JP5196736B2 - Apoptosis inducer and antitumor agent containing the same - Google Patents

Description

The present invention relates to an apoptosis-inducing agent and a medicament containing the same. More specifically, the present invention relates to an apoptosis-inducing agent suitable for treatment and / or amelioration of diseases in which a decrease or increase in the apoptotic system of a living body is considered to be involved in the onset of disease and progression of the disease, and a medicine containing the same.

Apoptosis is “natural death of cells”, ie, active cell death programmed in advance, and is a phenomenon different from necrosis (necrosis), which is passive cell death.
Once apoptotic signals are activated, complex and diverse biochemical reactions occur in the cells, producing various proteins and DNA-degrading enzymes that act on their own cells and cause cell death. In other words, apoptosis is not just a phenomenon of cell collapse, but is a physiological cell death that is indispensable for the maintenance of the life of an individual. In order to control not only the body shape in the development process but also the cellular society in mature individuals It plays an important role.

In recent years, it has become clear that apoptosis is involved in the onset and progression of various diseases due to advances in life science (see Non-Patent Document 1 and Non-Patent Document 2).
Specifically, diseases caused by decreased apoptosis include malignant tumors (follicular lymphoma, cancer with p53 mutation, breast cancer, prostate cancer, ovarian cancer, etc.), autoimmune diseases (systemic lupus erythematosus, certain threads) Examples include glomerulonephritis, rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, psoriasis), and viral infections (infections caused by herpes virus, adenovirus, poxvirus, etc.). On the other hand, diseases caused by increased apoptosis include AIDS, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration, polyglutamine disease, prion disease, etc.) And ischemic diseases (myocardial infarction, stroke, etc.).

  In recent years, research to develop and establish new drugs and therapeutic methods by analyzing the apoptotic phenomenon in these diseases at the molecular level and grasping the essence has been actively carried out all over the world.

However, many of the above-mentioned apoptosis-related diseases are still lacking effective drugs and treatment / prevention methods.
In particular, in malignant tumors (hereinafter also simply referred to as cancer), multifaceted efforts including prevention and screening methods have been undertaken in order to overcome them, but from 1981 to the present, It accounts for the number one cause of human death. Surgical therapy, radiation therapy, chemotherapy, immunotherapy, and combinations thereof are used as cancer treatment methods.

Among these, antitumor agents (hereinafter also referred to as anticancer agents) used for chemotherapy are classified into “cytotoxic anticancer agents” and “molecular target therapeutic agents” depending on the mechanism and origin.
However, although these “cytotoxic anticancer agents” act on cancer cells with certainty, there is a problem in that normal cells are also damaged to cause side effects, so there is a limitation in dosing and the expected effect cannot be brought out. Recently, “molecular target therapeutic drugs” (drugs that act on target molecules with high specificity for cancer) have been used in part, but side effects have been observed and are still not sufficient.

Furthermore, although many of these anticancer agents have apoptosis-inducing activity, in some cases, cancer cell populations that are resistant to apoptosis appear and allow proliferation. In order to deal with this, there is a method of analyzing the mechanism of apoptosis in more detail and searching for a target molecule, but there are many unknown parts.

Therefore, at present, in order to develop highly effective anticancer agents with fewer adverse drug reactions, search for compounds that do not damage normal cells, have high apoptosis activity against cancer cells, and are suitable as active ingredients of apoptosis inducers Is considered effective.
Edited by Hideaki Enomoto, Masayuki Miura, “A New Challenge in Apoptosis Research”, Experimental Medicine vol. 19, No. 13 (extra number), Yodosha, 2001, p.1764-1787 Edited by Haruki Suma, “Apoptosis Experiment Protocol”, separate volume of cell engineering, Shujunsha, 1994, p.26-30

  An object of the present invention is to provide a novel apoptosis inducer capable of selectively inducing apoptosis of pathogenic cells such as cancer and a medicament containing the same.

  As a result of intensive studies to solve the above problems, the present inventors have found that lactone derivatives and cyclic ketone derivatives having a specific structure have selective and powerful apoptosis-inducing activity against cancer cells. The invention has been completed.

That is, the present invention relates to the following matters, for example.
The first apoptosis inducer according to the present invention is characterized by containing a compound represented by the following formula (1) as an active ingredient:

(In formula (1), R represents an alkyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 3).
In the present invention, the apoptosis inducer preferably contains a compound in which n is 1 or 3 in the formula (1) as an active ingredient.

Further, in the present invention, the compound represented by the formula (1) is (11E, 13R) -10-oxo-11-octadecene-13-olide, (11E, 13S) -10-oxo-11-octadecene-13- It is at least one compound selected from the group consisting of orido, (13E, 15R) -12-oxo-13-octadecene-15-olide, and (13E, 15S) -12-oxo-13-octadecene-15-olide It is preferable.

  Further, the second apoptosis inducer according to the present invention is characterized by containing a compound represented by the following formula (2) as an active ingredient:

(In formula (2), Y represents OR ¹ or SR ¹ , R ¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and m represents an integer of 1 to 3)
The apoptosis-inducing agent of the present invention preferably contains, as an active ingredient, a compound in which Y is OR ¹ and R ¹ is a hydrogen atom in the formula (2).

  Further, in the present invention, the compound represented by the formula (2) is (R) -1- (2-oxycyclopentylidene) -2-decanol, (S) -1- (2-oxycyclopentylidene)- It is preferably at least one compound selected from the group consisting of 2-decanol.

  The medicament according to the present invention is characterized by containing the apoptosis inducer. In the present invention, the medicament is preferably used as an antitumor agent.

According to the apoptosis-inducing agent of the present invention, apoptosis can be selectively induced on pathogenic cells such as cancer and the growth of the pathogenic cells can be suppressed. Therefore, treatment and / or prevention of diseases caused by the cells The effect can be expected. Therefore, the medicament of the present invention containing the apoptosis inducer is a disease in which an abnormal decrease in the apoptosis system of pathogenic cells or an abnormal increase in the apoptosis system of normal cells is considered to be involved in the onset of disease and progression of disease ( Cancer, leukemia, autoimmune diseases, viral infections, neurodegenerative diseases, etc.), and is useful as an antitumor agent.

Hereinafter, the present invention will be specifically described.
The first apoptosis inducer of the present invention contains a compound represented by the following formula (1) as an active ingredient.

  Therefore, first, the compound represented by the following formula (1) will be described.

In formula (1), R represents an alkyl group having 1 to 6 carbon atoms. Specific examples of the alkyl group include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, Examples thereof include linear or branched alkyl groups such as isopentyl group, tert-pentyl group, neopentyl group, 2-methylbutyl group, 1,2-dimethylpropyl group, n-hexyl group and isohexyl group. Among these, from the viewpoint of improving the fat solubility of the compound, the straight chain having 1 to 6 carbon atoms such as methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, etc. A chain alkyl group is preferable, a linear alkyl group having 3 to 5 carbon atoms such as an n-propyl group, an n-butyl group, and an n-pentyl group is more preferable, and an n-propyl group or an n-pentyl group is further preferable.

Furthermore, in formula (1), n represents an integer of 1 to 3, and is preferably 1 or 3.
As is clear from the definition of the above formula (1), the compound has an asymmetric carbon atom. Therefore, the compound has optical isomers such as R-form, S-form, and racemate (hereinafter sometimes referred to as (+/-)). In the present invention, the compound is an optical isomer of the optical isomer. Any of these may be used regardless of the type. Moreover, these optical isomers may be used individually by 1 type, and may be used in combination of 2 or more type.

Specific examples of the compound represented by the above formula (1) (lactone derivative) include, for example, a compound in which n is 1 and R is an n-propyl group; (11E, 13R) -10-oxo-11-hexadecene -13-
Orido, (11E, 13S) -10-oxo-11-hexadecene-13-olido:
Compounds in which n is 1 and R is an n-pentyl group; (11E, 13R) -10-oxo-11-octadecene-13-olide, (11E, 13S) -10-oxo-11-octadecene-13-olide:
Compounds in which n is 3 and R is an n-propyl group; (13E, 15R) -12-oxo-13-octadecene-15-olide, (13E, 15S) -12-oxo-13-octadecene-15-olide:
Compounds in which n is 3, and R is an n-pentyl group; (11E, 13R) -10-oxo-11-dodecene-13-olide, (11E, 13S) -10-oxo-11-dodecene-13-olide: Etc. In addition, the racemic compound of the specific compound illustrated above is mentioned similarly.

Of these, (11E, 13R) -10-oxo-11-octadecene-13-olide, (11E, (13S) -10-oxo-11-octadecene-13-olide, (13E, 15R) -12-oxo-13-octadecene-15-olide, (13E, 15S) -12-oxo-13-octadecene-15-olide Etc. are more preferable.

The compounds mentioned above can be used singly or in combination of two or more.
In addition, the compound shown by the said Formula (1) is a well-known compound, The manufacturing method is also well-known. For example, in the formula (1), a compound in which n is 1 and R is an n-pentyl group can be isolated from an aqueous extract of corn, Matsushita et al.,
"Enantioselective syntheses of 10-oxo-11 (E) 0ctadecen-13-olide and related fatty
acid ", Tetrahedron Lett., 38 (1997) p.6055-6058. It can also be synthesized from linoleic acid according to the following synthesis scheme. In the following synthesis scheme, Ph is phenyl. Group, MEM is 2-methoxyethoxymethyl group, OTHP is tetrahydropyranyloxy group, Ac is acetyl group, DMAP is 4-dimethylaminopyridine, Et is ethyl group, PPTS
Represents pyridinium p-toluenesulfonate.

  Similarly, in the formula (1), a compound in which n is 3 and R is an n-propyl group can be synthesized according to the following synthesis scheme.

  The second apoptosis-inducing agent of the present invention contains a compound represented by the following formula (2) as an active ingredient. As is clear from the definition of the following formula (2), since the compound has an asymmetric carbon atom, the compound has optical isomers such as R-form, S-form, and racemate. Any of these may be used. Moreover, these optical isomers may be used individually by 1 type, and may be used in combination of 2 or more type.

  Hereinafter, the compound of the following formula (2) (cyclic ketone derivative) will be specifically described.

In formula (2), Y represents OR ¹ or SR ¹ , and R ¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec
Straight chain such as -butyl group, tert-butyl group, n-pentyl group, isopentyl group, tert-pentyl group, neopentyl group, 2-methylbutyl group, 1,2-dimethylpropyl group, n-hexyl group, isohexyl group, etc. Examples include branched alkyl groups.

Among these, from the viewpoint of improving the lipophilicity of the compound, R ¹ has 1 to 6 carbon atoms such as methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, and n-hexyl group. Are preferably a linear alkyl group or a hydrogen atom, more preferably a hydrogen atom. Furthermore, in said formula (2), m represents the integer of 1-3, Preferably it is 3.

As a specific and preferred example of the compound represented by the above formula (2) when Y represents OH or SH in the above formula (2), for example, a compound in which Y is OH and m is 1; (R) -1- (2-oxycyclopentylidene) -2-octanol, (S) -1- (2-oxycyclopentylidene) -2-octanol:
Compounds in which Y is OH and m is 2; (R) -1- (2-oxycyclopentylidene) -2-nonanol, (S) -1- (2-oxycyclopentylidene) -2-nonanol:
Compounds in which Y is OH and m is 3; (R) -1- (2-oxycyclopentylidene) -2-decanol, (S) -1- (2-oxycyclopentylidene) -2-decanol:
Compounds in which Y is SH and m is 1; (R) -1- (2-oxycyclopentylidene) -2-octanethiol, (S) -1- (2-oxycyclopentylidene) -2-octanethiol :
Compounds in which Y is SH and m is 2; (R) -1- (2-oxycyclopentylidene) -2-nonanethiol, (S) -1- (2-oxycyclopentylidene) -2-nonanethiol :
Compounds in which Y is SH and m is 3; (R) -1- (2-oxycyclopentylidene) -2-decanethiol, (S) -1- (2-oxycyclopentylidene) -2-decanethiol : And so on. In addition, the racemic compound of the specific compound illustrated above is mentioned similarly.

Among these, (R) -1- (2-oxycyclopentylidene) -2-decanol, (S) are excellent in terms of growth-inhibitory activity and apoptosis-inducing activity against cancer cells, particularly human leukemia cell line HL-60. ) -1- (2-oxycyclopentylidene) -2-decanol is more preferred.

The compounds mentioned above can be used singly or in combination of two or more.
The compound represented by the above formula (2) is a known compound, and its production method is also known.
For example, in the formula (2), a compound in which Y represents OH and m represents an integer of 1 to 3 is Ryozo Irye et al., “Synthesis of 2- (2-hydroxyalkylidene)
cyclopentanones and their bio-antimutagenic activity ", Biosci. Biotech. Biochem., 59 (3), 1995, p. 401-407. They can also be synthesized according to the following synthesis scheme.

  The amount of the compound represented by the formula (1) contained in the first apoptosis-inducing agent of the present invention and the amount of the compound represented by the formula (2) contained in the second apoptosis-inducing agent of the present invention were: The amount of the apoptosis-inducing activity is not particularly limited as long as it is an amount capable of obtaining the apoptosis-inducing activity for the pathogenic cells for the purpose of inducing apoptosis. Usually, the amount is 10 to 100% by mass in the total amount of the apoptosis-inducing agent. .

  That is, in the present invention, the compound represented by the formula (1) or the compound represented by the formula (2) may be used as an apoptosis-inducing agent per se, or according to the intended dosage form. In addition to the above compound, a composition containing a known additive or a known solvent used for normal formulation may be used as an apoptosis inducer.

  Moreover, the apoptosis inducer described above can be used as a medicament containing the compound represented by the formula (1) or the compound represented by the formula (2) as an active ingredient.

Since the compound is suitable as an apoptosis inducer for selectively inducing apoptosis of pathogenic cells and treating and / or preventing diseases caused by the cells, the compound contains the compound as an active ingredient. A medicine, ie, a medicine containing the apoptosis inducer, is a disease in which abnormal decrease in the apoptosis system of pathogenic cells or abnormal increase in the apoptosis system of normal cells is considered to be involved in the onset of disease or progression of disease (cancer, Leukemia, autoimmune diseases, viral infections, neurodegenerative diseases, etc.).

When the apoptosis-inducing agent is used as a pharmaceutical, either an orally or parenterally administered agent may be used, and the dosage form is not particularly limited, but is usually used in the form of a general pharmaceutical preparation. Such a preparation is prepared using, in addition to the active ingredient, a diluent or excipient such as a filler, a filler, a binder, a moistening agent, a disintegrant, a surfactant, a lubricant, etc., which are usually used. If necessary, carriers, stabilizers, absorption enhancers, etc. may be added and administered orally as tablets, powders, granules, capsules, syrups, etc., or suppositories, injections, You may administer parenterally as an external preparation, a drip, etc. Furthermore, the apoptosis-inducing agent and medicament of the present invention may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other medicines as necessary.

  The amount of the apoptosis derivative contained in the medicament of the present invention is not particularly limited, and is the amount of the compound represented by the formula (1) or the compound represented by the formula (2) contained in the apoptosis derivative as an active ingredient. Any amount that is suitable for managing the daily dose described below may be used.

  The dose of the medicament of the present invention varies depending on the weight, age, medical condition, etc. of the patient to be administered, but the amount of the active ingredient is usually 2 to 5,000 mg per day, divided into 1 to several times. It is good to administer.

EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited to these Examples.
In the following examples, the NMR spectrum is Bruker's DRX-500 spectrometer (500.15 MHz for ¹ H, and 125.75 MHz for ¹³ C) with TMS (tetramethylsilane) as an internal standard and Bruker's pulse program and data processing. Measured using the system. The MS spectrum was measured using a JEOL JEOL data processing system using a JEOL Ltd. JMS 700 spectrometer.

[Synthesis Example 1]
<(11E, 13R)-(+)-10-oxo-11-octadecene-13-olide, (11E, 13S)-(-)-10-oxo-11-octadecene-13-olide, and racemic (11E ) -10-Oxo-11-octadecene-13-olide (Synthesis of compounds (10-R), (10-S) and ((+/-)-10))>
(I) (13R) -12-hydroxy-13-[(R) -O-MEM-mandelyloxy-1-tetrahydropyra
Nyloxy] -10-octadecanone (3-R), (13S) -12-hydroxy-13-[(R) -O-MEM-mandelyloxy-1-tetrahydropyranyloxy] -10-octadecanone (3-S) Composition

Synthesis of (ia) (13R) -12-hydroxy-13-[(R) -O-MEM-mandelyloxy-1-tetrahydropyranyloxy] -10-octadecanone (3-R)
N-isopropylcyclohexylamine (1.03 g (7.3 mmol)) was added in THF (2 ml) and n-
To a solution of lithium N-isopropylcyclohexylamide (LICA) prepared by reacting with butyllithium (n-BuLi) (0.468 g, 7.3 mmol), 1-tetrahydropyranyloxy-10-undecanone ((2), 0.986 g, 3.65 mmol) in THF (3 ml) was added dropwise at −78 ° C. and stirred as such for 1 hour.

Then (R) -2-[(R) -O-MEM-mandelyloxy] -heptanal ((1-R), 1.28g, 3.65 m
mol) was dissolved in THF (3 ml) and slowly added dropwise. The mixture was stirred at -78 ° C for 30 minutes, and acetic acid (1 ml) was added to stop the reaction. The reaction product was extracted with ethyl acetate and saturated brine, and the ethyl acetate layer was washed with 2N hydrochloric acid, saturated aqueous sodium bicarbonate and saturated brine in that order, and dried over magnesium sulfate.

Then, the solvent was distilled off to obtain (13R) -12-hydroxy-13-[(R) -O-MEM-mandelyloxy] -1-tetrahydropyranyloxy-10-octadecanone ((3-R), 1.22 g , 2.0 mmol, 94% yield
)

(1-R): ¹ H NMR (CDCl ₃ ) δ: 0.87 (3H, t, J = 6.8 Hz, H7), 1.20-1.33 (6H, m, H4-H6), 1.71 (1H, bhex, J = 7.5 Hz, H3), 1.81 (1H, m, H3), 3.37 (3H, s, H6 '), 3.51-3.55 (2H, m, H5'), 3.69 (1H, ddd, J = 10.4, 5.6, 3.9 Hz, H4 '), 3.81 (1H, ddd, J = 10.5, 5.4, 4.0 Hz, H4'), 4.82 (1H, d, J = 7.1 Hz, H3 '), 4.91 (1H, d, J = 7.1 Hz , H3 '), 5.00 (1H, dd, J = 7.1, 3.0 Hz, H2), 5.34 (1H, s, H2'), 7.38 (3H, dt, H9'-H11 '), 7.49 (2H, d, J = 6.7 Hz, H7 '&H12'), 9.31 (1H, s, H1) ¹³ C NMR (CDCl ₃ ) δ: 13.88 (C7), 22.31 (C6), 24.39 (C4), 28.71 (C5), 31.29 ( C3), 58.97 (C6 '), 67.62 (C4'), 71.64 (C5 '), 76.71 (C2'), 78.89 (C2), 94.23 (C3 '), 127.45 (C9'& C11 '), 128.72 (C8'& C12 '), 128.94 (C10'), 135.89 (C7 '), 170.59 (C1'), 197.90 (C1) [α] _D = -26.7 ° (c 0.25, EtOH)
(3-R): NMR (CDCl ₃ ) δ _H : 0.75 (3H, t, J = 7.0Hz, 18), 0.88-1.4 (18H, 2, 3, 4, 5, 6, 14, 15, 16, 17), 1.4-1.6 (8H, 7, THP), 1.69, 1.83 (1H, m, 8), 2.38 (2H, t, J = 7.4 Hz, 9), 2.45-2.55 (2H, m, 11), 3.35 (3H, s, MEM), 3.38, 3.73 (1H, dt, 1), 3.45-3.52 (3H, m, MEM), 3.68 (1H, ddd, J = 11.2, 5.9 & 3.4 Hz, MEM), 3.77 (1H, ddd, J = 10.8, 6.0 & 3.3 Hz, MEM), 3.87 (1H, m, THP), 4.1 (1H, qint, J = 5.4 Hz, 12), 4.57 (t, J = 4.1 Hz, THP ), 4.79, 4.86 (1H, AB type, J = 7.1 Hz, MEM), 4.79-4.86 (m, 13), 5.23 (1H, s, mandelyl), 7.25-7.4 (3H, m, madelyl), 7.45 ( 2H, dd, J = 8 & 1.4 Hz,
δ _C : 13.86 (18), 19.70 (THP), 22.31 (17), 23.48 (8/3), 24.34 (3/8), 25.54 (THP), 26.23 (14), 29.13, 29.33, 29.39 , 29.42, 29.76, 29.89 (2, 4, 5, 6, 7, 15), 30.81 (THP), 31.40 (16), 43.72 (9), 44.66 (11), 58.91 (OMe), 62.29 (THP), 67.52 (1), 67.63 (MEM), 68.79 (12), 71.69 (MEM), 76.77 (13), 76.94 (mandelyl), 94.07 (MEM), 98.84 (THP), 127.44 (x2), 128.51 (x2), 128.73 (mandelyl), 136.33 (mandelyl), 170.57 (mandelyl), 211.18 (mandelyl) .HR-MS m / z: C ₃₅ H ₅₈ O ₉ : Calcd. For 622.4081; Measured, 622.4125.
Synthesis of (ib) (13S) -12-hydroxy-13-[(R) -O-MEM-mandelyloxy-1-tetrahydropyranyloxy] -10-octadecanone (3-S)
Using LICA solution (1.48 mmol) and 200 mg (0.74 mmol) of compound (2), (S) -2-[(R) -O-MEM-mandelyloxy] heptanal ((1-S), 260 mg, 0.74 (13S) -12-Hydroxy-13-[(R) -O-MEM-mandelyloxy] -1-tetrahydropyranyloxy-10, except that mmol) was used in the same manner as (ia) above. -Octadecanone ((3-S), 429 mg, 0.69 mmol, yield 93%) was obtained.

(1-S): ¹ H NMR (CDCl ₃ ) δ: 0.79 (3H, t, J = 7.0 Hz, H7), 1.02-1.20 (6H, m, H4-H6), 1.58-1.67 (1H, m, H3), 1.67-1.78 (1H, m, H3), 3.37 (3H, s, H6 '), 3.51 (2H, dt, J = 3.9, 5.5 Hz, H5'), 3.68 (1H, ddd, J = 11.0 , 5.7, 3.6 Hz, H4 '), 3.81 (1H, ddd, J = 10.8, 5.6, 3.5 Hz, H4'), 4.82 (1H, d, J = 7.1 Hz, H3 '), 4.89 (1H, d, J = 7.1 Hz,
H3 '), 4.99 (1H, dd, J = 4.4 Hz, H2), 5.36 (1H, s, H2'), 7.36 (3H, m, H9'-H11 '), 7.47 (2H, d, J = 6.4 Hz, H7 '&H12'), 9.52 (1H, s, H1) ¹³ C NMR (CDCl ₃ ) δ: 13.79 (C7), 22.22 (C6), 24.13 (C4), 28.56 (C5), 31.07 (C3), 58.97 (C6 '), 67.64 (C4'), 71.64 (C5 '), 76.57 (C2'), 78.74 (C2), 94.19 (C3 '), 127.47 (C9'& C11 '), 128.69 (C8'& C12 ') , 128.91 (C10 '), 135.97 (C7'), 170.54 (C1 '), 197.97 (C1) [α] _D = -53.0 ° (c 0.17, EtOH)
(Ii) (R) -13-acetoxy-10-oxo-1-tetrahydropyranyloxy-trans-11-octadecene (5-R), (S) -13-acetoxy-10-oxo-1-tetrahydropyranyl Oxy-trans-11-
Synthesis of octadecene (5-S)

(Ii-a) (R) -13-acetoxy-10-oxo-1-tetrahydropyranyloxy-trans-11-octadecene (5-R) compound (3-R) 1.26 g (2.02 mmol) mixed It melt | dissolved in the solvent (dimethylformamide (DMF) 30 ml-hexamethylphosphoric triamide (HMPA) 10 ml), sodium acetate (1.66 g, 20.2 mmol) was added, and it stirred at 55 degreeC for 16 hours. The reaction mixture was extracted with ethyl acetate and saturated brine, and the ethyl acetate layer was washed with 2N hydrochloric acid, saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over magnesium sulfate.

Thereafter, the solvent was distilled off to obtain an oily substance.
The oil was purified using silica gel chromatography (silica gel 4 g, eluent hexane: EtOAc = 7: 3) to give pure (R) -13-hydroxy-10-oxo-1-tetrahydropyranyloxy-trans- 11-octadecene ((4-R), 770 mg, 2.02 mmol, yield 100%) was obtained.

[α] _D = -6.25 ° (c = 0.70, EtOH). NMR (CDCl ₃ + Pyr.-d ₅ ) δ _H : 2.53 (2H, t, J = 7.4Hz, 9), 3.37 (1H, dt, J = 9.6 & 6.7Hz, 1), 3.73 (1H, dt, J = 9.6 & 6.7Hz, 1), 4.32 (1H, q, J = 6.1 Hz, 13), 4.54 (1H, t, J = 4.1Hz , THP), 6.32 (1H, dd, J = 15.9 & 1.5Hz, 11), 6.82 (1H, dd, J = 15.9 & 5.0Hz, 12); δ _C : 40.77 (9), 67.68 (1), 71.02 (13), 127.97 (11), 148.46 (12), 200.87 (10) .HR-MS: Calcd.for C ₂₃ H ₄₀ O ₃ (M ⁺ -H ₂ O),
364.2977; Measured, 364.2971.
The optical purity of the compound (4-R) is the NMR spectrum of the MTPA (α-methoxy-α- (trifluoromethyl) phenylacetic acid) ester (prepared using (R)-(-)-MTPA chloride) (δ _H 6.05 (0.4124H, d, J = 15.8Hz, H-11 of (R) -form), 6.22 (0.0396H, d, J = 15.8Hz, H-11 of
(S) -form), 6.48 (0.4153H, dd, J = 15.9 & 5.5 Hz, H-12 of (R) -form), 6.68 (0.0356H, dd, J = 15.9 & 6.0 Hz, H-12 of (S) -form). As a result, the optical purity of the R-type was 83.34%.

  Compound (4-R) 225 mg (0.59 mmol) was acetylated with acetic anhydride (2 ml) and pyridine (5 ml) to give (R) -13-acetoxy-10-oxo-1-tetrahydropyranyloxy-trans- 11-octadecene ((5-R), 250 mg, 0.59 mmol, yield 100%) was obtained.

NMR (CDCl ₃ + Pyr.-d ₅ ) δ _H : 2.22 (3H, s, OAc), 5.40 (1H, q, J = 5.5 Hz, 13) .δ _C : 21.05 (OAc), 72.70 (13), 170.15 (COO), 200.23 (CO). HR-MS: Calcd. For C ₂₅ H _4
3 O ₅ (M ⁺ -H), 423.3110; Measured, 423.3093.
(Ii-b) (S) -13-Acetoxy-10-oxo-1-tetrahydropyranyloxy-trans-11-octadecene (5-S) compound (3-S) 429 mg (0.69 mmol) was used Otherwise, (S) -13-hydroxy-10-oxo-1-tetrahydropyranyloxy-trans-11-octadecene ((4-S), 255
mg, 0.668 mmol, 97% yield, [α] _D = + 6.5 ° (c = 2.0, EtOH)). The purification was performed by silica gel column chromatography (silica gel 4 g, eluent hexane: EtOAc = 7: 3).

The NMR spectrum of the compound (4-S) was consistent with the NMR spectrum of the compound (4-R).
The optical purity of the compound (4-S) was 85.6% from the NMR measurement of MTPA ester prepared from (R)-(+)-MTPA chloride.

The compound (4-S) 315 mg (0.82 mmol) was then acetylated to give (S) -13-acetoxy-10-oxo-1-tetrahydropyranyloxy-trans-11-octadecene ((5-S), 348 mg, 0.82 mmol, 100% yield).

(Iii) (R) -13-acetoxy-10-oxo-trans-11-octadecenoic acid (6-R), (S) -13-acetoxy-10-oxo-trans-11-octadecenoic acid (6-S) Synthesis of

(Iii-a) Synthesis of (R) -13-acetoxy-10-oxo-trans-11-octadecenoic acid (6-R) Compound (5-R) 225 mg (0.53 mmol) in acetone (5 ml) at room temperature Jones oxidation (40 minutes). Subsequently, 2-propanol was added to the reaction solution to stop the reaction, and the reaction product was extracted with ethyl acetate and saturated brine. The organic layer was washed with 2N sulfuric acid and saturated brine in this order. The solvent was distilled off and (R) -13-acetoxy-10-oxo-trans-11-octadecenoic acid ((6-R), 188 mg, 0.53 mmol,
Yield 100%).

[α] _D = -16.2 ° (c = 0.30, EtOH) NMR (CDCl ₃ + Pyr.-d ₅ ) δ _H : 2.03 (2H, q, J = 7.3 & 3.9Hz, 14), 2.10 (3H, s , Ac), 2.35 (2H, t, J = 7.6Hz, 2), 2.51 (2H, dt, J = 14.1 & 7.3Hz, 9), 5.39 (1H, q, J = 9.2 & 5.5Hz, 13), 6.19 (1H, dd, J = 16.0 & 1.4Hz, 11), 6.68 (1H, dd, 16.0 & 5.4Hz, 12); δ _C : 20.36 (Ac), 33.64 (14), 34.41 (2), 40.73 ( 9),
72.72 (13), 129.26 (11), 143.01 (12), 170.20 (Ac), 177.32 (1), 191.04 (10).
HR-MS: Calcd.for C ₂₀ H ₃₅ O ₄ (M ⁺ -H), 339.2535; Measured, 339.2545.
(Iii-b) (S) -13-Acetoxy-10-oxo-trans-11-octadecenoic acid (6-S) (5-S) (315 mg, 0.743 mmol) iii-a), (S) -13-acetoxy-10-oxo-trans-11-octadecenoic acid ((6-S), 262 mg, 0.74 mmol, 100% yield, [α] _D = + 13.0 ° (c = 0.33, EtOH)).

The NMR spectrum of compound (6-S) was consistent with the NMR spectrum of compound (6-R).
(iv) (R) -13-acetoxy-10,10-ethylenedioxy-trans-11-octadecenoic acid (7-R), (S) -13-acetoxy-10,10-ethylenedioxy-trans-11 -Octadecenoic acid (7-S) synthesis

(iv-a) (R) -13-Acetoxy-10,10-ethylenedioxy-trans-11-octadecenoic acid (7-R)
Compound (6-R) 188 mg (0.53 mmol) was dissolved in synthetic benzene (8 ml), and ethylene glycol (0.3 ml) and pyridinium p-toluenesulfonate (PPTS, 150 mg) were added and refluxed for 8 hours. The reaction mixture was extracted with ethyl acetate and saturated brine, and washed with saturated brine. Then, it was dried with magnesium sulfate. Distilling off the solvent, a mixture of (R) -13-acetoxy-10,10-ethylenedioxy-trans-11-octadecenoic acid (7-R) and unreacted 92: 8 (186 mg, 88% yield) )

NMR (CDCl ₃ + Pyr.-d ₅ ) δ _H : 2.05 (3H, s, Ac), 2.34 (2H, t, J = 7.6Hz, 2), 3.60 to 3.97 (4H, m, 1 ', 2' ), 5.25, (1H, q, J = 12.9 & 6.5Hz, 13), 5.53 (1H, d, J = 15.7Hz, 11), 5.75 (1H, dd, J = 15.6 & 6.2Hz, 12); δ _C : 20.34 (OAc), 34.56 (2), 64.47, 64.58 (1 ', 2'), 73.61 (13), 108.84 (10), 129.63 (11), 131.75 (12), 170.27 (Ac), 190.88 ( 1). HR-MS: Calcd. For C ₂₀ H ₃₄ O ₄ (M ⁺ -C ₂ H ₄ O ₂ ), 338.2457; Measured, 338.2456.
(iv-b) (S) -13-Acetoxy-10,10-ethylenedioxy-trans-11-octadecenoic acid (7-S)
(S) -13-acetoxy-10,10-ethylenedioxide was prepared in the same manner as (iv-a) above except that 273 mg (0.77 mmol) of the synthetic compound (6-S) and ethylene glycol (0.43 ml) were used. Oxy-trans-11-octadecenoic acid ((7-S), 251 mg, 0.72 mmol, 94% yield) was obtained.

The NMR spectrum of the compound (7-S) coincided with the NMR spectrum of the compound (7-R).
(v) (R) -13-hydroxy-10,10-ethylenedioxy-trans-11-octadecenoic acid (8-R), (S) -13-hydroxy-10,10-ethylenedioxy-trans-11 -Octadecenoic acid (8-S) synthesis

(va) (R) -13-Hydroxy-10,10-ethylenedioxy-trans-11-octadecenoic acid (8-R)
186 mg (0.47 mmol) of the synthetic compound (7-R) was dissolved in 5% NaOH-65% EtOH mixed solution (3 ml) and stirred at room temperature for 2.5 hours. Ethyl acetate was added thereto, and the aqueous layer was adjusted to pH 3 with 2N sulfuric acid at 0 ° C.
The reaction was quickly extracted and washed with saturated brine. Next, after drying over magnesium sulfate, the solvent was distilled off to give (R) -13-hydroxy-10,10-ethylenedioxy-trans-11-octadecenoic acid
((8-R), 157 mg, 0.437 mmol, 94% yield).

[α] _D = -4.4 ° (c = 0.59, EtOH) NMR (CDCl ₃ + Pyr.-d ₅ ) δ _H : 3.82 to 3.96 (4H, m, 1 '
, 2 '), 4.15 (1H, q, J = 12.5 & 6.4Hz, 13), 5.56 (1H, d, J = 15.6Hz, 11), 5.83 (1H, dd, J = 15.6 & 6.2Hz, 12) ; δ _C : 64.50 (1 ', C2'), 71.83 (13), 108.99 (10), 129.78 (11), 134.31 (12), 177.00 (1) .HR-MS: Calcd. for C ₁₈ H ₃₂ O ₂ (M ⁺ -C ₂ H ₂ O ₂ ), 280.2402; Measured, 280.2412.
(vb) (S) -13-Hydroxy-10,10-ethylenedioxy-trans-11-octadecenoic acid (8-S)
Synthetic compound (7-S) 251 mg (0.63 mmol) was hydrolyzed with 5% NaOH-65% EtOH (3 ml) for 2.5 hours, and (S) -13-hydroxy- 10,10-ethylenedioxy-trans-11-octadecenoic acid ((8-S), 214 mg, 0.63 mmol, yield 100%) was obtained.

[α] _D = + 5.6 ° (c = 0.67, EtOH)
The NMR spectrum of the compound (8-S) coincided with the NMR spectrum of the compound (8-R).
(vi) (11E, 13R)-(+)-10-oxo-11-octadecene-13-olide (10-R), (11E, 13S)-(-)-10-oxo-11-octadecene-13- Synthesis of Orido (10-S), Racemic (11E) -10-Oxo-11-octadecene-13-Olide ((+/-)-10)

Synthesis of (vi-a) (11E, 13R)-(+)-10-oxo-11-octadecene-13-olide (10-R) Compound (8-R) 34.1 mg (0.096 mmol) under nitrogen flow While stirring a THF (7 ml) solution (containing Et ₃ N (22 mg, 0.21 mmol)), 2,4,6-trichlorobenzoyl chloride (47 mg, 1.9
mmol) was added thereto and further stirred at room temperature for 2 hours. To this, dry benzene (10 ml) was added and stirred, and the upper layer was collected and added to dry benzene (15 ml).

  Further, while stirring a dry benzene solution (15 ml) of DMAP (116.9 mg, 0.96 mmol), the above benzene layer was slowly added at 70 ° C. over 3 hours. After the addition, the mixture was further stirred for 1 hour. Subsequently, the reaction solution was washed with a saturated citric acid solution, a saturated aqueous sodium bicarbonate solution, and a saturated saline solution in this order, and dried over magnesium sulfate. Thereafter, the solvent was distilled off to obtain an oily product (36.1 mg).

  The resulting oily product was purified by silica gel preparative thin layer chromatography (TLC) (EtOAc: hexane = 1: 30) to give pure (11E, 13R)-(+)-10,10-ethylenedioxy. -11-octadecene-13-olide ((9-R), 17.0 mg, 0.050 mmol, yield 52.5%) was obtained.

[α] ¹⁸ _D = + 36.3 ° (c 0.31, EtOH) IR ν _max (film) cm ^-1 : 2925, 2860, 1816, 1728, 1627, 1456, 1360, 1050, 976. NMR (CDCl ₃ ) δ _H : 0.88 (3H, t, J = 6.7 Hz, H18), 1.19-1.35 (16H, m, H4-H8 & H15-H16), 1.55-1.77 (6H, m, H3, H9 & H14), 2.28 (1H, ddd, J = 3.9, 8.0, 14.6 Hz, H2), 2.36 (1H, ddd, J = 3.9, 9.0, 14.7 Hz, H2), 3.77-3.97 (4H, m, H1 '&H2'), 5.34 (1H, dt, J = 7.1, 7.9 Hz, H13), 5.63 (1H, d, J = 15.5 Hz, H11), 5.74 (1H, dd, J = 8.2, 15.5 Hz, H12) .δ _C : 13.96, 22.09, 22.52, 24.59, 24.95, 25.72, 25.75, 26.47, 26.51, 31.50, 33.89, 33.97, 37.27, 63.88, 64.73, 73.47, 108.55, 130.28, 133.60, 173.15.HR-MS m / z: Calcd. For C ₂₀ H ₃₄ O ₄ (M ⁺ ) 338.2457, Found 338.2491.
Subsequently, 17.0 mg (0.050 mmol) of the compound (9-R) was dissolved in 80% EtOH (5 ml), PPTS (10 mg) was added, and the mixture was stirred at 50 ° C. for 8 hours. The reaction mixture was extracted with ethyl acetate and saturated brine, washed with saturated brine, and dried over magnesium sulfate. The solvent was distilled off to obtain a crude product (17.0 mg).

  The resulting crude product was purified by TLC (EtOAc: hexane = 1: 8) to give pure (11E, 13R)-(+)-10-oxo-11-octadecene-13-olide ((10-R ), 13.0 mg, 0.044 mmol, yield 88.3%) as crystals. The NMR spectrum of compound (10-R) was consistent with that of literature.

mp. 63.0-64.0 ℃ (Hexane)
[α] ²⁰ _D = + 37.3 ° (c 0.72, EtOH). IR ν _max (nujol) cm ^-1 : 2925, 2850, 1724, 1682, 1662, 1617, 1456, 1337, 1249, 985. NMR (CDCl ₃ ) δ _H : 0.88 (3H, t, J = 6.5 Hz, H18), 1.19-1.43 (8H, m, H4-H7), 1.60-1.74 (6H, m, H3, H8 & H14), 2.30 (1H, ddd, J = 4.6, 8.3, 14.5 Hz H2), 2.39 (1H, ddd, J = 4.8, 8.1, 13.5 Hz, H9), 2.45 (1H, ddd, J = 4.4, 8.1, 14.0 Hz, H2), 2.58 (1H , ddd, J = 4.8, 8.2, 13.4 Hz, H9), 5.48 (1H, q, J = 6.9 Hz, H13), 6.43 (1H, dd, J = 1.0, 15.7 Hz, H11), 6.71 (1H, dd , J = 6.2, 15.7
Δ _C : 13.96, 22.46, 24.47, 24.81, 25.33, 25.43, 25.87, 26.23, 26.42, 31.48, 33.53, 34.44, 41.29, 72.16, 128.82, 143.29, 172.99, 201.74.HR-MS m / z : Calcd.for C ₁₈ H ₃₀ O ₃ 294.2195, Found 294.2185.
(vi-b) Synthetic compound (8-S) of (11E, 13S)-(-)-10-oxo-11-octadecene-13-olide (10 -S) was used except that (vi-a ) To give (11E, 13S)-(−)-10,10-ethylenedioxy-11-octadecene-13-olide ((9-S), yield 45.9%). [α] ¹⁹ _D = -35.8 ° (c 2.83, EtOH)
The NMR spectrum of the compound (9-S) coincided with the NMR spectrum of the compound (9-R).

Further, (11E, 13S)-(−)-10-oxo-11-octadecene-13-olide (10-S) was prepared in the same manner as in (vi-a) except that the compound (9-S) was used. ) Was obtained quantitatively. [α] ¹⁹ _D = -41.2 ° (c 0.60, EtOH), m. p. 60.5-63.0 ℃ (Hexane)
The NMR spectrum of the compound (10-S) coincided with the NMR spectrum of the compound (10-R).

Synthesis of (vi-c) racemic (11E) -10-oxo-11-octadecene-13-olide ((+/-)-10)
(11E) Racemic 10,10-ethylene in the same manner as (vi-a) above except that 13-hydroxy-10,10-ethylenedioxy-11-octadecenoic acid (compound (8)) was used Dioxy-11-octadecene-13-olide ((+/-)-9), yield 56.5%) was obtained.

In addition, racemic 10,10-ethylenedioxy-11-octadecene-13-olide ((+/-)-9)
In the same manner as in the above (vi-a), racemic (11E) -10-oxo-11-octadecene-13-olide ((+/-)-10), yield 94.5%) was obtained. It was.

The NMR spectrum of the racemic compound ((+/-)-10) coincided with the spectrum of the compound (10-R). mp 48.0-49.5 ℃ (Hexane, literature value 52 ℃)
[Synthesis Example 2]
<(13E, 15R)-(+)-12-oxo-13-octadecene-15-olide, (13E, 15S)-(-)-12-oxo-13-octadecene-15-olide (compound (20-R ), (20-S) synthesis)>
(i) (13E, 15R) -15-hydroxy-12-oxo-1-tetrahydropyranyloxy-13-octadecene (14-R), (13E, 15S) -15-hydroxy-12-oxo-1-tetrahydro Synthesis of pyranyloxy-13-octadecene (14-S)

(ia) (13E, 15R) -15-Hydroxy-12-oxo-1-tetrahydropyranyloxy-13-octa
Synthesis of decene (14-R) Under a nitrogen stream, diisopropylamine (0.351 ml, d = 0.722,2.51 mmol, 1.5 eq.) Was added to 4 ml of THF and cooled to −78 ° C., and this was cooled to n-BuLi 10% hexane. Solution (1.77 ml, 2.76 mmol, 1.32 eq.) Was added dropwise and stirred for 5 minutes, and then stirred at 0 ° C. for 30 minutes. Lithium diisopropylamide (LDA)
The solution (2.5 mmol) was cooled to −78 ° C., and 13-tetrahydropyranyloxy-2-tridecanone ((12), 500 mg, 1.68 mmol) dissolved in 3 ml of THF was added dropwise to enolize.

After stirring for 30 minutes, (2R)-(-)-2-[(R) -O-MEM-mandelyloxy] pentanal (11-R) (532 mg, 1.64 mmol, 0.98 eq.) Dissolved in 3 ml of THF was added dropwise, The aldol reaction was carried out with stirring for 30 minutes.

Thereafter, 0.5 ml of acetic acid was added to stop the reaction, the temperature was returned to room temperature, 50 ml of saturated brine was added, and the mixture was extracted with ethyl acetate. The organic layer was washed successively with 2N HCl, saturated aqueous NaHCO ₃ and saturated brine, dried (MgSO ₄ ) and concentrated to give a light yellow oily aldol reaction product ((13-R), 928 mg, 1.57 mmol, 96% )

Subsequently, the aldol reactant (13-R) was dissolved in DMF (20 ml) and HMPA (10 ml) without purification, sodium acetate (9.7 g) was added, and the mixture was stirred at 50 ° C. for 3 hours, and then at 70 ° C. Stir for 17 hours. Saturated brine was added to the reaction mixture, and the mixture was extracted with ethyl acetate (3 times). Combine the resulting organic layers and add 2N HCl,
The extract was washed successively with saturated aqueous NaHCO ₃ and saturated brine, dried (MgSO ₄ ), and concentrated to give a brown oil. This was purified with a silica gel column (7 g) to obtain (13E, 15R) -15-hydroxy-12-oxo-1-tetrahydropyranyloxy-13-octadecene ((14-R), 160 mg).

IR ν _max (film) cm ^-1 : 3580, 2925, 2850, 1724, 1670, 1626, 1460, 1437, 1408, 1130,1075, 978.NMR (CDCl ₃ ) δ _H : 0.95 (3H, t, J = 7.3 Hz, H18), 1.24-1.37 (16H, m, H3-H9 & THP), 1.37-1.64 (10H, m, H2, H10, H16-H17 & THP), 1.68-1.74 (1H, m, THP), 1.79-1.86 (1H, m, THP), 2.22-2.36 (1H, br, -OH), 2.54 (2H, t, J = 7.5 Hz, H11), 3.38 (1H, dt, J = 6.7, 9.6 Hz, THP), 3.47-3.51 (1H, m, H1), 3.72 (1H, dt, J = 6.9, 9.6
Hz, THP), 3.84-3.89 (1H, m, H1), 4.32 (1H, dt, J = 5.3, 6.1 Hz, H15), 4.57 (1H, t, J = 4.2 Hz, THP), 6.30 (1H, dd, J = 1.5, 15.9 Hz, H13), 6.79 (1H, dd, J = 5.0, 15.9 Hz, H14) .δ _C : 13.61, 18.52, 19.71, 24.20, 25.54, 26.25, 29.29, 29.40, 29.45, 29.47 , 29.53, 29.56, 29.77, 30.82, 38.92, 40.84, 62.35, 67.71, 71.05, 98.87, 128.04, 147.87, 200.77.HR-MS m / z: Calcd.for C ₂₃ H ₄₀ O ₃ (M ⁺ -H ₂ O 364.2977, Found
364.2954.
Optical purity: 90.4% ee (esterified with (S)-(+)-MTPA chloride and NMR analysis (C ₁₃ -H
Measured by))
(ib) (13E, 15S) -15-Hydroxy-12-oxo-1-tetrahydropyranyloxy-13-octa
Synthesis of decene (14-S)
(2S)-(-)-2-[(R) -O-MEM-mandelyloxy] pentanal (11-S) was used in the same manner as in (ia) above, except that compound (11-S) and ( 12) Aldol reaction followed by sodium acetate treatment and purification on silica gel column, (S) -isomer (13E, 15S) -15-hydroxy-12-oxo-1-tetrahydropyranyloxy-13-octadecene ( 14-S) (yield 21.1%)
.

Note that the NMR spectrum of the compound (14-S) coincided with the spectrum of the compound (14-R).
(ii) (13E, 15R) -15-acetoxy-12-oxo-13-octadecenoic acid (16-R), (13E, 15S) -15-acetoxy-12-oxo-13-octadecenoic acid (16-S) Synthesis of

(ii-a) (13E, 15R) -15-Acetoxy-12-oxo-13-octadecenoic acid (16-R) compound (14-R) 476 mg (1.24 mmol) in anhydrous pyridine (15 ml), acetic anhydride (7 ml) was added and stirred at room temperature for 16 hours. After adding ice pieces to decompose excess acetic anhydride, saturated brine was added and the mixture was extracted with ethyl acetate. The organic layer was washed successively with 2N HCl (3 times), saturated aqueous NaHCO ₃ and saturated brine, dried over MgSO ₄ and concentrated under reduced pressure to give a brown oil (678 mg).

The obtained brown oily substance was purified with a silica gel column (6 g) to give (13E, 15R) -15-acetoxy-12-oxo-1-tetrahydropyranyloxy-13-octadecene ((15-R), 512 mg, 97.3%) was obtained pure.

HR-MS m / z: Calcd. For C ₂₅ H ₄₄ O ₅ (M ⁺ ) 424.3189, Found 424.3196
Subsequently, the Jones reagent was added dropwise to a solution of the compound (15-R) (512 mg, 1.205 mmol) in acetone (10 ml) at room temperature until the brown color was maintained, followed by stirring for 1.5 hours. A few drops of 2-propanol were added thereto to decompose excess reagent, and then acetone was removed under reduced pressure. A saturated saline solution was added to the resulting aqueous solution and extracted with ethyl acetate (twice). The organic layer was washed successively with 2N sulfuric acid (3 times) and saturated brine, dried over MgSO ₄ and concentrated under reduced pressure to give (13E, 15R) -15-acetoxy-12-oxo-13-octadecenoic acid as a colorless oil ((16-R), 457 mg, crude yield 107%) was obtained. The obtained compound (16-R) was used in the next reaction without purification.

[α] ¹⁸ _D = + 17.4 ° (C 1.00, EtOH). IR ν _max (film) cm ^-1 : 3300, 2925, 2850, 1738, 1704, 1667, 1628, 1455, 1410, 1370, 1229. NMR ( CDCl ₃ ) δ _H : 0.93 (3H, t, J
= 7.4 Hz, H18), 1.24-1.41 (14H, m, H4-H9 & H17), 1.55-1.64 (6H, m, H3, H10 & H16), 2.09 (3H, s, OAc), 2.34 (2H, t, J = 7.5 Hz, H2), 2.53 (2H, t, J = 7.5 Hz, H11), 5.40 (1H, q, J = 5.8 Hz, H15), 6.19 (1H, dd, J = 16.0, 1.1 Hz, H13), 6.67 (1H, dd, J = 16.0,
Δ _C : 13.77, 18.31, 21.03, 23.99, 24.68, 29.01, 29.16, 29.21, 29.32, 29.33, 33.87, 35.98, 40.78, 72.53, 129.30, 142.99, 170.21, 178.98, 200.30.
HR-MS m / z: Calcd. For C ₂₀ H ₃₄ O ₅ (M ⁺ -C ₂ H ₂ O) 312.2301, Found 312.2286.
(ii-b) Synthesis of (13E, 15S) -15-acetoxy-12-oxo-13-octadecenoic acid (16-S) (14-S) 500 mg (1.30 mmol) was added to pyridine (15 ml) and acetic anhydride ( (13E, 15S) -15-acetoxy-12-oxo-1-tetrahydropyranyloxy-13-octadecene (15-S) (523 mg) except that the product was dissolved in 7 ml). 1.27 mmol, yield 97.7%).

The compound (15-S) (502 mg, 1.19 mmol) obtained was dissolved in acetone (10 ml), and the Jones reagent was added dropwise until the brown color was maintained, and the mixture was stirred at room temperature for 1.2 hours. ) To give oily (13E, 15S) -15-acetoxy-12-oxo-13-octadecenoic acid ((16-S), 459 mg) quantitatively.

[α] ¹⁸ _D = -19.5 ° (c 1.00, EtOH).
The NMR spectra of the compounds (16-S) and (16-R) matched.
(iii) (13E, 15R) -15-hydroxy-12,12-ethylenedioxy-13-octadecenoic acid (18-R), (13E, 15S) -15-hydroxy-12,12-ethylenedioxy-13 Of 2-octadecenoic acid (18-S)

(iii-a) (13E, 15R) -15-hydroxy-12,12-ethylenedioxy-13-octadecenoic acid (18-R) synthetic compound (16-R) 455 mg in benzene (20 ml), The mixture was refluxed for 48 hours in the presence of ethylene glycol (670 mg) and PPTS (300 mg). The product was extracted with ethyl acetate and saturated brine, and the organic layer was washed with saturated brine and dried over magnesium sulfate. The solvent was distilled off to obtain a crude product ((17-R), 515 mg). The crude product (17-R) was dissolved in 5% sodium hydroxide-65% ethanol solution, allowed to stand at room temperature for 2 hours, and then adjusted to pH 5 with 2N citric acid. The reaction product was extracted again with ethyl acetate and saturated brine, and the organic layer was washed with saturated brine and dried over magnesium sulfate. Thereafter, the solvent was distilled off to obtain (13E, 15R) -15-hydroxy-12,12-ethylenedioxy-13-octadecenoic acid ((18-R), 403 mg).

IR ν _max (film) cm ^-1 : 3580, 3300, 2925, 2850, 1706, 1645, 1460, 1408, 1039.
NMR (CDCl ₃ ) δ _H : 0.93 (3H, t, J = 7.2 Hz, H18), 1.24-1.41 (14H, m, H4-H9 & H17), 1.44-1.71 (8H, m, H3, H10, H11 & H16), 2.33 (2H, t, J = 7.4 Hz, H2), 3.85-3.98 (4H, m,
H1 '&H2'), 4.16 (1H, q, J = 6.4 Hz, H15), 5.56 (1H, d, J = 15.5 Hz, H13), 5.82 (1H,
dd, J = 6.1, 15.5 Hz, H14) .δ _C : 13.98, 18.57, 23.42, 24.69, 28.98, 29.13, 29.31,
29.34, 29.45, 29.70, 33.94, 38.45, 39.43, 64.55, 71.78, 109.00, 130.04, 134.00,
HR.MS m / z: Calcd. For C ₂₀ H ₃₆ O ₅ (M ⁺ ) 356.2563, Found 356.2572.
(iii-b) (13E, 15S) -15-Hydroxy-12,12-ethylenedioxy-13-octadecenoic acid (18-S) except that synthetic compound (16-S) was used (iii-a ), The crude product ((17-S
), 517 mg). Except that 517 mg of this crude product (17-S) was dissolved in 5% sodium hydroxide-65% ethanol solution and stirred at room temperature for 2 hours, (13E, 15S) ) -15-hydroxy-12,12-ethylenedioxy-13-octadecenoic acid ((18-S), 400 mg) was obtained.

The NMR spectrum of the compound (18-S) was consistent with the compound (18-R).
(Iv) (13E, 15R)-(+)-12-oxo-13-octadecene-15-orido (20-R), (13E, 15S)-(-)-12-oxo-13-octadecene-15- Synthesis of Orido (20-S)

(Iv-a) 250 mg of a synthetic compound (18-R ) of (13E, 15R)-(+)-12-oxo-13-octadecene-15-olide (20-R) , applying the Yamaguchi method, Converted to 12,12-ethylenedioxy-15-olide (19-R). That is, to a solution of 250 mg (0.701 mmol) of the compound (18-R) obtained by the previous reaction in anhydrous THF (7 ml), Et ₃ N (215 μl, d = 0.73, 1.54 mmol, 2.2 eq.) And 2,4,6-
Trichlorobenzoyl chloride (219 μl, d = 1.56, 1.40 mmol, 2.0 eq.) Was added, and the mixture was stirred at room temperature for 3 hours. After adding dry benzene (20 ml) and allowing to stand, the supernatant was collected and transferred to a dropping funnel containing benzene (250 ml) in a nitrogen stream for dilution.

This solution was slowly added dropwise over 5 hours to a dry benzene (50 ml) solution in which 4-DMAP (856 mg, 7.01 mmol, 10 eq.) Was dissolved and heated to 70 ° C., and the mixture was further stirred for 1 hour. The reaction mixture was washed successively with saturated aqueous citric acid (twice), saturated brine, saturated aqueous NaHCO ₃ (twice) and saturated brine (twice), dried over MgSO ₄ and concentrated under reduced pressure to give a yellow oil. (329 mg, crude yield 138%) was obtained. Purification by a silica gel column (3.5 g) yielded a colorless oily lactone compound (19-R) (126 mg, yield 53.2%).

Rf: 0.54 (5: 1); [α] ²² _D = + 21.3 ° (c 2.00, EtOH) HR-MS m / z: Calcd. For C ₂₀ H ₃₄ O ₄ (M ⁺ ) 338.2457, Found 338.2424]
Next, 116 mg (0.342 mmol) of the compound (19-R) was dissolved in 75% ethanol containing 0.7% PPTS, stirred at 50 ° C. for 8 hours, and (13E, 15R)-(+)-12-oxo-13 was obtained. -Octadecene-15-olide ((20-R), 78 mg, yield 70.3%) was obtained.

[α] ²² _D = + 31.4 ° (c 2.00, CHCl ₃ ) mp. 56.5-58.0 ° C (Hexane) IR ν _max (nujol) cm ^-1 : 2925, 2852, 1733, 1692, 1670, 1627, 1458, 1350 , 1243.NMR (CDCl ₃ ) δ _H : 0.93 (3H, t, J = 7.4 Hz, H18), 1.20-1.42 (14H, m, H4-H9 & H17), 1.60-1.76 (6H, m, H3, H10 & H16) , 2.32-2.44 (3H, m, H2 & H11), 2.61 (1H, dt, J = 13.2, 7.4 Hz, H11
), 5.47 (1H, dt, J = 6.1, 6.9 Hz, H15), 6.28 (1H, dd, J = 1.0, 16.0 Hz, H13), 6.69 (1H, dd, J = 5.8, 16.0 Hz, H14). δ _C : 13.76, 18.34, 24.67, 25.10, 26.47, 26.64, 27.19, 27.40, 27.50, 27.92, 34.39, 35.89, 39.81, 72.36, 129.26, 143.71, 172.97, 201.53.HR-MS m / z: Calcd. for C ₁₈ H ₃₀ O ₃ (M ⁺ ) 294.2195, Found 294.2178.
(iv-b) (13E, 15S)-(-)-12-oxo-13-octadecene-15-olide (20-S) except that the compound (18-S) was used, ) To give (13E, 15S) -12,12-ethylenedioxy-13-octadecene-15-olide (19-S) (yield 54.2%).

[α] ¹⁹ _D = -21.8 ° (c 2.00, EtOH)
Further, (13E, 15S)-(-)-12-oxo-13-octadecene-15-olide (20-S) was prepared in the same manner as in (iv-a) except that compound (19-S) was used. (Yield 78.4%).

[α] ²⁰ _D = -33.3 ° (c 2.00, CHCl ₃ ) mp. 56.0-58.0 ° C (Hexane)
Note that the NMR spectrum of the compound (20-S) coincided with the compound (20-R).
[Synthesis Example 3]
<(R) -1- (2-oxycyclopentylidene) -2-decanol, (S) -1- (2-oxycyclopentylidene) -2-decanol ((23-R), (23-S) Synthesis)
(I) Synthesis of 2-bromodecanal (25)

5 g of decanal (24) was dissolved in 500 ml of ether, 1.1 equivalents of bromine was added at room temperature, and the mixture was stirred for about 1 hour (if bromine remained, stirring took 1.5 to 2.0 hours. If the reaction did not proceed, HBr
1 ml of liquid was added. ). A large amount of water was added to the reaction solution to wash the ether layer, followed by washing in the order of sodium bisulfite and saturated brine, and then dried over magnesium sulfate. The ether was distilled off under reduced pressure to obtain bromodecanal (25).

(ii) (R) -2-[(R) -O-MEM-Mandelyloxy] decanal (26-R), (S) -2-[(R) -O-MEM-Man
Deryloxy] decanal (26-S) synthesis

(R) -O-MEM-mandelic acid sodium salt (1.1 equivalents) was added to the DMF-HMPA mixed solution (3: 2).
The mixture was stirred in a nitrogen stream at 1.5 ° C. for 1.5 hours to obtain a suspension in which almost 2/3 of the salt was dissolved, and 1 equivalent of 2-bromodecanal (25) was added all at once and heated for 1 hour. The reaction product was extracted by adding it to a mixed system of ether and saturated brine, and the ether layer was washed with 2N hydrochloric acid, aqueous sodium bicarbonate and brine in this order. After drying with magnesium sulfate, ether was distilled off to obtain a crude product (yield 99%). The crude product was separated and purified using flash column chromatography (solvent hexane: ethyl acetate = 3: 1, then 2: 1) using about 20 times the amount of silica gel, and (R)-(- ) -2-[(R) -O-MEM-Mandeloyloxy] decanal ((26-R), Yield 36.1%, 92% ee) and (S)-(-)-2-[(R) -O- MEM-mandelloyoxy] decanal ((26-S), yield 38.4%, 96% ee) was obtained. These optical purities were measured based on the area ratio of aldehyde group hydrogen on the NMR spectrum.

(26-R): ¹ HNMR (CDCl ₃ ) δ: 0.88 (3H, t, J = 7.1 Hz, H10), 1.24-1.34 (12H, m, H4-H9), 1.72 (1H, bhex, J = 7.7 Hz, H3), 1.79 (1H, m, H3), 3.37 (3H, s, H6 '), 3.51 (2H, m, H5'), 3.70 (1H, ddd, J = 11.0, 5.4, 3.9 Hz, H4 '), 3.80 (1H, ddd, J = 11.0, 5.5, 3.8 Hz, H4'), 4.82 (1H, d, J = 7.1 Hz, H3 '), 4.91 (1H, d, J = 7.1 Hz, H3' ), 5.00
(1H, dd, J = 8.2, 4.8 Hz, H2), 5.34 (1H, s, H2 '), 7.33-7.42 (3H, m, H9'-H11'), 7.49 (2H, dd, J = 7.8, 1.6 Hz, H8 '&H12'), 9.31 (1H, d, J = 0.5 Hz, H1) ¹³ C NMR (CDCl ₃ ) δ: 14.08 (C10), 22.3 (C9), 24.73 (C4), 28.77 (C7) , 29.12 (C6), 29.17 (C5), 29.24 (C3), 31.81 (C8), 58.97 (C6 '), 67.63 (C4'), 71.66 (C5 '), 76.73 (C2'), 78.90 (C2), 94.24 (C3 '), 127.45 (C9'& C11 '), 128.71 (C8'& C12 '), 128.93 (C10'), 135.92 (C7 '), 170.59 (C1'), 197.89 (C1) [α] _D =- 15.3 ° (c 0.60, EtOH) HR-MS m / z: Calcd.for C ₂₂ H ₃₄ O ₆ (M ⁺ ) 394.2356, measured 394.2386
(26-S): ¹ H NMR (CDCl ₃ ) δ: 0.88 (3H, t, J = 7.2 Hz, H10), 1.07-1.23 (10H, m, H5-H9), 1.27 (2H, quint, J = 6.8 Hz, H4), 1.64 (1H, m, H3), 1.70 (1H, m, H3), 3.37
(3H, s, H6 '), 3.51 (2H, m, H5'), 3.69 (1H, ddd, J = 11.0, 5.8, 3.6 Hz, H4 '), 3.81
(1H, ddd, J = 11.0, 5.0, 3.5 Hz, H4 '), 4.82 (1H, d, J = 7.1 Hz, H3'), 4.89 (1H, d, J = 7.1 Hz, H3 '), 4.99 ( 1H, dd, J = 8.9, 4.4 Hz, H2), 5.36 (1H, s, H2 '), 7.32-7.40 (3H, m, H9'-H11'), 7.48 (2H, dd, J = 7.7, 1.6 Hz, H8 '&H12'), 9.52 (1H, d, J = 0.5 Hz,
H1) ¹³ C NMR (CDCl ₃ ) δ: 14.10 (C10), 22.63 (C9), 24.47 (C4), 28.61 (C7), 28.95 (C6), 29.04 (C5), 29.17 (C3), 31.81 (C8) , 58.98 (C6 '), 67.65 (C4'), 71.65 (C5 '), 76.58 (C2'), 78.75 (C2), 94.21 (C3 '), 127.49 (C9'& C11 '), 128.70 (C8'& C12 ' ), 128.92 (C10 '), 136.00 (C7'), 170.54 (C1 '), 197.97 (C1)
[α] _D = -61.7 ° (c 0.60, EtOH) HR-MS m / z: Calcd. for C ₂₁ H ₂₆ O ₅ (M ⁺ -CH ₃ OH-2H ₂ ) 358.1780, measured 3581788
(iii) cyclopentanone and (R) -2-[(R) -O-MEM-mandelyloxy] decanal, (S) -2-[(R) -O-MEM-mandelyloxy] decanal ((26-R) , (26-S))

Cyclopentanone ((21), 0.583 mmol, 49 mg) in THF (1 ml) LDA (prepared from 0.76 mmol (76.5 mg) diisopropylamine and 0.76 mmol (48.6 mg) n-BuLi in 1 ml THF) 0.76 mmol Was enolized using (-78 ° C, 20 min). (S) -2-[(R) -O-MEM-Mandelyloxy] decanal ((26-R), 0.641 mmol, 235 mg) was added to this solution as a THF (2 ml) solution and cooled to -78 ° C. . The mixture was left as it was for 30 minutes, the reaction was stopped by adding acetic acid (1 ml), and the reaction product was extracted with ethyl acetate. The ethyl acetate layer was washed with 2N hydrochloric acid, aqueous sodium hydrogen carbonate and saturated brine, and dried over magnesium sulfate. The solvent was distilled off to obtain the aldol reactant (22-R) quantitatively.

In addition, aldol reaction product (22-S) was obtained quantitatively by the same procedure as above except that (S) -2-[(R) -O-MEM-mandelyloxy] decanal (26-S) was used. .
(22-R): NMR (CDCl ₃ + D ₂ O) δ _H [δ _C ]: 3.61 (1H, dd, J _{1-1 '} = 8.1 Hz, J _1-2 = 3.6 Hz, 1), 4.91 ( 1H, brdt, J _{1'-2 '} = 3.6, J _2'-3' = 9.8 & 2.6 Hz, 2) .δ _C : 50.773 (2), 72.862 (1), 76.512 (2), 170.528 (COO) , 222.615 (CO). Precise MS m / z: Calcd for C ₂₄ H ₃₄ O ₅ (M ⁺ -CH ₃ OCH ₂ CH ₂ OH): 402.2406; Measured, 402.2408.
(22-S): NMR (CDCl ₃ ) δ _H : 3.868 (1H, dd, J _{1-1 '} = 8.3, J _1-2 = 3.0 Hz, 1), 4.808 (1H, dt, J _2-3 = 10.1 & 2.7 Hz, J _1-2 = 3.0 Hz, 2). Δ _C : 73.10 (1), 76.35 (2), 170.81 (COO), 222.87 (CO). Precise MS m / z: Calcd. For C ₂₄ H ₃₄ O ₅ (M ⁺ -CH ₃ OCH ₂ CH ₂ OH): 402.2408; Measured, 402.2406.
(iv) (R) -1- (2-oxycyclopentylidene) -2-decanol, (S) -1- (2-oxycyclo
Synthesis of (pentylidene) -2-decanol ((23-R), (23-S))

The aldol reaction product ((22-R), 284 mg, 0.63 mmol) was stirred in a mixed solvent (DMF 12 ml + HMPA 8 ml) at 65 to 70 ° C. for 24 hours in sodium acetate (5 g) and a nitrogen stream. The reaction solution was put into a mixed system of ethyl acetate and saturated brine, and the reaction product was extracted with ethyl acetate. The ethyl acetate layer was washed with 2N hydrochloric acid, sodium bicarbonate water and saturated brine in this order, and dried over magnesium sulfate. The solvent was distilled off to obtain an oily reaction product (128 mg, 88% purity by NMR analysis). The obtained oily reaction product was purified by preparative thin-layer chromatography (hexane: ethyl acetate = 3: 1, developed three times) to obtain pure (R) -1- (2-oxocyclopentylidene) -2. -Decanol ((23-R), 68.3 mg, 44% yield) was obtained.

[α] _D = -19.3 ° (c = 0.88, EtOH)
In addition, except that the aldol reactant (22-S) was used, (S) -1- (2-oxocyclopentylidene) -2-decanol ((23-S), 83 mg) was obtained in the same manner as above. Yield 51%).

[α] _D = + 20.7 ° (c = 1.76, EtOH)
When the optical purity was measured using the compounds (23-R) and (23-S) as MTPA esters, they were 89% ee and 94% ee, respectively.

23-R MTPA ester;
δ _H 6.24 (dt, J = 8.9, 2.8 Hz, H1: enantiomer), 6.35 (dt, J = 9.0, 2.7 Hz, H1). The signal appearing at 6.24 ppm was based on the S-isomer, and the optical purity was 89% based on the integration ratio with the signal of 6.35 ppm.

23-S MTPA ester:
From the integral ratio of the signal appearing at 6.24 ppm to the signal appearing at 6.35 ppm (enantiomer), the optical purity was 94%.

Preparation of MTPA ester:
Add compound (23-R) (10 mg, 1.0 eq., 0.042 mmol) and pyridine (0.2 ml) in an insulator flask and dissolve well. (S)-(+)-α-Methoxy-α- (trifluoro Ethyl) phenylacetyl-chloride [(S)-(+)-MTPA-Cl] (119.4 mg, 7.0 eq., 0.4 mmol, d = 1.35) was added, and the mixture was sealed and stirred at room temperature overnight. Ice water was added to the reaction solution to stop the reaction, and the mixture was extracted with ethyl acetate and saturated brine. The ethyl acetate layer was washed successively with 2N hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine. The ethyl acetate layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to give (R) -2-[(R) -O-MTPA] -1- (2-oxocyclopentylidene) -2-decanol (15.0 mg , 0.033 mmol,
A crude yield of 78.0%) was obtained.

¹ H NMR (CDCl ₃ ) d: 0.88 (3H, t, J = 7.0 Hz, H10), 1.13-1.38 (12H, m, H4-H9), 1.59 (1H, m, H3), 1.76 (1H, m , H3), 1.99 (2H, m, H4 '), 2.36 (2H, dt, J = 8.1, 7.9 Hz,
H3 '), 2.61 (1H, dt, J = 7.8, 2.7, 17.2 Hz, H5'), 2.90 (1H, m, H5 '), 3.52 (3H, s, H9'), 5.53 (1H, ddd, J = 8.7, 8.0, 6.3, 5.5 Hz, H2), 6.24 (0.12H, dt, J = 8.9 Hz, H1
: enantiomer), 6.34 (1H, dt, J = 9.0, 2.7 Hz, H1), 7.29-7.42 (3H, m, H12'-H14 '), 7.42-7.54 (2H, m, H11'& H15 ')
¹³ C NMR (CDCl ₃ ) d: 14.08 (C10), 19.73 (C4 '), 22.63 (C9), 24.64 (C7), 27.08 (C5'), 29.11 (C6), 29.14 (C5), 29.31 (C4) , 31.80 (C8), 33.53 (C3), 38.26 (C3 '), 55.53 (C9'), 74.51 (C2), 125.91 (C8 '), 127.22 (C13'), 128.43 (C12 '&C14'), 129.64 ( C11 '&C15'), 129.66 (C1), 132.30 (C10 '), 139.95 (C1'), 166.12 (C6 '), 207.08 (C2')
23-S MTPA ester crude yield 81%: NMR
¹ H NMR (CDCl ₃ ) d: 0.88 (3H, t, J = 7.0 Hz, H10), 1.11-1.26 (11H, m, H4 & H5-H9), 1.26-1.38 (1H, m, H4), 1.61 (1H , m, H3), 1.80 (1H, m, H3), 1.96 (2H, m, H4 '), 2.35 (2H, q, J = 8.2 Hz, H3'), 2.54 (1H, dt, J = 7.8, 2.8, 16.1 Hz, H5 '), 2.87 (1H, m,
H5 '), 3.53 (3H, s, H9'), 5.51 (1H, dt, J = 14.5, 6.8 Hz, H2), 6.24 (1H, dt, J = 8.9, 2.8 Hz, H1), 6.35 (0.06H , dt, J = 9.0, 2.8 Hz, H1: enantiomer), 7.28-7.42 (3H, m, H12'-H14 '), 7.46-7.67 (2H, m, H11'& H15 ')
¹³ C NMR (CDCl ₃ ) d: 14.07 (C10), 19.74 (C4 '), 22.63 (C9), 24.90 (C7), 27.08 (C5')
, 29.15 (C6), 29.24 (C5), 29.36 (C4), 31.80 (C8), 33.51 (C3), 38.22 (C3 '), 55.50
(C9 '), 74.71 (C2), 84.60 (C7'), 124.41 (C8 '), 127.32 (C13'), 128.42 (C12 '&C14'), 129.68 (C11 '&C15'), 129.70 (C1), 132.12 (C10 '), 139.97 (C1'), 166.19 (C6 '), 206.97 (C2')
[Example 1]
<Apoptosis-inducing activity test>
Add human leukemia cell line HL-60 to RPMI1640 medium supplemented with 10% fetal bovine serum to prepare a cell suspension of 1 × 10 ⁵ cells / ml, and add 100 μL each to each well of a 96-well culture microplate. After injecting and culturing in a carbon dioxide incubator at 37 ° C. for 4 hours, a solution of compounds 1 to 6 synthesized in Synthesis Examples 1 to 3 was added to 10.0 μΜ (however, compound 3 (20-S) and compound 4 (20- R) was added to individual wells to a concentration of 10.5 μM) and further cultured for 2 or 4 hours. In addition, as a solution of the above compounds 1 to 6, a 1 mM solution (90% DMSO) prepared by dissolving compounds 1 to 6 in dimethyl sulfoxide (DMSO) and adding RPMI1640 medium in advance was used.

  Also, instead of the above compounds 1-6, camptothecin ((S)-(+)-Camptothecin, Mw 348.4, Sigma-Aldrich) was used as a positive control and added to the wells to 10 μg / mL. In the same manner as above, prepare cells cultured for 2 hours or 4 hours, and as a negative control, add only solvent (DMSO) to the wells, and otherwise incubate for 2 hours or 4 hours. I prepared what I did.

After culturing, the cells were collected, and 100 μL of 2% glutaraldehyde solution was added to the collected cells.
After fixing overnight at ℃, suspended in 20 μL of PBS (-) solution, added 2 μL of 10 μg / mL 4 ', 6-diaminidin-2-phenylindole dihydrochloride (DAPI, Boehringer Mannheim) solution, and stained with fluorescence The morphology of the nuclei was observed with a fluorescence microscope.

10 Observe ³ cells, count cells that are causing aggregation of chromatin, and determine the ratio (%) of chromatin-aggregated cells to the observed number of cells (10 ³ ).
%).

  The results are shown in Table 1.

From Table 1, the apoptosis-inducing activity of the group added with compounds 1 to 6 is 19.9 to 32.4% in the case of 2-hour contact culture and 40.7 to 52.2% in the case of 4-hour contact culture, which is more potent than the negative control. It was found to have activity. In addition, in the case of the positive control camptothecin (10 μg / mL) addition group, the apoptosis-inducing activity was 23.7% in the 2-hour contact culture (Experiment I).
) And 29.5% (Experiment II), 59.5% (Experiment I) and 60.0% (Experiment II) in 4-hour contact culture
The compounds 1 to 6 added group were found to have slightly lower or almost the same activity as the positive control group.

Next, the effect of normal human peripheral blood-derived mononuclear cells (PBMC) on apoptosis induction was examined.
That is, using a vacuum blood collection tube containing heparin, 20 mL of peripheral blood was collected from the vein of a healthy human male, dispensed into a test tube, and centrifuged (1300 rpm, 10 minutes, 25 ° C) to obtain a Buffy coat fraction ( Plasma layer and cell layer intermediate layer) were separated.

Add medium (RPMI1640 medium supplemented with 10% fetal bovine serum) to this fraction, and gently form a layer on the liquid surface of the test tube containing 4 mL of Ficoll-Paque liquid (Amersham Bioscience, Tokyo) in advance. Poured and centrifuged (1300 rpm, 30 minutes, 25 ° C.).

The generated cell layer (intermediate layer between the medium layer and Ficoll-Paque layer) was collected, and a medium was added to the cells, followed by centrifugation and cell washing.
This washing operation was repeated 3 times to obtain PBMC.

RPMI1640 medium supplemented with 10% fetal bovine serum is added to the obtained PBMC to prepare a cell suspension of 2 × 10 ⁵ cells / ml, and 200 μL each is injected into each well of a 96-well culture microplate, After culturing in a 37 ° C. carbon dioxide incubator for 4 hours, 10.
Each was added to individual wells at a concentration of 0 μM (wherein Compound 3 (20-S) and Compound 4 (20-R) were 10.5 μΜ), and further cultured for 2 hours.

The preparation method, culture method, and apoptosis evaluation method of each compound solution and positive control camptothecin solution used were the same as in HL-60.
The results are shown in Table 2.

From Table 2, the apoptosis inducing activity of the compounds 1 to 6 addition group against PBMC was 2.0 to 3.4%, which was clearly lower than the 17.5% of the positive control camptothecin.
Therefore, in comparison with the case of HL-60 in Table 1, it was shown that compounds 1 to 6 selectively act on cancer cells to induce apoptosis.

[Example 2]
<Proliferation inhibitory activity test during 72-hour contact culture for HL-60 cells>
Add human leukemia cell line HL-60 to RPMI1640 medium supplemented with 10% fetal bovine serum to prepare a cell suspension of 1 × 10 ⁵ cells / ml, and add 100 μL each to each well of a 96-well culture microplate. After injecting (n = 3) and culturing in a carbon dioxide incubator at 37 ° C. for 4 hours, 100 μL of a compound 1-6 solution prepared in advance as in Example 1 was added to each well (final concentration). 10.0 μM (however, Compound 3 (20-S) and Compound 4 (20-R) were 10.5 μM)), and further cultured in a 37 ° C. carbon dioxide incubator for 72 hours.

As a negative control, a culture was prepared in the same manner as above except that only the solvent (DMSO) was added to the wells. The DMSO concentration was adjusted to 1% in all the wells.
Separately, a culture was prepared as described above except that nothing was added.

Next, 20 μL / 200 μL of cell proliferation detection reagent WST-1 (Dojindo Laboratories) was added, and after further incubation for 3 hours, absorbance (450 nm-630 nm) was measured using a plate reader.
The results are shown in Table 3.

From Table 3, the absorbance of the group added with compounds 1 to 6 was 0 to 0.02, and it was confirmed that the proliferation of HL-60 cells was clearly suppressed.
[Example 3]
<Proliferation inhibitory activity test during 24-hour contact culture on HL-60 cells>
A solution of compounds 1 to 6 prepared in the same manner as in Example 1 was serially diluted with a 90% DMSO solution, and in the same manner as in Example 2, 2 × 10 ⁵ cell suspension / ml was added 200 μL at a time. Added to each well of a microplate (n = 3), incubated in a carbon dioxide incubator at 37 ° C for 24 hours, and then measured for absorbance (450nm-630nm), growth inhibition rate (%) and 50% growth inhibition The concentration (IC ₅₀ ) was calculated.

The growth inhibition rate (%) was determined on the basis of an absorbance of 0.53 when the compound concentration was 0 as a growth inhibition rate of 0%. The 50% growth inhibitory concentration (IC ₅₀ ) was calculated as the compound concentration when the growth inhibition rate was 50%.

  The results are shown in Tables 4 and 5.

[Example 4]
<Proliferation inhibition test during 48-hour contact culture for PBMC>
A solution of compounds 1 to 6 prepared in the same manner as in Example 1 was serially diluted with a 90% DMSO solution, and 2 × 10 ⁵ PBMC suspension / 200 μL was added to a microplate in the same manner as in Example 1. Were added to each well (n = 3) and cultured in a carbon dioxide incubator at 37 ° C. for 48 hours.

For comparison, an experimental group was prepared in which 2 × 10 ⁴ cells / 200 μL of HL-60 cells prepared in the same manner as in the above Example were used instead of PBMC.
8 hours before the end of the culture, 1 μCi of ³ H-thymidine (Amersham Bioscience) was added to each well.

After completion of the culture, the cells were collected on a glass filter and washed with PBS.
After drying the filter, the radioactivity incorporated into the cells was measured using a liquid scintillation counter.

Furthermore, inhibition rate (%) and 50% inhibition concentration (IC ₅₀ ) were calculated using radioactivity as an index. The suppression rate (%) was determined based on the radioactivity of 43,490 dpm and 46,407 dpm when the compound concentration was 0 as the suppression rate of 0%. IC ₅₀ was calculated as the compound concentration when the inhibition rate was 50%.

The results are shown in Tables 6 and 7. The upper value in Table 6 indicates the radioactivity uptake of PBMC, and the lower value indicates the uptake of HL-60 (average value of n = 3).

As shown in Table 6, the inhibitory action of the additive compound group on the amount of ³ H-thymidine incorporation of PBMC is clearly lower than that of leukemia cell HL-60.
The IC ₅₀ for HL-60 was higher than that for HL-60.

Thus, these results support that compounds 1-6 act selectively on cancer cells over normal cells.

Claims (6)

An apoptosis inducer comprising a compound represented by the following formula (1) as an active ingredient:

(In the formula (1), R represents an alkyl group having 1 to 6 carbon atoms, and n represents 3.) An apoptosis inducer comprising a compound represented by the following formula (2) as an active ingredient:

(In the formula (2), Y represents OR ^1, R ¹ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, m is an integer of 1-3.).   The compound represented by the formula (1) is selected from the group consisting of (13E, 15R) -12-oxo-13-octadecene-15-olide and (13E, 15S) -12-oxo-13-octadecene-15-olide. The apoptosis-inducing agent according to claim 1, which is at least one compound selected. The apoptosis inducer according to claim 2, wherein in the formula (2) , R ¹ is a hydrogen atom.   The compound represented by the formula (2) comprises (R) -1- (2-oxocyclopentylidene) -2-decanol and (S) -1- (2-oxocyclopentylidene) -2-decanol. The apoptosis inducer according to claim 2, which is at least one compound selected from the group. An antitumor agent comprising the apoptosis-inducing agent according to any one of claims 1 to 5.