WO2008026300A1

WO2008026300A1 - Neem seed-derived therapeutic agent for malignant tumor

Info

Publication number: WO2008026300A1
Application number: PCT/JP2007/000091
Authority: WO
Inventors: Takashi Suzuki; Toshihiro Akihisa; Keiichi Tabata; Taisuke Noto
Original assignee: Nihon University
Priority date: 2006-08-28
Filing date: 2007-02-15
Publication date: 2008-03-06
Also published as: JP2009274956A

Abstract

The object is to provide a novel therapeutic agent for malignant tumor. Disclosed is a therapeutic agent for malignant tumor or an apoptosis inducer which comprises a compound selected from epoxyazadiradione, gedunin, 17-epi-17-hydroxyazadiradione and 7-O-benzoylnimbocinol as an active ingredient.

Description

Specification

Neem seed-derived malignant tumor therapeutic agent

Technical field

[0001] The present invention relates to a malignant tumor therapeutic agent, and more particularly to a malignant tumor therapeutic agent comprising a plant-derived component as an active ingredient.

Background art

[0002] Plants have attracted attention as a pharmaceutical resource since ancient times, and various pharmaceuticals or pharmaceutical materials have been found in plants. Plant-derived malignant tumor treatments include paclitaxel (Taxol), an extract from yew plants; argentine camptothecin; Vincristine, vinblastine and the like, which are indole alkaloids, are known.

[0003] The extract of the Indian family (M eliaazadirachta L .; also known as Neem) contains a number of ingredients, which are astringents, antispasmodics, stomachic agents, tonics, animal repellents. It is known to be useful as an agent or the like (Patent Document 1). Further, Patent Document 2 describes that an extract of a plant of the family Sendanaceae has an antitumor activity, but describes that the molecular size of the active substance is around 5,000.

Patent Document 1: Japanese Patent Laid-Open No. 8_2 3 9 30 6

Patent Document 2: Japanese Patent Laid-Open No. 2 0 0 4 _ 2 5 6 4 2 6

Disclosure of the invention

Problems to be solved by the invention

[0004] An object of the present invention is to search for novel malignant tumor therapeutic agents from plants, and to solve the problems

[0005] The inventor of the present invention is directed to an organic solvent extract of a seed of Lindsendan as an antitumor agent. As a result of screening using the activity and apoptosis-inducing ability as indicators, it was found that a specific tetracyclic compound having a small molecular weight has excellent antitumor activity and apoptosis-inducing ability and is useful as a therapeutic agent for malignant tumors. completed.

[0006] That is, the present invention provides a therapeutic agent for malignant tumors comprising as an active ingredient a compound selected from epoxyazadiradione, gedunin, 17-epoxy_17-hydroxyazadiradione and 7_0_benzoylninpocinol. And an apoptosis inducer.

In addition, the present invention provides a malignant tumor therapeutic agent and an apoptosis inducer of a compound selected from epoxyazadiradione, gedunin, 17-epi_1 7-hydroxyazadiradione and 7_0_benzoylninposinol. It is intended to provide usage for

Furthermore, the present invention provides a treatment for a malignant tumor characterized by administering a compound selected from epoxyazadiradione, gedunin, 17-epi_1 7-hydroxyazadiradione and 7_0_benzoylninpocinol. The present invention provides a method and a method for inducing apoptosis.

The invention's effect

[0007] The therapeutic agent for malignant tumor of the present invention has an excellent antitumor activity, and its action is considered to be due to the ability to induce apoptosis of cancer cells. Therefore, it is useful as a new malignant tumor therapeutic agent.

BEST MODE FOR CARRYING OUT THE INVENTION

[0008] The malignant tumor therapeutic agent and apoptosis inducer of the present invention (hereinafter referred to as malignant tumor therapeutic agent, etc.) are epoxyazadiradione (1), gedunin (2), 17-epoxy_17-hydroxyazadiradione ( 3) and 7_0_benzoylnin Posinol (4) A compound selected from (4) is an active ingredient. These compounds are represented by the following formulas (1) to (4), respectively.

[0009] [Chemical 1]

Γ7-epi-17-hydroxyazadiradione (3) 7-benzoylninpocinol (4)

[0010] These compounds (1) to (4) can be obtained by extracting from seeds of neem. For example, after neem seeds are crushed and extracted with η_hexane, the extract is further extracted with methanol, and the methanol fraction is divided into a methylene chloride fraction and a water fraction. When fractionated by mass chromatography, the compounds (1) to (4) are obtained.

[0011] As will be apparent from Examples below, the compounds (1) to (4) have excellent cytotoxic activity against leukemia cells, malignant melanoma cells, and the like. The cytotoxic activity is based on apoptosis-inducing activity. Therefore, the compounds (1) to (4) or neem extracts containing these compounds are useful as therapeutic agents for malignant tumors in mammals including humans.

The malignant tumors that are the targets of the malignant tumor therapeutic agent of the present invention include blood and hematopoietic tissue tumors such as leukemia and lymphoma, and solid tumors. For solid tumors, the skin Examples include skin cancer, lung cancer, colon cancer, stomach cancer, breast cancer, prostate cancer, thyroid cancer and other epithelial cell cancers; and leiomyosarcoma and sarcomas such as osteosarcoma.

[0012] The medicament of the present invention comprises compounds (1) to (4), or an extract, an excipient, a binder, a lubricant, a disintegrant, a coating agent, an emulsifier, a suspending agent, a solvent, and a stable agent. One or more pharmaceutically acceptable carriers such as an agent, absorption aid, ointment base, and the like are added as appropriate, and dosage forms such as for oral administration, for injection, for rectal administration, and for external use are added by conventional methods. It is obtained by formulating.

Preparations for oral administration include granules, tablets, dragees, capsules, pills, solutions, emulsions and suspensions; preparations for injection administration are intravenous injection, intramuscular injection, subcutaneous injection Preparations for infusion, etc .; As preparations for rectal administration, suppository soft capsules and the like are preferable.

The medicament of the present invention can be administered as a preparation as described above to mammals containing baboons.

The medicament of the present invention is preferably administered about 1 to 500 mgZkg per day as compounds (1) to (4) 1 to 4 times.

Example

[0013] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0014] Example 1 (Extraction, fractionation and isolation of compounds (1) to (4))

Dry neem seeds (629 g) were crushed in a mortar, extracted with n-hexane [degreasing] (60-80 ° C, 2 LX 3 times), and after filtration, the solvent was distilled off under reduced pressure. Xanthine extract (80 g) was obtained. The degreased residue was further extracted with methanol (room temperature, 2 L × 3 times). After filtration, the solvent was distilled off under reduced pressure to obtain a methanol extract (206 g). The methanol extract (205 g) was subjected to solvent fractionation with methylene chloride monohydrate (1: 1) to obtain a methylene chloride fraction (36 g) and a water fraction (1 65 g).

[0015] The methylene chloride fraction (22 g) was fractionated by silica gel (gel weight 844 g) column chromatography. N-Hexane mono-vinegar as elution solvent Ethyl [1: 0 (2000 mL), 95: 5 (200 OmL), 9: 1 (250 OmL), 8: 2 (200 OmL), 7: 3 (350 OmL), 6:

4 (3 OO Om L), 1: 1 (55 O Om L), 4: 6 (2 OO Om L), 3: 7 (4 OO Om L), 2: 8 (35 O Om L), O: 1 (350 Om L) vZv] and methanol (300 OmL), 18 fractions [F r .A (0.0342 g), F r .B (1.551 73 g), F r .C ( 0. 4099 g), F r .D (0. 1 688 g), F r .E (0. 2902 g) .F r .F

(0. 1 941 g), F r .G (0 5273 g), F r .H (2. 37 1 O g), F r .I (1. 6291 g) .F r .J (2. 5473 g), F r .K (1.11 1 79 g) .F r .L (0.657 1 g) .F r .M (0. 2 329 g), F r .N (0. 4521 g ), F r .O (1.1 837 g), F r .P (0.1 483 g), F r .Q (2. 7239 g), F r .R (7 2787 g)] I drew it.

Fr. H (2. 2721 g) in methylene chloride fraction was added to silica gel (gel weight 1

50 g) Fractionation was performed using column chromatography. As elution solvent, n-hexane-ethyl acetate [9: 1 (700 mL), 7: 3 (45 OmL)

), 6: 4 (95 Om L), 55:45 (60 Om L), 1: 1 (700 m), 45:55 (50 Om L), 4: 6 (50 Om L), 3: 7 (600 mL), 2: 8 (60 OmL), 1 9 (50 OmL)], ethyl acetate (6

0 Om L), Ethyl acetate monomethanol [8: 2 (50 OmL)], MeOH

(650 ml), 15 fraction [F r. H-1 (22.4 mg), F r.

H-2 (5.8 mg), F r .H-3 (6.6 mg), Fr.H-4 (85

-5 mg), F r .H-5 (891 8 mg), F r.H—6 (1 1 1.2 mg), F r.H-7 (344.9 mg), F r.H- 8 (59.9 mg)

, F r .H -9 (1 01.4 mg), F r .H- 1 0 (1 85.4 mg),

F r .H— 1 1 (76. Omg), F r .H- 1 2 (1 04. Omg), F r .H- 1 3 (291.5 mg), F r .H- 1 4 (69 .4 mg), F r

H—15 (277. 3 mg)]. The methylene chloride fraction Fr. H-5 (891. 8 mg) was further fractionated using silica gel (gel weight 38 g). The elution solvent is n-hexane monoethyl acetate [8: 2 (58 OmL), 6: 4 (18 OmL), 4: 6 (19 OmL)], ethyl acetate (200 mL), ethyl acetate Using methanol [1: 1 (9 Om L)] and MeOH (8 Om L), 10 fraction [F r. H— 5— 1 (2. 9 mg), F r. H— 5— 2 ( 28. 4mg), F r. H— 5— 3

(3 91.5 mg), F r, H-5 ― 4 (1 72.

-5 (23.4 mg), F r .H-5 -6 (40.

-7 (65.8 mg), F r .H-5 -8 (29.

-9 (1 2-3 mg), F r .H-5-1 0 (1 1 Methylene chloride fraction F r. H— 5— 3 (98. 8 mg) and F r. H_5_ 4 (1 1 3 2 mg) for reversed phase HP LC (column: P egasi IO DS-II, 10 X 25 Omm. Manufactured by Senshu Science Co., Ltd .; eluent: methanol monohydrate = 7: 3 (0.1% acetic acid) Flow rate: 3. OmLZ min), and 27.3 mg of epoxyazadiradione (1; retention time 31.4 min) was isolated.

The methylene chloride fraction Fr. H-7 (344. 9 mg) was fractionated using silica gel (gel weight: 17 g) column chromatography. The elution solvent is n-hexane monoethyl acetate [7: 3 (310 mL), 6: 4 (280 mL), 1: 1 (17 OmL)], ethyl acetate (20 OmL), ethyl acetate-methanol. [1: 1 (1 60mL)], methanol (1 20mL), 8 fractions [F r. H-7-1 (1. 1 mg), F r. H-7 -2 (49. 4 mg ), F r.H-7 -3 (1 38.3 mg), F r.H-7 -4 (68.5 mg), F r.H-7 -5 (1 0.3 mg), F r. H-7-6 (11.7 mg), Fr. H-7-7 (8.2 mg), Fr. H-7-8 (26.8 mg)].

Methylene chloride fraction F r .H-7 -3 (1 20. 2mg), F r.H-7- 4 (61.9 mg) and Fr. I (191.8 mg) reverse phase HPLC (column: P egasi I ODS—II, 10 x 25 Omm; eluent: methanol-water = 7: 3; The flow rate was 3. OmLZ min) and 24.3 mg of gedunin (2; retention time 20.4 min) was isolated.

[0019] Reversed phase HP LC for methylene chloride fraction F r. K (1. 0860 g) (force ram: P egasi I ODS—II, 10 x 25 Omm; eluent: methanol monohydrate = 7: 3 ; Flow rate: 3. OmLZ min) 1 7-Epi 1 7 -Hydroxyazadiradione (3; 24.7 mg, retention time 1 0.2 min), 7 _0_ benzoylninposinol (4; 7.9 mg, retention time 29.6 minutes).

[0020] Compound (1) was identified by comparison of ¹ H NMR and MS data with literature value ¹⁾ .

Epoxy azaradione (1) ES I—MS mZz 489 [M + Na] +, 505 [M + K] +. ¹ H-NMR (600 MHz, CDCI ₃ ): δ

1. 04 (3 H, s, H 3 - 1 8), 1. 07 (3 H, s, H 3 _28), 1. 08 (3 H, s, H 3 - 29), 1. 21 (3 _{. H, s, H 3 _30} ), 1 22 (3 H, s, H 3 - 1 9), 2. 03 (3 H, s, OA c), 2. 1 8 (1 H, dd, J = 3.2, 1 3. OH z, H_5), 2.63 (1 H, dd, J = 3.8, 1 2. 7 H z, H-9), 3. 41 (1 H, s, H — 1 5), 3. 89 (1 H, s, H- 1 7), 4. 73 (1 H, t, J = 2.8 H z, H-7), 5. 88 (1 H, d , J = 1 0.4 H z, H_22), 6. 24 (1 H, m, H-22), 7. 16 (1 H, d, J = 1 0. OH z, H — 2), 7. 40 (1 H, t, J = 1.8 Hz, H-23), 7. 56 (1 H, m, H-21).

Compound (2) was identified by comparing ¹ H NMR and MS data with literature value ²⁾ .

Gedunin (2) High resolution ES I—MS mZz 505. 2235 [C ₂₈ H ₃ -0 ₇ N a [M + N a] +: 505. 2202]. H-NMR (40 _{OMH z, CDC I 3):} ά 1. 07 (3 H, s, H 3 _28), 1. 08 (3 H, s, H 3 - 29), 1. 1 6 (3 H, s, H 3 - 1 8), 1. 23 ( 3 H, s, H 3 - 1 9), 1. 25 (3 H, s, H 3 _30), 2. 1 1 (3 H

, s, OAc), 2. 1 7 (1 H, d d, J = 2.4, 1 3.4 H z, H ―

5), 2.49 (1H, dd, J = 6.1, 12.7Hz, H-9), 3

53 (1 H, s, H_ 1 5), 4.56 (1 H, d d, J = 2. 2, 3 3

H z, H-7), 5.62 (1 H, s, H- 1 7), 5.8 7 (1 H, d

J = 1 0. 2 H z, H-2) 0), 6. 34 (1 H, b r. S, H-22) 7

1 0 (1 H, d, J = 1 0. 2 H z, H- 1), F. 42 (2 H, d J

= 1.5 Hz, d 23).

Compound (3) is a known compound, but since there is no literature describing the spectral values, the structure was confirmed by analyzing two-dimensional (2D) -NMR.

1 7 _Epi 1 7 _Hydroxyazadiradione (3) White crystal. IR (KB r) V cm- ¹ : 3462 (0_H), 2960, 2878 (C_H), 1 736, 1 7 1 6 (C = 0 a, β—unsaturated enone), 1 667 (C = 0 ester), 1 591 (C = C), 1 245, 1 028 (C—O ester). [Hi] _D : _57 1 ° (c O. 21, black mouth form). High resolution ES I—MS mZz 489. 2246 [C ₂₈ H ₃₄ 0 ₆ N a [M + N a] +: 489. 2253]. U VA _max ^EtOH . nm (lo ε): 233 (4. 1 8) H-NMR (600MH z, CDCI 3): ά 1. 086 (3 H, s, H 3 - 28), 1. 093 (3 H, s, _{H 3 - 29), 1. 1} 9 (3 H, s, H 3 - 1 9), 1. 27 (3 H, s, H 3 - 1 8), 1. 37 (3 H, s, H 3 -30), 1.59 (2H, m, H ₂ _ 1 2), 1.69 (1 H, m, H) S_ 1 1), 1. 92 (1 H, m, Hd? -6), 1. 96 (3 H, s, O Ac), 1. 98 (1 H, m, HQ?-1 1), 2. 01 (1 H, m, H-6), 2. 21 (1 H, dd, J = 2. 7, 1 3.1 Hz, H_5), 2.43 (1 H, dd, J = 6. 9, 1 1.9 Hz, H-9), 5.34 (1 H, t, J = 3.1 H z, H-7), 5. 89 (1 H, d, J = 1 0. 3 H z , H-2), 6. 02 (1 H, s, H- 1 5), 6. 23 (1 H, dd, J = 0.7, 1. 9 H z, H-22), 7.1 2 (1 H, d, J = 1 0.3 H z, H- 1), 7. 1 9 (1 H, t, J = 1.4 H z, H-23), 7. 3 2 (1 H, t, J = 1. 7 H z, H-21). ³ C-NMR (15 OMH z, CDC I ₃ ): 51 5. 8 (C- 1 1), 1 9. 0 (C- 1 9), 20. 9

(-O-CO-Me), 21.2 (C—28), 23.5 (C_6), 25.0 (C- 1 8), 25.5 (C- 1 2), 27.0 ( C- 29), 27.

2 (C-30), 38.4 (C-9), 40.0 (C—10), 44.0 (C-4), 44.8 (C-8), 46.1 (C_5) , 51.8 (C— 1 3), 73.8 (C-7), 84.7 (C- 1 7), 19.5 (C—22)

, 1 21.0 (C- 15), 1 25.9 (C-2), 1 27.1 (C—20), 1 39.7 (C-23), 1 42.9 (C_21), 1 56. 7 (C_ 1), 1 699.7 (-O-CO-Me), 193.4 (C— 1 4), 203.9 (C-3), 206.4 (C-1 6).

[0023] Compound (4) was identified by comparing ¹ H NMR and MS data with literature value ³⁾ .

7 _0_Benzylninposinol (4) ES I _MS: mZz 51

3 [M + H] +, 535 [M + Na] +, 551 [M + K] +. ¹ H-NMR (

400MH z, CDC I ₃ ) δ ^. 00 (3 H, s, H-30), 1.03 (3 H, s, H-28), 1.1 1 (3 H, s, H-29) , 1. 31 (3

H, s, H- 1 9), 1. 42 (3 H, s, H_ 1 8), 3.41 (1 H, s, H- 1 7), 5. 63 (1 H, t, J = 2. 7 H z, H_7), 5. 9 2 (1 H, d, J = 1 0.2 H z, H-2), 5. 94 (1 H, s, H_ 1 5), 6. 20 (1 H, m, H-22), 7. 1 9 (1 H, d, J = 1 0.2 H z, H- 1), 7. 39 (4 H, m, H-21, H- 23, H_3 ', H-5'), 7. 54 (1 H, m, H_4,), 7. 91 (2H, dd, J = 1.0, 8.3 H z, H-2 ', H -6 ').

[0024] References 1) D. L ave ECL evy, Me I iane― Me Macin Re I ationshi P, T etrahedron 2 7 (1 6)

, 3 94 1 -394 7 (1 9 7 1)

2) S. A. K h a I i d H. D u d d e c k, M G S i e r r a

, I so o I a t i O n a n d C h a r a c t e r i z a t i o n o f a n A n t i m a I a r i a I A g e n t o f t h e N e e m

T r e e A z a d i r a c h t a i n d i c a J N at. P ro d., 52 (5) 9 2 2 ― 9 27 (1 9 89)

3) W. K r a u s R C r a m e r, G. S a w i t z k i, D.

Example 2 (Anti-tumor activity)

Antitumor activity was measured by the MT T (3- (4,5_dimethylthiazole_2_yl) -2,5-diphenyltetrazolium promide) method.

A 96-we II plate was seeded with 100 L of tumor cells (3 X 10 ³ ce II Zw el I) suspended in RPM I 1 640 medium and cultured for 24 hours (5% CO 2, 37 ° C, saturated) Condition). Thereafter, the test compounds were added to the (final concentration 1 X 1 0- 1 X 1 0- 5, 1 X 1 0_ 6 M) and respective One not a 0. 2 L of DMSO as control was applied to the 48 hours the tumor cells . Next, 10 L of 0.5% MTT solution was added, and 3 hours later, 100 L of a reaction stop solution (0.04 NHCIZ isopropanol) was added to stop the reaction. After pipetting well, absorbance at 570 nm (top) and 655 nm (bott orn) was measured with a microplate reader. The cell viability at each concentration of each specimen was determined as a percentage of the control group, and the EC ₅₀ value was calculated therefrom. As tumor cells, malignant melanoma CR L 1 579 and leukemia cells Jurkat cells were used. Cisbratin was used as a positive control. As a result, as shown in Table 1, the compounds (1) to (4) were found to have a strong antitumor activity equivalent to that of cisbratin, which is known as a potent antitumor agent.

[0027] [Table 1] Antitumor activity (EC ₅₀ : M)

Compound (1) Compound (2) Compound (3) Compound (4) Sysf. Latin

CRL1579 67.8 36.7 19.1 4.04 21.1 (Malignant melanoma)

Jurkat 6.68 6.00 20.9 2.06 5.58 (leukemia)

[0028] Example 3 (apoptosis induction ability)

Apoptosis-inducing ability was tested by nuclear staining with Hoec h st 33342 staining.

6-we II plate cells suspended in RPM I 1 640 medium (CRL 1 579: 2 xl 0 5 cel I Zw e II) were plated 2m L, 24 hours of culture _{(5% C0 2, 37 °} C, saturated Under wet conditions). Then, test compound (final concentration _{1 X 1 0 - 4, l} X l O- 5, 1 X 1 0- 6 M) was added to each and DMSO as a control by 4 L, allowed to act on cells for 24 hours. Next, 100 L of 0.05% Hoechst 33342 solution was added, and after 30 minutes, a phase contrast image and a fluorescence image were photographed with a fluorescence microscope to observe changes in cell morphology.

As a result, both compounds (3) and (4) were 1 to 10 M, and nuclear aggregation and fragmentation were observed. Therefore, cytotoxic activity against CRL 1 579 and Jurkat cells was induced by apoptosis induction. Turned out to be

Claims

The scope of the claims

[1] A therapeutic agent for malignant tumors comprising as an active ingredient a compound selected from epoxiazadiradione, gedunin, 17-epi_17-hydroxyzaziradione and 7_0-benzoylninpocinol.

[2] An apoptosis inducer comprising as an active ingredient a compound selected from epoxiazadiradione, gedunin, 17-epi_17-hydroxyzaziradione and 7_0-benzoylninpocinol.

[3] of a compound selected from the group consisting of epoxyazadiradione, gedunin, 17-epi _17-hydroxyzaziradione and 7_0_benzoylninposinol

Use for manufacturing malignant tumor therapeutics.

[4] of a compound selected from epoxiazadiradione, gedunin, 17-epi _17-hydroxyzaziradione and 7_0_benzoylninposinol

Use for the manufacture of apoptosis inducers.

[5] A method for treating a malignant tumor, comprising administering a compound selected from epoxiazadiradione, gedunin, 17-epi_17-hydroxyzaziradione, and 7_0-benzoylninposinol.

[6] A method for inducing apoptosis, comprising administering a compound selected from epoxiazadiradione, gedunin, 17-epi_17-hydroxyzaziradione and 7_0-benzoylninposinol.