JP2850716B2 - Novel vitamin D3 analogs - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel vitamin D ₃ analogs with modulating effect and differentiation inducing action of tumor cells in vivo calcium.

[0002]

Is the Background of the Invention in vivo metabolite of vitamin D _3,
It is known that 1α, 25-dihydroxyvitamin D ₃ , which is known as active vitamin D ₃ , has an action of promoting calcium absorption from the intestine and is effective as a therapeutic agent for bone lesions and the like. Recently, this active form of vitamin D ₃ and its analogs have a differentiation-inducing effect of returning cancerous cells to normal cells (Hirofumi Tanaka et al .: Biochemistry 55, 1323, 1923).
1983), and in fact, some of them have the effect of inhibiting the progression of cancer (KW Colton, et. Al., Lan .
cet ., Jan. 28 , p. 188, 1989). However, active vitamin D ₃ has a strong effect on calcium metabolism,
These are undesired side effects when using Class ₃ as an anticancer drug. General formula:

Embedded image (Wherein, R, R ₁ and R ₂ independently represent a hydrogen atom or an acyl having 1 to 4 carbon atoms). A hexafluorovitamin D derivative having a vitamin D ₃ -like activity represented by WO83 / 00335 (PCT / US82 / 00909)
It is described in.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have found that there are few side effects, excellent effects on calcium metabolism in a living body, that is, no hypercalcemia, Research was conducted for the purpose of creating a novel vitamin D ₃ analog having an excellent cell differentiation-inducing action, and a vitamin D ₃ having desired properties was found, thereby completing the present invention. An object of the present invention is to provide a novel vitamin D ₃ analog having a pharmacological action, particularly a calcium regulation based on a cell differentiation inducing action and an antitumor action. Another object of the present invention is to provide a method for producing the vitamin D ₃ analog. These objects and the advantages provided by the present invention will be apparent to those skilled in the art from the following description.

The novel vitamin D ₃ analog provided by the present invention has a general formula:

Embedded image (Where R ¹ is a hydrogen atom and R ² is a hydroxyl group, or R ¹ is a hydroxyl group and R ² is a hydrogen atom, X is a hydrogen atom, a hydroxyl group or a hydroxyl group protected by a hydroxyl-protecting group, and R ³ is a hydrogen atom Or a protecting group for a hydroxyl group). Here, as the protecting group for the hydroxyl group, a trimethylsilyl group,
Examples include silyl ether-based protecting groups such as a t-butyldimethylsilyl group and a t-butyldiphenylsilyl group.

Specific examples of the compound represented by the general formula [I] include: 26,26,26,27,27,27-hexafluoro-2
2S, 25-dihydroxyvitamin D ₃ [R ¹ is OH, R ²
Is H, R ³ is H and X is H. [I]] (Compound A ₁ ) 26,26,26,27,27,27-hexafluoro-2
2R, 25-dihydroxyvitamin D ₃ [Compound [I] wherein R ¹ is H, R ² is OH, R ³ is H and X is H] (Compound A ₂ ) 26,26,26,27,27,27 -Hexafluoro-1
α, 22S, 25-trihydroxyvitamin D ₃ [R ¹ is O
A compound wherein H and R ² are H, R ³ is H and X is OH
[I]] (Compound B ₁ ) 26,26,26,27,27,27-hexafluoro-1
α, 22R, 25-trihydroxyvitamin D ₃ [R ¹ is H, R ² is OH, R ³ is H and X is OH
[I]] 3-t- butyl (Compound B ₂₎ Compound A ₁ or Compound 3-t-butyldimethylsilyl ether compounds A ₂ 3-trimethylsilyl ether compounds A ₁ or Compound A ₂ A ₁ or Compound A ₂ diphenyl silyl ether compound B ₁ or the compound B ₂ 1α, 3- bis (trimethylsilyl ether) of compound B ₁ or the compound B ₂ 1α, 3- bis (t-butyldimethylsilyl) ether compound B ₁ or the compound B ₂ l [alpha] , 3-bis (t-butyldiphenylsilyl) ether, but is not limited thereto.

The compound [I] of the present invention can be produced by various methods. One example of the best method is shown below. That is, the general formula [II]:

Embedded image (Wherein, R ⁴ is OR ⁶ and R ⁵ is a hydrogen atom, or R ⁴ is a hydrogen atom and R ⁵ is OR ⁶ , R ⁶ is a hydroxyl-protecting group) and a ketone compound represented by the general formula Formula [III]:

Embedded image (Wherein, R ⁷ represents a protecting group for a hydroxyl group, Y represents a hydrogen atom or OR ⁷ and Ph represents phenyl), and subjected to a coupling reaction with an anion derived from a phosphine oxide represented by It is obtained by deprotection as required. Derivation of the phosphine oxide to the anion is achieved in the presence of a base, and the base used is n-
Alkyl lithium such as butyl lithium is preferred.

The above-mentioned coupling reaction between the compound [II] and the compound [III] is carried out at a low temperature, for example, -100 ° C to -50 ° C.
Preferably at −80 ° C. to −20 ° C., under an inert atmosphere (eg, under an argon atmosphere), in an ether solvent (eg, diethyl ether, tetrahydrofuran (THF), etc.) for 10 minutes to 24 hours, preferably 30 minutes. Perform for ~ 2 hours.
The resulting product [I] can be purified by a known method such as silica gel chromatography. Compound
Deprotection of the hydroxyl group from [I] can be performed by a known method.

The protecting group R for a hydroxyl group in the general formula [II]⁶
Is a silyl protecting group such as t-butyldimethylsilyl group or
Acyl protecting groups such as cetyl are preferred. Formula [II
I] a protecting group R for a hydroxyl group⁷Is t-butyldimethylsiloxane
Preferred are silyl-based protecting groups such as a ril group. Coupling above
The production method of the starting compound [III] used in the
Giorgini et al. (EG Baggiolini et al.),J.Am.Che
m.Soc., 104, 2945, 1982
It is disclosed in Japanese Unexamined Patent Publication No. 2-250844. On the other hand,
Another starting material [II] can be produced by the following reaction steps.
Can be. Here, R^FourIs t-butyldimethylsilicone
[II] (compound IIa) and R^FiveIs t
[II] which is -butyldimethylsilyl (compound
IIb) is prepared.

Embedded image

Embedded image (Where Ph is phenyl, Bn is benzyl, MO
M is methoxymethyl, TBDMS is t-butyldimethylsilyl, TBDSTf is t-butyldimethylsilyl triflate and PCC is pyridinium chlorochromate)

Following the above reaction steps, the starting material [IIa]
And [IIb] can be produced. First, the aldehyde compound (1) is reacted with the compound (A) to obtain a compound (2), and the compound (2) is oxidized to obtain a compound (3). The compound
Reaction of (3) with SmI ₂ to give compound (4)
Reduction of (4) gives compound (5). The compound (5) is isomerized to obtain compounds (6) and (7), and then the hydroxyl groups are protected to obtain compounds (8) and (10). The hydroxyl-protecting group is removed from the compounds (8) and (10), and finally the obtained compounds (9) and (11) are oxidized. Detailed reaction conditions of the above reaction step are described in Reference Examples 1 to 8 described below. Hereinafter, the present invention will be specifically described with reference to Examples and Test Examples, but the present invention is not limited thereto. Each compound listed in Examples and Test Examples is assigned a number, and this number corresponds to the compound number assigned to each compound in the reaction step.

[0010]

EXAMPLE 1 26,26,26,27,27,27-Hexafluoro- by the Wittig reaction of compound [II] with compound [III]
Synthesis of 1α, 22S, 25-trihydroxyvitamin D ₃ (Compound B ₁ ): R ⁷ is t-butyldimethylsilyl;
Compounds wherein Y is t-butyldimethylsilyloxy [I
II] (1060 mg) in anhydrous tetrahydrofuran (THF)
N-Butyllithium (2.5 M, hexane solution, 68 ml) was added dropwise to the (10 ml) solution at -78 ° C.
Stir for minutes. Compound [IIa] (8) wherein R ⁴ is t-butyldimethylsilyloxy and R ⁵ is a hydrogen atom is added to the reaction mixture.
6 mg) in anhydrous tetrahydrofuran (5 ml) are added in one portion. The mixture is warmed to room temperature and then stirred for 10 minutes. A saturated aqueous ammonium chloride solution was added to the reaction mixture,
Extract with ethyl acetate. The ethyl acetate layer is washed with water and dried over anhydrous magnesium sulfate. After evaporating the solvent, the residue was purified by column chromatography, and 26,26,26,27,2
7,27-Hexafluoro-1α, 22S, 25-trihydroxyvitamin D ₃ 1α, 22S, 25-tris (t-
Butyldimethylsilyl) ether is obtained. The tris (t-butyldimethylsilyl) ether obtained above is stirred at room temperature for 8 days in a methanol suspension of an acidic ion exchange resin (50 W × 4). The ion exchange resin is filtered off. After evaporating the solvent under reduced pressure, the residue was subjected to column chromatography (Si
Purification with O ₂ , eluent; ethyl acetate-n-hexane) gave the title compound (19.8 mg) as colorless needles. mp 187.7-181.8 ° C (ether-hexane). ¹ H-NMR (CD ₃ OD) δ: 0.59 (s, 3H), 0.94
(d, J = 6.6 Hz, 3 H), 1.20-3.70 (m, 25 H),
4.14 (m, 1H), 4.37 (m, 1H), 4.88 (s, 1H),
5.30 (s, 1H), 6.10 (d, J = 10.4 Hz, 1H), 6.
33 (d, J = 10.4 Hz, 1H). IR (KBr): 3418, 2943, 1214 cm ^-1 .

Reference Example 1 Synthesis of compound (2) by reaction of compound (1) with compound (A): solution of compound (A) (1.7 g) in anhydrous tetrahydrofuran
(5 ml) was added to a hexane solution of n-butyllithium (2.39).
M, 1.8 ml) at −78 ° C. and the mixture
Stir for 0 minutes. Next, compound (1) (1 g) was added to the mixture.
Of anhydrous tetrahydrofuran (5 ml) is added and the mixture is stirred at -78 ° C for 1 hour. The reaction mixture was added to a saturated aqueous ammonium chloride solution, and extracted with diethyl ether.
The ether layer is dried over anhydrous magnesium sulfate. After evaporating the solvent, the residue was purified by column chromatography (SiO ₂ ) to obtain compound (2) (1.91 g). Physical properties of compound (2) are as follows. IR (neat): 3529, 2936, 2876, 1218c
m ^-1 .

Reference Example 2 Synthesis of compound (3) by oxidation of compound (2): DM solution was added to a solution of oxalyl chloride (340 μl) in methylene chloride (20 ml).
A solution of SO (360 μl) in methylene chloride (2 ml) was added at −78 ° C.
And the mixture is stirred for 10 minutes. Then, a solution of compound (2) (1.32 g) in methylene chloride (10 ml) was added to the mixture.
Is added dropwise, and the mixture is stirred at -78 ° C for 15 minutes and at 45 ° C for 1 hour. Triethylamine (2.1 ml) was added to the reaction mixture.
Is added and the mixture is stirred at 0 ° C. for 20 minutes. The reaction mixture is added to a saturated aqueous solution of ammonium chloride and extracted with diethyl ether. The ether layer is dried over anhydrous magnesium sulfate. After concentration of the filtrate, the residue was purified by column chromatography (SiO ₂ ) to obtain compound (3) (839 mg). Physical properties of compound (3) are as follows. IR (neat): 2937, 2879, 1723, 1210c
m ^-1 .

Reference Example 3 Synthesis of compound (4) by reaction of compound (3) with SmI ₂ : Compound (3) (611 mg) obtained in Reference Example 2 was treated with anhydrous tetrahydrofuran (14 ml) and methanol (3.6 ml). mixture was added 0.1M tetrahydrofuran solution of nourishment of samarium (SmI ₂₎ (20ml) was at -78 ° C., 30 minutes the mixture at the same temperature, and stirred for 10 min at 0 ° C.. The reaction mixture is added to a saturated aqueous solution of ammonium chloride, extracted with diethyl ether, and the ether layer is dried over anhydrous magnesium sulfate. After evaporating the solvent, the residue was subjected to column chromatography (Si
O ₂ ) to give compound (4) (279 mg). Compound
The physical properties of (4) are as follows. Colorless oil. ¹ H-NMR (CDCl ₃ ) δ: 0.98 (s, 3H), 1.09
(d, J = 6.9 Hz, 3H), 1.15-2.90 (m, 17H),
3.45 (s, 3H), 3.72 (m, 1H), 4.35 (d, J = 1
2.5 Hz, 1 H), 4.61 (d, J = 12.5 Hz, 1 H), 4.
91 (s, 2H), 7.20-7.40 (m, 5H). IR (neat): 2935, 1717, 1213 cm ^-1 .

Reference Example 4 Synthesis of compound (5) by reduction of compound (4): To a solution (1 ml) of compound (4) (279 mg) obtained in Reference Example 3 in ethanol was added sodium borohydride (33 mg). The mixture is stirred at room temperature for 18 hours. The reaction mixture is added to ice water and extracted with diethyl ether. The ether layer is washed with water and dried over anhydrous magnesium sulfate. After evaporating the solvent, compound (5)
Get. This compound is used as it is in the next step.

Reference Example 5 Synthesis of Compounds (6) and (7) by Isomerization of Compound (5): To a dioxane solution (5 ml) of compound (5) obtained in Reference Example 4 was added concentrated hydrochloric acid (500 μl). The mixture is stirred at 65 ° C. for 6 hours. Dilute the reaction mixture with diethyl ether, then
The ether layer is washed with an aqueous sodium hydrogen carbonate solution and saturated saline, and dried over anhydrous magnesium sulfate. After evaporating the solvent, the residue was purified by column chromatography (SiO ₂ ) to obtain compound (6) (164 mg) and compound (7) (54 mg). Physical properties of compound (6) are as follows. ¹ H-NMR (CDCl ₃ ) δ: 0.94 (d, J = 6.8 Hz, 3
H), 0.98 (s, 3H), 1.10-2.45 (m, 17H), 3.
73 (m, 2H), 4.36 (d, J = 11.9 Hz, 1H), 4.6
3 (d, J = 11.9 Hz, 1 H), 6.58 (s, 1 H), 7.25
-7.40 (m, 5H). Physical properties of compound (7) are as follows. ¹ H-NMR (CDCl ₃ ) δ: 0.95 (d, J = 6.8 Hz, 3
H), 0.98 (s, 3H), 1.10-2.45 (m, 17H), 3.
73 (m, 2H), 4.34 (d, J = 11.9 Hz, 1H), 4.6
3 (d, J = 11.9 Hz, 1 H), 6.74 (s, 1 H), 7.25
-7.40 (m, 5H).

REFERENCE EXAMPLE 6 Synthesis of compound (8) by protecting hydroxyl group of compound (6): 2,6-lutidine (100 μl) and a solution of compound (6) (130 mg) obtained in reference example 5 in anhydrous methylene chloride were added. t-Butyldimethylsilyl triflate (120 μl) is added at 0 ° C. and the mixture is stirred at room temperature for 13 hours. The reaction mixture is added to ice water and extracted with methylene chloride. The methylene chloride layer is washed with water and dried over anhydrous magnesium sulfate. After evaporating the solvent, the residue was purified by column chromatography (SiO ₂ ) to obtain Compound (8) (164 mg). This compound is used as it is in the next step.

Reference Example 7 Synthesis of compound (9) by deprotection of the hydroxyl group of compound (8):
Compound (8) (164 mg) obtained in Reference Example 6 was dissolved in hydrogen gas at 5
Stir at room temperature and atmospheric pressure for 12 hours in the presence of% Pd-C catalyst (10 mg) / methanol (10 ml). After removing the catalyst by filtration and concentrating the filtrate, the residue was purified by column chromatography (SiO ₂ ) to obtain Compound (9) (120 mg). Physical properties of compound (9) are as follows. Colorless oil. ¹ H-NMR (CDCl ₃ ) δ: 0.07 (s, 3H), 0.09
(s, 3H), 0.92 (d, J = 6.9 Hz, 3H), 1.10 −
2.05 (m, 18H), 3.59 (m, 1H), 3.75 (s, 1
H), 4.09 (m, 1H). IR (neat): 3625,3296,2953,121
0,836,774 cm ^-1 .

Reference Example 8 Synthesis of Compound [IIa] by Oxidation of Compound (9): A solution of compound (9) (170 mg) obtained in Reference Example 8 in anhydrous methylene chloride.
(5 ml) was added dropwise to a suspension of pyridinium chlorochromate (400 mg) in anhydrous methylene chloride (5 ml) and the mixture was stirred at room temperature for 2 hours.
Stir for hours. The reaction mixture is extracted with diethyl ether, and the ether layer is washed with water and dried over anhydrous magnesium sulfate. After evaporation of the solvent, the residue is subjected to column chromatography.
(SiO ₂ ) to give Compound [IIa] (152 mg).
Physical properties of compound [IIa] are as follows. Colorless oil. ¹ H-NMR (CDCl ₃ ) δ: 0.07 (s, 3H), 0.08
(s, 3H), 0.65 (s, 3H), 0.90 (s, 9H),
0.94 (d, J = 6.9 Hz, 3H), 1.55-2.50 (m, 1
7H), 3.59 (m, 1H). IR (neat): 3400, 2950, 1694, 839,
774 cm ^-1 .

Test Example 1 Test Method for Inducing Cell Differentiation Intracellular cells derived from human colon cancer (HT-29) were used for tissue culture.
R inoculated in a 4-well plate and supplemented with 10% calf serum
Cultured in PMI / 1640. After about 24 hours, the culture supernatant was removed and 2 × 10 ⁻³ M sodium butyrate and 1α,
A culture solution containing 25-dihydroxyvitamin D ₃ or a vitamin D ₃ analog as a compound of the present invention is added (culture medium exchange), and the mixture is placed in a carbon dioxide incubator (37 ° C., 5% carbon dioxide-95).
% Air). The culture medium having the same composition was exchanged every two days, and on the seventh day, the number and the morphology of the mucus-producing cells were determined by the method of Augron et al. ( Cancer Res ., 44 , 396).
1 (1984)). Mucus production is found in normal colon (colon) cells, but is rarely found in these cancerous HT-29 cells. Therefore, the number of mucus-producing cells was counted as a quantitative marker that the cancer cells HT-29 came to express the traits of the induced normal cells,
The percentage with respect to the cell number of 200 was determined.

Test results

Table 1 Activity test measured value Test compound Concentration (M) Mucus-producing cells (%) * None (solvent ethanol only) 0 34 ± 71α, 25-dihydroxyvitamin D ₃ 10 ^-794 ± 51α, 25-dihydroxy vitamin ^{_{D 3 10 -8 40 ± 10 1α}} , 25- dihydroxyvitamin D ₃ 10 ^-9 29 ± 1 compound B ₁ 10 ^-7 93 ± 6 compound B ₁ 10 ^-8 92 ± 2 compound B ₁ 10 ^-9 77 ± 6 Compound B ₂ 10 ⁻⁷ 78 ± 2 Compound B ₂ 10 ⁻⁸ 42 ± 9 Compound B ₂ 10 ⁻⁹ 28 ± 10 *) n = 5 From the results in Table 1 above, it was found that Compound ₁ of the present invention provided It can be seen that more HT-29 cells are induced to differentiate into mucus-producing cells as compared to 25-dihydroxyvitamin D ₃ .

Continued on the front page (58) Fields investigated (Int. Cl. ⁶ , DB name) C07C 401/00 A61K 31/59 CA (STN) CAOLD (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] 1. A compound of the general formula [I]: (Where R ¹ is a hydrogen atom and R ² is a hydroxyl group, or R ¹ is a hydroxyl group and R ² is a hydrogen atom, X is a hydrogen atom, a hydroxyl group or a hydroxyl group protected by a hydroxyl-protecting group, and R ³ is a hydrogen atom vitamin D ₃ analogs represented by or a hydroxyl group is a protecting group). 2. The method according to claim 1, wherein the protecting group for the hydroxyl group is trimethylsilyl, t
The compound according to claim 1, which is selected from the group consisting of -butyldimethylsilyl and t-butyldiphenylsilyl. 3. 26,26,26,27,27,27-hexafluoro-1α, 22S, 25-trihydroxyvitamin D ₃ ; 26,26,26,27,27,27-hexafluoro-1
α, 22R, 25-trihydroxyvitamin D ₃ ; 26,26,26,27,27,27-hexafluoro-1
alpha, 22S, 25-trihydroxy vitamin D ₃ 1α, ₃
-Bis (trimethylsilyl) ether; 26,26,26,27,27,27-hexafluoro-1
α, 22R, 25- 1α trihydroxy vitamin D _3, 3
-Bis (trimethylsilyl) ether; 26,26,26,27,27,27-hexafluoro-1
alpha, 22S, 25-trihydroxy vitamin D ₃ 1α, ₃
-Bis (t-butyldimethylsilyl) ether; 26,26,26,27,27,27-hexafluoro-1
α, 22R, 25- 1α trihydroxy vitamin D _3, 3
-Bis (t-butyldimethylsilyl) ether; 26,26,26,27,27,27-hexafluoro-1
alpha, 22S, 25-trihydroxy vitamin D ₃ 1α, ₃
-Bis (t-butyldiphenylsilyl) ether; and 26,26,26,27,27,27-hexafluoro-1
α, 22R, 25- 1α trihydroxy vitamin D _3, 3
The compound according to claim 1, which is selected from -bis (t-butyldiphenylsilyl) ether.