JPH05163194A - Isocarbacyclin derivative, its production and photophilic labeling - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful for the photophilic labeling of prostacyclin receptors by reacting a Horrer-Emmons reagent with an isocarbacyclin compound in the presence of a basic catalyst and subsequently subjecting the reaction product to a reduction reaction, a protecting group- removing reaction, an azido-forming reaction and a hydrolysis reaction. CONSTITUTION:A Horner-Emmons reagent of formula I (Ya is H, trityl-protected amino group, azido; n is 1-7) is reacted with an isocarbacyclin derivative of formula II (R<2> is 1-5C lower alkyl) in the presence of a base, and the reaction product is subjected to a reduction reaction, if necessary, further to a trityl group-removing reaction, to an azido-forming reaction of the amine, and to a hydrolysis reaction to obtain the objective isocarbacyclin derivative consisting of a compound of formula III (R<1> is H, 1-5C lower alkyl; X is H, tritium; Y is H, azido, trityl-protected amino, amino), its epimer, their mirror image isomers or their mixture in an arbitrary ratio.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel isocarbacycline derivative, a method for producing the same and uses of the derivative. More specifically, it relates to a novel isocarbacycline derivative useful as a drug or a photoaffinity labeling agent for prostacyclin (PGI ₂ ) receptor protein, a method for producing the same, and use thereof.

[0002]

2. Description of the Related Art Prostacyclin is a local hormone mainly produced in the inner wall of blood vessels of arteries in the living body,
Its physiological activities such as platelet aggregation inhibitory activity, vasodilatory activity, etc. are important factors that regulate the cell functions of the living body,
Attempts are being made to use this directly as a medicine [Clinical Pharmacology of Prostacycli
n), Raven Press, NY, 1981].

However, since natural prostacyclin has an enol ether bond which is very easily hydrolyzed in the molecule, it is easily deactivated under neutral or acidic conditions, and it is a preferable compound because of its chemical instability as a drug. I can't say. Therefore, a chemically stable synthetic prostacyclin derivative having a physiological activity similar to that of natural prostacyclin has been intensively studied both inside and outside.

Among them, derivatives of prostacyclin in which the 6,9-position oxygen atom is substituted with a methylene group, ie, 9
(O) -methanoprostacyclin (carbacycline) is known as a prostacyclin that sufficiently satisfies chemical stability [Prostacyclin (Prostacyclin
cyclin), JR Vane and S. Bergstrom, Eds. Raven Pre
ss, NY, pp. 31-41], and is expected as a medicine. However, the biological activity of 6,9 (O) -methanoprostacyclin is weaker than that of natural prostacyclin, and its action selectivity cannot be said to be specific, so that it cannot be said to be a preferable compound.

On the other hand, isocarbacycline which is one of the double bond isomers of carbacyclin, that is, 9
(O) -Methano-Δ6 ^{(9α) -Prostaglandin} I ₁
It was discovered that these compounds have an inhibitory effect on platelet aggregation, and they have been expected to be applied as pharmaceuticals [Ikegami et al., Tetrahedron Lett., 24 , 34.
93 (1983), JP-A-59-137445].

However, little research has been conducted on prostacyclin receptor proteins.
This is due to the low density of prostacyclin receptor proteins on the cell membrane, the lack of useful antagonists, or the difficulty in solubilizing receptor proteins in homogenates while retaining receptor activity. To do.

[0007]

SUMMARY OF THE INVENTION The present inventors have paid attention to the above-mentioned various points, and are novel 9 (O) -methano Δ ^{6 (9α) -prostaglandins} I ₁ useful for drugs, photoaffinity labeling agents, diagnostics and the like. As a result of earnest research to find out (isocarbacycline), the inventors have found that isocarbacyclins having a phenyl group introduced into the ω chain can achieve such an object, and have reached the present invention.

[0008]

The present invention includes the following formula [I]:

[0009]

[Chemical 7]

[Wherein R ¹ is a hydrogen atom or C _{1 to} C ₅
Represents a lower alkyl group, X represents a hydrogen atom or a tritium atom, Y represents a hydrogen atom, an azido group, a trityl-protected amino group or an amino group, and n is 1 to 1
Represents 7. ] And its 15 epimer and its enantiomer or a mixture of them in any proportions.

In the above formula [I], R ¹ represents a hydrogen atom or a C _{1 to} C ₅ lower alkyl group. C _{1 to} C ₅
Examples of the alkyl group include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, pentyl group and the like. R ¹ is preferably a hydrogen atom or a C ₁ -C ₃ alkyl group, and particularly preferably a hydrogen atom or a methyl group.

In the above formula [I], X represents a hydrogen atom or a tritium atom. In the above formula [I], Y
Represents a hydrogen atom, an azido group, a trityl-protected amino group or an amino group. Preferred examples thereof include a hydrogen atom and an azido group, and the substitution position of the azido group is preferably the meta position or the para position, and particularly preferably the meta position.

In the above formula [I], n represents 1 to 7, preferably n is 2 to 4, and particularly preferably n.
Is 4.

In the compound represented by the above formula [I], the bicyclo [3.3.0] octane ring itself and the substituent bonded to the bicyclo [3.3.0] octane ring are bonded to each other. Existing carbon; carbon substituted with a hydroxyl group on the ω chain has stereoisomers due to the asymmetric environment, but in the present invention, any stereoisomers are included, and any of these It can be a stereoisomeric mixture. Further, the compound represented by the formula represents all of these stereoisomers and a mixture of these isomers at an arbitrary ratio, and a compound having a stereostructure represented by the formula is most preferred. ..

Preferred specific examples of the novel isocarbacyclines represented by the above formula [I] provided in the present invention include the compounds shown below. (1) 17-phenyl-18,19,20-trinorisocarbacycline (2) 18-phenyl-19,20-dinorisocarbacycline (3) 19-phenyl-20-norisocarbacycline (4) 20-phenyl-isocarbacycline (5) 17- (3-azidophenyl) -18,19,2
0-trinorisocarbacycline (6) 18- (3-azidophenyl) -19,20-dinorisocarbacycline (7) 19- (3-azidophenyl) -20-norisocarbacycline (8) 20 -(3-Azidophenyl) -isocarbacycline (9) 17- (4-azidophenyl) -18,19,2
0-trinorisocarbacycline (10) 18- (4-azidophenyl) -19,20-
Dinorisocarbacycline (11) 19- (4-azidophenyl) -20-norisocarbacycline (12) 20- (4-azidophenyl) -isocarbacycline (13) 17- (3-Tritylaminophenyl) -1
8,19,20-Trinorisocarbacycline (14) 18- (3-Tritylaminophenyl) -1
9,20-Dinorisocarbacycline (15) 19- (3-Tritylaminophenyl) -20
-Noriisocarbacycline (16) 20- (3-Tritylaminophenyl) -isocarbacycline (17) 17- (4-Tritylaminophenyl) -1
8,19,20-Trinorisocarbacycline (18) 18- (4-Tritylaminophenyl) -1
9,20-Dinorisocarbacycline (19) 19- (4-Tritylaminophenyl) -20
-Norisocarbacycline (20) 20- (4-tritylaminophenyl) -isocarbacycline (21) 17- (3-aminophenyl) -18,19,
20-trinorisocarbacycline (22) 18- (3-aminophenyl) -19,20-
Dinorisocarbacycline (23) 19- (3-aminophenyl) -20-norisocarbacycline (24) 20- (3-aminophenyl) -isocarbacycline (25) 17- (4-aminophenyl)- 18, 19,
20-Trinorisocarbacycline (26) 18- (4-aminophenyl) -19,20-
Dinorisocarbacycline (27) 19- (4-aminophenyl) -20-norisocarbacycline (28) 20- (4-aminophenyl) isocarbacycline (29) Methyl ester of (1) to (28) Examples include, but are not limited to, 15R isomers of bodies (30) (1) to (29), and enantiomers of (31) (1) to (30).

The novel isocarbacycline of the present invention represented by the above formula [I] is produced as follows.

That is, the following formula [II]

[0018]

[Chemical 8]

[In the formula, Ya represents a hydrogen atom, a trityl-protected amino group or an azido group, and n represents 1 to 7. ] The Horner-Emmons reagent represented by
]

[0020]

[Chemical 9]

[In the formula, R ² represents a C _{1 to} C ₅ lower alkyl group. ] The compound represented by the formula [IV]

[0022]

[Chemical 10]

[In the formula, R ² , n, and Ya are the same as defined above. ], A reduction reaction,
Further, a deprotection reaction of a trityl protected amine, if necessary,
Amine azidation reaction and, if necessary, hydrolysis reaction, the following formula [I]

[0024]

[Chemical 11]

[Wherein R ¹ is a hydrogen atom or C _{1 to} C ₅
Represents a lower alkyl group, X represents a hydrogen atom or a tritium atom, Y represents a hydrogen atom, an azido group, a trityl-protected amino group or an amino group, and n is 1 to 1
Represents 7. ] And the 15-epimer thereof and the enantiomers thereof or a mixture of them in any proportion, isocarbacycline is produced.

The reaction of the compound of the above formula [II] with the compound of the above formula [III] is carried out by using the phosphonate compound represented by the [II] as a base such as NaH, NaNH ₂ , LiN (iP).
r) ₂ , treated with CH ₃ ONa or the like and then reacted with an aldehyde compound represented by [III], a so-called Horn
er-Emmons reaction (New Experimental Chemistry Course 14 , p. 238;
Maruzen). At this time, as a solvent used in the reaction, for example, benzene, toluene, tetrahydrofuran (THF), diglyme, dimethoxyethane (DME), dimethylformamide (DMF), dimethylsulfoxide (DMSO) and the like are used.

The base is 0.1 to 10 equivalents, preferably 0.9 to 1.4 equivalents, and the aldehyde compound [III] is 0.1 to 10 equivalents, relative to the phosphonate compound [II].
It is preferable to use 0.9 to 1.4 times equivalent amount. The reaction temperature is 0 to 150 ° C., preferably 10 to 8 ° C.
It is 0 ° C. The reaction time varies depending on the compound, but it varies from 10 minutes to
It is about 24 hours. After completion of the reaction, the compound [IV] is subjected to usual post-treatment such as extraction or column chromatography.
Is obtained. The aldehyde derivative [III] as the raw material is, for example, as shown in the following chart 1, Sharpless oxidation of isocarbacycline methyl ester (1),
Acetylation of the hydroxyl group, epoxy cleavage, resulting tetraol body (4) by deacetylation, NaIO ₄ for this product
The aldehyde derivative (5) can be obtained by oxidative cleavage with.

[0028]

[Chemical 12]

[0029]

[Chemical 13]

The aldehyde derivative (5) may be directly used in the next Horner-Emmons reaction, but the aldehyde derivative (5) generated in the system by the oxidation of 4 may be used as it is without isolation.

The Horner-Emmons reagent of the above formula [II] can be synthesized from the corresponding ester compound, for example, by the route shown in Chart 2.

[0032]

[Chemical 14]

The obtained 6 can be converted to an azide by a Sandmeyer reaction after detritylation. Not only 6 but also 7 can be used in the Horner-Emmons reaction.

Thus, the compound represented by the above formula [IV] can be obtained. Such a compound [IV] can then be subjected to a reduction reaction, or optionally a deprotection reaction of a trityl-protected amine, an azidation reaction of an amine, or an optional hydrolysis reaction.

The reduction reaction can be carried out by a method known per se. A metal hydrogen complex compound is used as a reagent for the reduction reaction. Examples of such metal hydrogen complex compounds include aluminum hydride complex compounds and borohydride complex compounds. Examples of the aluminum hydride complex compounds include lithium aluminum hydride, lithium diethoxyaluminum hydride, lithium triethoxyaluminum hydride, lithium trimethoxyaluminum hydride, and trihydrated hydride.
Examples thereof include t-butoxyaluminum lithium, aluminum magnesium hydride, aluminum magnesium hydride chloride, sodium aluminum hydride, sodium triethoxyaluminum hydride, and sodium bis (2-methoxyethoxy) aluminum hydride. Examples of the borohydride complex compound include sodium borohydride, sodium trimethoxyborohydride, sodium sulfide borohydride, sodium cyanoborohydride,
Lithium borohydride, lithium cyanoborohydride, lithium triethylborohydride, calcium borohydride, potassium borohydride, zinc borohydride,
Examples include tetramethylammonium borohydride and the like. Among these metal hydrogen complex compounds, the borohydride complex compound is preferable as the reagent for the reduction reaction, and sodium borohydride is particularly preferable.

The reduction reaction using sodium borohydride is preferably carried out in the presence of lanthanide chlorides. Examples of such lanthanide chlorides include cerium trichloride, samarium trichloride, eurobium trichloride and the like, and cerium trichloride is particularly preferably used.

The reduction reaction is represented by the above formula [II] 2
-Chloro-4-hydroxy-2-cyclopentenones 1
It is carried out in the range of 1 to 100 equivalents, preferably 1 to 50 equivalents, in terms of hydride ions capable of generating a metal-hydrogen complex compound with respect to equivalents.

The lanthanide chloride used together with sodium borohydride is 0.2 to 50 mol, preferably 0.5 to 10 mol of lanthanide chloride based on 1 mol of sodium borohydride.

The reaction solvent varies depending on the reducing reaction reagent used, but is usually an alcohol such as methanol, ethanol, 2-propanol or t-butyl alcohol; an ether such as tetrahydrofuran, diethyl ether, dioxane, dimethoxyethane or diglyme; dimethyl. An aprotic polar solvent such as formamide, dimethyl sulfoxide and hexamethylphosphoric triamide; water, acetonitrile and the like are used alone or in a mixture at an arbitrary ratio. Alcohols such as methanol, ethanol, 2-propanol and t-butyl alcohol are preferably used, and methanol is particularly preferable.

The reaction temperature of the reduction reaction varies depending on the reagent used and the reaction solvent, but is preferably -100 to 100.
C., particularly preferably in the range of -20 to 50.degree.

The reaction time of the reduction reaction varies depending on the reagent used, the reaction solvent and the reaction temperature, but is usually within 5 hours, preferably 1 minute to 1 hour.

For the deprotection reaction of the trityl protected amine, for example, acetic acid, p-toluenesulfonic acid, pyridinium p-
It is preferable to use toluene sulfonate, a cation exchange resin or the like as a catalyst, and use, for example, water, methanol, ethanol or tetrahydrofuran, ethyl ether, dioxane, acetone, acetonitrile or the like in the coexistence of water, methanol, ethanol or the like as a reaction solvent. Be implemented. The reaction temperature is usually in the range of -78 ° C to + 50 ° C,
It is performed for 0 minutes to 3 days.

The azidation reaction of an amine can be carried out by a Sand meyer type reaction (New Experimental Chemistry Course 14, p. 1665; Maruzen) which is a method known per se.

The ester hydrolysis reaction is carried out, for example, with an aqueous solution of sodium hydroxide, lithium hydroxide, potassium hydroxide, calcium hydroxide or a water-alcohol mixed solution,
Or sodium methoxide, potassium methoxide,
It can be carried out by hydrolysis in a methanol or ethanol solution containing sodium ethoxide.

The desired product can be isolated and purified by a conventional method, that is, a general means such as extraction, chromatography and the like.

The thus-obtained isocarbacycline derivative of the present invention was shown to exhibit affinity for the PGI ₂ (prostacyclin) receptor protein.

Therefore, of the compounds represented by the formula [I] of the present invention, compounds having an azide group as Y can be labeled with photoaffinity for the prostacyclin (PGI ₂ ) receptor protein. The compounds are useful for biochemical evaluation of receptor proteins, and on the other hand, they are expected to be useful for diagnosis and the like.

Among the compounds of the present invention, those in which Y does not contain an azido group are useful as raw materials for at least a compound in which Y contains an azido group.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0050]

Example 1 Synthesis of 15-oxo-17- (3- (tritylamino) phenyl) -18,19,20-trinorisocarbacycline methyl ester;

[0051]

[Chemical 15]

13,14-Dihydroxy-13,14-
Dihydroisocarbacycline methyl ester 24.0
130 mg of sodium metaperiodate in a solution of mg (0.06 mmol) in methanol (0.1 ml) and ether (1 ml)
0.8 ml aqueous solution of (0.61 mmol) was added and the mixture was stirred for 1 hour at room temperature. Then, water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and methyl-5-{(1S, 5S,
A crude product of 6R, 7R) -6-formyl-7-hydroxybicyclo [3.3.0] -2-octen-3-yl} pentanoate was obtained and subjected to the next reaction.

2-oxo-4 (4- (tritylamino)
Dimethyl phenyl) butylphosphonate 52.3 mg (0.
10 mmol of DME-toluene mixed solvent (7: 1, 1.
5 mg) solution with 4 mg NaH (50% in oil)
(0.08 mmol) was added and stirred for 10 minutes. Then methyl-5-{(1S, 5S, 6R, 7R) obtained above
-6-Formyl-7-hydroxybicyclo [3.3.
0] -2-Octen-3-yl} pentanoate crude product DME-toluene mixed solvent (7: 1, 1.5 ml)
The solution was added and stirred at room temperature for 10 minutes. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 3: 1) to give 15-oxo-1.
7- (3- (Tritylamino) phenyl) -18,1
26.5 mg (59%) of 9,20-trinorisocarbacycline methyl ester were obtained.

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 1.2-2.7 (m, 19H), 3.0-3.1 (m, 1H), 3.66
(s, 3H), 3.87 (dd, 1H, J = 9.4, 7.0Hz), 5.00 (br.s, 1H),
5.30 (br.s, 1H), 6.08 (d, 1H.J = 15.8Hz), 6.15 (s, 1H), 6.
21 (d, 1H, J = 7.4Hz), 6.39 (d, 1H, J = 7.4Hz), 6.63 (dd, 1H, J
= 15.8, 8.4Hz), 6.82 (t, 1H, J = 7.4Hz).

[0055]

Example 2 Synthesis of 17- (3- (tritylamino) phenyl) -18,19,20-trinorisocarbacycline methyl ester

[0056]

[Chemical 16]

26.5 mg (0.035) of 15-oxo-17- (3- (tritylamino) phenyl) -18,19,20-trinorisocarbacycline methyl ester
mmol) to 13.0 mg of cerium (III) chloride heptahydrate
It was dissolved in a solution of (0.035 mmol) in methanol (0.5 ml), and 1.5 mg (0.0
40 mmol) was added and the mixture was stirred for 1 minute at room temperature. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure.
-(3- (Tritylamino) phenyl) -18,19,
A crude product of 20-trinorisocarbacycline methyl ester was obtained. The obtained oily substance was directly used in the next reaction.

¹ H-NMR (δppm, CDCl ₃ , 2
70MHz): 1.2-2.7 (m, 20H), 2.85-3.10 (m, 1H), 3.6
5 (s, 3H), 3.70-3.75 (m, 1H), 3.85-3.90 (m, 1H), 4.98-4.
99 (br.s, 1H), 5.28 (s, 1H), 5.40-5.55 (m, 2H), 6.16 (s, 1
H), 6.21 (d, 1H, J = 7.9Hz), 6.39 (d, 1H, J = 6.9Hz), 6.83
(t, 1H, J = 7.9Hz).

[0059]

Example 3 17- (3-aminophenyl) -18,1
Synthesis of 9,20-trinorisocarbacycline methyl ester

[0060]

[Chemical 17]

The crude product of 17- (3- (tritylamino) phenyl) -18,19,20-trinorisocarbacycline methyl ester obtained in Example 2 was treated with methanol (0.2 ml) and ether (0 ml). 0.5 ml), and 0.3 ml of acetic acid-water (9: 1) was added thereto.
After stirring at room temperature for 2 hours, the reaction was terminated by neutralizing with sodium hydrogen carbonate. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oil was subjected to silica gel column chromatography (hexane: ethyl acetate = 1: 2 → 1: 5) to give 17- (3- (aminophenyl) -18,19,20-trinorisocarbacyclinemethyl. Ester 4.7 mg (highly polar), (15
R) -17- (3-aminophenyl) -18,19,2
0-trinorisocarbacycline methyl ester 4.
2 mg (low polarity) and 3.7 mg of these mixtures were obtained.
The total yield was 12.6 mg (70% in 2 steps).

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 15S body: 1.2-2.8 (m, 20H), 2.9-3.1 (m,
1H), 3.67 (s, 3H), 3.77 (dd, 1H, J = 9.4, 6.9Hz), 4.11 (d
d, 1H, J = 12.9, 5.4Hz), 5.28 (s, 1H), 5.55-5.65 (m, 2H),
6.53 (d, 1H), 6.55 (s, 1H), 6.60 (d, 1H, J = 7.4Hz), 7.07
(t, 1H, J = 7.4Hz) .15R body: 1.2-2.8 (m, 20H), 2.9-3.1
(m, 1H), 3.67 (s, 3H), 3.7-3.8 (m, 1H), 4.13 (dd, 1H, J = 1
2.5, 5.5Hz), 5.29 (br.s, 1H), 5.55-5.68 (m, 2H), 6.54
(d, 1H), 6.56 (s, 1H), 6.62 (d, 1H, J = 7.4Hz), 7.07 (t, 1H,
J = 7.4-7.9Hz).

[0063]

Example 4 Synthesis of (15R) -17- (3-azidophenyl) -18,19,20-trinorisocarbacycline methyl ester

[0064]

[Chemical 18]

4.2 mg (0.0082 mmol) of (15R) -17- (3-aminophenyl) -18,19,20-trinorisocarbacycline methyl ester was added to ether (0.1 ml) and methanol (0.5 ml). ), Acetic acid-water (9: 1; 0.5 ml) mixed solvent, and the reaction system was adjusted to 0.
Cooled to ° C. Sodium nitrite 11.2mg here
(0.16 mmol) was added, and the mixture was stirred at 0 ° C for 15 minutes.
At this time, the reaction liquid was deep yellow. Then, 10.6 mg (0.16 mmol) of sodium azide was added, and the mixture was further stirred at 0 ° C for 15 minutes. Dilute the reaction system with ethyl acetate,
A neutralization reaction was carried out with a saturated sodium hydrogen carbonate aqueous solution, and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 2: 3) to give (15R) -17-
(3-azidophenyl) -18,19,20-trinorisocarbacycline methyl ester 3.7 mg (84
%) Was obtained.

Rf: 0.26 (1: 2 / hexane / ethyl acetate, silica gel) IR (cm ^-1 , CHCl ₃ ) 2108, 1712, 1605, 1506 ¹ H-NMR (δ ppm, CDCl ₃ , 270 MH)
z): 1.2-2.1 (m, 15H), 2.2-2.9 (m, 5H), 2.9-3.1 (m, 1
H), 3.67 (s, 3H), 3.76 (dd, 1H, J = 8.4, 16.8Hz), 4.0-4.3
(m, 1H), 5.29 (br.s, 1H), 5.4-5.7 (m, 2H), 6.95 (d, 1H, J =
8.1Hz), 7.18 (d, 1H, J = 8.1Hz).

[0067]

Example 5 17- (3-azidophenyl) -18,1
Synthesis of 9,20-trinorisocarbacycline methyl ester

[0068]

[Chemical 19]

17- (3-aminophenyl) -18,1
4.7 mg (0.0092 mmol) of 9,20-trinorisocarbacycline methyl ester was added to ether (0.1 m).
l), methanol (0.5 ml), acetic acid-water (9: 1;
0.5 ml) and the reaction system was cooled to 0 ° C. Sodium nitrite 11.2mg (0.16mmo
l) was added and stirred at 0 ° C. for 15 minutes. At this time, the reaction liquid was deep yellow. Then sodium azide 10.
6 mg (0.16 mmol) was added, and the mixture was further stirred at 0 ° C. for 15 minutes. The reaction system was diluted with ethyl acetate, neutralized with a saturated aqueous sodium hydrogen carbonate solution, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 2: 3) to give 17- (3-azidophenyl) -1.
2.6 mg (52%) of 8,19,20-trinorisocarbacycline methyl ester were obtained.

Rf: 0.20 (1: 2 / hexane / ethyl acetate, silica gel) IR (cm ^-1 , CHCl ₃ ) 2108, 1712, 1605, 1506 ¹ H-NMR (δ ppm, CDCl ₃ , 270 MH)
z): 1.2-2.1 (m, 15H), 2.2-2.9 (m, 5H), 2.9-3.1 (m, 1
H), 3.67 (s, 3H), 3.76 (dd, 1H, J = 8.4, 16.8Hz), 4.0-4.3
(m, 1H), 5.29 (br.s, 1H), 5.4-5.7 (m, 2H), 6.95 (d, 1H, J =
8.1Hz), 7.18 (d, 1H, J = 8.1Hz).

[0071]

Example 6 17- (3-azidophenyl) -18,1
Synthesis of 9,20-trinorisocarbacycline

[0072]

[Chemical 20]

17- (3-azidophenyl) -18,1
2.6 mg (0.0048 mmol) of methanol (0.50) of 9,20-trinorisocarbacycline methyl ester (0.5
0.1 ml of 3N lithium hydroxide was added to the solution, and the mixture was stirred at room temperature for 3 hours. The reaction system was acidified with an aqueous sodium hydrogen sulfate solution and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 1: 2 → 1: 5 → 1 :).
10-), 17- (3-azidophenyl) -18,
19,20-Trinorisocarbacycline 1.9mg
(75%) was obtained.

Rf: 0.25 (1: 4 / hexane / ethyl acetate, silica gel) ¹ H-NMR (δ ppm, CDCl ₃ , 270 MH)
z): 1.2-2.1 (m, 12H), 2.2-2.5 (m, 4H), 2.5-2.8 (m, 2
H), 2.9-3.1 (br, 1H), 2.3-3.1 (br, 2H), 3.76 (dt, 1H, J =
7.4, 7.4Hz), 4.06 (dd, 1H, J = 6.8, 13.8Hz), 5.29 (br.s,
1H), 5.4-5.7 (m, 2H), 6.85 (s, 2H), 6.98 (d, 1H, J = 7.9H
z), 7.26 (t, 1H, J = 8.2Hz).

[0075]

Example 7 Synthesis of 15-oxo-17- (4- (tritylamino) phenyl) -18,19,20-trinorisocarbacycline methyl ester

[0076]

[Chemical 21]

Dimethyl 2-oxo-4- (4- (tritylamino) phenyl) butylphosphonate 31.0 mg
(0.06 mmol) DME-toluene mixed solvent (7:
1, 1.5 ml) solution with NaH (50% in oil)
3 mg (0.06 mmol) was added and stirred for 10 minutes. Then, methyl-5-{(1S, 5S, 6R, 7R) -6-formyl-7-hydroxybicyclo [3.3.0] -2-octen-3-yl} pentanoate 6.4 mg (0.02
4 mmol of DME-toluene mixed solvent (7: 1; 0.5
ml) solution was added and stirred at room temperature for 10 minutes. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily matter was subjected to silica gel column chromatography (hexane: ethyl acetate = 3: 1) to give 15-oxo-17- (4- (tritylamino) phenyl) -1.
7.6 mg (42%) of 8,19,20-trinorisocarbacycline methyl ester was obtained.

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 1.2-2.8 (m, 20H), 3.0-3.25 (m, 1H), 3.66
(s, 3H), 3.88 (dd, 1H, J = 16.0, 9.0Hz), 4.93 (br.s, 1H),
5.30 (s, 1H), 6.15 (d, 1H, J = 15.8Hz), 6.27 (d, 2H, J = 8.4H
z), 6.69 (dd, 1H, J = 15.8, 8.9Hz), 6.73 (d, 2H, J = 8.4Hz),
7.15-7.45 (m, 15H).

[0079]

Example 8 Synthesis of 17- (4- (tritylamino) phenyl) -18,19,20-trinorisocarbacycline methyl ester

[0080]

[Chemical formula 22]

15-oxo-17- (4- (tritylamino) phenyl) -18,19,20-trinorisocarbacycline methyl ester 7.6 mg (0.010 mm
ol) to 6.0 mg (0.0 mg) of cerium (III) chloride heptahydrate
16 mmol) in methanol (0.5 ml), and sodium borohydride 1.0 mg (0.026 mmo)
l) was added and the mixture was stirred at room temperature for 5 minutes. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained 17- (4- (tritylamino) phenyl) -18,
The crude product of 19,20-trinorisocarbacycline methyl ester (7.3 mg) was directly used in the next reaction.

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 1.2-2.7 (m, 20H), 2.90-3.05 (m, 1H), 3.6
6 (s, 3H), 3.70-3.75 (m, 1H), 4.90 (br.s, 1H), 5.28 (s, 1
H), 5.50-5.60 (m, 2H), 6.28 (d, 2H, J = 8.4Hz), 6.73 (d, 2
H, J = 8.4Hz), 7.15-7.45 (m, 15H).

[0083]

Example 9 17- (4-aminophenyl) -18,1
Synthesis of 9,20-trinorisocarbacycline methyl ester

[0084]

[Chemical formula 23]

7.3 mg of the crude product of 17- (4- (tritylamino) phenyl) -18,19,20-trinorisocarbacycline methyl ester obtained in Example 8.
(0.0097 mmol) was dissolved in a mixed solvent of methanol (0.1 ml) and ether (0.3 ml), and 0.5 ml of acetic acid-water (9: 1) was added thereto. After stirring at room temperature for 2 hours, the reaction was terminated by neutralizing with sodium hydrogen carbonate.
Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 2: 3 → 1: 2 →
1: 4), 17- (4-aminophenyl) -1
There was obtained 4.0 mg (80%; mixture of 15R, 15S form) of 8,19,20-trinorisocarbacycline methyl ester.

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 1.2-2.7 (m, 21H), 2.95-3.10 (m, 1H), 3.6
7 (s, 3H), 3.70-3.80 (m, 1H), 5.29 (s, 1H), 5.50-5.70 (m,
2H), 6.64 (d, 2H, J = 7.5Hz), 6.99 (d, 2HmJ = 7.5Hz).

[0087]

Example 10 17- (4-azidophenyl) -18,
Synthesis of 19,20-trinorisocarbacycline methyl ester

[0088]

[Chemical formula 24]

17- (4-aminophenyl) -18,1
9,20-Trinorisocarbacycline 4.0 mg
(0.0078 mmol 15R, 15S mixture) was dissolved in a mixed solvent of ether (0.05 ml), methanol (0.05 ml) and acetic acid-water (9: 1; 0.1 ml), and the reaction system was heated to 0 ° C. Cooled. Sodium nitrite 10mg here
(0.14 mmol) was added, and the mixture was stirred at 0 ° C for 15 minutes.
Then add 20 mg (0.3 mmol) of sodium azide,
Stirred for an additional 15 minutes at 0 ° C. The reaction system was diluted with ethyl acetate, neutralized with a saturated aqueous sodium hydrogen carbonate solution, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure.
The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 2: 3-1: 2) to give 1
2.2-mg (8- (3-azidophenyl) -18,19,20-trinorisocarbacycline methyl ester)
6%; a mixture of 15R, 15S form) was obtained.

Rf: 0.34 and 0.45 (1: 2 hexane / ethyl acetate, silica gel) IR (cm ^-1 , CHCl ₃ ) 2108, 1730, 1605, 1506 ¹ H-NMR (δ ppm, CDCl ₃ , 270 MH)
z): 1.3-1.7 (m, 8H), 1.7-2.1 (m, 7H), 2.32 (t, 2H, J = 7.
2Hz), 2.4-2.5 (m, 1H), 2.68 (t, 2H, J = 6.9Hz), 2.9-3.1
(m, 1H), 3.67 (s, 3H), 3.7-3.9 (m, 1H), 4.0-4.2 (m, 1H),
5.29 (br.s, 1H), 5.4-5.7 (m, 2H), 6.95 (d, 2H, J = 8.4Hz),
7.19 (d, 2H, J = 8.4Hz).

[0091]

Example 11 17- (4-azidophenyl) -18,
Synthesis of 19,20-trinorisocarbacycline

[0092]

[Chemical 25]

17- (4-azidophenyl) -18,1
0.04 ml of 3N lithium hydroxide was added to a solution of 9,20-trinorisocarbacycline methyl ester (3.6 mg, 0.0067 mmol, mixture of 15R and 15S) in methanol (0.2 ml), and the mixture was stirred at room temperature for 3 hours. did. The reaction system was acidified with an aqueous sodium hydrogen sulfate solution and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 1: 2 → 1: 5 → 1: 10) to give 17-
(4-azidophenyl) -18,19,20-trinorisocarbacycline 2.4 mg (68%; 15R, 15
A mixture of S form) was obtained.

Rf: 0.23 and 0.18 (1: 4 hexane / ethyl acetate) IR (cm ^-1 , CHCl ₃ ) 2108, 1712, 1605, 1506 ¹ H-NMR (δ ppm, CDCl ₃ , 270 MH)
z): 1.2-2.1 (m, 15H), 2.2-2.9 (m, 5H), 2.9-3.1 (m, 1
H), 3.76 (dd, 1H, J = 8.4, 16.8Hz), 4.0-4.3 (m, 1H), 5.29
(br.s, 1H), 5.4-5.7 (m, 2H), 6.95 (d, 2H, J = 8.1Hz), 7.18
(d, 2H, J = 8.4Hz).

[0095]

Example 12 Synthesis of 15-oxo-19- (3- (tritylamino) phenyl) -20-norisocarbacycline methyl ester

[0096]

[Chemical formula 26]

13,14-Dihydroxy-13,14-
Dihydroisocarbacycline 24.0 mg (0.06 mm
ol) is a mixed solvent of tetrahydrofuran and ether (1:
1) Dissolve in 1 ml and add sodium metaperiodate 1
40 mg (0.65 mmol) of 0.5 ml aqueous solution was added, and the mixture was stirred for 2 hours at room temperature. Then, water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and methyl-5-{(1S,
A crude product of 5S, 6R, 7R) -6-formyl-7-hydroxybicyclo [3.3.0] -2-octen-3-yl} pentanoate was obtained and directly used in the next reaction.

2-oxo-6 (3- (tritylamino)
Phenyl) hexyl sulfonate dimethyl 65.6 mg
(0.12 mmol) DME-toluene mixed solvent (7:
1, 1 ml) solution with 7 mg NaH (50% in oil)
(0.14 mmol) was added and stirred for 10 minutes. Then, a methyl-5-{(1S, 5S, 6R, 7R) -6-formyl-7-hydroxybicyclo [3.3.0] -2-octen-3-yl} pentanoate crude product in DME-toluene mixed solvent 7: 1; 1 ml) solution was added and stirred at room temperature for 15 minutes. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 3:
It was subjected to 1) to obtain 39.3 mg (84%) of 15-oxo-19- (4- (tritylamino) phenyl) -20-norisocarbacycline methyl ester.

¹ H-NMR (δppm, CDCl ₃ , 2
70MHz): 1.2-2.5 (m, 23H), 3.0-3.15 (m, 1H), 3.67
(s, 3H), 3.85-4.00 (m, 1H), 4.97 (br.s, 1H), 5.31 (s, 1
H), 6.1-6.2 (m, 3H), 6.37 (d, 1H, J = 7.9Hz), 6.72 (dd, 1H,
J = 15.8, 7.4Hz), 6.80 (t, 1H, J = 7.5Hz), 7.15-7.40 (m, 15
H).

[0100]

Example 13 Synthesis of 19- (3- (tritylamino) phenyl) -20-norisocarbacycline methyl ester

[0101]

[Chemical 27]

31.9 mg (0.041 mmol) of 15-oxo-19- (3- (tritylamino) phenyl) -20-norisocarbacycline methyl ester was added to 15.0 mg (0) of cerium (III) chloride heptahydrate. 0.04 mmol) in a methanol (0.8 ml) solution, sodium borohydride (2.0 mg, 0.053 mmol) was added thereto, and the mixture was stirred for 1 minute at room temperature. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. Obtained 19- (3-
(Tritylamino) phenyl) -20-norisocarbacycline methyl ester crude product 33.5 mg (15
The mixture of R and 15S) was used as it was in the next reaction.

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 1.20-2.50 (m, 24H), 2.95-3.10 (m, 1H),
3.66 (s, 3H), 3.65-3.85 (m, 1H), 3.95-4.10 (m, 1H), 4.85
-5.05 (br.s, 1H), 5.29 (s, 1H), 5.45-5.60 (m, 2H), 6.14
(s, 1H), 6.18 (d, 1H, J = 7.4Hz), 6.37 (d, 1H, J = 7.4Hz), 6.
81 (t, 1H, J = 7.9Hz), 7.15-7.40 (m, 15H).

[0104]

Example 14 19- (3-aminophenyl) -20-
Synthesis of norisocarbacycline methyl ester

[0105]

[Chemical 28]

33.5 mg (0.041) of the crude product of 19- (3- (tritylamino) phenyl) -20-norisocarbacycline methyl ester obtained in Example 13 was obtained.
mmol) to methanol (0.2 ml), ether (0.5 m)
l) dissolved in a mixed solvent of acetic acid-water (9: 1)
0.5 ml was added. After stirring at room temperature for 7 hours, the reaction was terminated by neutralizing with sodium hydrogen carbonate. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 1: 2 → 1: 5) to give 1
9- (3-aminophenyl) -20-norisocarbacycline methyl ester 6.6 mg (highly polar) and its 15S compound and (15R) -19- (3-aminophenyl)
13.3 mg of a mixture with -20-norisocarbacycline methyl ester (low polarity) was obtained. Total yield 19.9mg
(86% in 2 steps).

By subjecting the mixture to silica gel column chromatography, the 15R form (low polarity) 8.
0 mg, 15S isomer (highly polar) 2.5 mg and a mixture of these 0.9 mg were obtained.

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 15S body: 1.2-2.6 (m, 24H), 2.90-3.10
(m, 1H), 3.67 (s, 3H), 3.66-3.85 (m, 1H), 4.05-4.15 (m, 1
H), 5.28 (s, 1H), 5.45-5.65 (m, 2H), 6.45-6.65 (m, 3H),
7.06 (dd, 1H, J = 8.4,7.4Hz) .15R body: 1.20-2.60 (m, 24H), 2.95-3.10 (m, 1H), 3.67
(s, 3H), 3.66-3.80 (m, 1H), 4.05-4.15 (m, 1H), 5.28 (s, 1
H), 5.45-5.65 (m, 2H), 6.45-6.65 (m, 3H), 7.06 (dd, 1H, J
= 8.4, 7.4Hz).

[0109]

Example 15 19- (3-Azidophenyl) -20-
Synthesis of norisocarbacycline methyl ester

[0110]

[Chemical 29]

4.5 mg (0.010 mmol) of 19- (3-aminophenyl) -20-norisocarbacycline was added to ether (0.1 ml), methanol (0.5 ml) and acetic acid-water (9: 1; 0). 0.5 ml) and the reaction system was cooled to 0 ° C. Sodium nitrite 10mg here
(0.15 mmol) was added, and the mixture was stirred at 0 ° C for 15 minutes.
Then, 10 mg (0.15 mmol) of sodium azide was added, and the mixture was stirred at 0 ° C for additional 15 minutes. The reaction system was diluted with ethyl acetate, neutralized with a saturated aqueous sodium hydrogen carbonate solution, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 1: 1) to give 19-
4.1 mg (88%) of (3-azidophenyl) -20-norisocarbacycline methyl ester was obtained.

Rf: 0.28 (1: 2 hexane / ethyl acetate, silica gel) IR (cm ^-1 , CHCl ₃ ) 2210, 1730, 1604 ¹ H-NMR (δppm, CDCl ₃ , 270 MH)
z): 1.2-1.8 (m, 13H), 1.8-2.1 (m, 4H), 2.1-2.4 (m, 5
H), 2.61 (t, 2H, J = 7.7Hz), 2.9-3.1 (br, 1H), 3.67 (s, 3
H), 3.77 (dd, 1H, J = 8.9, 17.8Hz), 4.08 (dd, 1H, J = 5.7, 1
1.4Hz), 5.28 (s, 1H), 5.4-5.6 (m, 2H), 6.83 (s, 1H), 6.8
5 (d, 1H, J = 8.9Hz), 6.95 (d, 1H, J = 7.4Hz), 7.26 (t, 1H, J =
7.4Hz).

[0113]

Example 16 Synthesis of (15R) -19- (3-azidophenyl) -20-norisocarbacycline methyl ester

[0114]

[Chemical 30]

4.0 mg (0.0091 mmol) of (15R) -19- (3-aminophenyl) -20-norisocarbacycline methyl ester was treated with ether (0.1 m).
l), methanol (0.5 ml), acetic acid-water (9: 1;
0.5 ml) and the reaction system was cooled to 0 ° C. Sodium nitrite (10 mg, 0.15 mmol) was added thereto, and the mixture was stirred at 0 ° C for 15 minutes. Then, 10.0 mg (0.15 mmol) of sodium azide was added, and the mixture was further stirred at 0 ° C for 15 minutes. The reaction system was diluted with ethyl acetate, neutralized with a saturated aqueous sodium hydrogen carbonate solution, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The resulting oily matter was subjected to silica gel column chromatography (hexane:
Ethyl acetate = 1: 1), (15R) -19- (3
-Azidophenyl) -20-norisocarbacycline methyl ester 4.1 mg (96%) was obtained.

Rf: 0.14 (1: 2 hexane / ethyl acetate, silica gel) IR (cm ^-1 , CHCl ₃ ) 2210, 1730, 1604 ¹ H-NMR (δ ppm, CDCl ₃ , 270 MH)
z): 1.2-1.8 (m, 13H), 1.8-2.1 (m, 4H), 2.2-2.5 (m, 5
H), 2.61 (t, 2H, J = 7.7Hz), 2.9-3.1 (br, 1H), 3.67 (s, 3
H), 3.76 (ddd, 1H), 4.10 (dd, 1H, J = 5.4, 10.8Hz), 5.29
(s, 1H), 5.4-5.6 (m, 2H), 6.83 (s, 1H), 6.85 (d, 1H, J = 7.9
Hz), 6.95 (d, 1H, J = 7.9Hz), 7.26 (t, 1H, J = 7.9Hz).

[0117]

Example 17 19- (3-Azidophenyl) -20-
Synthesis of norisocarbacycline

[0118]

[Chemical 31]

19- (3-azidophenyl) -20-norisocarbacycline methyl ester 4.1 mg (0.
0.18 ml of 3N lithium hydroxide was added to a solution of methanol (0.5 ml), and the mixture was stirred at room temperature for 2.5 hours. The reaction system was acidified with an aqueous sodium hydrogen sulfate solution and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure to obtain a yellow oily substance 19- (3-azidophenyl) -20-norisocarbacycline 4.0 mg (100%).

Rf: 0.28 (1: 4 hexane / ethyl acetate) ¹ H-NMR (δ ppm, CDCl ₃ , 270 MH)
z): 1.2-1.7 (m, 13H), 1.8-2.2 (m, 4H), 2.2-2.4 (m, 5
H), 2.66 (t, 2H, J = 7.4Hz), 2.9-3.1 (br, 1H), 3.73 (ddd, 1
H, J = 6.9, 13.8, 13.8Hz), 4.03 (dd, 1H, J = 6.4, 12.8Hz),
5.33 (s, 1H), 5.4-5.6 (m, 2H), 6.91 (br.s, 2H), 7.04 (d, 1
H, J = 7.4Hz), 7.32 (t, 1H, J = 7.4Hz).

[0121]

Example 18 Synthesis of (15R) -19- (3-azidophenyl) -20-norisocarbacycline

[0122]

[Chemical 32]

(15R) -19- (3-Azidophenyl) -20-norisocarbacycline methyl ester 4.1 mg (0.0088 mmol) of methanol (0.5 m
l) To the solution, 0.1 ml of 3N lithium hydroxide was added and stirred at room temperature for 2.5 hours. The reaction system was acidified with an aqueous sodium hydrogen sulfate solution and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and the solvent was distilled off under reduced pressure to give a yellow oil (15R) -19- (3-azidophenyl) -20-norisocarbacycline 4.0 mg (1
00%).

Rf: 0.35 (1: 4 hexane / ethyl acetate) ¹ H-NMR (δ ppm, CDCl ₃ , 270 MH
z): 1.2-1.7 (m, 13H), 1.8-2.2 (m, 4H), 2.2-2.5 (m, 3
H), 2.33 (t, 2H, J = 7.4Hz), 2.66 (t, 2H, J = 7.4Hz), 2.9-3.
1 (br, 1H), 3.6-3.8 (m, 1H), 4.05 (dd, 1H, J = 5.4, 10.8H
z), 5.34 (s, 1H), 5.5-5.7 (m, 2H), 6.91 (br.s, 2H), 7.03
(d, 1H, J = 7.9Hz), 7.31 (dt, 1H, 1.8, 7.9Hz).

[0125]

Example 19 15-oxo-17-phenyl-18,
Synthesis of 19,20-trinorisocarbacycline methyl ester

[0126]

[Chemical 33]

To a solution of 5.0 mg (0.02 mmol) of dimethyl 2-oxo-4-phenylbutylphosphonate in DME-toluene mixed solvent (7: 1, 0.2 ml) was added NaH (50%).
in oil) 1 mg (0.021 mmol) was added, and the mixture was stirred for 20 minutes. Then methyl-5-{(1S, 5S, 6
R, 7R) -6-Formyl-7-hydroxybicyclo [3.3.0] -2-octen-3-yl} pentanoate 1.73 mg (0.0065 mmol) of DME-toluene mixed solvent (7: 1; 0) 0.2 ml) solution and add 1 at room temperature
Stir for 0 minutes. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate =
3: 2), 15-oxo-17-phenyl-1
1.8 mg (70%) of 8,19,20-trinorisocarbacycline methyl ester was obtained.

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 1.20-2.50 (m, 14H), 2.80-3.00 (m, 5H),
3.00-3.15 (m, 1H), 3.67 (s, 3H), 3.80-3.95 (m, 1H), 6.18
(d, 1H, J = 15.8Hz), 6.72 (dd, 1H, J = 15.8, 8.9Hz), 7.10-
7.40 (m, 5H).

[0129]

Example 20 Synthesis of 17-phenyl-18,19,20-trinorisocarbacycline methyl ester

[0130]

[Chemical 34]

15-oxo-17-phenyl-18,1
1.8 mg (0.0045 mmol) of 9,20-trinorisocarbacycline methyl ester was added to cerium chloride (III
) Heptahydrate (2.0 mg, 0.005 mmol) was dissolved in a solution of methanol (0.2 ml), sodium borohydride (0.2 mg, 0.005 mmol) was added thereto, and the mixture was stirred at room temperature for 5 minutes. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained 17-phenyl-18,
The crude product of 19,20-trinorisocarbacycline methyl ester (15R, 15S isomer mixture) was directly used in the next reaction.

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 1.20-2.50 (m, 18H), 2.60-2.85 (m, 2H),
2.90-3.10 (m, 1H), 3.66 (s, 3H), 3.65-3.85 (m, 1H), 4.05
-4.20 (m, 1H), 5.29 (s, 1H), 5.50-5.70 (m, 2H), 7.15-7.4
0 (m, 5H).

[0133]

Example 21 Synthesis of 17-phenyl-18,19,20-trinorisocarbacycline

[0134]

[Chemical 35]

17-Phenyl-1 obtained in Example 20
Crude product of 8,19,20-trinorisocarbacycline methyl ester (0.0045 mmol 15R, 15S
6N lithium hydroxide (0.02 ml) was added to a solution of the mixture (body mixture) in methanol (0.2 ml), and the mixture was stirred at room temperature for 16 hours. Acidify the reaction system with aqueous sodium hydrogensulfate,
Extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain 17 mg of 17-phenyl-18,19,20-trinorisocarbacycline (98% 15R, 15S mixture).

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 1.20-2.85 (m, 20H), 2.90-3.10 (m, 1H),
3.65-3.85 (m, 1H), 4.05-4.20 (m, 1H), 5.29 (s, 1H), 5.50
-5.70 (m, 2H), 7.15-7.40 (m, 5H).

[0137]

Example 22 15-oxo-19-phenyl-20-
Synthesis of trinorisocarbacycline methyl ester

[0138]

[Chemical 36]

13,14-Dihydroxy-13,14-
Dihydroisocarbacycline 24.0 mg (0.06 mm
ol) was dissolved in a mixed solvent of ether (1 ml) and methanol (0.1 ml), and sodium metaperiodate 26 was added thereto.
0 mg (1.21 mmol) of 0.8 ml aqueous solution was added, and the mixture was stirred at room temperature for 5 hours. Then, water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to remove methyl-5-{(1S, 5
A crude product of S, 6R, 7R) -6-formyl-7-hydroxybicyclo [3.3.0] -2-octen-3-yl} pentanoate was obtained. The obtained oily substance was directly used in the next reaction.

29.0 mg (0.10 mmol) of dimethyl 2-oxo-6-phenylhexylphosphonate in a DME-toluene mixed solvent (7: 1, 1 ml) was added to NaH (50%).
in oil) 4.8 mg (0.10 mmol) was added and 10
Stir for minutes. Then here methyl-5 obtained above
-{(1S, 5S, 6R, 7R) -6-Formyl-7-
Hydroxybicyclo [3.3.0] -2-octene-3
A solution of a crude product of -yl} pentanoate in a DME-toluene mixed solvent (7: 1, 1 ml) was added, and the mixture was stirred at room temperature for 15 minutes. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 5: 1 →
3: 1), 15-oxo-19-phenyl-20
-Trinorisocarbacycline methyl ester 19.
Obtained 4 mg (76%).

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 1.20-2.70 (m, 23H), 3.00-3.15 (m, 1H),
3.67 (s, 3H), 3.85-4.00 (m, 1H), 5.30 (s, 1H), 6.18 (d, 1
H, J = 15.8Hz), 6.76 (dd, 1H, J = 15.8, 8.4Hz), 7.10-7.35
(m, 5H).

[0142]

Example 23 Synthesis of 19-phenyl-20-norisocarbacycline methyl ester

[0143]

[Chemical 37]

15-oxo-19-phenyl-20-norisocarbacycline methyl ester 19.4 mg
(0.046 mmol) of cerium (III) chloride heptahydrate 1
7.0 mg (0.046 mmol) of methanol (0.5 ml)
Dissolve in solution, here 2.0 mg sodium borohydride
(0.053 mmol) was added, and the mixture was stirred for 1 minute at room temperature.
Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained 19-phenyl-20-norisocarbacycline methyl ester crude product (15R, 15S
The mixture of bodies was used as such for the next reaction.

¹ H-NMR (δppm, CDCl ₃ , 2
70MHz): 1.20-2.70 (m, 23H), 2.90-3.10 (m, 1H),
3.67 (s, 3H), 3.65-3.80 (m, 1H), 3.95-4.15 (m, 1H), 5.28
(s, 1H), 5.40-5.60 (m, 2H), 7.10-7.35 (m, 5H).

[0146]

Example 24 Synthesis of 19-phenyl-20-norisocarbacycline

[0147]

[Chemical 38]

19-Phenyl-2 obtained in Example 23
7 drops of 3N lithium hydroxide was added to a methanol (0.5 ml) solution of a crude product of 0-norisocarbacycline methyl ester (0.046 mmol of a mixture of 15R and 15S compounds), and the mixture was stirred at room temperature for 2 hours. The reaction system was acidified with an aqueous sodium hydrogen sulfate solution and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the resulting oily product was subjected to silica gel column chromatography (hexane: ethyl acetate = 1: 2 →
1: 5 → 1: 10), 14.5 mg of 19-phenyl-20-norisocarbacycline (76% two steps,
A mixture of 15R and 15S) was obtained.

¹ H-NMR (δ ppm, CDCl ₃ , 2
70MHz): 1.20-1.70 (m, 12H), 1.80-2.10 (m, 4H),
2.20-2.50 (m, 5H), 2.50-2.65 (m, 2H), 2.90-3.10 (m, 1H),
3.65-3.80 (m, 1H), 3.95-4.15 (m, 1H), 5.28 (s, 1H), 5.4
0-5.60 (m, 2H), 7.10-7.35 (m, 5H).

[0150]

Example 25 Synthesis of 15-oxo-19- (3-azidophenyl) -20-norisocarbacycline methyl ester

[0151]

[Chemical Formula 39]

13,14-Dihydroxy-13,14-
Dihydroisocarbacycline methyl ester 45.0
mg (0.11 mmol) of THF-ether mixed solvent (1:
1, 1.5 ml) solution with sodium metaperiodate 241
0.5 mg (1.1 mmol) of 0.5 ml aqueous solution was added, and the mixture was stirred for 1 hour at room temperature. Then, water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and methyl-5-{(1S, 5S,
A crude product of 6R, 7R) -6-formyl-7-hydroxybicyclo [3.3.0] -2-octen-3-yl} pentanoate was obtained and subjected to the next reaction.

Dimethyl 2-oxo-6 (3-azidophenyl) butylphosphonate 54.0 mg (0.165 mmol)
DME-toluene mixed solvent (7: 1, 2.0 ml) solution of NaH (50% in oil) 8 mg (0.16 mmo)
l) was added and stirred for 10 minutes. Then, the crude product of methyl-5-{(1S, 5S, 6R, 7R) -6-formyl-7-hydroxybicyclo [3.3.0] -2-octen-3-yl} pentanoate obtained above DME-
A toluene mixed solvent (7: 1, 0.5 ml) solution was added, and the mixture was stirred at room temperature for 10 minutes. Water was added to the reaction system and extraction was performed with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was subjected to silica gel column chromatography (hexane: ethyl acetate = 3: 1) to give 15-oxo-19- (3-azidophenyl) -20-norisocarbacycline methyl ester 47.2 mg (92%). ) Got.

¹ H-NMR (δppm, CDCl ₃ , 2
70MHz): 1.2-2.70 (m, 23H), 3.00-3.15 (m, 1H), 3.
67 (s, 3H), 3.85-4.00 (m, 1H), 5.30 (s, 1H), 6.18 (d, 1H, J
= 15.8Hz), 6.72 (dd, 1H, J = 15.8, 8.9Hz), 6.83 (s, 1H),
6.85 (d, 1H, J = 7.9Hz), 6.95 (d, 1H, J = 7.9Hz), 7.26 (t, 1H,
J = 7.9Hz).

[0155]

Example 26 [15- ³ H] -19- (3- azido-phenyl) -20-synthesis of nor-isocarbacyclin

[0156]

[Chemical 40]

19- (3-Azidophenyl) -15-oxo-20-norisocarbacycline methyl ester was treated with [ ³ H] NaBH ₄ -in the same manner as in Example 13.
Was treated with _{CeCl 3 · 7H 2 O (60Ci} / mmol), then purified by ^{HPLC [15- 3 H] -19- (} 3- azido-phenyl) -20-nor-isocarbacyclin methyl ester (66.7μCi, 4 .4 nmol) was obtained. This was made into a methanol (2 ml) solution, 5N sodium hydroxide (0.12 ml) was added thereto, and the mixture was stirred at 25 ° C. for 4 hours. After the reaction, 10 mg of sodium hydrogensulfate was added and pH was adjusted to 3 years. Water (4 ml) and ethyl acetate (3 ml) were added for extraction. The aqueous layer was extracted twice with ethyl acetate (1 ml), and the organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced ^{pressure, [15- 3 H] -19- (} 3
-Azidophenyl) -20-norisocarbacycline (66.4 μCi, 99.6%) was obtained.

[0158]

Example 27 The molecular weight of PGI ₂ receptor protein was identified by the photoaffinity labeling method as follows.

[0159] in the cell membrane fraction of cancerous mast cells PGI ₂ receptor protein there are many (mastocytoma P-815 cell), synthesized in Example 26 [15- ³ H] -19-
(3-Azidophenyl) -20-norisocarbacycline was added, incubated, and then irradiated with ultraviolet rays. When a dodecyl sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on the UV-irradiated body, a clear band could be recognized around its molecular weight of 43 K from its fluorography. The intensity of this band was reduced by the addition of GTPrS and almost completely disappeared by the addition of iloprost. From the above results, the molecular weight of the PGI ₂ receptor protein was identified as 43K.

[0160]

Example 28 Azidophenyl derivative (Examples 6, 1
1, 17, 18) was evaluated for binding ability to prostacyclin receptor protein (mastcytoma P-815 cells) as follows.

200 μg of cell membrane component of mastcytoma P-815 cells (Hashimoto et al., Prostagulandins 1990, 40,
491) with 1 mM EDTA and 10 mM MgCl ₂ (1
00 μL, pH 6.0) in K ₃ PO ₄ buffer, [
³ H] iloprost (20 nM, 0.0028 μCi)
And various concentrations of the azidophenyl derivative (Example No. 6,
11, 17, 18) and incubated at 30 ° C. for 60 minutes. After the incubation, the cells were quickly filtered through a glass filter (Whatman GF / C) and washed 4 times with the buffer solution (2 ml) using the filter. The radioactivity of the glass filter at this time represents the total amount of [ ³ H] iloprost bound. Non-specific binding of [ ³ H] iloprost is
The radioactivity of the glass filter when 10 μM iloprost was used under the same conditions as above corresponds to that. [
^The specific binding amount of [ ³ H] iloprost receptor to a protein is obtained by subtracting the nonspecific binding amount from the total binding amount. The binding ability of azidophenyl derivatives to prostacyclin receptor proteins is [ ³
It was evaluated by measuring the inhibitory activity of the present compound on the binding of [H] iloprost to the receptor. As a result of the evaluation by this evaluation system, the compound [19- (3
-Azidophenyl) -20-norisocarbacycline] has the strongest binding ability.
(IC50 3nM)

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical indication location C07C 247/12 9160-4H (72) Inventor Manabu Negishi Gokasho Nishida 3-1, Grand, Uji, Kyoto Prefecture Domour Ujikinawa 413 (72) Inventor Hitoshi Hashimoto 3-11-2-103 Sakuranocho, Toyonaka City, Osaka Prefecture (72) Inventor Atsushi Ichikawa 10-1 Besshohonmachi, Takatsuki City, Osaka Prefecture -608 (72) Inventor Ito Seiji 5-18-7 Nomura, Kusatsu City, Shiga Prefecture

Claims (7)

[Claims] 1. The following formula [I]: [In the formula, R ¹ represents a hydrogen atom or a C ₁ -C ₅ lower alkyl group, X represents a hydrogen atom or a tritium atom, and Y represents a hydrogen atom, an azido group, a trityl-protected amino group or an amino group. And n represents 1 to 7. ] The compound represented by], its 15 epimer, and the isocarba cyclin derivative which is a mixture of those enantiomers or their arbitrary ratios. 2. The isocarbacycline derivative according to claim 1, wherein R ¹ is a hydrogen atom or a methyl group. 3. The isocarbacycline derivative according to claim 1, wherein n is 2 to 4. 4. The isocarbacycline derivative according to claim 1, wherein Y is an azido group. 5. The isocarbacycline derivative according to claim 1, wherein Y is a hydrogen atom. 6. The following formula [II]: [In the formula, Ya represents a hydrogen atom, a trityl-protected amino group or an azido group, and n represents 1 to 7. And a Horner-Emmons reagent represented by the following formula [III]: ## STR3 ## wherein R ² represents a C ₁ -C ₅ lower alkyl group. ] The compound represented by the following formula [IV]: [In the formula, R ² , n, and Ya are the same as defined above. ] Then, a compound represented by the following formula (1) is obtained, and then subjected to a reduction reaction, a deprotection reaction of a trityl-protected amine if necessary, an azidation reaction of an amine if necessary, and a hydrolysis reaction if necessary, Formula [I] [In the formula, R ¹ represents a hydrogen atom or a C ₁ -C ₅ lower alkyl group, X represents a hydrogen atom or a tritium atom, and Y represents a hydrogen atom, an azido group, a trityl-protected amino group or an amino group. And n represents 1 to 7. ] The manufacturing method of the compound represented by these, its 15 epimer, its enantiomer, or the isocarba cyclin derivative which is a mixture of them in arbitrary ratios. 7. The following formula [I ′]: [In the formula, R ¹ represents a hydrogen atom or a C _{1 to} C ₅ lower alkyl group, X represents a hydrogen atom or a tritium atom, Yb represents an azide group, and n represents 1 to 7. ] The photoaffinity labeling method of the prostacyclin receptor protein characterized by using the isocarbacycline derivative represented by these.