in (4) and DIEA (0.159ml,0.913mmol) was dissolved in dichloromethane (50ml) was dropped at a rate of 12.5ml per an hour into dichloromethane (100ml) containing HATU (174.Omg, 0.457mmol), which was then stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate: this was washed successively with 10% aqueous <BR> <BR> <BR> <BR> <BR> citric acid, brine, saturated aqueous sodiumhydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a <BR> <BR> <BR> <BR> <BR> chloroform -methanol mixture in a ratio of 30:1; yield TK340-6-A 19.6mg (20.2%) and TK340-6-B 17.Omg (17.5%).

1H-NMR: Fig.4 for TK340-6-A, Fig.5 for TK340-6-B.

Example 4 Cyclo-(Ile-MeVal-MeAla-B-Ala-L-OHex-Pro) (TK350-6-A) and Cyclo-(Ile-MeVal-MeAla-B-Ala-D-OHex-Pro) (TK350-6-B). o ¼NThO 00 + HO DCC H3 0 O H 0 0 pyrolidinopyridine HO;JJ CC F c NH-Z 0 H3 OPac H3CN Zn/AcON {CH3H00 WSCD/HOAt ~N/<O S Zru'AcOH N,CH1 w H 0 ¼CNH OH~~~ (1) Z-Ile-MeVal-MeAla- -Ala-D,L-OHex-OPac The Z-Ile-MeVal-MeAla-B-Ala-OH (0.500g,0.937mmol) was dissolved in DMF (3ml) containing H-D,L-OHex-OPac (0.234g, 0.937mmol) and 4-pyrrolidinopyridine (41.7mg, 0.281mmol) under ice-cooling. DCC (268mg, 1.03mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h. Ethyl acetate was added in reaction mixture and then the insoluble material was filtered off, and the filtrate was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure.

(2) Z-Ile-MeVal-MeAla-B-Ala-D,L-OHex-OH Z-Ile-MeVal-MeAla-B-Ala-D,L-OHex-OPac (719mg,0.937mmol) obtained in (1) was added to 90% aqueous acetic acid (30ml) containing zinc dust (3.06g,46.9mmol). The mixture was vigorously stirred for 1 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure.

The residue was dissolvedin ethyl acetate; this was washed with 10% aqueous citric acid. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a chloroform -methanol mixture in a ratio of 50 : 1 gave an oil.

(3) Z-Ile-MeVal-MeAla-B-Ala-D,L-OHex-Pro-Obzl The HCl Pro-OBzl (0.226g, 0.937mmol) was dissolved in DMF (15ml) containing Z-Ile-MeVal-MeAla-B-Ala-D,L-OHex-OH (0.719g, 0.937mmol) obtained in (2) and HOAt (0.140g, 1.03mmol) under ice-cooling. WSCD (0.225ml, 1.03mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 781mg.

(4) H-Ile-MeVal-MeAla-B-Ala-D,L-OHex-Pro-OH Z-Ile-MeVal-MeAla-B-Ala-D,L-OHex-Pro-OBzl (781mg, 0.937mmol) obtained in (3) was dissolved in MeOH-acetic acid-water in a ratio of 8:2:1 (100ml). The solution was bubbled with hydrogen in the presence of Pd-black for ih. The catalyst was filtered off, and the filtrate concentrated under reduced pressure, followed by lyophilization.

(5) Cyclo-(Ile-MeVal-MeAl-a-- -Ala-L-OHex-Pro) (TK350-6-A) and Cyclo-(Ile-MeVal-MeAla-B-Ala-D-OHex-Pro) (TK350-6-B) H-Ile-MeVal-MeAla-B-Ala-D,L-OHex-Pro-OH (100.Omg,0.159mmol) obtained in (4) and DIEA (0.166m1,0.954mmol) dissolved in dichloromethane (50ml) was dropped at a rate of 12.5ml per an hour into dichloromethane (100ml) containing HATU (181.0mg, 0.477mmol), which was then stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a chloroform-methanol mixture in a ratio of 30:1; yield TK350-6-A 22.4mg (23.1%) and TK350-6-B 23.2mg (23.9%).

1H-NMR: Fig.6 for TK350-6-A, Fig.7 for TK350-6-B.

Example 5 Cyclo-(Ile-MeVal-MeAla-B-Ala-OLeu-Oic) (TK70-5) w¼N\MWOH 0 pvroiidinopyridine N-CH, H O HOA DCC F< H 0 O tOp N-CH3 H 0 + HO DCC Pac ;½CN C; 0 yrolidinopyridinl O}i3 o < WSCD/HOAt ½ o MW0 M¼ON¼{N3yH OH HCl-H-L-Oic-OBzi Z ,H o H H/Pd < I W N-CH, H O O=r\N X XH j 0 | 9 S DIEA/HATU N-CH3 H ° o v °4 O H Nt X 9NJW < / Ej Hzp) o (1) Z-Ile-MeVal-MeAla-B-Ala-OLeu-OPac.

The Z-Ile-MeVal-MeAia-B-Ala-OH (1.50g,2.8lmmol) was dissolved in DMF (10ml) containing H-OLeu-OPac (0.703g, 2.81mmol) and 4-pyrrolidinopyridine (125.Omg, 0.843mmol) under ice-cooling.

DCC (639.0mg, 3.09mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h. Ethyl acetate was added in the reaction mixture and then the insoluble material was filtered off, and the filtrate was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a chloroform-methanol mixture in a ratio of 200 : 1; yield 1.29g (60.0%).

(2) Z-Ile-MeVal-MeAla-B-Ala-OLeu-OH Z-Ile-MeVal-MeAla-B-Ala-OLeu-OPac (1.29g,1.68mmol) obtained in (1) was added to 90% aqueous acetic acid containing zinc dust (5.50g,84.2mmol). The mixture was vigorously stirred for 2 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure.

Purification was performed by silica gel column chromatography using a chloroform-methanol mixture in a ratio of 50 : 1 ; yield 1.03g (94.0%).

(3) Z-Ile-MeVal-MeAla-B-Ala-OLeu-Oic-Obzl The HCl Oic-OBzl (45.6mg, 0.154mmol) was dissolved in DMF containing Z-Ile-MeVal-MeAla-B-Ala-OLeu-OH (100mg,0.154mmol) obtained in (2) and HOAt (23.lmg, 0.17mmol) under ice-cooling.

WSCD (0.0372ml, 0.17mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure: yield 126mg (92.1%).

(4) H-Ile-MeVal-MeAla-B-Ala-OLeu-Oic-OH Z-Ile-MeVal-MeAla-B-Ala-OLeu-Oic-OBzl (126mg, 0.142mmol) obtained in (3) was dissolved in MeOH-acetic acid-water in a ratio of 8:2:1 (30ml). The solution was bubbled with hydrogen in the presence of Pd-black for 1.5 h. The catalyst was filtered off, and the filtrate concentrated under reduced pressure, followed by lyophilization; yield 61.8mg(62.1%).

(5) Cyclo-(Ile-MeVal-MeAla-B-Ala-OLeu-Oic) (TK70-5) H-Ile-MeVal-MeAla-B-Ala-OLeu-Oic-OH (50.Omg,0.07lmmol) obtained in (4) and DIEA (0.0746ml,0.428mmol) dissolved in dichloromethane (20ml) was dropped at a rate of 10.0ml per an hour into dichloromethane (50ml) containing HATU (81.4mg, 0.214mmol), which was then stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a chloroform-methanol mixture in a ratio of 19:1: yield 25.3mg (52.0%).

1H-NMR:Fig.8.

Example 6 Cyclo-(Ile-MeVal-MeAla-B-Ala-OLeu-MeAla) (TK32-5).

(1) Z-Ile-MeVal-MeAla-B-Ala-OLeu-MeAla-Obzl The HCl MeAla-OBzl (70.8mg, 0.300mmol) was dissolved in DMF (5ml) containing Z-Ile-MeVal-MeAla-B-Ala-OLeu-OH (200mg,0.300mmol) and HOAt (46.2mg, 0.330mmol) under ice- cooling. WSCD (0.0743ml, 0.330mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 206mg (81.6%).

(2) H-Ile-MeVal-MeAla-B-Ala-OLeu-MeAla-OH Z-Ile-MeVal-MeAla-B-Ala-OLeu-MeAla-OBzl (206mg, 0.252mmol) obtained in (1) was dissolved in MeOH-acetic acid-water with ratio of 8:2:1 (40ml). The solution was bubbled <BR> <BR> <BR> <BR> <BR> <BR> with hydrogen in the presence of Pd-black for 0.5 h. The catalyst was filtered off, and the filtrate concentrated under reduced pressure, followed by lyophilization: yield 149.Omg(99.9%).

(3) Cyclo-(Ile-MeVal-MeAla-B-Ala-OLeu-MeAla) (TK32-5) H-Ile-MeVal-MeAla-B-Ala-OLeu-MeAla-OH (100mg,0.168mmol) and DIEA (0.176ml,1.01mmol) dissolved in dichloromethane (50ml) was dropped at a rate of 22.5ml per an hour into dichloromethane (llOml) containing HATU (192mg, 0.504mmol), which was then stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a chloroform-methanol mixture in a ratio of 19:1; yield 34.4mg (35.5%).

MS: m/z 582.2(M+H+) calculated as C29H5lN507 :581.4 1H-NMR:Fig.9.

Example 7 Cyclo-(Ile-MeVal-MeAla- -Ala-OLeu-MeVal) (TK33-5) (1) Z-Ile-MeVal-MeAla-B-Ala-OLeu-MeVal-Obzl The HCl MeVal-OBzl (79.5mg, 0.300mmol) was dissolved in a solution of Z-Ile-MeVal-MeAla-B-Ala-OLeu-OH (200mg,0.300mmol) and HOAt (46.2mg, 0.330mmol) in DMF under ice-cooling. WSCD (0.0743ml, 0.330mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 222mg (85.0%).

(2) H-Ile-MeVal-MeAla-B-Ala-OLeu-MeVal-OH Z-Ile-MeVal-MeAla-B-Ala-OLeu-MeVal-OBzl (222mg, 0.262mmol) obtained in (1) was dissolved in MeOH-acetic acid-water in a ratio of 8:2:1 (40ml). The solution was bubbled <BR> <BR> <BR> <BR> <BR> with hydrogen in the presence of Pd-black for 0.5 h. The catalyst was filtered off, and the filtrate concentrated under reduced pressure, followed by lyophilization; yield 162.0mg(99.2%).

(3) Cyclo-(Ile-MeVal-MeAla-B-Ala-OLeu-MeVal) (TK33-5) H-Ile-MeVal-MeAla-B-Ala-OLeu-MeVal-OH (100mg,0.161mmol) obtained in (2) and DIEA (0.168ml,0.963mmo1) dissolved in dichloromethane (50ml) was dropped at a rate of 25.0ml per an hour into dichloromethane (110ml) containing HATU (183mg, 0.482mmol), which was then stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using achloroform-methanol mixture in a ratio of 19:1; yield 33.2mg (34.1%).

MS: m/z 610.2(M+H+) calculated as C3lH55N507 : 609.4 lH NMR:Fig-10- Example 8 Cyclo-(MeIle-MeVal-MeAla-B-Ala-OLeu-Pro) (TK50-8) Z-Nlelle-OH HCl-H-&leVal-NleAla- -Ala-OPac - WSCD/NOA Z-ìvlelle-NteVal-lsleAla-B-Ala-OPac WSCD/HOAt Zn/AcOH > Z-;vlelle-ìvleVal-,SfeAla- -Ala-OH HO-Leu-OPac > Z-ìwlelle-i9leval-MeAla- -Ala-oLeu-opac DCC pyrolidinopyridine Zn/AcOH o Z-2vlelle-hleVal-MeAla- -Ala-OLeu-OH HCl-H-Pro-OBzl ~ Z-Melle-MeVal-MeAla- -Ala-OLeu-Pro-OBzl WSCD/HOAt H2/Pd o H-ì9lelle-»teval eAla- -Ala-oLeu-pro-oH 0 r0\ ~gN ° XJ ~~ ¼cH3Mi DfEA/HATU ~ O N- (1) Z-MeIle-MeVal-MeAla-B-Ala-Opac The HCl-H-MeVal-MeAla- -Ala-Opac (1.26g, 2.87mmol) was <BR> <BR> <BR> <BR> dissolved in DMF (5ml) containing Z-MeIle-OH (0.976g, 3.0lmmol) and HOAt (0.430g, 3.16mmol) under ice-cooling. WSCD (0.692ml, 3.16mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 18 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a toluene-ethyl acetate mixture in a ratio of 3: 1; yield l.9g.

(2) Z-MeIle-MeVal-MeAla-B-Ala-OH Z-Ile-MeVal-MeAla-B-Ala-Pac (1.9g, 2.87mmol) obtained in (1) was added to 90% aqueous acetic acid (100ml) containing zine dust (9.38g, 14.3mmol). The mixture was vigorously stirred for 2 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure.

Purification was performed by silica gel column chromatography using a chloroform-methanol mixture in a ratio of 50 : 1; yield 1.10g (70%).

(3) Z-MeIle-MeVal-MeAla-B-Ala-OLeu-Opac Z-MeIle-MeVal-MeAla-8-Ala-OH (0.626g,1.14mmol) obtained <BR> <BR> <BR> <BR> <BR> in (2) was dissolved in DMF (5ml) containing H-OLeu-OPac (0.286g, 1.14mmol) and 4-pyrrolidinopyridine (50.7mg, 0.34mmol) under ice-cooling. DCC (258.Omg, 1.25mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h. The ethyl acetate was added in reaction mixture and then the insoluble material was filtered off, and the filtrate was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a toluene-ethyl acetate mixture in a ratio of 2 : 1; yield 460mg (51.7%).

(4) Z-MeIle-MeVal-MeAla-B-Ala-OLeu-OH Z-MeIle-MeVal-MeAla-B-Ala-OLeu-OPac (460mg,0,589mmol) obtained in (3) was added to 90% aqueous acetic acid (100ml) containing zinc dust (1.93g,29.5mmol). The mixture was vigorously stirred for 2 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure.

The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification performed by silica gel column chromatography using a chloroform-methanol mixture in a ratio of 50 : 1 ; yield 0.390g (100%).

(5) Z-MeIle-MeVal-MeAla-B-Ala-OLeu-Pro-Obzl The HCl Pro-OBzl (0.142g, 0.589mmol) was dissolved in DMF (10ml) containing Z-MeIle-MeVal-MeAla- -Ala-OLeu-OH (0.390g, 0.589mmol) obtained in (4) and HOAt (88.2mg, 0.648mmol) under ice-cooling. WSCD (0.142ml, 0.648mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 22 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 0.333g (67%).

(6) H-MeIle-MeVal-MeAla-B-Ala-OLeu-Pro-OH Z-MeIle-MeVal-MeAla-B-Ala-OLeu-Pro-OBzl (333mg, 0.392mmol) obtained in (5) was dissolved in MeOH-acetic acid-water with ratio of 8:2:1 (40ml). The solution was bubbled with hydrogen in the presence of Pd-black for 1 h. The catalyst was filtered off, and the filtrate concentrated under reduced pressure, followed by lyophilization; yield 245.0mg(100%).

(7) Cyclo-(MeIle-MeVal-MeAla-B-Ala-OLeu-Pro) (TK50-8) H-MeIle-MeVal-MeAla-B-Ala-OLeu-Pro-OH (100mg, 0.16mmol) obtained in (6) and DIEA (0.167ml,0.960mmol) dissolved in dichloromethane (50ml) was dropped at the rate of 25.Oml per an hour into dichloromethane (110ml) containing HATU (182.5mg, 0.480mmol), which was then stirred at room temperature for 19 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a chloroform-methanol in a ratio of 30:1; yield 11.9mg (12.3%).

MS: m/z 608.2(M+H+) calculated as C31H53N507 : 607.4 1H-NMR:Fig.ll.

Example 9 Cyclo-(B-Ala-OPhe-Pro-B-Ala-OPhe-Pro) (TK510-7) HO-OPhe-OPac Boc- -Ala-OH - o Boc- -Ala-OPhe-OPac DCC 4-pyrrolidinopyridine Zn/AcOH Pro-OBzl - Boc- -Ala-OPhe-OH r Boc- -Ala-OPhe-Pro-OBzl WSCD/HOAt (a) HCI/dioxane HCI H- a -Ala-OPhe-Pro-OBzl (a) HCl H- -Ala-OPhe-Pro-OBzl H)¼/Pd Boc- -Ala-OPhe-Pro-OH WSCD/HOAt Boc- 2 -Ala-OPhe-Pro-OH + HCl H- 2 -Ala-OPhe-Pro-OBzl vN/T°cX l)H2/Pd N H O o O olS O H O0M/NBOHCN 2)HCl/dioxane si0' OBzi DIEA/HATU ~ H/< °R O 11 Nn t°eN'@ (1) Boc-B-Ala-OPhe-Opac The Boc-B-Ala-OH (1.50g,7.9mmol) was dissolved in DMF (10ml) containing H-OPhe-OPac (2.28g, 8.Ommol) and 4- pyrrolidinopyridine (0.351g, 2.4mmol) under ice-cooling. DCC (1.79g, 8.7mmol) was added to the solution, which was then stirred for 2 h, and then further stirred at room temperature for 20 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was disslved in ethyl acetate, which was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure;yield 3.5g.

(2) Boc- -Ala-OPhe-OH Boc-B-Ala-OPhe-OPac (3.5g,7.9mmol) obtained in (1) was added to 90 aqueous acetic acid containing zinc dust (25.8g,395mmol). The mixture was vigorously stirred for 2 h.

The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid.

The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a chloroform-methanol mixture in a ratio of 19 : 1 ; yield 1.17g (43.8%).

(3) Boc-B-Ala-OPhe-Pro-Obzl The HCl Pro-OBzl (0.624g, 2.58mmol) was dissolved in DMF containing Boc-B-Ala-OPhe-OH (0.87g, 2.58mmol) obtained in (2) and HOAt (0.386g, 2.84mmol) under ice-cooling. WSCD (0.62ml, 2.84mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h.

After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 1.35g.

(4) HClH-B-Ala-OPhe-Pro-Obzl Boc-B-Ala-OPhe-Pro-OBzl (0.675g, 1.28mmol) obtained in (3) was added to 4 N hydrogen chloride in 1,4-dioxane solution (6.4ml, 25.6mmol). The solution was allowed to stand at room temperature for 30 min. After removal of the solvent by concentration under reduced pressure, the residue was washed with ether; yield 0.557g (94.2%).

(5) Boc-B-Ala-OPhe-Pro-OH Boc-B-Ala-OPhe-Pro-OBzl (0.675g, 1.28mmol) obtained in (3) was dissolved in MeOH-acetic acid-water with ratio of 8:2:1.

The solution was bubbled with hydrogen in the presence of Pd-black for 2 h. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure, the residue was washed with hexane; yield 0.526g (94.1%).

(6) Boc-B-Ala-OPhe-Pro-B-Ala-OPhe-Pro-OBzl TheHClH-B-Ala-OPhe-Pro-OBzl (0.556g, 1.2lmmol) obtained in (4) was dissolved in DMF containing Boc-B-Ala-OPhe-Pro-OH (0.524g, 1.2lmmol) obtained in (5) and HOAt (0.181g, 1.33mmol) under ice-cooling. WSCD (0.291ml, 1.33mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 20 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 1.02g.

(7) HClH-B-Ala-OPhe-Pro-B-Ala-OPhe-Pro-OH Boc-B-Ala-OPhe-Pro-i3-Ala-OPhe-Pro-OBzl (1.02g, 1.2lmmol) obtained in (6) was dissolved in MeOH-acetic acid-water with ratio of 8:2:1. The solution was bubbled with hydrogen in the presence of Pd-black for 2 h. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure, the residue was dissolved in 4 N hydrogen chloride in 1,4-dioxane (6.05ml, 24.2mmol). The solution was allowed to stand at room temperature for 30 min. After removal of the solvent by concentration under reduced pressure. The residue was washed with ether; yield 0.750g (90.1%).

(8) Cyclo-(B-Ala-OPhe-Pro-B-Ala-OPhe-Pro) (TK510-7) HCl-H- -Ala-OPhe-Pro- -Ala-OPhe-Pro-OH (100mg, 0.145mmol) obtained in (7) and DIEA (0.152m1,0.87mmol) dissolved in dichloromethane (50ml) was dropped at the rate of 25.0ml per an hour into dichloromethane (150ml) containing HATU (166mg, 0.436mmol), which was then stirred at room temperature for 20 h. After removal ofthe solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine.

The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using developing a chloroform-methanol mixture in a ratio of 19:1; yield 52.lmg (53.7%).

MS: m/z 633.0(M+H+) calculated as C34H40N408 : 632.3 lH-NMR Fig-12- Example 10 Cyclo-(Ala-OPhe-Pro-Ala-OPhe-Pro) (TK610-7) HO-OPhe-OPac Boc-Ala-ON - » Boc-Ala-OPhe-OPac DCC 4-pyrrolidinopyridine - Zn/AcOH Pro-OBzl o Boc-Ala-OPhe-OH ' Boc-Ala-OPhe-Pro-OBzl WSCD/HOA: (a) HCl/dioxane HCIH-Ala-OPhe-Pro-OBzl (a) UCl-N-Ala-OPhe-Pro-OBzl H2/?d L Boc-Ala-OPhe-Pro-OH WSCDJHOAt Boc-Ala-OPhe-Pro-OH + HCl-H-Ala-OPhe-Pro-OBzl 0 0 -CY N l)H2/Pd NHO0 1)H2/Pd °4 0 HN 2)HCl/dioxane 0 X N,H 1 n 0 NI$$ 0 ' OH OBI 0 o 0\ X l > S DIEA/HATU N H O oe > O X o H < W (1) Boc-Ala-OPhe-OPac.

The Boc-Ala-OH (1.50g,7.9mmol) was dissolved in DMF (10ml) containing H-OPhe-OPac (2.28g, 8.Ommol) and 4- pyrrolidinopyridine (0.351g, 2.4mmol) under ice-cooling. DCC (1.79g, 8.7mmol) was added to the solution, which was then stirred for 2 h, and then further stirred at room temperature for 20 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, which was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure;yield 3.5g.

(2) Boc-Ala-OPhe-OH Boc-Ala-OPhe-OPac (3.5g,7.9mmol) obtained in (1) was added to 90% aqueous acetic acid containing zinc dust (25.8g,395mmol). The mixture was vigorously stirred for 2 h.

The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid.

The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a chloroform-methanol mixture in a ratio of 19 : 1 ; yield 1.35g (50.5%).

(3) Boc-Ala-OPhe-Pro-Obzl The HCl Pro-OBzl (0.716g, 2.84mmol) was dissolved in DMF containing Boc-Ala-OPhe-OH (1.00g, 2.96mmol) obtained in (2) and HOAt (0.444g, 3.26mmol) under ice-cooling. WSCD (0.714ml, 3.26mmol) was added to the~solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h.

After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 1.56g.

(4) HCl-H-Ala-OPhe-Pro-Obzl Boc-Ala-OPhe-Pro-OBzl (0.780g, 1.48mmol) obtained in (3) was added to 4 N hydrogen chloride in 1,4-dioxane solution (7.43ml, 29.7mmol). The solution was allowed to stand at room temperature for 30 min. After removal of the solvent by concentration under reduced pressure, the residue was washed with ether; yield 0.636g (93.2%).

(5) Boc-Ala-OPhe-Pro-OH Boc-Ala-OPhe-Pro-OBzl (0.780g, 1.48mmol) obtained in (3) was dissolved in MeOH-acetic acid-water with ratio of 8:2:1.

The solution was bubbled with hydrogen in the presence of Pd-black for 2 h. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure, the residue was washed with hexane; yield 0.643g (100%).

(6) Boc-Ala-OPhe-Pro-Ala-OPhe-Pro-OBzl The HCl H-Ala-OPhe-Pro-OBzl (0.636g, 1.38mmol) obtained in (4) was dissolved in DMF containing Boc-Ala-OPhe-Pro-OH (0.600g, 1.38mmol) obtained in (5) and HOAt (0.207g, 1.52mmol) under ice-cooling. WSCD (0.333m1, 1.52mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 20 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 0.838g.

(7) HCl H-Ala-OPhe-Pro-Ala-OPhe-Pro-OH Boc-Ala-OPhe-Pro-Ala-OPhe-Pro-OBzl (0.838g, 0.996mmol) obtained in (6) was dissolved in MeOH-acetic acid-water in a ratio of 8:2:1. The solution was bubbled with hydrogen in the presence of Pd-black for 2 h. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure, the residue was dissolved in 4 N hydrogen chloride in 1,4-dioxane (5.00ml, 19.9mmol). The solution was allowed to stand at room temperature for 30 min. After removal of the solvent by concentration under reduced pressure, the residue was washed with ether; yield 0.457g (66.7%).

(8) Cyclo-(Ala-OPhe-Pro-Ala-OPhe-Pro) (TK610-7) HCl H-Ala-OPhe-Pro-Ala-OPhe-Pro-OH (100mg, 0.145mmol) obtained in (7) and DIEA (0.112g, 0.87mmol) dissolved in dichloromethane (50ml) was dropped at a rate of 25.0ml per an hour into dichloromethane (150ml) containing HATU (166mg, 0.436mmol), which was then stirred at room temperature for 20 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a chloroform-methanol mixture in a ratio of 19:1; yield 37.Omg (38.1%).

MS: m/z 633.2(M+H+) calculated as C34H40N408 : 632.3 1H-NMR:Fig.l3.

Example 11 Cyclo-(Val-OPhe-Pro-Val-OPhe-Pro) (TK620-9) HO-OPhe-OPac Boc-Val-OH z Boc-Val-OPhe-OPac DCC 4-pylTolidinopyt ine Zn/A cO H Pro-O B zl t Boc-Val-OPhe-OH o Boc-Val-OPhe-Pro-OBzl WSCD/HOBt (a) HCl/dioxane HCI H-Vnl-OPhe-Pro-OBzl (a) NC N-Val-OPhe-Pro-OBzl HS Boc-Val-OPhe-Pro-OH WSCD/HOAt Boc-Val-OPhe-Pro-OH + HC1 H-Va -OPhe-Pro-OB zl 0 N01 < 4 01 l)H2/Pd N00 0 o))NH. oB$7oC)zi q H i?2) 2)HCI/dioxane oJxN Hfi < 0 2;9 DIEA/HATU f O 4 H N o (1) Boc-Val-OPhe-Opac The Boc-Val-OH (5.00g,23.Ommol) was dissolved in DMF (80ml) containing H-OPhe-OPac (6.54g, 23.Ommol) and 4- pyrrolidinopyridine (1.02g 6.90mmol) under ice-cooling. DCC (6.59g, 25.3mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 20 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, which was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a toluene-ethyl acetate mixture in a ratio of 5 : 1;yield 10.6g (95.2%).

(2) Boc-Val-OPhe-OH Boc-Val-OPhe-OPac (10.6g, 21.9mmol) obtained in (1) was added to 90% aqueous acetic acid containing zinc dust (71.7g, 1100mmol). The mixture was vigorously stirred for 2 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid.

The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was washed with hexane; yield 4.15g (51.9%).

(3) Boc-Val-OPhe-Pro-Obzl The HCl Pro-OBzl (0.794g, 3.28mmol) was dissolved in DMF (10ml) containing Boc-Val-OPhe-OH (1.20g, 3.28mmol) obtained in (2) and HOBt (0.488g, 3.61mmol) under ice-cooling. WSCD (0.79ml, 3.61mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine.

The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 1.96g (100%).

(4) HCl H-Val-OPhe-Pro-Obzl Boc-Val-OPhe-Pro-OBzl (0.980g, 1.64mmol) obtained in (3) was added to 4 N hydrogen chloride in 1,4-dioxane solution (16.4ml, 65.7mmol). The solution was allowed to stand at room temperature for 40 min. After removal of the solvent by concentration under reduced pressure, the residue was washed with ether; yield 0.686g (85.5%).

(5) Boc-Val-OPhe-Pro-OH Boc-Val-OPhe-Pro-OBzl (0.980g, 1.64mmol) obtained in (3) was dissolved in MeOH-acetic acid-water in a ratio of 8:2:1.

The solution was bubbled with hydrogen in the presence of Pd-black for 2 h. The catalyst was filtered off, and the filtrate concentrated under reduced pressure, the residue was washed with hexane; yield 0.710g (93.6%).

(6) Boc-Val-OPhe-Pro-Val-OPhe-Pro-OBzl The HCl-H-Val-OPhe-Pro-OBzl (0.324g, 0.700mmol) obtained in (4) was dissolved in DMF containing Boc-Val-OPhe-Pro-OH (0.343g, 0.700mmol) obtained in (5) and HOAt (0.105g, 0.771mmol) under ice-cooing. WSCD (0.169ml, 0.771mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 20 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 0.65g (100%).

(7) HCl H-Val-OPhe-Pro-Val-OPhe-Pro-OH Boc-Val-OPhe-Pro-Val-OPhe-Pro-OBzl (0.65g, 0.700mmol) obtained in (6) was dissolved in MeOH-acetic acid-water in a ratio of 8:2:1. The solution was bubbled with hydrogen in the presence of Pd-black for 1 h. The catalyst was filtered off, and the filtrate concentrated under reduced pressure, the residue was dissolved in 4 N hydrogen chloride in 1,4-dioxane (3.50ml, 14.0mmol). The solution was allowed to stand at room temperature for 30 min. After removal of the solvent by concentration under reduced pressure, the residue was washed with ether; yield 0.468g (85.2%).

(8) Cyclo-(Val-OPhe-Pro-Val-OPhe-Pro) (TK620-9) HCl H-Val-OPhe-Pro-Val-OPhe-Pro-OH (200mg, 0.255mmol) obtained in (7) and DIEA (0.266m1,1.53mmol) dissolved in dichloromethane (50ml) was dropped at the rate of 12.5ml per an hour into dichloromethane (200ml) containing HATU (291mg, 0.765mmol), which was then stirred at room temperature for 20 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a chloroform-methanol mixture in a ratio of 19:1; yield 121.0mg (65.0%). mp: 181.30C MS: m/z 689.0 (M+H+) calculated as C38H48N408 : 688. 4 W Amax(MeOH) nm (£):208(26781.3) 1H-NMR: Fig.14 IR(KBr): Fig.15.

Example 12 Cyclo-(Ile-OPhe-Pro-Ile-OPhe-Pro) (TK690-4) HO-OPhe-OPac Z-lle-ON > Z-lle-OPhe-OPac DCC 4-pyrrol id inopyridine Zn/AcOH Pro-OBzl Z-lle-OPhe-OH - + Z-Ile-OPhe-Pro-OBzl WSCD/HOBt H,/Pd H-Ile-OPhe-Pro-OH DIEA/HATU \ N H O O O=( O H N 19° X X 0 (1) Z-Ile-OPhe-Opac The Z-Ile-OH (2.80g,10.6mmol) was dissolved in DMF (20ml) containing H-OPhe-OPac (3.00g, 10.6mmol) and 4- pyrrolidinopyridine (0.47g, 3.17mmol) under ice-cooling. DCC (3.02g, 11.6mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 18 h. The insoluble material was filtered off, and the filtrate concentrated under reduced pressure. The residue was dissolved in ethyl acetate, which was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a toluene-ethyl acetate in a ratio of 5 1; yield 6.00g (100%).

(2) Z-Ile-OPhe-OH Z-Ile-OPhe-OPac (6.00g, 10.6mmol) obtained in (1) was added to 90% aqueous acetic acid containing zinc dust (34.7g, 530mmol). The mixture was vigorously stirred for 2 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid.

The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a toluene-ethyl acetate mixture in a ratio of 1 : 1; yield 2.31g (56.1%).

(3) Z-Ile-OPhe-Pro-Obzl The HCl-Pro-OBzl (1.47g, 6.09mmol) was dissolved in DMF <BR> <BR> <BR> <BR> <BR> (10ml) containing Z-Ile-OPhe-OH (2.31g, 5.95mmol) obtained in (2) and HOBt (0.91lg, 6.70mmol) under ice-cooling. WSCD (1.47ml, 6.70mmol) was added to the solution, which was then stirred for 30 min, and then additionally at room temperature for 16 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure, Purification was performed by silica gel column chromatography using a toluene-ethyl acetate mixture in a ratio of 10 : 1;yield 2.95g (86.1%).

(4) H-Ile-OPhe-Pro-OH Z-Ile-OPhe-Pro-OBzl (2.95g, 5.12mmol) obtained in (3) was dissolved in MeOH-acetic acid-water with ratio of 8:2:1. The solution was bubbled with hydrogen in the presence of Pd-black for 3 h. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure; yield 1.85g (96.1%).

(5) Cyclo-(Ile-OPhe-Pro-Ile-OPhe-Pro) (TK690-4) H-Ile-OPhe-Pro-OH (100mg, 0.266mmol) obtained in (4) and DIEA (0.277ml,1.60mmol) dissolved in dichloromethane (50ml) was dropped at a rate of 12.5ml per an hour into dichloromethane (200ml) containing HATU (303mg, 0.798mmol), which was then stirred at room temperature for 20 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a toluene-ethyl acetate mixture in a ratio of 1:1; yield 43.9mg (46.1%).

MS: m/z 717.5 (M+H+) calculated as C40H52N408 : 716.4 1H-NMR:-NMR: 1H-NMR:-NMR: Fig.l6Fig. 16 Fig.l6Fig. 16 Example 13 Cyclo-(Ile-D,L-OLeu-Lac-B-Ala-OPhe-Pro) (TK660-7) HO-OLeu-OPac Zn/AcON Z-lle-ON a Z-lle-OLeu-OPac r Z-lle-OLeu-OH DCC 4-pyrrolidinopyridine Lac-O Pac Zn/AcO H > Z-lle-OLeu-Lac-OPac o Z-lle-OLeu-Lac-OH DCC 4-pyrrolidinopyridine (1) HO-OPhe-OPac Zn/AcOH Boc- 2 -Ala-OH A Boc- 2 -Ala-OPhe-OPac a Boc- -Ala-OPhe-OH DCC 4-pyrrolidinopyridine HC1 H-Pro-Bzl HCl/dio.:ane a Boc- a -Ala-OPhe-Pro-OBzl a HCI H- a -Ala-OPhe-Pio-OBzl WSCD/HOBt (2) WSCD/HOAt (1)+(2) Z-lle-OLeu-Lac- -Ala-OPhe-Pro-OBzl H9/Pd o H-lle-OLeu-Lac- -Ala-OPhe-Pro-OH DIEA/HATU O g a MNTh O H Oo X O H) O (1) Z-Ile-D,L,-OLeu-Opac The Z-Ile-OH (5.32g, 20.lmmol) was dissolved in DMF (20ml) containing H-D,L-OLeu-OPac (5.02g, 20.lmmol) and 4- pyrrolidinopyridine (0.892g, 6.02mmol) under ice-cooling. DCC (5.74g, 22.lmmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 18 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, which was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a toluene-ethyl acetate mixture in a ratio of 1 : 1;yield 10.0g (100%).

(2) Z-Ile-D,L-OLeu-OH Z-Ile-D,L-OLeu-OPac (10.0g, 20.lmmol) obtained in (1) was added to 90% aqueous acetic acid containing zinc dust (65.7g, 1.Olmol). The mixture was vigorously stirred for 2 h. The insoluble material was filtered off, and the filtrate concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid.

The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a chroloform-methanol mixture in a ratio of 9 : 1;yield 6.23g (81.7%).

(3) Z-Ile-D,L-OLeu-Lac-Opac The Z-Ile-D,L-OLeu-OH (7.65g, 20.2mmol) obtained in (2) was dissolved in DMF (20my) containing Lac-OPac (4.20g, 20.2mmol) and 4-pyrrolidinopyridine (0.896g, 6.05mmol) under ice-cooling. DCC (5.77g, 22.2mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 18 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, which was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a toluene-ethyl acetate mixture in a ratio of 50 : 1;yield 2.00g (17.4%).

(4) Z-Ile-D,L-OLeu-Lac-OH Z-Ile-D,L-OLeu-Lac-OPac (2.00g, 3.51mmol) obtained in (3) was added to 90% aqueous acetic acid containing zinc dust (11.5g, 176mmol). The mixture was vigorously stirred for 2 h.

The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid.

The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a chroloform;yield 0.72g (45.4%).

(5) Boc-B-Ala-OPhe-Opac The Boc-B-Ala-OH (1.85g, 9.78mmol) was dissolved in DMF (10ml) containing HO-OPhe-OPac (2.78g, 9.78mmol) and 4- pyrrolidinopyridine (0.435g, 2.93mmol) under ice-cooling. DCC (2.80g, 10.8mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 18 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, which was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a toluene-ethyl acetate mixture in a ratio of 5 : 1;yield 4.08g (91.5%).

(6) Boc-B-Ala-OPhe-OH Boc-B-Ala-OPhe-OPac (4.08g, 8.96mmol) obtained in (5) was added to 90% aqueous acetic acid containing zinc dust (29.3g, 448mmol). The mixture was vigorously stirred for 2 h. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate; this was washed with 10% aqueous citric acid.

The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using a chloroform-methanol mixture in a ratio of 19 : 1; yield 2.84g (93.9%).

(7) Boc-B-Ala-OPhe-Pro-Obzl The HCl Pro-OBzl (2.03g, 8.42mmol) was dissolved in DMF containing Boc-B-Ala-OPhe-OH (2.84g, 8.42mmol) obtained in (6) and HOBt (1.25g, 9.26mmol) under ice-cooling. WSCD (2.03ml, 9.26mmol) was added to the solution, which was then stirred for 30 min, and then further stirred at room temperature for 16 h.

After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure; yield 3.89g (88.0%).

(8) HCl-H-B-Ala-OPhe-Pro-Obzl Boc-B-Ala-OPhe-Pro-OBzl (3.89g, 7.41mmol) obtained in (7) was added to 4 N hydrogen chloride in 1,4-dioxane solution (37.0ml, 148mmol). The solution was allowed to stand at room temperature for 1 h. After removal of the solvent by concentration under reduced pressure, the residue was washed with ether; yield 3.01g (88.4%).

(9) Z-Ile-D,L-OLeu-Lac-B-Ala-OPhe-Pro-Obzl TheHClH-B-Ala-OPhe-Pro-OBzl (0.733g, 1.59mmol) obtained in (8) was dissolved in DMF containing Z-Ile-D,L-OLeu-Lac-OH (0.72g, 1.59mmol) obtained in (4) and HOAt (0.239g, 1.75mmol) under ice-cooling. WSCD (0.384m1, 1.75mmol) was added to the solution, which was then stirred for 1 h, and then further stirred at room temperature for 18 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by silica gel column chromatography using chroloform;yield 1.24g (91.1%).

(10) H-Ile-D,L-OLeu-Lac-B-Ala-OPhe-Pro-OH Z-Ile-D,L-OLeu-Lac-B-Ala-OPhe-Pro-OBzl (1.24g, 1.45mmol) obtained in (9) was dissolved in MeOH-acetic acid-water with ratio of 8:2:1. The solution was bubbled with hydrogen in the presence of Pd-black for 3 h. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure, followed by liophylization; yield 0.878g (95.7%).

(11) Cyclo-(Ile-D,L-OLeu-Lac-B-Ala-OPhe-Pro) (TK660-7) H-Ile-D,L-OLeu-Lac-B-Ala-OPhe-Pro-OH (15Omg, 0.237mmol) obtained in (10) and DIEA (0.248m1,1.42mmol) dissolved in dichloromethane (50ml) was dropped at a rate of 12.5ml per an hour into dichloromethane (190ml) containing HATU (271mg, 0.712mmol), which was then stirred at room temperature for 18 h. After removal of the solvent by concentration under reduced pressure, the residue was dissolved in ethyl acetate; this was washed successively with 10% aqueous citric acid, brine, saturated aqueous sodium hydrogencarbonate, and brine. The organic layer was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed by thin layer silica gel chromatography using a toluene-ethyl acetate mixture in a ratio of 1:5; yield 120mg (82.7%).

1H-NMR: Fig.17.

Test Example 1 The effects of TK22-4 obtained in Example 1 were investigated as reported previously (Kurokawa M. and Tsunoo A.: Parasympathetic depression of vas deferens contraction in the guinea-pig involves adenosine receptors. J. Physiol., 407, 135-153, 1988; Tsunoo A. etal. : Neurallyevokedpotentiation of tonic contractions in the guinea-pig vas deferens involves adenosine receptors. J. Physiol., 433, 163-181, 1991).

Heart muscles isolated from male rats of the Sprague Dawley strain were set in a perfusion bath of a 0.8 ml volume. Tensions of muscles were recorded isometrically. Perfusion solutions were either a Krebs' solution bubbled with 95% O2 and CO2 , or a salt solution of the following composition (mM): NaCl, 140; KCl, 5; CaCl2, 2.6; MgCl2, 1.3; HEPES, 5; and glucose, 10. The salt solution was aerated. The solutions were kept at 36-37 C, and flowed at a rate of 3-4 ml/min. The pH of the solutions was 7.3-7.4. Isolated right atria contracted automatically.

Isolated papillary and trabeculaer muscles of right ventricles were stimulated at 1 or 2 Hz through a pair of platinum wires in the bath.

The relationship between TK22-4 concentration in the bath and the contractile force of right atria is shown in Fig. 18, and relationship betweeen TK22-4 concentration and intercontraction interval of the automatic atrial contractions in Fig. 19. The values are expressed as the ratios with respect to the control value before each drug application.

TK22-4 increased the contractile force of the atrial automatic contractions at 2-200 ILM (Fig. 18). The intercontraction intervals were not affected markedly at lower concentrations or prolonged at higher concentrations (Fig. 19) .

Similarly, TK22-4 caused an increase in contractile forces of the ventricular papillary and trabecular muscles.

The relationship between TK620-9 concentration and contractile force of right atria is shown in Fig. 20, and relationship between TK620-9 concentration and intercontraction interval of the automatic atrial contractions in Fig. 21. Similarly to TK22-4, TK620-9 increased the contractile forces of heart muscles and prolonged the intercontraction intervals.

Test Example 2 The inotropic activities of cyclodepsipeptides other than TK22-4 and TK620-9 are shown below. The values are expressed in the ratios with respect to the control value before each drug application. The concentration tested was 20 AM in all cases.

Table 1 Compounds Contractile forces Intercontraction interval TK330-6(Example 2) 2.99 1.23 TK340-6(Example 3) 2.74 1.3 TK70-5(Example 5) 3.4 1.23 TKS10-7(Example 9) 1.21 1.07 TK610-7(Example 10) 1.26 1.02 TK690-4(Example 12) 1.58 1.16 TK660-7(Example 13) 1.54 1.2 Test Example 3 The effects of TK620-9 on digoxin-induced aberrant contractions were examined. Digoxin(0.6 gM) induced aberrant contractions in right atria isolated from male guinea pigs (100-200 g). Before digoxin application, the atria contracted regularly in a sinus rhythm (Fig. 22A). In the presence of <BR> <BR> <BR> <BR> <BR> digoxin, contractions became aberrant in rhythm and contraction amplitude (Fig. 22B). In the simultaneous presence of digoxin and TK620-9(20 AM) , the rhythm and contraction amplitude of the contractions recovered to be regular (Fig. 22C).

Therefore, TK620-9 was found to inhibit digoxin-induced aberrant contractions.

Test Example 4 The vasodilating effect of TK22-4 was examined. Aorta strips isolated from rats were contracted continuously by a 50 mM KCl-containing solution in with NaCl was replaced by KCl.

TK22-4 (20 gM) caused a slow relaxation of the aorta strip, as shown in Fig. 23.

Test Example 5 The vasodilating effects of the invented compounds including TK22-4 are summarized in Table 2. The experimental conditions were the same as those of TK22-4. The amplitude of the contraction evoked by 50 mM KCl was measured as 1, and the amplitude of the relaxation evoked by the compounds was normalized to that of the contraction evoked by 50 mM KCl.

Table 2 Compounds (20 A£M) Relaxing ratio TK22-4 (Example 1) 0.05 TK330-6 (Example 2) 0.056 TK340-6 (Example 3) 0.229 TK70-5 (Example 5) 0.112 TK610-7 (Example 10) 0.02 TK620-9 (Example 11) r 0.022 Test Example 6 The effects of the cyclodepsipeptides on cyclic AMP contents in heart muscles were examined. Trabecular strips were isolated from the right ventricles of the Sprague Dawley rats.

The preparations were set in Magnus tubes filled with 10 ml Krebs-Henseleit solution which was bubbled with 95% °2 and 5% C 02. The upper ends of the preparations were connected with <BR> <BR> <BR> <BR> <BR> pressure transducers, and tensions were recorded isometrically.

The preparations were stimulated through electrodes in the tubes at 1 Hz with a 3 msec duration. The preparations were exposed to TK22-4(200 LLM), TK610-7(400 AM) and TK620-9(100 g <BR> <BR> <BR> <BR> <BR> <BR> M) for 10 min, and to isoproterenol(50 nM) for 5 min. Immediately after contractile measurements, the preparations were frozen in liquid nitrogen, and stored at -30 C. The frozen samples were homogenized in trichloroacetic acid by a microhomogenizer, centrifuged at 10000 rpm for 10 min, and the supernatents were obtained. The supernatents were washed 5 times with ether.

Measurements of cyclic AMP contents were carried out using cAMP EIA system of Amersham LIFE SCIENCE. Test compounds were dissolved in propylene glycol. Contraction amplitudes were expressed as the ratios with respect to the control value before each drug application. Significance was determined based on comparison with the results obtained.

Table 3 Test compounds Contractile ratio Cyclic AMP (meaniSE) (mean+SE) (pmol/mg w.w.) Propylene glycol 0.89+.0083 0.26i0.015 Isoproterenol 1.78i0.14*** 0.40i0.029*** TK22-4 (Example 1) 1.86+0.06*** 0.22+0.012* TK610-7 (Example 10) 1.66i0.12*** 0.30i0.07 TK620-9 (Example 11) 1.33i0.024*** 0.26+0.021 ***: P<0.001, *: P<0.05 Table 3 shows that isoproterenol causes a significant increase in the cyclic AMP content in the heart muscles, but that the cyclodepsipeptides do not even though they all cause significant increases in contractile force. TK22-4 causes a significant decrease in the cyclic AMP content. Therefore, the cyclodepsipeptides do not produce an increase in the cyclic AMP content in the heart muscles which is thought to cause arrhythmias, inspite of their positive inotropic effect.

Test Example 7 Hemodynamic effects of cyclodepsipeptides were examined.

The left ventricular pressure (LVP) was recorded from a male rat of the Sprague Dawley strain (350-450 g) through a catheter, and the maximal rising rate of LVP (LVdP/dt) was obtained.

TK22-4 (10 mg/kg) and TK610-7 (10 mg/kg) were dissolved in 0.1 ml of 60 % ethanol, and administered intravenously into a rat at time 0. <BR> <BR> <BR> <BR> <BR> <P> The effects of the compounds on LVdP/dt expressed as the ratios with respect to the control values before each drug administration are shown in Figs. 24 and 25. Both of TK22-4 and TK610-7 increased LVdP/dt persistently.

Test Example 8 The ouabain-induced arrhythmic model was used to examine <BR> <BR> <BR> <BR> <BR> an antiarrhythmic effect of the cyclodepsipeptides. Guinea pigs (350-450 g) were anesthesized by intraperitoneal urethane (1.5 g/kg), and a cannula was set into the trachea. For drug application, a polyethylene tube was inserted into the right carotid vein. Elecrocardiogram (lead 1l ) (ECG) was obtained from needle electrodes, and signals was fed into a preamplifier.

After the ECG became stable, TK22-4 (10 mg/kg) was injected into the carotid vein 15-18 min before ouabain infusion, and TK610-7(3 mg/kg) 5 min before ouabain infusion. Ouabain was continuously infused into the same vein by a microsyringe pump.

Test compounds were dissolved in a solvent (60 % ethanol), and disopyramide (3 mg/kg) was used as a positive control.

As shown in Fig. 26, TK22-4 and TK610-7 as well as disopyramide significantly prolonged the times for premature ventricular contractions, ventricular fibrillations and cardiac arrest to occur from the beginning of ouabain infusion.

INDUSTRIAL APPLICABILITY The cyclodepsipeptides described in this invention show an excellent cardiotonic, antiarrhythmic and vasodilating effect without an incease in the cyclic AMP contents in heart muscles. These results may open a new avenue for a cardiotonic agent without the arrhythmogenic activity which many cardiotonic agents have.